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cc, coiled-coil; dapi, ′, -di- amidino- -phenylindole; dtt, dithiothreitol; elisa, enzyme-linked immunosor- bent assay; er, endoplasmic reticulum; ergic, er–golgi intermediate compart- ment; fbs, fetal bovine serum; gapdh, glyceraldehyde -phosphate dehydrogenase; gfp, green fluorescent protein; gms, galloway-mowat syn- drome; gst, glutathione s-transferase; ha, hemagglutinin; hrp, horseradish per- oxidase; il- β, interleukin- β; lap, localization and affinity purification; lcl, lym- phoblastoid cell line; mbp, maltose-binding protein; pbs, phosphate-buffered saline; pi( )p, phosphatidylinositol -phosphate; pwwca, polyproline-wh -wh - connector-acidic; rnai, rna interference; rt-pcr, reverse transcription-pcr; sirna, small interfering rna; snx, sorting nexin; tnfα, tumor necrosis factor α; wasp, wiskott-aldrich syndrome protein; wmd, whamm membrane-interac- tion domain; wt, wild type. alyssa j. mathiowetza,†, emma bapleb,†, ashley j. russoa, alyssa m. coultera, eric carranoc, judith d. brownc, robert n. jinksd, andrew h. crosbyb,‡, and kenneth g. campellonea,* adepartment of molecular and cell biology and cdepartment of allied health sciences, institute for systems genom- ics, university of connecticut, storrs, ct ; binstitute of biomedical and clinical science, university of exeter medical school, rild wellcome wolfson centre, exeter ex dw, uk; ddepartment of biology and biological foundations of behavior program, franklin and marshall college, lancaster, pa volume september , whamm founder mutation impairs autophagy | from complications from renal failure. genome-wide single nucleo- tide polymorphism genotyping mapped the responsible locus to a small autozygous region on chromosome q . -q . , and par- allel whole-exome sequencing revealed two novel homozygous variants within the region: a base pair deletion (c. delt; p.phe- leufs* ) in the last exon of wdr and a base pair deletion (c. _ delataaaag) in exon of whamm (jinks et al., ). both variants cosegregated according to autosomal-reces- sive disease status in affected patients and extended family members, with only one affected patient homozygous for the wdr variant and heterozygous for the whamm variant. this in- dividual displayed the cardinal neurological features of gms but died of a nonrenal cause, and no data on kidney involvement were available. this case provides evidence that homozygosity for the amish wdr mutation is primarily responsible for the clinical pre- sentation in this cohort (jinks et al., ). however, the molecular and cellular phenotypes arising from the whamm mutation have not been explored. cells from amish gms patients are deficient in whamm expression the canonical whamm gene is composed of coding exons giv- ing rise to a . kb transcript (figure a). to examine whether the base pair deletion at the ′ end of exon alters whamm transcript levels, we cultured primary dermal fibroblasts from amish gms pa- tients and healthy amish individuals, isolated rna from the sam- ples, and performed reverse transcription-pcr (rt-pcr). whamm mrna levels in homozygous gms/gms mutant cells were present at – % of the levels in +/+ normal cells (figure b), suggesting that the whammgms variant encodes a less stable transcript. given the position of the base pair deletion, it may destabilize whamm mrna by several mechanisms. as examples, a simple frameshift would result in a premature stop codon and possible non- sense-mediated decay, while a defect in splicing might also result in transcript degradation. to explore the effect of the amish wham- mgms mutation on the gene transcript, we used several primer pairs to amplify portions of exons – using cdna from amish +/+ and gms/gms fibroblasts (supplemental figure s a). with a plasmid control and +/+ cdna sample, all primer pairs yielded pcr products corresponding to the predicted length of a whamm+ mrna (sup- plemental figure s b). with an amish gms patient sample, pcr products spanning exons – , – , – , and – were similar in size to those from the control cells, consistent with a simple base pair deletion (supplemental figure s b). in contrast, a primer pair in ex- ons and produced a smaller-than-expected pcr product specifi- cally with the gms sample (supplemental figure s b). sequencing of this product indicated that exon was followed by an alternative exon from a predicted whamm rna variant named x (xm_ ). incorporation of this cryptic exon also results in a frameshift and premature termination codon. we have thus termed the deletion and alternatively spliced transcripts whammgmsΔ and whammgmsx , respectively (figure a). wild-type whamm mrna encodes an amino acid protein consisting of a whamm membrane-interaction domain (wmd), a microtubule-binding coiled-coil (cc) region, and a polypro- line-wh -wh -connector-acidic (pwwca) segment that promotes actin nucleation in conjunction with the arp / complex (figure c). in contrast, whammgmsΔ and whammgmsx , if translated, would lead to truncated protein products that possess part of the cc re- gion but none of the pwwca motifs (figure c and supplemental figure s c). to examine whamm protein expression, we gener- ated lymphoblastoid cell lines (lcls) from control amish individuals, proteins, jmy can be recruited to the plasma membrane and en- hances epithelial cell motility (zuchero et al., ), but it also influ- ences other intracellular functions such as transport from the trans- golgi (schluter et al., ). finally, whamm is found on membranes of the secretory pathway, including the endoplasmic reticulum (er), cis-golgi, and er–golgi intermediate compartment (ergic), where it promotes membrane tubulation and trafficking (campellone et al., ; blom et al., ; russo et al., ). wasp-family pro- teins appear to be crucial for mammalian development, because targeted disruptions of the mouse genes encoding n-wasp, wave , or wash are embryonic lethal (campellone and welch, ; gomez et al., ). whether the whamm and jmy proteins, which are % similar to one another, are also essential has yet to be determined. recently whamm and jmy have been implicated in macroau- tophagy (autophagy) (coutts and la thangue, ; kast et al., ). autophagy is a mechanism of cytoplasmic digestion involving double membrane–bound compartments called autophagosomes that fuse with lysosomes and is crucial in cellular homeostasis, stress responses, and development (mizushima and levine, ; yang and klionsky, ; choi et al., ; carlsson and simonsen, ). roles for actin and the arp / complex in autophagy in mammalian cells were obscure until actin and cortactin, a stabilizer of arp / branchpoints, were found to influence this process (lee et al., ; aguilera et al., ). functions for wasp-family members have also emerged. wash can exert positive or negative effects on autophagy (xia et al., ; zavodszky et al., ), perhaps due to its participation in trafficking multiple types of endosomal cargo. moreover, whamm-mediated arp / activation can drive autopha- gosomal rocketing (kast et al., ), while jmy can interact with the autophagosomal protein lc and affect its expression and localiza- tion (coutts and la thangue, ). interestingly, actin assembly was also recently revealed to take place at the interior of forming au- tophagosomes, and to be dependent on the phospholipid phos- phatidylinositol -phosphate (pi( )p; mi et al., ). however, the connections between pi( )p, actin nucleation factors, and autopha- gosomal biogenesis, as well as the physiological importance of actin assembly in autophagy, have not come into focus. in the current study, we investigated the effects of a frameshift deletion in whamm that occurs alongside a wdr mutation in amish patients with galloway-mowat syndrome (gms; also called nephrocerebellar syndrome) (jinks et al., ). both founder muta- tions are coinherited in a homozygous manner in almost all cases due to their close proximity on chromosome . given that prelimi- nary characterizations of wdr have been undertaken (jinks et al., ), the current study aimed to define the outcome of the whamm mutation in amish gms patient cells and to uncover the mechanisms underlying whamm function during autophagy. results clinical features and genetic basis of amish gms amish gms is an autosomal-recessive condition that was initially clinically delineated in individuals (jinks et al., ) and is phe- notypically consistent with wdr -associated gms that occurs outside the amish community (colin et al., ; ben-omran et al., ; vodopiutz et al., ). shortly after birth, affected amish children typically presented with irritability and roving eye move- ments. psychomotor development was severely delayed. common associations included progressive microcephaly with cerebellar at- rophy, hypotonia, extrapyramidal movements, visual impairment with optic atrophy, multifocal seizures, and steroid-resistant ne- phrosis. premature death in childhood was common, most often | a. j. mathiowetz, e. baple, et al. molecular biology of the cell that recognize the c-terminal wwca do- main indicated that lcls from carriers ex- pressed approximately half the amount of full-length whamm compared with lcls from wild-type individuals (figure d). moreover, no full-length whamm was de- tected in lcls or fibroblasts from amish gms patients (figure d). to explore whether truncated whamm proteins might be expressed from the whammgmsΔ or x transcripts, we exam- ined amish gms patient fibroblasts by im- munofluorescence using antibodies to the whamm cc domain. while control cells exhibited a prominent golgi-like staining pattern as expected, cells from gms patients stained less intensely in the golgi region and displayed a more dispersed reticular signal (figure e). total whamm fluorescence in gms fibroblasts averaged less than half the level found in normal cells (figure e), de- spite an apparent increase in nuclear fluores- cence (figure a). it is not clear whether the residual whamm staining is due to expres- sion of the whamm(Δ ), whamm(x ), and/ or other proteins derived from alternative splicing, as any such truncations would retain nearly half of the cc region that is recog- nized by the fluorescently labeled antibody (campellone et al., ). nevertheless, ha (hemagglutinin)- and lap (localization and affinity purification)-tagged versions of whamm(Δ ) and/or whamm(x ) were ca- pable of transient expression in cos cells and stable expression in nih t cells follow- ing cdna transfections (figure , b–d). con- sistent with the staining pattern of endoge- nous whamm in gms patient cells, the tagged mutants localized more diffusely than tagged whamm(wt) in the transfected cell lines (figure , c and d). together these data indicate that amish gms patient cells lack the full-length whamm protein but appear to harbor a residual amount of at least one truncated whamm variant. amish whamm truncations do not promote actin nucleation but associate with microtubules the two best-characterized molecular func- tions of whamm are arp / -dependent actin nucleation via its pwwca segment (campellone et al., ) and microtubule binding via its cc region (shen et al., ). the whamm(Δ ) variant is predicted to contain the first amino acids of whamm followed by inappropriate residues after the frameshift, while whamm(x ) is pre- dicted to contain the first amino acids of whamm plus additional residues (figure c). therefore both truncated whamm derivatives lack roughly half of the microtubule- binding region and are completely devoid of the motifs required for heterozygous carriers of the whammgms and wdr gms variants, and gms patients homozygous for both mutations. consistent with expectations based on gene dosage, immunoblots using antibodies figure : cells from amish gms patients encode truncated whamm variants. (a) diagrams of the exon organization in wild-type whamm cdna and in the Δ and x variants are shown. start and stop codons are indicated in green and red, respectively. (b) rna was isolated from normal (+/+) or amish gms patient (gms/gms) fibroblasts and subjected to rt-pcr with primers to exons – of whamm or to β-actin. relative levels of whamm-specific products (mean + sem from three to four experiments) are shown. ***, p < . (t tests). (c) the -residue whamm(wt) protein includes a wmd that interacts with membranes, a cc region that binds microtubules (mts), and a c-terminal pwwca segment that promotes actin nucleation. the gms Δ and x variants include the n-terminal or amino acids of whamm followed by or additional residues after the respective frameshifts. (d) lymphoblastoid cell lines and skin fibroblasts from homozygous unaffected (+/+), heterozygous (+/gms), or homozygous affected (gms/gms) individuals were subjected to immunoblotting with anti-whamm (left), anti-wwca (right), and anti-tubulin antibodies. relative levels of whamm(wt) were determined by densitometry of a representative blot. (e) wild-type (+/+) or gms patient (gms/gms) fibroblasts were stained with antibodies to visualize whamm (green) and microtubules (red), and with dapi to label dna (blue). the fluorescence intensity of whamm staining (mean ± sem from – cells per genotype) was lower in patient cells, p < . (t test). scale bar: μm. volume september , whamm founder mutation impairs autophagy | sence of its known c-terminal actin- and arp / -binding motifs, whamm does not promote actin nucleation. for testing micro- tubule binding, we polymerized tubulin in vitro and performed low-speed and high- speed microtubule cosedimentation assays (shen et al., ) with mbp-whamm(wt), mbp-whamm(Δ ), and mbp-whamm(x ). despite the fact that the Δ mutant was missing part of its cc region, it copelleted with microtubules almost as well as wild- type whamm (figure c), indicating that this construct retains significant microtu- bule-binding activity. unfortunately, we were unable to assess whether the x mu- tant bound to microtubules, as this protein largely pelleted by itself (figure d), sug- gesting that the purified x variant is more prone to self-aggregation than the Δ pro- tein in vitro. overexpression of wild-type whamm at high levels in cos cells is known to cause microtubule bundling (campellone et al., ), so to explore whether the x mutant can interact with microtubules in cells, we coexpressed lap-whamm(wt) with mcherry-tagged whamm(x ). when coex- pressed with wild-type whamm, the x mutant localized to microtubule bundles (figure e), suggesting that it is capable of associating with microtubules. thus the two possible whamm truncations expressed by amish gms patients retain some ability to interact directly or indirectly with microtu- bules, but both completely lack the actin nucleation-promoting activity that is inher- ent to full-length whamm. amish gms fibroblasts have altered f-actin organization and ergic morphology whamm is the only wasp-family protein known to function at membranes of the early secretory system, as depletion and overexpression approaches indicate that whamm helps localize the cis-golgi near the centrosome and promotes tubulation of ergic-like membranes in an actin- and mi- crotubule-dependent manner (campellone et al., ; russo et al., ). to deter- mine whether patient cells display alterations in cytoskeletal or membrane organization, we stained cultured fibroblasts with probes to visualize microtubules and actin, as well as antibodies to detect organelles of the conventional secretory pathway. while amish gms patient fibroblasts exhibited normal-looking microtu- bule arrays (figure e and supplemental figure s ), many con- tained unusually thick or stellate f-actin bundles (figure , a and b), similar to the changes in stress fiber organization that were previ- ously observed in some cells treated with sirnas to whamm (gad et al., ). gms fibroblasts also harbored significantly fewer punctate, possibly vesicle-associated, f-actin structures than con- trol cells (figure , a and c). each of these alterations in actin is actin nucleation. to determine how cytoskeletal interactions are af- fected in each whamm variant, we purified maltose-binding pro- tein (mbp)-tagged versions of whamm(Δ ) and whamm(x ) in parallel to mbp-whamm(wt) (figure a), and compared their abili- ties to promote actin assembly and bind microtubules in vitro. for assessing actin assembly, we employed pyrene-actin polym- erization assays, which measure the bulk formation of fluorescent actin filaments over time (campellone et al., ). as expected, the full-length mbp-whamm(wt) protein accelerated actin polym- erization in conjunction with the arp / complex (figure b). con- versely, neither mbp-whamm(Δ ) nor mbp-whamm(x ) increased the rate of actin assembly (figure b), confirming that, in the ab- figure : truncated whamm proteins exhibit altered properties in cells. (a) fibroblasts from two healthy (+/+) amish individuals or amish gms (gms/gms) patients were fixed and stained with anti-cc antibodies to visualize whamm (green). nuclei, based on dapi staining, were denoted with dashed ovals. (b) cos cells were transfected with plasmids encoding a vector control, ha-whamm(wt), (Δ ), or (x ) and blotted with anti-ha or anti-tubulin antibodies. (c) cos cells transiently expressing lap-whamm(wt), (Δ ), or (x ) (green due to the gfp tag) were fixed and stained with phalloidin to visualize f-actin (red) and with dapi to label dna (blue). (d) nih t cells stably transfected with lap-whamm(wt) or (Δ ) or a lap vector control were fixed and stained as in c. all scale bars: μm. | a. j. mathiowetz, e. baple, et al. molecular biology of the cell amish gms cells have defects in autophagosomal biogenesis due to a loss in whamm function given the clinical severity of amish gms, we investigated whether aspects of membrane remodeling beyond ergic morphology were altered in patient fibroblasts. one function requiring dramatic membrane shape changes is autophagy, a catabolic process in which cytoplasmic components are enveloped in double membrane–bound autophagosomes that can fuse with lyso- somes, leading to the degradation of autol- ysosomal contents (mizushima and levine, ; yang and klionsky, ; choi et al., ; carlsson and simonsen, ). while whamm was recently shown to localize to autophagosomes and promote their actin- based motility (kast et al., ), the physi- ological importance of whamm in autoph- agy is not known. we examined patient fibroblasts for autophagosomes by staining the cells with antibodies to lc , the stan- dard marker of these organelles (kabeya et al., ). interestingly, while cells from healthy individuals contained lc -positive preautophagosomal puncta under normal growth conditions, patient fibroblasts were virtually devoid of lc -associated structures (figure , a and b). to test whether patient cells could form autophagosomes under conditions in which lysosome-mediated degradation was blocked, we treated fibro- blasts with chloroquine, a drug that inhibits vesicle acidification and causes autophago- somes to accumulate. while control cells exhibited the expected buildup of lc -pos- itive autophagosomes under these condi- tions (figure a), many patient cells lost adherence, with the remaining cells exhibit- ing a diffuse lc staining pattern and a vacuole-filled cytoplasm (figure , a and c). thus patient fibroblasts display striking de- fects in autophagosomal biogenesis and trafficking that are exacerbated when lyso- some-mediated degradation is inhibited. to determine whether patient fibro- blasts could form autophagosomes under autophagy-inducing conditions, we treated cells with rapamycin or subjected them to starvation before staining for lc . while fibroblasts from an amish gms patient contained some lc -positive autophagosomal struc- tures under both types of autophagy-inducing conditions, cells from a healthy individual contained significantly more (figure , d and e). when measured as a fraction of the cytoplasm, lc staining was - to -fold less abundant in patient cells (figure e). on av- erage, lc puncta were ∼ % smaller in patient cells versus con- trol cells following rapamycin treatment ( . μm , n = vs. . μm , n = ; p < . ). therefore patient fibroblasts have an autophagosomal biogenesis deficiency even when grown under pharmacological or nutritional conditions that ordinarily induce autophagy. consistent with a loss of nucleation activity resulting from the homo- zygous whammgms mutation. in accordance with the role of whamm in organizing membranes of the intermediate compart- ment and golgi (blom et al., ; campellone et al., ), the ergic (figure , d and e) and cis-golgi (supplemental figure s ) appeared significantly larger in patient cells when compared with control cells. in contrast, nuclear size was unchanged (figure f), as was er morphology, er exit-site distribution, and lysosome organi- zation (supplemental figure s ). collectively these results expand upon previous transient rnai studies of whamm and highlight several actin-associated and organelle-organizing activities that are disrupted in patient fibroblasts. figure : amish gms whamm truncations do not promote actin nucleation but can associate with microtubules. (a) purified mbp-whamm(wt), (Δ ), and (x ) were analyzed by sds–page and coomassie blue staining or anti-mbp immunoblotting. whamm is abbreviated whm. (b) actin was polymerized in the presence of the arp / complex and either mbp, mbp- whamm(wt), (Δ ), or (x ). the amount of f-actin, in arbitrary fluorescent units (au), formed over time is shown. (c) microtubules were incubated in the presence of mbp-whamm(wt) or (Δ ), and collected by centrifugation. proteins found in pellet (p) and supernatant (s) samples were separated by sds–page. tubulin was visualized by ponceau s staining, while whamm fusions were detected by anti-mbp blotting. (d) mbp-whamm(x ) was incubated in the presence or absence of microtubules and processed as in c. (e) cos cells transiently coexpressing lap-whamm(wt) and mcherry-whamm(x ) were fixed and stained with antibodies to visualize microtubules. volume september , whamm founder mutation impairs autophagy | fibroblasts. cells were transfected with plasmids encoding green fluorescent protein (gfp)-tagged whamm(wt) or wdr (wt), and again stained with anti- bodies to lc . in contrast to untrans- fected or gfp-wdr –expressing cells, gfp-whamm–expressing cells harbored many lc puncta under normal growth conditions and accumulated autophago- somes after treatment with chloroquine (figure f). these results indicate that whamm is sufficient to reestablish au- tophagosomal biogenesis in amish gms patient fibroblasts. to assess autophagic digestion in pa- tient samples, we used lcls for measuring the levels of p /sqstm , a protein that links polyubiquitinated proteins to lc in autophagosomes, and whose degradation serves as an indicator of autophagy (klionsky et al., ). by immunoblotting lcl ex- tracts for p and quantifying its abun- dance relative to tubulin and glyceraldehyde -phosphate dehydrogenase (gapdh) load- ing controls (figure a), we found that p levels were generally % higher in amish gms patient cells compared with cells from individuals with full-length whamm (figure b). notably, although lcls express the wild-type whamm protein in a gene dose– dependent manner (figure d), we have been unable to detect wdr by immunob- lotting in any of the lcl extracts, implying that these immortalized b-cells express little, if any, wdr protein. thus the accumula- tion of p is consistent with the idea that autophagic degradation is inefficient in gms patient lcls primarily because of the homo- zygous whamm mutation. as an independent measure of an au- tophagy-related process in lcls, we ex- amined unconventional secretion of interleukin- β (il- β). this cytokine is ex- pressed in immune cells in response to microbial products, and autophagosomal structures facilitate its export (dupont et al., ; zhang et al., ). for induc- ing il- β production, lcls from amish in- dividuals with wild-type whamm and pa- tients with gms were treated with escherichia coli, and the amount of il- β released into culture supernatants was measured by enzyme-linked immunosor- bent assay (elisa). compared with normal lcls, lcls from gms patients secreted less il- β (figure c). in contrast, levels of tumor necrosis factor α (tnfα), a cytokine exported via the conventional secretory apparatus, did not differ significantly (figure c). hence amish gms patients possess fibroblasts with defects in autophagosomal biogenesis and immune cells with im- paired autophagic functions. as amish gms patients have mutations in both whamm and wdr , we sought to determine whether reexpression of either wild-type protein could restore autophagosomal formation to figure : amish gms fibroblasts have altered f-actin organization and ergic morphology. (a) fibroblasts from two healthy (+/+) amish individuals or amish gms (gms/gms) patients were stained with phalloidin to visualize f-actin (green) and with dapi to label dna (blue). unusual orthogonal bundles of f-actin fibers and aggregates are highlighted with arrows. a lack of cytoplasmic f-actin puncta in gms cells is exemplified in the row of magnified images. (b) the percentage of cells with aberrant (orthogonal, aggregated, or stellate) f-actin stress fibers was quantified. each bar represents the mean (+ sem) from analyses of cells. ***, p < . (fisher’s exact test). (c) the percentage of cells with prominent cytoplasmic f-actin puncta in the juxtanuclear region was quantified. each bar represents the mean (+ sem) from analyses of cells. for cells with puncta, the number per cell was also quantified. each bar represents the mean (+ sem) from analyses of cells. ***, p < . (fisher’s exact test, anova). (d) wild-type or gms fibroblasts were stained with antibodies to detect ergic- (red) and with dapi to label dna (blue). (e, f) the areas occupied by the ergic, nucleus, and entire cell were calculated in imagej and converted to a ratio of organelle area/cell area. each bar represents the mean (+ sem) from analyses of cells. ***, p < . (anova). all scale bars: μm. | a. j. mathiowetz, e. baple, et al. molecular biology of the cell play a key role in autophagy in other cell lines. hela cells, when treated with a pool of sirnas to whamm, were recently shown to contain smaller autophagosomes that lack actin tails (kast et al., ). we also ex- posed hela cells to sirnas for depleting whamm or wdr (supplemental figure s , a and b). after starving the cells to in- duce autophagy and stalling flux with chlo- roquine, we assessed autophagosomal bio- genesis by immunoblotting cell extracts for lc and measuring its conversion from a slower-migrating immature form (lc -i) to a faster-migrating lipidated form (lc -ii) that associates with autophagosomal mem- branes and correlates with autophagosomal abundance (klionsky et al., ). compari- sons of cells grown in normal media versus buffer plus chloroquine revealed that lc -ii levels increased in control and wdr -de- pleted cells but not in whamm-depleted cells (supplemental figure s , c and d). to further explore the role of whamm and wdr in autophagy using a genetically stable cellular system, we used clustered regularly interspaced short palindromic re- peats (crispr)/cas -mediated genome ed- iting for creating whamm- or wdr -defi- cient cells. to generate human cell lines that lack whamm or wdr function, we made derivatives of ehap cells, a fully haploid fi- broblast-like cell line (essletzbichler et al., ). frameshift mutations were intro- duced into exon of whamm or exon of wdr , and immunoblotting indicated that expression of full-length whamm and wdr was lost in the respective wham- mmut and wdr mut cell lines (figure a). consistent with a deficiency in basal autoph- agy under standard growth conditions, the whammmut cell line displayed approxi- mately half the amount of lc -ii as the pa- rental cell line (figure , b and c). we next shifted cells from rich growth media to starvation conditions and quanti- fied lc conversion on immunoblots. while parental ehap cells and wdr mut cells showed similar levels of lc -ii after min of starvation, whammmut cells generated only half of the amount of lc -ii as the other two cell lines (figure , d and e). blocking flux with chloroquine resulted in a similar pattern of lc conversion (figure e), suggesting that the lipidation deficit in whamm-deficient cells is primar- ily due to less autophagosomal biogenesis instead of increased autophagosomal deg- radation. moreover, a time course revealed that the rate of lc lipidation between and min of starva- tion was slower in whammmut cells than in parental or wdr mut cells (figure f). thus genetic inactivation demonstrates a key role for whamm in autophagosomal biogenesis. depletion or inactivation of whamm inhibits lc lipidation and clearance of protein aggregates given the autophagy-related defects in cells of amish gms pa- tients, we next examined whether whamm and/or wdr might figure : amish gms fibroblasts exhibit defects in autophagosomal biogenesis due to a loss in whamm function. (a) fibroblasts from two healthy (+/+) amish individuals or amish gms (gms/gms) patients were grown in the absence or presence of chloroquine (cq) for h and stained with antibodies to detect lc a (red) and with dapi to label dna (blue). (b) the number of lc a puncta per cell, and the relative area within each cell occupied by lc + structures was quantified using imagej. each bar represents the mean (+ sem) from analyses of cells. ***, p < . (anovas). (c) the percentage of cells with large cytoplasmic vacuoles was quantified. each bar represents the mean (+ sem) from analyses of cells. ***, p < . (fisher’s exact test). (d) fibroblasts from a healthy individual or gms patient were grown in the presence of rapamycin (rm) for h and stained with antibodies to detect lc b (red) and with dapi to label dna (blue). (e) fibroblasts were treated with rapamycin for h or starved for min and stained as in d. the relative area within each cell occupied by lc + structures was quantified as in b. ***, p < . (t test). (f) fibroblasts from a gms patient were transfected with plasmids encoding gfp-whamm or gfp-wdr , grown in the absence or presence of cq, and stained as in a. normal lc staining was observed in / gfp-whamm-expressing cells and / gfp-wdr -expressing cells. all scale bars: μm. volume september , whamm founder mutation impairs autophagy | bodies to ubiquitin. the parental cell line contained many cytoplas- mic puncta indicative of small aggregates of polyubiquitinated pro- teins (figure g). whammmut cells also accumulated aggregates, but they appeared much larger (figure g) and occupied signifi- cantly greater areas of the cytoplasm than in ehap cells (figure h). collectively our transient and stable loss-of-function studies indicate that whamm is important for promoting lc lipidation and au- tophagosomal biogenesis so that cells may ultimately degrade cy- toplasmic components. whamm is recruited to nascent autophagosomes by binding to pi( )p to investigate the mechanism underlying whamm’s normal func- tion in autophagy, we first examined its localization. in most mam- malian cells, the majority of endogenous and tagged whamm is associated with juxtanuclear ergic/cis-golgi membranes (campel- lone et al., ; blom et al., ; russo et al., ). in retinal epi- thelial cells, tagged murine whamm is also detected on puncta adjacent to lc -positive vesicles and near regions of the er called omegasomes (kast et al., ). omegasomes are transient hotspots for autophagosomal biogenesis marked by the protein dfcp (axe et al., ). consistent with these findings, when we starved fibro- blasts, mcherry-whamm was located both at the golgi region and adjacent to gfp-dfcp puncta (figure a). we further found that mcherry-whamm and gfp-dfcp colocalized at some puncta (figure a, arrows) and at omegasome-shaped structures that asso- ciated with actin filaments (figure b), implying that whamm can promote actin nucleation at sites of autophagosomal biogenesis, not just at mature autophagosomes. indeed, staining with antibod- ies to lc revealed that gfp-whamm localized to subdomains of developing (open) phagophores in addition to complete (closed) autophagosomes (figure c). in contrast, wdr exhibited a dif- fuse nucleo-cytoplasmic distribution (supplemental figure s ) and was not enriched on autophagosomes (figure c). the observations that whamm is recruited to omegasomes and phagophores point to a direct role for this protein in membrane remodeling before ac- tin-based rocketing of mature autophagosomes. whamm localization to membranes of the secretory pathway is mediated by the wmd, which has been suggested to interact with multiple phospholipids in vitro (campellone et al., ). upon in- spection of this domain, we noticed that residues – resemble a phospholipid-binding r-[y/f]-xn-k-xn-r motif commonly present in px domain proteins (supplemental figure s a). further examination of the wmd using the hhpred algorithm indicated that whamm resi- dues – also share similarity with px domains from many sorting nexin (snx) proteins (supplemental figure s b), which often bind to pi( )p on endosomes (teasdale and collins, ). pi( )p is also a cru- cial component of autophagosomal membranes (roberts and ktista- kis, ) and was recently shown to be required for actin assembly at phagophores (mi et al., ). therefore we explored whether whamm could bind to this phospholipid by purifying an mbp- tagged version of the wmd and testing its capacity to bind to pi( )p and other lipids that were immobilized on nitrocellulose strips. af- ter probing the strips with soluble mbp-wmd or mbp alone and de- tecting bound protein with anti-mbp antibodies, we found that the wmd bound strongly to pi( )p and weakly to pi( )p and pi( )p (sup- plemental figure s c). similar phospholipid-binding patterns were observed for mbp-whamm(wt), mbp-whamm(Δ ), and mbp- whamm(x ) (supplemental figure s c). to better gauge the affinity of the interactions between whamm and membranes, we used the mbp-wmd to probe arrays containing different concentrations of lip- ids. again, the wmd bound to pi( )p and, to a lesser extent, pi( )p to determine whether this slower autophagosomal assembly im- pacts the eventual digestion of cargo, we induced the production of truncated misfolded proteins in ehap and whammmut cells using the translational inhibitor puromycin and stained the cells with anti- figure : lymphoblastoid cell lines from amish gms patients exhibit defects in autophagy-related processes. (a) lcls from two healthy (+/+) amish individuals or three amish gms (gms/gms) patients were cultured under normal growth conditions, collected, and subjected to immunoblotting with anti-p /sqstm , anti-tubulin, and anti-gapdh antibodies. (b) p band intensities were quantified relative to tubulin. each bar represents the mean (+ sd) from analyses of three experiments. *, p < . ; **, p < . (anova). (c) lcls from several healthy (+/+) or amish gms (gms/gms) patients were stimulated with e. coli, and the levels of il- β and tnfα in culture supernatants were measured by elisa. each bar represents the mean (+ sem) from four to six samples across two to three experiments. data represent two to three patients per genotype. **, p < . (anova). | a. j. mathiowetz, e. baple, et al. molecular biology of the cell supplemental figure s d), suggesting that, in addition to binding to one another, whamm and rab both bind to pi( )p. to assess whether the wmd was capable of binding to pi( )p in a different context, we mixed mbp-wmd and mbp with synthetic liposomes containing pi, pi( )p, or pi( )p and performed vesicle–protein cosedimentation assays. anti-mbp immunoblotting of pel- leted samples indicated that mbp-wmd, but not the mbp control protein, interacted with all phosphoinositide-containing lipo- somes (figure c). band quantification showed that slightly more mbp-wmd bound to pi( )p than to pi (figure d), sug- gesting that although the wmd acts as a promiscuous lipid-binding domain in vitro, it exhibits some preference for pi( )p. consis- tent with pi( )p binding, when we mixed al- exa fluor –labeled mbp-whamm(wt) with alexa fluor –labeled liposomes, whamm could be found on the pi( )p-con- taining vesicle structures (figure e). because the wmd bears resemblance to px domains, we finally tested whether resi- dues predicted to be part of this domain were required for pi( )p binding in vitro and for membrane localization in cells. we gen- erated a r a/f a mutant version of the wmd fused to mbp and purified it along- side the wild-type mbp-wmd (figure a). to compare the affinity of the wmd(rf/aa) mutant and wmd(wt) for pi( )p, we used the mbp-tagged constructs to probe arrays containing different amounts of this phos- pholipid. as predicted, based on the con- servation of r and f in the px consen- sus sequence, mutation of these residues to alanine resulted in a dramatically lower de- gree of wmd binding to pi( )p (figure b). even when pi( )p was present at its highest amount ( pmol), it bound approximately fourfold less wmd(rf/aa) than wmd(wt) (figure b and supplemental figure s e), further demonstrating that r and f are crucial for the interaction with pi( )p. to determine the localization of the wmd(rf/aa) mutant in cells, we expressed it as a gfp-fusion protein in cos fibroblasts in parallel to the wild-type gfp-wmd and gfp alone (figure c). under starvation conditions, gfp-wmd(wt) was found at the juxtanuclear golgi region and at lc -posi- tive autophagosomes (figure d). in con- trast, gfp-wmd(rf/aa) exhibited a diffuse cytoplasmic localization and was not present on lc -positive mem- branes (figure d), indicating that residues r and f are re- quired for whamm recruitment to autophagosomes. collectively our results give rise to a model for autophagosomal biogenesis in healthy cells in which pi( )p recruits whamm via its n-terminal px- like wmd, and the whamm wca domain drives actin assembly to promote membrane remodeling and lc incorporation. and pi( )p (figure a). at omegasomes, pi( )p is responsible for bind- ing dfcp and also for recruiting rab , a small g-protein that can positively regulate autophagosomal formation (zoppino et al., ; huang et al., ). because rab binds directly to whamm and re- cruits it to membranes (russo et al., ), we also tested whether recombinant rab could interact with pi( )p. indeed, glutathione s- transferase (gst)-tagged rab also bound to pi( )p (figure b and figure : targeted inactivation of whamm inhibits lipidation of lc and clearance of protein aggregates. (a) parental ehap cells or cells with mutations engineered in whamm or wdr were analyzed by immunoblotting for whamm, wdr , tubulin, and gapdh. (b) ehap and whammmut cells were analyzed by immunoblotting for lc b, tubulin, and gapdh. lc b was detected in its immature (lc -i) and mature lipidated (lc -ii) forms. (c) lc b-ii band intensities were quantified relative to tubulin. each bar represents the mean (+ sd) from analyses of three experiments. *, p < . (t test). (d, e) ehap cell derivatives were starved for min in the absence or presence of chloroquine (cq), and lc -ii levels were measured as in b and c. each bar represents the mean (+ sd) from analyses of three to five experiments. *, p < . ; ***, p < . (anova). (f) ehap cell derivatives were starved for the indicated times, and lc -ii levels were measured as in c. (g) parental or whamm-deficient cells were treated with puromycin for . h and stained with antibodies to visualize ubiquitinated protein aggregates (red) and with phalloidin to visualize f-actin (green). scale bar: μm. (h) the areas occupied by ubiquitinated protein aggregates and the entire cell were calculated using imagej and converted to a ratio. each bar represents the mean (+ sem) from analyses of – cells. **, p < . (t test). volume september , whamm founder mutation impairs autophagy | (jinks et al., ). in the current work, we showed that cells from amish children with gms who are homozygous for a base pair deletion in whamm lack a nucleation-profi- cient whamm protein and, as a result, have major defects in autophagy. taken together with experiments using independent ge- nome-edited cells and purified proteins, our results further revealed that whamm-medi- ated actin assembly is fundamentally impor- tant for autophagosomal biogenesis at a step that follows pi( )p synthesis but pre- cedes lc incorporation. in the absence of this pi( )p-whamm-arp / pathway for au- tophagosomal remodeling, cells accumu- late polyubiquitinated protein aggregates. a major question arising from these studies is whether defects in autophagy or other whamm-associated functions mod- ify the clinical outcomes in amish gms pa- tients, as any resulting phenotypes may be concealed beneath a particularly severe clinical picture that arises through the coin- cident wdr mutation. thus the direct medical relevance of findings using patient cells will likely remain unclear until individu- als are identified who display bi-allelic whamm mutations in the absence of wdr mutations. interestingly, it has been suggested that gms is not a single gene disorder and that the clinical features of gms can arise from mutations in genes other than wdr (rosti et al., ). moreover, since the initial description of amish gms, we have identified a second child in whom a recombination event led to heterozygosity for the whamm variant alongside the homozygous wdr muta- tion. this -year-old’s neurocognitive im- pairment is less significant than is typical for amish children with gms. hypotonia re- mains the predominant clinical feature; he is able ride a tricycle, walk with assistance, gesture to indicate needs, and has not de- veloped any renal impairment. how whamm heterozygosity may affect the clinical picture and/or cellular phenotypes in this patient or other amish gms patients remains to be determined. notably, the autophagy defects seen in amish gms patient fibroblasts were more severe than those in whamm-deficient hela or ehap cells, as only the gms cells vacuolated after treatment with chloroquine. this could be due to differences in cell type and tissue origin. however, it is also plausible that residual truncated whamm pro- teins exacerbate the autophagy defects in patient cells. another possibility is that differences among the amish, hela, and ehap genomes influence autophagy or related membrane-trafficking processes. for example, aberrant wdr function in amish gms cells may worsen their autophagy defect. although targeted loss- of-function and localization studies did not point to a direct role for wdr in autophagy, we have been unable to inactivate discussion for decades, wasp variants have been known to cause immunode- ficiencies, but mutations in other actin nucleation factors have sur- prisingly not been characterized in relation to human disease. gms is a genetic disorder featuring neurological abnormalities and pro- gressive kidney dysfunction, and several variants of the wdr gene are thought to be responsible for its clinical hallmarks (colin et al., ; ben-omran et al., ; vodopiutz et al., ; rosti et al., ). however, in parallel with those studies, we described coincident wdr and whamm mutations in all but one of amish individuals exhibiting clinical features consistent with gms figure : whamm localizes to omegasomes and subdomains of nascent autophagosomes. (a) cos cells transiently expressing mcherry-whamm (red) and gfp-dfcp (green) were starved and stained with dapi to label dna (blue). arrows indicate areas of whamm and dfcp colocalization; arrowheads highlight a region of whamm and dfcp juxtaposition. scale bars: μm. (b) cos cells transiently expressing mcherry-whamm (red) and gfp-dfcp (green) were starved and stained with phalloidin to label f-actin (pink). scale bars: . μm. (c) cos cells transiently expressing gfp-whamm or gfp-wdr (green) were starved and stained with antibodies to label lc (red). individual autophagosomal structures with punctate gfp-whamm localization to regions of membrane growth or curvature are highlighted in this composite. scale bars: . μm. | a. j. mathiowetz, e. baple, et al. molecular biology of the cell that wdr can localize to the mitotic spin- dle (jinks et al., ), it is tempting to spec- ulate that microtubule-dependent functions might be altered by mutations in whamm, wdr , or both genes. apart from microtubule binding, the best-characterized biochemical activity of whamm is promoting actin nucleation with the arp / complex (campellone et al., ). however, until recent years, little was known about actin assembly during autoph- agy in mammalian cells. the first nucleation factor studied in this process was cortactin (lee et al., ), which presumably influ- ences filament branching at autophago- somes. the wasp-family member wash is also involved in autophagy (xia et al., ; zavodszky et al., ), perhaps by control- ling trafficking to autophagosomes. more recently, whamm was shown to promote arp / -mediated autophagosomal rocket- ing (kast et al., ), while jmy was shown to interact with lc and also nucleate actin at autophagosomes (coutts and la thangue, ). finally, actin-capping pro- tein was found to regulate actin polymeriza- tion at autophagosomes downstream of pi( )p synthesis (mi et al., ). our current work both interconnects and expands on those studies. jmy and whamm were proposed to influence autophagosomal maturation be- cause a jmy sirna reduced the amount of autophagosomes in u os epithelial cells (coutts and la thangue, ), while whamm sirna treatment reduced au- tophagosomal size and movement in retinal epithelial cells (kast et al., ). expression of a whamm point mutant that should have a reduced affinity for arp / also de- creased the number of lc -positive au- tophagosomes in cells (kast et al., ). our results point to a crucial function for whamm early in biogenesis, between pi( )p generation and lc lipidation. this is supported by our find- ings that whamm and rab each bind to pi( )p, and that whamm inactivation delays the conversion of lc -i to lc -ii. how whamm facilitates lc conversion is not clear, but could be related to its ability to affect trafficking from the ergic, an important source of vesicle templates for lc lipidation (ge et al., , ). whamm-mediated actin assembly could also help shape the phagophore in a manner that promotes lc incorporation. indeed, the punctate localization of whamm at omegasomes and autopha- gosomal membranes is consistent with whamm being the missing factor that promotes actin nucleation inside forming autophago- somes downstream of pi( )p synthesis (mi et al., ). finally, we have highlighted the physiological importance of whamm through the use of amish gms patient cells, which lacked autophagosomal structures and possessed excess p under steady-state condi- tions, exhibited weak autophagic responses to rapamycin or starva- tion, and accumulated large vacuoles when lysosomal degradation was inhibited. wdr in a whammmut background, implying that wdr is es- sential in whamm-deficient cells. further studies are required to better understand the factors that impact autophagy in distinct cell lines. in addition to the cellular phenotypes attributable to whamm function in amish gms patient cells, changes in whamm expres- sion and whamm polymorphisms have been linked to several other conditions. whamm is one of biomarkers that are differen- tially expressed in children with systemic onset juvenile idiopathic arthritis (allantaz et al., ) and is one of genes associated with risk for lupus-like diseases in dogs (wilbe et al., ). the whamm and wdr genes are separated by only . mb, and several hu- mans with chromosome microdeletions encompassing whamm or both whamm and wdr exhibit developmental delay or intel- lectual disability (doelken et al., ; burgess et al., ). whether the specific loss of whamm and/or wdr function contributes to these illnesses has not been investigated. however, given that whamm is a microtubule-binding protein (shen et al., ) and figure : whamm binds to pi( )p in vitro. (a) different quantities of phospholipids immobilized on a nitrocellulose membrane were probed with purified mbp-wmd. bound protein was detected using antibodies to mbp and visualized by chemiluminescence. mbp- wmd binding was quantified by measuring the mean pixel intensity of protein-specific spots after normalizing to background staining. (b) different concentrations of pi( )p immobilized on a nitrocellulose membrane were probed with purified gst-rab a. bound protein was detected using antibodies to gst and quantified as in a. (c) synthetic liposomes containing % of the indicated phosphoinositides were incubated with mbp and mbp-wmd and collected by centrifugation. liposome-associated proteins were subjected to sds–page and detected by immunoblotting for mbp. (d) the amount of mbp-wmd pulled down was determined by densitometry. each bar represents the mean (+ sd) from three to four experiments. (e) pi( )p-containing biotinylated liposomes were mixed with alexa fluor –labeled mbp-whamm and alexa fluor –labeled streptavidin and examined microscopically. volume september , whamm founder mutation impairs autophagy | nisms of autophagy that influence health and disease. materials and methods human genetics and cell culture studies leading to the definition of the ge- netic basis of clinical gms in the amish were described previously (jinks et al., ). pri- mary patient cells were obtained from the blood and skin of human subjects as part of a previous study approved by the university of arizona irb (project # - - , ge- netic studies in the amish, irb ). subsequent cellular studies were approved by the university of connecticut irb (pro- tocol #h - , cell biology of a neurode- velopmental disorder, fwa ). pri- mary dermal fibroblasts isolated from two normal individuals and two amish gms pa- tients were cultured in dmem plus % fetal bovine serum (fbs) and antibiotic–antimy- cotic. phenotype quantification was per- formed with one set of gms fibroblasts only, because the second proliferated poorly and did not survive freeze–thaw cycles. lcls (european collection of authenticated cell cultures) were derived from two normal amish individuals, two heterozygous carri- ers, and three gms patients, and grown in rpmi plus % fbs and antibiotic–antimy- cotic. cos , nih t , and hela cells were cultured in dmem plus % fbs and antibi- otic–antimycotic. ehap cells (essletzbichler et al., ) and their derivatives were cul- tured in imdm plus % fbs and penicil- lin–streptomycin. all mammalian cells were grown at °c in % co . dna, rna, viruses, and insect cells key reagents are listed in supplemental table s . plasmids, bacmids, and baculovi- ruses encoding human whamm(wt) were described previously (campellone et al., ; shen et al., ; russo et al., ). plasmids encoding whamm(Δ ) were gen- erated by site-directed mutagenesis on whamm(wt) using primers gactctcttaaaagacttaaaaa- caagatgaagttg and caacttcatcttgtttttaagtctttta- agagagtc. plasmids encoding whamm(x ) were generated by replacing the appropriate region of whamm(wt) with a synthetic xhoi-saci fragment of x (integrated dna technologies). recombi- nant baculoviruses were generated using the bac-to-bac system (in- vitrogen). the plasmid for expression of gfp-wdr in mammalian cells was generated by pcr using a cdna clone template (open biosystems), primers atcatcggtaccatggatcctggggacgactgg and atcatcgcggccgcttatcagcggggggcacaaaggt, and cloned as a kpni-noti fragment into pkc-egfp-c (campellone et al., ). plasmids for expression of mbp, mbp-whamm(wmd), and gst-rab in insect cells were described elsewhere (russo et al., ). plasmids encoding the wmd r a/f a mutant were gen- erated using a synthetic codon-optimized wmd(rf/aa) fragment flanked by kpni-noti sites (integrated dna technologies). a autophagy is known to be up- or down-regulated in many dis- ease states (choi et al., ), but examples of genetic mutations that directly perturb this process to cause human illness are rare. in one example, recessive mutations in the epg gene, whose protein product appears to control autophagosome–lysosome fusion, give rise to a neurodevelopmental disorder called vici syndrome (byrne et al., ). a second example is a homozygous mutation in atg that causes an e d substitution and a neurodegenerative ataxia. atg normally functions in conjugating phosphatidylethanolamine to lc -i to create lc -ii during phagophore elongation, but cells from patients with the atg variant exhibit less lc lipidation (kim et al., ). thus far, that is the only known mutation in a core com- ponent of the autophagy machinery with physiological and clinical consequences. however, based on our current results with amish gms patients, further studies on the molecular functions of whamm and wdr should also provide important insights into the mecha- figure : point mutations in the wmd that disrupt binding to pi( )p in vitro prevent localization to autophagosomes in cells. (a) purified mbp-wmd(wt) and (rf/aa) were analyzed by sds–page and coomassie blue staining. (b) different quantities of phospholipids immobilized on a nitrocellulose membrane were probed with purified mbp-wmd(wt) or (rf/aa). bound protein was detected using antibodies to mbp and quantified by measuring the intensity of protein-specific spots after normalizing to background staining. the bar graph represents the mean amounts (+ sem) of mbp-wmd variants bound to pmol of pi( )p in three experiments. **, p < . (t test). (c) cos cells were transiently transfected with plasmids encoding a gfp vector control, gfp-wmd(wt), or gfp-wmd(rf/aa) and blotted with anti-gfp, anti-tubulin, and anti-gapdh antibodies. (d) cos cells transiently expressing gfp, gfp-wmd(wt), or gfp- wmd(rf/aa) were starved and stained with antibodies to lc b (red) and dapi to label dna (blue). arrows indicate areas of whamm localization to lc + puncta; the arrowhead highlights the golgi (g) region. insets depict individual autophagosomes. scale bar: μm. | a. j. mathiowetz, e. baple, et al. molecular biology of the cell ducing an inactivating mutation in exon of wdr in the above whamm mutant background failed (horizon genomics). all wdr mutants were found to be in-frame, implying that wdr is an es- sential gene in this whamm-deficient background. immunoblotting and quantification for preparation of protein extracts, cells were washed with phos- phate-buffered saline (pbs) and lysed in mm tris-hcl (ph . ), mm nacl, % triton x- , mm phenylmethylsulfonyl fluoride, and μg/ml aprotinin, leupeptin, pepstatin, and chymostatin, be- fore being mixed with sds–page sample buffer. protein samples were boiled and subjected to sds–page before being transferred to nitrocellulose membranes and stained with ponceau s. mem- branes were blocked in pbs with % milk before being probed with rabbit anti-whamm (shen et al., ), chicken anti-whamm(wca) (campellone et al., ), rabbit anti-mbp (shen et al., ), mouse anti–tubulin e (developmental studies hybridoma bank), mouse anti-gapdh (ambion), rabbit anti-lc b or anti-p /sqstm (cell signaling technology), rabbit anti-wdr (santa cruz biotech), or mouse anti-ha (covance) antibodies. following washes, mem- branes were treated with irdye- or horseradish peroxidase (hrp)-conjugated secondary antibodies for detection. infrared or chemiluminescent bands were visualized with a li-cor fc imager, and their intensities were quantified using li-cor image studio soft- ware. for measurement of lc lipidation, lc -ii bands were nor- malized to tubulin and/or gapdh bands in each lane. statistical significance and p values were evaluated using an unpaired stu- dent’s t test or one-way analysis of variance (anova) using graph- pad prism software. microscopy and quantification cells grown on mm glass coverslips were fixed with . % parafor- maldehyde for min. for whamm, microtubule, and lc stain- ing, cells were treated with % methanol for . min at − °c. we used the following primary antibodies: guinea pig anti-whamm(cc) or rabbit anti-gfp (campellone et al., ), mouse anti–tubulin e (developmental studies hybridoma bank), mouse anti–ergic- (alexis biochemicals), rabbit anti-lc a or anti-lc b (cell signaling technology), mouse anti–ubiquitin fk (enzo life sciences), rabbit anti-wdr (santa cruz biotechnology), rat anti-grp (novus bio- logicals), mouse anti–sec a or anti-gm (bd biosciences), and mouse anti-lamp (santa cruz biotechnology). we used secondary antibodies conjugated to alexa fluor , , or (invitrogen). dna was visualized with μg/ml dapi, and f-actin was detected using u/ml alexa fluor–conjugated phalloidins (invitrogen). cover- slips were mounted in prolong gold anti-fade (invitrogen). images were captured using × ( . na) or × ( . na) plan-apo ob- jective lenses on a nikon ti-e microscope equipped with an andor clara-e camera, and elements software. for measurement of fluo- rescence intensities, areas, and quantities, individual cells and/or organelles were outlined using imagej software. for intensity mea- surements, the mean pixel intensity of whamm staining in a cell was multiplied by its two-dimensional area and divided by the area of the cell. the intensity in control cells was set to . for organelle area measurements, an ergic, lc , polyubiquitin, or dapi signal was defined using a thresholding function in imagej and was di- vided by the area of the cell. numbers of f-actin and lc puncta per cell were counted manually. for measuring the percentage of cells with f-actin puncta, abnormal stress fibers, or chloroquine-in- duced vacuoles, sample identities were coded, and randomly cho- sen cells were scored as (negative) or (positive) for the specific phenotype. samples were decoded after quantification. statistical synthetic wmd(wt) fragment was synthesized as a control (inte- grated dna technologies). for quantification of human whamm mrna, cdna was prepared from skin fibroblasts with trizol reagent and the m-mlv reverse transcriptase (invitrogen) and subsequently amplified using primers ctccgtgctctgtcctcatcctctca (exon ) and ctaaccatcccactggccagggtcct (exon ). the whamm pcr product corresponds to nucleotides at the ′ end of the open reading frame. whamm levels were normalized to β- actin, as described previously (campellone et al., ). rt-pcr primers spanning exons – were used in supplemental figure s . all bacteria were cultured using lb media with appropriate antibiot- ics at °c. plasmids were maintained in e. coli xl- blue and puri- fied using standard kits (machery-nagel) before transfection. sf insect cells were grown in esf medium (expression systems) at °c and infected with baculoviruses encoding mbp-fusion proteins at a multiplicity of infection of ∼ (russo et al., ). transfections and chemical treatments standard mammalian cell transfections with dna or rna were per- formed using lipofectamine-ltx or rnaimax (invitrogen) (russo et al., ). dermal fibroblasts, however, were shifted to . x dmem, . x rpmi medium, . x f medium, % fbs, x non- essential amino acids, x glutamax, and antibiotic–antimycotic (all from invitrogen) at least h before transfection. nih t clones stably encoding lap, lap-whamm(wt), or lap-whamm(Δ ) were selected in media containing μg/ml g . for inducing lap expression, . mm sodium butyrate was added for h. control sirnas or stealth sirnas to whamm and wdr were purchased from thermo fisher scientific. treatments with hank’s buffered sa- line solution were used in starvation experiments, chloroquine (sigma) was used at μm, rapamycin (tocris) was used at μm, and puromycin (sigma) was used at μg/ml. genome editing hap fibroblasts are a nearly haploid human cell line that contains an immortalizing bcr-abl fusion and a single copy of all chromo- somes, except for a heterozygous mb fragment of chromosome , which is integrated on the long arm of chromosome (carette et al., ). this diploid portion encompasses genes, includ- ing whamm and wdr . crispr/cas -engineered ehap cells, which are fully haploid (essletzbichler et al., ), were therefore used for mutagenesis of whamm and wdr . using crispr/ cas -mediated recombination, a -nucleotide deletion was intro- duced into exon of whamm, and a -nucleotide insertion was introduced into exon of wdr (haplogen, now horizon genom- ics). mutations were confirmed by dna sequencing (haplogen, now horizon genomics). using fluorescence in situ hybridization probes, we additionally verified the homozygosity and integrity of chromosomes and in ehap derivatives (supplemental figure s a). briefly, whole-chromosome paint probes (rainbow scientific cytocell) were hybridized and washed per the manufacturer’s proto- col. slides were mounted in a : dilution of ′, -diamidino- -phe- nylindole (dapi):vectashield (vector laboratories).  it is also impor- tant to note that, after editing, haploid cells tend to eventually revert to diploidy, a phenotype that we confirmed with karyotype analyses (supplemental figure s b). briefly, mitotic cells were ar- rested at metaphase via treatment with . μg/ml colcemid for h, collected, swollen with a . m kcl solution, and subsequently fixed with a modified carnoy’s solution. slides were g-banded with trypsin and giemsa. all images were captured on an olympus ax fluorescence microscope equipped with appropriate filters and ana- lyzed with leica cytovision imaging software. our attempts at intro- volume september , whamm founder mutation impairs autophagy | nm gst-rab . after washes, bound protein was detected using rabbit anti-mbp or rabbit anti-gst antibodies (shen et al., ) in blocking buffer, followed by hrp-conjugated anti-rabbit antibodies in % milk, and visualized using enhanced chemiluminescence with film (ge healthcare) or with a li-cor fc imager. for protein binding quantification, densitometry was performed by measuring the mean pixel intensity of protein-specific spots in imagej and normalizing these values to background staining, or by using li-cor image stu- dio software. for liposome cosedimentation assays, μm synthetic polypiposomes (echelon biosciences) containing % polymeriz- able-pc, % polymerizable-pe, % polymerizable pi, pi( )p, or pi( )p, and % polymerizable biotin-pe were mixed with μm mbp and μm mbp-wmd at room temperature for min in mm tris (ph . ), mm mgcl , mm kcl, and % glycerol. liposomes and proteins were centrifuged in a tla rotor (beckman) for min at , rpm at °c. pellet fractions were resuspended in sample buffer and analyzed by sds–page and immunoblotting with anti-mbp antibodies. for protein binding quantification, den- sitometry was performed by measuring the mean pixel intensity of mbp-wmd bands in imagej and normalizing these values to back- ground staining. for microscopic visualization of whamm in asso- ciation with liposomes, mbp-whamm(wt) was covalently labeled with alexa fluor (invitrogen), and polypiposomes were detected with alexa fluor –streptavidin. cytokine secretion assays human lcls were seeded at × cells/well and allowed to grow to × cells/well in -well plates before stimulation with × e. coli xl -blue for h. the contents of each well were then trans- ferred to microfuge tubes and placed on ice for min before cen- trifugation at × g for . min at °c. supernatants were trans- ferred to fresh tubes, recentrifuged, and again transferred to fresh tubes before being frozen at − °c. ready-set-go elisa kits (ebioscience) were used to quantify levels of il- β and tnfα in once-thawed lcl supernatants. briefly, -well costar plates (corning) were coated with capture antibodies in pbs for > h at °c, blocked at °c using % fbs in pbs for h, and incubated with recombinant standards or lcl supernatants for h; this was followed by cytokine detection antibodies for h and avidin-hrp for min. well contents were developed with tetramethylbenzi- dine for – min before quenching with m h po and measure- ment of the colorimetric product at nm on a spectramax m plate reader (molecular devices). il- β and tnfα concentrations were calculated from optical density values using softmax pro ver- sion . . statistical analyses statistical parameters and significance are reported in the figures and figure legends. data were determined to be statistically signifi- cantly different when p < . by student’s t test, anova, or fish- er’s exact test in graphpad prism software as indicated in preceding sections. significance and p values were evaluated using an unpaired stu- dent’s t test, one-way anova, or fisher’s exact test using graphpad prism software. purified proteins mbp, mbp-whamm(wt), mbp-whamm(wmd), gst, and gst- rab preparations were described previously (russo et al., ), and stored at − °c in mm mops (ph . ), mm kcl, mm mgcl , mm egta, mm edta, and % glycerol. the Δ and x variants, as well as codon-optimized versions of wmd(wt) and wmd(rf/aa), were expressed and purified in an identical manner to other mbp fusions. mbp-whamm(wt) was covalently labeled using an alexa fluor labeling kit (invitrogen). all protein quanti- ties were measured using bradford assays (bio-rad), and purities were confirmed by sds–page analyses. actin assembly assays pyrene-actin assembly assays were performed essentially as de- scribed previously (russo et al., ). actin (cytoskeleton) was re- suspended in g-buffer ( mm tris, ph . , . mm cacl , . mm dithiothreitol (dtt), . mm atp) and subjected to gel-filtration chromatography using a superdex column (ge healthcare) to remove small filaments. actin ( . μm; % pyrene labeled) was then polymerized in the presence of nm arp / complex plus nm mbp, mbp-whamm(wt), mbp-whamm(Δ ), or mbp-whamm(x ). pyrene fluorescence (actin filaments) was measured using a horiba jobin yvon fluorolog- spectrofluorimeter capable of multiwave- length excitation/detection and equipped with a four-position sample changer. similar results were obtained using different prep- arations of g-actin in up to four experiments for each mbp fusion protein. microtubule cosedimentation assays porcine brain tubulin was purified as described previously (campel- lone et al., ) and diluted to μm in brb ( mm pipes, mm egta, mm mgcl ) containing % glycerol, . mm gtp, and mm dtt. tubulin was then polymerized at °c and stabilized with stepwise additions of taxol until reaching the final concentra- tions of μm microtubules and μm taxol (shen et al., ). samples ( μl) containing microtubules and fusion proteins were prepared by mixing brb , mm kcl, μm microtubules, and nm precleared mbp-whamm(wt) or mbp-whamm(Δ ) at °c. (mbp-whamm(x ) pelleted by itself in the preclearance step.) after min, μl of each sample was overlaid onto brb containing glycerol at % (low-speed assays) or % (high-speed assays). the samples were then centrifuged at , × g (low speed) in a benchtop microcentrifuge (eppendorf) or at , × g (high speed) at °c for min in a tla rotor (beckman) to sedi- ment the microtubules. supernatants were removed and precipi- tated with % trichloroacetic acid, and both the supernatant and pellet samples were mixed with sds–page sample buffer to equiva- lent volumes. lipid-binding assays for protein overlay assays, pip strip and pip array nitrocellulose membranes spotted with – pmol of immobilized phospholipids (echelon biosciences) were hydrated in pbs, and blocked in block- ing buffer ( % milk, % bsa, and . % tween- in pbs) for h. pip strips were probed with nm soluble mbp, mbp-whamm(wt), mbp-whamm(wmd), mbp-whamm(Δ ), mbp-whamm(x ), gst, or gst-rab in blocking buffer for h at room temperature. pip arrays were probed with nm soluble mbp-wmd variants or acknowledgments this work was supported by national institutes of health grant r - gm (k.g.c.), medical research council grants g and g (a.h.c., e.b.), and the newlife foundation (a.h.c., e.b.). we thank henry chen (university of connecticut) for assis- tance with elisas and kevin strauss (clinic for special children) for communication of clinical and genetic data. we also thank the amish families for their participation in these studies. | a. j. mathiowetz, e. baple, et al. molecular biology of the cell gomez ts, gorman ja, artal-martinez de narvajas a, koenig ao, billadeau dd ( ). trafficking defects in wash-knockout fibroblasts originate from collapsed endosomal and lysosomal networks. mol biol cell , – . huang j, birmingham cl, shahnazari s, shiu j, zheng yt, smith ac, campellone kg, heo wd, gruenheid s, meyer t, et al. 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( ). wash inhibits autophagy through suppression of beclin ubiq- uitination. embo j , – . yang z, klionsky dj ( ). eaten alive: a history of macroautophagy. nat cell biol , – . zavodszky e, seaman mn, moreau k, jimenez-sanchez m, breusegem sy, harbour me, rubinsztein dc ( ). mutation in vps associated with a genome-wide linkage scan of insulin level–derived traits the amish family diabetes study wen-chi hsueh, kristi d. silver, toni i. pollin, callum j. bell, jeffrey r. o’connell, braxton d. mitchell, and alan r. shuldiner , objective—serum insulin levels are altered in insulin resis- tance and insulin deficiency, states that are associated with the development of type diabetes. the goal of our study was to identify chromosomal regions that are likely to harbor genetic determinants of these traits. research design and methods—we conducted a se- ries of genetic analyses, including genome-wide and fine-map- ping linkage studies, based on insulin levels measured during an oral glucose tolerance test (ogtt) in nondiabetic partici- pants in the amish family diabetes study. indices of insulin secretion included the insulinogenic index and insulin at min postglucose load (insulin ), while indices of insulin resistance included homeostasis model assessment of insulin resistance (homa-ir) and fasting insulin. insulin area under the curve, a measure of both insulin secretion and insulin resistance, was also examined. results—all traits were modestly heritable, with heritability estimates ranging from . to . (all p � . ). there was significant genetic correlation between fasting insulin and homa-ir (�g � . , p � . ), as well as insulin and insulinogenic index (�g � . , p � . ), suggesting that common genes influence variation in these pairs of traits. sug- gestive linkage signals in the genome scan were to insulin on chromosome q (logarithm of odds [lod] . , p � . ) and to insulinogenic index on chromosome p (lod . , p � . ). fine-mapping study further refined our signal for insu- lin on chromosome (lod . at cm). conclusions—these results suggest that there may be dif- ferent genes influencing variation in ogtt measures of insulin secretion and insulin resistance. diabetes : – , t ype diabetes is a classic example of a complex disease that results from the interaction of mul- tiple genetic and environmental factors. one strategy to identify the genes regulating the type diabetes phenotype is to focus on related subclinical (intermediate) phenotypes, which are likely to be less genetically complex and involve fewer alleles. the patho- physiology of type diabetes involves defects in insulin sensitivity and/or insulin secretion thus making these measures excellent intermediate traits to study in order to dissect the genetic underpinnings of type diabetes. circulating levels of plasma insulin, measured in either the fasting state or in response to a glucose load, vary consid- erably in nondiabetic individuals, with higher levels pre- dicting future development of diabetes. several indices of insulin secretion and insulin resistance can be derived from insulin and glucose levels measured at different time points during an oral glucose tolerance test (ogtt). ogtt-derived measures of insulin secretion include the insulinogenic index and insulin at min following a -g oral glucose load (insulin ), while measures of insulin resistance include fasting insulin and the homeostasis model assessment of insulin resistance (homa-ir). insu- lin area under the curve (insulin auc) during the ogtt reflects elements of both insulin secretion and insulin resistance. several studies have investigated the genetic epidemiol- ogy of fasting insulin levels and ogtt-derived indices ( , ). genome-wide scans have identified several chromo- somal regions linked to these traits ( – ), but few have been replicated. the goal of this study was to characterize the genetic epidemiology of five insulin-related traits, including fasting insulin, insulin , insulinogenic index, homa-ir, and insulin auc. we first estimated their heri- tability, then assessed whether and to what degree these traits may share common genetic influence, and per- formed genome-wide and fine-mapping linkage analyses of these traits. we found that these insulin traits were significantly heritable and that there may be different genetic influences underlying these ogtt-derived mea- sures of insulin secretion and resistance. furthermore, our genome scan provides evidence of linkage on chromo- somes p and q to measures of insulin secretion. clinical characteristics of nondiabetic subjects are shown in table . the overall prevalence of impaired glucose tolerance (igt) and/or impaired fasting glucose (ifg) was % in this sample. insulin resistance–related measures in those with igt/ifg were significantly higher (p � . ) than in euglycemic individuals, whereas from the department of medicine, school of medicine, university of cali- fornia, san francisco, california; the department of medicine, university of maryland school of medicine, baltimore, maryland; axys pharmaceuticals, la jolla, california; and the geriatrics research and education clinical center, baltimore veterans administration medical center, baltimore, maryland. address correspondence and reprint requests to wen-chi hsueh, mph, phd, post st., c , san francisco, ca - . e-mail: wen-chi. hsueh@ucsf.edu. received for publication july and accepted in revised form july . published ahead of print at http://diabetes.diabetesjournals.org on july . doi: . /db - . c.j.b. is currently affiliated with emergen, inc., salt lake city, utah. additional information for this article can be found in an online appendix at http://dx.doi.org/ . /db - . auc, area under the curve; homa-ir, homeostasis model assessment of insulin resistance; ifg, impaired fasting glucose; igt, impaired glucose tolerance; ogtt, oral glucose tolerance test; str, short tandem repeat. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. brief report diabetes, vol. , october insulin secretion–related measures were similar between the two groups (data not shown). heritability (h ) estimates for insulin-related traits ranged from . to . (all p � . ) (table ). we next examined the extent to which these traits share common genetic influences in order to assist our interpretation of subsequent linkage analyses. as shown in table , high genetic correlations were observed between fasting insu- lin and homa-ir, suggesting that these two measures share very substantial genetic components (estimated �g � . , p � . ). these findings are not unexpected since homa-ir is derived from the fasting insulin. insulin had a strong genetic correlation with insulinogenic index (�g � . , p � . ). insulin auc was more genetically correlated with insulin than with any other trait (�g � . , p � . ). there was no significant genetic correla- tion between fasting insulin and insulin or the insulino- genic index. our genome-wide linkage analysis identified two chro- mosomal regions with suggestive evidence for linkage (defined as p � . or logarithm of odds [lod] � . ) to insulin secretion traits. the first region was for insulin (lod . , p � . ), occurring at cm on chromo- some q (nearest marker d s ) (fig. a), which was also supported by the two-point analysis of d s (lod . , p � . ). the second region was for insulinogenic index at cm on chromosome p (lod . , p � . ; nearest marker d s ) (fig. b), as supported by two-point analysis of d s (lod . , p � . ). similar results were obtained when the analysis was performed without adjustment for bmi. nine other chromosomal regions (on chromosomes q , q -q , p -p , q -q , q , q , q -q , p -p , and p ) showed linkage signals to one or more insulin traits with lod � . (p � . ) (table ). bivariate linkage analysis for the two pairs of highly correlated insulin secretion traits was conducted. the maximum lod score for (insulin � the insulinogenic index) was . at cm from the p-ter of chromosome . insulin � insulin auc showed a lower lod score ( . ) at cm, also on chromosome . these results suggest that the genes in this region may contribute more to the insulin pheno- type than to the other two traits. the complete genome scan results can be viewed in the online appendix (avail- able at http://dx.doi.org/ . /db - ). to follow up on the linkage signal to insulin on chromosome q, additional short tandem repeat (str) markers were genotyped to increase the information con- table plasma levels of traits in nondiabetic study subjects by sex* trait male female h � se† n age (years) . � . . � . — bmi (kg/m ) . � . . � . . � . ‡ fasting glucose (mmol/l) . � . . � . . � . glucose (mmol/l) . � . . � . . � . fasting insulin (mu/l) . � . . � . . � . § insulin (mu/l) . � . . � . . � . § insulinogenic index (units/g) . � . . � . . � . ‡ homa-ir (mu per mmol/l ) . � . . � . . � . § insulin auc (mu � l� � h� ) . � . . � . . � . § data are means � sd. *see ref. for additional characteristics of the amish family diabetes study. due to missing data points, sample sizes ranged from for fasting insulin to for insulin auc. †heritability � se, adjusted for effects of bmi, sex-specific age, and age . ‡p � . ; §p � . . table correlation coefficients* among insulin traits† fasting insulin homa-ir insulin insulinogenic index homa-ir �p . � . �g . � . ‡ �e . � . insulin �p . � . . � . �g . � . . � . �e . � . . � . insulinogenic index �p . � . . � . . � . �g . � . . � . . � . § �e . � . . � . . � . insulin auc �p . � . . � . . � . . � . �g . � . . � . . � . § . � . �e . � . . � . . � . . � . *�p, phenotypic correlation; �g, genetic correlation; �e, random environmental correlation. †all �p and �e are with p � . . ‡p � . ; §p � . . genome scan of insulin traits in the amish diabetes, vol. , october fig. . a: multipoint linkage analysis results on chromosome for insulin at min. b: multipoint linkage analysis results on chromosome for the insulinogenic index. w.-c. hsueh and associates diabetes, vol. , october tent in the region. these markers reduced the marker density from . to . cm and further refined the linkage peak to near d s (at cm, lod . ), � cm closer to the centromere compared with the original genome scan results, which was well within the -lod interval. insulin secretion and insulin sensitivity are important determinants of glucose homeostasis and diabetes. the current gold standard for quantifying insulin secretion is the acute insulin response to intravenous glucose (airg) test and, for insulin sensitivity, the hyperinsulinemic- euglycemic clamp. since it is not practical to obtain these labor-intensive and costly phenotypes in large numbers of subjects for genetic studies, many have used surrogate measures of insulin secretion and insulin sensitivity. pre- vious studies have shown that the correlation between insulin-related indices from ogtt investigated in this study and those measured in the clamp were moderate (r � . – . ) but significant ( , ). in contrast, bergman et al. ( ) showed that in african and hispanic americans, the �g between fasting insulin and homa-ir was high ( . ), while the �g between fasting insulin and insulin sensitivity (si) derived from an intravenous glucose toler- ance test and that between homa-ir and si were modest (�g � � . and � . , respectively). the authors inter- preted these findings to mean that compared with si, fasting insulin and homa-ir are not good proxy measure- ment of insulin resistance. however, another interpreta- tion is that there may be differential genetic influence on insulin resistance among different populations or that homa-ir and si may be measuring different aspects of insulin resistance. indeed, variation in the relative amount of hepatic versus muscular insulin resistance has been demonstrated in individuals with type diabetes, and fasting glucose and insulin mark hepatic insulin resis- tance, while si may be a better marker of muscular insulin resistance ( , ). our systematic genetic analyses of plasma insulin levels during a -h ogtt and indices derived from these mea- sures provide insights that are likely to be useful in the search for genetic influences on insulin secretion and sensitivity. we demonstrate modest levels of heritability for a number of insulin secretion–related traits that have not been previously reported, such as insulin auc, insuli- nogenic index, and insulin . the level of heritability for fasting insulin levels in the amish was relatively low (h � . ) compared with values reported in caucasians ( . – . ) ( , ), african americans ( . ) ( ), mexican americans ( . – . ) ( , ), pima indians ( . ) ( ), and asians ( . ) ( ), although not much different from that estimated in african americans and hispanics from the insulin resistance and atherosclerosis study (h � . – . ) ( , ). as expected, based on the fact that homa-ir is derived from fasting insulin, both of these traits are phenotypically and genetically highly correlated. these two traits may reflect relatively more of the insulin-resistance phenotype. homa-ir is commonly used in epidemiological studies as a proxy measurement of insulin resistance, yet it did not appear to be more informative compared with fasting insulin in our genetic analysis. this is likely due to the fact that fasting insulin is a primary component of homa-ir calculation, particularly in nondiabetic subjects for whom there is less variation in glucose levels. our results suggest that a simpler measurement (fasting insulin) may serve as well as a more complicated composite measurement (homa-ir) for genetic studies of insulin resistance. on the other hand, insulin shared a significant genetic component with the insulinogenic index— both measures are thought to be more related to insulin secretion. impor- tantly, there was no significant genetic correlation be- tween fasting insulin and insulin , suggesting that these two traits are likely to have different genetic influences. these findings are consistent with the biological concept that defects in insulin sensitivity and insulin secretion are genetically distinct. from our genome-wide linkage analysis, we identified two chromosomal regions, q for insulin and p for insulinogenic index, with suggestive evidence of link- age. for the linkage signal for insulin on chromosome q, the -lod support interval is a . -cm region defined by markers d s and d s . within this interval, there are genes ( named, predicted [ncbi build . ]). several genes within our region of linkage on q may potentially be associated with 	-cell development and function. some of the candidate genes in this region are involved with hormone secretion (scamp and scamp ) ( , ), others are growth factors (nrg ) ( ), while others such as isl are homologous to proteins known to be involved in 	-cell development (isl ) ( ). for the linkage signal for insulinogenic index on chromosome p, the -lod support interval is a -cm region defined by markers d s and d s . within this interval, there are genes ( named, predicted). a number of candidate genes for 	-cell development or function lie within the -lod region, including two genes encoding secretory vesicle associated membrane proteins vamp and vamp . it is somewhat surprising that there was little overlap of table lod scores � . (p � . ) for each insulin-related trait from multipoint linkage analyses chromosome trait position (cm) nearest str marker lod score q homa-ir d s . p insulinogenic index d s . q -q fasting insulin d s . p -p insulinogenic index d s . q -q homa-ir d s . q fasting insulin d s . insulin auc d s . q insulinogenic index d s . q -q insulin d s . p -p fasting insulin d s . q insulin d s . p fasting insulin d s . genome scan of insulin traits in the amish diabetes, vol. , october linkage signals for insulin and insulinogenic index on chromosomes and given that genetic correlation was high. however, these traits were not perfectly correlated and thus may not have an identical genetic basis. further- more, gene-by-gene or gene-by-environment interactions, which were not accounted for in estimates of genetic correlation, may contribute to the lack of significant overlapping linkage signals in these regions. thus far, genome-wide linkage studies of diabetes- related traits, including insulin traits, have been reported in several ethnic groups ( – ). studies conducted in jap- anese and mexican americans have also revealed linkage to type diabetes on chromosome q. in a japanese study ( ), a suggestive linkage signal to type diabetes was observed on q -q (maximum lod score � . ), whose -lod region overlaps with the -lod region for the linkage to insulin in the amish. a small linkage signal to type diabetes (two-point maximum lod score for d s � . ) was also observed in the same region in a study of mexican americans ( ). on the other hand, evidence for replication of our linkage on chromosome p to insulinogenic index (lod . or empirical p � . , at cm) is more limited. a meta-linkage analy- sis utilizing information from four ethnic groups of the national heart, lung, and blood institute family blood pressure program ( ) reported suggestive linkage signals to both fasting insulin and homa-ir (lod . – . or empirical p � . – . , at � – cm on the amish map). we also observed lod scores � . (corresponding to an empirical p � . ) on nine other chromosomal re- gions. several of these regions of linkage have also been reported in the literature. in a recent study, freedman et al. ( ) observed suggestive linkage to both fasting insulin and homa-ir in african americans at the same location on chromosome p (lod . , near d s , at � cm on the amish map) where we observed a modest linkage signal to fasting insulin (lod . at cm). we did not observe evidence for linkage to regions on chromosomes p ( ), q ( ), or p ( ), which were previously reported to harbor loci for insulin traits we examined. in summary, we observed evidence for linkage of insulin traits to regions of chromosome p and q — both regions with a number of 	-cell candidate genes. further examination of these regions through linkage disequilib- rium mapping and positional candidate gene analysis will be necessary to identify the genes and their functional variants, which should be relevant not only to their influences on insulin levels but also to susceptibility to type diabetes. research design and methods our study was based on members of the amish family diabetes study. details of subject recruitment have been previously reported ( ). the study protocol was approved by the institutional review board at the university of maryland, baltimore, and informed consent was obtained from each participant. phenotypes. after an overnight fast, a standard -h ogtt with blood sampling every min was administered to subjects without a prior history of diabetes. plasma glucose and insulin concentrations were assayed with standard protocols. fasting glucose levels ranged from . to . mmol/l in this population. total insulin auc during the -h ogtt was calculated using the trapezoid method. homa-ir index was calculated as [fasting insulin (mu/l) fasting glucose (mmol/l)]/ . . the insulinogenic index was calcu- lated as [(insulin � fasting insulin)/(glucose � fasting glucose)]. criteria for the diagnosis of type diabetes, igt, and ifg were adapted from american diabetes association recommendations. as the development and treatment of type diabetes can significantly alter insulin levels, only nondiabetic subjects were included in the analysis (n � ). genotypes. we typed str markers on autosomes using dna from leukocytes. these markers were from the abi prism linkage mapping set (perkin-elmer). overall genotyping rates across all strs were . � . % complete. the marker order and sex-averaged intermarker distances (mean . cm) were estimated from our data using cri-map ( ). the mean marker heterozygosity was . (range . – . ). based on initial linkage analysis results, additional str markers were genotyped on chromosome (between and cm) for a fine-mapping study. statistical analysis. values for traits with a significantly skewed distribution were transformed by their natural logarithm, and extreme outliers (value deviating from the mean by � sd) were excluded from analysis (n � – , depending on the trait). furthermore, to reduce the computational complex- ity, we divided the single large pedigree into smaller pedigrees (n � – ) for analysis. results from all analyses were adjusted for sex-specific age, age , and bmi. all analyses were conducted using a pedigree-based variance components method. we first estimated heritability then genetic correlation. bivariate modeling was used to partition the phenotypic correlation (�p) between a given pair of quantitative traits into their additive genetic (�g) (i.e., genetic correlation) and random environmental (�e) components ( ). for linkage analyses, the effect of a quantitative trait locus was estimated by modeling the covariance in a trait between individuals to be a function of the probability that they inherited both alleles at the marker locus from a common ancestor. both two-point and multipoint analyses were performed, and statistical significance was evaluated by likelihood ratio tests using the solar program ( ). multipoint identity-by-descent matrices were computed using the ko- sambi function in the loki program ( ). as the variance components methods can be susceptible to significant violations of the multivariate normality assumption, we used simulations to estimate the empirical proba- bility of obtaining false evidence for linkage. we derived the distribution of nominal lod scores under the null hypothesis of no linkage by simulating , unlinked markers, dropping them through the pedigrees, and conduct- ing linkage analysis with each of the , markers for each of the insulin traits. the probability of obtaining a false-positive result was defined as the proportion of replicates for which we obtained a specified lod score or higher. the p values obtained from the simulation study were then back- converted into lod scores by first converting them into corresponding � values and then by dividing the � values by ( ln ). all lod scores from quatitative trait locus analyses presented in this report were obtained from this simulation. acknowledgments this study was supported in part by a research grant from glaxowellcome, inc. and axys pharmaceuticals; national institutes of health grants r dk , k dk , u dk , r ag , r dk , and k ag ; and the american diabetes association. fund- ing and support was also provided by the university of maryland general clinical research center (grant m rr ), general clinical research centers program, na- tional center for research resources, and the baltimore veterans administration geriatric research and educa- tion clinical center. we thank the amish research clinic staff for their energetic efforts in study subject recruitment and charac- terization, drs. alejandro schäffer and richa agarwala for assistance in pedigree construction, and dr. pamela st. jean for helpful comments on the manuscript. this study would not have been possible without the outstanding cooperation of the amish community. references . mercado mm, mclenithan jc, silver kd, shuldiner ar: genetics of insulin resistance. curr diab rep : – , . stumvoll m, fritsche a, haring hu: clinical characterization of insulin secretion as the basis for genetic analyses. diabetes (suppl. ):s – s , . an p, freedman bi, hanis cl, chen yd, weder ab, schork nj, boerwinkle e, province ma, hsiung ca, wu x, quertermous t, rao dc: genome-wide linkage scans for fasting glucose, insulin, and insulin resistance in the national heart, lung, and blood institute family blood pressure program: w.-c. hsueh and associates diabetes, vol. , october evidence of linkages to chromosome q and q from meta-analysis. 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characterization and heritability analysis of the amish family diabetes study. diabetes care : – , . green p, falls k, crooks s: documentation for cri-map. version . . st. louis, mo, department of genetics, school of medicine, washington university, . williams-blangero s, blangero j: quantitative genetic analysis of skin reflectance: a multivariate approach. hum biol : – , . almasy l, blangero j: multipoint quantitative-trait linkage analysis in general pedigrees. am j hum genet : – , . heath sc: markov chain monte carlo segregation and linkage analysis for oligogenic models. am j hum genet : – , genome scan of insulin traits in the amish diabetes, vol. , october injuries among amish children: opportunities for prevention short report open access injuries among amish children: opportunities for prevention stephen strotmeyer * , abigail koff , joshua n. honeyman and barbara a. gaines abstract objectives: the purpose of this study was to examine the injury risk patterns among amish children, many of whom may be exposed to uncommon injuries and limited access to care due to their agrarian lifestyle and remote communities. design: retrospective chart review. methods: with irb approval, we performed a retrospective review of amish patients age ≤ years presenting to a level i pediatric trauma center between january , , and december , . data abstracted from the institutional trauma registry and electronic medical record were analyzed using descriptive statistics and univariate/ multivariate analysis. results: one hundred eighty-three amish children were admitted, and died from injuries. patients were . % male; the median age was (iqr – ); median injury severity score (iss) was (iqr – ), most injuries were the result of blunt force trauma ( . %). the most frequent mechanisms were falls ( . %), followed by animal-related ( . %), and buggy ( . %). most injuries occurred at home ( . %) or on a farm ( . %). hay hole falls were a unique source of injury with a high iss ( ; iqr – ). the overall median length of stay (los) was days (iqr – ), with animal-related injuries associated with the longest los ( days; iqr – . ). conclusions: the majority of injuries among amish children are due to falls. hay hole falls and animal-related injuries result in the highest iss and longest los. these findings identify the farm as a potential target for culturally appropriate interventions for risk modification. keywords: amish, injury, trauma, pediatric introduction trauma is the leading cause of death in children older than year (center for disease control and prevention ), and hospitalizations for pediatric trauma patients contribute over $ billion to medical expenditures in the united states [“children’s safety network report on preventing adolescent injury: the role of health plans.”] (jones ). currently, there is limited data in the literature on the etiology and impact of trauma in the pediatric amish community. pennsylvania and ohio are home to the largest collec- tion of amish settlements in the united states, with an estimated , amish children and adults. pennsylvania alone is home to of the largest amish settlements, with lancaster county home to the largest settlement in the country at an estimated , (young center for anabaptist and pietist studies ). while there is a range of cultural expression within the larger old order anabaptist community, amish beliefs often include avoid- ance of technology, conservative dress, and lifestyle, and adherence to traditional farming practices (rohrer and dundes ). the amish largely avoid allopathic health care and health insurance, believing the latter undermines the accountability of the community (kraybill et al. ). previous studies have investigated mechanisms of injury in the amish community, identifying the rural agrarian lifestyle and utilization of horse-drawn buggies as unique mechanisms of injury in this population (vitale et al. ). previous pediatric studies focused on farm-related injuries established that children in agrarian © the author(s). open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/ . /) applies to the data made available in this article, unless otherwise stated. * correspondence: stephen.strotmeyer@pitt.edu upmc children’s hospital of pittsburgh, penn avenue, faculty pavilion, - , pittsburgh, pa , usa full list of author information is available at the end of the article strotmeyer et al. injury epidemiology ( ) : https://doi.org/ . /s - - -x http://crossmark.crossref.org/dialog/?doi= . /s - - -x&domain=pdf http://orcid.org/ - - - http://creativecommons.org/licenses/by/ . / http://creativecommons.org/publicdomain/zero/ . / mailto:stephen.strotmeyer@pitt.edu communities often assume responsibility for farming and animal husbandry tasks, placing them at risk for po- tential work-related injury at a young age (tevis ). also, the farm and barn environments are often sites of play and recreation, increasing risk exposure (fisher et al. ). however, even within agrarian communities, amish children exhibit a unique spectrum of injury given cultural limitations on usage of technology and farming, as well as decreased access to care and health insurance (hubler, hubcey, ). one study focusing on the pediatric amish commu- nity specifically, identified falls as the leading cause of injury, specifically through hay holes (vitale et al. ), prompting further studies (engbrecht et al. ) and in- terventions (batra et al. ). haylofts are storage areas on the second story in barns in which hay is kept, and hay holes are holes in the floor of the lofts utilized to drop hay to the animals below. as a stand-alone children’s hospital, upmc children’s hospital of pittsburgh has a large catchment area over- lapping large amish populations (fig. .), we reviewed our experience with pediatric trauma in the amish population, focusing on the mechanism and outcome of the injury. methods following approval from the institutional review board of the university of pittsburgh school of medicine (pro ), a retrospective review of the inpatient medical record and the institutional trauma registry was performed. we identified all amish patients aged years and younger admitted to children’s hospital of pittsburgh for traumatic injuries between january , , and december , . from an initial cohort , patients were screened for inclusion into the study through a series of selection criteria (fig. .) including mention of “amish” in either mechanism of injury or location, mention of “buggy”, high frequency last names, and mentions of “horse”, “farm”, or “gun”. patient charts were then checked for the religion section of “patient information”. if it stated amish, they were ruled in. if it stated any other specific religion, they were ruled out. if it said unspecified or was blank, history & physical exam and progress notes were checked for a mention of amish in social history or assessment. data abstracted from the trauma registry and elec- tronic medical record included: age, sex, past medical/ surgical history, date of injury, time of injury, time from injury to presentation for care, postal code where injury occurred, location where injury occurred (e.g. home, farm, road), location of first medical care, mechanism (fall, mvc, animal, buggy, pedestrian, bicycle, machin- ery, recreational vehicle), documented use of protective equipment (if applicable), activation trauma level, injur- ies, injury severity score, initial gcs, radiologic studies obtained (trauma series, number of ct scans, number of mri), hospital admission, length of stay, icu admission, operative procedures, total hospital charges, disposition (e.g. home, rehab), and residual disability. prior to analysis, all data were exported to an elec- tronic spreadsheet for further data cleaning and coding. descriptive statistics were used to describe demographic information. that is, frequencies and proportions were reported for categorical variables, while the means and standard deviations or % confidence intervals (ci) were reported for continuous variables. paired student t- tests were used to determine if there were any significant differences between the observations. all statistical ana- lyses were performed using spss version [ibm corp. released . ibm spss statistics for windows, version . . armonk, ny: ibm corp.]. fig. geographic distribution of amish trauma patients strotmeyer et al. injury epidemiology ( ) : page of results between january , , and december , , amish children were evaluated for traumatic injury. children ranged in age from month to years (mean . , median , iqr – ). patients were . % male and . %, female (table .). while the catchment area for trauma referrals includes urban and rural environments throughout western pennsylvania, eastern ohio, west virginia, and western new york, over % of identified amish patients came from only four counties: mercer ( . %), jefferson ( . %), indiana ( . %), and crawford ( . %). most injuries occurred at home ( . %) or on a farm ( . %) with an additional . % occurring on the road- way. (fig. .) the most frequent mechanisms were falls ( . %), followed by animal-related ( . %), buggy ( . %), and gun-related ( . %) (fig. ). fourteen percent of patients met the criteria for the high- est level of trauma activation. of the level i activations, were animal-related in nature, were the result of hay hole falls, and were directly related to farm equipment. the median injury severity score (iss) was . (iqr – ; range to ). while the majority of patients ( . %) had a normal glasgow coma score (gcs), the next most fre- quent gcs was ( . % of patients). there did not seem to be a strong correlation between gcs and iss however, with the patients with the highest iss ( – ) having a gcs of to . interestingly, the fatally injured patients had iss of and , both with a gcs of . median in-hospital length of stay was days (iqr – ; maximum days). eighty-six patients stayed day in the hospital, with patients staying greater than days, and only patients staying greater than days in the hos- pital. fifty-one patients ( %) were admitted to the inten- sive care unit, with . % ( patients) staying from to days. only patients had an icu stay longer than days (maximum days). of those patients admitted to the icu, ( . %) had a skull fracture, and ( . %) had a cns injury. of the icu patients, ( %) suffered injuries to the lungs, and ( . %) suffered axial orthopedic in- juries. mechanism of injury (moi) for icu patients in- cluded falls (n = ; . %), of which ( . %) were associated with a hay hole. the next most frequent moa for icu stays was animal-related injuries (n = ; . %). most patients were discharged home ( . %), and pa- tients ( . %) were discharged to a rehabilitation center. two patients died of their injuries: a gsw to the head with an initial iss of , and a blunt head injury with an iss of . hay hole falls were a unique source of injury with a high iss ( ; iqr – ) (table .) the most commonly injured organ system was neurological with ( . %) injuries, of which ( . %) had associated skull fractures. of these pa- tients, ( . %) patients spent at least day in the icu. all patients had a hospital los less than days, with a majority ( , . %) staying days or less. fig. selection criteria: if patient met criteria for steps or , chart review was completed for mention of “amish”. patients were excluded if there was no mention of “amish” or if an alternative religion was explicitly mentioned. ai: already included in prior screen. moa: mechanism of action (of injury) table demographic counts gender male female age (in years) < – – – strotmeyer et al. injury epidemiology ( ) : page of of the animal-related injuries, a majority ( ) in- volved a horse, with being the result of kicking. the majority of patients ( ) were under years of age. animal-related injuries were associated with the longest los ( days; iqr – . ). eleven ( . %) patients spent at least day in the icu. there was a median iss of (iqr – ) with a maximum iss of , the highest of all patients in the study. of the -total animal-related injuries, ( %) resulted in neurological injuries, of which were skull fractures. orthopedic injuries were the second most common, with ( . %) injuries. discussion the results of our study seem to be in line with the histor- ical findings surrounding the etiology of trauma in pediatric patients. more specifically, our data support the epidemiological data surrounding the pediatric amish population, with the most frequent mechanisms of injury being falls, followed by animal-related injuries, and buggy/ transportation injuries. most likely due to their high agrar- ian lifestyle, our study identified farm-related mechanisms of injury common in this population. in comparison to the non-amish population, however, in which a majority fig. mechanism of injury fig. number of injuries by location and mechanism strotmeyer et al. injury epidemiology ( ) : page of of injuries are due to equipment (cogbill et al. ), a large amount of farm-related injuries were due to falls or animals. like a previous study of the epidemiology of pediatric amish trauma, our study identified hay hole falls as a significant and unique source of injury with a corresponding high iss. our study highlights potential areas for intervention and prevention. both falls, and animal-related injuries make up a large portion of the injuries in the pediatric amish population. previous data from penn state had identified the hay hole as a unique area for potential intervention, and with the support of the pennsylvania amish safety committee, a hay hole cover was devel- oped that was both culturally appropriate as well as feas- ible for the community. initial data from the dispersion of these covers was encouraging, with them being well received and utilized by the anabaptist community. our data suggest that while hay hole falls are a significant source of trauma, animal-related injuries result in a lon- ger length of stay, identifying another potential area for intervention. previously, multiple strategies have been aimed at pediatric farm-related injuries, but there has been little data published on the long-term effects of such intervention. one systematic review found some ef- fectiveness in regards to short term knowledge in school-based programs and safety day camps aimed at the intervention of acute pediatric agricultural injuries, but mixed results were seen with farm-based interven- tions (hartling et al. ). this study also found that retention and knowledge acquisition improved if accom- panied by education on pediatric developmental stages and/or a farm visit from a dedicated safety specialist. any potential intervention strategy would necessitate a partnership with the amish and anabaptist communi- ties. the trauma and acute care surgery department at lancaster general health has piloted interventions such as annual farm safety days and the amish safety com- mittee, in which amish leaders meet with a member of the benedum trauma program from children’s hospital of pittsburgh, that could serve as potential guides to in- terventions for our own communities of amish patients. limitations there are many limitations to our study. as a level trauma center, our patient population is often higher acuity and thus may not only underestimate the number of pediatric traumas but also may not illustrate alterna- tive mechanisms not seen at our institution due to their lower acuity. as a quaternary care center, our patient population is more severely injured, and thus may not be the most complete picture of the spectrum of severity of traumatic injuries. however, it is likely that our data are generalizable to the larger amish community given not only the large percentage of amish patients seen at our center but also the fact that our findings appear to be in line with findings of previous institutions. how- ever, a multi-institution study or a larger systematic re- view after further studies have taken place would aid in remedying the small sample size, single geographical area, and possible selection bias. the methods of selection introduce bias. since at present there is no reliable way to select amish patients from the trauma registry, our study utilized a series of “select in” methods to construct our data population. our use of “self-payer insurance” as the primary selec- tion criteria is reasonable, however, given it has been established previously that the amish community gener- ally do not carry health insurance, and instead pay out of pocket, thus labeled in our system as “self-payer”. conclusion the majority of injuries among amish children are due to falls. hay hole falls and animal-related injuries result in the highest iss and longest los. these findings iden- tify the farm as a potential target for culturally appro- priate interventions for risk modification. we hope to use the data collected from this study to in- form future studies, including outreach and educational interventions aimed at reducing the incidence of traumatic injuries and mitigating the severity of injuries that occur. abbreviations ai: already included in previous inclusion criteria; chp: children’s hospital of pittsburgh; gcs: glasgow coma scale; icu: intensive care unit; iqr: interquartile range; iss: injury severity scale; moa: mechanism of action; mvc: motor vehicle collision acknowledgments the authors would like to thank the members of the benedum trauma program, whose diligence in trauma data collection was considerable and appreciated: christine perlick, shannon shoemaker, kathleen oxenreiter, marianne miller, majorie vinski and melinda gallagher. we would also like to thank our clinical research coordinator, min shi. authors’ contributions jh, ak, bg conceived the research idea and developed the project. ak and jh collected and screened the dataset. ss programmed and analyzed and statistically interpreted the findings. all authors read and approved the final manuscript. funding the authors received no financial support for the research, authorship, or publication of this article. table number of injuries by location and mechanism mechanism of injury iss (iqr) los (iqr) falls ( – ) . ( . – . ) falls: hay-hole ( – ) . ( . – . ) buggy ( – ) . ( . – . ) animal-related ( – ) . ( . – . ) strotmeyer et al. injury epidemiology ( ) : page of availability of data and materials study data for the research resides at the benedum pediatric trauma program in the division of pediatric general and thoracic surgery at upmc children’s hospital of pittsburgh, university of pittsburgh. ethics approval and consent to participate ethical approval was obtained from the university of pittsburgh institutional research board under exempt review, pro . consent for publication not applicable. competing interests the authors declare that they have no competing interests. author details upmc children’s hospital of pittsburgh, penn avenue, faculty pavilion, - , pittsburgh, pa , usa. department of pediatrics, university of washington affiliated hospitals, seattle, wa , usa. hasbro children’s hospital, warren alpert medical school of brown university, dudley street, suite , providence, ri , usa. received: september accepted: november references batra ek, gross bw, jammula s, bradburn eh, baier rd, reihart mj, murphy d, moyer k, hess j, lackmann s, miller ja. preliminary results of a novel hay- hole fall prevention initiative. j trauma acute care surg. ; ( ): – . center for disease control. cdc childhood injury report. retrieved from https://www.cdc.gov/safechild/child_injury_data.html children’s safety network report on preventing adolescent injury: the role of health plans. retrieved from https://www.nihcm.org/pdf/injuryissuebrief.pdf. accessed sept . cogbill th, busch hm, stiers gr. farm accidents in children. pediatrics. ; ( ): – . engbrecht bw, kulaylat an, dias m, kendig jw, cilley re. childhood injuries due to hay-hole falls: a -year experience at a rural pediatric trauma center. pediatr emerg care. ; ( ): – . fisher km, hupcey je, rhodes da. childhood farm injuries in old-order amish families. j pediatr nurs. ; ( ): – . hartling l, brison rj, crumley et, klassen tp, pickett w. a systematic review of interventions to prevent childhood farm injuries. pediatrics. ; ( ):e – . hubler cl, hupcey je. incidence and nature of farm-related injuries among pennsylvania amish children: implications for education. j emerg nurs. ; ( ): – . jones mw. a study of trauma in an amish community. j trauma. ; ( ): – . kraybill db, johnson-weiner km, nolt sm. the amish. baltimore: johns hopkins university press; . rohrer k, dundes l. sharing the load: amish healthcare financing. inhealthcare , no. , p. . multidisciplinary digital publishing institute. tevis c. downsizing farm chores. successful farming. ; ( ): – . vitale ma, rzucidlo s, shaffer ml, ceneviva gd, thomas nj. the impact of pediatric trauma in the amish community. j pediatr. ; ( ): – . young center for anabaptist and pietist studies, e. c. ( ). twelve largest amish settlements, . retrieved from http://groups.etown.edu/ amishstudies/statistics/largest-settlements/. publisher’s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. strotmeyer et al. injury epidemiology ( ) : page of https://www.cdc.gov/safechild/child_injury_data.html https://www.nihcm.org/pdf/injuryissuebrief.pdf http://groups.etown.edu/amishstudies/statistics/largest-settlements/ http://groups.etown.edu/amishstudies/statistics/largest-settlements/ 	abstract 	objectives 	design 	methods 	results 	conclusions 	introduction 	methods 	results 	discussion 	limitations 	conclusion 	abbreviations 	acknowledgments 	authors’ contributions 	funding 	availability of data and materials 	ethics approval and consent to participate 	consent for publication 	competing interests 	author details 	references 	publisher’s note med genet ; : - cartilage-hair hypoplasia in finland: epidemiological and genetic aspects of patients outi makitie abstract cartilage-hair hypoplasia (chh) is an autosomal recessive form of metaphyseal chondrodysplasia characterised by short limbed short stature, hypoplastic hair growth, and impaired cell mediated immunity and erythrocyte production. the syndrome is exceptionally prevalent among the finns and among the old ord- er amish in the united states; sporadic cases have been reported from other countries. an epidemiological and genetic study of chh in finland showed patients, males and females, in families. eighteen ofthem had died, seven before the age of year. the living patients ranged in age from to years, median years. the incidence was estimated to be : live births. consanguinity was found in two families and interfamilial relationships in families. geographical distribution of the birth places of the patients and their great grandparents showed accumulation in a small area in western finland and regional clusters were seen in other parts of the country as well. the result ofthe segregation analysis was in accordance with recessive inherit- ance with reduced penetrance. (j med genet ; : - ) department of medical genetics, and children's hospital, university of helsinki, helsinki, finland. o mmkitie correspondence to dr makitie, department of medical genetics, helsinki university hospital, tukholmankatu f, sf- helsinki, finland. received february . accepted february . cartilage--hair hypoplasia (chh) is a form of metaphyseal chondrodysplasia characterised by disproportionate short limbed short stature and fine and sparse hair.' skeletal growth is retarded from birth; the median adult height is cm for males and cm for females.' cellular immunity is impaired as indicated by lymphopenia and decreased in vitro lympho- cyte reactivity; humoral immunity is intact. deficient erythrocyte production often leads to mild transient macrocytic anaemia, and, on occasion, to congenital hypoplastic anaemia. the disease was first described among the old order amish in the united states and it has also been found in the finns in exceptionally high numbers. sporadic cases have been reported in other countries. mckusick et ap concluded that the mode of inheritance is autosomal recessive with reduced penetrance, since the number of patients in the families was smaller than expected. as part of a multidimensional project on chh, an epidemiological and genealogical study was carried out in finland. patients and methods the material consisted of chh patients dia- gnosed and followed at the department of medical genetics or the children's hospital, helsinki university hospital, which is the major national centre for studying children with growth failure. in addition, a question- naire study was carried out in to in each of finland's health care districts in order to attain complete ascertainment. the questionnaire was sent to the senior physicians of paediatric, medical genetic, internal medi- cine, and orthopaedic units of all central hospi- tals (n = ) and district hospitals (n = ) as well as to physicians in charge in health centres (n = ). the questionnaire consisted of de- scriptions and illustrations of chh, diastro- phic dysplasia, and achondroplasia, the three most common chondrodysplasias in finland. information was requested on any such patients. in case of positive answers, the patients were contacted by their own doctors, and medical records, radiographs, and detailed information on the growth failure were obtained. a clinical study was performed at the children's hospital if chh was suspected. furthermore, chh patients were looked for and personally contacted at the meetings of the association of little people in finland. the diagnosis of chh was based on short limbed short stature, generalised laxity of joint ligaments and, in childhood radiographs, meta- physeal flaring and irregularities of the growth plates. hair hypoplasia was used only as a positive criterion; normal hair does not exclude chh (makitie and kaitila, unpublished data). there were seven patients who had normal hair but otherwise typical features of chh. two of them had a sib, and another two a relative, all with typical features of chh. since present urbanisation has mixed the possible regional clusters, differences in re- gional gene frequencies were assessed by the great grandparents' birth places. the patients' and their great grandparents' birth places and the great grandparents' identity were confirmed from the population register. detailed genea- logy was available for some families. for the incidence calculation the number of annual live births was obtained from the statistical year- book. information on abortions was obtained from parents and hospital records and was compared with the abortion frequency in the normal population. if the family had not been contacted recently, the number of sibs was verified from the population register. heights of the sibs were recorded from the child health o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ cartilage-hair hypoplasia in finland: epidemiological and genetic aspects of patients centres to find possible undiagnosed patients. segregation analysis was performed with the 'singles method"' and the 'a priori method'," which both assume truncate complete ascer- tainment, and with the 'sib method'," which assumes single incomplete ascertainment. the study was approved by the ethical com- mittee of the children's hospital. results the epidemiological study showed a total of finnish chh patients, males and females, in families. eighty patients had been followed at the department of medical genetics or the children's hospital. eighteen families with chh patients were added through the questionnaire study and one patient through the association of little people. eighteen of the patients had died, seven of them under the age of year. the living patients ranged in age from to years, median years. the incidence of chh calcu- lated for the year period to was : live births, which means about three new patients yearly (fig ). the frequency of spontaneous abortions was %, which is not higher than the abortion frequency in the normal population. there was no apparent relationship between the birth order and occurrence of chh. two female patients from two finnish fami- lies lived in sweden. since adequate informa- tion on their sibs or ancestors could not be obtained, they were not included in the segre- gation analysis. thus, the series consisted of finnish chh patients in families (fig ). fourteen families had two affected sibs and four families had three. altogether there were unaffected sibs in the families. using the correction for the 'singles method', the ratio of the affected sibs was - (se ). by the 'a priori method' the observed number of patients ( ) was lower than expected on the basis of recessive inheritance ( , se - ). the 'sib method' resulted in a ratio of - (se ) of the affected sibs. the patients' and their great grandparents' birth places showed an accumulation in a small area in western finland, southern ostroboth- nia. furthermore, the gene was more frequent in three other areas: the south-eastern corner of the country, northern savonia, and the most southern part of lapland (fig ). the incidence in the high frequency area of south- a alive (n = ) e dead (n= ) e - - clloh h h * . h. * * *o , ' * o o: * u. o s o o o - o * * * o * o o * u.. ho.m o m o ho.o/ 'o * o o i o o i o o * o o i o o o i o b/d ' o o hz el o o o o o ooe o o o o /' o o ao oqe ) ( h ej flue * * o- ' * , , .e. o h *,o iodi o oo ,sob o o * ow' o o u oyo o o ojz(i z' jzfo.oozouo ho * *ha o *,o'a o o * * affected o unaffected * spontaneous abortion figure the sibships with affected, unaffected, and spontaneous abortions. em ostrobothnia was : live births. con- sanguinity, not closer than third cousins, was found within two families, and interfamilial relationships in families (fig ). in more cases, however, possible remote relationships between the families could not be detected, since the ancestors were not traced back further than the great grandparents. six female patients were married to unaffec- ted males. two of these couples had two children and two had one child. all the chil- dren had been delivered by caesarean section and were healthy. none of the male patients was married or had children. discussion cartilage-hair hypoplasia (chh) is a rare inherited form of metaphyseal osteochondro- dysplasia which results in severe growth fail- ure. it is overrepresented among the religious sect of the old order amish in the united states and among the finns. at least chh patients have been recognised among the amish. the present study showed a total of chh patients among the finns. the number of chh patients among other natio- nalities is low: seven patients have been reported among the english,"-" eight among the dutch (van der burgt, personal communi- cation), and sporadic cases among the french, - germans, - danes algerians, italians, ' polish (van der burgt, personal communication), spanish," and mexicans.' a study of children with bone dysplasia in nigeria found no patients with chh." the specific pattern of rare hereditary dis- eases in finland, that is, overrepresentation of - - - - - - - - - - year of birth figure i cartilage-hair hypoplasia patients born in to . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ makitie a patients' birth figure geographical distribution of birth places of (a) the patients and (b) the great grandparents (one dot =family or great grandparent). the area denoted by the broken line belonged to finland before the second world war. some rare autosomal recessive disorders and lack of some others, is the result of national and regional isolation of the small population. most of the ancestors of chh families origi- nated from a small area in western finland with some regional clusters in a few eastern and south-eastern communities. these are sparsely inhabited rural areas which were permanently settled as late as the sixteenth century and are still isolated. even today most native inhabit- ants of these communities are descendants of a few settlers from to generations back and are therefore remotely related to one another. the break up of isolates by increas- ing migration to towns will in the future slowly even out the clustering of recessive genes and thereby result in a decrease in the incidence of chh. the patients' birth places, however, still gave evidence of accumulations similar to their great grandparents' birth places. calculated using the hardy-weinberg coefficient, the gene frequency for the whole country was - and for the high frequency area of southern ostro- bothnia - , and the carrier frequencies were : and : , respectively. however, this method is partly invalidated by uneven distri- bution of the gene in the finnish population. the present study showed consanguinity in two families and interfamilial relationships in families. however, by tracing the ancestors back further than the great grandparents, several other remote relationships within and between chh families would certainly have emerged. in the study of chh among the amish, the number of affected was higher for females than for males ( v ). similarly, females were overrepresented among the finnish patients ( v ). when the two groups are combined, the sex ratio is : (x = , ns). autosomal recessive inheritance was estab- lished in a study of chh in amish sibships. however, as the number of affected patients was significantly lower than expected on the basis of the recessive hypothesis ( v - , se ), reduced penetrance of about % was suggested. the other possible explanations discussed were contamination of the series by numerous sporadic, mimicking dominant conditions, non-random chromosome segrega- tion, gametic selection, and increased mortality in utero. the segregation analysis of fin- nish chh families was performed by methods of complete and incomplete ascertainment. the ascertainment was probably close to complete among young patients, but incomplete in the older age groups, since no patients over years of age were ascertained. as the ascertainment was somewhere between complete and incom- plete, the corrected ratio of affected patients lies between those derived by the two extreme methods, that is, between - (se ) and - (se ). the result is in accordance with reduced penetrance. five more patients in these families would have resulted in a ratio of - ('singles method'). the abortion frequency among the finnish chh families does not support increased mortality in utero. the lack of affected patients is too great to be explained figure pedigrees of chhfamilies with consanguinity (two families) or interfamilial relationships ( families). o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ cartilage-hair hypoplasia in finland: epidemiological and genetic aspects of patients by contamination by new dominant mutations and contamination by another recessive dis- order should not lead to lack of affected. the other explanations, non-random chromosome segregation and gametic selection, are hypo- thetical and highly improbable, but difficult to prove or disapprove. the reduced penetrance in chh may be explained by some homozygotes being so mildly affected that they are presumed to be healthy. among the amish this was found in two cases: very mild chh, only found radio- logically, in three children of affected parents, and thin hair, short and pudgy hands, and contracted pelvis in a female who was cm tall. furthermore the hair diameter in some healthy sibs overlapped that of the patients. in another study, lymphopenia and low response to lymphocyte stimulation were observed in a healthy sib who had no other clinical features of chh. in order to find mildly affected, undiagnosed persons among the finnish chh families, the relative height (deviation ofheight, in sd units, from mean height for age and sex) of healthy sibs was compared with the mid- parent relative height: no difference of more than - sd was observed. also, the midparent relative height was always within the mean ( . sd) of the normal population. the relative heights of the patients, in contrast, were always more than sd below the midparent relative height, and the adult relative heights ranged from - sd to - sd. childhood radio- graphs of the left knee were obtained from healthy sibs; none of them showed metaphyseal changes. one healthy sister had poor hair growth and wore a wig; her adult height was cm and her sitting height normal. no child- hood radiographs were available for her. more thorough clinical and biochemical studies of healthy sibs are needed to settle the question of reduced penetrance. the results of this study were consistent with those of studies of the amish chh patients. the number of affected persons in the families was lower than expected on the basis of a recessive hypothesis, and the affected females slightly outnumbered the affected males. no definite explanation was found and these features of the recessive chh syndrome remain to be elucidated further. i am grateful to professor reijo norio, dr ilkka kaitila, and professor jaakko perheen- tupa for their comments on the manuscript. this study was supported by grants from the university of helsinki, the finnish medical foundation, the paulo foundation, and the paivikki and sakari sohlberg foundation, helsinki, finland. i mckusick va. metaphyseal dysostosis and thin hair: a "new" recessively inherited syndrome? lancet ;i: . mckusick va, eldridge r, hostetler ja, ruangwit u, egeland ja, dwarfism in the amish ii. cartilage-hair hypoplasia. bull johns hopkins hosp ; : - . makitie , perheentupa j, kaitila i. growth in cartilage- hair hypoplasia. pediatr res ; : - . virolainen m, savilahti e, kaitila i, perheentupa j. cellu- lar and humoral immunity in cartilage-hair hypoplasia. pediatr res ; : - . polmar sh, pierce gf. cartilage hair hypoplasia: immuno- logical aspects and their clinical implications. clin immu- nol immunopathol ; : - . miikitie , rajantie j, kaitila i. anemia and macrocytosis, unrecognized features of cartilage-hair hypoplasia. acta paediatr scand (in press). kaitila i, perheentupa j. cartilage-hair hypoplasia. in: eriksson aw, forsius h, nevanlinna hr, workman pl, norio rk, eds. population structure and genetic disorders. london: academic press, : - . central statistical office of finland. statistical yearbook of finland . helsinki: valtion painatuskeskus, : . hemminki k, mutanen p, saloniemi i, niemi n-l, vainio h. spontaneous abortions in hospital staff engaged in sterilising instruments with chemical agents. bmj ; : - . li cc, mantel n. a simple method of estimating the segregation ratio under complete ascertainment. am j hum genet ; : - . hogben l. the genetic analysis of familial traits. i. single gene substitutions. j genet ; : - . hogben l. an introduction to mathematical genetics. new york: norton, . fisher ra. the effect ofmethods ofascertainment upon the estimation of frequencies. ann eugen ; : - . francomano ca, trojak je, mckusick va. cartilage hair hypoplasia in the amish: increased susceptibility to malignancy. am j hum genet ; : a. savage mo. metaphyseal dysplasia in siblings: a variant of cartilage-hair hypoplasia (case report). proc r soc med ; : . boothby cb, bower bd. cartilage hair hypoplasia. arch dis child ; : - . siggers dc, burke jb, morris b, normand ics, tanner jm, williamson daj. cartilage-hair hypoplasia. postgrad med j ; : - . maroteaux p, savart p, lefebvre j, royer r. les formes partielles de la dysostose metaphysaire. presse med ; : - . sacrez r, levy jm, godar g, castanier j. anemie de blackfan-diamond associe a des malformations multiples. med infantile ; : - . l'hirondel j, caen, daridon, tillet. anemie de blackfan- diamond et dysostose metaphysaire recessive autosomi- que. ouest med ; : - . rubie h, graber d, fischer a, et al. hypoplasie du cartilage et des cheveux avec deficit immunitaire combine. ann pediatr (paris) ; : - . le merrer m, briard ml, chauvet ml, maroteaux p. chondrodysplasie metaphysaire recessive autosomique et maladie de hirschspring. ann pediatr (paris) ; : - . wiedemann hr, spranger j, kosenow w. knorpel-haar- hypoplasie. arch kinderheilkd ; : - . beals rk. cartilage-hair hypoplasia. a case report. j bone joint surg (am) ; : - . lowry rb, wood bj, birkbeck ja, padwick ph. cartilage- hair hypoplasia. a rare and recessive cause of dwarfism. clin pediatr ; : - . seige m. metaphysare chondrodysplasie vom typ mcku- sick (knorpel-haar-hypoplasie). monatsschr kinder- heilkd ; : - . steffensen , stergaard pa. an inverted ratio for t- helper/suppressor cells, and selective deficiency of cell- mediated immunity, in a girl with cartilage-hair hypo- plasia. eur j pediatr ; : - . d'apuzzo v, joss e. metaphysare dysostose und hypopla- sie der haare: knorpel-haar-hypoplasie. beitrag zur statistischen erfassung der morphologie der haare. helv paediatr acta ; : - . ventruto v, pisciotta r, lambertini p, et al. su una rara forma ereditaria di condrodisplasia metafisaria: la ipopla- sia cartilagine-capelli. progr med ; : . pinto l, nobili b, scarano g, iolascon a. cartilage-hair hypoplasia: studio clinico et immunologico. pediatr napoli ; : - . ray hc, dorst jp. cartilage-hair hypoplasia. progr pediatr radiol ; : - . sanchez-corona j, garcia-cruz d, medina c, cantii jm, ramos-zepeda r, rivas f. increased adenosine deami- nase activity in a patient with cartilage-hair hypoplasia. ann genet (paris) ; : - . adeyokunnu aa. spectrum of bone dysplasias in african children: ibadan nigerian experience. in: papadatos cj, bartsocas cs, eds. skeletal dysplasias. new york: alan r liss, : - . norio r, nevanlinna hr, perheentupa j. hereditary dis- eases in finland; rare flora in rare soil. ann clin res ; : - . norio r. diseases of finland and scandinavia. in: roths- child h, ed. biocultural aspects of disease. new york: academic press, : - . norio r. heredity in the congenital nephrotic syndrome; a genetic study of finnish families with a review of reported cases. ann paediatr fenn ; (suppl ). mckusick va, eldridge r, hostetler ja, egeland ja. dwarfism in the amish. trans assoc am physicians ; : - . trojak je, polmar sh, winkelstein ja, et al. immunologic studies of cartilage-hair hypoplasia in the amish. johns hopkins medj ; : - . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ pgen. .. genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits the harvard community has made this article openly available. please share how this access benefits you. your story matters citation speliotes, elizabeth k., laura m. yerges-armstrong, jun wu, ruben hernaez, lauren j. kim, cameron d. palmer, vilmundur gudnason, et al. . genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits. plos genetics ( ): e . published version doi: . /journal.pgen. citable link http://nrs.harvard.edu/urn- :hul.instrepos: terms of use this article was downloaded from harvard university’s dash repository, and is made available under the terms and conditions applicable to other posted material, as set forth at http:// nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of- use#laa http://osc.hul.harvard.edu/dash/open-access-feedback?handle=&title=genome-wide% association% analysis% identifies% variants% associated% with% nonalcoholic% fatty% liver% disease% that% have% distinct% effects% on% metabolic% traits&community= / &collection= / &owningcollection / &harvardauthors=dd ca f d a b e&department http://nrs.harvard.edu/urn- :hul.instrepos: http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits elizabeth k. speliotes , , , *., laura m. yerges-armstrong ., jun wu ., ruben hernaez , , ., lauren j. kim ., cameron d. palmer , vilmundur gudnason , , gudny eiriksdottir , melissa e. garcia , lenore j. launer , michael a. nalls , jeanne m. clark , , , braxton d. mitchell , alan r. shuldiner , , johannah l. butler , , , marta tomas , , udo hoffmann , , shih-jen hwang , joseph m. massaro , , christopher j. o’donnell , , , dushyant v. sahani , veikko salomaa , eric e. schadt , stephen m. schwartz , , david s. siscovick , nash crn", giant consortium", magic investigators", benjamin f. voight , j. jeffrey carr , mary f. feitosa , tamara b. harris , caroline s. fox , , albert v. smith ., w. h. linda kao , ., joel n. hirschhorn , , ., ingrid b. borecki *., gold consortium" department of internal medicine, division of gastroenterology, university of michigan, ann arbor, michigan, united states of america, center for computational medicine and bioinformatics, university of michigan, ann arbor, michigan, united states of america, division of gastroenterology, massachusetts general hospital, boston, massachusetts, united states of america, broad institute, cambridge, massachusetts, united states of america, division of endocrinology, diabetes, and nutrition, department of medicine, university of maryland school of medicine, baltimore, maryland, united states of america, division of statistical genomics, department of genetics, washington university, saint louis, missouri, united states of america, department of epidemiology, johns hopkins bloomberg school of public health, baltimore, maryland, united states of america, division of general internal medicine, the johns hopkins hospital, baltimore, maryland, united states of america, department of internal medicine, washington hospital center, washington d.c., united states of america, laboratory of epidemiology, demography, and biometry, intramural research program, national institute on aging, national institutes of health, bethesda, maryland, united states of america, divisions of endocrinology and genetics and program in genomics, children’s hospital, boston, massachusetts, united states of america, icelandic heart association, kopavogur, iceland, university of iceland, reykjavik, iceland, laboratory of neurogenetics, national institute on aging, national institutes of health, bethesda, maryland, united states of america, welch center for prevention, epidemiology, and clinical research, baltimore, maryland, united states of america, geriatric research and education clinical center (grecc), veterans administration medical center, baltimore, maryland, united states of america, cardiovascular epidemiology and genetics, institut municipal d’investigació mèdica, barcelona, spain, ciber epidemiologı́a y salud pública, barcelona, spain, department of radiology, massachusetts general hospital, boston, massachusetts, united states of america, division of cardiology, massachusetts general hospital, boston, massachusetts, united states of america, framingham heart study, national heart, lung, and blood institute (nhlbi), framingham, massachusetts, united states of america, department of biostatistics, boston university school of public health, boston, massachusetts, united states of america, division of intramural research, national heart, lung, and blood institute, bethesda, maryland, united states of america, chronic disease epidemiology unit, department of health promotion and chronic disease prevention, national public health institute, helsinki, finland, pacific biosciences, menlo park, california, united states of america, cardiovascular health research unit, departments of medicine and epidemiology, university of washington, seattle, washington, united states of america, department of epidemiology, university of washington, seattle, washington, united states of america, departments of radiologic sciences, internal medicine-cardiology, and public health sciences, wake forest university school of medicine, winston-salem, north carolina, united states of america, department of genetics, harvard medical school, boston, massachusetts, united states of america abstract nonalcoholic fatty liver disease (nafld) clusters in families, but the only known common genetic variants influencing risk are near pnpla . we sought to identify additional genetic variants influencing nafld using genome-wide association (gwa) analysis of computed tomography (ct) measured hepatic steatosis, a non-invasive measure of nafld, in large population based samples. using variance components methods, we show that ct hepatic steatosis is heritable (, %– %) in family-based amish, family heart, and framingham heart studies (n = to , ). by carrying out a fixed-effects meta-analysis of genome-wide association (gwa) results between ct hepatic steatosis and , . million imputed or genotyped snps in , individuals from the old order amish, age, gene/environment susceptibility-reykjavik study (ages), family heart, and framingham heart studies, we identify variants associated at genome-wide significant levels (p, ) in or near pnpla , ncan, and ppp r b. we genotype these and other top ct hepatic steatosis-associated snps in subjects with biopsy-proven nafld from the nash clinical research network (nash crn). in comparisons with , healthy controls from the myocardial genetics consortium (migen), we observe significant associations with histologic nafld at variants in or near ncan, gckr, lyplal , and pnpla , but not ppp r b. variants at these five loci exhibit distinct patterns of association with serum lipids, as well as glycemic and anthropometric traits. we identify common genetic variants influencing ct–assessed steatosis and risk of nafld. hepatic steatosis associated variants are not uniformly associated with nash/fibrosis or result in abnormalities in serum lipids or glycemic and anthropometric traits, suggesting genetic heterogeneity in the pathways influencing these traits. plos genetics | www.plosgenetics.org march | volume | issue | e citation: speliotes ek, yerges-armstrong lm, wu j, hernaez r, kim l j, et al. ( ) genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits. plos genet ( ): e . doi: . /journal.pgen. editor: mark i. mccarthy, university of oxford, united kingdom received june , ; accepted february , ; published march , this is an open-access article distributed under the terms of the creative commons public domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. funding: this work was partially supported by nih grants t dk - to daniel podolsky (for eks), f dk - to eks, nih k dk - to eks, and r dk to jnh. the ages-reykjavik study is funded by nih contract n -ag- , the nia intramural research program, hjartavernd (the icelandic heart association), and the althingi (the icelandic parliament). the old order amish study was supported by nih research grants k dk , r ag , r hl , u hl , and u hl . partial funding was also provided by the diabetes research and training center of maryland (p dk ) and the nutrition and obesity research center of maryland (p dk ). lmy-a was supported by nih training grants t ag and f ar . rh was supported by the american diabetes association mentor-based postdoctoral fellowship program ( - -mn- ). the nhlbi family heart study was supported by niddk r dk (to ibb) and nhlbi r hl . this research was conducted in part using data and resources from the framingham heart study of the national heart lung and blood institute of the national institutes of health and boston university school of medicine. the analyses reflect intellectual input and resource development from the framingham heart study investigators participating in the snp health association resource (share) project. this work was partially supported by the national heart, lung, and blood institute’s framingham heart study (contract no. n -hc- ) and its contract with affymetrix for genotyping services (contract no. n -hl- - ). the nonalcoholic steatohepatitis clinical research network (nash crn) is supported by the national institute of diabetes and digestive and kidney diseases (niddk) (grants u dk , u dk , u dk , u dk , u dk , u dk , u dk , u dk , u dk ). several clinical centers use support from general clinical research centers or clinical and translational science awards in conduct of nash crn studies (grants ul rr , m rr , m rr , ulrr , m rr , ul rr , m rr , m rr ). the migen study was funded by the us national institutes of health (nih) and national heart, lung, and blood institute’s stampeed genomics research program. mt was supported by the beatriu de pinós postdoctoral fellowship, generalitat de catalunya ( bp-b , mt), and red heracles, isciii (rd / ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. competing interests: eric e. schadt is an employee of pacific biosciences. * e-mail: espeliot@med.umich.edu (eks); iborecki@wustl.edu (ibb) . these authors contributed equally to this work. " for membership information, please see acknowledgments. introduction nafld includes a spectrum of disease ranging from fatty infiltration of the liver (steatosis) to histologic evidence of inflammation (nonalcoholic steatohepatitis or nash), to fibrosis or cirrhosis, without a history of excessive alcohol ingestion [ , ]. nafld can lead to liver failure and is accompanied by substantial morbidity and mortality, with few known effective treatments [ ]. obesity is a primary risk factor for nafld, but not all obese individuals are affected [ ]. familial clustering of the disease has been identified [ – ], suggesting that nafld may be influenced by genetic variants. however, thus far only one genetic locus has been found to reproducibly associate with magnetic resonance measured steatosis [ , ]. liver attenuation measured using computed tomography (ct) is a quantitative measure that is inversely related to the amount of fat in the liver [ – ]. it is highly correlated (r = . ) with the macrovesicular hepatic steatosis and thus is a non invasive measure of nafld [ ]. the purpose of the present study was to determine the heritability of ct measured hepatic steatosis and to search for associated genetic variants in a meta-analysis of , individuals of european descent from the framingham heart study (fram), the old order amish study (amish), the family heart study (famhs), and the age, gene/environment suscep- tibility-reykjavik study (ages), which together comprise the gold (genetics of obesity-related liver disease) consortium (see table s ). to validate top associating variants for risk of histologically verified nafld, we utilized cases from the nash clinical research network (nash crn) that were genetically matched to healthy controls from the myocardial genetics consortium (migen) consortium(see table s ). we then further tested genome wide significant or replicating snps for associations with histologic nafld using the same cases from the nash clinical research network (nash crn) versus a different set of controls from the illumina control database (icont) (see table s ). further, we report the association of these snps with other metabolic traits using data from the global lipids genetics [ ], giant [ ], diagram [ ], and magic [ ] consortia, as well as investigate cis gene expression variation (eqtls) in liver, subcutaneous and visceral fat from bariatric surgery patients from massachusetts general hospital [ ](figure ). results we estimated the heritability of ct hepatic steatosis in three family-based cohorts. we found that the heritability of ct hepatic steatosis was . (standard error, se . ), . (se = . ), and . (se . ) in the amish, famhs, and fram cohorts respectively (n = – , ) (see materials and methods and table ). these data suggest that ct hepatic steatosis, like other measures of fat has a genetic basis and that a search for influential genetic variants is warranted. to identify specific genetic loci associated with ct hepatic steatosis, genome-wide association analyses were carried out in each of the four studies (see materials and methods and tables s , s ) and the results combined using a fixed effects meta-analysis (n = , in total). variants at three loci emerged as being associated with ct hepatic steatosis at genome-wide significance levels (p, ; table , figure a). these included rs in pnpla (p = . ), a locus previously reported as associated with magnetic resonance spectroscopy measured steatosis, [ ] and two additional novel loci: rs near ppp r b (rs , p = . ) and rs near ncan (rs , p = . ). the alleles associated with increas- ing ct hepatic steatosis ranged in frequency from . to . and together account for . % of the variance in hepatic steatosis (table ; range . – . %). after removing these genome-wide significant loci, a quantile-quantile plot of the results demonstrated an excess of low p-values compared to expectations under the null (figure b), suggesting that additional variants among those with moderately low p-values may also be associated with this trait. except for variants near pnpla , we did not observe any variants in the region of any of the previously reported liver function test associated regions [ ]. we could not assess whether the recently reported nafld associated variants near apoc [ ] associate with ct hepatic steatosis as they were not genotyped on the five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e affymetrix or illumina platforms used by our studies and these variants do not have proxies that we could use in hapmap to impute them. to determine whether snps with evidence of association with ct hepatic steatosis are also associated with histologic nafld, we genotyped snps (independent snps with p, - , with independence defined as pairwise r , . ; see table s for snp details in gold and each cohort) in subjects with biopsy- proven nafld from the nash crn (see table s ). using ancestry-informative genetic markers [ ], we had previously matched these cases to , healthy controls [ ] from the migen study [ ] that had undergone gwas genotyping and imputation (see table s ). forty-five of the snps passed genotyping and imputation quality control in the nash crn and migen data sets respectively (see table s ) and were tested for association with histologic nafld in this sample. two of the three variants with genome-wide significant associations to ct hepatic steatosis were also significantly associated with histologic nafld (corresponding to a false discovery rate (fdr) p, . ): rs in pnpla (or = . , p = . ) as we and others have recently reported [ , ] and rs in ncan (or = . , p = . ) which is a novel finding (table ; see table s ). the rs variant near ppp r b was not associated with histologic nafld in this sample (or = . , p = . ). of the remaining snps showing suggestive association with ct hepatic steatosis, rs in gckr (or = . , p = . ) and rs near lyplal (or = . , p = . ) were also significantly associated with histologic nafld (table ; see table s ). to confirm that the effects on histologic nafld observed in the nash crn/migen analyses were not due to the characteristics of the controls, we performed a separate analysis of the nash crn cases with an alternate set of controls from the illumina control database (icont; http://www.illumina.com/ science/icontroldb.ilmn). we found that the effects and p values of rs in pnpla (or = . , p = . ), rs in ncan (or = . , p = . ), rs near ppp r b (or = . , p = . ), rs in gckr (or = . , p = . ), and rs near lyplal (or = . , p = . ) were similar to the effects seen in migen establishing that these results are not dependent on the choice of control sample (see table s ). furthermore, assessment of imputation accuracy with the snps in these control sets indicates that imputed genotypes at the associated snps are likely to be highly accurate (see tables s , s ). the variants with the lowest p-values of association with ct hepatic steatosis at the pnpla (rs ), ncan (rs ), and gckr (rs ) loci are in high ld with or are themselves non- synonymous variants in pnpla (rs ; i m, r = ), ncan (rs ; p s, same as hepatic steatosis snp), and gckr (rs ; p l; r = . ) (figure ). the variants with figure . study design. meta-analysis of genome-wide association data was performed in stage across the cohorts shown. snps representing the best associating loci were genotyped in histology based nafld samples (stage ) from the nash crn matched to genome wide genotyped and imputed migen controls. the effects of the five nafld associated snps on nash crn/icont, metabolic phenotypes and eqtls in liver and adipose tissue were then performed (stage ). doi: . /journal.pgen. .g author summary nafld is a spectrum of disease that ranges from steatosis to steatohepatitis (nonalcoholic steatohepatitis or nash: inflammation around the fat) to fibrosis/cirrhosis. hepatic steatosis can be measured non-invasively using computed tomography (ct) whereas nash/fibrosis is assessed histo- logically. the genetic underpinnings of nafld remain to be determined. here we estimate that %– % of the variation in ct measured hepatic steatosis is heritable or genetic. we identify three variants near pnplal , ncan, and ppp r b that associate with ct hepatic steatosis and show that variants in or near ncan, gckr, lyplal , and pnpla , but not ppp r b, associate with histologic lobular inflam- mation/fibrosis. variants in or near ncan, gckr, and ppp r b associate with altered serum lipid levels, whereas those in or near lyplal and pnpla do not. variants near gckr and ppp r b also affect glycemic traits. thus, we show that nafld is genetically influenced and expand the number of common genetic variants that associate with this trait. our findings suggest that development of hepatic steatosis, nash/fibrosis, or abnormalities in metabolic traits are probably influenced by different metabolic pathways that may represent distinct therapeutic targets. five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e the lowest p-values of association with ct hepatic steatosis at lyplal and ppp r b lie downstream and upstream of the coding regions of these genes (figure ). in epidemiologic studies nafld is associated with increased central obesity, higher low density lipoprotein (ldl)- cholesterol and lower high density lipoprotein (hdl)-cholesterol levels, impaired fasting glucose, increased risk of diabetes and increased insulin resistance. [ ]in addition, variants in or near gckr, ncan, and ppp r b have been previously associated with lipid levels, gckr with glycemic traits and lyplal with abdominal obesity [ , – ]. therefore, we examined the associations of each of the ct hepatic steatosis-associated variants with serum ldl-cholesterol, hdl-cholesterol, triglycerides (tg), hour glucose levels, hour glucose levels controlled for body mass index (bmi), fasting glucose, homeostatic model for beta call function (homa-b), homeostatic model for insulin resistance (homa-ir), fasting insulin, bmi, waist to hip ratio (whr) controlled for bmi, and diabetes in the largest analyses of these traits available from the global lipids genetics [ ], giant [ ], diagram [ ], and magic [ ] consortia (see table , table s ) interestingly, we observed several distinct patterns of association. the allele associated with increasing ct hepatic steatosis at ncan was associated with lower triglycerides and plasma ldl-cholesterol levels. by contrast, the hepatic steatosis- increasing allele at gckr was associated with higher levels of plasma ldl-cholesterol and triglycerides, lower fasting glucose, lower fasting insulin, lower homa-ir, but increased hour glucose, increased hour glucose controlled for bmi, and whr controlled for bmi. the hepatic steatosis increasing allele at ppp r b was associated with increased hdl- and ldl- cholesterol levels and decreased fasting glucose.(table , figure ). the variants near pnpla and lyplal were not associated with any of the traits tested (see table , table s and figure ). for pnpla (rs ), ncan (rs ), and gckr (rs ) the variants with the lowest p-values of association with ct hepatic steatosis are either themselves missense snps or in high ld with missense snps. thus, the most parsimonious model of how they may act is by directly affecting protein structure or function. however, the variants with the lowest p-values of association with ct hepatic steatosis near lyplal and ppp r b fall in non-coding regions and thus for these (as well as the other three loci above) we tested whether they have effects on the expression of nearby genes in liver and adipose tissue from a sample of bariatric surgery patients [ ] (see table s ). we found that that the hepatic steatosis increasing variant (rs ) at the ppp r b locus increased liver mrna expression of ppp r b and aw _rc and decreased expression of ak . the hepatic steatosis increasing variant (rs ) at the gckr locus increased expression of c orf mrna in liver. in these cases the eqtl with the lowest p-value of affecting these transcripts in the region was the same or highly correlated with the allele that had the lowest p-value of association with ct hepatic steatosis consistent with the possibility that these snps may function by affecting expression of nearby genes. for all other cases, the eqtl with the lowest p-value of affecting transcript expression at the locus was not eliminated by controlling for the variant that had the lowest p- value of association with ct hepatic steatosis and thus in these cases, the data do not support an expression effect as mediating the association with steatosis. because alteration of ppp r b expression has been shown to affect serum lipid levels [ ] one possibility is that changes in expression of this gene could mediate its effect on hepatic steatosis. for gckr, the variant with the lowest p-value of association with ct hepatic steatosis is in high ld with a missense variant in gckr which has been shown to affect gckr function [ ]. thus, at gckr an alternate model of action of how the ct hepatic steatosis associated variant affects hepatic steatosis is via altering gckr function rather than via altering expression of c orf . further functional work will be needed to prove that these variants exert their effects on hepatic steatosis via these possible mechanisms. discussion we have identified variants in three novel loci (ncan, gckr, and lyplal ) and one previously reported locus (pnpla ) that are associated with both increasing ct hepatic steatosis and histologic nafld. ppp r b is associated with ct steatosis but not histologic nafld that includes individuals mostly with inflammation and fibrosis. these variants all have distinct patterns of effects on nafld and metabolic traits. we have shown that ct hepatic steatosis is heritable and that gwa meta-analysis led to the identification of variants associated not only with ct hepatic steatosis but, also, with more severe nash/fibrosis mostly present in the nash crn sample. because ct hepatic steatosis measurements can be obtained noninvasively, much larger sample sizes can be accumulated, thereby increasing power to identify variants that associate with nafld compared with only studying individuals that have histology diagnosed disease. follow-up association testing in samples with histologic phenotypes remains useful however. we did observe one variant near ppp r b that was associated with ct–assessed liver attenuation but not histology-proven nafld. possible reasons for why the variant near ppp r b is associated with ct liver steatosis but not histology-proven nafld include . it influences steatosis only, not progression to nash/fibrosis: . its association with ct fat may be a false positive: . the nash crn/migen sample is underpowered to see an effect on histologic nafld: or . the variant is associated with something other than fat reflected in the ct scan (eg. glycogen content). further work is needed to differentiate among these possibilities. table . characterization of family data for heritability estimation. study n n families design age range (years) heritability se amish founder population participants link to a single, -generation pedigree – . . family heart study -generational pedigrees – . . framingham heart study -generational pedigrees – . . n: total number of individuals with fatty liver phenotype; se: standard error; for all studies, solar software was used to estimate heritability [ ]. doi: . /journal.pgen. .t five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e t a b le . g e n o m e -w id e si g n if ic a n t o r re p li c a ti n g v a ri a n ts fr o m g o l d , n a s h c r n /m ig e n , a n d m e ta b o li c p h e n o ty p e a n a ly se s. l o c u s g o l d m e ta -a n a ly s is n a s h c r n /m ig e n a n a ly s is m e ta b o li c p h e n o ty p e s m e ta a n a ly s e s * * d ir . n = , n = / , n = , – , n = , – , s n p id c h r. p o s . n e a re s t e a e a f e ff e c t % v a r g o l d e a f a e a f b o r n a f l d n a f l d l d l h d l t g g lu c o s e h o m a -i r (m b ) g e n e (s e ) p ( % c i) p p p p rs p n p la g . . . . e - . . . . e - . e - . e - . e - . . ++ n n n n n . ( . – . ) rs n c a n t . . . . e - . . . . e - . e - . e - . e - . . ++ -n -n n . ( . – . ) rs ly p la l c . . . . e - . . . . e - . e - . e - . e - . . ++ n n n n n . ( . – . ) rs g c k r t . . . . e - . . . . e - . e - . e - . e - . e - . e - ++ +n +— . ( . – . ) rs p p p r b a . . . . e - . . . . e - . e - . e - . e - . e - . +n ++ n -n . ( . – . ) g o l d : g e n e ti c s o f o b e si ty -r e la te d l iv e r d is e a se ; n a s h c r n : n o n a lc o h o li c s te a to h e p a ti ti s c li n ic a l r e se a rc h n e tw o rk ; m ig e n : m y o c a rd ia l in fa rc ti o n g e n e ti c s c o n so rt iu m ; ** fr o m t h e g lo b a l l ip id s g e n e ti c s, g ia n t , m a g ic , a n d d ia g r a m c o n so rt ia ; c h r. c h ro m o so m e ; p o s. : p o si ti o n , b u il d ; m b : m e g a b a se ; e a : e ff e c t a ll e le ; e a f : e ff e c t a ll e le fr e q u e n c y ; e ff e c t: in c re a se in in v e rs e n o rm a li ze d fa tt y li v e r b y c o m p u te d to m o g ra p h y s e : s ta n d a rd e rr o r; % v a r- p e rc e n ta g e o f v a ri a n c e e x p la in e d ; g o l d p : p -v a lu e o f a ss o c ia ti o n in g o l d ; e a f a : f re q u e n c y o f th e e ff e c t a ll e le in c a se s fr o m th e n a s h c r n st u d y ; e a f b : f re q u e n c y o f th e e ff e c t a ll e le in c o n tr o ls fr o m th e m ig e n st u d y ; n a f l d : n o n a lc o h o li c fa tt y li v e r d is e a se ; o r n a f l d : o d d s ra ti o fo r th e p re se n c e o f n a f l d o n p a th o lo g y p e r e ff e c t a ll e le ; n a f l d p : f a ls e d is c o v e ry ra te p -v a lu e o f a ss o c ia ti o n fo r h is to lo g ic n a f l d ; l d l : lo w d e n si ty li p o p ro te in c h o le st e ro l; h d l : h ig h d e n si ty li p o p ro te in c h o le st e ro l; t g : tr ig ly c e ri d e s; g lu c o se : fa st in g g lu c o se ; h o m a -i r : h o m e o st a ti c m o d e l a ss e ss m e n t o f in su li n re si st a n c e ; p : p - v a lu e o f a ss o c ia ti o n ; d ir : d ir e c ti o n o f e ff e c t a ll e le fo r si g n if ic a n t a ss o c ia ti o n s in g o l d , n a s h c r n /m ig e n , l d l , h d l , t g , g lu c o se , h o m a -i r a n a ly se s re sp e c ti v e ly ; +/ re p re se n ts in c re a si n g /d e c re a si n g fa tt y li v e r in g o l d , a n d h a v in g a h ig h e r/ lo w e r o d d s o f h a v in g n a f l d in th e n a s h c r n /m ig e n a n a ly se s a n d h ig h e r/ lo w e r l d l , h d l , t g , g lu c o se , h o m a -i r re sp e c ti v e ly ; n re p re se n ts n o si g n if ic a n t e ff e c t; p n p l a : p a ta ti n -l ik e p h o sp h o li p a se d o m a in -c o n ta in in g p ro te in (h u g o g e n e n o m e n c la tu re c o m m it te e , h g n c : ); n c a n : n e u ro c a n (h g n c : ); l y p l a l : ly so p h o sp h o li p a se -l ik e (h g n c : ); g c k r : g lu c o k in a se re g u la to ry p ro te in (h g n c : ); p p p r b : p ro te in p h o sp h a ta se , re g u la to ry su b u n it b (h g n c : ). d o i: . /j o u rn a l.p g e n . .t five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e we show that some of the variants that are associated with increased ct hepatic steatosis have distinct patterns of effects on metabolic traits that, when taken together, give us insight into their functional clustering. for example, unlike the other three loci, variants in or near pnpla and lyplal do not affect any of the other metabolic traits and interestingly pnpla and lyplal - related proteins have been predicted to play a role in consecutive steps in triglyceride breakdown [ , ]. thus these could increase hepatic steatosis by preventing breakdown of triglycerides, as recently shown for pnpla (i m) [ ]. the apparent discor- dance between the strong effect on hepatic steatosis and modest, if any, effect on serum lipid levels suggests that these genes, if they are involved in lipid metabolism, exert their effects within the liver in ways that are not well reflected in serum measurements. thus, similarities in the pattern of pleiotropic effects on other traits may provide insights into the functional clustering of the genes that these variants effect. unlike pnpla and lyplal , variants near ncan(which encodes for an adhesion molecule [ ]), ppp r b (which encodes for a protein that regulates glycogen breakdown [ ]), and gckr (which, through inhibition of glucokinase, regulates glucose storage/disposal and provides substrates for de novo lipogenesis [ ]), are associated with distinct changes in serum and liver lipids as well as glycemic traits. indeed, these data may provide new insights into how obesity can lead to metabolic complications in some but not all individuals- some but not all of these individuals carry variants that predispose them both to liver fat deposition and to metabolic dysregulation. further, our data show that the alleles of snps that associate with increased liver steatosis are also associated with a diverse pattern of metabolic phenotypes including different combinations of increased or decreased serum ldl-cholesterol, increased serum hdl-cholesterol, increased serum tg, decreased serum fasting glucose and insulin, decreased insulin resistance, and increased whr adjusted for bmi. in addition, some hepatic steatosis-associated variants are not strongly associated with any of these metabolic traits (pnpla and lyplal ). these results indicate that hepatic steatosis is likely to be influenced by different metabolic pathways, based on these various patterns of association. thus it may be possible to resolve genetic heterogeneity in the etiology of hepatic steatosis, which may present unique opportunities for personalized therapies. compared with earlier efforts, this study is well-powered, using more than , individuals for discovery of variants that affect nafld. thus, noninvasive measures of hepatic steatosis such as figure . genome-wide association results for gold (stage ). a. manhattan plot showing the significance of association of all snps in the stage gold meta-analysis with ct hepatic steatosis. snps are plotted on the x-axis according to their position on each chromosome against association with ct hepatic steatosis on the y-axis (shown as -log p-value). snps that also associate with histology based nafld are in red, those that only associate with ct hepatic steatosis in blue. b. quantile-quantile plot of snps after stage gold meta-analysis (black) and after removing any snps within kb of pnpla , ppp r b, and ncan (red). doi: . /journal.pgen. .g five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e ct scanning can provide valuable information for use in population- and family-based studies aimed at identifying genetic risk factors for nafld. although the identities of nearby genes and effects on lipid levels provide important clues, functional studies will be needed to further understand the mechanisms by which these risk factors influence the development and progression of nafld. overall however, our work gives us new insights into the biology and genetics of nafld and opens up avenues for biological, diagnostic, and therapeutic research for this condition in humans. materials and methods ethics statement all work done in this paper was approved by local institutional review boards or equivalent committees. gold studies and genetic analyses each of the participating studies had the overarching objective of investigating cardiovascular disease and its risk factors. the studies are population based and of the are family studies. genome-wide snp data were available in each case, and the platforms and quality control measures are described in tables s and s . age gene-environment susceptibility—reykjavik study (ages-reykjavik). the ages-reykjavik study is a single center prospective population-based cohort nested in the original rykjavik study, a cohort of , randomly sampled persons living in reykjavik, iceland. the cohort included , men and women born between and . re-examination of a sample of surviving members of the reykjavik study was initiated in as the ages-reykjavik study. this study included imaging by computerized tomography, from which liver attenuation was measured from a mm thick slice at the level of the l /l vertebrae by calculating the average hounsfield unit in a region of interest with a diameter of cm located % of the distance from where a tangent from the mid-anterior of the spinal canal bisected a line between the second and third rib. four thousand seven hundred and seventy two individuals were assessed for hepatic steatosis using ct scanning. liver attenuation controlled for an external phantom was inverse normally transformed and residuals created from a linear regression model in proable [ ]/r with covariates of age, age , gender and drinks along with the snps in an additive genetic model (see tables s , s ). figure . effects on traits. direction of effect on ct fatty liver, histology nafld, lipid and glycemic traits of the best associating snps at the loci shown. direction is shown only for significant associations. ct: ct hepatic steatosis; ldl: low density lipoprotein cholesterol; hdl: high density lipoprotein cholesterol; tg: triglycerides; homa-ir: homeostatic model of insulin resistance; pnpla : patatin-like phospholipase domain-containing protein (hgnc: ); ncan: neurocan (hgnc: ); lyplal : lysophospholipase-like (hgnc: ); gckr: glucokinase regulatory protein (hgnc: ); ppp r b: protein phosphatase , regulatory subunit b (hgnc: ). doi: . /journal.pgen. .g figure . regional plots of genome-wide significant or replicating loci of association in gold. snps are plotted by position on chromosome against association with ct hepatic steatosis (–log p-value). the figures highlight the snp taken into stage (diamond). the snps surrounding the most significant snp are color-coded to reflect their ld with this snp as in the inset (taken from pairwise r values from the hapmap ceu database, www.hapmap.org). estimated recombination rates (from hapmap) are plotted in cyan to reflect the local ld structure. genes and the direction of transcription, are noted below the plots (data from ucsc genome browser, genome.ucsc.edu). coding snps in high ld with the best snp are noted with rs number and protein change. doi: . /journal.pgen. .g five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e the amish. subjects were identified from studies of cardiovascular health in the old order amish community in lancaster county: the amish family calcification study ( – ) [ ] and the amish longevity study ( – ) [ ]. in total, individuals had both genome-wide snp data and ct- assessed hepatic steatosis. thoracic electron-beam computerized tomography (ebct) scans were obtained as part of the amish family calcification study by an imatron c- ebct scanner. measurements from two regions-of-interest, the liver and spleen, were obtained. the spleen measurements were used as an attenuation standard. accu view (accuimage corp.) software was used to calculate the attenuation coefficient in hounsfield units for each region-of-interest. two . -cm region-of-interest measurements were obtained from the liver and one was obtained from the spleen. the average of the liver attenuation measurements divided by the spleen attenuation measurement was then calculated. the region-of-interest measurements were placed in such a manner that minimized measurements from vessels, focal lesions, areas of artifact or near the edges of the organs. the liver attenuation/spleen attenuation ratio was inverse normally transformed and association was tested with genotypes in an additive genetic model controlling for age, age , and gender and relatedness; alcohol is generally not consumed in this population. a (n- )-degree-of-freedom t test was used to assess the significance of the measured genotype. the polygenic component was modeled using the relationship matrix derived from the complete -generation pedigree structure, to properly control for the relatedness of all subjects in the study. the family heart study. the family heart study (https:// dsgweb.wustl.edu/projects/mp .html) recruited , families, half randomly sampled, and half selected because of an excess of coronary heart disease (chd) or risk factor abnormalities as compared with age- and sex-specific population rates [ ] from four population-based parent studies: the framingham heart study, the utah family tree study, and two atherosclerosis risk in communities centers (minneapolis, and forsyth county, nc). study participants belonging to the largest pedigrees were invited for a second clinical exam, at which time coronary artery calcification was assessed using computed tomography, which included imaging of the liver. a total of , caucasian subjects in extended families were examined; the heritability was estimated in this sample . a two- stage design was adopted for the gwas. in the first stage, , subjects were chosen, equally distributed between the highest and lowest quartiles of age- and sex-adjusted values for coronary artery calcification, assessed by ct scan. these subjects were chosen to be largely unrelated with subjects having or more siblings selected into the sample. we report association results based on subjects after excluding subjects ascertained from the framingham massachusetts to avoid any possible overlap with the framingham heart study participants. participants underwent a cardiac multidetector ct exam with four detectors using a standardized protocol as described previously [ ]. for participants weighing kg ( lbs) or greater, the milliamperes were increased by %. participants received two sequential scans of the heart with ecg gating in late diastole. a phantom with either or samples of calcium hydroxylapatite was included in each participants scan. ct images from all study centers were sent electronically to the central ct reading center located at wake forest university health sciences, winston salem, nc, usa. ct images were analyzed using medical image processing, analysis, and visualization (mipav) software (mcauliffe ) with custom programmed subroutines (a.k.a.‘‘plug-ins’’) coded at wake forest university health sciences. ct images of the chest were used to measure liver attenuation corresponding to superior aspects of the right and medial lobes or hepatic segments a, and using the couinaud system. an external calcium standard was used as a control for penetrance of the films. the liver attenuation was regressed on age, age , age , field center, phantom average, alcohol consumption and genetic principal components, by sex, using a stepwise procedure and retaining terms significant at the % level. we then applied an inverse normal rank transformation to the adjusted phenotype within sex strata and association was assessed assuming an additive model using proc mixed in sas to account for the siblings. the framingham heart study. the framingham heart study recruited , residents in from the population in framingham, massachusetts [ ]. these individuals have had serial examinations and collection of respective data since. in , , offspring from the original residents and their spouses were recruited into the offspring study and have been followed for four to eight years since [ ]. in , , third generation members and their spouses were enrolled [ ]. between and , , individuals from the offspring study and , individuals from third generation underwent multidectector computed tomograms on which we evaluated liver attenuation as previously described [ ]. inclusion criteria favored individuals who lived in the new england area and included families. minimum age was in men and in women. women of childbearing age were screened and pregnant women and individuals . kilograms were excluded from scanning. individuals with scans that could not be interpreted for hepatic steatosis or did not attend offspring examination as they lacked covariate data were not used for analysis. the average of the liver attenuation measures and a high density external calcium control were used to create a liver/phantom ratio to control for scan penetrance. for gwas analysis, inverse normally transformed liver attenuation/phantom ratio was used in a mixed linear model (controlling for relatedness) in r [ ]with covariates of age, age squared, gender, and alcoholic drinks ( oz = drink) with the first ten principal components (as determined in eigenstrat [ ]) as covariates. principal components were first generated using an unrelated sample of and then projected to the rest of the cohort. individuals who deviated from the mean of the principal components of more than six standard deviations were removed prior to analysis (n = ). heritability analyses. three of the four studies parti- cipating in this consortium were family studies and the family structure characteristics used for heritability are shown in table . liver attenuation adjusted for scan penetrance and then inverse normally transformed and corrected for age gender and number of alcoholic drinks (drinks in famhs, and fram only as the amish do not drink) was estimated in each of the studies and then heritability assessed using a variance components method as implemented in the software solar [ ]. despite the diverse character of these family studies, there was remarkable consistency in the estimates of the proportion of variance due to genetic effects, and the magnitude of the heritabilities is comparable to many complex quantitative traits and suggests that a search for underlying genetic variants is warranted. meta-analysis and gwas. association data from the four studies above were filtered for snps that had a minor allele frequency . % and for snps that had an imputation quality score of . . . all files were gc corrected after filtering and before meta-analysis. the inflation factor for the ages study was . , for the amish was . , for the family heart study was . , for the framingham heart study was . . meta-analysis was five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e conducted using a fixed effects model with a beta and standard error as implemented in metal (http://www.sph.umich.edu/ csg/abecasis/metal/). after meta-analysis, snps present in fewer than studies were eliminated from analysis. the inflation factor for the overall meta-analysis was . . the meta-analysis was gc corrected before the final p values were reported. the variation in ct hepatic steatosis explained by the tested snps was estimated from stage analyses using f ( – f) a , where f is the frequency of the variant and a is its additive effect in units of standard deviations from the meta analysis [ ]. selection of snps for validation/replication with histologic nafld to define independently associated snps, the ld was required to be r , . and the snps located at least megabase from each other. from among these, the snp with the strongest association was chosen for follow up (p, . ). two iplex pools consisting of snps were designed and were successfully genotyped in the nash crn samples. of these, only were imputed well in migen, and only these snps were analyzed. variants with a false discovery rate of q , . were considered associated with nafld. nash crn samples study: the nash crn samples were collected from eight different centers in the u.s. as previously described [ , ]. adults from both the database and the pivens trial (pioglitazone versus vitamin e versus placebo for the treatment of nondiabetic patients with nonalcoholic steatohepatitis) were used for analysis. briefly, individuals from the database were part of an observational study of nonalcoholic fatty liver disease. inclusion criteria included age . , histologic diagnosis for nafld, or histologic diagnosis for cryptogenic cirrhosis or suspected nafld on the basis imaging studies suggestive of nafld, or clinical evidence of cryptogenic cirrhosis. no subjects reported regular excessive use of alcohol within two years prior to the initial screening period. exclusion criteria included histologic evidence of liver disease besides nonalcoholic liver disease, known hiv positivity, and conditions that would interfere with study follow up. individuals in the pivens database were part of a multicenter placebo controlled study with three parallel groups examining the effects of pioglitazone vs. vitamin e vs. placebo on nafld. inclusion and exclusion criteria were as described previously [ , ]. for this analysis, we excluded individuals who did not describe their race as being white and non-hispanic. there were adults who matched these criteria. finally, individuals without histology available for central review were excluded, leaving adults for the current study. histology determination in nash crn histologic diagnoses were determined in the nash crn by central review by nash crn hepatopathologists using previously published criteria [ , ]. predominantly macrovesicular steatosis was scored from grade – . inflammation was graded from – and cytologic ballooning from – . the fibrosis stage was assessed from a masson trichrome stain and classified from – according to the nash crn criteria. individuals could contribute to more than one of these outcomes. the nash crn samples were genotyped and analyzed as described in tables s and s . analysis in nash crn/migen samples migen controls were matched to the nash crn samples for genetic background. as previously described, the migen samples were collected from various centers in the us and europe by the myocardial infarction genetics consortium (migen) [ ] as controls for individuals with early onset mi. the genetic ancestry the migen samples was explored by using the program eigenstrat [ ]; the first principal component was the most significant and correlated with the commonly observed northwest- southeast axis within europe [ ] and genetic ancestry along this principal component is correlated with reported country of origin in the migen sample [ ]. from this analysis, unlinked snps were chosen from the migen genotype data that were most strongly correlated with the first principal component. these snps were genotyped in the nash crn samples to enable matching of migen controls to the nash crn [ ] cases for genetic background. plink [ ] was used to match individuals based on identity by state (ibs) distance using a pairwise population concordance test statistic of . for matching. the snps selected for validation were tested in this case-control sample using logistic regression controlling for age, age , gender, and the first principal components as covariates in plink [ ]. we report the p-values, odds ratios and confidence intervals. icont samples we obtained , population based control samples with genotypes from illumina (see http://www.illumina.com/science/ icontroldb.ilmn). these individuals were used as controls in various case control analyses. individuals were removed as described in table s and , individuals were then used as controls for the nash crn/icont analyses. analysis in nash crn/icont samples the individuals from the nash crn described above were used as cases and , individuals from the icont database were used as controls. genome wide significant or replicating snps were tested in this case-control sample using logistic regression controlling for gender in plink [ ]. we report the p-values, odds ratios and confidence intervals. concordance analysis of imputed snps in migen and icont with the hapmap tsi sample to assess the concordance of imputed snps in the migen and icont samples we obtained the genotyped snps from the hapmap tsi (tuscans from italy) sample. using only the snps present on the affymetrix . platform (used to genotype migen) or only the snps present on the illumina platform (used to genotype icont samples) and the ld information from hapmap we imputed the remainder of the snps using mach( . . ) and compared the imputed calls to the actual genotypes stratified by imputation quality score (r hat). evaluation of effects on other metabolic traits to obtain data on whether ct hepatic steatosis snps affect other metabolic traits we obtained data from four consortia that had the largest and most powered analyses of these traits. association results for hdl-, ldl- cholesterol levels and triglycerides (tg) were obtained from publicly available data of the global lipids genetics consortium (http://www.sph. umich.edu/csg/abecasis/public/teslovich et al. ) [ ]. association results for fasting insulin, glucose, hr-glucose, homa-ir and homa-b were obtained from the magic investigators. association results for risk of type diabetes were obtained from the diagram consortium [ ]. association results for risk of bmi and waist to hip ratio controlled for bmi were obtained from the giant consortium [ ]. we used a conservative nominal p, . corresponding to five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e a bonferroni correction of phenotypes tested for snps to determine significance. expression qtl analyses the expression qtl analyses in liver, subcutaneous and omental fat tissue have been described in detail previously [ ]. tissue were obtained from patients who underwent bariatric surgery, and rna expression assessed using a custom agilent , feature microarray composed of , oligonucleotide probes targeting transcripts representing , known and predicted genes. patients were also genotyped on the illumina y snp genotyping arrays. snps were tested for cis-associations with transcripts within a mb region, assuming an additive effect of the ct hepatic steatosis increasing allele adjusting for age, race, gender, and surgery year using linear regression. cis-associations between each snp and the adjusted gene expression data were tested, and only associations with a nominal p-value , . corresponding to a bonferroni correction for gene transcripts x snps tested are shown in table s . conditional analyses were performed by conditioning the ct hepatic steatosis associated snp on the most significant cis-associated snp for that particular gene transcript and vice versa. supporting information table s study sample characteristics. (*) drinking alcohol is not practiced in amish culture and not measured this study. gold: genetics of obesity-related liver disease; nash crn: nonalcoholic steatohepatitis clinical research network; migen: myocardial infarction genetics consortium; icont: illumina control database; sd: standard deviation; p , p : th and th percentiles; phantom ld or hd- low or high density external hydroxyapetite ct control; median raw liver measures in hounsfield units; steatosis . % more than % steatosis on histology; nash: having histologic criteria for diagnosis of nonalcoholic steatohepatitis (nash); fibrosis: having histologic criteria for diagnosis of fibrosis. found at: doi: . /journal.pgen. .s ( . mb doc) table s genotyping and association information. imp’n: imputation; maf: minor allele frequency; hwe: hardy wein- berg equilibrium; gold: genetics of obesity-related liver disease; nash crn: nonalcoholic steatohepatitis clinical research network; migen: myocardial infarction genetics consortium; icont: illumina control database. found at: doi: . /journal.pgen. .s ( . mb doc) table s quality control. * sample genotyping success rate; i.e. percentage of successfully genotyped snps per sample. gold: genetics of obesity-related liver disease; nash crn: nonal- coholic steatohepatitis clinical research network; migen: myocardial infarction genetics consortium; icont: illumina control database; ibd pi hat: value for identical by descent of . . . found at: doi: . /journal.pgen. .s ( . mb doc) table s top genotyped hits from gold, ages, amish, family heart study, framingham heart study. gold: genetics of obesity-related liver disease; chr.: chromosome; pos.: position, build ; ea: effect allele; oa: other allele; eaf: frequency of the effect allele in the analyses (weighted average in gold); effect: increase in inverse normalized fatty liver by computed tomography se: standard error; p: p-value of association in the analyses; % var: % variance explained; p het: p-value for heterogeneity across studies; n: number of individuals in the analyses. found at: doi: . /journal.pgen. .s ( . mb doc) table s top genotyped hits in nash crn/migen analysis. nash crn: nonalcoholic steatohepatitis clinical research network; migen: myocardial infarction genetics consortium; chr. chromosome; pos.: position, build ; ea: effect allele; oa:other allele; eafa: frequency of the effect allele in cases from the nash crn study; eafb :frequency of the effect allele in controls from the migen study; impb: imputation quality score in migen; nafld: nonalcoholic fatty liver disease; or nafld: odds ratio for the presence of nafld on pathology per effect allele; p nafld: false discovery rate p-value of association for histologic nafld. found at: doi: . /journal.pgen. .s ( . mb doc) table s genome-wide significant or replicating variants in nash crn/icont analysis. nash crn: nonalcoholic steatohepatitis clinical research network; icont: illumina control database; ea: effect allele; oa:other allele; eafa: frequency of the effect allele in cases from the nash-crn study; eafb :frequency of the effect allele in controls from icont; impb: imputation quality score in icont; nafld: nonalcoholic fatty liver disease; or nafld: odds ratio for the presence of nafld on pathology per effect allele; p nafld: false discovery rate p-value of association for histologic nafld. found at: doi: . /journal.pgen. .s ( . mb doc) table s imputation r hat measures versus concordance to real genotypes in tsi individuals from hapmap . tsi: toscans in italy; r hat: imputation quality score from mach; n snps: number of snps used for concordance analysis; concordance: average concordance amongst the snps assayed. found at: doi: . /journal.pgen. .s ( . mb docx) table s imputation r hat measures in migen and icont versus concordance to real genotypes in tsi individuals from hapmap . impa: imputation quality score in migen; con- cordancea: average concordance of snps in tsi given imputation quality score in migen; impb: imputation quality score in icont; concordanceb: average concordance of snps in tsi given imputation quality score in icont. found at: doi: . /journal.pgen. .s ( . mb docx) table s effect of genome-wide significant or replicating variants on glucose, anthropometric and lipid traits. association results for high density lipoprotein (hdl)-, low density lipoprotein (ldl)- cholesterol levels and triglycerides (tg) were obtained from publicly available data of the global lipids genetics consortium (http://www.sph.umich.edu/csg/abecasis/public/ teslovich et al. ) association results for fasting insulin and glucose, hr-glucose, homa-ir and homa-b were obtained from the magic consortium (dupuis et al. nature genetics ). association results for risk of type diabetes were obtained from the diagram consortium (voight et al. nature genetics ). association results for risk of bmi and waist to hip ratio controlled for bmi were obtained from the giant consortium (speliotes et al. nature genetics ). bmi: body mass index; homa-ir: homeostasis model assessment insulin resistance; homa-b: homeostasis model assessment beta cell function; ea: five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e effect allele; oa: other allele; effect: the change in the trait per effect allele from the various studies; se: standard error in the effect from the various studies; p: p-value of association from the various studies; n: number of individuals in the analyses; or: odds ratio for the effect allele on diabetes; u % and l %- upper and lower % confidence levels for the or. found at: doi: . /journal.pgen. .s ( . mb doc) table s significant associations between genome-wide signif- icant or replicating snps and cis gene expression (cis -eqtls) in liver, omental fat and subcutaneous fat. snp: the fatty liver associating snp from gwas analysis. ea: effect allele (fatty liver increasing allele from gwas). effecta: direction of effect on the gene transcript expression level for the effect allele. p: p-value of association of the fatty liver snp with change in gene expression. padjb :p-value for the fatty liver snp after conditioning on the most significant snp for change in gene transcript. peak snpc: snp in the region that has the most significant eqtl p-value on expression of the gene transcript rsqd: the r squared correlation between the fatty liver snp and the peak snp. padje: p-value for the peak snp after conditioning on the fatty liver snp for change in gene transcript. na: peak snp is the same as the fatty liver associating snp. found at: doi: . /journal.pgen. .s ( . mb doc) acknowledgments we would like to thank dr. qiong yang for use of her r program for related analyses in the framingham heart study and dr. monty krieger for critically reviewing the manuscript. we would like to thank arun sanyal for serving as our liason to the nash crn. samples were provided by the nash crn for analyses in this paper. nash crn arthur mccullough, m.d.; diane bringman, r.n., b.s.n.; srinivasan dasarathy, m.d.; kevin edwards, n.p.; carol hawkins, r.n.; yao-chang liu, m.d.; nicholette rogers, ph.d.; p.a.-c.; ruth sargent, l.p.n.; margaret stager, m.d.; anna mae diehl, m.d.; manal abdelmalek, m.d.; marcia gottfried, m.d.; cynthia guy, m.d.; paul killenberg, m.d.; samantha kwan; yi-ping pan; dawn piercy, f.n.p.; melissa smith; naga chalasani, m.d.; prajakta bhimalli; oscar w. cummings, m.d.; ann klipsch, rn; lydia lee; jean molleston, m.d.; linda ragozzino; raj vuppalanchi, m.d.; brent a. neuschwander-tetri, m.d.; sarah barlow, m.d.; jose derdoy, m.d.; joyce hoffmann; debra king, r.n.; joan siegner, r.n.; susan stewart, r.n.; judy thompson, r.n.; elizabeth brunt, m.d.; joel e. lavine, m.d., ph.d.; cynthia behling, m.d.; lisa clark; janis durelle; tarek hassanein, m.d.; lita petcharaporn; jeffrey b. schwimmer, m.d.; claude sirlin, m.d.; tanya stein; nathan m. bass, m.d., ph.d.; kiran bambha, m.d.; linda d. ferrell, m.d.; danuta filipowski; raphael merriman, m.d.; mark pabst; monique rosenthal; philip rosenthal, m.d.; tessa steel; arun j. sanyal, m.d.; sherry boyett, r.n.; daphne bryan, m.d.; melissa j. contos, m.d.; michael fuchs, m.d.; martin graham, m.d.; amy jones; velimir a.c. luketic, m.d.; bimalijit sandhu, m.d.; carol sargeant, r.n., m.p.h.; kimberly selph; melanie white, r.n.; kris v. kowdley, m.d.; grace gyurkey; jody mooney, m.s.; james nelson, ph.d.; sarah roberts; cheryl saunders, m.p.h.; alice stead; chia wang, m.d.; matthew yeh, m.d., ph.d.; david kleiner, m.d., ph.d.; edward doo, m.d.; jay everhart, m.d., m.p.h.; jay h. hoofnagle, m.d.; patricia r. robuck, ph.d.; leonard seeff, m.d.; james tonascia, ph.d.; patricia belt, b.s.; fred brancati, m.d., m.h.s.; jeanne clark, m.d., m.p.h.; ryan colvin, m.p.h.; michele donithan, m.h.s.; mika green, m.a.; milana isaacson; wana kim; laura miriel; alice sternberg, sc.m.; aynur ünalp, m.d., ph.d.; mark van natta, m.h.s.; laura wilson, sc.m.; katherine yates, sc.m. giant consortium elizabeth k. speliotes, cristen j. willer, sonja i. berndt, keri l. monda, gudmar thorleifsson, anne u. jackson, hana lango allen, cecilia m. lindgren, jian’an luan, reedik mägi, joshua c. randall, sailaja vedantam, thomas w. winkler, lu qi, tsegaselassie work- alemahu, iris m. heid, valgerdur steinthorsdottir, heather m. stringham, michael n. weedon, eleanor wheeler, andrew r. wood, teresa ferreira, robert j. weyant, ayellet v. segrè, karol estrada, liming liang, james nemesh, ju-hyun park, stefan gustafsson, tuomas o. kilpeläinen, jian yang, nabila bouatia-naji, tõnu esko, mary f. feitosa, zoltán kutalik, massimo mangino, soumya raychaudhuri, andre scherag, albert vernon smith, ryan welch, jing hua zhao, katja k. aben, devin m. absher, najaf amin, anna l. dixon, eva fisher, nicole l. glazer, michael e. goddard, nancy l. heard-costa, volker hoesel, jouke-jan hottenga, åsa johansson, toby johnson, shamika ketkar, claudia lamina, shengxu li, miriam f. moffatt, richard h. myers, narisu narisu, john r.b. perry, marjolein j. peters, michael preuss, samuli ripatti, fernando rivadeneira, camilla sandholt, laura j. scott, nicholas j. timpson, jonathan p. tyrer, sophie van wingerden, richard m. watanabe, charles c. white, fredrik wiklund, christina barlassina, daniel i. chasman, matthew n. cooper, john-olov jansson, robert w. lawrence, niina pellikka, inga prokopenko, jianxin shi, elisabeth thiering, helene alavere, maria t. s. alibrandi, peter almgren, alice m. arnold, thor aspelund, larry d. atwood, beverley balkau, anthony j. balmforth, amanda j. bennett, yoav ben-shlomo, richard n. bergman, sven bergmann, heike biebermann, alexandra i.f. blakemore, tanja boes, lori l. bonnycastle, stefan r. bornstein, morris j. brown, thomas a. buchanan, fabio busonero, harry campbell, francesco p. cappuccio, christine cavalcanti-proença, yii-der ida chen, chih-mei chen, peter s. chines, robert clarke, lachlan coin, john connell, ian n.m. day, martin den heijer, jubao duan, shah ebrahim, paul elliott, roberto elosua, gudny eiriksdottir, michael r. erdos, johan g. eriksson, maurizio f. facheris, stephan b. felix, pamela fischer-posovszky, aaron r. folsom, nele friedrich, nelson b. freimer, mao fu, stefan gaget, pablo v. gejman, eco j.c. geus, christian gieger, anette p. gjesing, anuj goel, philippe goyette, harald grallert, jürgen gräbler, danielle ?m. greenawalt, christopher j. groves, vilmundur gudnason, candace guiducci, anna-liisa hartikainen, neelam hassanali, alistair s. hall, aki s. havulinna, caroline hayward, andrew c. heath, christian hengsten- berg, andrew a. hicks, anke hinney, albert hofman, georg homuth, jennie hui, wilmar igl, carlos iribarren, bo isomaa, kevin b. jacobs, ivonne jarick, elizabeth jewell, ulrich john, torben jørgensen, pekka jousilahti, antti jula, marika kaakinen, eero kajantie, lee m. kaplan, sekar kathiresan, johannes kettunen, leena kinnunen, joshua w. knowles, ivana kolcic, inke r. könig, seppo koskinen, peter kovacs, johanna kuusisto, peter kraft, kirsti kvaløy, jaana laitinen, olivier lantieri, chiara lanzani, lenore j. launer, cecile lecoeur, terho lehtimäki, guillaume lettre, jianjun liu, marja-liisa lokki, mattias lorentzon, robert n. luben, barbara ludwig, magic, paolo manunta, diana marek, michel marre, nicholas g. martin, wendy l. mcardle, anne mccarthy, barbara mcknight, thomas meitinger, olle melander, david meyre, kristian midthjell, grant w. montgomery, mario a. morken, andrew p. morris, rosanda mulic, julius s. ngwa, mari nelis, matt j. neville, dale r. nyholt, christopher j. o’donnell, stephen o’rahilly, ken k. ong, ben oostra, guillaume paré, alex n. parker, markus perola, irene pichler, kirsi h. pietiläinen, carl g.p. platou, ozren polasek, anneli pouta, suzanne rafelt, olli raitakari, nigel w. rayner, martin ridderstråle, winfried rief, aimo ruokonen, neil r. robertson, peter rzehak, veikko salomaa, alan r. sanders, manjinder s. sandhu, serena sanna, jouko saramies, markku j. savolainen, susann scherag, sabine schipf, stefan schreiber, heribert schunkert, kaisa silander, juha sinisalo, david s. siscovick, jan h. smit, nicole soranzo, ulla sovio, jonathan stephens, ida surakka, amy j. swift, mari-liis tammesoo, jean- claude tardif, maris teder-laving, tanya m. teslovich, john r. thompson, brian thomson, anke tönjes, tiinamaija tuomi, joyce b.j. van meurs, gert-jan van ommen, vincent vatin, jorma viikari, sophie visvikis-siest, veronique vitart, carla i. g. vogel, benjamin f. voight, lindsay l. waite, henri wallaschofski, g.bragi walters, elisabeth widen, susanna wiegand, sarah h. wild, gonneke willemsen, daniel r. witte, jacqueline c. witteman, jianfeng xu, qunyuan zhang, lina zgaga, andreas ziegler, paavo zitting, john p. beilby, i. sadaf farooqi, johannes hebebrand, heikki v. huikuri, alan l. james, mika kähönen, douglas f. levinson, fabio macciardi, markku s. nieminen, claes ohlsson, lyle j. palmer, paul m. ridker, michael stumvoll, jacques s. beckmann, heiner boeing, eric boerwinkle, dorret i. boomsma, mark j. caulfield, stephen j. chanock, francis s. collins, l. adrienne cupples, george davey smith, jeanette erdmann, philippe froguel, henrik grönberg, ulf gyllensten, per hall, torben hansen, tamara b. harris, andrew t. hattersley, richard five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e b. hayes, joachim heinrich, frank b. hu, kristian hveem, thomas illig, marjo-riitta jarvelin, jaakko kaprio, fredrik karpe, kay-tee khaw, lambertus a. kiemeney, heiko krude, markku laakso, debbie a. lawlor, andres metspalu, patricia b. munroe, willem h. ouwehand, oluf pedersen, brenda w. penninx, annette peters, peter p. pramstaller, thomas quertermous, thomas reinehr, aila rissanen, igor rudan, nilesh j. samani, peter e.h. schwarz, alan r. shuldiner, timothy d. spector, jaakko tuomilehto, manuela uda, andré uitterlinden, timo t. valle, martin wabitsch, gérard waeber, nicholas j. wareham, hugh watkins on behalf of procardis consortium, james f. wilson, alan f. wright, m.carola zillikens, nilanjan chatterjee, steven a. mccarroll, shaun purcell, eric e. schadt, peter m. visscher, themistocles l. assimes, ingrid b. borecki, panos deloukas, caroline s. fox, leif c. groop, talin haritunians, david j. hunter, robert c. kaplan, karen l. mohlke, jeffrey r. o’connell, leena peltonen, david schlessinger, david p. strachan, cornelia m. van duijn, h.-erich wichmann, timothy m. frayling, unnur thorsteinsdottir, gonçalo r. abecasis, inês barroso, michael boehnke, kari stefansson, kari e. north, mark i. mccarthy, joel n. hirschhorn, erik ingelsson, ruth j.f. loos magic investigators josée dupuis, claudia langenberg, inga prokopenko, richa saxena, nicole soranzo, anne u jackson, eleanor wheeler, nicole lglazer, nabila bouatia-naji, anna lgloyn, cecilia mlindgren, reedik mägi, andrew p morris, joshua randall, toby johnson, paul elliott, denis rybin, gudmar thorleifsson, valgerdur steinthorsdottir, peter henne- man, harald grallert, abbas dehghan, jouke jan hottenga, christopher s franklin, pau navarro, kijoung song, anuj goel, john r b perry, josephine m egan, taina lajunen, niels grarup, thomas sparsø, alex doney, benjamin f voight, heather m stringham, man li, stavroula kanoni, peter shrader, christine cavalcanti-proença, meena kumari, lu qi, nicholas j timpson, christian gieger, carina zabena, ghislain rocheleau, erik ingelsson, ping an, jeffrey o’connell, jian’an luan, amanda elliott, steven a mccarroll, felicity payne, rosa maria roccasecca, françois pattou, praveen sethupathy, kristin ardlie, yavuz ariyurek, beverley balkau, philip barter, john p beilby, yoav ben- shlomo, rafn benediktsson, amanda j bennett, sven bergmann, murielle bochud, eric boerwinkle, amélie bonnefond, lori l bonnycastle, knut borch-johnsen, yvonne böttcher, eric brunner, suzannah j bumpstead, guillaume charpentier, yii-der ida chen, peter chines, robert clarke, lachlan j m coin, matthew n cooper, marilyn cornelis, gabe crawford, laura crisponi, ian n m day, eco j cde geus, jerome delplanque, christian dina, michael r erdos, annette c fedson, antje fischer- rosinsky, nita g forouhi, caroline s fox, rune frants, maria grazia franzosi, pilar galan, mark o goodarzi, jürgen graessler, christopher j groves, scott grundy, rhian gwilliam, ulf gyllensten, samy hadjadj, göran hallmans, naomi hammond, xijing han, anna-liisa hartikainen, neelam hassanali, caroline hayward, simon c heath, serge hercberg, christian herder, andrew a hicks, david r hillman, aroon dhingorani, albert hofman, jennie hui, joe hung, bo isomaa, paul r v johnson, torben jørgensen, antti jula, marika kaakinen, jaakko kaprio, y antero kesaniemi, mika kivimaki, beatrice knight, seppo koskinen, peter kovacs, kirsten ohm kyvik, gmark lathrop, debbie a lawlor, olivier le bacquer, cécile lecoeur, yun li, valeriya lyssenko, robert mahley, massimo mangino, alisa k manning, marı́a teresa martı́nez-larrad, jarred b mcateer, laura j mcculloch, ruth mcpherson, christa meisinger, david melzer, david meyre, braxton d mitchell, mario a morken, sutapa mukherjee, silvia naitza, narisu narisu, matthew j neville, ben a oostra, marco orrù, ruth pakyz, colin na palmer, giuseppe paolisso, cristian pattaro, daniel pearson, john f peden, nancy l pedersen, markus perola, andreas f h pfeiffer, irene pichler, ozren polasek, danielle posthuma, simon c potter, anneli pouta, michael a province, bruce m psaty, wolfgang rathmann, nigel wrayner, kenneth rice, samuli ripatti, fernando rivadeneira, michael roden, olov rolandsson, annelli sandbaek, manjinder sandhu, serena sanna, avan aihie sayer, paul scheet, laura j scott, udo seedorf, stephen j sharp, beverley shields, gunnar sigurðsson, eric j g sijbrands, angela silveira, laila simpson, andrew singleton, nicholas l smith, ulla sovio, amy swift, holly syddall, ann-christine syvänen, toshiko tanaka, barbara thorand, jean tichet, anke tönjes, tiinamaija tuomi, andré guitter- linden, ko willems van dijk, mandy van hoek, dhiraj varma, sophie visvikis-siest, veronique vitart, nicole vogelzangs, gérard waeber, peter j wagner, andrew walley, g bragi walters, kim l ward, hugh watkins, michael n weedon, sarah h wild, gonneke willemsen, jaqueline c m witteman, john w g yarnell, eleftheria zeggini, diana zelenika, björn zethelius, guangju zhai, jing hua zhao, m carola zillikens, diagram consortium, giant consortium, global bpgen consortium, ingrid b borecki, ruth j f loos, pierre meneton, patrik kemagnusson, david m nathan, gordon h williams, andrew t hattersley, kaisa silander, veikko salomaa, george davey smith, stefan r bornstein, peter schwarz, joachim spranger, fredrik karpe, alan r shuldiner, cyrus cooper, george v dedoussis, manuel serrano-rı́os, andrew d morris, lars lind, lyle j palmer, frank b hu, paul w franks, shah ebrahim, michael marmot, w h linda kao, james s pankow, michael j sampson, johanna kuusisto, markku laakso, torben hansen, oluf pedersen, peter paul pramstaller, h erich wichmann, thomas illig, igor rudan, alan f wright, michael stumvoll, harry campbell, james f wilson, anders hamsten on behalf of procardis consortium, richard n bergman, thomas a buchanan, francis s collins, karen l mohlke, jaakko tuomilehto, timo t valle, david altshuler, jerome i rotter, david s siscovick, brenda w j h penninx, dorret i boomsma, panos deloukas, timothy d spector, timothy m frayling, luigi ferrucci, augustine kong, unnur thorsteinsdottir, kari stefansson, cornelia mvan duijn, yurii saulchenko, antonio cao, angelo scuteri, david schlessinger, manuela uda, aimo ruokonen, marjo-riitta jarvelin, dawn m waterworth, peter vollenweider, leena peltonen, vincent mooser, goncalo r abecasis, nicholas j wareham, robert sladek, philippe froguel, richard m watanabe, james b meigs, leif groop, michael boehnke, mark i mccarthy, jose c florez & inês barroso for the magic investigators. gold consortium gudny eiriksdottir, melissa e. garcia, vilmundur gudnason, tamara b. harris, lauren j. kim, lenore j. launer, michael a. nalls, albert v. smith,jeanne m. clark, ruben hernaez, w. h. linda kao, braxton d. mitchell, alan r. shuldiner, laura m. yerges-armstrong, ingrid b. borecki, j. jeffrey carr, mary f. feitosa, jun wu, johannah l. butler, caroline s. fox, joel n. hirschhorn, udo hoffmann, shih-jen hwang, joseph m. massaro, christopher j. o’donnell, cameron d. palmer, dushyant v. sahani, elizabeth k. speliotes. author contributions conceived and designed the experiments: eks cjo csf whlk jnh ibb. performed the experiments: eks lmya jw rh jlb mff. analyzed the data: eks lmya jw rh cdp ge meg ljl man sjh jmm bfv avs. contributed reagents/materials/analysis tools: eks vg jmc bdm ars mt uh jmm cjo dvs vs ees sms dss nash crn giant consortium magic investigators jjc tbh csf whlk jnh ibb. wrote the paper: eks lmya jw rh ljk csf whlk jnh ibb gold consortium. 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( ) plink: a tool set for whole-genome association and population-based linkage analyses. am j hum genet : – . five gwas loci associate with nafld plos genetics | www.plosgenetics.org march | volume | issue | e research article open access non-replication study of a genome-wide association study for hypertension and blood pressure in african americans srividya kidambi , *, soumitra ghosh , jane m kotchen , clarence e grim , shanthi krishnaswami , mary l kaldunski , allen w cowley jr , shailendra b patel , * and theodore a kotchen abstract background: a recent genome wide association study in african americans identified several single nucleotide polymorphisms that reached genome-wide significance for systolic blood pressure. we attempted to replicate these findings in an independent sample of unrelated african americans in the milwaukee metropolitan area; % were women and % were hypertensives. methods: we evaluated sixteen top associated snps from the above genome wide association study for hypertension as a binary trait or blood pressure as a continuous trait. in addition, we evaluated eight single nucleotide polymorphisms located in two genes (stk- and cdh- ) found to be associated with systolic and diastolic blood pressures by other genome wide association studies in european and amish populations. taqman mgb-based chemistry with fluorescent probes was used for genotyping. we had an adequate sample size ( % power) to detect an effect size of . - . for all the single nucleotide polymorphisms for hypertension as a binary trait, and % variance in blood pressure as a continuous trait. quantitative trait analyses were performed both by excluding and also by including subjects on anti-hypertensive therapy (after adjustments were made for anti- hypertensive medications). results: for all snps, no statistically significant differences were noted in the minor allele frequencies between cases and controls. one snp (rs ) showed borderline association (p = . ) with hypertension status using recessive model and systolic blood pressure (p = . ), but was not significant after adjusting for multiple comparisons. in quantitative trait analyses, among normotensives only, rs was associated with sbp (p = . ). in addition, several nominally significant associations were noted with sbp and dbp among normotensives but none were statistically significant. conclusions: this study highlights the importance of replication to confirm the validity of genome wide association study results. background hypertension is a major contributor to the global dis- ease burden with world-wide prevalence estimated to be ~ %, totaling ~ billion people [ , ]. currently, in the u.s., approximately million americans have hypertension, and the prevalence is particularly high in african americans. based on recent results of the national health and nutrition examination survey (nhanes), the age-adjusted hypertension prevalence is . % in non-hispanic blacks and . % in non-hispa- nic whites [ ]. adoption, twin, and family studies docu- ment a significant heritable component to blood pressure levels and hypertension [ - ] and indicate that the heritability of blood pressure is in the range of - % [ , ]. hypertension before the age of years occurs . times more frequently among persons with a positive family history of hypertension [ ].* correspondence: skidambi@mcw.edu; sbpatel@mcw.edu department of medicine, medical college of wisconsin, milwaukee, wi , usa full list of author information is available at the end of the article kidambi et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / © kidambi et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:skidambi@mcw.edu mailto:sbpatel@mcw.edu http://creativecommons.org/licenses/by/ . genome-wide association studies (gwas) are one strategy that is being exploited to identify the genetic contributions to hypertension. this strategy has been facilitated by the unraveling of the human genome, the hapmap project, and the availability of dense computer chips for genetic sequencing. in contrast to candidate gene approaches, gwas offers the potential to identify novel mechanisms in the pathophysiology of hyperten- sion. the first large gwas for hypertension, performed by the wellcome trust case control consortium (wtccc) among british subjects (with cases and controls) in the [ ], revealed no single nucleotide polymorphisms (snps) crossing the experimental threshold of statistical significance established at p < - . subsequently, several additional gwas have been published, including large-scale meta-analyses, demon- strating sporadic and inconsistent associations of snps with either blood pressure or hypertension [ - ]. notably, these studies have been carried out in predomi- nantly european and amish populations. in the first ever gwas in african americans (all subjects were residents of the washington dc area), adeyemo et al identified several snps reaching gen- ome-wide significance for systolic blood pressure in or near genes: pms , slc a , ywha , ipo , and cacana h [ ]. two of these genes, slc a (a sodium/potassium/calcium exchanger) and cacna h (a voltage-dependent calcium channel), are potential candidate genes for blood pressure regulation and the latter is also a drug target for a class of calcium chan- nel blockers. some of the significant snps were repli- cated in a sample of west africans [ ]. independent confirmation is necessary to validate these findings. therefore, we investigated the association of snps identified by adeyemo et al in an independent popula- tion of african american subjects from the mid-wes- tern united states. in addition, in these african american subjects, we also sought to replicate snp variants recently identified by gwas in european and amish populations, and further confirmed in the study by adeyemo et al [ , , ]. results a total of eligible subjects were genotyped of whom % (n = ) were women. fifty-three percent (n = ) of the subjects were normotensives, and % (n = ) were hypertensives (table ). of the hyper- tensives, % were on antihypertensive drug therapy. hypertensives were older, had higher bmi, waist cir- cumference, serum glucose, and cholesterol (p < . ) compared to normotensives. there were no significant differences in the gender composition or serum creati- nine concentration. hardy-weinberg equilibrium genotype distributions were in hardy-weinberg equili- brium for all the snps except two (rs and rs ) when all the subjects were considered together as well as when normotensives were analyzed separately. among hypertensives, in addition to rs and rs , rs was also not in hardy-weinberg equilibrium. case-control association analyses case-control association analyses were done separately for each snp with hypertensives as cases and normoten- sives as controls using plink association analyses. we did not observe any statistically significant difference in the allele frequency or in the genotype distribution of the snps between cases and controls (additional file : table s ). genetic association analyses utilizing domi- nant and additive models did not reveal any significant associations between snps that were tested and hyper- tension status (table - only additive model results are shown). one snp (rs ) showed borderline asso- ciation when recessive model was considered with a p- table demographic characteristics (mean ± sd) of cases and controls characteristic all hypertensives (n = ) untreated hypertensives (n = ) normotensives (n = ) p-value between normotensives and all hypertensives age (years) ± ± ± < . female (%) . sbp (mmhg) ± ± ± dbp (mmhg) ± ± ± bmi (kg/m ) ± ± ± < . waist circumference (cm) ± ± ± < . serum glucose (mg/dl) ± ± ± < . serum creatinine (mg/dl) . ± . . ± . . ± . . serum cholesterol (mg/dl) ± ± ± < . kidambi et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of value of . , but was not significant when adjusted for multiple comparisons and for age, gender, bmi and creatinine. quantitative trait analyses quantitative trait associations were carried out using plink between these snps and systolic and diastolic blood pressure on subjects both with and without inclu- sion of treated hypertensive subjects (additional file : table s ). blood pressures of treated hypertensives were adjusted for the effect of antihypertensive drug treatment before conducting the analyses. while this increased the sample size, the results were similar with both inclusion and exclusion of treated hypertensive subjects. neither systolic nor diastolic blood pressures were significantly dif- ferent among different genotypes of these snps after statistical adjustment for multiple comparisons. however, without this statistical adjustment one snp (rs ) that was previously associated with diastolic blood pres- sure in the report of adeyemo et al, showed association with systolic blood pressure (p = . ). in additional quantitative trait analyses, we evaluated normotensives and hypertensives separately (additional file : table s ). in normotensives only, snp rs was associated with systolic blood pressure (p = . ). while this was statistically significant after adjustment for multiple comparisons, it was no longer significant after adjustment for age, sex, bmi, serum creatinine. moreover, the association noted was with lower systolic blood pressure rather than previously associated hypertension status [ ]. in addition, snps rs and rs showed borderline associa- tion with systolic and diastolic blood pressures (p = . ) respectively, but these were not significant after adjustment for multiple comparisons. quantitative trait analyses that included only hyper- tensive subjects (untreated and treated) showed no sig- nificant associations of blood pressure with any of the snps after adjustment for multiple comparisons. however, the following several borderline associations were observed without statistical adjustment for multiple comparisons (additional file : table s ): a) snp rs was associated with systolic blood pressure (p = . ); b) snp rs was associated with systolic and diastolic blood pressure (p = . and . respec- tively); c) snp rs is associated with systolic and diastolic blood pressure (p = . ); d) snp rs was associated with systolic blood pressure (p = . ). the number of untreated hypertensive subjects was insufficient to permit quantitative trait analyses in this group of subjects. regression analyses multiple linear regression analysis did not show a statis- tically significant impact of any of the alleles or geno- types on systolic and diastolic blood pressure in all subjects category (including treated subjects with stepped-up blood pressure), in all untreated subjects (normotensives plus untreated hypertensives), in normo- tensives alone or in hypertensives alone (table ). inde- pendent variables included in the model were gender, age, body mass index, and serum creatinine. multiple logistic regression analysis was also conducted with the same independent variables as linear regression with hypertension as a dependent binary trait. no significant impact of either genotypes or alleles of all snps was noticed on the hypertension status. discussion in this sample of african americans, none of the snps evaluated were convincingly associated with hyperten- sion as a binary trait or with blood pressure level as a quantitative trait. thus, we were unable to confirm pre- viously reported associations between pms , slc a , ywha , ipo , and cacana h with systolic and table results of case-control genetic association analyses using additive model snp id gene symbol minor allele maf p-value rs p ha t . . rs (ac . ) c . . rs adh t . . rs (ac . ) g . . rs aldh a t . . rs (al . ) a . . rs * (rp - f . ) c . . rs slc a t . . rs * ywhaz t . . rs pms a . . rs * ipo t . . rs * cacna h t . . rs sv b g . . rs nrxn t . . rs abcc a . . rs prc c . . rs * stk g . . rs stk g . . rs stk c . . rs stk c . . rs cdh c . . rs cdh t . . rs cdh t . . rs * cdh a . . *p-values were obtained using modeling with fishers exact test for dominant/ recessive tests of association when the frequency of observations was < per group kidambi et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of diastolic blood pressure. however, among normotensives only, one snp (rs ) showed a significant asso- ciation with systolic blood pressure even after adjusting for multiple comparisons, but was no longer significant after adjustment for age, sex, bmi and serum creatinine. this snp was previously marginally associated with hypertension as a binary trait in the study by adeyemo et al, but had not reached genome-wide significance in their study either. it is located on chromosome in the intergenic region and its biological plausibility is unknown at this time. we observed several suggestive, but not significant associations. snp rs , located on chromosome in the intergenic region, was associated with systolic blood pressure in all subjects (treated and untreated). similarly, rs which is also located on chromo- some in the intergenic region, showed borderline asso- ciation with systolic blood pressure among normotensives. among hypertensives, borderline asso- ciations (p > . ) were noted between rs (in the intergenic region near the gene aldehyde dehydro- genase family, member a , aldh a ) and systolic blood pressure, and rs (in the intronic region of the gene tyrosine -monooxygenase/tryptophan - monooxygenase activation protein, zeta polypeptide, ywhaz) and systolic and diastolic blood pressure. the biological plausibility of these associations is currently unknown (additional file : table s ). similarly, ehret et al attempted to replicate six top- associated snps (rs ( q ), rs ( q ), rs ( p ), rs ( q ), rs ( q ) and rs ( q )) from wtccc in the family blood pressure program (fbpp) cohort [ , ]. dna was genotyped on , participants ( % afri- can americans, % european americans, % hispanic americans) and the results showed that only one of these six snps (rs on q ) was negatively associated with systolic and diastolic blood pressure among european americans. the same snp showed a significant but opposite effect with systolic blood pres- sure in hispanic americans and was not associated in african americans [ ]. no replication could be shown for hypertension status. the associations that were clo- sest to being suggestive could not be replicated, even in table results of logistic and linear regression analyses using minor allele as standard snp id htn ß ± se (p-value) untreated subjects ß ± se (p-value) sbp dbp rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs * - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs . ± . ( . ) . ± . ( . ) - . ± . ( . ) rs * . ± . ( . ) . ± . ( . ) - . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs * - . ± . ( . ) - . ± . ( . ) - . ± . ( . ) rs * - . ± . ( . ) - . ± . ( . ) - . ± . ( . ) rs . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) - . ± . ( . ) - . ± . ( . ) rs . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs * - . ± . ( . ) - . ± . ( . ) - . ± . ( . ) rs - . ± . ( . ) - . ± . ( . ) - . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs . ± . ( . ) . ± . ( . ) . ± . ( . ) rs - . ± . ( . ) . ± . ( . ) . ± . ( . ) rs * . ± . ( . ) . ± . ( . ) - . ± . ( . ) kidambi et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of a well characterized sample such as the fbpp suggesting the difficulty in replicating the findings of gwas [ ]. the most significant criticism of the wtccc was the use of young controls that could potentially become cases at a later age. inconsistent results among several small-scale gwas [ - , ] suggested that hypertension is polygenic dis- ease with multiple low frequency genes acting in har- mony to result in elevated blood pressure. to overcome some of these limiting factors, a large meta-analysis of charge and bpgen consortium results was conducted recently and several novel loci have been identified for blood pressure. although these associations have not been replicated in independent populations, these results may be more robust than previous gwas as the meta- analysis involved more than , subjects [ , ]. we did not choose to replicate these associations as these studies involved subjects primarily from european and asian indian ancestry. on a similar note, a large gwas for blood pressure among african americans, the candidate gene associa- tion resource (care) study, has recently been pub- lished. in a meta-analysis across five community-based cohorts, two novel loci were identified that reached sta- tistical significance: rs on chromosome with systolic blood pressure and rs on chromosome with diastolic blood pressure [ ]. however, neither of these associations were replicated in independent afri- can american samples, again highlighting the difficulty in extending the findings of gwas to independent populations. in addition, this study did not identify any of the loci identified by adeyemo et al. even though the evidence for a genetic contribution remains robust, the effect of environment may be a major modifier, and this may be the reason for inconsis- tent data. many common everyday activities can pro- foundly affect blood pressure, such as salt intake, exercise, stress, etc., and many of these are not quanti- tated in population-based studies. to minimize environ- mental and genetic variability, wang et al carried out a gwas of systolic and diastolic blood pressure in amish subjects [ ]. strong association signals with several common variants in a serine/threonine kinase gene (stk ) were found and they confirmed these associa- tions in an independent amish and non-amish cau- casian samples [ ]. adeyemo et al found that several snps in this gene were associated with the blood pres- sure ( / for systolic blood pressure and / for diastolic blood pressure) in african americans. variants in stk may influence blood pressure by increasing stk expression and consequently altering renal sodium excretion [ ]. we were unable to confirm the associations of top-associated snps in this gene for systolic and diastolic blood pressure in our cohort despite having adequate power. these associations were not tested in care study by fox et al. another novel locus contributing to blood pressure was identified by org et al in a gwas of a german population and is known as cadherin -heart (cdh ) [ ]. cdh is a calcium-dependent cell adhe- sion glycoprotein and may mediate interaction between cells in heart. several snps in this locus were also found to be associated with systolic and diastolic blood pressures among african americans by adeyemo et al. we tested the association of of these snps with hypertension as a binary trait and blood pressure as a quantitative trait in our sample. we confirmed a border- line association (insignificant when considered with multiple tests) of rs and rs with blood pressure as quantitative trait among hypertensive and normotensive subjects. again, this association was not tested in care study by fox et al. as evidenced by these studies, identification of genetic contributors to hypertension remains challenging for several reasons. population heterogeneity, environmental effects, and sample size all contribute to these failures. our study is also subject to these criticisms. in addition, as already eluded to, inheritance of blood pressure is polygenetic where in a single gene or combination of genes act in concert with above mentioned environmen- tal exposures to contribute only a modest effect on blood pressure. indeed, genetic variants identified by gwas contribute to only a small effect on blood pres- sure ( - mmhg). although this may have a population- based impact, this effect may not be discernible among individuals. moreover, the difficulty of obtaining a stan- dardized blood pressure phenotype cannot be over emphasized. we also acknowledge the limitations of using com- mercially available tag snps. while it is possible to identify genetic variation with tag snps without geno- typing every snp in the chromosomal region and are widely used in genome-wide association studies, it may make replication of target snps difficult due to differ- ences in linkage disequilibrium between two samples. for this reason, many studies have now adopted the technique of “local replication” in which snps sur- rounding tag snp are also targeted, especially for snps that are not directly replicated from previous studies [ ]. apart from inherent difficulties in identifying genetic determinants of hypertension due to the phenotype itself as well as methods, we acknowledge that the differences between the populations investigated by our study and adeyemo et. al. may account for non-replication of the gwas. since african americans as an ethnic group are quite diverse with different origins, we recruited only the individuals whose parents were both born in the kidambi et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of united states, with english as their first language, to achieve some uniformity in ethnicity. however, this may not have been sufficiently homogeneous population and the differences in the results may reflect different popu- lation admixture rates [ , ]. since we do not know the admixture rate in our population, we were unable to adjust for that while adeyemo et. al used genome-wide markers to compute principal components and used these in their models. these baseline differences in the african american population composition is also reflected in the allele frequencies of the various snps, e. g. cacna h ( . vs . ), ipo ( . vs . ), ywhaz ( . vs . ), slc a ( . vs . ), pms ( . vs . ) (additional file : tables ). there were several strengths to our study. cases and controls were clearly defined with normotensives having blood pressures in the lower third of the population dis- tribution of blood pressure (~ sd below the untreated hypertensive means) to eliminate the bias of cases mas- querading as controls or vice versa. subjects with sec- ondary hypertension were excluded including those with renal insufficiency. in addition, the mean age of the cases and controls are in mid-forties, which is suffi- ciently young to capture genetically determined blood pressure increases and old enough to capture most pri- mary hypertensives. however, it is still possible some mis-classification of cases and controls might have occurred. conclusions while they are relatively easy to perform, interpretation of the results of successful association studies is neither straightforward nor always replicable. in addition, over- estimation of genetic effect size in initial studies due to “winner’s curse” may cause follow-up studies to be underpowered and so to fail [ ]. results of the current study and others do not replicate previously identified hypertension-related snps in gwas. non-genetic fac- tors particularly environment, profoundly affect the impact of genes on blood pressure and hypertension and inherited phenotypes may potentially be a result of mechanisms without change in underlying dna sequence. consequently, in addition to focusing on replication of gwas findings, future studies may need to move beyond dna-based sequence approaches and may involve the evaluation of heritable changes in gene expression [ ]. methods subjects and measurements the study was approved by the froedtert memorial lutheran hospital/medical college of wisconsin insti- tutional review board. unrelated african americans subjects between the ages of - years were recruited from a variety of community resources and health care providers within the milwaukee area. subjects were defined as african americans based on self-identifica- tion, birth in the continental united states, both parents reported as being african americans, and english as the native language. all subjects were evaluated during a single outpatient visit and were considered to have hypertension if standardized outpatient measurement of systolic blood pressure was ≥ mmhg, diastolic blood pressure was ≥ mmhg and/or if they were tak- ing antihypertensive medications. blood pressures of normotensives were in the lower third of the distribu- tion of population blood pressures. pregnant subjects, subjects with diabetes mellitus (fasting blood glucose ≥ mg/dl ( . mmol/l) or random blood sugar > mg/dl ( mmol/l), and subjects with serum creatinine concentrations ≥ . mg/dl ( . μmol/l) were excluded. after subjects provided informed consent, standar- dized measurements of blood pressure and anthropo- metric measurements including height, weight, and waist circumference were acquired. blood pressures were taken by trained and certified personnel according to the american heart association guidelines by the shared care method using a sphygmomanometer [ ]. after being seated for at least minutes, readings were obtained from each arm using an appropriate sized arm cuff and the average of the arm with the higher measurement was used for the visit reading. waist cir- cumference was taken at the narrowest point between the umbilicus and superior iliac crest. serum glucose was measured with an automated glucose oxidase enzy- matic assay. plasma cholesterol was measured using an enzymatic procedure. selection of snps and genotyping snps associated with hypertension and blood pressure in the genome wide association study by adeyemo et al among aas were selected for the study [ ]. as shown in additional file : table s , these include the follow- ing: a) seven top-associated snps for hypertension as a binary trait b) five top associated snps for systolic blood pressure as a continuous trait and c) four top- associated snps for diastolic blood pressure. in addition, variants in genes (stk and chd ) identified in european and amish populations [ , ] and replicated by adeyemo et al [ ] in african americans were also selected for replication in the current study. among these snps, only a few had reached genome-wide signif- icance for systolic bp in the study by adeyemo et. al. there are the snps in or near the genes: pms (rs ), slc a (rs ), ywha (rs ), ipo (rs ), and cacana h (rs ). in addition, stk and chd gene kidambi et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of variants reached genome wide significance in non-afri- can american populations. dna was extracted manually from peripheral white blood cell pellets using reagents from qiagen (qiagen inc. valencia, ca, usa) and precipitated using isopro- panol [ ]. taqman mgb-based chemistry (applied biosystems, foster city, ca, usa) with fluorescent probes was used for all genotyping. genotypes were called using the manufacturer’s software and all data were inspected manually. in our laboratory, this method has ~ % concordance between duplicate samples, and produces a genotype for . % of all samples attempted with % validity. approximately . % of the samples were deemed indeterminate or failures. statistical methods continuous variables were reported as means ± standard deviation (sd). differences in the distributions of all selected phenotypes between hypertensive and normo- tensive subjects were determined either by student’s t- test or by wilcoxon rank sum test, depending upon the distribution of variables. both sas software version . (sas institute, cary, nc) and plink [ ] were used for analyses. plink was used to perform genetic asso- ciation analyses considering log-additive, dominant, and recessive models for both quantitative and qualitative traits. deviations of the observed allele frequencies from those expected under hardy-weinberg equilibrium were tested by the c -test. in case-control analyses, odds ratios for hypertension were calculated using the major allele as reference. when analyzing systolic and diastolic blood pressures as continuous traits, to account for the treatment effect amongst those on anti-hypertensive medications, we used stepped addition of blood pres- sures to treated subjects based on number of drugs a subject was taking. as previously described by others [ ], stepped increments of / mmhg, / mmhg, and / mmhg were added to measured systolic and diastolic blood pressures of treated subjects taking , , and ≥ drug classes, respectively. these stepped incre- ments were chosen to achieve an average increase of / mmhg in treated subjects. analyses for blood pressure as a continuous trait were also performed excluding subjects on antihypertensive medications. p-values < . were deemed as significant after adjustment for multiple tests. one-way anova was conducted to assess if there was a difference in the blood pressure among all genotypes for each snp. multiple linear regression analyses were carried out with systolic and diastolic blood pressures as continuous dependent variables and multiple logistic regression with hypertension as categorical dependent variable. snps were represented as categorical variables either as minor allele or as homozygous genotype of minor allele in both logistic regression and linear regression analyses. other independent variables included gender, age, bmi, and creatinine in the same model. power calculations were performed using the pro- gram quanto v . (james gauderman, university of southern california, usa) [ ]. power for unmatched case-control (case-control ratio = : ) studies was esti- mated using actual allelic frequencies of tested genetic markers ranging from . to . , a log-additive model, a disease prevalence of % and a type error rate of . . sample sizes were estimated for genetic effect sizes (odds ratios) of . to . with the log-additive model. due to different minor allele frequencies (mafs) for different snps, we listed the minimal effect size (odds ratio) that could be detected with the avail- able sample size for the qualitative trait analyses (additional file : table s ). to estimate power in a continuous trait analysis, systolic and diastolic blood pressures were used as a continuous outcome for inde- pendent subjects, with an additive model and a type error rate of . and an effect size of % variance. a sample size of was needed to detect a difference of mmhg of blood pressure. additional material additional file : table s . genotype distribution of all snps in cases and controls. table s - quantitative trait analyses. table s - quantitative trait analyses in normotensive and hypertensive subjects. table s - list of single-nucleotide polymorphisms (snps) genotyped. table s - results of current study in comparison to study by adeyemo et. al. table s - power calculation of effect size detectable based on minor allele frequency in case-control analyses. abbreviations nhanes: national health and nutrition examination survey; gwas: genome wide association study; snp: single nucleotide polymorphisms; wtccc: wellcome trust case control consortium; pms : post-meiotic segregation increased ; ipo : importin ; cacana h: calcium channel voltage- dependent: t type: alpha h subunit; bmi: body mass index; aldh a : aldehyde dehydrogenase family member a ; ywhaz: tyrosine - monooxygenase/tryptophan -monooxygenase activation protein zeta polypeptide; fbpp: family blood pressure program; dna: deoxyribonucleic acid; stk : serine threonine kinase ; cdh : cadherin - heart; charge: cohorts for heart and aging research in genomic epidemiology; global bpgen: global blood pressure genetics; care: candidate gene association resource; p ha : prolyl -hydroxylase alpha polypeptide ii; adh : alcohol dehydrogenase (class iv) mu or sigma polypeptide; htn: hypertension; chr: chromosome; rs: reference snp number; slc a : solute carrier family (na/ca/k exchanger) member ; sv b: synaptic vesicle glycoprotein b; nrxn : neurexin ; abcc : atp-binding cassette (abc) sub- family c (cftr/mrp): member ; prc : protein regulator of cytokinesis ; sbp: systolic blood pressure; dbp: diastolic blood pressure; maf: minor allele frequency. acknowledgements the authors would like to thank brian stogsdill for technical assistance this work was supported by national institutes of health grants hl and - m -rr- (general clinical research center) and scor grant p hl- . kidambi et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of http://www.biomedcentral.com/content/supplementary/ - - - -s .docx author details department of medicine, medical college of wisconsin, milwaukee, wi , usa. division of endocrinology, clement j. zablocki va medical center, milwaukee, wi , usa. department of pediatrics, medical college of wisconsin, milwaukee, wi , usa. institute for health and society, medical college of wisconsin, milwaukee, wi , usa. department of physiology, medical college of wisconsin, milwaukee, wi , usa. authors’ contributions sk participated in design the study, data acquisition, interpret and analyze the data and drafted the manuscript. sg helped interpret the data, jk participated in design and data acquisition, cg participated in design and data acquisition, shk helped data analyses and manuscript writing, mk assisted in data acquisition and review of the manuscript, ac participated in design of the study, sp participated in design, analyses and interpretation of the data and drafting manuscript, tk participated in design, data acquisition, interpretation and data analyses and drafted the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. received: august accepted: april published: april references . chobanian av, bakris gl, black hr, cushman wc, green la, izzo jl jr, jones dw, materson bj, oparil s, wright jt jr, et al: seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. hypertension , : - 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(editor ed.^eds.) , pp. a computer program for power and sample size calculations for genetic- epidemiology studies city; :a computer program for power and sample size calculations for genetic-epidemiology studies. pre-publication history the pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/ - / / /prepub doi: . / - - - cite this article as: kidambi et al.: non-replication study of a genome- wide association study for hypertension and blood pressure in african americans. bmc medical genetics : . kidambi et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract 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/ / /prepub 	abstract 	background 	methods 	results 	conclusions 	background 	results 	hardy-weinberg equilibrium 	case-control association analyses 	quantitative trait analyses 	regression analyses 	discussion 	conclusions 	methods 	subjects and measurements 	selection of snps and genotyping 	statistical methods 	acknowledgements 	author details 	authors' contributions 	competing interests 	references 	pre-publication history genotype-based changes in serum uric acid affect blood pressure genotype-based changes in serum uric acid affect blood pressure afshin parsa , eric brown , matthew r. weir , jeffrey c. fink , alan r. shuldiner , braxton d. mitchell and patrick f. mcardle division of nephrology, university of maryland school of medicine, baltimore, maryland, usa and division of medicine, university of maryland school of medicine, baltimore, maryland, usa elevated serum levels of uric acid consistently correlate with hypertension, but the directionality of the association remains debated. to help define this relationship, we used a controlled setting within a homogeneous amish community and the mendelian randomization of a nonsynonymous coding single-nucleotide polymorphism, rs (val ile), in the slc a gene. this gene expresses the glut transporter that also transports uric acid and is associated with lower serum uric acid levels. we studied the unconfounded association between genotype and blood pressure in amish adults, each placed for days on standardized diets, first with high sodium, followed by low sodium, with an intervening washout period. blood pressure, measured using -h ambulatory monitoring, during both diet periods was used as the primary outcome. all participants were free of diuretic or other antihypertensive medications and the relationships between glut genotype and both serum uric acid and blood pressure were assessed. each copy of the glut minor ile allele was found to confer a significant . mg/dl reduction in serum uric acid and was associated with a significant mean decrease in the systolic blood pressure of . and . mm hg on the high- and low- sodium diet, respectively. thus, a mendelian randomization analysis using variants in the glut gene indicates that a decrease in serum uric acid has a causal effect of lowering blood pressure. kidney international ( ) , – ; doi: . /ki. . ; published online december keywords: blood pressure; hypertension; polymorphisms; proximal tubule an association between serum uric acid (ua) and blood pressure (bp) has long been established; over years have passed since the observation that % of untreated hypertensive patients were hyperuricemic. numerous large-scale observational studies have shown persistent independent associations between serum ua and bp, as well as with various other cardiovascular (cv) outcomes. – however, the complex interplay among metabolic syndrome components, as well as renal function, has produced strong debate about the causality and directionality of the observed associations. indeed, although ua initially gained much attention as an important mediator of vascular disease, it subsequently was felt to be a secondary marker of disease and was dropped from standard metabolic panels. more recently, there has been resurgence in the interest of the likely causal association between serum ua and a variety of cv-related outcomes, most notably hypertension. for example, both animal and human studies have shown that the presence of hyperuricemia precedes the development of hypertension and that, after adjusting for multiple independent risk factors, hyperuricemia carries an increased relative risk of developing hypertension. , – however, these studies, although often providing compelling arguments, are not sufficient to fully resolve concerns regarding the temporality of ua change along with more subtle potential confounders and bp. there have also been some small trials showing an improvement in bp, c-reactive protein, and renal function in response to treatment with allopurinol. , however, it remains unclear whether these benefits were related to the ability of allopurinol to inhibit xanthine oxidase and/or lower serum ua. although diet is well known to modulate serum ua concentration, genetic variability is also significantly asso- ciated with serum ua levels. the gene most strongly associated with ua levels resides on chromosome and codes for glut (also known as slc a ), a recently discovered key urate transporter. genetic variants of glut have been strongly associated with reduced levels of serum ua in several caucasian cohorts, including ours, – and in non-caucasian cohorts. – specifically, in our cohort, out of a total of genotyped single-nucleotide poly- morphisms (snps), a missense variant that codes for a val to o r i g i n a l a r t i c l e http://www.kidney-international.org & international society of nephrology received march ; revised august ; accepted october ; published online december correspondence: afshin parsa, division of nephrology, university of maryland school of medicine, west baltimore street, mstf , baltimore, maryland , usa. e-mail: aparsa@medicine.maryland.edu kidney international ( ) , – http://dx.doi.org/ . /ki. . http://www.kidney-international.org mailto:aparsa@medicine.maryland.edu ile amino-acid substitution at position on the glut gene (glut val ile) was most significantly associated with a reduction in ua concentration of b . mg/dl in women and b . mg/dl in men per copy, and is associated with increased renal clearance of ua. , the discovery of the aforementioned glut val ile variant allows for an opportunity to study whether ua levels are, in fact, causal of changes in bp, by using a method known as mendelian randomization. the mendelian randomization principle relies on the tenet that alleles, and hence genotypes, are randomly assigned during gamete formation. the main advantage of this method is that gamete formation occurs before birth and is therefore unaffected by traditional confounders that occur after conception, such as diet, socioeconomic status, access to health care, and all other environmental factors. because relationships between geno- types and outcomes have only limited susceptibility to confounding and are not subject to reverse causality, genetic variation may be used to establish directionality and infer causality between a certain gene product and a specific outcome. therefore, mendelian randomization is akin to a randomized trial design with genetic variation used as a proxy for an exposure that leads to a given outcome. in a theoretical randomized trial design, inheritance of the glut ile allele would be analogous to randomly being assigned probenecid, a uricosuric agent, from birth, whereas inheritance of the wild-type genotype would be analogous to receiving placebo. in this paper, we set out to determine whether exposure to a genotype-associated lowering in serum ua concentration, while on standardized prepared high- and low-salt diets, and free of any diuretic or other antihyperten- sive medication use, underlies decreases in mean -h bp measurements. results sample characteristics the participants of the hapi (hereditary and phenotype intervention) heart study ranged in age from to years at the time of the study, and % were men. table gives the baseline clinical characteristics of the sample. association of the glut val ile genotype with serum ua level we previously performed a genome-wide association study of serum ua and found snps within the glut gene to be most strongly associated with serum ua. included in the genome-wide association study were glut variants. however, as these snps were not viewed as functional, we then identified four nonsynonymous snps within glut and genotyped those to see whether a putative snp could be identified. of these, a missense snp rs (val ile) in exon proved to be the best associated marker and the only independently associated one, when included with the other snps in the same regression model. on the basis of these findings, we selected this variant for our mendelian randomization analysis. no deviation from hardy–weinberg equilibrium was noted. the glut val ile genotype was strongly associated with serum ua levels (table ), and accounted for . % of the variance in our population. among homozygotes for the ile allele (ile/ile), serum ua levels were nearly mg/dl lower than for those with the wild- type (val/val) genotype; each copy of the ile allele conferred an b . mg/dl reduction in ua (p¼ . � � ). con- sistent with previous reports, , , , , this effect was more pronounced in women than in men (data not shown). this strong association between genotype and phenotype is consistent with previous evidence and supports the first condition for the use of mendelian randomization, as a weak genotype effect would otherwise yield low power to detect any effect. association of serum ua levels with bp consistent with previous studies, we found serum ua levels to be correlated with multiple measurements of bp, metabolic syndrome factors, and renal function. both our bp and serum ua measurements were approximately normally distributed and did not require normalization. regression coefficients and p-values from this analysis are shown in table . after standardized bp was taken on the initial clinic visit, subjects began days of a high-salt diet, followed by an - to -day washout period, and then days of a low-salt diet. serum ua levels were positively correlated with multiple measurements of baseline bp (taken during clinic visit one and a home visit following the washout period) and -h ambulatory bp measurements. however, it is important to remember that these associations may not represent valid estimates of true causal effects given that they are subject to both confounding and reverse causality. association of the glut val ile genotype with bp mean values and regression coefficients of baseline and -h bp measurements by genotype are presented in table . the right-most column of table shows the two-stage estimate of table | clinical characteristics (mean (s.d.)) of the old order amish enrolled in the hapi heart study, lancaster county, pennsylvania characteristic men (n= ) women (n= ) age (years) . ( . ) . ( . ) bmi (kg/m ) . ( . ) . ( . ) total cholesterol (mg/dl) . ( . ) . ( . ) triglycerides (mg/dl) . ( . ) . ( . ) sbp (mm hg) . ( . ) . ( . ) dbp (mm hg) . ( . ) . ( . ) diabetes (%) . . current smokers (%)a . . lipid-lowering meds (%)b . . antihypertensive meds (%)b . . abbreviations: bmi, body mass index; dbp, diastolic blood pressure; hapi, hereditary and phenotype intervention; meds, medication; sbp, systolic blood pressure. aindicates use of cigarettes, cigars, and pipes. b medication use assessed at the time of recruitment, before participants were asked to discontinue use, per our study protocol. kidney international ( ) , – a parsa et al.: genotype-based changes in ua affect bp o r i g i n a l a r t i c l e the serum ua–bp association derived from the mendelian randomization approach that uses the glut val ile genotype. it is important to note that our clinic visit one bp measurements (average of three repeated bp measurements at a single visit) were obtained when the individuals were on a liberal diet, and these were not associated with genotype under an additive model (table ). however, when the same participants were on standardized prepared high-salt diets (delivered to the home of each subject), we noted a significant additive genotype-mediated association with our -h mean bp measurements. moreover, this was internally replicated when the same individuals were on standardized low-salt diets where each ile allele was associated with a decrease in bp. our genotype-mediated effect estimate went from null effect (� . mm hg) per ile allele on liberal diet to � . mm hg on low-salt diet and � . mm hg on high- salt diet (table ). our power is over % to identify the observed effect with our given sample size. the potentially confounded associations between serum ua and renal function, body mass index, triglycerides, and glucose were nullified under the genotype-based approach (table ). association of serum ua with salt sensitivity as with bp readings, mean values and regression coefficients of measurements of salt sensitivity are also presented in table . genotype was not associated with salt sensitivity, defined as bp on high-salt diet minus bp on low-salt diet (p¼ . ). it should be noted that our relatively healthy and young caucasian population were mostly non-salt sensitive, further reducing our power for this secondary analysis. we did, however, note an attenuation of the effect of genotype on systolic bp when on a low-sodium vs. high-sodium diet (absolute effect estimate of . vs. . mm hg per ile allele). these results suggest potential effect modification or interaction between dietary salt intake and serum ua effect on bp. a formal test of interaction did not yield significant results; however, we must again acknowledge limited power to detect a statistically significant interaction given the sample size and modest difference in effect estimate size, as related to dietary modification of salt intake. discussion in this study, we used the mendelian randomization principle to show that decreases in serum ua concentration due to a missense snp in the glut gene are directly associated with lower level of bp. the strength of the association between change in serum ua level and systolic bp was significant, with a mg/dl change in serum ua being associated with an b – mm hg change in systolic bp, depending on salt intake. considering that the population distribution of serum ua is much wider than that conferred solely by glut variability, the population effect of ua on bp and cv disease outcomes may be quite substantial. the full spectrum of mechanisms by which glut affects serum ua level continues to be elucidated. it has recently been shown that glut is a urate transporter that localizes to both the apical and basolateral membranes of human renal proximal tubular cells in vitro; different splice variants affect trafficking of the transporter and dictate into which membrane it will be inserted. splice variant localizes to the basolateral membrane and functions as an efflux transporter for ua out of the tubular cell, whereas splice variant is located in the apical membrane and is involved in the rapid influx of ua into the proximal tubular cell. , given the location of these two splice variants, it is reasonable to postulate that glut may alter serum ua concentration table | observational estimates for uric acid adjusted for age and gender with individual phenotypes compared with corresponding genotype-based mean phenotype values and additive model point estimates for each ile allele and corresponding p-value serum ua correlations glut genotype-based approach trait (n= ) estimate (s.e.) p-value ile/ile mean (s.d.) val/ile mean (s.d.) val/val mean (s.d.) effect estimate additive p-value uric acid, mg/dl — — . ( . ) . ( . ) . ( . ) . ( . ) . � � estimated gfr (ml/min) � . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . body mass index (kg/m ) . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . triglycerides . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . glucose . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . clinic visit sbp . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . clinic visit dbp . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . high-salt -hr sbp a . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . high-salt -h dbp a . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . low-salt -h sbpa . ( . ) . � � . ( . ) . ( . ) . ( . ) . ( . ) . low-salt -h dbpa . ( . ) . . ( . ) . ( . ) . ( . ) . ( . ) . salt sensitivity -h sbp � . ( . ) . . ( . ) . ( . ) . ( . ) . ( . . salt sensitivity -h dbp . ( . ) . � . ( . ) � . ( . ) � . ( . ) � . ( . ) . abbreviations: ci, confidence interval; dbp, diastolic blood pressure; gfr, glomerular filtration rate; sbp, systolic blood pressure; ua, uric acid. estimates of serum ua correlations are based on mg/dl change in ua. no significant difference was noted between high- and low-salt diet sbp measures. all blood pressure measurements are given in mm hg. a the -h blood pressure measurements were available in a subset of of participants. bold indicates p-valueo . . kidney international ( ) , – o r i g i n a l a r t i c l e a parsa et al.: genotype-based changes in ua affect bp via regulation of proximal urate reabsorption. the finding that different snps in the glut gene modify fractional excretion of ua strengthens this theory. , , another possible mechanism for the effect of glut on serum ua concentration is via its role in fructose homeostasis. it has been well described that increased fructose levels are associated with increased production of ua. – given that ua transport by glut is facilitated by fructose, it is plausible to suggest that glut may also have a role in calibrating urate concentration in response to fructose. , although the exact pathway by which serum ua is associated with higher levels of bp is yet to be established, previous studies provide several potential mechanisms such as renal vasoconstriction with microvascular disease, , impaired vascular endothelial function or compliance, – and disturbances in sodium and volume homeostasis. , in an animal model, rats were rendered hyperuricemic by administration of the uricase inhibitor oxonic acid for weeks while on a low-salt diet; control animals were maintained on the same low-salt diet but did not receive oxonic acid. while still on a low-salt diet, the oxonic acid was then discontinued for weeks to allow the serum ua levels to return to normal. the rats were then randomized to a high- or low-salt diet to assess for salt sensitivity. only the rats that were previously hyperuricemic demonstrated an increase in bp on the high-salt diet (that is, salt sensitivity). in agreement with these findings, we observed suggestive evidence that a high-salt diet may accentuate the relationship of serum ua on bp. further investigation in humans will need to be performed in order to determine whether elevations in serum ua increase are associated with salt sensitivity in various populations. although the number of subjects studied here is relatively modest, there are several notable strengths to our study that should be discussed. foremost, all participants were on a standardized prepared diet before obtaining bp measure- ments, including an equivalent amount of dietary protein and carbohydrates. as the daily variability in serum ua in a noncontrolled setting is on the same order as that of the glut allele, an uncontrolled dietary setting could sig- nificantly attenuate findings associated with a modest increase in serum ua. second, we used -h averaged ambulatory bp measurements on high- and low-salt diets. in contrast, our regular standardized clinic bp measurements on a liberal diet did not demonstrate a statistically significant association between genotype. moreover, we have repeated -h bp measurements in all participants, showing internal replication. third, our culturally isolated founder population is quite homogeneous and not susceptible to population stratification bias. fourth, potential gene-by-environment interactions that are not controlled for in standard popula- tion-based studies (for example, salt and fructose) are accounted for in our experimental setting through our fixed-diet protocol. fifth, our population is relatively young and healthy, which may be more sensitive to vascular effects of serum ua compared with older population with more vascular disease burden. , finally, we used direct genotyp- ing of glut as opposed to imputation, further minimizing potential measurement error. in short, limitations related to potential uncontrolled effect modifiers, interactions, and measurement error as noted in our liberal diet and single- visit clinic-based bp measurements have been minimized by the nature of our selected population and controlled setting. not surprisingly, combining large number of heterogeneous population cohorts in an uncontrolled setting to explore the association between glut genotype and bp, where these limitations could not be mitigated, yielded null results, comparable to our findings when the participants were on a liberal diet along with similar single-visit standardized bp measurement. , in summary, a genetic variant strongly associated with lower levels of serum ua is also associated with decreases in bp, when tested in an experimental setting. the evidence from our mendelian randomization approach implies that this association is causal in nature, corroborating all the previous studies that strongly inferred a causal association between serum ua and bp. this study also highlights the potential importance of a controlled setting and homogenous population when examining secondarily mediated gene effects (for example, effect of genotype on ua and then secondarily on bp). these findings add impetus to the possible benefit of reduced serum ua control in hyperuri- cemic patients with cv disease risk factors, or established disease, and suggest that larger clinical trials of serum ua reduction are well warranted. materials and methods study sample the hapi heart study began recruitment in with the goal of identifying genes that interact with environmental exposure to alter risk of cv disease. the study was carried out in an old order amish community in lancaster county, pennsylvania, and the full hapi study included individuals aged x years who were relatively healthy. details of the study aims and recruitment procedures have been previously described. in brief, exclusion criteria included severe hypertension (bp / mm hg), malignancy, and kidney, liver, or untreated thyroid disease. the study protocol was approved by the institutional review board at the university of maryland school of medicine, and informed consent was obtained from each study participant. baseline clinical and serological measurements were obtained during an initial clinic visit at the amish research clinic in strasburg, pennsylvania. baseline clinic bp was measured in triplicate using a standard sphygmomanometer in the sitting position after min of rest, and the average of the three measurements was used for analysis. fasting blood samples were also drawn at this time. serum ua levels drawn at the screening exam were assayed by quest diagnostics (baltimore, md) and measured to the nearest . mg/dl. biochemical measurements relevant to this study include basic metabolic and lipid panels (quest laboratory, horsham, pa). dietary salt intervention. the dietary salt intervention study began immediately following the clinic visit and was completed in a subsample of individuals. study subjects were put on a high- sodium diet ( meq per day) for days, which is close to their customary diet, and then after a - to -day washout period, kidney international ( ) , – a parsa et al.: genotype-based changes in ua affect bp o r i g i n a l a r t i c l e were put on a low-sodium diet ( meq per day) for days. the potassium level was held constant at meq per day during both diets, as was the daily protein content at g and the total carbo- hydrate content at g. all meals were prepared in a specially outfitted kitchen and delivered to the home of the subjects. measurement of -h ambulatory bp. the -h bp measure- ments were recorded at -min intervals by an ambulatory bp monitor worn by the subjects on the last day of each diet. salt sensitivity was expressed on a continuous scale as the absolute difference between the average -h bp during the high- and low- salt diet. we used the mean of all recorded bp measurements during each -h period as our outcome measure. specific protocols for the hapi heart study measurements have been published. genotyping of the glut gene. participants were genotyped for the rs snp (val ile) using taqman snp genotyping assays (applied biosystems, foster city, ca) according to the standardized manufacturer’s protocol. replicate genotyping in % of the samples resulted in a % concordance rate. analytical approach mendelian randomization. the mendelian randomization approach exploits the fact that genotype precedes life events and is therefore not affected by lifestyle factors. as previously reviewed, mendelian randomization is an application of instrumental variable analysis, and with certain assumptions the genotype–phenotype relation can be used to attain unconfounded estimates of the relationship between the gene product and outcomes of interest. these assumptions include an adequately strong relationship between genotype and phenotype and the absence of alternate pathways from genotype to the outcome of interest (for example, pleiotropy, population stratification, linkage disequilibrium). the first assump- tion, a strong relation between genotype and phenotype, has been previously demonstrated for the snp rs on the glut gene (glut val ile) and serum ua levels. – , with regard to population stratification, our sample population of old order amish individuals is extremely homogenous with no evidence of population stratification. we also considered the possibility of pleiotropic effect of glut on hexose (that is, glucose/fructose) transport as a potential source of confounding. however, given that glut is one of the principle high-capacity ua transporters and a very low- capacity fructose transporter ( – -fold increase in ua transport capacity compared with glucose/fructose), it seems highly unlikely that fructose could account for these findings. , considering the low capacity for fructose transport, the effect of fructose would have to be unrealistically strong to mediate a noticeable change in phenotype. a more plausible scenario is that fructose, by altering the transport rate of ua by glut , , may account for some of the noted association between fructose intake and elevations in ua. , given the missense nature of this highly conserved snp, predicted exonic splicing enhancer function (http://compbio.cs.queensu.ca/ f-snp/), consistent strong linear association of glut snps with serum ua in numerous cohorts and ethnicities, and demonstrated role of glut as a major urate transporter, we are comfortable assuming that the associations noted in our result section are not secondary to linkage disequilibrium with another gene or other violations of the assumptions underlying mendelian randomization. statistical methods the principle of mendelian randomization is illustrated by con- trasting the correlations observed between serum ua levels and bp risk factors without consideration for the glut (val ile) genotype with those obtained between serum ua levels and bp risk factors using a two-stage approach that uses the genotype–out- come and genotype–ua regressions. the simple (potentially confounded) estimates were obtained by regressing serum ua levels (the independent variable) on each bp risk factor (the dependent variable) separately, after adjusting for age and gender. many of the participants of the hapi heart study are related. to address the correlations potentially existing in phenotype by virtue of the fact that subjects are related, we accounted for residual familial correlations in phenotype using a variance component regression framework. specifically, we modeled variation in the trait as a function of fixed covariates, a polygenic component, and a normally distributed error component. the polygenic component was derived from the kinship coefficient matrix, which describes the relationship of each pair of individuals in the sample. the software packages solar (southwest foundation for biomedical research, san antonio, tx) and mmap (university of maryland school of medicine, baltimore, md) were used for the analyses. disclosure all the authors declared no competing interests. acknowledgments this work supported by the nih research grant u hl and nih k rr (university of maryland mcrdp), the university of maryland general clinical research center grant m rr , and the mid-atlantic nutrition and obesity research center (p dk ). we thank the staff at the amish research clinic for their outstanding efforts and our amish research volunteers for their long- standing partnership in research. we also thank dr edward weinman for his thoughtful insights and review of the manuscript. references . cannon pj, stason wb, demartini fe et al. hyperuricemia in primary and renal hypertension. n engl j med ; : – . . fang j, alderman mh. serum uric acid and cardiovascular mortality the nhanes i epidemiologic follow-up study, - . national health and nutrition examination survey. jama ; : – . . mellen pb, bleyer aj, erlinger tp et al. serum uric acid predicts incident hypertension in a 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nakagawa t. the effect of fructose on renal biology and disease. j am soc nephrol ; : – . kidney international ( ) , – a parsa et al.: genotype-based changes in ua affect bp o r i g i n a l a r t i c l e 	genotype-based changes in serum uric acid affect blood pressure������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 	results������������������������������������� 	discussion���������������������������������������������� 	disclosure���������������������������������������������� 	acknowledgments������������������������������������������������������������� 	references���������������������������������������������� s jra .. research paper introduction to the special issue on the economics of religion jared rubin chapman university, argyros school of business & economics, argyros school of business & economics, one university drive, orange, california, - , usa corresponding author. e-mail: jrubin@chapman.edu (received july ; revised july ; accepted july ) the economics and political science of religion have blossomed into full-fledged fields in the last decade and a half. what was once a field on the far outskirts of economics and political science now regularly publishes in its top journals (see figure ). by , the field was large enough for iannaccone ( ) to write a survey of the shape of the field. the field was very much at its infancy at that time, and most of the best work was done by sociologists and/or published in sociology journals. this has changed significantly in the years since iannaccone’s survey. while the sociology of religion is still thriving as a field, the economics and political science of religion has become a distinct and impactful field of its own. institutionally, this change is mirrored by the growth of the asrec (association for the study of religion, economics, and culture) annual meetings. asrec, founded by iannaccone in , met for a few sessions at the annual meetings of the society for the scientific study of religion until . since then, it has held its own annual meetings, and it is mainly attended by economists and political scientists. the meetings have anywhere between and papers presented, most of top quality. indeed, most of the data points in figure were presented at asrec. there have been a number of sources of growth in the economics and political science of religion, but two stand out: economic history and islam. researchers in both areas have been active enough to warrant surveys of the specific fields within the economics of religion. kuran’s ( ) “islam and economic performance: historical and contemporary links” overviews recent works in the economics of islam, while becker et al.’s ( ) “religion in economic history: a survey” analyzes recent work done on the economic history of the three major abrahamic faiths. these trends were also noted in iyer’s ( ) survey updating iannaccone’s ( ) survey. some of these trends have to do with data availability, advanced econometric techniques, ocr, and the capacity to store massive amounts of data. there is simply much more we can say about religion, religious activity, and their interactions with economic and political phenomena than we could years ago. indeed, at least half of the articles in this special issue would have been exceedingly difficult, if not impossible, to write years ago. i believe there is a generational element to it, as © université catholique de louvain a reference list of the papers in figure is available at my website, https://www.jaredcrubin.com/links. journal of demographic economics ( ), , – doi: . /dem. . terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /dem. . downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core https://orcid.org/ - - - mailto:jrubin@chapman.edu https://www.jaredcrubin.com/links https://www.jaredcrubin.com/links https://www.cambridge.org/core/terms https://doi.org/ . /dem. . https://www.cambridge.org/core well. as the pioneers in the field—larry iannaccone, roger finke, rodney stark, eli berman, avner greif, rachel mccleary, robert barro, and timur kuran—showed that it was possible for the very best work to be well published and influential, graduate students slowly caught on to the fact that they could make a career out of studying the economics or political science of religion. i was one of those students (i received my ph.d. in ). the next generation of students—including, but certainly not limited to, sriya iyer, daniel hungerman, sascha becker, chris bader, mark koyama, noel johnson, jeanet bentzen, and jean-paul carvalho—built on the success of their predecessors, publishing important work while also helping to build the field. this has become a self-perpetuating cycle. the demand to study the economics and political science of religion has always been there for students. the problem used to be that it was an extremely risky career move. now, however, as more and more graduate students see the field as a path to a successful career, many of the best young minds are entering the field. in short, the state of the field is as strong as ever. one area in which relatively little recent work has been done in the economics and political science of religion is demography. there has certainly been some excellent work done in the economic demography of religion; iyer’s ( ) book demography and religion in india comes to mind. yet, the field as a whole has not taken off. this is a bit surprising, since demography was long one of the primary areas of study when the field was located closer to the sociology of religion. the effect of religion on family, fertility, gender, migration, and so on has long been a source of fascination. and with good reason. demographic characteristics are among the most important and direct mechanisms through which religion impinges on daily figure . economics and political science of religion articles published in top journals, – . jared rubin terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /dem. . downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core https://www.cambridge.org/core/terms https://doi.org/ . /dem. . https://www.cambridge.org/core life. there are so many unanswered, important questions out there that volumes could be written. this special issue is an attempt to begin to fill this void. this special issue contains seven articles, all of which are of extremely high quality. to be honest, i did not know what to expect when i agreed to edit this issue. i knew that there was a lot of work that could (and should) be done at the intersection of religion, demography, and economics or political science, but i was not sure how much was out there. to say that i was pleasantly surprised by the quality of submissions would be an understatement. the seven articles in this issue are all of the highest quality; some of them could have easily been published in the top journals in their discipline. they are all the types of articles i love: they address important questions while opening up many new ones. julia cagé and valeria rueda’s “sex and the mission: the conflicting effects of early christian investments on sub-saharan africa’s hiv epidemic” addresses an incredibly important issue: what is responsible for the spread and persistence of hiv in sub-saharan africa? hiv/aids has contributed to a demographic crisis on the continent; understanding its causes is of first-order importance. while there are many socio-economic causes, cagé and rueda look at the important historical legacy of religious missionaries. they hypothesize that missionaries may have had two counter-vailing effects that are still with us in the st century. on the one hand, they promoted the spread of religious norms that discouraged safe sex. on the other hand, they built health clinics and the health institutional infrastructure persisted. cagé and rueda find evidence in favor of both mechanisms; they sometimes offset, they sometimes do not. it is an important work revealing how historical legacies continue to impinge on health, demographic, and economic outcomes. two related papers that also address historical demographic issues are sascha o. becker and francesco cinnirella’s “prussia disaggregated: the demography of its universe of localities in ” and ran abramitzky and hanna halaburda’s “were jews in interwar poland more educated?” both of these papers are somewhat descriptive in nature, revealing the demographic patterns of different religious groups late- th and early- th century europe. one fascinating finding common to both is that the relationship between religion and education can switch depending on the urban–rural divide of adherents. indeed, it is possible to have a simpson’s paradox: abramitzky and halaburda find that urban polish jews are (slightly) less literate than urban christians and rural jews were less literate than rural christians, but jews as a whole were more literate than christians. the reason is that jews were so much more urban and urbanites had much higher literacy rates. becker and cinnirella find similar results in th century prussia. these results should encourage much more work. existing work on jewish and protestant literacy tends not to be disaggregated. these papers suggest that this may lead to misleading results. lisa blaydes and melina platas’s fascinating paper, “religion, family structure, and the perpetuation of female genital cutting in egypt,” explores the role religious leaders have played in slowing the spread of female genital cutting in egypt. comparing muslims and (christian) copts, they find that anti-fgc attitudes tended to become more common over time in both communities, but the rate of decline was much sharper among copts. blaydes and platas find that attitudes of religious leaders help explain these differences. more coptic leaders have come out against fgc, and more coptic women believe that their religious leaders do not favor fgc. these are important findings. they reveal the power that religious leaders can have to shape the attitudes of their members. journal of demographic economics terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /dem. . downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core https://www.cambridge.org/core/terms https://doi.org/ . /dem. . https://www.cambridge.org/core sriya iyer and melvyn weeks present a new theory, supported by a data analysis, for how to think about fertility and religion/ethnicity in “social interactions, ethnicity, religion and fertility in kenya.” they argue that ethnic spillovers swamp out religious and many other effects when it comes to fertility decisions. what is meant here by spillovers is that the fertility choices of one person tend to be influenced by what their co-ethnics choose. this is an important insight, and iyer and weeks provide an econometric methodology for teasing out the “direct” effects of ethnicity from the spillover effects. eli berman and zaur rzakhanov present another paper at the intersection of fertility and religion in “fertility, migration, and altruism.” their paper attempts to provide an explanation for why early jewish migrants to israel had much higher fertility rates (nearly one child per family!) than post-soviet immigrants. there are two possible reasons for this difference: selection (the two cohorts were different for some reason) or a behavioral response to the new environment (once in israel). berman and rzakhanov are able to decompose the two effects and they find that nearly all of the differences between the two cohorts can be explained by selection. they provide highly suggestive evidence that differences in intergenerational altruism can explain this result: earlier cohorts had greater levels of altruism, which in turn encouraged them to seek a better life for their offspring. the final paper, choon wang’s “religious prohibition and sacrifice: evidence from the amish restriction on high school education” harkens back to the canonical models in the economics of religion [especially iannaccone ( )]. wang asks why the amish have major restrictions on secondary education (past th grade), and whether these restrictions serve the welfare-enhancing purpose often found in religious groups. he presents a theory which shows how education restrictions can be welfare-enhancing by inducing those with the highest shadow price of religious investment (i.e., those with profitable outside opportunities) to leave the group. he tests this theory with an empirical analysis that exploits the u.s. supreme court decision in wisconsin vs. yoder, which exempted amish children from compulsory high school education. this decision made it possible for the amish to use education reduction as a screening mechanism. wang finds that the amish (who remained amish) who were of high school age after the decision in fact have lower levels of education, higher fertility, and lower earnings. these are all wonderful papers. it is an honor to have them in the special issue, and i hope they are only the beginning of what should be important research programs. i thank david de la croix and murat iyigun for allowing this special issue to happen. i did not know what to expect coming in, but i am thrilled with the end result (and i hope they are too!). i would also like to thank sébastien schillings for excellent and responsive copy-editing. the entire team at the journal of demographic economics has been excellent and supportive throughout the process. i hope you enjoy reading the articles in the special issue as much as i did. references becker, sascha o., jared rubin and ludger woessmann ( ) religion in economic history: a survey. in alberto bisin, and giovanni federico (eds.), handbook of historical economics. new york: elsevier, forthcoming. iannaccone, laurence r. ( ) sacrifice and stigma: reducing free-riding in cults, communes, and other collectives. journal of political economy ( ), – . jared rubin terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /dem. . downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core https://www.cambridge.org/core/terms https://doi.org/ . /dem. . https://www.cambridge.org/core iannaccone, laurence r. ( ) introduction to the economics of religion. journal of economic literature ( ), – . iyer, sriya ( ) demography and religion in india. oxford: oxford university press. iyer, sriya ( ) the new economics of religion. journal of economic literature ( ), – . kuran, timur ( ) islam and economic performance: historical and contemporary links. journal of economic literature ( ), – . cite this article: rubin j ( ). introduction to the special issue on the economics of religion. journal of demographic economics , – . https://doi.org/ . /dem. . journal of demographic economics terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /dem. . downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core https://doi.org/ . /dem. . https://www.cambridge.org/core/terms https://doi.org/ . /dem. . https://www.cambridge.org/core 	introduction to the special issue on the economics of religion 	references the effect of burns & wounds (b&w)/burdock leaf therapy on burn-injured amish patients | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / corpus id: the effect of burns & wounds (b&w)/burdock leaf therapy on burn-injured amish patients @article{kolacz theeo, title={the effect of burns & wounds (b&w)/burdock leaf therapy on burn-injured amish patients}, author={n. kolacz and mark t. jaroch and monica l bear and r. hess}, journal={journal of holistic nursing}, year={ }, volume={ }, pages={ - } } 	n. kolacz, mark t. jaroch, + author r. hess 	published 	medicine 	journal of holistic nursing purpose: the purposes of this pilot study were to measure pain associated with dressing changes, assess the presence of infection, and document healing times of burn-injured amish in central ohio using an herbal therapy consisting of burns and wounds™ ointment (b&w) and burdock (arctium ssp.) leaves. b&w contains honey, lanolin, olive oil, wheat germ oil, marshmallow root, aloe vera gel, wormwood, comfrey root, white oak bark, lobelia inflata, vegetable glycerin, bees wax, and myrrh. design: a… expand view on sage ahna.org save to library create alert cite launch research feed share this paper citationsbackground citations view all tables and topics from this paper 	 table 	 table 	 table 	burn injury 	lanolin 	glycerin 	lobelia inflata 	wheat germ oil 	plant leaves 	bees 	community 	aloe vera gel 	olive oil 	burdock root extract 	phytotherapy 	wounds and injuries 	sessile serrated adenoma/polyp 	ointments 	lobelia spicata leaf extract 	wounds, stab 	waxes 	symphytum uplandicum leaf extract 	aloe vera plant paper mentions news article how to use natural comfrey plant to heal bones and sports injuries faster (video) emaxhealth.com may citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency the management of paediatric burns with burns and wounds ointment and burdock leaves: a case series. 	shelby l. nathan, autumn d nanassy, b. burkey, w. davis, p. glat 	medicine 	journal of wound care 	 save alert research feed honey-based salve and burdock leaf dressings as an alternative to surgical debridement of a traumatic wound eschar 	adam schell, jonathan copp, k. bogie, r. wetzel 	medicine 	advances in wound care 	 	pdf save alert research feed histopathological evaluation of burdock ( arctium lappa ) root hydroalcoholic extract on wound healing 	f. ghorat, m. azizkhani, shahriar naji, a. g. ranjbary, farzad doostishoar 	medicine 	 	 	pdf save alert research feed amish and mennonite lay caregivers' experiences using the b&w burdock leaf treatment on burns and wounds: a qualitative study 	r. hess 	medicine 	 	 	pdf save alert research feed amish culture and their utilization of burns and wounds ointment for the treatment of burns. 	krystal melich trinkle 	medicine 	holistic nursing practice 	 	 save alert research feed wound healing and the use of medicinal plants 	aleksandra shedoeva, d. leavesley, zee upton, chen fan 	medicine 	evidence-based complementary and alternative medicine : ecam 	 	 	pdf save alert research feed traditional and ethnobotanical dermatology practices in romania and other eastern european countries. 	m. gilca, g. s. tiplica, c. sălăvăstru 	geography, medicine 	clinics in dermatology 	 	 save alert research feed bone regeneration by homeopathic symphytum officinale 	shahla vaezi, h. m. haghighi, s. farzad, sepideh arabzadeh, f. kalalinia 	chemistry 	regenerative engineering and translational medicine 	 save alert research feed application of predictive nursing care in elderly patients with fractures that underwent total hip arthroplasty procedures 	s. wang, yan qiu 	 	pdf save alert research feed oral health and medical conditions among amish children 	m. heima, m. harrison, p. milgrom 	medicine 	journal of clinical and experimental dentistry 	 	 	pdf 	view excerpt, cites background save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency evaluation of banana leaf dressing for partial thickness burn wounds. 	m. gore, d. akolekar 	medicine 	burns : journal of the international society for burn injuries 	 	 save alert research feed a multinational survey of the assessment of pain when removing dressings richard white 	r. white 	medicine 	 	 save alert research feed effectiveness of aloe vera gel compared with % silver sulphadiazine cream as burn wound dressing in second degree burns. 	m. shahzad, n. ahmed 	medicine 	jpma. the journal of the pakistan medical association 	 	 	pdf save alert research feed use of a new, flexible lipidocolloid dressing on acute and chronic wounds: results of a clinical study. 	s. meaume, j. pérez, + authors s. bohbot 	medicine 	journal of wound care 	 	 	pdf save alert research feed banana leaf dressing for skin graft donor areas. 	m. gore, d. akolekar 	medicine 	burns : journal of the international society for burn injuries 	 	 save alert research feed the properties of an "ideal" burn wound dressing--what do we need in daily clinical practice? results of a worldwide online survey among burn care specialists. 	h. selig, d. lumenta, m. giretzlehner, m. jeschke, d. upton, l. kamolz 	medicine 	burns : journal of the international society for burn injuries 	 	 save alert research feed banana leaves as an alternative wound dressing 	e. guenova, w. hoetzenecker, + authors m. moehrle 	medicine 	dermatologic surgery : official publication for american society for dermatologic surgery [et al.] 	 	 save alert research feed wound healing activities of different extracts of centella asiatica in incision and burn wound models: an experimental animal study 	j. somboonwong, mattana kankaisre, b. tantisira, m. tantisira 	medicine 	bmc complementary and alternative medicine 	 	 	 save alert research feed effects of aloe vera cream on posthemorrhoidectomy pain and wound healing: results of a randomized, blind, placebo-control study. 	f. eshghi, s. hosseinimehr, n. rahmani, m. khademloo, mohammad sina norozi, omolbanin hojati 	medicine 	journal of alternative and complementary medicine 	 	 	pdf save alert research feed honey in the treatment of burns: a systematic review and meta-analysis of its efficacy. 	m. wijesinghe, m. weatherall, k. perrin, r. beasley 	medicine 	the new zealand medical journal 	 	 	pdf save alert research feed ... ... related papers 	abstract 	tables and topics 	paper mentions 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue [pdf] autosomal recessive epilepsy associated with contactin mutation is different from familial cortical tremor, myoclonus and epilepsy. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /brain/awt corpus id: autosomal recessive epilepsy associated with contactin mutation is different from familial cortical tremor, myoclonus and epilepsy. @article{striano autosomalre, title={autosomal recessive epilepsy associated with contactin mutation is different from familial cortical tremor, myoclonus and epilepsy.}, author={p. striano and f. zara and s. striano and c. minetti}, journal={brain : a journal of neurology}, year={ }, volume={ pt }, pages={ e } } 	p. striano, f. zara, + author c. minetti 	published 	biology, medicine 	brain : a journal of neurology article sir, in a recent interesting paper, stogmann et al. ( ) described a consanguineous egyptian family with autosomal recessively inherited condition featuring focal epilepsy, neuropsychiatric features, borderline cognitive level, and myoclonus. exome sequencing in this family revealed a homozygous deletion (c. _ delg) leading to a frameshift in the coding region of cntn and segregating in the five affected family members. the gene cntn encodes for contactin , a… expand view on pubmed academic.oup.com save to library create alert cite launch research feed share this paper citationshighly influential citations background citations results citations view all topics from this paper 	myoclonus 	epilepsy 	tremor 	contactin 	epilepsies, partial 	cntn gene 	unverricht-lundborg syndrome 	voltage-gated potassium channel 	frameshift mutation function 	open reading frames 	membrane proteins 	homozygote 	glycosylphosphatidylinositols citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency reply: autosomal recessive epilepsy associated with contactin mutation is different from familial cortical tremor, myoclonus and epilepsy. 	e. stogmann, a. zimprich, f. zimprich 	psychology, medicine 	brain : a journal of neurology 	 	highly influenced 	pdf 	view excerpts, cites background and results save alert research feed intronic atttc repeat expansions in stard in familial adult myoclonic epilepsy linked to chromosome 	m. corbett, thessa kroes, + authors j. gécz 	biology, medicine 	nature communications 	 	 	pdf save alert research feed unravelling the enigma of cortical tremor and other forms of cortical myoclonus. 	a. latorre, l. rocchi, + authors k. bhatia 	biology, medicine 	brain : a journal of neurology 	 	 	pdf 	view excerpt, cites background save alert research feed cell adhesion molecules 	xia-jing tong, yan zou 	biology 	 save alert research feed advances in membrane proteins: building, signaling and malfunction 	yu cao, xian-cheng jiang, y. chen, y. xia 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency autosomal dominant cortical tremor, myoclonus and epilepsy: many syndromes, one phenotype 	p. striano, f. zara, s. striano 	biology, medicine 	acta neurologica scandinavica 	 	 	view excerpts, references results and background save alert research feed familial cortical myoclonic tremor with epilepsy: a single syndromic classification for a group of pedigrees bearing common features 	a. van rootselaar, i. n. van schaik, a. v. d. van den maagdenberg, j. koelman, p. callenbach, m. tijssen 	psychology, biology 	movement disorders : official journal of the movement disorder society 	 	 save alert research feed familial adult myoclonic epilepsy: recognition of mild phenotypes and refinement of the q locus. 	d. crompton, l. sadleir, + authors i. scheffer 	medicine 	archives of neurology 	 	 	pdf 	view excerpt save alert research feed familial cortical myoclonic tremor with epilepsy (fcmte): clinical characteristics and exclusion of linkages to q and p in a large french family. 	e. magnin, m. vidailhet, + authors p. labauge 	medicine 	revue neurologique 	 	 	view excerpt, references background save alert research feed recessive symptomatic focal epilepsy and mutant contactin-associated protein-like . 	k. strauss, e. puffenberger, + authors d. morton 	medicine 	the new england journal of medicine 	 	 	view excerpts, references background and results save alert research feed life‐threatening status epilepticus following gabapentin administration in a patient with benign adult familial myoclonic epilepsy 	p. striano, a. coppola, f. madia, m. pezzella, f. zara, s. striano 	medicine 	epilepsia 	 	 	 	view excerpt, references background save alert research feed exacerbation of partial seizures and onset of nonepileptic myoclonus with carbamazepine 	a. dhuna, a. pascual-leone, d. talwar 	psychology, medicine 	epilepsia 	 	 	view excerpts, references background save alert research feed when antiepileptic drugs aggravate epilepsy 	p. genton 	medicine 	brain and development 	 	 	view excerpts, references background save alert research feed cortical tremor 	a. ikeda, r. kakigi, n. funai, r. neshige, y. kuroda, h. shibasaki 	psychology, medicine 	neurology 	 	 	view excerpt, references background save alert research feed autosomal recessive cortical myoclonic tremor and epilepsy: association with a mutation in the potassium channel associated gene cntn 	e. stogmann 	biology 	 	 	highly influential 	view excerpts, references background and results save alert research feed ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue biomed centralbmc medical genetics ss open acceresearch article combinatorial mismatch scan (cms) for loci associated with dementia in the amish jacob l mccauley , daniel w hahs , lan jiang , william k scott , kathleen a welsh-bohmer , charles e jackson , jeffery m vance , margaret a pericak-vance and jonathan l haines* address: center for human genetics research and department of molecular physiology and biophysics, vanderbilt university medical center, nashville, tn, usa, center for human genetics and department of medicine, duke university medical center, durham, nc, usa, joseph & kathleen bryan adrc/division of neurology, duke university medical center, durham, nc, usa and scott & white, temple, tx, usa email: jacob l mccauley - jacob.mccauley@vanderbilt.edu; daniel w hahs - dan.hahs@freelinnovations.com; lan jiang - jiang@chgr.mc.vanderbilt.edu; william k scott - bscott@chg.duhs.duke.edu; kathleen a welsh-bohmer - kwe@duke.edu; charles e jackson - cejackson@swmail.sw.org; jeffery m vance - jeff@chg.duhs.duke.edu; margaret a pericak-vance - mpv@chg.duhs.duke.edu; jonathan l haines* - jonathan@chgr.mc.vanderbilt.edu * corresponding author abstract background: population heterogeneity may be a significant confounding factor hampering detection and verification of late onset alzheimer's disease (load) susceptibility genes. the amish communities located in indiana and ohio are relatively isolated populations that may have increased power to detect disease susceptibility genes. methods: we recently performed a genome scan of dementia in this population that detected several potential loci. however, analyses of these data are complicated by the highly consanguineous nature of these amish pedigrees. therefore we applied the combinatorial mismatch scanning (cms) method that compares identity by state (ibs) (under the presumption of identity by descent (ibd)) sharing in distantly related individuals from such populations where standard linkage and association analyses are difficult to implement. cms compares allele sharing between individuals in affected and unaffected groups from founder populations. comparisons between cases and controls were done using two fisher's exact tests, one testing for excess in ibs allele frequency and the other testing for excess in ibs genotype frequency for microsatellite markers. results: in all, dementia cases and normal controls were identified who were not related at least through the grandparental generation. the examination of allele frequencies identified markers ( %) nominally (p ≤ . ) associated with dementia; the most interesting (empiric p ≤ . ) markers were d s , d s , and d s . the examination of genotype frequencies identified markers ( %) nominally (p ≤ . ) associated with dementia; the most significant markers were both located on chromosome (d s and d s ). notably, one of these markers (d s ) demonstrated differences (empiric p ≤ . ) under both tests. conclusion: our results provide the initial groundwork for identifying genes involved in late-onset alzheimer's disease within the amish community. genes identified within this isolated population will likely play a role in a subset of late- onset ad cases across more general populations. regions highlighted by markers demonstrating suggestive allelic and/ or genotypic differences will be the focus of more detailed examination to characterize their involvement in dementia. published: march bmc medical genetics , : doi: . / - - - received: august accepted: march this article is available from: http://www.biomedcentral.com/ - / / © mccauley et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ - / / http://creativecommons.org/licenses/by/ . http://www.biomedcentral.com/ http://www.biomedcentral.com/info/about/charter/ bmc medical genetics , : http://www.biomedcentral.com/ - / / background with over million individuals affected with alzheimer's disease (ad) in the u.s., dementia of the alzheimer's type (dat) is the leading cause of dementia in the elderly. these current estimates are projected to triple over the next years as the population ages [ - ]. ad has a com- plex etiology with strong genetic and environmental determinants. tremendous evidence suggests the involve- ment of at least three genes in early-onset autosomal dom- inant ad. amyloid precursor protein (app on chromosome ) [ , ], presenilin i (psen on chromo- some ) [ - ], and presenilin ii (psen on chromosome ) [ , ] are all prominent early-onset alzheimer's dis- ease genes. understanding of the more common late- onset alzheimer disease (load), is centered on the role of one universally accepted risk gene, the apolipoprotein e locus (apoe) [ ]. the apoe ε allele (frequency approximately %) [ , ] acts in a dose-related man- ner to increase risk for load and decrease age-of-onset [ , ]. although its involvement is without question, apoe accounts for less than half of late-onset ad suscep- tibility [ ]. given the strong heritability of ad, other genetic factors are likely to be involved. multiple linkage screens have been conducted to elucidate additional regions harboring susceptibility genes for late-onset ad [ - ]. while regions on chromosomes , and are most consistently identified, candidate genes within those regions have yet to be clearly implicated in ad. mean- while, numerous other regions have been implicated but have not been the focus of detailed study due to the prom- inence of , , and . though numerous promising load candidate genes have been examined, the lack of replication across studies has made a definitive declaration of their involvement diffi- cult (reviewed in [ , ]). genetic heterogeneity is likely to be one of the underlying reasons for this lack of repli- cation. given this, one possible solution is to study popu- lations likely to be more genetically homogeneous, thereby enriching for a more homogeneous set of risk alle- les. the north american amish population is a relatively isolated, genetically well-defined homogeneous popula- tion, well-suited for this type of study. further detail regarding the establishment of the north american amish population has been described elsewhere [ - ]. while there may be a number of load susceptibility genes con- tributing to disease in the general population, the rela- tively homogeneous amish population is likely to contain a smaller set of risk alleles. one challenge in performing linkage analysis in amish pedigrees is to utilize the extensive pedigree information available while maintaining tractability of the computa- tions. due to their strong religious and cultural beliefs, the amish very rarely marry outside of their communities, thereby promoting a genetically isolated population [ - ]. this in turn has led to an elevated degree of consan- guinity, yielding family pedigrees that contain many loops that can often be traced back three or more genera- tions. in fact, through use of the anabaptist genealogy database (agdb), we find that % of our overall study population of individuals and more specifically / ( %) of the individuals used in this study can be traced back generations to a single founding couple. moreover, % of individuals within our entire amish sample ( ) belong to one very large extended pedigree when allowing parent-child and marriage links to be included [ ]. since load cannot be ascertained until late in life, affected individuals are usually only available for genotyping in a single generation. hence, by far, most of the individuals in the pedigree have unknown pheno- type and genotype status. it should be noted that there are limited methodologies available to analyze disease gene linkage utilizing such large complex pedigrees. one such method is simwalk which utilizes descent graph theory and markov chain monte carlo (mcmc) simulation to compute lod scores [ ]. this is a computationally demanding process and because of the uncertainty of mcmc convergence, the accuracy of the scores obtained may be difficult to assess. combinatorial mismatch scanning (cms) is an alterna- tive technique to search for ibs sharing in distantly related individuals from isolated founder populations where standard linkage and association analyses are difficult to implement. while several other methods could be imple- mented, this approach was used because of its simplicity in examining existing data. this method was also chosen because at the onset of analysis, we lacked the more detailed knowledge of the inter-relatedness of our sample often required to perform similar, but more sophisticated approaches within large inbred pedigrees [ - ]. this strategy is designed to circumvent the confounding issue of genetic heterogeneity, by examining affected and unaf- fected persons from relatively small founder populations [ ]. by genetically evaluating case and control individu- als selected from such a population, whose common ancestor is no more closely related than grandparents, some prevailing problems in allelic association studies of complex disease within generally outbred populations can be avoided. population stratification can lead to allelic association and be misinterpreted as linkage dise- quilibrium. in this approach, population stratification is less of an issue due to the relative isolation and common heritage of the study population. another difficulty facing genetic studies within large outbred populations is that these populations are likely to exhibit locus heterogeneity. within an isolated population, the probability that the risk allele of interest might have entered the gene pool only once or rarely, provides a great advantage. this in page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / turn will likely facilitate the distinction between true and spurious association. heath and colleagues highlight another advantage of examining isolated populations by alluding to the potential for detecting gene-gene interac- tions [ ]. these epistatic interactions may play a substan- tial role in complex disease, effectively hampering the ability to detect association using single locus methods within heterogeneous populations. with reference to this problem, there are good reasons to believe that epistatic (gene-gene) interactions are ubiquitous in complex dis- ease and may in fact be more important than single-gene effects [ ]. methods subjects and phenotypes the amish often have large sibships and extensive pedi- gree records that permit the accurate estimation of ibs gene sharing to be accurately evaluated. the estimated coefficient of inbreeding for the entire population is . , which is approximately equivalent to having sec- ond cousins as parents [ ]. this effect has led present- day amish to possess genes inherited identically from a common ancestor at rates higher than observed in the general population. by searching the anabaptist geneal- ogy database (agdb) with the query software ped- hunter, we have determined the level of relatedness of our sample more precisely [ , ]. we calculated the average kinship coefficient for our overall ascertained amish sam- ple to be . ± . (mean ± sem). this measure demonstrates a significant difference from the average kinship coefficients calculated for the within cases group ( . ± . , mean ± sem), the within controls group ( . ± . , mean ± sem), and the between cases and controls group ( . ± . , mean ± sem). these calculations provide us with additional confidence that our cases and controls are more distantly related to each other relative to our overall sample population. the subjects included in this study are a subset of individ- uals described in extensive detail elsewhere [ ]. briefly, individuals enrolled in the study each were assigned to one of three clinical impression categories; dementia (probable or possible alzheimer's disease); unclear (includes mild cognitive impairment (mci)); or unaf- fected (cognitively normal). participants were adminis- tered the mini-mental state exam (mmse) [ ], with possible scores ranging from to . all individuals scor- ing or greater were classified as cognitively normal/ unaffected. those scoring or less were classified as cog- nitively impaired and labeled as probable dementia. those who scored – had additional neuropsycholog- ical testing including the dementia rating scale (drs) [ ], the boston naming test (bnt) [ ], and a reading subtest from the wide range achievement test-revised (wrat-r) [ ]. persons were categorized as having mild cognitive impairment if their drs score fell below an age- adjusted threshold. each case was discussed and a consen- sus "final" diagnosis was determined using all available information. for analytical purposes, the cases were clas- sified as affected (demented), unclear (includes mci), and unaffected (cognitively normal). five amish pedigrees were included in this study. three families were from elkhart and lagrange counties in indi- ana, one extended family from adams county indiana, and one extended family from holmes county ohio. the extended pedigree from adams county has been the sub- ject of other previous and ongoing studies of dementia in the amish [ , ]. among the individuals who were genotyped, were classified as having dementia, were classified as unclear, and individuals were unaffected. to minimize chance ibs inheritance, individuals selected for the cms analysis were unrelated through the grandpa- rental generation [ ]. for this current study we identified dementia cases and cognitively normal individuals who met this requirement. this study was undertaken after institutional review board review and approval. molecular analysis following informed consent, blood samples were col- lected from each individual and genomic dna was extracted from blood using standard procedures. cell lines have been initiated on most sampled individuals. all dna samples were coded and stored at °c until used. markers were genotyped at both the vanderbilt and duke laboratories for all dna samples. laboratory personnel were blinded to pedigree structure, affection status, and location of quality control samples. duplicate quality control samples were placed both within and across plates and equivalent genotypes were required for all quality control samples to ensure accurate genotyping. at the vanderbilt laboratory, marker primer sequences were obtained from the genome database [ ] or designed with primer software [ ] and synthesized by invitrogen life technologies (carlsbad, ca). amplification was per- formed in a pcr express machine (thermohybaid, need- ham heights, ma) with the following conditions: °c- min.; °c- sec., at- sec., °c- sec. ( cycles); °c- min. pcr products were denatured for min. at °c and run on a % polyacrylamide gel (sequagel- ®from national diagnostics, atlanta, ga) for ~ hr. at w. gels were stained with a sybrgold®rinse (molecular probes, eugene, or) and scanned with the hitachi biosys- tems fmbioii laser scanner (brisbane, ca). marker geno- typing at the duke laboratory was performed using fluorescence imaging (molecular dynamics si fluorim- ager) and a semi-automated allele calling system [ ]. page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / hardy-weinberg equilibrium calculations were per- formed for each marker and mendelian inconsistencies were identified using pedcheck in the overall dataset [ ]. suspect genotypes were re-read by a different technician or re-run as necessary to reduce errors. all microsatellite markers were required to have > % of possible geno- types to be included in the analysis. statistical analysis comparisons between case and control genotype data for the microsatellite markers were conducted using the r software package to perform fisher's exact tests in r × c contingency tables [ - ]. each marker was examined for both allele and genotype differences between individ- uals affected with dementia and those without dementia. the first test was for ibs allele frequency inequality between the two classes, and the second test was for ibs genotype frequency inequality between the two classes. fisher's exact test computes the probability p that the pat- tern of alleles observed in the sample would be obtained if there were truly no difference between the allele fre- quencies among affected and unaffected individuals. while our current sample size is adequate to detect mod- erate to major effects (odds ratio of > with % power), it does not preclude our ability to detect smaller effects given that these power calculations are based on the assumptions of complete independence of samples and random sampling of the population, neither of which is true. to empirically evaluate the statistical significance of the p-values computed in the cms study, we permuted our dataset. we randomly re-assigned affection status for each of the individuals maintaining the original total of cases and controls. we then executed the fisher's exact test using the same allele and genotype data in the original dataset for each of the markers. the distribution of p-values obtained from fisher's exact test- ing on randomized sets of data was then created for both the allele and genotype comparisons to assess the empiric thresholds. we would expect the fisher's exact p- value to match the p-value within the large distribution. these permutations were needed to correct for any resid- ual bias from unrecognized kinship correlation present. results we tested microsatellite markers for differences in both allele frequency and genotype frequency between amish dementia cases and controls. we considered all pointwise p-values and have chosen to report only mark- ers demonstrating fisher's exact p-values < . for either allele or genotype frequency differences. this arbitrary threshold was chosen to limit the results to be displayed and to provide a reference point for discussion of markers demonstrating nominally significant (albeit within the null expectation given the number of markers examined) evidence of association to dementia within our popula- tion. as an example, table a shows the allele count data for marker d s . there are eight d s alleles in the sample with subjects being typed for alleles. in the example, the probability of this data being obtained if there were no underlying difference between the allele dis- tributions for the two classes is < . (table ). table b shows the genotype data for marker d s . note that out of the thirteen genotypes observed in the data only one genotype is present in both affected and unaffected classes. the probability of the data being obtained if there were no underlying difference between the genotype dis- tributions for the case and controls is < . (table ). markers demonstrating nominally significant (p ≤ . ) differences between cases and controls are listed in table table : comparison of allele and genotype frequencies for d s in dementia cases and controls allele counts alleles cases controls totals totals genotype counts genotypes cases controls totals / / / / / / / / / / / / / totals bold highlights allele demonstrating greatest difference page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / . there were out of markers ( %) demonstrating significant differences in allele frequency. the most signif- icant markers were d s , d s , and d s . when examining the markers for genotype frequency dif- ferences, out of markers ( %) were significantly different between our dementia cases and controls. the most significant markers were d s and d s . while there were seven markers (d s , d s , d s , d s , d s , d s , d s , and d s ) with p-values ≤ . for both the allelic and genotypic tests, only one marker (d s ) was significant at the empiric p ≤ . level for both tests. although all findings are uncorrected and there are no findings with genome-wide significance, markers in close proximity to those regions previously identified are of particular inter- est for future study. discussion we have detected a few microsatellite markers of particu- lar interest, which demonstrate significant differences between dementia cases and controls within our amish founder population using the combinatorial mismatch scanning approach. the cms concept is based on excess ibs allele/genotype sharing between individuals sharing a distant set of common founders [ ]. the most notewor- thy finding is on chromosome q . at approximately cm where we find evidence for both allele and geno- type differences between our dementia cases and controls for marker d s . in their large genome-wide linkage study of alzheimer's disease, blacker et al. detected a multipoint lod score of . at this same marker [ ]. in a recent study of consanguineous israeli-arab communities, farrer and colleagues found significant evidence for allele frequency differences between ad cases and controls at the closest marker (d s at cm) on chromosome run in their study [ ]. positive findings across three dis- tinct study populations suggest that a gene or genes within this region of chromosome may be involved in risk for dementia of the alzheimer's type (dat). thus future examination of this region on chromosome within our amish families is warranted. while another marker on chromosome q . (d s at cm) demonstrated genotype differences between dementia cases and con- trols, this location is novel with respect to other previous studies. we also found evidence of allele frequency differences on chromosome q . at d s ( cm). one study tested for association with ad in a geographically distinct finnish population descended from a small group of orig- inal founders [ ]. this group found significant associa- tion (empiric p = . ) at marker d s (also located at cm) within their ad sample. an interesting candi- date gene at this location is sst, the gene encoding soma- tostatin, which functions as a neurotransmitter in the central nervous system. somatostatin inhibits the release of glucagon, growth hormone, gastrin, insulin, and secre- tin. additional evidence for this region stems from our genome-wide linkage study within the amish population, for which we observed a suggestive two-point lod score of . at the nearby marker d s ( cm) [ ]. an additional marker demonstrating suggestive allele frequency differences is located on chromosome p . (d s at cm). hiltunen et al. had detected evidence for association at two nearby markers (d s and d s ) spanning the region contain- ing our significant results [ ]. icam- (intercellular adhesion molecule ), a previously-reported ad candi- date gene, also lies within this region of interest. pola et al. showed that the icam- k e gene polymorphism was associated with ad in an italian population [ ]. this association was not, however, supported in studies of the gene in finnish and spanish populations [ , ]. additional strong evidence from previous work indicates the presence of a late-onset ad locus within this region. a study by wijsman et al. provides substantial evidence for a locus at approximately cm affecting ad age at onset [ ]. while our study does not address age at onset, it further suggests the involvement of this region in ad. another region of relative interest is on chromosome q . at marker d s . this marker located on chro- mosome q at approximately cm lies between two markers (d s at cm and d s at cm) demonstrating highly suggestive evidence for linkage within our amish population [ ]. further evidence for this region stems from work by pericak-vance et al. where they detect modest evidence for linkage to a marker only cm away (d s , lod = . ) from d s (table ) [ ]. given that our data may violate assumptions (i.e. normal- ity and/or unrealized correlation) of the fisher's exact test, we determined the empiric p-value for our results through permutation. we performed the fisher's exact test on replicates containing the same original genotype data, but with randomized affection status. the resulting distribution of p-values was then used as an empiric meas- ure of significance for our results (table ). on the whole, the empiric p-value thresholds for our study showed the fisher's exact p-value to be somewhat more liberal than expected. we have previously performed a genome-wide linkage screen for dementia within this population; however the complex nature of the amish pedigrees provides a chal- lenge for linkage analysis, given the size and number of consanguineous loops within these extended families. accordingly the linkage analysis by itself does not allow page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / page of (page number not for citation purposes) table : microsatellite markers demonstrating nominally significant (p ≤ . ) empiric p-values for allele and genotype frequency differences between dementia cases and controls. microsatellite markers in close proximity to those demonstrating significance in this study and found to be either linked (lod ≥ ) or associated (p ≤ . ) in previous studies are also listed. chromosome map position (cm) mb location marker fisher's exact p-value empiric p-value max lod score study allele genotype allele genotype . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . hahs et al. . d s . ** hiltunen et al. . d s . . . . d s . hahs et al. . d s . hahs et al. d s . . . . . d s . hahs et al. . d s . . . . . d s . hahs et al. . d s . pericak-vance et al. ( ) d s . . . . . d s . . . . . d s . hahs et al. . d s . . . . . d s . * farrer et al. . d s . . . . . d s . blacker et al. . d s . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . d s . . . . . d s . . . . . d s . . . . . d s . . . . d s . . . . . d s . ** hiltunen et al. . d s . hahs et al. . d s . . . . . d s . . . . . d s . . . . bold denotes markers nominally significant (p ≤ . ) in both allele and genotype comparisions italics highlights markers empirically significant at p ≤ . *sas software was used to measure significant differences in allele frequency between dat cases and controls **pearson's chi-square was calculated and then empirical significance was determined through examination of replicated datasets. bmc medical genetics , : http://www.biomedcentral.com/ - / / taking full advantage of the data available to us. to exam- ine our data more thoroughly, we performed the combi- natorial mismatch scan. both this approach and the linkage analysis utilize the high level of inter-relatedness, within the amish population, to their advantage. the nature of the cms analysis, allowed us to examine these data without being computationally burdened by the size or family structure of our population. these two methods complement each other by allowing the examination of the same data using both a family-based approach and a "pseudo" case-control approach to identify regions across the genome which are potentially involved in ad suscep- tibility. we are fully aware of the limited power of our cur- rent sample; however, these analyses should be viewed as an adjunct to our recent genomic screen. conclusion we have reported several markers across the genome (chr , , , and ) to have significant allelic and/or gen- otypic frequency differences between dementia cases and controls within the combined amish communities of ohio and indiana. while the evidence presented here is not overwhelming for any specific region, these results must be viewed in conjunction with not only our genomic screen but with findings across other studies within addi- tional populations. in conclusion, our results provide the groundwork for future detailed study of these regions within our growing sample of amish individuals. competing interests the author(s) declare that they have no competing inter- ests. authors' contributions jlm directed and performed some of the analyses, col- lated the results, and was responsible for preparing and editing the manuscript and tables therein. dwh was involved in the drafting of the manuscript and providing input on the analyses. lj was responsible for data manage- ment and analysis. wks provided input for the analysis, helped in editing the manuscript, and provided financial support through grant funding. kaw was key in all clini- cal evaluations and provided input for the manuscript. cej has been a longtime consultant for ascertainment and recruitment from the amish community due to his exten- sive interaction within this isolated population. jmv coor- dinated the genotyping of the microsatellite markers used within this study. jlh and mpv are principal investigator (pi) and co-pi, respectively. both pis were instrumental in providing the infrastructure, aiding in the study design, providing input in the manuscript, and supporting this project as well as additional projects surrounding this manuscript through their grant funding. acknowledgements this work was funded through nih/nia grants ag , ag , ag , the claude pepper center (ag ), and a discovery grant from vanderbilt university. additional work was performed using the van- derbilt center for human genetics research core facilities, the vanderbilt general clinical research center (m rr- ), and the duke center for human genetics core facilities. we thank all of the family participants and the amish 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citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.gdb.org/ http://www-genome.wi.mit.edu/cgi-bin/primer/primer _www.cgi http://www-genome.wi.mit.edu/cgi-bin/primer/primer _www.cgi http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ - / / /prepub http://www.biomedcentral.com/ http://www.biomedcentral.com/info/publishing_adv.asp http://www.biomedcentral.com/ 	abstract 	background 	methods 	results 	conclusion 	background 	methods 	subjects and phenotypes 	molecular analysis 	statistical analysis 	results 	discussion 	conclusion 	competing interests 	authors' contributions 	acknowledgements 	references 	pre-publication history wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ er .pptx best-­‐effort	
  modeling	
  of	
   structured	
  data	
  on	
  the	
  web	
   alon	
  halevy	
   google	
   november	
   ,	
   	
   structured	
  data	
  and	
  the	
  web	
   •  a	
  huge	
  amount	
  of	
  structured	
  data	
  on	
  the	
  web	
   – reference	
  data,	
  hobbies,	
  products,	
  coffee,	
  …	
   – reac%ng	
  to	
  exis%ng	
  data	
   •  a	
  plahorm	
  for	
  geing	
  more	
  data	
  out:	
   – government	
  data,	
  crime,	
  water	
  condilons,	
  …	
   – being	
  proac%ve	
  	
   •  new	
  kinds	
  of	
  data	
  colleclon	
  and	
  management	
   – collaboralon,	
  crowd-­‐sourcing,	
  real-­‐lme	
  data,	
  crisis	
   response,	
  …	
   –  inven%ng	
  a	
  bright	
  future	
   goal:	
  structured	
  data	
  ecosystem	
   discover	
   import	
   clean	
   query	
   share	
   integrate	
   visualize	
   publish	
   communicate	
   outline	
   •  google	
  fusion	
  tables: 	
  	
   – a	
  database	
  management	
  service	
  for	
  the	
  web	
   •  webtables:	
   – discovering	
  a	
  (structured)	
  needle	
  in	
  an	
   (unstructured)	
  haystack	
   •  observalons	
  about	
  modeling	
  along	
  the	
  way	
   fusion	
  tables	
   google.com/fusiontables	
   •  goal:	
  an	
  easy-­‐to-­‐use	
  database	
  system	
  that	
  is	
   integrated	
  with	
  the	
  web.	
   •  key	
  features:	
   – easy	
  upload	
  (csv,	
  kml,	
  spreadsheets)	
   – sharing	
  (even	
  outside	
  your	
  company)	
   – visualiza/ons	
  front	
  and	
  center	
   – easy	
  publishing	
   •  goal	
   :	
  a	
  data	
  cloud	
  -­‐-­‐	
  discover	
  others’	
  data	
   and	
  combine	
  with	
  yours.	
  	
   crowd sourcing a	
  gis	
  in	
  the	
  cloud	
   •  that’s	
  not	
  what	
  we	
  set	
  out	
  to	
  do,	
  really.	
   •  challenges:	
   – trickling:	
  show	
  only	
  a	
  small	
  number	
  of	
  features	
   (points,	
  polygons)	
  from	
  a	
  large	
  data	
  set	
   – need	
  to	
  thin	
  polygons,	
  clip	
  to	
  the	
  window	
   – style	
  features	
  on	
  the	
  fly	
   – all	
  in	
  less	
  than	
   ms	
   and	
  the	
  credit	
  goes	
  to…	
   •  hector	
  gonzalez	
   •  jayant	
  madhavan	
   •  	
  sree	
  balakrishnan	
   •  heidi	
  lam	
  	
   •  hongrae	
  lee	
   •  warren	
  shen	
  	
   •  anno	
  langen	
  	
   •  rebecca	
  shapley	
  	
   •  anish	
  das	
  sarma	
   •  boulos	
  harb	
   •  fei	
  wu	
   •  cong	
  yu	
   •  spiros	
  papadimitriou	
   outline	
   ü google	
  fusion	
  tables: 	
  	
   – a	
  database	
  management	
  service	
  for	
  the	
  web	
   •  webtables:	
   – discovering	
  a	
  (structured)	
  needle	
  in	
  an	
   (unstructured)	
  haystack	
   discovery	
  =	
  incen-ve	
  to	
  publish	
   tables on the web goal:	
  search	
  for	
  structured	
  data	
   challenges:	
   •  finding	
  the	
  good	
  tables	
  on	
  the	
  web	
   •  understanding	
  their	
  semanlcs	
  	
   •  understanding	
  user’s	
  intenlons	
   the deep web store locations used cars radio stations patents recipes see “google’s deep web crawl”, vldb html lists the	
  needle	
  in	
  the	
  haystack	
   finding	
  high	
  quality	
  html	
  tables	
   vertical tables semantics embedded in surrounding text and sometimes, complicated webtables: exploring the relational web [cafarella et al., vldb , webdb ] •  in corpus of b raw tables, we estimate m are “good” relations –  single-table databases; schema = attr labels + types –  largest corpus of databases & schemas we know of •  the webtables system: –  recovers good relations from crawl and enables search –  builds novel apps on the recovered data searching tables is tricky [tweak document search, cafarella ] •  consider new cues in ranking: –  hits on left column –  hits on schema (where there is one) –  number of rows, columns –  hits on table body –  size of table relative to page •  ~ % increase in good results in top- results (compared to filtering google results for tables tree search amish quilts parking tickets in india horses modeling	
  challenge:	
  	
   	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  data	
  is	
  about	
  everything	
   even	
  if	
  we	
  had	
  a	
  kb	
  of	
  everything	
   [consider	
  freebase	
  as	
  example]	
   •  user:	
   –  aclon	
  movies	
   •  freebase:	
  	
   –  movies	
  and	
   genre=aclon	
   •  user	
   –  governor	
  of	
  california	
   •  freebase:	
   –  us	
  state	
  has	
  governing	
   posilons	
   –  governing	
  posilons	
   have	
  office	
  holder	
   –  office	
  holder	
  has	
   posilon	
   mismatch	
  between	
  kb	
  model	
  and	
  users’	
  conceptual	
  model!	
  	
   the	
  people’s	
  ontology	
   [open	
  informalon	
  extraclon]	
   mine	
  a	
  database	
  of	
  enlles	
  and	
  classes	
  from	
  the	
   web:	
   mine	
  binary	
  relalonships	
  	
   broad,	
  dirty,	
  but	
  uses	
  culturally	
  aware	
  terminology	
   recovering table semantics [venetis et al., vldb ] recovering binary relationships recovering	
  seman-cs:	
  be er	
  search	
   and	
  quality	
  filter	
   attribute correlations db raw crawled pages raw html tables recovered relations relation search inverted index job-title, company, date make, model, year rbi, ab, h, r, bb, avg, slg dob, player, height, weight … … attribute correlation statistics db •  . m distinct schemas •  . m attributes the	
  unreasonable	
  effec%veness	
  of	
  data	
  [halevy,	
  norvig,	
  pereira]	
   synonym discovery •  use schema statistics to automatically compute attribute synonyms –  more complete than thesaurus •  given input “context” attribute set c: .  a = all attrs that appear with c .  p = all (a,b) where a∈a, b∈a, a≠b .  rm all (a,b) from p where p(a,b)> .  for each remaining pair (a,b) compute: synonym discovery examples name! e-mail|email, phone|telephone, 
 e-mail_address|email_address, date|last_modified! instructor! course-title|title, day|days, course|course-#, 
 course-name|course-title! elected! candidate|name, presiding-officer|speaker! ab! k|so, h|hits, avg|ba, name|player! sqft! bath|baths, list|list-price, bed|beds, price|rent! conclusions	
   •  fusion	
  tables:	
  helping	
  get	
  the	
  ecosystem	
  started.	
  	
   •  search	
  for	
  structured	
  data	
  sets:	
  	
   – much	
  more	
  to	
  do!	
   – unify	
  with	
  other	
  search	
   – manually	
  created	
  ontologies	
  vs.	
  extracted	
  ones?	
   •  can	
  we	
  get	
  the	
  crowds	
  to	
  help?	
   – resolving	
  heterogeneity	
   – create	
  new	
  data	
  sets	
   •  can	
  we	
  help	
  domain-­‐specific	
  expert	
   communiles?	
   a	
  few	
  references	
   •  communicalons	
  of	
  the	
  acm:	
  feb	
   	
   •  deep	
  web:	
  vldb	
   	
   •  webtables:	
  vldb	
   ,	
   ,	
   	
   •  fusion	
  tables:	
  sigmod	
   ,	
  socc	
   	
   – google.com/fusiontables	
   •  principles	
  of	
  data	
  integralon	
  (doan,	
  halevy,	
   ives):	
  morgan	
  kaufmann,	
   .	
   •  the	
  infinite	
  emolons	
  of	
  coffee	
  (halevy):	
  next	
   month!	
   ucla ucla previously published works title high school completion rates among men with hemophilia permalink https://escholarship.org/uc/item/ b d xc journal american journal of preventive medicine, ( ) issn authors drake, john h soucie, j. michael cutter, susan c et al. publication date - - doi . /j.amepre. . . peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ b d xc https://escholarship.org/uc/item/ b d xc#author https://escholarship.org http://www.cdlib.org/ f u d d h p t m v a c r © high school completion rates among men with hemophilia john h. drake, msn, rn, j. michael soucie, phd, susan c. cutter, msw, mpa, ann d. forsberg, ma, mph, judith r. baker, mhsa, brenda riske, ms, mba, mpa background: the benefıts of a high school diploma are well documented. studies indicate that people with hemophilia have lower than average academic achievement, particularly if they have � bleeding episodes annually. purpose: this study compares the high school graduation rate of men with hemophilia to that of the u.s. population of men. methods: data were obtained from the universal data collection program, a surveillance project conducted by approximately hemophilia treatment centers in the nation. data from men aged � years were evaluated to determine high school graduation status and were analyzed by race/ethnicity and severity of hemophilia. these data were collected between and , and analysis was conducted in . results: men with hemophilia a had higher or similar high school graduation rates across all racial/ethnic groups and all levels of hemophilia severity, compared with u.s. men of the same age. graduation rates for black and hispanic men with hemophilia b were higher or similar to rates of u.s. men, but rates for whites were lower, especially among those with moderate and mild disease. however, when graduation rates were controlled for areas where amish populations reside, differ- ences in graduation rates for whites disappeared. conclusions: in this study, participants obtained hemophilia care at comprehensive hemo- philia treatment centers. this multidisciplinary, family-centered care emphasizes prevention of complications, encourages medically supervised disease management, and facilitates psychoso- cial development. the care aims to maximize the affected child’s participation in school. this care approach may partially explain the higher-than-expected high school graduation rates among the study population, which is affected by a rare, chronic, and potentially debilitating disorder. (am j prev med ; ( s):s –s ) © published by elsevier inc. on behalf of american journal of preventive medicine. i t i c a l m s t w w rom the gulf states hemophilia and thrombophilia center (drake), niversity of texas health science center at houston, houston, texas; ivision of blood disorders (soucie), national center on birth defects and evelopmental disabilities, cdc, atlanta, georgia; penn comprehensive emophilia and thrombosis center (cutter), hospital of the university of ennsylvania, philadelphia, pennsylvania; new england hemophilia cen- er (forsberg), university of massachusetts memorial hospital, worcester, assachusetts; division of pediatric hematology/oncology (baker), uni- ersity of california los angeles, los angeles, california; and hemophilia nd thrombosis center (riske), university of colorado denver, denver, olorado address correspondence and reprint requests to: john h. drake, msn, n, gulf states hemophilia and thrombophilia center, travis, suite , houston tx . e-mail: john.h.drake@uth.tmc.edu. a - / /$ . doi: . /j.amepre. . . published by elsevier inc. on behalf of american journal of preve ntroduction he benefıts of receiving a high school diploma or equivalency degree are well documented. u.s. census data for indicate that annual income s much higher for people with a high school diploma ompared to those without one ($ , vs $ , ). in ddition, those with a high school diploma are not only ess likely to be living below the poverty level but also ore likely be in good health, maintain a healthy life- tyle, and have health insurance. most studies of children with chronic illnesses, such as ype diabetes, epilepsy, and sickle cell disease, all of hich can affect cognitive function, show that children ith these conditions have lower levels of academic chievement than their healthy peers. – a study of per- ntive medicine am j prev med ; ( s)s –s s mailto:john.h.drake@uth.tmc.edu f t t w f t c m i c t i s a c m r n c h e t g t i i p a t a s g m t s m a l y s a s w a d h b a b a r p q p m m w c c h s t d t h u a s g t m d d s f t c s r d u d l w ( � s n a y p b d s drake et al / am j prev med ; ( s):s –s ormance on standardized tests in north carolina found hat students with various chronic medical condi- ions scored in the st percentile, whereas their state- ide peers scored in the rd percentile. however, dif- erences in achievement in that study were related more o socioeconomic issues and specifıc aspects of chronic onditions than to absences from school. the underlying causes of poorer academic perfor- ance for chronically ill children who are not cognitively mpaired are less well defıned than they are for those with ognitive impairments resulting from chronic illness. al- hough excessive absenteeism in children with chronic llness has been well documented for many years, re- earch results have been mixed regarding the impact of bsences on academic achievement in children with hronic illness. there are few studies that address academic achieve- ent in individuals with hemophilia. hemophilia is a are, chronic, inherited bleeding disorder in which ab- ormalities in blood clotting occur because of a defı- iency of factor viii or ix. about % of people with emophilia have the severe form of the disorder and xperience frequent, spontaneous joint hemorrhages hat, without appropriate treatment, can lead to pro- ressive joint disease and loss of functional ability. hose with no history of meaningful head trauma or ntracranial hemorrhage typically have no cognitive mpairment. nonetheless, studies involving children with hemo- hilia have revealed below average levels of academic chievement. in one study, standardized achievement ests were administered to children with hemophilia. lthough the participants received average grades in chool, % scored more than two levels below their rade on standardized reading tests, and % scored ore than two grade levels below on standardized math ests. these children missed an average of days of chool each year compared with an average of days issed by their peers. however, factors such as excessive bsenteeism and severity of disease were unrelated to ower academic achievement in that study. another study involving children aged – ears with severe hemophilia found that excessive ab- ences from school resulted in lower levels of academic chievement. patients with extenuating medical circum- tances, including treatment with inhibitors, and patients ith considerable developmental, mental, and/or psychi- tric disorders were excluded from the study. mean aca- emic achievement scores for the group were average. owever, when patients were separated into two groups ased on number of bleeding episodes, differences in cademic performance were notable. a higher number of o leeding episodes (� ) correlated with increased school bsences and below-average aptitude in math and eading. a -year longitudinal study of children with hemo- hilia found that, overall, participants had intelligence uotients (iqs) in the average range. however, partici- ants who experienced high levels of physical impair- ent from their hemophilia had lower academic achieve- ent and intellectual abilities, even after adjustments ere made for other intervening variables such as intra- ranial hemorrhage and hiv infection. the researchers oncluded that marked physical impairments resulted in indered ability to concentrate and learn, excessive ab- enteeism, and limited participation in non-academic ac- ivities that can enhance intellectual growth. the purpose of the current study was to use collected ata on participants of a public health surveillance system o compare high school graduation rates of men with emophilia with graduation rates of the general u.s. pop- lation of men. this project demonstrates the practical pplicability of iq ranges and academic achievement tudies in children and how this affects their high school raduation and ability to become productive members of he community. ethods ata for this project were collected as part of the universal ata collection program (udc), a surveillance system ponsored by the cdc and conducted by approximately ederally supported hemophilia treatment centers (htcs) hroughout the u.s. since . participants give informed onsent, and data are collected annually by htc staff using tandardized data forms. demographic information collected includes gender, ace/ethnicity, fırst digits of the zip code of current resi- ence, and month and year of birth; birth information was sed to calculate age at most recent htc visit. clinical and iagnostic data include hemophilia type (a or b) and base- ine factor activity (fa) level relative to normal. the fa level as used to categorize hemophilia severity as either mild fa %– %); moderate (fa %– . %); or severe (fa %). the highest education level achieved was obtained by elf-report during the most recent udc re-enrollment. inety percent of the data used were from udc evaluations fter , and % were from udc evaluations after . all men who had hemophilia a or b and were aged � ears were eligible to participate in the study. of the atients for whom data were available, were ineligible ecause their high school graduation status could not be etermined from information provided; after this exclusion, men remained in the study. no additional exclusions ccurred. subjects who reported completing years of www.ajpm-online.net s � t c b s w ( c t g r s r o h t h r h m l w u p h b t w e p b h w r w w y b a h e t a r f a b le . h ig h sc h o o l gr a d u a ti o n ra te s fo r u .s . m e n a n d fo r m e n w it h h e m o p h ili a (% [ % c i] u n le ss o th e rw is e in d ic a te d )a g e (y e a rs ) p o p u la ti o n n r a c e o r e th n ic it y p -v a lu e b a ll w h it e b la c k h is p a n ic o th e r u .s . m e n , . . . . n a m e n w it h h e m o p h ili a a , . ( . , . ) . ( . , . ) . ( . , . )* . ( . , . ) . ( . , . ) � . m e n w it h h e m o p h ili a b , ( . , . )* . ( . , . )* . ( . , . ) . ( . , . ) . ( . , . ) . – u .s . m e n , . . . . n a m e n w it h h e m o p h ili a a , . ( . , . ) . ( . , . ) . ( . , . ) . ( . , . ) . ( . , . ) � . m e n w it h h e m o p h ili a b . ( . , . )* . ( . , . )* . ( . , . ) ( . , . ) . ( . , . ) . u .s . m e n , . . . . n a m e n w it h h e m o p h ili a a , . ( . , . ) ( . , . ) . ( . , . ) . ( . , . ) . ( . , . ) � . m e n w it h h e m o p h ili a b , . ( . , . ) . ( . , . )* . ( . , . ) . ( . , . ) . ( . , . ) . e : d a ta in b o ld in d ic a te si gn ifi ca n tl y h ig h e r va lu e s co m p a re d w it h u .s . m e n . th e c i d o e s n o t in cl u d e th e va lu e fo r th e ge n e ra l m a le p o p u la ti o n a n d , th e re fo re , is si gn ifi ca n tl y d if fe re n t a t th e % le ve l o f si gn ifi ca n ce . n a ge d � ye a rs w h o vi si te d u .s . h e m o p h ili a tr e a tm e n t ce n te rs co m p a re d w it h u .s . m e n in th e sa m e a ge gr o u p s b a se d o n u .s . c e n su s d a ta fo r a lu e s fo r te st s o f d if fe re n ce in p e rc e n ta ge s o f h ig h sc h o o l gr a d u a ti o n a cr o ss ra ce gr o u p s fo r h e m o p h ili a p a ti e n ts gn ifi ca n tl y lo w e r va lu e s co m p a re d w it h u .s . m e n n o t a va ila b le drake et al / am j prev med ; ( s):s –s s a chooling or who had education beyond high school, such as year of college, were considered high school graduates. he proportion of subjects who completed high school was alculated for four categories of race and ethnicity (white, lack, hispanic, and other) and three levels of hemophilia everity. for each category, % cis were calculated. rates ere also calculated separately for each of three age groups � years, – years, and � years). these rates were ompared with those for the general population of men in he same age groups, based on u.s. census data. raduation rates with cis outside the bounds of the national ate for each category of age and race or severity were con- idered signifıcantly different at the % confıdence level. esults verall, men with hemophilia a had signifıcantly higher igh school graduation rates compared with u.s. men for he three age groups (table ). in contrast, those with emophilia b had signifıcantly lower overall graduation ates in two of the three age groups. white men with emophilia a tended to have rates similar to those of u.s. en, and white men with hemophilia b had uniformly ower rates than the u.s. population of men. black men ith hemophilia tended to have similar high school grad- ation rates as u.s. black men. hispanic men with hemo- hilia tended to have higher graduation rates than u.s. ispanic men; however, graduation rates for neither lack nor hispanic men showed variation by hemophilia ype. in general, men with hemophilia a, especially those ith mild disease, had graduation rates similar to or xceeding those of u.s. men (table ). however, the attern was strikingly different for men with hemophilia . although men with mild and severe disease tended to ave graduation rates similar to those of u.s. men, those ith moderate disease had signifıcantly lower graduation ates for all three age groups. rates across severity levels ithin age groups were signifıcantly different for men ith hemophilia b in the aged � years and aged � ears groups only. differences in rates by severity were of orderline signifıcance for men with hemophilia a in the ged � years group only. the amish population in the u.s. has a particularly igh prevalence of hemophilia b owing to a founder ffect. the amish have a high degree of homogeneity, and hose with hemophilia are primarily affected with mild nd moderate hemophilia b. they traditionally live in ural areas, and most boys in this population stop their ormal education after the th grade. to test whether the low graduation rates observed mong moderate hemophilia b patients might result pril t a a � � � n o t a m e b p -v * s i n a , f m g c d w h m w p e t a t s s g e m t g n t h p d t g r s s l l h r p t a a c p o m s t n a a b * t h a s drake et al / am j prev med ; ( s):s –s rom low high school graduation rates among amish en, the fırst digits of the zip code were used to identify eographic areas in which amish populations were con- entrated. graduation rates by severity level and resi- ence in a geographic area were then compared with and ithout amish residents for men aged � years who ad hemophilia b (table ). in areas with no amish residents, graduation rates for en with hemophilia b were similar to those for men ith hemophilia a (tables and ). in areas with amish opulations, graduation rates of men with mild and mod- rate hemophilia b were only . % and . %, respec- ively, well below the graduation rates for men with mild nd moderate hemophilia b in non-amish communities. he graduation rates for men aged – years with evere hemophilia b were higher than those for men with evere hemophilia a (table ). in addition, when men with hemophilia b living in the eographic areas with high concentrations of amish were xcluded from analysis, overall graduation rates among able . high school graduation rates by age group for u age (years) population mild � u.s. men . men with hemophilia a . ( . , . men with hemophilia b . ( . , . � – u.s. men . men with hemophilia a . ( . , . men with hemophilia b . ( . , . � u.s. men men with hemophilia a . ( . , . men with hemophilia b . ( . , . ote: boldface indicates significantly higher values compared with u. nd, therefore, is significantly different at the % level of significanc men aged � years who visited u.s. hemophilia treatment centers data for . p-values for tests of difference in percentages of high school gradu significantly lower values compared with u.s. men able . high school graduation rates among men with emophilia b (% unless otherwise indicated)a residence in area with amish residents n hemophilia b severity mild moderate severe yes . . . no . . . cmen aged � years who visited u.s. hemophilia treatment centers en with hemophilia b increased to levels similar to hose among men with hemophilia a for all three age roups (table ). graduation rates for all racial and eth- ic groups, and white men with hemophilia b, increased o rates nearly as high as those among white men with emophilia a; graduation rates among black and his- anic men with hemophilia b remained unchanged. iscussion his is a large nationwide study comparing high school raduation rates of men with hemophilia with graduation ates for the general u.s. population of men. the results uggest that men with hemophilia a graduate from high chool at a similar or higher rate than the national popu- ation of men. studies conducted years ago showed ower academic performance levels among children with emophilia. since that time, the recommended treatment egimen has changed from on-demand therapy to pro- hylactic therapy that prevents bleeding episodes and husdecreasesdayslostfromschool.presumably,improved ttendanceinschoolcontributestoahighergraduationrate. ll participants in this study attended federally supported omprehensive htcs, where they received multidisci- linary care. – the focus of this care addresses not nly medical treatment, but also psychosocial develop- ent of chronically ill patients, notably children with pecial healthcare needs. during childhood, a key priority of the hemophilia en and for men with hemophiliaa ophilia severity (% [ % ci]) p-valuebmoderate severe . . . ( . , . ) . ( . , . ) . . ( . , . )* . ( . , . ) � . . . . ( . , . ) . ( . , . ) . . ( . , . )* . ( . , . ) . . ( . , . ) . ( . , . ) . . ( . , . )* . ( . , . ) � . n. the ci does not include the value for the general male population pared with u.s. men in the same age groups based on u.s. census across levels of disease severity in hemophilia patients .s. m hem ) ) ) ) ) ) s. me e. com ation enter partnership with the family is to maximize the www.ajpm-online.net a a w w e b v a c r p c s a r c a f g h s r u t n a m c e r h g t i c u r t t c c r s d w i p a g g s t i h t t t g a d n t r drake et al / am j prev med ; ( s):s –s s a ffected child’s participation in school, both in academic nd non-academic arenas. hemophilia center clinicians ork directly with parents and the schools, particularly hen the child enters a new school. the purpose of these arly interventions is to identify and resolve potential arriers to the child’s regular school attendance. inter- entions include creating adaptations to reduce tardiness nd absenteeism due to acute or chronic mobility diffı- ulties and to eliminate potentially inappropriate school estrictions (e.g., prohibition from physical education). the ongoing coordinated involvement of the hemo- hilia center team, usually beginning at the child’s birth, an establish a pattern of assisting the family in under- tanding the importance of reducing barriers to school ttendance and, beginning in primary school, assisting in educing those barriers. as the child ages, the team fo- uses on assisting the child and family in transitioning to dult health care, work, and independence; this step ful- ılls healthy people national health objectives and oals of the division of services for children with special ealth care needs, a division of the health resources and ervices administration’s maternal and child health bu- eau, a federal agency that provides grant support for the .s. hemophilia center network. hemophilia center eams encourage adolescents to pursue careers that do ot require extensive manual labor; such work is inadvis- ble for men with hemophilia. because careers without anual labor tend to require higher education, the staff ounsels adolescents to remain in school; this ongoing ncouragement may be a factor in the higher graduation ates among children with hemophilia, especially among ispanics and blacks who have a high dropout rate in the eneral population. this study is limited because it is a descriptive analysis hat did not control for factors related to ses in compar- sons of patients with hemophilia, especially those of ra- ial and ethnic groups such as blacks and hispanics, with .s. men. for example, we know that poverty is a major isk factor for poor academic achievement. thus, a po- ential for bias exists and may explain the higher gradua- ion rates for blacks and hispanics in the udc data ompared with the general population of men in the ensus. geography may also play a role in the study esults. additionally, we cannot directly compare the re- ults with those of previously cited studies that used aca- emic performance measures, because the outcome here as high school graduation rates, which are not necessar- ly a measure of academic performance. the comprehensive-care, multidisciplinary team ap- roach of the federally supported htcs may be associ- ted with the higher-than-expected rates of high school raduation among the study population. advocacy roups for other chronic diseases and for children with pril pecial healthcare needs are moving toward adoption of he comprehensive care model. because all men partic- pating in the udc surveillance study were seen at an tc, whether adolescents with hemophilia not seen at he htcs also have a higher high school graduation rate han average is unknown. further research is warranted o determine whether differences exist in high school raduation rates among htcs in the national network nd, if so, to identify factors contributing to those ifferences. o fınancial disclosures were reported by the authors of his paper. eferences . bergman m. u.s. census bureau. college degree nearly dou- bles annual earnings, census bureau reports [press release]. washington dc: march . . cohen ra, martinez me, free hl. health insurance coverage: early release of estimates from the national health interview survey . hyattsville md: national center for health sta- tistics, . www.cdc.gov/nchs/data/nhis/earlyrelease/ insur .htm. . robert wood johnson foundation commission to build a healthier america. reaching america’s health potential among adults: a state-by-state look at adult health. www. commissiononhealth.org/documents/adulthealthchartbook fullreport.pdf. . usdhhs. national healthcare quality and disparities reports. ahrq publication no. - . rockville md: agency for healthcare research and quality, . . tara h, pots-datema w. chronic health conditions and student performance at school. j sch health ; ( ): – . . schatz j, finke rl, kellett jm, kramer jh. cognitive function- ing in children with sickle cell disease: a meta-analysis. j pedi- atr psychol ; : – . . holmes ds, o’brien b, greer t. cognitive functioning and academic achievement in children with insulin-dependent di- abetes mellitus. school psychology ; ( ): – . . fowler mj, johnson mp, atkins ss. school achievement and absence in children with chronic health conditions. j pediatr ; ( ): – . . charlton a, larcombe ij, meller st, et al. absence from school related to cancer and other chronic conditions. arch dis child ; : – . . woolf a, rappaport l, reardon p, ciborowski j, d’angelo e, bessette j. school functioning and disease severity in boys with hemophilia. j dev behav pediatr ; ( ): – . . shapiro ad, donfıeld sm, lynn hs, et al. defıning the impact of hemophilia: the academic achievement in children with hemophilia study. pediatrics ; :e . www.pediatrics. org/cgi/content/full/ / /e . . usner dw, donfıeld sm, sirois pa, gomperts ed, bale jf jr, mitchell wg. hemophilia morbidity, cognitive functioning, and academic achievement. j pediatr ; ( ): – . http://www.cdc.gov/nchs/data/nhis/earlyrelease/insur .htm http://www.cdc.gov/nchs/data/nhis/earlyrelease/insur .htm http://www.commissiononhealth.org/documents/adulthealthchartbookfullreport.pdf http://www.commissiononhealth.org/documents/adulthealthchartbookfullreport.pdf http://www.commissiononhealth.org/documents/adulthealthchartbookfullreport.pdf http://www.pediatrics.org/cgi/content/full/ / /e http://www.pediatrics.org/cgi/content/full/ / /e s drake et al / am j prev med ; ( s):s –s . u.s. census bureau. educational attainment in the u.s.: . www.census.gov/population/www/socdemo/education/ cps .html. . baker jr, crudder so, riske b, bias v, forsberg a. a model for a regional system of care to promote the health and well-being of people with rare chronic genetic disorders. am j public health . hoots wk. comprehensive care for hemophilia and related bleeding disorders: why it matters. curr sci ; : – . . grosse sd, schechter ms, kulkarni r, lloyd-puryear ma, strickland b, trevathan e. models of comprehensive multidis- ciplinary care for individuals in the u.s. with genetic disorders. ; ( ): – . pediatrics ; ( ): – . did you know? you can search over top health sciences journals online, including medline, via the ajpm website. visit www.ajpm-online.net today! www.ajpm-online.net http://www.census.gov/population/www/socdemo/education/cps .html http://www.census.gov/population/www/socdemo/education/cps .html 	high school completion rates among men with hemophilia 	introduction 	methods 	results 	discussion 	references letters to the editor gramme. this is in keeping with the tremor of other body parts, for example the wing- beating arm tremor of wilson's disease and task-specific tremors such as writing and other occupational tremors. we have examined clinically, three other patients with clear dystonic head tremor in whom head tremor was altered by the posi- tion of the head in the anterior-posterior plane. in none of the others could we demonstrate a vestibular (otolith) mecha- nism. despite these negative observations for a clear otolith influence upon head tremor electrophysiological measurements have shown that otolith spinal reflexes may be modulated by both head and body position. thus there still remains the possibility that a change in head or body position may in itself modify vestibular influences upon body movement. s mossman l cleeves l findley mrc human movement and balance unit, section of neuro-otology, national hospital for neurology and neurosurgery, london, uk correspondence to: dr mossman, department of neurology, wellington hospital, private bag, wel- lington south, wellington, new zealand. denny-brown d. clinical symptomatology of diseases of the basal ganglia. in: vinken p, bruyn g, eds. handbook of clinical neurology, vol . amsterdam: north holland publishing co, : - . bronstein a, rudge p, beechey a. spasmodic torticollis following unilateral viii nerve le- sions: neck emg modulation in response to vestibular stimuli. j neurol neurosurg psy- chiatry ; : - . cleeves l, findley l, marsden d. odd tremors. in: marsden d, fahn s, eds. movement disorders . butterworths (in press). podivinsky f. torticollis. in: vinken p, bruyn g, eds. handbook of clinical neurology, vol . amsterdam: north holland. : - . gresty m, barratt h, page n, rudge p. analysis of downbeat nystagmus. otolith vs semi- circular canal influences. arch neurol ; : - . dieterich m, brandt t, fries w otolith func- tion in man. brain ; : - . probable cases of mast syndrome in a non-amish family complicated forms of hereditary spastic paraplegia are rare. a year old flemish woman was admitted with a clinical picture of slowly progressive spastic paraplegia, dys- arthria, presenile dementia and mild atheto- sis. at the age of her gait became shuffling. before the age of she used a walking stick, since the age of years she has needed a walking frame and from the age of she has become more wheelchair bound. during the third decade (maybe earlier) dysarthria, apa- thy and negativism appeared. towards her th year, urinary incontinence began. on admission, aged , significant bradyphrenia and comprehension difficulties were noted and during the following years she presented a further mental deterioration. she is now bedridden and her speech restricted to rare, usually inappropriate, single syllable answers, which are sometimes repeated. she has diffi- culty swallowing fluids. she often shows spontaneous repeated slow turning of the head to the right and left and mild tortuous movements of the shoulders. except for a divergent strabismus there are neither oculo- motor abnormalities, nor fundoscopic anomalies. there is a slight bilateral facial weakness. the fine motor hand skills are lost , = proband; * = birth order in this sibship is uncertain figure pedigree without clear paresis of the upper limbs. the strength of leg muscles measures about to / . there were neither sensory deficits nor cerebellar signs. deep tendon reflexes in the upper limbs were increased slightly. knee jerks were unusually brisk and without clo- nus; ankle jerks were decreased. plantar responses were extensor. snout reflex and bilateral palmomental reflexes were present whilst corneomandibular reflexes were absent. the following laboratory investigations showed no significant abnormalities: routine blood examination (except for intermittent elevation of glucose with normal haemoglo- bin a c ), creatine kinase, copper, lipids, very long chain fatty acids ratios c /c and c /c , vitamin e, vitamin b , folate, cortisol, adrenocorticotropic hor- mone, thyroid hormones, arylsulfatase a and hexosaminidase a + b; the csf protein was mg/l with normal electrophoretic pat- tern. the eeg showed mild general slowing. nerve conduction studies and needle electro- myography showed a mild axonal polyneur- opathy. the somato-sensory evoked potentials demonstrated a slightly prolonged central conduction time. an electrocardio- gram was normal. mri of the brain showed diffuse cortico-subcortical atrophy, periven- tricular hyperintensities, thin corpus callo- sum and less marked atrophy of the brainstem and cerebellum. light microscopic and electron microscopic examination of conjunctiva and skin showed some mem- branous cytoplasmic body-like inclusions (professor j j martin, dr c ceuterick-de groote, university hospital antwerp). the family history revealed two similar cases (figure). there was no known con- sanguinity. the monozygotic twin has an almost identical medical history and clinical picture. she is able to stammer a few simple words. her answers are sometimes slightly more appropriate, particularly for old memories. her knee jerks and ankle jerks are both unusually brisk, and the plantar responses are extensor. one brother died at the age of . the medical records and relatives des- cribed difficulties with walking from the age of (maybe earlier). during the following decades he presented a progressive spastic paraparesis, dysarthria, mental deterioration and urinary and faecal incontinence; no deficits of sensation or coordination were demonstrated. there was dysphagia in his last years. death was due to pneumonia. the pedigree suggests an autosomal reces- sive inheritance. the neurodegenerative syn- drome in this family seems fully comparable to the mast syndrome, described in in an ohio amish isolate by cross and mcku- sick.`'- there appears to be no similar cases that have been described outside the amish population. marc d'hooghe department of neurology, algemeen ziekenhuis st j an, ruddershove , brugge, belgium cross he, mckusickva. the mast syndrome: a recessively inherited form of presenile de- mentia with motor disturbances. arch neurol (chic) ; : - . harding ae. classification of hereditary ataxias and paraplegias. lancet ;i: - . mckusick va. mendelian inheritance in man. baltimore: the johns hopkins university press, : . ultrasensitive tsh assay and anti-par- kinsonian treatment with levodopa we have recently reported the association of parkinson's disease and hyperthyroidism in a group of patients.' in that report, symp- toms of parkinson's disease were always significantly exacerbated by the development of hyperthyroidism and improved by its successful treatment.' we proposed that hyperthyroidism should be suspected in all parkinsonian patients when their condition deteriorates.' since clinical diagnosis of thy- rotoxicosis is difficult in parkinsonian patients, they should have a comprehensive thyroid examination and, if there is the slightest suspicion of hyperthyroidism, a hor- monal evaluation of thyroid function (free t , ultrasensitive tsh). thyroid hormone levels (t and t ) have been found to be normal in parkinsonian patients untreated or treated with levodopa. however, a decreased response of thyro- tropin (tsh) after stimulation by trh (thyrotropin releasing hormone) has been reported in parkinsonian patients treated with levadopa. such a decreased tsh response after trh stimulation is observed during hyperthyroidism, and is sometimes the only hormonal abnormality, especially in elderly patients with autonomous thyroid nodules. thus the decreased tsh response after trh-stimulation in patients treated with levodopa could be responsible for a false diagnosis of hyperthyroidism. these results, however, were obtained before the ultra- sensitive tsh determination with a mono- clonal antibody assay was available. a low ultrasensitive tsh level has the same sig- nificance as a decreased tsh response to trh, indicating an increased negative feed- o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn n p .b m j.co m / j n e u ro l n e u ro su rg p sych ia try: first p u b lish e d a s . /jn n p . . . o n d e ce m b e r . d o w n lo a d e d fro m http://jnnp.bmj.com/ research article 	 the	journal	of	clinical	investigation      http://www.jci.org  � variations in the g pc /abcb genomic region are associated with fasting glucose levels wei-min chen, , michael r. erdos, anne u. jackson, richa saxena, serena sanna, , kristi d. silver, nicholas j. timpson, torben hansen, marco orrù, maria grazia piras, lori l. bonnycastle, cristen j. willer, valeriya lyssenko, haiqing shen, johanna kuusisto, shah ebrahim, natascia sestu, william l. duren, maria cristina spada, heather m. stringham, laura j. scott, nazario olla, amy j. swift, samer najjar, braxton d. mitchell, debbie a. lawlor, george davey smith, yoav ben-shlomo, gitte andersen, knut borch-johnsen, , , torben jørgensen, jouko saramies, timo t. valle, thomas a. buchanan, , alan r. shuldiner, edward lakatta, richard n. bergman, manuela uda, jaakko tuomilehto, , oluf pedersen, , antonio cao, leif groop, karen l. mohlke, markku laakso, david schlessinger, francis s. collins, david altshuler, gonçalo r. abecasis, michael boehnke, angelo scuteri, , and richard m. watanabe , department of public health sciences and center for public health genomics, university of virginia, charlottesville, virginia, usa. genome technology branch, national human genome research institute, bethesda, maryland, usa. center for statistical genetics and department of biostatistics, university of michigan, ann arbor, michigan, usa. program in medical and population genetics, broad institute of mit and harvard, cambridge, massachusetts, usa. istituto di neurogenetica e neurofarmacologia, consiglio nazionale delle ricerche, cagliari, italy. division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland, usa. mrc centre for causal analyses in translational epidemiology, department of social medicine, university of bristol, bristol, united kingdom. steno diabetes center, gentofte, denmark. department of clinical sciences, diabetes and endocrinology, lund university, university hospital malmö, malmö, sweden. department of medicine, university of kuopio and kuopio university hospital, kuopio, finland. department of epidemiology and population health, non-communicable disease epidemiology unit, london school of hygiene and tropical medicine, university of london, london, united kingdom. gerontology research center, national institute on aging, baltimore, maryland, usa. social medicine department, university of bristol, bristol, united kingdom. research centre for prevention and health, glostrup university hospital, glostrup, denmark. faculty of health sciences, university of aarhus, aarhus, denmark. savitaipale health center, savitaipale, finland. diabetes unit, department of health promotion and chronic disease prevention, national public health institute, and department of public health, university of helsinki, helsinki, finland. department of medicine, division of endocrinology, and department of physiology and biophysics, keck school of medicine, university of southern california, los angeles, california, usa. south ostrobothnia central hospital, senäjoki, finland. department of genetics, university of north carolina, chapel hill, north carolina, usa. laboratory of cardiovascular science, national institute on aging, nih, baltimore, maryland, usa. unità operativa geriatria, istituto nazionale ricovero e cura anziari, rome, italy. department of preventive medicine, keck school of medicine, university of southern california, los angeles, california, usa. introduction glucose is the major source of energy in humans, with levels in  vivo determined by a balance of glucose absorption via the gut,  production primarily by the liver, and utilization by both insulin- sensitive and insulin-insensitive tissues ( ,  ). homeostatic control  of glucose levels involves complex interactions between humoral  and neural mechanisms that work in concert to regulate tightly  the balance between production and utilization to maintain a nor- nonstandard	abbreviations	used: abcb , atp-binding cassette, subfamily  b (mdr/tap), member  ; bwhhs, british women’s heart and health study;  dgi, diabetes genetics initiative; fusion, finland–united states investigation of  non–insulin-dependent diabetes mellitus genetics; g pc , glucose- -phosphatase  catalytic subunit  ; gwa, genome-wide association; ld, linkage disequilibrium;  metsim, metabolic syndrome in men; t dm, type   diabetes mellitus. conflict	of	interest: the authors have declared that no conflict of interest exists. citation	for	this	article: j. clin. invest. doi: . /jci . identifying	the	genetic	variants	that	regulate	fasting	glucose	concentrations	may	further	our	understanding	of	the	 pathogenesis	of	diabetes.	we	therefore	investigated	the	association	of	fasting	glucose	levels	with	snps	in	 	genome- wide	scans	including	a	total	of	 , 	nondiabetic	individuals	from	finland	and	sardinia.	we	found	a	significant	 association	between	the	snp	rs 	and	fasting	glucose	concentrations	(p	=	 . × – ).	this	association	was	fur- ther	investigated	in	an	additional	 , 	nondiabetic	individuals	of	mixed	european	descent	from	 	different	stud- ies.	the	combined	p	value	for	association	in	these	follow-up	samples	was	 . × – ,	and	combining	results	from	all	 studies	resulted	in	an	overall	p	value	for	association	of	 . × – .	across	these	studies,	fasting	glucose	concentra- tions	increased	 . – . 	mm	with	each	copy	of	the	major	allele,	accounting	for	approximately	 %	of	the	total	varia- tion	in	fasting	glucose.	the	rs 	snp	is	located	between	the	genes	glucose- -phosphatase	catalytic	subunit	 		 (g pc )	and	atp-binding	cassette,	subfamily	b	(mdr/tap),	member	 	(abcb ).	our	results	in	combination	 with	data	reported	in	the	literature	suggest	that	g pc ,	a	glucose- -phosphatase	almost	exclusively	expressed	in	 pancreatic	islet	cells,	may	underlie	variation	in	fasting	glucose,	though	it	is	possible	that	abcb ,	which	is	expressed	 primarily	in	liver,	may	also	contribute	to	such	variation. research article �	 the	journal	of	clinical	investigation      http://www.jci.org mal fasting glucose. elevations in blood glucose are diagnostic of  diabetes. type   diabetes mellitus (t dm) afflicts more than    million worldwide and is a leading cause of kidney failure, blind- ness, and lower limb amputations ( – ). even more modest eleva- tions in glucose concentration (so-called prediabetes) are associated  with cardiovascular disease and accelerated atherosclerosis ( ). in  individuals progressing toward future t dm, the fasting glucose  concentration appears to change only modestly over time until the  advent of β cell dysfunction, at which point the glucose concen- tration increases rapidly ( ,  ). many studies have shown that the  lowering of glucose levels in individuals with diabetes can prevent  or delay diabetes-related complications, providing further evidence  for the damaging effects of chronic glucose elevations. both genetic and environmental factors contribute to the patho- physiology of t dm ( – ). the contributions of environmental  exposures to t dm risk are best illustrated by results from the dia- betes prevention program ( ) and the finnish diabetes prevention  study ( ), in which t dm incidence was significantly reduced  by intensive lifestyle modification. however, the contribution of  genetic factors to t dm risk is not as well understood. recent  genome-wide association (gwa) studies have identified   novel  t dm susceptibility loci ( – ), generating new insights into the  genetic architecture underlying t dm. in contrast to disease sta- tus, even less is known about genetic variation that alters specific  t dm-related quantitative traits such as glucose and insulin con- centrations. as seen for t dm, identification of genetic variants  associated with t dm-related quantitative traits is likely to require  large sample sizes due to relatively small gene effect sizes. fasting  glucose concentrations have been shown to be heritable, with nar- row-sense heritability estimates ranging from  % to  % ( – ).  given the central role of glucose concentration in the pathogenesis  and diagnosis of t dm and its complications, gwa for glucose  concentrations provides an excellent opportunity to identify genes  underlying variation in glucose concentrations that may also repre- sent additional t dm susceptibility loci. an example of this comes  from the studies by weedon et al., who showed by metaanalysis and  large cohorts that variation in the glucokinase gene was associated  with both fasting glucose and birth weight ( ). gwa studies for t dm and adiposity were completed by the  groups undertaking the finland–united states investigation of  non–insulin-dependent diabetes mellitus genetics (fusion)  ( ,  ,  ) and the sardinia study of aging ( ,  ), respec- tively. both studies assessed fasting glucose in their respective  cohorts, allowing gwas for fasting glucose in each study and  combination of these results in a metaanalysis. the strongest  signals from the fasting glucose gwa metaanalysis were from  variants near genes for atp-binding cassette, subfamily b (mdr/ tap), member   (abcb ) and glucose- -phosphatase catalytic  subunit   (g pc ). this association was replicated in a series of   studies involving a total of  ,  individuals ( ,  – ), sug- gesting for what we believe is the first time that variation in one  of these genes may play a role in the regulation of fasting glucose  concentrations in humans. results subject demographics and clinical characteristics for the fusion  and sardinia samples are summarized in table  . because treat- ment for t dm affects fasting glucose concentrations, all analyses  in this report were restricted to nondiabetic subjects. initial review  of association results from both the fusion stage   and sardinia  gwa scans of a combined total of  ,  nondiabetic individuals  focused on snps that were genotyped in the sardinia study and  imputed in the fusion study. among these, rs  exhibited  the strongest evidence for association in both samples (sardinia,  p =  . × – ; fusion stage  , p =  . × – ; table  ), with a  metaanalysis p value of  . × – . given the strength of this initial  association, our follow-up efforts focused on rs . additional  independent associations from our fasting glucose gwa study are  presented in supplemental table   (supplemental material avail- able online with this article; doi: . /jci ds ). analyses were repeated once imputation was completed in both  the fusion stage   and sardinia samples. snp rs  and  other snps in strong linkage disequilibrium (ld; defined as r  >  .    in the fusion samples) constituted the   strongest association  results in the combined fusion/sardinia gwa for fasting glu- cose metaanalysis (figure  ). in fact,   snps associated with fast- ing plasma glucose with p ≤  × –  were located within a  . -kb   region on chromosome   (supplemental table  ). these snps  were located in an extended region of ld that spans   biologically  plausible candidate genes for glucoregulation (figure  ). the first  is  g pc ,  also  known  as  islet-specific  glucose- -phosphatase– related protein (igrp). g pc  is part of a larger family of enzymes  involved in hydrolysis of glucose- -phosphate in the gluconeogen- ic and glycogenolytic pathways ( ,  ). the second is abcb ,  a member of the mdr/tap subfamily of atp-binding cassette  transporters involved in multidrug resistance ( ,  ). table � subject demographics and clinical characteristics for individuals with rs genotype data study	 phenotyped		 geographic		 study	age		 bmi		 fasting		 	 subjects	 origin	 (years)	 (kg/m )	 glucose	(mm) fusion stage , finland . ( . ) . ( . ) . ( . ) fusion stage finland . ( . ) . ( . ) . ( . ) fusion additional spouses/offspring finland . ( . ) . ( . ) . ( . ) sardinia , sardinia, italy . ( . ) . ( . ) . ( . ) dgi , finland and sweden . ( . ) . ( . ) . ( . ) amish , usa . ( . ) . ( . ) . ( . ) metsim , finland . ( . ) . ( . ) . ( . ) caerphilly , united kingdom . ( . ) . ( . ) . ( . ) bwhhs , united kingdom . ( . ) . ( . ) . ( . ) inter , denmark . ( . ) . ( . ) . ( . ) values are reported as median (interquartile range). research article 	 the	journal	of	clinical	investigation      http://www.jci.org  � among all genotyped or imputed snps in this region, rs ,  which was genotyped in fusion stage  , and imputed and fol- lowed up by genotyping in sardinia, showed the strongest overall  evidence for association (sardinia, p =  . × – ; fusion stage  ,   p =  . × – ; supplemental table  ), with a metaanalysis p value  of  . × – . in addition, rs  and rs , both located in  intron   of abcb  and in perfect ld with each other (d′ =  . ,   r   =  . ),  showed  strong  evidence  for  association  with  fasting  glucose concentrations with metaanalysis p values of  . × –   and  . × – , respectively (supplemental table  ). rs  is  located  .  kb from rs  and  .  kb from rs  and is  in strong ld with both snps (d′ =  . , r  =  .  with rs ;  and d′ =  . , r  =  .  with rs ). rs  is  .  kb from  rs , is in high ld with rs  (d′ =  . , r  =  . ), and  is located in intron   of g pc . in contrast, rs  lies between  g pc  and abcb  and is in extended ld with abcb . in both  the sardinia and fusion stage   samples, each copy of the a  allele for rs  was associated with small increases in fast- ing glucose ( .  mm for sardinia and  .  mm for fusion  stage  ; table  ) that are clinically insignificant and accounted  for approximately  % of the variance in fasting glucose. similar  effect sizes were observed for rs  ( .  mm for sardinia  and  .  mm for fusion stage  ). we assessed the potential contribution of population stratifica- tion by computing the genomic control parameter ( ) indepen- dently for both studies. the genomic control values were  .  for  both fusion and sardinia, suggesting that population stratifi- cation and/or unmodeled relatedness did not contribute signifi- cantly to our observed association. analyses that included bmi  as a covariate did not significantly alter the association between  rs  and fasting glucose in the fusion stage   (p =  . × –   without bmi versus  . × –  with bmi) and sardinia samples  ( . × –  without bmi versus  . × –  with bmi) independently  or jointly (p =  . × –  without bmi versus  . × –  with bmi),  suggesting the association was not a consequence of adiposity,  which is known to induce insulin resistance and increase glucose  concentrations ( ). the association between rs  and fasting  glucose also remained significant after individual adjustment for  each of the   snps shown to be associated with t dm in our  recent gwa studies (supplemental table  ) ( – ) or when all   snps were included jointly in the model (p =  . × –  versus  p =  . × –  for fusion stage   samples and  . × –  versus  . × –  for sardinia samples). the   snps shown to be associ- ated with t dm were themselves not significantly associated with  fasting glucose concentrations in the fusion stage   or sardinia  samples (supplemental table  ). fusion investigators genotyped rs  in   stage   sam- ples and   additional spouses and offspring of t dm patients  included  in  stage  ;  this  snp  continued  to  show  evidence  for  association with fasting glucose (fusion stage  , p =  . × – ;  fusion stage   families, p =  . × – ; see table  ). the meta- analysis that combined results from the fusion stage   and    and sardinia studies resulted in a p value of  . × – , surpassing  standard thresholds for genome-wide significance. we also examined the association between rs  and fast- ing glucose in   follow-up samples (table  ). the characteristics  of these samples are summarized in table  . association between  rs  and fasting glucose was confirmed in the amish study  (p =  . × – ); the metabolic syndrome in men study (metsim;   p =  . × – ), the caerphilly study ( . × – ), the british wom- en’s heart and health study (bwhhs; p =  . × – ), and inter   (p =  . × – ; table  ), with fasting glucose concentrations increas- ing with each copy of the a allele in all studies. while evidence for  association in the diabetes genetics initiative (dgi) study was  not statistically significant (p =  . ; table  ), the results show a  trend in the same direction as observed in the other samples. when  the results from all follow-up studies were combined in a meta- analysis of  ,  samples, there was strong evidence for associa- tion between rs  and fasting glucose in both the follow-up  samples (n =  , , p =  . × – ; table  ) and in all gwa and  follow-up samples combined (n =  , , p =  . × – ; table  ).   in contrast, rs  did not show evidence for association with  t dm in the fusion stage   study (p =  . ), the dgi gwa sam- ples (p =  . ), or the metsim study (p =  . ). table � association between rs and fasting glucose in nondiabetic individuals 	 	 	 frequency	 mean	fasting	glucose	(mm)	(sd)	 effect	(se)	 effect	(se) study	 n	 c	allele	 cc	 ac	 aa	 mm	 standardized	 p	value gwa samples fusion stage , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – sardinia , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – . × – a fusion familiesb , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – follow-up samples fusion stage . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – dgi , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . amish , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – metsim , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – caerphilly , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – bwhhs , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – inter , . . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . × – . × – c . × – d glucose values are reported unadjusted for covariates. a gwa metaanalysis p value. bthese data represent the additional spouses and offspring com- bined with their fusion stage family members. c follow-up metaanalysis p value. d overall metaanalysis p value. research article �	 the	journal	of	clinical	investigation      http://www.jci.org figure    shows  the  results  of  a  metaanalysis  based  upon  the  effect size observed in each of the   studies. overall, fasting glucose  concentrations increased  .  mm ( % ci:  . – .  mm)   with each copy of the major allele. discussion we took advantage of gwa studies originally performed to identify  susceptibility genes for t dm (fusion) and aging-related traits  (sardinia) to also identify genes underlying variation in fasting  glucose concentration. both fusion and sardinia initially identi- fied rs  as being associated with fasting glucose levels. given  that both studies were performed in relatively homogeneous popu- lations of mixed european descent, it is unlikely that population  stratification accounted for the initial association. the estimated  genomic control ( ) values for fusion stage   and sardinia were  both  . , providing further evidence against the contribution of  population stratification to the observed association. in  the  sardinia  sample,  we  genotyped  snps  rs   and  rs  to validate the results based on imputation. the dis- crepancy rate per allele between the imputed and typed genotypes  at these   snps was  . % and  . %, respectively, and the associa- tion result with the actual genotypes was stronger than with the  imputed genotypes: p =  . × –  and  . × – , respectively adiposity may induce insulin resistance and thus alter glucose  concentrations ( ) independent of the effects of the snp on glu- cose concentrations per se. however, the association remained sig- nificant even when we included bmi as a covariate in the analysis,  suggesting adiposity is not a major contributor to the observed  association.  similarly,  in  the  follow-up  studies,  the  results  did  not change whether bmi was included or excluded as a covariate.  some known sex-specific effects, such as differences in fat distri- bution, could also confound our results. we found no sex-specific  effect modification in the fusion and sardinia samples. also, it  should be noted that we observed evidence for association between  rs  in the metsim and caerphilly samples that only includ- ed men and in the bwhhs that comprised women only. thus,  the lack of a sex-specific effect in fusion and sardinia is sup- ported by the independent associations observed in these samples.  subsequent analyses of the gwa data revealed rs  as having  the strongest evidence for association with fasting glucose in this  region and suggested, based on the snp location, that g pc  plays  a role in glucoregulation. however,   additional snps in strong ld  with rs  located in the adjacent abcb  also showed similar  evidence for association with fasting glucose. in the   follow-up studies, rs  continued to show associa- tion with fasting glucose, although marginal evidence for hetero- geneity among studies was noted (q =  . ; p =  . ; i  =  . %;  % ci:  . – . %) ( ). for example, the dgi samples did not  exhibit a significant association and the bwhhs samples, despite  being among the largest follow-up samples, showed only modest  evidence for association (table  ). these   studies yielded similar  effect size estimates ( .  for dgi and  .  for bwhhs) that  were smaller than in the other studies (table  ). differences in  both populations and sample ascertainment could be contribut- ing to the observed heterogeneity. when these   studies are not  considered, the heterogeneity estimate is reduced (q =  . ; p =  . ;  i  =  %;  % ci =  – . %) however, despite the variability in effect  size, the direction of the effect was the same in all studies. there are   biologically plausible candidate genes in the region  identified  by  our  association  analyses  that  may  affect  glucose  levels. although rs , which is located in intron   of g pc   just   bp proximal to exon  , showed the strongest evidence for  association in the gwa studies, snps in ld with rs  and  rs  that show similar levels of association with fasting glu- cose concentrations were located in intron   of abcb . abcb   is involved in atp-dependent secretion of bile salts and is almost  exclusively expressed in the liver. mutations in abcb  have been  shown  to  be  associated  with  intrahepatic  cholestasis  (omim  ) ( ) and drug-induced hepatotoxicity ( ,  ). in anti- lipid drug trials, bile acid sequestrants have been shown to lower  glucose concentrations and improve insulin sensitivity, presum- ably through reduction of triglyceride levels ( ). based upon these  observations, if abcb  were contributing significantly to varia- tion in fasting glucose, one might expect to also see associations  figure � fasting glucose association in the fusion and sardinia gwa metaanalysis. top panel shows evidence for association with fasting glucose under an additive genetic model for the com- bined fusion stage and sardinia metaanal- ysis. the –log(p value) for the test of associa- tion is plotted against genomic position (ncbi build ) for all genotyped (red circles, sardinia; blue circles, fusion) and imputed (gray circles) snps in the sardinia data. snps typed in both samples are indicated by red circles with blue centers. snps rs and rs were later typed in the sardinia samples, and the actual genotypes resulted in even stronger asso- ciation than shown here. bottom panel shows the ld pattern (r ) around g pc and abcb for the ceph (utah residents with ancestry from northern and western europe) sample from the hapmap. the scale at the bottom shows the magnitude of ld in colors, ranging from blue for low ld to red for high ld. research article 	 the	journal	of	clinical	investigation      http://www.jci.org  � with lipids or insulin sensitivity. however, rs , rs ,  rs , and rs  were not associated with lipid measure- ments in a metaanalysis of fusion stage   and sardinia samples  (p >  . ). also, none of these snps were associated with minimal  model-derived insulin sensitivity in fusion samples (p >  . ).  thus, our data do not support a role for abcb  in glucoregula- tion, and other evidence directly linking abcb  to regulation of  glucose concentrations is scarce. in contrast, g pc , the β cell–specific isoform of glucose- -phos- phatase is a highly relevant candidate gene for glucoregulation. the  mouse homolog g pc  has been previously implicated as an auto- antigen in the nod mouse model of type   diabetes ( ). wang et  al. recently generated g pc -null mice and noted that at   weeks  of age, fasting glucose concentrations had decreased approximately  % in both male and female g pc -null mice when compared with  wild-type mice ( ). this modest decrease in glucose concentration  was observed despite the absence of any differences in body weight,  fasting insulin, or fasting glucagon concentrations. the character- istics of these g pc -null mice closely paralleled our observations  that rs  and rs  were associated with modest chang- es in fasting glucose but not in bmi or fasting insulin, which are  consistent with the hypothesis that presence of a c allele results in  lower g pc  expression and therefore lower glucose concentrations.  interestingly, g pc  mrna levels appear to increase with increasing  glucose concentration in isolated mouse islets ( ). molecular cloning of g pc  identified   splice forms that differ  by the presence or absence of exon   in balb/c and ob/ob mice  and in insulinoma tissue ( ). the longer cdna including exon    has approximately  % homology with glucose- -phosphatase cat- alytic subunit (g pc) across a variety of species including humans  and is membrane bound in the endoplasmic reticulum ( ). the  corresponding g pc  splice forms have been observed in human  pancreas ( ). rs  is located in intron  , just   bp proximal  to exon  , raising the possibility that this variant may play a role in  whether the full-length transcript is formed. g pc  hydrolyzes  glucose- -phosphate  to  form  glucose  and  release a phosphate group. despite its similarity to g pc, g pc   is reported to have little to no hydrolase activity in humans ( ,  ,  ,  ). in normal and genetically obese mice, the splice form lack- ing exon   appears to be the most predominant observed in islets  ( ) and lacks sequences that may be critical for hydrolytic activity  ( ,  ), suggesting the full-length form of g pc  may have impli- cations for activity of g pc  and its potential role in glucoregula- tion. greater hydrolase activity has been reported in cell lines over- expressing the full-length form of g pc  ( ). also, in islets from  streptozotocin-treated mice, glucose cycling, an indicator of g pc   activity, was approximately  -fold higher compared with islets from  untreated mice ( ), and even greater increases were observed in  islets from ob/ob mice ( ,  ). the conversion of glucose to glu- cose- -phosphate is the critical step in stimulus-secretion coupling  for  insulin  secretion.  variation  in  g pc   may  increase  glucose  cycling in β cells, resulting in altered generation of atp, which  would have implications for insulin secretion. in addition, g pc - induced alterations in β cell glucose metabolism would also have  downstream effects on phosphoinositide  -kinase activity, which  regulates pancreas duodenum homeobox-  (pdx ) binding to the  insulin gene and subsequent insulin gene transcription ( ). the possible role for g pc  in altering glucose concentrations  raises the question of whether this gene also confers susceptibil- ity to t dm. we observed no association between fasting glucose  and rs  and rs  in individuals with t dm from the  fusion, dgi, and metsim studies (p >  . ). however, the anal- ysis of fasting glucose concentration in individuals with t dm  is confounded by diabetes pathology, treatment, and differential  response to therapy. therefore the lack of association with fast- ing glucose in individuals with t dm does not preclude g pc   as  contributing  to  susceptibility  to  t dm.  similarly,  when  we  tested  these  snps  for  association  with  t dm  in  the  fusion,  dgi, and metsim samples, we observed no evidence for associa- tion (p >  . ). further, the modest effect on glucose concentra- tions observed in our analysis of nondiabetic individuals suggests  we may lack sufficient power to detect association with t dm.  whereas the cumulative evidence would suggest that g pc  may  regulate fasting glucose concentrations and does not contribute  significantly  to  susceptibility  to  t dm,  larger  studies  may  be  required to elucidate the role of this gene in t dm susceptibility. variation in the promoter region of glucokinase (gck, rs )  has been shown to be associated with fasting glucose and impaired  insulin secretion ( – ) and may play a role in altering birth weight  ( ). these initial findings were confirmed in a comprehensive meta- analysis performed by weedon et al., demonstrating that rs   was associated with fasting glucose (meta p =  . × – ) and that the  presence of a maternal a allele for rs  was associated with  increased birth weight of the child (p =  . ) ( ). gck, an enzyme  that works counter to g pc , converts glucose to glucose- -phos- phate,  forming  the  critical  step  in  secretion-stimulus  coupling  in pancreatic β cells. in addition, the recent gwa study from the  dgi identified variation in glucokinase regulatory protein (gckr)  (rs ) to be associated with triglyceride levels ( ). gckr is an  allosteric regulator of gck in both liver and pancreatic islets whose  inhibitory  effect  is  enhanced  by  fructose- -phosphate  and  sup- pressed by fructose- -phosphate ( ). we found modest evidence  figure � effect size and % ci for rs are shown for the studies. the overall metaanalysis across these studies yielded an effect size of . mm ( % ci: . , . mm). research article �	 the	journal	of	clinical	investigation      http://www.jci.org for association between fasting glucose and rs  (fusion  stage  , p =  . × – ; sardinia, p =  . × – ; meta p =  . × – )   and no evidence for association between fasting glucose and rs   (fusion stage  , p =  . ; sardinia, p =  . ; meta p =  . ).   while these results provide evidence for association between varia- tion in gck and fasting glucose but not between gckr and fast- ing glucose in our studies, we cannot exclude the possibility that a  complex interaction among gck, gckr, and g pc  may regulate  fasting glucose levels. this will require further study. in conclusion, we used gwa to identify variation in both abcb   and  g pc  as genes that potentially contribute to variation in  fasting glucose concentrations in nondiabetic subjects of mixed  european descent. there is more literature with data supporting  a role for g pc , but in the absence of functional data, we cannot  discount the possibility that abcb  may also contribute signifi- cantly to variation in fasting glucose concentration. heritability  for fasting glucose has been estimated to be  %– % ( – ),   yet the variants we identified account for approximately  % of  the variance in fasting glucose, indicating that the majority of the  variability in fasting glucose remains unexplained. the remaining  variability is likely due to the effects of additional common genetic  variants of modest effect, less common genetic variants of mod- erate effect, and a variety of gene-gene and gene-environmental  interaction effects. it should also be noted that the magnitude of  the effect observed in our study is consistent with other reports of  quantitative trait associations ( – ). additional studies, likely with larger sample sizes, will be required  to identify additional genetic variants contributing to variation  in fasting glucose. the variants identified in our study are not  likely to be functional, but in ld with the functional variant(s).  additional fine mapping, sequencing, and functional studies will  be required to define the molecular mechanisms underlying our  observed association. methods the fusion and sardinia study samples and gwa genotyping have been  described in detail ( ,  ,  – ). here, we briefly review the study cohorts  and genotyping methods. we also describe briefly each of the   follow-up  samples. subject demographics and basic clinical characteristics for indi- viduals genotyped for rs  for each sample are described below and  summarized in table  . all protocols were approved by the institutional  review boards or research ethics committees at the respective institutions,  and informed consent was obtained from all subjects. fusion gwa study. the goal of the fusion study is to identify genet- ic variants that predispose to t dm or that determine the variability in  t dm-related quantitative traits. the study began as an affected sibling- pair family study ( ,  ), later augmented by large numbers of cases and  controls for association analysis ( ). the fusion gwa study was per- formed using a  -stage case-control design ( ). cases and controls were  approximately frequency matched on  -year age category, sex, and birth  province. all stage   dna samples were genotyped using the illumina  humanhap  beadchip version  . , resulting in data on  ,  snps  that passed quality control filters ( ). genotype data for an additional  .  million snps were estimated using an imputation procedure ( ).  the genotype imputation method uses stretches of chromosome shared  between  individuals  genotyped  at  relatively  low  density  in  our  studies  and individuals genotyped in greater density by the international hap- map consortium ( ) to estimate the missing genotypes. comparison of  imputed and measured genotypes yielded estimated error rates of  . %  (illumina) to  . % (affymetrix) per allele with an average concordance of  . %, consistent with expectations from hapmap data ( ). snps show- ing promising association with fasting plasma glucose in the stage   sam- ples were genotyped in the stage   dna samples by homogeneous massex- tend reaction using the massarray system (sequenom) ( ). because  treatment for t dm affects fasting glucose concentrations, all analyses  in this report were restricted to nondiabetic subjects. diabetes status was  confirmed by who criteria ( ) or confirmation of treatment for diabe- tes by medical record review. fasting plasma glucose concentrations were  available for  ,  stage   and   stage   samples. additional fusion  samples included nondiabetic spouses or offspring from fusion stage   families; fasting plasma glucose data were available for   individuals.  these   samples were genotyped using the applied biosystems taq- man allelic discrimination assays ( ) and yielded   samples with both  genotype and fasting glucose data. these samples were integrated into the  fusion stage   samples and independently analyzed to assess whether the  additional family members improved the evidence for association. we have  denoted this analysis fusion families. sardinia gwa study. the sardinia study is a longitudinal study of aging- related quantitative traits and comprises a cohort of  ,  individuals    years or older recruited from   towns in the lanusei valley in sardinia.  data from  ,  individuals with fasting serum glucose measurements  from this cohort were used for the gwa study;  ,  were genotyped using  the affymetrix  k snp mapping array, and an additional  ,  were  genotyped using the affymetrix  k snp mapping array ( ).  ,   snps passed quality control and were tested for association with fasting  serum glucose. we first used the genotyped snps in the  ,  individuals  to estimate genotypes for all the polymorphic snps genotyped by the hap- map consortium. taking advantage of the relatedness among individuals  in the sardinia sample, we then conducted a second round of computa- tional analysis to impute genotypes for analysis in the  ,  individuals  not genotyped with the  k snp array. in this second round, we identi- fied large stretches of chromosome shared within each family and proba- bilistically “filled-in” genotypes within each stretch whenever   or more  of its carriers was genotyped with the  k array set ( ,  ). for these  analyses,   non-sardinians and   of their family members (n =  )   and   individuals with known diabetes were excluded from the analysis,  resulting in a final sample size of  , . follow-up samples. the initial association identified in the metaanalysis  of the fusion and sardinia gwa studies was also tested in a series of  follow-up samples (table  ),   from fusion described above and   others,  which are described briefly below. dgi.  the  dgi  case-control  gwa  sample  consists  of  ,   cases  with  t dm and  ,  normoglycemic controls from finland and sweden and  has been previously described in detail ( ). fasting glucose measurements  were available for  ,  nondiabetic control subjects ( ,  unrelated sub- jects and   siblings). among these, fasting plasma glucose was measured in   subjects and fasting whole blood glucose was measured in   subjects.  whole-blood glucose concentrations were converted to equivalent plasma  values using a conversion factor of  .  ( ). all samples were genotyped  using the affymetrix genechip human mapping  k array set; results of  gwa of  ,  snps with fasting glucose levels (including snp rs )  are publicly available at www.broad.mit.edu/diabetes/scandinavs/index. html.  ,  individuals were available with both rs  genotype and  fasting glucose data. old order amish subjects. the old order amish study participants report- ed here were  ,  nondiabetic subjects from lancaster, pennsylvania,  usa, for whom fasting plasma glucose measurements were available. these  subjects were enrolled in ongoing family studies of complex diseases and  traits ( – ). genotyping for rs  was performed using the taqman  allelic discrimination assay ( ). research article 	 the	journal	of	clinical	investigation      http://www.jci.org  � metsim study.  subjects  were  selected  from  the  ongoing  metsim  study, which includes  ,  men, aged   to   years, randomly selected  from the population of the town of kuopio, eastern finland, finland  (population  , ). the present analysis is based on the first  ,  non- diabetic subjects examined for metsim with available fasting plasma  glucose values. genotyping was performed using the taqman allelic dis- crimination assay ( ). caerphilly study. the caerphilly study is a cohort study of white, euro- pean men (n =  , ;  . % born in the united kingdom), aged  –  years  at entry in  –  ( ), recruited from the town of caerphilly, united  kingdom, and   adjacent villages. men were selected using the electoral role  and general practitioner records. dna and fasting plasma glucose measure- ments used in this study relate to the first phase of data collection. bwhhs. the bwhhs consists of female participants, aged   to   years  and recruited between april   and march  . initially,  ,  women  were randomly selected from   british towns and were interviewed and  clinically examined. they also completed medical questionnaires ( ). genotyping for the caerphilly study and bwhhs was performed by  kbioscience using their f luorescence-based competitive allele-specific  pcr (kaspar) technology. the inter study. rs  was genotyped in  ,  danes for whom fast- ing plasma glucose values were available. this sample comprises part of the  population-based inter  sample of middle-aged people sampled at research  centre for prevention and health (glostrup, denmark; refs.  ,  ). geno- typing was performed using taqman allelic discrimination (kbioscience). statistics. association between fasting glucose and genotypes in the fusion  and sardinia studies was carried out using a regression framework in which  regression coefficients were estimated in the context of a variance compo- nent model to account for relatedness among individuals ( ). for fusion  samples, plasma glucose concentration was adjusted for sex, age, age , birth  province, and study group. analyses were carried out in nondiabetic individ- uals excluding those known to be taking medications that directly affect glu- cose concentration. similarly, sardinia serum glucose values were adjusted  for sex, age, and age . because diabetes-based exclusions were based only on  medical records and sardinia only measured fasting serum glucose, a small  number of undiagnosed new-onset diabetes cases may have been included  in the analysis. for both studies, analyses were repeated including bmi as  an additional covariate to assess whether adiposity significantly contributed  to the evidence for association. covariate-adjusted trait values were trans- formed to approximate univariate normality by applying an inverse normal  scores transformation; the scores were ranked, ranks were transformed into  quantiles, and quantiles were converted to normal deviates. a weighted z score–based fixed effects metaanalysis method was used to  combine results from the fusion and sardinia studies. in brief, for each  snp, a reference allele was identified and a z statistic summarizing the mag- nitude of the p value for association and direction of effect was generated for  each study. an overall z statistic was then computed as a weighted average  of the individual statistics, and a corresponding p value for that statistic was  computed. the weights were proportional to the square root of the num- ber of individuals in each study and scaled such that the squared weights  summed to  . for the metaanalysis of the effect size, the inverse variance was  used as weights for each study. for the fusion   families (fusion stage    plus additional fusion spouses and offspring) a regression-based analysis  under a variance components framework was used to appropriately account  for relationships among individuals ( ). because we did not have birth prov- ince information for the additional spouses and offspring, these analyses  were carried out adjusting for age, age , sex, and study group only. given the different sampling schemes, statistical analyses for the follow- up samples varied by study. the old order amish samples consisted of  large amish pedigrees, so the evidence for association between genotype  and  fasting  plasma  glucose  was  evaluated  using  variance  components  analysis implemented in solar to adjust for the relatedness of study sub- jects ( ,  ). plasma glucose levels were natural logarithm transformed for  analysis, and covariates included sex, age, and age . for the dgi study, glu- cose values were converted to z scores separately by sex, and tests for associ- ation were carried out using a regression framework with age and log(bmi)  included as covariates; genomic control was applied to account for related- ness ( ). for the metsim study, analyses were carried out identically as  in fusion, with the exception that birth province was not included as a  covariate. for the caerphilly and bwhhs studies, association was assessed  using a regression framework with age, age , and bmi as covariates. for  the inter  study, association was assessed using a regression framework  with age and sex as covariates. individuals with known diabetes at the time  of examination were excluded from the analyses. results from all follow- up studies were combined in a metaanalysis as described above. finally, a  metaanalysis that combined results from all gwa and follow-up studies  was performed as described above. acknowledgments we would like to thank the many research volunteers who generous- ly participated in the various studies represented in this study. for  the fusion study, we also thank peter s. chines, narisu narisu,  andrew g. sprau, and li qin for informatics and genotyping sup- port and the center for inherited disease research for the fusion  gwa genotyping. for the sardinia study, we thank the mayors of  lanusei, ilbono, arzana, and elini, the head of local public health  unit asl , and the residents of the towns for their volunteerism  and cooperation. in addition, we are grateful to the mayor and the  administration in lanusei for providing and furnishing the clinic  site. we thank the team of physicians — maria grazia pilia, danilo  fois, liana ferreli, marcello argiolas, francesco loi, and pietro  figus — and the nurses paola loi, monica lai, and anna cau, who  carried out the physical examinations and made the observations. we thank the former medical research council (mrc) epide- miology unit (south wales) who undertook the caerphilly study.  the department of social medicine, university of bristol, now acts  as custodian for the caerphilly database. we are grateful to all of  the men who participated in this study. for the bwhhs, we thank  all of the general practitioners and their staff who supported data  collection and the women who participated in the study. for the amish studies, we thank members of the amish com- munity for the generous donation of time to participate in these  studies and our field nurses, amish liaisons, and clinic staff for  their extraordinary efforts. we also acknowledge sandy ott and  john shelton for genotyping of amish dna samples. support for this study was provided by the following: ameri- can diabetes association (ada) ( - -ra-  to r.m. watanabe;  - -ra-  to a.r. shuldiner; and postdoctoral fellowships to  c.j.  willer  and  h.m.  stringham);  and  nih  grants  (dk   and u  da  to r.m. watanabe; dk  to m. boehn- ke;  dk   to  k.l.  mohlke;  dk   to  w-m.  chen;  r   dk , u  hl , and r  ag  to a.r. shuldiner;  r  hl  to b.d. mitchell; and r  dk  to k.d. sil- ver). d.a. lawlor is funded by a uk department of health career  scientist award, and n. timpson is funded by a studentship from  the mrc of the united kingdom. the  inter   study  was  supported  by  the  european  union  (eugene , lshm-ct- - ); the lundbeck founda- tion centre of applied medical genomics in personalized dis- ease prediction, prevention and care; the food study group/ research article �	 the	journal	of	clinical	investigation      http://www.jci.org the  danish  ministry  of  food,  agriculture  and  fisheries  and  ministry of family and consumer affairs ( - - ); and  the danish medical research council. this research was supported in part by the intramural research  program of the nih, national institute on aging, and the niddk.  additional support came from contract n -ag- -  from the  nia intramural research program for the sardinia (progenia)  team;  national  human  genome  research  institute  intramural  project number   z  hg  (to f.s. collins); university of  maryland general clinical research center (m  rr  ); johns  hopkins university general clinical research center (m  rr  ); the niddk clinical nutrition research unit of maryland  (p  dk ); and the department of veterans affairs and veter- ans affairs medical center baltimore geriatric research, education  and clinical center (grecc). the bwhhs receives core funding  from the united kingdom department of health policy research  program. the dna extraction and genotyping for bwhhs were  funded by the british heart foundation. the caerphilly study was  funded by the mrc of the united kingdom. funding for the caer- philly dna bank was from an mrc grant (g ). the united  kingdom mrc supports work undertaken in the centre for causal  analyses in translational epidemiology. the views expressed in this paper are those of the authors and  not necessarily those of any funding body or others whose support  is acknowledged. those providing funding had no role in study  design, data collection and analysis, decision to publish, or prepara- tion of the manuscript. received  for  publication  november  ,  ,  and  accepted  in  revised form april  ,  . address correspondence to: angelo scuteri, unità operativa geria- tria, istituto nazionale ricovero e cura anziari, rome, italy. phone:  - ; fax:  - - 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dank für ihre anwesenheit! herzlich willkommen! aufrichtigen dank für ihre anwesenheit! william o. douglas (richter des obersten gerichtshofes der vereinigten staaten von zu ) “if a parent keeps his child out of school beyond the grade school, then the child will be forever barred from entry into the new and amazing world of diversity that we have today.” (justice william o. douglas beim im jahre erfolgten ausdruck seines partiellen dissenses bezüglich des yoder- spruchs seitens des obersten gerichtshofes der vereinigten staaten) „wenn ein elternteil das eigene kind vom zugang zur schule nach der erfüllung der schulpflicht fernhält, dann wird dem kind der zugang zur neuen und erstaunlichen welt der diversität für immer versperrt bleiben, die wir heute haben.“ thema podiumsdiskussion „ziele schulischer erziehung – soll erziehung in den schulen autonomie bei den schülerinnen und schülern fördern oder nicht? “ ao. univ.-prof. dr. karl garnitschnig doz. ostr prof. mag. heide manhartsberger-zuleger mag. dr. gianluigi segalerba montag, . märz , ab : volkshochschule mariahilf, damböckgasse , wien arbeitsprämisse alles steht zur verfügung: powerpoint und entwurf (sie sind jedoch vorerst noch nicht von einem muttersprachler oder von einer muttersprachlerin korrigiert worden) selbstpräsentation viel studiert, aber leider vorerst (noch) nicht genug index • einführung • der ursprung: yoder . • positionen von amy gutmann • positionen von arneson-shapiro. • positionen von william galston. die zentrale frage • was und welches ist das ziel der erziehung? spezifikationen zur zentralen frage ( ) • wie sollte die erziehung sein: kritisch, sokratisch, autonomiefördernd? sollte erziehung einer bestimmten tradition unterworfen sein? • welche sind die rechte der kinder in sachen erziehung? • welche sind die rechte der eltern in sachen erziehung? • welche sind die pflichte der eltern in sachen der erziehung? • welche sind die pflichte des staates in sachen erziehung? • welche ist die verantwortung des staates in sachen erziehung? • welche ist die legitime eingriffsmacht des staates ins privatleben der familien? spezifikationen zur zentralen frage ( ) • bis zu welchem punkt weitet sich die eingriffsmacht des staates in sachen erziehung? • hat die familie oder der staat den primat in sachen der bestimmung der erziehungsprogramme? • sind die kinder besitz von den familien? • soll der staat lediglich minimale erziehungsniveaus stellen? soll hingegen der staat größere erziehungsniveau stellen? • existieren gruppenrechte in sachen erziehung, oder existieren ausschließlich die rechte der individuen (der kinder)? • soll die die erziehung eine art vorbereitung zum leben in der gesellschaft sein? • welches soll das programm der erziehung sein, auf dass eine künftige bürgerin / ein künftiger bürger eine gute (im sinne von informierter) bürgerin / ein künftiger (im sinne von informiertem) bürger sein kann? • sollen die erziehungsprogramme des staates den vorrang über die projekte der eltern haben, oder sollen die projekte der eltern den vorrang über jedwede staatszuständigkeit haben? personen ( ) - arneson, richard ( -): lehre an der university of san diego, california. - callan, eamon ( -): lehre an der stanford university; publikationen: “autonomy and schooling” (kingston, ontario, ); “creating citizens: political education and liberal democracy” (oxford ). - feinberg, j., ( - ): lehre an der brown university, an der princeton university, an der ucla, an der rockefeller university and an der university of arizona; publikationen: “freedom and fulfillment: philosophical essays” (princeton ). - galston, william a. ( -): lehre an den brooking institutions; vormaliger professor an der school of public policy an der university of maryland; publikationen: “liberal purposes : goods, virtues, and diversity in the liberal state” (cambridge, u.k., ); “liberal pluralism: the implications of value pluralism for political theory and practice” (cambridge, u.k., ). personen ( ) - gutmann, amy ( -): präsidentin of the university of pennsylvania; publikationen: “democratic education” (princeton ). - raz, joseph ( -): lehre von jura an der columbia university law school und am king’s college london; vormalige lehre von rechtsphilosophie an der university of oxford; publikationen: “the authority of law” (oxford ); “the morality of freedom” (oxford ). - shapiro, ian ( -): professor von politikwissenschaft an der yale university; publikationen: (hrsg.) “democracy’s place” (ithaka, n.y., ); (herausgegeben mit will kymlicka) “ethnicity and group rights” (new york and london ); “the real world of democratic theory” (princeton, n.j., ). wisconsin yoder vs. wisconsin ( u.s. , ) • drei amish kinder aus drei verschiedenen familien wurden von der schule in new glarus, wisconsin (der name der schule war new glarus high school) nach dem ende des achten schuljahres wegen des glaubensbekenntnisses der eltern zurückgezogen: die fortsetzung der schulpflicht hätte die kinder von der gemeinde entfernt und die freie ausübung des eigenen glaubensbekenntnisses gehindert. • die drei familien wurden von jonas yoder, einem der väter, die im streit involviert waren, vertreten. • die familien verloren den fall vor dem green county court. • der oberste gericht von wisconsin entschied zugunsten von yoder: er begründete die eigene entscheidung damit, dass der verpflichtende schulbesuch gegen das recht der eltern, das von der klausel der freien ausübung des eigenen bekenntnisses vom ersten zusatzartikel der verfassung der vereinigten staaten geschützt werde, verstieße. erster zusatzartikel der verfassung der vereinigten staaten der kongress soll kein gesetz erlassen, das eine einrichtung einer religion zum gegenstand hat oder deren freie ausübung beschränkt, oder eines, das rede- und pressefreiheit oder das recht des volkes, sich friedlich zu versammeln und an die regierung eine petition zur abstellung von missständen zu richten, einschränkt. berufung das board of school von wisconsin legte berufung bei dem obersten gerichtshof der vereinigten staaten ein. der fall yoder: schülerin und schüler • frieda yoder ( ) • barbara miller ( ) • vernon yutzy ( ) der fall yoder: eltern • jonas yoder and wallace miller waren mitglieder der old order amish religion. • adin yutzy war mitglied der conservative amish mennonite church. inhalt des ersten zusatzartikels der verfassung der vereinigten staaten • congress shall make no law respecting an establishment of religion, or prohibiting the free exercise thereof; or abridging the freedom of speech, or of the press; or the right of the people peaceably to assemble, and to petition the government for a redress of grievances.” • „der kongress soll kein gesetz erlassen, das eine einrichtung einer religion zum gegenstand hat oder deren freie ausübung beschränkt, oder eines, das rede- und pressefreiheit oder das recht des volkes, sich friedlich zu versammeln und an die regierung eine petition zur abstellung von missständen zu richten, einschränkt.“ amish auffassungen innerhalb der amish maßstäbe wurde die höhere erziehung als nicht notwendig im verhältnis zum leben erachtet, das für die amish kinder zugedacht war. darüber hinaus wurde die höhere erziehung als beschädigend für die seelische rettung der amish. die familien plädierten vor dem obersten gerichtshof der vereinigten staaten für die ausnahme aus der verpflichtenden erziehung. die entscheidung des obersten gerichtshofs der vereinigten staaten (mehrheitsmeinung, synthese) der oberste gerichtshof der vereinigten staaten entschied auf die folgende art und weise: • die amish kinder dürfen nicht von der verpflichteten erziehung nach dem . schuljahr betroffen werden. • das recht der eltern, das eigene glaubensbekenntnis auszuüben, wurde als überwiegend im vergleich zu den interessen des staates, die kinder zu erziehen, erachtet. begründung ( ) der oberste gerichtshof begründete die eigene entscheidung auf die folgende art und weise: • die staaten dürfen nicht die individuen zum schulbesuch zwingen, wenn der schulbesuch gegen die rechte verstößt, die vom ersten zusatzartikel bestimmt sind. in diesem falle hatte der staat wisconsin mit der ausübung von einem legitimen glaubensbekenntnis interferiert. • nicht all die glaubensbekenntnisse entsprechen der erfordernisse der religiösen klausel des ersten zusatzartikels. dafür ist evidenz erforderlich, dass sachliche religiöse ausübungen vorliegen; die tatsache, dass individuen bestimmte maßstäbe herbeigeführt haben, reicht dazu nicht. begründung ( ) • der amish lebensweg ist jedoch ein lebensweg, der von tiefen religiösen Überzeugungen charakterisiert wird, die von der bibel stammen. dieser lebensweg ist von ihrer religion determiniert, welche die ablehnung der weltlichen güter und das leben in einer biblischen einfachheit einschließt. die zeitgenössische höhere erziehung konfligiert scharf mit diesem lebensweg. • der oberste gerichtshof ist mit der ansicht vom staate wisconsin nicht einverstanden, dass die hinzukommende erziehung nach dem . schuljahr dazu notwendig sei, die bürgerinnen und bürger für das politische system der vereinigten staaten auf wirksame und produktive art und weise vorzubereiten. der staat wisconsin hat keine evidenz dazu verschafft, dass zwei jahre mehr an öffentlichen schulen einen großen oder entscheidenden vorteil zur vorbereitung für das öffentliche leben repräsentieren. begründung ( ) • zudem behauptete der oberste gerichtshof, dass die amish gemeinde sehr erfolgreich in der amerikanischen gesellschaft ist, dass sie eine selbstversorgende, gesetzestreue komponente der gesellschaft ist, die all die steuern bezahlt und jede art von öffentlicher wohlfahrt ablehnt. die amish kinder setzen ihre erziehung in form der berufsausbildung fort, nachdem sie die öffentliche schule verlassen haben. • der oberste gerichtshof fand keine evidenz dafür, dass die amish kinder, wenn sie die amish gemeinde ohne die zwei jahre von hinzukommender erziehung verlassen, ein ballast für die gesellschaft werden würden. begründung ( ) • im gegensatz dazu war sich der oberste gerichtshof darüber einig, dass die kinder dank dem leben in der amish gemeinde über einen guten beruflichen hintergrund verfügten. • es war die falsche annahme seitens des staates wisconsin, dass die amish kinder ungebildet wären. • die verpflichtende erziehung nach der volkshochschule entsprach einer neueren orientierung, die am beginn des . jahrhunderts mit dem ziel entstand, kinderarbeit zu verhindern und kinder mit einem bestimmten alter in der schule beizubehalten. die ansicht vom staate wisconsin über das verpflichten des schulbesuches war infolgedessen weniger substantial. begründung ( ) auf den dissens vom richter douglas antwortend, behauptete der oberste gerichtshof, dass die erhobene frage das interesse der eltern betraf, das eigene glaubensbekenntnis auszuüben; sie befasste nicht mit den rechten der kinder, die aus dem ersten zusatzartikel entstammen. das argument über das recht der kinder, das freie glaubensbekenntnis auszuüben, war in diesem falle irrelevant. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika (synthese) • u.s. supreme court • wisconsin v. yoder, u.s. ( ) • wisconsin v. yoder • no. - • argued december , • decided may , • u.s. • certiorari to the supreme court of wisconsin entscheidung des obersten gerichtshofes der vereinigten staaten von amerika (synthese) • syllabus • respondents, members of the old order amish religion and the conservative amish mennonite church, were convicted of violating wisconsin's compulsory school attendance law (which requires a child's school attendance until age ) by declining to send their children to public or private school after they had graduated from the eighth grade. the evidence showed that the amish provide continuing informal vocational education to their children designed to prepare them for life in the rural amish community. the evidence also showed that respondents sincerely believed that high school attendance was contrary to the amish religion and way of life, and that they would endanger their own salvation and that of their children by complying with the law. the state supreme court sustained respondents' claim that application of the compulsory school attendance law to them violated their rights under the free exercise clause of the first amendment, made applicable to the states by the fourteenth amendment. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika (synthese) respondents, members of the old order amish religion and the conservative amish mennonite church, were convicted of violating wisconsin's compulsory school attendance law (which requires a child's school attendance until age ) by declining to send their children to public or private school after they had graduated from the eighth grade. the evidence showed that the amish provide continuing informal vocational education to their children designed to prepare them for life in the rural amish community. the evidence also showed that respondents sincerely believed that high school attendance was contrary to the amish religion and way of life, and that they would endanger their own salvation and that of their children by complying with the law. the state supreme court sustained respondents' claim that application of the compulsory school attendance law to them violated their rights under the free exercise clause of the first amendment, made applicable to the states by the fourteenth amendment. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika (synthese) • held: • . the state's interest in universal education is not totally free from a balancing process when it impinges on other fundamental rights, such as those specifically protected by the free exercise clause of the first amendment and the traditional interest of parents with respect to the religious upbringing of their children. pp. u. s. - . • . respondents have amply supported their claim that enforcement of the compulsory formal education requirement after the eighth grade would gravely endanger if not destroy the free exercise of their religious beliefs. pp. u.s. - • . aided by a history of three centuries as an identifiable religious sect and a long history as a successful and self-sufficient segment of american society, the amish have demonstrated the sincerity of their religious beliefs, the interrelationship of belief with their mode of life, the vital role that belief and daily conduct play in the continuing survival of old order amish communities, and the hazards presented by the state's enforcement of a statute generally valid as to others. beyond this, they have • page u. s. • carried the difficult burden of demonstrating the adequacy of their alternative mode of continuing informal vocational education in terms of the overall interest that the state relies on in support of its program of compulsory high school education. in light of this showing, and weighing the minimal difference between what the state would require and what the amish already accept, it was incumbent on the state to show with more particularity how its admittedly strong interest in compulsory education would be adversely affected by granting an exemption to the amish. pp. u. s. - , u. s. - . • . the state's claim that it is empowered, as parens patriae, to extend the benefit of secondary education to children regardless of the wishes of their parents cannot be sustained against a free exercise claim of the nature revealed by this record, for the amish have introduced convincing evidence that accommodating their religious objections by forgoing one or two additional years of compulsory education will not impair the physical or mental health of the child, or result in an inability to be self-supporting or to discharge the duties and responsibilities of citizenship, or in any other way materially detract from the welfare of society. pp. u. s. - . • wis. d , n.w. d , affirmed. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika (synthese) . aided by a history of three centuries as an identifiable religious sect and a long history as a successful and self-sufficient segment of american society, the amish have demonstrated the sincerity of their religious beliefs, the interrelationship of belief with their mode of life, the vital role that belief and daily conduct play in the continuing survival of old order amish communities, and the hazards presented by the state's enforcement of a statute generally valid as to others. beyond this, they have page u. s. carried the difficult burden of demonstrating the adequacy of their alternative mode of continuing informal vocational education in terms of the overall interest that the state relies on in support of its program of compulsory high school education. in light of this showing, and weighing the minimal difference between what the state would require and what the amish already accept, it was incumbent on the state to show with more particularity how its admittedly strong interest in compulsory education would be adversely affected by granting an exemption to the amish. pp. u. s. - , u. s. - . . the state's claim that it is empowered, as parens patriae, to extend the benefit of secondary education to children regardless of the wishes of their parents cannot be sustained against a free exercise claim of the nature revealed by this record, for the amish have introduced convincing evidence that accommodating their religious objections by forgoing one or two additional years of compulsory education will not impair the physical or mental health of the child, or result in an inability to be self- supporting or to discharge the duties and responsibilities of citizenship, or in any other way materially detract from the welfare of society. pp. u. s. - . wis. d , n.w. d , affirmed. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika • holding • the wisconsin compulsory school attendance law violated the free exercise clause of the first amendment because required attendance past the eighth grade interfered with the right of amish parents to direct the religious upbringing of their children. supreme court of wisconsin affirmed. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika amish objection to formal education beyond the eighth grade is firmly grounded in these central religious concepts. they object to the high school, and higher education generally, because the values they teach are in marked variance with amish values and the amish way of life; they view secondary school education as an impermissible exposure of their children to a "worldly" influence in conflict with their beliefs. the high school tends to emphasize intellectual and scientific accomplishments, self-distinction, competitiveness, worldly success, and social life with other students. amish society emphasizes informal learning-through-doing; a life of "goodness," rather than a life of intellect; wisdom, rather than technical knowledge; community welfare, rather than competition; and separation from, rather than integration with, contemporary worldly society. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika formal high school education beyond the eighth grade is contrary to amish beliefs, not only because it places amish children in an environment hostile to amish beliefs with increasing emphasis on competition in class work and sports and with pressure to conform to the styles, manners, and ways of the peer group, but also because it takes them away from their community, physically and emotionally, during the crucial and formative adolescent period of life. during this period, the children must acquire amish attitudes favoring manual work and self-reliance and the specific skills needed to perform the adult role of an amish farmer or housewife. they must learn to enjoy physical labor. once a child has learned basic reading, writing, and elementary mathematics, these traits, skills, and attitudes admittedly fall within the category of those best learned through example and "doing" rather than in a classroom. and, at this time in life, the amish child must also grow in his faith and his relationship to the amish community if he is to be prepared to accept the heavy obligations imposed by adult baptism. in short, high school attendance with teachers who are not of the amish faith—and may even be hostile to it—interposes a serious barrier to the integration of the amish child into the amish religious community. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika the amish do not object to elementary education through the first eight grades as a general proposition because they agree that their children must have basic skills in the "three r's" in order to read the bible, to be good farmers and citizens, and to be able to deal with non-amish people when necessary in the course of daily affairs. they view such a basic education as acceptable because it does not significantly expose their children to worldly values or interfere with their development in the amish community during the crucial adolescent period. while amish accept compulsory elementary education generally, wherever possible they have established their own elementary schools in many respects like the small local schools of the past. in the amish belief higher learning tends to develop values they reject as influences that alienate man from god. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika although the trial court in its careful findings determined that the wisconsin compulsory school- attendance law "does interfere with the freedom of the defendants to act in accordance with their sincere religious belief" it also concluded that the requirement of high school attendance until age was a "reasonable and constitutional" exercise of governmental power, and therefore denied the motion to dismiss the charges. the wisconsin circuit court affirmed the convictions. the wisconsin supreme court, however, sustained respondents' claim under the free exercise clause of the first amendment and reversed the convictions. a majority of the court was of the opinion that the state had failed to make an adequate showing that its interest in "establishing and maintaining an educational system overrides the defendants' right to the free exercise of their religion." entscheidung des obersten gerichtshofes der vereinigten staaten von amerika there is no doubt as to the power of a state, having a high responsibility for education of its citizens, to impose reasonable regulations for the control and duration of basic education. see, e. g., pierce v. society of sisters, u. s. , ( ). providing public schools ranks at the very apex of the function of a state. yet even this paramount responsibility was, in pierce, made to yield to the right of parents to provide an equivalent education in a privately operated system. there the court held that oregon's statute compelling attendance in a public school from age eight to age unreasonably interfered with the interest of parents in directing the rearing of their offspring, including their education in church-operated schools. as that case suggests, the values of parental direction of the religious upbringing and education of their children in their early and formative years have a high place in our society. see also ginsberg v. new york, u. s. , ( ); meyer v. nebraska, u. s. ( ); cf. rowan v. post office dept., u. s. ( ). thus, a state's interest in universal education, however highly we rank it, is not totally free from a balancing process when it impinges on fundamental rights and interests, such as those specifically protected by the free exercise clause of the first amendment, and the traditional interest of parents with respect to the religious upbringing of their children so long as they, in the words of pierce, "prepare [them] for additional obligations." u. s., at . entscheidung des obersten gerichtshofes der vereinigten staaten von amerika it follows that in order for wisconsin to compel school attendance beyond the eighth grade against a claim that such attendance interferes with the practice of a legitimate religious belief, it must appear either that the state does not deny the free exercise of religious belief by its requirement, or that there is a state interest of sufficient magnitude to override the interest claiming protection under the free exercise clause. long before there was general acknowledgment of the need for universal formal education, the religion clauses had specifically and firmly fixed the right to free exercise of religious beliefs, and buttressing this fundamental right was an equally firm, even if less explicit, prohibition against the establishment of any religion by government. the values underlying these two provisions relating to religion have been zealously protected, sometimes even at the expense of other interests of admittedly high social importance. the invalidation of financial aid to parochial schools by government grants for a salary subsidy for teachers is but one example of the extent to which courts have gone in this regard, notwithstanding that such aid programs were legislatively determined to be in the public interest and the service of sound educational policy by states and by congress. lemon v. kurtzman, u. s. ( ); tilton v. richardson, u. s. ( ). see also everson v. board of education, u. s. , ( ). entscheidung des obersten gerichtshofes der vereinigten staaten von amerika the essence of all that has been said and written on the subject is that only those interests of the highest order and those not otherwise served can overbalance legitimate claims to the free exercise of religion. we can accept it as settled, therefore, that, however strong the state's interest in universal compulsory education, it is by no means absolute to the exclusion or subordination of all other interests. e. g., sherbert v. verner, u. s. ( ); mcgowan v. maryland, u. s. , ( ) (separate opinion of frankfurter, j.); prince v. massachusetts, u. s. , ( ). entscheidung des obersten gerichtshofes der vereinigten staaten von amerika as the society around the amish has become more populous, urban, industrialized, and complex, particularly in this century, government regulation of human affairs has correspondingly become more detailed and pervasive. the amish mode of life has thus come into conflict increasingly with requirements of contemporary society exerting a hydraulic insistence on conformity to majoritarian standards. so long as compulsory education laws were confined to eight grades of elementary basic education imparted in a nearby rural schoolhouse, with a large proportion of students of the amish faith, the old order amish had little basis to fear that school attendance would expose their children to the worldly influence they reject. but modern compulsory secondary education in rural areas is now largely carried on in a consolidated school, often remote from the student's home and alien to his daily home life. as the record so strongly shows, the values and programs of the modern secondary school are in sharp conflict with the fundamental mode of life mandated by the amish religion; modern laws requiring compulsory secondary education have accordingly engendered great concern and conflict. the conclusion is inescapable that secondary schooling, by exposing amish children to worldly influences in terms of attitudes, goals, and values contrary to beliefs, and by substantially interfering with the religious development of the amish child and his integration into the way of life of the amish faith community at the crucial adolescent stage of development, contravenes the basic religious tenets and practice of the amish faith, both as to the parent and the child. entscheidung des obersten gerichtshofes der vereinigten staaten von amerika the impact of the compulsory-attendance law on respondents' practice of the amish religion is not only severe, but inescapable, for the wisconsin law affirmatively compels them, under threat of criminal sanction, to perform acts undeniably at odds with fundamental tenets of their religious beliefs. see braunfeld v. brown, u. s. , ( ). nor is the impact of the compulsory-attendance law confined to grave interference with important amish religious tenets from a subjective point of view. it carries with it precisely the kind of objective danger to the free exercise of religion that the first amendment was designed to prevent. as the record shows, compulsory school attendance to age for amish children carries with it a very real threat of undermining the amish community and religious practice as they exist today; they must either abandon belief and be assimilated into society at large, or be forced to migrate to some other and more tolerant region. in sum, the unchallenged testimony of acknowledged experts in education and religious history, almost years of consistent practice, and strong evidence of a sustained faith pervading and regulating respondents' entire mode of life support the claim that enforcement of the state's requirement of compulsory formal education after the eighth grade would gravely endanger if not destroy the free exercise of respondents' religious beliefs. dissens von douglas ( ) • die erziehung der kinder ist nicht ausschließliche zuständigkeit der eltern. • die analyse des obersten gerichtshofes zieht ausschließlich die positionen der eltern und die positionen vom board of education of wisconsin in betracht. • die eltern versuchen nicht nur, das eigene recht auf die freie ausübung des glaubensbekenntnisses, sondern auch das recht der eigenen kinder. • die kinder sollten erhört werden. • ein kind könnte klavierspieler, astronaut oder ozeanograph werden. dafür wird er sich von der amish tradition entfernen sollen. • die entscheidung des obersten gerichtshofes gefährdet die zukunft der kinder, nicht die zukunft der eltern. dissens von douglas ( ) • wenn ein kind von den eigenen eltern verhindert wird, die schule nach der erfüllung der pflichtschule zu besuchen, wird dem kind unmöglich sein, die welt der varietät und neuigkeit kennenzulernen, in welcher wir heute leben. • das kind mag entscheiden, dass das leben in der amish gemeinde das beste ist, oder er kann gegen dieses leben rebellieren. • es ist das urteil der schülerinnen und schüler, nicht das urteil der eltern, das essentiell ist, wenn die kinder meister von ihrer eigenen zukunft sein sollten. • wenn das kind im leben der amish gemeinschaft von denjenigen eingespannt wird, die über das kind autorität haben und seine erziehung damit unterbrochen wird, kann das ganze leben des kindes gehemmt und entstellt. dissens von douglas ( ) “i agree with the court that the religious scruples of the amish are opposed to the education of their children beyond the grade schools, yet i disagree with the court's conclusion that the matter is within the dispensation of parents alone. the court's analysis assumes that the only interests at stake in the case are those of the amish parents on the one hand, and those of the state on the other. the difficulty with this approach is that, despite the court's claim, the parents are seeking to vindicate not only their own free exercise claims, but also those of their high-school-age children.... on this important and vital matter of education, i think the children should be entitled to be heard. while the parents, absent dissent, normally speak for the entire family, the education of the child is a matter on which the child will often have decided views. he may want to be a pianist or an astronaut or an oceanographer. to do so he will have to break from the amish tradition. dissens von douglas it is the future of the students, not the future of the parents, that is imperiled by today's decision. if a parent keeps his child out of school beyond the grade school, then the child will be forever barred from entry into the new and amazing world of diversity that we have today. the child may decide that that is the preferred course, or he may rebel. it is the student's judgment, not his parents', that is essential if we are to give full meaning to what we have said about the bill of rights and of the right of students to be masters of their own destiny. if he is harnessed to the amish way of life by those in authority over him and if his education is truncated, his entire life may be stunted and deformed. the child, therefore, should be given an opportunity to be heard before the state gives the exemption which we honor today.” positionen von amy gutmann • entwicklung und förderung der autonomie der kinder ist ein unentbehrliches ziel der erziehung. • autorität und paternalismus der eltern soll dem prinzip unterworfen sein, dass eine pluralität von lebensaussichten den kindern ermöglicht werden soll. desgleichen soll den kindern die erfahrung einer pluralität von sozialen gütern ermöglicht werden. die autorität der eltern ist keine unbestimmte macht: sie ist funktional zur Öffnung von lebensaussichten für die kinder. • es existiert ein recht der kinder auf erziehung. • es existiert ein recht der kinder auf sozialisierung. • die erziehungsinhalte hängen davon ab, welche inhalte dazu angemessen sind, ein vollständiges leben in der gesellschaft zu leben. • das recht der kinder auf erziehung bildet eine voraussetzung dafür, ein rationales lebewesen und ein vollständiger bürger von einer liberaldemokratischen gesellschaft zu werden. • die konsequenz des mangels einer formalen erziehung ist die unfähigkeit seitens der kinder, ihre zivilen und politischen rechte auszuüben. positionen von amy gutmann • primäre soziale güter, wie z.b. erziehung, bildung und dementsprechendes recht auf erziehung und auf bildung, existieren. • primäre güter sind nicht entbehrlich. • bürgerschaftliche erziehung ist eines der obersten güter. • die lebensdimension der kinder darf nicht auf die gemeinschaft oder auf die familie beschränkt werden. die kenntnis einer pluralität von modellen ist unentbehrlich. positionen von amy gutmann • gutmanns positionen befürworten die bürgerschaftliche erziehung, die entwicklung von autonomie in den kindern und ein schulsystem, welches das entstehen vom kritischen denken unterstützt. positionen von amy gutmann • es hat keinen sinn, von einer tatsächlichen freiheit zu sprechen, falls das erziehungssystem nicht befürwortet wird, welches die entwicklung der kinder fördert. • gutmann denkt, dass die eltern eine art von paternalismus im verhältnis zu den eigenen kindern ausüben dürfen. positionen von amy gutmann • kinder müssen als individuen erachtet werden, die noch nicht dazu fähig sind, all die entscheidungen bezüglich der eigenen zukunft zu treffen. eine art paternalismus kann bezüglich der zukunft der kinder und deren organisation nicht vermieden werden. • nichtsdestoweniger kann die ansicht, dass den eltern eine gewisse macht über die eigenen kinder zuerkannt wird, nicht bedeuten, dass die eltern die eigenen kinder auf das, was die eltern wollen, ausrichten dürfen. paternalismus sollte so ausgeübt werden, dass kinder mit einer pluralität von gütern und von lebenschancen vertraut gemacht werden. paternalismus, d.h. macht über die kinder ist der elternpflicht unterworfen, den eigenen kindern eine pluralität von lebensaussichten zu eröffnen. es handelt sich nicht um eine macht, die ohne weitere bestimmung ist. diese macht ist zur entwicklung und entwicklungsförderung der kinder funktional. positionen von amy gutmann • einmischung in der handlungsfreiheit der kinder lässt sich infolgedessen nur damit rechtfertigen, dass diese einmischung auf die gegenwärtigen oder auf die künftigen interessen der kinder bezogen wird und bezug nimmt. kinder sollen als potentiell vernünftige lebewesen angesehen werden, die dazu fähig sind, auf freie art und weise zwischen einer pluralität von konzeptionen des guten lebens zu wählen; zugleich müssen die kinder als lebewesen betrachtet werden, die dazu fähig sind, in einer demokratischen gesellschaft sich selbst zu regieren. dies bedeutet, dass nach gutmanns ansicht eine voraussetzung des ganzen prozesses darin besteht, dass eine bestimmte auffassung der kinder herrscht: kinder sind zumindest potentiell vernünftige lebewesen zu erachten: dieser anwachsenden rationalität soll rechnung getragen werden. kinder sind keine teigware. • gutmann unterstreicht die wichtigkeit von gütern wie einer erziehung, die den individuen ermöglicht, zwischen verschiedenen möglichkeiten zu wählen und gebildete bürgerinnen und bürger zu werden. ohne gebildete bürgerinnen und bürger zu sein, werden die individuen um eine orientierung bezüglich der welt, in welcher sie leben, bestohlen. die macht der eltern darf nur dazu verwendet werden, um die entwicklung der kinder zu fördern. positionen von amy gutmann • die bedeutung des kinderrechts auf erziehung innerhalb einer liberalen gesellschaft muss überprüft werden. wenn kinder rechte aufgrund ihrer bedürfnisse und interessen als künftige staatsbürgerinnen und bürger besitzen, ist eines von diesen rechten das recht auf erziehung oder, wie einige theoretiker sagen, „das recht auf sozialisierung“. der inhalt des kinderrechts auf erziehung wird davon abhängig sein, was für ein realisiertes leben innerhalb der gesellschaft nötig ist. kindern soll ermöglicht werden, zwischen verschiedene konzeptionen vom gut zu wählen und an der politik teilzunehmen, wenn sie es wollen. das kinderrecht auf verpflichtende erziehung ist eine voraussetzung dazu, ein rationales lebewesen und ein vollständiger bürger / eine vollständige bürgerin innerhalb einer liberaldemokratischen gesellschaft zu werden. positionen von amy gutmann • ohne schulische erziehung werden die kinder dazu unfähig sein, ihre zivilen und politischen rechte innerhalb der gesellschaft auszuüben. die behauptung, dass die eltern ein unbegrenztes recht auf die ausübung des eigenen glaubensbekenntnisses besitzen, enthält infolgedessen die behauptung des rechtes dazu, die eigenen kinder um zwei grundlegenden güter zu bestehlen, für welche der liberaldemokratische staat existiert: • das treffen von einer informierten wahl zwischen den komponenten des guten lebens und • die teilnahme an der demokratischen selbstregierung. positionen von amy gutmann • das recht auf eine verpflichtende schulerziehung bildet eine voraussetzung dafür, dass die individuen rationale lebewesen werden und vollständige bürgerinnen und bürger werden. • ohne die erfüllung dieser voraussetzung liegt keine möglichkeit vor, eine korrekte erziehung der individuen zu erlangen. das interesse der kinder ist nach der ansicht von gutmann, die eigenen potentialitäten zu entwickeln: dies kann ausschließlich erfolgen, nur wenn das diesem interesse entsprechende recht der kinder auf die schulische erziehung in die tat umgesetzt wird. ohne die schulische erziehung werden die kinder eine verminderung bezüglich ihrer eigenen entwicklung erleiden; sie werden nicht die gebührende mentale entwicklung erlangen können. dies ist eine deutliche verminderung in der freiheit: denn freiheit kann ohne erziehung vervollständigt werden. positionen von amy gutmann ohne erziehung verlieren die liberalen freiheiten vieles, um nicht zu sagen alles, von ihrem wert. positionen von amy gutmann • die auffassung, dass keine wirksame, keine tatsächliche freiheit existieren kann, wenn zumindest ein gewisser grad an erziehung nicht erlangt wird, ist ein fixer punkt in der meditation über erziehung seitens amy gutmanns. • eine person, die um die möglichkeit der erziehung bestohlen wird, ist eine person, die in der gesellschaft orientierungslos ist. eltern sollen nach der auffassung von gutmann die kinder mit einer pluralität von möglichen realisierungsweisen ausstatten. positionen von amy gutmann • eltern haben infolgedessen kein recht dazu, die eigenen kinder von umwelten zurückzuziehen, in denen sie zugang zu verschiedenen lebenschancen haben könnten. • obwohl eltern eine macht über die kinder haben, kann diese macht ihrerseits nicht implizieren, dass die eltern ihre kinder zu einem bestimmten gut dirigieren dürfen oder dass sie ihre kinder von der pluralität der lebensaussichten ausschließen dürfen. eltern sollen ihre eigenen kindern mit einer pluralität von gütern vertraut machen. gutmann erachtet als ein grundlegendes prinzip, dass die individuen das recht besitzen, über ihre zukunft zu entscheiden: das recht, über die eigene zukunft zu entscheiden, impliziert jedoch, dass die kinder mit einer pluralität von möglichkeiten vertraut werden sollten. ohne über eine pluralität von lebensmöglichkeiten zu verfügen, verfügen kinder eigentlich nicht über eine freie wahl. ohne schulsystem wird es den kindern nicht möglich sein, reife staatsbürgerinnen und staatsbürger zu werden. amy gutmann “to determine who ought to have educational power over children, i begin by looking for a standard of paternalism: a standard justifying interference with a child’s freedom of action by reference to the child’s present or future interests. i assume that a liberal justification of paternalism toward children must rest upon an equal consideration of the interests of children as potentially rational beings, beings capable of choosing freely among a range of competing conceptions of the good life and of intelligently governing themselves in a democratic society” (children, paternalism, and education: a liberal argument, pag. ). amy gutmann “the most obvious primary goods we as rational persons would want provided to us as children are adequate nutrition, health care, housing, familial affection, and an education adequate to choosing among available economic and social opportunities and to becoming informed, democratic citizens. yet, from the point of view of some citizens within our society, even this short list is controversial. many jehovah’s witnesses and christian scientists do not think that their children should be forced to take certain medicines or undergo certain medical treatments that are necessary to good health and normal physiological development, and sometimes to life itself. the old order amish do not believe that formal secondary education is a primary good in our society. but these examples need not upset the plausibility of a primary good standard of paternalism applied to children.” (children, paternalism, and education: a liberal argument”, p. ) amy gutmann “primary goods justifiably guide paternalistic interventions only if certain conditions hold. the subject’s reason or will must be too weak to guide his actions or evidently must have failed to do so. and we must be incapable of knowing enough about the subject’s more settled aims and preferences. (…) we know that children (at least below a certain age) lack adequate reason to make many important decisions for themselves, and we cannot take their aims or preferences to be settled.” (children, paternalism, and education: a liberal argument”, p. ) amy gutmann “rarely, if ever, does a family furnish its children with as broad a range of resources and opportunities to choose among free and adequate adult lives as does a wider society. while parents often do give their children some of the essential goods of life, they still have a duty to permit, if not to prepare, their children to choose among a range of conceptions of the good life that differ substantially from those held by the family. as citizens of a society offering a broad range of choices to its adult members, parents have an obligation to allow their children to be exposed to the choices available in their extra- familial society.” (children, paternalism, and education: a liberal argument”, p. ) amy gutmann “if my argument so far is correct, it will not suffice to claim that the amish community has the right to impose its standards upon amish children. even if the amish community were an economically and politically self-sufficient unit within our society, the amish would still have a duty to expose their children to a greater range of choice than they now do. if they were self-sufficient enough, it might be the case that no state surrounding the amish community would have a clear right to intervene in the education of amish children so as to enforce the duty of amish parents. the parental duty, nonetheless, would remain. of course, the wisconsin amish today are not a self- sufficient community, nor do they provide their children with as broad a range of choices among good lives as the larger society does.” (children, paternalism, and education: a liberal argument, pp. - ) amy gutmann “… we must briefly examine the meaning of a child’s right to education within a liberal society. if children have rights in virtue of their basic needs and interests as future adult citizens, one of those rights will be a right to education, or what some theorists have called “a right to socialization”. the content of children’s right to education will depend upon what is adequate for living a full life within their society – for being capable of choosing among available conceptions of the good and of participating intelligently in democratic politics if they so choose. i have no doubt that this criterion imposes substantially greater obligations upon us than we fulfill by our current educational, and other related, practices. but beyond this point, education ceases to be a right, even if it remains an important human good. on this understanding, a child’s right to compulsory education is a precondition to becoming a rational human being and a full citizen of a liberal democratic society. without any formal schooling, children will be incapable of intelligently exercising their civil or political rights within our society. the assertion on the part of the parents of an unconstrained right of free exercise therefore entails the assertion of a right to deprive their children of two fundamental goods for which a liberal democratic state exists: the making of an informed choice among components of the good life, and participation in meaningful democratic self-government.” (children, paternalism, and education: a liberal argument, pp. - ) amy gutmann without any formal schooling, children will be incapable of intelligently exercising their civil or political rights within our society. the assertion on the part of the parents of an unconstrained right of free exercise therefore entails the assertion of a right to deprive their children of two fundamental goods for which a liberal democratic state exists: the making of an informed choice among components of the good life, and participation in meaningful democratic self-government.” (children, paternalism, and education: a liberal argument, pp. - ) amy gutmann “without education, liberal freedoms lose a great deal, even if not all, of their value.” (children, paternalism, and education: a liberal argument, p. ) joel feinberg children are not legally capable of defending their own future interests against present infringement by their parents so that task must be performed for them, usually by the state in its role of parens patriae. american courts have long held that the state has a “sovereign power of guardianship” over minors and other legally incompetent persons, which confers upon it the right, or perhaps even the duty, to look after the interests of those who are incapable of protecting themselves. (the child’s right to an open future, p. ) joel feinberg the case against the exemption for the amish must rest entirely on the rights of amish children, which the state as parens patriae is sworn to protect. an education that renders a child fit for only one way of life forecloses irrevocably his other options. he may become a pious amish farmer, but it will be difficult to the point of practical impossibility for him to become an engineer, a physician, a research scientist, a lawyer, or a business executive. the chances are good that inherited propensities will be stymied in a large number of cases, and in nearly all cases, critical life decisions will have been made irreversibly for a person well before he reaches the age of full discretion when he should be expected, in a free society, to make them himself. (the child’s right to an open future, p. ) positionen von richard arneson – ian shapiro ( ) - es existieren bestimmte grenzen zur autorität der eltern. - die beziehungen zwischen eltern und kindern sollten als ein verhältnis von pflegschaft und von treuhänderschaft (treuhandschaft) interpretiert werden. - pflegschaft und treuhänderschaft schließen aus, dass kinder der besitz der eltern sind. - die patriarchalische annahme, dass kinder hab und gut der eltern sind, wird abgelehnt. - das recht der kinder auf erziehung übertrumpft das recht der amish gemeinschaft, das leben der eigenen gemeinschaft aufzubewahren. - erziehung sollte kinder und schüler für ein leben in der welt im generellen sinne, nicht für das leben in einer besonderen gemeinschaft mit dem ausschluss aller anderer lebensweisen vorbereiten. - erziehung sollte erziehung zur staatsbürgerschaft sein. erziehung sollte den schülerinnen und schülern die teilnahme am öffentlichen leben ermöglichen, wenn sie den wunsch haben, am öffentlichen leben teilzunehmen. positionen von richard arneson – ian shapiro ( ) • der staat hat eine präzise pflicht bezüglich erziehung. der staat darf sich mit dem machbaren minimum begnügen. • der grundlegende aspekt ist, dass erziehung als solche ein wert ist. ohne erziehung ist keine reife staatsbürgerschaft da. • der punkt, dass das element, welches die positionen von arneson-shapiro auszeichnet, besteht darin, dass sie die staatsbürgerkunde als das unersetzliche instrument erachten, um eine reife staatsbürgerschaft zu erlangen. • die teilnahme an der öffentlichen dimension bildet eine vernünftige obligation. arneson-shapiro zeigen keine gleichgültigkeit im verhältnis zur teilnahem oder nicht-teilnahme an der stimmabgabe. sie plädieren für ein aktives modell von teilnahme. • teilnahme am sozialen leben ist besser als rückzug vom sozialen leben. • gruppenrechte dürfen die individuelllen rechte nicht übertrumpfen. • die annahme, dass gruppenrechte das recht der individuen zur erziehung übertrumpfen können, darf nicht akzeptiert werden. • erziehung als erziehung zur autonomie. • maximisierung von optionen. • entwicklung der kritischen vernunft. arneson - shapiro arneson und shapiro bringen zahlreiche beobachtungen in ihrer eigenen studie zum ausdruck: • kinder sind nicht eigentum von ihren eltern. • jede form vom patriarchalismus wird in verhältnis zu den kindern abgelehnt. • kinder dürfen nicht für die ziele der eltern verwendet werden. • kinder dürfen nicht dazu verwendet werden, das Überleben einer glaubensgemeinschaft zu garantieren. • die interesse der kinder darf nicht anderen zielen untergeordnet werden. • kinder leben mit ihren eigenen eltern in einer beziehung von treuhänderschaft, nicht in einem verhältnis von unterordnung. • kinder haben das recht, eine erziehung zu bekommen, die sie zumindest mit einem gewissen grad an autonomie ausstattet. • wenn kinder nicht zur autonomie erzogen werden, sind sie nicht frei. arneson - shapiro • arneson und shapiro erklären, dass sie die grenzen der autorität der eltern bezüglich der erziehung der kinder, mit denen sie beauftragt worden sind, analysieren. statt von autorität der eltern über die kinder bevorzugen die zwei autoren, vom verhältnis der treuhänderschaft der eltern mit den kindern zu sprechen. sie lehnen die auffassung ab, dass kinder besitz der eltern sind. es liegen präzise grenzen in bezug auf die autorität der eltern mit ihren kindern. • die zwei autoren sehen die präsenz einer diskrepanz zwischen den rechten der eltern, die mit der ausübung der religion verbunden war, und den ansprüchen des staates, die auf die erziehung der kinder bezogen wird. die diskrepanz betrifft - das interesse der amish gemeinde, das in der ausübung des glaubensbekenntnisses liegt und in der möglichkeit der aufrechterhaltung der gemeinschaft, und - das interesse des staates, die erziehung der kinder zu fördern, und dies ganz unabhängig davon, welche konsequenzen dies für das Überleben der gemeinschaft haben kann. • die zwietracht liegt unter anderem darin, dass die amish einen höheren grad an erziehung als eine bedrohung für die gemeinschaft erachten, da höheren graden an erziehung eine höhere möglichkeit entspricht, dass die kinder die gemeinschaft verlassen. arneson - shapiro • an der amish erziehung kritisieren die zwei autoren, dass das erziehungssystem der amish auf den verbleib der kinder in der gemeinschaft abzielt. das erziehungssystem der amish ist dafür ausgedacht, dass die kinder für das leben in der amish gemeinschaft vorbereitet sind; eine generelle vorbereitung für ein leben außerhalb der gemeinschaft ist nicht vorgesehen. dafür versuchen die amish auf jede art und weise, die kinder von der externen welt zu isolieren; das erziehungssystem der amish gemeinschaft tendiert dazu, den kinder von jede form von kritischer auseinandersetzung mit den amish werten und glaubensprinzipien abzuraten. • im besonderen opponiert die amish gemeinschaft der hochschulerziehung, da sie die hochschulerziehung als eine bedrohung für die ganze lebensweise der amish gemeinschaft ansehen. es wird behauptet, dass mit vierzehn jahren die amish kinder alles wissen, um erfolgreich in der amish gemeinschaft zu leben: daher opponiert die amish gemeinschaft jeder weiteren form von schulischer erziehung, die außerhalb der gemeinschaft stattfinden sollte: dafür wird eine art von berufsausbildung bevorzugt, die innerhalb der gemeinschaft stattfindet. • die schulerziehung erweitert die interessen der kinder: daher wird sie als ein hindernis zur stabilität und aufrechterhaltung der gemeinschaft erachtet. innerhalb dieser konstellation wird jede form von kritischem denken vermieden: da die mischung mit dem leben außerhalb der gemeinschaft als eine gefahr für die rettung des individuums angesehen wird, wird die gemeinschaft alles machen, auf dass diese mischung nicht vorfällt. • das erziehungssystem existiert ausschließlich mit dem ziel, dass das weiterleben und Überleben der gemeinschaft gesichert werden kann. arneson - shapiro • an der amish erziehung kritisieren die zwei autoren, dass das erziehungssystem der amish auf den verbleib der kinder in der gemeinschaft abzielt. das erziehungssystem der amish ist dafür ausgedacht, dass die kinder für das leben in der amish gemeinschaft vorbereitet sind; eine generelle vorbereitung für ein leben außerhalb der gemeinschaft ist nicht vorgesehen. dafür versuchen die amish auf jede art und weise, die kinder von der externen welt zu isolieren; das erziehungssystem der amish gemeinschaft tendiert dazu, den kinder von jede form von kritischer auseinandersetzung mit den amish werten und glaubensprinzipien abzuraten. • im besonderen opponiert die amish gemeinschaft der hochschulerziehung, da sie die hochschulerziehung als eine bedrohung für die ganze lebensweise der amish gemeinschaft ansehen. es wird behauptet, dass mit vierzehn jahren die amish kinder alles wissen, um erfolgreich in der amish gemeinschaft zu leben: daher opponiert die amish gemeinschaft jeder weiteren form von schulischer erziehung, die außerhalb der gemeinschaft stattfinden sollte: dafür wird eine art von berufsausbildung bevorzugt, die innerhalb der gemeinschaft stattfindet. arneson - shapiro • die schulerziehung erweitert die interessen der kinder: daher wird sie als ein hindernis zur stabilität und aufrechterhaltung der gemeinschaft erachtet. innerhalb dieser konstellation wird jede form von kritischem denken vermieden: da die mischung mit dem leben außerhalb der gemeinschaft als eine gefahr für die rettung des individuums angesehen wird, wird die gemeinschaft alles machen, auf dass diese mischung nicht vorfällt. • das erziehungssystem existiert ausschließlich mit dem ziel, dass das weiterleben und Überleben der gemeinschaft gesichert werden kann. arneson - shapiro • die zweitracht zwischen den beiden anschauungen ist stärker, als es auf den ersten blick geglaubt werden könnte. die amish gemeinschaft ist auf der einen seite davon überzeugt, dass eine bestimmte lebenswese unentbehrlich ist, um die rettung der seele zu erlangen. auf der anderen seite liegt eine interpretation des lebens als eine wahl zwischen einer pluralität von lebensmöglichkeiten vor: diese interpretation des lebens entspricht einem modell von teilnahme am pluralismus des sozialen lebens: unterschiedliche lebensweise sind zugelassen, und keine spezifische art von leben wird als ein leben erachtet, das die personen zur rettung der eigenen seele führt. • die auffassung der staatsbürgerschaft, die arneson und shapiro vertreten, sieht vor, dass die individuen dazu fähig sind, auf eine informierte art und weise in wahlen abzustimmen, welche die mitglieder von legislativen versammlungen bestimmen. darüber hinaus werden bei diesen wahlen die personen gewählt, die die gesetze anwenden. die wählerinnen und wähler sollen die notwendige vorbereitung haben, um die entscheidung bei den wahlen zu treffen. • wählerinnen und wähler sollen eine adäquate kenntnis der politischen wissenschaft; sie sollten ein passendes verständnis der modernen weltgeschichte haben; sie sollten die geschichte der demokratischen institutionen und die kultur der eigenen gesellschaft kennen. sie sollten darüber hinaus über ein kritisches denken verfügen, die ihnen ermöglicht, sich die situationen der mitbürgerinnen und der mitbürger vorzustellen, die erfahrung der mitbürgerinnen und der mitbürger anzuschauen, die prinzipien und die attitüden der mitbürger und der mitbürgerinnen zu analysieren. • staatsbürgerinnen und staatsbürger sollten die fähigkeit dazu haben, sich selbst auf dem laufenden über die ereignisse zu halten, welche mit den entscheidungen der regierungen zusammenhängen könnten. arneson - shapiro • innerhalb einer demokratischen grundordnung, in welcher verschiedene glaubensbekenntnisse, weltanschauungen, gruppen, interessen vorliegen, entscheidungen zu treffen, die all diesen verschiedenheiten rechnung tragen, ist eine sehr schwierige aufgabe. arneson und shapiro behaupten, dass eine erziehung, welche dem niveau der einfachen alphabetisierung überlegen ist, notwendig ist, damit die kinder auf die verantwortung und auf die pflichten vorbereitet sein könnten, die mit der staatsbürgerschaft zusammenhängen: eine höhere schulerziehung löst nicht all die probleme, sie ist jedoch eine form von erziehung, die einige fertigkeiten verschafft, auf dass eine person die kompetenzen erlangen kann, die für die ausübung der staatsbürgerschaft notwendig sind. • bezüglich der tatsächlichen ausübung der staatsbürgerschaft sind arneson und shapiro der ansicht, dass eine gewisse pflicht zu wählen in einer funktionierenden demokratie existiert. die teilnahme an den wahlen ist notwendig, wenn das system richtig funktionieren soll. arneson und shapiro geben zu, dass fälle da sind, in welchen nicht zu wählen ist gestattet und sogar empfehlenswert. • der rückzug der amish von der gesellschaft könnte auch vertretbar sein: ein informiertes urteil bezüglich dieser fächer ist trotzdem erreichbar nur für personen, die verstehen, wie demokratische systeme funktionieren und welche themen im rahmen der demokratischen wahlen besprochen werden. es ist notwendig, dass eine person für eine kompetente staatsbürgerschaft erzogen wird, auf dass es bestimmt werden kann, ob es statthaft oder verpflichtend ist, am wahlprozess nicht teilzunehmen. damit es auf begründete art und weise entschieden werden kann, am wahlprozess nicht teilzunehmen, soll eine person die kompetenz besitzen, die mit dem vollständiger staatsbürgerschaft zusammenhängt. arneson - shapiro • ein aspekt, der arneson und shapiro am yoder-spruch kritisieren, besteht darin, dass die betroffenen eltern die these befürwortet hatten, dass die amish gemeinde das recht hat, die bedingungen für das eigene Überleben zu gewährleisten und die ausübung der eigenen religion aufzubewahren. • der ansatz, dass ein recht der gemeinschaft existiert, stellt nach der ansicht von arneson und shapiro einen übertriebenen anspruch für gruppenrechte dar. gruppen sind gruppen von individuen; vom recht, das Überleben der amish gemeinschaft zu garantieren, zu sprechen, zieht nicht in betracht, dass zwietracht bei den mitgliedern einer gruppe herrschen könnte: sie könnten voneinander verschiedene interessen und rechte haben. • wenn das interesse der amish kinder darin besteht, eine adäquate erziehung zu bekommen, und wenn die tatsache, dass die amish kinder eine adäquate erziehung bekommen, das bestehen der gruppe gefährden wird, dann entsteht und besteht eine zwietracht von interessen und von rechten zwischen gemeinschaft und kindern. arneson und shapiro sind der auffassung, dass in diesem falle kein grund angeführt werden kann, aufgrund dessen die interessen der kinder bezüglich ihrer eigenen erziehung zugunsten des wunsches der eltern, das Überleben der amish gemeinschaft zu garantieren, aufgeopfert werden sollte. das recht und das interessen der kinder auf eine angemessene erziehung darf unter keinen umständen zugunsten eines gruppenrechtes aufgeopfert werden. arneson - shapiro • der zwiespalt, die an die oberfläche auftaucht, besteht darin, dass die forderung der amish familien, die eigenen kinder aus der schule mit nach statt nach jahren zurückzuziehen, auf der auffassung basiert, dass längere schulperioden den verbleib der amish kinder in der gemeinschaft gefährdeten, so dass das Überleben an sich der gemeinschaft in gefahr stünde. das individuelle recht auf erziehung sollte daher zugunsten des gruppenrechts aufgeopfert werden, dass die gemeinschaft das recht zu Überleben hat. • es fehlt innerhalb dieser auffassung, dass das recht der kinder auf erziehung dem gruppenrecht opponieren könnte; es wird einfach nicht akzeptiert oder auch nur in betracht gezogen, dass bestimmte individuelle rechte dem gruppenrecht opponieren könnten. das grupperecht ist jeder möglichkeit vorgeordnet. • arneson und shapiro behaupten deutlich, dass ihre positionen nicht eine solche ist, die gegen die existenz der amish gemeinschaft ist. sie sind nicht dafür, dass die gemeinschaft vom staat entfernt werden sollte. sie akzeptieren nicht, dass den eltern ein recht zuerkannt wird, sich an den rechten der kinder auf eine adäquate erziehung zu vergreifen, auf dass die eltern die amish gemeinschaft aufbewahren können. das recht der eltern muss dem recht der kinder weichen. das recht der amish auf freie assoziation enthält nicht, dass kinder verletzungen bezüglich ihrer rechte wegen dieses rechts auf assoziation ertragen sollten. arneson - shapiro • arneson und shapiro sind der ansicht, dass das recht, eine eigene individuelle autonomie zu entwickeln, die richtige antwort auf die probleme repräsentiert, die vom yoder-spruch aufgeworfen worden sind. kinder haben das recht auf eine offene zukunft: das ziel der erziehung sollte innerhalb dieser sichtweise darin bestehen, dass die kinder für eine lebensweise vorbereitet werden, die sich aus rationaler autonomie zusammensetzt, nachdem sie erwachsen geworden sind. eine rationale selbständige lebensweise ist eine solche, die von sich selbst gewählt wird. die erziehung für die rationale autonomie schließt zwei erfordernisse ein: - den individuen sollte am anfang ihres erwachsen-werden ermöglicht werden, in der weitestmöglichen varietät von lebensweisen und konzeptionen des guten zu wählen. - individuen sollten fertigkeiten von kritischer reflexion bekommen werden, so dass sie am besten die fähigkeit erlangen, zwischen den alternativen lebensweisen zu wählen. • arneson und shapiro nennen diese zwei bedingungen die maximierung von optionen und die entwicklung der kritischen vernunft. eine offene zukunft kann garantiert werden, nur wenn eine entsprechende erziehung gegeben wird. wenn die erziehung geschlossen ist, wird keine möglichkeit zur offenen zukunft vorliegen. arneson - shapiro • arneson und shapiro sind der ansicht, dass mitglieder von einer demokratischen gesellschaft dazu verpflichtet sind, die verantwortungen, die mit einer demokratischen staatsbürgerschaft zusammenhängen, auszuführen. • um politische vorschläge zu beurteilen, sollen die bürgerinnen und bürger dazu fähig sein, eine kritische vernunft zu verwenden, um zu sehen, welche konsequenzen aus den verschiedenen vorschlägen herauskämen. innerhalb einer demokratie ist es notwendig, da die bürgerinnen und bürger von den entscheidungen der mehrheit betroffen werden, dass die bürgerinnen und bürger die fertigkeiten entwickeln, die zum verständnis und zur beurteilung der politischen entscheidungen, von denen ihre eigenen leben betroffen werden könnten und durch die sie das leben der anderen beeinflussen könnten, leiten. innerhalb einer demokratie sollten die kinder zur entwicklung des eigenen kritischen denkens erzogen werden; eltern und erziehen werden aufgefordert, instrumente zum kritischen denken den kindern zu geben. arneson und shapiro sind der auffassung, dass all diejenigen, die sich zu einem rückzug aus dem öffentlichen leben entschließen, dies machen können, nur wenn sie die instrumente haben, um eine vernünftige entscheidung zu treffen: auf dass eine solche entscheidung auf begründete art und weise getroffen wird, müssen die individuen die entsprechende kenntnisse haben. erst wenn die autonomie erreicht worden ist, kann gesagt werden, dass eine entscheidung zum rückzug aus dem öffentlichen leben eine authentische entscheidung ist, da diese entscheidung von einer autonomen person getroffen worden ist. arneson - shapiro • die inkompatibilität zwischen dem kinderrecht auf autonomie und dem gruppenrecht der amish ist infolgedessen klar: die amish sind der festen Überzeugung, dass sie ihre kinder dazu erziehen sollten, dass die kinder das leben der amish gemeinschaft annehmen und weiterverfolgen. eventuelle fertigkeiten, die aus kritischem denken bestehen, würden die kinder von einer identifizierung mit der amish lebensweise entfernen. infolgedessen würde das kritische denken für die kinder korrumpierend sein. • bezüglich der erziehung für autonomie an sich selbst sind arneson und shapiro der auffassung, dass autonomie in der entwicklung von fertigkeiten vom kritischen denken besteht. die autonome person ist nicht die person, die die eigenen gedanken selbständig erzeugt; die autonome person, um eine authentische autonome person definiert zu werden, ist diejenige, die dazu fähig ist, über die eigenen gedanken kritisch zu reflektieren; sie ist die person, die dazu fähig ist, ihre eigenen gedanken infolge des eigenen denkens und umdenkens zu modifizieren. das amish erziehungsprogramm ist insofern falsch, als es die kinder nicht zum kritischen denken trainiert; dieses erziehungsprogramm schließt nicht die möglichkeit ein, dass die kinder zum kritischen denken über ihre eigenen werte aufgefordert werden. ganz im gegenteil zielt das amish erziehungsprogramm darauf ab, das kritische denken einzugrenzen, so dass kinder glaubensprinzipien annehmen, ohne dass diese prinzipien der reflexion unterworfen werden. arneson - shapiro • arneson und shapiro sind der ansicht, dass mitglieder von einer demokratischen gesellschaft dazu verpflichtet sind, die verantwortungen, die mit einer demokratischen staatsbürgerschaft zusammenhängen, auszuführen. • um politische vorschläge zu beurteilen, sollen die bürgerinnen und bürger dazu fähig sein, eine kritische vernunft zu verwenden, um zu sehen, welche konsequenzen aus den verschiedenen vorschlägen herauskämen. innerhalb einer demokratie ist es notwendig, da die bürgerinnen und bürger von den entscheidungen der mehrheit betroffen werden, dass die bürgerinnen und bürger die fertigkeiten entwickeln, die zum verständnis und zur beurteilung der politischen entscheidungen, von denen ihre eigenen leben betroffen werden könnten und durch die sie das leben der anderen beeinflussen könnten, leiten. innerhalb einer demokratie sollten die kinder zur entwicklung des eigenen kritischen denkens erzogen werden; eltern und erziehen werden aufgefordert, instrumente zum kritischen denken den kindern zu geben. arneson und shapiro sind der auffassung, dass all diejenigen, die sich zu einem rückzug aus dem öffentlichen leben entschließen, dies machen können, nur wenn sie die instrumente haben, um eine vernünftige entscheidung zu treffen: auf dass eine solche entscheidung auf begründete art und weise getroffen wird, müssen die individuen die entsprechende kenntnisse haben. erst wenn die autonomie erreicht worden ist, kann gesagt werden, dass eine entscheidung zum rückzug aus dem öffentlichen leben eine authentische entscheidung ist, da diese entscheidung von einer autonomen person getroffen worden ist. synthese - es existieren bestimmte grenzen zur autorität der eltern. • - die beziehungen zwischen eltern und kindern sollten als ein verhältnis von pflegschaft und von treuhänderschaft (treuhandschaft) interpretiert werden. • - pflegschaft und treuhänderschaft schließen aus, dass kinder der besitz der eltern sind. • - die patriarchalische annahme, dass kinder hab und gut der eltern sind, wird abgelehnt. • - das recht der kinder auf erziehung übertrumpft das recht der amish gemeinschaft, das leben der eigenen gemeinschaft aufzubewahren. • - erziehung sollte kinder und schüler für ein leben in der welt im generellen sinne, nicht für das leben in einer besonderen gemeinschaft mit dem ausschluss aller anderer lebensweisen vorbereiten. • - erziehung sollte erziehung zur staatsbürgerschaft sein. erziehung sollte den schülerinnen und schülern die teilnahme am öffentlichen leben ermöglichen, wenn sie den wunsch haben, am öffentlichen leben teilzunehmen. • - der staat hat eine präzise pflicht bezüglich erziehung. der staat darf sich nicht mit dem machbaren minimum begnügen. • - der grundlegende aspekt ist, dass erziehung als solche ein wert ist. ohne erziehung ist keine reife staatsbürgerschaft da. synthese • der punkt, dass das element, welches die positionen von arneson-shapiro auszeichnet, besteht darin, dass sie die staatsbürgerkunde als das unersetzliche instrument erachten, um eine reife staatsbürgerschaft zu erlangen. • - die teilnahme an der öffentlichen dimension bildet eine vernünftige obligation. arneson-shapiro zeigen keine gleichgültigkeit im verhältnis zur teilnahem oder nicht-teilnahme an der stimmabgabe. sie plädieren für ein aktives modell von teilnahme. • - teilnahme am sozialen leben ist besser als rückzug vom sozialen leben. • - gruppenrechte dürfen die individuellen rechte nicht übertrumpfen. • - die annahme, dass gruppenrechte das recht der individuen zur erziehung übertrumpfen können, darf nicht akzeptiert werden. • - erziehung als erziehung zur autonomie. • - maximisierung von optionen. • - entwicklung der kritischen vernunft. arneson-shapiro in this chapter we take up a small piece of the newly discovered terrain: we explore the limits of parents’ authority in the education of children who have been committed to their charge. our use of this formulation, rather than parents’ authority over “their children,” prefigures one of the central claims we seek to defend and employ: that the relationship between parents and children is best thought as one of trusteeship; children are in no sense the property of their parents. although most people will find the claim thus stated unexceptionable, and few would go so far as to describe their children as their property, many of the convictions to which people find themselves drawn in thinking about the authority of parents over children nevertheless reflect the archaic idea that the child is the chattel of the parent (which once went hand in hand with the patriarchal idea that the wife is the chattel of the husband). (democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pp. - .) arneson-shapiro the free exercise interests in question were the interests of the amish parents in practicing their religion in their traditional way. but the state’s expressed interest concerned the education of amish children. on the face of it, there was a gap between the rights claimed by the parents, having to do with their practice of religion, and the claims of the state having to do with the children’s education (democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pp. ) arneson-shapiro on the amish principles “although the amish believe that vow of baptism must be taken voluntarily by a mature person, they go to great lengths in designing their system of education and acculturation to ensure that amish children will take the vow and join the church. herein lies the source of the half century of conflict between the amish and secular educational authorities which culminated in the yoder decision. the amish educational system is designed to prepare children for life in the amish community, not the outside world. to this end, the amish try to shield children from the secular world, and they actively discourage critical questioning of amish values and beliefs. they are particularly opposed to high school education, which they see as threatening to their entire way of life. by age fourteen the amish child knows everything necessary to live successfully in the amish community; as a result the amish oppose further schooling, preferring on-the-job vocational training that will ease children into the community.“ (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pp. - ) arneson-shapiro on the requirements for the participation to the public life “… we suppose that a significant aspect of citizenship includes the requirement that people have the capacity to vote in an informed way in elections that determine the membership of legislative assemblies, hence the content of the laws, as well as the identity of public officials and judges who execute and apply the laws. to be able to participate competently in democratic decision making, voters should have an adequate knowledge of contemporary science in its bearing on public issues, and understanding of modern world history and particularly the history of democratic institutions and the culture of their own society, and critical thinking skills that include the ability to represent the situation of others in imagination, to intuit their experience, and sympathetically to analyze and assess their attitudes, principles, and policy arguments. citizens should have the capacities to keep themselves briefed on current events relevant to governmental decisions to be made. in a diverse democracy composed of disparate creeds, faiths, races, world views, and concerns, arriving at fair and reasonable decisions about public policy is a task of delicate and complex judgment.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pp. ) arneson-shapiro on the level of education needed to participate to the public life: basic literacy is not sufficient “without entering into an extended consideration of what sort of education best prepares children for the responsibilities of democratic citizenship so conceived, we submit that education beyond basic literacy is needed to increase to an adequate level the likelihood that an individual will have the skills needed for democratic deliberation an the disposition to exercise these skills on appropriate occasions. in this regard although a high school education is not a panacea, it does provide some skills and knowledge needed to be a competent democratic deliberator.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pp. ) arneson-shapiro on the duty to participation to the democratic dimension “although we would not go so far as to argue that people necessarily have an obligation to vote in a democracy, there surely is a defensible obligation to vote in any tolerably functioning democracy. voting by many is necessary if the system is to function properly, and there is no obvious, fair way to select a subset of voters to whom the obligation does not apply. no doubt there are circumstances in which refraining from voting is permissible and even obligatory. for example, it may well be the case, for all that we say in this chapter, that the amish practice of withdrawal from society is justified. but informed judgment about these matters can only be made by people who understand how democratic systems work and what the issues at stake in democratic elections are. one needs to be educated for competent citizenship in order to determine whether it is permissible or obligatory to abstain from exercising this or that function of citizenship.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pag. ) arneson-shapiro on the contrasts between rights of parents and rights of communities “now the parents in yoder did not invoke a fiduciary obligation to their children in support of their actions. instead they appealed to a mixture of the right of the amish community to reproduce itself and free-exercise rights of the parents. the first of these rests on an exceedingly powerful claim for group rights, for which it is difficult to see a coherent justification. groups and classes are groups and classes of individuals, and talk of the “right” of the amish community to reproduce itself glosses over the reality that group members may have conflicting rights and interests. if it is in the interest of amish children to receive an adequate education, and their receiving it will threaten the existence of the group (assuming for now that this is true), then a conflict of this kind is evidently present. our claim is that in such an eventuality there is no defensible reason to sacrifice the interests of the children in their education to their parents’ desire to reproduce the amish community in the name of group rights.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pag. ) arneson-shapiro on the rights of children to education against the community rights “(…) it is a far cry from saying that adults have a right to violate the rights of children to an adequate education, or to violate the legitimate interests of third parties in the education of children, in order that they may associate freely as an amish community. if rights violations of either sort really are necessary conditions, then the claims of the amish adults must give way. this is not because the amish ought to be stamped out but because there is no reason that children and third parties should have to endure rights violations so that the amish may freely associate.” (see richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pag .) arneson-shapiro on the definition of autonomy “several commentators on wisconsin v. yoder and related issues in family law have found the key to the puzzle to be a strong value of individual autonomy which finds appropriate expression in the “child’s right to an open future.” on this view, the aim of education is to prepare children for lives of rational autonomy once they become adults. a “rationally autonomous” life is one that is self-chosen in a reasonable way. education for rational autonomy thus encompasses two requirements: ( ) upon onset of adulthood individuals should be enabled to choose from the widest possible variety of ways of life and conceptions of the good and ( ) individuals should be trained into habits and skills of critical reflections, so that they attain to the greatest feasible extent the capacity to choose rationally among these alternative ways of life. we refer to these two aspects of education for autonomy as ( ) the maximization of options and ( ) the development of critical reason.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pag. ) arneson-shapiro on the claims of amish community “the conflict between the child’s right to autonomy so conceived and the claims of the old order amish parents is clear and direct; for the amish straightforwardly believe that they should educate their children so that they embrace the traditional amish way and that skills of critical reasoning would alienate their children from wholehearted identification with the right way to live and would therefore be corrupting.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pag. ) arneson-shapiro on the general characters of autonomy and of education for autonomy “what marks education for autonomy is development of skills and habits of critical thinking. the autonomous person is not the person who bears allegiance only to spontaneously self-chosen values untainted by environmental influence. the autonomous person is, rather, one who is capable of standing back from her values and engaging in critical reflection about them and altering her values to align them with the results of critical reflection. the difficulty with the amish program of socialization is not that it instils prejudgments – for any educational program does that – but rather that it fails to train children in skills of critical thinking and to encourage them to place a positive value on engagement in critical thinking about one’s fundamental values. the amish acculturation program is expressly designed to limit critical thinking, to get children to accept things on faith without submitting them to reasoned reflection.” richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pp. - ) arneson-shapiro on the duties of the citizens “member of a democratic society are obligated to fulfil the responsibilities of democratic citizenship, which centrally involve the assessment of public policy proposals. to assess policy proposals as a citizen one must be able to exercise critical reason by imagining what can be said for and against candidate policies from the different points of view represented in the electorate. the claim that one ought to exercise critical reason in evaluating policies and candidates is in conflict with the claim that one is entitled to eschew critical reason altogether in forming and affirming a view of one’s ow individual good.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pag. ). arneson-shapiro on targets of the education for children “our argument has been that in a democracy, where citizens are affected by the collective actions of the majority, it is necessary that citizens develop the capacities needed to understand and evaluate the policies by which their lives might be affected and through which they might affect the lives of others. children in a democracy should be educated to an age when critical reason is developed and can be deployed, and third parties have a right to expect parents and other educators to try to provide children with such an education.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pag. ) arneson-shapiro on the relationships between person’s choices and education for autonomy “we do not go so far as to assert that citizens in a democracy are obligated to participate in political life. we do insist, however, that even if, when they become adults, those who accept the amish way of life choose to withdraw from participation in the political order, they need critical reasoning capacities if such a choice is to be authentic. it is because the amish acculturation program is explicitly designed to prevent the development of critical reason that the amish should have lost in yoder. to accept a person’s choice of an amish way of life, one must have some reasonable confidence in that person’s choice-making competence. this competence, we have argued, is developed in education for autonomy.” (richard arneson and ian shapiro, “democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: ian shapiro (ed.), “democracy’s place”, pag. ) positionen von william galston ( ) - es existiert, es liegt keine rangordnung von gütern vor. - es existiert, es liegt keine erste tugend vor. - es existiert kein maßstab für güter. - güter sind im verhältnis zueinander qualitative heterogen. - es existiert kein summum bonum: es existiert kein höchster wert für alle individuen. - es existiert keine erste tugend in der institutionen der gesellschaft. - es existieren unterschiedliche güter und unterschiedliche tugenden. deren wichtigkeit und deren relevanz ist nicht absolut. positionen von william galston ( ) - wichtigkeit und relevanz der unterschiedlichen güter und der unterschiedlichen tugenden lassen sich nicht auf absolute art und weise messen. sie lassen sich lediglich in bezug auf verschiedene umstände messen. - der staat darf ausschließlich minimale maßstäbe bestimmen. - der staat darf lediglich einen minimalen inhalt von erziehung bestimmen. - der staat muss eine politik von the state must pursue a politics of maximal machbarer unterkunft verfolgen. galstons positionen • galstons grundsatzauffassung besteht darin, dass die prinzipien einer liberaldemokratie präzise grenzen den maßstäben auferlegen, die ein staat legitim anwenden darf. die prinzipien von galston sind die folgenden: • - es existiert keine rangordnung für güter. • - es existiert keine erste tugend. • - es existiert kein maßstab für güter. • - güter sind im verhältnis zueinander qualitativ heterogen. • - es existiert kein summum bonum: es existiert kein absoluter, kein größter wert für all die individuen. • - es existiert keine erste tugend bei den sozialen institutionen. • - es existieren verschiedene güter und unterschiedliche tugenden, deren wichtigkeit and relevanz nicht absolut sind. • - wichtigkeit und relevanz der unterschiedlichen güter und der verschiedenen tugenden können nicht auf absolute art und weise, sondern lediglich mit bezug auf die unterschiedlichen umstände gemessen und beurteilt werden. galstons positionen • schon auf der basis der prinzipien von galston können wir bemerken, dass wir uns innerhalb einer vollständig anderen denkweise als die denkweise von arneson und shapiro befinden. während arneson und shapiro die mentale entwicklung und bildung, die durch das erlernen der staatsbürgerkunde erlangt wird, als einen absoluten wert erachten, sind die positionen von galston ohne zweifel auf die abwesenheit eines absoluten wertes, auf die abwesenheit jedweden absoluten wertes für die sozialen güter orientiert. • die sozialen güter haben einen verschiedenen wert je nach den unterschiedlichen umständen; ihr wert variiert mit den umständen. es fehlt darüber hinaus auch ein maßstab, durch welchen die verschiedenen güter gemessen werden können. wir können infolgedessen sehen, dass galston der staatsbürgerkunde keinen absoluten wert zuschreibt. galston interpretation stammt aus seiner interpretation vom liberalismus als verschiedenheit, die an sich selbst der interpretation des liberalismus als autonomie opponiert. • während liberalismus als eine interpretation des individuums und der gesellschaft, welche das entstehen und die förderung der autonomie unterstützt, für einige denker gilt, deutet galston liberalismus als eine theorie, die diversität in der gesellschaft respektiert und die bedingungen für das zusammenleben zwischen den unterschiedlichen gemeinschaften, die im selben staat leben, garantiert. der staat soll nach galstons auffassung nicht autonomie in den individuen fördern, sondern sich mit dem minimalen machbaren begnügen. autonomie ist nicht der absolute wert, auf den der staat und das erziehungssystem hinzielen sollten. bezüglich des yoder-spruchs bemerkt galston das folgende: galstons positionen • wiewohl der staat interesse an der verpflichtenden erziehung hat, ist dieses interesse nicht absolut im sinne, dass all die anderen interessen im vergleich zur erziehung vernachlässigt werden. • - der fall yoder betrifft die grundinteressen der eltern, die mit dem staat kollidiert, die religiöse zukunft und erziehung der kinder zu garantieren. • der eingriff des staates in die sphäre hat viele grenzen: • - demokratie ist kein trump für alle fälle. • - in einer liberalen sichtweise sollen die öffentlichen institutionen vorsichtig und zurückhaltend sein, wenn sie mit den privaten vereinen handeln. • - es existiert keine voraussetzung dafür, dass der staat dazu berechtigt ist, in der sphäre der vereine zu intervenieren, da sie ihre inneren angelegenheiten auf eine andere art und weise durchführen, die von den generellen öffentlichen prinzipien divergieren. galstons positionen • galston ist der ansicht, dass kein einzige form von legitimer entscheidungsfindung existiert. demokratische institutionen sind nicht unbedingt dazu berechtigt, dass sie in der sphäre der vereine intervenieren. der staat hat keine legitimation, in vereinen zu intervieren, nur weil diese vereine andere prinzipien als diejenigen haben, die in der öffentlichen sphäre mehrheitlich sind. • galston befürwortet eine interpretation des liberalismus, die mit dem pluralismus koinzidiert: die annahme einer pluralistischen aussicht impliziert, dass der staat zumindest am anfang eine strategie von nicht-eingriff in die inneren angelegenheiten der vereine verfolgen soll. • - das verhältnis zwischen freiwilligen vereinen und öffentlichen normen ist nach galstons auffassung eine quelle für zwietracht zwischen zeitgenössischen interpreten des liberalismus. einige behaupten, dass die sozialen güter dermaßen wichtig sind, dass der staat zu deren verteidigung in der sphäre der vereine intervenieren darf. • - galston ist der auffassung, dass die freien vereine wichtige menschliche güter erzeugen, so dass der staat die beweislast hat, wenn der staat zu intervenieren versucht. • - da wertepluralismus, freiheit und politischer pluralismus prinzipien der gesellschaft sein sollten, darf der staat nicht als eine bevollmächtige behörde angesehen werden. galstons positionen • ein widerstreit liegt zwischen den auffassungen vor, die über die verhältnisse zwischen öffentlichen normen und freiwilligen vereinen zum ausdruck gebracht werden. nach einer bestimmten interpretation vom liberalismus berechtigen die bedingungen für die verteilung der sozialen güter zwischen bürgerinnen und bürgern den staat zu einem eingriff in all die vereine, die bestimmte soziale güter (wie es genau für die erziehung und das schulsystem der fall ist) ausschließen. die möglichkeit der verwendung von sozialen gütern hat den vorrang über die unabhängigkeit und über die souveränität innerhalb dieser spezifischen auffassung. galston ist seinerseits gegensätzlicher ansicht: er ist der auffassung, dass der staat im rahmen der entscheidung über die eingriffslegitimität die beweislast hat. der staat muss beweisen, dass er die berechtigung zum eingriff hat. • - innerhalb einer liberaldemokratie liegt das prinzip einer trennung zwischen der hoheit der eltern und der hoheit des staates. • - der staat hat das recht, minimale maßstäbe zu bestimmen, wie es diejenigen sein könnten, dass die eltern die pflicht haben, ihre eigenen kinder zu erziehen. • - der staat hat das recht, minimale erziehungsinhalte zu bestimmen. • - eltern haben dementsprechend einen weiten und geschützten umfang von wahlen, die das thema betreffen, wie die pflicht zur erziehung ausgeführt werden muss. • - dieselben rechte stehen den freiwilligen vereinen zu. • - ein liberaler staat darf jedenfalls bestimmte handlungen nicht machen, auch wenn diese handlungen dazu gerichtet sind, gute bürgerinnen und bürger zu bilden. • - die berufung auf die erfordernisse der politischen bildung ist mächtig, aber sie ist nicht immer entscheidend, falls dieser berufung von ansprüchen widersprochen wird, die sich auf die autorität der eltern oder auf die freiheiten der individuen und der assoziationen stützen. galstons positionen • es bleibt infolgedessen bestehen, dass die eltern die pflicht besitzen, ihre kinder zu erziehen. • es bleibt bestehen, dass der staat das recht hat, minimale inhalte für die erziehung zu bestimmen. • galston betont trotzdem, dass eltern und vereine die wahl besitzen, wie diese pflicht in die tat umgesetzt werden soll. die eingriffsmacht des staates hat präzise grenzen. die erfordernisse des staates haben nicht unbedingt den vorrang im vergleich zu den rechten der eltern und der freiwilligen vereinen. die anspruch der eltern und der freiwilligen vereine haben nicht unbedingt eine kleinere macht als die macht, die der staat hat. • der wortschatz, den galston verwendet, ist interessant: galston spricht von minimalen maßstäben und von minimalen erziehungsinhalten. die zuständigkeit des staates weitet sich nur auf ein minimum aus. die zuständigkeit des staates ist limitiert. die eltern haben hingegen eine weite sphäre für ihre handlung. der staat muss den freiheiten der zuständigkeit eltern und der freiwilligen vereine weichen. die priorität wird von galston den eltern und den vereinen gewährt. galstons positionen • eine liberale ordnung die als eigenen ansatz die verpflichtung zum moralischen und politischen pluralismus hat, wird sich nach der auffassung von galston bezüglich der möglichkeit des eingreifens in die sphäre der vereine und der familien limitieren: die strategie des staates wird darauf gerichtet sein, die maximal machbare unterkunft zu verfolgen. die prinzipien der strategie werden die sicherheit der individuen und die bürgerliche einheit sein. • - galston gesteht ein, dass eine solche strategie einen preis haben wird. • - diese strategie wird innerhalb der verschiedenen gruppen die präsenz von praktiken erlauben, die auf abneigung stoßen, wie es z.b. für den patriarchalismus der fall ist. • - diese strategie wird darüber hinaus erlauben, dass zahlreiche vereine die bedingungen für ihre mitgliedschaft auf eine art und weise definieren, die als einschränkungen für die individuelle freiheit erachtet werden könnten. • - diese sind preise, die nach galstons ansicht bezahlt werden müssen, auf dass die individuelle und die assoziative freiheit nicht vermindert werden. galstons positionen • die interpretation der pflichte und der damit zusammenhängenden zuständigkeit des staates, die galston gibt, sieht als die ziele, welche der staat verfolgen muss, • - die sicherheit der individuen und • - die bürgerliche einheit an: der staat muss diese ziele verfolgen. • Über diese ziele hinaus sollte der staat nicht gehen. der staat muss akzeptieren, dass das bestehen der assoziationsfreiheit und die individuelle freiheit eine verminderung der individuellen freiheit mit sich bringt. galston individuiert die anwesenheit von zwei verschiedenen konzeptionen des liberalismus: eine konzeption basiert auf der liberalen autonomie, die andere auf der liberalen diversität. • - die konzeption, welche die liberale autonomie als den grundlegenden ansatz hat, hängt mit der aufklärung zusammen. • - diese konzeption zielt auf die befreiung durch vernunft aus der autorität ab. • - innerhalb dieses kontextes wird vernunft als die erste quelle der autorität angesehen. • - das leben, das unter dem modell der vernunft gelebt wird, ist der tradition und dem glauben überlegen. • - das ziel des lebens ist der selbst-determinierung über die externe determinierung gegeben. • - ausschließlich die individuelle reflexion wird als eine legitime quelle für die interpretation des guten und bösen im leben erachtet. • - die liberale diversität ist zum postreformatorischen projekt verbunden: es wird damit eine lösung dafür gesucht, die politischen konsequenzen der religiösen differenzen zu versöhnen. • - das ziel dieser position liegt nicht darin, die autonomie zu fördern, sondern das zusammenlebern zwischen verschiedenen gruppen zu ermöglichen. galstons positionen • liberale autonomie wird mit der kritischen Überprüfung von sich selbst, von der gesellschaft, von den traditionen verbunden. die liberale autonomie heißt Überprüfung. liberale diversität ist legitimierung der unterschiede zwischen individuen und zwischen gruppen. diversität impliziert einen pluralismus der ideen in der gesellschaft. • - die liberalen demokratien sind nicht bürgerliche republiken. der liberaldemokratische staat besitzt nicht vollmächte. • - Öffentliche ziele sind nicht unbedingt das prinzip, dass in allen umständen die handlungen der bürgerinnen und bürger leiten. • - galston erachtet nicht autonomie und diversität als kompatibel. • - je nach der annahme von autonomie oder von diversität als grundprinzip des staates, werden voneinander verschiedene interpretationen der erziehung, des assoziationsrechtes und der freien ausübung der religion entstehen. • - die förderung der autonomie darf nicht als das prinzip des staates genommen werden. • - im falle, dass autonomie als das ziel des staates interpretiert würde, würde dies mit all den lebensmodellen konfligieren, die autonomie nicht als den ersten wert erachten. • eine zentrale auffassung von galston ist, dass liberalismus den schutz der legitimierten diversität als aufgabe hat. ein liberaler staat darf nicht darüber stellung nehmen, ob vernunft oder tradition befolgt werden sollte. autonomie als prinzip würde die homogenisierung aller gruppe herbeiführen. autonomie würde die diversität zerstören. wertepluralismus ist galstons modell. galstons positionen nach galstons auffassung bedeutet respekt für die religiöse freiheit der kinder nicht, dass die eltern all die glaubensbekenntnisse als äquivalent erachten sollten und dann die kinder zwischen den verschiedenen glaubensbekenntnissen wählen lassen sollten. • bezüglich der amish gemeinschaft sagt galston aus, dass die amish gemeinschaft kein gefängnis ist. • galston erachtet nicht die tugenden, die mit der teilnahme am öffentlichen leben verbunden sind, als die die einzigen tugenden, welche die staatsbürgerschaft kennzeichnen. die gesetzestreue, verantwortung, toleranz sind ebenso wichtig für die funktionierung einer liberalen demokratie. • galston gibt zu, dass es der fall sein könnte, dass familien und gemeinden die grundrechte der bürgerinnen und bürger verletzen; in diesen fällen muss der staat intervenieren. der staat muss aber vorsichtig sein, wenn es darum geht, den bereich zu definieren, in welchem die uniformität durch zwang gesichert werden muss. galston schließt nicht aus, dass der staat intervenieren kann; in all den fällen, in welchen die grundrechte der bürgerinnen und bürger bedroht werden, soll der staat intervenieren. galston denkt eher, dass der staat die reichweite der möglichkeit der intervention eingrenzen muss. galston on public goods “objective goods cannot be fully rank-ordered. this means that there is no common measure for all goods, which are qualitatively heterogeneous. it means that there is no summum bonum that is the chief goods for all individuals. it means that there are no comprehensive lexical orderings among types of goods. it also means that there is no “first virtue of social institutions” but, rather, a range of public goods and virtues the relative importance of which will depend on circumstances.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. ) galston consider, finally the case of wisconsin v. yoder, decided by the supreme court (…). this case presented a clash between a wisconsin state law, which required school attendance until age sixteen, and the old order amish, who claimed that high school attendance would undermine their faith-based community life. the majority of court agreed with the amish and denied that the state of wisconsin had made a compelling case for intervening against their practices: “[h]owever strong the state’s interest in universal compulsory education, it is by no means absolute to the exclusion or subordination of all other interests. … [t]his case involves the fundamental interest of parents, as contrasted with that of the state, to guide the religious future and education of their children. (liberal pluralism: the implication of value pluralism for political theory and practice”, p. ) galston on the limits of public institutions “… from a liberal pluralist point of view, i argue, there are multiple types of legitimate decision making, and democracy is not trumps for all purposes. another example: from a liberal pluralist point of view, public institutions must be cautious and restrained in their dealings with voluntary associations, and there is no presumption that a state may intervene in such associations because they conduct their internal affairs in ways that diverge from general public principles.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. ) galston on the contrast between the principles of voluntary associations and civic norms “the relationship between voluntary associations and publicly enforced civic norms has emerged as a key point of disagreement among contemporary liberals. some argue that civic goods are important, or fragile, enough to warrant substantial state interference with civil associations. it is a mistake, they believe, to give anything like systematic deference to associational claims. i disagree. i begin with the intuition that free associations yields important human goods and that the state bears a burden of proof whenever it seeks to intervene. my accounts of value pluralism, expressive liberty, and political pluralism lend theoretical support to this intuition and help explain why we should not see state power as plenipotentiary.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. - ) galston on the rights of parents “first, in a liberal democracy, there is in principle a division of authority between parents and the state. the state has the right to establish certain minimum standards, such as the duty of parents to educate their children, and to specify some minimum content of education, wherever it may be conducted. but parents have a wide and protected range of choices as to how the duty to educated is to be discharged. suitably revised and extended, these considerations apply to the liberties of civil associations as well. second, there are some things the liberal stat may not do, even in the name of forming good citizens. the appeal to the requisites of civic education is powerful, but not always dispositive when opposed by claims based on the authority of parents or the liberties of individuals and associations.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. ) galston on the costs of liberty “a liberal polity guided (as i believe it should be) by a commitment to moral and political pluralism will be parsimonious in specifying binding moral public principles and cautious about employing such principles to intervene in the internal affairs of civil associations. it will, rather, purse a policy of maximum feasible accommodation, limited only by the core requirements of individual security and civic unity. that there are costs to such a polity cannot reasonably be denied. it will permit internal associational practices (for example, patriarchal gender relations) of which many strongly disapprove. it will allow many associations to define their membership in ways that may be seen as restraints on individual liberty. and it will, within limits, protect those whose words and way of life express deep disagreement with the regime in which they live. but unless liberty – individual and associational – is to be narrowed dramatically, these costs must be accepted.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. ) galston on liberal autonomy liberal autonomy, i shall argue, is linked to an historical impulse often associated with the enlightenment – namely, liberation through reason from externally imposed authority. within this context, reason is understood as the prime source of authority; the examined life is understood as superior to reliance on tradition or faith; preference is given to self-direction over external determination; and appropriate relationships to conceptions of good or of value, and especially conceptions that constitute groups, are held to originate only through acts of conscious individual reflections on and commitment to such conceptions . liberal diversity, by contrast, is linked to what i shall call the post-reformation project – that is, to the effort to deal with the political consequences of religious differences in the wake of divisions within christendom. (liberal pluralism: the implication of value pluralism for political theory and practice”, pp. - ) galston on autonomy and on diversity “by “autonomy” i mean individual self-direction in at least one of many senses explored by john locke, immanuel kant, john stuart mill, and americans writing in an emersonian vein. liberal autonomy is frequently linked with the commitment to sustained rational examination of self, others, and social practices – whence mill’s invocation of socrates as liberal hero. by “diversity” i mean, straightforwardly, legitimate differences among individuals and groups over such matters as the nature of good life, sources of moral authority, reason versus faith, and the like.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. ) galston on aims and ends of liberalism “properly understood, liberalism is about the protection of legitimate diversity. a liberal state need not and should not take sides on such issues as purity versus mixture or reason without tradition. to place an ideal of autonomous choice – let alone cosmopolitan bricolage – at the core of liberalism is in fact to narrow the range of possibilities available within liberal societies. in the guise of protecting the capacity for diversity, the autonomy principles in fact exerts a kind of homogenizing pressure on ways of life that do not embrace autonomy.” (see william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. ) galston on value pluralism “value pluralism is offered as an account of the actual structure of the actual structure of the normative universe. it advances a truth-claim about that structure, not a description of the perplexity we feel in the face of divergent accounts of what is valuable. so value pluralism is not to be confused with emotivism, noncognitivism, or humean arguments against the rational status of moral propositions. like monism, it advances a “realist claim about the metaphysical structure of value.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. ) galston on the difference between pluralism and relativism “pluralism is not the same as relativism. philosophical reflection supports what ordinary experience suggests – a nonarbitrary distinction between good and bad or good and evil.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. ) galston on the duties of the parents “does respect for a child’s religious freedom mean that the parent is required to treat all comprehensive views equally, taking the child on a tour of different faiths and secular philosophical outlooks and then saying, in effect, you choose? few parents, whatever their outlook, would accept this proposition; even fewer would endorse its enforcement by the state. and i do not see considerations weighty enough to warrant such a sharp break with established practices. at the very least, parents are entitled to introduce their children to what they regard as vital sources of meaning and value, and to hope that their children will come to share this orientation. one might als argue that instructing children within a particular tradition, far from undermining intellectual or religious freedom, may in fact promote it. knowing what it means to live within a coherent framework of value and belief may well contribute to an informed adult choice between one’s tradition of origin and those encountered late in life.” (william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. - ) galston on the plurality of civic virtues “… the active deliberative/participatory virtues are not the only virtues of citizenship we should care about. law-abidingness, personal and family responsibility, and tolerance of social diversity are also important for the successful functioning of contemporary liberal democracies. in these respects, among others, the amish score high. they may not be the best of citizens, but may we not say that they are good enough? at least they fulfil the political version of the hippocratic oath – to do no harm. i might add that if nonvoting and civic withdrawal are taken as sufficient evidence of parental and pedagogical failure warranting state intervention, then or indictment extends far beyond the minute numbers of amish to implicate more than half the families and graduates of public schools in the united states.” (see william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. - ) galston on the duties of the state “if families, schools, or local communities are acting in ways that violate the basic rights of citizens, then the state must step in. and if the result is resistance – even “massive resistance” in the face of compulsory school desegregation – that is the price that must be paid for defending the rightful claims of all citizens. my point is, rather, that the state must be parsimonious in defining the realm in which uniformity must be secured through coercion. an educational program based on an expansive and contestable definition of good citizenship or civi unity will not ordinarily justify the forcible suppression of expressive liberty.” (see william galston, “liberal pluralism: the implication of value pluralism for political theory and practice”, pag. - ) raz on the definition of autonomy “the ruling idea behind the ideal of personal autonomy is that people should make their own lives. the autonomous person is a (part) author of his own life. the ideal of personal autonomy is the vision of people controlling, to some degree, their own destiny, fashioning it through successive decisions throughout their lives.” (joseph raz, the morality of freedom, chapter , autonomy and pluralism, pag. ) raz on the value of autonomy “autonomy is a constituent element of the good life. a person’s life is autonomous if it is to a considerable extent his own creation. naturally the autonomous person has the capacity to control and create his own life. i called this the capacity sense of autonomy, for ‘autonomy’ is sometimes used to refer to that capacity alone. that capacity, which involves both the possession of a certain mental and physical abilities and the availability of an adequate range of options, is sometimes referred to as positive freedom.” (joseph raz, the morality of freedom, chapter , freedom and autonomy, pag. ) raz on the duties of the government as regards the promotion of autonomy ( ) “so if the government has a duty to promote the autonomy of people the harm principle allows it to use coercion both in order to stop people from actions which would diminish people’s autonomy and in order to force them to take actions which are required to improve people’s options and opportunities.” (joseph raz, the morality of freedom, chapter , freedom and autonomy, pag. ) raz on the duties of the government as regards the promotion of autonomy ( ) “but the autonomy principle is a perfectionist principle. autonomous life is valuable only if it is spent in the pursuit of acceptable and valuable projects and relationships. the autonomy principle permits and even requires governments to create morally valuable opportunities, and to eliminate repugnant ones. does not that show that it is incompatible with the harm principle? the impression of incompatibility is encouraged by the prevalent anti-perfectionist reading of the harm principle. that reading is at odds with the fact that the principle merely restricts the use of coercion. perfectionist goals need to be pursued by the use of coercion. a government which subsidizes certain activities, rewards their pursuits, and advertises their availability encourages those activities without using coercion.” (joseph raz, the morality of freedom, chapter , freedom and autonomy, pag. ) raz on the contrasts between autonomy and community cultures “one particular troubling problem concerns the treatment of communities whose culture does not support autonomy. these may be immigrant communities, or indigenous people, or they may be religious sects. it is arguable that even the harm principle will not defend them from the ‘cultural imperialism’ of some liberal theories. since they insist on bringing up children in their own ways they are, in the eyes of liberals like myself, harming them. therefore can coercion be used to break up their communities, which is the inevitable by-product of the destruction of their separate school, etc.?” (joseph raz, the morality of freedom, chapter , freedom and autonomy, pag. ) raz: transformation, not disintegration “the perfectionist principles espoused in this book suggest that people are justified in taking action to assimilate the minority group, at the cost of letting its culture die or at least be considerably changed by absorption. but that is easier said than done. time and again i have emphasized that people can successfully enjoy an autonomous life only if they live in an environment which supports suitable social forms. by hypothesis members of the autonomy-rejecting group lack this support in their communities. wrenching them out of their communities may well make it impossible for them to have any kind of normal rewarding life whatsoever because they have not built up any capacity for autonomy. toleration is therefore the conclusion one must often reach. gradual transformation of these minority communities is one thing, their precipitate disintegration is another. so long as they are viable communities offering acceptable prospects to their members, including their young, they should be allowed to continue in their ways.” (joseph raz, the morality of freedom, chapter , freedom and autonomy, pag. ) raz und autonomie bibliografie ( ) arneson, r., - shapiro, i., democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: democracy’s place, edited by i. shapiro, ithaca and london, , pp. - . callaan, e., autonomy and schooling, kingston, ontario, . callaan, e., creating citizens: political education and liberal democracy, oxford, . feinberg: the child’s right to an open future, in freedom and fulfillment: philosophical essays, princeton , pp. - . galston, w. a.,liberal purposes : goods, virtues, and diversity in the liberal state, cambridge, u.k., . galston, w. a., liberal pluralism: the implications of value pluralism for political theory and practice, cambridge, u.k., . gutmann, a., children, paternalism, and education: a liberal argument, in: philosophy & public affairs, vol. , no. (summer, ), pp. - . gutmann, a., democratic education, mill, j. s., on liberty, malden, massachusetts, . bibliografie ( ) • raz, j., the morality of freedom, oxford, . • shapiro, i., - arneson, r., democratic autonomy and religious freedom: a critique of wisconsin v. yoder, in: democracy’s place, edited by i. shapiro, ithaca and london, , pp. - . • shapiro, i. (ed.), democracy’s place, ithaca and london, . herzlichen dank für ihre aufmerksamkeit! gianluigisegalerba@gmail.com can- - .. therapeutics, targets, and chemical biology preclinical therapeutic efficacy of a novel pharmacologic inducer of apoptosis in malignant peripheral nerve sheath tumors vincent chau , , s. kyun lim , wei mo , chiachi liu , amish j. patel , ren�ee m. mckay , shuguang wei , bruce a. posner , jef k. de brabander , noelle s. williams , luis f. parada , , and lu q. le , abstract neurofibromatosis type i (nf ) is an autosomal disorder that affects neural crest-derived tissues, leading to a wide spectrum of clinical presentations. patients commonly present with plexiform neurofibromas, benign but debilitating growths that can transform into malignant peripheral nerve sheath tumors (mpnst), a main cause of mortality. currently, surgery is the primary course of treatment for mpnst, but with the limitation that these tumors are highly invasive. radiotherapy is another treatment option, but is undesirable because it can induce additional mutations. patients with mpnst may also receive doxorubicin as therapy, but this dna-intercalating agent has relatively low tumor specificity and limited efficacy. in this study, we exploited a robust genetically engineered mouse model of mpnst that recapitulates human nf -associated mpnst to identify a novel small chemical compound that inhibits tumor cell growth. compound (cpd ) inhibits growth of all available in vitro models of mpnst and human mpnst cell lines, while remaining nontoxic to normally dividing schwann cells or mouse embryonic fibroblasts. we show that this compound delays the cell cycle and leads to cellular apoptosis. moreover, cpd can reduce mpnst burden in a mouse allograft model, underscoring the compound's potential as a novel chemotherapeutic agent. cancer res; ( ); – . � aacr. introduction neurofibromatosis type i (nf ) is an autosomal dominant genetic disorder that affects in , people ( ). patients typically present with a constellation of signs, including caf�e- au-lait pigmentation spots, benign neurofibromas, axillary or inguinal freckling, optic gliomas, lisch nodules in the eye, bone abnormalities, and multiple additional less penetrant mani- festations ( ). furthermore, nf patients are predisposed to malignant peripheral nerve sheath tumors (mpnst; ref. ), which are a significant source of mortality. neurofibromas are benign tumors that grow on the skin (dermal) or inside the body (plexiform; ref. ). neurofibromas have complex cellularity, including schwann lineage cells, fibro- blasts, perineurial cells, mast cells, collagen deposits, and phys- ical proximity to peripheral nerves. plexiform neurofibromas appear in % of nf patients ( ), and although they retain the capacity to enlarge throughout life, many lines of evidence point to an embryonic origin ( ). dermal neurofibromas can arise from a type of adult stem cell called skin-derived precursors (skp; ref. ), whereas plexiform neurofibromas arise from embryonic neural crest lineage schwann cell progenitors ( ). whether the two cell types are related or the same remains unclear ( ). nf patients can also develop malignant tumors, including leukemia, rhabdomyosarcoma, and neuroblastoma in children ( ). the most common malignancy, however, is mpnst, which arises from and appears in, approximately % to % of patients with plexiform neurofibromas ( ). mpnsts are ini- tially difficult to detect, metastasize widely, and have a poor prognosis ( ). mutation of nf , which encodes a ras-gtpase activating enzyme ( ), is known to activate ras downstream effectors, including the erk, pi k, and mtor pathways ( – ). the two most common cancer-associated mutations present in mpnsts in addition to nf mutation are the tumor suppressors p ( – ) and cdkn ( , – ). mice harbor- ing mutated nf and p or nf and cdkn spontaneously develop mpnst ( , ). in addition to nf and its downstream effector ras, the conditional deletion of pten also leads to mpnst in mice ( ). in this study, we exploit a robust model of mpnst that histologically and molecularly recapitulates human mpnst. we used primary cells from these mpnsts to screen for compounds that specifically arrest tumor cell growth. such compounds would not only have potential as a platform for novel therapeutic development but also for probing the biology of mpnst. elucidating the mechanism of action of such compounds could identify mpnst specific pathways and point to therapeutic opportunities. we report a novel small authors' affiliations: departments of dermatology, developmental biology, and biochemistry, and harold c. simmons comprehensive cancer center, university of texas southwestern medical center at dallas, dallas, texas corresponding authors: lu q. le or luis f. parada, ut southwestern medical center, harry hines blvd, dallas, tx - . phone: - - , fax: - - ; e-mail: lu.le@utsouthwestern.edu or luis.parada@utsouthwestern.edu doi: . / - .can- - � american association for cancer research. cancer research www.aacrjournals.org of research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ molecule, compound (cpd or identification number sw ), which induces apoptosis in all models of mpnst tested but spares normal cells and tissues. moreover, we demonstrate that this compound can decrease the tumor burden of mpnst in a mouse allograft model. materials and methods cell and tissue samples s and snf . cells were a gift from karen cichowski. schwann cells (sciencell) were cultured in schwann cell medi- um (sciencell) andwere immortalized with myc-retrovirus( ). high-throughput screening smpnst and schwann cells ( cells/well) were seeded in -well plates and grown overnight. compounds (final con- centration of . mmol/l) were added and incubated for hours. celltiter-glo (promega) was added and luminescence was quantified using envision multilabel reader (perkin elmer). soft agar assay acellular layer of . % bacto-agar in mpnst media was plated. cells were trypsinized, pelleted, resuspended, mixed in : ratio with . % agar to a final density of � cells/ml, and plated on top. after incubation for days, colonies were stained with . ml . % crystal violet. cell-cycle analysis brdurd ( mmol/l) was added to cells for minutes. cells werecollected, resuspendedin ml pbs, fixed in ml ice-cold ethanol, and then stored overnight at �c. n hcl/triton x- was added to denature dna. after neutralization, samples were incubated with ml . % tween / %bsa/pbs, ml anti- brdu-fitc (bd biosciences), and ml of mg/ml rnase overnight. samples were pelleted, resuspended in ml mg/ml propidium iodide (sigma), and analyzed by flow cytometry. western blotting cells were collected, trypsinized, and lysed with ml radioimmunoprecipitation assay buffer. samples were run on %– % sds-page gel (bio-rad). the following primary anti- bodies were used: cyclin d , h (millipore), caspase- , perk, erk, pakt, akt (cell signaling technology), parp (novus biologicals and millipore), a-tubulin (sigma), and gapdh (santa cruz). annexin v staining floating and adherent cells were resuspended in binding buffer ( . mol/l hepes/naoh (ph . ), . mol/l nacl, . mmol/l cacl ), and processed according to manufacturer (macs miltenyi biotec). three hundred microliters of suspen- sion was added to ml annexin v antibody (macs miltenyi biotec) and . ml propidium iodide, and analyzed by flow cytometry. metabolic stability studies cpd levels for metabolic stability and pharmaco- kinetic studies were monitored by liquid chromatography (lc/ms-ms) with ab/sciex (applied biosystems) qtrap mass spectrometer coupled to shimadzu prominence lc. cpd was detected with the mass spectrometer in positive mrm (multiple reaction monitoring) mode by following the precursor to fragment ion transition . to . . an agilent c xdb mm packing column ( � . mm) was used for chromatography. for s studies, cpd ( mmol/l) was incubated in a ml incubation volume with mg of murine cd- s (combined cytosol and microsome) fractions (celsis/in vitro technologies) and phase i (the nadph regenerating system) cofactors (sigma) for to minutes. reactions were quenched by mixing the incuba- tion mixture with ml of methanol/ . ng/ml n-benzyl- benzamide/ . % formic acid. the quenched mixture was vortexed for seconds, incubated at room temperature for minutes, and spun for minutes at � g. super- natants were spun in a refrigerated microcentrifuge for minutes at , � g. the second supernatant was trans- ferred to an hplc vial and analyzed by lc/ms-ms. pharmacokinetic studies six- to -week-old ncr-nu/nu female mice were implanted with . � mpnst cells in the left flank, subcutaneously. when tumors reached mm , animals were injected with cpd at mg/kg in . ml % ethanol, % peg , % of a solution of % dextrose, ph . . blood was drawn at , , , , , , and , minutes using the anticoagulant, acd (acidified citrate dextrose) and plasma isolated by centrifuga- tion. one hundred microliters of plasma was mixed with ml acetonitrile containing . % formic acid and . ng/ml n- benzylbenzamide is. samples were vortexed seconds, incu- bated at room temperature for minutes, and spun twice at , � g at �c. amount of cpd present in plasma was quantified by lc/ms-ms to determine the rate of clearance from mouse blood. a cpd standard curve was generated using blank plasma spiked with known cpd concentrations and processed as described above. concentrations of cpd in each time-point sample were quantified using analyst . . . the limit of detection was defined as -fold above the signal obtained from blank plasma. the limit of quantitation (loq) was defined as the lowest concentration at which back calcu- lation yielded a concentration within % of theoretical. the loq for plasma was ng/ml and ng/ml for tumor. back calculation of points on the standard curve yielded values within % of theoretical over orders of magnitude ( , to or ng/ml). pharmacokinetic parameters were calculated using the noncompartmental analysis tool of winnonlin (pharsight). bioluminescent imaging smpnst-lucþ cells ( � cells/ ml) were injected in both flanks of nude mice. mice were injected with mg/kg cpd or vehicle ( % ethanol, % peg , and % of % dextrose in water) intraperitoneally twice per day for weeks. mice were injected with ml mg/ml d-luciferin potas- sium salt (perkin elmer) in . % sterile nacl, and total flux was quantified using ivis lumina ii (caliper life sciences) weekly. chau et al. cancer res; ( ) january , cancer researchof research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ immunohistochemistry tissues were fixed in % formalin and paraffin embedded. after sectioning, immunohistochemical studies were per- formed as previously described ( ). the following primary antibodies were used:s ( ), gap (abcam), ki (thermo scientific), caspase- (cell signaling technology), and parp (novus biologicals). results nf and p -deficient skps form mpnst nf -associated dermal and plexiform neurofibromas are histologically compatible despite having significantly different natural history and tumor progression properties. recently, we reported that skps are the cell-of-origin of dermal neurofibro- mas ( ). given the potential in mice for neural crest progenitors to give rise to mpnst after additional loss of the p tumor suppressor, we tested the consequences of dual nf and p loss of function (np) in skps. skps from mice with nf f/f;p f/f genotype were cultured and infected with adenovirus contain- ing cre-gfp (ad-cre) to induce tumor suppressor recombina- tion (fig. a, a; ref. ). the majority of infected cells displayed green fluorescence, indicating effective viral entry and expres- sion (fig. a, b). we confirmed effective cre-mediated recom- bination of all floxed alleles by pcr analysis (fig. ab). the nf �/�;p �/� cells (np skps) were reimplanted into the same mouse from which they were derived (autograft) and highly aggressive, malignant tumors developed in all cases (fig. a, c). these tumors could be successfully cultured as either spheres or adherent cells (data not shown) or allografted into nude mice(fig. a,d). thesetumors(termedsmpnst, for skp- derived mpnst) were assayed for human mpnst-associated genes, including embryonic schwann cell markers, s ( , ), krox ( ), plp ( ), and gap ( , ); developing glia marker, dhh ( ); and neural-crest-associated marker, snail ( ). the quantitative reverse transcriptase-pcr (qrt-pcr) figure . mpnsts can be generated from skps that are deficient in nf and p . a, skps were isolated from the skin of cis-nf f/f;p f/f mice, cultured, and infected with ad-cre-gfp (a). infected skps were screened for expression of gfp and genotyped for the deletion of nf (b). lane , nf f/f control. lane , wild-type nf þ/þ control. lanes and , nf �/� skps that were infected with ad-cre-gfp. these nf �/� ;p �/� skps were then autologously transplanted back into the original mouse (c). resultant tumors were transplanted into nude mice via xenotransplantation (d). qrt-pcr results (e). lanes – , mpnsts derived from cis-nf þ/�;p þ/� mice. lanes – , mpnsts derived from plp-creert-nf f/f;p f/f mice that had been injected with tamoxifen to induce recombination. lanes – , smpnsts. b, a, hematoxylin and eosin staining of smpnst. immunohistochemical staining for s b in smpnst (b) and skin (c), and for gap in smpnst (d) and skin (e). scale bars, mm. small-molecule inhibitor of mpnst www.aacrjournals.org cancer res; ( ) january , of research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ figure . identification of cpd by high-throughput, small-molecule screening and validation. a, summary of screening procedure. b, high-throughput screening dataforsmpnst cells(top) and schwann cells (bottom).intensity of blueor redsignal correlates withdegreeof inhibitionor inductionof atplevels by small molecules, respectively. c, structure of cpd . d, dose-response curves of cpd ( . , . , . , , . , , , and mmol/l) in smpnst cells, mef, schwann cells, and mpnst cells from multiple murine models. e and f, dose-response curves of cpd treatment on human mpnst cell lines, s and snf . , respectively, compared with mouse nih t cells and smpnst cells. g, dose-response curves of cpd treatment on wild-type schwann cells, compared with smpnst and mpnst from cis-nf f/f;p f/f mice. h, soft agar assay of cpd -treated ( . , . , , . , or mmol/l) smpnst cells compared with dmso. arrows indicate colonies, which may be out of plane in agar. scale bars, mm. i, quantification of h. j, soft agar assay of mpnst cells from cis-nf þ/� ;p þ/� mice and that were treated with dmso or cpd at . , . , , . , or mmol/l. arrows indicate colonies, which may be out of plane in agar. scale bars, mm. k, quantification of j. all values, mean � sd. the student t test used for significance testing (�, p < . ; ��, p < . ; ���, p < . ). chau et al. cancer res; ( ) january , cancer researchof research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ data demonstrated expression of all markers in all smpnsts analyzed (fig. ae). thus, smpnsts exhibit molecular char- acteristics of human nf associated mpnst. hematoxylin and eosin (h&e) staining of smpnsts showed presence of spindle-shaped cells that interweave in a poorly differentiated, wavy appearance, reminiscent of human mpnst histology (fig. b, a). in addition, the com- monly used mpnst markers, s and gap , were present (fig. b—, b–e). therefore, our results indicate that skps, cells that give rise to neurofibromas upon loss of nf , can also give rise to mpnst with additional loss of p . the facility of generating and explanting smpnsts made this a favorable system for performing chemical compound screens. novel mpnst inhibitory small molecule we hold the concern that over time in culture, tumor cells will drift from their original intrinsic cellular and molecular programs in unpredictable ways. these uncontrollable and unmeasurable changes may inadvertently alter fundamental properties of the original tumor cells. to mitigate these con- cerns, the relative facility of generating primary cultures from smpnsts afforded the advantage of propagating sufficient numbers of cells for a high-throughput screening while keeping the cell passage number low. we performed a limited high- throughput, small-molecule screen using smpnst cells pro- duced above in an extension of a related ongoing large-scale screen in our research group. we carried out a , com- pound screen on primary mouse glioblastoma multiforme cells that are deficient for nf , p , and pten (s. kyun lim and luis f. parada, unpublished data). this original glioblastoma multi- forme screen yielded , compounds, identified as inhibitory to glioblastoma multiforme cell growth. we reasoned that since smpnst cells are deficient in two of these same tumor suppressors, some compounds might have conserved smpnst cell growth inhibitory properties. a well-established luminescence assay for measuring atp levels (an indication of cellular metabolic activity) was adopted (celltiter-glo, promega), and performed hours after com- pound exposure. a total of , compounds exhibited more than % decrease in atp levels at . mmol/l (fig. a and b). to exclude compounds that exert general cell toxicity, or perturb the generic cell cycle or mitotic machinery, we count- er-screened the , compounds against myc-immortalized schwann cells ( , ) and eliminated compounds that showed more than % lower atp levels. the resultant compounds were rescreened on smpnsts with a cutoff at % inhibition. twenty-eight compounds passed this test and were further tested in dose response studies ranging from nmol/l to mmol/l. the twenty-first compound (fig. c; cpd or sw ) inhibited atp consumption of smpnst and all other models of mpnst tested (fig. d) with an ec of mmol/l. we extended the functional analysis of cpd to additional cell lines. human mpnst cell lines, s (fig. e) and snf . (fig. f), were assayed for dose-dependent growth, and ec concentrations of . and . nmol/l, respective- ly, were determined. in contrast, nih t cells and wild-type figure . effect of cpd on cell-cycle machinery. a, effect of cpd on cell-cycle distribution. smpnst cells were treated with dmso, d, or cpd ( . , . , , . , or mmol/l) for hours, pulsed with brdurd for minutes, and analyzed by flow cytometry. percentage of cells in each phase is shown. b–d, smpnst cells were treated with dmso, d, or with cpd ( . , . , , . , or mmol/l) for hours and rna harvested for qrt-pcr for cyclins (b), cdks (c), or cdkis (d). e, western blot analysis for cyclin d in smpnst cells that were treated with dmso, d, or cpd ( . , . , , and . mmol/l). all values, mean � sd. the student t test used for significance testing (�, p < . ; ��, p < . ; ���, p < . ). small-molecule inhibitor of mpnst www.aacrjournals.org cancer res; ( ) january , of research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ schwann cells showed a considerably higher dose resistance (fig. g). we also tested smpnst response to cpd in anchor- age-independent soft agar growth assays and found that cpd inhibited colony formation (fig. h and i). this effect was also observed in mpnst cells derived from- cis-nf þ/�;p þ/� mice (fig. j andk). thus, cpd hasgeneral toxic activity on nf and p -deficient mpnst cells but not on diverse nontumorigenic, mitotically active fibroblasts or schwann cells. cpd delays the cell cycle in smpnst to address the mechanism of action of cpd , we treated smpnst cells at . , . , , . , and mmol/l for hours, and pulsed with brdurd for minutes. subsequent flow cytometry analysis revealed a decreased percentage of cells in s-phase, and a corresponding increased percentage in g –g and g –m, compared with treatment with dmso alone (fig. a). several cell-cycle regulatory genes exhibited decreased mrna in the presence of cpd , including cyclin a , cyclin b , cyclin d , cyclin e, cdk , and cdk (fig. b and c). increased levels of cdkn a and cdkn a mrna were observed in a dose- dependent manner (fig. d). also, levels of cyclin d protein, which is known to interact with cdk / in regulating g –s progression, were decreased (fig. e; refs. , ). these results indicate that following cpd exposure, cells in s-phase are underrepresented, consistent with cell-cycle delay and an arrest of the mitotic machinery. we examined cpd -treated smpnst cells for apoptosis by annexin v-fitc–conjugated flow cytometry. tumor cells treated with cpd concentrations at or above . mmol/l showed a statistically significant increase in the percentage of apoptotic cells (fig. a). at the ec concentration of mmol/l, . � . % of the cells were annexin v-positive, compared with . % � . % in dmso-treated control cells. we also performed western blotting of cpd -treated smpnst cells and observed caspase- (fig. b) and parp (fig. c) cleavage, consistent with induced apoptosis. we further validated this mechanism in mpnst cells cultured from spon- taneous mpnsts from our cis-nf þ/�;p þ/� mouse model (fig. d), and in the human mpnst cell line, s (fig. e). other cell types, including myc-immortalized schwann cells (fig. f) and mef cells (fig. g), did not undergo caspase- cleavage. together, these data indicate that, in vitro, cpd figure . cpd induces apoptosis in vitro. a, annexin v staining as detected by flow cytometry. smpnst cells were treated with dmso, d, or cpd ( . , . , , and . mmol/l). smpnst cells were treated with staurosporine (st.) as positive control. b–g, western blotting for caspase- cleavage in smpnst cells (b), parp cleavage in smpnst cells (c), caspase- cleavage in mpnsts from cis-nf þ/�;p þ/� mice (d), caspase- cleavage in human mpnst cell line, s (e), caspase- cleavage in myc-sc (f), and caspase- cleavage in mef cells (g). each cell type was treated with staurosporine (st.) as a positive control. total caspase- (t. casp.- ) and cleaved caspase- (cl. casp.- ) were assayed in each case. all values, mean � sd. the student t test used for significance testing (�, p < . ; ��, p < . ; ���, p < . ). chau et al. cancer res; ( ) january , cancer researchof research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ small-molecule inhibitor of mpnst www.aacrjournals.org cancer res; ( ) january , of research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ induced mitotic arrest of mpnst cells that ultimately results in cellular apoptosis, and that this phenotype was not observed in untransformed dividing cells. cpd decreases tumor burden and induces apoptosis in vivo to determine whether cpd could be directly tested in vivo, we sought to define the metabolic properties in vitro according to previously published methods ( , ). to examine stability, cpd was incubated with purified, hepatic, enzymatic s fraction over a period of minutes, and measured over time (fig. a). thehalf-life inthes fraction is . minutes. we also incubated cpd with cultured hepatocytes and found that the half-life is . minutes (fig. b). these assays demonstrate that cpd would have reasonable stability in vivo. we next sought to define the pharmacokinetic properties of cpd according to previously published methods ( ). smpnst ( . � ) cells were injected subcutaneously into nude mice, and when tumors reached mm , a single dose of cpd was injected intraperitoneally at mg/kg. plasma and tumor samples were collected after , , , , , and minutes and assayed by mass spectrometry (fig. c). we observed that cpd rapidly distributes from the plasma into smpnst tissues and that the half-life in smpnst tissues is to hours once the compound is completely distributed. impor- tantly, cpd concentration remains above the ec concen- tration of mmol/l ( ng/ml) in the smpnsts for more than hours. these results indicate that cpd is successfully delivered to the tumor tissue in adequate concentrations and with an adequate half-life for in vivo studies. we next determined a dose tolerable for chronic in vivo administration of cpd . cpd dissolved in a mixture of ethanol, peg , and dextrose was delivered intraperitoneally at or mg/kg twice per day, into nude mice over the course of weeks for comparison with vehicle administration. these concentrations were selected because mg/kg is the maxi- mum solubility in the aforementioned formulation. over this period, the mice were weighed daily and observed for signs of toxicity. neither concentration of cpd produced measurable weight loss or distress in the mice (data not shown). therefore, we selected mg/kg twice per day intraperitoneally as the dosing regimen for efficacy studies. we then tested the effects of cpd administration on tumor burden in vivo. nude mice were injected with smpnst cells carrying a luciferase gene (mpnst-lucþ) to permit noninva- sive in vivo tumor growth assessment by luminescence. one week after allograft, recipient mice were intraperitoneally injected with either mg/kg of cpd or vehicle twice daily over weeks, and mice were imaged weekly. we observed that treatment with cpd reduced the tumor burden as measured by total flux normalized to baseline flux levels (fig. d and e). brdurd was injected into mice before necropsy. both brdurd and ki evaluation indicated a significant decrease in tumor cell proliferation following cpd treatment (fig. f–i). these results are consistent with our observations that cpd inhi- bits the cell cycle in vitro, resulting in a decrease in the proliferative capacity of smpnsts. consistent with in vitro data, cpd -treated smpnsts exhibited elevated activated caspase- and parp (fig. j– m). these results were confirmed by western blotting to detect cleaved caspase- and parp in tumors from our in vivo model (fig. n). taken together, our results demonstrate that cpd can be delivered to mice in concentrations to sufficiently penetrate smpnst tissue, and inhibit tumor development. to extend the previous observation that chronic cpd exposure did not appear to cause general toxicity, we examined various tissues following necropsy for histologic evidence of cpd -induced toxicity. in addition to tumor tissue (fig. a), we examined liver (fig. b), kidney (fig. c), brain (fig. d), skin (fig. e), and sciatic nerve (fig. f), and found no evidence of abnormal histology. activated caspase- could only be detected in tumor samples (fig. g) and not in other tissues examined (fig. h–l). in contrast to other chemotherapeutic agents such as doxorubicin that induce toxicity in a variety of tissues ( ), cpd appears to act most notably on malignant tumor tissue. interaction with other anticancer agents in addition to doxorubicin, mpnsts have been shown to be sensitive to pathway-specific inhibitors of mapk/erk ( ), pi k/akt ( ), mtor ( – ), and cxcr ( ). we therefore examined whether cpd in conjunction with doxorubicin or specific inhibitors, including u , a mek inhibitor ( ); ly , an akt inhibitor ( ); rapamycin, an mtor inhib- itor ( – ); and amd , a cxcr inhibitor ( ), might better inhibit smpnst cell growth as assessed by atp consumption ( ). we found the inhibition of the pi k pathway combined with cpd treatment resulted in a decrease in cellular growth and survival that is greater than either inhibitor alone (fig. a and b). phospho-akt western blots of smpnst cells treated with ly at mmol/l for or minutes verified effective pakt inhibition in smpnst cells (fig. c). inhibition of the mek/erk pathway combined with cpd treatment did not produce detectable additive or synergistic effects (fig. d). phospho-erk inhibition by u was verified by western blotting (fig. e). inhibition of the mtor pathway combined with cpd treatment did not produce detectable additive or synergistic effects (fig. f). in fact, smpnst cellular growth figure . effect of cpd in vivo. a and b, cpd was incubated with murine, hepatic s fractions (a) or murine hepatocyte (b) for indicated times. amount of cpd remaining was quantified by mass spectrometry. c, cpd was injected at mg/kg i.p. and quantified in plasma or tumor over time. pharmacokinetic parameters were quantified using the noncompartmental analysis tool in winnonlin (pharsight). d, smpnst-lucþ cells were injected subcutaneously in nude mice. cpd ( mg/kg) or vehicle was injected intraperitoneally twice per day for weeks. mice were imaged every days. images shown are from week . e, quantification of tumor images in d by normalizing total flux at week with baseline flux (n ¼ for vehicle, n ¼ for cpd ). f, brdurd staining of cpd - or vehicle-treated tumors. g, quantification of f. h, immunohistochemistry of ki . i, quantification of h. j, immunohistochemistry of cleaved caspase- . k, quantification of j. l, immunohistochemistry of cleaved parp. m, quantification of l. n, western blotting for caspase- and parp cleavage in tumor lysates. all microscopic images taken at � . scale bars, mm. all values, mean � sd. the student t test was used for significance testing (�, p < . ; ��, p < . ; ���, p < . ). chau et al. cancer res; ( ) january , cancer researchof research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ seemed relatively resistant to rapamycin, despite exhibiting decreased ps levels after rapamycin treatment (fig. g). none of the other tested reagents produced measurable additive or synergistic effects (data not shown). we extended the above studies of dual ly and cpd treatment to mpnst cells derived from cis-nf þ/�;p þ/� mice, and again, the combined effect of both inhibitors is greater than that of each individual inhibitor (fig. h). we tested the effects of both inhibitors on phosphorylation of akt and found that pakt was inhibited only when ly is present (fig. c and i). thus, in contrast to published reports of successful application of mek inhibi- tors to suppress nf -based solid tumors and leukemias, in the context of cp , coinhibition of the pi k pathway was most effective in impeding tumor cell growth and survival. discussion malignant peripheral nerve tumors are the major source of mortality in nf . like most malignant sarcomas, these tumors are resistant to all current chemotherapeutic and radiother- apeutic strategies. it has long been recognized that mpnsts harbor tumor suppressor mutations in addition to nf loss. notably, loss-of-function mutations of the p tumor suppres- sor have been reported ( – ). this has led to the successful generation of spontaneous mouse models of mpnst by muta- tion of both nf and p tumor suppressor genes ( , ). a further refinement of mpnst modeling came about by iden- tifying skps as a source for dermal neurofibromas in the skin and by the capacity of skps to engender plexiform neurofi- bromas when placed in the sciatic nerve ( ). additive mutation of p together with nf turned skps into progenitors of mpnst, thus facilitating high-throughput screens for chemical compounds that might inhibit early passage cell growth. we identified cpd as a small molecule with inhibitory properties on cultured mouse and human mpnstcells. it was a surprise that this compound originating from a , chem- ical compound library, had, without chemical modification, a repertoire of desirable in vivo properties, including low micromolar ec , adequate stability, manageable toxicity, and adequate accumulation in solid tumor tissue. these serendip- itous favorable properties allowed allograft analysis of figure . apoptotic effect of cpd is specific to tumor tissue. a–f, h&e staining of tumors (a), liver (b), kidney (c), brain (d), skin (e), and sciatic nerve (f) from mice treated with either cpd or vehicle. g–l, immunohistochemistry for cleaved caspase- was only detected in tumor tissue (g), but not in liver (h), kidney (i), brain (j), skin (k), or sciatic nerve (l). all images taken at � . scale bars, mm. small-molecule inhibitor of mpnst www.aacrjournals.org cancer res; ( ) january , of research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ antitumor efficacy and demonstrated a significant reduction in tumor burden by this compound. future efforts to assess the effectiveness of cp on human mpnst xenografts will pro- vide additional insight into the potential for this compound as a therapeutic agent. at present we cannot identify the specific mechanism whereby cpd exerts its inhibitory action on mpnst cells. compound exposure to tumor cells results in a cell-cycle delay manifested by depletion of certain cell-cycle–related molecules and eventually leading to cellular apoptosis. a similar course of events is seen when tumor-bearing mice are treated with compound. however, the precise molecular target and mech- anistic mode of compound action remains a topic of investi- gation. empirical chemical compound screens to identify cancer cell inhibitory compounds have been applied for several decades. a significant proportion of such empirically derived cancer cell inhibitory small molecules that have reached the clinic have turned out to mechanistically impinge on various aspects of cell division, including mitotic spindle machinery, cell-cycle machinery, and dna replication and repair machin- ery. these cellular properties are not unique to cancer cells and are shared by many normally dividing cells in the body that are cpd only cpd only u only cpd + ly r l u sa m p le /r l u d m s o ly only * * * * * * * * * * . . . r l u sa m p le /r l u d m s o . . . r l u sa m p le /r l u d m s o . . . . r l u sa m p le /r l u d m s o . . . . . . . . cpd + ly . . . cpd + u . . . . – – –– – . . cpd + rapamycin . . . . . combined ( µmol/l ly ) combined ( µmol/l u ) cpd ( nmol/l) cpd ( nmol/l) + ly ( µmol/l)ly ( µmol/l) dmsoa c f h i g d e b combined ( µmol/l u ) combined ( µmol/l ly ) combined ( µmol/l ly ) cpd only cpd only ly only cpd only cpd (µmol/l) ly (µmol/l) cpd + ly rapamycin only r a p a m yc in - m in r a p a m yc in - m in d m s o combined ( . µmol/l rapamycin) ly (min) ly only pakt akt gapdh pakt akt gapdh perk d m s o u erk h ps d m s o s tubulin d combined ( µmol/l ly ) figure . cpd interacts with pi k inhibitor. a, atp assay following combined treatment of cpd and ly in smpnst cells. synergism determined by loewe method. b, phase contrast images of smpnst cells treated with dmso, cpd alone, ly (ly) alone, or a combination of both inhibitors. images taken at � . scale bars, mm. c, western blotting for pakt in smpnst cells treated with mmol/l ly for or minutes. d, atp assay following combined treatment of cpd and u in smpnst cells. e, western blotting for perk in smpnst cells treated with mmol/l u for minutes. f, atp assay following combined treatment of cpd and ly in smpnst cells. g, western blotting for ps in smpnst cells treated with . mmol/l ( nmol/l) rapamycin for or minutes. h, atp assay following combined treatment of mpnst cells from cis-nf þ/� ;p þ/� mice with cpd and ly . synergism determined by loewe method. i, western blotting for pakt and akt in smpnst cells treated with cpd alone, ly alone, or with both inhibitors. all graphs plotted in micromolar concentrations. chau et al. cancer res; ( ) january , cancer researchof research. on april , . © american association for cancercancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .can- - http://cancerres.aacrjournals.org/ essential for organ health. chemotherapeutic toxicity to nor- mal cells represents a major impediment to effective cancer treatment. in this regard, our screen departs from traditional high-throughput screens in that we limited our cells assayed to early passage cells rather than to established cell lines. it is also noteworthy that the cellular specificity of cpd renders it relatively innocuous to the normally dividing cells we tested (mefs, myc-schwann cells, and wild-type skps). this feature is consistent with the idea that the compound target(s) relates to the "cancer state" of the mpnst cells rather than to more general properties of mitotically active cells. although we currently do not know whether cpd is also effective against the benign plexiform neurofibromas, it is entirely possible that these slowly dividing tumor cells may also be susceptible to cpd activity if they share common pathways with mpnsts that are potential target(s) of cpd . recent advances in mpnst research have pointed to addi- tional potential therapeutic targets, including the cxcr cyto- kine axis ( ), the mtor pathways ( ), and the ras/mek/erk pathways( , – ).inthese studies,amd andrapamycin have been shown to inhibit mpnsts from the cis-nf þ/�;p þ/� -mouse model, and erk inhibitors, such as u , have been verified in a variety of human and mouse derived mpnsts. interestingly,whensuchcompoundsweretestedinconjunction with cp , we found no additive value. instead, blockade of the pi k pathway did cooperate with cpd action to inhibit tumor growth.thisintriguingresultpoints tothe potentialplasticity of cancer cells and to the complexity of tumor pathway interac- tions. for example, it may be that cpd activity somehow unveils a synthetic dependence on pi k signaling that is not present in the absence of compound. continued studies of cpd , its specific mode of action, and the mechanistic basis for its activity on mpnst cells but not on nontumorigenic cells may shed light on novel anticancer pathways. disclosure of potential conflicts of interest v. chau has ownership interest in patent pending for cpd . no potential conflicts of interest were disclosed by the other authors. authors' contributions conception and design: v. chau, s.k. lim, w. mo, l.f. parada, l.q. le development of methodology: v. chau, s.k. lim, w. mo, l.f. parada, l.q. le acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): v. chau, s.k. lim, w. mo, c. liu, a.j. patel, s. wei, b.a. posner, n.s. williams, l.f. parada, l.q. le analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): v. chau, a.j. patel, r.m. mckay, s. wei, b.a. posner, n.s. williams, l.f. parada, l.q. le writing, review, and/or revision of the manuscript: v. chau, s.k. lim, r.m. mckay, s. wei, b.a. posner, l.f. parada, l.q. le administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): v. chau, s.k. lim, w. mo, s. wei, b.a. posner, l.q. le study supervision: v. chau, w. mo, l.f. parada, l.q. le input on chemistry, selection of chemical compounds from library screen, and proofreading manuscript: j.k. de brabander acknowledgments the authors thank michael white for helpful discussions throughout this project and karen cichowski for sharing the human mpnst cell lines. the authors also thank all members in luis parada's laboratory and in lu le's laboratory for helpful discussions. grant support this work was partially supported by funding from texas neurofibromatosis foundation, the national cancer institute (grant# r ca ; l.q. le), the nih (grant# p ns ; l.f. parada), cprit (rp ; l.f. parada), and the simmons cancer center support grant# p ca - . l.q. le is a disease- oriented clinical scholar and holds a career award for medical scientists from the burroughs wellcome fund. l.f. parada is an american cancer society research professor. the costs of publication of this article were defrayed in part by the payment of page charges. this article must 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% (table i). the risk of multifactorial disease, however, varies from one disease to another and from family to family. within a family the risk depends on the severity of the disorder in the proband, the number of affected family members, and the contribution from environmental risk factors. table i-rissfor common polygenic diseases ofaduls disorder in proband risk for first degree relatives (%) coronary heart disease for male relatives for female relatives diabetes mellitus - epilepsy - hypertension manic depressive psychosis - psoriasis - schizophrenia thyroid disease number ofgene loci implicated in polygenic disease for any particular disease we need to ascertain the genes that may participate. for a complex disorder such as coronary heart disease, in which plasma lipoproteins, the coagulation system, and the cellular elements of the blood and arterial wall all play a part, the number of genes may be large. i have taken one component of this problem as an example; table ii lists genes that may be associated with the regulation of plasma cholesterol concentration. the list is not comprehensive because there are proteins that affect plasma cholesterol concentrations which have not even been identified. the issues are, firstly, to establish which ofthese genes has a major effect on the genetic variance in plasma cholesterol concentration-the extent to which each gene contributes, if at all, has still to be established-and, secondly, to identify new loci that contribute. for other disorders such as diabetes and manic depression the list of candidate genes is short since few of the gene loci have been identified. table ii-genes associated with lipid metabolism class gene apo-ai apo-aii apo-aiv apolipoproteins apo-bapo-ci apo-cii apo-cili apo-e hmg coa reductase lecithin cholesterol acyl transferase fatty acyl-coa cholesterol acyl transferase endothelial lipoprotein lipase enzymes hepatic triglyceride lipase fatty acid snthetase phosphatadic acid phosphohydrolase cholesterol ester hydrolase cholesterol -a hydrolase transfer proteins lipid transfer proteins low density lipoprotein receptors iapo-e thigh density lipoprotein candidate genes how can we establish whether a specific gene contributes to a disorder? as an example, let us considermanic depressive psychosis, which may be due to abnormal dopamine metabolism. the enzyme tyrosine hydroxylase has a key regulatory role in dopamine bio- synthesis. perhaps a mutation of the tyrosine hydroxylase gene on chromosome contributes to this disorder. this suggestion can be tested using probes for the tyrosine hydroxylase gene or nearby flanking genes. if a mutation of the tyrosine hydroxylase gene causes the disease cosegregation of a specific allele of the gene with the disease within affected families could be tracked with restriction fragment length polymorphisms. if cosegregation does not exist the tyrosine hydroxylase gene can be eliminated as a suspect in those families. on the other hand, if consistent cosegregation does exist a causal role for the gene in the disease is strongly supported. remarkably, in a pennsylvania amish kindred a locus identical with or adjacent to the tyrosine hydroxylase gene has been associated with this disease.' the tyrosine hydroxylase locus does not, however, provide the whole answer to manic depressive illness. genetic heterogeneity exists and at least two other loci are also implicated. one has been localised near the fragile site on the x chromosome, and there is good evidence for at least one other locus in the genome. - classic and reverse genetics classicgenetic studiesofchromosome deletionsandtranslocations may provide clues to help identify occult loci responsible for genetic disease. alzheimer's disease occurs as early as the fourth decade in individuals with trisomy of the short arm of chromosome division of molecular medicine, mrc clinical research centre, harrow, middlesex hai uj james scott, msc, frcp, head ofdivision, consultant physician o n a p ril b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://w w w .b m j.co m / b r m e d j (c lin r e s e d ): first p u b lish e d a s . /b m j. . . o n s e p te m b e r . d o w n lo a d e d fro m http://www.bmj.com/ british medical journal volume september (down's syndrome). interestingly, the gene that codes for the alzheimer's amyloid protein has been localised to the short arm of chromosome . this gene codes for a membrane protein, possibly a neurotransmitter receptor.' in hereditary alzheimer's disease the gene is duplicated. the similarity between this condition and down's syndrome is remarkable. with a common disorder such as senile dementia (prevalence in in people over the age of ) we may expect that common polymorphisms of the alzheimer's amyloid protein gene might be found to predispose to the disease. congenital heart disease is also common in patients with down's syndrome; this may provide a clue to the localization of one of the genes that lead to this common birth defect. classic genetics has recently provided a candidate locus on chromosome for a gene that may contribute to schizophrenia. also the study ofx chromosome linked cleft palate and tied tongue has provided a locus for this disorder. in many genetic diseases the phenotype provides no information about the locus or the biochemical abnormality responsible for the disease. in monogeneic disorders the logical extension of the candidate gene approach is to use random deoxyribonucleic acid (dna) probes and restriction fragment length polymorphism segregation analysis to pinpoint the chromosomal locus responsible for the disease. this approach has been successful in identifying loci for cystic fibrosis' (and a candidate gene has been discovered) and for adult polycystic kidney disease." for polygenic disease, where a common phenotype is produced by the interaction of a number of genes, the use of this approach is much more difficult. if clinical material is chosen carefully to maximise single gene effects, however, there is no reason why this approach should not work. the study of large families such as the pennsylvania amish with manic depression is a clear example.' for the other loci implicated in manic depression a search of the genome using random probes is entirely reasonable. for many diseases this may be the only approach. mouse models the study ofrecombinant inbred strains ofmice provides another valuable tool for analysing the complex patterns of inheritance found in polygenic disease and for identifying occult loci. the genetics of insulin dependent diabetes mellitus have been the subject of intensive study in man. genetic predisposition combined with other (presumably environmental) factors leads to islet destruction and clinical diabetes. the presence of specific alleles in the hla gene complex (hla dr and dr ) is important for the development of the disease. hints about the other gene loci that participate have come from the study of non-obese diabetic mice." in these animals three recessive loci on different chromosomes have been implicated. one of these loci is the mhc complex (h- ), another is the thy-i locus, and the third has not yet been localised. the thy locus in man is on the long arm of chromosome . in addition, a dominant locus associated with the control of t cell hyperplasia has been implicated. this model is close to human insulin dependent diabetes and is likely to point to the important loci concerned. studies of mouse lipoprotein metabolism have also provided information about the regulation of human plasma lipoprotein concentrations.' ' a mouse locus has been identified which regulates the concentration of plasma high density lipoprotein. it is distinct from genes coding for the apolipoprotein, structural components of high density lipoprotein. these studies indicate the presence of a new locus that may be important for determining the risk of atherosclerosis. genetic epidemiology genetic epidemiology has provided statistical methods for establishing the contribution of a particular locus to a trait, such as plasma cholesterol concentration." variation at the apo-e and apo-b loci has been shown to- contribute % of the total genetic variance which affects plasma cholesterol concentrations.' another risk factor for coronary heart disease is plasma fibrinogen concentration. genetic variation at the fibrinogen locus has recently been showntro account for % of the total phenotypic variance in plasma fibrinogen concentrations.' if these observations can be extended to other polygenic traits we can be optimistic that the detailed study of a few loci is likely to allow the refinement of diagnostic tools for predicting the risk of a particular disorder. genetic polymorphism and susceptibility to disease the differences between humans in traits such as skin colour, height, intelligence, and blood pressure extend to the ability to handle environmental insults such as exposure to infectious agents and chemical carcinogens and excessive consumption of saturated fat and cholesterol. individual differences are produced by the dna sequence variation that exists throughout the genome. the dna between two individuals varies every - base pairs." dna sequence variation is more frequent in the dna between genes because natural selection conserves sequences that regulate gene expression and code for proteins. protein polymorphism can be detected by isoelectric focusing, which identifies charged amino acid variants. charge variants affect one fifth of all proteins (hence genes)." isoelectric focusing certainly underestimates protein polymorphism because it does not detect uncharged amino acid changes; only four of the essential amino acids are charged. moreover, variation in the dna sequences concerned with regulation of gene expression will not be detected. common alleles (variants at a single locus) or polymorphisms form the basis of human diversity, including the ability to handle environmental challenge. such alleles are likely to have increased in prevalence owing to positive selection acting on variants that confer selective advantage in the heterozygous state. for example, the genetic polymorphisms that contribute to the common polygenic diseases such as coronary heart disease, essential hypertension, and diabetes mellitus almost certainly have a high prevalence in the population. perhaps the variants which once conferred selective advantage for our hunter-gatherer ancestors by maintaining blood pressure and blood glucose and plasma cholesterol concentrations in a hostile environment now respond to overnutrition by predisposing to the major diseases that affect modern man. one ofthe best researched examples ofpolymorphism that affects susceptibility to disease occurs at the apo-e locus.' the presence of the e allele (which occurs in % of people) increases plasma cholesterol concentration by about % compared with the e wild type allele (which occurs in % of people), whereas the e allele (which occurs in %) decreases cholesterol concentration by %. these small changes in plasma cholesterol concentration are sufficient to produce a substantial enrichment of the e allele with advancing age and decrease of the e allele in the aging population, presumably as a result of an increased number of deaths from myocardial infarction. alleles at the apo-b locus similarly affect plasma cholesterol concentration and the risk of myocardial infarction.' a most important example of genetic polymorphism which leads to susceptibility to infection with the human immuno- deficiency virus and progression to the acquired immune deficiency syndrome (aids) has recently been described at the a' globulin locus (vitamin d binding factor). ' selection acting on these alleles could act rapidly in the absence of a cure for aids. rare alleles may also contribute to genetic diversity and susceptibility to disease. homozygosity for a defect of the gene cystathionine synthetase causes collagen malformation and vascular thrombosis. heterozygosity (prevalence in ) for the same rare allele has now been associated with increased risk of atherosclerotic peripheral vascular disease and cerebrovascular disease. identification of polymorphic alleles that predispose to polygenic disease charge variants may be identified by isoelectric focusing and direct protein sequencing. occasionally, close association (linkage o n a p ril b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://w w w .b m j.co m / b r m e d j (c lin r e s e d ): first p u b lish e d a s . /b m j. . . o n s e p te m b e r . d o w n lo a d e d fro m http://www.bmj.com/ british medical journal volume september disequilibrium) can be shown between a restriction fragment length polymorphism and a mutation causing disease. the first such association was found between an hpai restriction fragment length polymorphism and the mutation causing sickle cell disease. further examples of such polymorphisms associated with pre- disposition to polygenic disease have now been described. these include those associated with increased plasma concentrations of lipoproteins' and fibrinogen," increased risk of cardiovascular disease, and the risk of amyloidosis complicating juvenile arthritis. an alternative and in many cases more searching approach is to identify restriction fragment length polymorphism haplotypes (restriction fragment length polymorphisms in linkage disequilibrium at the same locus) that are associated with disease traits. ultimately, there is no substitute for identifying precise mutation by dna sequence analysis. once a mutation has been identified its detection by amplification of genomic dna and the use of allele specific oligonucleotide probes makes diagnosis a relatively trivial matter. linkage and linkage disequilibrium two or more gene loci that exist in close proximity on the same chromosome and cosegregate in. family studies are said to show genetic linkage. iflinked genes confer susceptibility to a disease this wil have implications for the level of risk, particularly if disease susceptibility alleles -are -in linkage disequilibrium. linkage dis- equilibrium is best illustrated by the hla complex. the hla genes are located on the short arm ofchromosome and are made up of fotr closely linked loci (a, b, c, and d). the products of these genes are proteins concerned with the recognition of foreign antigens. each hla locus. consists of multiple alleles, of which an individual may inherit any two of possibly . certain hla genes occur together more often than would be predicted by chance alone. hla al and b in north european white populations are at least four times as common as would be expected by chance and are described as being in linkage disequilibrium. it is highly likely that this association was selected during the evolutionary history of the north european white population; it may have conferred protection against the plague. an example of selection may be seen among the ancestors of dutch settlers in surinam: most of the original settlers succumbed to a typhoid epidemic, and only those with particular hla antigens survived. new examples of linkage disequilibrium between disease susceptibility alleles are likely to emerge, especially where selection has favoured their close association. the example of polymorphisms which produce a relatively high blood pressure and high blood glucose and serum cholesterol concentrations in order to combat a hostile environment has already been given. the future before the year linkage and physical maps of the entire human genome will have been established and much of the genome will have been sequenced." most of the genes implicated in common polygenic disease of adults and common birth defects of children will have been characterised, and the mutant alleles which predispose to disease will have been fully identified. by use of procedures for amplifying dna and allele specific oligonucleotide probes it should be possible to sreen for polymorphisms associated with a variety ofcommon diseases in'a single reaction. screening in early life for genes which predispose to the common ailments later in life has clear advantages but poses ethical and practical problems." for a disorder which can be prevented such as coronary heart disease it is a remarkable bonus to know and to treat early. predictive screening may lead to modification of lifestyle or the introduction ofspecific treatment. difficulties can be envisaged, however: termination of pregnancy, lifestyle, health insurance, or occupational decisions could be based on relatively minor and ill judged genetic risks. and will it pay? for monogeneic disorders it is certainly cheaper to screen antenatally than to care for the chronically handicapped. ' for common diseases of adults the answer is also likely to be yes-for example, in terms of health care alone coronary heart disease has been estimated to cost £ billion a year, and this does not count the even greater cost to industry and to commerce. the bill could probably be halved by appropriate preventive measures. i thank miss lesley sargeant for typing the manuscript. references egeland ja, gerhard ds, pauls dl, et al bipolar affective disorders linked to dna markers on chromosome . nature ; : - . baron m, risch n, hamburger r, et al. genetic linkage between x-chromosome markers and bipolar affective illness. nature ; : - . detera-wadleigh sd, berrettini wh, goldin lr, boorman d, anderson s, gershon es. close linkage of c-harvey-ras-i and the insulin gene to affecave disorder is rued out in three north american pedigrees. nature ; : - . hodgkinson s, sherrington r, gurling h, et al. molecular genetic evidence of heterogeneity in manic depression. nature ; : - . mendlewicz j, 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kj, martin jb, wilson jd, eds. principles ofiernal medicine. new york: mcgraw-hill, : - . o n a p ril b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://w w w .b m j.co m / b r m e d j (c lin r e s e d ): first p u b lish e d a s . /b m j. . . o n s e p te m b e r . d o w n lo a d e d fro m http://www.bmj.com/ draft merits and perils of targeted neonatal echocardiography- based hemodynamic research: a position statement journal: canadian journal of physiology and pharmacology manuscript id cjpp- - .r manuscript type: review date submitted by the author: -sep- complete list of authors: kharrat, ashraf; mount sinai hospital, paediatrics mcnamara, patrick; university of iowa weisz, dany; university of toronto jain, amish; university of toronto is the invited manuscript for consideration in a special issue: connecting maternal fetal newborn physiology keyword: https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft merits and perils of targeted neonatal echocardiography-based hemodynamic research: a position statement ashraf kharrat , , patrick j. mcnamara , dany weisz , , amish jain , . department of paediatrics, university of toronto, toronto, on, canada. . department of paediatrics, mount sinai hospital, toronto, on, canada. . department of pediatrics, university of iowa, iowa city, ia, united states. . newborn and developmental paediatrics, sunnybrook health sciences centre, toronto, on, canada. address correspondence to: dr. amish jain mbbs phd department of paediatrics, room - , mount sinai hospital, university avenue, toronto, ontario, m g x , canada. phone: x email: amish.jain@sinaihealthsystem.ca funding source: none. financial disclosure: the authors have no relevant financial relationships to disclose. conflict of interest: the authors have no potential conflicts of interest to disclose. page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft graphical abstract [see separate file]. figure : graphical abstract highlighting merits and considerations in tne research. introduction in the neonatal setting, point-of-care ultrasound is being used increasingly to help clinicians with the evaluation of heart function. practices in neonatology, particularly with regards to acute and chronic hemodynamic managements, were traditionally more driven on dogma and predefined thresholds and not always supported by demonstrable physiology. for the first time, targeted neonatal echocardiography (tne) provided neonatal intensivists with a bedside tool which made real-time assessment of neonatal hemodynamics status feasible in even the tiniest of babies. this opened the door towards more targeted physiological driven practices, allowing us to test historical approaches to clinical problems in a more precise way. over the last ten years, evidence of the use tne and its effect on clinical management has accumulated. while tne is helping to advance bedside decision-making, it also fosters an entirely new field of scientific interrogation. the wide availability of tne has made it feasible for academicians to ask a variety of novel research questions pertaining to neonatal hemodynamics. however, despite the standardization of tne training and the creation of a formalized curriculum, little attention has been paid to the establishment of an empirical framework to adjudicate scientific investigation. in this position statement, we reflect on the evolution of tne in canadian nicus, appraise its strengths and limitations, and suggest guiding principles for clinicians and researchers to consider as they take this field forward. page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft merits in the evolution of tne clinicians have been using functional echocardiography in neonatal care in parts of europe and australasia for over two decades, while in north america, it has been more recently adopted. the first tne program in canada was established in at the hospital for sick children in toronto (el-khuffash, herbozo, jain, lapointe, and mcnamara ). while for a few years that remained the only tne program in canada, over the last years, a number of formal and standardized tne programs have emerged in several nicus across the country. this was in part facilitated by the endorsement of tne by the american society of echocardiography which published a comprehensive practice and training guideline in (mertens et al. ) and resulted in a strong collaboration between neonatologists and pediatric cardiologists. the european group also published their guidelines for neonatologist performed echocardiography (npe) in (boode et al. ). this consensus-based guideline differed from the ase in a few aspects, most importantly in that it does not mandate a period of training in a pediatric echocardiography laboratory. instead, trainers of npe are recommended to spend months acquiring experience in infants with congenital heart disease, under the supervision of a pediatric cardiologist. despite these variations, both groups strongly advocate for neonatologists with an interest in functional echocardiography to establish close clinical and academic links with their pediatric cardiology colleagues, and prescribes a minimum scanning exposure that trainees should have with neonates with structural heart defects other than patent ductus arteriosus and patent foramen ovale. one of the impacts of these consensus documents is the spread of tne in clinical practice. at the time of writing this review, at least tertiary nicus in canada are page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft known to have tne capabilities, with more american centres coming on board. while consensus-based guidelines are excellent resources, it is important to note that at this time, they are not backed by a large body of evidence. we encourage clinicians, researchers and administrators to continue to build the evidence base for allowing the future refinement of tne training guidelines. impact on clinical management a number of studies have now confirmed that tne in tertiary neonatal care frequently impacts clinical decision-making. from canada, three centres have published their impact. in a study conducted on outborn patients, a review of infants showed that tne was associated with a change in clinical management among % of cases (el-khuffash et al. ). a subsequent study from calgary examining the utility of tnes from patients showed that tne altered clinical management in approximately % of cases in the first week of life, and in % of cases thereafter (harabor & soraisham ). more recently, we examined the role of tne in a large tertiary perinatal centre and found that tne resulted in a significant impact on clinical management, particularly infants with high illness severity and non-pda pathologies (papadhima et al. ). outside of canada, corredera et al from spain demonstrated that in their unit, tne was associated with a modified management plan in % of cases, usually as it pertains to pda management (corredera et al. ). however, there are some limitations to the current literature. these studies are retrospective in nature, and assume that changes in clinical management after a tne were a direct result of the tne consult itself. the intent of the attending clinicians in the absence of a page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft tne consult cannot be accounted for. similarly, in cases where there was no clinical change after tne, a presumption may be made that the tne had no clinical impact. however, in real life, confirmation of a clinical diagnosis and treatment pathway by tne is important, and may prevent subsequent alterations in clinical management. there is therefore a need to formally evaluate the utility of tne in neonatal care in a prospective manner. impact on patient outcomes even though there is a great deal of evidence showing the impact of tne on clinical practice, its ability to influence patient outcomes remains under-evaluated. the few studies published in this field are retrospective in nature, and include a small number of patients with a specific disease process. shah and kluckow, in a retrospective case-control study, found that in preterm infants born after preterm prolonged rupture of membranes, the availability of tne was associated with earlier identification of pulmonary hypertension, which was linked to a prompter initiation of treatment and improved survival (shah & kluckow ). in a study of a similar design, jain et al compared preterm infants undergoing pda ligation who were assessed routinely with tne post-ligation and treated with milrinone for low left ventricular output with a historical cohort, and found that the cases had improved postoperative cardiorespiratory stability (jain et al. ). a more recent study examined the impact of integrated evaluation of hemodynamics on infants, and found that tne was associated with a shorter time to recovery in patients with late-onset compromised systemic circulation (amer, kalash, seshia, and elsayed, ). given the paucity of currently available data from prospective studies, there is clearly an urgent need for high-quality and comprehensive evaluation of the direct impact of tne on page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft patient outcomes. such studies will not only justify the adoption of this technology in nicus, but will target its use to the most in-need population and at the most appropriate time, maximizing its impact. one of the areas where good-quality retrospective studies have been conducted is in the role of tne in identifying malpositioned catheter tips of central venous lines. there have been a few retrospective studies confirming its utility (harabor and soraisham ; weisz, poon, james, and mcnamara ), and these findings have been confirmed by several prospective studies which compared tne and radiography assessment of umbilical venous catheter placement, and found that tne may be a more optimal in determining line malpositioning (franta, harabor, and soraisham ; karber, nielsen, balsam, messina, and davidson ; pulickal et al. ). a great deal of academic work is still needed to be done in this field to understand its utility in clinical and research settings. considerations for the future: perils the role of tne in physiologic studies typically, physiological studies in the neonatal field are conducted using animal models. such experimental studies are crucial in providing mechanistic insights into disease pathophysiology and identify therapeutic targets. increasingly, non-invasive methods such as tne are also being incorporated into animal experiments, as they provide the desirable ability to provide sequential assessments. while this has opened a new field to researchers, there are certain limitations to its translatability to human subjects. these include inter-species page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft differences, the effect of the interplay of multiple co-existing disease processes, and the influence of concurrent therapies. in addition, the tne measurements used in these animal studies have not undergone rigorous validation. nevertheless, the use of tne has opened the door to appraise physiologic alterations in humans. this is especially important in neonatology, due to the limited feasibility of continuous invasive monitoring. unfortunately, its translatability into day-to-day clinical practice has lagged behind, due to some key obstacles. the patient population in the nicu can be highly variable. both the physiological processes and the effect of diseases on them may vary with gestational maturity. for instance, the immature myocardium of the preterm heart is known to have an intrinsic diastolic dysfunction that may not be seen in term infants. hence, there is a need to have robust gestational age-specific normative data. this is still missing for a number of tne measurements. the existent normative data may also need to be re-evaluated, given the major advances in perinatal care over the last two decades, such as the increased uptake of magnesium sulphate, prophylactic indomethacin, antenatal steroids, delayed cord clamping (demarini, dollberg, hoath, ho, and donovan ; wu, azhibekov, and seri ). another limitation of the currently available normative data is the lack of consideration of postnatal age. for example, data acquired on day for a -week infant may not necessarily be applicable to a two-week old -week infant. further, normative data might apply to certain developmental phases for the preterm infant, but not others. for example, the extremely preterm infant is innately less tolerant of hemodynamics disturbances in its transitional period, and the consequences of low cardiac output states may lead to significant intraventricular hemorrhages. in addition, infants who tend to receive tne typically do so for a medical indication; therefore, it may be challenging to apply retrospective data acquired from presumed healthy infants. ideally, page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft normative data should be established at a population level with a large cohort size, and be validated against long-term clinical outcomes. this has yet to be done. another factor to consider with using normative data especially applicable to hemodynamic principles is that expected normal ranges may vary in well and disease states. for example, a left ventricular output obtained from a healthy infant at rest may not be “safe” for a sick infant with a higher metabolic demand. lastly, while some hemodynamic parameters have undergone rigorous validation by comparing against gold standards, several others that are used in day-to-day clinical and research settings remain untested. with the availability of cardiac magnetic resonance imaging (mri), such validation studies may now be feasible, even in neonates. recent advancements in cardiac mri techniques now offer a non-invasive surrogate of traditionally invasive gold-standard investigations, however, at this time, this modality is generally only used for stable newborn infants, despite hemodynamically unstable infants being the typical population of interest. the need for academic rigor so far, most studies using tne methodology have used a small number of patients, and have focused on the description of the hemodynamic physiology observed in disease states. while this has been helpful in furthering our understanding of hemodynamic pathophysiology, at least at a macro level, it is our opinion that in the future, studies need more rigorous methodology to have a greater clinical impact. this should start at the stage of constructing the research plan itself. it is key, as in any other field of research, that careful planning and consultation be undertaken at this stage. consideration should be given to formulating a clear and objective hypothesis and stating relevant primary and secondary outcomes. page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft one of the limitations of many of the previous tne-based studies is the absence of justifiable sample sizes, which are often chosen out of convenience. this is understandable for descriptive physiological studies, however in order to generate research findings that are more generalizable and widely applicable, as well as have a long-standing impact on practice change, it is imperative that studies are adequately powered to detect differences in clinically meaningful outcomes. in the absence of having appropriate sample sizes, research findings need to be validated in secondary cohorts prior to their clinical implementation. one way to generate a large sample size is to have a multi-centre tne network. at this time, a national tne database does not exist in canada, but is a potential solution to increasing the feasibility of larger and more rigorous studies. for such a network to produce reliable results, it will require participating centres to standardize the types of measurements to be obtained and the techniques required in doing so. intra- and inter-person variability is a known barrier in tne- based research. in addition, careful consideration should be given to identify potential sources of bias, and mitigating strategies should be incorporated from early in research planning. one unique obstacle in tne-based research is the difficulty in blinding the operator. due to resource limitations, tnes are typically performed and/or analyzed by clinicians with expertise, who may be also either aware of or directly involved in patient management. one way to overcome this challenge is for the research scans and analysis to be performed by trained sonographers, or by careful blinding of personnel. other factors to consider are the handling of repeat measures and the effect of varying postnatal age at the time of tne assessments. data acquired at multiple time-points on the same patient should not be considered independent observations, as this may page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft introduce bias related to clustering. most contemporary statistical software packages provide the capability to control for repeated measures. one of the unique features of tne-based research is the ability to acquire a large number of variables from a single scan. while this allows for the opportunity to corroborate research results, it also increases the potential for type i error (i.e. identifying statistically significant but erroneous findings), which may be misleading. it is therefore important to adjust for multiple comparisons during statistical analysis. further, unexpected secondary findings should be re- confirmed in subsequent studies. it is with these considerations that we advise tne researchers to foster close collaboration with experts in clinical epidemiology. such collaboration would be mutually beneficial, as physiology-driven epidemiological studies could have a major long- lasting impact on patient care. next steps targeted neonatal echocardiography provides many opportunities to expand our knowledge of sick and healthy infants across all gestational ages. to continue to move this field forward, future work should cultivate a more refined approach to hemodynamic problems by using validated tne techniques on important clinical cardiovascular disease states that require mechanistic assessments. more focused effort should be spent in corroborating echocardiographic findings with the clinical picture, by harmonizing the extraction of tne data with clinical variables. commonly used cardiovascular therapies should be studied with more emphasis, as there are many holes in our current knowledge of their physiological mechanisms of action across the various gestational and postnatal age groups. finally, following infants with page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft hemodynamic disturbances to assess both their short- and long-term outcomes will help fill in gaps in our understanding of the repercussions of abnormal cardiovascular health states. conclusions over the last two decades, and with the efforts of national and international leaders, tne is being widely endorsed in the neonatal community with a growing number of people being trained in its use. for this field to continue advancing its impact, clinicians and researchers of the future need to be well trained in both scanning, and its strengths and limitations in analysis. tne research should be designed and executed in a methodologically rigorous fashion. ultimately, through the combination of tne-based research and clinical integration, the goal of neonatal care of providing the right therapy, for the right patient, at the right time may be better realized. page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft references amer, r., kalash, r., seshia, m. m., and elsayed, y. n. . the impact of integrated evaluation of hemodynamics on management of preterm infants with late-onset compromised systemic circulation. am j perinatol, ( ), – . https://doi.org/ . /s- - boode, w. p. de, singh, y., gupta, s., austin, t., bohlin, k., dempsey, e., and groves, a. . recommendations for neonatologist performed echocardiography in europe : consensus statement endorsed by european society for paediatric research (espr) and european society for neonatology (esn), ( ), – . https://doi.org/ . /pr. . corredera, a., rodríguez, m. j., arévalo, p., llorente, b., moro, m., and arruza, l. . functional echocardiography in neonatal intensive care: -year experience in a unit in spain. anales de pediatría (english edition), ( ), – . https://doi.org/ . /j.anpedi. . . demarini, s., dollberg, s., hoath, s., ho, m., and donovan, e. . effects of antenatal corticosteroids on blood pressure in very low birth weight infants during the first hours of life. j perinatol, ( ), – . el-khuffash, a., herbozo, c., jain, a., lapointe, a., and mcnamara, p. j. . targeted neonatal echocardiography (tnecho) service in a canadian neonatal intensive care unit: a -year experience. j perinatol, ( ), . https://doi.org/ . /jp. . franta, j., harabor, a., and soraisham, a. s. . ultrasound assessment of umbilical venous catheter migration in preterm infants: a prospective study. arch dis child fetal neonatal ed, ( ), f –f . https://doi.org/ . /archdischild- - page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft harabor, a., and soraisham, a. . rates of intracardiac umbilical venous catheter placement in neonates. j ultrasound med, ( ), – . https://doi.org/ . /ultra. . . harabor, a., and soraisham, a. s. . utility of targeted neonatal echocardiography in the management of neonatal illness. j ultrasound med, ( ), – . https://doi.org/ . /ultra. . . jain, a., sahni, m., el-khuffash, a., khadawardi, e., sehgal, a., and mcnamara, p. j. . use of targeted neonatal echocardiography to prevent postoperative cardiorespiratory instability after patent ductus arteriosus ligation. j pediatr, ( ), – . karber, b. c. f., nielsen, j. c., balsam, d., messina, c., and davidson, d. . optimal radiologic position of an umbilical venous catheter tip as determined by echocardiography in very low birth weight newborns. j neonatal perinatal med, ( ), – . https://doi.org/ . /j.jpeds. . . mertens, l., seri, i., marek, j., arlettaz, r., barker, p., mcnamara, p., et al. . targeted neonatal echocardiography in the neonatal intensive care unit: practice guidelines and recommendations for training. eur j echocardiogr, ( ), – . https://doi.org/ . /ejechocard/jer papadhima, i., louis, d., purna, j., deshpande, p., diambomba, y., lee, s., et al. . targeted neonatal echocardiography (tne) consult service in a large tertiary perinatal center in canada. j perinatol, . https://doi.org/ . /s - - -y pulickal, a. s., charlagorla, p. k., tume, s. c., chhabra, m., narula, p., & nadroo, a. m. . superiority of targeted neonatal echocardiography for umbilical venous catheter tip localization: accuracy of a clinician performance model. j perinatol, ( ), . https://doi.org/ . /jp. . page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft shah, d. m., and kluckow, m. . early functional echocardiogram and inhaled nitric oxide: usefulness in managing neonates born following extreme preterm premature rupture of membranes (pprom). j paediatr child health, ( ), – . https://doi.org/ . /j. - . . .x weisz, d. e., poon, w. b., james, a., and mcnamara, p. j. . low cardiac output secondary to a malpositioned umbilical venous catheter: value of targeted neonatal echocardiography. ajp reports, ( ), . https://doi.org/ . /s- - wu, t.-w., azhibekov, t., and seri, i. . transitional hemodynamics in preterm neonates: clinical relevance. pediatr neonatol, , – . https://doi.org/ . /j.pedneo. . . page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology draft evidence to support beneficial impact on patient outcomes in certain pathologies demonstrable positive impact on clinical management comprehensive practice and training guidelines practical limitations in conducting physiological studies bias in research studies due to small sample sizes and repeat measures need for academic rigor merits in tne evolution perils and future considerations need for:  physiology-driven epidemiological studies  prospective evaluation of impact  correlation with patient outcomes page of https://mc .manuscriptcentral.com/cjpp-pubs canadian journal of physiology and pharmacology seasonality of mood and behavior in the old order amish journal of affective disorders ( ) – contents lists available at sciverse sciencedirect journal of affective disorders - http://d n corr and anx building fax: þ e-m journal homepage: www.elsevier.com/locate/jad research report seasonality of mood and behavior in the old order amish uttam k. raheja a,b, sarah h. stephens c, braxton d. mitchell c, kelly j. rohan d, dipika vaswani a, theodora g. balis a, gagan v. nijjar a,b, aamar sleemi a,b, toni i. pollin c, kathleen ryan c, gloria m. reeves e, nancy weitzel c,f, mary morrissey f, hassaan yousufi a, patricia langenberg g, alan r. shuldiner c,h, teodor t. postolache a,b,e,i,n a mood and anxiety program, department of psychiatry, university of maryland school of medicine, baltimore, md, usa b saint elizabeths hospital, psychiatry residency training program, washington, dc, usa c division of endocrinology, diabetes and nutrition, department of medicine, university of maryland school of medicine, baltimore, md, usa d department of psychology, university of vermont, burlington, vt, usa e division of child and adolescent psychiatry & university of maryland child and adolescent mental health innovations center, university of maryland school of medicine, baltimore, md, usa f amish research clinic of the university of maryland, lancaster, pa, usa g department of epidemiology and public health, university of maryland school of medicine, baltimore, md, usa h geriatric research and education clinical center, veterans administration medical center, baltimore, md, usa i national center for the treatment of phobias, anxiety and depression, washington, dc, usa a r t i c l e i n f o article history: received september accepted october available online november keywords: seasonal affective disorder seasonality amish epidemiology /$ - see front matter & elsevier b.v. a x.doi.org/ . /j.jad. . . esponding author at: university of marylan iety program, department of psychiatry, room , baltimore, md , u . ail address: tpostolache@psych.umaryland.ed a b s t r a c t background/objective: we examined seasonality and winter seasonal affective disorder (sad) in the old order amish of lancaster county, pennsylvania, a unique population that prohibits use of network electric light in their homes. methods: we estimated sad using the seasonal pattern assessment questionnaire (spaq) in amish adults and compared the frequencies of sad and total sad (i.e., presence of either sad or subsyndromal-sad) between men and women, young and old, and awareness of (ever vs. never heard about) sad. heritability of global seasonality score (gss) was estimated using the maximum likelihood method, including a household effect to capture shared environmental effects. results: the mean ( sd) gss was . ( . ). prevalence was . % ( % ci: . – . ) for sad and . % ( % ci: . – . ) for total sad. heritability of gss was . . (se) (p¼ . ) after adjusting for age, gender, and household effects. limitations: limitations include likely overestimation of the rates of sad by spaq, possible selection bias and recall bias, and limited generalizability of the study. conclusions: in the amish, gss and sad prevalence were lower than observed in earlier spaq-based studies in other predominantly caucasian populations. low heritability of sad suggests dominant environmental effects. the effects of awareness, age and gender on sad risk were similar as in previous studies. identifying factors of resilience to sad in the face of seasonal changes in the amish could suggest novel preventative and therapeutic approaches to reduce the impact of sad in the general population. & elsevier b.v. all rights reserved. . introduction changes in mood and behavior with changes in season, referred to as seasonality, have been recognized since ancient times (wehr and rosenthal, ). winter seasonal affective disorder (sad) was first defined in by rosenthal et al. ll rights reserved. d school of medicine, mood west baltimore street, mstf sa. tel.: þ ; u (t.t. postolache). ( ) as a syndrome characterized by recurrent episodes of depression in the autumn and winter with remission in the spring and summer. while patients with sad must have at least one episode of major depression according to the rosenthal criteria [ibid.] or two episodes of major depression in two consecutive years according to dsm-iv-tr criteria (american psychiatric association, ), a form with shorter or milder forms of depression, often with predominant neurovegetative symptoms (sleep and appetite changes), has been described as subsyndromal sad (s-sad) (kasper et al., a). the dual vulnerability hypothesis of sad (lam et al., ) proposes that sad is a result of chronobiological vulnerability and www.elsevier.com/locate/jad www.elsevier.com/locate/jad dx.doi.org/ . /j.jad. . . dx.doi.org/ . /j.jad. . . dx.doi.org/ . /j.jad. . . http://crossmark.dyndns.org/dialog/?doi= . /j.jad. . . &domain=pdf http://crossmark.dyndns.org/dialog/?doi= . /j.jad. . . &domain=pdf http://crossmark.crossref.org/dialog/?doi= . /j.jad. . . &domain=pdf mailto:tpostolache@psych.umaryland.edu dx.doi.org/ . /j.jad. . . u.k. raheja et al. / journal of affective disorders ( ) – vulnerability to affective dysregulation. a tendency toward an extension of nocturnal duration of melatonin secretion in response to shortened day length (photoperiod) (wehr et al., ) and delays of circadian rhythms in response to limited (or reduced) exposure to morning light in fall and winter (lewy et al., ) represent the major chronobiological mechanisms proposed for sad. symptoms of sad resemble seasonal changes in photoperiodic mammals in response to shortening of photoperiod, including hyperphagia and weight gain, hypersomnia, anergia, and decreased libido (wehr, ; workman and nelson, ). these symptoms of sad respond favorably to treatment with bright light (eastman et al., ; lewy et al., ; terman et al., ; golden et al., ). like photoperiodic mammals, patients with sad, but not healthy controls, have a longer duration of melatonin secretion in winter than in summer (wehr et al., ). lewy et al. ( ) have reported that light can suppress secretion of melatonin in humans, provided it is of sufficiently high intensity (about lx). more recent studies have demon- strated that exposure to even low levels of light corresponding to ordinary room light (about lx) can affect the circadian pace- maker and suppress melatonin secretion (boivin et al., ; zeitzer et al., ; gooley et al., ). it is therefore likely that, in modern societies, exposure to electric light may mask the exposure to longer nights in winter and thereby possibly reduce the observed prevalence of sad. in fact, to our knowledge, all epidemiological studies of sad to date have been conducted on populations that use network electric lighting (lighting powered by the electric grid) (magnusson, ). by corollary, one could postulate that a population with limited use of electric light would be more susceptible to the seasonal effects of long nights and would potentially have a higher prevalence of sad. the old order amish of lancaster, pennsylvania ( n), are such a population. there are approximately , old order amish in lancaster county, about half of whom are over the age of years, constituting a rural, primarily agrarian community (kraybill, ). the old order amish religion prohibits technol- ogies – such as the use of telephones in the home, driving cars, and wrist watches – that are perceived as colliding with their beliefs and practices [ibid.]. this community has not linked to the electric power grid and does not use electric light in the home; rather they use relatively low-intensity propane-powered gas lighting and candles (scott and pellman, ). the amish, thus, represent a convenient population to conduct a study on sad in which the masking effects of artificial light on natural photoper- iod are minimized. the role of genetic factors in sad was initially suggested by an increased prevalence of affective disorders and sad in first degree relatives of sad patients. since then, a number of studies have suggested a genetic contribution for seasonality (rosenthal et al., ; willeit et al., ; thierry et al., ; roecklein et al., ). for instance, a study of adult twins in an australian registry reported a % heritability of seasonality (madden et al., ). to our knowledge, there is no study of heritability of seasonality in a population with limited exposure to modern artificial lighting, such as the amish. we thus conducted a cross-sectional study of seasonality of mood and prevalence of sad based on the seasonal pattern assessment questionnaire (spaq) (rosenthal et al., ) in the old order amish. we hypothesized that the global seasonality score (gss), a measure of magnitude of seasonal behavioral changes, and the prevalence of sad would be higher in the amish than in other populations previously studied at similar latitudes (kasper et al., b; rosen et al., ). as reported in previous epidemiological studies (magnusson, ), we further hypothe- sized that women would have a higher prevalence of sad than men, and that awareness (whether or not the subject had ever heard about sad) and younger age would be associated with higher gss and frequency of sad. since all lancaster amish are related through a well-documented -generation genealogy, we also estimated the heritability of gss in the amish. . methods . . procedures and sample selection we mailed the seasonal pattern assessment questionnaire (spaq) (rosenthal et al., ) to amish individuals, aged and older, who had previously participated in studies of cardiovas- cular, metabolic, and bone health conducted at the university of maryland (streeten et al., ; hseuh et al., ; mitchell et al., ; rampersaud et al., ). a letter was included with direc- tions to complete the questionnaire and return it in a pre-stamped, addressed envelope. a $ bill was included as a token of apprecia- tion. the letter sent to the participants explicitly stated that their completing the questionnaire would document their consent to participate in this study. this study and all previous studies were approved by the institutional review board of the university of maryland school of medicine. the seasonal pattern assessment questionnaires were mailed in may, with a second mailing to non-responders to the first in september, . a total of questionnaire responses, representing a response rate of . %, were received back before december , and are included in the dataset. . . seasonal pattern assessment questionnaire (spaq) the spaq (rosenthal et al., ) is a research and screening tool that is widely used in studies of seasonality and sad. the spaq evaluates severity of global seasonal changes, degree of functional impairment with those changes (‘‘problem’’), and seasonal pattern (summer vs. winter) if the other two criteria are met. the gss was calculated based on responses to the six parameters of seasonality (sleep duration, social activity, mood, weight, appetite, and energy level) as rated on a (‘‘no change’’) to (‘‘extremely marked change’’) scale reflecting degree of change across the seasons (kasper et al., b). in a few cases where responses to one or more of the six items were left blank, the gss was estimated using a proportion based calculation. a convenience sample analysis of test–retest reliability of the gss and problem rating score (prs) on the spaq in study subjects yielded satisfactory results (gss, a¼ . , po . ; prs, a¼ . , po . ) (kuehner et al., in press). sad was defined according to three criteria: (a) gss score, (b) ‘‘problem’’ and (c) seasonal pattern (magnusson, ). subjects were classified as having sad if they had a gss of or higher, if they experienced seasonal changes as a problem to at least a moderate degree, and if they reported a fall–winter pattern of mood disturbance, i.e., if they felt worst during one or more months from september to february. subjects were classified as having s-sad if they had a gss of at least but the problem score was less then moderate (i.e., mild or none) or if they had a gss of or and considered seasonal changes as at least a mild problem. for subjects who met gss and problem criteria for either sad or s-sad, and had a predominantly fall/winter pattern with one or more months falling outside the fall/winter range, we used the following criterion to classify the pattern: if subjects felt worst during months in both fall–winter (september–february) and spring-summer (march–august), then they were classified as having sad or s-sad only if the number of months in which they felt worst during the fall–winter period (september–february) exceeded the number of months in which they felt worst during u.k. raheja et al. / journal of affective disorders ( ) – the spring–summer period (march–august) by at least two. if subjects met gss and problem criteria for sad or s-sad but felt worst for several consecutive months that overlapped the cut-off between summer and fall (i.e., august/september) or winter and spring (i.e., february/march), then they were classified as having a winter pattern when at least two of these consecutive months were in the fall or winter and only one of these months was in the spring or summer. for example, if a participant indicated that he or she felt worst in january, february, and march or in august, september, and october, he or she was considered to have a winter pattern. awareness was assessed by a single question on the spaq (‘‘have you ever heard about seasonal affective disorder (s.a.d.)?’’) to which the subject responded by checking either ‘‘yes’’ or ‘‘no.’’ . . statistical analysis we compared the distribution of variables (e.g., age, bmi) between groups (e.g., men vs. women, aware vs. not aware) using linear models that accounted for family structure because many of the study subjects were related. this was accomplished using a variance components model that included the relationship matrix as a random effect. the heritability of gss was estimated by quanti- tative genetic procedures (falconer and mackay, ; hsueh et al., ) with adjustments made for age, gender, and household, the latter included to allow for an environmental effect shared among household members. heritability was defined as the proportion of the total trait variance attributable to the additive effect of genes and was estimated by modeling the phenotypic covariance (condi- tional upon covariate effects) between any two individuals in the pedigree as a function of their degree of biological association. all parameter effects, including heritability, were estimated using the maximum likelihood method with the solar software package (texas biomedical research institute, san antonio, texas) (almasy and blangero, ). table frequency of sad and total sad in those aware and not aware of sad. aware of sad (n¼ ) not aware of sad (n¼ ) adjusted p-value, conditional on family structure* mean age (years) . . . . . men (%) . (n¼ ) . (n¼ ) . mean gss . . (n¼ ) . . ( ) . sad (%) . (n¼ ) . (n¼ ) . total sad (%) . (n¼ ) . (n¼ ) . *age adjusted for sex, sex adjusted for age, and gss and sad adjusted for age and sex. . results . . demographic characteristics of sample (table ) the sample included women ( . %) and men ( . %). the mean age ( sd) was . ( . ) years and mean body mass index (bmi) was . ( . ) kg/m . out of all respondents, ( . %) had heard of sad before. there was no significant difference in gss in respondents who completed two spaq mailings. . . characteristics of sad and total sad in the amish table shows mean characteristics of the study sample by gender. the mean gss ( sd) was . ( . ) and was slightly table mean gss and frequencies of seasonal affective measures in amish men and w men (n¼ ) wom mean age . . . mean bmi . . . mean gss . . . % reporting awareness . (n¼ ) . % with sad . (n¼ ) . (n % with total sad . (n¼ ) . (n total sad¼sadþsubsyndromal-sad. n all p-values adjusted for age (except age). higher, although not significantly so, in women ( . . ) com- pared to men ( . . ). there were cases of sad in the sample ( . %; % ci . – . ). the number of total sad (defined as subjects with either sad or s-sad) cases was ( . %; % ci . – . ) with a higher prevalence in women than in men ( . % vs. . %, po . ). there was little difference in age between those with (n¼ ) and without (n¼ ) sad ( . vs. . years., p¼ . ), although subjects with total sad (n¼ ) were significantly younger than those without total sad (n¼ ) ( . vs. . years, p¼ . ). awareness of sad was similar between men and women (table ). subjects aware of sad tended to be younger, albeit not significantly ( . vs. . years, sex-adjusted p¼ . ), and were more likely to have sad ( . % vs. . %, p¼ . ) and total sad ( . % vs. . %, p¼ . ) compared to subjects not aware of sad. . . heritability heritability of gss was estimated to be . % (n¼ ; p¼ . ; % ci¼ . – . %) after controlling for age and gender. the heritability estimate was unaltered when adding household effect as a random effect to the model (h ¼ . %; p¼ . ; % ci¼ . – . %). . discussion to our knowledge, this is the first study on seasonality and its heritability in the amish population and also the first sad study in a population with limited exposure to bright electric light. global seasonality scores and prevalence of sad in the amish were found to be lower than expected based on earlier studies in other populations (magnusson, ). studies performed in different countries, continents, and hemispheres show sad pre- valence estimates from zero in the philippines, a tropical country, to over % in denmark (dam et al., ), with a significant correlation between prevalence and latitude in north america, omen. en (n¼ ) total (n¼ ) p-value* . . . . . . . o . . . . . (n¼ ) . (n¼ ) . ¼ ) . (n¼ ) . ¼ ) . (n¼ ) . u.k. raheja et al. / journal of affective disorders ( ) – but only a non-significant trend in european studies [mersch et al., ]. certain populations appear to be less vulnerable to sad. for instance, sad is less common in icelanders ( . %) (magnússon and stefánsson, ) than in residents of maryland ( . % and . % (kasper et al., b; rosen et al., )), new york ( . %), and new hampshire ( . %) in the us (rosen et al., ), even though iceland is at a higher latitude. interestingly, sad prevalence is also low in individuals of icelandic descent in canada ( . %) (magnússon and axelsson, ) (again at higher latitude than in us studies, suggesting that there may be resi- lience to sad in icelanders). in finland, one study found that sad is more common in finns than in lapps (saarijärvi et al., ), possibly suggesting resilience in the latter group. in switzerland, the country of origin of the amish founders (cross, ), the prevalence of sad is relatively low ( . %) (wirz-justice et al., ). contrary to our primary hypothesis, the frequencies of sad ( . %) and s-sad ( . %) were found to be lower in the amish than in other populations at similar latitudes (kasper et al., b; rosen et al., ), and were even lower than in popula- tions living in temperate climates with previously documented low frequencies of sad such as icelanders, swiss, and lapps (magnússon and stefánsson, ; magnússon and axelsson, ; saarijärvi et al., ; wirz-justice et al., ). in fact, the observed prevalence of sad in this amish sample is the lowest in all spaq-based studies of sad prevalence conducted in pre- dominantly caucasian populations (magnusson, ). while it is possible that the use of a mailed pen and paper questionnaire lowered response rates in the amish, this is an unlikely scenario because the response rate of our study is quite similar to the response rates in other studies using mailed ques- tionnaires (approximately % for mailed spaqs (magnusson, )), and the prevalence of sad was higher in those previous studies. the response rates in our study match well with the response rates for general physician questionnaires (cummings et al., ). however, amish-specific cultural factors may have played a role in answering the spaq. the amish may be more stoical than other populations and may be less inclined to report emotional or behavioral problems, especially in an impersonal context inherent to pen and paper questionnaire completion. this may affect the way in which they respond to questions regarding whether seasonal issues are a ‘‘problem’’ for them and the months in which they ‘‘feel worst.’’ to take these factors into consideration, we considered individuals who had gss values and problem ratings that met the criteria for sad or s-sad but who did not indicate the months during which they felt worst. by considering the months in which these participants indicated they felt ‘‘best’’ and assuming the inverse of these months as the months in which they felt worst, we were able to identify new ‘‘cases’’ of sad, bringing the number of cases from ( . %) to ( . %). the number of cases of total sad increased from ( . %) to ( . %). these prevalence rates are still notably lower than most other predominantly caucasian populations studied. one could hypothesize that a lower awareness of sad and its treatment in the amish could not only be the result of lower rates of sad, but could also contribute to underreporting of problems and thus contribute to a lower rate of sad. indeed, only % of the surveyed amish were aware of sad, in contrast to approximately half of the non-amish respondents in montgomery county, md more than years earlier (kasper et al., b). if not a response bias, perhaps routine patterns of exposure to sunlight play a role in contributing to this low prevalence of sad. while light intensity during early morning and late evening in winter is likely to be reduced in the amish (although there are no direct measurements reported to date), it is likely that light exposure during daytime is greater in the amish because they spend more time working outdoors in general, although fewer than half are now farmers (kraybill, ). it is conceivable that people who spend more time working outdoors, such as the amish, might have a lower likelihood of developing sad. in addition, actigraphic studies of physical activity in the amish of lancaster county have found much higher levels of physical activity in this population (rampersaud et al., ; evans et al., ). exercise has potential beneficial effects in patients with non-seasonal depression (rimer et al., ) and possibly also in seasonal depression (partonen et al., ) and thus, physical work may contribute to the low prevalence of sad in the amish. our primary hypothesis that the amish would have higher seasonality and more cases of sad was based on the photoperiod hypothesis of sad, which states that sad is a result of shortened photoperiod in the winter. in animals, the effects of photoperiod are mediated by changes in duration of nocturnal melatonin secretion which converts the calendar signal (duration of external night) (wehr, ). in humans, the duration of nocturnal mela- tonin secretion is longer in winter than in summer in patients with sad but not in healthy individuals in their regular habitat (wehr et al., ). extension of the photoperiod with light therapy is efficacious for treating sad (rosenthal et al., ), further providing evidence for the photoperiodic hypothesis. another dominant theory, i.e., the phase shift hypothesis of sad, posits that sad is a result of phase delay of endogenous circadian rhythms in relation to the external clock, the sleep- wake cycle, or other rhythms (lewy et al., ) due to insuffi- cient exposure to morning light in winter. according to the well replicated phase response curve to light exposure, exposure to bright light in the morning results in a phase advance whereas exposure to bright light in the evening induces a phase delay, with mid-day exposure having no effect (van cauter et al., ; khalsa et al., ). there is evidence for (lewy et al., , , ; terman et al., ) and against (wirz-justice et al., ) the phase shift hypothesis. in particular, studies attempting to unmask sleep and environment from circadian factors in sad resulted in conflicting results. for instance, a ‘‘constant routine’’ protocol (which unmasks endogenous circadian rhythms by studying subjects in a controlled setting for h to minimize the influence of masking effects) confirmed phase delays of temperature and cortisol in hypersomnic seasonal depression (avery et al., ). in contrast, going against the phase-delay hypothesis, a study utilizing a forced desynchrony routine (a gold-standard protocol to unmask endogenous circadian rhythms while minimizing effects of sleep deprivation) found no difference in the temperature and variations of mood with circadian phase or sleep-wake cycles between subjects with and without sad (koorengevel et al., ), failing to provide support for the phase delay hypothesis. the strongest evidence supporting the phase shift hypothesis is the consistently reported superiority of morn- ing light as compared to evening light for treatment of sad (golden et al., ). because exposure to artificial lights in the evening can result in phase delays, one possible speculative, and somewhat paradoxical, explanation of our results is that in modern humans electric light exposure in the evening is contributing to, rather than alleviating, seasonal changes in mood. boivin and czeisler ( ) have shown that even light of low intensity equivalent to room light (likely above the light intensities generated in the amish homes) can result in phase shifting effects. it could be that a greater number of individuals in modern populations, but not in the amish, would tend to be phase-delayed in the winter in part because of extended exposure to evening bright light, and, according to the phase shift hypothesis, to be more likely to develop seasonal depression. this novel hypothesis could be formally tested in future studies. the heritability of seasonality was found to be . % after adjustment of age, sex, and household. it is possible that u.k. raheja et al. / journal of affective disorders ( ) – environmental factors play a larger role in seasonality in the amish than genetic factors. the limitations of heritability should also be kept in mind. for example, heritability is not constant and may change over the course of time due to changes in environmental factors or gene-environment interactions (visscher et al., ). the limitations of the study include the likely overestimation of the rates of sad by spaq in comparison to clinical diagnostic criteria (levitt and boyle, ; steinhausen et al., ). thus, it is possible that the actual rate of sad in the amish population is even lower than that found in our study. it should be emphasized that the spaq is a screening and research tool, not a diagnostic tool. the diagnosis would need to be verified by clinical interview or questionnaires, which was not done in our study. also, there is the potential for selection bias and recall bias with mailed questionnaires. obviously, the results of this study, which was done in a very specific population, have limited generalizability. further studies examining the level of exposure of the amish to light and, possibly, of genes potentially involved in sad would help elucidate the reasons for the low prevalence of sad in the amish. . conclusion this was, to our knowledge, the first study in a population without regular exposure to network electric lighting. our hypothesis that the prevalence of sad in the amish would be higher than in other populations due to their limited exposure to electric lighting was not supported. in fact, contrary to expecta- tion, the amish had the lowest prevalence of sad in all spaq- based population studies in caucasians (mersch et al., ; magnusson, ). similarly, heritability of gss in the amish was found to be relatively low. further studies involving clinical interviews and non-amish controls would be required to confirm this first report and potentially to identify the mechanisms responsible for the lower seasonality score, heritability of season- ality scores, and prevalence of sad in the old order amish. this may lead to identifying new factors of resilience to sad, which may better inform future preventative and curative approaches. role of funding source k mh - (pi postolache). conflict of interest the authors declare they have no conflicts of interest and nothing to disclose. acknowledgements we thank the staff of the amish research clinic of the university of maryland for their overall support and the trainees of the mood and anxiety program for their help with references, mailings and data management. references almasy, l., blangero, j., . variance component methods for analysis of complex phenotypes. cold spring harbor protocols ( ), pdb.top . review. pubmed pmid: ; 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(pt ), – , pubmed pmid: ; pubmed central pmcid: pmc . 	seasonality of mood and behavior in the old order amish 	introduction 	methods 	procedures and sample selection 	seasonal pattern assessment questionnaire (spaq) 	statistical analysis 	results 	demographic characteristics of sample (table ) 	characteristics of sad and total sad in the amish 	heritability 	discussion 	conclusion 	role of funding source 	conflict of interest 	acknowledgements 	references - - .pdf ucla ucla previously published works title effects of eicosapentaenoic acid and docosahexaenoic acid on lipoproteins in hypertriglyceridemia. permalink https://escholarship.org/uc/item/ gg p s journal current opinion in endocrinology, diabetes, and obesity, ( ) issn - x authors patel, amish a budoff, matthew j publication date - - doi . /med. peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ gg p s https://escholarship.org http://www.cdlib.org/ copyright © wolters kluwer health, inc. unauthorized reproduction of this article is prohibited. ce: swati; med/ ; total nos of pages: ; med currentopinion effects of eicosapentaenoic acid and docosahexaenoic acid on lipoproteins in hypertriglyceridemia amish a. patel and matthew j. budoff purpose of review the treatment of hypertriglyceridemia (htg) with v- fatty acid preparations adds a novel therapy to reduce cardiovascular disease. this review examines the effects of eicosapentaenoic acid (epa) and docosahexaenoic acid on lipoproteins and the cardioprotective effects in htg. recent findings the evidence that v- fatty acid therapy at prescription strength is effective and safe at lowering triglyceride levels is growing. although epa/docosahexaenoic acid formulations did lower triglyceride levels, an increase in low-density lipoproteins was observed and outcome data were mixed. more recent trials have shown that decreased levels of low-density lipoprotein can be achieved with epa preparations. although the cardiovascular outcomes data are not fully available, meta-analysis of available data reports protection against vascular disease. summary the addition of v- fatty acid treatment should be considered in patients with severe htg as well as high-risk patients for atherosclerotic disease. emerging data are supportive, but long-term outcome studies are still underway. keywords hypertriglyceridemia, lipoproteins, v- fatty acids introduction cardiovascular disease (cvd) remains to be a prom- inent focus of research worldwide [ ]. indicators that lead to cvd and the strategies for prevention and treatment remain the central components. major clinical factors of cvd risk include high- plasma cholesterol and high triglycerides. due in part to the definitive link between raised concen- trations of low-density lipoprotein (ldl) cholesterol and cvd, decades of research has been focused on approaches to lower plasma ldl cholesterol [ , ]. the result has been the highly effective use of statins in cvd treatment and prevention [ ]. we now understand that the treatment of ldl cholesterol alone, however, does not completely reduce cvd risk [ , ]. hypertriglyceridemia (htg) represents a risk fac- tor for atherosclerotic cvd although, contrasting observations has been reported [ – ]. the national cholesterol education program – adult treatment panel iii defined normal triglyceride level as less than mg/dl, borderline high as – mg/dl, high as – mg/dl, and very high triglyceride as at least mg/dl [ ]. a growing body of evidence links htg with cvd [ ]; however, the role of triglyceride in the cvd risk assessment remains con- troversial. the american college of cardiology/ american heart association guidelines on the treat- ment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults did not directly address triglyceride, as the panel felt that concrete data are still lacking to make formal recommendations [ ]. owing to the interrelationship of triglyceride with other lipoproteins such as low levels of high-density lipoprotein (hdl) and increased levels of ldl, los angeles biomedical research institute, harbor-ucla medical center, torrance, california, usa correspondence to matthew j. budoff, md, los angeles biomedical research institute, harbor-ucla medical center, torrance, ca, usa. tel: + ; fax: + ; e-mail: mbudoff@labiomed.org curr opin endocrinol diabetes obes , : – doi: . /med. - x copyright ! wolters kluwer health, inc. all rights reserved. www.co-endocrinology.com review copyright © wolters kluwer health, inc. unauthorized reproduction of this article is prohibited. ce: swati; med/ ; total nos of pages: ; med elevated triglyceride levels have been felt to represent a marker rather than an independent risk factor for cvd [ ]. recent theories suggest that elevated triglyceride is a reflection of triglyceride in atherogenic triglyceride-rich lipoprotein remnants, which consists of chylomicron remnants and very low-density lipoprotein (vldl) remnant particles [ ]. the triglyceride-rich lipoproteins rem- nants are thought to generate toxic substances and damage the endothelium when in contact with endo- thelial lipoprotein lipase [ ]. owing to the limita- tion of statin therapy on triglyceride and the recognition of the possible role of triglyceride on cvd, an interest is now focused on v- fatty acid preparations [ ]. the two v- fatty acids of focus are eicosapen- taenoic acid (epa) and docosahexaenoic acid (dha), which are essential in human physiology. both epa and dha are mostly obtained by consuming oily fish, such as salmon, albacore tuna, mackerel, herring, and sardines or by fish oil supplements [ ]. numerous studies are reporting the potent triglyceride-lowering effects and potential benefits to protect against cvd [ ]. we intend to review the effects of epa and dha on lipoproteins in htg. hypertriglyceridemia the causes of htg can be classified into two major categories, primary and secondary. primary htg results from genetic defects leading to disordered triglyceride metabolism, such as familial combined hyperlipidemia and familial htg, familial dysbetali- poproteinemia, apolipoprotein c- deficiency, and lipoprotein lipase deficiency [ ]. secondary htg is most commonly caused by diets high in carbo- hydrates or high glycemic index content, high alco- hol consumption, uncontrolled diabetes mellitus, and hypothyroidism. medications such as nonselec- tive b-blockers, tamoxifen, oral estrogens, glucocorti- costeroids, thiazide diuretics, propofol, and antiretroviral drugs have been implicated as well [ , ]. high levels of triglyceride (! mg/dl) have been well established as a risk factor for pancreatitis, and currently is the third leading cause after alcohol and gallstones [ ]. in the presence of triglyceride levels ( – mg/dl), treatment is recommended to reduce cvd risk [ ]. many therapeutic strategies to lower triglyceride levels are recommended but must be in conjunction with changes in dietary habits such as restriction to caloric intake, reduction of fat ingestion, and absti- nence from alcohol. the current pharmacological interventions to lower triglyceride levels include sta- tins, fibrates, nicotinic acid, and v- fatty acids [ , ]. fibrates are often recommended as first-line with v- fatty acids as adjunct therapy. most com- mon fibrates include fenofibrate and gemfibrozil. although they are not benign therapies and the rate of adverse events observed are dependent on the choice of fibrate administered. gemfibrozil increases risk of myopathy when used with a statin and fibrates are associated with increased gallstones [ ]. niacin has been shown to increase hdl, decrease triglyceride, and reduce rate of cvd events but the most common adverse effect of cutaneous vasodilatation or flushing is reported in up to % of patients receiving therapy [ ]. this prevents dose titration or it decreases adherence by patients [ ]. in contrast, the frequency of adverse effects observed has generally been similar in v- fatty acids treatment group and the placebo group [ , ]. the most common adverse events being gastrointestinal (nausea, diarrhea, and mild gastrointestinal disturb- ances) and no serious safety issues were identified [ , ]. the v- fatty acids (epa and dha) are thought to reduce triglyceride levels primarily by promoting fatty acid degrading via peroxisomal b-oxidation, inhibiting lipogenesis in the liver, and accelerating clearance triglyceride from the plasma by increasing fractional clearance rate of vldl [ ]. various types of v- in prescription form currently exist on the us market, v- fatty acid in ethyl ester formulation with both epa and dha, v- fatty acid with mostly pure ethyl ester of epa termed icosapent ethyl and lastly the v- fatty acid in free fatty acid form of epa and dha [ ]. effects of eicosapentaenoic acid and docosahexaenoic acid on lipids the pharmacological doses of v- fatty acids (at least g/day) significantly reduce triglyceride levels, but appear to affect other lipoproteins, including ldl [ ]. the increase in ldl was observed in patients with very high triglyceride levels and following the use of dha alone compared with studies using epa alone [ ]. the initial available pharmaceutical formulation of v- was composed of epa % and dha % in their ethyl ester form. it was used as an adjunct to diet key points " v- fatty acid consistently lowers triglyceride levels. " treatment of htg protects against vascular disease. " v- fatty acids add a novel therapy to at risk patients. lipids www.co-endocrinology.com volume " number " month copyright © wolters kluwer health, inc. unauthorized reproduction of this article is prohibited. ce: swati; med/ ; total nos of pages: ; med for the treatment of severe htg, triglyceride at least mg/dl [ , ]. a g/day formulation taken for weeks significantly reduced triglyceride levels by %, vldl by %, and increased hdl by %, but it also significantly increased ldl by % (p¼ . ) [ ]. in another study involving patients with severe htg taking g/day for weeks reduced triglyceride levels by . %, vldl by . %, and increased hdl by . %, but also seen was a significant increase in ldl by . % (p¼ . ) [ ]. a recently food and drug administration- approved v- in the free fatty acid formulation containing epa % and dha % was used for the treatment of adults with severe htg. in the double-blinded, randomized trial of epanova for lowering very high triglycerides, patients with severe htg (triglyceride! mg/dl but $ mg/dl) were tested at three doses: , , and g/day versus olive oil as the control [ ]. serum triglyceride levels were decreased significantly in all three doses compared with controls by . , . , and . %, respectively. non-hdl cholesterol was decreased as well by . , . , and . %, respectively. ldl was significantly increased with g/day by . % (p < . ) and by . % with g/day (p < . ) compared with controls, but not with g/day [ ]. the multi-center, placebo-controlled, randomized, double-blind, -week study with an open-label extension study studied the triglyceride- lowering effect of icosapent ethyl, which is an v- fatty acid with % pure ethyl ester of epa [ , ]. the study specifically looked at the effect of icosapent ethyl in severe htg patients who had triglyceride levels at least mg/dl and the impact on other lipoproteins, which included ldl, vldl, hdl, non-hdl, total cholesterol, and lipoprotein- associated phospholipase a [ ]. the study included patients ( . % with triglyceride ! mg/dl and . % with triglyceride > mg/dl) who were randomized to icosapent ethyl g/day, icosapent ethyl g/day, or placebo over a -week period. the results reported that g/day reduced triglyceride by . % and g/day reduced triglyceride levels by . % in patients with triglyceride at least mg/dl. in patients with triglyceride higher than mg/dl, g/day reduced triglyceride levels by . %, whereas g/day reduced triglyceride levels by . %. overall, icosapent ethyl g/day decreased triglyceride levels by . % (p¼ . ) and g/day decreased trigly- ceride levels by . % (p < . ) [ ]. further reductions were reported in patients with baseline triglyceride higher than mg/dl and with concur- rent statin therapy. icosapent ethyl g/day with statin decreased triglyceride levels by . % (p¼ . ) and g/day reduced triglyceride levels by % (p¼ . ). this suggests a possible syner- gistic relationship between icosapent ethyl and statins. icosapent ethyl did not simultaneously increase ldl levels [ ]. in follow-up analysis the reported effects of ico- sapent ethyl on lipoprotein particle concentration and size showed that g/day significantly reduced the concentration of the large vldl particles by . % (p¼ . ), small ldl by . % (p < . ), total ldl by . % (p¼ . ), and total hdl by . % (p < . ). however, the g/day did not have the statistically significant reductions on these lipoprotein parameters [ ]. in another multicenter, placebo-controlled, randomized, double-blinded, -week clinical trial named the anchor study; it assessed the safety and efficacy of icosapent ethyl in patients who were at high risk for cvd. the study included patients who had adequate control of ldl on statin therapy and persistently elevated triglyceride level at least mg/dl but less than mg/dl [ ]. the anchor study patients were randomized to icosa- pent ethyl g/day, g/day, or placebo and the primary end point was triglyceride level change from baseline to the end of the -week period. the secondary included percentage change in ldl, non-hdl, vldl, apolipoprotein-b, and phospholipase a . selected exploratory end points included total cholesterol, hdl, and high- sensitivity c reactive protein. the g/day group had a reduction in triglyceride levels by . % and the g/day group had a reduction in triglyceride levels by . %. icosapent ethyl g/day decreased ldl by . % (p¼ . ), whereas the g/day group did not significantly reduce ldl. in addition g/day table . effects of eicosapentaenoic acid and docosahexaenoic acid on triglyceride and low-density lipoprotein in hypertriglyceridemia patients [ – , , ] epaþdha (ethyl esters) epaþdha (free fatty acid form) epa (ethyl esters) epa (ethyl esters) g/day g/day g/day g/day g/day g/day g/day g/day tg & . % & . % & . % & . % & . % & . % & . % & . % ldl þ . % þ . % þ . % þ . % & . % & . % & . % & . % dha, docosahexaenoic acid; epa, eicosapentaenoic acid; htg, hypertriglyceridemia; ldl, low-density lipoprotein; tg, triglyceride. effects of eicosapentaenoic acid and docosahexaenoic acid patel and budoff - x copyright ! wolters kluwer health, inc. all rights reserved. www.co-endocrinology.com copyright © wolters kluwer health, inc. unauthorized reproduction of this article is prohibited. ce: swati; med/ ; total nos of pages: ; med decreased total cholesterol ( . %), apolipoprotein b ( . %), very-low density lipoprotein cholesterol ( . %), lipoprotein-associated phospholipase a ( . %), and high-sensitivity c-reactive protein ( . %) versus placebo (p < . for all compari- sons) [ ]. the current formulations of v- fatty acids in the studies mentioned report the effects of different doses. the higher dose of g/day had the greatest effect on triglyceride levels compared to the g/day formulation. the initial formulation of epa and dha studied the g/day effects, whereas the randomized trial of epanova for lowering very high triglycerides, multi-center, placebo-controlled, randomized, double-blind, week study with an open label extension, and anchor studied and reported the effects of both doses (table ). cardiovascular disease outcomes cardiovascular outcome trials of v- fatty acids have resulted in inconsistent results [ ]. the japan epa lipid intervention study observed a decrease of % in major cardiovascular events (mace) [ ] and in an italian study, the gissi prevenzione investigators a % decrease of mace was seen [ ]. although the risk and prevention study, the outcome reduction with initial glargine intervention study and the omega trial did not report significant reduction in rate of mace [ – ]. a meta-analysis consisting of clinical trials of which included partici- pants reported that v- fatty acid treatment may protect against vascular death, but did not signifi- cantly affect the rate of arrhythmia, cerebrovascular events or sudden death [ ]. it is important to note that combination v- fatty acids (epaþdha) have consistently failed to prevent atherosclerotic cardiovascular disease in the presence of statins. currently, underway are two large international car- diovascular outcome studies to evaluate the clinical efficacy of v- fatty acid therapy: the reduction of cardiovascular events with epa – intervention trial (reduce-it) and the statin residual risk reduction with epanova in high cardiovascular risk patients with hypertriglyceridemia (strength) [ , ]. they are expected to complete december and june , respectively. both studies are designed to evaluate the safety and efficacy of v- fatty acids in combination with statin therapy, particularly in high-risk patients with htg. conclusion the reduction of high or very high triglyceride with epa or dha is associated with different effects on ldl. treatment with either lowered triglyceride levels, but dha was more often associated with an increases in ldl and epa on the other hand, decreased ldl or resulted in no significant change of ldl. ongoing trials are currently underway to evaluate the efficacy of v- fatty acids on the prevention of cvd. acknowledgements none. financial support and sponsorship none. conflicts of interest there are no conflicts of interest. references . nowbar an, howard jp, finegold ja, et al. global geographic analysis of mortality from ischaemic heart disease by country, age and income: statistics from world health organisation and united nations. int j cardiol ; 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: e . . clinicaltrials.gov. a study of amr to evaluate its ability to reduce cardio- vascular events in high risk patients with hypertriglyceridemia and on statin, in: the primary objective is to evaluate the effect of g/day amr for preventing the occurrence of a first major cardiovascular event (reduce-it); . https:// clinicaltrials.gov/ct /show/nct [accessed january ]. . clinicaltrials.gov. outcomes study to assess statin residual risk reduction with epanova in high cv risk patients with hypertriglyceridemia (strength); . https://clinicaltrials.gov/ct /show/nct [accessed january ]. effects of eicosapentaenoic acid and docosahexaenoic acid patel and budoff - x copyright ! wolters kluwer health, inc. all rights reserved. www.co-endocrinology.com biomarkers for simplifying hts d cell culture platforms for drug discovery: the case for cytokines feature drug discovery today �volume , numbers / �april perspective p e rs p e ct iv e � f e a t u r e biomarkers for simplifying hts d cell culture platforms for drug discovery: the case for cytokines yinzhi laia, amish asthanaa and william s. kisaalita, williamk@engr.uga.edu in this review, we discuss the microenvironmental cues that modulate the status of cells to yield physiologically more relevant three-dimensional ( d) cell-based high throughput drug screening (hts) platforms for drug discovery. evidence is provided to support the view that simplifying d cell culture platforms for hts applications calls for identifying and validating ubiquitous three-dimensionality biomarkers. published results from avascular tumorigenesis and early stages of inflammatory wound healing, where cells transition from a two-dimensional ( d) to d microenvironment, conclusively report regulation by cytokines, providing the physiological basis for focusing on cytokines as potential three-dimensionality biomarkers. we discuss additional support for cytokines that comes from numerous d and d comparative transcriptomic and proteomic studies, which generally report upregulation of cytokines in d compared with d culture counterparts. introduction it is now a well-accepted view that traditional flat-surface cell culture dishes do not adequately represent the natural context of the cell and their use in cell-based assays might, in some cases, result in less accurate drugs’ effects predictions [ – ]. fig. a shows the possible cues that might affect the status of a cell, grouped along che- mical, physical, and spatial and/or temporal ‘dimensions’ [ , ]. recently, numerous three- dimensional ( d) cell-based assay platforms have been commercially introduced with more contexts to better mimic the complex in vivo microenvironment. the premise behind these products is that mimicking the in vivo micro- environment yields physiologically more rele- vant cells that provide physiologically more relevant drug responses [ ]. - / /$ - see front matter � elsevier ltd. all rights reser several studies have provided evidence in support of this premise. for example, d multi- cellular tumorspheroids(mcts)havebeen shown to mimic in vivo situations closely [ , ]; they were also able to recapitulate fully the cell adhesion- mediated drug resistance (cam-dr) of emt tumors [ ], which was induced in vivo but was lacking in cultured monolayers of the same cells. also, weaver et al. [ ] showed the phenotypic transformation of malignant cells in a d collagen matrix upon treatment with integrin antibodies; however, this phenomenon, also observed in vivo, was not observed in traditional monolayer cul- tures. in another study, which highlights the relevance of d culture format, two tumorigenic cell lines showed protease-independent amoe- boid movement within a d collagen, challenging the traditional screening for anti-metastatic ved. doi: . /j.drudis. . . agents against proteolytic activity with two- dimensional ( d) cultures [ ]. in drug develop- ment, drug-induced liver injury or hepatotoxicity is a major deciding factor behind the approval, non-approval or limitation in the usage of the drug by the fda. usage of hepatoblastoma cell line (hepg ) cultured in d monolayers is the current gold standard in early screening of drug candidates for hepatotoxicity activity. however, cells in this format either entirely lack or express acutely low levels of many drug metabolizing enzymes [cytochrome p s (cyps)] and trans- porters found in hepatocytes in vivo [ ]. by contrast, dhepatocyte-likecellculturesachieved in bioreactors have been shown to emulate in vivo characteristics to such a high degree that they can be utilized to fabricate a functional bioartificial organ for transplantation [ , ]. www.drugdiscoverytoday.com http://dx.doi.org/ . /j.drudis. . . s t l p w w s b c h c h y w b s p o m e l [ n t t a o c c a s perspective drug discovery today �volume , numbers / �april [()td$fig] d geometric property (three-dimensionality) cell–matrix adhesion (including polarity and mechanical inputs) growth factors (cytokines and hormones) (a) soft (b) chemical matrix composition physical matrix characteristics mechanical stress and fluid flow nutritional status (e.g. o and nutrients) stiff d autoregulation (niche formation) cell–cell junctions and communication (including polarity and mechanical inputs) chemical s p a ti a l/ te m p o ra l (t o p o g ra p y a n d d if fe re n ti a ti o n ) (d) (e) (e .g . m at rix p ro pe rty an d fo rc e) ph ys ic al (c) drug discovery today figure microenvironmental cues and cellular phenotype outcomes. (a) the possible cues that might affect the status of a cell. the major players are spatial and/or temporal, physical and chemical cues [ , ]. (b–e) phalloidin-stained f-actin. (b, d) show muscle cells cultured on soft and stiff flat surfaces, respectively [ ]. (c, e) show skbr malignant breast cancer cells grown in d lrecm [ ] and d glass surfaces [ ], respectively. scale bars = mm (c) and mm (d). p e rsp e ctiv e � f e a t u r e an increase in studies highlighting the phy- iological relevance of the d culture format over raditional monolayers have formed the intel- ectual basis behind the founding of new com- anies such as dbiotek (http:// ww. dbiotek.com), kiyatec inc. (http:// ww.kiyatec.com), cellasic (http://www.cella- ic.com) and bellbrook labs (http://www.bell- rooklabs.com), which have since launched ommercial d products for cell-based assay igh throughput screening (hts). examples of ompanies with hts application products that ave been marketed for more than at least ears include: invitrogen corporation (http:// ww.invitrogen.com/ d-cellculture), glycosan iosystems (http://www.glycosan.com) and urmodics (http://www.surmodics.com). more roducts are in the pipeline, increasingly relying n developments in microfabrication and icrofluidics technologies. for example, huh t al. [ ] successfully reconstituted organ-level ung function on a chip and wang and kisaalita ] are developing a nanofibrous intercon- ected microwell platform for neural networks. although there might be exceptions, intui- ively, the more complex the culture system is, he more cues it can capture and, thus, the more ble it is to mimic the in vivo microenvironment f the cell. however, as the complexity in the ulture system increases, so does the screening ost per compound. product, cell maintenance nd readout instrumentation can cost more for d platforms. with the industry drive to reduce creening costs, an optimal balance between www.drugdiscoverytoday.com cost and in vivo microenvironment emulation is needed. it might not be prudent to pursue a ‘perfect’ cell culture system that prices itself out of adoption in state-of-the-art screening laboratories. so the question that begs an answer is: among the three cue group dimen- sions outlined in the first paragraph, can any one of them carry more weight and, as such, con- stitute the major driver in designing less com- plex cell-based hts platforms? to answer this question, we examine the microenvironment cues below in more detail. microenvironmental cues chemical cues can be subdivided into short- and long-range categories [ ]. short-range chemistry refers to the chemistry of the surface. to be able to emulate the in vivo phenotype, the approach adopted by most researchers is to present a substrate to the cell that chemically resembles the extracellular matrix (ecm). long- range chemistry refers to diffusible chemical species that are found in the ecm that contri- bute to the maintenance or control of the cell behavior and/or phenotype outcome. the list includes basic nutrients, growth factors, cyto- kines and other morphogens, as well as metalloproteinases. although their use in, for example, controlling differentiation is not well understood, long-range chemical cues are the easiest factor to manipulate, typically by including them in culture medium. a key attri- bute of chemical cues is that they can be orthogonal to the other two major cues; that is, they can be altered without affecting other properties of the platform. physical cues of the material or scaffolding in or on which the cells grow and differentiate have been characterized in terms of softness, stiffness, pliability or young’s modulus (terms used inter- changeably to express stiffness of an isotropic elastic material). when physical properties were first investigated with hydrogels, they were intertwined with chemical cues; high water content in the hydrogel makes the hydrogel softer, but simultaneously decreases the che- mical ligands density (i.e. the number of adhe- sion ligands for cell surface receptors per unit area of the substrate surface). to address this problem, researchers developed a novel hydro- gel system in which they were able to alter the stiffness of the hydrogel without altering ligand density [ ]. this type of culture system made it possible to study the effect of the physical properties of the scaffold without interfering with the chemical effects. however, most studies on substrate physical properties have been conducted on flat surfaces. initially, ‘ d cell cultures’ mainly referred to culture system scaffolds that constituted com- plex architecture at micro and/or nano scales, which included only the spatial context. the term now has a more comprehensive meaning that entails the three ‘dimensions’ outlined in fig. a. however, closer examination reveals that spatial cues are highly intertwined with the other cues. for example, by introducing micropores and/or nanofibers into the cell culture platform, http://www. dbiotek.com/ http://www. dbiotek.com/ http://www.kiyatec.com/ http://www.kiyatec.com/ http://www.cellasic.com/ http://www.cellasic.com/ http://www.bellbrooklabs.com/ http://www.bellbrooklabs.com/ http://www.invitrogen.com/ d-cellculture http://www.invitrogen.com/ d-cellculture http://www.glycosan.com/ http://www.surmodics.com/ onmental factor to achieve more platform drug discovery today �volume , numbers / �april perspective p e rs p e ct iv e � f e a t u r e the surface area of the construct can increase significantly, thus increasing the amount che- mical ligands that can be deposited on it. the thickness of the porous or fibrous scaffolds can also affect the concentration of the culture medium by generating a gradient of long-range chemical cues, such as oxygen and growth fac- tors. additionally, the porous and fibrous archi- tecture might alter the physical properties of the material, reducing its strength and altering the anisotropism of the material. for example, the strength of fibrous scaffolds along the fiber direction is higher than the direction that is perpendicular to the fiber, and the strength in both directions is lower than the bulk property of the material [ ]. mathematical models have been used to study chemical gradients induced by d cell cultures (e.g. see [ ]). however, few d cell culture studies have included physical property measurements in their studies. it is true that architecture and/or geometry have a profound impact on cell behavior. to be able to engineer a platform that more accurately emulates the in vivo microenvironment, it is important to isolate the contributions of the different cues to the phenotypic outcome. consider morphology for example; the right panel of fig. shows f-actin staining from muscle cells on soft (fig. b) and stiff (fig. d) flat surfaces [ ], skbr malignant breast cancer cells in d laminin-rich extracel- lular matrix (lrecm) (fig. c) [ ] and on glass d surfaces (fig. e) [ ]. the soft surface and d cell cultures can both make cells more roundish and smaller in smaller size when compared with the same cells grown on rigid d surfaces. the f- actin organization is also similar between soft surfaces and d cell cultures: in both cases, the f- actin does not form stress fibers and is more abundant at the edge of the cells. why do two different factors have similar effects on a cellular outcome? the young’s modulus for lrecm (also called matrigel in other studies) ranges from pa to pa as concentration changes from % to % [ ]. in addition, the d lrecm culture had a lrecm coating as thick as mm (thick in comparison to the typical cell size of mm). it is reasonable to speculate that the cells cultured in d lrecm interacted with a soft tissue-mimicking microenvironment and the morphological changes observed might have resulted mainly from pliability changes. in many d culture systems, cells cluster together and form multicellular aggregates [ – ]. under such circumstances, most individual cells within the multicellular spheres are exposed to their neighboring cell surfaces, which are soft. therefore, it is possible that the influence of the material physical properties in such cases is limited, which raises the question; if softness is all a cell requires to become round and subse- quently mimic its in vivo counterpart, why is there a need to create a complex d architec- ture? alternatively, the rigidity effect of the material has been bypassed by simple fabrica- tion of micropost arrays ( . – . mm) onto a polydimethylsiloxane (pdms) material; the rigidity of the altered material is reflected by the resulting d-like cellular morphology from cells cultured on the highest micropost substrate [ ]. above, we have briefly summarized current understanding of microenvironmental cues. unfortunately, this understanding is not suffi- cient to guide the design of the simplest plat- form that provides the desired emulation of the in vivo situation. a common practice is the trial- and-error approach, in which the platform designer focuses on a complex physiological relevance (cpr) outcome as an indication of the capture of one or several cues sufficient for the desired outcome. cpr here means in vitro emulation of in vivo structure and/or function in d that is not possible in d cultures, such as bile canaliculi-like structures [ ] and albumin secretion [ ] by hepatocytes. there are many cells whose phenotypes are altered by culture in d structures but, unlike hepatocytes, their cpr is not readily discernible. for such cells, as the platform architecture is simplified, it is difficult to determine the simplest form that maintains the desired emulation of the in vivo situation. there is a need for simple technology to establish the culture status trajectory that results in formation of three-dimensionality that leads to cpr. such a technology will enable the establishment of a balance between a simplistic architectural design and the complex microenvironmental features. as mentioned above, some cells might not require a complex platform to grow in or on; rather, they might initially need the minimal essential cues to become competent enough to produce endogenous ecm, which then provides the remaining or additional essential cues. this has been observed in several studies; for example, tumor cells grown in porous d poly(lactide-co-glycolide) (plg) and matrigel scaffolds showed upregulation of interleukin (il)- compared with d cultures [ ]. even though both systems have a varying degree of com- plexity and provide different microenviron- mental cues, the outcome (elevation in il- level) was similar and physiologically justified as it depicts the angiogenic capability of the cells. alternatively, il- could have been added to the culture systems as a biochemical microenvir- complexity; however, it would have been redundant as the cells can produce it themselves if the essential initial minimal factors are present. this example underscores the need to better understand the required essential cues and their thresholds that yield cpr or in vivo emulation outcomes. this will enable simplification of the platform without giving up the physiologically relevant behavior of the cells. however, the task of specifying such complex microenvironments is daunting. a simpler approach is to generalize the measurement of the developmental com- petence that leads to cpr outcomes in terms of ubiquitous three-dimensionality biomarkers [ ]. as the platform architecture design is simplified, it will be possible, with such a measure, to know when the trajectory toward cpr outcomes is being compromised by the introduced simpli- fication(s). to address the question of what the biomar- kers might be, we reviewed the current d culture literature with respect to finding che- mical entities that are produced differentially between d (minimal or no in vivo-like micro- environment) and d (more in vivo-like micro- environment providing cpr) systems across a variety of cells derived from different tissue types and d architectural platforms. this search indicates cytokines as the most probable family of compounds to provide the badly needed biomarkers. we summarize the arguments below for the case for cytokines. searching for three-dimensionality biomarkers cytokines are soluble, low molecular weight, extracellular protein mediators that usually act at short range between neighboring cells. they are crucial intercellular regulators and mobilizers of cells engaged in innate and adaptive inflam- matory host defenses, cell growth, differentia- tion, cell death, angiogenesis, and development and repair processes. they have been assigned to various family groups based on the structural homologies of their receptors and can be broadly classified into families such as colony stimulating factors, interleukins, interferons, transforming growth factors (tgf), tumor necrosis factors (tnf), platelet-derived growth factors (pdgf) and chemokines. although cytokines have been extensively studied in the field of immunology and oncology, they have been overlooked by tissue engineers. however, these same small proteins might have the power to revolutionize the field. although the evidence for their existence in d cultures is compelling, they have not yet been looked at as candidates for potential biomarkers. however, we suggest www.drugdiscoverytoday.com perspective drug discovery today �volume , numbers / �april [()td$fig] d, rgd- alginate d, alginate d, rgd- alginate d, rgd- alginate d, alginate d, rgd- alginate v e g f ( p g / c e ll s / h ) il - ( p g / c e ll s / h ) * * * * (a) (b) drug discovery today figure cytokine production by tumor cells (oral squamous cell carcinoma, oscc- ) in alginate hydrogels containing covalently coupled rdg peptides at a density that correlates to the number of rdg sites in tumors in vivo. the d system resulted in enhanced il- production (a); however, this was not the case for vegf (b). (*, p � . ; **, p � . ; black and gray bars represent and days in culture, respectively). adapted, with permission, from [ ]. p e rsp e ctiv e � f e a t u r e that they are the ideal family to explore for identification and validation follow-up studies for the following three main reasons. first, when cells are transitioned from a d monolayer to a d microenvironment, they become surrounded by homotypic neighbors, forming a loosely bound disorganized aggre- gate. in vivo, such a scenario is encountered only during avascular tumorigenesis or early stages of inflammatory wound healing, which are both similar in nature and regulated by cytokines [ ]. therefore, in vitro, the cells growing in d relate to any of those two models, depending on their type (malignant or primary, respectively), therefore explaining the upregulation of their cytokine levels. second, several d– d comparative tran- scriptomic studies with cells from the four main tissue types (nerve, muscle, connective and epithelial) cultured on a variety of platforms, have reported the upregulation of cytokines and their receptors in d cultures. for example, klapperich and bertozzi [ ] reported upregu- lation of seven cytokines [il- , chemokine (c-x-c motif) ligand (cxcl)- , cxcl , cxcl , cxcl , vascular endothelial growth factor (vegf) and leukemia inhibitory facto (lif)] by a human fetal lung fibroblast (imr- ) cultured in a collagen– glycosaminoglycan (collagen/gag) d mesh. the mesh was prepared by freeze drying and heat enabled cross-linking of the polymer and had an average pore size of – mm. in addition, ghosh et al. [ ] reported upregulation of six cytokines [cxcl – , il- , macrophage inflammatory protein- (mip- a) and angiopoe- tin-like ] by a melanoma cell line (na ) cultured www.drugdiscoverytoday.com on poly- -hydroxyethyl methacrylate (poly- hema) plates when compared with d surfaces. the polymer coating prevented cells from attaching to the plastic surface leading to the formation of mcts. third, transcriptomic findings such as those in the above examples have been supported by studies at the protein level. for example, enzerink et al. [ ] has shown that clustering of fibroblasts induces chemokine (ccl - , cxcl - and cxcl ) secretion in five different fibroblast cell lines cultured in agarose. in addition, fischbach et al. [ ] cultured tumor cells in a d and d rgd-alginate system and reported a dramatic increase in il- levels; however, no significant vegf differences were reported between d and d cultures (fig. ). in the same study, cells grown in alginate gels having rgd peptides (spatial, biophysical and biochemical cues) produced a higher level of il- compared with those lacking the adhesion peptide (only spatial and biophysical cues), although both produced higher levels when compared with d (lacking all three cues). this shows that changes in the microenvironment are conveyed directly by the difference in the level of cytokine pro- duction. in another study by the same group, the same cells also showed an upregulation of cytokines when grown in matrigel (lrbm) com- pared with their d counterparts. this compar- ison is particularly important as cells grown on matrigel have already been shown to produce a cpr outcome (formation of mammary gland acinus and milk-like secretions into the lumen) [ ], which mean that matrigel provides the relevant microenvironmental cues. taken together, these studies suggest that the upre- gulation of cytokines in d compared with d cultures is not a random differential response but a potentially ubiquitous biomarker for three- dimensionality. further studies to establish this view firmly are needed. cytokines are particularly attractive as bio- markers for several practical reasons. first, they are secreted in the media, making it easy for their detection to be amenable for hts readout. second, they are expressed in a wide range of cells from the four tissue types (muscle, con- nective, epithelial and nerve), suggesting the potential for their ubiquity as opposed to being cell or tissue specific. third, their temporal expression suggests the use of profiles as opposed to single-time measurements, which increases their robustness as biomarkers. concluding remarks simplifying d cell culture platforms for hts applications calls for the identification and validation of ubiquitous three-dimensionality biomarkers. our review of comparative tran- scriptomic and proteomic literature strongly suggests the cytokine family as having the greatest potential to yield compounds that can serve as three-dimensionality biomarkers. how- ever, narrowing the candidates to a practical few still remains to be done. references weaver, v.m. et al. 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( ) constitutive p -activated kinase (pak) activation in breast cancer cells as a result of mislocalization of pak to focal adhesions. mol. biol. cell , – yinzhi lai, amish asthana, william s. kisaalita cellular bioengineering laboratory, faculty of engineering, driftmier engineering center, university of georgia, athens, ga , usa www.drugdiscoverytoday.com p e rs p e ct iv 	biomarkers for simplifying hts d cell culture platforms for drug discovery: the case for cytokines 	introduction 	microenvironmental cues 	searching for three-dimensionality biomarkers 	concluding remarks 	references genome-wide association study for coronary artery calcification with follow-up in myocardial infarction genetics genome-wide association study for coronary artery calcification with follow-up in myocardial infarction christopher j. o’donnell, md, mph*; maryam kavousi, md, msc*; albert v. smith, phd*; sharon l.r. kardia, phd*; mary f. feitosa, phd; shih-jen hwang, phd; yan v. sun, phd, ms; michael a. province, phd; thor aspelund, phd; abbas dehghan, md, phd; udo hoffmann, md, mph; lawrence f. bielak, dds, mph; qunyuan zhang, phd; gudny eiriksdottir, msc; cornelia m. van duijn, phd; caroline s. fox, md, mph; mariza de andrade, phd; aldi t. kraja, phd; sigurdur sigurdsson, msc; suzette e. elias-smale, md; joanne m. murabito, md, scm; lenore j. launer, phd; aad van der lugt, md, phd; sekar kathiresan, md; the cardiogram consortium; gabriel p. krestin, md, phd; david m. herrington, md, mhs; timothy d. howard, phd; yongmei liu, md, phd; wendy post, md, ms; braxton d. mitchell, phd; jeffrey r. o’connell, phd; haiqing shen, phd; alan r. shuldiner, md; david altshuler, md, phd; roberto elosua, md, phd; veikko salomaa, md, phd; stephen m. schwartz, phd; david s. siscovick, md, mph; benjamin f. voight, phd; joshua c. bis, phd; nicole l. glazer, phd, mph; bruce m. psaty, md, phd; eric boerwinkle, phd; gerardo heiss, md, phd; stefan blankenberg, md; tanja zeller, phd; philipp s. wild, md; renate b. schnabel, md; arne schillert, phd; andreas ziegler, dr rer nat habil dipl-stat; thomas f. münzel, md; charles c. white, phd; jerome i. rotter, md; michael nalls, phd; matthijs oudkerk, md, phd; andrew d. johnson, phd; anne b. newman, md, mph; andre g. uitterlinden, md, phd; joseph m. massaro, phd; julie cunningham, phd; tamara b. harris, md, ms*; albert hofman, md, phd*; patricia a. peyser, phd*; ingrid b. borecki, phd*; l. adrienne cupples, phd*; vilmundur gudnason, md, phd*; jacqueline c.m. witteman, phd* background—coronary artery calcification (cac) detected by computed tomography is a noninvasive measure of coronary atherosclerosis, which underlies most cases of myocardial infarction (mi). we sought to identify common genetic variants associated with cac and further investigate their associations with mi. methods and results—computed tomography was used to assess quantity of cac. a meta-analysis of genome-wide association studies for cac was performed in men and women from independent community-based cohorts, with replication in additional independent cohorts (n� ). we examined the top single-nucleotide polymorphisms (snps) associated with cac quantity for association with mi in multiple large genome-wide association studies of mi. genome-wide significant associations with cac for snps on chromosome p near cdkn a and cdkn b (top snp: rs ; p� . � � ) and p (top snp: rs , within the phactr gene; p� . � � ) replicated for cac and for mi. additionally, there is evidence for concordance of snp associations with both cac and mi at a number of other loci, including q (mras gene), q (col a /col a genes), and p (sort gene). conclusions—snps in the p and phactr gene loci were strongly associated with cac and mi, and there are suggestive associations with both cac and mi of snps in additional loci. multiple genetic loci are associated with development of both underlying coronary atherosclerosis and clinical events. (circulation. ; : - .) key words: cardiac computed tomography � coronary artery calcification � coronary atherosclerosis � genome-wide association studies � myocardial infarction received august , ; accepted august , . a list of the institutions and affiliations for the authors of this report may be found in the appendix at the end of this article. *drs o’donnell, kavousi, smith, kardia, harris, hofman, peyser, borecki, cupples, gudnason, and witteman contributed equally to this work. guest editor for this article was gregory y.h. lip, md. the online-only data supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi: . /circulationaha. . /-/dc . correspondence to christopher j. o’donnell, md, mph, nhlbi’s framingham heart study, mount wayte ave, suite , framingham, ma . e-mail odonnellc@nhlbi.nih.gov; and patricia a. peyser, phd, school of public health, university of michigan, washington heights, no. , ann arbor, mi . e-mail ppeyser@umich.edu © american heart association, inc. circulation is available at http://circ.ahajournals.org doi: . /circulationaha. . d ow nloaded from http://ahajournals.org by on a pril , http://circ.ahajournals.org/lookup/suppl/doi: . /circulationaha. . /-/dc http://circ.ahajournals.org/lookup/suppl/doi: . /circulationaha. . /-/dc atherosclerosis in coronary arteries underlies mostcases of myocardial infarction (mi) and other clinical coronary heart diseases (chd). chd comprises the lead- ing cause of death in western countries. extent of coronary atherosclerosis may be determined with noninva- sive, high-resolution computed tomography (ct) to mea- sure coronary artery calcification (cac). cac quantity is heritable, significantly higher in people with a parental history of chd, correlates with increased burden of subclinical coronary plaque, and predicts incident chd in multiple ethnic populations after adjustment for other traditional chd risk factors. , , clinical perspective on p genome-wide association (gwa) studies identified common genetic variations influencing risk of mi, includ- ing a strongly replicated association on chromosome p , – as well as strong associations with phactr and � other loci. , recently, a large meta-analysis of gwa studies for coronary disease phenotypes in the coronary artery disease genome wide replication and meta-analysis (cardiogram) consortium reported a total of loci including the previously known loci for mi. neither chromosome p nor any other locus associated with chd has been shown to be associated with the cac quantity at a genome-wide significance level. we conducted a meta-analysis to identify loci underlying variation in extent of cac. we further assessed whether single-nucleotide polymorphisms (snps) associated with cac quantity were also associated with mi and whether snps previously shown to be associated with mi are associated with cac quantity. methods setting we conducted a meta-analysis of gwa data in participants of european ancestry from large cohorts. the study was performed in the cohorts for heart and aging research in genomic epidemiology (charge) consortium including data from the age, gene/ environment susceptibility–reykjavik study (ages-reykjavik), , the framingham heart study (fhs), – the rotterdam study i (rs i), and the rotterdam study ii (rs ii). , in addition, participants from the genetic epidemiology network of arteriopathy study (genoa) were included. each study received institutional review board ap- proval, and all participants gave written informed consent. these cohorts are described in the online-only data supplement. measures cac measurement different cohorts used different ct scanners to assess cac, as described in the online-only data supplement. total calcium score, based on the sum of the individual coronary arteries (left main, left anterior descending, circumflex, and right coronary arteries), was quantified by the agatston method and used as cac quantity in analyses. prior studies by others confirm the highly significant association between cac scores obtained by the different scanners used in the present study. , because the scanning techniques yield similar results and the available evidence for prediction of cvd risk is similar regardless of use of electron beam ct or multidetector ct, consensus clinical guidelines recommending clin- ical use of ct allow for the use of either electron beam ct or multidetector ct. genotyping and imputation different discovery cohorts used different genotyping platforms: illu- mina cnv for ages-reykjavik, affymetrixhuman k and gene-centric k for fhs, illumina k version for rs i and ii, and affymetrix . for genoa. each study imputed genotype data to . million nonmonomorphic, autosomal snps with the use of hapmap haplotypes (ceu population, release , build ) with the imputation software mach (http://www.sph.umich.edu/csg/abecasis/mach/) and snps that passed quality control criteria (described in the online-only data supplement). all studies imputed genotype dosage, from to , which is the expected number of alleles. extensive quality control analyses were performed as described in the online-only data supplement. statistical analyses we conducted gwa analyses in each discovery cohort independently. each study evaluated population substructure in their cohort, primarily with the use of principal components from eigenstrat. (http://gene path.med.harvard.edu/�reich/eigenstrat.htm). to reduce nonnor- mality, total cac score was natural log transformed after adding and was adjusted for age and sex variation. data were analyzed with the use of linear regression in ages-reykjavik and rs i and rs ii and with linear mixed-effects models to account for family covariance structure in fhs and genoa with an additive genetic model. fixed-effects meta-analysis was conducted with the inverse variance weighted ap- proach in metal (http://www.sph.umich.edu/csg/abecasis/metal). genomic control was applied to each cohort before meta-analysis. the inflation of the association test statistic, �gc, was as follows: . for ages-reykjavik, . for fhs, . for genoa, . for rs i, and . for rs ii. tests for homogeneity of observed effect sizes across cohorts were conducted with the use of metal. the analyses were repeated with further adjustment for total cholesterol, high-density lipoprotein cholesterol, type diabetes mellitus, hypertension status, current smoking, and use of statins. the analyses were also repeated with exclusion of individuals with a prior mi. the association of each snp with cac score � versus � , a threshold that is an independent predictor of clinical events, was also investigated in each discovery cohort. details of the analyses are found in the online-only data supplement. the a priori threshold for genome-wide significance was � � , and a p value � � � but � � � was considered moderate evidence for association. with a sample size of , a minor allele frequency of . , an additive model with a mean of and sd of [mean and sd similar to residuals from log(cac score� ) adjusted for age and sex], and � at � � , we had at least % power to detect a �-coefficient of � . or �� . in analysis of cac quantity. replication replication cohorts included participants from the family heart study, the multi-ethnic study of atherosclerosis (mesa), and the amish family calcification study. details about these cohorts, cac measures, genotyping, and statistical analysis are provided in the online-only data supplement. meta-analysis was repeated for the replication cohorts alone and then for the discovery and replication cohorts combined. association with mi to test for association with mi, we chose a set of snps associated with cac quantity, at a p value of � � , that were nonredundant at a linkage disequilibrium threshold of r � . among the hapmap ceu using snap. the studies, including partici- pants from studies independent of the charge consortium, were the heart and vascular health (hvh) study, myocardial infarction genetics consortium (migen), gutenberg heart study/athero- gene study (cadomics), and charge consortium. these studies, including participants ( with mi), are described in tables iia and iib in the online-only data supplement. a bonferroni-adjusted p value of . � � was used as the threshold for significance. circulation december / , d ow nloaded from http://ahajournals.org by on a pril , http://www.sph.umich.edu/csg/abecasis/mach/ http://genepath.med.harvard.edu/≈reich/eigenstrat.htm http://genepath.med.harvard.edu/≈reich/eigenstrat.htm http://www.sph.umich.edu/csg/abecasis/metal results cac discovery characteristics of the discovery cohorts are presented in table . the cohort differences in the distribution of cac reflect the cohort differences in the age and sex distributions. figure provides a plot of the meta-analysis p values by chromosome position. forty-eight snps located on chromo- some p near cdkn b and cdkn a and snp on chromosome p attained genome-wide significance. table lists the genome-wide significant snps as well as those considered moderately associated with cac quantity. figure a provides a plot of meta-analysis p values for snps near rs , the variant most strongly associated with cac quantity in our meta-analysis as well as with chd in previously reported gwa study results. the strong association on p in the phactr gene represents a new finding for cac. the most strongly associated snps, rs and rs , are modestly correlated (r � . ) and reside bp apart (table ). figure b provides a plot of meta-analysis p values for snps near rs . figure ia and ib in the online-only data supplement provides plots of observed �log (p) versus expected �log (p) with and without the genome-wide significantly associated snps in the analysis. table presents results for snps in other loci—col a / col a , serpini, hal, cdc , and irs —moderately asso- ciated with cac quantity. table iii in the online-only data supplement shows the results for the most strongly associated snps in each individual cohort, and table iv in the online-only data supplement presents results of all snp associations for cac quantity with p� � � from the meta-analysis, the associations when those with prior mi are excluded, and the results of tests for homogeneity. the results are similar when those with prior mi are excluded, and there is no evidence for significant heterogeneity. none of the snps in table or table iv in the online-only data supplement have a p� . for the test for snp heterogeneity. table . baseline participant characteristics of the discovery cohorts in the meta-analysis of genome-wide association studies of coronary artery calcification characteristic ages-reykjavik (n� ) fhs (n� ) genoa (n� ) rs i (n� ) rs ii (n� ) years of cac measurements – – – – – scanner type mdct, detector mdct, detector ebct (c- ) ebct (c- ) mdct, or detector age, y . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) women, % mean cac score ( ) ( ) ( ) ( ) ( ) maximum cac score detectable cac, % . . . . . cac score � , % . . . . . cac score � , % . . . . . hypertension, % . . . . . diabetes mellitus, % . . . . . current cigarette smoker, % . . . . . former cigarette smoker, % . . . . . total cholesterol, mmol/l . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) hdl cholesterol, mmol/l . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) triglycerides, mmol/l . ( . ) . ( . ) . ( . ) . ( . ) na body mass index, kg/m . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) waist circumference, cm ( ) ( ) ( ) ( ) ( ) prevalent mi, % . . . . values are mean (sd) or no. (%). ages-reykjavik indicates age, gene/environment susceptibility study–reykjavik; fhs, framingham heart study; genoa, genetic epidemiology network of arteriopathy; rs, rotterdam study; cac, coronary artery calcification; hdl, high-density lipoprotein; mi, myocardial infarction; mdct, multidetector computed tomography; ebct, electron beam computed tomography; and na, not available. figure . plot of �log (p) for association of single-nucleotide polymorphisms and chromosomal position for all autosomal single-nucleotide polymorphisms analyzed in the age- and sex- adjusted model of coronary artery calcification quantity in the meta-analysis of independent discovery cohorts. o’donnell et al genomics of coronary artery calcification d ow nloaded from http://ahajournals.org by on a pril , the results for snp associations were also similar after adjustment for additional chd risk factors in addition to age and sex (data not shown). in addition, inferences for the cac threshold of were similar to those for cac quantity (table v in the online-only data supplement). cac replication and association with mi table presents the results in the replication cohorts for the snps with the strongest association in the discovery cohorts. the meta-analysis p value for the combined discov- ery and replication cohorts remained genome-wide signifi- cant and lower than the discovery p value for rs and rs and moderately associated (p� � � ) for rs and rs (col a /col a ). table presents association of selected snps with mi. in addition to known associations near cdkn b and phactr with mi, several loci recently reported to be associated with mi, , including rs (intronic snp in col a ), rs (nearest celsr and also near sort ), and rs (near mras), had concordant associations (same direction of effect for same allele) with both cac and mi in our meta-analysis, although the associations were not below a bonferroni-adjusted p value of . � � (table ). when we examined the association of the same snps in the gwa study results from the cardiogram consortium, a total of of snps were associated with coronary disease after bonferroni adjustment, including the aforementioned snps as well as rs (in col a ), rs (near psrc ), and rs (in mras); for the snps, the direction of effect was the same for cac and coronary disease (table vi in the online-only data supplement). twelve of snps strongly associated with mi in recent gwa studies were available in the discovery cohorts (table ). in addition to the known associations with snps in p and p , other loci—cxcl , mras, lpa, and celsr / psrc /sort —were associated with cac quantity (after bonferroni adjustment for tests). when we examined the association of the snps in the cardiogram consor- tium that are significantly associated with coronary disease and available in our discovery gwa studies for cac, a total of of snps were associated with cac after bonferroni adjustment, with the same direction of effect for cac and coronary disease for the snps (table vii in the online-only data supplement). discussion our replicated cac association of rs on chromo- some p and our novel replicated associations of rs on p (the phactr locus), as well as the strong evidence for association in the combined discovery and replication cohorts for rs , provide evidence that these loci contribute to variation in coronary atherosclerotic calcified plaque. although both loci have been associated with mi and now with cac, the specific causal variants have not been elucidated for either locus, and our gwa study is not able to provide evidence for causal association with nearby genes. snps in p reside � bp away from and are poorly correlated (ie, in low linkage disequilibrium) with the nearest protein-coding genes, the cell cyclin- dependent kinase inhibitors ckdn b and cdkn a. snps associated with both cac and mi overlap with the upstream portion of a newly annotated antisense noncoding rna (also known as anril or dq ). the mechanism of asso- ciation of anril is not known; however, the strength and consistency of snp associations with cac and mi strongly suggest involvement in formation of calcified atherosclerotic lesions in coronary arteries. a recent report implicates the p risk interval in regu- lation of cardiac cdkn a/b expression, suggesting that this region may affect atherosclerosis progression via vascular cell proliferation. of note, snps in this region are also strongly associated with abdominal aortic aneurysms , and vascular stiffness but not with carotid intimal-medial wall thickness determined by ultrasonography. , our newly identified association between cac and phactr snps, similar to the association between mi and these snps, may represent a novel pathway for development of clinical atherosclerosis. phactr in p is an inhibitor table . top snp association results for coronary artery calcification quantity in the meta-analysis of discovery cohorts snp chromosome position closest reference gene* distance from nearest transcript† �-coefficient se p coded allele frequency coded allele noncoded allele rs ‡ (cdkn b) . . . � � . c g rs § phactr � . . . � � . a g rs § phactr . . . � � . t c rs col a . . . � � . a g rs serpini � . . . � � . t c rs hal � . . . � � . a g rs (cdc ) . . . � � . c g rs (irs ) � . . . � � . t g snp indicates single-nucleotide polymorphism. *genes for snps that are outside the transcript boundary of the protein-coding gene are shown in parentheses [eg, (cdkn b)]. †distance in base pairs from nearest start or stop site for transcription. ‡an additional snps with p� . � � were located near rs in a -bp region of chromosome between position and . see table iii in the online-only data supplement. §snps rs and rs reside bp apart and are only moderately correlated (r � . , d�� . ). circulation december / , d ow nloaded from http://ahajournals.org by on a pril , of protein phosphatase , a ubiquitous serine/threonine phos- phatase known to regulate multiple cellular processes through dephosphorylation of different substrates. the exact mech- anism of association of this locus or pathway with cac and mi is unknown. in addition, snps on q . near mras, on p near celsr /psrc /sort , and in cxcl and lpa showed concordant association with cac and mi, although the cac associations did not reach genome-wide signifi- cance. , the m-ras protein encoded by mras ( q ) belongs to the ras superfamily of gtp-binding proteins and is expressed in the heart and aorta and may be involved in adhesion signaling, which is important in the atherosclerotic process. the locus containing the celsr (cadherin egf lag seven-pass g-type receptor ) gene also harbors psrc and sort . in recent very large gwa studies, as in prior smaller gwa studies, snps in this locus were shown to be associated with mi and with circulating low-density lipoprotein cholesterol levels. in previous work, rs , which is highly correlated with rs (d�� . ), was strongly associated with human liver transcript concentrations of the nearby genes sort , celsr , and psrc . in our cac meta-analysis, rs had the same direction of effect with continuous cac (p� . � � ) and with cac � (p� . � � ) as reported for mi (data not shown). a recent investiga- tion, incorporating both mouse and human models, dem- onstrated that a nearby common noncoding polymorphism at the p locus (rs , d�� . ) that serves as a proxy for rs directly implicates hepatic sort gene expression in the causal mechanism of altered plasma low-density lipoprotein and risk for mi. these findings are consistent with a genetic component to the association of low-density lipoprotein cholesterol levels with both cac and mi. a new and potentially interesting finding is the concordant association with snps in the col a /col a locus at figure . observed �log(p) and recombi- nation rates by chromosomal position for all associated single-nucleotide polymor- phisms near rs near cdkn b on p . (a) and rs near phactr on p (b). results from the genome- wide association analysis of single-nucleo- tide polymorphisms vs age- and sex- adjusted coronary artery calcification quantity in the meta-analysis of indepen- dent discovery cohorts are shown. associ- ation plots were conducted with the use of snap. top single-nucleotide polymor- phisms of interest and p values in each region are indicated (blue diamonds). color coding indicates the strength of link- age disequilibrium of each single-nucleo- tide polymorphism with the top single-nu- cleotide polymorphism in each region: red (r � . ), orange (r � . ), yellow (r � . ), and white with no color (r � . ). o’donnell et al genomics of coronary artery calcification d ow nloaded from http://ahajournals.org by on a pril , q . although not reaching genome-wide significance, the region was moderately associated with both cac and clini- cally apparent mi. importantly, col a /col a is a new gene identified in the cardiogram consortium. type iv collagen is a basement-membrane collagen that does not form ordered fibrillar structures but instead forms mole- cules cross-linked together at the ends. the col a and col a genes are transcribed divergently by the same promoter. mutations in col a have been implicated in several rare familial conditions including porencephaly, man- ifested by cystic cavities of the brain ; small arterial vessel disease and cerebral hemorrhage ; and a syndrome including hereditary angiopathy, nephropathy, and aneurysms. a recent gwa study for vascular stiffness measures reported a strong replicated association of snps in col a with arterial stiffness. a prior candidate gene case-control study in japan provided the first unreplicated report of an associa- tion of snps in col a with mi at a modest level of table . association of top coronary artery calcification quantity snps in the replication panel of cohorts and combined with the discovery cohorts snp chromosome position closest reference gene* distance from nearest transcript† coded allele replication discovery�replication‡ �-coefficient (se) p �-coefficient (se) p rs (cdkn b) c . ( . ) . � � . ( . ) . � � § rs phactr a � . ( . ) . � � � . ( . ) . � � § rs phactr t . ( . ) . � � . ( . ) . � � § rs col a a . ( . ) . � � . ( . ) . � � rs serpini t . ( . ) . � � � . ( . ) . � � rs hal a � . ( . ) . � � � . ( . ) . � � rs (cdc ) c . ( . ) . � � . ( . ) . � � rs (irs ) t � . ( . ) . � � � . ( . ) . � � *genes for single-nucleotide polymorphisms (snps) that are outside the transcript boundary of the protein-coding gene are shown in parentheses [eg, (cdkn b)]. †distance in base pairs from nearest start or stop site for transcription. ‡§combined p values marked with § are below the genome-wide threshold; other combined p values are not significant at a genome-wide level. table . association of top coronary artery calcification quantity snps with myocardial infarction in a meta-analysis of studies snp chromosome position closest reference gene* distance from nearest transcript† �- coefficient se p coded allele frequency coded allele noncoded allele associations with p� . � � rs (cdkn b) � . . . � � . c g rs ‡ phactr � . . . � � . a g associations with . � � �p� � � rs col a . . . � � . a t rs celsr . . . � � . a t rs § mras � . . . � � . t g rs col a . . . � � . a g rs hdac . . . � � . t c rs adcy � . . . � � . a c rs (psrc ) . . . � � . a g rs � (dkfzp n ) � . . . � � . a g rs § mras . . . � � . c g rs ¶ dkfzp e � . . . � � . a g rs (ppic) � . . . � � . t c rs (ell ) . . . � � . a t rs ‡ phactr . . . � � . t c rs (sorcs ) . . . � � . t c *genes for single-nucleotide polymorphisms (snps) that are outside the transcript boundary of the protein-coding gene are shown in parentheses [eg, (cdkn b)]. †distance in base pairs from nearest start or stop site for transcription. ‡snps rs and rs reside bp apart (r � . , d�� . ). §snps rs and rs reside bp apart (r � . , d�� ). �hypothetical protein loc . ¶hypothetical protein loc . circulation december / , d ow nloaded from http://ahajournals.org by on a pril , significance (p� . ). although to date there have been no reports of common variants in the col a /col a locus having genome-wide significant associations with cac, our findings for cac taken together with findings from the cardiogram consortium provide evidence in support of a role for variants in this locus in both coronary atherosclerosis and clinically apparent mi. not all individuals with high cac quantity develop mi, and some cases of mi occur in the absence of high levels of cac. , , in comparing snps for mi with the cac gwa, we note that there is evidence for association of other snp reported to be associated with mi, rs near cxcl on q . (p� . � � in our cac gwa meta-analysis). in contrast, there was no evidence for association with cac quantity of several other snps associated with mi, including snps on q . near wdr , p . near ldlr, q . near kcne , p . near pcsk , q . near sh b , and q . near hfn a (table ). rs on q near mia was not assessed in our meta-analysis because it is not localized in hapmap, although there are nonsignificant associations with cac quantity of snps within a -bp interval (eg, rs ; p� . ). when we examined the association of the snps in the cardio- gram consortium that are significantly associated with coronary disease and available in our discovery gwa studies for cac, a total of of snps were associated with cac after bonferroni adjustment, including snps in p and p as well as snps near cxcl , sort , mras, col a /col a , and adamts ; the remainder of the snps were not significantly associated with cac quantity (table vii in the online-only data supplement). the absence of a genetic association may be due to limited power or confounding factors. however, the fact that some loci are risk factors for both cac and cad and others are not raises the possibility that some but not all genetic mechanisms for cad are strongly related to the presence and burden of coronary atherosclerosis. it is notable that a recent report of a gwa study for coronary atherosclerosis detected by angiography in selected cases of diseased patients reported novel genome-wide associations with adamts and abo and also reported concordance of association with several other loci including p , phactr , mras, and sort . strengths of the present study include data from large community-based studies, similarity in cac assessment and quality control measures across cohorts, and similarity in imputation strategies and analytical methods. limita- tions include heterogeneity in the cohorts and differences in actual ct scanners. moreover, we have modest statis- tical power to detect associations with low-frequency snps or poorly imputed snps. this limitation in power may have affected our ability to identify additional snps associated with both cac and mi. examination of larger cohorts with cac may detect more genome-wide signifi- cant associations. in addition, identified associations with genomic regions require further follow-up studies to es- tablish the actual functional variants and elucidate actual mechanisms of association. finally, we cannot generalize our findings to individuals of non-european ancestry. the associations of snps in p and p in the discov- ery and replication studies for cac and in the mi studies, as well as the association with snps in or near other genes including mras, col a /col a , and sort , suggest that the common mechanism of some genetic loci underlying mi is development of early, underlying coronary atherosclerosis. investigations to understand mechanisms underlying the ge- netic associations with coronary atherosclerosis may ulti- mately suggest new strategies for prediction, prevention, and treatment of chd. table . association results for coronary artery calcification quantity for snps with strong association with premature-onset mi snps associated with early mi in past gwa studies snps associated with cac in the meta-analysis of discovery cohorts cac (continuous) snp chromosome position gene(s) risk allele mi gwa study p �- coefficient se p rs cdkn a, cdkn b g . � � . . . � � rs phactr c . � � . . . � � rs cxcl c . � � . . . � � rs mras t . � � . . . � � rs lpa g . � � . . . � � rs celsr , psrc , sort t . � � . . . � � rs wdr c . � � . . . rs ldlr g . � � . . . rs slc a , mrps , kcne t . � � . . . rs pcsk t . � � . . . rs sh b t . � � � . . . rs hnf a t . � � � . . . snp indicates single-nucleotide polymorphism; mi, myocardial infarction; gwa, genome-wide association; and cac, coronary artery calcification. o’donnell et al genomics of coronary artery calcification d ow nloaded from http://ahajournals.org by on a pril , appendix the following is a list of institutional affiliations for the authors of this article: from the cardiology division, department of medicine, massa- chusetts general hospital, harvard medical school, boston, ma (c.j.o’d., s.k.); national heart, lung, and blood institute’s fra- mingham heart study, framingham, ma (c.j.o’d., s.h., c.s.f., j.m. murabito, a.d.j., j.m. massaro, l.a.c.); national heart, lung and blood institute, bethesda, md (c.j.o’d., s.h., c.s.f., a.d.j.); department of epidemiology, erasmus medical center, rotterdam, netherlands (m.k., a.d., c.m.v.d., a.g.u., s.e.e.-s., a.h., j.c.m.w.); department of radiology, erasmus university medical center, rotterdam, netherlands (s.e.e.-s.); netherlands genomics initiative-sponsored netherlands consortium for healthy aging, rotterdam, netherlands (m.k., a.d., c.m.v.d., a.g.u., a.h., j.c.m.w.); icelandic heart association, kopavogur iceland (a.v.s., t.a., g.e., s.s., v.g.); department of epidemiology, school of public health, university of michigan, ann arbor (s.l.r.k., y.v.s., l.f.b., p.a.p.); division of statistical genomics, depart- ment of genetics, washington university school of medicine, st. louis, mo (m.f.f., m.a.p., q.z., a.t.k., i.b.b.); university of iceland, reykjavik, iceland (t.a., v.g.); department of radiology, massachusetts general hospital, harvard medical school, boston, ma (u.h.); division of endocrinology, metabolism, and diabetes, department of medicine, brigham and women’s hospital, harvard medical school, boston, ma (c.s.f.); division of biomedical statistics and informatics, mayo clinic, rochester, mn (m.d.a.); boston university school of medicine, boston, ma (j.m. mu- rabito); laboratory of epidemiology, demography, and biometry, intramural research program, national institute on aging, national institutes of health, bethesda, md (l.l., t.b.h.); department of radiology, erasmus medical center, rotterdam, netherlands (a.v.d.l., g.p.k.); program in medical and population genetics, broad institute of harvard and massachusetts institute of technol- ogy, cambridge, ma (s.k., d.a., b.f.v.); wake forest university school medicine, winston-salem, nc (d.m.h., t.d.h., y.l.); department of medicine and epidemiology, johns hopkins hospi- tal, baltimore, md (w.p.); university of maryland school of medicine, baltimore (b.d.m., j.r.o’c., h.s., a.r.s.); veterans administration medical center, baltimore, md (a.r.s.); center for human genetic research, massachusetts general hospital, boston, ma (d.a., b.f.v.); cardiovascular epidemiology and genetics, institut municipal d’investigacio medica, and ciber epidemiolo- gia y salud publica, barcelona, spain (r.e.); chronic disease epidemiology unit, department of health promotion and chronic disease prevention, national public health institute, helsinki, fin- land (v.s.); cardiovascular health research unit, departments of medicine and epidemiology, university of washington, seattle (s.m.s., d.s.s., j.c.b., n.l.g.); departments of epidemiology, medicine, and health services, university of washington, seattle (b.m.p.); university of texas health science center, human ge- netics center and institute of molecular medicine, houston (e.b.); school of public health, university of north carolina at chapel hill (g.h.); department of medicine ii, university medical center mainz, mainz, germany (s.b., t.z., p.s.w., r.b.s., t.f.m.); institut für medizinische biometrie und statistik, universitüt zu lübeck, universitätsklinikum schleswig-holstein, campus lübeck, lübeck, germany (a.s., a.z.); department of biostatistics, boston university school of public health, boston, ma (c.c.w., l.a.c.); medical genetics institute, cedars-sinai medical center, los ange- les, ca (j.i.r.). laboratory of neurogenetics, intramural research program, national institute on aging, national institutes of health, bethesda, md (m.n.); center for medical imaging–north east netherlands, department of radiology, university medical center groningen, university of groningen, groningen, netherlands (m.o.); center for aging and population health, department of epidemiology, university of pittsburgh, pittsburgh, pa (a.b.n.); department of internal medicine, erasmus medical center, rotter- dam, netherlands (a.g.u.); department of biostatistics, boston university, boston, ma (j.m. massaro); and genotyping shared resource, mayo clinic college of medicine, rochester, mn (j.c.). acknowledgments see the online-only data supplement. disclosures dr ziegler reports receiving honoraria and ownership interest �$ k. drs rotter, murabito, borecki, bielak, kardia, peyser, and witteman report receiving government research grants relevant to the research topic. the other authors report no conflicts. references . rosamond w, flegal k, furie k, go a, greenlund k, haase n, hailpern sm, ho m, howard v, kissela b, kittner s, lloyd-jones d, mcdermott m, meigs j, moy c, nichol g, o’donnell c, roger v, sorlie p, stein- berger j, thom t, wilson m, hong y. heart disease and stroke statis- tics— update: a report from the american heart association sta- tistics committee and stroke statistics subcommittee. circulation. ; 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: – . . teslovich tm, musunuru k, smith av, edmondson ac, stylianou im, koseki m, pirruccello jp, ripatti s, chasman di, willer cj, johansen ct, fouchier sw, isaacs a, peloso gm, barbalic m, ricketts sl, bis jc, o’donnell et al genomics of coronary artery calcification d ow nloaded from http://ahajournals.org by on a pril , aulchenko ys, thorleifsson g, feitosa mf, chambers j, orho-melander m, melander o, johnson t, li x, guo x, li m, shin cy, jin gm, jin ky, lee jy, park t, kim k, sim x, twee-hee or, croteau-chonka dc, lange la, smith jd, song k, hua zj, yuan x, luan j, lamina c, ziegler a, zhang w, zee ry, wright af, witteman jc, wilson jf, willemsen g, wichmann he, whitfield jb, waterworth dm, wareham nj, waeber g, vollenweider p, voight bf, vitart v, uitterlinden ag, uda m, tuomilehto j, thompson jr, tanaka t, surakka i, stringham hm, spector td, soranzo n, smit jh, sinisalo j, silander k, sijbrands ej, scuteri a, scott j, schlessinger d, sanna s, salomaa v, saharinen j, sabatti c, ruokonen a, rudan i, rose lm, roberts r, rieder m, psaty bm, pramstaller pp, pichler i, perola m, penninx bw, pedersen nl, pattaro c, parker an, pare g, oostra ba, o’donnell cj, nieminen ms, nickerson da, montgomery gw, meitinger t, mcpherson r, mccarthy mi, mcardle w, masson d, martin ng, marroni f, mangino m, mag- nusson pk, lucas g, luben r, loos rj, lokki ml, lettre g, langenberg c, launer lj, lakatta eg, laaksonen r, kyvik ko, kronenberg f, konig ir, khaw kt, kaprio j, kaplan lm, johansson a, jarvelin mr, cecile jwj, ingelsson e, igl w, kees hg, hottenga jj, hofman a, hicks aa, hengstenberg c, heid im, hayward c, havulinna as, hastie nd, harris tb, haritunians t, hall as, gyllensten u, guiducci c, groop lc, gonzalez e, gieger c, freimer nb, ferrucci l, erdmann j, elliott p, ejebe kg, doring a, dominiczak af, demissie s, deloukas p, de geus ej, de fu, crawford g, collins fs, chen yd, caulfield mj, campbell h, burtt np, bonnycastle ll, boomsma di, boekholdt sm, bergman rn, barroso i, bandinelli s, ballantyne cm, assimes tl, quertermous t, altshuler d, seielstad m, wong ty, tai es, feranil ab, kuzawa cw, adair ls, taylor ha jr, borecki ib, gabriel sb, wilson jg, holm h, thorsteinsdottir u, gudnason v, krauss rm, mohlke kl, ordovas jm, munroe pb, kooner js, tall ar, hegele ra, kastelein jj, schadt ee, rotter ji, boerwinkle e, strachan dp, mooser v, stefansson k, reilly mp, samani nj, schunkert h, cupples la, sandhu ms, ridker pm, rader dj, van duijn cm, peltonen l, abecasis gr, boehnke m, kathiresan s. biological, clinical and population relevance of loci for blood lipids. nature. ; 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: – . . gould db, phalan fc, breedveld gj, van mil se, smith rs, schimenti jc, aguglia u, van der knaap ms, heutink p, john sw. mutations in col a cause perinatal cerebral hemorrhage and porencephaly. science. ; : – . . gould db, phalan fc, van mil se, sundberg jp, vahedi k, massin p, bousser mg, heutink p, miner jh, tournier-lasserve e, john sw. role of col a in small-vessel disease and hemorrhagic stroke. n engl j med. ; : – . . plaisier e, gribouval o, alamowitch s, mougenot b, prost c, verpont mc, marro b, desmettre t, cohen sy, roullet e, dracon m, fardeau m, van agtmael t, kerjaschki d, antignac c, ronco p. col a mutations and hereditary angiopathy, nephropathy, aneurysms, and muscle cramps. n engl j med. ; : – . . tarasov kv, sanna s, scuteri a, strait jb, orru m, parsa a, lin pi, maschio a, lai s, piras mg, masala m, tanaka t, post w, o’connell jr, schlessinger d, cao a, nagaraja r, mitchell bd, abecasis gr, shuldiner ar, uda m, lakatta eg, najjar ss. col a is associated with arterial stiffness by genome-wide association scan. circ cardiovasc genet. ; : – . . yamada y, kato k, oguri m, fujimaki t, yokoi k, matsuo h, watanabe s, metoki n, yoshida h, satoh k, ichihara s, aoyagi y, yasunaga a, park h, tanaka m, nozawa y. genetic risk for myocardial infarction determined by polymorphisms of candidate genes in a japanese popu- lation. j med genet. ; : – . . reilly mp, li m, he j, ferguson jf, stylianou im, mehta nn, burnett ms, devaney jm, knouff cw, thompson jr, horne bd, stewart af, assimes tl, wild ps, allayee h, nitschke pl, patel rs, martinelli n, girelli d, quyyumi aa, anderson jl, erdmann j, hall as, schunkert h, quertermous t, blankenberg s, hazen sl, roberts r, kathiresan s, samani nj, epstein se, rader dj. identification of adamts as a novel locus for coronary atherosclerosis and association of abo with myo- cardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies. lancet. ; : – . clinical perspective coronary artery calcification, detected by computed tomography, is a noninvasive measure of coronary atherosclerosis and an independent predictor of myocardial infarction. we conducted a genome-wide association study with a discovery sample of nearly participants of european original and a replication sample of � participants of european origin. we report strong evidence for genetic variants in p near the cdkn b and cdkn a genes and in p within the phactr gene. additionally, we found evidence for concordance of single-nucleotide polymorphism associations with both coronary artery calcification and some but not all other loci known to be associated with myocardial infarction, including q (mras gene), q (col a /col a genes), and p (sort gene). these findings reinforce the important role of variation at multiple genes in the pathogenesis of coronary atherosclerosis. although the functional mechanism of many of these genetic associations remains to be determined, our comprehensive study lays the groundwork for future studies to determine the role of several genes in treatment, prediction, and prevention of coronary atherosclerosis and resulting myocardial infarction and other clinically apparent forms of coronary heart disease. circulation december / , d ow nloaded from http://ahajournals.org by on a pril , acute esophageal necrosis in an alcoholic after successful resuscitation from cardiac arrest case report acute esophageal necrosis in an alcoholic after successful resuscitation from cardiac arrest amish shah, viveksandeep thoguluva chandreskar, ravi doobay, arundeep kahlon, and ioana amzuta internal medicine department, state university of new york upstate medical university, e adams st, syracuse, ny , usa gastroenterology department, state university of new york upstate medical university, e adams st, syracuse, ny , usa pulmonary/critical care department, state university of new york upstate medical university, e adams st, syracuse, ny , usa correspondence should be addressed to amish shah; shaham@upstate.edu received april ; revised may ; accepted may ; published june academic editor: shiro kikuchi copyright © amish shah et al. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction. we present a patient who presented to the icu after successful resuscitation from cardiac arrest who was subsequently diagnosed with aen. case presentation. a -year-old female presented after cardiac arrest in which return of spontaneous circulation was achieved within minutes after the initiation of cpr. she was intubated on the scene and found to have coffee ground emesis in her bathroom when found unresponsive. due to the hemodynamically significant gi bleed, patient was started on iv proton pump inhibitor, octreotide, and levophed. subsequent endoscopy showed diffuse severe mucosal changes characterized by blackness, erythema, friability, granularity, inflammation, and decreased vascular pattern in the middle third of the esophagus and in the lower third of the esophagus. discussion. aen is a rare syndrome with a prevalence ranging from . to . % of egd. this patient is especially rare as this patient was female and had aen in the middle esophagus along with lower esophagus. the pathophysiology in this patient is hypothesized that she had cardiac arrest secondary to acute upper gi hemorrhage. subsequent low-flow state (cardiac arrest) in addition to being in the setting of severe alcohol abuse led to esophageal necrosis. . introduction acute esophageal necrosis (aen) is a rare syndrome char- acterized by diffuse circumferential black appearance of esophageal mucosa that affects the distal esophagus on endoscopy [ ]. the prevalence of this syndrome ranges from . to . % of endoscopies [ ]. there is a high mortality rate of % found in this population [ ]. we are presenting a unique case of a patient who presented to the icu after successful resuscitation from cardiac arrest who was subsequently found to have been diagnosed with aen during endoscopy. . case presentation a -year-old female with unknown past medical history except for alcohol abuse (daily use of - pints of hard liquor) and other illicit drugs (history of pcp, benzodiazepine, and methadone abuse) presented as a transfer from an outside hospital after cardiac arrest. return of spontaneous circula- tion was achieved within minutes after the initiation of cpr. she was noted to have coffee ground emesis in her bathroom when found unresponsive and was intubated on the scene. on admission to the icu, she was afebrile with a temper- ature of . ∘f, tachycardic with a pulse of /min, tachypneic with a respiratory rate of /min, and was hypotensive (a low blood pressure as low as / ). she required pressor support with norepinephrine. additionally, she was started on intravenous pantoprazole and octreotide for concerns of an ongoing gi bleed with the source unclear. gastroenterology service was consulted on admission. an emergent upper gi endoscopy was performed which revealed diffuse severe mucosal changes characterized by black interspersed with erythematous mucosa which was friable and granular and had decreased vascular pattern in the middle third and lower third of the esophagus concerning hindawi case reports in gastrointestinal medicine volume , article id , pages https://doi.org/ . / / https://doi.org/ . / / case reports in gastrointestinal medicine figure : middle esophagus showing discoloration, erythema, friability (with contact bleeding), granularity, hemorrhagic appear- ance, inflammation, nodularity, altered texture, and a decreased vascular pattern. figure : moderate size hiatal hernia, esophageal enlargement diffusely with hyperdense material noted centrally within the stomach and distal esophagus. given the history, this is concerning for gastric/distal esophageal hemorrhage but remains nonspecific. acute sternal fracture was also found. for ischemia (figure ). no esophageal varices were seen and stomach appeared normal. ct thorax (figure ) revealed diffusely enlarged esophagus with hyperdense material noted in the distal esophagus, fundus of the stomach, concerning for hemorrhage into the stomach. there was also an acute sternal fracture with multiple rib fractures likely secondary to car- diopulmonary resuscitation. patient was continued on sup- portive care with intravenous fluids, pantoprazole, octreotide, and blood transfusions. despite aggressive resuscitation, the patient was diagnosed with anoxic brain injury. she received comfort and care by the family and eventually passed away within few days. autopsy was not performed due to family’s refusal. . discussion aen also known as black esophagus is a rare syndrome with a prevalence ranging from . to . % of egd [ ] that was first described by goldenberg et al. [ ]. with mortality rate as high as % in this population, patients often go undiagnosed until an autopsy is performed [ , , ]. there are multiple comorbidities associated with aen. among them, those placing a patient at highest risk of devel- oping aen include diabetes mellitus, malignancy, hyperten- sion, alcohol abuse, and coronary artery disease. there is also a gender difference in the incidence of aen. men are four times more commonly affected [ ]. most commonly, patients will present with hematemesis and/or melena [ ]. additionally, they will have other symp- toms suggestive of sepsis including lactic acidosis, tachycar- dia, and hypotension. the accepted hypothesis behind the pathophysiology is a two-hit hypothesis [ , ]. there is an initial event that predis- poses the patient to subsequent injury. we hypothesize that the initial event of chronic alcohol abuse leads to decreased gastric defenses and increased acidic gastric contents in the esophagus. her acute upper gi hemorrhage in this setting of severe alcohol abuse (first hit) likely led to cardiac arrest due to hypovolemia. this low-flow state (second hit) may have been exacerbated by acute sternal fracture due to chest compression during cardiopulmonary resuscitation. the middle esophageal blood supply is derived from the bronchial arteries, right third or fourth intercostal arteries, and numerous small esophageal arteries off the descending aorta. in addition to the cardiac arrest, the acute hemorrhage found on the ct thorax along with acute sternal fracture, it is hypothesized that the cardiopulmonary resuscitation and sternal fracture may have severed these bronchial and intercostal arteries leading to decreased blood supply to the middle esophagus. the influx of gastric contents in the esoph- agus where gastric defenses were already compromised due to alcohol abuse and low-flow state leads to the necrosis seen in our patient. our patient, retrospectively, fits this hypothesis. endoscopy is the gold standard in diagnosing aen. findings on ct thorax are largely nonspecific and, therefore, endoscopy is always necessary to diagnose aen. as seen in the ct thorax for our patient, hemorrhage was noted as hyperdense material, but these findings were nonspecific for aen. the treatments for patients with aen are aggressive resuscitation and time. there is no reversal agent or surgery to reverse the damage. if caught early enough and resuscitation is provided via iv fluids and blood transfusions, aen can be reversed [ ]. despite aggressive resuscitation, mortality rate is still high at % for this condition. our patient’s presentation of aen was unique in two distinct ways; she was female (it is four times more common in males) and necrosis was found in the middle and lower esophagus. normally, only the distal third esophagus is found to be necrotic as its blood supply is comparable to a watershed area [ ]. . conclusion only one major retrospective review regarding aen has been published which reviewed documented case reports from to , which was highlighted in a much-cited article by gurvits et al. in [ ]. further research needs to be conducted as many statistics and conclusions have been made case reports in gastrointestinal medicine based off of the patients found in that review article. as almost years have passed since that review article, and being an extremely rare condition, further research must be done to help triage patients who may benefit from a high suspicion of acute esophageal necrosis to decrease the mor- tality and morbidity from this condition. consent informed patient consent was obtained for publication of the case details from the bereaved family. disclosure abstract of this manuscript was presented at american col- lege of gastroenterology annual scientific meeting in las vegas, nv in october . conflicts of interest no financial support or conflicts of interest exist. references [ ] y. s. haviv, c. reinus, and j. zimmerman, “‘black esophagus’: a rare complication of shock,” american journal of gastroenterol- ogy, vol. , no. , pp. – , . [ ] a. geller, h. aguilar, l. burgart, and c. j. gostout, “the black esophagus,” the american journal of gastroenterology, vol. , no. , pp. – , . [ ] g. e. gurvits, “black esophagus: acute esophageal necrosis syndrome,” world journal of gastroenterology, vol. , no. , pp. – , . [ ] s. p. goldenberg, s. l. wain, and p. marignani, “acute necrotiz- ing esophagitis,” gastroenterology, vol. , no. , pp. – , . [ ] m. tsokos and h. herbst, “black oesophagus: a rare disor- der with potentially fatal outcome - a forensic pathological approach based on five autopsy cases,” international journal of legal medicine, vol. , no. , pp. – , . [ ] k. unuma, k. harada, t. funakoshi, and k. uemura, “sudden death of an alcoholic elderly man with acute esophageal necrosis (black esophagus),” forensic science international, vol. , no. - , pp. e –e , . [ ] r. w. postlethwait and a. wendell musser, “changes in the esophagus in , autopsy specimens,” journal of thoracic and cardiovascular surgery, vol. , no. , pp. – , . [ ] g. e. gurvits, a. shapsis, n. lau, n. gualtieri, and j. g. robilotti, “acute esophageal necrosis: a rare syndrome,” journal of gastroenterology, vol. , no. , pp. – , . [ ] g. e. gurvits, k. cherian, m. n. shami et al., “black esophagus: new insights and multicenter international experience in ,” digestive diseases and sciences, vol. , no. , pp. – , . [ ] p. katsinelos, i. pilpilidis, s. dimiropoulos et al., “black esoph- agus induced by severe vomiting in a healthy young man,” surgical endoscopy, vol. , no. , pages, . [pdf] electrophoretically deposited reduced graphene oxide platform for food toxin detection. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /c nr d corpus id: electrophoretically deposited reduced graphene oxide platform for food toxin detection. @article{srivastava electrophoreticallydr, title={electrophoretically deposited reduced graphene oxide platform for food toxin detection.}, author={s. srivastava and v. kumar and md azahar ali and p. solanki and a. srivastava and g. sumana and p. saxena and amish g joshi and b. d. malhotra}, journal={nanoscale}, year={ }, volume={ }, pages={ - } } 	s. srivastava, v. kumar, + authors b. d. malhotra 	published 	materials science, medicine 	nanoscale reduced graphene oxide (rgo) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. here, the chemically active rgo has been synthesized and deposited onto an indium tin oxide (ito) coated glass substrate by the electrophoretic deposition technique. this novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin b (anti-afb ) for food toxin (afb ) detection… expand view on pubmed rsc.org save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all topics from this paper 	aflatoxins 	indium tin oxide 	aflatoxin b 	stannic oxide 	graphene 	incontinentia pigmenti achromians 	wakefulness 	x-ray photoelectron spectroscopy 	spectroscopy, fourier transform infrared 	uv-vis spectrum citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced 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photographic interpretation to evaluate the safety of home environments preventive medicine reports ( ) – contents lists available at sciencedirect preventive medicine reports journal homepage: http://ees.elsevier.com/pmedr using photographic interpretation to evaluate the safety of home environments carlee lehna phd, aprn-bc a,b,⁎, stephanie twyman bsn, ba, rn a, john myers phd b a university of louisville school of nursing, united states b school of medicine, department of pediatrics, united states ⁎ corresponding author at: s. floyd st., louisville, k e-mail address: c lehn @louisville.edu (c. lehna). http://dx.doi.org/ . /j.pmedr. . . - /© published by elsevier inc. this is an op a b s t r a c t a r t i c l e i n f o article history: received june received in revised form august accepted august available online august in the us there were , home fires resulting in deaths, , injuries, and $ . b lost. the purpose of this study was to evaluate the content-validity of photographs taken in the home for use as an educational instrument to teach about “safe” and “unsafe” fire safety practice for adults and older adults. a total of home fire safety experts were provided photographs to evaluate home fire safety practice. initially, a krippendorff's alpha was calculated for the first questions to evaluate inter-rater reliability, and differences in demographics were evaluated. unique codes and themes for the last three questions were identified and inter-rater reliability examined. a majority of re- spondents were female (n = , . %), college educated (n = , . %), nurses (n = , . %), or worked for a fire department (n = , . %). their mean age was . years and they had . years of experience. the first questions had high inter-rater reliability (krippendorff α = . ). no significant differences existed between the strata of the demographic variables (all p-values n . ). similarly, based on the codes and themes identified, the last three questions had moderate-to-good inter-rater reliability (krippendorff α = . ). providing photographs as a ‘seek-and-find’ or ‘what's wrong with this picture?’ tools and simplified visual images is an excellent way to aid recognition of unsafe home fire safety environments. education through non-traditional visual methods increases the possibility of change for diverse low-literacy populations. © published by elsevier inc. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). keywords: photographs home fire safety instrument development diverse low-literacy populations . introduction nationally, , residential fires were reported in which in- cluded deaths, , injuries, and $ . b lost (united state fire administration (usfa), ). cooking ( , ) and heating ( , ) were the leading causes of fire (united state fire administration (usfa), ). deaths were related to carelessness ( ), electrical mal- function ( ), smoking ( ), and intentional reasons ( ) (united state fire administration (usfa), ). injuries were caused by cooking ( ), open flame ( ), and carelessness ( ) (united state fire administration (usfa), ). carelessness ($ b) and electrical mal- function ($ m) accounted for the greatest fire dollar losses (united state fire administration (usfa), ). though preventable, residential fires are significant causes of death, injury, and dollars lost in the united states. assessment of home fire safety (hfs) practices most frequently in- volved checking for fire alarm location(s), and functionality (gielen et al., ; miller et al., ; parker et al., ; sidman et al., ). less often was tap hot water temperature tested (parker et al., ), carbon monoxide alarms presence assessed (mcdonald et al., ), presence and practice of fire escape plans determined (yang et al., ), or participant knowledge regarding fire safety obtained (parker y , united states. en access article under the cc by-nc et al., ). the united states fire administration's home fire safety checklist examines hfs from multiple practice perspectives (united states fire administration). the checklist has eight subscales (i.e., smoke alarms, cooking safety, electrical and appliance safety, carbon monoxide (co) alarms, candle safety, smoking safety, heating safety, and home escape plans) with three to six items per subscale (united states fire administration). to date, no reported evidence of reliability or validity is available for this instrument. the practice of hfs is com- plex, needing a comprehensive approach using an instrument with ev- idence of reliability and validity. in two previous studies by the authors on hfs education, the team studied parents of newborns (with and without special needs) (lehna et al., a) and older adults (community active and homebound) (lehna et al., b). participants were asked to watch a -minute hfs dvd; complete a pre, post, and -week follow-up phone call. in addition, in-home fire safety checks using the home fire safety checklist were conducted (lehna et al., a; lehna et al., b). at the time of the hfs check some participants of the parent study provided researchers permission to take photographs of “safe” and “un- safe” home fire hazards within their residence. initially, to examine the content validity of the photographs, two fire inspectors independently rated the photographs (inter-rater reliability). the local fire inspectors rated all the photographs twice, two-weeks apart in time (test-retest re- liability) (lehna et al., c). inter-rater reliability between the two -nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). http://creativecommons.org/licenses/by-nc-nd/ . / http://dx.doi.org/ . /j.pmedr. . . mailto:c lehn @louisville.edu http://dx.doi.org/ . /j.pmedr. . . http://creativecommons.org/licenses/by-nc-nd/ . / http://www.sciencedirect.com/science/journal/ http://ees.elsevier.com/pmedr c. lehna et al. / preventive medicine reports ( ) – fire inspectors was . , and the intra-rater reliability was . . when community participants and hfs team members rated the photographs the inter-rater reliability rating was . using krippendorff's alpha coef- ficient (lehna et al., c). as community members rated the photographs for content validity, team members found that the photographs evoked discussion between older adult participants about similar situations from their past (lehna et al., c). including photographs in a hfs education program could potentially increase knowledge retention adding to improvement in hfs practice, especially for those who were older, had lower literacy, or for whom english was a second language. with the exception of the two studies (lehna et al., a; lehna et al., b), assessment of hfs practices remains fragmented and dis- jointed. researchers mainly focus on smoke alarm placement and func- tionality (gielen et al., ; miller et al., ; parker et al., ; sidman et al., ). the research team is attempting to shift the para- digm to prevention (eradicating unsafe practices) from a traditional re- actionary approach (fire alarms). there was a need to further evaluate the effectiveness of the photographs obtained in the two previous stud- ies (lehna et al., a; lehna et al., b) with both a local and a na- tional group of burn prevention experts. photograph . unsafe - candle in shallow container; surrounding flammable materials potential fire hazard. . methods . . measure for the current project focusing on photographic instrument develop- ment, photographs were grouped according to the united states fire administration's home fire safety checklist (united states fire administration) and presented in two formats: as a powerpoint presenta- tion and as a poster. expert participants used either presentation format for their evaluation. participants rated whether photographs were “safe”, “unsafe”, and “unable to determine”. while n = ( %) photo- graphs were anticipated to be viewed as unsafe, n = ( . %) were an- ticipated to be viewed as safe. the authors purposively set the allocation rate at : . the ordering of photographs was randomly determined (see table ). `for of the photographs, the home fire safety respondents sim- ply recorded if they felt the practice in the photograph was “safe” or “unsafe.” for the three additional photographs, the practice was clearly unsafe and the respondents answered an open-ended question to de- scribe the unsafe practice. examples of some of the unique hfs photo- graphs include: unsafe cooking practices (see photograph ); unsafe candle safety (see photograph ); and safe fire escape practice (see photograph ). photograph . unsafe cooking practices. . . procedure this anonymous survey was institutional review board exempt. re- turn of the completed forms considered consent. initially, a krippendorff's alpha was calculated for the first questions to evaluate inter-rater reliability, and differences in demographics were evaluated. unique codes and themes for the last three questions were identified and inter- rater reliability examined (photographs and ). . . sample participants were recruited at the american burn association annual conference and meeting. at the burn prevention committee photograph . safe – in-home posted fire escape plan. photograph . unsafe - electrical strip without surge protector, cords under mat. table demographics of expert panel. categorical variable n = (%) female ( . %) education hs/ged ( . %) college ( . %) advance degree (masters, phd, md) ( . %) profession fire department staff ( . %) clinician (rn, md) ( . %) ot/pt ( . %) other (law, research, admin, etc.) ( . %) burn prevention ( . %) continuous variable mean (sd) age . ( . ) yrs. experience . ( . ) table photograph alphas. photo krippendorff α c. lehna et al. / preventive medicine reports ( ) – meeting, the powerpoint presentation was presented to members and photographs were rated. the powerpoint presentation also was emailed to all members to have experts rate at their home location. the poster was available in the exhibit room at the burn prevention booth for any participant to judge. completed response sheets were either submitted in-person or by email to the principal investigator (pi). locally, fire inspector participants were recruited from an urban cen- tral fire state headquarters during roll call. recruitment also occurred at a monthly safe kids coalition meeting where members completed the evaluation while either viewing the powerpoint presentation or exam- ining the poster. . results a majority of respondents were female (n = , . %), college ed- ucated (n = , . %), nurses (n = , . %) or worked for a fire de- partment (n = , . %). their mean age was . years and they had . years of burn prevention experience (see table ). the first questions had high inter-rater reliability (krippendorff α = . ). table shows the individual alphas for the respective photographs. as shown in table , a great deal of agreement in response/view (safe vs. unsafe) of the individual photographs occurred (average agree- ment of . %). no significant differences existed between the strata of the demographic variables and responses of safe vs. unsafe (all p- values n . ). photograph . unsafe - flammable materials and chemicals on dryer. based on the codes and themes identified, the final open-ended questions had moderate-to-good inter-rater reliability (krippendorff α = . ). participants who responded (n = , %), correctly iden- tified in photograph two themes, the power strip was without the surge protector (n = , . %) and cords were running under the mat (n = , . %). for photograph , participants described two themes, the fire hazard was flammable chemicals (n = , . %) and potentially flammable materials on the dryer/washer (n = , . %). for photograph , the respondents had one theme, a wiring/cord issue (overload, too many wires/cords, etc.; n = , . %). . discussion this study further extends previous mixed method predominantly qualitative (ethnographic focus) support of photographic analysis ob- tained during hfs checks (lehna et al., c) through further valida- tion with experts. there is a paucity research on fire safety practice within individual homes. currently, hfs education programs lack com- prehensive approaches for teaching and assessing home practice, often focusing on one or two aspects of hfs (e.g., smoke alarms, carbon mon- oxide alarms, scald prevention) (gielen et al., ; miller et al., ; parker et al., ; sidman et al., ; mcdonald et al., ; yang et al., ). findings from this study, moderate to strong evidence of . . . . . . . . . . . . . . . . . . . . . . . . table most popular responses and agreement for individual photographs. photograph most popular response percent of same response among panel n = (%) safe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) safe ( . %) unsafe ( . %) safe ( . %) unsafe ( . %) unsafe ( . %) safe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) safe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) unsafe ( . %) safe ( . %) c. lehna et al. / preventive medicine reports ( ) – inter-rater reliability and validity, provide evidence to support use of “safe” or “unsafe” photographs for the basis of future hfs curriculum development (e.g., story book or powerpoint formats). use of multi- method education strategies has been found to be successful in teaching parents. reimer and kagan in a series of three consecutive studies developed and tested the effectiveness of a burn prevention curriculum for amish children (reimer and kagan, a; reimer and kagan, b; reimer and kagan, ). by survey, mothers identified burn hazards unique amish communities (reimer and kagan, a). using the survey infor- mation an education curriculum was developed which involved teachers telling stories and running through scenarios with an open- viewed doll house as a game to identify home burn hazards. change in burn prevention knowledge was measured through pre- and post-test- ing (reimer and kagan, b; reimer and kagan, ). using a cur- riculum based on this unique community's burn prevention needs in different formats (storytelling and open-viewed doll house) was shown to be highly effective and culturally accepted (reimer and kagan, b; reimer and kagan, ). sinha and colleagues evaluated the effectiveness of “tales of burn safety” comic book with two groups of school aged children, one from west virginia (n = ) and the other from west bengal (n = ). change in student learning was evaluated by pre- and post-testing (sinha et al., ). in implementing a multi-learning approach (words with supporting graphics) cues are both written and visual for learners which the authors found to be effective. . conclusion using ‘seek-and-find’ or ‘what's wrong with this picture?’ tools and simplified visual images aid in recognition of unsafe home environments. fire and burn prevention education through multi- and non-traditional methods for older adults, migrant and refugee popula- tions, or those having low literacy increases the possibility of change. transparency document the transparency document associated with this article can be found, in online version. conflict of interest statement all authors declare no conflict of interest. acknowledgements funding was provided by the federal emergency management agency fire prevention & safety grant # emw- -fp- . to the american burn association burn prevention committee; the louisville fire department; and safe kids louisville, led by kosair children's hospital and children's hospital foundation office of child advocacy at kosair children's hospital. references gielen, a.c., shields, w., frattaroli, s., et al., . enhancing fire department home visit- ing programs: results of a community intervention trial. jbcr e –e . lehna, c., fahey, e., janes, e., et al., a. home fire safety education for parents of new- borns. burns ( ), – . http://dx.doi.org/ . /j.burns. . . . lehna, c., coty, m.b., fahey, e., et al., b. intervention study about the changes in home fire safety knowledge in urban older adults. burns ( ), – . http://dx.doi. org/ . /j.burns. . . . lehna, c., twyman, s., fahey, e., et al., c. “worried about them when we left:” a mixed-methods essay. qual. rep. ( ), – available at: http://www.nova.edu/ ssss/qr/qr / /lehna .pdf. mcdonald, e.m., gielen, a.c., shields, w.c., et al., . residential carbon monoxide (co) poisoning risk: correlates of observed co alarm use in urban households. j. environ. health ( ), – . miller, t.r., berger, g., ballesteros, m.f., bhattacharya, s., gielen, a.c., sheppard, m.s., . increasing smoke alarm operability through theory-based health education: a ran- domized trial. j. epidemiol. community health , – . parker, e.m., gielen, a.c., mcdonald, e.m., et al., . fire and scald burn risks in urban communities: who is at risk and what do they believe about home safety? health ed. research ( ), – . reimer, m.t., kagan, r.j., a. development of a burn prevention teaching tool for amish children. jbcr ( ), – . http://dx.doi.org/ . /bcr.ob e c. reimer, m.t., kagan, r.j., b. pilot testing of a burn prevention tool for amish children. jbcr ( ), – . http://dx.doi.org/ . /bcr. b e . reimer, m.t., kagan, r.j., . multicenter testing of a burn prevention teaching tool for amish children. jbcr ( ), – . http://dx.doi.org/ . /bcr. b e a. sidman, e.a., grossman, d.c., mueller, e.a., . comprehensive smoke alarm coverage in lower economic status homes: alarm presence, functionality, and placement. j. community health , – . sinha, i., patel, a., sun kim, f., maccorkle, m.l., watkins, j.f., . comic books can edu- cate children about burn safety in developing countries. jbcr ( ), e –e . http://dx.doi.org/ . /bcr. b e c f. united state fire administration (usfa), . residential building fire trends ( – ). usfa fire estimates summary. fema retrieved january , from: https://www.usfa.fema.gov/downloads/pdf/statistics/res_bldg_fire_estimates.pdf. united states fire administration, d. home fire safety checklist retrieved on january from http://www.usfa.fema.gov/downloads/pdf/home_safety_checklist.pdf. yang, j., peek-asa, c., allareddy, v., zwerling, c., lundell, j., . perceived risk of home fire and escape plans in rural households. am. j. prev. med. ( ), – . http://dx.doi.org/ . /j.pmedr. . . http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://dx.doi.org/ . /j.burns. . . http://dx.doi.org/ . /j.burns. . . http://www.nova.edu/ssss/qr/qr / /lehna .pdf http://www.nova.edu/ssss/qr/qr / /lehna .pdf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://dx.doi.org/ . /bcr.ob e c http://dx.doi.org/ . /bcr. b e http://dx.doi.org/ . /bcr. b e a http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf http://dx.doi.org/ . /bcr. b e c f https://www.usfa.fema.gov/downloads/pdf/statistics/res_bldg_fire_estimates.pdf http://www.usfa.fema.gov/downloads/pdf/home_safety_checklist.pdf http://refhub.elsevier.com/s - ( ) - /rf http://refhub.elsevier.com/s - ( ) - /rf 	using photographic interpretation to evaluate the safety of home environments 	 . introduction 	 . methods 	 . . measure 	 . . procedure 	 . . sample 	 . results 	 . discussion 	 . conclusion 	transparency document 	conflict of interest statement 	acknowledgements 	references pdf hosted at the radboud repository of the radboud university nijmegen the following full text is a publisher's version. for additional information about this publication click this link. http://hdl.handle.net/ / please be advised that this information was generated on - - and may be subject to change. http://hdl.handle.net/ / hum genet ( ) : - tuija sulisalo • ineke van der bürgt • david l. rimoin jaques bonaventure • david sillence • john b. campbell david chitayat • charles i. scott • albert de la chapelle pertti sistonen • ilkka kaitila genetic homogeneity of cartilage- received: july abstract cartilage-hair hypoplasia (chh) is an ¿utoso- mal recessive metaphyseal chondrodysplasia character­ ized by short stature and hypoplasia of the hair. associ­ ated pleiotropic features include deficient erythrogenesis, impaired t-cell mediated immunity, hirschsprung’s dis­ ease, and an increased risk of malignancies. chh is most prevalent among the old order amish in the united states and among the finns, but sporadic families have been described among many other populations. we have previously mapped the gene for chh to the short arm of chromosome in finnish and amish families. the chh locus resides close to d s within an interval of . cm flanked by d s and d s . in order to investigate the genetic homogeneity of chh in various populations, we studied nine families with no genealogical connections to t. sulisalo (ce ) * a. de la chapelle • i. kaitila department of medical genetics, p.o. box , fin- university of helsinki, helsinki, finland i. van der bürgt department of human genetics, university hospital nijmegen, nijmegen, the netherlands d. l. rim oin cedars-sinai medical center, los angeles, usa j. bonaventure cnrs, hospital necker, paris, france d. sillence the children’s hospital, sidney, australia j. b. campbell arnold palmer hospital for children & women, orlando, usa d. chitayat the hospital for sick children, toronto, canada c. i. scott alfred i. dupont institute, delaware, usa p. sistonen finnish red cross blood transfusion service, helsinki, finland i. kaitila department of medical genetics, helsinki university central hospital, helsinki, finland © springer-verlag hypoplasia either amish or finns. no recombinants were detected be­ tween the chh gene and any of the three closest marker loci studied, suggesting that chh in these families results from mutation(s) at the same locus as in the amish and finnish families. introduction cartilage-hair hypoplasia or mckusick type metaphyseal chondrodysplasia (m im no. ) is an autosomal re­ cessive disorder resulting in short-limbed short stature and poor hair growth (mckusick et al. ; mákitie and kaitila ). associated pleiotropic features include de­ ficient erythropoiesis, impaired t-cell mediated immunity, intestinal problems such as hirschsprung’s disease, and an increased risk of malignancies. although the pathogenesis of chh is unknown, defective proliferation of t- and b- lymphocytes and fibroblasts, and defective erythrogenesis suggest a more generalized defect in cellular proliferation. chh was first described among the old order amish in the united states (mckusick et al. ) and was later also shown to be a relatively common cause of short stature among the finns (perheentupa ). gene frequencies of . and . have been estimated in these populations, respectively (mckusick et al. ; mákitie ). data on gene frequencies among other populations are not avail­ able, but sporadic families have been described among many populations, including the dutch, polish, french, germans, danes, algerians, italians, spanish, non-amish americans, and the mexicans (reviewed in makitie ). we have previously assigned the gene for chh to chromosome by linkage in finnish and amish families (sulisalo et al. , a). the chh gene has been mapped to an area of approximately . cm flanked by d s and d s , and is close to d s (sulisalo et al. b). no recombinations have been detected between the chh gene and d s , and a statistically significant linkage disequilibrium was observed between these loci in both amish and finnish families (sulisalo et al a, b). luria-delbrück method-based calculations (hastbacka et d s d s d s i r^ \ i u v j nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd fig. pedigrees of the families studied. the haplotypes for d s , d s , and d s are shown for all individuals stud­ ied. the phase could not be determined for the marker alleles in all cases. square male, circle female, open symbol unaffected, filled symbol affected, all symbols with diagonal deceased, nd not de­ termined t able two-point tod scores between the c h h gene and three nearest marker loci in nine c h h fam ilies for w hich neither finnish nor a m ish ancestors are know n al. ; lehesjoki et al. ) suggest an approximate distance of . cm between c h h and d s (sulisalo et a l b). here, we have studied nine c h h families for which neither amish nor finnish ancestors are known. in addi­ tion, seven sporadic cases were studied. these families originate from the netherlands, poland, france, croatia, the united states, and canada. materials and methods patients the pedigrees o f the families ¿ire shown in fig. . families and come from the netherlands, fam ily from poland, family from france, fam ily from australia (originally from croatia), families - and - from the united states, and family from canada. diagnosis was performed by lv.d.b. in families - (van der burgt et al. ), by j.b. in family , by d.s. in family , by d .l.r, in families - and - , by c.i.s. in family , by d .c . in family , and by j.b.c. in family . the diagnostic criteria are short-limbed short stature, generalized laxity of joint ligaments, and metaphyseal tiring and irregularities o f the growth plates in childhood radiographs. poor hair growth is used only as a positive criterion (m ckusick et al. ; miikitie and kaitila ). d n a samples d n a was extracted from blood or lymphoblastoid cell lines ac cording to standard methods. polymerase chain reaction and detection of microsatellite markers the polymerase chain reaction protocols for the detection of mi- crosatellite markers have been described previously (sulisalo et al, ). microsatellite markers detecting d s , d s * and d s were studied (weissenbach et al. ; w ilkie et al. ). linkage analysis pairwise linkage analysis was performed using the m l in k pro­ gram of the l in k a g e package (lathrop et al. ). m ultipoint analysis against a fixed marker map of three loci, d s , d s , and d s , was carried out using the l nkm ap pro­ gram and assuming a constant female/male sex difference ratio of . and distances as previously reported (sulisalo et al. b). the c h h gene was considered fully penetrant and the frequency was set to . . the marker allele frequencies were calculated from the normal chromosomes of finnish ch h families (sulisalo et al. b), heterogeneity was tested using the a-test as implemented in program h o m o g (ott ). results and discussion locus e . . . . . . . . d s i . . , . . . . . d s . . . . . . . . d s . . . . . . . . nine potentially informative families were included in ifi vadous populations. the linkage analysis (families - ). the distribution of marker alleles in these families is shown in fig. , the two-point lod scores between the c h h gene and three closest marker loci are shown in table . no recombinations were detected between the c h h gene and markers in any of the informative meioses, suggesting genetic homogeneity of c h h in various populations. the highest lod score of . at a zero recombination fraction was detected between the c h h gene and d s . the m axim um lod scores for d s and d s reached . and . , respectively. the maximum m ultipoint lod score of . was obtained at d s . no evidence o f heterogeneity was found (h o ­ m o g ), although the sample size was limited and all meioses were not fully infoi*mative for all markers stud­ ied. the genetic homogeneity o f c h h validates the use of closely linked markers for prenatal diagnosis in families from various populations. we have previously observed a statistically significant association between the c h h gene and alleles at d s in both finnish and a m ish families (sulisalo et al. a, b). in finnish families, c h h is associated with allele , and in am ish families, with allele . o f the c h h chro­ mosomes studied here, carried allele , carried al­ lele , seven carried allele , and one carried allele at d s . alleles , , and are common in the normal chromosomes of the finnish and a m ish c h h families, and in the centre d ’etude du polymorphisme h um ain (ceph) reference families (frequencies vary from . to . ; data not shown). a m ong the finnish chromosomes, allele is the most com m on ( % ) and alleles ( %) and ( %) the second and third most common (sulisalo et a l b). a m ong the am ish and the ceph families, however, the most com m on allele is ( % in both se­ ries) and alleles and are the second and third most common (sulisalo et al. a; c eph data not shown). as c h h is associated with a common allele at d s among both the finns and the am ish, no definitive con­ clusions can be drawn about the origin o f the c h h muta­ tions in the families studied here. however, as most of the latter patients are heterozygous for alleles at d s , it can be concluded that either there are many different c h h mutations and a c h h mutation is a more common occurrence than has been assumed, or only a few muta­ tions exist but recombinations or mutations at markers have changed the original marker alleles. characterization of the gene will be required to determine whether a single or different c h h mutations are responsible for the disease a cknow ledgem ents w e thank sophie m arc (genethon, paris, france) for .sharing the allele frequency data on d s in ceph families. m aryann priore and d aniel c ohn (cedars-sinai medical center, los angeles, u sa ), and judith hall (british colum bia childrens hospital, vancouver, canada) are acknowledged for collaborative help. this study was financially supported by the march o f dim es birth defects foundation (# -fy - ), the sigrid juselius foundation, the academy o f finland, and the paulo foundation. part o f the study was carried out at the folkhalsan in ­ stitute of genetics. references burgt van der, haraldsson a , oosterwijk jc , essen aj van, wee- maes c, hamel b ( ) cartilage hair hypoplasia, metaphy­ seal chondrodysplasia type m ckusick: description of seven pa­ tients and review o f the literature. a m j med genet : - hastbacka j, chapelle a de ia, k aitila i, sistonen p, weaver a, lander e ( ) linkage disequilibrium m apping in isolated populations: diastrophic dysplasia in finland. nature genet : - lathrop g m , lalouel jm , julier c, ott j ( ) strategies for m ul­ tilocus linkage analysis in humans. proc natl acad sci u s a : - lehesjoki a-e, k oskiniem i m , norio r, tirrito s, sistonen p, lander e, chapelle a de ia ( ) localization of the epm gene for progressive m yoclonus epilepsy on chromosome : linkage disequilibrium allows high resolution mapping. hum m ol genet : - mckusick va, eldridge r, hostetler ja, ruangw it u, egeland ja ( ) dwarfism in the amish. . cartilage-hair hypoplasia. bull johns hopkins hosp : - makitie o ( ) cartilage-hair hypoplasia in finland - epidem i­ ological and genetic aspects of patients. j med genet : - makitie o, kaitila i ( ) cartilage-hair hypoplasia - clinical manifestations in finnish patients. e u rj pediatr ; — ott j ( ) analysis o f human genetic linkage, rev. ed. johns hopkins university press, baltimore, m d perheentupa j ( ) kolme periytyvaii kasvuh&iriota (in finnish). duodecim : - sulisalo t, sistonen p, hastbacka j, wadelius c, m akitie o, cha­ pelle a de la, kaitila i ( ) cartilage-hair hypoplasia gene assigned to chromosome by linkage analysis. nature genet : - sulisalo t, francomano ca, sistonen p, maher jf, m ckusick v a , chapelle a de ia, kaitila i ( a) high resolution genetic mapping of the cartilage-hair hypoplasia gene in am ish and finnish families. genomics : - sulisalo t, klockars j, makitie o, francomano c a , chapelle a de la, kaitila i, sistonen p ( b) high resolution linkage-dise- quilibrium mapping of the cartilage-hair hypoplasia gene. a m j hum genet : - weissenbach j, gyapay g, dib c, vignal a, morissette j, mil- iasseau p, vaysseix g, lathrop m ( ) a second-generation linkage map of the human genome. nature : - w ilkie pj, krizman db, weber jl ( ) linkage map of human chromosome microsatellite polymorphisms. genomics : - [pdf] highly conductive poly( , -ethylenedioxypyrrole) and poly( , -ethylenedioxythiophene) enwrapped sb s nanorods for flexible supercapacitors. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /c cp a corpus id: highly conductive poly( , -ethylenedioxypyrrole) and poly( , -ethylenedioxythiophene) enwrapped sb s nanorods for flexible supercapacitors. @article{reddy highlycp, title={highly conductive poly( , -ethylenedioxypyrrole) and poly( , -ethylenedioxythiophene) enwrapped sb s nanorods for flexible supercapacitors.}, author={b. n. reddy and m. deepa and amish g joshi}, journal={physical chemistry chemical physics : pccp}, year={ }, volume={ }, pages={ - } } 	b. n. reddy, m. deepa, amish g joshi 	published 	medicine 	physical chemistry chemical physics : pccp composites of poly( , -ethylenedioxypyrrole) or pedop and poly( , -ethylenedioxythiophene) or pedot enwrapped sb s nanorods have been synthesized for the first time for use as supercapacitor electrodes. hydrothermally synthesized sb s nanorods, several microns in length and - nm wide, offer high surface area and serve as a scaffold for coating conducting polymers, and are a viable alternative to carbon nanostructures. fibrillar morphologies are achieved for the pedop-sb s and pedot… expand view on pubmed npl.csircentral.net save to library create alert cite launch research feed share this paper citationsbackground citations view all figures and topics from this paper 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure view all figures & tables 	electrode 	natural graphite 	polymers 	light emitting diode device component 	nanorods 	nanostructured materials 	one thousand 	behavior 	electron transport 	electric capacitance citations citation 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-ethylenedioxythiophene) film electropolymerized in an aqueous sodium dodecyl sulfate and lithium tetrafluoroborate medium 	chun li and, t. imae 	chemistry 	 	 	pdf save alert research feed improvement of the electrosynthesis and physicochemical properties of poly( , -ethylenedioxythiophene) using a sodium dodecyl sulfate micellar aqueous medium 	n. sakmeche, s. aeiyach, j. aaron, m. jouini, j. lacroix, p. lacaze 	chemistry 	 	 save alert research feed supercapacitors based on polymeric dioxypyrroles and single walled carbon nanotubes 	merve ertas, ryan m. walczak, rajib das, a. rinzler, j. reynolds 	materials science 	 	 save alert research feed ultrathin mno nanorods on conducting polymer nanofibers as a new class of hierarchical nanostructures for high-performance supercapacitors 	han jie, liya li, p. fang, guoxin rong 	materials science 	 	 save alert research feed high-performance asymmetric supercapacitor based on graphene hydrogel and nanostructured mno . 	hongcai gao, fei xiao, c. ching, hongwei duan 	materials science, medicine 	acs applied materials & interfaces 	 	 save alert research feed ... ... related papers 	abstract 	figures and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue brief genetics report genetic variation in adiponectin receptor and adiponectin receptor is associated with type diabetes in the old order amish coleen m. damcott, sandra h. ott, toni i. pollin, laurie j. reinhart, jian wang, jeffrey r. o’connell, braxton d. mitchell, and alan r. shuldiner , adiponectin receptor (adipor ) and adiponectin re- ceptor (adipor ) are newly identified receptors for adiponectin, an adipocytokine with anti-inflammatory and insulin-sensitizing properties. we screened for polymorphisms by performing sequence analysis on all eight exons, splice junctions, and � kb of the � flanking regions of each receptor. we detected single nucleotide polymorphisms (snps) in adipor and snps in adipor . we genotyped these snps in amish subjects with type diabetes (n � ), impaired glucose tolerance (igt) (n � ), and normal glucose tolerance (n � ) to test for association with type diabetes. three intronic snps in adipor were signif- icantly associated with type diabetes (p � . – . ; odds ratio [or] . – . ) and in high linkage disequilibrium (r � . – . ). in adipor , we found that five snps delineated one large haplotype block (r � . – . ) spanning > kb of the gene and promoter region, which was strongly associated with the com- bined type diabetes/igt trait (p < . ; or . – . ). to our knowledge, these data provide the first evidence for association between variation in the adi- ponectin receptors and type diabetes. diabetes : – , a diponectin is an adipocytokine that is involved in lipid and glucose metabolism, energy ho- meostasis, and inflammatory pathways ( – ). circulating concentrations of adiponectin are reduced in subjects with type diabetes ( – ). low plasma adiponectin levels are observed in rodent models of obesity and insulin resistance ( , ), and adiponectin- deficient mice exhibit diet-induced insulin resistance ( , ). the insulin-sensitizing effects of adiponectin ap- pear to be mediated by increased fatty acid oxidation and glucose uptake ( , ) and suppression of gluconeogene- sis ( , ) through activation of amp-activated protein kinase ( , ) and peroxisome proliferator–activated re- ceptor-� ( ). recently, two receptors, adiponectin receptor (adi- por ) and adiponectin receptor (adipor ), were cloned, each consisting of eight exons that are located on chromosomes q . and p . , respectively. in hu- mans, adipor is ubiquitously expressed, with highest levels in the skeletal muscle, while adipor is predomi- nantly expressed in skeletal muscle and liver ( ). yam- auchi et al. ( ) showed that activation of amp-activated protein kinase and peroxisome proliferator–activated re- ceptor-� by adiponectin is mediated by adipor and adipor . in addition, normal glucose-tolerant mexican- american subjects with a family history of type diabetes exhibit significantly lower levels of adipor and adi- por mrna in skeletal muscle compared with those without a family history ( ). civitarese et al. ( ) also observed that mrna expression of both receptors was positively correlated with glucose disposal, while adi- por expression was positively correlated with plasma adiponectin levels. these studies suggest that downregu- lation or altered function of adipor and adipor may be responsible for development of insulin resistance in peripheral tissues. therefore, we hypothesized that ge- netic variation in adipor and adipor may contribute to increased susceptibility to type diabetes. we ad- dressed this question by screening adipor and adi- por for polymorphisms and testing for association between the observed sequence variation and type diabetes in the old order amish. in adipor , dna sequence analysis in amish indi- from the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland; and the geriatric re- search and education clinical center, veterans administration medical cen- ter, baltimore, maryland. address correspondence and reprint requests to alan r. shuldiner, md, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood st., room , baltimore, md . e-mail: ashuldin@medicine.umaryland.edu. received for publication october and accepted in revised form march . additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. adipor , adiponectin receptor ; adipor , adiponectin receptor ; afds, amish family diabetes study; igt, impaired glucose tolerance; ld, linkage disequilibrium; maf, minor allele frequency; ngt, normal glucose tolerance; ogtt, oral glucose tolerance test; snp, single nucleotide polymorphism. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , july fig. . gene structure, location of polymorphic sites, and pairwise ld among snps in adipor (a) and adipor (b). the upper portion of the figures show the gene structure and location of polymorphisms identified by sequencing in the amish. the base pair positions of the variants were determined from a sequence obtained from the may assembly of genome browser (university of california, santa cruz; http:// genome.ucsc.edu/) and based on numbering from the translation initiation site. the asterisks denote snps with mafs < . in the amish. snps that were not genotyped in the full sample due to technical difficulties are marked with a dagger (†). the lower portion of the figures shows a schematic of the pairwise ld, calculated as r , among the common snps (maf > . ). the dotted lines connect each snp name and position with the corresponding cell in the ld matrix. increasing level of ld is shown by darker grayscale. adipor and adipor and diabetes in the amish diabetes, vol. , july viduals detected five snps previously reported in dbsnp (http://www.ncbi.nlm.nih.gov/projects/snp/). figure a shows the adipor gene structure, snps identified, and pairwise linkage disequilibrium (ld) (r ). we genotyped all five snps in individuals enrolled in the amish family diabetes study (afds) ( subjects with type diabetes, subjects with impaired glucose tolerance [igt], and control subjects with normal glucose toler- ance [ngt]). two of the snps, rs and rs , are located bp apart in intron and were in perfect ld (r � ); therefore, only one of them (rs ) was included in the analyses. genotype frequencies of the two intronic snps, rs and rs , differed signifi- cantly between subjects with type diabetes and those with ngt (p � . and p � . , respectively), with the minor allele being the risk allele for type diabe- tes (odds ratio [or] . and . , respectively) (table and online appendix fig. [available at http://diabetes. diabetesjournals.org] for genotype frequencies in type diabetes and igt cases and ngt control subjects). the intronic snps span � kb of the gene from intron to intron and were in high ld (r � . ) (fig. a). the two snps flanking the intronic snp cluster, rs and rs , were not significantly associated with type diabetes (p � . and p � . , respectively) nor were they in ld with each other or the intronic snps, suggesting that the intronic snps mark a type diabetes susceptibil- ity allele or haplotype. we used haploscore to estimate four snp haplotype frequencies and to identify haplotypes associated with type diabetes. none of the haplotypes provided stronger evidence for association with type diabetes than the single snps (online appendix table ). in addition to the case/control analysis, we examined the effects of adipor snp genotype on bmi and plasma glucose and insulin during a -h oral glucose tolerance test (ogtt) in an expanded set of nondiabetic afds subjects, but found no association (data not shown). we previously reported evidence for linkage to type diabetes on chromosome q -q in the amish ( ), which raises the question of whether adipor contrib- utes to this linkage. while we cannot rule out that possi- bility, adipor resides � mb downstream from the -lod (logarithm of odds) support interval for our peak linkage to chromosome and is thus unlikely to be the cause of the linkage. we identified snps by sequence analysis in adipor (fig. b) and genotyped of the snps in afds subjects. four snps (ar p , ar pid, rs , and rs ) were eliminated due to difficulties with geno- typing assays. ar p , ar pid, and rs had minor allele frequencies (mafs) � . , and rs had an r � with rs in the extended sequencing set (n � alleles); thus, these snps were not pursued further. three snps were in perfect ld (r � ), including rs , rs , and rs (estimated from alleles), so only rs was included in the analy- ses. one large haplotype block spanning � kb of the promoter, exon through exon , and � . kb of exon (� kb total) contains three common snps (rs , rs , and rs ) that are in high ld (r � . ) and show strong association with the combined type diabetes/igt trait (p � . ; or . – . ), with the minor allele being the risk allele (table and fig. b; see online appendix fig. for genotype frequencies in type diabetes and igt cases and ngt control subjects). this block is interrupted by one snp (rs ) that is neither in ld with the large haplotype block (r � . – . ) nor associated with the type diabetes/igt trait (p � . ; or . ). two additional snps located � of the large haplo- type block (rs and ar – f) were significantly associated with type diabetes/igt (p � . and p � . , respectively), but not in high ld with each other or the snps in the large haplotype block. the haplotype analysis revealed one haplotype (gtgtt) that was signif- icantly associated with the combined type diabetes/igt trait (p � . ; global p value � . ) (fig. ). this haplotype contains the risk alleles for each of the single type diabetes/igt-associated snps. these results should be interpreted with caution since haploscore does not account for relatedness between subjects. quantitative trait analysis of adipor snps in an expanded set of nondiabetic subjects revealed signif- icant associations between rs and mean plasma glucose levels and total glucose area under the curve during the ogtt (p � . – . and p � . , respec- tively, for recessive model). carriers of the g risk allele for rs exhibited significantly higher plasma glucose levels at each -min interval from to min and total glucose area under the curve during the ogtt (online appendix fig. ), providing additional evidence that the haplotype containing the g allele of rs influences glucose homeostasis and type diabetes risk. there was table allele frequencies and results of association analysis in type diabetes and igt cases and ngt control subjects for snps in adipor snp name snp location (bp)* major/minor allele minor allele frequency type diabetes vs. ngt type diabetes � igt vs. ngt type diabetes (n � ) igt (n � ) ngt† (n � ) or p value‡ or p value‡ rs � , c/t . . . . . . . rs � a/g perfect ld (r � ) w/rs rs � t/g . . . . . . . rs � , a/g . . . . . . . rs � , g/c . . . . . . . *base pair location from the atg start site. †all snps fit the expectations of hardy-weinberg equilibrium in the ngt group. ‡p values are based on genotype frequencies, and the ors were computed by comparing the odds of disease between subjects carrying one copy of the minor allele and subjects not carrying any copies of the minor allele. p values � . are shown in bold. c.m. damcott and associates diabetes, vol. , july no association between snps in adipor and bmi or plasma insulin during the ogtt. although these type diabetes/igt-associated snps do not disrupt any obvious transcription factor– binding sites ( ) or splice sites, these variants could possibly alter expression or mrna stability. we explored the possibility of epistasis between polymorphisms in the receptors but found no evidence for interaction between snps in adipor and adipor . in a recent report, wang et al. ( ) screened adipor (but not adipor ) for sequence variation in caucasian and african-american subjects but found no association between the snps they detected (including the five snps we examined) and type diabetes, insulin sensitivity, or insulin secretion. the discrepancy between their study and ours may be a result of low power or underlying population differences. alternatively, it is possible that we have detected a false-positive. we did not correct for multiple comparisons in our analyses. if the highly conser- vative bonferroni correction were used in our case/control analysis, the significantly associated adipor snps would become borderline to nonsignificant whereas the adipor snps would remain significant at the p � . level. this observation strengthens the evidence that vari- ation in adipor contributes to increased susceptibility to type diabetes. in summary, we have extensively screened the two adiponectin receptors to search for polymorphisms asso- ciated with type diabetes in the old order amish. we identified multiple polymorphisms in both adipor and adipor that were significantly associated with type diabetes and related traits, providing evidence that genetic variation in the receptors, particularly adipor , plays a role in type diabetes susceptibility. alternatively, these snps may be in ld with an undetected functional snp in the receptors or a neighboring gene. further analysis in other populations, as well as functional studies, will be necessary to further elucidate the role of polymorphism in adipor and adipor in the pathogenesis of type diabetes. research design and methods the afds was initiated in with the goal of identifying susceptibility genes for type diabetes in the old order amish. details of the afds design, recruitment, phenotyping, and pedigree structure have been described previ- ously ( ). briefly, probands with previously diagnosed type diabetes (onset age – years) and, where possible, all first- and second-degree relatives of probands and spouses � years of age were recruited. phenotypic charac- terization of study participants included medical and family history, anthro- pometry, and an ogtt with insulin levels. total glucose and insulin areas under the curve during the ogtt were calculated using the trapezoid method. bmi was calculated as weight in kilograms divided by the square of height (in meters). this study includes additional subjects recruited subsequent to the report by hsueh et al. ( ) and excludes all subjects with ngt � years of fig. . adipor haplotype frequencies and asso- ciation analysis in cases with type diabetes/igt and ngt control subjects. *haplotypes with esti- mated frequencies > . in case and control sub- jects are shown. redundant snps (r � ) and rare snps (maf < . ) were removed from the haplotype analysis. type diabetes risk alleles are shaded. p values < . are shown in bold. †global p value � . . table allele frequencies and results of association analysis in type diabetes and igt cases and ngt control subjects for snps in adipor snp name snp location (bp)* major/minor allele minor allele frequency type diabetes vs. ngt type diabetes � igt vs. ngt type diabetes (n � ) igt (n � ) ngt† (n � ) or p value‡ or p value‡ rs � , t/c perfect ld (r � ) with rs rs � , t/g . . . . . . < . rs � a/t . . . . . . . ar – � , c/t . . . . . . . rs � , t/c perfect ld (r � ) with rs (estimated from alleles) rs � , g/a . . . . . . . ile ile � , c/a . . . . . . . rs � , g/a . . . . . . . rs � , c/t . . . . . . < . rs � , c/t . . . . . . . rs � , a/t . . . . . . . ar – e � , a/g . . . . . . . ar – f � , t/c . . . . . . . *base pair location from the atg start site. †all snps fit the expectations of hardy-weinberg equilibrium in the ngt group. ‡p values are based on genotype frequencies, and the ors were computed by comparing the odds of disease between subjects carrying one copy of the minor allele and subjects not carrying any copies of the minor allele. p values � . are shown in bold. adipor and adipor and diabetes in the amish diabetes, vol. , july age in order to increase the probability of their capacity for diabetes resistance. informed consent was obtained from all study subjects, and the institutional review board at the university of maryland school of medicine approved the study protocol. sequence analysis and genotyping. direct sequencing was used to screen the eight exons with splice junctions (� bp of the intronic flanking sequence) of both adipor and adipor and , bp and , bp of the region � of the initiation codons of adipor and adipor , respectively, for genetic variation in participants in the afds (eight subjects each with type diabetes, igt, and ngt). this sequencing set provides % power to detect at least one copy of the minor allele for snps with allele frequencies � . . subsequent to pcr amplification both strands were sequenced on an abi dna sequencer and analyzed using sequence analysis . software (applied biosystems, foster city, ca). all identified variants were genotyped in individuals enrolled in the afds ( subjects with type diabetes, with igt, and with ngt) with the exception of ar p , ar pid, rs , and rs , which were not genotyped due to technical difficulties. to obtain better estimates of mafs and ld for these four snps, an additional subjects were sequenced. genotyping for the other snps was completed using the snpstream ultra high throughput genotyping platform (beckman coulter, fullerton, ca) ( ) and the pyrosequencing psq hs a system (pyrosequencing, uppsula, sweden). the error rate, based on blind replicates for the snps examined, was – %. statistical analysis. before analysis, genotypes were checked for mendelian consistency using the pedcheck software program ( ) in the extended amish pedigree. mendelian errors were resolved or removed before analysis. allele frequencies were calculated by gene counting. all snps conformed to hardy-weinberg expectations. we evaluated the association between snp genotype and disease status (type diabetes versus ngt and type diabetes/igt versus ngt) under the additive genetic model using a variance component approach, in which we modeled the probability that the subject was a case or control as a function of the individual’s age, sex, and genotype, conditional on the correlations in phenotype among relative pairs. statistical testing was accomplished using the likelihood ratio test ( ). the or was computed by comparing the odds of disease between subjects carrying one copy of the minor allele and subjects not carrying any copies of the minor allele. the association analyses were carried out using the solar software program ( ). in similar fashion, we also evaluated the effect of genotype on bmi and plasma glucose and insulin levels during an ogtt in an expanded set of nondiabetic ngt (n � ) and igt (n � ) afds subjects. we compared the likelihood of a model in which the trait values were allowed to vary by genotype (unconstrained model) to that in which the genotype effects were constrained to be zero using the likelihood ratio test. within each model, we simultaneously estimated the effects of age, sex, and family relationship ( ). insulin values were transformed by their natural logarithms (ln) to reduce skewness. quantitative trait analyses were conducted using the solar program ( ). pairwise ld (r ) was computed using the zaplo software program ( ). haploscore was used to estimate and compare haplotype frequencies between case and control subjects ( ). acknowledgments this work was supported by research grants t -ag , r -dk , k -dk , u -dk , k -ca , the university of maryland general clinical research center, grant m rr , the general clinical research centers program, the national center for research re- sources, the national institutes of health, and the balti- more veterans administration geriatric research and education clinical center. we gratefully acknowledge our amish liaisons and field workers and the extraordinary cooperation and support of the amish community, without which these studies would not be possible. references . maeda k, okubo k, shimomura i, funahashi t, matsuzawa y, matsubara k: cdna cloning and expression of a novel adipose specific collagen-like factor, apm (adipose most abundant gene transcript ). biochem bio- phys res commun : – , . hu e, liang p, spiegelman bm: adipoq is a novel adipose-specific gene dysregulated in obesity. j biol chem : – , . ouchi n, kihara s, 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open access revelation of graphene-au for direct write deposition and characterization shweta bhandari , melepurath deepa , amish g joshi , aditya p saxena and avanish k srivastava * abstract graphene nanosheets were prepared using a modified hummer’s method, and au-graphene nanocomposites were fabricated by in situ reduction of a gold salt. the as-produced graphene was characterized by x-ray photoelectron spectroscopy, ultraviolet-visible spectroscopy, scanning electron microscopy, and high-resolution transmission electron microscopy (hr-tem). in particular, the hr-tem demonstrated the layered crystallites of graphene with fringe spacing of about . nm in individual sheets and the ultrafine facetted structure of about to nm of au particles in graphene composite. scanning helium ion microscopy (him) technique was employed to demonstrate direct write deposition on graphene by lettering with gaps down to nm within the chamber of the microscope. bare graphene and graphene-gold nanocomposites were further characterized in terms of their composition and optical and electrical properties. introduction graphene, structurally known as a monatomic layer of allotropic-carbon atoms in a hexagonal honeycomb two- dimensional lattice system, has always been a potential candidate for various applications due to its remarkable structural, physical, and electronic properties [ - ]. the zero density of state at the fermi level without an energy gap offered by graphene, and a linear, rather than parabolic, energy dispersion around the fermi level has been well understood in the past. the material has also been investigated in a combination with other car- bon structures to yield composites with superior proper- ties [ , ]. the composites of metal nanoparticles on graphene sheets also provide a new way to develop catalytic, mag- netic, and optoelectronic materials. moreover, adhesion of such metal nanoparticles to the graphene prevents their aggregation in dry state [ ]. recently, kamat et al. [ ] have used solution-based approach of chemical reduction of aucl - ions in graphene suspensions to fabricate gold (au) nanoparticles-graphene hybrid assemblies. in yet another report, goncalves et al. [ ] demonstrated how presence of oxygen functionalities at the graphene surface provides reactive sites for the nucleation and growth of au nanoparticles (aunps). these graphene/au nanocomposites act as potential substrates for surface-enhanced raman scattering. min et al. [ ] have also used a surface-chemistry-based approach for investigating the influence of surface func- tionalization on the growth of au nanostructures on graphene thin films by utilizing various pyrene deriva- tives containing different functional groups. but in comparison to these reports, the work pre- sented here highlights a simpler route to obtain stable au nanoparticles-graphene nanocomposites. it also demonstrates the capability of direct labeling on nano- composite by use of scanning helium ion microscopy (him). the demonstration of imaging by helium (he) ions is relatively a new technique to characterize the surfaces at sub-nanoscale with extraordinary additional advantages of in situ ion lithography, nano-patterning, device proto- typing, fabrication of quantum dots, beam-induced chemistry, and milling at nanoscale [ , ]. such a diverse usage is possible due to the light mass of the he ion and high speed, which results in smaller interaction volume with the surface layers and therefore in better resolution and potential milling feature size. from the perspective of sputtering and patterning, the result is a reduced proximity effect in the surface layer. the light ion mass results in low energy transfer and hence a rela- tively lower sputtering yield compared to gallium. * correspondence: aks@nplindia.ernet.in national physical laboratory, council of scientific and industrial research, dr. k.s. krishnan road, new delhi, , india full list of author information is available at the end of the article bhandari et al. nanoscale research letters , : http://www.nanoscalereslett.com/content/ / / © bhandari et al; licensee springer. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:aks@nplindia.ernet.in http://creativecommons.org/licenses/by/ . exploiting the method of nano-patterning of graphene with helium ions leads considerable promise for a num- ber of applications in nanoscale electronics, optoelectro- nics, and mechanics. it has been emphasized [ - ] that in an application like high-speed field-effect transis- tors, there is a strong need for graphene to be patterned at the nanoscale. patterned graphene can form complex extended geomenies and can be readily contacted elec- trically, yielding a well-controlled connection between microscale and nanoscale systems and devices. experimental section hydrogen tetrachloroaurate (haucl ) was purchased from aldrich (st. louis, mo, usa). sodium borohy- dride (nabh ) was acquired from merck (darmstadt, germany). inorganic transparent electrodes of sno :f- coated glass (pilkington, sheet resistance of Ω/sq) were cleaned in a soap solution, % hcl solution, dou- ble-distilled water, acetone, and trichloroethylene (in that order) prior to use. deionized water (resistivity ≈ . mΩ cm) obtained through milli-q system, nitric acid (hno ) (merck), sulfuric acid (h so ) (merck), and toluene (spectrochem, hyderabad, india)were used as solvents. preparation of acid-functionalized graphene for acid functionalization of graphene, a solution with h so :hno in a : volume ratio ( ml h so and ml hno ) and g graphite powder was made in a flask and refluxed at °c for h. the resulting solution was washed with deionized water till the ph was reduced to or . as a result, a black colored solution of acid-functionalized graphene was obtained. preparation of au-graphene nanostructures to fabricate au-graphene nanostructures, au nanoparti- cles were synthesized in situ in graphene suspension by the reduction of gold(iii) complex by nabh . a concen- trated aqueous solution of . m nabh was first mixed with acid-functionalized graphene suspension in toluene. with continuous stirring, mm of haucl was then introduced into this suspension. after continuously stir- red for h, the resulting au-graphene composites were collected by centrifugation and washed with water for three times. characterization techniques fourier transform infrared spectra for the films were recorded in reflection mode with a perkin elmer gx optica spectrophotometer at °c, rh ≈ % to %. i-v measurements of films were carried out on keithley high-current electrometer characteriza- tion system. absorbance (a) spectra were recorded in the - to -nm wavelength range in a perkin elmer lambda spectrophotometer (perkin elmer, ferdi- nand-porsche-ring, rodgau, germany). x-ray photoelec- tron spectroscopy (xps) spectra were recorded for the as-synthesized graphene samples using a perkin elmer model phi, maple grove, minnesota, u.s.a operating at a base pressure of . × - torr at k with a non-monochromatized alka line at , . ev, an analyzer pass energy of ev kept for core level spectra and a hemispherical sector analyzer capable of -mev resolution. the overall instrumental resolution was about . ev. the core level spectra were deconvo- luted using a non-linear iterative least squares gaussian fitting procedure. for all fitting doublets, the fwhms were fixed accordingly. surface morphology of the graphene sheets was stu- died employing a variable pressure scanning electron microscopy (sem), model: zeiss evo ma carl zeiss smt ag, germany. nanostructural imaging at high magnifications was carried out using hr-tem model: fei-tecnai g f stwin fei, achtseweg noord gg eindhoven, netherlands (operated at the elec- tron accelerating voltage of kv). hr-tem speci- mens were prepared by dispersing the graphene films on copper grid of . mm in diameter having a - mesh pore size. further, the surface topography of gra- phene and graphene-au composite films was analyzed by him (model: zeiss orion carl zeiss, nts corpora- tion way, peabody ma , u.s.a.). the he ion cap- ability of the microscope was used to perform the experiments of nanoscale patterning on the surfaces of graphene. results and discussion uv-vis spectral response the successful synthesis of graphene and au nanoparti- cles decorated graphene was confirmed by ultraviolet- visible (uv-vis) spectroscopy (figure ). the uv-vis spectrum of graphene in toluene shows two absorption peaks, one at nm corresponding to π-π* transitions of aromatic c-c bonds and the other at nm which is attributable to n®π* transitions of c=o bonds [ ]. when au nanoparticles were decorated onto the gra- phene, a broad peak in the visible range was observed corresponding to the surface plasmon absorption of au nanoparticles. in order to study the effect of graphene concentration in the synthesis of au nanoparticles, we have also synthesized and recorded uv-vis spectra at three different concentrations of functionalized graphene in the bath. as concentration of graphene was increased, the peak shows a red shift from nm at . g l- to nm at . and . g l- . the quenching in the peak intensity was also observed which is clearly visible in the inset of figure . this is probably attributable to the increase in au nanoparticle size that further controls bhandari et al. nanoscale research letters , : http://www.nanoscalereslett.com/content/ / / page of the surface plasmon absorption, with increase in the concentration of functionalized graphene [ ]. also, charge transfer from au nanoparticles to graphene resulted in a decrease in electron density which even- tually contributes to the red shift of the surface plasmon absorption [ ]. it is highly probable that this charge transfer is playing role in the stability of this nanocomposite. x-ray photoelectron spectroscopy the formation of stabilized au-graphene nanocompo- site was further confirmed by the xps spectra as shown in figure . various compendia of peak attribu- tions of c s and o s are listed in table . c s com- plex envelope is constituted of five contributions confirming the acid functionalizing of the graphene. peak at . ev in o s spectra owing to c-o-au bond confirms the stabilized au-graphene nanocompo- site. the n s peak at . ev shows clearly the func- tionalization of graphene by acid treatment. the signature of au doublet was found with two distinct state of au( f / ) and au( f / ) [ ] due to the spit- orbit splitting. the binding energy values are somewhat lower. similar trend was observed by li et al. [ ] for ag/graphene nanocomposites where the effect was attributed to electron transfer from ag to graphene due to smaller wave function of ag than graphene [ , ]. interaction between au and c=o of graphene also contributes to the electron transfer [ ], and the result corroborates with that of uv results. the bind- ing energy difference between the two states found . figure uv-vis absorption spectra of pure acid-functionalized graphene and au-graphene nanocomposites. in toluene containing different concentrations of functionalized graphene. inset shows the magnified view of surface plasmon absorption peaks. figure core level spectra of au-graphene nanocomposite. with solid lines signifying the deconvoluted contributions of (a) c s, (b) o s, (c) au f and (d) n s. bhandari et al. nanoscale research letters , : http://www.nanoscalereslett.com/content/ / / page of ev, which confirms the au in charged au+ state. deconvolution was performed on c( s), o( s) and au ( f) xps core spectra are shown in figure . au( f) deconvoluted spectra was composed of four peaks (fig- ure c). the resolved peaks related to au ( . and . ev) exhibit the metallic nature of au, while au+ state ( . and . ev) probably due to the interaction with the negatively charged graphene around au induces a positive charge. the contribution of various spices of core level spectra is listed in table . microstructural features induced during synthesis crumpled, folded, layers of bare graphene can be seen in the sem image shown in figure a. the sem image of bare graphene displayed in figure b shows stacks of graphene layers, bound by van der waals forces. the thick edges of the sheets therein (inset of figure b) reveal that the layers are atop each other with a thickness of about . µm. hr-tem was employed to study the graphene and au-graphene nanocomposites to investigate the microstructure of graphene as well as the size, shape, and distribution of au nanoparticles in the graphene matrix (figure ). a conventional folded microstructure of thin graphene sheets was observed throughout the specimen (figure a). the thickness of these sheets varies between to nm, whereas the size of these sheets is on an average between nm to µm (figure a). a significant observation was made by resolving the graphene sheets at lattice scale. the magnified regions, marked as a and b (as indicated in figure b), are displayed in fig- ure c,d, respectively. figure c exhibits a cluster of graphene sheets with well-resolved fringes showing the crystalline nature of individual sheets at lattice scale, whereas figure d further reveals the lattice fringe spa- cing of about . nm from a single sheet of a gra- phene. a good distribution of au nanoparticles in the matrix phase of graphene has been delineated in the graphene-au composite materials with a good interface between the matrix and the nanoparticle. an inset in figure a exhibits the presence of carbon decorated with ultrafine dispersion of au nanoparticle in a gra- phene-au nanocomposite. moreover, a faceted mor- phology of au nanoparticle with the edges of about nm clearly shows that the nanoparticle of au is crys- talline with preferred orientation (inset in figure a). since au is characterized by a face-centered cubic crystal structure, the hexagonal-shaped particles are presumably due to the preferred growth along the planes of a cubic crystal. the planes of au with table deconvoluted contributions of various core level spectra present in au-graphene nanocomposite peaks binding energy (ev) attributions peak area (%) c s (fwhm = . ev) . c-c in graphene . c-oh in graphene . . c-o-c in graphene . . c=o in graphene . . c(o)o in graphene . o s (fwhm = . ev) . c(o)o in graphene . . c=o in graphene . . c-o-au of composite . . c=o in graphene . . c-oh in graphene . au f (fwhm= . ev) . , . au f (of au ) . . , . au f (of au+) . *fwhm: full width at half maximum figure sem images of graphene. showing (a) an aggregate of nanosheets and (b) stacks of the sheets. inset shows the edges of individual sheets. bhandari et al. nanoscale research letters , : http://www.nanoscalereslett.com/content/ / / page of graphene of c-axis growth of carbon lattice also justify a distinct orientation relationship and therefore a crys- tallographic compatibility between the carbon as matrix and the au as second phase. nano-patterning on the helium ion microscopy nano-patterning by use of a high-resolution microscope is a fast developing method which facilitates in situ examination of the microstructure and direct write of arbitrary patterns on the given nanomaterial. him is showing the capability to create smaller structures than possible with other technique [ - ]. in the present work, him has been employed to write national physi- cal laboratory in hindi language in devnagari script in the form of deposited carbon (figure a). combining a high-brightness gas field ion source with unique sample interaction dynamics, the he ion microscope provides images with unique contrast and complementary infor- mation to existing charged particle imaging instruments such as the sem and tem. formed by a single atom at the emitter tip, the he probe can be focused to below . nm offering the highest recorded resolution for sec- ondary electron images. the small interaction volume between the helium beam and the sample also results in images with stunning surface detail. besides high-resolu- tion imaging, the collimated beam of he ions can be manipulated for nano-patterning on even two-dimen- sional nanostructured materials like graphene. the unique combination of sub-nanometer high-resolution surface microscopy and in situ nano-scaled structural buildings elucidates a new field which is so far relatively unexplored in fabrication and process control of funda- mentally important nano-objects like graphene. in the present work, the text ("national physical laboratory” in figure hr-tem micrographs of acid-functionalized grapheme. (a) sheets with wrinkle contrast, (b) different layers of graphene and (c, d) lattice scale fringes of graphene resolved from two different regions as marked a and b in (b). figure he ion microscopy. showing (a) nano-patterning by direct write deposition and (b) distribution of gold particles marked with a set of arrows in graphene. bhandari et al. nanoscale research letters , : http://www.nanoscalereslett.com/content/ / / page of hindi language) was created by deposition of carbon. this pattern, in the form of a bitmap, was opened up in orion software carl zeiss nts llc, corporation way, peabody ma , u.s.a. subsequently, the user defined the size of the overall pattern, and the pixel size was scaled accordingly. the ion dose per pixel was also variable, being set in proportion to the gray level in the bitmap (up to levels). lettering with gaps down to nm was observed. in this process, the direction of scan- ning was also user selectable. in another set of experi- ments, him was used to study the distribution of au in the matrix of graphene (figure b). we have noticed that spherical au nanoparticles of size in the range of to nm are uniformly distributed in the matrix. a thin layer of graphene on the surface of individual au nanoparticles is also inferred due to the presence of a glazy contrast on au surfaces. it is important to men- tion that there is no deterioration at the boundaries between the matrix and the second phase. electrical properties the i-v characteristics of au-graphene and functiona- lized graphene were recorded in the following configura- tion: sno :f/au-graphene/aluminum as shown in figure where respective solutions were drop casted on the sno :f-coated glass substrates. the aunps decoration was having a beneficial effect on the electronic conduc- tivity of graphene. both the films show ohmic contact with the substrate in to + v potential region as can be seen from the linear variation of current with applied bias. for the au-graphene composite, the conductivity was determined to be . s cm- which is much higher than blank functionalized graphene film where the value was estimated to be . s cm- which validates the role of interaction with aunps in the enhancement of con- ductivity. in the literature, the value of pristine graphene has been reported to be . s cm- [ ]. here, probably due to functionalization, the value is lower. in au-gra- phene, enhanced coupling occurred between aunps as they attached themselves onto the defect sites of gra- phene surface, hereby increasing the charge transfer between the two. functional groups present on the gra- phene sheets served as anchors for adsorption of nano- particles and the positively charged aunps as depicted earlier in xps could easily adsorb on these negative sheets through electrostatic attraction. moreover, even the inherent electronic conductivity of the metal nps are higher and all these attribute to the increased con- ductivity in the au-graphene nanocomposite. conclusion a simple modified hummer’s method was used to fabri- cate graphene and graphene-au nanocomposites. a signif- icant change in i-v characteristics between bare graphene and its au incorporated nanocomposites has been noticed. an important in situ direct write deposition on nanosheets of graphene has been demonstrated by employing he ions inside the chamber of the microscope. acknowledgements the authors thank the director, npl, new delhi for his guidance and encouragement. one of the authors (aks) acknowledges the csir travel grant to visit usa in february . m/s carl zeiss nts (usa) is gratefully acknowledged for extending the facility of helium ion microscopy to carry out the experiments of nano-patterning. mr. k. n. sood is acknowledged for sem measurements. author details national physical laboratory, council of scientific and industrial research, dr. k.s. krishnan road, new delhi, , india department of chemistry, indian institute of technology hyderabad, hyderabad, , india authors’ contributions sb carried out the graphene preparation and interpretation of results. md did the lectrical measurements. agj carried out xps measurements and interpretations. aps assisted in synthesis of material. aks initiated the idea of working on present topic and subsequently compiled the data. competing interests the authors declare that they have no competing interests. received: december accepted: june published: june references . hermando dh, guinea f, bratas a: spin-orbit coupling in curved graphene, fullerenes, nanotubes, and nanotube caps. phys rev b , : . . wallac pr: the band theory of graphite. phys rev , : . . eda g, fanchini g, chhowalla m: large-area ultrathin films of reduced graphene oxide as a transparent and flexible electronic material. nature , : . . slonczewski jc, weiss pr: band structure of graphite. phys rev , : . . vincenzo dpd, mele ej: self-consistent effective-mass theory for intralayer screening in graphite intercalation compounds. phys rev b , : . . 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reduction of ag(cn) - on silver and platinum colloidal nanoparticles. langmuir , : . . brust m, walker m, bethell d, schiffrin dj, whyman r: synthesis of thiol- derivatised gold nanoparticles in a two-phase liquid-liquid system. j chem soc chem commun , : . . lopez-salido i, lim dc, dietsche r, bertram n, kim yd: electronic and geometric properties of au nanoparticles on highly ordered pyrolytic graphite (hopg) studied using x-ray photoelectron spectroscopy (xps) and scanning tunneling microscopy (stm). j phys chem b , : . . wu xj, zeng xc: periodic graphene nanobuds. nano lett , : . . deng zw, chen m, wu lm: novel method to fabricate sio /ag composite spheres and their catalytic, surface-enhanced raman scattering properties. j phys chem c , : . doi: . / - x- - cite this article as: bhandari et al.: revelation of graphene-au for direct write deposition and characterization. nanoscale research letters : . submit your manuscript to a journal and benefi t from: convenient online submission rigorous peer review immediate publication on acceptance open access: articles freely available online high visibility within the fi eld retaining the copyright to your article submit your next manuscript at springeropen.com bhandari et al. nanoscale research letters , : http://www.nanoscalereslett.com/content/ / / page of http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.springeropen.com/ http://www.springeropen.com/ 	abstract 	introduction 	experimental section 	preparation of acid-functionalized graphene 	preparation of au-graphene nanostructures 	characterization techniques 	results and discussion 	uv-vis spectral response 	x-ray photoelectron spectroscopy 	microstructural features induced during synthesis 	nano-patterning on the helium ion microscopy 	electrical properties 	conclusion 	acknowledgements 	author details 	authors' contributions 	competing interests 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top 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rigney, bradley n delman, j mocco, amish doshi, puneet belani neuroimaging to cite: kihira s, schefflein j, chung m, et al. j neurointervent surg ; : – . diagnostic, molecular and interventional radiology, mount sinai health system, new york, new york, usa department of neurosurgery, the mount sinai health system, new york, new york, usa correspondence to dr shingo kihira, diagnostic, molecular and interventional radiology, mount sinai health system, new york, ny , usa; shingokihira@ gmail. com received april revised april accepted april published online first  may  © author(s) (or their employer(s)) . no commercial re- use. see rights and permissions. published by bmj. abstract background authors have noticed an increase in lung apex abnormalities on ct angiography (cta) of the head and neck performed for stroke workup during the coronavirus disease (covid- ) pandemic. objective to evaluate the incidence of these cta findings and their relation to covid- infection. methods in this retrospective multicenter institutional review board- approved study, assessment was made of cta findings of code patients who had a stroke between march and april , at six hospitals across new york city. demographic data, comorbidities, covid- status, and neurological findings were collected. assessment of covid- related lung findings on cta was made blinded to covid- status. incidence rates of covid- related apical findings were assessed in all code patients who had a stroke and in patients with a stroke confirmed by imaging. results the cohort consisted of a total of patients with mean±sd age of . ± . years and . % ( / ) were male. among all code patients who had a stroke, % ( / ) had covid- related lung findings. rt- pcr was positive for covid- in . % ( / ) of these patients with apical cta findings. among patients who had a stroke confirmed by imaging, . % ( / ) had covid- related apical findings. rt- pcr was positive for covid- in all ( / ) of these patients with apical findings. conclusion the incidence of covid- related lung findings in stroke cta scans was % in all code patients who had a stroke and . % in patients with a stroke confirmed by imaging. stroke teams should closely assess the lung apices during this covid- pandemic as cta findings may be the first indicator of covid- infection. introduction coronavirus disease (covid- ) is a clinical manifestation of severe acute respiratory syndrome coronavirus- (sars- cov- ), which was first detected in wuhan, china in december of . covid- has since spread worldwide and was declared a global pandemic on march , . as of april , , at least confirmed cases of covid- had been detected in the state of new york, with at least succumbing to covid- related deaths. the symptoms initially reported with covid- were related to pneumonia, such as cough, fever, and dyspnea. however, subse- quent studies have since shown that the severity of symptoms can vary greatly, from essentially asymptomatic to life- threatening complications, – with manifestations transcending the respiratory system to include gastrointestinal disease, head- aches, and altered mental status. neurologic symptoms are prevalent in covid- patients, with one report suggesting they can be seen in approximately % of hospitalized covid- patients. most recently, poyiadji et al reported that acute hemorrhagic necrotizing encephalopathy can be a manifestation of covid- infection. the tendency of respiratory infections to increase the short- term risk of acute ischemic stroke – may offer some explanation for the variety of neuro- logical symptoms seen in this patient population. although the exact pathway remains unestab- lished, it is believed that the systemic inflammatory response to the infection, rather than the microbe itself, is responsible for the elevated stroke risk. proposed pathways include prothrombotic state, alterations in endothelial function, plaque insta- bility, and rupture. – additionally, recent studies specific to sars- cov- have shown that the virus can cause a cytokine storm, which may induce acute ischemic strokes. ct angiography (cta) of the neck and head that is obtained in acute workup for stroke will typically include the lung apices. with the recent alarming increase in incidence of covid- infection, the authors observed abnormal findings in the apices that appeared more frequently than prior to the pandemic. recent studies showing diagnostic utility of chest ct for covid- , – prompted the authors to investigate whether evalu- ation of the lung apical portions captured on cta may have utility in affecting patient care during the covid- pandemic. in this study, we aim to assess ( ) what proportion of all code patients who had a stroke have lung findings on cta, ( ) what proportion of patients with confirmed stroke have lung findings on cta, and ( ) how these find- ings correspond to covid- status diagnosed by reverse transcriptase polymerase chain reaction (rt- pcr) from nasal swabs. methods study population this multicenter retrospective study included patient populations of six hospitals spread across three new york city boroughs. expedited insti- tutional review board approval was obtained with a waiver of informed consent. between march and april , , ‘code stroke’ was activated on a total of patients presenting with acute o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://jnis.bmj.com/ http://www.snisonline.org http://orcid.org/ - - - http://crossmark.crossref.org/dialog/?doi= . /neurintsurg- - &domain=pdf&date_stamp= - - http://jnis.bmj.com/ of kihira s, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - neuroimaging figure typical ct appearance of covid- infection. typical ct for covid- pneumonia: axial cta image of the lung apex in a patient in their s who presented with sudden aphasia and limb paresthesia and retrospectively found to have cough and fever for week, shows bilateral peripheral ground- glass opacities in the upper lobes (arrows), some with a rounded morphology in the left lung. this patient tested positive for covid- on rt- pcr. figure indeterminate ct appearance of covid- infection. indeterminate ct for covid- pneumonia: axial cta image of the lung apex in a patient in their s who presented with weeks of headaches, myalgias, and worsening dysarthria, shows central, perihilar ground- glass opacities in the left upper lobe (arrows). the unilateral central and unilateral distribution makes this appearance ‘indeterminate’. this patient tested positive for covid- on rt- pcr. neurological symptoms. all ‘code stroke’ patients were included in this analysis if they underwent head and neck cta in addi- tion to non- contrast head ct during their acute stroke workup. patients were excluded if they ( ) did not obtain a head and neck cta scan such as for medical contraindication (n= ) or ( ) did not have sufficient clinical data in our electronic medical record system (n= ). scanning parameters standard emergent stroke protocol was performed on a - slice lightspeed vct scanner (general electric (ge) healthcare, waukesha, wisconsin, usa) with helical rotation time of ms and tube current of – ma. scanning was timed to acquire a . mm axial slice thickness with field of view from the level of the aortic arch to the vertex based on the initial scout radiograph. an kv tube voltage was used for all patients with matrix of and with a pitch of . . angiography was performed with non- ionic iodinated contrast medium (isovue mg i/ml, optiray, guerbet) at an injection rate of ml/s. axial and coronal maximum- intensity projection images were reconstructed from initial axial images at a slab thickness of . mm and . mm reconstruction increment for axial and coronal maximum- intensity projection images. data collection demographic characteristics, vascular risk factors, vital signs, laboratory values, and covid- status were collected from our institutional electronic medical record system. vascular risk factors included the diagnoses of hypertension, coronary artery disease, diabetes mellitus type , atrial fibrillation, congestive heart failure, dyslipidemia, current or former smoking status, evidence of prior stroke, and overweight/obese body mass index. covid- infection was confirmed by in- house in vitro rt- pcr of nasopharyngeal swabs performed on the roche cobas / systems (roche diagnostic, basel, switzerland). patients were considered covid- negative if the rt- pcr was negative. nasal swab was repeated typically up to three times in patients with high suspicion for covid- infection if they presented with typical covid- symptoms or if imaging (chest x- ray or ct chest) was incongruent with initial rt- pcr results. the presence or absence of stroke was confirmed on ct and/ or mri by a board- certified fellowship trained neuroradiologist. characterization of apical lung findings on cta lung apices included in each cta field of view were evaluated for the presence or absence of ground- glass opacity (ggo) and consolidation by a board- certified and fellowship- trained cardiothoracic radiologist blinded to the covid- status of the patient. ground- glass opacity is defined as an area of increased attenuation in the lungs on ct, through which bronchial and vascular markings are preserved. consolidation refers to more dense opacification of the lungs with obscuration of underlying vascular markings. parenchymal findings were further charac- terized as either ‘typical’ (figure ), ‘indeterminate’ (figure ), or ‘atypical’ (figure ) based on a recent expert consensus statement for radiology reporting of covid- chest ct findings. from this consensus, ‘typical’ findings suggest high likelihood for covid- infection, but because of the community prevalence, ‘indeterminate’ was also grouped as suggestive for covid- in the setting of the pandemic. thus, ‘typical/indeterminate’ find- ings were grouped and considered as radiologically positive for covid- related findings. ‘atypical’ was considered unlikely to be related to covid- infection. results clinical characteristics of patient population our patient cohort consisted of a total of patients with mean±sd of age of . ± . years with . % ( / ) male (table ). of all code patients who had a stroke . % ( / ) had acute infarction on imaging. of the patients, ( . %) were identified as covid positive from nasal swab rt- pcr. the majority of patients worked up for acute stroke presented with altered mental status ( %) and dysarthria ( . %). o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://jnis.bmj.com/ of kihira s, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - neuroimaging figure atypical ct appearance of covid- infection. atypical ct for covid- pneumonia: axial cta image of the lung apex (a) in a patient in their s who presented with sudden altered mental status and syncope, shows completely right upper lobe consolidation (arrows). corresponding anteroposterior chest x- ray examination (b) shows that the right upper lobe consolidation represents lobar collapse (arrows) secondary to mucus plugging. this patient tested negative for covid- on rt- pcr. table demographic characteristics study population variables total cohort (n= ) confirmed stroke* (n= ) age (mean±sd) . ± . . ± . male gender . % ( ) . % ( ) stroke* . % ( ) % ( ) covid related apical findings on cta† . % ( ) . % ( ) ground- glass opacity . % ( ) . % ( ) consolidation . % ( ) . % ( ) covid (+) on nasal swab rt- pcr . % ( ) . % ( ) diabetes type ii . % ( ) . % ( ) hypertension . % ( ) . % ( ) coronary artery disease . % ( ) . % ( ) congestive heart disease . % ( ) . % ( ) dyslipidemia . % ( ) . % ( ) atrial fibrillation . % ( ) . % ( ) prior stroke . % ( ) . % ( ) bmi (mean±sd) . ± . . ± . smoking status . % ( ) . % ( ) current . % ( ) . % ( ) former . % ( ) . % ( ) nih stroke scale (mean±sd) . ± . . ± . common presenting neurological symptoms altered mental status . % ( ) . % ( ) headache . % ( ) . % ( ) dysarthria . % ( ) . % ( ) syncope/unresponsiveness . % ( ) . % ( ) facial droop . % ( ) . % ( ) numbness . % ( ) . % ( ) *stroke that is confirmed on imaging. †apical findings on cta indicative of covid- infection include typical and indeterminate findings. note that indeterminate findings are considered secondary to covid- infection during pandemic. covid- related lung findings on head and neck cta of all code patients who had a stroke, % ( / ) of patients had covid- related apical lung findings (table ). all ( / ) patients with covid- related lung findings had a component of ggo. consolidations were seen in . % ( / ) of patients with covid- related apical findings. subsequent rt- pcr revealed that . % ( / ) of all code patients who had a stroke tested positive for covid- status, and . % ( / ) of patients with covid- related apical findings tested posi- tive for covid- on rt- pcr. among patients with confirmed stroke, . % ( / ) of patients had covid- related apical findings with all ( / ) apical findings having a component of ggo and only . % ( / ) with consolidations. rt- pcr was positive for covid- in all ( / ) these patients with covid- related apical findings. discussion as the primary role of head and neck cta during a ‘code stroke’ is to assess vascular pathology, such as a large vessel occlusion, aneurysm, or arteriovenous malformation, the lung apices may garner less attention and be regarded as having less clinical rele- vance. however, the authors have noticed an alarming increase in incidental findings in the apices of the lungs on cta prob- ably associated with an increase in covid- cases during the pandemic. in this study, we demonstrated that % of all code patients who had a stroke and . % of patients with confirmed stroke had lung apical findings on cta related to covid- and corresponding with rt- pcr findings. thasan et al had previously reported incidental findings in the lung apices on cta in . % of patients with confirmed strokes. in this study, we assessed only for covid- related findings. the alarming rate of these lung findings draws attention to the importance of careful evaluation of the lung apices in patients who had a stroke. these findings may be the first and only indicator of a patient’s covid- status during early emergency or inpa- tient evaluation, since standard rt- pcr from a nasal swab may take – hours or more for results. furthermore, a patient presenting to the hospital with a suspected stroke is likely to undergo emergent cta well before rt- pcr results are known or even performed. in addition, with low sensitivity and reli- ability of rt- pcr, management could be altered significantly if imaging findings suggest a positive incongruent covid profile. the importance of these observations to the endovascular surgeon and stroke neurologist are multifold. as these patients presented with predominantly neurologic symptoms, this study o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://jnis.bmj.com/ of kihira s, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - neuroimaging attests to the wide range of symptomatology associated with covid- infection and raises awareness that if lung findings are observed, these patients should be placed in isolation or in a desig- nated covid investigation area and tested for covid- . if these findings are observed in the lung apices, the stroke team should wear appropriate personal protective equipment when revisiting the patient. stroke neurologists and endovascular surgeons should be aware of the significance of apical findings on cta and consider extra precautions when evaluating these patients. additionally, members of the stroke team may benefit from understanding the characteristic features of covid- related lung findings and differentiate them from 'atypical' findings as lung findings for covid- may be diverse. in this study, we characterized the appearance of covid- related findings and demonstrated that these predominantly include ggos. although chest ct has been shown to be superior to chest x- ray examina- tion and has a sensitivity of up to %, the utility of apical evaluation on neck cta has not been previously evaluated. recent studies have shown that the presence of ggos in the periphery of lung parenchyma correlates with duration of the covid- infec- tion with a change in characteristic pattern with time. there are several limitations to our study. first, lung apices were evaluated by only one cardiothoracic rater. having multiple reviewers score apical lung findings would improve the reli- ability of our assessment and reduce operator bias. additionally, we used the 'gold standard' of rt- pcr from a nasal swab, for which the sensitivity and specificity for diagnosis of covid- has not been definitively established. despite the test’s approval under emergency use authorization, recent studies have shown that it may have sensitivity as low as % for the diagnosis of covid- . furthermore, we acknowledge that chest imaging findings in covid- patients are not specific and may overlap with other viral (particularly, influenza), bacterial, and autoimmune/inflammatory processes (eg, alveolar hemorrhage in patients with systemic lupus erythematosus) as imaging mani- festations are probably from a systemic inflammatory response to the infection rather than the microbes themselves. however, given the ongoing covid- pandemic and waning of the influ- enza season, these non- specific findings are more likely to be related to covid- infection, due to a higher pretest proba- bility at this time. finally, this study was conducted in new york city, which is an epicenter of the pandemic. thus, the incidence rate reported in our study may not necessarily be applicable to all institutions. however, given the spread of covid- and ongoing pandemic, we believe that this study serves to raise awareness to the stroke team, which is applicable at all locations experiencing the pandemic. in conclusion, to our knowledge this is the first study to evaluate the incidence of covid- related apical lung find- ings on head and neck cta scans during acute stroke workup. the alarming incidence of these findings should raise awareness for the stroke neurologists and endovascular surgeons to check the lung apices on cta and consider extra precautions when evaluating patients with characteristics findings for covid- . this may become especially relevant when imaging is the first tool to suggest covid- infection, and early recognition can facilitate more prompt isolation and treatment strategies. the alarming rate of lung apical findings in code patients who had a stroke may even prompt consideration for extension of the cta protocol beyond the apices to confidently rule out patients in concert with a negative nasopharyngeal swab or for detection of covid- in patients with chest imaging manifestations outside the lung apices. ongoing efforts to improve early covid- diagnosis and patient care may benefit from extending head/ neck cta protocol during code strokes to capture the entire chest during the covid- pandemic. contributors study design: sk, js, mc, jm, pb. data collection: sk, js, mc, km, br. statistical analysis: sk, pb. manuscript writing: sk, js, mc, km, br, bnd, jm, ad, pb. funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not- for- profit sectors. competing interests none declared. patient consent for publication not required. ethics approval institutional review board (irb) of the mount sinai school of medicine, in accordance with mount sinai’s federal wide assurances (fwa# , fwa# ) to the department of health and human services, approval was obtained on an expedited basis with a waiver of informed consent. irb approval number: # - . provenance and peer review not commissioned; externally peer reviewed. data availability statement all data for this manuscript are included in the manuscript. there is no supplementary information. this article is made freely available for use in accordance with bmj’s website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non- commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. orcid id shingo kihira http:// orcid. org/ - - - references huang c, wang y, li x, et al. clinical features of 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[advances in the research of cytokine storm mechanism induced by coronavirus disease and the corresponding immunotherapies]. zhonghua shao shang za zhi ; :e . hirano t, murakami m. covid- : a new virus, but a familiar receptor and cytokine release syndrome. immunity . doi: . /j.immuni. . . . [epub ahead of print: apr ]. ai t, yang z, hou h, et al. correlation of chest ct and rt- pcr testing in coronavirus disease (covid- ) in china: a report of cases. radiology : . bernheim a, mei x, huang m, et al. chest ct findings in coronavirus disease- (covid- ): relationship to duration of infection. radiology : . fang y, zhang h, xie j, et al. sensitivity of chest ct for covid- : comparison to rt- pcr. radiology : . hansell dm, bankier aa, macmahon h, et al. fleischner society: glossary of terms for thoracic imaging. radiology ; : – . simpson s, kay fu, abbara s, et al. radiological society of north america expert consensus statement on reporting chest ct findings related to covid- . endorsed by the society of thoracic radiology, the american college of radiology, and rsna. radiology ; :e . kanesa- thasan r, cox m, patel m, et al. actionable vascular and other incidental findings on cta in patients undergoing acute stroke intervention. neuroradiol j ; : – . li y, yao l, li j, et al. stability issues of rt- pcr testing of sars- cov- for hospitalized patients clinically diagnosed with covid- . j med virol . doi: . / jmv. . [epub ahead of print: mar ]. wong hyf, lam hys, fong ah- t, et al. frequency and distribution of chest radiographic findings in covid- positive patients. radiology : . kanne jp, little bp, chung jh, et al. essentials for radiologists on covid- : an update- radiology scientific expert panel. radiology : . winichakoon p, chaiwarith r, liwsrisakun c, et al. negative nasopharyngeal and oropharyngeal swabs do not rule out covid- . j clin microbiol ; . doi: . /jcm. - . [epub ahead of print: apr ]. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://dx.doi.org/ . /cma.j.cn - - http://dx.doi.org/ . /j.immuni. . . http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /ryct. http://dx.doi.org/ . / http://dx.doi.org/ . /jmv. http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /jcm. - http://jnis.bmj.com/ 	incidental covid- related lung apical findings on stroke cta during the covid-  pandemic 	abstract 	introduction 	methods 	study population 	scanning parameters 	data collection 	characterization of apical lung findings on cta 	results 	clinical characteristics of patient population 	covid- related lung findings on head and neck cta 	discussion 	references mra versus dsa for the follow-up imaging of intracranial aneurysms treated using endovascular techniques: a meta-analysis of ahmed su, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - review mra versus dsa for the follow-up imaging of intracranial aneurysms treated using endovascular techniques: a meta-analysis syed uzair ahmed,   j mocco, xiangnan zhang, michael kelly, amish doshi, kambiz nael, reade de leacy   neuroimaging to cite: ahmed su, mocco j, zhang x, et al. j neurointervent surg ; : – . ► additional material is published online only. to view please visit the journal online (http:// dx. doi. org/ . / neurintsurg- - ). division of neurosurgery, saskatoon, saskatchewan, canada the mount sinai health system, new york, new york, usa department of neurosurgery, icahn school of medicine at mount sinai, new york, new york, usa royal university hospital, university of saskatchewan, neurosurgery, saskatoon, saskatchewan, canada department of radiology, icahn school of medicine at mount sinai, new york, new york, usa correspondence to dr reade de leacy, neurosurgery, icahn school of medicine at mount sinai, new york , usa; reade. deleacy@ mountsinai. org received march revised april accepted april published online first  may  © author(s) (or their employer(s)) . no commercial re-use. see rights and permissions. published by bmj. abstract background treated aneurysms must be followed over time to ensure durable occlusion, as more than % of endovascularly treated aneurysms recur. while digital subtraction angiography (dsa) remains the gold standard, magnetic resonance angiography (mra) is attractive as a non-invasive follow-up technique. two different mra techniques have traditionally been used: time-of-flight (tof) and contrast-enhanced (ce) mra. we analysed data from studies comparing mra techniques with dsa for the follow-up of aneurysms undergoing endovascular treatment. subgroup analysis of stent-assisted coiling (sac) and flow diversion (fd) techniques was completed. methods comprehensive searches using the embase, pubmed, and cochrane databases were performed and updated to november . pooled sensitivity and specificity were calculated using aneurysm occlusion status as defined by the raymond–roy occlusion grading scale. results the literature search yielded unique titles. forty-three studies were included. for tof-mra, sensitivity and specificity of all aneurysms undergoing endovascular therapy were % and %, respectively. for ce-mra, the sensitivity and specificity were % and %, respectively. for sac and fd techniques, sensitivity and specificity of tof-mra were % and %, respectively. ce-mra had sensitivity and specificity of % and %. conclusion mra is a reliable modality for the follow- up of aneurysms treated using endovascular techniques. while the data are limited, mra techniques can also be used to reliably follow patients undergoing fd and sac. however, clinical factors must be used to optimize follow-up regimens for individual patients. introduction intracranial aneurysms have a global prevalence of approximately %. aneurysm-related morbidity and mortality primarily arises from their rupture (or re-rupture), and treatment strategies aim to secure aneurysms to eliminate this risk. endovascular treatment of intracranial aneurysms has evolved significantly over the last decade and now includes coil occlusion, with or without stent assistance, as well as parent vessel reconstruction using flow-di- verting stents. treated aneurysms must be followed over time to ensure durable occlusion as more than % of endovascularly treated aneurysms recur, with % requiring retreatment. while digital subtraction angiography (dsa) remains the gold standard test for diagnosis of aneurysm recurrence, it is an invasive imaging tech- nique with several associated risks. these include the risks of ionizing radiation exposure, nephro- toxicity from iodine-based contrast agents, cerebral thromboembolism, as well as iatrogenic arterial damage. reported rates of neurological compli- cations are . – . %. since durability of the aneurysm treatment must be confirmed over time, requiring multiple diagnostic studies, the risks asso- ciated with dsa follow-up are amplified. higher costs associated with dsa are also relevant in this context, and must be accounted for in devising follow-up regimens. magnetic resonance angiography (mra) has been used in the follow-up of endovascularly treated cerebral aneurysms as it is a non-invasive technique and reduces some of the risks associated with serial dsa examinations. two different mra techniques have traditionally been used: time-of- flight (tof) and contrast-enhanced (ce) mra. of these, tof-mra has the advantage of not requiring intravenous contrast agent administration. while meta-analyses have previously assessed the diagnostic performance of mra techniques compared with dsa, these have been limited by the quality of the data. furthermore, no studies have previously assessed the diagnostic quality of mra in cases of stent-assisted coiling or flow diversion. the most recent of these have been the meta-analyses by van amerongen et al and menke et al. both authors conclude that mra has moderate-to-high diagnostic performance when compared with dsa. however, the body of evidence has since increased. furthermore, studies assessing stent-assisted coiling and flow diversion have also been performed. our meta-analysis aims to include newer studies, with a significant increase in the available cases analyzed, and include available evidence on stent-assisted coiling and flow diversion cases as these techniques become increasingly used by neurointerventionalists. methods study acquisition we carried out comprehensive database searches using the embase, pubmed, and cochrane data- bases. the search was updated to november , . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://jnis.bmj.com/ http://www.snisonline.org http://orcid.org/ - - - http://orcid.org/ - - - http://crossmark.crossref.org/dialog/?doi= . /neurintsurg- - &domain=pdf&date_stamp= - - http://jnis.bmj.com/ of ahmed su, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - neuroimaging figure study inclusion flow diagram. study inclusion acquired studies were screened for appropriateness for inclu- sion in the meta-analysis. the titles and abstracts were used to exclude studies that did not compare mra and dsa in the evaluation of endovascularly treated aneurysms. review articles, meta-analyses, conference abstracts, comments, and editorials were also excluded. for the remaining articles, full-text versions were obtained and analysed for inclusion. inclusion criteria were: ( ) studies comparing at least one mra technique and dsa for the evaluation of aneurysms treated using an endovas- cular approach; ( ) studies that used a raymond–roy or compa- rable grading scale to assess aneurysm occlusion; and ( ) studies that provided suitable data to construct × contingency tables. data acquisition included studies were searched for occlusion status data for the meta-analysis. further data for qualitative and subgroup anal- ysis were also acquired, including number of patients, aneu- rysms, and studies included, patient age, time to follow-up, time between follow-up modalities, and modality specifics (mri magnetic field strength, d vs d dsa). study quality methodological quality of the included studies was assessed using the grades of recommendation, assessment, develop- ment, and evaluation (grade) method. – specific care was taken to apply the grade criteria to studies of diagnostic test accuracy. the assessment was done by consensus between the researchers. data analysis studies were included if they provided data regarding accu- racy of mra for detecting residual aneurysmal flow, as defined using the raymond-roy or a comparable scale. these data were used to construct × contingency tables. meta-disc software (http://www. hrc. es/ investigacion/ metadisc_ en. htm) was used for statistical analysis. pooled sensitivity and specificity with % confidence intervals were calculated and subgroup analyses were o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://www.hrc.es/investigacion/metadisc_en.htm http://jnis.bmj.com/ of ahmed su, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - neuroimaging table sensitivity and specificity for time-of-flight magnetic resonance angiography (tof-mra) and contrast-enhanced (ce)-mra versus digital subtraction angiography (dsa) tof-mra ce-mra sensitivity ( % ci) specificity ( % ci) positive lr ( % ci) negative lr ( % ci) sensitivity ( % ci) specificity ( % ci) positive lr ( % ci) negative lr ( % ci) recanalization . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) residual neck . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) residual dome . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) figure pooled sensitivity and specificity for aneurysm recanalization. (a) contrast-enhanced magnetic resonance angiography (ce-mra) sensitivity. (b) ce-mra specificity. (c) time-of-flight (tof)-mra sensitivity. (d) tof-mra specificity. carried out. forest plots and summary receiver operating charac- teristic curves were used to present the data. results study inclusion the search method yielded , , and records in the pubmed, cochrane, and embase databases, respectively. after removal of duplicates, records were screened using titles and abstracts. seventy-nine records were then used to obtain full- text articles, which were reviewed using the inclusion and exclu- sion criteria. – after full-text review, studies were excluded (figure ). one study was a review article and was excluded. three studies were excluded as they did not assess follow-up of patients treated with endovascular techniques, and a further three were excluded as all patients did not receive mra and dsa at follow-up. eight studies were excluded as there was no comparison of mra and dsa, – and two because tof and ce-mra data could not be separated. sixteen studies were excluded as they did not contain enough informa- tion for × contingency tables, – – and three were excluded because the data were used in another study. hence, studies remained and were included in the analysis (figure ): studies assessed coiling, – of which studies included cases of stent assistance ( – % of cases) ; five studies assessed stent-assisted coiling – ; and two studies assessed flow diversion. of the studies assessing intracranial stent use, five addressed the status of the parent vessel. grade assessment the methodological quality of the included studies was assessed using the grade criteria, as applied to studies of diagnostic test accuracy. since the included studies were comparing the diag- nostic accuracy of mra to dsa using a cohort, all were initially granted the maximal starting score of . one study included separate cohorts of patients treated with flow diversion and coiling, and both were assessed separately. six studies were rated down for indirectness and two for inconsis- tency. seventeen studies had deductions made due to quality issues such as using non-consecutive patients or non-blinded assessment of tests. – overall, studies ( %) were given a score of , ( %) were scored , ( %) studies scored , while ( %) study scored . data analysis overall sensitivity and specificity of tof-mra and ce-mra are presented in table and summarized in figure . for tof-mra, sensitivity for any recanalization was % ( % ci % to %) and specificity was % ( % ci % to %). ce-mra had similar sensitivity and slightly higher specificity at % ( % ci % to %) and % ( % ci % to %), respectively. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://jnis.bmj.com/ of ahmed su, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - neuroimaging table subgroup analysis of different treatment techniques comparing sensitivity and specificity of time-of-flight magnetic resonance angiography (tof-mra) and contrast-enhanced (ce)-mra treatment tof-mra ce-mra sensitivity ( % ci) specificity ( % ci) positive lr ( % ci) negative lr ( % ci) sensitivity ( % ci) specificity ( % ci) positive lr ( % ci) negative lr ( % ci) coiling . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) stent- assisted coiling + flow diversion . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) figure pooled sensitivity and specificity for aneurysm recanalization for cases of flow diversion and stent-assisted coiling. (a) constrast-enhanced magnetic resonance angiography (ce-mra) sensitivity. (b) ce-mra specificity. (c) time-of-flight (tof)-mra sensitivity. (d) tof-mra specificity. neck residuals were more difficult to detect using either tech- nique, with sensitivity of % ( % ci % to %) using tof-mra and % ( % ci % to %) using ce-mra. table summarizes the subgroup analysis for coiled aneu- rysms compared with stent-assisted coiling and flow diversion techniques. for these techniques, sensitivity and specificity of tof-mra was % ( % ci % to %) and % ( % ci % to %), while ce-mra showed sensitivity and spec- ificity of % ( % ci % to %) and % ( % ci % to %), respectively (figures and ). studies that included stent-assisted coiling in their coiling cohort were excluded from this subgroup analysis. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://jnis.bmj.com/ of ahmed su, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - neuroimaging figure summary receiver operating characteristic for flow diversion and stent-assisted coiling cases. (a) contrast-enhanced magnetic resonance angiography (ce-mra). (b) time-of-flight (tof)-mra. table subgroup analysis of various imaging and study characteristics study characteristic tof-mra ce-mra sensitivity ( % ci) specificity ( % ci) positive lr ( % ci) negative lr ( % ci) sensitivity ( % ci) specificity ( % ci) positive lr ( % ci) negative lr ( % ci) d dsa . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) d dsa . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . t mra . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) t mra . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) grade – . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) grade – . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) prospective . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) retrospective . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) ce, constrast-enhanced; grade, grades of recommendation, assessment, development, and evaluation; mra, magnetic resonance angiography; tof, time-of-flight. further subgroup analyses were performed by distinguishing different study characteristics: dsa technique ( d vs d), mri field strength ( . t vs t), strength of evidence, and study design (prospective vs retrospective). these results are summa- rized in table . discussion the results of this study show that mri techniques remain reli- able in the detection of residual aneurysms after endovascular treatment. furthermore, subgroup analysis shows high sensi- tivity and specificity for stent-assisted coiling and flow diversion techniques. while both sensitivity and specificity are integral to the assessment of a diagnostic test, the clinical context of the study will help determine which characteristic is of greater value. aneurysm follow-up using mra alone will have to provide a high degree of sensitivity in order to capture all recanalizations. the overall sensitivity and specificity of mra is higher than that found previously by van amerongen et al and menke et al. in the study by van amerongen et al, sensitivity and specificity of ce-mra for any residual were % and %, respectively, compared with % and % for tof-mra. furthermore, both the sensitivity and specificity of ce-mra were higher than that for tof-mra in our study, unlike the previously reported literature. contrast timing and a narrow scanning interval were posited as potential reasons for lower rates of ce-mra sensi- tivity and specificity in the past, and overall improvements in these scanning characteristics may be the reasons for this improvement. rates of detection of aneurysm dome residuals were higher than those for neck residuals using both mra techniques. this was an expected finding, but was not observed in previously reported meta-analyses. one possible explanation for this was o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://jnis.bmj.com/ of ahmed su, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - neuroimaging insufficient data specifying the degree of residual aneurysm, as evidenced by wider % cis in previous meta-analyses. the overall sensitivity and specificity of dome residuals has increased significantly, but % cis remain wide for both tof-mra and ce-mra. the sensitivity and specificity of mra techniques for detecting aneurysm residuals in patients undergoing stent-assisted coiling and flow diversion was comparable to the coiling-only group in our subgroup analysis. this was an unexpected finding, since stent-associated artifacts are felt to interfere with assessment of residual aneurysm flow. more false negatives are expected with tof compared with ce-mra, and this is borne out by the lower tof sensitivity compared with ce-mra for aneu- rysms treated with intracranial stents. however, stent use does not appear to affect tof sensitivity compared with coiling-only. tof sensitivity was higher for the stent-assisted/flow diversion group since fewer false positives were identified. this result lends reassurance to the follow-up of these patients using mra. however, only seven studies were available for the stent-assisted and flow diversion subgroup, and further study is required to confirm this finding. it is also important to note that nine studies with varying incidence of stent deployment were excluded from this subgroup analysis as they did not separately report cases of stent-assisted coiling. the status of the parent vessel is another question that arises in cases of intracranial stent placement. of the seven studies that assessed intracranial stent deployment, five addressed the patency of the parent vessel. in another study, tof-mra was felt to be incapable of accurately assessing parent vessel patency and only dsa data were reported. of the five studies that did compare mra and dsa, one rated the ability of mra to assess in-stent stenosis as poor in > % of cases. one study reported significantly higher rates of in-stent stenosis with ce-mra, with false positives found in % of cases. attali et al and akkaya et al both found perfect sensitivity with both tof-mra and ce-mra for in-stent stenosis, but low rates of specificity for both tof-mra ( % and %) and ce-mra ( % and %), respectively. van amerongen et al described five possible sources of heterogeneity in their data, including publication bias, enrollment methods, dsa technique used as the reference standard, mri magnet field strength, and study quality. these reasons also apply to our meta-analysis. significantly higher sensitivity and specificity were found for grade – studies than for grade – studies. furthermore, retrospective studies showed higher sensitivity and specificity than prospective studies for both tof-mra and ce-mra. a further potential source of heterogeneity is the time interval between the reference dsa and the mra study. the mean inter- vals were ≤ days in studies; – days for two studies; – days for four studies; and > days in one study ( days). six studies did not specify this interval. a significant time interval between the studies will artificially raise or lower the sensitivity and specificity if the aneurysm occlusion status changes within this time period. this change will also depend on which test was performed earlier. two processes in the time course of aneu- rysm evolution after endovascular treatment are important in this regard: ( ) the progressive thrombosis and occlusion of an aneurysm with residual flow at the end of the procedure; and ( ) aneurysm recanalization after initial occlusion. further investi- gation, especially involving flow diversion, should minimize the interval between comparison studies in order to minimize this source of heterogeneity. applicability of the data to clinical practice is a valid concern. some aneurysms, such as paraclinoid carotid and carotid cave aneurysms, may be more difficult to assess and poten- tially reduce the diagnostic performance of mra techniques. other factors such as aneurysm size are also potential imped- iments to diagnostic performance. the studies included in the meta-analysis do not specify the aneurysm morphology beyond the vessel location, and thus this information cannot be gleaned from this meta-analysis. however, most of the included studies ( / , see online supplementary materials) included consecu- tive patients, thus providing an accurate sampling of aneurysms treated in a clinical setting. further studies should aim to address these factors when assessing mra performance. the inter- and intra-rater reliability was fair-to-high in the studies that addressed this topic. however, there were few observers in all of the included studies. a larger study of inter-rater reliability of angiographic occlusion of coiled aneurysms found fair concordance (kappa= . ) between a multicenter group of neurosurgeons and neuroradiologists. further research in this domain may benefit from voxel-based assessment of aneurysm recanalization, which has recently been investigated. conclusion this study demonstrates the reliability of mra techniques in the follow-up of aneurysms treated using endovascular tech- niques, with excellent rates of sensitivity and specificity for both tof-mra and ce-mra. sensitivity and specificity were both higher for dome residuals, which are more likely to require retreatment, than for neck residuals. follow-up of aneurysms treated with intracranial stents, with or without adjunctive use of coils, also showed high rates of sensitivity and specificity for both techniques. further studies may add to this body of knowledge and may also provide data to assess the parent vessel following intracranial stent placement. contributors sua and rdl: study design, data acquisition and analysis, manuscript preparation, and editing. jm: study design, manuscript preparation, and editing. xz: data analysis, manuscript preparation, and editing. mk, ad, and kn: study design, and manuscript editing. all authors were involved with manuscript final approval and agree to be accountable for all aspects of the work. funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests none declared. patient consent for publication not required. provenance and peer review not commissioned; 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: – . . nawka mt, sedlacik j, frölich a, et al. multiparametric mri of intracranial aneurysms treated with the woven endobridge (web): a case of faraday’s cage? j neurointerv surg ; : – . . akkaya s, akca o, arat a, et al. usefulness of contrast-enhanced and tof mr angiography for follow-up after low-profile stent-assisted coil embolization of intracranial aneurysms. interv neuroradiol ; : – . . jp f, liu l, zhao h, et al. the efficacy of enterprise stent-assisted coil embolization in the treatment of intracranial wide necked aneurysms by magnetic resonance angiography. int j clin experiment med ; : – . . ikemura a, yuki i, suzuki h, et al. time-resolved magnetic resonance angiography (tr-mra) for the evaluation of post coiling aneurysms: a quantitative analysis of the residual aneurysm using full-width at half-maximum (fwhm) value. plos one ; :e . . zuckerman sl, lakomkin n, magarik ja, et al. evaluation of previously embolized intracranial aneurysms: inter-and intra-rater reliability among neurosurgeons and interventional neuroradiologists. j neurointerv surg ; : – . . ernst m, buchholz a, bourcier r, et al. voxel based analysis of recurrence dynamics in intracranial aneurysms after coiling. j neurointerv surg ; : – . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n m a y . d o w n lo a d e d fro m http://dx.doi.org/ . /s - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.ejrnm. . . http://dx.doi.org/ . / .str. . . b http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /j.inat. . . http://dx.doi.org/ . /j.wneu. . . http://dx.doi.org/ . /rct. b e e http://dx.doi.org/ . /rct. b e e http://dx.doi.org/ . /inr- - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /neu. http://dx.doi.org/ . /j.neurad. . . http://dx.doi.org/ . /neurintsurg- - http://dx.doi.org/ . /neurintsurg- - http://dx.doi.org/ . / .str. . .a http://dx.doi.org/ . /s - - -z http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /ajnr.a http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . / s http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . / . . http://dx.doi.org/ . / . . http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /rct. b e bd http://dx.doi.org/ . /s http://dx.doi.org/ . / http://dx.doi.org/ . /neurintsurg- - http://dx.doi.org/ . /j.jocn. . . http://dx.doi.org/ . /j.jocn. . . http://dx.doi.org/ . / http://dx.doi.org/ . / http://dx.doi.org/ . /kjr. . . . http://dx.doi.org/ . /j.neurad. . . http://dx.doi.org/ . /con. http://dx.doi.org/ . /neurintsurg- - http://dx.doi.org/ . /neurintsurg- - http://dx.doi.org/ . / http://dx.doi.org/ . /journal.pone. http://dx.doi.org/ . /neurintsurg- - http://dx.doi.org/ . /neurintsurg- - http://jnis.bmj.com/ 	mra versus dsa for the follow-up imaging of intracranial aneurysms treated using endovascular techniques: a meta-analysis 	abstract 	introduction 	methods 	study acquisition 	study inclusion 	data acquisition 	study quality 	data analysis 	results 	study inclusion 	grade assessment 	data analysis 	discussion 	conclusion 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ [pdf] a systematic review and meta-analysis of the prevalence of chronic widespread pain in the general population | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /j.pain. corpus id: a systematic review and meta-analysis of the prevalence of chronic widespread pain in the general population @article{mansfield asr, title={a systematic review and meta-analysis of the prevalence of chronic widespread pain in the general population}, author={kathryn e mansfield and j. sim and j. jordan and k. jordan}, journal={pain}, year={ }, volume={ }, pages={ - } } 	kathryn e mansfield, j. sim, + author k. jordan 	published 	medicine 	pain abstract chronic widespread pain (cwp) is common and associated with poor general health. there has been no attempt to derive a robust prevalence estimate of cwp or assess how this is influenced by sociodemographic factors. this study therefore aimed to determine, through a systematic review and meta-analysis, the prevalence of cwp in the adult general population and explore variation in prevalence by age, sex, geographical location, and criteria used to define cwp. medline, embase, cinahl, and… expand view on wolters kluwer europepmc.org save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all topics from this paper 	widespread chronic pain 	confidence intervals 	estimated 	pooled sample 	genetic heterogeneity 	conflict (psychology) 	eligibility determination paper mentions observational clinical trial mixed methods study protocol_chronic pain and marginalized populations a mixed-methods sequential explanatory design study. the first quantitative phase will be a multi-language survey that includes questions related to pain status, patient beliefs, pain… expand 	conditions	chronic pain, marginalization, social 	intervention	other high point university february - december blog post drug dealer, md: how doctors were duped, patients got hooked and why it’s so hard to stop – book review lse business review april blog post long read review: drug dealer, md: how doctors were duped, patients got hooked and why it’s so hard to stop by anna lembke lse review of books march citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency chronic widespread pain prevalence in the general population: a systematic review 	p. andrews, m. steultjens, j. riskowski 	medicine 	european journal of pain 	 	 	highly influenced 	 	view excerpts, cites methods and background save alert research feed prevalence of chronic pain in the uk: a systematic review and meta-analysis of population studies 	a. fayaz, p. croft, r. langford, l. donaldson, g. jones 	medicine 	bmj open 	 	 	pdf save alert research feed prevalence of hoarding disorder: a systematic review and meta-analysis. 	a. postlethwaite, s. kellett, d. mataix-cols 	medicine 	journal of affective disorders 	 	 	pdf save alert research feed distribution and prevalence of musculoskeletal pain co-occurring with persistent low back pain: a systematic review 	c. Øverås, m. johansson, + authors j. hartvigsen 	medicine 	bmc musculoskeletal disorders 	 	pdf 	view excerpt, cites background save alert research feed global and regional estimates of dental pain among children and adolescents—systematic review and meta-analysis 	k. pentapati, sravan kumar yeturu, h. siddiq 	medicine 	european archives of paediatric dentistry : official journal of the european academy of paediatric dentistry 	 	 	pdf 	view excerpt save alert research feed prevalence of primary angle closure glaucoma in the last years: a meta-analysis and systematic review 	nan zhang, jiaxing wang, biyue chen, y. li, bing jiang 	medicine 	frontiers in medicine 	 	 	view excerpt, cites methods save alert research feed the impact of chronic widespread pain on health status and long-term health predictors: a general population cohort study 	c. sylwander, i. larsson, m. andersson, s. bergman 	medicine 	bmc musculoskeletal disorders 	 	 	highly influenced 	 	view excerpts, cites background and results save alert research feed prevalence of posttraumatic stress disorder in persons with chronic pain: a meta-analysis 	j. siqveland, a. hussain, j. lindstrøm, t. ruud, e. hauff 	medicine 	front. psychiatry 	 	 	 save alert research feed neuropathic pain prevalence of older adults in an urban area of iran: a population-based study 	reza salman roghani, a. delbari, m. asadi-lari, v. rashedi, j. lökk 	medicine 	pain research and treatment 	 	 	pdf 	view excerpt, cites background save alert research feed a cross-sectional study of factors associated with the number of anatomical pain sites in an actual elderly general population: results from the pains + cohort 	e. dragioti, b. larsson, l. bernfort, l. levin, b. gerdle 	medicine 	journal of pain research 	 	 	pdf 	view excerpts, cites background and results save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency chronic musculoskeletal pain, prevalence rates, and sociodemographic associations in a swedish population study. 	s. bergman, p. herrström, k. hoegstroem, i. petersson, b. svensson, l. jacobsson 	medicine 	the journal of rheumatology 	 	 	view excerpts, references background save alert research feed epidemiology of chronic non-cancer pain in europe: narrative review of prevalence, pain treatments and pain impact 	k. reid, j. harker, + authors j. kleijnen 	medicine 	current medical research and opinion 	 	 	pdf save alert research feed [the prevalence of chronic generalized pain and its relationship to demographic characteristics and mental status]. 	o. n. storozhenko, o. lesniak, g. macfarlane, j. mcbeth 	medicine 	klinicheskaia meditsina 	 	 	view excerpts, references background save alert research feed prevalence and long-term predictors of persistent chronic widespread pain in the general population in an -year prospective study: the hunt study 	ingunn mundal, r. gråwe, j. bjørngaard, o. linaker, e. fors 	medicine 	bmc musculoskeletal disorders 	 	 	 	view excerpt save alert research feed chronic musculoskeletal pain rarely presents in a single body site: results from a uk population study. 	d. carnes, s. parsons, + authors m. underwood 	medicine 	rheumatology 	 	 	pdf save alert research feed the prevalence and associated features of chronic widespread pain in the community using the 'manchester' definition of chronic widespread pain. 	i. hunt, a. silman, s. benjamin, j. mcbeth, g. macfarlane 	medicine 	rheumatology 	 	 	pdf save alert research feed [prevalence of chronic pain in germany. a representative survey of the general population]. 	w. häuser, g. schmutzer, a. hinz, a. hilbert, e. brähler 	medicine 	schmerz 	 	 save alert research feed prevalence of chronic pain in a representative sample in the united states. 	j. hardt, clemma jacobsen, j. goldberg, r. nickel, d. buchwald 	medicine 	pain medicine 	 	 	pdf 	view excerpts, references methods save alert research feed the epidemiology of chronic syndromes that are frequently unexplained: do they have common associated factors? 	v. aggarwal, j. mcbeth, j. zakrzewska, m. lunt, g. macfarlane 	medicine 	international journal of epidemiology 	 	 	pdf 	view excerpts, references background save alert research feed chronic widespread pain in the population: a seven year follow up study 	a. papageorgiou, a. silman, g. macfarlane 	medicine 	annals of the rheumatic diseases 	 	 	pdf 	view excerpts, references background save alert research feed ... ... related papers 	abstract 	topics 	paper mentions 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators blog posts, news articles and tweet counts and ids sourced by altmetric.com terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue notes from the field: multidrug-resistant tuberculosis among workers at two food processing facilities — ohio, – morbidity and mortality weekly report mmwr / august , / vol. / no. us department of health and human services/centers for disease control and prevention notes from the field: multidrug-resistant tuberculosis among workers at two food processing facilities — ohio, – amish talwar, md , ; rebekah stewart, msn, mph ; sandy p. althomsons, ma, mhs ; jessica rinsky, phd ; david a. jackson, md , ; maria e. galvis ; philip graham ; moises a. huaman, md ; james karrer ; karthik kondapally, mbbs ; sarah mitchell, ms ; jonathan wortham, md ; sietske de fijter, ms during – , the ohio department of health (odh) reported three cases of multidrug-resistant tuber- culosis (mdr tb)* in persons who worked in two food processing facilities. the national tuberculosis molecular surveillance center† performed whole genome sequencing of a mycobacterium tuberculosis isolate from each patient; phylo- genetic analysis revealed the isolates were genetically identical. prompted by concern for mdr tb transmission associated with these workplaces and surrounding communities, odh began an investigation in february . cdc was invited to assist with the investigation and deployed a team to ohio on april , . the cdc-odh team, which included representatives from cdc’s division of tuberculosis elimination and the national institute for occupational safety and health (niosh), reviewed medical and employment records, conducted prin- cipal informant interviews, and conducted a tour of one of the facilities (facility a) where the three patients worked. the third patient also worked at a second facility (facility b), which had closed as part of an unrelated business restructuring before the cdc-odh team could begin its investigation; facility a remained operational throughout the investigation. a separate niosh team had visited facility b before it closed to conduct a health hazard evaluation following notification that one the facility’s employees had mdr tb; observations from that visit were used to guide the exposure assessment of facility b employees. the index case occurred in a person born in one of the countries designated by the world health organization as having a high prevalence of mdr tb ( ). according to available work schedules, during the index patient’s infectious period, the second and third patients had worked for at least days and days,§ respectively, on the same food production line as the index patient. the investigation team was unable * mdr tb is a form of tuberculosis caused by m. tuberculosis resistant to isoniazid and rifampin, two cornerstone drugs used in the first-line tb treatment regimen. † https://www.cdc.gov/tb/topic/laboratory/default.htm. § these reflect minimum counts because complete daily employment records were unavailable. to find any other potential transmission venues or common exposures among the three patients. no additional cases of mdr tb related to this group of patients were identified. however, contacts of the three mdr tb patients were identified, including who were workplace contacts; the majority of contacts were non–u.s.-born persons. contacts were prioritized according to levels of possible tb exposure; contacts, including workplace and personal contacts, had the highest risk of exposure (high-priority contacts).¶ as of april , , a total of ( %) of the high-priority workplace contacts had been tested for tb infection, ( %) of whom had positive results for a tuberculin skin test or interferon-g release assay test, both of which test for tb infection. among those with positive test results, ( %) began latent tuberculosis infec- tion treatment (table). among the overall u.s. population, an estimated % of non–u.s.-born persons have a positive tuberculin skin test in the united states, and % have a posi- tive interferon-g release assay result ( ). the higher percentage of positive tb test results at the workplace provides evidence for likely workplace transmission. based on principal informant interviews, likely contributors to the low level of tb testing and treatment for infection among contacts included difficulties in communication, perceived barriers to care, and mistrust of government authorities. after the investigation concluded on april , , all three patients with mdr tb disease had either recovered or were continuing to recover, and no additional cases have been identified. odh continues to work with its local partners to facilitate tb testing and treatment of contacts with latent tb infection and to monitor for new cases. mdr tb is rare in the united states (< % of tb cases annu- ally since ) ( , ); in , there were mdr tb cases in the united states out of a total of , tb cases ( ). although the tb transmission source for the index patient remains uncer- tain, the low prevalence of mdr tb in the united states and the absence of other genotype-matched tb cases in the national tb molecular surveillance database indicate that the patient was likely infected in the patient’s country of origin. given the non- specific signs and symptoms of tb, health care providers should consider tb when examining persons with cough, chest pain, ¶ high-priority contacts include named contacts and workplace contacts with documented direct exposure to an mdr tb patient, health care workers with documented direct exposure to an mdr tb patient when the patient was contagious and not under airborne infection isolation, and contacts with risk factors for tb, such as human immunodeficiency virus infection, diabetes mellitus, end stage renal disease, or immunosuppression. https://www.cdc.gov/tb/topic/laboratory/default.htm morbidity and mortality weekly report mmwr / august , / vol. / no. us department of health and human services/centers for disease control and prevention table. tuberculosis (tb) care cascade for high-priority* contacts of three patients with multidrug-resistant tb — ohio, april contact type no. of high- priority contacts no. (%) tested† tested, with positive tb test result† tested, with positive tb test result and started on ltbi treatment workplace ( ) ( ) ( ) facility a ( ) ( ) ( ) facility b ( ) ( ) § ( ) personal¶ ( ) ( ) ( ) total ( ) ( ) ( ) abbreviation: ltbi = latent tuberculosis infection. * includes named contacts and workplace contacts with documented direct exposure to a multidrug-resistant (mdr) tb patient, health care workers with documented direct exposure to an mdr tb patient when the patient was contagious and not under airborne infection isolation, and contacts with risk factors for tb, such as human immunodeficiency virus infection, diabetes mellitus, end stage renal disease, or immunosuppression. † includes five contacts who were tested with interferon-γ release assay (quantiferon-tb gold in-tube test), three of whom had positive test results (all personal contacts). § initiation of treatment was pending drug-susceptibility testing results, as of april , . ¶ includes contacts who spent substantial time with patients at home. hemoptysis, weight loss, fever, chills, night sweats, weakness, fatigue, or loss of appetite, especially when the person has tb risk factors, including birth in areas with high rates of tb.**,†† in addition, providers should consider prompt molecular detec- tion of drug-resistance testing for tb patients with risk factors for drug-resistant tb.§§,¶¶ finally, public health agencies need to facilitate engagement with communities with higher rates of tb to build trust, which is important for successful disease investigations. activities might include communicating in a culturally sensitive manner with community members, offer- ing patients incentives for getting tested or treated, providing transportation to clinics, using mobile clinics, and conducting communitywide education efforts. ** https://www.cdc.gov/tb/topic/basics/signsandsymptoms.htm. †† https://www.cdc.gov/tb/topic/basics/risk.htm. §§ risk factors for drug-resistant tb include failure to adhere to or complete tb treatment, incorrect tb treatment (i.e., incorrect dose or length of treatment prescribed), prior tb treatment, residence in areas of the world where drug-resistant tb is common, and known contact with patients with drug-resistant tb. ¶¶ https://www.cdc.gov/tb/topic/drtb/default.htm. acknowledgments hamilton county public health personnel, ohio; butler county general health district personnel, ohio; ohio department of health personnel; paul regan, cdc; marie de perio, mark m. methner, kevin h. dunn, duane r. hammond, national institute for occupational safety and health, cdc. corresponding author: amish talwar, atalwar@cdc.gov, - - . division of tuberculosis elimination, national center for hiv/aids, viral hepatitis, std, and tb prevention, cdc; epidemic intelligence service, cdc; national institute for occupational safety and health, cdc; hamilton county public health, ohio; butler county general health district, ohio; ohio department of health. all authors have completed and submitted the international committee of medical journal editors form for disclosure of potential conflicts of interest. no potential conflicts of interest were disclosed. references . world health organization. global tuberculosis report . geneva, switzerland: world health organization; . https://www.who.int/tb/ publications/global_report/en/ . miramontes r, hill an, yelk woodruff rs, et al. tuberculosis infection in the united states: prevalence estimates from the national health and nutrition examination survey, – . plos one ; :e . https://doi.org/ . /journal.pone. . cdc. tuberculosis in the united states – : national tuberculosis surveillance system. atlanta, ga: us department of health and human services, cdc; . https://www.cdc.gov/tb/statistics/surv/surv / pdf/ -surveillance-report-slideset.pdf . curry international tuberculosis center; california department of public health. epidemiology and background. in: chen l and schecter gf, eds. drug-resistant tuberculosis: a survival guide for clinicians. rd ed. san francisco, ca: curry international tuberculosis center; . . cdc. tuberculosis: reported tuberculosis in the united states, . atlanta, ga: us department of health and human services, cdc; . https://www.cdc.gov/tb/statistics/reports/ /national_data.htm https://www.cdc.gov/tb/topic/basics/signsandsymptoms.htm https://www.cdc.gov/tb/topic/basics/risk.htm https://www.cdc.gov/tb/topic/drtb/default.htm mailto:atalwar@cdc.gov https://www.who.int/tb/publications/global_report/en/ https://www.who.int/tb/publications/global_report/en/ https://doi.org/ . /journal.pone. https://www.cdc.gov/tb/statistics/surv/surv /pdf/ -surveillance-report-slideset.pdf https://www.cdc.gov/tb/statistics/surv/surv /pdf/ -surveillance-report-slideset.pdf https://www.cdc.gov/tb/statistics/reports/ /national_data.htm wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ embracing microbes in exposure science journal of exposure science & environmental epidemiology ( ) : – https://doi.org/ . /s - - - article embracing microbes in exposure science william w nazaroff received: june / revised: august / accepted: september / published online: september © springer nature america, inc. abstract although defined more broadly, exposure science has mainly focused on exposures to environmental chemicals and related stressors, such as airborne particulate matter. there is an opportunity for exposure science to contribute more substantially to improving public health by devoting more attention to microorganisms as key stressors and agents in exposure. the discovery that pathogenic microbes cause disease in humans precipitated a revolution in public health science and disease prevention. with a continued global urgency to address spread of pathogenic microbes, contributions of microorganisms to both infectious and noninfectious processes merit more attention from the exposure science community. today, discoveries of the importance of the human microbiome as a determinant of health and disease are precipitating a second revolution. emerging knowledge creates a major opportunity to expand the scope of exposure science to incorporate the human microbiome as a target and modulator of exposure. a study committee of the national academies of sciences, engineering, and medicine has defined a research strategy to address health risks that pertain to the interaction of environmental chemicals with the human microbiome. some aspects of this strategy pose important challenges and opportunities for the exposure science community. keywords environmental chemical ● health risk ● human microbiome ● infectious disease infectious agents, other microbiologic stressors, and exposure science among the greatest achievements of humankind is the understanding that infectious microbes cause disease. the consequent development and application of that under- standing has contributed mightily to improvements in public health. historical examples highlight the scale of importance. the black death (plague), caused by yersinia pestis and peaking during the middle of the th century, was responsible for the deaths roughly million people in eurasia. stenseth et al. [ ] conclude that for today and future conditions, “plague should be taken much more seriously by the international community than appears to be the case.” the flu pandemic, involving the h n influenza virus, infected million people worldwide and resulted in deaths of about million [ ]. in each of these cases, the mortality totals are similar in scale to those associated with the world wars of the th century. in reviewing the history of tuberculosis, “an ancient scourge,” daniel [ ] suggests that “mycobacterium tuberculosis may have killed more persons than any other microbial pathogen.” in the struggle to understand and effectively respond to diseases caused by microorganisms, many important sci- entific and technological contributions can be noted. an example is john snow’s demonstration of a role for drinking water contamination in the cholera outbreak in london [ ]. investigations of microbial agents as causes of infectious disease, such as tuberculosis, were central in the formulation of the henle-koch postulates, the “classical point of reference in relating causative agents to disease.” [ ] robert koch was awarded the nobel prize in medicine or physiology in “for his investigations and dis- coveries in relation to tuberculosis” (https://www. nobelprize.org/nobel_prizes/medicine/laureates/ /). in efforts to battle cholera outbreaks, max von pettenkofer, the “founder of modern hygienic science,” made important contributions to public health, including “promotion of sanitary reforms, adequate pressurized water supply, and a sufficient sewage network.” [ ] * william w nazaroff nazaroff@berkeley.edu department of civil and environmental engineering, university of california, berkeley, ca - , usa () ;, : () ;,: http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf https://www.nobelprize.org/nobel_prizes/medicine/laureates/ / https://www.nobelprize.org/nobel_prizes/medicine/laureates/ / mailto:nazaroff@berkeley.edu the scientific achievements of the th century in understanding the roles of microbes as the causes of infectious diseases were followed by major technological developments throughout the th century to prevent and treat infectious disease. a prime example is the use of chemical disinfection for municipal drinking water, building on seminal investigations of disinfection kinetics [ ] and chemistry [ ]. in , readers of the british medical journal voted “sanitation (clean water and sewage dis- posal)” as the “most important medical milestone since .” [ ] other major achievements improving public health in response to the challenge posed by infectious microorganisms include the widespread use of antibiotics [ ] and the development of vaccines based on immunology [ ]. more mundane, yet still important, are handwashing and other hygienic practices in health-care settings [ , ]. despite major progress, understanding pathways of infectious disease transmission and how to control them remain important topics of scientific and public health concern. these issues are of keen interest with regards to specific diseases, such as influenza [ ], tuberculosis [ ], and sars [ ]. they are relevant to specific infrastructure components that may contribute to exposure pathways, as in the case of premise plumbing and its roles in legionnaire’s disease and pulmonary nontuberculous mycobacterial dis- ease [ , ]. infectious disease transmission is also a major concern in high-risk environments such as passenger air- craft [ ] and health-care facilities [ ]. given the importance of pathogenic organisms as causes of disease and given the importance of infectious disease in the realm of environmental and public health, it is surprising that the exposure science community devotes relatively little attention to the subject. the topic certainly lies within scope. referring to defi- nitions [ ] in the official glossary of the international society of exposure analysis (now the international society of exposure science), exposure is the “contact between an agent and a target.” an agent is “a chemical, biological, or physical entity that contacts a target.” and the target is “any biological entity that receives an exposure or a dose (e.g., a human, human population, or a human organ).” this set of definitions clearly provides for exposure science to include characterizing the nature, scope, and conditions that influence human exposure to pathogenic microbes. in , the national academies published exposure science in the st century: a vision and a strategy [ ]. that document defines exposure science as “the collection and analysis of quantitative and qualitative information needed to understand the nature of contact between recep- tors (such as people or ecosystems) and physical, chemical, or biologic stressors.” that report frequently uses the phrase (or close variants), “physical, chemical, or biologic stres- sors.” yet, it is almost silent on any of the manifold specific exposure issues that would arise in considering pathogenic microorganisms. consider, too, the history of work published in this journal. as of june , the web of science catalogued articles published in the journal of exposure science and environmental epidemiology (jesee) and its pre- decessor (with “science” replaced by “analysis”). of these, only about % are selected in a search in which the topic is “microbe” or “microbial” or “bacteria” or “fungus” or “virus.” to its credit, a special anniversary release of jesee, published in , included, among the sixteen brief arti- cles, two ( %) that were specifically microbial. they addressed, respectively, anthrax and the h n pan- demic flu virus. (online source: https://www.nature.com/ jes/articles?type = exposure-science-digests.) chemical methods for managing health risks from pathogens require contributions from exposure science to properly balance microbiological and chemical hazards. among noteworthy examples in this regard are exposures to disinfection byproducts in drinking water [ ]. such con- cerns contributed to a transition in water quality engineer- ing, substituting chloramines for chlorine as residual disinfectants. triclosan was developed and introduced as a broad-spectrum antibacterial agent in health-care settings [ ], but then more broadly applied in personal care and household products, raising a host of chemical toxicity concerns, which have contributed to an international call for limits on its production and use [ ]. a third example is the finding that heavy use of bleach in household cleaning is associated with increased risk of nonallergic asthma [ ]. in each of these cases, chemical exposures are clearly inter- twined with microbial concerns. in addition to infectious microbial agents, other biologic stressors contribute to adverse health risks. issues related to exposure science are not yet well resolved in many such cases. for example, dampness in buildings is an important risk factor for adverse respiratory outcomes [ ]. until now, it has not been possible to determine the underlying cause for the associations, and it remains unclear whether the most important exposures are microbial or chemical in nature. a case could be advanced that understanding the cause isn’t so important if an effective remedy is to remediate the underlying dampness. that pragmatic approach misses important opportunities for synergistic insights that can result from deeper understanding of the causal relationships. exposure science can make considerable contributions in improving knowledge about health risks associated with infectious agents and other biological stressors. for exam- ple, exposure analyses can be improved. many studies acknowledge the importance of exposure yet rely upon qualitative descriptors or weak proxy indicators such as the abundance of a stressor in an environmental medium w. w. nazaroff https://www.nature.com/jes/articles?type=exposure-science-digests https://www.nature.com/jes/articles?type=exposure-science-digests without adequate attention to the extent of contact between the target and that medium. exposure science can contribute improved mechanistic descriptions of the source-to-intake pathways in ways that would support mathematical mod- eling for risk assessment and risk management. exposure science can effectively contribute improved knowledge about exposures that occur through multiple pathways, for instance in assessing nosocomial viral infection risk [ ]. exposure science also is well-positioned to incorporate explicit consideration of human factors for parameters such as inhalation and ingestion rates. central tendency, popu- lation variability, and dependence on underlying factors (such as age, sex, and activity level) may be important considerations for quantifying exposures. likewise, expo- sure science is well suited to provide explicit consideration of human activity patterns [ ], illuminating where people are and when, essential information for properly relating a stressor’s abundance in an environmental medium to the consequent contact that constitutes an exposure. exposure science can play an important role in outbreak investigations, in epidemiological studies of patterns of infectious disease, and in the evaluation of technical and administrative interventions for public health protection. exposure science is particularly well positioned to con- tribute quantitative evidence needed to support rational tradeoffs when evaluating chemical interventions to protect public health against infectious agents [ ]. these aspects pertain closely to current value-added opportunities for exposure science. a key point to consider is this: while exposure science is designed to address microbial agents as biologic stressors, contributions are occurring at a lower rate than would be justified by public health significance in infectious and noninfectious disease risk assessment and risk management. second revolution: healthy human microbiome a second scientific revolution is underway in our under- standing of the dependence of human health on micro- organisms. the first revolution, considered in the previous section, was precipitated by the discovery that pathogenic microbes cause disease in humans. the second revolution is demonstrating that health depends in many important dimensions on proper composition and functioning of the human microbiome. awareness that humans have associated microbiota extends back at least several decades. however, under- standing the nature of these microbes and their importance for their human hosts has shifted rapidly. luckey wrote in the early s about intestinal microecology, noting the evolutionary linkage between enteric bacteria and the mammalian alimentary tract. drawing on experimental investigations of germ-free animals, luckey stated that “microbes are dispensable. life is possible without germs.” [ ] that opinion doesn’t prevail today. the idea that intestinal microbes might be health bene- ficial and that diet could influence their composition is identified with two terms: probiotic and prebiotic. con- sidering farm animals as well as humans, fuller [ ] pro- vides a scientific review of probiotics and offers this definition: “a live microbial feed supplement which bene- ficially affects the host animal by improving its intestinal microbial balance.” fuller concludes that, “there is good evidence that the complex microbial flora present in the gastrointestinal tract of all warm-blooded animals is effec- tive in providing resistance to disease.” precipitated in part by the advent of molecular approa- ches for measuring microbes, including quantitative pcr and next-generation sequencing, we are now seeing an explosion of scientific attention focusing on the micro- organisms associated with the human body. the earliest article in web of science with “human microbiome” in the title was published in . ley et al. [ ] wrote then that, with advances in metagenomics, “we start to see ourselves as supra-organisms whose genome evolved with associated microbial genomes.” in less than a half century, we are seeing a complete reversal from luckey’s “microbes are dispensable” [ ] perspective to a view in which we study ourselves “as an integral and dependent part of our microbe- dominated world.” [ ] let’s take a closer look at the human microbiome. first, how many human cells and how much bacterial matter are associated with humans? sender et al. [ ] have estimated that the number of bacterial cells ( trillion) is comparable to the number of human cells ( trillion). however, the number of nucleated human cells is estimated to be only % of the total, i.e. trillion, indicating that— by count— the human is clearly outnumbered by associated bacteria. furthermore, ley et al. [ ] observe that the human- associated microbial genes exceed our human genes by a factor of . here is a useful working definition: [ ] “the human microbiome is an all-encompassing term that refers to all microorganisms on or in the human body, their genes, and surrounding environmental conditions.” several major reviews have been published on the role of the human microbiome in health and disease, highlighting the property of resilience [ ], characterizing the “ranges and diversity of both taxonomic compositions and functional potentials” [ ] along with influential factors, and emphasizing the roles of appropriate microbial exposure during early life in shaping an effectively active immune system [ ]. the human microbiome exhibits enormous diversity along several axes. one portion of the diversity is biogeo- graphical: the microbiota inhabiting different portions of the body vary markedly. with regard to exposure science, the embracing microbes in exposure science most important parts of the human microbiome are likely to be those that are in most intimate contact with environ- mental media: the gastrointestinal tract, the respiratory tract, and the skin. by mass and by cell number, the dominant subsystem of the human microbiome is found in the gut. this component is also the most thoroughly studied with regard to health [ , ]. in their review, lynch and pedersen [ ] note that “gut microbiota dysbiosis—imbalances in the composition and function of these intestinal microbes— is associated with diseases ranging from localized gastroenterologic dis- orders to neurologic, respiratory, metabolic, hepatic, and cardiovascular illnesses.” marchesi et al. [ ] draw parallels between the gut microbiome and an immune system, “a collection of cells that work in unison with the host and that can promote health but sometimes initiate disease.” among the diseases and disorders considered in their review are “metabolic syndrome and obesity-related disease, liver disease, inflammatory bowel disease, and colorectal can- cer.” a fascinating feature of the gut microbiome is its influence in the two-way communication channel with the brain, referred to as the “gut-brain axis.” [ ] the gut microbiome is suspected to play “key role in the biological and physiological basis of neurodevelopmental, age-related and neurodegenerative disorders.” [ ] it has been posited that the dysbiosis of the gut microbiome may have central nervous system consequences contributing, e.g., to depres- sion when the intestinal barrier function is disrupted [ ]. “recent research has also linked microbial dysbiosis to neurological disorders, such as parkinson’s and alzhei- mer’s diseases, multiple sclerosis, and autism.” [ ] the mature gut microbiome is established during infancy and influenced by early-life exposures. “cessation of breast- feeding was identified as a major factor determining gut microbiota maturation.” [ ] focusing on differences between populations and over time, rook et al. [ ] write that the “immune system evolved to require input from at least three sources that we collectively term the ‘old friends’”. these include commensal microbes from mothers and other family members; organisms acquired from early- life environmental exposures; and the types of subclinical infections that could have persisted in “small isolated hunter-gatherer groups.” gensollen et al. [ ] speak to the importance of early life exposure conditions, writing that, “microbial colonization of mucosal tissues during infancy plays an instrumental role in the development and education of the host mammalian immune system. these early-life events can have long-standing con- sequences: facilitating tolerance to environmental exposures or contributing to the development of disease in later life, including inflammatory bowel disease, allergy, and asthma.” stein et al. [ ] have compared immunity and asthma risk in amish and hutterite farm children. these populations are from similar genetic stock and both rely on agrarian lifestyles. however, the amish follow more tradi- tional farming practices whereas the hutterites farm in a more industrialized manner. the prevalence of asthma and allergic sensitization in hutterite children was similar to that in the general us population, whereas it was – × lower in amish children. house dust in amish homes had elevated endotoxin levels. when instilled intranasally in mice, dust extracts from amish homes were found to inhibit airway hyperreactivity. not long ago, the prevailing view was that the human respiratory tract was sterile. that view might seem sur- prising, given what we now understand about the ability of microbes to inhabit and populate challenging micro- environments throughout the biosphere. notwithstanding considerable sampling challenges, we now know that the respiratory tract is well populated by microorganisms [ , ]. however, the specific understanding of the role of the lung microbiome in health and disease is trailing investi- gations of the gut microbiome. dickson et al. [ ] high- lighted “respiratory dysbiosis” as a factor in the “pathogenesis of exacerbations of chronic lung disease.” in a recent review, man et al. [ ] write, “the microbiota of the respiratory tract probably acts as a gatekeeper that provides resistance to colonization by respiratory patho- gens. the respiratory microbiota might also be involved in the maturation and maintenance of homeostasis of respira- tory physiology and immunity.” a specific area of concern is that certain respiratory tract microbes may con- tribute in a positive feedback cycle to inflammatory pro- cesses [ ]. the skin microbiome is characterized, among other features, by substantial variation across body sites accord- ing to local physiology, clustered into dry, moist, and sebaceous (oily) [ ]. the skin is an environmentally harsh and nutrient poor environment compared with the gut, and so the microbial biomass of the skin is considerably lower than that of the gut. the microbial communities on adult skin appear stable over years-long periods. initial coloni- zation differs between babies born through the vaginal canal as compared to those born by caesarian section. puberty is a time when the skin microbiota undergoes considerable change in composition. some common disorders are asso- ciated with dysbiosis in the skin microbiome, including acne, eczema, and chronic wounds. built environment and the human microbiome people spend % of their time indoors [ ]. do indoor environmental factors influence the human microbiome? a recent report of the national academies of sciences, engi- neering and medicine developed a “research agenda for indoor microbiology, human health, and buildings.” [ ] among the w. w. nazaroff stated research goals was to “elucidate the immunologic, physiologic, or other biologic mechanisms through which microbial exposures in built environments may influence human health.” the study committee reported that, “questions remain about the extent to which indoor microbiomes influence the composition and function of the human microbiome … and what that may mean for health outcomes.” there is ample evidence to support a concern that indoor environmental exposures to infectious agents can cause disease, even for healthy individuals. indoor environmental reservoirs govern the risk of legionnaires’ disease [ ] and probably contribute to the spread of norovirus [ ]. strong evidence has emerged that indoor airborne transmission is important for certain viral infectious diseases such as sars [ ] and influenza [ ]. evidence has also emerged that building microbiological factors can meaningfully interact with the human microbiome in the case of hospitalized premature infants [ ] and for individuals with seriously compromised immune systems [ ]. strong evidence is emerging that the microbiome of building occupants influ- ences the microbiology of indoor environments. for example, luongo et al. [ ] found that the sex of inhabitants could be discerned from bacterial sequencing of dust col- lected from dormitory rooms. lax et al. [ ] reported that household microbiota was “identifiable by family” and that “humans sharing a home were more microbially similar than those not sharing a home.” lehtimäki et al. [ ] stu- died skin microbiota and found an age-dependent difference between rural and urban children. given the pace of recent progress, we can anticipate the emergence of stronger evi- dence in the near future regarding the nature and extent to which microbiology of built environments interacts with the human microbiome [ – ]. nexus: environmental chemicals, human microbiome, health risk the concern that exposure to environmental chemicals can pose health risks combined with emerging knowledge that the human microbiome is an important agent in health and disease leads to a critical question: might interactions between the human microbiome and environmental che- micals influence human health risk? the us environmental protection agency and the national institute of environ- mental health sciences commissioned the us national academies of sciences, engineering, and medicine (nasem) to convene a study committee to address this topic. the primary charge: “to develop a research strategy to better understand the interactions between environmental chemicals and human microbiomes … and the implications of those interactions on human health risk.” [ ] the committee’s report can be freely downloaded from the national academies press. what follows is a brief summary and update, viewed through an exposure science lens. in reviewing the existing state of knowledge, the com- mittee identified several mechanisms of potential interest. exposure to an environmental chemical might directly influence the human microbiome in ways that impact health risk. scientific evidence to support the plausibility of this concern has emerged from recent studies. for example, the vulnerability of gut microbiota to alterations induced by chemical exposure has been demonstrated in the case of noncaloric artificial sweeteners [ ]. impaired glucose tol- erance, of concern in relation to type diabetes, was shown to be a consequence of the changes. more specifically relevant for environmental chemicals, hu et al. [ ] found that low dose exposures to common environmental chemi- cals—diethyl phthalate, methyl paraben, and triclosan— altered the composition of the gut microbiome in adolescent rats. jin et al. [ ] have reviewed the influence on gut microbiota in relation to health of “environmental pollutants including antibiotics, heavy metals, persistent organic pol- lutants, pesticides, nanomaterials, and food additives.” the microbiome also could modulate exposure to environmental chemicals, e.g., by altering the chemical form in ways that would affect their absorption, distribution, metabolism, and elimination (adme) [ – ]. claus et al. [ ] describe the capabilities of gut microbiota to metabo- lize environmental chemicals in terms of specific enzymatic families. they conclude that “there is a body of evidence suggesting that gut microbiota are a major, yet under- estimated element that must be considered to fully evaluate the toxicity of environmental contaminants.” although the majority of research attention concerning the environmental chemical–human microbiome–health risk nexus has focused on the gut microbiome, evidence is also emerging about other microbiome subsystems. for exam- ple, adar et al. [ ] reviewed the evidence concerning microbiome interactions with inhaled pollutants. they reported: “the respiratory microbiome has been shown to influence chronic lung disease exacerbations, and increasing evidence indicates a role in disease development. research also suggests that the respiratory microbiome could plau- sibly metabolize inhaled pollutants or modulate host inflammatory responses to exposure.” the research strategy put forward by the nasem committee emphasizes three subcomponents of the human microbiome associated, respectively, with the gut, the respiratory tract, and the skin. two categories of processes were the primary focus: effects that environmental chemi- cals might have on the composition and especially function of the human microbiome; and the roles that the micro- biome might play in modulating human exposures to environmental chemicals. also, being responsive to the embracing microbes in exposure science statement of task, the research strategy was explicitly attentive to the variation and variability in the microbiome, where variation captures differences in central tendency associated with factors such as body site, age, and sex, and variability describes the potentially continuous differences across populations in composition and functional attributes once other parameters are fixed. highlights of the research strategy priorities advanced in the committee’s report include the following: [ ] investigating “…the effects of environmental chemicals on the human microbiome and consequent changes to human health. the question is whether environmental-chemical exposures or doses that are in the range of known or anticipated human exposures can induce microbiome perturbations that modulate adverse health effects.” understanding “… the effects of the human microbiome on exposure to environmental chemicals. specifically, what is the role of a microbiome in modulating absorption, distribution, metabolism (activation or inacti- vation), and elimination (adme) of environmental chemicals?” considering implications of variation and variability in microbiomes for assessing risks from chemical expo- sures. “the human microbiome structure and function vary with, for example, body site, life stage, genetics, geography, and health status. the human microbiome also differs from microbiomes of animal species.” exposure science would play a key role in research that aims to illuminate the relationships among environmental chemicals, the human microbiome, and health risk. to that end, here are some key exposure-science findings quoted from the committee’s report: [ ] “adequate consideration of the roles of the human microbiome will improve understanding of the health risks posed by exposures to environmental chemicals.” “characterization of animal and human exposure and health risk has advanced through the use of biomonitor- ing, biomarkers, and physiologically based pharmacoki- netic models. those methods have not been consistently applied to or do not encompass aspects known to be important for the microbiome, such as life stage, sex, and disease state.” “there is a need to expand the scope of exposure science to incorporate the emerging understanding of the roles of the human microbiome and its components as agents that influence exposures to and risks posed by environmental chemicals.” the nasem report presented illustrations of several environmental chemicals for which incorporating full consideration of the human microbiome might alter the way that exposure is assessed or interpreted. three examples are briefly recapitulated here. formaldehyde is a toxic air contaminant and common indoor air pollutant [ ]. iarc has judged that there is “sufficient evidence in humans for the carcinogenicity of formaldehyde. formaldehyde causes cancer of the naso- pharynx and leukaemia.” [ ] formaldehyde is also used as a preservative for biological specimens. these features lead one to question whether some of the physiologic response to inhalation exposure for formaldehyde might be modu- lated by microbiota in the upper respiratory tract. “what is particularly germane is whether exposures to formaldehyde at concentrations encountered (or potentially encountered) in the environment interact with the microbiota in the upper airways in a manner that materially influences associated health risks, considering both irritancy responses associated with acute exposures and cancer risk associated with cumulative exposures.” [ ] phthalates are a class of compounds widely used in consumer products. among the health concerns associated with phthalate exposures are insulin resistance [ ], meta- bolic syndrome [ ] and reproductive and developmental toxicity [ ]. exposures can occur via multiple pathways, including ingestion, inhalation, and dermal absorption [ , ]. a recent study demonstrated that transdermal uptake from air could make a meaningful contribution to exposure to diethyl phthalate and di(n-butyl) phthalate [ ]. there are clues that microbes might influence exposure to phthalates. for example, it has been demonstrated that the skin is more permeable to the metabolite mono ( -ethylhexyl) phthalate (mehp) than to the parent compound di ( -ethylhexyl) phthalate (dehp) [ ]. nakamiya et al. [ ] have shown that microbes extracted from house materials can convert dehp to mehp. it is an open question whether skin microbiota carry out a similar conversion process. triclosan presents a challenging case for exposure sci- ence. its widespread incorporation into household and personal care products such as soaps and toothpastes have contributed to a high level of exposure intimacy [ ]. concerns have been raised about triclosan as a possible endocrine disrupting compound [ ] and as a contributor to antibacterial resistance in environmental media, such as indoor dust [ ]. as a broad spectrum antibacterial agent, there is also a basis for health-risk concerns in which microbiota in the human microbiome are an exposure target. scientific understanding of health risks associated with tri- closan as an environmental chemical is still developing. in a recent review, goodman et al. [ ] concluded that “the current body of epidemiologic literature does not allow a meaningful weight of evidence assessment due to the methodological limitations of individual studies and lack of inter-study consistency.” evidence for concern is w. w. nazaroff accumulating, however. ribado et al. [ ] conducted a randomized intervention study of triclosan and triclocarban (tc) in personal care products in households with new babies. they found that “antibiotic resistant species from the phylum proteobacteria … were enriched in stool sam- ples from mothers in tc households after the introduction of triclosan-containing toothpaste.” they reported that— independent of treatment —“infants with higher triclosan levels also showed an enrichment of proteobacteria spe- cies.” bever et al. [ ] studied triclosan in breast milk in relation to personal care product use by mothers. they reported that “bacterial diversity in the fecal microbiome of the infants exposed to breast milk with detectable triclosan levels differed compared to their peers exposed to milk containing non-detectable amounts.” using a mouse model, yang et al. [ ] found that brief exposure, “at relatively low doses, causes low-grade colonic inflammation, increases colitis, and exacerbates colitis-associated colon cancer in mice.” they attribute adverse effects of triclosan, in part, to “modulation of the gut microbiota.” “given widespread human exposure, research to investigate the effects of tri- closan on the human microbiome and to answer such questions as whether early-life exposure to triclosan is predisposing infants to adverse health outcomes seems warranted.” [ ] way forward for exposure science exposure science is an important domain and a young discipline. evidence of importance include the existence of this journal, an associated international society (the inter- national society of exposure science, ises), and consensus reports of the national academies focused on the discipline [ ]. evidence of youth include the relatively recent dates of founding of this journal ( ) and of ises ( ). with young disciplines, efforts toward systematization are necessary, as illustrated by the effort to define an exposure science ontology [ ]. paradigm shifting discoveries and discernment should also be anticipated, as in the case of the exposome [ , ]. at the time exposure science began, knowledge about microbes as agents of infectious disease was already well established, whereas knowledge about health risks asso- ciated with environmental chemicals was sparse. conse- quently, one might view the almost exclusive focus of exposure science on environmental chemicals as a rational approach. however, in the succeeding decades, accounting for progress made and recognizing the relative importance of the topics for public health, it seems worthwhile to adjust the balance, explicitly expanding the areas of concern in exposure science to include exposure aspects pertaining to infectious and noninfectious microbes as agents and as biologic stressors. over a time-scale comparable to the age of exposure sci- ence, knowledge about the microbes associated with humankind has undergone a revolutionary transformation, the ultimate outcomes of which are not yet clear. in the early s, luckey’s view may have prevailed: although microbes were common and prevalent on and in humans, they were nonessential. by the late s, an understanding of our dependence on microbiota as factors influencing our health had begun to take root. in the past few decades, we have seen a strong shift: human-associated microbes are no longer thought of being apart from us, but rather are increasingly recognized as an integral part of us, the human microbiome. the ramifications of this radical shift in perspective have not yet been absorbed in the exposure sciences. ises operates as a “global community of exposure sci- ence professionals.” to achieve its vision “to better our world, its ecosystems, and inhabitants,” much more atten- tion is needed to the microbial aspects of exposure science. in this perspective, i have aimed to provide a constructive critique, grounded in the belief that exposure science has much to offer and much to gain by increased attention to microbial aspects of its endeavors. acknowledgements preliminary study supporting the preparation of this article was undertaken in conjunction with the author’s service on the committee on advancing understanding of environmental- chemical interactions with the human microbiome, national acad- emy of sciences, engineering and medicine. financial support from the alfred p. sloan foundation (chemistry of indoor environments program) is gratefully acknowledged. compliance with ethical standards conflict of interest the author declares that he has no conflict of interest. references . stenseth nc, atshabar bb, begon m, belmain sr, bertherat e, carniel e, et al. plague: past, present, and future. plos med. ; 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: – . . rappaport sm. implications of the exposome for exposure sci- ence. j expo sci env epid. ; : – . w. w. nazaroff 	embracing microbes in exposure science 	abstract 	infectious agents, other microbiologic stressors, and exposure science 	second revolution: healthy human microbiome 	built environment and the human microbiome 	nexus: environmental chemicals, human microbiome, health risk 	way forward for exposure science 	compliance with ethical standards 	acknowledgments 	references am. j. hum. genet. : – , a novel nemaline myopathy in the amish caused by a mutation in troponin t jennifer j. johnston, richard i. kelley, , thomas o. crawford, d. holmes morton, richa agarwala, thorsten koch, alejandro a. schäffer, clair a. francomano, and leslie g. biesecker medical genetics branch and genetic disease research branch, national human genome research institute, information engineering branch, and computational biology branch, national center for biotechnology information, national institutes of health, bethesda; kennedy krieger institute and department of pediatrics and department of neurology and pediatrics, johns hopkins university, baltimore; the clinic for special children, strasburg, pa; and konrad-zuse-zentrum für informationstechnik, berlin the nemaline myopathies are characterized by weakness and eosinophilic, rodlike (nemaline) inclusions in muscle fibers. amish nemaline myopathy is a form of nemaline myopathy common among the old order amish. in the first months of life, affected infants have tremors with hypotonia and mild contractures of the shoulders and hips. progressive worsening of the proximal contractures, weakness, and a pectus carinatum deformity develop before the children die of respiratory insufficiency, usually in the second year. the disorder has an incidence of ∼ in among the amish, and it is inherited in an autosomal recessive pattern. using a genealogy database, automated pedigree software, and linkage analysis of dna samples from four sibships, we identified an ∼ -cm interval on chromosome q . that was homozygous in all affected individuals. the gene for the sarcomeric thin-filament protein, slow skeletal muscle troponin t (tnnt ), maps to this interval and was sequenced. we identified a stop codon in exon , predicted to truncate the protein at amino acid , which segregates with the disease. we conclude that amish nemaline myopathy is a distinct, heritable, myopathic disorder caused by a mutation in tnnt . introduction the nemaline myopathies (nm [mim , ]) are neuromuscular disorders characterized by muscle weakness and rod-shaped “nemaline” inclusions in skel- etal muscle fibers (north et al. ). nm has been di- vided into three clinical subtypes—severe neonatal, mild congenital, and adult-onset—varying in their age at onset, rate of progression, and other associated features. child- hood forms of nemaline myopathy segregate in either an autosomal recessive or an autosomal dominant pattern. mutations in genes encoding three sarcomeric proteins, including nebulin (neb [mim ]) (pelin et al. ), a-tropomyosin (tpm [mim ]) (laing et al. ; tan et al. ), and a-actin (acta [mim ]) (nowak et al. ), have been shown to cause different forms of hereditary nm. since , we have treated or obtained clinical information on infants and young children from nuclear families with an appar- ently unique form of nm, which we call amish nemaline myopathy (anm), among the old order amish of lan- caster county, pennsylvania. the purpose of this study received june , ; accepted for publication august , ; electronically published august , . address for correspondence and reprints: dr. jennifer j. johnston, rm. d- , bldg. , center drive, national institutes of health, bethesda, md - . e-mail: jjohnsto@nhgri.nih.gov q by the american society of human genetics. all rights reserved. - / / - $ . was to characterize the genetics of the anm phenotype and to determine the molecular cause of the disorder. subjects and methods subjects we collected information on the mode of presentation, clinical manifestations, affection status of first-degree relatives, age at death, and the genetic relationships of nuclear families having a child affected by anm in the old order amish genealogy. four nuclear families with anm, chosen on the basis of accessibility, under- went phlebotomy and were included in the genotyping for the whole-genome scan. all participants in the latter four families were evaluated clinically by one or more of the authors of the present article, and affection status was ascertained before the study was initiated. clinical data on children from other families were obtained from clinic reports, through home visits, or by history from the families ascertained by community outreach at the clinic for special children. the study was reviewed and approved by institutional review boards of the national institutes of health and johns hopkins university. pedigree analysis because manual genealogy analysis and pedigree drawing are error prone, we used the amish genealogy database version . (agarwala et al. ) and the johnston et al.: tnnt gene mutation in nemaline myopathy associated software, pedhunter (agarwala et al. ), to connect the nuclear families with an affected child into “all-shortest-paths” pedigrees. all known fam- ilies were used for the pedigree analysis, to increase the likelihood of finding the true inheritance path of the mutant allele. we found four all-shortest-paths pedigrees with different founder couples at the top. each all-short- est-paths pedigree connects the same of obligate carrier parents back to a founder couple, one of whom may have been a carrier. we then formulated a steiner tree problem to extract a minimal pedigree (also called a “steiner” pedigree) that has the smallest number of meioses needed to provide one path of inheritance from the founder couple down to each carrier parent (agar- wala et al. ). we used the sophisticated branch-and- cut–based steiner tree software of koch and martin ( ) to solve to optimality all four instances corre- sponding to the different all-shortest-paths pedigrees. from the four possible solutions, we chose the one with the smallest number of individuals. for genetic linkage analyses, the subpedigree of the chosen steiner pedigree was pruned, removing families and leaving the four sampled nuclear families, their ancestors, and any par- ent-child links among their ancestors used in the full steiner pedigree. genotyping human genomic dna was isolated from epstein-barr virus–immortalized lymphoblastoid lines or fibroblast cell lines, using puregene dna isolation kits (gentra- systems). polymorphic microsatellite markers from the abi prism linkage mapping set, version , were used for genotyping. additional markers were identified from genetic maps and were used in fine mapping. these ad- ditional markers were ordered as labeled map pairs (re- search genetics) or as custom-labeled oligonucleotides (life technologies). for initial homozygosity mapping, three pools of genomic dna were created, representing parents, unaffected siblings, and affected siblings. all genotyping reactions were performed using the standard protocol from pe biosystems, with individual reaction components and a reduced reaction volume of ml. pcr products were pooled according to supplied pro- tocols and were loaded on an abi automated se- quencer (filter set d: % longranger acrylamide gel [fmc bioproducts]), and the data were analyzed using abi genescan . and abi genotyper . software (pe biosystems). linkage analysis the genetic linkage analyses were performed using the fastlink software package, version . p (lathrop et al. ; cottingham et al. ; schäffer et al. ). loops were broken using the method of becker et al. ( ). some multipoint runs were done in parallel (gupta et al. ), using a shared-memory computer and the p parallel programming system (butler and lusk ). in the initial genome scan, we used only four of the five loops (three of the four families) and did two-point analyses on each marker, using equal allele frequencies. for the linkage results shown on chromo- some , we used all five loops (four families). we used equal allele frequencies in the calculations, because the sample size is sufficiently small and biased toward af- fected individuals and their parents to preclude reason- able estimates of allele frequencies. in all analyses, we assumed that anm is a fully penetrant phenotype in- herited in an autosomal recessive pattern, with a disease allele frequency of . . this allele frequency was used to increase the effectiveness of homozygosity mapping, reducing the probability that another allele would be introduced into the pedigree. for comparison purposes, the multipoint linkage was also calculated using an allele frequency of . (square root of disease frequency). the genethon map of the chromosome markers that ul- timately proved to be the correct region (numbers in parentheses are the genetic map position [in cm]) is d s ( . )–d s ( . )–d s (∼ )– d s ( . )–d s ( . )–[d s , d s ( . )]–d s ( . )–d s ( ). analysis with blast searches (altschul et al. ) of the sequence- tagged-site content of bacterial artificial chromosomes (bacs), with genbank accession numbers ac and ac , showed that they both contain d s and d s but not d s , supporting the order of markers shown. sequence analysis the coding exons of tnnt , including the flanking intron sequence, were amplified from genomic dna from an affected individual, an obligate carrier, and a control. genomic dna ( ng) was amplified in a -ml pcr reaction containing pmol each of for- ward and reverse primer, . units amplitaq dna polymerase (perkin-elmer), and . mm dntp mix (pharmacia biotech). the pcr reaction buffers and annealing temperatures were oligonucleotide-pair spe- cific. reactions for primer pair f/ r contained mm tris-hcl ph . , mm kcl, and . mm mgcl , with an annealing temperature of c. re- actions for f/ r, f/ r, f/ r , and f/ r con- tained mm tris-hcl ph . , mm kcl, . mm mgso , and # pcr enhancer (life technologies), with an annealing temperature of c; reactions for f/ r were run under identical conditions to f/ r, except for the addition of x pcr enhancer. initial denaturation for all sets was min at c, followed by cycles of s at c, s at the appropriate annealing temperature, min at c, and a final extension for min at c. using oligonucleotides am. j. hum. genet. : – , figure pedigree used for the whole-genome–scan linkage analysis. the pedigree consisted of four nuclear families, which included six affected patients. these four families are connected in a single subpedigree with five loops. the full pedigree was generated computationally from an old order amish genealogy database and represents a portion of the simplest pedigree that connects affected nuclear families to a putative founder couple who were born in the first half of the th century. figure haplotype analysis of affected persons. to determine the locus boundaries using an analysis of recombinants of affected individuals only, marker d s provides the centromeric boundary and marker d s provides the telomeric boundary. the pedigree designations refer to the generation number in roman numerals from the pedigree; the individual number is arabic, numbered from left to right. the allele numbers are the standard ceph alleles, and “m” signifies the mutant tnnt allele. f/ r, exon was amplified from genomic dna in control individuals and additional family members with anm. reactions were run under iden- tical conditions to f/ r, except for the absence of pcr enhancer. pcr products were purified and se- quenced as described elsewhere (johnston et al. ). primer sequences named for the tnnt introns from which they are derived are listed in the appendix. results phenotype analysis all neonates with anm have tremors that are evident at birth or within a few days of birth and that involve most skeletal muscle groups, especially the muscles of the jaw and lower limbs. these tremors subside over the first – mo of life. mild proximal contractures present at birth gradually worsen with age, such that hip abduction is often limited to < by age mo. progressive muscle atrophy and weakness develop simultaneously with the contractures. gross motor development is limited to roll- ing side to side, but forearm and hand function is normal, as is apparent intelligence. a characteristic, severe pectus carinatum deformity, with rigidity of the chest wall, de- velops along with the proximal contractures. the neu- romuscular examinations of parents and normally devel- oping siblings are unremarkable. however, several siblings with normal outcomes have had abnormal trem- ors both in utero and for several weeks after birth. the neurological examinations and, in one case, tests for hy- poglycemia and hypocalcemia, were otherwise normal in these siblings. these findings suggest the presence of a subtle carrier effect, but persons with this effect have been considered unaffected, and we have not included samples from any of these individuals in our study. all routine laboratory chemistries and metabolic stud- ies have been normal in affected patients. although there often are terminal signs of right-side congestive heart failure, there is no evidence of primary cardiac involve- ment, and one patient who was studied at age mo johnston et al.: tnnt gene mutation in nemaline myopathy table two-point lod scores for nm phenotype and markers on chromosome marker lod scorea at v p . . . . . . d s ` . . . . . d s . . . . . . d s ` . . . . . d s . . . . . . d s . . . . . . d s . . . . . . d s . . . . . . d s . . . . . . d s ` . . . . . a the five-loop version of the pedigree was used for these cal- culations. recombination fractions are shown only up to . , be- cause the markers are very close to each other and we expect the disease gene to be close to these markers. figure three-point linkage analysis using two chromosome genetic markers. calculations were done using disease allele fre- quencies of . and . . had a normal electrocardiogram and echocardiogram. four children in their first year had quadriceps muscle biopsies with both light and electron microscopic ex- aminations. light microscopy showed prominent type i fiber disproportion, z-band streaming, and abundant centrally placed refractile rods, as well as areas of my- ofibrillar disruption and myofiber degeneration (data not shown). pedigree and linkage analysis the affected families included nuclear families with one or more children affected with anm. these families included probands, with a total of affected chil- dren and unaffected siblings. the steiner pedigree used for genetic linkage analysis is shown in figure . prior to performing the full genomewide scan, we used linkage analysis to exclude the gene for neb as a can- didate for this disorder (johnston et al. ). in ad- dition, we performed a homozygosity screen using mark- ers from the abi linkage set surrounding candidate genes for a-tropomyosin and troponin t , among others. the region on chromosome surrounding the gene for tro- ponin t was followed up with additional markers, but the data were not sufficiently convincing (see below). in our analyses of the genome scan data, six regions merited higher resolution genotyping because they had either a marker with a two-point lod score . or two ad- jacent markers with scores . . of these six regions, only the chromosome region, containing tnnt , had a marker with a two-point lod score . and a multipoint lod score . . we found the two highest- scoring adjacent markers—d s and d s —to be the only markers in the study that are homozygous for the same allele in all six genotyped affected individ- uals. however, both markers are also homozygous for the disease-associated allele in two of the eight obligate carrier parents. haplotypes of affected individuals are shown in figure . two-point lod scores are shown in table . a three-point lod score curve using markers d s , d s , and disease is shown in figure ; the peak lod score using a disease allele frequency of . is . , and the peak lod score using a disease allele frequency of . is . . these values are con- sistent (to the third significant digit) throughout the in- terval d s –d s . this region of homozygosity was considered inconclusive on the original candidate linkage analysis. in retrospect, that assessment can be attributed to the small region of homozygosity (∼ cm), a double crossover in one nuclear family, and dearth of informative markers in the region. these data strongly support the hypothesis that anm is inherited in an au- tosomal recessive pattern and is linked to the chromo- some region containing the gene tnnt , suggested by others to be a candidate for nm (laing ). mutation analysis of tnnt because troponin t (tnt) is a sarcomeric protein, we considered tnnt to be a candidate for the anm dis- ease gene and sequenced the coding region, including the intron/exon borders, in one affected individual, an obligate carrier, and a control. a homozygous nonsense mutation was present in the dna from the affected in- dividual in exon , g t (genbank entry s ), resulting in a stop codon e x (fig. ). although tnnt transcripts undergo alternative splicing of exons and , producing up to four isoforms (samson et al. ), exon is present in all known splice products. the change was present in a heterozygous state in the am. j. hum. genet. : – , figure sequence traces of exon of the tnnt gene in an unaffected control, a carrier parent, and an affected patient. the carrier is heterozygous for the g t substitution, and the affected individual is homozygous for this change. the nucleotide and predicted amino acid sequence is shown above the traces, with the stop codon labeled by an asterisk (*). obligate carrier and absent from the control sample. all available samples from the full pedigree were ana- lyzed by sequencing, and the mutation was found to segregate with the disease state. the mutation was not found in non-amish control chromosomes nor in chromosomes from amish individuals drawn from nuclear families unaffected by anm. because genetic control samples must be acquired from unrelated indi- viduals, it is impossible to gather true amish controls, since all amish persons are related. discussion anm is a distinct form of nm that segregates in an autosomal recessive pattern. with the identification of a mutation in tnnt (gene encoding slow skeletal muscle tnt), it becomes the fourth form of nm to be associated with an abnormality of a protein component of the thin filament of the sarcomere. nemaline bodies, the hallmark of nm, are histochemically and ultra- structurally similar to the z disk of the sarcomere and are often found in physical continuity with the z disk in nm. it is notable, therefore, that all four of the genes associated with nm code for protein components in- tegral to the thin filament rather than protein com- ponents found solely in the z disk. however, one of the gene products responsible for nm—nebulin—is thought to be an important regulator of the assembly and organization of the z disk (pelin et al. ). the other three gene products associated with nm—a-actin (nowak et al. ), a-tropomyosin (laing et al. ; tan et al. ) and, with this article, slow skeletal tnt—interact with one another and ultimately regu- late the development of muscle contraction. like the more heterogeneous adult forms of nm, the prolifer- ation of nemaline bodies in each of these latter three disorders may be a secondary consequence of abnormal regulation of force within the sarcomere (north et al. ; michele et al. ). the mapping of this phenotype was facilitated by the structure of the amish population. the old order amish are a genetic isolate with a high rate of consan- guinity and multiple disorders with proved or apparent founder effects. the old order amish trace their an- cestry to – european immigrants of the mid to late s. in the amish genealogy database, version . , the average inbreeding coefficient is . (agar- wala et al. ). the genetic origins of this population and their strict endogamy predispose them to manifest rare disorders that are inherited in an autosomal reces- sive pattern. these same genetic and demographic fea- tures of the old order amish advance the identification of rare disease-causing genes that are found within this group. the technique of whole-genome linkage analysis via identification of regions of homozygosity among af- fected individuals is particularly suited to this popula- tion (mckusick ) and was a productive approach in this instance to map, clone, and identify a nonsense mutation in tnnt , coding for slow skeletal tnt as the cause of amn. the identified mutation of tnnt predicts a trun- cation of the tnt protein at amino acid , removing the c-terminal amino acids that include the principal site of interaction with the other two components of the troponin complex, troponin i (tni) and troponin c (tnc). in addition, a second site of interaction with tropomyosin, which is dependent on ca concentra- tion, is also in the deleted part of the protein. the re- maining n-terminal portion of tnt forms the whole of the troponin tail region, which anchors the complex to tropomyosin. a schematic diagram of these interactions is shown in figure (cohen ; flicker et al. ). there are at least three potential consequences of this mutation: the mutant message may be subject to accel- johnston et al.: tnnt gene mutation in nemaline myopathy figure thin filament structure showing troponin complex, tropomyosin, and actin. tnc, tni, and the c-terminal portion of tnt bind near cys of tropomyosin. the n-terminal portion of tnt extends along the c-terminal half of tropomyosin. the predicted trun- cation in anm would delete a part of the c-terminal end of the tnt molecule that interacts with tni, tnc, and tropomyosin. erated nonsense-mediated decay (maquat ), the protein may be unstable or improperly localized, or the truncated protein may be translated in normal abun- dance and appropriately bound to tropomyosin in the thin filament but deficient in the ability to interact prop- erly with tni and tnc. we speculate that the recessive inheritance pattern in anm may be indicative of ac- celerated nonsense-mediated decay or protein instabil- ity, compared with the dominant inheritance pattern seen in the family with nm segregating a missense mu- tation in tpm (laing et al. ) and the families segregating missense mutations for acta (nowak et al. ). production of aberrant sarcomeric proteins may be less well tolerated than haploinsufficiency of these same proteins. in any of these cases, this abnormality of tnt is likely to disturb the coupling of excitation to contraction within muscle fibers. at rest, and with low concentra- tions of ca , tni interacts with a-actin and a-tropo- myosin to block the atpase of actomyosin and thus inhibit contraction. with muscle fiber depolarization, ca released from the sarcoplasmic reticulum is bound to tnc, and in the presence of tnt, a conformational change of the complex releases the inhibitory effect of tni upon actomyosin. hypertrophic cardiomyopathy (hcm), like nm, has been associated with mutations of protein com- ponents of the thin filament of the sarcomere, in- cluding cardiac tnt (laing ). a variety of de- letion, splice site, and missense mutations in the gene for cardiac tnt (tnnt ) cause familial hcm (perry ). a splice-site mutation in intron of tnnt , seen in one individual with familial hcm, is pre- dicted to lead to production of a truncated protein. transfection of this mutation into a quail myotube expression system resulted in disorganized myofibrils and a greatly diminished ca -activated force of con- traction (watkins et al. ; perry ), indicating that expression of a truncated cardiac tnt may have a dominant negative effect on muscle. we hypothesize that mutations in tnnt manifest in an autosomal recessive pattern, whereas tnnt mutations segre- gate in an autosomal dominant pattern, because skel- etal and cardiac muscle may have different pheno- typic thresholds. it is interesting to note that mutations in the cae- norhabditis elegans tnt (mup- ) gene lead to myocyte dysfunction suggestive of the phenotype seen in anm. the recessive inheritance pattern of human tnnt mu- tations is consistent with that seen in the c. elegans mutants (both truncation and null mutants). worms ho- mozygous for either a null allele or an amino terminal truncation mutation of mup- show a phenotype with apparent hypercontraction and ultimate detachment of body wall muscles (myers et al. ). prior to de- tachment, the embryos show uncoordinated, slow twitching, rather than the vigorous rolling typical of wild-type embryos. mutations affecting voltage-depen- dent muscle ca release, tnc, or sarcomere assembly are epistatic to mup- , which suggests that the mup- phenotype is not due to sustained ca -independent contraction but is caused by aberrant regulation of con- traction (mcardle et al. ). in vitro studies of human fast skeletal tnt show that truncated tnt protein does not bind tni/tnc as effi- ciently as wild-type tnt (jha et al. ). the authors propose that, in the absence of tnt tethering tni/tnc to tropomyosin, inhibition of contraction is not estab- lished normally after contraction, leading to the phe- notypic twitching and ultimate muscle detachment. although we have treated anm patients with myo- tonia-limiting drugs, such as carbamazepine and mex- ilitene, in an attempt to limit the progression of the chest deformity and associated respiratory insufficiency, such treatments have had minimal benefit. further under- standing of the cellular pathogenesis of anm and study of the identified mutation in potential animal models should stimulate research into effective treatments for this relentlessly progressive disorder. acknowledgments we wish to thank ralph kuncl for identification of nemaline myopathy in biopsy specimens. we would also like to thank joie davis for her contribution to the fieldwork. am. j. hum. genet. : – , appendix primer sequences f tccctaagacccaggaatccg f cggtcctctaacagcctctcacg r ggcttaaaggaccgggcctgg f tgaccctcttcctcctttacagc r tctgcctttctcacaccttgtctc f ctacctcactggacggcacatc r gcctcgtgtttgacactaatcagc f gctgattagtgtcaaacacgaggc r ggcatccggctgaagaagtaac f tgcgtttctgctggttctgatg r ctcagcccctcctccgttagg f ctgtggtcccaacatggaaatgg r cctttatccccctgtctcacacc f tgaacaaaatggtgccgacaac r acccaggattccgagagcagag f tgcttcctttgggcagggag r acatccaggaggggaactgc electronic-database information accession numbers and urls for data in this article are as follows: genbank, http://www.ncbi.nlm.nih.gov/genbank/index.html (for bac clones ac and ac and for tnnt sequence s ) généthon, ftp://ftp.genethon.fr/pub/gmap/nature- /data/ data_chrom (for microsatellite markers from the chro- mosome region) online mendelian inheritance in man (omim), http://www .ncbi.nlm.nih.gov/omim/ (for nm [mim , ], neb [mim ], tpm [mim 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troponin t that causes hypertrophic cardiomyo- pathy: evidence for a dominant negative action. j clin invest : – this peer-reviewed article has been accepted for publication but not yet copyedited or typeset, and so may be subject to change during the production process. the article is considered published and may be cited using its doi . /s . epidemiology & infection is published by cambridge university press. risk factors for toxoplasma gondii seropositivity in the old order amish markon, a. o. , ryan k. a. , , wadhawan a. , , pavlovich m. , , groer m.w. , punzalan c. , gensheimer k. , jones j. , daue m.l. , , , dagdag a. , donnelly p. , peng x. , , pollin t. , , , mitchell b.d. , , , postolache t.t. , , u.s. food and drug administration, center for food safety and applied nutrition (cfsan), office of analytics and outreach (oao), division of public health informatics and analytics (dphia), college park, md, usa. division of endocrinology, diabetes and nutrition, department of medicine, university of maryland school of medicine, baltimore, md, usa. program for personalized and genomic medicine, university of maryland school of medicine, baltimore, md, usa. mood and anxiety program, department of psychiatry, university of maryland school of medicine, baltimore, md, usa. saint elizabeth’s hospital, psychiatry residency training program, washington, dc, usa. college of nursing, university of south florida college of nursing, tampa, fl, usa. geriatrics research and education clinical center, veterans affairs medical center, baltimore, md, usa. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms amish research clinic, university of maryland school of medicine, lancaster, pa, usa. rocky mountain mental illness research education and clinical center (mirecc), veterans integrated service network (visn) , military and veteran microbiome: consortium for research and education (mvm-core), denver, co, usa. mental illness research, education and clinical center (mirecc), veterans integrated service network (visn) , va capitol health care network, baltimore, md, usa. corresponding authors: main: teodor t. postolache: tpostola@som.umaryland.edu andré o. markon andre.markon@fda.hhs.gov disclaimer: the findings and conclusions in this study are those of the authors and do not necessarily represent the official position of the u.s. food and drug administration (fda). data availability: the data cannot be shared because of the original protocol and amish consent. a collaborative effort could be considered and interested individuals should contact the corresponding author. -word summary: risk of infection with the major foodborne pathogen, toxoplasma gondii, varies by individual, geographic, sociocultural, and economic factors. we assessed the risk of toxoplasmosis, focusing on food safety and environmental factors, among the old order amish, a relatively homogenous population. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. mailto:tpostola@som.umaryland.edu mailto:andre.markon@fda.hhs.gov https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms summary toxoplasma gondii is an important human disease-causing parasite. in the us, t. gondii infects > % of the population, accrues economic losses of us$ . billion/year, and ranks as the second leading culprit of foodborne illness-related fatalities. we assessed toxoplasmosis risk among the old order amish, a mostly homogenous population with high prevalence of t. gondii seropositivity, using a questionnaire focusing on food consumption/preparation behaviours and environmental risk factors. analyses were conducted using multiple logistic regression. consuming raw meat, rare meat, or unpasteurized cow or goat milk products were associated with increased odds of seropositivity [unadjusted ors: . , . , . , and . , respectively). in separate models by sex, consuming raw meat, or consuming unpasteurized cow or goat milk products, were associated with increased odds of seropositivity among women; washing hands and touching meat with decreased odds of seropositivity among women [adjusted or (aor): . ]; and cleaning cat litterbox with increased odds of seropositivity among men (aor: . ). this is the first study to assess associations between behavioural and environmental risk factors and t. gondii seropositivity in a us population with high seroprevalence for t. gondii. our study emphasizes the importance of proper food safety behaviours to avoid risk of infection. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms introduction the apicomplexan parasite toxoplasma gondii (t. gondii) infects one-third of the world’s population, disproportionately affects socioeconomically disadvantaged groups, and ranks among five neglected parasitic infections targeted in the united states (us) by the centers for disease control (cdc) for public health action[ , ]. some estimates indicate that in the us ~ , new toxoplasmosis cases occur annually and that . million are currently infected, amounting to infection among . % (age-adjusted: . %) of the population [ ]. other sources note that although > million men, women, and children in the us may harbour the parasite without symptoms because the immune system prevents occurrence of the full-blown illness, consequences of infection can be severe for the immunocompromised and for women infected during or shortly before pregnancy [ ]. the burden of t. gondii infection by foodborne routes, which is estimated to account for about half of all us cases, exceeds $ . billion and equates to the loss of , quality-adjusted lifedays (qalds)[ ]. according to scallan at al. ( ), . % of us-acquired foodborne t. gondii infections will result in hospitalizations, and . % will result in death, leading to an estimated deaths annually, making toxoplasmosis the second leading cause of foodborne illness fatality in the us [ ]. sexual reproduction of t. gondii occurs in its definitive felid hosts, including cats, while asexual reproduction occurs in intermediate hosts, including humans and nearly all warm-blooded vertebrates. the parasite has three infectious stages[ ] - : sporulated oocysts that contain sporozoites, tachyzoites that invade and multiply rapidly within the https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms cells of intermediate hosts, and bradyzoites that slowly replicate in tissue cysts (life cycle detailed in figure ). tissue cysts are most often found in the central nervous system; the eyes; and smooth, skeletal, or cardiac muscles. while bradyzoites in tissue cysts characterize chronic infection, tachyzoites characterize primary acute or reactivated infections, which can trigger adaptive igg immune responses[ , ]. in reactivation, tissue cysts rupture and release bradyzoites that transform back into destructive tachyzoites , . toxoplasmosis may present in immunocompetent individuals with painless lymphadenopathy; mononucleosis-like symptoms, including fever, malaise, sore throat, and maculopapular rash; ocular infections in some older populations; or no symptoms in pregnant women[ ] , . severe clinical manifestations, such as pneumonitis, chorioretinitis, or multi-organ involvement, occur most often in reactivated and primary infections among immunocompromised individuals and in congenital infections[ , ]. hiv-positive individuals often present with life-threatening toxoplasmic encephalitis, characterized by headache, confusion, weakness, focal neurologic involvement, and seizures[ , ]. previously, chronic infections were not associated with clinical manifestations[ ], but recent studies have linked chronic infection to schizophrenia[ - ] and suicidal behaviour [ , ]. multiple behavioural, socioeconomic, and demographic factors may contribute to the risk of toxoplasmosis. studies focusing on behavioural risk factors often use a combination of immunoassays to determine infection and questionnaires to conduct epidemiologic assessment of potential exposures. while the questionnaires themselves are often tailored to specific populations, many ask similar questions regarding behaviour-related exposures: most notably, eating different meats[ - ] (porcine, https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms bovine, lamb/mutton, other); handling or eating raw or undercooked meat[ , ]; consuming dairy/ dairy products[ - ]; eating unwashed or improperly washed fruits and/or vegetables[ , , ]; gardening and/or engaging in contact with soil[ , , , ]; living with pets, such as cats and/or dogs at home[ , , ]; drinking water (treatment, source, other characteristics)[ , , ]; and even hunting/consuming wild game (venison, boar, other)[ , ]. in the us, those below the federal poverty threshold have higher t. gondii seropositivity[ ], while higher early-life socioeconomic status (ses) has been shown to be related to seronegativity[ ]. other risk factors for higher seroprevalence, specifically in the us, include foreign country of birth, hispanic and non-hispanic black race/ethnicity, lower than high school education levels, crowded housing conditions, rurality/urbanicity, and employment type[ , ]. the old order amish (ooa) population in the us, by virtue of its homogeneous characteristics, provides a unique opportunity to assess relationships between t. gondii seropositivity and specific behavioural and environmental (including food-related) risk factors. t. gondii seropositivity prevalence levels among the ooa exceed those found among the general us population[ ], and the geographic, ethnic, and socioeconomic homogeneity of the ooa population [ ] helps to control for potential confounding that can otherwise complicate risk factor assessment. in addition, the amish lifestyle presents the prospect of evaluating potential associations between t. gondii seropositivity and culturally specific meal-preparation practices, such as slaughtering, smoking, curing, and canning meats[ ]. these distinctive characteristics, in conjunction with antibody testing and data collection using ooa lifestyle-specific questions, allowed this study to pursue the objective of advancing understanding of https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms relationships between the prevalence of t. gondii seropositivity and behavioural and environmental (including food-related) risk factors of infection, while minimizing confounding by other potential sociodemographic and behavioural determinants. methods study population the amish wellness study the amish wellness study (aws), an ongoing community-based investigation conducted by the university of maryland baltimore and approved by the university of maryland baltimore institutional review board, was established in to offer health screening and collection of dna, blood samples, and basic medical information to address specific research questions. criteria for participation included membership in the ooa community of lancaster county, pennsylvania; age of ≥ years; and an informed consent. a registered nurse obtained multiple clinical measures of general health (e.g., weight, height, waist and hip circumference, blood pressure, measures of respiratory health). an amish liaison facilitated administration of the study questionnaire in person to assess several aspects of each participant’s health and well-being, including mood, sleep, and medical, personal and family histories; as well as to evaluate life-style factors. a university of maryland amish research clinic research nurse confirmed completion of each questionnaire. a fasting blood sample was drawn for measurement of lipids and glucose, and from this sample, we also measured t. gondii igg antibodies for this study. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms for the present study, we sent the amish toxoplasma risk factor questionnaire (atrfq) to a convenience sample from the aws (n = , nonrandomly selected, based on availability to participate), between december , and january , . we evaluated participant responses to the atrfq, along with their demographic information and t. gondii antibody results from the aws. immunoglobulin g serointensity and seropositivity testing for this study, the laboratory of maureen groer at the university of south florida in tampa determined t. gondii antibody seroprevalence using the enzyme-linked immunosorbent assay (elisa) (ibl international, männedorf, switzerland) that tests for rh factor common to all t. gondii serotypes. concentrations of ≥ iu/ml were defined as seropositive, and of - iu/m as equivocal. the equivocal samples were tested a second time, and if the second testing yielded samples that tested positive for concentrations of ≥ iu/ml on the second test were also considered positive as previously reported [ ]. amish toxoplasma infection risk factor questionnaire (atrfq) the atrfq was developed by researchers at the university of maryland baltimore with input from epidemiologists at fda’s center for food safety and applied nutrition (cfsan) and co-author jeffrey jones, based on current risk factor literature and subject matter expertise. the questionnaire was adapted after incorporating input from sessions of review and feedback by a group of amish liaisons, sessions of feedback by nurses experienced in working with the amish, and individual researcher comments and questions to maximize comprehension and cultural sensitivity. the https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms questionnaire consisted of questions focusing primarily on food consumption and preparation, as well as other environmental risk factors, such as water sources, pet ownership and contact, and contact with soil. the pet component specifically asked about cat/ kitten ownership, cat breeding, cat litter cleaning, where the cats live (outdoors vs indoors), where the cats/ kittens eat, type of food they eat, visits of not- owned cats, type of cats seen in the garden or yard, cats for breeding, feral cats (domesticated and returned to the wild), and stray cats (lost or abandoned). . it was mailed to study participants between december , and january , with a cover letter, response envelope, and us$ compensation. the cover letter informed participants that they could keep the $ even if they decided not to participate. responses from the returned questionnaire received by april , were entered into an access database. returned envelopes received after april , were not included in the analytical sample used for this study. statistical analyses the analytical sample included those with both t. gondii serological measures and atrfq responses. analyses were performed to describe the analytical sample (table ) and to assess the strength and significance of associations with examined behavioural and environmental (including food-related) risk factors through models estimating odds of t. gondii seropositivity (tables - ). in addition to age and sex derived from the parent aws survey, the atrfq variables addressed contact with soil, pets (including cats and litter boxes), and shoes/feet before touching food; farm vs. other-than-farm residence and time at residence; drinking untreated or treated water and drinking water source/filtration; working with animals/raw meat/horses; consuming https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms locally-produced, cooked, cured, frozen, canned, raw and/or rare meats; consuming raw oysters; consuming unpasteurized goat and/or cow milk/products; washing produce; washing hands/utensils after raw meat contact; and eating outside the home. bivariate analyses, including chi-square (Χ ) and t-tests, compared seropositive to seronegative to identify significant or borderline significant (p value < . and < . ) variables. for those variables, we then specified comparison reference groups and estimated unadjusted odds ratios (uors), % confidence intervals (cis), and p values (tables and ). we included the variables that exhibited significance in the bivariate uor analysis (p value < . ) in the final unconditional multiple logistic regression models by sex to estimate adjusted odds ratios (aors), % cis, and p values, evaluating the associations between each of the predictor variables and the outcome of t. gondii seropositivity as defined above (table ). these analyses were performed using sas . (cary, nc), as well as the x calculator in mcdonald ( )[ ]. we also adjusted the results for relatedness (heritability) and shared households by applying a variance component approach using solar . . software (san antonio, tx) to assess significance (p value < . ) [ ]. results of the questionnaires mailed to study participants, ( %) were returned. t. gondii serology was assessed in . % (n= ) of those who returned questionnaires. we limited our analytical sample to the amish subjects with both serology test results and questionnaire response data ( . % of the who were mailed and returned the questionnaire also had serology results). https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms more than half ( . %) of the study participants tested positive for t. gondii igg antibodies (table ). nearly two-thirds of participants ( . %) were female among both the seropositive and seronegative groups. the age distribution/n was similar among seropositive individuals, but not among seronegative individuals or the whole study sample. mean and median ages were higher among seropositive than among seronegative individuals, and there were more seropositive than seronegative males. in addition to sex, the Χ test results from the first step of the bivariate analysis indicated significant/borderline significant associations between t. gondii seropositivity (p values < . ) and the category-based variables capturing farm vs. other-than-farm residence and time at residence; working with horses; consuming unpasteurized cow milk/products; consuming cured, rare, and raw meats; pet contact; and washing hands/utensils after raw meat contact, as well as the frequency-based variables addressing how often unpasteurized cow milk/products, raw meats, rare meats, and meals outside the home were consumed. of those variables, the uor estimates from the second step of the bivariate analysis showed significance (p value < . ) for pet contact, consumption of meat and unpasteurized milk/products, and handwashing behaviours (table ), as well as for frequency of consumption of unpasteurized cow milk/ yoghurt, rare meat, raw meat, and meals outside the home (table ). the four meat-eating behaviours, as well as the two variables addressing the frequency of consumption, were all associated with t. gondii seropositivity. the odds of seropositivity from eating rare and raw meat compared to not eating rare and raw meat https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms were . ( % ci: . , . ) and . ( % ci: . , . ), respectively, while seropositivity among those who reported eating compared to not eating raw ground beef was greater (or: . , % ci: . , . ). only one individual reported eating raw ground pork and was seropositive. odds of seropositivity comparing consumption of unpasteurized milk to not consuming raw milk was . (or: . , % ci: . , . ). among the behaviours related to pets, only cleaning a cat’s litterbox was found statistically significantly associated with t. gondii seropositivity (or: . , % ci: . , . ). because of the significant difference found between sex and seropositivity in the bivariate analysis, separate models by sex were used to estimate the adjusted associations between the other variables also identified as significantly associated with seropositivity (table ). several of the associations between t. gondii seropositivity and behavioural and environmental (including food-related) risk factors did not retain significance in the adjusted models by sex. however, the separate adjusted models by sex showed that significance persisted nominally for cleaning the litterbox (among men only), eating raw meat (among women only), and washing hands after touching raw meat (protective in women only). consuming raw goat or cow milk was significantly associated with seropositivity among women only (or: . , % ci: . , . ; or: . , % ci: . , . , respectively, where only respondents indicated consumption of unpasteurized goat milk products, and indicated intake of unpasteurized cow milk products). washing versus not washing hands after touching raw meat was also significantly associated with seropositivity among women only (or: https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms . , % ci: . , . ). these adjusted significant results held, even after adjustment for heritability and shared household. discussion this study found that members of the ooa community of lancaster, pennsylvania have a high seroprevalence of t. gondii antibodies, with over % of study participants testing seropositive, exceeding the levels reported in many other us populations, although similar to levels reported in java, indonesia ( . %)[ ] and among the nanvik inuit of canada ( . %)[ ]. the jones et al. ( ) us-based analysis of the - national health and nutrition examination survey (nhanes) that included a wave of participants aged ≥ years found a much lower seroprevalence of . % [ % confidence limit (cl): . %, . %] prior to adjustment for age and an age-adjusted seroprevalence of . % ( % cl: . %, . %)[ ]. as these studies vary in terms of recruitment and analytical methodologies, we suggest caution when interpreting these differences. the uniqueness of our study population may further complicate comparisons—as previously mentioned, the ooa community is an ethnically, behaviourally, and culturally distinct group. we are not aware of any studies assessing the seroprevalence of t. gondii in other amish populations and have found only one study focusing on a similar population. alvarado-esquivel et al. ( ) conducted a t. gondii risk factor and seroprevalence analysis among mennonites in durango, mexico, an isolated and mostly rural population of ethnic germanic descent, with cultural and behavioural aspects that differ from the surrounding communities[ ]. they found lower seroprevalence ( . %) among the mennonites of durango[ ], compared to the % seroprevalence rate that we have observed in the ooa https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms community of lancaster, pennsylvania. also, the seroprevalence of t. gondii igg antibodies in the general population of durango city, mexico was reported to be . %, which was remarkably less than the seroprevalence in the mennonite community living in the same city[ ]. this difference in seroprevalence may relate to diverse factors, including food sources, diet, unique group practices, geographic characteristics, and other factors that differ between this mennonite community and the ooa. the rural location of residence may contribute to the higher seroprevalence among the ooa compared to other populations. seroprevalence studies often consider living in a rural environment a risk factor because of the presence in rural settings of potentially infected animals that can transmit the parasite to humans in a variety of ways[ ]. the finding of higher seroprevalence among rural compared to urban populations is consistent with the results from the united kingdom, germany, and ireland[ , , ]. in addition, a study by muñoz-zanzi et al. found that children residing on farms in wisconsin were more likely seropositive than those who did not reside on farms[ ]. in their multivariate analysis of the - wave of nhanes, jones et al. reported significantly lower seropositivity among non-hispanic blacks living in metropolitan areas (> million population) (or: . , % ci: . , . ) than among non-hispanic blacks living in non-metropolitan areas[ ]. yet, the risk of t. gondii infection was not different among residents of metropolitan and non-metropolitan areas for populations of non-hispanic whites or mexican-americans[ ]. our study population lacked a proper urban counterpart to assess the impact of rurality/urbanicity, so we cannot exclude the possibility that there is simply high seroprevalence in the general study area. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms several of the food- and food safety-related risk factors found significantly associated with t. gondii seropositivity in this study are consistent with results from other studies. first, consumption and handling of raw and/or undercooked meats was associated with t. gondii seropositivity for both beef and pork, as previously seen in the literature[ , - ]. while the practice of cleaning a cat’s litterbox was also observed to be a risk factor among males in our study, our findings highlight the importance of the foodborne transmission route for t. gondii. in addition, washing hands was found protective of t. gondii seropositivity in our study, especially among women. our findings emphasize the importance of proper food safety behaviours, such as cooking meat to a safe temperature prior to eating/ not consuming raw meat, which is effective at diminishing risk of contracting the parasite. also, people should avoid accidental contamination by thoroughly washing hands before and after handling meats, and properly washing preparation surfaces and utensils (such as knives) with soap and hot water after use. washing of hands and/or use of gloves during gardening, may also help prevent contamination[ ]. our study also found that consumption of unpasteurized milk products was associated with risk of seropositivity. while intake of these products provides a possible route of exposure to the parasite[ - ], consumption rates are low among the us population[ , , ], and thus could explain, in part, the increased seropositivity among the amish. this study provided novel insights into risk factors for infection with a highly prevalent parasite in a unique us population. among several strengths, the study population is known for its ethnic and socioeconomic homogeneity, which may have helped to minimize confounding by unmeasured variables. second, response rates https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms were high for both the aws questionnaire and the atrfq. third, to counteract the possibility that the participants’ close family and household aggregation could have produced spurious associations, the study adjusted post-hoc for heritability and household in the analytical sample, without any loss of statistical significance for any of the positive findings reported in our study. among the study’s important limitations, the findings may not be generalizable to other populations in the us or elsewhere, given the unique characteristics of the culturally distinct ooa[ ]. in addition, we saw different distributions between age groups, which may be due to cumulative seropositivity and may have biased some of the observed associations. our study was also only included adults (age y or older), which means that we are unable to assess whether infections were acquired during childhood. consequently, we are unable to access certain risk factors and are also unable to provide recommendations to prevent these potential exposures. this study is also limited by a relatively small sample size given the number of risk factors analysed. although the high seropositivity in the ooa may have mitigated sample size issues, the low incidence of certain behaviours may have masked the statistical significance of their potential association with seropositivity. we presented our findings, both formally and informally, to the ooa community in lancaster, pa. we discussed the different t. gondii risk factors identified in our analysis and specifically addressed the observed sex-based differences. direct feedback from the ooa community and the findings themselves have and will continue to be used to develop content for future education efforts, including food safety. we plan on applying for grants to develop additional educational materials, train amish https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms educators, and conduct future studies to assess the possible relationships between knowledge and practice among the oaa. for example, future research efforts include developing a study to assess toxoplasma risk factors and foetal infection, morbidity, and mortality, and possibly establishing a longitudinal study to look at a variety of other important genetic, epigenetic, proteomic, clinical, and environmental factors, with sampling data for t. gondii in food, soil, water, and farm and domestic animals, as well as neuroimaging and other markers of physical and neuropsychiatric health and functioning. conclusion this study found t. gondii seropositivity among more than half of the participants from a unique ooa community with culturally specific meal preparation practices. the study highlights significant behavioural and environmental (including food-related) risk factors associated with infection, such as consuming undercooked meats and unpasteurized cow milk products, as well as protective behaviours, such as eating meals outside of the home and washing hands after contact with raw meat, showing the importance of proper food safety practices for meal preparation, which is of growing relevance to the consumers in the current general us population, as well. future research will focus on characterizing specific microorganism traits, including serotypes, markers of infection, and genetic, physiological, and clinical associations with t. gondii seropositivity, as well as advancing understanding of education and outreach needed to https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms promote protective behaviours and reduce the burden of toxoplasmosis from foodborne and other routes. acknowledgments: the authors thank gursharon nijjar, christopher chen, phillip miljanic, hassan yousufi, nupur hegde, winny mwaura, hira mohyuddin, sonia y. postolache, thea postolache, iqra mohyuddin, sunghee flores, hanna king, christopher a. lowry, lisa a. brenner, andrew hoisington, jeffrey o’connell, amish liaisons and nurses and staff of the amish research clinic of the university of maryland, lancaster, pa, who went out of their way to support this project. the authors also thank the entire amish community for collaborating in this project. we further would like to thank dolores hill, dietmar fuchs and christopher hunter for their expert advice on this project. this work was supported by the u.s. food and drug administration through the cooperative agreement fdu. (ttp). additional funding for this study was provided by the mid-atlantic nutrition obesity research center (norc) pilot & feasibility project (postolache, pi), a sub-award of the parent grant p dk (simeon i. taylor, program director), and, in part, by the va merit review csr&d grant i cx - a (postolache, pi). the views, opinions and findings contained in this article belong to the authors and should not be construed as an official position of the us food and drug administration, the us department of veterans affairs, or the national institutes of health. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms conflicts of interest: the authors declare that they have no conflict of interest related to the publication of this paper. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms references ( ) jones jl, parise me, fiore ae. neglected parasitic infections in the united states: toxoplasmosis. american journal of tropical medicine & hygiene ; ( ): - . 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( ) kraybill d, johnson-weiner j, nolt s. the amish: the john hopkins university press, . ( ) cuyun carter gb, et al. dietary intake, food processing, and cooking methods among amish and non-amish adults living in ohio appalachia: relevance to nutritional risk factors for cancer. nutrition & cancer ; ( ): - . ( ) duffy ar, et al. toxoplasma gondii serointensity and seropositivity: heritability and household- related associations in the old order amish. international journal of environmental research and public health ; ( ). ( ) mcdonald jh. handbook of biological statistics ( rd ed.). baltimore, maryland: sparky house publishing, : pp. - . ( ) almasy l, blangero j. variance component methods for analysis of complex phenotypes. cold spring harbor protocols ; ( ): pdb top . ( ) alvarado-esquivel c, et al. seroepidemiology of toxoplasma gondii infection in a mennonite community in durango state, mexico. journal of parasitology ; ( ): - . ( ) alvarado-esquivel c, et al. seroepidemiology of toxoplasma gondii infection in general population in a northern mexican city. journal of parasitology ; ( ): - . ( ) wilking h, et al. prevalence, incidence estimations, and risk factors of toxoplasma gondii infection in germany: a representative, cross-sectional, serological study. scientific reports ; : . ( ) taylor mr, et al. community study of toxoplasma antibodies in urban and rural schoolchildren aged to years. archives of disease in childhood ; ( ): - . ( ) muñoz-zanzi c, williams-nguyen j, belongia ea. a sero-survey of toxoplasmosis in farm and non-farm children from wisconsin, united states, – . bmc public health ; ( ): . ( ) jones jl, et al. toxoplasma gondii infection in the united states: seroprevalence and risk factors. american journal of epidemiology ; ( ): - . ( ) costard s, et al. outbreak-related disease burden associated with consumption of unpasteurized cow's milk and cheese, united states, - . emerging infectious diseases ; ( ): - . ( ) boughattas s. commentary on: "detection of toxoplasma gondii in raw caprine, ovine, buffalo, bovine, and camel milk using cell cultivation, cat bioassay, capture elisa, and pcr methods in iran". frontiers in microbiology ; : . https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms table . demographic characteristics of old order amish participants, toxoplasma gondii serostatus and risk factor study, - . characteristic total n (%) ( %) seropositive n (%) ( . ) seronegative n (%) ( . ) p value* age (y) - ( . ) ( . ) ( . ) < . - ( . ) ( . ) ( . ) - ( . ) ( . ) ( . ) - ( . ) ( . ) ( . ) - ( . ) ( . ) ( . ) + ( . ) ( . ) ( . ) mean (sd) . / . ( . ) . / . ( . ) . / . ( . ) < . sex female ( . ) ( . ) ( . ) < . male ( . ) ( . ) ( . ) body mass index mean (sd) . ( . ) . ( . ) . ( . ) . t. gondii titre concentration (iu/ml) ** median (sd) . ( . , . ) . ( . , . ) . ( . , . ) < . sd, standard deviation; t. gondii, toxoplasma gondii. *p value from x test of independence for age and sex[ ]; from sas . (cary, nc) for t-test; italics denote significance at p < . . **t. gondii titre concentration: seropositive ≥ iu/ml; seronegative < iu/ml. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms table . bivariate analysis of unadjusted associations (unadjusted odds ratios) of serostatus and category-based behavioural risk and protective factors of old order amish participants, toxoplasma gondii serostatus and risk factor study, - . behavioural risk or protective factor seropositive of those positive for behaviour n/n (%) seropositive of those negative for behaviour n/n (%) unadjusted odds ratio % ci p value pets (ref: no pets) has pets / ( . ) / ( . ) . . - . . has cats / ( . ) / ( . ) . . - . . has kittens / ( . ) / ( . ) . . - . . has contact with cats / ( . ) / ( . ) . . - . . cleans the litterbox / ( . ) / ( . ) . . - . . meat consumption (ref: no consumption) ate rare meat / ( . ) / ( . ) . . - . . ate raw meat / ( . ) / ( . ) . . - . . ate raw ground pork* / ( . ) / ( . ) ∞ ∞ < . * ate rare ground beef / ( . ) / ( . ) . . - . . unpasteurized milk consumption** (ref: no consumption) consumed unpasteurized cow or goat milk products* / ( . ) / ( . ) . . - . . washing hands (ref: not always washing hands after touching raw meat) always washing hands after touching raw meat / ( . ) / ( . ) . . - . . *only respondent indicated raw ground pork intake. **only respondents indicated consumption of unpasteurized goat milk products; indicated intake of unpasteurized cow milk products. italics denote significance at p < . . https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms table . bivariate analysis of associations (unadjusted odds ratios) of serostatus and frequency- based behavioural risk and protective factors of old order amish participants, toxoplasma gondii serostatus and risk factor study, - . behavioural risk or protective factor never < /week ≥ week daily unadjusted odds ratio % ci p value consumption of: unpasteurized cow milk/ yoghurt / ( . ) / ( . ) / ( . ) / ( . ) . . - . . rare meat / ( . ) / ( . ) / ( . ) / ( . ) . . - . . raw meat / ( . ) / ( . ) / ( .) / (n/a) . . - . . less than once a month once a month or more unadjusted odds ratio % ci p value location of meal consumption: outside the home / ( . ) / ( . ) . . - . . italics denote significance at p < . . https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms table . adjusted associations (adjusted odds ratios) of toxoplasma gondii seropositivity and age and behavioural risk and protective factors by sex of old order amish participants, toxoplasma gondii serostatus and risk factor study, - male female factor adjusted odds ratio % ci p value adjusted odds ratio % ci p value age female . . – . < . male . . – . . pets has pets . . – . . . . – . . has kittens . . – . . . . – . . has cats . . – . . . . – . . cleaned the litterbox . . – . . . . – . . meat consumption* ate rare meat . . – . . . . – . . ate raw meat . . – . . . . – . . ate rare ground beef . . – . . . . – . . unpasteurized milk consumption** consumed unpasteurized cow or goat products** . . - . . . . - . . consumed unpasteurized cow milk products . . - . . . . - . . washing hands always washing hands after touching raw meat . . – . . . . – . . italics denote significance at p < . . *only respondent indicated raw ground pork intake; results, therefore, not shown. **only respondents indicated consumption of unpasteurized goat milk products; indicated intake of unpasteurized cow milk products. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms a mems electrostatic particle transportation system - micro electro mechanical systems, . mems . proceedings., the eleventh annual international w a mems electrostatic particle transportation system a. desai, s.w. lee, and y.c. tai caltech micromachining laboratory, electrical engineering - , california institute of technology, pasadena, ca usa abstract we demonstrate here an electrostatic mems system capable of transporting particles - pm in diameter in air. this system consists of -phase electrode arrays covered by insulators (figs. i , ). extensive testing of this system has been done using a variety of insulation materials (silicon nitride, photoresist, and teflon), thickness ( - pm), particle sizes ( - opm), particle materials (metal, glass, polystyrene, spores, etc), waveforms, frequencies, and voltages. although previous literature [ - claimed it impractical to electrostatically transport particles with sizes - pm due to complex surface forces, this effort actually shows it feasible (as high as % efficiency) with the optimal combination of insulation thickness, electrode geometry, and insulation material. moreover, we suggest a qualitative theory for our particle transportation system which is consistent with our data and finite-element electrostatic simulations. regime the electrostatic forces that can be exerted on the particle are of the same magnitude as the adhesive forces between the particle and the solid surface. numerous researchers have noted this size range cut- off; for example, novick et a [ ] have noted that the sub pm range presents a different regime where surface adhesive forces and particle charging hinder the successful engineering of a robust particle transport system. the goal of this work then, is to reexamine this difficult task and design a dielectrophoretic (dep) particle transportation system [ - capable of moving particles below pm in air with low voltages. our work emphasizes a three-phase electrode array with specific insulative films. it is found that these films enable us to not only repeatably demonstrate motion for and uni particles, but reveal a novel phenomenon - the strong dependence of particle transportation efficiency with film thickness. in addition, computer simulation of dep force on the particle is consistent with our observations and hypothesis. introduction fabrication the ability to transport and manipulate particles in air is desirable in many instruments such as airborne particle samplers, particle sorters, and electrostatic particle- cleaning apparatuses. there are many different ways to transport particles larger than pm such as forced air jets, centrifuges and other mechanical means. however for particles ranging from to pm, there is still no efficient way to transport them because complex surface forces, instead of gravitational force, dominate. this attribute has long been a bottleneck for the development of automated airborne pollutant samplers for biological spores, dust particles, and chemical agents because their sizes fall into the - ym range. in the past, successful transportation of sub pm particles has been accomplished in the liquid medium, but not on a solid surface in air. in air, moesner and higuchi [ ] and balachandran et a [ ] have demonstrated motion of larger particles with voltages up to a few kv. unfortunately, in the sub pm the particle transportation chip was developed in using standard microfabrication processes. the process basically consists of depositing two insulation layers and two conductive layers for a three-phase network of electrodes. a cross-sectional view of a finished electrode panel is shown below. (fig. ) i $ i $ fig. i : electrode cross-section the fabrication sequence begins with a silicon substrate coated with - pm of thermal oxide. next, a of aluminum is thermally evaporated and patterned to form the electrodes. plasma-enhanced chemical vapor deposition (pecvd) silicon nitride is deposited to form - - -x/ /$ . ieee a l y m insulation layer between the " and "d metal layers. vias are etched into this silicon nitride layer for connection of the "d a layer to the lst. next, the znd ai layer is deposited and patterned. in addition to the above process a similar process using gold instead of aluminum and low temperature thermal oxide instead of pecvd silicon nitride was also developed. finally, another insulation coating ranging from pm to pm is deposited to change particle adhesion characteristics. details on the insulation material (photoresist and teflon) will be discussed in the experimental section. cross-sectional views of the process steps are shown below. thermal oxidation sioz si insulator deposition & patterning siu, s st metal deposition & patterning sin si sio, nd metal deposition & patterning -- sin si sio, final insulation coat insulator sin si sio, fig. : process flow for electrode chip with the above process, various electrode designs were fabricated. pictures below are examples of radial and linear geometries that were designed. example of the voltage patterns required for each phase is illustrated in the schematic below. (fig. ) fxn generator signal ov phase (v ) ov ioilv n phase (v ) ov f-- one cycle _____) fig. : typical applied waveform other voltage patterns such as sinusoidal waves, triangular waves, bi-polar traveling waves, and the inverse of the waveforms shown in fig. were also utilized for sustained particle motion. particles can be made to change direction by switching phases and , and step (altemate) from one electrode to another by tuming off one of the phases. particle forces dielectrophoretic force much work has been done in investigating the dep force on particles ranging from dust to biological cells. the equation, from jones[s], for the special case for a lossless dielectric sphere is f~~~ = n s , ~ ~ ~ , the dep force is a function of e ~ , the permittivity of the surrounding medium, r , particle radius, and eo, the electric field. the constant k is defined as ( ) k=----- € - e + e l where e is the pemiittivity of the sphere. the important factor to note is that the dep force is a strong function of the particle volume ( r ) and the gradient of the electric field squared (ve;). it is the latter term that makes eq. difficult to compute in closed form for spherical particles. adhesion force ... ........ .. ......-"....i_.." radial linear fig. : various electrode structures the electrode structures were operated with -phase circuit capable of waveforms from typically to v,, with frequencies from . hz to okhz. an the adhesion forces between a ym-sized particle and a surface are mostly van der waals forces, electrostatic forces (image force), meniscus forces, and also gravitational forces.[ ] it is impossible to calculate the exact contribution of these various forces that govern the adhesion of particles in this size range. however, the calculation of these forces on micro-particles, even in vacuum is approximate in order of magnitude at best.[ ] in air, these theoretical foimulas become more inaccurate because of irregularities of surfaces, molecular adsorption, and stray electronic charging. nevertheless, we use these formulas to gain an understanding of the magnitude of the forces involved. many scientists have experimentally studied these phenomena, and have come up with estimates for adhesive forces.[ ] because of the irregularity of particle and surface contact areas, experimental results for this range of particles are often denoted in percentages of particles that are removed vs. adhered. large variation is common with these percentages due to the statistical nature of the phenomena. surface coating pecvd silicon nitride az teflon (af s) experimental frequency max . - . - % - , % a variety of particle sizes and materials were tested with this particle transportation chip. most of the experiments were performed on linear electrode and spacing widths of pm and pm (pitch and width spacing is identical). table summarizes the types of particles tested. table i : particles tested microsp heres borosilicate glass polystyrene dvb paper mulberry pollen* . - . . - . *refers to hydrated size testing was conducted in the following fashion. first, the electrode panel was "turned on" and then particles were released on the electrode panel with the aid of a micromanipulator probe tip. as the probe tip neared the electrode panel surface, the traveling electric field force was sufficient to detach particles from the probe tip onto the surface of the transport chip. multiple sets of experiments for each parameter (particle type, size, insulation, electrode spacing) were performed in this fashion. particle efficiency data was gathered by noting the ratio of particles transported to the edge of the linear electrode array vs. the total number of particles on the structure. the laboratory environment was standard room temperature, t and - % relative humidity. fig. : particles before applying waveform fig. : particle transported to edge of electrodes observations two major trends were noted during testing. regardless of the type of final insulation films and their varying thicknesses, the transportation efficiency increased with increasing voltage. secondly, the type of final surface coating also had a significant effect on particle efficiency and threshold voltage, the minimum voltage level to exhibit particle motion. experiments using silicon nitride as the insulation layer resulted in poor performance. large threshold voltages were needed, and moreover, particles would exhibit motion for few seconds and then stop moving. motion would resume only when the voltage level was increased further. this was observed for voltage levels as high as v. any further increase in voltage larger than v resulted in breakdown of the insulation layer between the '' and "d metal layers on the chip. in comparing different insulation materials, the effect was remarkably different. for example, photoresist film (hardbaked az ) resulted in maximum efficiencies for glass particles of approximately %, but teflon film@] resulted in efficiencies of %. table summarizes the behavior of different surface coatings. table : linear pm spacing structure, pm glass particles in order to ascertain which adhesive forces (meniscus vs. van der waal vs. coulombic) contribute most to particle stiction, various surface modification treatments were performed. for example, the silicon nitride insulation was annealed at t for hrs in order to remove surface moisture, and also, the chip was heated up to c while the particles were transported. the nitride surface was also silanized to increase surface hydrophobicity with a coating of trimethylcholorsilane (sigma corp.). however, the above treatments did not result in any signilicant improvement. increase in surface roughness has also been known to reduce particle adhesion.[ ] consequently, the resist surface was roughened with o plasma which changed the characteristics of the motion. all the particles did not move in the same direction of the traveling wave, but rather, in a random fashion. this effect was probably due to the large amount of negative charge implanted on the resist surface from the plasma. since no significant improvement was observed with the previous treatments, it is concluded that meniscus forces are not dominant factors in contributing to particle stiction surface treu tmen t contact hurnzonic efect potential full sinusoidal wave bipolar wave plasma roughening positive voltage wave tmcs coating ground voltage wave table : surface and waveform tests as mentioned previously, on silicon nitride, particles would only exhibit momentary motion when voltage level was constantly increased. this type of phenomenon is most likely associated with particle to surface contact charging. however, some sources also point out that the difference in contact potentials between the particle material and the substrate material can causing particle charging during detachment. [ , ] therefore, to investigate if this type of charging was occurring, we also fabricated electrode panels with low temperature oxide (lto) instead of si,n,. nevertheless from our measurement data, we did not find any noticeable difference between lto and nitride for glass particles. different waveforms such as the inverse of the waveform in fig. , bipolar, and triangular were also investigated to ensure particle motion was due to dep force and not just particle charging. when the traveling wave in fig. was utilized, the particles traveled in the same direction as the waveform, and when this same waveforms was inverted (i.e. positive loov traveling pulse) the particle also moved in the same direction. from this observation, we can conclude that particle motion is dielectrophoretic and not due to charging. however, the most significant and remarkable effect on performance was noted when insulation thickness was increased. transport efficiency variation also is a strong function of final insulation film thickness. fig. shows the variation of transportation efficiency vs. insulation height for spm glass spheres for photoresist and teflon. - ~ ~ -~ - _ _ transport efficiency vs. insulation thickness ~ ( p t i sjoz_pajticles) i - r % - -- - insulation thickness (pm) fig. : optimal thickness effect in addition, fig. s also shows the effect of frequency. as frequency increases the efficiency improves, and then saturates. for photoresist, motion cuts off at -lkhz, and for teflon motion was observed only up to okhz due to limitation of our circuit. this frequency window can be explained due to the dynamics of particle movement. there is a finite time associated with particle stepping from one electrode to another. this time involves particle acceleration and deceleration from one electrode to the next. at the cut- off frequency, the particle is unable to keep up with the traveling wave. traveling wave frequency effect i ( pm sioz particles on teflon) ' %. % % % % so% % i i i teflon thickness (pm) fig. : frequency effect on efficiency discussion understanding the interaction between the dep forces and adhesive forces on the particle is the key to interpretation of the experimental observations. from the experimental data, it seems quite possible that the image force contribution is the dominant adhesive force and decreasing this image force, f', leads to higher transportation efficiency. the image force arises when particles are charged and are in close proximity to a conductor.[ ] in fact, the presence of an optimal thickness with the insulative films supports this hypothesis because f' is reduced when the distance from the particle to the electrodes is increased. fy = f,”(x,y) + fy (x,y,p)+f,ft f,(x,y): f,(x,y.p): image force due charges on particle fax,y): friction force, (p,* f,(x,y)} f i stiction forces driving force from applied voltage fig. : schematic of particle forces fig. [ ] illustrates the various forces acting on the particle. the downward force on the particle, fy, arises from three sources: the image force, f,’ , the stiction force, f;‘, and also the y-component of the dep force, f f . the horizontal force, fx, can be separated into four parts: ) the dep force, fxd, ) f,’, the x- component of the image force, ) the frictional force, ff, and ) f,”. however, ff is inherently related to fy through the frictional coefficient, pq. because of this relationship, any change in insulation thickness also has an effect on the net horizontal force on the particle. to verify this downward component of the dep force, fxd and f: on a single particle have been computed using maxwell, a -d finite element simulation.. (fig.[ lo]) the simulation parameters are the following: spin sioz particle, pm electrode width and spacing, l ov wave, particle distance from electrode is pm, and the particle is uncharged. the electrodes and a particle are drawn to scale on the x-axis. the x coordinate of each data point represents the position of the particle center with respect to the electrodes, and the y-axis shows magnitude of the force. from fig. , we see that although the horizontal or x-component of the dep force, f,”, due to the voltage waveform is responsible for particle transport, the dep y- component, f,,, acts downward thus adding to the frictional force. in other words, the dep force not only exerts a horizontal force but a downward force as well. dep force simulation for pm sioz particle . oc- .oor,-o - .noe-o l - m ~ - n o o f - n o - (i e+ - . f- - e- - i. oe- - . e- - - - - - - - particle coordinate (um) - ~ - __ - fig. : dep force on particle likewise in agreement with electrostatic theoiy, other simulation results show that f,’ diminishes as the height from the electrodes is increased, and it is greatest when the particle is on top of the electrode. superposition of these forces, image, dep, and stiction gives us total downward force. the fem results corroborate the phenomena observed in experiment. in our experiments variation of this vertical distance which is controlled by the thickness of the insulation film, clearly affects the performance. if the insulation is too thin, the downward forces dominate and efficiency drops. if the insulation film is too thick the driving force is not enough to overcome the adhesive forces. therefore at a specific thickness, fx is maximized and thus gives the highest transportation efficiency. however, each insulation film has its own optimal thickness (fig. ) because van der waals, meniscus force, and frictional coefficient between the particle and the insulation surface differ from film to film. conclusion we have shown that particles of - i o pin size range can be manipulated electrostatically with voltages below v in a standard room environment. in contrast to the work done in the past in this field, our work shows that it is possible to move pm-sized particles in air. furthermore, we have shown that particle charging is a significant cause of adhesion on our mems transportation system, and we have discovered a simple way to reduce its negative effect on particle transportation performance. our goal is to incorporate this mems transportation system in an automated particle sampler. acknowledgements the funding for this project was provided by darpa, microflumes contract. n - -c- . the authors would also like to thank mr. x.q. wang and mr. w. hsieh for their knowledge and assistance with parylene and teflon films. references [ i . ] d.w. cooper, h.l. wolfe, j.t.c. yeh, r.j. miller, “surface cleaning by electrostatic removal of particles,” aerosol science and technology, . v.j. novick, c.r. hummer, p.f. dunn, “minimum dc electric field requirements for removing powder layers from a conductive surface,” journal of applied physics, vol. , - , . vol. , - , . [ .] f.m. moesner, t. higuchi, “devices for particle handling by an ac electric field,” ieee conference proc. mems . [ .] w. machowski, w. balachandran, smieee, d. hu, “influence of electrode geometry on transport and separation efficiency of powders using travelling wave field techniques,” ieee, . [ .] t.b. jones, “electromechanics of particles,” cambridge university press, new york, . [ .] c.n. davies, “aerosol science,” academic press, london and new york, . [ .] a.d. zimon, “adhesion of dust and powder,” consultants bureau, new york, . [s.] t. hsu, w.h. hsieh, k.furutani, y.c. tai, “a thin film teflon electret technology for microphone applications.” hilton head solid-state sensor & actuator workshop. [ .] g.m.colver, “dynamic and stationary charging of heavy metallic and dielectric particles against a conducting wall in the presence of a dc applied electric field,” journal of applied physics, vol. , no. , november . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ mergedfile 	
   	
   development of allergic immunity in early life clare m lloyd & sejal saglani , inflammation, repair & development section, national heart and lung institute, faculty of medicine, imperial college, london; dept of paediatric respiratory medicine, royal brompton hospital, royal brompton harefield nhs foundation trust, london, uk contact: inflammation, repair & development section national heart and lung institute sir alexander fleming building faculty of medicine imperial college south kensington london sw az. uk + c.lloyd@imperial.ac.uk 	
   	
   running title: allergic immunity in early life abstract the growth and maturity of the peripheral immune system and subsequent development of pulmonary immunity in early life is dictated by host, environmental and microbial factors. dysregulation during the critical window of immune development in the postnatal years results in disease which impacts on life-long lung health. asthma is a common disease in childhood and is often preceded by wheezing illnesses during the preschool years. however, the mechanisms underlying development of wheeze and how and why only some children progress to asthma is unknown. human studies to date have generally focused on peripheral immune development, with little assessment of local tissue pathology in young children. moreover, mechanisms underlying the interactions between inflammation and tissue repair at mucosal surfaces in early life remain unknown. disappointingly, mechanistic studies in mice have predominantly used adult models. this review will consider the aspects of the neonatal immune system which might contribute to the development of early life wheezing disorders and asthma, and discuss the external environmental factors which may influence this process. key words: asthma, inflammation, wheezing, immune development 	
   	
   introduction asthma is a childhood onset disease, with . million children currently affected in the uk. children diagnosed with asthma by age years already have a deficit in lung function and increased bronchial responsiveness as neonates ( ). furthermore, longitudinal birth cohort studies have shown wheezing and bronchial hyper-responsiveness in early childhood are predictors of newly diagnosed asthma in early adulthood ( ), and a prior diagnosis of asthma is associated with an increased risk for copd among never smokers( ). the fundamental pathophysiological features of allergic asthma in both adults and children include airway hyperresponsiveness, resulting in airway obstruction that is usually reversible, chronic inflammation that is predominantly eosinophilic and structural changes of the airway wall, termed remodelling. we have shown eosinophilic inflammation and structural airway remodelling ( ) are already established during the preschool years and become maximal and equivalent to the changes seen in adulthood by school age ( ). birth cohort and cross- sectional studies of preschool children with wheezing have uncovered strong associations between lung function and immune responses in early life. furthermore, children who apparently "outgrow" early wheezing illnesses remain at increased risk for relapse or recurrence during midlife( ). although approximately one-third of all infants and preschool children have wheezing disorders, we do not know why some but not all pre-school children wheeze; and why some wheezy children develop asthma, whereas others spontaneously remit. no current medication, including inhaled corticosteroids (ics), can prevent the development of asthma. therefore, a focus on the understanding of the early life pulmonary immune environment is essential not only to prevent the development of asthma, but also in the long term to contribute to the prevention of copd ( ). this review will consider the impact of various intrinsic and extrinsic factors which may impact the development of wheezing and progression to asthma (fig ). 	
   	
   severe asthma in children although the majority of children with asthma achieve good control with only low-moderate doses of maintenance inhaled steroid therapy, approximately % remain poorly controlled despite maximal prescribed therapy. this small proportion with persistent poor control are a significant clinical challenge as they utilise up to % of all healthcare resources for asthma ( ). all children with on-going asthma symptoms despite maximal conventional treatments are described as having problematic severe asthma ( ). the sub-group in whom persistent symptoms result from a failure of basic asthma management have “difficult asthma” (da) ( ), whilst those who remain symptomatic despite these factors having been addressed have “severe therapy resistant asthma” (stra) ( ). a common definition and guidelines for management of severe asthma have recently been proposed for all patients aged years and over ( ). although a significant advantage of common guidelines is the ability to trial novel treatments using similar criteria, some key differences underlying the evolution and pathophysiology of severe asthma between children and adults must be considered, and an automatic extrapolation of findings from adult clinical trials to children may not always be appropriate. pathophysiology of paediatric asthma asthma is characterised by eosinophilic inflammation, airway hyperresponsiveness (ahr) and structural airway remodelling, including increased thickness of the subepithelial reticular basement membrane, increased airway smooth muscle mass and angiogenesis ( ). all of these changes are established in children by school-age, and are present regardless of disease severity ( ),( ) ( ). asthma has traditionally been thought of as a disease of type immunity due to increased levels of il- , il- and il- . as such therapy has been directed towards mitigating the effects of these cytokines ( , ). however, asthma is notoriously heterogeneous, with numerous clinical and pathological phenotypes ( ) and 	
   	
   there is evidence that not all patients fit this restrictive, th biased categorisation of disease pathology ( ). in particular, patients with severe disease may not all have a th phenotype. a feature of severe asthma in young children is the early presence of airway remodelling – including thickened basement membrane ( ). it has been proposed that a normal inflammatory response to harmful stimuli is followed by a phase of repair and regulation, whereas during chronic disease repeated cycles of inflammation result in exaggerated repair culminating in tissue remodelling. however, increasing evidence now confirms that at mucosal surfaces, the barrier epithelial cells and underlying stromal cells are active immunologically and cooperation with immune effector cells results in development of inflammation and repair in parallel, or in disease exaggerated inflammation and abnormal remodelling ( ). using both our neonatal model of inhaled allergen exposure and paediatric bronchoscopic airway samples we have shown early and parallel onset of inflammatory and structural changes is apparent in airway mucosal diseases such as preschool wheezing and asthma ( ) ( , ) and cystic fibrosis ( ). specifically, we were among the first to show an absence of tissue eosinophilic inflammation or remodeling in infants with recurrent respiratory symptoms and wheeze at a year of age ( ), but increased inflammation and remodeling by years ( ) which increased further with age and was established to a similar degree to adults by school-age ( , ). early life events that result in altered tissue repair are likely to be sustained across the life-course and result in down-regulation of cell defence mechanisms and impact the ageing trajectory. although associations between inflammation and remodelling are apparent during early life mucosal diseases, nothing is known about the interactions between the maturing immune system and remodelling pathways, and whether repair pathways are more easily triggered in early life and thus lead to exaggerated repair.	
   th mediators in paediatric severe asthma children with stra have a persistent airway luminal and tissue eosinophilia and airway remodelling despite high dose maintenance steroid therapy. another key feature of 	
   	
   childhood severe asthma is the presence of severe atopy with multiple aero-allergen sensitisation. this has been found repeatedly in cohorts as a feature of severe disease ( ) ( , ). however, in our study, in which evidence of type inflammation was sought in induced sputum supernatant and bronchoalveolar lavage fluid (two different platforms) and immunohistochemistry of bronchial biopsies, evidence for the presence of il- , il- and il- was scarce ( ). furthermore, in a separate unrelated cohort, the paediatric severe asthma research programme (sarp), found neither type nor type inflammation dominated in children with severe asthma ( ). the airway pathology is therefore present despite a relative absence of elevated levels of th cytokines. using an age appropriate neonatal mouse model of inhaled allergen challenge, we have shown that the th cytokines, il- and il- in particular, are steroid sensitive ( ). an explanation for the paucity of th mediators in paediatric stra may be that since significant care is taken to ensure patients are compliant with maintenance steroid therapy, levels of th mediators may be dampened. innate mediators and severe asthma with fungal sensitisation the fact that children with stra remain symptomatic, even while on inhaled steroids, and in % of cases while on maintenance oral steroids ( ) indicates that other molecular pathways underlie disease pathology. although il- , il- and il- levels were difficult to detect, we did find an increased frequency of patients with elevated il- + cells in the submucosa of the airway wall ( ). il- is an innate cytokine derived from the airway epithelium that has been associated with the initiation and potentiation of the pathobiology of allergic asthma ( , ). we have shown using endobronchial biopsies from children with stra that submucosal il- expression was associated with increased thickness of the reticular basement membrane, a specific feature of airway remodelling ( ). moreover, in vitro culture of primary fibroblasts from the stra patients’ biopsies showed increased secretion of collagen following il- stimulation compared to controls, even in the presence of the steroid budesonide. intranasal administration of ril- to non-allergic mice resulted in increased collagen production in the lung, as well as the expected increase in ahr. using 	
   	
   our neonatal murine model of inhaled house dust mite exposure, we showed levels of il- remained elevated in allergen challenged mice, despite steroid therapy, and this was associated with persistent ahr and remodelling. interestingly, levels of il- were significantly reduced. thus it was apparent that il- was relatively steroid resistant and was associated with airway remodelling, both of which are features of severe disease in children with stra ( ). conversely, remodelling was absent in hdm-exposed st -/- mice that lack a functional receptor for il- . the fact that il- , but not il- , was maintained following steroid treatment of neonatal hdm-exposed mice, as well as in biopsies from children with stra suggests that il- is resistant to the actions of steroids. this is important because il- is a cytokine with wide ranging effects on a variety of cells and is thought to bridge innate and adaptive immune pathways ( ). numerous experimental models have shown that il- induces the development of type innate lymphoid cells (ilc s) ( ) ( ). these cells belong to a recently discovered population that displays none of the usual lineage markers for leukocytes but produce effector cytokines. like effector t cells, ilcs have now been classified according to the cytokines that they secrete and the transcription factors that they express ( ). ilc cells are defined by the production of type cytokines il- , il- and il- as well as expression of gata . we have shown that children with stra have significantly elevated levels of ilc cells (defined as lineage-cd +crth +) in their blood, broncho- alveolar lavage and in induced sputum ( ). although present in the blood, numbers were very small, and there were significantly higher proportions of ilc in the airways, demonstrating the propensity of these cells to remain at local tissue sites. although as a group, children with stra had increased tissue expression of il- , the heterogeneity of the disease was reflected in the spread of il- levels within the group, thus highlighting the need to identify sub-phenotypes of patients in whom any mediator, such as il- , is the predominant driver of disease. having shown using our adult murine model that exposure to the fungal allergen alternaria alternata resulted in il- driven exacerbation of allergic airways disease ( ), we investigated the role of this mediator in the very specific clinical sub-phenotype of severe asthma with fungal sensitisation (safs) ( ). we have 	
   	
   shown that children with safs had evidence of worse allergy than stra without safs, despite being prescribed more steroid treatment ( ). using our neonatal mouse model we compared disease severity following inhaled hdm exposure to alternaria exposure, and also showed higher serum ige levels and greater eosinophilia in animals sensitised to alternaria, which was maintained despite administration of steroids. although levels of th cytokines were not different between hdm and alterrnaria exposure, il- was significantly higher in the alternaria exposed neonatal mice. moreover, on dividing patients into those with and without fungal sensitisation we showed il- was increased in bal, and immunohistochemistry of bronchial biopsies in children with safs ( ). interestingly, these patients also had higher levels of mmp- which is known to be important in regulation of extracellular matrix molecules such as collagens within tissues. to date, trials of anti-fungal agents have been disappointing in patients with safs ( ), but these data suggest this sub- phenotype may be the most amenable to therapeutics that block the action of il- . neutrophils in childhood severe asthma unlike adults, in whom neutrophilic asthma is a recognised steroid resistant phenotype of severe disease ( ), we have shown that children with stra do not have increased neutrophils or mast cells in their airways or in bronchial biopsies ( , ). however, we have recently demonstrated that intra-epithelial neutrophils are present in some children with stra ( ). although children with stra had increased intra-epithelial neutrophils compared to controls, what was most interesting was that the epithelial neutrophil expression was high in a sub-group of stra patients and low in others. challengingly, the neutrophil-high children had better asthma outcomes (higher fev percent predicted, better symptom control) while being prescribed lower maintenance inhaled steroid doses. however, despite the presence of intra-epithelial neutrophils, levels of tissue and luminal il- a were not increased in stra, while, epithelial expression of the il- r was increased and stimulation of primary bronchial epithelial cells with il- a led to steroid resistant il- secretion ( ) . 	
   	
   interestingly, children who grow up in cattle farms and are protected from the development of asthma also have increased peripheral neutrophils ( ). these data highlight two critical issues pertaining to discovery of novel therapies for paediatric stra. the first is the inability to extrapolate findings from adult studies to children and the need for age appropriate mechanistic studies, and secondly that targeting inflammatory mediators or cells without confirmation of findings from experimental models in human tissue is unlikely to be successful as has been demonstrated by initial trial of blocking il- in adult patients ( ). clinical features and risk factors specific for paediatric disease preschool wheeze and its progression to asthma an important clinical phenotype that is very distinct and unique to children is that of wheezing in preschool children. cohort studies have shown that there are several clinical phenotypes of wheezing in preschool children with different outcomes by school-age( ). preschool wheezers who develop asthma have a permanent reduction in lung function by school-age ( ), persisting into adulthood ( ) and increasing susceptibility to chronic obstructive pulmonary disease (copd) ( ). the factors that initiate asthma are very different from those that perpetuate the disease. no current medication, including inhaled corticosteroids (ics), can prevent progression from preschool wheeze to asthma, but ics are able to control the symptoms of established asthma. thus investigating the molecular mechanisms underlying the inception of preschool wheeze and its progression to asthma is essential to identify therapeutic targets that will prevent early and sustained lung function abnormalities. early viral infections ( ), bacterial colonization ( ) and allergen sensitization ( ) are important in causing wheeze and the subsequent development of asthma. these early life exposures, coupled with genetically determined susceptibility, have a major impact on the natural history of the disease. cumulatively, the data highlight the critical nature of this early period in which the maturing immune system meets new environmental exposures, and subsequent immune/inflammatory responses in the lung are initiated. this underscores 	
   	
   the need for animal models of asthma to include young, as well as adult animals, particularly when considering the induction of disease. two clinical phenotypes of preschool wheeze have been described ( ). in episodic (viral) wheeze, children only wheeze with respiratory infections (usually viral), and do not have symptoms at other times. in multiple trigger wheeze, children wheeze both with infections, and are symptomatic between infections. importantly, the natural history and clinical management of each phenotype is different ( , ). multiple trigger wheezers are more likely to be atopic, respond to regular inhaled steroids, develop an early and permanent reduction in lung function and go on to develop asthma in school age. conversely, episodic wheezers are less atopic, only respond to intermittent therapy at the time of symptoms, and are less likely to develop asthma. however, the distinctive molecular mechanisms that result in development of each of these wheezing subtypes and the factors that dictate progression to allergic asthma remain unclear. pathology of preschool wheeze preschool multiple trigger wheezers have a distinct airway inflammatory profile to episodic wheezers ( ). multiple trigger wheezers have evidence of eosinophilic airway inflammation ( ). children with this phenotype tend to have a good response to treatments for allergic asthma, in particular they have reduced symptoms and exacerbations if treated with maintenance low dose inhaled steroids ( ). although the inflammatory profile is eosinophilic, numbers of mast cells are similar in wheezers and non-wheezers. little is known about the cytokines driving preschool multiple trigger wheeze, although increased expression of il- in the submucosa has been reported ( ). since leukotriene-receptor antagonists have variable benefit, there are few other therapeutic targets currently available for those with severe preschool wheeze that persists despite high-dose inhaled steroids. it is known that in addition to eosinophilia, features of airway remodelling, specifically increased thickness of the reticular basement membrane, are already present in children with persistent, severe wheeze ( , ). however, neither of these features are predictive of 	
   	
   asthma development by school-age. the only pathological abnormality that predicts future asthma is airway smooth muscle( ), but at present, no biomarkers that represent smooth muscle function in preschoolers are known, so this feature cannot be used to identify future asthmatics; and in any event, we lack interventions to prevent the evolution of preschool wheeze to asthma. early sensitisation ( ), in particular to inhalant and perennial allergens with high levels of specific ige ( ), is an important risk factor determining progression to asthma in school age and early, multiple sensitisation predicts a severe disease trajectory ( ). in addition, genetic susceptibility is an important factor that contributes to childhood severe asthma ( ). several genes identified from gwas have specifically been associated with childhood asthma including il- , which has also been associated with severe disease in both children ( ) and adults ( ). in addition, when the sub-group of children with severe exacerbations are considered, il- was again identified as a susceptibility locus. it is clear that there are significant early changes in lung pathology that occur in children with these wheezing phenotypes, but what events precipitate these changes and crucially what factors mediate progression and propagation of disease through the life course are not well understood. given that it is recognised that asthma and probably preschool wheeze, reflect the pathological consequences of a disordered immune system, it seems important to consider the immense changes that occur to the immune system during this early life period, and how these may impact the lungs, which also continue to develop postnatally. development of immunity in early life newborn babies are particularly vulnerable to infection in early life because the immune system is not yet developed, but is shaped and educated during this immediate postnatal period. although transfer of maternal antibodies across the placenta provides some 	
   	
   protection against pathogens, the potential antigenic burden is very high compared to the relatively sterile semi-allogeneic environment in utero. it was perceived that babies were born in a state of immune tolerance in order to protect them from developing overwhelming immune responses upon exposure to the wealth of pathogens encountered on leaving the protected in utero environment. this antigenic burden can be intrinsic as well as extrinsic - as birth triggers exposure to previously unseen, benign self-antigens to which the neonate must develop tolerance in order to prevent later autoimmune reactions. similarly, immune responses must be restrained upon exposure to environmental antigens, such as allergens. robust responses to these antigens would lead to hyper-inflammation that would be dangerous and destructive to organs such as the lungs that are still developing postnatally. thus, immune reactions against most antigens are kept under tight control, but mature adaptive immune responses can be mobilised when the baby is faced with life threatening, dangerous pathogens ( ). it is apparent that early life represents a window of both vulnerability and opportunity that impacts both immune development and tissue homeostasis, therefore it is important to understand how immunity develops in the normal lung in order to try and appreciate how allergic immunity develops. innate immune mechanisms are compromised during the first months of life. there is evidence that expression of tlrs that recognise key molecular signatures on the surfaces of pathogens are either absent or expressed at very low levels in the new-born. in fact, it has been documented that tlr and are largely absent from the murine foetal lung, but expression is rapidly upregulated after birth ( ). human studies are much harder to perform, but although data in human neonatal lung are rare, nasal mucosal explants taken from young children showed enhanced allergen-induced t cell reactivity and proliferation, increased production of th cytokines, il- production and tlr expression in response to tlr stimulation ( ). investigators have also taken advantage of cord blood as well as some studies using peripheral blood from babies. a comprehensive longitudinal profiling study of innate immunity over the first years of life revealed qualitative and quantitative 	
   	
   differences in tlr responses in neonates compared to those in adults. comparison of responses to tlr stimulation in adult and neonatal peripheral blood determined that monocytes and dendritic cells from neonates have a reduced capacity to secrete il- p , ifn-α and ifn-c, but a greater facility to secrete il- than adult cells( ). these findings suggest that rather than neonates being in a state of immune suppression responses to tlr stimulation are enhanced at birth, but differences are age specific and progression is not linear. studies with human cord blood have shown that perinatal tlr responses are increased – particularly in newborns who subsequently develop allergic disease. neonates from allergic mothers, or those that subsequently developed allergies showed increased secretion of tnfα following tlr , , stimulation and enhanced il- after tlr, , , ligands. crucially, however, t cell derived ifnγ responses to mitogen or allergen were suppressed compared to non-allergic subjects ( ). subsequent longitudinal analysis of tlr responses in allergic versus non allergic children during the first years of life revealed significant differences over time. from birth non-allergic children exhibit increased production of il- , tnfα and il in response to tlr stimulation over the first five years of life ( ). in comparison, allergic children show enhanced cytokine production at birth but this decreased over time so that by age , this hyperresponsiveness to stimuli had declined and cytokine responses were reduced in comparison to similar aged non-allergic children. a comprehensive study of immune development in urban preschool children determined that early life environmental exposures are vital in the stimulation of cytokine responses ( ). importantly, although cytokine production increased with age, these responses at birth were poorly predictive for later responses at aged or . interestingly, exposure to common environmental allergens, such as cockroach, mouse or dust mite was associated with enhanced cytokines responses at age years, including production of ifn-α and il- . however, reduced lps-induced il- responses as birth were related with recurrent wheeze, while rsv-induced il- , allergen-induced il were both associated with atopy. clearly the cytokine balance during early life is critical in the development of allergic 	
   	
   responses and is influenced by a variety of external factors. in future, it will be important to determine which cells secrete particular cytokines and establish how this fluctuates over time as well as determine whether the cellular source of the cytokine during development influences pathology. for example, development of allergic disease in the first year of life in high risk children has been associated with reduced responses of t regulatory cells to microbial stimuli ( ). collectively, studies show that tlr patterning occurs during the perinatal period and that the mother’s allergic status influences this. although these differences in tlr responses may protect the neonate from over-exuberant inflammation to microbial infection, they may contribute to the development of inappropriate reactions to environmental antigens such as allergens. neutrophils are a vital component of the innate immune system and are the most prevalent leukocyte in human blood. however, both neutrophil numbers and functionality are reduced in neonates compared with adults ( ). neonatal neutrophils have reduced expression of tlr , defective myd and p signalling ( ), and lower levels of cd b/cd which reduces their capacity for transmigration and chemotaxis ( ). in addition, neonatal neutrophils have reduced phagocytic capacity and impaired intracellular killing ( ). collectively, these deficiencies in numbers, mobilization and function increase the susceptibility of neonates to sepsis. pulmonary macrophages in early life the lungs are colonised with macrophages in the early postnatal period. macrophages are the most predominant leukocyte within the lung and there are at least two distinct populations resident at homeostasis: airway macrophages (am) that are resident within the airspaces and interstitial macrophages (im) that are situated within the lung parenchyma. each are characterized by their unique location, properties and functions, and populations may be identified on the basis of their expression patterns of the integrins cd b and cd c ( ). ams have high surface expression of cd c but lack cd b expression, distinguishing them from macrophages present in other tissue compartments. conversely, 	
   	
   ims express high levels of cd b but only low levels of cd c. ams have a critical influence in maintaining immunological homeostasis and are able to sense the local environment to maintain suppression or initiate the appropriate host defence response. in contrast, ims are thought to exert a regulatory function within the lung tissue. whilst am employ non-specific lines of defence (such as high phagocytic ability, the secretion of antimicrobials, nitric oxide (no), tumour necrosis factor (tnf)-α and interferon (ifn)-γ), it has been suggested that interstitial macrophages have a greater capacity to release specific cytokines associated with the adaptive immune response, such as interleukin (il)- ( ). although once thought to be derived from mononuclear phagocyte precursors recruited from the bone marrow it is now accepted that airway macrophages derive from yolk sac macrophages and are established prenatally. experiments with mice show that monocytes do not make a significant contribution to tissue macrophage populations under homeostatic conditions and furthermore, following depletion of lung macrophages, repopulation of the lung occurred by proliferation in situ, rather than replacement from the bone marrow ( - ). given the recognition that tissue residency has a specific impact on macrophage phenotype, experiments examining cord blood monocytes are unlikely to be reflective of airway and lung macrophage phenotypes. however, these samples are particularly difficult to obtain and there is scant information regarding phenotypes of interstitial macrophages in newborns or how they impact on development of allergic immunity. some studies have used bronchial lavage samples from babies requiring mechanical ventilation during chronic infections, and suggested that phenotypic maturation of monocyte populations was associated with gestational age. preterm infants had increased proportions of non-classical cd +cd + monocytes at birth, and immature macrophage phenotypes were associated with progression from respiratory distress syndrome to chronic lung disease of prematurity ( ). preterm infants with resolving lung injury had greater proportions of mature macrophages, than those with progressive lung disease. since these types of studies rely on lavage of ventilated, intubated babies, there is no information regarding the phenoptype of 	
   	
   macrophages during normal human perinatal development or on the impact of phenotype on development of asthma in subsequent childhood. perinatal dendritic cells in the lung neonatal murine lungs show fewer conventional dendritic cells, coupled with a lower ratio of cd + to cd b+ dc ( ). however, neonatal lungs matured and were able to prime adult naïve cd t cells as effectively as adult dc. interestingly, both adult and neonatal bcg-primed dc induced th cytokine responses from neonatal lymph node t cells, indicating that th skewing is an intrinsic feature of neonatal t cells ( , ). similarly, the ratio of plasmacytoid (pdc) to myeloid dendritic cells (mdc) is reversed in human cord blood dendritic subsets compared to those in adult peripheral blood ( ). both mdc and pdc were found to exhibit a more immature phenotype compared to those from adult blood, with lower expression of key markers such as mhc class ii and icam as well as the costimulation molecules cd and cd . in vitro stimulation assays using tlr ligands such as lps, poly i:c or cpg showed that neonatal (cord blood) mdc failed to mature to the extent of adult cdc ( , ). the failure of neonatal dc to initiate efficient th responses may rest with a specific defect in mdc whereby il- (p ) is transcriptionally repressed at the chromatin level ( ). human neonatal apc also show impaired production of type i interferons in response to tlr agonists. allergic children show higher numbers of tlr +dc at birth, and these enhanced levels are maintained throughout the first year of life. this is important because these cells are known to produce enhanced levels of inflammatory cytokines such as il- ( ). collectively, these defects in innate immune signalling may contribute to the propensity of neonatal t cells to generate immune reactions involving type cytokines rather than type and with persistent allergen exposure may contribute to the development of allergic disease and asthma in children. t helper cells in early life 	
   	
   early life exposure history shapes development of the immune system - however it is not known whether pathology progresses because of an imbalance in regulatory cells vs. effector cells, and if so how microbial exposures affect this imbalance. this is important since accumulating evidence suggests that these early life microbial exposures are linked to subsequent development of wheeze and asthma ( ). although the ontogeny of cd subsets has been examined in peripheral blood during the first year of life there is less information regarding the phenotypes of key lymphoid cells within the pulmonary mucosa of neonates. the relatively limited exposure to antigen in utero dictates that newborns are more reliant on innate immune pathways for protection against infections. however, neonatal mice have been shown to be able to mount mature adaptive immune responses in vivo ( , ). foetally derived cd + t cells with an effector memory phenotype have been shown to be present in cord blood ( ). these cells develop during foetal life within the uterine environment which is thought to be relatively sterile, but occur even in the absence of pathology or infectious history. importantly, they exhibit a variety of different effector inflammatory functions associated with cd t helper cells at birth. moreover, foetal cells are thought to be an important source of type- cytokines in early life in both mice and humans ( , ). nasal associated lymphoid tissue (nalt) is established before birth, while bronchus associated lymphoid tissue (balt) develops postnatally ( ). this immature immune system is shaped following postnatal exposure to bacteria, viruses and allergens, influencing its development and may result in skewing towards health or disease. the majority of studies to date have used peripheral blood cells to examine early life immune cell phenotypes. these studies have determined that a t helper (th ) cell preference is required for a healthy pregnancy ( ), but also that this skew is maintained during the neonatal period, reducing gradually during the first years of life ( ). in contrast, atopic children retain these foetally derived allergen-specific responses ( ). this deviation from the physiological in utero th 	
   	
   skewing, with exaggerated th responses in either pregnancy or the first months of life has been associated with an increased risk of subsequent childhood asthma or wheeze ( ). interestingly, a skewed th cytokine response was initiated from either adult or neonatal bcg-primed dc using neonatal lymph node t cells, indicating that th skewing is an intrinsic feature of neonatal t cells ( ). in fact, it seems that this skewing of neonatal t cell immunity towards type reactions limits inflammatory damage while permitting colonisation with neonatal commensal microbes in the intestine. one of the major effector functions of human neonatal t cells has been shown to be production of the chemokine cxcl , which can activate antimicrobial neutrophils and γδt cells. importantly these t cells were found to be rare in adults – highlighting the distinct nature of the neonatal immune system and provides a potential protective mechanism against bacterial infection in newborns ( ). similarly, γδt cells from newborns show enhanced pleiotropic functional responses when compared to αβt cells, and importantly lack the characteristic deficit in ifnγ production. therefore, in the absence of a mature αβt cell compartment, γδt cells are poised at birth to contribute to both immuno-protection and immuno-regulation ( ). although the mechanisms that facilitate persistence of the neonatal type environment are unclear, transcriptional profiling of circulating th cells in mice and humans has revealed sets of genes that are likely to confer pathological features to th cells that may be either unique to specific allergic diseases such as asthma or rhinitis, while others maybe common to a range of atopic disorders ( , ). interestingly, th cells from asthmatic patients specifically displayed increased expression of genes which were predicted to support enhanced th survival, proliferation/activation and cytokine production. furthermore, the il- /st axis seems to selectively licence memory th cells to promote allergic type inflammation via production of il- and thus stimulate eosinophilic inflammation ( , ). these pathogenic effector th cells seem to be a specific subpopulation of th cells with enhanced pro-inflammatory function and have been described in patients with eosinophilic 	
   	
   gastrointestinal disease as well as atopic dermatitis ( ). the latter patient population were children, but the point during immune development when this subpopulation develops has yet to be determined. t cell phenotypes are widely acknowledged to be plastic with receptor expression and cytokine production influenced by a wide range of intrinsic and extrinsic factors – including allergen exposure as well as age, so it is possible that the balance of subsets changes over the lifespan according to particular exposures. in the future, it will be important to examine the temporal shifts in th subsets of the lifespan in order to identify pathways for novel treatment. regulation of inflammation at mucosal sites is controlled by specialized populations of tissue resident regulatory t cells that express rorgt or gata - key transcription factors normally associated with effector th populations ( ) ( ). described in murine models of gut inflammation, these regulatory populations are held in balance by the local microbiota. in addition, expression of the il- receptor is thought to define a population of t regulatory cells in the gut that is important for accumulation and maintenance of t regulatory cells in an inflammatory environment ( ). as yet, it is unclear whether they are also present at human mucosal sites – including the lung. normal pregnancy is associated with skewing towards a type immune environment which is thought to afford protection to the developing foetus, and maybe due to an altered ratio of effector to regulatory t cells ( ). although this inequity normally corrects during the first year of life, there is evidence that prolongation of the type bias increases the risk of allergy and atopic diseases. functional foxp + cells with enhanced expression of pd- are generated following in vitro activation of naïve cord blood derived cd ro+cd +cd+ t cells with apc, compared to cells derived from adult peripheral blood ( ). longitudinal analysis of infant peripheral blood has determined that perinatal th cells have an enhanced propensity to develop into foxp + t regulatory cells in the first months of life but that th cells can already be induced at months ( ). additionally, the proportion of both resting naıve t regulatory cells and activated tregs isolated from peripheral blood increase markedly from birth to months of age ( ). in 	
   	
   contrast little is known regarding the phenotypes of cells within the airways of infants. however, it is important to remember that neonatal thymectomy in mice results in development of autoimmune disease, highlighting the importance of a competent regulatory population even early in life. cd +cd + t cells are important for the development of neonatal tolerance to transplantation antigens. innate lymphoid cells in early life innate lymphoid cells (ilcs) are considered to play an indispensable role in immune homeostasis as well as the initiation, regulation and resolution of inflammation ( ). ilcs are thought to exert particular influence at mucosal surfaces including the airways and gastrointestinal (gi) tract, with ilc s promoting repair following influenza infection in the lung, and ilc s mediating tissue repair following intestinal inflammation ( ). they are also key players mediating interactions between innate and adaptive type immune pathways ( ). despite the proposed importance of these effector cells in immune regulation and disease, and the intimate link with t helper cells, nothing is known about their role in immune maturation and early life development. recent evidence indicates that the ilc population undergoes rapid expansion in the neonatal lung during the first few weeks of life ( ) ( ),( ). factors that may result in accentuated or prolonged th skewing include maternal allergy ( ) but environmental exposures are also critical as has become apparent from the farming exposure studies ( ). growing up in close proximity to cows seems to provide protection and incidence of allergic diseases are greatly reduced in children who have grown up in farming communities ( ). similarly, diet has a profound influence on the systemic microbiome which impacts the developing immune system and thus the trajectory towards disease or health ( ). although it is apparent that the composition of inhaled exposures has a direct impact on the airway immune profile, mechanistic studies in experimental laboratory models in the context of a developing immune system are scarce, while studies in children have predominantly used peripheral blood rather than cells directly isolated from the 	
   	
   lung. recent data in mice suggests that enhanced neonatal skewing of th immunity after exposure to allergens results from hyperactivity of the il- -pathway ( ). il- production during the postnatal phase of lung development is postulated to drive th type immunity, thus lowering the threshold for innate immune response to allergen. similarly, it has been shown that the process of birth is enough to trigger il- production from the lungs of alveolar epithelial cells of newborn mice which drives the expansion of il- r expressing innate lymphoid cells (ilc), concomitant with il- driven polarization of airway macrophages into an m phenotype ( ). it is thought that this homeostatic type pathway delays antibacterial effector responses, as well as prolonging the in utero derived type environment. however, it is not known whether human neonates experience a similar wave of hyperactivity in il- responses. thus, it is not known how the balance between effector t cells or innate cell subsets differ in the lung following exposure to viruses, bacteria or allergens in early life. we have recently shown that ilc s are present in the airways of children with severe therapy resistant asthma ( ). proportions of ilc were determined by flow cytometry in bal samples from children with stra and compared to samples collected from children undergoing investigations for recurrent lower respiratory tract infections. whereas ilc were extremely rare or absent in this latter group, they were present in bal from asthmatic children. interestingly, there were relatively higher proportions of ilc in the airway samples (either bal or sputum) compared to peripheral blood. these are descriptive studies, but mechanistic studies in mouse models have focused solely on the use of adult models with environmental exposures in the context of a mature immune system. however, since it is known that the neonatal immune system is physiologically skewed towards type immunity, and an exaggeration of this skewing results in later disease, it is possible that in early life ilcs are less important in disease initiation, or that they may be protective. experiments using adult mouse models have suggested that ilcs are vital for the development of an effective adaptive t cell response during classical type responses initiated by allergens or parasitic worm infections ( ) ( ). however, given that 	
   	
   the neonatal immune landscape is different from that of the adult it is likely that there will be distinct differences in responses to allergens and cytokines in neonatal mice compared to adult mice. indeed, accumulation of pulmonary ilcs at homeostasis occurs postnatally and relatively small numbers are present at birth. conversely, ilc are thought to be critical in limiting inflammation and promoting tissue repair in order to maintain homeostasis ( ). although ilc are able to secrete a number of factors that are associated with wound repair, such as amphiregulin ( ), mechanistic studies have shown that they are involved in development of pulmonary fibrosis following bleomycin injury and in patients with liver or dermal fibrosis, rather than in the specific repair of tissue following inflammation ( - ). it will be important in the future to delineate how ilc are able to contribute to the wound healing process without causing tissue remodelling. it is possible that those ilc associated with driving allergic pathology may represent a different subset or that their function may be context specific. this is a particularly important point in early life, when the lung is still developing, so the phenotypic characteristics of ilc during pulmonary immune development and their functional role in repair or resolution of inflammation needs to be understood. external influences on the developing immune system although the mechanisms underlying development of early life wheeze are not well understood it is clear that a wide variety of external, environmental influences affect the developing neonatal immune system and therefore the development of sensitivity to allergens as well as to pathogens. it is clear that the postnatal period represents a critical window for development of aberrant immune responses, but the maternal environment has a significant impact. one of the major influences on the development of neonatal immunity and subsequent maintenance of immune homeostasis is our relationship with the microbes that live upon and within us. all of our mucosal surfaces, the skin, gut and lungs are colonized by dynamic communities of microbes that are integral for our well being. these local microbiota are essential for energy harvest from food sources, for metabolism and most importantly 	
   	
   when considering neonatal development, the training and education of local and systemic immunity ( ). it is clear that robust immune health is associated with a flourishing and diverse microbiota, but each of these microbial ecosystems is dynamic and is exquisitely sensitive to the environment provoked by changes in diet, intake of drugs and hormonal influences. a lack of diversity and resilience in the microbiota has been associated with the rise in autoimmune and allergic diseases documented in the developed world. for the neonate the first few years of life are associated with the acquisition of a competent microbiota, since the uterine environment is relatively sterile. this period represents a critical window for immune development, since the composition of the microbiome is affected by the birth process itself, whether the baby is breastfed, weaning as well as a whole host of external environmental influences such as living in a home with siblings and or pets, spending time in day care and living in an urban or rural environment ( ). each of these factors will impact on the diversity of the airway microbiota and therefore the education and training of the immune system. we have shown that age at first allergen exposure is critical in determining the degree of allergic immune responses generated. neonatal mice exposed to inhaled house dust mite from day of life had significantly more eosinophilia, ahr and type immune responses compared to adult mice ( ). moreoever, if allergen challenge was commenced at day , then responses were negligible and the mice appeared protected. the formation of the lung microbiota is a key parameter in this process. during the first weeks after birth, the bacterial load in the lungs increased, and there was a shift in the airway bacterial phyla from a predominance of gammaproteobacteria and firmicutes towards bacteroidetes. the changes in the microbiota were associated with the decreased aeroallergen responsiveness seen at day and the emergence of a helios(-) treg cell subset that required interaction with programmed death ligand (pd-l ) for development. this demonstrated that the airway microbiota induces t regulatory cells early in life, and if its development dysregulated during a critical period of development postnatally, can lead to sustained susceptibility to allergic airway inflammation in adulthood ( ). 	
   	
   maternal influences although once considered a sterile environment the advent of more sensitive methods of detection has shown that the womb contains commensal microorganisms which may affect development of the foetal immune system, but this concept is not universally accepted ( , ). given that the foetus is nourished by the mothers blood supply it is understandable that key aspects of the mother’s wellbeing will affect the foetus. these exposures will affect the growth and development of the foetus, but may also influence subsequent development of disease. maternal diet exerts a significant influence on the developing foetus. a recent study determined that the nature of maternal diet influenced epigenetic imprinting of immune cells and lung stromal cells in utero. enhanced intake of dietary fibre by the mother was associated with increased levels of acetate which primed foxp t regulatory cell mediated protection against development of asthma in mice, but critically a similar axis was observed in humans ( ). similarly, a recent study showed that supplementation during the last trimester of pregnancy with fish oil-derived fatty acids was sufficient to reduce the risk of persistent wheeze or asthma and lower respiratory tract infections by one third ( ). deficiency in certain vitamins, particularly vitamins d and a is associated with increased asthma risk. vitamin d has wide ranging effects on immune cells, particularly development and functional capacity of t regulatory cells ( ). specifically, lower levels of serum vitamin d have been associated with increased airway remodeling and worsened asthma control in children with severe asthma ( ). we have shown using a neonatal model of inhaled allergen exposure, that offspring from mother mice that were fed a vitamin d deficient diet had exaggerated airway eosinophilia and th cells with reduced t regulatory cells. in addition, if pups were supplemented with a vitamin d sufficient diet at weaning this resulted in a significant reduction in serum ige levels, reduced pulmonary eosinophilia and airway remodelling ( ). interestingly, a recent clinical trial determined that supplementation with vitamin d during pregnancy elicited a modest reduction in incidence of recurrent wheeze and 	
   	
   asthma in the offspring ( ). although mouse models have determined that maternal vitamin a (retinoic acid) affects development of lymphoid tissue inducer cells – thus affecting the size of secondary lymphoid organs and setting the threshold for immune responses in the offspring, supplementation studies in humans have not shown efficacy in prevention of development of asthma ( ). only a weak, inverse association was observed for maternal intake of vitamin a and e with childhood allergic rhinitis ( ). conversely, maternal obesity has profound effects on immune responses in the baby, giving rise to systemic inflammation as well as asthma. maternal prenatal bmi is associated with increased incidence of wheezing and asthma ( ). there are multiple longitudinal studies that support the hypothesis that maternal, or paternal, smoking has a negative impact on pulmonary health in babies and greatly enhances the odds of developing recurrent wheeze or asthma ( - ). cigarette smoke exposure has been shown to increase expression of specific micrornas in the blood concomitant with lower numbers of t regulatory cells in maternal and cord blood, which was associated with an increased risk of atopic disease ( ). infants from smoking mothers also display attenuated innate tlr-mediate immune responses compared to infants from nonsmoking mothers ( ). similarly, a number of different studies have shown that traffic related air pollution not only affects lung growth, but is associated with an increased prevalence of asthma as well as wheezing ( ). maternal stress is a significant risk factor for wheezing in early life as well as development of asthma ( , ). this may be due to transfer of glucocorticoid hormone across the placenta, which has wide ranging effects on the immune system ( ). although it has been suggested that maternal stress impacts development of adaptive immune responses in neonates, with reduced humoral immunity and reduced capacity for th responses, recent studies suggests that the effects may only be transient ( , ). postnatal influences 	
   	
   immune homeostasis at mucosal surfaces is achieved by maintaining a balance between promoting tolerance to environmental particles and mounting rapid immune responses to pathogens including viruses and bacteria. increasing evidence has shown that postnatal microbial colonization with commensal bacteria is vital in shaping the developing immune system, particularly at mucosal surfaces ( ). neonatal colonization by microbes begins soon after birth, is influenced by gestational age, the mother’s microbiota, route of delivery at birth and use of antibiotics. indeed prolonged use of antibiotics also has profound effects on the development of the neonatal immune system ( ). as the neonate transitions from the relative low microbe environment of the womb into the external world, rapid colonisation of mucosal surfaces by microbes takes place. these microbes and their metabolic products are arguably the strongest stimulators of early immune development and play a vital role in the maturation and education of the developing neonatal immune system. the mode of delivery of the infant has a profound effect on the microbial load experiences by the baby in the first few days and weeks of life. caesarean and vaginal births result in different species of microbes that colonise the infant from the beginning, reflecting the normal flora of the skin and the vagina respectively ( ). these microbes initially colonise the gastrointestinal tract and this intestinal microbiome undergoes dynamic changes during the immediate postnatal period, and this is associated with functional development of the immune system. as well as the differences in microbial exposure, the very nature of labour including high levels of circulating hormones, the pressure on the baby of uterine contractions and the stress of passage through the birth canal itself impacts multiple aspects of the neonatal immune system. collectively, these factors elicit the release of a number of pro-inflammatory cytokines, including il- , il- , ifnγ and tnfα which are all measurable in both the maternal circulation and cord blood ( ). epidemiologic evidence suggests that children born via caesarian delivery have an increased risk for a range of metabolic and immune diseases, including atopic diseases, compared to than those born via vaginal delivery. a recent clinical study showed that 	
   	
   exposure of infants delivered by c-section exposed to maternal vaginal fluids soon after birth restored the skin, gut and oral microbial communities to a level comparable to that shown in babies after a vaginal birth ( ). although the authors did not assess the subsequent long- term consequences on health or development of immunity it is proof of concept that the microbiota can be manipulated postnatally in babies delivered by c-section. this concept is not universally accepted however, and an alternative study has suggested that the microbiota of each organ reflects that of its mother – regardless of the route of delivery. longitudinal sampling of infants revealed that the microbiota structure and function expands and diversifies considerably from birth through the first few months. thereafter it resembles the microbiota from the corresponding maternal body site – irrespective of the mode of delivery or other prenatal factors ( ). breast-feeding is an important factor in neonatal immune development, apart from the obvious nutritional benefits. human milk contains a variety of different proteins that aid digestion, facilitate maturation of the neonatal gut while also stimulating neonatal immunity and providing early antimicrobial protection during this period of immune development ( ),( ). as well as providing leukocytes and complement proteins from the mother, breast milk contains regulatory cytokines such as il- and tgfβ, which promote tolerance to antigens, including those in food, in early life ( , ). other immune-modulatory molecules include immunoglobulins, lysozyme – which can degrade gram-positive bacterial cells walls and kappa-casein that blocks pathogen binding to the gut wall, as well as dietary nucleotides and lipids. multiple epidemiologic studies have shown that in developed countries a history of breast feeding is associated with reduced risk of severe lower respiratory infections, obesity as well as other important childhood diseases, but the effect on asthma or wheezing is debated ( ). this is possibly because of a lack of clarity surrounding the diagnosis of both asthma and wheeze in many studies, but it seems that breast feeding for at least months, compared to formula feeding may delay development of atopic dermatitis, cows milk allergy and wheezing in early childhood ( ). 	
   	
   influence of early life infections the developing immune system is shaped by the antigens that are encountered in the immediate postnatal period, resulting in colonisation and establishment of the pulmonary microbiota and subsequent expression of pattern recognition receptors (prrs) on resident cells such as epithelial cells and airway macrophages. numerous epidemiologic studies have shown that growing up in a farming environment is protective against development of asthma and childhood wheeze ( ). the strongest protective effect has been seen in farming communities which retain traditional farming practices with families living in close contact with animals ( , ). amish and hutterite farming communities share a genetic heritage and many other cultural aspects of life – apart from the close contact that the amish have with their farm animals. however, whereas, incidence of allergies, wheeze and asthma are incredibly low in the amish communities, incidence in hutterite children is similar to that in urban communities. importantly, the strongest protection was afforded to those children that are exposed to farm dust in utero ( ). recent analysis showed that although the incidence of asthma is fold lower in the amish population compared to the hutterites, the level of dust in their homes was much higher ( ). there were significant differences in the proportions and phenoytpes of innate immune cells between the two groups - with lower eosinophils and higher neutrophils in the amish, and the converse in the hutterites. cell surface markers such as cxcr and cd b were raised on neutrophils from the hutterite children and their monocytes had raised levels of hla-dr. these data indicate that the farm dust that the amish were exposed to from very early in life has a significant influence on the developing immune system impacting on subsequent allergic diseases. respiratory infections are common in childhood and the combination of infections encountered during this developmental period has an important influence on the immune status of the lung. respiratory infections with viruses or bacteria are the most common 	
   	
   triggers of exacerbations in asthmatic children but there is also evidence that they may be precipitating factors in the development of wheeze as well as asthma. rhinovirus is the most commonly attributed driver of viral induced exacerbations. however, infants who develop wheeze concomitant with hrv infection are at a significantly enhanced risk for subsequent asthma. it has been suggested that host genotype is a significant factor and it is notable that variants within the q locus are associated specifically with asthma in children who experiences hrv wheezing illness ( ), and expression of ormdl and gsdm – two genes found at this locus - were identified in gwas studies as asthma risk genes ( ). cadherin-related family member (cdhr ) was recently identified as an asthma risk gene in children with severe asthma and exacerbations ( ). this is of particular interest because expression of cdhr on epithelial cells facilitates both binding and replication of rhinovirus- c in those cells that would normally be unable to support infection ( ). moreover, a coding snp associated with enhanced cell-surface expression of cdhr protein is related to increased risk of wheezing illnesses and hospitalizations for childhood asthma. although the most common infection in childhood is rhinovirus, rsv is also very common in babies, and can lead to significant asthma risk. it has been shown that babies that experience very early infection with rsv (at less than months of age) have higher th responses with increased levels of il- ( ). however, although viruses have been described as causative agents in the development of wheeze and asthma, a prospective study in children indicates that the reverse is true. prospective, repeated characterization of a birth cohort demonstrated that allergic sensitization during early life predisposes children to more severe viral respiratory illnesses and wheezing but that wheezing respiratory illnesses do not increases the risk for subsequent development of allergic sensitization ( ). serial viral infections are a significant risk for wheezing and asthma but in addition certain bacterial infections can also enhance risk. a prospective study collected hypopharyngeal aspirates in babies at one month and incidence of wheeze monitored for the next years (bisgaard ( ). those neonates colonised with s.pneumoniae, h influenzae or m.catarrhalis, either alone or in combination, were found to be at increased risk 	
   	
   for recurrent wheeze and asthma by years of age. thus the nature, pattern and severity of infections experienced during childhood influences development of wheezing illness and asthma in some children, particularly if they have a susceptible genetic background. summary and future directions. early life immunity is critical in determining lifelong health. myriad influences on development of the pulmonary immune system and the combination of factors encountered by the neonate during this window determines heath or disease. all children encounter respiratory infections from birth, yet only a proportion develop asthma. the combination of genetic together with intrinsic and extrinsic factors dictate whether the immune response is appropriately tailored to clear infection versus an exaggerated response which changes the course of pulmonary immunity, resulting in disease (fig ). the challenge in determining the molecular mechanisms underlying this process is to accurately phenotype patients and use age-appropriate and tissue specific clinical samples in studies. this represents a significant challenge, but with the advent of novel technologies, which allow analysis of gene and protein expression at the micro level, this should be achievable. similarly, it is imperative that we use appropriate preclinical models that will enable accurate translation of findings to children. moving forward in our goal of more effective treatment of childhood allergic disease will require a shift in mind-set with respect to clinical trial design and more effective collaboration between industrial partners, clinicians and academic researchers. 	
   	
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   airways	remodelling immune	development microbiome infection genetics lausanne wheeze/	 asthma allergen wheeze protective signals harmful signals diverse microbiome exposure to farm dust pets siblings diet rich in pufa, fibre serial viral infections pollution smoke exposure poor diet allergen exposure asthma ? gene x environment interactions untitled a population-based study of kcnh p.arg his and bipolar spectrum disorder kevin a. strauss , , ,{,∗, sander markx ,{, benjamin georgi , steven m. paul , robert n. jinks , toshinori hoshi , ann mcdonald , michael b. first , wencheng liu , abigail r. benkert , , adam d. heaps , yutao tian , aravinda chakravarti , maja bucan and erik g. puffenberger , , { clinic for special children, strasburg, pa, usa, franklin & marshall college, lancaster, pa, usa, lancaster general hospital, lancaster, pa, usa, department of psychiatry, columbia university, new york, new york, usa, department of genetics, perelman school of medicine and, department of physiology, university of pennsylvania, philadelphia, pa, usa, departments of neuroscience, psychiatry and pharmacology, weill cornell medical college of cornell university, new york, new york, usa, biological foundations of behavior program, franklin & marshall college, lancaster, pa, usa and center for complex disease genomics, mckusick-nathans institute of genetic medicine, johns hopkins university school of medicine, baltimore, md, usa received february , ; revised and accepted june , we conducted blinded psychiatric assessments of amish subjects ( +++++ years) from four families with prevalent bipolar spectrum disorder, identified potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger amish study of major affective disorder (asmad) cohort, and studied mutant potassium channels in neurons. fourteen of amish had bipolar spec- trum disorder. the only candidate allele shared among them was rs , a non-synonymous variant of kcnh (c. g>a, p.arg his). kcnh c. g>a and nine other potentially pathogenic variants were sub- sequently tested within the asmad cohort, which consisted of subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar disorder, bipolar spectrum disorder and any major affective disorder). kcnh c. g>a had the highest enrichment among individuals with bipolar spec- trum disorder (x . ) and the strongest family-based association with bipolar (p . ), bipolar spectrum (p . ) and any major affective disorder (p . ). in vitro, the p.arg his substitution allowed normal expression, trafficking, assembly and localization of herg /kv . channels, but altered the steady-state volt- age dependence and kinetics of activation in neuronal cells. although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of herg-type potassium channels, electrophysiological data) implicates neuronal herg /kv . potassium channels in the pathophysiology of bipolar spectrum disorder. such a find- ing, if corroborated by future studies, has implications for mental health services among the amish, as well as development of drugs that specifically target herg /kv . . introduction mental illness afflicts – % of people worldwide ( ). mood disorders—including bipolar disorder, bipolar spectrum dis- order and major depressive illness—account for at least half of this global mental health burden ( ). in north america, % of medical disability in persons aged – years is attributable to psychiatric illness ( ) and in the usa, suicides outnumber homicides two to one ( ). our failure to prevent serious psychi- atric morbidity results in part from insufficient understanding of † equal contributors. ∗ to whom correspondence should be addressed at: clinic for special children, bunker hill road, strasburg pa, , usa. tel: + ; fax: + ; email: kstrauss@clinicforspecialchildren.org # the author . published by oxford university press. this is an open access article distributed under the terms of the creative commons attribution non-commercial license (http://creativecommons.org/ licenses/by-nc/ . /), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. for commercial re-use, please contact journals.permissions@oup.com human molecular genetics, , vol. , no. – doi: . /hmg/ddu advance access published on june , http://creativecommons.org/licenses/by-nc/ . / http://creativecommons.org/licenses/by-nc/ . / its root causes ( ). here, the application of genetics holds promise as a means to identify individuals predisposed to psychi- atric disease ( ), but genetic studies of mental illness have thus far produced few specific risk alleles that help clinicians care for patients ( ). the clinic for special children (csc) is a non-profit commu- nity health center that serves uninsured amish and mennonite (plain) communities of pennsylvania (usa) and surrounding states ( ). although the csc has historically focused on pediat- ric health, bipolar and other affective disorders pervade every aspect of family and community life ( ) and it is increasingly ap- parent that adult-onset mental disorders can be associated with prodromal symptoms during childhood, including disturbances of mood, attention and thought ( ). the csc invests heavily in genetic strategies that allow prevention of disability and disease ( ). this concept is germane to the diagnosis and treat- ment of mental disorders, for which early detection of specific risk alleles in youth could enable more timely and effective psy- chiatric care ( ). endogamous populations such as the old order amish provide distinct advantages for investigating the genetic bases of mental illness ( , ). the amish study of major affective disorder (asmad), initiated in by egeland and collea- gues, has tracked several large, multi-generation pedigrees with high prevalence of bipolar spectrum disorders ( ). despite three decades of sustained and valuable research, the asmad cohort has revealed no definitive genetic risk factors for major affective disease ( ). however, a recent study of asmad subjects (n ¼ ) that combines microsatellite and high-density single nucleotide polymorphism (snp) geno- types with whole-genome sequence data implicates dozens of rare alleles that may interact to determine risk for bipolar disorder ( ). traditional linkage analysis is less informative in the asmad cohort given multiple, unexpected lines of interrelatedness within an endogamous group such as the amish ( ). mapping susceptibility alleles for mental disorders in any population poses additional challenges: (a) behavioral phenotypes such as bipolar disorder are, by their nature, incompletely penetrant and variable in expression both within and between individuals; (b) a single genetic variant can have pleiotropic effects on psychopathology that change over the lifespan ( , ); (c) cat- egorization of mental illness often depends critically on self- reporting of remembered subjective experience, vulnerable to errors of both omission and commission; and ( ) instruments currently used to categorize mental disorders (e.g. diagnostic and statistical manual of mental disorders, dsm) are based on phenomenology rather than firm biological constructs ( , ), and thus do not capture the full phenotypic spectrum (i.e. endophenotypes) associated with any particular susceptibil- ity allele ( , , ). these facts are especially problematic when using conven- tional statistical paradigms to identify rare variants of clinical significance in small, endogamous groups ( ). recognizing this, we developed a strategy that depends on multiple, conver- ging lines of evidence to evaluate a complex phenotype within a narrow genetic context. we first applied an approach common- ly used to investigate mendelian disorders ( , ), searching whole-exome data for low-frequency alleles shared among closely related amish individuals with bipolar spectrum disorder ( , ). we then used these findings to independently test for genetic associations within the larger asmad cohort ( ), and finally conducted functional studies of mutant potas- sium channels in neuronal cells. based on our statistical and functional results, kcnh c. g.a (p.arg his; rs ) emerges as a strong candidate for bipolar disease risk among the pennsylvania amish. this corroborates findings from a recent genome-wide association (gwa) study of an independent cohort of taiwanese patients, which isolated kcnh as one among four genes likely to be associated with bipolar disorder ( ). to support the genetic data, we provide functional evidence that p.arg his alters the electrophysiological properties of herg /kv . -mediated po- tassium currents in neuronal cells. taken together, these findings suggest that functional variation of herg-type neuronal potas- sium channels ( – ), and herg /kv . in particular, may have a role in the pathogenesis of bipolar disorder and schizophre- nia. because our association data do not reach genome-wide sig- nificance, the main finding should be viewed as provisional until confirmed or refuted by future studies. results exome variants in core amish families a – d we initially studied four old order amish sibships with a high prevalence of bipolar disorder (fig. ). families a – d consisted of amish subjects (mean age + years, range – years, % female) who underwent independent, blinded psy- chiatric assessment. phenotype was characterized on four levels (table ): ( ) structured clinical interview for dsm-iv-tr (scid) diagnosis; ( ) a sub-categorization of de- pressive, manic and psychotic symptom clusters; ( ) a designa- tion of multidomain affected if at least two of three symptom clusters (i.e. mania, depression and psychosis) were present; and ( ) a detailed breakdown of specific symptoms (supplemen- tary material, table s ). fourteen of amish subjects from families a – d (fig. ) met dsm-iv-tr criteria for at least two of three symptom clus- ters (mania, depression or psychosis) and were designated as multidomain affected. they comprised diverse axis diagnoses (table and supplementary material, table s ): bipolar with psychotic features (n ¼ ), bipolar with psychotic features (n ¼ ), bipolar disorder not otherwise specified (n ¼ ), schizoaffective disorder (n ¼ ), schizophrenia with major de- pressive disorder (n ¼ ), and recurrent major depression com- plicated by somatoform disorder and substance-induced psychosis (n ¼ ). seven of these subjects were chosen for exome sequencing (indicated with asterisks in fig. ) and shared a total of exome variants. because our study design lacked power to detect common var- iants associated with small or modest effects, we restricted our focus to low-frequency variants with potentially higher patho- genicity (fig. ). we first excluded alleles with minor allele fre- quency . % in control plain exomes; this narrowed the list to variants. we then excluded synonymous and intronic changes which further reduced the number to ‘candidate’ variants (table and fig. ). to perform association analyses, all sub- jects from families a – d and all subjects from the asmad cohort were genotyped for these variants (figs . and ). human molecular genetics, , vol. , no. http://creativecommons.org/licenses/by-nc/ . / http://creativecommons.org/licenses/by-nc/ . / http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/ddu /-/dc candidate variants in three of genes (krt , utp c, nek ) had minor allele frequencies . % in genomes project, european controls, or the exome variant server. among variants in the genes, three (kcnh , muc , aldh a ) were predicted to be pathogenic by sift, polyphen- and mutation taster, and two of these (kcnh , muc ) were absent in all three non-plain control exome datasets ( genomes project, european controls, exome variant server; table ). muc p.cys phe was not associated with bipolar disorder in the asmad pedigree [family- based association test (fbat) p-value ¼ . ]. moreover, mucin- has no known function in neurons and is not expressed in human brain (http://proteinatlas.org/). the muc variant was there- fore considered an unlikely candidate. association of kcnh c. g>a with psychiatric illness in the asmad cohort kcnh c. g.a (rs ) was the only candidate exome variant carried by all subjects from families a – d who were multidomain affected based on the presence of at least two of three symptom clusters (i.e. mania, depression and psychosis) (table and supplementary material, table s ). moreover, kcnh c. g.a was deemed the most likely pathogenic variant based on multiple converging lines of evidence, including: (a) results from independent gwa and whole-genome sequencing studies ( , ); (b) expression pattern of kcnh in areas of the brain that are believed to mediate mood and cognition ( ); figure . a (upper panel): individuals from four families underwent blinded, independent psychiatric assessments using the structured clinical interview for dsm-iv (scid), research version. exome sequencing was done on subjects designated with a red asterisk. families a – c (blue enclosures) were interviewed during the first phase of the study and family d (green enclosure) was recruited later. black symbols indicate individuals who met dsm-iv-tr criteria for at least two of three symptom clusters—mania, major depression, psychosis—and were considered multidomain affected with bipolar spectrum disorder. gray symbols indicate individuals who met diagnostic criteria for depressive illness (recurrent or single episode) uncomplicated by mania or psychosis. the ‘‡’symbol indicates subjects who were unavailable for interviews or declined to participate. b (lower panel): during the second phase of the study, samples from the asmad were used to test associations of exome variants with bipolar spectrum disorder (eighteen asmad samples were individuals from families a and c and thus excluded from the replication analysis). all asmad subjects were genotyped for candidate exome variants and categorized as unaffected (n ¼ ) or affected (n ¼ ) by major affective illness; the latter category was then subdivided into the increasingly restrictive designations of bipolar spectrum disorder (n ¼ ) and bipolar disorder (n ¼ ). human molecular genetics, , vol. , no. http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/ddu /-/dc (c) evidence that antipsychotic drugs block the herg /kv . channels encoded by kcnh ( ); (d) the proposed role of other potassium channel subunits in bipolar disorder and schizophrenia ( – ); and e) the conservation of nucleotide guanine , corre- sponding to amino acid arginine , across all species from homo sapiens to caenorhabditis elegans (phylop . ) (table ). to further test this observation, we obtained de-identified dna and clinical data for asmad samples. individuals from aforementioned families a and c (fig. ) were represented in the asmad cohort, but were excluded from the replication analysis. fifty-four asmad subjects had minor or incompletely characterized psychiatric phenotypes and were also excluded. we grouped the remaining subjects into the following over- lapping clinical categories, as depicted in figure : bipolar dis- order (n ¼ ), bipolar spectrum disorder (n ¼ , including bipolar , bipolar and bipolar disorder not otherwise specified), any major affective disorder (n ¼ , including major depres- sive disorder, recurrent), and unaffected by major affective illness (n ¼ ). among these individuals, we investigated association of the candidate variants with psychiatric diagno- ses using three complementary methods: (a) a simple x analysis of allele distribution with phenotype; (b) the fbat, which mea- sures transmission distortion of alternative alleles to affected and unaffected siblings in pedigrees ( ) and (c) the efficient mixed- model association expedited method (emmax), which controls and corrects for relatedness between subjects ( ). kcnh c. g . a (rs ) behaved in a manner dif- ferent from all other variants (table and fig. ). table lists nominal (uncorrected) x calculations as well as fbat and emmax p-values for the candidate exome variants. kcnh table . phenotypes and genotypes of amish study subjects from families a – d human molecular genetics, , vol. , no. c. g.a had the highest enrichment in subjects with affective disorders (x for bipolar ¼ . ; bipolar spectrum ¼ . , any af- fective disorder ¼ . ), lowest emmax p-value for bipolar and bipolar spectrum disorders (p ¼ . ) and lowest fbat p-value for bipolar (p ¼ . ), bipolar spectrum (p ¼ . ) and any major affective disorder (p ¼ . ) (table and fig. ). the statistical results presented in table do not alone provide sufficient evidence of association after correcting for multiple tests. we nevertheless pursued kcnh c. g . a further based on (a) the weight of evidence from multiple sources ( , – , , , – ); (b) recognition that our cohort size and study design lacked power to generate an un- equivocal signal for any true positive association (discussed below); and (c) the important implications that a true positive as- sociation would have for design of preventative mental health services among amish communities as well as future drug devel- opment for patients with bipolar disorder and related psychiatric disorders. we thus turned to studies of herg arg his expres- sion and function in neurons. expression and function of kcnh arg his when overexpressed in mouse and human neuroblastoma cells, wild-type and herg /kv . arg his potassium channel protein subunits had similar abundance, core and mature glycosylation and localization to the plasma membrane (fig. a – f and suppl- ementary material, fig. s ). wild-type and arg his mixed monomers co-localized in a pattern indistinguishable from that of wild-type proteins alone, suggesting appropriate intracellular traf- ficking and formation of mature heteromers (supplementary material, fig. s ). depolarization of neuro- a cells transfected with wild-type kcnh elicited outward currents that progressively diminished in size with depolarization to . mv (fig. g), a pattern char- acteristic of herg/kv channels with fast c-type inactivation ( ). in cells transfected with herg arg his , the following differences were observed: (a) when currents were normalized to the maximal current size in each cell, fractionally smaller currents were observed through arg his channels at a given voltage , mv (fig. i); (b) greater depolarization was required to elicit currents through the arg his channel; the normalized conductance (g/gmax) curve, proportional to the probability that the channel is open, was shifted � mv in the positive direction (fig. j); and (c) upon depolarization, current kinetics through the arg his channel were slower (fig. k), but the deactivation kinetics at a negative voltage were essentially indistinguishable between the two channel types (fig. l). together, the results suggest that the p.arg his muta- tion slows the activation process of herg /kv . channels and thereby shifts the overall voltage dependence of activation in the positive direction. discussion kcnh , herg-type potassium channels and mental illness by studying a few amish families to search for low-frequency, relatively penetrant bipolar risk alleles, we discovered a specific missense variant of kcnh (c. g.a) that appears to segregate with bipolar spectrum disorder among a subset of penn- sylvania amish families. in our view, the most important conclu- sions to be drawn from our results are that the kcnh c. g.a allele, uniquely present in all affected patients among the original cohort of , clearly distributes in a way differ- ent from all nine other rare and potentially pathogenic exome var- iants tested within the larger asmad cohort (table and fig. ), and significantly alters potassium channel currents in neuronal cells. given the relatively small sample size used and incomplete penetrance of the bipolar spectrum phenotype, the genetic evi- dence is alone insufficient to provide definitive proof of associ- ation. however, we believe kcnh c. g.a warrants further investigation based on the cumulative weight of evidence from multiple sources, its high degree of specificity, and the poten- tial public health implications for amish communities. the kcnh c. g.a variant (rs ) was recently highlighted as one of potentially pathogenic missense variants figure . among seven amish individuals with bipolar spectrum disorder, we identified a total of exome variants, of which remained after filtering out synonymous and intronic changes. focusing on low-frequency alleles with potentially high pathogenicity, we excluded exome variants with minor allele frequency (maf) . % among population-specific control exomes. only of these variants were present in all seven individuals. these ‘candidate’ alleles were then used to test for associations with bipolar spectrum disorder and broader diagnostic categories within the extended core pedigree (families a – d, n ¼ ) and the larger asmad cohort (n ¼ ), respectively. human molecular genetics, , vol. , no. http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/ddu /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/ddu /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/ddu /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/ddu /-/dc table . ten exome variants among the seven affected amish individuals chosen for exome sequencing allele frequency predicted effects on protein function chr position ref alt dbsnp gene class codon change amino acid change phylop score all plain exomes (n ¼ ) amish exomes (n ¼ ) genomes evs ceu sift polyphen- mutationtaster g a rs aldh a missense gcg/gtg a v . . . . . . damaging probably damaging disease-causing c t rs kcnh missense cgc/cac r h . . . . . . damaging probably damaging disease-causing g c rs xirp missense aga/aca r t . . . . . . tolerated na polymorphism c a . muc missense tgt/ttt c f . . . . . . damaging probably damaging polymorphism a g rs alg b missense att/gtt i v . . . . . . tolerated benign polymorphism g t rs ccdc missense gat/tat d y . . . . . . damaging; low confidence possibly damaging polymorphism g a rs csrnp missense acg/atg t m . . . . . . damaging; low confidence possibly damaging polymorphism g c rs krt missense ccc/gcc p a . . . . . . damaging probably damaging disease-causing a g rs utp c missense act/gct t a . . . . . . tolerated benign na t g rs nek missense aaa/caa k q . . . . . . damaging probably damaging polymorphism the highest phylop value is indicated by orange fill. alleles that were not detected in non-plain exomes are designated with green fill, and blue fill indicates alleles predicted to have damaging effects on protein function. asmad, amish study of major affective disorder; ceu, control european exomes; evs, exome variant server. h u m a n m o le c u la r g e n e tic s, , v o l. , n o . in whole-genome sequence analysis of asmad extended fam- ilies ( ). a parallel, independent gwa study of taiwanese patients identified a different kcnh variant (rs ) as one of four alleles associated with bipolar (empirical p-value ¼ . ; n ¼ ) ( ). again, the statistical signal for rs fell short of genome-wide significance among tai- wanese patients, but this allele nevertheless behaved in a way not likely to be observed by chance. available data also implicate other potassium channels genes (kcnh and kcnj ) in bipolar disorder and schizophrenia ( ), localize herg-type channels to the brain’s limbic circuits ( , ), and demonstrate a role for altered potassium currents in mania and the therapeutic actions of lithium ( – ). these converging lines of evidence, com- bined with genetic and electrophysiological data detailed in this report, suggest that variation of neuronal herg-type potassium channels ( – ), and specifically herg /kv . , might con- tribute to mental illness in certain individuals. kcnh and mechanisms of mental illness herg /kv . , encoded by kcnh , belongs to the ether-á- go-go-related (erg) family of voltage-gated potassium chan- nels expressed throughout the mammalian brain, especially in limbic and cortical areas associated with mood and cognition ( ). heterologously expressed herg arg his is processed to the plasma membrane in neuroblastoma cells, but the histidine substitution at a highly conserved cytoplasmic arginine shifts voltage dependence of activation in the positive direction and table . association testing of exome variants with affective disorders in the asmad cohort (n ¼ ) a gene chromosome variant bipolar disorder bipolar spectrum disorder any major affective disorder x fbat p emmax p x fbat p emmax p x fbat p emmax p aldh a a v . . . . . . . . . kcnh r h . . . . . . . . . xirp r t . . . . . . . . . muc c f . . . . . . . . . alg b i v . . . . . . . . . ccdc d y . . . . . . . . . csrnp t m . . . . . . . . . krt p a . . . . . . . . . utp c t a . . . . . . . . . nek k q . . . . . . . . . a the nominally most significant value from each column is shaded blue (bipolar disorder), red (bipolar spectrum) or purple (any major affective disorder). figure . testing for the association of rare candidate alleles with bipolar (bp , circles), bipolar spectrum (bps, squares), and any major affective disorder (any aff, triangles) among subjects from the amish study of major affective disorder cohort. fbat p-values (abscissa) and x distribution (ordinate) were calculated for each of the rare candidate gene variants detected by exome sequencing. nine of these variants (aldh a , xirp , muc , alg b, ccdc , csrnp , krt , utp c and nek ) are plotted in gray. kcnh c. g.a, represented with red symbols, shows the strongest association with affective disorders and shows an unusual distribution behavior among the variants. for graphical clarity, fbat is transformed to the log ; dotted lines indicate arbitrary thresholds of p ≤ . and x ≥ for fbat and chi-square testing, respectively. human molecular genetics, , vol. , no. slows activation kinetics; thus the mutation is predicted to increase excitability of neuronal cells in vivo. penetrance and severity of mental illness were similar among kcnh c. g.a heterozygotes and homozygotes. this may reflect the heterotetrameric nature of erg channels (e.g. other erg subunits may partially substitute for erg ) and/or a high degree of potassium channel redundancy in the nervous system that attenuates the biological impact of modest functional abnor- malities of any one channel subunit ( ). potassium channel dysfunction appears mechanistically im- portant in animal models of mania and may be relevant to the actions of lithium ( ). kcnh is expressed in mammalian mid- brain, where its blockade prolongs plateau potentials in bursting dopaminergic neurons and may in turn alter mesolimbic dopa- mine release ( ). certain typical and atypical antipsychotic drugs inhibit herg /kv . ( ) and lithium is believed to exert mood-stabilizing effects in part by modulating potassium currents, either by reducing voltage-gated potassium channel open events or by inhibiting gsk ß kinase-mediated channel phosphorylation ( ). in murine models of mania (kcnd / kv . knockout; clockd ), genetic deletion or experimental manipulation of potassium currents attenuates physiological and behavioral correlates of mania and dose-dependently increases phosphorylation of gsk b in prefrontal cortex and hippocampus ( ). the latter mechanism is thought to be shared among all effective mood-stabilizing drugs ( ). figure . left panel: localization of overexpressed kcnh wild-type and arg his in neuro- a cells immunostained under non-permeabilizing conditions (see materials and methods) with mouse monoclonal anti-v igg a ( : ), followed by alexafluor -conjugated goat anti-mouse igg a ( : ). nuclei were stained with ’, -diamidino- -phenylindole (dapi, . mg/ml) (blue fluorescent signal). (a and b) kcnh wild-type and arg his with the v epitope tag inserted in the s – s extracellular loop localize to the plasma membrane in non-permeabilized neuro- a cells (single confocal images). (c and d) maximum projection z-stack images of the cells shown in a and b. (e) left—confocal image of neuro- a cells transiently overexpressing arg his s -v -s -kcnh . right—orthogonal projection of a section through the cell in the center of the left image demonstrating membrane localization for arg his s -v -s -kcnh . (f) western blot of transiently overexpressed wild-type and arg his s -v -s -kcnh fusion proteins in neuro- a cells from the same transfections used for a – d. s -v -s -kcnh fusion proteins migrated as a core glycosylated and mature glycosylated doublet at � kda. b-actin was labeled as a loading control. primary antibodies: anti-v mouse monoclonal igg a ( : ) and anti-b actin ( : ). secondary antibody: goat anti-mouse igg hrp-conjugated ( : ). data are representative of four independent transfections. right panel: electrophysiological characteristics of wild-type (wt) and arg his herg (kcnh ) currents. (g) representative currents from a neuro- a cell transiently expressing wt herg channels. (h) representative currents from a neuro- a cell transiently expressing arg his herg channels. (i) scaled peak current – voltage (i/imax) curves for wt (blue) and arg his (red) channels. the results are normalized to the maximal current size in each cell. the data points are connected by lines for an illustrative purpose only. n ¼ and for wt and arg his, respectively. (j) normalized conductance (g/gmax) as a function of voltage for wt (blue) and arg his (red). the half-activation voltage (v . ) and the apparent equivalent charge movement were – . + . mv and . + . e for wt and . + . mv and . + . e for arg his. n ¼ and for wt and arg his, respectively. the v . values for arg his are statistically different from those for wt (p , × – ). the equivalent charge numbers are indistinguishable between the groups (p ¼ . ). the smooth curves are boltzmann fits to the pooled results. kinetics of ionic currents at mv (k) and – mv (l). currents are scaled to facilitate comparison. the sweep width represents the mean + sem. in (k), n ¼ and for wt and arg his, respectively. in (l), n ¼ and for wt and arg his, respectively. human molecular genetics, , vol. , no. a community-based approach to psychiatric genetics among people afflicted with serious mental disorders, conserva- tive estimates suggest that only – % in developed nations and – % in less-developed nations are diagnosed and treated appropriately ( ). such is the case in amish communities ( ), where treatment for psychiatric disease may only occur in re- sponse to crises like intractable mental anguish, emergent hospi- talization, violence, or the threat of suicide ( ). the treatment gap ( ) in amish as well as other communities results from mul- tiple factors, including social stigma, a dire shortage of profes- sional resources and abiding ignorance of underlying disease mechanisms and their developmental expression ( ). for many patients, the first signs of mental illness surface during childhood or adolescence, while there remains a window for effective intervention ( ). at present, identification of presymptomatic individuals who might later develop major psychiatric disease is based on a combination of family history, prodromal symptoms, and concerning patterns of behavior ( ). underlying this effort is the simple notion that recognizing a predilection for mental illness allows medical and psychosocial interventions to be implemented proactively ( ). indeed, in- formed prevention has proven the key practical benefit that genetic knowledge confers on clinical practice ( ), and it is widely believed that mental health services can be improved by a firmer grounding in genetics and developmental biology ( ). effective treatment strategies for bipolar disorder will largely depend on the identification of biological markers suffi- ciently specific to determine who is at risk ( ). the csc has invested heavily in the discovery of such markers—typically rare and highly penetrant alleles—that can guide the design of population-specific surveillance and prevention programs ( ). despite the presumed genetic complexity of bipolar disorder ( ), we hypothesized that one or more rare alleles might exert strong pathogenic effects within certain endogamous demes ( ). this strategy allowed us to identify kcnh c. g.a as a potential risk factor for bipolar spectrum disorder within a subgroup of pennsylvania amish families. population-specific risk alleles and overlapping psychiatric phenotypes our observations suggest that kcnh c. g.a, and pre- sumably other psychiatric risk alleles, can have pleiotropic effects and do not segregate solely with a single categorical psychiatric phenotype (e.g. bipolar disorder). kcnh c. g.a carriers have prevalent psychotic symptoms and diverse, overlapping axis diagnoses (including schizoaffective disorder, schizophre- nia and major depression). this is not particularly surprising; within the general population, most mental disorders are thought to arise from the combinatorial effects of multiple alleles and their interaction with epigenetic and life events ( ). it is also in- creasingly evident that a single allele can segregate with different categorical psychiatric diagnoses (e.g. bipolar disorder or schizo- phrenia) ( , ). this basic model surely also applies to genetic isolates like the amish, but within such populations it is compara- tively easier to identify low-frequency alleles with stronger pathogenic effects and to document the full range of psychiatric phenotypes that segregate with a particular allele within extended families ( ). in the asmad cohort, kcnh c. g.a segregates in nuclear families and is found in % of patients with a bipolar spectrum diagnosis. however, within these families it appears to be relatively penetrant and might therefore be clinically ac- tionable ( ). further research is needed to verify this, delineate what other alleles may predispose amish individuals to mental illness, map their distribution among the various amish demes, and determine how they might interact with kcnh c. g.a to affect disease expression. such knowledge could lead to personalized pharmacological therapies and, for the first time within this community, preventative mental health care ( ). conclusions, limitations and future directions major limitations of the present study are its small size and narrow focus. by restricting our analysis to amish cohorts, we may have identified a variant unique to this population. however, a recent independent gwa study suggests an associ- ation between bipolar illness and a different kcnh variant in a cohort of ethnically homogeneous taiwanese patients ( ). observations from amish and taiwanese cohorts reveal how we might advance the field of complex disease genetics through the investigation of ‘common’ phenotypes in relatively small, en- dogamous groups ( , ). an association between kcnh c. g.a and bipolar spectrum disorder, even if limited to a few genetic isolates, informs the underlying biology of mood regulation and can suggest more widely applicable treatment strat- egies (i.e. new drug targets). for certain rare pathogenic alleles discovered in small, isolated populations, conventional statistical thresholds for genome-wide significance may be difficult if not impossible to achieve. for example, a recent review suggests that studies sufficiently powered to identify rare variants of clinical significance should include discovery sets of cases or more ( ), a number representing roughly half the amish population of pennsylvania ( ). moreover, the pennsylvania old order amish are more accurately understood as many separate founder populations; the several reproductively isolated demes within the state are defined by different allele distributions ( ). germane to this point, kcnh c. g.a only segregated in a minority of the nuclear families within the asmad cohort, and therefore will be only one of many bipolar risk alleles within the population as a whole. these considerations underscore the importance of using mul- tiple or different sources of evidence to optimize investigations of complex and incompletely penetrant phenotypes within small genetic isolates. despite limitations inherent in the genetic data, we pursued kcnh c. g.a further for three reasons. first, this allele segregated differently from nine other rare, potentially pathogenic variants in two amish cohorts (core families a-d and the larger asmad pedigree); while recognizing this result could be by chance, we were persuaded by the nominal differences represented in table and figure . second, potassium channels in general, and herg channels in particular, have a plausible causative role in bipolar spectrum based on a large body of knowledge about their function in neurons ( , ), distribution within the central nervous system ( ), and pharmacological interactions with lithium and antipsychotic drugs ( , , ). finally, our interest in kcnh c. g.a was strengthened human molecular genetics, , vol. , no. by the recent finding of a potential association between kcnh and bipolar illness in an independent taiwanese cohort ( ), although the latter study also only demonstrated nominal, not genome-wide, significance (empirical p value ¼ . ; n ¼ ). our observations, together with the evidence for genetic heterogeneity from analysis of whole-genome sequence and imputed genotypes of asmad extended families ( ), sets the stage for a diverse genetic landscape of bipolar disease risk even within a population as seemingly ‘uniform’ as the pennsyl- vania amish ( , ). moreover, this study highlights the chal- lenges of statistical analyses using small, endogamous groups to study a phenotype that is: (a) incompletely penetrant, (b) vari- able in expression and (c) by its very nature, difficult to categor- ize with certainty. nevertheless, efforts to link genetic variants to bipolar illness will continue at a rapid pace ( ). our experience suggests that future studies should better delineate subtypes of this complex behavioral disorder by combining systematic discovery of genetic variants with multisystem analyses of quantitative traits that more deeply and reliably characterize the psychopathology ( ), and will likely rely on convergent evidence from multiple sources. multidimensional research strategies within small founder populations could be crucial to these efforts. materials and methods phenotypic assessments the study was approved by the institutional review board of lancaster general hospital and all patients consented in writing to participate. study subjects underwent independent, blinded psychiatric assessment using the structured clinical interview for dsm-iv-tr (scid), research version (http:// scid .org/) ( ). for each subject, supplemental information was collected from at least two closely related individuals (e.g. parent, sibling or child) and in some cases, hospital records. phenotype was characterized on four levels as described above, and phenotypic assessments, including final scid dsm-iv-tr diagnoses, were determined by uniform consensus among three blinded interviewers (a.m., m.f., s.m.). the asmad began in ( ). a five-member psychiatric board blinded to familial ties, pre-existing diagnoses and treat- ment used strict research diagnostic and dsm-iii/iv criteria to develop consensus diagnoses for each subject. uniform as- sessment procedures were applied longitudinally for more than three decades of follow-up, and samples were donated to the coriell cell repository (coriell institute for medical research, camden nj). genomic and statistical methods we performed exome sequencing on a subgroup of amish sub- jects as previously described (broad institute, boston, ma) ( ). our aim was to identify low-frequency alleles with relatively high penetrance; thus exome data were filtered to exclude syn- onymous and intronic changes as well as variants with minor allele frequency . % in two different, but overlapping, sets of population control exomes (designated ‘plain’ exomes: control amish and mennonite exomes combined and control amish exomes). ten candidate variants passed filtering criteria and were verified by sanger sequencing (table ). for each variant, we obtained a measure of conservation (phylop) from the university of california santa cruz genome browser (http://genome.ucsc.edu/) and modeled potentially damaging effects on protein structure in silico using sift (http://sift.jcvi. org/), polyphen- (http://genetics.bwh.harvard.edu/pph /) and mutationtaster (http://mutationtaster.org/). asmad samples (n ¼ ) were genotyped using the illu- mina omni . snp array platform. in addition, all samples were genotyped for each of the candidate variants using high- resolution melt analysis (lightscanner , biofire diagnostics; lightcycler , roche diagnostics). estimates of pair-wise relatedness of the asmad subjects were obtained based on illumina omni . snp array data. a x statistic was used to assess distribution of candidate alleles among individuals with and without mood disorders, and association of these variants with psychiatric diagnoses was tested using the fbat ( ) and ef- ficient mixed-model association expedited (emmax) methods ( ). the bonferroni correction was applied for multiple com- parisons. fbat p-values were log transformed to construct figure . functional studies of kcnh arg his in cell lines all cell lines were obtained from american type culture collection (http://atcc.org/). we cloned wild-type kcnh (also known as herg or kv . ; nm_ . ) from human adherent retinal pigment epithelium cells (arpe- ), introduced c. g.a by site directed mutagenesis, and overexpressed verified constructs in human neuroblastoma (sh-sy y), mouse neuroblastoma (neuro- a) and transformed human embryonic kidney (hek- t) cell lines for immuno- fluorescence and western blotting (supplementary methods). to assess membrane localization of kcnh subunits, the v epitope tag (gkpipnpllgldst) was inserted between amino acids and of the s -s extracellular loop of kcnh and indirect immunofluorescence labeling was performed under non-permeabilizing conditions. briefly, s -v -s kcnh fusion proteins overexpressed in neuro- a cells were labeled with mouse monoclonal anti-v ( : ) (life technologies) at c for min in dmem with % fetal bovine serum and washed three times before fixation (see supplementary methods for details). neuro- a cells overexpressing wild-type or kcnh arg his (n-terminal epitope tags) were tested by patch-clamp experiments using the whole-cell configuration. we recorded ionic currents at room temperature with an axopatch a amp- lifier (molecular devices), elicited currents by s pulses applied every s from a holding potential of mv, and analyzed results using custom routines implemented in igor pro (wave- metrics) (supplementary methods). supplementary material supplementary material is available at hmg online. acknowledgements the clinic for special children board of directors allowed interviews and genetic studies to be conducted on-site and human molecular genetics, , vol. , no. http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/ddu /-/dc donated the clinic’s material and professional resources to the study. dr stacey gabriel and the biological samples, genotyp- ing and sequencing platform at the broad institute (boston, ma) kindly donated exome sequencing services. dr alan shuldiner of the university of maryland amish research clinic generously shared control exome data. amos and rebecca smoker assisted with study design and subject recruitment. donald kraybill and jean endicott provided important cultural context for interpret- ation of scid data. dr sara hamon provided independent statis- tical analyses and made valuable comments about manuscript content. the authors thank wade edris, penn state college of medicine for assistance with confocal microscopy. we thank dr egeland for her tireless effort to study bipolar disorder in the old order amish of lancaster county, pa and for her dona- tion of dna samples to the coriell. finally, we are especially in- debted to the individuals afflicted with severe mental illness who agreed to participate in this study. conflict of interest statement. none declared. funding s.m.p., w.l., b.g. and m.b. were supported by national insti- tutes of health grant ro -mh- - . t.h. and y.t. were supported in part by national institutes of health grant r -gm- . r.n.j., e.g.p. and k.a.s. were supported by hhmi undergraduate science education awards and . r.n.j. was also supported by the center for re- search on women and newborn health, and by connectcare . funding to pay the open access publication charges for this article was provided by the clinic for special children. references . world health organization. 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>> /allowpsxobjects true /checkcompliance [ /none ] /pdfx acheck false /pdfx check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ . . . . ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ . . . . ] /pdfxoutputintentprofile (none) /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname () /pdfxtrapped /false /createjdffile false /description << /enu () >> >> setdistillerparams << /hwresolution [ ] /pagesize [ . . ] >> setpagedevice untitled d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il rspb.royalsocietypublishing.org research cite this article: kovach rp et al. vive la résistance: genome-wide selection against introduced alleles in invasive hybrid zones. proc. r. soc. b : . http://dx.doi.org/ . /rspb. . received: june accepted: october subject areas: evolution, genomics keywords: hybridization, natural selection, introgression, invasive species, genomics, temperature author for correspondence: ryan p. kovach e-mail: rkovach@usgs.gov electronic supplementary material is available online at https://dx.doi.org/ . /m .fig- share.c. . & the author(s) published by the royal society. all rights reserved. vive la résistance: genome-wide selection against introduced alleles in invasive hybrid zones ryan p. kovach , brian k. hand , paul a. hohenlohe , ted f. cosart , , matthew c. boyer , helen h. neville , clint c. muhlfeld , , stephen j. amish , , kellie carim , shawn r. narum , winsor h. lowe , fred w. allendorf and gordon luikart , northern rocky mountain science center, us geological survey, missoula, mt , usa flathead biological station, university of montana, polson, mt , usa institute for bioinformatics and evolutionary studies, department of biological sciences, university of idaho, moscow, id , usa fish and wildlife genomics group, division of biological sciences, university of montana, missoula, mt, usa montana fish, wildlife, and parks, kalispell, mt , usa trout unlimited, boise, id , usa wildlife biology program, university of montana, missoula, mt , usa hagerman genetics laboratory, columbia river inter-tribal fish commission, hagerman, id , usa rpk, - - - evolutionary and ecological consequences of hybridization between native and invasive species are notoriously complicated because patterns of selection acting on non-native alleles can vary throughout the genome and across environments. rapid advances in genomics now make it feasible to assess locus-specific and genome-wide patterns of natural selection acting on inva- sive introgression within and among natural populations occupying diverse environments. we quantified genome-wide patterns of admixture across mul- tiple independent hybrid zones of native westslope cutthroat trout and invasive rainbow trout, the world’s most widely introduced fish, by genotyp- ing individuals from populations using species-diagnostic loci. a significantly greater proportion of the genome appeared to be under selec- tion favouring native cutthroat trout (rather than rainbow trout), and this pattern was pervasive across the genome (detected on most chromosomes). furthermore, selection against invasive alleles was consistent across popu- lations and environments, even in those where rainbow trout were predicted to have a selective advantage (warm environments). these data corroborate field studies showing that hybrids between these species have lower fitness than the native taxa, and show that these fitness differences are due to selection favouring many native genes distributed widely throughout the genome. . introduction biological invasions are increasing due to environmental change and human translocation of species [ ], and these invasions can have severe negative impacts on native species through ecological and evolutionary processes. invasions can lead to hybridization between historically allopatric species, thereby producing complex conservation, evolutionary and legal consequences [ – ]. the negative consequences of hybridization between native and invasive species are numer- ous, including genomic extinction and loss of biodiversity [ ], ecological and genetic homogenization [ ], outbreeding depression [ ], and increased inva- siveness of the non-native taxon [ , ]. however, interspecific hybridization can also benefit native species through heterosis and adaptive introgression [ – ], facilitating colonization of novel environments [ ], and adaptation to environmental change [ , ]. http://crossmark.crossref.org/dialog/?doi= . /rspb. . &domain=pdf&date_stamp= - - mailto:rkovach@usgs.gov https://dx.doi.org/ . /m .figshare.c. https://dx.doi.org/ . /m .figshare.c. http://orcid.org/ http://orcid.org/ - - - £ temp (°c) km n – – > jocko blackfoot flathead st mary figure . map of the study areas, including the populations across four major river basins (shaded dark grey) in montana, usa, and british columbia, canada. stream watersheds occupied by each population are colour-coded (see legend) based on maximum average august stream temperatures at each location. lake populations are denoted by a red dot. rspb.royalsocietypublishing.org proc. r. soc. b : d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il in practice, the fitness outcomes of invasive hybridization are variable, as the strength and direction of selection frequently vary across environmental gradients [ ] and throughout the genome [ ]. for example, environmental conditions often influ- ence spatial and temporal dynamics of invasive introgression [ , ], as well as patterns of selection acting on hybrids [ ]. additionally, natural selection can favour introgression of single genes even when there is near-lethal selection against overall genome-wide admixture [ ]. because of the complex relationship between environ- mental variation, admixture and natural selection, there is limited understanding of the net adaptive consequences of invasive introgression in nature [ , ], and it has been par- ticularly challenging to characterize genome-wide patterns of admixture and natural selection in non-model organisms. however, recent advances in genomics now make it feasible to assess genome-wide patterns of invasive introgression and selection within and between natural populations occupying diverse environments [ , ]. this richer understanding of the architecture and mechanisms influencing invasive intro- gression is necessary for evaluating the implications of invasive hybridization and making informed conservation decisions [ ]. the ecological, evolutionary and conservation problems associated with invasive hybridization are evident where native westslope cutthroat trout (oncorhynchus clarkii lewisi) and invasive rainbow trout (o. mykiss) come into contact due to large-scale human releases of rainbow trout. despite extensive hybridization where these species come into human-induced sympatry [ ], intensive field studies suggest there can be strong selection against rainbow trout admixture [ , ]. however, preliminary genomic data indicate that selection may favour the introgression of several ‘super-inva- sive’ rainbow trout alleles [ ]. furthermore, other lines of evidence, including measurements of thermal tolerances [ ], metabolic traits [ ] and spatial patterns in admixture [ ], suggest that rainbow trout and their hybrids may have fitness advantages in warmer conditions, potentially leading to adaptive introgression from invasive to native trout in warm environments. we used genetic samples collected from populations occupying thermally diverse habitats (figure ; electronic rspb.royalsocietypublis d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il supplementary material, table s ) and species-diagnostic loci [ ] to assess genome-wide patterns of admixture between rainbow trout and westslope cutthroat trout. we addressed three questions: (i) what are patterns of natural selection acting on invasive admixture? (ii) do patterns of selection vary depending on temperature? (iii) is natural selection acting for or against rainbow trout admixture concentrated or dispersed throughout the genome? hing.org proc. r. soc. b : . material and methods (a) sample collection we obtained tissue samples from juvenile trout in streams and lakes located in montana, usa and british columbia, canada. all sampling locations were chosen to represent local breed- ing populations. we strategically included locations spanning environmental gradients (i.e. putative selective pressures) thought to influence local adaptation in salmonid fishes [ – ], and spatial patterns of hybridization between rainbow trout and west- slope cutthroat trout [ , – ]. specifically, we included populations inhabiting both lake and stream environments and also locations with widely varying temperatures (figure ; electronic supplementary material, table s ). in total, we obtained genetic samples from four independent river systems—the north fork flathead river, blackfoot river, jocko river and st mary river—with the highest number of populations in the north fork flathead and blackfoot river basins. the vast majority of populations ( of ) were located near the edge of the hybrid zone between native westslope cut- throat and invasive rainbow trout, where hybridization was present but admixture proportions were relatively low ( pro- portion rainbow trout ancestry less than . ; electronic supplementary material, table s ). it is in these populations with low admixture where power was highest to detect rainbow trout alleles at higher than expected frequencies, potentially due to natural selection [ ]. the remaining two populations were highly admixed, such that the original westslope cutthroat popu- lations were genomically extinct [ , ]. throughout this region, admixture began sometime in the early twentieth century (initiation of rainbow trout stocking), but some populations were probably pure westslope cutthroat trout until as late as the early twenty-first century [ , ]. thus, admixture in any one population has been occurring for approximately – gen- erations (assuming an approx. year generation time). (b) rad genotyping detailed methods for identifying diagnostic loci between rainbow trout and westslope cutthroat trout are described elsewhere [ ]. in short, we extended previous efforts [ , ] and used rad sequen- cing to identify fixed species-diagnostic snp loci that differentiated pure westslope cutthroat trout and pure rain- bow trout individuals of diverse origins. specifically, we used radseq to genotype individuals from populations contain- ing various amounts of rainbow trout ancestry (electronic supplementary material, table s ). libraries for rad sequencing were prepped from genomic dna according to standard protocols using the restriction enzyme sbfi and unique bp barcodes for each sample [ ]. we multiplexed rad libraries (at equal concen- tration) in six sequencing lanes on an illumina hiseq machine. we conducted initial processing of the sequence data from all individ- uals into rad loci using several modules from the stacks software package, v. . [ ]. we used process_radtags from stacks to sort read pairs by barcode and remove any pairs in which the forward read did not contain both a correct barcode and the remaining six bases of the sbfi recognition sequence. paired-end reads from the same individual were used to ident- ify pcr duplicates. the random shearing step in traditional rad sequencing produces staggered paired-end reads, so that any set of read pairs with identical sequences across both the forward and reverse reads are likely pcr duplicates from a single original geno- mic dna fragment [ ], and were removed using the stacks program clone_filter. the remaining read pairs from each individ- ual were aligned to the rainbow trout reference genome [ ]. we used the alignment software bowtie v. . . [ ] for end-to-end alignment without allowing gaps, and permitted one high-quality nucleotide mismatch per bp. given the recent whole-genome duplication in salmonid fishes [ ], we used multiple methods to identify diagnostic snp loci from sequence differences in duplicated genomic regions (homeo- logues and paralogues) [ ]. first, we retained only those loci that aligned uniquely to a single genomic location. we also enabled the deleveraging and removal algorithms in stacks to filter out highly repetitive loci that were also likely paralogous [ ]. we assigned diploid genotypes at each nucleotide position in each individual using the bounded maximum-likelihood method, with a mini- mum phred quality-score threshold of at each nucleotide, the upper bound of the sequencing error rate set to . , and a likelihood ratio significance level of a ¼ . . for quality control, we further filtered the data to remove all individuals with greater than % missing data and all loci that were genotyped in fewer than eight individuals (after individ- uals with missing data were removed) in each population. our final dataset included individuals genotyped at puta- tively diagnostic loci (electronic supplementary material, table s and figure s ). we did not test for linkage disequilibrium or confirmation to hardy – weinberg proportions at these loci, because we expected extensive physical linkage and some non- random mating due to recent and ongoing admixture between the species. (c) data analysis to minimize false positives, and the likelihood that false signals of natural selection were due to shared (ancestral or derived) poly- morphisms [ ], we used a highly conservative strategy for identifying loci that exhibited evidence for selection favouring or resisting rbt admixture. relative to previous efforts [ ], we used an extended baseline ( panel of non-hybridized individuals) for identifying fixed diagnostic polymorphisms between rainbow trout and westslope cutthroat trout [ ]. nevertheless, pure west- slope cutthroat trout populations are rare in many portions of their historical range; our westslope cutthroat trout individuals were therefore from a limited number of extant populations, lar- gely originating in the south fork flathead river and the clark fork river. this limited our ability to detect low-frequency poly- morphisms that are ancestral in westslope, thus we anticipated that alleles shared with rainbow trout would be present at some markers. shared polymorphisms at putatively diagnostic loci can be mistaken for loci under selection. indeed, after increasing the baseline ascertainment panel used previously to identify the larger set of putative diagnostic markers used in this study [ ], several alleles that were candidates for selection favouring rainbow trout [ ] were flagged as polymorphisms persisting in pure west- slope cutthroat trout. those loci were removed from the dataset. we used long’s heterogeneity test [ ] to identify outlier loci having rainbow trout allele frequencies that significantly deviated from random expectation in each population (see [ ] for more details). long’s test accounts for sampling variation as well as genetic drift across loci, making it ideal for identifying loci poten- tially under selection in hybrid zones [ ]. rainbow trout alleles with higher frequencies relative to selectively neutral loci are candidates for selection favouring rainbow trout, whereas loci that are resistant to rainbow trout admixture have rainbow rspb.royalsocietypublishing.org proc. r. soc. b : d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il trout allele frequencies that are lower than expected by chance (e.g. electronic supplementary material, figure s ). samples from populations that were nearly pure rainbow trout or westslope cut- throat trout (electronic supplementary material, table s ) provide little information about positive or negative selection, respectively, acting on rainbow trout admixture. therefore, we used reduced subsets of populations to test for positive and negative selection. specifically, we grouped populations into three categories: ( ) populations where we could detect positive selection favour- ing rainbow trout admixture (where proportion rainbow trout admixture was less than . , n ¼ ); ( ) populations occupying warm habitats (mean august temperature more than c), where we could detect positive selection favouring rainbow trout admixture (a subset of category , n ¼ ); and ( ) populations where we could detect negative selection (resistance) acting against rainbow trout admixture ( proportion rainbow trout admixture more than . , n ¼ ). seven of the eight locations where we could detect negative selection against rainbow trout using long’s test were in warm environments. we predicted that there would be more evidence for selection favouring rainbow trout admixture in warm environments. within each category, we used a fisher’s combined test to calculate an overall test statistic and associated probability that a particular marker deviated from neutral expectation. p-values were adjusted based on the false discovery rate (a benjamin and yekutieli (by)-adjusted p-value [ , ]) and a ¼ . . we further required that each candidate locus with a by-adjusted p-value of less than . demonstrate consistent outlier behaviour in multiple populations and river basins. for the entire set of populations where we could detect selection favouring rainbow trout (i.e. category , above), we required that outliers demon- strate evidence ( p , . without correction for multiple tests) of directional selection favouring rainbow trout in at least one population from the north fork flathead river and one from the blackfoot river, and directional selection for rainbow trout in at least four populations overall (i.e. % of populations). we used similar criteria for directional selection favouring rainbow trout in warm environments (category ), but reduced the overall number of populations where a locus had to exhibit evidence for selection favouring rainbow trout to two (i.e. % of populations in warm environments). although we required fewer populations for the latter tests, the actual proportion of populations where we required that each locus exhibit a con- sistent pattern of selection was nearly identical: % for all populations, % for warm environments only. the same criteria were used for populations where we could detect selection favouring westslope cutthroat trout (category ): there had to be evidence for resistance to rainbow admixture in at least one population from each of the north fork flathead and blackfoot river systems (i.e. consistent evidence for selection had to be detected in % of populations). by requiring evidence for direc- tional selection in both the north fork flathead and blackfoot rivers, we attempted to minimize error associated with shared ancestral polymorphisms. snp loci with significant fisher’s combined p-values that were outliers in multiple populations in the north fork flathead or the blackfoot, but not both, were flagged as loci that may have shared polymorphisms and were removed from subsequent genomic cline analyses. some of these markers may actually be diagnostic (not shared) and subject to natural selection, but we opted to be conservative in the face of various complexities that could produce false signals of selection. as a complementary method to long’s heterogeneity test, we used genomic clines to identify snp loci potentially under natural selection [ ]. unlike long’s test, which is unable to detect selection favouring rainbow trout or westslope cutthroat trout in population samples where overall rainbow trout admixture proportions were near or , genomic cline analyses use locus-specific and overall genome-wide admixture proportions across individuals within each population to identify signatures of natural selection. specifi- cally, a logit-logistic model was used to fit locus-specific rainbow trout allele frequencies against overall genome-wide admixture across all populations. the same analysis was also conducted for the subset of all populations inhabiting warm environments (n ¼ ; electronic supplementary material, table s ). outliers were identified based on unusual combinations for the estimated parameters defining the slope (u) and cline centre (v) for each locus relative to the observed distribution of slope and cline- centre estimates across all loci. in general, steeper clines indicate loci that are resistant to admixture (i.e. selection acting against rain- bow trout admixture), whereas shallower, intermediate clines represent rainbow trout alleles potentially favoured by selection [ ]. we also examined locus-specific allele frequencies for each outlier cline to confirm the general pattern of selection. the r pack- age hiest was used to fit genomic clines and identify statistically significant outlier loci. all p-values were adjusted using the by-adjusted false discovery rate and a ¼ . . although long’s tests and genomic cline analyses are similar, there are fundamental differences between the methods, including the unit of analysis (i.e. differences in allele frequencies within a population versus differences in cline shape across populations, respectively) and the underlying data used for each analysis (i.e. the set of populations). we also used more restrictive criteria when evaluating long’s tests. outlier loci from long’s tests needed to have consistent signatures of selection across all populations and evidence for selection in independent river basins; the same criteria were not applied to the genomic cline analyses. thus, results from the two approaches are supportive rather than overlapping. we used multiple tests to assess overarching patterns of selection acting across the genome and across populations. we used x -tests to compare the total proportion of outlier loci that were candidates for selection favouring or resisting rainbow trout admixture using data from all appropriate populations, and for those populations inhabiting warm environments. chi-square tests were performed on results from long’s test and the genomic cline analysis individually, and all outlier loci combined (i.e. ident- ified from both tests). for long’s tests, we considered the total proportion of populations where an snp exhibited evidence for favouring or resisting selection as a measure of strength of evidence (e.g. more populations is stronger evidence). a nucleotide blast using sequence data for each candidate rad locus and the rainbow trout genome were used to identify whether outlier snps were located within or proximate to genes of known function. we compared the rad loci alignment coor- dinates with start and end coordinates for the predicted gene annotations in the rainbow trout genome [ ]. we collected the gene annotations whose start and/or end coordina- tes either overlapped or were within kbp of the start or end coordinate of the outlier rad loci. the degree of linkage disequili- brium (ld) between rad markers and functional loci has not been quantified, and the state of the genome assembly did not allow us to estimate ld extent among our rad markers. however, we anticipate a relatively large extent of ld across physically linked loci for westslope cutthroat trout versus rainbow trout alleles, given the recent history of admixture [ ], so that kb is expected to be relatively conservative for identifying potential genes. we required not only that each locus and gene were on the same chromosome, but that they also shared the same scaffold or contig, because the assembled chromosomes contain sequence gaps of unknown size. to find detailed descriptions for the pre- dicted genes in the genome annotations, we used mrna sequences corresponding to the coding sequence (cds) and untranslated regions (utrs) of the genes as queries in a blastn alignment [ ]. for each query, we collected the blast hit with the highest bitscore. when more than one hit had the highest bitscore, we collected the one that was first in blast output order. table . the number of outlier snps where there was evidence for selection favouring or resisting rainbow trout (rbt) admixture. outlier snps are grouped according to statistical methods used to detect selection: long’s heterogeneity tests (lt), genomic cline analysis for all populations (gc), genomic cline analysis for populations inhabiting warm environments (gc warm), all unique snps detected as outliers in any analysis (un) (i.e. at least one analysis including lt, gc or gc warm), and the subset of snps that were consistently detected as significant outliers in all three analyses (all). direction of selection lt gc gc warm un all favouring rbt resisting rbt . – . . – . . – . . – . . – . direction of selection proportion of populations where snp was an outlier nu m be r of o ut li er s n p s favouring rbt favouring rbt—warm resisting rbt figure . the number of outlier snps revealing evidence of selection favour- ing (red and black bars), or resisting (blue bars) rainbow trout (rbt) admixture. counts of outliers in each category are binned according to the proportion of populations where each snp was identified as an outlier (i.e. higher proportions represent snps that were identified as a candidate for selection in a larger proportion of populations). results from outlier tests focused on detecting selection favouring rainbow trout are reported for all populations (black bars), and a subset of nine populations occupying warm environments (more than c mean august temperature; red bars). rspb.royalsocietypublishing.org proc. r. soc. b : d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il . results (a) patterns of natural selection acting on invasive admixture there was consistent evidence for selection against rainbow trout admixture across different populations and hybrid zones (table and figure ). rainbow trout alleles at snp loci (table ) occurred at lower than expected frequencies— evidence for selection acting against admixture—within multiple independent populations (figure ; electronic sup- plementary material, figure s ). selection against rainbow trout admixture was detected at snp loci in each of three of the eight populations where we could detect selection favour- ing westslope cutthroat trout ( % of populations); snps were outliers in four of those eight populations ( % of populations) and four snps were outliers in five to seven populations ( – % of populations; figure ). similarly, there were snps where rainbow trout allele fre- quencies were significantly lower than the genome-wide average across populations, as represented by genomic clines (figure a). patterns of selection against rainbow admixture gen- erally took two forms. most outlier loci had lower than expected rainbow trout allele frequencies across a range of genome-wide admixture values (the ‘shallow’ clines in the lower right quadrant of figure ). a subset, however, were fixed for the rain- bow trout allele in the two highly admixed populations (gold creek and abbott creek), but the rainbow trout allele was lar- gely absent in other populations—the ‘steep’ clines centred at genome-wide proportion rainbow trout admixture ( prbt) � . (figure ). the five loci with this second pattern were all flagged as outliers using long’s test because they had lower than expected frequencies of the rainbow trout allele in – % of populations. there were no populations where the rainbow trout allele was at higher than expected frequencies, suggesting that this pattern reflects, in part, the lack of samples from populations with prbt between . and . . there were considerably fewer snps that exhibited patterns suggestive of selection favouring rainbow trout admixture (table ). using long’s heterogeneity test and data from all populations where we could detect directional selec- tion favouring rainbow trout admixture (n ¼ populations), eight snps satisfied our criteria (e.g. at least one population from two drainages showing selection) for selection favouring the rainbow trout admixture (table ). four of those snps were significant outliers in four populations ( % of populations), two were significant outliers in five populations ( % of populations) and two were significant outliers in six popu- lations ( % of populations; figure ). when using genomic data from all populations, there were four snp loci with sig- nificant genomic clines suggestive of selection favouring rainbow trout admixture (i.e. the rainbow trout allele was at higher than expected frequencies across genome-wide values of prbt). regardless of method, populations used in the analyses or thermal environment, there were substantially more outlier snps where selection appeared to resist rainbow trout admix- ture (table ). in all cases, the overall proportion of snps where there was evidence for selection opposing rainbow trout alleles was substantially higher than the proportion of snps with rainbow trout alleles that were favoured by selection (all pair-wise tests across analyses p , . ). snps that were can- didates for directional selection acting against rainbow trout were identified as outliers in more populations than snps that were candidates for selection favouring rainbow trout (figure ). the subset of snps that were consistently identified as outliers in each analysis (n ¼ ) were all candidates for selection acting against rainbow trout (table ). (b) patterns of natural selection in warm habitats patterns of selection among populations inhabiting warm environments mirrored those obtained from populations inhabiting the entire thermal spectrum. we only detected one snp where there was evidence for selection favouring rainbow trout in populations inhabiting warm environments (n ¼ populations; figure ). however, this same snp was also ident- ified as an outlier using the data from all populations, suggesting that there were no rainbow trout alleles potentially favoured by selection specifically in warm environments. five lo cu s- sp ec if ic p r b t genome-wide prbt genome-wide prbt (a) (b) . . . . . . . . . . . . . . . . . . . . figure . locus-specific genomic clines that exhibited outlier patterns of introgression representative of selection favouring (red) and resisting (blue) rainbow trout admixture across (a) all populations, and (b) rainbow trout admixture for a subset of the nine populations inhabiting warm environments (more than c mean august temperature). prbt is the individual-level proportion of rainbow trout admixture. the bold black genomic cline represents genome-wide average across all putatively neutral loci. . . sx nn chromosome number pr op or ti on o f ou tl ie r s n p s direction of selection favouring rbt resisting rbt figure . the distribution of outlier snps favouring (red) or resisting (blue) rainbow trout (rbt) admixture across the genome (i.e. chromosomes). the proportion of outlier snps is relative to the number of snps mapped to each chromosome, and includes all unique snps identified across all analyses (table ). the labels on the x-axis represent the autosomal chromosomes, sex chromosomes (sx) and rad tags on scaffolds that are not yet mapped to specific chromosomes (nn). there is no genome assembly for chromosome . rspb.royalsocietypublishing.org proc. r. soc. b : d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il genomic clines were suggestive of natural selection favouring rainbow trout admixture in warm environments. by contrast, clines were suggestive of selection opposing rainbow trout admixture (figure b), with a subset (n ¼ ) of these outlier clines exhibiting steep slopes centred at prbt � . , all of which appeared to be under selection resisting rainbow trout admixture similar to the pattern described above (i.e. fixed for the rainbow trout allele in highly admixed populations but absent from other upstream locations). for these loci, long’s tests confirmed that rainbow trout allele frequencies were at lower than expected frequencies within populations, and were never at higher than expected frequencies within any population (i.e. there was evidence for selection opposing rainbow trout admixture). in summary, there was no evidence for higher rates of adaptive rainbow trout introgression in the warmest streams and lakes. instead, the proportion of snps with rainbow trout alleles potentially favoured by selection in the warmest environments was nearly identical to (genomic cline analyses, figure b; p ¼ . ) or slightly less than (long’s heterogeneity tests, figure ; p ¼ . ) the proportion of snps with rainbow trout alleles potentially favoured by selection in any thermal environment. (c) genomic distribution and potential functionality of outlier loci rainbow trout segments where selection resisted admixture were distributed widely across chromosomes (mapped to of the assembled autosomal chromosomes and to the pair of sex chromosomes), as well as the unmapped portions of the genome (figure ). there was some evidence for concentrated selection against rainbow trout segments on chromosome , but the majority of outlier snps mapped to known chromosomes were found on globally ordered but non-anchored portions of the genome [ ], making it impossible to identify specific chromoso- mal regions potentially under selection. forty of the rainbow trout genome segments potentially under selection (favouring or resisting rainbow trout admixture) were located in or near described gene annotations (electronic supplementary rspb.royalsocietypublis d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il material, table s ). specifically, of the rad loci aligned com- pletely inside gene annotations. the rest (n ¼ ) were within kbp of annotated genes. highest-scoring blast alignments generally yielded salmo salar sequences ( out of ). after removing redundant gene annotations, there were unique gene descriptions with potential functions that varied widely from cellular metabolism to gene expression to disease resistance (electronic supplementary material, table s ). hing.org proc. r. soc. b : . discussion genomic data from hybridizing populations of native and invasive trout strongly suggest that natural selection consist- ently acts against non-native alleles throughout the genome, across populations and in different environments. the con- sequences of invasive hybridization, particularly fitness outcomes, are notoriously complicated and frequently contro- versial because it has been challenging to describe patterns of natural selection acting on non-native genetic introgression [ , ]. here, we show the applied value of genomics for illumi- nating patterns of selection acting in invasive hybrid zones and further clarify the implications of hybridization for this species of conservation concern. more generally, these data underscore that widespread admixture between native and non-native species can largely reflect large-scale propagation and releases of the non-native species, not positive selection favouring the invasive taxon. consistent evidence for selection acting against invasive rainbow trout genomic segments throughout the genome cor- roborates detailed field studies showing reduced fitness in rainbow trout and their hybrids. in one population, fish with proportion rainbow trout ancestry more than . suffered from nearly a % reduction in reproductive success relative to pure westslope cutthroat trout [ ]. similarly, selection coeffi- cients (s) acting against rainbow trout genotypes were high (s � . ) in two streams with contrasting thermal and hydro- logic regimes [ ]. data presented here suggest that selection consistently acts against rainbow trout across the genome in other populations and—more importantly—separate river basins with independent invasion histories. this pattern of selection against rainbow trout admixture is striking given that we strategically sampled warm environ- ments in multiple independent river basins. spatial patterns of admixture and laboratory thermal tolerances suggest that rainbow trout may have a selective advantage under warmer temperatures. however, we found no evidence that there were higher rates of adaptive introgression in the warmest environments. the data presented here and in [ ] highlight that spatial patterns in admixture should be interpreted care- fully with respect to selection. higher levels of admixture in low-elevation, warmer environments [ – ] do not necess- arily imply that rainbow trout and hybrids have higher relative fitness than westslope cutthroat trout. this pattern may simply indicate that selection acting against rainbow trout is not as severe or consistent in these environments, or that human releases of rainbow trout in certain areas over- whelmed selective regimes. rainbow trout were stocked routinely and abundantly throughout the early part of the twentieth century, and these stocking events occurred predomi- nantly in lower elevation rivers and lakes that often are warmer than those at higher elevation [ , , ], suggesting that spatial patterns in rainbow trout admixture probably reflect stocking and subsequent dispersal, more than selection favouring rain- bow trout in certain environments [ , ]. we detected positive selection favouring rainbow trout admixture at several loci, but there were no loci where rainbow trout alleles were consistently swept to high frequency across multiple populations. given the strong selection regimes that appear to be acting against rainbow trout admixture, it may take many generations of recombination before beneficial rain- bow trout alleles can successfully overcome genome-wide selection against rainbow trout admixture [ , ]. further- more, most adaptive traits are highly polygenic and, thus, most adaptive snps are presumably of relatively small effect [ , ], which probably impedes rapid adaptive introgression from rainbow trout to westslope cutthroat trout during early stages of invasive admixture. hybridization can act as a valuable source of evolutionary novelty [ ], and a stimulus for species invasions, particularly in plants [ ]. however, evidence for adaptive introgression between native and invasive vertebrate species remains scarce [ ] (but see [ ]). using an extensive baseline of geneti- cally pure individuals from both species during diagnostic snp discovery and a conservative approach to identify loci potentially under selection, we found that rainbow trout alleles previously inferred to be favoured by selection [ ] appear to be shared polymorphisms (alleles) within both species (see material and methods). separating signals of selection from shared polymorphisms remains a significant challenge in hybridization genomics, even for model systems (e.g. homi- nids) [ ]. we caution that patterns of adaptive introgression should be interpreted carefully, and recommend that future genomic studies use conservative methods including robust baselines for initial snp discovery and replication across inde- pendent hybrid zones to avoid false positives due to shared polymorphisms between species. this work has several caveats, which represent opportu- nities for future research. first, the rainbow trout reference genome remains a work in progress, the majority of which is anchored to chromosomes with unknown order, or is entirely unmapped [ ]. a more complete genome assembly is needed to better delineate specific regions of the genome under natural selection, and thereby clarify the genomic basis of fitness differ- ences between species. for example, this would help clarify whether the signal of selection on chromosome is highly con- centrated (i.e. one or several genes of large effect resulting in selection acting on a single rainbow trout haplotype block), or dispersed. it also remains unclear whether the signature of selec- tion favouring westslope cutthroat trout represents extrinsic selection acting on portions of the genome associated with local adaptation, or consistent intrinsic selection resulting from hybrid incompatibilities between species [ , ]. finally, we fil- tered our data to remove potentially paralogous snps during snp discovery and mapping [ ], a necessary strategy, but one with unknown consequences for understanding genomic signatures of selection in organisms like salmonid fishes with residual tetraploidy in their genomes [ , ]. future progress on the rainbow trout reference genome and emerging method- ologies for discovery and genotyping of snps in paralagous regions [ ] will provide additional insight into selection in this system and many others. although adaptive introgression is gaining interest as a process that may have some positive evolutionary and conservation outcomes [ , ], the consequences of human- mediated hybridization are likely to vary widely and rspb.royalsocietypublishing.org proc. r. so d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il remain challenging to predict [ , , ]. our data highlight that native genotypes are often favoured by natural selection [ , ], and thus management strategies focused on minimiz- ing the deleterious impacts of invasive hybridization here and in similar contexts are prudent until an adaptive basis of introgression can be established. this study shows that combining genome-wide data from multiple populations on the leading edge of an invasive hybrid zone can help eluci- date overarching patterns of selection across the genome. more specifically, our findings provide strong genome-scale evidence of selection against invasive hybridization across environmentally heterogeneous wild populations. data accessibility. the genotypes used in all analyses can be found at dryad: http://dx.doi.org/ . /dryad.ss . authors’ contributions. g.l., r.p.k., w.h.l., f.w.a., c.c.m. and m.c.b. designed the study. r.p.k., b.k.h., p.a.h., s.r.n., t.f.c., s.j.a. and h.h.n. contributed to laboratory and data analyses. k.c., m.c.b. and c.c.m. provided samples. r.p.k. drafted the manuscript and all co-authors provided input. competing interests. we declare we have no competing interests. funding. funding was provided by nsf (deb- ), nih (p gm ), the usgs national climate change and wildlife science center, the usgs mendenhall fellowship, bonneville power administration grant no. to montana, fish, wildlife and parks, and an individual donation (larry garlick) to trout unlimited. acknowledgements. we thank ben fitzpatrick for helpful advice on hiest. jeff good and two anonymous reviewers provided helpful comments on the manuscript. disclaimer. any use of trade, firm or product names is 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(doi: . / ) http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /czoolo/ . . http://dx.doi.org/ . / - ( ) -i http://dx.doi.org/ . / - ( ) -i http://dx.doi.org/ . /j. - x. . .x http://dx.doi.org/ . /hdy. . http://dx.doi.org/ . /t - . http://dx.doi.org/ . /f - http://dx.doi.org/ . /f - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /pnas. http://dx.doi.org/ . /pnas. http://dx.doi.org/ . /f - http://dx.doi.org/ . /f - http://dx.doi.org/ . / . . http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /j. - x. . .x http://dx.doi.org/ . /j. - x. . .x http://dx.doi.org/ . /mec. http://dx.doi.org/ . /nrg http://dx.doi.org/ . /ncomms http://dx.doi.org/ . /nmeth. http://dx.doi.org/ . /jhered/esv http://dx.doi.org/ . /jhered/esv http://dx.doi.org/ . /nrg http://dx.doi.org/ . /aos/ http://dx.doi.org/ . /aos/ http://dx.doi.org/ . /s - - -y http://dx.doi.org/ . /s - - -y http://dx.doi.org/ . /ece . http://dx.doi.org/ . /ece . http://dx.doi.org/ . /j.tree. . . http://dx.doi.org/ . /s - ( ) - http://dx.doi.org/ . /s - ( ) - http://dx.doi.org/ . /f - http://dx.doi.org/ . /f - http://dx.doi.org/ . / - . http://dx.doi.org/ . /j.tree. . . http://dx.doi.org/ . / - - - http://dx.doi.org/ . / - - - http://dx.doi.org/ . /cobi. http://dx.doi.org/ . /cobi. http://dx.doi.org/ . /eva. http://dx.doi.org/ . / http://dx.doi.org/ . /annurev-genet- - http://dx.doi.org/ . /annurev-genet- - http://dx.doi.org/ . / - - - http://dx.doi.org/ . / - - - http://dx.doi.org/ . /mec. http://dx.doi.org/ . /mec. http://dx.doi.org/ . /cobi. http://dx.doi.org/ . /annurev.es. . . http://dx.doi.org/ . /annurev.es. . . http://dx.doi.org/ . / 	vive la r&eacute;sistance: genome-wide selection against introduced alleles in invasive hybrid zones 	introduction 	material and methods 	sample collection 	rad genotyping 	data analysis 	results 	patterns of natural selection acting on invasive admixture 	patterns of natural selection in warm habitats 	genomic distribution and potential functionality of outlier loci 	discussion 	data accessibility 	authors’ contributions 	competing interests 	funding 	acknowledgements 	disclaimer 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ journal of abnormal psychology , vol. , no. , - copyright by the american psychological association, inc. - x/ /$ . short reports cultural factors considered in selected diagnostic criteria and interview schedules steven lopez and joseph a. nunez university of southern california researchers have argued that diagnostic criteria and interview schedules inadequately reflect cul- tural influences in the definition and expression of psychopathology. in this study widely used diagnostic criteria and interview schedules for schizophrenia, affective disorders, and personality disorders were examined to assess the extent to which they refer to cultural factors. the results indicated that of instruments referred to cultural influences in psychopathology at least once. the consideration of cultural factors, however, was primarily limited to the identification of delu- sions and hallucinations in schizophrenia. very few cultural references were made in the diagnostic instruments of affective and personality disorders. the clinical implications of these findings are discussed with respect to the evaluation of cultural minority group members residing in the united states. specific recommendations are offered to increase the attention given to culture in diagnostic instruments and to increase our understanding of how culture influences psychopathology. this study assesses the extent to which frequently used diag- nostic criteria and interview schedules consider cultural factors in the identification of schizophrenic, affective, and personality disorders. although some authors have discussed how selected diagnostic instruments fail to address cultural influences (alar- con, ; egeland, hostetter, & eshleman, ; klerman, vaillant, spitzer, & michaels, ; swartz, ben-arie, & teg- gin, ), none have systematically looked at the extent to which a wide range of diagnostic instruments address the role of culture in psychopathology. such an examination should re- flect the relative importance given to culture in the classification of mental disorders. schizophrenic and affective disorders were chosen because much of the cross-cultural psychopathology research concerns these two diagnostic categories (draguns, , ; klein- man & good, ; marsella, ). personality disorders were selected because of the recent attention given to their cultural nature (alarcon, ; klerman et al., ). moreover, sub- jects in current investigations of these disorders are at times drawn from cultural minority groups, including blacks (robins et al., ), hispanics (karno et al., ), and the amish (egeland et al., ). given the use of these instruments with cultural minority groups as well as with international popula- tions, questions of cross-cultural validity are raised. method instruments the diagnostic criteria and structured interview schedules selected for this study were the following: the diagnostic and statistical manual of mental disorders (dsm-ih; american psychiatric association, ), the feighner criteria (feighner et al., ), the flexible who (carpenter, strauss, & bartko, ), the new haven schizophrenic index (nhsi; astrachan et al., ), the research diagnostic criteria (rdc; spitzer, endicott, & robins, ), taylor and abrams ( ), the diagnostic interview schedule (dis; robins, helzer, croughan, & ratcliff, ), present state examination (pse; wing, cooper, & sar- torius, ), the schedule for affective disorders and schizophrenia (sads; spitzer & endicott, ), the structured clinical interview for rw-///(scid; spitzer & williams, ), and the structured inter- view for dsm-ii personality disorders (sidp; stangl, pfohl, & zim- merman, ). we chose these instruments because they are among the most frequently used in the study of schizophrenia, major affective disorders, and personality disorders. the international classification of diseases: clinical modification (u.s. department of health and hu- man services, ) was considered for review, but we decided to ex- clude it because it is based on a classification scheme that offers no spe- cific criteria for mental disorders. self-report measures were also ex- cluded because of their large number and because they frequently do not have explicit diagnostic criteria. including such measures would have gone beyond the desired focus of this investigation. this study was completed while steven lopez was a recipient of a ford foundation postdoctoral fellowship. during this time he was affiliated with the spanish speaking psychosocial clinic, neuropsychi- atric institute, university of california, los angeles. correspondence concerning this article should be addressed to steven lopez, department of psychology, university of southern california, los angeles, california - . procedure joseph a. nunez carefully read the diagnostic criteria and structured interview schedules and identified the instruments' direct references to possible cultural influences in judging the presence of symptomatology regarding schizophrenic, affective, and personality disorders. in addi- tion, introductory comments about the perceived role of culture in psy- chopathology were noted. for the present study, culture generally refers to the distinctive body of customs, beliefs, and institutions characteris- short reports tic of a racial, ethnic, religious, or national group. social factors (socio- economic status) and patient variables (sexual orientation, age) are also important in the diagnosis of mental disorders and could be included in a broad definition of culture. for this investigation, however, these factors were not considered to be cultural in nature unless the authors of a given instrument referred to them as cultural (e.g., taylor & abrams, ). in general, we attempted to identify the instruments' perspective regarding how cultural factors should be considered in the identification and diagnosis of psychopathology. reliability check to assess the reliability of the rater's judgments, an advanced under- graduate psychology student, blind to the study's purpose, was in- structed to carefully read the instruments and available instructions and to identify the direct cultural references. the two raters concurred on of cultural references ( %). except for dsm-lii, the following pairs of ratings were based on the entire set of criteria or interview schedule: dsm-ih (american psychiatric association, , pp. - , - , - , - ) ( : ), feighner criteria ( : ), flexible who ( : ), nhsi ( : ), rdc ( : ), taylor and abrams ( : ), dis ( : ), pse ( : ), sads ( : ), scid ( : ), and sidp ( : ). this level of interrater agreement was judged adequate for this research. results overall, the selected diagnostic instruments minimally rec- ognized how cultural factors can influence the expression and definition of schizophrenic, affective, and personality disorders. of the six sets of diagnostic criteria, the rdc makes three cul- tural references, the feighner criteria makes none, and the re- maining four sets of criteria (dsm-ih, flexible who, nhsi, and taylor and abrams) make only one cultural reference each. the review of interview schedules revealed similar findings; the pse and sads refer to cultural factors on six and five occa- sions, the scid refers to culture only once, and the dis and sidp fail to acknowledge cultural influences altogether. con- sidering the sets of diagnostic criteria and interview schedules as a group, of the instruments consider culture at some level. however, the number of references are very few relative to the total number of symptoms designated for a particular disorder. an examination of the diagnostic criteria and interview schedules by disorder revealed the type of cultural references made. in the diagnosis of schizophrenia, of the diagnostic instruments (dsm-iii, nhsi, pse, rdc, sads, and scid) that offer criteria for the disorder contain some reference to cul- ture and its potential role in properly identifying delusions and hallucinations. the main point of these cultural references is that diagnosticians should make sure that the patient's particu- lar belief or perceptual experience is not shared by other mem- bers of his or her cultural group. the rdc and its companion structured interview (sads) also point out cultural factors that should be taken into account when assessing formal thought disorder. interviewers are cautioned that some speech or think- ing patterns considered to be representative of thought disorder could be representative of normal speech or thinking for some groups. with respect to the diagnostic instruments that have criteria for affective disorders, only the rdc, sads, and scid refer to possible cultural influences. the cultural basis of hallu- cinations and delusions is again mentioned here. in addition, rdc and sads indicate that bereavement may not represent a depressive disorder if all features of the bereavement are com- monly seen in members of the subject's subcultural group in similar circumstances. in regard to personality disorders, none of the diagnostic instruments makes reference to possible cul- tural influences. two of the diagnostic instruments (flexible who and pse) make explicit the assumption that a transcultural descrip- tion of mental disorders can be formulated. although the au- thors of these instruments indicated that cultural factors can affect nuances in the expression of mental illness, they stated that diagnostic criteria can be used cross-culturally because there are enough common elements across cultures. discussion overall, these findings indicate that the lack of attention given to cultural factors in diagnostic instruments goes beyond what has been previously noted for dsm-iii (alarcon, ; kler- man et al., ), rdc (egeland et al., ), and the pse (swartz et al., ). it is fair to say that the currently used sets of diagnostic criteria and interview schedules for schizophrenic, affective, and personality disorders pay little attention to cul- tural factors. some general recommendations are offered in an attempt to address the limited consideration of culture. at the very least, each set of diagnostic criteria and each interview schedule should have a general statement pointing out that cultural val- ues, beliefs, and practices can influence the definition and ex- pression of psychopathology. the need for such a statement is supported by the growing cross-cultural psychopathology litera- ture (al-issa, ; draguns, ; fabrega, , ; kleinman & good, ; marsella & white, ) as well as by the more limited u.s. minority group research (adebimpe, ; cuellar & roberts, ). the inclusion of this statement should alert diagnosticians and interviewers to seriously con- sider the cultural background of the patient. we also recom- mend that references to cultural factors be made for specific disorders and symptoms, and whenever possible, examples per- taining to specific cultural groups be cited. comments such as the following might be included in diagnostic instruments: black patients' depressive symptoms may not be properly iden- tified as depressive in nature (adebimpe, ; simon, fleiss, gurland, stiller, & sharpe, ), and pressured speech may be inaccurately perceived as thought disorder among the amish (egeland et al., ). these and related comments may prompt evaluators to be more cautious in applying the available criteria when evaluating these patient groups. in terms of research, we recommend that investigators exam- ine the phenomenology, course, and outcome of the major dis- orders for u.s. minority groups. cross-cultural studies to date indicate the important role culture plays in the definition and expression of psychopathology. however, we are only just begin- ning to understand how cultural factors influence the psychopa- thology of the major minority groups in the united states— blacks (adebimpe, l),hispanics (cuellar & roberts, ), asians (chin, ), and american indians (manson, shore, & bloom, ). in addition, it is important that investigators and clinicians who work with specific cultural groups follow the lead short reports of egeland and her colleagues in systematically assessing whether available criteria are appropriate. if criteria or inter- view schedules are not appropriate, then ways to modify these instruments should be explored. references adebimpe, v. r. ( ). overview: white norms and psychiatric diag- nosis of black patients. american journal of psychiatry, , - . alarcon, a. d. ( ). a latin american perspective on dsm-iii. american journal of psychiatry, , - . al-issa, i. ( ). (ed.). culture andpsychopathology. baltimore: uni- versity park press. american psychiatric association. ( ). diagnostic and statistical manual of mental disorders ($r& ed.). washington, dc: author. astrachan, b. m., harrow, m., adler, d., brauer, l., schwartz, a., schwartz, c, & tucker, g. ( ). a checklist for the diagnosis of schizophrenia. british journal of psychiatry, , - . carpenter, w. x, strauss, j. s., & bartko, j. j. ( ). flexible system for the diagnosis of schizophrenia: report from the who interna- tional pilot study of schizophrenia, science, , - . chin, j. l. ( ). diagnostic considerations in working with asian- americans. american journal oforthopsychiatry, , - . cuellar, i., & roberts, r. e. ( ). psychological disorders among chi- canos. in j. l. martinez & r. h. mendoza (eds.), chicanopsychology ( nd ed., pp. - ). new york: academic press. draguns, j. g. ( ). psychological disorders of clinical severity. in h. c. triandis & j. g. draguns (eds.), handbook of cross-cultural psychology: psychopathology (vol. , pp. - ). boston: allyn & bacon. draguns, j. g. ( ). assessing mental health disorders across cul- tures. in p. b. pedersen, n. sartorius, & a. j. marsella (eds.), mental health services: the cross-cultural context (pp. - ). beverly hills, ca: sage. egeland, j. a., hosteller, a. m., & eshleman, s. k., iii. ( ). amish sludy, iii: the impacl of cultural factors on diagnosis of bipolar ill- ness. american journal of psychiatry, , - . fabrega, h. ( ). problems implicit in the cultural and social study of depression. psychosomatic medicine, , - . fabrega, h. ( ). culture and psychiatric illness: biomedical and eth- nomedical aspects. in a. j. marsella & g. m. white (eds.), cultural conceptions of mental health and therapy (pp. - ). dordrecht, the netherlands: d. reidel. feighner, j. p., robins, e., guze, s. b., woodruff, r. a., winokur, g., & munoz, r. ( ). diagnostic criteria for use in psychiatric research. archives of general psychiatry, , - . karno, m., jenkins, j., de la selva, a., santana, f, telles, c., lopez, s., & mintz, j. ( ). expressed emotion and schizophrenic outcome among mexican-american families. journal of nervous and mental disease, , - . kleinman, a., & good, b. (eds.). ( ). culture and depression. berkeley: university of california press. klerman, g. l., vaillant, g. e., spitzer, r. l., & michaels, r. ( ). a. debate ondsm-ltt. american journal of psychiatry, , - . manson, s. m., shore, j. h., & bloom, j. d. ( ). the depressive experience in american indian communities: a challenge for psychi- atric theory and diagnosis. in a. kleinman & b. good (eds.), culture and depression (pp. - ). berkeley: university of california press. marsella, a. j. ( ). depressive experience and disorder across cul- tures. in h. c. triandis & j. g. draguns (eds.), handbook of cross cultural psychology: psychopathology (vol. , pp. - ). boston: allyn & bacon. marsella, a. j., & white, g. (eds.). ( ). cultural conceptions of men- tal health and therapy. dordrecht, the netherlands: d. reidel. robins, l. n., helzer, j. e., croughan, j., & ratcliff, k. s. ( ). na- tional institute of mental health: diagnostic interview schedule. ar- chives of general psychiatry, , - . robins, l. n., helzer, j. e., weissman, m. m., orvaschel, h., gruen- berg, e., burke, j. d., & regier, d. a. ( ). lifetime prevalence of specific psychiatric disorders in three sites. archives of general psy- chiatry, , - . simon, r. j., fleiss, j. l., gurland, b. j., stiller, p. r., & sharpe, l. ( ). depression and schizophrenia in hospitalized black and white mental patients. archives of general psychiatry, , - . spitzer, r. l., & endicott, j. ( ). the schedule for affective disor- ders and schizophrenia ( rd ed.). new \brk: new york state psychi- atric institute, biometrics research division. spitzer, r. l., endicott, j., & robins, e. ( ). research diagnostic criteria ( rd ed.). new york: new york state psychiatric institute, biometrics research division. spitzer, r. l., & williams, j. b. w. ( ). structured clinical interview for dsm-iii. new york: new york state psychiatric institute, bio- metrics research division. stangl, d., pfohl, b., & zimmerman, m. ( ). a structured interview for dsm-iii personality ( nd ed.). iowa city: university of iowa col- lege of medicine, department of psychiatry. swartz, l., ben-arie, o, & teggin, a. f. ( ). subcultural delusions and hallucinations: comments on the present state examination in a multi-cultural context. british journal of psychiatry, , - . taylor, m. a., & abrams, r. ( ). a critique of the st. louis psychiat- ric research criteria for schizophrenia. american journal of psychia- try, , - . u.s. department of health and human services. ( ). international classification of diseases: clinical modification ( nd ed.). washing- ton, dc: author. wing, j. k., cooper, j. e., & sartorius, n. ( ). the measurement and classification of psychiatric symptoms. london: cambridge uni- versity press. received december , accepted january , • short reports fig.-patient at time of presentation, showing ascites. normal serum bilirubin and no evidence ofhepatocellular dysfunction or biliary obstruction. total serum proteins normal at - g/ ml, lipoprotein electro- phoresis showed a raised b fraction, serum cholesterol was high at mg/ ml, and serum triglycerides increased at mg/ ml. these abnormally high values persisted, as did the ascites, after the child's diabetes was brought under control; and at this stage her thyroid function was investigated. her height at cm was well below the rd centile, though bone age was only marginally less than chrono- logical age. serum protein-bound iodine was ,ug/ ml (normal - ,ug/ ml) and serum thyroxine - jug/ ml (normal - - ug/ ml). the -hour uptake after i ( ilci) was % (normal range - %/'), but when repeated after administration of units thyroid-stimulating hormone, -hour uptake was only - %. the absence of any extra response to thyroid-stimulating hormone indicates an absence of thyroid reserve in the patient. no thyroid antibodies were shown in either the patient or her mother. after a diagnosis of hypothyroidism, her progress on a small dose of thyroxine ( mg daily) has been encouraging. she has grown cm in months, ascites and abdominal distension are no longer evident, and her skin and hair are less dry and coarse. her mother reports she is generally more lively and interested. discussion this case is presented not only because the combination of down's syndrome with diabetes mellitus and hypothyroidism is rare, but also because of the unusual presentation of this child's disturbance of thyroid function. thyroid function in down's syndrome is usually normal (marks and hamlin, ), but both hyperthyroidism (kurland et al., ) and hypothyroidism have been described. the latter is less common, and until only cases had been reported (hayles, hinrichs, and tauxe, ). the triple combination of hypothyroidism, down's syndrome, and diabetes mellitus is even more rare and there are only two previously described cases (daniels and simon, ; litman, ), who were aged months and years at the time the full picture became evident. investigations in this child have shown that she was hypothyroid rather than totally deficient in thyroid function. the presenting symptom of ascites is unusual in children, but fluid accumulation in the peritoneum and elsewhere is a well-recognized clinical feature of adult myxoedema. on admission the provisional diagnosis in this case was mauriac syndrome (guest, ), but it is doubtful if this condition of hepatomegaly, stunting of growth, and poorly controlled diabetes mellitus is an entity. rather, this patient shows a rare cause of that unusual condition. summary a child with down's syndrome who subsequently developed diabetes mellitus and hypothyroidism before the age of years is described. the principal presenting feature of her disturbed thyroid function was ascites. references daniels, d. m., and simon, j. l. ( ). down's syndrome, hypothyroidism, and diabetes mellitus. journal of pediatrics, , . guest, g. m. ( ). the mauriac syndrome. diabetes, , . hayles, a. b., hinrichs, w. l., and tauxe, w. n. ( ). thyroid disease among children with down's syndrome (mongolism). pediatrics, , . kurland, g. s., fishman, j., hamolsky, m. w., and freedberg, a. s. ( ). radioisotope study of thyroid function in mongoloid subjects, including observations in parents. journal of clinical endocrinology, , . litman, n. n. ( ). down's syndrome, hypothyroidism, and diabetes mellitus. journal of pediatrics, , . marks, j. f., and hamlin, m. ( ). free thyroxine levels in children with mongolism. journal of pediatrics, , . w. abdel shaheed and l. rosenbloom* royal liverpool children's hospital, liverpool. *correspondence to dr. l. rosenbloom, alder hey children's hospital, eaton road, liverpool l ap. cartilage hair hypoplasia cartilage hair hypoplasia was first described by mckusick in . while studying the old amish sect he noticed an association between short-limbed dwarfism and sparseness of hair, and later reported o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n n o ve m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ short reports cases (mckusick et al., ). since then several cases with no amish ancestry have been published. however, though two variants of this rare condition were shown in britain recently (savage, ), no other cases have been published in britain. case report a male was the product of the third pregnancy of a young mother. there was no history of consanguinity in the parents and, apart from hydramnios, the pregnancy had been normal. he weighed - kg ( oth centile) at birth, and crown to heel length was cm (below the rd centile). an oesophageal tube was passed without difficulty into the stomach. both upper and lower limbs were short in relation to the trunk, but there was no clinical or radiological evidence of achondroplasia. at weeks of age he was admitted to the radcliffe infirmary with large intestinal obstruction, which was relieved by sigmoid colostomy. a biopsy showed hirschsprung's disease and at months of age the aganglionic segment was resected and the colostomy closed (mr. malcolm gough). at n years of age he was readmitted for assessment of his short stature and particularly for investigations appropriate to the cartilage hair hypoplasia syndrome, which seemed a likely diagnosis. his height remained below the third centile and his weight had fallen below this level, falling away from the centile lines. on examination he was strikingly short, height cm and weight kg. he had very fine and sparse fair hair, and the lower limbs appeared disproportionately shorter than the upper limbs (fig. ). his hands were fig. .-the patient aged years. note increased lumbar lordosis and fine sparse hair. short and broad, and it was not possible to extend fully either elbow. the sternum was abnormally prominent, and there was marked bowing of the legs. no hyper- extensibility of the joints could be shown. his intellectual development was considered to be within normal limits for his age. investigations. x-rays. the skeleton showed generalized changes of typical metaphyseal dysostosis, i.e. widening, cupping, and defective mineralization in the metaphyses of the tubular bones (fig. ). fig. .-x-ray of left lower limb of patient showing widening, cupping, and defective mineralization of the metaphvses. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n n o ve m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ short reports hair. amino acid analysis of the scalp hair (dr. r. j. pollitt) gave normal results. electron microscopical examination (dr. r. p. r. dawber) showed that the majority of the hairs had a diameter approximately i of normal, but the cuticular scale pattern was within normal limits. biochemical. the following were performed with normal results: lipid tolerance test, microscopical examination for fat globules in stools, blood urea and electrolytes, urinary amino acid chromatography, and mucopolysaccharide screening test. discussion the main features of the syndrome are (i) short limbed dwarfism with bow legs and increased lumbar lordosis; (ii) minor skeletal abnormalities of anterior protrusion of the sternum, hyper- extensibility of joints, limitation of extension of elbows; (iii) fine sparse hair; and (iv) autosomal recessive inheritance. associated features in some patients are hirschsprung's disease, malabsorption syndrome, and poor resistance to some virus infections. skeletal disorder. cartilage hair hypoplasia is one of several disorders in which the abnormality of the growth plate known as metaphyseal dysostosis leads to short stature (irwin, ). the other main disorders are ( ) jansen type; rare; associated with mental and motor retardation; pattern of inheritance unknown. ( ) schmidt type; dwarfism mild; skeletal deformities slight; intelligence average; an autosomal dominant. ( ) spahr type; similar to schmidt type but an autosomal recessive. thus cartilage hair hypoplasia differs from these disorders in the markedly short stature, and differs from the jansen type in the absence of severe intellectual deficit. the localized skeletal abnormalities present in cartilage hair hypoplasia (smith, ), i.e. short broad hands, lack of full extension of elbow joints, prominence of the sternum with flaring of the bony rib cage, bow legs, and increased lumbar lordosis, may be present in the other varieties. hair. the other striking feature which distinguishes this from the other types of meta- physeal dysostosis is the very short, fine, sparse scalp hair. coupe and lowry ( ) have analysed hair from typical cases and found a normal amino acid content, but a thin structure with lower tensile strength and the lack of a central pigment core, which is present in normal hair. the hair from our patient shows the first two features. associated alimentary tract disorders. in the report of mckusick et al. ( ) one of the patients died of coeliac disease and in others malabsorption was suspected. there was no evidence of malabsorption in our patient, though full investigation to exclude this was not carried out. two of mckusick's patients had hirschsprung's disease, which was the presenting feature in our case. associated severe viral infections. of mckusick's patients died of chickenpox and others were so severely affected that a diagnosis of smallpox was seriously considered. lux et al. ( ) extensively investigated children with cartilage hair hypoplasia who had severe varicella and found them to have chronic neutropenia and disordered cellular immunity. burke et al. ( ) mentioned patients with exocrine pancreatic insufficiency and neutropenia who had metaphyseal dysostosis (cases and ) and one who had hirschsprung's disease. no mention was made of the state of the hair. our patient has so far had no clinical evidence of a poor response to infection. this is another of the many rare genetic disorders in which correct diagnosis is advantageous to the patient and his family even though the condition itself is untreatable, as the parents can be informed of the poor prognosis for linear growth but the fairly good prognosis for intellectual development, and the genetic implications for further sibs can be explained. the physician will be aware of the possibility of malabsorption, hirschsprung's disease, and severe illness with viral infection. summary a patient with cartilage hair hypoplasia associated with hirschsprung's disease is described. the syndrome is a rare cause of short-limbed dwarfism, and is inherited as an autosomal recessive. apart from small stature, the main features are fine, sparse scalp hair and characteristic radiological changes of metaphyseal dysostosis. we are grateful to drs. r. j. pollitt (university of sheffield, department of psychiatry), r. p. r. dawber (st. john's hospital for diseases ofthe skin), t. j. ryan, consultant dermatologist, and j. c. mclamon, consultant radiologist (united oxford hospitals) for their help; and thank mr. malcolm gough, consultant surgeon (united oxford hospitals) for information on the surgical aspects of this patient. references burke, v., colebatch, j. h., anderson, c. m., and simons, m. j. ( ). association of pancreatic insufficiency and chronic neutropenia in childhood. archives of disease in childhood, , . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n n o ve m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ short reports coupe, r. l., and lowry, r. b. ( ). abnormality of the hair in cartilage-hair hypoplasia. dermatologica, , . irwin, g. a. l. ( ). cartilage-hair hypoplasia (chh) variant of familial metaphyseal dysostosis. radiology, , . lux, s. e., johnston, r. b., august, c. s., say, b., penchaszadeh, v. b., rosen, f. s., and mckusick, v. a. ( ). chronic neutropenia and abnormal cellular immunity in cartilage-hair hypoplasia. new england journal of medicine, , . mckusick, v. a. ( ). metaphyseal dysostosis and thin hair; a 'new' recessively inherited syndrome ? lancet, , . mckusick, v. a., eldridge, r., hostetler, j. a., ruangwit, u., and egeland, j. a. ( ). dwarfism in the amish. ii. cartilage-hair hypoplasia. bulletin of the johns hopkins hospital, , . savage, m. . ( ). metaphyseal dysplasia in siblings: a variant of cartilage-hair hypoplasia. proceedings of the royal society of medicine, , . smith, d. w. ( ). recognizable patterns of human mal- formation, p. . saunders, philadelphia and london. c. b. boothby* and b. d. bower department of paediatrics, radcliffe infirmary, oxford. *correspondence to dr. c. b. boothby, west suffolk general hospital, bury st. edmunds, suffolk ip jx. the following articles will appear in future issues of this journal: looking back: bray's discovery of pathogenic esch. coli as a cause of infantile gastroenteritis. j. bray. personal comment: paediatrics and child psychiatry: the case for collaboration. p. pinkerton. assessment of techniques for measurement of blood pressure in infants and children. a. m. elseed, e. a. shinebourne, and m. c. joseph. exercise-induced bronchial lability and atopic status of families of infants with wheezy bronchitis. p. konig and s. godfrey. sex chromosome aberrations and speech development. m. garvey and d. e. mutton. serum protein and transferrin determinations to distinguish kwashiorkor from iron deficiency anaemia. a. e. j. masawe and j. rwabwogo-atenyi. diaphragmatic paralysis in the newborn. d. anagnostakis, e. economou-mavrou, a. moschos, p. vlachos, and d. liakakos. treatment with new synthetic analogue of vasopressin in diabetes insipidus. h. nash. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n n o ve m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ valence band and core-level analysis of highly luminescent zno nanocrystals for designing ultrafast optical sensors amish g. joshi, sonal sahai, namita gandhi, y. g. radha krishna, and d. haranatha� national physical laboratory, council of scientific and industrial research, dr. k. s. krishnan road, new delhi , india �received december ; accepted february ; published online march � the detailed surface analysis such as survey scan, core-level, and valence band spectra of highly luminescent zno:na nanocrystals were studied using the x-ray photoelectron spectroscopy to establish the performed presence of na+ ions. the observed increase in band gap from . �bulk� to . ev �nano�, is attributed to the quantum confinement of the motion of electron and holes in all three directions. the photoluminescence and decay measurements have complemented and supported our study to design an efficient and ultrafast responsive optical sensing device. © american institute of physics. �doi: . / . � sensors or detectors for various kinds of optical radia- tions are known in the literature , but they are deficient in many ways. for example, the photoresponse of known semi- conductor sensors in ultraviolet �uv� and x-ray radiation range is limited due to nonradiative surface recombinations. the radiation with energy greater than few hundred mega- electron-volt above the band gap will be absorbed � nm deep near the surface. the photogenerated free carriers due to the absorption recombine nonradiatively near the surface of the semiconductor. however, in the visible/infrared re- gion the absorption length of the radiation is significantly larger and the recombination occurs deeper in the material away from the surface. the carrier recombination at the sur- face could be avoided either chemically passivating the sur- face or using heterojunction schemes, which is evidenced in many iii–v semiconductors devices. but the above two schemes fail if the incident radiation is of very high energy such as uv, x-ray, synchrotron radiation etc. hence, there is a serious pursuit globally for newer materials that are radia- tion sensitive, have higher brightness that should match commonly-used photodetector, minimum self-absorption, re- duced light scattering, and very short recombination time, suitable for designing ultrafast optical sensors. phosphors are the best materials to detect uv radiation but their prac- tical usage for sensing has been limited due to their longer decay/lifetimes and light scattering. however, nanophos- phors of the same kind could very well satisfy the prerequi- sites of an optical sensor due to their highest brightness lev- els and shortest possible lifetimes dictated by their sizes. a combination of an efficient nanophosphor and a known op- toelectronic detector could yield an advanced optical sensor with increased signal to noise ratio, is the main objective of our work. high brightness zno nanophosphors having size � nm were the best choice in the current study as they exhibit two luminescence bands in the uv and green regions of the electromagnetic spectrum. this makes the material versatile to be used to design an efficient optical radiation detector covering a broad wavelength range. uv emission from zno is caused by band-to-band and exciton transitions whereas; the origin of green photoluminescence �pl� band is still on debate. – spatial confinement of charge carriers could be the possible reason for substantial increase in the photoluminescence. considering this property in view we investigated the surface states of alkali doped-zno nanocrys- tals �ncs� by surface sensitive spectroscopic techniques to design an efficient nanosecond optical sensor. an improved sol-lyophilization process was used for the synthesis of ultrafine zno:na ncs with no surfactant. this method allows for the synthesis of ultrafine nanocrys- tals with no surfactant. the details and the associated advan- tages of the process are described elsewhere. in a typical experiment ethanolic stock solutions of zinc acetate and so- dium hydroxide �naoh� were allowed to react at room tem- perature �� ° c�. the nucleation and growth rate of the zno ncs were monitored by adjusting the ph, relative hu- midity and the reacting temperatures. the ncs were precipi- tated out by adding a low relative polarity solvent such as n-hexane, washed and dried below ° c. in another experi- ment, calculated amount of high � . %� purity zno powder �from sigma-aldrich� was mixed with naoh and annealed in air for h at ° c to form a reference bulk-zno:na sample. these two representative bulk and nano-zno:na samples were chosen for the present study. figure shows the x-ray diffraction �xrd� pattern of the zno:na nc powder and from the line broadening the particles are estimated to be ultrafine and have wurtzite phase. the inset of fig. portray the transmission electron microscopy �tem� image taken at a magnification of kx and the photograph of a vial containing highly luminescent zno:na nc powder kept under uv � nm� radiation. a�electronic mail: haranath@nplindia.org. fig. . �color online� powder xrd pattern of zno:na nc’s. insets show the tem micrograph and a vial containing zno:na nc powder irradiated under uv � nm� light. applied physics letters , � � - / / � �/ / /$ . © american institute of physics , - downloaded sep to . . . . redistribution subject to aip license or copyright; see http://apl.aip.org/about/rights_and_permissions http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . incorporation of na+ in zno samples were quantitatively determined by atomic absorption spectroscopy and for bulk and nano-zno:na, it was estimated to be . % and . %, respectively, with an error of � %. further, the values were compared to the results from inductively coupled plasma atomic emission spectroscopy reported by kshirsagar et al. using x-ray photoelectron spectroscopy �xps� and core-level spectra we tried to characterize the sur- face of zno:na samples at � � − torr with a non- monochromatized al k� x-ray source. survey spectra in fig. shows sharp peaks of c s � . ev�, o s � ev�, zn� p / �, and zn� p / � at and ev; whereas peaks related to na were observed at na� s� � ev�, na� s� � ev�, and na� p� � ev�. the weak peaks ob- served for ncs indicate their poor crystallinity. the core level xps spectra of zn, o, and na have shown symmetric profiles depicting uniform bond structure and emphasize that both bulk and nc’s do exist in same phase. the core level spectra �fig. �a�� of zn shows two distinct zn� p / � and zn� p / � states at . �nano� and . ev �bulk�; and at . �nano� and . ev �bulk�, respectively. the separation of the states in ncs and bulk is and . ev, respectively, and the slight shift in binding energies are due to variation from site to site substitution of na in zno sys- tems. figure �b� shows o� s� core level spectra. the peaks x and x� observed at . and . ev are the contri- butions from oxygen of zno and partially from na–o–na bonding. interestingly there is an appearance of shoulder at higher binding energies � ev�, which is denoted as y and y� for bulk and nano-phases, which is attributed to the pres- ence of oh group at zno surface. the peak intensity of shoulder peak y� is higher than that of y, which clearly manifests the adsorption of more oh groups in nanophase due to larger surface to volume ratio. our results agree very fig. . �color online� xps survey scan spectra of bulk and nc’s of zno:na recorded with a photon energy of al k� �hv = . ev�. fig. . �color online� core level xps spectra of �a� zn �b� o and �c� na in bulk and nc’s of zno:na acquired at the pass energy of ev. fig. . �color online� vb xps spectra of bulk and nc’s of zno:na ac- quired at the pass energy of ev. fig. . �color online� room temperature photoluminescence �pl� and pho- toluminescence excitation �ple� spectra for bulk and nc’s of zno:na. - joshi et al. appl. phys. lett. , � � downloaded sep to . . . . redistribution subject to aip license or copyright; see http://apl.aip.org/about/rights_and_permissions well with ab initio calculations where x and x� are related to bridging oxygen, whereas y and y� to central oxygen of zno structure. figure �c� shows na � s� high resolution spectra of bulk and ncs which appear almost symmetric and further establishes the presence of na+ ions. figure shows the valence band �vb� xps spectra of zno:na and the weak peaks around fermi level, ef, are a kind of non-local states in the band gap possibly caused by defects. peak a and a� observed at � ev involves o p orbitals and the part of o p orbitals hybridized with zn s ones. peak b and b� for bulk and nano observed at � and . ev, respectively, is mainly attributed to the zn d band. in nc, the zn d moves to lower energy by � . ev with respect to bulk. in peak regions of a and a�, the uppermost valence-band edges �vbe� could be easily separated out. the vbe was linearly fitted to determine the shift, �ev, as shown by diagonal lines in fig. . the tangent to the fitted curve was drawn and intersect is defined as vbm. it is evi- dent from fig. that the vbm of nano-zno shifts toward higher binding energy by � . ev than bulk-zno. the band gaps estimated are . and . ev for bulk and nano- zno:na, respectively, which are in excellent agreement with the values derived from xrd and tem observations. since the spectroscopic methods such as optical absorp- tion, photoluminescence excitations etc., are accurate to determine the crystallite band gap, we performed detailed luminescence studies. figure shows the typical photolumi- nescence excitation �ple� and pl spectra of ncs and bulk phosphor samples. pl studies showed a clear superiority of brightness levels in zno:na ncs over their bulk counterpart. the zno:na ncs synthesized by the present method exhibit a distinct excitation peak in the uv region at � nm � . ev�, when monitored at nm, which is a clear indi- cation of smaller size of the ncs. however, the bulk phos- phor having larger crystallite sizes, when monitored at nm, exhibited the excitation peak around nm � . ev� that corresponds to bulk band gap of zno system. the dif- ference in ple peak position �or the band edge� for nc and bulk-zno:na samples is mainly due to different mean sizes. the broad green emission at � nm is weakly observed in the bulk-zno:na and is assigned to defect levels associ- ated with oxygen vacancies or zinc interstitials, , whereas the confinement effects dominate the luminescence phenom- enon in zno ncs. there could be many interdependent fac- tors, such as electron-phonon coupling, lattice dislocation, localization of charge carriers due to interface effects and point defects that are incrementing the pl emission. since, the sizes observed for zno ncs are � nm, one more de- gree of freedom along with size dependency exists leading to intense ple and pl intensities over their bulk counterparts. this is the property related to the sensitivity where most of the optical sensors lack in the uv to visible region. added that the performance of an optical sensor would be optimum if the incident photon energy is transferred at a faster rate from host crystal to dopant. to check the possibility of using zno:na ncs for designing ultrafast uv sensor, we have investigated the dynamics of bound excitons for bulk and nano-zno:na powders at their peak wavelengths i.e., at nm emission and nm excitation for bulk; and nm emission and nm excitation for ncs, using a time cor- related single photon counting technique. table i shows the results of exciton lifetimes of both bulk and nano-zno:na powders that varied from few milli to nanoseconds, respec- tively. the lifetime components �fast and slow� are calcu- lated at the / th part of the initial decay curves for bulk and zno:na nc. it is inferred that size dependency and quantum confinement are responsible for faster decay com- ponents and donor-acceptor pair type of recombination for longer lifetimes, are for bulk materials. in summary, the surface states of highly luminescent zno:na ncs were studied using xps. the vbm of ncs shift by � mev from fermi energy level as compared to that of bulk-zno:na, which is due to the quantum confine- ment effects aroused due to size restrictions. the fastest rate of energy transfer from host to the activator in zno:na ncs suggest the strong possibility of designing advanced uv sen- sor that could be operated in nanoseconds. the authors �ng, ygrk� acknowledge csir and ugc, india for their fellowships. t. a. dickinson, j. white, j. s. kauer, and d. r. walt, nature �london� , � �. j. w. gardner and p. n. bartlett, sens. actuators b - , � �. c. j. murphy, anal. chem. , a � �. a. k. burrell, k. c. ott, j. c. gordon, r. e. del sesto, and t. m. mccles- key, us patent no. / a � �. d. haranath, s. sahai, and p. joshi, appl. phys. lett. , � �. s. cho, j. ma, y. kim, y. sun, g. k. l. wong, and j. b. ketterson, appl. phys. lett. , � �. c. m. mo, y. h. li, y. s. liu, y. zhang, and l. d. liu, j. appl. phys. , � �. e. g. bylander, j. appl. phys. , � �. r. n. bhargava, d. gallagher, x. hong, and a. nurmikko, phys. rev. lett. , � �. d. haranath, s. sahai, a. g. joshi, b. k. gupta, and v. shanker, nano- technology , � �. s. kumar, v. gupta, and k. sreenivas, nanotechnology , � �. r. n. bhargava, d. haranath, and a. mehta, j. korean phys. soc. , � �. v. chhabra, b. kulkarni, and r. n. bhargava, us patent no. � march �. s. d. kshirsagar, v. v. nikesh, and s. mahamuni, appl. phys. lett. , � �. k. kotsis and v. staemmler, phys. chem. chem. phys. , � �. m. kunat, st. gil girol, u. burghaus, and ch. wöll, j. phys. chem. b , � �. table i. the results pertaining to exciton lifetimes of both bulk and nano-zno:na powders are shown below. s. no. size determination using ple peak wavelength �nm� exciton lifetime components referencexrd tem ple fast slow . nm . nm . nm . ns . ns this study . nm . nm . nm . ns ns . nm . nm . nm ns ns . nm . nm . nm �s �s � �m � �m � �m ms ms this study - joshi et al. appl. phys. lett. , � � downloaded sep to . . . . redistribution subject to aip license or copyright; see http://apl.aip.org/about/rights_and_permissions http://dx.doi.org/ . / a http://dx.doi.org/ . /ac v http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . /physrevlett. . http://dx.doi.org/ . /physrevlett. . http://dx.doi.org/ . / - / / / http://dx.doi.org/ . / - / / / http://dx.doi.org/ . / - / / / http://dx.doi.org/ . /jkps. . http://dx.doi.org/ . / . http://dx.doi.org/ . /b h http://dx.doi.org/ . /jp z homozygous mutation of mtpap causes cellular radiosensitivity and persistent dna double-strand breaks open homozygous mutation of mtpap causes cellular radiosensitivity and persistent dna double-strand breaks nt martin*, , , k nakamura , u paila , j woo , c brown , ja wright , sn teraoka , s haghayegh , d mccurdy , m schneider , h hu , ar quinlan , ra gatti , , and p concannon , the study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in dna damage responses to ionizing radiation. in the present study, a mutation in mitochondrial poly-a-polymerase (mtpap), not previously recognized for its role in the dna damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. cell lines derived from two patients with the homozygous mtpap missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtpap cdna into mtpap-deficient cell lines. further analysis of the cellular phenotype revealed delayed dna repair, increased levels of dna double-strand breaks, increased reactive oxygen species (ros), and increased cell death after irradiation (ir). pre-ir treatment of cells with the potent anti-oxidants, a-lipoic acid and n-acetylcysteine, was sufficient to abrogate the dna repair and clonogenic survival defects. our results firmly establish that mutation of the mtpap gene results in a cellular phenotype of increased dna damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ros homeostasis. cell death and disease ( ) , e ; doi: . /cddis. . ; published online march subject category: cancer xcind syndrome (x-ray sensitivity, cancer predisposition, immunodeficiency, neurologic involvement, and dna double- strand break repair deficiency) is a pleiotropic syndrome of radiosensitivity disorders resulting from mutation of dna damage recognition and repair genes. , ataxia-telangiecta- sia (a-t) is the archetypal xcind syndrome disorder and results from mutation of the ataxia-telangiectasia-mutated (atm) protein kinase gene. a-t patients manifest both cellular and clinical hypersensitivity to ionizing radiation, which results from disruption of the nuclear function of atm in the recognition and response to dna double-strand breaks (dsbs). , additional radiosensitivity disorders have been identified since a-t was first characterized, and, to date, have involved mutations in dna dsb recognition or repair genes. , recognition and repair of dsbs is a complex and dynamic process involving at least two main repair pathways, non-homologous end joining (nhej) and homologous recombination (hr) repair. the mrn complex (meiotic recombination homolog (s. cerevisiae), rad , and nijmegen breakage syndrome (nbs )) and atm are initial sensors of dsbs and are recruited to nuclear foci surrounding breaks. loss of a single member of the mrn complex results in cellular radiosensitivity and clinical features that overlap with a-t. – recognition of dsbs by the mrn complex and atm initiates a signaling cascade leading to the activation of cell cycle checkpoints and the recruitment of other repair proteins, such as h a histone family, member x, phosphory- lated serine (g-h ax) and p -binding protein ( bp ), to the sites of dsbs. – g-h ax forms nuclear foci that persist throughout the repair process and disengage once the repair process has been completed. the initial recruitment of mrn, atm, and g-h ax is followed by recruitment of the bp repair protein via a chromatin ubiquitin ligase cascade. – bp ucla department of pathology and laboratory medicine, macdonald research laboratories, los angeles, ca, usa; ucla biomedical physics interdepartmental graduate program, los angeles, ca, usa; department of public health sciences, center for public health genomics, university of virginia, charlottesville, va, usa; genetics institute, university of florida, gainesville, fl, usa; ucla department of pediatrics, los angeles, ca, usa; carle physician group, urbana, il, usa; ucla department of human genetics, los angeles, ca, usa and department of pathology, immunology and laboratory medicine, university of florida, gainesville, fl, usa *corresponding author: nt martin, ucla department of pathology and laboratory medicine, macdonald research laboratories, charles e young dr. south, room - , los angeles, ca - , usa. tel: + ; fax: + ; e-mail: namartin@mednet.ucla.edu received . . ; revised . . ; accepted . . ; edited by a finazzi-agró keywords: radiosensitivity; mtpap; dna repair; sequencing; reactive oxygen species; dna damage abbreviations: bp , p -binding protein ; a-t, ataxia-telangiectasia; a-la, a-lipoic acid; atm, ataxia-telangiectasia mutated; brca , breast cancer , early onset; csa, clonogenic survival assay; dchf-da, dichloro-dihydro-fluorescein diacetate; ddr, dna damage response; dhe, dihydroethidium; dsb, dna double-strand break; fccp, protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; g-h ax, h a histone family, member x, phosphorylated serine ; gy, gray; hr, homologous recombination; ir, irradiation (ionizing); indel, insertion-deletion; lcl, lymphoblastoid cell line; mrn, protein complex; mre , nbs , rad ; mtpap/ mtpap, mitochondrial poly-a-polymerase; nac, n-acetyl cysteine; nbs , nijmegen breakage syndrome ; nca, neutral comet assay; nhej, non-homologous end joining; oligo, oligomycin; rad , rad homolog (s. cerevisiae); rad , rad recombinase; ros, reactive oxygen species; sf%, survival fraction percentage; smc , structural maintenance of chromosomes ; tm, tail moment; wt, wild type; xcind, x-ray sensitivity, cancer predisposition, immunodeficiency, neurologic involvement, dna repair defect citation: cell death and disease ( ) , e ; doi: . /cddis. . & macmillan publishers limited all rights reserved - / www.nature.com/cddis http://dx.doi.org/ . /cddis. . mailto:namartin@mednet.ucla.edu http://www.nature.com/cddis is important for modulating the choice of repair pathway between nhej, the dominant dsb repair mechanism, and hr, an error-free repair mechanism active in late s and g phase cells because it requires homologous chroma- tids. , when bp is displaced from the sites of dsbs by the breast cancer susceptibility protein, brca (breast cancer , early onset), end resection via ctbp-interacting protein (ctip) and the mrn complex occurs to initiate homologous recombination repair. – recently, reduction of ctip levels was shown to disrupt brca focus accumulation leading to dsb repair defects and cellular radiosensitivity. the dna repair protein rad recombi- nase (rad ) is also recruited to the sites of dsbs after brca localization when hr repair is occurring. , although much of what is known about the cellular response to ionizing radiation is focused on molecules that accumulate at the sites of damage, the cellular environment would also be expected to have an important supporting role in facilitating these responses and maintaining genomic stability. ionizing radiation generates dsbs via direct interaction with dna. many more breaks are generated from reactive oxygen species (ros) that also cause oxidative and mitochondrial stress. , cellular ros homeostasis and atp levels are required for supporting dna repair, and protecting against dsbs generation, and mitochondria are the main mediators of these levels. – in addition, mitochondria act as central modulators of apoptotic cell death, which has an important role in removing cells with persistent or oncogenic dna lesions. , thus, mitochondria would be expected to have a central role in the dna damage response (ddr). several studies focusing on mutations in mitochondrial genes have indicated dysfunction of mitochondrial atp production, dysregulation of dsb repair gene expression, and genomic instability. , – however, the exact mechanism by which mitochondrial dysfunction, such as that induced by mutation of genes important for mitochondrial function, can reduce survival after radiation exposure remains unclear. – in the present study, exome sequencing identified a homozygous a g dna variant encoding the missense change n d in the mitochondrial poly-a-polymerase (mtpap) gene in two siblings of an amish family affected with spastic ataxia. lymphoblastoid cell lines (lcls) derived from these siblings were radiosensitive, based on colony survival fraction post-irradiation (ir). although the homozy- gous a g mtpap variant was previously associated with spastic ataxia in an amish pedigree, the listed features of the affected individuals did not include radiosensitivity. therefore, the association of homozygous a g with sensitivity to ionizing radiation was tested by transfecting the patient-derived lcls with wild-type (wt) mtpap and assaying clonogenic survival. the mtpap variant lcls were also assessed for levels of dna damage and repair, ros, mitochondrial respiration, and apoptosis in order to elucidate pathways by which mtpap influences cellular responses to ionizing radiation. results radiosensitivity segregated with bi-allelic mtpap mutations in rs family. two affected siblings from a consanguineous amish family were tested for reduced colony survival levels post-ir, a radiosensitive phenotype, because they displayed a clinical phenotype similar to a-t. both affected siblings, rs - and rs - , were characterized by ataxia with initiation tremors. growth failure, developmental delay, and spasticity were also noted, whereas no immune deficiencies or tumors have been observed to date. there was at-like reduced colony survival in rs - and - . unaffected siblings (rs - , - , - ) had normal colony survival post-ir (figure a). the neutral comet assay (nca) is another useful method for assessing dna dsb repair fidelity and correlates well with the clonogenic survival assay (csa). rs - and - demon- strated an at-like dsb repair defect, consistent with the csa result, and the unaffected siblings of the family were found to have normal levels of dna repair (figure b). the at-like cellular and clinical phenotype strongly suggested a diagnosis of a-t, however, atm protein expression was normal. exome sequencing was employed figure cellular radiosensitivity and dna repair defect segregated with affected members of rs family with a homozygous mutation of mtpap. (a) the survival fraction (sf%) measured by the csa (at gy) is reduced in the two members of the rs family, rs - and - , who are homozygous for the mtpap mutation. the ‘#’ indicates sf% representative of wt and highly radiosensitive (a-t) cells used for comparison in clinical radiosensitivity testing based on sun et al. the ‘$’ denotes patients for whom we were not able to generate an lcl. the ‘&’ denotes that we were unable to obtain dnas for mtpap sequencing analysis; however, the father is an obligate heterozygote. (b) the neutral comet assay, measuring predominately dna dsbs, reveals an at-like dna repair defect in rs - and - . results are displayed as percent dna repair (% repair) post- gy and represent the return of comet tails to baseline levels at h post-ir. an asterisk denotes statistical significance at po . mtpap mutation causes radiosensitivity nt martin et al cell death and disease as an untargeted approach to identify candidate causative mutations in other genes. at the time of initial sequencing, only an lcl derived from rs - was available. comparison of the rs - exome sequence to the human reference sequence confirmed the absence of atm mutations, but identified coding variants relative to the reference, of which were unique to rs - . based upon the consanguinity in the rs family, the variants were filtered for homozygosity, yielding coding variants. variants predicted to be deleterious because they would result in gain or loss of a stop codon, a frameshift or a non-synonymous substitution at a conserved amino-acid position, and were not observed in either the nhlbi exome sequencing project or the genomes project were identified. this yielded hornerin and mtpap as candidate variants. these two genes were manually examined to determine which might have a role in the ddr or in cerebellar development or function. the homozygous missense mutation (c. a g, n d) in the mtpap gene was singled out because it had been previously associated with spastic ataxia and optic atrophy and was the only one of the two possible variants predicted to be deleterious by polyphen. , the a g mtpap mutation, and lack of atm mutations, was later confirmed in rs - once a cell line could be derived. the unaffected siblings (rs - , - , - ) and mother were heterozygous for the mtpap mutation, suggesting autosomal recessive inheritance (supplementary figure ). mutation of mtpap was causally linked to the radiosensitive and persistent dna damage phenotype by mtpap transfection. mtpap is nuclear encoded, poly-adenylates mitochondrial transcripts, and, unlike nuclear polyadenylation polymerases, has a variable affect on transcript stability. , , thus, it was unclear how mutation of mtpap might result in radiosensitivity. the at-like radiosensitivity (figure a) and dna repair defect (figure b and supplementary figure ) observed in rs - and - were rescued when wt mtpap cdna was transfected into the patient’s lcls, confirming a causal link between mtpap, cellular radiosensitivity, and defective dna repair. increased post-ir dna dsb induction in rs - and - . the similarities in clinical phenotype between rs - and rs - and a-t patients led us to consider that mutation of mtpap might impact the atm signaling cascade. we screened a panel of atm kinase activity biomarkers, including atm autophosphorylation and downstream phosphorylation of structural maintenance of chromosomes (smc ), krab-associated protein (kap ), and nbs , which are necessary for detection of dsbs and transmission of the ddr signal. , , , phos- phorylation of atm, smc , kap , and nbs in cell lysates from rs - and - was wt-like post-ir, indicating that mtpap does not likely impact early atm kinase signaling (data not shown). we have shown that monitoring g-h ax, bp , and brca foci kinetics post-ir can be used to characterize ddr defects in radiosensitive patients of unknown etiology by comparing kinetic curves to those of known radiosensitivity disorders. rs - and - displayed normal basal levels of g-h ax foci and normal repair kinetics at , , and h; however, the h post-ir response was increased in both lcls, suggesting increased levels of dsb induction (figure a). similarly, increased dsb levels post-ir in rs - and - were noted by the nca (figure b). increased levels of bp foci were also observed h post-ir and persisted at h (supplementary figure a). finally, brca and rad foci were assessed as markers of hr. , a trend toward reduced brca foci- positive cells, compared with wt, at h was observed followed by a significant reduction in foci numbers at h post- ir, suggesting either a defect in recruitment of brca to dsbs or a shift away from initiating hr (supplementary figure b). consistent with reduced hr repair, rad foci were also reduced at h post-ir (supplementary figure c). hr is only active in the late s and g phases of the cell cycle and, thus, we hypothesized that cell cycle perturbations might impact the reductions observed in brca and rad foci numbers at h. cell cycle distribution was assessed in rs - and - and was found to be wt-like before and after ir (supplementary figure ). similarly, the fidelity of the s-phase and g /m phase checkpoints were wt-like (supplementary figures and ). thus, our data suggest that the reductions in brca and rad foci were not due to figure transfection of wt mtpap into rs - and - rescued radiosensitivity and dna repair defects. (a) transfection of wt mtpap cdna ( þ ) into rs - and - rescued the radiosensitive (i.e., reduced sf%) phenotype measured by the csa (at gy). a ( þ ) denotes transfection with wt mtpap cdna and ( � ) denotes transfection with an empty vector. (b) wt mtpap ( þ ) or control plasmid ( � ) was transfected into rs - and - ; the wt mtpap plasmid rescued the dna dsb repair defect as measured by the nca (at gy). ‘% dna repair’ represents the return to baseline tail moment (tm) measure of dna damage h post-ir and is the tmno ir/tm h. an asterisk denotes statistical significance at po . mtpap mutation causes radiosensitivity nt martin et al cell death and disease large differences in cell cycle distribution, nor checkpoint activation, and that cell cycle control was not significantly impacted by mutation of mtpap. normal mitochondrial respiration, increased ros levels, and increased apoptosis in rs - and - post-ir. mitochondria are closely involved in the ddr by at least three mechanisms: ( ) maintaining ros homeostasis, ( ) mediating apoptotic signals, and ( ) producing energy needed for dsb repair. , owing to the known function of mtpap in the mitochondria, we postulated that mutation of mtpap might result in dysfunctional mitochondria and subsequent disruption of the ddr. mitochondrial respiration levels were assessed with the rezasurin dye. contrary to our working model, basal mitochondrial respiration was normal in rs - and - (figure a). mitochondrial bioenergetics were further probed by measuring the oxygen consumption rate after addition of oligomycin (oligo; atp synthase inhibitor), fccp (protonophore carbonyl cyanide p-(trifluoromethoxy)- phenylhydrazone; respiratory chain uncoupler), and rotenone/myxothiazol (r/m; mitochondrial respiration inhibi- tors). basal respiration (time – ), atp-linked respiration (time – ), and total mitochondrial respiration levels (time – ) were largely similar to wt levels in rs - and - . however, maximal mitochondrial respiration levels (time – ), induced by addition of fccp, were greatly increased in rs - and - (figure b). transfection of wt mtpap cdna into rs - and - partially abrogated the increased maximal respiration after addition of fccp phenotype (time - ), and returned respiration levels closer to wt levels (supplementary figure ). abnormal ros levels have been linked to reduced colony survival and dsb repair, and mitochondria are known to be the main producers, and reducers, of many ros. , dichloro-dihydro-fluorescein diacetate (dchf-da), a dye that can react to a broad array of ros moieties, was used to assess ros levels in rs - and - . increased ros levels were observed in rs - and - post-ir, similar to those of a-t lcls (figure c and supplementary figure a). these data would suggest that the antioxidant responses in rs - and - were not functioning optimally, post-ir. however, given the increased maximal respiration in rs - and - , we postulated that some of the ros may be entering the cytosol through leaky or dysfunctional mitochondria. superoxide is the main ros moiety that could leak from dysfunctional respiration and dihydroethidium (dhe) dye, a cell permeable dye preferentially detecting superoxide radicals, was used to assess superoxide levels. increased basal superoxide levels were observed in rs - and - , further supporting the notion of mitochondrial dysfunction (supplementary figure b). superoxide levels were not significantly induced in wt-lcls post- gy, but there were significant increases in superoxide levels post-ir in rs - and - (supplementary figure b). transfection of wt mtpap cdna into rs - and - partially abrogated the increased basal superoxide levels observed in rs - and - , and rescued the ir-induced increase in superoxide levels (supplementary figure c). owing to technical limitations (i.e., fluorescent spectra overlap between the gfp tag for the cdna constructs and dchf-da), we were unable to also demonstrate rescue of the ir-induced ros levels with this more general dye for ros in rs - and - . consistent with cells undergoing oxidative stress, increased apoptosis levels were found in rs - and - post-ir (figure d). antioxidants, a-lipoic acid (a-la) and n-acetylcyseine, rescued mtpap-deficient cellular radiosensitivity and dna repair phenotype. the increased levels of ros were intriguing, but it was unclear whether this contributed to the cellular phenotype of rs - and - . to test this, a-la, a potent antioxidant, was used to lower ros levels. g-h ax foci levels were assessed at -h post-ir to determine whether increased levels of foci in rs - and - could be returned to wt levels by a-la. indeed, pre-treatment with a-la at concentrations mm reduced g-h ax foci levels to wt levels (figure a). interestingly, a-la treatment had no effect on g-h ax foci levels in wt-lcls at the concentrations tested. when assessed by the nca, the increased levels of dsbs initially induced post-ir in rs - and - were also abrogated by pre-ir treatment with a-la, confirming protection against elevated ir-induced damage (supplementary figure ). the dna repair defect measured figure increased induction of dna damage post-ir in rs - and - . (a) g-h ax irradiation-induced foci (irif) were increased at h (post- gy) in rs - and - compared with wt levels. the results are expressed as the percentage of cells positive for four or more foci/nuclei (% pos). wt is a representative wt response. asterisks denote statistical significance at po . . (b) dna dsb levels were assessed immediately post- and - gy. increased levels of dsbs were observed in rs - and - . asterisks indicate statistical significance at the po . level comparing rs - and - (n ¼ , each) to both wt and wt (n ¼ , each) mtpap mutation causes radiosensitivity nt martin et al cell death and disease by the nca was also rescued in rs - and - by pre-ir treatment with mm a-la (figure b). further, pre-ir a-la treatment rescued the radiosensitive phenotype, but had no affect on wt or a-t lcls (figure c). the lack of survival fraction percentage (sf%) improvement with a-la treatment in a-t lcls is consistent with previous findings and further elucidates the differential dependency of radiosensitivity and ros levels in wt, a-t, and mtpap-deficient lcls. recently, however, a-la has been shown to modulate atp synthase levels and mitochondrial dna content, which might also modulate ddrs. to rule out phenomena specific to a single antioxidant, a-la, we tested whether n-acetylcis- teine (nac), another potent antioxidant, could also abrogate the radiosensitive cellular phenotype in rs - and - . pre-treatment of cells with nac protected against increased dna dsb induction (supplementary figure a), rescued the delay in dna repair (supplementary figure b), and restored clonogenic survival to wt levels in rs - and - (supplementary figure c). discussion we describe two amish siblings (rs - and - ) with a homozygous mtpap mutation that had been previously associated with spastic ataxia in an old order amish population and can now be causally linked to radiosensitivity and modulation of the ddr. identification of mtpap as a candidate gene responsible for the defects observed in these patients was initially enabled by exome sequencing of dna from these patients. however, as is common with exome sequencing, many variants were identified that were poten- tially disease causing. with the expectation of homozygosity in the disease-causing variant, the variant list was narrowed and led to selection of the n d missense mutation in mtpap as a candidate radiosensitivity gene because it was previously shown to segregate with spastic ataxia in the old world amish population; a similar phenotype to rs - and - . this would be the first radiosensitivity associated gene identified by exome-sequencing approaches in patients. colony survival testing indicated that lcls derived from rs - and - were radiosensitive, a new clinical phenotype for spastic ataxia associated with mtpap mutation. the nca further indicated a dna repair defect in rs - and - . crucially, transfection of wt mtpap into rs - and - cells abrogated both the dsb repair defect and the reduction in clonogenic survival, confirming that mutation of mtpap is responsible for the cellular radiosensitivity phenotype. mtpap is a nuclear-encoded mitochondrial polyadenylation polymerase that was not an obvious candidate for causing radiosensitivity. , examination of atm expression and kinase activity indicated that it was unlikely that mutation of mtpap disrupts atm-mediated signaling or is required for the early recognition of dsbs. increased g-h ax and bp foci at h, and persistent bp foci at h were observed, suggesting disruption of the ddr early post-ir, but indepen- dent of the initial sensing of dsb damage by atm. increased induction of dna damage post-ir relative to wt levels was suspected in rs - and - , and was confirmed by increased comet tails measured by the nca. these results suggest that mutation of mtpap may affect the induction of dsbs rather than their recognition or repair, a potential deviation from the classical presentation of human radiosensitivity disorders. interestingly, general ros levels and superoxide radical levels were also increased in rs - and - . it has been known that mitochondrial function is important for maintaining ros homeostasis before and after exogenous insults and figure rs - and - lcls had normal mitochondrial respiration but increased levels of ros and apoptosis post-ir. (a) mitochondrial respiration was measured by monitoring metabolism of the rezasurin dye and indicated wt-like, or normal, respiration in rs - and - . the results are displayed as relative fluorescent units (rfus) of the metabolized rezasurin dye. wt is a representative wt-lcl response. at is a representative a-t cell with a characteristic mitochondrial defect. (b) mitochondrial bioenergetics were profiled using the xf extracellular flux analyzer, and rs - and - had increased maximal respiration levels after addition of fccp, compared with wt-lcls. wt is a representative wt-lcl response. the oxygen consumption ratio (ocr) was measured as an indicator of mitochondrial respiration. (c) reactive oxygen species (ros) levels were assessed using dchf-da dye (rfu). increased ir-induced levels of ros were noted in rs - and - . asterisks denote statistical significance at the po . level comparing the þ ir condition of the indicated sample to both of the wt and wt þ ir conditions. (d) apoptosis was determined by facs analysis of cells positive for annexin v staining h post- gy, and indicates increased post-ir apoptosis in rs - and - . asterisks indicate statistical significance at the po . level mtpap mutation causes radiosensitivity nt martin et al cell death and disease that, when dysfunctional, mitochondria can leak superoxide radicals into the cytosol and can impair antioxidant response mechanisms. these ros levels can impact the number and complexity of dsbs through the induction of single or many clustered single-strand breaks. the increased level of bp foci and decrease in both brca and rad foci post-ir may indicate a shift away from hr repair, which may fail with clustered complex breaks, and a preference toward nhej repair. based on the known function of mtpap, we postulated that the increased ros levels were due to dysregulation of mitochondria function. contrary to our prediction, basal respiration levels were wt-like in rs - and - . however, using the cellular bioenergetics assay, we found that there was a significant difference in maximal respiration levels in rs - and - compared with wt levels when the respiratory chain decoupling agent, fccp, was added to cells. the mitochondrial respiratory chain produces ros moieties and, when deregulated, can leak ros, especially superoxide radicals, into the cellular compartment producing a state of oxidative stress. transfection of wt mtpap cdna into rs - and - partially reduced maximal mitochondrial respiration, basal superoxide levels, and ir-induced increases in superoxide levels to wt-like levels. these findings suggested that mutation of mtpap is, indeed, disrupting mitochondrial function and resulting in increased levels of ros before and after ir. because of technical limitations, we were unable to demonstrate a causal link between generally increased post-ir ros levels measured by dchf-da, a dye sensitive to multiple ros moieties, and mutation of mtpap. ionizing radiation predominately induces different ros moieties than superoxide, namely the hydroxyl radical, and it is likely that post-ir increases in ros levels measured by the dchf-da dye reflect ros moieties other than superoxide released from dysfunctional mitochondria. the increased levels of ros measured by dchf-da, while reflecting the increase in superoxide levels in rs - and - , also may reflect reduced antioxidant response pathways to ir-induced hydroxyl radicals or other ros that stem from other pathways, such as ros induced by dna damage. nevertheless, the data presented in this study supports a working model whereby mutation of mtpap results in mitochondrial dysfunction and unbalanced ros homeostasis through increased levels of pre-ir and post-ir ros. we explored the importance of ros for modulating the rs - and - cellular phenotype by pre-treating lcls with the antioxidants, a-la and nac. pre-treatment with a-la and nac abrogated the increased dna damage levels, reduction in repair, and radiosensitivity in rs - and - . these results are consistent with our hypothesis that increased ros levels negatively impact upon the response to ionizing radiation in cells with mtpap mutations. the possibility of treating patients with mtpap mutations with a-la and nac is clinically intriguing as well. both nac and a-la are fda-approved antioxidants that come in many formulations and represent attractive potential therapeutic approaches for patients harboring mutations in the mtpap gene to protect against dsbs and chromosomal instability induced by oxidative stress. , in the studies reported herein, pre-treatment of wt and a-t lcls with a-la had no affect on dna repair nor clonogenic survival, whereas previous studies have indicated that antioxidants can improve other aspects of the a-t cellular phenotype. , similar to a-la, nac has been shown to modulate dna damage levels at higher concentrations than those used in this study, but that nac does not improve clonogenic survival. the contrasting response to a-la between a-t and mtpap-deficient patient cells underscores the differential causes of the radiosensitive phenotype in these patients. , figure anti-oxidant a-lipoic acid (a-la) reduced initial g-h ax foci levels, rescued repair defect, and abrogated radiosensitivity in rs - and - . (a) g-h ax foci were assessed -h post- gy after overnight treatment with a-la or vehicle control (dmso). pre-ir treatment with a-la mm abrogated the increased g- h ax foci phenotype in rs - and - , restoring wt-like levels of g-h ax foci- positive cells. one asterisk indicates statistical significance at the po . level comparing a-la-treated samples to vehicle control samples for each cell line. two asterisks indicate significance at the po . level comparing vehicle control samples across cell lines. (b) pre-ir treatment of rs - and - with mm a-la abrogated the post-ir dna repair defect measured by the nca. comets resulting from unrepaired dsbs were assessed post- gy and results are presented as ‘% repair’, which is the return to baseline levels of damage -h post-ir (tmno ir/tm h). asterisks indicate statistical significance at the po . level when comparing a-la-treated samples to vehicle control samples. (c) cellular radiosensitivity, indicated by reduced sf% post- gy, was rescued in rs - and - by pre-treatment with mm a-la. asterisks denote statistical significance at the po . level when comparing a-la-treated samples to vehicle control samples mtpap mutation causes radiosensitivity nt martin et al cell death and disease the a-t lcls illustrate a classic model of radiosensitivity and xcind, whereby increased levels of ros are secondary to a core dsb recognition and repair defect arising from mutation of a core ddr gene. thus, modulating ros levels has little impact on dsb repair or clonogenic survival. conversely, the data for rs - and - indicate that ros levels increased dsb induction, resulted in reduced repair, and led to reduced clonogenic survival in these cells. the data suggest a potentially atypical mechanism of radiosensitivity and xcind, where there is not mutation of a core ddr gene but, rather, a mutation in a gene leading to amplified induction of dna damage by ionizing radiation. this might also account for the absence of immunodeficiency and cancer in mtpap-deficient patients that would be expected if these patients followed a more classical at-like presentation of the xcind syndrome. materials and methods cell culture, ir, and antioxidant treatment. lcls were derived from lymphocytes isolated from whole blood submitted for radiosensitivity testing as previously described. lcls were cultured in rpmi media with % fbs, % streptomycin/penicillin/glutamine (invitrogen, carlsbad, ca, usa) and maintained at c in % co . cells were harvested for experiments when growing under log growth phase conditions and ir was carried out using a mark iv cs- sealed- source irradiator at a dose rate of b . gy/min. where indicated, lcls were treated overnight with a-la in dmso or nac in h o (sigma-aldrich, st. louis, mo, usa) before ir. wt mtpap plasmid and transfection. the open reading frame of human mtpap ( bp) was pcr amplified from wt-lcls using the following primers: xba-mtpap forward ( -gactctagaatggcggttcccggcgtggggc tct- ) and mlu-mtpap reverse ( -gacacgcgttcatgtctgagtactaattgt tctc- ) and subcloned into a ta-vector using the manufacture’s protocol (invitrogen) to generate the wt mtpap plasmid. the resulting product was sequenced to confirm wt mtpap cdna sequence. the ta-cloned mtpap cdna was then digested with xbai and mlui restriction enzymes and the purified fragment was ligated into xbai-mlui-digested (new england biolabs, ipswich, ma, usa), sap-treated (clontech, mountain view, ca, usa), lenti plasmid vector containing gfp (ucla vector core, los angeles, ca, usa). once the plasmid was generated, b � lcls were transiently transfected with mg of lenti- mtpap/gfp (wt mtpap) or lenti-gfp control (ctl) expression constructs using electroporation ( v, mf) in a cell-porator (invitrogen). cells were harvested for experiments two days after transfection. transfection efficiencies were estimated at b % by fluorescent microscopy imaging of gfp-positive cells. csa. the csa for cellular radiosensitivity testing was performed on lcl samples as previously described. briefly, lcls were seeded in a -well plate. the plates were treated with gy or sham-ir and returned to the incubator for – days. wells positive for at least a single colony of cells (i.e., five generations) were scored as positive and results were compared with sham-treated plates to determine the sf%. neutral comet assay. the nca for assessing dna repair capacity of lcls has been previously optimized by nahas et al. the comet assay kit (trevigen inc, gaithersburg, md, usa) was used under neutral conditions according to the manufacturer’s specifications. lcls were treated with gy or sham treated and collected at h (no-ir), min, and -h post-ir. lcls were re-suspended in % low-melting-point agarose (sigma-aldrich) and plated on -well comet assay slides. once the agarose solidified, the slides were added to a bath of cell lysis solution (trevigen inc) overnight at c. the following day, the samples (i.e., the free dna fragments) on the slides were electrophoresed and stained using sybr gold (invitrogen). comets were visualized using an olympus fluorescent microscope (olympus, tokyo, japan) equipped with an axiovision camera and software (zeiss, oberkochen, germany). the tail moments (tms) of comets were scored using cometscore software (tritek, sumerduck, va, usa). percent repair (% repair) was determined by monitoring the return to baseline tm levels (tmno ir/tm h). where no specific wt-lcl line is denoted, % repair values are normalized to multiple wt samples used across experimental replicates to avoid any unexpected idiosyncrasies specific to a particular wt line. initial induction of dna breaks (i.e., without time for dsb repair) was also measured by the nca. lcls were treated with and gy and immediately placed on ice after ir to assess the level of induced damage. samples were then re-suspended in % low-melting-point agarose and assayed as described above to determine the initial induced levels of dsbs, and the results are reported as the tm without ir ( gy) and post- and - gy. ir-induced foci assay. the g-h ax, bp , rad , and brca (millipore, billerica, ma, usa; santa cruz biotechnology, santa cruz bio- technology, santa cruz, ca, usa and novus biologics, littleton, co, usa, respectively) ir-induced foci assay was performed as previously described. for g-h ax and bp foci, cells were treated with gy and collected at , , , and h. cells were collected at , , and h post- gy for assessment of brca foci. rad foci were assessed h post-ir, as previously described. cells were plated on coverslips, fixed with % paraformaldehyde, permeabilized with . % triton-x (sigma-aldrich), blocked in % fbs, and incubated with primary antibodies ( : ) for h at room temperature. the coverslips were washed, blocked with % fbs, and incubated with a alexafluor- secondary antibody ( : , invitrogen) for min at room temperature. coverslips were washed a final time and mounted on slides using prolonggold anti-fade reagent containing dapi (applied biosystems, grand island, ny, usa). foci were imaged using an olympus fluorescent microscope equipped with an axiovision camera and software. cells were scored as positive if they contained four or more foci/nuclei and results are presented as percent positive cells (% pos). rezasurin assay. rezasurin is reduced to a fluorescent dye as mitochondria respire and has been used to monitor mitochondrial respiration in radiosensitive cells. , cells were grown at low density, collected, and plated on a -well plate. before plating, cells were resuspended in fresh media containing mm of the rezasurin dye (sigma-aldrich). the plates were measured at , , , and h post-plating to monitor the relative increase in fluorescence, which is indicative of respiration levels. fluorescence levels were measured on a spectramax m plate reader (molecular devices, sunnyvale, ca, usa). ros assessment and annexin v assay. dchf-da was used to assess the presence of a broad array of cellular ros levels, as previously described. dhe preferentially detects superoxide moieties and was used to assess superoxide levels. cells were collected and re-suspended in � pbs at c containing mm dchf-da dye (sigma) for min or for min in rpmi media containing mm dhe dye (life technologies, carlsbad, ca, usa). for the dchf-da dye, samples were treated with gy or sham treated and immediately placed on ice and analyzed within min by facs (fl- ). samples incubated with dhe were also sham treated or irradiated with gy and analyzed by facs (fl- ) within min. annexin v has been previously described to label apoptotic cells for rapid analysis by facs. the flow cytometry-based annexin v apoptosis kit was used per manufacturer’s protocol (bd biosciences, san jose, ca, usa). cells were collected h post- gy and re-suspended in � binding buffer containing annexin v and propidium iodide. cells positive for annexin v staining were scored as positive for apoptosis. mitochondrial bioenergetics assay. the mitochondrial bioenergetics assays were performed on the xf extracellular flux analyzer (seahorse biosciences, billerica, ma, usa) as previously described. , briefly, � cells were re-suspended in rpmi media and added to each poly-d-lysine-coated well in an xf analyzer -well plate. four wells (i.e., four replicates) were used for each cell line and condition measured. the plate was then centrifuged for min and incubated at c and % co for min to allow suspension cells to attach to the plate before analysis. measurements were carried out after addition of oligo, fccp, and rotenone/myxothiazol at concentrations of . mm for each compound to act as an atp synthase inhibitor, respiratory chain uncoupler, and mitochondrial respiration inhibitors, respectively. a -min mix and -min wait period preceded measurement of the oxygen consumption rate during a -min measurement period for each cycle of the xf analyzer. exome sequencing and variant calling. at the time of initial sequencing, only one lcl was available, rs - . exonic sequences from rs - were captured using agilent sureselect v (agilent, santa clara, mtpap mutation causes radiosensitivity nt martin et al cell death and disease ca, usa) and submitted for base pair, paired end sequencing on an illumina hiseq instrument (illumina, san diego, ca, usa). a total of gb of sequence was obtained and aligned to the human reference genome (grch ). the median read depth was x; . % of the exome was sequenced to a depth of x or greater. after alignment to the human reference genome, duplicate molecules arising during pcr were removed with the markduplicates utility in picard (version . ; source forge, http://picard.sourceforge.net). in the interest of improved insertion-deletion (indel) discovery, alignments supporting candidate indels were realigned with the gatk (version . . ; broad institute, http:// broadinstitute.org/gatk) indelrealigner utility. both single-nucleotide and indel polymorphisms were identified with the gatk unifiedgenotyper (version . . ) using default settings and all variants were annotated and prioritized with gemini. statistics. unless otherwise noted, data are presented as the mean of three independent measurements (n ¼ ) and error bars represent ± standard deviation. statistical analysis was determined by the student’s t-test for two independent samples of equal variance and p-values of po . were considered significant. conflict of interest the authors declare no conflict of interest. acknowledgements. we thank dr. laurent vergnes for his technical assistance with the mitochondrial bioenergetics assay and ms. brandi woo for her technical help profiling the dna damage response in rs - and - . flow cytometry was performed in the ucla jonsson comprehensive cancer center (jccc) and center for aids research flow cytometry core facility that is supported by national institutes of health 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genome analysis toolkit: a mapreduce framework for analyzing next-generation dna sequencing data. genome res ; : – . . paila u, chapman ba, kirchner r, quinlan ar. gemini: integrative exploration of genetic variation and genome annotations. plos comput biol ; : e . cell death and disease is an open-access journal published by nature publishing group. this work is licensed under a creative commons attribution-noncommercial- sharealike . unported license. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/ . / supplementary information accompanies this paper on cell death and disease website (http://www.nature.com/cddis) mtpap mutation causes radiosensitivity nt martin et al cell death and disease http://creativecommons.org/licenses/by-nc-sa/ . / http://www.nature.com/cddis 	homozygous mutation of mtpap causes cellular radiosensitivity and persistent dna double-strand breaks 	main 	results 	radiosensitivity segregated with bi-allelic mtpap mutations in rs family 	mutation of mtpap was causally linked to the radiosensitive and persistent dna damage phenotype by mtpap transfection 	increased post-ir dna dsb induction in rs - and - 	normal mitochondrial respiration, increased ros levels, and increased apoptosis in rs - and - post-ir 	antioxidants, α-lipoic acid (α-la) and n-acetylcyseine, rescued mtpap-deficient cellular radiosensitivity and dna repair phenotype 	discussion 	materials and methods 	cell culture, ir, and antioxidant treatment 	wt mtpap plasmid and transfection 	csa 	neutral comet assay 	ir-induced foci assay 	rezasurin assay 	ros assessment and annexin v assay 	mitochondrial bioenergetics assay 	exome sequencing and variant calling 	statistics 	acknowledgements 	notes 	references maturational patterns of systolic ventricular deformation mechanics by two-dimensional speckle tracking echocardiography in preterm infants over the first year of age maturational patterns of systolic ventricular deformation mechanics by two-dimensional speckle tracking echocardiography in preterm infants over the first year of age philip t. levy , , afif el-khuffash , , meghna d. patel , colm r. breatnach , adam t james , aura a. sanchez , cristina abuchabe , sarah r rogal , mark r. holland , patrick j mcnamara , amish jain , orla franklin , luc mertens , aaron hamvas , and gautam k. singh department of pediatrics, washington university school of medicine, saint louis, mo, usa department of pediatrics, goryeb children’s hospital, morristown, nj, usa department of neonatology, the rotunda hospital, dublin, ireland school of medicine (department of paediatrics), royal college of surgeons in ireland, dublin, ireland department of pediatrics, university of minnesota, minneapolis, minnesota, usa department of radiology and imaging sciences, indiana university purdue university, indianapolis, in, usa division of neonatology & department of physiology, the hospital for sick children, toronto, canada department of paediatrics, mount sinai hospital, toronto, ontario, canada department of cardiology, our lady's children's hospital crumlin, dublin, ireland division of cardiology, the labatt family heart centre, the hospital for children, toronto, canada pediatrics, northwestern university feinberg school of medicine, chicago, il, usa abstract background—we aimed to determine the maturational changes in systolic ventricular strain mechanics by two-dimensional speckle tracking echocardiography in extreme preterm neonates from birth to one year of age, and discern the impact of common cardiopulmonary abnormalities on the deformation measures. methods—in a prospective multi-center study of extreme preterm infants (< weeks gestation at birth), left ventricle (lv) global longitudinal strain and systolic strain rate (gls, glsrs), interventricular septal wall (ivs) gls and glsrs, right ventricle free wall longitudinal s and sr (rv fwls, fwlsrs), and segmental ls (sls) in the rvfw, lvfw and ivs were serially measured at days , , – , weeks and weeks post-menstrual age (pma), and one year corrected age (ca). premature infants who developed bronchopulmonary dysplasia (bpd) or had echocardiographic findings of pulmonary hypertension (ph) were analyzed separately. correspondence address: philip t. levy one children’s place campus box -nwt st. louis, mo phone: - - fax: - - levy_p@kids.wustl.edu. philip t. levy and afif el-khuffash equally contributed to writing the first draft of the manuscript publisher's disclaimer: this is a pdf file of an unedited manuscript that has been accepted for publication. as a service to our customers we are providing this early version of the manuscript. the manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. hhs public access author manuscript j am soc echocardiogr. author manuscript; available in pmc july . published in final edited form as: j am soc echocardiogr. july ; ( ): – .e . doi: . /j.echo. . . . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t results—in uncomplicated preterm infants (n= , %), lv gls and glsrs remained unchanged from day – to one year ca (p= . and . ). rv fwls, fwlsrs and ivs gls and glsr significantly increased over the same time period (p < . for all measures). a significant base-to-apex (highest to lowest) sls gradient (p < . ) in the rvfw and a reverse apex-to-base gradient (p < . ) existed in the lvfw. in infants with bpd and/or ph (n= , %), rv fwls and ivs gls were significantly lower (p < . ), lv gls and glsrs were similar (p= . ), and ivs sls persisted as an rv dominant base-to-apex gradient from weeks pma to one year ca. conclusions—this study tracks the maturational patterns of global and regional deformation by dste in extreme preterm infants from birth to one year ca. the maturational patterns are ventricular specific. bpd and ph leave a negative impact on rv and ivs strain, while lv strain remains stable. keywords cardiac function; prematurity; strain imaging; echocardiography introduction ventricular performance is an important prognostic determinant of clinical status and long- term outcome in preterm neonates. – ventricular mechanics begin to undergo maturational changes in the early and late postnatal periods that can have a long-term impact on cardiac function beyond the first year of age. , the exposure of an immature preterm heart to a sustained increase in hemodynamic load of postnatal circulation, at a time in the development when the heart primarily supports a low resistance circulation, induces myoarchitectural adaptation that may lead to ventricular remodeling. , the proper evaluation of ventricular function in preterm infants by echocardiography has been limited by the lack of reliable quantitative parameters. furthermore, there is paucity of longitudinal studies on prematurity-related alterations in the maturation of cardiac function beyond the early neonatal period. the establishment of sensitive indices of cardiac function in birth cohorts affected by prematurity and its common cardiorespiratory complications is a necessary prerequisite for the clinical adoption of a normative references patterns for use in evaluating pathologic changes and progression. myocardial strain is a measure of tissue deformation and strain rate is the rate at which deformation occurs. longitudinal deformation by two-dimensional speckle tracking echocardiography ( dste) has been validated as a reproducible measure of ventricular function in premature infants. – initial data indicate that measuring deformation values in this population could have clinical implication, as they appears to have superior prognostic value for assessing and potentially predicting major adverse cardiopulmonary events when compared with conventional measurements (i.e. shortening and ejection fraction). , maturational patterns of dste derived longitudinal strain measures during the transitional period through the first month of age have recently been established in preterm infants. , , – however, the evolution of ventricular strain mechanics from birth to one year of age for clinical application has not been comprehensively described in a large longitudinal preterm cohort. , disturbances in myocardial function may also impact levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t neonatal morbidity and mortality, but there is limited information on how different prematurity associated cardiopulmonary conditions, such as bronchopulmonary dysplasia (bpd), pulmonary hypertension (ph), and a persistent patent ductus arteriosus (pda), influence the normal changes in longitudinal cardiac function. since the right ventricle (rv) and left ventricle (lv) are embryologically and structurally distinct and their functional roles change in the postnatal period, we hypothesized that ( ) prematurity related maturational changes in rv and lv deformation measures would have uniquely different trajectories; and ( ) prematurity associated cardiopulmonary conditions would influence changes differently in lv and rv mechanics. accordingly, we aimed to determine the maturational (age- and weight-related) changes in lv, rv, and interventricular septum (ivs) strain mechanics by dste in healthy uncomplicated preterm infants not affected by significant cardiopulmonary abnormalities, and study the influence of the cardiopulmonary abnormalities on the maturational changes in myocardial deformational indices from birth through one year of corrected age (ca). methods study population all data were prospectively obtained as part of an observational research study that included patients who were enrolled between august and january at hospitals affiliated with two academic institutions (washington university school of medicine, saint louis children’s hospital, and royal college of surgeons in ireland, rotunda hospital). two hundred and thirty nine preterm infants (born / to / weeks gestation) were recruited at birth and longitudinally followed until one year corrected age (ca). the preterm infants enrolled from the washington university site were among infants participating in the prematurity and respiratory outcomes program (prop, clinical trials number: nct ). infants with any suspected congenital anomalies of the airways, congenital heart disease (except atrial septal defects), chromosomal anomalies, intrauterine growth restriction (iugr) or small for gestational age (sga, birth weight < th centile for gestation) were excluded from the healthy uncomplicated cohort arm of the study. at both centers, reference values and maturational patterns of rv fractional area of change from these cohorts have been recently published, but deformation imaging by dste has not been reported. , at the washington university school of medicine site, a small proportion of the deformation data was previously used to test feasibility and reporducbility. , at the royal college of surgeons in ireland site, deformation imaging by tissue doppler has been assessed in the transitional period and up to weeks post- menstrual age (pma). , the institutional review board of washington university and the ethical committee on human research at royal college of surgeons approved the protocol. written informed consent was obtained from the parents or guardians of all participants. inclusion criteria in uncomplicated cohort only infants with ‘cardio-respiratory healthiness’ were classified as healthy uncomplicated infants in this study. , in the early neonatal period, a large proportion of premature infants levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t present with acute respiratory failure that often require some sort of respiratory support up to weeks pma, making it difficult to determine a true definition of ‘respiratory healthiness’. , respiratory disease syndrome and the need for invasive and non-invasive ventilation are common in extreme preterm birth in the early postnatal period. bpd, defined as the need for persistent supplemental oxygen support at weeks’ pma, is recognized as the most significant respiratory consequence of premature birth in the late postnatal period. if preterm infants still required any respiratory support at or beyond weeks pma, they were excluded from the uncomplicated cohort. we assessed for the contributions of bpd, as defined by a modified definition of the national institutes of health (nih) bpd workshop , in a sub-analysis. infants with any of the following echocardiographic signs of late onset ph, identified at any time point from weeks pma through one year ca were excluded: an estimated right ventricular systolic pressure (rvsp) greater than mm hg, a ratio of rvsp to systemic systolic blood pressure greater than . , any cardiac shunt with bidirectional or right-to-left flow, unusual degree of right ventricular hypertrophy or dilatation, or ventricular septal wall flattening. , they were assessed in a separate analysis. since the incidence of ph ranges from – % of infants with bpd , we performed stepwise regression to analyze the influence of ph and bpd on ventricular strain patterns. the significance of a pda, and its impact on long term cardiorespiratory health, remain areas of ongoing debate in neonatology. even the persistent patency of the pda remains an ongoing clinical conundrum, as most premature neonates who fail to close their pda in the first week of age or even by the time of discharge will undergo spontaneous closure a few weeks later. prolonged patency is associated with numerous adverse outcomes, but the extent to which these adverse outcomes are attributable to the hemodynamic consequences of ductal patency, if at all, has not been established. however, infants with moderate to large pda, based on its size relationship to the left pulmonary artery (lpa), have a times greater likelihood of requiring treatment for clinically and hemodynamically significant pda than those with small pda. , we, therefore, excluded any infant with a moderate to large hemodynamically significant pda (hspda) in the first week of age, and any size pda from weeks and beyond, but a separate sub-analysis was performed for this cohort. for this study a hspda was defined as a pda diameter of > . mm with the presence of flow reversal in the descending aorta and a left atrial to aortic root ratio (la:ao) of > . . in addition, we used the relationship of the pda to lpa to define size and clinical significance (large = pda/lpa ratio > , moderate = pda/lpa ratio < but > . , and small = pda/lpa ratio < . ). combined, these approaches allowed us to properly assess pda characteristics, signs of pulmonary over circulation, and left heart-loading condition. finally, infants that underwent pharmacological or surgical intervention at any time point in their neonatal course to close a pda were also excluded from this cohort. echocardiographic examination echocardiograms were performed at six time points from birth to one year ca, (figure ): at washington university school of medicine site, the echocardiograms were performed serially at day (n= ), day , (n= ), weeks pma (n= ), weeks pma (n= ), levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t and one year ca (n= ). at the royal college of surgeons in ireland site, echocardiograms were performed at day (n= ), day , (n= ), day – (n= ), and weeks pma (n= ), (figure ). we chose three time points in the first week of age to capture the physiological changes that occur during the transitional period in preterm infants. the timings of the echocardiograms at weeks pma, weeks pma and one year ca were carefully selected to avoid the early postnatal period of clinical and cardiopulmonary instability and early mortality associated with extreme preterm birth. choosing to study all infants at a common pma and ca optimizes the determination of the impact of gestational and chronological age on cardiac function at a specific developmental stage, and allows for the analysis of measures by post-gestational weeks from birth. the infants’ antenatal, delivery and demographic characteristic were obtained. echocardiograms were performed using the same commercially available ultrasound imaging system (vivid and ; general electric medical systems, milwaukee, wisconsin) at each center. one designated trained pediatric cardiac sonographer at each center obtained all the echocardiographic images using a phased array transducer ( . – mhz). the echocardiographic images were acquired using a standardized image acquisition protocol in decubitus position during restful period without changing the position of the infant or disturbing the hemodynamic condition to minimize heart rate and respiratory variation during image acquisition. the image data were digitally stored in raw dicom cine-loop format for offline analysis. heart rate and blood pressure readings were recorded at the time of each echocardiogram. strain analysis two-dimensional speckle tracking imaging—myocardial mechanics were analyzed by the quantification of lv, rv, and ivs longitudinal strain (ls, %) and systolic strain rate (srs, %/sec) using previously published image acquisition and data analysis protocols from our laboratories. , a frame rate to heart rate ratio (fr/hr) between . and . frames/sec per bpm was utilized to optimize myocardial speckle tracking and mechanical event timing. lv global ls (lv gls) and srs (lv glsrs) were calculated by averaging all values of the regional peak ls and srs obtained from segments in two-chamber, apical long-axis, and four- chamber apical views. rv free wall ls (rv fwls) and srs (rv fwlsrs) were calculated as the average of the three segmental longitudinal strain (sls) measures in the rv fw from the rv focused apical four-chamber view. lv sls at the apex, mid- and basal ventricular levels was calculated by averaging each segment from the two-chamber, apical long-axis, and four- chamber apical views of the lvfw. rv sls was obtained from the segments at the apical, mid-ventricular, and basal levels of the rvfw from the rv focused apical -chamber view. although ivs strain is incorporated in lv gls, we decided to also measure ivs global and sls with a new region of interest that only covered the width of the ivs from the trigonal crux at the basal septal side of the triannular plane to the apical point at the apical junction of the ivs and free walls. this separate region of interest would allow us to determine if the ivs strain has its own unique trajectory, distinct from the lv. in this model, ivs gls is averaged from all the values obtained from -segments in the two-chamber, apical long-axis, and four- chamber apical views along the ivs wall only. peak strain for each index was measured as end-systolic strain at the closure levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t of the aortic valve. two observers, who were blinded to the maternal and infant clinical and cardio-respiratory conditions, analyzed the strain imaging using vendor customized commercially available software (echopac; general electric medical systems, waukesha, wi, usa, version ). reproducibility of strain imaging in preterm infants we along with other groups have previously demonstrated that dste-derived ls imaging of the rv, lv, and ivs is highly feasible and reproducible in premature infants using specific cardiac image acquisition and postprocessing data analysis protocols. – , , , these protocols have improved the image acquisition and reduced variance that has resulted in improved reliability of strain as measure of ventricular function in preterm infants. reproducibility of sls has not been comprehensively analyzed in preterm infants. , we assessed sls reproducibility using intra- and inter-observer variability analysis (bland altman plot analysis, intraclass correlation coefficient, and coefficient of variation) in % of the images at each time point. two observers performed offline analysis using the same measurement protocol and were blinded to the patients’ clinical status, and one another. statistical analysis all data are expressed as mean ± sd or as percentages. continuous variables of strain imaging were tested for normality using the kolmogorov-smirnov test and a histogram illustration of the data. analysis of variance and student t tests were used to compare the changes in deformation values from birth to one year ca in the preterm infants and to compare the patterns between uncomplicated preterm infants and those with bpd, ph, and/or a persistent pda, respectively. all outcome variables with non-normal distributions were analyzed in simple comparisons using wilcoxon rank sum tests or kruskal-wallis one- way analysis of variance for tests with more than two independent groups. chi-square tests (or fisher exact test as appropriate) were used to assess the association between categorical variable. two-way anova with repeated measures was used to compare change over time between infants with and without bpd, pda, and ph. percentile charts (mean ± sd) were created using linear regression to assess the independent effect of postnatal age (in weeks) and weight (at time of echocardiogram) on each strain measurement, while adjusting for gestational age at birth and gender. finally, generalized logistic regression models were developed to identify risk factors for bpd and/or ph at weeks pma using a stepwise variable selection. the statistical analysis was performed using spss version . (spss, inc., chicago, il). results study population characteristics two–hundred and thirty-nine infants with a median gestation of . weeks (iqr . – . ) and birthweight of g (iqr – , ) were recruited in this study ( patients from the washington university site and patients from the royal college of surgeon site). of the patients, infants ( % with equal distribution amongst centers) died prior to hospital discharge and were excluded from the analysis, leaving infants with data to be analyzed. ninety-five ( %) were female and ( %) were delivered by caesarean levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t section. two hundred and thirteen ( %) received at least one course of antenatal steroids and all infants received postnatal surfactant replacement therapy. there were relatively lower rates of chorioamnionitis (n = , %), pre-eclampsia (n = , %) and antepartum hemorrhage (n = , %). complete studies were available for > % of infants on days , , – , and at weeks pma. fifty-eight infants were transferred to peripheral hospitals prior to weeks pma leaving infants ( %) available for assessment at that time point (figure ). although echocardiographic data was unavailable at weeks for the transferred infants, their respiratory outcome data at weeks pma was obtained. at one year ca, ( % of the eligible infants from the washington university cohort) returned for an echocardiogram. the clinical and demographic characteristics of the preterm patients are summarized and compared by time point in table . bpd was diagnosed in % of all infants (n= / ). there were echocardiographic signs of ph in ( %) at and week pma. on day – , infants ( %) had a pda of which ( % of total cohort) were classified as hspda. none of the infants underwent pda treatment during the first week of age. however, infants ( %) eventually received pharmacological therapy and ( %) underwent surgical intervention to close the pda. of the remaining infants who did not receive any intervention to augment its closure, a pda was evident in infants ( %) at weeks pma, infants ( %) at weeks pma. none of the infants received inotropes or administration of inhaled nitric oxide. therefore, after meeting all inclusion criteria, patients ( %) infants were classified as “healthy uncomplicated preterm infants” (figure ). table compares the maternal and infant characteristics between those infants classified as healthy uncomplicated and infants classified with cardio-respiratory disease. maturational patterns of myocardial strain in uncomplicated preterm infants in uncomplicated preterm infants (n= ), a time-specific maturational pattern revealed that rv fwls and fwlsrs had stable pattern from day and and their magnitudes increased by day – , p < . . (table ). rv fwls and fwlsrs continued increasing in magnitude from day – to one year ca (− . % ± . to − . % ± . , p < . and − . % ± . to − . ± . p < . , figure a). ivs gls and glsrs followed the same pattern as rv strain mechanics (figure b, table ). the magnitudes of lv gls and lv lsrs increased from day to day – (− . % ± . to − . % ± . and − . /s ± . to− . /s ± . , p < . for both) and then remained stable through one year ca (− . % ± . to . % ± . , p= . and − . ± . to − . ± . , figure c). maturational changes in lv, rv, and ivs strain were further analyzed to produce percentile charts (mean ± sd) related to gestational age at birth and postnatal age- and weight-related changes. rv fwls and ivs gls were linearly associated with gestational age (r= . and . , p= . ), while lv gls and fwls were not (r= . and . , p > . ). step-wise regression analysis of the effects of gender, birth weight, change in postnatal weight, total oxygen days, length of stay, and common neonatal morbidities (necrotizing enterocolitis, intraventricular hemorrhage, and retinopathy of prematurity, table ) on maturation of ls revealed that at all time points increasing rv fwls and ivs gls were associated with both increasing weight (r = . , . . p < . ) and postnatal age (r = . , . , p < . ), levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t table . lv, rv, and ivs srs imaging followed similar patterns as strain values, when adjusted for weight and postnatal age (tables and ). maturational patterns of regional myocardial strain in uncomplicated preterm infants rv fw had a persistent base-to-apex sls (highest to lowest) gradient from birth to one year ca (p < . at all time points). similar to the rv fwls, the magnitude of each sls value at the basal-, mid-, and apical- ventricular regions increased throughout maturation (p < . for each level). ivs sls had an initial base-to-apex gradient on day and day (p = . and p = . , respectively) that switched to an apex-to-base gradient (highest to lowest) at day – (p < . ) that persisted to one year ca (p < . ). lv fw had a stable apex-to-base longitudinal strain gradient over the same time period (p < . for all time points), (table ). confounding cardiopulmonary factors bpd. infants with bpd (n = , %) had a significantly lower gestational age than those without bpd [median (range); . ( . – . ) vs. . ( . – . ) weeks, p < . ] there were no significant differences in measures of lv, rv, or ivs strain and srs between groups during the first week of age (all p < . ). however, the magnitude of rv fwls and ivs gls were significantly lower in preterm infants with bpd compared to uncomplicated preterm infants at weeks (p= . and p < . , respectively), and these patterns persisted to one year ca (p < . for both, figure a and b). the association between bpd and low rv fwls (p = . ) and low ivs gls (p = . ) remained significant when adjusting for gestational age, gender, and postnatal steroid use. despite the differences between groups, the magnitudes of rv fwls and ivs gls increased, but at a slower rate from weeks to one year ca in preterm infants with bpd (slope of change comparison between infants without bpd and with bpd for rv fwls and ivs, . % vs. . , p< . and . % vs. . , p< . , respectively), (figure a). rv fw maintained a base- to-apex sls gradient from weeks to one year ca (p < . at each time point), but the magnitude of each segment was decreased in preterm infants with bpd compared to uncomplicated preterm infants (p= . ). on the other hand, lv gls, glsr, fwls, and fwlsrs all remained stable (p= . , p= . , p= . , p= . respectively) with a persistent fw apex-to-base sl gradient in preterm infants with and without bpd at each time point (p < . for all), (figure c). the individual ivs strain segments were decreased in premature infants with bpd when compared to uncomplicated preterm infants at all time points (p < . for each segment at each time period), (table ). there was also a persistent base-to- apex ivs sls gradient (reflective of the rv pattern) by day – (p < . ) that did not reverse to the expected apex-to-base ivs sls gradient (reflective of the lv pattern) in infants with bpd, even by one year ca (p = . ), (table ). gestational age at birth and a persistent ivs sls base-apex-pattern at day – were significantly associated with the development of bpd in this analysis, (rr, . ; % ci, . – . , p= . and rr, . ; % ci, . – . , p < . , respectively). infants who received antenatal steroids had a higher lv gls [− . ± . ) vs. − . ± . ) %, p= . ] at day . this relationship remained significant when adjusting for gestation on linear regression (β = . , p= . ), but disappeared after day . postnatal steroids were levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t administered in infants ( %) at least one time during their hospital course. the majority (n= / , %) of the infants received postnatal steroids to treat lung disease beyond the first month of age, and these infants were also diagnosed with bpd. the four infants who did not develop bpd received steroids in the first month of age to facilitate a trial of extubation. there was no statistical difference in the maturational patterns of strain between those infants that did and did not receive postnatal steroids (p= . ), although the study was not properly powered to answer this question. ph—based on echocardiographic evidence, we found an overall incidence of ph of % (n= ) at weeks pma, % (n= ) at weeks pma, and % (n= ) at one year ca. overall, % (n= ) of the preterm infants with ph at weeks pma returned for follow up at one year ca, of whom % (n= ) did not carry a diagnosis of bpd. none of the infants with ph at weeks pma required oxygen or respiratory support at one year ca. lv gls, glsrs and fw sls patterns were unchanged between the infants with and without the evidence of ph at and weeks pma (p > . for all). all the infants with ph at and weeks pma continued to have persistently decreased rv fwls and ivs gls (p= . and p= . ), with an altered ivs base-to-apex sls gradient at one year ca (reflective of the rv pattern) when compared to those infants without ph, even after adjusting for the presence of bpd (β= . , p< . ), (table ). the day – risk factor of a persistent base-to-apex gradient (rr, . ; % ci, . – . , p= . ) was associated with late ph at weeks pma. there were three infants at weeks pma and infants at weeks pma that were not diagnosed with bpd, but had echocardiographic signs of ph. these infants also had lower values of rv fwls and ivs gls (p= . and p= . ), an ivs base-to-apex (reflective of the rv pattern) sls gradient (p < . ), and preserved lv gls (p= . ) with an apex-to-base sls gradient that persisted to one year ca (p < . ), when compared to uncomplicated infants.. pda—the influence of a hspda present at day – on the deformation measurements was assessed. thirty-three infants (out of , %) were classified as having a hspda: their pda diameter ( . [ . – . ] vs [ – . ] mm, p< . ), and la:ao ratio [ . ± . vs. . ± . , p< . ] were all higher than infants without a hspda (and all hspda infants had flow reversal in the abdominal aorta). on day – , infants with a hspda had higher lv gls [− . % ± . ) vs. − . % ± . ), p< . ] and higher rv fwls [− . ± . ) vs. − . ± . ), p= . ], figure a and c). there was no difference in lv or rv srs between the two groups (p > . for all measures) at any time points in the first week of age (figure b and d). a hspda maintained its independent effect on lv gls when adjusting for lv srs (hspda β= . , p< . and lv srs β= . , p< . ). however, the association between a hspda and rv fwls was lost when adjusting for rv srs (hspda β= . , p= . and rv srs β= . , p= . ). there was no difference in the maturational patterns of rv, lv, or ivs gls or sls between infants who still had pda at weeks pma (n= , %), weeks pma (n= , %), or one year ca (n= , %), (adjusted for age, gender, and pda size) and those who did not have pda at these time points (p = . , p= . , p= . , respectively). levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t feasibility and reproducibility the reproducibility analysis for rv, lv, and ivs sls measures is summarized in appendix . the measurements were feasible in % of the images using the methods previously described by our group for image acquisition. for all measurements (intra- and inter- observer variability), the bias ranged between % and %, coefficients of variations ranged between % and %, and intraclass correlation coefficient ranged between . and . . discussion in a prospective multi-center longitudinal study of a large cohort of premature infants (≤ weeks at birth), we evaluated ventricular mechanics with dste derived deformational indices from birth to one year ca to determine the maturational patterns of postnatal cardiac adaptation. the main findings of this study are that: ( ) rv longitudinal strain has a distinct regional magnitude distribution with a base-to-apex gradient and an incremental progression, while lv strain has an apex-to-base magnitude gradient with relatively unchanged progression through the neonatal period and infancy in uncomplicated preterm infants; and ( ) preterm infants who develop bpd and/or ph have similar lv strain patterns but with decreased magnitudes of rv and ivs strain that track lower to those in uncomplicated preterm infants through the late neonatal period and infancy, even after clinical resolution of bpd and ph. the functional evaluation of each ventricle by echocardiography in preterm infants is an area of ongoing research, but has been limited by the lack of reliable quantitative parameters that can be used for the assessment of both rv and lv function. the complex three- dimensional structure of the rv, with thin wall and high compliance, is evolutionally and embryologically different from the thick-walled and highly contractile lv. despite these morphological differences, deformation imaging by dste has emerged as a reliable technique to assess global and regional myocardial function in each ventricle and permits a more direct assessment of myocardial function in preterm infants during this transitional period and through one year ca that could not be previously obtained with conventional modalities. , in addition, deformation imaging has been mostly limited to the early postnatal period in preterm infants. , – , , – , , , we leveraged the large cohort of preterm infants in a multi-center format to study the impact of prematurity on the maturation of cardiac function in the preterm infants. this is the largest study to longitudinally assess strain imaging with dste in preterm infants up to one year ca. we showed that myocardial mechanics in both ventricles undergo unique longitudinal maturational changes in preterm infants, and that this maturation trajectory is influenced by body weight and age. we also studied the impact of different cardiopulmonary diseases’ influence on these adaptive processes. maturational patterns of ventricular strain in uncomplicated preterm infants rv fwls and ivs gls—myocardial function undergoes unique longitudinal changes in preterm infants that are specific to each ventricle. rv fwls, and ivs gls remained relatively stable from day to day , but then physiologically increased from day – to one-year ca. nasu et al. found no change in rv strain patterns over the first hours of age levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t (ranged between - . % and − . %). schubert et al observed similar values in the first week of age (− . % ± . ). interestingly, deformation indices were also higher in the rv than the lv beginning at weeks pma and persisting to one year ca, which is in concordance with previous studies in children and neonates. , , in this study, the increase of rv and ivs strain in uncomplicated preterm infants was influenced by both postnatal increase in weight (in grams) and post-gestational age (in weeks), figure . the relative higher values of rv strain and its progressive increase throughout the late neonatal period and infancy (when compared to lv strain) is probably related to the more longitudinal orientation of rv fibers with a predominant longitudinal contraction pattern in the rv compared to the lv. , the higher rv strain is also reflected in the increased metabolic demand of rv myocardium that leads to a larger percentage ratio of collagen matrix to myofibers when compared to the lv myocardium. despite the overall reduction in cardiomyocyte endowment in the preterm neonate, the increased extracellular matrix (ecm) deposition in the rv and the changing postnatal loading conditions result in different properties of deformation between the lv and rv. , the decreasing afterload that results from a postnatal drop in pulmonary vascular resistance also leads to an increase in rv deformation over time. in a small comparative study of preterm and term infants from birth to six months of age, schubert et al. observed an increasing upward trend in rv and ivs strain values. in larger studies, both hefler and cerznick and james et al , observed that tissue doppler- derived rv and ivs strain values rose significantly from birth to days of age and weeks pma, respectively. with dste-derived strain, czernik et al. also observed a rise in ivs gls over the first days of age. our findings are consistent with these recent reports that had shorter follow-up time. lv gls—compared to rv strain patterns, lv gls remained relatively unchanged from day – through one year ca. we did observe an increase in lv gls from day to day – . lv strain values do not change markedly with age or heart rate in uncomplicated children, despite the alteration in loading conditions and changes in myocardial properties that occur during early childhood. there are a few studies in neonates that have demonstrated similar stable maturational pattern of global lv strain from birth through six months of age, , – , however, none of these studies followed the infants to one year ca. nasu et al. performed eight serial echocardiograms over the first hours of age and demonstrated no change in lv strain. czernik et al. and de waal et al. each serially observed stable dste-derived lv gls in preterm infants from birth to days of age. hirose et al. demonstrated that clinically healthy preterm infants (delivered at < weeks gestation) also have constant dste-derived lv strain measures from days of age to near term equivalent age ( months ca). schubert et al. observed that in a small heterogeneous cohort of preterm infants ( % had bpd, and % had septicemia), and term infants that there was relatively uniform lv strain values from birth to six month of age. these results coincide with the findings of helfer and czernik and james et al , whose groups used tissue doppler-derived strain imaging to demonstrate relative stability in lv strain imaging in preterm infants from birth through days and weeks pma, respectively. two recent meta-analyses defined reference values of dste-derived lv gls in health levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t term-born neonates that were also stable from birth to one year of age. , although we observed a slight increase in lv gls from day to day – , the impact of this early transitional period on lv strain patterns remains unclear from individual studies because of the variable methodologies and timings of scans employed in all those studies described above. all of the previous studies in preterm infants only measured lv deformation from the apical four-chamber view. , , – , this is the first study to calculate the true “global” strain as an average from the three apical views. we further speculate that this difference is one reason for the disparity in reported values between studies, as well as the observed increase in lv gls from day to day – in our study. lv gls is defined by different lv segmentation models, and it is recommended that each segment should be evaluated in multiple views to assess complete wall motion. – , despite these recommendations, deformation imaging is still reported only from the apical - chamber view. however, despite that lack of uniform consensus on “which approach is more accurate or correlates more efficiently with health and disease,” it appears that by the end of the first week of age there is relative stability in lv strain patterns that persists throughout maturation, irrespective of which approach is utilized. segmental ls—in the rvfw there is a base-to-apex sls gradient that reflects the base- to-apex alignment of the dominant deep longitudinal layers of the rv, and allows for greater longitudinal shortening. in this study and the report by schubert et al. there was a base- to-apex sls gradient in the rvfw that was preserved throughout maturation. in the lvfw there exists an apex-to-base sls gradient in children that occurs because of two primary reasons: ( ) torsional mechanisms of lv deformation is greatest toward the apex, as the right-handed helix in the subendocardium and the left-handed helix in the subepicardium converge toward the apex to form the “vortex of the double helical loop;” and ( ) the electric excitation of cardiac motion begins in the apex and travels to the base. , coinciding with previous reports by czernik et al. and de waal et al. , there existed a stable apex-to-base lv dominant sls gradient in the lvfw that reflects the relatively constant geometry of the normal heart with maturation. in the ivs, there was also a persistent apex-to-base gradient (lv-dominant pattern ) in uncomplicated preterm infants that increased from day – to one year ca. a series of meta-analysis defined similar reference ranges and physiological developmental patterns of sls at the apical-, mid-, and basal- ventricular levels of the lv (apex-to-base) and rv (base-to-apex) fws in healthy term-born children less than one year of age. , alteration of these “physiological” gradients have the potential to discern clinical changes in regional myocardial function in patients with different disease processes. , clinical implications of deformation imaging in neonates children with bpd and ph are much more likely than their gestational age counterparts to develop long-term respiratory morbidity that may affect the cardiovascular system. the extent to which prematurity-related alterations in cardiopulmonary structures affect their function has remained unclear due to the lack of early detection by a reliable biomarker of the disease, making the clinical management, intervention planning, and outcomes levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t prediction for these patients challenging. we believe evaluation of cardiac function in preterm infants by speckle-tracking deformation imaging may now provide a marker (that was not previously available with conventional imaging) to longitudinally track cardiopulmonary disease over the first year of age. with an accepted physiological maturation patterns of ventricular strain in uncomplicated preterm infants based on postnatal weight and age over the first year age, we feel that these myocardial deformation parameters can provide a valid basis that allows comparison among studies and between health and disease. bpd and respiratory support improved survival of extremely premature babies has led to increased recognition of rv dysfunction and ivs abnormalities in infants with bpd. , , in this study we found a significant decrease in rv fwls and ivs gls in infants with bpd compared to infants without bpd (uncomplicated preterm infant), which remained significant after adjusting for the gestational age at birth, gender, and postnatal steroid use. in preterm infants with bpd, the magnitude of rv fwls increased from weeks to one year ca, but remained lower and tracked inferior when compared to infants without bpd by one-year ca, even when they no longer displayed clinical evidence of lung disease with oxygen requirement or respiratory support. helfer and czernik observed that tissue doppler-derived rv global strain measures increased over the first month of life, but the rate of increase was also slower in infants who developed bpd. these findings suggest that rv function, as characterized by deformation values, remains subnormal in infants with bpd. in contrast to rv strain measure, lv gls was preserved from day – through one year ca, irrespective of neonatal lung status. czernik et al. also demonstrated that lv gls by dste remained relatively constant from birth to days of life in infants with and without bpd. subtle differences in lv sls values were seen at the mid-ventricular level in the early neonatal period that disappeared by one month of age. similarly, james et al. demonstrated that bpd negatively impacted rv fwls, but that lv tissue doppler deformation values were unaffected by bpd. we also demonstrated that there was a preserved physiological segmental apex-to-base strain gradient in infants with and without bpd, suggesting that both global and regional longitudinal lv function remained preserved in the setting of lung disease. however, infants who developed bpd not only had decreased ivs gls from weeks pma to one year ca, but a persistent base-to-apex (reflective of an rv dominant pattern) ivs strain gradient that never reversed, even by one year of age. infants without bpd demonstrated an ivs apex-to-base sls gradient, reflective of an lvfw pattern. mechanical ventilation can alter the pre- and after-load conditions and affect deformation values. in this study, % (n= ) of infants were on mechanical ventilation at weeks pma and % (n= ) of the infants at weeks pma. there were no infants on respiratory support at one year ca. we were unable to properly investigate the effect of mechanical ventilation on deformation changes because nearly all of the mechanically ventilated infants ( %, n= ) at weeks and all infants at weeks pma developed bpd. we found an interesting association between the use of antenatal steroids and lv function on the first day levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t of age. infants whose mothers received antenatal steroids had higher lv gls and srs, which was independent of gestation. no effect was observed on rv function. preterm infants may suffer from a combination of adrenocortical insufficiency and down regulation of cardiovascular adrenergic receptors. therefore, it is possible that the administration of antenatal steroids can reverse this phenomenon leading to improved function. ph—we utilized a broad echocardiogram-based definition of ph described by mourani et al. and found an overall incidence of ph of % at weeks pma and % at weeks pma. infants with bpd had a higher incidence of ph at weeks pma ( % vs. %) and at weeks pma ( % vs. %) when compared to those infants without bpd. infants with ph at and weeks pma had lower values of rv fwls and ivs gls, with preserved lv gls. ivs sls displayed a persistent base-to-apex gradient (rv dominant) in infants with ph that did not reverse by weeks pma. the base-to-apex pattern that was present at day – was associated with a higher risk of late ph. mourani et al. observed that ventricular septal wall flattening at days of age (as an early sign of pulmonary vascular disease) was also associated with increased risk of late ph. similar to mourani et al., we also found that % of infants without bpd had detectable pulmonary hypertension at -week corrected gestational age. the patterns of decreased rv fwls and ivs gls persisted in the infants without bpd, but with ph from weeks to one year ca. as highlighted by farrow et al., this observation suggests that a primary vascular injury coupled with rv dysfunction may occur in some extreme preterm infants, independent of the lung disease. although we only observed that one infant had echocardiographic signs of ph at one year ca, the infants with ph on their week pma echocardiograms that returned for a one-year ca follow-up (n= of , none of which required oxygen or respiratory support at one year ca ) still had decreased rv fwls and ivs gls and altered sls base-to-apex pattern. interestingly % (n= ) of the infants with ph at weeks pma that returned at one year ca did not carry a diagnosis of bpd and the patterns of altered strain mechanics persisted in this small sub-cohort as well. these findings might suggest that while some of the mechanisms of bpd and ph overlap, impairments of the developing pulmonary circulation in the extreme preterm infant without bpd may not be severe enough to be clinically recognized as pulmonary hypertension, but may still lead to cardio-respiratory morbidity. whether this ph is clinically significant alone or predicts long-term morbidity in infants with bpd will require further study. pda—the true influence of persistent pda on ventricular strain patterns beyond the early neonatal period is an area of ongoing research. , , , , , infants with a pda accompanied by evidence of systemic hypoperfusion (diastolic flow reversal in the descending aorta, and pulmonary over-circulation (increased la:ao and increased ejection fraction) had a higher lv gls by day – . however, lv srs remained similar in the two groups. de waal et al. also found higher lv gls and srs in infants with a pda. it is well documented that a hspda significantly increases lv preload, and leads to an increase in lv output. the increase in preload is accompanied by an increase in s but not srs. this adds further support to the relative lack of load dependency of srs illustrated in animal models. in our regression model, both hspda presence (and the accompanying increased preload), levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t and lv srs (representative of inherent contractility) had an independent effect on lv gls. therefore, lv longitudinal strain measurements must be interpreted taking into consideration loading conditions. in this study, the infants who did not undergo pharmacological or surgical intervention to augment closure of the pda, but still had a persistent pda beyond weeks, did not show any differences in their strain values when compared to infants without a pda. furthermore, infants who received pharmacological or surgical intervention had relatively stable values of rv, lv, and ivs fws and fwsrs values in the late postnatal period when compared to infants without a pda. schubert et al. also found no significant associations between the speckle-tracking outcome variables at three months of corrected age and the diagnosis of pda. we suspect that the effects of a pda on lv strain imaging during the transition period and early neonatal period that has been observed in previous studies, may either dissipate over time, , may be more of a true reflection in myocardial diastolic performance, or are unlikely to be due solely to the presence of pda. the importance of these findings is that they support the notion that most premature neonates who fail to close their pda after the first week of age may likely not experience significant cardiac morbidity as evidence by similar lv and rv strain patterns. study limitations and future directions in this study we only evaluated longitudinal strain imaging, as the feasibility and reproducibility of radial and circumferential strain has not been fully described in preterm infants beyond the transitional period. , although longitudinal strain remains the most reproducible quantitative tool of the three to assess lv function in preterm infants during the transitional period and later neonatal period, , future work is now needed to assess the feasibility and reproducibility of circumferential and radial deformation at multiple time points in the first year of age and understand their maturational patterns in the context of uncomplicated preterm infants and those exposed to a pda, at risk for bpd and or ph. both systolic and diastolic strain rate imaging provide unique insight into myocardial contractility and loading conditions, but we did not assess early and late diastolic strain rate with dste, because they too lack reproducibility in preterm infants. finally, we demonstrated reduced inter- and intra- variability of segmental strain data in the lvfw, rvfw and ivs when compared to the reproducibility of global strain imaging in preterm infants. the difference in variability between global and regional strain may be due to the notion that in the small heart of a preterm infant each individual segment may not comprise a lot of speckles, and coupled with the elevated baseline heart rates in neonates, will lead to smoothing over of segments. to some extent, this may be mitigated by keeping the frame rate to heart rate ratio (fr/hr) between . and . frame/sec per bpm to optimize myocardial speckle tracking and mechanical event timing. however, until further validation of regional strain values is performed amongst different vendor platforms, its clinical use may be limited to the research arena. levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t conclusions this study tracks the maturational patterns of ventricular mechanics with global and regional deformation imaging by dste in extreme preterm infants during the first year of age. the maturational patterns are ventricular specific. bpd and ph appear to leave a negative impact on rv and ivs strain, while lv strain remains stable through the first year of age, independent of birth weight, postnatal age, and clinical status. this study suggests that dste derived strain can be used as a complementary modality to assess systolic ventricular function in neonates. acknowledgments this study was supported by grants from premature and respiratory outcomes program (nih u hl , u hl ), nih r hl , pediatric physician scientist training grant (nih t hd - ), and postdoctoral mentored training program in clinical investigation (nih ul tr ). afif el-khuffash is funded by multiple sources: eu fp / - grant (agreement no. , the hip trial); the friends of the rotunda research grant (reference: for/equipment/ ); health research board mother and baby clinical trials network ireland (ctn- - ). abbreviations dste two-dimensional speckle tracking echocardiography bpd bronchopulmonary dysplasia ca corrected age fwls free wall longitudinal strain gls global longitudinal strain ivs interventricular septum ls longitudinal strain lv left ventricle pda patent ductus arteriosus pma post-menstrual age ph pulmonary hypertension rv right ventricle srs systolic strain rate references . breatnach cr, levy pt, james at, franklin o, el-khuffash a. novel echocardiography methods in the functional assessment of the newborn heart. neonatology. ; : – . 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[pubmed: ] . el-khuffash a, weisz d, mcnamara p. reflections of the changes in patent ductus arteriosus management during the last years. arch dis child fetal neonatal ed. ; :f – . [pubmed: ] . ferferieva v, van den bergh a, claus p, jasaityte r, veulemans p, pellens m, et al. the relative value of strain and strain rate for defining intrinsic myocardial function. am j physiol heart circ physiol. ; :h – . [pubmed: ] appendix . reproducibility analysis for segmental longitudinal strain bland altman intraclass correlation cv absolute bias (%) loa ( % ci) p (%) left ventricle apex % − . – . . ( . – . ) . . % mid-ventricle % − . − . . ( . – . ) . . % base % − . – . . ( . – . ) . . % right ventricle apex % − . – . . ( . – . ) . . % mid-ventricle % − . – . . ( . – . ) . . % base % − . – . . ( . – . ) . . % interventricular septum apex % − . – . . ( . – . ) . . % mid-ventricle % − . − . . ( . – . ) . . % base % − . – . . ( . – . ) . . % levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t highlights • two-dimensional speckle tracking echocardiography ( dste) derived myocardial strain is a feasible and reproducible imaging modality that can be used to characterize systolic ventricular function in premature infants. • this study establishes ventricular specific systolic strain maturational patterns by dste in a large cohort of extreme preterm infants from birth through one year corrected age (ca). • common cardiopulmonary morbidities, such as bronchopulmonary dysplasia and pulmonary hypertension appear to leave a negative impact on rv strain, while lv strain remains stable through the first year of age. • with the establishment of the range of maturational patterns of strain mechanics and associated variations up to one year ca, deformation imaging by dste may now be implemented in preterm infants as a means to identify cardiovascular compromise earlier, guide therapeutic intervention, monitor treatment response, and improve overall outcome. levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t figure . enrollment and follow-up of study participants bpd, bronchopulmonary dysplasia; ph, pulmonary hypertension; pda, patent ductus arteriosus. * / infants were excluded from the healthy uncomplicated cohort because they either were: a) diagnosed with bpd, defined as the requirement any respiratory support at weeks pma, and based on a modified nih workshop definition ; b) evidence of ph on echocardiogram at and/or weeks pma, defined as any infant with any conventional echocardiographic signs identified, by an estimated rv systolic pressure > than mm hg, a ratio of rvsp to systemic blood pressure > than . , any cardiac shunt with right-to-left flow, unusual degree of rv hypertrophy or dilatation, or any degree of ventricular septal wall flattening; or c) evidence of a hemodynamically significant pda by day – , (defined by pda characteristics, signs of pulmonary over circulation, and left heart-loading condition), or any pda at and week pma. there was significant overlap between these four categories. ** there were infants recruited for this study ( infants from the washington university school of medicine site in saint louis, usa and infants from the royal college of surgeons in ireland site in dublin, ireland). echocardiograms were performed at day (n= ), day (n= ), weeks pma (n= ), weeks pma (n= ), and one year ca (n= ) in saint louis, usa. echocardiograms were performed at day (n= ), day (n= ), day (n= ), and weeks pma (n= ) in dublin, ireland. levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t figure . maturational patterns of ventricular deformation over the first year of age (a) rv fw longitudinal strain (rv fwls%): in all infants rv fwls % increased from day – to one year ca (p < . ). rv fwls % was higher and tracked superior in the uncomplicated preterm infants (blue circles with blue solid line) when compared to the preterm infants who developed bpd (red squares, dotted red lines) on echocardiogram at the time of evaluation. (b) interventricular septal (ivs) global longitudinal strain (gls, %): in all infants ivs gls increased from weeks pma to one year ca (p < . ). ivs gls was higher and tracked superior in the uncomplicated preterm infants when compared to the preterm infants who developed bpd on echocardiogram at the time of evaluation. (c) lv global longitudinal strain (lv gls): lv gls was preserved and relatively unchanged from weeks pma to one year ca (p= . ), irrespective on neonatal cardiopulmonary disease. levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t figure . left and right ventricle strain and systolic strain rate in infants with and without a hemodynamically significant patent ductus arteriosus on day – (a) lv gls. on day – , infants with a hspda (green diamond, dotted green lines) had higher lv gls than the healthy uncomplicated cohort (blue circle, solid blue line); (b) lv glsrs. there was no difference in the magnitude of lv glsrs between cohorts at any time point in the first week of age; (c) rv gls. on day – , infants with a hspda (green diamond, dotted green lines) had higher rv gls than the healthy uncomplicated cohort (blue circle, solid blue line); (d) rv glsr. there was no difference in the magnitude of rv glsrs between cohorts at any time point in the first week of age. *= p < . . lv: left ventricle; rv: right ventricle; srs: systolic strain rate; fwls, free wall longitudinal strain; fwlsrs, free wall longitudinal systolic strain rate; gls, global levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t longitudinal strain; glsrs, global systolic longitudinal strain rate; hspda: hemodynamically significant patent ductus arteriosus. levy et al. page j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page ta b le d em og ra ph ic a nd c li ni ca l ch ar ac te ri st ic s of p re te rm i nf an ts b as ed o n ti m in g of e ch oc ar di og ra m e va lu at io n t im in g of e ch oc ar d io gr am s d ay d ay d ay – w ee k s p m a w ee k s p m a o n e ye ar c a t ot al # i nf an ts a t ea ch t im e po in t * h ea lt hy u nc om pl ic at ed i nf an ts ** r es p ir at or y r r ( br ea th s/ m in ) ** ± ± ± ± ± ± in va si ve m ec ha ni ca l ve nt il at io n ( % ) ( % ) ( % ) ( % ) ( % ) b ro nc ho pu lm on ar y dy sp la si a ( % ) ( % ) ( % ) ( % ) ( % ) ( % ) c ar d io va sc u la r p d a ( % ) ( % ) ( % ) ( % ) ( % ) hs p d a n a n a ( % ) n a n a n a p ul m on ar y hy pe rt en si on n a n a ( . % ) ( . % ) ( . % ) ( % ) h r ( be at s/ m in ut e) * * ± ± ± ± ± ± s b p ( m m h g) * * ± ± ± ± ± ± d b p ( m m h g) * * ± ± ± ± ± ± m a p ( m m h g) * * ± ± ± ± ± ± d at a ar e pr es en te d as m ea n ± s d , o r nu m be r (p er ce nt ag e) ; n a , n ot a pp li ca bl e p m a , p os t- m en st ru al a ge ; c a , c or re ct ed a ge r r , r es pi ra to ry r at e; h r , h ea rt r at e; s b p, s ys to li c b lo od p re ss ur e; d b p, d ia st ol ic b lo od p re ss ur e; m a p, m ea n ar te ri al b lo od p re ss ur e * t he re w er e in fa nt s re cr ui te d fo r th is s tu dy ( in fa nt s fr om t he w as hi ng to n u ni ve rs it y s ch oo l of m ed ic in e si te i n s ai nt l ou is , u s a a nd i nf an ts f ro m t he r oy al c ol le ge o f s ur ge on s in i re la nd s it e in d ub li n, i re la nd ). e ch oc ar di og ra m s w er e pe rf or m ed a t d ay ( n= ), d ay ( n= ), w ee ks p m a ( n= ) , w ee ks p m a ( n= ) , a nd o ne y ea r c a ( n= ) in s ai nt l ou is . e ch oc ar di og ra m s w er e pe rf or m ed a t d ay ( n= ) , d ay ( n= ) , d ay ( n= ), a nd w ee ks p m a ( n= ). ** a ll t he d em og ra ph ic a nd c li ni ca l ch ar ac te ri st ic s ar e fr om t he u nc om pl ic at ed p re te rm i nf an ts . t he i nf an ts e xc lu de d fr om t hi s co ho rt i nc lu de d: . m od er at e- la rg e hs p d a a t d ay – o r pr es en ce o f p d a a t an y ti m e po in t fr om w ee ks p m a t o on e ye ar c a , o r re ce iv ed s ur gi ca l or m ed ic al a ug m en ta ti on t o cl os e p d a . , , . b p d , r eq ui ri ng r es pi ra to ry o r ox yg en s up po rt a t w ee ks p m a . . e ch oc ar di og ra ph ic s ig ns o f pu lm on ar y hy pe rt en si on – d ay s of a ge a nd b ey on d. j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page ta b le m at er na l an d in fa nt c ha ra ct er is ti c co m pa ri so ns b et w ee n co ho rt s a l l u n co m p li ca te d c oh or t * in fa n ts w it h e it h er b p d , p h , a n d /o r p d a p - v al u e n = n = n = b ir th w ei gh t (g ) [ , ] [ , ] [ , ] . b ir th w ei gh t st ra ta ( g) – (n = ) [ , ] [ , ] [ . ] . – (n = ) [ , ] [ , ] [ , ] . – (n = ) [ , ] [ , ] [ , ] . g es ta ti on al a ge [ , ] [ , ] [ , ] < . g en de r (f em al e) ( % ) ( % ) ( % ) . r ac e . w hi te ( % ) ( % ) ( % ) b la ck ( % ) ( % ) ( % ) a si an ( % ) ( % ) ( % ) o th er ( % ) ( % ) e th ni ci ty . h is pa ni c or l at in o ( % ) ( % ) ( % ) n ot h is pa ni c or l at in o ( % ) ( % ) ( % ) m at er na l s m ok in g ( % ) ( % ) . a nt en at al c or ti co st er oi ds ( % ) ( % ) ( % ) . s ur fa ct an t r ep la ce m en t t he ra py ( % ) ( % ) ( % ) . m ul ti pl es ( % ) ( % ) ( % ) . c es ar ea n se ct io n ( % ) ( % ) ( % ) . m at er na l co m pl ic at io ns g es ta ti on al d m ( % ) ( % ) ( % ) . g es ta ti on al h t n ( % ) ( % ) ( % ) . p ro lo ng ed r up tu re o f m em br an es ( % ) ( % ) ( % ) . c ho ri oa m ni on it is ( % ) ( % ) ( % ) . p re ec la m ps ia ( % ) ( % ) ( % ) . p la ce nt al a br up ti on ( % ) ( % ) ( % ) . j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page a l l u n co m p li ca te d c oh or t * in fa n ts w it h e it h er b p d , p h , a n d /o r p d a p - v al u e n = n = n = n ec ro ti zi ng e nt er oc ol it is ( % ) ( % ) ( % ) . r o p t hr es ho ld ( s ta ge o r hi gh er ) ( % ) ( % ) ( % ) . iv h ( g ra de o r ) ( % ) ( % ) ( % ) . t ot al o xy ge n da ys ( n ic u ) [ , ] [ , ] [ , ] < . l en gt h of s ta y (n ic u ) [ , ] [ , ] [ , ] < . d at a ar e pr es en te d as m ed ia n [i nt er qu ar ti le r an ge ], o r nu m be r (p er ce nt ag e) b p d , b ro nc ho pu lm on ar y dy sp la si a; p h , p ul m on ar y hy pe rt en si on ; p d a , p at en t du ct us a rt er io su s r o p, r et in op at hy o f pr em at ur it y; i v h , i nt ra ve nt ri cu la r he m or rh ag e * h ea lt hy u nc om pl ic at ed c oh or t, d ef in ed a s pr et er m i nf an ts w it ho ut b ro nc ho pu lm on ar y dy sp la si a, e ch oc ar di og ra ph ic s ig ns o f pu lm on ar y hy pe rt en si on a t o r w ee ks p m a , a nd /o r a he m od yn am ic al ly si gn if ic an t pa te nt d uc tu s ar te ri os us a t d ay – o r an y si ze p d a a t o r w ee ks p m a . j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page table deformation measurements over the first week of age. day day day – p entire cohort number of infants lv gls (%) − . ± . − . ± . * − . ± . * † < . lv glsrs ( /s) − . ± . − . ± . * − . ± . * † < . ivs gls (%) − . ± . − . ± . − . ± . *† < . ivs glsr ( /s) − . ± . − . ± . − . ± . † . rv fwls (%) − . ± . − . ± . − . ± . . rv fwlsrs ( /s) − . ± . − . ± . * − . ± . * † < . healthy uncomplicated cohort number of infants lv gls (%) − . ± . − . ± . * − . ± . † < . lv glsrs ( /s) − . ± . − . ± . * − . ± . † < . ivs gls (%) − . ± . − . ± . − . ± . *† < . ivs glsr ( /s) − . ± . − . ± . − . ± . † . rv fwls (%) − . ± . − . ± . − . ± . * . rv fwlsrs ( /s) − . ± . − . ± . − . ± . † . values are presented as mean (standard deviation). one way anova with repeated measures was used to assess the change in values over time. * = p < . compared with previous day measurement; † = p < . compared with day measurement (with bonferroni adjustment). rv, right ventricle; lv, left ventricle; ivs, interventricular septum fwls, free wall longitudinal strain; fwlsrs, free wall longitudinal systolic strain rate gls, global longitudinal strain; glsrs, global systolic longitudinal strain rate j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page ta b le v en tr ic ul ar d ef or m at io n in h ea lt hy u nc om pl ic at ed p re te rm i nf an ts b y w ei gh t w ei gh t (k il og ra m s) in fa n ts * ( n ) r v f w l s ( % ) r v f w l s r s ( /s ec ) l v g l s ( % ) l v g l s r s ( /s ec ) iv s g l s ( % ) iv s g l s r s ( /s ec ) . – . − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . . – . − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . . – . − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . . – . − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . . – . − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . . – . − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . . – . − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . d at a ar e pr es en te d as m ea n ± s d . * h ea lt hy u nc om pl ic at ed c oh or t, d ef in ed a s pr et er m i nf an ts w it ho ut b ro nc ho pu lm on ar y dy sp la si a, e ch oc ar di og ra ph ic s ig ns o f pu lm on ar y hy pe rt en si on a t o r w ee ks p m a , a nd /o r a he m od yn am ic al ly si gn if ic an t pa te nt d uc tu s ar te ri os us a t d ay – o r an y si ze p d a a t o r w ee ks p m a . a t d ay t he re w er e n= , d ay , n = , d ay – , n = , w ee ks p m a , n = , w ee ks p m a , n = , a nd o ne y ea r c a , n = . r v , r ig ht v en tr ic le ; l v , l ef t ve nt ri cl e; i v s , i nt er ve nt ri cu la r se pt um f w l s , f re e w al l lo ng it ud in al s tr ai n; f w l s r s, f re e w al l lo ng it ud in al s ys to li c st ra in r at e g l s , g lo ba l lo ng it ud in al s tr ai n; g l s r s, g lo ba l sy st ol ic l on gi tu di na l st ra in r at e j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page ta b le v en tr ic ul ar d ef or m at io n in u nc om pl ic at ed p re te rm i nf an ts b y ag e be yo nd t he f ir st w ee k . a ge ( w ee k s) in fa n ts * (n ) r v f w l s ( % ) r v f w l s r s ( /s ec ) l v g l s ( % ) l v g l s r s ( /s ec ) iv s g l s ( % ) iv s g l s r s ( /s ec ) – − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . – − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . – − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . – − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . – − . ± . − . ± . − . ± . − . ± . − . ± . − . ± . d at a ar e pr es en te d as m ea n ± s d . * u nc om pl ic at ed , d ef in ed a s in fa nt s w it ho ut b ro nc ho pu lm on ar y dy sp la si a, e ch oc ar di og ra ph ic s ig ns o f pu lm on ar y hy pe rt en si on a t o r w ee ks . t he re w er e u nc om pl ic at ed p re te rm i nf an ts a t w ee ks p m a , un co m pl ic at ed p re te rm i nf an ts a t w ee ks p m a , a nd un co m pl ic at ed p re te rm i nf an ts a t on e ye ar c a . p m a , a nd /o r a he m od yn am ic al ly s ig ni fi ca nt p at en t du ct us a rt er io su s at d ay – o r an y si ze p d a a t o r w ee ks p m a . r v , r ig ht v en tr ic le ; l v , l ef t ve nt ri cl e; i v s , i nt er ve nt ri cu la r se pt um f w l s , f re e w al l lo ng it ud in al s tr ai n; f w l s r s, f re e w al l lo ng it ud in al s ys to li c st ra in r at e g l s , g lo ba l lo ng it ud in al s tr ai n; g l s r s, g lo ba l sy st ol ic l on gi tu di na l st ra in r at e j am soc echocardiogr. author manuscript; available in pmc july . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t levy et al. page ta b le r eg io na l ve nt ri cu la r de fo rm at io n pa tt er ns i n pr et er m i nf an ts a t on e ye ar c or re ct ed a ge b as e m id -v en tr ic le a p ex p - va lu e h ea lt h y u n co m p li ca te d p re te rm i n fa n ts * r v s l s ( % ) − . ± . − . ± . − . ± . < . l v s l s ( % ) − . ± . − . ± . − . ± . < . iv s s l s ( % ) − . ± . − . ± . − . ± . < . p re te rm i n fa n ts w it h b p d a n d /o r p h r v s l s ( % ) − . ± . − . ± . − . ± . . l v s l s ( % ) − . ± . − . ± . − . ± . < . iv s s l s ( % )* * − . ± . − . ± . − . ± . < . d at a ar e pr es en te d as m ea n ± s d . r v , r ig ht v en tr ic le ; l v , l ef t ve nt ri cl e; i v s , i nt er ve nt ri cu la r se pt um s l s , s eg m en ta l lo ng it ud in al s tr ai n b p d , b ro nc ho pu lm on ar y dy sp la si a; p d a , p at en t du ct us a rt er io su s, * h ea lt hy u nc om pl ic at ed , d ef in ed a s in fa nt s w it ho ut b ro nc ho pu lm on ar y dy sp la si a, e ch oc ar di og ra ph ic s ig ns o f pu lm on ar y hy pe rt en si on a t o r w ee ks p m a , a nd /o r a he m od yn am ic al ly s ig ni fi ca nt p at en t du ct us a rt er io su s at d ay – o r an y si ze p d a a t o r w ee ks p m a . ** iv s s l s w it h pr et er m i nf an ts w it h b p d a nd /o r p h r em ai ns i n an a pe x- to -b as e pa tt er n (h ig he st t o lo w er s) , r ef le ct iv e of r v r eg io na l gr ad ie nt . j am soc echocardiogr. author manuscript; available in pmc july . 	abstract 	introduction 	methods 	study population 	inclusion criteria in uncomplicated cohort 	echocardiographic examination 	strain analysis 	two-dimensional speckle tracking imaging 	reproducibility of strain imaging in preterm infants 	statistical analysis 	results 	study population characteristics 	maturational patterns of myocardial strain in uncomplicated preterm infants 	maturational patterns of regional myocardial strain in uncomplicated preterm infants 	confounding cardiopulmonary factors 	ph 	pda 	feasibility and reproducibility 	discussion 	maturational patterns of ventricular strain in uncomplicated preterm infants 	rv fwls and ivs gls 	lv gls 	segmental ls 	clinical implications of deformation imaging in neonates 	bpd and respiratory support 	ph 	pda 	study limitations and future directions 	conclusions 	references 	appendix . reproducibility analysis for segmental longitudinal strain 	table t 	figure 	figure 	figure 	table 	table 	table 	table 	table 	table wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ troponin i ser- phosphorylation sustains troponin ca + sensitivity in an acidic environment a wednesday, february , of cardiac troponin c’s defunct ca þ -binding site (site ). in this study, we combined in vitro and in situ structural and functional techniques to elucidate the role this mutation may play in the modulation of troponin’s function. we used nuclear magnetic resonance to solve the structure and characterize the backbone dynamics of the regulatory lobe of troponin c with this mutation. the overall structure and dynamics of troponin c was not significantly altered by l q; however there was a slight rearrangement of site making it more similar to trout cardiac troponin c, which also has a glutamine at position at residue and displays increased ca þ sensitivity. backbone dynamics mea- surements indicated that q was more flexible than l . the structure and function of l q was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. the structure and/or orientation of the regulatory lobe of troponin c was slightly perturbed by l q in relaxing conditions and was unaffected at activating ca þ concentrations. the ca þ sensitivity of the structural change and contractility were both unaltered by the l q mutation, suggesting that while this may cause a small change in the structure of troponin c, this does not translate to a large functional effect in cardiac muscle. -pos board b troponin i ser- phosphorylation sustains troponin ca d sensitivity in an acidic environment benjamin r. nixon, shane d. walton, jonathan p. davis, brandon j. biesiadecki. physiology and cell biology and davis heart and lung research institute, the ohio state university, columbus, oh, usa. a hallmark of cardiac ischemia is decreased intracellular ph which can affect a number of cellular processes. such an acidic environment alters cardiac troponin (tn) myofilament regulation to decrease ca þ sensitive force produc- tion. tn also undergoes cardiac ischemia-induced ampk troponin i (tni) ser- phosphorylation. we recently characterized the effects of tni ser- phos- phorylation demonstrating that it blunted the functional effects of canonical tni ser- / phosphorylation; however, the role of ser- phosphorylation in ischemia remains unknown. as an initial step, we sought to investigate the effect of acidic ph on myofilament regulation in the presence of tni ser- phos- phorylation alone and in combination with ser- / phosphorylation. we first investigated the effect of in vivo cardiac ischemia on levels of tni ser- and ser- / phosphorylation. exposure to minutes of regional ischemia re- sulted in elevation of both tni ser- and ser- / phosphorylation. next we determined the effects of tni ser- pseudo-phosphorylation (s d) on the myofilament by measuring troponin c (tnc) ca þ binding properties at normal and acidic ph. results demonstrate acidic ph decreases steady- state ca þ binding to tnc in reconstituted thin filaments across all tn (wt, s d, s / d, and s / / d) such that tni s d ca þ sensitivity at ph . is similar to wt at ph . decreasing the ph had no effect on ca þ disso- ciation such that compared to wt, s / / d remained fast while s d was slowed. we conclude that tni ser- phosphorylation imparts resistance to acidic ph-induced myofilament ca þ desensitization while retaining increased tn ca þ dissociation when in combination with ser- / phosphorylation suggesting the potential for an increase in force while maintaining accelerated ca þ dissociation. future investigations are aimed at examining the effect of tni ser- and ser- / phosphorylation on protease cleavage of tni. -pos board b deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue bin wei, yingru lu, j.-p. jin. physiology, wayne state university, detroit, mi, usa. loss of slow skeletal muscle troponin t (sstnt) due to a nonsense mutation at codon glu in exon of the tnnt gene causes a severe form of recessive nemaline myopathy (amish nemaline myopathy, anm). to investigate the pathogenesis and muscle pathophysiology of anm, we studied the phenotypes of partial and total loss of sstnt in tnnt gene targeted mice. an insertion of neomycinr cassette in intron of tnnt caused approximately % decrease in sstnt protein expression whereas deletion of exons - using cre-loxp approach resulted in total loss of sstnt as that seen in the muscle of anm pa- tients. in diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that sstnt deficiency resulted in significantly decreased levels of other slow fiber-specific myofilament proteins while fast fiber-specific myofilament proteins were increased. histology studies revealed that sstnt deficiency caused significant atrophy of type i slow fibers and a hypertrophic growth of type ii fast fibers. along with the slow fiber atrophy and the changes in myofilament protein isoform contents, sstnt deficiency in soleus muscle shifted the force-frequency relationship toward the fast muscle type and significantly reduced the tolerance to fatigue. sstnt deficient soleus muscle also exhibited a significant number of smaller size central nuclei type i fibers, indicating an adaptive regeneration. sstnt deficient mouse soleus muscle contained apparently normal number of spindles, in which intrafusal fibers were positive for type i myosin with a trend of atrophic morphology. the results demonstrate the essential function of sstnt in skeletal muscle and the causal effect of its loss on the pathology of anm. -pos board b attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp thomas j. longyear , matthew a. turner , brandon j. biesiadecki , joseph lopez , jonathan p. davis , edward p. debold . university of massachusetts, amherst, ma, usa, the ohio state university, columbus, oh, usa. repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, which defines fatigue. the decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains unclear. we sought to gain novel insight into the decrease in velocity by determining if the depressive effect of acidosis could be altered by ) introducing ca þþ -sensitizing mutations into troponin (tn) or ) by agents that directly affect myosin function, including inorganic phosphate (pi) and -deoxy-atp (datp) in an in vitro motility assay. acidosis reduced regulated thin filament velocity (vrtf) at both maximal and sub- maximal caþþ levels in a ph-dependent manner. a truncated construct of the inhibitory subunit of tn, r , and a caþþ-sensitizing mutation in the caþþ- binding subunit of tn, v q, increased vrtf at sub-maximal ca þþ under acidic conditions, but had no effect on vrtf at maximal ca þþ levels. in contrast, both mm pi and replacement of atp with datp reversed much of the acidosis- induced depression of vrtf at saturating ca þþ ( . . control, . . withpi, . . withdatp, . . withbothpi anddatp),withthecombined effectfully restoringthevrtf to thevalue under control conditions.interestingly, despite producing similar magnitude increases in vrtf, the combined effects of pi and datp were additive, suggesting different underlying mechanisms of action. these results suggest that the major mechanism by which acidosis slows vrtf is through directly slowing myosin’s rate of detachment from actin. -pos board b the effect of truncated troponin components on activation of lethocerus flight muscle belinda bullard , bogos agianian , gian-felice de nicola , annalisa pastore , kevin leonard . department of biology, university of york, york, united kingdom, molecular biology and genetics, democritus university of thrace, alexandroupolis, greece, national institute of medical research, london, united kingdom, european bioinformatics institute, cambridge, united kingdom. indirect flight muscle (ifm) of lethocerus is activated by periodic stretches at a constant priming concentration of calcium. the muscle is unusually stiff and stress is transmitted to the thick and thin filaments by kettin, which reinforces links between both filaments and the z-disc. the activating effect of stress on thin filaments is likely to affect troponin. the isoforms of troponin in ifm differ from those in other muscles. tnt has a c-terminal extension not present in vertebrate tnt; tnh is an isoform of tni with a c-terminal extension rich in pro and ala; tnc is present in two isoforms: f binds a single calcium in the c-lobe and is needed for stretch-activation; f binds one calcium in both n- and c-lobes and is needed for isometric force. under conditions of low ionic strength, native fibres have a force-pca curve that shows high calcium- sensitivity and low cooperativity (pca = . , nh = . ). fibres with f alone have a pca curve similar to that of cardiac muscle, (pca = . , nh = . ). a fragment of f without the n-lobe (f -ct) inhibits stretch-activation; therefore the n-lobe of f is necessary, although it does not bind calcium or tnh. f -ct is displaced by f and isometric force is restored, but not stretch-activation. we hope to show the effect of replacing endogenous troponin in fibres with a com- plex containing tnt truncated at the c-terminus, tnh with tni sequence but without the pro-ala extension, and either f or f . this will show how impor- tant the ifm isoforms of troponin are to the stretch-activation response. -pos board b changes in the orientation of the myosin light chain domain (lcd) associated with thick filament-based regulation of skeletal muscle luca fusi, zhe huang, malcolm irving. randall division of cell and molecular biophysics, king’s college london, london, united kingdom. the dependence of myosin lcd orientation on temperature, myofilament lattice spacing and sarcomere length was determined using fluorescence 	troponin i ser- phosphorylation sustains troponin ca + sensitivity in an acidic environment 	deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue 	attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp 	the effect of truncated troponin components on activation of lethocerus flight muscle 	changes in the orientation of the myosin light chain domain (lcd) associated with thick filament-based regulation of skelet ... physical activity and the association of common fto gene variants with body mass index and obesity original investigation physical activity and the association of common fto gene variants with body mass index and obesity evadnie rampersaud, msph, phd; braxton d. mitchell, phd; toni i. pollin, phd; mao fu, phd; haiqing shen, phd; jeffery r. o’connell, phd; julie l. ducharme, md; scott hines, md; paul sack, md; rosalie naglieri, md; alan r. shuldiner, md; soren snitker, md, phd background: common fto (fat mass and obesity asso- ciated) gene variants have recently been associated with body mass index (bmi) and obesity in several large studies. the role of lifestyle factors (such as physical activity) in those with an underlying fto genetic predisposition is unknown. methods: to determine if fto variants are associated with bmi in old order amish (ooa) individuals, and to further determine whether the detrimental associa- tions of fto gene variants can be lessened by increased physical activity, a total of healthy ooa adults were selected from the heredity and phenotype intervention (hapi) heart study, an investigation of gene � environ- ment interactions in cardiovascular disease, for whom ob- jective quantified physical activity measurements were available and for whom single-nucleotide polymor- phisms (snps) in fto were genotyped. results: twenty-six fto snps were associated with bmi (p = . to �. ), including rs (p � . ) and rs (p � . ), snps in moderate linkage dis- equilibrium in the ooa (d� = . ; r = . ). stratified analyses of rs revealed its association with bmi to be restricted entirely to those subjects with low sex- and age-adjusted physical activity scores (p � . ); in contrast, the snp had no effect on those with above- average physical activity scores (p = . ), with the genotype � physical activity interaction achieving statis- tical significance (p = . ). similar evidence for interac- tion was also obtained for rs . conclusions: our results strongly suggest that the in- creased risk of obesity owing to genetic susceptibility by fto variants can be blunted through physical activity. these findings emphasize the important role of physi- cal activity in public health efforts to combat obesity, par- ticularly in genetically susceptible individuals. arch intern med. ; ( ): - o besity and related co- morbid conditions rep- resent a global public health burden and ac- count for a growing por- tion of health care spending in the indus- trialized world. it is widely acknowledged that there is a substantial genetic contri- bution to body mass index (bmi), and re- cently, robust associations of common vari- ants in intron of the fat mass and obesity associated (fto) gene with bmi, percent- age of body fat, and obesity were identi- fied in large studies of white adults and chil- dren. - owing to the high frequency of the obesity-associated fto variants (about % allele frequency for the most strongly associated single-nucleotide polymor- phisms [snps] in european populations) and their impact (each “risk allele” is as- sociated with a . -kg increase in body weight), these variants carry a population- attributable risk for obesity of greater than % in the studied populations. , the function of fto is incompletely un- derstood, although recent work has dem- onstrated that this gene codes for a protein expressed in the hypothalamus, a center of energy balance, and adipose tissue, where it is localized in cell nuclei and may be in- volved in demethylation of dna and per- haps other, as-yet unidentified, functions. in addition to genetic factors, lifestyle factors, including diet and physical inac- tivity, are important contributors to weight gain and obesity. although physical ac- tivity has been shown to facilitate weight loss and weight maintenance in obese sub- jects, there is great interindividual varia- tion in response. it is unknown whether lifestyle factors, such as physical activity, can attenuate weight gain and obesity in author affiliations: department of medicine, university of maryland (drs rampersaud, mitchell, pollin, fu, shen, o’connell, ducharme, hines, sack, naglieri, shuldiner, and snitker), and the geriatric research and education clinical center, baltimore veterans administration medical center (dr shuldiner), baltimore. dr rampersaud is now with the miller school of medicine, miami institute for human genetics, university of miami, miami, florida. (reprinted) arch intern med/ vol (no. ), sep , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / those with an underlying fto genetic predisposition. the specific issues addressed in this report are whether fto variants are associated with bmi in the old order amish (ooa), a population in which moderate to high levels of physical activity are common and, if so, whether the detrimental associations of bmi-associated fto gene vari- ants can be lessened by increased physical activity. methods study participants study participants were members of the ooa community in lancaster county, pennsylvania. the ooa are a rural-living, closed, founder population of european origin, well known for eschewing many modern conveniences; they do not own cars or use electricity in their homes. most ooa men are farmers or work in physically demanding occupations such as black- smithing and carpentry. women are homemakers, working with- out the aid of modern appliances and often taking care of many children. similarities in tradition, formal education, and geo- graphic location among the ooa make for a relatively homo- geneous lifestyle, including dietary habits. individuals in- cluded in this report took part in the heredity and phenotype intervention (hapi) heart study, an investigation of gene � envi- ronment interactions in cardiovascular disease of adult ooa individuals in generally good health, who were recruited from to . subjects provided blood samples for dna analy- sis and underwent a wide panel of physiological tests, includ- ing -day measurement of physical activity by accelerometry. the protocol was approved by the institutional review board of the university of maryland, baltimore. subjects gave in- formed consent before participation. phenotypic assessment all study subjects underwent a detailed clinical examination at the amish research clinic in strasburg, pennsylvania. height and weight were measured by trained nurses in subjects with- out shoes and in light clothing using a stadiometer and cali- brated scale. body mass index was calculated as weight in ki- lograms divided by height in meters squared. subjects with a bmi of or greater and less than were defined as being overweight, and those with a bmi of or greater were de- fined as obese. additional measures of other obesity-related traits were obtained, including waist circumference, measured to the nearest . cm using an inelastic tape, and in a subset of individuals, body composition (fat mass, lean mass, and de- rived percentage of body fat) as determined by dual energy x-ray absorptiometry (dexa). objective measurement of physical activity was obtained over consecutive -hour days using actical activity monitors (ver- sions . and . ; mini mitter co inc, bend, oregon) worn on the hip. these devices incorporate an accelerometer, sensitive to . times gravity in multiple directions, electronic cir- cuitry, and a memory. acceleration of the device is integrated and expressed as a number—activity counts—for each - second recording interval that is stored in memory until the device is returned and data uploaded. activity can be ex- pressed as raw activity counts, inherently independent of body size, or as its associated energy expenditure (in kilocalories per day) using formulas validated against gas exchange. acceler- ometry is a well-established method to quantify physical ac- tivity. of the hapi heart study participants, physical ac- tivity measurements were available for subjects, of whom had valid genotype data. genotyping genomic dna isolated from whole blood was genotyped with the affymetrix genechip human mapping k array set (af- fymetrix, santa clara, california). total genomic dna ( ng) was digested with nspi or styi enzymes and processed accord- ing to the affymetrix protocol. the genechip genotyping analy- sis software (gtype version . ; affymetrix) was used for au- tomated genotype calling as part of the genechip operating software platform. the gtype-generated chip files were re- analyzed using the bayesian robust linear model with maha- lanobis (brlmm) genotype calling algorithm (free, download- able software available at http://www.affymetrix.com/support /technical/whitepapers/brlmm_whitepaper.pdf) and a confidence threshold for call quality of . . as an initial quality control measure, brlmm-generated chip files with genotype call rates of less than % for both enzymes were excluded from further analysis. for this study, snps that were either monomorphic or that deviated from hardy-weinberg equilibrium using a cut point of p � . were excluded from analysis. the fto gene, located on chromosome q . , is approxi- mately kilobases (kb) in length and contains exons. the affymetrix k arrays contain snps within the region of this gene. the mean genotype call rate was . % for these snps. statistical analyses association analyses of fto snps and trait variables were per- formed using a variance component approach. non–normally distributed variables were natural log-transformed prior to analy- ses. we modeled bmi and related traits as a function of mea- sured environmental covariates, additive genetic associations, and a residual error component. the associations of age, age , sex, and interaction terms for age and sex were estimated jointly with genotype associations. genotype was scored using an ad- ditive model to allow for allele dosage effects. parameter esti- mates were obtained by maximum likelihood methods, and the significance of association was tested by a likelihood ratio test. we accounted for the relatedness of ooa study subjects by es- timating parameter effects conditional on residual correla- tions in bmi (or similar trait) between related individuals. the association analyses were performed using the solar soft- ware program. pairwise linkage disequilibrium (ld) correlation statistics (r ) were computed using haploview beta software, version . . mul- tilocus haplotype analyses were performed using haplostats soft- ware, , which estimates haplotype frequencies using an ex- pectation-maximization algorithm in situations in which the haplotype phase is ambiguous. global and haplotype-specific score statistics and permutation-based p values were calculated after adjustments were made for sex, age, and age . these analyses did not take into account the relatedness of study subjects. we evaluated the associations of fto snps on bmi after strati- fication of the sample according to “high” and “low” physical activity strata. because the daily number of physical activity ac- celerometer counts was generally lower in women than in men and decreased with age in both sexes (data not shown), this strati- fication was performed in a sex- and age-specific fashion. after logarithmic transformation of mean total physical activity counts, subjects were dichotomized into the high- or low-activity stra- tum depending on whether their age-, age - and sex-specific re- siduals were greater than or less than . in addition to these strati- fied analyses, we determined whether the association of genotype on bmi was modified by physical activity levels by constructing a regression model that included the following independent variables: sex, age, age , age � sex, age � sex, snp genotype, ln-transformed physical activity counts, and an interaction term (reprinted) arch intern med/ vol (no. ), sep , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / (ln-transformed physical activity counts � genotype). the pres- ence of an interaction between physical activity and snp geno- type on bmi was assessed by a likelihood ratio test, in which we compared a model with the interaction term (full model) with a model without the interaction term (nested model). as in pre- vious analyses, these analyses were performed using variance com- ponents analyses in the solar software program to account for the correlations in bmi among family members. for the conversion of accelerometer counts into activity en- ergy expenditure (in kilocalories per day), we used actical com- puter software (version . ), provided by the manufacturer of the accelerometers. this software applies a validated regres- sion equation to accelerometer counts on a per-epoch basis. results a total of subjects with both physical activity and genotype data were included in our analyses. the mean (sd) age was . ( . ) years, and the sample included slightly more men than women ( % vs %). the mean bmi was higher in women ( . [ . ]) than in men ( . [ . ]). the prevalence of overweight and obesity in ooa men was . % and . %, respectively, and in ooa women was . % and . %, respectively. mean physi- cal activity in men and women amounted to ( ) and ( ) � counts/d, respectively. fto snp associations with obesity and related traits we analyzed snps falling in a -kb interval that spanned fto (data are available from the authors at http: //medschool.umaryland.edu/endocrinology/afdspublic .asp). of these snps, were associated with bmi (p = . to �. under an additive genetic model). rs , the associated snp originally reported by frayling et al, was modestly associated with obesity (p = . ) and not associated with bmi (p = . ) in the ooa, although stron- ger associations with this snp were seen with total fat mass (p = . ) and percentage of body fat (p = . ). figure shows snps in the region surrounding rs . these snps all fall within an -kb re- gion spanning from within intron to within intron of the fto gene. the snp most strongly associated with bmi in the ooa was rs , a common snp with a similar risk allele frequency ( . ) as in hapmap (http:// www.hapmap.org) genotype samples of white individu- als ( . ). each a allele of rs was associated with a . increase in bmi (p � . ), corresponding to a mean (sd) increase in weight of . ( . ) kg per allele (table ). individuals with the a allele for rs were at increased risk of being obese (odds ratio [or], . ; p = . ) and of being overweight (or, . ; p = . ). the a allele of rs was also associated with greater waist circumference (p = . ) and weight (p = . ). in a smaller subset of subjects, the same directional associations were seen with total fat mass (p � . ) and with percentage of fat mass (p = . ). sex-, age-, and age -adjusted physical activity levels did not dif- fer significantly according to genotype (p = . ). similar associations were observed with the c allele of rs , the second most significant snp in the ooa. in the ooa, each risk allele for rs was as- sociated with a mean (sd) increase in bmi of . ( . ) (p � . ). the snps rs and rs are . kb apart in intron and are in moderate ld in the ooa (d� = . ; r = . ). we examined the relationship between the best snps identified from our analyses with rs . in the ooa, this snp is in strong ld with rs (d� = ; r = . ) but not with rs (d� = . ; r = . ) (figure ). together, the snps (ordered from � to � rs - rs -rs ) defined possible haplotypes ( with frequencies � %), of which were associated with bmi (global permuted p = . ) (table ). the gta hap- lotype had a frequency of . % and was associated with decreased bmi (permuted p � . ), whereas the comple- mentary act haplotype had a frequency of . % and was associated with increased bmi (permuted p = . ). the less frequent aca haplotype ( . %) was also as- sociated with increased bmi (permuted p = . ). these results suggest that in the ooa, regardless of rs allele (a or t), rs and rs define a hap- lotype that is associated with increased bmi. analyses stratified by physical activity levels we next assessed the relationship of rs with bmi and obesity, stratified according to physical activity level (figure ). among those within the lower half of the physical activity distribution (n = ), the rs a allele was strongly associated with greater bmi (a mean [sd] increase of . [ . ] for each risk allele; p � . ). in contrast, bmi was not significantly associated with the rs genotype in subjects in the upper half of the physical activity distribution (n = ) ( . [ . ] in- crease in bmi per risk allele; p = . ). rs was also associated with being obese in the low-activity group (or, . for each risk allele; p = . ) but not in the high- activity group (p = . ). a formal test for interaction re- vealed significant evidence that increased physical ac- tivity levels blunted the association between the rs a allele on bmi (interaction p = . ). in figure , we graphically illustrate, using the predicted values from the linear regression of residualized physical activity on bmi and rs genotype, that the difference in bmi across rs genotypes is large in less physically active in- dividuals but small and not statistically significant in more physically active individuals. the impact of increased physical activity on bmi reduction is most apparent for individuals with the rs aa genotype. similarly, the rs c allele was associated with a . ( . ) increase in bmi per risk allele in the low- activity group (p = . ) but only a . ( . ) increase in bmi in the high-activity group (p = . ) (interaction p = . ) (figure ). the rs c allele was associ- ated with obesity in the low-activity group (or, . for each risk allele; p = . ) but not in the high-activity group (p = . ). the level of physical activity in the strata can be described by the energy expenditure at the th and th centiles of the distribution, respectively. for women, energy expenditure was and kcal/d (reprinted) arch intern med/ vol (no. ), sep , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / . . . . . . . . –l og ( p va lu e) a b c rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs bmi block ( kb)block ( kb) block ( kb) x x block ( kb) block ( kb) figure . linkage disequilibrium (ld) structure and fto single-nucleotide polymorphism (snp) associations with body mass index (bmi). a, for each snp surrounding rs , we show –log (p value) for the additive model in the old order amish (ooa). b, the ld (r ) of fto in ooa white subjects. c, the ld (r ) of fto in the hapmap (http://www.hapmap.org) genotype of white individuals. the association of rs with bmi was first reported in european white individuals and subsequently in french white individuals ; rs was the snp most highly associated with bmi in a sardinian population ; rs was associated with type diabetes mellitus (t dm) in european white subjects. - zeggini et al later found that the association disappeared after adjustment for bmi. in an independent sample of ooa subjects (of whom had t dm), rs was associated with t dm (p = . ), and this association was abolished after adjustment for bmi (p = . ) (data not shown). rs , rs , and rs all reside in a cluster of snps in high ld in hapmap genotypes of white individuals. black squares indicate r � . ; gray squares, . � r � . ; white squares, r � . . kb indicates kilobases. (reprinted) arch intern med/ vol (no. ), sep , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / at the th and th centiles, respectively, whereas for men energy expenditure was and kcal/d at the same centiles. thus, in this population, a mean activity level of calories for women and calories for men separates the high- and low-activity strata, which differ in their phenotypic expression of the at-risk fto genotypes. comment our study replicates the association between variants in the fto gene and obesity-related traits (eg, bmi, body weight, waist circumference, and percentage of body fat) recently reported by others. - , - furthermore, we have shown that the association of genotype on body compo- sition is much smaller and not statistically significant in subjects having higher physical activity levels. the snps most strongly associated with bmi in our study were rs and rs , both of which are common in the ooa ( . and . , respectively) and in non- ooa white individuals ( . and . , respectively). these snps are in higher ld in the ooa (d� = . ; r = . ) than in non-ooa european white individuals (hapmap genotypes from white individuals, d� = . , r = . ). greater ld in the ooa compared with the gen- eral white population might be expected because the ooa are a relatively young founder population. although an association of rs with obesity (p = . � − based on an additive model) has previously been reported in a study of french adults, to our knowledge, the associa- tion of rs with bmi or obesity has not been de- scribed. in our analyses, a common haplotype defined by both snps (rs , a allele; rs , c allele) seems to jointly confer risk to increased bmi. this seems to be independent of rs , the snp originally found to be associated with bmi in multiple european white populations , (table ). at least in the ooa, these snps may both be marking a common, yet unknown func- tional allele or may themselves have functional effects on body size through an as-yet undetermined mechanism. further fine mapping with additional genotyping to help localize the true functional variant was beyond the scope of the current study. a primary finding of this study is that of a gene � envi- ronment interaction between variants in the fto gene and physical activity. using objective-, age-, and sex- adjusted physical activity measures obtained for con- secutive -hour days, we were able to investigate the genotype associations of fto variants in ooa subjects with high and low physical activity levels. unlike ques- tionnaires, the actical device provides an objective esti- mate of physical activity that is unaffected by study par- ticipant recall bias. we found that snp associations with bmi and related measures were limited to less physically active individuals. in the less active group, table . clinical characteristics of the study population by fto rs genotypes a aa (n = ) ag (n = ) gg (n = ) p value characteristic women, % . . . . age, y . ± . . ± . . ± . . bmi . ± . . ± . . ± . �. waist circumference, cm . ± . . ± . . ± . . weight, kg . ± . . ± . . ± . . height, cm . ± . . ± . . ± . . overweight ( � bmi � ), % . . . . obese (bmi � ), % . . . . total daily accelerometer counts/ , median ( %, % percentiles) ( , ) ( , ) ( , ) physical activity b − . ± . . ± . . ± . . fat and lean mass c (n = ) (n = ) (n = ) total fat mass, kg . ± . . ± . . ± . �. total lean mass, kg . ± . . ± . . ± . . total % fat . ± . . ± . . ± . . abbreviation: bmi, body mass index (calculated as weight in kilograms divided by height in meters squared). a data are given as mean ± sd except where noted; “n” indicates the number of samples. b group means for age- and sex-residualized daily physical activity counts. c measures of body composition as determined by dual-energy x-ray absorptiometry (dexa). table . haplotypes for fto snps rs , rs , and rs haplotype frequency haplotype score p value a gta . − . �. gca . − . . gct . − . . ata . . . act . . . aca . . . abbreviation: snp, single-nucleotide polymorphisms. a global p = . ; df = . p values were calculated by score test after adjustment for sex, age, and age , using haplostats software. , statistical analysis was restricted to samples completely genotyped at all snps (n = ). (reprinted) arch intern med/ vol (no. ), sep , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / being aa homozygous for the rs fto variant was associated with an increase in bmi of . , a slightly higher estimate than that obtained by frayling et al for rs in united kingdom (uk) wellcome trust case control subjects with type diabetes mellitus, and much higher than the same group’s estimate for rs in uk type diabetes mellitus genetics con- sortium controls. by contrast, in the more physically active ooa stratum, the associations of the fto vari- ants were much smaller and not statistically significant. because the fto genotype was not associated with physical activity, this finding suggests a strong moderat- ing effect of physical activity on the deleterious effects of fto variants, consistent with published findings based on self-reported physical activity data. when men and women were evaluated separately without consid- eration of covariates, the association of rs genotype reached nominal statistical significance in women only, not in men. however, there was no evi- dence that the effect of genotype on bmi differed sub- stantially between men and women, particularly when the level of physical activity was included in the regres- sion model (ie, the genotype by sex interaction term was not statistically significant; p = . ). recent work , has demonstrated that fto codes for a protein expressed in the hypothalamus, the function of which is impacted in many obesity-related genetic defects in humans. because fto is down-regulated by krebs cycle intermediates, it is conceivable that this protein is involved in incompletely understood nutrient sensing pathways, which are pivotal to central regula- tion of energy intake. thus, a mechanism whereby increased physical activity can negate the association of fto variants with fat accretion could be through pertu- bation of energy flux resulting in alterations in expres- sion of fto. aa ag gg genotype m ea n bm i a p < . n = n = n = aa ag gg genotype p = . n = n = n = cc ct tt genotype p = . n = n = n = rs cc ct tt genotype m ea n bm i b p = . n = n = n = rs figure . mean body mass index (bmi), calculated as weight in kilograms divided by height in meters squared, in high and low physical activity groups according to fto single-nucleotide polymorphism genotypes for rs (a) and rs (b). high activity was defined as age-, age -, and sex-specific residuals greater than , and low activity was defined by residuals less than . interaction p values: rs , p = . ; rs , p = . . . . . . . . . . . . . – . – . – . – . . . . . residualized physical activity bm i aa ga gg genotype figure . predicted body mass index (bmi), calculated as weight in kilograms divided by height in meters squared, as a function of residualized age- and sex-specific ln-transformed physical activity accelerometer counts according to fto rs genotypes. on the left side of the plot (low physical activity), bmi levels are strikingly dissimilar between rs genotypes. in contrast, on the right side of the plot, similar bmi levels can be seen across genotypes, particularly in subjects with very high levels of physical activity. (reprinted) arch intern med/ vol (no. ), sep , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / activity levels in the “high-activity” stratum were ap- proximately kcal higher than in the “low-activity” stratum, which, depending on body size, corresponds to about to hours of moderately intensive physical ac- tivity, such as brisk walking, house cleaning, or garden- ing. although this seems to be a large amount of physi- cal activity, the ooa demonstrate that this level of activity was typical of an agrarian lifestyle without modern ma- chinery. of course, our cross-sectional study is un- suited to determine the amount of activity required to negate the effect of an fto-related genetic predisposi- tion to weight gain; however, in a retrospective analysis in which weight regain was measured as a function of physical activity energy expenditure, schoeller et al found that the addition of min/d of moderate activity or min/d of vigorous activity to a sedentary lifestyle was sufficient for weight maintenance. prospective in- tervention studies will be necessary to define these para- meters more accurately. in conclusion, we have replicated the associations of common snps in the fto gene with increased bmi and risk to obesity in the ooa. furthermore, we provide quan- titative data to show that the weight increase resulting from the presence of these snps is much smaller and not statistically significant in subjects who are very physi- cally active. this finding offers some clues to the mecha- nism by which fto influences changes in bmi and may have important implications in targeting personalized life- style recommendations to prevent obesity in genetically susceptible individuals. accepted for publication: march , . correspondence: evadnie rampersaud, msph, phd, miller school of medicine, miami institute for human genetics, university of miami, po box (m- ), miami, fl (erampersaud@med.miami.edu). author contributions: study concept and design: rampersaud, mitchell, shen, shuldiner, and snitker. ac- quisition of data: fu, ducharme, hines, sack, naglieri, shuldiner, and snitker. analysis and interpretation of data: rampersaud, mitchell, pollin, fu, o’connell, shuldiner, and snitker. drafting of the manuscript: rampersaud, mitchell, and snitker. critical revision of the manuscript for important intellectual content: rampersaud, mitchell, pollin, fu, shen, o’connell, ducharme, hines, sack, naglieri, shuldiner, and snitker. statistical analysis: rampersaud, mitchell, pollin, shen, o’connell, and snitker. obtained funding: shuldiner. administrative, technical, and material support: fu, ducharme, and sack. study supervision: mitchell, shuldiner, and snitker. financial disclosure: none reported. references . frayling tm, timpson nj, weedon mn, et al. a common variant in the fto gene is associated with body mass index and predisposes to childhood and adult obesity. science. ; 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( ): - . . westerterp kr. physical activity assessment with accelerometers. int j obes relat metab disord. ; (suppl ):s -s . . almasy l, blangero j. multipoint quantitative-trait linkage analysis in general pedigrees. am j hum genet. ; ( ): - . . barrett jc, fry b, maller j, daly mj. haploview: analysis and visualization of ld and haplotype maps. bioinformatics. ; ( ): - . . lake sl, lyon h, tantisira k, et al. estimation and tests of haplotype-environment interaction when linkage phase is ambiguous. hum hered. ; ( ): - . . schaid dj, rowland cm, tines de, jacobson rm, poland ga. score tests for association between traits and haplotypes when linkage phase is ambiguous. am j hum genet. ; ( ): - . . zeggini e, weedon mn, lindgren cm, et al; wellcome trust case control con- sortium. replication of genome-wide association signals in uk samples reveals risk loci for type diabetes. science. ; ( ): - . . scott lj, mohlke kl, bonnycastle ll, et al. a genome-wide association study of type diabetes in finns detects multiple susceptibility variants. science. ; 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(reprinted) arch intern med/ vol (no. ), sep , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / mission of insomnia among elderly adults: an epidemiologic study of , per- sons over three years. sleep. ; (suppl ):s -s . . ganguli m, reynolds cf, gilby je. prevalence and persistence of sleep com- plaints in a rural older community sample: the movies project. j am geriatr soc. ; ( ): - . . quan sf, katz r, olson j, et al. factors associated with incidence and persis- tence of symptoms of disturbed sleep in an elderly cohort: the cardiovascular health study. am j med sci. ; ( ): - . . foley dj, monjan aa, izmirlian g, hays jc, blazer dg. incidence and remission of insomnia among elderly adults in a biracial cohort. sleep. ; (suppl ): s -s . . mendelson wb. long-term follow-up of chronic insomnia. sleep. ; ( ): - . . kish l. survey sampling. new york, ny: john wiley & sons inc; . . morin cm, beaulieu-bonneau s, leblanc m, savard j. self-help treatment for insomnia: a randomized controlled trial. sleep. ; ( ): - . . leblanc m, mérette c, savard j, baillargeon l, morin cm. prospective data on insomnia incidence and risk factors in a population-based sample of good sleepers. sleep. in press. . bastien ch, vallières a, morin cm. validation of the insomnia severity index as a clinical outcome measure for insomnia research. sleep med. ; ( ): - . . buysse dj, reynolds cf, monk th, berman s, kupfer dj. the pittsburgh sleep quality index: a new instrument for psychiatric practice and research. psychia- try res. ; ( ): - . . buysse dj, ancoli-israel s, edinger je, lichstein kl, morin cm. recommenda- tions for a standard research assessment of insomnia. sleep. ; ( ): - . . blais fc, gendron l, mimeault v, morin cm. assessment of insomnia: valida- tion of three questionnaires. encephale. ; ( ): - . . agresti a. an introduction to categorical data analysis. new york, ny: john wiley & sons inc; . . vollrath m, wicki w, angst j. the zurich study, viii: insomnia: association with depression, anxiety, somatic syndromes, and course of insomnia. eur arch psy- chiatry neurol sci. ; ( ): - . . jansson m, linton sj. psychological mechanisms in the maintenance of insom- nia: arousal, distress, and sleep-related beliefs. behav res ther. ; ( ): - . . american academy of sleep medicine. international classification of sleep dis- orders: diagnostic and coding manual. nd ed. westchester, il: american acad- emy of sleep medicine; . . edinger jd, bonnet mh, bootzin rr, et al; american academy of sleep medi- cine work group. derivation of research diagnostic criteria for insomnia: report of an american academy of sleep medicine work group. sleep. ; ( ): - . correction errors in funding/support, role of the sponsor, and additional contributions: the original investigation titled “physical activity and the association of com- mon fto gene variants with body mass index and obe- sity,” which was published in the september , , is- sue of the archives ( ; [ ]: - ), contained omissions of the funding/support, role of the sponsor, and additional contributions paragraphs. the follow- ing information should have been included: funding/support: the hapi heart study receives funding from national institutes of health (nih) grant u hl . partial funding for this study was pro- vided by the clinical nutrition research unit of mary- land, grant p dk ; the university of maryland general clinical research center, grant m rr ; the johns hopkins university general clinical re- search center, grant m rr ; and the geriatric research and education clinical center, baltimore vet- erans administration medical center. dr rampersaud was funded by a postdoctoral nih/national heart, lung, and blood institute–sponsored nrsa training grant t hl . role of the sponsor: as part of the progeni net- work, this project was overseen by an nih-appointed data safety and monitoring board. additional contributions: we thank the amish study participants and our amish research clinic and laboratory staff for their extraordinary efforts. (reprinted) arch intern med/ vol (no. ), mar , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / functional variants in mbl are associated with type diabetes and pre-diabetes traits in pima indians and the old order amish yunhua l. muller, robert l. hanson, li bian, janel mack, xiaolian shi, ruth pakyz, alan r. shuldiner, , william c. knowler, clifton bogardus, and leslie j. baier objective—mbl encodes the mannose-binding lectin, which is a key player in the innate immune system and has recently been found to play a role in insulin resistance and development of type diabetes and gestational diabetes mellitus. to assess the role of mbl in diabetes susceptibility, this gene was analyzed in the pima indian population, which has a high prevalence of type diabetes. research design and methods—nineteen tag single nucleotide polymorphisms (snps) were genotyped in a popula- tion-based sample of , full-heritage pima indians, and se- lected snps were further genotyped in independent samples of native american (n � , ) and old order amish (n � ) subjects. results—two variants, a promoter snp (rs ) at � bp with a risk allele frequency of . and a gly asp (rs ) with a risk allele frequency of . , were associated with type diabetes in the full-heritage pima indians (odds ratio . per copy of the g allele for rs , p � . , and . per copy of the glycine allele for rs , p � . , adjusted for age, sex, birth year, and family membership). these associations replicated in an independent native american sample ( . , p � . , for rs ) and a caucasian sample, the old order amish ( . , p � . , for rs and . , p � . , for rs ). among pima indians with normal glucose tolerance, the diabetes risk allele glycine of gly asp was associated with a decreased acute insulin response to an intravenous glucose bolus infusion (p � . , adjusted for age, sex, percent body fat, glucose disposal under physiological insulin stimulation, and family membership). conclusions—our data suggest that the functional variants in mbl contribute to type diabetes susceptibility in both native americans and the old order amish. diabetes : – , m annose-binding lectin (mbl) is a liver-derived serum lectin involved in the innate immune defense. upon binding to specific carbohy- drate structures on various microorganisms, mbl may utilize mbl serine protease (masp)- to activate the third pathway of complement (lectin pathway) and thereby opsonophagocytosis ( ). serum mbl levels have been shown to be strongly correlated with the presence of variants within the mbl gene. missense polymorphisms at codon (resulting in a glycine to aspartic acid), codon (resulting in a glycine to glutamic acid), and codon (resulting in an arginine to cysteine) impair oligomer formation, leading to reduced serum levels of functional mbl. in addition, three pro- moter polymorphisms at position � bp g � c (h/l), � bp g � c (y/x) and � bp c � t (p/q) influence the expression of mbl ( – ). deficiency of mbl has been associated with immunode- ficiency, autoimmune disorders such as systemic lupus erythematosus, and rheumatoid arthritis ( , ). recent studies have further implicated mbl deficiency in the development of type diabetes ( ), gestational diabetes mellitus ( ), diabetic nephropathy ( ), and insulin resis- tance and obesity ( ). based on the biological role of mbl , this gene was investigated as a potential suscepti- bility gene for type diabetes in pima indians. research design and methods subjects in the present study are part of a longitudinal study of the etiology of type diabetes among the gila river indian community in arizona, where most of the residents are pima indians or of the closely related tohono o’odham tribe. diabetes status was determined by an oral glucose tolerance test according to the criteria of the world health organization ( ). the initial genetic study was conducted in a population-based sample of full-heritage pima indians (n � , ), where , subjects had type diabetes ( % male, age at the last exam � years, and bmi � kg/m ) and , subjects were nondiabetic ( % male, age at the last exam � years, and bmi � kg/m ). independent replication was assessed in , subjects from the same longitudinal study who were of mixed native american heritage (reported heritage, on average, was one-half pima and three-quarters native american). the replication sample had subjects with type diabetes ( % male, age at the last exam � years, and bmi � kg/m ) and , nondiabetic subjects ( % male, age at the last exam � years, and bmi � kg/m ). additional replication was assessed in a case-control sample from the old order amish ( diabetic subjects and subjects with normal glucose tolerance) as previously described ( ). metabolic quantitative traits. among the full-heritage pima indians, subjects ( % male, age � years, and bmi � kg/m ) had undergone detailed metabolic testing for risk factors that predict type diabetes. these individuals were determined to be nondiabetic, and acute insulin response was only analyzed in subjects who had normal glucose tolerance. glucose tolerance was determined by a -g oral glucose tolerance test (ogtt) with measurements of fasting and , , , and -min plasma glucose and from the diabetes molecular genetics section and,diabetes epidemiology and clinical research section, the phoenix epidemiology and clinical research branch, the national institute of diabetes and digestive and kidney disease, the national institutes of health, phoenix, arizona; the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland; and the geriatric research and education clinical center, veterans administration medical center, balti- more, maryland. corresponding author: leslie baier, lbaier@phx.niddk.nih.gov. received october and accepted may . published ahead of print at http://diabetes.diabetesjournals.org on june . doi: . /db - . © by the american diabetes association. readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. see http://creativecommons.org/licenses/by -nc-nd/ . / for details. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. brief report diabetes, vol. , august diabetes.diabetesjournals.org insulin concentrations ( ). the acute insulin response to intravenous glucose was measured on a separate day from the ogtt. blood samples were collected prior to a -g glucose intravenous bolus infusion and at , , , , , and min after infusion. the acute insulin response was calculated as the mean increment in plasma insulin concentrations from – min ( ). insulin sensitivity was assessed using the hyperinsulinemic-euglycemic clamp tech- nique as previously described ( , ). body composition was estimated by underwater weighing until january and by dual-energy x-ray absorpti- ometry (dpx- ; lunar radiation) thereafter ( ). snp identification and genotyping. dna from pima indians ( nondi- abetic and aged � years; diabetic with onset age � years) was sequenced using a big dye terminator (applied biosystems) on an automated dna capillary sequencer (model ; applied biosystems). genotyping was done using the snplex genotyping system -plex (applied biosystems) on an automated dna capillary sequencer (model ; applied biosystems) for the native american samples and taqman genotyping assays (applied biosys- tems) for the old order amish samples. statistical analysis. in the pima study, statistical analyses were performed using the software of the sas institute (cary, nc). the general association of genotypes with type diabetes was assessed by logistic regression analysis and was adjusted for covariates (age, sex, and birth year). the model was fit with a generalized estimating equation technique to account for correlation among siblings. genotype was analyzed as a numeric variable representing the number ( , , ) of copies of a given allele. the association of quantitative traits with genotypes was analyzed by linear regression using the generalized estimating equation procedure to account for correlation among siblings. p values were adjusted for potential confounding covariates. in the replication study, which included individuals of mixed ancestry, the individual estimate of european admixture was also used as a covariate. these estimates were derived by the method of hanis et al. ( ) from informative markers with large differences in allele frequency between populations ( ). linkage disequilibrium (ld) and haplotype blocks were estimated by haploview (version . ). for the amish study, the odds ratio (or) is derived from a logistic regression model, while the p value is based on the normal-liability threshold model implemented in solar to account for relationships among individuals. cis (and the approximate se) are test based ( , ). results and discussion sequencing of the mbl gene (all four exons, three introns, and � kb of the upstream region) in pima indians identified variants. three were previously known vari- ents which predicted missense substitutions—rs (arg cys), rs (gly asp), and rs (gly glu)— commonly referred to as a/d, a/b, and a/c, respectively ( – ). three known promoter snps, rs , rs , and rs , previously classified as � g � c (h/l), � g � c (y/x), and � c � t (p/q), respectively, were also identified ( ). from these snps in the mbl gene, tag snps were selected based on the tagger algorithm (haploview . , using r � . to indicate redundancy [supplementary fig. , available in the online appendix at http://diabetes. diabetesjournals.org/cgi/content/full/db - /dc ]). to additionally analyze variation flanking mbl (� kb flanking each side of the sequenced region, chromosome : – ), database snps that serve as tag snps in the chinese hapmap (r � . ; minor allele frequency � . ) were genotyped in pima indians, and it was determined that seven snps could serve as tag snps (defined above) in pima samples (supplementary fig. ). all tag snps ( in mbl and flanking) were genotyped in the sample of , full-heritage pima indi- ans for association analysis with type diabetes (table ). the missense snp rs (gly asp, designated a/b) and the promoter snp rs (designated h/l) were in high ld (d� � . ; r � . ) and were associated with type diabetes (p � . and . , respectively, adjusted for age, sex, birth year, and family membership; table ). these two tag snps captured several additional snps within intron and the region near mbl . to determine whether the association with type diabetes in the full-heritage pima indians could be replicated in other native americans, rs and rs were further genotyped in a nonoverlapping sample of , subjects who were predominately of mixed native american heritage. the promoter snp rs reproducibly associated with type diabetes (p � . , adjusted for age, sex, birth year, and heritage) (table ). combining the initial and replication samples (n � , ) provided the strongest evidence for associa- tion with type diabetes for rs (p � . 	 � ) (table ). the combined sample also showed a significant association for rs (p � . ) (table ), but this significant association was largely driven by the initial full-heritage pima sample, with only a nonsignificant asso- ciation in the same direction (p � . ) identified in the largely mixed-heritage replication group. to determine whether variants in mbl had a signifi- cant effect on diabetes in non–native american popula- tions, rs and rs were genotyped in an amish sample of diabetic subjects and subjects with normal glucose tolerance. consistent with the native american samples, rs was associated with type diabetes in the amish (or . ; p � . , adjusted for age, sex, and family structure) (table ), but the frequency of risk allele g was lower in the amish than in pima indians ( . vs. . , respectively). the snp rs (gly asp) was also associated with type diabetes in the amish (or . , adjusted p � . ) (table ), but the frequency of risk allele glycine was comparable in the amish and pima indians ( . vs. . ). in contrast, these associations do not appear to replicate in the large cau- casian diabetes genetics replication and meta-analysis (diagram) ( ). neither of these snps were directly genotyped in genome-wide association (gwa) studies from the diabetes genetics initiative (dgi) and the well- come trust case control consortium (wtccc), which are two of the three large studies from which the meta- analysis was derived; however, a proxy, rs , which we determined to have an r � with rs (based on our genotyping of caucasians), was not associated with type diabetes in diagram (p � . , table ). combining the negative diagram data together with the positive amish and native american data rendered the overall combined association nonsignificant (p � . ) (table ). data across a larger genomic region encompassing mbl could also be obtained from prior gwa studies in pima indians ( ), dgi ( ), wtccc ( ), and diagram ( ). for example, a � kb region encompassing the � . kb mbl (chromosome : – ) yielded snps that were previously genotyped in a gwa study of pima indians. only one gwa study snp (rs ) was located within mbl (intron ). the pairwise ld pattern of these snps and their associa- tion with early-onset type diabetes in pima subjects who were analyzed in this prior gwa ( early-onset diabetes case and control subjects) are shown in supplemen- tary fig. a and b. gwa study snp rs , which is in near-perfect ld with the promoter rs (r � . ), was associated with early-onset type diabetes (defined as diabetes onset � years of age) in the gwa study (adjusted p � . ), as were several nearby gwa study snps (adjusted p � . – . ) (supplementary fig. ), which were in high ld among themselves (r � . – . ) but in low ld with rs (r � . – . ). in y.l. muller and associates diabetes.diabetesjournals.org diabetes, vol. , august contrast with rs , these additional gwa study snps (tagged by rs ) had only a borderline association with type diabetes (defined as diabetes at any age) in the current larger study of , full-heritage pima subjects (rs , p � . ) (table ). gwa study data across this region in the dgi and wtccc studies and diagram are shown in supplementary fig. . overall, there were no consistent associations with type diabetes across the region in any of these caucasian studies. when gwa study results of pimas were compared with other pop- ulations in the mbl region, rs was not significant in diagram. snps tagged by rs in caucasians had a borderline significance with type diabetes in diagram (p � . ) (supplementary fig. ) but was nearly monomorphic (minor allele frequency � . ) and therefore uninformative in full-heritage pima indians, while snp rs , which had a borderline association with type diabetes in wtccc (p � . ) (supplementary fig. ), was not associated with type diabetes in pima indians (table ). table associations of mbl tag snps with type diabetes in pima indians snp location risk/ non-risk full-heritage pima indian (n � , ) replication mixed heritage (n � , ) combined (n � , ) af or ( % ci) padditive af or ( % ci) padditive or ( % ci) padditive rs �-flanking t/c . . ( . – . ) . rs �-flanking c/t . . ( . – . ) . rs �-flanking c/t . . ( . – . ) . rs �utr t/c . . ( . – . ) . novel: �utr �utr a/g . . ( . – . ) . rs leu leu c/g . . ( . – . ) . rs intron a/g . . ( . – . ) . rs intron a/g . . ( . – . ) . rs gly glu (a/c) g/a . . ( . – . ) . rs gly asp (a/b) g/a . . ( . – . ) . . . ( . – . ) . . ( . – . ) . rs arg cys (a/d) c/t . . ( . – . ) . rs promoter (p/q) c/t* . . ( . – . ) . . . ( . – . ) . . ( . – . ) . rs promoter (y/x) g/c* . . ( . – . ) . rs promoter (h/l) g/c* . . ( . – . ) . . . ( . – . ) . . ( . – . ) . 	 � novel: promoter promoter g/a . . ( . – . ) . rs �-flanking c/t . . ( . – . ) . rs �-flanking a/g . . ( . – . ) . rs �-flanking c/t . . ( . – . ) . rs �-flanking a/c . . ( . – . ) . nineteen tag snps (r � . ) were selected from snps that span mbl (� . kb) and approximately kb flanking each side of the gene. allele frequency (af) is presented as frequency of the risk allele. or is expressed as per copy of the risk allele (and thus, by definition, is � ). the risk allele is underlined where the p value is � . . *genotypes were determined according to the reverse strand of the snp database sequence. sequences flanking the two novel snps are as follows: novel: promoter, tttcatggatgggtgtgtgc g/a�tgcatgcacgtgtctgtgtg; novel: �utr, catgactgcacagtaatttc g/a�tctgtttataaacattgtat. table association of promoter rs and rs (gly asp) with type diabetes in pima indian, amish, and diagram subjects rs rs or ( % ci) p phet or ( % ci) p phet native american full-heritage pima indian . ( . – . ) . . ( . – . ) . replication mixed heritage . ( . – . ) . . ( . – . ) . combined . ( . – . ) . 	 � . . ( . – . ) . . caucasian amish . ( . – . ) . . ( . – . ) . diagram* . ( . – . ) . combined . ( . – . ) . . native american and caucasian combined . ( . – . ) . . a combined test was conducted by the inverse variance method. ors are per copy of the risk allele identified in the full-heritage pima sample. p is for the null hypothesis that the or � . phet is the p value for the null hypothesis that the ors are the same for the combined groups. *diagram results based on rs , which has an r of . with rs . mbl and type diabetes diabetes, vol. , august diabetes.diabetesjournals.org the observation that specific mbl snps had replicated associations with type diabetes in native americans and a small group of amish subjects, who are of european descent but were not associated with diabetes in the large diagram, is unexpected. it is possible that the associa- tion in the amish is a false positive. alternatively, both the pima indians and the old order amish represent far more homogeneous populations compared with the study pop- ulation of diagram. it is also possible that susceptibility genes for common diseases may have larger effects in these populations compared with others as a result of segregation of high-penetrance alleles that are rare or nonexistent in the general population, gene-gene or gene- environment interactions, or the absence of other suscep- tibility genes whose effects could mask other genes. to aid in validating the positive associations with type diabetes, we further investigated whether these variants in mbl were associated with metabolic risk factors that predict type diabetes among nondiabetic, full-heri- tage pima indians. for both rs and rs , the allele associated with higher risk for diabetes (g and glycine, respectively) was associated with a higher -h plasma glucose concentration (adjusted p � . and . , respectively) and higher -h plasma insulin concen- tration (adjusted p � . and . , respectively) during an oral glucose tolerance test (table ). the risk allele glycine for rs was additionally associated with a lower acute insulin response to an intravenous glucose bolus infusion (adjusted p � . ) among sub- jects who had normal glucose tolerance (n � ) (table ). however, neither snp was associated with insulin- stimulated glucose uptake. previous functional studies have shown that serum mbl levels are greatly influenced by variants within the mbl gene. three promoter variants (rs [h/l], rs [y/x], and rs [p/q]) and three missense variants (rs [a/d], rs [a/b], and rs [a/c]) were previously associated with mbl deficiency ( , ). several of these variants are in high ld, so a limited number of haplotypes are present in humans. hypa (g-g-c-gly) haplotype carriers have the highest serum concentration of mbl, typically from , to , �g/l, whereas lypb (c-g-c-asp) haplotype carriers have the lowest serum concentration: – �g/l ( , ). since rs (y/x) and rs (p/q) are very rare in pima indians (minor allele frequency � . ), these high versus low serum level haplotypes can essentially be determined from a two-snp haplotype of the promoter rs (h/l) and missense rs (a/b) in pima indians. haplotype analysis for rs and rs was performed in the combined sample of , predominately pima indians. the haplotype g-glycine (ha), carrying both the g nucleotide (designated h allele) of the promoter rs and glycine (designated a allele) of rs , was associated with increased risk for type diabetes in pima indians (haplotype frequency of g-glycine . in diabetic vs. . in nondiabetic; or . [ % ci . – . ], p � . , adjusted for age, sex, and birth year [heritage estimate in the replication group]). because this haplotype g-glycine is highly concordant with the g (h) allele of rs (r � . ), it is difficult to statistically distin- guish the single genotypic versus haplotypic effects. when both snps are included in a single model, there is a significant association with rs (or . [ % ci . – . ]; p � . ) conditional on the effect at rs , but there is little association with rs table metabolic characteristics of full-heritage nondiabetic subjects grouped by genotypes of promoter rs or rs (gly asp) variants rs (g/c) rs (g/a) g/g g/c c/c padditive g/g (gly/gly) g/a (gly/asp) a/a (asp/asp) padditive nondiabetic subjects n percent body fat � . � . � . . � . � . � . . bmi (kg/m ) � . � . � . . � . � . � . . fasting plasma glucose (mg/dl) � . � . � . . � . � . � . . -min plasma glucose (mg/dl) � . � . � . . � . � . � . . -h plasma glucose (mg/dl) � . � . � . . � . � . � . . log fasting plasma insulin (�u/ml) . � . . � . . � . . . � . . � . . � . . log -h plasma insulin (�u/ml) . � . . � . . � . . . � . . � . . � . . log glucose disposal rate under low-dose insulin clamp (mg � kg embs� � min� ) . � . . � . . � . . . � . . � . . � . . normal glucose tolerant subjects n log acute insulin response (�u/ml) . � . . � . . � . . . � . . � . . � . . log -min plasma insulin (�u/ml) . � . . � . . � . . . � . . � . . � . . data are means � se unless otherwise indicated. plasma insulin concentrations (fasting, h, and min), rates of glucose disappearance during the low-dose insulin stimulation, and the acute insulin response were log transformed before analyses to approximate a normal distribution. the p value for percent body fat was adjusted for age, sex, and family membership. the p value for the acute insulin response was adjusted for age, sex, percent body fat, and rate of glucose disappearance during the low-dose insulin stimulation. all remaining p values (except for age) were adjusted for age, sex, percent body fat, and family membership. embs, estimated metabolic body size. y.l. muller and associates diabetes.diabetesjournals.org diabetes, vol. , august conditional on the effect at rs ( . [ . – . ]; p � . ). it thus appears that the promoter rs is the stronger predictor of diabetes, with little additional infor- mation from rs . the prevalence of type diabetes was plotted in the full-heritage pima indian (n � , ) (fig. a) and the replication native american (n � , ) (fig. b) groups according to the genotypes of rs and rs . ordering the genotypic groups according to their associa- tion with serum mbl levels showed that subjects homozy- gous for both g and glycine alleles had a higher prevalence of diabetes than did subjects homozygous for the c allele and either homozygous or heterozygous for the aspartic acid alleles (ptrend � . 	 � in the combined analysis of , predominately pima subjects, adjusted for age, sex, and birth year, and in the replication group, heritage). in caucasians, eskimos, africans, south americans, and native americans, the g-glycine (ha) haplotype is associ- ated with higher mbl serum level ( , – ). the present study demonstrates that an allele for mbl , which arises predominately from the promoter snp rs (g allele), predicts a higher serum level of mbl and is associated with increased risk for type diabetes, increased -h plasma glucose and -h plasma insulin, and decreased insulin secretion in some popula- tions. consistent with our observations, high mbl levels have recently been reported to be associated with high a c levels in the strong heart study, a longitudinal study of cardiovascular disease among native americans ( ). snp rs is in high ld with other snps that map within intron and a flanking region of mbl ; therefore, the contribution of other functional snps cannot be ruled out. nevertheless, both high ld and diabetes association were restricted to the region in and near mbl , suggesting that evidence for association with type diabetes is more likely derived from mbl rather than other genes in the region. although the physiologic mechanisms underlying the association of mbl levels with diabetes are unknown, it has previously been shown that mbl plays a dual role in modifying inflammatory responses ( ). deficiency of mbl has been linked to increased risk of developing type diabetes ( ), insulin resistance, and obesity ( ) as a result of a chronic infectious state or low-grade inflamma- tion. mbl could also affect metabolic pathways through stimulating fatty acid oxidation in skeletal muscle ( ) or reducing release of tumor necrosis factor-
, interleukin- , and interleukin- ( ). in contrast, increased mbl levels could lead to an overly activated complement system, thereby inducing inflammatory damage or interweaving a complex autoimmune process ( ). consistent with the latter effect, high mbl levels have been associated with increased risk for insulin resistance in pregnancy ( ) and late-onset of rheumatoid arthritis ( ). however, our study indicates that mbl variants are more likely to influence type diabetes via an effect on insulin secretion rather than on insulin action, suggesting that inflammatory dam- age in pancreatic �-cell function may be involved. addi- tional studies are needed to investigate the impact of this gene on specific type diabetes related–pathways and disease susceptibility in non–native american groups. acknowledgments this work was supported by the intramural research program of the national institute of diabetes and diges- tive and kidney disease, the national institutes of health. the amish study was supported by national insitutes of health grants r dk and p dk (to the clinical nutrition research unit of maryland) and p dk (to the baltimore diabetes research and train- ing center). li bian was supported by an ada mentor grant awarded to clifton bogardus. no potential conflicts of interest relevant to this article were reported. yunhua l. muller researched data, wrote manuscript. robert l. hanson reviewed/edited manuscript, contributed to discussion. li bian researched data, contributed to discus- sion. janel mack researched data. xiaolian shi researched data. ruth pakyz researched data. alan r. shuldiner re- viewed/edited manuscript, contributed to discussion. william p re va le nc e of d ia be te s (% ) genotypes n= n= n= n= p re va le nc e of d ia be te s (% ) genotypes cc/x* cg /g lyasp cg /g lyg ly g g /g lyg ly cc/x* cg /g lyasp cg /g lyg ly g g /g lyg ly n= n= n= n= a b fig. . prevalence of type diabetes in the full-heritage pima indian group (a) (n � , ) and the replication mixed heritage group (b) (n � , ) according to genotypes of promoter rs and rs (gly asp). *either homozygous or heterozygous for the asp alleles. mbl and type diabetes diabetes, vol. , august diabetes.diabetesjournals.org c. knowler reviewed/edited manuscript, contributed to dis- cussion. clifton bogardus reviewed/edited manuscript, con- tributed to discussion. leslie j. baier wrote manuscript, contributed to discussion. references . dommett rm, klein n, turner mw. mannose-binding lectin in innate immunity: past, present and future. tissue antigens ; : – . turner mw. the role of mannose-binding lectin in health and disease. mol immunol ; : – . madsen ho, garred p, thiel s, kurtzhals ja, lamm lu, ryder lp, svejgaard a. interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. j immunol ; : – . davies ej, teh ls, ordi-ros j, snowden n, hillarby mc, hajeer a, donn r, perez-pemen p, vilardell-tarres m, ollier we. a dysfunctional allele of the mannose binding protein gene associates with systemic lupus erythema- tosus in a spanish population. j rheumatol ; : – . garred p, madsen ho, marquart h, hansen tm, sørensen sf, petersen j, volck b, svejgaard a, graudal na, rudd pm, dwek ra, sim rb, andersen v. two edged role of mannose binding lectin in rheumatoid arthritis: a cross sectional study. j rheumatol ; : – . araujo j, brandão la, guimarães rl, santos s, falcão ea, milanese m, segat l, souza pr, de lima-filho jl, crovella s. mannose binding lectin gene polymorphisms are associated with type diabetes in brazilian children and adolescents. hum immunol ; : – . megia a, gallart l, fernández-real jm, vendrell j, simón i, gutierrez c, richart c. mannose-binding lectin gene polymorphisms are associated with gestational diabetes mellitus. j clin endocrinol metab ; : – . 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genetics and disease associations. rev immunogenet ; : – . kilpatrick dc. mannan-binding lectin concentration during normal human pregnancy. hum reprod ; : – y.l. muller and associates diabetes.diabetesjournals.org diabetes, vol. , august wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ religious barriers to measles vaccination review religious barriers to measles vaccination eric wombwell • mary t. fangman • alannah k. yoder • david l. spero published online: october � springer science+business media new york abstract in , the united states has experienced an increase in measles activity, the most since the elimination of the virus in . the measles infection occurs in unvaccinated individuals. communities and individuals choose to not vaccinate for a number of reasons, primarily citing religious and philosophical motives. objections based upon religion most often center on the use of aborted human fetus tissue used in the rubella component of the combined vaccine products, and animal derived gelatins used in vaccine production. objections among religious communities may also not be faith based, rather in some cases concerns related to lack of safety and efficacy of the vaccination result in refusal. keywords measles � vaccines � religion � faith � mmr vaccine introduction due to aggressive vaccination programs measles has not been an endemic disease in the united states (us) since [ ]. however, due to international travel and the global economy the measles virus has been imported to the us from endemic areas of the world, leading to sporadic outbreaks especially among unvaccinated populations [ ]. in , the us has experienced the largest amount of measles activity since the elimination of the virus with different outbreaks and cases officially reported as of august , [ , ]. a majority of the us outbreaks occurring in were imported from the philippines where the disease is still endemic. individuals traveling from endemic regions of the world import the virus while they are still contagious and introduce it to others. measles, or rubeola, is an extremely contagious acute respiratory disease that is caused by the measles virus. the virus is a single stranded rna virus whose only natural host are humans; it is a member of the genus morbillivirus which is part of the paramyxoviridae family. there are several immunoglobulins that can be tested for in the blood to confirm a measles case. symptoms of the infection include fever, cough, runny nose and conjunctivitis which are then followed by a rash that can appear over the entire body. the characteristic measles rash usually appears days after the individual is exposed to the virus. sec- ondary complications include ear infections, pneumonia, encephalitis and in the most severe cases death. serious complications associated with measles occur more fre- quently in pediatric and elderly populations. in , the infection was responsible for an estimated , , deaths worldwide [ ]. the incubation period is approximately – days long. infected individuals are considered con- tagious days prior to the presentation of rash and e. wombwell (&) � a. k. yoder � d. l. spero division of pharmacy practice and administration, school of pharmacy, university of missouri - kansas city, charlotte street, kansas city, mo - , usa e-mail: wombwelle@umkc.edu e. wombwell department of pharmacy, centerpoint medical center, independence, mo, usa m. t. fangman department of preventive medicine, university of kansas medical center, kansas city, ks, usa m. t. fangman city of kansas city, missouri health department, office of epidemiology and community health monitoring, kansas city, mo, usa j community health ( ) : – doi . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf continue to shed virus for days following presentation of the rash. transmission of the virus occurs through the air via aerosolized droplets from breathing, sneezing or coughing. the virus is highly contagious; nearly anyone exposed to the virus without immunity will contract mea- sles. the r (basic reproductive number) has been reported to range from to . this high level of transmissibility underscores the need for continued vaccinations, especially among travelers entering parts of the world where measles remains endemic. many communities have made themselves particularly vulnerable to outbreak by choosing not to vaccinate for a myriad of reasons, most notably religious and philosophi- cal [ ]. when the measles virus infects an individual in one of these unvaccinated communities it is highly transmis- sible and often spreads to all members. the purpose of this paper is to describe the religious objections as they spe- cifically relate to the measles vaccination. methods a comprehensive literature search was conducted in august to identify peer-reviewed publications authored on religious objections to vaccination specifically pertaining to the measles vaccine. the search used medline through ovid, google scholar, and pubmed. searches were performed using the keywords ‘‘measles’’, ‘‘vaccines’’, ‘‘religion’’, ‘‘faith’’, ‘‘measles vaccine’’, ‘‘measles, mumps, rubella vaccine’’, ‘‘measles-mumps-rubella vaccine’’, and ‘‘mmr vaccine’’. after individually searching these terms, they were combined in a number of ways using ‘and’. from this search, the terms were exploded and focused in order to map the terms to the subject headings. articles regarding the manufacturing/production of the mmr vaccine, measles outbreaks, and religious views on vaccination were obtained. citations contained in the reference sections of relevant articles were also reviewed to expand the literature search to include additional citations. the authors reviewed only english language papers. measles vaccine vaccination against measles is available in two commer- cially available vaccines, m-m-r � ii (measles, mumps, and rubella virus vaccine live) and proquad � (measles, mumps, rubella, and varicella virus vaccine live), both of which are manufactured by merck & co, inc. m-m-r � ii is indicated for individuals months or older, whereas proquad � is indicated only for children months through years of age [ , ]. history of vaccine the first measles vaccine was licensedin the us in .while both the live attenuated vaccine and inactivated vaccines were available originally,theinactivatedvaccine waswithdrawndue lack of efficacy and the appearance of atypical measles in some recipients. multiple live attenuated strains of the vaccine have been licensed; however, only the edmonston–enders strain remains available. the edmonston–enders strain provides a more attenuated strain that results in significantly fewer reac- tionsthanotherstrains.in ,thecombinedmeasles-mumps- rubella vaccine was licensed, with the combined measles- mumps-rubella-varicella vaccine following in [ ]. efficacy after administration of the measles vaccination via m-m-r � ii, antibodies are detectable in % of patients in about days. in addition, immunity occurs within days and persists for at least years. it is thought that most individuals experience permanent immunity to each virus [ ]. similar antibody and immunity response is seen in proquad � [ ]. perhaps, the best way to evaluate the efficacy of measles vaccinations is the dramatic decrease in the number of cases since vaccine initiation and approval. over a % reduction in deaths was seen from the pre-vaccine era to [ ]. components and manufacturing the components and processing of m-m-r � ii and pro- quad � may include substances that are of human and animal origin leading to religious based vaccination objections. examples of such components include human diploid cells, human albumin, bovine excipients, or porcine excipients. human diploid cells human diploid cells are immature cells that contain large quantities of chromosomes. these cells play a critical role in vaccine production as they provide a reliable medium for the replication of multiple viruses. additionally, human diploid cells may be artificially and indefinitely propagated [ ].two specific strains of human diploid cells, wi- and mrc- , are excellent for vaccine production. viruses, including measles, cannot replicate independently; there- fore, wi- and mrc- human fetal fibroblasts allow for preparation of viral vaccines [ ]. both cell lines were obtained from fetuses that were aborted for reasons not associated with vaccine production. the wi- line was isolated from fetal lung cells from a fetus that was aborted because the parents felt they had too many children [ ]. the mrc- line was isolated from fetal lung cells from -week fetus that was aborted due maternal psychiatric reasons [ ]. j community health ( ) : – the measles and mumps viruses do not require human dip- loid cells to replicate. rather, these viruses are propagated in chick embryo cell cultures for both the m-m-r � ii and proquad � [ , ]. however, the varicella virus present in proquad � is propagated in the mrc- cells during pro- duction [ ]. furthermore, the rubella virus (known as strain ra / ) was isolated from kidney fibroblasts obtained from a fetus aborted due to rubella. the isolated strain was then serially propagated into human diploid cells, specifically wi- cells [ ]. the ra / strain is present in both the m-m-r � ii and proquad � vaccines [ , ]. recombinant human albumin and human albumin the growth medium for varicella, in proquad � , contains human albumin [ ]. human albumin is obtained via frac- tionation of the human plasma from blood donations [ ]. in contrast, the growth medium for measles, mumps, and rubella contains recombinant human albumin [ ]. recom- binant human albumin does not contain blood or human plasma; currently, rice and yeast are used for large-scale production of recombinant human plasma [ ]. bovine serum the growth medium for measles, mumps, and rubella is supplemented with bovine serum. m-m-r � ii contains less than parts per million (ppm) fetal bovine serum per dose [ ]. proquad � contains . mcg bovine calf serum per dose [ ]. hydrolyzed gelatin hydrolyzed gelatin, which is often incorporated in vaccines in order to stabilize and preserve active ingredients or act as a solvent, may be of porcine or bovine origin. the hydrolyzed gelatin is obtained from collagen of the skin, bone, or other components of pigs or cattle [ ]. m-m-r � ii contains . mg of hydrolyzed gelatin per dose [ ]. pro- quad � contains mg of hydrolyzed gelatin per dose [ ]. as discussed, the animal and human components of the m-m-r � ii and proquad � are essential to the production of the vaccines in their current formulation and directly con- tribute to vaccines’ role in the prevention of measles. yet these same components can present religious objections due to their derivation from human and animal sources. religious views on the use of measles vaccination judaism there are several branches within the jewish faith, which include conservative judaism, secular judaism, reform judaism, orthodox judaism and reconstructional judaism. each branch interprets the teachings of the torah, the main scripture of the religion, differently. along with the torah there are other scriptures, texts and writings that express the history, laws, traditions, ethics and philosophy of the jewish faith. to what extent these teachings are followed or how they are interpreted makes up the different sects within the religion [ , ]. in judaism there is a supreme value placed on human life and preserving it at all costs; the physician and the patient both have an obligation to provide and seek health so that one can further the practice of the religion and live the life god intended [ ]. in the torah there are several references to the duty of physicians to heal. it is said ‘‘and heal he shall heal’’ (exodus : ) which has been interpreted as god giving physicians authorization to heal or even obligating them to heal when they are able. the phrase ‘‘[d]o not stand idly by the blood of your neighbor’’ (leviticus : ) has been interpreted as mandating physicians to provide care to patients when they are ill. this same verse from leviticus has been used by jewish scholars to encourage smallpox vaccinations in past eras [ ]. several jewish dietary laws restrict what can be con- sumed by followers of the faith. food that is considered suitable for eating is termed kosher; more conservative and orthodox jews are more likely to follow these dietary restrictions. certain animal products from pork, shellfish or improperly slaughtered animals are considered non-kosher and shouldn’t be consumed according to jewish law. these restrictions could be applied to vaccines that contain por- cine gelatins, such as both measles vaccine formulations. however, several jewish authorities have only limited the use of porcine components to orally administered medi- cations, not products that are given intravenously. the use of porcine components in a vaccine therefore do not violate jewish law [ , ]. among the jewish branches, orthodox jews have expe- rienced the greatest number of documented measles out- breaks due to declining vaccination rates within specific communities [ ]. orthodox jews adhere most thoroughly to jewish law and tend to isolate themselves into communities of orthodox families living within a specific geographic location. they often utilize the same jewish schools and interact socially with each other without much interaction with outsiders of the community and faith. these factors create pockets of unvaccinated jewish communities which are susceptible to infection when the measles virus is intro- duced. a measles outbreak occurred in antwerp, belgium among an orthodox jewish community, infected individuals were interviewed, and reasons for non-vaccination were not found to be due to religious beliefs. rather, due to the advice of their primary care providers, or due to fears of side effects or allergies to the vaccination itself [ ]. j community health ( ) : – hinduism hinduism has over million adherents worldwide, with the majority of indian and nepali people subscribing to hinduism [ ]. hinduism considers itself to be the eternal tradition. its roots can be traced to between , and bc [ , ]. hinduism has no one individual founder, doctrine, or scripture and is thus often referred to as ‘a way of life’ or ‘a family of religions’ [ ]. hindus advocate nonviolence and respect for life [ , ] with many hindus practicing vegetarianism as an extension of this belief. hindu’s believe in a circle of life, known as reincarnation, with the ultimate reincarnation occurring in the form of a cow. thus, cows are highly regarded in hindu culture and eating beef is banned [ ]. hindu’s medical ethics stem from a principle of nonviolence and respect for animal life forms. at least two issues may arise for hindu’s concerning the measles vaccine. both measles vaccine formulations con- tain trace bovine components [ , ]. because hindu’s believe divinity permeates all things, including plants and animals, and place special emphasize on the sanctity of the bovine species, there may be concerns with the use of cows in the creation of the vaccine [ , ]. additionally, hin- dus, in general, are opposed to abortion [ ]. the measles vaccines are formulated with the rubella vaccine, which is derived from cell lines obtained from an aborted fetus [ , ]. the wi- cell line was obtained from a deliberately aborted baby in sweden [ , ]. with no overall faith law or doctrine, the issue of vaccine development with bovine components produced in combination with aborted fetus tissue, may be of concern to individual hindus. christianity christians are followers of jesus christ, the son of god, and use the bible as their sacred text. christianity traces its roots to the first century and has approximately . billion adherents worldwide, representing nearly a third of the world’s population [ ]. in general, there is no prohibition of food sources or preparation for individuals practicing christianity. how- ever, christians in general do believe in the sanctity of life and may have objections to the use of aborted fetus tissue in the production of the vaccine. different christian denominations will be further explored as to possible objections to use of the vaccine. roman catholic christians the roman catholic tradition is the original form of christianity. all other christian denominations are dece- dents of the roman catholic faith with differences arising due to different retained and rejected aspects of catholi- cism. the roman catholic church is headed by the pope, who is given the ultimate church authority on earth. the pope is advised and supported by cardinals, archbishops, etc. and various councils and advisory boards. as relates to the measles vaccine, the church’s stance was published in a statement on june th, by the center of bioethics [ ]. catholics believe abortion is immoral. involvement with vaccines derived from aborted fetal tissue carries differing moral weight for consumers, marketers, and vaccine producers [ ]. looking specifically at consumers, use of the measles vaccine confers protection to individu- als, their children, and the population against disease. this is good. however, use of vaccine derived from aborted fetus tissue creates a situation of ‘‘passive cooperation’’ in which a population consensus is reached allowing vaccine production to occur in an otherwise immoral manner [ ]. as such, catholics have an ethical obligation to promote development of an alternative live rubella vaccine and the support of the church to make conscientious objections to vaccines with which there are moral problems [ ]. cath- olics are encouraged to support development of vaccines derived from non-aborted tissue sources [ , , – ]. catholics may obtain the mmr vaccine for their children because of the protective effects of the vaccine but they are obligated to lobby for development of a morally acceptable alternative. protestant christianity like roman catholics, protestant christians do not tend to have objections to the use of the measles vaccine except for possible concerns with components of the vaccine origi- nating from aborted fetus tissue. specific protestant denominations may have additional unique concerns. for instance, christian scientists believe disease is not a reality and diseases can be treated with prayer [ ]. as such, they may have a fundamental issue with vaccines in general, including but not specific to the measles vaccine. in addi- tion, dutch reformed congregations believe vaccines pre- vent an individual from fully relying on god for their health [ – ]. again, this may lead to a general, but not specific, lack of measles vaccine usage due to religious reasons. amish in recent years, amish communities throughout the us have experienced outbreaks of disease due to a lack of vaccination and community immunity [ – ]. the amish are a group of christian fellowships which began during the protestant reformation in sixteenth-century europe. amish accept basic christian beliefs but also have some j community health ( ) : – special interpretations and emphases that have emerged throughout their history. their spirituality has been shaped by their interpretation of the bible as well as several other written sources [ ]. immunizations are not prohibited by amish religious doctrine. however, there are large communities that do not receive scheduled immunizations [ , – ], exemplified by measles outbreaks in . immunization rates are low within the faith primarily due to poor access to care, and concerns about vaccine safety. only – % of respondents from a study of amish who objected to vaccination declined having their children vaccinated for religious reasons. rather, the primary reasons cited were concerns for safety. the same study observed that % of the exempting parents stated that they would consider vacci- nating their children if ‘‘they knew the shots were safe to give’’ [ ]. the amish therefore represent a religious population that could benefit from improved education as a driver for increased immunization rates [ , – ]. jehovah’s witnesses jehovah’s witnesses is a christian denomination started in the late s that is currently led by the the watch tower bible and tract society. since , the watch tower society has instructed its followers to refuse transfusions of whole blood and certain blood components considered violations of god’s law - derived from the interpretation of several scriptural passages. their blood doctrine has undergone multiple changes since , principally in , , and . by abstaining from blood, wit- nesses express their faith that only the shed blood of jesus can redeem them and save their life. in this view, those who respect life as a gift from god do not try to sustain life by taking in blood, even in an emergency [ , , ]. the watch tower society denounced vaccination from the s through the s, due to similar scriptural passages cited for the avoidance of blood transfusions. an early leader of the jehovah’s witnesses, c. j. woodworth, believed vaccination caused animal blood cells to be injected into humans, thus members were banned from having vaccinations around this time, under penalty of excommunication [ ]. the society revised this doctrine in the december , , issue of the watchtower, saying that those passages did not apply to vaccination. ‘‘after consideration of the matter, it does not appear to us to be in violation of the everlasting covenant made with noah, as set down in genesis : , nor contrary to god’s related commandment at leviticus : - . most certainly it cannot reasonably or scripturally be argued and proved that, by being vaccinated, the inoculated person is either eating or drinking blood and consuming it as food or receiving a blood transfusion. vaccination does not bear any relationship to or any likeness to the intermarriage of angelic ‘‘sons of god’’ with the daughters of men, as described in genesis : - . neither can it be put in the same class as described at leviticus : - , which forbids the mingling of humans with animals. it has nothing to do with sex relations.’’ the watchtower, -dec- . in s, the society moved into a stance of neutrality, neither endorsing nor prohibiting vaccination. moving towards the new millennium, awake! magazine published statements supporting the clinical value of vaccination. ‘‘in the world’s developed countries, new vaccines dramatically decreased the toll of measles, mumps, and german measles. a mass polio vaccination campaign, launched in , was so successful that cases of the disease in western europe and north america plummeted from , in that year to fewer than , in . smallpox, a major killer disease, was eradicated worldwide.’’ awake! -feb- . the decision of whether to vaccinate themselves or their family is currently a decision made by the individual witness. islam the foundation of islam belief includes muhammad, a prophet, who received revelations from a monotheistic god which were recorded in the book of qur’an. the qur’an serves as the written form of god’s word and is considered to be sacred, reverent, and the ultimate revelation of god. in addition to the qur’an, muslims use the supplementary hadith collections to serve as a model for daily life. the hadith qudsi includes the direct words of god; whereas, the hadith sharif are the words and deeds of the prophet muhammad [ ]. islamic scholars, known as muhtahids, may also issue opinions or ruling in regards to the inter- pretations of the qur’an (fatwas) [ ]. although fatwas are not considered so be authoritative, they offer valuable principles which may be applied to daily living, including modern medicine. both theological and social issues may be present for muslims regarding the m-m-r � ii and proquad � vac- cines. theological issues may include use of porcine components, while social issues may include concerns for safety. both vaccines contain hydrolyzed gelatin of porcine origin. the qur’an and tradition indicate certain animal products are absolutely forbidden, while others are j community health ( ) : – permitted (halal) or forbidden (haram) depending on how they perished. food containing porcine gelatin derived from skin or bone is forbidden (haram); while, gelatin from beef or fish is permitted [ ]. in , a seminar was held by the islamic organization for medical sciences in order to address specific concerns of food and drugs. it was noted that gelatin derived from pigs has undergone extensive alterations through chemical reactions and follows the accepted constructs of ‘transformation’ [ ]. the trans- formation from impermissible to permissible is known as istihala [ ]. therefore, the following recommendation was made: ‘‘the gelatin formed as a result of the trans- formation of the bones, skin and tendons of a judicially impure animal is pure, and it is judicially permissible to eat it’’ [ ]. it is important to note that shafis, hanbalis, and some malikis do not accept istihala for porcine derivatives in vaccines, as they believe istihala applies only to natural transformations [ ]. the qur’an also states that ‘‘whoever is forced [by necessity], neither desiring [it] or trans- gressing [its limit], there is no sin upon him’’ (qur’an surah , verse ). islamic law considers situations of exceptional circumstances through the ‘‘law of necessity’’ [ ]. however, controversy still remains among islamic juriconsults about defining what constitutes a dire neces- sity, especially when it comes to modern medicine and vaccines [ ]. the multiple ethical and legal beliefs asso- ciated with vaccines, including the measles vaccination, leads to varying beliefs among islamic individuals. discussion this article is intended to provide discussion regarding religious and philosophical beliefs related to the accept- ability of the measles vaccine. recent measles outbreaks in the us have been linked to religious communities that have chosen not to vaccinate. this paper examines the faith belief basis contributing specifically to refusal of the m-m- r � ii and proquad � vaccines which contain the measles vaccine. interestingly, refusals based upon religion most often center on the use of aborted human fetus tissue used in the rubella component of the combined vaccine products, and animal derived gelatins used in vaccine production. of the major religions practiced in the world, this paper focuses on judaism, hinduism, christianity, roman catholicism, protestant christianity, amish, jehovah’s witnesses and islam. in review of the jewish and amish faiths, there are no restrictions on the use of the m-m-r � ii and proquad � vaccines; however, some communities within these faiths have chosen not to vaccinate due to safety and efficacy concerns. the hindu faith does not explicitly pro- hibit the use of vaccines, but followers of the faith may object to vaccination due to its derivation from fetal cells or containing bovine components. in general there are no laws or writings prohibiting the use of vaccines in christianity, but objections to the m-m-r � ii and proquad � vaccines arise due to the rubella virus component originating from aborted human fetus tissue. similarly, roman catholicism professes the act of abortion to be immoral and thus supports the development of a vaccine product derived from non-aborted tissue. the jehovah’s witness faith has had a number of revisions on the topic of vaccine use in general; originally vaccines were considered blood products and were not to be administered. recently the faith has retracted this viewpoint and now leaves it up to the individual to vaccinate or not. in islam the primary concern is the use of a porcine based gelatin in vaccine production. there is not a uniform state- ment in regards to this issue yielding varying beliefs among islamic individuals. limitations of this paper include its focused nature, not every of the over , estimated world religions could be covered, thus religions with known, current or historical, objections or measles outbreaks were emphasized. fur- thermore, the authors do not provide detailed theological descriptions for the faith based objections, but rather describe for the healthcare professional more generally the beliefs that impact vaccine acceptance. in reviewing faith objections it became clear that in some instances the reli- gious ideology were not actually what contributed to vac- cination objection. for instance in judaism and amish faiths vaccine refusal is often a result of concerns related to lack of safety and efficacy of the vaccination. additionally, in a number of faiths the final discretion is left upon the individual to make a judgment—hinduism, islam, protes- tant christianity. because vaccines are not % effective, an estimated – % (dependent on the vaccine-preventable disease) of the population must be immunized in order to exert community immunity. therefore, suboptimal rates of immunization lead to gaps in the protection from com- munity immunity, opening the potential for outbreaks amongst the general population [ ]. the advisory com- mittee on immunization practices (acip) recommends, doses of mmr vaccine routinely for children with the first dose administered at age through months and the second dose administered at age through years before school entry. two doses are recommended for adults at high risk for exposure and transmission (e.g., students attending colleges or other post-high school educational institutions, health-care personnel, and international travelers) and dose for other adults aged c years [ ]. this review should serve as a resource for healthcare professionals when encountering religious objections to mmr vaccination. objections based upon religion most j community health ( ) : – often center on the use of aborted human fetus tissue used in the rubella component of the combined vaccine pro- ducts, and animal derived gelatins used in vaccine pro- duction. objections among religious communities may also not be faith based, rather in some cases concerns related to lack of safety and efficacy of the vaccination result in refusal. these groups represent opportunities for education to enhance vaccination rates and reduce risk for outbreaks within unvaccinated communities. conflict of interest the authors of this manuscript have no conflicts of interest to disclose. there was no funding involved in this study or the writing of this manuscript. references . katz, s. l., & hinman, a. r. 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( ). introduction to world religions. lanham, md: fortress press. . world health organization, regional office for the eastern mediterranean. ( ). statement arising from a seminar held by the islamic organization for medical sciences concerning the use of pork gelatin. http://www.immunize.org/concerns/porcine.pdf. . padela, a., furber, s., kholwadia, m., & moosa, e. ( ). dire necessity and transformation: entry-points for modern science in islamic bioethical assessment of porcine products in vaccines. bioethics, ( ), – . . fine, p., eames, k., & heymann, d. l. ( ). ‘‘herd immunity’’: a rough guide. clinical infectious diseases, ( ), – . . mmwr, june , , vol , #rr- prevention of measles, rubella, congenital rubella syndrome, and mumps, summary: recommendations of the acip. j community health ( ) : – http://childrenshealthcare.org/?pageid= http://childrenshealthcare.org/?pageid= http://childrenshealthcare.org/?pageid= http://childrenshealthcare.org/?pageid= http://www.britannica.com/ebchecked/topic/ /amish/ /beliefs-and-way-of-life http://www.britannica.com/ebchecked/topic/ /amish/ /beliefs-and-way-of-life http://www.jwfacts.com/watchtower/medical.php http://www.jwfacts.com/watchtower/medical.php http://www.immunize.org/concerns/porcine.pdf 	religious barriers to measles vaccination 	abstract 	introduction 	methods 	measles vaccine 	history of vaccine 	efficacy 	components and manufacturing 	human diploid cells 	recombinant human albumin and human albumin 	bovine serum 	hydrolyzed gelatin 	religious views on the use of measles vaccination 	judaism 	hinduism 	christianity 	roman catholic christians 	protestant christianity 	amish 	jehovah’s witnesses 	islam 	discussion 	conflict of interest 	references a novel, layered phase in ti&#x ;#x ;rich srtio<sub> </sub> epitaxial thin films uc berkeley uc berkeley previously published works title a novel, layered phase in ti-rich srtio epitaxial thin films. permalink https://escholarship.org/uc/item/ h d xp journal advanced materials (deerfield beach, fla.), ( ) issn - authors lee, sungki damodaran, anoop r gorai, prashun et al. publication date - - doi . /adma. peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ h d xp https://escholarship.org/uc/item/ h d xp#author https://escholarship.org http://www.cdlib.org/ © wiley-vch verlag gmbh & co. kgaa, weinheim wileyonlinelibrary.com c o m m u n ic a t io n a novel, layered phase in ti-rich srtio epitaxial thin films sungki lee , anoop r. damodaran , prashun gorai , nuri oh , jarrett a. moyer , ji-hwan kwon , naheed ferdous , amish shah , zuhuang chen , eric breckenfeld , r. v. k. mangalam , paul v. braun , peter schiffer , moonsub shim , jian-min zuo , elif ertekin , and lane w. martin* s. lee, dr. a. r. damodaran, dr. z. chen, prof. l. w. martin department of materials science and engineering university of california berkeley, berkeley , california , usa e-mail: lwmartin@berkeley.edu dr. p. gorai, n. ferdous, prof. e. ertekin department of mechanical science and engineering university of illinois urbana-champaign, urbana , illinois , usa n. oh, dr. j.-h. kwon, dr. a. shah, dr. e. breckenfeld, dr. r. v. k. mangalam, prof. p. v. braun, prof. m. shim, prof. j.-m. zuo department of materials science and engineering and materials research laboratory university of illinois urbana-champaign, urbana , illinois , usa dr. j. a. moyer, prof. p. schiffer department of physics university of illinois urbana-champaign, urbana , illinois , usa doi: . /adma. characterization methods, these approaches push the edge of materials control. using these techniques it is possible to pro- duce exotic new phases that do not exist in the bulk, [ , ] syn- thesize artifi cial heterostructures, [ , ] and control materials at the unit-cell level to enable new states of matter. [ , ] although these approaches provide unprecedented control, they are lim- ited in the geometries of phases that can be created (i.e., lay- ered heterostructures) and the cost, complexity, and lack of scalability of the continuous in situ monitoring makes large scale use challenging. in this work, we explore a new, self-assembly route to produce novel phases, whereby we combine systems with a tendency for spontaneous phase separation with non-equilibrium deposi- tion techniques and thin-fi lm epitaxy. the goal is to explore the evolution of a classical equilibrium concept (i.e., eutectic mate- rials) under non-equilibrium growth conditions that are kineti- cally limited and infl uenced by epitaxial relationships. to do this, we focus on a model oxide eutectic system: srtio –tio . the equilibrium srtio –tio eutectic phase diagram has been known for decades, [ ] but this work is motivated by a number of recent studies. first, directional solidifi cation of this eutectic system has produced exotic, split-ring resonator-like tio fea- tures embedded in a srtio matrix. [ ] second is the use of complex shuttered thin-fi lm deposition processes and epitaxy to study new members of the sr n + ti n o n + ruddlesden–popper (rp) homologous series and their properties. [ , ] it should be noted that most work on the ti-rich side of the phase dia- gram has been done in the context of bulk eutectic systems and what little work has been done on the ti-rich materials as fi lms has shown only the production of amorphous tio x inclusions in a srtio matrix. [ ] most thin-fi lm work in this system has focused on nearly stoichiometric srtio or sr-excess phases. this begs the question of whether non-equilibrium growth techniques and thin-fi lm epitaxy can be used to produce novel phases and/or nanostructures akin to those observed in sr-rich srtio on the ti-rich side of the phase diagram. here, we study the growth, structure, and properties of the ti-rich portion of the srtio –tio phase diagram in and around the eutectic composition. we observe that the non-equilibrium nature of the growth process results in fi lms that greatly exceed the ther- modynamic solubility limit of ti in srtio (≈ . mol% of ti in bulk srtio [ ] as compared with ≈ mol% of ti in srtio in the current study) and the eventual formation of a layered, ti- rich phase with nominal chemical formula sr ti o . scanning transmission electron microscopy (stem)-based studies map out the structure and valence state of this phase, fi rst-principles self-assembled oxide nanostructures produced, for example, via eutectic phase separation, spinodal decomposition-like routes, and other pathways [ – ] have drawn considerable interest for their varied properties. such approaches leverage innate chemical and thermodynamic driving forces that favor the spontaneous separation of two phases into an equilibrium state characterized by potentially complex micro- and nano- structures (i.e., layered structures, vertically aligned rods in a matrix, etc.) and exotic composite properties. [ , , ] researchers have demonstrated the ability to control the geometry, shape, and size of such phase-separated structures by tuning material composition, [ ] growth temperature, [ ] strain state, [ ] growth, and cooling rates. [ , – ] deterministic and ordered materials self-assembly in these systems is, however, a challenge. in particular, it is diffi cult to access submicron feature sizes in eutectic systems due to the high processing temperatures and fast cooling rates that must be used. [ – ] although spinodal decomposition-like routes have been used to produce nanoscale features, producing long-range ordered arrays of features via this approach is diffi cult. [ ] on the other hand, non-equilibrium approaches, including modern thin-fi lm growth techniques, can be applied to sys- tems to produce atomically and chemically precise artifi cial heterostructures and nanostructures. [ , ] leveraging advances in deposition techniques and the development of in situ adv. mater. , , – www.advmat.de www.materialsviews.com http://doi.wiley.com/ . /adma. wileyonlinelibrary.com © wiley-vch verlag gmbh & co. kgaa, weinheim c o m m u n ic a t io n approaches explore the phase stability and electronic properties, and studies of the dielectric, optical, thermal, and magnetic properties reveal diminished dielectric permittivity (and low dielectric loss), an enhanced bandgap, glass-like thermal con- ductivity, and the potential for d anti-ferromagnetism. in the equilibrium phase diagram ( figure a), [ ] the eutectic composition for the srtio –tio system is located at mol% sro/ mol% tio . films of the srtio –tio system were grown on srtio ( ), ( ), and ( ) substrates via pulsed- laser deposition from targets of the following compositions (where the fi rst and second numbers refer to the mole per- centage of sro and tio , respectively): : , : , : , : , : , : , : , : , and : . details of the growth process are provided in the supporting information. x-ray dif- fraction studies of all fi lms (focusing on ≈ nm thick fi lms on srtio ( ) substrates) reveal only a single set of diffraction peaks and no evidence of phase separation or multiple phases (figure s , supporting information). atomic-force microscopy studies reveal no evidence of phase separation with the surface remaining relatively smooth across all compositions (figure s , supporting information). only upon in-depth cross-sectional high-angle annular dark-fi eld (haadf)-stem imaging of the atomic structure of the fi lms did clear differences emerge (see figure s , supporting information for details) and only when we reach the eutectic composition of mol% sro/ mol% tio does the stem imaging reveal the presence of an unexpected layered phase with an out-of-plane periodicity of ≈ . nm (figure b). such a layered phase was observed on ( )-ori- ented versions of srtio , (laalo ) . –(sr altao ) . (lsat), laalo , and nb-doped srtio substrates, but not on ( )- and ( )-oriented substrates (where complex and disordered struc- tures are observed, figure s , supporting information). high-resolution haadf-stem imaging ( figure a) and nano-area electron diffraction (naed) (figure b) were com- pleted to assess the atomic structure of the phase (see sup- porting information for details). both the stem imaging and the naed patterns reveal a periodicity of ≈ . nm along the out-of- plane direction (ca. times longer than that of the srtio { } d-spacing). along the in-plane direction, fi rst- and second-order diffraction peaks (figure b) reveal periodicities of ≈ . and ≈ . nm that correspond to sr–sr and sr–o interatomic spac- ings, respectively. comparison with the srtio substrate (inset, figure a,b) reveals that the layered phase is composed of layers with local coordination akin to that in srtio that are separated by a monolayer of periodically aligned ti- and o-ions (that exhibit reduced z-contrast and are consistent with the highly ti-rich nature of the fi lm composition). average structural information about the layered phase was obtained using on-axis (about the -diffraction peak of the substrate) and off-axis (about the - and -diffraction peaks of the substrate) x-ray reciprocal space mapping (rsm) studies (figure c–e). here, we index the fi lm assuming that the a , b , and c lattice parameters are parallel to the [ ], [ ], and [ ] of the substrate, respectively. the order of the diffrac- tion condition is determined by comparison to the measured periodicities from the stem imaging and naed patterns. on-axis rsm studies (figure c) show the presence of a fi lm peak (indexed as the -diffraction condition) that reveals an out-of-plane lattice spacing c ≈ . nm (commensurate with the . nm obtained from the stem and naed analysis). off-axis rsm studies about the -diffraction condition of the substrate (figure d) show the presence of a fi lm peak indexed to be the -diffraction condition that enables calculation of the in-plane spacing b ≈ . nm (≈ . % expanded as com- pared with the substrate in the same direction). off-axis rsm studies about the -diffraction condition of the substrate (figure e) reveal the presence of a set of fi lm peaks indexed to be the - and -diffraction conditions that enables cal- culation of the in-plane lattice spacing a ≈ . nm (≈ . % expanded as compared with the substrate lattice spacing in the same direction). φ -scans about the - and -diffraction con- ditions of the substrate and fi lm (figure f), respectively, reveal the epitaxial relationship to be [ ] f //[ ] s and [ ] f //[ ] s and, from stem/naed, the out-of-plane [ ] f is found to be tilted by ≈ . ° from the [ ] s (where f and s refer to fi lm and substrate, respectively). in summary, the unit cell of the layered adv. mater. , , – www.advmat.de www.materialsviews.com figure . self-assembled, layered eutectic srtio –tio fi lm. a) equilibrium eutectic phase diagram of sro–tio system (adapted with per- mission. [ ] copyright , american ceramic society. printed with permission of the american ceramic society (www.ceramics.org).). b) haadf-stem image of the resulting fi lm of eutectic composition ( mol% sro/ mol% tio ) grown on a srtio ( ) substrate. inset shows alignment of the structure. wileyonlinelibrary.com© wiley-vch verlag gmbh & co. kgaa, weinheim c o m m u n ic a t io n phase is: a = . nm, b = . nm, c = . nm, α = γ = °, and β = . °. note that this unit cell corresponds to two for- mula units (i.e., sr ti o ) to fully encompass the complex symmetry of the system. from the stem imaging, it is possible to extract the atomic positions (of the cations) and to produce a model of the pro- posed structure. although it is not possible to resolve all of the o-ions, the cation arrangement suggests that all ti–o bonding should occur within octahedra (tio ) and that all o-sites are fully occupied to both compensate charge and coordination (a detailed discussion of the atomic model and simulated diffrac- tion patterns is provided in table s and figure s and s in the supporting information). based on the extracted inter atomic dis- tances, an atomic model can be constructed and the simulated diffraction pattern [ ] for this initial model matches that obtained in the naed studies well, suggesting a good initial under- standing of the structure (figure s c, supporting information). disambiguation of the structure, however, requires addi- tional steps. briefl y, the stem imaging provides a projection of the atomic columns, but does not reveal the density or exact make-up of atoms in those columns. thus, there were a number of potential candidate structures consistent with the image. we reiterate that no ti-rich srtio phases are reported within the vicinity of the eutectic composition in the equilibrium phase diagram. although not reported on the equilibrium phase dia- gram, two ti-rich phases have been reported in the literature: sr ti o [ ] (corresponding to mol% sro/ mol% tio ) and sr ti o [ ] (corresponding to mol% sro/ mol% tio ). both phases are known to exist only as minor phases, are thought to be metastable, and are off in composition ( and mol% defi cient in tio , respectively) from the eutectic composition studied herein. nonetheless, we have considered the possibility that these phases (or a phase possessing these nominal chemical formulas and a structure commensurate with that observed here) could be the novel, layered phase. a detailed description of these candidate phases is provided (figure s , supporting information). differentiation of the potential structures for the layered phase is achieved by chemical analyses. first, from rutherford backscattering spectrometry (figure s , supporting informa- tion) the sr:ti ratio for the fi lms is determined to be ≈ : . that matches (within the experimental error) the sr ti o phase. adv. mater. , , – www.advmat.de www.materialsviews.com figure . structural characterization of the novel, layered phase. a) haadf-stem image after smoothing process (along the [ ] zone axis of the substrate) of the novel, layered phase. inset shows a corresponding image along the same zone axis for the srtio substrate. b) naed pattern from the novel, layered phase. inset shows a corresponding naed pattern along the same zone axis for the srtio substrate. rsm studies about the c) -, d) -, and e) -diffraction conditions of the substrate. f ) φ -scans about the - and -diffraction conditions of the substrate and fi lm, respectively, revealing the epitaxy of the fi lm/substrate system. wileyonlinelibrary.com © wiley-vch verlag gmbh & co. kgaa, weinheim c o m m u n ic a t io n thus, from the atomic-level imaging, x-ray diffraction, and chemical analyses, the novel, layered phase produced during the growth of the eutectic composition materials is determined to be a new phase – sr ti o . the proposed unit cell for this phase is highlighted by the presence of layers with local coor- dination akin to that in srtio that are separated by a mono- layer of periodically aligned ti- and o-ions. we also note that in the unit cell of this structure that there are three types of octahedral bonding including two corner-, four edge-, and eight face-sharing octahedra (figure s b, supporting information). this specifi c complex and close-packed tio octahedra arrange- ment is not reported in any published tio x phase, but corner-, edge-, and face-sharing octahedra are all possible in tio x sys- tems. the appropriate charge balance of this structure could be achieved (theoretically, assuming nearly complete oxidation) with three possible combinations of ti + :ti + :ti + species occur- ring in ratios of : : , : : , and : : . previous studies suggest that face-sharing octahedra in ti-based systems require the presence of ti + [ ] and since eight of the total octahedra in the unit cell are face-sharing, we expect that ti + must populate the majority of the ti-sites, suggesting that the structure should possess a : : ratio of ti + :ti + :ti + . the average valence state of ti in sr ti o was probed via stem-based electron energy loss spectroscopy (eels) studies of the ti– l , and o– k edge spectra ( figure a). we observe a shift (to lower energies) and broadening of the ti– l , peaks for the novel, layered phase as compared with the srtio substrate consistent with what has been observed in oxygen-defi cient srtio −δ [ ] and ti o (that possess only ti + valence states) [ ] thereby confi rming the presence of a mixed ti + /ti + valence state. we also note that the intensity of the α peak from the o– k edge spectra is signifi cantly diminished indicating that the sr–o bonding contributes less to the spectra [ ] which is, again, consistent with sr ti o (especially the tio x inter-layers) pos- sessing less sr–o bonding compared with srtio . armed with this detailed structural and chemical data, we further explored the structure and stability of the sr ti o phase via density functional theory (dft) using hybrid func- tionals (see supporting information for details). considering the possibility of several valence states for the ti-ions within the structure, it is possible that the tio octahedra may be sus- ceptible to energetically favorable internal distortions. small perturbations of the tio octahedra may not be resolvable via experiment alone and we used the dft approaches to assess the lowest energy structure of the sr ti o phase. the simu- lations reveal that the structure is locally stable, and confi rm the presence of slight octahedral distortions, for which we fi nd an energy recovery of ≈ . ev per unit cell in comparison to the undistorted structure. this is a reasonably large energy recovery in comparison to the atomization energy, which we fi nd to be ≈ ev per unit cell. several distinct distortions are present, corresponding to the various ti + and ti + environ- ments for the octahedra. a schematic illustrating the lowest- energy structure is provided (figure b,c). from this experi- mentally and dft-optimized structure, we can thus compare the experimentally observed atomic structure (figure d) to a simulated z-contrast stem image (figure e), as well as a sim- ulated selective area diffraction pattern (figure f) to the actual naed pattern. this comparison shows excellent and nearly ideal matching (including matched modulation of the intensity across the diffraction pattern); thereby confi rming the extracted and optimized structure is representative of the real structure of the material. based on the structural analysis that suggests a new struc- ture and phase, it is important to understand the mechanism for and nature of the evolution of this exotic phase. clearly the structure that is formed deviates, somewhat dramatically, from the traditional eutectic system from which it was grown, and thus it warrants comparison with other exotic structures observed in materials – namely crystallographic shear struc- tures (css). css can be considered as an example of so-called non-conservative defect structures – or defect structures that change the composition of the parent material – and have been found in a range of materials. [ – ] in transition metal oxides, adv. mater. , , – www.advmat.de www.materialsviews.com figure . atomic models and spectroscopy of the novel, layered phase. a) eels profi le from the novel, layered phase (orange) and srtio substrate (blue) revealing the presence of a mixture of ti + /ti + in the former. schematic illustrations of b) multiple unit cells (along the [ ]) and c) a d view of a single unit cell of the proposed (experimentally and dft-optimized) structure for sr ti o revealing the various types of tio octahedra bonding including corner- (blue), edge- (violet), and face-sharing (cyan). d) smoothed z -contrast stem image of the novel layered phase. e) simulated z-contrast stem image of the same area of the novel layered phase confi rming excellent matching of the extracted structure. f ) simulated diffraction pattern for the model structure overlaid on the top of the naed diffraction pattern revealing excellent matching. the blue circle marks the diffrac- tion condition. wileyonlinelibrary.com© wiley-vch verlag gmbh & co. kgaa, weinheim c o m m u n ic a t io n special non-conservative defect structures occur whereby a reduction of the oxygen content results in a disruption of the underlying structural framework whence the structure is effec- tively cleaved, a fraction of the oxygen ions are removed, and the structure is placed back together, now sheared (or shifted) with respect to one another thereby creating a so-called crystal- lographic shear plane. [ , ] while early work on css focused on binary oxide materials, more recent work has explored css in ternary, anion-defi cient perovskites [ , – ] and others have gone on to extend the idea to cation-excess systems such as the rp homologous series. [ – ] in a similar spirit, it is possible to discuss sr ti o within the confi nes of css. assuming that the parent material is in fact srtio and noting that the full unit cell for our system has been determined to be sr ti o , we would then want to compare this to unit cells of the parent srtio (i.e., sr ti o ). sr ti o could be produced upon the removal of sr- and o-ions from the unit cells of srtio (or . sr and . o per unit cell of srtio ). the sr and o are preferentially removed from ( ) in the structure (producing layers of tio x with no sr atoms) and the remaining perovskite layers are sheared by ½[ ]. such a comparison with css may provide an alternative way of thinking about this system, but is important to point out a number of ways that the sr ti o differs from traditional css systems. first, as noted from the analysis above, sr ti o is a fully oxidized system (i.e., with the exception of a small, equilibrium density of oxygen vacancies common to all oxide materials, all available oxygen sites in the structure are fully occupied) like in the case of the rp phases. in sr ti o the mixed ti + and ti + valence state originates from the coor- dination environment of the ti-ions in the tio x -layers, not from large-scale oxygen defi ciency. this is different from the vast majority of work on css that has focused on signifi cantly anion-defi cient systems. second, the nature of the non-stoichi- ometry is both opposite to and vastly larger in magnitude in sr ti o than in previously examined systems. in the rp sys- tems, for instance, there is a -site excess and in the worst case that excess is only : a : b ratio whereas in sr ti o there is b -site excess with a much larger : a : b ratio. finally, typical css systems lack long-range order to the css since there is a degenerate family of shear planes. this typically causes the material to possess complex rotations of the css giving rise to what some have called waves, hairpins, and Γ-shapes as in the case of the a n b n o n − homologous series of perovskite-based css. [ ] in the sr ti o phase, however, we have observed only one variant of the potential css that runs parallel to the growth plane of the fi lm in a highly ordered, periodic fashion across the sample. in the end, articulation of the structure within the parlance of css may help shed some light on the mechanism under- lying the formation of this structure. ultimately it appears to be an extreme type of css that is likely infl uenced by a number of factors. first, thin-fi lm epitaxy and substrate orientation appear to play a critical role since growth of fi lms simultaneously on srtio ( ), ( ), and ( ) substrates reveals that only the ( )-oriented substrates give rise to the sr ti o phase. this likely arises from the fact that the tio x layers in the structure, which possess sixfold symmetry, would be diffi cult to form on the fourfold or twofold symmetric cubic and rectangular lattices of ( )- and ( )-oriented substrates (in other words, the ( ) surface is the only orientation that can accommodate the css). second, the fi lm composition is found to be important since a deviation of only ≈ mol% in tio composition can destroy the phase (figure s , supporting information). finally, the kinetics of the growth process appear to play a role. truly this is a non-equilibrium structure and the degree of order in the sr ti o increases as the growth rate is reduced suggesting that the formation of the phase requires suffi cient time for the diffusion/rearrangement of the constituent ions. the system is likely metastable in the sense that the sr ti o was found to be stable at growth temperatures < °c, above which the fi lm decomposes into two phases (rutile tio and srtio ). having established the structure, we proceed to explore the electronic structure and physical properties of sr ti o including optical absorption, dielectric permittivity and loss, thermal conductivity, and magnetism. again, due to the pres- ence of mixed-valence cations [ti + ( d ) and ti + ( d )], special attention was given to probing the electronic structure and potential for spin ordering. in structures with face-, edge-, and corner-sharing octahedra, superexchange and direct exchange mechanisms compete with each other and the prevailing spin confi guration is determined by a competition between different exchange interactions that are affected by electron occupancy of orbitals, bond angles, and cation–cation separation. [ – ] from inspection of the crystal structure, two spin confi gurations are possible. in the fi rst, direct exchange interactions within both pairs of ti-ions participating in face-sharing octahedra drive localization of the excess electrons. in this confi guration, a direct exchange interaction j is expected to be large within a pair of face-sharing octahedra, but weaker from one pair to the next. in the second, the ti-ions (in the tio x plane) par- ticipating in edge-sharing octahedra carry the excess electrons and superexchange mechanisms support a d ordered spin confi guration. conventional dft simulations [ ] predict that the structure is not spin-polarized and, although there is a large gap between the nominal valence and conduction bands, the fermi level is located above the conduction band edge and the system is predicted to be metallic (which it is not). additionally, rather than distinct ti + -and ti + -ions, the extra electrons are shared equally amongst all ti-ions (in the tio x plane) in a metallic state. moving to hybrid dft calculations, which we expect to be more accurate in this case, we are able to assess the possibility of charge and spin ordering. for a standard choice of hybrid parameters, [ ] we predict that an anti-ferromagnetic check- erboard-like spin confi guration, in which the excess electrons are localized on the ti-ions within the tio x plane ( figure a), is more stable than the non-magnetic one by ≈ . ev/unit cell. examination of the distribution of up- and down-spin electron density within the tio x plane (figure b) shows the nature of the anti-ferromagnetic order and how the oxygen ions along the octahedral shared edges mediate the anti-ferromagnetic ti confi guration via superexchange, indicating that the second scenario considered above prevails. it should also be noted, that the spin-down electron distributions are observed to extend asymmetrically out of the plane toward the neighboring ti atoms across the shared octahedral face, suggesting that mild elements of the fi rst scenario (a ferromagnetic coupling across adv. mater. , , – www.advmat.de www.materialsviews.com wileyonlinelibrary.com © wiley-vch verlag gmbh & co. kgaa, weinheim c o m m u n ic a t io n face sharing octahedra due to direct exchange) are also present, but weaker than the anti-ferromagnetic order within the plane. finally, the electronic density of states (figure c) reveals three distinct sets of states are present in the vicinity of the fermi energy. the lower set of states (nominally the valence band), exhibits an oxygen p character while the intermediate and upper (nominally conduction band) exhibit a ti d char- acter. the intermediate states are highly localized to the edge- sharing ti atoms in the tio x plane and likely are states offset from the conduction bands, stabilized in part by exchange interactions leading to the anti-ferromagnetic confi guration. [ ] the primary gap is calculated to be . – . ev. subsequent experimental study confi rms many of these observations. magnetic measurements of the sample cooled from k in a magnetic fi eld and in zero fi eld reveal no fer- romagnetic ordering either in- or out-of-the-plane of the fi lm from to k, but a broad peak in the magnetic susceptibility was observed in both fi eld cooled and zero fi eld cooled measure- ments around k, which could be indicative of anti-ferromag- netic ordering in the material and is consistent with the predic- tions of anti-ferromagnetic order from the hybrid dft studies. transmittance and refl ectance studies of sr ti o /laalo ( ) heterostructures also confi rm the predictions of the hybrid dft studies. a linear fi t of αhv (where α is the absorption coeffi - cient, h is planck constant, and υ is the frequency of photon) as a function of the photon energy ( hυ ) ( figure a) reveals a direct bandgap for sr ti o of ≈ . ev. the decreased transmittance in sr ti o between and nm is due to the laalo sub- strate with complex twin structures that refl ect the visible light diffusively and decreases the transmittance. see the supporting information for details of these measurements. likewise, probes of electronic conduction reveal a highly insulating material (albeit with some anisotropy). when measured in the out-of-plane geometry, nm thick fi lms revealed low leakage current densities of < . µa cm – even under applied electric fi elds in excess of kv cm – while measurements in the in-plane geometry revealed leakage currents at least -times larger, but still indicative of an insulating material (figure s , supporting information). such obser- vations are consistent with what might be expected based on the highly anisotropic structure, which could limit conduction out- of-the-plane of the fi lm and based on what has been observed mixed ti + /ti + tio x com- pounds where the conductivity can be larger than in purely ti + systems. [ ] subsequent probes of the out-of-plane dielectric per- mittivity ( ε r ) and loss tangent of sr ti o / nb-doped srtio ( ) heterostructures at room temperature as a function of frequency have also been completed (figure b). the dielectric permittivity of the sr ti o fi lms was found to be essentially frequency-inde- pendent with a value of with low loss tangents ( . at khz) in the frequency range – khz. this low dielectric per- mittivity may be expected considering that the sr ti o phase consists of srtio -like layers ( ε r ≈ ) and layers of face-sharing octahedra tio x layers (which are structurally similar to ti o that has a ε r ≈ ) [ ] that effectively produce a nanoscale, layered dielectric. lastly, the thermal conductivity ( κ ) of the sr ti o fi lms was measured in the fi lm-normal direction by time-domain thermorefl ectance (tdtr) [ ] (see figure s , supporting information for details). we note that the thermal conduc- tivity of the sr ti o fi lms shows a signifi cantly lower value of κ ≈ . w m-k – compared with parent phases srtio and tio that range between . and . w m – k – [ , ] whereas the speed of sound of the layered fi lm remains essentially the same as the parent phases. previously, low κ has been observed in artifi cially synthesized srtio -superla- ttice systems ( κ ≥ . w m – k – for (srtio ) m /(catio ) n and κ ≥ . w m – k – for (srtio ) m /(batio ) n superlattices) [ ] and in sr n + ti n o n + rp-phases ( κ ≥ . w m – k – ), [ ] but not as low as that observed in the sr ti o fi lms. we hypothesize that the glass-like thermal conductivity arises from two main contributions: i) signifi cant phonon scattering at the bounda- ries between srtio -like and tio x layers in this phase and ii) a low contribution of electronic thermal conductivity from low free electron concentration. this is not surprising considering that sr ti o has a periodic spacing of . nm in the out-of- plane direction (excellent for scattering portions of the heat car- rying phonons) and that the electrical conductivity is extremely low as we observed from the leakage experiment. the ability to produce highly ordered layered structures with subnanom- eter length-scales could open up many opportunities for low thermal transport applications. in conclusion, we have synthesized a new phase sr ti o that self-assembled into a highly ordered, layered structure that consists of layers with local order akin to that of srtio sepa- rated by close-packed, tio x layers by combining non-equilib- rium growth techniques, thin-fi lm epitaxy, and a system with an innate chemical/structural instability. we observe that the layered sr ti o phase is realized only at the eutectic composi- tion of the srtio –tio system, when growth takes place on adv. mater. , , – www.advmat.de www.materialsviews.com figure . spin confi guration, orbital charge density, and electronic density of states of the novel, layered phase. schematic illustration of a) the proposed spin confi guration in which the edge-sharing, planar tio octahedra exhibit a d electron valence and a checkerboard anti-ferromagnetic spin confi guration and b) the charge density of the orbitals corresponding to this confi guration according to hybrid dft calculations, showing the occupation of orbitals to minimize electron overlap. c) corresponding electronic density of states for the system. wileyonlinelibrary.com© wiley-vch verlag gmbh & co. kgaa, weinheim c o m m u n ic a t io n ( )-oriented perovskite substrates, and when the kinetics of the growth process are appropriately controlled. it should be noted, however, that although the phase is found to form at the eutectic composition, the role of the innate eutectic tendency of this system in stabilizing this layered phase is not fully under- stood at this point and is clearly a matter for future studies in other systems. in-depth x-ray and electron diffraction studies and imaging have allowed for the precise identifi cation of the structure and unit cell of this phase. subsequent chemical and spectroscopic studies were used to confi rm these studies. the sr ti o phase possesses properties that are unique from the parent srtio and tio phases including a larger optical bandgap of . ev, reduced dielectric constant of , low dielec- tric loss, and a glass-like thermal conductivity of . w m – k – . this unique approach to the formation of high-ordered lay- ered materials could represent a new modality to achieve novel states of matter in other systems possessing similar chemical/ structural instabilities and can provide new insights into the design of hierarchical structures by self-assembly. supporting information supporting information is available from the wiley online library or from the author. acknowledgements s.l., z.c., p.v.b., and l.w.m. acknowledge the support of the air force offi ce of the scientifi c research under grant fa – – – . a.r.d. acknowledges support from the army research offi ce under grant w nf- – – . e.b. acknowledges support from the national science foundation under grant dmr- . p.g, n.f, and e.e. acknowledge support from the strategic research initiatives program of the college of engineering at the university of illinois at urbana- champaign. n.o. and m.s. acknowledge support from the national science foundation under grant che- . the work was carried out, in part, in the materials research laboratory central research facilities, university of illinois. received: august , revised: october , published online: december , [ ] r. l. ashbrook , j. am. ceram. soc. , , . 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[ ] k. h. lee , s. w. kim , h. ohta , k. koumoto , j. appl. phys. , , . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ degrowth and critical agrarian studies full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=fjps the journal of peasant studies issn: - (print) - (online) journal homepage: https://www.tandfonline.com/loi/fjps degrowth and critical agrarian studies julien-françois gerber to cite this article: julien-françois gerber ( ) degrowth and critical agrarian studies, the journal of peasant studies, : , - , doi: . / . . to link to this article: https://doi.org/ . / . . © the author(s). published by informa uk limited, trading as taylor & francis group published online: jan . submit your article to this journal article views: view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=fjps https://www.tandfonline.com/loi/fjps https://www.tandfonline.com/action/showcitformats?doi= . / . . https://doi.org/ . / . . https://www.tandfonline.com/action/authorsubmission?journalcode=fjps &show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=fjps &show=instructions https://www.tandfonline.com/doi/mlt/ . / . . https://www.tandfonline.com/doi/mlt/ . / . . http://crossmark.crossref.org/dialog/?doi= . / . . &domain=pdf&date_stamp= - - http://crossmark.crossref.org/dialog/?doi= . / . . &domain=pdf&date_stamp= - - degrowth and critical agrarian studies julien-françois gerber international institute of social studies, erasmus university rotterdam, the hague, netherlands abstract degrowth refers to a radical politico-economic reorganisation that leads to smaller and more equitable social metabolisms. degrowth posits that such a transition is indispensable but also desirable. however, the conditions of its realisation require more research. this article argues that critical agrarian studies (cas) and degrowth can enrich each other. the agrarian question and the growth question should be addressed in concert. while degrowth should not fall into the ‘agrarian myth’, cas should not embrace the ‘myth of growth’, even when green and socialist. ideas of one philosopher and four agrarian economists are presented, with illustrations from bhutan, cuba and north america, hoping to offer a preliminary research agenda for ‘agrarian degrowth’. keywords sustainability; anarchism; marxism; agrarian and environmental justice; bhutan; cuba . introduction compounding growth, environmental degradation, and widespread alienation are the three most dangerous contradictions for our time. – david harvey ( , – ) if the spectre of communism was haunting europe in the mid-nineteenth century, even though ‘all the powers of old europe [had] entered into a holy alliance to exorcise this spectre’ (marx and engels [ ], ), it is perhaps the spectre of degrowth that is today haunting the industrialised world, while all the imperial powers tend to deny the urgency of the situation (akbulut et al. ). for the economic élites, the lack of growth is a most frightening idea, but for other people, degrowth may represent a way out, a ‘con- crete utopia’ (latouche ; muraca ) from which alternatives can be rethought. the current state of our biosphere does not indeed look good, and the measures taken are grossly insufficient. we are reaching the peak of raw materials that are foundational to our economies, such as oil and phosphorous, while myriads of ecosystems have been damaged beyond repair and are no longer able to absorb our emissions and waste, as evident in the phenomenon of climate change (steffen et al. ). on the socioeconomic front, things look hardly better. disconnected from ecological reality, capitalism continues to deploy its inbuilt tendency to grow and seek new accumulation opportunities, both vir- tually (financialization) and materially (extraction and production). debts and derivatives have reached unparalleled levels worldwide, increasing the risk of economic meltdown © the author(s). published by informa uk limited, trading as taylor & francis group this is an open access article distributed under the terms of the creative commons attribution-noncommercial-noderivatives license (http://creativecommons.org/licenses/by-nc-nd/ . /), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. contact julien-françois gerber gerber@iss.nl the journal of peasant studies , vol. , no. , – https://doi.org/ . / . . http://crossmark.crossref.org/dialog/?doi= . / . . &domain=pdf&date_stamp= - - http://creativecommons.org/licenses/by-nc-nd/ . / mailto:gerber@iss.nl http://www.tandfonline.com (durand ). relative poverty is on the rise everywhere (wir ), while global gdp shows no sign of absolutely decoupling from ecological impacts (hickel and kallis ). not unsurprisingly, then, more and more people and movements are starting to question the world’s trajectory of maldevelopment (to borrow samir amin’s word), not only from an environmental and economic perspective, but also politically and existentially. among these different mobilizations, degrowth is steadily emerging as a central counter-narrative. this article argues that critical agrarian studies (cas) and degrowth can bring essential elements to each other, and that the bridges have so far remained too rare (but see roman-alcalá ; gomiero ; scheidel, ertör, and demaria forthcoming). the word ‘degrowth’ only appeared four times in jps, and always in passing (except in martínez- alier et al. , where it occupies a small paragraph). in reverse, a recent overview of the field of degrowth (d’alisa, demaria, and kallis ) does not explicitly mention any distinct cas sources and remains sketchy on agricultural issues. this lack of communi- cation is surprising because the agrarian question, after all, is about the deployment of capitalist growth in the countryside and about how to transition to egalitarian alternatives. additionally, the political economy of land and food is a central concern for degrowth scholars, and so have been other traditional strongholds of cas, like agroecology and pro- ductive ‘sovereignties’ as in food sovereignty. but beyond their mutual overlaps, the two fields can also usefully address each other’s blind spots. degrowthers should be wary not to fall into various versions of the ‘agrarian myth’ and base their alternative models on naïve ideas about ‘peasant economies’. similarly, cas scholars should be careful not to endorse the pervasive ‘myth of growth’ – even when green and socialist – according to which the constant development of the productive forces ultimately leads to more welfare and can decouple from ecological impacts with appropriate technologies. building on these elements, the present article seeks to bring the fields of degrowth and cas closer, or to bridge the agrarian question with the growth question. i define the latter with the following basic interrogations: what are the effects of growth ‘on the ground’? who benefits from it and until what point? what are the alternatives and who will support them? to me, the growth question needs to be incorporated at the core of the agrarian questions (see also gerber and veuthey ). i will start with a short exposition of some of the key problems with growth and with a brief presentation of what degrowth is and is not. after that, the core of the article will explore fundamental connections between cas and degrowth through the contribution of one philosopher and four agrarian political economists. i contend that the approach of these five authors, taken together, provide an already fairly coherent glimpse at what research and activism in ‘agrarian degrowth’ might look like. the key ideas are simone weil’s notion of rootedness within limits, alexander chayanov’s multiple balances of repro- duction, joseph kumarappa’s democratic decentralisation, nicholas georgescu-roegen’s social metabolism, and joan martínez-alier’s alliance between environmental justice and degrowth. these ideas will, as much as possible, be illustrated with empirical examples, and especially with vignettes from bhutan, north america and cuba. i am not normatively endorsing every aspect of each of these authors’ work or of each case study; my goal is today’s version of the ‘agrarian myth’ refers to an unspecified ‘older time’ when undifferentiated peasant communities generated eco-friendly knowledges, values and practices, while the subsequent fall from grace came with urbanization, globalisation and the rise of the modern state and its doctrines of development and modernisation (brass ). j.-f. gerber simply to show that they all raise crucial questions for ‘agrarian degrowth’. i then suggest some lines for future research and end with a few remarks. . where are we and what next? the crisis consists precisely in the fact that the old is dying and the new cannot be born; in this interregnum a great variety of morbid symptoms appears. – antonio gramsci (quoted in weis , ) . . what is wrong with economic growth? the ideology of growth – or growthism – is at the core of capitalism. growthism sustains capitalism politically because it allows avoiding redistribution by giving the impression that everyone will continually benefit from it. growthism pacifies class struggle while jus- tifying existing structures of inequality (schmelzer ). this ideological function of growth can be observed in mainstream economic theory too, as for example in the kuznets curve narrative (palma ). the latter acknowledges that problems of inequality and ecological destruction may increase during the initial stages of developments, but it posits their resolution in the near future thanks to accrued economic growth. in the west, growth was instrumental to diffuse demands of the workers’ movement, in the east, to excuse the lack of democracy and worker control (dale ), and in the south, to justify dispossession and extractivism (gerber and raina b). today, gdp growth remains the key stabilising mechanism of capitalist economies. apologists of growth regularly claim that it will be able to ‘dematerialize’, and hence become ‘green’, with the advancement of cleaner or more efficient technologies and with renewable energies (see e.g. unep ). however, to date, no country has experienced an absolute reduction in material use while growing (taking trade into account) and no trends indicate that this will occur anytime soon (hickel and kallis ). as a result, it is highly improbable to envisage effective climate mitigation scenarios that involve growth (jackson ). some scenarios estimate that it is possible, but their models typically assume some kind of ‘magical’ technical fix after (kallis et al. ). furthermore, the relationship between growth and well-being are complex and open to debate. in industrialised countries, the increase in self-reported well-being typically stag- nated somewhere between and , or even turned into a negative trend, despite a steady growth in gdp since then (layard ). good levels of well-being are achieved by countries such as cuba and vietnam – two countries led by communist parties – at a frac- tion of the output, energy and resource use of countries like the usa. however, even these lower levels of resource use could not be extended to the entire world over the long-term. no country currently satisfies social well-being standards while staying within its share of pla- netary boundaries (o’neill et al. ). but nothing indicates that this is an impossible goal, even in the developing world. some radical changes are thus clearly needed. . . what is degrowth? degrowth is a field of scientific research and activism that starts with the recognition that the flows of material and energy used in the global economy are unequally distributed and often massively oversized, especially in industrialised countries. a radical reorganisation the journal of peasant studies and resizing of our economies is thus needed if equity and sustainability are to be taken seriously. the fundamental degrowth proposition is that it is possible to live well in ega- litarian societies with much smaller resource throughputs (latouche ; pallante ; kallis ; schmelzer and vetter ). the ideas behind degrowth are old and have roots in many parts of the world. but as a slogan in social movements, the word itself was first used in france (‘décroissance’) in the early s and it then rapidly spread elsewhere. it became a focus of scientific research at the end of the same decade. the term was originally not a concept but a ‘missile word’ that would trigger discussions on limits, hit at the core of capitalist moder- nity, and re-politicise the false technocratic and pro-market consensus around ‘sustainable development’ and ‘green growth’. there are four immediate sources to the modern notion of degrowth (demaria et al. ): first, the radical western environmental movement of the s and s, with two famous women in particular, rachel carson who wrote silent spring in and donella meadows who coordinated the limits to growth report in ; second, the political, cultural and existential critique of capitalist modernity, as in the works of erich fromm and cornelius castoriadis; third, the heterodox current of ecological economics, and particularly the work of nicholas georgescu-roegen as we will see below; and fourth, the critique of ‘development’ seen not as a liberation process but as the continuation of western capitalist hegemony, with the works of arturo escobar or ivan illich. degrowth thus goes much beyond the ecological critique of gdp growth and includes a broader reflection on what constitutes an existentially meaningful mode of co-existing. its answer has to do with concepts like sharing, commoning, caring, healing, horizontality, conviviality and simplicity – notions that can all have subversive implications when taken seriously. defined in this way, the ideas behind degrowth go also beyond the euro- pean origin of the label and beyond the four modern sources i just mentioned. it is obviously in industrialised countries that metabolic flows and imperialism need to be tackled the most urgently, but growthism and the resulting maldevelopment have also taken solid roots in the global south. there is therefore scope for creative and radical measures beyond growthism in the global south as well (see e.g. gerber and raina b; kothari et al. ). contemporary systems of production/consumption have been the object of perhaps four major critiques from the radical left. in a nutshell, the latter has problematised capitalism as in marxism, capitalism and any state as in anarchism, developmentalism as in post-development theory, and, most recently, growthism as in degrowth. of course, one does not have to stick to one critique since they may all complement each other well. but from this perspective, the degrowth critique is arguably broader than the classical marxist one because it includes the critique of any growthism. ‘growth is the child of capitalism’, noted kallis ( , ), ‘but the child outdid the parent, with the pursuit of growth surviving the abolition of capitalist relations in the word ‘degrowth’ was first used in the modern sense in by french political ecologist andré gorz (demaria et al. ), but one could look for older related concepts (since ‘degrowth’ is so closely linked to gdp). the term ‘post-indus- trialism’, for example, was coined in by ceylonese philosopher ananda coomaraswamy (marien ; calmé and taleb ). going much further, latouche ( ) argued that lao tzu ( th c. bc) is an early proponent of degrowth in his defence of simplicity and autonomy, his refusal of the superfluous, and his critique of existing power. the word has been criticised for being economistic, ‘negative’ and/or eurocentric (see e.g. drews and antal ). j.-f. gerber socialist countries’. today, very different political movements and regimes share the ima- ginary of permanent economic growth. however, this has not been the case of anar- chism and post-development theory, two currents of thinking that have had a profound influence on degrowth. just like marxism classically seeks to generate ‘socialism’ or ‘communism’, what sort of systemic alternative does degrowth seek to give birth to? the labelling exercise is open to discussion and many degrowthers would not find it useful, but ‘eco-municipalism’ could be a candidate. the term is regularly seen in the social ecology literature (gerber and romero ) and was praised by latouche ( ). it originally refers to a multi-level network of egalitarian and sustainable communal entities governed by direct democracy. the term resonates well with other non-western candidates like shri- vastava and kothari’s ( ) ‘radical ecological democracy’. the term also nicely trans- cends the urban and the rural as well as the local and the nation-state while excluding none. *** before pursuing further, i will try to briefly clarify six common misunderstandings about degrowth. first, degrowth is not just about reversing gdp growth rates. this phenomenon already has a name and it is called a recession. the point of degrowth is not to have ‘less of the same’, but to organise appropriation, extraction, production, distribution, consumption and waste differently. second, degrowth is not about imposing ‘austerity’ everywhere and shrinking ‘everything’. some items (like local products) will surely be consumed and pro- duced much more in a degrowth society, and many economic activities will increase in such a society (like agroecology, urban gardening, and so on). third, degrowth is not anti-technology. after almost two centuries of extraordinary technological progress (and also disruptions), degrowthers simply advocate for the right to choose what to take and what to leave behind. fourth, degrowth is not just about celebrating the local. small can be beautiful, but not always, as we will also see below. in fact, serious post-growth thinking can only be world-systemic – and therefore internationalist – because socio- metabolic flows are globally so unequally distributed and dimensioned. fifth, degrowth is not soluble in capitalism. there are different currents within post-growth thinking (e.g. steady state economics, agrowth, post-gdp) and not all of them are post-capitalist, but degrowth is. finally, degrowth is not anti-marxist, but it is against a certain marxism that has a blind faith in industrial productivism, technological progress, and modern science as the only valid source of knowledge. to give just one example, leon trotsky was a formidable thinker, but his futurist eagerness of sounds today rather frightening: faith merely promises to move mountains; but technology, which takes nothing ‘on faith’, is actu- ally able to cut down mountains and move them. up to now this was done for industrial purposes (mines) or for railways (tunnels); in the future this will be done on an immeasurably larger scale, according to a general industrial and artistic plan. man will occupy himself with re-registering mountains and rivers, and will earnestly and repeatedly make improvements in nature. in the end, he will have rebuilt the earth, if not in his own image, at least according to his own taste. (quoted in foster , ) since critical agrarian studies (cas) have been profoundly influenced by marxism, the next section on simon weil will continue exploring the relations between marxism, degrowth and cas. the journal of peasant studies . bridging cas and degrowth: one philosopher and four agrarian economists we see the need to dream for more radical change than contemporary politics offers. – patel and moore ( , ; quoted in borras , ) . . simone weil: rootedness within limits marxism and the important branch of cas that claims the marxist tradition have usually not, of course, been about degrowth but about socialist growth. however, this does not imply that the critique of endless growth has never been made by the revolutionary left. the thought of simone weil ( – ), a french philosopher, activist and mystic, is an early and remarkable example of this. already in the s, she denounced the violence of industrialism that ‘uproots’ workers and peasants, denies limits, and negates the material and immaterial ‘necessities’ of the body and the soul. after graduating in philosophy and while working in revolutionary trade-unionism, she spent long periods of time as a factory worker and as an agricultural labourer in order to better understand the condition of the working class. she took part in the spanish revolu- tion alongside the anarchists and participated in the french resistance before dying in london at age . her short and busy life did not, however, prevent her from writing extensively. she can be seen as a precursor not only of political ecology but also of degrowth – albeit without, of course, using the term. her own political evolution shows how her ‘degrowth’ ideas were nourished by marxism, but also how they began to differ- entiate in some important respects (azam and valon ). weil’s thought is therefore an entry point into the relationship between marxism and degrowth as well as into degrowth’s existential critique of industrial modernity, including in the rural world. against marx, she did not believe that the constant development of the productive forces is the key progressive power behind human history, a ‘materialist’ idea that reverses hegel’s unfolding of the spirit as the main engine of history. instead, weil ( [ ]) proposed to take materialism really seriously and thus came to emphasise limits and ener- getics. she problematised the contradiction between capitalist accumulation, on one hand, and the limited availability of natural resources and the irreversible degradation of energy on the other hand (see georgescu-roegen, below). ‘[t]o hope that the develop- ment of science will one day bring about, in some sort of automatic way, the discovery of a source of energy which would be almost immediately utilisable for all human needs, is simply day-dreaming’ (weil , ). she also noted, anticipating the idea of the anthro- pocene, that the industrial society has become a ‘force of nature’ changing the face of the earth (weil , ). in her critique of technocratic industrialism, weil argued that ‘the worker’s complete subordination to the enterprise and to its managers is founded on the factory’s structure and not on the system of property’ (p. ). she criticised trotsky, both in print and in person, saying that the bolshevik élite would become just as oppressive as the capitalists it was supposed to replace. as an alternative, she advanced the notion of ‘rootedness’ built around autonomous communities, cooperatives and interconnected smaller production weil was not afraid to use the word ‘soul’ in a metaphysical sense. she was deeply interested in spirituality and read exten- sively on christianity, hinduism (she learned sanskrit), mahayana buddhism, taoism as well as on ancient greek and egyptian religions. j.-f. gerber units (weil [ ]). such a system would have both collective as well as regulated private ownership rights. those who work the land, she thought, should be able to control their plot and manage it themselves. in a way that may sound unfashionable today, she defended the potential ‘grandeur’ of work, of efforts, seen as a central element of being in the world and engaging with it, biophysically as well as spiritually. she did not, thereby, seek to celebrate ‘artisan’ or ‘peasant’ work per se, although she had a great respect for it, but to radically change the nature of labour, very much along marx’s critique of alienation. people should control the process and fruit of their labour, including the techniques it involves, as in illich’s ( ) idea of convivial technology. weil’s notion of ‘rootedness’ involves the need to be soundly situated in time, space and social relationships. many peasants in europe and the (neo-)colonies, she noted, have been forcefully uprooted by agriculture’s industrialisation, and those who stay are uprooted too, through debt, contracts and a widespread ‘inferiority complex’ (weil , ). farming, for her, should somehow subjectively reclaim ‘the wonderful cycle whereby solar energy, poured down into plants, […] becomes concentrated in seeds and fruits, enters into man […] and spends itself on preparing the soil’, supporting the sustainable cycle of life (p. ). however, weil refused the conservative forms of rootedness, as expressed in the ‘land’ or the ‘nation’. she replaced the land with the ‘field’, and the nation with ‘friendship’. her idea of rootedness is about ‘a re-localisation of effective and affective solidarities’ (latouche , ). rootedness refuses violence and speed, and embraces limits, cooperation, decentralisation, autonomy, friendship and beauty – the material and immater- ial ‘necessities’ of a degrowth society (azam and valon , ). *** broadly speaking, the problem of limits has been underestimated by marxist cas scholars who have devoted more time and energy to deconstruct ‘green populism’ and/or ‘essenti- alist ecofeminism’ than to elaborate new and creative insights on viable alternatives. it is not rare to find cas researchers that seem to be supporting socialist development models that could not be generalised within planetary boundaries. bernstein ( , ), however, acknowledged the deficiency of cas on these questions and noted – in a way that would have pleased simone weil – that the ‘“orthodox” materialist conceptions of the develop- ment of the productive forces in capitalist agriculture’ show ‘an inherited weakness’ when they embrace ‘such development as forever “progressive”’. reviewing three decades of cas, bernstein and byres ( , ) recognise that ‘very few contributions engaged with the- orizing issues of technical change’ and with analysing ‘the ecological conditions of farming and environmental change’. there is thus, according to bernstein ( , ), an urgent need to reduce the intellectual deficit of cas on the relationships between technological and ecological change, or, in other words, between ‘growth’ and ‘nature’. since then, cas contributions in agroecology, extractivism and the metabolic rift have expanded, but biophysical analyses and the theorization of broader alternatives remain scarce. weis ( ) is among the welcome exceptions in cas, clearly exposing the ecologi- cal and social costs of the productive forces in today’s ‘most advanced’ agriculture, and seeking a way out. in a very degrowth way, he wrote that she specified: ‘the principle of private property is violated where the land is worked by agricultural labourers and farm- hands under the orders of an estate-manager, and owned by townsmen who receive the profits’ (weil , ). the journal of peasant studies the need to radically restructure agriculture is at the very core [of] any hope of making the ‘human relation to the earth’ more ecologically sustainable. it is also, simultaneously, at the core of attempts to build more socially just and humane societies. agricultural systems must be vastly more labour-intensive and biodiverse, and geared towards much less meat pro- duction, as finite biophysical overrides diminish and in order to reduce the untenable environ- mental and atmospheric burden of industrial methods. (p. ) this does not mean, however, that weis embraced any version of the agrarian myth, as ‘agrarian social relations have rarely been equitable or even sustainable’ (p. ). yet there is no reason to think that work in urban or rural industries represents a real progress for the many smallholders who leave the sector. if class and non-class forms of oppression can be overcome, farm work ‘has an unmatched potential to generate autonomous, skill- ful, experimental, healthy and meaningful work’ (p. ). table outlines some of the differences between marxist cas and degrowth research. the purpose of the table is to show strengths/weaknesses on both sides in order to promote more dialogue and integration. i would like to make the basic point that cas and degrowth would gain from considering ‘socialist degrowth’ and hence enrich each other’s theory and praxis. along this line, kallis ( a) convincingly argued that if social- ism is serious about curbing the exploitation of ‘the soil and the worker’ (marx , ), it can only go post-growth (see also the entire special issue in capitalism nature socialism on these questions: andreucci and engel-di mauro ). in a line pioneered by simone weil and others, research in degrowth is an attempt to move beyond economistic analyses and to include socio-ecological relations as well as existential questions (latouche ). accordingly, degrowth research would not only table . rough differences/complementarities between the research agendas of cas (within the marxist tradition) and degrowth. research in critical agrarian studies (marxist) … research in degrowth … focuses on the deployment of capitalism in agriculture and on its relation with industrial growth and socialism (agrarian question) focuses on the deployment of growth(ism) in society and ecosystems, and on its relation with post-growth politics (growth question) debunks the agrarian myth debunks the (green) growth myth sees the development of productive forces and technology as generally progressive sees the development of productive forces and technology as a central object of critical enquiry focuses on the first contradiction of capitalism (clash between capital and labour) focuses on the second contradiction of capitalism (clash between capital accumulation and living conditions [o’connor ]) emphasises problems of economic reproduction, especially through processes of appropriation, production and distribution emphasises problems of socio-ecological reproduction, especially through processes of extraction, consumption and excretion (waste) problematizes exchange value and abstract labour (as potentially exploitative) problematizes use value and concrete labour (as potentially meaningless and unsustainable) evaluates agriculture’s performance in monetary and production terms evaluates agriculture’s performance in socio-metabolic terms, including all its ‘side-effects’ is politically sceptical of ‘the community’ (seen as differentiating and hence divided) is politically open to ‘the community’ (seen as central to work and living conditions) supports, normatively, agrarian justice initiatives and class struggle supports, normatively, environmental justice initiatives and prefigurative politics as ‘incubators’ argues that marx is central in laying out the processes of capital accumulation, crises and class struggle agrees but argues that ‘marx cannot help much in understanding cooperation and self-organisation from below’ (o’hearn and grubačić , ) j.-f. gerber problematise exchange values and abstract labour as potential sites of exploitation; it would also critically examine the actual usefulness and implications of ‘use values’ (as in new technologies and products) as well as the existential nature of concrete labour and community relations. this renewed interest in the quality of use value, work, and social relations can not only be linked to weil’s central concerns, but also to harvey’s ( ) critique of ‘widespread alienation’ and to graeber’s ( ) recent take on ‘bullshit jobs’. to reopen these fundamental questions is essential to the degrowth project and may, to some extent, resonate with the chayanovian arts of balancing. . . alexander chayanov: the multiple balances of (re)production chayanov ( – ) was a russian economist and sociologist who theorised the ‘peasant economy’ and who, without a doubt, remains one of the most influential and debated authors in cas. chayanov considered himself a marxist, along a neo-narodnik line, and believed that the ‘peasant economy’ represents a non-capitalist system that requires its own economic theory. he championed the development of agriculture on the basis of cooperative peasant households instead of the standard soviet policy of large-scale state farms. after two arrests followed by five years in a labour camp, chayanov was arrested again in and executed the same day (shanin ). chayanov’s analysis of the family farm can nourish aspects of degrowth theory and praxis (garcia ). he noted that because the economy of middle peasants is organised outside the traditional profit/wage couple of capitalism, its economic logic does not purely rely on quantitative necessities. instead, it crucially depends on qualitative balances defined within the family farm and its context. chayanov ( ) mentioned two key balances: the labour/ consumer and the utility/drudgery balance. they both indicate that labour increases until it meets the needs of the household, and then it stops, largely because the extra efforts (the ‘drudgery’) are not worth it. there is thus no clear process of accumulation (or growth) as in the capitalist firm (see also sahlins ). in other words, the middle peasant family farm has no inbuilt growth imperative and can potentially live well without it. chayanov advanced many hypotheses that could have been investigated further (or corrected) had he lived longer. a few contemporary scholars – like teodor shanin or jan douwe van der ploeg – have continued this task. van der ploeg ( ) has, for example, identified several other balances that are essential to the family farm, like the delicate balance between the farm’s and its ecosystem’s reproduction (a balance also dis- cussed in toledo’s pioneering article, ) or the balance between internal resources (fos- tering autonomy) and external ones (fostering dependence). chayanov ( ) attributed a strategic importance to the degree of commodification of the household and was well aware that debts or taxes may completely destabilise the family farm. this disruption may come from the market, but also from the state (as also discussed in e.g. scott ). ploeg mentioned as well the balance between scale and intensity, between the short and the long term, between the known and the unknown, between innovation and conservatism, and between the peasant family and the community. in sum, for the chayanovians, farming is about finding the right balances for fulfilling human needs – it is not necessarily about growing. the resource base of the farm represents an organic unity controlled by those directly involved in it, but embedded in the broader politico- institutional-cultural context (sahlins ). the journal of peasant studies but chayanov did not stop at the farm level. he also theorised a cooperative-based economic system where different types of production are associated with their appropri- ate scale (chayanov ). forests, for example, can be well managed at the regional level, pastures at the community level, and salad production at the household level. such a multiscale model does not follow a dogmatic ‘small is beautiful’ principle (as also noted by bernstein ) sometimes seen among the proponents of the degrowth movement. chayanov’s greatest contribution to degrowth is perhaps to help us imagine an economy that is free from the fundamental categories of capitalism and simultaneously based on the popular knowledge of balancing, defining the right scales, and seeking autonomy (garcia ). but the link with post-growth has never been mentioned by today’s chayanovian scholars, probably because it would have been counterproductive to add another ‘controversial’ topic to their already ‘controversial’ agenda of defending smallholders and arguing that they can ‘feed the world’ (j.d. van der ploeg, pers. comm.). this is unfortunate because degrowth, as we have seen, does not imply that ‘everything’ would need to shrink. while capitalist farming would indeed shrink, other elements would need to multiply, and very much so, like cooperatives and commons. *** like chayanov’s russia, bhutan is today largely an agrarian economy. agriculture occupies almost per cent of the population, and among them, middle peasants are the majority. bhutan is an important case study for degrowth research, not only because it may offer a good setting for investigating chayanovian balances and their evol- ution, but also because of its alleged ‘new development paradigm’ that is also said to be about balancing different objectives. bhutan is one of the last countries to be currently, and probably partially, transitioning to capitalism. one can thus observe in ‘real time’ processes of differentiation and resist- ance as well as persisting ‘traditional’ family farms and artisans whose logic may corre- spond to a chayanovian model of slow or no growth. feudal-like relations of production (including serfdom and slavery) were abolished between and . up until then, most peasants were owners of their land but heavily taxed in kind and labour (t. wangchuk ). from the s onwards, bhutan’s cautious development has been orchestrated by state planning through a series of five-year plans. in , the country became a constitutional monarchy (phuntsho ). while a small capitalist class has been able to emerge, largely originating from the aristoc- racy, there are important spheres of the country’s economy that do not seem to follow a capi- talist logic of production (basu ). traditional (non-capitalist) institutions are still strong (s. wangchuk ) and petty commodity production is important. like in neighbouring aruna- chal pradesh studied by harriss-white, mishra, and upadhyay ( ), bhutan never experi- enced an important enclosure movement or widespread rural indebtedness, two key in chayanov’s first novel, published in , one can see an outline of his agrarian utopia which combines narodnik, anar- chist and socialist-cooperativist elements (chayanov ). it describes a peasant eden where large cities have been replaced by a network of rural communities, the industry does not override agriculture, cooperatives have become the central way of producing and consuming, and the role of the state, while important, is to stay in the background and to focus on a few key resources like fossil fuels. however, in , chayanov published another utopian text, the possibilities of agriculture, which takes a completely different path and speculates about the future power of industrial agriculture, off-ground and without family farms. ‘such drastic change in ideals could be explained by the change of political situation in soviet russia and the inner decision of chayanov [who refused to emigrate in – ] to cognitively cooperate with the soviet regime’ (raskov , ). j.-f. gerber mechanisms which would have accelerated processes of social differentiation (gerber ). the countryside is largely free from large-scale agribusiness investments and from a promi- nent class of commercial landlords. the introduction of a full-fledged market economy could thus be unrealistic in these conditions, and this could be bhutan’s greatest chance, if only the situation is correctly understood and creatively used. despite a tight dependency on the indian economy, bhutan is trying to avoid some of the negative consequences of neoliberal globalisation. the government has thus invited various post-growth thinkers to provide advice (rgob ). the results so far contain contradictory but also inventive elements. on one hand, bhutan’s gdp has been growing at a fast rate over the past three decades, but largely because of a series of growth peaks following important state-run hydroelectric projects (mitra and jeong ). on the other hand, the country has put in place a number of policies limiting gdp growth and seeking to enhance welfare and sustainability. one can cite: free edu- cation and healthcare; severe restrictions on foreign investments; no wto membership; no outdoor advertising; heavy taxes on car imports; limits on mass tourism (and ban on alpinism); limits on mining; half of the country under protected areas; constitutional per cent of forest cover; declared willingness to generalise organic agriculture. bhutan has also developed the concept of gross national happiness (gnh) which has attracted a lot of attention. gnh was formalised after a particularly ‘unhappy’ period in the country’s history. in the late s, bhutan’s external situation was tense: its debts to india were swelling while india had turned neoliberal around and annexed sikkim in (following strong anti-monarchy movements). fearing a possible loss of sovereignty, the bhutanese government sought to reinforce the buddhist ngalop identity shared by much of the élite (western bhutan), to the detriment of other groups in the country, especially the hindu lhotshampa minority (southern bhutan). a violent crisis followed, leading to the eviction of thousands of lhotshampas out of the country (hutt ; phunt- sho ). gnh began to enter the country’s policy arena about a decade after these events, partly as a resistance to neoliberal globalisation threatening existing structures, and partly, possibly, as an attempt to rebuild the country’s image but also to alleviate many internal wounds (phuntsho ). today, gnh remains a contested notion. it encompasses different interpretations, more or less radical (gerber and raina a). for some bhutanese, gnh is a loosely-defined ‘green growth’ and a brand name to fit every occasion. for others, gnh is synonymous with the gnh index, a sophisticatedindicatorseekingtomeasurewell-beingholistically and intended to replace gdp in guiding development policies (ura et al. ). and still for others, albeit a minority, gnh is not just a new indicator but a philosophy of social flourishing integrating as a result, characteristically, the government has been unable to find a bhutanese labour force willing to carry out unplea- sant work like road construction, and the necessary workforce of landless peasants is hired from india. some of their contributions can be found here: http://www.newdevelopmentparadigm.bt/ / / /contributions-to- the-ndp-report/ (accessed on february ). the gnh index is based on nine domains – i.e. living standards, education, health, environment, community vitality, time- use, psychological well-being, good governance, and cultural resilience and promotion – measured with fine-tuned indicators. the indicators have sufficiency thresholds which are benchmarks of ‘how much is enough’ for a ‘good life’. they are based on international or national standards, normative judgements, or the outcome of participatory meetings. concretely, a gnh screening tool has been implemented to help the gnh commission – a powerful committee orches- trating the economy’s planning process – in assessing policies and projects for their compatibility with the gnh index (hayden ). the journal of peasant studies http://www.newdevelopmentparadigm.bt/ / / /contributions-to-the-ndp-report/ http://www.newdevelopmentparadigm.bt/ / / /contributions-to-the-ndp-report/ outer and inner needs and seeking sufficiency with respect to economic growth – a position that gets close to degrowth provided it is egalitarian and democratic (hayden ). one of the main architects of gnh, karma ura, wrote that ‘it is possible that a gnh state is analytically different from socialist, liberal or free market states. [our] strategy [is] to take the country from being a late starter in modernization directly to a sustainable society’ (ura , – ). more research is needed to substantiate this claim – that chayanov would probably not have rejected – and clarify the nature of growth and post-growth in bhutan, from the family farm to the state level. . . joseph cornelius kumarappa: decentralisation and autonomy kumarappa ( - ) was an indian economist, the founder of ‘gandhian economics’, but an original theorist in his own right and a man of action. after studying economics in london and at columbia university in new york, he joined the indian nationalist move- ment in the s. he was in charge of gandhi’s schemes of village reconstruction and helped create and run the all india village industries association (aivia), the organisation that was intended to promote and actualise gandhi’s economic ideas (kumarappa ). his extensive travels, many surveys, and work with aivia gave him a deep understanding of the conditions of rural india. among his numerous books, why the village movement ( ) and economy of permanence ( ) – written while in jail for more than a year during the quit india period – are arguably his best known publications, and they inspired several of the forefathers of degrowth, including ivan illich and ernst schumacher. at the heart of his views lie the concepts of autonomy, non-violence, and the idea of a ‘natural order’ to be cared for, and defined as a moral web of connections between all sentient and insentient beings. these concepts serve as the ontological foundation for his critique of the moral and material dimensions of industrial modernity. in a nutshell, his theory and praxis can be seen – at their mature stage – as an effort to promote a decentralised and self-sufficient economy that would abolish class and caste distinctions and that would seek to keep the emancipatory possibilities of modernity while building on the strengths of peasant and artisan production (govindu and malghan ). his focus on the village economy was thus, as he specified, ‘no ancestor worship’ (quoted in govindu and malghan , ), but an attempt to protect the autonomy and dignity of the indian villager by enabling control over his or her own economic destiny. similarly to chayanov, kumarappa proposed the model of an agrarian economy that would rebalance the countryside and cities, based on the smallest possible scale at which a decentralised economy could fulfil the basic needs of its members. accordingly, he suggested regional units of , people with ‘balanced cultivation’ of cereals, pulses, vegetables, dairy, and so on, ensuring a daily diet of about calories and cotton for making clothing. he recognised that some sectors such as the railways had to be centralised and was also aware of the limitations of existing practices in the village economy, but for him ‘[t]he remedy is not to abandon cottage units but to bring the light of science to cottage workers’ (kumarappa , ). any reformulation of modernity – like in the degrowth movement today – has to begin by examining the notion of ‘standards of living’. for kumarappa, ‘high’ vs. ‘low’ standards are repeatedly not describing a gradation in the quality of life but a difference between ‘complex’ vs. ‘simple’ material standards where ‘complex’ ones are artificially created by j.-f. gerber the market and tend to undermine sufficiency and autonomy. both capitalist and state élites, he argued, shape for their own benefits normative ideals of what constitutes the ‘good life’, ideals that one has to question radically. kumarappa was critical of deriving market prices from individual preferences and proposed instead that valuation standards had to be found at a deeper level, in the moral economy. the distance between the pro- ducer and consumer hinders the moral assessment of an exchange, he observed, and this is one of the key reasons behind his defence of local production and consumption. more- over, he noted that an economy that encourages unrestricted accumulation is antithetical to the ‘natural order’, thereby clearly resonating with degrowth. a critic of aivia ridiculed its approach, calling it ‘a cloak of tattered patches’ that stitched kumarappa’s ‘economic notions’ together with ‘elements of ruskin, william morris, proudhon, bakunin and tolstoy … [to] try to furnish a swadeshi [“made in india”] alternative to the imported ideas of socialism’; kumarappa simply replied that indeed ‘certain aspects of our reasoning can be paralleled from anarchists and others but that is not sufficient cause to hang us’ (quoted in zachariah , , ). kumarappa’s work refutes the common assumption that degrowth ideas are an invention of industrial- ised countries. he anticipated by several decades many contemporary notions on the topic, and the left is slowly rediscovering the relevance of his thought (guha ; gerber and raina b). shrivastava and kothari’s ( ) ‘radical ecological democracy’ could be seen as a contemporary take on some of kumarappa’s core ideas. but as it is often the case with ‘pro-peasant’ thinking, right-wing farmers’ movements have also sometimes misused kumarappa’s idea for their own purposes (see e.g. brass ). *** are there today networks of agrarian communities that would embody elements of kumarappa’s ideas and exemplify degrowth in practice? would the amish of north america be a candidate? the amish form a network of religious and mostly agrarian muni- cipalities which hold simplicity and mutual aid as core values and reject many aspects of capitalist modernity. they represent without a doubt a rich and complex experience that degrowth theory and praxis can learn from. because ‘after all,’ wrote daly ( , ), ‘any group that can make a good living from small farms for a century without government aid and without mining the soil might be doing something right’. in the sixteenth century, anabaptism – the theological foundation of the movement – was a radical ideology, ‘the left wing of the reformation’, which was seen by kautsky ( ) as the apex of medieval communism. the first anabaptists were urban intellectuals in zurich who rejected state-based religion and advocated pacifism, egalitarianism, com- munity life, the free religious choice of adults (against the baptism of children) as well as a return to the new testament. after intense persecutions (due in part to revolutionary lean- ings during the german peasant war), the movement became agrarian and a number of schisms divided them into various orders of mennonites, hutterites and amish. unlike the hutterites, the amish do not collectivise their resources nor do they pool their income. they engage in local entrepreneurial capitalism and may hire labourers, mostly from within the community. class differentiation is thus visible and economic inequality has appeared within the communities. according to hurst and mcconnell’s ( , ) survey, ‘nine out of ten [amish respondents] considered the accumulation of personal material wealth to be a threat to the internal harmony of the community’. this was seen as conflicting with their adoption of simplicity as a core principle through the journal of peasant studies plain cloths, organic farming and a selective rejection of modern technology (like cars, tractors, radios, tv, internet). this does not mean, however, that all modern technologies are rejected. electricity is produced locally and used for refrigerators, lamps and sewing machines. in a way that would certainly please kumarappa, every new technology, before being adopted, gives rise to careful discussions supported by an exegesis of the sacred texts. marglin ( , ) argued that the key criteria when discussing technology is the extent to which it might undermine the community: ‘it is clearly a balancing act’, he wrote, and not an easy one, as ‘amish communities have fractured on the issue of tech- nological choice’. in order to balance their commitment to simplicity with their insertion into the wider society, the amish make a useful distinction between ownership and access (kraybill, johnson-weiner, and nolt ). amish households may for example rent the services of a non-amish driver to deliver goods, or if they have a stand in a large market house, they may use the lights and refrigeration made available to them by the building’s non-amish owner. ‘some observers see hypocrisy in this use-but-don’t-own approach, but the amish see a deep consistency’, argued nolt ( , ). private owner- ship of technology, from an amish perspective, quickly runs against the community as it promotes unnecessary individualism. in contrast, to have to pay for access to services or technology is rooted in the ideal of co-dependence, humility, and practical limits, seen as a healthy way of being in the world, but it also allows some flexibility in terms of technological use. it is of course debatable to what extent the amish are self- reliant and autonomous, as much benefit seems to be gained from their insertion in the larger world. the same remark applies to many neo-rural intentional communities – with various degrees of political radicalism – that have been looking for an alternative, simpler, and self-sufficient lifestyle free from wage labour. they are, without a doubt, present in the degrowth movement, and this is why calvário and otero ( , ) described back-to- the-landers as ‘actors of a diversified strategy of socio-ecological change towards a degrowth future’. while these experiences could be theorised as ‘incubators’ for larger transformations (see below), some authors have argued that alternatives that reinforce the notions of consumer (like food) sovereignty, state failure, and self-reliant ordered com- munities are in fact reproducing neoliberal subjectivities and practices (see e.g. guthman ; argüelles, anguelovski, and dinnie ); others have argued that by remaining local and by not targeting state power, ‘from below’ alternatives are basically toothless if not counterproductive (see e.g. brass ). research on degrowth has little choice but to go back to these old questions that continue to divide marxist and anarchist orientations. community economies like the amish raise important kumarappan questions about ‘the ongoing negotiations between the individual and the community, between freedom and regulation, and between tra- dition and modernity’ (halteman , ), as well as between genders, classes, and between growth and no-growth. a science article on the energetics of amish agriculture concluded that the amish are not quite degrowthers when it comes to population expansion as their birth rate is very high. bernstein ( ) convincingly suggested tackling the leftist taboo of population growth more openly, while martínez-alier ( ) explicitly connected feminist neo-malthusianism with degrowth (masjuan and martínez-alier ). j.-f. gerber the amish experience should make us more confident about the future if [fossil] energy should become progressively scarcer. it is often said that the amish provide a vignette of early america; is it also possible that they may provide an image of the future? (johnson, stoltzfus, and craumer , ) . . nicholas georgescu-roegen: the solar economy georgescu-roegen ( - ) was a romanian-american mathematician and economist who is seen as the father of ecological economics and a key intellectual influence in the degrowth movement. after studying statistics in bucharest, paris and london, he spent a short but decisive time at harvard where he became the protégé of joseph schumpeter. harvard was a vibrant place at the time and his colleagues included wassily leontief, nicho- las kaldor, paul samuelson, paul sweezy and oskar lange. schumpeter offered him a pos- ition in and asked him to work with him on a book, but georgescu-roegen declined and decided to return to romania to ‘help his native land become a happier place for all’ (georgescu-roegen , ). it is in the romanian agrarian economy of the interwar period that georgescu-roegen truly discovered economic problems. from to , he held several important posts in the government, but after the victory of the stalinists and the following repression, he managed to flee to turkey with his wife and then to the united states, where he stayed until the end of his life, mostly at vanderbilt university. his work on agrarian economies (georgescu-roegen , ) signalled the begin- ning of his radical epistemological critique of economics based on the principles of ther- modynamics, ecology and the role of institutions. in agreement with chayanov (whom he quoted in , some years before he was revived and translated in the west), georgescu- roegen argued that the theories of capitalism – whether neoclassical or marxist – do not neatly apply to non-capitalist economies. the latter have their own institutions that one has first to understand. of course, it is still possible that propositions coming from the different theories of capitalism are relevant to them, but the validity of each proposition must be empirically reconfirmed. the difficulty of building a general analytical framework for agrarian economies, he argued, mainly stems from their immense variability. like chayanov, however, georgescu-roegen suggested a few general principles of agrarian economies. the one that is perhaps the most relevant to degrowth relates to the agriculturalist’s dependence on biotic resources, which have a radically different potential than mineral resources. from this, georgescu-roegen developed what became the central idea of his magnum opus, the entropy law and the economic process ( ), namely that the common terms of ‘production’ and ‘consumption’ obscure the lavoisian fact that nothing is created or destroyed in the economy, but every- thing is transformed. the second law of thermodynamics – the entropy law – tells us that energy always tends to be degraded to ever poorer qualities. accordingly, economic pro- cesses irreversibly transform valuable (low entropy) natural resources into valueless (high entropy) waste and emissions. there are mainly two sources of low entropy available to humans: the finite stocks of mineral resources like fossil fuels and the flow of solar radiation that will be available to us for another four billions of years. whereas stocks can be extracted at high rates, solar energy arrives at a constant and fixed rate, beyond human control. this natural asymmetry helps explain the historical opposition between urban and rural life: the frenetic pace of the journal of peasant studies modern cities is associated with industrial growth fuelled by mineral resources, while the slower tempo of rural life is associated with a zero or slow growth structured around the reception of solar flow. given that biotic resources depend on the cyclical rhythms of nature, they cannot be used continuously and this explains why economic activities in agrarian economies must be diversified. in contrast, the flow of mineral resources from stocks (e.g. oil) allows an industrial organisation of production in line, which makes it poss- ible to use production funds at their full capacity and to have specialisation (georgescu- roegen ). georgescu-roegen showed that the mechanisation of agriculture is unsustainably mimicking the industrial logic: machinery, chemical fertilisers and pesticides all rely on mineral resources, rendering modern agriculture – and the industrialised food systems associated with it – as dependent on non-renewable stocks as the industrial sector. this is, georgescu-roegen ( , ) explained, ‘in the longrun a move against the most elementary bioeconomic interest of the human species’. the implications for sustainability are thus clear and radical: the kind of economic growth that is needed is not a stationary one – as his student herman daly was already arguing in – but a ‘declining’ or a degrowing one ( , ). georgescu-roegen suggested a utopian ‘minimal bioeco- nomic program’ in eight points: after opening all borders and equalising the conditions to ‘arrive as quickly as possible at a good (not luxurious) life’ for everyone, the global objec- tive is to ‘gradually lower [the world’s] population to a level that could be adequately fed only by organic agriculture’ ( , ; see also martínez-alier ). *** any glimpse into aspects of such a transition is worth examination, and cuba’s special period of the s could be one such instance. it represents ‘the largest conversion from conventional agriculture to organic and semi-organic farming that the world has ever known’ (murphy , ). for almost a decade, it involved deep socioeconomic changes and, unlike other similar crises like the russian, it succeeded in some important respects that could echo elements of a degrowth transition (boillat, gerber, and funes-monzote ; borowy ). by , cuban agriculture was a showcase of the green revolution, heavily relying on one crop – sugarcane – and importing per cent of its food. while this model guaranteed a good standard of living to the cuban population, it was dangerously dependent on the soviet union, politically authoritarian, and ecologically quite destructive. after the collapse of the soviet union, cuba brutally lost per cent of its trade relations. many industrial complexes closed, and transportation and electricity consumption were reduced to a minimum. in brief, the crisis forced the cubans to take seriously georgescu-roegen’s ideas (although the cuban leadership probably never mentioned him), namely to produce and consume locally, to refrain from credits, to replace tractors with oxen, agro-chemicals with bio-pesticides, industrial fertilisers with compost, machinery with manual labour, long-distance with face-to-face communication, and, above all, to live a simple, low-consumption lifestyle (borowy ). large state farms were transformed into cooperatives, the basic units of cooperative production (ubpcs), seen as more flexible, but it turned out difficult to convert farm workers into peasants, and the ubpcs have had mixed results. the peasant families who own the land and group together in cooperatives to share farm machinery proved to be much more rapidly adaptable to the crisis than the ubpcs and other collective j.-f. gerber farms. the facts that decisions are taken at the family level and that ‘their members typi- cally exhibit a strong sense of belonging to, and caring for, the land’ (rosset et al. , ) made them initially very quick and very open to alternative ecological methods. the growth of urban agriculture and gardening is also a well-known outcome of the crisis (altieri et al. ), and so was the movement of city dwellers to the countryside, sup- ported by the government, to reinforce organic production. cuban authorities took several other measures (e.g. legalisation of the dollar, boost of tourism) that were ‘conservative’ since their goal was to get back to a more standard growth model. in any case, by the end of the s, the acute agricultural crisis was over, while food was being produced with much less inputs and equipment. what is more, borowy ( ) documented surpris- ing improvements in public health indicators including maternal and infant mortality, obesity, and mortality from diabetes, tumours, strokes and external causes. the ‘degrowth period’ brought about lifestyle changes with tangible health benefits. it is encouraging to see how cuba has been able to substitute fossil fuels with renew- able energy and with positive effects on health and sustainability. but one should not ignore the important role of a solid welfare state and a low level of inequality, which would make the generalisation of the cuban successes difficult. also, the special period was never intended as the beginning of a new era and the country is now back on the track of economic growth, largely based on tourism. however, agroecological production is still thriving in a context where food continues to be partly imported (machín sosa et al. ; ipes ). georgescu-roegen’s goal was to be as rigorous as possible when assessing ecological sustainability. his recommendations were accordingly oriented towards a downscaling of the social metabolism rather than towards ‘green new deals’ that promote a world-scale transition to renewable energies but without emphasising the need for a differentiated reduction in levels of consumption. such proposals – like pollin’s ( ) one, which is also an unfortunate attack on degrowth – are quickly biophysically naïve and end up justifying the ideology of growthism that created the problems in the first place. for pollin, degrowth is ‘utterly unrealistic’ and the left cannot afford to dream that another world is possible given the urgency of climate change. ‘we are [thereby] asked to accept’, remarked kallis ( b) in his reply to pollin, ‘that the only game in town is capit- alism, and that questioning capitalism and its destructive pursuit of growth is a luxurious waste of time. if not now, then when, one might wonder?’ biophysically speaking and with current technologies, a wind-hydro-solar economy could only support much smaller econ- omies, and a transition to renewables would therefore have to be a degrowth transition. . . joan martínez-alier: the politics of degrowth the catalan economist and political ecologist joan martínez-alier ( -) is one of the most influential figures of the degrowth movement and a former board member of jps. there are a number of similarities between his intellectual path and georgescu- roegen’s. like the latter, he spent more than a decade studying agrarian economies, especially in spain, cuba and peru; and like georgescu-roegen, agrarian questions led him to be interested in ecological sustainability and confirmed his dissatisfaction with the ‘leninist’ lens to apprehend rural problems – a dissatisfaction sustained by his own anarchist sympathies. in an observation that echoes georgescu-roegen’s ( ) views the journal of peasant studies on the great diversity of rural institutions, martínez-alier ( , ) wrote that ‘the variety of rural social formations makes it difficult to accept that anything very specific can be said about peasant politics in general’. much more than georgescu-roegen, however, martí- nez-alier has been interested in grassroots conflicts over processes of exploitation, dispos- session and contamination. in the s and early s, he studied struggles over agrarian production relations and he is engaged today in the immense work of compil- ing a global inventory of ecological distribution conflicts (see www.ejatlas.org). in the peru of the early s, martínez-alier became for the first time in contact with indigenous peasants who explicitly rejected a certain modernisation and fought to pre- serve their traditions, communities and identities – something that would resonate with post-development theory a decade later. in addition, it is in peru that he began to immerse himself in ecological questions, at first through the ecological anthropology of andean peasants, and later on, through the untold history of agricultural energetics and ecological economics in general. the resulting book (martínez-alier ) provided an intellectual lineage to the nascent field of ecological economics. the book’s epilogue politicises socio-metabolic analyses, criticises the ‘the belief in economic growth’ (p. ), and puts forward ‘ecological neo-narodnism’ – later called the ‘environmentalism of the poor’ – a neglected current of environmentalism that became his research and activist focus for the next three decades, culminating in another influential volume in (martínez-alier ). this current of environmentalism – also referred to as the environmental justice move- ment – combines ecological concerns and social justice. it literally fights the impacts of economic growth and their unequal distribution. the environmentalism of the poor typically involves impoverished, working-class or indigenous populations, struggling against the state or the corporate sector. the chipko movements against state plantations in india, the seringueiros against large-scale ranchers in brazil, and the ogoni struggle against shell in nigeria are emblematic cases of this current. taken as a whole, the myriads of such conflicts on extraction, transport and waste disposal represent one of the most powerful socio-political forces in the global south today, and they are on the rise as the metabolism of industrialised regions requires ever more energy and materials (see e.g. gerber ; martínez-alier et al. ; conde ; scheidel et al. ). however, this massive mobilisation strength has arguably failed, so far, to translate into an equal theoretical strength, despite the fact that many creative concepts have been forged through socio-environmental activism, such as the ‘ecological debt’, ‘climate justice’, ‘biopiracy’, ‘food sovereignty’ or ‘land grabbing’ (martínez-alier et al. ). this is not to say, also, that environmental justice movements lack broader con- ceptual frameworks – like buen vivir or swaraj – that may influence their interpretation of problems and guide alternative projects (kothari et al. ). yet, overall, many one of martínez-alier’s main interests was in sharecropping that he described as ‘akin to piece-work’ (martínez-alier , ). he argued that sharecropping tends to be more profitable for the landlord than wage labour because it rests on both class exploitation and the peasants’ self-exploitation. sharecropping is thus neither ‘inefficient’ (from a conventional per- spective) nor ‘semi-feudal’ (from a certain marxist perspective). with josé manuel naredo, martínez-alier rediscovered the work of sergei podolinsky in agricultural energetics and engels’ rather negative reaction to it (martínez-alier and naredo ; martínez-alier ). podolinsky was a ukrainian narodnik marxist who could have, they argued, bridged marxism and ecology for the first time in a systematic way (but see foster and burkett , for a diverging opinion). podolinsky was critical of industrial agriculture on energetics ground. he understood agriculture as a system of energy transformation and was familiar with thermodynamics. j.-f. gerber http://www.ejatlas.org grassroots movements remain local and narrow in their conceptual scope. the resulting fragmentation can obstruct wider synergies and the broader societal alternatives that can be imagined and constructed. this is why martínez-alier ( ) suggested that there is an important potential alliance between the large global environmental justice movement and the ideas of the emerging degrowth movement. he has promoted this alli- ance on analytical grounds – i.e. there is a convergence of material interests between the two movements – but also on normative grounds, as degrowth can offer a radical political project to environmental justice. *** martínez-alier emphasises concrete socio-ecological struggles as key sites where ‘degrowth’ demands are being formulated. for him, such conflicts are ‘degrowth in practice’, since they de facto seek to prevent growth from taking place, be it in the form of a new highway, a new power plant, a new plantation, or more contamination with industrialisation. but is it the case that environmental justice movements and degrowth are natural allies? a recent special issue in ecological economics explored this relationship and revealed different opinions (akbulut et al. ). some authors argued that there is a clear affinity between the two movements. alcock ( , ), for example, analysed the new rural reconstruction movement (nrrm) in china and argued that ‘the links between the sources of degrowth and nrrm theory as well as how that theory is put into practice mean that the nrrm can be seen as a type of degrowth movement’. refuting the charge of western-centrism, the author calls for radical knowledge co-production by scholar-activists aware of ‘the specifics of different countries and localities’. similarly, forst ( ) reviewed first nations sovereignty and environmental struggles in british columbia, canada, and found that ‘many of the core and repeated aims found within degrowth conversations parallel grassroots first nations goals’ (p. ). in contrast, some authors have been more sceptical about the ‘naturalness’ of the alli- ance. rodríguez-labajos et al. ( ) argued that there are ‘significant differences’ between the two movements and that the policies proposed by degrowthers are often perceived as western-centric (like reducing working time). more attention should therefore be placed on the specificities of places and histories before any substantial alliance can be built. scheidel and schaffartzik ( ) wrote that environmental justice protesters and degrowthers have not exactly the same aims: while the former often seek to protect ‘tra- ditional livelihoods and ways of living’, the latter seeks ‘new livelihoods and new ways of living, within alternative societies’ (p. ). many grassroots resistance movements may indeed start with the defence of a local status quo, hence the normative quest for a radical theory able to transcend this. the key point, from a degrowth perspective, is to transform nimby movements (not in my backyard) into niaby movements (not in anyone’s backyard) actively seeking broader transformations. muradian ( , ), for his part, argued ‘that the degrowth movement reflects the values of a particular social group, namely the well-educated european middle class that share pro- gressive-green-cosmopolitan values’, a situation that ‘creates significant barriers for its disse- mination among lower-income social groups in other parts of the world’. if one defines degrowth as an undifferentiated call for ‘voluntary frugality’ for all, then such ideas will under- standably not speak to many people. the present article has, hopefully, offered a different view on what degrowth is all about. having said this, i can only agree with muradian that degrowth is still largely today a white middle-class movement of the global north. yet the journal of peasant studies marx and engels were also white middle-class northerners. if it took some time for marxism to establish some connections with the world’s working class, could it be that it will also take some time for degrowth (or other labels for the same ideas) to establish some connections with various agrarian, urban and environmental justice movements? vía campesina seems hesitant to endorse degrowth, but this does not mean that things will not change (martínez-alier ). in , for example, the movement wrote a petition that was clearly degrowth-oriented, saying that ‘we need to replace capitalism with a new system that seeks harmony between humans and nature and not an endless growth model that the capitalist system promotes in order to make more and more profit’ (quoted in roman-alcalá , ). similarly, degrowth ideas are still rarely discussed among workers’ organisations. yet again, a clear pro-degrowth stance appeared in the french confédération nationale du travail (cnt) in : ‘the defense of the environment implies the fight against capitalism; our labour class union is ecologist and in favour of degrowth’ (quoted in bayon , ). kallis ( ) suggested a gramscian framework for understanding the dialectical role of the state and the grassroots in a degrowth transition. for him, grassroots alternatives, little by little, generate a counter-hegemony that influences the ‘common sense’: alternative economies are not then just microcosms or prefigurations of a degrowth world. they are incubators, where people perform every day the alternative world they would like to construct, its logic rendered common sense. […] as they expand, they undo the common senses of growth and make ideas that are compatible with degrowth hegemonic, creating the conditions for a social and political force to change political institutions in the same direction. (p. , his italics) in turn, alternative policies open space for new alternative practices that reinforce the new common sense in a kind of virtuous cycle. the point here is to emphasise that grassroots alternatives are essential but insufficient in themselves (buch-hansen ). after a certain point, food sovereignty, to take just one example, faces structural constraints (kallis ): it is limited by access to land, by legislative rules that prioritise agribusinesses, by price dumping, or even by the erosion of the welfare state and the rising costs of health or education, which may all push people towards more profitable cash crops monocultures. in the necessary upscaling process, ‘bottom-bottom’ networks for environmental and agrarian justice (borras and franco ) could become platforms where a new ‘common sense’ and new ‘bottom-up’ post-growth politics and policies could emerge. but more empirical research is needed, as i will propose next. . a research agenda on agrarian degrowth it is clear that […] capitalist expansion has now nearly reached the point where it will be halted by the actual limits of the earth’s surface. […] we are in a period of transition; but a transition towards what? – simone weil ( , ) as here, levien et al.’s ( ) introduction to a jps special issue on agrarian marxism con- cluded with suggestions for future research. in their penultimate paragraph, the authors wrote this: finally, there is the question of socialism itself, and [of] socialist alternatives, experiments and praxis. this draws something like a blank in contemporary marxian analysis. it’s all very well to j.-f. gerber dig back into russia in the s, or maoism in the s, but there is not much that is forward looking. (p. ) that contemporary marxian research has little new to say about ‘forward looking’ socialism is indeed quite dismaying, and it seems clear that this should constitute the starting point of a degrowth research agenda in cas. we need to go back to the messiness of concrete practical-political ‘alternatives, experiments and praxis’. the vignettes presented in this article were intended as stimulants for further research on such ‘experiments’. i will now try to identify more systematically what a research agenda in ‘agrarian degrowth’ could look like. the suggested lines of inquiries follow the definition of the growth question i proposed above. . . what are the effects of growth and who benefits? the political economy/ecology of growth is a fundamental area of future research in ‘agrarian degrowth’, an area that would immediately speak to both cas and degrowth scholars. in what ways has growthism – be it neoliberal, social-democrat or communist – shaped the countryside? to what extent has it lessened/increased ‘poverty’? what kind of ‘wealth’ does it create and for whom? ‘[india’s] pattern of dazzling economic growth’, noted walker ( , ), ‘has been propelled by a powerful “reinforcing mech- anism” through which “inequality drives growth and growth fuels further inequality”’. such mechanisms must be clarified in order to demystify the current common sense that growth ultimately leads to more welfare. . . what are the limits to (agrarian) economies and how to deal with them? the extractive and commodity frontiers are another big topic for research in ‘agrarian degrowth’. these frontiers epitomise the ever shifting ‘limits to growth’ and they need more theoretical and empirical investigations. does agroecology represent a way to stay within planetary boundaries? the kind of agriculture that would match a degrowth society needs to be conceptualised more rigorously. infante amate and gon- zález de molina ( , ) suggested a degrowth strategy for agriculture and food based on four ‘rs’: ‘re-territorialization of production, re-localization of markets, re-vegetaria- nisation of diet, re-seasonalisation of food consumption’ (see also altieri and toledo ). gomiero ( ), for his part, argued that biotech-based agriculture such as genetically modified crops are not suitable for a degrowth society. building on these initial efforts, research in agrarian degrowth needs to develop appropriate socio-meta- bolic approaches (see e.g. padró et al. ), and reversely, analyses of the social metab- olism must be enriched with cas insights into its politico-institutional determinants which are often grossly under-incorporated (gerber and scheidel ). more research on the socio-political metabolism of rural systems would represent the royal road for rig- orous assessments of what is possible/desirable to maintain over the long term. giam- pietro and mayumi ( ), for example, used a state-of-the-art metabolic approach to investigate biofuel production and end up with a demolition of the ambitions of large-scale biofuel production from agricultural crops. the journal of peasant studies . . what are the alternatives and who will support them? how do social movements – agrarian, indigenous or environmental – ‘undo’ growth in practice? how do they contest (if at all) the capitalist growth imaginary? how likely are they to become the ‘natural’ allies of the degrowth movement? a systematic study of the alternative narratives and practices that emerge from these movements would be much needed (see e.g. holt-giménez ; martínez-alier et al. ; borras ). in a world where the vast majority of those working in agriculture are ‘small’ farmers, what can degrowth learn from pre-, semi-, non- or anti-capitalist smallholders? what is the place of ‘small’ farmers in a degrowth transition? what kind of politico-institutional context would make them ‘degrowth-compatible’? (see e.g. paech ). the central kumarappan ideas of autonomy, decentralisation and simplicity are to some extent prac- ticed in contemporary ‘community economies’ (see e.g. gibson-graham ). how and under what conditions may their successes be scaled up? and more generally, what can degrowth learn from the anthropology and history of non-capitalist and non-growing economies? the cuban experience with ‘degrowth’ also relates to the role of crises, which needs more attention. crises of capitalism must be understood, as moore ( ) emphasised, in the fra- mework of its total conditions of reproduction, including the contradictory relations of capital and nature. ecological crises are thus likely to also play a major role in generating alternatives. how do rural economies adapt to crises like resource depletion or recessions? ‘involuntary declines are not degrowth in themselves, and countries in recession or depression are not degrowth experiments, unless communities make a virtue out of neces- sity, building low-impact livelihoods that enhance wellbeing and equality’ (kallis et al. , ). this is an important area for further research in ‘agrarian degrowth’, with plenty of possible fieldwork sites, from greece to venezuela. under what conditions do crises reinforce authoritarian populism instead of creative responses like in cuba of the s? cas is in a strong position to examine some of the key proposed policies for degrowth – such as those concerned with commoning, decommodification, post-extractivism and resource extraction caps, debt audit, and tax and subsidy reforms. what can we learn from past and current implementations? ‘reclaiming the commons’, for example, is a stra- tegic point for many land-based social movements; but to what extent do they thereby succeed in transcending capitalist relations of private property and reconnect inhabitants with their territories? if a viable post-capitalist degrowth alternative is to be organised, new ownership institutions will need to be designed (gerber and steppacher ). for harvey ( , ), ‘the long-range perspective is to displace private property arrangements by greater and greater degrees of commoning […] including in some instances the creation of alternative common property rights or even non-markets rights’. along a similar line, amin ( , ) argued that a transition to socialism requires an agrarian reform not based on standard private property. what harvey and amin have in mind here is the necessity to design post-capitalist ownership arrangements that do not foster the same deleterious dynamics as capitalism. another crucial example of degrowth policies to be investigated from an ‘agrarian degrowth’ perspective concerns the organisation of work. reductions of working hours, in particular, are included in all major degrowth scenarios (latouche ; kallis ; schmelzer and vetter ). they are meant to prevent unemployment in non-growing j.-f. gerber economies and free up time for reproductive, social, creative and recreational activities. yet small-scale and organic agriculture typically requires more work (sorman and giampie- tro ). woodhouse ( , ) summarised the issue as follows: ‘while there is evi- dence that smaller-scale production is more efficient in terms of energy use, it generally involves lower labour productivity, and hence lower earnings, than either large-scale agri- culture or non-farm work’; in this context, can small-scale organic farmers survive without non-farm activity and hence without losing autonomy? how is work to be organised in post-growth agriculture? finally, new (and old) values are said to be expressed in various alternative initiatives like the slow food movement, permaculture, vegetarianism/veganism, radical homemakers, back-to-the-landers, alternative conceptions of the good life, eco-spiritualities, etc. what is the broader political potential of these initiatives and movements? to what extent are they ‘incubators’ for further and deeper transformations, or simply the object of new forms of commercialization? are we witnessing the uneven but overall encouraging emer- gence of a ‘new consciousness’ (see e.g. dussel ) for which the current spread of author- itarian populism is just a confirmation in reverse? can this new consciousness (revealed also in the sudden growth of environmentalism among young people, including their school strikes and mass demonstrations) come into alliance with the revival of indigenous militancy in many areas of the world around ecological and cultural issues? values of respect for nature and sacredness of nature are frequently claimed by indigenous peoples and also by peasants. about per cent of all environmental conflicts registered in the ejatlas (almost cases by october ) are led at least partly by indigenous people (j. martí- nez-alier, pers. comm.). is this new consciousness the foundation for a degrowth transition? . concluding remarks the less you are and the less you express your life; the more you have and the greater is your alienated life. […] everything which the economist takes away from you in terms of life and humanity, he replaces for you in the form of money and wealth. – karl marx ( [ ], , his italics) one should distinguish between a myth as a false idea and a myth as a conveyor of sym- bolic meanings. even if the agrarian myth and the myth of growth are factually untenable, they may nonetheless both symbolise very real needs to resist the alienating tendencies of capitalist modernity. the agrarian myth is a tale of lost harmony with one another and with nature, while the myth of growth could be seen as a compensation for the impossibility to ‘express our lives’, as marx’s quote alludes to. if this is true, then the left should take these two myths seriously, as an unconscious expression of distress, and should gently help people see where and how these fundamental needs can be actualised in the real world. degrowth could be a direction of choice for that. this article argued that cas and degrowth can enrich each other, not only in the pro- blematization of the causes, nature and effects of capitalist growth in agriculture, but also in the empirical analyses of alternative experiences and proposals at different levels. i par- ticularly focused on the work of one philosopher and four agrarian economists which, in my view, can show what a research and activist agenda in ‘agrarian degrowth’ might look like (even if these authors did not necessarily intend to do so). simplifying their respective contributions to the extreme, one could say that weil offers fragments of a the journal of peasant studies rooted consciousness for degrowth, chayanov suggests basic elements for a degrowth economy, kumarappa theorises a governance for degrowth, georgescu- roegen offers its ecological foundation, while martínez-alier provides an initial political subject/movement. all five thinkers became critical of capitalism, of large-scale industrialisation and of politi- cal centralisation – but none of them became dogmatically localist or anti-technology. all five authors have been influenced by marx – but all of them have followed their own path, often highly critical of official communist party lines. all five, except georgescu- roegen, have expressed anarchist sympathies. all five, except chayanov, have praised agroecological practices and/or the ecological soundness of ‘traditional farming’. and they would possibly all five doubt that wage labourers are more easily radicalizable than smallholders when it comes to getting involved in what they regard as progressive politics. having said this, all five thinkers may underestimate the extent to which petty commodity producers are entrenched – consciously or not – in capitalist relations of pro- duction, for example through various forms of debt arrangements. thirty years ago, utsa patnaik ( , ) wrote a critique of chayanov’s theory of peasant production ( ) complaining that the book is blind to class relations and broader systemic considerations. she also criticised georgescu-roegen along the same line, and her points were largely valid as georgescu-roegen’s article of (that she was concerned with) is indeed weak on class. one can only regret that georgescu-roegen ( ) did not take the oppor- tunity of his reply to patnaik, surprisingly short and awkward, to agree with at least some of her points, but to add that cas would gain from taking ecological relations as seriously as social relations. this would not only reinforce the critique of capitalism and the elaboration of viable alternatives; it would also help bring closer cas and degrowth. acknowledgments thank you to bengi akbulut, murat arsel, jun borras, federico demaria, wendy harcourt, shivani kaul, joan martínez-alier, rajeswari raina and arnim scheidel for numerous discussions on these questions. three anonymous reviewers were extremely useful in sharpening the argumentation and are gratefully acknowledged. all remaining shortcomings are mine. this article is dedicated to joan martínez-alier. disclosure statement no potential conflict of interest was reported by the author. references akbulut, b., f. demaria, j.-f. gerber, and j. martínez-alier. . “who promotes sustainability? 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[ ]. oppression and liberty. london: routledge. weil, s. [ ]. the need for roots. london: routledge. weis, t. . “the accelerating biophysical contradictions of industrial capitalist agriculture.” journal of agrarian change ( ): – . wir. . world inequality report. accessed february , . https://wir .wid.world/files/ download/wir -full-report-english.pdf. woodhouse, p. . “beyond industrial agriculture? some questions about farm size, productivity and sustainability.” journal of agrarian change ( ): – . zachariah, b. . developing india: an intellectual and social history. oxford: oxford university press. julien-françois gerber is assistant professor at the international institute of social studies in the hague. after completing his phd in barcelona and postdoc at harvard university, he lived in india and bhutan before moving to the low countries. he has published on the relationships between the economy and the conditions for sustainability, flourishing, alienation and resistance. he is also interested in bridging science and activism and has been active in the degrowth movement. j.-f. gerber https://wir .wid.world/files/download/wir -full-report-english.pdf https://wir .wid.world/files/download/wir -full-report-english.pdf 	abstract 	 . introduction 	 . where are we and what next? 	 . . what is wrong with economic growth? 	 . . what is degrowth? 	 . bridging cas and degrowth: one philosopher and four agrarian economists 	 . . simone weil: rootedness within limits 	 . . alexander chayanov: the multiple balances of (re)production 	 . . joseph cornelius kumarappa: decentralisation and autonomy 	 . . nicholas georgescu-roegen: the solar economy 	 . . joan martínez-alier: the politics of degrowth 	 . a research agenda on agrarian degrowth 	 . . what are the effects of growth and who benefits? 	 . . what are the limits to (agrarian) economies and how to deal with them? 	 . . what are the alternatives and who will support them? 	 . concluding remarks 	acknowledgments 	disclosure statement 	references doi: . / am. j. hum. genet. : – , report linkage analysis of a complex pedigree with severe bipolar disorder, using a markov chain monte carlo method chad garner, , l. alison mcinnes, , , susan k. service, ,* mitzi spesny, eduardo fournier, pedro leon, and nelson b. freimer , , ,* department of integrative biology, university of california berkeley, berkeley; neurogenetics laboratory, center for neurobiology and psychiatry, and department of psychiatry, university of california san francisco, san francisco; and school of medicine and cell and molecular biology research center, university of costa rica, san jose, costa rica recently developed algorithms permit nonparametric linkage analysis of large, complex pedigrees with multiple inbreeding loops. we have used one such algorithm, implemented in the package simwalk , to reanalyze previously published genome-screen data from a costa rican kindred segregating for severe bipolar disorder. our results are consistent with previous linkage findings on chromosome and suggest a new locus on chromosome that was not identified using traditional linkage analysis. a single large pedigree can provide a powerful sample for mapping complex traits; compared with a collection of independent nuclear families, a single pedigree may contain more linkage information and less etiologic het- erogeneity and yields a greater possibility of identifying genotyping errors. large pedigrees from recently founded population isolates may be particularly valua- ble, as affected individuals in such populations are more likely to share common ancestry than in admixed populations. the increase in power associated with the large-ped- igree study design comes at the cost of computational feasibility, and, until recently, pedigree size and consan- guinity were limiting factors for both model-based and model-free linkage analysis. investigators who collected large complex pedigrees traditionally had to break up their samples into smaller family units that available algorithms could handle. an example is the extended old order amish pedigree that has been investigated in a series of linkage studies of bipolar disorder (bp) (ege- received december , ; accepted for publication january , ; electronically published february , . address for correspondence and reprints: dr. nelson b. freimer, center for neurobehavioral genetics, university of california los angeles, gonda center, room , charles e. young drive south, box , los angeles, ca - . e-mail: nfreimer @mednet.ucla.edu * present affiliation: center for neurobehavioral genetics, univer- sity of california los angeles, los angeles. � by the american society of human genetics. all rights reserved. - / / - $ . land et al. ; ginns et al. ). although genea- logical information has shown that this sample of individuals (including affected with bp) could be rep- resented as a single, highly consanguineous -genera- tion kindred, for linkage analyses, the family has been broken into smaller pedigrees, each covering, at most, five generations. none of these analyses has produced unequivocal localization of bp genes. as a result of the perceived failures in identification of linkage using large pedigrees, mapping studies of com- plex traits now mainly use less-powerful nuclear-family study designs. software packages have recently become available, however, that use new algorithms that can compute linkage statistics on highly complex pedigrees. we used one such package, simwalk (sobel and lange ), to reanalyze data from a previously published genome screen (freimer et al. a; mcinnes et al. ) for severe bp (bp-i) in a kindred from the genet- ically isolated costa rican population. the previous analyses of the kindred used a model-dependent method, assuming a nearly dominant mode of inheritance. with the algorithms available at the time of the previous anal- ysis, it was necessary to analyze the kindred as two fam- ilies without including inbreeding loops. in contrast, the simwalk analysis reported here takes advantage of the power provided by the full-pedigree structure. in addi- tion, simwalk , which uses markov chain monte carlo (mcmc) methods to compute allele-sharing statistics, provides a model-free (or nonparametric) analysis; this type of analysis is more robust than model-dependent fi gu re f u ll c o st a r ic an k in d re d .a ff ec te d in d iv id u al s ar e sh o w n w it h b la ck en ed sy m b o ls .g en ea lo gi ca li n fo rm at io n is re p re se n te d fo r ge n er at io n s. a ll in d iv id u al s in th e fi rs t se ve n ge n er at io n s w er e co n si d er ed p h en o ty p e u n k n o w n . t h e co n sa n gu in eo u s m ar ri ag es (t h ic k m ar ri ag e li n es ) in cl u d e se ve n se co n d -c o u si n m ar ri ag es (o n ce o r tw ic e re m o ve d ), tw o fi rs t- co u si n m ar ri ag es (o n ce o r tw ic e re m o ve d ), an d o n e th ir d -c o u si n m ar ri ag e. reports table locations, allele-sharing statistics, and p values for all markers tested on chromosome q marker locationa allele- sharing statistic p d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . d s . . . a locations are taken from the most centromeric marker, d s . table locations, allele-sharing statistics, and p values for five markers on chromosome q marker locationa allele- sharing statistic p d s . . d s . . . d s . . . d s . . . d s . . . a locations are taken from the most centromeric marker, d s . analysis when the mode of inheritance is unknown, as is the case with bp. although there are powerful methods for computing exact nonparametric linkage statistics (lander and green ; kruglyak and lander ), these methods could not accommodate the size and com- plexity of the costa rican bp kindred, thus necessitat- ing the application of a stochastic method such as simwalk . figure shows the pedigree as analyzed in the present study, with all known connections specified. we iden- tified the eight great-grandparents for each affected in- dividual to verify that there were no connections be- tween these individuals closer than those depicted in the figure. given the demographic history of the costa rican population, it is likely that there are still unknown re- mote connections between these individuals; however, such distant connections would not likely substantially affect the linkage analysis (l. a. mcinnes and n. b. freimer, unpublished data). we reanalyzed genotypes from the markers in the genomewide linkage analysis of the kindred. the marker selection and genotyping procedures for the genomewide data have been described elsewhere (freimer et al. a; mcinnes et al. ). marker allele frequencies were estimated from the families using known relation- ships among the individuals but without linkage to the disease phenotype (boehnke ), by means of the pro- gram ilink (lathrop and lalouel ) and using the simplified pedigree structure from freimer et al. ( b). nonparametric linkage analysis was performed using simwalk . simwalk uses mcmc methods to sample from the complete distribution of underlying inheritance patterns proportional to their likelihood, which is cal- culated from the observed genotype data. statistic d, calculated by simwalk , measures the extent of allele sharing among affected relative pairs as the average across the sampled inheritance patterns. a large value of the statistic indicates a high degree of identity-by- descent allele sharing among the affected relatives. we chose statistic d over other nonparametric statistics cal- culated by simwalk because it is generally powerful when the model of inheritance is unknown and because similar statistics have been studied by others (weeks and lange ; whittemore and halpern ). all marker information was used in this multipoint computation of allele-sharing statistics. empirical p values are obtained by comparing the observed value of the statistic to that found under the null hypothesis, which is generated by repeated sampling of marker data simulated with a gene- dropping algorithm, without linkage to the phenotype. sobel and lange ( ) suggest that p values from this procedure will be slightly conservative; thus, statistical significance will be potentially understated. all the markers showing nominal p values !. in the current analysis were on chromosomes q and q. markers on q had provided, by far, the strongest ev- idence of any portion of the genome for linkage in the prior analysis of these data (mcinnes et al. ), and the majority of affected individuals shared a marker hap- lotype in this region (freimer et al. a). table shows the relative locations, allele-sharing statistics, and significance levels for all markers tested on chro- mosome q in the current analysis; each of these mark- ers showed allele-sharing statistics that were sd above the genomewide mean, with a range of . – . sd. two regions within q contained clusters of markers for which allele-sharing statistics re- am. j. hum. genet. : – , sulted in p values !. . these two clusters of markers (from d s to d s and from d s to d s ) correspond to the q segments highlighted in the prior analyses of these data (freimer et al. a). five consecutive markers, spanning ∼ cm of chro- mosome q, showed evidence for linkage in the current nonparametric analysis. the allele-sharing statistics for markers d s , d s , d s , d s , and d s had p values of . , . , . , . , and . , respectively (table ). our prior parametric anal- yses of the genome-screen data (mcinnes et al. ) found no evidence for linkage to q. the six markers now providing such evidence only did so when analyzed with the data from neighboring markers. visual exam- ination of the genotypes of the individual markers showed that there is not a clear association between their alleles and bp, suggesting that the evidence in q derived from an informative haplotype rather than from infor- mation at individual markers. visual inspection also sub- sequently confirmed that the majority of affected indi- viduals in the kindred shared a single haplotype over this region of q (data not shown). we carried out tests to assess the sensitivity of the results observed for the five consecutive markers showing p values !. on chro- mosome q, to the prespecified marker allele frequencies (data not shown). these additional tests showed that the results in q were not sensitive to the allele frequency used. in the prior analysis, markers on chromosomes and provided linkage evidence that surpassed a predefined threshold (lod . in the combined pedigrees) (mc- innes et al. ). in the current analysis, neither of these locations showed linkage evidence at a nominal signif- icance of . the variability in these results betweenp ! . the two analyses is difficult to evaluate, given the dif- ferences in the methods of analysis and pedigree struc- ture used. by reanalyzing the costa rican pedigrees as a single kindred using simwalk , we continue to detect the most suggestive linkage evidence identified in the original analyses, that for q -q . the fact that a previously undetected region on q was identified with the new methods demonstrates the utility of haplotype infor- mation in linkage analysis of genome-scan data from large complex pedigrees. we suggest that similar anal- yses should be applied to genotype data from other such pedigrees—for example, the old order amish bp kindred. acknowledgments this work was supported by the national institutes of health (nih) grants mh- , to l.a.m., and mh- and mh- , to n.b.f.; by fundacion de la universidad de costa rica para la investigacion (fundevi); and by the vice rectory of research of the university of costa rica. c.g. is supported by nih grant gm- . we thank the wellcome trust centre for human genetics, for the use of computer resources, and eric sobel and lodewijk sandkuijl, for helpful comments. we thank the families who participated in this pro- ject and costa rican institutions that made this work possible: hospital nacional psiquiatrı́co, hospital calderon guardia, caja costarricense de seguro social, archivo nacional de costa rica, and iglesia catolica de costa rica. a complete list of genomewide results can be obtained from n.b.f. references boehnke m ( ) allele frequency estimation from data on relatives. am j hum genet : – egeland ja, gerhard ds, pauls dl, sussex jn, kidd kk, allen cr, hostetter am, and housman de ( ) bipolar af- fective disorders linked to dna markers on chromosome . nature : – freimer nb, reus vi, escamilla ma, mcinnes la, spesny m, leon p, service sk, smith lb, silva s, rojas e, gallegos a, meza l, fournier e, baharloo s, blankenship k, tyler d, batki s, vinogradov s, weissenbach j, barondes s, sankuijl l ( a) genetic mapping using haplotype, association and linkage methods suggests a locus for severe bipolar disorder (bpi) at q -q . nat genet : – freimer nb, reus vi, escamilla m, spesny m, smith l, service s, gallegos a, meza l, batki s, vinogradov s, leon p, sand- kuijl l ( b) an approach to investigating linkage for bipolar disorder using large costa rican pedigrees. am j med genet : – ginns ei, ott j, egeland ja, allen cr, fann cs, pauls dl, weissenbachoff j, carulli jp, falls km, keith tp, paul sm ( ) a genomewide search for chromosomal loci linked to bipolar affective disorder in the old order amish. nat genet : – kruglyak l, lander es ( ) complete multipoint sib pair analysis of qualitative and quantitative traits. am j hum genet : – lander es, green p ( ) construction of multilocus genetic linkage maps in humans. proc natl acad sci usa : – lathrop g, lalouel j ( ) easy calculations of lod scores and genetic risks on small computers. am j hum genet : – mcinnes la, escamilla ma, service sk, reus vi, leon p, silva s, rojas e, spesny m, baharloo s, blankenship k, peterson a, tyler d, shimayoshi n, tobey c, batki s, vinogradov s, meza l, gallegos a, fournier e, smith l, barondes s, sandkuijl l, freimer n ( ) a complete genome screen for genes predisposing to severe bipolar disorder in two costa rican pedigrees. proc natl acad sci usa : – sobel e, lange k ( ) descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker- sharing statistics. am j hum genet : – weeks de, lange k ( ) the affected-pedigree-member method of linkage analysis. am j hum genet : – whittemore as, halpern j ( ) a class of tests for linkage using affected pedigree members. biometrics : – 	linkage analysis of a complex pedigree with severe bipolar disorder, using a markov chain monte carlo method 	acknowledgments 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( 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the ongoing creationism/ evolution controversy has been the proposal to institute opt- out policies that would allow creationist parents to exempt their children from any instruction involving evolution. by way of an explanation of some of the philosophical issues at play in the debate over evolution and the nature of science, this article shows the educational folly of such policies. if evolution is taught properly, it should not be possible to opt out of it without opting out of biology. moreover, if intelligent design creationist criticisms of evolution and scientific naturalism were taken as the basis for opting out, then the effect would be even more radical and would require opting out of science entirely. keywords evolution education . intelligent design creationism . nature of science . opt-out policies opting out of evolution in , a proposed bill in alberta, canada included a provision that would require schools to notify parents in advance if a class dealt explicitly with religion, sexuality, or sexual orientation and allow them to have their child excluded from the discussion. the provision made head- lines when the provincial premier in alberta confirmed that this would allow parents to exclude their child from a class when evolution came up. said one opposition leader, this was going to hurt alberta’s international image and make it “sound like arkansas.” (cbc news a) a similar controversy had arisen in the united states in oklahoma earlier in the year. a proposed “science education and academic freedom act,” which followed the script of other so-called “academic freedom” bills that intelligent design creationists (idcs) were lobbying for, included language that said students could not be “penalized in any way because [they] subscribe to a particular position” on scientific theories. the key aim of the bill was to allow creationist material that would critique evolution and other scientific theories, and opponents of the bill pointed out that it also would allow students to opt out even of particular test questions if they objected to evolution (cavanagh ). in florida, the river of life church, an evangelical christian church in niceville, assists its parishioners in asking for their children to opt out of a wide variety of school activities that they disapprove of. by clicking check boxes on its helpful web page, the church automatically generates a letter for parents to print which instructs a school to excuse their child from activities such as halloween, “sexual education,” “homosexual, or toler- ance training,” or “green days, saving the earth.” another check box creates the following opt-out request for any class discussions on evolution: while many of our local teachers seek to be sensitive to this topic, the text books and matierials [sic] often pertrayed [sic] it more as fact, rather then [sic] popular scientific theory. this presentation is often a biased one, refusing to look at even the possibilities that other views present. and it is biased even in the face of evidance [sic] to the contrary. i do not want my child to be apart [sic] of such discussions. please provide an alternative activity for my child (river of life church ). r. t. pennock (*) lyman briggs college and depts. of philosophy & computer science, e- holmes hall, michigan state university, east lansing, mi , usa e-mail: pennock @msu.edu evo edu outreach ( ) : – doi . /s - - - there are a variety of reasons why creationists want to be able to have their children opt out of evolution. one basic reason seems simple, at least at first glance. they see evolution as contradicting their understanding of the story of creation as it is told in their sacred text, and they want to teach that religious account instead. this is true, as far as it goes, but in fact, their objections to evolution go far beyond a dispute about the facts of biological origins. as i have explained in detail elsewhere, at stake for most creationists is morality itself and the possibility of meaning and purpose to life (pennock ). but they don’t stop with evolution; most creationists also reject key findings in geology, astronomy, anthropology, and many other sciences as well. even more radically, the new creationists also reject essential elements of scientific methodology, which they claim to be a naturalistic metaphysical dogma. instead of natural science, they propose a theistic science that is based in their understanding of biblical truth. as intelligent design creationist william dembski puts it, “any view of the sciences that leaves christ out of the picture must be seen as fundamentally deficient.” (dembski , p. ) no science teacher or administrator with integrity could allow creationism to be taught in a science class, but given the headaches that creationists cause, it is not hard to see how one might be tempted to allow an opt-out policy as a practical expediency. many schools already have policies that allow students to opt out of sex education classes, for instance. why not meet creationists halfway and treat evolution the same way? eugenie scott and glenn branch have addressed this question of opt-out policies specifically including evolution (policies that they wittily label with the pointed acronym oopsie), showing some of the reasons why this proposed “compromise” is ill advised. at the very least, having students bob in and out whenever “the dreaded e-word” comes up is disruptive to the course as a whole. leaving aside the educational loss to students themselves and the additional burden on teachers, such a policy would harm school districts as well, since students who do not learn about evolution will perform worse on the statewide examinations and in colleges. but more significantly, given that evolution pervades or at least ought to pervade biology education, there is no alternative to learning about it and no activity that would be an adequate substitute (scott and branch ). this last point deserves to be developed further; that is the task of the first part of this essay. creationists seriously mislead the public about the foundational significance of evolution within biology by portraying it as a “controversial theory.” the problem is not just that they make factual errors about evolution; even more significant are their conceptual errors about the nature of evolutionary biology and about science. understanding these philosophical issues behind the creationism controversy can not only show the educational folly of such opt-out policies, but also help instructors teach about evolution and science in general more effectively. to be clear, this paper will not take a stand on the question of whether students are or should be legally allowed to specifically opt out of evolution. one can, of course, foresee the morass of practical problems that opening the door to such personal exemptions would cause for schools. but one might argue that the law should nevertheless allow for at least deep-seated religious exemptions in a similar way that the supreme court allowed the old order amish, whose distinctive and comprehensive religious way of life is fundamentally at odds with the modern world, to opt out of compulsory education past eighth grade (wisconsin v. yoder, ). this may seem extreme, but we will later see a significant sense in which the creationists’ religious objections are similarly comprehensive and deep-seated. however, addressing the legal question would take us too far afield. rather than arguing whether students are or should be allowed to opt out of just evolution, our question is whether they should be able to do so. my contention is that, if evolution is taught properly, then it should not be possible for a student to opt out of it without opting out of biology as a whole. moreover, i want to argue that when the systematic religious objections that creationists have to evolution are fully understood, one sees that the issue involves not just biology but all of science. appreciating some of the philosophical aspects here will help science educators teach their subject in a manner that will better serve all their students. teaching evolution the right way so how should evolutionary biology be taught? it should be taught like any science should be taught, namely, so as to reveal the nature of science. the main point for teachers to keep in mind is that science focuses on investigating the causal structure of the natural world. it is the lawful relations between causes and effects that determine the regularities and patterns that we aim to explain. for example, the molecular patterns that chemists investigate are the result of combinations of chemical elements that interact in ways that become more and more understandable as these scientists uncover the relevant chemical processes. biology works the same way; it focuses on patterns of biological phenomena and the biological causal processes that produce them. faced with some interesting phenome- non, be it the geographical distribution of species or the functioning of organs in the body, biologists will seek to explain the relevant pattern by looking for the causes that evo edu outreach ( ) : – produced it. the interesting thing about biology is that there are two different kinds of causes that are relevant. the first kind involves the immediate nearby causes of the pattern— what is producing a particular effect right now, for instance. these are called the proximate causes. by themselves, proximate causes sometimes seem disjointed, but a second form of explanation helps unify them by pointing to deeper historical causes. philosophers usually call these “ultimate” causes, though that term of art is somewhat misleading to non-professionals because such causes need not be ultimate in the vernacular sense of the term (sometimes the term “distal” is used instead). in biology, these are the evolutionary causes of a pattern. there are a variety of ways in which evolutionary causes come into play, but two of the most important are the principles of common descent and natural selection. biolog- ical patterns that would otherwise seem puzzlingly contin- gent are suddenly brought together and made understandable once evolutionary history is taken into account. it is common descent that explains, for example, the distinctive patterns of biogeography from the level of continents to that of islands. and it is natural selection that explains the striking universal patterns of adaptation between organisms and environment. put simply, every proximate biological cause has an ultimate evolutionary cause that helps explain how it came to be, which is why evolutionary theory is a basic unifying explanatory framework for all of biology. it is in this sense that one should understand the famous quotation from theodosius dobzhansky that nothing in biology makes sense except in the light of evolution (dobzhansky ). scott and branch had this exactly right. “a teacher who tries to present biology without mentioning evolution” they quipped, “is like a director trying to produce hamlet without casting the prince” (scott and branch , p. ). when biology is taught properly, one should find evolution across its entire curriculum. for various reasons, biology textbooks have not always been organized in a way that clearly shows this. they explain evolution explicitly in one dedicated chapter or section but elsewhere simply take it for granted. profes- sional biologists already understand how evolution is foundational to biological explanation and so do not always make it explicit. however, one happy result for science education of creationist attacks is that these have awakened biologists to the need to improve textbooks and classroom instruction to make this fact more obvious. scientists are working now to generate pedagogically useful examples and to demonstrate practically how teachers can better highlight evolution throughout the biological disciplines. we may look forward to more textbooks where evolution will appear explicitly not just in its own section, but also throughout discussions of organismal, cellular, and molec- ular biology. taught properly, evolution should be visible everywhere in biology education. it is in this straightfor- ward, structural sense that a student taking any biology course should not be able to opt out of evolution—when biology is taught in an integrated fashion, evolution is everywhere and is thus unavoidable. revealing the nature of science however, teaching biology properly to reveal its underlying explanatory framework is only one part of what we need to do. as i have argued previously, the best science teaching reveals not just the science of nature but also the nature of science (pennock ). one especially important aspect of this idea is that teaching evolution properly, so as to reveal its role as a foundational explanatory framework in biology, immedi- ately illuminates how scientific explanations work. science does not stop with mere description; it is not just a catalog of facts. although one still occasionally hears a scientist repeat the old claim that science does not explain but only describes, this is a long-discredited view of science. in science, one explains effects by citing causes, and that is exactly what is going on when one explains biological patterns in terms of common descent, natural selection, or some other from the suite of causes that are part of evolutionary theory. a second issue has to do with the sense in which the term theory is used in science. unlike the colloquial meaning of the term, theory is not the opposite of fact. the opt-out request letter from the river of life evangelical church provides just one example of this common misun- derstanding. rather, as we have seen above, theory refers to the framework of causal laws and concepts that help explain particular empirical phenomena. once hypotheses about these have been tested and confirmed, then they are no less factual than the things that they explain. the idea of testability deserves special emphasis when we teach about science, for it is the key feature of scientific methodology. too often the way that science is taught—as lists of facts and formulae to be memorized—gives students a false impression about why scientists accept these findings. unlike religious dogma, it is not by authority that scientific conclusions are to be accepted. to the extent that students are told only what the conclusions are, they will likely fail to appreciate what is distinctive about science, namely the means by which these conclusions were tested and confirmed by evidence. this point is also related to the important notion of observability in science. creationists seem to believe that the only way to test something is by observing it directly, and they think that evolution isn’t observable. they are wrong on both counts. evo edu outreach ( ) : – first of all, the idea of observation in science is not the naïve notion that creationists assume when they recom- mend that students challenge their instructors who teach about the evolution by asking them “were you there?” (simon ). the faulty assumption behind this imperti- nent challenge is that one has to observe something with one’s own eyes for it to qualify as science. if direct observation is the only way to test a hypothesis, then no scientist is in any position to know how creation occurred— only god is. that is the creationist view, of course. this seriously misunderstands the relationship between testability and evidence in science. after all, most of the most important discoveries in science involve things that cannot be seen directly. the real way in which observability is a requirement in science is not that scientists may countenance only observable entities, but that scientists have to rely upon observational evidence. to give just a simple example, scientists do not need to observe sub-atomic particles directly to conclude that they exist; rather, they confirm their existence (and much more about their particular properties) by checking their hypotheses about them against predicted, observable patterns of physical data. confirming evolutionary hypotheses, even about the distant past, works in exactly the same way. second, creationists are wrong even about the basic facts regarding the evidence for evolution: in many cases, evolution is directly observable. the evolutionary processes that darwin discovered continue to operate and may be observed all around us. my colleague richard lenski, whose own long-term investigation of evolving populations of e. coli set a new standard for direct evolutionary experimentation, likes to point out that such observations of evolution in action are not really new but have been taking place since darwin’s own time. he tells, for instance, of rev. william dallinger, a methodist minister who also was skilled in microbiology, who conducted a six-year evolutionary experiment using protozoa and excitedly wrote to darwin about how his findings “palpably demonstrate your great doctrine” (lenski ). today, investigating evolution in action is becoming more and more important for practical purposes in fields ranging from agriculture to medicine to zoology. evolutionary science and society: educating a new generation (cracraft & bybee ) is a useful resource for educators to get a sample of this research, as is david mindell’s the evolving world (mindell ). moreover, we are now at a point where, by instantiating the evolutionary mechanism directly in an artificial-life environment in a computer, evolutionary hypotheses can be tested even more precisely and effectively. one of my own efforts along these lines has been to develop and test an educational version of such an evolving a-life system— avida-ed—that allows students in undergraduate courses to observe evolution in action for themselves (pennock b; speth et al. ). creationism consistently fails to follow science’s meth- odological rules and so does not begin to qualify as science. this failure starts with its most basic claim that biological complexity is explained by the design of a supernatural agent, not evolutionary or indeed any natural causes. but appeal to miraculous powers is no scientific explanation. because by definition, the miraculous has no limits, it rules out no observable pattern—any data may follow in any situation. something that could “explain” anything in any situation really explains nothing at all. the problem is not just that special creation is a bad scientific theory; creationism offers no positive theory at all. and it is not just that creationists are notoriously slippery about their views; rather, there is no conceptual friction at all, to continue the metaphor, by which one may hold onto a purported supernatural power and say what likelihood it confers upon any possible state of affairs. it thus fails to be testable either by direct or indirect observational evidence. it is because of such considerations that scientific method- ology is restricted to appeals to natural causes; methodo- logical naturalism is a basic principle of science because it is essential for the very idea of empirical evidence (pennock ). this is not to say that supernatural powers do not or could not possibly exist. science is not in the business of proving or disproving metaphysical claims. religious belief in a supernatural creator may rest upon theological argu- ments or upon faith, but it is wrong to pretend, as creationists do, that supernatural design is proven by science. indeed, this defining element of creationism is enough to allow a reasonable person to determine that it is not science but a religious view. this is exactly what the courts have determined every time that the issue has been tried, whether the form of creationism being considered is creation-science or intelligent design. the ruling against intelligent design creationism in the kitzmiller v. dover case should be mandatory reading for anyone who wants to see why creationism does not belong in public schools (kitzmiller v. dover ). creationism: a religious controversy having consistently failed to satisfy the courts that including their views in public school science classes is constitutional, creationists now lobby for schools to “teach the controversy” and have students learn the “strengths and weaknesses” of evolution under the guise of “academic freedom” (matzke et al. ; pennock a; branch and scott ). their strategy here, of course, is to open the door to their usual objections to evolution without mention- evo edu outreach ( ) : – ing their movement by name. this approach trades on a basic ambiguity about the nature of the controversy. the proper response to such efforts is “what controver- sy?” anyone who understands the nature of science and the centrality of evolution within biology knows that there is not a scientific controversy about evolution as creationists claim. there is a creationism/evolution controversy, but it is a re- ligious controversy. however, it is not a religious controver- sy in the simplistic sense of “science vs. religion” as the story is commonly played out in the media. as we shall see, it is a controversy of religion vs. religion; in particular, the crea- tionism controversy is based in fringe sectarian views that oppose one another as well as mainstream theology. the defining element of creationism is the rejection of evolution in favor of some supernatural act of creation. in a simple sense, that does make creationism a religious attack on science, but the controversy is really much more complicated and much more interesting, for it is critical to remember that creationists come in a wide variety of stripes. young-earth creationists (yecs) such as the well-known “creation-science” advocates, who hold that the world is not much more than , years old, are probably the most common anti-evolutionists today, but old-earth creationists (oecs) see no theological problem with accepting the standard geological chronology of billions of years. intelli- gent design creationism (idc) attempted to unite yecs and oecs under a “big tent” of anti-evolutionism by agreeing to temporarily set aside their fundamental disagreement about the age of the earth, but this political alliance is fragile and already beginning to come apart at the seams. idc leader william dembski, who for many years refused to state his own view on the matter, was recently forced to publicly announce “i am an old earth creationist” and defend himself against a theological challenge to his religious orthodoxy by a baptist pastor who accused him of being a theistic evolutionist (dembski ). and of course, there are non-christian creationists who have their own preferred re- ligious account of creation. the above (incomplete) taxonomy of varieties of creationism shows the fallacy of suggesting that one should teach “both sides” of the “controversy.” there are any number of non-scientific views whose proponents are eager to have them included once the integrity of science classes is compromised by any form of creationism. the point to remember is that such sectarian religious views disagree with each other as much as they disagree with evolutionary science. it cannot be emphasized enough that avoiding irreconcilable religious differences in the public and political arena is a central reason for not allowing the establishment by the government of any religious view. this is as much for protection of freedom of religious belief as it is for the protection of the prerequisites for civil action in a democracy. the taxonomy also shows a logical problem with the basic form of argument that creationists use. if there were only two possibilities, then one could support the second simply by refuting the first. this is the way creationists try to set up the issue, so that they only have to disprove evolution and do not have to give positive evidence for their own view. in the past, creationists did this by presenting what they called a “dual model,” with creation-science and “evolution-science” as the only candidates. idcs followed the same approach, though they relabeled the models as “design theory” and “darwinism.” but in no case is this an accurate account of alternatives. creationists hope to win by default with only negative arguments, but given this false dichotomy, their argument does not get off the ground (pennock ). the problem of the false dichotomy shows up elsewhere as well, such as in the way that creationists set up the controversy in terms of science versus religion. the main way they do this is by treating evolution as though it were equivalent to atheism. this too is a long-standing view, going back to nineteenth-century calvinist theologian charles hodge who wrote a book asking the question “what is darwinism?” and answering: it is atheism (hodge ). creation-science advocates said much the same thing and so do id creationists today, though in a more devious way, by trying to build it into the very definition of evolution (e.g., johnson ). idc’s building atheism into the definition of evolution is devious not only because it is surreptitiously replacing science’s methodological naturalism with a metaphysical view, but also because it inaccurately implies that there is no way to accept both evolution and belief in god. in fact, mainstream christian theology sees no problem in accom- modating evolution. many statements from religious denominations and organizations that attest to this are helpfully compiled in the national center for science education’s book voices for evolution (sager ). the significant point is that idcs reject this mainstream christian view, again creating a false dichotomy. as william dembski put it “design theorists are no friends of theistic evolution” [emphasis in original] (dembski ). the creationism controversy is not a simple matter of science versus religion but rather is a classic example of religion versus religion. teaching evolution the wrong way the considerations in the above sections should help science educators think not only about the right way to teach evolution, but also about how not to teach it (pennock a). evolution is a science, not a religion, and when evo edu outreach ( ) : – taught properly, this should be clear. idcs are wrong to build a metaphysical form of atheism into the definition of evolution. science educators must be careful, therefore, not to accept the creationists’ way of framing evolution and not to inadvertently present it as a metaphysical view—that really would be quite inimical to science’s methodological rules. if one were to teach evolution in this significantly wrong way, then creationists would have a legitimate complaint and would soon be joined by the mainstream moderates. however, it would not be very difficult for science teachers to avoid this mistake if science education regularly included some basic instruction about the nature of science. with a little care, there should be no legitimate reason for opt-out policies for any but the most extreme creationists. as we have seen, the idc objection to evolution is, at least if held consistently, religiously very deep-seated. it is a comprehensive worldview that opposes not just evolution but the entire scientific framework and advocates a pre- modern theistic science. the notre dame philosopher and early idc advocate alvin plantinga articulated this kind of view in a paper about the teaching of creation and evolution in the schools. parents, he said, might have comprehensive beliefs that are opposed to those of science. they have a basic right, he claims, not to have their children taught comprehensive beliefs that contradict their own (plantinga ). plantinga used this as part of an argument for requiring parents’ alternative beliefs, such as creationism, to be included in science classes, rather than as an argument for allowing a student to opt out of evolution, but his idea could easily be redeployed in favor of allowing opt-out policies. i have previously discussed the problems with the specifics of plantinga’s argument (pennock ) and will not repeat those here. my point here is just the straightforward one that idc is, in plantinga’s sense, a comprehensive religious belief. one sees this, for instance, in the leaked idc manifesto the “wedge document”—its call for a revolutionary theistic science and a radical transformation of modern culture that is based in their understanding of the bible is the governing principle of the id movement (discovery_institute ; downey ). seen in this way, this is indeed very similar to the case of the amish as adjudicated in wisconsin v. yoder ( ). again, i am not taking up the question here of whether students are or should be legally allowed to opt out. i simply want to point out what this comes to, given what we have seen about evolution and the nature of science. if methodological naturalism is a problem, it is not in any special sense a problem for evolution, for it is essential to all of science. the idc attack on science’s naturalistic methodology is an attack on science itself. if this is the basis for an opt-out policy, then it means opting out of science entirely. conclusion in the alberta case with which we began this article, the concern about the possibility of an opt-out policy for evolution was quickly resolved. the alberta minister for human rights stepped in to explain that the opt-out proposal involved being able to opt out of religious instruction, not out of some particular part of the curriculum because of religious beliefs. so the thought that somebody can get out of evolution using the fact that it's against their religious beliefs is not correct....evolution is not a part of religious studies, it's part of science curriculum, and there is nothing that will change that going forward (cbc news, b). this is a rational response. evolution should be taught robustly as a core part of the science curriculum. it is a well- confirmed conclusion of standard scientific methodology, and for most mainstream christians, it is accepted as such. so long as evolution is not taught improperly as a metaphysically atheist view, there should be no danger of mistaking it for a part of religious studies. the canadians got this exactly right. unfortunately, the controversy is not likely to be solved so simply in the united states. in oklahoma, the proposed legislation that might have led to schools having to heed such requests failed to pass the senate education committee, but just barely, in a narrow seven to six vote (hoberock ). in niceville, fl, the river of life church continues to help its members try to opt out of evolution. given the extreme and expansionist views that drive the creationist movement, we should not expect that creationists will give up their battle. perhaps the time will come when the courts conclude that creationist parents should be allowed to have their children opt out of evolution. what i have argued in this essay is that if evolution is taught properly, opting out of evolution really means opting out of biology. and if parents object not just to evolution, but also to science’s naturalistic methodology, as id creationists do, then they need to know that this means opting out of science entirely. such a radical departure from the real world makes the amish look like amateurs. references branch g, scott ec. the latest face of creationism. scientific american. ; ( ): – . cavanagh s. oklahoma evolution. february: education week; . . cbc news. evolution classes optional under proposed alberta law. cbc news, toronto, canada. a. http://www.cbc.ca/canada/ calgary/story/ / / /cgy-bill-evolution-law-alberta-classes- teachers.html. accessed april . evo edu outreach ( ) : – http://www.cbc.ca/canada/calgary/story/ / / /cgy-bill-evolution-law-alberta-classes-teachers.html http://www.cbc.ca/canada/calgary/story/ / / /cgy-bill-evolution-law-alberta-classes-teachers.html http://www.cbc.ca/canada/calgary/story/ / / /cgy-bill-evolution-law-alberta-classes-teachers.html cbc news. proposed alberta law doesn't make evolution classes optional: minister. cbc news, toronto, canada. b. http:// www.cbc.ca/canada/calgary/story/ / / /cgy-evolution- alberta-human-rights.html. accessed may . cracraft j, bybee rw, editors. evolutionary science and society: educating a new generation. washington dc: biological sciences curriculum study; . dembski wa. what every theologian should know about creation, evolution, and design. access research network. . http:// www.arn.org/docs/dembski/wd_theologn.htm. dembski wa. intelligent design: the bridge between science and theology. downers grove: intervarsity press; . dembski wa. comment to johnny t. helms. in: our sovereign joy. . http://oursovereignjoy.blogspot.com/ / /book-review- william-dembskis-end-of.html. accessed dec . discovery_institute. the wedge strategy. . http://www.stephen jaygould.org/ctrl/archive/wedge_document.html. accessed may . dobzhansky t. nothing in biology makes sense except in the light of evolution. am biol teach. ; : – . downey r. discovery’s creation. seattle weekly news, seattle, washington. february . hoberock b. school science bill is killed. tulsa world, tulsa, ok. feb . hodge c. what is darwinism? new york: scribner, armstrong and company; . johnson pe. darwin on trial. washington, d.c.,: regnery gateway; . kitzmiller, et al. v. dover area school district, et al. . ( f. supp. d , docket no. cv ) lenski r. evolution in action: a -year salute to charles darwin. in: maloy s, kolter r, editors. microbial evolution years after the origin of species. washington, dc: asm press; . matzke nj, gross pr. analyzing critical analysis: the fallback antievolution strategy. in: scott ec, branch g, editors. not in our classrooms: why intelligent design is wrong for our schools. boston: beacon press; . p. – . mindell dp. the evolving world. cambridge: harvard university press; . pennock rt. tower of babel: the evidence against the new creationism. cambridge: ma, the mit press; . pennock rt. should creationism be taught in the public schools? sci educ. ; ( ): – . pennock rt. on teaching evolution and the nature of science. in: cracraft j, bybee r, editors. evolutionary science and society: educating a new generation. colorado springs, co: biological sciences curriculum study; . pennock rt. how not to teach the controversy about creationism. in: jones ll, reiss mj, editors. teaching about origins: taking account of creationism. new york: peter lang; a. p. – . pennock rt. learning evolution and the nature of science using evolutionary computing and artificial life. mcgill j educ. b; ( ): – . plantinga a. creation and evolution: a modest proposal. in: pennock rt, editor. intelligent design creationism and its critics: philo- sophical, theological, and scientific perspectives. boston: the mit press; . p. – . river of life church. partents [sic] opt out letter. . http://www. theriverfamilychurch.com/publicschool/optout.php. accessed dec . sager c. ( ). voices for evolution, rd edition. national center for science education, berkeley, ca. . http://www.lulu.com/ items/volume_ / / / /print/ .pdf. scott ec, branch g. overcoming obstacles to evolution education: the oopsie compromise—a big mistake. evol educ outreach. ; : – . simon s. their own version of a big bang. los angeles times, los angeles, ca. february . speth eb, long t, pennock rt, ebert-may d. using avida-ed for teaching and learning about evolution in undergraduate introduc- tory biology courses. evol educ outreach. ; ( ): – . wisconsin v. yoder. u.s. . certiorari to the supreme court of wisconsin. . evo edu outreach ( ) : – http://www.cbc.ca/canada/calgary/story/ / / /cgy-evolution-alberta-human-rights.html http://www.cbc.ca/canada/calgary/story/ / / /cgy-evolution-alberta-human-rights.html http://www.cbc.ca/canada/calgary/story/ / / /cgy-evolution-alberta-human-rights.html http://www.arn.org/docs/dembski/wd_theologn.htm http://www.arn.org/docs/dembski/wd_theologn.htm http://oursovereignjoy.blogspot.com/ / /book-review-william-dembskis-end-of.html http://oursovereignjoy.blogspot.com/ / /book-review-william-dembskis-end-of.html http://www.stephenjaygould.org/ctrl/archive/wedge_document.html http://www.stephenjaygould.org/ctrl/archive/wedge_document.html http://www.theriverfamilychurch.com/publicschool/optout.php http://www.theriverfamilychurch.com/publicschool/optout.php http://www.lulu.com/items/volume_ / / / /print/ .pdf http://www.lulu.com/items/volume_ / / / /print/ .pdf 	should students be able to opt out of evolution? some philosophical considerations 	abstract 	opting out of evolution 	teaching evolution the right way 	revealing the nature of science 	creationism: a religious controversy 	teaching evolution the wrong way 	conclusion 	references doi: . /j.bbabio. . . p/ import and assembly of mitochondrial proteins nikolaus pfanner institute for biochemistry and molecular biology, university of freiburg, germany e-mail: nikolaus.pfanner@biochemie.uni-freiburg.de mitochondria contain about different proteins. % of the proteins are synthesized as precursors on cytosolic ribosomes. the precursors are imported via the translocase of the outer mitochon- drial membrane (tom complex) and are subsequently sorted into the four mitochondrial subcompartments, outer membrane, inter- membrane space, inner membrane and matrix. (i) cleavable preproteins are transported from the tom complex to the presequence translocase of the inner membrane (tim complex). the presequence translocase-associated motor (pam) drives trans- location into the matrix. (ii) hydrophobic inner membrane proteins are transferred through the intermembrane space by a chaperone complex (small tim proteins) and inserted into the inner membrane by the tim complex. (iii) the mitochondrial import and assembly machinery (mia) directs small proteins into the intermembrane space and promotes the formation of disulfide bonds. (iv) beta- barrel proteins are transported from the tom complex to the sorting and assembly machinery of the outer membrane (sam complex). doi: . /j.bbabio. . . p/ new functions for novel mitochondrial transporters ferdinando palmieri university of bari, italy e-mail: fpalm@farmbiol.uniba.it a strikingly large number of mitochondrial dna (mtdna) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. these mitochondrial dis- orders were the first to be investigated after the small mtdna had been sequenced in the 's. only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, hhh syndrome, aspartate/glutamate isoform deficiency, amish microce- phaly and neonatal myoclonic epilepsy; these disorders are characterised by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabo- lism. defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phos- phate and adp, are responsible for diseases characterised by defective energy production. herein, all the mitochondrial carrier- associated diseases known to date are reviewed for the first time. particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders. doi: . /j.bbabio. . . p/ the water oxidizing enzyme a. william rutherford ibitec-s, ura cnrs, cea saclay, gif-sur-yvette, france e-mail: alfred.rutherford@cea.fr photosystem ii, the water oxidising enzyme of photosynthesis, put the energy (or at least a major fraction of it) into the biosphere and the oxygen into the atmosphere. it is certainly one the most influential and important enzymes on the planet. the aim of our research is to understand how this enzyme works as ) a solar energy converter and ) the only known thermodynamically efficient catalyst for oxidizing water. the information obtained is used in the design of artificial catalysts and photocatalysts. a chemical catalyst that has the thermodynamic efficiency of the enzyme could greatly improve the efficiency of ) water electrolysis and photolysis for fuel (e.g. h ) production and ) the reverse reaction, oxygen reduction, in fuel cells. there is therefore a great interest in understanding the mechanism of this enzyme and in reproducing aspects of its function in artificial systems. i will describe our current knowledge of photosystem ii, including some recent experimental studies, as well as recent efforts in our joint saclay/orsay program aimed at producing bio-inspired water oxidizing catalysts. doi: . /j.bbabio. . . p/ the structure of purple bacterial antenna complexes: from single molecules to native membranes richard j. cogdella, alastair t. gardinera, mads gabrielsena, aleks w. roszaka, june southalla, tatas brotosudarmoa, neil w. isaccsa, hideki hashimotob, juergen baierc, silke oellerichc, martin richterc, juergen koehlerc, francesco franciad, giovanni venturolid, dieter oesterhelte adivision of biochemistry and molecular biology, ibls and department of chemistry, university of glasgow, glasgow g qq, uk bcrest-jst and department of physics, graduate school of science, osaka city university, - - sugimoto, sumiyoshi-ku, osaka - , japan cexperimental physics iv, university of bayreuth, d- bayreuth, germany ddepartment of biology, university of bologna, bologna, italy edepartment of membrane biochemistry, max-planck institute for biochemistry, martinsried, germany e-mail: r.cogdell@bio.gla.ac.uk the photosynthetic unit of purple photosynthetic bacteria typically contains two types of light-harvesting complexes, called lh and lh . these antenna complexes are constructed on a modular principle. they are circular or elliptical oligomers of dimers of two low-molecular weight, hydrophobic apoproteins, called a and b, that bind bacteriochlorophylls and carotenoids non-covalently. the lh complex surrounds the reaction centre and, depending on the species, is either a monomer or a dimer. the lh complexes are arranged around the lh -rc complexes. this plenary lecture will present the current status of structural studies on these pigment- protein complexes, based upon a combination of x-ray crystal- lography and single molecule spectroscopy. then an overall view of how they are arranged in their native photosynthetic membranes will be presented. doi: . /j.bbabio. . . p/ catalysis of substrate conversion and electron transfer by mitochondrial complex i judy hirst medical research council dunn human nutrition unit, cambridge, cb xy, uk e-mail: jh@mrc-dunn.cam.ac.uk s abstracts / biochimica et biophysica acta ( ) s –s http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . mailto:nikolaus.pfanner@biochemie.uni-freiburg.de mailto:fpalm@farmbiol.uniba.it mailto:alfred.rutherford@cea.fr mailto:r.cogdell@bio.gla.ac.uk mailto:jh@mrc-dunn.cam.ac.uk wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ considerations of marrow cellularity in -dimensional dosimetric models of the trabecular skeleton wesley e. bolch, phd ; phillip w. patton, phd ; didier a. rajon, ms ; amish p. shah, bs ; derek w. jokisch, phd ; and benjamin a. inglis, phd department of nuclear and radiological engineering, university of florida, gainesville, florida; department of physics and astronomy, francis marion university, florence, south carolina; and center for structural biology, university of florida brain institute, university of florida, gainesville, florida dose assessment to active bone marrow is a critical feature of radionuclide therapy treatment planning. skeletal dosimetry models currently used to assign radionuclide s values for clin- ical marrow dose assessment are based on bone and marrow cavity chord-length distributions. accordingly, these models cannot explicitly consider energy loss to inactive marrow (adi- pose tissue) during particle transport across the trabecular mar- row space (tms). one method to account for this energy loss is to uniformly scale the resulting tms absorbed fractions by reference values of site-specific marrow cellularity. in doing so, however, the resulting absorbed fractions for self-irradiation of the trabecular active marrow (tam) do not converge to unity at low electron source energies. this study attempts to address this issue by using nuclear magnetic resonance microscopy images of trabecular bone to define -dimensional ( d) dosi- metric models in which explicit spatial distributions of adipose tissue are introduced. methods: cadaveric sources of trabec- ular bone were taken from both the femoral heads and humeral epiphyses of a -y-old male subject. the bone sites were sectioned and subsequently imaged at a proton resonance frequency of mhz ( . t) using a d spin-echo pulse sequence. after image segmentation, voxel clusters of adi- pocytes were inserted interior to the marrow cavities of the binary images, which were then coupled to the egs radiation transport code for simulation of active marrow electron sources. results: absorbed fractions for self-irradiation of the tam were tabulated for both skeletal sites. substantial variations in the absorbed fraction to active marrow are seen with changes in marrow cellularity, particularly in the energy range of – kev. these variations are seen to be more dramatic in the humeral epiphysis (larger marrow volume fraction) than in the femoral head. conclusion: results from electron transport in d models of the trabecular skeleton indicate that current meth- ods to account for marrow cellularity in chord-based models are incomplete. at kev, for example, the eckerman and stabin model underestimates the self-absorbed fraction to active mar- row by %. at mev, the model of bouchet et al. overesti- mates this same value by %. in the energy range of – kev, neither model accurately predicts energy loss to the active bone marrow. thus, it is proposed that future extensions of skeletal dosimetry models use d transport techniques in which explicit delineation of active and inactive marrow is feasible. key words: active marrow; adipocyte; skeletal dosimetry; nu- clear magnetic resonance microscopy; marrow dosimetry j nucl med ; : – in radionuclide therapies for the treatment of neoplastic disease, risk of marrow toxicity may be quantified through the assessment of the absorbed dose to sensitive tissues within the bone marrow ( ). computational skeletal dosim- etry models are thus used in clinical applications of radio- nuclide therapy in which a distinction between active (red) and inactive (yellow) marrow is necessary. this binary classification of target tissues is in stark contrast to the complex microanatomy of real marrow. in this study, -di- mensional ( d) tomographic images of trabecular bone structure are used to introduce target and nontarget regions that explicitly distinguish active from inactive marrow in a manner related to their clinical and histologic definitions. histology of marrow tissues bone marrow is a connective tissue found within cancel- lous, or trabecular, bone that is divided into irregular inter- connected spaces by the bone trabeculae. the tissues of bone marrow may be grouped into categories: (a) the hematopoietic cellular component (granulocytic, erythroid, and megakaryocytic series), (b) bone marrow stromal cells and the extracellular matrix, (c) venous sinuses and other blood vessels, and (d) various other support cells ( , ). separating the osseous tissues of the bone trabeculae and the marrow is a thin layer of connective tissue, the en- dosteum, populated with osteoblasts and osteoclasts. the former are responsible for osteoid deposition and bone formation, whereas the latter are involved in bone resorption and remodeling. spongiosa is defined as the combined tis- received apr. , ; revision accepted sep. , . for correspondence or reprints contact: wesley e. bolch, phd, department of nuclear and radiological engineering, university of florida, gainesville, fl - . e-mail: wbolch@ufl.edu marrow cellularity in skeletal dosimetry • bolch et al. sues of both the bone trabeculae and the marrow tissues interior to the cortical bone cortex of the skeletal site. the hematopoietic component is supported by a micro- environment of randomly distributed stromal cells, a com- plex extracellular matrix (ecm), and vascular structures that branch unevenly throughout the marrow space. stromal cells include adipocytes, fibroblast-like reticulum cells, en- dothelial cells, and the osteoblasts and osteoclasts of the endosteum. the hematopoietic cell series are supported within marrow chords delineated by the fibroblast-like stro- mal cells and the stromal matrix. the ecm, which is produced by the stromal cells, contains various substances, including collagen, fibronectin, laminin, thrombospondin, hemonectin, and proteoglycans. in addition to their struc- tural role, these substances also present growth factors to the hematopoietic progenitor cells ( – ). the vascular struc- tures of bone marrow are largely composed of thin-walled venous sinuses ( , ). these sinuses receive blood from the nutrient artery and the periosteal capillary network ( ). the former reaches the bone marrow through penetrations of the cortical bone cortex, whereas the latter provides articulation between the marrow sinuses and the haversian canals of the cortex. various topographic patterns of hematopoietic cell dif- ferentiation are found to occur within the marrow space. for example, precursor cells within the granulocytic series are arranged along the endosteal surface of the trabeculae with maturation occurring toward regions central to the marrow cavities ( , ). higher concentrations of hemonectin have been found along the endosteal regions of marrow ( ), and this protein is particularly adherent to granulocytic precur- sors ( , ). in contrast, erythroid elements are organized into small groups (erythroblastic islands) present within the central regions of the marrow extending back to the first fat space (adipocytes adjacent to the bone trabeculae). mature megakaryocytes and their precursors are found uniformly throughout the central regions of the marrow space and are associated with the venous sinuses. their presence along the bone trabeculae is considered abnormal. other cells, which support the function and health of the marrow tissues, include lymphocytes, plasma cells, mast cells, and macrophages. b- and t-lymphocytes are found in small aggregates throughout the marrow space and increase in number with age. plasma cells may occur individually or in - or -cell clusters and are found more frequently in layers around blood vessels within the marrow. mast cells are distributed throughout the marrow with a tendency for a perivascular and paratrabecular distribution. macrophages are also found uniformly throughout the marrow space, including the luminal surfaces of the venous sinusoids. marrow cellularity clinical and pathologic assessment of bone marrow func- tion includes, among other assays, an assessment of the bone marrow cellularity defined as the percentage of bone marrow volume occupied by hematopoietic cells ( , ). bone marrow cellularity generally decreases with age in normal, healthy individuals at rates that vary with skeletal site. in the femoral head and neck, for example, nominal values of marrow cellularity are % in the newborn, % in the y old, and only % in the adult ( , ). reference values for the marrow within the lumbar spine at these same ages are %, %, and %, respectively. in a study by ballon et al. ( ), in vivo -point chemical-shift mri was applied as a noninvasive assay of marrow cellularity in the posterior iliac crests of volunteers and patients. in the healthy subjects, marrow cellularity ranged from a low of . % � . % to a high of . % � . %. in the same study, marrow cellularity measured in patients with acute myelogenous leukemia (aml) ranged from . % � . % to . % � . %. a subsequent study by these authors reported marrow cellularities within the posterior iliac crests ranging from % (aml remission) to . % � . % (chronic myelogenous leukemia, chronic phase) ( ). for of the cases studies, the nuclear magnetic resonance (nmr)-derived cellularities were later confirmed by bone marrow biopsy. in terms of the microanatomy of marrow tissues, a defi- nition of marrow cellularity based on the volume fraction of active marrow would, in principle, exclude the volume associated with the marrow stromal cells and ecm, the blood sinuses and other vessels, and the marrow support cells (categories b– d above). however, most of these cells and structures are of smaller or comparable size and spatial location to the hematopoietic cell series, thus making any differentiation of their occupied marrow volume difficult to achieve in the clinical setting. fortunately, the relative num- ber and size of the adipocytes within the marrow space are easily distinguished and may be used to provide a more clinically achievable assessment of marrow cellularity. in this manner, marrow cellularity may be defined as the fraction of marrow space not occupied by adipocytes: �marrow cellularity� � � �fat fraction�. eq. within this clinical working definition, one may then define active marrow—needed to define the source and target regions in radiation transport models of skeletal do- simetry—as the portion of trabecular marrow space not occupied by adipocytes. this definition is consistent with mri assessments of marrow cellularity and with clinical conditions in which hypercellularity or hypocellularity is accompanied by proportional decreases or increases, re- spectively, in marrow fat composition. previous methods of including marrow cellularity in skeletal dosimetry models most skeletal dosimetry models currently used in clinical medicine are fundamentally based on the seminal work of spiers and his students at the university of leeds some y ago ( – ). in these studies, optical scanning of high- the journal of nuclear medicine • vol. • no. • january resolution radiographs of trabecular bone slices were im- aged giving rise to chord-length distributions across marrow cavities and bone trabeculae. dosimetry models subse- quently developed using these chord distributions could only assess radiation absorbed dose averaged across the entire marrow space. explicit consideration of active and inactive cellular components of the marrow were not in- cluded within the transport models when one considers the method by which the chord distributions were measured and assembled (i.e., radiographs showing only bone trabeculae and marrow spaces). nevertheless, estimates of total active marrow mass were included in the final reporting of dose conversion factors (dose per unit activity concentration in the bone matrix) by the leeds’ group. a reference value of , g of active marrow ( , g of total marrow) within reference man was used as taken from international com- mission on radiological protection (icrp) publication ( ). the spiers’ data were subsequently used by snyder et al. ( , ) in the development of radionuclide s values within mird pamphlet no. . the icrp used single-value approximations of absorbed fractions from the data of spiers in icrp publication ( ). explicit consideration of inactive and active marrow again was made only through the assignment of a total active marrow mass within the full skeleton. the skeletal dosimetry model of mirdose uses the icrp publication model for bone tissue dosimetry ( ). more recent revi- sions to the icrp reference man skeletal data have revised downward the estimate of total active marrow to , g, thus acknowledging that in many adult trabecular bone sites, the marrow is only partially active ( ). recently, eckerman and stabin ( ) and bouchet et al. ( , ) presented more refined models of absorbed dose to skeletal tissues. both of these models are chord-based mod- els of radiation transport and both use the original marrow and trabeculae chord distributions measured by spiers and his students. as such, these models cannot explicitly con- sider the influence of energy lost to adipocytes as an elec- tron traverses a given marrow cavity. for example, consider the assignment of an absorbed fraction to the trabecular active marrow (tam) for an electron source located within the active marrow tissues of bone site j: �j�tam tam�. because the eckerman and stabin model and the bouchet et al. model use the spiers’ chord-length distributions, the exact spatial location of the active tissues of the marrow cannot be considered. consequently, the monte carlo sim- ulations of electron transport yield only an estimate of the absorbed fraction to the trabecular marrow space (tms) for a radiation source emitted within the tms: �j�tms tms�. as such, both models are forced to make approximations to assess the self-absorbed fraction to only the active marrow regions of the skeletal site. in the eckerman and stabin model, a uniform scaling of the tms self-absorbed fraction is made across all electron energies. in their model, the marrow cellularity factor (cf) (fraction of marrow space occupied by active marrow) is taken as the appropriate weighting factor. in the bouchet et al. model, no weighting factor is applied. consequently, these authors make the following assignments at all electron energies: �j�tam tam� � cfj . �j�tms tms� eckerman and stabin model. eq. �j�tam tam� � �j�tms tms� bouchet et al. model. eq. note that in both cases, these absorbed fractions would be divided by the same reference man mass of active marrow in assigning radionuclide s values unique to each skeletal site. at very low electron energies, the bouchet et al. model correctly approaches the limiting value of unity as the electron source energy is totally imparted within the target region. in this case, no scaling of the tms absorbed fraction is warranted. however, at high electron energies, electrons traverse or more marrow cavities, and the fraction of electron energy lost to the active marrow tissues is more closely approximated by the fraction of tissue that is active within those cavities. consequently, a uniform scaling of the tms self-absorbed fraction is appropriate at high elec- tron energies as is done in the eckerman and stabin model. at intermediate energies (as yet to be determined), both models would appear to be incorrect as the bouchet et al. model would overestimate the self-dose to active marrow, and the eckerman and stabin model would underestimate that same dose. however, in both models, energy lost to the bone trabeculae would be accounted for and would be reflected in the energy-dependent profile of the absorbed fraction at all values of cellularity. again, a dosimetry model that explicitly includes active and inactive marrow regions during particle transport would, in theory, give a correct profile of the absorbed fraction energy dependence without the need for scaling factors or other approximations. the goal of this study was to create such a model using d images acquired through nmr microscopy. materials and methods bone site harvesting a -y-old male cadaver, cm in total height and kg in total mass, was purchased from the state of florida anatomical board (gainesville, fl). the cause of death was a large cardio- vascular accident, which included edema and cardiomyopathy. no change in trabecular microstructure is expected from this cause of death. more than bone samples were removed and stored frozen at � °c until nmr imaging sessions were conducted. of these various skeletal sites, the femoral head and humeral epiphysis were selected for study because microstructural data on the latter have marrow cellularity in skeletal dosimetry • bolch et al. not been reported previously; data from the femoral head are thus used as a surrogate in current reference man models. jokisch ( ) has reported nmr microscopy and dosimetry of the thoracic vertebrae. ct scanning the right femoral head and right humeral epiphysis harvested from the male cadaver were imaged on a lightspeed qx/i ct scanner (general electric medical systems, milwaukee, wi) op- erated at kv and ma. helical scans, reconstructed to an in-plane spatial resolution of � �m and a slice thickness of . mm, were obtained for the femoral heads. for the humeral epiphysis, the in-plane spatial resolution was � �m for each . -mm slice. in addition to identifying the best location to physically section the trabecular regions of the skeletal samples, the ct images allow one to determine the thickness of the cortical bone cortex, a parameter of importance in macrostructural trans- port models of the skeleton ( ). the ct images indicated that the male femoral head sample has a cortex thickness of . � . cm. furthermore, the ct images show that the spongiosa within the male femoral head is best modeled as a sphere with a . -cm inner diameter. the spongiosa of the male humeral epiphysis is modeled as a . -cm inner-diameter sphere with a cortical thick- ness of . � . cm. in each case, the spheric representations of these samples neglect the relatively small regions that articulate with the femoral or humeral necks. the d models thus developed characterize only the local dosimetry of the marrow tissues within these skeletal sites. sample preparation after ct scanning, each skeletal sample was physically sec- tioned. two pieces were taken (rectangular prisms approximately � � cm in size) from both the right femoral head and the right humeral epiphysis of the male subject. bone sample sizes were such that the bone prisms represented approximately % of the total spongiosa within the femoral head. for the humeral epiphysis, the sectioned bone prisms represented % of the spon- giosa. considering that rectangular samples were taken from a spheric bone site, these percentages are considered acceptable in modeling the trabecular structure of these skeletal sites. each femoral head bone prism was suspended in a container filled with an aqueous solution of . % sodium hypochlorite. the container was placed on a magnetic stirrer, and the solution was circulated for – h. the samples were removed from the solution, rinsed with hot water, and reimmersed in a new solution until the marrow was no longer visible within the sample. next, the marrow spaces of each bone prism were filled under vacuum with gado- linium-doped water and taken to the advanced mri and spectros- copy facility of the university of florida brain institute for nmr imaging. for the humeral epiphysis sample, marrow-intact imag- ing was performed. visual inspection of the ct images from each of these samples showed that the fractional bone volume in each sample was lower than that in the corresponding femoral heads. marrow-intact imaging was thus selected because we feared that marrow digestion might comprise the trabecular lattice. recently, we have shown that reproducible dosimetry can be achieved in marrow-intact and in marrow-free specimens ( ). nmr microscopy all nmr images were acquired on a -cm wide-bore avance imaging spectrometer (bruker medical, karlsruhe, germany), op- erating at a -mhz proton resonance ( . -t magnetic field strength) using a -mm-diameter quadrature birdcage coil of length . cm. a conventional d spin-echo pulse sequence was used to obtain fully d images of each sample. the field of view of each imaging session was . � . � . cm with matrix dimensions of � � . therefore, the resulting spatial resolution of the d image was � � �m. table lists the repetition time, echo time, receiver gain, and number of aver- ages used in each session. details of image processing techniques applied to the nmr microscopy images, by which d binary images of the bone trabeculae and marrow spaces are obtained, have been reported ( ). also given in table are the marrow volume fractions for each bone sample. for this male subject, the volume fraction of marrow was found to be higher in the humeral epiphysis than in the femoral head. explicit consideration of adipocyte clusters in marrow spaces the final step before radiation transport is to further classify the marrow voxels of the d images into active and inactive marrow, with the latter defined as the presence of adipocyte cell clusters. first, a random marrow voxel is selected within the image and subsequently reclassified as a voxel of adipose tissue. two ran- domly selected neighboring marrow voxels, both of which share a common face with the initial voxel, are then additionally reclas- sified. in this manner, adipocyte clusters of voxels are randomly placed within the marrow spaces of the d image. this process is continued until an overall targeted marrow cellularity is attained. the model is somewhat approximate in that voxel dimensions within these current images are cubes �m on edge, whereas true adipocytes are approximately spheric cells with a mean diameter of � �m ( ). the clustering pattern chosen in this model is based on visual observations of biopsy slides of normal bone marrow ( ). figure is a simulated -dimensional slice with %, %, %, and % marrow cellularity imposed on an image extracted from a d nmr image. for the femoral head and humeral epiph- ysis, a reference cellularity factor of . ( % active marrow and % adipose tissue) is recommended by the icrp (table of icrp publication ( )). our work investigates marrow cellu- larities ranging from % to % (fat fractions ranging from . to . , respectively). the atomic composition of osseous tissue is taken from that given in international commission on radiation units and measurements (icru) publication for cortical bone ( ). similarly, the atomic compositions of active and inactive marrow are taken from those given in icru publication for red marrow and yellow marrow, respectively. table parameters of nmr imaging sessions used for bone samples sectioned from -year-old male cadaver sample tr (ms) te (ms) rg no. of averages marrow volume (%) right femoral head prism . . prism . . right humeral epiphysis , . tr 	 repetition time; te 	 echo time; rg 	 receiver gain. the journal of nuclear medicine • vol. • no. • january egs macrostructural transport model in this study, macrostructural transport models of the femoral head and the humeral epiphysis are considered during egs electron transport simulations ( ). electrons are transported within the region of interest defined within the original d nmr image. as they escape the region of interest, they are reintroduced through identical and adjacent copies of the image ( , ). addi- tionally, the macroscopic dimensions of the bone site, including the thickness of the cortical bone cortex and the inner diameter of the spongiosa, are also considered. once a particle reaches the edge of the spongiosa, it is no longer transported within the voxels of the nmr image but is instead transported within a spheric shell of cortical bone. if the particle reenters the trabecular region, it is reintroduced within the voxel transport geometry. if the particle exits the exterior side of the cortical shell, it is transported within a surrounding region of soft tissue, thus allowing for the potential reentry to the bone site. in this study, only the tam is considered as the source and target region. other regions defined in the model are the trabecular bone volume (tbv), the trabecular bone endosteum (tbe), the cortical bone cortex, the trabecular inactive marrow (tim), and the surrounding soft tissues. details of the techniques to model the endosteum within digital images of trabecular bone have been reported by jokisch et al. ( ). ten sets of , monoenergetic electrons of initial energies between and mev are transported. coefficients of variation on the reported absorbed fractions are . %. results comparisons of absorbed fractions for self-irradiation of active marrow figures and display absorbed fractions calculated using the macrostructural transport model for self-irradia- tion of the active marrow within the femoral head and humeral epiphysis, respectively, of the -y-old man. five values of marrow cellularity are assumed: %, %, %, %, and %. for each marrow cellularity, the absorbed fraction begins at unity for low-energy electrons in which total energy absorption occurs within the tam source re- gion. as the electron source energy increases, more and more kinetic energy is lost to adipocytes within the marrow cavities. at increasingly higher energies, energy is addition- ally lost to the surrounding bone trabeculae. at approxi- mately several hundred kiloelectron volts, steady-state val- ues are approached, as predicted under models that transport the electrons within infinite regions of trabecular bone ( , , ). in the macrostructural models of the femoral head and humeral epiphysis, however, a downturn in the absorbed fraction profile is seen at energies above mev as electrons leave the spongiosa of the skeletal site and deposit their residual energies within the surrounding cortical bone. the variations in absorbed fraction with changes in cel- lularity are slight at low electron energies, yet they progres- sively increase with increasing source energy to values proportional to the reduction in total active marrow mass. for example, figure shows that the absolute difference in the self-absorbed fractions at kev for bone samples with % and % marrow cellularity is % (ratio of . ). this absolute difference increases to % at kev (ratio of . ). at high electron energies, the absolute difference declines to about % (ratio of . ), as an increasing larger portion of the electron energy is lost to the cortical bone at both marrow cellularities. absorbed fractions for the re- maining marrow cellularities follow similar patterns. for bone sites with a larger marrow volume fraction, as seen in the humeral epiphysis for the male subject, the figure . two-dimensional region of interest selected from d nmr image of trabecular bone sample sectioned from male femoral head. four values of marrow cellularity are displayed: % (a), % (b), % (c), and % (d). gray voxels rep- resent simulated adipocyte cell clusters defining inactive marrow tissue. marrow cellularity in skeletal dosimetry • bolch et al. self-absorbed fraction to the active marrow is systematically higher. figures and show that the self-absorbed fraction to the active marrow at % cellularity is . in the humeral epiphysis and only . in the femoral head at kev (ratio of . ). at mev, these values are . and . , respectively (ratio of . ). these differences are important because current clinical models of skeletal dosim- etry implicitly assume that the absorbed fraction profile in the femoral head (for which the spiers’ microstructural chord-length data are available) can be used to estimate the dosimetry of the humeral epiphysis (for which no micro- structural data have yet been published). table displays the absorbed fraction data for active marrow sources and targets for the femoral head and hu- meral epiphysis taken from the male cadaver. values are given for marrow cellularities ranging from % to % in increments of %. comparisons of specific absorbed fractions for self- irradiation of active marrow in figure , the self-absorbed fractions for electron sources in the active marrow of the male femoral head are shown to separate from one another at high energies in direct proportion to the reduction in active marrow target mass. consequently, it is instructive to normalize the ab- sorbed fractions at each value of marrow cellularity by the corresponding tam target mass. as defined in the mird schema, the resulting values of specific absorbed fraction, �(tam tam), are directly proportional to the mean dose to active marrow per particle emission i: d� tami � ãtam . �i . ��tam tam�i, eq. where ãtam is the cumulated activity in the active marrow, and �i is the mean energy emitted per decay. values of �(tam tam) in the male femoral head are shown in figure . electron absorbed fractions calculated for %, %, %, %, and % marrow cellularity using macro- structural transport model for femoral head of -y-old man. values shown are aver- age absorbed fractions of separate bone samples sectioned from femoral head ( % variation at high energies). figure . electron absorbed fractions calculated for %, %, %, %, and % marrow cellularity using macro- structural transport model for humeral epiphysis of -y-old man. the journal of nuclear medicine • vol. • no. • january figure . for high-energy electron emitters (energies ex- ceeding several hundred kiloelectron volts), the mean dose per decay to active marrow is shown to be independent of the marrow cellularity. at high energies, the electrons more or less uniformly traverse the marrow cavities resulting in a uniform density distribution of imparted energy to all mar- row tissues. at high energies, the active and inactive mar- row components experience the same absorbed dose inde- pendent of their respective volume fractions within the marrow space. however, as one considers lower and lower energy emissions within the active marrow, the spatial sep- aration of the active and inactive marrow tissues becomes increasingly important, particularly at low cellularities (high fat fractions). figure shows that, for -kev electrons, the mean dose per decay to active marrow at % cellularity is a factor of higher than that in marrow of % cellularity. for marrow at % cellularity, the dose per decay delivered by -kev electrons is a factor of . lower than that at % cellularity. for low-energy emitters in the energy range of – kev, the specific absorbed fractions, and thus the radionuclide s values, vary considerably with marrow cel- lularity. as discussed later in this article, these variations in radionuclide s values with marrow cellularity might, under certain conditions, be accompanied by compensatory changes in radionuclide uptake, thus leaving the active marrow absorbed dose per administered activity invariant with changes in patient marrow cellularity. as noted earlier, the original spiers’ chord-length mea- surements for reference man did not include data for the humeral epiphysis. thus, it is instructive to compare values table absorbed fractions of energy for monoenergetic electrons emitted within tam irradiating tam of femoral head and humeral epiphysis of -year-old male cadaver for %– % marrow cellularity energy (mev) femoral head*† marrow cellularity (%) humeral epiphysis† marrow cellularity (%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . *values reported for femoral head are averages calculated from different bone samples sectioned from bone site. †all values were calculated using macrostructural transport model. figure . specific absorbed fractions for monoenergetic electrons calculated for %, %, %, %, and % marrow cellularity using macrostructural transport model for femoral head of -y-old man. marrow cellularity in skeletal dosimetry • bolch et al. of �(tam tam) within the humeral and femoral heads of the male subject using our data from d transport sim- ulations. for the -y-old man, the dosimetry at high ener- gies within the humeral epiphysis is shown in table to be only slightly higher than that in the femoral head (ratios of . – . ). at low energies, however, the mean dose per decay in the humeral epiphysis is only three fourths of the dose per decay in the femoral head. in all cases, these ratios are independent of the marrow cellularity and are strictly a function of the relative volumes of spongiosa within the skeletal sites as well as the relative magnitudes of bone and marrow volume fractions (bone trabeculae thinning). the combination of these parameters determines the total mass of active and inactive marrow within the marrow cavities. the ratios of total marrow mass in these bone sites (humeral epiphysis to femoral head) are . for the male subject used in this study. comparisons with published methodologies for electron energies between and kev, the slope of the relationship between absorbed fraction and energy can vary greatly with the marrow cellularity and the marrow volume fraction (figs. and ). consequently, scaling of the tms self-absorbed fractions by a constant reference cellularity does not represent the true energy profile over this energy range. furthermore, the assumption that the tam self-absorbed fraction equals the tms self-absorbed fraction neglects the fact that energy is lost to fat cells in this or at higher electron energies. figure displays the ab- sorbed fraction profiles for a tam source and target in the femoral head of the male subject using published meth- odologies and compares those results with values obtained through direct radiation transport in the digital nmr im- ages. in these comparisons, the radiation transport results use a reference cellularity of . for the femoral head ( ) (fig. , solid line, no data points). two additional values table ratios of specific absorbed fraction �(tam tam) within humeral epiphysis to corresponding value in femoral head of -year-old male cadaver energy (mev) marrow cellularity (%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ��tam tam�humeral epiphysis ��tam tam�femoral head spongiosa volume ratio 	 . . tms mass ratio 	 . . figure . electron absorbed fractions for self-irradiation of tam within femoral head of -y-old man. three dosimetry methodologies are compared. method of bouchet et al. ( , ) assumes that tam self-absorbed fractions are equal to tms self-absorbed fractions, whereas method of eckerman and stabin ( ) scales tms self-absorbed fractions by reference cellularity factor ( . for femoral head). tam self-absorbed fractions reported in this study are calculated directly using macrostructural transport model of egs transport code (solid line, no data points). dashed lines indicate potential variations in absorbed fraction with changes in individual marrow cellularity either lower than that of reference man ( %) or higher than that of reference man ( %). the journal of nuclear medicine • vol. • no. • january (fig. , dashed lines) are shown for % cellularity (lower than average) and % cellularity (higher than average). self-absorbed fractions for the tms are calculated in this work using an infinite trabecular region transport model as representative of the bouchet et al. model. next, this curve is then uniformly scaled by a value of . at all energies to produce absorbed fractions representative of the eckerman and stabin model. eckerman and stabin ( ) only report electron absorbed fractions as averaged across all skeletal sites; consequently, values unique to the femoral head are not available for comparison. still, the above approach, using internally consistent data from the nmr image, fairly compares the methodologies. figure shows that the method used by eckerman and stabin greatly underestimates the absorbed fraction to active marrow for electron energies below kev. in fact, at kev, their method underestimates the tam absorbed frac- tion by as much as % as defined in their scaling approach. however, at electron energies above kev, their method produces results that differ by % from the absorbed fractions calculated in our work, provided that the assumed reference cellularity of % is appropriate to the patient in question. as shown by the dashed lines in figure , the absorbed fraction can vary over a factor of . at high energies for corresponding cellularity variations ranging from % to %. at mev, their method begins to overestimate the tam self-absorbed fractions by a factor of . in that their model does not allow for electron energy escape to the surrounding cortical bone (macrostructural vs. infinite trabecular-region transport). the method of bouchet et al. ( , ) is consistent with the absorbed fractions calculated by direct transport at all marrow cellularities only at extremely low electron ener- gies. at kev, this method overestimates the absorbed fraction at reference cellularity by a factor of . and overestimates by as much as a factor of . at mev. in the energy range of – kev, neither method accurately predicts the fraction of electron energy absorbed within the active tissues of the marrow cavities. in the methods of bouchet et al. ( , ) and eckerman and stabin ( ), absorbed fractions are divided by the active marrow mass in reference man ( . � total marrow mass) to obtain corresponding values of �(tam tam). con- sequently, discrepancies in �(tam tam) shown in figure are reflected in corresponding values of �(tam tam) as shown in figure . as discussed previously, the specific absorbed fraction is shown to be invariant with changes in marrow cellularity at energies of several hundred kiloelectron volts and above. consequently, the method of eckerman and stabin preserves values of mean dose to active marrow per electron emission with changes in mar- row cellularity for high-energy electron sources. however, as the source energy decreases, the model of bouchet et al. provides accurate dosimetry at energies of � kev, pro- vided that the assumed reference cellularity is justified for that particular patient. if the patient’s marrow cellularity is lower than normal ( % in the extreme), the dose per low-energy electron emission is shown to be a factor of . times higher than predicted by bouchet et al. and a factor of times higher than that predicted by the eckerman and stabin model. discussion in the definition of marrow cellularity given by equation , radiation absorbed dose to active marrow would neces- sarily represent the energy per unit mass averaged across not only the hematopoietic stem cells (pleuripotent hema- topoietic stem cell, myeloid stem cell, and lymphoid stem cell) but also across their various hematopoietic progeny, the stromal cells, the ecm, and the support cells of the marrow space. the adequacy of this definition depends on the purpose of the dose estimate. for short-term effects, the above definition may be considered appropriate in that many of the cells in marrow components b– d (histology of figure . electron-specific absorbed fractions for self-irradiation of tam within femoral head of -y-old man. values are obtained by dividing each curve in figure by corresponding mass of active marrow. dashed lines indicate potential variations in specific absorbed fraction with changes in individual marrow cellularity either lower than that of reference man ( %) or higher than that of reference man ( %). marrow cellularity in skeletal dosimetry • bolch et al. marrow tissues section) directly support hematopoiesis. for example, reticulum cells induce proliferation of my- eloid and b-lymphoid progenitor cells ( ), whereas endo- thelial cells play a role in regulating the trafficking and homing of the hematopoietic cells as they reach maturity and are released into circulation ( , ). if, however, one is interested in long-term risks of marrow irradiation, then a direct dose assessment to only the stem cells of marrow component a is desired. unfortunately, the explicit location of the stem cells within the irregular network of the trabec- ular marrow spaces remains highly uncertain, and one must again resort to an assessment of the average dose across all active marrow tissues. in either case, an explicit segmenta- tion of the adipose tissue within the marrow provides a more histologically correct and achievable geometry for active marrow dose assessment. a dosimetric model that makes a distinction among active and inactive marrow can use pa- tient-specific information on marrow cellularity derived ei- ther invasively through marrow biopsies or noninvasively through mri ( , , ). in addition to changes in cellular- ity with the patient’s age, damage to marrow tissues through either disease or chemotherapy may alter marrow cellularity within the skeletal regions of the patient. thus, the presence of marrow cellularity as an independent model parameter permits improved patient specificity in the resulting dose estimates. one must also consider active marrow as a potential source region. as outlined by sgouros ( ) and sgouros et al. ( ), radiochemicals may localize in active marrow either through equilibrating within the extracellular fluid volume of the marrow space (nonspecific uptake) or through bind- ing to cellular marrow components (specific uptake). in either case, a d delineation of the cellular-level locations of these source regions within the marrow cavities is diffi- cult to define in the clinical setting as well as to implement in more macroscopic radiation transport models in which only the tbv and tms are defined through microimaging. the use of equation to partition the tms into its inactive (adipocyte) and active volumes again provides an accept- able solution. the clinical significance of this new approach to skeletal dosimetry may be assessed using the mird schema for determining the mean absorbed dose to the active marrow for radiation emission i: d� tami � ãtam . �i . ��tam tam�i mtms . cf � ãtam . �i . ��tam tam�i mtam , eq. where �i indicates the mean energy per electron emission i within the active marrow tissues. if the active marrow mass term, mtam, in the denominator is positioned under the first term of the numerator, then equation can be rewritten in terms of the mass concentration of cumulated activity in the active marrow [ã]tam: d� tami � ãtam mtam . �i . ��tam tam�i � 
ã�tam . �i . ��tam tam�i. eq. alternatively, one may position the mass term under the absorbed fraction and rewrite equation in terms of specific absorbed fractions: d� tami � ãtam . �i . ��tam tam�i mtam � ãtam . �i . ��tam tam�i. eq. for nonspecific uptake in skeletal tissues, the radiophar- maceutical equilibrates quickly within the marrow plasma and the extracellular fluid volume regions, which are both assigned to active marrow in the present model. here, the total cumulated activity is proportional to the mass of active marrow, and thus [ã ]tam remains constant (for a given administered activity a ) and is independent of marrow cellularity. according to equation , the mean absorbed dose to active marrow is directly proportional to the absorbed fraction for marrow self-irradiation. figure shows that for low-energy electron sources, values of �(tam tam) are fairly independent of cellularity, and thus patient-specific assessments of marrow cellular- ity are not needed for marrow dose estimates (reference values will suffice). if, however, high-energy electron sources are used, the active marrow dose becomes very much a function of the patient marrow cellularity. efforts to assess marrow cellularity in the patient are then war- ranted. for specific binding to marrow components, as for the case of radiolabeled antibodies, the importance of knowing the patient’s marrow cellularity in providing an accurate dose estimate depends on whether the binding sites have been saturated. for uptake in active marrow below satura- tion, the cumulated activity in active marrow, ãtam, will be fixed for a given value of a . according to equation , the mean dose to active marrow in this case is propor- tional to the specific absorbed fraction for marrow self- irradiation. furthermore, figure reveals that values of �(tam tam) are strong functions of marrow cellu- larity only for low-energy electron sources. consequent- ly, patient-specific assessments of marrow cellularity, with corresponding corrections to the specific absorbed fraction (and thus the radionuclide s value), should be made for specific uptake of low-energy emitters below binding saturation. if the antibody has reached binding saturation, the value of [ã]tam once again remains con- stant for a given value of a , and the conclusions drawn above for nonspecific uptake then apply. the journal of nuclear medicine • vol. • no. • january conclusion this study extends previous work in nmr microscopy– based skeletal dosimetry in which image voxels delineating the marrow tissues are further partitioned into regions of active and inactive marrow, with the latter defined as clus- ters of adipocytes. absorbed fractions are then calculated within these images for active marrow electron sources and for marrow cellularities ranging from % to % (fat fractions from %– %), a range that corresponds to values potentially seen in patient populations ( ). transport cal- culations are based on d nmr microscopy images from trabecular bone samples acquired from the femoral head and humeral epiphysis of a -y-old male subject. the absorbed fraction profiles are then compared between bone sites in this individual and against data from published transport methodologies. the d electron-transport simulations indicate that the eckerman and stabin model used in mirdose accurately describes specific absorbed fractions for self-irradiation of the active marrow for source energies exceeding kev. however, this model substantially underestimates the dose per electron emission at energies below several tens of kiloelectron volts. below kev, the model of bouchet et al. accurately portrays the specific absorbed fraction for self-irradiation of active marrow, provided that the refer- ence cellularity selected for reference man is accurate for the patient in question. if the patient marrow cellularity differs from the reference value of %, neither model is particularly accurate at electron energies below kev. radionuclide s values cannot be compared in this study because reference active marrow masses are given only for the upper half of the femur and the humerus in the bouchet et al. and eckerman and stabin models. consequently, additional nmr imaging of the femoral and humeral necks is required, as well as an expansion of the macrostructural transport model, before comparable tissue masses and ra- dionuclide s values would be available from d transport simulations. patient-specific information on marrow cellularity is clearly an important parameter that might influence the estimates of cumulated activity in active marrow and the magnitude of the radionuclide s value. simple mass scaling of the reference man radionuclide s value is not sufficient for total patient specificity because marrow cellularity should also be considered as an independent model param- eter in the assignment of absorbed fractions and radionu- clide s values. additional factors to consider are the overall size of the skeletal site relative to that in reference man and the bone volume fraction within the spongiosa. this work indicates that d microimaging of trabecular bone, as done through nmr microscopy or other methods, can be a valu- able tool in efforts to expand the existing database of reference man s values for skeletal dosimetry in ways that will explicitly consider these important factors. acknowledgments this work was supported in part by u.s. department of energy nuclear engineering education research grant de-fg -id with the university of florida. references . sgouros g, stabin m, erdi y, et al. red marrow dosimetry for radiolabeled antibodies that bind to marrow, bone, or blood components. med phys. ; : – . . gatter k, brown d. an illustrated guide to bone marrow diagnosis. malden, ma: blackwell science ltd.; . . naeim f. pathology of bone marrow. baltimore, md: williams & wilkins; . . verfaillie c, hurley r, bhatia r, mccarthy jb. role of bone marrow matrix in normal and abnormal 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a spouse in the amish: a population-based retrospective cohort study ari seifter, sarabdeep singh, patrick f mcardle, kathleen a ryan, alan r shuldiner, , braxton d mitchell, , alejandro a schäffer to cite: seifter a, singh s, mcardle pf, et al. analysis of the bereavement effect after the death of a spouse in the amish: a population-based retrospective cohort study. bmj open ; :e . doi: . /bmjopen- - ▸ prepublication history and additional material for this paper is available online. to view these files please visit the journal online (http://dx.doi.org/ . / bmjopen- - ). as and ss contributed equally. received july revised december accepted december for numbered affiliations see end of article. correspondence to dr alejandro a schäffer; schaffer@helix.nih.gov abstract objective: this study investigates the association between bereavement and the mortality of a surviving spouse among amish couples. we hypothesised that the bereavement effect would be relatively small in the amish due to the unusually cohesive social structure of the amish that might attenuate the loss of spousal support. design: population-based cohort study. setting: the usa. participants: amish couples born during – located in pennsylvania, ohio and indiana. all the participants are deceased. outcome measures: the survival time is ‘age’; event is ‘death’. hazard ratios (hrs) of widowed individuals with respect to gender, age at widowhood, remarriage, the number of surviving children and time since bereavement. results: we observed hrs for widowhood ranging from . to . over the study period (nearly all differences significant at p< . ). mortality risks tended to be higher in men than in women and in younger compared with older bereaved spouses. there were significantly increased mortality risks in widows and widowers who did not remarry. we observed a higher number of surviving children to be associated with increased mortality in men and women. mortality risk following bereavement was higher in the first months among men and women. conclusions: we conclude that bereavement effects remain apparent even in this socially cohesive amish community. remarriage is associated with a significant decrease in the mortality risk among amish individuals. contrary to results from previous studies, an increase in the number of surviving children was associated with decreased survival rate. introduction it has been consistently established that widowed individuals exhibit an increased mortality compared with married indivi- duals. – this excess mortality risk, referred to as the ‘bereavement effect,’ is strongest in the first few years following widowhood, among men who outlive their wives and among younger widows/widowers. factors proposed to explain this phenomenon include the acute grief and stress of bereave- ment, shared environmental risk factors between spouses, marital selection, economic hardship and loss of social support. these influences are difficult to isolate and independently assess. several studies have shown that a larger social network size and a greater level of social support are gen- erally associated with lower rates of mortality and morbidity. in a study based on the utah population database, mortality risk among widowers decreased with membership in the church of latter day saints (lds, a.k.a. mormon church) and with increasing numbers of children. these factors were interpreted as proxies for social support. in addition, a study in washington county, maryland, indicated that living alone was a risk factor for increased mortality in widowed populations. loss of social support following the death of a spouse is generally more pro- found in widowers than in widows, but there is currently no empirical evidence that this strengths and limitations of this study ▪ owing to the availability of remarriage status of the surviving spouses, we could reproduce and quantify the decreased bereavement effect asso- ciated with remarriage. ▪ owing to the unavailability of data on causes of death, it was not possible to determine the rela- tionship between differential measures of social support and certain causes of death. ▪ the number of surviving children was simplified to be a time-independent covariate although it may change between the death of the first parent and the death of the second parent. seifter a, singh s, mcardle pf, et al. bmj open ; :e . doi: . /bmjopen- - open access research o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n ja n u a ry . d o w n lo a d e d fro m http://dx.doi.org/ . /bmjopen- - http://dx.doi.org/ . /bmjopen- - http://crossmark.crossref.org/dialog/?doi= . /bmjopen- - &domain=pdf&date_stamp= - - http://bmjopen.bmj.com/ difference mediates the higher mortality consistently observed among widowed husbands compared with widowed wives. the amish maintain a cultural identity distinct from mainstream american culture that is characterised by traditional dress, a plain lifestyle and non-adoption of modern technology (eg, electricity, cars and tele- phones), german dialect, separate school system and ultra-conservative anabaptist religious practices. a central tenet of amish culture throughout its history in the usa has been social cohesiveness with emphasis on family and community. members of this tight-knit society have extraordinary social support from cradle to grave, including community financing of medical costs and support during times of need. the community financing of medical costs became increasingly forma- lised during the th century to the point where it is now a formal system of self-insurance called ‘amish aid’. to gain a different perspective on potential influences of social support on bereavement, we assessed the bereavement effect in an amish population, whose culture is characterised by its core beliefs in community and social cohesion. examples of the strong degree of social support within the amish include community- managed health insurance, community ties through membership in the church, and the sharing of ultracon- servative cultural beliefs and lifestyles. we reasoned that the high level of social support in the amish popu- lation might mitigate the bereavement effect. previous studies used covariates such as education (homogeneous in the amish), health habits, remarriage, church visits, neighbourhood interaction and household size to study the relationship between bereavement and social support. we estimated bereavement effects in widows and widowers separately and further assessed the mortality risks as a function of age at widowhood and time since bereavement (tsb). we used cox proportional hazard (cph) models to analyse the association of bereavement and mortality of widowed husbands and wives. we considered remarriage and the number of sur- viving children as additional potential modifiers of the bereavement effect. material and methods data source the anabaptist genealogy database (agdb) v. was used to study the effect of bereavement on the amish population. the agdb is a computerised genealogy database of the amish and some mennonite popula- tions, last updated in . this database includes infor- mation about the family relationships and the birth, marriage and death dates of children of amish indivi- duals from north america, mostly located in pennsylvania, ohio and indiana. the ‘individual table’ of agdb contains information about indivi- duals. the ‘relationship table’ includes information figure a flow diagram representing all the steps performed for filtering couples from a total of couples available in anabaptist genealogy database (agdb). in the flow diagram, each couple is counted as excluded only once, even if multiple exclusion criteria apply. ‘unknown spouse’ refers to entries in the agdb relationship table in which at least one parent is unknown; almost all of these entries are for adopted children for whom at least one of the biological parents is unknown. as agdb is used primarily in genetic studies (unlike this study), the distinction between biological and adoptive relationships is stored. ‘birth year too late’ means that the birth year of the husband or wife is known and is > . ‘dates not recognised by r’ are invalid dates such as the june, which got into agdb due to errors in the original sources. ‘implausible birth or death dates’ refer to a few individuals who are shown as married but have lifespans of less than years likely due to typos in the birth year in the original sources. seifter a, singh s, mcardle pf, et al. bmj open ; :e . doi: . /bmjopen- - open access o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n ja n u a ry . d o w n lo a d e d fro m http://bmjopen.bmj.com/ about amish couples. an individual who is married multiple times participates in multiple relation- ship table entries. there are relationship entries among the entries concerning children for whom one or both biological parents are unknown (figure ). the amish people rarely give birth out of wedlock, and a vast majority of the mating samples we used are documented as marriages in the three original sources from which agdb is derived. however, there is less avail- ability of marriage dates than birth dates; we did not wish to exclude couples with missing marriage dates. for this study, we included couples in which both spouses met four conditions: ( ) they were born prior to january ; ( ) their birth and death dates were recorded; ( ) they were in their first marriage and ( ) they did not die on the same day. constraint ( ) excludes couples who may have died from a shared dis- aster (eg, accident). a total of couples met the inclusion criteria. the oldest person included was born on february , and the youngest on december . see figure for how the exclusion criteria were applied. our dataset included couples with information on their dates of birth, dates of death, the number of surviving children, family ids and remarriage status after widowhood. for some analyses, these couples were first partitioned into three cohorts based on the husband’s birth year (prior to and – ) and based on husband’s and wife’s birth year ( – ) and sex. for the tsb analyses, we did not partition into cohorts because the number of widow(er)s who died within months after being bereaved is small (n= ). the characteristics of these couples are shown in table . statistical analyses similar to several past analyses, we used the cph model where the response variable or survival time is ‘age at death’ and the event is ‘death’. the model is used to study the association of widowhood and mortality rates in the surviving spouse, while adjusting for covariates such as education, health habits, age in years, the number of children and remarriage. in some of our analyses, we adjusted for remarriage and the number of children as covariates; we did not adjust for education or health habits. husbands and wives were always analysed separately. the main covariate, widowhood, was monitored as a time-dependent covariate, by assigning a value of for each time period the individual was widowed, and otherwise. the remarriage covariate was also a time- dependent covariate, by assigning a value for the widowed period if the individual remarried, and other- wise. the number of surviving children was a time- independent covariate and counted at the beginning of the widowhood period of the surviving spouse. the cov- ariate was divided into three categories as follows: =number of surviving children is ≤ , =number of sur- viving children is between and and =number of sur- viving children is > . the categories ≤ children, – children and > children are separate. these boundaries were chosen to ensure categories that were roughly balanced in size. to evaluate any possible bias by count- ing the number of surviving children at the death of the first spouse, we also estimated how many couples changed categories between the death of the first spouse and the death of the second spouse due to death of one or more children during the widowhood period. survival analyses were performed using the r pro- gramming environment. all estimates and cis were obtained using the coxph function available in the survival package. representation of survival data for cph analysis we show below examples of how we represented the sur- vival data for cph analysis using a time modelling approach. to represent the survival data columns, the standard approach is to convert the couple’s demo- graphic information, date of death, date of birth, remar- riage and the number of surviving children in columns table characteristics of spouse pairs according to birth cohort of husband cohorts pre- – – number of couples number (%) of wives out-surviving their husband ( . ) ( . ) ( . ) number (%) of husbands out-surviving their wife ( . ) ( . ) ( . ) mean husband age at widowhood . . . mean wife age at widowhood . . . mean widowed husband survival in years . . . mean widowed wife survival in years . . . mean husband age at death . . . mean wife age at death . . . mean age difference husband-wife . . . mean number of children . . . number (%) of widowed husbands remarried ( . ) ( . ) ( . ) number (%) of widowed wives remarried ( . ) ( . ) ( . ) seifter a, singh s, mcardle pf, et al. bmj open ; :e . doi: . /bmjopen- - open access o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n ja n u a ry . d o w n lo a d e d fro m http://bmjopen.bmj.com/ representing start time, stop time, event status and all of the included covariates (widowhood, remarriage and the number of surviving children). here (start, stop) is an interval of risk, open on the left and closed on the right; the event column is set to if the participant had an event (death) at time stop, and is otherwise. to illus- trate the subtleties, we consider a hypothetical example of three wives (id , id and id ) from the cohort to . ▸ id : born on january ; widowed on january at age ; got remarried; the number of surviv- ing children= and eventually died on january at age . ▸ id : born on january ; widowed on january at age ; never got remarried; the number of surviving children= and died on january at age . ▸ id : born on january ; never widowed and never remarried; the number of surviving children= and died on january at age . the data used for the cph considering widowhood as a single time-dependent covariate is presented in table . the following model was used to estimate the association between widowhood and mortality. hðtÞ ¼ h ðtÞ exp½bw � this model can be run with additional covariates and results that adjust for the ability to remarry and for the number of surviving children presented. timing of widowhood the above model considers widowhood as a single time- dependent covariate, evaluating the association between mortality and being widowed. one can further ask whether that association varies over the life course by con- sidering the impact of age at widowhood on mortality. we addressed this issue following the approach of mineau et al by adding terms for age at widowhood into the model as covariates (< , – , – , – and > ) to allow the hr to vary according to age at widowhood (see table ). these terms were included in the model as time- dependent dummy variables, created to represent the widowhood experience of each individual across the five age windows spanning the individual’s age: w< , w – , w – , w – and w +. furthermore, we also addressed the impact of remarriage and the number of surviving children on the mortality of amish individuals. the following model can be used to estimate the asso- ciation between each of the five dummy variables and mortality: hðtÞ ¼ h ðtÞ exp h x bjwj þ b r þ b c i with j ¼ ; . . . ; where w ,…,w dummy variables are associated with w< , w – , w – , w – , w + columns provided in table . time since bereavement next, we evaluated the association between mortality and tsb or widowhood. we followed the approach of schaefer et al by considering the following tsb ranges: – , – , – , – , – , – and > months. this approach allowed us to estimate hr according to tsb (see table ). the columns tsb – , tsb – , etc in table are time-dependent covariates that change with the survival time associated with widowed husbands and wives. we did not account for remarriage in this analysis because of missing remarriage dates. the following model was used to study the association between mortality and tsb: hðtÞ ¼ h ðtÞ exp h x bjwj i with j ¼ ; . . . ; where w ,…,w dummy variables are associated with the tsb – , tsb – , tsb – , tsb – , tsb – , tsb – and tsb> columns provided in table . results we initially partitioned the couples from agdb into three cohorts ranging in size from (husband’s birth year – ) to (husband’s birth year – ). wives were more likely to die after their husbands in all three cohorts while husbands had a higher mean age at death than their wives. the average age differences between the husbands and wives for all cohorts are shown in table . the number of remarried wives was far smaller than the number of remarried hus- bands (n= widowed husbands vs widowed wives). the number and proportion of remarried hus- bands and wives were increased in the more contempor- ary cohorts (see table ). in contrast to other populations from the th and early th centuries, the majority of widowed husbands did not remarry, making it interesting to study the effect of remarriage in the amish population. hrs the overall hrs, and their % cis, associated with widow- hood are displayed in figure according to birth cohort. table data structure for cox proportional hazard model that does not estimate the effect for different ages, but instead estimates only widowed versus non-widowed id start stop event w r c (age at widowhood) (age at death) (age at widowhood) (age at death) (age at death) c, number of surviving children; r, remarriage. seifter a, singh s, mcardle pf, et al. bmj open ; :e . doi: . /bmjopen- - open access o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n ja n u a ry . d o w n lo a d e d fro m http://bmjopen.bmj.com/ the significance of these hrs is indicated with the help of p values (*p< . and **p< . ) on the top of each block in figure . these hrs were estimated using the cph model and design provided in table . as expected, widowhood was associated with an increased mortality for husbands and wives following the death of their spouse, with hrs ranging from . to . (all hrs significantly greater than except for widowed husbands in cohort – ). the impact of widowhood was disproportion- ately greater on surviving husbands than on surviving wives in the pre- and – cohorts, although there was little difference in mortality between widowed hus- bands versus wives in the – . there was no clear trend showing that the hrs changed across cohorts in either surviving husbands or wives. figure shows the hrs along with % cis, and range of p values for the model in which age at widow- hood is accounted for (design model in table ). these results reveal markedly higher bereavement effects in each age at widowhood group compared with the results shown in figure , consistent with the notion that the bereavement effect may be diminished over time and most pronounced in the years proximal to the death of the spouse. in nearly all age at widowhood cat- egories, the bereavement effect is stronger in widowed husbands than in widowed wives. the number of children surviving when the first spouse died (with pairwise comparisons – vs ≤ ; > vs ≤ ) was included as a time-independent covariate in the models whose results are shown in figure . in general, there was a very weak association between the number of surviving children and mortality in husbands and wives. contrary to our expectations and a prior study, in each case, the higher number of surviving chil- dren was not significantly associated with lower mortality in husbands and wives (see figure ). furthermore, the results in figure show that the effect of bereavement decreases if the amish individual remarries. there are two potential sources of bias in analyses involving the number of surviving children covariate. couples in which one spouse died at age < are likely not to have as many children as couples in which both parents survived to at least age . to quantify this bias, we repeated the analysis in figure by excluding all the couples who got widowed before age (data not shown). the results show that there is no significant table data structure for cox proportional hazard model that estimates the association between widowhood at given ages and mortality id start stop event w< w – w – w – w + r c c, number of surviving children; r, remarriage. table data structure for cox proportional hazard model that estimates the association between widowhood with respect to time since bereavement and mortality id start stop event tsb – tsb – tsb – tsb – tsb – tsb – tsb> id id . id . id id id id id id id . id . id id id id id id start and stop columns are in years and tsb columns are in months. tsb, time since bereavement. seifter a, singh s, mcardle pf, et al. bmj open ; :e . doi: . /bmjopen- - open access o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n ja n u a ry . d o w n lo a d e d fro m http://bmjopen.bmj.com/ change in the hrs versus figure . the maximum change for any of the hrs related to the number of sur- viving children was . (data not shown). on the other end of the age spectrum, there is a potential source of bias as more children may die, the longer the surviving parent lives. as the number of chil- dren was divided into three categories ≤ , – and > , any possible bias could arise only when the death of a child shifted the number of surviving children from a higher category to a lower category. over all couples, the number of widows or widowers whose number of surviv- ing children changed to a lower category from the date of widowhood to the date of death was only (< % of all couples). when the analyses corresponding to figure were repeated excluding these couples, the hrs were essentially unchanged. the maximum change was . ; sometimes the hr increased slightly and sometimes it decreased slightly (data not shown). hrs and % cis for the tsb analysis are shown in figure . the significant p values are indicated in figure . these results were obtained using the cph model and the design is defined in table . the results show that there is a high risk of mortality for recently widowed husbands and figure hrs of widowed husbands and wives versus their married counterparts (design provided in table ); * and ** on top of the blocks representing the significance of hrs with p< . and p< . , respectively. figure hrs of widowed husbands and wives versus their married counterparts according to time since bereavement (months; design provided in table ); * and ** on the top of the blocks represent the significance of hrs with p< . and p< . , respectively. figure hrs of widowed husbands and wives versus their married counterparts according to age at widowhood (design provided in table ); nsc : number of surviving children ( – vs ≤ ); nsc : number of surviving children (> vs ≤ ); * and ** on top of the blocks representing the significance of hrs with p< . and p< . , respectively. seifter a, singh s, mcardle pf, et al. bmj open ; :e . doi: . /bmjopen- - open access o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n ja n u a ry . d o w n lo a d e d fro m http://bmjopen.bmj.com/ wives. furthermore, the hazard is higher (not significant) in wives versus husbands during the first months follow- ing bereavement. the range of p values provided in figures – indicates the significance of each of the hrs. for example, in figure , the p value= . associated with the hr= . (men; tsb range < months) strongly indicates that the hr is signifi- cantly > . similarly, the p value= . associated with the hr= . (men; tsb – months) weakly indicates the sig- nificance of the hr > . graphical checks of the overall adequacy of the cph models were performed. based on the cox-snell residuals plot, the final model gave a reasonable fit to the data (data not shown). the deviance residual plots revealed no obvious outliers in the data (data not shown). furthermore, the wald test statistic was used to test the fit of the final model, and according to this test statistic, the final model fits the data reasonably well (see online supplementary tables s –s ). discussion this is the first comprehensive study to evaluate the rela- tionship between bereavement and social support in the amish population. this study provides evidence that amish widows and widowers have an increased mortality risk compared with married cohort members. although it is difficult to determine whether this effect is of equivalent magnitude as that observed in studies of other popula- tions, the most recent studies of the bereavement effect using the cph suggest that our findings are generally con- sistent with data from other populations. several previous studies on american, european and middle eastern populations have found that mortality is magnified in individuals widowed at a younger age and that widowers have a higher mortality risk than widows. the lds study is closest to our study because of the large family sizes and the population selected as a religious isolate. the effects of bereavement on mortality with respect to gender and the age at widow- hood ranges observed in the amish are also largely consist- ent with those observed in the lds population, and other populations such as finland and israel (figure ). as expected, widowers showed higher hrs than widows, except for the – cohort (figure ). in the present study, the association between bereave- ment and mortality is greater in the first months for men and women (figure ), consistent with previous findings. the mortality risks in the first months are lower in the amish (figure ) compared with some, but not all, studies. one common pattern observed in this and other studies is that the bereavement effect is higher in the first months and later life. we did a regression analysis for trend in the data of figure and there is no significant declining trend after the first months. we speculate that the higher mortality during the first months might reflect acute effects related to the loss of a spouse, while the higher mortality in later life might reflect decreased sur- vival from ageing-related diseases that is unmasked in the absence of spousal support. one strength of our study is that we could evaluate the effect of remarriage because there were a substantial number of widowed individuals who did and did not get remarried (table ). shor et al noted the difficulty in studying the effect of remarriage in populations where all individuals are deceased because “in previous decades, widowed men almost always remarried”. as suggested by previous studies, remarriage after the death of a spouse significantly influences the bereavement effect because it is associated with an increased survival of men and women across all cohorts. this association is likely influenced by the fact that men or women who get remarried are in suffi- ciently good physical and mental health to do so. furthermore, the survival rate is higher for men compared with women, possibly reflecting support in the amish society for men getting remarried. interestingly, more children at the time of the death of the first spouse was associated with an increased risk of death, though the hr for having > surviving chil- dren compared with ≤ was not significantly greater than . . this result does not support the hypothesis that more surviving children confer a survival advantage to parental longevity, as perhaps by providing social support for their parents. spouses in the amish society may also provide a unique emotional, psychological and social support to each other which cannot be provided by their surviving children. the lack of protective associ- ation was similarly observed when the number of surviv- ing children was considered as a linear or as a categorical variable (data not shown). this contrasts with the data from the utah population database, in which increasing numbers of children were associated with a decreased hr. we considered the number of sur- viving children as a separate term (table ), but did not evaluate the interaction of the number of surviving chil- dren with widowhood. the implication of these findings is that differential familial support following bereavement does not appear to be the key factor affecting mortality increases in widowed amish populations. one potential explanation is that spouses, as ‘attachment figures,’ provide a unique, irreplaceable social support which must be con- sidered independently from other ancillary support pro- viders, such as children. data suggesting that relative mortality risk is also elevated in divorced and never- married populations may support this hypothesis. in addition, quality of social support has significantly greater effects on well-being than quantity of support in elderly populations of both sexes. in adults aged and older, lower reported social support has been associated with decreased life satisfac- tion and increased depressive symptoms. family members and close friends should be especially vigilant during the sensitive acute period following the loss of a spouse, when relative mortality risk is the highest. seifter a, singh s, mcardle pf, et al. bmj open ; :e . doi: . /bmjopen- - open access o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n ja n u a ry . d o w n lo a d e d fro m http://bmjopen.bmj.com/ there are two limitations of our data. first, the number of surviving children covariate was simplified because it was treated as a time-independent covariate, although some children die before their parents, so it could be considered as time-dependent instead. second, the causes of death were unavailable. future research is needed to study the effect of the loss of a child on the mortality of husbands and wives by considering the number of surviving children as a time-dependent cov- ariate. also, research is needed to determine whether differential measures of social support may be associated with certain causes of death in widowed populations. divorce in the amish population is sufficiently rare that this was not a major potential source of error. our study results indicate that remarriage plays an important role in improving the survival rate of men and women in the amish population. contrary to the results from previous studies, an increase in the number of surviving children was associated with a decreased sur- vival rate of amish individuals. author affiliations department of medicine and program for personalized and genomic medicine, university of maryland school of medicine, baltimore, maryland, usa national center for biotechnology information, national library of medicine, national institutes of health, dhhs, bethesda, maryland, usa geriatric research and education clinical center, veterans administration medical center, baltimore, maryland, usa acknowledgements the authors would like to thank two reviewers for excellent suggestions that led us to improve the manuscript. contributors bdm conceived the study. as and ss did the research and data analysis, with substantial guidance from pfm, bdm and aas. as and ss wrote the manuscript, with substantial editing by pfm, bdm and aas. aas managed the project with assistance from bdm. aas manages the anabaptist genealogy database (agdb) project at the national institutes of health and kar managed the agdb data at the university of maryland medical school during the period of this project. ars provided resources and advice. all authors proofread and approved the manuscript. funding this work was supported by research grants r hl , r dk , r ag , r hl , r ar and u hl from the national institutes of health. this research was supported in part by the intramural research programme of the national institutes of health, nlm and the geriatric research and education center, baltimore veterans administration medical center. competing interests none. ethics approval the construction and maintenance of anabaptist genealogy database (agdb) are covered under an irb-approved human subjects protocol at the national institute of health (nih), leslie biesecker, principal investigator. national human genome research institute institutional review board at the national institutes of health. provenance and peer review not commissioned; externally peer reviewed. data sharing statement data from the anabaptist genealogy database (agdb) are available to investigators (including ars and various others not participating in this study) who have an irb-approved protocol to study the amish or other anabaptist groups. investigators can request access to agdb by writing to aas and to dr biesecker. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. see: http:// creativecommons.org/licenses/by-nc/ . / references . jagger c, sutton cj. death after marital bereavement—is the risk increased. stat med ; : – . . schaefer c, quesenberry c, wi s. mortality following conjugal bereavement and the effects of a shared environment. am j epidemiol ; : – . . johnson nj, backlund e, sorlie pd, et al. marital status and mortality: the national longitudinal mortality study. ann epidemiol ; : – . . shor e, roelfs dj, curreli m, et 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springer, . . tableman m, kim js. survival analysis using s: analysis of time-to-event data. st edn. new york, ny: texts in statistical science, chapman and hall/crc, . . lichtenstein p, gatz m, berg s. a twin study of mortality after spousal bereavement. psychol med ; : – . . mendes de leon cf, kasl sv, jacobs s. widowhood and mortality risk in a community sample of the elderly: a prospective study. j clin epidemiol ; : – . . peritz e, bialik o, groen jj. mortality by marital status in the jewish population of israel. j chronic dis ; : – . . hart cl, hole dj, lawlor da, et al. effect of conjugal bereavement on mortality of the bereaved spouse in participants of the renfrew/ paisley study. j epidemiol community health ; : – . . diamond lm. contributions of psychophysiology to research on adult attachment: review and recommendations. pers soc psychol rev ; : – . . antonucci tc, akiyama h. an examination of sex differences in social support among older men and women. sex roles ; : – . . newsom jt, schulz r. social support as a mediator in the relation between functional status and quality of life in older adults. psychol aging ; : – . seifter a, singh s, mcardle pf, et al. bmj open ; :e . doi: . /bmjopen- - open access o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n ja n u a ry . d o w n lo a d e d fro m http://creativecommons.org/licenses/by-nc/ . / http://creativecommons.org/licenses/by-nc/ . / http://www.r-project.org/ http://www.r-project.org/ http://bmjopen.bmj.com/ basic science investigations adipocyte spatial distributions in bone marrow: implications for skeletal dosimetry models amish p. shah, ms ; phillip w. patton, phd ; didier a. rajon, ms ; and wesley e. bolch, phd , department of biomedical engineering, university of florida, gainesville, florida; department of health physics, university of nevada–las vegas, las vegas, nevada; and department of nuclear and radiological engineering, university of florida, gainesville, florida few studies have been conducted to quantify the spatial distributions of adipocytes in the marrow cavities of trabec- ular bone. nevertheless, such data are needed for the devel- opment of -dimensional ( d) voxel skeletal models where marrow cellularity is explicitly considered as a model param- eter for dose assessment. in this investigation, bone marrow biopsies of the anterior iliac crest were examined to deter- mine the size distribution of adipocyte cell clusters, the per- centage of perimeter coverage of trabecular surfaces, and the presence or absence of adipocyte density gradients in the marrow space, all as a function of the biopsy marrow cellu- larity ( %– %). methods: biopsy slides from patients were selected as designated by the hematopathologist as either normocellular or with no evidence of disease. still- frame video image captures were made of – regions of interest per biopsy specimen, with subsequent image analy- sis of adipocyte spatial characteristics performed via a user- written matlab routine. results: a predictable shift was found in cluster size with decreasing marrow cellularity from single adipocytes to clusters of � cells; the percentage of -cell clusters remained relatively constant with changing cellularity. also, a nonlinear increase in trabeculae perimeter coverage was found with increasing fat tissue fraction at marrow cellularities between % and %. finally, it was demonstrated that only in the range of %– % marrow cellularity was a slight gradient in adipocyte concentration indicated with adipocytes localized preferentially toward the trabecular surfaces. conclusion: electron transport simula- tions were conducted in different d voxel models of tra- becular bone for sources localized in the active marrow (tam), bone volume (tbv), bone endosteum (tbe), and bone surfaces (tbs). voxel model simulations demonstrated that absorbed fractions to active marrow given by the icrp model (mirdose ) are exceedingly conservative for both tbv and tbs sources, except in the case of high-energy particles (� kev) at high values of marrow cellularity (� %). values of both �(tam tbv) and �(tam tbs) given by the eckerman and stabin model (mirdose ) were shown to be reasonably consistent with d voxel model simulations at the reference cellularity of %, except in the case of low-energy emitters (� kev) on the bone surfaces. key words: active (red) marrow; adipocyte; skeletal dosimetry; nuclear magnetic resonance microscopy; marrow dosimetry j nucl med ; : – the need for improved patient specificity of skeletal dosimetry models is widely recognized in the field of ra- dionuclide therapy. patient specificity in the absorbed dose to active (red) marrow requires separate assessments of the amount of radioactivity taken up within skeletal tissues of the patient and the absorbed dose per particle emission (s value), considering the patient’s internal skeletal structure (normal, osteopenic, or osteoporotic trabecular lattice), ex- ternal skeletal structure (size of the spongiosa and bone cortex), and marrow composition (cellularity). extensive efforts have been made in patient-specific assessments of activity uptake in active marrow, including estimation of marrow activity concentration as a function of the periph- eral blood concentration ( ) or via direct imaging of skeletal regions with high active-marrow content ( – ). little re- search, however, has been focused on improving the patient specificity of radionuclide s values. considerations of patient marrow cellularity may be an important factor in improving correlations between marrow dose and hematopoietic toxicity. to this end, chemical-shift mri techniques have been proposed whereby marrow cel- lularity can be assessed in vivo ( , ). skeletal dosimetry models can then be applied in which marrow cellularity is not simply a fixed constant per skeletal site but is permitted to vary continuously as measured in the patient, allowing for potential changes due to damage from prior chemotherapy or other treatments ( ). although individual patient mea- surements of marrow cellularity would be ideal, economic and practical constraints might limit this approach. in these cases, clinical implementation of mr-based cellularity ad- justments of s values could be based on standard cellularity values representing mean values for different patient and received aug. , ; revision accepted dec. , . for correspondence or reprints contact: wesley e. bolch, phd, department of nuclear and radiological engineering, university of florida, gainesville, fl - . e-mail: wbolch@ufl.edu the journal of nuclear medicine • vol. • no. • may population groups (e.g., those receiving prior chemother- apy, women in their s, and so forth). rozman et al. ( ) conducted one of the first comprehen- sive studies of human marrow adipocytes. in that study, biopsy specimens taken from the anterior iliac crest were examined using stereologic methods. adipocyte diameters were found to be normally distributed with mean diameters that increased linearly with increasing marrow fat fraction: � -�m diameter at % fat fraction (cellularity, %) and � -�m diameter at % fat fraction (cellularity, %). increases in marrow fat fraction were found to depend on both increases in the size of individual adipocytes and increases in the number of adipocytes within the marrow cavities. adipocyte size accounted for . % of the fat tissue fraction, whereas both size and cell number explained . % of the fat tissue fraction. the group found similar results for biopsies of abnormal marrow ( ). allen et al. ( ) later confirmed many of the observations made by rozman et al. ( , ) through observations of marrow in human ribs. these authors, however, found that the adipocyte diameters were better characterized as pro- portional to the cube root of the fat tissue fraction, thus predicting mean diameters of , , and �m at fat tissue fractions of %, %, and %, respectively. additionally, these authors quantified frequency distributions of distances between individual adipocytes and the nearest trabecular bone surface. although many observations were consistent with a uniform distribution of adipocytes in the marrow cavities, some specimens displayed a propensity for adipo- cyte localization closer to the trabecular surfaces, with lower concentrations interior to the marrow cavities. this formation of a “first fat layer” ( % coverage of bone surfaces by definition) can have important consequences for active marrow dose assessments by low-energy particle emissions either within the volume or on the surfaces of the bone trabeculae. the purpose of this study was to provide the experimental data needed to implement histologically realistic distribu- tions of marrow adipocytes within -dimensional ( d) nu- clear magnetic resonance (nmr)-based voxel models of trabecular bone. in these models, image segmentation tech- niques are used to separate bone voxels from marrow space voxels in the d skeletal image. the marrow voxels are then further differentiated into voxels of active (red) marrow and inactive (yellow) marrow, the latter representing adipocytes and their cell clusters. specific investigations of this study include the characterization of adipocyte cell cluster sizes, the percentage of trabecular bone surfaces (tbs) covered by adipocytes, and the changes in local marrow cellularity as a function of distance interior to the marrow cavity. materials and methods selection of bone marrow biopsy specimens bone marrow biopsy slides, archived from earlier examinations of the anterior iliac crest, were obtained from the hematopathol- ogy section, department of pathology, at the university of florida shands hospital. selection criteria were established to identify specimens representing normal spatial distributions of adipocytes within their marrow cavities. biopsies designated by the hemato- pathologist as either normocellular or with no evidence of disease were entered into the study. the former designation indicates biopsies that fall within accepted age-dependent ranges of cellu- larity for disease-free individuals. the latter designation excluded specimens showing either inadequate cell maturation or displaying abnormalities such as tumors, myelodysplasia, neoplasia, or lym- phoma. biopsies with normal pathology, but which contained some areas of hypercellularity or hypocellularity, were also in- cluded. the study population included patients in both diag- nostic categories: females with normocellular marrow (age, – y), females with no evidence of disease (age, – y), males with normocellular marrow (age, – y), and males with no evidence of disease (age, – y). image acquisition and processing image capture was conducted using a digital photo camera (model dkc- ; sony corp., san diego, ca) attached to an eclipse e optical microscope (nikon, melville, ny) in the university of florida hematopathology laboratory. each biopsy sample was first examined for regions of interest (rois) that included clear delineations of active marrow, adipose tissue, and trabecular bone. areas of exclusion included regions of blood spillage, incomplete or irregular biopsies, or abundance of trabec- ular or cortical bone either torn or grouped from the extraction process. once acceptable rois were identified, the images were still-framed, recorded, and saved in jpeg format for subsequent use with a user-written matlab (the mathworks, inc., natick, ma) routine for spatial measurements (adipocyte cluster sizes, trabeculae perimeters, and so forth). distance measurements were calibrated against reference markings on a stage micrometer (fish- er scientific, pittsburgh, pa) at equivalent magnification ( � or �). marrow cellularity for each biopsy specimen was deter- mined, as in clinical practice, via visual inspection using a chart of reference biopsy images indexed at cellularity increments of %. marrow cellularities within selected rois were thus assigned to the nearest % ( %, %,. . . %, %) via reference image interpolation. between and specimens, each with or rois per specimen, were examined at each value of cellularity between % and %. representative images at % and % are shown in figure . frequency distribution of adipocyte cluster size (study ) in this first study, image rois were reviewed to measure the frequency distribution of adipocyte cluster size interior to the figure . video capture images of bone marrow biopsy specimens. (a) marrow at % cellularity. (b) marrow at % cellularity. adipocyte spatial distributions in marrow • shah et al. marrow cavities as a function of the marrow cellularity. cluster sizes ranged from single isolated cells to groupings of up to cells. clusters of � cells were difficult to quantify with respect to cell number and were thus assigned to a single category of or more cells. for cellularities � % (fat fractions, � %), adipocyte cluster size became visually indeterminate in the marrow spaces. percentage of trabecular surfaces covered by adipocytes (study ) in this second study, measurements were made to quantify the presence and extent of the first fat layer on the surfaces of the bone trabeculae. for each patient image, a matlab routine was first used to measure the total perimeter for all trabeculae visible within the roi ( . �m/pixel at � magnification). next, a reduced trabeculae perimeter was estimated that contained only those sec- tions of the trabecular surfaces that were in immediate contact with surface adipocytes (single cells or cell clusters). ratios of the adipocyte-covered perimeter to the total trabeculae perimeter were calculated and then indexed to the marrow cellularity of the biopsy specimen. marrow cellularity as function of distance from trabecular surfaces (study ) a third study was conducted to quantify any spatial preference for adipocyte cluster formation within the marrow cavities at differing marrow cellularities. in particular, the study sought to determine if adipocyte clusters were uniformly distributed in the marrow cavities or preferentially distributed either toward or away from the trabecular surfaces. for each image roi, contours were drawn at distances of , , and �m from all trabecular surfaces. next, visual inspections of marrow cellularity were made within each marrow region ( – , – , – , and � �m from the bone–marrow interface). values of local cellularity and their gradients were then indexed to the global cellularity measured across the entire image roi. bone sample acquisition and nmr microscopy in this study, previously segmented and filtered nmr micros- copy images of the femoral head and humeral epiphysis of a -y-old male subject and a -y-old female subject were used in the simulation of adipocyte spatial distributions and the resulting �-particle (electron) dosimetry of the active marrow. details re- garding sample harvesting, sample preparation, and nmr micros- copy image acquisition and processing are given in bolch et al. ( ) and in patton et al. ( ). the images were acquired on a -cm wide-bore avance imaging spectrometer (bruker medical, karlsruhe, germany) operated at a -mhz proton resonance ( . -t magnetic field strength) using a -mm-diameter quadrature birdcage coil of length . cm. a conventional d spin-echo pulse sequence was used to obtain fully d images of the sample at a spatial resolution of � � �m. voxels in the d binary image were assigned to either the bone trabeculae or to the bone marrow. bone endosteum was further defined as that portion of marrow surface voxels mm from the surface of an adjacent bone voxel. the residual voxel volumes were assigned to bone marrow, thus creating a -�m boundary of endosteum separating the osseous tissues of the bone trabeculae and the tissues of the marrow cavity. methods for adipocyte introduction within voxel models of trabecular bone in the previous study by bolch et al. ( ), marrow voxels in the binary d nmr images were partitioned into those belonging to either trabecular active marrow (tam) or trabecular inactive mar- row (tim), the later identifying marrow adipose tissue. regions of tim were modeled in bolch et al. as randomly positioned -voxel groupings placed uniformly within the marrow cavities ( -voxel method). in this study, additional methods are explored: random and defined. the former randomly tags marrow voxels in the original binary image as adipose tissue voxel at a time. in the defined method, the spatial arrangement of the tim voxels is forced to conform to measurements made on the biopsy specimens (studies – ) (assuming no skeletal-site dependence on their spatial distribution). first, clusters of marrow voxels on the trabecular surfaces are tagged as tim according to both the cluster size distribution of study and the percentage of perimeter cov- erage of study . it is assumed that values of percentage of area coverage in the d bone images are equivalent to measurements of percentage of perimeter coverage in the -dimensional ( d) biopsy images. second, clusters of tim voxels in the interior of the marrow cavities are created in a manner consistent with the cluster size distribution of study and any cellularity gradients observed in study . additional clusters are created until an overall targeted marrow cellularity is achieved. to verify the accuracy of the algorithms of the defined method, d slices were extracted through the resulting d image and values of cluster size distri- bution and percentage of perimeter coverage of trabecular surfaces were tabulated and compared with the measured values of studies and . figure shows representative d slices through the femoral head of the -y-old man at values of marrow cellularity via the defined method. final d images of trabecular bone, with both active and inactive marrow regions present, were then coupled to the egs radiation transport code. particle transport ( , histories) was initiated for electron sources ( kev to mev) originating in the tam, trabecular bone endosteum (tbe), tbs, or trabecular bone volume (tbv). particle histories of were simulated so that errors on the absorbed fraction to the tam were held to � % in each case. results frequency distribution of adipocyte cluster size (study ) figure displays the relative number of adipocyte clus- ters as functions of both the cluster cell size and the marrow figure . representative slices from d segmented nmr images of -y-old male femoral head. black, white, and gray pixels represent bone trabeculae, active marrow, and adipose tissue, respectively, with the latter implemented using the defined method of voxel tagging. images are shown for % marrow cellularity (a), % cellularity showing only bone sur- face adipose voxels (b), and % cellularity showing only mar- row space adipose voxels (c). scale of each panel is . mm on edge. the journal of nuclear medicine • vol. • no. • may cellularity of the biopsy sample. at extremely high marrow cellularities (� %), adipocytes are found primarily as isolated cells (� . ) or as clusters of or cells (� . and � . , respectively). as the cellularity of the bone marrow decreases, the relative number of single adipocytes decreases accordingly (down to � . at %– % mar- row cellularity), whereas the relative number of -cell clus- ters remains fairly constant at between . and . across all cellularities considered. concurrently, increases are noted in the relative proportions of multicell clusters with decreasing cellularity at each cluster size of � cells. these frequency distributions were constructed with approxi- mately or biopsy subjects per % marrow cellularity interval, with the %– % grouping having subjects and the %– % grouping having only subjects. at cellu- larities of � %, the density of adipocytes was such that individual cell clusters could not be identified. figure gives a comparison of the measured cluster size distributions with corresponding distributions of voxel cluster sizes in the d nmr images for the -y- old male femoral head as implemented via the defined and random methods of marrow voxel tagging. at high values of marrow cellularity ( %– % in fig. a), the defined method is shown to provide a slightly better depiction of the measured distribution than a sim- ple random tagging of voxels in the marrow spaces, particularly for the larger cell clusters (� cells). as the cellularity of the bone marrow decreases ( %– % in fig. b), the defined and random methods of mar- row voxel tagging yield comparable distributions, with both giving fairly good approximations to the measured distributions of adipocyte cluster sizes. in general, the defined method of voxel tagging was shown to be slightly superior to the random distribution at marrow cellularities of � %. both methods were comparable at figure . frequency distribution of adi- pocyte cluster cell size as function of mar- row cellularity from % to %. figure . comparison of relative number of adipocytes per cluster within marrow cavity as measured in marrow biopsies (�) and as simulated in nmr microscopy images using defined (■ ) and random (u) methods of voxel tagging. com- parisons are made at marrow cellularities of %– % (a) and %– % (b). adipocyte spatial distributions in marrow • shah et al. cellularities of � %, indicating that adipocytes are po- sitioned randomly in the marrow spaces. percentage of trabecular surfaces covered by adipocytes (study ) figure displays measured values (closed circles) of the percentage of bone trabeculae perimeter that is in contact with marrow adipocytes as a function of the marrow cellu- larity. at a marrow cellularity of %, � % of the trabec- ular perimeter in the biopsy images is covered by adipo- cytes. this perimeter coverage increases to � % as the marrow cellularity drops to %, after which the rate of perimeter coverage is noted to increase more rapidly until � % of the perimeter is covered by adipocytes at a mar- row cellularity of %. the rate of coverage then decreases slightly as marrow cellularity further declines (and the fat fraction approaches %). for comparison, the percentage of trabecular surfaces covered by adipose voxels in the d nmr images is shown using the random and the defined methods (the latter designed to reproduce mea- sured values of perimeter coverage). not surprisingly, the random method of voxel tagging yields a uniformly increasing percentage of trabecular perimeter coverage by adipose voxels with decreasing marrow cellularity. devia- tions from this random method are noted in the marrow cellularity range of %– %. the measurements are con- sistent with the initial appearance of a first fat layer on the trabecular surfaces at marrow cellularities at and below � % (fat fractions, � %). the first fat layer ( % pe- rimeter coverage) is thus fully established at a cellularity of � %. marrow cellularity as function of distance from trabecular surfaces (study ) in this study, the radial distribution of marrow cellularity as a function of distance into the marrow space is assessed. figure displays measurements of local marrow cellularity within radial distance intervals from the bone trabeculae surface ( – , – , and – �m) as a function of the global marrow cellularity assigned to the entire bi- opsy roi. no statistically significant gradients in local marrow cellularity are noted at global cellularities of � % and � %. for the former (fat fractions, � %), the spatial distribution of adipocytes and their clusters appears to be uniformly distributed within the marrow cavities. for the latter (fat fractions, � %), the density of adipose tissue is sufficiently high to conceal any gradient in adipocyte spatial position in the marrow cavities. nevertheless, at intermedi- ate values of global cellularity ( %– %), the data are suggestive of a gradient in local cellularity as mean values in the first radial interval ( – �m) are statistically smaller than mean values in the third radial interval ( – �m) (p � . ). in this range of global cellularity, it appears that adipocytes are modestly localized preferen- tially toward the trabecular surfaces and less so within the interior regions of the marrow cavities. these observations are reasonably consistent with both the data of figure (which shows that the formation of the first fat layer nears completion at a global cellularity of � %) and the data of allen et al. ( ) on adipocyte spatial positioning in the marrow spaces of the human rib. electron sources in active marrow transport simulations in different nmr microscopy voxel models of trabecular bone were conducted to deter- mine the degree to which the method of marrow voxel tagging would impact values of absorbed fraction to active marrow. results are shown in figures a and b for electron sources emitted in the active marrow of the femoral head of the -y-old man and -y-old woman, respec- figure . percentage of bone trabeculae perimeter that is in immediate contact with marrow adipocytes (f – biopsy mea- surements). with a random method of tagging adipose marrow voxels, percentage of trabeculae perimeter coverage is shown to increase linearly with decreasing marrow cellularity ( – fat fraction). figure . variations in local marrow cellularity as function of both distance from bone trabeculae and global marrow cellu- larity of biopsy sample. values shown are means and -� errors for replicate measurements for different bone trabeculae and for up to different biopsy samples. data are plotted at means of distance intervals in staggered fashion to better display data error bars. the journal of nuclear medicine • vol. • no. • may tively. data points display values of the absorbed fraction at values of marrow cellularities as taken from bolch et al. ( ) using the -voxel method of defining the tim. shown in comparison are absorbed fraction data for transport sim- ulations in which the tim is modeled using either the defined method or the random method of voxel tag- ging. at energies of � kev, all methods yield essen- tially identical results, with the single exception of % marrow cellularity in the female femoral head. at energies below � kev, the defined method generally yields absorbed fraction values � %– % higher than those calcu- lated under the -voxel or random method of adipose voxel tagging. only at these low-emission energies would slight differences in target and nontarget geometries influ- ence patterns of energy deposition. comparisons of voxel- based assessments of �(tam tam) with those given by chord-based models were given previously in bolch et al. ( ) and have resulted in revisions to the eckerman and stabin model for marrow sources ( ). electron sources in tbv when the electron emissions in trabecular bone are uni- formly distributed within the volumes of the bone trabecu- lae, the absorbed fraction of energy to active marrow, �(tam tbv), is shown to have an energy dependence as displayed in figure . figures a and b give results using the defined method of adipose voxel tagging for the -y-old male femoral head and humeral epiphysis, respec- tively. corresponding data for these same skeletal sites in the -y-old woman are shown in figures c and d, respectively. in each voxel model, the marrow cellularity is allowed to vary from % (the icrp reference value) to % as indicated. for each case, the electron emission energy must exceed � – kev for a meaningful fraction of the particles to escape the bone trabeculae, penetrate the -�m endosteal layer, and deposit their residual energy to voxels denoting hematopoietically active regions of the marrow tissues. at energies exceeding � kev, the ab- sorbed fraction profiles begin to decrease as electrons es- cape the trabecular spongiosa and deposit their residual energy to the surrounding cortex of cortical bone. for the male subject, maximum values of absorbed fraction to ac- tive marrow are noted to be higher in the humerus than in the femoral head, primarily because of the higher marrow volume fraction in the former ( . vs. . ) with its re- sulting thinner trabeculae (spongiosa volumes are both � cm ). these same upward shifts in absorbed fraction are seen in the female subject for these bone sites, although they are less dramatic. electron sources in tbe and on tbs other bone-seeking radiopharmaceuticals localize not within the volumes of the trabeculae but at the surfaces of the bone–marrow interface. here, one can consider the localization to occur either (a) uniformly within the -�m tbe (e.g., cellular incorporation within the osteoblasts) or (b) on the tbs (e.g., interface between of the mineral bone and endosteal layer). figures a– d show values of both �(tam tbe) and �(tam tbs) at values of marrow cellularity for humerus and femoral head of both the -y- old-old male and the -y-old female subject as indicated in each panel. in the energy range of – kev, values of �(tam tbe) steadily increase as more electron energy escapes from the endosteal layer to irradiate regions of the active marrow on the emission side of the bone trabeculae. as the electron emission energy increases further to � – kev, a second upturn in �(tam tbe) is noted. here, electrons emitted toward the interior of a bone trabecula now have sufficient energy to penetrate the trabecula and endosteal layer and, thus, emerge on its far side to irradiate additional voxels of active marrow. at energies exceeding several hundred kev, values of �(tam tbe) merge with corresponding values of �(tam tbv) as the energy dis- tribution patterns become independent of the emission site in bone. figure . comparison of electron absorbed fraction data, �(tam tam), for self-irradiation of tam within femoral head of -y-old man (a) and that of -y-old woman (b). at each value of marrow cellularity, simulation results are shown for all methods of adipocyte introduction within marrow cavities of d nmr image: defined method (dashed curves), random method (solid curves), and -voxel method used previously by bolch et al. ( ) (data symbols). adipocyte spatial distributions in marrow • shah et al. corresponding values of �(tam tbs) are shown in figures a– d as dashed lines which merge with values of �(tam tbe) at energies of � kev. for electron source energies of � kev, the choice of a tbe or tbs source strongly influences values of absorbed fraction to active marrow. for emissions on the tbs, the electron emission energy must exceed � kev for a meaningful fraction of the particles to penetrate the -�m endosteal layer and deposit their residual energy to tissues of the active marrow. as with the tbe sources, a second upturn is at emission energies of – kev as particles begin to penetrate the bone trabeculae and irradiate other regions of active marrow. discussion comparisons can be made between the cellularity-depen- dent voxel-based transport models developed in this study and published models: the icrp model ( ), the eckerman and stabin model ( ), and the bouchet et al. model ( ). these models are based on chord-length distri- butions measured at the university of leeds on a -y-old male subject ( – ). in the latter models, values of absorbed fraction to the marrow spaces determined via transport simulations have been scaled by a reference cel- lularity of % as given by the icrp ( ). in figures a– d, the icrp model specifies an energy-independent value of . for all skeletal sites in reference man, with no consideration of marrow cellular- ity. in comparison with the voxel-based transport models, the absorbed fraction to active marrow is significantly over- estimated by the icrp- model for low-energy emitters (� kev) across all values of marrow cellularity. at energies of � kev, this energy-independent model pro- vides a rough approximation of �(tam tbv) only at marrow cellularities of � %– %. in contrast, the cellularity-scaled models of eckerman and stabin and of bouchet et al. show excellent agreement with values of �(tam tbv) from the voxel-based trans- port model at the same reference marrow cellularity ( %). exceptions are noted for (a) the male femoral head, where the chord-based models yield absorbed fractions � %– % higher than those given by the voxel-based model (e.g., figure . absorbed fractions to tam, �(tam tbv), for electron sources emitted uniformly within tbv at values of marrow cellularity: %, %, %, and %. data are shown for skeletal sites and individuals as indicated in a–d. for comparison, values are shown as well for icrp model (energy-independent dashed line) and both the eckerman and stabin ( ) and the bouchet et al. ( ) models (energy-dependent dashed line). the journal of nuclear medicine • vol. • no. • may possibly thinner trabeculae in the leeds subject); and (b) electron emission energies exceeding � mev, where par- ticle escape from the skeletal site is not accounted for within the chord-based models (e.g., infinite trabecular region transport as discussed in patton et al. ( ) and in jokisch et al. ( )). reference values of �(tam tbs) are shown in figures a– d from all chord-based models for electron sources on the bone surfaces. in the icrp model, a simple half-space is implied in which only % of isotropic particle emissions reach the marrow space, where full energy dep- osition to active marrow is then assumed. as with the tbv sources, the icrp value of . is noted to be exceed- ingly conservative except in the case of high-energy emit- ters (� kev) in the humeri (figs. b and d) and female femoral head (fig. c) at high values of marrow cellularity ( %– %). in contrast, values of �(tam tbs) given by the eck- erman and stabin and the bouchet et al. models show good to excellent agreement with voxel-model transport results at energies of � kev. discrepancies are noted, however, for electron emitters below � kev for the eckerman and stabin model. at these energies, the eckerman and stabin model shows little-to-no energy dependence, as energy dep- osition events to the trabecular endosteum are not differen- tiated from those to the surrounding active marrow. conse- quently, their absorbed fractions at low energies begin at . (an absorbed fraction of . multiplied by the ref- erence marrow cellularity of %). in the voxel-based trans- port models, electron emissions on the tbs must penetrate either the -�m endosteal layer or adipose voxels of the first fat layer before they can participate in energy deposi- tion to the hematopoietically active marrow. both the voxel-based and chord-based models discussed above are constructed using an extremely limited number of subjects; nevertheless, the voxel-based modeling data shown above tend to support the use of cellularity-scaled chord-based models from either eckerman and stabin ( ) (electrons of � kev) or from bouchet et al. ( ) (all energies). further research is needed, however, to quantify figure . absorbed fractions to tam for electron sources emitted uniformly within either tbe or tbs at values of marrow cellularity: %, %, %, and %. data are shown for skeletal sites and individuals as indicated in a–d. for comparison, values are shown as well for icrp model (energy-independent dashed line) and both the eckerman and stabin ( ) and the bouchet et al. ( ) models (energy-dependent dashed line). adipocyte spatial distributions in marrow • shah et al. the full extent to which individual variability in bone min- eral density (e.g., bone trabeculae thicknesses) can influence dose to active marrow. in addition to their ability to incorporate changes in marrow cellularity and skeletal site size, voxel-based skel- etal dosimetry models provide the potential for reporting (a) dose–volume histograms of active marrow dose and (b) active marrow dose as a function of distance from the bone–marrow interface ( ). these capabilities are not available to chord-based dose models. in voxel-based mod- els, cellularity-dependent patterns of adipocyte clustering, trabeculae surface coverage, and adipocyte concentration gradients reported here could potentially play key roles in altering dose gradients for bone–volume and bone–surface sources. finally, quantification of these dose gradients for various radionuclides, coupled with a knowledge of stem and progenitor cell concentrations within the marrow cavi- ties ( ), might provide an improved basis for predicting marrow response in radionuclide therapies. conclusion experimental data have been collected on the spatial distributions of adipocytes in normal human bone marrow as needed to define the inactive (yellow) marrow regions in d voxel models of trabecular bone. these studies included measurements of (a) size distributions of adipocyte cell clusters, (b) the fractional perimeter coverage of bone tra- beculae perimeter by marrow adipocytes, and (c) the pres- ence or absence of cellularity gradients within the marrow cavity. study revealed a predicable shift in cluster size with decreasing marrow cellularity from single adipocytes to clusters of � cells; the percentage of -cell clusters remained relatively constant with changing cellularity. study revealed a small nonlinear variation in trabeculae perimeter coverage with increasing fat tissue fraction at marrow cellularities between % and %. study dem- onstrated that only in the range of %– % marrow cel- lularity was a slight gradient in adipocyte concentration indicated with adipocytes localized preferentially toward the trabecular surfaces. these data were subsequently used to assemble d images of trabecular bone with both active (red) and inactive (yellow) marrow delineated in patterns consistent with the marrow biopsy measurements. as dem- onstrated in figure , however, electron dosimetry of the skeletal site remains fairly independent of the method by which adipocyte clusters are introduced. at image resolu- tions of � �m, the -voxel and random methods provide reasonable alternatives to the defined method of adipocyte spatial characterization in the d images. for both bone volume and bone surface electron sources, absorbed fractions to active marrow given by the icrp model (mirdose ) were shown to be exceedingly conser- vative except in the case of high-energy emissions (� kev) and at high values of marrow cellularity (� %). values of both �(tam tbv) and �(tam tbs) given by the eckerman and stabin model (mirdose ) were shown to be reasonably consistent with d voxel model simulations at the reference cellularity of %, except in the case of low-energy emitters (� kev) on the bone sur- faces. the data of figures and further highlighted the need for careful specification of skeletal source location for those bone-seeking radiopharmaceuticals with low-energy particles (� kev) in their emission spectrum, as esti- mates of absorbed fraction to active marrow can vary greatly at these energies. acknowledgments we thank drs. keith eckerman and michael stabin for providing absorbed fraction data for their chord-based skeletal dose models. we further thank them, as well as dr. derek jokisch, for their helpful comments and suggestions during the preparation of this article. this work was supported in part by u.s. department of energy nuclear engineering education research (neer) grants de-fg - id and de-fg - id with the university of florida. references . sgouros g. bone marrow dosimetry for radioimmunotherapy: theoretical con- siderations. j nucl med. ; 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. . whitwell jr, spiers fw. calculated beta-ray dose factors for trabecular bone. phys med biol. ; : – . . beddoe ah, darley pj, spiers fw. measurements of trabecular bone structure in man. phys med biol. ; : – . . icrp. basic anatomical and physiological data for use in radiological pro- tection: the skeleton. icrp publication . oxford, u.k.: international com- mission on radiological protection; . . jokisch dw, bouchet lg, patton pw, rajon da, bolch we. beta-particle dosimetry of the trabecular skeleton using monte carlo transport within d digital images. med phys. ; : – . . kvinnsland y, skretting a, bruland os. radionuclide therapy with bone-seeking compounds: monte carlo calculations of dose-volume histograms for bone mar- row in trabecular bone. phys med biol. ; : – . . lord bi. the architecture of bone marrow cell populations. int j cell cloning. ; : – . adipocyte spatial distributions in marrow • shah et al. singular dyt phenotypes in association with new thap frameshift mutations hal id: hal- https://hal.archives-ouvertes.fr/hal- submitted on dec hal is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. the documents may come from teaching and research institutions in france or abroad, or from public or private research centers. l’archive ouverte pluridisciplinaire hal, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. singular dyt phenotypes in association with new thap frameshift mutations arnaud blanchard, agathe roubertie, marion simonetta-moreau, vuthy ea, coline coquart, melissa y. frederic, gael gallouedec, jean-paul adenis, isabelle benatru, michel borg, et al. to cite this version: arnaud blanchard, agathe roubertie, marion simonetta-moreau, vuthy ea, coline coquart, et al.. singular dyt phenotypes in association with new thap frameshift mutations. movement disor- ders, wiley, , ( ), pp. - . � . /mds. �. �hal- � https://hal.archives-ouvertes.fr/hal- https://hal.archives-ouvertes.fr . wong sh, steiger mj, larner aj, fletcher na. hereditary myo- clonus dystonia (dyt ): a novel sgce gene mutation with intrafamilial phenotypic heterogeneity. mov disord ; : – . . misbahuddin a, placzek m, lennox g, taanman jw, warner tt. myoclonus-dystonia syndrome with severe depression is caused by an exon-skipping mutation in the epsilon-sarcoglycan gene. mov disord ; : – . . doheny do, brin mf, morrison ce, et al. phenotypic features of myoclonus-dystonia in three kindreds. neurology ; : – . . asmus f, hjermind le, dupont e, et al. genomic deletion size at the epsilon-sarcoglycan locus determines the clinical phenotype. brain ; : – . singular dyt phenotypes in association with new thap frameshift mutations dyt dystonia is an autosomal dominant disorder with incomplete penetrance (� %) characterized by early-age onset (median, years) and slight female pre- dominance. the upper limb is a common site of onset, with progressive extension of the disease to other body parts. the cranial region is affected in almost two thirds of patients, and the functional repercussions of the disease are perceived by patients as mainly a result of disturbances in this region and particularly to the speech problems present in more than half of the patients; functional impairment is moderate overall (patients remain ambulatory). thap gene mutations have now been identified in numerous dyt families. here we describe new thap mutations identified in non-amish patients with primary non-dyt dystonia. patients were selected among those followed in outpatient clinics by trained neurologists from the french dystonia net- work. eight index patients were recruited from polish outpa- tient clinics through an international collaboration. a set of independent index patients with primary non-dyt dys- ------------------------------------------------------------ *correspondence to: gwenaelle collod-béroud, inserm, u , montpellier, france; gwenaelle.collod-beroud@inserm.fr arnaud blanchard and agathe roubertie contributed equally to this work. relevant conflicts of interest/financial disclosures: nothing to report. this study was supported by grants from the french ministry of health (national phrc -a - ), amadys-lfcd, alliance france dystonie, lions club, french dystonia network, université montpellier and inserm. arnaud blanchard and vuthy ea are supported by a grant from the ministère de l’enseignement supérieur et de la recherche (mesr). full financial disclosures and author roles may be found in the online version of this article. published online april in wiley online library (wileyonlinelibrary.com). doi: . /mds. fig. . family tree demonstrating index cases with sgce deletion (iii: and iii: ) who had cognitive impairment and mild myoclonus dystonia (black fill). the father of index cases had the same sgce deletion (ii: ) but had adult-onset psychosis without myoclonus dystonia. three of his siblings had a history of psychosis without movement disorders (ii: , ii: , and ii: ), but were unavailable for clinical examination or sgce testing. tonia were included. patients with a mean age at onset of . . years were diagnosed as having ‘‘generalized, segmental, or multifocal dystonia’’ ( % of patients), or ‘‘oromandibular or cervical dystonia’’ ( . %). moreover, as clinical expression of dyt has been redefined as ‘‘broad and overlapping with non-dyt dystonia subtypes,’’ we also included patients with ‘‘blepharospasm’’ ( . %) or with ‘‘other focal dystonia’’ such as writer’s cramp ( . %). sixty- three percent were isolated cases, and autosomal dominant transmission with sometimes incomplete penetrance was observed in families. informed consent and blood samples were collected and dna extracted from peripheral lympho- cytes according to standard procedures. direct sequencing of the thap coding regions and their exon boundaries was carried out. in the group of ‘‘generalized, segmental, or multi- focal dystonia,’’ we identified novel thap heterozygous mutations (c. _ delct deletion [p.pro argfsx ] in patient ; c. dupa insertion [p.arg lysfsx ] in patient ) (supplementary figure ). the same thap dele- tion was identified in the dystonic sibling of patient (patient ). these new frameshift mutations result in the formation of premature stop codons at positions and , respectively, and suggest, as previously reported, a loss-of- function mechanism by haploinsufficiency. the clinical picture of these patients (table ) fits with the clinical phenotype associated with thap mutations, but some clinical aspects are singular. in patient , dystonia initially implicated the right upper limb at the age of , with progressive axial involvement; at years of age, the right upper limb and the cervical region were clearly dystonic, with writer’s cramp and torticolis. the patient was not treated. at the age of the patient reported spontaneous, complete remission (cervical and upper limb). this remission was transient, and after a free interval of years, without any obvious triggering factor, dystonia rapidly recurred with right arm and cervical involvement. afterward, dystonia did not spread to other body parts; upper limb dystonia remained moderate and stable, and disability was mainly due to cervical involvement. no worsening or remission has been reported since the age of , except for partial improvement after tri- hexiphenydyl treatment and botulinum toxin injections in the neck. such transient improvement, or ‘‘honeymoon,’’ is not uncommon in other primary dystonias, particularly in cervical dystonia, but usually occurs within the first years after onset. to our knowledge, such a honeymoon has never been observed in other known dyt patients. patient is remarkable because of the lower limb onset of the disease at an early age, thus mimicking dyt dystonia. nevertheless, upper limb onset is reported in more than half of dyt patients, and cranial onset occurs in one quarter of the cases (table ). in patients and , who are siblings, dystonia became progressively generalized, with prominent cranial involvement, as described in almost half the dyt patients reported in the literature. although the patients did not report any family history of motor disabilities or movement disorders (except for the sibling of patient ), unfortunately, other family members could not be examined or tested for thap mutations. patient was thus considered a sporadic case. the occurrence of cognitive and psychiatric disorders is an area of uncertainty in rare genetic forms of dystonia. standar- dized cognitive and psychiatric assessments were not performed in our patients; they were all university graduates, and their past medical histories was uneventful for psychiatric disturban- ces. paisan-ruiz reported cognitive changes in dyt patient. cognitive or psychiatric functions among thap mutation carriers will need to be better analyzed. in conclusion, thap implication in primary dystonia is rare in non-amish patients (about . % of tested dystonic patients reported in the literature). the dyt phenotype over- laps with that of other forms of primary early-onset dystonia, especially dyt dystonia. good candidates for thap screen- ing might be patients with onset in childhood or during adoles- cence who present cervicocranial and upper limb involvement or generalized dystonia and cranial involvement. acknowledgments: we thank all patients and family mem- bers for participation in this study. note that during the reviewing process, several new thap mutations were reported (zittel et al, groen et al., söhn et al, cheng et al, and de carvalho et al). arnaud blanchard, ms, , agathe roubertie, md, phd, , , marion simonetta-moreau, md, phd, vuthy ea, ms, , coline coquart, melissa y. frederic, phd, , table . clinical characteristics of carriers of thap mutations patient patient patient amish families* n ¼ non-amish patients** n ¼ sex f m m f ( %) f ( . %) age at onset (y) median, . ( – ) – age at last examination (y) median, ( – ) – family history þ þ no . site at onset patientsa upper limb þ þ ( %) of ( . %) lower limb þ ( %) of ( . %) cervical ( %) of ( . %) cranial ( %) of ( . %) site at examination patientsb upper limb þ þ þ ( %) of ( . %) lower limb þ þ no ( %) of ( . %) cervical þþ þ þ ( %) of ( . %) cranial þ þ no ( %) of ( . %) speech þ þ no ( %) of ( . %) distribution g g s fo: ( %) fo: / ( . %) s: ( %) s: / ( . %) mu: ( %) mu: / ( . %) g: ( %) g: / ( . %) sex: f, female; m, male. dystonia distribution: g, generalized; fo, focal; mu, multifocal; s, segmental. * families, patients. ** families, sporadic cases, patients including our patients. , – a patients, as data were not available for patients reported by djarmati et al. b patients, as data were not available for patients reported by houlden et al. gael gallouedec, md, jean-paul adenis, md, isabelle benatru, md, michel borg, md, pierre burbaud, md, phd, patrick calvas, md, phd, laura cif, md, philippe damier, md, phd, alain destee, md, phd, laurence faivre, md, phd, lucie guyant-marechal, md, piotr janik, md, phd, samer janoura, md, alexandre kreisler, md, phd, , anna lusakowska, md, phd, sylvie odent, md, phd, anna potulska-chromik, md, phd, monika rudzińska, md, phd, stephane thobois, md, phd, isabelle vuillaume, md, phd, christine tranchant, md, sylvie tuffery-giraud, phd, , philippe coubes, md, phd, bernard sablonnière, md, phd, mireille claustres, md, phd, , , , gwenaelle collod-béroud, phd, , * inserm, u , montpellier, france; université montpellier , ufr médecine, montpellier, france; chu montpellier, hôpital gui de chauliac, service de neuropédiatrie, montpellier, france; hôpitaux de toulouse, pôle neurosciences, toulouse, france; chu montpellier, hôpital arnaud de villeneuve, laboratoire de génétique moléculaire, montpellier, france; chu dupuytren, service de neurologie, limoges, france; chu dupuytren, service d’ophtalmologie, limoges, france; chu de dijon, hôpital général, service de neurologie, dijon, france; chu de nice, service de neurologie, nice, france; chu pellegrin, service de neurophysiologie, clinique, bordeaux, france; chu toulouse, hôpital purpan, service de génétique médicale, toulouse, france; chu montpellier, hôpital gui de chauliac, service de neurochirurgie, montpellier, france; chu nantes, cic , service de neurologie, nantes, france; chu de lille, service de neurologie, et pathologie du mouvement, lille, france; chu de dijon, hôpital d’enfants, centre de génétique, dijon, france; chu de rouen, hôpital charles nicolle, département de neurologie, rouen, france; department of neurology, medical university of warsaw, poland; chg roanne, service de neurologie, roanne, france; service de génétique clinique, chu de rennes, hôpital sud, rennes, france; department of neurology, jagiellonian university medical college, poland; hospices civils de lyon, hôpital neurologique, service de neurologie c, lyon, france; chru de lille, département de biochimie, et de biologie moléculaire, lille, france; hôpitaux universitaires de strasbourg, service de neurologie, strasbourg, france references . fuchs t, gavarini s, saunders-pullman r, et al. mutations in the thap gene are responsible for dyt primary torsion dystonia. nat genet ; : – . . bressman sb, raymond d, fuchs t, heiman ga, ozelius lj, saunders-pullman r. mutations in thap (dyt ) in early-onset dystonia: a genetic screening study. lancet neurol ; : – . . koukouni v, martino d, arabia g, quinn np, bhatia kp. the en- tity of young onset primary cervical dystonia. mov disord ; : – . . paisan-ruiz c, ruiz-martinez j, ruibal m, et al. identification of a novel thap mutation at r amino-acid residue in sporadic patients with early-onset dystonia. mov disord ; : – . . bonetti m, barzaghi c, brancati f, et al. mutation screening of the dyt /thap gene in italy. mov disord ; : – . . djarmati a, schneider sa, lohmann k, et al. mutations in thap (dyt ) and generalised dystonia with prominent spasmodic dyspho- nia: a genetic screening study. lancet neurol ; : – . . houlden h, schneider sa, paudel r, et al. thap mutations (dyt ) are an additional cause of early-onset dystonia. neurology ; : – . . xiao j, zhao y, bastian rw, et al. novel thap sequence var- iants in primary dystonia. neurology ; : – . pantothenate kinase–associated neurodegeneration: clinical description of patients and identification of new mutations pantothenate kinase–associated neurodegeneration (pkan) is a rare autosomal recessive neurodegenerative dis- order of childhood onset, characterized by progressive dysto- nia and iron accumulation in the brain, mainly in the globus pallidus and the pars reticulata of the substantia nigra. patients have mutations in the gene encoding pantothenate kinase (pank ), a key regulatory enzyme in the biosyn- thesis of coenzyme a. we ascertained patients from unrelated algerian fami- lies between and ; all were examined at different stages of the disease and presented with early-onset, typical, – and rarely described clinical pkan features (table and sup- plementary fig. ). hyperactivity (video) and attention deficit rarely described in previous series but observed in most of our patients were inaugural signs of the disease, as demonstrated by the absence of any other clinical signs in patient f p at the age of years. pigmentary retinopathy, present in all our patients, was also considered as an early sign because it was al- ready detectable in the neurologically asymptomatic patient (f p ). the phenotype thereafter extended with abnormal falls ------------------------------------------------------------ additional supporting information may be found in the online version of this article. *correspondence to: meriem tazir, department of neurology, chu mustapha bacha and laboratoire de neurosciences, université d’alger, algérie, algeria; meriem.tazir@sante.dz relevant conflicts of interest/financial disclosures: nothing to report. this work received support from the ministère de la santé, de la population et de la réforme hospitalière (algeria), the ministère de l’enseignement supérieur et de la recherche scientifique (algeria), the agence national de la recherche (france, to a.d. and g.s.), the association française contre les myopathies (afm, france; to h.a.) and the verum foundation (germany; to a.b.). full financial disclosures and author roles may be found in the online version of this article. published online march in wiley online library (wileyonlinelibrary.com). doi: . /mds. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top 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published with open access at springerlink.com abstract an auto-inflammatory syndrome consequent to samhd mutations involves cerebral vasculopathy char- acterized by multifocal stenosis and aneurysms within large arteries, moyamoya, chronic ischemia, and early- onset strokes (sams). while this condition involves the innate immune system, additional clinical features mimic systemic lupus erythematosus. mutations in this gene can also cause a subset of the rare genetic condition aicardi- goutières syndrome. to date, no established therapy suc- cessfully prevents disease progression. we report a corticosteroid-dependent sams patient, a -year-old male of old order amish ancestry, with diffuse cerebral arteriopathy identified through contrast brain magnetic res- onance arteriography (mra) and mri. he received sub- cutaneous adalimumab every weeks for months with minimal response. then, he started intravenous tocilizumab ( mg/kg/dose) every weeks. he sustained steadily normal- izing cerebral vasculopathy and lab abnormalities resolved, allowing prednisone reduction. we conclude that the cerebral vasculopathy of the homozygous samhd mutation- mediated auto-inflammatory disease sams responded favor- ably to tocilizumab infusion therapy. keywords aicardi-goutières syndrome . cerebral vasculopathy .moyomoya .samhd mutation .tocilizumab introduction cerebral vasculopathy associated with mutations in the samhd gene can invoke early-onset stroke [ ]. mutations in this gene can also cause the rare genetic condition, aicardi- goutières syndrome (ags, mim ), which bears a phe- notypic resemblance to this cerebral vasculopathy occurring in the amish population. ags involves increased production of interferon (ifn)-α [ ]. loss-of-function mutations in any of six ifn-stimulated genes (trex , rnaseh a, rnaseh b, rnaseh c, samhd , and adar) are the identified cause of ags [ ]. these mutations involve the in- nate rather than the adaptive immune system. ags presents with a phenotypic overlap of early-onset encephalopathy, mimicking congenital viral infection (cerebrospinal fluid lym- phocytosis and elevated ifn-α levels, loss of white matter, and basal ganglia calcifications), and features of systemic lu- pus erythematosus (sle), including cytopenias, oral ulcers, arthritis, perniosis with cutaneous erythematous lesions, and autoantibodies [ , ]. ags can involve a subset of children that develop early-onset sle [ ]. intracerebral large artery disease and chronic progressive arthropathy with distal joint contractures have been reported in ags associated with mu- tation of ags samhd protein [ , ]. further, ags associ- ated with heterozygous trex mutations involves ulcerating acral skin lesions suggestive of chilblain lupus (lupus pernio) [ ]. heterozygous samhd mutation can also be associated with progressive arthropathy, distal joint contractures, painful oral ulcers, and chilblains [ ]. we describe a patient with a similar clinical spectrum who does not have ags, rather homozygous samhd mutation which led to cerebral vasculopathy. affected patients within described pedigrees carrying this mutation develop multifocal cerebral stenosis and aneurysms within large arteries, chronic ischemic changes and moyamoya morphology, leading to * michael henrickson michael.henrickson@cchmc.org division of rheumatology, cincinnati children’s hospital medical center, burnet ave., cincinnati, oh - , usa ddc clinic for special needs children, middlefield, oh, usa clin rheumatol ( ) : – doi . /s - - - http://orcid.org/ - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf early-onset strokes [ ]. we report the first successful reversal of this cerebral vasculopathy with tocilizumab infusion therapy. case report the -year-old male patient mm, of old order amish an- cestry, was previously reported with autosomal recessive ho- mozygous samhd mutation (x- in the original study) [ ]. briefly, he developed symmetric bdry^ polyarthritis at age years, hoarseness at age years requiring vocal nodule resection, bilateral hand and foot pernio at age years, and a clinical course characterized by familial short stature, pho- tosensitivity, and persistent acral vasculopathy presenting as episodic raynaud’s disease and progressive, bilateral hand and foot sclerodactyly. peripheral vascular examination re- vealed diminished bilateral carotid korotkoff sounds, without bruits of his bilateral carotid or subclavian arteries, or abdom- inal aorta. table summarizes salient data from his disease and treatment course. mm underwent contrast brain magnetic resonance arteri- ography (mra) and contrast brain mr imaging (mri) based upon familial risk associated with his sister’s encephalopathy and the desire to identify potential vasculopathy prior to sim- ilarly devastating functional consequences. mm’s initial stud- ies performed at age years were abnormal. on mra, he had diffuse narrowing of his bilateral internal carotid arteries and irregular, narrowed segments of his bilateral anterior ce- rebral arteries’ a and m segments (fig. , a–c). his bi- lateral thalamostriate vessels had increased flow, with mild asymmetry in the early arterial phase of the perfusion study, consistent with a compensated perfusion pattern despite ex- tensive fibromuscular changes of arteriopathy. his brain mri had no evidence of cerebrovascular occlusive disease; howev- er, he had bilateral, diffuse arteriopathic changes of his distal internal carotid, anterior, and middle cerebral arteries. figure depicts the serial contrast cerebral mra findings during his treatment course. figure depicts the temporal sequence of mm’s medication interventions. based upon adalimumab’s capacity to penetrate the central nervous system’s blood brain barrier and the putatively pro- tective role of functional samdh in mediating the pro- inflammatory tumor necrosis factor (tnf)-α response [ ], mm started mg sc adalimumab every other week just prior to obtaining an interval brain mri/mra. mm underwent serial, biannual, contrast brain imaging (mri and mra) including d time-of-flight mra of the circle of willis, to assess therapeutic efficacy, and accompanying lab- oratory biomarkers of inflammation: erythrocyte sedimenta- tion rate (esr), immunoglobulin g (igg) and total protein. throughout his pre-biologic or biologic therapy clinical course, the c-reactive protein never increased, nor did he manifest anemia of chronic disease. compared to his baseline study (month ), contrast brain mri and mra obtained at onset of adalimumab therapy table data summary elapsed time / / / / / / / / / / / / / / / / / / years . . . . . . . . months . . . . . . cumulative (months) . . . . . . . medication (mg/dose) methotrexate (oral, weekly) prednisone (oral, daily) adalimumab (sc every weeks) tocilizumab (monthly infusion number) ( nd) ( rd) ( th) ( th) ( th) lab results (range) hemoglobin ( – g/l) . esr ( – mm/h) immunoglobulin g ( . – . g/l) . . nd . nd nd nd . nd total protein ( – g/l) nd a platelets ( – × /l) interleukin- ( – . pmol/l) . . joint count active joints limited joints nd no data a / / total protein clin rheumatol ( ) : – (month ) indicated a mixed picture. vasculopathy progressed with mild narrowing of his vertebral arteries’ distal segments and bilateral globus pallidus early collateralization consistent with moyamoya. cerebrovascular disease remained unchanged with persistent bilateral distal internal carotid ar- tery narrowing and indicated mild improvement of the anterior cerebral a segments and proximal middle cerebral arteries (m segments). there were equivocal changes of mild basilar artery narrowing and subtle abnormalities of the internal cap- sules’ posterior limb (arising from the anterior cerebral a and m segments). there was also increased signal intensity of the corticospinal tracts, extending into his cerebral peduncles and pons. the second study obtained during adalimumab therapy at month indicated stable vasculopathy without progressive change in these previously identified abnormalities (fig. , a–c). during a -month course of adalimumab therapy, there was mild evidence of disease advancement (including evolving moyomoya) but predominant stability while he remained on a daily -mg prednisone dose. however, the therapeutic goal was amelioration of identified extra- and intracranial medium- and small-vessel vasculitis with elimination of corticosteroid dependence. failure to meet these goals compelled the deci- sion to change biologic therapy. serum interleukin- (il- ) was elevated ( . pmol/l) [normal – . ] after months of adalimumab therapy. with this evidence, he discontinued adalimumab at month and started tocilizumab mg iv every weeks at month ( . mg/kg/dose). although mm did not appreciate a clinical change in his perniosis or articular disease after his first two month- ly tocilizumab infusions, he had appreciable improvement fig. serial contrast magnetic resonance cerebral arteriography fig. treatment course clin rheumatol ( ) : – in his cerebral vasculopathy. this coincided with temporal deterioration in unilateral hand perniosis requiring escala- tion in his prednisone dose to mg/kg/day during his second month of tocilizumab therapy. he tapered predni- sone successfully to . mg/kg/day by his third monthly infusion, when he obtained repeat brain imaging. following two tocilizumab infusions, the mra at month indicated similarly stable findings with the exception of normalization of his a and m cerebral artery seg- ments (fig. , a–c). at month , the second mra obtained during tocilizumab therapy indicated stable in- creased signal intensity within the bilateral internal cap- sules’ posterior limbs; other previous abnormalities remained stable, and the a and m cerebral artery seg- ments stayed normal (fig. , a–c). at month , repeat mra following consecutive tocilizumab infusions re- vealed stable, bilateral distal internal carotid artery narrowing, continued a and m segment improvement, resolution of globus pallidus communicating artery collateralization (moyamoya), and normalization of the previously increased bilateral cortical spinal tract signal intensity (fig. , a–c). discussion the auto-inflammatory disease sams, an acronym for cerebral stenosis, aneurysm, moyomoya, and stroke, pro- duces a heterogeneous phenotype of diverse clinical pre- sentations, including cerebral palsy, stroke, developmental delay, failure to thrive, chilblains, and arthritis. early fea- tures include mild intrauterine growth restriction, infantile hypotonia, and irritability, followed by failure to thrive and short stature. during cold weather months, affected patients experience a spectrum of vascular abnormalities of their acral regions including raynaud’s disease and chilblain lesions (lupus pernio). they may also have a low-pitch hoarse voice, glaucoma, migraine headache, and arthritis characterized by joint tenderness and restrict- ed motion. patients do not have hypertension; their car- diopulmonary, hepatic, and renal function is normal. the elevated esr, igg, neopterin, and tumor necrosis factor-α (tnf-α) found in these patients are consistent with in- flammatory disease. cerebral vasculopathy is a major hallmark found in all affected individuals typified by ste- noses and aneurysms. distinctive neuroimaging findings include chronic ischemic changes, multifocal stenoses of the large intracranial arteries, including some with moyamoya morphology and evidence of prior acute in- farction and hemorrhage [ ]. clinical outcomes in affected patients are largely dependent on cerebral vasculopathic pro- gression. indeed, the early-onset or recurrence of cerebral vasculopathy-associated strokes always predicts severe func- tional impairment and poor cognitive outcomes. while the specific pathophysiology is uncertain, aberrant processes involving the innate immune system may support one or several potential mechanisms. defective metabolism of intracellular nucleic acids can activate a cell-intrinsic autoim- mune response, pivotal to host capacity to discriminate be- tween self and non-self (e.g., viral) nucleic acids. nucleic acid recognition is an essential innate immune strategy for detect- ing viral infection. functional regulation involves prevention of self-activated innate immunity, including negative regula- tion of the ifn-stimulatory dna (isd) response. virtually all ifn-mediated antiviral immunity occurs via two types of nucleic acid detection systems: toll-like receptors (tlrs) and cytosolic sensors [ ]. because flawed distinction of viral from self nucleic acids can occur, defective clearance of self- derived nucleic acids may lead to severe, ifn-associated au- toimmunity [ ]. activation of tlrs on autoreactive b cell lymphocytes is a major contributing mechanism. specific tlrs predispose to autoantibody production in murine models of lupus and autoimmunity. such activation of tlr- dependent ifn production also occurs in human autoimmuni- ty (e.g., psoriasis) [ ]. in each circumstance, accumulated nucleic acids are detected by non-cell autonomous mecha- nisms rather than cell-dependent detection [ ]. cytosolic nucleic acid sensors can initiate autoimmunity if flawed control mechanisms exist. negative regulators of the isd response play a major role in preventing self-activation of innate immunity via cell-intrinsic components. one important negative regulatory mechanism in part involves ′ repair exo- nuclease (trex ) induction. trex is a cytosolic toll-like receptor-independent, antiviral pathway that detects dna and triggers immune activation through transcription factor ifn regulatory factor [ ]. cell-intrinsic initiation of autoimmu- nity has distinct requirements for regulation and unique mech- anisms that precipitate lymphocyte-dependent autoimmunity. autoimmunity may be triggered by cell-intrinsic initiation of the isd pathway in trex -deficient mice, preceding a tlr- dependent contribution to autoantibody production [ ]. disease pathogenesis may reflect samhd protein loss of function instead of partially functional protein expression [ ]. the samhd gene mutation localizes to chromosome q . -q involving a pathogenic, homozygous splice- acceptor site mutation (c. – a > g) in intron . the samhd gene consists of coding exons and encodes a protein of amino acids. this identified mutation results in exon mrna transcription skipping, leading to formation of an aberrant protein. there are two functional domains in the samhd protein: a sterile alpha motif (sam) (residuals – ) and an hd domain (residuals – ) [ ]. the sam domain has the ability to bind rna [ ]. the hd domain recurs in an enzyme superfamily with phosphohydrolase ac- tivity which is involved in nucleic acid metabolism [ ]. clin rheumatol ( ) : – samhd is a dgtp-stimulated triphosphohydrolase; it con- verts deoxynucleoside triphosphates (dntp) to deoxynucleoside and inorganic triphosphate. this gene de- creases dntp to minimal levels that do not support reverse transcription, preventing viral infection [ , ]. samhd also plays a significant role in clearance of cellular waste and maintaining ifn homeostasis [ ]. while this suggests samhd may function as a nuclease, samhd , like trex , is also another potential negative regulator of the isd response. both genes may have a similarly protective, immunomodulatory role in preventing self-activation of in- nate immunity. finally, samhd may further provide a protective role in mediating tnf-α pro-inflammatory responses [ ]. in a case-control study of ags, a phenotypically similar inflammatory disorder caused by mutations in any of six ifn- stimulating genes (samhd , trex , rnaseh a, rnaseh b, rnaseh c and adar), % ( / ) of affect- ed patients vs. % ( / ) of controls had a positive ifn score, reflecting increased expression of these genes [ ]. clinically, ags presents as an infantile encephalopathy, manifesting as progressive microcephaly and psychomotor retardation; % of patients die in early childhood consequent to its leukodys- trophy and microangiopathy. a leading hypothesis regarding ags is that the accumulation of intracellular nucleic acids triggers an auto-inflammatory response and consequent in- creased astrocytic ifn-α production [ ]. in ags, chronic ifn-α exposure results in altered gene expression for proteins involved in the stability of brain white matter (atf , eif bα, cathepsin d, cystatin f), an increase of antigen-presenting genes (human leukocyte antigen class i) and downregulation of pro-angiogenic factors and other cytokines, i.e., vascular endothelial growth factor and interleukin- (il- ). these ef- fects on therapeutic targets for ags and other ifn-α- mediated encephalopathies, may primarily involve down- stream ifn-α signaling cascade effectors rather than only ifn-α [ ]. the relationship of increased il- production and ifn-α signaling in the context of samhd mutation disease is un- clear. however, the model of viral infection provides context for indirect evidence of increased il- expression consequent to ifn-α priming. during viral infection, host cells are alerted by the presence of circulating class i ifn, preparing them for response if they become infected. when a cell recognizes double-stranded rna (dsrna), this indicates the cell is likely already infected with virus. however, when a cell encounters either il- β or tnf-α, the cell receives warning about the host’s escalating inflammatory response. this warning does not indicate direct viral infection of the cell. instead, the class i ifn priming phenomenon promotes a vigorous response to direct viral infection more than il- β or tnf-α provide. while ifn-α does not directly induce il- expression, prior cellular exposure to ifn-α synergistically enhances il- expression in response to the viral dsrna. the magnitude and kinetics of il- induction vary in these situations since class i ifns selectively enhance response to dsrna [ ]. systemic vasculitides with increased il- expression in- clude takayasu’s arteritis [ – ], acute henoch-schönlein purpura [ ], giant cell arteritis [ , ], acute kawasaki dis- ease [ – ], and various large vessel vasculitides [ – ]. tocilizumab therapy provides a promising role in achieving disease remission for refractory neuro-behçet’s disease, re- lapsing or refractory giant cell arteritis, and large vessel vas- culitis secondary to takayasu’s arteritis, cogan syndrome, and relapsing polychondritis [ , – ]. tnf inhibition (typically infliximab) has circumstantial evidence suggesting efficacy in open label use for large vessel vasculitis secondary to refractory takayasu’s arteritis, relaps- ing polychondritis, behçet’s disease (also including adalimumab), and relapsing polychondritis. however, this ap- proach has not been effective for large vessel vasculitis due to giant cell arteritis as demonstrated in randomized controlled trials or a prospective trial for granulomatosis with polyangi- itis [ , ]. adalimumab stabilized our patient’s cerebral vasculitis without remarkably reversing progressive arteriopathy. results are mixed regarding the efficacy of tnf inhibitors for the therapy of lupus pernio. tnf inhibitors can induce this disease [ ]. however, lupus pernio secondary to sarcoidosis responded swiftly to adalimumab in one case report [ ]. tocilizumab has not been reported for this use. neither adalimumab nor tocilizumab were effective for our patient’s sams-associated pernio. conclusions there is no established, uniformly successful treatment strat- egy for sams, an auto-inflammatory disease consequent to homozygous samhd mutation. relapses remain common with prednisone tapering. traditional corticosteroid-sparing, non-biologic immunosuppressive medications used in the treatment of rheumatic diseases involving adaptive immunity, e.g., methotrexate, are ineffective in the treatment of sams cerebral vasculopathy, a disease involving the innate immune system. based on the effectiveness of tocilizumab in control- ling a variety of active large vessel vasculitides including takayasu’s arteritis and giant cell arteritis, tocilizumab pro- vides a novel therapeutic option for sams. following its initiation, mm obtained disease control within months, followed by substantial reversal of much of his cerebral vas- culopathy after nearly a year of tocilizumab treatment. with this apparent efficacy and durability, tocilizumab may offer a means of controlling cerebral vasculopathy from the auto- inflammatory disease sams. clin rheumatol ( ) : – acknowledgements none. compliance with ethical standards funding source no funding was secured for this study. financial disclosure authors have no financial relationships relevant to this article to disclose. disclosures none. open access this article is distributed under the terms of the creative commons attribution . international license (http:// creativecommons.org/licenses/by/ . /), 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/archdermatol. . http://dx.doi.org/ . /archdermatol. . 	tocilizumab reverses cerebral vasculopathy in a patient with homozygous samhd mutation 	abstract 	introduction 	case report 	discussion 	conclusions 	references asmscience | peroxisomes mount first-line antiviral defense toggle navigation toggle navigation more toggle navigation toggle navigation 	 personal account sign-in / register access provided by: carnegie mellon university (# ) asm member status join asm 	 sign-in as administrator 	 	 username: password: sign in need login help? 	 sign out 	 	 www.asm.org 	 membership 	 subscriptions 	 contact us 	 home 	 search/browse selected topics coming soon! concepts/taxonomy search content by concept advanced search advanced search and filter tools 	 journals/magazines open access journals microbiology resource announcements™ journal of microbiology & biology education® mbio® msphere® msystems® subscription journals antimicrobial agents and chemotherapy® applied and environmental 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albicans can be a benign inhabitant of the human digestive tract or an invasive pathogen; researchers exploring the surface proteins of this yeast find that both types express many of the same surface proteins (see p. ). (image © david m. phillips/photo researchers, inc.) /docserver/fulltext/microbe/ / /coverfig.gif /docserver/fulltext/microbe/ / /coverfig.gif microbe magazine, cover image cover imagecover: scanning electron micrograph of candida albicans. c. albicans can be a benign inhabitant of the human digestive tract or an invasive pathogen; researchers exploring the surface proteins of this yeast find that both types express many of the same surface proteins (see p. ). (image © david m. phillips/photo researchers, inc.) full text loading... /deliver/fulltext/microbe/ / /znw a.html?itemid=/content/journal/microbe/ . /microbe. . . &mimetype=html&fmt=ahah related content /content/journal/microbe/ . /microbe. . . pub_author -contenttype:journal -contenttype:contributor -contenttype:concept -contenttype:institution related content from pubmed carol potera 	 personal account sign-in / register access provided by: carnegie mellon university (# ) asm member status join asm 	 sign-in as administrator 	 username: password: sign in need login help? 	 sign out 	 share tools 	 export citations 	 bibtex 	 endnote 	 zotero 	 plain text 	 refworks 	 mendeley 	 recommend to library these fields are mandatory: * librarian details name: * email: * your details name: * email: * department: * why are you recommending this title? select reason: i need to refer to this publication frequently this publication is an essential resource for my studies/research i'll refer my students to this publication 	 i'm an author/editor/contributor to this publication 	 i'm a member of the publication's editorial board 	 other:   eventtype:personalisation;jsessionid:muuqa uodczdedzybfymt_u.asmlive- - - - ;itemid:http://asm.metastore.ingenta.com/content/journal/microbe/ . /microbe. . . ;timestamp: invalid site public key invalid site private key invalid request cookie incorrect. try again. unabled to verify parameters could not contact recaptcha for validation i am not a robot * recommend — recommend this title to your library thank you your recommendation has been sent to your librarian. 	 request permission/order reprints access key 	 free content 	 open access content 	 subscribed content back to top about us 	about asm 	contact us 	terms of use 	privacy policy 	copyright & licenses 	public access policy 	permission & reprints resources 	 for authors 	 for librarians 	 for advertisers 	 for instructors 	 for reviewers 	 for the press publications 	 individual member journal subscriptions 	 journal of microbiology & biology education® 	microbiology spectrum® 	ecosal plus® 	books 	colloquia reports 	faq series 	protocols 	curriculum archive american society for microbiology © this is a required field please enter a valid email address please check the format of the address you have entered. approval was a success invalid data an error occurred approval was partially successful, following selected items could not be processed due to error - .. mammographic breast density—evidence for genetic correlations with established breast cancer risk factors julie a. douglas, marie-hélène roy-gagnon, chuan zhou, braxton d. mitchell, alan r. shuldiner, heang-ping chan, and mark a. helvie departments of human genetics and radiology, university of michigan medical school, ann arbor, michigan; and department of medicine, university of maryland medical school, baltimore, maryland abstract previous twin and family studies indicate that the familial aggregation of breast density is due (in part) to genetic factors. whether these genetic influences are shared with other breast cancer risk factors, however, is not known. using standard film-screen mammogra- phy, we screened women, including pairs of sisters, from the old order amish population of lancaster county, pennsylvania. we digitized mam- mograms and quantified the dense and nondense areas of the breast using a computer-assisted method. information about other breast cancer risk factors was collected via questionnaires and a physical exam. using pedigree-based variance component methods, we estimated the genetic contributions to several breast cancer risk factors, including breast density, and eval- uated the evidence for shared genetic influences between them. after adjusting for covariates, genetic effects accounted for > % of the total variance of each risk factor (p < . ), including breast density, and the dense and nondense areas of the breast were significantly genetically correlated with parity [genetic correlation (rg) = - . ; p = . ] and age at menarche (rg = - . ; p = . ), respectively. the nondense area of the breast and, in turn, breast density, expressed as a ratio of dense area to total area, were also genetically correlated with most measures of adipo- sity but in opposite directions (rg z . ; p < - for nondense area). we conclude that the genetic compo- nents that influence breast density are not independent of the genetic components that influence other breast cancer risk factors. this shared genetic architecture should be considered in future genetic studies of breast density. (cancer epidemiol biomarkers prev ; ( ): – ) introduction breast cancer is the most common cause of cancer-related mortality in women worldwide ( ). among breast can- cer risk factors, increased breast density, as measured from a mammogram, is one of the strongest but perhaps least understood ( ). mammographic breast density refers to the radiographic dense areas on a mammogram and is a measure of the amount of fibroglandular tissue in the breast. studies have repeatedly shown that women with dense tissue in > % of the breast are at a - to -fold increased risk of developing breast cancer com- pared with women with little to no breast density ( , ). some studies also suggest that breast cancer risk is directly associated with ( - ) and may be even better predicted by ( ) the absolute amount of dense tissue. at present, however, the most commonly used quanti- tative measure of breast density is the ratio of dense area to total area. while breast density (measured as a ratio) may be a useful prognostic indicator of breast cancer risk, there are several undesirable consequences of using it in the context of etiologic research ( ). for example, ratios can be difficult to interpret because of the potential confounding due to the nondense compo- nent of the denominator, which reflects the amount of fat in the breast. still, only a few studies have separately measured and analyzed the dense and nondense com- ponents, and even fewer have compared the inferences made from absolute versus relative measures of breast density ( , ). twin and family studies have established evidence for a significant genetic influence on breast density. for example, in a study on monozygotic and dizygotic twin pairs from the united states and australia, unmeasured genes accounted for > % of the variation in percent ( ) [and absolute ( )] breast density after adjustment for age and other covariates. although the mode of inheritance of breast density is likely to be complex, vachon and colleagues ( ) previously implicated the transmission of a major gene for percent breast density in a study of , women from multigenerational breast cancer families. in a subsequent genomewide scan based on women from of these families, vachon et al. ( ) also recently reported significant evidence of linkage for percent breast density on chromosome p. although f candidate genes are located within the -lod (log of odds) support interval surrounding their chromosome cancer epidemiol biomarkers prev ; ( ). december received / / ; revised / / ; accepted / / . grant support: nih (grant ca ), fashion footwear charitable foundation of new york/qvc presents shoes on sale, gladys e. david endowed fund, and elizabeth caroline crosby research award (j.a. douglas). requests for reprints: julie a. douglas, room , buhl building, e. catherine street, ann arbor, mi - . phone: - - ; fax: - - . e-mail: jddoug@umich.edu copyright d american association for cancer research. doi: . / - .epi- - on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ p peak, to our knowledge, none have been tested for association with breast density. indeed, candidate gene studies of breast density are still in their infancy, with a relatively small number of genes examined and few, if any, clear associations. in addition to having a documented genetic compo- nent, breast density is known to vary with age, reproductive and menstrual history, and measures of body size. studies have consistently shown that breast density is inversely associated with age and, among women of the same age, is lower in those who are parous, have had a larger number of live births, or are postmenopausal ( ). as a ratio, breast density is also inversely associated with several measures of body size, including body mass index (bmi) and weight ( ). finally, observational studies and clinical trials indicate that breast density is higher in women who are using combination (estrogen plus progestin) hormone replace- ment therapy ( , ). many of these breast density – associated traits are also well-recognized breast cancer risk factors, with several exhibiting associations in the same direction as their effects on breast density. for example, nulliparity and hormone replacement therapy use are associated with increased breast density and breast cancer risk ( - ). although these relationships are thought to reflect hormonal exposures, the actual biological pathways by which these risk factors operate on breast density (and breast cancer risk) are not known. because breast density is associated with several breast cancer risk factors that also have significant heritable components, we hypothesized that some of the observed phenotypic correlations between them are attributable (in part) to genetic factors. we address these hypotheses here in the context of an ongoing family- based genetic study of breast density in women from the old order amish population of lancaster county, pennsylvania. although the overall goal of our study is to identify genes that influence breast density, the aim of the current investigation is to estimate the genetic contributions to individual differences in breast density and associated breast cancer risk factors and assess the evidence for shared genetic influences between them. whether shared genetic factors influence these traits is relevant to ongoing and future genetic studies of breast density, including our own. materials and methods overall study design. we recruited women from the old order amish population, with an emphasis on pairs of sisters, as part of an ongoing study to identify the genetic factors that influence mammographic breast density. participants were identified by word-of-mouth and door-to-door interviews. between june and october , we had approached a total of , women, including ( %) who had participated, ( %) who had declined, ( %) who did not meet our eligibility criteria, and ( %) who withdrew after consenting. for the present investigation, our final analysis-ready sample included women. all old order amish women were eligible to participate if they were z years of age at the time of interview and had at least one living sister who was also z years of age. women were excluded if they (a) were pregnant or lactating in the previous months, (b) had ever been diagnosed with breast or ovarian cancer, (c) had one or both ovaries removed, or (d) used exogenous sex steroid hormones in the previous months. to study natural variation in breast density, it was necessary to exclude women who had taken exogenous hormones and/or whose endogenous hormone production may have been medically altered. although suspension of exogenous hormone use for f weeks seems to reverse the mammographic breast density increase associated with its use ( ), we elected to apply the more conservative -month exclusion criteria. the impact of using this more stringent threshold is likely minimal since < % of our participants reported having ever used exogenous hormones. measurement of mammographic breast density. women who had not had a mammogram in the previous months were screened via standard, two-view film screen mammography at a mammography qualified standard act – approved site (n = ). for women who had had a mammogram in the previous months (n = ), we requested medical record release of their most recent mammogram and report. craniocaudal views were digitized with a lumisys laser film scanner at a pixel size of . � . mm and , gray levels. a single radiologist (m.a.h.) measured total breast area and absolute dense area from a digitized craniocaudal view of the right and/or left breast using interactive thresholding and our computer-assisted program mammographic density estimator ( ). addi- tional technical details of our approach ( ), including an evaluation study ( ), are described elsewhere. for comparative purposes, relative density was calculated as the ratio of the dense area to the total area, and the nondense area was calculated as the difference between the total and dense areas. based on data from the first women, agreement between percent breast density estimates from the left and right craniocaudal views was high, with a mean absolute difference of . %, a root mean squared error of %, and a within-individual r of . . when we rescored an f % random sample of films distributed across the range of percent breast density ( right craniocaudal views and left cranio- caudal views), the intrareader variability was equally low, with a mean absolute difference of . %, a root mean squared error of . %, and a within-individual r of . for percent density. agreement between estimates for left and right craniocaudal views and intrareader variability were comparable for absolute dense area. because our measurements of breast density were highly reproducible and differences in estimates from both breasts did not exceed differences from repeated readings of the same breast, we present results below using the right craniocaudal view for women and (for technical reasons) the left craniocaudal view for the remaining women. questionnaires and anthropometric measurements. information on medical, reproductive, menstrual and family history, and medication use was collected using standardized questionnaires adapted for use in this population. medical history information included physi- cian-diagnosed cancer, osteoporosis, diabetes, heart attack, stroke, and surgeries. reproductive or menstrual information included current and past frequency of mammographic breast density cancer epidemiol biomarkers prev ; ( ). december on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ menstrual bleeding, childbearing and breastfeeding history, and ages at menarche, first birth, and meno- pause. family history was limited to the number of first- degree relatives by relationship type, history of breast or ovarian cancer, age at diagnosis, and, if deceased, age and cause of death. history of breast or ovarian cancer in paternal and maternal grandmothers and age at diagno- sis were also sought. because smoking (especially among women) and alcohol consumption are infrequently practiced among the amish ( ), we did not collect this information. trained nurses measured height and weight using a stadiometer and calibrated scale, with shoes removed and in light clothing. bmi was calculated as weight (kilogram) divided by the square of height (square meter). waist circumference was measured at the level of the umbilicus, and hip circumference was measured at the widest protuberance across the pelvis. we defined participants as postmenopausal if they reported having natural menopause and no menstrual bleeding in the previous months, and we defined women as premenopausal if they reported having menstrual bleeding in the previous months. women who reported a hysterectomy (n = ) were defined as post- and premenopausal if they were > and v years of age, respectively, the ages at which natural menopause had occurred in f % and f % of participants. in other words, after excluding participants who reported a hysterectomy, natural menopause had occurred in f % of women who were > years of age ( of ) and in f % of women who were v years of age ( of ). participants reported no other form of surgical meno- pause besides hysterectomy (without oophorectomy). statistical analysis. in total, we analyzed three breast measures (dense area, nondense area, and percent density), four reproductive or menstrual traits (number of live births and ages at menarche, first birth, and menopause), and four measures of body size (height, weight, bmi, and waist circumference). before conducting the quantitative genetic analyses described below, we assessed the distributions of all traits and, where necessary, transformed them to approximate univariate normality. a logarithm transformation was applied to the dense area of the breast, percent breast density, age at menarche, bmi, weight, and waist circumference, and power transformations were applied to the nondense area of the breast ( . ) and age at menopause ( ). all other variables were left untransformed. we used standard variance and covariance component models and pedigree- based maximum likelihood methods ( , ) as imple- mented in solar (sequential oligogenic linkage analysis routines) ( ) to estimate trait heritabilities and to investigate the genetic and environmental correlations between pairs of traits. pedigree relationships were determined from the anabaptist genealogy database (version . ; ref. ) by including genealogic information on the parents and grandparents of the study participants. to estimate heritability, we partitioned variation in each trait, for example, dense area of the breast, into a component due to individual-specific covariates, includ- ing age and menopausal status, the additive genetic variance (ra ), which captures the effects of unmeasured genes, and an individual-specific environmental compo- nent or residual error. the heritability (h ) of each trait was estimated by the ratio of the variance attributable to the additive genetic effects (ra ) and the phenotypic variance after adjustment for covariates (radj ). we assessed the significance of particular components, for example, ra , using standard likelihood ratio tests, that is, by comparing the likelihood of a model in which the component was estimated to the likelihood of a model in which the component was constrained to be zero. given our sibling pair design, we were unable to distinguish and therefore estimate genetic dominance and shared sibling environ- ment. we estimated the proportion of the total phenotypic variance explained by the additive genetic variance as the product of the heritability estimate and minus the proportion of the variance explained by the covariates (rc ), that is, ( - rc ) (h ). to evaluate the evidence for genetic effects jointly influencing breast density and other breast cancer risk factors, we used bivariate variance component models to partition the phenotypic correlation (qp) between each pair of traits, for example, dense area and number of live births, into components attributable to the same additive genetic effects (qg or genetic correlation) and the same environmental effects (qe or environmental correlation). briefly, based on the heritabilities of the two traits (h and h ), the phenotypic correlation be- tween the traits can be expressed as a weighted sum of their genetic and environmental correlations, namely, qp = qg [(h h )] / + qe [( - h )( - h )] / . the genetic correlation (qg) captures the extent to which the same genes influence both traits, whereas the environmental correlation (qe) captures the extent to which the same environmental factors influence both traits. because significant genetic and/or environmental correlations can arise from nonsignificant phenotypic correlations, for example, when the genetic and environmental correla- tions have opposite signs, we analyzed each pair of traits without regard to their overall phenotypic correlation. using likelihood ratio tests, we evaluated two hypothe- ses involving the genetic correlation. first, we tested whether the genetic correlation was zero (qg = ). rejection of this hypothesis suggests that one or more of the same genetic factors influence both traits. second, we tested whether the genetic correlation was or - (qg = or - ). rejection of this hypothesis suggests that there exist one or more unique genetic factors that influences one trait but not the other. lastly, we also tested whether the environmental correlation was zero (qe = ). rejection of this hypothesis suggests that one or more of the same environmental factors (unmeasured or unadjusted for) influence both traits. all statistical tests were necessarily one sided, and p values < . were considered statistically significant. no adjustments for multiple comparisons were made. we assessed the impact of outliers on the estimates of heritability and genetic and environmental correlation by examining the change in estimates after excluding extreme values, which we defined by > sds from the mean. all analyses (except where noted previously) were conducted using version . of the statistical analysis system programming language (sas institute). human subjects approval. the institutional review boards at the universities of michigan and maryland approved all aspects of the protocol, and all participants gave written informed consent, including permission to release their medical records. cancer epidemiology, biomarkers & prevention cancer epidemiol biomarkers prev ; ( ). december on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ results for this investigation, our sample included women from distinct sibships, with to women per sibship. of these sibships, %, %, and % were composed of two, three, and four or more participants, respectively. table summarizes the number of pairwise relationships among all women after merging in genealogical information on their parents and grandparents. in total, there were pairs of first-degree relatives, including sister-sister and mother-daughter pairs, and , more distantly related pairs. table describes selected characteristics of the participants. all women were between the ages of and years, with a mean of years. there were and pre- and postmeno- pausal women, respectively. after excluding the postmenopausal women who reported previous surgical removal of their uteri, the average age at natural menopause was f years (fsd). fewer than % of all participants reported previous use of exogenous hormones, and none had taken hormones in the previous months (per our exclusion criteria). most women were parous ( %), with an average of live births. mean (fsd) dense area, nondense area, and propor- tion of dense area were f cm , f cm , and . f . , respectively. as expected, breast density was higher in premenopausal women than in postmenopaus- al women (data not shown) and was inversely associated with age (fig. ). age and menopausal status were significantly correlated with log-transformed dense area and percent density and accounted for % and % of the variation in each trait, respectively (table ). after adjusting for these covariates, the heritability of dense area was % and was significantly different from zero (p < - ). of the total variation in dense area, age and menopausal status accounted for %, additive genetic factors accounted for % [( - . )( . )], and % remained unexplained. similarly, of the total variation in percent density, covariates explained %, additive genetic factors accounted for %, and % remained unexplained. the heritability of the nondense area of the breast was % (p < - ) after adjusting for covariates. after transformation and adjustment for age and menopausal status, the dense and nondense areas were both genetically and environmentally correlated. the genetic and environmental correlations (fse) were . f . and - . f . , respectively, and both were significantly greater and less than zero (p = . and . , respectively), suggesting the presence of shared genetic and environmental factors exerting similar and opposite effects, respectively, on the dense and nondense areas of the breast. at the same time, the genetic correlation between these two areas was significantly < , suggesting that there also exist unique genetic factors influencing each of these traits. based on these estimates and estimates of the trait heritabilities, the corresponding phenotypic correlation between the dense and nondense areas was close to zero ( . ), consistent with the within- individual partial r (data not shown). heritability estimates for the other breast cancer risk factors were also significantly different from zero, with % to % of the total variation in each trait attributable to additive genetic effects (table ). estimates of the genetic correlation between each of these traits and each breast measure are given in table (after transformation and adjustment for age and menopausal status). all genetic correlations were significantly different from f (data not shown). in addition, the dense area of the breast was inversely related to and significantly genet- ically correlated with the number of live births (- . f . ; fse; p = . for test of qg = ). thus, the inverse correlation between the dense area of the breast and live birth number may be attributable (in part) to genetic factors. similarly, the nondense area of the breast was significantly genetically correlated with age at menarche (- . f . ; p = . for test of qg = ). as expected, the nondense area of the breast was positively and strongly genetically correlated with most body size measures (p < - for test of qg = ), including weight (qg = . ), bmi (qg = . ), and waist circumference (qg = . ). similar genetic correlations were observed for percent breast density but in the opposite direction. the genetic correlations between the remaining pairs of traits were low and not significantly different from zero. estimates of the environmental correlation between each breast measure and each of the other breast cancer risk factors are also given in table (after transforma- tion and adjustment for age and menopausal status). the dense area of the breast was positively and significantly environmentally correlated with age at menarche ( . f . ; p = . ) and height ( . f . ; p = . ) and negatively and significantly table . number of pairwise relationships among study participants relationship pair no. of pairs sister-sister mother-daughter aunt-niece double first cousins first cousins , first cousins, once removed half first cousins half first cousins, once removed second cousins half second cousins note: pedigree relationships were determined by merging in the parents and grandparents of the study participants (n = ). table . selected characteristics of study participants (n = ) mean f sd range age (y) f - premenopausal* ( ) age at menarche (y) f - age at natural menopause (y) f - ever used hormones* ( ) reproductive factors parous* ( ) number of live births f c - age at first birth (y) f c - ever breast fed* ( ) c body size measures height (cm) f - weight (kg) f - bmi (kg/m ) f - waist circumference (cm) f - *number (and percentage). cbased on parous women. mammographic breast density cancer epidemiol biomarkers prev ; ( ). december on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ environmentally correlated with bmi (- . f . ; p = . ). similarly, the nondense area of the breast was positively and significantly environmentally corre- lated with the number of live births ( . f . ; p = . ), as well as most body size measures, including weight ( . f . ; p = . ), bmi ( . f . ; p = . ), and waist circumference ( . f . ; p = . ). the environmental correlations between percent breast density were similar and in the same (opposite) direction as they were for the dense (nondense) area. the environmental correlations between the remaining pairs of traits were low and not signifi- cantly different from zero. together, these results suggest that there exist individual-specific but unmeasured environmental factors that contribute to the correlations between several pairs of these traits. we repeated all heritability and genetic and environ- mental correlation analyses after removing individuals with extreme values. with the exception of the environ- mental correlation between percent breast density and age at first birth, all heritability and correlation estimates from these analyses were within se of the original estimates. discussion with their unique cultural customs and relatively similar environmental exposures, a well-defined, genetically closed population structure, and extensive genealogic records, the old order amish provide an ideal context in which to study the genetic contributions to breast density. of particular relevance to studying breast density, the old order amish population is character- ized by a very low prevalence of exogenous hormone use, including oral contraceptives and hormone replace- ment therapy, and high parity. still, our results suggest that breast density varies widely in the old order amish population, with values that are comparable with other highly parous populations. for example, in a sample of hispanic women (two thirds of whom were postmenopausal and three fourths of whom reported three or more live births), lopez et al. ( ) reported an overall mean of . % for percent breast density, with a range of . % to . %. similarly, in our sample of women (approximately two thirds of whom were also postmenopausal and three fourths of whom reported five or more live births), the mean and range of percent breast density were . % and . % to %, respectively. to our knowledge, our study is the first non-twin study to estimate the genetic contributions to the dense and nondense areas of the breast and the first study to examine the contribution of genetic factors to the correlation between breast density and other breast cancer risk factors. we found that the dense and nondense areas of the breast were significantly heritable in our sample, with % and % of the total variance, respectively, attribut- able to additive genetic effects. although these estimates are consistent with the significant genetic influences reported by stone et al. ( ), comparisons of heritability are always ill advised. for example, with respect to the environmental factors that impact breast density, the women in this sample likely share relatively similar environments. thus, all one can infer from a relatively higher (or lower) estimate of heritability is that there is less (or more) environmental variation relative to the genetic variation in this sample. we note that screening and adjusting for other significant covariates (in addition to age and menopausal status) did not meaningfully alter our estimates of the heritability of absolute breast density. in fact, age at menarche and number of live births were the only other covariates significantly correlated with log- transformed dense area, and together, they explained no more than an additional % of the variation in this trait. after including all four covariates in our model, the heritability of the dense area of the breast was % (versus % with adjustment for age and menopausal status only). figure . interindividual variability in dense area (a) and percent breast density (b) by age (n = ). horizontal black bars, median; boxes, interquartile range; whiskers, . times the interquartile range. cancer epidemiology, biomarkers & prevention cancer epidemiol biomarkers prev ; ( ). december on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ we also found that other breast cancer risk factors were significantly heritable and that some of their associations with breast density were due to an under- lying structure of shared genetic (and environmental) effects. particularly noteworthy is our finding that breast density and live birth number are genetically correlated. it has been commonly hypothesized that the inverse association between these two traits is due to a decrease in the proliferative activity of the parous epithelium and, in turn, that the subsequent decreased risk for breast cancer is due to the differentiation (during pregnancy) of lobular type to cells, which are assumed to be less susceptible to malignant transformation. our results are consistent with this hypothesis and suggest that a significant component of this association may be due to genetic factors that influence breast density and live birth number (or fertility) in opposite directions. because the old order amish discourage the use of contra- ceptives and share a relatively uniform socio-cultural and -economic background, we were in a unique position to study this relationship. of note, our finding of a significant genetic component to fertility is also consis- tent with a recent report by pluzhnikov et al. ( ), who studied both components of reproductive fitness (fertility table . heritability estimates (h ) for breast measures and other breast cancer risk factors trait h f se p proportion of total variance explained by covariates genes dense area . f . . � - . . percent density . f . . � - . . nondense area . f . . � - . . age at menarche . f . . � - < . . number of live births . f . . � - < . . age at first birth . f . . � - < . . age at natural menopause . f . . � - . . height . f . . � - . . weight . f . . � - . . bmi . f . . � - . . waist circumference . f . . � - < . . note: data in second column are after adjustment for age and menopausal status. dense area, percent density, age at menarche, weight, bmi, and waist circumference were log transformed. nondense area and age at natural menopause were power transformed ( . and , respectively). other variables were not transformed. table . genetic and environmental correlations (rg and re, respectively) between breast measures and other breast cancer risk factors trait trait qg f se ue f se dense area age at menarche - . f . . f . * number of live births - . f . c - . f . age at first birth - . f . . f . age at natural menopause . f . - . f . height - . f . . f . c weight . f . - . f . bmi . f . - . f . c waist circumference . f . - . f . percent density age at menarche . f . . f . number of live births - . f . - . f . c age at first birth - . f . . f . age at natural menopause . f . - . f . height - . f . . f . c weight - . f . * - . f . b bmi - . f . * - . f . b waist circumference - . f . b - . f . * nondense area age at menarche - . f . * . f . number of live births - . f . . f . c age at first birth . f . - . f . age at natural menopause . f . . f . height . f . - . f . weight . f . b . f . * bmi . f . b . f . b waist circumference . f . b . f . * note: data are adjusted for age and menopausal status. dense area, percent density, age at menarche, weight, bmi, and waist circumference were log transformed. nondense area and age at menopause were power transformed ( . and , respectively). *p < . for test of null hypothesis that qg = (or qe = ), that is, correlation due to additive genetic factors is zero (or correlation due to unmeasured environmental factors is zero). cp < . for test of null hypothesis that qg = (or qe = ), that is, correlation due to additive genetic factors is zero (or correlation due to unmeasured environmental factors is zero). bp < . for test of null hypothesis that qg = (or q e = ), that is, correlation due to additive genetic factors is zero (or correlation due to unmeasured environmental factors is zero). mammographic breast density cancer epidemiol biomarkers prev ; ( ). december on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ and mortality) in the hutterites and found significant familial correlations in family size. at present, however, the genes that influence fertility in human populations are unknown, partly owing to the difficulty of controlling for the influence of nongenetic factors. our results suggest it may be ill advised to adjust for live birth number in the genetic analysis of breast density given the strong genetic correlation between them. based on samples of unrelated women, boyd et al. ( ) and haars et al. ( ) previously showed that the inverse correlations of various measures of adiposity with breast density, expressed as a percentage of total breast area, are due to positive correlations with the nondense area of the breast. our data are consistent with these observa- tions and suggest that many of these correlations may have a common and strong genetic basis. specifically, in our sample, several measures of body size exhibited strong and significant positive genetic correlations with the nondense (but not dense) area of the breast. for example, approximately two thirds of the phenotypic correlation between the nondense area of the breast and weight ( . ) was due to the same genetic factors after adjusting for age and menopausal status. thus, any genetic analysis of percent breast density will be strongly confounded by adiposity. one such example is provided by vachon et al. ( ), who recently reported that their linkage evidence on chromosome p for percent breast density nearly doubled after adjustment for bmi. although vachon et al. ( ) recognized that percent breast density was genetically correlated with bmi in their sample ( . ), they were unable to analyze the dense and nondense areas separately because only percent density was characterized. in our sample, the nondense area of the breast was also significantly (negatively) genetically correlated with age at menarche. age at menarche was, in turn, significantly (negatively) genetically correlated with each of the adiposity measures described above (data not shown). together, these correlations are consistent with findings from a recent study by wang et al. ( ), who reported significant negative genetic correlations between several obesity phenotypes, including bmi, and age at menarche. as described by wang et al. ( ), these findings are biologically consistent with documented differences in hormonal concentrations and fat distribution in women who experience early versus late menarche. in addition to identifying significant genetic correla- tions between the dense and nondense areas of the breast and other breast cancer risk factors, we also found that the environmental correlations were significantly differ- ent from zero for several trait pairs. for example, the dense area of the breast was positively environmentally correlated with age at menarche and height. these findings imply the existence of other important cova- riates that were either not included in our models or, more likely, not measured in our study and are consistent with the individual-specific effects noted in our univariate analyses. for example, f % of the total variability in the dense area of the breast was unex- plained by measured covariates and unmeasured addi- tive genetic factors. factors that may have contributed to this unexplained variation (and environmental correla- tion with other traits) include exposures that may have occurred earlier in life, for example, dietary intake and hormones. indeed, some of the hormonal factors that influence height also seem to regulate mammary gland development ( ). based on an analysis of monozygotic and dizygotic twins, stone et al. ( ) previously reported a negative genetic correlation between the dense and nondense areas of the breast [- . f . (fse) after a logarithm transformation and adjustment for covariates]. in our sample, however, the genetic correlation between these areas was positive ( . f . after transformation and adjustment for covariates). in other words, data from stone et al. ( ) suggest that there exist common genetic influences that act in opposite directions on the dense and nondense areas, whereas the data presented here suggest that these shared genetic influences operate in the same direction. it is interesting to note that the within-individual correlation between the dense and nondense areas was also remarkably different between our two studies [after adjustment for age, . in our sample versus - . in the sample of stone et al. ( )] but consistent with our study-specific environmental corre- lations, which were similar in sign and magnitude (- . f . in our sample and - . f . in their sample). because our parameterizations, populations of inference, and study designs are not directly comparable, it is difficult to reconcile these differences. data from the present study add to the accumulating evidence that breast density has a strong heritable component and provide new evidence that part of this heritable component is shared with other breast cancer risk factors. still, we acknowledge several study limi- tations. first, given our study design, we were unable to examine the influence of shared environments. for example, to the extent that shared childhood environ- ments contribute to correlations in breast density between sisters, we may have overestimated the genetic contributions to individual differences in (and correla- tions between) breast density and other breast cancer risk factors. second, our findings may not generalize to other populations, particularly given the unique reproductive practices of the old order amish. despite this, our study participants were similar in many other ways to the u.s. female caucasian population as determined by our analysis of age-matched data from the - nation- al health and nutrition examination surveys (data not shown). third, we were unable to examine (with confidence) the relationship between breast density and an important breast cancer risk factor, namely, family history of breast cancer. irregular medical care practices in this population make it difficult to obtain and/or verify information on family cancer history. fourth, with our modest sample size, we were underpowered to examine the extent to which genetic variances and correlations were menopausal specific. tentative exam- ination of menopausal-specific estimates of heritability and genetic and environmental correlations, however, suggests that the relative contributions of genetic and nongenetic factors were similar in pre- and postmeno- pausal women (data not shown). in summary, our results indicate that breast density varies widely in the old order amish population and is strongly influenced by genetic factors. our results also suggest that the genetic and environmental factors that influence breast density are not independent of the genetic and environmental factors that influence other breast cancer risk factors. these findings are being used cancer epidemiology, biomarkers & prevention cancer epidemiol biomarkers prev ; ( ). december on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ to inform our ongoing genetic investigation of breast density in the old order amish. the evidence presented here for shared genetic influences on breast density and other breast cancer risk factors may lead to more powerful searches for the loci and genes that influence breast density. indeed, the power to identify loci that influence breast density may be increased by jointly analyzing genetically correlated traits ( , ). disclosure of potential conflicts of interest no potential conflicts of interest were disclosed. acknowledgments the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked advertisement in accordance with u.s.c. section solely to indicate this fact. we thank the members of the amish community for their out- standing support and participation in this study; the members of the amish research clinic for their dedicated recruitment and fieldwork efforts; the members of dr. margarita shultz’s radiology clinic for their expert mammography services; and terry gliedt, jennifer greene, lubomir hadjiiski, albert levin, kristen maas, and cris van hout at the university of michigan for their technical assistance with data management and entry, pedigree construction, figure preparation, and digitization. references . kamangar f, dores gm, anderson wf. patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. j clin oncol ; : – . . couzin j. breast cancer. dissecting a hidden breast cancer risk. science ; : – . . boyd nf, byng jw, jong ra, et al. quantitative classification of mammographic densities and breast cancer risk: results from the canadian national breast screening study. j natl cancer inst ; : – . . byrne c, schairer c, wolfe j, et al. mammographic features and breast cancer risk: effects with time, age, and menopause status. j natl cancer inst ; 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: – . . almasy l, dyer td, blangero j. bivariate quantitative trait linkage analysis: pleiotropy versus co-incident linkages. genet epidemiol ; : – . . klei l, luca d, devlin b, roeder k. pleiotropy and principal components of heritability combine to increase power for association analysis. genet epidemiol ; : – . mammographic breast density cancer epidemiol biomarkers prev ; ( ). december on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ ; : - . cancer epidemiol biomarkers prev julie a. douglas, marie-hélène roy-gagnon, chuan zhou, et al. correlations with established breast cancer risk factors evidence for genetic−−mammographic breast density updated version http://cebp.aacrjournals.org/content/ / / access the most recent version of this article at: cited articles http://cebp.aacrjournals.org/content/ / / .full#ref-list- this article cites articles, of which you can access for free at: citing articles http://cebp.aacrjournals.org/content/ / / .full#related-urls this article has been cited by highwire-hosted articles. access the articles at: e-mail alerts related to this article or journal.sign up to receive free email-alerts subscriptions reprints and .pubs@aacr.orgdepartment at to order reprints of this article or to subscribe to the journal, contact the aacr publications permissions rightslink site. (ccc) click on "request permissions" which will take you to the copyright clearance center's .http://cebp.aacrjournals.org/content/ / / to request permission to re-use all or part of this article, use this link on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/content/ / / http://cebp.aacrjournals.org/content/ / / .full#ref-list- http://cebp.aacrjournals.org/content/ / / .full#related-urls http://cebp.aacrjournals.org/cgi/alerts mailto:pubs@aacr.org http://cebp.aacrjournals.org/content/ / / http://cebp.aacrjournals.org/ modern amish farming as ecological agriculture modern amish farming as ecological agriculture katharine v. blake enrico a. cardamone steven d. hall glenn r. harris susan m. moore environmental studies program st. lawrence university canton, new york, usa critics of industrial agriculture and advocates of ecological agriculture have cited amish farming as a model of stewardship and sustainability. amish farming in st. lawrence county, new york, embodied ecological agriculture in some respects but not others. in comparison with non-amish neighbors, amish farms were smaller in scale, more diverse, and less integrated into the market economy. on the other hand, use of fertilizers and pesticides for crop production appeared to differ in kind, not amount. amish farmers relied primarily on their own experience, not trade magazines or the local cooperative extension, for agricultural information. the high use of petro- leum-based inputs may have reflected the newness of amish settlement in st. lawrence county, a lack of awareness of the ecological impacts of these substances, or a shift away from traditional practices. in the self-sufficiency of their lives based on subsistence and diversity, these amish otherwise exemplified the productive and self-regulatory characteristics of ecological agriculture. keywords amish agriculture, amish pesticide use, ecological agriculture, new york state agriculture, stewardship, sustainability for many decades, the amish way of life has been recognized as one of simplicity, where necessity and practicality guide decision making and lifestyle. in light of this perception, it is only logical that amish agriculture would be thought to represent "ecological agri- culture." a philosophy of reaping what one sows and savoring the simple pleasures de- rived from daily labor is reflected in amish agricultural practices (kolodge, ). the religious constructs of amish society contribute to this notion by making it detrimental to harm the land or destroy something not made by human hands. it can be said that the amish strive for a harmony among god, nature, family, and community (berry, ; hosteller, ). ecological agriculture encompasses the principles of organic farming, in which only natural processes and products are used as inputs to grow crops. beyond organic farming, ecological agriculture places an additional focus on the larger environmental, social, and economic impacts of farming practices (goering et al., ). ecological farming ulti- mately attempts to achieve a diverse, healthy, and productive ecosystem in which biologi- received february ; accepted december . the authors gratefully acknowledge the constructive suggestions of four anonymous review- ers, as well as the assistance of nancy alessi, who helped prepare the manuscript for publication. address correspondence to glenn r. harris, environmental studies program, st. lawrence university, canton, ny , usa. e-mail: ghar@music.stlawu.edu. society & natural resources, : - , copyright © taylor & francis - / $ . + . d ow nl oa de d by [ l ib r i] a t : d ec em be r k.v. blake etal. cal processes and cycles dominate. the emphasis is on agricultural methods that are sus- tainable as well as organic. a sustainable farm is one in which the ecosystem on the farm can reach a sort of permanence (soule and piper, ), accomplished when nutrient in- puts and eventual outputs are balanced and do not exhaust the natural resources of the farm. the resulting dynamic equilibrium can adapt to changes and is considered to be ecological because the sustainable farm operates much as a natural system would. the maintenance and stabilization of the dynamic equilibrium on the farm occurs as a result of diversity in crops and farm animals, the avoidance of potential pollution, and the re- placement of petroleum products with renewable resources. in addition, alternative means of weed and pest control are preferred to the use of herbicides and insecticides. alterna- tive means may include crop rotation, integrated pest management, cultivation, or biolog- ical pest control (national research council, ). such practices contribute to ecologi- cal farming because they have been shown to enhance biological interactions rather than suppress them, while at the same time limiting external inputs. in addition to sustainability, the concept of stewardship was important to earlier crit- ics of industrial agriculture (jackson, ). stewardship of the land is fundamental to amish religion; stewardship is a form of good behavior, and eternal life is a reward for good behavior. both sustainability and stewardship are enhanced by self-sufficiency, which minimizes reliance on the outside world by deflecting external social and eco- nomic influences from the amish culture. several authors have portrayed amish agricul- ture along these lines, but works by wendell berry ( , , ) are especially noteworthy for praising the sustainability and stewardship of amish agriculture. when one sees an amish farm and compares it with a modern industrialized farm, there are obvious differences between the two. berry ( ) has described amish land as varied, interesting, healthy-looking farm country, containing a great deal of natural and agricultural life. in contrast, he has portrayed industrialized farms as deserts of aban- doned buildings and machinery, existing on overworked fields with low crop yields. it is not, according to berry, uncommon to hear of amish farmers who brought overworked and unproductive fields back to life and maintained them using methods that have not changed through the centuries. in light of land improvement because of amish practices, it might be concluded that their methods are ecologically sound. a close reading of berry, however, may lead one to question how completely modern amish agriculture follows the tenets of ecological agri- culture. in an essay titled "seven amish farms," berry ( ) discussed why manure was important on one particular amish farm and how it decreased dependence on chemi- cal fertilizer. it was conceded, nevertheless, that some chemical fertilizer was used as a starter for corn and oats. this essay also revealed how herbicides were utilized to control weeds. hay purchased for extra horses that were housed during the winter brought weed seed that had to be controlled. berry summarized and compared the costs and efficiencies of this particular farm with non-amish agriculture, carefully downplaying throughout the use of both pesticides and fertilizers on the amish farm. a similar analysis was reported by logsdon ( ), who described how he pursued the matter following discussion after a softball game between some amish and non- amish farmers. logsdon compared the costs of producing corn on a single amish farm with the costs on a typical non-amish farm. although costs were clearly much lower on the amish farm, it is interesting to note that chemical fertilizers accounted for % and pesticides and herbicides for % of the total amish expenses. in comparisons of amish and non-amish farming, it is obvious that there are several differences with respect to di- versity, technology, use of petroleum, self-sufficiency, harmony with nature, and involve- d ow nl oa de d by [ l ib r i] a t : d ec em be r amish farming as ecological agriculture ment of family and community. however, in celebrating these differences, writers like berry and logsdon have tended to minimize amish involvement in certain controversial practices that are of importance to the environment, especially the use of pesticides and fertilizers. many researchers have conducted studies regarding the culture of amish settlements (e.g., hostetler, , ; reschly and jellison, ). investigators have been espe- cially interested in the ways that amish communities have adopted facets of modern cul- ture and the ways that amish society has adapted to both internal and external change (e.g., ericksen et al., ; foster, ; schwieder and schwieder, ). with respect to agriculture, ediger ( ) noted that some amish in iowa and ohio used steel-wheeled tractors. place ( ) described how amish farmers in lancaster county, pennsylvania, had selectively adopted certain modern innovations, such as mechanical milkers, veteri- nary services, and artificial cattle insemination, although they continued to use horses and mules to pull field machinery. only a few studies have attempted to characterize amish agriculture and compare it with that of the non-amish. these studies have examined attitudes toward environmental issues in farming, as well as energy and land use patterns in amish and non-amish agri- culture. previous investigations of amish agriculture in ohio are particularly pertinent to the present study, because most of the amish farmers in the research presented in this ar- ticle migrated from ohio to northern new york. stinner, paoletti, and stinner ( ) discussed amish culture and agriculture based on the results of a two-summer study of a single amish farm in holmes county, ohio. they described amish agriculture as one based on "moderation, simplicity of life, frugal- ity, neighborliness, family stability, and financial common sense" (p. ) and suggested that these characteristics are derived from the strength of community. jackson ( ) also studied a single amish farm in holmes county, focusing on soil characteristics, in com- parison with a single non-amish farm that used no-till agricultural methods. cropland on the amish farm exhibited higher rates of water infiltration, higher levels of alkaline-phos- phatase activity, less compaction, and more organic matter than the non-amish farm. it was concluded that traditional horse-drawn farming may cause less soil degradation than no-till farming advocated by the usda soil conservation service to reduce erosion of topsoil. both of these studies involved only one amish farm; despite difficulties of gener- alizing, they are included here because of the particular relevance of holmes county to the northern new york community studied in this research. in another study conducted in three counties of ohio, non-amish and amish farmers were surveyed regarding the threat of groundwater pollution from chemical use and their attitudes toward agricultural practices that might reduce pollution (sommers and napier, ). it had been hypothesized that the lower levels of formal education, younger average age, and lesser degree of farming experience among amish farmers would lead to lower levels of knowledge about the threat of groundwater contamination from chemical fertilizers, insecticides, and herbicides. however, amish farmers were found to be significantly more aware of the potential of chemicals to pollute groundwater and more open to changing their methods to reduce the threat of pollution than non- amish farmers. for example, the one farmer studied by stinner et al. ( ) in holmes county also demonstrated a receptivity to technology that supported conservation, such as the horse-drawn no-till planter. the energy consumption and efficiency of amish agriculture in relation to non-amish were the subject of a study in mifflin county, pennsylvania (craumer, ). forty-seven amish farmers were interviewed for comparison with data from about , non-amish d ow nl oa de d by [ l ib r i] a t : d ec em be r k.v. blake etal. farms throughout pennsylvania taken from the pennsylvania dairy farm business analysis. agricultural input and output were quantified in terms of fossil fuel use, ex- pressed in kilocalories. an efficiency level (energy ratio) was calculated by dividing energy output by input. the amish were % to % more efficient than the non-amish. non- amish farms used as much as two times the amount of total energy to produce kg of milk as the amish farms. the amount of human labor for amish versus non-amish farming was also quantified. the amish required % to % more energy in human labor to produce the same amount of milk as the non-amish. the use of draft animals and human labor in place of machinery allowed for greater fossil fuel efficiency on amish farms. a similar study, published a few years earlier, showed similar results (johnson et al., ). land use patterns of amish agriculture were also categorized in some of the studies cited above. amish farms were smaller because of limitations imposed by the use of human and animal labor rather than machinery. amish farms were also smaller than non- amish ones because of the diversified interests and activities of amish farmers. amish men were called upon by their beliefs "to be good husbands, fathers, and neighbors," (stinner et al., , p. ). the proportion of farmland devoted to pasture and feed grains was smaller for amish fanners than for non-amish farmers, % to % versus % in one study (craumer, ). however, amish cropland produced a greater diversity of grains and was conserved by a more frequent rotation of crops. the single amish fanner in the study by stinner et al. ( ) used non-cropland for timbering, beekeeping, maple sugaring, and gardening, but this pattern of diversity has not been universal. according to stoltzfus ( ), for example, some amish farmers in illinois took to specializing in one crop or animal because of economic conditions in that region about two decades ago. regardless of location, all amish communities are bound to upholding the specific belief system, called the ordnung or ottning. in northern new york, as elsewhere, the ordnung prescribes certain behavior in relation to daily life and the outside world. most amish communities are based on agriculture because of their belief that "god created the world and enjoyed it and then created human beings whom he commanded to harness and cultivate the earth" (johnson-weiner, , p. ). the earth, therefore, is viewed as a sa- cred trust. nature is not worshipped, but the amish take comfort as christians in a close relationship with nature. religion can serve to reinforce certain practices in farming; farming activities, in turn, have spiritual ramifications. the bible teaches that "some soil favors good seed and some favors weeds; so too, they [the amish] believe, some occupa- tions favor a christian life" (johnson-weiner, , p. ). farming is therefore more than an occupation; it is a way of protecting the religion and values of amish culture. ac- cording to savells ( ), in a study of amish in indiana, iowa, ohio, pennsylvania, and tennessee, agriculture has helped to maintain culture by providing opportunities for amish youths, few of whom have left their communities or religion. case study: st. lawrence county, new york although previous studies of amish farming have focused on communities in ohio, pennsylvania, and elsewhere, no prior research has been conducted on amish agricultural practices in northern new york. motivated by the availability of relatively inexpensive property, amish communities became established in two different locations in st. lawrence county in the early s. this paper focuses on the heuvelton-de peyster community, a settlement of the swartzentruber amish. this community is a homoge- neous settlement comprising families who originated primarily in holmes county, ohio (johnson-weiner, ). d ow nl oa de d by [ l ib r i] a t : d ec em be r amish farming as ecological agriculture st. lawrence county has a climate generally well suited for agriculture. the growing season is relatively long, with little variability from year to year. the growing season ranges from to days, with an average temperature between °f and °f. the average precipitation for a growing season is in., a figure only slightly lower than those of other important agricultural areas in eastern north america. early growing season temperatures are frequently low, however, and the soil tends to remain cool late into the spring. conse- quently, the planting of corn and oats is often delayed (nobe and conklin, ). methods the study area contained parts of five towns within st. lawrence county: canton, de kalb, de peyster, lisbon, and oswegatchie. possible candidates for participation in the study were identified in a windshield survey of the area. both amish farms and neighbor- ing non-amish farms were selected. although the amish farms were of primary interest, an equal number of non-amish farms was also identified to provide a control group for comparison. figure identifies the location of the amish and non-amish farms that eventually participated in the study. the two groups were intermixed throughout the study area, and the distribution within each town, as shown in figure , was approxi- mately the same. statistical analysis demonstrated that the numeric distribution of amish versus non-amish farms was not significantly different within towns. it was hoped that by sampling both amish and non-amish farms within the same geographic area, certain environmental factors that could affect farming (e.g., soil types, topography, localized weather) would be controlled. in , an economic classification divided st. lawrence county into various land classes according to net incomes that farms in the county were likely to yield in the future (nobe and conklin, ). although this economic classification was compiled a number of years ago, it provided the only measure of anticipated success for agriculture in the county based on natural features of the soil, topography, and drainage. moreover, it described the anticipated conditions and prospects for farming during a period that unfolded as the amish began to move into this region of st. lawrence county. table shows the distribution of farms within the economic land classes that com- pose the study area. approximately two-thirds of both amish and non-amish farms were in classes and . land class comprised areas in which farm incomes were generally low, yet high enough to meet the minimum requirements for continued operation for an expected years. many farms existing at the time of designation were small to medium in size, with crop yields frequently limited by adverse soil conditions. land class consisted of areas where medium incomes were expected from farming in the future. here, farms were gener- ally larger, maintained higher production rates, and were more efficient in the utilization of farm capital. the soils in these areas were thought to be adequate to support a well-adjusted agriculture for at least two more generations. land classes further demarked by sub-class -x had "sufficiently strong land resources to promise increased incomes if farms are increased in size, more adequately equipped with buildings and machinery, and more intensively oper- ated" (nobe and conklin, , p. ). areas delineated by sub-class -y were thought to be "progressively more handicapped in their competition with other areas in the same class as farming became more intensive" (p. ). two non-amish farms each were in land class , which comprised areas where the chances of continued farming were considered to be virtu- ally nonexistent, and land class , which consisted of areas projected to have very high in- come expectancies for at least one generation. the distributions of amish versus non-amish farms were not found to be significantly different across economic land classes. d ow nl oa de d by [ l ib r i] a t : d ec em be r k. v. blake etal. key • amish *non-amish town boundary scale . m i l e \ figure . distribution of amish and non-amish farms in study region, st. lawrence county, new york. data on farm characteristics, agricultural activities, and farm inputs and outputs were collected by use of a structured questionnaire. prior to creating the questionnaire, litera- ture concerning amish culture and history, and articles pertaining to amish farming, were carefully reviewed by the authors so as to gain a greater understanding of the sub- ject matter. an initial questionnaire was developed for use on a pilot group before data for the actual study were collected. after seven pilot interviews, the questionnaire was re- vised so as to collect more accurate and detailed information about certain topics. the re- vised questionnaire consisted of questions. specific items inquired about amounts of d ow nl oa de d by [ l ib r i] a t : d ec em be r amish farming as ecological agriculture table distribution of study farms across economic land classes land class (very low) -x -y (very high) total note. x = . ; number of amish farms p = . . number of non-amish farms land farmed, land leased, and plans for expansion; crop types and acreage over the past three years; animals on the farm over the past two years; products marketed and locations of markets; methods of pest control during the last growing season, including insecticide and herbicide use (types, amounts, and frequency of applications); fertilizer use and amounts over the past year; crop rotation; and sources of agricultural information. field interviews were conducted in person with amish farmers and non-amish farmers during the autumn of . of those who were approached for interviews, three amish farmers and two non-amish farmers chose not to take part in the study. the overall re- sponse rate was therefore %. there were approximately sixty-five amish farms in the swartzentruber community of heuvleton-depeyster. the sample of amish farmers therefore represented about % of the total membership of this settlement. with the exception of a few answers that could not be easily translated into quantita- tive terms, most of the information from the questionnaires was coded so it could be en- tered into a minitab database. nonquantifiable answers were summarized and evaluated separately. elementary statistical analyses were employed to evaluate the data; t tests were used to compare amish with non-amish, and chi-square tests were used to analyze frequency counts in certain tables. results farm characteristics figure summarizes amish and non-amish agriculture in relation to amount of land farmed. consistent with research on agriculture elsewhere, the amount of land farmed by each amish farmer in st. lawrence county was small enough to be worked properly by a team of horses. the difference in size between amish and non-amish farming operations was similar to that found in pennsylvania (craumer, ) as well as to that found in ohio (sommers and napier, ). in addition to their own farmland, as depicted in fig- ure , of the amish farmers leased an average of . acres, whereas of the non-amish farmers leased an average of . acres. eight amish farmers wanted to ex- pand between and acres, and seven non-amish farmers desired to add between and acres. d ow nl oa de d by [ l ib r i] a t : d ec em be r k.v. blake etal. - - - acres - - - - ' • • ' • • ' • / - - - - - - years — j mean acres farmed mean length of ownership in years t statistic = . (p = . ) t statistic = . (p = . ) figure . comparison of acres farmed and length of ownership for amish and non-amish farms. beyond this obvious difference in farm size, amish and non-amish agriculture var- ied in terms of both amount and type of cropland. the amount of land in various crops and pasture is summarized for in table . at least of the amish farmers planted three different crops: corn, oats, and hay. in contrast, only six of the non- amish farmers grew three or more crops, including oats. similar data were reported for and . table presents the types of animals reported on farms participating in this study. at least of the amish farms had the following animals in : dairy cattle, heifers, pigs, chickens, and horses. although livestock production is labor-intensive, requiring daily attention to maintain animals properly, it was handled easily on amish farms by ef- ficient use of family labor, which was plentiful because of the large size of amish fami- table amounts of land in crops and pasture for amish and non-amish farms in crop corn oats hay peas sorghum wheat barley pasture number of farms (%) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) amish mean number of acres (sd) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) non-amish number of farms (%) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) mean number of acres (sd) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) note. x = . ;p = . d ow nl oa de d by [ l ib r i] a t : d ec em be r amish farming as ecological agriculture lies. non-amish farmers had a larger number of dairy cattle, but far fewer nondairy ani- mals than the amish farmers. in addition to the animals cited above, a handful of both amish and non-amish farmers had turkeys, ducks and geese, goats, beef cattle, sheep, and fallow deer. each of these animal types was found on only one or two farms. data for showed a similar pattern in the number and diversity of animals. although they were not systematically recorded as part of this study, obvious differ- ences in agricultural machinery were observed during the course of interviewing. amish respondents were particularly interested in talking about this aspect of their farms. clearly, their machinery was rudimentary. they relied on horse-drawn equipment in the field, whereas stationary, petroleum-powered engines were used occasionally around farm outbuildings, for example, in cold-water systems for cooling milk and on equipment for lifting hay into barns. farm products another major difference between amish and non-amish farmers was in the diversity of products generating revenue for the farm. one non-amish farmer offered products for sale on a roadside stand, in comparison with of the amish farmers. both the non- amish and amish farmers offered a variety of products, including strawberries, aspara- gus, squash, peas, tomatoes, cucumbers, corn, beets, potatoes, carrots, and pumpkins. the amish farmers also included maple syrup, honey, and eggs among the items for sale. the self-sufficiency of amish farms could be seen in the wide diversity of fruits and vegeta- bles offered for sale to the public. the variety and fluctuating amounts of products found at the average amish roadside stand reflected cultural values and family orientation that define amish agriculture in this region. food grown in the garden was primarily for sub- sistence, and only extra items that were not useful to the family were sold at the stand. amish farmers explained that their roadside stands did not serve as a major source of in- come, but merely provided a few dollars from items not consumed by the family. twenty-six of the non-amish farmers produced milk, averaging . pounds per day. this milk was shipped to cooperatives and cheese factories located within the table number of animals on amish versus non-amish farms in animal dairy cows heifers bulls pigs chickens horses other note, x number of farms (%) ( ) ( ) ( ) ( ) ( ) ( ) ( ) = . ; p = . . amish mean number of animals (sd) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) non-amish number of farms (%) ( ) ( ) ( ) ( ) ( ) ( ) ( ) mean number of animals (sd) . ( . ) . ( . ) . ( . ) ( ) . ( . ) . ( . ) . ( . ) d ow nl oa de d by [ l ib r i] a t : d ec em be r k.v. blake etal. county. the amish farmers were not involved in milk production to the same extent as their non-amish neighbors. only of amish farmers produced milk for sale, averag- ing . pounds per day. amish farmers sent their milk to a cheese factory used exclu- sively by amish farmers throughout the area and also located within the county. because of the growth of amish settlements in recent years, a second cheese plant was under con- struction at the time of the survey. milk production on a per cow basis was slightly higher for non-amish farmers ( . pounds per cow per day) than for amish farmers ( . pounds per cow per day). in terms of livestock sales, both amish and non-amish farmers sold pigs and dairy cattle, primarily as heifers. these sales were handled by companies that brought cattle to local and regional auctions. ten amish farmers sold on the average . cows in , whereas ten non-amish farmers sold on the average . cows in the same year. in terms of hogs, four amish farmers marketed an average of . animals in , and one non- amish fanner sold , animals. amish farmers did not engage in livestock sales in- volving other animals. on the other hand, two additional non-amish farmers engaged in the sale of beef, with one marketing beef cattle in and the other marketing an un- specified number. finally, one additional non-amish farmer sold an uncertain amount of baled hay. these data reveal the differing philosophies underlying amish and non-amish agri- culture. although both groups raised a variety of animals in the aggregate, each amish farmer was individually diverse, whereas each non-amish farmer specialized in one, or at most two, animals. furthermore, as reflected in market sales, amish farmers raised far fewer animals for sale. the diversity of animals on amish farms was mostly for personal consumption by their families; the motive behind animal production was primarily self- sufficiency and subsistence. the non-amish farmers specialized and marketed a far larger number of certain animals for profit in the marketplace. soil maintenance table presents the type and amount of fertilizer applied in by farmers participat- ing in this study. this table shows that amish and non-amish used different types of fer- tilizers to a significant extent. amish farmers used - - on their fields to a much larger degree than non-amish farmers; while triple ( - - ) was utilized solely by table fertilizer applications on amish and non-amish farms in fertilizer nitrogen/phosphorous/ potassium (as percent of total) / / / / / / other note. y* = . ; n = amish number of farms (%) ( ) ( ) ( ) ( ) . . mean lbs/acre . . . non-amish number of farms (%) ( ) ( ) ( ) ( ) mean lbs/acre . . . . d ow nl oa de d by [ l ib r i] a t : d ec em be r amish farming as ecological agriculture non-amish, and triple ( - - ) was employed by about the same number in each group. in addition, more than a dozen other substances were reported as fertilizing agents during the interviews. all but a few of these fertilizers were utilized by only one of either the amish or non-amish farmers. the formulae of inorganic fertilizers refer to percentages of the three most important elements of plant nutrition. the first number represents the percentage of nitrogen, the second stands for phosphorous, and the third denotes potassium as potash. of all ele- ments in soil, these three are most readily depleted by the growing of crops, especially com, and they are the ones that must be replenished in the largest amounts. amish farm- ers spread fertilizer containing less nitrogen because levels of this nutrient can be main- tained by applications of animal manure. although virtually all farmers with livestock probably disposed of animal manure on their fields as a form of waste disposal, it was interesting to note how many farmers thought of manure as fertilizer. the question on the survey was open-ended, asking re- spondents to list various fertilizers used to maintain soil and assist in the growing of vari- ous crops. without a specific prompt about manure as a form of fertilizer, of the amish farmers thought to mention animal waste, compared with only of the non- amish fanners. this difference in perception can be attributed to the disparity in underly- ing philosophies of agriculture: the self-sufficient nature of amish farming reinforces an ideology emphasizing the cycles and dynamic equilibrium of ecological agriculture based on permanence. amish farmers are therefore apt to think of animal waste products as fertilizers. all amish and most non-amish farmers spread inorganic fertilizer only in the spring. in addition, some amish farmers ( ) and some non-amish farmers ( ) applied manure only in the spring. the remaining amish and eight non-amish farmers spread manure in seasons other than spring. in some cases, fall and winter applications were pre- ferred. in other cases, manure was spread as a form of waste disposal whenever facilities for storage became insufficient. in general, most farms were characterized by similar crop rotations. the main differ- ence was in the greater frequency with which oats were interjected into the cycle by amish farmers, to provide feed for horses. most ( ) of the amish farmers employed a standard three-year rotation of corn-oats-hay, or a six-year cycle of corn-corn-oats-hay- hay-hay, with an occasional fourth or more years of hay added, lengthening the cycle. the length of time that a field would be planted in hay depended on the quality of hay produced from year to year. one amish farmer was uncertain about his crop rotation, be- cause he had been farming in the region for only one year at the time of the study. seven- teen of the non-amish farmers rotated crops. eleven farmers utilized a crop rotation of corn and hay in three- to four-year shifts. five farmers incorporated a three-crop rotation, which sustained an eight- to -year cycle of two to three years of corn, followed by one year of oats and then five to seven years of hay. the last farmer employed a three-year cycle rotating corn, peas, and hay on an annual basis. insect and weed control insect and weed control differed somewhat between amish and non-amish farmers. overall, the amish used about the same amounts of pesticides as the non-amish, but the specific types of chemicals varied, as did the proportions. more pronounced differences were observed in relation to the use of herbicides than to the use of insecticides. with respect to insecticides, amish farmers used them on their gardens, including two that also used them on cropland; one additional amish farmer used them on cropland d ow nl oa de d by [ l ib r i] a t : d ec em be r k.v. blake etal. only. in contrast, only two non-amish farmers used insecticides on cropland. on gardens, insecticides were applied once or twice daily; on cropland, insecticides were applied one to three times during the growing season. the insecticide used most commonly was car- baryl (brand name: sevin). carbaryl is a relatively nonpersistent chemical in wide use, al- though it is a known teratogen, a suspected carcinogen, and a strong toxicant for a variety of animals, including bees, worms, crustaceans, fish, and birds (briggs, ). the larger number of amish who used insecticides on their gardens compared with the number of non-amish may be attributable to a larger proportion of amish farmers having gardens, but this possibility could not be tested because the questionnaire did not inquire about the practice of gardening. a more likely explanation is that amish partici- pants in the survey thought of gardens as part of their farms, whereas non-amish partici- pants did not. in particular, gardens are essential for amish subsistence, and a large num- ber of amish farmers in the study sold surplus vegetables at roadside stands. gardens may be important for non-amish farmers, but they are unlikely to be necessary for exis- tence, and they are certainly not part of the agricultural enterprise. thus, when asked about pest control, amish respondents thought to include what they did in their gardens, whereas non-amish participants did not. table summarizes weed control for corn crops during the growing season. the number of farmers using herbicides was not appreciably different for the amish ( of ) and the non-amish ( of ). however, considerable differences were reported in the types and varieties of herbicides preferred by the two groups. most of the amish farmers applied atrazine only to control weeds in corn. although these farmers appeared to apply more atrazine per acre than their non-amish neighbors, this difference was not statistically significant. any difference, furthermore, was counterbalanced by the simulta- neous use of several other herbicides by more of the non-amish farmers. of the amish farmers who applied herbicides in cornfields, % used only one chemical (atrazine), tables weed control methods associated with corn production on amish and non-amish farms in control method none cultivation herbicide use atrazine cyanazine (bladex) glyphosate (round-up) metolachlor (dual) pendimethalin (prowl) simazine (princep) , -d (amine) other amish number of farms (%) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) mean lbs/acre . . . . . non-amish number of farms (%) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) mean lbs/acre . . . . . . . note, x = . ; p = . . d ow nl oa de d by [ l ib r i] a t : d ec em be r amish farming as ecological agriculture % used two chemicals, and % used three chemicals. in contrast, only % of the non- amish farmers used just one chemical, % used two chemicals, % used three chemi- cals, and one used four different chemicals to control weeds in com. in terms of non- chemical means of controlling weeds in corn, the same proportion of amish farmers, of , mentioned the use of cultivation as non-amish farmers ( of ). the greater dependency of amish farmers on atrazine, simazine (brand name: prin- cep), and , -dichlorophenoxyacetic acid or , -d (brand name: amine) is troublesome. of the herbicides listed in table , these three are the most dangerous in terms of harm to the environment and human health. atrazine is a known carcinogen and immunotoxin; simazine causes long-term damage to the kidney, liver, thyroid, and testes, with conse- quent disturbances in sperm production; and , -d is a known carcinogen, teratogen, and immunotoxin, a suspected fetotoxin, and a toxicant to the kidney, liver, and central ner- vous system. atrazine and , -d also create serious ecological impacts for a wide variety of nontarget species (briggs, ). the greater frequency with which oats were planted by amish farmers may have dic- tated a certain need for some herbicides and fertilizers, because oats demand more of these petroleum-based inputs than hay. of the amish farmers who grew oats, nine used , -d and three used glyphosate (brand name: round-up). according to craumer ( ), the amish in mifflin county, pennsylvania, also required such inputs for the growing of oats. one amish fanner in holmes county, ohio, also justified his use of her- bicides by the need to grow feed grain for animals housed during the winter (stinner et al., ). source of agricultural information figure compares the places where amish and non-amish fanners learned about agri- cultural practices and innovations. it was important to inquire about where farmers thought they had obtained their knowledge about agriculture, because cultural differences seed magazines cooperative neighbors experience distributors extension chi-squara - . (p - o.o ) figure . primary sources of agricultural information for amish and non-amish farmers. d ow nl oa de d by [ l ib r i] a t : d ec em be r k. v. blake et al. have been reported to play an important role in encouraging or discouraging interaction with others. the present results, in fact, seem to support this view. amish farmers de- pended primarily on their own experience, and, to a lesser extent, on neighbors and seed distributors. on the other hand, none of the non-amish farmers reported that they relied primarily on neighbors for agricultural information. the non-amish, instead, used the co- operative extension of cornell university and subscribed to farming magazines. the results here reinforce the observations of other investigators, who have found that the amish are reticent to seek help from government programs and agencies as part of a deliberate strategy to separate themselves from non-amish. for example, place ( ) found that amish farmers in lancaster county, pennsylvania, do not solicit aid from the county conservation district or the usda soil conservation service in relation to plans for managing animal manure and other farming practices. however, many amish in lancaster county do consult with the cooperative extension of pennsylvania state uni- versity, viewing it as a resource for information rather than a government agency, and others subscribe to agricultural periodicals. although all amish both harbor suspicions about government and desire to maintain the integrity of their culture through community self-sufficiency, there are clearly differences among communities, with the swartzentru- ber amish of northern new york apparently among the more conservative settlements. discussion in general, it was found that amish farmers in upstate new york differ from their non- amish counterparts in terms of scale (small versus large), diversity (high versus low), technology (low versus high), and philosophy (self-sufficiency for subsistence versus production for the market economy). amish farms are smaller and more diverse than non-amish farms. amish farmers grow a wider variety of vegetables and raise a wider variety of animals than non-amish farmers. the primary focus of amish agriculture is on self-sufficiency rather than production for profit. amish farmers are concerned only with generating enough output to live consistently with their beliefs and to get children started out on their own farms. non-amish farmers raise animals primarily to participate in the regional marketplace, particularly with respect to dairy cattle and milk production. in contrast to non-amish farmers, most amish have only enough cows to yield the amount of milk that the family will consume. some amish farmers do produce surplus milk for the manufacturing of cheese at an amish-operated plant, and a few sell heifers and hogs, as well, but these quantities are far smaller than those of their non-amish neighbors. a trend toward diversity and self-sufficiency is also evident in vegetable gardens. what is produced is to be eaten by the family, with any amount left over to be sold at roadside stands. both amish and non-amish fanners use inorganic fertilizers and synthetic pesti- cides. although the two groups use somewhat different kinds of fertilizers and pesticides, the overall amounts are approximately the same. more importantly, the environmental impacts of substances used by both groups are equally problematic in terms of ecological impacts and human health effects. the use of these substances dispels the notion that the amish in this region are organic farmers. amish agriculture in st. lawrence county, new york is not appreciably different from amish agriculture in other northeastern states (pennsylvania and ohio), to the ex- tent that comparisons can be made on the basis of published literature. in certain respects, the swartzentruber amish in northern new york are comparatively conservative, for ex- ample, in their very limited contact with any governmental agencies. on the other hand, it seems that the amish in st. lawrence county utilize synthetic pesticides and inorganic d ow nl oa de d by [ l ib r i] a t : d ec em be r amish fanning as ecological agriculture fertilizer to a greater extent than their brethren elsewhere. however, the literature on amish agriculture is not only sparse, but also somewhat dated, some of it having been published to years ago. one possibility is that amish farming everywhere has been increasingly adopting higher agricultural inputs. amish farming in st. lawrence county is congruent with the definition of ecological agriculture in several aspects. by having small and diverse farms, these amish farmers have avoided the specialization and monocultures characteristic of industrial agriculture. on the other hand, the use of petroleum-based fertilizers and pesticides contradicts the principle of sustainability embedded in ecological agriculture. although these amish farmers perceive animal manure as a source of nutrients for maintaining soil, their re- liance on fertilizers such as - - and triple is not indicative of a self-sufficient sys- tem that recycles internally, like the biological processes of a productive and self-regulat- ing ecosystem. the use of insecticides and herbicides, such as carbaryl, atrazine, simazine, and , -d, obviously violates the notion of stewardship. quite clearly, ecologi- cal agriculture prefers organic means of pest control to prevent chemical contamination of the environment. one question is whether the dynamic equilibrium of amish agricul- ture can be sustained if the amish continue to rely so heavily on petroleum-based inputs. because the amish in this area prefer their own experience as a source of information to guide their agricultural practices, they may be in a good position to recognize early warn- ing signs of trouble and to adjust accordingly. why do the amish of st. lawrence county embrace petroleum-based inputs such as fertilizers and pesticides? this question suggests several possibilities for future research. in light of the expectation that the amish would be found to practice ecological agricul- ture, it came as a surprise that pesticides and fertilizers were widely used in this commu- nity. therefore the focus here has been on documenting the extent of this use rather than rigorously engaging the more intriguing question of why. in further studies, the present authors expect to explore at least three explanations for use of fertilizers and pesticides by amish farmers in st. lawrence county. first, the use of these substances may portend a fundamental shift away from traditional practices; amish farming in this particular community may be in the early stages of conversion to an indus- trial style of agriculture. alternatively, the use of these substances may indicate a tempo- rary strategy rather than a profound change. the swartzentruber amish may not be suffi- ciently established in the heuvelton-de peyster area to have developed the productive and self-regulating characteristics of ecological agriculture. they started to settle in this area only about years ago, having an average length of residency of about nine years. fertil- izers may be necessary only to enhance poor soil conditions or to revitalize soils depleted by the exhaustive practices of the non-amish farmers who previously occupied the land. herbicides may be necessary only to control weeds until a generation or two of amish farmers have established the dynamic equilibrium of a permanent system that minimizes the need for petroleum-based inputs. in northern new york, however, the climate and per- haps the soil are more problematic for labor-intensive farming than in holmes county, ohio. finally, the use of fertilizers and pesticides may not be accompanied by an aware- ness of the associated ecological disruptions. consistent with an ethic of stewardship, amish farmers in ohio have expressed a willingness to modify agricultural practices that cause environmental problems (sommers and napier, ; stinner et al., ). under this third hypothesis, amish farmers in st. lawrence county can be expected to reduce or abandon inorganic fertilizers and synthetic pesticides if and when they become aware of the ecological problems these substances create. anecdotal evidence, based on preliminary conversations, suggests that all of these explanations are plausible. d ow nl oa de d by [ l ib r i] a t : d ec em be r k.v. blake etal. amish farming has been praised by both critics of industrial agriculture and advo- cates of ecological agriculture. it is often assumed that the amish practice organic farm- ing and that they represent an appropriate model for ecological agriculture based on sub- stainability and stewardship. however, a closer examination of their current farming methods, with particular attention to the use of fertilizers and pesticides, brings this as- sumption into question. although modern amish farming subscribes to many of the tenets of ecological agriculture, amish practices in relation to fertilizers and pesticides may not, in the future, be significantly different from those of the non-amish engaged in industrial agriculture. references berry, wendell. . the unsettling of america: culture and agriculture. san francisco, ca: sierra club books. berry, wendell. . seven amish farms. in the gift of the good land: further essays cultural and agricultural, ed. wendell berry, pp. - . san francisco, ca: north point press. berry, wendell. . the amish way. blair & ketchum's country journal ( ): - . briggs, shirley a. . basic guide to pesticides: their characteristics and hazards. washington, dc: hemisphere. craumer, p. r. . farm productivity and energy efficiency in amish and modern dairy fanning. agriculture and environment : - . ediger, marlow. . amish adaptations to modern society: technical, medical, and legal. sociol- ogy and social research : . ericksen, eugene p., julia a. ericksen, and john a. hosteller. . the cultivation of the soil as a moral directive: population growth, family ties, and the maintenance of community among the old order amish. rural sociology : - . foster, thomas w. . occupational differentiation and change in an ohio amish settlement. ohio journal of science ( ): - . goering, peter, helena norberg-hodge, and john page. . from the ground up: rethinking in- dustrial agriculture. london: zed books. hostetler, john a. . amish society. baltimore: johns hopkins university press. hostetler, john a. . a new look at the old order. rural sociologist : - . jackson, mary. . amish agriculture and no-till: the hazards of applying the usle to unusual farms. journal of soil and water conservation : - . jackson, wes. . new roots for agriculture. san francisco, ca: friends of the earth. johnson, warren a., victor stoltzfus, and peter craumer. . energy conservation in amish agriculture. science : - . johnson-weiner, karen m. . the plain people among us: the north country amish. the quarterly ( ): - . kolodge, craig. . amish farming: a modern day paradox. small farm news (davis, ca), (january-february). logsdon, gene. . a lesson for the modem world: amish economics. whole earth review, : - . national research council. . alternative agriculture. washington, dc: national academy press. nobe, kenneth c., and howard e. conklin. . an economic classification of farm areas: st. lawrence county, new york. cornell economic land classification leaflet . ithaca, ny: new york state college of agriculture. place, elizabeth. . land use. in the amish and the state, ed. donald b. kraybill, pp. - . baltimore: the johns hopkins university press. reschly, steven d., and katherine jellison. . production patterns, consumption strategies, and gender relations in amish and non-amish farm households in lancaster county, pa, - . agricultural history : - . d ow nl oa de d by [ l ib r i] a t : d ec em be r amish farming as ecological agriculture savells, jerry. . economic and social acculturation among the old order amish in select com- munities: surviving in a high-tech society. journal of comparative family studies : - . schwieder, elmer, and dorothy schwieder. . the paradox of change in the life style of iowa's old order amish. international review of modern sociology (l): - . sommers, david g., and ted l. napier. . comparison of amish and non-amish farmers: a diffusion/farm structure perspective. rural sociology : - . soule, judith d., and jon k. piper. . farming in nature's image: an ecological approach to agriculture. washington, dc: island press. stinner, deborah h., m. g. paoletti, and b. r. stinner. . in search of traditional farm wisdom for a more sustainable agriculture: a study of amish farming and society. agriculture, ecosystems, and environment : - . stoltzfus, victor. . amish agriculture: adaptive strategies for economic survival of community life. rural sociology : - . d ow nl oa de d by [ l ib r i] a t : d ec em be r   open peer review f  faculty reviews are written by members of the prestigious  . they aref  faculty commissioned and are peer reviewed before publication to ensure that the final, published version is comprehensive and accessible. the reviewers who approved the final version are listed with their names and affiliations. any comments on the article can be found at the end of the article. review better understanding of childhood asthma, towards primary prevention – are we there yet? consideration of pertinent  literature [version ; peer review: approved] michal gur ,   fahed hakim , lea bentur , pediatric pulmonary institute and cf center, rappaport children's hospital, rambam health care campus, haifa, israel rappaport faculty of medicine, technion–israel institute of technology, haifa, israel abstract asthma is a chronic disease, characterized by reversible airway obstruction, airway inflammation and hyper-reactivity. the prevalence of asthma has risen dramatically over the past decade, affecting around , ,  people. the etiology is multifactorial, with genetic, epigenetic, developmental and environmental factors playing a role. a complex interaction between the intrauterine environment, the developing immune system, the infant's microbiome and infectious organisms may lead to the development of allergic sensitization and asthma. thus, a large number of studies have investigated the risk factors for childhood asthma, with a meticulous search of modifiable factors that could aid in primary prevention. we present a current literature review from  - , as well as older classic publications, on the pathogenesis and the potential modifiable factors for primary prevention of asthma. no ideal preventive measure has yet been found. rather, creating favorable prenatal and postnatal environments, minimal exposure to hostile environmental factors, prevention of infections in early life, allergic desensitization and nutritional modifications could possibly reduce asthma inception. in the era of personalized medicine, identifying individual risk factors and tailoring specific preventive measures is warranted. keywords asthma, wheezing, environmental, factors, prevention , ,    reviewer status   invited reviewers  version published  dec    , university of manitoba, winnipeg,allan becker canada , vanderbilt university school oftina v hartert medicine, nashville, usa , vanderbilt university schoolchristian e lynch of medicine, nashville, usa    dec  ,  (f  faculty rev):  (first published: )https://doi.org/ . /f research. .    dec  ,  (f  faculty rev):  (latest published: )https://doi.org/ . /f research. . v page of f research , (f faculty rev): last updated: jul https://f research.com/browse/f -faculty-reviews http://f .com/prime/thefaculty https://f research.com/articles/ - /v https://f research.com/articles/ - /v https://f research.com/articles/ - /v https://orcid.org/ - - - https://f research.com/articles/ - /v https://doi.org/ . /f research. . https://doi.org/ . /f research. . http://crossmark.crossref.org/dialog/?doi= . /f research. . &domain=pdf&date_stamp= - -    lea bentur ( )corresponding author: l_bentur@rambam.health.gov.il  no competing interests were disclosed.competing interests:  the author(s) declared that no grants were involved in supporting this work.grant information:  ©   gur m  . this is an open access article distributed under the terms of the  , whichcopyright: et al creative commons attribution licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.  gur m, hakim f and bentur l. how to cite this article: better understanding of childhood asthma, towards primary prevention – are we  f research  ,  (f  faculty rev):  (there yet? consideration of pertinent literature [version ; peer review: approved] )https://doi.org/ . /f research. .    dec  ,  (f  faculty rev):  ( ) first published: https://doi.org/ . /f research. . page of f research , (f faculty rev): last updated: jul http://creativecommons.org/licenses/by/ . / https://doi.org/ . /f research. . https://doi.org/ . /f research. . introduction asthma is a chronic disease, characterized by episodes of reversible airflow obstruction. the prevalence of allergic diseases and asthma has risen substantially over the past decades. currently, it is the most common non-communicable disease, affecting around , , people, especially in developed countries, leading to enormous public health costs . in a recent study, the total annual health care expenditure attributable to asthma for school-aged children in the united states was . billion us dollars . currently, no available therapeutic regimens can cure asthma, and the burden of asthma will continue to be driven by the increased but, as yet, poorly explained prevalence . population-based birth cohorts on asthma and allergies may provide insights into the development and natural history of the diseases. over birth cohorts have been initiated in the last years . these birth cohorts have improved our understanding of asthma inception, progressions and persistency. thus, they may help in targeting the ambitious and important goal of primary pre- vention of asthma. the tucson birth cohort, developed in by martinez et al. is the “classic” and most utilized model. this cohort proposed three categories of wheezing phenotypes at early age: transient wheezers (wheezing symptoms before years, no wheezing at age ), late onset wheezers (no wheeze until years, wheezing at age ), and persistent wheeze (wheezing in the first years, wheezing at years). this latter group of persistent wheezers can be divided into non-atopic and atopic. one third of all children aged or younger had lower respiratory tract illnesses with wheezing; however, by the age of close to percent of these children no longer had wheezing symptoms. transient early wheezers were found to have lower levels of lung function compared to other groups ofwheezers, possibly reflecting congenitally smaller airways . understanding the trajectory of early wheezing may help identify early predictors of later childhood persistent asthma. this is of utmost impor- tance in identifying preventive interventions that could potentially reduce the inception of asthma . the etiology of asthma is multifactorial; genetic, epigenetic, developmental and environmental factors play a role, as do the interactions between them , . two important risk factors have been identified: the development of allergic sensitization and wheezing respiratory tract illnesses at an early age. since both allergic sensitization and viral/bacterial illnesses occur in children who do not develop asthma, it is crucial to identify genetic and environmental factors that activate, interfere with and direct the immune system toward the development of asthma . moreover, it has been found that environmental factors affecting a critical period in lung development (during pre- and postnatal periods) are associated with the development of allergic diseases and asthma. epigenetic pathways could mediate the gene- environ- ment interactions and may explain the impact of external environ- mental factors on disease development. taking into account the significant burden of asthma, it is crucially important to find preventive measures. strategies targeting asthma prevention can be primary (e.g. infants at high- risk for asthma) or secondary, dealing with children who have developed allergic sensitization or the first manifestations of allergic diseases (e.g. eczema or wheezing) . however, the heterogeneity and complex natural history of the disease serve as a barrier for the use of a single prevention strategy and suggest the importance of individualized risk assessment and multiple prevention measures with personalized primary prevention strategies . this review discusses research from the past years, focusing on childhood asthma and primary preventions. we conducted a pubmed search of observational studies and clinical trials, including systematic reviews and meta-analyses, from to . we also included older classic publications; thus, the search included studies dealing with potentially modifiable factors (both prenatal and postnatal) implicated in the inception of asthma. environmental factors, early life respiratory infections, host factors and nutritional interventions will also be discussed. environmental factors favorable environment the “farm effect”. the importance of favorable environmen- tal exposures in the development of asthma was demonstrated by epidemiologic studies showing significant protection from asthma and allergic diseases in children raised on traditional dairy farms. in particular, it was found that children who lived on farms were exposed to a greater variety of environmental microorgan- isms than controls. endotoxin, a cell wall component of gram negative bacteria, along with peptidoglycan, is widespread in stables and other farming environments and exposure to such microbial compounds was found to be inversely correlated with the risk of atopy . higher diversity of microorganism expo- sure was correlated with a reduced prevalence of asthma and atopy . compelling data exist showing that the “farm effect” is not only present in childhood but also exerts an effect during pregnancy, influencing the developing fetal immune system. the farming environment has been linked with lower rates of asthma in offspring, although early-life farm exposure was shown to boost this effect . a recent study with the amish community using tra- ditional farming and the hutterite community using industrialized farming evaluated asthma prevalence in children with similar genetic ancestries and lifestyles . asthma prevalence and allergic sensitization was and times lower in the amish community, whereas median endotoxin levels in amish house dust was . times higher. the authors used a murine model of experimen- tal allergic asthma to assess the effects of dust extracts found in amish and hutterite households on immune and airway responses. experiments in humans and mice indicated that the traditional farm environment guards against asthma via interaction with and molding of the innate immune response. hence, it is evident that altering innate immune signaling, as by farm environment, may be the primary target of asthma protection. however, the inter- ventions required to create a farm effect without living on a farm are as yet unknown. pets. several prospective birth cohort studies found a decreased prevalence of atopic disease in children having daily contact with pets, in particular cats and dogs, during early infancy . exposure to two or more dogs or cats in the first year of life was associated with a significantly lower risk of atopy (adjusted or . , % ci . - . ) . another study found that living with page of f research , (f faculty rev): last updated: jul a cat was inversely related both to having a positive skin test to cat (rr, . [ . – . ]) and incidence of physician-diagnosis of asthma (rr, . [ . – . ]). the effect was most pronounced among the children with a family history of asthma. ownership of a dog resulted in weaker protective trends . a pooled analysis of individual participant data of prospective european birth cohorts of , children concluded that pet ownership in early life did not appear to either increase or reduce the risk of asthma . however, a large nationwide cohort study ( , , children) in sweden found that dog exposure during the first year of life was associated with a decreased risk of asthma in school-aged children (or . , % ci . - . ) and in children years or older (hr . , % ci . - . ). exposure to farm animals was correlated with a lowered risk of asthma in both children of school and preschool age. these results were independent of parental asthma . however, the effect of exposure to dogs and farm animals was less pronounced in children younger than years of age. these young children may reflect a group of children with transient wheeze and not persistent asthma . taken together, most (but not all) studies support the theory of protective effects of having pets on asthma development. day care attendance. the effect of day care attendance on asthma development is complex. in a study involving , children fol- lowed since birth as part of the tucson study, children with older siblings or day care attendance were more likely to have frequent wheezing at the age of years, but less likely to have frequent wheezing from the age of to years . different results were obtained in a birth cohort of children . in this study, day care attendance at months was associated with an increased risk of asthma [or . , % ci . - . ]. a multivariate logis- tic model showed that day care attendance and number of lower respiratory infections at months were associated with asthma [or . ( . - . ); or . ( . - . ), respectively]. never- theless, day care attendance of greater than . hours per week was associated with a lower risk of asthma [or . ( . - . )]. hence, day care during infancy can either increase or reduce the risk of asthma. prevention of allergic sensitization there is a clear association between asthma and allergy . most school age children and adolescents with asthma also have allergic sensitization. early allergic sensitization is a risk factor for asthma development. in children of preschool age, researchers have found a sequential relationship of allergic sensitization followed by virus-induced wheezing. exposure to allergens may increase asthma severity in susceptible patients (for example, an asthmatic patient exposed to dust mite allergens may develop increased airway hyper-reactivity) . therefore, prevention of allergic sensitization is a major path for primary prevention of asthma. a cochrane review in found that multifaceted interven- tions (reducing exposure to both inhalant and food allergens) resulted in a significant decrease in asthma compared to usual care (< years: or . , % ci . to . ; > years: or . , % ci . to . ). monofaceted interventions (reducing exposure to either inhalant or food allergens) did not produce significant effects . another interventional study assessed the effectiveness of a multifaceted intervention program for the primary prevention of asthma in high-risk infants. the interventions were initiated shortly before birth and applied until year of age, and included avoidance of house dust mites, pets, environmental tobacco smoke, encouragement of breast-feeding, and delayed introduc- tion of solids. these measures resulted in a -fold reduction in the risk of asthma (adjusted or, . ; % ci . - . ) in the children who did not develop atopy by year of age; however, in children with early, persistent atopy, the risk of asthma was not reduced . moreover, attempts to reduce exposure to house dust mites, known to be strongly associated with atopic sensitization and the development of asthma in childhood, were not found to be effective in the primary prevention of disease . lynch et al. examined a birth cohort and a nested case-control study to assess the factors linking contact with allergens and bacteria in the first years of life with the inception of recurrent wheeze and atopy. as the authors put forward, increasing allergen expo- sure in the first years correlated with allergic sensitization, and allergic sensitization correlated with recurrent wheeze. however, there was a negative association between contact with cockroach, mouse and cat allergens in the first year and recurrent wheeze (or . , . , and . , respectively, p≤ . ) . overall, the reported nature of the relationship between exposure and sensitization varies widely among studies, from no signifi- cant association to a simple linear dose–response relationship or a ‘bell-shaped’ dose–response model with a protective effect of high allergen exposure. a recent study assessed the potential role of immunotherapy to alter the natural course of allergic march from allergic sensitization to asthma. the study included children ( – years), with grass pollen allergic rhinocon- junctivitis and no medical history or signs of asthma. children were double blinded and randomly allocated to receive immu- notherapy or placebo for three years and were followed for two additional years . the study showed reduced risk of experienc- ing asthma symptoms and using asthma medication, but did not show an effect on the time to onset of asthma. hence, the relationship of atopic desensitization and asthma is complex and is likely determined by the type of allergen, the timing, pattern, route of exposure (inhaled, oral, transcutane- ous), dose, as well as other environmental factors and individual genetic predispositions . thus, the role and the measures of prevention of sensitization in the development of ige-mediated sensitization asthma are yet to be determined. hostile environment air pollution. air pollutants, representing a complex exposure to inorganic and organic components, are known to exacerbate asthma symptoms and might play a role in initiation of this disease. particulate matter (pm) carries both environmental pollutants, such as polycyclic aromatic hydrocarbons (pahs)— formed during incomplete combustion of fossil fuels and oil products—as well as agents causing immune stimulation—such as pollens, endotoxin and fungal spores. air pollutants probably page of f research , (f faculty rev): last updated: jul cause oxidative injury to the airways, leading to inflammation, remodeling, and an increased risk of sensitization. the idea that air pollution can cause exacerbations of pre-existing asthma is supported by evidence-based studies, but evidence suggests that air pollution might cause new-onset asthma as well, both in chil- dren and in adults . in a recent epidemiologic review, schulz et al. concluded that early life and school-age exposures to air pollution has a negative impact on lung function, at least up to adolescence . in a canadian birth cohort study, postnatal exposure to traffic-related air pollution increased the risk for the development of atopy to any allergens (adjusted or . ; % ci . - . ) . gauderman et al. found an association between improvements in air quality in southern california and measurable improve- ments in lung-function development in children . consequences of exposure to pollutants, pre- or postnatal, represent a complex interaction between the environment, the host and epigenetic factors. in children, pah exposure has been associated with changes in dna methylation, as well as impaired function of regulatory t cells . epigenetic processes translate environmen- tal exposures into regulation of the identity, gene expression profile, and activity of specific cell types that participate in the pathophysiology of the disease. further discussion of the effects of air pollution is beyond the scope of this review. tobacco smoke. there is evidence indicating a consistent det- rimental effect of prenatal exposure and postnatal environmen- tal smoking on childhood wheezing illnesses. environmental tobacco smoke (ets) during critical periods of lung development (prenatally, i.e. during pregnancy, and during early life) is consid- ered a substantial risk factor for childhood allergic diseases. ets induces over-expression of toll-like receptors on the surface of the airway epithelium, increases oxidative stress and activates dendritic and innate lymphoid cells through the production of cytokines, such as il- , il- and il- . the result is a higher susceptibility to allergen sensitization and a further risk for asthma . genetic polymorphism on chromosome q was found to be predictive of childhood-onset asthma, and the risk was further increased by early-life exposure to environmental tobacco smoke , . in a meta-analysis, ets was associated with an increased risk of elevated specific ige (or . ; % ci . - . ) and posi- tive skin prick test (or . ; % ci . - . ). the relationship was stronger in young children and in prospective studies . in another meta-analysis of prospective studies, ets was found to increase the risk of wheeze by age years by %; prenatal smoking was related to a % increased risk. the risk of asthma decreased with age ( % at age – years, % at – years) . in a cohort study of , mother–child pairs, children of moth- ers exposed to passive smoking whilst pregnant but no other smoking exposure had an increased tendency to develop wheeze up to years old (or . ; % ci . – . ) in comparison with control pairs. exposure to passive smoke postnally, in addi- tion to their mothers’ prenatal passive exposure, further increased the risk (or . ; % ci . – . ). the risk was highest with passive neonatal exposure in addition to prenatal active smoking (or . ; % ci . – . ) . thus, ets was found to be an important but avoidable risk factor for the development of allergic disease in children . smoking is a modifiable risk factor in asthma. there is definitely a need for robust measures to reduce prenatal and postnatal smoking as a strategy for primary prevention of asthma. early life respiratory infections infections childhood asthma and infant respiratory viral infection are the most frequent chronic and acute illnesses of childhood, respec- tively. over the years, it has been possible to make links between these diseases and spot common clinical traits. early life respira- tory syncytial virus (rsv) and human rhinovirus (hrv) lower respiratory tract infections (lrtis) have been found to be strongly associated with increased asthma risk. during early infancy, rsv is a more common cause of severe lrti. with advancing age, the situation is reversed, with hrv becoming more common. in spite of continuous research, the role of these respiratory viruses in the inception of asthma is still under debate . respiratory syncytial virus (rsv). severe rsv bronchioli- tis requiring hospitalization is considered a risk factor for future asthma. in the rsv bronchiolitis in early life study, % of patients hospitalized with rsv had a physician diagnosis of asthma at age years . another study found that % of infants hospital- ized for rsv bronchiolitis had asthma at age years, compared to % in controls . there is a direct relationship between the severity of the initial rsv bronchiolitis and the risk of subsequent asthma, while environmental factors may further augment the risk . thus, the association between early life rsv ltri and later wheezing is consistent across most studies with large effect sizes and severity dose-response relationship. the pathway from early-life infections with rsv to asthma is the result of complex interactions between the specific type of the virus, genetic, and environmental factors. rsv induces persistent airway damage and bronchial hyper-responsiveness . it has been suggested that rsv infection affects th /th balance in early childhood, thereby inducing an atopic state and may, therefore, be involved in the inception of asthma . there is data that suggest that in utero exposure to rsv is followed by dysregulation of neurotrophic pathways, predisposing to postnatal airway hyperreactivity upon reinfection with the virus . palivizumab (monoclonal antibody) prophylaxis in the first year of life reduces recurrent wheezing in children aged to years . palivizumab resulted in a relative reduction of % ( % ci - ) in the total number of wheezing days during the first year of life . however, the substantial cost, the need to treat a large population early in life (before rsv infection), and the need for parenteral administration limit its widespread use . while there is evidence that palivizumab prophylaxis reduces wheezing, its long-term impact on the development of atopic asthma remains controversial. in a follow-up study of children aged to years, palivizumab administration resulted in a % reduction in wheezing in the families of non-asthmatics and an % reduction in wheezing in non-atopic families, but no pro- tection was achieved in atopic families . similarly, in a recent page of f research , (f faculty rev): last updated: jul prospective multicenter observational cohort study of pre- term infants, palivizumab prophylaxis administration significantly reduced subsequent physician diagnosis of recurrent wheezing up to years, but did not reduce the incidence of atopic asthma, casting doubt on the suggestion that rsv prevention may decrease atopic asthma inception . carroll et al. looked at infants at high-risk for severe rsv and whether a link could be found between better adherence to immunoprophylaxis and decreased childhood asthma. analysis revealed that % or greater adher- ence decreased the odds of asthma compared to those with % or less adherence (or . ; % ci . - . ) . recently, in two prospective birth cohort studies, hrv infection was significantly more common among infants administered with rsv immuno- prophylaxis (or, . ; % ci . - . ) . taken together, the impact of palivizumab on the development of atopic asthma after rsv infection is still controversial. a definitive large-scale randomized clinical trial (rct) measur- ing the effect of the prevention of rsv on childhood asthma is yet to be done. a large study evaluated children aged to months at the beginning of a lrti. administration of azithromycin significantly reduced the odds of progres- sion to severe lrti (hr . ; % ci . - . , p= . ) . a relatively small double-blind placebo-controlled trial found a protective effect of azithromycin therapy during rsv bronchi- olitis on subsequent recurrent wheeze . currently, a larger study assessing asthma prevention following rsv by azithromycin is being conducted (nct ). other strategies to prevent rsv are under investigation. there are some efforts to develop a specific vaccination against rsv. recently, a genetically engineered, live attenuated vaccine was found to be effective in a phase i study . another innovative approach includes immunization of pregnant women that potentially will decrease airway hyperreactivity upon postnatal reinfection with the virus . in conclusion, although there is a clear association between rsv and asthma, the role of strategies to prevent rsv (palivizu- mab, azithromycin, vaccine) in asthma prevention has yet to be confirmed. human rhinovirus (hrv). hrv has been increasingly recognized as an etiologic factor in preschool wheeze, as well as a significant risk factor in the development of asthma . episodes of wheezing during which hrv was found in the upper airways have been found to be a strong predictor of subsequent asthma . hrv wheezing illnesses were found to increase the risk of asthma by -fold at years of age . lee et al. analyzed , samples collected from infants enrolled in the coast (childhood origins of asthma) cohort study; these infants had an increased risk for developing allergies and asthma. hrv species a and c were about times more likely to cause moder- ate to severe illness. these results firmly indicate that antiviral therapies aiming at lowering hrv-related morbidity in those infants at high risk ought to be directed towards hrv-a and hrv-c species. as with rsv, the mechanism by which severe early-life hrv causes future asthma remains uncertain. postulated mechanisms are airway epithelial injury and/or creating the appropriate pro- inflammatory allergenic milieu; alternatively, wheezing viral lrti may serve as a marker for asthma susceptibility. several studies suggest that the initial wheezing hrv lrti may serve as a marker for asthma tendency, while early-life severe rsv bronchiolitis may have a causative role in the development of asthma . thus, preventive measures are aimed at decreasing the incidence and severity of infections caused by hrv. a recent study identified day-care attendance (or . ; % ci . - . ), high eosinophil blood counts (or . ; % ci . - . ) and expo- sure to tobacco smoke (or . ; % ci . - . ) as significant risk factors for hrv lrti. hence, restricting children’s exposure to tobacco smoke may limit dissemination of viruses to younger children, counteract severe respiratory diseases, and thus may reduce sequelae. an rct assessed the efficacy of oral corticosteroid treatment during the first hrv lrti to reduce the frequency of subsequent wheezing within months. the study failed to show reduction in post hrv wheezing; however, children with a high viral load treated with prednisolone had a longer time to the next wheezing episode compared to placebo . strategies for developing an effective vaccine or for preventing viral contact and invasion by forming a barrier on the host mucosa are being developed; however, currently there is no approved strategy against hrv. limiting viral spread is the main available protective measure. host factors the microbiome the whole host-microbe system benefits from the essential ecosystem services provided by microbial communities (micro- biota) and the host environment in which they reside. such services include making vital resources, nutrient bioconver- sion, and guarding against harmful microbes. disease can arise from a shortfall in these beneficial functions or the maladaptive functions introduced by pathogenic microbes. with the advent of s rrna sequencing, a strong associa- tion between the host microbiome and asthma has emerged. the microbiome can be modulated by various environmental fac- tors, including diet, prebiotic and probiotic, and early-life microbial exposures. recently, the nasopharyngeal microbiome of healthy infants was compared to infants with confirmed rsv. the abundance of the dominant genera was significantly different between the groups, suggesting that rsv alters the infant nasopharyngeal microbiome and, thereby, may contribute to asthma development . affecting the microbial dysbiosis was suggested as a target for the prevention of asthma . herein, we will discuss the potential role of a prebiotic and probiotic diet in the prenatal and postnatal period. page of f research , (f faculty rev): last updated: jul prenatal. prenatal prebiotic and probiotic supplementation may alter maternal gut bacteria and influence maternal immune function. there is an inconsistent effect on the offspring of mothers supplemented during pregnancy, and a comprehensive world allergy organization review found a lack of evidence to support the use of prebiotics during pregnancy . maternal urinary tract infection during pregnancy increased the likelihood of asthma (or . ; % ci . - . ) . several studies have reported an association between the use of over- the-counter antipyretics during pregnancy or infancy and an increased risk (or . ; % ci . - . ) of asthma in early childhood, but not mid-childhood . in a recent review, the maternal use of antibiotics or paracetamol during pregnancy was suggested as a modifiable risk factor for childhood asthma . postnatal. microbial gut colonization typically starts at the time of birth, and is influenced by the bacterial load of maternal microbiota, type of delivery (cesarean section vs. vaginal delivery), feeding (formula vs. breast-feeding), and the use of antibiotics. in a retrospective cohort study including , births, there was no difference in the risk of asthma following planned com- pared to unscheduled cesarean surgery. however, compared to vagi- nal delivery, planned cesarean delivery resulted in a small increase in the risk of asthma requiring hospital admission (adjusted hr . ; % ci . - . ) and salbutamol inhaler prescription at age years (adjusted hr . ; % ci . - . ) . in another study, cesarean section increased the risk of childhood asthma by % in univariate analysis and % after adjusting for other environmental exposures and covariates . infants born via cesarean section have higher rates of clostridum, klebsiella, bacteroides, and several other species at age to months, which predispose them to a higher risk for asthma, atopy and allergic rhinitis . in the newborn period, the gut microbiota plays a crucial role in maintaining the structure and function of the mucosal immune system. gut-associated mucosal lymphoid tissue becomes reac- tive to pathogenic bacteria but tolerant to “beneficial” bacteria. t regulatory cells (treg) are the key players in immunological tolerance; changes in their number or function are associated with the development of allergy . in a recent systematic review, the early microbiota of children who later developed allergies showed lower bacterial diversity; moreover, a predominance of firmicutes, a higher prevalence of escherichia coli, clostridium difficile, and a lower prevalence of lactobacillus were found . in the canadian healthy infant longitudinal development (child) study, arrieta et al. evaluated the gut microbiota of individuals. they found that those at risk of asthma showed transient gut microbial dysbiosis in their first days of life, with reduced levels of faecalibacterium, veillonella, lachnospira, and rothia. moreover, when these four bacterial taxa were introduced to germ-free mice, they showed decreased airway inflammation, suggesting a causal role of these bacteria in asthma development . several animal and human studies have examined the role of the postnatal use of prebiotics and probiotics in the prevention of allergic diseases. a meta-analysis reported data from , children ( , in the probiotic group, , controls). infants treated with probiotics had a significantly lower risk of eczema (rr . ; % ci . - . ). no significant difference in terms of prevention of asthma (rr . ; % ci . - . ) or wheezing (rr . ; % ci . - . ) was found. another meta-analysis concluded that controlled studies have not yielded sufficient evidence to date to recommend prebiotics and probiotics for the primary prevention of asthma . perinatal and postnatal suggested modifiable behaviors were natural childbirth, breastfeeding, increased outdoor activities, diet and the judicious use of antibiotics and antipyretics. these measures may help restore the neonatal microbiome and may reduce the risk for allergic diseases . nutritional prevention dietary interventions during pregnancy are attractive because they are inexpensive and follow the accepted practice of folic acid supplementation . several nutritional supplements have been investigated; we will discuss the main recent findings. current evidence suggests that maternal diet during pregnancy influences the developing immune system of the fetus. interest- ingly, maternal weight gain or obesity during pregnancy was found not only to increase maternal asthma exacerbations but also the risk for childhood asthma . thus, efforts to prevent childhood asthma focus on early prenatal and postnatal interventions. the mediterranean diet (md) comprises fruit, vegetables and grains, a moderate intake of dairy products and a low intake of meat. this diet has been suggested to have a potential protective role in asthma. in a recent pilot study involving pregnant women, the introduction of md was feasible and acceptable, with % of participants retaining the diet . in a meta-analysis, md was associated with a lower prevalence of “asthma ever” (or - . ; % ci . – . ), as well as “current wheeze” (or - . ; % ci . – . ) and “current severe wheeze” (or - . ; % ci . – . ) . thus, although md has been suggested to be beneficial to general health, its impact on asthma prevention is still not proven and requires a large epidemiological study; assessing the mechanism, the relevant window of exposure and addressing specific components of the diet is warranted. vitamins most studies focus on vitamin d supplementation and the results are inconclusive. vitamin d has the ability to regulate inflam- mation and modulate immune responses and cell growth. experimental data suggest that vitamin d may affect the devel- oping lung and immune system during the prenatal and postnatal periods. additionally, observational studies have suggested an association between maternal intake of vitamin d, cord blood vitamin d levels and persistent and recurrent wheezing in early childhood . in an rct, pregnant danish women receiving iu/d of vitamin d during the third trimester of pregnancy were randomized to receive an additional iu/d or placebo. follow-up of the children (n= ) was completed when the page of f research , (f faculty rev): last updated: jul youngest child reached age years. persistent wheeze was diag- nosed in similar rates ( % and %) of children whose moth- ers received supplemental vitamin d and placebo, respectively (hazard ratio . ; % ci . - . ; p= . ). the results suggest that iu/d during the third trimester of pregnancy cannot reduce the risk of persistent wheeze in the offspring through age years. in the vitamin d antenatal asthma reduction trial (vdaart) rct, pregnant women at risk of having children with asthma were randomized to , international units (iu)/d vitamin d or placebo plus iu/d of vitamin d. supplementation with iu/d resulted in a % reduction of recurrent wheeze or asthma (hazard ratio . ; % ci . - ) that did not reach statistical significance (p= . ) . a secondary analysis of the data revealed that the largest protective effect was found in women with higher initial vitamin d levels who were randomized to the intervention group (adjusted or . ; % ci . - . ) . this suggests that higher vitamin d levels in early pregnancy may be required for asthma/recurrent wheeze prevention in early life. a meta-analysis reporting data from , children demon- strated that prenatal supplementation of vitamin d significantly decreased recurrent wheeze (rr . ; % ci . - . ). postnatal vitamin d administration was found to be associated with a reduction in upper respiratory tract infections ; however, its role in primary asthma prevention remains unclear. thus, a large randomized double-blind controlled study is currently being conducted to assess the role of postnatal vitamin d supplementa- tions on multiple end points, including allergy, atopy and asthma later in life (nct ) . vitamin c and e studies have not undergone meta-analysis due to high heterogeneity; these vitamins did not appear to have a signifi- cant preventive effect on recurrent wheeze . to conclude, currently there is no sufficient evidence regarding any of the vitamins in the inception and prevention of asthma. larger double-blind studies are required to recommend their routine use. antioxidants and fish oil antioxidants reduce reactive oxygen species and there are several reports about the inverse associations between intake of antioxidants and allergic diseases. total antioxidant capacity (tac) assesses the combined activity of all the dietary antioxidants. a study in , swedish children found that higher dietary tac was inversely associated with the sensitization to aeroallergens (or . ; % ci . - . ) and the risk of allergic asthma (or . ; % ci . - . ). interestingly, the relationship was modi- fied by exposure to air pollution. a stronger inverse association between dietary tac and allergy was observed in children with low exposure to air pollution. the authors postulated that high tac may not be enough to counteract the high oxidative stress caused by air pollution . more than a decade ago, hodge et al. examined the relation between certain food consumption and asthma. they found a significantly reduced risk of current asthma in children who ate fresh, oily fish (or . ; % ci . - . ; p< . ) . later on, observational studies suggested an association between low levels of dietary fish-oil derived fatty acids (fa) and the risk of asthma and wheezing. in an rct, pregnant women were assigned to receive . gr of fish oil or placebo (olive oil). in the treatment group, there was a . % reduction in the risk of persistent wheeze or asthma. the effect was most prominent in the women with low fa levels at randomization; moreover, the effect of the intervention remained at age years, suggesting that it is not restricted to the “transient wheezers” . taken together, dietary interventions may aid in the primary prevention of asthma. antioxidants, md and fish oil seem to have a beneficial effect. vitamin d is associated with asthma, but evidence for its role in primary prevention is still lacking. other vitamins studied (such as c and e) failed to show a beneficial effect. stress stress has been suggested to modify the normal lung morpho- genesis and maturation during pregnancy and the postnatal period. stress may affect neuroendocrine, autonomic and immune function programming, thereby leading to increased asthma inception. prenatal maternal stress was found to alter innate and adaptive immune response in cord blood mononuclear cells, suggesting that prenatal stress may impact the expression of allergic diseases and increase the risk for later childhood wheezing , . postnatally, maternal behavior was found to influence the development of hypothalamic-pituitary-adrenal (hpa) responses to stress in rodents, mediated by changes in glucocorticoid receptor (gr) expression . dreger et al. found that exposure to maternal distress restricted to the first year of life resulted in a % increase in cortisol levels in children; beyond the postna- tal period, response to stress differed according to the presence of asthma . a recent study in a high-risk birth cohort found that maternal stress at age and years and maternal depression at any age were positively associated with recurrent wheeze (p< . and p≤ . , respectively) . moreover, both active and passive stressors in asthmatic patients were associated with an increased activation of the sympathetic nervous system . taken together, the data suggest that prenatal as well as postnatal maternal distress may contribute to asthma development in children; thus, lowering early life stress may help decrease asthma. conclusions asthma is a chronic inflammatory disease, and genetic, infectious, nutritional and environmental factors play a role in its pathogen- esis. in recent years, there has been some advance in the concept of primary prevention of asthma. however, there is no consensus on the relative importance of risk factors associated with asthma inception and none of the primary prevention or intervention strate- gies investigated has provided sufficient evidence to lead to wide- spread implementation in clinical practice. current findings suggest that the major preventing measures during pregnancy are avoiding of passive and active smoking and the possible modification of maternal microbiome (e.g. lifestyle, diet, nutritional supplements). avoiding unnecessary caesarean delivery is the main perinatal page of f research , (f faculty rev): last updated: jul measure that may affect asthma. postnatally, the most important measures are preventing severe neonatal respiratory infection, increasing favorable environment and behaviors (e.g. mimicking farm residence, breastfeeding) and decreasing hostile environ- ments (e.g. smoking and air pollution). unfortunately, gaps in knowledge still exist. the exact immune pathways that predis- pose certain infants (and not others) to asthma following early life viral infections are not fully understood. additionally, although there is a clear association between allergy and asthma, its role in the primary prevention of asthma is under debate. moreover, none of the suggested therapies or interventions can serve as a sole solution for the prevention of asthma inception. instead, the combination of several pre- and postnatal factors, such as creat- ing a favorable environment with minimal exposure to a hostile environment, attempts to beneficially affect the maternal and infant microbiome, with prevention of infections in early life, is expected to be more effective. this should be achieved by extensive educational and public health efforts to reduce tobacco smoking and air pollution, to implement dietary interventions in pregnant women, and to encourage breastfeeding and childhood vaccinations. future research should focus on the prevention of rsv and hrv, possibly by vaccine development. moreover, in the era of person- alized medicine, a test that would recognize the specific asthma phenotype and endotype of each patient, as well as his or her own risk factors, would enable tailoring specific preventive measures for the individual patient. competing interests the authors declared that they have no competing interests. grant information the author(s) declared that no grants were involved in supporting this work. references f recommended . jackson dj, hartert tv, martinez fd, et al.: asthma: nhlbi workshop on the primary prevention of chronic lung diseases. ann am thorac soc. ; (suppl ): s – . pubmed abstract | publisher full text | free full text . sullivan pw, ghushchyan v, navaratnam p, et al.: the national cost of asthma among school-aged children in the united states. ann allergy asthma immunol. ; ( ): – .e . pubmed 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first-trimester prenatal diagnosis of pyruvate kinase deficiency in an indian family with the pyruvate kinase-amish mutation genetics and molecular research ( ): - ( ) funpec-rp www.funpecrp.com.br first-trimester prenatal diagnosis of pyruvate kinase deficiency in an indian family with the pyruvate kinase-amish mutation p.s. kedar , s. nampoothiri , s. sreedhar , k. ghosh , k. shimizu , h. kanno and r.b. colah institute of immunohaematology, indian council of medical research, k.e.m. hospital campus, parel, mumbai, india department of pediatric genetics, amrita institute of medical sciences and research center, ernakulam, cochin, india department of bioscience and bioinformatics, kyushu institute of technology, iizuka, fukuoka, japan department of transfusion medicine and cell processing, tokyo women’s medical university, tokyo, japan corresponding author: r.b. colah e-mail: colahrb@gmail.com abstract. pyruvate kinase (pk) deficiency is a rare red cell gly- colytic enzymopathy. the purpose of the present investigation was to offer prenatal diagnosis for pk deficiency to a couple who had a pre- vious child with severe enzyme deficiency and congenital non-sphe- rocytic hemolytic anemia. pk deficiency was identified in the family by assaying the enzyme activity in red cells. chorionic villus sampling was performed in an -week gestation and the mutation was located in exon of the pklr gene characterized by polymerase chain re- genet. mol. res. ( ): - ( ) received december , accepted june , published june , case report genetics and molecular research ( ): - ( ) www.funpecrp.com.br p.s. kedar et al. introduction red cell pyruvate kinase (pk, ec. . . . ) deficiency is the most common en- zyme abnormality in the embden meyerhoff pathway of glycolysis in humans. pk defi- ciency is associated with hereditary non-spherocytic hemolytic anemia and is transmitted as an autosomal recessive disorder (jacobasch and rapoport, ). the clinical severity of this disorder varies widely, ranging from a mildly compensated anemia to severe child- hood anemia, though in some cases it does not manifest itself until adulthood. affected newborns usually present unconjugated hyperbilirubinemia and may require exchange transfusion. pk deficiency was first described by valentine et al. in , and since then, approximately cases have been described in the literature. the human pklr gene spans bp of dna located on the short arm of chromosome and comprises exons (pissard et al., ). prenatal testing for pyruvate kinase deficiency is sometimes requested by parents, who already have an affected child. prenatal diagnosis can be done accurately by analyz- ing fetal dna for the mutation causing the enzyme deficiency. we had earlier screened for mutations in the pklr gene in a nine-year-old child with severe pk deficiency and identi- fied a homozygous ga substitution at nucleotide , changing arginine to histidine at amino acid residue . both parents were heterozygous for this mutation. this muta- tion has been reported previously among the old order amish of pennsylvania (bowman et al., ) and the molecular defect was identified (kanno et al., ), where a ga transition at nucleotide was found. soon after the initial diagnosis, the mother of the propositus became pregnant. after genetic counseling, the family requested prenatal diagnosis, which was possible as the mutations causing pk deficiency in this family were already identified. case report the propositus presented neonatal jaundice at birth and had received an exchange transfusion. he had been transfused times until the age of years, but no diagnosis was action and using restriction endonuclease digestion with the mspi en- zyme, which was confirmed by dna sequencing on the abi dna sequencer. both the parents were heterozygous for the ga [ arghis] mutation in exon and the proband was homozygous for this mutation. the fetus was also heterozygous for this mutation and the pregnancy was continued. prenatal diagnosis allowed the parents with a severely affected child with pk deficiency to have the reproductive choice of having the fetus tested in a subsequent pregnancy. key words: pyruvate kinase deficiency, pyruvate kinase-amish, neonatal jaundice, prenatal diagnosis, india genetics and molecular research ( ): - ( ) www.funpecrp.com.br pyruvate kinase deficiency in an indian family established. at years, he underwent splenectomy and subsequently was maintaining a hemoglobin level between and g/dl. at that time, he was referred to us for diagnosis as a case of unexplained non-spherocytic hemolytic anemia. at the time of investigation, his hb level was . g/dl and reticulocyte count was %. red blood cell pk activity was assayed according to the standard procedure (beutler, ), and pk was found to be markedly reduced ( . iu/g hb at °c) with a - to -fold elevation in , -diphospho- glycerate ( , -dpg). , -dpg level was determined by the chromotropic acid method (ea- ton et al., ). pk deficiency causes accumulation of few glycolytic intermediates such as phosphoenol pyruvate, -phosphoglycerate, -phosphoglycerate, and , -dpg in the red cell. several previous investigators have also reported a good correlation between in- creased levels of , -dpg and pk deficiency (lestas et al., ). the parental red blood cell pk activities (father with . iu, mother with . iu/g hb, at °c) were consistent with heterozygosity for pk deficiency. the normal reference range of pk activity was es- tablished using healthy normal control samples and ranged from . to . iu/g hb. dna analysis showed the propositus to be homozygous and his parents heterozygous for the ga mutation. in the next pregnancy, chorionic villus sampling (cvs) under ul- trasound guidance was performed at weeks gestation, after obtaining informed consent from the couple, and the villus tissue was cleaned under a dissecting microscope and used for molecular analysis. mutation analysis genomic dna was extracted from peripheral blood of the parents and the cvs using the qiagen mini-columns (hilden, germany). the ga mutation was identified using re- striction endonuclease digestion with the enzyme mspi (new england biolabs, uk). a -bp dna fragment of exon of the pklr gene was amplified by the polymerase chain reaction (pcr) using ng dna and ng each of primers pk ex- f and pk ex- r ( ’ gac taa cat tct ggc acc tg ’ and ’ aag ctc cat ctg gac att cc ’, respectively). dna was initially denatured at oc for min followed by cycles of denaturation at oc for s, annealing at °c for s, and primer extension at °c for s, with final extension at °c for min. a volume of μl of the pcr product was digested with mspi ( u) at °c overnight. dna fragments were visualized by uv-transillumination after separation by elec- trophoresis on an % polyacrylamide gel and staining with ethidium bromide. dna sequence analysis of exon was performed on the abi prism sequencer to confirm the mutation (kanno et al., ). figure a shows the results of restriction enzyme digestion with mspi and the dna sequencing of the cvs and the proband. the cvs showed fragment sizes of and bp and was heterozygous for the ga mutation. both parents were also heterozygous for this mutation, while the affected child showed the absence of the -bp band. the -bp band (lane ) corresponds to the undigested pcr product. this is due to heteroduplex forma- tion between normal and mutant dna resulting in the abolition of the mspi recognition site preventing cleavage. the dna sequence electropherogram confirmed that the fetus is hetero- zygous (figure c). figure b and d show dna sequencing of exon in a normal individual and the homozygous child, respectively. genetics and molecular research ( ): - ( ) www.funpecrp.com.br p.s. kedar et al. discussion pk deficiency, although rare, is probably the most common cause of hereditary non- spherocytic hemolytic anemia, often causing severe anemia from infancy or early childhood. prenatal diagnosis by assaying pk activity in fetal blood is not always accurate and molecular analysis is required. the mutations in the pklr gene resulting in pk deficiency are many and varied; however, once the mutation in the parents and an affected child born earlier is charac- terized, prenatal diagnosis can be easily done in the first trimester of pregnancy by cvs and dna analysis. we describe the cellular and molecular studies of erythrocyte pk deficiency in a family originating from south india where the mutation in both the parents was the same as in the dutch amish population of pennsylvania. nucleotide sequencing of the patient’s pk gene showed a point mutation, cgc to cac, corresponding to nucleotide from the translational initiation site of the r type pk (r-pk) mrna, causing a single amino acid substitution from arg to his at the th amino acid residue of the r-pk gene. it has been shown earlier that the substituted arg figure . prenatal diagnosis of pyruvate kinase deficiency due to a codon ( ga) mutation. a. mspi digest of exon of the pklr gene on % polyacrylamide gel electrophoresis. lane , pcr amplified undigested dna; lane , father (heterozygous); lane , mother (heterozygous); lane , chorionic villus sample (heterozygous); lane , proband (homozygous); lane , marker viii (roche); b. dna sequencing of exon in a normal individual. c. dna sequencing of exon of the chorionic villus sample. d. dna sequencing of exon of the proband. genetics and molecular research ( ): - ( ) www.funpecrp.com.br pyruvate kinase deficiency in an indian family residue is located in the c domain of the pk subunit that is essential for both intersubunit contact and allosteric regulation. as this enzyme shows catalytic activity only as a dimer or tetramer, it is rational that the structural alteration would result in severe pk deficiency (kanno et al., ). pk deficiency hemolytic anemia, described in the old order amish of pennsylvania (bowman et al., ), was much more severe than that reported by tanaka et al. ( ). the mutation was subsequently characterized by kanno et al. ( ). the affected individual in this report had severe hemolytic anemia which could have led to death in the first years of life if not treated by transfusions and splenectomy. in the family referred to us, the propositus was also homozygous for the ga mutation. as we had already characterized the mutation, it allowed a rapid and specific prenatal diagnosis by mspi restriction analysis which was also confirmed by dna sequencing. the fetus was found to be heterozygous and the pregnancy was continued. there are only a handful of reports on prenatal diagnosis of pk deficiency. there are two reported cases where prenatal diagnosis was done using amniotic fluid cells in one case and cord blood in the other by pcr and restriction enzyme analysis as well as linkage analysis using two polymorphic sites linked to the pklr gene (baronciani and beutler, ). in , prenatal diagnosis of pk-monder due to a frame shift mutation in the pklr gene causing severe hereditary non-spherocytic anemia was done in france (rouger et al., ). in india, pk deficiency had remained an under-diagnosed disease. in the last five years we encountered cases of pk deficiency (colah and kedar, ; kedar et al., ), and hence, this erythro-enzymopathy is not that uncommon in india. fetal tissue in our case was obtained by cvs to enable diagnosis in the first trimester of pregnancy. the possibility of phenotypic variation in pk deficiency should be borne in mind when counseling families at risk about the likely clinical effects in an affected child. prospects for treatment of severe pk deficiency are limited as it is necessary to correct the enzyme deficiency in red cells. in the long term, this may be achieved by gene therapy. until then, the ability to offer early prenatal diagnosis to families at risk for this often lethal disease will remain an important option. references baronciani l and beutler e ( ). prenatal diagnosis of pyruvate kinase deficiency. blood : - . beutler e ( ). red cell metabolism: a manual of biochemical methods. grune and stratton, new york. bowman hs, mckusick va and dronamraju kr ( ). pyruvate kinase deficient hemolytic anemia in an amish isolate. am. j. hum. genet. : - . colah rb and kedar ps ( ). red cell enzymopathies in india. in: hematology today (agarwal mb, ed.). ashirwad hematology centre, mumbai, - . eaton jw, brewer gj and grover rf ( ). role of red cell , -diphosphoglycerate in the adaptation of man to altitude. j. lab. clin. med. : - . jacobasch g and rapoport sm ( ). hemolytic anemias due to erythrocyte enzyme deficiencies. mol. aspects med. : - . kanno h, ballas sk, miwa s, fujii h, et al. ( ). molecular abnormality of erythrocyte pyruvate kinase defi- ciency in the amish. blood : - . kedar ps, warang p, colah rb and mohanty d ( ). red cell pyruvate kinase deficiency in neonatal jaundice cases in india. indian j. pediatr. : - . lestas an, kay la and bellingham aj ( ). red cell -phosphoglycerate level as a diagnostic aid in pyruvate kinase deficiency. br. j. haematol. : - . genetics and molecular research ( ): - ( ) www.funpecrp.com.br p.s. kedar et al. pissard s, max-audit i, skopinski l, vasson a, et al. ( ). pyruvate kinase deficiency in france: a -year study reveals new mutations. br. j. haematol. : - . rouger h, girodon e, goossens m, rouger h, et al. ( ). pk mondor: prenatal diagnosis of a frameshift muta- tion in the lr pyruvate kinase gene associated with severe hereditary non-spherocytic haemolytic anaemia. prenatal diagn. : - . tanaka kr, valentine wn and miwa s ( ). pyruvate kinase (pk) deficiency hereditary nonspherocytic hemo- lytic anemia. blood : - . valentine wn, tanaka kr and miwa s ( ). a specific erythrocyte glycolytic enzyme defect (pyruvate kinase) in three subjects with congenital non-spherocytic hemolytic anemia. trans. assoc. am. physicians : - . growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging aaron r. jeffries, reza maroofian, claire g. salter, , , barry a. chioza, harold e. cross, michael a. patton, , emma dempster, i. karen temple, , deborah j.g. mackay, faisal i. rezwan, lise aksglaede, diana baralle, , tabib dabir, matthew f. hunter, , arveen kamath, ajith kumar, ruth newbury-ecob, angelo selicorni, amanda springer, , lionel van maldergem, vinod varghese, naomi yachelevich, katrina tatton- brown, , , jonathan mill, andrew h. crosby, and emma l. baple , institute of biomedical and clinical science, university of exeter medical school, rild wellcome wolfson centre, royal devon and exeter nhs foundation trust, exeter, ex dw, united kingdom; genetics research centre, molecular and clinical sciences institute, st. george’s university of london, london sw re, united kingdom; human genetics and genomic medicine, faculty of medicine, university of southampton, southampton, so yd, united kingdom; wessex clinical genetics service, princess anne hospital, southampton, so ya, united kingdom; department of ophthalmology and vision science, university of arizona school of medicine, tucson, arizona , usa; department of clinical genetics, copenhagen university hospital, blegdamsvej b, copenhagen n, denmark; northern ireland regional genetics centre, clinical genetics service, belfast city hospital, belfast, bt ab, united kingdom; monash genetics, monash health, clayton, victoria, vic , australia; institute of medical genetics, university hospital of wales, cardiff, cf xn, united kingdom; north east thames regional genetics service and department of clinical genetics, great ormond street hospital, london, wc n jh, united kingdom; university hospitals bristol, department of clinical genetics, st michael’s hospital, bristol, bs eg, united kingdom; uoc pediatria asst lariana, como, italy; department of paediatrics, monash university, clayton, victoria, vic , australia; centre de génétique humaine and clinical investigation center (inserm), université de franche-comté, , besançon, france; clinical genetics services, new york university hospitals center, new york university, new york, new york , usa; division of genetics and epidemiology, institute of cancer research, london sm ng, united kingdom; south west thames regional genetics service, st. george’s university hospitals nhs foundation trust, london sw qt, united kingdom; peninsula clinical genetics service, royal devon and exeter hospital, exeter, ex ed, united kingdom germline mutations in fundamental epigenetic regulatory molecules including dna methyltransferase alpha (dnmt a) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. we pro- filed genome-wide dna methylation patterns in dnmt a c. g > a; p.(arg gln) carriers in a large amish sibship with tatton-brown–rahman syndrome (tbrs), their mosaic father, and tbrs patients with distinct pathogenic de novo dnmt a variants. this defined widespread dna hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. tbrs patients also displayed highly accelerated dna methylation aging. these findings were most marked in a carrier of the aml-asso- ciated driver mutation p.arg cys. our studies additionally defined phenotype-related accelerated and decelerated epi- genetic aging in two histone methyltransferase disorders: nsd sotos syndrome overgrowth disorder and kmt d kabuki syndrome growth impairment. together, our findings provide fundamental new insights into aberrant epigenetic mecha- nisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders. [supplemental material is available for this article.] dna methylation is an essential epigenetic process involving the addition of a methyl group to cytosine. it is known to play a role in many important genomic regulatory processes, including x-chromosome inactivation, genomic imprinting, and the repres- sion of tumor suppressor genes in cancer, mediating transcription- al regulation as well as genomic stability (jones ). three catalytically active dna methyltransferases (dnmts) are involved corresponding authors: e.baple@exeter.ac.uk, a.h.crosby@exeter.ac.uk, j.mill@exeter.ac.uk article published online before print. article, supplemental material, and publi- cation date are at http://www.genome.org/cgi/doi/ . /gr. . . freely available online through the genome research open access option. © jeffries et al. this article, published in genome research, is available un- der a creative commons license (attribution . international), as described at http://creativecommons.org/licenses/by/ . /. research : – published by cold spring harbor laboratory press; issn - / ; www.genome.org genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from mailto:e.baple@exeter.ac.uk mailto:a.h.crosby@exeter.ac.uk mailto:j.mill@exeter.ac.uk http://www.genome.org/cgi/doi/ . /gr. . http://www.genome.org/cgi/doi/ . /gr. . http://genome.cshlp.org/site/misc/terms.xhtml http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / http://genome.cshlp.org/site/misc/terms.xhtml http://genome.cshlp.org/ http://www.cshlpress.com in the methylation of cytosine: dnmt , which is mainly respon- sible for the maintenance of dna methylation over replication, and dnmt a and dnmt b, which generally perform de novo methylation of either unmethylated or hemimethylated dna. an absence of these enzymes in mice results in embryonic (dnmt and b) or postnatal (dnmt a) lethality (okano et al. ), confirming their essential roles in development. in line with knockout mouse models, pathogenic variants affecting the chromatin binding domains of dnmt have been shown to cause two separate progressive autosomal dominant adult-onset neuro- logic disorders (klein et al. ). biallelic pathogenic variants in dnmt b have been associated with immunodeficiency, centro- mere instability, and facial anomalies (icf) syndrome (jiang et al. ). to date, dnmt a has been linked to a number of physiological functions, including cellular differentiation, malig- nant disease, cardiac disease, learning, and memory formation. somatically acquired pathogenic variants in dnmt a are associat- ed with > % of acute myeloid leukemia (aml) cases, whereas het- erozygous germline pathogenic loss-of-function variants have been found to underlie tatton-brown–rahman syndrome (tbrs; also known as dnmt a-overgrowth syndrome, omim ) (challen et al. ; tatton-brown et al. ). tbrs is character- ized by increased growth, intellectual disability (id), and dysmor- phic facial features. more recently, heterozygous gain-of-function dnmt a missense variants affecting the dnmt a pwwp domain have been shown to cause microcephalic dwarfism and hypermethylation of polycomb-regulated regions (heyn et al. ). there is an emerging group of epigenetic regulatory mole- cule-associated human growth disorders in which the underlying molecular defect is a disruption to the dna methylation and his- tone machinery. there are now over disorders identified within this group, which can be further subgrouped into diseases result- ing from disruption of the “writers,” “readers,” and “erasers” of epigenetic modifications (bjornsson ). example disorders in each group include kabuki, sotos, and weaver syndromes (“writ- ers”); smith-magenis, rett, and bohring–opitz syndromes (“read- ers”); and wilson–turner and cleas–jensen syndromes (“erasers”). the final subgroup occurs because of disruption of chromatin remodelers, with example resulting disorders including charge and floating–harbor syndromes. neurological and cognitive impairment are common features of these conditions, suggesting that precise epigenetic regulation may be critical for neuronal homeostasis. however, a true understanding of the pathogenic mechanism underlying these conditions remains poorly understood. in the current study, we investigated the methylomic conse- quences of a dnmt a pathogenic variant (nc_ . : g. c > t; nm_ . :c. g > a; p.(arg gln)) in a large amish family comprising four individuals affected with tbrs arising as a result of a mosaic pathogenic dnmt a variant in their father (xin et al. ). the occurrence of multiple affected and unaffected individuals in the same sibship, together with the combined genetic and environmental homogeneity of the amish, permitted an in-depth investigation of the genome-wide patterns of dna methylation associated with pathogenic variation in dnmt a. we subsequently extended our analyses to other (non-amish) tbrs patients harboring distinct pathogenic de novo dnmt a variants, as well other methyltransferase-associat- ed overgrowth and growth deficiency syndromes, defining altered epigenetic profiles as common key themes of these growth disorders. results reduced dna methylation at key sites involved in morphogenesis, development, and differentiation in tbrs patients dnmt a encodes a dnmt with both de novo and maintenance activity (okano et al. ; chen et al. ). we first looked for global changes in dna methylation in whole blood obtained from dnmt a c. g > a; p.(arg gln) carriers, using the methylation-sensitive restriction enzyme–based luminometric methylation assay (luma) (karimi et al. ) to quantify dna methylation across gc-rich regions of the genome, finding no ev- idence for altered global dna methylation (luma: mean dnmt a c. g > a carriers = . , wild type = . , t-test p- value = . ). we next quantified dna methylation at , autosomal sites across the genome using the illumina k array. globally, a subtle decrease in mean dna methylation was noted in available age/sex-matched dnmt a heterozygous c. g > a; p.(arg gln) individuals compared with their matched unaffect- ed sibling samples, although this was not statistically significant (wilcoxon rank-sum test p-values for two matched pairs = . and . ) (supplemental fig. s ). in contrast, an analysis of site- specific dna methylation differences in dnmt a c. g > a; p.(arg gln) carriers (including the mosaic father) versus wild- type individuals in the amish pedigree identified differen- tially methylated positions (dmps; benjamini–hochberg false discovery rate [fdr] < . ) (fig. a,b; supplemental table s ), of which dmps were characterized by a > % change in dna methylation. supplemental figure s also highlights dna methyl- ation levels at these dmps across all carriers and control individu- als profiled in this study. technical validation of illumina k array data was performed using bisulfite pyrosequencing for three top-ranking dmps, confirming significant differences in dnmt a c. g > a; p.(arg gln) carriers at each of the tested loci (supplemental fig. s ). the dmps identified were highly enriched for sites character- ized by reduced dna methylation in dnmt a c. g > a; p.(arg gln) heterozygotes (n = dmps, . %, sign-test p-value < . × − ). although there were no statistically signifi- cant differences between dna methylation-based blood cell com- position estimates derived from our data (supplemental table s ), we examined the extent to which the identified dmps were poten- tially influenced by cell-type differences between dnmt a c. g > a; p.(arg gln) carriers and wild-type family mem- bers. there was a highly significant correlation (r = . , p-value < . × − ) (supplemental fig. s ) in effect sizes at the dmps between models, including and excluding cell types as co- variates, indicating that the observed patterns of differential dna methylation are not strongly influenced by cell-type varia- tion. we used dmrcate (peters et al. ) to identify spatially cor- related regions of differential dna methylation significantly associated with the dnmt a c. g > a; p.(arg gln) variant, identifying autosomal differentially methylated regions (dmrs) (for an example dmr, see supplemental fig. s ), all characterized by hypomethylation in dnmt a c. g > a; p.(arg gln) carriers apart from one -bp dmr that showed increased dna methylation (supplemental table s ). the mean size of the identified dmrs was bp (range = – bp), span- ning an average of six probes (supplemental fig. s ). we next investigated whether dnmt a p.(arg gln)-asso- ciated dmps are enriched in specific genic locations (see methods). we found a modest enrichment of dmps in regions ≥ bp jeffries et al. genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/ http://www.cshlpress.com upstream of the transcriptional start site (chi-squared yates-correct- ed p-value = . ) and more prominent enrichment in intergenic regions (chi-squared yates corrected p-value = . × − ) (sup- plementalfig.s ).dmpswerealsosignificantlyenrichedincpgis- land shore regions (chi-squared yates-corrected p-value = × − ) (supplemental fig. s ). we also examined dmp occurrence in ex- perimentally determined cancer and reprogramming-specific dmr locations (doi et al. ), finding a . -fold and b a c d e figure . tbrs dnmt a variants are associated with widespread dna hypomethylation. (a) simplified pedigree indicating the genotyping of individuals in the amish family investigated: (+/−) heterozygous carriers of the dnmt a c. g > a p.(arg gln) variant; (+/− mosaic) the dnmt a c. g > a p.(arg gln) mosaic father; (−/−) wild-type individuals. black shading indicates individuals with a phenotype consistent with tbrs, gray shading, the father with macrocephaly and mild intellectual impairment; and white shading, unaffected individuals. each of these samples was profiled on the illumina k dna methylation array. (b) volcano plot showing site-specific dna methylation differences (x-axis) and −log p-values (y-axis) from an analysis comparing amish dnmt a c. g > a; p.(arg gln) pathogenic variant carriers and wild-type family members using the illumina k array. red values indicate the differentially methylated positions (dmps) detected at a benjamini–hochberg fdr < . . (c) top gene ontology enrich- ment analysis categories associated with the dmps identified in dnmt a c. g > a; p.(arg gln) pathogenic variant carriers versus wild-type family members. (d) comparison of dnmt a c. g > a; p.(arg gln) identified dmps (log fold change) relative to other dnmt a tbrs-associated variants assessed in this study (all variants grouped and measured relative to controls). pearson correlation coefficient = . , p-value < . × − . (e) boxplot illustrating the dna methylation changes observed in association with the dnmt a tbrs variants studied at the dmps identified in the amish dnmt a c. g > a p.(arg gln) carriers. the predicted protein consequence of each dnmt a variant studied is indicated: pink indicates in-frame deletion; yellow, single-nucleotide variant; cyan, duplications predicted to result in a frameshift; green, amish c. g > a; p.(arg gln) variant. epigenetic aging abnormalities in growth disorders genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/ http://www.cshlpress.com . -fold overrepresentation (chi-squared yates-corrected p-value = . × − and chi-squared yates-corrected p-value < . × − , respectively), as well as predicted enhancer elements that showed a . -fold overrepresentation (chi-squared yates-corrected p-value = . × − ). we then undertook gene ontology analysis, ac- countingfor the background distribution of probes on the illumina k array, to functionally annotate the dna methylation differences observed in the dnmt a c. g > a; p.(arg gln) carriers. the dmps identified in this study showed a significant overrepresentation in functional pathways related to morphogenesis, development, and differentiation (top hit go: , multicellular organism development, contains genes associated with dmps; fdr q-value = . × − ) (fig. c; supplemental table s ). we also performed a functional overlap analysis to identify cell- or tissue-specific chromatin signals associ- atedwiththesedmpsusingeforge(breezeetal. ).significant overlap (fdr q-value < . ) was found with dnase i sensitivity hotspots, most apparent with pluripotent cells in encode (the encode project consortium ; davis et al. ) and fetal tis- sues within the nih roadmap epigenomics consortium data set (supplemental data s ; roadmap epigenomics consortium et al. ). chromatin states fromthe nih roadmap epigenomics con- sortium data set show an enrichment of dmps in regions defined as active transcriptional start sites in brain tissue and embryonic stem cells. of particular interest, given the established importance of dnmt a during embryonic development, eforge analysis of blood cell types highlighted an enrichment of dmps in regions characterized by repressed polycomb and enhancer activity. to provide additional evidence to support the notion that dnmt a c. g > a; p.(arg gln) carriers show disruption to developmental pathways, we used the genomic regions enrichment of annotation tool (great) (mclean et al. ) to explore functional pathways enriched in genes annotated to dnmt a c. g > a; p.(arg gln)–associated dmrs. this re- vealed a significant effect on genes implicated in developmental pathways (first ranked go biological process = skeletal system de- velopment, fold enrichment = . , binomial fdr q-value = . × − ), with a specific enrichment for homeobox protein domain encoding genes (interpro; fold enrichment = . , binomial fdr q-value = . × − ), fundamental for normal developmen- tal processes. an enrichment for malignancy terms was also noted (from the molecular signatures database; first ranked term = genes with promoters occupied by pml-rara fusion protein in acute promyelocytic leukemia [apl] cells nb and two apl primary blasts, based on chip-seq data, fold enrichment = . , binomial fdr q-value = . × − ) (see supplemental data s ; liberzon et al. ). to establish whether these dmps are a consistent feature of tbrs, we profiled a further non-amish patients carrying dis- tinct previously published dnmt a pathogenic variants (table ; fig. ) using the illumina epic dna methylation array. examination of the dmps identified in dnmt a c. g > a; p.(arg gln) carriers revealed that the majority of dmps were common to all of the tbrs patients regardless of the underlying causative dnmt a variant (fig. d), with a pearson correlation co- efficient of . (p-value < . × − ) for effect sizes across all dmps. each variant showed some heterogeneity in effect size (fig. e), with dnmt a c. c > t p.(arg cys) associated with the greatest overall changes in dna methylation. this data leads us to conclude that tbrs patients show loss of methylation at sites annotated to key genes involved in development and growth pathways, mirroring the well-characterized overgrowth and neurocognitive features that characterize this disorder. dnmt a mutations are associated with highly accelerated epigenetic aging, particularly the cardinal aml driver mutation p.arg cys dna methylation at a specific set of cpg sites, representing a so-called “epigenetic clock,” has been shown to be strongly table . tbrs dnmt a variants are associated with epigenetic age acceleration id nucleotide change protein change chronological age (yr) epigenetic age (yr) epigenetic age acceleration (fold change) epigenetic age acceleration (percentage increase) single nucleotide variants c. t > a p.(ile asn) . . . % c. g > a p.(gly ser) . . . % c. t > c p.(cys arg) . . % c. t > c p.(leu pro) . . . % c. c > t p.(pro leu) . . . % c. c > t p.(arg cys) . . . % c. g > a p.(arg his) . . . % c. g > a p.(arg gln) – . – . . %a c. a > g p.(asn asp) . . . % c. c > t p.(arg cys) . . . % c. t > c p.(phe ser) . . . % c. c > t p.(pro leu) . . . % in-frame deletions c. _ deltgg p.(trp del) . . . % c. _ deltct p.(phe del) . . . % duplications resulting in a frameshift c. dupa p.(arg fs) . . . % c. _ duptctt p.(ser fs) . . % dnmt a genotype (p.(arg gln), shown in blue; p.(arg cys), shown in red), chronological age, predicted epigenetic age, and percentage of age acceleration calculated for tbrs syndrome cases included in this study. aepigenetic age acceleration taken from the linear regression model applied to four individuals carrying the mutation. jeffries et al. genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/ http://www.cshlpress.com correlated with chronological age (horvath ). deviations from chronological age have been associated with several measures of ac- celerated biological aging and age-related phenotypes (johnson et al. ; levine et al. ; marioni et al. ; chen et al. ). we investigated the dna methylation age of dnmt a c. g > a p.(arg gln) carriers using the dna age calculator (horvath ; http://dnamage.genetics.ucla.edu/), finding that dnmt a c. g > a; p.(arg gln) carriers show evidence for highly accelerated aging—an increase of ∼ % beyond their chro- nological age—compared with wild-type family members (ancova p-value = . ) (fig. a). only one of probes used in the epigenetic clock (horvath ) overlapped with the dmps significantly associated with the dnmt a c. g > a; p.(arg gln) pathogenic variant, leading us to conclude that this finding represented a true acceleration of epigenetic age. furthermore, compared with an extensive number ( ) of wild- type control samples profiled in a previous study from our group (hannon et al. ), dnmt a c. g > a; p.(arg gln), carri- ers were consistent outliers for epigenetic age, suggesting their profiles fall outside the normal distribution of variance observed in the general population (supplemental fig. s ). consistent with this, the mosaic amish father was found to have an interme- diate level of epigenetic age acceleration, with a % increase over his chronological age. this age acceleration was a cumulative pro- cess as indicated by the increased slope of dnmt a c. g > a; p.(arg gln) carriers versus wild-type. epigenetic age could therefore be predicted by the linear regression model as follows: epigenetic age = . + . × chronological age. the cumulative increase of epigenetic age relative to chronological age is also nota- ble compared with a recent meta-analysis of longitudinal cohort data that shows the trajectory of epigenetic age in different popula- tions progresses at a slightly slower rate compared with increasing chronological age (marioni et al. ). we next looked for evidence of elevated epigenetic aging in tbrs patients carrying one of the additional de novo dnmt a pathogenic variants. all tbrs patients showed accelerat- ed epigenetic aging, although the position and type of each variant result in differing degrees of accelerated epigenetic aging (table ). the greatest rate of epigenetic age acceleration (> %) was ob- served in association with the germline p.(arg cys) substitu- tion, somatic mutation of dnmt a arg being the most commonly associated with aml. altered epigenetic aging in methyltransferase-associated human growth disorders to determine whether altered epigenetic aging is a characteristic of other growth disorders associated with disruption of epigenetic regulatory molecules, we extended our study using publicly avail- able illumina k dna methylation data. we first analyzed the data from individuals with sotos syndrome, a congenital over- growth syndrome that results from mutation of the epigenetic modifier nsd (supplemental table s ), a lysine histone methyl- transferase (kurotaki et al. ; qiao et al. ). consistent with dnmt a pathogenic variant carriers, these individuals are characterized by an epigenetic age acceleration of ∼ % (linear re- gression model r = . , p-value = . × − ) (fig. b,d). we then examined data from kabuki syndrome patients carrying path- ogenic variants in the kmt d gene (supplemental table s ), which also encodes a lysine histone methyltransferase (ng et al. ; butcher et al. ). kabuki syndrome is a multisystem dis- order. patients typically present with postnatal growth deficiency (rather than overgrowth), a characteristic facial gestalt, id, and other variable phenotypic features. although there is more hetero- geneity in epigenetic age when compared with the nsd patho- genic variant carriers, there was a significant reduction in epigenetic age of ∼ % seen across these individuals (linear regres- sion model r = . , p-value = . ) (fig. c,d). discussion to date, individuals have been described with the overgrowth condition tbrs. within this group, a wide variety of germline dnmt a pathogenic variants have been reported, including missense, eight stop-gain, seven frameshift and two splice site var- iants, two in-frame and five whole-gene deletions (including a set of identical twins) (tatton-brown et al. ; okamoto et al. ; tlemsani et al. ; hollink et al. ; kosaki et al. ; lemire et al. ; shen et al. ; spencer et al. ; tatton-brown et al. , ; xin et al. ). clinically, the predominant aa prc /eed-ezh complex s-adenosyl-l-methionine - - - aa posi�ons pwwp add mtasedomains interac�ons dnmt and dnmt b aa aa aa aa aa aa aa aascale: missense variant in-frame dele�on frameshi� variant figure . schematic representation of dnmt a. the positions of the disease-associated variants included in this study are indicated relative to the pro- tein domain architecture. pwwp, proline-tryptophan-tryptophan-proline domain; add, atrx-dnmt -dnmt l domain; mtase, methyltransferase domain; aa, amino acid. epigenetic aging abnormalities in growth disorders genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/ http://www.cshlpress.com features of tbrs are overgrowth, a characteristic facial gestalt, and neurocognitive impairment. these features show phenotypic overlap with conditions associated with germline pathogenic var- iants in other epigenetic regulatory genes, including sotos and weaver syndromes caused by variants in nsd and ezh histone methyltransferases, respectively (tatton-brown et al. ). these genes encode essential epigenetic regulatory proteins, with a dual somatic/germline role in the pathogenesis of hematological malig- nancies and overgrowth syndromes with variable degrees of intel- lectual impairment (tatton-brown et al. ). the majority of dnmt a pathogenic variants in tbrs have been found to be de novo, with five individuals inheriting the pathogenic variant from two mosaic parents (tlemsani et al. ; xin et al. ) and two individuals inheriting the patho- genic variant from their affected father (lemire et al. ). extensive studies of the role of dnmt a in hematopoietic stem cell (hsc) differentiation are also reported, including the regular occurrence of somatic dnmt a variants in patients with acute myeloid leukemia (aml). the most common somatic pathogenic variant reported in patients with aml affects the amino acid resi- due arg . to date, pathogenic variants predicted to affect this residue have been described in the germline of tbrs patients, five with p.(arg his) and seven with p.(arg cys) (tlemsani et al. ; hollink et al. ; kosaki et al. ; shen et al. ; spencer et al. ; tatton-brown et al. ). despite these studies, the underlying biological mech- anism and outcomes of dnmt a gene mutation in tbrs, as well as the potential risks of hematological malignancy, re- main largely unclear. here we investigated variation in dna methylation associated with a germline heterozygous dnmt a mis- sense pathogenic variant c. g > a; p.(arg gln), affecting the catalytic mtase domain, in a large amish family comprising four children with tbrs, un- affected siblings, and their mosaic father who displayed an intermediate clinical phenotype (xin et al. ). affected in- dividuals were characterized by wide- spread hypomethylation, with dmps enriched in the vicinity of genes/reg- ulatory regions associated with growth and development, tissue morphogene- sis, and differentiation. the magnitude of hypomethylation typically exceeded %, a level often considered to show biological significance (leenen et al. ). the accelerated epigenetic age ob- served did not appear to be driven by overlap of the dnmt a c. g > a; p.(arg gln) variant–associated dmps with the probes that comprise the epige- netic clock as shown by overlap with only one out of the probes used in the epigenetic age estimation (horvath ). although the relevance of blood cells to understanding the etiology of tbrs is not yet known, we hypothesize that our findings will be generalizable across cell types given the ubiquitous developmental expression of dnmt a and given that many age-associated dmps are shared across different cell types (zhu et al. ). nevertheless, it would still be prudent to undertake epigenetic age assessment of other tissues from tbrs patients to determine whether epigenetic agetrulyisaccelerated across all cell typesorafindingthatislimited to blood. although dysregulation of growth control has been linked to numerous developmental disorders and malignancy, the specific molecular basis of this relationship is not fully understood. the as- sessment of dmps associated with the dnmt a c. g >a; p.(arg gln) variant identified an enrichment of pluripotent and fetal dnase i sensitivity hotspots, as well as brain and embry- onic stem cell–associated chromatin sites according to the encode project consortium and epigenomic roadmap consortium data sets (breeze et al. ). similarly, functional an- notation based on gene ontology terms showed an overrepresen- tation of pathways related to morphogenesis, development, and differentiation annotations. dnmt a loss of function has previ- ously been reported to result in up-regulated multipotency genes a b c d w figure . altered epigenetic aging is observed in methyltransferase-associated human growth disor- ders. (a) scatter plot comparing “dna methylation age” derived from the illumina k data (y-axis) and actual chronological age (x-axis) in dnmt a c. g > a p.(arg gln) pathogenic variant carriers (red) versus wild-type family members (blue). green indicates the mosaic individual. the linear regression model is also shown. (b) scatter plot comparing dna methylation age versus chronological age in patients with sotos syndrome. in-frame legend illustrates the different nsd pathogenic variants studied. (c) scatter plot comparing dna methylation age versus chronological age in patients with kabuki syn- drome. in-frame legend illustrates the different kmt d pathogenic variants studied. (d) boxplot compar- ing the epigenetic age acceleration rates found in association with tbrs dnmt a variants, kmt d kabuki syndrome variants, and nsd sotos syndrome variants. each age acceleration observation is plot- ted as a circle. the dotted red line denotes no age acceleration. jeffries et al. genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com and impaired differentiation of neural stem cells and hscs (wu et al. ; challen et al. ; jeong et al. ) compared with a gain-of-function dnmt a variant that may increase cellular dif- ferentiation (heyn et al. ). it is thus conceivable that tbrs-as- sociated dnmt a variants may promote increased proliferation of stem/progenitor cell pool, resulting in increased cell numbers dur- ing organ morphogenesis and clinical overgrowth. our finding of altered epigenetic outcomes in tbrs prompted us to consider similar investigations in other growth disorders as- sociated with epigenetic dysfunction: sotos syndrome, a neurode- velopmental disorder with features overlapping tbrs and with association with overgrowth in childhood owing to histone meth- yltransferase nsd gene alterations, and kabuki syndrome, a dis- tinct neurodevelopmental disorder associated with poor growth and histone methyltransferase kmt d gene alterations. this work defined clear aberrations in epigenetic aging appropriate to the specific nature of each condition. in both overgrowth condi- tions, tbrs and sotos syndrome, we identified accelerated epige- netic aging as measured by the dna methylation age calculator (horvath ). conversely, patients with kabuki syndrome, clin- ically characterized by poor growth, displayed decelerated epige- netic age. epigenetic age has been strongly correlated with chronological age in unaffected individuals in previous studies of a variety of tissue types (hannum et al. ; horvath ). the observation of accelerated epigenetic aging in both tbrs and sotos syndrome potentially results from reduced methyltrans- ferase activity in addition to increased cell turnover associated with the overgrowth seen with these disorders, with the converse being the case for kabuki syndrome. accelerated epigenetic aging has been associated with age-related clinical characteristics and mortality in epidemiological studies. for example, accelerated epi- genetic age in lymphocytes correlates with reduced physical and cognitive function in the elderly and with increased overall mor- tality independent of other variables such as bmi, sex, and smok- ing status (marioni et al. ; chen et al. ). the molecular basis of tbrs has only been determined relatively recently, and as such, most of the affected individuals reported are children and young adults. there is therefore still only very limited data available relating to the progression and prognosis of this disorder, meaning that it is not yet possible to determine whether there might be any clinical evidence of multimorbidity indicative of pre- mature aging or a reduction in average life span in tbrs. further long-term natural history studies of tbrs patients will be extreme- ly helpful for determining the clinical implications of the epige- netic age acceleration observed as a feature of this disorder. accelerated epigenetic age has previously been reported in as- sociation with specific diseases such as huntington’s disease (+ . yr) (horvath et al. ), down syndrome (+ . yr) (horvath et al. ), and werner’s syndrome (+ . yr) (maierhofer et al. ). the accelerated epigenetic aging described in association with these disorders is an average increase in epigenetic age, which is relatively consistent throughout lifespan. a distinguishing feature of carriers of the amish dnmt a c. g > a; p.(arg gln) var- iant is the year-on-year or cumulative increase of accelerated epige- netic aging over life time course, in other words, a true acceleration of epigenetic aging. although it was not possible to undertake these studies for other dnmt a variants, this may be indicative of a similar effect on cumulative epigenetic age acceleration over the life course in tbrs. it is also noted that the gene encoding dnmt l is located on chromosome ; given the previous report of an average dna methylation age acceleration of . yr in blood and brain tissue in individuals with down syndrome (horvath et al. ) and the role of dnmt l in stimulating dnmt a de novo methylation, further investigations are needed to explore the potential relevance of this observation. there are currently only four reported cases of an aml tumor carrying the dnmt a p.arg gln substitution. biochemical measurements of dnmt a show that mutations at both the arg and arg residues result in reduced methyltransferase activity, with a greater degree of reduction resulting from arg variants compared with arg variants ( . -fold difference) (holz-schietinger et al. ). given this reduced methyltransfer- ase activity, we may expect to observe more pronounced changes in dna methylation in patients with germline variants affecting arg compared with variants affecting other amino acid resi- dues such as arg . our data reflected this notion, with alteration arg displaying markedly greater methylation changes com- pared with the other dnmt a mutations investigated in this study. currently, available literature suggests that the risk of hema- tological malignancy in tbrs individuals may vary depending on the specific pathogenic variant underling their condition (hollink et al. ). the significantly advanced epigenetic age that we ob- served in association with p.arg cys may explain why hemato- logical malignancy has to date only been reported in two tbrs patients, one harboring this germline variant and the second the p.tyr ser variant, the latter not being assessed in this study (hollink et al. ; tatton-brown et al. ). in summary, our findings identify widespread dna hypome- thylation in genes involved in morphogenesis, development, dif- ferentiation, and malignancy in tbrs patients. tbrs patients also displayed highly accelerated dna methylation aging. our studies additionally defined phenotype-related altered epigenetic aging in two histone methyltransferase disorders: nsd sotos syn- drome overgrowth disorder and kmt d kabuki syndrome growth impairment. taken together, these findings provide important new insights into the role of dnmt a during development and of relevance to hematological malignancy, and define perturba- tion to epigenetic machinery and biological aging as common themes in overgrowth and growth deficiency syndromes. methods genetic and clinical studies the phenotypic features of the four affected siblings (three females and one male, aged – yr) (fig. a, individuals iii: , iii: , iii: , and iii: ) include macrocephaly, tall stature, hypotonia, mild to moderate id, behavioral problems, and a distinctive facial appear- ance. whole-genome snp genotyping and exome sequencing of dna samples taken with informed consent under regionally ap- proved protocols excluded pathogenic variants in known genes, or candidate new genes, associated with neurodevelopmental dis- orders. subsequent studies defined a heterozygous c. g > a variant in dnmt a, resulting in a p.(arg gln) substitution, as the cause of the condition. full clinical details are previously de- scribed (xin et al. ). further testing revealed mosaicism for the dnmt a c. g > a variant in the father, and xin et al. ( ) showed pathogenic variant load varied in different tissue types. dna methylation profiling genomic dna from blood was sodium bisulfite converted using the ez- dna methylation kit (zymo research) and dna meth- ylation quantified across the genome using the illumina epigenetic aging abnormalities in growth disorders genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com infinium humanmethylation array (illumina k array) (illumina). the additional dnmt a pathogenic variants were profiled using the illumina infinium epic array (illumina). the bioconductor package watermelon (pidsley et al. ) in r . . (r core team ) was used to import idat files, and after check- ing for suitable sodium bisulfite conversion (bisulfite control probe median > %), the dna methylation data were imported and quantile normalized using the dasen function in watermelon and methylation beta values produced (ratio of intensities for methylated versus unmethylated alleles). probes showing a detec- tion p-value > . in at least % of samples or a beadcount < in % of samples were removed across all samples. any samples showing low quality, indicated by a detection p-value > . in ≥ % of probes within a sample, were removed from analysis. probes containing common snps within bp of the cpg site were removed (minor allele frequency > %). nonspecific probes and probes on the sex chromosomes were also removed (chen et al. ; price et al. ). identification of dmps dmps were identified using a limma-based linear model based on pathogenic variant genotype and sex as a covariate (smyth ) and a benjamini–hochberg fdr of % applied (benjamini and hochberg ). when the epigenetic age was used as a covariate, a similar level of dmps were detected ( dmps) with an % overlap to the limma model without age as a covariate. changes in methylation were calculated based on comparison between dnmt a c. g > a; p.(arg gln) carriers versus wild-type in- dividuals in the amish pedigree. the additional dnmt a path- ogenic variants were assessed relative to seven wild-type control samples run on the same epic array run. blood cell counts were unknown and so were estimated using the dna methylation age calculator (horvath ; koestler et al. ) and assessed in the linear model. to identify dmps, the package dmrcate was used with the same limma-based design (peters et al. ). gene ontology and functional enrichment analyses gene ontology enrichment analysis was performed using genes annotated to fdr corrected dmps using the gometh function of the missmethyl package (phipson et al. ), which takes into account potential bias of probe distributions on the beadchip ar- ray. kegg pathway analysis was performed using the gsameth command of missmethyl and kegg annotation files from the bioconductor keggrest package (http://bioconductor.org/ packages/release/bioc/html/keggrest.html). regional enrich- ment analysis based on illumina annotations was performed using a chi-squared test with yates correction in r. dmrs were function- ally annotated using the webtool great (http://great.stanford .edu/public/html/). the top p-value–ranked dmps were also annotated using the eforge tool (https://eforge .altiusinstitute.org/) to perform functional overlap analysis for identifying any cell- or tissue-specific epigenetic signals. quantification of global dna methylation global dna methylation measurements were made using the luminometric methylation assay (luma) (karimi et al. ) based on cleavage by a methylation-sensitive restriction enzyme followed by polymerase extension assay via pyrosequencing on the pyromark q (qiagen). peak heights were obtained using the pyro q cpg . . software and a t-test applied in r . . . global methylation estimates from the illumina k array were assessed through r . . using summary statistics and a wilcoxon rank-sum test on two pairs of samples matched for age and sex. dna methylation age estimation epigenetic age calculations were made using the dna methylation age calculator (https://dnamage.genetics.ucla.edu/) for illumina k data, and illumina epic arrays were assessed using the agep function of the watermelon bioconductor package, the latter based on the original calculator developed by steve horvath ( ). accelerated age was calculated for the amish tbrs dnmt a c. g > a; p.(arg gln) carriers and wild-type family members and sotos syndrome and kabuki syndrome patients and compared with data from control individuals taken from a previous study (hannon et al. ), using linear models of recorded chronolog- ical age and calculated epigenetic age. estimates of age acceleration for the additional tbrs cases were calculated by dividing the calculated epigenetic age with their chronological age. additional nsd sotos syndrome patient illumina dna methylation files were obtained from geo accession gse , with corresponding chronological ages derived from the associated paper (choufani et al. ). kmt d kabuki syndrome dna methylation data and chronological age were obtained from geo accession gse (butcher et al. ). validation of dmps using bisulfite-pyrosequencing bisulfite pyrosequencing was used to validate specific differentially methylated cpg sites originally identified using the illumina k array. primers, designed using pyromark assay design soft- ware (qiagen), and pcr conditions are provided in supplemental table s . bisulfite conversion was performed on ∼ ng of dna using a bisulfite-gold kit (zymo research). pcr was performed with hot firepol dna polymerase (solis biodyne) for min at °c followed by cycles of sec at °c, sec at annealing temperature (shown in supplemental table s ), and sec at °c. a final extension of min at °c was then applied. dna methylation was then assessed using the resulting bisulfite pcr amplicons, together with a pyrosequencing primer on the pyromark q system (qiagen) following the manufacturer’s standard instructions and the pyro q cpg . . software. data access the raw and processed primary data sets generated in this study have been submitted to the ncbi gene expression omnibus (geo; https://www.ncbi.nlm.nih.gov/geo/) under accession num- ber gse . r scripts are provided as supplemental code s and at the following repository: https://github.com/arjeffries/ tbrs . acknowledgments we thank the amish families for participating in this study and the amish community for their continued support of the windows of hope project. the work was supported by the newlife foundation for disabled children (ref: sg/ - / , to c.g.s., a.r.j., a.h.c., and e.l.b.), medical research council (mrc) grant g (to e.l.b.), mrc grant g (to a.h.c.), and mrc grants mr/ m / and mr/k / (to j.m.). author contributions: e.l.b., a.h.c., and j.m. conceived the study. r.m. and b.a.c. performed the genetic analysis. a.r.j. and r.m. performed luma global methylation assay. a.r.j. performed the microarray-based dna methylation analysis and all associated statistical analyses. a.r.j. and e.d. performed the pyrosequencing jeffries et al. genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://great.stanford.edu/public/html/ http://great.stanford.edu/public/html/ http://great.stanford.edu/public/html/ http://great.stanford.edu/public/html/ http://great.stanford.edu/public/html/ https://eforge.altiusinstitute.org/ https://eforge.altiusinstitute.org/ https://eforge.altiusinstitute.org/ https://eforge.altiusinstitute.org/ https://eforge.altiusinstitute.org/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc https://github.com/arjeffries/tbrs https://github.com/arjeffries/tbrs https://github.com/arjeffries/tbrs https://github.com/arjeffries/tbrs https://github.com/arjeffries/tbrs http://genome.cshlp.org/ http://www.cshlpress.com and associated analysis. a.r.j., c.g.s., r.m., a.h.c., j.m., and e.l.b. wrote the manuscript. h.e.c., m.a.p., i.k.t., d.j.g.m., f.i.r., k.t.-b., l.a., d.b., t.d., m.f.h., r.n.-e., a.k., a.k., a.s., a.s., l.vm., v.v., and n.y. contributed samples and clinical data. references benjamini y, hochberg y. . controlling the false discovery rate: a prac- tical and powerful approach to multiple testing. j r stat soc ser b : – . doi: . / bjornsson ht. . the mendelian disorders of the epigenetic machinery. genome res : – . doi: . /gr. . breeze ce, paul ds, van dongen j, butcher lm, ambrose jc, barrett je, lowe r, rakyan vk, iotchkova v, frontini m, et al. . eforge: a tool for identifying cell type-specific signal in epigenomic data. cell rep : – . doi: . /j.celrep. . . 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/wellcomeopenres. . tlemsani c, luscan a, leulliot n, bieth e, afenjar a, baujat g, doco-fenzy m, goldenberg a, lacombe d, lambert l, et al. . setd and dnmt a screen in the sotos-like syndrome french cohort. j med genet : – . doi: . /jmedgenet- - wu h, coskun v, tao j, xie w, ge w, yoshikawa k, li e, zhang y, sun ye. . dnmt a-dependent nonpromoter dna methylation facilitates transcription of neurogenic genes. science : – . doi: . / science. xin b, cruz marino t, szekely j, leblanc j, cechner k, sency v, wensel c, barabas m, therriault v, wang h. . novel dnmt a germline mu- tations are associated with inherited tatton-brown–rahman syndrome. clin genet : – . doi: . /cge. zhu t, zheng sc, paul ds, horvath s, teschendorff ae. . cell and tissue type independent age-associated dna methylation changes are not rare but common. aging (albany ny) : – . doi: . /aging . received september , ; accepted in revised form may , . jeffries et al. genome research www.genome.org cold spring harbor laboratory press on july , - published by genome.cshlp.orgdownloaded from https://www.r-project.org/ https://www.r-project.org/ https://www.r-project.org/ https://www.r-project.org/ https://www.r-project.org/ https://www.r-project.org/ http://genome.cshlp.org/ http://www.cshlpress.com . /gr. . access the most recent version at doi: : - originally published online june , genome res. aaron r. jeffries, reza maroofian, claire g. salter, et al. associated with abnormalities of epigenetic aging growth disrupting mutations in epigenetic regulatory molecules are material supplemental http://genome.cshlp.org/content/suppl/ / / /gr. . .dc references http://genome.cshlp.org/content/ / / .full.html#ref-list- this article cites articles, of which can be accessed free at: open access open access option.genome researchfreely available online through the license commons creative .http://creativecommons.org/licenses/by/ . / commons license (attribution . international), as described at , is available 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sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ variation in the lamin a/c gene variation in the lamin a/c gene associations with metabolic syndrome nanette i. steinle, rasa kazlauskaite, ikhide g. imumorin, wen-chi hsueh, toni i. pollin, jeffrey r. o’connell, braxton d. mitchell, alan r. shuldiner objective—metabolic syndrome is associated with increased risk for cardiovascular disease and type diabetes mellitus (t dm). the lamin a/c (lmna) gene, mutations of which cause rare syndromes of severe insulin resistance and dyslipidemia, is located on chromosome q -q , a region linked to t dm in several genome wide scans, including in the old order amish. to determine whether polymorphisms in lmna influence susceptibility to metabolic syndrome and its constituent components. methods and results—we performed dna sequence analysis of lmna. six single-nucleotide polymorphisms (snps) were identified: c. c�t (intron ), c. g�t (intron ), a a (c. t�c; exon ), c. g�a (intron ), c. c�t (intron ), and h h (c. c�t; exon ). in participants from the amish family diabetes study, the h h polymorphism of lmna was associated with metabolic syndrome diagnosed according to national cholesterol education program atp iii criteria and also higher mean fasting triglyceride and lower mean high-density lipoprotein-cholesterol concentrations. however, no differences in allele frequencies were observed for any snp among participants with t dm or impaired glucose homeostasis (igh) and normoglycemic controls. haplotype analysis showed that � % of individuals carried of common lmna haplotypes. there were no significant differences in haplotype frequencies among subjects with metabolic syndrome t dm, igh, and controls. conclusion—sequence variation in lmna may confer modest susceptibility for development of metabolic syndrome and dyslipidemia in the amish. (arterioscler thromb vasc biol. ; : - .) key words: lamin a/c � lipids � triglyceride � metabolic syndrome � insulin resistance � positional cloning � chromosome q -q metabolic syndrome is characterized by the presence of � of the following characteristics: abdominal obesity, ele- vated triglyceride concentrations, low high-density lipoprotein (hdl), high blood pressure, and elevated fasting glucose. on the basis of estimates from the third national health and nutrition examination survey, the prevalence of metabolic syndrome is % among adults in the united states � years of age. metabolic syndrome is associated with increased risk of type diabetes mellitus (t dm) and cardiovascular disease. evidence from the west of scotland study indicates that men with metabolic syndrome had . -fold increased risk of a cardiovascular event and . -fold increased risk of developing diabetes. the mechanisms that cause metabolic syndrome are poorly understood but likely involve insulin resistance, which is associated closely with abdominal obesity and is an independent risk factor for cardiovascular disease. indeed, a large body of evidence supports the notion that adipose tissue factors may mediate several of the metabolic changes that occur with metabolic syndrome. metabolic syndrome, along with its indi- vidual components, is likely to be under the influence of both environmental and genetic factors. to localize genes contributing to t dm, cardiovascular disease, and related traits, including obesity and metabolic syndrome, we conducted a genome-wide linkage analysis in large old order amish families from lancaster county, pa. , evidence for linkage with t dm, impaired glucose toler- ance, or impaired fasting glucose was observed on chromo- some q -q (logarithm of odds� . ; p� . ). this region is a gene-rich region that includes � genes. excellent positional candidate genes include apoa (apoli- poprotein a ), pbx (pre–b-cell leukemia transcription factor ), slc a (solute carrier family [thiamine trans- porter] member ), insrr (insulin receptor–related receptor gene), kcnj and kcnj (potassium inwardly-rectifying channel, subfamily j, members and ), pea (phos- phoprotein enriched in astrocytes ), rxrg (the � subunit received may , ; revision accepted june , . from the department of medicine (n.i.s., t.i.p., j.r.o., b.d.m., a.r.s.), university of maryland school of medicine, baltimore, md; the department of medicine (r.k.), rush medical college, chicago, ill; the department of biology (i.g.i.), valdosta state university, georgia; the department of medicine (w.-c.h.), university of california, san francisco; and the geriatric research and education clinical center (a.r.s.), veterans administration medical center, baltimore, md. correspondence to nanette steinle, md, division of endocrinology, diabetes, and nutrition, university of maryland school of medicine, w redwood st, room , baltimore, md . e-mail nsteinle@medicine.umaryland.edu © american heart association, inc. arterioscler thromb vasc biol. is available at http://www.atvbaha.org doi: . / .atv. . .c d ow nloaded from http://ahajournals.org by on a pril , of the retinoid x receptor), crp (c-reactive protein), hyplip (familial combined hyperlipidemia gene), casq (calsequestrin ), , vamp (vesicle-associated membrane protein ; m sabra, s ott, j lee, jr o’connell, bd mitchell, ar shuldiner, unpublished data, ), usf (upstream stimulatory factor ), , and lmna (lamin a/c). this region is approximately to cm from p-telomere and corresponds to a region of chromosome that has been linked to t dm in several other populations, including pima indians, utah mormons, british whites, french whites, and chinese. familial partial lipodystrophy is a condition associated with severe insulin resistance, diabetes, dyslipidemia, and atherosclero- sis, making lmna an excellent positional candidate gene for metabolic syndrome, t dm, and related traits. lmna, located on chromosome q . , encodes a nuclear envelope protein that exists as a heterodimer of lamin a and lamin c and is expressed in several cell types including liver, fat, muscle, and pancreas. the gene contains coding exons. alternative splicing within exon of lmna results in coding either the lamin a or lamin c protein. mutations in lmna have been shown to result in a number of disorders, including emery–dreifuss muscular dystrophy (online mendelian inheritance in man [omim] ), , dilated car- diomyopathy with conduction defects (omim ), familial partial lipodystrophy (omim ), – mandibuloacral dyspla- sia (mad; omim ), huchinson–gilford progeria (omim ), – and autosomal dominant and recessive forms of charcot marie tooth disease (omim ). – the mechanism whereby mutations in lmna lead to these diverse phenotypes is presently unknown. the aim of this study was to search for novel sequence variation in lmna and to determine whether variants in this gene influence susceptibility to metabolic syndrome and its constituent components. we sequenced lmna in amish subjects diagnosed previously with t dm from the amish family diabetes study (afds). these individuals were selected from families showing evidence for linkage to the region of interest on chromosome . we then performed association and haplotype analyses in the full afds cohort to determine whether the polymorphisms identified in lmna are associated with diabetes, metabolic syndrome, and related traits in the amish. we found that the polymorphism h h in exon was associated with metabolic syndrome as well as with higher fasting triglyceride and lower hdl-cholesterol concentrations, suggesting that lmna variation may confer modest susceptibility for development of metabolic syndrome and dyslipidemia in the amish. research design and methods subjects subject recruitment for the afds began in . details of the afds design, recruitment, phenotyping, and pedigree structures have been described previously. briefly, probands were defined as individuals with previously diagnosed diabetes with the age at diagnosis between and years. all first- and second-degree family members � years of age were recruited around the diabetic probands. if another diabetic individual was identified in the family (eg, aunt or uncle), the family was expanded further to include the first- and second-degree relatives of that individual � years of age. the afds participants were divided into families, although all of these families can be joined into a single pedigree by including ancestors going back to generations. – none of the subjects had known muscular dystrophy, structural or cardiac conduction abnormalities, progeria, or familial partial lipodys- trophy, conditions that have been associated previously with mutations in the lmna gene. data regarding medical, social, and family history were obtained during an interview at our amish research clinic or the subject’s home. height, weight, and waist circumference were measured. body mass index (bmi) was calculated as weight (kilograms) divided by height squared (m ) to estimate total adiposity. waist circumference was measured by a single observer. for this procedure, subjects were asked to remove any unnecessary clothing. the subject stood erect with the abdomen relaxed, arms at sides, and feet together. the measurer faced the subject and placed an inelastic tape around him/her in a horizontal plane at the level of the natural waist, which is the narrowest part of the torso, as seen from the anterior aspect. in obese subjects, the smallest horizontal circumference was measured in the area between the ribs and iliac crest. the measurement was taken at the end of a normal expiration, without the tape compressing the skin, and recorded to the nearest . cm. blood pressure was measured in the left arm with standard sphygmomanometer while the subject was seated after minutes of rest. a single observer made duplicate measurements, and the average of these was recorded. fasting lipid profile (hdl-cholesterol and triglyceride concentrations) was assayed by quest diagnostics (baltimore, md; interassay coefficient of variation . % for hdl-cholesterol; . % for triglycerides). a -hour -g oral glucose tolerance test (ogtt) was performed on subjects with no previous history of diabetes. the t dm diagnosis was defined on the basis of the ogtt using the plasma glucose criteria of the american diabetes association ( hour glucose � . mmol/l or fasting glucose � mmol/ l). subjects were also considered to have diabetes if they were currently taking medications for diabetes treatment. subjects were excluded if there was a history of type diabetes. additionally, we defined impaired glucose homeostasis (igh) as hour glucose � . mmol/l and � . mmol/l or fasting glucose � . mmol/l and � mmol/l. metabolic syndrome was defined using the national cholesterol education program (ncep) atp iii guidelines. it is characterized by the presence of � of the following characteristics: abdominal obesity (waist circumference � cm for men and � cm for women), elevated triglycerides (� . mmol/l), low hdl (� . mmol/l for men and � . mmol/l for women), high blood pressure (� / mm hg), and elevated fasting glucose (� . mmol/l). clinical characteristics of amish subjects are shown in table . the mean age of diagnosis of diabetes in the afds cohort was . � . years (range to years), and the mean bmi was . � . kg/m (range . to . kg/m ). the afds subjects selected for screening for mutations in lmna in this study were individuals with diabetes who were members of different nuclear families that showed evidence for linkage to the area of interest on chromosome . the mean age of these subjects was . � . years (range to years). the study was conducted in accordance with the human subject standards of the institutional review board of the university of maryland, and informed consent was obtained from all subjects. laboratory methods whole blood was collected from the subjects, and genomic dna was isolated using maxipreps according to the instructions of the manufacturer (qiagen). all exons as well as the exon–intron junctions of lmna were amplified by polymerase chain reaction (pcr). primers for dna amplification and sequencing were derived using the published sequence for lmna (genbank ac ; table i, available online at http://atvb.ahajournals.org). dna se- quencing was performed using an applied biosystems auto- mated dna sequencer (perkin–elmer). pcr products were se- quenced in both directions, visually inspected, and compared with the genbank sequence of human lmna. genotyping was completed using an orchid snpstream uht genotyping system (orchid bioscience). first, the target genomic sequences containing the single-nucleotide polymorphisms (snps) of interest were amplified in a -plex pcr reaction. after enzy- matic clean-up, pcr products were subjected to an extension steinle et al lmna gene and metabolic syndrome d ow nloaded from http://ahajournals.org by on a pril , reaction using �-tagged extension primers and fluorescent dye– labeled terminators. in a thermal cycled extension step, the primers hybridized to the specific amplicons base adjacent to the snp site and were extended by base at the � end with a fluorescently labeled nucleotide. these reaction products were transferred to an array plate, where each of the extension products in the multiplex reaction was sorted by hybridization of the unique �-tag sequence to its complementary probe immobilized in a miniarray within each well. the orchid snpscope reader imaged the microarray plates. the -color detection allowed snp detection by comparing signals from the fluorescent dyes. the image signals were then transferred to genotyping software that translated the images of the arrays into genotype calls. the error rate on the basis of blind replicates was . % to % for the snps examined in the present study. analysis before analysis, genotypes were checked for mendelian consistency, and inconsistencies, which were detected in � . % of genotypes, were removed from analysis. genotype frequencies of all snps were tested for consistency with hardy–weinberg expectations by the � test. we then followed a -tiered analytic approach, first performing association analyses with each snp individually and then consider- ing snps jointly as haplotypes. because mutations in lmna have been associated previously with diabetes and dyslipidemia, we examined the relationship of lmna polymorphisms identified in the amish with the metabolic syndrome and its component traits. for the quantitative traits associated with metabolic syndrome (ie, glucose, obesity, and lipid levels), we estimated mean trait levels according to lmna geno- types, whereas for the qualitative traits (metabolic syndrome, t dm, and abnormal glucose tolerance), we compared disease prevalence across genotypes. subjects currently taking insulin (n� ), �-hydroxy-�-methylglutaryl coenzyme a reductase inhibitors or other lipid-lowering medications (n� ), aspirin (n� ), angioten- sin-converting enzyme inhibitors, or other blood pressure–lowering agents (n� ), or estrogens (n� ) were excluded from analysis (n� subjects taking � of the above). to account for the relatedness among family members, we used the measured genotype approach, in which we estimated the likelihood of specific genetic models given the pedigree structure. for example, we compared the likelihood of a full model, which allowed for genotypic- specific means, to that of a nested model in which genotypic means were restricted to be equal to each other. parameter estimates were obtained by maximum likelihood methods, and the significance of association was tested by likelihood ratio tests. within each model, we simulta- neously estimated the effects of age and sex. triglyceride values were transformed by their natural logarithms (lns) to reduce skewness. all analyses were conducted using the sequential oligogenic linkage analysis routines program. the strength of linkage disequilibrium between all pair-wise combinations of snps and haplotypes were inferred for each individual using an expectation maximization algorithm imple- mented into the zaplo software program. additional association analyses were then performed treating each haplotype as a super-al- lele as described above. results in sequence analysis of relatively unrelated amish t dm subjects ( alleles), we identified snps, including c. g�t and c. c�t (intron ); a a (c. t�c; exon ); c. g�a (intron ); c. c�t (intron ); and h h (c. c�t; exon ). this exon snp is also designated as rs and has been identified as genbank no. x c t previous- ly. although this snp is silent, it is of particular interest because it occurs at the last nucleotide in exon , where alternate splicing determines the relative amounts of lamin a and lamin c transcripts (figure). the exon a a (c. t�c) snp is also a silent mutation. the intronic snps do not predict any obvious functional sequelae. the frequencies of the minor alleles at each of the snps ranged from . to . (table ). the distribution of geno- types for all snps was consistent with hardy–weinberg equi- librium. the degree of linkage disequilibrium was strong be- tween the intron snps (r � . ; d�� ), moderate between the intron snps and snps at exon , intron , and intron , and among the latter snps (r � . to . ; d�� . to ) and weaker between the exon snp and all others (r � . to . ; d�� . to ; see table ). the prevalence rates for t dm and igh were . % and . %, respectively, in this sample. allele and genotype fre- quencies did not differ between t dm cases and controls for any of the snps, nor was there evidence for association of any of the snps with the composite trait t dm/igh (table ). we examined the relationships between bmi and metabolic syndro- me–related traits and lmna sequence variants. mean levels of obesity and lipid measures are also shown in table according to the exon h h (c. c�t) genotype. triglyceride concentrations were higher and hdl concentrations lower in subjects with the t allele (p� . and . , respectively). there was also a significant association with metabolic syn- drome as defined by ncep atp iii guidelines (p� . , odds ratio . ; ci . to . ). corresponding analyses of the exon a a (c. t�c) snp showed higher fasting glucose among those with the c allele than those with the tt genotype (p� . ). no significant association with obesity measures, lipid traits, or blood pressure was seen for this snp. analysis of the intronic snps showed no significant association with obesity, lipid traits, or metabolic syndrome (data not shown). as a secondary analysis of the exon variant h h (c. c�t), we evaluated whether the effects of the exon schematic of the genomic structure of lmna. lmna is composed of exons. alternate splicing within exon results in distinct mrna transcripts corre- sponding to lamin a ( amino acids) and lamin c ( amino acids). the mature protein lamin a/c functions as a heterodimer. also shown are snps detected in our mutation screen and their locations. note that c�t is a silent mutation in exon at the alter- nate splice site. promoter is not shown. nls indicates nuclear location signal; utr, untranslated region. arterioscler thromb vasc biol. september d ow nloaded from http://ahajournals.org by on a pril , ct/tt genotype on metabolic syndrome and related traits differed between men and women or between younger or older individuals. there was little evidence that the genotype effect differed between men and women for any trait. for triglycerides, age-stratified analyses indicated that the geno- type effect differed little between younger and older men, with the ct/tt genotype associated with a . % and . % increase in ln triglyceride levels in men younger and older than age , respectively. however, in women, the effect of the ct/tt genotype was restricted almost entirely to those � years of age; among these older women, being exposed to the t allele was associated with a . % increase in ln triglyceride levels compared with only a . % increase for women � years of age. this interaction effect did not achieve statistical significance (p� . ). we next considered the possibility that haplotypes defined by allelic combinations at these snps might show associations with either disease status or with quantitative trait levels. because of marked linkage disequilibrium between markers (table ), most frequent haplotypes (c -t - c -g -c -t and t -t - c -g -c -t ) together accounted for � % of the haplotypes in the amish. the estimated frequen- cies of the first most common haplotypes are . and . , respectively. there were no significant differences in haplotype frequencies among subjects with t dm or metabolic syndrome and controls, nor were there any differences in glucose, insulin, obesity, or lipid traits between haplotypes. discussion mutations in lmna have been found in diseases involving muscle and fat cells and include emery–dreifuss muscular dystrophy, , dilated cardiomyopathy with conduction de- fects, familial partial lipodystrophy, – mad, and char- cot marie tooth disease, – as well as premature aging. – familial partial lipodystrophy is associated with severe insu- lin resistance, diabetes, dyslipidemia, and atherosclerosis. our genome-wide scan in the amish and those of several others – provide strong evidence for the existence of � t dm susceptibility genes on chromosome q -q , the region in which lmna resides. these observations led us to sequence all of the exons and exon–intron boundaries of lmna in subjects with diabetes from amish families, pro- viding evidence for linkage to chromosome q -q . al- though our mutation screen detected snps, none resulted in obvious alterations in protein structure, and none were associated with t dm or igh. we did not screen for mutations in regulatory regions or the entirety of all introns, and thus cannot rule out the possibility that significant mutations may be present in these regions. however, lack of association of any haplotype with t dm or related traits make this possibility less likely. thus, lmna is unlikely to be the gene on chromosome q -q detected through linkage analysis contributing to risk of t dm. modest associations with a common variant in exon of lmna (h h) have been shown to be associated with adipose tissue metabolism and obesity phenotypes. – in canadian oji-cree and inuit, this variant was associated with table . clinical characteristics of amish subjects trait means women (n� ) men (n� ) age (years) . � . . � . bmi (kg/m ) . � . . � . waist circumference (cm) . � . . � . hdl (mmol/l) . � . . � . ln (tgs; mmol/l� ) . � . . � . systolic blood pressure (mm hg) . � . . � . diastolic blood pressure (mm hg) . � . . � . metabolic syndrome (%) . . t dm (%) . . t dm/igh (%) . . tgs indicates triglycerides. table . means (ses in parentheses) for metabolic syndrome–related traits in afds subjects with and without the exon h h polymorphism trait means exon (h h) polymorphism age and sex adjusted x and p value non-t allele carriers n� t allele carriers n� x p bmi (kg/m ) . ( . ) . ( . ) . . waist circumference (cm) . ( . ) . ( . ) . . hdl (mmol/l) . ( . ) . ( . ) . . ln (tgs; mmol/l� ) . ( . ) . ( . ) . . systolic blood pressure (mm hg) . ( . ) . ( . ) . . diastolic blood pressure (mm hg) . ( . ) . ( . ) . . fasting glucose . ( . ) . ( . ) . . metabolic syndrome (%) . . . . t dm (%) . . . . t dm/igt (%) . . . . tgs indicates triglycerides. metabolic syndrome defined according to ncep atp iii guidelines (see text). steinle et al lmna gene and metabolic syndrome d ow nloaded from http://ahajournals.org by on a pril , bmi, waist-to-hip ratio, leptin, and body fat percentage. , additionally, this variant has been studied in a japanese cohort and in pima indians. , in pimas, no evidence for association of h h with t dm, bmi, total cholesterol, hdl-cholesterol triglycerides, or indices of insulin sensitiv- ity and secretion was found. however, pimas with the t allele had significantly greater subcutaneous fat cell size. in the japanese cohort, this variant was associated with higher fasting insulin, triglyceride, total cholesterol concentrations, and lower hdl-cholesterol concentrations. amish individuals who were exposed to the t allele of the c. c�t variant had higher triglyceride concentrations and lower hdl-cholesterol and were more likely to have the presence of metabolic syndrome. thus, it is possible that this variant plays a modest role in modulating the metabolic syn- drome, particularly lipid components. although this snp encodes a silent mutation h h, it is the terminal exonic nucleotide of exon , the site of alternate splicing for lamin a and c mrna. this polymorphism could alter mrna splicing and thus the relative amounts of lamin a and c mrna and protein. indeed, the silent lmna g g snp has been shown to cause aberrant mrna splicing and to cause hutchinson–gilford progeria. in summary, screening of lmna revealed snps. one snp, h h (c. c�t), showed significant association with metabolic syndrome and with lower hdl-cholesterol and higher triglyceride concentrations. these associations are consistent with other studies reporting association of this same variant with metabolic syndrome traits. none of the snps or haplotypes examined showed evidence of associa- tion with t dm or igh. we conclude that genetic variation in lmna may play a modest role in susceptibility to meta- bolic syndrome and dyslipidemia in our population but is unlikely to be the gene on chromosome q -q detected through linkage analysis contributing to risk of t dm. acknowledgments this work was supported by a scientist development grant of the american heart association (n.i.s.), national institutes of health grants k dk and r dk , an american diabetes association research award (a.r.s.), and the university of maryland general clinical research center. a.r.s. is also supported by the baltimore veterans administration geriatric research and educa- tion clinical center. we thank drs richa agarwala and alejandro schäffer for their assistance in pedigree construction using the anabaptist genealogy database. we also wish to acknowledge demian lewis, sandy ott, and dr arnel tagle for technical assistance, and dr holmes morton and caroline morton for their support in establishing and maintaining 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article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ dc .. comparison of bmi and physical activity between old order amish children and non-amish children kristen g. hairston, md, mph julie l. ducharme, md margarita s. treuth, phd wen-chi hsueh, phd ania m. jastreboff, md, phd kathy a. ryan, mph xiaolian shi, ms braxton d. mitchell, phd alan r. shuldiner, md soren snitker, md, phd objectivedthe old order amish (ooa) is a conservative christian sect of european origin living in pennsylvania. diabetes is rare in adult ooa despite a mean bmi rivaling that in the general u.s. non-hispanic white population. the current study examines childhood factors that may contribute to the low prevalence of diabetes in the ooa by comparing ooa children aged – years with national health and nutrition examination survey (nhanes) data and children from maryland’s eastern shore (es), a nearby, non-amish, rural community. we hypothesized that pediatric overweight is less common in ooa children, that physical activity (pa) and bmi are inversely correlated, and that ooa children are more physically active than es children. research design and methodsdwe obtained anthropometric data in ooa children and es children ( non-hispanic white, non-hispanic black, hispanic). pa was measured by hip-worn accelerometers in all es children and in ooa children. instru- mentation in ooa children was identical to es children. resultsdooa children were approximately . times less likely than non-hispanic white es children and nhanes estimates to be overweight (bmi $ th percentile, centers for disease control and prevention). time spent in moderate/vigorous pa (mvpa) was inversely correlated to bmi z-score (r = . , p = . ). pa levels did not differ by ethnicity within the es group, but ooa children spent an additional min/day in light activity ( vs. , p = . ) and, impressively, an additional min/day in mvpa ( vs. , p , . ) compared with es children. in both groups, boys were more active than girls but ooa girls were easily more active than es boys. conclusionsdwe confirmed all three hypotheses. together with our previous data, the study implies that the ooa tend to gain their excess weight relatively late in life and that ooa children are very physically active, both of which may provide some long-term protection against diabetes. diabetes care : – , t he old order amish (ooa) is a conservative christian sect of euro- pean origin living in rural areas of lancaster county, pennsylvania. we have previously reported ( ) that the preva- lence of diabetes in adult ooa is only approximately half of that in the general u.s. population of european ancestry. this is an intriguing finding because the mean bmi of adult ooa ( . kg/m ) ri- vals that of the adult general u.s. popula- tion of european ancestry ( . kg/m ) ( ). by contrast, our anecdotal observa- tions from the same lancaster county amish community suggest that ooa chil- dren are very rarely overweight compared with non-amish u.s. children. if correct, our anecdotal observations imply that the ooa generally tend to gain their excess weightatalaterstageinlifethanthegeneral, non-amish u.s. population. gaining one’s excess weight relatively late in life may be protective against diabetes, as several pro- spective studies of adults indicate that the number of years lived with obesity is a pre- dictor of diabetes risk ( , ) independently of age and current bmi. thus, to follow up on our report on ooa adults, we designed the ooa childhood obesity study to col- lect data in ooa children and adolescents (in the following, children and adolescents willcollectively be referred to as “children”). if ooa children are indeed much more rarely overweight than their non-ooa peers, this observation would pave the way for studies involving this population to better understand the role of weight- gain trajectory in the etiology of disease. the first aim was to compare the age- and sex-adjusted bmi of ooa children with es- timates from the national health and nu- trition examination survey (nhanes). the second aim was to test the hy- pothesis that physical activity (pa) and bmi are correlated in the ooa. although it is often taken for granted that pa and bmi are causally connected, the two are not always correlated on a population level. reports indicate that pa has impor- tant bmi-independent effects on diabetes risk ( ), and it has been argued ( ) that pa is the most proximal behavioral factor influencing insulin resistance. pa was measured objectively by hip-worn pa monitors (accelerometers). another relevant question is whether pa levels in amish children differ from those of their non-amish peers. ooa life is guided by the ordnungdrules that reg- ulate many aspects of life to be consistent with ooa values, which are religious de- votion, family, and community cohesion. many modern technologies are banned, including electric power, telephones, self-powered farm equipment, and per- sonal automobiles. these voluntary con- straints result in a lifestyle resembling that of farming americans or europeans a cen- tury or two ago. although one might c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c from the university of maryland school of medicine, baltimore, maryland; the department of physical therapy, university of maryland eastern shore, princess anne, maryland; the university of california at san francisco school of medicine, san francisco, california; and yale university school of medicine, new haven, connecticut. corresponding author: soren snitker, ssnitker@medicine.umaryland.edu. received may and accepted september . doi: . /dc - k.g.h. and j.l.d. contributed equally to this work. © by the american diabetes association. readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. see http://creativecommons.org/ licenses/by-nc-nd/ . / for details. care.diabetesjournals.org diabetes care, volume , april e p i d e m i o l o g y / h e a l t h s e r v i c e s r e s e a r c h o r i g i n a l a r t i c l e mailto:ssnitker@medicine.umaryland.edu http://creativecommons.org/licenses/by-nc-nd/ . / http://creativecommons.org/licenses/by-nc-nd/ . / think that these circumstances promote greater levels of pa in ooa children compared with non-amish children, the null hypothesis is favored by studies ( , ) finding similar pa levels in other groups whose environments differ greatly (e.g., individuals residing in the u.s. and nigeria). to compare ooa children with a relevant control, we amended the pro- tocol of the ooa childhood obesity study by adding, as a third aim, a com- parison of pa levels in the ooa children with non-amish children residing in maryland’s eastern shore (es). the es is a nearby, rural community with similar topography and climate. one of the inves- tigators, who had collected pa data in the es a few years prior ( ), provided the protocol, instrumentation, and data to allow a direct comparison. the final ooa participants were enrolled in the amended protocol. research design and methods study populations the study enrolled ooa children aged – years from lancaster county, pennsylvania from to . re- quirements for participation were willing- ness to wear an accelerometer around the waist for days and to undergo a physical examination. the recruitment team con- sisted of a research nurse and an amish liaison, who visited the homes of children to inquire about interest in the study. to ensure that the sample was as representa- tive as possible, homes to visit were se- lected without prejudice regarding the body composition or presumed pa level of the children living there. to minimize the possibility of self-selection bias on the basis of body composition or pa level, our recruitment narrative described the stud- ies in a way that did not imply judgment regarding body type or pa level. informed parental consent and minor assent were obtained before any study procedure. the study was approved by the institutional review board of the university of mary- land baltimore. the es children were recruited in as part of a school-based pa assess- ment study ( ). participants were girls and boys residing on the es of maryland and attending one elementary and one combined middle and high school. of enrolled children, were excluded because of incomplete accelerometry data. the final es sample (n = ) included girls and boys aged – years. the es children were % non-hispanic white (n = ), % non- hispanic black (n = ), and % hispanic (n = ). the methods and consent and as- sent procedures of the es study have been published previously ( ) and are largely similar to those of the ooa study. the es study was approved by the institu- tional review board of the johns hopkins bloomberg school of public health and by the county school board of education. the ooa and es samples were com- pared with contemporaneous national estimates in a published summary ( ) of data from the nhanes – cy- cle. nhanes is a complex, multistage probability sample of the u.s. civilian, noninstitutionalized population, conducted by the national center for health statis- tics of the centers for disease control and prevention (cdc). its – cycle included , children. measurements the ooa children underwent a brief physical examination, including mea- surements of height (shorrboard ica stadiometer; olney, md) and weight (calibrated electronic scale). the es chil- dren were examined according to an essentially identical protocol ( ). bmi was calculated as weight/height in meters squared (kg/m ). bmi values were con- verted to z-scores (z-bmi) and percentiles based on the cdc age- and sex- specific tables using algorithms and pa- rameters provided on the cdc web site ( ). because it is difficult to visualize the physical manifestation of a given z-bmi value, we also calculated the variable body weight above the cdc age- and sex- specific median by subtraction of the cdc age- and sex-specific th percentile from individual body weight. pa by actical the actical pa monitor (version . ; mini mitter co., inc., bend, or) was used throughout the study in ooa children. the actical device has been validated by heil ( ) against gas exchange in children and adolescents aged – years. epoch length was set for s and software ver- sion . was used for programming. the device was worn on the lateral aspect of the hip, held in place by a neoprene belt around the body, recording for at least consecutive days, h a day. to keep the belt dry, participants were permitted to remove it (and thus the monitor) for showering or bathing. the actical analysis incorporated the first complete days of wear (from midnight to mid- night). noncompliance with the h-a- day wear requirement was detected by the presence of extended periods of zero counts. a day was considered incomplete due to nonwear if an incident of subse- quent zero counts lasted for more than h. however, up to three nights of non- wear was tolerated (in this context, “night” was defined by the child’s habitual sleep and wake times). noncompliance was rare; only children had more than incomplete days and were ex- cluded from analysis. four recordings were excluded as a result of obvious de- vice malfunction. fifty-seven children did not have actical recordings because of temporary device shortages. during these times of high demand, actical devices were assigned randomly to participating children. usable actical recordings were available in children ( boys and girls). in addition to total daily counts, activity energy expenditure was calculated on a per-minute basis, using heil’s equations ( ), as implemented in the actical software, to derive time spent at defined levels of pa intensity (seden- tary, light, moderate, and vigorous). using a cutoff point between light and moderate activity of $ . kcal z kg z min as proposed by puyau et al. ( ) for children and adolescents, we re- port time spent in a collapsed category of moderate and vigorous pa (mvpa). sedentary activity was defined as periods with an activity count of less than counts/min for consecutive min. the activity measures were expressed as daily means. pa by actiwatch all es children and the final ooa children wore the actiwatch pa monitor (mini mitter co., inc.), which is physi- cally similar to the actical but has slightly different electronics and software. all actiwatch devices used for the ooa had been used in the es study (provided by m.s.t.) or had a serial number in the same range (i.e., starting with “v ”). all ooa children who wore an actiwatch also si- multaneously wore an actical according to the protocol used before the actiwatch was added. the simultaneous use of acti- watch and actical monitors allowed us to examine whether pa levels in the final children were representative of the entire ooa cohort. the ooa actiwatch proto- col was developed in collaboration with m.s.t. to be identical to that used in the diabetes care, volume , april care.diabetesjournals.org bmi and pa in ooa children es protocol ( ). the following applies to actiwatch studies in both es and ooa: the device was worn on the lateral aspect of the hip and held in place by a neoprene belt around the body. actiware rhythm . software was used to program the devices for an epoch length of min and upload data. these data were considered to be complete if ; % of the day ( , min) was recorded for at least of the days with of the days on a weekend. an excel spreadsheet macro, developed in the laboratory of dr. nancy butte (u.s. de- partment of agriculture children’s nutri- tion research center, baylor college of medicine, houston, tx), was used to cal- culate amount of time spent at defined lev- els of pa. we used the actiwatch cutoff points proposed by puyau et al. ( ), which are based on a threshold between light and moderate activity of $ . kcal z kg z min for children and adolescents and report time spent in a collapsed cate- gory of mvpa. statistical analysis analyses were performed with sas . software (sas institute inc., cary, nc). we compared continuous variables be- tween ooa and es children using anova to adjust for age and sex. prevalence of high bmi-for-age (defined as bmi $ th percentile for age and sex) was compared between groups using logistic regression. actical data from ooa children were used to assess the relation of pa levels to bmi in this group; this question has previously been examined in the es children ( ). to account for multiple ooa subjects who were recruited from the same household, a mixed model was used, adjusting for family as a random effect. each of the families was num- bered from to . the family number was defined as a class variable in an sas proc mixed model with the bmi mea- sure of interest as the dependent variable and the pa measure of interest and the family number as the independent vari- ables. the family number was specified as a random effect. actiwatch data from ooa and es children were used to compare pa levels between groups. a t test was used to examine whether the dual-device (i.e., simultaneously wear- ing actical and actiwatch) ooa children were representative of the greater (n = ) ooa sample. adjusted point esti- mates are given as least-squares means using the lsmeans statement in proc glm. results physical characteristics of ooa and es children the age range was – years in the ooa sample and – years in the es sample, as the latter included children aged years. mean age was . . and . . years in the ooa and es chil- dren, respectively (p = . ). es children had higher mean bmi ( . . vs. . . kg/m ), z-bmi ( . . vs. . . ), and body weight above the age- and sex-specific cdc median ( . . vs. . . kg; all p , . ). table reports the characteris- tics of the ooa children and es children according to age-group. within the es, mean body weight was . kg higher in non-hispanic black participants than in non-hispanic white participants after adjustment for age and sex (p = . ). thus, to produce the most conservative estimate of the effect of the ooa/es environmental differences on bmi, we performed an analysis limited to the es non-hispanic white subgroup (n = ). the prevalence of a bmi $ th percentile was . % in the ooa and . % in es non-hispanic whites (p , . ); age- and sex- ad- justed odds ratio of . ( % ci . – . ). in the group aged years and younger, the prevalence was . % in the ooa, . % in es non-hispanic whites (p , . ), and . % in nhanes non-hispanic whites. in the group aged years and older, the prev- alence was . % in the ooa, . % in es non-hispanic whites (p , . ), and . % in nhanes non-hispanic whites. correlates of pa in the ooa children actical recordings were not available for of the ooa children because of device shortages during times of increased demand, noncompliance, or device mal- function (see research design and methods). the subjects who had no actical data were slightly older than the that did ( . . vs. . . years, p = . ), but there were no differences in sex distribution ( vs. % girls, p = . ) or z-bmi ( . . vs. . . , p = . ). correlates of pa were ex- amined in the ooa children who had actical data. compared with ooa girls, ooa boys collected significantly more total average daily actical counts ( vs. counts/day, p , . ), spent more time in mvpa ( vs. min/day, p , . ), and spent less time in sedentary activity ( vs. min/day, p = . ). the ooa children belonged to nuclear families (mean number of participating siblings per family was . . [range – ]). in separate mixed table dphysical characteristics for ooa and es children ages – p ages – p ooa es* ooa vs. es ooa es ooa vs. es sex girls boys age (years) . . . . . . . . . . body weight (kg) . . . . , . . . . . , . above cdc age and sex median (kg) . . . . . . . . . , . bmi (kg/m ) . . . . , . . . . . , . z-bmi . . . . , . . . . . , . weight $ th percentile for age and sex ( . ) ( . ) , . ( . ) ( . ) , . data are shown as n (%) or means sd. *the youngest children in es group were years old (n = ). care.diabetesjournals.org diabetes care, volume , april hairston and associates models, adjusting for family as a random effect, z-bmi (b = . , p = . ), and body weight above the cdc age- and sex- specific median (b = . , p = . ) were both in- versely correlated with time spent in mvpa (fig. ), but not with time spent in sedentary activity (p = . – . ). when the above models with relative body size as the dependent variable were expanded to include sex as an indepen- dent variable in addition to mvpa (and family number), there was no significant effect of sex (p = . – . ). pa in ooa and es children the entire cohort of es children was used for comparison with the actiwatch- wearing ooa children because neither total actiwatchactivitycounts,timespentinlight activity, nor time spent in mvpa differed by race or ethnicity in the es participants (total counts [non-hispanic white], [non-hispanic black], [hispanic] counts/day; p = . for non-hispanic white vs. non- hispanic black). ooa children collected approximately . times as many total actiwatch counts as es children ( vs. counts/ day; p , . ). compared with the es, the ooa spent more time in both light activity ( vs. min/day, p = . ) and mvpa ( vs. min/day, p , . ). a multivariate regression analysis of dif- ferences in pa between ooa and es, adjusted for age and sex, produced least-squares means that deviated mini- mally from the crude means, demon- strating that minor imbalances in sex and age between the ooa and es groups were inconsequential for the group com- parisons (least-squares means [ % ci] total counts in ooa vs. es: [ – ] vs. [ – ] counts/day, p , . ; light activity [ – ] vs. [ – ] min/day, p = . ; mvpa [ – ] vs. [ – ] min/ day, p , . ). although ooa girls col- lected fewer totalcounts and spentless time in mvpa than ooa boys, ooa girls col- lected more total counts than es boys ( vs. counts/ day; p . ) and spent more time in mvpa than es boys ( vs. min/day, p . ). all statistically significant comparisons of pa between the ooa and the multiracial/ethnic group of es children remained significant when the es group was limited to the non- hispanic white children. table and fig. provide further stratification by age-group and sex. to examine whether the ooa actiwatch studiesdwhich were done in march and aprildwere subject to sea- sonal bias or sampling error, we took ad- vantage of the following circumstances: ) all ooa children wearing the actiwatch had simultaneously been wearing actical devices, and ) the actical devices had been used year-round in the greater ooa sample. thus, we could compare the actical counts of the dual device– wearing children with the means of the actical counts of the entire ooa sam- ple. the actical total counts in the dual device–wearing children did not differ from the means of the entire ooa sample in any of the four strata defined by sex and age-group (p = . – . ), indicating that the pa levels of the dual device– wearing children were indeed represen- tative of the greater ooa sample. as an additional analysis, we exam- ined differences between weekend and nonweekend days. in the ooa children, total activity counts on saturdays were within % of the monday through fri- day average and on sundays were . % below the monday through friday aver- age (p , . ). in the es, total activity counts on saturdays were within % of the monday through friday average and on sundays were . % below the monday through friday average (p = . ). conclusionsddiabetes is only half as prevalent in adult ooa as it is in non-amish americans of european ori- gin, despite a similar mean bmi in both populations ( ). because the number of years lived with obesity is a risk factor for diabetes independent of age and cur- rent bmi ( , ), we wanted to investigate whether the trajectory of weight gain in the ooa is delayed relative to that of other americans. if so, this might be a fac- tor contributing to the decreased risk for developing diabetes in this population ( ). our study compared anthropomet- ric observations from ooa children with those of non-hispanic white participants from a multiracial/ethnic co- hort of non-amish, rural children liv- ing nearby. indeed, our study confirmed that ooa children are very rarely over- weight compared with non-amish rural children of european background, in whom the prevalence of a high bmi ap- proximates national survey estimates from nhanes. incidentally, the ooa children were extremely close to the his- torical populations on which the current cdc norms are based ( ), as indicated by a mean z-bmi of ; . or a mean body weight only to kg above the age- and sex-specific cdc median. our observa- tions are consistent with the notion that the ooa experience a delayed accretion of excess body weight compared with other americans, a phenomenon that may protect against the development of diabetes. furthermore, we found that total pa and mvpa were inversely correlated to excess weight in the ooa. some ( , , ), but not all, pediatric studies have made concordant findings in the entire sample or a subgroup. treuth et al. ( ) found such a relationship in the es children, but only in girls; in the ooa, there was no effect of sex on these relationships. we believe that the rela- tively wide range of pa levels in the ooa, the use of an objective measure of figure da: bmi z-score as a function of time spent in mvpa in ooa children (r = . , p = . ). the correlation was robust to adjustment for age by partial correlation (r = . , p = . ). b: body weight above the age- and sex-specific cdc median as a func- tion of time spent in mvpa in ooa children (r = . , p , . ). the correlation was robust to adjustment for age by partial corre- lation (r = . , p , . ). (a high- quality color representation of this figure is available in the online issue.) diabetes care, volume , april care.diabetesjournals.org bmi and pa in ooa children pa, and limited variability in other envi- ronmental factors may have enhanced our ability to demonstrate a true correlation between pa and body composition. re- garding the direction of causality, some prospective studies of adults provide evi- dence that acquisition of excess weight precedes a decrement in activity (i.e., that obesity causes sedentary behavior [ ]), although the traditional explana- tion that sedentary behavior causes obe- sity is probably also true ( ). lastly, we compared pa between the ooa and the es children living nearby. there was no effect of race or ethnicity within the es group. in ooa children, compared with es children, time spent in light activity was up from to min/ day, and more dramatically, time spent in mvpa doubled from to min/day. these findings agree with those of esliger et al. ( ), who reported high levels of pa in a sample of amish children residing in canada. the magnitude of the differences in pa between the ooa and the es is in- triguing because other studies ( , ) com- paring groups whose environments were thought to differ crucially found that the groups had similar levels of pa. the exact origins of the differences between ooa and es children are unclear, but certain circumstances deserve mention. the ooa lifestyle affects the whole family, in- volving the children in household or farming chores from an early age. ooa children also seem to spend a substantial amount of time in outdoor play with their siblings and neighbors, facilitated by the large size of the average ooa nuclear family. ooa children do not use comput- ers or electronic games, nor do they watch television. ooa children attend amish one-room schoolhouses and almost always go outside for recess. even the youngest ooa students use active transportation to get to school, generally walking in a group. bicycles are banned, but some use a foot- propelled scooter, which is less energy- efficient than a bicycle. by contrast, the es almost universally travel to school and other destinations by bus or car. the high level of pa in the ooa is notable because despite public recom- mendations to increase pa ( – ), little is known about how to produce such an increase in youths ( ). although it would not be realistic to propose that non-amish individuals adopt the ooa lifestyle, it is possible that future studies, mapping the nature of activities under- taken by ooa youths and the attitudes of their parents, may provide novel ideas for the design of interventions at the in- dividual and community level to increase pa in non-amish boys and girls. ooa girls were easily more active than es boys. some challenges and limitations of our study deserve mention. first, only the actiwatch-wearing ooa children were used in the ooa-to-es pa compar- ison. because these actiwatch-wearing ooa children were nested in the group of actical-wearing ooa children and wore the two devices simultaneously, we could determinethatthepalevelsoftheactiwatch- wearing ooa children were representative of the greater ooa sample (n = ). the overall comparison with the es children showed that these ooa children spent twice as much time in mvpa as the es children. however, because of small cell sizes (n = ; ) in the ooa when we split the actiwatch-wearing children into four strata by age-group and sex, we have in- cluded fig. for orientation only and re- frained from stratified statistical tests to support any suggested age- or sex-specific trends. second, the data collection in the ooa children took place to years after the data collection in the es chil- dren. nevertheless, we find it unlikely that this temporal difference is a major contributor to the vast differences in bmi and pa observed between the ooa and the es. lastly, although the ooa and es studies were performed by overlapping groups of investigators using similar pro- tocols and instrumentation, the bmi data for the general u.s. population were obtained from nhanes, which is very different in size and scope from our local studies. however, ignoring the excellent national estimates that nhanes provides would be foolish. the nhanes data sup- port our conclusion that ooa children are rarely overweight compared with other groups. in summary, together with our pre- vious study of adult ooa, the current study implies that the ooa tend to gain their excess weight relatively late in life table dpa as assessed by actiwatch monitors for ooa and es children ages – years p ages – years p ooa es* ooa vs. es ooa es ooa vs. es sex girls boys actiwatch counts ( /day) , . , . time spent in light activity (min/day) . . time spent in mvpa (min/day) , . , . data are shown as n or means sd. *the youngest children in es group were years old (n = ). figure dtime spent in light activity (a) and mvpa (b) in ooa and es children by age-group and sex. *the es group included boys aged years. mean data are presented with the sd. care.diabetesjournals.org diabetes care, volume , april hairston and associates and that ooa children are very physically active, both of which may provide some long-term protection against diabetes. acknowledgmentsdsupport was provided by the nutrition and obesity research center (norc) of maryland (p dk ), the geriatric research and education clinical center, baltimore veterans administration medical center, a postdoctoral national in- stitutes of health/national institute on ag- ing training grant t -ag- (j.l.d.), a postdoctoral award from the american diabetes association (k.g.h.), and grant k -ag- (w.c.h.). the es study was supported by the cdc agency for toxic sub- stances and disease registry project s - . no potential conflicts of interest relevant to this article were reported. k.g.h. designed the study; collected, managed, and analyzed data; and wrote, re- viewed, and edited the manuscript. j.l.d. collected, managed, and analyzed data, and wrote, reviewed, and edited the manuscript. m.s.t. collected, managed, and analyzed data, and reviewed and edited the manuscript. w.-c.h. collected data and reviewed and edited the manuscript. a.m.j. collected data and reviewed and edited the manuscript. k.a.r. and x.s. managed and analyzed the data. b.d.m. reviewed and edited the manuscript. a.r.s. designed the study and reviewed and edited the manuscript. s.s. designed the study, man- aged and analyzed the data, and wrote the manuscript. s.s. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the in- tegrity of the data and the accuracy of the data analysis. the authors extend their gratitude to all study participants and to the extraordinary efforts of the amish research clinic staff. references . hsueh wc, mitchell bd, aburomia r, et al. diabetes in the old order amish: characterization and 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kleber, graciela delgado, günther silbernagel, anne u. jackson, emil v. appel, niels grarup, joshua p. lewis, , may e. montasser, , claes landenvall, , harald staiger, , , jian’an luan, timothy m. frayling, michael n. weedon, weijia xie, sonsoles morcillo, , maría teresa martínez-larrad, mary l. biggs, , yii-der ida chen, arturo corbaton-anchuelo, kristine færch, juan miguel gómez-zumaquero, , mark o. goodarzi, jorge r. kizer, , heikki a. koistinen, , , aaron leong, , lars lind, cecilia lindgren, , fausto machicao, , alisa k. manning, , , gracia maría martín-núñez, gemma rojo-martínez, , , jerome i. rotter, david s. siscovick, , , , joseph m. zmuda, zhongyang zhang, , manuel serrano-rios, ulf smith, federico soriguer, , , torben hansen, torben j. jørgensen, , , allan linnenberg, , , oluf pedersen, mark walker, claudia langenberg, robert a. scott, nicholas j. wareham, andreas fritsche, , , hans-ulrich häring, , , norbert stefan, , , leif groop, , jeff r. o’connell, , michael boehnke, richard n. bergman, francis s. collins, karen l. mohlke, jaakko tuomilehto, , , , winfried märz, , , peter kovacs, michael stumvoll, bruce m. psaty, , , , , johanna kuusisto, markku laakso, james b. meigs, , , josée dupuis, , erik ingelsson, , and jose c. florez , , genome-wide association study of the modified stumvoll insulin sensitivity index identifies bcl and fam a as novel insulin sensitivity loci diabetes ; : – | doi: . /db - genome-wide association studies (gwas) have found few common variants that influence fasting measures of insulin sensitivity. we hypothesized that a gwas of an integrated assessment of fasting and dynamic measures of insulin sensitivity would de- tect novel common variants. we performed a gwas of the modified stumvoll insulin sensitivity index (isi) within the meta-analyses of glucose and insulin-related diabetes research center (diabetes unit), massachusetts general hospital, boston, ma center for human genetic research, massachusetts general hospital, boston, ma department of medicine, harvard medical school, boston, ma department of medical sciences, uppsala university, uppsala, sweden data coordinating center, boston university school of public health, boston, ma university of eastern finland and kuopio university hospital, kuopio, finland department of biostatistics, boston university school of public health, boston, ma department of biostatistics, harvard t.h. chan school of public health, bos- ton, ma cardiovascular health research unit, university of washington, seattle, wa department of medicine, university of washington, seattle, wa department of biostatistics, university of washington, seattle, wa department of biostatistics, university of liverpool, liverpool, u.k. wellcome trust centre for human genetics, university of oxford, oxford, u.k. estonian genome center, university of tartu, tartu, estonia department of medicine, university of leipzig, leipzig, germany department of genomics of common disease, imperial college london, london, u.k. oxford centre for diabetes, endocrinology and metabolism, university of oxford, oxford, u.k. fifth department of medicine, medical faculty mannheim, heidelberg university, heidelberg, germany division of angiology, department of internal medicine, medical university of graz, graz, austria diabetes volume , october g e n e t ic s /g e n o m e s /p r o t e o m ic s /m e t a b o l o m ic s http://crossmark.crossref.org/dialog/?doi= . /db - &domain=pdf&date_stamp= - - traits consortium. discovery for genetic association was performed in , individuals, and replication was attempted for the most significant novel loci in , independent individuals. association with isi was tested in models adjusted for age, sex, and bmi and in a model analyzing the combined influence of the geno- type effect adjusted for bmi and the interaction effect between the genotype and bmi on isi (model ). in model , three variants reached genome-wide signi- ficance: rs (nyap ; p = . ), rs (bcl ; p = . ), and rs (fam a ; p = . ). the association at nyap was eliminated by conditioning on the known irs insulin sensitivity locus; the bcl and fam a associations department of biostatistics and center for statistical genetics, university of michigan, ann arbor, mi the novo nordisk foundation center for basic metabolic research, faculty of health and medical sciences, university of copenhagen, copenhagen, denmark division of endocrinology, diabetes, and nutrition, university of maryland school of medicine, baltimore, md program for personalized and genomic medicine, university of maryland school of medicine, baltimore, md department of clinical sciences, diabetes and endocrinology, lund university diabetes centre, malmö, sweden department of immunology, genetics and pathology, science for life labora- tory, uppsala university, uppsala, sweden department of internal medicine, division of endocrinology and diabetology, angiology, nephrology, and clinical chemistry, university hospital tübingen, tübingen, germany german center for diabetes research (dzd), tübingen, germany institute for diabetes research and metabolic diseases, helmholtz center munich, university of tübingen, tübingen, germany mrc epidemiology unit, university of cambridge school of clinical medicine, cam- bridge, u.k. university of exeter medical school, exeter, u.k. ciber pathophysiology of obesity and nutrition, madrid, spain department of endocrinology and nutrition, hospital regional universitario de málaga, málaga, spain spanish biomedical research centre in diabetes and associated metabolic disorders (ciberdem), instituto de investigación sanitaria del hospital clínico san carlos (idissc), madrid, spain institute for translational genomics and population sciences, departments of pediat- rics and medicine, labiomed at harbor-ucla medical center, torrance, ca steno diabetes center, gentofte, denmark instituto de investigación biomédica de málaga (ibima), málaga, spain sequencing and genotyping platform, hospital carlos haya de málaga, málaga, spain division of endocrinology, diabetes and metabolism, cedars-sinai medical center, los angeles, ca department of medicine, albert einstein college of medicine and montefiore medical center, bronx, ny department of epidemiology and population health, albert einstein college of med- icine, bronx, ny department of health, national institute for health and welfare, helsinki, finland minerva foundation institute for medical research, biomedicum u, helsinki, finland department of medicine and abdominal center: endocrinology, university of helsinki and helsinki university central hospital, helsinki, finland division of general internal medicine, massachusetts general hospital, boston, ma broad institute of the massachusetts institute of technology and harvard univer- sity, cambridge, ma department of endocrinology and nutrition, hospitales regional universitario y virgen de la victoria de málaga, málaga, spain ciber de diabetes y enfermedades metabólicas asociadas (ciberdem), madrid, spain department of epidemiology, university of washington, seattle, wa the new york academy of medicine, new york, ny department of epidemiology, graduate school of public health, university of pittsburgh, pittsburgh, pa department of genetics and genomic sciences, icahn school of medicine at mount sinai, new york, ny icahn institute for genomics and multiscale biology, icahn school of medicine at mount sinai, new york, ny the lundberg laboratory for diabetes research, department of molecular and clinical medicine, sahlgrenska academy at the university of gothenburg, gothenburg, sweden department of public health, faculty of health and medical science, university of copenhagen, copenhagen, denmark faculty of medicine, aalborg university, aalborg, denmark research center for prevention and health, the capital region of denmark, co- penhagen, denmark department of clinical experimental research, rigshospitalet, glostrup, denmark department of clinical medicine, faculty of health and medical science, university of copenhagen, copenhagen, denmark institute of cellular medicine, newcastle university, newcastle upon tyne, u.k. finnish institute for molecular medicine, university of helsinki, helsinki, finland diabetes and obesity research institute, cedars-sinai medical center, los angeles, ca medical genomics and metabolic genetics branch, national human genome research institute, national institutes of health, bethesda, md department of genetics, university of north carolina, chapel hill, nc chronic disease prevention unit, national institute for health and welfare, helsinki, finland centre for vascular prevention, danube-university krems, krems, austria diabetes research group, king abdulaziz university, jeddah, saudi arabia dasman diabetes institute, dasman, kuwait clinical institute of medical and chemical laboratory diagnostics, medical university of graz, graz, austria synlab academy, synlab services gmbh, mannheim and augsburg, germany integrated research and treatment (ifb) center adipositydiseases, univer- sity of leipzig, leipzig, germany epidemiology and health services, university of washington, seattle, wa group health research institute, seattle, wa group health cooperation, seattle, wa department of medicine, university of eastern finland and kuopio university hospital, kuopio, finland framingham heart study, national heart, lung, and blood institute, fra- mingham, ma department of medical sciences, molecular epidemiology and science for life laboratory, uppsala university, uppsala, sweden department of medicine, division of cardiovascular medicine, stanford university school of medicine, stanford, ca corresponding author: geoffrey a. walford, gwalford@partners.org. received february and accepted july . this article contains supplementary data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc . g.a.w., s.g., and d.r. contributed equally as first authors. e.i. and j.c.f. contributed equally as senior authors. the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. © by the american diabetes association. readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. more information is available at http://www.diabetesjournals .org/content/license. diabetes.diabetesjournals.org walford and associates mailto:gwalford@partners.org http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://www.diabetesjournals.org/content/license http://www.diabetesjournals.org/content/license were independent of known cardiometabolic loci. in con- clusion, we identified two novel loci and replicated known variants associated with insulin sensitivity. further stud- ies are needed to clarify the causal variant and function at the bcl and fam a loci. genome-wide association studies (gwas) have identified common genetic variants associated with type diabetes ( ), a disease marked by a reduction in b-cell function and insulin sensitivity ( ). while both b-cell function and in- sulin sensitivity traits are partly heritable, gwas have demonstrated relatively few single nucleotide polymor- phisms (snps) associated with insulin sensitivity ( ). traits used to estimate insulin sensitivity from fasting measurements in prior large gwas, including fasting insulin and the homa–insulin resistance (homa-ir), demonstrate approximately half the heritability of traits that incorporate both fasting and dynamic assessments of insulin sensitivity following a glucose load ( ). more- over, there is only modest genetic correlation between homa-ir and measures of insulin sensitivity by euglyce- mic clamp, which is considered the gold standard measure of peripheral insulin sensitivity ( , ). thus, an alternative approach to discover new common genetic variants asso- ciated with insulin sensitivity is to perform gwas using a dynamic measure of whole-body insulin sensitivity. as an example, a recent gwas identified a novel insulin sen- sitivity locus at nat using euglycemic clamp and insulin suppression test techniques in , subjects, with repli- cation in another , individuals ( ). however, these direct, whole-body measures of insulin sensitivity are time- and resource-intensive interventions, which limits the feasible sample size of such experiments. indices de- rived from an oral glucose tolerance test that integrate fasting and dynamic measures of insulin sensitivity rea- sonably approximate euglycemic clamp measures and can be applied in existing large cohorts with glycemic traits, potentially increasing the statistical power to detect novel variant associations. we tested the hypothesis that a well-powered gwas would detect common genetic variants for the modified stumvoll insulin sensitivity index (isi). insulin sensitivity assessed by the euglycemic-hyperinsulinemic clamp (aver- age glucose infusion rate/average plasma insulin concen- tration [m/i]) has a stronger correlation with the isi than with homa-ir (r = . vs. . , respectively) ( ). in ad- dition, the isi is well correlated (r = . ) with m/i, even when calculated using only fasting insulin values and glu- cose and insulin values min after a -g oral glucose load ( ); this modified version is widely available in existing cohorts, providing a larger sample size for association anal- yses than the sample size that would be available if indices requiring additional time points were used. we further hypothesized that a subset of these common genetic var- iants would influence the isi independently or through their effect on bmi. thus we tested the association of the modified isi in statistical models without adjusting for bmi, in statistical models adjusting for bmi, and in a validated model ( , ) analyzing the combined influence of the genotype effect adjusted for bmi and the interaction effect between the genotype and bmi on isi. research design and methods cohort descriptions the cohorts participating in the meta-analyses of glucose and insulin-related traits consortium (magic) contrib- uted a total of , individuals to the analyses. detailed information on the study cohorts and methods is provided in supplementary table . all participants were of white euro- pean ancestry from the united states or europe and did not have diabetes. all studies were approved by local research ethic committees, and all participants gave informed consent. modified stumvoll isi missing trait data were not imputed, and outliers were not excluded from analyses. the isi was calculated as previously described ( ), according to the following formula: : � : insulin h½pmol=l� � ð : insulinfasting½pmol=l� � ð : glucose h½mmol=l� � discovery effort: gwas cohorts that were able to contribute genome-wide geno- typing results during the course of the project were in- cluded in the discovery effort. these were the framingham heart study (fhs), sorbs, the finland–united states investigation of niddm (fusion), the cardiovascular health study (chs), ludwigshafen risk and cardiovascular health (luric) study, the uppsala longitudinal study of adult men (ulsam), and metabolic syndrome in men (metsim) study. for the discovery gwas, all samples with call rates , % were excluded, and snps departing from hardy-weinberg equilibrium (at p , ), genotype rate , %, or minor allele frequency , % were excluded. poorly imputed snps were excluded if r , . or proper- info was , . . each snp was tested for association with isi in three different additive genetic models: model was adjusted for age and sex; model was adjusted for age, sex, and bmi; and model analyzed the combined influence of the genotype effect adjusted for bmi and the interaction effect between the genotype and bmi on isi ( , ). the associations in model result from a test with two de- grees of freedom. when no interaction is present, the additional degree of freedom results in a modest loss of statistical power. when interaction is present, however, the statistical power of the model is greater ( ). to ad- just for differences in insulin measurement between novel insulin sensitivity loci diabetes volume , october http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc cohorts, effect estimates were normalized to the sd of the isi in each cohort (supplementary table ). a ro- bust estimate of the standard error was calculated in the interaction analysis using probabel, quicktest, or generalized estimating equations using the r geepack package. an inverse-variance meta-analysis using metal was performed on the b coefficient/sd from each cohort. following meta-analysis, snps with total sample size less than , (approximately half of the maximum sample size) or with heterogeneity p values # (a value chosen to take into account multiple hypothesis testing but below the level of strict bonferroni correction) in the meta-analysis of the discovery cohorts were re- moved. genomic correction of cohort-specific association statistics (i.e., correction for each individual study) was performed. in total, up to . million snps were meta- analyzed for association with isi in the discovery effort. selection of snps for replication candidate snps for replication were identified by their association p value # in one or more of the analysis models. for gene loci with multiple replication candidates, the snp with the lowest p value and any other snp in low linkage disequilibrium (ld; r , . ) with the index snp in europeans were retained. using these filters, unique candidate snps from loci were identified for replica- tion. the snp annotation and proxy search site was used to find up to three proxies in high ld (r . . ) in europeans for each candidate snp. replication effort cohorts that did not contribute to the discovery effort but were able to contribute association results during the course of the project were included in the replication effort. these were the european network on functional genomics of type diabetes (eugene ) study, amish studies, the relationship between insulin sensitivity and cardiovascular risk study (risc), the tübingen family study for type diabetes (tübingen), inter study, the segovia study, the pizarra study, the botnia study, the birth cohort, and the ely study. genotype data were obtained using in silico data from preexisting gwas or de novo genotyping. in replication cohorts, snps with a minor allele count (mac) , were excluded. additional details of the replication cohort effort are provided in sup- plementary table . combined meta-analysis we required the absence of heterogeneity in the combined analysis of discovery and replication cohorts (p . ) as well as nominal significance (p , . ) in the replication effort and genome-wide significance (p , ) in the combined meta-analysis for statistical evidence of association between a novel snp and the isi. to assess the effect of removing lower-frequency snps in model , a sensitivity anal- ysis was performed using the mac , filter on a cohort- wise basis in both the discovery and replication cohorts. assessment for association of known insulin sensitivity loci with isi the associations of published insulin sensitivity loci were tested for association with the isi in the discovery cohorts. loci associated with fasting insulin without ( ) and with adjustment for bmi ( , ), with fasting insulin using the approach in model ( ), and with direct measures of in- sulin sensitivity were included in these analyses ( ). the published results for associations with fasting insulin with or without bmi adjustment (n = ; , – , ) ( , ) or exploiting potential bmi-by-gene interaction (model ; n = ; , ) ( ) used the same statistical approach as in the current study but were derived in a sample size ap- proximately three to six times larger than that of the cur- rent study discovery cohort (n = ; , ). the sample sizes of the published fasting insulin analyses were much greater because only fasting insulin and bmi phenotypes were required for cohort participation. to analyze the as- sociation with fasting insulin and isi in a comparable sam- ple, we also examined the subset of discovery cohorts that contributed to the current assessment of isi and prior assessments of fasting insulin: fhs, sorbs, fusion, and chs. in models and , only data from fhs, sorbs, and fusion were analyzed because participant-level bmi data were not available in chs. a binomial sign test was used to determine whether the expected direction of the effect for these published loci with isi occurred more often than by chance. conditional analyses and assessment of the association of top findings with direct measures of insulin sensitivity findings that reached genome-wide significance were assessed for association with direct measures of insulin sensitivity in the genetics of insulin sensitivity (genesis) consortium ( ). direct measures of insulin sensitivity were inverse normal transformed m value in cohorts with eugly- cemic insulin clamp assessments and inverse normal trans- formation of the steady state plasma glucose from cohorts with an insulin suppression test. these two traits are highly correlated (r = . ; p , . ) ( ), and tests of association with the direct measure of insulin sensitivity showed no evidence of heterogeneity (p value for hetero- geneity = . for the bcl variant and . for the fam a variant). therefore, we did not perform sepa- rate tests of association in the smaller subsets of data with either the m value or the insulin suppression test pheno- type. statistical models were adjusted for age, sex, and bmi. the top findings of the isi analyses were also assessed in a magic association analysis from manning et al. ( ) with fasting insulin using the approach in model . these isi variants were only available in the discovery cohort from manning et al. (n = , for rs ; n = , for rs ). we also performed association analyses with fasting insulin and isi in a subset of the discovery cohort—fhs, sorbs, and fusion—to ascertain values in a comparable sample. diabetes.diabetesjournals.org walford and associates http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc approximate conditional analyses were performed to understand whether known loci contributed to the associ- ations of novel findings with the isi ( ). these analyses were based on the summary-level statistics from the meta- analysis and the estimated ld using individual-level geno- type data from the fhs discovery cohort. the software implementation for this approach does not incorporate the interaction term from model , and therefore condi- tional analyses were not performed in model . results the demographic characteristics of the participants in- cluded in the discovery and replication efforts are pre- sented in table . in total, the discovery, replication, and combined meta-analyses included up to , , , , and , participants, respectively. using a variance component approach implemented in solar software ( ), the heritability of the isi (h r se) in related fhs participants (n = , ) was very similar without or with adjustment for bmi ( . . %; p = . and . . %; p = . , respectively). within the ulsam discovery cohort, the spearman corre- lation between the isi and m value from the euglycemic- hyperinsulinemic clamp was . (fig. ), consistent with reports from the literature ( ); the spearman correlation between isi and fasting insulin was . (fig. ). when tested in the full discovery cohort, of loci previously associated with fasting insulin in the literature ( ) (p = . for binomial sign test) and of loci previously associated with fasting insulin after adjusting for bmi in the literature ( , ) (p = . for binomial sign test) showed the expected direction of effect with the isi in the discovery cohorts (supplementary table ). when these associations were examined in a subset of the current study discovery cohort (supplementary table ), statistical significance was reduced, but effects at each loci remained in the expected direction ( of loci for isi vs. fasting insulin without bmi adjustment, p = . for binomial sign test; of loci for isi vs. fasting insulin with bmi adjustment, p = . for binomial sign test). using a variant in ld with rs (rs ; r = . ), we also found the expected direction of effect with isi in the discovery cohorts (n = , ) at the nat locus (model ; b= . ; p = ) ( ). the qq plots for models , , and are shown in sup- plementary figs. – , respectively. measures of genomic control were consistent with low inflation (model lgc = . ; model lgc = . ; model lgc = . ). while genomic control was used to correct for each individual study, no additional corrections were applied to the meta- analysis results. the separate results of the discovery and replication results for model (adjusting for age and sex), model (adjusting for age, sex, and bmi), and model (adjusting for age, sex, and bmi and analyzing the com- bined influence of the genotype effect adjusted for bmi and the interaction effect between the genotype and bmi on isi) are shown in supplementary table . four snps selected from the discovery effort reached nominal signif- icance (p , . ) in the replication analyses: rs (nyap ) in models , , and ; rs (bcl ) in mod- els and ; rs (fam a ) in model ; and rs (pfdn ) in model . although the association with rs (cdh ) reached nominal significance in the replication effort for model , the association was in the opposite direction of effect, as in the discovery analyses; consequently, the association of this variant also had high heterogeneity in the combined meta-analysis. we compared the b coefficients for the snps iden- tified in the discovery effort (rs [cdh ] was excluded given its high heterogeneity) with fasting in- sulin and isi in a subset of the discovery cohort. pearson correlations between the b for fasting insulin and the b for isi were . in model , . in model , and . (for snp effect) and (for interaction) in model . the results of the combined discovery and replication cohort meta-analyses in each of the three models are shown in table and supplementary table . no association reached genome-wide significance in model . in model , rs (nyap ; p = . ) and rs (bcl ; p = . ) achieved genome-wide significance. in model , rs (nyap ; p = . ), rs (bcl ; p = . ), and rs (fam a ; p = . ) reached genome-wide signifi- cance. in model , rs (arhgap ; p = . ) also reached genome-wide significance but did not achieve nominal significance in the replication cohort, and rs (pfnd ) had high heterogeneity in the com- bined meta-analysis of discovery and replication cohorts (p for heterogeneity = . ); therefore associations with these snps were not included as trustworthy findings. hence, rs (nyap ), rs (bcl ), and rs (fam a ) were the three snps that reached our a priori requirements for claiming statistical evidence. the association at rs (nyap ) was in ld (r = . ) with previously reported results at the known insulin sensitivity signal rs (irs ) ( ), and the associa- tion with the isi in model was greatly reduced by con- ditioning rs on rs in the discovery cohort (b = . . ; p = . to b = . . ; p = . ). thus, this snp was considered a reflection of the known irs signal and not an independent signal. the associations for rs (bcl ) and rs (fam a ) with the isi were consistent across the discov- ery and replication cohorts (supplementary figs. and , respectively). when stratifying by bmi, the effect of the minor allele (a) at rs (fam a ) on insulin sen- sitivity was negative at lower bmi and became positive and stronger with increasing bmi (fig. ), and the effect of the major allele (t) at rs (bcl ) on isi was more negative with increasing bmi (fig. ). the genomic inflation of models and was low and slightly higher in model . because the same individuals were used in each model, inflation in model was unlikely novel insulin sensitivity loci diabetes volume , october http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc t a b le — c o h o rt a n d p a rtic ip a n t d e m o g ra p h ic s c o h o rt p a rtic ip a n ts (n ) f e m a le (% ) a g e (ye a rs) b m i (kg /m ) f a stin g g lu c o se (m m o l/l ) f a stin g in su lin (p m o l/l ) s tu m vo ll is i ([m m o l * p m o l]/[kg * m in * l ]) d isc o ve ry f h s , . . . . . . . . . . s o rb s . . . . . . . . . . f u s io n . . . . . . . . . . c h s , . . . . . . . . . . l u r ic . . . . . . . . . . u l s a m . . . . . . . . . . m e t s im , . . . . . . . . . . r e p lic a tio n e u g e n e . . . . . . . . . . a m ish s tu d ie s . . . . . . . . . r is c . . . . . . . . . t ü b in g e n , . . . . . . . . . . in te r , . . . . . . . . . . s e g o via . . . . . . . . . . p iza rra . . . . . . . . . . b o tn ia s tu d y , . . . . . . . . . . b irth c o h o rt . . . . . . . . . . e ly s tu d y , . . . . . . . . . . c o n tin u o u s re su lts a re sh o w n a s m e a n s d . a d d itio n a l in fo rm a tio n fo r e a c h c o h o rt c a n b e fo u n d in s u p p le m en ta ry t a b le . diabetes.diabetesjournals.org walford and associates http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc figure —correlation of isi with the m value from the insulin clamp (a) and fasting insulin (b) in ulsam. insulin sensitivity was measured within the ulsam discovery cohort (n = , ) using a hyperinsulinemic-euglycemic clamp (m value), the modified stumvoll isi, and fasting insulin. the ulsam cohort contains only men, and individuals with known diabetes were excluded from these analyses. for the compar- ison of the m value with isi, the pearson correlation was . and the spearman correlation was . , which are consistent with prior published reports. for the comparison of the isi with fasting insulin, the pearson correlation was . and the spearman correlation was . . novel insulin sensitivity loci diabetes volume , october t a b le — m e ta -a n a lys is re s u lts fo r va ria n t a s s o c ia tio n w ith th e is i s n p c h ro m o so m e l o c u s a lle le (e ffe c t/ o th e r) f re q u e n cy o f th e e ffe c t a lle le m o d e l , b s e (p va lu e ) m o d e l , b s e (p va lu e ) m o d e l , b s e (m a in /in te ra c tio n ) (jo in t p va lu e ) n (m in im u m / m a xim u m ) rs n y a p c /g . . . ( . ) . . ( . ) . . / . . ( . ) , / , . rs g p t /g . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs a r r d c t /c . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs t o x a /c . . . ( . ‡ ) . . ( . ) . . / . . ( . ‡ ) , / , rs m r p l a /t . . . ( . ) . . ( . ) . . / . . ( . ) , / , . rs a d a m t s t /c . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs a r h g a p a /g . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs b c l t /c . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs f a m a a /g . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs e l t d t /c . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , . rs n r g a /g . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs f g f a /t . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , rs c d h t /c . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , . rs f a m b a /g . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , rs is x a /g . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , rs a g b l t /c . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , rs p f d n a /g . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , . rs p p a r d a /g . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , rs k ia a a /g . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs n p a s t /g . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , . rs v s n l a /g . . . ( . ) . . ( . ) . . / . . ( . ) , / , . rs t x n d c t /g . . . ( . ) . . ( . ) . . / . . ( . ) , / , rs s h g l t /c . . . ( . ) . . ( . ) . . / . . ( . ‡ ) , / , . m o d e l is a d ju ste d fo r a g e a n d se x; m o d e l is a d ju ste d fo r a g e , se x, a n d b m i; m o d e l a sse sses th e c o m b in e d in flu e n c e o f th e s n p e ffe c t a d ju ste d fo r b m i a n d th e in te ra c tio n e ffe c t b e tw e e n th e g e n o typ e a n d b m i o n is i. f o r m o d e ls a n d , th e e ffe c t (b ), s e , a n d p va lu e s fo r th e s n p a re sh o w n . f o r m o d e l , th e b a n d s e a re p ro vid e d fo r th e s n p a n d th e in te ra c tio n ;a p va lu e is p ro vid e d fo r th e jo in t in flu e n c e o f th e s n p a n d in te ra c tio n e ffe c t.n sa m p le size fo r th e c o m b in e d a n a lysis o f d isc o ve ry a n d re p lic a tio n c o h o rts.e ffe c t size s a re p re se n te d a s th e s d p e r e ffe c t a lle le . ‡ p va lu e fo r h e te ro g e n e ity in th e c o m b in e d a n a lysis o f d isc o ve ry a n d re p lic a tio n c o h o rts (p # ). diabetes.diabetesjournals.org walford and associates to arise from population stratification. we performed an additional sensitivity analysis that applied the mac , filter to both the discovery and replication cohorts (supplementary table ), which tended to reduce the sta- tistical significance of associations with high heterogene- ity and slightly reduced the statistical significance of the association at the fam a locus in model without markedly reducing the magnitude of effect or affecting heterogeneity (b = . . ; p = . ; p for heterogeneity = . to b = . . ; p = . ; p for heterogeneity = . ). the sample size for the fam a locus association in model was individuals fewer when the mac filter was applied in the discovery cohorts versus when the minor allele frequency filter was applied, and the resulting loss in power was likely respon- sible for the slight reduction in statistical significance. conditioning the results at either variant with known signals at least mb away did not attenuate the as- sociation with the isi in the discovery cohorts of model (a full description is provided in supplementary table ). the rs (fam a ) variant was not associated with fasting insulin using model in a separate gwas result ( ) or with direct measures of insulin sensitivity in genesis. the major allele (t) of rs (bcl ), which was associated with lower insulin sensitivity in this study, was also associated with a trend toward higher fasting insulin in a separate gwas result using model (snp effect . . ; interaction effect . . ; p = . ; n = , ) ( ). similar trends were observed when the variant was tested for association with isi and fasting insulin in the same discovery cohort subset (supplementary table ). discussion in a study of over , participants, we found novel, independent, genome-wide significant associations for the isi at rs (bcl ) and rs (fam a ). strengths of this study’s design include a large sample size, individuals with glycemic and metabolic phenotyp- ing, high-quality genomic data, and use of traditional and contemporary statistical models to account for the influence of bmi on insulin sensitivity. in addition, our approach targeted a phenotype not previously examined in gwas: the modified stumvoll isi. by incorporating glucose and insulin measures before and after a glucose load, this phe- notype captures information that fasting assessments such as homa-ir or insulin alone would not. indeed, the cor- relation between isi and the m value is higher than that between the m value and fasting insulin ( ), which has been used in prior genetic studies of insulin sensitivity ( , ). at the same time, the use of measures obtained at only two time points (fasting and min into an oral glucose tolerance test) permitted the assembly of the large sample size required to achieve adequate statistical power. several findings serve as positive controls for our results and demonstrate that the isi is a robust measure of fasting and whole-body insulin sensitivity. first, we observe a strong correlation of isi with direct measures of insulin sensitivity. second, we show that the isi can detect genetic influences on measures of fasting insulin sensitivity ( , , ), generally ascribed to hepatic physi- ology, as well as on measures of whole-body insulin sen- sitivity, which also incorporates contributions from muscle and adipose tissue. integrated measures of insulin sensitivity may have clinical relevance, since a reduction in peripheral insulin sensitivity may be an early contrib- utor to the development of type diabetes ( – ). consistent with prior genetic explorations of insulin sensitivity ( ), the association of variants at the bcl and fam a loci became stronger and genome-wide sig- nificant after accounting for the effect of bmi on isi. notably, the isi can be calculated with or without bmi in the formula, and the correlation of the isi with m/i is figure —the effect of rs (fam a ) on insulin sensitiv- ity by bmi category. the effect of the minor allele (a) at rs (fam a ) on the isi is shown by bmi category. at a low bmi (< kg/m ), the effect is negative. at each category of increasing bmi above kg/m , the effect is positive and stronger. figure —the effect of rs (bcl ) on insulin sensitivity by bmi category. the effect of the major allele (t) at rs (bcl ) on the isi is shown by bmi category. at each category of increasing bmi, the effect is negative and stronger. novel insulin sensitivity loci diabetes volume , october http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc greater when bmi is included (r = . vs. . ) ( , ). we note that the effect of these loci on insulin sensitivity is modest, consistent with published findings on other common genetic variants for glycemic traits, such as glucose ( ) and fasting insulin ( , , ). yet our findings are meaningful because they provide a more complete understanding of the contri- bution of common genetic variations to insulin sensitivity. the existing literature bolsters our finding of bcl as a novel candidate insulin sensitivity locus. the major allele (t) at rs , which was associated with lower in- sulin sensitivity in our analysis, has been previously asso- ciated with type diabetes in a multiethnic gwas (odds ratio . [ % ci . – . ]; p = . ) ( ) in analyses adjusted for bmi. further, this same variant was recently associated with higher bmi-adjusted waist-to-hip ratio in women (b = . ; p = . ; n = , ) but not in men (b = . ; p = . ; n = , ) ( ). all these findings suggest that the metabolically deleterious effects of the bcl locus become more evident after adjust- ing for bmi. last, we find that the statistical association of rs (bcl ) is stronger with the isi than with fast- ing insulin ( ). notably, the published fasting insulin re- sults were from a study much larger than ours. the ability of the isi to detect a genome-wide significant finding in a smaller sample suggests that the bcl locus may have a greater influence on insulin sensitivity when fasting and postprandial phenotypes are assessed together. the mechanism by which bcl influences insulin sensi- tivity remains unclear. the bcl family of proteins regulate apoptosis through control of mitochondrial permeability ( ). mouse models suggest that inhibiting bcl improves glucose tolerance through effects on pancreatic b-cells ( ). conversely, pharmacological inhibition of the bcl pro- tein causes hyperglycemia among a subset of patients with chronic lymphocytic leukemia ( ), but the mechanism of this observation is unknown. by contrast, there is little direct published literature to support the role of fam a in in- sulin sensitivity. we found that the association of the minor allele (a) at the fam a locus with reduced insulin sensi- tivity was detected at bmi , kg/m . this may suggest the variant is more deleterious among individuals with lower levels of adiposity. while bcl and fam a are the closest genes to rs and rs , respectively, we have not excluded other genes in the region (supplemen- tary figs. and ). additional in silico findings at the bcl and fam a variants are provided in supplemen- tary table . we recognize limitations to our study. first, anal- yses were performed exclusively in white individuals of european ancestry. exploring these loci in other racial and ethnic groups is necessary. second, we used an es- timate of whole-body insulin sensitivity derived from measures of glucose and insulin after a glucose load, rather than direct measures of insulin sensitivity. the wide availability of the isi provided increased statistical power of the association analyses relative to that of other indices that are better correlated with euglycemic measures of insulin sensitivity, such as the matsuda index ( ). as- sessment of our novel findings in the genesis consortium suggests that the isi may be capturing different informa- tion on insulin sensitivity than that provided by the insulin clamp or the insulin suppression test, or that the power in the genesis analyses was limited to detect this associa- tion. third, conditional analyses could not be performed in model , which would have been the best method of assess- ing the dependence of the signals at bcl and fam a . however, the ld for each variant with other known glu- cose and insulin loci in the region was low, and the nom- inally significant associations of the bcl and fam a variants with isi were stable after conditioning in model , suggesting that analyses in model would have prob- ably confirmed secondary loci. fourth, given our desire for the early dissemination of these results, no experimental attempts at determining the causal gene and mechanisms of action in our novel candidate insulin sensitivity loci were performed. in conclusion, we identified two novel candidate insulin sensitivity loci through a gwas of the modified stumvoll isi. our results demonstrate that the isi is a robust measure of fasting and whole-body measures of insulin sensitivity and suggest that genetic variation in the fam a and bcl loci influence insulin sensitiv- ity. while further functional work is needed to clarify the causal genes and mechanisms of action of these loci, our work and the published literature provide support for genes in these loci having an effect on human glycemic metabolism. acknowledgments. the authors thank all the participants of each cohort for their cooperation and contribution to this study. for fhs, this research was conducted in part using data and resources from the fhs of the national heart lung, and blood institute of the national institutes of health and boston university school of medicine; the analyses reflect intellectual input and resource development from the fhs investigators participating in the snp health association resource (share) project. a full list of principal chs investigators and institutions can be found at chs-nhlbi.org. for ulsam, the authors thank the snp&seq technology platform in uppsala (www.genotyping.se) for excellent genotyping. computations were performed on resources provided by snic through uppsala multidisciplinary center for advanced computational science (uppmax) under project b . for sorbs, the authors thank knut krohn (microarray core facility, institute of pharmacology, university of leipzig) for genotyping support and joachim thiery (institute of laboratory medicine, clinical chemistry and molecular diagnostics, university of leipzig) for clinical chemistry services. for luric, the authors thank the luric study team members who are either temporarily or permanently involved in patient recruitment and sample and data handling. furthermore, the authors thank the laboratory staff at the ludwigshafen general hospital and the universities of freiburg, ulm, and graz. for amish studies, the authors gratefully thank their amish community and research volunteers for their long-standing partnership in research, and they acknowledge the dedication of their amish liaisons, field workers, and the amish research clinic staff, without whom these studies would not have been possible. for the ely study, the authors are grateful to the staff of st. mary’s surgery, ely, u.k., and the study team. funding. this work was supported by national institutes of health grant dk (to g.a.w.). grant support was provided to cohorts. for metsim, the diabetes.diabetesjournals.org walford and associates http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://diabetes.diabetesjournals.org/lookup/suppl/doi: . /db - /-/dc http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org http://chs-nhlbi.org study was funded by the academy of finland (grants and ). for sorbs, the work was supported by grants from the german research council (dfg-sfb , “obesity mechanisms,” a , c , b , and spp to / - ), the german diabetes association, and the diabetes hilfs- und forschungsfonds deutschland (dhfd). this work was further supported by the federal ministry of education and research (bmbf), germany, fkz ( eo , ad - e to p.k.), and by the boehringer ingelheim foundation. for fhs, the study was supported by the national heart, lung, and blood institute (contract nos. n - hl- - [supporting its contract with affymetrix, inc., for genotyping services], n -hc- , hl , and hhsn i) and the national institute of diabetes and digestive and kidney diseases (r dk to j.d., u - dk to h.c. and j.d., r dk and k dk to j.m.). a portion of this research utilized the linux cluster for genetic analysis (linga‐ii) funded by the robert dawson evans endowment of the department of medicine at boston university school of medicine and boston medical center. a.k.m. was supported by american diabetes association research foundation (grant - -mn- ). for chs, research was supported by national heart, lung, and blood institute contract nos. hhsn c, hhsn c, n hc , n hc , n hc , n hc , n hc , n hc , and n hc and grant nos. u hl , hl , and hl , with additional contri- bution from the national institute of neurological disorders and stroke. additional support was provided by r ag from the national institute on aging (nia). for ulsam, the project was supported by the knut and alice wallenberg foundation (wallenberg academy fellow), the european research council (erc starting grant), the swedish diabetes foundation (grant - ), the swedish research council (grant - ), and the swedish heart-lung foundation ( ). a.p.m. is a wellcome trust senior fellow in basic biomedical science (grant wt from the nordic center of excellence in disease genetics). for luric, the study was supported by the th framework program (atheroremo, grant agreement no. , and riskycad, grant agreement no. ) of the european union and by the interreg-iv-oberrhein-program (project a , genetic mechanisms of car- diovascular diseases) with support from the european regional development fund (erdf) and the wissenschaftsoffensive. m.e.k. and w.m. are supported by the german federal ministry of education and research as part of the competence cluster of nutrition and cardiovascular health (nutricard). for inter , the study was financially supported by research grants from the danish research council, the danish centre for health technology assessment, novo nordisk, the research foundation of copenhagen county, ministry of internal affairs and health of denmark, the danish heart foundation, the danish pharmaceutical association, the augustinus foundation, the ib henriksen foundation, the becket foundation, and the danish diabetes association. for fusion, the study was supported by the national institute of diabetes and digestive and kidney diseases (dk , dk , dk ) and the national human genome research institute (zia-hg ). h.a.k. has received funding from the academy of finland (support for clinical research careers, grant no. ). work on the amish studies was supported by national institutes of health awards k gm (from national institute of general medical sciences) to j.p.l. and hl (from national heart, lung, and blood institute) to j.r.o. the botnia study has been financially supported by grants from the sigrid juselius foundation, the folkhälsan research foundation, the nordic center of excellence in disease genetics, a european union grant (exgenesis, ga fp - ), the signe and ane gyllenberg foundation, the swedish cultural foundation in finland, the finnish diabetes research foundation, the foundation for life and health in finland, the finnish medical society, the paavo nurmi foundation, the helsinki university central hospital research foundation, the perklén foundation, the ollqvist foundation, the närpes health care foundation, and the ahokas foundation. the dgi study was further supported by a swedish research council linné grant ( - ) and by the ministry of education in finland, the municipal health care center and hospital in jakobstad, and the health care centers in vasa, närpes, and korsholm. the tübingen study was supported in part by a grant from the german federal ministry of education and research to the german center for diabetes research (dzd e.v.). for the ely study, j.l., c.lang, r.a.s., and n.j.w. acknowledge support from the medical research council (mc_uu_ / ). the ely study was funded by the medical research council (mc_u ) and diabetes uk. genotyping in the ely and fenland studies was supported in part by a medical research council– glaxosmithkline pilot program grant (g ). for the birth cohort and inter studies, work was support by the novo nordisk foundation center for basic metabolic research, an independent research center at the university of copenha- gen partially funded by an unrestricted donation from the novo nordisk foundation (www.metabol.ku.dk). work on the pizarra study was support by instituto de salud carlos iii pi / and instituto de salud carlos iii ps / . for segovia, this work was supported by the fondo europeo para el desarrollo regional, red de centros rcmn (c / ) grants feder fd / , the instituto de salud carlos iii-retic rd / / , madrid, spain (fis / ); ciber in di- abetes and associated metabolic disorders (instituto de salud carlos iii, minis- terio de ciencia e innovación); and the madrid autonomous community (moir s /bmd- ), and by educational grants from eli lilly lab, spain; bayer pharmaceutical co., spain; and the fundación mutua madrileña , spain. duality of interest. m.e.k. (luric) has received lecture fees from astrazeneca. g.s. (luric) is member of an amgen advisory board (thousand oaks, ca). w.m. (luric) is employed by synlab services gmbh and holds shares of synlab holding gmbh. he has received grants from siemens diagnostics, aegerion pharmaceuticals, amgen, astrazeneca, danone, sanofi/- genzyme, pfizer, basf, and abbott diagnostics. b.m.p. (chs) serves on the data safety and monitoring board of a clinical trial funded by the manufacturer (zoll lifecor corp.) and the yale open data access project steering committee funded by johnson & johnson. no other potential conflicts of interest relevant to this article were reported. author contributions. g.a.w. (massachusetts general hospital [mgh]) and j.c.f. (mgh and fhs) conceived the study. g.a.w. (mgh), s.g. (ulsam), and d.r. (fhs) wrote the manuscript under the guidance of j.d. (fhs), e.i. (ulsam), and j.c.f. (mgh, fhs); the manuscript was finalized based on detailed comments from other authors. s.g. (ulsam) and d.r. (fhs) constructed the figures and performed the meta-analysis of data from all cohorts. a.k.m. (fhs) performed analysis. s.g. (ulsam), d.r. (fhs), a.le. (fhs), a.p.m. (fhs), w.x. (genetics of insulin sensitivity, genesis), and z.z. (genesis) performed in silico analyses for follow-up studies. d.r., (fhs), h.c. (fhs), c.-t.l. (fhs), j.h. (fhs), a.p.m. (ulsam), n.g. (inter and birth cohort ), j.p.l. (amish), h.s. (tübingen), j.l. (ely study), m.t.m.-l. (segovia), m.l.b. (chs), y.-d.i.c. (chs), a.-c.a. (segovia), j.m.g.-z. (pizarra), m.o.g. (chs), l.l. (ulsam), f.m. (tübingen), g.m.m.-n. (pizzara), j.i.r. (chs), m.s.-r. (segovia), r.a.s. (ely study), n.j.w. (ely study), m.l. (metsim), j.d. (fhs), c.m.l. (ulsam), and c.lang. (ely study) performed genotyping within cohorts. a.s. (metsim, eugene ), m.e.k. (luric), g.d. (luric), g.s. (luric), a.c.-a. (segovia), k.f. (inter ), j.r.k. (chs), h.a.k. (fusion), a.li. (inter and birth cohort ), c.lang. (ely study), g.r.-m. (pizzara), d.s.s. (chs), t.h. (inter and birth cohort ), t.j. j. (inter and birth cohort ), o.p. (inter and birth cohort ), r.a.s. (ely study), n.j.w. (ely study), a.f. (tübingen), n.s. (tübingen), r.n.b. (fusion), j.t. (fusion), p.k. (sorbs), m.s. (sorbs), j.k. (metsim), j.b.m. (fhs), j.d. (fhs), and e.i. (ulsam) performed phenotyping within cohorts. p.k. (sorbs), c.land. (botnia study), s.g. (ulsam), d.r. (fhs), h.c. (fhs), c.-t.l. (fhs), j.h. (fhs), r.a.j. (chs), k.r. (chs), a.p.m. (ulsam), r.m. (sorbs), a.t. (sorbs), i.p. (sorbs), a.u.j. (fusion, metsim), e.v.a. (inter and birth cohort ), n.g. (inter and birth cohort ), j.p.l. (amish), m.e.m. (amish), h.s. (tübingen), j.l. (ely study), t.m.f. (risc), m.n.w. (risc), w.x. (risc), s.m. (pizarra), m.t.m.-l. (segovia), m.w. (risc), and j.r.o. (amish) analyzed data within cohorts. a.li. (inter and birth cohort ), c.lang. (ely study), j.h. (metsim), m.s.-r. (segovia), u.s. (eugene ), f.s. (pizarra), t.h. (inter and birth cohort ), t.j.j. (inter and birth cohort ), o.p. (inter and birth cohort ), r.a.s. (ely study), n.j.w. (ely study), a.f. (tübingen), h.u.h. (tübingen), n.s. (tübingen), l.g. (botnia study), j.r.o. (amish studies), m.b. (fusion), r.n.b. (fusion), f.s.c. (fusion), k.l.m. (fusion), j.t. (fusion), w.m. (luric), p.k. (sorbs), m.s. (sorbs), b.m.p. (chs), j. k. (metsim), m.l. (metsim), and e.i. (ulsam) were the cohort principal investigators. g.a. w. is the guarantor of this work and, as such, had full access to the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. novel insulin sensitivity loci diabetes volume , october references . morris ap, voight bf, teslovich tm, et al.; wellcome trust case control con- sortium; meta-analyses of glucose and insulin-related traits consortium (magic) in- vestigators; genetic investigation of anthropometric traits (giant) consortium; asian genetic epidemiology network–type diabetes (agen-t d) consor- tium; south asian type diabetes (sat d) consortium; diabetes genetics replication and meta-analysis (diagram) consortium. large-scale association analysis provides insights into the genetic architecture and pathophysiology of type diabetes. nat genet ; : – . kahn se. the relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of type diabetes. 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insulin sensitivity and insulin release in relation to glycemia and glucose tolerance in , finnish men. diabetes ; : – diabetes.diabetesjournals.org walford and associates doi: . /j.bpj. . . a monday, march , provide valuable insights into its mode of action. the molecular framework of dfbp resembles that of levosimendan, thus it was chosen to mimic levosimen- dan to establish how the ctnc-ctni binding equilibrium is modulated. we have utilized d { h, n} hsqc and d { h, c} hsqc nmr spectroscopy to examine the binding of dfbp to cntnc�ca þ in the absence and presence of ctni - and of ctni - to cntnc�ca þ in the absence and presence of dfbp. the results show that dfbp and ctni - bind cntnc�ca þ concurrently and the affinity of dfbp for cntnc�ca þ is increased ~ - - fold by ctni - . we are in the process of determining the nmr solution structure of cntnc�ca þ�ctni - �dfbp. this structure will contribute to the understanding of the mechanism of action of levosimendan in the therapy of heart disease. it will also provide a structural basis for the design of ca þ- sensitizing drugs in general. -pos board b decreased fatigue tolerance in diaphragm muscle of slow troponin t knockdown mice hanzhong feng, bin wei, j.-p. jin. northshore university healthsystem and northwestern university feinberg school of medicine, evanston, il, usa. the loss of slow skeletal muscle troponin t (tnt) results in a severe type of nemaline myopathy in the amish (anm). the genes encoding tnt and tropo- nin i (tni) are closely linked in pairs in which the -enhancer region of the slow tnt gene overlaps with the cardiac tni gene. in a mouse line with the en- tire cardiac tni gene deleted, a partial destruction of the slow tnt gene pro- moter produces a knockdown effect. by crossing with transgenic mouse lines that over-express a core structure of cardiac tni (ctni-nd) under the control of cloned alpha-mhc promoter, we rescued the postnatal lethality of the car- diac tni gene-deleted mice with no detrimental cardiac phenotypes or leaking expression in non-cardiac tissues. the double transgenic mice exhibited de- creased expression of slow tnt mrna and protein in adult diaphragm muscle. functional analysis of isolated muscle strips showed that the slow tnt deficient (stnt-kd) diaphragm had significantly decreased fatigue tolerance evident by the faster decrease in force and slower rate of recovery as compared with that in wild type controls. as a consequence of slow tnt deficiency, the stnt-kd di- aphragm muscle contained a higher proportion of fast tnt, decreased slow tni with increased fast tni, and decreased type i myosin with increased type ii my- osin. consistent with the switch toward fast myofilament contents, the stnt- kd diaphragm muscle produced higher specific tension in twitch and tetanic contractions as well as shorter time to develop peak tension in twitch contrac- tions. the decreased fatigue tolerance of stnt-kd diaphragm muscle explains the terminal respiratory failure seen in virtually all anm patients and this dou- ble transgenic mouse model provides a useful experimental system to study the pathogenesis and treatment of anm. -pos board b troponin isoforms and stretch-activation of insect flight muscle uros krzic , gian de nicola , vladimir rybin , annalisa pastore , kevin leonard , wolfgang linke , belinda bullard . embl, heidelberg, germany, nimr, london, united kingdom, ebi, cambridge, united kingdom, university of muenster, muenster, germany, department of biology, university of york, united kingdom. oscillatory contraction of insect indirect flight muscle (ifm) is activated by si- nusoidal length changes. work done by oscillating fibres is measured from the area of loops on a length-tension plot. at [ca þ] above mm, progressively less oscillatory work is produced because fibres contract isometrically and are unable to relax fully after each cycle of oscillation. periodic stretches during oscillations activate fibres through the action of tnc f , which binds one ca þ in the c-lobe. activation of isometric contraction by ca þ acts through f , which binds ca þ in both n- and c-lobes. lethocerus ifm fibres substituted with f gave oscillatory work, which did not decline at high [ca þ], while fibres substituted with f produced more isometric tension as [ca þ] was increased. varying proportions of f and f gave maximal work with an f :f ratio of : , which is higher than the in vivo ratio of : . the structure of f , and the interaction with tni, were determined by nmr. the n-lobe of f is in the closed conformation in apo and ca þ- bound forms and does not bind tni. unexpectedly, the c-lobe is open in both states, and binds the n-terminal domain of tni independently of ca þ. the affinity of f and f for a complex containing tropomyosin, tnt and tnh (lethocerus tni) were measured by isothermal calorimetry in the presence of ca þ. the affinities of f and f for the complex were . mm and nm respectively. this difference is likely to be due to a single tni binding site on f and two sites on f . stretch may be sensed by an extended c-terminal domain of tnh, and transmitted to the c-lobe of f , resulting in a change in the interac- tion of the tni inhibitory domain and actin. -pos board b tracking of qdot conjugated titin antibodies in single myofibril stretch experiments reveals ig-domain unfolding at physiological sarcomere lengths anika grützner , pallav kosuri , julio m. fernandez , wolfgang a. linke . university of münster, münster, germany, columbia university, new york, ny, usa. the mechanical characteristics of titin in muscle sarcomeres were previously studied by us in single myofibril stretch experiments, where the extensibility of i-band titin segments was usually measured under static conditions. here we investigated the behavior of i-band titin during and after stretch of single rabbit psoas myofibrils in real-time. the focus was on titin’s proximal ig-do- main region, whose stretch dynamics were analyzed by labeling the myofibrils specifically in the n a-titin domain using antibody-conjugated quantum dots, which stained the periphery of the myofibril but did not enter the myofilament lattice. qdot labels were tracked to obtain the stretch-dependent change in epi- tope distance (across z-disc) and sarcomere length (sl) over time. in contrast to what was expected from the current titin extensibility model, at sarcomere lengths of . and . mm, titin’s proximal ig-domain region elongated contin- uously, in proportion to the half i-band length. already at ~ . mm sl the prox- imal ig-segment length exceeded the value expected if all ig-domains remain folded. our results suggest that ig-domains unfold in parallel with pevk-titin extension at physiological sarcomere lengths and under relatively low forces. by reducing the antibody-qdot concentration, we succeeded in observing titin ig-domain dynamics in myofibrils at the single-molecule level. -pos board b constitutive phosphorylation of cardiac myosin binding protein-c increases the probability of myosin cross-bridge interaction with actin brett a. colson , tanya bekyarova , matthew r. locher , carl w. tong , daniel p. fitzsimons , patricia a. powers , thomas c. irving , richard l. moss . university of wisconsin medical school, madison, wi, usa, illinois institute of technology, chicago, il, usa. protein kinase a-mediated (pka) phosphorylation of cardiac myosin binding protein-c (cmybp-c) accelerates the kinetics of cross-bridge cycling and ap- pears to relieve the tether-like constraint of myosin heads imposed by cmybp-c (colson et al., , circ res., : - ). we favor a mechanism in which phosphorylation of the pka sites in cmybp-c modulates cross- bridge kinetics by regulating the proximity and interaction of myosin with actin. to test this idea, we used synchrotron low-angle x-ray diffraction and mechanical measurements in skinned myocardium isolated from a mouse model with phosphomimetic substitutions in cmybp-c, i.e., the ctsd mouse. the substitutions were introduced by transgenic expression of cmybp-c with ser-to-asp mutations on a cmybp-c null background. western blots showed that expression of ctsd cmybp-c was % of wild-type (wt), and the heart weight to body weight ratio was similar ( . . mg/g) in ctsd and wt mice. expression of wt cmybp-c on the knockout background served as con- trol (i.e., the ctwt mouse). skinned myocardium from ctsd and ctwt mice exhibited similar maximum active forces (mn/mm : . . vs . . ), ca þ-sensitivities of force (pca : . . vs . . ), and maximum rates of force development (ktr, sec - : . . vs . . ; kdf, sec - : . . vs . . ). i /i intensity ratios and d lattice spacings determined from equatorial reflections from ctsd and ctwt myocardium were used to determine the effect of constitutive cmybp-c phosphorylation on the distribution of cross-bridge mass between the thick and thin filaments and on interfilament lattice spacing. the results suggest that interactions between cmybp-c and the s domain of myosin heavy chain are dynamically regulated by phosphorylations in the cmybp-c motif. (aha-predoctoral fellowship (bac); nih-hl-r - ) -pos board b obscurin interacts with a novel isoform of myosin binding protein c-slow to regulate the assembly of thick filaments maegen a. borzok, rebecca hu, amber l. bowman, john strong, robert j. bloch, aikaterini kontrogianni-konstantopoulos. university of maryland, baltimore, md, usa. obscurin is a multidomain protein composed of adhesion and signaling do- mains that plays key roles in the organization of contractile and membrane structures in striated muscles. we used adenoviral-mediated gene transfer to overexpress its extreme nh -terminus in developing myofibers, followed by immunofluorescence and ultrastructural methods to study its effects in sarco- merogenesis. we found that overexpression of obscurin’s second immunoglob- ulin domain (ig ) inhibits the assembly of a- and m-bands, but not z-disks and 	decreased fatigue tolerance in diaphragm muscle of slow troponin t knockdown mice 	troponin isoforms and stretch-activation of insect flight muscle 	tracking of qdot conjugated titin antibodies in single myofibril stretch experiments reveals ig-domain unfolding at physiological sarcomere lengths 	constitutive phosphorylation of cardiac myosin binding protein-c increases the probability of myosin cross-bridge interaction with actin 	obscurin interacts with a novel isoform of myosin binding protein c-slow to regulate the assembly of thick filaments intrahepatic cholestasis in two omani siblings associated with a novel homozygous atp b mutation, c. c>g (p.l v) | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /sjg.sjg_ _ corpus id: intrahepatic cholestasis in two omani siblings associated with a novel homozygous atp b mutation, c. c>g (p.l v) @article{narchi intrahepaticci, title={intrahepatic cholestasis in two omani siblings associated with a novel homozygous atp b mutation, c. c>g (p.l v)}, author={h. narchi and suhailah alhefeiti and fatmah althabahi and j. hertecant and a. knisely and a. souid}, journal={saudi journal of gastroenterology : official journal of the saudi gastroenterology association}, year={ }, volume={ }, pages={ - } } 	h. narchi, suhailah alhefeiti, + authors a. souid 	published 	medicine 	saudi journal of gastroenterology : official journal of the saudi gastroenterology association we report two omani brothers with intrahepatic cholestasis that resolved with supportive care. in one, cholestasis began in infancy; in the other, only at the age of months. whole exome sequencing identified a novel homozygous variant, c. c>g (p.l v) in atp b . those attending patients with cholestasis from the arabian peninsula should be aware of this mutation and of the variation in its phenotypic effects.  view on wolters kluwer doi.org save to library create alert cite launch research feed share this paper citationsbackground citations view all figures and topics from this paper 	 figure 	intrahepatic cholestasis 	homozygote 	whole exome sequencing citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency disease mutation study identifies essential residues for phosphatidylserine flippase atp a 	kuanxiang sun, w. tian, wenjing liu, yeming yang, xianjun zhu 	chemistry, biology 	 	 	view excerpts, cites background save alert research feed disease mutation study identifies critical residues for phosphatidylserine flippase atp a 	kuanxiang sun, w. tian, x. li, wenjing liu, y. yang, xianjun zhu 	chemistry, medicine 	biomed research international 	 	pdf 	view excerpts, cites background save alert research feed references showing - of references a novel truncation mutation in atp b gene in progressive familial intrahepatic cholestasis. 	a. sharma, u. poddar, shikha agnihotry, r. aggarwal 	medicine 	indian pediatrics 	 	 	pdf save alert research feed characterization of atp b gene mutations and a hot-linked mutation found in chinese children with progressive intrahepatic cholestasis and low ggt 	l. liu, x. wang, z. wang, q. zhu, j. wang 	medicine 	journal of pediatric gastroenterology and nutrition 	 	 save alert research feed progressive familial intrahepatic cholestasis. genetic basis and treatment. 	e. jacquemin 	medicine 	clinics in liver disease 	 	 	 save alert research feed progressive familial intrahepatic cholestasis. 	g. m. cavestro, l. frulloni, + authors f. di mario 	medicine 	acta bio-medica : atenei parmensis 	 	 	pdf save alert research feed characterization of mutations in atp b associated with hereditary cholestasis 	l. klomp, j. c. vargas, + authors l. bull 	biology, medicine 	hepatology 	 	 	 	view excerpts, references background save alert research feed fic disease: a spectrum of intrahepatic cholestatic disorders. 	s. v. van mil, l. klomp, l. bull, r. houwen 	biology, medicine 	seminars in liver disease 	 	 	view excerpt, references background save alert research feed benign recurrent intrahepatic cholestasis progressing to progressive familial intrahepatic cholestasis: low ggt cholestasis is a clinical continuum. 	n. a. v. van ooteghem, l. klomp, g. p. van berge-henegouwen, r. houwen 	medicine 	journal of hepatology 	 	 	view excerpt, references background save alert research feed familial cholestasis: progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy. 	wendy l van der woerd, s. v. van mil, j. stapelbroek, l. klomp, s. v. d. van de graaf, r. houwen 	medicine 	best practice & research. clinical gastroenterology 	 	 	view excerpt, references background save alert research feed atp b deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport 	coen c. paulusma, a. groen, + authors r. oude elferink 	biology, medicine 	hepatology 	 	 	 	view excerpts, references background save alert research feed genetic cholestatic liver diseases: the example of progressive familial intrahepatic cholestasis and related disorders. 	c. baussan, d. cresteil, + authors e. jacquemin 	medicine 	acta gastro-enterologica belgica 	 	 save alert research feed related papers 	abstract 	figures and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue cellular fate of truncated slow skeletal muscle troponin t produced by glu nonsense mutation in amish nemaline myopathy* cellular fate of truncated slow skeletal muscle troponin t produced by glu nonsense mutation in amish nemaline myopathy* received for publication, december , , and in revised form, january , published, jbc papers in press, january , , doi . /jbc.m xin wang‡§, qi-quan huang§, mark t. breckenridge§, aihua chen§, thomas o. crawford¶, d. holmes morton�, and jian-ping jin‡§** from the ‡section of molecular cardiology, evanston northwestern healthcare and northwestern university feinberg school of medicine, evanston, illinois , the §department of physiology and biophysics, case western reserve university, cleveland, ohio , the ¶departments of neurology and pediatrics, johns hopkins university, baltimore, maryland , and the �clinic for special children, strasburg, pennsylvania a nonsense mutation at codon glu in exon of slow skeletal muscle troponin t (tnt) gene (tnnt ) causes an autosomal-recessive inherited nemaline my- opathy. we previously reported the absence of intact or prematurely terminated slow tnt polypeptide in amish nemaline myopathy (anm) patient muscle. the present study further investigates the expression and fate of mutant slow tnt in muscle cells. intact slow tnt mrna was readily detected in patient muscle, indicating unaf- fected transcription and rna splicing. sequence of the cloned cdnas revealed the single nucleotide mutation in two alternatively spliced isoforms of slow tnt mrna. mutant tnnt cdna is translationally active in esche- richia coli and non-muscle eukaryotic cells, producing the expected truncated slow tnt protein. the mutant mrna was expressed at significant levels in differenti- ated c c myotubes, but unlike intact exogenous tnt, truncated slow tnt protein was not detected. transfec- tive expression in undifferentiated myoblasts produced slow tnt mrna but not a detectable amount of trun- cated or intact slow tnt proteins, indicating a muscle cell-specific proteolysis of tnt when it is not integrated into myofilaments. the slow tnt-( – ) fragment has substantially lower affinity for binding to tropomyosin, in keeping with the loss of one of two tropomyosin- binding sites. our findings suggest that inefficient in- corporation into myofilament is responsible for the in- stability of mutant slow tnt in anm muscle. rapid degradation of the truncated slow tnt protein, rather than instability of the nonsense mrna, provides the protective mechanism against the potential dominant negative effect of the mutant tnt fragment. striated muscle contraction is initiated by a series of ca �- induced allosteric changes in troponin and tropomyosin. the conformational changes in these thin filament regulatory pro- teins allow the interaction of myosin in the thick filament to form a strong cross-bridge with f-actin, activate myosin atpase, and generate contractile force ( ). troponin is a com- plex of three component proteins: tnc (the ca �-binding sub- unit), tni (the inhibitory subunit), and tnt (the tropomyosin- binding subunit) ( – ). in anchoring the troponin complex to tropomyosin and actin, tnt occupies an organizer position and role for this ca � signaling machinery ( , ). higher vertebrates have evolved three different types of muscle, and three homologous tnt genes exist that encode isoforms of tnt specific to each type: slow skeletal muscle tnt (tnnt ), fast skeletal muscle tnt (tnnt ), and cardiac tnt (tnnt ) ( – ). in adult animals, these tnt isoforms are spe- cifically expressed within their respective muscle fiber types. whereas the three tnt isoforms have diverged in structure, each shows evolutionary conservation across the vertebrate phylum ( ), suggesting that each isoform possesses a differ- entiated functional role. a nonsense mutation at codon glu in exon of slow skeletal muscle tnt gene tnnt is found to cause an autoso- mal-recessive inherited form of nemaline myopathy named amish nemaline myopathy (anm) ( ). this is the first skele- tal muscle troponin disease identified. whereas most human skeletal muscles contain mixed fast and slow fibers ( ) and express both fast and slow tnts ( ), the fact that loss of only one isoform of tnt causes lethal myopathy provides the first evidence for the critical role of the functionally diverged fiber type-specific tnt isoforms. this mutation is predicted to pro- duce a truncated slow tnt protein (slow tnt-( – )), but muscle of anm patients showed no intact or truncated slow tnt ( ). this raises a question about the expression of mutant tnnt or fate of transcribed mutant slow tnt gene products in anm muscle cells. imaging and structural studies suggest that tnt adopts an extended conformation ( – ). studies using proteolytically cleaved or genetically engineered tnt fragments have identi- fied two protein-binding domains of tnt. the c-terminal (t ) region binds to the central region of tropomyosin and interacts with tni, tnc, and f-actin ( – ). the central region of tnt has another tropomyosin-binding site that binds to the tropo- myosin head-to-tail overlap junction in the thin filament ( – ). the crystal structure of partial human cardiac troponin complex including the c-terminal domain of tnt agrees with * this work was supported in part by national institutes of health niams grant ar- and nichd grant hd- (to j.-p. j.). the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. the nucleotide sequence(s) reported in this paper has been submitted to the genbanktm/ebi data bank with accession number(s) ay and ay . ** to whom correspondence should be addressed. tel.: - - ; fax: - - ; e-mail: jpjin@northwestern.edu. the abbreviations used are: tnc, troponin c; tni, troponin i; tnt, troponin t; anm, amish nemaline myopathy; cmv, cytomegalovirus; fbs, fetal bovine serum; mab, monoclonal antibody; mhc, myosin heavy chain; rt, reverse transcription; dmem, dulbecco’s modified eagle’s medium. the journal of biological chemistry vol. , no. , issue of april , pp. – , © by the american society for biochemistry and molecular biology, inc. printed in u.s.a. this paper is available on line at http://www.jbc.org this is an open access article under the cc by license. http://creativecommons.org/licenses/by/ . / these protein binding data ( ). the anm nonsense mutation in codon glu of the slow tnt gene results in a deletion of the c-terminal amino acids of the protein, removing the major- ity of the t domain that contains the tnc, tni, and c-termi- nal tropomyosin-binding sites. nonetheless, the truncated slow tnt-( – ) fragment retains the central tropomyosin-binding site that participates in the anchoring of troponin complex to the thin filament ( ). despite encoding a potentially deleterious functional struc- ture, a single copy of the mutant slow tnt allele does not appear to affect anm carriers ( ). this is in marked contrast to a human cardiac tnt truncation mutation, deleting the c-terminal amino acids, which causes a dominantly inher- ited familial hypertrophic cardiomyopathy ( ). in addition to increasing our understanding of the molecular pathology of anm, investigation of the expression and fate of mutant slow tnt may also provide information about the protective mech- anism that effectively eliminates mutant slow tnt from anm muscle cells. in the present study, we found that the mutant slow tnt mrna was readily detected in patient muscle, indicating un- affected transcription and rna processing. sequence of the cloned cdna reveals the single nucleotide mutation in two alternatively spliced slow tnt isoform mrnas. the mutant cdna can be expressed in escherichia coli and non-muscle eukaryotic cells to produce truncated slow tnt protein at high levels. transfective expression of the mutant slow tnt cdna in c c myotubes produces significant amounts of the mutant mrna, but no detectable truncated slow tnt. furthermore, transfective expression in undifferentiated myoblasts do not produce a detectable amount of truncated or intact slow tnt proteins, indicating a muscle cell-specific proteolysis of tnt when it is not integrated into myofilaments. these results demonstrate instability of slow tnt-( – ) specifically within muscle cells, suggesting that the loss of tni, tnc, and one tropomyosin-binding site restricts incorporation of mutant slow tnt into the myofilament, which in turn leads to accelerated degradation. therefore, rapid degradation of tnt-( – ) pro- tein, rather than loss of the nonsense mrna, is responsible for the absence of a dominant effect of this mutation. experimental procedures cloning of mutant human slow tnt cdna from anm patient mus- cle—a frozen diagnostic muscle biopsy obtained from the quadriceps muscle of a -week-old anm patient was used for rna isolation. this investigation was determined to be exempted research under section iv c criteria by the johns hopkins hospital institutional review board. this patient was previously confirmed for homozygous mutation at codon glu in the slow tnt gene ( ) and the muscle sample showed no detectable intact or truncated slow tnt protein ( ). as described previously ( ), total rna was isolated from � mg of muscle tissue using the trizol reagent (invitrogen) according to the manufacturer’s protocol. two �g of the muscle rna was used to syn- thesize cdna by reverse transcription (rt) using an oligonucleotide primer ( �-t v- �, v � a, c, or g) complementary to the poly(a) tail of mrna in an anchored manner. from the cdna mixture copied from all poly(a)� mrna, cdna encoding slow skeletal muscle tnt was ampli- fied by pcr using oligonucleotide primers synthesized according to the human slow tnt gene sequence (genbanktm accession number nt_ . , position . in the chromosome genomic contig). the forward primer sequence corresponds to a part of exon flanking the translation initiation codon plus an ndei cloning site and the reverse primer sequence corresponds to a part of exon flanking the translation termination codon with an ecori cloning site incorporated. the pcr products were verified by a secondary pcr using a slow tnt-specific internal reverse primer paired with the exon forward primer, cut at the ndei and ecori sites built at the ends of the cdna, and cloned into topo pcr vector (invitrogen). after trans- formation of host e. coli cells, colonies containing the recombinant plasmid were identified by pcr using slow tnt-specific primers and the plasmid dna was purified for sequencing the cdna insert by the dideoxy chain termination method at a service facility. mutagenesis in mouse slow tnt cdna—we have previously cloned full-length mouse cdna encoding the alternatively spliced high and low molecular weight slow tnt isoforms ( ). using the cdnas cloned in paed plasmid ( ) as template, recombinant pcr was used to create a stop codon at glu . a pair of complementary mutagenesis oligonu- cleotide primers was synthesized to reproduce the g to t mutation found in anm patients ( ). the primer sequences and the mutagenesis strategy are shown in fig. . an ecori restriction endonuclease cutting site was constructed in the primer sequences downstream of the mutant stop codon to introduce a unique genotyping marker for the transgene product. the mutagenesis primers were used together with the t and pet reverse primers flanking the �- and �-ends of the mouse slow tnt cdna in the vector sequence to construct the mutant cdna by a three-step recombinant pcr procedure. the reconstituted full-length slow tnt cdna containing the point mutation and ecori site was digested with restriction enzymes xbai and xhoi and ligated into com- patibly cut paed vector dna. after transformation of jm e. coli cells, colonies containing the recombinant plasmids were identified by pcr using t primer in the �-flanking region of the vector sequence and a reverse primer specific to the mouse slow tnt cdna insert. the pcr-positive cdna clones were further screened by mini-scale expression in e. coli. briefly, the transformed jm e. coli colonies were expanded as . � – -cm smear cultures on lb/ampicillin agar plates (� smears per -mm plate). approximately one-third of each smear was transferred using a toothpick to a . -ml microcentrifuge tube containing �l of : phenol:chloroform and �l of te buffer ( mm tris-hcl, ph . , and mm edta). the tubes were touch spun to get the bacteria into the chemical mixture and then vortexed for s. the tubes were then spun in a microcentrifuge at top speed at room tem- perature for min. one �l of the aqueous layer that contains nucleic acid extracted from the bacteria was used to transform bl (de )plyss e. coli. cells from to transformations can be plated in separate areas on an -mm lb/ampicillin/chloromphenicol plate for culture at °c overnight. from each transformation, multiple bacterial colonies were collected to start -ml cultures in lb/ampicillin/chloromphenicol media containing . mm isopropyl -thio-�-d-galactopyranoside. after being cultured at °c with shaking for h, the induced bacteria were collected by centrifugation, lysed in sds gel sample buffer, and used for sds- fig. . construction of mutant mouse slow tnt cdna and expression vectors. a forward and a complementary reverse mu- tagenesis primers (primers and ) were synthesized containing the g to t point mutation in codon and a downstream ecori site to generate a unique marker for the mutant cdna. three-step recombi- nant pcr mutagenesis was carried out on mouse slow tnt cdna templates previously cloned in paed plasmid. the two dna frag- ments produced by the first step pcr using primers and paired with pet reverse and t primers, respectively, were annealed to form full length mutant cdna that was further amplified by pcr using t and pet reverse primers for cloning into paed vector. the predicted rt-pcr products from the mutant and wild type slow tnt mrna using exon forward and exon reverse primers are outlined. the size of the dna fragments represents that of the low molecular weight isoform lacking the -bp encoded by exon (fig. b). truncated slow troponin t in nemaline myopathy page and western blot analysis as described below. after confirming expression of the anticipated slow tnt fragment, the recombinant plas- mid dna was purified from the original jm cultures and the slow tnt cdna insert was sequenced as above to verify the mutagenesis as well as authenticity. sds-page and western blotting—muscle samples or bacterial ex- tracts were homogenized in sds-page sample buffer containing % sds. after being heated at °c for min and clarified by spinning in a microcentrifuge at top speed and room temperature for min, the total protein extracts were resolved by polyacrylamide gel using the laemmli buffer system. the resulting gels were stained with coomas- sie blue r- to reveal the resolved protein bands. duplicate gels were electrically transferred to nitrocellulose membranes as previously de- scribed ( ). after blocking in tris-buffered saline containing % bovine serum albumin, the nitrocellulose membranes were incubated with (a) monoclonal antibody (mab) ct recognizing slow and cardiac tnt ( ), (b) mab t against fast tnt (ref. , a gift from dr. jim lin, univer- sity of iowa), (c) rabbit polyclonal antibody raised against fast tnt, or (d) mab tni- against tni ( ). the membranes were then washed with high stringency using tris-buffered saline containing . % triton x- and . % sds, incubated with alkaline phosphatase-labeled anti- mouse or anti-rabbit igg second antibody (sigma), and washed again. the blots were developed in -bromo- -chloro- -indolylphosphate/nitro blue tetrazolium substrate solution as previously described ( ) to reveal tnt or tni bands. expression and purification of truncated human slow tnt—cdna encoding mutant human slow tnt was subcloned into the paed prokaryotic expression vector as described for the mutant mouse slow tnt cdnas. by transformation of bl (de )plyss e. coli cells, large scale protein expression was carried out in � ty media containing mg/liter ampicillin and . mg/liter chloroamphenicol with vigorous shaking at °c. when a nm reached � . , the cultures were in- duced by . mm isopropyl -thio-�-d-galactopyranoside and further incubated for – h. the induced bacterial cells were collected by centrifugation and lysed in mm tris-hcl, ph . , containing mm edta, mm �-mercaptoethanol by passing a french cell press at , p.s.i. three times. the cell lysis was fractionated by ammonium sulfate precipitation and the fraction between and % saturation at °c was dialyzed against . mm edta containing mm �-mercapto- ethanol. following dialysis, the fraction was brought to m urea, . mm edta, mm �-mercaptoethanol, mm tris-hcl, ph . , clarified by centrifugation, and chromatographed on a de- cellulose anion-ex- change column equilibrated in the same buffer. the column was eluted with a linear kcl gradient ( – mm) in the same buffer and the protein peaks were analyzed by sds-page. the fractions containing human slow tnt-( – ) fragment were dialyzed against . mm edta and concentrated by lyophilization. the truncated slow tnt was fur- ther purified by sephadex g- gel filtration chromatography in m urea, . m kcl, and mm imidazole-hcl, ph . , as described previ- ously ( ). both high and low molecular weight variants of truncated human slow tnt were purified by this procedure. construction of eukaryotic expression vectors—the mutant mouse slow tnt cdna was isolated as an ndei/xhoi fragment and subcloned into bamhi/xhoi cut pcdna . (�) eukaryotic expression vector (in- vitrogen) downstream of the cytomegalovirus (cmv) promoter for ex- pression in non-muscle and undifferentiated muscle cells. recombinant plasmids with slow tnt cdna inserted in the sense orientation were selected by pcr using a cmv promoter-specific forward primer and a slow tnt cdna-specific reverse primer. the positive plasmids were verified by restriction endonuclease mapping. to construct an expression vector specific to differentiated muscle cells, the mutant mouse slow tnt cdnas were inserted into the xhoi site of a promoter-less pcdna . (�) plasmid upstream of the sv intron sequence. the cmv promoter in the pcdna . (�) had been deleted by nhei and nrui digestion and self-ligation. a . -kb mouse genomic dna fragment containing the �-myosin heavy chain (�-mhc) promoter ( ) (a gift from dr. jeffrey robbins, university of cincinnati) was then inserted into the noti site upstream of the mutant mouse slow tnt cdna. the rational for using �-mhc promoter that is active in differentiated c c myocytes ( ) for the expression of exogenous slow tnt cdna is to allow the transgene to be independent of the endoge- nous slow tnt gene regulation. the recombinant plasmids were screened by pcr and verified by restriction enzyme mapping. transfection of monolayer cell cultures—for use in cell transfection experiments, the recombinant pcdna . (�) plasmid dna was pre- pared in large quantities from transformed jm e. coli using the qia-well cartridge (qiagen) following the manufacturer’s instructions. transfection of human kidney epithelial cell line (atcc crl- ), mouse fibroblast cell line nih t (atcc crl- ), and mouse skeletal myoblast cell line c c (atcc crl , ref. ) was carried out using the lipofectaminetm transfection reagent (invitrogen) follow- ing the manufacturer’s instructions. two to five �g of the recombinant supercoil plasmid dna in – �l of te buffer was mixed with – �l of lipofectamine in dmem and incubated at room temperature for min. the lipofectamine-dna complex was gently mixed with ml of dmem without fetal bovine serum (fbs) and added to monolayer cell cultures at – % confluent after removing the old medium. after incubation at °c in % co for h, ml dmem containing % fbs was added and the culture was continued for h before change to fresh culture media containing % fbs. for transient transfective expres- sions, the cells were suspended using the versene solution ( . mm edta, . mm nacl, . mm kcl, . mm na hpo , . mm kh po , ph . ) ( ) and washed three times with phosphate-buffered saline, ph . , for the examination of slow tnt mrna and protein expression h after transfection. the elimination of trypsin digestion from the collection of cells for sds gel and western analysis avoided artificial enzymatic degradation of the cellular proteins. to establish stable transfection of c c cells, the cells transfected with the g resistant recombinant pcdna . plasmid were cultured in dmem containing % fbs plus g ( �g/ml, icn biomedical, inc.). results from testing the tolerance of non-transfected c c cells to g showed that this cell line is sensitive to g . in culture media containing �g/ml g , all cells died after days of culture. the c c cell colonies resistant to g were individually picked up from the culture dish by trypsin digestion in small cylinders greased to the dish. the cells were expanded for extracting dna to verify the trans- fection by pcr as described above. the c c cell lines stably trans- fected with the mutant slow tnt cdna were expanded and stored in dmem containing % fbs and % me so in liquid nitrogen for later characterization. in vitro differentiation of c c myocytes—c c is an immortalized myoblast cell line derived from adult mouse skeletal muscle ( ). the cells were purchased from american type culture collections and cultured in dmem containing % fbs, penicillin ( �g/ml), and streptomycin ( �g/ml) at °c in % co . the untransfected or transfected c c cells were placed for in vitro differentiation in dmem as above with % horse serum instead of fbs. the expression of tnt isoforms was examined by western blots in both undifferentiated c c myoblasts and differentiated myotubes. as described above, the cells in the culture dish were suspended using the versene solution and washed three times with phosphate-buffered saline. sds gel sample buffer was added to dissolve the cells and to extract total cellular protein by vortexing. after heating and centrifugation of the samples, sds-page and western blotting were carried out as above. restriction endonuclease analysis to identify and quantify the mu- tant slow tnt mrna expressed in c c cells—as described above, total rna was extracted from the stably transfected c c cells using the trizol reagent and rt-pcr was carried out to amplify the entire coding region of mouse slow tnt mrna (fig. ). to obtain a quantita- tive comparison between the endogenous and exogenous slow tnt cdnas, the pcr program was designed based on preliminary experi- ments for a pre-saturated level of amplification (data not shown). the total pcr product containing both endogenous and exogenous slow tnt cdnas were digested with ecori at the site built in the exogenous cdna but absent in the endogenous cdna (fig. ). agarose gel electro- phoresis was carried out to determine the restriction pattern, and densitometry analysis of the ethidium bromide-stained bands was used to quantify the relative levels of endogenous and exogenous slow tnt mrna. the original rna extract equivalent to -fold of the amount used for the rt-pcr was used in control pcr to avoid samples with any cellular dna contamination. expression of intact exogenous tnt in c c cells—we have previ- ously cloned and characterized a . -kb mouse genomic dna containing the slow skeletal muscle tnt gene promoter ( ). to evaluate the compatibility of c c cellular environment in expressing exogenous tnt cdna, we used the mouse slow tnt promoter to direct an expres- sion of chicken fast skeletal muscle tnt in differentiated c c myo- tubes. a cdna encoding chicken breast muscle tnt ( ) was con- structed downstream of the mouse slow tnt promoter using the pcdna . backbone as described above. stably transfected cell lines were established and in vitro differentiation was carried out as above. expression of chicken fast tnt in differentiated c c myotubes was examined by western blotting using a polyclonal antibody, ratnt, raised against chicken breast muscle tnt ( ) and an mab, b , spe- cific to the chicken tnt isoform expressed ( ) together with mab ct control of endogenous cardiac and slow tnt expression. truncated slow troponin t in nemaline myopathy protein binding assay—to investigate the binding affinity of the anm truncated slow tnt to tropomyosin, enzyme-linked immunosor- bent assay solid phase protein binding experiments ( ) were applied to compare binding of the truncated and intact slow tnt to �-tropomyosin. intact mouse slow skeletal muscle tnt was expressed from cloned cdna and purified as described previously ( ). �-tropomyosin was purified from rabbit heart as described previously ( ). intact slow tnt, high and low molecular weight truncated slow tnt proteins, were dissolved individually at �g/ml in buffer a ( mm kcl, mm mgcl , mm pipes, ph . ) and coated onto triplicate wells of microtiter plates by incubation at °c overnight. after washes with buffer a plus . % tween (buffer t) to remove the unbound tnt, the plates were blocked with buffer t containing . % bovine serum albumin (buffer b). the plates were then incubated with serial dilutions of rabbit �-tropomyosin in buffer b at room temperature for h. after washes with buffer t, the bound tropomyosin was quantified via an anti- tropomyosin mab ch ( ), a gift from dr. jim lin, university of iowa, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin second antibody (sigma), and h o / , �-azinobis-( -ethylbenzthiazo- linesulfonic acid) substrate reaction using the enzyme-linked immu- nosorbent assay procedure described previously ( ). the enzyme- linked immunosorbent assay results were recorded by an automated microplate reader (bio-rad benchmark) and the a nm values from the linear range of color development were used to plot the tnt-tropo- myosin binding curves. bovine serum albumin-coated wells were used to produce a negative control curve. data analysis—dna and protein sequence analysis was carried out using the dna star computer programs. densitometry of sds-page gels and western blots was performed on digital images scanned at d.p.i. and analyzed using nih image program version . . statis- tical analysis was carried out by student’s t test. results the mutant slow tnt mrna is present at significant amounts in anm patient muscle—because the limited quan- tity of diagnostic biopsy muscle samples precludes the use of northern blot to detect and quantify the amount of slow tnt mrna, we applied rt-pcr to examine the mrna with the opportunity to clone cdna from the mutant mrna. slow tnt cdna was obtained from patient rna on the first trial of rt-pcr using standard conditions. the easy detection of slow tnt mrna suggests that the slow tnt mrna was present at significant amounts in the muscle of the homozygous anm patient. expression of cdna clones in e. coli yields protein products with the predicted sizes that are recognized by the anti-slow tnt mab ct but not the anti-fast tnt mab t (fig. ). the results verified the authenticity of the cloned cdna and demonstrate that the ct epitope is preserved in the truncated slow tnt, confirming that the negative ct western blots of anm patient muscle biopsy samples indicates the absence of truncated slow tnt ( ). sequences of the anm slow tnt cdnas showed for the first time at the mrna level the presence of the g to t mutation, at nucleotide from the translation initiation codon, which converted codon glu into a stop codon (fig. a). the two normally occurring alternatively spliced variants of slow tnt mrna are both present in the anm muscle—like the cardiac and fast skeletal muscle tnt genes, the slow skel- etal muscle tnt gene contains alternatively spliced exons in the �-variable region ( ). alternative splicing of exon pro- duces a high molecular weight and a low molecular weight isoform of human slow tnt ( , ). sequences of multiple original slow tnt cdnas cloned by rt-pcr from the anm muscle revealed two alternative splicing variants including or excluding exon (fig. b). both variants were cloned at sig- nificant frequencies. the results indicate that the glu non- sense mutation in exon did not affect the alternative splicing of exon in the �-region. sequences of the alternatively spliced high and low molecular weight anm slow tnt mutants have been submitted to the genbanktm/ebi data bank with acces- sion numbers ay and ay , respectively. the mutant slow tnt mrna can be effectively translated in e. coli—to investigate whether the mutant slow tnt mrna is translatable, we first examined its capacity as a translation template in e. coli. the sds gel and western blots in fig. a show that the high and low molecular weight variants of the mutant human slow tnt and the engineered mouse counter- parts were expressed in e. coli at significant levels. the appar- ent molecular weight of the human and mouse slow tnt frag- ments produced in e. coli as reflected by sds gel mobility is in agreement with that predicted from the cdna sequences (ta- ble i). both the high and low molecular weight protein products were confirmed as slow tnt by their recognition by mab ct (fig. a). the successful expression of truncated slow tnt demonstrates that the mutant mrna is normally translatable until the nonsense mutation-generated stop codon (fig. a). the effective expression of mutant cdna in e. coli produced sufficient truncated slow tnt for functional analysis. fig. b demonstrates the purity of the high and low molecular weight variants of the truncated human slow tnt produced. the high and low molecular weight slow tnt fragments behaved simi- larly in all steps of the purification procedure, consistent with their similar physical properties as predicted from their amino acid composition (table i). nonetheless, the high molecular weight slow tnt fragment was eluted from the de anion exchange column at a slightly higher kcl concentration than that for the low molecular weight variant, in agreement with their charge difference because of the additional acidic residues encoded by the exon sequence (fig. b, table i). expression of endogenous tnt in c c cells—we used c c mouse myocytes to examine the expression of mutant fig. . cloning of mutant tnnt cdna from anm muscle. a, the coding region of slow tnt cdna was cloned from anm muscle mrna by rt-pcr using the exon forward and exon reverse primers and subcloned into paed vector for expression in e. coli. b, western blots of total protein extracts from transformed e. coli cultures and control human skeletal muscle using mab ct against slow tnt and mab t against fast tnt verified authenticity of the anm slow tnt cdna. the results also demonstrate that both the high (h) and low (l) molecular weight anm slow tnt fragments retain the ct epitope. truncated slow troponin t in nemaline myopathy slow tnt mrna and truncated protein in skeletal muscle cells. although the c c cell line has been widely used in muscle studies and is known to form differentiated myotubes in cul- ture with abundant myofilaments and muscle-specific protein expression ( , ), the expression of tnt isoforms in c c cells has not been characterized. the western blots in fig. demonstrate that very little tnt was expressed in confluent c c myoblasts before switching to % horse serum media. with differentiation, c c myocyte-myotubes express cardiac, slow, and fast tnt isoforms. cardiac tnt is expressed earliest during in vitro differentiation and slow tnt expression follows. fast tnt expression reaches its highest level after longer du- ration in the differentiation media at a time when the expres- sion levels of cardiac and slow tnt decline. the early expres- sion of cardiac tnt during c c cell differentiation mimics developmental regulation in which cardiac tnt is expressed in embryonic skeletal muscle ( , ). a similar pattern is seen in the transition of tni isoforms (fig. ). the expression of endog- enous slow tnt in c c cells justifies its use as an experimen- tal system to study the expression and fate of the mutant slow tnt mrna and protein. multiple alternatively spliced fast tnt protein bands were detected at various levels in differentiated c c cells. how- ever, only adult cardiac tnt and high molecular weight slow tnt were detected throughout the course of in vitro differenti- ation (fig. ). in addition to reflecting the adult muscle origin of the c c cell line ( ), this observation suggests that alter- native splicing of the transcripts of the three homologous tnt genes is differentially regulated in c c cells. whereas the regulatory mechanism remains to be investigated, this feature does not preclude the use of the c c cell line as a muscle cellular environment to study the fate of mutant slow tnt fig. . glu nonsense mutation in two alternatively spliced variants of slow tnt mrna cloned from anm muscle. a, exon sequence of slow skel- etal muscle tnt cdna cloned from the mrna of a homozygous anm patient muscle demonstrates the g to t nucleo- tide substitution at position from the translation initiation codon, converting codon glu into a premature stop codon. b, amino acid sequence alignment of the two classes of truncated slow tnt pre- dicted from the anm cdna sequences demonstrates alternative splicing of exon to produce the high and low molecular weight protein variants (table i). fig. . expression of truncated slow tnt in e. coli and purified pro- teins. a, sds-page using a % gel with a acrylamide:bisacrylamide ratio of : and western blots using mab ct dem- onstrated the expression of high (h) and low (l) molecular weight truncated hu- man and mouse slow tnt proteins in e. coli. b, the sds-page gel and ct western blot show the purified high and low molecular weight truncated human slow tnts. truncated slow troponin t in nemaline myopathy mrna and protein, because overexpression of mutant slow tnt cdna does not involve rna splicing. transfective expression of mutant tnnt cdna in c c myotubes produces mutant slow tnt mrna but no detectable truncated protein—we established multiple stably transfected c c cell lines to test the expression of mutant slow tnt cdna. pcr on dna extracted from the cells confirmed the presence of the �-mhc promoter-directed slow tnt transgene (data not shown). rt-pcr using the exon forward and exon reverse primer pair that is common to the wild type and mutant mrnas (fig. ) with non-saturating cycling numbers readily detected slow tnt cdna from total rna extracted from transfected c c cells that had undergone days of differen- tiation in culture. digesting the pcr-amplified slow tnt cdna with ecori produced fragments specific to the exogenous mu- tant slow tnt mrna as well as uncut cdna from endogenous slow tnt mrna (fig. ). the results show that expression levels of mutant mrna can reach – % that of the total slow tnt mrna (exogenous plus endogenous). despite the presence of mutant slow tnt mrna at levels comparable with or higher than that of endogenous slow tnt in the transfected c c cells, no truncated slow tnt protein could be detected in the total protein extracts (fig. a). because the mutant mrna is translatable as shown in e. coli expression (fig. a), the results suggest that no significant amount of truncated slow tnt was accumulated in the myocytes. it is notable that, although truncated slow tnt protein was not found, overexpression of this exogenous mutant slow tnt mrna diminishes the level of endogenous slow tnt. shown in fig. b, an inverted correlation was found between the levels of exogenous slow tnt mrna (relative to total slow tnt mrna) and endogenous slow tnt protein (relative to the level of car- diac tnt that is used as a differentiation state control). this observation suggests that overexpression of slow tnt mrna may have a negative feedback effect on the level of endogenous slow tnt gene expression. expression of exogenous slow tnt mrna in the present study was directed by �-mhc promoter and, therefore, not subject to this feedback regulation. intact exogenous tnt can be expressed in differentiated but not undifferentiated c c cells and anm truncated slow tnt can be produced in non-muscle cells—to verify the ca- pacity of c c cells for expressing exogenous tnt, we estab- lished stable transfective expression of a cdna encoding chicken fast skeletal muscle tnt under control of the cloned mouse slow tnt gene promoter. the western blots in fig. a table i physical properties of the truncated human and mouse slow tnt the molecular weight (mr) and isoelectric point of the slow tnt proteins were calculated from amino acid sequences using the dna star computer program. the data for intact human ( , ) and mouse ( ) slow tnt isoforms were deduced from previously published sequences. molecular weight isoelectric point high mr low mr high mr low mr human intact , . , . . . human truncated , . , . . . mouse intact , . , . . . mouse truncated , . , . . . fig. . expression of tnt and tni isoforms in c c cells dur- ing in vitro differentiation. confluent c c cells cultured in dmem containing % horse serum were harvested on various days of differen- tiation. total cellular protein extracted (shown in the amido black- stained nitrocellulose membrane, as expected, the high molecular weight proteins were transferred less effectively) was examined by western blotting to monitor the expression of tnt isoforms. cardiac and slow tnt expression was examined using mab ct , fast tnt was examined using polyclonal antibody ratnt, and tni was examined using mab tni- . the results show that cardiac tnt was expressed in c c cells during the earlier phase of in vitro differentiation. the expression of slow tnt followed and fast tnt was expressed latest with alternatively spliced isoforms. a similar transition pattern was seen for tni isoforms. fig. . expression of mutant slow tnt mrna in transfected c c cells. the expression of mutant low molecular weight slow tnt isoform mrna in stably transfected c c cell lines after days of in vitro differentiation was examined by rt-pcr and . % agarose gel electrophoresis. the upper panel shows the total slow tnt cdna (endogenous and exogenous) amplified. the endogenous slow tnt expressed in c c cells is mainly the high molecular weight isoform (fig. ). the -bp and -bp high and low molecular weight cdnas ran together in the . % gel. the total cdna was digested with ecori that specifically cleaves the exogenous -bp mutant but not the wild type endogenous cdna (fig. ). the lower panel agarose gel demonstrates that the mutant slow tnt cdna was detected in the transfected cells at various levels as compared with that of endoge- nous wild type cdna. normalized by the fragment sizes, densitome- try analysis was performed to quantify the exogenous versus endog- enous slow tnt mrna in each sample. truncated slow troponin t in nemaline myopathy showed high level expression of chicken tnt in differentiated c c myotubes. the successful expression of avian tnt in mouse myocytes demonstrates the feasibility of expressing in- tact exogenous tnt in differentiated c c cells. to verify that the anm mutant slow tnt mrna can be translated in eukaryotic cells, fig. b shows that transient transfection of the cmv promoter-directed expression con- struct produced significant amounts of the anm truncated slow tnt in epithelial cells and t fibroblasts. these results indicate that the mutant slow tnt mrna is translat- able in eukaryotic cells and the truncated slow tnt protein can be accumulated to a significant level in non-muscle cells. the results in fig. b further show that cmv promoter- directed transient transfection can express intact slow tnt in and t cells but not in undifferentiated pre-confluent c c myoblasts. rt-pcr on dnase-treated rna extracted from the transient transfected c c cells confirmed the ex- pression of mutant slow tnt mrna (data not shown). no fig. . overexpression of mutant slow tnt mrna in c c cells did not produce detectable truncated slow tnt but re- duced the level of endogenous slow tnt. a, western blots using mab ct showed that no truncated slow tnt protein was detectable in the stably transfected c c myotubes despite the significant amounts of mutant slow tnt mrna (fig. b). high (h) and low (l) molecular weight truncated slow tnt proteins expressed in e. coli were used as control. b, the correlation between the level of mutant slow tnt mrna relative to the total slow tnt mrna (fig. ) and the level of endogenous wild type slow tnt protein normalized with cardiac tnt expressed in each transfected c c cell line (panel a) was examined. the result shows an inverted correlation, suggesting an inhibition of endogenous slow tnt expression by overexpression of mutant slow tnt mrna (p � . ). fig. . transfective expression of intact exogenous tnt in c c cells and expression of truncated and intact slow tnt in non-muscle cells. a, two stably transfected c c lines were examined along with controls to demonstrate the expression of chicken breast muscle tnt in differentiated c c cells under the direction of the mouse slow tnt promoter. the western blot using a polyclonal anti- tnt antibody ratnt shows significant amounts of chicken tnt ex- pressed in c c mouse myotubes. the feasibility of expressing intact exogenous tnt in c c myotubes was confirmed by western blot using mab b specific to the chicken tnt isoform. the less intense western staining of b versus that of the polyclonal ratnt reflects the nature of single versus multiple epitope binding. b, ct western blots show that transient transfection of the cmv promoter-directed expression vectors produced significant amounts of the anm truncated slow tnt (slow tnt – ) in human epithelial cells and t mouse fibro- blasts. like that in differentiated c c myotubes (fig. a), transfec- tive expression using cmv promoter failed to produce truncated slow tnt protein in undifferentiated c c myoblasts. parallel transient transfections produced significant levels of intact slow tnt in and t cells but not undifferentiated c c cells, suggesting a muscle cell-specific protein degradation. in contrast to the low level tnt ex- pression in confluent c c cells at the beginning of differentiation (fig. ), undifferentiated c c showed no endogenous tnt. truncated slow troponin t in nemaline myopathy endogenous tnt was detected in undifferentiated c c cells (fig. b), indicating the absence of myofilaments. the results demonstrate a muscle cell-specific rapid degradation of intact as well as truncated slow tnt proteins in the absence of myo- filaments. because intact exogenous tnt is stable in differen- tiated c c cells that contain myofilaments (fig. a), forma- tion of troponin complex and/or association with the thin filaments may be required for tnt protein to be stable in muscle cells. loss of high affinity binding of truncated slow tnt to tropomyosin—the truncated slow tnt produced by the anm glu nonsense mutation lacks the c-terminal tni-, tnc-, and tropomyosin-binding sites ( ) and is unable to form troponin complex. however, it retains the central tropomyosin-binding site ( ) and can potentially interact with tropomyosin or com- pete with intact tnt, theoretically producing a dominant neg- ative effect in anm carriers. the results in fig. show that binding of the truncated slow tnt to tropomyosin is almost non-detectable in the solid phase protein binding assay, whereas a saturable binding was found between intact slow tnt and tropomyosin. there was no difference between high and low molecular weight truncated variants. because the solid phase enzyme-linked immunosorbent assay protein binding experiments are done with repeated washing, these results only reflect the loss of high affinity binding ( ), although low affinity binding between truncated slow tnt and tropomyosin is expected ( ). discussion slow tnt gene transcription and rna processing are preserved in anm muscle—the anm tnnt mutation was first discovered through genetic linkage analysis and genomic dna sequencing ( ). the present study provides data to show the normal expression of the mutant transcript in anm mus- cle. thus neither nonsense mutation-mediated mrna decay ( ), nor mutation-induced aberration of splicing can explain the absence of truncated slow tnt in anm muscle. moreover, the mutation does not alter the native pattern ( ) of alternative splicing of the rna transcript of the slow tnt gene, further demonstrating the absence of mutational effects upon splicing. the apparently normal transcription and rna processing as well as the abundance of the mutant slow tnt mrna in anm muscle provides a potential therapeutic target, using somatic therapies to correct the slow tnt protein deficiency. this find- ing suggests that reagents that promote translational read- through of aberrant nonsense mutations, such as the aminogly- coside antibiotic gentamycin ( ), may be of potential benefit as a specific treatment. further testing of this hypothesis would be necessary before contemplating human trials. two-site binding to tropomyosin is essential to the incorpo- ration of tnt into myofilament—predicted from previous stud- ies on various tnt fragments ( , ), the anm truncated slow tnt still contains a tropomyosin-binding site and may compete with intact tnt in the anm muscle cell. this raised the in- triguing question as to why anm heterozygotes do not show a muscle phenotype. the comparison of binding affinity of intact and truncated slow tnt to tropomyosin demonstrates that slow tnt-( – ) does not bind tropomyosin strongly (fig. ). this result supports a hypothesis that the two-site binding to tro- pomyosin is necessary to anchor tnt and the rest of the tropo- nin complex to the thin filament. the tropomyosin-binding site in the c-terminal domain of tnt, lost in the truncated slow tnt-( – ) fragment, is not thought to be of high affinity ( ). therefore, the high affinity binding of tnt to tropomyosin must be a consequence of both sites acting together. this requirement in forming a stable thin filament structure suggests further that the two-site connec- tion between tnt and tropomyosin is constant during muscle activation and relaxation cycles, supporting an elongated con- formation of tnt in the thin filament assembly ( , ). this two-site anchoring requirement may also be a structural base for the observation that the strong binding between tnt and tropomyosin is a corporative process in the absence of f-actin ( ). rapid degradation of unincorporated tnt in myocytes is responsible for the absence of truncated slow tnt protein in anm muscle and prevents dominant negative effect—to un- derstand the missing link between abundant mutant mrna and lack of truncated slow tnt protein in anm muscle cells, we first tested whether the mutant mrna can be translated into protein. expression of mutant human tnnt cdna in e. coli and non-muscle eukaryotic cells yields easily detectable levels of the truncated slow tnt protein, proving the stability and capacity for active translation of the mrna. however, expression of mutant slow tnt mrna in c c myocytes failed to produce a detectable amount of the truncated slow tnt protein. these results suggest that muscle cell-specific protein degradation is responsible for the absence of truncated slow tnt protein in anm muscle. skeletal muscle protein is a major source of the energy supplied to the organism during the catabolic state ( ). muscle cells may thus have a high intrinsic proteolytic activity. none- theless, transfective expression could produce significant amounts of intact exogenous tnt in differentiated but not undifferentiated c c cells (fig. ). the data that successful accumulation of tnt in muscle cells requires integrity of tnt as well as the presence of myofilaments suggests that the inability of slow tnt-( – ) to form troponin complex and/or strongly bind tropomyosin may be the reason for its sensitivity to degradation. therefore, the intrinsic high proteolytic activity of muscle cells may be specifically directed to non-incorporated myofilament proteins. striated muscle contraction is based on the highly organized sarcomere structure. a high constitutive proteolytic activity against unincorporated sarcomeric proteins may represent a general mechanism of surveillance by which the highly orga- nized, multimeric myofilament structure is protected against a potential “weakest link” produced by incorporation of a dam- fig. . low binding affinity of the truncated slow tnt to tro- pomyosin. solid phase protein binding assays showed that the binding of truncated slow tnt to rabbit �-tropomyosin was undetectable by the high strangency binding assay, in contrast to the saturable binding curve of intact slow tnt. the high and low molecular weight (mw) variants showed similar low binding affinity. truncated slow troponin t in nemaline myopathy aged sarcomeric protein. damaged or improperly processed proteins that are not incorporated into or dissociated from the myofilaments will be degraded rapidly. although the single site binding between the truncated slow tnt and tropomyosin is much weaker than that of intact slow tnt (fig. ), even a low affinity interaction with tropomyosin may affect physiological function of the muscle if a high level of truncated slow tnt is accumulated in muscle cells. a previous report has shown that the slow tnt-( – ) fragment does not bind tnc or tni and cannot reconstitute troponin complex. when used to reconsti- tute myofilaments, the truncated slow tnt caused severe loss of ca � regulation of actomyosin atpase ( ). these authors also showed that when present at a high concentration, the slow tnt-( – ) fragment could displace endogenous tnt in myofibril in vitro. therefore, the muscle cell-specific degrada- tion of truncated slow tnt converts a potentially dominant mutation (which is a characteristic of most sarcomeric struc- tural protein mutations) to a recessive mutation. adult skeletal muscle-derived stem cells may retain a po- tential to express embryonic genes—it is known that cardiac tnt is expressed in embryonic skeletal muscle cells ( ). the presence of cardiac tnt as well as embryonic isoforms of fast skeletal muscle tnt in fetal and neonatal anm patient muscle is proposed as the molecular basis for the fact that anm pa- tients have normal muscle function at birth but develop fea- tures of myopathy during infancy, correlating with postnatal down-regulation of the fetal tnt isoforms ( ). the potential compensation of fetal tnt to the lack of slow tnt in anm neonates suggests a therapeutic target. although the c c myoblast cell line was established from adult mouse skeletal muscle ( ), the expression of cardiac tnt at significant levels in the early state of in vitro differentiation (fig. ) indicates that the developmental pattern of skeletal muscle gene regu- lation can be recapitulated in certain settings. the potential for adult skeletal muscle-derived stem cells to re-express cardiac tnt suggests another possible means to compensate for the absence of functional slow tnt ( ). it is also interesting to note that multiple alternatively spliced fast tnt protein bands were detected at various levels in differentiating c c cells. because the high molecular weight fast tnts represent acidic embryonic isoforms ( ) that may be able to compensate for the loss of slow tnt in embryonic anm muscle ( ), this finding suggests that promoting embryonic alternative splicing path- ways for the fast tnt mrna may also provide compensation to the lack of slow tnt in anm muscle. investigating the mech- anisms may identify approaches to promote the expression of cardiac tnt and/or embryonic fast tnt in anm patient muscle as a treatment of this lethal disease. acknowledgments—we thank stephen chong for technical assist- ance, dr. jim lin for 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( ) biophys. j. , a truncated slow troponin t in nemaline myopathy 	cellular fate of truncated slow skeletal muscle troponin t produced by glu nonsense mutation in amish nemaline myopathy* 	experimental procedures 	results 	discussion 	acknowledgments 	references response of obligate heterozygotes for phytosterolemia to a low-fat diet and to a plant sterol ester dietary challenge copyright © by lipid research, inc. this article is available online at http://www.jlr.org journal of lipid research volume , response of obligate heterozygotes for phytosterolemia to a low-fat diet and to a plant sterol ester dietary challenge peter o. kwiterovich, jr., , * shirley c. chen, † donna g. virgil,* amy schweitzer,* dagmar r. arnold,* and lisa e. kratz* lipid research/atherosclerosis division,* department of pediatrics, johns hopkins university, north broadway, baltimore, md ; and nutrition science group, † unilever bestfoods na, sylvan avenue, englewood cliffs, nj abstract twelve obligate heterozygotes from two kindreds were ascertained through phytosterolemic probands homo- zygous for molecular defects in the atp binding cassette (abc) half transporter, abcg . the response of these het- erozygotes to a step diet low in fat, saturated fat, and cho- lesterol, and to . g daily of plant sterols (as esters) was de- termined in protocol i ( weeks) and protocol ii ( weeks) during three consecutive feeding periods: step /placebo spread; step /plant sterol spread; and step /placebo spread (washout). at baseline, half the heterozygotes had moderate dyslipidemia and one-third had mildly elevated campesterol and sitosterol levels. on the step /placebo spread, mean ldl cholesterol decreased significantly, . % in protocol i (n � ), and . % in protocol ii (n � ). substitution with plant sterol spread produced a significant treatment ef- fect on ldl levels in protocols i and ii. conversely, the mean levels of campesterol and sitosterol increased % and %, respectively, during the use of plant sterol spread for weeks in protocol i, an effect mirrored for weeks in protocol ii. during the placebo spread washouts, ldl lev- els increased, while those of plant sterols decreased to base- line levels in both protocols. in conclusion, phytosterolemic heterozygotes respond well to a step diet, and their re- sponse to a plant sterol ester challenge appears similar to that observed in normals. —kwiterovich, jr., p. o., s. c. chen, d. g. virgil, a. schweitzer, d. r. arnold, and l. e. kratz. response of obligate heterozygotes for phytoster- olemia to a low-fat diet and to a plant sterol ester dietary challenge. j. lipid res. . : – . supplementary key words lipoproteins • carotenoids • fat soluble vi- tamins • plant sterol ester enriched spread • margarine studies in humans with inherited disorders of choles- terol metabolism have provided unique and important insights into the mechanisms underlying both hyper- cholesterolemia and premature atherosclersosis ( ). this has led to a more precise understanding of the effects of both dietary restriction of cholesterol and of pharmaco- logical agents on reducing plasma cholesterol levels and preventing cardiovascular disease ( – ). for example, both dietary and drug treatments that lower the pool of cholesterol in the liver lead to an up-regulation of ldl re- ceptors and to a decrease of plasma ldl cholesterol ( ). the effect of dietary cholesterol on plasma cholesterol levels is modest, due in part to the incomplete absorption of cholesterol by the intestine ( , ). for example, bosner et al. ( ) found that the mean ( sd) cholesterol absorp- tion in humans was . ( . )% with a range from . % to . %. this protective barrier in the normal human in- testine is even more efficient for dietary plant sterols, such as sitosterol and campesterol, with estimates for sitosterol absorption ranging from . % to . %, and for campes- terol from . % to % ( ). ostlund and coworkers ( ), using serum to establish unequivocal absorption into the systemic circulation and mass spectrometry for definitive identification of labeled tracers, recently reported lower estimates of absorption, namely, . % for sitosterol and . % for campesterol. the plant sterols are structurally very similar to choles- terol, except that they always contain substitutions at the c position, and the mechanism that selectively prevents most of their intestinal absorption is not clear. the plant sterols, due to this structural similarity to cholesterol, pre- vent cholesterol absorption, most likely by precipitating cholesterol and competing for space in mixed micelles ( ). an increased fecal excretion of cholesterol occurs, the hepatic concentration of cholesterol decreases, ldl receptors up-regulate, and the blood levels of total and ldl cholesterol subsequently fall ( – ). plant sterols were first given in high doses of g to g per day to treat hypercholesterolemic patients. more recently, more mod- est doses of phytosterols, such as g daily, have been in- corporated into margarine and given to normal or moder- to whom correspondondence should be addressed. e-mail: pkwitero@jhmi.edu manuscript received december and in revised form march . published, jlr papers in press, april , . doi . /jlr.m -jlr this is an open access article under the cc by license. http://creativecommons.org/licenses/by/ . / journal of lipid research volume , ately hypercholesterolemic subjects to lower their ldl cholesterol levels about % ( – ). recently, novel insights into the mechanisms affecting cholesterol and plant sterol absorption have been gained through the study of patients with the rare ( � one in a mil- lion) autosomal recessive disorder, phytosterolemia ( ). patients with phytosterolemia have markedly elevated ( � -fold) plasma levels of plant sterols ( – ). this re- sults from two metabolic abnormalities. first, patients with phytosterolemia hyperabsorb plant sterols, for example, between % and % of the average of mg to mg of plant sterols consumed daily ( , , ). second, unlike normal humans in whom almost all of any ab- sorbed plant sterol is quickly secreted into the bile ( , ), phytosterolemic homozygotes excrete only a fraction of the plant sterols into the bile ( , , ). phytosterolemic subjects also absorb a higher fraction of dietary choles- terol than normals, and they secrete less cholesterol into the bile ( , , ). the cholesterol pool in the liver in- creases despite a low cholesterol synthetic rate, ldl re- ceptors down-regulate, and ldl levels increase. the liver secretes the excess sterols into the blood on vldl, which is converted into ldl, the major carrier of sterol in blood ( ). about % to % of the sterol on ldl in phy- tosterolemia is plant sterol ( – ), and a similar propor- tion of these sterols is found in human tissues ( ). as a result of these two metabolic abnormalities, patients with phytosterolemia often have elevated levels of ldl ste- rol similar to those heterozygotes for familial hypercholes- terolemia (fh); however, phytosterolemic subjects develop xanthomas (cholesterol deposits in skin and tendons) in the first decade of life, while fh heterozygotes do so in the third and fourth decades. phytosterolemic homozygotes can also develop premature coronary artery disease at an earlier age than fh heterozygotes ( – , ). phytosterolemic pa- tients often develop aortic stenosis, an unusual finding in fh heterozygotes, and more typical of fh homozygotes ( ). unlike individuals with fh or other forms of hyper- cholesterolemia, phytosterolemic subjects respond dramati- cally to restriction in dietary cholesterol and plant sterols, and to bile acid sequestrants ( , , ), often with the to- tal and ldl sterol levels falling to within the normal range. the molecular defects responsible for homozygous phy- tosterolemia are caused by two mutant alleles either in the gene that encodes the atp binding cassette (abc) half transporter, abcg , or in the gene encoding the half transporter abcg ( , ). these two genes are on chro- mosome p, where they are located in a head-to-head ori- entation. abcg and abcg are expressed exclusively in human liver and intestine, the sites of the two metabolic abnormalities in phytosterolemia. in mice, diets contain- ing high cholesterol markedly increase the expression of abcg and abcg mrna in liver and intestine ( ). the current hypothesis, therefore, is that abcg and abcg have two normal functions: first, to limit the ab- sorption of cholesterol and plant sterols, and second, to promote their excretion from the liver into the bile. while it is clear that consuming plant sterols is con- traindicated in phytosterolemic homozygotes, little infor- mation is available on the effect of increasing dietary plant sterols on the plasma total and ldl cholesterol, and plant sterol levels in the more common ( � in ) het- erozygotes for phytosterolemia ( , ). the intake of di- etary plant sterols can be increased significantly using margarines containing either unsaturated plant sterol es- ters or saturated plant sitostanol esters, both of which lower total and ldl cholesterol levels in normal humans about – % ( – ), and have similar effects on sup- pressing cholesterol absorption ( ). since unsaturated sterol esters have been shown to increase the average plasma sitosterol and campesterol levels in normals about -fold while saturated sitostanol esters decrease these lev- els to below the average ( ), we selected unsaturated ste- rol esters as the dietary challenge. our objectives were to characterize the clinical and bio- chemical phenotype of obligate heterozygotes for phy- tosterolemia and their response to an unsaturated plant sterol ester-enriched spread, as judged by their change in ldl cholesterol levels and the extent of their increase in plant sterols levels. methods study participants sixteen members of two families with homozygous phytoster- olemic probands participated in the study. an amish family was ascertained through the sudden death of an -year-old boy who had xanthomas and extensive atherosclerosis at autopsy. of the siblings of this amish proband, five were found to be phy- tosterolemic homozygotes ( , ). of the amish obligate heterozygotes (two parents and children of the homozy- gotes), participated in this study. two siblings and two spouses of the amish homozygotes also participated as familial controls. both parents of the two original phytosterolemic homozygotes described by bhattacharyya and connor ( ) were also studied. the two families were not related to each other, and each had different mutations in the gene for the abcg half transporter ( ). the amish phytosterolemics were homozygous for a mis- sense mutation (arg for gly, g a) in a residue that was con- served in mouse and human abcg ( ). the original phy- tosterolemic patient was homozygous for a nonsense mutation ( g–a) in exon that introduced a premature terminal sig- nal at codon , terminating abcg ( ). the subjects were healthy males (n � ) and females (n � ), aged to years. informed consent was obtained for each subject. the johns hop- kins joint committee on clinical investigation approved the study. the subjects were free of active surgical or medical illnesses. subjects were excluded from participating in the study if they had any of the following conditions: types i, iii, or v hyperlipo- proteinemia; secondary hyperlipoproteinemia; body mass index � . kg/m ; were pregnant or lactating women; or used oral hypolipidemic therapy, systemic corticosteroids, androgens, or thyroid hormones (except stable-dose replacement therapy for � months prior to enrollment). study protocols the flow diagrams for the two study protocols are schemati- cally summarized in fig. . protocol i was of weeks duration and consisted of seven outpatient visits. five subjects were also sampled weeks after the conclusion of protocol i (an eighth kwiterovich et al. response of phytosterolemic heterozygotes to plant sterols visit). protocol ii was of weeks duration and consisted of eight outpatient visits. each study was single blind and placebo con- trolled. study design in both protocol i and protocol ii, each subject received an in- struction at the first visit by a nutritionist on a low total-fat, satu- rated-fat, and cholesterol diet following the guidelines of the american heart association ( ). the revised guidelines placed increased emphasis on foods and an overall eating pattern. the goal is a total fat intake of % of calories or less, a saturated fat intake of � % of calories, and a daily cholesterol intake of � mg. while these guidelines collectively replace the “step ” designation used earlier, we simply refer to this diet here as the step diet. this step diet was followed throughout both proto- col i and protocol ii. in protocol i, the first visit was followed by a week baseline period; after weeks of a stabilization period (fig. a), a placebo spread [four servings ( g per serving) of a % fat spread] was incorporated into the step diet. after weeks on the placebo spread, each subject entered a week treatment period, during which a plant sterol spread [four serv- ings ( . g plant sterols per serving) in the form of sterol esters] was incorporated into the step diet. protocol i concluded with a week washout period during which time the subjects reverted back to the use of the placebo spread as part of their step diet. in protocol ii, the subjects were given a placebo spread [two servings ( g each) daily of a % fat spread] at their first visit to be incorporated into their step diet. after weeks on pla- cebo spread and diet, each subject entered a week treatment period, during which time the plant sterol spread [two servings ( g each) daily, each containing . g of plant sterols in the form of sterol esters] was incorporated into the step diet. pro- tocol ii concluded with a week washout period, during which time the subjects reverted back to the use of the placebo spread in their step diet. for both protocols, evaluation included a physical examina- tion and an electrocardiogram at baseline and final visit, vital signs at each visit, urinary pregnancy measurement at baseline, and safety laboratory evaluation (chemistry and hematology pro- files and urinalysis) at visits one, three, four, and seven in proto- col i, and at visits one, five, and eight in protocol ii. thyroid- stimulating hormone was measured in each subject at baseline. plasma levels of total cholesterol, total triglycerides, and hdl cholesterol were determined at each visit in both protocols. the concentrations of the plasma plant sterols were measured at each visit in both protocols except at week , visit two in protocol i. the plasma levels of apolipoprotein b (apob), apoa-i, and lipo- protein [a] (lp[a]) were assessed at each visit in both protocol i and protocol ii. retinol, tocopherols, and carotenoids were de- termined in plasma at visits three, four, five, six, and seven in pro- tocol i, and at each visit in protocol ii. the test margarines were specially prepared spreads (unilever bestfoods, na, baltimore, md). one was a plant sterol spread, the other was a placebo spread. the plant sterol spread was forti- fied with phytosterol (ester) concentrates derived from vegetable oil distillates. the sterols were a mixture of sitosterol, campes- terol, brassicasterol, and stigmasterol ( ). the vegetable oil ste- rols were esterified with fatty acids from sunflower oil to an ester- ification degree of %. the two spreads were provided in the form of g individual packs for protocol i. for protocol ii, the spreads were provided in tubs, along with a measuring spoon that provided a g serving. the placebo and study spreads had identical fat levels ( %, or . g/serving) and fatty acid compo- sitions. the chemical composition of the spreads has been de- scribed in detail ( ). the plant sterol spread contained . g of phytosterol in each serving in protocol i, and . g of phy- tosterol in each serving in protocol ii. thus, the daily total intake of phytosterol was . g in both studies, but the package size was different. compliance with study spread consumption was evaluated by patient interview and by day food records. compliance was re- corded as a percentage of scheduled intakes of study product consumed. compliance was � % in both protocols. the intakes of dietary cholesterol and plant sterols, total fat, saturated fat, and unsaturated fat were estimated for each subject using a day food record before, during, and after the sterol- containing test spread. the day food records were then ana- lyzed using nutritionist pro, first data bank, san bruno, ca. the subjects were also asked to report the frequency, kind, and amount of supplements, including vitamins that they used at each visit. statistical methods the effect of the three study diets (placebo spread, plant ste- rol ester spread, placebo spread) on the dependent variables (plasma levels of total cholesterol, total triglycerides, hdl cho- lesterol, ldl cholesterol, the ratio of ldl-hdl cholesterol, campesterol, sitosterol, lp[a], apob, apoa-i, the ratio of apob- apoa-i, retinol, tocopherols, and carotenoids) were determined in protocol i and protocol ii using a one-way mixed model fig. . protocol i is schematically summarized in a and protocol ii is summarized in b. journal of lipid research volume , anova for repeated measurements (sas proc mixed). in this model, individual differences were accounted for using baseline (home diet) as a covariate ( ), and by including a random sub- ject effect in the model. the remaining time series (residual) correlation was modeled as one parameter autoregressive ( ). we used the tukey-kramer multiple comparison test ( ) to de- termine which of the study diets significantly affected the depen- dent variables. additional posthoc comparisons of the same vari- ables for subjects at baseline and at the end of the placebo spread run-in period were determined using paired student’s t -tests ( ). laboratory methods total cholesterol, total triglycerides, and direct hdl choles- terol were measured in a hitachi chemistry analyzer in the johns hopkins lipoprotein analytical laboratory with coeffi- cients of variability (cvs) of � %. the lipid standardization program of the centers for disease control, atlanta, ga, certified the laboratory. ldl cholesterol was calculated by the friedewald formula ( ). the plasma levels of total apob and apoa-i were measured in a bering immunonephelometer with cvs of % ( ). we measured lp[a] by elisa, as described, with a cv of % ( ). lipoprotein phenotypes were defined as follows. first, an ele- vated plasma level of ldl cholesterol or triglycerides was de- fined as a value � th percentile, and a low level of hdl choles- terol as a value � th percentile, using the age- and sex-specific cut points from the lipid research clinics program ( ). a type iia phenotype was defined as an elevated level of ldl choles- terol with normal triglycerides, and a type iib as elevated levels of ldl cholesterol and triglycerides. a type iv phenotype was defined as an elevated triglyceride level with a normal ldl cho- lesterol level. hyperapob phenotype was defined as an elevated level of apob ( � . � mol/l in adults, � . � mol/l in chil- dren) in the presence of a normal ldl cholesterol level. a nor- mal phenotype was defined as the absence of any of these dyslipi- demic phenotypes. campesterol and sitosterol were measured using selected ion monitoring gas liquid chromatography/mass spectrometry (gc/ ms) as described ( ), except to increase the plasma sample vol- ume to � l to permit more accurate measurement of the trace quantities of phytosterols in plasma of normal individuals. this method detects small amounts ( � . � mol/l) of plant sterols. the plasma levels of lutein, � -cryptoxanthine, � -cryptoxan- thine, lycopene, � -carotene, � -carotene, total carotenoids, reti- nol, 	 -tocopherol, and � -tocopherol were determined using hplc as described ( , ). results the biochemical and clinical characteristics of the study population are summarized in table . twelve are obligate phytosterolemic heterozygotes ( children of four amish homozygotes and two parents of the original phytosterolemic homozygotes). four additional amish subjects were studied as familial controls: two adult sib- lings (ii- and ii- ) of the homozygous proband, and two spouses (ii- and ii- ) of two other homozygotes. nine males and seven females were studied. at baseline, six of the obligate phytosterolemic heterozygotes (type iib, n � ; type iv, n � ; hyperapob, n � ), and both the amish proband’s siblings (type iia, n � ; hyperapob, n � ) had a dyslipidemia (table ). both the spouses were normal. to interpret the plant sterol levels in the study popula- tion at baseline, plasma sitosterol and campesterol levels (mg/dl) were determined in unrelated control sub- jects. these controls included normal mennonites, who were unlikely to carry the mutant allele for phytoster- olemia, but who share a similar diet and life style with the amish. seventeen other caucasian controls who were not mennonites were also studied. the mean (sd) sitosterol and campesterol levels ( � mol/l), respectively, in the con- trol subjects were: ) adult mennonite (n � ), . table . clinical and biochemical characteristics of the study population subjects sex age tc tg hdl cholesterol ldl cholesterol apob apoa-i sitosterol campesterol lp[a] lipoprotein phenotypes mmol/l � mol/l mg/dl obligate heterozygotes i- m . . . . . . . . hyperapob i- f . . . . . . . . normal ii- f . . . . . . . . type iv ii- m . . . . . . . . normal ii- m . . . . . . . . normal ii- f . . . . . . . . normal ii- f . . . . . . . . normal ii- m . . . . . . . . normal ii- m . . . . . . . . type iib ii- f . . . . . . . . hyperapob ii- m . . . . . . . . type iv ii- m . . . . . . . . type iv proband’s siblings ii- m . . . . . . . . type iia ii- m . . . . . . . . hyperapob spouse controls ii- f . . . . . . . . normal ii- f . . . . . . . . normal apo, apolipoprotein; lp[a], lipoprotein[a]; tc, total cholesterol; tg, triglycerides. the lipoprotein phenotypes were determined as described in methods. kwiterovich et al. response of phytosterolemic heterozygotes to plant sterols ( . ), and . ( . ); ) mennonite, – years (n � ), . ( . ), and . ( . ); ) mennonite, – years (n � ), . ( . ), and . ( . ); and ) adult cauca- sian (n � ), . ( . ), and . ( . ). the menno- nite controls were used for the amish participants and the caucasian controls for the parents of the original phy- tosterolemic homozygotes. four of the amish obligate heterozygotes had both plasma sitosterol and campesterol levels that were two sd above the mean of the controls. one sibling (ii- ) of the amish homozygous proband also had levels of sitosterol and campesterol that exceeded these cutpoints. both the amish spouses and the other two obligate heterozygotes (i- and i- ) from the original family had plasma plant sterol levels that were within the normal ranges. individual responses of obligate heterozygotes to modified-fat and plant sterol ester challenge we first examined the individual responses of the obli- gate heterozygotes to the step diet and to the plant ste- rol ester-enriched margarine to determine the patterns of responses to the dietary interventions. individual responses in ldl cholesterol levels. the change in ldl cholesterol levels in each obligate heterozygote during protocol i is summarized in fig. a . the pattern of de- crease in ldl cholesterol levels from baseline to the end of the placebo spread period, with an additional fall dur- ing the plant sterol spread period, followed by an increase during the washout placebo spread period, was internally consistent, with each subject generally following this pat- tern. five of the subjects were also sampled weeks af- ter the conclusion of the week period of protocol i, and ldl cholesterol values remained stable (n � ) or in- creased (n � ) (fig. a). in the longer-term protocol ii, there was again a down- ward trend in ldl cholesterol in each subject from base- line to week on the step /placebo spread diet (fig. b). after weeks of the substitution of the plant sterol spread for the placebo spread, the subject (ii- ) with high- est ldl cholesterol at baseline had the greatest decrease in ldl cholesterol (fig. b). the decrease in ldl choles- terol in the remaining six obligate heterozygotes was less accentuated, and one subject (i- ) had an increase in ldl cholesterol on the plant sterol ester spread (fig. b). dur- ing the week placebo spread washout period, however, the individual ldl cholesterol values increased, as ex- pected, toward levels that were similar to those observed at baseline (fig. b). individual responses in plant sterol levels. the response of the plasma plant sterols in the obligate heterozygotes to the chal- lenge of . g per day of dietary plant sterols in the test margarine is summarized in fig. a – d . in protocol i, both sitosterol (fig. a) and campesterol (fig. c) increased during the plant sterol spread phase, reaching a maxi- mum after weeks of treatment, and declining thereaf- ter on the placebo spread to values similar to those at baseline. however, at the end of protocol i, three subjects (ii- , ii- , and ii- ) had campesterol levels � � mol/l; the sitosterol levels in each of these three subjects were � � mol/l. protocol ii was therefore designed to assess for a longer period of time the effect of the plant sterol spread on the sitosterol and campesterol levels. in proto- col ii, subjects ii- and ii- again had the largest increase in sitosterol and campesterol on the test margarine (fig. b, d). both these subjects also had sitosterol and campes- terol levels at baseline that exceeded sd above the mean of the age-appropriate controls (table ). the sitosterol levels did not exceed � mol/l on the plant sterol spread, and fell to about � mol/l on the placebo spread during the washout period, a value close to baseline (fig. b). the campesterol levels exceeded � mol/l in each of these two subjects on the plant sterol spread (fig. d), but fell during the washout period on the placebo spread, and were about � mol/l at weeks and weeks of protocol ii, close to the baseline values (fig. d). each of the rest of the subjects in protocol ii had an increase in si- tosterol and campesterol on the plant sterol spread that then fell during the washout with the placebo spread to values similar to those observed at baseline. fig. . individual responses of ldl cholesterol to pla- cebo spread and plant sterol ester spread in obligate phy- tosterolemic heterozygotes in protocol i (a) and in proto- col ii (b). journal of lipid research volume , group response of obligate heterozygotes for phytosterolemia to step diet and to a plant sterol ester challenge changes in nutrient intake. the group data for the obligate heterozygotes studied in protocol i was next assessed, focus- ing on the plasma levels of ldl cholesterol and plant ste- rols. an important first step was to characterize the baseline (home) diet of these subjects, to document that they had modified their dietary intake as a result of their instruction on a step diet (see methods), and that they continued to follow this diet throughout the three study periods ( table ). the study group lowered their mean intakes of satu- rated fat and cholesterol significantly (table ). total fat was also lowered from . % of calories to . % of calo- ries, but this change was not significant. the modest in- crease in polyunsaturated fat was significant (table ). these changes were maintained throughout the study (ta- ble ), and there were no significant differences in these variables across the three experimental diet periods (by anova, see methods) (table ). during the three experi- mental periods, a significant difference was observed for energy intake, due mainly to decreased carbohydrate in- take (table ). there was an � -fold increase in plant ste- rols during the plant sterol ester spread period (table ). the group in protocol ii was very similar to that in pro- tocol i in regard to their marked increase in dietary plant sterols during the plant sterol ester spread period. the mean nutrient intakes for the seven obligate heterozy- gotes in protocol ii differed, however, from those of the obligate heterozygotes in protocol i in several ways. first, at the initial visit on the home diet, their mean in- takes of total fat ( . %), saturated fat ( . %), and cho- lesterol ( mg/day) were already at the step goals. there were no significant differences in protocol ii be- tween these nutrients (or any others) when the home diet and the end of the placebo run-in period were compared (data not shown). nevertheless, the group in protocol ii decreased their total fat and saturated fat to % and . % of calories, a change that was maintained through- out the plant sterol ester spread period. in protocol ii, during the placebo washout, the study group increased their protein, total-fat, saturated-fat, monounsaturated-fat, polyunsaturated-fat, and cholesterol intakes significantly, compared with the plant sterol ester spread study period, but they were still within the step goals. fig. . individual responses of plasma levels of sitosterol (top) and of campesterol (bottom) to placebo spread and plant sterol es- ter spread in obligate phytosterolemic heterozygotes in protocol i (a, c) and protocol ii (b, d). table . nutrient intakes of obligate heterozygotes for sitosterolemia (n � ) based on day diet records (protocol i) variables and study periods home diet a (baseline, week ) study diets placebo spread (run-in, weeks , ) plant sterol ester spread (weeks , ) placebo spread (washout, week ) p e energy, kcal , , c , d , cd . protein, g/day . carbohydrate, g/day c d c . fat, g/day . cholesterol, mg/day b . saturated fat, g/day b . monounsaturated fat, g/day . polyunsaturated fat, g/day b . plant sterols, mg/day d , c d . a mean se. b are significantly different from the corresponding values at the end of the run-in period (week ), p � . . c,d,cd least squares mean se. for the three study diet periods, values in a row with different superscripts dif- fer, p � . . e p values for treatment effect, as determined in methods. kwiterovich et al. response of phytosterolemic heterozygotes to plant sterols group response of plasma ldl cholesterol levels. there were sig- nificant falls in ldl cholesterol of . % from baseline to weeks in protocol i and of . % from baseline to weeks in protocol ii (tables , ). there was a significant treatment effect of the plant sterol-enriched margarine on ldl cholesterol levels in both protocols i and ii (tables , ). during the plant sterol spread period, ldl cholesterol in protocol i decreased another . % but did not fall fur- ther in protocol ii. during the washout periods of both protocols i and ii, the substitution of the placebo spread for the plant sterol spread produced significant increases in the mean ldl cholesterol levels of . % and . %, re- spectively (tables , ). these data taken together sug- gested that the plant sterol spread was responsible for the significant treatment effect on ldl cholesterol observed in both studies (tables , ). group response of plasma plant sterol levels. in both protocol i and protocol ii, there was a significant treatment effect of the plant sterol ester spread on plasma levels of both si- tosterol and campesterol (tables , ). the mean plasma campesterol levels increased about -fold in both protocol i and protocol ii during the plant sterol ester spread pe- riod. the mean increase in the sitosterol level on the plant sterol ester spread was about half that seen with campes- terol (tables , ). in the washout period, when the pla- cebo spread was substituted for the plant sterol spread, both the mean campesterol and sitosterol levels decreased to those observed in the placebo run-in periods in proto- cols i and ii (tables , ). despite differences in the dura- tion of the plant sterol treatment and the placebo washout period in the two protocols, the qualitative pattern and the quantitative extent of response were very similar. table . lipids and apolipoprotein concentrations of obligate heterozygotes for sitosterolemia (n � ) at baseline, and in response to study diets (protocol i) study diets variables and study periods home dieta (baseline, week ) placebo spread (run-in, weeks , ) plant sterol ester spread (weeks , ) placebo spread (washout, week ) overall p e total cholesterol, mmol/l . . b . . cd . . d . . c . triacylglycerols, mmol/l . . . . . . . . . hdl cholesterol, mmol/l . . . . . . . . . ldl cholesterol, mmol/l . . b . . cd . . d . . c . ldlc/hdlc . . . . . . . . . campesterol, �mol/l . . . . d . . c . . d . sitosterol, �mol/l . . . . d . . c . . d . lp[a], mg/dl . . . . . . . . . apob, �mol/l . . b . . . . . . . apoa-i, �mol/l . . b . . . . . . . apob/apoa-i . . . . . . . . . a mean se. b values are significantly different from the corresponding values at the end of the run-in period (week ), p � . . c,d,cd least squares mean se. for the three study diet periods, values in a row with different superscripts differ, p � . . e p values for treatment effect, as determined in methods. table . lipids and apolipoprotein concentrations of obligate heterozygotes for sitosterolemia (n � ) at baseline, and in response to study diets (protocol ii) study diets variables and study periods home dieta (baseline, week ) placebo spread (run-in, week ) plant sterol ester spread (weeks , , ) placebo spread (washout, weeks , , ) overall p e total cholesterol, mmol/l . . b . . d . . d . . c . triacylglycerols, mmol/l . . . . d . . c . . c . hdl cholesterol, mmol/l . . . . d . . c . . c . ldl cholesterol, mmol/l . . b . . d . . d . . c . ldlc/hdlc . . b . . . . . . . campesterol, �mol/l . . . . d . . c . . d . sitosterol, �mol/l . . b . . d . . c . . d . lp[a], mg/dl . . . . . . . . . apob, �mol/l . . . . d . . d . . c . apoa-i, �mol/l . . . . d . . c . . c . apob/apoa-i . . . . . . . . . a mean sd. b values are significantly different from the corresponding values at the end of the run-in period (week ), p � . . c,d least squares mean se. for the three study diet periods, values in a row with different superscripts dif- fer, p � . . e p values for treatment effect, as determined in methods. journal of lipid research volume , group response in other biochemical lipid variables. in protocol i, the mean plasma levels of both total cholesterol and apob decreased significantly after the step /placebo spread run period (table ). there was a significant treatment ef- fect for the plant sterol ester spread for total cholesterol, but that for the apob did not reach statistical significance (table ). the plasma level of apoa-i decreased signifi- cantly after the step /placebo spread run-in, but that of hdl cholesterol did not (table ). there was no signifi- cant treatment effect on either the hdl cholesterol or apoa-i levels in protocol i (table ). in protocol ii, the plasma levels of total cholesterol also decreased significantly after the step /placebo spread, and a significant treatment effect was noted for total cho- lesterol. in contrast to protocol i, apob, triglycerides, hdl cholesterol, and apoa-i also manifested a significant treatment effect (table ). there was no effect of either the step diet or the plant sterol ester spread on lp[a] lipoprotein levels (ta- bles , ). response of familial controls to the plant sterol ester challenge the individual responses of the four familial controls to the plant sterol ester treatment were also assessed and compared with responses in unrelated subjects from the published literature ( , , – ) where a similar dose of plant sterol esters was used and both ldl cholesterol and plant sterols were measured (table ). the average fall in ldl cholesterol in the familial controls was . %, similar to the high end of the range of unrelated subjects in the literature ( ). the average plasma sitosterol and campesterol levels increased . % and . % in the fa- milial controls, within the ranges previously reported in unrelated subjects (table ). fat-soluble vitamins and carotenoids. if plant sterols displace fat-soluble vitamins and carotenoids from bile acid mi- celles, their use might theoretically decrease the absorp- tion of these fat-soluble nutrients and potentially promote deficiencies in such nutrients ( , , ). we therefore also examined whether the use of plant sterol in a dose of . g/day significantly decreased carotenoids, retinol, and tocopherols. ten variables were measured in protocols i and ii (lutein, �- and �-cryptoxanthine, lycopene, �- and �-carotene, total carotenoids, retinol, and �- and 	-tocoph- erols). the data are presented for protocol ii because these variables were measured at baseline and could be used as covariates in the statistical analysis. protocol ii was also longer than protocol i, and therefore any effect of the plant sterol esters more pronounced. after correction for plasma total cholesterol levels, significant treatment ef- fects of the plant sterol ester were found for plasma levels [mean (sem) as �g/ mg cholesterol] of �-cryptoxan- thine, lycopene, and �-carotene (table ). there was no significant effect on any of the other variables examined (table ). discussion we report here the response of obligate heterozygotes from two well-characterized families with phytosterolemia to . g daily of plant sterol via the consumption of a plant sterol esters-enriched spread, as judged by the change in their plasma levels of ldl cholesterol and plant sterols. we used an aba study design that provided two control periods (a), one before and one after the plant sterol es- ter challenge (b; aba switchover). each obligate het- erozygote served as his or her control. table . comparisons of mean plasma levels of ldl-cholesterol, sitosterol and campesterol responses to intake of plant sterol ester-containing margarine of subjects from published literature and individual responses of familial controls in the current study percent change in plasma levels after treatment with plant sterol esters subjects n design (duration) daily intake of plant sterol (background diet) ldl cholesterol sitosterol campesterol published studies (reference) mildly hypercholesterolemic adults ( ) incomplete latin square ( weeks) . g (habitual diets) � . � . � . normal to mildly hypercholesterolemic adults ( ) , b two arm parallel ( weeks) . g (step ) � . � . � . hypercholesterolemic adults with or without statins ( ) cross-over ( weeks) . g (recommended diet for hypercholesterolemics) � . � . � . hypercholesterolemic adults ( ) cross-over ( weeks) . g (habitual diets) � . � . � . normal to mildly hypercholesterolemic adults ( ) two arm parallel ( weeks) . g (habitual diets) � . � . � . current study (familial controls)a aba switchoverc . g (step ) adult sibling of homozygote protocol i � . d � . d � . d adult sibling of homozygote protocol i � . � . � . spouse of homozygote protocol ii � . � . � . spouse of homozygote protocol ii � . � . � . a see table for subject descriptions and fig. for study design. b n � for the ldlc measurement; n � for the plant sterol measurements. c there were two control periods (a), one before and one after the plant sterol ester challenge (b). d comparison of values at the end of the lead-in period with control placebo spread and at the end of the treatment period with plant sterol ester spread. kwiterovich et al. response of phytosterolemic heterozygotes to plant sterols the study population responded to a step diet with an average decrease in ldl cholesterol of . % in proto- col i and of . % in protocol ii, both highly statistically significant. in protocol i, the ingestion of about . g of plant sterols daily produced a significant additional reduc- tion of . % in ldl cholesterol over that achieved by a step diet. such a significant fall was not seen in protocol ii. since the blood levels of the plant sterols increased sig- nificantly in protocol ii, and in a pattern similar to proto- col i (fig. ), lack of compliance to the ingestion of the test margarine does not explain the failure to find a de- crease in ldl. the simplest explanation is that the obser- vation was due to intraindividual heterogeneity, which might become of more importance, given the smaller sam- ple size in protocol ii. for example, in one subject (i- ) in protocol ii, the ldl cholesterol levels increased notably during the plant sterol ester period (fig. b). the mean dietary intakes of total fat, saturated fat, and cholesterol were lower at baseline and during the placebo run-in pe- riod in protocol ii than in protocol i, but both groups fol- lowed a step diet throughout all the study periods. moreover, in both protocol i and protocol ii, there was a significant increase in ldl cholesterol in the placebo spread washout period, indicating that the plant sterol- enriched spread had an effect independent of the step diet. while sources of error, such as instrument effect, un- derestimate or overestimate of dietary intakes, and adher- ence effect (compliance bias) can influence the estimates of the intake of these nutrients ( ), the changes in these the intakes of total fat, saturated fat, and cholesterol were consistent across our study population and both protocols. the moderate dyslipidemia in the obligate heterozy- gotes, observed at baseline here and elsewhere ( , ), might reflect some moderate down-regulation of the ldl receptor in these subjects, which might accompany in- creased sterol absorption. further metabolic studies will be required to answer this issue. while the response of ldl cholesterol to the dietary changes employed here are of relatively low magnitude, on a population basis even a mean decrease of only % to % may have a significant effect on the overall incidence of coronary artery disease (cad) ( – ). conversely, the mean plasma levels of campesterol and sitosterol increased % and %, respectively, during the use of the plant sterol-enriched spread in protocol i, an effect that was mirrored in protocol ii. while such changes were highly statistically significant, they were of similar magnitude to those seen in normal and mildly hy- percholesterolemic unrelated subjects in other studies of plant sterol esters ( , , – ) and in our familial con- trols (table ). it must also be noted that the levels of plant sterols remained a minute fraction of the total blood sterols (tables and ). the greater increase in the plasma level of campesterol than sitosterol in the obligate heterozygotes given the plant sterol-enriched spread may be related to the fact that humans have a greater intestinal absorption of campesterol than of sitosterol, a differential related to the decreased uptake of plant sterols by intesti- nal cells with an increasing number of carbon atoms at c ( , , ). in that regard, campesterol has one carbon, while sitosterol has two carbons at c . once the effect of the plant sterol-enriched spread reached a plateau, no further increase was observed in either protocol i or pro- tocol ii, suggesting that the obligate heterozygotes elimi- nated the plant sterols sufficiently to prevent their accu- mulation. this tenet was supported by the fact that the mean plasma levels of both campesterol and sitosterol re- turned to baseline levels during the placebo spread wash- out period. in normal humans, ostlund et al. ( ) re- ported that the mean (sd) half-life of plasma sitosterol was . ( . ) days and that of plasma campesterol . ( . ) days. despite the relatively long half-life of these plant sterols, one would predict that these levels would re- turn to baseline by the time we sampled our obligate het- erozygotes on the placebo spread in the washout period, unless there was a continued secretion of plant sterols ab- table . plasma carotenoids and fat-soluble vitamin concentrations (�g/ mg cholesterol)a of obligate heterozygotes for sitosterolemia (n � ) at baseline, and in response to study diets (protocol ii) study dietse variables and study periods home dietb (baseline, week ) placebo spread (run-in, week ) plant sterol ester spread (weeks , , ) placebo spread (washout, weeks , , ) overall p f lutein . �-cryptoxanthine . �-cryptoxanthine c d cd . lycopene cd d c . �-carotene c d d . �-carotene . retinol . . �-tocopherol , , , , . 	-tocopherol , , , , . a corrected for total cholesterol. b mean se. c,d,cd values at baseline and at the end of the run-in period (week ) are not different. e least squares mean se. for the three study diet periods, values in a row with different superscripts differ, p � . . f p values for treatment effect, as determined in methods. journal of lipid research volume , normally accumulated in liver. these observations are consistent with the metabolic findings of salen and co- workers ( ), who reported that the somewhat increased plant sterol absorption in heterozygotes for phytoster- olemia was offset by rapid elimination from the liver to prevent accumulation of plant sterols. protocol ii examined the effect of the plant sterol spread on the plant sterol levels for a longer period of time, and also followed the participants for a greater time on the placebo spread to ensure that the increase in the plant sterols observed in protocol i actually reached a pla- teau and then fell to the baseline levels and remained there. very similar qualitative and quantitative patterns of increase in the mean plant campesterol and sitosterol lev- els, followed by a prompt decrease and stabilization, were observed in protocol ii as in protocol i (tables , ). the mean plasma sitosterol and campesterol levels (mg/ dl) determined by gc/ms (see methods) in our control groups were similar to those reported by others ( , , , ). for example, the range of total plasma plant sterols in human serum under normal conditions was found to be be- tween . to . �mol/l. stalenhoef et al. ( ) reported mean plasma levels of . �mol/l for campesterol and . �mol/l for sitosterol in healthy subjects; the campes- terol levels varied . -fold (range . to . �mol/l) and the sitosterol levels varied . -fold (range . to . �mol/ l). the mean baseline levels of sitosterol of . �mol/l and of campesterol of . �mol/l in our obligate heterozygotes were about -fold higher than those found in our age- and population-specific controls. four of the amish obligate heterozygotes had both sitosterol and campesterol levels that were two sd or higher above the mean for our controls. however, these values ranged from . to . �mol/l, and were clearly not elevated to a range intermediate between normals and phytosterolemic homozygotes (� �mol/l). thus, while theoretically heterozygotes might have a % deficiency in the intestinal absorption and hepatic elimina- tion of plant sterols leading to a more significant increase in their plasma plant sterols, this does not appear to be the case, and the function of the normal abcg allele appears to compensate for the mutant allele, thereby leading to the recessive phenotype ( – ). the implication for the development of cad when plant sterol levels are in the range of . to . �mol/l is not known. in a cross-sectional study, glueck and co- workers ( ) reported such modestly elevated levels in probands and relatives from families with premature cad. however, these observations may be confounded by dietary intake of sterols and age factors. berge et al. ( ) have recently found that the plasma levels of campesterol and sitosterol are heritable, and that two common dna sequence variations (d h and t k) in the abcg gene are associated with lower concentrations of these plant sterols. thus, the abcg gene appears to influence the plant sterol levels in the general population. in fact, a higher ratio of plasma plant sterols to cholesterol levels appears to be correlated positively with the fractional ab- sorption of cholesterol but negatively with cholesterol synthesis ( ). in the scandinavian simvastatin survival study, those patients with cad who had a lower ratio of plasma plant sterols to cholesterol, and consequently a greater biosynthesis of cholesterol, were found to have a greater reduction in plasma cholesterol levels with treat- ment, and reduced recurrences of cad events ( ). reduced cholesterol solubilization in bile acid micelles has been proposed as an important mechanism in the in- hibition of absorption of cholesterol by phytosterols ( ). since campesterol and sitosterol are more hydrophobic than cholesterol, they have a higher affinity than choles- terol for micelles, thus restricting the solubility of cho- lesterol. this should affect both dietary and biliary cho- lesterol absorption, and our observation that the plant sterols appear effective even in the face of a reduced-cho- lesterol diet supports this mechanism. one study ( ) found that the addition of g/day of sitostanol to a low- cholesterol diet was not efficacious in lowering plasma cholesterol in moderate hypercholesterolemic men, an observation that might be explained by the likelihood that the stanols used in this study were not in solution. the absorption of cholesterol and plant sterols by intes- tinal cells was previously postulated to be nonspecific, but now a high-affinity receptor-mediated mechanism has been proposed ( ). if this hypothesis is correct, then plant sterols might also inhibit the high-affinity receptor- mediated uptake of cholesterol, as well as displace choles- terol from micelles. either of these two mechanisms might lead to decreased cholesterol absorption and esterifica- tion in the intestine, less subsequent excretion into the blood stream on chylomicrons, decreased hepatic uptake of cholesterol from chylomicron remnants, induction of ldl receptors, and reduction in ldl cholesterol levels. the decrease in ldl cholesterol is most likely not as ef- fective as it could be, due to the compensatory increase in hepatic cholesterol biosynthesis ( , ). the identification of a high-affinity receptor that nor- mally mediates the absorption of cholesterol and plant sterols has been elusive. if abcg and abcg co-coordi- nately functioned as such a receptor, one might expect mutations in the genes for these half abc transporters to produce decreased intestinal absorption of sterols. the opposite occurs in the phytosterolemic homozygotes, and therefore it has been postulated that abcg and abcg normally mediate the egress of cholesterol and plant ste- rol from inside the intestinal cell back out into the intesti- nal lumen. in the intestine, cholesterol feeding up-regu- lates the genes for abgg and abcg , an effect that is regulated by the oxysterol receptors, liver x-activated re- ceptor (lxr)�, and lxr�, transcription factors that regulate the expression of genes involved in cholesterol efflux, storage, catabolism, and elimination ( ). in enterocytes, lxr agonists also markedly up-regulate the gene for abca , a full-sized abc transporter that medi- ates the efflux of cholesterol from cells ( ). however, mutations in abca cause tangier disease ( ), and it is unlikely that abca is the primary receptor that regulates sterol absorption. scavenger receptor class b type i (sr- bi) is another cell surface receptor postulated to play a role in cholesterol absorption ( ). ezetimibe, a drug that kwiterovich et al. response of phytosterolemic heterozygotes to plant sterols inhibits the absorption of cholesterol in humans ( ), binds with high affinity to sr-bi, and overexpression of sr-bi in chinese hamster ovary cells resulted in increased cholesterol uptake, which was blocked by ezetimibe ( ). however, sr-bi knockout mice have normal cholesterol absorption, indicating that sr-bi may have a role in cho- lesterol absorption, but is not essential for it to occur ( ). we used the amount of test margarine that one is likely to encounter in individuals attempting to lower their total and ldl cholesterol an additional amount over that obtained with a low total-fat, saturated-fat, and cholesterol diet. we did not examine, therefore, the dose-response relationship between the amount of dietary plant sterol and the ldl cholesterol level. others ( ) found no evidence for a statis- tically significant dose-response relationship between cho- lesterol lowering and three different, relatively low, intake levels of plant sterols ( . , . , and . g/day). the ab- sence of a clear dose dependency may be due to the com- pensatory increase in cholesterol synthesis that occurs after ingestion of higher dosages of plant sterols ( , ). in a double-blind, randomly-crossed-over controlled feeding study of mildly hypercholesterolemic adults fed . g plant sterols daily, judd et al. ( ) found that base- line levels were a significant predictor of the final concen- trations for all blood lipids and lipoproteins. we also ob- served in both protocol i and protocol ii that those obligate heterozygotes for phytosterolemia with the high- est baseline ldl cholesterol levels had the greatest fall in ldl cholesterol during the treatment period. those with the lowest ldl levels also had a response, however, and the response does not appear to be simply a function of the baseline ldl levels. furthermore, in examining a treat- ment effect of plant sterol esters, differences in baseline ldl levels were taken into account (see methods). our pediatric subjects, � years of age, responded similarly to the young and older adults in the study. tammi et al. ( ) found that doubling dietary plant sterol intake in - month-old children almost doubled the serum plant ste- rol concentrations. becker et al. ( ) previously reported that children with heterozygous fh responded to either sitosterol or sitostanol with a significant reduction of ldl cholesterol. in agreement with at least a half dozen other reports ( , , ), we found no significant treatment effect of the plant sterol esters on the levels of hdl cholesterol or apoa-i, its major apolipoprotein, in protocol i. the treat- ment effect on the levels of hdl cholesterol and apoa-i in protocol ii may have been due to the increase in hdl and apoa-i in the placebo washout period when the di- etary fat content of the diet increased. the reduction in ldl cholesterol that we observed was accompanied by a significant fall in the apob levels, in agreement with a number of previous reports ( ). this observation further suggests that treatment with a plant sterol-enriched mar- garine decreases the number of atherogenic ldl parti- cles, and is not simply decreasing the core cholesteryl es- ter content. in agreement with a number of other studies ( – , ), we found no evidence that the use of a plant sterol es- ter-enriched food decreased significantly the plasma lev- els of total carotenoids, retinol, and tocopherols. even though we observed significant reductions of �-cryptoxan- thine, lycopene, and �-carotene, the levels were within the broad limits of the normal population ranges. thus, in re- gard to plasma carotenoids and fat-soluble vitamins, there appears to be no problem based on these relatively short- term studies in a special population of subjects. a concern has also been raised that a plant sterol-enriched marga- rine may decrease the availability of �- and �-carotene, even after correcting for changes (decreases) in ldl-c levels ( ). speculatively, this may decrease the availability of such antioxidants and promote the oxidation (and thus atherogenicity) of ldl, but this study provides no infor- mation in that regard except that the levels of �-carotene did decrease significantly on the plant sterol spread. a larger cohort of older obligate heterozygotes for phy- tosterolemia will be required to determine if such carriers are at high risk for cad. future longer-term studies will also be required in large free-living populations to deter- mine if small increases in plasma plant sterols might be as- sociated with the development of atherosclerosis. we be- lieve that this is unlikely, given the good prognosis of populations that consume diets low in animal fat and en- riched with plant products. normal humans, and appar- ently obligate heterozygotes for phytosterolemia, have a remarkable capacity to excrete plant sterols from the liver into bile, thus preventing their accumulation in the body. the authors thank dr. beverly clevidence for performing the analyses of the plasma carotenoids, retinol, and tocopherols, and dr. matthew kramer for help with the statistical analyses. both dr. clevidence and dr. kramer are with the united states department of agriculture. we thank dr. gert meijer for his helpful discussions about the study. dr. richard kelley oversaw the analyses of the plant sterol levels and provided critical re- view of the manuscript. we also thank dr. d. holmes morton, christine l. hendrickson, donna robinson, and lee ann drum of the clinic for special children in strasburg, pa; dr. brian henry and jo ingraham of the due west family medical clinic, due west, sc; and dr. alvin graber and valerie havi- land of nappanee medical clinic, nappanee, in for their gen- erous support in the conduct of both protocols. the authors thank sharon blackburn for providing the graphic arts. this study was partly supported by an unrestricted research grant from unilever bestfoods. references . goldstein, j. l., and m. s. brown. . the cholesterol quartet. science. : – . . kwiterovich, p. o., jr. . the effect of dietary fat, antioxidants and pro-oxidants on blood lipids and lipoproteins and atheroscle- rosis. j. am. diet. assoc. 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staab, and k. von bergmann. . treatment of se- vere familial hypercholesterolemia in childhood with sitosterol and sitostanol. j. pediatr. : – . 	response of obligate heterozygotes for phytosterolemia to a low-fat diet and to a plant sterol ester dietary challenge 	methods 	study participants 	study protocols 	study design 	statistical methods 	laboratory methods 	results 	individual responses of obligate heterozygotes to modified-fat and plant sterol ester challenge 	group response of obligate heterozygotes for phytosterolemia to step diet and to a plant sterol ester challenge 	response of familial controls to the plant sterol ester challenge 	discussion 	references development and evaluation of novel snp assays for population genetic studies of westslope cutthroat trout and genetic identification of related taxa development and evaluation of novel snp assays for population genetic studies of westslope cutthroat trout and genetic identification of related taxa n . r . c a m p b e l l , * s . j . a m i s h , † v . l . p r i t c h a r d , ‡ k . m . m c k e l v e y , § m . k . y o u n g , § m . k . s c h w a r t z , § j . c . g a r z a , ‡ g . l u i k a r t †, ¶ a n d s . r . n a r u m * *columbia river inter-tribal fish commission, f national fish hatchery rd, hagerman, id , usa, †division of biological sciences, fish and wildlife genomics group, university of montana, missoula, mt , usa, ‡southwest fisheries science center and the university of california, santa cruz, shaffer rd, santa cruz, ca , usa, §usda forest service, rmrs, forestry sciences laboratory, e. beckwith avenue, missoula, mt , usa, ¶flathead lake biological station, university of montana, polson, mt , usa abstract dna sequence data were collected and screened for single nucleotide polymorphisms (snps) in westslope cutthroat trout (oncorhynchus clarki lewisi) and also for substitutions that could be used to genetically discriminate rainbow trout (o. mykiss) and cutthroat trout, as well as several cutthroat trout subspecies. in total, expressed sequence tag-derived loci were sequenced and allelic discrimination genotyping assays developed from of the variable sites. another putative snps in westslope cutthroat trout were identified by restriction-site-associated dna sequencing, and seven of these were developed into assays. twelve o. mykiss snp assays that were variable within westslope cutthroat trout and previously published snp assays were also included in downstream testing. a total of assays were tested on six westslope cutthroat trout populations (n = per population), as well as collections of four other cutthroat trout subspecies and a population of rainbow trout. all assays were evaluated for reliability and deviation from hardy–weinberg and linkage equilibria. poorly performing and duplicate assays were removed from the data set, and the remaining assays were used in tests of population differentiation. the remaining markers easily distinguished the various subspecies tested, as evidenced by mean gst of . . a smaller subset of the markers (n = ; average gst = . ) was useful for distinguishing the six populations of westslope cutthroat trout. this study increases by an order of magnitude the number of genetic markers available for the study of westslope cutthroat trout and closely related taxa and includes many markers in genes (developed from ests). keywords: admixture, hybridization, kaspar, oncorhynchus clarki, population genomics, rainbow trout, snp, taqman, westslope cutthroat trout received january ; revision received april ; accepted april introduction cutthroat trout (oncorhynchus clarki) are native to cold water environments in western north america from alaska to new mexico, usa. in the late miocene and early pliocene, cutthroat trout spread throughout por- tions of western north america via paleohydrological connections influenced by orogeny and climate (smith et al. ). subsequent isolation in major river basins led to the evolution of up to subspecies that are geographi- cally, phenotypically and genetically distinct (behnke ). the coastal subspecies (o. c. clarki) inhabits lakes and streams west of the cascade crest and includes both resident and anadromous populations (behnke ). the remaining three major lineages (westlope, yellow- stone group and lahontan group) are found in streams and lakes of the rocky mountains on both sides of the continental divide (behnke ; wilson & turner ). all cutthroat trout are sensitive to changes in their environment, and human impact on water temperature and quality has caused the decline of many populations (shepard et al. ). introductions of non-native species to their habitat have also supplanted native cutthroat trout populations by way of competition for resources, predation and interbreeding (griffith ; young ; ruzycki et al. ; muhlfeld et al. ). these effects correspondence: nathan r. campbell, fax: - - ; e-mail: camn@critfc.org � blackwell publishing ltd molecular ecology resources ( ) doi: . /j. - . . .x have led to the extinction of two cutthroat subspecies (o. c. alvordensis and o. c. macdonaldi) and another three subspecies, lahontan (o. c. henshawi), paiute (o. c. selen- iris) and greenback (o. c. stomias) cutthroat trout are listed as threatened under the us endangered species act (esa). moreover, declining populations of wests- lope (o. c. lewisi), yellowstone (o. c. bouvieri), bonneville (o. c. utah), colorado (o. c. pleuriticus) and rio grande (o. c. virginalis) cutthroat trout subspecies are of conservation concern although not currently protected under the esa. the most widely distributed of the cutthroat trout subspecies is the westslope cutthroat trout (behnke ). historically, this subspecies ranged from the rocky mountains of southern canada in the north to central idaho in the south (both east and west of the continental divide) with isolated populations in the eastern cascade range of british columbia, washington and oregon (behnke ). however, recent estimates have revealed that genetically pure westslope cutthroat trout now inhabit only % of their native range (cosewic ; may ). despite these dismal statistics, many popula- tions of this subspecies still persist across a wide geo- graphical area suggesting that a great deal of genetic diversity still exists. genetic tools to better understand the genetic relationships between westslope cutthroat trout populations and their habitats could be an impor- tant tool in their conservation and are the main focus of this study. genetic analyses within cutthroat trout subspecies using microsatellite markers have revealed that popula- tions are often highly genetically distinct from one another, even within the same drainage (e.g. taylor et al. ; wofford et al. ; drinan et al. ). these sub- stantial genetic differences are attributable to physical barriers to fish passage that reduce gene flow, and rela- tively small breeding populations with high rates of genetic drift (waples et al. ; fausch et al. ; young ). human impacts on fish passage and water quality have likely led directly to the isolation of wild cutthroat to headwater streams (fausch et al. ) and, in many streams, introduction of rainbow trout and subsequent hybridization has further isolated pure cutthroat trout to small tributary streams. rainbow trout and cutthroat trout are sister species, and hybridization between them is common when they share habitat. hybridization with introduced rainbow trout has been described as the primary threat to the sur- vival of many cutthroat trout subspecies because of diminished fitness and reproductive success of hybrids and their offspring (leary et al. ; allendorf & leary ; muhlfeld et al. ). mechanisms for reduced fit- ness may include genetic incompatibilities such as chro- mosomal rearrangements, possible differences in the numbers of chromosomes (rainbow trout: n = – ; cut- throat trout: n = – ) and the disruption of co-adapted gene complexes, which contribute to outbreeding depres- sion (leary et al. ). despite these potentially deleteri- ous effects, rainbow trout dna can spread rapidly through a cutthroat trout population because of the rela- tively high survival and reproductive success of first gen- eration (f ) hybrids (muhlfeld et al. ). owing to the difficulty in the phenotypic identification of hybrids, molecular and genetic markers such as allozymes, aflps and microsatellites have been used to study hybridization (allendorf & leary ; hitt et al. ; pritchard et al. ). more recently, however, diagnostic single nucleotide polymorphisms (snp) loci have become available for detection of invasive rainbow trout dna (kalinowski et al. ; hohenlohe et al. ; pritchard et al. ). in this study, we identified variation in cutthroat trout dna sequence data and developed assays for snps in westslope cutthroat trout and for substitutions that dis- tinguish westslope cutthroat trout from other cutthroat trout subspecies and from rainbow trout. variable sites were identified by sequence alignment and assays were developed for the most polymorphic loci, focusing on snps that were variable within westslope cutthroat trout or substitutions diagnostic between taxa. another seven snp loci were developed based on restriction-site-associ- ated dna (rad) sequence data generated using rainbow and cutthroat trout samples (hohenlohe et al. ). all assays were evaluated by genotyping a set of cutthroat trout samples from populations and a single rainbow trout population. assays with poor plot quality (i.e. geno- type resolution) and significant deviations from hardy– weinberg proportions were removed from the data set. the remaining assays were evaluated for their ability to distinguish populations of westslope cutthroat trout and identify cutthroat trout subspecies and hybrids with rain- bow trout. methods snp discovery: ascertainment panel salmonid dna sequences, mostly from mrna tran- scripts, were collected from online database entries in tigr and ncbi. sequences containing + bases of -prime un-translated region (utr) were chosen preferentially as these tend to contain more variation than coding sequence. primers were designed using either primer (http://frodo.wi.mit.edu/primer /) or the embedded primer design tool in ncbi’s database to target gene regions – bp in length. all primer pairs (n = ) were initially tested for amplification of a single product using dna from two westslope cutthroat � blackwell publishing ltd n . r . c a m p b e l l e t a l . trout. pcr conditions were · multiplex master mix (qiagen, valencia, ca, usa), nm primers and – ll of extract dna in a ll reaction. thermal cycling con- ditions were [ �c for min ( �c for s, �c for s, �c for s, repeated cycles) and �c hold]. amplified products were size separated on a % agarose gel and stained using sybr-safe reagent (life technolo- gies, grand island, ny, usa). primer pairs that produced clean pcr products (n = ) were used to screen an ascertainment panel of individuals (table s ). westslope cutthroat trout were the best represented subspecies in the ascertainment panel, and samples were chosen from collections from different drainages in idaho, montana and british columbia. another two sam- ples each of yellowstone, bonneville and coastal cut- throat trout were included in the ascertainment panel as well as one doubled haploid rainbow trout (table s ). prior to pcr, all dna extracts were quantified and individuals from the same drainage ⁄ collection were duplexed (two individual extracts combined into one aliquot) to increase the number of screened genomes while keeping sequencing costs low. ascertainment samples were then pcr amplified using multiplexed primers such that each reaction pro- duced four products and with the same pcr and cycling conditions as in the initial amplification test. pcr prod- ucts were cleaned by treatment with exonuclease and shrimp alkaline phosphatase (new england biolabs, ipswich, ma, usa) to remove unincorporated dntps and primers. sequencing reactions were performed in one direction using the big dye v. . sequencing kit (life technologies) and with ll of each product (as the sequencing primers are different for each amplicon, there is no need to separate them prior to sequencing). the reaction products were purified by ethanol-edta-naac- etate precipitation prior to sequencing by capillary elec- trophoresis using a automated dna analyzer instrument (life technologies). chromatogram data were analysed with sequencing analysis v. . (life tech- nologies), and sequencher v. . (gene codes corpora- tion, ann arbor, mi, usa) was used to align and edit the data. observed snps and small indels were recorded and minor allele frequencies calculated for each. sequences containing sites chosen for assay design were submitted to dbsnp (tables s and s ). snp discovery: ascertainment panel a second sanger sequencing snp discovery effort using a distinct ascertainment panel (table s ) focused on vari- ation within westslope cutthroat trout and between cut- throat trout subspecies. an expanded version of this same panel was used previously for identification of sites diagnostic between rainbow and cutthroat trout (pritchard et al. ). initial pcr was performed using primer pairs, designed from randomly chosen nuclear tentative consensus sequences generated from o. mykiss expressed sequence tags reported to the harvard ⁄ dfci gene index project (http://compbio.dfci. harvard.edu/tgi/tgipage.html). pcr, subsequent visuali- zation, product purification and sequencing were as described in pritchard et al. . variable sites were identified by visual examination of sequence data aligned using sequencher v . . (gene codes corporation, ann arbor, mi, usa). sequences containing sites chosen for assay design were submitted to dbsnp (tables s and s ). it is noted there is no overlap between the pritchard et al. assays and the assays reported here as that study focuses strictly on diagnostic markers between o. mykiss and o. clarki. assays reported here are either variable within westslope cutthroat trout or diagnostic between cutthroat subspecies. snp discovery: ascertainment panel restriction-site-associated dna sequence data generated on an illumina gaii instrument for identification of diag- nostic snps between rainbow and cutthroat trout (hohenlohe et al. ) had also identified snps within westslope cutthroat trout. rad-tagged sequences from westslope cutthroat trout samples included fish from wild populations from both sides of the continental divide in montana (table s ). strict filtering based on observed heterozygosity and deviations from hardy– weinberg (hw) proportions was applied to remove homologs (described in hohenlohe et al. ). fifty of the loci with highest minor allele frequency that were sequenced in at least ten of the samples were identified for assay design. a search with these -bp sequences against a draft of the o. mykiss genome (m. miller per- sonal communication – miller.michael.ryan@gmail.com) was used to generate enough flanking sequence for designing assays. assay design and genotyping suitable snp and diagnostic sites were used to design either taqmantm (life technologies) or kaspar (kbioscience, herts, england, uk) genotyping assays, which use different chemistry, by submitting sequence data to each company. both types of assay were designed for some of the snp sites, but only one for most sites. these assays, along with another eight previously pub- lished diagnostic assays (harwood & phillips ), were used to genotype collections of approximately fish each representing each of the following species ⁄ subspe- cies: rainbow trout (one collection), westslope (six collections), yellowstone (two collections), bonneville � blackwell publishing ltd s n p d e t e c t i o n a s s a y s f o r c u t t h r o a t t r o u t (one collection), coastal (one collection) and lahontan cutthroat trout (one collection). all genotyping was per- formed using the ep system with . dynamic geno- typing arrays (fluidigm). taqmantm assay genotyping was modified from the manufacturers’ suggested proto- cols to include a sample preamplification step and an increase to cycles of pcr. the sample preamplification used · qiagen multiplex master mix, nm pooled primers and ll of extract dna in a ll reaction, [thermal cycler conditions: �c– min ( �c– s, �c– min, cycles) and �c–hold]. kaspar assay genotyping used reagents and thermal cycling conditions as recommended by the manufacturer and also included a preamplification step. data analysis the program genepop (raymond & rousset ) was used to evaluate deviations from hw proportions and test for statistically significant linkage disequilibrium (ld), after corrections for multiple tests with b-y false discovery rate (narum ). assays that produced poor quality genotyping plots or significant deviations from hw proportions in multiple collections were removed from further testing. the program genetix (belkhir et al. ) was used to calculate gst and evaluate genetic differentiation by factorial correspondence analysis (fca). individual assignment tests were performed using the program geneclass (piry et al. ) to assess the ability of this set of assays to assign individuals back to their collection of origin. program settings used a bayesian method (rannala & mountain ) for individual assignment and a score threshold of . . results of the primer pairs producing clean pcr fragments, also generated adequate sequence data for snp detection. another rad-tagged dna fragments (hohenlohe et al. ) were examined for identification of snps within westslope cutthroat trout. there were sites chosen for assay design with either variation among westslope cutthroat trout samples or that were putatively diagnostic between westslope cutthroat trout and either rainbow trout or other cutthroat trout subspecies. of these, a total of were successfully converted into genotyping assays (taqmantm = ; kaspar = ; both = ). assay primer ⁄ probe sequences are listed in supplemental tables s and s . the designed assays were then evaluated by genotyp- ing samples from cutthroat trout collections and a sin- gle rainbow trout collection (table ). these samples were also genotyped using rainbow trout assays and previously published diagnostic assays (supplemental tables s and s ). assays with poor quality genotyping plots, low genotyping success and duplicated loci were removed from further analysis (n = ). mitochondrial snps were collapsed into haplotypes, and the unique haplotypes observed were coded into single genotypes. significant deviations from hwe within multiple popu- lations were observed in eight snp loci. two of these were sex-linked snps and were retained, because the observed heterozygote deficits were expected, and the other six were removed from further analysis. statistically significant ld was detected in of pairwise tests for one or more of the six westslope cutthroat trout collections (p-value adjusted for multiple tests < . ). seven of these locus pairs, involving total snps, were in significant ld in at least two of the collec- tions (supplemental table s ). assays were excluded based on significant ld only for fca and assignment testing (exclusion of four assays resolved all seven signif- icant pairs). only westslope cutthroat trout were used for this analysis because of the lack of variation within the other subspecies collections. several pairs of loci known to be from the same sequence were not in significant ld table assignment test among collections of cutthroat trout subspecies and rainbow trout using geneclass collection drainage n % correct assignment ave. confidence (%) wilson cr. (o. mykiss) salmon r. . . dog cr. (o. c. clarki) hood r. . . glenwood hat. (o. c. utah) hatchery stock . . l. lenore (o. c. heshawi) hatchery stock . . barnes cr. (o. c. bouvieri) south fork snake r. . . gibson cr. (o. c. bouvieri) portneuf r. . . trout cr. (o. c. lewisi) kootenai r. . . mcguire cr. (o. c. lewisi) kootenai r. . . schwartz cr. (o. c. lewisi) upper clark r. . . mccabe cr. (o. c. lewisi) blackfoot r. . . flat cr. (o. c. lewisi) lower clark r. . . cache cr. (o. c. lewisi) salmon r. . . n, number of individuals; ave. confidence, average of the likeli- hood score for correct assignment calculated for each individual within a population by geneclass . � blackwell publishing ltd n . r . c a m p b e l l e t a l . because of the diagnostic nature (near fixation between taxa) of one or both of the sites. likewise, lack of hetero- zygosity within the westslope cutthroat trout collections may have masked ld for many other pairs of loci. the remaining genotypes were used to evaluate the utility of these assays to differentiate populations of westslope cutthroat trout as well as distinguish cutthroat trout subspecies from one another and from rainbow trout. analysis of the gst values for each locus reveals that more than half of the assays are informative only for differentiation between the tested species ⁄ subspecies (fig. and supplemental tables s and s ). this leaves markers with significant differentiation between the six included westslope cutthroat trout collections (gst > . ), with the average gst of . , suggesting a high degree of differentiation between these six westslope cut- throat trout collections. average heterozygosity within these collections for that subset of snps ranged from . to . indicating that heterozygosity within indi- vidual westslope cutthroat trout populations is quite var- iable. the population with the lowest observed heterozygosity was mcguire cr. in the upper kootenai river drainage. the trout cr. population from the lower kootenai river, however, showed much higher heterozy- gosity (hobs = . ) implying isolation of the mcguire cr. population from the lower kootenai river gene pool. the genotyped collections separated extremely well using this set of markers as evidenced by the fca plots (fig. ) and assignment tests (table ). the large number of markers that were informative for differentiation between both the different cutthroat subspecies and between cutthroat ⁄ rainbow trout allowed the major lin- eages to be easily distinguished from one another (fig. a). five fish from the coastal cutthroat collection were identified as rainbow trout, and four fish were iden- tified as rainbow ⁄ cutthroat trout hybrids by their inter- mediate positions on the fca graph. the misidentified and hybrid fish were then removed from the data set for fca and assignment testing of westslope cutthroat trout collections. separation of westslope cutthroat trout collections on the fca plot was less defined (fig. b), as it relied on variation in a much smaller subset of snps in more genetically similar groups. the first two axes of the westslope fca, however, only explained . % of the total variation observed with these markers. subsequent assignment testing showed that every individual fish could be correctly assigned back to their collection of ori- gin using only genetic data from these markers (table ). moreover, the confidence of the assignments was high and no misassignments were observed even between populations from the same lineage, including the two populations of yellowstone cutthroat trout for which dif- ferences in allele frequency of % or more occur in only of the markers. supplemental tables s and s contain minor allele frequencies and population differentiation information that describes the utility of each marker. discussion this study used multiple methods for snp discovery that were influenced by different ascertainment biases. for example, one of the sanger sequencing efforts used an ascertainment panel directed at finding snps within the westslope cutthroat trout subspecies while the other included roughly equal representation of all extant cut- throat trout subspecies. as each panel contained individ- uals from several subspecies, it was possible to obtain both putatively diagnostic markers and snps that were variable within westslope cutthroat trout from both efforts. however, there was significant ascertainment bias for variation in a small number of westslope cut- throat trout populations when the second panel was used, because of the limited number and origin of wests- lope cutthroat trout samples included in discovery (pritchard et al. ). a total of snp sites variable within westslope cutthroat trout were chosen from this sequencing panel. likewise, limited representation of the cutthroat trout subspecies in all three panels made the identification of truly diagnostic markers from sequence fig. graph of gst for each of the single nucleotide polymorphism loci. grey bars indicate gst when all collections are included and black dots indicate gst within only the westslope cutthroat trout populations. � blackwell publishing ltd s n p d e t e c t i o n a s s a y s f o r c u t t h r o a t t r o u t data unreliable. of the sites chosen from sequence data as putatively diagnostic, only had fixed geno- types in all subsequently genotyped taxa. however, although not strictly diagnostic, many of these markers were nearly so, given the low frequency of alternate alleles, and were still powerful for population identifica- tion. the occurrence of alternate diagnostic alleles may also be due to limited introgression within some of the tested populations. moreover, as only a single population of rainbow trout was genotyped, the representation of o. mykiss diversity was far from exhaustive. previous studies using microsatellite loci have shown marked genetic distinctions among westslope cutthroat trout populations (young et al. ; drinan et al. ). these genetic differences, often even within the same watershed, have led to recommendations for stream-level management (young et al. ). although population coverage is limited, our results also reveal strong genetic differentiation among westslope cutthroat trout popula- tions. we also observed low heterozygosity within some populations, suggesting a risk of inbreeding depression, but it is unclear the extent to which this is owing to ascer- tainment bias. the average heterozygosity of westslope cutthroat trout informative snps (hobs = . ) is some- what lower than that observed in similar snp panels for populations of anadromous, less genetically isolated sockeye and coho salmon (hobs = . and . ; campbell & narum ). this is likely a result of limited migration and gene flow in relatively isolated popula- tions of westslope cutthroat trout. the development of informative genetic markers for the study of nonmodel organisms is the most costly (a) (b) fig. factorial correspondence analysis plots generated using the program genetix ( markers). (a) individuals from all species ⁄ subspecies collections. f rainbow ⁄ cutthroat trout hybrids are indicated in the red ellipse. (b) individuals in the six westslope cutthroat trout populations. � blackwell publishing ltd n . r . c a m p b e l l e t a l . segment of most population genetics studies. once a set of markers has been developed and validated, the costs associated with population genetic analysis decreases significantly. here, we describe a set of snp and diagnostic marker genotyping assays and their relative utility in differentiation of populations of westslope cutthroat trout and diagnosis for several cutthroat trout subspecies and rainbow trout. the snp assays evaluated in this study will be useful for a broad range of research questions, including westslope cutthroat trout popula- tion structure, detection of other cutthroat trout subspe- cies and identification of introgression and hybrids between related taxa. acknowledgements we would like to thank matt campbell, denise hawkins, jessica metcalf, kirk patten, mary peacock, dennis shiozawa, christian smith, chris rossel, robb leary and montana fish wildlife and parks for supplying cutthroat trout samples for genotyping. thanks also to sally painter and megan moore for help with dna extraction and genotyping. funding for this study was provided by the bonneville power administra- tion, the rocky mountain research station, the us fish and wildlife service (lahontan national fish hatchery and abernathy fish technology center) and the national oceanic and atmospheric administration (southwest fisheries science center). references abadı́a-cardoso a, clemento ac, garza jc ( ) discovery and charac- terization of single nucleotide polymorphisms in steelhead ⁄ rainbow trout, oncorhynchus mykiss. molecular ecology resources, (suppl. ), – . allendorf fw, leary rf ( ) conservation and distribution of genetic variation in a polytypic species, the cutthroat trout. conservation biol- ogy, , – . behnke rj ( ) native trout of western north america. american fisher- ies society, bethesda, md. behnke rj ( ) trout and salmon of north america. the free press, new york, ny. belkhir k, borsa p, chikhi l, raufaste m, bonhomme f ( ) genetix . , logiciel sous windowstm pour la génétique des populations. montpel- lier, france, laboratoire génome, populations, interactions, cnrs umr , université de montpellier ii. brunelli jp, thorgaard gh ( ) single-nucleotide polymorphisms associated with allozyme differences between inland and coastal rainbow trout. transactions of the american fisheries society, , – . campbell nr, narum sr ( ) development of novel single- nucleotide polymorphism (snp) assays for sockeye and coho salmon and assessment of available snps to differentiate stocks within the columbia river. molecular ecology resources, (suppl. ), – . castaño-sanchez c, smith tpl, wiedmann rt et al. 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( ) characterizing diversity in salmon from the pacific northwest. journal of fish biology, (suppl. a), – . wilson wd, turner tf ( ) phylogenetic analysis of the pacific cutthroat trout (oncorhynchus clarki ssp.: salmonidae) based on partial mtdna nd sequences: a closer look at the highly frag- mented inland species. molecular phylogenetics and evolution, , – . wofford jeb, gresswell re, banks ma ( ) influence of barriers to movement on within-watershed genetic variation of coastal cutthroat trout. ecological applications, , – . young mk(ed.) ( ) conservation assessment for inland cutthroat trout. us forest service general technical report. rm- . rocky mountain research station, fort collins, co, pp. young mk ( ) generation-scale movement patterns of cutthroat trout (oncorhynchus clarkii pleuriticus) in a stream network. canadian journal of fisheries and aquatic sciences, , – . young sf, mclellan jg, shaklee jb ( ) genetic integrity and microgeo- graphic population structure of westslope cutthroat trout, oncorhyn- chus clarki lewisi, in the pend oreille basin in washington. environmental biology of fishes, , – . study design, sample collection, and oversight by ks mckelvey, mk young, mk schwartz, jc garza, g lui- kart and sr narum. snp identification using sanger sequencing was performed by nr campbell and vl pritchard. snps were identified within rad sequencing data by sj amish and g luikart. assay design, genotyp- ing, and assay evaluation was completed by nr camp- bell, sj amish, and vl pritchard. data accessibility snp loci: dbsnp accessions ss – ss . the accessions are matched with reported assay names in supplemental data tables. snp detection assays: tables with primer ⁄ probe sequences uploaded as online supplemental material. genotype data: genotypes at all tested loci for indi- viduals in all collections, genepop files used for analy- sis, and related raw data available in the dryad database (doi: . /dryad. m h m p). supporting information additional supporting information may be found in the online version of this article. table s describes each of the taqmantm assays, the panel used for snp discovery, their minor allele frequencies for each popu- lation tested, and their informativeness for distinguishing popu- lations (gst). table s describes each of the kaspar assays, the panel used for snp discovery, their minor allele frequencies for each popu- lation tested, and their informativeness for distinguishing popu- lations (gst). table s describes the ascertainment panels used for snp dis- covery. table s statistically significant linkage disequilibrium (ld) between marker pairs within or more populations. please note: wiley-blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. � blackwell publishing ltd n . r . c a m p b e l l e t a l . bone morphogenic protein- induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney* bone morphogenic protein- induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney* received for publication, november , , and in revised form, december , published, jbc papers in press, december , , doi . /jbc.m michael zeisberg‡, amish a. shah, and raghu kalluri§ from the center for matrix biology, department of medicine, beth israel deaconess medical center and harvard medical school, boston, massachusetts and division of nephrology, children’s hospital and harvard medical school, boston, massachusetts in the kidney, a unique plasticity exists between epi- thelial and mesenchymal cells. during kidney develop- ment, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (met). in the injured adult kidney, renal epithelia contribute to the generation of fibro- blasts via epithelial-mesenchymal transition, facilitat- ing renal fibrosis. recombinant human bone morpho- genic protein (bmp)- , a morphogen that is essential for the conversion of epithelia from condensing mesen- chyme during kidney development, enhances the repair of tubular structures in the kidney. in this setting, bmp- inhibits epithelial-mesenchymal transition in- volving adult renal epithelial tubular cells and de- creases secretion of type i collagen by adult renal fibro- blasts. in search of a mechanism behind the ability of bmp- to repair damaged renal tubules, we hypothe- sized that systemic treatment with bmp- might induce met involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. here we report that bmp- induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of e-cadherin expression and decreased motility, mimicking the effect of bmp- on embryonic metanephric mesenchyme to generate epithelium. in ad- dition, we provide evidence that bmp- -mediated repair of renal injury is associated with met involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. collectively, these findings suggest that adult renal fibroblasts might retain parts of their original em- bryonic imprint and plasticity, which can be re-engaged by systemic administration of bmp- to mediate repair of tubular injury in a fibrotic kidney. a distinction between epithelial and mesenchymal cells was appreciated in the late th century based on their morphology and topic organization ( ). interconversion of mesenchymal and epithelial cells during embryonic development via epithe- lial-mesenchymal transitions (emt) and mesenchymal to ep- ithelial transition (met) are well established mechanisms in the development of multicellular organisms ( ). capacity of epithelial and mesenchymal cells to interconvert during devel- opment is essential, because formation of mesenchymal cells from epithelial cells (emt) enables these cells to migrate through an extracellular environment and settle in distinct areas to form organs ( , ). in adults, mesenchymal and epithelial cells have been tra- ditionally considered as terminally differentiated ( , ). how- ever, more recent studies demonstrate that emt is a common event among adult epithelial cells ( , ). it has now been established that adult epithelial cells of the kidney, eye, lung, peritoneum, and colon can undergo emt in chronic inflamma- tory diseases associated with fibrosis, in addition to emt as- sociated with carcinogenesis in a broad range of epithelia ( , ). in animal models of kidney fibrosis, about % of fibroblasts derive via emt, and inhibition of emt can prevent the pro- gression of fibrosis, demonstrating that emt is an important contributor to the progression of renal disease ( ). the kidney has evolved as a model system to study embry- onic and adult cellular plasticity between epithelia and mesen- chyme ( ). kidney development depends on a program of recip- rocal inductive interactions between two mesodermal derivatives, the ureteric bud and the metanephric mesenchyme ( , ). in mice, at � . days postcoitum, the ureteric bud contacts the metanephric mesenchyme, inducing these cells to condense and aggregate ( ). subsequently, this induced popu- lation undergoes mesenchymal to epithelial transition (met) to form a comma-shaped node, which elongates to form an s-shaped tubular epithelial structure ( , ). further, morpho- genesis and differentiation of this s-shaped tubule results in the formation of the glomerulus, the distal and proximal tubule elements associated with the mature nephron ( ). in the adult kidney, tubular epithelial cells can convert into fibroblast via emt ( ). during kidney fibrosis, conversion of renal tubular epithelial cells into myofibroblasts/fibroblasts is considered un- favorable, since it leads to disruption of polarized renal tubular epithelial layers and an increase in fibrotic scar formation ( ). therefore, emt in the kidney is of significant interest as a therapeutic target, and in this regard, inhibition of emt by systemic administration of bone morphogenic protein (bmp)- , * this work was supported in part by national institutes of health (nih) grants dk and dk and a research fund from the beth israel deaconess medical center for the center for matrix biol- ogy. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. ‡ supported by nih gi fellowship training grant t dk (to beth israel deaconess medical center). § to whom correspondence should be addressed: harvard medical school, center for matrix biology, beth israel deaconess medical cen- ter; dept. of medicine, dana , brookline ave., boston, ma . tel.: - - ; fax: - - ; e-mail: rkalluri@ bidmc.harvard.edu. the abbreviations used are: emt, epithelial to mesenchymal tran- sition; bmp, bone morphogenic protein; rhbmp- , recombinant human bone morphogenic protein- ; fsp , fibroblast-specific protein- ; met, mesenchymal to epithelial transition; ntn, nephrotoxic serum nephri- tis; imdm, iscove’s modified dulbecco’s medium. the journal of biological chemistry vol. , no. , issue of march , pp. – , © by the american society for biochemistry and molecular biology, inc. printed in u.s.a. this paper is available on line at http://www.jbc.org this is an open access article under the cc by license. http://creativecommons.org/licenses/by/ . / results in regression of established lesions in the kidney and improved renal function ( ). the family of bmps contains several different members and in general, bmps control morphogenic pathways at different stages of development ( , ). bmp- , also sometimes referred to as osteogenic protein- , was originally identified as a potent osteogenic factor purified from bone ( ). different studies have demonstrated a specific role for bmp- during mamma- lian kidney development ( , ). although bmp- is expressed in many organized regions of the early embryo, defects in bmp- -deficient mice are largely restricted to kidney and eye ( , ). bmp- -deficient mice die shortly after birth due to severe kidney dysplasia ( , ). in the developing kidney, bmp- is expressed initially in the ureteric bud ( ). as devel- opment proceeds, expression is next observed in the metaneph- ric mesenchyme and then in the early tubules derived from the mesenchyme ( ). bmp- -deficient kidneys show a gradual cessation of nephrogenesis, associated with a reduction of ure- teric bud and the loss of metanephric mesenchyme, suggesting that bmp- activities are essential for morphogenesis during the later stages of kidney development ( , ). bmp- is considered a principal regulator of met in kidney development and a survival factor for the metanephric mesenchyme ( , ). in adults, bmp- is robustly expressed in the kidney ( ). however, both acute and chronic renal injury are associated with substantial decrease in the expression of bmp- ( – ). several recent reports unequivocally demonstrate that admin- istration of exogenous recombinant human bmp- (rhbmp- ) enhances recovery of the kidney in animal models of acute and chronic renal injury ( , , , – ). these studies collec- tively suggest that the therapeutic effect of rhbmp- is due to its direct effect on tubular epithelial cells and interstitial fibro- blasts ( ). in previous studies, we demonstrate that bmp- can inhibit emt involving tubular epithelial cells, resulting in potential repair of injured tubules ( ). in addition, we demon- strate that rhbmp- can reduce secretion of type i collagen from adult renal fibroblast ( ). however, the precise action and mechanism of bmp- activity on fibroblasts are still un- known. here we report that bmp- can induce met and po- tentially facilitate the repair of tubular epithelial structures in injured kidneys. experimental procedures cell culture—the human renal fibroblast cell line tk was main- tained in dulbecco’s modified eagle’s medium (invitrogen) supple- mented with % fetal bovine serum, units/ml penicillin, and units/ml streptomycin ( ). the cells were cultured with % co and % humidity. for experiments, the medium was replaced with iscove’s modified dulbecco’s medium (imdm; invitrogen) without fetal bo- vine serum. in cell culture experiments, rhbmp- , dissolved in mm sodium acetate containing % mannitol, was used (curis, inc., cambridge, ma) ( ). induction of emt-dependent crescentic nephritis—the protocol for inducing crescentic glomerulonephritis in mice, modified for studies to avoid rapid lethality from acute renal failure while ensuring adequate tissue injury for analysis, was used as described in our previous publi- cation ( , ). briefly, cd mice weighing � g were preimmunized by subcutaneous injection of �g of normal sheep igg (sigma) in freund’s complete adjuvant (sigma). after days, experimental mice were injected intravenously with �l of nts for consecutive days (nts was a generous gift from dr. salant (boston university)). nts was generated by immunization of sheep with rat glomeruli as de- scribed previously ( ). control groups (n � mice/group) were sacri- ficed , , and weeks post-nts injection to obtain representative kidney specimen corresponding to the time course of ntn. treatment with �g/kg bmp- was started at weeks (n � mice/group) postinduction of ntn. for in vivo studies, recombinant human bmp- homodimer, noncovalently attached to prodomain protein (referred to as soluble rhbmp- ) was used (curis, inc., cambridge, ma) as de- scribed previously ( ). treated mice were sacrificed weeks postinduc- tion of ntn, and kidneys and plasma were obtained for future analysis. histologic assessment of renal injury—tissue of kidneys, liver, heart, spleen, and brain was fixed in % paraformaldehyde and embed- ded in paraffin. sections were stained with hematoxylin and eosin, masson’s trichrome stain, and periodic acid-schiff stain. extent of renal injury was estimated by morphometric assessment of the tubulointer- stitial injury and glomerular damage in a double-blinded manner. the relative interstitial volume was evaluated by morphometric analysis using a -mm graticule fitted into the microscope. the relative inter- stitial volume was defined as the cortical region outside tubules and glomeruli ( ). five randomly selected cortical areas, which included glomeruli, were evaluated for each animal ( ). the relative interstitial volume was defined as the cortical region outside tubules and glomeruli. immunocytochemistry—for immunocytochemistry, human renal fi- broblasts (tk ) were seeded into -well chamber slides (nalge nunc international, naperville, il) and stimulated according to the experi- mental protocol. at the end of incubation, cells were washed twice with phosphate-buffered saline and fixed with ethanol/acetic acid ( : , v/v) at � °c for min ( ). the cells were subsequently washed again, blocked with % bovine serum albumin in phosphate-buffered saline/ tween for min at room temperature, and incubated for h with primary monoclonal antibodies to e-cadherin (transduction laborato- ries). cells were then washed again and incubated for h at room temperature with fluorescein isothiocyanate-conjugated secondary an- tibodies (jackson immunoresearch). the stained cells were covered with vectashield® mounting medium containing �, -diamidino- -phe- nylindole (vector laboratories, burlingame, ca) and coverslips. the staining was visualized by fluorescence microscopy, and representative pictures were documented using zeiss axiovision software. immunohistochemistry—immunohistochemical staining was per- formed as described previously ( ). briefly, -�m sections were obtained using a cryostat. sections were fixed in % acetone at � °c for min before the sections were incubated with primary antibodies to fsp and e-cadherin (sigma) at °c overnight. subsequently, the fig. . bmp- induces condensation of adult renal fibroblasts. human renal fibroblasts were grown for imdm control medium (a, c, and e) or in medium containing ng/ml rhbmp- (b, d, f). bmp- treatment induced formation of cell aggregates within the confluent cell monolayer (b and c, arrows). f, fibroblasts within these islands dis- played abundant expression of the epithelial marker e-cadherin (green staining) (the circles mark the outer border of the cell islands). e, without bmp- treatment, fibroblasts ( �, -diamidino- -phenylindole- stained nuclei are seen in blue), did not express e-cadherin. a–d, bright field microscopy. e and f, immunofluorescence staining. original mag- nification was � (a and b) or � (c–f). met and adult renal fibroblasts slides were washed three times in tris-buffered saline and incubated with donkey-derived secondary antibodies conjugated with rhodamine or fluorescein isothiocyanate (jackson immunoresearch, west grove, pa). after four washes with tris-buffered saline, the slides were mounted with vectashield mounting medium (vector laboratories) and coverslipped. the staining was analyzed using a fluorescence micro- scope (zeiss). fsp antibody was a gift from dr. eric g. neilson. analysis of mouse e-cadherin promoter fragment activity in epithe- lial cells—for analysis of mouse e-cadherin promoter activity, a frag- ment containing the regulatory region of the e-cadherin promoter was ligated into the pxp luciferase vector ( ). this truncated construct (� e-cadherin pxp ) displayed enhanced specificity for epithelial cells compared with the full-length promoter fragment, which exhibits activity in epithelial cells as well as in fibroblasts ( , ). this con- struct (� ecad/luc)was used to transfect tk adult renal fibro- blasts, and stable clones were obtained. tk cells were co-transfected with �g each of � e-cadherin pxp construct and pneo�-gal plasmid (stratagene, la jolla, ca) using a calcium phosphate co-pre- cipitation method. cells were tested for luciferase activity, staining with antibodies to luciferase (promega, madison, wi), and �-galacto- sidase activity was assessed by an o-nitrophenyl-�-galactopyranoside substrate-based assay kit (stratagene). single-cell-derived cell lines were established by limited dilution in selection medium that contained �g/ml g ( ). a selection of cells that contained the � e-cadherin pxp construct was confirmed by staining with antibody to luciferase. stable transfected tk cells were stimulated in imdm that contained bmp- in a -well plate when they were – % con- fluent for h. measurement of luciferase activity was performed using the bright-glo™ luciferase assay system (promega) according to the manufacturer’s recommendations. migration assay—migration assays using a boyden chamber were performed as described previously ( ). briefly, polyvinyl-pyrrolidone- free polycarbonate membranes with -�m pores (neuro probes, inc.) were coated with type i collagen on both sides ( �g/ml). the bottom wells of chamber were filled with imdm medium containing supple- ments according to the specific experimental protocol. wells were cov- ered with the coated membrane sheet, and , -fibroblasts/well, which had been serum-starved for h, were added into the upper chamber. the boyden chamber was incubated for h at °c to allow possible migration of cells through the membrane into the lower cham- ber. membranes were stained with hema ® stain according to the manufacturer’s recommendations (biochemical sciences, inc., swedes- boro, nj). cells that migrated through the membrane were counted using a counting grid, which was fitted into an eyepiece of a phase- contrast microscope. all experiments were repeated at least three times. under-agarose cell spreading assay—under-agarose cell motility assays were performed as described with modifications ( ). -well plates were coated with fibronectin for h at °c. -cm-thick agarose gels ( % low melt agarose in dulbecco’s modified eagle’s medium) were cast into each well. after the gel had solidified, we punched a central hole (� cm) surrounded by four additional holes (� . cm). adult renal fibroblasts were stained with green (pkh ; sigma) or red (pkh ; sigma) fluorescent dyes according to the manufacturer’s recommenda- tions. equal uptake of the dyes was confirmed by fluorescence micros- copy, before the cells were treated for h with rhbmp- or maintained in serum-free imdm control medium. cells/ �l of serum-free medium were plated into the central cavity in the center of the gel, and the surrounding holes were each filled with �l of medium contain- ing % fetal calf serum to create a chemotactic gradient. plates were incubated for h in a cell culture incubator under standard conditions to allow the cells to migrate. the cells were fixed in % paraformalde- hyde, and the cells were visualized under a fluorescent microscope (zeiss axiovert). cells that migrated from the central hole underneath the gel were counted using a fluorescent microscope. statistical analysis—all values are expressed as mean � s.e. unless specified. analysis of variance was used to determine statistical differ- ences between groups using sigma-stat™ software (jandel scientific, san rafael, ca). further analysis was carried out using t test with bonferroni correction to identify significant differences. a level of p � . was considered statistically significant. results bmp- decreases the expression of type i collagen in adult renal fibroblasts ( ). in experiments here, treatment of adult fig. . regulation of e-cadherin expression by bmp- in adult renal fibroblasts. a, schematic representation of the proximal e-cadherin promoter fragment (� /� ), indicating the e-boxes, the ccaat box, and the gc-rich region. b, adult renal fibroblasts (tk ) were transfected with the indicated promoter fragment construct, treated with bmp- ( . – ng/ml) for h prior to lysis, and analyzed for luciferase activity. bmp- up-regulated e-cadherin promoter activity in a dose-dependent manner with a maximum induction at a concentration of ng/ml. c, adult renal fibroblasts were treated with bmp- , and the expression of e-cadherin, pax , and wnt was analyzed by immunoblot. the blots were stripped and reprobed with specific antibodies to actin to control for equal loading. bmp- -mediated increase of e-cadherin protein was associated with increased expression of wnt and pax . the experiments were repeated three separate times. representative blots are shown here. met and adult renal fibroblasts renal fibroblasts with bmp- at the optimum dose of ng/ml resulted in the formation of epithelial cell aggregates within the – % confluent fibroblast monolayer (fig. , b and d). immunocytochemistry analysis of bmp- -treated fibro- blasts revealed that cells within the aggregated/condensed ar- eas expressed abundant levels of the epithelial marker e-cad- herin (fig. f) and cytokeratin (data not shown) as compared with the untreated fibroblasts (fig. e). e-cadherin staining was diffusely distributed within the cells as well as some dis- tribution within the typical junctional regions, suggesting that the phenotype represents an early stage of met. several different studies have demonstrated that e-cadherin is an important determinant for maintenance of the epithelial phenotype ( , ). e-cadherin is an epithelial cell-specific in- tercellular adhesion molecule, which by itself can induce met if overexpressed in mesenchymal cells ( ). such studies have led to the speculation that e-cadherin is a potential epithelial master gene ( ). in order to further analyze the regulation of e-cadherin expression in adult renal fibroblasts, we performed stable transfections using a cdna fragment of the mouse e- cadherin promoter (� /� ) conjugated to the luciferase gene (lux), which contains all of the reported key regulatory elements for function in epithelial cells (fig. a) ( , ). the � /� mouse e-cadherin promoter construct (� e-cad- herin pxp ) is active only in epithelial cells with minimal activity in fibroblasts, as compared with the full-length pro- moter, which displays robust activity in both epithelial cells and fibroblasts ( , ). in dose-response studies, rhbmp- induces e-cadherin promoter activity with maximal induction ( . -fold) observed at ng/ml in tk fibroblast cells (fig. b). similar levels of induction ( -fold) of e-cadherin promoter activity, associated with epithelial-mesenchymal plasticity, has been reported previously ( , , ). formation of e-cadherin-expressing epithelial cell aggre- gates, as observed in our studies, mimics bmp- -mediated condensation of the embryonic metanephric mesenchyme, which represents the initial step in the process that leads to formation of the tubular epithelium ( , , , ). to confirm this notion, we examined the cells for the expression of pax and wnt . wnt encodes a secreted molecule, which is a puta- tive early marker for epithelium formation ( ). in the devel- oping kidney, it appears in the condensing mesenchyme and persists in primary vesicles, but it is down-regulated in the s-shaped bodies ( ). the epithelium of the adult nephron does not express wnt ( ). pax is expressed in induced mesen- chyme and in the early epithelial derivates, and it is rapidly down-regulated in mature kidney ( ). incubation of adult renal fibroblasts with the optimum dose of bmp- ( ng/ml) induced expression of both pax and wnt , further suggesting that bmp- induces formation of epithelial cell aggregates in adult renal fibroblasts, as it does in the embryonic metanephric condensing mesenchyme ( ). because fibroblasts are highly mobile, whereas epithelial cells are not, regulation of cell motility is an important aspect of the plasticity between epithelium and mesenchymal cells ( – , ). in order to determine the functional consequence of met involving adult renal fibroblasts, we evaluated the effect of bmp- treatment on cell motility. we first used a two-compartment boyden chamber migration assay, which allows evaluation of haptotactic migration (mi- gration from the upper compartment into the bottom compart- ment due to direct stimulation of the cells in the upper com- partment) and chemotactic migration (migration of cells from the upper chamber into the bottom chamber induced by a growth factor gradient from the bottom compartment) ( ). direct stimulation with rhbmp- in the upper chamber re- duced haptotactic migration of adult renal fibroblasts in a dose-dependent manner. however, this effect was statistically insignificant (data not shown). rhbmp- used as a chemoat- tractant in the bottom chamber had no effect on migration of adult renal fibroblasts (data not shown). in the boyden cham- ber assay, a single cell suspension is allowed to migrate for h; therefore, an inhibitory effect on cell motility was probably not realized, since h may be too short a period for met to occur and for epithelial cell junctions to assemble. in order to further validate the role of bmp- on the motility of human renal fibroblasts, we next used an under-agarose cell motility assay, which allows evaluation of cell motility over a -h period. we treated human renal fibroblasts for h with bmp- to induce met. we then stained bmp- -treated fibro- fig. . regulation of adult renal fibroblasts motility by bmp- . a, schematic illustration of the under-agarose cell motility assay. agarose gels were cast into -well plate, and a central hole surrounded by holes was punched into each well. a suspension of untreated fibroblasts (stained in green) and of bmp- treated fibroblasts (stained in red) was filled in the central hole, whereas the surrounding holes were filled with media containing % fetal bovine serum (fbs) to create a chemotactic gradient. the fibroblasts were allowed to migrate under the gel for h toward the chemotactic gradient. the images display representative areas of the periphery (upper image) and the center region of the central hole. the dashed line indicates the edge of the center hole; cells that are to the right of the line have migrated underneath the gel toward the chemotactic gradient. direct comparison of untreated control fibroblasts (green) and bmp- -treated fibroblasts (red) demonstrates that bmp- decreased the migratory capacity of adult renal fibroblasts. the bmp- -treated cells remained mainly in the center region of the hole. b, the bar graph summarizes cell counts of fibroblasts per visual field at � magnification that have migrated underneath the gel. experiments were performed three independent times in triplicate. met and adult renal fibroblasts blasts with a red fluorescent cell tracker dye and untreated control fibroblasts with green fluorescent dye. both cell popu- lations were then mixed at equal number, and the cell suspen- sion was placed into the middle hole in an agarose-filled tissue culture dish. the cells were then allowed to migrate from the center toward a fetal calf serum gradient for h, allowing for direct comparison between the bmp- -treated (red) fibroblasts and the untreated (green) control fibroblasts (fig. ). whereas the bmp- -treated fibroblasts remained in the center of the well, the control fibroblasts migrated toward the fetal bovine serum gradient (fig. ). in summary, these findings suggest that bmp- induces the formation of epithelial cell aggregates in adult renal fibroblasts in association with decreased cell motility. administration of rhbmp- accelerates recovery of acute renal failure and inhibits progression of fibrosis in the kidney ( , , – , ). we have previously demonstrated that administra- tion of rhbmp- results in regression of chronic renal disease associated with restoration of the tubular epithelium architec- ture ( ). in these studies, we could demonstrate that bmp- protects tubular epithelium by inhibition and potential reversal of emt ( ). we speculated that met of fibroblasts could mediate decrease of interstitial fibroblasts and additionally contribute to the repair of renal tubular epithelial structures. in order to test this hypothesis, we used a mouse model of nephrotoxic serum nephritis that, when untreated, leads to severe renal fibrosis after weeks ( ). this model is responsive to rhbmp- therapy and leads to regression of renal fibrosis and repair of damaged tubular structures ( ). we initiated treatment with �g/kg rhbmp- (established as optimal therapeutic dosage in previous fig. . reversal of chronic renal injury by bmp- in a mouse model of nephrotoxic serum nephritis. a, normal mouse kidney. the arrow points to normal interstitium between tubules with tubular epithelial cells. b, kidneys of untreated control mice week after nts injection. at this stage, the kidneys display moderate interstitial dis- ease associated with widening of the interstitial area (arrows). the blue color represents ecm accumulation as detected with masson’s trichrome stain. c, kidneys of untreated control mouse weeks after nts injection. the arrows show widening of the interstitial space, representative of the fibroblast accumulation and inflammatory cells. injury of tubular epithelial cells results in tubular atrophy (arrow- heads) d, kidneys of rhbmp- -treated mice, weeks after ntn. the rhbmp- treatment was started weeks after initiation of disease (compare with fig. c). the kidney specimen displayed vastly de- creased interstitial space (arrows) and improved tubular structure (ar- rowhead). e, summary of morphometric assessment of interstitial vol- ume. progression of interstitial fibrosis was associated with an increasing interstitial volume until week of the disease. the intersti- tial volume was decreased as compared with the untreated control after weeks of disease. fig. . bmp- mediated in an in vivo renal injury model. a–d, immunofluorescence double staining for e-cadherin (red) and fsp (green). a, in normal mouse kidney tubular epithelial cells express e-cadherin (a, red staining), whereas interstitial fibroblasts can be detected by fsp staining (a, arrows). in the normal mouse kidney, cells that co-express fsp and e-cadherin are not present. b, after the first week of ntn, the kidneys display increased numbers of fsp - positive fibroblasts. tubules with decreased levels of e-cadherin stain- ing appear. cells that co-express e-cadherin and fsp are not detect- able. c, after weeks post-nts-injection, tubular cells with de novo expression of fsp and diminished expression of e-cadherin, indicating emt, are robustly present. fsp -positive fibroblasts are increased in the interstitium. cells that co-express e-cadherin and fsp cannot be found within the interstitium. d, administration of rhbmp- (starting after weeks of ntn, until after week ; compare d with c) restored normal tubular architecture with e-cadherin expression. a novel pop- ulation of cells that co-express e-cadherin and fsp , indicating an intermediate phenotype of epithelial cell and fibroblasts, is now present within the interstitium (arrows). e, fibroblasts were identified by stain- ing with fsp antibody, and the percentage of fibroblasts of total cells within the kidney cortex was assessed. the graph displays the average of six different kidneys in each group. the progression of fibrosis in the untreated kidneys was associated with a significant increase of inter- stitial fibroblasts. treatment with rhbmp- resulted in a significant reduction of fibroblasts as compared with untreated control. f, kidneys were evaluated for the presence of cells in the interstitium that stained positive for both e-cadherin (ecad) and fsp . the graph summarizes the average of six different kidneys in each group. e-cadherin/fsp double-positive interstitial cells were exclusively present in the bmp- -treated kidneys. met and adult renal fibroblasts studies) after weeks of disease, when fibrotic lesions have been established and we evaluated the kidneys after weeks of treat- ment ( ). treatment with rhbmp- starting from week resulted in improved excretory renal function, decrease of interstitial fi- broblasts, and repair of tubular injury (fig. ). immunofluores- cence double staining with fsp antibodies (fibroblast marker) and e-cadherin (epithelial marker) confirmed that in the normal kidney, e-cadherin was exclusively expressed by epithelial cells and fsp was exclusively expressed by fibroblasts (fig. ). pro- gression of the disease was associated with an increase in the number of interstitial fibroblasts (that express fsp but not e-cadherin) and emt of epithelial cells (indicated by co-expres- sion of e-cadherin and fsp in cells within the tubular epithe- lium) (fig. ). bmp- -induced repair of renal injury was associ- ated with a significant decrease of interstitial fibroblasts (fig. ). in addition, decrease of fibroblasts and improvement of tubular architecture were associated with detection of a novel cell popu- lation that co-expressed fsp and e-cadherin within the inter- stitium, indicating a cell phenotype that reveals it as a fibroblast and also an epithelial cell (fig. ). such cells were not present in the untreated control kidneys and only present in the bmp- - treated kidneys, in association with decreased interstial fibro- blasts and improved tubular structure. these findings suggest that these novel cells are probably interstitial fibroblasts re- sponding to rhbmp- treatment by undergoing met. discussion it is known that during kidney development, the metaneph- ric mesenchyme converts to epithelium (met) and that during adult kidney disease renal epithelia can become fibroblasts via emt ( , , , ). whereas the adult kidney possesses sub- stantial intrinsic capacity to repair epithelial damage, the po- tential of adult renal fibroblasts to contribute to epithelial regeneration by converting into epithelial cells has not been explored. bmp- is required for formation of epithelia during kidney development, and several studies have demonstrated that treatment with bmp- enhances the repair of tubular epithe- lial injury ( , ). whereas we previously demonstrated that bmp- inhibits emt involving adult renal epithelial cells, we now provide evidence that bmp- induces formation of epithe- lial aggregates in adult renal fibroblasts, which mirrors the influence of bmp- on embryonic metanephric mesenchyme. these findings suggest that adult renal fibroblasts have re- tained at least some part of their embryonic epithelial imprint and plasticity, which can be potentially utilized to mediate repair of kidney injury by generating epithelial cells. acute renal failure is associated with re-expression of pax- , a principal regulator of epithelial development in the kidney, within the tubular epithelium ( ). in addition, renal injury is associated with increased expression of wnt- , mainly in inter- stitial fibroblasts ( ). furthermore, it is well established that renal injury is associated with decreased bmp- expression, a regulator of met during embryonic development ( , , , ). these studies provide evidence that embryonic developmental programs are still likely to be active in the adult injured kidney. in our studies, bmp- induces phenotypic changes in adult renal fibroblasts, which mimics the effect of bmp- on embry- onic metanephric mesenchymal cells ( , , , ). decreased type i collagen expression, decreased motility, increased e- cadherin expression, and formation of cell aggregates represent an early stage of met ( , , , ). this is further validated by increased expression of pax and wnt , both of which are present within condensing metanephric mesenchyme but are lost during tubule formation ( ). because bmp- induces for- mation of epithelial aggregates in both embryonic and adult renal mesenchymal cells, further studies are required if combina- tions of growth factors such as fibroblast growth factor- , tumor necrosis factor-�, and leukemia inhibitory factor, which induce formation of tubules in the metanephric mesenchyme, are required to induce similar changes in adult renal fibroblasts ( ). administration of exogenous bmp- mediates repair of chronic lesions in a mouse model of nephrotoxic serum nephri- tis. here we provide evidence that bmp- -mediated repair is associated with potential met involving interstitial fibro- blasts. whereas the method of immunofluorescence double staining does not exclude the possibility that the cells detected here are epithelial cells undergoing emt, this is highly un- likely for the following reasons. first, we could never detect e-cadherin/fsp double-positive cells in the untreated ntn control mice. second, treatment with bmp- mediates repair of renal injury, and enhanced emt would be counterproductive. third, our in vitro results confirm that bmp- induces expres- sion of e-cadherin in adult renal fibroblasts. an unequivocal role for met in the repair of renal injury remains unclear. it also remains unclear whether all of the renal fibroblasts retain the capacity to acquire an epithelial phenotype or whether only a subpopulation of fibroblasts main- tain their embryonic plasticity. nevertheless, our studies offer evidence for the capacity of adult fibroblasts to undergo met and probably contribute to the emergence of healthy epithelial cells in the repaired renal tubular structures. references . lilie, f. r. 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( ) am. j. physiol. renal physiol. , f –f met and adult renal fibroblasts 	bone morphogenic protein- induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney* 	experimental procedures 	results 	discussion 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email 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(wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ nemaline myopathy caused by tnnt mutations in a dutch pedigree original article nemaline myopathy caused by tnnt mutations in a dutch pedigree w. ludo van der pol , jolien f. leijenaar , wim g. m. spliet , selma w. lavrijsen , nicolaas j. g. jansen , kees p. j. braun , marcel mulder , brigitte timmers-raaijmakers , kimberly ratsma , dennis dooijes ,a & mieke m. van haelst ,a rudolf magnus institute of neuroscience, department of neurology and neurosurgery, university medical center utrecht, ea, utrecht, the netherlands department of medical genetics, university medical center utrecht, ea, utrecht, the netherlands department of pathology, university medical center utrecht, ea, utrecht, the netherlands department of pediatrics, pediatric intensive care, wilhelmina children’s hospital, university medical center utrecht, ea, utrecht, the netherlands keywords nemaline myopathy, tnnt , troponin t correspondence mieke van haelst, department of medical genetics, university medical center, utrecht, the netherlands. tel: + - ; fax: + - ; e-mail: m.vanhaelst@umcutrecht.nl funding information no funding information provided. athese authors contributed equally. received: july ; revised: october ; accepted: october doi: . /mgg . abstract nemaline myopathy (nm) is genetically heterogeneous disorder characterized by early onset muscular weakness and sarcoplasmatic or intranuclear inclusions of rod-shaped z-disk material in muscle fibers. thus far, mutations in seven genes have been identified as cause of nm. only one single tnnt nonsense mutation has been previously described that causes autosomal recessive nm in the old order amish with a very specific clinical phenotype including rapidly progressive contractures. here, we report a patient who is compound heterozy- gous for a c. + g>a mutation and an exon deletion in the tnnt gene. this report confirms the specific clinical phenotype of tnnt nm and docu- ments two new tnnt mutations outside the old order amish. introduction nemaline myopathy (nm [mim , ]) is a congenital myopathy characterized by early onset muscu- lar weakness and rod-like inclusions of z-disk material in muscle fibers. typical cases have a congenital onset with hypotonia, predominantly proximal weakness of the extremities and respiratory insufficiency, often accompa- nied by facial weakness with sparing of the extraocular muscles (wallgren-pettersson et al. ; nance et al. ). mutations in seven genes have been described to cause nm; alpha-tropomyosin (tpm ), nebulin (neb), alpha-actin (acta ), beta-tropomyosin (tpm ), kelch- repeat, and btb/poz domains containing protein (kbtbd ), cofilin- (cfl ), and troponin t (tnnt ) (kiphuth et al. ; wallgren-pettersson et al. ; nance et al. ). with the exception of tnnt , muta- tions in these genes have a global distribution. homozy- gosity for a single nonsense tnnt nm mutation was identified in the old order amish and is associated with a very specific clinical phenotype (johnston et al. ). amish nm (anm) has a relatively high incidence among amish and is, unlike other nm, characterized by early onset but subsiding tremors of the jaw and lower limbs, progressive contractures of the shoulders and hips, and a pectus carinatum. respiratory insufficiency characteristi- cally starts in the second year of life (johnston et al. ). here, we report the first patients outside the old order amish community with clinical features strongly reminiscent of anm. the proband is the first child of healthy nonconsan- guineous caucasian parents. he was born after an ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. this is an open access article under the terms of the creative commons attribution license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. uneventful pregnancy of weeks with a birthweight of g. at the age months, he presented with general- ized stiffness, rigid spine, and reduced cervical rotation range. passive movements of the shoulders, hips, and knee joints were severely limited and precluded assess- ment of muscle strength. muscles of extremities felt atro- phic, hand movements were normal. reflexes could not be elicited and there were no tremors. he had a marked kyphosis, a rigid spine and thorax, and marked pectus carinatum, but no other dysmorphic features. disease course was uneventful until he was admitted at the age of years with pneumonia and respiratory insufficiency. there had been serious feeding difficulties in the weeks before admission. chest x-rays showed infiltrative changes in the right lung and herniation of the stomach and intestines in the thoracic cavity. treatment consisted of tracheostomy, chronic ventilation, and surgical correc- tion of his diaphragmatic hernia followed by gastrostomy. he was discharged with chronic home mechanical ventila- tion. the family history reported two paternal cousins (their mother is the sister of the proband’s father), a boy and a girl from nonconsanguineous parents, who died aged and years, respectively, due to respiratory insuf- ficiency. they had a history of severe hypotonia and mus- cle weakness. contractures of elbows and knees were reported in the boy and severe pectus carinatum with ky- phosis and contractures of elbows and ankles in the girl. they had a postmortem diagnosis of nm after revision of the muscle biopsies. magnetic resonance imaging of the muscles and spine of the proband showed marked kyphosis (fig. a) and increased t signal of the solei and glutei in the lower leg, adductor muscles, and vasti of the upper leg, with relative sparing of the rectus femoris, sartorius, gracilis, and ham- strings (fig. b). muscle biopsy from the lateral vastus showed marked variation in fiber size with smaller type than type fibers and nemaline rods in the gomori stain and electron microscopy images (fig. c and d). molecular analysis revealed no abnormalities in acta , cfl , tpm , tpm , and kbtbd . a new het- erozygous pathogenic mutation was identified in intron of the tnnt gene (c. + g>a). this mutation was inherited from the father. maternal tnnt sequence analysis did not reveal a pathogenic mutation. molecular analysis of the dna of the two deceased paternal cousins showed homozygosity for the pathogenic c. + g>a mutation confirming a postmortem diagnosis of tnnt nm. the c. + g>a mutation is located at the invariant splice donor site of exon , predicting to result in tnnt mrna exon skipping. to test this, we examined figure . magnetic resonance imaging (mri) and muscle biopsy results. mri showed marked kyphosis (a), and diffuse fatty infiltration of the anterior compartment of the upper leg (white arrows) with relative sparing of the rectus femoris, gracilis, and sartorius and posterior compartment (black arrows) (b). gomori trichrome staining (c) showed variation in muscle fiber size and the presence of nemaline rods (arrows). electron microscopy ultrastructural analysis (d) also showed nemaline rods (arrows). ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. nemaline myopathy caused by tnnt mutations w. ludo van der pol et al. tnnt expression in the father of the proband. total rna was isolated from paternal fresh blood samples using the pax protocol (qiagen benelux, venlo, the netherlands) and subjected to reverse transcriptase-pcr. the obtained cdna products were pcr amplified using primers specific for tnnt exons and . pcr frag- ments were size separated using % agarose gel electro- phoresis. in addition, the pcr fragments were used for direct sequence analysis. primers and pcr details are available upon request. in addition to a normally sized pcr fragment including exons - - - , the father of the proband showed a nucleotides smaller pcr fragment (fig. a) which was shown to lack exon and consists of exons - - only (fig. b). these results confirm that the c. + g>a splice donor site mutation functionally results in exon skipping (r. - del). the resulting mrna is an in-frame mrna, but is predicted to lack functionally important amino acids (p.(asp _ile del)) from the tnnt troponin domain (fig. ). to identify a second pathogenic tnnt mutation in the proband we performed gene dosage analysis, using quantitative real- time pcr. this revealed a heterozygous tnnt exon deletion. dna analysis of the proband’s mother con- firmed that she was carrier for this deletion. in conclusion, in addition to the previously reported amish g t founder mutation in exon (nm_ . :c. g>t p.(glu *)), this study identifies two new tnnt nm mutations in three related individuals outside the old order amish. most of the clinical features are very similar to anm, in particular the severe figure . c. + g>a mutation leads to tnnt mrna exon skipping. (a) agarose gel showing exon to exon rt-pcr products of control individual (lane ), heterozygous father of index patient (lane ), and water blank (lane ). lane m: bp size ladder. arrow indicating rt-pcr product from mutated allele with deletion of exon ( bp) from mrna. (b) control: chromatogram of rt-pcr fragments from lane (a). patient: chromatogram from rt-pcr fragments from lane (a). rt-pcr analysis of heterozygous father (“patient” sample, lane ) shows mix of exon –exon mrna fragments from the normal allele and exon –exon fragments (arrow) showing exon skipping from the mutated allele. (c) schematic presentation of inferred effects on tnnt protein composition of the c. g>a and exon deletion mutations described in this study. both mutations lead to truncated tnnt protein (or, alternatively to absense of mutated protein through nmd). for comparison, the position of the original amish c. g>t p.(glu *) mutation is shown. ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. w. ludo van der pol et al. nemaline myopathy caused by tnnt mutations progressive contractures of the extremities, severe pectus carinatum, rigid chest deformities and respiratory insuffi- ciency in the proband. in addition, the proband from our study showed severe kyphosis and diaphragmatic hernia- tion. this latter feature has not been reported before in nm and may reflect thinning and weakness of the dia- phragm in the course of the disease. hypotrophy of the diaphragm has also been observed in tnnt knockout mice (feng et al. ). the cases reported here show an overall slightly milder phenotype than the anm cases. trembling of the jaw or lower extremities as reported in the anm patients were not seen in the dutch cases. besides, the anm patients died at the age of , whereas the index case is still alive and the cousins died at the ages of and , respectively. these differences may reflect ‘normal’ clinical variability in the condition or, alterna- tively, may reflect different functional effects of the sepa- rate mutations on residual tnnt protein function. the anm cases were all homozygous for a nonsense mutation in exon of tnnt (fig. c). in these patients, in all likelihood mutated transcripts from both tnnt alleles would be targeted for degradation by non- sense-mediated decay (nmd), resulting in total absence of functional tnnt protein and likely a very severe phe- notype. in the cases described in our report, both deceased cousins were homozygous for a tnnt splice site muta- tion resulting in exon skipping from the tnnt mrna resulting in shortened in-frame transcripts. these short- ened transcripts miss an important part of the function- ally important troponin domain. the proband from our study has an exon deletion in addition to the exon skipping mutation. this exon deletion could lead to absence of mutated transcripts through nmd or, alterna- tively to c terminally shortened transcripts. for both mutations described in our study, we cannot exclude that these shortened transcripts are translated into shortened tnnt protein with some residual activity. this possible residual protein activity may explain phenotypic differ- ences between the anm cases and the ones presented in our study. whether clinical differences are real and attributable to genotypic differences is something we feel is too specula- tive to conclude from these small numbers of patients. more cases need to be reported to further delineate a pos- sible genotype–phenotype correlation. our findings show that nm caused by tnnt muta- tions is not restricted to the amish population and that the clinically relevant spectrum of tnnt mutations also includes partial gene deletions. the characteristic clinical phenotype with chest deformities and progressive contrac- tures may be important in directing tnnt mutation analysis in nm patients. acknowledgment we thank the family members for their participation. conflict of interest none declared. references feng, h. z., b. wei, and j. p. jin. . deletion of a genomic segment containing the cardiac troponin i gene knocks down expression of the slow troponin t gene and impairs fatigue tolerance of diaphragm muscle. j. biol. chem. : – . johnston, j. j., r. i. kelley, t. o. crawford, d. h. morton, r. agarwala, t. koch, et al. . a novel nemaline myopathy in the amish caused by a mutation in troponin t . am. j. hum. genet. : – . kiphuth, i. c., s. krause, h. b. huttner, g. dekomien, t. struffert, and r. schr€oder. . autosomal dominant nemaline myopathy caused by a novel alpha-tropomyosin mutation. j. neurol. : – . nance, j. r., j. j. dowling, e. m. gibbs, and c. g. b€onnemann. . congenital myopathies: an update. curr. neurol. neurosci. rep. : – . wallgren-pettersson, c., c. a. sewry, k. j. nowak, and n. g. laing. . nemaline myopathies. semin. pediatr. neurol. : – . ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. nemaline myopathy caused by tnnt mutations w. ludo van der pol et al. book reviews of some new diseases, and a reminder of diseases already described in the amish population. much of the first half of the book is concerned with nosology, and an excellent chapter is that by p. e. becker on the spastic paraplegias and spinocerebellar ataxias. he has brought interest and order to a difficult branch of neurology. some of the other chapters on nosology are tedious and wordy. however there are some unusual topics covered in this book. for example, there are useful descriptions of some viral infections of the central ner- vous system, some of which give rise to malformations such as neural tube defects, cerebellar degeneration, and aqueduct stenosis; while others lead to the degenera- tive-occasionally familial-disorders of scrapie, kuru, and creutzfeldt-jacob disease. in another chapter, w. e. nance discusses the aetiology of anencephaly spina bifida, mentioning the facts that only a small pro- portion of like-sexed twins are concordant for neural tube malformation, that maternal half-sibs have a recurrence risk as high as that of full sibs, and that the increased incidence in second and third-degree relatives is largely confined to matrilineal relatives. dr nance suggests that the explanation for these observations is that in- heritance is through cytoplasmic factors. chapters which are more clinically oriented include good ones on tay-sach's disease, refsum's disease with an account of dietary treatment in two patients, and descriptions of some of the childhood leucodystrophies. of particular interest in the case reports in the later part of the volume are those autosomal recessive conditions associated with mental retardation, where diagnosis and subsequent genetic counselling are so important. such disorders include an oculocerebrofacial syndrome where, in addition to retardation there is microcomea, optic atrophy, and small mandible; an example of the dyggve- melchoir-clausen syndrome with mental deficiency; and familial megalencephaly. in all this is a helpful and interesting volume in the birth defects series. sarah bundey genes, dreams and realities. by sir macfarlane bumet. (pp. ; illustrations. c .) aylesbury: medical and technical publishing co. ltd. . all that sir macfarlane bumet writes must be treated with respect and in this book he discusses several of the most important biological issues of the day: the human applications of the new biology; the implications of the treatment of genetic disease; the immunological basis of carcinogenesis and of ageing; and the possibilities of population control. on most of these issues his con- clusions are pessimistic and many readers will regard him as being unduly so; but will find it a valuable exercise to think out just where they feel he may be mistaken. on a more technical level the book is noteworthy in empha- sizing the author's view of the importance of somatic mutation, as opposed to mutation in the germ cells, as a cause of disease. he thinks that basic work on molecular biology is now largely completed except for the elucidation of the structure of ribosomes in relation to their function of translation and a complete specification of an rna bacteriophage. he regards the chance of discovery of practical methods of direct gene replacement in man as infinitely small. he does however think that there is a reasonable chance of culturing and transforming some of the patient's own cells and returning them to the body so that they can compensate for genetically determined deficiencies. he notes the difficulty that many doctors still have in accepting that a disease has a genetic cause and reminds us that this was once equally true for the concept that some diseases were due to invasion by microorganisms. he is doubtful of the ethics of treating children with disorders such as pku and notes 'it will probably be many years before the logical solution can be accepted that infants with gross genetic defects of metabolism should be treated as those with no brains (anencephalic monsters) ... and not allowed to survive'. most medi- cal practitioners perhaps would agree only if the detec- tion and destruction of those affected could be readily achieved early in pregnancy, always provided of course that this is what the parents wanted. he makes a good case for the origin of cancers from somatic mutation and the reviewer was interested to learn that tumours in pure line strains of mice induced by carcinogenic chemicals are each antigenetically different. bumet is sceptical of the view that viruses play much part in the aetiology of cancer on the grounds that he can see no selective ad- vantage to a virus in inducing cancer in the host. he thinks that immune mechanisms control much potential cancer and offer the best hope for a cure. he believes however that 'little further advance can be expected from laboratory science in the handling of the 'intrinsic' type of disability and disease'. on population problems he is equally pessimistic. he believes it necessary to get back to a global population of between one and two million until a greater proportion of solar energy can be harnessed by collectors in geo- stationary orbit which can beam energy onto the earth's surface. the control of population he regards as quite the most important priority. 'family planning with its slogan of the children you want when you want them is absolutely inadequate; it is population control or chaos.' however he does not believe that any demo- cratic country will ever initiate effective policies of population control so that 'current civilization will de- stroy itself and that a second civilization rebuilt in cen- turies or more millennia from pockets of survivors will probably again end in another catastrophe'. c. . carter o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n m a rch . d o w n lo a d e d fro m http://jmg.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ “clustering of exemptions” as a collective action threat to herd immunity ‘clustering of exemptions’ as a collective action threat to herd immunity thomas may medical college of wisconsin, watertown plank road, p.o. box , milwaukee, wi - , usa ross d. silverman department of medical humanities, southern illinois university, school of medicine, springfield, il, usa abstract in this paper, we examine the phenomenon of ‘clustering of exemptions’ to childhood vaccination, and the dangers this poses both to those exempted as well as the general population. we examine how clusters of exemptions might form through collective action as described by thomas schelling, and how religious groups who live in close proximity to one another can “self-select” in a way that exacerbates this phenomenon. given the growing number of exemptions and the increasing visibility of the anti- vaccine movement, policy makers must be vigilant for dangerous clustering in order to avoid loss of herd immunity. keywords: clustering of exemptions; herd immunity; vaccination . introduction herd immunity is a concept that is at the foundation of any public health vaccination program. no vaccine is % effective, so the eradication, elimination or radical reduction of epidemics relies on the protection provided when a large enough percentage of a given population is immune, so as to prevent potential outbreaks of vaccine-preventable disease from getting started. in this way, even those for whom vaccination is not effective are protected through the unlikelihood that they will be exposed to the disease (since an outbreak cannot get a “foothold” where herd immunity is achieved). along with those who are vaccinated but do not achieve immunity to vaccine-preventable diseases, children exempted from mandatory vaccination are protected through herd immunity. the greater the number of people not immune, however, the greater the chances become that the protection provided through herd immunity will be lost. in this context, the medical literature has recently begun to focus on the potential harmful effects of the anti-vaccination movement and expanding exemptions to mandatory childhood vaccination [ ]. for example, one study found that drops in vaccination rates due to this movement in the uk, japan, sweden, russia, ireland, italy, the former west germany and australia have resulted in pertussis incidence – times greater than in countries where high vaccine coverage was maintained [ ]. the level of vaccination required in order to achieve herd immunity varies by disease, but generally ranges from to % [ ]. although historically, the level of exemptions to vaccination in the us has not been high enough to pose a threat (not exceeding % for any state prior to ), the exemption rate is rising. in michigan, the percentage of children entering the school system who were exempt from vaccination this is the author's manuscript of the article published in final edited form as: may, t., & silverman, r. d. ( ). “clustering of exemptions” as a collective action threat to herd immunity. vaccine, ( – ), – . http://dx.doi.org/ . /s - x( ) - http://dx.doi.org/ . /s - x( ) - requirements exceeded % for the first time in [ ]. in colorado, the percentage of those seeking exemptions rose by % between and . in order to avoid loss of protection through herd immunity, the literature calls for greater public and parental education about the risks associated with being vaccinated versus not being vaccinated, in order to counter the influence of the anti-vaccination movement [ ]. education concerning the relative risks of opting out of vaccination is mitigated, however, when the basis of exemption is not related to risk assessment, but to religious beliefs. in particular, one important area related to this concern has not received proper attention: the phenomenon of “clustering” and how this might exacerbate the effects of the anti-vaccination movement. clustering, as this relates to vaccination, is a phenomenon in which the proportion of people who seek exemption to mandatory vaccination is higher in a particular locality than it is for the broader population. it is this phenomenon that we examine in this paper. . the phenomenon of clustering and critical mass the phenomenon of clustering is attributable to a number of things. for example, a child may have an adverse reaction to vaccination; when this happens in a small community, it may become a focus of that community’s attention, leading to a higher than normal exemption rate. most importantly for our purposes, people who share religious beliefs that object to vaccination will, in many cases, live in close proximity to each other. for example, in ohio, the amish population is concentrated in nine of the state’s counties, with of those counties being contiguous [ ]. whatever the reasons that motivate some individuals to seek exemption to mandatory childhood vaccination, there is a danger of clustering once exemption begins to take hold in a particular community. as we will see below, this can create conditions in which exemption to vaccination multiplies. in a classic book titled micromotives and macrobehavior [ ], thomas schelling examines the ways in which the behavior of some individuals effect the behavior of others. the foundational concept schelling works from is the concept of “critical mass.” this concept involves an understanding of behavior as becoming widespread and self-sustaining once enough people have begun to engage in this behavior. the examples schelling gives are as follows: i walk across the lawn if that seems to be what others are doing; i sometimes double-park if it looks as though everybody is double-parked…. if a few people get away with smoking in a non-smoking section… so many others will light up… [ ]. schelling goes on to observe: what is common to all these examples is the way people’s behavior depends on how many are behaving a particular way… [ ]. the influence of the behavior of some people on the behavior of others may well lead to a multiplying effect once a critical mass is reached. an example schelling uses to illustrate this phenomenon involves students deciding whether to take a course for a letter grade or pass–fail [ ]: if this option is available to all students, there will be some who take the course for a grade and some who take it pass–fail regardless of what others do. many students, however, will choose according to how many others are choosing in that particular way—this is called the “intermediate group.” the actual number of other students required to influence the “intermediate group” will differ, however, among the members of the intermediate group. nonetheless, if a critical mass of students who choose one option independent of the actions of others is reached, the intermediate group can soon begin to choose in a common way. schelling explains that if the number of students who choose to take the course pass–fail is great enough to induce those who require only a minimum number of others in order to choose pass–fail, then these students will be influenced to choose this option. when these “intermediate group” students requiring only a minimal level of others are added to those who choose pass–fail independent of the behavior of others, however, it forms a group of enough students choosing pass–fail to induce more “intermediate group” students to choose the pass–fail option. the addition of these students to those choosing pass–fail, in turn, induce even more intermediate group students to choose this option, and so on until all but the students who will choose to take the course for a letter grade regardless of the behavior of others are taking the course pass–fail. this phenomenon is known as “tipping” [ ]. how do the above phenomena relate to threats to herd immunity? because those whose religious or personal views create a willingness to seek exemption to mandatory childhood vaccination often live, as we discussed earlier, in proximity to others who share these outlooks, the possibility of reaching a “critical mass” of exemptors large enough to undermine herd immunity is made more likely. herd immunity, though not requiring % compliance with mandatory vaccination, nonetheless requires a very high percentage of the population to be vaccinated (since not all of those who are vaccinated will achieve immunity). based on national and state averages, the number of people who seek exemption from mandatory childhood vaccination is, on average, very low [ ]. there are “clusters” of exemptions in local communities, however, that are much higher than the national or state averages. for example, while the percentage of people exempted from mandatory childhood vaccination is only . % nationally, utah has a rate of exemption twice that percentage ( . %). furthermore, one county in utah (washington county), has an exemption rate nearly six times the national average ( . %). this high exemption rate undermined herd immunity for this county, resulting in an outbreak of measles lasting viral generations and cases, half of which were contracted by people who had been vaccinated [ ]. returning to schelling’s analysis, even if we assume that most people will not fall into an “intermediate group” for seeking exemption to vaccination (unlike the students in schelling’s example), but instead will fall into the group that will choose vaccination for their child regardless of the behavior of others, the tendency of those who might be induced to seek exemption, to live in proximity to others who might also be induced to seek exemption, creates conditions in which a critical mass of exemptors could result in “tipping” that undermines herd immunity. that is, even if we assume that the “intermediate group” for seeking exemption to childhood vaccination is small, the tendency of those who do fall into this intermediate group to live in proximity to others in this intermediate group poses a potential “tipping” phenomenon that could undermine herd immunity. evidence for the results of clustering can be seen in recent outbreaks of pertussis and rubella among amish populations, and in the period – , when outbreaks of measles among persons with religious exemptions were documented, resulting in more than cases and at least deaths. this phenomenon is not unique to the us: a measles outbreak in the netherlands also exemplifies this phenomenon. what began with a cluster of children enrolled in a religious school whose members routinely decline vaccination, grew into a -month-long outbreak, with reported cases. these cases were largely concentrated in the communities in which members of this religious organization reside, and the vast majority of those who contracted the disease— of the cases—were found to have been eligible for vaccination but had declined for religious reasons. in addition, five percent of those who contracted the disease whose vaccination status was known had received at least one dose of the measles mumps and rubella (mmr) vaccine [ ]. while vaccination of children in the netherlands is recommended rather than compulsory for entrance into schools, most children are vaccinated. however, in some communities with significant numbers of members of this religious group coverage rates were as low as %. . local communities and critical mass central to the understanding of what dangers the phenomena schelling describes pose to herd immunity, is a recognition that the number of “others” needed to induce “tipping” may be related to the make-up of the community one lives in. while some behaviors will be influenced by the absolute number of other people who engage in that behavior, schelling observes that other behaviors are “undoubtedly” influenced by the proportion of other people who engage in that behavior, rather than absolute numbers [ ]. for these behaviors, the make-up of a particular community becomes of great importance. if a community is composed of people that disproportionately tend to display a particular characteristic, the tipping phenomenon is increased when related to proportionate numbers. for example, consider attendance at an “optional” class field trip: if the attendance of some is influenced by the proportion of other class members who attend, it might matter if the course is one that is a “general required course” or one attracting only students who have an inherent interest in the subject. if the latter, the students may have “self-selected” according to characteristics that will bias the proportion who attend the field trip. regarding this type of attendance phenomenon, schelling states: by separating away half the population, and specifically the half least likely to attend, we have doubled the influence of everybody who attends—doubled the percentage that he or she represents [ ]. in brief, when a group of people is composed in a way that tends to select for a certain characteristic or characteristics, the influence of those characteristics becomes greater. this phenomenon is known as “separating populations.” states schelling: if it is proportions that matter—smoking cigarettes or wearing turtlenecks… depending on the fraction of the relevant population that does so—there is the possibility of dividing or separating populations. if people are influenced by local populations—the people they live with or work with or play or eat with or go to school with or ride the bus with… any local concentration of the people most likely to display the behavior will enhance the likelihood that, at least in that locality, the activity will reach critical mass [ ]. exemption to mandatory childhood vaccination is often based on religious beliefs that are not “mainstream” in the society as a whole. often, this type of shared religious belief will result in communities whose make-up is “self-selected” for this characteristic. the amish, christian scientists, and jehovah’s witnesses are but a few examples of religious groups whose members are likely (relative to the population as a whole) to seek exemption to childhood vaccination. the fact that members of groups whose shared beliefs tend to make them open to seeking exemption also tend, as we have seen at the outset of this paper and in outbreaks described in the previous section, to live in proximity to other members of these groups, create conditions in which a clustering of exemptions sufficient to undermine herd immunity might occur. the dangers that the ‘clustering’ phenomenon poses goes beyond the groups that opt out of vaccination. for example, in colorado public health records confirmed that, for % of vaccinated children who contracted measles between and , the exposure source was unvaccinated children; however, because two-thirds of the measles exposure sources during this time period were unknown, it is reasonable to deduce that the actual percentage of vaccinated children who contracted measles from unvaccinated neighbors was significantly higher. awareness of this danger is essential for physicians advising parents in communities susceptible to clustering, and for local authorities who determine whether to grant exemption to mandatory childhood vaccination in a particular community. given the growing numbers of exemptions, it is imperative that physicians and policy makers be vigilant for dangerous “clustering” that might undermine herd immunity. in communities susceptible to the clustering phenomenon, difficult decisions may need to be made to deny exemptions, even where current rates of exemption fall below a level that threatens herd immunity, in order to avoid a snowballing of exemptions that would threaten not only those who seek exemption, but some percentage of those who undergo vaccination. references [ ] (a) feikin dr, et al. individual and community risks of measles and pertussis associated with personal exemptions to immunization. jama ; ( ): – ; (b), salmon d, et al. health consequences of religious and philosophical exemptions from immunization laws. jama ; ( ): – . [ ] gangarosa ej. impact of anti-vaccine movements on petiussis control: the untold story. lancet ; ( ): . [ ] http://www.bt.cdc.gov/agent/smallpox/overview/intro-to-smallpox.pdf. [ ] bauza m, fewer kids get immunized. detroit news, may , p. a . [ ] abramson j, pickering l. us immunization policy. jama ; ( ): – . [ ] american religion data archive, http://www.thearda.com/ [ ] schelling tc. micromotives and macrobehavior. new york (ny): w.w. norton & company; . [ ] schelling tc. micromotives and macrobehavior. new york (ny): w.w. norton & company; . p. . [ ] schelling tc. micromotives and macrobehavior. new york (ny): w.w. norton & company; . p. . [ ] schelling tc. micromotives and macrobehavior. new york (ny): w.w. norton & company; . p. – . [ ] schelling tc. micromotives and macrobehavior. new york (ny): w.w. norton & company; . p. – . [ ] national vaccine advisory committee. report of the nvac working group on philosophical exemptions. in: national vaccine program office, minutes of the national vaccine advisory committee, january ; atlanta, georgia. [ ] edwards gl, et al. measles outbreak—southwestern utah, . morb mortal weekly report ; ( ): – . [ ] measles outbreak—the netherlands, april– january. morb mortal weekly report ; ( ): – . [ ] schelling tc. micromotives and macrobehavior. new york (ny): w.w. norton & company; . p. . [ ] schelling tc. micromotives and macrobehavior. new york (ny): w.w. norton & company; . p. . lowed by cell death and permanent disability [ , , ]. crigler and najjar first characterized fatal hereditary non- hemolytic jaundice in [ ]. they described six infants in three families; five children died of kernicterus by months of age, and the remaining patient died at age years, several months after suffering devastating brain injury. crigler-najjar disease is rare in most populations, but a loss-of-function (type i) ugt a mutation is prevalent among the amish and mennonite groups of pennsylvania and the midwestern united states. while the development of effective phototherapy systems has altered the course of this previously lethal disease [ , ], cnd patients remain vulnerable to brain injury throughout their life span [ ], and the high disease incidence within our local plain commu- nities necessitated the development of a systematic treatment protocol. we report here uniformly good neurological outcomes for all cnd patients managed under this protocol over the last years. patients and methods patients twenty cnd patients, ages . – years, were managed over a -year period and represent patient-years of follow-up (table ). over this same time period, we were consulted about additional children (not included in the - patient cohort) who suffered kernicterus. data from these cases are included under the section case summaries to illustrate various concepts. for the patients from amish or mennonite communities, only exon of ugt a was sequenced to determine the homozygous c→a genotype. for the three remaining patients, all five exons of ugt a were sequenced according to previously described methods [ ]. written informed consent to dna testing was obtained from the parents of all patients. clinical management the mainstay of therapy was an overhead array of ten philips w special blue (bb) fluorescent tubes . in recent years, two other phototherapy systems were used: portable light emitting diode (led)-based panels (stanford portabed system [ ]) and a custom-made “lightbox” housing fluorescent tubes (combined tl and bb). for newborns, a bb- or led-based light source was placed – cm from the skin for – h/day with – % of the body surface exposed. for older children and adults using the bb panel and portabed systems, light was positioned – cm from the skin and delivered at night for – h with – % of the skin exposed. mirrors were placed at the sides and head of the bed, and all patients used white sheets. lightbox therapy was provided for shorter daytime intervals ( – h) with about % of skin exposed. ursodiol ( – mg/kg/day) and a lipid-rich bedtime snack were used to stimulate bile acid-dependent bile flow and increase hepatic clearance of lumirubin, the water-soluble hepatically-excreted bilirubin photoproduct [ ]. irradiance was measured at the skin surface using three different instruments: the biliblanket meter ii (ohmeda medical, laurel, md.), joey dosimeter (respironics, pittsburgh, pa.), and a relatively inexpensive light meter (model lm , meterman test tools, everett, wash.) adapted at stanford university and calibrated against the biliblanket meter ii [ ]. the dose of light in watt-hours per nanometer (w-h/nm) was estimated as the product of skin- level irradiance (μw/cm /nm), surface area exposed (cm ), table clinical and biochemical summary of patients with crigler-najjar disease patient information mean (range) current age (years) ( . – ) age treatment started (days) ( – ) number of non-surgical hospitalizationsa . ( – ) clinical parameters percentage of patients (n= ) exchange transfusionb ursodiol therapy cholecystectomy liver transplantation neurological impairment visual impairment biochemical parameters means, sd (range) reference range bilirubin (mg/dl)c ± ( – ) . – . albumin (g/dl) . ± . ( . – . ) . – . bilirubin:albumin (mol:mol) . ± . ( . – . ) not applicable alanine transaminase (iu/l) ± ( – ) – alkaline phosphatase (iu/l) ± ( – ) – γ-glutamyl transpeptidase (iu/l) ± ( – ) – aover % of non-surgical hospitalizations were for neonatal hyper- bilirubinemia, the remainder were for infectious illnesses. overall hospitalization rate was . hospitalizations per patient per year bexchange transfusions were performed on two patients during the newborn period, before they came under our care in . no exchange procedures were performed within the cohort after cbilirubin in mg/dl× . = bilirubin in μmol/l; albumin in g/dl× = albumin in μmol/l overhead bb panels were constructed by floyd martin under the guidance of dhm. the biliblanket ii meter-based light meter and led-based portabed system were designed and constructed by hjv and colleagues. the led light panel research and development was financed by a grant from the dutch crigler-najjar association. the upright lightbox was originally designed and built by alex carmichael (australia) utilizing tl tubes. a modified bb tube- based lightbox unit, used for this study, was constructed by fm. hue test [ ]. five of these subjects were from our cohort (ages – years) and five were managed at the sophia children’s hospital in rotterdam, the netherlands (ages – years). none of the cnd patients tested wore eye shields during phototherapy. three age-matched healthy siblings (ages – years) were used as controls. results diagnosis all seventeen patients of amish or mennonite descent were homozygous for a c→a mutation in exon of the ugt a gene that resulted in a stop codon (y x) and complete absence of transferase activity. among the remain- ing patients, two had severe phenotypes (with genotypes c. [ c→t] + [ c→t] and c. [ - g→a] + [ _ delinst]) and one had a milder cn variant (with genotype c. [ a→g] + [ a→g]). patients for whom records were available began receiving phototherapy at ± days of age, before molecular confirmation of the cnd diagnosis. in the newborn period, no patient had evidence of infection, hepatopathy, or hemolysis. bilirubin rose – mg/dl/day , and the hour-specific bilirubin at presenta- tion was equal to or greater than the th percentile for healthy term neonates [ ] (fig. a). for high-risk neonates (e.g. sibling affected with cnd or parents known to be carriers), mutation detection from cord blood was typically completed within the first h of life. phototherapy we detected no serious health consequences for individuals exposed to > , lifetime hours of high-intensity blue light. for patients homozygous for the y x mutation, phototherapy maintained total bilirubin at a mean value of ± mg/dl (range: – mg/dl) and a bilirubin:albumin molar concentration ratio of . ± . (range: . – . ). major determinants of individual bilirubin values were the total light dose (irradiance × treated surface area × time) and patient age. despite consistent light dosing, baseline total bilirubin increased at a rate of . mg/dl/year (r= . , fig. b), such that the bilirubin:albumin molar ratio approached . by age years. daily illumination time ( – h) and percentage body surface treated ( – %) were similar among children and adults with cnd, whereas irradiance varied considerably. using the ohmeda biliblanket ii meter, an overhead bank with ten fresh w bb tubes irradiated skin with – μw/cm /nm at a distance of cm. the joey dosimeter measured considerably higher irradiance values from a common light source at a similar distance (fig. a). for fluorescent bb systems, major determinants of skin- level irradiance were source distance, hours of use (tube “freshness”), and the nature of surrounding materials. energy output was also influenced by the length ( –foot vs. foot) and number of tubes in the array, their axis of orientation to the body, and tube temperature; irradiance was highest when a large number of w ( -foot) tubes were oriented parallel to the long axis of the body and kept cool by a separate system of fans. tube irradiance diminished by – % after about , h ( months) of use, but this was not visually detectable. families that used light meters on a regular basis more effectively controlled the many variables influencing light dose. irradiance provided by the portabed ( – μw/cm / nm) was roughly equivalent to overhead bb light banks. major advantages of the led panels were portability, source longevity, and sleep under cover, but the system remains prohibitively expensive at this time. the upright “light box” was most effective, delivering a uniform circumferential irradiance that was . - to -fold higher than that achieved with either the overhead bb panel or portabed (fig. b). advantages of the light box system were a high surface area exposure, capacity for a large number of tubes, close proximity of light source to skin, and controlled use of reflective surfaces. because it constrained the patient during waking hours, it was only practical for about h of use per day. all three systems generated considerable heat, necessitating the use of cooling fans and warranting special attention to patient hydration. hospitalizations the overall hospitalization rate was . hospital admis- sions per patient per year, and six patients in the cohort ( %) were never hospitalized. over % of all hospi- talizations were for neonatal hyperbilirubinemia and occurred within the first weeks of life. remaining hospitalizations were for various non-surgical and surgical indications. the major reason for non-surgical hospitaliza- tion was infectious illness, which was often associated with transiently increased bilirubin and decreased albumin. daily light dose could be increased in the hospital by adding a second high-intensity unit and increasing expo- sure time to > h/day. however, given the high efficacy of home phototherapy units, patients were not hospitalized for any intensification of phototherapy per se, but for intrave- nous albumin therapy, more careful clinical and biochem- ical monitoring, and the treatment of complicating factors our hospital laboratory routinely reports bilirubin values in mg/dl and albumin in g/dl, whereas other laboratories use units of μmol/l. to interconvert these units: bilirubin in mg/dl × . = bilirubin in μmol/l; albumin in g/dl × = albumin in μmol/l. throughout the manuscript, calculated energy doses are based on direct light meter readings over the spectral range found effective toward photodegrading bilirubin ( – nm). this range encompasses the blue light absorption spectrum of bilirubin. these measurements are valid for comparing relative light energies. however, for more precise calculation of total light energy striking a biological material, such as the retina, we use “full width at half max” (fwhm), defined as the spectral width at half-maximal light intensity. this is also called the “bandwidth” and for practical purposes takes into account the gaussian distribution of energy over the detectable range. (e.g., vomiting, anorexia, dehydration, dysnatremia, etc.). there were no complications of albumin administration, and no patient received plasmapharesis while under our care. ten of twenty patients ( %) developed cholelithiasis and underwent elective laproscopic cholecystectomy; eight of these patients were being treated with ursodiol prior to developing gallstones. neurological outcome serial neurological examinations of cnd patients showed no evidence of hearing loss, cranial nerve injury, dystonia, cerebellar dysfunction, gait abnormalities, or peripheral neuromuscular disease. children and adults with cnd had normal intellectual function as assessed by school performance and social interactions. visual acuity was / – in both eyes for non-spectacled cnd patients over years of age and, compared to unaffected siblings, cnd patients did not have an increased need for corrective lenses. the farnsworth-munsell color discrimination error score was ± for cnd patients and ± for controls (p= . , non-significant), where error scores of < are consistent with normal color vision. liver transplantation four patients (ages , , , and years) had whole orthotopic liver transplants performed under standard protocol at the thomas e. starzl institute (pittsburgh, pa.). mean post-transplantation follow-up time was months (range: – months). total bilirubin was normal within h of allograft perfusion, but staining of skin and sclerae did not resolve completely for – weeks. the youngest of these patients had significant post- transplant complications, including cmv hepatitis, ebv viremia, acute and chronic allograft rejection, widespread opportunistic infection of the small bowel, and post- transplant lymphoproliferative disease. case summaries five clinical cases are grouped by clinical paradigm and briefly summarized. cases , , and are patients from the present cohort. two additional cases ( and ) are amish cnd patients from midwestern states managed elsewhere. mechanisms of kernicterus are introduced for further discussion (see figs. , ). fig. a the rate of rise of bilirubin over the first h of life may be normal in cnd patients but continues to rise to dangerous levels by day of life . using the th percentile hour-specific bilirubin [ ] as a screening cutoff value for pre- dicting severe jaundice (peak bilirubin > mg/dl), all of these cnd patients would have been identified for targeted follow-up and the early institution of pho- totherapy. b in our cnd pa- tients the molar ratio of bilirubin to albumin is elevated from . to . during the neonatal peri- od, reaches a nadir by age years, and then rises progres- sively to adulthood at a rate of . mol:mol/year (r= . ). this is due to a progressive . mg/dl/year (range: . – . mg/dl/year) rise of bilirubin unrelated to patient compliance. based on the chemistry of bili- rubin-albumin binding in healthy individuals, we assign the “safe” bilirubin:albumin molar ratio at or below . in older children and adults (de- noted by the dashed line) [ ] type i crigler-najjar disease in the newborn period case . prospective management of cnd a mennonite newborn had a sibling and several cousins homozygous for the y x mutation. phototherapy with an overhead bb light bank was started at home when jaundice was apparent on day of life . cord blood was sent to our clinic for targeted mutation testing, and the diagnosis of cnd was confirmed on day of life , at which time total bilirubin was mg/dl ( μmol/l), albumin was . g/dl ( μmol/l), and the bilirubin:albumin molar ratio was . . she was managed throughout the first year of life with daily overhead phototherapy and ursodiol ( mg/kg/day). during that time, total bilirubin ranged from . to . mg/dl ( – μmol/l) and the bilirubin:albumin ratio ranged from . – . . growth and development were normal, and she had no hospitalizations. she is now years of age, receiving phototherapy h per day, and healthy. case . poor neonatal risk assessment, inadequate therapy, and kernicterus in an amish infant from the midwest (not in the -patient cohort) was delivered at fig. a using two different light meters, the ohmeda bili- blanket meter ii and the respironics joey dosimeter, irradiance measurements were taken at -cm increments from an overhead bank of bb tubes (philips w, -foot long) used routinely at a community hos- pital neonatal intensive care unit (nicu). bb tubes in use were aged, putting out only about % of the power pro- vided by fresh tubes, irradiance decreased as a function of dis- tance from the source, and light meters which should yield roughly equivalent values dif- fered by as much as two- to threefold. b the graph displays hourly light power within the - to -nm bandwidth as the product of skin-level irradi- ance (μw/cm /nm, biliblanket meter ii) and average treatable body surface area with each type of system, estimated for a pa- tient with total body surface area of m ( , cm ). because irradiance values from the same source vary considerably using different light meters, the graph displays a relative, rather than absolute, comparison of light power home by a lay midwife. jaundice was first apparent on day of life but was not measured until he was hospitalized at days of age with lethargy and severe hyperbilirubinemia ( mg/dl, μmol/l). liver disease and hemolysis were excluded. phototherapy decreased total bilirubin to mg/dl over h. he was discharged without phototherapy, and bilirubin again rose to a range of – mg/dl. he was first examined by us at months of age, and found to be stuporous, irritable, and hypotonic. he had generalized axial dystonia, athetoid movements, poor head control, and feeding impairment. muscle mass and power were normal, deep tendon reflexes were diminished, and ankle clonus was absent. babinski reflexes were strongly plantarflexion. brain stem auditory evoked potentials were absent. a brain mri showed enlarged cerebrospinal fluid spaces over the frontal and temporal lobes and prominent sulci suggestive of diffuse cortical atrophy. the basal ganglia were normal in size. there were symmetric t signal hyperintensities in the internal and external pallidi, nucleus accumbens, substantia nigra, and hypothalamus. at present, he is deaf and has severe generalized dystonia. hospital management case . prevention of kernicterus in a cnd patient during an infectious illness a -year-old amish girl with cnd was admitted to our hospital with streptococcal pharyngitis, fever, vomiting, dehydration, rising total bilirubin ( mg/dl, μmol/l), and a high bilirubin:albumin molar ratio ( . ). she was managed according to an inpatient protocol (table ) over a -h period. continuous phototherapy fig. bilirubin in the cerebral circulation is bound to albumin and to a lesser extent red blood cells (rbc). unbound bilirubin (b) is the fraction that interacts with cerebral membranes, moving continuously between intravascular and extravascular binding sites. cumulative bilirubin deposition on endothelial membranes interferes with a variety of cellular processes, perhaps by altering the properties of membrane- associated and -embedded proteins such as ion pumps (nak-atpase), nutrient transporters (e.g.,glut , lat ), and metabolic enzymes. the uptake of bilirubin by brain can be expressed by the equation [ ]: d = (f)(b)(t)[ − fe(−k t)], where d is amount of bilirubin per weight of brain tissue, f is cerebral blood flow, b is total bilirubin concentration (> % of which is albumin-bound), e is the dimensionless value . , and t is duration of exposure. the last term expresses the relative quantity of bilirubin extracted by the brain for any given total plasma bilirubin value. it depends on the fraction of bilirubin unbound (f) and the dissociation constant of the bilirubin-albumin complex (k ) relative to the transit time of blood moving through brain capillaries (t). total bilirubin-binding capacity of blood [ ] is a function of albumin concentration, the number of high-affinity sites (n) per albumin molecule, and affinity of those sites for bilirubin (kab = k /k ); where kab = [b]/(b[na−b]). a variety of drugs, preservatives, and endog- enous anions interact with albumin to either reduce the number of high- affinity binding sites (l ) or increase the dissociation rate of the complex (l ; increase k , decrease kab). in experimental animals, fractional extraction and brain uptake of bilirubin is invariably – % lower than predicted by this model [ ]. this may reflect both the unmeasured effect of erythrocyte binding and the important role of brain endothelial proteins in bilirubin extrusion (e.g. p-glycoprotein, mdr ). brain bilirubin extrusion matures postnatally and may increase considerably in chronically jaundiced patients (approximately w-h/nm/day), four albumin infusions ( g/kg/dose), and enteral ursodiol ( mg/kg/day) lowered the bilirubin:albumin molar ratio to . . she recovered uneventfully. case . acute kernicterus during an infectious illness in a patient with cnd an amish boy with cnd (not in the - patient cohort) had chronic bilirubin levels of – mg/dl receiving – h of phototherapy per day. albumin levels were not monitored. bilirubin levels periodically increased to – mg/dl during illnesses. growth and development were normal. at years of age he developed streptococcal pharyngitis and was prescribed amoxicillin as an outpatient. the following day his speech became slurred and he stopped eating. four days later he presented to a midwestern hospital deeply jaundiced, stuporous and mute, unable to swallow or stand, and febrile. deep tendon reflexes were increased, he had ankle clonus, and babinski reflexes were plantarflexion. total bilirubin was mg/dl ( μmol/l), albumin was . g/dl ( μmol/l), and the bilirubin:albumin ratio was . . serum chemistries were normal, including glucose ( mg/dl), bicarbonate ( meq/l), and blood urea nitrogen ( mg/dl). an eeg showed diffuse slowing and paroxysms of generalized polyspike activity. a brain mri was normal. at the midwestern hospital, he was managed with contin- uous phototherapy, intravenous crystalloid, albumin, and phenobarbital. the bilirubin level decreased to his baseline value, but neurological function did not improve. he died at home a few weeks later. liver transplantation case . liver transplant in a -year-old mennonite man with cnd and increasing bilirubin levels the first of our patients to undergo a liver transplant had total bilirubin levels that ranged from to mg/dl (molar ratio to albumin: . – . ) despite rigid compliance with photo- therapy, treatment with ursodiol, and cholecystectomy at age years. post-transplant, the bilirubin decreased to normal within h, but yellow discoloration of the skin and sclera persisted for weeks. liver explant showed portal tract fibrosis, inflammation, ductal proliferation, and patchy bridging fibrosis. crystallized plugs of biliru- bin were seen in small degenerating ductules. centrolob- ular hepatocytes showed focal hepatocellular giant cell transformation, pseudoacinar formation, leathery degen- eration, and cholestasis. discussion diagnosis and management of cnd in the newborn period our data demonstrate that cnd patients can be detected by universal screening procedures for neonatal hyperbiliru- binemia [ , , ] (fig. a). whether or not a jaundiced newborn has cnd does not change their basic need for timely detection and effective treatment; in all of our patients phototherapy was started well before molecular confirmation of the diagnosis (case ). thus, brain injury in neonates with cnd does not result from delayed di- agnosis per se, but from poor recognition and inadequate treatment of “idiopathic” neonatal hyperbilirubinemia (case ). cnd patients can be managed safely to prevent death and disability table compares our study to various population measures from five previously published clinical surveys. crigler and najjar’s classic description preceded the advent of effective phototherapy, and all of their patients developed brain injury and eventually died [ ]. the combined data from four recent surveys [ , – ] suggest that – % of cnd patients suffer neurological injury ranging from mild to severe, – % of patients will need one or multiple exchange transfusions, and – % die of complications related to the disease. in contrast, we had no brain injuries or deaths extending over patient-years and did not perform a single exchange procedure since we began caring for children with cnd in . long-term biochemical data suggest that liver transplantation or some other curative procedure may ultimately be necessary to manage cnd patients safely (fig. b, case ). nevertheless, a systematic fig. in vitro studies [ ] show a progressive rise in free bilirubin when the bilirubin:albumin molar ratio exceeds . (dotted line) and a sharp rise in free bilirubin when the ratio exceeds . (i.e., sat- uration, dashed line). maximum bilirubin:albumin molar ratio was recorded during hospitalizations of our cnd patients with transient exacerbation of hyperbilirubinemia (open circles). none of these patients developed kernicterus. from our consultation records and published cases, we found cases of brain injury in patients for whom bilirubin and albumin values were available (solid circles). maximum recorded bilirubin:albumin molar ratio was significantly higher in patients that developed kernicterus ( . ± . vs. . ± . , p< . ) approach to neonatal screening, light dosing, and kernicterus prevention can assure that children and adults proceed to transplantation in good neurological health. providing effective phototherapy effective phototherapy for any patient requires education of health care providers and parents, phototherapy systems that are in good working order, a supply of fresh bb tubes or an energetic equivalent (e.g., tl s, leds), and the use of calibrated and affordable light meters [ ]. variables that influence light dose can be monitored intermittently by a nurse, physician, or parent, and allow patients to derive adequate phototherapy from a variety of sources in the hospital or at home. to be meaningful in terms of efficacy, a phototherapy order should specify light source and number, distance from the patient’s skin, and nature of surrounding materials (e.g., white sheets and mirrors), with a goal to achieve a measured irradiance over a percentage of skin surface for a specified period of time. a physician’s order for phototherapy in “hours per day” is incomplete, and an order for “double” or “triple” phototherapy is uninformative. one good light is usually sufficient, provided it is used correctly. surprisingly, many hospital practices use phototherapy systems without regularly monitoring or recording their performance. data in fig. a was obtained from a w fluorescent bb system in use at the nicu of a local community hospital. it revealed visually imperceptible aging of the tubes. in hospital settings, tubes may only be replaced when they no longer function, such that phototherapy systems operate for long periods emitting well below their energy potential. furthermore, fluorescent devices are often fitted ( : ) with cool-white bulbs to attenuate the blueness of bb light; this makes the system less effective. figure a also shows that despite measurement within a similar bandwidth and peak sensitivity, irradiance readings from different commercial light meters can vary as much as two- to threefold, while all meters show the decay of energy as a function of distance from the light source. light power calculations also demonstrate the comparatively low efficacy of fiber-optic blankets and halogen-quartz spotlights, which are inappropriate for managing patients with cnd or severe idiopathic jaundice (fig. b). these observations have several practical implications: ( ) light meter(s) in use at any facility should be calibrated at a determined distance against each phototherapy system operating at maximum output; ( ) any light source should be placed as close to the skin as possible; ( ) phototherapy systems should be accompanied by a tag or logbook that records the date of bulb/tube replacements and peak irradiance measurement with fresh lights at a standardized distance; ( ) for an infant who genuinely needs phototherapy, home treatment with a fiberoptic blanket is inappropriate. other factors reduce the dose of light achieved with a given source, including plexiglass barriers, pigmented (light- absorbing) materials, and objects that shield the skin. we find that isolettes are not necessary for well-hydrated term or near-term infants receiving phototherapy. in neonates, the thorax, outer arms, and head constitute the majority of table historical trends in crigler-najjar disease (nr not reported, na not applicable) reference year of publication number of patients age in years mean, standard deviation (range) bilirubin (mg/dl) mean, standard deviation (range) exchange transfusion (%) brain damage (%) mortality (%) liver transplant (%) transplant age, mean (range) crigler and najjar [ ]a ( . – . ) . ( . – . ) na van der veere et al. [ ]b . ± . ( . – . ) nr . ( – ) suresh and lucey [ ]c (< – ) . ± . ( . – ) shevel [ ]d nr nr nr nr nr na nazer [ ]e nr . ( – ) nr present studyf ± ± ( – ) ( – ) acrigler and najjar’s original report preceded the advent of exchange transfusion and phototherapy bbrain injured patients (n= ) were transplanted later ( . ± . years) than those without brain damage (n= , . ± . years) cinformation is compiled from an international questionaire involving patients from four continents. there is patient overlap with other studies, including our amish cohort ( of the patients reported). in the non-amish subgroup, neurological disability was present in of patients ( %). mean ± standard deviation bilirubin values (in mg/dl) varied by sub-group: neonatal peak, . ± . ; neonatal typical, . ± . ; postnatal typical, . ± dthe % incidence of neurological injury is based on an extensive review of published articles ( – ) by these authors, who further subdivided adverse outcomes into four discrete patterns of disability einformation from affected siblings, not included in the -patient cohort, is included in the paper by nazer et al. all died with bilirubin- induced brain injury, resulting in a total brain injury rate of / ( %) and a disease-related mortality of %. four patients required more than one exchange transfusion, for a total of exchange procedures among of the patients reported fprior to coming under our care, two patients in the cohort required exchange transfusion as neonates, and one patient developed kernicterus. however, the outcomes reported here were observed for patients under our care from to the present treatable skin surface, these but may be obscured by unnecessary diapers, monitor leads, and iv boards. we place term newborns with cnd in an open crib, naked, lying on an open diaper over bright white sheets. with a bb or led system placed – cm from the skin, the newborns appear comfortable, feed well, and maintain core body temperatures of °– °f. anticipating and preventing kernicterus patients with all variants of cnd remain vulnerable to brain injury throughout life, particularly during intercurrent illnesses, after injuries, or during surgery (case ). routine laboratory measurements can only be used to estimate the magnitude of the whole body bilirubin load and its rate of change, but for any individual patient a variety of other clinical and physiological details more fully characterize the potential for brain injury (figs. , ). these multiple factors vary from one individual to the next and in a single individual depending on age and clinical conditions. it is thus impractical and misleading to assign rigid guidelines for identifying patients “at risk” for kernicterus. rather, we base treatment decisions on general biochemical indices con- sidered together with particulars of time and circumstance. our treatment protocol in table is based on the pathophysiologic model illustrated in fig. [ , , , , , ] and is designed to lower total body exchangeable bilirubin and prevent its movement to extravascular sites. in jaundiced infants and adults, the total body bilirubin pool is very large and almost exclusively bound to proteins and lipid membranes, while the lower unbound bilirubin concentra- tion [ , ] is characterized by continual flux between two competing reservoirs – a finite number of intravascular binding sites and a much larger number of lower-affinity sites on endothelia of brain and other organs. as bilirubin passes through the circulation, the fraction deposited in the brain and other tissues depends only on its molar relationship with high-affinity albumin binding sites and the dissociation rate of the bilirubin-albumin complex relative to the time it takes blood to transit a tissue capillary bed [ , ]. according to this model, when high-affinity bilirubin binding sites on albumin approach saturation, or are occupied by competing ligands, bilirubin can shift rapidly from intravascular to tissue binding sites (fig. ). in clinical practice, the most important factor affecting bilirubin-albumin binding is the presence of organic anions which either compete directly for the bilirubin binding site or allosterically reduce its affinity for bilirubin [ ]. numerous exogenous and endogenous organic anions interfere with bilirubin-albumin binding, including intravenous and oral medications, drug metabolites pro- duced in vivo, commonly used preservatives, and endog- enous organic acids and free fatty acids. drugs which generally do not interfere with the binding equilibrium are cationic, glycosylated, or present in blood at protein- bound concentrations of < μmol/l [ ]. the number and complexity of bilirubin-albumin-drug interactions under- scores the point that all medications and intravenous solutions should be used cautiously in patients with hyperbilirubinemia, and only after a focused review of drug information in the physicians’ desk reference, medical literature, and/or a comprehensive pharmacology text. based on available resources [ , , , – , , , , , , – , , , , – , , ], we compiled a list of commonly used drugs, infusible substances, and endogenous metabolites in the appendix. drugs are listed based on probable safety for use in hyperbilirubin- emic patients. however, it is important to recognize that in vitro studies are not a perfect predictor of drug-albu- min-bilirubin in living patients. for example, drugs in the “variable safety” column show weak displacing activity in vitro which may become clinically significant under certain circumstances. for these compounds, lower dosing and slower infusion rates minimize displacing potential. important clinical drugs for which no data are available are listed to guide further studies in this important area. caring for the aging cnd population adolescence is a particularly important period of vulner- ability for kernicterus in patients with cnd. as patients mature, phototherapy increasingly interferes with lifestyle, social opportunities, and the formation of intimate relation- ships. baseline total bilirubin increases with age and approaches dangerous levels in young adulthood (fig. b, case ). contrary to other studies [ , ], we could not identify a relationship between age and compliance with phototherapy as a plausible explanation. the progressive rise in bilirubin is likely influenced by several physiolog- ical variables. first, “treatable” body surface area decreases relative to bilirubin volume of distribution as patients age (i.e., plasma in the cutaneous vascular bed accessible with light represents a progressively smaller proportion of total extracellular volume and distributed blood flow). second, bilirubin distributes to peripheral extravascular binding sites over time, creating a large tissue “reservoir” in equi- librium with the intravascular pool. evidence of this was found in all four transplanted patients, in whom cutaneous and scleral icterus took up to weeks to dissipate following liver transplantation, despite normal bilirubin levels within – post-operative hours. finally, hepatobiliary clear- ance of lumirubin may become rate-limiting in some patients, as evidenced by cholestatic changes in two of four liver explants. whatever the causes of rising bilirubin with age, it is apparent that most of our cnd patients will face liver transplantation over the next – years. liver transplanta- tion is currently the only clinically robust way to replace ugt a and hepatic transferase activity [ ]. viral and non- viral gene transfection techniques are far from human trials, and when these methods reach clinical maturity they may not be safer, cheaper, or even more effective than liver trans- plantation. fortunately, there is now considerable experience with liver transplantation for primary metabolic disorders as well as reductions in peri-operative mortality and medica- tion-related morbidity over recent years [ , , ]. how- ever, patient from our cohort suffered life-threatening post- transplant complications. thus, despite its metabolic ef- ficacy, liver transplantation is not an easy solution for patients with cnd. conclusion: lessons learned from cnd timely recognition of hyperbilirubinemia followed by effective phototherapy makes exchange transfusions and prolonged hospitalizations unnecessary. the core principles of management for neonates with idiopathic hyperbilirubin- emia should be the same as those for cnd: pre-symptomatic detection, timely risk assessment, scheduled follow-up monitoring, phototherapy guided by measurements of light power, treatment of maximum body surface, and the use of emergency protocols that emphasize bilirubin biodistribu- tion (table , figs. a, a, , ) [ ]. a precise formulation of kernicterus mechanisms remains obscure, but a great deal is known about bilirubin, its distribution in the body, and interaction with the brain. we believe this knowledge is sufficient to prevent brain injury in most, if not all, jaundiced patients. the most significant obstacle to the prevention of kernicterus is not scientific ignorance, but inadequate education of parents and health care providers, and a failure to translate established concepts into practice [ , , , , ]. acknowledgements we extend thanks to cnd patients and their parents from our local community for their support of this work and their hopes for future progress. we thank the outstanding nursing, pharmacology, and biomedical engineering staffs at lancaster general hospital for providing patients exceptional clinical care. floyd and katie martin were instrumental in gathering light measurements in the field and tim weaver, m.d., helped gather data for fig. . special thanks to charles e. ahlfors, m.d., for a technical review and helpful comments. finally, thank you to the dutch crigler-najjar association (huizen, the netherlands), for funding development of the portabed led system. appendix potential bilirubin-albumin displacing interactions (see text for references) safety class (see note) anti-inflammatory/antipyretic acetaminophen • aspirin • dexamethasone • ibuprofen • indomethacin • ketorolac • naproxen • phenacetin • prednisolone • safety class (see note) salicylate, sodium • antimicrobial acyclovir • amoxicillin • amoxicillin-clavulanate • amphotericin b • amphotericin, liposomal ampicillin • ampicillin-sulbactam • azithromycin azlocillin • aztreonam • carbenicillin • cefazolin • cefalothin • cefepime • cefixime • cefmetazole • cefonicid • cefoperazone • ceforanide • cefotaxime • cefotetan • cefoxitin • cefpodoxime proxetil • ceftazidime • ceftizoxime • ceftriaxone • cefuroxime • cefuroxime axetil • cephalexin • cephapirin • cephradine • ciprofloxacin • clarithromycin • clindamycin • dicloxacillin • doxycycline • erythromycin • erythromycin es-sulfisoxazole • fusidic acid • gangcyclovir • gentamicin • imipenem • imipenem-cilastatin • isoniazid • levofloxacin • lincomycin • linezolid • meropenem • methicillin • metronidazole • minocycline • safety class (see note) nafcillin • nitrofurantoin • oxacillin • penicillin g • penicillin v • piperacillin • piperacillin-tazobactam • rifampin • streptomycin • sulfisoxazole • sulphamethoxazole • sulphasalazine • tobramycin • trimethoprim • trimethoprim-sulfa (bactrim) • vancomycin • cardiovascular drugs atropine • bretylium tosylate • digoxin • disopyramide • dobutamine • dopamine • edrophonium chloride • enalapril • epinephrine • hydralazine • isoproterenol • lidocaine • nitroprusside • procainamide • propanalol • verapamil • contrast agents diatrizoate sodium • iodate sodium • iodipamide sodium • iopanoic acid • meglumin ioglycamate • metrizamide • metrizoate sodium • diuretics acetazolamide • bumetanide • chlorothiazide • ethacrynic acid • furosemide • hydrochlorothiazide • mannitol • spironolactone • neuroactive drugs aminophylline • amitryptyline hcl • safety class (see note) atomoxetine • bupropion • carbamazepine • chloral hydrate • clonazepam • codeine • desipramine hcl • diazepam • ethosuximide • etomidate • fentanyl • fluoxetine/norfluoxetine • inhaled anesthetics • imipramine hcl • ketamine • lorazepam • meperidine • methylphenidate • midazolam • morphine • naloxone • nortryptyline • olanzapine • oxazepam • paroxetine • phenobarbital • phenytoin • primidone • propofol • risperidone • theophylline • thiopental • valproic acid • venlafaxine • neuromuscular blocking agents neostigmine • pancuronium • rocuronium • succinylcholine • vecuronium • preservatives/metabolites [a] n-acetyl-dl-tryptophan • n-acetyltyrosine • benzoic acid (benzoate sodium) • caprylic acid • hippurate (from benzoic acid) -hyroxybenzoylglycine • miscellaneous bicarbonate • calcium chloride • calcium gluconate • carnitine • clofibrate • safety class (see note) heparin • intralipid/free fatty acids [b] • magnesium sulfate • prostaglandin e • tin mesoporphyrin • probably safe for clinical use considered unsafe safety variable: eg. drug dosing and combinations insufficient data a) acetyltryptophan (stabiliser in hsa i), acetyltyrosine (component of some tpn amino acid mixtures), caprylic acid, hexanoic acid (stabiliser in hsa i and ii) b) plasma free fatty acids are increased by fasting and infusions of intralipid, epinephrine, or heparin references . american academy of pediatrics, subcommittee on hyper- bilirubinemia ( ) management of hyperbilirubinemia in the newborn infant or more weeks of gestation. pediatrics : – . ahlfors ce, herbsman o ( ) unbound bilirubin in a term newborn with kernicterus. pediatrics : – . ahlfors ce, wennberg rp ( ) bilirubin-albumin binding and neonatal jaundice. semin perinatol : – . andersen dh, blanc wa, crozier dn, silverman wa ( ) a difference in mortality rate and incidence of kernicterus among premature infants allotted to two prophylactic antibac- terial regimens. pediatrics : – . bhutani vk, donn sm, johnson lh ( ) risk management of severe neonatal hyperbilirubinemia to prevent kernicterus. clin perinatol : – . bhutani vk, johnson l, sivieri em ( ) predictive ability of a predischarge hour-specific serum bilirubin for subsequent significant hyperbilirubinemia in healthy term and near-term newborns. pediatrics : – . brodersen r ( ) determination of the vacant amount of high-affinity bilirubin binding site on serum albumin. acta pharmacol toxicol : – . brodersen r ( ) bilirubin. solubility and interaction with albumin and phospholipid. j biol chem : – . brodersen r, ebbesen f ( ) bilirubin-displacing effect of ampicillin, indomethacin, chlorpromazine, gentamicin, and parabens in vitro and in newborn infants. j pharm sci : – . brodersen r, funding l ( ) binding of bilirubin and long- chain fatty acids to human serum albumin with general remarks on displacement of firmly bound ligands. scand j clin lab invest : – . brodersen r, friis-hansen b, stern l ( ) drug-induced displacement of bilirubin from albumin in the newborn. dev pharmacol ther : – . cashore wj ( ) bilirubin metabolism and toxicity in the newborn. in: polin ra, fox ww (eds) fetal and neonatal physiology. w.b. saunders, philadelphia, pp – . cashore wj, oh w, brodersen r ( ) bilirubin-displacing effect of furosemide and sulfisoxazole. an in vitro and in vivo study in neonatal serum. dev pharmacol ther : – . chalasani n, chowdhury nr, chowdhury jr, boyer td ( ) kernicterus in an adult who is heterozygous for crigler- najjar syndrome and homozygous for gilbert-type genetic defect. gastroenterology : – . chowdhury jr, wolkoff aw, chowdhury nr, arias im ( ) hereditary jaundice and disorders of bilirubin metabolism. in: scriver cr, beaudet al, sly ws, valle d (eds) the metabolic and molecular bases of inherited disease. mcgraw-hill, new york, pp – . christakis da, rivara fp ( ) pediatricians’ awareness of and attitudes about four clinical practice guidelines. pediatrics : – . cooper-peel c, brodersen r, robertson a ( ) does ibuprofen affect bilirubin-albumin binding in newborn infant serum? pharmacol toxicol : – . cremer rj, perryman pw, richards dh ( ) influence of light on the hyperbilirubinaemia of infants. lancet : – . crigler jf jr, najjar va ( ) congenital familial nonhemo- lytic jaundice with kernicterus. pediatri : – . ebbesen f, brodersen r ( ) comparison between two preparations of human serum albumin in treatment of neonatal hyperbilirubinaemia. acta paediatr scand : – . ennever jf ( ) phototherapy for neonatal jaundice. in: polin ra, fox ww (eds) fetal and neonatal physiology. w.b. saunders, philadelphia, pp – . farnsworth d ( ) the farnsworth-munsell hue dichotomous tests for colour vision. j opt soc amer : – . fink s, karp w, robertson a ( ) ceftriaxone effect on bilirubin-albumin binding. pediatrics : – . fink s, karp w, robertson a ( ) effect of penicillins on bilirubin-albumin binding. j pediatr : – . gartner lm, herrarias ct, sebring rh ( ) practice patterns in neonatal hyperbilirubinemia. pediatrics : – . guentert tw, frey bm, luedin e, heinzl s, brodersen r ( ) increase of plasma nonesterified fatty acid concentration and decrease of albumin binding affinity after intravenous injection of glycocholate-lecithin mixed micelles. j lab clin med : – . honore b, brodersen r ( ) albumin binding of anti- inflammatory drugs. utility of a site-oriented versus a stoi- chiometric analysis. mol pharmacol : – . ivarsen r, brodersen r ( ) displacement of bilirubin from adult and newborn serum albumin by a drug and fatty acid. dev pharmacol ther : – . jarnerot g, andersen s, esbjorner e, sandstrom b, brodersen r ( ) albumin reserve for binding of bilirubin in maternal and cord serum under treatment with sulphasalazine. scand j gastroenterol : – . johnson lm, bhutani vk, brown ak ( ) system-based approach to management of neonatal jaundice and prevention of kernicterus. j pediatr : – . kinney hc, armstrong dd ( ) perinatal neuropathology. in: graham di, lantos pl (eds) greenfield’s neuropathology. arnold, london, p – . krukow n, brodersen r ( ) toxic effects in the gunn rat of combined treatment with bilirubin and orotic acid. acta paediatr scand : – . kuang aa, rosenthal p, roberts jp, renz jf, stock p, ascher nl, emond jc ( ) decreased mortality from technical failure improves results in pediatric liver transplantation. arch surg : – ; discussion – . lucey jf ( ) the future demise of exchange transfusions for neonatal hyperbilirubinemia. dev med child neurol : – . martin e, fanconi s, kalin p, zwingelstein c, crevoisier c, ruch w, brodersen r ( ) ceftriaxone-bilirubin-albumin interactions in the neonate: an in vivo study. eur j pediatr : – . meropol sb, luberti aa, de jong ar, weiss jc ( ) home phototherapy: use and attitudes among community pediatri- cians. pediatrics : – . nazer h, al-mehaidib a, shabib s, ali ma ( ) crigler- najjar syndrome in saudi arabia. am j med genet : – . ostrea em, jr., bassel m, fleury ca, bartos a, jesurun ca ( ) influence of free fatty acids and glucose infusion on serum bilirubin and bilirubin binding to albumin: clinical implications. j pediatr : – . park ws, chang ys, chung sh, seo dw, hong sh, lee m ( ) effect of hypothermia on bilirubin-induced alterations in brain cell membrane function and energy metabolism in newborn piglets. brain res : – . puffenberger eg, hu-lince d, parod jm, craig dw, dobrin se, conway ar, donarum ea, strauss ka, dunckley t, cardenas jf, melmed kr, wright ca, liang w, stafford p, flynn cr, morton dh, stephan da ( ) mapping of sudden infant death with dysgenesis of the testes syndrome (siddt) by a snp genome scan and identification of tspyl loss of function. proc natl acad sci usa : – . rand eb, olthoff km ( ) overview of pediatric liver transplantation. gastroenterol clin north am : – . rasmussen lf, ahlfors ce, wennberg rp ( ) displace- ment of bilirubin from albumin by indomethacin. j clin pharmacol : – . rasmussen lf, ahlfors ce, wennberg rp ( ) the effect of paraben preservatives on albumin binding of bilirubin. j pediatr : – . robertson a, brodersen r ( ) effect of lactate, pyruvate, acetone, acetoacetate, and beta-hydroxybutyrate on albumin binding of bilirubin. j pediatr : – . robertson a, brodersen r ( ) effect of drug combinations on bilirubin-albumin binding. dev pharmacol ther : – . robertson a, fink s, karp w ( ) effect of cephalosporins on bilirubin-albumin binding. j pediatr : – . robertson a, karp w, brodersen r ( ) bilirubin displacing effect of drugs used in neonatology. acta paediatr scand : – . robertson af, baker jp ( ) lessons from the past. semin fetal neonatal med : – . robinson pj, rapoport si ( ) binding effect of albumin on uptake of bilirubin by brain. pediatrics : – . roger c, koziel v, vert p, nehlig a ( ) mapping of the consequences of bilirubin exposure in the immature rat: local cerebral metabolic rates for glucose during moderate and severe hyperbilirubinemia. early hum dev : – . schauer r, stangl m, lang t, zimmermann a, chouker a, gerbes al, schildberg fw, rau hg ( ) treatment of crigler-najjar type disease: relevance of early liver trans- plantation. j pediatr surg : – . shevell mi, majnemer a, schiff d ( ) neurologic perspectives of crigler-najjar syndrome type i. j child neurol : – . suresh g, lucey jf ( ) lack of deafness in crigler-najjar syndrome type : a patient survey. pediatrics :e . van der veere cn, sinaasappel m, mcdonagh af, rosenthal p, labrune p, odievre m, fevery j, otte jb, mcclean p, burk g, masakowski v, sperl w, mowat ap, vergani gm, heller k, wilson jp, shepherd r, jansen pl ( ) current therapy for crigler-najjar syndrome type : report of a world registry. hepatology : – . vreman hj, wong rj, murdock jr, stevenson dk ( ) in vitro efficacy of an led–based phototherapy device (neo- blue™) compared to traditional light sources. pediatr res : a . vreman hj, wong rj, stevenson dk ( ) phototherapy: current methods and future directions. semin perinatol : – . wadsworth sj, suh b ( ) in vitro displacement of bilirubin by antibiotics and -hydroxybenzoylglycine in newborns. antimicrob agents chemother : – . walker pc ( ) neonatal bilirubin toxicity. a review of kernicterus and the implications of drug-induced bilirubin displacement. clin pharmacokinet : – . wennberg rp ( ) the blood-brain barrier and bilirubin encephalopathy. cell mol neurobiol : – 	management of hyperbilirubinemia and prevention of kernicterus in patients with crigler-najjar disease 	abstract 	introduction 	patients and methods 	patients 	clinical management 	vision testing 	results 	diagnosis 	phototherapy 	hospitalizations 	neurological outcome 	liver transplantation 	case summaries 	type i crigler-najjar disease in the newborn period 	case . prospective management of cnd 	case . poor neonatal risk assessment, inadequate therapy, and kernicterus 	hospital management 	case . prevention of kernicterus in a cnd patient during an infectious illness 	case . acute kernicterus during an infectious illness in a patient with cnd 	liver transplantation 	case . liver transplant in a -year-old mennonite man with cnd and increasing bilirubin levels 	discussion 	diagnosis and management of cnd in the newborn period 	cnd patients can be managed safely to prevent death and disability 	providing effective phototherapy 	anticipating and preventing kernicterus 	caring for the aging cnd population 	conclusion: lessons learned from cnd 	appendix 	potential bilirubin-albumin displacing interactions (see text for references) 	references << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (none) /calrgbprofile (srgb iec - . ) /calcmykprofile (iso coated) /srgbprofile (srgb iec - . ) /cannotembedfontpolicy /error /compatibilitylevel . /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /perceptual 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{background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ assessment of sex-specific genetic and environmental effects on bone mineral density lillian b. brown, elizabeth a. streeten, alan r. shuldiner, , laura a. almasy, patricia a. peyser, and braxton d. mitchell n department of epidemiology, university of michigan school of public health, ann arbor, michigan division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland geriatric research and education clinical center (grecc), veterans administration medical center, baltimore, maryland department of genetics, southwest foundation for biomedical research, san antonio, texas although it is widely accepted that genes contribute significantly to the variation in bone mineral density (bmd), the nature of the genetic contribution is poorly defined. there are large gender differences in bmd, although whether sex-specific genetic effects influencing variation in bmd contribute to these differences is not known. to address this issue, we studied subjects from large families participating in the amish family osteoporosis study. bone mineral density was measured at the hip and spine by dual energy x-ray absorptiometry (dxa). we used variance decomposition procedures to partition variation in bmd into genetic and environmental effects common to both sexes and to men and women separately. after accounting for covariate effects, the heritability of bmd ranged from . to . in men and . to . in women. the residual environmental variance in bmd at the spine, but not hip, was significantly higher in men than in women (p o . ), reflecting a greater variance in bmd due to unexplained non-genetic factors in men. in contrast, there were no significant differences between men and women in the magnitude of the genetic variance in bmd, nor did the genetic correlation in bmd between men and women differ significantly from one. overall, these analyses do not provide evidence for sex-specific genetic effects, suggesting that many of the genes influencing variation in bmd should be detectable in both men and women. & wiley-liss, inc. key words: sex; bone mineral density; genetics; variance; interaction; heritability grant sponsor: national institutes of health; grant numbers: ro -ar , ro -ag , ro -hl , and m rr awarded to the university of maryland general clinical research center. ncorrespondence to: braxton d. mitchell, phd, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, w. redwood street, room , baltimore, md . e-mail: bmitchel@medicine.umaryland.edu received december ; accepted march published online may in wiley interscience (www.interscience.wiley.com) doi: . /gepi. introduction osteoporosis is a debilitating disease character- ized by low bone mineral density (bmd) that is associated with significant morbidity and mortal- ity. according to the third national health and nutrition examination survey (nhanes iii), % of u.s. women over age have low bone mass and % of white postmenopausal women have osteoporosis at the femoral neck (hip) [looker et al., ]. osteoporotic fractures are one of the most common causes of disability and contribu- tors to medical care costs in many regions of the world [cummings and melton, ]. large prospective studies have shown that almost all types of fracture are increased in adults with low bmd [cummings et al., ]. fractures result in functional impairment, including impaired basic activities of daily living, subsequent nursing home care, the loss of ambulatory ability, and loss of the ability to live independently [cummings and melton, ; gullberg et al., ]. longitudinal, family, and twin studies suggest that variation in bmd is strongly influenced by genes. women with a maternal history of hip fracture have lower bmd than women without a history of such fracture [seeman et al., ] and are themselves twice as likely to suffer a hip fracture [cummings et al., ; zmuda et al., ]. bmd is highly correlated between twin pairs [eisman, ; christian et al., ; pocock et al., ] and among related family members [jouanny et al., ; matkovic et al., ; ferrari et al., ; lutz, ; seeman et al., ]. genetic epidemiology : – ( ) & wiley-liss, inc. overall, twin and family studies suggest that after considering covariates, – % of variance in bone density measurement is genetically deter- mined [eisman, ; krall and dawson-hughes, ; prentice, ]. the nature of the genetic contribution to bmd is poorly defined. there are strong gender differ- ences in bmd that contribute to a substantially higher fracture risk among women than men. moreover, the gender differences are not constant across the age range as, for example, in the immediate post-menopausal period, when bone loss is accelerated in women as compared to men of the same age [hunter and sambrook, ]. given the large genetic effects on bmd, it is possible that different (although overlapping) subsets of genes contribute to variation in bmd in men and women and/or that the genetic determinants of bmd may be modulated by sex- specific hormonal, environmental, and nutritional factors. evidence for sex-specific genetic effects on bmd, however, is scant. in the experimental mouse model, orwoll et al. [ ] examined peak bmd in males and females from different inbred recombinant strains and observed signifi- cant gender by strain interactions, with males having higher bmd than females in some strains, but lower in others. in subsequent quantitative trait linkage (qtl) analysis, these researchers then identified six distinct chromosomal regions linked to variation in bmd in male mice only, five regions linked in female mice only, and two linked regions that were shared between the genders [orwoll et al., ]. whether these qtls will, in fact, turn out to have differential effects on bmd between male and female mice remains to be seen, as does their potential relevance to bmd in humans. in humans, naganathan and colleagues also provided evidence for the presence of sex-specific effects on bmd by showing that the correlations in bmd at the forearm and spine, as measured by ultrasound, were higher in same sex dizygotic (dz) twin pairs than in opposite sex dz twin pairs [naganathan et al., ]. the intraclass correla- tion for the forearm bmd measurement was . in both female (n¼ pairs) and male (n¼ pairs) dz twin pairs, compared to . in opposite sex twin pairs (n¼ pairs). estimated correlations between female dz twin pairs, male dz twin pairs, and opposite sex dz twin pairs at the spine were . , . , and . , respectively. addition- ally, linkage studies carried out in at least two populations have reported stronger evidence for linkage to qtls influencing bmd in one sex than in the other [karasik et al., ; kammerer et al., ] although these results are difficult to interpret since the hypothesis of sex-specific linkage was not formally tested. aside from these few studies, the issue of sex-specific genetic effects has not been extensively addressed. the goal of our study is to explore more fully potential sex-specific differences in the relative influence of genetic and environmental factors on bmd. to address this issue, we have analyzed data collected from amish individuals from large extended families. these families were recruited through the amish family osteoporosis study (afos), a study consisting of subjects enrolled from very large families from lancaster county, pennsylvania. using variance decomposi- tion procedures, we quantified genetic effects on bmd that are common to both sexes and to men and women separately. specifically, we consid- ered the following questions: ( ) is the magnitude of the genetic variation larger in one sex than in the other?; ( ) is the magnitude of the residual environmental variation larger in one sex than in the other?; and ( ) is there evidence for sex- specific genetic effects on bmd (i.e., is there a subset of genes that influences variation in bmd in both sexes jointly and another subset of genes that influences variation in each sex separately)? methods subjects and measurements the afos began in with the goal of identifying the genetic determinants of osteoporo- sis. individuals believed to be at risk for osteo- porosis by virtue of their fracture history or prior bone density measurements were recruited into the study as index cases. these individuals were recruited by word-of-mouth, a community-wide mailing, advertisements in an amish newspaper, or by referral from local physicians. the diagnosis of osteoporosis in these individuals was verified by measurement of bmd using dual energy x-ray absorptiometry (dxa). individuals found to have a t score of � . or less in either the hip or spine were designated as probands. we then invited the probands’ spouses and all first-degree relatives aged years and over to participate in the study. in addition, we recruited into the study the first- degree relatives of any other examined individual (e.g., spouses) having a t score of � . or lower at the spine or hip on our bone densitometry test. brown et al. between the initiation of recruitment in and january , a total of individuals were enrolled into the afos, including probands and their relatives. of those enrolled, complete information was obtained on subjects, includ- ing women and men. using the extensive genealogical records maintained on the amish [beiler, ; agarwala et al., , ], these individuals could be combined into a single -generation pedigree. study participants were evaluated at the amish research clinic in strasburg, pa, by qualified nurses known to the participants. a medical interview included past medical history, family history of medical pro- blems including fractures, and specific details regarding previous fractures, history of medica- tion use, and menstrual and reproductive history for women. height was measured using a stadiometer and weight was recorded with the participant in standard amish clothing but with- out shoes. the mineral content at the lumbar spine and hip was measured by dexa by a registered nurse certified in bone densitometry (hologic w, hologic, inc., bedford, ma). bmd was determined by dividing the total bone mineral content (g) by the projected area of the region scanned (cm ). for this report, we have restricted analysis of bmd to measures obtained at the spine, femoral neck, and total hip. total hip bmd is defined as the sum of the bone mineral content at the femoral neck, trochanter, and intertrochan- ter sites divided by the total area of these three sites. the protocol for the afos was approved by the institutional review board at the university of maryland. informed consent was obtained from all subjects prior to participation. analytical methods we carried out a series of statistical analyses using a full pedigree-based variance component approach for the purpose of partitioning variation in bmd into selected components [almasy and blangero, ]. in the basic model, the level of bmd, y, for individual i was modeled as yi¼mþ p bixij þgiþei, where m is the mean bmd, xij is the j-th covariate for the ith individual, bj is its regression coefficient, and gi and ei represent the random deviations from m for individual i that are attributable to additive genetic and residual error effects, respectively. the residual error component includes true random error, measure- ment error, and any non-additive genetic compo- nents. the effects of gi and ei are uncorrelated and normally distributed with mean zero and var- iances sg and se , respectively. the se term can be considered the residual environmental variance, that is, the remaining environmental variance after accounting for the effects of measured covariates. maximum likelihood methods were used to simultaneously estimate the mean and variances as well as the covariate and genetic effects. all models included the following covari- ates: age, age , height, and bmi. these factors were selected as covariates because they were each independently associated with one or more bmd measures in a sex-specific preliminary analysis. the significance of particular compo- nents can be assessed by comparing the likelihood of a model with the component of interest estimated to the likelihood of a model in which the component effect is constrained to be zero. the full and restricted models are then compared by likelihood ratio test, which produces a test statistic that is asymptotically distributed as a w distribution with the degrees of freedom equal to difference in number of parameters between the two models. using this variance component modeling approach, we then considered various hypotheses concerning the sex-specific variances in bmd. our initial goal was to compare the genetic variance between the two sexes. we addressed this issue in two ways, first by comparing the ‘‘relative’’ proportion of the variance attributable to genetic effects in men and women separately and, second, by comparing the absolute magnitude of the genetic effect. the proportion of the total phenotypic variation in bmd that could be attributable to additive genetic effects (sg /sp ) corresponds to ‘‘narrow’’ sense heritability (h ) since it reflects the degree of additive genetic variance only. we tested whether the heritabilities in bmd differed between men and women (i.e., h men¼h women) by comparing the difference between the heritability estimates in the two sexes with the estimated variance of the difference. estimating the absolute magnitude of the genetic variance required a further partitioning of the variance. following the approach of blangero and colleagues, [blangero, ; martin et al., b; towne et al., ], we expanded the basic variance component model to allow the genetic variances in male and female bmd to differ when men and women were considered together. briefly, the expected genetic covariance sex-specific effects on bmd between a male and female relative pair is defined as: covðgm; gfÞ ¼ frg�sg�m�sg�f where f is the coefficient of kinship between the two individuals, rg is the genetic correlation between the expressions of the trait in the two sexes, and sg-m and sg-f are the genetic standard deviations for men and women, respectively. with the additional terms as defined above, we constructed a general model that partitioned variance in bmd into the following terms: an overall mean scaled to the value in males, a coefficient corresponding to the effect of sex (bsex), coefficients for age and sex*age (bage and bage*sex), coefficients for age and sex*age (bage and bage *sex), coefficients for height (bheight), and body mass index (bbmi), male and female genetic standard deviations (sg-m and sg-f), male and female residual environmental standard deviations (se-m and se-f), and the genetic correlation between males and females (rg). genetic correlations reflect the degree to which the genetic effect on bmd in men correlates with the genetic effect on bmd in women [falconer and mackay, ]. interaction terms of sex with age and age were included because of the well- established differences between men and women in the relationship between age and bmd. this expanded model allowed us to test several explicit hypotheses related to sex by gene interac- tions. first, we considered if the magnitude of the genetic effect was similar between the sexes by testing whether the genetic standard deviations were similar between men and women (i.e., h : sg-m¼ sg-f). rejection of this hypothesis implies that genes account for a larger proportion of the variance in one sex than in the other. in similar fashion, we tested if the magnitude of the residual environmental effect was similar between the sexes (i.e., h : se-m¼ se-f). the third hypothesis that we tested was whether the magnitude of the genetic correlation was significantly less than one (i.e., h : rg¼ ). a genetic correlation between men and women that is significantly less than one implies that a different gene or suite of genes contributes to variance in bmd in men and women. if there is a sex � gene interaction, then the expectation is that rg o and sg-m a sg-f. as before, significance testing was conducted using the likelihood ratio test. specifically, we compared likelihoods between models in which values of sg-m and sg-f were allowed to differ (full model) and in which they were constrained to be the same (restricted model). similarly, we compared the likelihood between a model in which rg was estimated (full model) to that in which its value was constrained to be one (restricted model). when testing models for which the value of a particular parameter was con- strained to a boundary value (e.g., rg¼ ), the p value was based on a : mixture of a w distribution and a point mass at zero [self and liang, ]. results basic characteristics of the study population are shown in table i. the mean age of the men and women was approximately years. men were on average four to five inches taller than women (mean standard deviation; . . vs. . . in.) and lbs heavier ( . . vs. . . lbs). bmd measurements at the spine (l -l ), femoral neck, and total femur were significantly higher in men compared to women (p¼ . at the spine; p o . at the femoral neck and total hip). the overall (phenotypic) variance in bmd was also significantly greater in women than in men at all three bmd sites (p o . by the levene test for equality of variances). the numbers of relative pairs, both same sex and total, who were phenotyped and included in the analyses are shown in table ii. the sample included , female-female pairs ( mother- daughter, sister-sister, aunt-niece, and first cousin pairs) and male-male pairs ( father-son, brother-brother, uncle-nephew, and first cousin pairs). overall, there were , same sex and , opposite sex relative pairs, for a total of , total relative pairs, including same sex and opposite sex pairs combined. table i. characteristics (mean sd) of amish family osteoporosis study participants variable men (n¼ ) women (n¼ ) age-adjusted p value age (yrs) . . . . . height (in) . . . . o . weight (lb) . . . . o . bmi (kg/m ) . . . . o . bmd (g/cm ) spine (li-l ) . . . . . femoral neck . . . . o . total femur . . . . o . brown et al. the heritabilities of bmd at the spine, femoral neck, and total femur were estimated after accounting for the effects of the measured covariates age, age , height, and bmi. the residual heritability of bmd ranged from . (spine) to . (total femur) in men and from . (femoral neck) to . (spine and total hip) in women. at each site, the estimated residual heritability in bmd was significantly larger in women than in men (p o . at all sites) (data not shown). to gain insights into the factors contributing to variation in bmd in men and women, we partitioned the total variance in bmd into components attributable to measured covariates (e.g., age, height, and bmi), the additive effects of genes, and to unmeasured, or residual, environ- mental factors. results of these analyses are shown in table iii. in men, measured covariates accounted for very little ( . %) of the total variation in spine bmd, and from to % of the variation in hip bmd. the additive effects of genes accounted for to % of the total variation in bmd, in contrast to the to % of the residual variation in bmd as described above. thus, % of the total variation in spine bmd in men could not be accounted for by genes or measured covariates. in contrast, in women, the measured covariates accounted for % of the total variation in spine bmd and to % of the total variation in hip bmd. the additive effects of genes accounted for an additional % of the variation in spine bmd in women and to % of the variation in hip bmd. thus, only to % of the total variation in bmd in women could not be accounted for by genes and/or measured covariates. after estimating the proportion of the total phenotypic variation attributable to genetic and environmental effects in men and women, we next tested several additional hypotheses, includ- ing whether the magnitude of the genetic and environmental variances differed between men and women and whether the genetic correlation in bmd differed between men and women. to accomplish these goals, we performed a more complete partitioning of bmd into its constituent genetic and residual environmental components. in these analyses, we allowed the genetic and residual environmental variances in bmd be- tween males and females to differ, and also estimated the genetic and residual environmental correlations in bmd between men and women. results from the full model, in which all para- meters were estimated, are shown in table iv. following estimation of the full set of model parameters, we performed a series of nested tests table ii. number of relative pairs included in the sample of amish subjects relative pair class number parent-offspring mother-daughter father-son all pairsa sibling-sibling sister-sister brother-brother all pairsa , avuncular aunt-niece uncle-nephew all pairsa , cousin-cousin ( st cousin only) female-female male-male all pairsa , total numbers of pairsb female pairs , male pairs total same sex pairs , totala , aincluding same sex and opposite sex pairs. bdoes not include grandparent-grandchild, grand avuncular, nd cousins, and more distantly related pairs. table iii. components of variance for bone mineral density (bmd) men (n¼ ) women (n¼ ) bmd site measured covariatesa geneticb residual environment measured covariatesa geneticb residual environment spine . . . . . . femoral neck . . . . . . total femur . . . . . . ameasured covariates include age, age , height, and bmi. bnote that the genetic contribution to total phenotypic variation presented in this table differs from the heritability presented in the text, which is estimated as the genetic contribution to residual (not total) phenotypic variation once covariate effects are accounted for. sex-specific effects on bmd in which we constrained values of selected parameters, which enabled us to test, first, whether the magnitude of the genetic variance in bmd differed between men and women; second, whether the magnitude of the residual environ- mental variance in bmd differed between men and women; and third, whether the genetic correlation in bmd between male and female relative pairs differed from one. with respect to the first hypothesis, we observed that the genetic sd did not differ significantly between men and women (sg-m vs. sg-f: spine: . vs. . ; femoral neck: . vs. . ; total hip: . vs. . ; p . for all). in contrast, we observed marginal evidence (p¼ . ) for a larger residual environ- mental sd in men than in women for spine bmd ( . vs. . ), although not for bmd at either of the two hip sites. with respect to the third hypothesis, we observed that the genetic correla- tion between men and women did not differ significantly from one (rg¼ . , . , and . for spine, femoral neck, and total hip). thus, our analyses provided modest evidence for a sex by residual environment interaction at one of the bmd sites (spine), but no evidence for a gene by sex interaction. nor was there evidence for sex- specific genetics effects on bmd. discussion the hypothesis that genetic and/or residual environmental effects on bmd might differ between men and women is motivated in part by the well-known gender differences that are observed in bmd. typically, men have higher bmd than women, and women experience a significantly higher rate of bone loss around the time of menopause compared to men of similar age. there are many possible explanations for these sex differences, including the sex differ- ences present in levels of androgens and estro- gens, which are thought to influence bone development and/or decrease bone resorption [vanderschueren and bouillon, ; gallagher, ]. genes may potentially play a significant role in influencing variation in steroid production and regulation, including the changes in these processes that occur over time. evaluating the gender-specificity of genetic effects on bmd is not straightforward. although the hypothesis that a particular allele is more strongly associated with bmd in one sex than the other is easily tested in the context of conventional table iv. model parameters estimated from variance partitioning of bone mineral density (bmd) parameter spine bmd ( � , in g/cm ) neck bmd ( � , in g/cm ) total hip bmd ( � , in g/cm ) m . . . b(age) � . � . � . b(sex) . . � . b(agensex) � . � . � . b(age ) � . . � . b(age nsex) � . � . � . b(height) . . . b(bmi) . . . sg-m . . . sg-f . . . se-m . . . se-f . . . rg . . . ll (full model) � . � . � . hypothesis : h : genetic variance equal between the sexes parameterization: sg-m¼sg-f ll (nested model): � . � . � . w . . . p . . . conclusion accept h accept h accept h hypothesis : h : environmental variance equal between the sexes parameterization: se-m¼se-f ll (nested model): � . � . � . w . . . p . . . conclusion: reject h accept h accept h hypothesis : h : genetic correlation similar between males and females parameterization: rg¼ ll (nested model): � . � . � . w . . . p . . . conclusion accept h accept h accept h amodel parameters: m¼mean bmd; b¼regression coefficients (for age, agensex, age , age nsex, height, and body mass index); sg-m¼genetic sd in males; sg-f¼genetic sd in females; se-m¼environmental sd in males; se-f¼environmental sd in females; rg¼genetic correlation in bmd between men and women. bmi¼body mass index; bmd¼bone mineral density; ll¼log-likelihood. p values for hypothesis based on a : mixture of a w and a point mass of zero. brown et al. genetic association studies, such analyses consider only the effect of an individual gene. linkage studies conducted in both mice [orwoll et al., ] and humans [karasik et al., ; kammerer et al., ] have revealed the presence of qtls and/or regions of linkage that are more strongly linked in one sex than in the other, although none of these studies has explicitly tested whether evidence for linkage could be excluded in the other sex. to our knowledge, only one other study has formally sought to identify gender-specific genetic effects in humans, with these researchers inferring the presence of such effects on the basis that the correlation in bmd was significantly higher in same sex dz twins than in opposite sex dz twins [naganathan et al., ]. however, these data are also consistent with several other interpretations, including the presence of a com- mon environmental component that was more frequently shared among same sex compared to opposite sex twin pairs. interestingly, the amish population does not appear to be at particularly high risk for fracture, and, in fact, rates of non-traumatic hip fracture are somewhat lower in the amish than in their non- amish counterparts, an observation consistent with mean bmd in the amish also being slightly higher [streeten et al., ]. nevertheless, the unique attributes of the old order amish (ooa) make this population an attractive one for attempting to dissect out the genetic contributions to phenotypic variation. amish families typically tend to be very large, so that there are a large number of sibling relative pairs available for analysis. moreover, the amish have a strong interest in their genealogies, and accurate record- keeping dating back many generations allows the large amish families to be linked into a single pedigree. finally, the relatively homogenous en- vironment of the ooa may allow for a more clear elucidation of the genetic factors contributing to bmd. comparison of trait heritabilities between men and women, whether expressed as the contribu- tion of genes to total trait variability or residual trait variability, provides only limited insight into possible gender differences in genetic effects. the reason for this is that heritabilities are proportions, and their values are influenced both by the magnitude of the genetic variance and the contribution of residual environmental factors to the total variance. we, thus, tested several addi- tional hypotheses, including whether the magni- tude of the genetic variance differed between men and women and whether the genetic correlation in bmd differed between men and women. the fact that the magnitude of the genetic standard deviation did not differ significantly between men and women indicates only that the contribu- tion of genes to the variance in absolute terms is relatively similar between men and women in this study group; this test does not address whether it is the same gene or suite of genes that influences variation in bmd in both sexes. however, this latter possibility (i.e., that the same subset of genes influences variation in bmd in men as in women) is consistent with the finding that the estimated genetic correlation did not differ significantly from one. our results do not support the existence of sex-specific genetic effects on bmd. however, this conclusion does not preclude the possibility that there might be individual loci that have sex-specific effects on bmd; rather the net contribution of sex-specific genetic effects across all loci is likely to be small. two additional caveats should be considered. first, this study population consists of families ascertained on probands with a history of hip fracture and/or osteoporosis. thus, the mean bmd in the study population is slightly lower than what would have been observed in randomly ascertained families. however, how this selection scheme might impact detection of sex-specific genetic effects on bmd is unclear. a second, and perhaps more limiting, issue is that we have also pooled subjects across a wide range of ages for our analyses. while pooling subjects of different ages produces large numbers of same sex relative pairs suitable for analysis, this strategy may dilute sex-specific genetic and residual environmental effects whose expression is age-dependent. in this regard, a more compre- hensive evaluation of this issue might include analysis of – -year-old men and women in order to evaluate sex-specific genetic and envir- onmental influences on acquisition of peak bone mass, or alternatively, to restrict analysis to post- menopausal women and older men to evaluate potential sex-specific genetic and environmental influences on bone loss. unfortunately, our study sample was not sufficiently large to consider sex- specific analyses in young and old groups separately as there were only males and females aged – years and males and females aged years and over. these totals provided an insufficient number of relative pairs in these strata to obtain the robust partitioning of sex-specific effects on bmd the variances required to estimate sex-specific genetic effects. despite the unique ancestral history of the amish and their relative protection against hip fracture, it seems reasonable to expect that the basic conclusions reached by this study pertaining to sex-specific genetic effects should be general- izable to other populations. the ooa in lancaster county are descendents of migrants from western europe and are thus derived from the same overall gene pool as the overall u.s. and european caucasian populations. perhaps more impor- tantly, the analytic approach we followed empha- sizes the additive effects of genes. although generalizability of these findings in the amish would be limited if there were amish-specific mutations in genes that had very large effects on bmd, we are not aware that this is the case. in our initial survey we did, in fact, identify several individuals with very low bmd who upon subsequent evaluation were found to carry muta- tions in col a , the gene encoding type collagen alpha and responsible for osteogenesis imperfecta. however, these individuals were excluded from the current analysis. it is possible that there may be gender differ- ences in genetic effects influencing other aspects of bone morphology not captured by bmd. male- female differences in a variety of indices related to hip geometry and structure have been reported [beck et al., ; kaptoge et al., ], many of which are also related to hip fracture indepen- dently of bmd [gluer et al., ; partanen et al., ; beck et al., ; karlsson et al., ]. genetic influences on some of these measures have been reported [jian et al., ; liu et al., ; koller et al., ], although the sex- specificity of these effects has not yet been investigated. detection and identification of sex-specific genetic effects when they exist is a worthwhile pursuit because such effects may provide impor- tant insights into disease etiology (e.g., genes expressed in only one sex or the other may operate on sex-specific risk factors) and also because appropriately modeling these effects should the- oretically enhance our ability to identify the genes underlying them. for example, knowledge of sex- specific genetic effects may motivate one’s choice of candidate genes, or improve ability to localize individual loci through linkage analysis by ex- plicit modeling of sex-specific linkage compo- nents [martin et al., a]. the absence of evidence for sex-specific genetic effects on bmd in our study offers the hope that at least many of the loci influencing variation in bmd should be detectable in both men and women. acknowledgments we thank drs. richa agarwala and alejandro schaffer for their assistance in pedigree construc- tion using the amish genealogical database. we also thank dr. holmes morton and caroline morton for their support in establishing and maintaining ties with the amish community. finally, we gratefully acknowledge our amish liaisons and field workers and the extraordinary cooperation and support of the amish community without which these studies would not have been possible. this work was supported in part by the university of maryland general clinical research center, grant m rr , general clinical research centers program, national center for research resources (ncrr), nih. references agarwala r, biesecker lg, hopkins ka, francomano ca, schäffer aa. . software for constructing and verifying pedigrees within large genealogies and an application to the old order amish of lancaster county. genome res : – . agarwala r, schäffer aa, tomlin jf. . towards a complete north american anabaptist genealogy ii: analysis of inbreeding. hum biol : – . almasy l, blangero j. . multipoint quantitative-trait linkage analysis in general pedigrees. am j hum genet : – . beck tj, 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reduced incidence of hip fracture in the old order amish. j bone miner res : – . towne b, blangero j, siervogel rm. . genotype by sex interaction in measures of lipids, lipoproteins, and apolipoproteins. genet epidemiol : – . vanderschueren d, bouillon r. . androgens and bone. calcif tissue int : – . zmuda jm, cauley ja, ferrell re. . recent progress in understanding the genetic susceptibility to osteoporosis. genet epidemiol : – . sex-specific effects on bmd wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ william a. fischel – professor of economics and robert c. & hilda hardy professor of legal studies skip to content 	 		advanced features of this website require that you enable javascript in your browser. thank you!	 	 	education 	degree finder 	departments & programs 	global learning 	graduate schools 	libraries 	lifelong learning 	summer at dartmouth 	the undergraduate experience 	research 	research news 	faculty experts 	schools & departments 	research support 	life & community 	explore the green 	arts across campus 	athletics & recreation 	diversity 	health & wellness 	technology 	outdoors 	residential life 	service 	student groups & activities 	sustainability 	more 	admissions 	schools 	centers 	global 	arts 	athletics 	giving search dartmouth college explore dartmouth 	 		 		 			 			 				 						william a. fischel 					 						professor of economics and robert c. & hilda hardy professor of legal studies 					 			 		 		 		 		 			 				 			 						 		 		 		 			primary menu 			menu 			 			 				 					 				 					search for: 					 				 				 			 	home 	working papers 	curriculum vita 	contact 	 				 					search for: 					 				 				 			 	 		 		 			 			 	 	 		 		 			autobiographical essay march 			 		 		 			photo credit janice g. fischel    i have been a professor in the dartmouth college economics department since and am also the robert c. and hilda hardy professor of legal studies, emeritus. i retired from teaching in . the courses i most recently taught were economics (survey for nonmajors) and economics (urban and land use). my continuing interests are in public economics, particularly local government structure and behavior. my most recent work in this vein is "counting on counties," which offers a theory of american county government formation and its regional geographic variations. climate and race are a large part of it.      unlike most other economists, i have relied primarily on sole-author books to develop my scholarship. their common theme holds that local governments should be thought of as active economic agents rather than passive “creatures of the state.” my most recent book is “zoning rules! the economics of land use regulation,” published by the lincoln institute of land policy in july . it is an update and extension of my book, “the economics of zoning laws,” which argued that zoning is the product of rational, if not always admirable, economic calculation by voters in american municipalities. a prize-winning undergraduate thesis by kihara kiarie explored zoning in an inverted way by examining land use in houston, texas, the only large city that lacks zoning, and a copy of his work is available here. the centerpiece of  “zoning rules!” is its economic explanation of how zoning evolved. it shows that zoning’s goals shifted from municipal “good housekeeping”  to “growth control” as a result of inflation and environmental activism in the s. this was the product of what i call “the rise of the homevoters,” the local political movement in which homeowners displaced the pro-growth factions in local government. the anti-growth shift accounts for the higher housing costs of the west and the northeast compared to the rest of the united states. my current research examines the link between zoning pioneers such as edward bassett (his autobiography is here) and my thesis that cheap motor vehicles provided the impetus for zoning in the - era. a general economic theory of local government behavior was the subject of  “the homevoter hypothesis” (harvard university press in ). because homeowners have so much of their net worth wrapped up in their houses, they pay close attention to the many things that local governments can do to enhance or detract from their value. this provides a political side for the famous vote-with-your-feet model of local government, the tiebout hypothesis. the homevoter model explains why proposition , the california voter initiative that limited property taxes, was caused by serrano v. priest, which undermined voter incentives to support local school taxes. i also organized a conference and edited a book of essays, “the tiebout model at fifty” (lincoln institute of land policy, ), that probes the implications of charles tiebout’s durably famous model. “regulatory takings” (harvard university press, ) investigated the constitutional ways by which the excesses of zoning might be curbed by the judiciary without infringing on the creativity and autonomy of local governance. of special interest during this phase of my career were follow-ups on regulatory takings cases to see what happened afterwards, as in the photographic essays (lucas essay) (lucas update) ) on lucas v. south carolina coastal council, u.s. ( ). a rethinking of regulatory takings is in my investigation of the facts of  miller v. schoene, the case of the cedar trees that made apples go bad. it has made me less optimistic about the doctrine’s viability. my book, “making the grade” (university of chicago press), explores the economic evolution of american public school districts, the local government boundaries that home buyers care most about. the book explains the transformation of education from one-room schools, which were ungraded, to the age-graded schools we now reflexively think of as “real school.” there were over , districts in , but they now number fewer than , (here is a map of american school districts in , courtesy of sarah battersby). i regard their transformation as an example of market-like spontaneous order, the most subtle of which is summer vacation. voters voluntarily surrendered one-room schools and their tiny districts because they retarded children’s access to high school. inverted support for this is provided by the continuing embrace of one-room schools by the old-order amish: “do amish one-room schools make the grade? the dubious data of wisconsin v. yoder” (university of chicago law review ). my writing has been shaped by several year-long leaves at universities on the west coast. my wife, janice g. fischel,  (moravian ‘ ) and i have been at the university of california at davis ( - ); at santa barbara ( - and - ); and at berkeley’s law school ( - ). we also spent a year in seattle, where i visited the daniel j. evans school of public affairs at the university of washington ( - ).  our permanent home is in hanover, new hampshire, in a neighborhood within swimming distance of vermont. i served on the hanover zoning board from to , and i have rejoined the board in my retirement. janice and i are both natives of bethlehem, pennsylvania, though i grew up in nearby lower saucon township and attended hellertown high school (class of ). i graduated from amherst college in and got a phd in economics from princeton university in . i studied some law (but do not have a law degree) at vermont law school and at what its graduates fondly called “henry manne’s summer camp.” i enjoy back-road bicycling and year-round hiking. janice and i have for years followed dartmouth men’s basketball, and i have most recently served as an academic advisor to the team. a short memoir that relates my basketball experience to economics is available here. we are also architecture enthusiasts and enjoy walkabouts in new york and other cities. janice makes and sells photographic note cards, which you can view at http://www.jgfischel.com. our son, josh, graduated from amherst college (class of ) and received his master’s degree from the gerald r. ford school of public policy at the university of michigan in . in , he married cameren cousins, a middlebury graduate who received an mba in sustainability from antioch in . both are teachers and live in acton, massachusetts with their son, cabot earnest fischel, born in . cabot, janice, and bill cabot, cameren, and josh. acton, mass. march 			 		 		 			 		 		 	 	 			 		 		 	 	 	 	 	 		 		footer content 				 		 			 								 			 		 	 		 			 			 		 		 	 	 	my dartmouth 	students 	faculty 	staff 	alumni 	families 	find it fast 	campus map 	directory 	events 	news 	visit 	resources 	dartmouth at a glance 	accessibility 	administrative offices 	emergency preparedness 	careers 	sexual respect & title ix connect with us 	facebook 	instagram 	twitter 	youtube 	copyright © trustees of dartmouth college 	• 	privacy 	• 	a-z index 	• 	site or accessibility feedback wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm 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)).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ doi: . /s - ( ) - leds in navigationm a r k e t a n a l y s i s iii-vs review the advanced semiconductor magazine vol - no - november from buoys to boats hb-led for navigation last year i described the revolution that hb leds brought to the navigational buoy industry. since then, hb-leds have been used as sealed units in marker buoys by the local fishermen in new england and now they are being extended to the boating public. the latest benefit being provided by the ever improving led is that of naviga- tional safety in the form of led based watertight housings and snap-in replace- ments for prior generations of marine navi- gational bulbs. this boating utility is derived from estab- lished led benefits such as improved brightness, long lifetime, convenience and the lower power consumption. in some markets this also leads to lower costs. according to the founders of orca green marine technology corp (ogm), otto (hobie) caldwell and meg littlefield, the argo navis series of lighting products were conceived during a harvest moon regatta in the fall of --- idyllic sound- ing circumstances that turned out to be in reality a grueling race. the company, based in kemah,texas and founded in november , has wasted little time in bringing a range of naviga- tional lighting products to market, start- ing with a low power, bi-colour led nav- light ( . amp at v dc) in june . these navigational products are very versatile from the user point of view, since they can be powered by either a v battery, or relatively low cost aa, c or d battery packs and still meet coast guard regulations of horizontal and ver- tical sector visibilities that exceed nautical miles. the almost % one colour output of leds of the correct wavelength, plus strict criteria for led brightness and viewing angle, allows the lamps to easily meet the coast guard ‘ colregs’ (without the use of filters, therefore no filter deteriora- tion --- darkening, cracking or clouding - -- occurs with age). the latest ogm products include com- plete led-based lights, such as the argo navis unit, in a waterproof housing (see figure ). designed as a bow light, it can mount at the front of a boat for years at a time. its rugged, waterproof housing will take countless waves in rough seas (a resistance to feet of salt water for one hour is specified!). also included in the argo navis series are snap-in led bulb-replacement boards to convert older bulb serviced navigational lamps into long-life led fixtures (see figure .) and stern lights that meet specifications for power and sail boats up to feet in length. the typical sailor’s response has been very positive (and sounds a little like last month’s amish story) with such comments as ‘the energy to run two w nav-light incandescent bulbs (at dr. alan mills reporting from san francisco. pelicans on the phoenix - boats such as these in san diego are likely to take new navigational led lights with alacrity ©gail purvis pp - .qxd / / : page v) for hours uses as much energy as my on-board refrigerator uses for the day’. needing about volts to operate, leds draw much less power and greatly increase the safety factor by extending battery life between charges by solar, diesel or other sources --- a benefit that is really valuable for long distance sailing --- such as a month without charging! one of the key features of the new orca green product line is the flexibili- ty of their novel, constant current led power supplies for which patent protec- tion has been applied. using small surface mount components, these switching regulators occupy a dime-sized area and are integrated into the lighting fixture, one for each colour in the bi-colour bow light. they can make possible a constant current of milliamps and give a constant led brightness across a to v input volt- age range. this feature, the company believes, will provide the best marine lighting-market options and a distinct market advantage in the near term.this power regulator also makes the ability to use standard and relatively inexpensive . v battery packs in an emergency an added safety feature. the next product in line will be the trianchor light, a weatherproof tri- colour/anchor light (red, white and green), which is expected to be released in november or early december. in addition to providing the red and green (port and starboard) and white (stern) navigational identification, it gives the required all around white lighting for riding at anchor. when anchored, the low power drain of the white leds (only . watts or . amp compared with w for bulb operat- ed lamps) provides a time benefit by reducing the frequent need to run the engine to recharge the battery. it also makes solar recharging of the bat- tery a realistic option especially for long distance sailing.the trianchor unit is designed to sit at the masthead for years at a time unattended, instead of someone having to climb a or ft ( m) mast, every year or two to replace a bulb. its small size (only ” high) will also be an advantage for masthead mounting. with the number of small boats around the world that could benefit from the introduction of the argo navis led navigation lights being in the millions, this market must surely become a significant demand segment of the future for high brightness leds and add another growth market to the hb-led holster. so all you boating enthusiasts check out the benefits of led marine lighting at www.orcagreen.com and help in the growth of another power conserving and user-friendly hb-led market. leds in navigation m a r k e t a n a l y s i s www.three-fives.com figure the argo navis series uses a patent-pending design for power regulation, which ensures the lights burn at full brightness as battery voltage drops from v down to v figure bulb replacement inserts give ‘snap-in" led power efficiencies without having to replace their existing housings. pp - .qxd / / : page article association between variants in the genes for adiponectin and its receptors with insulin resistance syndrome (irs)-related phenotypes in mexican americans d. k. richardson & j. schneider & m. j. fourcaudot & l. m. rodriguez & r. arya & t. d. dyer & l. almasy & j. blangero & m. p. stern & r. a. defronzo & r. duggirala & c. p. jenkinson received: december /accepted: june / published online: august # springer-verlag abstract aims/hypothesis the aim of this study was to examine whether genetic variation in adipoq, adipor and adipor may contribute to increased susceptibility to components of the insulin resistance syndrome (irs). materials and methods we genotyped single-nucleotide polymorphisms (snps) in adipoq, adipor and adi por in mexican american subjects (n= ) and per- formed an association analysis of irs-related traits. results of the eight snps examined in the adipoq gene, rs and rs exhibited significant associations with bmi (p= . and p= . ), fasting specific insulin (p= . and p= . ), sum of skin folds (ss) (p= . and p= . ) and homeostasis model assessment of insulin sensitivity (homa-%s) (p= . and p= . ). two other snps, rs and rs , were signifi- cantly associated with ss (p= . and p= . ). snp rs of adipor was significantly associated with bmi, ss and waist circumference (p= . , p= . and p= . ). fourteen of the adipor snps were found to be significantly (p< . ) associated with fasting plasma triglyceride concentrations. four of these snps (rs , rs , rs and rs ) were in high pairwise linkage disequilibrium (r = . ) and were strongly associated with fasting triglyceride levels (p= . , p= . , p= . and p= . ). adjusting for the effects of bmi and homa-%s on triglyceride concentrations increased significance to p= . for snp rs . bayesian quantitative trait nucleotide analysis was used to examine all possible models of gene action. again, snp rs provided the strongest statistical evidence of an effect on triglyceride concentrations. conclusions/interpretation these results provide evidence for association of snps in adipoq and its receptors with multiple irs-related phenotypes. specifically, several ge- netic variants in adipor were strongly associated with decreased triglyceride levels. keywords adiponectin . adiponectin receptors . association study. genetics of type diabetes mellitus . insulin resistance syndrome . single-nucleotide polymorphisms abbreviations ampk adenosine ′-monophosphate-activated protein kinase bqtn bayesian quantitative trait nucleotide fpg fasting plasma glucose fsi fasting specific insulin homa-%β homeostasis model assessment of β-cell insulin secretion homa-%s homeostasis model assessment of insulin sensitivity hwe hardy–weinburg equilibrium diabetologia ( ) : – doi . /s - - - d. k. richardson (*) : m. j. fourcaudot : l. m. rodriguez: r. a. defronzo : c. p. jenkinson division of diabetes mc , department of medicine, the university of texas health science centre at san antonio, floyd curl drive, san antonio, tx - , usa e-mail: richardsond @uthscsa.edu j. schneider : t. d. dyer : l. almasy : j. blangero : r. duggirala : c. p. jenkinson southwest foundation for biomedical research, san antonio, tx, usa r. arya : m. p. stern division of clinical epidemiology, department of medicine, university of texas health science centre at san antonio, san antonio, tx, usa irs insulin resistance (metabolic) syndrome ld linkage disequilibrium lep fasting leptin maf minor allele frequency mga measured genotype approach pai- plasminogen activator inhibitor- safds san antonio family diabetes study snp single-nucleotide polymorphism ss sum of skin fold thickness trl triglyceride-rich lipoproteins wc waist circumference introduction type diabetes mellitus is a chronic metabolic disorder characterised byhyperglycaemia, insulin resistance, impaired beta cell function [ , ] and a variety of cardiovascular risk factors, including hypertension, elevated triglycerides, reduced hdl cholesterol, increased plasminogen activator inhibitor- (pai- ) levels, and endothelial dysfunction [ , ]. collectively, these metabolic and cardiovascular risk factors have been referred to as the insulin resistance syndrome (irs) or metabolic syndrome. insulin resistance in type diabetes results from impaired insulin action in skeletal muscle and liver [ , ]. also, adipocytes are resistant to the anti-lipolytic action of insulin [ , ], and the resultant increase in plasma nefa exacerbates the insulin resistance in muscle and liver [ – ]. the prevalence of type diabetes and the irs is rapidly and relentlessly increasing and, although major risk factors for these disorders have been identified [ ], the genetic factors responsible for type diabetes mellitus and the irs remain unclear. genome-wide scans have demonstrated linkage between a number of chromosomal regions and diabetes or diabetes-related traits [ , ]. candidate gene studies and a number of single-nucleotide polymorphisms (snps) have also been associated with type diabetes and/or related diseases, including obesity and insulin resistance [ , ]. adiponectin (encoded by the gene adipoq) is a hormone that is expressed only by adipocytes [ ] and regulates energy homeostasis and glucose and lipid metab- olism [ ]. rodent and human studies have demonstrated that adiponectin levels are decreased in insulin-resistant states, such as type diabetes and obesity [ – ]. administration of adiponectin to rodents with obesity and diabetes had insulin-sensitising effects [ ] and reduced triglyceride accumulation in muscle [ ]. adiponectin acts through its receptors, adipor and adipor . it was initially thought that adipor was primarily expressed in skeletal muscle, whereas adipor was predominantly expressed in liver [ ]. however, we recently showed that adipor is highly expressed in human muscle and may be the predominant isoform through which adiponectin exerts its insulin-sensitizing effects in skeletal muscle [ ]. adiponectin receptors mediate enhanced fatty acid oxida- tion and glucose uptake, increase adenosine ′-mono- phosphate-activated protein kinase (ampk) activity, and interact with peroxisome proliferator-activated receptor (ppar) pathways [ , ]. common polymorphisms in the adipoq gene have been studied in several populations, including japanese [ ] and europeans [ ]. these studies suggest that genetic variation in the adipoq gene may predispose humans to insulin resistance and/or components of the irs. addition- ally, snp + and snp + polymorphisms of the adipoq gene have been shown to predict conversion from impaired glucose tolerance to type diabetes in the study to prevent non-insulin-dependent diabetes mellitus (stop-niddm) [ ]. in contrast, common adipoq polymorphisms were not associated with type diabetes in pima indians [ ]. studies of snps in the adipor and adipor genes failed to find any association with type diabetes in northern europeans or african americans [ ], or in the japanese population [ ]. however, genetic variants in both adipor and adipor genes were found to be associated with type diabetes in the old order amish population [ ]. more recently, it was reported that variation in adipor may affect insulin sensitivity and liver fat content in europeans [ ]. to date, no data have been published on the effects of genetic variation in the adipoq, adipor and adipor genes on type diabetes and the irs in mexican americans, a group at high risk of these diseases [ ]. in this study, we conducted a preliminary survey of these three candidate genes, selecting snps from the public snp database (dbsnp) and from a review of the literature. we chose a subset of snps in the adipoq gene and all available snps in the recently discovered adipor and adipor genes. we genotyped these common polymorphisms in a mexican american population sample, and performed association analyses with irs-related phenotypes. subjects and methods the san antonio family diabetes study all study participants were mexican americans from the san antonio family diabetes study (safds) [ , ]. briefly, probands for the safds were identified by diabetes status in an earlier epidemiological survey, the san antonio heart study, which has been extensively described elsewhere [ ]. all first, second and third degree relatives of the probands, aged from – years, were diabetologia ( ) : – invited to participate in the study. metabolic, anthropomet- ric, demographic and medical history information was obtained on individuals ( with type diabetes) distributed across low-income mexican american pedigrees [ , ]. all procedures were approved by the institutional review board of the university of texas health science centre at san antonio, and all subjects gave informed consent prior to their participation. for this study, we used phenotypic information from those subjects without diabetes (n= individuals, depend- ing on availability of data), to avoid metabolic derange- ments secondary to type diabetes or treatment for type diabetes. the following ten irs-related phenotypes, which have been described in detail elsewhere [ ], were considered for the present study: fasting plasma glucose (fpg), fasting specific insulin (fsi), homeostasis model assessment of β-cell function (homa-%β) and insulin sensitivity (homa-%s, an inverse measure of insulin resistance), fasting hdl cholesterol, fasting triglyceride concentrations, fasting leptin (lep), bmi, waist circumfer- ence (wc) and sum of skin fold thickness (ss). the sum of eight skinfold measures was used as a measure of overall subcutaneous adiposity. phenotype-specific distinct outliers were excluded from the analyses (e.g. triglyceride values > mmol/l). where indicated, the data were log-trans- formed to normalise distributions. snp genotyping genomic dna was isolated from whole blood (qiagen, chatsworth, ca, usa). snps were selected from the ncbi dbsnp database and from previous studies that reported association with diabetes, obesity and/or irs-related traits. snps were obtained by either the assay-on-demand or assay-by-design service and genotyped according to manufacturer’s instructions (applied biosystems, foster city, ca, usa). alleles were scored using the allelic discrimination software sequence detection system v . (applied biosystems). all snps were tested for mendelian pedigree inconsistencies. for all snps genotyped, our mean rate of success for genotyping was > %. primers and probes used for the snp genotyping are available on request. statistical genetic analysis we performed association analysis in our complex pedi- gree-based data using the measured genotype approach (mga) within the variance components (vc) analytical framework implemented in the program solar (http:// www.sfbr.org/solar/, last accessed in july ) [ ]. the vc-based approach accounts for the non-independence among family members. in this analytical technique, vcs are modelled as random effects (e.g. additive genetic effects and random environmental effects), whereas the effects of measured covariates such as age and sex are modelled as fixed effects on the trait mean. the vcs, the association parameters, and the other covariate effects (e.g. age and sex terms) were estimated, simultaneously, by maximum likelihood techniques. a likelihood function based on multivariate normal density was numerically maximised to obtain parameter estimates. prior to performing mga, the quantitative transmission disequilibrium test [ ], as implemented in solar, was used to examine hidden population stratification, using the notation of within (w) and between (b) family components. in mga, generally, the marker genotypes were incorporated in the mean effects model as a measured covariate, assuming additivity of allelic effects [ , ]. using the notation of within (w) and between (b) family components, a significant test of b=w vs b= and w= reflects a significant difference between the genotypic means (table ). all analyses included age and sex terms as covariates, if found to be significant. the tests of association and population stratifi- cation and the related hypothesis testing are detailed in table . the nested models were compared using the likelihood ratio test. twice the difference between the log- likelihoods of these models yields a test statistic that is asymptotically distributed, approximating a χ distribution with df. a p value ≤ . is considered significant. using solar, linkage disequilibrium (ld) between snp pairs was estimated using the absolute value of the correlation coefficient∣ρ∣. however, for the purpose of discussion, r values are reported to describe the pairwise ld patterns. the bayesian quantitative trait nucleotide model the bayesian quantitative trait nucleotide (bqtn) analytical technique is employed to analyse snps to find the responsible nucleotide variants (the qtns [ , ]) influencing a given phenotype. given complete snp data for a gene, this statistical technique can be used to identify the sequence variants that are either potentially functional or that exhibit the highest disequilibria with such potential functional sites. the bqtn model is a simple extension of the classical variance component model, which aims to disentangle the genetic architecture of a quantitative trait [ ]. this technique has been described in detail elsewhere [ ]. bayesian model selection/model averaging since a candidate gene may contain a number of snps that could generate several possible competing models of qtn action, we employed a bayesian model selection/model diabetologia ( ) : – http://www.sfbr.org/solar/ http://www.sfbr.org/solar/ averaging approach to analyse the snp data simultaneously in order to estimate the probability that each snp has a direct effect on the phenotype. this technique has been described in detail elsewhere [ , ]. the bqtn analysis can be extended to analyse the haplotype data. for a given individual, the most probable haplotypes were generated using the program merlin [ ], as implemented in solar. the bqtn analytical procedures are implemented in the computer program solar [ ]. conditional linkage/qtn analysis to examine whether the associated snp(s) found by the qtn analysis can account for our reported linkage of triglyceride concentrations at chromosome p . , which is close to the adipor locus (chromosome p . ), we combined the qtn analysis with our identity-by-descent (ibd)-based variance component linkage analysis. if a variant, or set of variants, in the adipor gene is responsible for the observed linkage signal, linkage analysis conditional on a fixed-effect mga of the polymorphism will yield an expected logarithm of the odds ratio in favour of linkage (lod) score near zero. alternatively, if the associated polymorphism is in less than complete linkage disequilibrium with the true functional site, linkage analysis will generally yield a non-zero lod score. this method has been described in detail elsewhere [ ]. results the clinical characteristics of the subjects are shown in table . we genotyped snps (adipoq= , adipor = and adipor = ), and, of these, (adipoq= , adi por = , and adipor = ) were polymorphic and in hardy–weinberg equilibrium (hwe) in our dataset. snps that were not in hwe were discarded from further analysis. schema of genomic structure and variants of the adipoq, adipor and adipor genes are shown in fig. . the minor allele frequencies (mafs) of the snps, categorised by diabetes status, are shown in table . the frequency of the minor alleles varied from less than to %. there were no significant associations with type diabetes (table ). we performed association analysis using mga, after finding no statistical evidence of hidden population stratifi- cation. the significant results (p≤ . ) of the association analyses are summarised in table . of the eight snps examined in the adipoq gene, four exhibited significant associations with several irs-related phenotypes. the minor allele for snps rs and rs were significantly associated with an increase in bmi (p= . and p= . ), fsi (p= . and p= . ) and ss (p= . and p= . ), and a decrease in homa-%s (p= . and p= . ). two other snps, rs and rs , were significantly associated with ss (p= . and p= . ). for all snps genotyped in the adipoq gene, the pairwise ld (r ) ranged from to . . the highest pairwise ld found among the eight adipoq snps was between snp pair rs –rs (r = . ), followed by snp pair rs –rs (r = . ), as shown in fig. a. the average pairwise ld among the adipoq snps was . . snp rs of adipor was significantly associat- ed with bmi, ss and wc (p= . , p= . and p= . , respectively). the minor allele was associated with an increase in bmi, ss and wc, as shown in table . correlations between snp pairs were estimated and are shown in fig. b. the pairwise ld in the adipor gene (r ) ranged from to . . the highest ld was between snp pair rs –rs (r = . ), as shown in fig. b. the average pairwise ld among the adipor snps was . . of note, of the adipor snps were significantly (p< . ) associated with fasting plasma triglyceride con- centrations. the mean triglyceride concentrations per genotype are depicted in table for the significantly associated snps. the majority of the minor alleles, with the exception of rs and rs , were associated with decreased triglyceride levels. figure c shows the overall pattern of ld in the adipor gene, and table highlights snps that were in strong ld with each other. the average proportion of shared variation between snps, measured by the pairwise correlation, was . , with snp- specific values ranging from to . . of particular note, four of these snps (rs , rs , rs and rs ) were in high pairwise ld (r = . ) and were strongly associated with triglyceride levels (p= . , p= . , p= . and p= . ). table association/linkage disequilibrium and population stratification analyses and hypothesis testing test hypothesis inference population stratification b, w (both parameters are estimated) vs b=w (parameters are estimated to be equal) significant b≠w indicates the presence of population stratification mga b=w (parameters are estimated to be equal) vs b= , w= (both parameters are fixed to ) significant b=w refers to significant difference between genotypic means diabetologia ( ) : – given our previous findings of common genetic influen- ces on irs-related phenotypes, including obesity, insulin resistance and triglyceride concentrations [ ], we verified whether adjustment for measures of obesity and insulin resistance may affect the observed patterns of association between variants in adipor and triglyceride concentra- tions. thus, we repeated the association analysis of triglyceride and adipor snp data after accounting for the effects of bmi and homa-%s. all of the initial findings were observed again, but with increased levels of significance, most notably for snp rs (p= . ), with the exception of snp rs , where the p value changed from . to . . following the findings of the association analysis of the snps in adipor using mga (table ), we performed the bqtn analysis using ten of the snps in adipor to obtain a more plausible model, given the data. the snps that exhibited redundancy owing to relatively high pairwise lds (r > . ) were excluded from the bqtn analysis. the remaining ten non-redundant snps resulted in the examination of , possible additive gene action models. the model with only snp rs provided the strongest evidence for the observed associations, with estimated posterior probability of > . . the bqtn analysis was extended to analyse the predicted adipor haplotypes. of the observed haplotypes, haplotypes had frequencies > %, with a cumulative frequency of %. the haplotypes generated , possible models to be exam- ined by bqtn analysis; however, haplotypic information did not improve the association. to determine whether rs has relevance to our reported linkage of fasting triglyceride concentrations at chromosome p . [ ], which is close to the adi por locus (chromosome p . ), we re-analysed the original triglyceride data using information from our recent genome scan data generated by the centre for inherited disease research (cidr). using the total fasting triglycer- ide dataset, for which snp rs data were also available, we found modest evidence for linkage (lod= . ) of plasma triglyceride levels to a genetic location between markers d s and gata d at p . . when we re-analysed the data, conditional on snp rs , the lod score was reduced to . . thus, about % of the evidence for linkage at p . was explained by the sequence variation at rs . discussion we have previously shown that the mexican american population is at relatively high risk of obesity, type diabetes and the irs. the genetic variation in candidate genes adipoq, adipor and adipor could influence variation in such disease conditions and related traits. in the present study we provide evidence that adipor poly- morphisms are strongly associated with decreased plasma triglyceride levels. of the polymorphisms that we geno- typed in the adipor gene, our strongest association was with fasting triglyceride levels, most notably snps rs , rs , rs and rs , where we observed high pairwise correlation between all snps (r = . ). these four snps with relatively high maf of . – . % appear to have substantial influence on variation in circulating triglyceride levels, and the evidence for association continues to be significant after bonferroni’s correction for multiple testing. given that there is some correlation between phenotypes and between snps, the bonferroni correction is rather conservative. to further investigate the potential functional relevance of the variants in the adipor gene, we performed a bqtn analysis, in which we compared all possible models of gene action. table characteristics of the safds subjects without diabetes distributed across families distributional properties of the phenotypes variable sample size mean±sd or % skewness kurtosis number – – – women % – – age (years) . ± . – – fasting specific insulin (pmol/l) ± . . fasting glucose (mmol/l) . ± . . . ln homa-%s . ± . . − . ln homa-%β . ± . − . − . ln triglycerides . ± . . − . hdl cholesterol (mmol/l) . ± . . . leptin (ng/ml) . ± . . . bmi (kg/m ) . ± . . . sum of skinfold thickness (mm) . ± . . − . waist circumference (mm) ± . . diabetologia ( ) : – this analysis provided strong statistical support for a single snp model (i.e. rs ) for the observed association with triglyceride concentrations. genetic variants in the adipor gene were found to be associated with type diabetes in the old order amish population [ ]. of the eight snps typed (rs , rs , rs , rs , rs , rs , rs and rs ), three (rs , rs and rs ) in the amish population were significantly associated with combined type diabetes and impaired ′ ′ rs (+ )* rs (+ ) * rs (+ ) * rs (+ ) * rs ( − , )† rs ( − , ) * rs ( − , ) * rs (− , )* atg stop rs ( − , ) *a b ′ ′ rs (− ) * rs (− , ) * rs (− ) * rs (+ , ) * rs (+ , )* rs (+ , ) * rs (+ , ) rs (+ , ) rs ( + , ) rs (+ , ) rs (+ , ) rs (+ , ) † atg stop c ′ ′ rs ( + , ) rs (+ , )* rs (+ , )* rs (+ , ) rs (+ , ) rs (+ , )* rs (+ , )* rs ( + , ) rs ( + , ) rs (+ , )* rs ( + , ) rs (+ , )* rs ( − , ) * rs (− , )* rs ( + ) † rs (+ , ) † rs (− , ) rs (− , ) rs ( − , ) * rs (− , )* rs (− , )* rs (− , )* rs ( − , ) * rs ( − , )* rs (− , )* rs ( − , ) * rs ( − , )* rs (+ , ) * rs ( + , )† rs (+ , )* rs ( + , )* rs ( + , ) rs (+ , )* rs ( + , )* rs (+ , )† rs (+ , )† rs (+ , )* rs ( + , )* atg stop fig. genomic structure, and location of snps in the adipoq (a), adipor (b) and adipor (c) genes. exons are shown as boxes, and introns as lines connecting the boxes. numbers in parenthesis indicate location relative to the a of the atg of the start codon. the asterisks (*) denote snps that were polymorphic in this population (maf > . ). the snps that were not in hwe are denoted by a dagger (†) diabetologia ( ) : – glucose tolerance, while in the present study, these snps were associated with fasting triglyceride concentrations. interestingly, the minor allele for rs in the present study, which was significantly associated with decreased fasting triglyceride, was found to be the major allele in the amish population. in contrast, a study in a japanese population demonstrated no association between variants (including five that we examined: rs , rs , rs , rs and rs ) in adipor and type diabetes mellitus [ ]. likewise, no association between variants (including five that we examined: rs , rs , rs , rs and rs ) in adipor and glucose and lipid metabolic parameters were found in a european population [ ]. we previously reported modest evidence for linkage of fasting triglyceride concentrations to a genetic location on chromosome p, which is close to the adipor locus (chromosome p . ) [ ]. to determine whether rs could account for our linkage signal, we re- evaluated linkage on chromosome p, conditional on the measured genotype effect. by including rs geno- types as a covariate in the model, the lod score for fasting table minor allele frequencies of snps by diabetes status gene snp rs no. position relative to atg starta major/minor allele frequency % (with diabetes)b frequency % (without diabetes)b adipoq rs − , c/t . . adipoq rs − , c/t . . adipoq rs − , c/g . . adipoq rs − , a/g . . adipoq rs t/g . . adipoq rs g/t . . adipoq rs a/g . . adipoq rs g/a . . adipor rs − , g/a . . adipor rs − g/a . . adipor rs − c/a . . adipor rs , a/g . . adipor rs , t/a . . adipor rs , c/g . . adipor rs − , c/t . . adipor rs − , a/c . . adipor rs − , c/a . . adipor rs − , t/a . . adipor rs − , t/c . . adipor rs − , t/c . . adipor rs − , c/t . . adipor rs − , c/g . . adipor rs − , a/g . . adipor rs − , a/g . . adipor rs − , a/g . . adipor rs , c/g . . adipor rs , a/t . . adipor rs , c/t . . adipor rs , a/g . adipor rs , c/t . . adipor rs , c/t . . adipor rs , t/g . . adipor rs , g/a . . adipor rs , t/c . . adipor rs , c/t . . adipor rs , c/t . . adipor rs , a/t . . adipor rs , g/a . . anumbers indicate location relative to the a of the start codon (atg), which is + bthe allele frequencies were not significantly (p< . ) different between the subjects with and without diabetes based on a chi squared test diabetologia ( ) : – triglyceride concentrations was reduced from . to . . thus, about % of the evidence for linkage at p . was explained by the sequence variation at rs , in turn suggesting that this may be one of several functional variants or in strong ld with the true functional variant(s). the majority of the minor alleles for the polymorphisms in adipor were associated with decreased fasting triglyc- eride and, thus, may provide some protection against developing the irs and atherosclerosis. two adipor snps, rs and rs , that were in strong ld with each other (r = . ), were associated with increased fasting triglyceride. this seemingly paradoxical finding is not unexpected, given that independent variants in the same gene may have independent effects on traits. the molecular mechanisms by which adiponectin exerts its insulin-sensitising effects on liver and muscle appear to be related to an increase in fatty acid oxidation, an effect that is mediated, at least in part, by its activation of ampk [ ]. in skeletal muscle, adiponectin acts through its receptors (adipor and adipor ) to phosphorylate ampk, which, in turn, phosphorylates acetyl coa carbox- ylase. this leads to an increase in fatty acid oxidation [ , ] and a reduction in toxic intracellular lipid metabolites that inhibit insulin signalling. adiponectin also decreases hepatic glucose output by reducing the expression of enzymes involved in gluconeogenesis in the liver [ , ], thereby contributing to the regulation of whole-body glucose homeostasis. as a result of these processes, a reduction in tissue triglycerides and toxic lipid metabolites is observed in both skeletal muscle and liver [ , , ], and this contributes to improved insulin signal transduction. the effects of adiponectin on muscle and liver were recently reviewed elsewhere [ ], and it was suggested that liver is the primary site of adiponectin bioactivity. table significant associations between genetic polymorphisms in adipoq, adipor and adipor genes and irs-related phenotypes gene snp rs no. phenotype major/majora major/minora minor/minora direction of change p value adipoq rs bmi (kg/m ) . ± . . ± . . ± . ↑ . adipoq rs bmi (kg/m ) . ± . . ± . . ± . ↑ . adipoq rs fsi . ± . . ± . . ± . ↑ . adipoq rs fsi . ± . . ± . . ± . ↑ . adipoq rs homa-%s . ± . . ± . . ± . ↓ . adipoq rs homa-%s . ± . . ± . . ± . ↓ . adipoq rs ss (mm) . ± . . ± . . ± . ↑ . adipoq rs ss (mm) . ± . . ± . . ± . ↑ . adipoq rs ss (mm) . ± . . ± . . ± . ↓ . adipoq rs ss (mm) . ± . . ± . . ± . ↑ . adipor rs bmi (kg/m ) . ± . . ± . . ± . ↑ . adipor rs bmi (kg/m ) . ± . . ± . –b ↓ . adipor rs ss (mm) . ± . . ± . . ± . ↑ . adipor rs wc (mm) . ± . . ± . . ± . ↑ . adipor rs c ln tg . ± . . ± . . ± . ↓ . adipor rs d ln tg . ± . . ± . . ± . ↑ . adipor rs ln tg . ± . . ± . . ± . ↓ . adipor rs d ln tg . ± . . ± . . ± . ↑ . adipor rs e ln tg . ± . . ± . . ± . ↓ . adipor rs e ln tg . ± . . ± . . ± . ↓ . adipor rs f ln tg . ± . . ± . . ± . ↓ . adipor rs c ln tg . ± . . ± . . ± . ↓ . adipor rs c ln tg . ± . . ± . . ± . ↓ . adipor rs ln tg . ± . . ± . . ± . ↓ . adipor rs e ln tg . ± . . ± . . ± . ↓ . adipor rs e ln tg . ± . . ± . . ± . ↓ . adipor rs ln tg . ± . . ± . . ± . ↓ . adipor rs e ln tg . ± . . ± . . ± . ↓ . adipor rs homa-%s . ± . . ± . . ± . ↑ . tg fasting triglyceride concentrations amean trait concentrations by genotype category, after adjusting for age and sex effects, using the measured genotype approach [ ] bno minor/minor homozygotes are observed in our model cthree markers are in strong ld with each other (r ≥ . ) dtwo markers are in strong ld with each other (r = . ) efour markers are in strong ld with each other (r = . ) ftwo markers are in strong ld with each other (r = . ) diabetologia ( ) : – fig. linkage disequilibrium (ld) between snp pairs within adipoq (a), adipor (b) and adipor (c) genes. snps are labelled on the y-axis, and the locations (bp) within the gene are shown on the x-axis. pairwise ld is estimated using the correlation coefficient ∣ρ∣, and depicted in the figure by the colour intensity of the shaded box, as shown in the legend. the diagonal represents a comparison of each snp against itself (i.e. ∣ρ∣= . ) diabetologia ( ) : – adipor is abundantly expressed in human liver [ ], and an important question raised by our results is whether adiponectin acts through the hepatic adipor to influence triglyceride biosynthesis and homeostasis. there has been considerable interest in the role of kinetic defects in triglyceride-rich lipoprotein (trl) metabolism in the development of atherosclerosis and dyslipidaemia (e.g. increased fasting triglycerides and low hdl cholesterol), as it relates to type diabetes and the irs [ , ]. it has been suggested that adiponectin concentration may play an independent role in regulating trl metabolism, aside from its link with abdominal fat, insulin resistance and dyslipi- daemia [ ]. these observations have relevance to a recent finding where a significant correlation was demonstrated between the expression of adipor and adipor mrna in human skeletal muscle and in vivo parameters of glucose and lipid metabolism [ ]. adipor mrna levels correlated positively and independently only with fasting triglyceride concentrations [ ], whereas adipor mrna expression was positively correlated with serum insulin and c-peptide concentrations, first-phase insulin secretion, and elevated plasma triglyceride and cholesterol concentrations [ ]. given this independent association of adipor gene expression with fasting triglyceride concentrations, but not with bmi, insulin or glucose, and our previous findings of common genetic influences on irs-related phenotypes (e.g. obesity, insulin resistance and fasting triglyceride) [ ], we examined whether adjustment for measures of obesity and insulin resistance may affect the observed patterns of association between variants in adipor and fasting triglyceride. the sample size was reduced (n= ) because of the requirement of common covariate information. all the initial findings were observed again, but with increased levels of significance (with the exception of snp rs , as noted above). for example, the p value for association of snp rs with fasting triglyceride concentrations was . . these results, together with previous findings [ ], indicate that adiponectin and its receptors, especially adipor , play an important role in regulating fasting plasma triglyceride levels, and that they could have a potential role in vldl metabolism. several studies have shown that the adipoq gene is associated with irs-related phenotypes [ – ]. of particular note, a haplotype including two snps at positions − , (rs ) and − , (rs ), both located in the promoter sequence of the adipoq gene, was shown to be strongly associated with adiponectin concentrations and type diabetes in the french population [ ]. our results provide additional evidence that variants of the adipoq gene are significantly associated with irs-related traits. we genotyped snps in the adipor gene (fig. ), and six of these were polymorphic and in hwe in our dataset. of the six polymorphic snps, one was significantly associated with irs-related traits. interestingly, snp rs of adipor was significantly associated with three obesity traits: ss, bmi and waist circumference. similar to our findings, variants in adipor were found to be associated with type diabetes in the old order amish population [ ]. the following five snps were evaluated in both studies: rs , rs , rs , rs and rs . in the amish population, allele/genotype frequencies of snps rs and rs differed significantly between subjects with type diabetes vs. those with normal glucose tolerance. however, there were no significant associations between adipor snps and type diabetes, insulin sensitivity or insulin secretion in northern europeans, african americans [ ] or a japanese population [ ]. in contrast, a recent study in a european population demonstrated significant association between several snps (including the two we examined, rs and rs ) and insulin sensitivity/ liver fat [ ]. in conclusion, this study demonstrates that poly- morphisms in adipor , adipor and adipoq are associated with several irs-related phenotypes in mexican americans, including plasma triglyceride levels, ss, bmi and waist circumference. of particular note, multiple genetic variants in adipor were strongly associated with decreased triglyceride levels. our findings of multiple strong associations between adipor polymorphisms and plasma triglyceride con- centrations may have important implications for athero- genesis and/or dyslipidaemia, owing to the potential influence of adipor genetic 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. hamsten a, silveira a, boquist s et al ( ) the apolipoprotein ci content of triglyceride-rich lipoproteins independently predicts early atherosclerosis in healthy middle-aged men. j am coll cardiol : – . staiger h, kaltenbach s, staiger k et al ( ) expression of adiponectin receptor mrna in human skeletal muscle cells is related to in vivo parameters of glucose and lipid metabolism. diabetes : – . populaire c, mori y, dina c et al ( ) does the − promoter variant of apm gene contribute to the genetic risk for type diabetes mellitus in japanese families? diabetologia : – diabetologia ( ) : – 	association... 	abstract 	abstract 	abstract 	abstract 	abstract 	introduction 	subjects and methods 	the san antonio family diabetes study 	snp genotyping 	statistical genetic analysis 	the bayesian quantitative trait nucleotide model 	bayesian model selection/model averaging 	conditional linkage/qtn analysis 	results 	discussion 	references << /ascii encodepages false /allowtransparency false 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family size and reproductive span in anabaptist populations by j. c. stevenson, p. m. everson a n d m. h. crawford abstract the anabaptist amish, hutterite and mennonite peo- ples trace their origins to the reformation. although they share cer- tain beliefs, such as adult baptism and the separation of church and state, each group is culturally unique. the hutterite and amish are highly fertile and their populations exhibit stable rates of growth. these demographic characteristics reflect communal living among the hutterites and labor intensive farming practices among the amish. the mennonites are the most receptive anabaptist group to outside socioeconomic influences and provide a demographic con- trast to the more conservative amish and hutterites. demographic data collected during a study of aging in mennonite population sam- ples from goessel and meridian, kansas, , and henderson, ne- braska, , formed the basis of a cohort analysis in order to assess fertility change over time. completed family size has decreased significantly in all three communities since . since the early 's the mean age of the mother at first birth has fluctuated but the mean age of mother at the birth of the last child is decreasing significantly for the communities of goessel and henderson, thus effectively shortening the reproduc- tive span. the pattern is somewhat different for meridian, the most conservative of the three communities. the amish, hutterite and mennonite peoples trace their origins to the reformation (dyck ). the three groups organized separately around the charismatic personalities of jacob ammann, jacob hutter and menno simons, respectively. all three anabaptist groups advocate adult baptism and separation of church and state. the latter idea was con- sidered treasonous in most areas of europe in the 's. adherence to this concept of separation of church and state resulted in persecution and department of anthropology, western washington university, bellingham, wa department of anthropology, university of washington, seattle, wa ^laboratory of biological anthropology, university of kansas, lawrence, ks human biology, february , vol. , no. , pp. - © wayne state university press, / stevenson, everson a n d crawford frequent forced migration. the movement began in switzerland spread- ing to parts of germany and holland, then to prussia, czechoslovakia, later to poland and russia. migration to the americas began in the 's (dyck ). how- ever, many amish moved in the 's coming primarily from switzer- land. the ethnically german hutterites migrated from russia in the 's settling first in south dakota. although mennonites were even- tually tolerated in europe during the late 's and 's, economic concerns due to discrimination and increasing militarism caused migra- tion to north america. migrants represented two categories: swiss-south german and dutch-prussian-russian. in the late 's, and continuing through the early s, , to , swiss and german mennon- ites first settled in pennsylvania. during the 's and the 's, ap- proximately , dutch-prussian-russians arrived in the united states midwest and canada. the cultural heterogeneity of the mennonites may be partially responsible for the more liberal attitudes of many contem- porary mennonites relative to hutterites and amish. published demographic studies of mennonites are few (allen and redekop ; harder ; yoder ), and there is little documen- tation of fertility change over time. thus, in this study, reproductive his- tories for women, representing population samples from one nebraska and two kansas mennonite communities, will be analyzed by cohort and compared to other anabaptists. the amish and hutterites, anabaptist "cousins" of the mennon- ites, are of considerable biological and demographic interest because of remarkably stable levels of mortality and fertility with little or no in or out migration from the group since the beginning of this century until the s (cook ; cross and mckusick ; eaton and mayer ; ericksen et al. ; hamman et al. ; sheps ; and tietze ). birth control is not condoned, so that fertility is high. the mean family sizes of . and . for the amish (ericksen et al. ) and hutterites (eaton and mayer ), respectively, have remained unchanged for at least or years. in addition, mortality is low. thus, growth rates of both groups are very high (cook ; cross and mckusick ; eaton and mayer ; friedmann ; laing ; peter ). the constancy of this growth coupled with maximum or near maximum fer- tility, particularly of the hutterites, has received much attention from social scientists interested in determining the limits of human fecundity or "natural fertility" (e.g. espenshade ; henry ; and robinson ). other researchers have utilized the excellent genealogical records of the anabaptist groups and their relative isolation to explore either topics in medical genetics (e.g. mckusick ) or additional aspects of family size and reproductive span / population structure such as inbreeding, genetic drift and group fissioning (hurd , a, b; mange ; mckusick et al. ; morgan and holmes ; steinberg et al. ). the mennonites, can provide an interesting contrast to the hut- terites and amish. although a heterogeneous group, the mennonites are, in general, the most receptive to the outside world (dyck ). today, except for old colony mennonites in mexico (allen and redekop ) and other conservative mennonite groups, the fertility levels of the men- nonites are the lowest of the anabaptists. yoder ( ) found that . to . children was the average per married woman over the age of in a census of the (old) mennonite church (mennonite general conference, organized ). when single women are averaged in, the mean drops to around to . children per woman. pollack ( ; also described in hurd, b) compared old amish, mennonites and non-mennonites and demonstrated the existence of a positive relationship between reli- gious conservatism and number of live births per completed family in plain city, ohio. thus, lower fertility is expected for the mennonites relative to the amish and hutterites. the focus here will be on the pat- tern in completed family size and reproductive span in these groups over time. the populations studied three communities are represented in this study: goessel, and meridian, located in kansas, and henderson, nebraska. the three com- munities are related historically, but the composition of each is unique. goessel and henderson mennonites are descendants of peoples liv- ing in the th and th century netherlands primarily, plus switzerland and southern germany. ancestry can also be traced to prussia and russia due to local conversions during their later travels (crawford and rogers ; rogers ). political and economic changes in russia convinced many of the members of one russian village, alexanderwohl, of the ukraine molotschna colony, to migrate to the u.s. in . after arriv- ing in the united states they split into factions, in part, because of com- petition between railroad agents for land sales. thus, one group settled west of lincoln, nebraska, in today's town of henderson. another group, new alexanderwohl, settled in the rural areas around today's goessel, kansas, in the counties of harvey, marion and mcpherson. most of the individuals in this study are from churches affiliated with the general conference mennonite division which was founded in with the intent of uniting all of the american mennonites / stevenson, everson a n d crawford (dyck ; van meter ). not as strict as many of the smaller groups, they only require adherence to the fundamental doctrines of the mennonite faith. new alexanderwohl was the first congregation to affiliate in and was followed by tabor, hillsboro, johannestal, lehigh, brudertal and goessel. in this study, % of the participants from the goessel community represent alexanderwohl, goessel and tabor churches. fifteen per cent are from other mennonite churches, and % are non-mennonites. a few smaller groups have remained separate. one such church is the holdeman or church of god in christ, represented in this study by the meridian congregation, which is located near hesston, kansas. "holdemans" are the most conservative mennonites in this study with plain dress and beards worn by the men. all meridian participants are members of this church and reside in rural areas surrounding the church or in the towns of hesston or moundridge, kansas. ethnically, the holdeman members are a mixture of the descendants of the prussian-russian immigrants and american mennonites from indiana and pennsylvania (crawford and rogers ). the henderson mennonites of nebraska are descendants of inhab- itants from a number of russian villages of the molotschna colony in- cluding alexanderwohl (crawford and rogers ; rogers ; voth ). disputes about church worship soon surfaced, probably along vil- lage lines. the largest group organized the bethesda church (now gen- eral conference), but a reform movement led to families breaking away to form the evangelical mennonite brethren church in . in addition, an earlier reform predating the immigration from the molotschna colony led to the organization of the mennonite brethren in . presumably members from this church are descendants of molotschna colony im- migrants and american mennonites. individuals in this study represent all three churches, respectively, in the following proportions: , and %. seven per cent are in a miscellaneous category which includes both non-mennonites and out-of-state mennonites. materials and methods the demographic data from the communities of goessel and merid- ian, kansas, and henderson, nebraska, were taken as part of a larger interdisciplinary aging study which is described in crawford and rogers ( ). interview and questionnaire data were collected at health clin- ics in goessel and meridian churches in kansas, during january, , and in the bethesda church of nebraska during january, . merid- family size and reproductive span / ian was a small sample which included % of the adults residing in the community. however, % of the families were represented by this sam- ple (sirijaraya ). the cornell medical index questionnaire provided reproductive histories for , and women from goessel, hender- son and meridian, respectively. the goessel clinic sample included % of the entire alexanderwohl church membership. this clinic sample was further supplemented by mailed questionnaires in order to obtain a larger sample of the community in . the town of henderson consisted of persons and a total sample of ( %) participated in the study. mennonites from the three communities are culturally homogenous, and thus, sample sizes approximating % should be representative. household surveys provided additional information about na- tal and reproductive families of household heads and their respective spouses. individuals were encouraged to consult family records in order to complete the household surveys. complete family histories were then used to reconstruct families for individuals born as early as the 's, and these reconstructed families were used to calculate completed family size for ten-year cohorts. reproductive histories were used to estimate mean age at menarche, mean age of mother at first and last child and mean reproductive span. completed family size is calculated as the to- tal number of children born to a married woman. reproductive span is calculated by subtracting the birth year of the last child from the birth year of the first child and adding one (tietze ). mothers with a sin- gle child are given a reproductive span value of one. this measure of reproductive span is not a potential measure; rather, it is an estimate of the actual span of childbearing. results the mean completed family size by community by decade, from to , for goessel, henderson and meridian are presented in table and graphically portrayed in figure . most of the women born in the 's have not completed their childbearing by - so the figures for this cohort are conservative. in each of the three communities, goessel, henderson, and meridian, the mean completed family sizes for the - cohort are significantly greater than the mean completed family sizes for the - cohort (t = . , p < . , t = . , p < . , t = . , p < . , one-tail tests, respectively). there were no significant differences in comparisons of mean completed family size between henderson and goessel for the same cohorts. sample sizes are smaller for meridian so means fluctuate. however, the trend to / stevenson, everson a n d crawford table . mean completed family size, by community, by decade of woman's birth, - decade of goessel henderson meridian woman's birth n x s.d. n x s.d. n x s.d. - . . . . . . - . . . . . . - . . . . . . - . . . . . . - . . . . . . - . . . . . . - . . . . . . - . . . . . . d e c a d e of w o m a n ' s b i r t h figure . mean completed family size, by community, by decade of woman's birth, - . decreasing family size is apparent in all three communities, decreasing from a high of to children among the women in the - cohort, to to children characterizing the women in the - cohort. summary measures of the reproductive span, by decade, of women born from - for goessel and henderson, and to for meridian, are presented in table . statistical comparisons for meridian could not be made due to the small sample sizes; however, trends are noted. family size and reproductive span / table . summary measures of reproductive span, by community, by decade of woman's birth, - . decade of mean age mean age mean age mean woman's at at at reproductive birth menarche first birth last birth span by community n x sd n x sd n x sd n x sd - goessel . . . . . . . . henderson . . . . . . . . meridian — id* — — id* — — id* — — id* — - goessel . . . . . . . . henderson . . . . . . . . meridian . . . . . . . . - goessel . . . . . . . . henderson . . . . . . . . meridian . . . . . . . . - goessel . . . . . . . . henderson . . . . . . . . meridian . . . . . . . . - goessel . . . . . . . . henderson . . . . . . . . meridian . . . . . . . . *id insufficient data. the mean age at menarche decreases in all three communities. within goessel the mean age at menarche decreases . years from to . thus, the mean age at menarche for the - cohort is significantly higher than the - and - cohorts (t = . , . , p = . , . , one-tail tests, respectively), and the mean ages at menarche for the - and - cohorts are significantly higher than for the - cohort (t = . , . , p = . , . , one-tail tests, respectively). within henderson, the mean age at menarche drops . years from to but fluctuates. there is a significant difference between the - and - cohorts when the mean age at menarche rises (t = . , p = . , one- / stevenson, everson a n d crawford tail test). however, the overall trend continues downward although the mean age at menarche for the - cohort is significantly higher than among the - and - cohorts (t = . , . , p = . , . , respectively). finally, the mean age at menarche for meridian women decreases . years from to . the mean age at first birth has fluctuated since the early 's hov- ering in the mid- 's. there are no discernable trends within communi- ties. however, goessel women tend to be older than henderson women at the birth of the first child for all cohort comparisons, - , - , - , - , - (t = . , . , . , . , . , p = . , . , . , . , . , respectively, two-tail tests). the mean age at last birth fluctuates around the age of for merid- ian women, but decreases . and . years, respectively, from to for the women of goessel and henderson. within goessel the mean age at last birth drops significantly every decade for all but the last cohort, - versus - , - vs. - , - vs. - , (t = . , . , . , p = . , . , . , respec- tively, one-tail tests). within henderson significant decreases are found between two pairs of cohorts, - vs. - , - vs. - , (t = . , . , p = . , . , respectively, one-tail tests). the mean age at last birth for goessel and henderson differ significantly only when the first two cohorts are compared, - , - , (t = . , . , p = . , . , respectively, two-tail tests). thus, goes- sel and henderson women become more alike over time with respect to mean age at last birth. the mean reproductive span has shortened . and . years for goessel and henderson women over the period - , and one year for meridian women from to . the most dramatic decreases occur among goessel women during the periods - vs. - , and - vs. - , (t = . , . , p = . , . , one-tail tests, respectively). mean reproductive span in henderson declines less so that significant differences are found only when the - , - , and - cohorts are compared to the - cohort (t = . , . , . , p = . , . , . , one-tail tests, respectively). discussion since the early 's, the mean age at menarche has decreased suggesting improved health and nutrition (danker-hopfe, ; malina, family size and reproductive span / ). the mean age at first birth has fluctuated slightly with no ob- vious trends although remaining consistently higher for goessel when compared to henderson women. the mean age at first birth increased slightly (although not significantly) for the women of goessel and hen- derson born during the decade - , but these are women who bore children during the depression and world war ii. the mean age at menarche also rises for henderson women during the depression years. the mean age at first birth decreased in later cohorts from both commu- nities and significantly so for goessel women although it is apparently rising again for women born in the 's. for henderson, the mean age at marriage averaged . , . , . , . and . years for women born in the decades - , - , - , - and - , respectively. the age at marriage also increased slightly for women born in the decade - and again for those born in the 's. however, the mean age at last child has decreased steadily (and most dramatically for the goessel community) so that the reproductive span has decreased , and % for goessel, henderson and merid- ian, respectively. the mean completed family size, presented in table , indicates how this shortened reproductive span has also resulted in reduced completed family size, although this is slightly more evident for goessel and henderson than for the more conservative community of meridian. the subtle differences in measures of reproductive span be- tween goessel and henderson women are not reflected in differences in mean completed family size. further study should reveal if these con- sistent differences reflect cultural differences in achieving desired family size. this demographic shift experienced by mennonites in the last thirty or forty years is also being predicted for the more culturally buffered amish and hutterite populations (laing ; morgan ; peter ). the demographic stability from approximately to the 's reflects the isolation and self-sufficiency of the hutterites and amish. both practice an agrarian lifestyle, are relatively uniform in socioeconomic circumstances and receive adequate medical care, but there are significant differences. the hutterites are communal and have utilized sophisticated farm machinery since the 's (hostetler , ). the amish live as single families and resist such technological innovations as automobiles, telephones and electricity (hostetler ). refusal to practice birth control, coupled with a high standard of living, has led to a dramatic increase in the population size of both groups. the hutterite population has increased from approximately immigrants in to approximately , individuals residing in colonies in (peter ). in the amish, the population growth has been / stevenson, everson a n d crawford at a slightly slower rate but with similar results. for example, between and , the number of amish church districts in lancaster county, pennsylvania, increased from to (ericksen et al. ). this population explosion has been supported among hutterites by the communal acquisition of capital and the purchase of new land, followed by group fissioning. as needed, the amish family also purchases additional farmland for its offspring. however, difficulty in obtaining land due to its scarcity, inflation, and increasing political restrictions has led several researchers to predict a decline in the fertility of both the hutterites and amish (eaton ; eaton and mayer ; ericksen et al. , ; peter ). there are a limited number of positions of responsibility in a hutterite colony and the inability to create new positions through group fissioning is likely to produce internal discontent (clark ; peter ). this dissatisfaction may result in a decline in family size, and seems to be happening for north american hutterites. peter ( ) demonstrates a decrease in the population growth rate from . % per year to . %. in an analysis of census data for alberta hutterites, laing ( ) demonstrates that the crude birth rate declined from . per thousand in to . per thousand in . laing also found a decrease in the age-specific fertility of the hutterite women. the conversion to contemporary farm technology has reduced the need for labor, and thus, the average size of hutterite colonies has been reduced. a number of strategies have been adopted to reduce fertility. hutterite women married at . and men at . years in (cook ), whereas, laing ( ) estimated that in mean age at marriage for alberta hutterite women had risen to . years and for men . years. peter ( ) has predicted a greater increase in the ages at marriage in order to account for the . % reduction in the birth rate from - . however, boldt and roberts ( ) argue that peter's predictions may be too high and that other means of reducing fertility may be in use, such as birth control. in addition, the percentage of never-married women is increasing, and women who do marry are having fewer children (laing ). the age specific rates decline most in women over the age of years. there is little evidence for a decline in amish fertility over time except for those who leave the congregation (ericksen et al. ). the mean age at marriage is below years for the amish women who marry by age and is over for their husbands (ericksen et al. ). amish men tend to marry earlier, while amish women marry at the same time as their u.s. neighbors. there has been little or no change since the late 's although smith ( ) finds a slight decrease in age of marriage for family size and reproductive span / southeastern pennsylvanian amish men and women. amish fertility has been lower than that of hutterites due primarily to a more rapid decline in fertility for amish women at older ages. sterilization and abstinence are likely explanations for this fertility differential. thus, the fertility decline is not yet evident for the amish. an age/sex distribution from a "nebraska" amish population of mifflin county, pennsylvania, for december, (hurd b) does not differ significantly from the age/sex distribution for holmes county, ohio, amish in (n = ) (cross and mckusick ). the impact of population pressure is probably more immediately felt in hutterite colonies rather than in the individual families of the amish. the amish have more job opportunities due to labor intensive practices (ericksen et al. ). because land may be more difficult to obtain among the amish, individuals may be initially absorbed into occupations such as carpentry, cabinet-making, blacksmithing and harness repair. thus, fewer amish families depend directly on farming before there is a change in fertility. about % of holmes county amish are either part or full-time farmers, whereas, ericksen et al. ( ) find that only % of amish males of lancaster county, pennsylvania, are directly involved with farming. the amish may be temporarily buffered from the demographic changes being experienced presently by the hutterites. ericksen et al. ( ) observed that it is difficult to give up farming and to remain amish. there is an increasing rate of defection from the amish church in families least able to purchase more land. in addition, those who defect exhibit a slight decrease in fertility. to counteract this trend, remaining amish are either going to have to locate new families outside of lancaster county (which causes additional problems because it is difficult to maintain family ties and thus amish identity), and/or reduce fertility. from the late 's to the 's, a period of time when amish and hutterite fertility has remained stable, mean completed family sizes for the three mennonite communities of this study have been decreasing. during that time mennonite fertility has also been sensitive to changes in the outside world. for example, note the later mean age at first birth (and for mean age at marriage for henderson women) for women born in the years to and the associated drop in mean completed family sizes for the communities of goessel and henderson. women born in the 's are reducing family size, and the mean age at first birth is slightly higher. reduction in completed family size is also occurring through a reduced reproductive span, and like the hutterites and amish, the reduction is primarily at the end of the childbearing years. the mean age at last child has lowered dramatically in goessel and has been relatively low in henderson since the early 's. thus, mennonites / stevenson, everson a n d crawford reduce fertility in the later years more and sooner than do the hutterites and amish. meridian, the most conservative mennonite community of the three, does not exhibit this pattern. the reduction in mean completed family size is due primarily to a slight elevation in the age of mother at first birth rather than due to a decreased mean age of mother at the birth of the last child. presumably, the most "liberal" mennonites of this study, the goessel and henderson communities, are more similar to their non- mennonite neighbors. however, there are no comparable measures avail- able for the kansas-nebraska census population. the census does provide an indirect measure of fertility, "children-ever-born". this mea- sure enumerates the number of children ever born per married woman to years of age. for the kansas-nebraska census population, these age groups consist of individuals who were born in the decades - , - , - , and - , with values of . , . , . , and . , respectively (u.s. bureau of census , , ). thus, the reduction in fertility of the goessel mennonites makes them more similar to their non-mennonite neighbors. in addition, mennonite women of goessel, henderson and merid- ian are using the contraceptive pill in order to both delay the birth of the first child and decrease the fertility of their later reproductive years (see table ). most of the women born in the late 's and 's have used the pill in the later reproductive years, and virtually all of the women born in the 's are using the pill in order to delay the birth of the first child. a small percentage ( - %) of women born in the 's and 's have also had tubal ligations. eaton and mayer ( ) predicted that hutterite women might eventually adopt birth control as they acculturated to american values, and boldt and roberts ( ) have suggested the use of contraception in order to explain the recent decrease in the rate of growth of the hutterites. however, to date, there is no pub- lished information to support the use of birth control by either amish or hutterite women. the use of the birth control pill by the more con- servative meridian women suggests that the pill may also be employed by contemporary amish and hutterite women. another artificial means of reducing the length of the reproductive span is through the surgical removal of the ovaries and/or uterus. table presents the proportions of women having hysterectomies by decade of birth, and the percentages are relatively high for all three communities. of those goessel and henderson women who also gave their ages at the time of the surgery, to % were years old or younger. these high frequencies suggest voluntary birth control. eaton and mayer ( ) also noted high rates of hysterectomies in hutterite women. family size and reproductive span / table . use of birth control pills, by community, by decade of woman's birth, - . decade of woman's birth, by community % have used sample size - goessel . henderson . meridian . - goessel . henderson . meridian . - goessel . henderson . meridian . - goessel . henderson . meridian . - goessel . henderson . meridian . - goessel . henderson . meridian . conclusions a cohort analysis indicates that the reproductive span of mennon- ite women has been shortened. the mean age of the mother at first birth is rising, while the mean age of mother at the birth of the last child is decreasing for the communities of henderson and goessel. thus, the reproductive span for both communities is being shortened most dra- matically as a result of a decrease in the age of the mother at the birth of the last child. meridian is the most conservative of the three mennonite communities and the women in that sample have not reduced either the completed family size or the reproductive span by as much as the women / stevenson, everson a n d crawford table . hysterectomies, by community, by decade of woman's birth, - . decade of woman's birth % total sample % < # with by community hysterectomy size age age - goessel henderson meridian id* id id id - goessel henderson meridian id id - goessel henderson meridian id id - goessel henderson meridian id id - goessel id id henderson id id meridian id id *id insufficient data in the goessel and the henderson samples. the slight reduction in the mean length of the reproductive span for meridian women was accom- plished primarily through the increase of the mother's age at first birth. the birth control pill is also utilized, plus there is a high rate of hys- terectomies for women years and younger. the demographic shift for these three mennonite communities begins for women born in the 's and 's. although a similar demographic transition in the amish and hutterites is anticipated as a result of the social changes accompany- ing population pressures due to historically high fertility, the preliminary evidence for this shift is apparent only for the hutterites. in sum, the fer- tility measures of contemporary mennonite women indicate that demo- graphically they are beginning to resemble the kansas-nebraska census population, instead of the other more conservative anabaptist groups. family size and reproductive span / acknowledgements this research was supported in part by n.i.h. grant ago - and a p.h.s. research career development award k de - . we are grateful to the goessel, meridian and henderson congregations, without whose support and participation this study would not have been possible. we also wish to thank ms. meredith uttley and dr. laurine rogers for their assistance in data analysis and data collection, and mr. lucky tedrow and mr. gene hoerauf of western washington university for their assistance in preparing the figure. ms. eileen smith's help in preparation of the final manuscript is acknowledged. literature cited allen, g. and c. redekop individual differences in survival and reproduction among old colony mennonites in mexico: progress to october . eug. quart. : - . boldt, e. d. and l. w. roberts the decline of hutterite population growth: causes and consequences—a critique. can. ethnic stud. : - . clark, p. leadership succession among the hutterites. canadian rev. sociol. and anthrop. : - . cook, r. c. the north american hutterites: a study in human multiplication. pop. bull. : - 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(ed.) henderson mennonites, from holland to henderson. henderson centennial committee, service press, inc., henderson, nebraska. y o d e r , m. l. findings from the mennonite census. menn. quart. rev. : - . © yokoo et al. this work is published and licensed by dove medical press limited. the full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the creative commons attribution – non commercial (unported, v . ) license (http://creativecommons.org/licenses/by-nc/ . /). by accessing the work you hereby accept the terms. non-commercial uses of the work are permitted without any further permission from dove medical press limited, provided the work is properly attributed. for permission for commercial use of this work, please see paragraphs . and of our terms (https://www.dovepress.com/terms.php). cancer management and research : – cancer management and research dovepress submit your manuscript | www.dovepress.com dovepress o r i g i n a l r e s e a r c h open access to scientific and medical research open access full text article http://dx.doi.org/ . /cmar.s extrahepatic metastasis risk of hepatocellular carcinoma based on a-fetoprotein and tumor staging parameters at cross-sectional imaging takeshi yokoo amish d patel naama lev-cohain amit g singal adam c yopp ivan pedrosa department of radiology, department of internal medicine, department of surgery, university of texas southwestern medical center, dallas, tx, usa background: extrahepatic metastases have important implications in the clinical management of hepatocellular carcinoma (hcc). the purpose of this study was to validate tumor staging parameters and serum afp as risk factors of hcc metastasis. patients and methods: in this retrospective case–control study, patients with a new diagnosis of hcc (n= ), median age years (range – years), and male-to-female ratio of / were divided into a “no-met” group (n= ) without extrahepatic metastasis or a “met” group with extrahepatic metastases (n= ). metastasis risk factors based on tumor staging parameters (size, number, infiltration, and vascular invasion) and serum afp level were calculated as odds ratio (or). sensitivities of the risk factors as metastasis screening tests were also calculated. results: afp > mg/ml, index tumor size > cm, and vascular invasion individually had strong association with metastasis, with or ( % confidence interval) of . ( . – . ), . ( . – . ), and . ( . – . ), respectively, but with moderate sensitivities as metastasis screening tests, with . % ( . – . ), . % ( . – . ), and . % ( . – . ), respec- tively. composite multiparametric criteria, eg, a logical union of ) tumor size outside of milan criteria, ) afp threshold > mg/ml, and ) vascular invasion, had excellent or up to . ( . – . ) with screening sensitivity . % ( . – . ). conclusion: serum afp, tumor size, and vascular invasion are strongly associated with extrahepatic metastasis of hcc, especially when combined into a multiparametric metastasis prediction criterion. keywords: hepatocellular carcinoma, risk factor, a-fetoprotein, stage, metastasis introduction hepatocellular carcinoma (hcc) is currently eighth leading cause of cancer-related deaths in the usa and the fastest growing cancer in mortality. treatment recom- mendation depends on the patient’s clinical status (eg, liver function and performance status) and tumor stage. , patients with advanced-stage hcc, defined by the presence of vascular invasion and/or extrahepatic metastasis, are generally not considered candidates for curative treatment and are usually treated with systemic or palliative therapy. the prognosis of advanced-stage hcc is poor with median survival < year, in contrast to the ~ % -year survival of early-stage hcc. – extrahepatic metastasis (“metastasis” hereafter) occurs in one-third of patients with hcc, , with the most common sites being lung, lymph nodes, bone, and adre- nal glands. , metastases have important management implications, as locoregional therapies (eg, ablation, resection, and liver transplantation) are no longer effective correspondence: takeshi yokoo department of radiology, university of texas southwestern medical center, inwood road, ne . b, dallas, tx - , usa tel + fax + email takeshi.yokoo@utsouthwestern.edu journal name: cancer management and research article designation: original research year: volume: running head verso: yokoo et al running head recto: extrahepatic metastasis risk of hcc doi: http://dx.doi.org/ . /cmar.s c a n ce r m a n a g e m e n t a n d r e se a rc h d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -d e c- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / http://www.dovepress.com/permissions.php www.dovepress.com www.dovepress.com www.dovepress.com https://www.facebook.com/dovemedicalpress/ https://www.linkedin.com/company/dove-medical-press https://twitter.com/dovepress https://www.youtube.com/user/dovepress cancer management and research : submit your manuscript | www.dovepress.com dovepress dovepress yokoo et al controlling extrahepatic disease. , , , metastasis is also an independent predictor of poor survival. – therefore, it is crucial to determine the presence of metastasis at the time of initial hcc diagnosis, as initiation of appropriate therapy will determine survival. however, exhaustive metastasis workup, which may include chest ct and bone scintigraphy, may be costly, time consuming, and unnecessary for those with low risk of metastasis. several noninvasive prognostic parameters of hcc have been proposed, including tumor size and serum afp levels. tumor size is an independent predictor of hcc progression, metastasis development, and overall survival. , – high levels of afp are independently associated with metastasis risk and poorer prognosis. – other parameters, such as vas- cular invasion and the number of tumors, also have survival implications. , accordingly, these parameters are integral part of various hcc staging systems. , – to our knowledge, however, no specific criteria have been proposed for hcc metastasis risk stratification. we hypothesize that the tumor staging parameters (eg, tumor size, number, infiltration, vascular invasion, and afp) are associated with synchronous or metachronous metastasis in patients with hcc. the purpose of this study was to vali- date the tumor staging parameters, either as single-parametric criteria or as multi-parametric criteria, as risk factors of hcc metastasis. if validated, such criteria may allow rapid metas- tasis risk stratification at the time of diagnostic imaging and facilitate timely management decisions including the need for comprehensive metastasis workup. patients and methods study design and patient population this retrospective case–control study at a tertiary-care public hospital was approved by the university of texas south- western medical center’s investigational review board. data were de-identified in compliance with health insur- ance portability and accountability act. the need to obtain informed consent was waived. a review of an hcc clinic database was conducted to identify consecutive patients with a new diagnosis of hcc between january and december . hcc diagnosis was made either by direct tissue sampling or dynamic contrast-enhanced cross-sectional imaging (ct or mri) per routine clinical care, interpreted by staff pathologists and radiologists, respectively. two authors (ags and acy) adjudicated each case to confirm that it met diagnostic criteria by histology or the american associa- tion for the study of liver disease. routine evaluation of metastatic disease included chest x-ray and abdominal-pelvic ct (if not already performed). additional imaging, such as chest ct and bone scan, was performed at the discretion of the treating physician. by chart review, patients were divided into the following two cohorts: ) “no-met” cohort comprised patients without documented metastasis at the time of hcc diagnosis and during the first -month follow-up period from the initial diagnosis and ) “met” cohort with documented metastasis at the time of initial diagnosis or detected during the first -month follow-up. a single index imaging study (ct or mri) was selected for each patient. for those in the no-met group, the index study was the initial imaging examination leading to the hcc diagnosis. for those in the met group, the index imaging study was the pretreatment imaging exami- nation closest to the time of metastasis diagnosis. patients were excluded from the study if the image/clinical data were incomplete, or determination of the hcc metastasis status was compromised: ) no documented metastasis but follow-up period < months; ) no available pretreatment ct or mri; ) systemic hcc treatment during the follow-up period; ) index ct/mri images not available; ) no contem- poraneous pretreatment serum afp, defined as < months of the index ct/mri; and ) history of any other cancers. the inclusion–exclusion criteria and the number of patients are graphically summarized in figure . data collection for each patient, the index study’s images and its radiology report were reviewed. an axial image series that best depicted the tumor boundary was selected. the size of the dominant (index) tumor, either meeting the aasld imaging criteria or biopsy proven, was measured as maximum axial diameter. other nonindex tumors were measured, if they met the imaging criteria or had similar imaging features as the biopsy-proven index tumor. for diffuse infiltrative hcc, its size was coded as cm due to difficulty in delineating the tumor boundary. presence of vascular invasion (ie, involving either portal vein or hepatic vein) and infiltrative morphology were determined based on the official radiology report, in order to maintain consistency with the patient’s clinical management decisions. the afp level (mg/ml) and other demographic and clini- cal data including the age, sex, and child-turcotte-pugh stage were recorded. etiology of liver disease, including hepatitis c (positive serum antibody or rna), hepatitis b (positive surface antigen), alcohol-related liver disease (alcohol intake > g/day for ≥ years), nonalcoholic steatohepatitis (negative work-up for other etiologies in the presence of the metabolic syndrome), and others/unknown, was noted. c a n ce r m a n a g e m e n t a n d r e se a rc h d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -d e c- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com cancer management and research : submit your manuscript | www.dovepress.com dovepress dovepress extrahepatic metastasis risk of hcc a database was constructed using microsoft excel (micro- soft corporation, redmond, wa, usa), which recorded tumor size, number, afp, infiltration, and vascular invasion status, as well as the metastasis status (met vs no-met). metastasis risk criteria various staging parameters were considered as candidate metastasis risk factors and included afp, tumor size, num- ber, vascular invasion, and infiltrative morphology. these parameters were primarily derived from the following hcc staging systems: american joint commission on cancer tnm system, barcelona clinic liver cancer system, cancer of the liver italian program score, and chinese university prognostic index. although not strictly a staging system, the milan criteria for liver transplant eligibility was also considered. okuda et al and japan integrated staging score were not considered separately, as the former did not include absolute size threshold and the latter used the tnm system for local staging. although infiltrative morphology is not a part of any existing staging system, its metastasis risk was evaluated as it is associated with poor prognosis. , the continuous variables (ie, afp, index tumor size, and number of tumors) were stratified into discrete intervals (ie, interval data) according to the threshold values used in different staging systems as follows: afp intervals – , – , and > mg/ ml; index tumor size – , – , and > cm; and number of tumors , – , and > . bivariate variables (vascular invasion and infiltrative morphology) were treated as categorical data. statistical analysis statistical analyses were performed using r version . . . summary statistics was calculated for demographic and clinical data, and the differences between the no-met and met cohorts were assessed using mann–whitney u test for contin- uous data and chi-square test for categorical data. multivari- ate logistic regression was performed to identify statistically significant independent risk factors of metastasis. rather than constructing a computationally demanding regression model, however, we considered a series of simple single- or multi- parametric criteria based on previously proposed threshold values. this approach is conceptually analogous to the milan criteria, which incorporates several parameters (tumor size, number, vascular invasion, and extrahepatic metastasis) into a single decision-rule using logical (union or intersection) operations. for each single- and multiparametric risk criteria, the association with metastasis was assessed by constructing the standard × contingency table and calculating the odds ratio (or) with % confidence intervals (cis). treating the risk criteria as a diagnostic test, the sensitivity, specificity, accuracy, and positive and negative predictive values were also calculated with their respective % cis. the metastasis sensitivity between the single- and multiparametric criteria was compared using exact binomial test using r’s diagnostic test comparison for paired study design package (dtcom- pair). p-values < . were considered statistically significant, after benjamini–hotchberg adjustment for multiple testing when appropriate. figure flowchart of inclusion and exclusion criteria (white and gray, respectively) with the number of subjects meeting each criterion in parentheses. abbreviation: hcc, hepatocellular carcinoma. follow-up < year ( ) no cross-sectional imaging ( ) no contemporaneous afp ( ) systemic therapy ( ) new diagnosis of hcc ( ) no metastasis at diagnosis ( ) metastasis at diagnosis ( ) metastasis detected within year ( ) no metastasis during year follow-up ( ) no metastasis cohort ( ) metastasis cohort ( ) c a n ce r m a n a g e m e n t a n d r e se a rc h d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -d e c- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com cancer management and research : submit your manuscript | www.dovepress.com dovepress dovepress yokoo et al results patient population the summary statistics of the study population (n= ) is summarized in table , including the demographics, clinical data, and the tumor staging parameters. there was no significant difference in the patient age, sex, race, or liver disease etiology between the met and no-met cohorts. patients with metastases tended to have more advanced cirrhosis (cpt stage b or c). all tumor stag- ing parameters were significantly worse in the met cohort than in the no-met cohort, with greater afp values and index tumor size, as well as multifocal disease, vascular invasion, and infiltrative tumor being more frequent. as expected, liver-directed hcc treatment was more common in the no-met cohort. single-parametric criteria the metastasis risk of each staging parameter is summarized in table . all staging parameters were associated with metastasis with variable strength, with ors ranging . – . . multifocality had the weakest and vascular invasion had the strongest association with metastasis. the criteria with great- est sensitivity for extrahepatic metastasis were index tumor size > cm ( . %), milan size criteria ( . %), and afp > mg/ml ( . %). criteria with greatest specificities were infiltrative tumor ( . %), number of tumors > ( . %), and vascular invasion ( . %). logistic regression multivariate logistic regression results are summarized in table . milan size criteria were excluded from this analysis table demographic and clinical data patient characteristics total no met met p demographics number of patients ( %) ( . %) ( . %) median age (range) (years) ( – ) ( – ) ( – ) . sex (male/female) / / / . race caucasian . black hispanic others etiology of liver disease hcv . hbv alcohol nash others/cryptogenic child-turcotte-pugh stage a . b c unknown tumor staging parameters afp median (iqr) ( – ) ( – ) , ( – , ) < . index tumor size (iqr) (cm) . ( . – . ) . ( . – . ) . ( . – . ) < . single/multiple / / / < . vascular invasion (-/+) / / / < . infiltrative tumor (-/+) / / / < . loco-regional therapy treatment (yes/no) / / / < . liver transplantation chemoembolization thermal ablation surgical resection radioembolization notes: no met, patients with no documented metastasis within months of follow-up; met, patients with synchronous or metachronous metastasis within months of follow-up. etiology – others, cryptogenic cirrhosis (n= ) and wilson’s disease (n= ). (-/+) indicates feature absent/present. abbreviations: hbv, hepatitis b virus; hcv, hepatitis c virus; iqr, interquartile range; nash, nonalcoholic steatohepatitis c a n ce r m a n a g e m e n t a n d r e se a rc h d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -d e c- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com cancer management and research : submit your manuscript | www.dovepress.com dovepress dovepress extrahepatic metastasis risk of hcc for its obvious correlation (by design) with tumor size and number. after correction for covariates, the following risk factors were independently associated with metastasis: afp level, index tumor size, and presence of vascular invasion, all with adjusted p-values < . . no significance was detected for the number of tumor nodules and infiltrative morphology, after correcting for effects of afp, tumor size, and vascular invasion. multiparametric criteria of all possible logical combination of the independent risk factors (afp, tumor size, and vascular invasion), only those that would improve sensitivity by “union” operations (to be used as metastasis screening test) were analyzed and results are shown in table . other logical combinations of risk fac- tors, or other choices of threshold values and ranges, were not exhaustively considered to control the total number of simultaneous tests and associated penalty on the adjusted p-values. all combination criteria of afp, size, and vascular invasion performed well with high sensitivities ranging from . to . % for the detection of extrahepatic metastasis, with variable specificities and ors. multiparametric criteria including “milan size or vascular invasion or afp > mg/ ml” had the highest sensitivity and or of . % and . , respectively. the sensitivity of this multiparametric criteria was significantly better (p< . ) than those of milan size criteria, vascular invasion, or afp > mg/ml alone as single- parametric criteria. therefore, for patients determined to be within the milan criteria based on imaging, the presence of extrahepatic metastatic disease is virtually excluded if afp is also < mg/ml. comparison of criteria the exact binomial test p-values for the differences in diagnostic sensitivity between the single-, two-, and three- parametric criteria are shown in table . depending on the tumor size parameter (> cm, > cm, or milan size criteria), two-parametric criteria with combination of size parameter table single-parametric criteria for metastasis criteria threshold odds ratio sensitivity specificity accuracy ppv npv afp (mg/ml) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) index tumor size (cm) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) number of tumors > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) milan size criteria a . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) vascular invasion – . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) infiltrative tumor – . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) notes: data in parentheses are wilson’s % confidence interval. amilan size criteria: one tumor < cm or up to three tumors < cm with no extrahepatic disease or vascular invasion. abbreviations: npv, negative predictive value; ppv, positive predictive value. table tumor staging parameters and multivariate logistic regression risk factor categories # patients odds ratio ( % ci) unadj p-value adj p-value a-fetoprotein (mg/ml) < – > ( . %) ( . %) ( . %) . ( . – . ) . . index tumor size (cm) < – > ( . %) ( . %) ( . %) . ( . – . ) < . < . number of tumors – > ( . %) ( . %) ( . %) . ( . – . ) . . vascular invasion absent present ( . %) ( . %) . ( . – . ) . . infiltrative morphologya absent present ( . %) ( . %) . ( . – . ) . . notes: aalthough infiltrative morphology is not a part of existing staging system, its metastasis risk was evaluated as it is a marker of poor prognosis. the p-values were adjusted using the benjamini–hochberg method for multiple comparisons. abbreviations: ci, confidence interval; unadj, unadjusted; adj, adjusted. c a n ce r m a n a g e m e n t a n d r e se a rc h d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -d e c- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com cancer management and research : submit your manuscript | www.dovepress.com dovepress dovepress yokoo et al and either afp > mg/ml or vascular invasion performed significantly better, and three-parametric criteria with com- bination of all size parameter, afp > mg/ml, and vascular invasion performed significantly better than two-parametric criteria. discussion in patients with new diagnosis of hcc, extrahepatic metas- tasis can profoundly impact treatment options and prognosis. determination of metastasis risk using readily available imaging and serum afp data may facilitate timely manage- ment decision-making, including the need for exhaustive metastasis workup. furthermore, correct risk stratification of these patients may help avoid unnecessary morbidity and cost associated to loco-regional therapy. the purpose of this retrospective study in patients with new diagnosis of hcc was to validate the association between tumor staging param- eters and synchronous/metachronous metastases, ultimately to enable rapid metastasis risk stratification based on imaging findings in conjunction with afp. among the staging parameters in existing hcc staging systems, we found that index tumor size, vascular invasion, and afp are independently associated with metastasis. logical combinations of these parameters tended to be more strongly associated with metastases, often with significantly higher sensitivity for metastasis detection. in particular, the combination of milan size criteria, vascular invasion, and afp > mg/ml had the highest or, sensitivity, and negative predictive values; in those patients in whom these criteria were simultaneously negative, metastasis was exceedingly rare. the current national comprehensive cancer network (nccn, version . ) recommendation for confirmed hcc cases includes a chest ct and optional whole-body bone scintigraphy for metastatic workup. utility of whole- body pet imaging has been investigated, , but its use as routine metastasis workup tool remains controversial. up to / ( %) of our patients in the met cohort received loco-regional therapy, illustrating the difficulty of predict- ing extrahepatic metastasis at initial diagnosis with current staging approaches. our study suggests that only a subset of patients with hcc is higher risk for metastasis who may require formal metastasis workup. metastasis risk may be easily assessed based multiphasic liver ct or mri find- ings in conjunction with serum afp. in low-risk patients, it may be reasonable to refer immediately to liver-directed therapy, thereby shortening time to therapy and saving cost of additional imaging studies. in our study population, for t ab le m ul ti pa ra m et ri c cr it er ia fo r m et as ta si s c ri te ri a t h re sh o ld o d d s ra ti o s en si ti vi ty s p ec ifi ci ty a cc u ra cy p p v n p v in de x tu m o r > c m o r v i – . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) in de x tu m o r > c m o r v i – . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) m ila n si ze c ri te ri aa o r v i – . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) ) . ( . – . ) in de x tu m o r > c m o r a fp > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) in de x tu m o r > c m o r a fp > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) m ila n si ze c ri te ri aa o r a fp > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) in de x tu m o r > c m o r v i o r a fp > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) in de x tu m o r > c m o r v i o r a fp > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) m ila n si ze c ri te ri aa o r v i o r a fp > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) > . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) . ( . – . ) n o te s: a m ila n si ze c ri te ri a: o ne t um o r < c m o r up t o t hr ee t um o rs < cm w it h no e xt ra he pa ti c di se as e o r va sc ul ar in va si o n. d at a in p ar en th es es a re % c is . a b b re vi at io n s: n pv , n eg at iv e pr ed ic ti ve v al ue ; p pv , p o si ti ve p re di ct iv e va lu e; v i, va sc ul ar in va si o n. c a n ce r m a n a g e m e n t a n d r e se a rc h d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -d e c- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com cancer management and research : submit your manuscript | www.dovepress.com dovepress dovepress extrahepatic metastasis risk of hcc example, the total cost saving would have been $ , in medicare us dollars (or ~$ /patient), assuming the standard metastasis workup consisting of chest ct without contrast and whole body bone scan. patients otherwise not meeting the low-risk criteria may benefit from comprehensive metastasis workup, and any extrahepatic abnormality found on imaging should be scrutinized with high suspicion of metastatic disease, especially those with high afp > mg/ ml, vascular invasion, multifocal, or infiltrative tumor(s), due to their moderate to high specificity for metastases. due to retrospective design, this study has several limita- tions. first, patient assignment into the no-met cohort was based on -month metastasis-free survival. this requirement was necessary because patients did not undergo uniform metastasis workup; the decision to obtain chest ct, bone scintigraphy, or pet in addition to routine chest radiograph was made at the discretion of the treatment provider, as per nccn guideline at the time of their clinical care. as this may have led to under-detection of subclinical metastases at initial diagnosis (ie, verif ication bias), we extended months of clinical and/or imaging observation to allow initially undetected metastasis to declare itself over time. however, this requirement resulted in exclusion of ~ / of the potentially eligible patients who were lost during follow-up. therefore, patients with very aggressive tumors or decompensated cirrhosis may have been underrepresented, as they were not likely to have survived long enough to meet the inclusion/exclusion criteria. second, the potential therapy effect on metastasis development and detection could not be completely addressed. patients already receiving systemic therapy at the time of diagnosis were excluded to prevent the table pairwise sensitivity comparison p-values between single-, two-, and three-parametric criteria metastasis prediction criteria index tumor > cm or vi index tumor > cm or afp > mg/ml index tumor > cm or vi or afp > mg/ml index tumor > cm . ( . ) . ( . ) . ( . ) index tumor > cm or vi – . ( . ) . ( . ) index tumor > cm or afp > mg/ml – – . ( . ) index tumor > cm or vi index tumor > cm or afp > mg/ml index tumor > cm or vi or afp > mg/ml index tumor > cm < . (< . ) < . (< . ) < . (< . ) index tumor > cm or vi – . ( . ) . ( . ) index tumor > cm or afp > mg/ml – – . ( . ) milan size criteria or vi milan size criteria or afp > mg/ml milan size criteria or vi or afp > mg/ml milan size criteria . ( . ) < . (< . ) < . (< . ) milan size criteria or vi – . ( . ) . ( . ) milan size criteria or afp > mg/ml – – . ( . ) note: values within parentheses indicate benjamini–hochberg adjusted p-values for multiple comparisons. abbreviation: vi, vascular invasion. confounding effect of systemic therapy on metastases. major- ity of the no-met cohort and minority of the met cohort under- went liver-directed therapy. while complete response after such loco-regional therapy likely would not affect the evolu- tion of extrahepatic metastases, partial, stable, or progressive disease could potentially pose additional risk of subsequent metastasis development. however, we believe that this was only a minor concern, since the majority of the met cohort had metastatic disease before initiation of therapy ( out of in met cohorts), and the possible confounding effect by treatment outcome is expected to be small. this study did not investigate cirrhosis as a risk factor of metastasis. the data on cirrhosis were incomplete, because determination of the cirrhosis status using a reference standard method (random liver biopsy; no serum markers or elastography techniques were available at the time of this study) was often not needed for clinical care. the hcc patient population at this public safety-net hospital has been overwhelmingly (> %) those with known or suspected cirrhosis, and there- fore, this population would not have allowed meaningful sub-analysis, even if the cirrhosis data were available. finally, this study was conducted in a public safety-net hospital in a large metropolitan area, where patients could present with more advanced hcc in theory compared to insured popula- tion undergoing routine hcc surveillance. , the rate of metastatic disease in this population, however, was similar to previous reports. , also, estimates of the or, sensitivity, and specificity are independent of disease prevalence and hence our results may be generalizable to other patient populations. our population was composed entirely of cirrhotic patients, most due to chronic hepatitis c infection; this may influence c a n ce r m a n a g e m e n t a n d r e se a rc h d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -d e c- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com cancer management and research : submit your manuscript | www.dovepress.com dovepress dovepress yokoo et al the frequency of metastasis and afp threshold level. – due to these limitations, the metastasis screening criteria need to be further validated prospectively in an independently sampled population. conclusion this retrospective study validated that tumor staging param- eters are associated with metastasis risk in patients with new diagnosis of hcc. patients with low metastasis risk may be identified based on afp, tumor size, and absence of vascular invasion. in these patients, comprehensive metastasis workup may not be needed, thereby facilitating timely delivery of treatment and eliminating cost for further diagnostic imag- ing studies. acknowledgments this study was conducted with support from the center for translational medicine, nih/ncats grant num- ber ul tr , and nih/ncats g rant number kl tr . the content is solely the responsibility of the authors and does not necessarily represent the official views of the center for translational medicine, ut southwestern medical center, and its affiliated academic and health care centers, the national center for advancing translational sciences, or the national institutes of health. disclosure ags is on the speakers’ bureau and a consultant for bayer. acy is on the speakers’ bureau for bayer and received research funding from novartis, merck, and peregrine. the authors report no other conflicts of interest in this work. references . u.s. cancer statistics working group [webpage on the internet]. united states cancer statistics: – incidence and mortality web-based report. u.s. department of health and human services, centers for disease control and prevention and national cancer institute; 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'candidate genes' whose involvement in the disease is suspected a priori may be used as probes. for example, genes coding for various enzymes involved in the monoamine neurotransmitter systems might be relevant to psychiatric disorders. another strategy is to concentrate attention upon probes that label a particular part of the genome, such as a single chromosome, when the disease gene is believed to be located in that area. unknown pieces of dna selected at random may also be used as probes and their identities and position in the human genome determined only if linkage is established. it was this technique that gusella et al. ( ) employed successfully to locate the gene for huntington's disease on chromosome . the excitement generated by findings in huntington's disease was tempered for psychiatrists by the realization that none of the common psychiatric disorders show such a clear and unambiguous pattern of inheritance. however, recent work on both alzheimer's disease and affective disorder has removed many of the doubts about the extent to which molecular genetics will be applicable to the study of mental illness. address for correspondence: dr m. j. owen, department of biochemistry and molecular genetics, st mary's hospital medical school, london w pg. psm https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core editorial: polymorphic dna markers in mental disease alzheimer's disease the existence of several large families in which alzheimer's disease (ad) appears to be segregating as an autosomal dominant suggested that the disease might be amenable to genetic analysis using rflp linkage techniques. furthermore, associations between down's syndrome and ad led to speculation that the genetic abnormality might be located on chromosome . people with down's syndrome commonly develop the neuropathological stigmata of ad between the ages of and , and the prevalence of down's syndrome is reported to be increased among relatives of probands with ad (oliver & holland, ). over the last few years, therefore, a considerable effort has been made to determine whether there is linkage between ad and gene markers for loci on chromosome . this has recently culminated in the demonstration of linkage by a large international group centred in boston (st george-hyslop et al. ). these investigators used several polymorphic dna markers spanning the long arm of chromosome to study the inheritance of the disease in four large pedigrees. linkage analysis revealed that two of these probes (designated d s/ and d s /d sll) are linked to the expression of ad with a highly significant probability. ad, unlike many psychiatric illnesses, is associated with characteristic neuropathological findings. the molecular composition of neuritic plaques, neurofibrillary tangles and amyloid angiopathy has been the focus of much recent investigation. this work has converged strikingly upon the genetic studies. the first breakthrough was the isolation of the amyloid protein found in the core of senile plaques and in the blood vessels of those with ad. this protein, which is also abnormally deposited in down's syndrome, has been termed a protein or / -amyloid. needless to say, a good deal of excitement was generated when the gene coding for a protein was located on chromosome close to the linked probes of the boston group (rang et al. ; goldgaber et al. ; tanzi et al. ). furthermore, it has been claimed that a small portion of chromosome containing the a gene is duplicated in ad so that, as in down's syndrome, three copies are present (delabar et al. ). taken together, these data represent strong circumstantial evidence that the a gene is the locus of the defect causing ad. however, evidence that this is not the case has come from two studies in which the a locus was only weakly linked to the disease. moreover, in several families, there was recombination between the a gene and ad (van broeckhoven et al. ; tanzi et al. ). these findings suggest that the disease locus is on the neighbouring portion of chromosome . it should be remembered that the majority of sufferers do not appear to have a family history of the disorder. these may all be sporadic cases. alternatively, since ad is predominantly a disease of the elderly, its inheritance might be masked by the death of predisposed relatives from unrelated disease (breitner et al. ). this means that genetic forms of the illness might be much more common than is at first apparent. finally, it is to be expected that the understanding of familial disease will ultimately throw light upon the pathophysiology of sporadic cases. affective disorder research into the molecular genetics of affective disorder has not benefited from a clue such as that provided for alzheimer's disease by the association with down's syndrome. however, much has been gained from the study of the old order amish community in pennsylvania. this population is highly suitable for genetic research as families are large, emigration rare and paternity relatively certain. moreover, religious convictions proscribe the use of alcohol or drugs and thereby facilitate unambiguous diagnosis of affective disorder. segregation analysis of data from the families of probands with bipolar disease provided evidence for a single genetic locus with autosomal dominant transmission. initial studies of rflps in a large amish pedigree using a number of probes suggested that the disease might be linked to two markers on the short arm of chromosome , the insulin gene (ins) and the cellular oncogene ha-ras- (hras ) (gerhard et al. ). these two markers have recently been employed in a more extensive study of the same pedigree which has produced quite convincing evidence for linkage (egeland et al. ). this suggests that, in this pedigree at least, https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core editorial: polymorphic dna markers in mental disease a dominant gene which confers a strong predisposition to bipolar affective disorder lies on the tip of the short arm of chromosome . it is of considerable interest that the structural gene for tyrosine hydroxylase is located upon the same segment of the chromosome. this enzyme catalyses an important step in the synthesis of two neurotransmitters, dopamine and noradrenaline, which have both been implicated in the pathogenesis of mental illness. however, the generality of findings in the amish has already been called into question by two studies, one of american and the other of icelandic pedigrees (detera-wadleigh et al. ; hodgkinson et al. ), which managed to rule out linkage of ins and hras to bipolar affective disorder. this discrepancy is probably not due to methodological differences, but rather suggests that there is more than one gene responsible for the condition. further support for genetic heterogeneity comes from recent studies showing linkage of bipolar illness to various x chromosome markers in israeli and belgian pedigrees (baron et al. ; mendelwicz et al. ). it seems likely, therefore, that there are at least three genes predisposing to bipolar disorder. several interesting issues arise from the demonstration of genetic heterogeneity. first, we might ask whether this is associated with clinical heterogeneity. there do not appear to have been gross clinical differences between the patients in the above studies. however, it might be possible to detect more subtle differences in symptoms or variations in course or outcome associated with different genetic defects. the second question, which might well turn out to be related to the first, concerns the degree of similarity between the various disturbances of brain function that result from the different genetic lesions. for example, each gene might code for a sub-unit of the same complex molecule, such as a receptor. conversely, they might have quite different effects whose consequences eventually converge upon the final common pathway to affective disorder. implications in the wake of these recent findings, study of the molecular pathology of mental illness is likely to begin in earnest. advances have already been made in some diseases causing mental handicap and this is likely to prove an especially fruitful area for molecular genetics (whatley et al. ). research workers are also starting to focus their attention upon other psychiatric disorders with a presumed genetic component, such as schizophrenia and anxiety. this would seem, therefore, to be an appropriate time to consider some of the goals and consequences of such research. diagnostic the discovery of dna polymorphisms which are tightly linked to an illness will, in some instances, allow pre-symptomatic diagnosis. we shall discuss the implications of this by reference to the three conditions in which significant advances have already been made; huntington's disease, alzheimer's disease and affective disorder. in the case of huntington's disease, pre-symptomatic diagnosis is possible with a high degree of accuracy because the probe is tightly linked to the disease locus and there is apparently % penetrance. however, there are several problems. first, the test cannot be applied to everyone at risk; at least one living grandparent must be available. in one study this criterion was fulfilled for only % of individuals at risk (harper, ). secondly, the test is not % accurate because there is up to a % chance of recombination occurring between the marker and the disease locus. moreover, although heterogeneity has not been demonstrated, it cannot be excluded with certainty. thirdly, by no means all those at risk will wish to be tested as no preventive therapy can be offered. fourthly, those identified as probable carriers of the gene might not be able to cope with the burden of this knowledge. careful counselling and long-term support will therefore be needed (crawfurd & harris, ). finally, the results of such a predictive test will have implications for others, such as prospective spouses, employers and insurers, and this might lead to ethical problems (crawfurd & harris, ). - https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core editorial: polymorphic dna markers in mental disease the marker test is likely to be more useful for pre-natal diagnosis where the foetus now represents the third generation. however, in most cases the parent will not have reached the age of risk and the test will therefore only determine whether or not the foetus has the same % risk as the parent (harper, ). it has been argued that, given the low rate of spontaneous mutation in huntington's disease, a combination of pre-natal diagnosis with selective termination and genetic counselling should ensure a dramatic fall in the prevalence of the disorder (harper, ). however, many will wonder whether it is ethically acceptable to abort a foetus, which, even if affected, (and the odds usually will be only : ) could be expected to live quite healthily for upwards of - years. in the case of alzheimer's disease, as we have seen, there is uncertainty about the degree of aetiological heterogeneity. in any case, the majority of sick individuals do not seem to have a family history of the disorder and it seems unlikely, therefore, that tests based upon linkage will be useful in more than a few, rare, multiply-affected families. even if it becomes possible to detect the disease allele pre-symptomatically, this will be of little clinical value until some form of preventive therapy is available because few carriers of the disease gene will consider % risk to offspring of an illness of such late onset sufficient grounds to desist from reproduction. such pre-disposed individuals will, of course, be of considerable interest to research workers concerned with the early features of the illness and in trials of prophylactic therapy. however, the ethical issues will have to be very carefully considered. as far as pre-natal diagnosis is concerned, even if a suitable test were to become available, the late onset of the disease would make termination ethically unacceptable to many. in affective disorder, genetic heterogeneity alone will limit the usefulness of chromosome markers for pre-symptomatic diagnosis. moreover, even in the amish pedigree penetrance is incomplete so that there is no guarantee that an individual with the disease gene will ever become unwell. in addition, the same clinical and ethical considerations apply to possible pre-symptomatic diagnosis of affective disorder that apply to alzheimer's disease. finally, few would consider bipolar affective disorder sufficient grounds for termination of pregnancy even if it were possible accurately to detect individuals at high risk. therapy we believe that the greatest benefits that will accrue from the application of molecular genetics to mental disorder will come not from the use of markers for diagnosis but from the insights into pathological mechanisms that will result. the progressive characterization of the genome in the region of the marked locus should eventually lead to the precise identification of the genetic defect. this work is now well under way for huntington's, alzheimer's and affective disorder. once a genetic abnormality has been identified it should be possible to determine its immediate biochemical consequences. from here we may hope to follow the sequence of changes that finally results in the phenotype so preparing the ground for more rational and effective therapeutic strategies. michael j. owen a n d stephen a. whatley professor w. a. lishman and drs r. m. murray, v. l. nimgaonkar and a. j. holland made valuable comments on the manuscript. references baron, m., risch, n., hamburger, r. mandel, b., kushner, s., crawfurd, d. i. o. & harris, r. 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( ). neuro- psychiatric disorders in the new genetics. in bridges between neurology and psychiatry (ed. e. h. bridges and m. r. trimble). churchill livingstone: edinburgh. https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core mutation of herc causes developmental delay with angelman-like features original article mutation of herc causes developmental delay with angelman-like features gaurav v harlalka, emma l baple, harold cross, simone kühnle, monica cubillos-rojas, konstantin matentzoglu, michael a patton, karin wagner, roselyn coblentz, debra l ford, deborah j g mackay, barry a chioza, martin scheffner, jose luis rosa, andrew h crosby ▸ additional data are published online only. to view these files please visit the journal online (http://dx.doi. org/ . /jmedgenet- - ). centre for human genetics, st. george’s, university of london, london, uk department of ophthalmology and vision science, college of medicine, university of arizona, tucson, arizona, usa department of biology and konstanz research school chemical biology, university of konstanz, konstanz, germany departament de ciències fisiològiques ii, idibell, campus de bellvitge, universitat de barcelona, l’hospitalet del llobregat, barcelona, spain windows of hope genetic study, holmes county, ohio, usa verde valley guidance clinic, cottonwood, arizona, usa division of human genetics, university of southampton, southampton, uk correspondence to dr andrew h crosby, centre for human genetics, st george’s university of london, cranmer terrace, sw re london, uk; acrosby@sgul.ac.uk gvh, elb, hc contributed equally. sk, mc-r contributed equally. ahc, jlr, ms and bac all contributed equally received october revised november accepted november published online first december to cite: harlalka gv, baple el, cross h, et al. j med genet ; : – . abstract background deregulation of the activity of the ubiquitin ligase e ap (ube a) is well recognised to contribute to the development of angelman syndrome (as). the ubiquitin ligase herc , encoded by the herc gene is thought to be a key regulator of e ap. methods and results using a combination of autozygosity mapping and linkage analysis, we studied an autosomal-recessive neurodevelopmental disorder with some phenotypic similarities to as, found among the old order amish. our molecular investigation identified a mutation in herc associated with the disease phenotype. we establish that the encoded mutant herc protein has a reduced half-life compared with its wild-type counterpart, which is associated with a significant reduction in herc levels in affected individuals. conclusions our data implicate a model in which disruption of herc function relates to a reduction in e ap activity resulting in neurodevelopmental delay, suggesting a previously unrecognised role of herc in the pathogenesis of as. introduction angelman syndrome (as, (mim )) and prader–willi syndrome (pws, (mim )) are distinct neurodevelopmental conditions caused by abnormalities at the q –q imprinted region. both conditions present with characteristic develop- mental, neurological and behavioural phenotypes. most commonly these disorders result from a – mb deletion of the imprinted region, which is either paternal in origin (pws) or maternal in origin (as), with clustered breakpoints at either of two centromeric sites and one telomeric site. homologous recombination between chromo- some-specific low-copy repeats (duplicons) is the mechanism underlying a number of genetic disor- ders. sequences homologous to the herc (hect domain and rcc -like domain , mim , nm . ) gene, encoding a giant kda protein, form the basis of the low-copy repeats associated with the q – q breakpoints. evolutionary evidence is suggestive of a herc duplicon-mediated process leading to an increased tendency for chromosomal rearrangements to occur in this region. although as and pws deletion patients are hemizygous for herc , to date there has been no evidence that this gene contributes to either phenotype. – patients with classical as present with severe mental retardation and profound speech impairment with only minimal use of words, although non-verbal and receptive communication skills are higher than expressive language skills. a movement disorder ranging from ataxia to clumsiness or unsteadiness and a characteristic behaviour pattern of hyperactiv- ity, a happy personality and episodes of inappropriate bursts of laughter are typically seen. a wide based gait with pronation at the ankles and arms held at shoulder level and flexed at the elbows are frequent findings. hypotonia and feeding difficulties are common in infancy with increased tone in the lower limbs and hyperactive reflexes developing later. seizures are present in over % of as patients and comprise a diversity of types; an eeg pattern of – hz large amplitude slow wave bursts is typical and seizures decrease in frequency in adulthood. brain structure is normal by ctor mri imaging. scoliosis, strabismus and hyperactivity are asso- ciated features of the condition. dysmorphic fea- tures are subtle and include microcephaly (by the age of years), prominent chin, macrostomia, hypopigmentation of skin, hair and eyes (deletion patients only). the clinical features of as have been reviewed previously. – recently we demonstrated physical and functional interactions of herc with e ap, the primary mol- ecule implicated in the pathogenesis of as. most sig- nificantly herc was shown to stimulate the e ubiquitin-protein ligase activity of e ap implicating it as a regulator of e ap and raising the intriguing possibility that herc may play an important role in the as phenotype. in the current study, we investigated a unique and recognisable inherited neurodevelopmental condition with some pheno- typic similarities to as, present at high frequency among the ohio old order amish community, which we show to be due to herc mutation. methods subjects blood or buccal samples were obtained from affected children, parents and unaffected siblings. dna and rna were extracted by standard proce- dures. a single . mm diameter skin biopsy was taken from affected individuals xi: , xi: , x:i and x: and from unaffected individuals. all samples harlalka gv, et al. j med genet ; : – . doi: . /jmedgenet- - developmental defects konstanzer online-publikations-system (kops) url: http://nbn-resolving.de/urn:nbn:de:bsz: - erschienen in: journal of medical genetics ; ( ), . - s. - https://dx.doi.org/ . /jmedgenet- - http://dx.doi.org/ . /jmedgenet- - http://dx.doi.org/ . /jmedgenet- - http://dx.doi.org/ . /jmedgenet- - http://dx.doi.org/ . /jmedgenet- - were obtained with approved informed consent (university of arizona irb ). genotyping and linkage analysis single-nucleotide polymorphism (snp) microarray genotyping was performed using illumina human cytosnp- v . and . k arrays. marker saturation was carried out using existing and newly generated microsatellite markers (primer sequences available on request). alleles were size-fractionated using % polyacrylamide gels and dna was visualised by silver staining. multipoint linkage analysis performed with simwalk under a model of autosomal recessive inheritance with full penetrance, using a disease allele frequency estimated at . . the pedi- gree was deconstructed to facilitate computational efficiency. mutation analysis unique intronic primers were designed for herc exons as well as exons of other genes located in the critical interval. the repetitive nature of herc prohibited amplification from genomic dna; as such the remaining exons were amplified from cdna using unique exonic primers. primers were designed using online primer software based on sequences from the online university of california, santa cruz (ucsc) genome browser database and ordered from sigma-aldrich (optimum pcr conditions are available on request). rt-pcr was carried out using primescript one step rt-pcr kit v. , takara according to the manufacturer’s instructions. purified pcr amplification products were sequenced using dye-terminator chemistry and electrophoresed on an abi xla capillary sequencer (applied biosystems). mutation ana- lysis was carried out using finch tv . . (created by geospiza inc.) and gene tool . . . (created by bio tools inc). both strands of each product were sequenced. seventy nine anonymised regional control and european control dna samples were screened by bidirectional sequencing for herc exon . cell lines, plasmids and transfections primary fibroblasts, hek t cells and u os cells were cul- tured in dmem (sigma aldrich) with % fetal bovine serum (fcs), mm glutamine (invitrogen) and % penicillin and streptomycin. expression constructs (transient transfection experiments) encoding ha-tagged wild-type (wt) e ap (isoform ), the ha-tagged catalytically inactive p.cys ala mutant of e ap, myc-tagged p.ile ser ring b mutant, ha-tagged herc and his-tagged ubiquitin were described previously; myc-herc ( – ) was kindly provided by t. ohta. the cdnas encoding the p.pro leu herc mutants (full-length or amino acid residues: – ), were generated by a pcr-based approach (further details will be provided upon request) and expressed as n-terminally ha-tagged or myc-tagged forms from pcdna . for transient expression, hek t and u os cells were trans- fected with the respective constructs in the presence of a reporter construct encoding β-galactosidase by lipofection (lipofectamine or ltx) according to the manufacturer’s instructions (invitrogen). protein extracts were prepared – h after trans- fection as described and transfection efficiency was determined by measuring β-galactosidase activity. extracts were analysed by polyacrylamide gel electrophoresis (page) and immunoblotting as previously described. protein levels were normalised and expressed as percentage of controls. the antibodies used were: anti-herc (bd biosciences); anti-e ap (santa cruz biotechnology, inc); anti-xpa (kamiya biomedical company); anti-myc (roche); anti- α tubulin (calbiochem); anti-ran; horse- radish peroxidase-conjugated secondary antibodies (invitrogen). ubiquitination and degradation assays for ubiquitination of p.ile ser ring b mutant within cells, one cm plate of hek t cells was transfected with expression constructs encoding ha-tagged p.ile ser ring b mutant ( mg), e ap ( . mg), his-tagged ubiquitin ( . mg) and herc or the p.pro leu herc mutant ( . mg, . mg or . mg) as indicated (figure ). twenty-four hours after transfec- tion, % of the cells were lysed under non-denaturing condi- tions to determine expression levels of e ap, p.ile ser ring b mutant and the two forms of herc . the remaining cells were lysed under denaturing conditions, and ubiquitinated proteins were purified. for cycloheximide assays using ectopically expressed herc and p.pro leu herc mutant, hek t cells were transfected with mg of the herc expression constructs indicated. twenty-four hours post transfec- tion, cells were trypsinised, pooled and equal aliquots seeded onto × cm plates. after additional h, cells were treated with mg/ml cycloheximide (dissolved in meoh) in the absence or presence of mm mg (dissolved in dimethyl sulfoxide (dmso)) and harvested at the times indicated. protein extracts were prepared and analysed by sds-page followed by western blot analysis. levels of the various ha-tagged proteins (e ap, p.cys ala e ap mutant, herc and p.pro leu herc mutant) and myc-tagged p.ile ser ring b mutant were determined by western blot analysis. the antibodies used for detection were the mouse monoclonal ha (hiss diagnostics, freiburg, germany) for ha-tagged proteins and the mouse monoclonal e (abcam) for myc-taggedp.ile ser ring b mutant protein. results fifteen affected individuals, aged between months and years, were identified (figure ). cardinal features include global developmental delay, hypotonia, delay in ambulation between ages – years, an unstable gait frequently with a broad base and arms held upwards and bent at the elbows with brisk walking or running. failure to acquire normal speech was present in all affected individuals. those with extreme poverty of speech use gesturing and communication aids effectively and under- standing is far greater than language ability. seizures were reported in / affected cases. neuroimaging was only available for / individuals, in three cases absence of the posterior half of the corpus callosum was reported, the remaining three were said to be unremarkable. affected individuals were frequently reported to have poor concentration and hyperactivity. dysmorphic features are subtle and include plagiocephaly, prognathism, narrow palate, elon- gated hallux, sandal gap and excessive pronation of the feet occasionally requiring surgery. the clinical features are detailed in tables a and b. interestingly the majority of affected individuals in the amish community have bright blue eyes due to the coincident close prox- imity of the causative founder mutation to a snp rs in herc that influences oca expression and is associated with eye colour (data not shown). assuming that a founder mutation was responsible we under- took a genome wide microarray scan using illumina human cytosnp- v . and . k arrays using dna from the affected individuals initially available (viii: , viii: , ix: , ix: , x: , x: ). this identified a single notable homozygous region shared by all affected individuals of . mb on chromosome harlalka gv, et al. j med genet ; : – . doi: . /jmedgenet- - developmental defects q . –q . , delimited by markers rs and rs , likely to correspond to the disease locus. subsequent microsatellite marker analysis (see online supplemen- tary figure s ) confirmed autozygosity across this region and refined and precisely positioned the disease locus (lodmax . ). the putative disease locus is predicted to contain transcript-encoding regions, of which three are hypothetical, two are micrornas and are pseudogenes. while screening the remaining six genes a non-synonymous base variant was identi- fied in exon of herc (nm_ . :c. c>t), pre- dicted to encode a herc protein with substitution of proline by leucine at amino acid position (see online supplementary figure s ). this substitution is not a known snp and is not present in the genomes project or the exome variant server, nhlbi go exome sequencing project ( subjects). genotyping of the full family revealed that the sequence variant cosegregated appropriately for a recessive disorder among the entire extended family comprising seven nuclear families, with affected individuals being homozygous, parents being heterozy- gous carriers and unaffected siblings being either wt or heterozy- gous carriers of the variant (figure a). the c. c>t variant was also found to cosegregate in a second amish family compris- ing four affected individuals from a distinct amish deme (figure b). analysis of control chromosomes from unaffected indi- viduals from the same ohio amish community identified two heterozygous carriers of the sequence variant. rna was extracted from the whole blood of two of the affected cases (ix: and ix: ) for cdna synthesis and sequencing, which verified the presence of the variant in the rna transcript. given the proximity of the herc gene to the as/pws imprinting control region, we analysed methylation status at the snrpn imprinted locus by methylation specific pcr; no abnor- malities were detected (data not shown). we next investigated the functional consequences of the herc mutation. western blot analysis revealed a profound effect of the mutation on protein levels, with herc protein being almost undetectable in fibroblasts derived from four affected individuals in comparison with three healthy controls. this effect seems specific for herc because protein levels of known herc interacting proteins such as e ap or xpa, or the unrelated protein α-tubulin were not significantly altered (figure a). the dramatic reduction of herc protein levels figure (a) and (b) pedigree drawings of the two amish families. harlalka gv, et al. j med genet ; : – . doi: . /jmedgenet- - developmental defects table a a comparison of the clinical findings of individuals homozygous for herc c. c>t (pedigree a) viii: viii: ix: ix: ix: x: x: x: x: xi: xi: gender f m f m f f f f f m m age (years) . . . . . . growth parameters birth weight sds n/k n/k n/k . . − . − . − . . . − . ofc sds − . . n/k . n/k − . n/k − . − . − . − . development speech words < words ss ss ss < words words – ss limited words walked (years) . . – . – intellectual disability moderate moderate/severe moderate moderate moderate moderate moderate moderate/severe moderate mild moderate neurology childhood hypotonia + + + + + + + + + + + gait arms raised+bent (run) arms raised+bent (run) bb arms raised+bent (run) bb arms raised +bent arms raised+bent (run) n/a arms raised + bent clumsy n/a foot pronation − − n/k + + + + n/a + + n/a seizures − − − absence gm − − − − − − xgm neuroimaging ct normal n/k n/k n/k ct normal ct normal n/k ct absent post cc n/k n/k mri absent post cc hyperactivity + − + + + + + − + + − behavioural characteristics affectionate aggression repetitive behaviour affectionate sociable affectionate sociable affectionate sociable affectionate sociable interactive flaps hands repetitive behaviour irritable head banging physical anomalies strabismus + − − − − − − + + − + high narrow palate + n/k + + + + + n/k + n/k n/k feet small small elongated hallux sg ot elongated hallux n/k elongated hallux sg n/k sg ot sg ot elongated hallux sg elongated hallux sg other physical findings scoliosis freq om mvp basal skin syndactyly all digits freq om freq om ngt fed until years tga bb, broad base; cc, corpus callosum; ct, computerised tomography; freq, frequent; gm, grand mal seizures; mri, magnetic resonance imaging; mvp, mitral valve prolapse; n/k, not known; n/a, not available; ofc, occipital frontal circumference; ot, overlapping toes; om, otitis media; post, posterior; sg, sandal gap; sds, standard deviation scores; ss, short sentences; tga, transposition of the great arteries. h arlalka g v, et al. j m ed g enet ; : – . doi: . /jm edgenet- - d e ve lo p m e n ta l d e fe cts may relate to reduced expression at the mrna level associated with reduced transcription rate or mrna stability, or reduced stability of the translated protein product. we therefore evaluated herc mrna levels by quantitative pcr and tested the effect of a proteasome inhibitor cocktail on p.pro leu herc levels in patient primary fibroblasts. this revealed no significant alteration in mrna levels (data not shown). evaluation of herc levels in patient fibroblasts following treatment with pro- teasome inhibitors was suggestive of increased turnover, although this could not be established conclusively due to limited availabil- ity of suitable patient cellular material (data not shown). consequently, we analysed the turnover of the mutant protein by transiently expressing wt herc as well as p.pro leu mutant herc in hek tcells. this revealed more rapid degradation of the p.pro leu herc mutant (figure b) indicating that the mutant protein has a shorter half-life than wt herc . a possible explanation for the apparent instability of the p.pro leu herc mutant may be provided using structural data from the protein data bank. herc is a member of the rcc (regulator of chromosome condensation ) superfamily, each of which contains at least one rcc -like domain (rld). within its amino acid sequence, herc contains three rld domains and a c-terminal hect domain (homologous to e ap carboxy terminus) with e ligase activity. the p.pro leu amino acid substitution affects a proline residue of the rld domain of herc highly conserved in all species examined (see online supplementary figure s a,b) and among all human rcc superfamily proteins. all rlds are thought to consist of a seven-bladed β-propeller structure similar to rcc . although no structural data is available for rld of herc , given its similarity to rld , it is highly likely to form such a β-propeller structure. in silico superposition of rcc and predicted herc rld structures ( jmol/superpose/ firstglance) permitted us to extrapolate the position of p in the rld domain of herc to the fourth propeller blade (figure ). substitution of the highly conserved pro , located within the fourth blade of the propeller, is likely to disrupt the loop between the fourth and the fifth blades and may result in a conformational change affecting protein structural stability and lead to increased turnover rate. to investigate this further, we transfected u os cells with wt myc-herc (residues – ) as well as p.pro leu mutant myc-herc (residues – ) gene constructs. this confirmed a profound reduction of protein levels associated with substitution of pro (figure c), further evidence that protein instability is the likely cause of reduced herc levels in patient fibroblasts. in light of the observation that herc stimulates e ap, we next examined the effect of the p.pro leu herc substitution on e ap e ligase activity. given that known substrates of e ap are either not detectable in primary fibroblasts (activity-regulated cytoskeleton-associated protein (arc); unpublished data) or particularly short-lived (ring b), we studied e ap-mediated ubiquitination of a ring b mutant in transient transfection experiments. this revealed that wt herc as well as p.pro leu herc mutant stimulate e ap-mediated ubiquitination of ring b in a dose- dependent manner (figure ), however mutant herc was found to be less active than wt herc . similar results were obtained for autoubiquitination of e ap (data not shown). although the e ap activation difference detected was moderate, and may not in itself be functionally significant, the dose- dependent nature of herc on e ap activation is likely to exacerbate effects of the p.pro leu herc mutation. discussion taken together our genetic and cellular data implicate herc in a human disease phenotype which, although a distinct entity, has some as-like features. table b a comparison of the clinical findings of individuals homozygous for herc c. c>t (pedigree b) ix: ix: ix: ix: gender m m f f age (years) . . . . growth parameters birth weight sds n/k − . − . − . ofc sds n/k n/k − . − . development speech ss ss – < words walked (years) . – – intellectual disability mild/moderate mild/moderate moderate mild/moderate neurology childhood hypotonia n/k n/k + + gait n/k bb n/k n/a foot pronation + + n/a + seizures fc none fc − neuroimaging mri absent post cc n/k n/k n/k hyperactivity − − n/a − behavioural characteristics n/a sociable physical anomalies strabismus − − − − high narrow palate n/k n/k n/k n/k feet n/k n/k sg sg other physical findings bb, broad base; cc, corpus callosum; freq, frequent; fc, febrile convulsion; mri, magnetic resonance imaging; mvp, mitral valve prolapse; n/k, not known; n/a, not available; ofc, occipital frontal circumference; om, otitis media; ot, overlapping toes; post, posterior; sds, sd scores; ss, short sentences; sg, sandal gap; tga, transposition of the great arteries. harlalka gv, et al. j med genet ; : – . doi: . /jmedgenet- - developmental defects there are reported members of the human rcc protein superfamily, six of these within the herc subgroup. a number of proteins within this family have been associated with a diverse set of human diseases, until now however no herc sub- group members had been causally linked. human and mouse herc protein display % homology and % similarity and herc homozygous and hemizygous mutations have been iden- tified as the cause of the mouse rjs syndrome (runty, jerky, sterile) also known as jdf ( juvenile development and fertility- ). affected mice are % smaller than their normal counterparts from birth, have abnormal gait and display behavioural abnor- malities, reflecting similarities with features seen in our affected subjects. none of the affected individuals that we have seen have shown any significant differences from their healthy siblings in terms of birth weight or subsequent growth, nor have any puber- tal abnormalities been reported. we have not observed any reduction in life expectancy; however the oldest affected individ- ual we have seen is years old. there does however appear to be some cognitive and behavioural decline in two of our oldest patients. currently there is no mouse model of p.pro leu herc , and production of such a future model may allow a more direct phenotypic comparison. all as cases associated with ube a mutations reported to date are predicted to affect the e ligase activity of e ap. the previously reported interaction between herc and e ap results in stimulation of e ap e ligase activity and involves herc rld , whereas the variant reported here affects rld . our data indicate a profound effect of the p.pro leu mutation on herc protein levels, which likely relate to reduced half-life resulting from distortion of the structure of the herc molecule. the modest effect on e ap stimulation asso- ciated with the herc p.pro leu mutation described here figure (a) levels of herc are diminished in fibroblasts from affected individuals. lysates from fibroblasts of wild-type (wt) controls and affected individuals were analysed by immunoblot with the indicated antibodies. the levels of herc were normalised with respect to α-tubulin as is indicated in the right panel. (b) the p.pro leu herc mutant has a shorter half-life than the wt protein. hek t cells with a stable knockdown of endogenous herc expression (unpublished) were transfected with expression constructs encoding ha-tagged forms of wt herc and p.pro leu mutant herc . after h, cells were treated with cycloheximide in the absence or presence of mm mg , harvested at the times indicated, and protein levels analysed by western blot analysis using the antibodies indicated. (c) u os cells were transfected with the indicated constructs and analysed by immunoblot. the right panel shows the levels of expression with respect to ran. harlalka gv, et al. j med genet ; : – . doi: . /jmedgenet- - developmental defects may in part explain why the developmental difficulties observed in our subjects are not as profound as those seen in as patients in whom there is likely a more significant loss of e ap activity. seizures are reported in excess of % of as patients. although seizure activity was only reported in / subjects, we speculate that this may indicate that a subtle decrease in e ap activity lowers the threshold for seizure activity. microcephaly is not a feature of this condition, however this feature of as is most commonly associated with microdeletions and less so with other molecular mechanisms leading to as. notable similarities between as and the individuals with herc mutation described here include hypotonia in childhood, a similar arms raised posture when walking particularly at speed, excessive pro- nation of the ankles, clumsiness and poor concentration. as individuals, most commonly women, frequently develop scoli- osis with age. although we only observed three adult females, it is noteworthy that one had developed severe scoliosis. strabismus is another feature commonly seen in as and was reported in / affected individuals. importantly a number of significant differences between the two conditions were also apparent. although the parents of our amish subjects did not report inappropriate bouts of laughter, many did remark on the sociability and loving nature of their children, however behav- ioural difficulties, aggression and stereotypies were also reported. further distinguishing features include lack of the clas- sical as facial gestalt and no reports of ataxia. it is interesting that in three out of six of our subjects with neuroimaging data there was absence of the posterior part of the corpus callosum, which in association with the other clinical features described may be helpful in terms of reaching a diagnosis in patients with this condition. finally although both conditions are charac- terised by significant speech abnormalities, as individuals rarely develop any meaningful speech, whereas a number of indivi- duals homozygous for the herc mutation have developed the ability to communicate effectively with language. good non- verbal communication skills and relatively preserved receptive language ability are features of both conditions. it was notable that those subjects who had received early and intensive speech and language therapy appeared to have made significantly more progress in language attainment. in the current report, we describe a novel neurodevelopmental syndrome with some features reminiscent of as arising due to figure superposition of human rcc (cyan) and human herc rld (light blue) lateral (a,c,d) and bottom (b) views of the β-propeller. high conserved proline, equivalent to proline of herc , is shown in rcc (yellow) and in rld (red). the n-termini and c-termini of rcc are indicated. the figure was generated and edited using the superpose and firstglance in jmol programmes. protein data bank entries: a for rcc and kci for herc rld . harlalka gv, et al. j med genet ; : – . doi: . /jmedgenet- - developmental defects herc mutation. although as had not been suspected in any of the patients described here and we recognise that there are important differences between the two conditions, it remains an intriguing possibility that more deleterious mutations in the herc gene may be detected in more severely affected patients who display a more significant phenotypic overlap with as. acknowledgements we are very grateful to the amish families for partaking in this study, and to the amish community for their continued support of the windows of hope project. we thank g. pons for his help with protein structure programmes and t. ohta for the myc-herc plasmid. contributors ahc designed the genetics experiments. gvh, elb, bac and djgm performed the genetics experiments. ahc, jlr, ms and elb designed the functional cell biology experiments. sk, mcr, km, jlr and ms performed the cell biology experiments. elb, hc, dlf, map, kw and rc contributed clinical data to the manuscript. ahc, elb, bac, jlr and ms wrote the manuscript. funding the work was supported by mrc grant (g ), newlife foundation (ahc, gvh and elb), mrc clinical research training fellowship (g to elb), the deutsche forschungsgemeinschaft (sche / - to ms), and by micinn (ministerio de ciencia e innovación) grant (bfu - ) to jlr. mcr is supported by a doctoral fellowship from generalitat de catalunya. competing interests none. ethics approval university of arizona. provenance and peer review not commissioned; externally peer reviewed. data sharing statement any unpublished data from this study is available on request from the corresponding author. web references the urls for data presented herein are as follows: online mendelian inheritance in man (omim), http://www.omim.org/. clustalw , http://www.ebi.ac.uk/tools/msa/clustalw /. jalview, http://www.jalview.org. jmol, http://www.jmol.sourceforge.net. firstglance in jmol, http://www.bioinformatics.org/ firstglance/fgij/. superpose, http://www.wishart.biology.ualberta.ca/superpose/. primer , http://www.frodo.wi.mit.edu/. genomes project, http://www. genomes.org. exome variant server, nhlbi go exome sequencing project (esp), seattle, wa,: http://evs.gs.washington.edu/evs/) (accessed, november, ). accession numbers the databank accession number for herc reported in this paper is nm_ . . references ji y, eichler ee, schwartz s, nicholls rd. structure of chromosomal duplicons and their role in mediating human genomic disorders. genome res ; : – . amos-landgraf jm, ji y, gottlieb w, depinet t, wandstrat ae, cassidy sb, driscoll dj, rogan pk, schwartz s, nicholls rd. chromosome breakage in the prader-willi and angelman 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and services can be found at: these include: material supplementary .dc http://jmg.bmj.com/content/suppl/ / / /jmedgenet- - supplementary material can be found at: references #biblhttp://jmg.bmj.com/content/ / / this article cites articles, of which you can access for free at: service email alerting box at the top right corner of the online article. receive free email alerts when new articles cite this article. sign up in the collections topic articles on similar topics can be found in the following collections ( )movement disorders (other than parkinsons) notes http://group.bmj.com/group/rights-licensing/permissions to request permissions go to: http://journals.bmj.com/cgi/reprintform to order reprints go to: http://group.bmj.com/subscribe/ to subscribe to bmj go to: http://jmg.bmj.com/content/ / / http://jmg.bmj.com/content/suppl/ / / /jmedgenet- - .dc http://jmg.bmj.com/content/suppl/ / / /jmedgenet- - .dc http://jmg.bmj.com/content/ / / #bibl 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otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ the hygiene hypothesis: immunological mechanisms of airway tolerance the hygiene hypothesis: immunological mechanisms of airway tolerance eline haspeslagh , , , ines heyndrickx , , hamida hammad , and bart n lambrecht , , available online at www.sciencedirect.com sciencedirect the hygiene hypothesis was initially proposed as an explanation for the alarming rise in allergy prevalence in the last century. the immunological idea behind this hypothesis was a lack of infections associated with a western lifestyle and a consequential reduction in type immune responses. it is now understood that the development of tolerance to allergens depends on microbial colonization and immunostimulatory environmental signals during early-life or passed on by the mother. these environmental cues are sensed and integrated by barrier epithelial cells of the lungs and possibly skin, which in turn instruct dendritic cells to regulate or impede adaptive t cell responses. recent reports also implicate immunoregulatory macrophages as powerful suppressors of allergy by the microbiome. we propose that loss of adequate microbial stimulation due to a western lifestyle may result in hypersensitive barrier tissues and the observed rise in type allergic disease. addresses laboratory of immunoregulation and mucosal immunology, vib center for inflammation research, technologiepark , b- ghent (zwijnaarde), belgium department of biomedical molecular biology, ghent university, technologiepark , b- ghent (zwijnaarde), belgium department of internal medicine, ghent university, de pintelaan k , b- ghent, belgium department of pulmonary medicine, erasmusmc, ’s-gravendijkwal , ce rotterdam, the netherlands corresponding authors: hammad, hamida (hamida.hammad@ugent. be), lambrecht, bart n (bart.lambrecht@ugent.be) current opinion in immunology , : – this review comes from a themed issue on allergy and hypersensitivity edited by onur boyman, alexander eggel and mario noti https://doi.org/ . /j.coi. . . - /ã the authors. published by elsevier ltd. this is an open access article under the cc by-nc-nd license (http://creative- commons.org/licenses/by-nc-nd/ . /). introduction allergic sensitization is characterized by the presence of allergen-specific immunoglobulin e (ige) in serum. exposure to allergens via inhalation, ingestion or contact with the skin can lead to diseases such as asthma, hay current opinion in immunology , : – fever, eczema and, in some cases, to systemic anaphylaxis. during the last years, allergies have emerged in a very rapid way and their prevalence is still on the rise. nowa- days, more than % of children are allergic, up to % of children suffer from asthma and allergic rhinitis, and – % of children have developed food allergy. it is still not entirely clear why asthma prevalence is so high, but the rapid time frame of its origination and expansion suggests that environmental or behavioral changes in western lifestyle are involved. a modern lifestyle is associated with dysbiosis an important evolution of the last years is a successful decrease of infectious disease burden, due to the massive introduction of hygiene measures, antibiotics, and vac- cines. in , strachan observed that growing up in large families with more older siblings decreased the chances of developing hay fever or eczema [ ]. he postulated that the recent increase in allergy incidence was a result of ‘declining family size, improvements in household ame- nities, and higher standards of personal cleanliness’, which had reduced ‘the opportunity for cross infection in young families’. the original ‘hygiene hypothesis’ was thus introduced. since then, this hypothesis has been supported by numerous studies, especially in murine models, showing that exposure to bacteria, viruses, hel- minths or microbe-derived products could protect from allergy (reviewed in [ ], [ �� ]). however, it should be kept in mind that not all pathogens are protective; for instance, respiratory syncytial virus (rsv) or rhinovirus are associ- ated with a higher risk to develop wheeze and asthma up to adulthood [ ]. changes in lifestyle can also heavily influence the com- position and diversity of the microbiome at several muco- sal surfaces. these microbial communities have co- evolved with and within the human body for millions of years, and, consequently, the human immune system has been calibrated and fine-tuned so to maintain and shape symbiotic relationships with them (reviewed in [ ]). two theories, the ‘old friends’ and the ‘biodiversity’ hypotheses, have been proposed by rook and by haah- tela as a more accurate, or at least complementary, expla- nation for the recent allergy pandemic [ , ]. they stipu- late that the reason for the increased incidence in allergic disorders is a reduced exposure to such beneficial symbi- otic bacteria or parasites. indeed, several studies have www.sciencedirect.com mailto:hamida.hammad@ugent.be mailto:hamida.hammad@ugent.be mailto:bart.lambrecht@ugent.be https://doi.org/ . /j.coi. . . https://smart.servier.com/ https://smart.servier.com/ http://crossmark.crossref.org/dialog/?doi= . /j.coi. . . &domain=pdf http://www.sciencedirect.com/science/journal/ hygiene hypothesis haspeslagh et al. reported that alterations in the composition of the skin, the nose or the gut microbiome are associated with eczema, asthma and food allergy [ – ]. these changes do not affect a single commensal, but rather reflect a reduced total microbial diversity [ ], and they may be caused by several factors, including sibling order in the family [ ], exposure to animals [ ], and other early-life events [ ]. the importance of a healthy microbiome in controlling allergies was further substantiated in mice, with germ-free mice being especially prone to develop overt allergic (airway) disease, a phenotype reverted by microbial recolonization [ , ]. however, other studies showed that germ-free mice are not universally more susceptible to house dust mite driven asthma, and that only selected strains of lung microbiota seem to suppress asthma [ ]. during the last years, the body of correla- tive epidemiological studies has expanded vastly, and is the subject of many excellent reviews. here, we will zoom in on recent advances in the search for the under- lying immunological mechanisms explaining the observed effects. microbes induce protective regulatory dcs and t cells allergies are generally aberrant immune reactions to innocuous antigens, orchestrated by t helper (th ) cells and type innate lymphoid cells (ilc s). in the case of asthma, this type cell activity leads to mucus hyper- secretion, goblet cell hyperplasia, smooth muscle cell hyperreactivity, and the infiltration and/or activation of eosinophils, mast cells and basophils, ultimately culmi- nating in breathing difficulties and airway remodeling [ ]. dendritic cells (dcs) are always found at the body’s barriers, and because they express a wide range of pattern recognition receptors (prrs), they can sense the envi- ronment for the presence of danger signals [ ]. our group has shown that th responses to house dust mite (hdm) allergens were induced by irf -dependent cdc s in the lungs and in the skin [ , � ] (figure ). these cdc s capture the hdm allergens in the airways and migrate to the draining lymph nodes, requiring ilc - derived il- , where they present the allergens to naı̈ve t cells [ ]. it is easy to imagine that environmental changes sensed at the level of the lungs, the skin but also of the gut will modify the context of allergen recognition by dcs, and either protect against or enhance allergic responses. chronic helicobacter pylori infection has been inversely linked to asthma in humans and can effectively protect mice from ova-induced asthma [ , ]. in mice, h. pylori infection induced the accumulation of cd + cdcs in the lungs, which were required for the protec- tion, as was their il- production [ ]. in a recent study, semi-therapeutic h. pylori extract treatment also reduced airway allergy, shifted the cd b+/cd + dc ratio in the lungs, and reduced the antigen processing by lung and www.sciencedirect.com lymph node dcs [ ]. other studies demonstrated pro- tective modulation of in vitro bone-marrow derived dc cultures (bmdcs). a synthetic tlr /tlr -agonist induced lps-tolerance and il- production in bmdcs, whereas the cowshed lactococcus lactis instigated a th - polarizing program, both rendering the bmdcs unable to sensitize mice to ova-allergen upon adoptive transfer [ , � ]. trompette et al. recently found that feeding mice a fiber- rich diet changed the composition of the lung and gut microbiome, the latter metabolizing the fiber into circu- lating short-chain fatty acids (scfa’s) [ ]. the increased scfa levels protected the mice from allergic lung inflammation. mechanistically, the scfa’s altered dc precursor generation in the bone marrow, and the dcs subsequently seeding the lungs had a higher phago- cytic capacity and were impaired in polarizing th cells. additional studies have supported the protective effect of dietary fiber supplementation on allergic asthma devel- opment in mice [ ], and on wheeze in human infants when the fiber was given to the pregnant mother [ ]. one mechanism by which the dcs in microbe-exposed animals can confer protection, is by inducing the genera- tion of regulatory t cells (tregs). microbial colonization in -week old mice was shown to be necessary for the transient upregulation of pd-l on lung cd b + dcs, and the expansion of a specific pulmonary treg subset [ ]. pd-l blockade in neonates resulted in exaggerated responsiveness to hdm through adulthood, suggesting a crucial role for this microbial-induced dc–treg axis for immunological tolerance. in another mouse model of h. pylori-mediated asthma protection, the helicobacter infec- tion inhibited tlr-induced dc maturation and repro- grammed the dcs towards a foxp + treg-polarizing phenotype [ ]. the bacterial component flagellin b, given semi-therapeutically together with allergen, could also inhibit murine allergic asthma symptoms in a dc� and cd + treg-dependent manner [ � ]. although helminths are prototypical inducers of type immunity, they have been correlated with reduced allergen skin prick test reactivity, and to some degree with asthma protection (reviewed in [ ]). a general explanation for this non-intuitive association is that hel- minths induce a so-called ‘modified th response, with immunoregulatory cells such as tregs complementing the th -arm of immunity, and regulating the response to bystander antigens such as aeroallergens. therefore, sev- eral groups have tried to find helminth-derived products with immunomodulatory properties that could be used to suppress th immunity. for instance, an anti-inflamma- tory protein (- ; aip- ) from the parasitic hookworm was identified to suppress murine airway allergy in a dc- dependent and treg-dependent manner [ ]. in another study, the helminth-derived immunomodulator current opinion in immunology , : – allergy and hypersensitivity figure treg th tlr il- il- gm-csf ccl il- α th irf + cd b+ ox l notch l il- allergens (hdm) endotoxin farming environment microbial colonization rsv second hand smoke neonatal lungs h. polygyrus hes microbial colonization h. pylori l. lactis scfas flagellin b aip- th th il- r il- tlr tlr rm avcystatin herpes current opinion in immunology proposed model of airway tolerance. in the absence of immunoregulatory pathways, epithelial barrier cells readily respond to allergen binding on their pattern recognition receptors, among which tlr , by the secretion of inflammatory mediators (il- a, il- , il- , gm-csf, ccl , and others). these mediators license antigen-bearing irf + cd b+ conventional dendritic cells (cdc s) to polarize naı̈ve t cells to t helper (th ) cells in the lung-draining lymph nodes. neonatal and germ-free mice are especially prone to develop such th responses. respiratory syncytial virus (rsv) infection and second hand cigarette smoke, two known asthma risk factors, increase il- secretion and may thereby stimulate this pathway. exposure to endotoxin, farm dust or microbial colonization blunts the epithelial response by increasing the expression of negative regulators. epithelial il- release is also inhibited by helminth-derived excreted and secreted products (hes). dcs devoid of epithelial activation signals do not induce t cell activation (th ). other protective factors impede t cell activity by influencing the maturation, antigen presentation, or phagocytic capacity of dcs. some protective factors induce dcs that provoke regulatory t cell (treg) activity or t helper (th ) activity. regulatory macrophages (rm) can also induce tregs, or block dc-mediated th -polarization. abbreviations: hdm, house dust mite; tlr , toll like receptor ; il- r, il- receptor; h. pylori, helicobacter pylori; l. lactis, lactococcus lactis; scfa, short chain fatty acids; aip- , anti-inflammatory protein . avcystatin was demonstrated to induce regulatory macrophages that protected against experimental asthma upon adoptive transfer [ ]. regulatory alveolar macro- phages from bone marrow origin were recently also impli- cated in long-lasting protection conferred by a latent current opinion in immunology , : – murine gammaherpesvirus infection, a model for epstein- –barr virus infection in mice [ � ]. the regulatory macro- phages induced by the infection replaced the long-lived and self-replenishing alveolar macrophages that are gen- erated shortly after birth, and became long-lived as well. www.sciencedirect.com hygiene hypothesis haspeslagh et al. this potentially explains the long-lasting effects of microbial stimuli in the lungs on allergy suppression. allergic asthma is initiated by aberrant immune responses at barrier tissues to initiate an allergen-specific th response, cdc s need to be instructed by barrier epithelial cells (ecs) lining the airways. barrier ecs are permanently exposed to environmental insults or innocuous signals and, like dcs, they are well-equipped to integrate these signals via a range of prrs (reviewed in [ ]). activation of prrs on ecs by allergens induces nf-kb activation and ros production, resulting in the secretion of a wide range of inflammatory mediators, among which the cytokines il- , il- and tslp. dcs react to these cytokines by ox l and notch ligand upregulation, and downregula- tion of il- production, an activation state that favors th polarization in the lung-draining lymph node [ , ]. interestingly, the barrier tissue of the skin also constitutes a possible entry route for aeroallergens [ � ]. thus, barrier cells act very upstream in the inflammatory cas- cade of events leading to allergic sensitization (figure ). our group has previously reported that the prr toll-like receptor (tlr ) on airway ecs was critically necessary to mount a th -mediated asthmatic response to hdm [ ]. strikingly, several hdm allergens have the intrinsic capacity to facilitate or amplify tlr signaling by bind- ing directly to proteins of the tlr signaling complex or to its ligands [ , ]. however, tlr is best known as the receptor for lps, also termed endotoxin, a component of gram-negative bacteria. it is difficult to reconcile how a receptor specialized in bacterial sensing can contribute to th immunity and allergy, especially given the fact that high endotoxin levels in children’s mattresses are protec- tive against atopic sensitization and asthma in humans and mice [ – ]. another study also associated house dust endotoxin levels with a significantly reduced risk of allergic sensitization or eczema, specifically in children with a polymorphism in the cd gene [ ]. in fact, a body of epidemiological studies have convincingly corre- lated a traditional farming environment, where endotoxin levels are high, with protection against hay fever, allergic sensitization, and asthma (reviewed in [ ]). in a hallmark study, children growing up on agricultural hutterite and amish farms in the us were compared, and the latter were found to have six times less chance of developing atopy and asthma [ �� ]. these two farming populations share a similar genetic ancestry and lifestyle. farming practices, however, differ, and the amish house dust contained almost times more endotoxin than the house dust from hutterite farms. only the transfer of the amish dust intranasally to mice inhibited subsequent experi- mental asthma development. we have recently confirmed that farm dust collected from bavarian farms (in which farming practices resemble the amish’s ones), and lps, conferred protection against experimental asthma. this www.sciencedirect.com protection was mediated by an increased epithelial expression of tnfaip (better known as a ), a nega- tive regulator of the nf-kb pathway, which blunted the epithelial cell response to hdm and downstream dc activity [ ]. a similar tolerance to lps mediated by a induction was demonstrated in intestinal ecs [ ]. inter- estingly, a expression was very low in neonatal rats, spontaneously increased shortly after birth coinciding with microbial colonization, and could be downregulated by treatment with antibiotics. it remains to be investi- gated if the gut or lung microbiota can similarly influence expression of a and other negative regulators in airway ecs. ec modulation has recently been demonstrated for heligmosomoides polygyrus, a helminth often confirmed to protect against murine allergy [ ]. secreted and excreted products (hes) of this parasite inhibited il- release by ecs and thereby suppressed alternaria-induced airway allergy [ �� ]). mechanistically, the h. polygyrus alarmin release inhibitor (hpari), a kda protein, binds to activated il- and at the same time tethers il- to the dna of necrotic cells, thus inhibiting il- action in a dual manner and inhibiting innate eosinophilic airway inflammation [ �� ]. other molecules secreted by the parasite are more related to tgfb and can induce a foxp + treg population with immunoregulatory poten- tial [ ]. a dysregulated immune response at the skin can cause atopic dermatitis (ad), which in itself is a risk factor to accumulate more allergies later in life, among which asthma, a process known as ‘the atopic march’. in fact, ad and asthma share several risk factors. ad is strongly correlated with changes in the skin microbiome, the most well-known being pertinent staphylococcus aureus coloni- zation of allergic skin. in a recent study, il- ra�/� mice spontaneously developed ad with naturally occur- ring skin dysbiosis and a compromised skin barrier, and antibiotic treatment ameliorated skin inflammation [ ]. a similar dysbiosis–ad axis has also been demonstrated in adam �/� mice [ ]. topical treatment with non- pathogenic bacteria, on the other hand, can alleviate cutaneous inflammation in murine ad [ ]. it has become clear in recent years that tonic sensing of skin commensals heavily shapes host dc and t cell functions [ – ]. it remains to be investigated what the relative importance is of passive barrier integrity and active sig- naling through keratinocyte prrs, also poised to rapidly respond to innate immune ligands, in this microbe- immune cell cross-talk [ ]. it will also be of great interest to study how environmental exposures influence the skin microbiome and/or the immune threshold of skin epithelium. one intriguing observation is that allergies tend to develop early in life. in the same time window, and even in utero, protective effects of environmental factors and the microbiome are also the strongest [ , ]. we current opinion in immunology , : – allergy and hypersensitivity recently demonstrated that the lung environment in neonatal mice is strongly type -skewed, with a gradual increase in il- release by lung ecs, and with the spontaneous recruitment of several th -associated innate immune cells, peaking weeks after birth [ �� ]. this spontaneous wave of early type immunity is likely to be caused by the mechanical stress induced by the breathing patterns [ ], but also by the constant remodeling necessary to build up new lung structures. interestingly, this period is prone to favor stronger th sensitization to inhaled allergens [ �� ], but also to favor lower immunity to bacteria [ ]. many environmental factors, like second hand smoking or rsv infection are known to facilitate th sensitization in children. these triggers have in common that they induce high levels of il- [ , ]. it is tempting to speculate that these risk factors act by prolonging or amplifying the epithelial cytokine response to allergens during early-life, and that combined early-life exposures thus define the final threshold for ec activation. conclusion effects of microbes on inducing treg cells, th cells and allergen cross-reactive antibody responses, are well- observed. in addition, we propose a model in which environmental and microbial stimuli are sensed and inte- grated by barrier tissues of the lung, the skin and the gut, resulting in a tonic dc activation status promoting either inflammatory or tolerogenic immunity. together with direct effects on dcs and t cells, most protective stimuli thus seem to converge in the same central tolerogenic immune pathways. fully understanding the fundamental immunological pathways underlying these protective triggers, their relative contribution, and how they interact, should hopefully allow us to pinpoint, modify or newly develop prophylactic or therapeutic therapies to cure asthma. conflict of interest statement nothing declared. acknowledgements research in the lambrecht and hammad lab is supported by an advanced european research council (erc) grant, an fwo flanders excellence of science grant, and the world without asthma (awwa) program of the dutch lung foundation. figure includes derivative material from servier medical art (https://smart.servier.com/) by servier, available under a creative commons attribution . unported license (https:// creativecommons.org/licenses/by/ . /). references and recommended reading papers of particular interest, published within the period of review, have been highlighted as: � of special interest �� of outstanding interest . strachan dp: hay fever, hygiene, and household size. bmj , : - . current opinion in immunology , : – . smits hh, hiemstra ps, prazeres da costa c, ege m, edwards m, garn h et al.: microbes and asthma: opportunities for intervention. j allergy clin immunol , : - . . �� mcsorley hj, blair nf, smith ka, mckenzie an, maizels rm: blockade of il- release and suppression of type innate lymphoid cell responses by helminth secreted 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with dysbiosis 	microbes induce protective regulatory dcs and t cells 	allergic asthma is initiated by aberrant immune responses at barrier tissues 	conclusion 	conflict of interest statement 	references and recommended reading 	acknowledgements [pdf] left atrial appendage remodeling after lariat left atrial appendage ligation | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /circep. . corpus id: left atrial appendage remodeling after lariat left atrial appendage ligation @article{kreidieh leftaa, title={left atrial appendage remodeling after lariat left atrial appendage ligation}, author={bahij kreidieh and f. rojas and p. schurmann and a. dave and a. kashani and m. rodr{\'i}guez-ma{\~n}ero and m. valderr{\'a}bano}, journal={circulation: arrhythmia and electrophysiology}, year={ }, volume={ }, pages={ – } } 	bahij kreidieh, f. rojas, + authors m. valderrábano 	published 	medicine 	circulation: arrhythmia and electrophysiology background—left atrial appendage (laa) ligation with the lariat device is being used for stroke prevention in atrial fibrillation. residual leaks into the laa are commonly reported after the procedure. little is known about the anatomic laa remodeling after lariat ligation. methods and results—in an exploratory study, we evaluated laa -dimensional geometry via computed tomographic scan in consecutive patients before lariat closure and after a minimum of days post procedure. thirteen… expand view on wolters kluwer ahajournals.org save to library create alert cite launch research feed share this paper citations view all figures, tables, and topics from this paper 	 figure 	 table 	 figure 	 table 	 figure 	 figure 	 figure 	 figure view all figures & tables 	hemorrhage 	left atrial appendage 	ligation 	heart atrium 	hypertensive disease 	cerebrovascular accident 	ethanol 	diabetes mellitus 	thromboembolism 	vascular diseases 	atrial fibrillation 	heart failure 	blood clot 	classification 	anatomical orifice 	liver diseases 	sex factors 	bleeding tendency 	body cavities 	small citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency incomplete closure of the left atrial appendage: implication and management 	a. aryana, a. d'avila 	medicine 	current cardiology reports 	 	 save alert research feed anatomy and physiologic roles of the left atrial appendage: implications for endocardial and epicardial device closure. 	nicholas y tan, omar z yasin, a. sugrue, a. el sabbagh, t. foley, s. asirvatham 	medicine 	interventional cardiology clinics 	 	 save alert research feed role of the left atrial appendage in systemic homeostasis, arrhythmogenesis, and beyond. 	ghulam murtaza, bharath yarlagadda, + authors d. lakkireddy 	medicine 	cardiac electrophysiology clinics 	 	 save alert research feed why follow-up examinations after left atrial appendage closure are important: detection of complications during follow-up and how to deal with them 	n. wunderlich, gabriele c. lorch, jörg honold, j. franke, harald küx 	medicine 	current cardiology reports 	 save alert research feed percutaneous left atrial appendage closure: current devices and clinical outcomes 	lluis asmarats, j. rodés-cabau 	medicine 	circulation. cardiovascular interventions 	 	 	 save alert research feed clinical significance of leaks following left atrial appendage ligation with the lariat suture delivery device. 	r. lee 	medicine 	jacc. cardiovascular interventions 	 	 	 save alert research feed sex differences in atrial fibrillation—update on risk assessment, treatment, and long-term risk 	c. bai, n. madan, shaza a. alshahrani, n. aggarwal, a. volgman 	medicine 	current treatment options in cardiovascular medicine 	 	 save alert research feed anatomical and electrical remodeling with incomplete left atrial appendage ligation: results from the laala‐af registry 	m. turagam, d. atkins, + authors d. lakkireddy 	medicine 	journal of cardiovascular electrophysiology 	 	 save alert research feed epicardial versus endocardial closure: is one better than the other? 	krishna akella, bharath yarlagadda, + authors d. lakkireddy 	medicine 	cardiac electrophysiology clinics 	 	 save alert research feed correlation between laa morphological features and computational fluid dynamics analysis for non-valvular atrial fibrillation patients 	b. m. fanni, k. capellini, + authors s. celi 	medicine 	 	 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency anatomic analysis of the left atrial appendage after closure with the lariat device. 	k. bartuś, remo morelli, w. szczepański, b. kapelak, j. sadowski, r. lee 	medicine 	circulation. arrhythmia and electrophysiology 	 	 	 save alert research feed left atrial thrombus after appendage ligation with lariat. 	matthew s baker, j. paul mounsey, a. gehi, e. chung 	medicine 	heart rhythm 	 	 save alert research feed late-occurring left atrial appendage thrombus after ligation using lariat 	alexander g. truesdell, chinmay patel, b. maini 	medicine 	journal of interventional cardiac electrophysiology 	 	 	pdf save alert research feed left atrial thrombus formation after successful left atrial appendage ligation: case series from a nationwide survey. 	d. lakkireddy, a. vallakati, + authors y. m. reddy 	medicine 	journal of the american college of cardiology 	 	 	 save alert research feed percutaneous left atrial appendage suture ligation using the lariat device in patients with atrial fibrillation: initial clinical experience. 	k. bartuś, frederick t. han, + authors r. lee 	medicine 	journal of the american college of cardiology 	 	 	pdf save alert research feed does the left atrial appendage morphology correlate with the risk of stroke in patients with atrial fibrillation? results from a multicenter study. 	l. di biase, p. santangeli, + authors f. gaita 	medicine, sociology 	journal of the american college of cardiology 	 	 	pdf save alert research feed left atrial thrombus after appendage closure using lariat. 	evaldas giedrimas, a. lin, b. knight 	medicine 	circulation. arrhythmia and electrophysiology 	 	 	 save alert research feed early safety and efficacy of percutaneous left atrial appendage suture ligation: results from the u.s. transcatheter laa ligation consortium. 	matthew j. price, douglas n. gibson, + authors m. valderrábano 	medicine 	journal of the american college of cardiology 	 	 	pdf save alert research feed left atrial thrombus after complete left atrial appendage exclusion with lariat device 	k. koranne, r. fernando, s. laing 	medicine 	catheterization and cardiovascular interventions : official journal of the society for cardiac 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homozygous deletion in kvlqt associated with jervell and lange-nielsen syndrome homozygous deletion in kvlqt associated with jervell and lange-nielsen syndrome qiuyun chen, phd; danmei zhang, md; robert l. gingell, md; arthur j. moss, md; carlo napolitano, md; silvia g. priori, md, phd; peter j. schwartz, md; eileen kehoe, bs; jennifer l. robinson, ms; eric schulze-bahr, md; qing wang, phd; jeffrey a. towbin, md background—long-qt (lqt) syndrome is a cardiac disorder that causes syncope, seizures, and sudden death from ventricular arrhythmias, specifically torsade de pointes. both autosomal dominant lqt (romano-ward syndrome) and autosomal recessive lqt (jervell and lange-nielsen syndrome, jlns) have been reported. heterozygous mutations in potassium channel genes, kvlqt , kcne (mink), and herg, and the cardiac sodium channel gene scn a cause autosomal dominant lqt. autosomal recessive lqt, which is associated with deafness, has been found to occur with homozygous mutations in kvlqt and kcne in jlns families in which qtc prolongation was inherited as a dominant trait. methods and results—an amish family with clinical evidence of jlns was analyzed for mutations by use of single-strand conformation polymorphism and dna sequencing analyses for mutations in all known lqt genes. a novel homozygous -bp deletion in the s transmembrane segment of kvlqt was identified in affected members of this amish family in which both qtc prolongation and deafness were inherited as recessive traits. this deletion represents a new jlns-associated mutation in kvlqt and has deleterious effects on the kvlqt potassium channel, causing a frameshift and the truncation of the kvlqt protein. in contrast to previous reports in which lqt was inherited as a clear dominant trait, parents in the jlns family described here have normal qtc intervals ( . and . seconds, respectively). conclusions—a novel homozygous kvlqt mutation causes jlns in an amish family with deafness that is inherited as an autosomal recessive trait. (circulation. ; : - .) key words: long-qt syndrome n deafness n jervell and lange-nielsen syndrome n potassium channel the inherited long-qt syndrome (lqt) is characterizedby prolonged qt interval on ecgs, syncope (sudden loss of consciousness), seizures, and sudden death from ventricular arrhythmias, specifically torsade de pointes. , both autosomal dominant (romano-ward syndrome, rws) , and autosomal recessive (jervell and lange-nielsen syndrome, jlns) forms of lqt have been reported. four genes for rws have been mapped to chromosomes p . (lqt ), – q – (lqt ), p – (lqt ), and q – (lqt ), and lqt genes (lqt , lqt , and lqt ) were subsequently identified, – including the potas- sium channel genes kvlqt (lqt ) and herg (lqt ) and the cardiac sodium channel gene scn a (lqt ). recently, mutations in kcne (mink) were also found to cause rws. jlns is rare and is associated with congenital sensorineu- ral deafness. it was first described in by jervell and lange-nielsen in a norwegian family in which of the children were affected by both lqt and congenital sensori- neural deafness but the parents appeared normal. three of the affected children died suddenly at the ages of , , and years. since then, several other cases of autosomal recessive lqt have been reported. – in some cases, the heterozygous parents had moderate qtc prolongation despite the fact that the syndrome was inherited as a recessive trait. in , neyroud et al and splawski et al reported the identifica- tion of homozygous mutations of kvlqt in jlns. in both studies, lqt appeared to be inherited in an autosomal dominant fashion (ie, at least of the parents was affected by lqt), and deafness was inherited in an autosomal recessive received february , ; revision received october , ; accepted december , . from the departmentx of pediatrics (cardiology) (q.c., d.z., q.w., j.a.t.) and molecular and human genetics (j.a.t.), baylor college of medicine, texas children’s hospital, houston, tex; children’s hospital at buffalo, buffalo, ny (r.l.g.); department of medicine (a.j.m., e.k., j.l.r.,) and department of community and preventive medicine (j.l.r.), university of rochester medical center, rochester, ny; department of cardiology, university of pavia, and policlinico s. matteo, ifccs, pavia, italy (c.n., s.g.p., p.j.s.); and hospital of the university of munster, munster, germany (e.s.-b.). guest editor for this article was harry dietz, md, johns hopkins university, baltimore, md. correspondence to jeffrey a. towbin, md, department of pediatrics (cardiology), baylor college of medicine, one baylor plaza, room e, houston, tx . e-mail jtowbin@bcm.tmc.edu © american heart association, inc. circulation is available at http://www.circulationaha.org d ow nloaded from http://ahajournals.org by on a pril , fashion. recently, mutations in kcne (mink) also have been found to cause jlns, establishing mink as a new gene for jlns. in this article, we describe a homozygous deletion of bp within the second transmembrane domain of kvlqt (s ) in a family with individuals affected with lqt and deafness. this deletion causes a frameshift and premature termination and leads to a nonfunctional kvlqt potassium channel. methods jlns patient evaluation the jlns family described here was identified in an amish community and referred for molecular genetic studies. only the nuclear family allowed evaluation. consanguinity was denied by the parents. informed consent was obtained from all participants or their guardians in accordance with standards established by local institu- tional review boards. phenotypic characterization was performed as described previously, with the bazett qt correction for heart rate (ie, qtc). single-strand conformation polymorphism and dna sequence analysis genomic dna was prepared from peripheral blood lymphocytes or lymphoblastoid cell lines derived from epstein-barr virus–trans- formed lymphocytes from the jlns family and from control individuals. polymerase chain reaction (pcr) for single-strand conformation polymorphism (sscp) analysis was performed as previously described. normal and abnormal sscp conformers were cut directly from dried gels, eluted in ml of distilled water at °c, and reamplified. pcr products were fractionated in . % fmc nusieve low-melting-temperature agarose gel and purified. purified pcr products ( fmol) were sequenced directly by cycle sequencing with the cyclisttm exo-pfu dna sequencing kit (stratagene). for each sequencing reaction, mci of a- s-datp and . u of the exo-pfu enzyme were used. pcrs were carried out in a perkin- elmer system- thermocycler with the following profiles: cycle at °c for minutes, cycles of °c for seconds, °c for seconds, and °c for minute. restriction fragment length polymorphism analysis genomic dna was pcr amplified to yield a -bp fragment. one of the primers was end labeled with [g- p]atp with t polynucle- otide kinase (neb) under standard conditions and included in a -ml pcr reaction as described for sscp. at the end of the pcr reaction, ml of the reaction mixture was digested in a -ml reaction under standard conditions with either bgli (which is unique to the wild-type pcr fragment) or mspi (which is unique to the mutant pcr). after incubation at °c for . hours, ml of formamide buffer was added. the mixture was heated at °c for to minutes and cooled immediately on ice, and ml was loaded onto % urea-denaturing polyacrylamide gels (acrylamide:bisacryl- amide : ). the gels were run in tris borate edta buffer at w, dried on filter paper (schleicher and schuell), and exposed to x-ray film (kodak). results phenotypic characterization phenotypic analysis of this amish family revealed deafness in both children but normal hearing in the parents (as defined by clinical evaluation only), indicating that the deafness in this family is inherited as an autosomal recessive trait. ecg analysis of this family revealed that the deaf male and female children are both affected by lqt, having qtc intervals of . and . second, respectively (figure ). no ventricular tachycardia was documented in these children. the father and mother, however, have borderline qtc intervals of . and . second, respectively. no provocative testing on the parents (ie, electrophysiology study with pharmacological provocation, stress testing) was performed, however. there- fore, with respect to the clinical expression of qtc prolon- gation in this family, there is autosomal recessive inheritance with subclinical manifestations in the heterozygous parents. with respect to the ecg findings, however, the trait is inherited as an incomplete dominant. these studies are consistent with the diagnosis of jlns in this family as well. mutational analysis screening for mutations in kvlqt with sscp and dna sequencing analysis identified an abnormal sscp conformer in the male patient of this amish family (proband, figure a) but not from . control individuals. sequence analysis of the abnormal sscp conformer revealed a -bp deletion in the s transmembrane domain of kvlqt (figure b). this mutation results in a frameshift and premature termination of kvlqt . cosegregation of a homozygous deletion in kvlqt with jlns to determine whether the -bp deletion was homozygous in the affected individuals, the dna segment that was amplified from genomic dna of each affected person was sequenced. the sequencing patterns, including the deletion of the nucleotides aa from both affected individuals, were identical to that of the sscp abnormal band (data not shown), indicating that both affected individuals are homozygous for the -bp deletion. the sequencing patterns of both parents are identical to that of the sscp abnormal band flanking the -bp deletion but are a mixture of overlapping sequencing panels within the -bp deletion region (data not shown). these data suggest that the parents are heterozygous for the -bp deletion figure . pedigree structure of amish family affected by jlns and associated ecgs of each individual. affected female indi- viduals are indicated by filled circles; male individuals, by filled squares; and individuals with borderline qtc interval, by half- filled circles and half-filled squares. qtc values are given in sec- onds under pedigree symbols. note that parents (individuals a and b) have normal qtc intervals, whereas offspring (individuals c and d) have definitively abnormal qtc intervals, consistent with autosomal recessive inheritance of lqt (and deafness). chen et al march , d ow nloaded from http://ahajournals.org by on a pril , and that homozygous deletion of the bp in kvlqt cosegregates with jlns. to further confirm that the affected individuals are ho- mozygous and that the parents are heterozygous for the -bp deletion, restriction fragment length polymorphism analysis was performed. in the analysis, genomic dna of all family members was used to produce the end-labeled dna frag- ments by pcr (see methods). the dna fragments were then digested with an allele-specific restriction digestion enzyme, bgli or mspi. bgli cuts only the wild-type allele, whereas mspi cuts only the mutant allele. as shown in figure c, dna fragments from both affected individuals were com- pletely cut by mspi but remained intact after exposure to bgli, confirming that affected individuals in the family are ho- mozygous for the -bp deletion. in contrast, dna fragments from both parents are partially cut by either bgli or mspi, further suggesting that they are heterozygous for the -bp deletion. discussion in this study, we identified a new homozygous deletion in kvlqt cosegregating with the patients of an amish family affected by both lqt and deafness. the deletion is located in the s transmembrane domain of kvlqt , resulting in a frameshift of the predicted amino acid sequence and prema- ture stop of kvlqt . because the mutation leads to a frameshift of kvlqt starting from the middle of domain s , it is expected to produce a nonfunctional potassium channel because most transmembrane domains and the pore region of kvlqt are deleted. the -bp deletion may also result in instability of the mutant rna or lead to a truncated (ie, shortened) protein that is unable to be incorporated into the membrane to form a functional channel. together with the recent identification of other kvlqt mutations in patients with jlns, , these data further confirm that homozygous mutations in kvlqt result in jlns. the s transmembrane domain of kvlqt has been reported to contain mutations by previous authors. splawski et al identified a homozygous insertion mutation in s that caused a frameshift, disrupting the coding sequence of kvlqt after s and leading to a premature stop codon and a truncated protein lacking the pore region in a family with jlns. others, such as chouabe et al and tanaka et al, reported missense mutations in s , with the clinical pheno- type resulting in a variable phenotype, ranging from mild to moderate to severe rws. in addition, donger et al identi- fied s mutations with widely varying clinical findings in families with rws, including several gene carriers with borderline qtc. hence, the clinical phenotype seen with mutations in the s transmembrane domain is heterogeneous but, in most cases, appears to be mild. chouabe et al performed biophysical analysis of a variety of kvlqt mutants, including mutations in s , and found that no matter where the mutation occurred, the general rule is that the only discernable effect is a reduction in current density, corre- sponding to a dominant-negative suppression of kvlqt function. jlns mutations studied also produced a dominant- negative effect, but the extent of the inhibition was lower than in rws patients. hence, jlns mutations produce no func- tional channels and have little effect on expression of wild- type subunits with the relatively normal findings in heterozy- gotes. therefore, depending on the severity of the dominant- negative effect of the different mutations, the disease is either dominant or recessive. in the latter case, the reduction in the current normally carried by the kvlqt subunit is so high that the defect becomes apparent in other tissues expressing this protein, the inner ear being most evident. tyson et al identified a family with normal hearing and normal qtc in the parents ( and ms) of a child with jlns, whereas in other families, the same mutation caused qt prolongation ( ms) in parent. it is likely that modification of the clinical phenotype occurs because of other genetic influences (ie, modifier genes) and environmen- tal influences and that, in some cases, qtc depends on these influences. as noted previously by vincent et al, qtc values in kvlqt mutation carriers may range from normal ( to ms) to severely abnormal, and this may vary even within families. in contrast to the previous reports by neyroud et al and splawski et al in which at least parent had clearly prolonged qtc ( . and . second, respectively), both parents in this study have normal or borderline qtc intervals of . to . second. thus, the clinical expression of lqt in this family is inherited as an autosomal recessive trait figure . identification of -bp deletion in kvlqt in proband of amish family. a, sscp analysis showing abnormal conformer in proband, lane m, as indicated by arrows. b, dna sequence analyses of normal (wild-type) and abnormal (sscp) conformers revealed a -bp (ct) homozygous deletion in proband. deletion occurs in s transmembrane segment of kvlqt , leading to frameshift and premature termination of kvlqt .c, restriction fragment length polymorphism analysis showing homozygous -bp deletion in kvlqt cosegregating with jlns in amish family. the -bp deletion in s transmembrane segment of kvlqt leads to change in susceptibility of dna to mspi (right) instead of bgli (left). pedigree of amish family is shown above each digestion panel. empty square indicates unrelated normal individual. s indicates substrate from pcr used for restriction digestion; p, products generated after restriction digestion. kvlqt deletion causes autosomal recessive lqt d ow nloaded from http://ahajournals.org by on a pril , with subclinical manifestations in heterozygotes (ie, the parents), which is closely related to the original descriptions of jlns by jervell and lange-nielsen, levine and woodworth, and fraser et al. , with respect to the ecg findings, however, the trait is inherited as an incomplete dominant trait, whereas deafness appears to be inherited as an autosomal recessive trait. this finding further extends our understanding of the clinical, genetic, and molecular genetic aspects of lqt. acknowledgments this work was supported by a scientist development award from the american heart association (dr wang); the caroline weiss law grant for research in molecular medicine (dr wang); the aber- crombie cardiology fund of texas children’s hospital (dr wang); deutsche forschungsgemeinshaft (dr schulze-bahr); funds of the european union, biomed z program (dr priori, schwartz, and schulze-bahr); the texas children’s hospital foundation endow- ment for pediatric cardiac research (dr towbin); and nih grants r hl- and r hl- (drs moss, towbin, and schwartz). we acknowledge the secretarial and administrative sup- port of valerie r. price and amy a. gentry. references . schwartz pj, periti m, malliani a. the long qt syndrome. am heart j. ; : – . . moss aj, schwartz pj, crampton rs, tzivoni d, locati eh, maccluer j, hall wj, weitkamp l, vincent gm, 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genetic heteroge- neity in romano-ward long qt syndrome: analysis of families. circulation. ; : – . . jiang c, atkinson d, towbin ja, splawski i, lehmann mh, li h, timothy k, taggart rt, schwartz pj, vincent gm, moss aj, keating mt. two long qt syndrome loci map to chromosome and with evidence for further heterogeneity. nat genet. ; : – . . schott j, charpentier f, peltier s, foley p, drouin e, bonheur j, donnelly p, vergnaud g, bachner l, moisan j, marec hl, pascal o. mapping of a gene for long qt syndrome to chromosome q – . am j hum genet. ; : – . . wang q, curran me, splawski i, burn tc, millholland jm, vanraay tj, shen j, timothy kw, vincent gm, de jager t, schwartz pj, towbin ja, moss aj, atkinson dl, landes gm, connors td, keating mt. posi- tional cloning of a novel potassium channel gene: kvlqt mutations cause cardiac arrhythmias. nat genet. ; : – . . curran me, splawski i, timothy kw, green ed, keating mt. a molecular basis for cardiac arrhythmia: herg mutations cause long qt syndrome. cell. ; : – . . wang q, shen j, splawski i, atkinson d, li z, robinson jl, moss aj, towbin ja, keating mt. scn a mutations associated with an inherited cardiac arrhythmia, long qt syndrome. cell. ; : – . . splawski i, tristani-firouzi m, lehmann mh, sanguinetti mc, keating mt. mutations in the hmink gene cause long qt syndrome and suppress iks function. nat genet. ; : – . . levine sa, woodworth cr. congenital deaf-mutism, prolonged q-t interval, syncopal attacks and sudden death. n engl j med. ; : – . . fraser gr, froggatt p, james tn. congenital deafness associated with electrocardiographic abnormalities, fainting attacks and sudden death. q j med. ; : – . . fraser gr, froggatt p, murphy t. genetical aspects of the cardioauditory syndrome of jervell and lange-nielsen (congenital deafness and electro- cardiographic abnormalities). ann hum genet. ; : – . . neyroud n, tesson f, denjoy i, leibovici m, donger c, barhanin j, faure s, gary f, coumel p, petit c, schwartz k, 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article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ on the supposed moral harm of selecting for deafness on the supposed moral harm of selecting for deafnessbioe_ .. melissa seymour fahmy keywords genetic selection, procreation, ethics, deafness, preimplantation genetic diagnosis abstract this paper demonstrates that accounting for the moral harm of selecting for deafness is not as simple or obvious as the widespread negative response from the hearing community would suggest. the central questions addressed by the paper are whether our moral disquiet with regard to selecting for deafness can be adequately defended, and if so, what this might entail. the paper considers several different strategies for accounting for the supposed moral harm of selecting for deafness and concludes that the deaf case cannot be treated in isolation. accounting for the moral harm of selecting for deafness necessarily entails moral implications for other cases of procreation and procreative decision-making, including unassisted coital reproduction. the lesson to be learned from the deaf case is that we need norms that govern not just the use of reproductive technology, but procreation and procreative decision-making in all of its various forms. we could be said to live in an age of unprecedented reproductive opportunity. the success of the human genome project combined with reproductive technolo- gies like in vitro fertilization (ivf) and preimplantation genetic diagnosis (pgd) make possible procreative choices unimaginable in the past. these developments in science and technology have introduced the possibility for new kinds of reproductive decision-making. potential reproducers can select in favor of a desirable genetic trait. for instance, parents with three sons might select in favor of a daughter. the same technologies can be utilized to select out or against undesirable genes, such as those responsible for diseases like tay-sachs or cystic fibrosis. this paper will closely consider the ethics of one contro- versial case of procreative selection: selecting for genetic deafness. deaf couples who regard deafness as a distinctive culture rather than a disability may have a strong desire to parent deaf children. if the desire is strong enough, these couples may seek to utilize genetic counseling ser- vices and reproductive technologies to help them inten- tionally reproduce a deaf child rather than leave the matter to reproductive chance. the response from the hearing community to this unusual procreative prefer- ence has been largely one of disbelief, moral disquiet, and in many cases outrage and condemnation. why would anyone want to ensure that her child is deaf ? the deaf case challenges the limits of procreative liberty and the ethic of nondirective genetic counseling. in a time when we are just beginning to consider the prospect of selecting our children on the basis of genetic features, and not without some considerable degree of reservation, the desire to select for a deaf child is likely to be altogether alien to most of us. however, if we are to condemn the decision to reproduce intentionally a deaf child, if we are to suggest that such a selection lies outside the boundaries of the reasonable exercise of procreative liberty, and if we are to refuse genetic counseling and other services to deaf couples with such intentions, then we must account for the moral harm of deliberately reproducing a deaf child. address for correspondence: melissa seymour fahmy, university of georgia – philosophy dept., peabody hall, athens, georgia , united states. email: meseymou@uga.edu bioethics issn - (print); - (online) doi: . /j. - . . .x volume number pp – © blackwell publishing ltd., garsington road, oxford ox dq, uk and main street, malden, ma , usa. todd highlight the central questions this paper will address are whether our moral disquiet with regard to selecting for deafness can be adequately defended, and if so, what this might entail. i will proceed by evaluating several different ways of accounting for the supposed moral wrongness of selecting for deafness. my aim in this paper is neither to defend the rights of deaf reproducers nor to vindicate the moral disquiet of the hearing community, but rather to utilize this controversial case as an opportunity to glean some general insights into the ethics of procreation. what we shall discover, upon closer examination, is that we cannot treat the deaf case as an isolated case. accounting for the moral harm of selecting for deafness necessarily entails moral implications for other cases of procreation and procreative decision-making, including unassisted coital reproduction. . harm to the child the first thing to note is that the ordinary account of harm will not be useful in the deaf case. that is to say, if deliberately selecting for deafness constitutes a moral harm, it cannot be accounted for in terms of some harm done to the particular child that comes into being as a result of the selection. as many authors have acknowl- edged, the child in question is not injured or made worse off by the selection or resulting birth, given that the only alternative for the child would be not to exist at all. this is an important difference between genetic selection and genetic manipulation. in cases of selection, the resulting child has not been made deaf, or designed to be deaf; rather, a particular gamete or embryo has been chosen in virtue of its possessing the genetic marker for deafness. as julian savulescu puts it, ‘the deaf child is harmed by being selected to exist only if his or her life is so bad it is not worth living.’ cases where a life is so bad that it is not worth living are quite rare, and the life of a deaf person is not one of these cases. . the child’s right to an open future dena davis has proposed an alternative analysis of the supposed moral harm of selecting for deafness. according to davis, ‘deliberately creating a deaf child counts as a moral harm, because it so dramatically curtails the child’s right to an open future.’ a child’s right to an open future is the term coined by joel feinberg to refer to the collection of ‘anticipatory autonomy rights’ or ‘rights-in-trust’ pos- sessed by children. a right-in-trust is a right which cannot be exercised by a child, but is saved for the child until he or she is an adult. though the right cannot be exercised as such, it nonetheless can be violated in advance by an adult. ‘the violating conduct,’ feinberg explains, ‘guarantees now that when the child is an autonomous adult, certain key options will already be closed to him.’ for example, while a -year-old possesses neither the capacity nor the right to procreate, his or her ability to exercise this autonomy right in the future can be seriously compro- mised before adulthood is reached. an adult who has been sterilized in childhood will not be able to exercise (or elect not to exercise) her right to procreate. sterilization thus constitutes a violation a child’s right to an open future. davis maintains that selecting for deafness similarly violate a child’s right to an open future. according to davis, the decision to select for deafness violates a child’s right to an open future insofar as it ‘confines her forever to a narrow group of people and a limited choice of careers.’ i think we must take issue with the notion that a decision to select for deafness constitutes a violation of the child’s right to an open future. that this charge is this simple but provocative fact has been acknowledged and explored by many authors. see d. parfit. . reasons and person. oxford: oxford university press; b. steinbock & r. mcclamrock. when is birth unfair to the child? hastings cent rep ; : – ; d. brock. the non-identity problem and genetic harms. bioethics ; : – ; and d. davis. . genetic dilemmas: reproductive technology, parental choices, and children’s futures. new york: routledge. j. savulescu. deaf lesbians, designer disability, and the future of medicine. br med j ; : . d. davis. . genetic dilemmas: reproductive technology, parental choices, and children’s futures. new york: routledge: . j. feinberg. . the child’s right to an open future. in whose child? children’s rights, parental authority, and state power. w. aiken & h. lafollette, eds. totowa, n.j.: littlefield, adams, & co.: . i am borrowing this example from dena davis. see davis, genetic dilemmas, . d. davis. . genetic dilemmas: reproductive technology, parental choices, and children’s futures. new york: routledge: (emphasis mine). see also d. davis. genetic dilemmas and the child’s right to an open future. hastings cent rep ; : – . davis’s assessment invites two interesting and important questions which i do not intend to pursue in this paper. the first concerns the extent to which a deaf child will experience fewer future options than a hearing child. ascertaining the kinds of personal and professional options that will be available to an adult who was born deaf will depend on a myriad of factors including educational opportunities, technological developments, and social con- ditions, and thus requires empirical analysis well beyond the scope of this paper. the second question concerns how restrictive an action or decision need be before it can be considered a violation of a child’s right to an open a future. my critique of davis’s charge does not depend on suitably answering either of these questions. on the supposed moral harm of selecting for deafness © blackwell publishing ltd. todd highlight todd highlight untenable is perhaps best demonstrated by comparing the case of selecting for deafness with the decision made by amish parents to end the formal education of their chil- dren after the eighth grade, a decision which davis takes to be an example of a failure to respect the child’s right to an open future. in the s amish parents in wisconsin sought exemp- tion from a state law mandating school attendance for all children under the age of . the amish maintained that exemption was necessary for the free exercise of their religious beliefs which are deeply intertwined with a par- ticular way of life. education beyond the eighth grade is unnecessary to prepare an amish child for a life in the amish community and is likely to be antithetical to this end. ending a child’s formal education after the eighth grade, however, woefully under-prepares a child for life outside of the amish community and thus invites the charge that the child’s right to an open future has been violated. what i wish to draw attention to is the direct causal connection between the parental decision to end a child’s formal education and the narrowing of the child’s future options, thus rendering some options ‘practically impos- sible.’ had the amish children remained in school, more options (educational, professional, social, economic, etc.) would have been available to them upon reaching adult- hood and assuming their autonomy rights. because a wider future was possible for these children prior to the parental decision, the decision can be said to render their future less open than it would have otherwise been. the same cannot be said with regard to selecting for deafness. the decision that purportedly curtails the child’s right to an open future is simultaneously the deci- sion which allows the child to exist and to have any future whatsoever. in the absence of gene therapy, there is no possible future in which the child in question could exist without also being deaf. the decision to select for deaf- ness thus, unlike the amish case and the sterilization case, cannot be said to deprive the resulting child of opportu- nities he or she would have otherwise been able to pursue. contrary to davis’s analysis, the parents who select for deafness do not constrain the ability of their children to make a wide variety of choices; rather, they elect to give birth to a child with impaired abilities that may entail more limited future options. the difference is subtle but significant. because the decision to select for deafness does not impose constraints on the child’s ability to exer- cise autonomy rights in the future, the decision cannot be said to violate that child’s right to an open future. the analysis of the supposed moral harm of selecting for deafness which appeals to a child’s right to an open future encounters the same difficulties as the harm analy- sis discussed above. this is not surprising if we under- stand a rights violation to be a kind of harm. while the right to an open future cannot ground a moral objection to the decision to select in favor of deaf- ness, it does appear to be applicable to the parental decision not to fit a deaf child with a cochlear implant. respecting a child’s rights-in-trust requires significantly more than not interfering with the child’s normal devel- opment. respecting a child’s right to an open future also entails substantial positive requirements to provide the child with the care, education, and discipline necessary to enable the child to exercise meaningfully his or her autonomy rights in the future. if fitting a child at an early age with a cochlear implant significantly improves his or her ability to acquire speech skills and to participate more widely in the larger hearing culture, then a strong case can be made for stipulating that parents have an obligation to seek out this treatment for a deaf child. an implant could allow the child, when an adult, to make a meaningful choice to live in the deaf community, in the hearing community, or biculturally in both worlds. the implications of the right to an open future argu- ment for parents of (existing) deaf children merit men- tioning insofar as there is a potential to think that acknowledging a responsibility to seek treatment for a deaf child via cochlear implant decides the issue with regard to the decision to select for deafness. if parents have a moral obligation to provide their deaf child with this device, then it might appear that the decision to select for deafness is self-defeating. i think it is a mistake to draw this conclusion. a deaf child with a cochlear implant is still a deaf child. that is to say, a genetically deaf child with a cochlear implant still shares deafness in common with her parents; she will have experiences of see d. davis. : – and : – . the kinds of opportunities that will be available to the child when she is an adult will depend largely on the parental decisions made after the child’s birth, decisions regarding the child’s education and whether or not to seek out medical interventions. these kinds of decisions might legitimately violate the child’s right to an open future. the point i wish to make, however, is that selection per se is not confining. this is one of the ways that respecting a child’s right to an open future differs from respecting an adult’s autonomy rights. the advantages and disadvantages of cochlear implants raise a number of interesting and important questions that are well beyond the scope of this paper. this is why i have chosen to phrase the position in hypothetical terms. if providing deaf children with cochlear implants allows them opportunities that translate into a significantly more open future, then, in virtue of a child’s right to an open future, there is reason to think that parents have an obligation to seek this treatment for their deaf children. melissa seymour fahmy © blackwell publishing ltd. todd highlight deafness that no hearing child has, and when she is an adult, she can choose not to use the implant and live the deaf lifestyle exclusively. acknowledging an obligation to outfit a deaf child with a cochlear implant may dissuade some deaf couples from seeking to reproduce deaf children or from repro- ducing at all. acknowledging an obligation of this kind may also pose a significant threat to deaf culture, analo- gous to the way that mandatory education laws pose a threat to traditional amish culture. however, acknowl- edging an obligation of this kind does not establish that selecting in favor of deafness is prima facie morally wrong. the failure of the harm analysis and the right to an open future analysis to account for the supposed moral wrongness of selecting for deafness suggests a need to reframe the locus of moral evaluation. there are at least two additional avenues to pursue. one could frame the moral evaluation in terms of the state of affairs that ensue from the decision to select for deafness. alternatively, one could frame the moral evaluation in terms that assess the character of the individuals who desire to make this kind of procreative selection. i will consider both of these alternatives in the following sections. . the introduction of avoidable suffering and/or limited opportunity one way to avoid the difficulties encountered by the pre- viously considered analyses is to locate the moral harm in selecting for deafness more generally in the ensuing state of affairs rather than in some injury done to a particular individual. a state of affairs analysis of the supposed moral harm maintains that the decision to select for deaf- ness is morally problematic insofar as the decision brings about some harm, such as limited opportunity, which could have been avoided by selecting a different child with normal abilities. according to this analysis, it is morally good to prevent the avoidable harm, but prevent- ing the harm cannot be said to be done for the sake of the child who would experience it. this is because the harm can be avoided only by preventing the existence of the impaired child. this is the kind of analysis that dan brock suggests can explain cases of wrongful disability – cases where the harm of a disability can be prevented only by preventing the existence of the particular disabled individual. a state of affairs analysis of the supposed moral harm does not fail in the way that the previously considered analyses do, which is to say that i think it offers at least a plausible account of the supposed moral wrong in select- ing for deafness. a state of affairs analysis, however, has implications for an ethics of procreation that extend well beyond the deaf case. if selecting for deafness constitutes a moral harm, insofar as it brings about some undesirable state of affairs that could have been avoided, then it would seem to be the case that, in addition to an obliga- tion not to pursue the undesirable state of affairs as an end, we must also acknowledge an obligation to avoid unintentionally bringing about the same state of affairs. this is typical of the common understanding of obliga- tions of nonmaleficence. while directly and intentionally generating some harm may be the more egregious wrong, failing to prevent foreseeable harm is far from innocent. taking reasonable precaution to prevent foreseeable harm is thus thought to be among an agent’s moral responsibilities. consider the case brock describes of a woman who is told by her physician that she should not attempt to become pregnant at present because she has a condition that, while fully treatable, would likely result in mild retardation in her child if she were to conceive prior to completing the one month treatment. the woman, impa- tient and unwilling to postpone pregnancy, disregards her physician’s warning and gives birth to a mildly retarded child. the case is vexing for the reasons articulated in the previous sections. mental retardation is not so bad that it renders a life not worth living, and the only way to prevent this impairment would be to prevent the existence of the particular child with the impairment. thus it is not the case that it would have been better for the child in question, had the mother postponed pregnancy. while the mother’s actions certainly appear blameworthy, their wrongness cannot be explained in terms of some harm done to her child. brock suggests that we can account for the morally troubling nature of the mother’s action by appealing to a non-person-affecting principle of harm prevention. the woman in the example acts wrongly, not because she a state of affairs analysis need not claim that deafness itself is a harm. the analysis can acknowledge that the harm of limited opportunity is the product of both the limited sensory abilities of the deaf person and the resources available to the deaf person within a particular society. d. brock. the non-identity problem and genetic harms. bioethics ; . see also a. buchanan, d. brock, n. daniels & d. wikler. . from chance to choice: genetics and justice. cambridge: cambridge university press: – . see brock. : ; and a. buchanan et al. : . see brock, op. cit. note , p. for a more complete account of the principle he proposes. on the supposed moral harm of selecting for deafness © blackwell publishing ltd. todd highlight todd highlight harms her child, but rather, because she fails to prevent harms (the harms of limited opportunity and suffering which ensue from mild retardation) that could have been avoided without incurring substantial burdens or costs. the fact that she did not intend to create a mildly retarded child does not absolve her from moral culpability. what does this mean for the case we are considering? if selecting for deafness constitutes a moral wrong insofar as it brings about some harm that could have been avoided (via the substitution of a different child), then there is reason to think that agents are obliged to avoid creating a deaf child, even unintentionally. individuals who have a family history of deafness would have to make use of genetic screening and other technologies in order to ensure that any child they produce does not experience suffering or limited opportunity that is ‘avoid- able by substitution.’ of course, this conclusion is by no means limited to genetic deafness. what is relevant to the moral evaluation we are considering is not deafness per se, but rather the fact that deafness results in limited opportunity for the child. other genetic anomalies that entail comparable or more severe forms of suffering or limited opportunity would also engender a similar obligation to prevent the existence of persons with these anomalies. here we are confronted with a difficult threshold question. how much anticipated suffering and/or limited opportunity is enough to suggest a moral obligation to avoid this harm by substituting one potential child for another, or by forgoing procreation altogether? i will not pursue this question here. my aim in this paper is far more modest. my intention is simply to draw out what would follow from an analysis of selecting for deafness that accounts for the supposed moral wrongness of this selection in terms of the introduction of avoidable harm. the idea that we may be obliged to avoid procreation where there is a chance of passing on some genetic con- dition is not new. laura purdy has argued for an obliga- tion to avoid reproduction if there is a nontrivial chance of passing on a genetic condition which is incompatible with a minimally satisfying life. the conditions purdy has in mind include tay-sachs disease and huntington’s disease. in focusing on the notion of a ‘minimally satis- fying life’ purdy sets the threshold rather low. her argu- ment would not ground on obligation to prevent the existence of deaf persons, insofar as we agree that deaf- ness does not preclude living a minimally satisfying life. it’s worth noting that the deaf case would set the thresh- old quite high. if deafness entails sufficient limited oppor- tunity to engender an obligation to prevent it, then we may have to acknowledge comparable obligations to prevent the existence of persons with down syndrome, cystic fibrosis, cerebral palsy, etc. this list would only expand as our knowledge of human genetics increases. of course, the harm to be avoided must be weighed against the costs and burdens such avoidance would require. in the case that brock considers, the woman could have avoided the harm of mild retardation by simply postponing pregnancy for a month. preventing the existence of persons with genetic diseases and impair- ments, however, is rarely so simple. the options open to potential parents – ivf and pgd, prenatal screening and selective abortion, gamete donors, and adoption – all entail potentially significant physical, financial, and emo- tional burdens. notably, the burden of being denied the opportunity to select one’s child on the basis of genetic features is rather trivial when compared with the more significant burdens entailed in actively endeavoring to prevent the existence of any impaired child. this reinforces the notion that the obligation to refrain from deliberately pursuing the birth of an impaired child (if there is such an obligation) is more stringent than the obligation to prevent the birth of any such child. the point i wish to make here is that a state of affairs analysis of the supposed moral wrongness of selecting for deafness cannot ground an obligation of the first kind without also committing us to some obliga- tion of the second kind. making sense of these obliga- tions will require answering the difficult threshold question i noted earlier, as well as determining the kinds of burdens that can be reasonably imposed on prospec- tive parents. an alternative approach to accounting for the sup- posed moral harm of selecting for deafness is open to those who find the state of affairs analysis troubling for one reason or another. this alternative locates the wrong- making feature in some aspect of the character of the decision-maker, rather than in some harm that results from the selection. i will consider several different ver- sions of this approach in the remaining sections of the paper. . parental & civic responsibility bonnie steinbock and ron mcclamrock have pro- posed a principle of parental responsibility to account for cases where, even though the child’s life is not so bad that nonexistence would be preferable, the decision to forgoing procreation need only entail forgoing children biologically related to oneself, not forgoing parenthood or childrearing. l. purdy. . reproducing persons: issues in feminist bioethics. ithaca: cornell university press: – . melissa seymour fahmy © blackwell publishing ltd. todd highlight procreate nonetheless appears to be unfair to the child. for instance, most teenagers lack the necessary resources, skills, and maturity to provide their children with mini- mally decent parenting. a teenager, who elects nonethe- less to have a baby, elects to create a child under conditions that are adverse to its well-being. convinced that the wrong-making feature in such cases cannot be given in terms of harm done to the resulting child, and unsatisfied by the state of affairs analysis, steinbock and mcclamrock suggest that a principle of parental respon- sibility provides us with a more appropriate moral critique. the principle of parental responsibility claims ‘that prospective parents are morally obligated to consider the kinds of lives their offspring are likely to have, and to refrain from having children if their lives will be suffi- ciently awful.’ the standard set by the principle is minimal. individuals are obliged to avoid procreation only in cases where the potential child is not likely to have ‘a decent chance at a happy life.’ the principle insists ‘only that it is wrong to bring children into the world when there is good reason to think that their lives will be terrible. it does not suggest that people should not have children unless conditions are ideal, still less that only conventional childrearing circumstances are morally permissible.’ steinbock and mcclamrock’s principle of parental responsibility, while defensible in its own right, does not appear to be of much help to us in resolving the question of selecting for deafness. deafness is not so bad that it renders a life awful, terrible, or unhappy. furthermore, it is significant that the parents we are considering in the deaf case are selecting for a child who shares a condition with them. deaf parents, especially those born deaf, are in a position to judge well the life prospects for their potential children. the term irresponsible does not seem an accurate description of the decision to select for deafness. given that genetic selection of this kind will likely require sig- nificant assistance from others and that selection of this kind is likely to be met with at least some resistance, it follows that selecting for deafness cannot be pursued casually or impulsively. this kind of procreation stands in sharp contrast to the not uncommon case of unplanned and accidental pregnancies, as well as the case brock describes where a woman gives birth to a mildly retarded child because she refuses to postpone pregnancy for the duration of her medical treatment. but perhaps there is a better way to describe what is problematic about the decision to select in favor of deaf- ness. it might be said that such a decision is objectionable in virtue of what it expresses, namely too little regard for the good that may be described as (for lack of a better description) the normal functioning of human capacities, which include hearing. i think this is a promising alterna- tive, though at present i am unconvinced that it is substan- tive enough to ground moral condemnation. it should be noted, however, that the same critique might also be applied to those who are aware of their risk of transmitting a debilitating condition to the next generation and yet fail to take precautions to prevent transmission. before moving on to examine more demanding paren- tal ideals, i’d like to consider a different sense in which the decision to select for deafness might be thought to be irresponsible. one could argue that it is irresponsible to deliberately create a child who will require significantly greater public assistance in order to pursue normal devel- opment and a happy and meaningful life. the kind of responsibility i have in mind here might be called civic responsibility. according to some reports, educating a deaf child currently costs the public close to three times what it costs to educate a hearing child. those who deliberately select in favor of deafness and then make use of publicly funded special facilities and services could be accused of exploiting public resources that were not designed for the purpose of accommodating unique pro- creative preferences. one response to this charge insists that the cost of educating a deaf child must be weighed against the child’s unique social contribution. according to deaf mother sharon duchesneau, deaf children make society more diverse, and this diversity in turn renders a society more humane. even if we grant the premise that diversity does render a society more humane, it is unclear how this benefit compares to the additional burden placed on public resources. furthermore, those who are inclined to sympathize with the impaired may take an entirely differ- ent view toward impairment that is deliberately pursued rather than accidental. the civic responsibility analysis of the supposed moral harm of selecting for deafness has implications similar to those that follow from the introduction of avoidable harm analysis. if deliberately selecting for a deaf child constitutes a failure of civic responsibility, then this sug- gests that civic responsibility may also require us to avoid steinbock & mcclamrock. when is birth unfair to the child? hast- ings cent rep ; : . ibid: . b.p. tucker. deaf culture, cochlear implants, and elective disabil- ity. hastings cent rep ; : . l. mundy. . a world of their own. the washington post march. on the supposed moral harm of selecting for deafness © blackwell publishing ltd. todd highlight todd highlight the birth of persons with impairments that require sub- stantial public assistance. as we noted earlier, there is reason to think that the obligation not to deliberately pursue an end may be more stringent than an obligation to avoid the same end, especially when the costs of pre- vention are taken into consideration. if the costs of pre- vention were minimal, however, and right now they are not, then i believe that the civic responsibility account of the moral harm of selecting for deafness would strongly suggest an obligation to avoid the birth of impaired children. . parental virtues: openness to the unbidden and unconditional parental love another way of accounting for the supposed moral wrongness of selecting for deafness is to argue that the selecting agents exhibit attitudes and dispositions that are hostile to certain parental ideals or virtues. in this section, i will consider two parental virtues that might be said to be lacking in an agent who selects in favor of deafness: openness to the unbidden and unconditional love. openness to the unbidden michael sandel has recently raised objections to an emerging new eugenics which, he contends, threatens to replace an ethic of giftedness with an ethic of mastery. according to sandel, . . . parenthood, more than other human relationships, teaches what the theologian william f. may calls an ‘openness to the unbidden.’ may’s resonant phrase helps us see that the deepest moral objection to enhancement lies less in the perfection it seeks than in the human disposition it expresses and promotes . . . even if this disposition did not make parents tyrants to their children, it would disfigure the relation between parent and child, and deprive the parent of the humility and enlarged human sympathies that an openness to the unbidden can cultivate. william f. may maintains that parenthood teaches open- ness to the unbidden. this seems right. the virtues of parenthood, whatever they may be, are likely to be acquired through practice. just as the warrior not only demonstrates but learns courage on the battlefield, the parent cultivates openness through her experiences with the unbidden qualities of her child. the virtue of open- ness thus should not be thought of as a qualification for parenthood but, rather, as a moral good that one, ideally, gains by the experience. to make the case against genetic selection, what needs to be shown is that the practice of selection does, as sandel suggests, pose a special threat to the parent-child relationship or to the cultivation of the parental virtue of openness to the unbidden. this is not easily demonstrated. parental hubris does not depend on biotechnology. inap- propriate parental expectations, expectations hostile to the virtue of openness, are not new. one can presume to know more about one’s child than is appropriate without actively selecting genetic traits. biological parents, for instance, know that their children share their genes and this alone may be sufficient to encourage inappropriate assumptions and expectations. parental hubris might lead one to think that because he is my son, he is going to be a great athlete or attend yale. even if we did select our children’s genetic traits, there would still be every reason to think that parent- hood teaches openness to the unbidden. this is because children are so much more than their genetic features. only the most naïve parents would presume to know what they are getting simply because they had a hand in selecting some of their child’s genetic features; and a naïve parent would receive a lesson in humility soon enough. parenthood might still be a school for humility, perhaps even more so. thus we can agree with sandel that openness to the unbidden is a disposition worth affirming, without concluding that the practice of genetic selection deprives the parent of an opportunity to cultivate this virtue. one might argue that while the selecting agent may well develop the virtue we’re calling openness to the unbidden, she is, in fact, less open to the kind of child she creates than the agent who does not select. this is certainly true. the challenge, however, is to articulate what is morally problematic about being less than com- pletely open in this way. here we risk confusing open- ness as a parental virtue with openness as a procreative virtue. as a parental virtue, it is fairly easy to see how openness to the unbidden will be of value both for the child’s sake and for the sake of the parent-child rela- tionship. it is unclear whether openness to the unbidden in procreation is itself a virtue or whether there is reason to think that a parent who is less open about the kind of child she creates will be less open in relation to the child she does create. m. sandel. the case against perfection: what’s wrong with designer children, bionic athletes, and genetic engineering. atlantic monthly : [emphasis mine]. see also m. sandel. . the case against perfection. cambridge: belknap press: – . melissa seymour fahmy © blackwell publishing ltd. unconditional parental love a related concern is that genetic selection is inconsistent with the ideal of unconditional parental love. this ideal maintains that parents should love their children, and act lovingly toward them, simply because the child is their child. this is to say, parental love should not be contin- gent on the child’s particular genetic features, abilities, physical appearance, performance, etc. the parental love objection maintains that ‘setting conditions on which child to create amounts to setting conditions on our love for whatever child we get, for it sets conditions on which child will receive that love.’ this objection takes issue with most cases of selection both in favor of and against particular genetic features. like sandel’s critique, the parental love objection has a much broader target than the case of selecting for deafness. the ideal of unconditional parental love depends on there being some existing child or potential child to receive that love. unconditional love is not an attitude or feeling one could have toward the entire class of one’s potential progeny. this suggests that unconditional love, unlike openness to the unbidden, is not a virtue that can be expressed in the decision to select or not to select. so the objection cannot be that potential parents fail to embody this ideal when they make the decision to select for or against a particular genetic trait. in order to make its case against selection, what the parental love objection must establish is that the practice of selection somehow makes it less likely that the resulting child will be an object of unconditional love. i believe that the strongest case for this position can be made by arguing that the process of selection encourages parental expectations which could lead to disappoint- ment in the future. genetic selection, at least at present, requires potential procreators to undergo procedures that are costly, time-consuming, and invasive. because of their investment, selecting parents may feel cheated or disap- pointed if the child they create is not the child they intended to create. these negative feelings may then con- stitute an impediment to unconditional parental love. the randomness of unselective procreation insulates parents from these particular affective impediments, whereas selecting parents put themselves at risk. parents may also feel disappointed if their child does not fulfill expectations indirectly associated with genetic selection. for instance, a deaf parent who engages in genetic selec- tion may strongly desire that her child live as a member of the deaf community. expectations of this nature might also constitute an impediment to unconditional parental love. two things should be noted here. first, the connection sketched above is only a speculative one, not a necessary one. selecting parents may very well love their children unconditionally even if they bear little or no resemblance to the children they intended to create. second, that selec- tion potentially endangers the ideal of unconditional love does not by itself establish that selection is morally rep- rehensible. this is due to the fact that the connection is an uncertain one, and because what we risk by selecting must be balanced against what we gain, as well as what is risked by not selecting. for these reasons, i believe that the parental love objection is too weak to provide an adequate account of supposed moral harm of selecting for deafness. conclusion i have endeavored to demonstrate that accounting for the moral harm of selecting for deafness is not as simple as the widespread negative response from the hearing com- munity would suggest. my interest in exploring this topic is not to vindicate the decision to select for deafness, but rather to uncover some general conclusions about how we ought to think about the ethics of procreation. the first lesson to be learned from the deaf case is that, in an age of unprecedented reproductive opportunity, we need norms that govern not just the use of reproductive tech- nology but procreation and procreative decision-making in all of its various forms. identifying such norms will be challenging; and the biggest challenge may well be over- coming our bias toward unassisted coital reproduction. if selecting for deafness is morally reprehensible because it introduces some preventable harm or places undue finan- cial burdens on the public, then we must evaluate unse- lective coital reproduction by the same standard. if selecting for deafness is morally reprehensible because it violates a principle of parental openness, then selecting against disease states that are compatible with a life worth living must be condemned on the same grounds. while there may be many good reasons for not impos- ing external restrictions on coital reproduction, good reasons for privileging this method of procreation in ethical analysis are much harder to come by, and will be harder still to come by as different forms of intervention become less costly and more accessible. we should also bear in mind that the norms of parenthood may be poor candidates for the norms which govern procreation. as we have seen, sometimes the norms of parenthood fit j. davis. selecting potential children and unconditional parental love. bioethics ; : – . i am assuming here that a fertilized egg is not yet a child, but once implanted, will become a child some time prior to birth. on the supposed moral harm of selecting for deafness © blackwell publishing ltd. rather awkwardly when applied to procreative decisions (e.g. openness to the unbidden), and sometimes they do not fit at all (e.g. unconditional parental love, respecting the child’s right to an open future). the second lesson to be learned from the deaf case is that parental decisions made post-implantation and post- birth are far more important and potentially problematic than selection decisions. this is true in the deaf case, as well as in cases of selecting for a particular sex, or so-called beneficent selection. post-implantation deci- sions can directly harm or benefit the child, as well as expand or confine her future, in ways that selection cannot. decisions made post-birth also have a far greater potential for developing and expressing parental virtues or vices. the challenge here will be to prevent the ethics of procreation, striking in its novelty, from overshadowing the ethics of parenthood. acknowledgements the author is grateful to the poynter center for the study of ethics and american institutions at indiana university for the opportunity to participate in an interdisciplinary seminar on the ethics and politics of childhood in – , to the seminar participants for inspiring this paper, and to daniel farnham, rené jagnow, piers stephens, and sarah wright for their comments on an earlier draft of the paper. melissa seymour fahmy is assistant professor of philosophy at the university of georgia. her research interests include kantian ethics, moral psychology, and various topics in bioethics. see j. savulescu. procreative beneficence: why we should select the best children. bioethics ; : – ; and j. davis. selecting potential children and unconditional parental love. bioethics ; : – . melissa seymour fahmy © blackwell publishing ltd. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ neutron structural studies on the superconducting „nd −xcax…„ba . la . …cu oz system amish g. joshi, ,a� r. g. kulkarni, w. b. yelon, ram prasad, and m. r. gonal national physical laboratory, dr. k.s. krishnan road, new delhi , india department of physics, saurashtra university, university road, rajkot , india graduate center for material research, university of missouri, rolla, missouri , usa metallurgy division, bhabha atomic research center, trombay, mumbai , india �received october ; accepted march ; published online april � we have investigated the influence of ca ion substitution on the structural and superconducting properties of �nd −xcax��ba . la . �cu oz system. magnetization, x-ray diffraction, and neutron diffraction studies have been carried out on a series of compounds with x = . – . . the superconducting transition temperature tc, determined from magnetization measurements, increases with increasing ca + substitution. neutron diffraction studies reveal that these compounds crystallize in a tetragonal structure �space group p / mmm�. a detailed analysis of the neutron diffraction data reveals that ca and la ions are intermixed at the nominal ba and nd sites. while a major fraction of ca ions occupy the usual nd site, a small fraction occupies the ba site. consequently, the corresponding amount of la substitutes at the nominal nd site. the intermixing of ca and la sites randomizes the chain site oxygens leading to a tetragonal structure despite an oxygen content close to . for all the ca doped samples. further increase in ca content leads to change in its coordination from sixfold to eightfold at x � . . © american institute of physics. �doi: . / . � i. introduction the normal state transport properties of oxide supercon- ductors are unusual and systematic studies of them are there- fore important for understanding high tc superconductivity. yba cu o −� �y � superconductor has been extensively studied; replacing y by r �r = rare earth� does not affect superconductivity except when r is ce, tb, or pr. the hole concentration p is an important parameter in the p-type ce- ramic high tc materials. the hole concentration can be var- ied by changing the concentration of substituents at different crystallographic sites �i.e., y, ba, or cu� or by the ordering of oxygens in basal plane. it is well known that in r +xba −xcu o −�, the light rare earth element �lr = la to nd� can occupy the ba site as well as the r site. for lr elements the ionic radii approach that of ba, , allowing for a large degree of substitution by r for ba without forming second phase, which results in oxygen defects on the anti- chain site. nd has an ionic radius closest to that of ba and, therefore, exhibits the greatest amount of solubility with ba �x � . �. when trivalent lr occupies the divalent ba site, charge balance requires oxygen for each r is incorporated into the structure. in the fully oxygenated orthorhombic structure the extra oxygen occupies the antichain site �o� ��. – since the basal plane acts as a charge reservoir, the extra oxygen results in modification of the charge distribu- tion in the structure. superconductivity is suppressed due to the localization of holes. – accompanying the lr + substi- tution on the ba + site is a change in the crystal structure from orthorhombic to tetragonal �o-t� and an increase in the flatness of the cuo plane. a comparison of substituting magnetic ions �such as eu +, pr +, nd +� �refs. , , and � and nonmagnetic ions �such as la +� �refs. – � at ba site leads to the conclusion that besides magnetic pair breaking, there is a considerable contribution to the tc originating from other effects such as defect scattering, carrier localization, or hole filling. by studying la substituted nd compounds, we have shown that increasing la concentration in nd�ba −ylay�cu oz � � y � . � causes a reduction in tc due to hole filling, and a structural phase transition �o-t� is observed at y = . . a similar structural transformation is observed with nonmagnetic la + at ba +, with magnetic rare earth r �r = eu, ho, dy� substitutions. , , this tc is expected to revive when divalent ca is substituted for trivalent rare earth r. when ca is incorporated in �nd . ca . �ba cu o −�, the sample is observed to be more metallic at gpa and k. on the other hand, thermo- electric power measurements have shown that la doping in nd −xlaxba cu oy causes a decrease in hole concentration smaller than that of ca doping in nd −xcaxba cu oy, which suggests that some la �� % � would enter in to the ba site with the generation of additional bacuo +v phase. our present efforts are to understand the effect of simultaneous substitution of ca and la at the nd and ba sites, respec- tively, on structure and superconductivity of �nd −xcax� �ba . la . �cu oz system. if all the la atoms occupy the ba site, the oxygen content would have to increase to maintain charge balance. as ca � . Å, in eightfold coordination� is slightly larger than nd � . Å�, there should be a prefer- ence for ca to occupy the ba-site rather than the nd site, which is solely based on lattice strain arguments. a�author to whom correspondence should be addressed. electronic mail: amish@mail.nplindia.ernet.in. journal of applied physics , � � - / / � �/ / /$ . © american institute of physics , - downloaded jan to . . . . redistribution subject to aip license or copyright; see http://jap.aip.org/jap/copyright.jsp http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . however, this preference is not observed and is expected to be due to some intersite mixing of ca and la at the ba and nd sites, respectively. for this reason neutron diffraction studies were performed to determine the relative site occu- pations of the ca and la ions. systematic investigations of neutron diffraction and magnetization measurements of compounds having the stoichiometric compositions �nd −xcax��ba . la . �cu oz with x = . to . have been studied in detail. the interrelationship between the supercon- ducting transition temperature �tc� and structural changes has also been discussed in the context of the carrier concen- tration. ii. experimental details the �nd −xcax��ba . la . �cu oz, series of compounds with � x � . , were synthesized via a solid state reaction, the details of which are given in ref. . powder x-ray dif- fraction patterns of the compounds were obtained in the � range ° � � � ° using cu k� radiation. room temperature neutron diffraction patterns were ob- tained at the university of missouri research reactor facil- ity using a position sensitive detector �psd� diffractometer. this diffractometer is equipped with a si � � monochro- matic, horizontally curved with about – m radius, and vertically segmented with . m radius and with nine asym- metrically cut blades. for collecting the diffraction data, the sample was contained in a thin-walled vanadium can of about – mm diameter and about cm long and rotated about the long axis to average out any preferred orientations. the data were obtained in a � range of ° � � � ° �at . ° intervals�, using a psd �with five detector elements� that covered a range of ° at a time. neutrons of wave- length of . Å were used for the diffraction experi- ments. magnetization measurements were performed using a superconducting quantum interference device magnetometer �quantum design� in the temperature range of . – k and an applied field of oe. iii. results and discussion x-ray powder diffraction studies were performed on se- ries of compounds �nd −xcax��ba . la . �cu oz with x = . – . , showed them to be single phase. a fit of a typical neutron diffraction pattern is shown in fig. . figure shows the typical zero-field-cooled �zfc� and field-cooled �fc� magnetization data of �nd −xcax��ba . la . �cu oz with x = . , . , . , and . with an applied filed of oe. in both zfc and fc states data were acquired during warming of the sample. the superconducting transition temperature �tc� is defined as the onset of diamagnetic response. it is seen from figs. and that tc increases with increasing ca concentra- tion, which is in agreement with an earlier report. for the sample with x = . , the diamagnetic signal is small, suggest- ing that superconductivity is not a bulk property of the sample. it is evident from the figure that the meissner frac- tion increases with increasing ca content, which may indi- cate that a superconducting phase is forming while more nd + is replaced by ca +. however, neither x-ray diffraction nor neutron diffraction shows any evidence of another phase at room temperature. the neutron diffraction data were analyzed by rietveld refinement procedure using the generalized structural analy- sis system �gsas� program. the structure refinement carried out with orthorhombic structure, space group pmmm. it was realized that lattice parameters a and b are nearly the same, exhibit possible formation of tetragonal phase. therefore, neutron diffraction data on all samples were refined on the basis of a tetragonal structure �space group p / mmm�. neu- tron scattering factors used were �in units of femtometers� . for la, . for ca, . for ba, . for nd, . for cu, and . for o. structural parameters such as atomic coordinates, fractional occupancies, and the thermal param- eter �ui� for different atoms �including various oxygen sites fig. . typical observed �dots� and the calculated �solid curve� neutron diffraction patterns for �nd −xcax��ba . la . �cu oz samples with x = . , . , . , and . at room temperature. the difference pattern for each sample is shown at the bottom. - joshi et al. j. appl. phys. , � � downloaded jan to . . . . redistribution subject to aip license or copyright; see http://jap.aip.org/jap/copyright.jsp assigned to the standard r- structure�, obtained from ri- etveld analysis, are listed in table i. in r- -type compounds, the unit cell is considered as an assembly of three abo -type pervoskite cells stacked one on top of the other with the central atom being r, in the middle cell, and ba in other two. this would result in a chemical formula rba cu o , and would contain a three- dimensional network of cu–o bonds. the lower dimension- ality and oxygen content o −� arise from the missing oxygen atoms surrounding the r-site, cu–o chains in the basal planes. the r and the two ba atoms occupy crystallographi- cally distinct sites with eightfold and tenfold oxygen coordi- nations, respectively. there are two different types of cu atoms, labeled as cu� � � , , � for the cu in the basal plane and cu� � � , , z� for the cu in the two cu–o planes. the cu� � atoms have strong covalent bonds to four o atoms in a square planner configuration, while cu� � atoms are bonded to five oxygens in pyramidal symmetry. however, cu – o planes extend indefinitely in two directions �in the ab-plane�, while cu–o chains extend indefinitely in only one direction �the b-axis direction�. the oxygen sites in the cu – o planes are identified as o� � � , , z� and o� � � , , z�. the oxygen site in the ba–o plane is designated as o� � � , , z� and is often called the “apical oxygen.” the oxygen sites in basal plane, often called as cu–o chains, are designated as o� �� , , � �along b-axis� and o� �� , , � �along a-axis�. in orthorhombic r- , the o� � sites are fully occupied, while the o� � sites are nearly vacant, giving rise to b � a. the o-t transition occur either by removing o� � �oxygen deficient r- with oxygen stoichiometry � . � or by filling the o� � sites �e.g., trivalent la at ba site, resulting in oxygen stoichiometry � . �. , moreover, an interesting situation can occur via redistribution of o� � and o� � with oxygen stoichiometry close to . , which is the unique case for the present case of �nd −xcax��ba . la . �cu oz. in the tetragonal system with a = b �cu� � – o� � = cu� � – o� ��, the o� � and o� � are in- distinguishable, and the same is true for o� � and o� � sites in the cu – o planes. the observed variations of lattice parameter and cell vol- ume with ca content are shown in fig. . it is observed that the lattice parameter a and the cell volume both decrease up to x = . . the decrease in lattice parameter reflects the de- crease in cu� �–o� � distance and indicates a decrease in flatness of the cu – o plane with the decrease in “buckling angle” �cu� �–o� �–cu� ��, which helps to revive tc from k �x = . � to k �x = . �. initially structure refinement model assumed with nd/r and ba / la ratios fixed by initial stoichiometry, but site oc- cupancies were allowed to vary. in the subsequent analysis of neutron diffraction data, it was assumed that nd and ba fully occupy their normal sites. after fixing nd and ba occupan- cies, the ca and la occupancies were allowed to vary freely both at the actual sites �ca at nd site and la at ba site� and the nominal sites �la at nd site and ca at ba site� with the condition that combined fractional and nominal at r site �ca at nd + la at nd site� and ba site �la at ba + ca at ba� add up to unity �i.e., full occupancy�. in order to maintain a mean- ingful and reliable variation in ca and la occupancies at the two sites, this procedure was adopted uniformly for all the samples. the variation in occupancies for la and ca at dif- ferent sites is shown in figs. �a� and �b�, respectively. the intermixing behavior of la and ca occupancy variation with r �r = nd, dy, and y� through neutron diffraction has also been observed in la- , la- , and r- systems. – it is well known that ca substitution at the r-site de- creases the oxygen content of r- compounds; however, la substitution at the ba-site increases the oxygen content. , analysis of the neutron diffraction data reveals that some ca atoms occupy the actual nd-site while some occupy the ba site. consequently, corresponding amount of la substitutes at the nd site. the oxygen content, obtained from refinement of neutron data, is found to be close to . fig. . �color online� zfc and fc magnetization of �nd −xcax� �ba . la . �cu oz samples with x = . , . , . , . , . , . , and . as a function of temperature in an applied filed of oe showing superconduct- ing transition temperature. - joshi et al. j. appl. phys. , � � downloaded jan to . . . . redistribution subject to aip license or copyright; see http://jap.aip.org/jap/copyright.jsp for all ca-doped �nd −xcax��ba . la . �cu oz compounds with x = . – . . the cross substitution of la at the nd site and ca at the ba site randomizes the chain site oxygen’s bringing the o� � and o� � site occupancies of oxygen in the basal planes to same level and forces the compound to crys- tallize in a p / mmm tetragonal structure. here it is worth mentioning that the substitution of ca + for nd + can increase the number of mobile holes provided the oxygen content remains nearly constant. an increased number of holes oxidizes the cu – o layer. the oxidation of cu – o layer removes electrons from the x − y orbitals, which have antibonding character in the in-plane cu–o bonds. therefore, as number of holes �nh� increases, the in- plane cu� �–o� � bond length �rcu–o� is shortened. , the effective cu valence, estimated from the overall oxygen con- table i. lattice parameters, cell volume, fractional occupancies, positional, and thermal parameter for �nd −xcax��ba . la . �cu oz system obtained from rietveld refinements of the neutron diffraction data. for the tetragonal refinements, o� � and o� � are equivalent, and o� � and o� � are equivalent. numbers in parentheses are statistical standard deviations of the last significant digits. parameter x = . x = . x = . x = . x = . x = . x = . space group p / mmm p / mmm p / mmm p / mmm p / mmm p / mmm p / mmm a �Å� . � � . � � . � � . � � . � � . � � . � � b �Å� . � � . � � . � � . � � . � � . � � . � � c �Å� . � � . � � . � � . � � . � � . � � . � � v �Å � . � � . � � . � � . � � . � � . � � . � � nd � , , � ui � �Å � . � � . � � . � � . � � . � � . � � . � � nnd . . . . . . . nca . � � . � � . � � . � � . � � . � � nla . . � � . � � . � � . � � . � � . � � ba � , , z� z . � � . � � . � � . � � . � � . � � . � � ui � �Å � . � � . � � . � � . � � . � � . � � . � � nba . . . . . . . nla . . � � . � � . � � . � � . � � . � � nca . � � . � � . � � . � � . � � . � � cu� � � , , � ui � �Å � . � � . � � . � � . � � . � � . � � . � � n . . . . . . . cu� � � , ,z� z . � � . � � . � � . � � . � � . � � . � � ui � �Å � . � � . � � . � � . � � . � � . � � . � � n . . . . . . . o� � � , , � ui � �Å � . � � . � � . � � . � � . � � . � � . � � n . � � . � � . � � . � � . � � . � � . � � o� � � , ,z� z . � � . � � . � � . � � . � � . � � . � � ui � �Å � . � � . � � . � � . � � . � � . � � . � � n . . . . . . . o� � � , ,z� z . � � . � � . � � . � � . � � . � � . � � ui � �Å � . � � . � � . � � . � � . � � . � � . � � n . . . . . . . o� � � , , � ui � �Å � . � � . � � . � � . � � . � � . � � . � � n . � � . � � . � � . � � . � � . � � . � � total oxygen content/formula unit . � � . � � . � � . � � . � � . � � . � � cu valence . . . . . . . fig. . �color online� variation in lattice parameter and cell volume with ca concentration. - joshi et al. j. appl. phys. , � � downloaded jan to . . . . redistribution subject to aip license or copyright; see http://jap.aip.org/jap/copyright.jsp tent �z�, obtained from neutron diffraction data is listed in table i, which increases with increasing ca concentration. simultaneous increase in tc with cu valence is observed and showed maximum tc of k at x = . . the introduction of ca into �nd −xcax��ba . la . �cu oz alters the normal and superconducting properties substantially. the ionic radii of ca + in sixfold and eightfold coordination are significantly different. the ionic size of nd + in eightfold coordination is . Å, which matches more closely with that of ca + in sixfold � . Å� than that of eightfold coordination � . Å�. the decrease in lattice parameter a and cell volume up to x = . indicates that ca at the nd site would have preferred a sixfold coordination. the lattice parameter c remains nearly unchanged which is in good agreement with earlier reports of the possibility of sixfold coordination in those samples , with ca + substitution at r site in fully oxygen annealed y- systems. as ca content increases in the regime x � . , some ca + might change its coordination from sixfold to eightfold, which occupy ba site and corresponding amount of la occupy at nd site �figs. �a� and �b�; table i�. therefore, lattice parameter a and cell volume increase due to the fact that la + and ca + in eightfold coordination are bigger than nd, which indicate an increase in the interplane cu� �–o� � bond distances and suggest a decrease in the number of holes in cu – o planes, which also results in an increase in the buckling angle. as the buckling angle in- creases, the cu – o planes become flatter which results in suppression of superconductivity. magnetization and neutron diffraction results may be summarized as follows. � � the superconducting transition temperature tc increases with increasing ca content up to x = . . � � with progressive ca doping at the nd site, the lattice parameter a decreases, but c remains nearly unchanged, suggesting that ca + may adopt sixfold coordination. � � ca + may change its coordination from sixfold to eight- fold with simultaneous increases in both lattice param- eter and cell volume for further increase in ca content x � . . it is evident from fig. that tc increases with increasing x up to x = . and thereafter, decreases slightly for x � . . the most spectacular proof that ca doping enhances super- conductivity is the phase �nd . ca . ��ba . la . �cu oz, which exhibits a tc of k, whereas the ca-free cuprate nd�ba . la . �cu oz does not superconduct down to k and shows tc on = k. the substitution of ca for nd intro- duces oxygen vacancies and nonstoichiometry, which lead to hole doping. therefore, this implies that the reduction in tc from k for pristine nd- to k for nd�ba . la . �cu oz is compensated by appropriate hole doping with ca. hence, the oxide displaying the optimum tc 	 k at x = . is identified as a compensated oxide whose tc lies close to that of pristine nd- �tc = k�. further increase in x from . to . causes tc to decrease from k �x = . � to . k �x = . � due to excess hole doping from ca. iv. conclusions we conclude that ca doping enhances superconductivity for doping level up to x = . ; thereafter it shows a decrease in tc with increasing x due to excess hole doping. neutron diffraction data suggest that there is intermixing of ca and la occupancies. at higher doping level x � . indicates pos- sibility of eightfold coordination of ca. p. karen, o. braaten, and a. kjekshus, acta chem. scand. , � �. k. osamura and w. zhang, z. metallkd. , � �. y. matsui, s. takekawa, and n. iyi, jpn. j. appl. phys., part , l � 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whangbo, d. b. kang, and c. c. torardi, physica c , � �. m. h. whangbo and c. c. torardi, science , � �. a. manthiram, s. j. lee, and j. b. goodenough, j. solid state chem. , � �. v. p. s. awana and a. v. narlikar, phys. rev. b , � �. - joshi et al. j. appl. phys. , � � downloaded jan to . . . . redistribution subject to aip license or copyright; see http://jap.aip.org/jap/copyright.jsp http://dx.doi.org/ . / - ( ) -q http://dx.doi.org/ . /bf http://dx.doi.org/ . /bf http://dx.doi.org/ . / - ( ) - http://dx.doi.org/ . /physrevb. . http://dx.doi.org/ . /physrevb. . http://dx.doi.org/ . /s - ( ) - http://dx.doi.org/ . / . http://dx.doi.org/ . /s - ( ) - http://dx.doi.org/ . /bf http://dx.doi.org/ . / - ( ) - http://dx.doi.org/ . / - ( ) - http://dx.doi.org/ . /physrevb. . rev_iss_web_acem_ _ - .. educational advance emergency medicine residents’ self- assessments play a critical role when receiving feedback richard bounds, md, colleen bush, md, amish aghera, md, nestor rodriguez, md, r. brent stansfield, phd, and sally a. santen, md, phd (the merc at cord feedback study group) abstract objectives: emergency medicine (em) faculty often aim to improve resident performance by enhancing the quality and delivery of feedback. the acceptance and integration of external feedback is influenced by multiple factors. however, it is interpreted through the “lens” of the learner’s own self-assessment. ideally, following an educational activity with feedback, a learner should be able to generate and act upon specific learning goals to improve performance. examining the source of generated learning goals, whether from one’s self-assessment or from external feedback, might shed light on the factors that lead to improvement and guide educational initiatives. using a standard oral board scenario, the objective of this study was to determine the effects that residents’ self-assessment and specific feedback from faculty have on not only the generation of learning goals but also the execution of these goals for performance improvement. methods: in this cross-sectional educational study at four academic programs, senior em residents participated in a standardized oral board scenario. following the scenario, residents completed a self-assessment form. next, examiners used a standardized checklist to provide both positive and negative feedback. subsequently, residents were asked to generate “smart” learning goals (specific, measurable, attainable, realistic, and time-bound). the investigators categorized the learning goals as stemming from the residents’ self-assessments, feedback, or both. within weeks, the residents were asked to recall their learning goals and describe any actions taken to achieve those goals. these were grouped into similar categories. descriptive statistics were used to summarize the data. results: a total of learning goals were initially generated (mean � sd = . � . per resident). forty-seven percent of the learning goals were generated by the residents’ self-assessments only, while % were generated by the feedback given alone. residents who performed poorly on the case incorporated feedback more often than high performers when generating learning goals. follow-up data collection showed that residents recalled learning goals, of which were acted upon. on follow- up, the numbers of learning goals from self-assessment and feedback were equal ( % each, of ), while the greatest number of reportedly executed learning goals came from self-assessments and feedback in agreement ( %). conclusions: following feedback on an oral board scenario, residents generated the majority of their learning goals from their own self-assessments. conversely, at the follow-up period, they recalled an increased number of learning goals stemming from feedback, while the largest proportion of learning goals acted upon stemmed from both feedback and self-assessments in from the departments of emergency medicine, christiana care health system (rb), newark, de; michigan state university (cb), east lansing, mi; maimonides medical center (aa), new york, ny; the university of wisconsin school of medicine and public health (br), madison, wi; and the university of michigan medical school (rbs, sas), ann arbor, mi. received february , ; revision received may , ; accepted may , . presented at the american college of emergency physicians scientific assembly research forum, denver, co, october ; and the society for academic emergency medicine annual meeting innovations in emergency medical education, chicago, il, may . the authors have no conflicts of interest or financial support to disclose. the study was initiated as a collaborative mentored mul- ticenter project through the medical education research certificate (merc) program, a joint faculty development program through the association of american medical colleges and the council of emergency medicine residency directors. supervising editor: lalena yarris, md. address for correspondence and reprints: richard bounds, md; e-mail: richbounds@gmail.com. doi: . /acem. pii issn - © by the society for academic emergency medicine issn - agreement. this suggests that educators need to incorporate residents’ self-assessments into any delivered feedback to have the greatest influence on future learning goals and actions taken to improve performance. academic emergency medicine ; : – © by the society for academic emergency medicine e ducators in the medical field often struggle with the efficient delivery of valuable feedback that reliably motivates learners to improve their per- formance. previous research has shown that effective formative feedback comes from a credible source and is focused on the task as opposed to the individual. – feedback delivery in medical education is challenged by numerous factors, including a lack of training for faculty and the need to protect the self-esteem of our learners and preserve a positive working relationship. many educators measure the value of feedback by looking at the learner’s improvement process and satis- faction with the feedback rather than actual results and changes in behavior. , the generation of specific goals by the learner serves as a powerful method for shifting the focus from the process of improvement, toward actual results and desired outcomes. – although learn- ing goals should be created through reflection, it is criti- cal that learners incorporate feedback from evaluators into the process, and evidence suggests that learners struggle with the interaction between self-assessment and feedback. , unfortunately, negative feedback may be consciously or unconsciously rejected and thereby less likely to be incorporated into the generation of learning goals. , in one study, learners’ perceptions of their own abilities were more likely to result in the generation of learning goals than was the actual feedback. these findings are somewhat troubling given the literature suggesting that physicians are unable to accurately self-assess. in fact, the least skilled and most overconfident physicians who would benefit the most from constructive feedback have shown the worst accuracy in self-assessment. this concern was supported in a recent study also demon- strating that many low performers did not generate learning goals that were concordant with their areas of weakness. to improve our residents’ performance, we need to further explore how feedback and self-assess- ment are each incorporated into the generation of learning goals and how the two interact in the mind of the learner. – this study evaluated the source of learning goals by emergency medicine (em) residents after participating in a standard oral board examination, performing their own self-assessments, and receiving specific feedback on their performance. the objective of this study was to investigate the contributions of self-assessments and external feedback and how the two interact, in the for- mation of learning goals, as well as the reported follow- through on those goals, for performance improvement. we expected self-assessments to play a significant role in the formation of learning goals, with faculty feedback playing a greater role, especially when the two perspec- tives contradicted one another. we also determined whether other factors affected the generation of learn- ing goals, such as quality of feedback provided, the high or low performance of the resident, and the correlation between faculty assessments and the learners’ self- assessments. methods study design this was a multicenter observational, cross-sectional, educational intervention study using an oral board sce- nario as a basis for self-assessment, feedback, and development of learning goals. this study was reviewed and approved by the local institutional review board at each of the four sites. outcomes were deidentified and kept confidential. all participants signed written consent. study setting and population this study was conducted at four em residency programs, led by one investigator at each site. all post- graduate year (pgy)- and above residents who were available on the designated days of study enrollment were offered the opportunity to participate in the oral board case, and volunteered. interns (pgy- resi- dents) were excluded from the study due to their limited experience with the oral board format. thirty residents were in pgy- ( %), and residents were in pgy- ( %). three residents from one site were in pgy- or - as part of a dual training program (em and inter- nal medicine). study protocol the four investigators have primary teaching appoint- ments and administered the oral board scenario and feedback. the investigators worked together to develop the study protocol during a national certification pro- gram for researchers in medical education. as a tool for the study of the interaction between feedback and self- assessment, a single case oral board scenario was taken from the council of emergency medicine residency directors oral board case bank. by consensus, a case of cardiac arrest due to ventricular fibrillation was selected and modified by the study group to include all six accreditation council for graduate medical educa- tion core competencies and incorporate certain skills that would challenge residents at all levels of training (see data supplement s , available as supporting infor- mation in the online version of this paper). the investi- gators felt that all pgy- and above em residents should demonstrate competency in advanced cardiac life support (acls) protocols and resuscitation, as well as communication with a cardiologist and a patient’s family. more advanced aspects of the case, such as bounds et al. • em resident feedback recognition of qt interval prolongation and an under- standing of the pathophysiology of digoxin toxicity in the setting of hypokalemia, were added to challenge the senior residents. for the primary outcome of learning goals generated by the resident, we used the “smart” framework as a guide: specific, measurable, attainable, realistic, and time-bound. the application of smart learning goals has been employed successfully in business and gen- eral education for many years, and the literature regarding learning goals in medical education supports its utility. , , the study group created a structured feedback form consisting of both a nationally validated quantitative scoring system and a novel qualitative feedback check- list (see data supplement s , available as supporting information in the online version of this paper). to stan- dardize the feedback across investigators, specific posi- tive and negative feedback phrases for each critical action of the case were developed by group consensus. positive feedback included phrases such as “accu-check performed promptly” and “recognized prolonged qt on ekg,” while negative feedback included points such as “does not obtain confirmatory cxr after intubation” and “does not speak to patient’s wife after patient is sta- bilized.” the investigators agreed to strictly use these scripted positive and negative phrases in their feedback delivery, and those points verbally delivered were docu- mented under “things done well” and “points for improvement.” in addition, the american board of emergency medicine (abem) oral board assessment form was used to generate a quantitative score across eight separate domains. these domains included skills such as data acquisition, problem solving, and interper- sonal relations, and each was scored using a scale of to , with being the highest. prior to study initiation, the protocol was pilot-tested by the investigators with two recent graduates from each of the sites. following each pilot test, the subject was shown the critical actions and feedback checklist, assessment forms and learning goals were discussed openly, and his or her input was used to modify the case and the feedback checklist to develop response process and internal structure validity evidence. the five categories for the subsequent learning goals (discussed below) were developed based on these pilot data. the four investigators administered the examination at their institutions with their own em residents. inves- tigators guided each resident through the standardized protocol individually. first, each resident participated in the oral board case scenario, with the investigator as the examiner. for self-assessment, after the case, each resident scored his or her own performance using the abem oral board evaluation form, then had minutes to note specific strengths and weaknesses in his or her performance (see data supplement s , available as sup- porting information in the online version of this paper). of note, the resident was not given access to the list of critical actions or the feedback checklist, so that the self-assessments could be generated based solely on reflection. the play of the case scenario might have pro- vided some real-time feedback, however, as the patient improved when critical actions were met, but decom- pensated when errors were made or critical steps were not taken. while the resident completed the self-assess- ment form, the examiner completed the feedback check- list and abem scoring form (data supplement s ). forms were not shared, and the resident was asked to avoid discussing the self-assessment with the examiner. the examiner then verbally provided two to four spe- cific positive feedback phrases from the “things done well” section of the form and two to four “points for improvement.” this number from two to four for each was chosen by group consensus based on experience, as well as the coursework on medical education research, revealing that overwhelming the learner with “too much” feedback proves counterproductive. follow- ing the self-assessment and feedback delivery, the resi- dent was asked to generate smart learning goals in writing based on the entire experience (see data sup- plement s , available as supporting information in the online version of this paper). clear definitions and examples of smart goals, adapted from a study by chang et al., were provided and each resident was asked to read them prior to listing his or her learning goals (table ). last, the resident was asked to rate the effectiveness of the feedback received from the exam- iner using a five-question feedback rating form adapted from a study by eva et al. on the generation of learn- ing goals. an eight-point likert rating scale from “worst” to “best” was used for each question, and the fifth question on “overall quality” was used for the sta- tistical analysis (see data supplement s , available as supporting information in the online version of this paper). immediately following data collection at each site, the investigators reviewed and categorized the raw data. the feedback checklists and assessment forms were uploaded onto a cloud platform and at least three inves- tigators reviewed the data by conference call. the team reviewed the forms for completeness, came to consensus on interpreting the source of the residents’ learning goals, and categorized them as stemming from the self- assessment or the examiner’s feedback. separate catego- ries were created for those learning goals that came from both the self-assessment and the feedback in agreement, as well as categories for one in disagreement table smart learning goals and examples “smart” learning goals examples specific “i will be able to clearly hear systolic murmurs in adult and pediatric patients.” measurable “i will improve my in-training exam score by % over the next year.” achievable “i will read two to four articles per month on important medical topics.” realistic “i will overcome my hesitancy to discuss my differential diagnosis on rounds.” time-bound “i will improve % in weeks and achieve my goal by may” academic emergency medicine • october , vol. , no. • www.aemj.org with the other. in addition, two separate groups were created for the purposes of data analysis: total associ- ated with self-assessment and total associated with feed- back. the former group included three categories: self- assessment only, self-assessment and feedback in agree- ment, and self-assessment in disagreement with the feedback. the latter group was the sum of learning goals from feedback only, from self-assessment and feedback in agreement, and from feedback in disagreement with self-assessment. for follow-up, within to weeks of the oral board case, the residents were given a form and asked to recall their learning goals and describe any actions taken toward achieving those goals. for example, if the resident wrote the learning goal, “i will review the acls protocols for unstable tachycardia,” did she remember that goal on follow-up, and did she actually study the acls protocols in the past couple of weeks? the learning goals recalled and reportedly acted upon at follow-up were categorized in a similar fashion to the initially generated learning goals so that the initial and follow-up data could be compared. data analysis descriptive statistics (frequency tables, % confidence intervals) were used to summarize the data. to deter- mine the summary self-assessment ratings and faculty ratings of the oral board case, the scores from each domain (data acquisition, problem solving, patient man- agement, etc.) from the abem forms were summed. to address concerns of violation of normality and homo- scedasticity, the error variance of the model did not differ significantly from normal (shapiro-wilk w = . , p = . ), and the model did not show signs of hetero- scedasticity (breusch-pagan test = . , p = . ). linear regression compared the quantitative self-assessment and faculty assessment scores. we estimated each resi- dent’s likelihood of incorporating feedback into his or her learning goals using residual maximal likelihood (reml). these estimates were log-likelihood ratios where a likelihood ratio of . meant that a resident was % as likely as the average resident to use feed- back in generating learning goals. we ran pearson’s correlations between perceived quality of feedback, self- assessment, faculty assessment ratings, and the likeli- hood of using faculty feedback for the generation of learning goals. finally, quality of feedback, self-assess- ment, and faculty ratings were entered into a regression with likelihood of incorporating feedback into learning goals as the dependent variable. results source of learning goals of the residents offered an opportunity to participate in the study at the four sites, % (n = ) volunteered. the enrolled subjects generated a total of learning goals (mean � sd = . � . per resident), which were categorized by the investigators according to the source, whether from self-assessments or feedback (table ). the majority of learning goals were associated with the residents’ own self-assessments ( %). surprisingly, fewer than half of the learning goals were generated based on faculty feedback. residents almost never incor- porated feedback that was in disagreement with their own self-assessments; however, they sometimes gener- ated learning goals based on self-assessments that con- tradicted the feedback provided by the examiners ( %). the relationship between faculty assessment and resident self-assessment scores there was some agreement between faculty scores and resident self-scoring, as one might expect given the standardized domains of the abem oral board assess- ment form. linear regression of self-assessment by fac- ulty assessment reached statistical significance, although the correlation was weak (r = . , p < . ). factors affecting the generation of learning goals a minority of our residents incorporated feedback into their learning goals. we analyzed different factors in an attempt to determine which residents were more or less likely to integrate external feedback. first, we sought to determine whether residents use feedback based on perceived quality. we compared the residents’ ratings of feedback quality to their likelihood of using the feed- back and found no significant relationship (r = . , p = . ). this suggests that learners are not more likely to use feedback that they deem of higher quality. another secondary hypothesis was that residents who score themselves highly (i.e., are more self-confi- dent) would be less likely to integrate external feedback. however, a relationship between self-assessment scores and incorporation of feedback was also not significant (r = . , p = . ). next, we compared the faculty rating (as opposed to self-assessment rating) to the incorporation of feed- back. did those residents who were high performers (by faculty ratings) use feedback less often? here, we found a negative relationship between faculty ratings and the use of feedback (r = – . , p < . ). this sug- gests that high performing residents were less likely to incorporate feedback, and poor performers tended to use feedback for incorporation into their learning goals. table sources of learning goals goal category no. (%) by goal (n = ) no. (%) by resident* (n = ) self-assessment only ( ) ( ) feedback only ( ) ( ) self-assessment and feedback, in agreement ( ) ( ) feedback, in disagreement with self-assessment ( ) ( ) self-assessment, in disagreement with feedback ( ) ( ) total associated with self-assessment ( ) ( ) total associated with feedback ( ) ( ) *each resident documented multiple learning goals. bounds et al. • em resident feedback because the correlation between faculty rating and feedback utilization was the only significant relationship found, we controlled for self-assessment scores and quality of feedback ratings in case these might serve as confounders. all three variables were analyzed in a lin- ear regression model to generate a likelihood ratio of . (table ). in other words, a -point higher score by faculty rating (out of points total, as eight domains were scored to each) makes the resident % less likely to integrate feedback into a particular learning goal. follow-up on learning goals and actions taken following a period of to weeks, subjects were asked which learning goals they were able to recall from the case, if any, and what actions they had taken to improve performance. seventy-two residents initially generated a total of learning goals. at to weeks, of the initial residents responded to the follow-up question- naire, and this group recalled a total of learning goals (mean = . learning goals per resident). of those, % ( of ) were reportedly acted upon (mean = . learning goals per resident). the sources of learning goals recalled and acted upon are summarized in table . although the origins of immediate learning goals were heavily weighted toward self-assessments, there was a shift toward feedback on the follow-up recall and actions taken. feedback that agreed with self-assess- ments led to the greatest number of actions taken to improve future performance. discussion we conducted a multicenter observational cross-sec- tional study of em residents taking an oral board exam- ination, performing a structured self-assessment, and receiving feedback from evaluators. we were surprised to find that the initial learning goals generated by the residents based on the experience were more strongly influenced by their own self-assessments than by faculty feedback. the follow-up actions taken, on the other hand, more often integrated faculty feedback as long as it agreed with the residents’ self-assessments. one’s self-assessment for a given task is influenced by multiple factors, including prior experience, confi- dence, and the context of the activity. , studies have shown that physicians’ self-assessments share very little association with external measures of objective perfor- mance. , , our study’s findings agreed, in that we found a very weak association between faculty scores and resident self-assessment scores. this suggests that residents whom faculty rated poorly tended to overesti- mate their performance while those rated highly tended to underestimate their performance. given the inaccu- racy of self-assessments, some authors have called into question the use of self-assessment tools in medical edu- cation and their value to performance improvement and patient care. on the contrary, we found that self- assessments are integral to the residents’ goals and plans to improve and thus have value that requires greater recognition. our study aimed to separate self-assessments and faculty feedback to determine the roles each of these table linear regression model* variables (intercept) likelihood ratios estimate standard error t-value p-value self-assessment . . . . . quality of feedback . . . . . faculty assessment . – . . – . < . *adjusted r = . . table sources of learning goals recalled and acted upon on follow-up questionnaires goal category goals recalled residents recalling goals goals executed residents executing goals n for column self-assessment only ( ) ( ) ( ) ( ) feedback only ( ) ( ) ( ) ( ) self-assessment and feedback, in agreement ( ) ( ) ( ) ( ) feedback, in disagreement with self-assessment ( ) ( ) ( ) ( ) self-assessment, in disagreement with feedback ( ) ( ) ( ) ( ) total associated with self-assessment ( ) ( ) ( ) ( ) total associated with feedback ( ) ( ) ( ) ( ) data are reported as n (%). academic emergency medicine • october , vol. , no. • www.aemj.org play, and how they interact, in the development of learning goals and in the reported execution of those goals. we found that the vast majority of learning goals were generated from the residents’ own self-assess- ments, while fewer than half of the initial learning goals incorporated faculty feedback. on the other hand, on subsequent follow-up, the actions taken to improve showed that faculty feedback had a greater influence than initially measured. the total learning goals report- edly executed showed an equal influence between self- assessments and feedback, and the strongest stimulus came from agreement between feedback and self- assessments. despite the known inaccuracy of self- assessments in physician evaluation, it is clear that we must at least consider these self-assessments when pro- viding feedback if we hope to influence performance. our results support the findings of prior studies, which have shown that the feedback evaluators provide is always interpreted through the “lens” of the residents’ self-assessments. our results indicate that the learning goals created by residents are based more on their self-assessments, while their actual behaviors and actions integrate exter- nal feedback, as long as it agrees with their self-assess- ments. it is possible that self-assessments play a greater role in motivating self-directed learning, such as learn- ing goals generation, while feedback plays a greater role in changing actual behaviors. although not conclu- sive, our results are hypothesis-generating and might lead to subsequent studies where a similar protocol is used with detailed recording of actual actions taken and repeat testing to objectively assess for improvement. in our analysis to determine which residents incorpo- rate our feedback into their learning goals, we found no significant relationship with quality of feedback rating or self-assessment scores. we did find an inverse rela- tionship between faculty scoring and feedback integra- tion, indicating that higher performers were less likely to use feedback from evaluators. the etiology of this is unknown. this could be explained by the underlying self-confidence of those high performers or, conversely, the greater interest in feedback on the part of lower performers. alternatively, the specific feedback pro- vided to higher performers may have been less action- able and perceived as less relevant. meanwhile, lower performers may have received feedback that was more critical to management. this is an area that requires further research. given these findings, educators might consider inte- grating more self-assessments into the various training modalities of the residency curriculum. we found that agreement between self-assessments and feedback led to the most actions reportedly taken to improve. per- haps once the learner’s self-assessment is communi- cated to the evaluator, the feedback can then be modified to make acceptance and integration more likely. feedback could be carefully molded into the framework of the learner’s own conclusions: positive points might be reinforced, while constructive feedback could be focused on the specific task to preserve one’s self-esteem. feedback integration is an area that is ripe for further research and exploration in many areas of resident education and even clinical care. limitations we chose an oral board examination as the tool for measurement of the variables of interest; this may not be generalizable to other contexts. it is possible that em residents had difficulty generating smart learn- ing goals, despite being provided with a clear frame- work and written examples. however, one of the new em practice-based learning and improvement mile- stones specifically requires residents to “implement learning plans.” em residents may require training in the development of learning goals and learning plans. regarding our data analysis, the categorization of residents’ learning goals according to the source was somewhat subjective. we minimized this by using clear definitions of categories and by ensuring that a group of at least three investigators simultaneously reviewed the self-assessments, feedback checklists, and learning goals and reached consensus agreement in all cases. for each site’s data analysis, the investigator who served as the examiner was on the conference call participating in the categorization; this may have introduced some bias, but we found this necessary, at times, to interpret what the resident wrote on the form and place the responses into context given the investigator’s knowledge of the encounter. for the fol- low-up data, of the original subjects completed the questionnaire, with not participating, mostly due to off-service or away rotations; this may have introduced bias. the actions taken were self-reported by the residents, and there was no mechanism to ver- ify the completion of those actions in this study proto- col. perhaps a future study that retests the subjects with another oral board case could more objectively assess for performance improvement. last, the multi- center nature of this study, while strengthening the validity of our findings, did introduce a potential for variability in style of examination administration and delivery of feedback between examiners. this variabil- ity was minimized by the group’s creation of a struc- tured feedback checklist. conclusions as teachers in graduate medical education, we too often focus on the quality and delivery of feedback. a grow- ing body of literature is demonstrating the need to focus more on the receiving end—the learners’ recep- tion and integration of our feedback. while this study found that the majority of initial learning goals gener- ated stemmed from the residents’ own self-assessments, most of the actions taken to improve after a follow-up period were from feedback and self-assessment in agreement. in addition, higher performers were less likely to use evaluator feedback than lower performers. these findings support the evidence that we, as educa- tors, need to gain an understanding of how residents assess their own performance, so that feedback might be modified and delivered in a way that is interpretable in the context of the residents’ self-assessments. although these self-assessments may sometimes be inaccurate, they lay the foundation upon which to 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http://www.acgme.org/acgmeweb/. accessed jul , . supporting information the following supporting information is available in the online version of this paper: data supplement s . ventricular fibrillation cardiac arrest. data supplement s . post-test evaluator assessment. data supplement s . post-test self-assessment. data supplement s . learning goals. data supplement s . feedback rating form. academic emergency medicine • october , vol. , no. • www.aemj.org clinical transplantation home glucometer monitoring markedly improves diagnosis of post renal transplant diabetes mellitus in renal transplant recipients amish shah, gwen kendall, richard a. demme, jeremy taylor, adel bozorgzadeh, mark orloff, ashok jain, peter abt, and martin s. zand , background. definitions of de novo posttransplant diabetes mellitus (ptdm) have varied widely in the renal trans- plant literature, and most have not used the american diabetes association (ada) definition of diabetes (fasting plasma glucose [fpg] � mg/dl on two occasions, or a casual plasma glucose level � mg/dl). most patients are monitored for ptdm by -hour fpg levels drawn for clinic visits. in contrast, we describe the diagnosis of ptdm by home glucometer monitoring methods. we screened consecutive nondiabetic renal transplant recipients for ptdm by ada criteria and home glucometer monitoring during the first months posttransplant results. of patients with impaired fasting glucose levels of – mg/dl, ( %) met ada criteria for diabetes mellitus of based on home glucometer monitoring. the incidence of de novo ptdm was % during this period. predictors of ptdm in a cox proportional hazards model were race and acute rejection, with a trend towards bmi. clinic visit fpg levels did not differ between ptdm and non-ptdm patients. all diagnoses were made based on prelunch or supper fpg � mg/dl conclusions. overnight fpg are inadequate for diagnosis of ptdm. all renal transplant recipients with impaired fpg should, at minimum, have home fpg testing. (transplantation ; : – ) de novo posttransplant diabetes mellitus (ptdm) occursin – % of renal transplant recipients, with the inci- dence increasing over time after transplantation ( ). the pathogenesis of ptdm is multifactorial, with corticosteroid administration ( ), calcineurin inhibitors ( , ), sirolimus ( ), obesity ( , , ), race ( ), hepatitis c infection ( ) and age ( ) all implicated as causative factors. the pathophysiology of ptdm involves both islet cell dysfunction ( ) and insulin resistance ( , , ). compared with normoglycemic trans- plant recipients, patients with ptdm have an increased risk of graft loss, cardiovascular morbidity, and premature death with a functioning allograft ( , ). timely diagnosis and accurate assessment of the prev- alence of ptdm have been confounded by narrow definitions of posttransplant diabetes mellitus including “a new require- ment for insulin therapy for more than days” ( – ), and “a new requirement for insulin or oral hypoglycemic agents” ( ). few reports in transplant recipients have used the ada- accepted definition of diabetes (fasting plasma glucose [fpg] � mg/dl on two occasions, or a single random blood glu- cose level � mg/dl), and only a handful have addressed the presence of impaired glucose tolerance (fpg – mg/dl) ( , , , ). indeed, it is only recently that the trans- plant community has endorsed the standard clinical criteria for diagnosis of diabetes mellitus ( ) established by the world health organization ( ) and the american diabetes association ( ). most renal transplant patients are monitored for ptdm based on -hour fpg levels drawn concurrently with their tacrolimus, sirolimus, or cyclosporine trough or c- levels, although these levels may not accurately reflect overall glycemic control. in patients with type diabetes, some re- ports suggest that overnight fpg levels correlate poorly with hba c levels, whereas postprandial and suppertime glucose levels are a better indicator of glycemic control ( ). in this report, we describe the results of home fpg monitoring in conjunction with the ada criteria for diagnosis of posttrans- plant diabetes in a cohort of renal transplant recipients. methods human subjects’ protection this study was approved by the research subjects re- view board at the university of rochester medical center. division of nephrology, department of medicine, university of rochester medical center, rochester, ny. division of solid organ transplantation, department of surgery, univer- sity of rochester medical center, rochester, ny. address correspondence to: martin s. zand, m.d., ph.d., division of nephrology, university of rochester medical center, elmwood ave- nue, box , rochester, ny . e-mail: martin_zand@urmc.rochester.edu. received february . revision requested march . accepted april . copyright © by lippincott williams & wilkins issn - / / - doi: . / .tp. . .f transplantation • volume , number , september , research data were coded such that subjects could not be identified, directly or through linked identifiers, in compli- ance with the department of health and human services regulations for the protection of human subjects ( cfr . (b) ( )). data collection from november through february , all adult (age � years) kidney and kidney pancreas transplant recip- ients with morning clinic fasting plasma glucose levels of – mg/dl after the second week posttransplant were given home glucometers (one-touch; lifescan, milpitas, ca), test strips, and lancets. patients were instructed in clinic on proper use of the glucometer, calibration, timing of test- ing, and recording of results. glucometer calibration was ver- ified in clinic by manufacturer’s instructions, and validated by comparison with plasma glucose levels from the clinical laboratory on their next return visit. preprandial (breakfast, lunch, and supper) and bed- time glucose levels were recorded for days. a diagnosis of diabetes mellitus was made by the american diabetes asso- ciation criteria, as recommended by the american society of transplantation from – ( , , ): fpg � mg/dl on two occasions or a single random blood glucose level � mg/dl. impaired plasma glucose was defined as a -hour fpg level of – mg/dl. during the days fol- lowing any steroid treatment for acute rejection, diagnosis of diabetes was suspended. to determine the total incidence of ptdm during the same time period, we also performed a retrospective review of all kidney and kidney pancreas transplants (n� ). demo- graphic data collected included the age, gender, ethnicity, type of transplant, pretransplant diagnosis of diabetes, hepa- titis c serology, weight, body mass index, family history of diabetes, time to diagnosis after transplantation, presence of delayed graft function, mean and cumulative tacrolimus doses, mean and cumulative steroid doses, use of antibody induction therapy, and modality and duration of dialysis prior to transplantation. a family history of diabetes was de- fined as the presence of diabetes mellitus in a first degree relative. delayed graft function was defined as the need for dialysis within the first seven days posttransplant. immunosuppression regimen during the period covered by this study, all patients received tacrolimus, mycophenolate mofetil, and prednisone based immunosuppression, with the exception of one patient receiving modified cyclosporine. patients were discharged from the hospital on mg/day of prednisone, which was tapered to mg/day after weeks, mg/day after weeks, and then down to mg/day at week posttransplant. goal serum tacrolimus levels were – ng/ml (imx assay) dur- ing the first weeks, – ng/ml during weeks – , – ng/ml during weeks – , and – ng/ml thereafter. only patients received antibody induction therapy. statistical analysis statistical analysis was performed using the statistica software package (statsoft ; statistica for windows, tulsa, ok). mean tacrolimus levels during the first days posttransplant were derived by multiplying the number of days between the previous tacrolimus level by the next ta- crolimus level. the mean level was then calculated by adding all interval weighted values and then dividing by the total number of days. demographic data was compared using fisher’s exact t test ( tailed) for categorical variables, and the mann-whitney u test for continuous variables. a cox pro- portional hazards analysis was conducted to determine the relative risk of developing posttransplant diabetes mellitus. the proportion of patients diagnosed with ptdm by the ada criteria versus the alternative definition of a de novo need for insulin greater than days were compared using the mcnemar test for paired proportions. the study was pow- ered to detect a % difference in these proportions (�� . , �� . , n� ). results diagnosis of posttransplant diabetes mellitus in all, consecutive renal transplant recipients were screened for impaired glucose tolerance – days post- transplant (fig. ). renal transplant recipients without a pre- transplant history of diabetes mellitus ( %) who presented to the outpatient clinic were classified according to the ada diagnostic criteria: normoglycemic (fpg � mg/dl), impaired (fpg – mg/dl) and diabetic (fpg � mg/dl). five patients ( %) presented with overt ptdm, whereas another ( %) had fpg levels within the im- paired range. all of patients with impaired fpg were pre- scribed glucometers and asked to check their preprandial breakfast, lunch, supper and prebedtime plasma glucose lev- els at home for consecutive days. patients were trained in clinic on proper glucometer use and calibration. twenty-three patients with impaired glucose tolerance had three days of complete home glucometer readings, and were used for statistical analysis of blood glucose patterns. figure b shows the mean preprandial plasma glucose levels in the patients. ptdm was diagnosed in ( %) patients with ipg by at least two preprandial plasma glucose levels of mg/dl or greater. almost uniformly, the diagnoses were based on preprandial lunch and suppertime levels. only one patient was a simultaneous kidney-pancreas recipient, while the remaining received kidney transplants. six patients had fewer than full days of glucometer readings, and five of these were also diagnosed with ptdm and severe hyperglycemia within hr. these individuals had presuppertime pg levels of � mg/dl and were immediately started on therapy. all patients with ipg had symptoms of diabetes by interview, defined as polyuria, polydipsia, and unexplained weight loss. however, the nondiabetic cohort frequently reported poly- uria ( %), polydipsia ( %), and weight loss ( %). thus, attributing these symptoms to diabetes mellitus in the early posttransplant period was problematic. risk factors for ptdm in patients with glucose intolerance factors which might predict differences between the impaired plasma glucose (ipg), euglycemic and ptdm sub- groups were studied in the patients for whom we had days of complete preprandial fpg levels. table shows the demographics for both groups, and univariate analysis indi- cated that ipg patients who developed ptdm tended to be transplantation • volume , number , september , older, received antibody induction therapy, and have had a rejection episode. there was a trend towards having a positive family history of diabetes, which did not reach statistical sig- nificance. a cox proportional hazards analysis confirmed these risk factors, as well as identifying family history of dia- betes and body mass index (bmi) � kg/m (table ). duration of hemodialysis for longer than months prior to renal transplantation was associated with a decreased risk of ptdm. in neither analysis did the mean steroid dose, mean tacrolimus level, or delayed graft function correlate with the figure . (a) classification of renal transplant study cohort based on american diabetes association criteria for diag- nosis of diabetes and glucose intolerance. patients in this cohort were screened within - days posttransplant. (b) results of home glucometer testing in the glucose intolerant cohort (clinic fpg - mg/dl). clinic, preprandial breakfast (br), lunch (lu), supper (su) and bedtime (ev) reflect three measurements on consecutive days for each patient. clinic fpgs reflect two measurements on two separate clinic visits within days. results are mean � standard deviation. table . characteristics of patients with fasting plasma glucose levels - mg/dl within days posttransplant non-ptdm ptdm p value n non-caucasian % % . age . � . . � . male . % . % ns body mass index (kg/m ) . � . . � . ns family history of diabetes . . % . mean tacrolimus level (ng/ml) . � . . � . ns mean steroid dose (mg/d) – days . � . � . ns acute rejection . % . % . delayed graft function . % . % ns antibody induction . % . % . hepatitis c virus . % . % ns cadaveric organ . % . % . duration of dialysis (months) . � . . � . ns ptdm, posttransplant diabetes mellitus. © lippincott williams & wilkins shah et al. development of ptdm in the ipg group. the presence of hepatitis c infection was not associated with development of ptdm, although the number of such patients was quite small and the analysis lacked power to detect significant differences. unfortunately, pretransplant hemoglobin a c levels were not uniformly available for this cohort and thus not included in the analysis. prevalence of and risk factors for ptdm in the entire study cohort we next examined differences in the entire patients between all patients who developed ptdm (n� ), and those who did not. this cohort included the five patients with overt ptdm on fasting clinic labs (fig. ), the patients diagnosed within the first days posttransplant ( patients with complete -day glucometer readings and the five pa- tients with incomplete glucometer data; figure ), and an additional group of four patients who were diagnosed with ptdm after the day posttransplant glucometer study pe- riod. figure b shows the kaplan-meier plot of the percent- age of patients without a pretransplant diagnosis of diabetes who remained free of ptdm over the – days of the glu- cometer study, and from – days posttransplant. the majority of patients in this series presented within the first months posttransplant. of the patients who met ada cri- teria for ptdm during the first years posttransplant, only five ( %) would have been diagnosed with ptdm by the oft used criteria of “a de novo requirement for insulin lasting greater than days” (p� . ). univariate analysis of the ptdm versus euglycemic subgroups revealed statistically sig- nificant differences between the two groups in non-cauca- sian race, bmi, acute rejection, and induction therapy (table ). a cox proportional hazards model showed that non- white ethnicity/race, bmi� , age� years, induction ther- apy, rejection episodes, and family history were all associated table . cox proportional hazards model for development of ptdm in patients with fasting plasma glucose - mg/dl factor odds ratio range p value induction . ( – ) . body mass index � . ( – ) . non-caucasian . ( – ) . rejection . ( – ) . dialysis � months . ( . – ) . family history ns delayed graft function ns hepatitis c virus ns prednisone � g ns tacrolimus � ng/ml ns age � years ns peritoneal dialysis ns cadaveric graft ns ptdm, posttransplant diabetes mellitus. figure . (a) follow-up of post- transplant diabetes regimens. this co- hort of patients included patients with ptdm diagnosed within the first days posttransplant (n� ) of glucom- eter monitoring, as well as an addi- tional patients who were diagnosed with ptdm after days posttrans- plant. although some patients came off insulin therapy, the majority of pa- tients required ongoing oral or insulin therapy for glycemic control. (b) kaplan-meier graph showing the pro- portion of patients without diabetes pretransplant that remained diabetes- free after kidney or kidney-pancreas transplantation. transplantation • volume , number , september , with an increased odds of developing ptdm. again, pre- transplant duration of dialysis � months lowered the odds of developing ptdm (table ). resolution of ptdm all patients who were diagnosed with ptdm by home glucometer monitoring were started on hypoglycemic ther- apy at the time of diagnosis, either oral hypoglycemic agents or insulin. this was done as part of the standard of care for management of ptdm, and not as a study intervention. pre- vious reports in the literature have noted the resolution of ptdm, as defined by loss of the need for insulin therapy ( ). we were thus interested in whether ptdm resolved in this cohort, as more appropriately defined by loss of the need for both oral agents and insulin to maintain euglycemia. while patients who initially required insulin therapy were eventu- ally weaned to oral hypoglycemic agents, only one patient became euglycemic as assessed by both preprandial glucom- eter monitoring and hga c level of . ; all other patients continued to require oral hypoglycemic therapy. discussion this study challenges the previously published conclu- sions that ptdm is a condition of limited frequency, is most appropriately diagnosed by the de novo requirement for over days of insulin therapy, and resolves once euglycemia can be maintained off insulin. only recently has the renal trans- plant community endorsed the international standards for diagnosis of diabetes mellitus. prior to this, the presence of numerous definitions for ptdm in the literature has, unfor- tunately, prevented an accurate assessment of the true mag- nitude of posttransplant diabetes mellitus ( , ). we believe that the use of such non-standard definitions of diabetes in renal transplantation has likely led to the under-diagnosis and under-treatment of ptdm throughout the transplant community. similarly dubious criteria for “resolution” of ptdm, irrespective of clinical measures of hyperglycemia or the need for oral hypoglycemic agents, have led to a false sense that ptdm is a short-lived condition limited to the peri- transplant period. our findings support the utility of preprandial glucose screening when monitoring renal transplant recipients for ptdm. when hr fasting glucose levels are within the “im- paired glucose tolerance” range of the ada criteria, a sub- stantial number of these patients will have prelunch and pre- suppertime levels greater than mg/dl that are diagnostic of diabetes mellitus. this pattern of hyperglycemia, charac- terized by a rising glycemic levels throughout the day, but – hr fasting euglycemia, was seen in all subjects with ptdm. it is interesting that some studies in non-transplant type diabetics have not shown a similar pattern ( ). one possible explanation for this difference may be the effect of prednisone which, when taken in the morning, causes an in- crease in insulin resistance which peaks between lunch an suppertime. alternatively, the morning dose of tacrolimus may have impaired insulin secretion at times which coincide with peak oral carbohydrate intake. further study of glycemic patterns in patients on steroid free, tacrolimus based immu- nosuppression regimens will be necessary to separate these effects. it is likely that a significant number of dialysis patients table . demographics of all patients without a pretransplant diagnosis of diabetes non-ptdm ptdm p value n non-caucasian . % . % . age . � . . � . ns male . % . % ns bmi (kg/m ) . � . .l � . . family history of diabetes . % . % ns mean tacrolimus level (ng/ml) . � . . � . ns mean steroid dose (mg/d) – days . � . . � . ns acute rejection . % . % . delayed graft function . % . % ns antibody induction . % . % . hepatitis c virus . % . % ns cadaveric organ . % . % ns duration of dialysis (months) . � . . � . ns ptdm, posttransplant diabetes mellitus. table . cox proportional hazards model for development of posttransplant diabetes mellitus in the entire cohort factor odds ratio range p value non-caucasian . ( . – . ) . age � . ( . – . ) . induction . ( . – . ) . rejection . ( . – . ) . family history . ( . – . ) . body mass index � . ( . – . ) . dialysis � months . ( . – . ) . delayed graft function ns hepatitis c virus ns prednisone � g ns tacrolimus � ng/ml ns peritoneal dialysis ns cadaveric graft ns © lippincott williams & wilkins shah et al. have occult type diabetes, which is then unmasked after renal transplantation. while the primary pathophysiology of type diabetes is insulin resistance, �-cell insulin secretion also declines over time ( – ). the clinical appearance of overt diabetes is often delayed as end stage renal disease in- creases the half-life of plasma insulin by % ( ). thus, esrd patients may progressively lose islet cells, but maintain euglycemia for some time. this may account for our finding that a duration of dialysis greater than months lowered the odds of developing ptdm. we hypothesize that this group may have lost enough additional �-cell mass during the years on dialysis to become overtly diabetic despite an in- crease in insulin half life. after kidney transplantation, how- ever, the restoration of renal insulin metabolism, coupled with steroid induced insulin resistance ( ) and calcineurin inhibitor impairment of insulin secretion ( ) all combine to reveal previously undiagnosed diabetes. although we found that home glucose monitoring markedly improves diagnosis of ptdm, our study cohort was restricted to patients with impaired fasting glucose levels and had a modest sample size. the former approach may miss patients with early type diabetes, who may have quite nor- mal fasting glucose levels, but postprandial hyperglycemia ( , ). indeed, several investigators have noted that when only fasting blood glucose is measured, impaired glucose tol- erance may remain undetected in some subjects ( ). the latter issue of modest sample size makes the risk of a type i error somewhat more likely. thus, our results suggest that a larger prospective study of pre- and postprandial glucose lev- els, with appropriate statistical power, should be undertaken. within the endocrinology community, the definitions of diabetes mellitus and impaired glucose tolerance, as well as the recommended methods for diagnosis, have been evolv- ing. indeed, after this study had been completed, the ada changed their definition of impaired glucose tolerance to an -hour fasting plasma glucose level of between - mg/dl ( ). this change was made to bring the definitions of im- paired fasting glucose (ifg; -hour fasting plasma glucose reading) and impaired glucose tolerance (igt; using oral glu- cose tolerance testing) closer in line in diagnosis of type diabetes ( ). one caveat to our study is that we were not able to capture patients who would meet the newer ada criteria for ifg. in addition, the who and ada criteria differ with respect to the need for an abnormal oral glucose tolerance test to diagnose diabetes. the who criteria focus on an abnor- mal ogtt, while the ada criteria do not recommend ogtt for routine clinical use. while our study did not perform ogtts, all of our patients met ada criteria for diagnosis of diabetes based on random blood glucose levels � mg/dl. both ogtt and fpg monitoring capture the vast majority of patients with type diabetes, and combined testing has a higher sensitivity and specificity ( ). with respect to screen- ing for ptdm, the addition of ogtt or combined pre- and postprandial plasma glucose testing to home glucometer monitoring would likely detect more cases of ptdm. further study will be necessary to determine the sensitivity and spec- ificity of home monitoring with respect to the ogtt. almost all patients diagnosed with ptdm in this study continued to require either insulin or oral hypoglycemic ther- apy, suggesting that the diagnosis of ptdm was not an arti- fact of early peritransplant steroid induction therapy. several recent studies have defined the resolution of ptdm as revert- ing to a state of noninsulin dependence ( – ). this defini- tion obscures the true issue of whether the physiologic state of diabetes mellitus persists in these patients, but can be man- aged with oral hypoglycemic agents. consistent with our re- sults is the finding that the incidence of ptdm continues to increase for years after renal transplantation ( , ). if ptdm truly regressed, one should see an improvement in the prev- alence of ptdm over time in a cohort of posttransplant pa- tients. this work does not address the use of home glucose monitoring to optimize glycemic control in renal transplant recipients. although preprandial glucose testing has been the standard for self-management of glycemic control in diabetes mellitus, there is increasing evidence to support the use of postprandial monitoring to adjust oral hypoglycemic and in- sulin therapy ( , ). our results and those of others ( , ) suggest that preprandial glycemic patterns in steroid treated transplant recipients on tacrolimus may differ from those seen in diabetic patients not taking these medications. fur- ther study will be necessary to determine whether postpran- dial glycemic monitoring is a useful adjunct in this popula- tion. based on our findings, we conclude that renal trans- plant recipients with -hr fasting plasma glucose levels in the impaired glucose tolerance range ( – mg/dl) should, at minimum, undergo preprandial home glucose monitoring. acknowledgments the authors would like to acknowledge the assistance of the transplant coordinators barbara byer, mary kremer, kathy stewart, joanne monaghan, leah bryan, and jennifer timm in the acute and continuing care renal transplant clinics at the university of rochester medical center. references . kasiske bl, snyder jj, gilbertson d, matas aj. diabetes mellitus after kidney transplantation in the united states. am j transplant ; 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(suppl_ ): s . . borch-johnsen k, colagiuri s, balkau b, et al. creating a pandemic of prediabetes: the proposed new diagnostic criteria for impaired fasting glycaemia. diabetologia ; ( ): . . cosio fg, pesavento te, osei k, et al. post-transplant diabetes melli- tus: increasing incidence in renal allograft recipients transplanted in recent years. kidney int ; ( ): . . suzuki h, fukushima m, usami m, et al. factors responsible for devel- opment from normal glucose tolerance to isolated postchallenge hy- perglycemia. diabetes care ; ( ): . . abrahamson mj. optimal glycemic control in type diabetes mellitus: fasting and postprandial glucose in context. arch intern med ; ( ): . © lippincott williams & wilkins shah et al. case report open access virilizing ovarian steroid cell tumor in a year old south indian female: a case report shihas salim , ghanshyam palamaner subash shantha , amish dilip patel *, anita a kumar , prasanthi ganeshram , nikita mehra , anish george rajan , tarun joseph and lavangi sudhakar address: department of general medicine, sri ramachandra university, chennai, india and department of obstetrics and gynecology, sri ramachandra university, chennai, india email: ss - shihas.salim@gmail.com; gpss - drpssghanshyam@yahoo.co.in; adp* - amishdoc@gmail.com; aak - ghanindia@gmail.com; pg - preshg@gmail.com; nm - nikitamehra @hotmail.com; agr - dr.anish.rajan@gmail.com; tj - tarunjoseph @gmail.com; ls - lavangisudhakar@gmail.com * corresponding author published: may received: january accepted: april cases journal , : doi: . / - - - this article is available from: http://casesjournal.com/casesjournal/article/view/ © salim et al; licensee cases network ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract virilism is the masculinization and enhancement of male secondary sexual characteristics in females. the etiology is usually of adrenal or ovarian origin. here we report a case of virilizing leydig cell type, steroid cell tumor of the left ovary, in a year old female who presented with clinical signs and symptoms of virilization: deepening of voice, hirsutism (ferriman-gallwey score ), clitoromegaly, and androgenic alopecia. on further evaluation, laboratory investigations revealed hyperandrogenism in the male range. basal testosterone values were elevated. folicle stimulating hormone and luteinising hormone levels were within normal limits. dexamethasone suppression test did not alter cortisol or testosterone levels. an ovarian mass was confirmed radiologically. following a total abdominal hysterectomy with bilateral salpingoophorectomy, histopathological studies confirmed a left sided steroid-cell ovarian tumor, leydig cell type (stage t n m ), which proved to the etiology of virilization in this patient. post-operatively her serum testosterone levels declined with near-complete reversal of symptoms over time. introduction hirsutism is defined as excessive male-pattern hair growth. virilization refers to a condition in which the androgen levels are sufficiently high to cause additional signs and symptoms such as deepening of the voice, breast atrophy, increased muscle bulk, clitoromegaly, and increased libido [ ]. virilizing ovarian tumors account for . % of all ovarian tumors [ ]. here we report a -year-old woman who presented to the gynecology outpatient department with signs and symptoms of virilization. after investiga- tions and histopathological examination, the patient was found to have a steroid-cell ovarian tumor, leydig-cell type, which proved to the etiology of virilization in this patient. page of (page number not for citation purposes) http://www.biomedcentral.com/info/about/charter/ http://casesjournal.com/casesjournal/article/view/ http://creativecommons.org/licenses/by/ . case presentation a -year-old multiparous woman from south india was referred to our gynecology department with a two year history of progressive deepening of voice, an increase in facial and body hair, and frontal balding; her symptoms have worsened over the past six months. she attained menarche at the age of years, and has had irregular menses lasting to days, every three or four months since then. she is a mother of two healthy boys, aged and years, both of whom were delivered vaginally without complications. the patient has been amenorrheic for the past four and a half years. there is no history suggestive of hypothyroidism or hyperprolactinemia. there is no significant family history, other than the fact that her mother attained menopause at the age of years. she is a known hypertensive on treatment with calcium channel blockers. she is not on any other medications. on examination, the patient was moderately built with a body mass index of kg/m . she had a low-pitched female voice. head and neck examination showed male- pattern alopecia and obvious facial hair (figure ). there was no thyromegaly. the patient had severe hirsuties affecting the chest, forearms, thighs and anterior abdom- inal wall, with a ferriman gallwey score of [ ]. she was afebrile, with a pulse rate of beats per minute and a blood pressure of / mmhg. chest examination revealed bilateral breast atrophy with periareolar hair. on abdominal examination, there were no visible striae; the abdomen was soft, non-tender, with no obvious free fluid or mass on palpation. pelvic examination revealed an enlarged clitoris (figure ), a nabothian follicle over the cervix, and an anteverted, small sized uterus with bilateral forniceal fullness. other systems examination were unremarkable. blood investigations showed a free testosterone level of ng/dl and a serum total testosterone of ng/dl (normal value: - ng/dl). serum cortisol, - hydroxyprogesterone, prolactin, fsh, lh, thyroid hor- mone levels, blood sugars, and all other blood parameters were within normal limits (table ). radiological imaging comprised of a chest x-ray, an ultrasonogram of the abdomen, a computed tomography (ct) scan, and a magnetic resonance imaging (mri) scan. the chest x-ray was normal. the ultrasound of the abdomen revealed polycystic ovaries. the ct-scan of the abdomen and pelvis showed a bulky uterus ( . × . cm) with a mass lesion ( × . × . cm) in the region of the fundus/left adnexa. an mri pelvis with contrast showed two separate, well- defined oval lesions, each one adjacent to either internal iliac vessel (right: × . × . cm; left: . × × . cm). the lesion on the left side showed patchy enhancement after instillation of the contrast, and was seen indenting the uterine fundus. both the ovaries were not visualized separate from either lesion. in view of the patient’s age and multiparity, the management involved a staging laparotomy followed by total abdominal hysterectomy, bilateral salpingoophorectomy, and infracolic omentect- omy. the specimen was sent for histopathological examination. figure . androgenic alopecia and excess facial hair. this photo shows growth of coarse hair over the face and male pattern baldness- characteristic of virilization. stubble is also seen over the chin and mandibular areas. figure . clitoromegaly. an enlarged clitoris is visualized. page of (page number not for citation purposes) cases journal , : http://casesjournal.com/casesjournal/article/view/ macroscopically, both the ovaries were enlarged, tan- brown in color, and stony hard in consistency (right: × cm; left: × cm). the right ovary revealed a uniform solid grey-tan color covering the cut surface. the cut surface of the left ovary showed a solid grey-tan color with irregular grey-brown areas (figure ). on microscopy, there was stromal hyperplasia of both ovaries, with the left ovary showing a well differentiated steroid cell tumor. crystalloids of reinke were seen in plenty, thus establish- ing the tumor to be of leydig cell subtype (figure ) [ , ]. the tumor capsule was intact and there was no evidence of vascular invasion. peritoneal fluid cytology did not reveal any malignant cells. intra-operatively, no significant lymph nodes were noted. thus, tumor was staged t n m . the preoperative elevated serum testosterone level returned to normal. there was a gradual reversal of her symptoms over time; however, the deep voice remains persistent. the patient is being followed up on a regular basis. discussion virilizing ovarian tumors account for less than % of all ovarian neoplasms [ ]. sex cord-stromal tumors, are derived from the sex cord and stromal components of the developing gonad [ ]. steroid cell tumors are a subtype of ovarian sex cord-stromal tumors that are composed entirely of steroid-secreting cells and account for . % of all ovarian neoplasms [ ]. steroid cell tumors are usually virilizing, frequently secreting testosterone; however, they may be endocrinologically inert or estrogenic. as many as one-quarter of steroid tumors exhibit malignant behavior [ ]. steroid cell tumors are further subclassified into stromal luteoma, leydig cell tumors (hilar and nonhilar), and steroid cell tumors that are not otherwise specific [ ]. most pure leydig cell tumors of the ovary arise from the hilus cells, or hilar-leydig cells, which have the morphologic features of testicular leydig cells and can be found in the ovarian hilus in more than % of adult women. these tumors are classified under the category of steroid cell tumors (lipid cell tumors) because they may be difficult or impossible to differ- entiate histologically from ovarian tumors of other steroid cell types [ ]. table . laboratory investigations parameters values hb . g/dl random blood sugar mg/dl tsh . miu/ml estradiol pg/dl fsh . miu/ml lh . miu/ml serum cortisol < . mcg/dl - hydroxyprogesterone . ng/dl serum prolactin ng/dl serum testosterone pre-operative ng/dl post-operative ng/dl free testosterone (pre-operative) ng/dl . ng/dl figure . macroscopic appearance of the ovarian mass. cut section of left ovary showing a solid, grey-tan color with grey-brown patches. figure . microscopic appearance of the ovarian mass. a periodic acid-schiff stained, histopathological specimen of the ovarian tumor ( x magnification), showing crystalloids of reinke. page of (page number not for citation purposes) cases journal , : http://casesjournal.com/casesjournal/article/view/ the clinical manifestations are to a large extent determined by the age of presentation, hormonal activity, and virilizing properties of the tumor. virilization or hirsutism is encountered with three fourths of leydig cell tumors [ ]. the clinical presentation may take many forms, including abdominal pain, abdominal distention, and bloating. however, the more noticeable presentations are those associated with the hormonal activity and virilizing properties of the tumor [ ]. signs and symptoms of masculinizing tumors usually take place in two definite phases, an early phase of defeminization and a subsequent phase of masculinization. typically, a menstruating female will first notice oligomenorrhea or amenorrhea. there is regression of the breasts and external genitalia, atrophy of the uterus and adnexa, and loss of the female body contour. this is followed by hirsutism, acne, clitoral enlargement, increased libido, sterility, enlargement of the larynx, deepening of the voice, and temporal alopecia [ , ]. on the other hand, these tumors may produce little or no androgenic activity and could, in fact, show some evidence of estrogenic effect [ , ]. with regard to blood investigations, detecting the source of the androgenic tumor is a process of exclusion. in our patient, the high serum free and total testosterone confirmed the presence of a virilizing neoplasm. a dexamethasone suppression test failed to alter basal values, moreover, -hydroxyprogesterone levels were also within normal limits; this ruled out a potential adrenal source of the androgens [ , ]. on radiological imaging, appearances of virilizing tumors of the ovary depend, to some extent, on the tumor type. virilizing steroid cell tumors of the ovary are usually one sided and often very small, measuring only slightly bigger than the normal ovary [ , ]. they are usually confined to the ovary at presentation, predominantly solid or mostly solid, non-calcified, and not associated with ascites. small steroid cell tumors have been described as slightly hypoechoic or hyperechoic (compared to the ovary) with high diastolic flow on doppler interrogation. they may be difficult to identify on radiological imaging, in part because they are isoechoic to the uterus on ultrasound and isoattenuating on ct [ , ]. techniques such as mri with phased array coils, or color doppler imaging, can possibly detect smaller tumors than more conventional imaging methods [ , , ]. in our patient, abdominal ultrasound examination was unremarkable except for the presence of ovarian cysts. a ct-scan and an mri helped in confirming the existence of a mass lesion involving the left adnexa. it was the post-operative histopathological study, however, that helped in clinching the final diagnosis as the presence of reinke’s crystalloid is diagnostic for leydig cell tumors [ , , ]. though steroid cell tumors of leydig-cell subtype are usually benign, there is still a potential risk for malignant transformation [ , , ]. surgery remains the mainstay in the management of these neoplasms as, in addition to overcoming the above risk, however small, the removal of the tumor is usually followed by near-complete regression of the presenting symptoms. initially, the signs of defeminization, such as flattened breasts and loss of fat around the hips, are reversed. subsequent to this, the virilizing effects disappear slowly; the hypertrophied clitoris and the deepening of the voice, however, frequently persist [ , ]. conclusion a patient who presents with virilism should be investi- gated systematically to determine if the high testosterone levels are of an adrenal or ovarian origin. in this patient, hematological investigations ruled out an adrenal cause. radiological imaging proved to find that the cause of virilization was of an ovarian etiology. surgical interven- tion was sought and the diagnosis of virilizing ovarian tumor was made by histopathologic examination. patient’s perspective it was a harrowing experience for me when i started balding and growing excessive hair all over my body. my fears increased as my friends and relatives started noticing a change in my voice. when the doctors told me i had a tumor, i felt my outcome was bleak. after the operation, my physical appearance improved, and my social life has returned to normal. i am grateful to the doctors for all they have done. list of abbreviations fsh, follicle stimulating hormone; lh, luteinizing hormone; ct, computer tomography; mri, magnetic resonance imaging; hb, hemoglobin; tsh, thyroid stimulating hormone. consent written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. competing interests the authors declare that they have no competing interests. authors’ contribution ss, gpss, adp, aak, pg, nm, tj, ls, agr were involved in the patient care, acquisition of data, analysis and inter- pretation of data, review of literature, drafting and revising the manuscript. gpss, ss and adp revised the manuscript for important intellectual content. all authors read and approved the final manuscript. page of (page number not for citation purposes) cases journal , : http://casesjournal.com/casesjournal/article/view/ references . david a ehrmann: hirsutism and virilization. in harrison’s principles of internal medicine. th edition. edited by anthony s fauci, dennis l kasper, dan l longo, eugene braunwald, stephen l hauser, larry j jameson, joseph loscalzo: new york, mcgraw-hill medical; : - . . young rh, scully re: steroid cell tumors of the ovary. in obstetric & gynecological pathology.edited by fox h, wells m: spain, churchill livingstone; : - . . ferriman dm, gallwey jd: clinical assessment of body hair growth in women. j clin endocrinol , : - . . robert e scully: classification of human ovarian tumors. environmental health perspectives , : - . . scully re: ovarian tumors. a review. am j pathol , ( ): - . . faraj g, di gregorio s, misiunas a, faure an, villabrile p, stringa i, petroff n, bur g: virilizing ovarian tumor of cell type not otherwise specified: a case report. gynecol endocrinol , : - . . young rh, scully re: sex-cord stromal steroid cell and other ovarian tumors with endocrine, paraendocrine, and para- neoplastic manifestations. in blaustein’s pathology of the female genital tract. th edition. edited by kurman rj: new york, springer- verlag; : - . . wang ph, chao ht, lee rc, lai cr, lee wl, kwok cf, yuan cc, ng ht: steroid cell tumors of the ovary: clinical, ultrasonic, and mri diagnosis - a case report. eur j radiol , ( ): - . . william e hill, john fj clark: functional ovarian tumors: a ten year study at freedmen’s hospital. j natl med assoc , ( ): - . . aimakhu ve, adeleye ja, hendrickse m, de von hendrickse jp, iloabachie g: masculinizing tumors of the ovary. j natl med assoc , ( ): - . . derksen j, nagesser sk, meinders ae, haak hr, van de velde cj: identification of virilizing adrenal tumors in hirsute women. n engl j med , : - . . azziz r, dewailly d, owerbach d: clinical review : nonclassic adrenal hyperplasia: current concepts. j clin endocrinol metab , : - . . monteagudo a, heller d, husami n, levine ru, mccaffrey r, timor- tritsch ie: ovarian steroid cell tumors: sonographic charac- teristics. ultrasound obstetrics gynecology , : - . . wang ph, chao ht, lee rc, lai cr, lee wl, kwok cf, yuan cc, ng ht: steroid cell tumors of the ovary: clinical, ultrasonic, and mri diagnosis- a case report. eur j radiol , : - . . outwater ek, marchetto b, wagner bj: virilizing tumors of the ovary: imaging features. ultrasound obstet gynecol , ( ): - . page of (page number not for citation purposes) cases journal , : http://casesjournal.com/casesjournal/article/view/ do you have a case to share? submit your case report today • rapid peer review • fast publication • pubmed indexing • inclusion in cases database any patient, any case, can teach us something www.casesnetwork.com 	abstract 	introduction 	case presentation 	discussion 	conclusion 	patient’s perspective 	list of abbreviations 	consent 	competing interests 	authors’ contribution 	references j clin exp dent. ; ( ):e - . oral manifestations of ellis-van creveld syndrome e journal section: oral medicine and pathology publication types: case report oral manifestations of ellis-van creveld syndrome. a rare case report juan-francisco peña-cardelles , david a. domínguez-medina , jorge a. cano-durán , daniel ortega-con- cepción , josé-luis cebrián dds, oral medicine postgraduate in complutense university of madrid. oral and maxillofacial surgery service, alcorcon southern hospital md, oral and maxillofacial surgery service, alcorcon southern hospital dds, oral medicine postgraduate in complutense university of madrid dds, oral medicine postgraduate in complutense university of madrid phd, md, dds. head of oral and maxillofacial surgery section of the la paz university hospital. co-director of the service of maxillofacial surgery and dentistry of hospital la luz. head of the oral and maxillofacial surgery service, alcorcon southern hospital correspondence: universidad rey juan carlos avenida de atenas s/n, alcorcón, madrid juanfranciscopenacardelles@gmail.com received: / / accepted: / / abstract ellis-van creveld syndrome (evc) or chondroectodermal dysplasia is an autosomal recessive disorder, characte- rized by dwarfism, polydactyly, hypoplastic fingernails and congenital heart defects, finding in most of the cases orofacial anomalies. we describe a clinical case of a year old male patient diagnosed with evc who visited our maxillofacial private consultation at alcorcon southern hospital, presenting typical oral manifestations such as dental agenesis, delayed eruption, hypoplasia of the enamel, dental dysmorphism, taurodontism and supernumerary teeth. evc syndrome is a rare disease and requires a multidisciplinary approach. oral features are constant and requires the jointly performance of odontologist and maxillofacial surgeon aiming to get an appropriate treatment sequence surgery-orthodontics in order to achieve a suitable functional result to improve the quality of life of these patients. key words: ellis-van creveld syndrome, chondroectodermal dysplasia, oral manifestations, craniofacial mani- festations. doi: . /jced. http://dx.doi.org/ . /jced. introduction ellis-van creveld syndrome or chondroectodermal dysplasia is a rare disorder, autosomal recessive ( - ), characterized by dwarfism, postaxial polydactyly of the hands and feet, severe dystrophy of the fingernails and congenital heart defects in about - % of the cases ( - ). first description of the syndrome was made by the pe- diatricians richard ellis and simon van creveld in . it is the result of a genetic defect located in chromosome p . ( ) the prevalence varies from / in general population to / in amish population (lancaster, pennnsylvania, usa) ( , ). orally, the syndrome features consist of teeth of abnor- article number: http://www.medicinaoral.com/odo/indice.htm © medicina oral s. l. c.i.f. b - eissn: - email: jced@jced.es indexed in: pubmed pubmed central® (pmc) scopus doi® system peña-cardelles jf, domínguez-medina da, cano-durán ja, ortega-con- cepción d, cebrián jl. oral manifestations of ellis-van creveld syndrome. a rare case report. j clin exp dent. ; ( ):e - . http://www.medicinaoral.com/odo/volumenes/v i /jcedv i p .pdf j clin exp dent. ; ( ):e - . oral manifestations of ellis-van creveld syndrome e mal form (microdontia, conical teeth , dens in dente, taurodontism), supernumerary teeth, hypoplasia of the enamel, neonatal teeth in about % of the cases, as well as premature exfoliation of the teeth, malocclusion, multiple frenula, absent vestibular sulcus, submucous clefts, hypertrophic frena and labial frenula, dystrophic philtrum ( - ). the present article describes the case report of a child patient diagnosed with evc, presenting the major spec- trum of oral features described in literature. our patient was object of maxillofacial surgical treatment as a first stage sequence that will include combined orthodontic and restauration odontology. we emphasize the impor- tance of a multidisciplinary approach for the correct ma- nagement of these patients dental problems. case report a year old male patient was brought by his father, to our maxillofacial private consultation at alcorcon sou- thern hospital, referred from public medical institution, seeking for evaluation and possibility of treatment, the reason of consult was the abscense of eruption of the permanent superior incisors. the diagnosis of evc was made at birth, in the public institution where he was re- ferred from. there was no family background of the di- sease, he had healthy parents and brothers. the parents are originally from morocco and consanguinity related in second degree. among the patient’s medical background, he presented heart congenital disease: interventricular communica- tion that closed spontaneously and interauricular com- munication, surgically corrected at the age of three. at the age of four, surgical correction of epispady was performed and at the the age of nine, surgical correction of both inferior limbs axis was performed “genu valgum deformity”. at the clinical exploration, he presented short disarmo- nic stature ( cm), both superior and inferior short limbs with bilateral hexadactyly and hypoplastic finger- nails. facial exploration, revealed normo-brachicephaly, nor- mal eyelid fissures, wide nasal tip and narrow nostrils (fig. ). intraoral exploration revealed, agenesis of lateral su- perior and inferior incisors [ , , , ], dysmorfism compatible with conical teeth at inferior incisors and canines [ , , , ], fusion between a superior incisor ( ) and a supernumerary tooth (mesiodens). as for the soft tissues, multiple frenum, macroglossia and glosos- quissis can be find (fig. ). furthermore, retention of maxillary incisors was pre- sent, due to the presence of a central supernumerary too- th. for this purpose, the retention was treated by extrac- tion of the included central supernumerary tooth, under general anesthesia. radiografically, certain degree of taurodontism was pre- sent at the permanent superior first molars and more dis- cretely at the permanent inferior first molars (fig. ). in total central supernumerary teeth were present. maxi- llary compression was also evidenced it is remarkable the maloclussion with a tendency to a class iii and anterior crossbite. discussion in our reported case, there was no family medical patho- logic background related, although there may be a his- tory of consanguinity in up to % of cases. among the published cases of the last decade, descri- bing oral manifestations of the syndrome, the prevalence was . % of men and , % of women (table ). in the international literature, it is characteristic a tetrad, present in our patient, consisting of: dwarfism, bilateral fig. : a. extraoral vision, b. intraoral vision: polidactyly in both hands, as well as severe neil displasia. j clin exp dent. ; ( ):e - . oral manifestations of ellis-van creveld syndrome e fig. : intraoral vision a. right side view, b. front view, c. left side view, d. superior occlusal view, e. inferior occlusal view. fig. : orthopantomography and computer tomography. both were performed with a diag- nostic purpose prior to the surgery of mesiodens extraction. polydactyly of the hands, ectodermal dysplasia (disorder in fingernails, teeth) and cardiac congenital malforma- tions ( , - , ). it is remarkable the varied spectrum of oral features involving both soft tissues and teeth, that are constant (tables - ), nevertheless there are unusual findings like the presence of taurodontism (fig. ) also present in our case. it is important to comment that development of taurodontism and conical form of teeth could be due to one single genetic alteration or multiple ones and be important to make differential diagnosis with other sy- ndromes. j clin exp dent. ; ( ):e - . oral manifestations of ellis-van creveld syndrome e p at ie nt s c os ta h an em an n ja e t a l. ( ) t un a e b e t a l. ( ) a lv es -p er ei ra d e t a l. ( ) sh ai k s et al . ( ) p ed ro r l et a l. ( ) g ok ul ra j s et a l. ( ) v ee na k m e t al . ( ) a m in ab ad i n a e t a l. ( ) t ah ri ri an d e t a l. ( ) sa sa la w ad ss e t a l. ( ) g ho sh s et a l. ( ) k al as ka r r e t a l. ( ) n (% ) se x f m m m f m m m f f f f f f f f f f f , % m , % h a ge , o ra l m an if es ta tio ns n eo na ta l t ee th - - - - - - - - - u u + u u - - + - - ( , % ) a ge ne si s / o lig od on ti a + + + + + + + + + + + + + + + + + + + ( % ) l at e er up tio n + + + + + + + + - u u u + + + + + + + ( , % ) c ar ie s of ra pi d ev ol ut io n - - - - - - - - + u - + u - + - u u - ( , % ) c le ft li p - - - - - - - - - u - - + - - - + u - ( , % ) l at e d en ta l d ev el op m en t + + + + + + + + - u u + u + + + u u + ( , % ) e na m el h yp op la si a - + + - + - - + + + u u u - + - + - - ( , % ) m al oc cl us io n + u u u u u u u u + + + u + + u u u - ( , % ) d en ta l t ra ns po si tio n - - - - - - - - - - - - - - - - u u - ( % ) a cc es so ry b ra ke s + + + + + + + + + + + - + - + + + + + ( , % ) d en ta l d ys m or ph is m + + + + + + + + + + + + + + + - + + + ( , % ) t au ro do nt is m + - - - + - + - + - + + - + + + + + + ( , % ) su pe rn um er ar y - - - - - - - - - - - - - - + - u u + ( , % ) 	 t ab le : c li ni ca l c as es p ub lis he d in p ub m ed th at c ol le ct o ra l m an if es ta tio ns in th e la st ye ar s ( - ) . a br ev ia tu ra s: p re se nt , + . n ot p re se nt , - .f em al e, f . m al e, m . u nk no w n, u . j clin exp dent. ; ( ):e - . oral manifestations of ellis-van creveld syndrome e tetrada feature case report disproporcionate dwarfism • • disarmonic low size • • rhizomelic short upper and lower extremities • • small hands and feet • • bilateral transverse palmar fold • • marked interphalangeal grooves bilateral postaxial polydactyly in hands • • bilateral complete axial polydactyly (hands and feet) • • bilateral brachydactyly (hands and feet) ectodermal dysplasia • • severe nail dysplasia • • agenesies of . , . , . , . and . • • conoid teeth (incisors and canines) • • taurodontism in first permanent molars • • mesiodens congenital heart malformations • • affection of the a-v channel with presence of single auricle • • interventricular communication • • primal ostrium defect • • systolic murmur of regurgitation • • tricuspid insufficiency • • mitral insufficiency 	 table : tetrad characteristic in the case report. oral manifestations according to literature oral manifestations in the patient neonatal teeth unknown partial anodontia present agenesis of upper lateral incisors present agenesis of lower lateral incisors present late eruption present caries of rapid evolution present cleft lip not present late dental development present enamel hypoplasia present malocclusion present dental transposition not present accessory brakes not present dental dysmorphism present taurodontism present 	 table : oral manifestations of evc syndrome in literature vs case report ( , - , ). another remarkable finding is the presence of malo- clussion, specifically prognatism of the mandible ; our patient presented a tendency to a maloclussion class iii and anterior crossbite, due to maxillary compression and partial retention of permanent central incisors (figs. , ). in reference to the alterations of the dental eruption, an interesting fact present in this case is the poor root deve- j clin exp dent. ; ( ):e - . oral manifestations of ellis-van creveld syndrome e lopment in permanent first molars and in inferior cani- nes, despite the patient’s age. in general, the statistics shown in table , have coincided with literature, we also have another data reflecting results that help us to understand better the evc. case reports articles published in the last ten years describes oral ma- nifestations evidence that dental agenesis has been seen in all cases ( %), the dental dimorphism in a total of cases ( , %), accessory frenums in cases ( , %) and late eruption in the , % of cases, all of them pre- sent in the studied case. however, presence of supernu- merary teeth is a characteristic which is in the present case but only in of the published cases ( , %). conclusions evc syndrome is an infrequent entity and requires a multidisciplinary approach of specialists such as den- tist and oral and maxillofacial surgeon. it is important the maintenance of the dental health and to perform a correct dental diagnosis in order to establish an optimal treatment sequence. references . costa hanemann ja, de carvalho f, carvalho e, carnevalli b. oral manifestations in ellis-van creveld syndrome: report of a case and review of the literature. j oral maxillofac surg. ; : - . . tuna eb, koruyucu m, kürklü e, Çifter m, gençay k, seymen f, tüysüz b. oral and craniofacial manifestations of ellis-van creveld syndrome: case series. j craniomaxillofac surg. ; : - . . baujat g, le merrer m. ellis-van creveld syndrome. orphanet j rare dis. ; : . . alves-pereira d, berini-aytés l, gay-escoda c. ellis-van creveld syndrome. case report and literature review. med oral patol oral cir bucal. ; :e - . . shaik s, raviraj j, dirasantchu s, venkata ss. ellis-van creveld syndrome with unusual oral and dental findings: a rare clinical entity. dent res j. ; : - . . pedro rl, andrade lh, maia lc. the importance of oral-clinical findings for the correct diagnosis of ellis-van creveld syndrome. gen dent. ; :e - . . gokulraj s, mohan n, raj jb, ahamed sy, arokiaraj cj, subbu- lakshmi ac. ellis-van creveld syndrome in siblings: a rare case re- port. j pharm bioallied sci. ; :s -s . . veena km, jagadishchandra h, rao pk, chatra l. ellis-van cre- veld syndrome in an indian child: a case report. imaging sci dent. ; : - . . aminabadi na, ebrahimi a, oskouei sg. chondroectodermal dysplasia (ellis-van creveld syndrome): a case report. j oral sci. ; : - . . tahririan d, eshghi a, givehchian p, tahririan ma. chondroecto- dermal dysplasia: a rare syndrome. j dent. ; : - . . sasalawad ss, hugar sm, poonacha ks, mallikarjuna r. ellis van creveld syndrome. bmj case rep. ; . pii: bcr . . ghosh s, setty s, sivakumar a, pai km. report of a new syndro- me: focus on differential diagnosis and review of ellis-van creveld, curry-hall, acrofacial dysostosis, and orofacial digital syndromes. oral surg oral med oral pathol oral radiol endod. ; : - . . kalaskar r, kalaskar ar. oral manifestations of ellisvan creveld syndrome. contemp clin dent. ; :s –s . conflict of interest the authors have declared that no conflict of interest exist. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ mitochondrial carrier structure and diseases stress conditions [ ]. the transporter contributes to virulence of pathogens such as staphylococcus aureus and helicobacter pylori. we utilize putp of escherichia coli as a model to explore structure and molecular mechanism of function of sssf proteins. here, we present a model of the helix bundle of putp obtained by molecular modeling constrained by experimentally determined intra- molecular distances and template restraints derived from the ten- helix core of the vsglt crystal structure [ ]. for this purpose, deer distance measurements between spin labels attached to helix ends were conducted and mean interspin distances were determined. fitting algorithm based on matrix geometry in combination with prediction of spin label conformations by a rotamer library approach [ ] resulted in an ensemble of helix bundle structures. the central structure of the ensemble showed a core structure with a fold similar to that of the vsglt template. furthermore, analysis of spin label motility and environmental polarity by cwepr yielded information on secondary structure elements and structural rearrangements of external loop (el) of putp upon sodium and/or l-proline binding. the results support the idea that el controls access to the sodium and/or l-proline binding site(s) similar as previously proposed for el of leut [ ]. references [ ] h. jung, febs lett. ( ) – . [ ] s. faham, a. watanabe, g.m. besserer, d. cascio, a. specht, b.a. hirayama, e.m. wright, j. abramson, science ( ) – . [ ] y. polyhach, e. pordignon, g. jeschke, phys. chem. chem. phys. ( ) – . [ ] d.p. claxton, m. quick, l. shi, f.d. de carvalho, h. weinstein, j.h. javitch, h.s. mchaourab, nat. struct. mol. biol. ( ) – . doi: . /j.bbabio. . . p mitochondrial carrier structure and diseases pierri ciro leonardo, palmieri ferdinando department of biosciences, biotechnology and pharmacological sciences, laboratory of biochemistry and molecular biology, university of bari, italy e-mail: ciroleopierri@gmail.com to date eleven disorders are known to be caused by defects of mito- chondrial carriers, a family of proteins that shuttle a variety of metabolites across the inner mitochondrial membrane. mutations of mitochondrial carrier genes are responsible for carnitine/acylcarnitine carrier deficien- cy, ornithine carrier deficiency (hhh syndrome), aspartate/glutamate isoform deficiency (global cerebral hypomyelination), aspartate/ glutamate isoform deficiency (ctln and niccd), amish microcephaly, early epileptic encephalopathy, congenital sideroblastic anemia, pic defi- ciency, adp/atp carrier isoform deficiency, neuropathy with bilateral striatal necrosis and adpeo (autosomal dominant progressive external ophthalmoplegia). structural, functional and bioinformatics studies have revealed the existence in mitochondrial carriers of a substrate-binding site in the internal carrier cavity, of two gates that close the cavity alternatively on the matrix or cytosolic side of the membrane, and of two sets of prolines and glycines in the six transmembrane a-helices located strategically between the substrate-binding site and the two gates. the key role played by these mitochondrial carrier areas is supported by the observation that they host most of the disease-causing missense mutations of the mitochondrial carriers. references [ ] e. pebay-peyroula, c. dahout-gonzalez, r. kahn, v. trézéguet, g.j. lauquin, g. brandolin, nature ( ) – . [ ] a.j. robinson, e.r. kunji, proc. natl. acad. sci. u. s. a. ( ) – . [ ] f. palmieri, biochim. biophys. acta ( ) – . [ ] f. palmieri, c.l. pierri, febs lett. ( ) – . [ ] f. palmieri, mol. aspects med. (in press). http://dx.doi.org/ . /j.mam. . . . doi: . /j.bbabio. . . p insights into the mechanism of the na+/ca + exchanger from atomistic molecular dynamics simulations fabrizio marinelli, josé d. faraldo-gómez theoretical molecular biophysics group, max planck institute of biophysics, max-von-laue strasse , frankfurt am main, germany e-mail: fabrizio.marinelli@biophys.mpg.de na+/ca + exchangers (ncx) and potassium-dependent na+/ ca + exchangers (nckx) are two related families of transporters involved in ca + signaling that function by extruding cytosolic ca + (and k+ for the potassium-dependent transporter) in exchange for extracellular na+ [ ]. previous studies have established that this exchange process is electrogenic and with a defined stoichiometry, and have identified specific acidic aminoacids believed to be crucial for ion binding and translocation [ – ]. recently the crystal structure of the ncx from methanococcus jannaschii was determined at . Å resolution [ ], revealing an intriguing transmembrane topology consisting of inverted structural repeats, and the presence of four putative ion binding sites formed by highly conserved residues. notwithstanding these groundbreaking insights, based on the struc- ture alone several ion occupancy states can be hypothesized that would be compatible with the experimental exchange stoichiometry. moreover, in the crystal the protein adopts a unique outward facing conformation, which does not immediately explain how ion binding to the protein facilitates the necessary outward-to-inward conforma- tional transition. here, we use extensive molecular simulations and molecular modeling to investigate the occupancy and specificity of the ion binding sites in ncx_mj, and the microscopic mechanism by which na+ and ca + are exchanged across the membrane. references [ ] v. frank, j. lytton, physiology ( ) – . [ ] k. kang, t.g. kinjo, r.t. szerencsei, p.p.m. schnetkamp, j. biol. chem. ( ) – . [ ] t.m. kang, d.w. hilgemann, nature ( ) – . [ ] j. liao, h. li, w. zeng, d.b. sauer, r. belmares, y. jiang, science ( ) – . doi: . /j.bbabio. . . p the evolutionary history of membrane-integral pyrophosphatases supports na+ as the ancestral coupling ion in membrane bioenergetics heidi h. luoto , alexander a. baykov , reijo lahti , anssi m. malinen department of biochemistry and food chemistry, university of turku, fin- turku, finland abstracts s http://dx.doi.org/ . /j.bbabio. . . http://dx.doi.org/doi: . /j.mam. . . http://dx.doi.org/doi: . /j.mam. . . http://dx.doi.org/ . /j.bbabio. . . http://dx.doi.org/ . /j.bbabio. . . association of the vitamin d metabolism gene cyp a with coronary artery calcification association of the vitamin d metabolism gene cyp a with coronary artery calcification haiqing shen, lawrence f. bielak, jane f. ferguson, elizabeth a. streeten, laura m. yerges-armstrong, jie liu, wendy post, jeffery r. o’connell, james e. hixson, sharon l.r. kardia, yan v. sun, min a. jhun, xuexia wang, nehal n. mehta, mingyao li, daniel l. koller, hakan hakonarson, brendan j. keating, daniel j. rader, alan r. shuldiner, patricia a. peyser, muredach p. reilly, braxton d. mitchell objective—the vitamin d endocrine system is essential for calcium homeostasis, and low levels of vitamin d metabolites have been associated with cardiovascular disease risk. we hypothesized that dna sequence variation in genes regulating vitamin d metabolism and signaling pathways might influence variation in coronary artery calcification (cac). methods and results—we genotyped single-nucleotide polymorphisms (snps) in gc, cyp b , cyp a , and vdr and tested their association with cac quantity, as measured by electron beam computed tomography. initial association studies were carried out in a discovery sample comprising amish subjects, and snps nominally associated with cac quantity ( snps in cyp a , p� . to . ) were then tested for association with cac quantity in independent cohorts of subjects of white european ancestry (genetic epidemiology network of arteriopathy study [n� ] and the penn coronary artery calcification sample [n� ]). one of the snps, rs , was associated with cac quantity in both the genetic epidemiology network of arteriopathy (p� . ) and penn coronary artery calcification (p� . ) studies. in all populations, the rs c allele was associated with lower cac quantity. metaanalysis for the association of this snp with cac quantity across all studies yielded a p value of . � � . conclusion—a common snp in the cyp a gene was associated with cac quantity in independent populations. this result suggests a role for vitamin d metabolism in the development of cac quantity. (arterioscler thromb vasc biol. ; : - .) key words: calcification � coronary artery disease � epidemiology � gene mutations � vitamin d metabolism coronary artery calcification (cac) is a measure ofcoronary subclinical atherosclerosis that predicts risk for cardiovascular disease (cvd) in the general population. the potential role of circulating levels of vitamin d in influencing the initiation or progression of cac is of great clinical interest given the large proportion of the population deficient in vitamin d , the fact that the vitamin d endocrine system is essential for calcium homeostasis, and that low vitamin d levels have been associated with risk for cvd. however, circulating levels of vitamin d metabolites have not been consistently associated with cac, although causal relation- ships may not be apparent from simple cross-sectional studies. see accompanying article on page the effects of vitamin d are myriad, including enhance- ment of cellular differentiation and immunomodulation, in addition to the well-known effects on calcium homeostasis. the effects of vitamin d are mediated by activation of the nuclear vitamin d receptor (vdr), which regulates transcrip- tion of a large number of genes in many tissues. the metabolism of vitamin d, providing active , (oh) d to the received on: june , ; final version accepted on: september , . from the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, md (h.s., e.a.s., l.m.y.-a., j.l., j.r.o., a.r.s., b.d.m.); department of epidemiology, university of michigan school of public health, ann arbor, mich (l.f.b., s.l.r.k., y.v.s., m.a.j., p.a.p.); the cardiovascular institute (j.f.f., n.n.m., b.j.k., d.j.r., m.p.r.), biostatistics and epidemiology (x.w., m.l.), and the institute for translational medicine and therapeutics, school of medicine (n.n.m., m.p.r.), university of pennsylvania, philadelphia, pa; geriatric research and education clinical center, department of veterans affairs medical center, baltimore, md (e.a.s., a.r.s.); division of cardiology, school of medicine (w.p.) and department of epidemiology, bloomberg school of public health (w.p.), johns hopkins university, baltimore, md ; human genetic center, school of public health, the university of texas health science center at houston, houston, texas (j.e.h.); department of medical and molecular genetics, school of medicine, indiana university, indianapolis, ind (d.l.k.); center for applied genomics, children’s hospital of philadelphia, philadelphia, pa (h.h.). correspondence to braxton d. mitchell, phd, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood st, rm , baltimore, md . e-mail bmitchel@medicine.umaryland.edu © american heart association, inc. arterioscler thromb vasc biol is available at http://atvb.ahajournals.org doi: . /atvbaha. . d ow nloaded from http://ahajournals.org by on a pril , vdr, requires the involvement of several key proteins in a tightly regulated process. vitamin d (made in the skin keratinocytes in response to uvb light or from exogenous supplements) is sequentially hydroxylated by the mitochon- drial cytochrome p enzymes -hydroyxlase (the product of cyp r ) in the liver and -� hydroxylase (the product of cyp b ) in the kidney (among other tissues) into the active metabolite , (oh) d. after , (oh) d binds to the ubiq- uitous vdr, the retinoid x receptor is recruited, forming a heterodimer complex that regulates gene transcription by interacting with vitamin d response elements in the promoter region of genes. vitamin d metabolites are transported through the circulation by vitamin d binding protein (gc). concentrations of , (oh) d are tightly regulated by both -hydroxylase and the catabolic enzyme -hydroxylase (gene cyp a ). cyp a is induced by both , (oh) d and (oh)d and is one of the most highly inducible genes in humans, capable of increasing its transcription by - fold. the high inducibility of cyp a is likely to be a critical factor in the large therapeutic window of vitamin d. thus, the key genes involved in vitamin d metabolism pathway include cyp r , cyp b , cyp a , vdr, and gc. we hypothesized that dna sequence variation in these genes and their levels might influence cac quantity varia- tion. to test this hypothesis, we obtained genotypes of single-nucleotide polymorphisms (snps) in available genes (gc, cyp b , cyp a , and vdr) of the major genes involved in the regulation of vitamin d levels and tested their association with cac quantity. initial association studies were carried out in a discovery sample comprising amish subjects, and snps nominally associated with cac quantity in this cohort were then tested for association with cac quantity in independent cohorts of subjects who were also of white european ancestry (the genetic epidemiology net- work of arteriopathy [genoa] study and the penn coro- nary artery calcification [penncac] sample). populations and methods discovery population (amish) the discovery sample comprised participants in the amish family calcification study (afcs), a community-based study initiated in to identify the joint determinants of bone mineral density and vascular calcification. details of the recruitment procedures have been published previously. many related individuals were recruited (indeed, all amish are related), and recruitment efforts were made without regard to cvd health status in this generally healthy population. because vascular calcification occurs infrequently in young adulthood, only men aged years and older or women aged years or older were recruited into the afcs. the protocol was approved by the institutional review board of the university of maryland and other participating institutions. informed consent, including permission to contact relatives, was obtained before participation. all afcs participants underwent a detailed medical history interview and assessment of potential risk factors for cvd at the amish research clinic in strasburg, pennsylvania. physical exam- inations were conducted in the early morning following an overnight fast. blood samples were obtained for biochemical measurements and dna extraction. images of the coronary arteries were obtained by electron beam computed tomography using an imatron c- scanner. the protocol included to -mm contiguous transverse slices between the aortic root and the apex of the heart, gated to % of the r-to-r interval (the cycle between consecutive r waves) and obtained during a single breath hold. the extent of calcification in the thoracic aorta was assessed by scanning between the superior aspect of the aortic arch and the superior pole of the kidney at -mm intervals. the presence of calcification was defined as a density � hounsfield units with area � mm . cac was quantified using the agatston method, which incorporates both density and area. the sum of the scores in the left main, left anterior descending, circumflex, and right coronary arteries was considered the cac score. all scans were scored by a single experienced cardiologist. a total of afcs study participants were genotyped using the humancvd beadchip v (illumina, inc., san diego, calif), which includes � snps within � cvd candidate genes. gc, cyp b , and vdr were considered in the chip design as group genes, with denser snp coverage (inclusive of the intronic and exonic untranslated regions and kb of the proximal promoter regions), whereas cyp a was considered a group gene, with sparser snp coverage (inclusive of intronic, exonic, and flanking untranslated regions). in the chip design, tag snps were selected from group genes to capture known variation across all of the representative hapmap populations and seattlesnps (where avail- able) for snps having minor allele frequency � . and an r of at least . . tag snps included in group genes were designed to capture known variation for snps having minor allele frequency � . and an r of at least . . carriers for the known monogenic mutation r q in apolipoprotein b- (n� ), responsible for familial defective apob- , were excluded from the analysis. r q has been associated with cac quantity in the amish. the humancvd beadchip included , , , and snps in the gc, cyp b , cyp a , and vdr genes, respectively. the initial genotype calls were generated with the cluster file provided by illumina beadstudio software. individuals with call rates less than % were excluded (n� ). snps with a low call rate (� %) were excluded. of successfully genotyped snps, , , , and were monomorphic in the amish across gc, cyp b , cyp a , and vdr, respectively, and snps ( from cyp a and from vdr) were excluded because of low call rate. this left a total of snps ( in gc, in cyp b , in cyp a , and in vdr included in this analysis [snp names are provided in supplemental table i, available online at http://atvb.ahajournals.org]). the observed distri- bution of genotypes was tested for deviation from hardy-weinberg equilibrium using the pearson � test. replication populations genoa study the family blood pressure program, established by the national heart, lung, and blood institute in , joined existing research networks that were investigating hypertension and cvd. one of the family blood pressure program networks is genoa, which recruited sibships with hypertensive adults to investigate genetic contributions to hypertension and hypertension-related target organ damage. sibships containing at least individuals with clinically diagnosed essential hypertension before age were recruited from rochester, minnesota. participants were diagnosed with hyperten- sion if they had either ( ) a previous clinical diagnosis of hyperten- sion by a physician with current antihypertensive treatment or ( ) an average systolic blood pressure � mm hg or diastolic blood pressure � mm hg based on the second and third readings at the time of their clinic visit as stipulated by the joint national committee- guidelines. exclusion criteria were secondary hyper- tension, alcoholism or drug abuse, pregnancy, insulin-dependent diabetes mellitus, active malignancy, hypercalcemia, renal insuffi- ciency (creatinine� . mg/dl), and known metabolic bone disease other than osteoporosis. after identifying the hypertensive sibships, all members of the sibship were invited to participate regardless of their hypertension status. between december and february , of the original genoa study participants in the rochester field center returned to undergo risk factor and target organ damage measurement. individ- shen et al association of cyp a with cac d ow nloaded from http://ahajournals.org by on a pril , uals with a history of coronary revascularization (n� ) were not eligible for measurement of cac. after further excluding partici- pants with a history of myocardial infarction (n� ), stroke (n� ), or positive angiogram (n� ); those who self-reported not being of european ancestry (n� ); and those missing risk factor data (n� ), there were genoa participants with cac and risk factor measures. participants were imaged with an imatron c- electron beam computed tomography scanner (imatron inc., south san francisco, calif) as previously described. cac was defined as a hyperattenu- ating focus in a coronary artery area � . mm and having a density � hounsfield units (hu) with area � mm . quantity of cac was defined as the total cac score summed from the major epicardial arteries using the method of agatston et al. the average cac score from sequential scans was used. snps to be tested for replication (rs , rs , and rs ) had previously been genotyped in the genoa study on the affymetrix genome-wide human snp array . platform. all passed the overall quality control filters (eg, snp call rate � %). snp imputation was performed using mach, version . . (http:// www.sph.umich.edu/csg/abecasis/mach), based on the publicly available phased haplotypes from hapmap release , build , ceu population. association analysis of genoa snp (rs ) was based on the imputed genotype dosage that ranges from to , which is the expected number of effective alleles. the imputed genotype of rs had an imputation quality score of . and r of . with a genotyped snp. of the genoa participants with cac and risk factor data, had genotype data available for analyses. these participants were in sibships, including singletons, sibships of size , sibships of size , sibships of size , sibships of size , sibships of size , sibship of size , sibships of size , sibship of size , and sibship of size . the study was approved by the institutional review boards of all the participating institutions, and participants gave written informed consent. penncac the penncac sample included subjects of white european ancestry recruited from separate parallel studies: the study of inherited risk of coronary atherosclerosis (n� ), the penn diabetes heart study (n� ), and the philadelphia area metabolic syndrome network (n� ). penncac samples were genotyped using the humancvd beadchip v (illumina, inc.). the study of inherited risk of coronary atherosclerosis is a cross-sectional study of factors associated with cac in a community-based sample of asymptomatic subjects and their fami- lies. subjects were healthy adults aged to years who had a family history of premature coronary artery disease. subjects were excluded if they reported evidence of coronary artery disease on screening questionnaire, reported a history of diabetes mellitus, or had a serum creatinine level � . mg/dl. the penn diabetes heart study is an ongoing, cross-sectional community-based study of type diabetic subjects without clinical evidence of coronary heart disease or overt chronic kidney disease. subjects were aged to years, had a clinical diagnosis of type diabetes (defined as fasting blood glucose � mg/dl, -hour postprandial glucose � mg/dl, or use of oral hypoglycemic agents/insulin in a subject older than ), and had a negative pregnancy test if female. subjects were excluded if they had evidence of clinical coronary heart disease, a clinical diagnosis of type diabetes (insulin use before age ), a serum creatinine � . mg/dl, or weight � lbs. the philadelphia area metabolic syndrome network is a cross- sectional study of patients with or more metabolic syndrome risk factors. philadelphia area metabolic syndrome network partici- pants were recruited between and via the university of pennsylvania health system primary care providers, word of mouth in the community, and penn health fairs for cvd risk factors. subjects were aged to years. subjects with known type diabetes or clinical atherosclerotic cvd were excluded. the study of inherited risk of coronary atherosclerosis, penn diabetes heart study, and philadelphia area metabolic syndrome network are all are single-center studies that used the same general clinical research center, nursing staff, computed tomography scan- ner, and research laboratories. all study subjects were evaluated at the general clinical research center at penn, as previously de- scribed. cac scores were determined by electron beam computed tomography (imatron) according to the method of agatston from continuous -mm thick computed tomograms. all penn study pro- tocols were approved by the penn institutional review board, and all subjects provided written informed consent. statistical analysis because the distribution of cac scores was positively skewed, the scores were natural log-transformed after adding . to evaluate the distribution of log-transformed cac scores after regressing out the effects of age, sex, and age�sex, we obtained the residualized values of transformed cac score in the afcs sample. the residu- alized values were approximately normally distributed. we followed a -stage approach in which we first identified a small number of promising snps in the amish discovery set for follow-up, which we would then take forward for more rigorous statistical testing in the independent replication cohorts. our criteria for identifying snps of potential interest in the amish discovery sample was that they be associated with cac quantity at a liberal threshold of p� . . to establish statistical significance, we then required that these snps be associated in both of the replication cohorts at probability values taking into account the number of snps tested in each cohort (ie, bonferroni-corrected significance thresholds of . / � . .) finally, we combined evidence for association across all studies in a metaanalysis, using a bonferroni correction for snps. in the amish discovery sample, association analyses were carried out using a variance component approach suitable for analysis of large pedigrees. the approach used a mixed-effect model to assess the effect of genotype (included as a fixed effect) on cac score while adjusting for covariates (age, sex, age�sex) and including a polygenic component (as a random effect) that accounted for the correlations in phenotype existing among related individuals. the polygenic component was modeled using the relationship matrix derived from the complete amish pedigree structure. these analyses were carried out using mixed model analysis for pedigree software developed by of the coauthors (j.r.o.). pairwise linkage disequi- librium (ld) correlation statistics (r ) were computed for the amish group using the haploview software (http://www.broad.mit.edu). the snps that nominally associated with cac score in the amish sample were then tested for association in the genoa and penncac samples. in genoa, association analysis was carried out in r using linear mixed-effect models and included a random effect for family structure (ie, sibship). the effect of the cyp a snps on log-transformed (cac score� ) was assessed with a random effect for sibship to account for the family structure (siblings) with adjustment for age, sex, and an age�sex interaction. the covariance structure was defined as compound symmetry. in penncac, the snp association with log-transformed (cac score� ) was tested using a linear regression model with cyp a genotype included as an independent variable and age, sex, and sex�age included as covari- ates. the analysis was carried out using the plink program, version . . for snps tested for association in all cohorts, we combined association results across cohorts in a metaanalysis using a weighted fixed effects metaanalysis approach as implemented in the metal program (http://www.sph.umich.edu/csg/abecasis/metal/index.html). results the characteristics of participants in the amish discovery sample (n� ) and the genoa (n� ) and penncac (n� ) replication samples are summarized in table . mean ages of participants in the amish and penncac samples were comparable ( to years) and slightly arterioscler thromb vasc biol december d ow nloaded from http://ahajournals.org by on a pril , younger than the mean age in the genoa sample ( years). the proportion of men in the samples ranged from % to %, and the mean body mass index was . kg/m in the amish compared with . and . kg/m in penncac and genoa. by design, there was a substantially higher propor- tion of subjects with diabetes in the penncac sample ( . %) compared with the amish ( . %) or the genoa ( . %) samples. by design, there was also a substantially higher proportion of subjects with hypertension in the genoa sample ( . %) compared with the amish ( . %) or penncac ( . %) samples. in the genoa sample, blood pressure measurements were recorded on blood pressure– lowering treatment. table presents the sizes of the genes, the numbers of snps genotyped, and summary results of the association testing. a total of snps in gc ( exons, bp), snps in cyp b ( exons, bp), snps in cyp a ( exons, bp), and snps in vdr ( exons, bp) were successfully genotyped and tested for association. we estimated the proportion of allelic variation in the genes captured by these snps in the hapmap ceu population. the snps in gc, snps in cyp b , and snps in vdr (both ibc group genes) captured %, %, and %, respectively, of all alleles having a frequency of . or higher at an r � . . the snps in cyp a (a group gene) captured % of all alleles having a frequency of . or higher at an r � . . there was no statistical evidence for deviation from hardy-weinberg equilibrium for any of the successfully genotyped snps in the amish based on a threshold of p� . ( . / ). in the discovery sample, no snps in the gc, cyp b , or vdr gene were associated with cac score at even a nominal probability value of . (see supplemental table i for individual snp association results). in contrast, of the snps in cyp a were associated with cac score, with nominal probability values ranging from . to . . the snps in cyp a nominally associated with cac quantity were to bp apart from each other. snp rs was located in haplotype block and snps rs , rs , and rs in another block (all pairwise ld among rs , rs , and rs : r � . to . , d�� . ). rs was not in ld with the other snps (pairwise ld between rs and other snps: r � . , d�� . ). the figure shows the ld (r ) structure of the snps in cyp a in the amish sample and probability values for association of these snps with cac score. the snps in cyp a with nominal probability value � . in the amish were then tested for association with cac score in the genoa and penncac populations. one of the snps, rs , was associated with cac score in both the genoa (p� . ) and penncac (p� . ) pop- ulations (table ). in all populations, the c allele (minor allele frequency ranging from . to . across the table . clinical characteristics of the study populations discovery sample afcs (n� ) replication samples genoa (n� ) penncac (n� ) age, years . ( . ) . ( . ) . ( . ) male, n (%) ( . ) ( . ) ( . ) body mass index, kg/m . ( . ) . ( . ) . ( . ) systolic blood pressure, mm hg . ( . ) . ( . ) . ( . ) diastolic blood pressure, mm hg . ( . ) . ( . ) . ( . ) total cholesterol, mg/dl ( ) . ( . ) . ( . ) low-density lipoprotein cholesterol, mg/dl ( ) . ( . ) . ( . ) high-density lipoprotein cholesterol, mg/dl ( ) . ( . ) . ( . ) triglycerides, median (interquartile range), mg/dl ( to ) . ( to ) ( to ) hypertension, n (%) ( . ) ( . ) ( . ) diabetes, n (%) ( . ) ( . ) ( . ) current smoker, n (%) ( . ) ( . ) ( . ) presence of cac, n (%) ( . ) ( . ) ( . ) cac score, median (interquartile range) ( to ) . ( to ) ( to ) for blood pressure measurements, some hypertension subjects were under blood pressure treatment. values are mean (sd) unless otherwise indicated. table . description of vitamin d metabolism candidate genes and summary of association results with cac in populations gc cyp b cyp a vdr gene size,* bp exon count (coding exons count) ( ) ( ) ( ) ( ) no. of tagging snps (discovery sample) no. of snps (p� . ) discovery sample afcs replication sample genoa . . . . . . . . . upenn . . . . . . . . . *only the translated region is shown. shen et al association of cyp a with cac d ow nloaded from http://ahajournals.org by on a pril , populations) was associated with lower cac score. a formal test of heterogeneity indicated no significant differences in effect size across the populations (het p value� . ). combining the probability values in a metaanalysis for the rs –cac score association across all studies yielded a metaanalysis probability value of . � � . com- bining results from the genoa and penncac samples only (that is, excluding results from the amish discovery sample) yielded a probability value of . � � . there was no evidence for association of cac score with any of the other snps in the genoa and penncac samples. the relationship between rs and traditional risk factors was tested in afcs. this snp was not significantly associated with any of the blood pressure or serum lipid phenotypes, nor was it associated with variation in serum (oh)d levels (data not shown). consistent with these results, the association of rs with cac quantity was essentially unchanged, with additional adjustment for low- density lipoprotein cholesterol and diabetes. discussion we have identified a common snp, rs , in cyp a that is associated with quantity of cac. the gene product of cyp a is the major enzyme responsible for the catabolism of , (oh) d and (oh)d. our data thus suggest that genetic variability in vitamin d homeostasis may contribute to the pathogenesis of cac. cyp a plays a pivotal role in maintaining vitamin d homeostasis. deletion of cyp a in mice causes figure. the association between cyp a tagging snps and cac score [ln(cac score � ), adjusted for age, sex, age�sex, and family structure] in the amish. pairwise ld (r ) values are shown. cyp a gene and exons are shown. arterioscler thromb vasc biol december d ow nloaded from http://ahajournals.org by on a pril , , (oh) d excess and hypercalcemia with severe bone mineralization defects and ectopic vascular calcification (renal calcium deposition) after chronic treatment of , (oh) d. on the other hand, transgenic rats that constitutively express cyp a develop atherosclerotic lesions in the aorta, which greatly progress with high-fat and high-cholesterol feeding. although there is much circumstantial evidence, the role of vitamin d in cardiovascular health remains controversial. there is some epidemiological evidence in humans support- ing a role of vitamin d metabolites in the development of cac. watson et al measured serum , (oh) d in asymptomatic individuals with high risk of coronary heart disease and patients with familial hypercholesterolemia and reported, in both groups, an inverse correlation between serum , (oh) d and cac. doherty et al reported that serum , (oh) d independently and inversely predicted cac quantity in a sample of asymptomatic subjects with risk factors for coronary heart disease. low (oh)d levels were also associated with subsequent development of cac in the multi-ethnic study of atherosclerosis. by contrast, there was no association between serum (oh)d levels and coronary calcification in a smaller study of patients undergoing angiography. in this amish population, there is no correlation between season-adjusted (oh)d levels and prevalent cac. the discordant results observed could be due to limited power to detect associations of small magni- tude, differences in patient characteristics among studies, or differences in assays used in these studies. although (oh)d is the best marker of vitamin d stores, , (oh) d is the active metabolite, and (oh)d levels might not reflect circulating , (oh) d levels. in addition, the unknown local , (oh) d production in the vasculature or in macrophages and other cell types involved in plaque formation might be more important than circulating levels for pathogenesis of vascular calcification. this is the first demonstration of association between cyp a gene variants and vascular calcification in human population studies. cyp a snps or haplotypes have also been associated with other diseases in humans, including cancer and asthma. in their asthma study, wjst et al reported that the cyp a haplotype that was moderately associated with asthma was also associated with , (oh) d, (oh)d, and ige. a recent genome-wide association analysis based on � individuals identified an associ- ation between rs , located � . kb centromeric of cyp a , and (oh)d levels. the snp is . kb away from rs , the snp associated with cac quantity in the present study, and there is little ld between rs and rs (hapmap ceu r � . , d’� . ). despite the important role of cyp a in vitamin d metabolism, the mechanism linking rs to coronary calcification remains unclear. in neither the amish study nor an independent sample of premenopausal white women from indiana (data not shown) was there any evidence for an association of rs with (oh)d levels. however, although an association of rs with (oh)d levels would have provided a clear potential mechanism linking this snp to variation in cac levels, there are several ways through which this snp could affect cac without influenc- ing mean (oh)d levels. for example, this snp or a snp in high ld with this snp could influence the conversion of active , (oh) d into its inactive metabolite without affect- ing (oh)d levels. one way to test this hypothesis would be to assess the association of rs with serum , (oh) d, but unfortunately we do not have these mea- surements in our study. another potential mechanism is that this genetic variant may affect -hydroxylase expression and vitamin d metabolism locally in the endothelium, and these effects may not be correlated with circulating levels of vitamin d metabolites. in summary, a common variant in the cyp a gene was associated with cac quantity in independent populations. this result suggests a role for vitamin d metabolism in coronary atherosclerosis. future studies should elucidate the underlying mechanisms and signaling pathways that entwine vitamin d metabolism and vascular health. these studies may lead to novel screening and therapeutic options for the identification and treatment of individuals at increased risk for cvd events. sources of funding the amish component of this study was supported by national institutes of health research grants u -hl and r - hl , f ar , university of maryland general clinical research center grant m rr , an american heart associ- ation scientist development grant ( n), and an american heart association grant-in-aid ( e). partial funding was also provided by the mid-atlantic nutrition and obesity research center (p dk ) and the baltimore department of veterans affairs medical center geriatric research and education clinical center. genoa was funded by national institutes of health research grants r -hl , u -hl (genetic determi- nants of high bp in three racial groups), and r -hl and table . the association between cyp a snps and cac score* in populations afcs (n� ) genoa (n� ) penncac (n� ) metaanalysis alleles† coded_f � (se) p coded_f � (se) p coded_f � (se) p p rs c/g . � . ( . ) . � � . � . ( . ) . � � . � . ( . ) . . � � rs c/t . � . ( . ) . � � . . ( . ) . . � . ( . ) . . rs a/g . � . ( . ) . � � . . ( . ) . . � . ( . ) . . rs c/t . � . ( . ) . � � . . ( . ) . . � . ( . ) . . coded_f: frequency of coded allele. *ln(cac� ), adjusted for age, sex, age�sex, and family structure in afcs and genoa. †coded/noncoded allele. shen et al association of cyp a with cac d ow nloaded from http://ahajournals.org by on a pril , by a national institutes of health general clinic research center grant (m -rr ) awarded to mayo clinic rochester. the penncac resource was supported by a clinical and translational science award (ul rr ) from the national center for research resources and a diabetes and endocrine research center award (p -dk ), both to the university of pennsylvania. dr reilly is supported by r -dk from the national institutes of health. the study of indiana women was supported by national institutes of health research grants p -ag- and ul -rr- . disclosures none. references . detrano r, guerci ad, carr jj, bild de, burke g, folsom ar, liu k, shea s, szklo m, bluemke da, o’leary dh, tracy r, watson k, wong nd, kronmal ra. coronary calcium as a predictor of coronary events in four racial or ethnic groups. n engl j med. ; 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: – . arterioscler thromb vasc biol december d ow nloaded from http://ahajournals.org by on a pril , resignation syndrome: is it a new phenomenon or is it catatonia? full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=imhn issues in mental health nursing issn: - (print) - (online) journal homepage: https://www.tandfonline.com/loi/imhn resignation syndrome: is it a new phenomenon or is it catatonia? sandra p. thomas (editor) to cite this article: sandra p. thomas (editor) ( ) resignation syndrome: is it a new phenomenon or is it catatonia?, issues in mental health nursing, : , - , doi: . / . . to link to this article: https://doi.org/ . / . . published online: jul . submit your article to this journal article views: view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=imhn https://www.tandfonline.com/loi/imhn https://www.tandfonline.com/action/showcitformats?doi= . / . . https://doi.org/ . / . . https://www.tandfonline.com/action/authorsubmission?journalcode=imhn &show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=imhn &show=instructions https://www.tandfonline.com/doi/mlt/ . / . . https://www.tandfonline.com/doi/mlt/ . / . . http://crossmark.crossref.org/dialog/?doi= . / . . &domain=pdf&date_stamp= - - http://crossmark.crossref.org/dialog/?doi= . / . . &domain=pdf&date_stamp= - - issues in mental health nursing , vol. , no. , – https://doi.org/./.. from the editor resignation syndrome: is it a new phenomenon or is it catatonia? sandra p. thomas, phd, rn, faan, editor, issues in mental health nursing have you heard of uppgivenhetssyndrom, a new syndrome affecting refugee children and adolescents in sweden? the case of georgi, a russian refugee whose case is described in an article in the new yorker (aviv, ), provided my first glimpse of the syndrome and prompted me to search the scientific literature to learn more. cases of refugee chil- dren becoming apathetic, then stuporous, and then uncon- scious, began to be reported in the early ’s, the num- ber of cases swelling to more than by (aviv, ). most often, the children were from ethnic minorities, such as roma or uyghur, or fleeing persecution because of their fam- ily’s religion (as in the case of georgi). the children, gener- ally between ages – , who exhibited the apathetic syndrome began to require tube feeding and diapering; they were unre- sponsive to physical stimuli and did not appear to even react to painful stimuli (bodegard, ). electroencephalogram and computed tomography of the skull and brain revealed no strik- ing abnormalities, nor did laboratory screenings such as toxi- cology (sallin, lagerkrantz, evers, engstrom, hjern, & petrovic, ). the story of georgi is illustrative. according to premorbid descriptions of childhood friends and family, georgi was pop- ular, athletic, and well integrated into swedish society (hav- ing arrived from russia at the age of ). at the age of , just before he would have begun th grade, the migration board denied (for a second time) the family’s petition for asy- lum in sweden, after which georgi became “sullen and aloof ” (aviv, ). another blow was the deportation of a teammate from afghanistan. the full-blown onset of his resignation syn- drome occurred several months later when the migration board informed the family of upcoming deportation back to russia: he stopped speaking, eating, and appeared to be in a deep sleep (aviv, ). after a brief hospitalization, he was discharged to home care, bedfast and requiring tube feeding. georgi’s condi- tion continued to deteriorate over the following months, with one doctor stating “the boy is alive but barely” (aviv, , p. ). as cases of this syndrome increased, vigorous debate about etiology and diagnoses occurred among swedish physicians and psychiatrists, with consideration of anorexia nervosa, selective mutism, depression, malingering, and states of conversion and dissociation (sallin, et al., ). the long and stressful migra- tory process of the refugee families gained favor as the etiology with the swedish board of health and welfare, which coined the term “resignation syndrome” and advised that “a permanent residency permit is considered by far the most effective treat- ment … the turning point will usually be a few months to half a year after the family receives permanent residence” (cited in aviv, , p. ). a family system perspective appears useful, because resignation syndrome has not been diagnosed in unac- companied minors (sallin et al., ). to date there has been little research on resignation syn- drome, but sallin et al. ( ) dispute that the condition is really new, pointing out that its characteristics fit with catato- nia. its clinical picture is consistent with the cardinal symptoms of pediatric catatonia (immobility, mutism, withdrawal, refusal to ingest). they also argue for culture-bound psychogenesis, since the syndrome has only been found in refugee children in sweden whose families face deportation. comparisons can be made to amish girls displaying conversion disorder symp- tomatology and to hopeless concentration camp detainees who exhibited similar resignation behavior. sallin et al. argue that if the standard treatment for pediatric catatonia were employed (benzodiazepines and ect), a prompt response of the patient would validate their hypothesis that resignation syndrome is really catatonia. this treatment approach, however, has not been tried. some children with resignation syndrome have remained bedridden for as long as years (aviv, ). the available evidence, to date, indicates remission of the syndrome if the refugee families are not deported. in recovered children, neu- rological examination is normal with no apparent functional deficits (sallis et al., ). new cases of resignation syndrome declined after the swedish migration board, facing public out- rage about the media stories on “apathetic children,” revised its policies. outcomes of deported children have not been system- atically studied, but aviv ( ) relates that one child who had been deported to serbia was found still unconscious months later. to return to the story of georgi, his family was granted permanent residence in sweden. two weeks afterward, georgi opened his eyes; three days later, he took water from a spoon; four days later, he attempted to turn his body. still later, he returned to school and began to interact normally with classmates—even joking with them. when visited by rachel aviv in his home in november, , he conversed easily with her about topics such as sports. he described his many months in bed as feeling as though he had been trapped in a “glass cage” down in the ocean, which would shatter if he moved, causing drowning. when asked by aviv if he was aware that swedish residency had been granted to his family because of his condi- tion, he replied, “i don’t think that i wanted to do this. not if i start to think about how i felt in the glass cage” (aviv, , p. ). ©  taylor & francis group, llc https://doi.org/ . / . . https://crossmark.crossref.org/dialog/?doi= . / . . &domain=pdf&date_stamp= - - s. p. thomas no one contends that these children “resign” voluntar- ily. but there are many unanswered questions about this syndrome. whether it is a new phenomenon or a permutation of catatonia, the suffering of these children and their families is profound. further exploration of contributing factors such as traumatization and individual predispositions may shed light on preventive interventions for the syndrome. manuscripts on any aspect of this topic would be welcome. references aviv, r. ( , april ). letter from sweden: the apathetic. the new yorker, – . bodegard, g. ( ). pervasive loss of function in asylum-seeking children in sweden. acta paediatrica, , – . sallin, k., lagerkrantz, h., evers, k., engstrom, i., hjern, a., & petro- vic, p. ( ). frontiers in behavioral neuroscience, ( ). doi: . /fnbeh. . . https://doi.org/ . /fnbeh. . 	references of all theory, rather than a flaw of rawls’s particular instance of it. if that is so, then freeden is moving towards a criticism, not simply of the narrowness or inappropriateness, or unreality, of rawls, but of political theory as a species of thinking about politics. freeden concludes this selection by speculating, tantalizingly briefly, on whether, since thinking is an activity, the conventional thought/action distinction might be replaced. he does not pursue the point. perhaps though the proliferation of hints and allusions, as well as of more substantially pursued arguments, is a necessary and desirable characteristic of a body of work that, by its very refusal of rigid system, continuously raises new and important questions of both interpretation and theory. rodney barker department of government, london school of economics, uk. identity in democracy amy gutmann princeton university press, princeton & oxford, , pp. isbn: x. contemporary political theory ( ) , – . doi: . /palgrave.cpt. amy gutmann’s identity in democracy is a recent addition to the important and continually expanding volume of scholarship dedicated to examining and effectively responding to the conceptual and practical challenges associated with ‘identity politics’ in contemporary liberal democracies. in this erudite and interesting study, gutmann employs normative arguments and empirical evidence to reveal ‘the good, the bad, and the ugly of identity politics’ (p. ). in the course of doing so, she hopes to provide a useful answer to the following question: how is the achievement and maintenance of democratic justice — understood as a combination of civic equality, individual liberty, and equality of opportunity — either facilitated or hindered by the presence of identity groups and their active involvement in the political process, and what can be done to help minimize the ability of such groups to impede the realization of such a goal? according to gutmann, neither the proponents nor the opponents of identity groups have yet successfully articulated the complex relationship between such groups and democracy. the actual role of identity groups in democratic politics has been problematically ignored by political scientists and book reviews contemporary political theory subjected to exaggerated criticism by popular commentators. the result is that ‘both academic and popular discourse’ have neglected to engage certain ‘basic questions about the political ethics of identity groups in democracy’ (p. ). with identity in democracy, gutmann hopes to redress that deficiency. it is impossible to do justice to gutmann’s study within the confines of this brief review. i will therefore restrict my efforts to reiterating certain conclusions essential to gaining a general understanding of her argument. gutmann begins by outlining what she considers to be a number of critical features of identity politics that have yet to be properly understood or analysed. foremost among such features is the distinctive character of identity groups. existing discourse has failed to offer a satisfactory definition of identity groups; in particular, it typically neglects to distinguish between identity groups and interest groups. according to gutmann, the defining difference between the two is that, whereas the latter emerge solely as a consequence of a shared instrumental goal that precedes their formation, the former arise because of a mutual identification (voluntary or otherwise) among individuals who share certain social markers, such as gender, race, class, ethnicity, nationality, religion, disability, sexual orientation, age, or ideology. though ‘identity and interests are often closely intertwined’ (p. ) and identity groups might and often do pursue self-interests, it is, gutmann argues, incorrect to reduce such groups to mere instruments whose establishment and maintenance is solely for the purpose of furthering such interests. she notes that, unlike interest groups, many identity groups — such as the national association for the advancement of colored people (naacp), for example — also purposely pursue the interests of non-members. also absent from the debate is the acknowledgment that no single identity group can effectively represent the ‘entire person’; individuals may and generally do identify with others for a number of reasons beyond those captured by a single identity group. in order to demonstrate both the significance of the above-noted deficiencies and the value of rectifying them, gutmann distinguishes four different types of identity groups present in contemporary liberal democracies — cultural (e.g. the pueblo, the basques, the old order amish), voluntary (e.g. the jaycees, the boy scouts, the knights of columbus), ascriptive (e.g. naacp, the national organization for women), and religious (e.g. jews, muslims, hindus, christians) — and explores certain political controversies and court cases associated with each. for example, she critiques the willingness of theorists and judges alike to both presume ‘that culture shapes individual identity in a comprehensive way’ (p. ) and, subsequently, to assign political and legal primacy to the claims of the group, rather than to their individual members, thereby, ironically, allowing cultural identity groups to limit the freedom and opportunity of their members inequitably. book reviews contemporary political theory gutmann concludes that identity groups are intrinsically neither good nor bad for democratic politics. when their presence serves to combat negative stereotypes and provides a vehicle for the effective political engagement and influence of disadvantaged people, identity groups can help facilitate the realization of the ideal of democracy. however, identity can also be employed as a weapon to undermine the pursuit and achievement of democratic justice, as when the ku klux klan or moral majority promote intolerance and oppression of certain races and those who embrace alternative lifestyles. not surprisingly, trying to determine ‘reasonable’ and generally acceptable criteria to distinguish between identity groups that should be encouraged vs those that should be discouraged can be an extremely difficult and, indeed, ugly task. yet, democratic justice requires that such determinations be made. for gutmann, the presence of identity groups and their active involvement in democratic politics becomes unacceptably problematic only when such groups assign primacy to the advancement of their own particular interests regardless of whether doing so may impede the pursuit and attainment of socio-political justice and peace. the preceding summary offers only the briefest of overviews of some of gutmann’s central arguments. identity in democracy constitutes a thoughtful and provocative analysis of the relationship between identity groups and democratic justice in contemporary liberal democracies. in an era when the interaction between identity and democracy seems to be increasing both in frequency and importance, gutmann’s book admirably demonstrates the complicated relation- ship between the two. further, though gutmann is a political theorist by vocation, there is much in this text that will be of interest to scholars and practitioners in a variety of disciplines, including, for example, law and sociology. not all readers will be persuaded to agree with each of gutmann’s substantive judgments, but no one will be left without food for thought. shaun p. young york university, canada. the politics of property. labour, freedom and belonging laura brace edinburgh university press, edinburgh, , viiiþ pp. isbn: . contemporary political theory ( ) , – . doi: . /palgrave.cpt. laura brace offers a much needed re-examination of the modern notion of property in this informative and clearly written book. her main point is that book reviews contemporary political theory 	identity in democracy antifreeze protein hydration waters: unstructured unless bound to ice commentary antifreeze protein hydration waters: unstructured unless bound to ice sean m. marksa and amish j. patela, how do fish, insects, and other organisms survive in frigid polar environments? they do so with the help of remarkable molecules known as antifreeze proteins (afps), which suppress freezing and associated cell death despite being present at concentrations of less than wt % ( ). in contrast, automotive antifreeze needs roughly to wt % of the additive to function ( ). afps are able to suppress freezing at such low concentrations because, unlike antifreeze, they do not rely on altering the inherent structure of water; instead, they bind to nascent ice nuclei and prevent them from growing ( ). thus, being able to recognize and preferentially bind ice in a vast excess of water is the key to afp function ( ). however, in the absence of any chemical differences between water and ice, how do afps discriminate between them? moreover, both water and ice are composed of a tetrahedral network of hydrogen bonds, so even the structural differences be- tween them are subtle. indeed, how afps are able to perform what has been touted as one of the most chal- lenging molecular-recognition tasks in all of biology has long been a source of amazement and intrigue ( , ). in pnas, hudait et al. ( ) clarify important aspects of the ice-recognition puzzle by using molecular simulations to study tmafp, a hyperactive insect afp. what makes this puzzle even more fascinating is that a wide array of organisms, ranging from bacteria to insects and fishes, has independently evolved afps that display substantial differences in their sequences, structures, and ice-binding sites (ibs) ( , ). in other words, there is not one, but a diversity of motifs that can confer afps with their ice-binding abilities. what, then, are the characteristic features of ibs, and how do they enable afps to bind ice? early suggestions focused on hydrophilic moieties, which could hydrogen-bond with ice; the rationale was that if the hydrophilic groups on the ibs were spaced to match the lattice spacing of ice, the bind- ing between the afp and ice would be particularly favorable ( , ). in other words, a complementarity in the spacing of hydrophilic groups on the afp surface and the crystal planes of ice was believed to enable afps to bind, and integrate themselves into, the ice lattice. however, as numerous afps from diverse organ- isms were discovered, and their crystal structures solved, it became clear that hydrophobic residues featured prominently on the ibs of afps ( ). even hy- drophilic residues, such as threonine, had hydropho- bic groups (−ch ), which protruded outward from the ibs (fig. b). the presence of hydrophobic groups on the ibs suggested that although hydrogen bonding and lattice matching could help afps bind ice, they were not sufficient by themselves. it also raised the question that, given that hydrophobes do not interact favorably with water or with ice, what role might they have in facilitating the binding of afps to ice? the hydration waters of a small hydrophobic group are constrained by having to hydrogen-bond around the hydrophobe, which is unfavorable from an entropic standpoint, so it was suggested that the release of such constrained waters from hydrophobic groups on the ibs might facilitate afp–ice binding ( ). thus, both hydrophilic and hydrophobic groups seem to be important in conferring afps with an affinity for ice. mutagenesis studies have further shown that a single mutation, whether it replaces a hydro- phobic group with a hydrophilic one or vice versa, can result in the loss of afp function ( ). such seem- ingly whimsical behavior has also been observed in molecular-simulation studies, which probe the “ice philicity” of extended surfaces by interrogating their propensities for nucleating ice. in particular, patey and coworkers found that kaolinite, a clay mineral, is only able to nucleate ice if it has the right amount of flexibility ( ); molinero and coworkers observed that the roughness of a graphitic surface influences its ice philicity ( ); and michaelides and coworkers showed that neither lattice matching (to ice) nor sur- face hydrophilicity alone is sufficient to confer ice philicity, but that appropriate values of both are needed ( ). collectively, these studies suggest that the affinity of an afp surface for ice depends not only on its ability to hydrogen-bond with ice but also on adepartment of chemical & biomolecular engineering, university of pennsylvania, philadelphia, pa author contributions: s.m.m. and a.j.p. wrote the paper. the authors declare no conflict of interest. published under the pnas license. see companion article on page . to whom correspondence should be addressed. email: amish.patel@seas.upenn.edu. published online august , . – | pnas | august , | vol. | no. www.pnas.org/cgi/doi/ . /pnas. c o m m e n t a r y d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://crossmark.crossref.org/dialog/?doi= . /pnas. &domain=pdf http://www.pnas.org/site/aboutpnas/licenses.xhtml mailto:amish.patel@seas.upenn.edu www.pnas.org/cgi/doi/ . /pnas. many other properties, such as its curvature, roughness, and flexibility, in a nontrivial manner ( – ) (fig. c). although no clear unifying features appear to enable afps to preferentially bind ice, could the affinity of an afp for ice be reflected in certain properties of its hydration waters? using mo- lecular simulations of diverse afps, sharp and coworkers showed that the orientational distribution of water molecules in the vicinity of ibs display certain key differences, which persist across differ- ent classes of afps ( – ); nutt and smith reported a subtle increase in tetrahedral order in the hydration shell of a hyperactive insect afp ( ); and both molecular simulations and terahertz spectroscopy measurements have reported slower relaxation dy- namics of the afp hydration waters ( – ). in each case, the waters in the afp hydration shell seem to, in one way or another, bear some resemblance to those in ice. do such seemingly ice-like waters enable afps to bind to ice and, if so, how? the crystal structure of a bacterial afp, mpafp, solved by davies and coworkers, provided an important clue by presenting, for the first time, a clear molecular picture of the afp–ice binding interface ( , ). although afp hydration waters can be resolved in the x-ray structure, they are usually perturbed by protein–pro- tein contacts in the crystal and tend not to be characteristic of waters that mediate afp–ice binding. however, mpafp crystal- lizes in a unique manner with its ibs well removed from any pro- tein–protein contacts, so that the afp–ice interface is clearly visible in the crystal structure ( , ). in what davies and coworkers called the anchored clathrate (ac) motif, the afp uses both hy- drophobic and hydrophilic groups on its ibs to bind ice ( ). in the ac motif, water molecules adopt a highly ordered structure, form- ing a clathrate-like shell around the hydrophobic groups of the ibs and participating in hydrogen bonds with its hydrophilic groups (fig. d). based on the clear evidence of structured waters at the afp–ice interface, and the fact that the ibs hydration waters are somewhat ice-like (in solution), it was suggested that a highly structured layer of clathrate-like waters exists at the ibs of some afps, which then enables the proteins to recognize and bind ice ( , ). molinero and coworkers explore this tantalizing suggestion in a series of recent articles ( , , ). in ref. , the authors studied a number of hyperactive afps derived from insects and found that afp binding to ice can occur not only through the ac motif but also through other similar motifs, wherein structured waters bridge the afp surface and ice. given the diversity of ibs ob- served across different afps, this result makes sense. although both experiments and simulations highlight the presence of struc- tured waters at the afp–ice interface, does this structuring precede binding, and, if so, could the preordering of waters by a surface be used as a signature of its affinity for ice? to answer these questions, hudait et al. ( ) chose to study tmafp, a hyper- active insect afp, which displays the best lattice matching with hexagonal ice and may thus be the most likely to structure waters into the ac motif in solution. using molecular simulations with three different water models, and multiple ways of characterizing the structuring of ibs hydration waters, the authors conclusively show that the ibs does not display ice-like or ac-like order in solution; rather, the ac motif forms only after the afp has moved next to the ice surface and aligned itself in an orientation that is optimal for binding to ice ( ). these results suggest that a preor- dering of their hydration waters is not needed for ice recognition by afps. support for these findings is also provided by nmr studies, wherein a fast (subnanosecond) exchange of waters is observed between the hydration shell of tmafp and the bulk ( ); by contrast, ac waters ought to relax much more slowly, akin to those in ice. more support comes from earlier work by molinero and coworkers, which showed that poly(vinyl alcohol), a flexible polymer that does not preorder water, nevertheless binds ice ( ). as more afps belonging to different classes are studied, and as even more afps are discovered, it will be interesting to see if an afp capable of preordering its hydration waters will be found. ice-nucleating proteins (inps) represent another class of proteins that are ice-philic; although they are structurally and chemically similar to afps, inps are much bigger in size and can even cluster, thereby providing large surfaces capable of nucleating ice. although tmafp does not structure its waters in solution, might the larger inps or their clusters be able to do so? to answer this question, hudait et al. ( ) use model afp-like surfaces spanning a range of sizes; inp monomers are represented by rectangular surfaces with a fixed nanoscopic width and increasing lengths, whereas inp clusters are mimicked using square surfaces. the authors find that large rectangular surfaces do not structure their hydration waters in solu- tion, but that as the size of the square surfaces is increased, they become more effective at ordering water. these findings suggest that it is not easy for surfaces to structure their hydration waters into ice-like or clathrate-like order; only large extended surfaces, like the ones assembled by inp clusters, may be successful in doing so. to facilitate experimental validation of their findings, the authors also used their simulations to predict vibrational spectra of the oh stretch of water; however, they find that neither infrared nor raman spectra can discriminate between ac waters at the afp–ice interface and the hydration waters of the afp in solution ( ). despite the remarkable progress made in understanding afps, their most sought-after secrets remain elusive for now—both what b d flexibility hydrogen bonding lattice matching c hydro- phobicity affinity for ice a water ice fig. . (a) afps have evolved to recognize and bind ice in a vast excess of water. (b) the ibs of typical afps display both hydrophilic (blue) and hydrophobic (red) groups ( ). (c) the affinity of a surface for ice depends sensitively on a number of surface properties, including its ability to hydrogen-bond with ice, correspondence with the ice lattice, flexibility, and the presence of nonpolar groups ( , – ). adapted from ref. . (d, left) although tmafp binds ice through the ac motif, (d, right) it does not preorder water in solution. adapted with permission from ref. . marks and patel pnas | august , | vol. | no. | d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , the molecular signatures of surface ice philicity are and how they enable afps to bind ice continue to mystify. however, the work of hudait et al. ( ) shows that the preordering of waters in solution, whether in the ac motif or otherwise, is not necessary for afps to bind ice. importantly, the authors demonstrate how molecular sim- ulations can build upon the wealth of knowledge provided by struc- tural studies, and how they are beginning to make meaningful contributions in furthering our understanding of afps. with such synergistic advances, we are hopeful that it will not be long before the secrets of afps are uncovered. a fundamental understanding of ice philicity could also have far-reaching practical implications and may lead to the discovery of synthetic molecules and materials that are even more potent at binding ice than afps; such materials would find use in diverse contexts, ranging from the preservation of organs for transplant to increasing the freeze tolerance of crop plants and the transportation of frozen foods ( ). acknowledgments a.j.p. thanks kim sharp for introducing him to afps and for numerous insightful discussions. this work was supported by national science foundation grants dmr , cbet , and che , charles e. kaufman founda- tion grant ka - , american chemical society petroleum research fund grant -dni , and alfred p. sloan research foundation fellowship fg- - (all to a.j.p.) and by a computational science graduate fellowship from the department of energy (to s.m.m.). jia z, davies pl ( ) antifreeze proteins: an unusual receptor–ligand interaction. trends biochem sci : – . kelland ma ( ) history of the development of low dosage hydrate inhibitors. energy fuels : – . knight c, devries a ( ) ice growth in supercooled solutions of a biological “antifreeze”, afgp – : an explanation in terms of adsorption rate for the concentration dependence of the freezing point. phys chem chem phys : – . prabhu n, sharp k ( ) protein–solvent interactions. chem rev : – . hudait a, et al. ( ) preordering of water is not needed for ice recognition by hyperactive antifreeze proteins. proc natl acad sci usa : – . sharp ka ( ) a peek at ice binding by antifreeze proteins. proc natl acad sci usa : – . davies pl ( ) ice-binding proteins: a remarkable diversity of structures for stopping and starting ice growth. trends biochem sci : – . devries al, lin y ( ) structure of a peptide antifreeze and mechanism of adsorption to ice. biochim biophys acta : – . zielke sa, bertram ak, patey gn ( ) simulations of ice nucleation by kaolinite ( ) with rigid and flexible surfaces. j phys chem b : – . lupi l, hudait a, molinero v ( ) heterogeneous nucleation of ice on carbon surfaces. j am chem soc : – . fitzner m, sosso gc, cox sj, michaelides a ( ) the many faces of heterogeneous ice nucleation: interplay between surface morphology and hydrophobicity. j am chem soc : – . gallagher kr, sharp ka ( ) analysis of thermal hysteresis protein hydration using the random network model. biophys chem : – . yang c, sharp ka ( ) the mechanism of the type iii antifreeze protein action: a computational study. biophys chem : – . yang c, sharp ka ( ) hydrophobic tendency of polar group hydration as a major force in type i antifreeze protein recognition. proteins : – . nutt dr, smith jc ( ) dual function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations. j am chem soc : – . xu y, gnanasekaran r, leitner dm ( ) analysis of water and hydrogen bond dynamics at the surface of an antifreeze protein. j at mol opt phys : – . meister k, et al. ( ) long-range protein-water dynamics in hyperactive insect antifreeze proteins. proc natl acad sci usa : – . meister k, et al. ( ) observation of ice-like water layers at an aqueous protein surface. proc natl acad sci usa : – . garnham cp, campbell rl, davies pl ( ) anchored clathrate waters bind antifreeze proteins to ice. proc natl acad sci usa : – . hudait a, odendahl n, qiu y, paesani f, molinero v ( ) ice-nucleating and antifreeze proteins recognize ice through a diversity of anchored clathrate and ice- like motifs. j am chem soc : – . naullage pm, lupi l, molinero v ( ) molecular recognition of ice by fully flexible molecules. j phys chem c : – . modig k, qvist j, marshall cb, davies pl, halle b ( ) high water mobility on the ice-binding surface of a hyperactive antifreeze protein. phys chem chem phys : – . zeng h, wilson ld, walker vk, ripmeester ja ( ) effect of antifreeze proteins on the nucleation, growth, and the memory effect during tetrahydrofuran clathrate hydrate formation. j am chem soc : – . the dill research group ( ) energy landscapes. available at dillgroup.org/#/landscapes. accessed july , . | www.pnas.org/cgi/doi/ . /pnas. marks and patel d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://dillgroup.org/#/landscapes www.pnas.org/cgi/doi/ . /pnas. merging the religious congregations and membership studies: a data file for documenting american religious change r e s e a r c h n o t e merging the religious congregations and membership studies: a data file for documenting american religious change rachel bacon • roger finke • dale jones received: october / accepted: april / published online: may � the author(s) abstract the decennial religious congregations and membership studies are a popular data source for analyzing local religious composition and diversity, but several methodological challenges hinder merging the datasets for longitudinal analyses. in this paper, we introduce strategies for addressing four of the most serious challenges: religious mergers and schisms, changes in membership stan- dards within certain groups, missing data and changes in county boundaries. in doing so we successfully merge the , , and collections and build new longitudinal datasets of congregational and membership counts at the state and county levels. these changes increase religious group representation from to , reduce bias from missing data, allow for the more reliable inclusion of – million adherents in each year, and improve overall ease of use. we also document instances when corrections were not possible and alert readers to the limitations of the merged files when measuring change among certain groups. the new longitudinal files are accessible from thearda.com. keywords religion census � religious change � data � longitudinal analyses the decennial religious congregations and membership studies collected by the association of statisticians of american religious bodies are perhaps the most heavily used datasets on american religion. these data provide counts of adherents and congregations belonging to dozens of religious groups at the state and county levels. scholars of varied backgrounds rely on these data to measure and analyze & rachel bacon rjb @psu.edu the pennsylvania state university, oswald tower, university park, pa , usa church of the nazarene global ministry center, prairie star parkway, lenexa, ks , usa rev relig res ( ) : – https://doi.org/ . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf https://doi.org/ . /s - - - local religious composition, diversity, and the contextual effects of religion (lim ). the four most recent of these datasets (i.e. , , , ) have been collectively downloaded more than , times from the association of religion data archives (thearda.com) over the past years. moreover, religious denominations and local congregations, as well as the media and community planners, use the files extensively for research, journalism, and planning. in short, the collections have proven invaluable to a diverse group of users for mapping and analyzing religious variations across counties, states and the nation. despite the popularity of these collections, they are seldom used to measure changes in religious group size or the composition and diversity of geographic regions overtime. several practical and methodological challenges deter researchers from merging the datasets. this paper introduces methodological strategies to address four of the most significant challenges for merging the collections: religious mergers and schisms, changes in membership standards within certain groups, missing data, and changes in county boundaries. relying on these strategies we merge the , , and collections and build two datasets of congregational and membership data across time: one for counties and another for states. when possible, we make corrections or offer improved estimates to standardize measures and units of analysis over time. when corrections are not possible, however, we alert readers to the limitations of the merged files. before we address the challenges of merging the data collections, however, we begin with a brief overview of the data collection procedures used. documenting how the data are collected helps to clarify and identify the challenges that emerge when we attempt to merge the collections. collecting congregational and membership data the congregational and membership studies conducted in , , and each provide a county-by-county enumeration of religious bodies in the u.s.. although similar collections also were conducted in and (national council of churches ; johnson et al. ), we have not included them because the level of participation was lower and the measures used were less standardized than the later collections. the remaining four datasets relied on procedures and measurement criteria that were largely standardized. nevertheless, we highlight several important differences between the remaining four collections. in each of the decennial studies, a study representative contacted all of the major religious groups and asked them to report on the number of congregations, members and adherents in their religious group for each county in the u.s.. we focus on congregational counts and total adherents because these measures are more consistently reported across religious groups and are more standardized than membership (membership counts are also unavailable in the online versions of the both files can be downloaded from thearda.com at: http://www.thearda.com/archive/files/ descriptions/rcmsmgcy.asp and http://www.thearda.com/archive/files/descriptions/rcmsmgst. asp. rev relig res ( ) : – http://www.thearda.com/archive/files/descriptions/rcmsmgcy.asp http://www.thearda.com/archive/files/descriptions/rcmsmgcy.asp http://www.thearda.com/archive/files/descriptions/rcmsmgst.asp http://www.thearda.com/archive/files/descriptions/rcmsmgst.asp and datasets). for many groups, especially christian groups practicing adult baptism, full membership status was reserved for adults. in contrast, adherents is a more inclusive measure defined as ‘‘all members, including full members, their children and the estimated number of other participants who are not considered members’’ (quinn et al. ). not all religious groups directly reported both membership and adherent data. when religious groups reported only adult members, the data collectors estimated the number of total adherents using a county-level multiplier. most religious groups who directly reported adherents agreed to the standard definition, but several groups changed their counting methodology over the years, usually to make their adherent counts more accurate and more comparable to other groups. data collectors also commissioned independent studies for groups that were difficult to count accurately, such as the amish and most non-christian groups. these independent studies were not always conducted by the same principal investigators (pis) from one decade to the next and employed different methodologies. among those groups affected by changes in measurement, therefore, counts differ substantially between the datasets and may not reliably reflect changes in size. we also call attention to the fact that the scope of the data collections has expanded over time, resulting in varying levels of data collection efforts allocated to specific groups. for the and collections the pis appealed only to christian denominations and jewish groups, capturing the vast majority of white americans attending local congregations. later collections expanded to include independent christian congregations as well as non-christian groups. despite the pis’ efforts to include as many groups as possible, the level of participation varied across the datasets, with participating in , in , in , and in . as shown in greater detail in online supplement appendix a (all appendices are available at thearda.com), however, only of the cumulative participating groups have both adherent and congregation data in all four decades. the considerable variation in participation can be partly explained by the expanded scope of the collection procedures in later decades. furthermore, some groups merged, split, or simply reported their data differently from one decade to the next, while other groups simply participated in some years, but not others. the result is that most groups have missing data for at least year. finally, we should note that the official name of the datasets and the sponsoring institutions have varied from year to year. the collection was assembled by the glenmary research center and the final three collections were conducted by the association of statisticians of american religious bodies. the and reported church members [total population/(total population - children years and under)]. in the collection, groups reported their own adherents while an additional groups reported members and had adherents estimated by data collectors using the county-level multiplier (grammich et al. ). the documentation identifies groups because the american lutheran church, lutheran church in america, and association of evangelical lutheran churches reported separately in the publication, but were merged in the stand-alone dataset available on the arda website. the organizations helping to fund one or more of the collection have included: the lilly endowment inc., john templeton foundation, national council of the churches of christ in the u.s.a. (new york), rev relig res ( ) : – datasets were entitled ‘‘church and church membership in the united states,’’ reflecting their focus on christian groups. the dataset was changed to ‘‘religious congregations and membership study’’ and the dataset was revised to the ‘‘u.s. religion census: religious congregations and membership study.’’ to reduce confusion, throughout this paper we refer to the four datasets as the religion census data, and the final dataset as the merged or longitudinal file. the religion census data offer great promise for conducting longitudinal and spatial analyses on religious change in america, but merging the files and offering comparable data over time poses multiple challenges. below we review these challenges, offer recommendations on how some can be addressed and review the limitations that will remain. addressing the challenges in this paper, we address four major challenges in merging the four religion census datasets. first, we devise a new scheme to address inconsistency in variable names across the datasets because of mergers, schisms or changes in how a group’s data were aggregated over time. second, we document how some groups changed their standards for reporting adherent and/or congregation counts between collections and we clearly delineate between the counts that can be rectified and those that cannot. third, we reduce the amount of missing data by offering adherent estimates for groups missing adherent and/or congregation data for a single year and by providing comparable estimates overtime for groups involved in mergers or schisms. fourth, we account for changes in the geographic boundaries of counties, allowing for a comparable unit of analysis over time. in addressing the problems, we produce a merged dataset of the four collections that readily permit trend analysis of adherent and congregation counts, although with some caveats that we clearly identify. accounting for mergers, schisms, and other aggregations religious group schisms occur when a group splits into two or more groups, and a merger occurs when two or more religious groups become one group, which makes it difficult to consistently identify the same group of adherents over time. this problem is further compounded by measurement errors, such as accidentally omitting one of the religious groups involved in the merger, schism or some other aggregate group change. since religious groups are geographically concentrated and therefore non-randomly distributed (land et al. ; stump ; finke and stark ), the unintentional omission of a group in longitudinal analyses could footnote continued association of statisticians of american religious bodies, glenmary research center, glenmary home missioners, aid association for lutherans, southern baptist convention, lutheran council in the u.s.a., african methodist episcopal zion church, church of the nazarene, american baptist churches in the usa, national association of free will baptists, presbyterian church (u.s.a.), united church of christ, wisconsin evangelical lutheran synod and evangelical lutheran church in america. rev relig res ( ) : – introduce significant bias. we address both of these problems in the new longitudinal file. we begin by replacing the old naming scheme for variables with one that is more consistent across the data collections. in the stand-alone versions of the religion census data available online, the variable names assigned to individual religious groups are different in each dataset. data are presented in ‘‘wide’’ format (i.e. each row is a county and the dozens of columns contain the counts of each religious group’s adherents and congregations separately) and users must rely on the name of the religious group to match them across datasets. although most groups retain the same name for each year they participated, their name can change due to mergers, splits or changes in how the data were reported. in the case of schisms, adherents who previously belonged to one religious group are split among two or more groups after the schism. in mergers, the opposite occurs. in addition, some groups in the religion census data did not formerly split or merge, but agreed to report together in year and separately in another. to alleviate the burden of identifying groups across the datasets for users, we first replaced the old variable names with the unique three-number codes assigned to each religious group by the religion census data collectors. second, we created new aggregate cases that combine all groups affected by a schism, merger, or other group count change into a single group. the full listing of religious group names and their accompanying codes are in appendix a and the ‘‘notes’’ column identifies which groups are combined on account of a merge, split or change in aggregated reporting. our solution resulted in eight new aggregate groups created from individual religious groups: (a) a total of five groups present in the religion census data were affected by a schism and were combined into two new groups for the longitudinal file. (b) six groups experienced a formal merger and were combined into two new groups. (c) sixteen groups chose to report together in year but separately in another, and we subsequently combined them into four new groups. table identifies the affected religious groups and their sizes. newly combined groups are now assigned the three-digit code that matches the largest group among them, followed by the letter ‘‘b’’ to indicate that multiple groups were combined to make the new group. data in the merged file are also presented in ‘‘long’’ format (i.e. rows now represent religious groups in a specific county and year, and only two columns are used to identify adherent and congregation counts). groups that would otherwise be dropped because they do not appear to participate across the datasets are now included, resulting in nearly two million additional adherents across all four datasets. in this way, the longitudinal file includes both the combined groups and the individual counts of each religious group that was included in the combined group. once we identified the groups that needed to be combined, however, some data remained missing. in five instances, one of the groups included in a combined group the merged file also includes a variable that flags whether each case is included elsewhere in a combined group or is itself a combined group. rev relig res ( ) : – table adherents of groups affected by schisms, mergers, or other changes in aggregation code schisms episcopal church groups combined b , , , , , , , , episcopal church , , , , , , , , the anglican church in north america , a evangelical lutheran church in america groups combined b , , , , , , , , evangelical lutheran church in america , , , , , , , , lutheran congregations in mission for christ , north american lutheran church , a merges mennonite groups combined b , , , , conservative mennonite conference , , mennonite church , , the mennonite church general conference , , mennonite church usa , , serbian orthodox church in the usa groups combined b , , serbian orthodox church in the usa , , serbian orthodox church in the usa (new gracanica metropolitanate) , a changes in aggregate reporting friends groups combined b , , , , friends (quakers) , , , friends central yearly meeting friends united meeting , friends—evangelical , friends—independent yearly meetings friends—unaffiliated local meetings a friends—conservative friends—general conference , friends—dual general conference and united meeting , bruderhof and hutterian groups combined b , , , hutterian brethren , , , a bruderhof communities, inc. orthodox church in america groups combined b , , orthodox church in america: territorial diocese , , orthodox church in america: albanian orthodox archdiocese orthodox church in america: romanian orthodox episcopate of america , rev relig res ( ) : – was missing adherent counts in year, so we estimated the missing adherent counts in these cases (please see the section on missing data for methodological details). because schisms are often regionally based, this correction reduces the geographic bias that would exist if they were omitted. in other cases, a few of the schismatic groups simply did not participate. for example, the alliance of baptists (aob) split from the southern baptist convention (sbc) in and is included in the religion census collection. we chose not to combine the aob counts with the sbc because the aob only provided data on the number of congregations in and provided no data at all in and . individual congregations also are known to switch affiliations or become independent churches at a smaller scale than formal schism (marcum ; chaves ), making it impossible to track the adherents of congregations who leave the denomination to another group in the data. most of these omissions, however, are small and will have little impact on the total counts. overall, our adjustments for known schisms, mergers and other aggregations allow for meaningful comparisons over time for more groups and greatly ease the use of the longitudinal files. group measurement changes a second major challenge for constructing a longitudinal file is when some groups change the criteria they use for counting adherents or congregations from one collection to the next. as noted earlier, the criteria or methods used for collecting the data were sometimes changed in an effort to improve the counts or make their measurement comparable to other groups in the collection. by making the change, however, the adherent or congregation counts for the group were no longer comparable across all four collections. this is a significant concern because it affects four of the largest religious groups in the country and because it is often the most difficult to correct. table continued code orthodox church in america: bulgarian diocese moravian church in america, alaska and north province combined b , , , , moravian church in america, alaska province moravian church in america (unitas fratrum) north province , , , , total adherents added after adjustments , , , , a adherent count is estimated for these groups the omission of the aob from sbc enumerations in the and subsequent collections is unlikely to be associated with significant bias. in , there were aob congregations compared to , congregations in the sbc. rev relig res ( ) : – table lists all of the groups where measurement criteria or methods changed for at least one time point, reports on the size of the group, and offers recommendations on when over time comparisons can be made. below we offer additional information and additional precautions for each of these groups. before we review these details, however, we first explain how we were able to offer corrections for the measurement changes of the united methodist church. for the united methodist church (umc), there was a change in adherent measurement between and . prior to the collection, the umc reported membership, but not adherent counts. because adherents were not directly reported, the religion census data collectors estimated adherents using the membership count and their standard county-level multiplier (see footnote ). in , however, umc statisticians directly reported their own adherent totals for each congregation, and this shift yielded more adherents than the old estimate. the county-level estimation would have identified . million adherents in rather than the . million reported by umc statisticians (see table ). since the umc submitted both membership and adherent counts to the religion census in , it is possible to use the old county-level estimation formula to create an adherent count consistent with earlier datasets. we also requested from the umc statisticians a direct reporting of adherent counts for , which made it possible to use the direct-reporting measurement for and . both the ‘‘new’’ and ‘‘old’’ versions of umc adherents for and are included in the longitudinal file. in the case of the umc, the measurement change was relatively simple and we were able to standardize the adherent count with earlier years using membership and a county-level multiplier. other groups have more complicated changes and we were unable to standardize their counts across all four datasets. like the umc, the measurement of roman catholic church adherents switched to a more direct-reported adherent count in . unlike the umc, however, earlier counts relied on some survey-based estimates, which could not be standardized with the new count method. the southern baptist convention and church of latter-day saints changed their definition of congregations or adherents and, unlike the umc, were unable to provide counts to us based on their old/new definition. for eastern orthodox groups and the wesleyan church, we are unable to determine the methodology used in some years, which calls into question measurement consistency across time. among the remaining cases of measurement change, religion census data collectors commissioned independent studies to improve the accuracy of measurement. differences in data collection methods and strategies used by these independent studies contribute another source of measurement inconstancy over time. each group has a unique measurement history that limits comparisons between two or more collection years. we describe these histories in more detail here: there are additional non-christian groups (i.e. buddhist, jain, muslim, and hindu) whose data were collected by different independent pis or with differing procedures between years, and only have counts for and . we do not discuss those groups here due to their limited participation, but we do include notes about their measurement in appendix a. more detailed descriptions of the methodologies used to count all groups in each collection are available in the appendices of the religion censuses’ data publications (quinn et al. ; bradley et al. ; jones et al. ; grammich et al. ). rev relig res ( ) : – table groups with measurement changes and recommendations for comparisons possible over-time comparisons catholic adherents , , , , , , , , , , ; caution when using congregations , , , , all years groups same group is measured in all years united methodist adherents (old) , , , , , , , , all years adherents (new) – – , , , , , congregations , , , , all years groups same group is measured in all years latter-day saints adherents , , , , , , , , , , ; caution when using congregations , , , , all years groups same group is measured in all years southern baptists adherents , , , , , , , , all years congregations , , , , , , ; caution when using groups same group is measured in all years independent churches adherents – , , , , , , , ; do not compare with congregations – , , ; do not compare with groups – , charismatic/ non-charismatic black protestants adherents , , , , – , , no years definitively comparable congregations – , no years definitively comparable groups – no years definitively comparable jewish groups adherents , , , , , , , , ; do not compare with or rev relig res ( ) : – • the catholic church in , some adherent counts were available at the parish-level, but in many areas the data collectors relied on diocese-level vital and sacramental statistics or on county-level estimates made using the american religious identification survey (jones et al. ). the shift to congregational- based direct reports in greatly reduced the number of catholic adherents. the number of catholics appears to decline from . million in to . million in , but the figure reported to the yearbook of american and canadian churches (yacc) suggests an increase from . to . million over the same time period (lindner , ). some survey estimates of catholics do identify a recent flattening in adherents, but most do not (grammich ), which suggests the decline captured in the religion census is a data artifact. table continued possible over-time comparisons congregations , ; do not compare with or groups jewish count split into four groups for old order amish adherents , , , , no years definitively comparable congregations no years definitively comparable groups additional amish group reported with the old order amish in , but separately in orthodox groups adherents , , , , , , , ; do not compare with or congregations , ; do not compare with or groups same groups present in and the wesleyan church adherents – , , , no years definitively comparable. congregations – no years definitively comparable groups – same group is measured in all years rev relig res ( ) : – • church of latter-day saints (lds) for the collection, the lds church reported a total of . million adherents. this total excluded members who were baptized, but not associated with a specific congregation. a more inclusive count is the . million members reported by the church in its almanac and to the yacc (lindner ). for the collection, the lds church changed its procedures to include the previously excluded baptized persons and reported an adherent total of . million, a figure more consistent with what it has reported elsewhere (lindner ) and more comparable to how other denominations count adherents in the religion census. • the southern baptist convention (sbc) the sbc reported adherents in a consistent way over time, but changed how they defined congregations. prior to , the congregation count did not include small ‘‘mission’’ gatherings, although adherents attending ‘‘missions’’ were counted. the sbc revised their methodology to include their mission congregations and therefore became consistent with other religious groups (grammich et al. ). as a result, there is a sudden increase of sbc congregations between and despite negligible growth in the number of adherents. • eastern orthodox groups in and , four orthodox groups directly reported adherent counts and an additional seven groups provided congrega- tional counts for , but they provided no documentation regarding counting methodology (quinn et al. ; bradley et al. ). for the and collections, however, data on a larger number of eastern orthodox groups were collected by a single pi using a more standardized definition of ‘‘adherents that included both adults and children known to participate in services at the local parish’’ (jones et al. ). as a result, the counts for several orthodox groups change suddenly between and . • the wesleyan church the documentation of data collection methodologies are available for and , but not for , and a significant change in estimation is suggested between decades. in , the wesleyan church provided adherent counts based on their own record-keeping, reflecting ‘‘those who have some contact with the church through its various departments’’ (jones et al. ). in , they estimated that adherents were simply percent more than the average attendance (grammich et al. ). • black protestant groups only four historically black protestant groups directly reported their own counts in the collection (quinn et al. ). in , three black protestant groups participated and an independent study estimated the size of black baptist churches, but the estimates did not include any congregational counts (bradley et al. ). black protestant groups are entirely absent in the collection, although finke and scheitle ( ) did create a state-level multiplier based on survey data to estimate the size of all black protestant adherents for that year. relying on congregational estimates from the online infogroup database on religious organizations, the collection included membership estimates for eight black protestant denominations and congregational estimates for . adherent counts were not available for all black protestant congregations, and so data collectors estimated a membership of for those congregations (grammich et al. ). rev relig res ( ) : – • jewish groups the collection included direct counts of conservative and reform judaism who were members of the united synagogues of america (quinn et al. ). the count also has direct reporting, but from an expanded four branches of judaism (conservative, reconstructionist, reform and orthodox) (grammich et al. ). in and , however, the data collectors relied on the american jewish yearbook to identify adherents and the jewish almanac yellow pages and phone directories to identify congregations (bradley et al. ; jones et al. ). • independent churches no efforts were made to count independent churches in and their measurement changed significantly between and . in and , the megachurch research center gathered information on large, independent churches for the religion census by calling the churches via telephone, identifying about two million adherents in each year (bradley et al. ; jones et al. ). in , a more exhaustive study using internet listings was possible, identifying million more adherents than in the and religion censuses (grammich et al. ). • old order amish adherents in and were estimated using an arbitrary congregational size based on the age of the church (quinn et al. ; bradley et al. ). in , the data were supplied from the mennonite publishing house and were more congregationally specific in estimation (jones et al. ). for , an exhaustive independent study was commissioned to conduct an accurate census using amish settlement directories (grammich et al. ). in summary, longitudinal analyses specific to these groups is limited. some of the caveats against overtime comparisons are negligible, depending on research questions and analytic plans. for instance, catholic and lds adherent data are comparable between and , and the congregational data is comparable through . researchers who wish to include the united methodists in their analyses must choose between the slightly less accurate county-level umc adherent estimates for a full – comparison, or use the newer adherent reports that are limited to and . similarly, some comparisons can be made among the amish between and , the jewish and independent church counts between and , and many orthodox groups can be compared between and . we strongly advise against making any comparisons at all among black protestant groups or the wesleyan church. missing data when merging the data, a third challenge is accounting for missing data in one or more of the collections. the vast majority of groups in the religion census have missing data in at least one of the datasets. due to the expanding scope of the for the collection, determined as per congregation for settlements established before and those established in and were assigned adherents per congregation. settlements established in and had per congregation (bradley et al. ). we include a flag variable in the merged file to identify groups with measurement problems that could prevent over-time comparisons. rev relig res ( ) : – collections as well as individual group’s partial or non-participation in one or more years, the pattern of missing data in the religion census is diverse. using two missing data strategies, we were able to estimate the missing data of religious groups. of these, groups become available across all four datasets, groups are limited to a or -year comparison, and the remaining four groups are included in an aggregate grouping (as noted in the merger and schism section). these strategies increased group participation across all four datasets from to , and from to in a -year comparison between and . appendix b lists all the affected groups and their new estimates. the first missing data strategy uses simple linear interpolation and extrapolation to estimate the number of adherents and congregations among groups who are missing both data types in only one of the four datasets. we limited our estimates to these groups in the interest of retaining as many groups as possible without compromising the reliability and validity of the counts. linear interpolation estimates the values between two points in time, and extrapolation estimates values beyond them, by continuing the line formed between the points. an interpolation and extrapolation strategy is commonly used in economics research to fill in missing data for entire variable values. past research has used interpolation/extrapolation with the religion census data to estimate values of total adherence in the years between and beyond the decennial datasets (hillary and hui ; dyreng et al. ; boone et al. ). the main assumption in this strategy is that adherent and congregation size has a linear relationship with time. this is the case for the vast majority of groups in the religion census data, and studies on church growth and decline usually observe a linear relationship with time (finke and stark ; blau et al. ; hadaway and roozen ; marcum ). because we limited our use of the interpolation/extrapolation strategy to select groups with nearly complete data, we believe that the estimates are reasonable within the assumption of linear growth/decline. we estimated the counts of groups via extrapolation for or , and another nine groups had their data estimated via interpolation for either or , adding a cumulative . million adherents to the longitudinal file (see table ). the second missing data strategy was applied to groups. these groups had congregation counts but did not have adherent counts in either the or dataset. we estimated adherent counts in the missing year by multiplying their congregation counts by the observed adherent-to-congregation ratios (i.e. congre- gation size) within each county in the other year. we focused on the – time period because the majority of adherent-only missing data occurs in these full group participation is reserved for groups with both adherent and congregation data. this count includes groups with changes in measurement (e.g. catholics). we used the ‘‘ipolate’’ command with the epolate option in stata , which uses the following general formula: y ¼ y �y x �x ðx � x Þ þ y : for some groups, the aggregate count does not appear linear. this is because the formula is applied at the county level. the group may be in decline in some counties, but growing in others, resulting in an overall shift in size contrary to expectations. in the merged file, a flag variable identifies the counts that are estimated using each missing data method. rev relig res ( ) : – years. this focus also improves the representation of eastern orthodox groups, for whom and are the only years when their measurement is comparable. estimations of adherents based on congregation-size have been used in the religion census data collections before, such as the membership size applied to some black protestant congregations in the dataset (grammich et al. ). such assigned congregation sizes, however, are relatively arbitrary and applied to counties of all characteristics. our strategy improves on past estimations by using data from another year to inform the congregation size unique to each county, thereby capturing a more accurate picture of each group’s geographic distribution and the addition/subtraction of adherents as congregations open or close. we used a few variations of the congregation-based strategy depending on the needs of the group. the adherents of three eastern orthodox groups were estimated in based on their adherent-to-congregation ratios in . another groups were missing adherents in , and so we used their congregation size from as the multiplier. for counties where there were congregations in year, but not another, we assigned a conservative ‘‘small church’’ size of adherents per congregation in that county. we also adjusted our congregation-size strategy to estimate the adherent counts of the four groups affected by mergers and schisms. these four groups are: the serbian orthodox church in the usa (new gracanica metropolitanate), friends unaffiliated local meetings, the anglican church in north america (acna), and the north american lutheran church (nalc). in appendix b, a value of ‘‘m’’ indicates that these groups’ data are merged with another group for that particular year. these four groups lack not only adherent counts in both and , but their congregation counts are also missing in one of the years. to address the data limitations, we drew the adherent to congregation ratios from larger religious groups with which they share history and ideology (e.g. other eastern orthodox or friends groups). in doing so, we are assuming that the groups with missing data have distributions similar to other groups with whom they share a theology and history, which is generally supported in the literature on religious regions (stump ; table total adherents added using missing data estimations total strategy , , , , , , strategy , , , total , , , , , , , totals are rounded; individual estimates are kept un-rounded there are groups that have congregation counts but are missing adherents in . we decided not to estimate these groups for two reasons: ) they do not have sufficient participation in and to allow comparisons beyond , or ) they are an eastern orthodox group for whom measurement changes in disallow comparisons before and after . adherents in = (adherents in /congregations in ) congregations in . adherents in = (adherents in /congregations in ) congregations in . rev relig res ( ) : – bauer ). for the serbian orthodox church, we used the ratio of all other orthodox groups that participated in the dataset and multiplied the ratio by the number of serbian congregations. for the friends unaffiliated local meetings we used the ratio from all other participating friends groups in . for the acna and nalc, we used all evangelical protestants. both the acna and the nalc have national-level statistics about their congregation and membership size on their official websites, which provide additional insight into the size that the congrega- tions of each group tend to be. their national-level congregational size was weighted by the geographical representation of other evangelical protestants in the dataset. the congregation-size estimate recovers nearly million adherents across and , and both strategies together add nearly . million across all years of data (table ). the two missing data strategies are not without caveats. the congregation-size strategy assumes that the average congregation size within the county for each group is the same over two decades. in reality, it is foreseeable that the addition or loss of a congregation could alter the average congregation size in a county, and recent evidence suggests that smaller congregations are more likely to close than large ones, which are becoming even larger in recent decades (chaves ). these factors indicate that change in average congregation size between decades does occur, and our approach masks the co-existence of small and large congregations that open or close at different rates within the same county. among the schism/ merger groups, we further assumed that the congregation size and/or distribution of the group matched that of ideologically similar religious groups, although there are likely counties where this is not the case. our interpolation and extrapolation techniques also provide relatively simplistic estimates of missing data, where the only relationship considered is a linear one with time. extrapolation also cannot predict future geographic expansion, but this problem is limited to only two groups for whom we extrapolated values in the dataset. the simplicity of our missing data methods, however, has future value; it is relatively easy for data users to update them using the religion census when it becomes available. we recommend that researchers use the missing data estimates within larger religious group traditions or families, such as measuring the size of all evangelical or friends groups, rather than to accurately represent the growth or decline of the specific religious group. overall, we believe our estimates improve the data by increasing group representation and we chose evangelical protestants as the larger category for the acna and nalc because the groups are more conservative than their mainline ‘parent’ denominations, and also because the evangelical adjustment produced estimates closer to each group’s national membership than the mainline adjustment would have. evangelical protestants in the dataset were identified using reltrad categorizations as a guide (steensland et al. ). we used the following formula: missing group adherents = group’s national congregation size (county evangelical protestant congregation size/national evangelical protestant congregation size) group congregations. the national congregation size for each group came from their official statistical reports available online. the acna’s most recent online report (anglican church in north america ) identified adherents per congregation; we used a size of as a more conservative estimate. the national congregation size for the nalc was in (north american lutheran church ); we used a size of as a more conservative estimate. rev relig res ( ) : – allowing for the adherent counts of groups involved in mergers or schisms to be included in aggregate groupings appropriate for longitudinal analyses. standardizing areal units whereas the first three challenges addressed how to offer comparable counts of congregations and adherents over time, the final challenge is concerned with the unit of analysis. while no changes in state boundaries have occurred in many decades, a total of counties are affected by changes between and that are problematic for data users. for instance, the creation of a county results in the re- allocation of other counties’ population into the new county, while the dissolution of a county results in its population being allocated to one or more surrounding counties. the and religion census datasets also are distinct from the and datasets in how they treat independent cities in virginia. in and , many (but not all) of the religious group counts in independent cities were attributed to their surrounding counties, making it appear that the independent cities have missing data and that the surrounding counties had more adherents than actual (quinn et al. ; bradley et al. ). in and , the counts in independent cities have adherent/congregation counts separate from their surround- ing counties. this problem makes it impossible to compare the counts in virginia independent cities and surrounding counties from / to / without aggregating them. to keep counties consistent across all four waves, we created aggregate county units to standardize their boundaries. for instance, the creation of broomfield county in colorado in took territory from four surrounding counties. we therefore merged all five of the counties for – to maintain consistent boundaries. likewise, counties that were dissolved over the time period were combined with the counties they dissolved into for the years prior to their dissolution. south boston city and clifton forge city counties in virginia were among the counties in the u.s. that dissolved over the time period, as are several territories in alaska. secondly, we addressed the problem of the independent cities in virginia, which affected county and county-equivalents. we merged the independent cities with their ‘parent’ counties in and , just as they appear in and . each of the county mergers are detailed in appendix c. in the original dataset, there are counties. after implementing the necessary county mergers between and , there are counties and county- equivalents in the new longitudinal file. the longitudinal file is not available with de-aggregated counties. aggregated county counts were necessary to calculate missing data and could not be deconstructed afterwards without additional bias. researchers may contact the authors to inquire about altering the data to accommodate fewer aggregations (e.g. examining only – data doesn’t require virginia independent city aggregations). rev relig res ( ) : – summary of improvements and remaining limitations table summarizes the consequences of our many adjustments. along with making the data more comparable overtime, the adjustments also sharply increased the number of adherents that could be included from each collection in the final merged files. the most substantial increases were for the white protestant groups. if we were to use the religion census datasets without any adjustments, several protestant groups would be omitted due to schisms, mergers, aggregations, measurement changes and missing data. accounting for schisms, mergers and other group aggregations allows for the full inclusion of the episcopal church, the evangelical lutheran churches in america, several additional mennonite groups, all of the friends groups and the moravian church in america (see table ). as shown in table , adjusting for mergers and schisms adds between six and nine million additional adherents in each year of the dataset, particularly benefiting the table summary of adjustments unadjusted a total , , , , , , , , mainline , , , , , , , , evangelical , , , , , , , , adjustments: schisms, mergers, and aggregations total , , , , , , , , mainline , , , , , , , , evangelical , , , , , , , , united methodist b total , , , , , , , , mainline , , , , , , , , evangelical , , , , , , , , missing data total , , , , , , , , mainline , , , , , , , , evangelical , , , , , , , , county merges total , , , , , , , , mainline , , , , , , , , evangelical , , , , , , , , a only includes groups with consistent adherent measures in all years b uses the ‘‘old’’ county-level adherent estimate mainline and evangelical categorization was based on the reltrad specification detailed by steensland et al.( ). appendix a identifies the reltrad category assigned to each group and a reltrad variable is also included in the longitudinal file. rev relig res ( ) : – mainline aggregate count. including the united methodist church after adjusting for their measurement change also greatly increases mainline adherents by about million each year. including the missing data estimates (net those involved in the mergers and schisms) adds approximately . million more adherents each year. we also can see the benefit of the county mergers in the last section of table . by including the merged county units, we added between two and three million protestant adherents to the dataset. taken altogether, the changes allowed for the more reliable inclusion of an additional – million protestant adherents in each year. despite these improvements, limitations in utilizing all years of data for longitudinal analyses remain. we did not estimate adherent and congregation counts of all the groups who had missing data, and not all groups involved in schisms are present in the years following their founding. the most significant limitations, however, are the unamendable measurement changes among some of the largest religious groups. adherent comparisons across all years are mostly limited to predominantly white mainline and evangelical christian denominations. if the analyses are limited to fewer years of data or to congregational counts, however, the new longitudinal files still can be used to study additional groups, such as catholics, jews, mormons, or some of the orthodox christian groups (see table ). unfortunately, the historically african-american denominations are not fully represented in any of the collections. limitations remain, but the new longitudinal files offer important improvements for using the religions censuses for overtime research. conclusion the decennial religious censuses now collected by the association of statisticians of american religious bodies have been used extensively by researchers; but the complexities of merging the files have prevented most from using the files for explaining religious change over time. schisms and mergers, changing geographical boundaries, modified methods of reporting and various sources of missing data have posed challenges that made the merging both complex and time consuming. some of the challenges required demographic training, others required an extensive knowledge of the specific groups being studied. addressing these challenges required both expertise and time. we have made several changes to the , , and religion census datasets in order to address challenges associated with merging the data collections into a longitudinal file. these changes included alterations to variable names, the creation of new combined groups to correct for schisms and mergers, introducing alternative counts of the united methodist church, providing estimates for missing data and merging county units in the county-level file. collectively, these changes produced new longitudinal files that increase religious group representation, reduce bias from missing data, improve the file’s ease of use and are readily accessible from thearda.com. rev relig res ( ) : – we have issued several warnings on the limitations of the data and on which groups can be meaningfully compared over time, but this shouldn’t distract from the many opportunities the merged files offer. for a core group of religious groups, meaningful comparisons among adherents and congregations now can be made using all four collections. for many of the remaining groups, comparable data is now available for at least two points in time. this allows researchers to explore how groups change in size and geography over time and how these changes are related to other social, demographic and economic changes. overall, we believe that our efforts greatly improve the quality of the data by reducing missing data and making the data more comparable over time, and will simplify the process of accessing and using a merged file for all users. acknowledgements we thank lauren arieux for providing the alternative and united methodist adherent counts, and daniel olson for sharing information about the religion census datasets. this project was made possible through the support of the lilly endowment, inc. and the john templeton foundation. the opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the lilly endowment, inc. or the john templeton foundation. funding was provided by lilly endowment, inc. (grant no. - ), john templeton foundation (grant no. ). open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, dis- tribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. references anglican church in north america. . about the anglican church in north america. http://www. anglicanchurch.net/media/acna_fact_sheet_ - - .pdf. accessed oct . bauer, john t. . u.s. religious regions revisited. the professional geographer ( ): – . blau, judith r., kent redding, and kenneth c. land. . ethnocultural cleavages and the growth of church membership in the united states, – . sociological forum ( ): . https://doi. org/ . /bf . boone, jeff p., inder k. khurana, and k.k. raman. . religiosity and tax avoidance. 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. census: religious congregations and membership study. lenexa: association of statisticians of american religious bodies. hadaway, kirk c., and david a. roozen. . church and denominational growth. nashville, tn: abingdon press. hillary, giles, and kai wai hui. . does religion matter in corporate decision making in america? journal of financial economics ( ): – . johnson, douglas w., paul r. picard, and bernard quinn. . churches and church membership in the united states: an enumeration by region, state, and county. washington, dc: glenmary research center. jones, dale e., sherri doty, clifford grammich, james e. horsch, richard houseal, mac lynn, john p. marcum, kenneth m. sanchagrin, and richard h. taylor. . religious congregations and membership in the united states : an enumeration by region, state and county based on data reported for religious bodies. nashville, tn: glenmary research center. land, kenneth c., glenn deane, and judith r. blau. . religious pluralism and church membership: a spatial diffusion model. american sociological review ( ): – . lim, chaeyoon. . counting the faithful: measuring local religious contexts in the united states. journal for the scientific study of religion ( ): – . lindner, eileen w. . yearbook of american and canadian churches. in yearbook of american and canadian churches, ed. national council of the churches of christ. nashville, tn: abingdon press. lindner, eileen w. . yearbook of american and canadian churches. in yearbook of american and canadian churches, ed. national council of the churches of christ. nashville, tn: abingdon press. marcum, john p. . w(h)ither the mainline? trends and prospects. review of religious research : – . national council of churches. . churches and church membership in the united states: an enumeration and analysis by counties, states and regions. new york: national council of churches. north american lutheran church. . about the nalc. https://www.thenalc.org/about-us- /. accessed oct . quinn, bernard, herman anderson, martin b. bradley, paul goetting, and peggy shriver. . churches and church membership in the united states, . washington, dc: glenmary research center. steensland, brian, jerry park, mark regnerus, w. lynn robinson, bradford wilcox, and robert woodberry. . the measure of american religion: toward improving the state of the art. social forces : – . stump, roger w. . the effects of geographical variability on protestant church membership trends, – . journal for the scientific study of religion ( ): – . rev relig res ( ) : – https://www.thenalc.org/about-us- / 	merging the religious congregations and membership studies: a data file for documenting american religious change 	abstract 	collecting congregational and membership data 	addressing the challenges 	accounting for mergers, schisms, and other aggregations 	group measurement changes 	missing data 	standardizing areal units 	summary of improvements and remaining limitations 	conclusion 	acknowledgements 	references amish (rural) vs. non-amish (urban) infant fecal microbiotas are highly diverse and their transplantation lead to differences in mucosal immune maturation in a humanized germfree piglet model | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /fimmu. . corpus id: amish (rural) vs. non-amish (urban) infant fecal microbiotas are highly diverse and their transplantation lead to differences in mucosal immune maturation in a humanized germfree piglet model @article{dhakal amishv, title={amish (rural) vs. non-amish (urban) infant fecal microbiotas are highly diverse and their transplantation lead to differences in mucosal immune maturation in a humanized germfree piglet model}, author={s. dhakal and l. wang and l. antony and jennifer rank and pauline bernardo and shristi ghimire and kathy bondra and christina siems and yashavanth shaan lakshmanappa and s. renu and bradley t. hogshead and s. krakowka and mike kauffman and j. scaria and j. lejeune and z. yu and g. renukaradhya}, journal={frontiers in immunology}, year={ }, volume={ } } 	s. dhakal, l. wang, + authors g. renukaradhya 	published 	biology, medicine 	frontiers in immunology the gut microbiome plays an important role in the immune system development, maintenance of normal health status, and in disease progression. in this study, we comparatively examined the fecal microbiomes of amish (rural) and non-amish (urban) infants and investigated how they could affect the mucosal immune maturation in germ-free piglets that were inoculated with the two types of infant fecal microbiota (ifm). differences in microbiome diversity and structure were noted between the two types… expand view on pubmed frontiersin.org save to library create alert cite launch research feed share this paper citationshighly influential citations background citations view all figures, tables, and topics from this paper 	 table 	 figure 	 table 	 figure 	 figure 	 table 	 figure 	 figure 	 table 	 figure 	 figure 	 figure view all figures & tables 	microbiota (plant) 	microbiota (environment) 	feces 	mucous membrane 	lymph nodes 	immune system 	fecal microbiota transplantation 	anatomical maturation 	metagenomics 	intestinal microbiome 	intestines 	phylum (taxon) 	fecal occult blood test 	submucosa 	biologic development 	progressive disease paper mentions news article convivir con animales refuerza nuestras defensas la vanguardia october news article cuddling with farm animals can help young kids develop healthier immune systems bangor daily news august news article how babies benefit from exposure to farm animals medical daily july news article baby's immune system may be boosted by keeping livestock at home technology networks july news article why growing up on a farm is good for your immune system mother nature network july news article early exposure to farm animals may lead to robust immune system development the medical news july news article keeping livestock in the yard might help your baby's immune system infection control today july news article keeping livestock in the yard just might help your baby’s immune system environmental news network july show more citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency impact of microbiota transplant on resistome of gut microbiota in gnotobiotic piglets and human subjects 	h. liu, h. wang 	biology, medicine 	frontiers in microbiology 	 	 	 	view excerpts, cites background save alert research feed perinatal environment shapes microbiota colonization and infant growth: impact on host response and intestinal function 	m. selma-royo, m. calatayud arroyo, + authors m. collado 	biology, 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	 	 	highly influenced 	pdf 	view excerpts, cites background save alert research feed establishing or exaggerating causality for the gut microbiome: lessons from human microbiota-associated rodents 	j. walter, anissa m. armet, f. shanahan 	biology, medicine 	cell 	 	 	 	view excerpts, cites background save alert research feed the human microbiome in the st century 	e. rackaityte, s. lynch 	biology, medicine 	nature communications 	 	 	pdf save alert research feed references showing - of references sort byrelevance most influenced papers recency diversity of the intestinal microbiota in different patterns of feeding infants by illumina high-throughput sequencing 	wenguang fan, g. huo, + authors j. chen 	biology, medicine 	world journal of microbiology & biotechnology 	 	 	view excerpts, references background and results save alert research feed inter-species transplantation of gut microbiota from human to pigs 	xiao-yan pang, x. hua, + authors liping zhao 	biology, medicine 	the isme journal 	 	 	pdf 	view excerpts, references methods and background save alert research feed distinct distal gut microbiome diversity and composition in healthy children from bangladesh and the united states 	a. lin, e. bik, + authors u. singh 	biology, medicine 	plos one 	 	 	pdf 	view excerpts, references results and background save alert research feed human microbiota-associated swine: current progress and future opportunities 	mei wang, s. donovan 	biology, medicine 	ilar journal 	 	 	pdf save alert research feed impact of diet in shaping gut microbiota revealed by a comparative study in children from europe and rural africa 	c. de filippo, d. cavalieri, + authors p. lionetti 	biology, medicine 	proceedings of the national academy of sciences 	 	 , 	pdf 	view excerpts, references results save alert research feed a pig model of the human gastrointestinal tract 	q. zhang, g. widmer, s. tzipori 	biology, medicine 	gut microbes 	 	 	highly influential 	 	view excerpts, references 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	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue ssri facilitated crack dancing case report ssri facilitated crack dancing ravi doobay, lili sun, amish shah, pardeep masuta, and zachary shepherd internal medicine, suny upstate medical university, e adams st, syracuse, ny , usa correspondence should be addressed to ravi doobay; doobayr@upstate.edu received march ; revised march ; accepted april ; published april academic editor: pablo mir copyright © ravi doobay et al. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. choreoathetoid movement secondary to cocaine use is a well-documented phenomenon better known as “crack dancing.” it consists of uncontrolled writhing movements secondary to excess dopamine from cocaine use. we present a -year-old male who had been using cocaine for many years and was recently started on paroxetine, a selective serotonin reuptake inhibitor (ssri) for worsening depression four weeks before presentation. he had been doing cocaine every weeks for the last three years and had never “crack danced” before this episode. the authors have conducted a thorough literature review and cited studies that suggest “crack dancing” is associated with excess dopamine. there has never been a documented case report of an ssri being linked with “crack dancing.” the authors propose that the excess dopaminergic effect of the ssri lowered the dopamine threshold for “crack dancing.” there is a communication with the raphe nucleus and the substantia nigra, which explains how the ssri increases dopamine levels. this is the first documented case of an ssri facilitating the “crack dance.” . introduction choreoathetoid movement secondary to cocaine use also known as the “crack dance” is a documented phenomenon that is surprisingly more common in the younger patient population [ ]. choreoathetosis literally means the occur- rence of involuntary movements with twisting and writhing [ ]. we present a first-time occurrence of the “crack dance” one month after beginning paroxetine, a selective serotonin reuptake inhibitor (ssri) in the context of cocaine abuse. we propose that the excess dopaminergic effect of the ssri lowered the threshold for the choreoathetoid movements. this is the first documented case report of the “crack dance” being associated with an ssri. . case presentation we present a -year-old male with a past medical history of hypertension, major depression, and cocaine abuse who presented with involuntary head, jaw, tongue, facial, shoulder, trunk, and leg movements; they were rhythmic and chor- eiform. voluntary movements would worsen the choreoathe- tosis. he did cocaine three days before presentation. he initially complained of fevers, chills, and diaphoresis two days before presentation. he then noticed abnormal move- ments of his head, which then progressed into uncontrolled movements of his arms, shoulder, trunk, and legs. he denied chest pain, dyspnea, nausea, vomiting, diarrhea, dysuria, or leg swelling. he had been recently put on paroxetine by his primary care physician four weeks ago for major depression. he claimed to have not done any other recre- ational drugs, which was supported by a urine toxicology screen. he presented with a temperature of . ∘c, pulse of , rr of , bp of / mmhg, and saturating % on room air. a comprehensive metabolic panel and thyroid stimulating hormone were within normal limits. there were no pertinent positives on physical exam other than the uncontrolled movements; he remained alert and oriented at all times. apart from the choreoathetosis the neurological examination was within normal limits. there was no parkin- sonian or dystonic features to suggest an alternate diagnosis. he received diphenhydramine and benztropine from the emergency department physicians without relief. toxicology was consulted and concluded these were indeed choreoa- thetoid movements secondary to cocaine use. there was no head imaging ordered as the diagnosis was established. he was admitted to medicine and symptoms resolved with iv hindawi case reports in neurological medicine volume , article id , pages https://doi.org/ . / / https://doi.org/ . / / case reports in neurological medicine lorazepam and intravenous fluids in hours. during these hours the uncontrolled movements were continuous. . discussion the differential diagnoses for acute adult-onset nonheredi- tary choreas are autoimmune (systemic lupus erythemato- sus, polyarteritis nodosa, behcet’s disease, sjögren’s syn- drome, sydenham’s chorea, and antiphospholipid syndrome), metabolic (hyponatremia, hypoglycemia, hypocalcemia, and hyperthyroidism), infectious (neurosyphilis, lyme disease, and aids), drugs (neuroleptics, amphetamines, lithium, and digoxin), and malignancy with basal ganglia involvement [ ]. the symptoms resolved with iv benzodiazepines, fluids, and symptomatic management which suggests a cocaine induced choreoathetosis, especially with the patient’s history. cocaine is known to have stimulating effects on behavior. it acts by blocking the reuptake of certain catecholamines such as dopamine, norepinephrine, and serotonin by blocking transporter receptors [ ]. more specifically, cocaine acts on dopamine transporter receptors thereby blocking dopamine reuptake which results in a rapid increase in dopamine availability at the synaptic cleft [ ]. studies have shown that chronic cocaine use may lead to a compensatory response to overcome the dopaminergic overstimulation [ ]. cocaine also decreases tyrosine hydroxylase activity, an enzyme used in production of dopamine, and postsynaptic dopamine receptor sites which would overall decrease the availability of dopamine and its effect. however, failure to engage in this mechanism and downregulate dopamine results in the occurrence of choreoathetoid movements [ ]. while ssris by definition inhibit serotonin reuptake from brain synapse, they also have an affinity for norepinephrine and dopamine receptors. in our patient, it is likely that with such chronic history of cocaine abuse, his recent regular usage of paroxetine synergistically added to the effect of increasing dopamine at the synapse thereby lowering the threshold for the development of choreoathetosis. there are compelling reasons why excess dopamine in the basal ganglia causes chorea. sutamtewagul et al. ( ) showed a similar presentation in a -year-old male after taking consecutive doses of “bath salts,” a well- known street drug, intravenously [ ]. the active compounds in bath salts are , -methylene-dioxypyrovalerone or - methylmethcathinone which inhibit dopamine reuptake [ ]. stork and cantor ( ) documented choreoathetosis sec- ondary to pemoline use [ ]. pemoline is an oxazolidine derivative that is a dopaminergic agonist [ ]. it is used to treat attention deficit disorder. two three-year-old males ingested multiple pemoline tablets and were found to be in choreoa- thetosis [ ]. they were treated with iv benzodiazepines and the symptoms resolved in hours [ ]. there was no history of movement disorder or family history of movement disor- ders in these patients [ ]. also choreoathetoid movements induced by levadopa is very well documented in parkinson patients [ ]. we suspect in this particular patient that the ssri caused release of serotonin in the raphe nucleus which has communication with the substantia nigra [ ] leading to increased dopamine resulting in uncontrolled movements better known as “crack dancing” [ ]. in conclusion choreoathetosis secondary to cocaine use also known as the “crack dance” is secondary to an excess dopaminergic effect. we present a first-time “crack dance” facilitated by an ssri in a -year-old male who has done cocaine every weeks for the last three years. we propose that the ssri started by his pcp four weeks ago increased the dopamine activity above the threshold for choreoathetosis. this is the first case report linking ssri use to “crack dancing.” there are two reasons why the authors believe the ssri induced the choreoathetosis: it was four weeks after the initiation of the ssri which is when it starts to take effect; also the patient had done cocaine for many years and this was a first-time occurrence of choreoathetosis. this case also demonstrates the potential risk of ssri use in chronic cocaine users predisposing them to involuntary movements. there are only a handful case reports of choreoathetosis secondary to cocaine use and there has never been a documented association with ssris. we present the first ssri facilitated “crack dance.” disclosure all authors had access to the data and a role in writing the manuscript. this is an original case report. there were no funds received from any source including pharmaceutical industry funds and there was no source of support in the form of equipment, drugs, or grants. conflicts of interest the authors declare that there are no conflicts of interest regarding the publication of this paper. references [ ] g. bartzokis, m. beckson, d. a. wirshing, p. h. lu, j. a. foster, and j. mintz, “choreoathetoid movements in cocaine dependence,” biological psychiatry, vol. , no. , pp. – , . [ ] o. suchowersky and j. muthipeedika, “a case of late-onset chorea.,” nature clinical practice. neurology, vol. , no. , pp. – , . [ ] g. sutamtewagul, v. sood, and k. nuggent, “sympathomimetic syndrome, choreoathetosis, and acute kidney injury following “bath salts” injection,” clinical nephrology, vol. , no. , pp. – , . [ ] c. m. stork and r. cantor, “pemoline induced acute choreoa- thetosis,” journal of toxicology. clinical toxicology, vol. , no. , pp. – , . [ ] c. markham, “the choreoathetoid movement disorder induced by levodopa,” clinical pharmacology and therapeutics, vol. , no. , pp. – , . [ ] m. a. bunin and r. m. wightman, “quantitative evaluation of -hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission,” journal of neu- roscience, vol. , no. , pp. – , . treatment of advanced stage nsclc with low dose gemcitabine and carboplatin in patients above age of years: p - journal of thoracic oncology • volume , number , supplement , august th world conference on lung cancer copyright © by the international association for the study of lung cancers toxicities were fatigue; nausea; asthenia, vomiting and arthralgia/ myalgia. more than forty percent of the patients received nd line therapy and, from those, % were responsive to the treatment. maintenance therapy with gemcitabine in perfusion for minutes versus for minutes both combined with carboplatin, showed similar response rates and safety profiles. p - nsclc: cytotoxic chemotherapy posters, tue, sept treatment of advanced stage nsclc with low dose gemcitabine and carboplatin in patients above age of years parikh, purvish m.; prasad, narayan; menon, hari; prabhash, kumar; vora, amish; agarwal, jayprakash; mistry, rajesh; pramesh, cs; tandon, s; desai, subhash tata memorial hospital, mumbai, india background: nsclc is a common malignancy worldwide-including developing countries like india. the standard treatment of advanced stage nsclc is platinum doublet chemotherapy, one of the most ac- tive being gemcitabine- platinum combination. gemcitabine requires conversion by deoxycytidine kinase into its active metabolite. this rate limiting step can be circumvented by prolonging infusion duration. methods: in the present study we treated elderly patients (age or more) having advanced stage nsclc with low dose prolonged infu- sion gemcitabine ( mg/m over hours, day and day ) and stan- dard dose carboplatin (auc- , day only) repeated every weeks for maximum cycles. results: there were ( %) males and females. of them were between the age of and yrs, between the age of and whereas patients were above the age of yrs. ps was in and in cases. stage wise distribution was ( . %) with stage iv and ( . %) with stage iiib. histopathology was adenocarcinoma in , squamous cell in and not specified in the remaining patients. of these , had significant co-morbidities. chemotherapy was administered by medical oncologists in patients and others (other oncologists/ non oncologists) in the remaining cases. all cycles were completed in ( . %) of patients - completed all cycles without any dose reduction, completed cycles with dose reduc- tion and could be goven less than cycles of ct. partial response was seen in ( . %), stable disease in ( %) and progressive disease in ( . %). there was no treatment related mortality. the median overall survival was months (range - months). there were significant differences among patients treated by medical oncolo- gists (group ) and by others (group ). the overall rr was . % ( / ) in group and . % ( / ) in group . the median survival was months in group and months in group (p value = . ), the overall rr was and the medial os was months. these differences were statistically significant. conclusions: we conclude that low dose prolonged infusion gem- citabine and standard carboplatin combination is an effective treatment for patients above the age of years with advanced stage nsclc. dose intensity impacts on rr as well as overall survival and is best optimized when chemotherapy is administered by medical oncologists. p - nsclc: cytotoxic chemotherapy posters, tue, sept phase ii trial of weekly docetaxel and gemcitabine for chemotherapy-naive patients with advanced non-small cell lung cancer hong, joon sik kim, yujin kyung, sun young an, chang hyeok lee, sang pyo park, jeong woong jeong, sung hwan park, se hoon cho, eun kyung shin, dong bok division of hematology and oncology, internal medicine, gachon university gil medical center, incheon, korea division of pulm- onology, internal medicine, gachon university gil medical center, incheon, korea background: docetaxel and gemcitabine combination chemotherapy has been reported to be active against non-small cell lung cancer (nsclc) and myelosuppression was the most common dose-limit- ing toxicity. this prospective phase ii study was designed to test the hypothesis that better tolerance and increased dose intensity might be achieved if patients are treated with weekly administration schedule. methods: consenting patients with stage iiib/iv or recurrent nsclc received first-line chemotherapy with docetaxel mg/m and gem- citabine mg/m on days , and . treatment was repeated every weeks, for up to cycles. results: of the patients who started treatment, only patients ( %) completed planned cycles of therapy. other than the comple- tion of all planned treatment cycles, the main reasons for treatment discontinuation were toxicity ( %) and progressive disease ( %). the most frequently encountered toxic effects were anemia ( % of pa- tients), nausea and vomiting ( %), fatigue ( %) and anorexia ( %). one patient died of bilateral pneumonitis shortly after the adminis- tration of first cycle. disease control (objective response and stable disease) in the itt population was achieved in % of patients and the overall response rate was % ( % ci, to %). with a median follow-up duration of . months, the median progression-free survival was . ( % ci, . to . ) months and the -year survival was %. conclusion: weekly schedule of docetaxel and gemcitabine has mod- est activity in advanced nsclc. this regimen offered no potential advantages over standard treatment approaches. j october , volume , no. journal of the american college of cardiology inside this issue jacc white paper d s s r p l i c t i i r d s s p i l c o v d c s w acc white paper stent thrombosis avid r. holmes, jr, dean j. kereiakes, scot garg, patrick w. serruys, gregory j. dehmer, tephen g. ellis, david o. williams, takeshi kimura, david j. moliterno tent thrombosis (st) with either drug-eluting stents (des) or bare-metal stents (bms) emains catastrophic and, although infrequent, modifies the risk-benefit equation of ercutaneous coronary intervention. this white paper by holmes and colleagues reviews the iterature regarding st and divides the risk factors into the following categories: ) the stent, ncluding its geometry, polymer, and drug; ) the patient, including clinical presentation and omorbidities; ) the procedure, including residual dissection or incomplete expansion; and ) the extent and duration of antiplatelet therapy and the patient response to this therapy. he authors hope that improved understanding of these risk factors will facilitate the dentification of optimal preventive strategies. clinical research nterventional cardiology egistry shows similar outcomes at to years for cabg or pci for left main disease uk-woo park, young-hak kim, sung-cheol yun, jong-young lee, won-jang kim, soo-jin kang, eung-whan lee, cheol-whan lee, jae-joong kim, suk-jung choo, cheol-hyun chung, jae-won lee, eong-wook park, seung-jung park ark and colleagues evaluated the long-term safety and effectiveness of percutaneous coronary ntervention (pci) compared with coronary artery bypass grafting (cabg) for unprotected eft main coronary artery (lmca) disease. in the -year follow-up cohort of ontemporaneous bare-metal stents or cabg, there were no differences in the adjusted risks f death or the composite of death, q-wave myocardial infarction, or stroke, while target essel revascularization (tvr) was times more likely. similarly, for the -year follow-up of rug-eluting stents (des) or cabg, there were no significant differences for death or the omposite outcome, but the rate of tvr was times higher in the des group. pci with tent implantation has similar long-term mortality and major adverse event rates compared ith cabg, but higher rates of tvr. (continued on page a- ) http://linkinghub.elsevier.com/retrieve/pii/s http://linkinghub.elsevier.com/retrieve/pii/s october , (continued) a- a j e w s a ( c a m u o h v j t b f e a m f t t e cute coronary syndromes prior aspirin use does not increase risk of acs onathan d. rich, christopher p. cannon, sabina a. murphy, jie qin, robert p. giugliano, ugene braunwald hile aspirin is clearly beneficial for the treatment of an acute coronary syndrome (acs), ome studies have suggested worse outcomes in those who were taking aspirin prior to their cs. rich and colleagues evaluated , acs patients from a merged database of timi thrombolysis in myocardial infarction) trials. prior aspirin users were older, had more oronary risk factors, and were more likely to have previously been diagnosed with coronary rtery disease. unadjusted analyses showed worse outcomes for aspirin users, but after ultivariate analysis, there was no difference in total mortality at day or by the last follow- p visit. prior aspirin use identifies patients with greater baseline risk but does not affect utcomes in acs patients. eart rhythm disorders optical mapping of the human sinus node adim v. fedorov, alexey v. glukhov, roger chang, geran kostecki, hyuliya aferol, william j. hucker, oseph p. wuskell, leslie m. loew, richard b. schuessler, nader moazami, igor r. efimov he site of origin and pattern of excitation within the human sinoatrial node (san) has not een directly mapped. fedorov and colleagues optically mapped the san in preparations rom human hearts and reconstructed the -dimensional structure of the san with histology. xcitation originated in the middle of the san, and then slowly spread within the san. fter a conduction delay within the san, the atrial myocardium was excited via superior, iddle, and/or inferior sinoatrial exit pathways. the san structure was functionally insulated rom the atrium by connective tissue, fat, and coronary arteries, except for these exit pathways. his study maps the pattern of excitation within the human san, and the exit pathways into he right atrium. ditorial comment: miguel valderrábano, amish s. dave, p. (continued on page a- ) http://linkinghub.elsevier.com/retrieve/pii/s http://linkinghub.elsevier.com/retrieve/pii/s http://linkinghub.elsevier.com/retrieve/pii/s x october , (continued) a- i r n j e d b t f f h h i s f j e d o s o p e p h z t c d d a a p e maging of coronary calcium cac score improves coronary risk prediction aimund erbel, stefan möhlenkamp, susanne moebus, axel schmermund, nils lehmann, andreas stang, ico dragano, dietrich grönemeyer, rainer seibel, hagen kälsch, martina bröcker-preuss, klaus mann, ohannes siegrist, karl-heinz jöckel, for the heinz nixdorf recall study investigative group rbel and colleagues used data from over , subjects without known coronary artery isease at baseline to determine net reclassification improvement (nri) and risk prediction ased on coronary artery calcification (cac) scoring in comparison to traditional risk factors. he baseline risk was categorized into low, intermediate, and high according to the ramingham risk score (frs) or adult treatment panel iii guidelines, and subjects were ollowed for years. reclassifying frs -year intermediate-risk (defined as % to % and % to %) subjects with cac � to the low-risk category and with cac � to the igh-risk category yielded an nri of % and %, respectively. cac scoring results in a igh reclassification rate in the intermediate-risk cohort. maging of coronary calcium cac score and -year coronary risk uzette e. elias-smale, rozemarijn vliegenthart proença, michael t. koller, maryam kavousi, rank j. a. van rooij, myriam g. hunink, ewout w. steyerberg, albert hofman, matthijs oudkerk, acqueline c. m. witteman lias-smale and colleagues followed over , subjects with a mean age of years for years to examine the effect of coronary calcium on the risk of -year hard coronary heart isease (chd) events. a total of % of subjects initially classified as intermediate-risk based n their framingham risk scores were reclassified by means of coronary artery calcium (cac) core: to high-risk for a cac � and to low-risk for cac � . in a general population f elderly persons at intermediate chd risk, cac scoring is a powerful method to reclassify ersons into more appropriate risk categories. ditorial comment: daniel duprez, p. re-clinical research doxycycline improves survival in a mouse model of cardiac proteinopathy anqiao zheng, mingxin tang, qingwen zheng, asangi r. k. kumarapeli, kathleen m. horak, ongwen tian, xuejun wang ransgenic (tg) cardiac overexpression of missense mutant �b-crystallin (cryabr g) auses aberrant protein aggregation and cardiomyopathy, recapitulating key features of human esmin-related cardiomyopathy (drc). zheng and colleagues treated tg mice with either oxycycline or placebo. doxycycline treatment significantly attenuated cardiac hypertrophy nd improved survival. in cell culture, doxycycline suppressed the formation of both protein ggregates and oligomers. doxycycline decreases the toxicity of aberrant cryabr g roduction and may be useful for human drc. ditorial comment: francisco villarreal, wilbur y. w. lew, p. http://linkinghub.elsevier.com/retrieve/pii/s http://linkinghub.elsevier.com/retrieve/pii/s http://linkinghub.elsevier.com/retrieve/pii/s http://linkinghub.elsevier.com/retrieve/pii/s http://linkinghub.elsevier.com/retrieve/pii/s document resume ed fl author paulston, christina bratt; paulston, rolland g. title language and ethnic boundaries. pub date sep note p.; paper presented at the scandinavian symposium on bilingualism ( st, esbo, finland, september r , ) edrs price bf-$ . hc-$ . plus postage. descriptors *acculturation; bias; biculturalism; bilingual education; *bilingualism; blacks; cultural pluralism; *educational policy; english language); ethnic groups; *ethnic relations; immigrants; *language attitudes; language instruction; minority groups; non english speaking; socialization; sociolinguistics identifiers canada; language maintenance; language shift; lapps; latin america; sweden abstract the paper examines the phenomenon of group bilingualism, the origin of the contact situations which lead to it, and the role of language in maintaining ethnic boundaries, especially in revitalization movements. language shift and language maintenance are seen as indicators of the degree to which ethnic boundaries are being maintained. many ethnic groups are discussed and the case of the swedish lapps examined at length. (author) *****************************************************************#***** documents acquiree by eric include many informal unpublished * materials not available from other sources. eric makes every effort * * to obtain the best copy available. nevertheless, items of marginal * * reproducibility are often encountered and this affects the quality * * of the microfiche and hardcopy reproductions eric makes available * * via the eric document reproduction service (edrs). edrs is not * responsible for the quality of the original document. reproductions * * supplied by edrs are the best that can be made from the original. * *********************************************************************** language and ethnic boundaries christina bratt pau st= departrrent of general linguistics rolland g. paulston international and dave cl:trent education program university of pittsburgh paper presented at the first scandinavian conference on bilingualism est.:), finland september - , u s department df health. education i. welf ar e national institute of education cument has been repro- ouceo exactly as receive!) from the person or organization origin- ating it points of view or opinions sta teo not necessarily repre sent official national institute of eoucation position on policy recently, in examining a colleague's research on the canadian immer- sion programs, it struck me forcibly what a bother the canadian language situation is, what a tremendous outpouring of energy and money -- and strife. i contented how much easier it would all be if the franco- canadians would just give up their french. she looked at me quizzically, grinned, and said: "of course." nbw,rothat simple exchange illustrates a number of issues that need to be considered in a discussion of bilingual- ism. the reason for her initial puzzlanent los that she for a moment took my remark as a scholarly statement about optative language policy goals for french speaking canada, although she knew very well that i was a supporter of cultural pluralism and concomitant bilingual education, and so she was puzzled. in other words, she was well acquaintedwith the par- ticular bias with which i approach bilingualim and bilingual education. i am in favor of it. it is not only mbias; it is shared by every lin- guist and anthropologist ilulw of who does scholarly work on bilingualisn and bilingual education. in all likelihood, it is a bias which is shared by trost if not all of the participants at this conference. it is not a bias for which i apologize; rather i find it tota , justified. rut there is the crux; the burden is upon me to demonstrate such a justifica- tion and to do so on the basis of data and empiric- facts. i find much writing and research on bilingualism and bilingual education flawed by a lack of objectivity, which influences the research designs as well as leads to unwarranted conclusions and speculations. the first scandinavian- conference on bilingualism is an appropriate tine to acknowledge our bias, because it is only by acknowledging it that we can adequately oontrol for it in oar work as well as allow the reader a more accurate interpretation of our scholarly opinions. once my aolleague realized what i neant, she agreed. language diver- sity within ivations frevently leads to a minter of problem, especially in the educational sector, which may not occur in a monolingual state. atterpts at solutions frequently carry a high econanic cost; e.g. federal funding alone in the unitedr.states for bilingual educatio.n in is $ million. a cannon rejoinder among my colleagues is "good, let them build one less atanic submarine." in other words, the perception and evaluation of the social results of ethnic groups in contact become a natter of priorities. it is undeniable, just as my colleague did not atterpt to deny it, that social life in canada would be simpler with only one language. but to her mind as to mine, increased efficacy and econany of ozurminication do not justify the enforced loss of cultural identity and way of life of a people, a choice of priorities which ultimately is based on moral values. frovently reason has little to do with attenpts at solutions . it is not reasonable for students to riot and get killed because the authorities want to teach one language rather than another in the schools, yet i think we all understand the black reaction to the introduction of afrikaans in- stead of english in south africa. afrikaans is a very pcmerful symbol for the hated oppression, and as i write this, the riots are continuing. clearly afrikaans in and by itself was not the real reason behind the re- volt, but rather the reason lay in the nature of the relationship between the subordinate blacks and the daninant whites. in our discussions on bilingualisn and bilingual situations, we need to recognize that often the problem has nothing or little to do with language m, se. the present language situation in the province of quebec is a result of a power struggle between the economically daninant anglo canadians and the politically dominant franco canadians. through legal measures, the french have been able to enforce a knowledge of french as a requisite for access to a number of jobs. as a result about % of the english speaking children in. montreal enter kindergarten in french immersion programs, (programs in which frendh is used as a median of instruction ), with the hope by their parents that they will learn sufficient french in school to be able to qualify for future positions. ianquage is used by the french as a mechanism for maintaining ethnic boundaries in order to deny english- speaking canadians access to scarce jobs. language can similarly be used for the mailnumance oi ethnic boun- daries in order to keep memberswithin the group. the trilingual old order amish is an example of a group who uses language for the maintenance of group boundaries both to keep their members in and outsiders out. the pennsylvania cld order amish is a protestant religious group characterized by: horse and buggies for transportation, no electricity in their homes, farm animals for farming, the occupa- tion most engaged in, education only to the eighth grade, plain dress, refbsal to accept government benefits such as social security, and the use of pennsylvania dutch, agerman dialect from the speech of german rhenish palatinate, german, and english (foder : - ). german is used for sermons, prayers, and bible reading fram the luther bible, the translation from the latin. since pennsylvania law re- quires school attendance until , many old order amish spend an addi- tional year after eighth grade in a special intensive progrsm learning german, thus fulfilling the legal requirement:while serving their own purpose. english is learnt in school and is used in any exchange uith "english" or "gay" persons, as non-mothers are referred to. english is also used for noir-religious reading. pennsylvania dutch is the mother - - tongue and is spoken in the home, always at church related social activi- ties and often to other church members even in the "english" world, such as in the grocery store (yoder ). members have a strong feeling that german is the better form of the language, richer, deeper, nore capable of expressing deep thoughts than either pennsylvania dutch or english the deep thoughts in one's life are those relating to god. so take away german, and one has taken away that aspect of his life. (yoder : ). for the deeply religious amish, it is clear that the functional distribu- tion of language use contributes to the motivation for stayirtgwithin the church and for resisting the obvious temptations to join one of the less conservative churches, like that of the mennonites, who do not have the same extreme restrictions on ( aily life bbr do the miannonites make similar use of language and indeed their present day high school genera- tion is monolingual in english. in this paper, i would like to examine the phenomenon of group bi- lingualism, the origin of the contact situations which lead to it, and the role of language inraintaining ethnic boundaries, especially in revitalizationmovrerents. we need to examine such issues because we will newer be able to understand the nature of bilingualism if we consider it as a unifimmaphercmenon. bilingualisrmay be a universal condition but it serves a variety of functions which need to be considered for an ade- quate understanding of the social oansequences of group bilingualism. furthermore, group bilingualism nore often than not is not stable, and becomes the majorrrechanim of language shift,'a phenomenon which is poorly understood (fishman ; lieberson et al. ). revitalization movements are,likely to be a rechanism for language maintenance or lan- guage revival. language shift and languagernainterance, with or without concomitant bdlingualism, are of course indicators of the degree to which ethnic boundaries are beingmaintained. gaarder (ia.d.) rakes the crucial distinction between elitist bdlin- lingualimnand folk bilingualign. elitist bilingualism is the hallmark of intellectuals and the learned in most societies, and'one might add, of upper class membership in many societies as it certainly is in scandinavia. it is a matter of choice. not so with folk bilingualism which is the re- sult of ethnic groups, in contact and competition within a single state, where "one of th peoples became bilingual involuntarily in order to sur- vive" (p. ). elitist bilingualism is not likely to be a mechanism for language shift ,orinaintenance, and in this paper i will only consider folk bilingualism. in an earlier paper (c. b. paulston b), i drew on schermerhorn's um "inductive typology" of comparative ethnic relations in an attempt to analyze the consequences of bilingual education in north pmerica, the direct result of ethnic groups in contact, and i would briefly like to review it here. schernertzorn points out that "the probability is over- whelming that when two groups with different cultural histories establish contacts that are regular rather than occasional or intermittent, one of the two groups will typically assune daninance over the other," ( : ), and he says elsewhere it is the nature of this dominance whidh is the major factor in ethnic relations ( : ff). the central question then in comparative research in ethnic relations (immediate causal factor of a group's bilingual status) is 'what are the conditions:that foster or pre- vent the integration of ethnic groups into their environing societies?" ( : ). the percentage of members of a group who become bilingual tan be seen as a concomitant condition of the degree of integration. scher- merhorn sees three major causal factors as determining the nature of the relationship between ethnic groups and the process of integration into the environing society. the first refers to the origin of the contact situation between "the subordinate ethnic and dominant groups, such as annexation, migration, and colonization," the second to "the degree of enclosure (institutional separation. or segmentation) of the subordinate group or grows from the society-wide network of institutions and asso- ciations," and the third to "the degree of control exercised by dominant groups over access to scarce resources by subordinate groups in a given society" ( : ). iieberson, dalto and johnston in a quantificationally very sophis- ticated article on "the cborse of mother-ubngue diversity in nations" ( ) point out the failure of developmental factors, such as urbaniza- tion, to account for cross-national changes in language diversity. they consider the very rapid language shift in the united states: "for the descendants of literally tens of millions of immigrants, english becane the mather.tongue in a matter of a fed generations (lbaberson and curry ). it is reasonable to akk how it came about that the shift was so rapid in the united states compared with that in the vast majority of nations" ( : )._ they conclude, like schermerhorn, that one must con- sider the origin of the contact situation and go on to "develop a theory which suggests that the course of race and ethnic relaticos will be dif- ferent in settings where the subordinate group is indigenous as opposed to those where the migrant populations are subordinate" ( : ). they consider, similarly to schermerhorn to whom they refer, four groups: ( ) indigenous supexordinate, ( ) nigrant superordinate, ( ) indigenous subordinate, and ( ) migrant subordinate. they find it unlikely that much, if any, mother-tongue shift will occur among the first two groups. "almost certainly a group enjoying both political and economic dominance will be in a position to ensure that its linguistic position is maintained. bilingualism may occur, but this is not the same as mother-tongue shift: at the very most, one can normally expect only an extremely slow rate of mother-tongue change among such groups" ( : ). the role of swedish in finland illustrates that point and gives us an example of an ethnic group in demographic and political decline which uses its native tongue to maintain its boundaries for ethnic survival.(r. g. paulston b). like the english-speakingcanadiams in quebec, the swedish-speakimg finns lack political power in finland and so are vulnerable to any legal mea- sures the finnish-speaking majority might institute in regards to swedish. presumably the status of swedish as a lingua franca in scandinavia (skutnabb-eangas ) and strong eeelings of scandinavian solidarity have contributed to the finns' tolerance toward swedishibut its continued role in finland is best explained by the former superordinate status of its mother-tongue speakers. subordinate groups who are indigenous at the time of contact, either through colonization as in the case of the american indians or through annexation as in the case of the chicanos in the u. s. southwest, are un- likely to change rapidly. migrant subordinate groups are the only groups likely to show rapid rates of mother..tongue shift, as the recent migrant finntwaworking-class populationinsweden illustrates in so rapid a shift that there is anecdotal evidence of difficulties of communication between parents and children. in the united states, as lieberson et al. show, the immigrant experience was one of extra-ordinarily rapid shift. in contrast, within the same nation and with access to the same educational institution of public schooling, the indigenous subordinategzoups have changed at a much slower rate. in , % of the whites in louisiana still reported french as their mother-tongue although the state had been purchased almost years before, from france in . in new mexico, conquered in , nearly % of the native parentage population (third or later generation) reported also in , spanish as their mother- tongue, which means that, since a fair proportion of this population was not of spanish origin, much more than half of the spanish-speaking popu- lation had not shifted (liebexson et al. ). in contrast to louisiana, the southwest has a steady trickle of new immigrants legal and illegal, frail btbxico, and no one really knows the exact rate of language shift, but thompson ( ) calculates that in texas spanish has remained the mother-tongue for eighty percent of the third generation. the indian population probably has been the slowest to become bilin- gual. liebe/son et al cite census data which show that as recently as slightly more tha % of the indian population could not speak english. many of those who did speak english also maintained their indian mother tongue, and lieberson et al concludes that "it is clear that nother-tongue shift was far slower than for the subordinate immi- grant groups" ( : ). the degree to which these populations become bilingual in mother- tongue and english varies, and the fact that many don't has resulted in national recognition of that problem in the form of federal legislation of the so called bilingual education act of . the official intent of this act is the more efficient teaching of english through the trarr sitional use of the mother tongue. the implementation of these programs, as i have written about elsewhere (c. b. paulston, in press), is accom- panied by considerable strife by school personnel who cite their own immigrant emperience background as example that there is no need for bi- lingual education. the militant minority ethnic groups, the chicanos, the puerto ricans, the navajos, refuse to accept the assimilationist goals of the programs, and instead talk about (andwhere they can implement) bi- lingual/bicultural maintenance programs. it is a clear group conflict situation, played out in the educational sector, where the ethnic groups are insisting on their rights to naintain their ethnic boundaries, much to the disapproval of the dominant anglos. these findings on language shift through a bilingual generation and languagerraintenance with or without concomitant bilingualism raise some issues which are important for a wore accurate under ningcsf k-aata of bilingualism. they also illustrate, i think, the akt ce f parative approach in this field of study. tne first tintio ' crne, toimird is why would the imadgrant experience result n . ch livioye shift with no apparent educational problems when the plq,tgenotls ing countered such difficulties; why have the latter maii"ed t e tongue and what is the mechanism of language maint-n" xt nkr, think, that themohanism of language shift in the un tki state° n ideo bilingualism: .a k, immigrant languages disappear because they cli?mkbt tr'",". ` frcm one generation to the next. typical-w the states the first generation:prefers to speek't e porr'%' ' mh tongue, the second generation is bilingualf_ the claims english as its mother tongue, learrdsfax,&"%e'isroll language mainly through contact with the gr"'%ret, ?; :), spanish language seems to be an exception.ca'noson mat is not clear are the factors which resulted jla e enguaosilnten, itti 'kance or sloc# rate of shift:of the indigenous popuictic j j the role of mother tongue language in the ethnic sliacj-t groa° ° mly do these groups insist =maintenance bilingual : o role k-athetr accept the transitional assimilation goals? in tdc xender. th i shall attempt to deal udth these issues. language shift can be seen as an indicator of ihrantioa . e environing society, and we can rephrase the first que i% aligh did the immigrants to the united states integrate int° th% large sn..ety wore rapidly and ccmpletely than did the indigenotis g"? on et al point out, one reason was that the indigenous g n airecla (n q. set of social and cultural institutions in situ throllorquch thel tempted to pursue their preconsiest activities. anotior e..asor ° they tended to be spatially isolated. these two reasons are both subsumed under sole="ala vat n of degree of enclosure. the less the two groups share uulttiral \t±.- - - tutions like the same churchese the same schools, the same jobs the higher the degree of enclosure within that society. schermrhorn points out that we do not have a very clear idea of the degree of enclosure of plural societies which are the result of annexation. in plural societies, "institutions of kinship, religion, the economy, education, recreation and the like are parallel but different in structure and norms ordinarily this is cavolnxleelby differences in language and sometimes by race as weal" ( : ). the relationship between degree of enclosure and the role of language in ethnic boundary maintenance processes is not under- stood, and i cannot think of any study which has eksimincx this particular problem. clearly it is an important topic for future investigation. the degree of control by-the anglo dominant group over access to goods and services also influenced the situation. the contact situations within the same nation between the anglo americans and the chicanos, the puerto ricans, the amerindians, were all the result of military conquest. the chicanos were segregated to one part of town and only given access to menial type jobs. the indians were isolated on reservations where no opportunity for jobs existed. the immigrants, on the other hand, were given access to jobs. brudner's thesis ( ) that jobs select language learning strategies is one that i have never found an exception to. when jobs were available which required a knowledge of english, the ethnic minority members became bilingual. without access to rewards, english was and is not salient. schermerhorn also posits three intervening or contextual variables which modify the effect of the independent variables. the most important is the agreement or disagreemera: between dominant and subordinate groups on collective goals for the latter, such as assimilation or pluralism. schermrhorn sets up a paradigm of which one purpose is to "specify the social contexts that can serve as intervening variables in answer to the - - scientific query, 'under what conditions?" ( : ). he bases his discussion on wirth's typology of the different policies adopted by minority groups in response to their unprivileged position. these policies he called assimdlationist, pluralist, secessionist, and militant. briefly, assimilation- ist policy seeks to merge the minority members into the_sdder_society__by_abandoning_their_orrat_cultural_ distinctiveness and adopting their superordinatas' values and style of life. the pauralist strategy solicits tolerance fram the dominant group that will allow the subordinates to retain much of their culcural distinctiveness. the secessionist:minority aims to separate or detach itself from the superor- dinates so as to pursue an independent existence. finally, the militants . . . intend to gain control over the dominants who currently have the asceedency ( : ). schemmrhorn points out that assimilation and plural= really refer to cultural aspects while secession and militancy refer to structural. to clarify this problem it is well to insist on the analytic distinction betwben culture and social structime. culture signifies the ways of action learned through socialization, based on norms and values that serve as guides or standards for that behaviaf. social structure, on the otherhand, refers to "the set of crystallized social rela- tionships which its (the society's) members have with each other which places them in groups, large or small, permanent or temporary, formally organ- ized or unorganized, and which relates them to the major institutional activities of the society, such as economic and occupational life, religion, mar- riage and the family, education, goverment, and recreation" (gordon, : - ). ( : ) in order to dealwiththe difficulty of applying cultural features to conditions which involve social features, he suggests the paired con- cepts of centripetal and centrifugal trendsiirsocial life. "centripetal tendencies refer both to cultural trends sudh as acceptance of common values, styles of life, etc., as well as structural features like in- creased participation in a common set of groups, associations, and in- stituticns" ( : ) . to keep the two aspects distinct, he calls the first assimilation, the latter incorporation. - - centrifugal tendencies among subordinate groups are those that foster separation from the dominant group or fnam societal bonds in one respect or another. culturally this most frequentlyneems retention and preservation of the group's distinctive traditions in spheres like language, religion, recreation, etc., together with the particularistic values associated with them: wirth's cultural requirements are needed, so there are demands for endogamy, separate associa- tions,- and_even at tirmaq a_restricted_range of occu- pations ( : - ). schernerhorn's major point is that integration, which involves the satisfaction of the ethnic group's modal tendency, whether it be centri- petal or centrifugal, depends on the agreement or congruence of views by the dominant and subordinate groups on the goals of the latter: cbngruent and incongruent orientations toward centri- petal and centrifugal trends of subordinates as viewed by themselves and superordinates. superordinates subordinates superordinates subordinates ass tion incorporation cf cp poroad segregation with resistance cp = centripetal trends cf = centrifugal trends ( : ) cf cf cultural piuralism autonomy tending toward integration tending toward conflict ilorced assamilation with resistance - - the immigrants' goals were clearly those cf assimilation; they had voluntarily left "the old country" with its frequently unsatisfactory conditions behind. the indigenous groups, in contrast, did not seek con- tact with ths cibminantanglos but found it imposed on them; their groups in their entirety were brought into the environing society with their culture intact. tbdgy many subordinate ethnic groups in the united states do not want to abandon their cultural distinctiveness; rather they want access to goods and services, to the institutional privileges held by the english speak:l. g mr:ddle class, i.e. econamicincorporationbut not assimilation. one important aspect of resisting assimilation is the maintenance of the mother tongue, in thesameway as language dhift is an important aspect of assimilation. ihe goals for all non-englibh speaking groups as seen by the domin- ant group have always been assimilation, the acceptance of "the american creed," the socialization into american ways and values. since this was also the goal of the immigrants, they willingly acquiesced to the assimi- lation process, and the relationship between the dominant group and the immigrants is best characterized by cell a a situation tending toward integration. anything less became considered unpatriotic, and i have more than once when i criticized same feature cf american life been told "if you don't like it here, why don't:mug° back to where you came from." the indigenousgmoups, on the other hand, tended to resist assimila- tion, and their situation is sydbolimd by cell d. it is slight wonder that, in a situation characterized by conflict, in which they resisted assimilation at the same time as theywere denied access to goods and services and were separated institutionally from the english-speaking group, members of indigenous groups did not shift to ehglish to the same degree and at the same rate as the immigrant group. the degree to which the indigenous groups became bilingual probably depended mostly on access - - to jobs which required a knowledge of english; the degree to which they maintained the mother tongue probably depended most on the resistance against assimilation. the affluent amish, who are an exception to the inmigrant experience of rapid language shift, exemplify both these points. they are perfectly bilingual as their business dealings with the "gay" ---liorid-necessitate-a-knowledge of english-i-which-ithey-learn-primarily-in-- the public schools. 'ihey have stubbornly resisted assimilation into anerican mainstream culture for religious reasons and not only have they maintained the mother tongue but also add standard german as an addition- al language, crucial to the transmittance of the group's basic values. however, the use of census data to establish language shift as lieberson et al. do masks the variation of language use between the various immigrant groups. furthermore, assimilation is not necessarily an irreversible process. the united states has lately experienced a re- surgence of ethnic awareness which brings into question the goal of can- plete assimilation for these ethnic groups. elazar and friedman discuss this new developrent of ethnic reaffirmation in anerican society in their perceptive diovin up: ethnic succession in anerica ( ) . they point out that ethnic identity has often been seen as a problem that must sat...- - i= be overcare. social scientists have often considered religious and ethnic grows as "vestiges of a priznitiv e. past that are destined to dis- appear" ( : ) , but recent "writers on the 'new pluralism' have argued that racial, religious, and ethnic groups are a basic canponent of our social structure" (p. ) who affect our institutions and at tines are ifore pouerful than econanic forces in their influence. as a result of the migration process, there has been a pattern of ethnic division of labor in the united states; e.g. the irish have been drawri to local politics and civil service, the slavic groups constitute a large part of labor in the coal mines and steel mills while the jews have - - been.active inter alia in family style businesses. elazar and friedman point out that large groups within the american populacecontinueto be "ethnic outs" in 'various stages of their own struggle to become 'ethnic ins.'" these groups of italian, slovak, polish, jewish, greek hungarian, and ukrainian background "are still struggling for recognition, upward ofiffibdest gains laboriously achieved after years of struggle (p. ). it is no coincidence that (with the excep- tion of jewish which is not a language) all of those languages are still natively spokenitypittsburgh and mark group boundaries which are iso- morphic with limited access to social rewards. the capacity of the "outs" to change their status, say elazar and friedman, depend on their potential social and political power, their willingness to use unorthodox tactics, the extent ofopposition, and their ability to evoke sympathy (p. ). they have befbre them the exmmple of the blacks. black gains during the 's and 's came about through ethnic solidarity and nere obtained primarily through the institution- alization of their own political strength" (p. ), which during the sixties resulted in highly visible ethnic confrontations. the results of these ethnic confrontations led other groups to make demands -- as groups. these demands come at a difficult time. from post-world war ii through the late 's, americelaystained rapid expansion and development of what has been called 'the metropolitan frontier.' there was a dramatic use of social, economic, and technical opportunities which the civil rights novement helped blacks to be able to take advan tage of. jobs, status and the good things of life were within the reach cl growing numbers of mexi- cans in an expanding economy (p. ). this expansion has now come to an end, and the present recession has exacerbated poverty and joblessness among the minorities. unemployment has contributed to ethnic solidarity as the experience seems to show that competition for rewards is more successful when carried out by groups than - - by individuals, and the group boundaries have been those of ethnicity. (r. g. paulston c).. one resource of ethnic groups which can be used in stressing ethnic awareness and identity of the members is the original mother tongue. pittsburgh radio stations, as do others in the country, carry radio pro- grams in italian, slovak, croatian, etc. the slok carramuty here, for example, offers a non-formal course in slovak culture, cuisine, music, and language. its students are from all social classes and all ages. many polish (for instance) families are changing their surname fram the anglofied version back to the original polish. at the university of pittsburgh, a number of students of polish, greek, hungarian, etc. back- ground, who have completely shifted to english, are laboriously studying the language of their grandparents. clearly this language learning will serve no immediate practical purpose but it does serve to reaffirm their ethnic identity and to reinforce old boundaries which in their case had become eliminated. the most extrema form of ethnic mobilization occurs in what wallace ( ) has termed revitalization movements, "deliberate, organized, con- scious efforts by members of a society to construct a more satisfying culture" ( : ). for wallace, this process involved a cultural trans- formation of the group. for the purposes of this paper, i will extere the term to include ethnic revivalmoverrents as well (which may not be involved in a cultural transformation) since wallace's concept of "revo- lutionary phase" ( : - ) applies to both movements. in his "schools in revolutionary and conservative societies" ( ), wallace discusses the learning priorities of the two types of society: mat a man is expected to do in his life will, in part, depend-on whether he lives in a revolution- ary, conservative, or reactionary society. and what he is expected to do determines what he is expected to learn ( : ). - - he outlines the following model of learnimg priorities: learning priorities in revolutionary, conservative, and readtiammry societies. °°°°. ' tectinic revolutionary phase 'wallace : ) technic morality morality chni intellect intellect cbnservative phase i social ) history reactionary phase wallace assigns very specific neanings to the terms technic, morality and intellect. by technic he refers to learning as a process of "reliability increase of action" through stimulus, reinforcemnt ard motivation; technic is learning "haw to." morality, on the cther hand, stresses nhat." mbr- ality concerns one particular kind of socially approved value: this kind of value is the conception that one's own behaviour, as well as the behaviour of others, should not merely take into consideration the attitude of the community, but should actively advance, or at least not retard, its welfare ( : ). although most commonly practiced in the humble endurance of dismal- fort by inconspicuous people, it is 'most conspicuously emeapliftedby such heroic actions as the soldier's throwilvhdnmelf on a handgrenade in order to smother the blast and save his buddies" ( : ). the criterion for morality is its potential for sacrifice, and all ethnic groups in the re- volutionary phase have sacrificial heroes as leaders, i.e. leaders who are willing to risk freed= or life for thecause; cesar chavez and la causa is a good exanyle. by intellect, wallace refers to jacques barzun's metaphor in his the house of intellect ( ) as an "establishment." mllace cites barzun: flculthe image of a hae-and its economy, one-can sob-- wh an inquiry into the institution of intellect nust include. themin topics are: the state of the language, - - the system of schooling, the neans and objects of communication, the supplies of money for thought and learning, and the code of feeling and conduct that goes with them. when the general tendency of these arrangements makes for order, logic, clarity, and speed of communication, one may say that a tra- dition of ihbellect exists ( : ). wallace ooncludes his discussion of intellect by pointing out that it is the_only truly universal too sahichis_t capable_of trinintaining mid restoring hunan arrangements against the erosions of time, capable of recognizing and solving new problems as well as learning answers to old ones" ( : ) . a group, like the nation of islam (in common parlance often referred to as the black muslims), or an entire society, like cuba, may enter a revolutionary phase when they perceive their present circumstances and state of affairs as intolerable to support further. the regressive re- sponse of individuals in the stage preceding a revitalization movement typically inclilapq "alcoholism, extreme passivity and indolence, the development of highly ambivalent dependency relationships, intragroup violence, disregard of kinship and sexual mores, irresponsibility in public officials, states of depression and self-reproach, and probably a variety of psychosomatic and neurotic disorders" ( : ). it is no accident that abstinence, hard nonrk, independence, black brotherhood, the importance of the family unit, responsibility, and black pride are some of the values most revered by. embers of the nation of islam ( . e. mohagmad ; see also bilalian news). as is typical and necessary in revitalization movements, the nation of islam has its charismatic leaders, especially the late elijah mohanrnad, who formulated "the nature of the existing culture's deficiencies, the nature of a desirable goal culture, and the nature and mode of operation of the transfer culture. this foundation must be more than an exercise of intellect: it must be passionately moral" ( : ). it speaks for - - the explicatory power of wallace's theoretical framework of revitaliza- tionmovenents that it was formulated years before the creation of the nation of islam, yet it perfectly accounts for its de;elopment. groups undergoing a revolutionary phase will always stress moral learning, and conflicts are certain to arise when a revitalization move- ----ment-takes-place-within-a amservative society-where-technic has-the highest learning priority, i.e. "in conservative societies, schools pre- pare people not for sacrifice but for jobs" (r. g. patiqtan : ). indeed wherever possible, the nation of islam has its own private schocas in order to be abae to implement its own learning priorities of nmoral transformation of the population" (wallace : ). the success of a revitalization movenent within a larger society, such as the economic and cultural succesa of the nation of islam, depends on the larger society's tolerance for cultural pauralism. the extreme respectability of the members in dress and an emphasis on moral values which are not in conflict with those of the mainstream culture presumably have contri- buted to this success. nor has there been any attempt to use language, so called non-standard negro english or black english, in the process of defining the group's new identity. the success of the nation of islam contrasts sharply with the fate of another black revitalizationmoverent, the black panther party. the second learning priority in a revolutionary phase is intellect, and says wallace, the moral intellectuals often appear as fanatics tathe conser- vative society. it is interesting to speculate that the failure of the black panthers partially was due to an emphasis on intellect rather than co, morality and to the lack of a consistently outlined goal culture as the nation of islam very carefully had done: the panthers, homever, like many other groups before them had a number of shortcomings. probably the main one was the lack of a thorough radical analysis and coupling that with a strong, organized set of strategies - - and programs. as with many other groups, publicity and their image may have clouded their own ideas of what they wanted to do. but, when as they sometimes seemed to be doing, the leaaprehip changed its views so often, with not enough outside feedback to guide them, they did amazingly well . . .(mason : ). the very point is that leadership in revitalizationmovenents is mess- ianic and does not depend on outside feedback nor is the moral teaching of goals frequently changed. the black panthers were militant; they carried loaded weapons, while still legal; cited nelcolm the assasin- ated black leader, mao tte-tung and marx; and some with eldridge cleaver called for underground terrorist-type activities. huey newton, a co- founder, titled his book to die for the people: the wtitings of huey p. newton ( ). the larger society considered them as fanatics and showed no tolerance: cleaverioent eventually into exile for many years, and newton was jailed for the murder of a policeman (le was freed in after two years in jail). says mason, "the trial of huey p. newton it- self is regarded by many educators, as well as lawyers, ordinary black citizens, etc. as truly revealing the racist character of the legal system" ( : ). the writings of the black panthers are also in impeccable standard english but intheir speeches there are occasional occurences of black english for stylistic effect, to mark group solidaricy. ethnic groups typically use its mother tongue or dialect to such purpose, and the re- liactance in both black revitalization movements to use black english reflects its stigmatized status as a former creole. 'lb members of the nation of islam, black english is associated with the conditiond of life before their cultural transformation and plays no part in the group's moral teaching. it should be recognized that this is an unusual situa- tion in revitalization movesrents. language skills in the official language can ordinarily be seen as an aspect of technic, an aspect of preparation for jobs which is the major priority of learning in a conservative society. the mother tongue, on the other hand, is an aspect of moral learning,,reaffirming the soli- darity and cultural uniqueness of the ethnic group, underscoring the need to teadh the moral values of good and evil, right and wrong, the values of the old gods, in the language in wbidh those values were originally transmitted._reaffirmation_of.sultural_values_are_freguently-a-part the moral teadhing, especially among ethnic groups wbo prior to the re- , vitalization novement have been taught by the dominant group to have nothing but contempt for theirdwn culture. the conflict over learning pxiorities explains the extreme impor- tance of control over local educational institutions. i have frequently 'heard commented among ny colleagues that the best bilingual sdhools are those that are under community control -- be it navajo or chicano.: i am not certain what 'best" means in this connection. in my discussion of the erickson report ( ). in an earlier paper, i pointed out that "rhe,- toric about cultural pluralism accounts for little if the objectives are not implemented (c. b. paulston a: ); the commurity-run navajo sdhool, as neasured by the achievement test batteries fran the california test bureau, was markedly inferior to the gavernment-run sdhool academically. i was at the time only interested in investigating the learning of eng- lidh language skills, but even so that statement and the evaluation itself -- dhows our typical tendency to assess and evaluate the sdhooling of groups undergoing a revitalization movement with moral learning as the priority, in terms of the standards of the conservative society -- the standards of tedhnic. the following case study of "ethnic revival and educational conflict in swedish lapland" (r. g. paulston a) illustrates the importance of autonomy over eaucational programs by the group in a revitalization phase. it illustrates onde again the tendency to intolerance of cultural pluralism - - by the larger society except when it sees its own purposes furthered; consistently, when the national economy favored reindeer herding, the collective goals for the lapps as seen by swedish officials included the use of lappish and support of same culture; otherwise, the goals were swedish and assimilation. we also see here the typical support (maintenance and/or revival) of the mother tongue in ethnic revival movements. swedish goverment relations with the lapps began with the lapp- mark edict of which sought to open the lapp's traditional homeland to swedish settlers and the state church. this document promulgated a policy of minority integration, along with descriptions of stereotypi- callninority attributes, that has continued in large part down to the present day (cited in ruong b: ). it states that: ( ) lapps should devote themselves to reindeer breeding in the mountain areas, a task for which they are best suited. ( ) swedish settlers shouldhave the right to take land from the forest lapps, to hunt and fish on lapp lands, and to burn and cleariviii land. ( ) the lapps are a barbaric people from else- where without legal title to the land they use. they are lazy and useless in war. in the following centuries, two themes pervaded ethnic relations. the dominant theme called for lapps to withdraw before the advance of western economic penetration. lapps might indeed join this advance by rejecting their language and other core cultural traditions and becoming swedes. the second and lesser theme, that the lapp minority should be pre- served and protected, largely through paternalistic efforts of the swe- dish state church, became increasingly important in the th century as traditional lapp nomad society disintegrated under the onslaught of ex- panding scandinavian societies. with a cultural-revitalizationmovemnt - - led by the minister iars laestadius the ensuring stzenhenip'j \%cial responsibility, and the increased value of reindeer herkng til° .\ tional'econamy, educational policy for the lapps shifra woes° nitn e ), acculturation to socialized isolation (nordberg ; ,'"" about world war i, what might be called the "oamsevrik)ist° ia°ny had gained predominance and became embodied in the tiomkt sch°° inara of . the ,act's rationale, both economic and earatt n , i° ntly, apparent in the position stated by the bishop of lule" leacr.a. school reform and the religious head of the lappuork (ced in i )°n b: ): lapps ladk the physical attributes necesed regular, heavy manual labor and therefore fp'k into deep poverty and misery when they adopt' settled way of life. if, on the other herld/m?)ey continue with reindeer breeding, they cat? eria- upon a secure source of livelihood. it st oor ngly, of national economic interest th,he lapps retain their inherited source of livey-"ood. the vast tundra and mountain areas of northern scan( nia could' k that time, be most eoditinically exploited as pastam reinele°' herding required that the lapps live a nomadic exietalla% thns' tl\ question of "what is an appropriate education for r-apv - .iis tex ? rg came clearly linked to the importance of..reindeerbteekilg far tile ilk tional econany. or as stated in the school re-w. yopp be a lapp, for then he serves his motherland best." ish policycontinued,especially with the reindeer d'otik)e inner possible to keep the lapp childreri within lapp culttlre :hd the ° kc economy (ruong b). following wotld war ii, the conservationist polio cl/re irlde .alvy attadk from lapps sicing greater educational pport ), ancl ° .. . e swedish gacernmentwhich had replaced the church in tr° oontro- schooling. the swedish missionary society's educatl&i'l efforts . focused almost entirely on the lapp's folk high sch(n %undod' av - - world war ii ( ) and located above the artic circle at jokknokk after . in this residential "folk college," same youthin their late teens received training in general citizenship, local and national history, reindeer breeding, and other practical subjects. cbntinuing the "lapp will be lapp" orientation, students were encouraged not to let the two- year course seduce them away fram reindeer herding. a, policy statatent at that time, for example, stressed that: not by following sweden and things swedieh, but by prandting your mother tongue will you be best fitted to contribute to the common treasures of the father- land. as lapps it is through following ycur own ways of living and by remaining faithful to your cul- ture that you will keep your place as a part of the swedieh nation (ftmag a: ). many young lapps leaving the lapp foik high.sdhool returned to the herding way of life, and a number became leaders in subsequent ethnic organizational development, as in the swedish reindeer herders associa- tion founded in . others became sdhool teachers and taught same children in settled communities and in nomad sdhools. a thitd very small group moved into nainstzeara swedish society and culture through subsequent professional training. but a number of these assimilated lapps maintained feelings of ethnic solidarity with their same origins and a concern for greater social justice in same attempts to fend off powerful state and, private interests seeking the continued economic ex- ploitation of lapland's rich natural wealth cf ninerals, timber, water, and scenery (wallmark ). thus, by the 's swedish authorities offered two distinct educe- tional programs for same children. for those who lived by reindeer herding, the nbmad schools, which had become fixed and residential, con- tinued to stress skills and values thought necessary for a nomadic life. for children of the growing number of same employed in forestry, agricul- ture, and fishing, regular primary and less frequently, secondary sdhools were built in rural areas -- sdhools taught entirely in swedish and atidende. by swedish youth, schoolswhere the iapp language and cul- turaimere viewed as inferior, amildiere lapp ethnic identity was un- desirable and, when possible, to be denied. school failure of lapp childrem was and is conmon. in their public school experiences, gemmrations of sane youth have learned that being a lapp and speaking iappish was strongly associated with defeat, contempt and poverty (with ; ostlund ). with increasiivpmessure fran the swedish social cemocratic government for the acculturation and out-migration of same youth, with continued serious infringement of forest and pasture land by "clear-cutting" forestry practices, by tourism, by mining and water- power develixerit interests, et al., the lapps, a distinct people with thousands of years of traditions as-an ethnic community, began to seriously doub* : eir ability to survive in the early 's ( bllmark ; park ; otm-s ; lundegard ). until recently, upwardlyinobile lapps could avoid career defeat only by "paying" with their identity. one was either a lapp, a swede, a norwegian, or a finn. the few sane who adhieved assimilation, or any same bar that natter, rarely protested this cost-mechanism (por- sanger ). a few exceptions can be bound, mostly among teachers, or isolated individualswlx, by paying the price had cane to knad the ideal democratic ideology of the doninant segment, and by simple trans- fer had came to seethe piiiiçation process as an injustice to a cul- ,; tural minority midheint by attempting to point out what might be viewed as a moral injustice according to the dominant group ideo- logy, they jeopardized their careers were denounced as threats to na- tional security by their superiors, and were generallyviewed with alarm by *apps who rightfully feared the consequences of such public -- \ ps p tof ethr -° relkictus and internal colo &la fevi fr°irptski such non-. controversial otni") acti-vir" foy l-t ,lafts &z efforts -to maintain swe., ellktty . rd/es nrveci- t° .t"h laza:want seguent. foll vice 'kr a of opc°rt - challgee t the ra- tional and in level neif ° toile - ° to ef- forts of lano slin.x"t eft irliklvido-ts arki illstitu- tions in the 'zir%te a ---'e v fr*tk, ois collective efv- toge" the first 'ulre '°- °se identi- fied with lapo clapn ouet t° % dref° seqpus e"rcrai kld cul- tural probler -/k u from p°k-tio ° *': ity 'e th% danal&" s taneously,that the same , d occuloi rapid econani ploigro/a &ire- tterl f ,ral s%ourc;es in traditiona r/ kreas hacj n- / 'trirntal °ffect. ori the herding econckgfr (el%r ) at idle j/terrinonal arid t-ional- levels, wartiko e" by riazi. to ettetktnate the akii.zh et.h- llnajp i% sule f°.t -the. tirlitecnic minority, ft so i ktioos work on human - -gli e ped tt'lrl -the e bl'itt of 'ate %rival to. .i p 'el at on e stat c)fhane and to s und,er-priviles rifities / th% scar - -t n co° trie (finerstad adina, ) . accortirdf ciao." snent ecave itiore -` ' of alp : fosed injustiwe sys uritp, ° trio influential pj teactie°' tncal ecia sts % ottita sage a con°"els so, a's to a renent ktioriale on ) and program ( - e /e"-. )j attk t has since tk -b` mle s )--- ° kl-re" kovemep %ght to rtikt ti) stion mobilize minok a ty tal-- ore" -z nevi orgarlizcations lciep/p , sureal re ).stan that embody tko ethnic activis - - acculturation (eidheim ). these groups in turn, have brought pressure on institutions of the dominant society to respond favorably to lapp demands for altered ethnic relations, for a more culturally pluralistic soaietywhexe same cultural survival and same perticipetion will be accepted as legitimate national concerns. lapp organizations in the past decade, for example, have called for a number of basic changes in the nine- year omprehensive school that most of their children attend. this reform program places priorities on bilingual instruction in lappish and swedish during the early grades, the creation of bilingual teacher- trainingpangxams, a revised curriculum including same history and cul- tural themes, and a secondary school progrimeepecially adapted to the needs of reindeer-herding culture and managetrent. the conflict of interests that arise from intensifying government efforts to assimilate and incorporate the lapps into the smedishttil- fare state, and lapp efforts to mcve towards a more autonomous situa- tion in a more culturally pluralistic society are clearly evident in the schools. same students despite recent special educational arrangements, continue as a group to be under-achievers, withdrawn, and seemingly unable to succeed in national schools. a, well-known lapp educator, the norwegian anton eoem, contends that the causes are to be found in competing and conflicting ideologies -- i.e. , those of the ethnic movement, and those of the-national society: the investments in special teacher training, text- books, and literature in lappish, introductionof lappish language and culture as subjects are efforts to raise the efficiency of teaching within the es- tablishej system. they are not efforts to adapt the school to the particular needs and values of lappish society. therefore, one will find different stanr dards. in fact, the more efficient the teaching, the greater the discrepancy between goals of educa- tion at home and in the school . . the main - - results are a cultural and social gap between the most successful pupils and the local lapp society, and a barrier betmeen losers and the nationwide system (ho= ). same movement activists have come to recognize that formal school- ing, despite gestures toward bilingual instruction, is essentially about the business of assimilation and social control, and not the strengthening of cultural pluralism. they have, accordingly, in the past few years sought to develop an alternative educational setting wheremovement ideology could be developed to shape goals, new learning, and action strategies. the lapp pak high school, a residential school for young adults at jokkmokk in the forest lapp area above the arctic circle, has begun to carry out this function but only with protracted conflict with the local commune authorities who reject the legitimacy of "lapp power" slogans, the same ethnic-revivalrmyvenant, and the emer- gence of a lapp-controlled folk school (lidroth ). despite local pressures and forceful arguments by a number of cbmmune leaders to change the school's name and orientation from the lapp's folk high school to something like "jokkmokk commune's f. h. s.," a small core of same activists have, during the past several years, used the school to develop an educational strategy seekingtorrobilize individual same into a politicized ethnic-interest group ( stlund ). although the same remain aminority on the folk high school's board of directors and must share control with representatives of the provincial government and the antagonistic local commune, a number of notable innovations have been secured, albeit with growing animosity betwzan lapp students and the swedish population both at jokkmokk and throughout northern sweden. studies of how ethnic groups mobilize for political action suggests a number of basic problems that each group must solve (barth ). these include questions about the distinc- tiveness of the group, and the need for agreement on standards by - - which group members can judge themselves and others and determine who lies within the ethnic boundary, and who lies without. these concerns, as well as problems of commaication, decisionmaking, authority and the legitimate use of power, of ideology and discipline, and of the in- doctrination necessary to keep ideology alive have all been addressed by educational activities at the lapp's fhs. before examining these activities, it may be useful to note that ethnicity has previously been viewed here as a phenomenon useful in the categorization ofpeople. at the folk school, ethnicity has also been a tool employed strategi- cally by movement activists seeking to assist same youth to negotiate new individual identities, commitmnts to struggle, as well as the learning of organizational and communications skills needed to advance the movement's manifest goals of same cultural revival and ethnic- minority survival. where the swedish missionary society used the lapp's folk high school during the 's and 's to further the national ethnic policy of socialized isolation, today, lapp activists fight to gain complete control of the school and use it to address their problems and dreams of survival as an ethnic minority in a pluralistic society. where earlier many of the reindeer herder's association leaders had their first training in formal organizaticmal life and social science at the school, today special courses are offered that focus on prob- lems of collective actionandmimority mobilization. these critical seminars are taught and attended by lapps of different socio-economic backgrounds and seek to unify the lapps as an ethnic group. lapps from the mountains, the forests, and the cities with differing professions and life-styles meet, discuss, and develop movement ideology and action strategies to strengthen ethnic identification and to gain greater ac- ceptance from a strongly ethnocentric swedish society. - - courses in the lapp language are also provided and related to the movement's literary and journalistic activities. classes in lapp his- tory and handicrafts, in reindeer management, and admdnistrative tech- niques all view same culture from perspectives that seek to raise stu- dents' consciousness of ethnic membership and the legitimacy of the group's dreams of survival and autonomy. the critical seminars, es- pecially, have frequently been starting points for reformulating the movement's ideological framework, i.e., the "iworld vi.emr" from dhich most lapp political actions follow. the joint nordic lapp ctilture policy statement of entitled, in english, "we re lapps and want to remain lapps" clearly indicates efforts in statihg themmament's argument-and goals (svenska samernas riksforbund ). it is also a document that owes much to the educational and political work of same movement activists and supporters at the lapp's folk.eigh school. the lapp's folk high school today presents an example of an educe- tional institution actively seeking to advanoe a prccess of ethnic re- vival, confrontation and resistance to acculturatio that is for the most part viewed unfavorably by members of the dominant society. at the local level, swedes press for rapid acculturati% and incorporation through, if necessary, forced assimilation. at the national level, assimilation is sought instead through a policy of "tvgkulturell utbildning," or bicultural education where swedish kid same stodies will supposedly be given equal value and emphasis.(norrbottenslgn ). so far, same activists have been largely able to subvert this pcaicy at jokkmokk and stress the latter. but as this tolerant situation may end at any time, the ethnic moverrent is pressing fot a state-supported rural folk high sdhool undei complete same control ( stlund : - ). while most lapps are sympathetic to the moverethes broad goals, many express concern about the possible consequences of conflict. one of their older leaders has put it thus: "we are not a warlike people - - and we don't fight. our culture is primitive, and i suppose we have to give in.to the stronger one." many younger lapps reject the re- signation of their elders and, under the rubric of "lapp power," seek to fight back: ne are not norwegians, swedes, or finns. we are dif- ferent and we intend to stay that way." it is this small activist group which seeks to make the lapp's folk high school its own, much to the consternation of many fearful lapps, the jokkmokk commune, and the national board of education (lidrothc ; sammallaht, ). conclusions it is clear that language maintenance of an ethnic group rein- forces the boundaries between that group and the larger society. boun dary maintenance reinforces the ethnic identity of a group and is undertaken for a number of reasons: religious, as in the case cf the amish;politimeconomical, ai in the case of the french speaking canadians; or economic, as in the case of the immigrant anerican italian, slovak, polish, etc. groups. often, the major function of language in boundary maintenance is to enable a group to resist assimi- lation: this is as true of the former superordinate, migrant finlands- svenskarna as it is of the subordinate, indigenous lapps natio both use language as a weapon in the fight for ethnic survival. the degree to which a group beccaes bilingual depends partly on the larger society's willingness to let that group assimilate, to grant that group access to the social institutions, and partly on the availability cf jabs which require a knowledge of the official lanr guage. the canadian french immersionprograns form a very good example of how job language requirements influence language learning strategies. group bilingualism is frequently accompanied by language shift to the official language when there are ample, material rewards in so doing. institutional enclosure nay not be an issue as long as the ' - - parallel institutions are open. the french canadians, who massively shifted to english (and gave cause to the legal measures) in spite of parallel institutions of church school, recreation, etc., are a case in point. many migrant superordinate groups do not seem able to main- tain their original mother tongue once they are (legally-pcaitically) separated fram the home culture as in the case of the normans and the franks; probably demographic factors were also at issue (nlomason and kaufman n.d..). spanish certainly has been maintained in colonized latin-america. it is a temptation to claim that language shift takes place when the socio-economical and political rewards of a nation, which are accessible, favor such a shift, but it is likely to be a simplification. it is a claim which is likely to account for the majority of situations of language shift but-it will not necessarily account for situations where one would expect language shift .but does not find it. it may be that the spatial isolation of the lapps can account for the maintenance of lappish, but it is more likely to be an expression of ethnic identity which will not be surrendered, a mecban- ism of ethnic boundary maintenance. linguists do not have a clear understanding of these issues, and my contention is that in crder to understand the nature of bilingual- ism we need to consider the relationship of these issues. in this paper i have indicated the direction i believe such a discussion should take, but it is very obvious that this paper is only a beginning. . this paper is co-authoredinthat the same case study is written by r. g. paulston and the rest of the paper by c. b. paulston. the case study is a much shortened version of the original "eth- nic revival and education cbnflict in swedish lapland." . fbr references to the research on the canadian inwersion pro- grams, see merrill swain's "bibliography: research on immer- sion education for the majority child." j. the following excerpts are from r. g. paulston's "ethnic re- vival and educational cbnflict in swedish lapland," cbmpara- tive education review, : , , - . permission to reprint is gratefully acknowledged. . it should be pointed out somewhere that many members of the eth- nic groups discussed in this paper do shift languages and assimr ilate into the larger society, and that this discussion cipals with what sometimes amounts to a minority within a minority. balalipn news; fornerly moharrnad speaks, published weekly. chicago, l.linois. barzun, j. . the house of intellect. new york: random house. barth, f., ed. . ethnic groups and boundaries. boston: little, brown and company. brudner, l. . 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"diglossia within the old order amish speech com . munity of lancaster, pennsylvania." ms. university of pitts- burgh, department of general linguistics. outcomes of physician‐staffed versus non‐physician‐staffed helicopter transport for st‐elevation myocardial infarction outcomes of physician-staffed versus non-physician-staffed helicopter transport for st-elevation myocardial infarction sverrir i. gunnarsson, md; joseph mitchell, md; mary s. busch, rn; brenda larson, rn; s. michael gharacholou, md; zhanhai li, phd; amish n. raval, md background-—the effect of physician-staffed helicopter emergency medical service (hems) on st-elevation myocardial infarction (stemi) patient transfer is unknown. the purpose of this study was to evaluate the characteristics and outcomes of physician- staffed hems (physician-hems) versus non-physician-staffed (standard-hems) in patients with stemi. methods and results-—we studied stemi patients transferred by either physician-hems (n= ) or standard-hems (n= ) for primary or rescue percutaneous coronary intervention at hospitals between and . data were collected from electronic medical records and each institution’s contribution to the national cardiovascular data registry. baseline characteristics were similar between groups. median electrocardiogram-to-balloon time was longer for the standard-hems group than for the physician-hems group ( vs minutes; p= . ). the standard-hems group was more likely than the physician- hems group to receive nitroglycerin ( % vs %; p< . ) and opioid analgesics ( . % vs . %; p< . ) during transport. in-hospital adverse outcomes, including cardiac arrest, cardiogenic shock, and serious arrhythmias, were more common in the standard-hems group ( . % vs . %; p= . ). after adjusting for age, sex, killip class, and transport time, patients transferred by standard-hems had increased risk of any serious in-hospital adverse event (odds ratio= . ; % ci= . – . ; p= . ). in-hospital mortality was not statistically different between the groups ( . % in the standard-hems group vs . % in the physician-hems group; p= . ). conclusions-—patients with stemi transported by standard-hems had longer transport times, higher rates of nitroglycerin and opioid administration, and higher rates of adjusted in-hospital events. efforts to better understand optimal transport strategies in stemi patients are needed. (j am heart assoc. ; :e . doi: . /jaha. . .) key words: acute myocardial infarction • outcome • percutaneous coronary intervention • st-segment elevation myocardial infarction • treatment p rimary percutaneous coronary intervention (pci)improves survival in patients with st-elevation myocar- dial infarction (stemi) and is the optimal treatment when performed expeditiously. over % of patients experiencing stemi in the united states initially present to hospitals without pci capability and thus are at risk for delayed reperfusion. to ensure rapid interfacility transport for pci, regional systems of care are recommended. – helicopter emergency medical services (hems) are com- monly used to transport patients from non-pci centers (ie, stemi referral hospitals) to pci centers (ie, stemi receiving hospitals). hems have been shown to be feasible, safe, and reduce transport time to the receiving facility. – thus, hems are important in rural or urban areas where ground transport times are predicted to be too long. the hems flight crew typically consists of paramedics and flight nurses who are skilled at performing a variety of advanced cardiac life support procedures. however, only � % of hems in the united states will also include an emergency-medicine–trained physician as part of the flight crew. local resources, cost, and hospital affiliations are factors that currently influence hems avail- ability and crew composition. there are limited data evaluating the composition and outcome of hems and no data comparing different hems from the division of cardiovascular medicine, departments of medicine (s.i.g., j.m., b.l., a.n.r.) and biostatistics and medical informatics (z.l.), university of wisconsin, madison, wi; mayo clinic health system-franciscan healthcare, la crosse, wi (m.s.b., s.m.g.); division of cardiology, mayo clinic, rochester, mn (s.m.g.). correspondence to: amish n. raval, md, facc, fscai, faha, division of cardiovascular medicine, department of medicine, university of wisconsin school of medicine and public health, university of wisconsin hospital and clinics (site of research), csc h / , highland ave, madison, wi . e-mail: anr@medicine.wisc.edu received october , ; accepted december , . ª the authors. published on behalf of the american heart association, inc., by wiley blackwell. this is an open access article under the terms of the creative commons attribution-noncommercial-noderivs license, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. doi: . /jaha. . journal of the american heart association original research d ow nloaded from http://ahajournals.org by on a pril , info:doi/ . /jaha. . http://creativecommons.org/licenses/by-nc-nd/ . / systems on transport times, treatments administered during patient transport, and in-hospital events in patients with stemi. as such, practice guidelines do not address the issue of crew composition during interfacility transfer. the primary objective of this study was to evaluate the characteristics, treatment patterns, in-transport, and in-hospital outcomes of stemi patients transferred byphysician-staffed hems (physician-hems group) versus nonphysician (standard-hems group) flight crews. methods the institutional review boards of both the university of wisconsin hospital and clinics (uwhc) and mayo clinic approved this study. patient demographic information, in-transport treatments and events, and in-hospital outcomes were abstracted from institutional contributions to the national cardiovascular data registry (ncdr), electronic medical records, and the hems flight records. stemi was defined based on criteria: ( ) electrocardiogram (ecg) evidence of st-elevation based on the american heart association/ american college of cardiology (aha/acc) stemi guidelines ; ( ) a culprit artery identified by emergency invasive coronary angiography; and ( ) performance of either primary pci or pci following fibrinolytic administration (ie, rescue pci). in-transport adverse events were defined as death, cardiac arrest, serious arrhythmia (ie, rapid supraventricular arrhyth- mias requiring direct current cardioversion, sustained ventric- ular tachycardia or ventricular fibrillation, or brady-arrhythmias requiring external pacing), or endotracheal intubation. data pertaining to the use of adjunctive medications, additional procedures, and vital signs were also recorded. in-hospital outcomes were reviewed by trained abstractors using standard definitions established by the ncdr action registry and included death, stroke, cardiac arrest, bleeding, cardiogenic shock, new requirement for dialysis, recurrent myocardial infarction (mi), and vascular complications. helicopter emergency medical services within both institutions’ stemi systems of care, hems are requested directly by the stemi referral hospital. the choice of commercial hems is dependent upon availability, local contractual affiliation, and regional emergency department staff preference. the majority of stemi transfers destined for the uwhc are managed by uw med flight, a physician-staffed service. the physician-staffed hems crew is comprised of an emergency-medicine–trained flight physician, a flight nurse, and a pilot. this service utilizes ec- model helicopters (american eurocopter, grand prairie, tx), which have a maximum speed of knots (kts). non-physician-staffed hems that transport to uwhc (react of rockford, il; flight for life, milwaukee and fond du lac, wi; and medlink air of la crosse, wi) consist of a pilot and either flight paramedics or a flight paramedic and a flight nurse. these hems utilize either ec- model or bk- helicopters (american eurocopter). the ec- and bk- models have maximum speeds of and kts, respectively. all hems and stemi referring and receiving hospitals follow a standardized antithrombotic protocol, which includes aspirin mg, clopidogrel mg loading dose, and weight- adjusted heparin; however, deviation from the protocol can occur when clinically appropriate. the stemi protocol suggests that nitroglycerin and opioids (morphine or fentanyl) may be administered as clinically indicated. statistical analysis continuous variables were analyzed using the wilcoxon rank- sum test. categorical variables were tested using the chi- square test. fischer’s exact was applied in those instances where the counts of observed outcomes were numerically small or the expected cell counts < . logistic regression models were constructed to evaluate independent predictors of outcomes. all tests were considered statistically significant if p< . . all analyses were performed using statistical package for the social sciences (spss) software (version . ; ibm inc., chicago, il). results from august through december , a total of stemi patients were transferred for primary or rescue pci. of those, patients were transported by physician-hems and patients by standard-hems. patient characteristics there was no significant difference in baseline age or frequency of cardiovascular disease risk factor between the physician-hems and standard-hems groups (table ). there was no significant difference in the baseline killip class between the groups (p= . ). the right coronary artery (rca) was the most common culprit artery in both groups. however, the physician-hems group was more likely to have the left anterior descending (lad) coronary artery as the culprit for stemi (p= . ; table ). in-transport events in transport mortality was % in both groups. there was no significant difference in the frequency of serious adverse events, such as cardiac arrest, arrhythmia, intubation or need for transcutaneous pacing, direct current cardioversion, or doi: . /jaha. . journal of the american heart association physician vs non-physician stemi transport outcomes gunnarsson et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , cardiopulmonary resuscitation (table ). the standard-hems group was more likely than the physician-hems group to receive intravenous analgesics, such as fentanyl or morphine and nitroglycerin, during transport ( . % vs . % and . % vs . %; p< . for both; table ). of the patients who received nitroglycerin during transport, ( . %) had rca as culprit vessel. in the standard-hems group, of ( . %) of the patients who had occluded rca received nitroglycerin compared to of ( . %) patients in the physician- hems group (p< . ). the standard-hems group was more likely to receive fluid bolus and anti-thrombotic medication in-flight. the median first electrocardiogram-to-balloon (ecg b) time was longer for the standard-hems group compared to the physician-hems group ( vs minutes; p= . ; table ). there was no statistical difference in flight distance, ground time, and flight time between the groups. table . patient characteristics* physician- hems (n= ) standard- hems (n= ) p value age, y . � . . � . . male, n (%) ( . ) ( . ) . diabetes mellitus, n (%) ( . ) ( . ) . active smoker, n (%) ( . ) ( . ) . hypertension, n (%) ( . ) ( . ) . dyslipidemia, n (%) ( . ) ( . ) . previous mi, n (%) ( . ) ( . ) . initial vital signs median sbp, mm hg (iqr) ( – ) ( – ) . median dbp, mm hg (iqr) ( – ) ( – ) . median hr, rate per minute (iqr) ( – ) ( – ) . killip class . i, n (%) ( . ) ( . ) ii, n (%) ( . ) ( . ) iii, n (%) ( . ) ( . ) iv, n (%) ( . ) ( . ) culprit coronary artery . lad, n (%) ( . ) ( . ) rca, n (%) ( . ) ( . ) left circumflex, n (%) ( . ) ( . ) other, n (%) ( . ) ( . ) rescue pci, n (%) ( . ) ( . ) . dbp indicates diastolic blood pressure; hr, heart rate; iqr, interquartile range; lad, left anterior descending; mi, myocardial infarction; pci, percutaneous coronary intervention; physician-hems, physician-staffed helicopter emergency medical service; rca, right coronary artery; sbp, systolic blood pressure; standard-hems, non-physician-staffed helicopter emergency medical service. *continuous variables are expressed as means�sd (standard deviation) or median�iqr. table . in-transport events, procedures and medications physician- hems (n= ) standard- hems (n= ) p value events death, n (%) na cardiac arrest, n (%) ( . ) ( . ) . serious arrythmia, n (%) ( . ) ( . ) . procedures intubation, n (%) ( . ) ( . ) . dccv, n (%) ( . ) ( . ) . transcutaneous pacing, n (%) ( . ) ( . ) . cpr, n (%) ( . ) ( . ) . medications analgesics, n (%) ( . ) ( . ) < . paralytics, n (%) ( . ) . fluid bolus, n (%) ( . ) ( . ) . intravenous metoprolol, n (%) ( . ) ( . ) . vasopressors, n (%) ( . ) ( . ) . anti-arrhythmics, n (%) ( . ) ( . ) . anti-thrombotics, n (%) ( . ) ( ) < . nitroglycerin, n (%) ( . ) ( . ) < . cpr indicates cardiopulmonary resuscitation; dccv, direct current cardioversion; physician-hems, physician-staffed helicopter emergency medical service; standard- hems, non-physician-staffed helicopter emergency medical service. table . transportation metrics* physician- hems (n= ) standard- hems (n= ) p value flight distance, nautical miles (iqr) ( – ) ( – ) . ground time, minute (iqr) ( – ) ( – ) . flight time, minute (iqr) ( – ) ( – ) . ecg b, minute (iqr) ( – ) ( – ) . d b, minute (iqr) ( – ) ( – ) . d b indicates time from arrival at percutaneous coronary intervention (pci) referring hospital to balloon inflation; ecg b, time from first electrocardiogram acquisition to balloon inflation; flight time, time from helicopter takeoff to landing; ground time, time from helicopter landing at pci referring hospital to take-off; iqr, interquartile range; ps-hems, physician-staffed helicopter emergency medical service; standard-hems, non-physician-staffed helicopter emergency medical service. *continuous variables are expressed as median�iqr. doi: . /jaha. . journal of the american heart association physician vs non-physician stemi transport outcomes gunnarsson et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , in-hospital outcomes there was no statistical difference in the rate of in-hospital death between the standard-hems and physician-hems groups ( . % vs . %; p= . ; table ). the rate of any serious in-hospital adverse event was higher in the standard- hems group compared to the physician-hems group ( . % vs . %; p= . ), including cardiac arrest ( . % vs . %; p= . ), cardiogenic shock ( . % vs . %; p= . ), and need for intra-aortic balloon counter-pulsation ( . % vs . %; p= . ). of the patients who sustained any in-hospital adverse outcome (n= ), of ( %) in the standard-hems group were given nitroglycerin versus of ( . %) of those in the physician-hems group (p= . ). similarly, of ( %) of the patients who were given analgesics in the standard-hems group sustained an in-hospital adverse event compared to of ( %) of those in the physician-hems group (p= . ). median length of hospital stay was not statistically different between the groups ( days for both groups; p= . ). after adjusting for age, sex, killip class, and transport time, patients transferred by standard-hems had an increased risk of any serious in-hospital adverse event (odds ratio [or]= . ; % ci= . – . ; p= . ; table ). discussion to our knowledge, this is the first study to compare physician- staffed to non-physician-staffed hems for patients with stemi. the main findings from our study are that patients transferred by standard-hems: ( ) were more likely to receive opioid analgesics during transport; ( ) were more likely to receive nitroglycerin and intravenous fluids; and ( ) had a nearly -fold higher adjusted risk of in-hospital adverse outcomes. these differences were not associated with a statistically significant difference in mortality between the groups. a previous study showed that nonphysician hems trans- port of patients with acute coronary syndrome was safe. however, that study was not designed to compare different methods of transportation. a survey of flight nurses showed that physicians made unique and important contributions to the care of % of patients with mi. outcomes comparison before and after implementation of physician-staffed hems has been done for trauma transport. one study found that physician-staffed hems was associated with lower mortality of > blunt trauma patients. contemporary series have shown several benefits of having physician-staffed crews for regional trauma systems, includ- ing faster transport time. , we think that the observed association between standard- hems and higher rates of adverse in-hospital outcomes, such as cardiac arrest and cardiogenic shock, might be explained by several factors. first, the standard-hems group had slightly longer ecg b time, but the effects of early revascu- larization on survival are well known. given that the ground time and flight time were not significantly different between the groups, this could be because of shorter transfer time after landing at the receiving pci hospital. or, this could be table . in-hospital outcomes* physician- hems (n= ) standard- hems (n= ) p value death, n (%) ( . ) ( . ) . iabp, n (%) ( . ) ( . ) . cardiogenic shock, n (%) ( . ) ( . ) . bleeding, n (%) ( . ) ( . ) . tamponade, n (%) ( . ) . ventricular free wall rupture, n (%) ( . ) . need for dialysis, n (%) ( . ) . ventilator-associated pneumonia, n (%) ( . ) ( . ) . cardiac arrest, n (%) ( . ) ( . ) . serious arrhythmia, n (%) ( . ) ( . ) < . recurrent mi, n (%) ( . ) ( . ) . stroke, n (%) ( . ) . any adverse event, n (%) ( . ) ( . ) . median los, days (iqr) ( – ) ( – ) . median lvef, % (iqr) ( – ) ( – ) . iabp indicates intra-aortic balloon counterpulsation; iqr, interquartile range; los, length of stay; lvef, left ventricular ejection fraction; mi, myocardial infarction; physician- hems, physician-staffed helicopter emergency medical service; standard-hems, non- physician-staffed helicopter emergency medical service. *continuous variables are expressed as median�iqr. table . predictors of any adverse in-hospital outcomes of st-elevation patients transferred by helicopter emergency medicine services* or % ci p value age . . to . . sex . . to . . killip class . . to . < . ground time . . to . . flight time . . to . . non-physician-staffed hems . . to . . hems indicates non-physician-comprised helicopter emergency medicine flight crews; or, odds ratio. *any adverse in-hospital outcome was defined as having any of the following: death, stroke, cardiac arrest, need for intra-aortic balloon pump, bleeding, cardiogenic shock, new requirement for dialysis, recurrent myocardial infarction, serious arrhythmia, or vascular complication. doi: . /jaha. . journal of the american heart association physician vs non-physician stemi transport outcomes gunnarsson et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , explained by a delay from first ecg to hems arrival. second, the standard-hems group was more likely to receive intra- venous nitroglycerin and opioid analgesics, such as morphine and fentanyl. this finding could suggest that flight paramedics and nurses are more likely to give these medications routinely compared with flight physicians. interestingly, in this study, almost % of the patients who received nitroglycerin had rca as the culprit vessel. previous studies have shown that nitroglycerin can cause hypotension and worse outcomes, particularly in the setting of right ventricular infarction. , in a recent study, stemi patients who were given morphine demonstrated decreased response to antiplatelet agents, possibly attributed to delayed gastric transit and impaired absorption. this effect could increase risk of thrombosis and recurrent mi. the aha/acc stemi guidelines recommend morphine for chest pain and pulmonary edema, but this medication has been linked to increased mortality. third, the standard-hems group was more likely receive intravenous fluid bolus, which could lead to pulmonary edema in patients who already have a propensity for high left ventricular filling pressure. baseline demographics and cardiovascular disease risk factors were similar in the groups and thus are unlikely to explain the difference in outcomes. also, baseline killip class— a strong predictor of adverse outcomes —was not signifi- cantly different between the groups. the same was also true when we compared the demographics and patient manage- ment transferred to university of wisconsin without a physician (n= ) and the mayo la crosse group (n= ). the physician- hems group had a higher rate of lad occlusion than the standard-hems group. we think that this difference is also unlikely to have affected our results because lad occlusion has been shown to be associated with worse prognosis, which is contrary to our findings given that the physician-hems group had lower rates of in-hospital adverse outcomes. our study has several limitations that are worth mention- ing. first, this is a retrospective analysis and is potentially subject to documentation and recall bias. second, the physician-hems group was larger than the standard-hems group. this is because the majority (> %) of stemi patients transferred by helicopter to the university of wisconsin is by physician-staffed hems. third, the overall number of patients in our study is modest, but still larger than similar studies on helicopter transport of stemi patients. however, we had enough clinical events to construct robust models for multivariable adjustment. the patient cohorts received treat- ment by different care teams at two hospitals, which is a potential confounder despite similar baseline characteristics. also, the indications for medications (such as nitroglycerin and analgesics) given during transfer were unavailable, and thus we could not assess appropriateness of medication administration. furthermore, we were unable to compare troponin con- centrations (to estimate infarct size) in the groups because university of wisconsin uses troponin-t, but mayo clinic uses troponin-i. however, we had information on postprocedure left ventricular ejection fraction (lvef), and this was not different between the groups. because of study design, we could not report compliance with medications such as anti- thrombotics or beta-blockers before transfer. however, the vast majority of the patients received recommended medical therapy during their acute hospitalization for stemi. last, a more-detailed analysis of the hems crews (eg, physician/ registered nurse [rn] vs rn/paramedic or paramedic/ paramedic) may have given additional insight into crew composition and its association with outcomes. in conclusion, our findings suggest a higher rate of in-hospital adverse outcomes in stemi patients transferred by non-physician-staffed hems. however, there was no difference in the rate of adverse events during transport. the higher risk of in-hospital adverse outcomes could be related to medications such as nitroglycerin or intravenous analgesics in-flight or because of other patient- or transport- related unmeasured confounders. the data are retrospective and thus only hypothesis generating. however, the results could have important clinical implications if confirmed in prospective or randomized trials. one potential implication is a novel recommendation for physician-staffed hems in stemi transfer systems. a cost-benefit analysis would be helpful before such implementation because of increased cost related to the addition of a physician to the flight crew. finally, our results underscore the importance of judicious use of nitroglycerin and intravenous analgesics for stemi patients. further investigation of administration of these medications during transport is needed. sources of funding this project was supported, in part, by the university of wisconsin institute for clinical and translational research (uw ictr), funded through an nih clinical and translational science award (ctsa), grant number ul tr , ncats, and 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warriors’ (vol. i, p. ), ‘akin to young olympian athletes’ who ‘thank their gods for such worthy opponents, and engage in the communicative-strategic agon anew’ each day (vol. ii, p. ). tully’s studies are of those special actors capable of playing the political game otherwise, of exploiting the gaps in the prevailing system of rules in order to amend them. this is not a kind of freedom that will suit everybody’s interests equally well. if tully’s books claimed to supply nothing more than an anthropological investigation of the resistance of domination by certain political actors then this need not be a problem. however, he is unequivocal that these studies form the basis of a normative ‘public philosophy’ which is ‘oriented to freedom before justice’, so that ‘the multiplicity of practices of governance in which we act together do not become closed structures of domination under settled forms of justice but are always open to practices of freedom’ (vol. i, p. ). egalitarians and rawlsians are entitled to ask how we are to prevent political freedom simply trumping their ambitions for instituting schemes for the fair allocation of material resources, designed to take account of the essentially arbitrary native endowments of physical and intellectual capacities across society. tully’s frequently invoked paean to ‘free and equal’ citizens (vol. i, pp. , , – , inter alia) ends up being little more than a slogan given his lack of attention to the latter term. for that, he would have had to give greater consideration to the category of justice, something he rules out on the basis of his critique of habermas. ben holland lecturer in international relations, university of nottingham, uk the liberal conscience: politics and principle in a world of religious pluralism lucas swaine columbia university press, new york, , pp., isbn: - contemporary political theory ( ) , – . doi: . /cpt. . it is a common belief that the gap dividing liberals and theocrats is unbridgeable. liberals stand for religious toleration, freedom of association reviews r macmillan publishers ltd. - contemporary political theory vol. , , – and fundamental individual rights; theocrats are deemed to be partisan and intolerant, to hold a totalizing view of society and to disregard the rights and liberties protected by liberal institutions. liberals are also in favor of a secular polity with no mingling between religion and politics. theocrats, on the contrary, call for an overcoming of the separation between these two realms so as to allow political institutions to be imbued with religious values. above all, liberals pride themselves on having realized the ideal of public reason in the modern world, whereas theocrats are contemptuously dismissed as unreason- able zealots from whom no sense can be expected. in this book swaine sets out to challenge the view that the divide between liberalism and theocracy is philosophically intractable. he argues that a solution of the conflict is possible, though only partially, and that failure to address the problems posed by theocracy will eventually result in a legitimation crisis of liberal democracy, with the risk that extremist groups feeling disenfranchised from secular government might resort to violence to assert their religious practices and ways of life. but there are moral reasons as well as prudential ones to seek to assuage the resentful alienation of theocrats. liberal government lacks in fact a theoretically consistent normative blueprint for dealing with them. it systematically fails to produce adequate explanations for its interference in the lifestyles of theocratic communities, and this is a moral shortcoming of liberalism itself. swaine meets the challenge of theocrats first of all by recognizing the inherent rationality of their otherworldly values, thus paving the way for religious reasons to be admitted into public debate. theocrats, he contends, can be persuaded to endorse the institutions of liberal democracy only if the attempt is made by liberals to reach them ‘with arguments that speak to their deeply held religious values’ (p. ). so, one of the dilemmas facing the theocrats concerns the right path to salvation. to the extent that theocrats demand strict institutions to foist a religious way of life upon the members of their communities, they implicitly acknowledge that people can lead wayward lives. a theocratic polity based upon draconian regulations becomes therefore necessary to bring into line those who take deficient paths. but in a pluralist society the theocrat has no guarantee that he holds the right religious doctrine nor is his community ever provided with the assurance that its pursuit of the good is not being hijacked by corrupted authorities. thus, to avoid spiritual disaster, swaine argues that the theocrat is rationally committed to three cardinal principles of liberty of conscience stating that conscience ought to be free (a) to reject lesser religious doctrines; (b) to embrace the good; and (c) to distinguish between true and false conceptions of the good. liberty of conscience is just one of the arguments that might appeal to theocrats and lead them to affirm liberal institutions. in order to guarantee theocrats religious free exercise, swaine also recommends that theocratic reviews r macmillan publishers ltd. - contemporary political theory vol. , , – communities be recognized as a semisovereign status within the overarching legal structure of liberal democracy. a regime of quasi-sovereignty goes a long way to prevent theocratic communities from being entirely absorbed within the laws of a liberal polity, but falls short of full sovereignty to ensure that their subjects are not treated in illiberal ways. under swaine’s legal framework, quasi-sovereign theocratic communities are obliged to respect a minimal set of basic rights for their members. they are required to provide food, shelter, clothing and education to all of them, with no exception, to abstain from corporal punishment, to practice policies that are not discriminatory towards women and to grant freedom of exit. it is precisely at this juncture in the book, where actual policies are laid down to flesh out the content of a semisovereign regime of theocratic autonomy, that swaine’s proposals betray their liberal imperialist bias. swaine’s attempt to establish limits on the claims of theocracy that would respect individual autonomy without violating the moral and religious fabric of the theocratic communities is certainly an admirable endeavor. but in order for us to assess the normative conditions he sets out for the resolution of the theocratic dilemmas, theocracy must be graspable from a non-religious place that happens to fall within the purview of the liberal subject. here the language of rights of liberalism looms in the horizon as a kantian transcendental a priori framework against which the claims of theocracy are to be negotiated. as a result, religion is privatized and ideologically depoliticized. besides, far from being constitutive of the identity of the theocratic communities the book wishes to protect, religion is degraded to the status of a legal concession, which, at best, one has the right to reclaim when its exercise is impaired. swaine’s solution may work for what he dubs ‘retiring theocrats’, that is for those theocratic communities, such as the amish or the mormons, ensconced within liberal democracy that have already largely embraced its values, but is utterly inadequate to deal with more ambitious and refractory theocrats, such as ‘the muslim other’, for whom he himself in his understated imperial insolence does not rule out war on terror as a more appropriate measure. giuseppe tassone faculty of arts and social sciences, university of balamand, deir el-balamand, el-khoura, lebanon reviews r macmillan publishers ltd. - contemporary political theory vol. , , – 	the liberal conscience: politics and principle in a world of religious pluralism, [pdf] heritability of blood pressure responses to cold pressor test in a chinese population. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /ajh. . corpus id: heritability of blood pressure responses to cold pressor test in a chinese population. @article{mei heritabilityob, title={heritability of blood pressure responses to cold pressor test in a chinese population.}, author={h. mei and d. gu and t. rice and j. hixson and j. chen and c. jaquish and q. zhao and c. chen and ji-chun chen and c. gu and t. kelly and j. he}, journal={american journal of hypertension}, year={ }, volume={ }, pages={ - } } 	h. mei, d. gu, + authors j. he 	published 	medicine 	american journal of hypertension background genetic determinants of blood pressure (bp) responses to the cold pressor test (cpt), a phenotype associated with risk of hypertension and cardiovascular disease has not been well studied. methods we examined the heritability of bp response to cpt in , subjects from families in rural north china. bp was measured before and at , , , and min after the participants immersed their hand in ice water for min. heritabilities of baseline bp and responses at min, maximum… expand view on pubmed academic.oup.com save to library create alert cite launch research feed share this paper citationsbackground citations view all figures, tables, and topics from this paper 	 figure 	 table 	cold pressor test 	cardiovascular diseases 	hypertensive disease 	diastolic blood pressure 	diastole 	bivariate normal distribution 	area under curve 	sample variance citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency blood pressure reactivity to the cold pressor test predicts hypertension among chinese adults: the gensalt study. 	q. zhao, dongfeng gu, + authors j. he 	medicine 	american journal of hypertension 	 	 	pdf save alert research feed polymorphisms of ace are associated with blood pressure response to cold pressor test: the gensalt study. 	j. huang, shufeng chen, + authors d. gu 	medicine 	american journal of hypertension 	 	 	pdf save alert research feed genetic correlation of blood pressure responses to dietary sodium and potassium intervention and cold pressor test in chinese population 	h. mei, t. rice, + authors j. he 	medicine 	journal of human hypertension 	 	 	pdf save alert research feed blood pressure responses to dietary sodium and potassium interventions and the cold pressor test: the gensalt replication study in rural north china. 	q. zhao, d. gu, + authors j. he 	medicine 	american journal of hypertension 	 	 	pdf save alert research feed genome-wide linkage and regional association study of blood pressure response to the cold pressor test in han chinese: the genetic epidemiology network of salt sensitivity study 	x. yang, d. gu, + authors t. kelly 	medicine, biology 	circulation. cardiovascular genetics 	 	 	 save alert research feed individual sensitivity of cold pressor, environmental meteorological factors associated with blood pressure and its fluctuation 	yaqin ni, q. miao, ruizhi zheng, ying miao, x. zhang, y. zhu 	medicine 	international journal of biometeorology 	 	view excerpts, cites background save alert research feed association between genetic variants of the add and gnb genes and blood pressure response to the cold pressor test in a chinese han population: the gensalt study 	l. wang, shufeng chen, + authors d. gu 	biology, medicine 	hypertension research 	 	 	pdf save alert research feed genome-wide association study identifies novel loci associated with blood pressure responses to interventions in han chinese 	j. he, t. kelly, + authors d. gu 	biology, medicine 	circulation. cardiovascular genetics 	 	 	 	view excerpt, cites background save alert research feed family history of hypertension is associated with anthropometric and nitric oxide bioavailability alterations in adolescents 	t. astorino, julien s baker, + authors m. moraes 	 	pdf 	view excerpt, cites background save alert research feed association of angiotensin-converting enzyme gene polymorphism and enzymatic activity with essential hypertension in different gender 	q. zhang, mingyu cong, + authors j. li 	medicine 	medicine 	 	 	 	view excerpt, cites background save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency quantitative genetic analysis of blood pressure response during the cold pressor test. 	a. choh, s. czerwinski, + authors r. siervogel 	medicine 	american journal of hypertension 	 	 	pdf save alert research feed genetic influences on blood pressure response to the cold pressor test: results from the heredity and phenotype intervention heart study 	marie-hélène roy-gagnon, m. weir, + authors j. douglas 	medicine 	journal of hypertension 	 	 	 save alert research feed genetic influences on blood pressure with the cold-pressor test: a twin study 	a. busjahn, h. faulhaber, r. viken, r. rose, f. luft 	medicine 	journal of hypertension 	 	 save alert research feed cardiovascular reactivity to the cold pressor test as a predictor of hypertension 	marilyn s. menkesf, k. matthews, + authors t. pearson 	medicine 	hypertension 	 	 	pdf save alert research feed relation between cold pressor test and development of hypertension based on -year follow-up. 	f. kasagi, m. akahoshi, k. shimaoka 	medicine 	hypertension 	 	 save alert research feed the heritability of blood pressure: an investigation of pairs of twins using the cold pressor test. 	m. l. mcilhany, j. shaffer, e. a. hines 	medicine 	the johns hopkins medical journal 	 	 save alert research feed the heritability of blood pressure: an investigation of pairs of twins using the cold pressor test. 	mcilhany ml, shaffer jw, hines ea 	medicine 	 	 save alert research feed cold pressor test as a predictor of hypertension 	t. pramanik, p. regmi, p. adhikari, p. roychowdhury 	medicine 	 	 save alert research feed cardiovascular risk factors in a french-canadian population: resolution of genetic and familial environmental effects on blood pressure by using extensive information on environmental correlates. 	l. pérusse, t. rice, c. bouchard, g. vogler, d. rao 	geography, medicine 	american journal of human genetics 	 	 save alert research feed maximum-likelihood generalized heritability estimate for blood pressure in nigerian families. 	c. rotimi, r. cooper, + authors r. ward 	medicine 	hypertension 	 	 	 save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue jbmr .. journal of bone and mineral research volume , number , published online on october , ; doi: . /jbmr. � american society for bone and mineral research reduced cox- expression in aged mice is associated with impaired fracture healing amish a naik, chao xie, michael j zuscik, paul kingsley, edward m schwarz, hani awad, robert guldberg, hicham drissi, j edward puzas, brendan boyce, xinping zhang, and regis j o’keefe abstract: the cellular and molecular events responsible for reduced fracture healing with aging are unknown. cyclooxygenase (cox- ), the inducible regulator of prostaglandin e (pge ) synthesis, is critical for normal bone repair. a femoral fracture repair model was used in mice at either – or – wk of age, and healing was evaluated by imaging, histology, and gene expression studies. aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed re- modeling, and altered expression of genes involved in repair and remodeling. cox- expression in young mice peaked at days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. in situ hybridization and immunohistochemistry showed that cox- is expressed primarily in early cartilage precursors that co-express col- . cox- expression was reduced by % and % in fractures from aged mice compared with young mice on days and , respectively. local administration of an ep agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of cox- during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. the findings suggest that cox- /ep agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging. j bone miner res ; : – . published online on october , ; doi: . /jbmr. key words: fracture, aging, cyclooxygenases, prostaglandin e , endochondral ossification introduction although increased age has been a known risk factorfor a decreased rate of fracture healing for > yr, little progress has been made toward understanding the mechanisms involved.( – ) the rate of bone repair is pro- gressively reduced with aging from the pediatric popula- tion to the elderly.( , ) delayed healing results in an in- creased duration of immobilization, increases the risk of joint stiffness, and is associated with increased morbidity. ( ) delayed healing also increases the risk of inadequate frac- ture alignment.( ) however, the most significant clinical problem is the development of nonunion. several studies have established that aging results in an overall reduction in union in numerous fractures.( – ) this population not only has an increased risk of fracture caused by reduced bmd but also has reduced fracture healing potential. thus, for any given fracture, the morbidity is greater for the aging population. several studies have evaluated gene expression during fracture repair and have established differences in the rate of fracture healing and the pattern of gene expression be- tween young and aged animals. stabilized -wk-old and - yr-old rat femur fractures had gene expression examined over a -wk period. whereas fracture union was estab- lished in young rats wk after fracture, union was consis- tently absent in aged rats after wk.( ) fractures in aged rats had reduced expression of indian hedgehog (ihh) and bmp- . ( ) other studies have established that fractures in aged mice have delayed expression of bone and cartilage matrix genes, such as col , aggrecan, and osteocalcin.( ) a recent study of nonstabilized fractures in mice at wk, mo, and mo of age showed decreased fracture callus volume, delayed maturation of the cartilage callus, and reduced expression of col and colx in aged animals. ( ) the cyclooxygenases have an important function in fracture repair with evidence suggesting a critical role during all stages of healing.( – ) cox- is transiently expressed in a number of tissues during periods of inflam- mation.( , ) cyclooxygenase- (cox- ) and - (cox- ) catalyze the rate-limiting step in the conversion to arach- idonic acid to prostaglandin h and ultimately to the pro- duction of prostaglandin e (pge ). pge binds to four receptor subtypes (ep –ep ). the ep receptor (ep ) and ep receptor (ep ) are gs-protein–coupled receptors that are expressed on mesenchymal stem cells, chondrocytes, the center for musculoskeletal research, university of rochester, rochester, new york, usa; department of pediatrics, uni- versity of rochester, rochester, new york, usa; institute for bioengineering and bioscience, georgia institute of technology, atlanta, georgia, usa; department of orthopaedics, university of connecticut school of medicine, storrs, connecticut, usa. the authors state that they have no conflicts of interest. and osteoblasts. ( – ) activation of ep and ep stimu- lates the production of camp and activation of protein kinase a (pka) signaling.( ) prior work from our labo- ratory has shown that stem cells along the periosteum are essential for bone repair,( , ) whereas others have estab- lished that ep -mediated signaling is critical for periosteal bone formation. delivery of pge using an alzet pump along the periosteal surface resulted in abundant periosteal bone formation in wildtype, ep / , ep / , and ep / mice. in contrast, ep / mice had no bone formation.( ) additionally, pge failed to stimulate the production of bone nodules in cultures of bone marrow–derived mesen- chymal stem cells (mscs) from ep -deficient mice.( ) the development of agonists that target specific ep receptors raises the possibility of targeted therapies to potentially accelerate bone repair. previously, we showed that deficiency of cox- in a murine model results in a delay in fracture healing.( ) a stabilized mouse femur fracture model was used to deter- mine whether different levels of cox- expression could account for the reduced rate of fracture healing observed with aging. our findings established reduced cox- ex- pression in fractures in aged mice. we further showed that the delayed fracture healing observed in aged mice could be rescued with local delivery of an ep selective agonist. the findings establish a role for cox- expression in the delayed fracture healing observed with aging. materials and methods experimental animals all animal studies were done in accordance and with approval of the university committee on animal re- sources. fifty-two-week-old female c bl/ j mice were obtained from the national institutes on aging (bethesda, md, usa). c bl/ j, - to -wk female mice were pro- cured from jackson laboratories (bar harbor, me, usa). stock aged animals were rederived from jackson labora- tory breeders in , , and . this routine practice by the nia was done to eliminate genetic drift that could have occurred between the two populations of mice. femur fracture model mice received anesthesia using ketamine and xylazine. the skin and underlying soft tissues over the left knee was incised lateral to the patellar tendon. the tendon was dis- placed medially, and a small hole was drilled into the distal femur using a -gauge needle. a stylus pin from a g quincke type spinal needle (bd medical systems, franklin lakes, nj, usa) was inserted into the intra- medullary canal and clipped. the wound was sutured closed. fractures were created using a three-point bending einhorn device as previously described.( ) mice were given six subcutaneous injections of mg/kg buprenor- phine (abbot laboratories, abbott park, il, usa) every h to control pain. a faxitron system (faxitron x-ray, wheeling, il, usa) was used to take x-ray images at the time of surgery, -week intervals, and at the time of death. histology and analysis mice were killed at , , , , , , , , , or days after fracture. a normal mid-diaphysis femoral bone seg- ment was used as a nonfractured day control. femurs were disarticulated from the hip and trimmed to remove excess muscle and skin. specimens were stored in % neutral buffered formalin for days. the tissues were infiltrated and embedded in paraffin. alcian blue and orange g along with tracp staining was done as previously described. ( , , ) histomorphometric analysis (n = animals per group) was done using a standardized eyepiece grid to measure tissue areas within the fracture callus. samples were cut at four levels spanning ; mm through the callus, with mm between each level. each cross-hatch was categorized as not callus (not counted), callus (quantified), and a specific tissue type. a total area of the external callus and areas of individual tissue types such as new bone (mineralized tis- sue), total cartilage, immature proliferative cartilage, hy- pertrophic cartilage, and mesenchyme were quantified. bone was defined as areas of new woven bone. cartilage was defined as tissues staining blue for proteoglycan. hy- pertrophic cartilage was clearly defined by cellular mor- phology, and other nonhypertrophic areas of cartilage were considered immature cartilage. finally, mesenchyme was defined as areas containing spindle-shaped fibroblasts with- out alcian blue staining. cortical bone was excluded from the histomorphometric analysis. the target tissue area was divided over the area of the external callus. in the rescue experiment, aged mice were treated with either vehicle or a nonprostanoid ep selective agonist, cp (pfizer, groton, ct, usa). cp (cp ) was freshly prepared in normal saline containing % ethanol. cp ( ml) was injected at the fracture site twice daily for a total daily dose of mg/kg/d. quantitative real-time pcr the fracture callus and mm of normal bone margin was carefully excised using a scalpel. these samples were frozen in liquid n , pulverized using a nitrogen-cooled mortar and pestle apparatus (bel-art, scienceware, pequannock, nj, usa), and purified for total rna using the trizol system (invitrogen, carlsbad, ca, usa).( ) the concentration of stock rna was determined using a spectrophotometer. cdna was synthesized from . mg of rna per callus using a commercial first-strand cdna synthesis kit (invitrogen, carlsbad, ca, usa). cdna from n = mice from each group was pooled. rt-pcr analyses were performed using murine specific primers for col a , colx, osteocalcin, bmp- and - , cox- , rankl, and opg expression (see table for specific sequences). the col a and colx mrnas encode for the collagen matrix proteins type ii collagen and type x collagen, respectively. col a is maximally expressed during chondrocyte proliferation and colx peaks during terminal differentiation of chondrocytes. osteocalcin is ex- pressed in fully mature osteoblasts. bmp- and bmp- are genes that encode for two subtypes of the bone morphoge- netic proteins that are involved chondrogenesis and bone formation.( ) rankl stimulates osteoclast maturation and bone remodeling, an important phase of fracture repair.( ) naik et al. finally, opg mrna encodes for osteoprotegerin a se- creted decoy receptor for rankl, which serves to regulate osteoclastogenesis.( ) qpcr reaction was performed us- ing sybergreen (abgene, rochester, ny, usa) in a rotorgene real-time pcr machine (corbett research, carlsbad, ca, usa). all genes were compared with a standard b-actin control. data were assessed quantitatively using two-way analysis of covariance comparing relative levels of transcript expression as a function of time and age. immunohistochemistry immunohistochemistry was done as previously de- scribed. ( ) sections were deparaffinized using xylene and rehydrated using graded alcohols. the endogenous per- oxidase was quenched using % hydrogen peroxide for min. nonspecific binding epitopes were blocked using : normal goat serum. slides were incubated at c overnight with a : dilution of mouse primary cox- antibody (cayman chemical, ann arbor, mi, usa). sections were rewarmed, rinsed in pbs, incubated with a : goat anti- rabbit secondary antibody, rinsed again in pbs, and finally incubated with a : horseradish peroxidase (hrp) streptavidin for min. sections were counterstained with hematoxylin and rinsed. the rankl goat anti-mouse antibody (santa cruz biotechnologies, santa cruz, ca, usa) incubation was done using a : dilution factor. the immunohistochemical data were qualitatively assessed. in situ hybridization femurs prepared for in situ hybridization were fixed in % paraformaldehyde by intracardiac injection and immersion at c for days. all samples were decalcified in % edta for days before sectioning. the mouse legs were dissected by disarticulating the femoral head from the hip and immediately immersed in fixative. excess muscle and pins were carefully removed. tissues were placed in paraffin and sectioned. in situ hybridization was performed using the mrnalocator kit (ambion, foster city, ca, usa). pro- teinase k digestion was done at c for min, and the hybridization reaction was carried out at c overnight. plasmids for the p-utp labeled riboprobes were provided by jill helms (col , colx, and osteocalcin)( ) and adam sapirstein (cox- ).( ) sections were observed using both light- and dark-field microscopy and assessed qualitatively. mct on death, femurs were disarticulated from the hip and fixed in % neutral buffered formalin (nbf). for vascular perfusion studies, mice were killed and given serial intracar- diac injections of heparinized saline, % nbf, and a lead chromate microfil perfusion reagent (flow tech, carver, ma, usa). the whole mice were soaked in % nbf overnight at c. the fractured limbs were disarticulated at the hip, and excess soft tissue was removed. calluses were further fixed in nbf for additional days, and decalcified in % edta for days. the samples were scanned twice, before and after decalcification, in a vivact scanner (scanco medical ag, bassersdorf, switzerland) at high resolution with a . -mm voxel size. an integration time of ms, a current of ma, and an energy setting of kv was used. the threshold was chosen using d evaluation of several slices in the transverse anatomic plane so that mineralized callus and vascular con- trast reagent were identified but surrounding soft tissue was excluded. an average threshold of was optimal and used uniformly for all samples. next, each sample was contoured around the external callus and along the edge of the cortical bone. all mineralized tissues above threshold between these two boundaries were included. thus, external soft tissues and cortical bone including the marrow cavity were excluded. contouring of images was done every axial slices proxi- mally to distally until the callus was not visible. because all the soft tissue was removed, it was possible to detect the entire callus volume by lowering the threshold. the volume of the mineralized tissue or vascular bed was divided by the volume of the whole external callus. we used an n = sample size for each group. data sets were examined using statistical assessments including anova. statistics statistical analysis was performed as previously de- scribed. ( ) the results were described as mean ± se. statis- tical significance was determined using two-way anova and student’s t-tests. these tests determined significance between young/old mice and the values at different time points. p < . was considered significant. data analysis was performed using graphpad prism version . (graphpad software, san diego, ca, usa). results fractures in aged mice have delayed radiographic healing, decreased bone formation, vascularization, and remodeling radiographs at various time points during fracture heal- ing were obtained (fig. a). calcified callus and evidence of table . list of oligonucleotide primer sequences for real-time pcr gene sequence b-actin -agatgtggatcagcaagcag- -gcgcaagttaggttttgtca- cox- -cacagcctaccaaaacagcca- -gctcagttgaacgccttttga- col a -actggtaagtggggcaagac- -ccacaccaaattcctgttca- colx -accccaaggacctaaaggaa- -ccccaggataccctgttttt- osteocalcin -cttggtgcacacctagcaga- -ctccctcatgtgttgtccct- bmp- -tggaagtggcccatttagag- -gcttttctcgtttgtggagc- bmp- -tgagcctttccagcaagttt- -cttcccggtctcaggtatca rankl -caccatcagctgaagatagt- -ccaagatctctaacatgacg- opg -agtccgtgaagcaggagtg- -ccatctggacattttttgcaaa- cox- expression is reduced in aged fractures bone union were observed in the fractures of young mice at days and , respectively, but were delayed in fractures from aged mice until and days after fracture (fig. a). the radiographic delay in fracture healing was con- sistently observed (fig. b; n = young and n = aged mice). remodeling also occurred earlier in young mice (fig. a). by days after fracture, clear evidence of re- modeling was observed, and radiographic remodeling was nearly complete by days. in fractures from aged mice, limited evidence of radiographic remodeling was observed days after fracture. mct was performed on mice harvested at , , and days after fracture. previously it has been shown that vas- cularization is a sensitive marker of the completion of en- dochondral bone formation and is important in the pro- gression of fracture repair.( – ) for this reason, the fractures were perfused with lead chromate microfil so that the vascularization within the callus could be determined in fractures in young and aged mice. mct showed delayed bone formation, fracture union, and vascularization in fractures from aged mice compared with fractures from young mice. similar to the data observed in radiographs, fracture healing was complete in young mice by days after fracture. in contrast, healing was delayed until days in aged fractures, and callus vascularization was delayed (figs. c and d). quantitative assessment of these pa- rameters showed that mineralization was increased in fractures in young mice compared with aged mice by . - and -fold at and days, respectively (fig. e). how- ever, mineralization was similar by days after fracture. similarly, vascularization was increased . - and . -fold in fractures from young compared with aged mice at and days but reached equivalent values by days after fracture (fig. f). these findings suggest a delay in the completion of endochondral ossification in aging fractures. fractures in aged mice have delayed chondrogenesis and completion of endochondral ossification, impaired bone remodeling, and altered gene expression histology sections of the fractures confirmed a delay in fracture healing and remodeling in aged mice (fig. ). whereas cartilage formation was observed in both young fig. . aged mice have a radiographic de- lay in fracture healing. serial radiographs of representative young and aged mice at vari- ous times after fracture. young mice showed evidence of callus mineralization by day , which was delayed to day in aged mice (a, *). by day in young mice and day in aged mice, the callus showed union (black arrows). remodeling was delayed in aged mice compared with young mice (a, white arrows). the delay in fracture repair was consistently observed in day fractures (n = ) (b). fractures were harvested, and high- resolution mct scans were performed on young (n = ) and aged (n = ) mice. for vascular analysis, mice were perfused with a lead chromate microfil contrast reagent be- fore decalcification. representative scans are shown for days , , and for calcified callus (c) and vascularization (d) for young and aged mice, respectively. mean calculated mineral (e) and vascular (f) volumes were obtained. mineral volume analysis showed lower mineral accretion in aged mice, par- ticularly on days and (c and e). on days and , aged mice have a significant reduction in the proportion of blood vessels in their calluses compared with young mice (d and f). statistical comparisons were per- formed using anova and are denoted by symbols: *p < . and **p < . . naik et al. and aged mice on day , cartilage undergoes more rapid maturation and endochondral bone formation is complete by day in young mice. in contrast, cartilage is present in aged fractures at days and areas of cartilage remains through days. histomorphometry was performed at each time point to quantify the amount of mesenchyme, cartilage, and bone present in the sections (figs. m– o). fractures in aged mice had reduced callus formation early and delayed chondrogenesis, bone formation, and remod- eling (figs. m– o). total bone area was significantly higher in the fractures of young mice on days , , and but was lower at days and after remodeling of the fracture. in contrast, aged animals continue to have a net increase in bone area until day (fig. m). in young animals, peak cartilage area is observed at day and is almost entirely replaced by bone by day . however, in aged animals, abundant cartilage persists at day . frac- tures in both young and aged mice have calluses almost entirely composed of bone by day (fig. n). delayed bone formation and chondrogenesis result in delayed peak total callus area and remodeling in aged animals (fig. o). histology showed more rapid remodeling in fractures in young mice (fig. ). by days, young mice have evidence of extensive remodeling, and at days, most of the initial woven bone within the callus has been remodeled. by days, the fractures in young mice have essentially under- gone complete remodeling and the femur is achieving a morphology similar to the nonfractured bone. these events are markedly delayed in the fracture callus from aged mice. extensive remodeling is not observed until days, and at days, fracture callus in aged mice remains enlarged and contains areas of woven bone (fig. ). expression of genes involved in matrix accumulation, bone formation, and remodeling is altered in fractures from aged mice total rna was harvested from fractures at various times between and days to examine molecular events underlying the delayed healing observed in aged mice. in the fractures from young mice, col a appeared earlier, and peak levels were observed by days – compared with maximal expression at day in aged mice (fig. a). similarly, maximal expression of the chondrocyte matu- ration marker, colx, occurred from days to in fractures from young mice, whereas aged mice had maximal ex- pression occurring at day . maximal col a and colx expressions were significantly higher in fractures from young mice (fig. b), consistent with the increase in car- tilage observed in young mice by histomorphometry. moreover, both col a and colx expressions disappear in young mice by day , signifying completion of the endochondral phase of fracture repair, whereas cartilage matrix gene expression persists until day in aged mice. fig. . (continued). cox- expression is reduced in aged fractures fig. is /c early and late peaks of osteocalcin expression were ob- served (fig. d). an early minor peak occurred at day when initial intramembranous ossification occurs along the periosteal bone surface. osteocalcin levels were slightly higher in fractures from aged mice at this early peak com- pared with those observed in young mice. however, by through days, osteocalcin levels were significantly in- creased in fractures from young mice. peak expressions were reached in both young ( – days) and aged mice ( days), and although higher in young mice, the difference was not statistically significant. at days, osteocalcin expres- sion was elevated in fractures from aged mice compared with fractures from young mice. altogether, the osteocalcin expression levels are consistent with a more robust early formation of bone in fractures from young mice, consistent with their earlier completion of endochondral ossification expressions of bmp- and bmp- were examined in the fracture calluses, and distinct temporal patterns of ex- pression were observed (figs. e and f). in fractures harvested from young mice bmp- , expression was ele- vated early during the endochondral phase of fracture re- pair, with peak expressions present between and days, with a subsequent decrease during the bone formation phase of repair. in contrast, bmp- expression was highest at days, corresponding to the peak of osteocalcin ex- pression and bone formation. bmp- and bmp- had an overlapping pattern of ex- pression in aged mice (figs. e and f). bmp- expression was maximal in aging fractures between and days compared with – days in young mice. similarly, bmp- expression was elevated between and days in contrast to the single peak of expression that occurred in young mice at days. this is consistent with less distinct cartilage and bone formation phases of fracture healing observed in the aged mice. finally, consistent with the diminished bone remodeling observed on radiographs and histological sections, the pattern of rankl and opg expressions was markedly altered in fractures from aged mice (figs. g and h). peak rankl expression occurs surprisingly early in the process of fracture repair and is observed at the onset of chondrogenesis. young mice had an abrupt peak in fig. . fractures in aged mice have delayed cartilage and bone formation. histology sec- tions were prepared from young and aged mice harvested at various times after fracture (a–l). by day , calluses were composed of abundant mature cartilage in young mice compared with largely immature cartilage in aged mice. by day , most callus tissue consisted of new bone in young animals, whereas aged animals showed a persistence of cartilage and delayed completion of en- dochondral ossification. on days , , and , young mice had more advanced remod- eling compared with aged animals. histo- morphometric analysis of total callus, bone, and cartilage areas were completed in n = young and n = aged mice, respectively, with three levels analyzed per sample. histomor- phometry showed delayed formation of car- tilage, bone and total callus area in aged mice (m–o). statistical comparisons were per- formed using anova and significant dif- ferences are denoted by symbols: *p < . and **p < . . naik et al. fig. is /c rankl expression at days with a gradual decline to basal levels by days, suggesting that the induction of osteoclastogenesis and remodeling is one of the early mo- lecular events to occur in fracture repair. a similar abrupt peak in opg expression was observed in fractures in young mice but occurred at day after fracture and thus immediately followed the induction in rankl. in con- trast, fractures in aged mice showed a less brisk but more prolonged elevation of rankl from days to after fracture. interestingly opg gene expression pattern mir- rored that observed with rankl, but with peak levels occurring between days and at days in fractures from aged mice. these findings show that rankl and opg expressions are temporally linked, with maximal rankl expression occurring well before the peak of bone re- modeling and immediately followed by an increase in the expression of the decoy receptor, opg. cox- is altered in fractures in aged mice cox- has previously been shown to be an important anabolic agent for fracture repair.( , , ) in normal bone harvested from the femoral shaft, basal levels of cox- expression was similar in young and aged bone tissue (supplemental fig. ). after fracture, cox- expression was induced early and preceded the onset of chondrogenesis (fig. h). in fractures from young mice, cox- was increased by days and had a sharp peak of maximal expression at days. cox- levels declined rapidly and returned toward baseline levels by – days. in fractures from aged mice, the magnitude cox- induction was markedly less and the peak ex- pression was less distinct so that a much lower level of maximal cox- expression was sustained between and days. fig. . fracture in aged mice have altered patterns of gene expression during the chondrogenesis, bone formation, and remodeling phases of repair. total rna was harvested and pooled from n = young and aged mice at various points. real-time rt-pcr was performed and normalized to b-actin expression. values from young mice are shown in the white bars and aged mice in the black bars. the bars at the top of each figure denote the period of time in which maximal expression of each gene occurred in the fractures from both young (white bar) and aged (black bar) mice. peak expression of col a , cox- , rankl, and opg occur in fractures in young mice during the chondrogenic phase of fracture healing between days and (a, f, g, and h). colx and bmp- are maximal at day during the peak of endochondral bone formation (b and d). finally, osteocalcin and bmp- gene expression (c and e) are maximal at day , consistent with the peak of primary bone formation on the cartilage template. in contrast, fractures have mice have an altered pattern of gene expressions. cox- gene expression was diminished on days , , and during the phase of chondrogenesis (h). other genes were delayed, had reduced or delayed maximal expression, or had a broader duration of expression. statistical comparisons were performed using anova and significance denoted by symbols: *p < . and **p < . . peak expression periods are denoted by the bars at the top of the figure. the maximal value measured in young and old mice for each gene is statistically different (p < . ; a, b, e, f, and h) in all cases except for panels in which ‘‘ns’’ appears with the bars (c, d, and g). cox- expression is reduced in aged fractures the finding that maximal cox- expression occurs at the onset of chondrogenesis suggests a connection with this process. in situ hybridization and immunohistochemistry were performed to determine whether cox- and col a were co-expressed in a chondroprogenitor population. fe- mur sections harvested from young mice between and days after fracture were hybridized to antisense probes specific for cox- and col a , and x-ray film based auto- radiography was performed (fig. a). the autoradiograph shows abundant expression of col a in fracture callus and in the distal femoral growth plate. maximal expression occurred between and days, but lower levels of ex- pression were observed at and days after fracture. cox- hybridization overlapped with areas of col a ex- pression (fig. a). however, cox- maximal expression occurred at the earlier times ( – days) when fracture callus is composed of a less differentiated chondroprogenitor cell population. to determine the cell populations expressing cox- , emulsion-based autoradiography was performed in day fractures (fig. b). both cox- and col a expression was co-localized in populations of chondroprogenitor cells that were transitioning from a flat mesenchymal cell morphol- ogy to a rounded chondrocyte in the process of becoming embedded within the matrix. thus, chondroprogenitors and early chondrocytes are the major source of cox- expression in early fracture repair. to confirm the findings and to examine the relative ex- pression of cox- in young and aged mice, immunohis- tochemical staining for cox- was performed on fractures from young and aged mice between and days (fig. ). in fractures from young mice, strong cox- staining was observed in mesenchymal cells and immature chondrocytes at and days after fracture. by days, cox- staining was minimal in cartilage, and at this point, localized pri- marily to the periosteum and osteoblast population. a different pattern of expression was observed in fractures from aged mice; cox- expression was relatively less at and days but persisted in cartilage through days, consistent with the altered gene expression pattern ob- served in fractures from aged mice. cox- and rankl are co-expressed in chondroprogenitor cells in fracture callus it has previously been established that pge induces the expression of rankl, a factor essential for the induction of osteoclasts.( ) immunohistochemistry was performed in -day fracture calluses from young mice to examine expression of rankl and cox- because both genes are highly expressed in fractures at that time (figs. g, h, and a). fracture sections stained with anti-rankl and anti- cox- show an essentially identical localization of these key signals. both proteins are localized in immature chondrocytes, osteoblasts, and vascular endothelium but not to hypertrophic chondrocytes. to evaluate whether the differences in rankl ex- pression in fracture calluses of young and aged mice was associated with altered osteoclast-mediated bone resorp- tion, tracp staining was performed on callus tissues be- tween and days and qualitatively assessed (fig. ). in young mice, tracp+ osteoclasts were evident by day along the periosteal surface. abundant remodeling of woven bone was apparent at day throughout the callus. in contrast, the appearance of tracp+ osteoclasts was delayed until days in fractures from aged mice and was not extensive in the callus tissue until days after fracture. local delivery of an ep r agonist (cp ) rescues the delayed fracture healing phenotype in aged mice the radiographic, histological, and molecular charac- terization of fractures in aged mice suggests that an early disruption in the progression of healing persists and results in a sustained delay of the entire reparative process. be- cause cox- is an important early anabolic gene and is markedly reduced in aged fractures, we examined whether the local delivery of the ep agonist cp could com- pensate for the delayed fracture repair that occurs in aged mice. cp ( mg/kg/injection) or vehicle was injected into the fractures of aged mice twice daily between and days after fracture and compared with fractures in young mice that received vehicle injections. tissues harvested after days showed healing in young mice with minimal remaining cartilage and abundant bone formation (fig. a). fractures in aged mice treated with cp had reduced cartilage and increased bone compared with vehicle-trea- ted control fractures (fig. a). histomorphometry showed that fractures in vehicle-treated young mice were com- posed of % bone, % cartilage consisting of % im- mature cartilage, and only % undifferentiated mesen- chyme. in contrast, fractures in vehicle-treated aged mice were composed of % bone, % cartilage ( % imma- ture), and % undifferentiated mesenchyme. addition of cp increased bone formation to % and reduced the composition of cartilage to % ( % immature) and % undifferentiated mesenchyme (fig. b; p < . ). consis- tent with data shown in fig. , day vehicle-treated fractures in aged mice have increased callus area compared with vehicle-treated young mice (fig. c). whereas callus area was similar in vehicle and cp -treated aged mice, cp significantly increased bone area, consistent with the more rapid completion of endochondral ossification (fig. d). the total bone area was similar to that observed in the vehicle-treated young mice. thus, cp accelerates the endochondral phase of bone repair in aged mice and results in a pattern of healing that mimics fracture healing in young mice. in -day fractures, untreated (fig. ) and vehicle-treated (fig. ) fracture calluses in young and aged mice have similar total callus and bone areas (figs. and ). cp treatment for days resulted in significantly enhanced callus size and bone formation in aged mice compared with the vehicle-treated young and aged mice with fractures. these findings suggest that, in the later phases of fracture repair, cp may have additional effects on the osteoblast population that results in increased total bone formation and callus area. thus, in addition to its early effects of accelerating callus maturation, cp also results in in- creased in callus size and bone formation at later times. naik et al. fig. . cox- mrna is expressed in chon- droprogenitor cells in healing fractures. in situ hybridization was performed on fractures obtained from young mice between and days after fracture using murine specific sense and antisense riboprobes for cox- and col a . detection was with kodak biomax mr x-ray film. cox- expression occurs in regions where early chondrocyte precursors express col a . cox- expression subse- quently declines as chondrocytes mature (figure a). microscopic expression was de- termined in histological sections of day fracture callus and confirms the co-expression of cox- and col a in early chondro- progenitor cell populations (figure b). s- d represents use of the sense probe on day tissue sections, which acts as a negative control. fig. . cox- protein localizes to chon- droprogenitors and the early chondrocyte population. immunohistochemical staining for cox- was performed on fracture cal- luses from young (left; y) and aged mice (right; a) between and days. consistent with mrna data, cox- protein was most abundantly expressed in chondroprogenitors. in young mice at day , the cox- signal almost completely disappears from cartilage and is limited to the periosteum and osteo- blasts. aged mice have fewer cartilage cells staining positive on days and but have persistent expression in cartilage through days and . cox- expression is reduced in aged fractures figs. , is /c discussion during fracture healing, there is a sequential series of highly linked events that include mesenchymal prolifera- tion, chondrogenesis, chondrocyte maturation and termi- nal differentiation, vascularization, primary bone forma- tion, and remodeling. ( ) because these events are tightly linked, cellular and molecular alterations that occur early during the healing response may result in an alteration in the timing of subsequent events. thus, the early cell, tissue, and molecular events that occur immediately after bone injury have particular importance. the phenotype of fracture repair in aged mice is con- sistent with altered early gene expression. compared with young mice, aged mice have delayed formation of callus and chondrogenesis, suggesting a decrease in the rate of proliferation and differentiation of mesenchymal chon- droprogenitors. maximal expression of col a occurred later, and gene expression persisted for a longer duration. colx, a marker of mature chondrocytes, ( , ) was maximal at – days in fractures from young mice and at day in fractures at aged mice. furthermore, fractures in aged mice had reduced peak levels of colx expression and expression persisted through days, showing failure to efficiently complete endochondral bone formation. mature, calcified cartilage acts as a template for bone formation and re- modeling. consistent with the delay and prolongation of the cartilage phase of fracture repair, osteocalcin gene peak expression was reduced and expression also persisted in aged callus. finally, vascularization and fracture union were delayed and the rate of remodeling was reduced in aged fractures. these findings are consistent with previous descriptions of delayed fracture healing in older mice and rats.( , – ) cox- has been shown to be a critical regulator of bone repair in multiple animal models.( , , – ) cox- ex- pression is rapidly induced in fractures, consistent with our observation of expression in day calluses, with peak ex- pression by days in young mice. cox- –deficient mice have a fracture healing phenotype that is similar to aged mice, including a slower rate of chondrogenesis, chondro- cyte maturation, delayed vascularization, and reduced bone formation. in cox- / mouse fractures, there is persistence of undifferentiated mesenchyme, suggesting that cox- is necessary for normal bone and cartilage differentiation in healing tissues.( ) in vitro studies have confirmed that cox- and its me- tabolite, pge , enhance the differentiation of mscs into both cartilage and bone.( , , , ) addition of pge to limb bud mscs in high-density cultures enhances chondrogene- sis. ( ) this has been observed in primary avian and murine cultures and mesenchymal cell lines and involves signaling through pka.( , , ) similarly, pge has been shown to stimulate osteoblast differentiation. ( , ) bone marrow msc cultures isolated from cox- / mice have reduced osteoblast differentiation. however, this can be compen- sated by the addition of pge to the cultures, suggesting that pge is a key metabolite in osteoblast differentiation. ( ) whereas a role for cox- in bone repair has been extensively described, the cell populations responsible for cox- expression in fracture callus have not been defined. in situ hybridization experiments showed co- localization of cox- and col a in the early chondropro- genitor population. morphologically, these were flattened fig. . fractures in aged mice have delayed osteoclast formation. high-power images, and , show immunostaining using anti-cox- and anti-rankl antibodies in chondroprogenitors and immature chondro- cytes at days after fracture. endothelial cells (arrow) also stained for cox- and rankl (a). tissues were harvested from young and aged mice at , , , or days after frac- ture, and sections were stained for tracp (b–i). the sections from young mice show tracp+ osteoclasts by day along the per- iosteal surface (b). osteoclasts increase through day when the entire callus is in- terspersed with tracp + osteoclasts (f). by day , remodeling is advanced, and the number of osteoclasts is reduced (h). in contrast, osteoclasts do not appear until day in fractures from aged mice, and resorp- tion area appears maximal at days (c, e, g, and i). naik et al. fig. is /c fibroblastic-appearing mesenchymal progenitors in the process of becoming embedded within a chondroid matrix. cox- expression was also present in immature chon- drocytes but was absent in cells with a hypertrophic phe- notype. interestingly, cox- expression is absent in the growth plate, consistent with lack of a developmental phenotype in cox- / mice. thus, the expression of cox- seems to be unique to reparative cartilage. cox- expression was also present in the osteoblast population, although gene expression studies suggest that the highest levels of expression were associated with the initial carti- lage phases of endochondral bone repair. whereas prior work has established that osteoblasts express cox- , these are the first experiments to show that cartilage is a major source of cox- during fracture repair. cox- expression was reduced in the fracture callus of aged mice, suggesting that the delayed healing in these mice may be caused by a functional decrease in this enzyme. pge- is the major metabolite of cox- in most tissues and activates one of four receptors, ep , ep , ep , and ep , that collectively are associated with the protein kinase c (pkc) and pka signaling pathways. numerous genes have been shown to be activated by pge , including bmp- and rankl, two critical factors in bone repair.( , – ) in young fractures, bmp- and rankl both have peak expression during the early cartilage period of fracture repair and are expressed in phase with cox- . ep and ep agonists have been shown to enhance bone formation in several bone repair models and may have anabolic ef- fects when combined with bmp- therapy.( , , – ) in an ectopic bone formation model, bmp- and selective ep agonists had a synergistic effect.( ) in rat growth plate chondrocytes, combined activation of ep and ep re- ceptors resulted in enhanced proliferation and induction of col a expression.( ) the observation that both anabolic and remodeling genes have peak expressions during the early chondrogenic period of fracture repair further sup- ports the notion that initial cellular and molecular events drive the overall repair process. aged mice have reduced and temporally prolonged ex- pressions of both bmp- and rankl. bmp- is regulated by cox- and pge through ep . ( ) undifferentiated fig. . administration of an ep agonist to fractures in aged mice accelerates fracture repair and increase bone formation. fractures in young mice were injected with vehicle ( ml twice per day), whereas fractures in aged mice received injection of either vehicle or a selective ep agonist (cp ; mg/kg/d). mice (n = per group) were harvested and days after fracture, and tissues were pre- pared for histology. representative sections from day are shown in a. histomor- phometry was used to measure the relative populations of undifferentiated mesenchyme, immature cartilage, hypertrophic cartilage, total cartilage, and new bone at day (b). total callus and bone area was measured on days and (mm ) (c and d). cp ac- celerated the completion of endochondral bone formation and resulted in reduced car- tilage and increased bone formation com- pared with the vehicle-treated aged fractures. the anabolic effects of cp on bone for- mation persisted to day in aged animals. these changes compensated for the age-re- lated effects on fracture healing (b–d). sta- tistical comparisons were performed using anova, and significance is denoted by symbols: *p < . and **p < . . cox- expression is reduced in aged fractures fig. is /c human mscs constitutively express more cox- , pge , and bmp- than mature osteoblasts.( ) when treated with selective cox- and ep inhibitors, the induction of bmp- in these cells was suppressed. ( ) similarly rankl is also regulated by pge . ( , , ) osteoblasts, stromal cells, and fibroblasts all express increased rankl after treatment with pge . ( , , , ) this effect is primarily caused by activation of the ep receptor, which activates the pka signaling pathway. gain of ep function stimulated rankl expression, whereas loss of function prevents rankl expression in pge -treated cells. ( ) rankl has been shown to be expressed in chondrocytes, and our laboratory recently has established that bmp- signaling is a potent inducer of rankl expression in chondro- cytes.( ) however, direct induction of rankl by pge has not been examined in a chondrocyte population. al- together, regulation of bmp- and rankl by cox- / pge is consistent with an early gene that regulates critical subsequent steps including cell differentiation and subse- quent remodeling. the likely source of the mesenchymal precursor popu- lation is the periosteum. prior work from our laboratory using a murine bone/periosteal cell transplant model clearly established that periosteal cells undergo prolifera- tion and subsequent chondrogenesis in response to in- jury.( ) in a murine model in which pge was delivered to the periosteal surface through an alzet pump, periosteal bone formation occurred in wildtype and ep , ep , and ep knockout mice, but not in ep -deficient mice.( ) this suggests that the periosteal stem cell population is partic- ularly responsive to ep signaling. for this reason, we examined whether local delivery of an ep receptor ago- nist could compensate for the reduced rate of fracture re- pair observed in aged mice. local injection of an ep agonist to the fracture site of aged mice compensated for the reduced fracture repair observed with aging. day fractures were selected for detailed analysis because this is the time point in which fractures in young animals are completing the final stages of endochondral ossification and only the final vestiges of hypertrophic cartilage remain. in contrast, aged mice con- tinued to have abundant immature cartilage. fractures re- ceiving the ep agonist had a significant reduction in both immature and hypertrophic cartilage and more efficient completion of endochondral ossification. this effect was readily observed at day , when endochondral bone for- mation is being completed in young mice and fracture union is occurring. as a result, ep agonist–treated fractures in aged mice had increased bone formation and developed a histological phenotype that was similar to that observed in young mice. our gain of function findings are consistent with prior work showing that ep knockout mice have delayed endochondral bone formation and fracture healing. ( ) a second finding observed in aged mice with fractures treated with the ep agonist was that the total amount of fracture callus was increased in mice treated with cp for days. unlike the observations at days with cartilage maturation, the increase in callus area and bone formation occurred in comparison with both vehicle-treated young and aged mice with fractures. one possible explanation for these findings is that, in the later phases of fracture repair, cp may have additional effects on the osteoblast popu- lation that results in increased total bone formation and callus area. thus, once endochondral bone has formed, ep may have an additional role in the proliferation, re- cruitment, or retention of osteoblastic cell populations in- volved in fracture repair. prior work has established that ep stimulates osteoblast differentiation and matrix pro- duction.( , , , ) furthermore, ep knockout mice were observed to have reduced bone with aging and decreased osteoblastogenesis, consistent with an important role in bone formation.( ) this study focused on the expression pattern of cox- in fractures, the reduction in cox- expression in fractures in a model of aging, and the potential of cox- /ep gain of function to compensate for and accelerate fracture repair in the setting of aging. the work did not address the issue of whether an ep receptor agonist has the potential to stimulate repair in young mice, and this remains an impor- tant issue. the molecular events associated with nonunions in young subjects are not understood and it is possible the ep receptor gain of function may have a role in promoting normal and delayed fracture repair in young individuals. the experiments used only a single dose of the ep agonist, cp . cp has previously been shown to restore trabecular bone mass and strength in ovariectomized rats. ( , ) based on the doses used in rats, we chose a daily injection of mg/kg/d, which represents the maximal ef- fects in these animal models. because the goal of these experiments was to show the concept that a ep gain of function can compensate for the reduced rate of fracture healing observed in aged mice, experiments designed to determine the relative potency of different concentrations of cp were not completed. similarly, we did not study the relative potential of other factors, such as bmp- or pth, to compensate for reduced fracture healing in the aging model. pth and bmp- are important and perhaps overlapping or integrated pathways that have been shown to regulate bone repair.( , , – ) similar to ep , the pth receptor is a g-coupled protein receptor that activates the protein kinase a signaling pathway.( ) whereas these agents may have an important role in fractures in aging, the current studies focused on the role of ep receptor sig- naling. future studies will need to determine the relative effectiveness of these and other agents. altogether, the experiments showed that the impaired fracture healing with aging involves essentially all stages of the process and suggest that altered expression of early genes involved in fracture repair affect the entire healing cascade. these findings define cox- /ep signaling as an important potential therapeutic target to improve fracture healing in the aging population. acknowledgments the authors acknowledge the assistance of krista ca- nary, barbara stroyer, angela sp lin, david e vizurraga, and pfizer pharmaceuticals (groton, ct). the authors also thank dr mei li for her helpful insights during manuscript naik et al. preparation. this study was funded by public health service awards r ar , p ar , and t ar and the howard hughes medical institute. references . nieminen s, nurmi m, satokari k healing of femoral neck fractures; influence of fracture reduction and age. ann chir gynaecol : – . . hammer r, edholm p, lindholm b 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e-mail: regis_okeefe@urmc.rochester.edu received in original form january , ; revised form august , ; accepted october , . naik et al. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ ucla ucla previously published works title hypotension following patent ductus arteriosus ligation: the role of adrenal hormones. permalink https://escholarship.org/uc/item/ g z h journal the journal of pediatrics, ( ) issn - authors clyman, ronald i wickremasinghe, andrea merritt, t allen et al. publication date - - doi . /j.jpeds. . . peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ g z h https://escholarship.org/uc/item/ g z h#author https://escholarship.org http://www.cdlib.org/ hypotension following patent ductus arteriosus ligation: the role of adrenal hormones ronald i. clyman, md , andrea wickremasinghe, md , t. allen merritt, md, mha , tabitha solomon, md , patrick mcnamara, md , amish jain, md , jaideep singh, md, mph , alison chu, md , shahab noori, md , krishnamurthy sekar, md , pascal m. lavoie, md, phd , joshua t. attridge, md , jonathan r. swanson, md , maria gillam-krakauer, md , jeff reese, md , sara demauro, md, msce , brenda poindexter, md , sue aucott, md , monique satpute, md , erika fernandez, md , and richard j. auchus, md on behalf of the patent ductus arteriosus ligation/hypotension trial investigators* departments of pediatrics and cardiovascular research institute, university of california san francisco department of pediatrics, loma linda university, loma linda, ca department of pediatrics, hospital for sick children, toronto, canada department of pediatrics, university of chicago, chicago, il department of pediatrics, university of oklahoma, oklahoma city, ok department of pediatrics, children’s & women’s health centre of british columbia, vancouver, canada department of pediatrics, university of virginia, charlottesville, va department of pediatrics, vanderbilt university, nashville, tn department of pediatrics, children’s hospital of philadelphia and university of pennsylvania perelman school of medicine, philadelphia, pa department of pediatrics, indiana university, indianapolis, in department of pediatrics, johns hopkins university, baltimore, md department of pediatrics, university of new mexico, albuquerque, nm department of pediatrics, department of medicine, university of michigan, ann arbor, mi © mosby, inc. all rights reserved. corresponding author: ronald i. clyman, m.d., box , hsw , university of california, san francisco, parnassus ave, san francisco, ca - , phone: ( ) - , fax: ( ) - , clymanr@peds.ucsf.edu. *a list of members of the pda ligation/hypotension trial investigators is available at www.jpeds.com (appendix). reprint request author: ronald i. clyman, m.d. the authors declare no conflicts of interest. publisher's disclaimer: this is a pdf file of an unedited manuscript that has been accepted for publication. as a service to our customers we are providing this early version of the manuscript. the manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. nih public access author manuscript j pediatr. author manuscript; available in pmc june . published in final edited form as: j pediatr. june ; ( ): – .e . doi: . /j.jpeds. . . . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t abstract objective—to test the hypothesis that an impaired adrenal response to stress might play a role in the hypotension that follows patent ductus arteriosus (pda) ligation. study design—we performed a multicenter study of infants born at < weeks gestation who were about to undergo pda ligation. serum adrenal steroids were measured three times: before and after a cosyntropin ( . microgram/kg) stimulation test (performed prior to the ligation), and at – hours after the ligation. a standardized approach for diagnosis and treatment of postoperative hypotension was followed at each site. a modified inotrope score ( x dopamine (μg/kg/min) + x dobutamine) was used to monitor the catecholamine support an infant received. infants were considered to have catecholamine-resistant hypotension if their highest inotrope score was > . results—of infants enrolled, ( %) developed hypotension and ( %) developed catecholamine-resistant hypotension. low post-operative cortisol levels were not associated with the overall incidence of hypotension following ligation. however, low cortisol levels were associated with the refractoriness of the hypotension to catecholamine treatment. in a multivariate analysis: the odds ratio for developing catecholamine-resistant hypotension was or= . , ci= . – , p= . . low cortisol levels (in infants with catecholamine-resistant hypotension) were not due to adrenal immaturity or impairment; their cortisol precursor concentrations were either low or unchanged and their response to cosyntropin was similar to infants without catecholamine-resistant hypotension. conclusion—infants with low cortisol concentrations following pda ligation are likely to develop postoperative catecholamine-resistant hypotension. we speculate that decreased adrenal stimulation, rather than an impaired adrenal response to stimulation, may account for the decreased production. keywords cortisol; hydrocortisone; surgery; dopamine; newborn between -to- % of preterm infants having surgical ligation of the patent ductus arteriosus (pda) develop systemic hypotension and hemodynamic deterioration approximately -to- hours after the surgical procedure ( – ). the factors responsible for postoperative hypotension are unclear. retrospective studies have not identified differences in the surgical, anesthetic, or intraoperative fluid management among the infants who develop postoperative hypotension ( , ). indices of impaired myocardial performance are often observed prior to the development of hypotension ( – ), and prophylactic administration of milrinone, begun shortly after the surgery, appears to reduce the incidence of postoperative cardiorespiratory instability. however, some infants continue to develop post-ligation hypotension despite early treatment of low ventricular output with milrinone ( , ). treatment with volume expanders and catecholamine infusions can usually correct the post- ligation hypotension. however, approximately % of infants develop catecholamine- resistant hypotension. in these infants, a “low stress-dose” of hydrocortisone can normalize clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t their blood pressure ( , , ). currently, there is no information about the ability of the premature infant to increase cortisol production after surgery. preterm infants are known to have an altered adrenal response to hypotension and postnatal stress during the first week of life: in % the cortisol levels are increased with postnatal stress ( , ) but in % the levels are either similar to or lower than those of healthy infants ( – ). the low cortisol concentrations do not appear to be due to low corticosteroid binding globulins ( ), nor are they explained by a diminished pituitary response to stress in hypotensive preterm infants because corticotropin releasing hormone causes a robust increase in acth, but only a blunted cortisol response in the same infants ( , – ). these findings suggest that the premature adrenal gland might not respond appropriately to elevated acth levels ( , , ). sick preterm infants have high circulating concentrations of -hydroxyprogesterone, -deoxycortisol, and cortisone. this pattern suggests that the activity of the enzymes that convert cortisol precursors to cortisol or from cortisol to cortisone may be altered ( – ). although adrenal function usually returns to normal by days after birth ( , ), several reports suggest that the abnormal accumulation of cortisol precursors and impaired production of cortisol may persist for several weeks in preterm infants ( , ). two reports suggest that infants at risk for developing peri-operative hypotension may have depressed cortisol responses to cosyntropin (acth – ) stimulation ( , ). therefore, we designed a prospective study to test the following hypotheses: ) infants who develop hypotension after pda ligation have a diminished post-operative cortisol response compared with infants who are normotensive; ) infants who develop catecholamine- resistant hypotension have the most diminished cortisol response and have a limited ability to convert cortisol precursors to cortisol (or a more rapid conversion of cortisol to cortisone); and, ) preoperative measurements of cortisol precursors or metabolites (before and after cosyntropin stimulation) will be able to predict which infants are likely to develop post-ligation cardiopulmonary deterioration. methods between may , and february , infants were enrolled at sites with institutional review board approval and parental consent. infants were eligible for the study if they were: ( ) delivered between / – / weeks gestation; and ( ) scheduled for pda ligation. the criteria to determine the need for ligation were not standardized between the centers. the decision to perform the ligation was made by the clinical care teams. infants were excluded from the trial if they had: ( ) major or lethal congenital or chromosomal abnormalities; ( ) congenital heart defects (excluding patent ductus arteriosus and small atrial or ventricular septal defects); or ( ) had received hydrocortisone or dexamethasone within days of the planned surgery. the pda left-to-right shunt was scored (moderate or large) with an echocardiogram prior to ligation as previously described ( ). the following clinical measures of cardiorespiratory status were recorded prospectively (starting hour prior to surgery, until hours after clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t surgery): arterial blood gases, blood pressures, heart rates, hematocrits, number of fluid boluses, and the amount, duration and type of vasopressor support, and the anesthetics, analgesics and sedatives the infants received. steroid measurements three serum samples ( . ml blood/sample) were collected for steroid metabolite measurements. two samples were obtained no more than hours before the surgery as part of a cosyntropin (acth – ) stimulation test: ) a baseline value, and ) a value obtained minutes after intravenous cosyntropin ( . microgram/kg). previous studies found that % of healthy preterm infants have normal stimulated cortisol responses minutes after this dose of cosyntropin whereas sick, ventilated infants have significantly lower cortisol responses ( , , ). a third serum sample for steroid metabolites was obtained between – hours after the surgery. this time-point was chosen because post-ligation hypotension usually is present by -to- hours after the ligation ( – ). (note: the third sample was collected before the first hydrocortisone dose, if hydrocortisone was started before the – hour sample time point. steroid assays were performed by ultra-performance liquid chromatography tandem mass spectrometry as previously described ( ). cortisol, cortisone, -hydroxyprogesterone, - deoxycortisol, -deoxycortisol, deoxycorticosterone, corticosterone, progesterone, and androstenedione were assayed simultaneously in a single run. values measured by this technique have correlation coefficients between . and . when compared with those obtained by radioimmunoassy ( ). serum cortisol concentrations had a skewed distribution in our population. therefore, we expressed the cortisol values as both absolute and log (base ) transformed values. because the adequacy of an infant’s cortisol response for maintaining blood pressure depends on both the circulating cortisol level and the stress experienced by the infant, we defined a “decreased cortisol response” after ligation as one in which the cortisol level was in the lower-third (tertile) of the postoperative cortisol concentrations found in the study population. in our study, all infants underwent the same operation. therefore, by using this definition we could match the appropriateness of the cortisol response to the level of stress that the infant was experiencing. surgical and postoperative management all infants received a muscle relaxant (pancuronium, rocuronium, vecuronium or cisatracurium) and fentanyl anesthesia ( infants also received ciboflurane, propofol, or ketamine). left mid-axillary thoracotomies were performed through the fourth intercostal space and the ductus were ligated using metal clips. morphine or fentanyl infusions were begun after the ligation and tapered over to hours depending on an infant pain profile score. at one study site ( infants), a milrinone infusion was started shortly after the ligation as part of an institutional protocol designed to prevent postoperative hypotension ( , ). clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t management of hypotension in study infants because vasopressor management of hypotension was the primary outcome, the hypotension treatment regimen needed to be directive rather than at the discretion of the clinicians. a standardized approach that determined when volume expanders and vasopressors would be initiated, and the rate at which they would be increased, was agreed upon by all participating centers. blood pressure (bp) was measured directly with an arterial line and transducer, or noninvasively using the oscillometric method. an arterial line and transducer were used to measure blood pressure continuously in all infants receiving catecholamine infusions or hydrocortisone for blood pressure support. hypotension was defined as “mean bp less than the rd percentile for postmenstrual age ( , ) that lasted more than minutes”. operationally this meant that infants were considered to be hypotensive, and require treatment for their hypotension, if their mean blood pressure was less than [(postmenstrual age in mm hg) − ( to mm hg)]. when infants failed to maintain an adequate bp (defined as “bp greater than the hypotensive range”), no more than fluid boluses (isotonic saline ml/kg per dose) could be given initially to correct presumed hypovolemia. if the fluid boluses were unsuccessful in maintaining an adequate bp, catecholamine support could be added. the choice of dopamine or dobutamine was left to the clinical neonatologist. infusion of dopamine or dobutamine was started at a rate of μg/kg/min. the dose could be increased by . μg/kg/min every minutes until an adequate bp was achieved. combinations of dopamine and dobutamine could also be used. we used a modified inotrope score ( ) to measure the amount of catecholamine support that an infant received to maintain an adequate bp. the modified inotrope score was calculated as the sum of all catecholamines corrected for potency: ( x dopamine (μg/kg/ min) + x dobutamine + x epinephrine). if a combination of dopamine and/or dobutamine (with an inotrope score > ) failed to maintain an adequate bp, epinephrine and/or a “low stress dose” of hydrocortisone could be added. the “low stress dose” of intravenous hydrocortisone could not be used unless the catecholamine infusion rates had an inotrope score > . hydrocortisone was started at mg/kg/day and could be increased up to mg/kg/day to maintain an adequate bp. the results of the steroid measurements were not available during the study and were not used in the decision to start hydrocortisone treatment. the severity of the hypotension was defined by the maximum support needed to maintain an infant’s bp above the hypotensive range: minimal = volume boluses alone; mild = maximum inotrope score less than ; moderate = maximum inotrope score -to- ; and severe (or catecholamine-resistant) hypotension = maximum inotrope score greater than . catecholamine resistant hypotension was classified as an inotrope score > based on the study centers’ experience prior to the study (namely, that infants who failed to maintain an adequate bp with dopamine infusions ≤ μg/kg/min usually required dopamine infusions > μg/kg/min). as a result, most infants who failed to maintain an adequate bp with clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t dopamine infusions ≤ μg/kg/min were started on hydrocortisone for additional treatment rather than increase the amount or number of catecholamine infusions. statistical analyses the χ test was used to compare categorical risk factors and outcomes, and the student t- test or mann-whitney test was used to compare the means or medians of continuous variables. an adjusted multivariable logistic regression model was used to determine the effects of specific predictive variables on the outcome of interest. we first identified the perinatal and neonatal risk factors that were most associated with hypotension using bivariate analyses (table i). a model was built for hypotension through forward selection. variables were added to the model in order of increasing statistical significance. variables were dropped from the model if their p-value rose to ≥ . after the addition of other variables. after the models were built, the steroid predictive variables were individually forced into the models to evaluate their effect when adjusted by the other predictors. odds ratios (or) and % confidence intervals (ci) were calculated using the multivariable model. a p-value of < . for the predictive variables was considered significant. all analyses were performed using stata (college station, tx) statistical software. results ninety-five infants were enrolled in the study prior to pda ligation; infants were normotensive throughout the post-operative period, and infants developed hypotension: infants had minimal hypotension (i.e., were treated with volume boluses alone ( ± ml/ kg)); also received catecholamines (dopamine-alone, %; dobutamine-alone, %; dopamine and dobutamine, %). six infants had mild, moderate, and severe/ catecholamine-resistant hypotension. twelve of the infants with catecholamine-resistant hypotension were treated with hydrocortisone. peak inotrope scores occurred during the first hours after surgery in infants ( %), between – hours in ( %), between – hours in ( %), and between – hours in ( %). infants who developed hypotension were more likely to be younger, weigh less, and be receiving dopamine at the time of ligation. they were also more likely to have received prolonged resuscitation at birth ( min apgar ≤ ) (table i). there was a significant increase in cortisol concentrations following the ligation among infants who remained normotensive during the post-operative period (median cortisol pre- ligation (interquartile range) = . ( . – . ) ng/ml; post-ligation = . ( . – . ) ng/ml, p< . ). this was also true for the study population as a whole (median cortisol pre- ligation (interquartile range) = . ( . – . ) ng/ml; post-ligation = . ( . – ) ng/ml, p< . ). we hypothesized that hypotensive infants would have lower post-operative cortisol concentrations than normotensive infants. however, we found no significant difference in postoperative cortisol concentrations between the two groups (table ii). in fact, infants who clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t developed minimal/mild or moderate hypotension had significantly higher post-operative cortisol concentrations than normotensive infants (table ii). although low post-operative cortisol concentrations were not associated with the overall incidence of hypotension, they were associated with its severity and resistance to catecholamine treatment. infants who developed catecholamine-resistant hypotension had significantly lower cortisol concentrations than normotensive infants or infants with milder degrees of hypotension (table ii). in our multivariable analysis (that included the previously defined nonsteroidal risk factors; table i), low cortisol concentrations at – hour after ligation was a significant and independent risk factor for developing catecholamine-resistant hypotension (orcortisol lower tertile = . , ci= . – , p= . ). similarly, the risk for developing catecholamine-resistant hypotension decreased significantly with increasing postoperative cortisol concentration (orcortisol (log ) = . , ci= . – . , p= . ). we hypothesized that infants with catecholamine-resistant hypotension would have increased concentrations of cortisol precursors and metabolites during the postoperative period. however, we found just the opposite: post-ligation precursor and metabolite levels were either unchanged or significantly decreased ( -deoxycortisol, -deoxycortisol, cortisone) in infants who developed catecholamine-resistant hypotension (table iii). neither the cortisol nor other steroid measurements, performed prior to ligation (at baseline or after the cosyntropin stimulation test), were predictive of which infants would be at risk for developing post-ligation catecholamine-resistant hypotension (table iii). discussion we hypothesized that an immature or impaired adrenal response to stress (with low levels of cortisol and high levels of cortisol precursors) might be responsible for the post-operative hypotension that often follows pda ligation. our findings do not support this hypothesis. in fact cortisol concentrations in infants who developed post-operative hypotension (responsive to catecholamine or volume resuscitation) were significantly higher than cortisol concentrations measured in normotensive infants (table ii). although low cortisol levels do not appear to be associated with the overall incidence of hypotension, they do appear to be related to the refractoriness of the hypotension to catecholamine treatment. infants who developed hypotension that was resistant to catecholamine treatment had significantly lower cortisol concentrations than normotensive infants or infants with milder degrees of hypotension (that responded to catecholamine and volume resuscitation) (table ii). our findings also do not support the hypothesis that impaired adrenal production might be responsible for low cortisol values in infants who develop catecholamine-resistant hypotension. cortisol precursors and metabolites were significantly decreased, rather than increased, in infants who developed catecholamine-resistant hypotension (table iii). in addition, infants who developed catecholamine-resistant hypotension had the same increase in cortisol and cortisol-precursors after cosyntropin (exogenous acth – ) as infants who never developed catecholamine-resistant hypotension (table iii). in hindsight, these findings should not be so surprising because the blunted, immature adrenal response to clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t cosyntropin, seen shortly after birth in preterm infants, usually normalizes by the end of the second week ( , ). in our study, the average age at ligation was weeks ( % of the infants who developed post-ligation catecholamine-resistant hypotension were more than weeks old when they were ligated). we suggest that decreased adrenal stimulation (perhaps secondary to poor integration of cerebral signals or decreased hypothalamic or pituitary output) is a better explanation for the low postoperative cortisol concentrations observed in infants with catecholamine-resistant hypotension. because we found that postoperative catecholamine-resistant hypotension was not caused by adrenal impairment, it should come as no surprise that the tests performed prior to the ligation (e.g., cosyntropin stimulation and cortisol precursor measurements), which were designed to identify infants with adrenal impairment, did not identify infants at risk for developing postoperative catecholamine-resistant hypotension. our study has certain limitations. the size of the pda was not a criterion for study entry, and pre-ligation echocardiograms were not interpreted by the same sonographer. this design might have altered our ability to detect a relationship between pda size and postoperative hypotension (if one existed). however, previous single center retrospective studies have not detected a relationship between these two variables. because we investigated a single surgical procedure and a specific anesthesia regimen, our findings may not be applicable to other surgeries in the newborn period. our study was not a controlled treatment trial, and therefore our data cannot tell us whether low cortisol values play a role in the development of catecholamine-resistant hypotension or simply are predictive of its occurrence. we speculate that when post-operative hypotension occurs, low cortisol concentrations contribute to its refractory nature and lead to the development of catecholamine-resistant hypotension: of the infants with inotrope scores greater than were treated with hydrocortisone ( . ± . mg/kg/d); the inotrope scores in these infants dropped by ± points (from ± to ± ) during the next hours. in contrast, other infants with scores greater than , and infants with scores between – , were not treated with hydrocortisone at the same time after ligation; their inotrope scores either did not change or increased during the next to hours (from ± to ± ) (p< . ). only a future, prospective controlled trial will elucidate the contribution of low cortisol production to the severity of postoperative hypotension. in conclusion, we found that low postoperative cortisol concentrations were not associated with the incidence of postoperative hypotension following pda ligation. they were, however, associated with the severity of hypotension when it occurred, and its resistance to catecholamine treatment. we speculate that decreased adrenal stimulation, rather than adrenal immaturity or impairment, may be responsible for the decreased cortisol concentrations in these hypotensive infants. acknowledgments supported by the thrasher research fund, national institutes of health/national center for research resources (ctsi ul tr through the university of california, san francisco), and the jamie and bobby gates foundation. mass spectrometry was supported by the michigan nutrition obesity center (dk ) and michigan regional comprehensive metabolomics research core (u dk ). clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t abbreviations pda patent ductus arteriosus rss respiratory severity score bp blood pressure acth adrenocorticotropic hormone references . moin f, kennedy ka, moya fr. risk factors predicting vasopressor use after patent ductus arteriosus ligation. am j perinatol. ; : – . 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[pubmed: ] appendix additional members of the pda ligation/hypotension trial investigators include: clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t study coordinating center--university of california san francisco, san francisco, ca: scott fields, pharmd, and neonatal clinical research center nurses. steroid measurement center--university of michigan, ann arbor, mi: susan matthews, phd, robert chomic, ms, stephen c. brown, phd. study sites--hospital for sick children, toronto, canada: afif el-khuffash md, jenny luc, rrt; university of chicago, chicago, il: michael schreiber, md; university of oklahoma, oklahoma city, ok: michael mccoy, ms, aprn; children’s & women’s health centre of british columbia, vancouver, canada: jennifer claydon, msc, nadine lusney, rn, kristi finlay, rn; university of virginia, charlottesville, va: amy e. blackman, rn; vanderbilt university, nashville, tn: amy law-beller, rn; indiana university, indianapolis, in: leslie dawn wilson, bsn, ccrc; university of new mexico, albuquerque, nm: kristi watterberg, md, theresa wussow rn; duke university, durham, nc: michael cotten, md, kim fisher, ph.d., fnp-bc, ibclc. clyman et al. page j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t clyman et al. page table patient demographics and risk factors associated with postoperative hypotension hypotension none (n= ) minimal/mild/moderate/severe (n= ) p-value* gestation –wks . ± . . ± . - gestation ≤ wks (%) . birth weight -gm ± ± - sex – male (%) - preterm labor (%) - prom > hr (%) - betamethasone > hr (%) - preeclampsia (%) - chorioamnionitis (%) - diabetes (%) - race -caucasian (%) - caesarean section (%) - apgar score ( min) ≤ (%) - apgar score ( min) ≤ (%) . ** rds (%) - surfactant (%) - pulmonary hemorrhage (%) . dopamine during st hr after birth (%) . sepsis (onset ≤ d) (%) - sepsis (onset > d) (%) - hydrocortisone treatment prior to study (%) - indomethacin or ibuprofen prior to study (%) - ivh ≥ grade prior to study (%) - nec/perforation prior to study (%) - acth stimulation test-time before surgery - hr ± ± - postnatal age at ligation - wks . ± . . ± . . postmenstrual age at ligation (wks) . ± . . ± . . weight at ligation - gm ± ± . ** pda size (large) prior to ligation (%) - rss prior to ligation . ± . . ± . - inotrope score preligation . ± . . ± . . ** fentanyl dose during ligation – μg/kg ± ± - ligation duration - min ± ± - values represent mean ± standard deviation or percent (%). * p-values ≥ . are not shown. j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t clyman et al. page ** variables used in the final multivariable model for catecholamine-resistant hypotension. note: other variables were dropped from the model if their p-value rose to ≥ . during forward selection. j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t clyman et al. page table postoperative cortisol concentrations: comparisons between different groups of normotensive and hypotensive infants patient groups postoperative cortisol concentrations cortisol–ng/ml (interquartile range) cortisol-log( ) (±sd) cortisol concentrations in lower tertile cortisol concentrations in upper tertile normotension compared with hypotension (minimal/mild/mod & catechol-resistant) normotension (n= ) . ( – ) . (± . ) % % hypotension (minimal/mild/mod & catechol- resistant) (n= ) ( . – ) . (± . ) % % hypotension (minimal/mild/mod) compared with normotension hypotension (minimal/mild/mod) (n= ) . * ( – ) . * (± . ) % % * normotension (n= ) . * ( – ) . * (± . ) % % * hypotension (catechol-resistant) compared with normotension hypotension (catechol-resistant) (n= ) . * ( . – ) . * (± . ) % * % normotension (n= ) . * ( – ) . * (± . ) % * % hypotension (catechol-resistant) compared with hypotension (minimal/mild/mod) hypotension (catechol-resistant) (n= ) . * ( . – ) . * (± . ) % * % * hypotension (minimal/mild/mod) (n= ) . * ( – ) . * (± . ) % * % * hypotension (catechol-resistant) compared with normotension or hypotension (minimal/mild/mod) hypotension (catechol-resistant) (n= ) . * ( . – ) . * (± . ) % * % * normotension or hypotension (minimal/mild/ mod) (n= ) . * ( – ) . * (± . ) % * % * values represent median (interquartile range) of absolute cortisol values, mean (± standard deviation) of the log (base ) transformed cortisol values, or percent (%) of infants with cortisol values in the lower or upper third of the distribution of postoperative cortisol concentrations. * p-values < . when comparing the two patient groups. hypotension severity is defined by the maximum support needed to maintain bp above the hypotensive range (see methods): minimal = volume boluses alone; mild = maximum inotrope score < ; moderate = maximum inotrope score -to- ; and catecholamine-resistant hypotension = maximum inotrope score > . j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t clyman et al. page t a b le s er um s te ro id c on ce nt ra ti on s as so ci at ed w it h c at ec ho la m in e- r es is ta nt h yp ot en si on h yp ot en si on n on e, m in im al , m il d , o r m od er at e (n = ) c at ec h ol am in e- re si st an t (n = ) m ed ia n ( n g/ m l) in te rq u ar ti le r an ge ( n g/ m l) m ed ia n ( n g/ m l) in te rq u ar ti le r an ge ( n g/ m l) p -v al u e* c or ti so l- pr e . . – . . – - c or ti so l- a c t h . – . – - c or ti so l- po st op . – . . – . o h p -p re . . – . . . – . . o h p -a c t h . . – . . . – . - o h p -p os to p . . – . . . – . - c or ti so ne -p re – – - c or ti so ne -a c t h – – - c or ti so ne -p os to p – . – . -d eo xy co rt is ol -p re . . – . . . – . - -d eo xy co rt is ol -a c t h . . – . . . – . - -d eo xy co rt is ol -p os to p . . – . . . – . . -d eo xy co rt is ol -p re . . – . . . – . - -d eo xy co rt is ol -a c t h . . – . . . – . - -d eo xy co rt is ol -p os to p . . – . . . – . . p ro ge st er on e- pr e . . – . . . – . - p ro ge st er on e -a c t h . . – . . . – . - p ro ge st er on e- po st op . . – . . . – . - -d o c -p re . . – . . . – . - -d o c -a c t h . . – . . . – . - -d o c -p os to p . . – . . . – . - c or ti co st er on e- pr e . . – . . . – . - c or ti co st er on e- a c t h . . – . . – - c or ti co st er on e- po st op . . – . . . – . . j pediatr. author manuscript; available in pmc june . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t clyman et al. page h yp ot en si on n on e, m in im al , m il d , o r m od er at e (n = ) c at ec h ol am in e- re si st an t (n = ) m ed ia n ( n g/ m l) in te rq u ar ti le r an ge ( n g/ m l) m ed ia n ( n g/ m l) in te rq u ar ti le r an ge ( n g/ m l) p -v al u e* a nd ro st en ed io ne -p re . . – . . . – . - a nd ro st en ed io ne -a c t h . . – . . . – . - a nd ro st en ed io ne -p os to p . . – . . . – . . d h e a s -p re – – - d h e a s -a c t h – – - d h e a s -p os to p – – - * m an n- w hi tn ey t es t w as u se d to c om pa re t he v ar ia bl es . p -v al ue s ≥ . ar e no t sh ow n. d ef in it io ns : pr e, v al ue s ob ta in ed p ri or t o a c t h s ti m ul at io n te st ( pr io r to l ig at io n) ; a c t h , v al ue s ob ta in ed m in a ft er a c t h s ti m ul at io n (p ri or t o li ga ti on ); p os to p, v al ue s ob ta in ed b et w ee 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specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ a population-based study of parkinsonism in an amish community | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / corpus id: a population-based study of parkinsonism in an amish community @article{racette aps, title={a population-based study of parkinsonism in an amish community}, author={b. racette and l. good and a. kissel and s. criswell and j. perlmutter}, journal={neuroepidemiology}, year={ }, volume={ }, pages={ - } } 	b. racette, l. good, + authors j. perlmutter 	published 	medicine 	neuroepidemiology background: parkinson’s disease (pd) is a neurodegenerative disorder with unknown cause. genetic mutations account for a minority of cases but the role of environmental factors is unclear. methods: we performed a population-based screening for pd in subjects in an amish community over age . pd was diagnosed using standard clinical criteria and the unified parkinson disease rating scale motor subsection (updrs ). community prevalence was calculated. we constructed a community pedigree and… expand view on pubmed karger.com save to library create alert cite launch research feed share this paper citationsbackground citations methods citations view all tables and topics from this paper 	 table 	 table 	 table 	secondary parkinson disease 	parkinsonian disorders 	neurodegenerative disorders 	physiological sexual disorders 	mental disorders 	subsection - htmllinktype citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency parkinson’s disease-related motor and 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accept & continue hindawi publishing corporation biomed research international volume , article id , pages http://dx.doi.org/ . / / review article why control activity? evolutionary selection pressures affecting the development of physical activity genetic and biological regulation j. timothy lightfoot huffines institute of sports medicine and human performance, health and kinesiology department, texas a&m university, blocker building, tamu, college station, tx , usa correspondence should be addressed to j. timothy lightfoot; tlightfoot@hlkn.tamu.edu received october ; accepted november academic editor: jaakko kaprio copyright © j. timothy lightfoot. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the literature strongly suggests that daily physical activity is genetically and biologically regulated. potential identities of the responsible mechanisms are unclear, but little has been written concerning the possible evolutionary selection pressures leading to the development of genetic/biological controls of physical activity. given the weak relationship between exercise endurance and activity levels and the differential genomic locations associated with the regulation of endurance and activity, it is probable that regulation of endurance and activity evolved separately. this hypothesis paper considers energy expenditures and duration of activity in hunter/gatherers, pretechnology farmers, and modern western societies and considers the potential of each to selectively influence the development of activity regulation. food availability is also considered given the known linkage of caloric restriction on physical activity as well as early data relating food oversupply to physical inactivity. elucidating the selection pressures responsible for the genetic/biological control of activity will allow further consideration of these pressures on activity in today’s society, especially the linkages between food and activity. further, current food abundance is removing the cues for activity that were present for the first , years of human evolution, and thus future research should investigate the effects of this abundance upon the mechanisms regulating activity. . introduction it has been a relatively short period of time since the first suggestions were made that physical activity may have a genetic control component [ ]. since that time, significant strides have been made in understanding the basis of both genetic and biological regulatory mechanisms of physical activity. estimates of the amount of physical activity regulated by genetics are variable, with both human and animal studies suggesting that genetics is responsible for between % and % of activity [ , ] with larger studies suggesting that this number is closer to % [ , ]. further, a deep and extensive set of studies have suggested that inherent biological pathways (e.g., sex steroids) have a marked control over physical activity [ – ]. whether variability in this biological control of activity is controlled by genetics or an alternative biological mechanism is currently unclear, and thus this paper will refer to “genetic/biological” control. thus, the purpose of this hypothesis paper is to propose a conceptual framework for considering why there might be genetic/biological regulation of physical activity and the potential selection pressures that drove the evolution of physical activity regulation. additionally, as the scope of this paper is limited, the reader is referred to other reviews for a full discussion of the factors influencing the variability of physical activity heritability [ – ]. . exercise endurance and physical activity appear to have evolved separately it is generally accepted that homo sapiens initially evolved the anatomical and physiological capability for endurance running approximately , – , years ago [ , ]. however, it is unclear whether the genetic control of physical http://dx.doi.org/ . / / biomed research international activity is a derivation from the selected traits that allowed endurance running or whether physical activity evolved as a separate trait. as we have noted elsewhere [ ], it is tempting to suggest that physiological characteristics that increase endurance (e.g., mitochondrial density, fiber type) might also be key components leading to higher voluntary physical activity levels, and thus both exercise endurance and physical activity would have evolved in lockstep with each other. however, two independent lines of evidence suggest that exercise capacity and activity levels did not evolve together. first, correlations of exercise capacity and activity levels in humans generally suggest that while the relationship is positive, there is only a low to moderate association between activity and endurance in adults (𝑟 = . to . ; [ – ]). in particular, studies reviewed by lamb and brodie [ ] demonstrated that the wide variability in the relationship between physical activity and endurance is potentially due to the use of various populations and differing recall methods for activity, as well as the use of submaximal or other indirect measures of exercise capacity. in children, the literature is not much clearer, with the largest analysis of available data (𝑛 = studies and comparisons; [ ]) noting that the median relationship between physical activity and exercise capacity in children and adolescents was extremely low (𝑟 value < . ). results from studies using more objective measures of physical activity in children have shown higher associations in - to -year-old children (𝑟-values = . – . ; [ ]) and – -year-old children (𝑟-values = . – . ; [ ]). however, in early studies relating direct measures of exercise capacity and accelerometer-based measurements of activity in adults, the association between exercise capacity and physical activity appears to be relatively weak, with males showing no correlation (𝑃 = . , 𝑟 = . , 𝑛 = , and age = . ± . years) and females showing a significant but weak correlation (𝑃 = . , 𝑟 = . , 𝑛 = , and age = . ± . years; [ ]). thus, while there appears to be methodological constraints on the earlier data, recent data still shows a moderate association at best between exercise capacity and activity levels with these associations dropping precipitously when used to fit prediction equations (i.e., 𝑟 values). animal models, which allow the objective measurement of activity and exercise capacity without many of the environmental influences and confounds present in human models, have supported the low/moderate correlation (𝑟 = . – . ) between exercise capacity and daily activity levels [ – ]. thus, even though there are methodological concerns with the data, the predominant view from the literature is that while there is a positive relationship between activity level and exercise capacity, that relationship is weak and certainly cannot be used to predict either activity level or functional capacity. a second line of developing evidence that suggests that physical activity and exercise capacity did not evolve together involves the actual genetic mechanisms that underlie inherent exercise capacity and inherent physical activity level. while delineation of actual genetic mechanisms for either pheno- type is still lacking for both animals and humans, genomic loci (i.e., qtl) associated with various indices of exercise capacity and physical activity appear to be separate and distinct [ , – ]. these distinct qtl suggest that genetic regulation of these traits arises through differing pathways. thus, while there may be pathways common to both phe- notypes, the low/moderate association between activity and exercise capacity in humans and animals, as well as the differing genomic loci associated with each trait, suggests that the underlying physiological regulation of inherent exercise capacity and inherent activity level of mammals evolved separately. . potential selection pressures for the evolution of physical activity regulation the evolution of a physiological system is necessarily linked to genetic selection pressure [ ], and the current literature is silent as to what selection pressure would have driven the evolution of systems to regulate physical activity. while hunter/gatherers were well known for having irregular, but sometimes extensive, hunting/foraging ranges [ , ], their overall activity patterns were not uniform (e.g., [ ]). recent data have suggested that total daily energy expenditure demands (not corrected for body weight) of hunter/gatherers were not different than modern, western-based lifestyles [ , ]. further, comparison of energy expenditure by weight between pretechnology farmers and hunter-gatherer popu- lations does not show significant differences in daily energy expenditures (tables and and figure ). whereas current western populations show decreased energy expenditures when corrected by weight, it can be argued that the higher energy expenditure required by either hunting/gathering and/or pretechnology farming could have been a selection pressure driving the development of activity regulation. however, some investigators dispute that energy expenditure requirements have decreased [ , ] which casts questions on the potential role that energy expenditure played in evolving activity regulation. while it is unclear whether energy expenditure would have been a selection pressure in the evolution of physical act- ivity control mechanisms, comparisons of required daily acti- vity (i.e., duration of activity) in nontechnology dependent agricultural societies (table ) show that the activity levels exhibited by both males and females in these populations were at least -fold higher than activity levels shown in hunter/gatherer populations (figure ). for example, panter- brick [ , ] characterized a nepali agropastoralist com- munity (the tamang) living at , to , m that exhib- ited food self-sufficiency through manual farming and live- stock rearing with little to no technology use. using both direct observations and indirect respirometry, panter-brick observed that the men worked an average of . ± . hours/ day, while the women worked . ± . hrs/day. more recently, bassett and colleagues [ ] measured physical activity lev- els in a north american labor-intensive, non-technological amish farming community. in this population, bassett and colleagues observed that the men averaged vigorous, moder- ate, or walking activity for . hrs/day and women averaged . hrs/day and only sat . hr/day ( % of the day) and biomed research international table : physical activity energy expenditures of various hunter/gatherer populations. populations sex tee aee aee/wt foraging range/day(km) weight (kg) ju/’hoansi (africa)a,b m . . a . ju/’hoansi (africa)a,b f . . a . ache (paraguay)b m . . . ache (paraguay)b f . . . hadza (tanzania)c m . . . . hadza (tanzania)c f . . . . average hunter/gatherer (±sd) m ( ) . ( . ) . ( . )∗ . ( . ) . ( . )∗ average hunter/gatherer (±sd) f ( ) . ( . ) . ( . )∗ . ( . ) . ( . )∗ average western populationc m ( ) . ( . ) . ( . ) . ( . )d ( . ) average western populationc f ( ) . ( . ) . ( . ) . ( . )d . ( . ) tee: total energy expenditure (kcal/day); rmr: resting metabolic rate (kcal/day); aee: activity energy expenditure = tee-rmr; aee/wt: activity energy expenditure divided by weight (kcal/kg/d); data from a[ ]; b[ ]; c[ ]; dvalues calculated using average daily step counts for men and women [ ] and average step lengths for men [ ] and women [ ]. ∗significantly different𝑃 < . between hunter/gatherer and average western population. values for average western population tee, aee, and aee/wt used in statistical analysis derived from artificial dataset derived from means, standard deviations, and subject numbers as reported in [ ]. table : physical activity energy expenditures of various agricultural populations. populations sex tee aee aee/wt weight (kg) tamanga (nepal) m . . . tamanga (nepal) f . . . devarishi kuppamb,c,j (tamil nadu, india) m . . . devarishi kuppamb,c,j (tamil nadu, india) f . . . gambianb,k (gambia) m . . . gambianb,d,e(gambia) f . . . mossib,f (upper volta) m . . . mossib,g (upper volta) f . . . senegalb,h (senegal) m . . . senegalb,h (senegal) f . . . amishi (canada) m . . . amishi (canada) f . . . aymaral (bolivia) m . . . aymaral (bolivia) f . . . average farming populations (±sd) m ( ) . ( . ) . ( . )† . ( . )† average farming populations (±sd) f ( ) . ( . ) . ( . )† . ( . )† tee: total energy expenditure (kcal/day; average between dry and wet season where available); rmr: resting metabolic rate (kcal/day); aee: activity energy expenditure =tee-rmr; aee/wt: activity energy expenditure divided by weight in kcal/kg/d; data from a[ , ]; breviewed by [ ]; data from c[ ]; d[ ]; e[ ]; f[ ]; g[ ]; h[ ]; i[ ]: bmrs estimated using formula ( . ml/kg/min o )∗ . ; j[ ]; k[ ] values derived from ph.d. thesis [ ]; l[ ].†significantly different𝑃 < . between farming and western populations. values for western tee, aee, and aee/wt used in statistical analysis derived from artificial dataset derived from means, standard deviations, and subject numbers as reported in [ ]. no significant differences between hunter/gatherer and farming populations. biomed research international female male sex western population hunter/gatherer pretechnology farmers a ct iv ity e ne rg y ex pe nd itu re by w ei gh t ( kc al /d ay /k g) ∗ ∗ figure : activity energy expenditure by weight. the amount of energy expended on nonbasal activity and standardized by weight of population. hunter/gatherer population estimates using ju/’hoansi [ ], ache [ ], and hadza [ ]. pretechnology farmer values from populations in figure [ – , , , , , , ]. western population data from [ ]. ∗significantly lower (𝑃 < . ) than hunter/gatherer and pretechnology farmers. there were no statistical differences between hunter/gatherers and pretechnology farmers. values for western aee/wt used in statistical analysis derived from artificial dataset derived from means, standard devi- ations, and subject numbers as reported in [ ]. (h ou rs /d ay ) n un oa ju /’ ho an si h ad za ∗ ta m an g m os si d ev ar is hi k up pa m a m is h se ne ga l populations male female figure : total time spent in vigorous, moderate, or walking activity on a daily basis in hunter/gatherer (nunoa, ju/’hoansi, hadza) or nontechnological agriculture-based populations (tamang, mossi, devarishi kuppam, amish, and senegali). data from [ – , , , , , , ]. ∗hadza activity time based on estimates from [ ] which provide the only known total daily activity time estimates for this population. . hr/day ( % of the day), respectively (figure ). this extensive physical activity pattern was reflected in their total steps per day where the amish men averaged , steps/day and the women averaged , steps/day. given the extensive data from both panter-brick and bassett’s groups, as well as from other nontechnological farming populations (table and figure ), there is little doubt that non-technological subsistence farming required extensive, long-duration, and low-intensity physical activity on a daily basis. the sustained agricultural activity requirement may not have required higher total daily energy expenditures than hunting/gathering (figure ), but the extensive time require- ments that were – -fold higher than hunting/gathering (figure ) would have required the physiological capability to complete lower intensities of exercise for much longer time frames than in hunting/gathering populations. the differing time requirements across which the energy was expended would have stressed different substrate systems—especially in farmers—favoring those individuals that could store and metabolize fats for longer duration activity. thus, the ability to be physically active for long periods of the day and the requisite requirement to produce calories from fat stores could have been a significant genetic selection pressure in the development of biological/genetic control of physical activity. further, in those early populations that adopted agriculture, individuals that were predisposed to higher levels of motivation and physical capability for daily activity would have been more successful and would have had a greater chance of reproductive success [ ]. in essence, a farmer could not have been lazy and insure that his genes would be passed on to future generations because his family would not survive. whether the genetic selection pressure linked to the development of biological control of physical activity was energy expenditure or duration of activity, ultimately, both factors link back to the availability of food. while estimates of average hunter/gatherer foraging ranges can appear extensive (e.g., table ), hunter/gatherers did not range far and had reduced energy expenditure when food was close at hand. when food became difficult to get or the hunting/foraging ranges became lengthy, hunter/gatherers simply moved to more fertile sites where food was more abundant [ ]. for farmers, because they were bound to a specific loca- tion, without physical activity, there was no food. in fact, food availability appears to have a direct causative effect on physical activity that is exhibited in both animal and human models, especially in the area of caloric restriction. numerous studies report that short-term caloric restriction decreases rodent activity, but long-term caloric restriction actually increases physical activity (e.g., [ ]). this same phenomenon appears in nonhuman primates (e.g., rhesus monkeys; [ ]) with a concomitant increase in metabolic efficiency of movement. further, it has been suggested that this caloric restriction-related hyperactivity also occurs in humans. casper [ ] hypothesized that, in the majority of anorexia nervosa (an) patients that present hyperactivity (suggested to range from % to % of an; [ , ]), the increased activity is a result of the hypocaloric nature of an, which differs from the lethargy seen in semistarvation biomed research international states. casper suggested several potential physiological path- ways that govern this human caloric restriction and related hyperactivity. for example, casper [ ] uniquely suggests that mutations in the “foraging” gene first found in drosophila (dgcalpha ; [ ]) can increase foraging locomotion in fruit flies [ ] and honey bees [ ] and may be involved in the regulation of the increased activity in an patients. further, the gene homologous to dgcalpha in rodents and humans is “guanylate cyclase , soluble, alpha ” (gucy a ), which is one of the genes that encodes soluble guanylyl cyclase (sgc), the most sensitive receptor for nitric oxide [ ]. further, the mouse homolog of gucy a (i.e. gucy a ) is located on chrm. downstream of a known physical activity-related qtl [ ]. the involvement of gucy a , or any other genetically-based mechanism regulating activity, would support epling and pierce’s early speculation [ ] that an patients represent a natural selection of individuals who become active during food shortages, leading to an increased chance of food finding even at the risk of negative caloric balance. garland and kelly [ ] also suggested that individ- uals with higher foraging behavior could be an example of a directed natural selection. thus, in individuals with foraging behavior more suited to the available food supply, the alleles responsible for this higher locomotor activity may be favored more highly in reproduction [ ]. conversely, to our knowledge, there have been no direct studies designed to determine if excess caloric intake directly decreases activity in human and/or animal models. indi- rectly, several studies suggest that, with overfeeding, physical activity levels decrease. for example, in an elegant study, levine and colleagues [ ] showed that overfeeding both lean and obese human subjects , kcal/day above their weight maintenance needs resulted in significant decreases in free-living walking in both groups. schmidt et al. [ ] directly measured spontaneous physical activity levels (i.e., neat) in obesity-prone and obesity-resistant individuals and observed decreases in spontaneous physical activity in the obesity-prone individuals three days after overfeeding (but not in obesity-resistant individuals). anecdotally, it has been observed [ ] that male baboons are markedly less active (e.g., reduced climbing, laying close to sugar-source) when their caloric intake was significantly increased through the availability of a sweetened beverage containing water, high fructose corn syrup, and artificial fruit flavoring [ ]. supporting these observations are indirect results strongly suggesting, in both adults and children, that decreased phys- ical activity was driven by an increased adiposity as opposed to adiposity being an effect of decreased activity [ , ]. neither ekelund and colleagues [ ] or metcalf et al. [ ] pro- posed potential causative biological mechanistic ties between overfeeding and inactivity, instead preferring to speculate on potential biomechanical and physical discomfort of increased weight prohibiting activity. however, other meta-analyses and animal studies have shown no relationship between body mass and activity levels (e.g., [ – ]) suggesting that it is not body weight per se decreasing activity, but rather the increase in caloric intake. supporting this indirect evidence of a tie between overfeeding and a decrease in physical activity is a potential mechanistic pathway. it has long been known that removal of sex hormones and subsequent reduction in testosterone or estrogen levels results in large decreases in activity (e.g., [ , ]) that can be rescued with administration of testosterone and/or estrogen which is mediated primar- ily through androgenic receptor pathways [ ]. recently, bouchard et al. [ ] showed conclusively that overfeeding in humans significantly decreases androgenic production— especially in males. therefore, hypothetically, this reduction in androgenic production from overfeeding could result in a reduction in physical activity through established pathways. thus, while it is not currently known whether increased caloric availability decreases the drive for activity, there are tentative evidence and potential hypothetical mechanisms that strongly support further research into this question. there are some significant limitations to the preceding discussion that should be considered in interpretation of these data. in particular, the quantification of daily activity levels in both human and animals continues to undergo refinement, and the limitations of older methods should be appreciated [ ]. thus, the use of older studies that used less than optimal methods of activity measurement, such as sur- vey or observational methods, may need to be reconsidered. for example, much of the extant hunter/gatherer activity data is based on observational or estimated activity levels and can be open to question. an example of this limitation is the recent publication of direct measures of energy expenditures collected in a hadza population by pontzer et al. [ ] which contradict earlier observations in the hadza which noted marked swings and inconsistencies in hadza activity patterns [ ]. further complicating the issue of valid activity measure- ment is the rapid diminishing of the opportunity to collect data on peoples that represent hunter/gatherer lifestyles. lee, who is considered the leading expert on the ju/’hoansi, has observed the creeping influence of western lifestyle and the diminishment of hunting/gathering in the ju/’hoansi is due to wide access to motorized transport, other food sources, and reduction in available foraging range [ ]. thus, if modern data were collected on the ju/’hoansi, whether this data truly represented a paleolithic hunter/gatherer lifestyle would be a fair question—as it is with the hadza data of pontzer and colleagues [ ]. therefore, it is important to use the best data available in populations that best represent the target populations and we have strived to do so in this paper. as scientists work to understand the identities of the genetic and biological mechanisms that control physical activity, it is important to also work to develop an under- standing of the evolutionary selection pressures that have led to these activity regulation mechanisms. at this point, it is unclear what the specific genetic selection pressures were that caused the development of genetic/biological regulation of activity, but there are suggestions that physical activity evolved separately from endurance capability (figure ). fur- ther, while energy expenditure may be an attractive candidate for genetic selection pressure, data suggests that total daily energy expenditure has not significantly changed, but rather, the duration of daily activity required to procure food radi- cally changed with the adoption of agriculture approximately , years ago. additionally, the suggestions of an inverse link between caloric intake and physical activity would add biomed research international paleolithic era development of agriculture technology/diet-enabled sedentarism years ago-future evolution of predisposition to endurance capability (trait) evolution of predisposition to long duration activity (trait) removal of activity requirements for daily survival requirement of sustained activity at higher physiological requirement of sustained activity at moderate physiological states traits not required for species survival become “ancestral traits”? homo sedentarius? physical activity required for food/evolution of activity regulation ? c ur re nt so ci et y food abundance/de-evolution of activity regulation? , – , years ago , – , years ago states for - hours for – hours figure : potential selection pressures on activity regulation in humans and possible future effects of food abundance. a strong biological cause/effect relationship that would both help explain evolution of genetic/biological regulation of activity and could further explain the precipitous declines in physical activity currently seen in most nations [ ]. again, the reader is cautioned that at this point in the maturity of the physical activity regulation literature, the above facts and hypotheses appear to provide the most probable—yet still hypothetical—explanation of the selection pressures influ- encing the evolution of physical activity regulation. further studies directly addressing these hypotheses, especially those using animal models and experimental evolution models [ ], may provide the best pathway toward conclusively establishing the evolutionary selection pressures on physical activity regulation. . applications and future directions while we have looked backward to discuss potential past causes/pressures that drove the evolution of physical activity in humans (figure ), it is imperative that we also look forward to consider potential areas of needed research, especially given the large health and economic consequences of the current downward trend of physical activity world- wide [ – ]. with the general acceptance of a continu- ing evolutionary change pattern in homo sapiens amongst evolutionary biologists [ ], it is interesting to speculate as to the effect of our current technology- and diet-enabled sedentarism on the genetic regulation of physical activity. as zimmer noted in [ ], predicting the outcome of evolution is difficult, especially human evolution where there are myriad factors influencing the selection of different traits. but as scientists, we should consider whether our current proclivity toward sedentarism—for example, troiano and colleagues objectively observed less than . % of adults in the united states were moderately active more than mins per day [ ]—will drive our evolution toward physiological mechanisms that allow us to remain inactive, yet healthy. theoretically, environment drives selection toward traits that increase reproductive fitness. for the first time in the his- tory of homo sapiens, we live in an era where our ability to be active or have high exercise capacity does not impact our abil- ity to obtain food. our current technology- and diet-enabled environment in most cases has removed the need to stay fit and be active on a daily basis. most of us neither have to hunt and gather or grow our own food. however, since the majority of hypokinetic chronic diseases do not significantly impact health until long after the reproductive cycle of most humans has begun, as long as one can find a reproductive partner, the embracing of a technology- and diet-enabled sedentarism would not affect societal reproduction as a whole. further, if it is assumed that the majority of individuals in a society embrace technology- and diet-enabled sedentarism, those individuals that are fit and active will become a smaller minority of the population and while potentially drawn to each other and finding health benefits in such a pairing, will find no reproductive advantage by daily exercise or activity. while the underlying genetic code that predisposes to a higher daily drive to be active will be transmitted to offspring, the environmental drive requiring the expression of that drive will be removed. thus, in the long term, if our current technological- and diet-enabled sedentarism continues, while the mechanisms that predispose and regulate physical activity will be transmitted to our offspring, these mechanisms may fall into the category of ancestral genes that are no longer required for species survival as a whole [ ]. further, it will be interesting to observe whether genetic variants eventually evolve that enable homo sapiens to physiologically deal with sedentarism—such as altered metabolic mechanisms to handle the increased fat and sugar loads characteristic of a modern diet. whether and how homo sapiens adapt and evolve for this new environment—perhaps into homo sedentarius (figure )—will be an interesting topic of study and observation for years to come. biomed research international . summary and conclusions evidence suggests that daily physical activity is significantly influenced by genetic mechanisms. however, these mecha- nisms and the actual site of physiological regulation of phys- ical activity at this point are somewhat unclear. this paper’s goal was to provide—given the current literature—a concep- tual framework that can be used to guide future investigations targeting the delineation of the genetic regulation of physical activity. first, it is unclear as to what environmental selective pressure resulted in the evolution of genetic mechanisms to control physical activity. while it is tempting to speculate that the need for ancient hunters/gatherers to run/walk long dis- tances may have been a selective pressure, daily activity above what was needed to provide food would have put strains on energy balance within the individual and impacted the collective tribe’s food supply. the acceptance of widespread agriculture demanded longer periods of activity (generally at lower intensities) and thus suggests that the longer required periods of activity inherent in farming might have provided a selection pressure. indeed, it is often noted that lazy farmers were dead farmers. given the known tie between food avail- ability and activity, especially in animals, it is possible that food availability was the underlying selection pressure for the evolution of activity-regulating mechanisms. indeed, both hunter/gatherer populations and farming populations show a negative relationship between food availability and activity. if food was scarce, activity increased and if food was available, activity decreased. thus, food availability becomes a factor in the reason to be active. whether food availability was the actual selection pressure for evolving regulation of physical activity is unknown but could be potentially studied given the multiple available methods of experimentally invoking evolution (e.g., [ ]). the value of continued research and thought regarding the selection pressures responsible for activity regulation is to consider how modern lifestyle and food availability may impact those regulatory mechanisms. with plentiful food for the majority of the earth’s population, the requirement for physical activity to provide sustenance is markedly reduced, and thus the requirement to be physically active does not impact the survival of the species. therefore, in the future, it will be interesting to observe whether the removal of these potential selection pressures will affect not only physical activity levels, but also the regulation of physical activity in homo sapiens. conflict of interests the author declares that there is no conflict of interests regar- ding the publication of this paper. acknowledgments funding for much of the data underlying this paper was originally provided by nih through ro ar and r dk , as well as a start-up grant from texas a&m university. further, thanks go to d. ferguson, m. dawes, e. schmitt, h. vellers, and b. 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[ ] n. venkataraman, a. l. cole, p. ruchala et al., “reawakening re- trocyclins: ancestral human defensins active against hiv- ,” plos biology, vol. , no. , article e , . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ outcomes of physician‐staffed versus non‐physician‐staffed helicopter transport for st‐elevation myocardial infarction outcomes of physician-staffed versus non-physician-staffed helicopter transport for st-elevation myocardial infarction sverrir i. gunnarsson, md; joseph mitchell, md; mary s. busch, rn; brenda larson, rn; s. michael gharacholou, md; zhanhai li, phd; amish n. raval, md background-—the effect of physician-staffed helicopter emergency medical service (hems) on st-elevation myocardial infarction (stemi) patient transfer is unknown. the purpose of this study was to evaluate the characteristics and outcomes of physician- staffed hems (physician-hems) versus non-physician-staffed (standard-hems) in patients with stemi. methods and results-—we studied stemi patients transferred by either physician-hems (n= ) or standard-hems (n= ) for primary or rescue percutaneous coronary intervention at hospitals between and . data were collected from electronic medical records and each institution’s contribution to the national cardiovascular data registry. baseline characteristics were similar between groups. median electrocardiogram-to-balloon time was longer for the standard-hems group than for the physician-hems group ( vs minutes; p= . ). the standard-hems group was more likely than the physician- hems group to receive nitroglycerin ( % vs %; p< . ) and opioid analgesics ( . % vs . %; p< . ) during transport. in-hospital adverse outcomes, including cardiac arrest, cardiogenic shock, and serious arrhythmias, were more common in the standard-hems group ( . % vs . %; p= . ). after adjusting for age, sex, killip class, and transport time, patients transferred by standard-hems had increased risk of any serious in-hospital adverse event (odds ratio= . ; % ci= . – . ; p= . ). in-hospital mortality was not statistically different between the groups ( . % in the standard-hems group vs . % in the physician-hems group; p= . ). conclusions-—patients with stemi transported by standard-hems had longer transport times, higher rates of nitroglycerin and opioid administration, and higher rates of adjusted in-hospital events. efforts to better understand optimal transport strategies in stemi patients are needed. (j am heart assoc. ; :e . doi: . /jaha. . .) key words: acute myocardial infarction • outcome • percutaneous coronary intervention • st-segment elevation myocardial infarction • treatment p rimary percutaneous coronary intervention (pci)improves survival in patients with st-elevation myocar- dial infarction (stemi) and is the optimal treatment when performed expeditiously. over % of patients experiencing stemi in the united states initially present to hospitals without pci capability and thus are at risk for delayed reperfusion. to ensure rapid interfacility transport for pci, regional systems of care are recommended. – helicopter emergency medical services (hems) are com- monly used to transport patients from non-pci centers (ie, stemi referral hospitals) to pci centers (ie, stemi receiving hospitals). hems have been shown to be feasible, safe, and reduce transport time to the receiving facility. – thus, hems are important in rural or urban areas where ground transport times are predicted to be too long. the hems flight crew typically consists of paramedics and flight nurses who are skilled at performing a variety of advanced cardiac life support procedures. however, only � % of hems in the united states will also include an emergency-medicine–trained physician as part of the flight crew. local resources, cost, and hospital affiliations are factors that currently influence hems avail- ability and crew composition. there are limited data evaluating the composition and outcome of hems and no data comparing different hems from the division of cardiovascular medicine, departments of medicine (s.i.g., j.m., b.l., a.n.r.) and biostatistics and medical informatics (z.l.), university of wisconsin, madison, wi; mayo clinic health system-franciscan healthcare, la crosse, wi (m.s.b., s.m.g.); division of cardiology, mayo clinic, rochester, mn (s.m.g.). correspondence to: amish n. raval, md, facc, fscai, faha, division of cardiovascular medicine, department of medicine, university of wisconsin school of medicine and public health, university of wisconsin hospital and clinics (site of research), csc h / , highland ave, madison, wi . e-mail: anr@medicine.wisc.edu received october , ; accepted december , . ª the authors. published on behalf of the american heart association, inc., by wiley blackwell. this is an open access article under the terms of the creative commons attribution-noncommercial-noderivs license, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. doi: . /jaha. . journal of the american heart association original research info:doi/ . /jaha. . http://creativecommons.org/licenses/by-nc-nd/ . / systems on transport times, treatments administered during patient transport, and in-hospital events in patients with stemi. as such, practice guidelines do not address the issue of crew composition during interfacility transfer. the primary objective of this study was to evaluate the characteristics, treatment patterns, in-transport, and in-hospital outcomes of stemi patients transferred byphysician-staffed hems (physician-hems group) versus nonphysician (standard-hems group) flight crews. methods the institutional review boards of both the university of wisconsin hospital and clinics (uwhc) and mayo clinic approved this study. patient demographic information, in-transport treatments and events, and in-hospital outcomes were abstracted from institutional contributions to the national cardiovascular data registry (ncdr), electronic medical records, and the hems flight records. stemi was defined based on criteria: ( ) electrocardiogram (ecg) evidence of st-elevation based on the american heart association/ american college of cardiology (aha/acc) stemi guidelines ; ( ) a culprit artery identified by emergency invasive coronary angiography; and ( ) performance of either primary pci or pci following fibrinolytic administration (ie, rescue pci). in-transport adverse events were defined as death, cardiac arrest, serious arrhythmia (ie, rapid supraventricular arrhyth- mias requiring direct current cardioversion, sustained ventric- ular tachycardia or ventricular fibrillation, or brady-arrhythmias requiring external pacing), or endotracheal intubation. data pertaining to the use of adjunctive medications, additional procedures, and vital signs were also recorded. in-hospital outcomes were reviewed by trained abstractors using standard definitions established by the ncdr action registry and included death, stroke, cardiac arrest, bleeding, cardiogenic shock, new requirement for dialysis, recurrent myocardial infarction (mi), and vascular complications. helicopter emergency medical services within both institutions’ stemi systems of care, hems are requested directly by the stemi referral hospital. the choice of commercial hems is dependent upon availability, local contractual affiliation, and regional emergency department staff preference. the majority of stemi transfers destined for the uwhc are managed by uw med flight, a physician-staffed service. the physician-staffed hems crew is comprised of an emergency-medicine–trained flight physician, a flight nurse, and a pilot. this service utilizes ec- model helicopters (american eurocopter, grand prairie, tx), which have a maximum speed of knots (kts). non-physician-staffed hems that transport to uwhc (react of rockford, il; flight for life, milwaukee and fond du lac, wi; and medlink air of la crosse, wi) consist of a pilot and either flight paramedics or a flight paramedic and a flight nurse. these hems utilize either ec- model or bk- helicopters (american eurocopter). the ec- and bk- models have maximum speeds of and kts, respectively. all hems and stemi referring and receiving hospitals follow a standardized antithrombotic protocol, which includes aspirin mg, clopidogrel mg loading dose, and weight- adjusted heparin; however, deviation from the protocol can occur when clinically appropriate. the stemi protocol suggests that nitroglycerin and opioids (morphine or fentanyl) may be administered as clinically indicated. statistical analysis continuous variables were analyzed using the wilcoxon rank- sum test. categorical variables were tested using the chi- square test. fischer’s exact was applied in those instances where the counts of observed outcomes were numerically small or the expected cell counts < . logistic regression models were constructed to evaluate independent predictors of outcomes. all tests were considered statistically significant if p< . . all analyses were performed using statistical package for the social sciences (spss) software (version . ; ibm inc., chicago, il). results from august through december , a total of stemi patients were transferred for primary or rescue pci. of those, patients were transported by physician-hems and patients by standard-hems. patient characteristics there was no significant difference in baseline age or frequency of cardiovascular disease risk factor between the physician-hems and standard-hems groups (table ). there was no significant difference in the baseline killip class between the groups (p= . ). the right coronary artery (rca) was the most common culprit artery in both groups. however, the physician-hems group was more likely to have the left anterior descending (lad) coronary artery as the culprit for stemi (p= . ; table ). in-transport events in transport mortality was % in both groups. there was no significant difference in the frequency of serious adverse events, such as cardiac arrest, arrhythmia, intubation or need for transcutaneous pacing, direct current cardioversion, or doi: . /jaha. . journal of the american heart association physician vs non-physician stemi transport outcomes gunnarsson et al o r ig in a l r e s e a r c h cardiopulmonary resuscitation (table ). the standard-hems group was more likely than the physician-hems group to receive intravenous analgesics, such as fentanyl or morphine and nitroglycerin, during transport ( . % vs . % and . % vs . %; p< . for both; table ). of the patients who received nitroglycerin during transport, ( . %) had rca as culprit vessel. in the standard-hems group, of ( . %) of the patients who had occluded rca received nitroglycerin compared to of ( . %) patients in the physician- hems group (p< . ). the standard-hems group was more likely to receive fluid bolus and anti-thrombotic medication in-flight. the median first electrocardiogram-to-balloon (ecg b) time was longer for the standard-hems group compared to the physician-hems group ( vs minutes; p= . ; table ). there was no statistical difference in flight distance, ground time, and flight time between the groups. table . patient characteristics* physician- hems (n= ) standard- hems (n= ) p value age, y . � . . � . . male, n (%) ( . ) ( . ) . diabetes mellitus, n (%) ( . ) ( . ) . active smoker, n (%) ( . ) ( . ) . hypertension, n (%) ( . ) ( . ) . dyslipidemia, n (%) ( . ) ( . ) . previous mi, n (%) ( . ) ( . ) . initial vital signs median sbp, mm hg (iqr) ( – ) ( – ) . median dbp, mm hg (iqr) ( – ) ( – ) . median hr, rate per minute (iqr) ( – ) ( – ) . killip class . i, n (%) ( . ) ( . ) ii, n (%) ( . ) ( . ) iii, n (%) ( . ) ( . ) iv, n (%) ( . ) ( . ) culprit coronary artery . lad, n (%) ( . ) ( . ) rca, n (%) ( . ) ( . ) left circumflex, n (%) ( . ) ( . ) other, n (%) ( . ) ( . ) rescue pci, n (%) ( . ) ( . ) . dbp indicates diastolic blood pressure; hr, heart rate; iqr, interquartile range; lad, left anterior descending; mi, myocardial infarction; pci, percutaneous coronary intervention; physician-hems, physician-staffed helicopter emergency medical service; rca, right coronary artery; sbp, systolic blood pressure; standard-hems, non-physician-staffed helicopter emergency medical service. *continuous variables are expressed as means�sd (standard deviation) or median�iqr. table . in-transport events, procedures and medications physician- hems (n= ) standard- hems (n= ) p value events death, n (%) na cardiac arrest, n (%) ( . ) ( . ) . serious arrythmia, n (%) ( . ) ( . ) . procedures intubation, n (%) ( . ) ( . ) . dccv, n (%) ( . ) ( . ) . transcutaneous pacing, n (%) ( . ) ( . ) . cpr, n (%) ( . ) ( . ) . medications analgesics, n (%) ( . ) ( . ) < . paralytics, n (%) ( . ) . fluid bolus, n (%) ( . ) ( . ) . intravenous metoprolol, n (%) ( . ) ( . ) . vasopressors, n (%) ( . ) ( . ) . anti-arrhythmics, n (%) ( . ) ( . ) . anti-thrombotics, n (%) ( . ) ( ) < . nitroglycerin, n (%) ( . ) ( . ) < . cpr indicates cardiopulmonary resuscitation; dccv, direct current cardioversion; physician-hems, physician-staffed helicopter emergency medical service; standard- hems, non-physician-staffed helicopter emergency medical service. table . transportation metrics* physician- hems (n= ) standard- hems (n= ) p value flight distance, nautical miles (iqr) ( – ) ( – ) . ground time, minute (iqr) ( – ) ( – ) . flight time, minute (iqr) ( – ) ( – ) . ecg b, minute (iqr) ( – ) ( – ) . d b, minute (iqr) ( – ) ( – ) . d b indicates time from arrival at percutaneous coronary intervention (pci) referring hospital to balloon inflation; ecg b, time from first electrocardiogram acquisition to balloon inflation; flight time, time from helicopter takeoff to landing; ground time, time from helicopter landing at pci referring hospital to take-off; iqr, interquartile range; ps-hems, physician-staffed helicopter emergency medical service; standard-hems, non-physician-staffed helicopter emergency medical service. *continuous variables are expressed as median�iqr. doi: . /jaha. . journal of the american heart association physician vs non-physician stemi transport outcomes gunnarsson et al o r ig in a l r e s e a r c h in-hospital outcomes there was no statistical difference in the rate of in-hospital death between the standard-hems and physician-hems groups ( . % vs . %; p= . ; table ). the rate of any serious in-hospital adverse event was higher in the standard- hems group compared to the physician-hems group ( . % vs . %; p= . ), including cardiac arrest ( . % vs . %; p= . ), cardiogenic shock ( . % vs . %; p= . ), and need for intra-aortic balloon counter-pulsation ( . % vs . %; p= . ). of the patients who sustained any in-hospital adverse outcome (n= ), of ( %) in the standard-hems group were given nitroglycerin versus of ( . %) of those in the physician-hems group (p= . ). similarly, of ( %) of the patients who were given analgesics in the standard-hems group sustained an in-hospital adverse event compared to of ( %) of those in the physician-hems group (p= . ). median length of hospital stay was not statistically different between the groups ( days for both groups; p= . ). after adjusting for age, sex, killip class, and transport time, patients transferred by standard-hems had an increased risk of any serious in-hospital adverse event (odds ratio [or]= . ; % ci= . – . ; p= . ; table ). discussion to our knowledge, this is the first study to compare physician- staffed to non-physician-staffed hems for patients with stemi. the main findings from our study are that patients transferred by standard-hems: ( ) were more likely to receive opioid analgesics during transport; ( ) were more likely to receive nitroglycerin and intravenous fluids; and ( ) had a nearly -fold higher adjusted risk of in-hospital adverse outcomes. these differences were not associated with a statistically significant difference in mortality between the groups. a previous study showed that nonphysician hems trans- port of patients with acute coronary syndrome was safe. however, that study was not designed to compare different methods of transportation. a survey of flight nurses showed that physicians made unique and important contributions to the care of % of patients with mi. outcomes comparison before and after implementation of physician-staffed hems has been done for trauma transport. one study found that physician-staffed hems was associated with lower mortality of > blunt trauma patients. contemporary series have shown several benefits of having physician-staffed crews for regional trauma systems, includ- ing faster transport time. , we think that the observed association between standard- hems and higher rates of adverse in-hospital outcomes, such as cardiac arrest and cardiogenic shock, might be explained by several factors. first, the standard-hems group had slightly longer ecg b time, but the effects of early revascu- larization on survival are well known. given that the ground time and flight time were not significantly different between the groups, this could be because of shorter transfer time after landing at the receiving pci hospital. or, this could be table . in-hospital outcomes* physician- hems (n= ) standard- hems (n= ) p value death, n (%) ( . ) ( . ) . iabp, n (%) ( . ) ( . ) . cardiogenic shock, n (%) ( . ) ( . ) . bleeding, n (%) ( . ) ( . ) . tamponade, n (%) ( . ) . ventricular free wall rupture, n (%) ( . ) . need for dialysis, n (%) ( . ) . ventilator-associated pneumonia, n (%) ( . ) ( . ) . cardiac arrest, n (%) ( . ) ( . ) . serious arrhythmia, n (%) ( . ) ( . ) < . recurrent mi, n (%) ( . ) ( . ) . stroke, n (%) ( . ) . any adverse event, n (%) ( . ) ( . ) . median los, days (iqr) ( – ) ( – ) . median lvef, % (iqr) ( – ) ( – ) . iabp indicates intra-aortic balloon counterpulsation; iqr, interquartile range; los, length of stay; lvef, left ventricular ejection fraction; mi, myocardial infarction; physician- hems, physician-staffed helicopter emergency medical service; standard-hems, non- physician-staffed helicopter emergency medical service. *continuous variables are expressed as median�iqr. table . predictors of any adverse in-hospital outcomes of st-elevation patients transferred by helicopter emergency medicine services* or % ci p value age . . to . . sex . . to . . killip class . . to . < . ground time . . to . . flight time . . to . . non-physician-staffed hems . . to . . hems indicates non-physician-comprised helicopter emergency medicine flight crews; or, odds ratio. *any adverse in-hospital outcome was defined as having any of the following: death, stroke, cardiac arrest, need for intra-aortic balloon pump, bleeding, cardiogenic shock, new requirement for dialysis, recurrent myocardial infarction, serious arrhythmia, or vascular complication. doi: . /jaha. . journal of the american heart association physician vs non-physician stemi transport outcomes gunnarsson et al o r ig in a l r e s e a r c h explained by a delay from first ecg to hems arrival. second, the standard-hems group was more likely to receive intra- venous nitroglycerin and opioid analgesics, such as morphine and fentanyl. this finding could suggest that flight paramedics and nurses are more likely to give these medications routinely compared with flight physicians. interestingly, in this study, almost % of the patients who received nitroglycerin had rca as the culprit vessel. previous studies have shown that nitroglycerin can cause hypotension and worse outcomes, particularly in the setting of right ventricular infarction. , in a recent study, stemi patients who were given morphine demonstrated decreased response to antiplatelet agents, possibly attributed to delayed gastric transit and impaired absorption. this effect could increase risk of thrombosis and recurrent mi. the aha/acc stemi guidelines recommend morphine for chest pain and pulmonary edema, but this medication has been linked to increased mortality. third, the standard-hems group was more likely receive intravenous fluid bolus, which could lead to pulmonary edema in patients who already have a propensity for high left ventricular filling pressure. baseline demographics and cardiovascular disease risk factors were similar in the groups and thus are unlikely to explain the difference in outcomes. also, baseline killip class— a strong predictor of adverse outcomes —was not signifi- cantly different between the groups. the same was also true when we compared the demographics and patient manage- ment transferred to university of wisconsin without a physician (n= ) and the mayo la crosse group (n= ). the physician- hems group had a higher rate of lad occlusion than the standard-hems group. we think that this difference is also unlikely to have affected our results because lad occlusion has been shown to be associated with worse prognosis, which is contrary to our findings given that the physician-hems group had lower rates of in-hospital adverse outcomes. our study has several limitations that are worth mention- ing. first, this is a retrospective analysis and is potentially subject to documentation and recall bias. second, the physician-hems group was larger than the standard-hems group. this is because the majority (> %) of stemi patients transferred by helicopter to the university of wisconsin is by physician-staffed hems. third, the overall number of patients in our study is modest, but still larger than similar studies on helicopter transport of stemi patients. however, we had enough clinical events to construct robust models for multivariable adjustment. the patient cohorts received treat- ment by different care teams at two hospitals, which is a potential confounder despite similar baseline characteristics. also, the indications for medications (such as nitroglycerin and analgesics) given during transfer were unavailable, and thus we could not assess appropriateness of medication administration. furthermore, we were unable to compare troponin con- centrations (to estimate infarct size) in the groups because university of wisconsin uses troponin-t, but mayo clinic uses troponin-i. however, we had information on postprocedure left ventricular ejection fraction (lvef), and this was not different between the groups. because of study design, we could not report compliance with medications such as anti- thrombotics or beta-blockers before transfer. however, the vast majority of the patients received recommended medical therapy during their acute hospitalization for stemi. last, a more-detailed analysis of the hems crews (eg, physician/ registered nurse [rn] vs rn/paramedic or paramedic/ paramedic) may have given additional insight into crew composition and its association with outcomes. in conclusion, our findings suggest a higher rate of in-hospital adverse outcomes in stemi patients transferred by non-physician-staffed hems. however, there was no difference in the rate of adverse events during transport. the higher risk of in-hospital adverse outcomes could be related to medications such as nitroglycerin or intravenous analgesics in-flight or because of other patient- or transport- related unmeasured confounders. the data are retrospective and thus only hypothesis generating. however, the results could have important clinical implications if confirmed in prospective or randomized trials. one potential implication is a novel recommendation for physician-staffed hems in stemi transfer systems. a cost-benefit analysis would be helpful before such implementation because of increased cost related to the addition of a physician to the flight crew. finally, our results underscore the importance of judicious use of nitroglycerin and intravenous analgesics for stemi patients. further investigation of administration of these medications during transport is needed. sources of funding this project was supported, in part, by the university of wisconsin institute for clinical and translational research (uw ictr), funded through an nih clinical and translational science award (ctsa), grant number ul tr , ncats, and the herman and gwendolyn shapiro summer research program, and the university of wisconsin school of medicine and public health department of medicine. disclosures none. references . keeley ec, boura ja, grines cl. primary angioplasty versus intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of randomised trials. lancet. ; : – . doi: . /jaha. . journal of the american heart association physician vs non-physician stemi transport outcomes gunnarsson et al o r ig in a l r e s e a r c h . blankenship jc, skelding ka, scott td, berger pb, parise h, brodie br, witzenbichler b, gaugliumi g, peruga jz, lansky aj, mehran r, stone gw. predictors of reperfusion delay in patients with acute myocardial infarction undergoing primary percutaneous coronary intervention from the horizons- ami trial. am j cardiol. ; : – . . jacobs ak, antman em, faxon dp, gregory t, solis p. development of systems of care for st-elevation myocardial infarction patients: executive summary. circulation. ; : – . . mathews r, peterson ed, li s, roe mt, glickman sw, wiviott sd, saucedo jf, antman em, jacobs ak, wang ty. use of emergency medical service transport among patients with st-segment-elevation myocardial infarction: findings from the national cardiovascular data registry acute coronary treatment intervention outcomes network registry-get with the guidelines. circulation. ; : – . . o’gara pt, kushner fg, ascheim dd, casey de, chung mk, de lemos ja, ettinger sm, fang jc, fesmire fm, franklin ba, granger cb, krumholz hm, linderbaum ja, morrow da, newby lk, ornato jp, ou n, radford mj, tamis-holland je, tommaso cl, tracy cm, woo yj, zhao dx, anderson jl, jacobs ak, halperin jl, albert nm, brindis rg, creager ma, demets d, guyton ra, hochman js, kovacs rj, kushner fg, ohman em, stevenson wg, yancy cw. accf/aha guideline for the management of st-elevation myocardial infarction: a report of the american college of cardiology foundation/american heart association task force on practice guidelines. circulation. ; :e –e . . topol ej, fung ay, kline e, kaplan l, landis d, strozeski m, burney re, pitt b, o’neill ww. safety of helicopter transport and out-of-hospital intravenous 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Øllgaard l, h€oiby p, rasmussen ls. impact of a physician-staffed helicopter on a regional trauma system: a prospective, controlled, observational study. acta anaesthesiol scand. ; : – . . dalby m, bouzamondo a, lechat p, montalescot g. transfer for primary angioplasty versus immediate thrombolysis in acute myocardial infarction: a meta-analysis. circulation. ; : – . . ferguson jj, diver dj, boldt m, pasternak rc. significance of nitroglycerin- induced hypotension with inferior wall acute myocardial infarction. am j cardiol. ; : – . . isis- (fourth international study of infarct survival) collaborative group. isis- : a randomised factorial trial assessing early oral captopril, oral mononitrate, and intravenous magnesium sulphate in , patients with suspected acute myocardial infarction. lancet. ; : – . . farag m, srinivasan m, gorog d. morphine use impairs thrombotic status in patients with st-elevation myocardial infarction undergoing primary percuta- neous coronary intervention. j am 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(abstract only). . meine tj, roe mt, chen ay, patel mr, washam jb, ohman em, peacock wf, pollack cv, gibler wb, peterson ed. association of intravenous morphine use and outcomes in acute coronary syndromes: results from the crusade quality improvement initiative. am heart j. ; : – . . degeare vs, boura ja, grines ll, o’neill ww, grines cl. predictive value of the killip classification in patients undergoing primary percutaneous coronary intervention for acute myocardial infarction. am j cardiol. ; : – . . califf rm, pieper ks, lee kl, van de werf f, simes rj, armstrong pw, topol ej. prediction of -year survival after thrombolysis for acute myocardial infarction in the global utilization of streptokinase and tpa for occluded coronary arteries trial. circulation. ; : – . doi: . /jaha. . journal of the american heart association physician vs non-physician stemi transport outcomes gunnarsson et al o r ig in a l r e s e a r c h wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ doi: . / am. j. hum. genet. : – , an unstable trinucleotide-repeat region on chromosome implicated in spinocerebellar ataxia: a common expansion locus john b vincent, ,* maria l. neves-pereira, andrew d. paterson, etsuko yamamoto, sagar v. parikh, fabio macciardi, hugh m.d. gurling, steve g. potkin, carlos n. pato, antonio macedo, maria kovacs, marilyn davies, jeffrey a. lieberman, herbert y. meltzer, arturas petronis, and james l. kennedy neurogenetics section and bipolar clinic, clarke division, centre for addiction and mental health, university of toronto, toronto; molecular psychiatry laboratory, windeyer institute of medical sciences, department of psychiatry and behavioural sciences, university college london medical school, london; department of psychiatry, university of california at irvine, irvine; department of psychiatry, state university of new york at buffalo, buffalo; department of psychiatry, faculty of medicine, and center for neuroscience, university of coimbra, coimbra, portugal; university of pittsburgh school of medicine, department of psychiatry and western psychiatric institute and clinic, pittsburgh; department of psychiatry, case western reserve university, cleveland; department of psychiatry, university of north carolina, chapel hill; and department of psychiatry, vanderbilt university, nashville larger cag/ctg trinucleotide-repeat tracts in individuals affected with schizophrenia (scz) and bipolar affective disorder (bpad) in comparison with control individuals have previously been reported, implying a possible etio- logical role for trinucleotide repeats in these diseases. two unstable cag/ctg repeats, sef - b and erda , have recently been cloned, and studies indicate that the majority of individuals with large repeats as detected by repeat-expansion detection (red) have large repeat alleles at these loci. these repeats do not show association of large alleles with either bpad or scz. using red, we have identified a bpad individual with a very large cag/ ctg repeat that is not due to expansion at sef - b or erda . from this individual’s dna, we have cloned a highly polymorphic trinucleotide repeat consisting of (cta)n (ctg)n, which is very long (∼ , bp) in this patient. the repeat region localizes to chromosome q , within . cm of fragile site fra c. repeat alleles in our sample were unstable in ( . %) of meioses. large alleles ( repeats) were observed in ( . %) of , patients with psychosis, borderline personality disorder, or juvenile-onset depression and in (. %) of healthy controls. very large alleles were also detected for centre d’etude polymorphisme humaine (ceph) reference family . this triplet expansion has recently been reported to be the cause of spinocerebellar ataxia type (sca ); however, none of our large alleles above the disease threshold occurred in individuals either affected by sca or with known family history of sca. the high frequency of large alleles at this locus is inconsistent with the much rarer occurrence of sca . thus, it seems unlikely that expansion alone causes sca ; other genetic mechanisms may be necessary to explain sca etiology. introduction trinucleotide-repeat expansion (tre) is associated with a number of neurological disorders and, in most of these diseases, provides a molecular basis for the observation of genetic anticipation. this has led many researchers to look for evidence of tre as a possible etiological cause for neuropsychiatric diseases such as bipolar affective disorder (bpad) and schizophrenia (scz). although an- received april , ; accepted for publication december , ; electronically published march , . address for correspondence and reprints: dr. john vincent, de- partment of genetics, room , hospital for sick children, university avenue, toronto, on m g x , canada. e-mail: jvincent@genet.sickkids.on.ca * present affiliation: department of genetics, hospital for sick chil- dren, toronto. � by the american society of human genetics. all rights reserved. - / / - $ . ticipation has been reported for both bpad and scz in many studies (mcinnis and margolis ), the perva- sive presence of ascertainment biases and lack of an ap- propriate statistical test means that it would be impru- dent to draw any firm conclusions from these findings (paterson et al. ). although early reports have sug- gested that dna from bpad and scz patients contains significantly longer stretches of cag/ctg repeats, as determined by repeat-expansion detection (red; lind- blad et al. ; morris et al. ; o’donovan et al. , ), several studies have contradicted these findings (petronis et al. a; vincent et al. , , b; laurent et al. ; li et al. ; zander et al. ; parikh et al., ). it is interesting to note that the sizes of trinucleotide repeat reported to be as- sociated with psychosis are, in general, larger than repeat expansions associated with huntington disease or spi- nocerebellar ataxias (scas). more recently it has been am. j. hum. genet. : – , table patient sample sources, numbers, and diagnostic criteria collection center diagnostic group n diagnostic instrument and criteria toronto schizophrenia scid/dsm-iiir italy schizophrenia sads/rdc portugal schizophrenia opcrit cleveland schizophrenia sads/rdc long island schizophrenia sads/rdc irvine schizophrenia dsm-iiir toronto schizophrenia scid/dsm-iv toronto bipolar affective disorder scid/dsm-iv london bipolar affective disorder sads/rdc cleveland borderline personality disorder scid/dsm-iv cleveland juvenile-onset psychosis scid/dsm-iv pittsburgh juvenile-onset depression k-sads/rdc demonstrated that large repeats at two specific loci, sef - b at q . (breschel et al. ) and erda at q . (nakamoto et al. ; ikeuchi et al. ), are responsible for the majority of the large-repeat tracts detected by red, and the distribution of large repeats at these two loci is similar in bpad, scz, and unaffected populations (lindblad et al. ; sidransky et al. ; vincent et al. b). although there is no clear evidence that expansion at either locus may be pathogenic, be- cause expansions in either moderate or large range at sef - b do not segregate in scz or bpad families (breschel et al. ; sirugo et al. ), to date, ex- pansions repeats at erda have been identified in a single family with childhood-onset depression (vin- cent et al. a). these cases represent only a small fraction of families examined. to eliminate any possible role of other large tres in bpad and scz, it is nec- essary to identify and analyze the remaining large repeats that occur in our bpad and scz sample. we identified a bpad individual, a , with a large red product but no large repeats at sef - b or erda (parikh et al. ). red evidence from this individual suggested the presence of a large stretch of cag/ctg repeats bp. in the present study, we describe the cloning and characterization of this large trinucleotide repeat, the screening for large repeats at this locus in psychosis and control populations, and association and linkage disequilibrium studies for scz and bpad at this locus. since this repeat was cloned, it has emerged that expansion at this repeat region has recently been reported to be the cause of spinocerebellar ataxia type (sca [mim ]; koob et al. ). data presented in this article, however, suggest that ex- pansion at this locus is common in the background population. subjects and methods patient and control sample selection description of patient source and diagnostic methods are given in table . all patients were screened for ab- sence of major medical and neurological disorders. of the , patients screened for large-repeat alleles, % were caucasian, % asian, % black, and % either of mixed ethnicity or with no information available. fifty-three percent were female, % male, and for % information was not available. mean age was . years (� . years, sd). the control dna samples were obtained from staff members and students at the clarke institute (toronto), case western reserve university (cleveland), university of lexington (kentucky), uni- versity of coimbra (portugal), and members of the pub- lic responding to advertisements, the majority of whom had been assessed for absence of psychiatric illness. of the control individuals analyzed, % were cau- casian, % asian, % black, and % either of mixed ethnicity or for whom information on ethnicity was una- vailable. forty-six percent were female, % male, and % sex unknown, and the mean age was . years (� . years). ninety-three proband-mother-father trios from the toronto bpad sample and trios from the toronto and italy scz samples were available for genotyping for transmission disequilibrium analysis. lo- cal ethical committee approval was obtained, blood was drawn after written informed consent was obtained from each subject, and dna was extracted according to stan- dard procedures. dna from lymphoblastoid cell lines for ceph pedigrees , , , , , , , , and were purchased from bios corp. dna samples for the old order amish pedigree and for ceph pedigree were pur- vincent et al.: sca : a common expansion locus figure southern blot of ecori-digested genomic dna hy- bridized with a large ( . – -kb), nonspecific cag/ctg repeat probe. lane shows a strong band at . – kb (arrow) for individual a . lanes – and contain dna from other bpad and scz individuals. figure red on primary lgt clones (a–j) picked from ge- nomic library (screened with cag/ctg probe) generated from indi- vidual a (red from genomic dna: lane number). myotonic dys- trophy positive control is shown in lane dm. size marker (sequamark size ladder; research genetics) is shown in lane m. chased from the coriell institute, and the most recent diagnostic information was made available by e. i. ginns and j. egeland. genomic library construction and screening the method used for cloning was similar to the di- rect strategy (sanpei et al. ), except that a very- large-repeat probe ( . – kb) generated by asymmetric pcr (petronis et al. b) was used for screening at very high stringency. genomic dna from bpad indi- vidual a (previously identified as giving large red products for the triplet cag/ctg but without large alleles at either of two commonly expanded repeats at sef - b or erda ; vincent et al. b) was digested with ecori and cloned into lgt (stratagene). the li- gated vector was packaged by means of maxplax pack- aging extracts (epicentre technologies) and plated with nm escherichia coli cells to give ∼ pfu, . # with average insert size . kb. the library was screened with a . – -kb cag/ctg probe generated by tem- plate-independent pcr using the complementary prim- ers [cag] and [ctg] , as described elsewhere (petronis et al. b). southern hybridization of ecori-digested genomic dna with use of the same probe revealed a strong band for a at . – kb (fig. ). after secondary screening, positive plaques were selected and dna prepared according to standard procedures. red anal- ysis (schalling et al. ; vincent et al. ) was per- formed on the clones, confirming the presence of a large cag/ctg repeat in clone l a (fig. ). a second library was constructed in the same way from genomic dna from another individual with large red products (with- out large alleles at sef - b or erda ). clone l - a was identified as an unexpanded version of l a. sequence analysis the . -kb ecori insert from clone l a was sub- cloned into m and sequenced by use of a li-cor long reader . sequence analysis of clone l - a and of alleles at this locus was performed on an abi prism (applied biosystems), with lgt forward and re- am. j. hum. genet. : – , verse primers and specific primers acag, actg, and aext (genbank accession af , ′ to ′ nucleotides – , – , and – , respectively). pcr genotyping.—pcr genotyping was performed with the primers acag and actg; �c for min followed by cycles of �c for s, �c for s, �c for s under standard conditions, followed by % polyacrylamide gel electrophoresis, blotting onto hybond-n� (amersham), hybridization using p- end-labeled [ctg] oligonucleotide followed by autoradiography. southern hybridization analysis.—to screen efficiently for the presence or absence of large expansions that may not amplify sufficiently for pcr detection, all dna sam- ples that were apparent homozygotes and all those that failed to amplify were checked by southern hybridiza- tion. this was performed according to standard pro- cedures, with ecori-restricted genomic dna ( mg), and the . -kb ecori insert from clone l - a as the hybridization probe. this insert contains a much shorter repeat stretch than clone l a, thus permitting much less cross-hybridization with other repeat loci. allele-specific oligonucleotide (aso) analysis.— filters from the genotype analysis were stripped ( . % sds, �c) and reprobed with either aso ( ′-tac- tactgctgc- ′) or aso ( ′-tactgctactgc- ′). aso and aso were ′ end-labeled with polynucle- otide kinase and p-gatp. hybridization was performed with use of amasino buffer at �c, and filters were washed at . �c in # ssc, . % sds. statistical analysis allele distributions for patient and control samples were compared by means of a rank-sum test (mann- whitney). preferential transmission of alleles in the trios was analyzed by means of an extended transmission/ disequilibrium test (etdt; sham and curtis ). linkage analysis for the old order amish pedigree was performed by mlink from the fastlink suite (ter- williger and ott ). test for association was per- formed by means of x analysis (spss . ). chromosomal localization the trinucleotide repeat was localized to chromosome by pcr screening of the nigms somatic cell hy- brid panel (rodent/human hybrid). subchromosomal localization was performed by pcr screening the genebridge radiation hybrid panel (research genetics) and anchored ceph yacs. northern blot and cdna screening human multiple-tissue northern blots and human adult and fetal brain cdna libraries hl b and hl a (clontech) were screened with probes flanking the repeat region according to the manufacturer’s instructions. results cloning and characterization of l a southern hybridization, followed by stringent wash- ing, of ecori-digested genomic dna with a large, non- specific cag/ctg repeat probe ( . – kb) revealed a strong signal band at . – kb (fig. ). ecori-digested genomic dna from this patient was used to generate a lgt /genomic library, which was then screened for re- peat containing clones by use of the large cag/ctg repeat probe. of clones identified, one contained a large cag/ctg repeat (fig. ). the red analysis dis- played in figure shows ligation products upward of [cag] ; however, this analysis used a large excess of the l a template dna. titration of the template dna was performed, and, at pg, the ligation product size is closer to [cag] . this clone, l a, contained a . - kb ecori fragment. the . -kb fragment was subclon- ed into m and sequenced and showed a stretch of cta repeats followed directly by ctg repeats (nt – ; genbank af ). a second clone, l - a , was identified from a second genomic library from another individual, which contains the same flanking sequence, and was used as confirmation of the sequence. the single insert from this clone was only bp long and contained only cta/ctg repeats. the ∼ -bp discrepancy in size arises from an apparent ecori poly- morphism (af , nt ), which was born out by genomic southern hybridization for a , which showed a -kb size difference between normal and ex- panded alleles for ecori and only a . -kb size differ- ence for psti and hindiii digests (fig. a, b). the south- ern hybridization evidence also confirmed that the repeat size is much larger in the genomic dna (∼ cta/ ctg repeats) than in l a ( repeats: cta ctg repeats), suggesting that contraction of the repeat oc- curred during the cloning procedure. no size mosaicism was observed for the large repeat allele in lymphocyte dna. in normal alleles, the cta repeat has either eight or nine copies, whereas the ctg repeat varies from to copies. pcr genotyping was performed for , caucasian, asian, and black unrelated individ- uals from the combined patient and control sample. dis- tribution of alleles (scored as the sum of cta and ctg repeats) is shown in figure a and shows interethnic differences. patient a , as well as a diagnosis of bpad i, also suffers from familial tremor, asthma, eczema, and thyroiditis. the sole sibling of a , who also has tremor but is unaffected by bpad, does not possess an expanded allele. clone l - a has several single-base-pair dis- figure southern hybridization analysis, which was performed with use of (a) ecori and (b) hindiii on bpad proband a (lane ), sibling of a (lane ), and unaffected individuals (lanes – ) and ecori (c) on scz trio, proband (lane p), father (lane f), and mother (lane m) and (d) on ceph pedigree . family member ceph numbers shown (bottom). lane s represents standard control dna. am. j. hum. genet. : – , figure a, distribution of alleles for different ethnic groups ( , caucasian, asian, and black unrelated individuals from the combined patient-control sample). b, sca allele distribution for unaffected caucasian control individuals and caucasian psychosis and depression individuals (scz [ ], schizoaffective disorder [ ], bpadi [ ], bpadii [ ], borderline personality disordern = n = n = n = [ ], juvenile-onset depression [ ], and major psychosis [ ]).n = n = n = crepancies from sequence af : a c instead of t at nt and a c instead of t at nt . the sequence for sca , af (koob et al. ) , which appears to be same repeat region, according to sequence, local- ization, and allelic distribution, also has cs instead of ts at these positions and also lacks a t at nt of af . our repeat region is referred to as sca , for consistency. ceph control ceph pedigree was screened for the repeat by use of pcr. the paternal grandmother, father, vincent et al.: sca : a common expansion locus and three sons appeared to have only a single allele, although the father and sons were clearly obligate het- erozygotes. very large alleles for these individuals were shown by southern hybridization (fig. d). repeat sizes were determined by means of semi-log calibration curves for the -kb ladder (gibco brl). the paternal grand- mother (gm , aged years) has ∼ repeats, which expands to ∼ repeats in her son (gm ; aged years) and then contracts to ∼ , ∼ , and ∼ repeats in his sons (gm , gm , gm , aged , , and years, respectively). pcr amplification and sequencing showed gm to have cta uninterrupted repeats followed by uninter- rupted ctg repeats. because the ceph pedigree dnas are extracted from lymphoblastoid cell lines and it is not known how stable the repeats are over many passages, the relationship to repeat length in lymphocyte dna is unclear. localization of the repeat region and linkage analysis for bpad in the old order amish the repeat region was localized to within . cr of sts marker wi- . a tiling path of yac clones around wi- was screened for the repeat region by pcr. ceph yacs g and f were positive for the repeat. the rpci- bac library was screened by hybridization with the acag/ actg pcr product and was positive for clone h_nh j . this re- gion maps to q . - . . according to the gb map (genemap ’ ), this maps between anchored markers d s and d s , . – . cm or ( . – . cr) from the p telomere. a number of recent studies have shown positive results for parametric and nonparametric linkage analyses for bpad and scz on q (barden and morissette, ; blouin et al. ; maximum lod score [mls] . at q , ∼ cm distal to sca ). the serotonin receptor htr a, which is a strong candidate gene and for which association to bpad and scz has been reported (gutierrez et al. ; williams et al. ), maps to q , . cm and cr proximal to the repeat. linkage to bpad in the ped- igrees from the old order amish (zmax dominant = . at d s , ∼ cm proximal to sca ; ginns et al. ) and in the national institute of mental health genetics bipolar initiative pedigrees ( at d s , ∼ p = . cm distal to sca ; stine et al. ) has been reported; however, we found no evidence for linkage of sca to either bp i (dominant: mls = � . ; recessive: mls = � . ) or bp i and ii (dominant: mls = � . ; reces- sive: mls = � . ) in amish pedigree . expansion screening in psychosis and control populations we screened , dnas from unrelated patients di- agnosed with psychosis (scz spectrum or bipolar dis- order), juvenile-onset depression, or borderline person- ality disorder (table ) and unrelated controls unaffected with psychiatric illness for expansion at sca . pcr genotyping was used initially and, for con- firmation of expanded alleles, apparent homozygotes or cases of failed amplification, southern hybridization analysis was used. six apparent homozygotes from the patient population, and five from the controls, could not be excluded for expansion, because of poor restriction digestion of dna. seven patients and eight controls that failed to pcr-amplify were excluded for expansion. fourteen patients ( . %) were identified as having large alleles (� repeats [age at interview, in years]: [ ], [ ], [ ], [ ], [ ], [ ], [age not available], [ ], [ ], [ ], [ ], [ ], , [ ], and , [ ] repeats) and (. %) with intermediate-sized alleles (� repeats: [ ], [ ], [ ], [ ] [ ], [ ], [ ], [ ], and [ ] repeats). five controls ( . %) were shown to have large alleles (� repeats: [ ], [ ], [ ], [ ], and [age not available] repeats) and two ( . %) with intermediate alleles (� repeats: [ ] and [age not available] repeats). analysis of the various subgroups according to diagnosis and ethnic group shows the highest clustering of large alleles in scz caucasians (� repeats: , , , , , , , and , repeats; [ . %] of ). x comparison of frequency of large alleles in control and affected groups did not reach sig- nificant levels. association and transmission/disequilibrium analysis for scz and bpad at sca patients and controls for each major ethnic group were analyzed separately, because interethnic difference in al- lele distribution is evident (fig. a). there was no sig- nificant difference in distribution of alleles for un- related caucasian individuals with psychosis (including scz [ ], schizoaffective disorder [ ], bpadn = n = i [ ], bpad ii [ ], borderline personalityn = n = disorder [ ], juvenile onset depression [ ],n = n = and juvenile-onset major psychosis [ ]) and n = unrelated caucasian control individuals (fig. b; mann- whitney rank sum test: -tailed ). subdivision ofp = . the patient group according to diagnosis revealed no significant differences in distribution in comparison with the control group. in a smaller but more closely matched subgroup, bpad individuals and control in- dividuals matched pairwise for age, sex, and ethnicity, the difference in distribution of alleles nearly reaches am. j. hum. genet. : – , table extended transmission/disequilibrium test (etdt; sham and curtis, ) for bpad and scz trios at sca group and value (n) sca alleles: n = n[tac] � n′[tgc] – bpad trios ( ) transmitted untransmitted x . . . . . . . p valuea . . . . . . . sca alleles: n = n[tac] � n′[tgc] scz trios ( ): transmitted untransmitted x . . . . . . . . p valuea . . . . . . . . note.—statistics are not shown for alleles for which fewer than observations were made. a p values are not corrected for multiple testing. significance (mann-whitney rank sum test: -tailed ). we tested bpad trios and scz trios forp = . evidence of transmission disequilibrium at sca (table ). we observed no significant preferential transmission of alleles for either bpad ( , df, ) or x = p = . scz ( , df, ) using an extended trans- x = p = . mission/disequilibrium test (sham and curtis ); however, much larger numbers would be required to exclude transmission disequilibrium for the rarer alleles. the sca repeat is highly polymorphic (observed het- erozygosity . , in caucasian control individuals). fourteen intergenerational instabilities were observed ( / ), including ceph pedigree , for sca : from maternal transmissions (� , � , � , � , � , � , � ; mean change � repeat units), from paternal transmissions (� , � , � , � , � ; mean change � repeat units), and where parental origin of the unstable allele was unclear. the smallest allele for which intergenerational instability was observed was repeats (maternal transmission). in one trio, an unaf- fected father was identified with a stretch of re- peats, which was transmitted to a son diagnosed with scz with a decrease in repeat number to ∼ repeats (fig. c). another trio was identified in which a daughter affected with bpad received an allele with repeats from the mother (unaffected) with repeats. aso analysis all pcr-amplified alleles tested ( control alleles, patient alleles) hybridized with the aso oligo- nucleotide (af variant). no positive hybridiza- tion was observed for the aso oligonucleotide (af variant). alleles that were too large to pcr amplify could not be checked by this approach. northern blot analysis and cdna screening no rna bands or cdna clones corresponding to the sca repeat region were identified from a wide range of tissues, including heart, brain, lung, liver, pancreas, kidney, and skeletal muscle. discussion we have identified, cloned, and characterized an unsta- ble trinucleotide repeat that, along with sef - b and erda , is responsible for the major proportion of the large red products that have been observed in our (and other) studies of cag/ctg repeats in bpad and scz populations. this repeat locus was cloned inde- pendently by koob et al. ( ) and named sca . large alleles at sca are, however, relatively infrequent, at ∼ % in comparison with ∼ % and % for sef - b and erda , respectively. sequence analysis of the clones isolated from the genomic library from individual a revealed a high enrichment for large cag/ctg re- peats and included two clones for sef - b (breschel et al. ) and two clones for cagh (margolis et al. ) as well as the new unstable repeat sca . it is clear that this method of cloning represents a useful ap- proach for identification of new cag/ctg repeats from the genome. large alleles at sef - b have been demonstrated in two of the three ceph pedigrees ( and ; bres- chel et al. ) that were reported to have expansion at the red locus, which was identified and mapped to chromosome by linkage analysis (schalling et al. ). the third ceph pedigree thought to have cag/ ctg expansion at the red locus, , does not have large alleles at sef - b, and, in fact, we have observed vincent et al.: sca : a common expansion locus very large alleles at sca in this pedigree for three of four children tested, the father, and the paternal grand- mother. thus, it appears that the original red locus consists of two loci, one on chromosome and the other on chromosome . the sca locus appears to be frequently unstable ( %; / transmissions), even for relatively small alleles. the majority of increases in repeat size occur during transmission of maternal alleles, and contrac- tions occur predominantly in male transmissions. in ceph pedigree , a maternal transmission results in an increase from ∼ to ∼ repeats, and three paternal unstable transmissions result in contractions from ∼ to ∼ , , and repeats. one un- stable paternal transmission observed in an scz trio resulted in a contraction from ∼ to ∼ repeats. it is difficult to gauge the respective contributions of the ctg repeats and cta repeats in the expanded al- leles, although both are enlarged in clone l a ([cta] [ctg] ). we assume that the ctg repeat is most likely the more dynamic of the two repeats, judg- ing by the available evidence (the high degree of het- erogeneity of the ctg repeat size compared with that for cta at sca and the relative lengths of the two repeats in clone l a). the cta repeat is enlarged to copies in the clone l a, but it is unclear whether this is expanded further in the large allele of proband a . the trinucleotide-repeat locus has now been ascribed to sca (koob et al. ). although no homologies were found for the sequence flanking the repeat, and our northern blot analysis and cdna library screening did not show any evidence of transcripts, koob et al. ( ) have identified a transcript from cerebellar polya rna that contains the cta;ctg repeat in the ′ utr. the inheritance pattern of disease in sca appears complex. penetrance of expanded alleles appears to be dependent on size of repeat and, probably, age, and pathogenic alleles appear to be mainly of maternal or- igin, possibly because of parental origin effect, whereby the paternal transmissions tend toward contraction and maternal transmissions tend toward expansion. despite the differential parental effect on repeat instability lead- ing to an apparent maternal penetrance bias in the large kindred, imprinting has been ruled out, because four cases in other families are reported in which sca is transmitted paternally and biallelic expression of the repeat is also demonstrated in the cerebellum (koob et al. ). it is, however, worth noting that the htr a gene, which is believed to exhibit polymorphic imprint- ing (bunzel et al. ), is close to sca (as close as . cm in male meioses; genetic location database). the authors conclude that the bias in maternal versus paternal transmissions of disease alleles is due to the differential instability for maternal and paternal meio- ses, which we also observed among our trio samples and ceph pedigrees. the repeat sizes in ceph pedigree (fig. d) neatly demonstrate the mutation dynam- ics during maternal and paternal transmissions. the rate of very large alleles for sca is significantly higher in our study ( %; independent cases with alleles � repeats of , independent cases and controls studied) in comparison to the frequency of scas ( . %, or / , ; koob et al. ). even in the very narrow pathogenic repeat range defined by koob et al ( ) of – ctg repeats or – combined repeats, our data still reveal . % of affecteds and . % of controls with repeats in this range. these data suggest that an etiologic factor other than expansion at sca is required for the development of ataxia. the genbank sequences for the sca trinucleotide repeat from koob et al. ( ) and from the present study (af and af ) differ at the junction between the two re- peats (af : [tac]n tgctac[tgc]n; af : [tac]n[tgc]n). our sequence analysis and also aso analysis failed to identify any alleles, either normal or expanded, with the af version. this suggests that the af variant at this position may be as- sociated with ataxia; however, no patients with sca have been tested to confirm this theory. another pos- sibility might be that the ratio of expansion of the cta and ctg triplets, as well as expansion itself, is impor- tant for onset of disease. it is of note that the sca sequence (koob et al. ; af ) has only uninterrupted cta triplets and ctg triplets, whereas clone l a (af ) has cta triplets and ctg triplets. thus, cta repeats represent % of the total repeat in af compared with . % in af . the observation of a large sca allele, repeats long, with several interruptions and stably trans- mitted from an unaffected parent (quan et al. ) suggests that repeat content, as well as size, may be important where genotype-phenotype correlations are inconsistent. sequence analysis of repeats in expansions in sca patients compared with expansions in unaf- fected individuals at the sca locus may be required. another alternative could be that all the very-large- repeat individuals detected in this study are nonpene- trant for sca , because of epigenetic modification, so that the expanded allele is not fully expressed. this could result from either ( ) sca undergoing methyl- ation of large repeat alleles, causing gene silencing, as occurs at fmr in fragile x syndrome or ( ) chromatin rearrangement or nucleosome repositioning around the expanded allele, preventing or impeding transcription of the gene. nucleosome repositioning/chromatin re- arrangement caused by large tre raises the possibility that large expansion at sca could affect expression of other nearby genes. the finding of a higher frequency of expansion alleles for sca among individuals with psychosis in comparison with controls may imply a role am. j. hum. genet. : – , for sca or a nearby gene as a susceptibility locus for psychosis, and further studies are implicated. because the expansion alleles are relatively rare in affected and control populations (frequency ∼. ) and the predicted effect relatively modest (odds ratio ∼ . ), a very large n ( , ) will be required to achieve statistical sig- nificance at an % power level. studies of families with psychosis and large sca repeats may be a more re- alistic approach for determination of the relative risk of large repeats for onset of psychosis. sca is clearly anomalous in comparison to the other tre spinocerebellar ataxias in that ( ) the triplet is ctg rather than cag, ( ) the repeat is noncoding, ( ) there is a bias toward expansion in maternal transmis- sion and contraction in paternal transmission, ( ) a much larger range of expansions is observed, in patients and controls, and ( ) expansion is relatively frequent in the background population. further work is necessary to determine whether, and to what degree, tre at sca actually plays a role in sca and whether expansion is a susceptibility factor for psychosis. acknowledgments much gratitude is owed to jennifer skaug, seema khan, barbara kallam, and jo-anne herbrick, at the centre for ap- plied genomics, and the hospital for sick children, for help with the sequencing and mapping work. we also thank dr. vural ozdemir for providing patient dna samples. the efforts of tasha cate at centre for addiction and mental health in compiling much of the demographic data are much appreci- ated. this work was supported by funding from the scottish rite schizophrenia research program, the ontario mental health foundation (omhf), the medical research council of canada (mrc; grant mt ), and the national alliance for research on schizophrenia and depression. partial support was also obtained from national institute for mental health grants mh and poimh . the bpad trio collec- tion was funded by axys pharmaceuticals. j.b.v. is a medical research council/ schizophrenia society of canada research fellow; a.d.p. is an mrc research fellow, and a.p. holds an omhf new investigator fellowship. electronic-database information accession numbers and urls for data in this article are as follows: genbank database, http://www.ncbi.nlm.nih.gov/genbank /genbankoverview.html genemap’ , http://www.ncbi.nlm.nih.gov/genemap / genetic location database, http://cedar.genetics.soton.ac.uk/ public_html/ldb.html online mendelian inheritance in man (omim), http://www .ncbi.nlm.nih.gov/omim (for sca [mim ]) references barden n, morissette j ( ) chromosome workshop. psychiatr genet : – blouin j-l, dombroski ba, nath sk, lasseter vk, wolyniec ps, nestadt g, thornquist m, et al ( ) schizophrenia susceptibility locus on chromosomes q and p . nat genet : – breschel ts, mcinnis mg, margolis rl, sirugo g, corne- liussen b, simpson sg, mcmahon fj, et al ( ) a novel, heritable, 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oyake m, sasaki h, wakisaka a, et al ( ) identification of spinocerebellar ataxia type gene using a direct identification of repeat expansion and cloning technique, direct. nat genet : – schalling m, hudson tj, buetow kh, housman de ( ) direct detection of novel expanded trinucleotide repeats in the human genome. nat genet : – sham pc, curtis d ( ) an extended transmission/disequi- librium test (tdt) for multi-allele marker loci. ann hum genet : – sidransky e, burgess c, ikeuchi t, linblad k, long rt, phi- libert ra, rapoport j, et al ( ) a triplet repeat on q accounts for most expansions detected by the repeat-expan- sion-detection technique. am j hum genet : – sirugo g, pakstis aj, kidd kk, matthysse s, levy dl, holz- man ps, parnas j, et al ( ) detection of a large ctg/ cag trinucleotide repeat expansion in a danish schizo- phrenia kindred. am j med genet : – stine oc, mcmahon fj, chen l-s, xu j, myers da, mackinnon df, simpson s, et al ( ) initial genome screen for bipolar disorder in the nimh genetics initiative pedigrees: chromosomes , , , , and x. am j med genet : – terwilliger jd, ott j ( ) handbook of human genetic link- age. johns hopkins university press, baltimore vincent jb, kalsi g, klempan t, tatuch y, sherrington rp, breschel t, mcinnis mg, et al ( ) no evidence of ex- pansion of cag or gaa repeats in schizophrenia families and monozygotic twins. hum genet : – vincent jb, klempan t, parikh ss, sasaki t, meltzer hy, si- rugo g, cola p, et al ( ) frequency analysis of large cag/ctg trinucleotide repeats in schizophrenia and bi- polar affective disorder. mol psychiatry : – vincent jb, kovacs m, krol r, barr cl, kennedy jl ( a) intergenerational cag repeat expansion at erda in a family with childhood onset depression, schizoaffective dis- order and recurrent major depression. am j med genet : – vincent jb, petronis a, strong e, parikh sv, meltzer hy, lie- berman j, kennedy jl ( b) analysis of genome-wide cag/ctg repeats, and at sef - b and erda in schizo- phrenia and bipolar affective disorder. mol psychiatry : – williams j, spurlock g, mcguffin p, mallet j, nothen mm, gill m, aschauer h, et al ( ) association between schizophrenia and t c polymorphism of the -hydrox- ytryptamine type a-receptor gene. lancet : – zander c, schurhoff f, laurent c, chavand o, bellivier f, samolyk d, leboyer m, et al ( ) cag repeat sequences in bipolar affective disorder: no evidence for association in a french population. am j med genet : – 	an unstable trinucleotide-repeat region on chromosome implicatedin spinocerebellar ataxia: a common expansion locus 	introduction 	subjects and methods 	patient and control sample selection 	genomic library construction and screening 	sequence analysis 	statistical analysis 	chromosomal localization 	northern blot and cdna screening 	results 	cloning and characterization of t a 	localization of the repeat region and linkage analysis for bpad in the old order amish 	expansion screening in psychosis and control populations 	association and transmission/disequilibrium analysis for scz and bpad at sca 	aso analysis 	northern blot analysis and cdna screening 	discussion 	references intermittent hypoxia during recovery from neonatal hyperoxic lung injury causes long-term impairment of alveolar development: a new rat model of bpd rapid report intermittent hypoxia during recovery from neonatal hyperoxic lung injury causes long-term impairment of alveolar development: a new rat model of bpd anastasiya mankouski, , crystal kantores, mathew j. wong, , julijana ivanovska, amish jain, , eric j. benner, stanley n. mason, a. keith tanswell, , , richard l. auten, and robert p. jankov , , division of neonatology, department of pediatrics, neonatal-perinatal research institute, duke university medical center, durham, north carolina; physiology & experimental medicine program, hospital for sick children research institute, toronto, ontario, canada; faculty of medicine, department of pediatrics, university of toronto, toronto, ontario, canada; and faculty of medicine, department of physiology, university of toronto, toronto, ontario, canada submitted october ; accepted in final form november mankouski a, kantores c, wong mj, ivanovska j, jain a, benner ej, mason sn, tanswell ak, auten rl, jankov rp. intermittent hypoxia during recovery from neonatal hyperoxic lung injury causes long-term impairment of alveolar development: a new rat model of bpd. am j physiol lung cell mol physiol : l –l , . first published december , ; doi: . / ajplung. . .—bronchopulmonary dysplasia (bpd) is a chronic lung injury characterized by impaired alveologenesis that may persist into adulthood. rat models of bpd using varying degrees of hyperoxia to produce injury either cause early mortality or spontane- ously recover following removal of the inciting stimulus, thus limiting clinical relevance. we sought to refine an established rat model induced by exposure to % o from birth by following hyperoxia with intermittent hypoxia (ih). rats exposed from birth to air or % o until day were recovered in air with or without ih (fio � . for min every h) until day . animals exposed to % o and recovered in air had no evidence of abnormal lung morphology on day or at – wk. in contrast, % o -exposed animals recovered in ih had persistently increased mean chord length, more dysmorphic septal crests, and fewer peripheral arteries. recovery in ih also increased pulmonary vascular resistance, fulton index, and arterial wall thickness. ih-mediated abnormalities in lung structure (but not pulmonary hypertension) persisted when reexamined at – wk, accompanied by increased pulmonary vascular reactivity and de- creased exercise tolerance. increased mean chord length secondary to ih was prevented by treatment with a peroxynitrite decomposition catalyst [ , , , -tetrakis( -sulfonatophenyl)- h, h-porphyrin iron (iii) chloride, mg/kg/day, days – ], an effect accompanied by fewer inflammatory cells. we conclude that ih during recovery from hyperoxia-induced injury prevents recovery of alveologenesis and leads to changes in lung and pulmonary vascular function lasting into adulthood, thus more closely mimicking contemporary bpd. chronic neonatal lung injury; bronchopulmonary dysplasia; oxygen toxicity, pulmonary hypertension despite many advances in neonatal care that have led to improved survival of infants born extremely preterm, develop- ment of bronchopulmonary dysplasia (bpd) and associated chronic pulmonary hypertension (pht) remain common ad- verse outcomes in this population ( ). a cardinal feature of contemporary bpd is an inhibition or arrest of alveolar devel- opment ( ). lung findings in fatal bpd typically include “emphysematous” distal lung structure with fewer lung units, areas of septal thickening, microvascular dysplasia/hypopla- sia, and inflammation ( ). remodeling of pulmonary resis- tance arteries due to smooth muscle hyperplasia and distal extension of smooth muscle into normally nonmuscular arteries is a pathognomonic structural finding of associated chronic pht ( ). consequences of moderate-severe bpd include persistent emphysema-like lung changes with airway obstruction and respiratory insufficiency ( , , ) leading to potentially lifelong morbidity [premature lung “aging” ( )] and an in- creased risk of severe comorbidities, including chronic, pro- gressive pht and poor neurodevelopmental outcomes ( , , , ). animal models have provided insights into the pathogen- esis of bpd and/or associated pht and the regulation of alveologenesis ( , ). chronic exposure of neonatal rats to hyperoxia is a clinically relevant injury ( ), leading to changes in lung structure and function that mimic bpd ( ). however, to maximize clinical relevance, abnormal lung pa- thology should persist into adult life. limitations of current rat hyperoxia models are that they are either rapidly lethal [� % o for � days ( , )] or, as described herein, spontaneously recover upon removal from the inciting stimulus ( % o for days). intermittent hypoxia (ih) is another clinically rele- vant injury, replicating severe desaturation episodes observed in infants with evolving or established bpd ( ). in this study, we sought to refine a rat chronic lung injury model secondary to exposure to % o by following the hyperoxia exposure with ih, hypothesizing that ih may prevent recovery and lead to changes in lung structure and function that persist into adulthood. materials and methods materials. oxygen exposure chambers and automated controllers (oxycycler model a xov) were from biospherix (parish, ny). alcohols, organic solvents, paraformaldehyde, permount, and super- frost/plus microscope slides were from fisher scientific (whitby, ontario, canada). weigert’s resorcin-fuchsin stain was from rowley address for reprint requests and other correspondence: r. p. jankov, hospital for sick children, . peter gilgan centre for research and learning, bay st., toronto, on, canada m g a (e-mail: robert.jankov@ sickkids.ca). am j physiol lung cell mol physiol : l –l , . first published december , ; doi: . /ajplung. . . - / copyright © the american physiological society http://www.ajplung.orgl downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . http://doi.org/ . /ajplung. . . http://doi.org/ . /ajplung. . . mailto:robert.jankov@sickkids.ca mailto:robert.jankov@sickkids.ca biochemical (danvers, ma). anti-�-smooth muscle actin (catalog no. ma - ) was from thermo scientific. anti-myeloperoxidase; (catalog no. a ) was from dako agilent (mississauga, ontario, canada). anti-cluster of differentiation (cd) (catalog no. mca r) was from serotec (raleigh, nc). anti-nitrotyrosine (cat- alog no. – ) was from emd millipore (billerica, ma). avidin- biotin-peroxidase complex immunohistochemistry kits and , - diaminobenzidine staining kits were from vector laboratories (burlingame, ca). biotinylated goat anti-mouse igg (catalog no. ) was from cell signaling technologies (beverly, ma). , , , -tetrakis( -sulfonatophenyl)- h, h-porphyrin iron (iii) chloride (fetpps) and peroxynitrite were from cayman chem- ical (catalog no. ; ann arbor, mi). unless otherwise specified, all other chemicals and reagents were from bioshop (burlington, ontario, canada). injury model. the animal care committee of the hospital for sick children research institute approved this study. timed-pregnant sprague-dawley rats were from taconic farms (germantown, ny). litters were born in the exposure chambers and continuously exposed to hyperoxia ( % o ) or air ( % o ) until postnatal day (pnd) . to avoid potential confounding effects of sex misdistribution and variations in litter size, litters were adjusted on the day of birth to consist of pups: males and females (sex identified by anogenital distance). to avoid maternal toxicity and consequent undernutrition of pups ( ), dams were swapped daily between air and hyperoxia chambers. from pnd , litters were recovered in air with or without ih ( % o for min every h) until pnd . this regimen did not cause mortality or growth restriction, which can independently aug- ment lung injury ( ). in preliminary experiments, intermittent % o had no effect on lung injury, and longer ( -min) periods in % o caused growth restriction (data not shown). for each exposure group, four litters were used ( litters in total). from pnd , animals from each group (equal sex distribution) remained in room air until – wk of age. an additional eight litters ( litters/exposure group) were treated with fetpps ( mg·kg� ·day� daily ip; litters) or . % saline vehicle ( litters), as previously described ( ), from pnd to . a schematic illustration of the injury model and interventions is provided in fig. . pulmonary vascular resistance. two-dimensional echocardiogra- phy and pulsed-wave doppler ultrasound were performed as previ- ously described ( ). to evaluate pulmonary vascular reactivity, measurements were made after a -min exposure to % o . right ventricular hypertrophy. right ventricular hypertrophy (rvh) was quantified using the fulton index (right ventricle/left ventricle � septum dry weight ratio), as previously described ( ). exercise tolerance. adult rats ( – wk of age) were trained daily on an exer- / animal treadmill (columbus instruments, columbus, oh) over a -day period, as previously described ( ). histological studies. lungs from six animals from each group ( male and female from each of separate litters) were air-inflated and perfusion-fixed at constant pressure, embedded in paraffin, sec- tioned, and stained with hematoxylin-eosin, for elastin, or immuno- stained for inflammatory cells, as previously described ( , , ). for all analyses, measurements were carried out on four noncontigu- ous left lung sections per animal by an observer blinded to group identity. analysis of mean chord length (lm), tissue fraction, counts of peripheral arteries (vessels of external diameter – �m with both internal and external elastic laminae visible), and counts of elastin- stained secondary crests were conducted as previously described ( , , ) from random nonoverlapping low-power fields imaged from each section. lm was normalized to lung volume, quantified by water displacement ( ). tissue fraction normalized to lung volume, a measure of the volumetric proportion of tissue compared with total area, was performed using the point-counting method, as described by weibel ( ). septal crests were considered dysmor- phic if elastin at the septal tip was frayed (vs. compact and dense for normal crests). measurement of arterial medial wall area, arterial muscularization, immunostaining for -nitrotyosine, cd , and myeloperoxidase, and inflammatory (macrophage and neutro- phil) cell counts were all performed as previously described ( , , , ). a nitrotyrosine staining index, representing the per- centage of tissue area reaching a preset staining intensity threshold (normalized to tissue area), was derived as previously described in detail ( ). data presentation and statistical analysis. values are shown as means � se. analyses were performed using sigma plot . (systat software, san jose, ca). statistical significance was determined by one- or two-way anova, then using tukey’s post hoc test, where significant (p 	 . ) intergroup differences were found. results recovery in ih alters lung morphology. exposure to % o for days leads to decreased numbers of secondary crests and peripheral vessels ( ). as shown in fig. , these changes were no longer present after recovery in air until pnd . recovery in ih caused no significant changes in body weight, wet lung weight, or lung volume (measured by displacement) at pnd (data not shown). recovery of % o -exposed animals in ih prevented recovery of alveologenesis, as measured by in- creased lm (fig. a) and decreased tissue fraction (fig. b). in addition, we observed fewer peripheral arteries (fig. c) and nondysmorphic secondary crests (fig. f) and more dysmor- phic secondary crests (fig. f). interestingly, ih in animals exposed to air from pnd to also caused smaller, but significant, changes in lm (fig. a), tissue fraction (fig. b), and numbers of peripheral arteries (fig. c). recovery in ih causes pht. exposure to % o for days leads to changes of pht, including arterial wall thick- ening and rvh ( ). these changes were no longer present after recovery in air until pnd (fig. ). in contrast, recovery of % o -exposed animals in ih increased the pulmonary vascular resistance (pvr) index (fig. a), rvh (fig. b), arterial medial wall area (fig. c), and increased numbers of fully muscularized arteries (fig. e). ih in animals exposed to air from pnd to also increased arterial medial wall area (fig. b), whereas increases in air or % o air or intermittent hypoxia day day day . % nacl or fetpps interventions exposures - weeks air fig. . schematic illustration of exposures and interventions. l intermittent hypoxia prevents recovery of alveologenesis ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . fig. . changes in lung morphology secondary to intermittent hypoxia. rat pups were exposed to normoxia (air; open bars) or hyperoxia ( % o ; closed bars) from postnatal days to followed by recovery in normoxia (air) or intermittent hypoxia (intermittent hypoxia) until postnatal day . a—c: morphometric analyses of mean chord length (a), tissue fraction (b), and peripheral artery counts (c) (n � animals/group). d and e: representative photomicrographs of hematoxylin and eosin (d) (bar length � �m) and elastin-stained sections (e) (bar length � �m). arrows point to peripheral arteries. f: counts of normal and dysmorphic secondary crests (n � – animals/group). g: representative photomicrographs of elastin-stained sections with arrows pointing to secondary crests. bar length � �m. graph bars represent means � se *p 	 . , by anova, compared with all other groups. #p 	 . , by anova, compared with air-air group. l intermittent hypoxia prevents recovery of alveologenesis ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . fig. . changes in markers of pulmonary hypertension secondary to intermittent hypoxia. rat pups were exposed to normoxia (air) or hyperoxia ( % o ) from postnatal days to followed by recovery in normoxia (air) or intermittent hypoxia (intermittent hypoxia) until postnatal day . a: pulmonary vascular resistance index (n � – animals/group). b: fulton index (right ventricular hypertrophy; n � animals/group). c: percentage medial wall area (arterial wall remodeling; n � animals/group). d: representative high-power photomicrographs of elastin-stained pulmonary arteries (bar length � �m). e: muscular- ization of pulmonary arteries (n � – animals/group). f: representative high-power photomicrographs of �-smooth muscle actin-stained pulmonary arteries (bar length � �m). graph bars represent means � se. *p 	 . , by anova, compared with air-air and % o -air groups. †p 	 . , by anova, for nonmuscular arteries, compared with air-air and % o -air groups. ‡p 	 . , by anova, for fully muscularized arteries compared with all other groups. l intermittent hypoxia prevents recovery of alveologenesis ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . pvr (fig. a) and the proportion of fully muscularized arteries (fig. e) were not significant. recovery in ih causes long-term changes in lung structure, pulmonary vascular reactivity, and exercise capacity. morpho- metric analyses were conducted on animals between and wk of life (young adulthood) following recovery in room air from pnd (fig. ). young adult animals that were exposed to % o -ih had persistently increased lm (fig. a) and fewer peripheral arteries (fig. b). as young adults, animals exposed to % o -ih no longer had evidence of pht [pvr and fulton indexes were comparable to air-exposed controls (p � . ); data not shown]. however, there was increased pulmonary vascular reactivity, shown by a greater pvr index in response to acute hypoxia (fig. d), in animals who recov- ered in ih, whether or not they were initially exposed to hyperoxia. as previously reported ( ), female rats ran a greater distance compared with male rats across all groups; therefore, data were stratified by sex. exercise capacity was fig. . lung structure, pulmonary vascular reactivity, and exercise tolerance in early adulthood. rat pups were exposed to nor- moxia (air; open bars) or hyperoxia ( % o ; closed bars) from postnatal days to followed by recovery in normoxia (air) or intermittent hypoxia (intermittent hypoxia) until postnatal day . thereafter, animals recovered in room air until early adulthood ( wk). a and b: morphometric analyses of mean chord length (a) and peripheral artery counts (b). c: representative low-power photomicrographs of elastin-stained sections (bar length � �m). d: pulmonary vas- cular resistance index following acute expo- sure to % o . e: exercise capacity. bars represent means � se for n � – animals/ group. *p 	 . , by anova, compared with all other groups. #p 	 . , by anova, compared with air-air group. †p 	 . , by anova, compared with air-air and % o -air groups. ‡p 	 . , by anova, compared with air-air and % o -air groups for the same sex. ¶p 	 . , by anova, compared with all other groups for the same sex. l intermittent hypoxia prevents recovery of alveologenesis ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . decreased in both male and female young adult animals ex- posed to % o -ih (fig. e). body weight has been shown in rats to greatly influence endurance, independent of sex ( ). therefore, a likely contributing factor to the sex difference in exercise capacity was the greater body weight of males ( � vs. � g for females; p 	 . by t-test). in accord, distance run was no longer significantly different between sexes when indexed to body weight (data not shown). there were no differences in body weight within either sex between exposure groups (p � . ; data not shown). l intermittent hypoxia prevents recovery of alveologenesis ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . treatment with the peroxynitrite decomposition catalyst fetpps prevents lung structural changes, lung inflammation, and impaired exercise tolerance secondary to ih. clinical and experimental evidence strongly implicates the reactive nitrogen species, peroxynitrite, as a critical mediator of chronic neonatal lung injury ( , ). we treated a subset of animals with fetpps from pnd to to determine the contribution of peroxynitrite to ih-mediated lung injury following exposure to % o . when compared with vehicle-treated animals at pnd , treatment with fetpps reduced -nitrotyrosine immuno- reactivity secondary to % o -ih (fig. a), normalized lm (figs. b), and increased the proportion of nondysmorphic secondary crests (fig. c). these effects were associated with attenuation of lung inflammation, with fewer tissue macro- phages (fig. d) and neutrophils (fig. e). preventive effects of fetpps extended into young adulthood, preserving lm (fig. f) and increasing exercise capacity (fig. g). discussion much of our understanding of the pathogenesis of bpd comes from animal models ( , ). the usefulness of such models is in large part determined by the extent to which they replicate the pathophysiology in human infants ( ). unlike the original pathological description, so-called “new bpd” lacks the florid airway damage and fibrosis, characterized instead by arrested alveolar development. term rodents are highly suit- able as models for neonatal lung injury, since alveolar devel- opment is a postnatal event, as it is in prematurely born humans ( ). rats are the preferred rodent species for modeling chronic pht associated with bpd, since vascular remodeling develops more readily than in mice ( ). in addition, mice lack respira- tory bronchioles, leading to a simplified acinar structure with fewer airway generations than in humans and rats ( ). it is now recognized that inhibition of alveolar development in human neonates with bpd may be permanent and potentially progres- sive, thereby acting as an important antecedent for adult lung diseases such as copd ( ). therefore, it is critical for animal models to replicate permanence of lung injury and for preclin- ical studies to incorporate examination of the long-term impact of interventions. the major novel findings of this study were that arrest of alveologenesis secondary to % o spontaneously resolves, which was prevented by a consecutive “second hit” with ih. intermittent hypoxemic episodes are frequently observed in ex-premature infants with established or evolving bpd ( , ). changes lasting into young adulthood secondary to ih included persistent emphysematous lung structure, reduced vascularity, and decreased exercise capacity. such changes are consistent with a permanent reduction of “lung units,” com- prising alveoli and accompanying distal vessels, thus limiting respiratory capacity and reserve ( ). in addition, while changes of chronic pht improved over time, as reported in most children with bpd-associated pht ( ), we observed hyper- reactivity of the pulmonary vasculature to acute hypoxia, which has been described in young adult survivors of pphn ( ) and may also be present in childhood survivors of bpd ( ). there were no apparent sex differences in the immediate or long-term effects of % o -ih on the above parameters (data not shown); however, we did not power the present study for the purpose of stratifying data by sex. mechanisms known to contribute to % o -induced lung injury include oxidative and nitrative stress, downstream of inflammatory cells and inflammatory mediators, including in- terleukin- and cyclooxygenase- ( , – ). in human in- fants with bpd, -nitrotyrosine levels (a stable marker of reactive nitrogen species, such as peroxynitrite) from circulat- ing and lung-derived proteins are a direct marker of disease severity ( , ). our current findings employing fetpps indi- cate that peroxynitrite also plays a critical role in persistent lung inflammation and abnormal structure mediated by ih. possible downstream effects of peroxynitrite-induced inhibi- tion of alveologenesis, not yet explored, include nitration- mediated changes in expression and function of growth factors ( , ) and mediators of angiogenesis ( , ). pulmonary vascular hyperreactivity has also been described in piglets exposed to brief postnatal hyperoxia, secondary to changes in thromboxane receptor function ( ) and to increased oxidative stress ( ). in conclusion, exposure of juvenile rats to ih during recov- ery from hyperoxia-induced chronic neonatal lung injury caused abnormalities in lung structure and function to persist into young adulthood, thus more closely mimicking contem- porary bpd. we propose that this model could be useful in deriving new insights into the pathogenesis of clinically rele- vant injuries and for preclinical studies incorporating exami- nation of longevity of effects on lung structure and function. grants this work was supported by operating funding from the canadian institutes of health research (mop- to r. p. jankov and mop- to a. k. tanswell) and by infrastructure funding from the canada foundation for innovation (to r. p. jankov). a. mankouski was supported by a t award from the national institute of child health and human development ( t - hd- - ) and by grants from the jean and george brumley, jr., neonatal-perinatal research institute and the derfner-children’s miracle net- work. a. jain was supported by a clinician-scientist training program award from the hospital for sick children research training centre and by a queen elizabeth ii/heart and stroke foundation of ontario graduate scholarship in science and technology. m. j. wong was supported by a graduate scholar- ship from the department of physiology, university of toronto, and by a fig. . effects of treatment with , , , -tetrakis( -sulfonatophenyl)- h, h-porphyrin iron (iii) chloride (fetpps). rat pups were exposed to normoxia from postnatal days to (control; open bars) or % o from postnatal days to followed by intermittent hypoxia from postnatal days to (experimental; closed bars). from postnatal days to , pups received daily injections of vehicle or mg·kg� ·day� fetpps. a: -nitrotyrosine immunostaining index and representative photomicrographs of -nitrotyrosine immunohistochemistry (immunoreactive -nitrotyrosine shown as brown stain; bar length � �m). b—e: mean chord length (b) and counts of normal/dysmorphic secondary crests (c), tissue macrophages (d), and tissue neutrophils (e) at day . representative photomicrographs of cd - or myeloperoxidase-stained sections are shown below each graph [arrows pointing to macrophages (brown) or groups of neutrophils (black); bar lengths � �m]. f and g: mean chord length (f) and exercise capacity (g) in young adult animals following recovery in room air until wk of life. bars represent means � se for n � – animals/group. *p 	 . , by anova, compared with all other groups. #p 	 . , by anova, compared with all other groups. †p 	 . , by anova, compared with air-air-exposed, vehicle-treated group. ‡p 	 . , by anova, compared with all other groups for the same sex. l intermittent hypoxia prevents recovery of alveologenesis ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . lorne phenix graduate award from the cardiovascular sciences collaborative program, faculty of medicine, university of toronto. disclosures no conflicts of interest, financial or otherwise, are declared by the authors. author contributions a.m., c.k., m.j.w., j.i., a.j., e.j.b., and s.n.m. performed experiments; a.m., c.k., and r.p.j. analyzed data; a.m., r.l.a., and r.p.j. interpreted results of experiments; a.m. and r.p.j. prepared figures; a.m., r.l.a., and r.p.j. drafted manuscript; a.m., c.k., m.j.w., j.i., a.j., e.j.b., s.n.m., a.k.t., r.l.a., and r.p.j. edited and revised manuscript; a.m., c.k., m.j.w., j.i., a.j., e.j.b., s.n.m., a.k.t., r.l.a., and r.p.j. approved final version of manuscript. references . ambalavanan n, morty re. searching for better animal models of bpd: a perspective. am j physiol lung cell mol physiol : l –l , . doi: . /ajplung. . . . an hs, bae ej, kim gb, kwon bs, beak js, kim ek, kim hs, choi jh, noh ci, yun ys. pulmonary hypertension in preterm infants with bronchopulmonary dysplasia. korean circ j : – , . doi: . 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http://dx.doi.org/ . / . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ cup_jbs_ .. j. biosoc. sci., ( ) , – , © cambridge university press, . this is an open access article, distributed under the terms of the creative commons attribution licence (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: . /s x first published online feb some uses of models of quantitative genetic selection in social science michael d. weight and henry harpending department of anthropology, university of utah, salt lake city, ut, usa summary. the theory of selection of quantitative traits is widely used in evolutionary biology, agriculture and other related fields. the fundamental model known as the breeder’s equation is simple, robust over short time scales, and it is often possible to estimate plausible parameters. in this paper it is suggested that the results of this model provide useful yardsticks for the description of social traits and the evaluation of transmission models. the differences on a standard personality test between samples of old order amish and indiana rural young men from the same county and the decline of homicide in medieval europe are used as illustrative examples of the overall approach. it is shown that the decline of homicide is unremarkable under a threshold model while the differences between rural amish and non-amish young men are too large to be a plausible outcome of simple genetic selection in which assortative mating by affiliation is equivalent to truncation selection. introduction in genetics quantitative traits like stature, body weight, milk yield in cattle, yield per acre of a field crop or the outcomes of pencil and paper tests for cognitive ability or personality, are traits that can be measured on a continuous scale. the theory of the evolution of quantitative traits under selection is well established in plant and animal breeding and in evolutionary biology (lande, ; falconer, ; walsh & lynch, ). the origin of quantitative genetic theory is r. a. fisher ( ), who modelled a quantitative trait as the additive effects of a large number of genetic loci, allowing for dominance within each locus, and random environmental effects. fisher’s paper ended the controversy between mendelians and biometricians, the two competing approaches to understanding the transmission of heritable traits in the early twentieth century. many regard this paper, after darwin’s origin of species, as the beginning of modern evolutionary biology (provine, ). in practice fisher’s basic model is simple and robust despite potential complications and elaborations. sufficient conditions for the key results are the stochastic corresponding author. email: michael.weight@utah.edu https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at mailto:michael.weight@utah.edu https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core independence of genotypes and environments, the absence of dominance and epistasis (non-additive interactions between genes at different genomic locations) and a linear regression of genotypic values on phenotypes (nagylaki, ). under these assumptions the breeder’s equation relates the change in the average value of some quantitative trait in response to selection as: r=h s: in this equation r, the response to selection, is the difference between the average phenotype of parents and the average phenotype of offspring after a single generation of selection. the term h is the narrow-sense heritability, or the fraction of phenotypic variance in the population attributable to the additive effects of genes. finally, the selection differential, s, denotes the difference between the average phenotype in the population and the average phenotype of parents of the next generation. given two terms of the breeder’s equation, one can estimate the third. agriculture favours estimating heritability through selection experiments. breeders or experimenters fix the selection differential by choosing parents from the population under study. the response r is obtained by measuring the offspring after selection. the heritability of the trait h immediately follows from the breeder’s equation. such experiments cannot be done in the social sciences. instead, heritability is estimated from similarities among relatives. complications can occur in the simple theory. for example, epistatic interactions between loci violate the simple set of assumptions. remarkably, these tend to cancel each other over time. crow ( ) suggested that natural selection generally favours changes that lead to the simple additive model given by the breeder’s equation. complexities have cast doubt over human heritability estimates. for example, a standard source of data has been pairs of identical and fraternal twins. heritability estimates then assumed that identical twins separated at birth are genetically identical and raised in random environments. challenges to this assumption arise from many sources: shared uterine environment of identical twins before birth, non-random placement of adopted twins and others (kamin & goldberger, ). data from the human genome project render many criticisms of twin studies moot. identical twins were of interest because they shared more or less the same genome, but suspicions of shared environment persisted. today the state of technology is to type approximately a million single nuclear polymorphisms (snps) in a large sample of subjects and measure the relationship between individuals by the number of snps they share. realized kinship to the order of second cousins and lower is assessed for tens of thousands of typed subjects. this technology renders studies of real pedigree relationship nearly obsolete while simultaneously validating the conclusions about human heritability estimates that had been so widely denounced in the twentieth century (see visscher et al., ; yang et al., ; davies et al., ; and lee & chow, , for further discussion). thought experiment assortative mating can be equivalent to directional selection. assortment for a trait generates either distinct groups or a continuum: those with more of the trait at one m. d. weight and h. harpending https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core extreme and those with less of the trait at the other, within a population. to illustrate this consider a simple thought experiment about assortative mating for stature in a population. assign average stature of . cm for males and . cm for females, each with a standard deviation of . cm. these figures are close to observed values in north america. suddenly assortative mating by height occurs such that those taller than the population mean mate only with others also above the mean while those shorter than the mean mate only with others shorter than the mean. first, it is assumed that the differences between male and female stature reflect fixed differences in the development of the two sexes. the data can then be normalized for each sex and stature in standard deviation units (sds) measured. the pooled population has a mean of zero and a standard deviation of . . the new units, called z-scores, are obtained by subtracting the population mean from everyone’s stature ( . cm from each male, . cm from each female) and dividing by the standard deviation of . cm. after imposition of the mating rule the population separates into two populations: talls and shorts. with this normal distribution assumption the overall normal distribution has been split into two distributions. the mean of a left half-normal distribution is − . sds and that of the right half + . sds. the difference in population means is therefore . sds, or . cm in the original units. the new subpopulations mate at random within themselves. the two sets of parents differ by . sds. after reproduction, assuming an additive heritability of . for stature, the group difference will be, following the breeder’s equation, . × . = . sds, corresponding to . × . = . cm. after the first generation of reproduction the genotypic group means differ by . cm without further movement between groups. before the new mating system the overall variance of stature was . . after a single generation of the new mating system the between-subpopulation variance is . or . on the standard deviation scale. the fraction of stature variance that is between groups is now %. the variance of stature within each of the two subpopulations is the complement or . . back on the scale of centimetres, the standard deviation within populations is the square root of . ( . ), corresponding to . cm. richard lewontin ( ) famously estimated the fraction of genic variance among continental human groups to be about % for a collection of presumably neutral genetic markers. that estimate of the ratio of between-group to total variance has remained constant as the number of loci available to estimate it grew from dozens to hundreds of thousands. after a single generation of this thought experiment the stature variance between groups at genetic loci influencing stature is between two and three times as great as the overall neutral genic variance among major human populations. yet there is absolutely no selection in this thought experiment. everything is selectively neutral. on the other hand, from the perspective from within subpopulations, there has been strong selection imposed by the mating rule allocating persons to groups according to stature. the mechanism of selecting according to a threshold is called truncation selection. it is, for example, the most common selection rule in animal and plant breeding. here, the threshold was at the mean height of the overall population. it is worth pointing out that after assortative mating the genetic variance within each group is somewhat smaller than what it otherwise could be as a result of negative linkage disequilibrium between causal sites (bulmer, ). this may seem somewhat paradoxical because of the overall positive linkage disequilibrium between causal sites models of selection https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core in the total population (including both talls and shorts) induced by the assortative mating itself (fisher, ; crow & felsenstein, ; nagylaki, ). the reader may consult lee and chow ( ) for an intuitive explanation of the so-called bulmer effect of negative disequilibrium. if random mating prevails within each group after assortment, then the bulmer effect decays; the genetic variance within each group will thus increase. the most important feature of the genetics to keep in mind is that the classical increase in the overall genetic variance due to assortative mating, first described by fisher himself, corresponds to the divergence in means between the two groups formed by the assortment process. the mating rule in this thought experiment about the effects of truncation selection acted on a biometric trait. suppose, instead, individuals in the population were similarly partitioned according to a threshold along a behavioural trait. bailey ( ) pointed out that behavioural traits like religiosity, extraversion, neuroticism and even divorce are all moderately heritable. indeed, eric turkheimer in an essay titled ‘three laws of behaviour genetics and what they mean’ (turkheimer, ) gives ‘all human behavioural traits are heritable’ as his first law of behaviour genetics. rejecting the null hypothesis that behaviours are not heritable is therefore no longer of much interest (bailey, ; turkheimer, ). the genetic effects of the mating rule in the thought experiment on behavioural traits therefore mirror those illustrated using height. despite this, the social sciences have generally neglected quantitative genetic models even though many other quantitative traits like iq (herrnstein & murray, ; jensen, ), the propensity to develop mental illnesses like bipolar disorder and schizophrenia (owen & o’donovan, ; barnett & smoller, ; wray & visscher, ), aggression and antisociality (barker et al., ) and other behavioural traits have significant social and economic consequences. if the rule had its origin as a purely culturally transmitted preference then the effect on the genome would be exactly the same as those described in the thought experiment. purely learned preferences or socially imposed assortments would ‘pick up’ any relevant genes, giving an identical genetic effect. pervasive social transmission of these preferences would therefore probably amplify the heritability of behavioural traits. consider new popular social traits that rapidly spread like a new religious movement, tofu consumption or crack cocaine use. initially there may be no genetic basis for the cultural success of these traits, but suppose the trait fostered reproductive success. standing genetic variation favouring the trait is then subject to selection. consequently, the traits would become more heritable as initially rare genes present within the population became more common. an older example of this effect is due to waddington ( ). he found that flies treated with ether were prone to develop a second thorax. without the ether treatment the trait, bithorax, was absent. however, bithorax began to appear even in the absence of ether after generations of selection for flies vulnerable to the ether treatment. alleles that slightly increased the probability of bithorax, but not enough to bring out this phenotype without the large effect of the ether treatment, were apparently positively selected during this experiment. selection then raised their frequencies high enough to trigger bithorax in the absence of ether. the observed heritability of bithorax increased through a process that waddington called genetic assimilation. though superficially like lamarckian inheritance of acquired characteristics, genetic assimilation has a well-studied quantitative genetic basis. m. d. weight and h. harpending https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core genetic accommodation (west-eberhard, ; braendle & flatt, ) is a generalization of genetic assimilation. even if the hypothetical examples of tofu or crack fostered reproductive success through purely social mechanisms, individuals whose biochemistry was congenial to the new trait would participate more easily and their genotypes would increase in frequency. the preference would become heritable even though it was not initially. the idea that cultural adaptation renders genetic adaptation irrelevant is widespread in anthropology. instead, genetic accommodation suggests that the pervasiveness of cultural transmission in our species has increased overall rates of genetic adaptation (see e.g. hawks et al., ; cochran & harpending, ; laland et al., ; richerson et al., , for further discussion of cultural pressures increasing the rate of human genetic adaptation). the old-order amish assortative mating are there concrete ethnographic examples of assortative mating about a trait analogous to height with the above kind of strong effect? there are several candidates. cochran et al. ( ) proposed that a similar mechanism operated in the evolution of european ashkenazi jews during the middle ages: the ashkenazim were a population closed to immigration, but with the possibility of emigration, that experienced strong selection for economic and managerial success. the parsi of india, a closed class of managers, scholars, musicians and other economically and culturally successful individuals (nelson, ), are another distinct population in which assortment may have been important in their evolution. recently charles murray in coming apart ( ) proposed that assortative mating by education, socioeconomic status and iq is splitting america into distinct and different classes – the ‘coming apart’ of the title. the old-order amish, an anabaptist sect in north america, also follow the pattern of negligible inward, but significant outward, gene flow, making them a potential candidate for selection by assortment. a model is described in which there is an underlying trait that we call aq (amish quotient) by analogy with iq derived from cognitive tests. it is postulated in this model that aq predicts the probability of an individual remaining in the community when adolescents decide to remain and be baptized or to decline membership and emigrate. specifically the measure of aq is a personality measure associated with the plain and simple lifestyle of the amish, postulated to indicate an individual’s underlying affinity for membership in the community. under the proposed mechanism of selection the aq within the amish population would increase each generation as those with lower aqs are truncated each generation through ‘boiling-off’ or defection. it is therefore predicted that declining boiling-off rates over time (see below) are a consequence of this selection mechanism increasing overall population aq each generation within the amish population. the model performs well relative to alternative models of amish defection and retention (see ericksen et al., ; meyers, , ; and greksa, , for review). while old-order endogamy is well described and understood, the trade-offs and constraints of defection are unclear. reductions in defection rates over the last century (meyers, , ; hostetler, ; greksa, ) need explanation. previous models of amish defection emphasize rapid population growth in regions with finite resources, models of selection https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core particularly expensive farm land, and the resulting trade-off between culturally sanctioned high fertility and the ability of families to support their own farms (markle & pasco, ; ericksen et al., , ; wasao & donnermeyer, ). other models focus on the effects of occupational shifts consequent to economic constraints on farming. these models predict that increasing exposure to non-amish lifestyles may tempt youth into defection or interfere with parents’ ability to transmit amish identity (meyers, ; greksa, ). unfortunately predictions of these more familiar approaches to amish defection are difficult to quantify and corroborate. endogamy as the most traditional and ‘plain’ anabaptist group, the old-order amish are pacifists who reserve baptism for believing adults and have strict shunning practices of deviant church members (hostetler, , ; roth, ). such non-hostile ethnocentrism and social solidarity introduce a near absolute boundary that culturally uncouples the amish from the ‘english’, their word for non-amish (huntington, ; savells, ). familiar amish norms like carrying out intragroup discussions in pennsylvania dutch, favouring horse-and-buggy over automobiles, traditional ‘humble’ clothing, absolute pacifism and forsaking appliances like telephones, televisions, refrigerators and indoor toilets, are means to signal this uncoupling from their broader society (fuchs et al., ; kraybill, ). genealogical and genetic analysis reveals the amish have been significantly isolated since old-order founding populations immigrated in the th and th centuries (hostetler, ; kraybill, ; pollin et al., ; hurst & mcconnell, ; lee et al., ). arguably the old-order amish are the most culturally and reproductively isolated anabaptist group in the united states (nolt, ; hostetler, ; kraybill, ). following this isolation, the old-order substitute communal aid for secular public support. initiatives like welfare, social security, life insurance, barn insurance (hurd, personal communication), government agricultural subsidies or public schools (if amish schools are available) are therefore prohibited or strongly discouraged (hostetler, , ; fuchs et al., ; hurst & mcconnell, ; kraybill, ) in the interests of communal self-reliance. amish substitutes for institutional support are primarily mediated through kin networks so extended families bear the emotional and financial burden of the welfare of their members (hurd, ; hostetler, ; hurst & mcconnell, ). children, especially males, rely on kin support to start businesses and farms when they begin their own families (ericksen et al., , ; hurd, ; wasao & donnermeyer, ). highly fertile amish mothers draw on relatives to help with their large families (hewner, ; kraybill, ). costs of disabled, ill, and senior members are mitigated by family and community support (hewner, ). the amish are a healthy population with low incidence of infectious disease, low infant and adult mortality rates, high fertility and high standards of living (mckusick et al., a, b; cross, ; cross & mckusick, ; fuchs et al., ; hewner, ; greksa, ). the pf wittmer ( ) reported the results of a personality questionnaire administered to amish and non-amish, - to -year-old men from daviess county, indiana. m. d. weight and h. harpending https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core the questionnaire was a widely used personality assessment called pf (for sixteen personality factors) developed in the s by raymond cattell and others at the university of illinois (cattell & eber, ). it went through several revisions over the years. each form has questions that are scored to yield sixteen numbers, or factors, supposed to represent the subjects’ sixteen dimensions of personality. these are listed in table along with the adjectives in common use that supposedly describe each trait. no use is made of the details of the traits in this paper: the sixteen numbers for each person in the study are simply regarded as self-reports of interesting characteristics. the test and the scoring system are designed to maximize the independence of the factors so the scores are not redundant indicators of the same trait. in some of the literature factor b is not reported nor used since it assesses intelligence rather than a personality trait. here b is included since these data are treated in the same way as genetic markers like snps would be treated, i.e. the authors are agnostic about the interpretation and meaning of the factors. to make sense out of the array of scores for the amish and non-amish sample a conventional method for data reduction is used called principal components analysis (pca). this provides a least-squares best two-dimensional picture of differences among subjects and differences (i.e. correlations) among variables. the process used is analogous to that of estimating iq. an important finding from cognitive testing is that cognitive ability is essentially one-dimensional. for example, people with high mathematical ability also have large vocabularies. one empirical result of this is that almost anything constructed to test ability functions as a suitable proxy for a proper iq test. sats, gres, mcats and afqts are, for example, nearly interchangeable with iq tests (frey & detterman, ; beaujean et al., ). in addition, the united states general social survey (gss) surveys a large sample of americans every few years. the results are conveniently available on the web (general table . factors assessed in the f questionnaire trait label high low a outgoing, warmhearted reserved, detached b abstract thinking, fast learner concrete thinking, slow learner c unemotional, calm emotional, changeable e assertive, dominant humble, co-operative f cheerful, lively sober, taciturn g conscientious, persistent expedient, undisciplined h venturesome, socially bold shy, retiring i tough-minded, self-reliant tender-minded, sensitive l suspicious, sceptical trusting, accepting m imaginative, bohemian practical, conventional n shrewd, discreet forthright, straightforward o guilt-prone, worrying resilient, self-assured q radical, experimental conservative, traditional q self-sufficient, resourceful group-dependent, affiliative q controlled, compulsive undisciplined, lax q tense, driven relaxed, tranquil models of selection https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core social survey, ). each round of the survey presents each respondent with ten words, asks for their definitions, and then tabulates the variable wordsum that varies from to for that subject. wordsum is a useful proxy for iq in the sense that differences among groups along the variable replicate reported differences among the same groups (jensen, ) from more elaborate tests. the pca of the results from wittmer’s study may reveal an analogous one-dimensional personality trait corresponding to the aq. scattergrams along principal components (groups) and principal co-ordinates (traits) from the pf results are shown in figs and . figure shows patterns among group means, the amish and non-amish from wittmer’s study, along with a collection of other populations culled from the literature. details are given in the caption of fig. . the scatter of traits in the left panel is not very informative. there is no clear pattern, presumably reflecting the design goal of independence among the sixteen factors. the scatter among groups, in the right panel, is more interesting. there are three groups that are on the edge of the scatter: amish, chinese nurses and candidates for high-level management positions in the uk. the outstanding (an) and ordinary (en) chinese nurses (see zhang et al., , for data) are nearly identical to each other on these first two dimensions. generic english (uk), according to data taken from bartram ( ), are more similar to indiana rural young men than they are to high-level managers in the uk. figure shows a broad perspective on group differences, especially the difference between the british norms and the non-amish indiana rural males. fig. . principal components analysis (pca) of pf score differences among groups of - to -year-old indiana men. the left panel portrays similarities (i.e. correlations) among the sixteen traits listed in table . the right panel portrays differences among means of several large samples. am are young amish males from indiana; in are non-amish males from the same indiana county; an and en are outstanding and ordinary chinese nurses, respectively; mk are candidates for high- level manager jobs in the uk; and uk are british norms of the pf test. notice the lack of pattern in the left panel, indicating the relative independence of the sixteen traits. several of the labels have been nudged on the figure to avoid overlaps. m. d. weight and h. harpending https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core the pf test itself is proprietary and widely used in personnel selection. comparable data in the literature are scarce, accounting for this strange choice of chinese nurses and uk managers. they were simply chosen by convenience to convey a sense of the magnitude of group differences. even more dismaying is that a standard way to present pf results is to normalize everyone in a sample to a fixed mean. consequently much of the published pf data are not comparable between groups. the interest in fig. is to show individual differences among young rural indiana men. the scatter shows a nearly complete separation of the amish and non-amish along the horizontal axis (the first principal component). the uk mean is shown for comparison in the right panel as a diamond. remarkably, the overall uk mean is near the centre of the non-amish scatter, implying that they are not very different. the amish are strongly different. in a genetic model the differences are partially the accumulated effects of amish voluntary endogamy along with boiling-off of the young amish in each generation who decide to leave the amish community. the scatter of traits in the left panel of fig. helps identify those traits distinguishing the amish and non-amish along the horizontal axis. traits g, q and i on the extreme left of the horizontal axis are in the space of those with the highest aq. the pf scores show that amish describe themselves, albeit indirectly, as conscientious and persistent (g), fig. . principal components analysis (pca) of individual pf scores of - to -year-old indiana men. the left panel portrays similarities among traits, and the right panel among individuals along with the overall uk population. trait labels are given in table . amish young men are shown as circles, non-amish as squares and the large diamond shows the centroid of the uk standard for comparison. the two panels are dual, so proximity between a trait in the left panel and a subject in the right indicates a high value of that trait in that subject. for example the amish subjects have high values of pf trait g, which is called ‘conscientiousness’ and ‘persistence’ in the literature. non-amish young indiana males have relatively higher scores on trait q , called ‘radical’ and ‘experimental’. models of selection https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core tough-minded and self-reliant (i), and controlled (q ). correspondingly they describe themselves as low in q and e, scoring respectively as conservative/traditional and humble/co-operative. these imputed self-characterizations agree remarkably well with stereotypes of the amish that they hold themselves according to ethnographies. the authors are agnostic about the meaning and uses of the pf and other similar tests except as indirect self-descriptions by subjects. a model of (self-)selection under this model the horizontal axis in fig. is a numeric scale of a trait aq identifying where an individual is in a suite of traits differentiating young amish men from their non-amish neighbours. movement out of the amish community is open to anyone, especially around adolescence. what would be the consequences for the amish community if individuals on the low end of aq were especially prone to leaving the population? it is informative to bring a quantitative genetics model to the phenomenon as a well understood baseline for evaluating competing models. in this spirit it is assumed that aq is a partially heritable quantitative trait. there are enough data on behavioural or psychological trait heritabilities to sensibly estimate the additive heritability of aq, written h , at . . it can be seen below that the conclusions are unaltered if the heritability is set to either a plausible limit of . or . . boiling-off rates vary among communities but . (i.e. % of youth choose to leave per generation) is a reasonable contemporary value. following standard practice in quantitative genetics it is now assumed that aq has an underlying normal or gaussian distribution in the amish population. the % boiling-off rate is equivalent to losing the bottom % of that distribution each generation. it is also assumed that the boiling-off mechanism is equivalent to truncation selection, but other selection schemes will have some equivalent alternative truncation point. with % with the lowest aq leaving each generation, the average aq of those who remain is . standard deviations if the population mean before emigration was . in other words this amount of self-selective emigration leaves behind a population whose average aq is . standard deviations greater than it was before the emigration. assume that mating occurs exclusively within the amish. fixing the heritability of aq at . means that the offspring will regress halfway back to their parental mean. the next generation of children will therefore have an average aq of . standard deviations greater than their parents did before emigration. the process of selective emigration repeats each generation, increasing the mean amish aq by one-tenth of a standard deviation per generation. with years per generation, ‘amishness’ will increase by a full standard deviation in ten generations, or years, unless changes in gene frequency lead to changes in heritability. this is substantial social evolution on a time scale of a few centuries. the modelled aq of an individual is simply the value of that individual along the x-axis of the right panel of fig. . the computed standard deviations of aq for the amish and non-amish subjects are . and . respectively, giving the within-group standard deviation to be . units. interestingly the variances within groups, i.e. the squared within-group standard deviations, differ very little. it is not an unusual finding m. d. weight and h. harpending https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core in experiments that selection has only small effects on the variance of a selected population. falconer and mackay ( ) discussed the outcome of experiments at length in their chapter . the means of the two groups are − . (amish) and . (non-amish) units. their difference, corresponding to the distance between the two group means, is . units. our model implies a difference of this magnitude requires generations, or years, which is substantially longer than the amish have been in the united states. this may suggest that the heritability of aq is higher than the assumed . , that the boiling-off rate was higher in the past (for which there is support), or that the process has been going on longer than ten generations. this last possibility is unlikely. currently no old-order exist in europe. persecution and political sanctions in the th and th centuries had spread european amish families too far apart to form cohesive communities, forcing early european amish to eventually assimilate with non-amish (gascho, ; hostetler, ; crowley, ; nolt, ; roth, ). thus the magnitude of amish endogamy was probably negligible in europe compared with amish endogamous mating in the united states. if correct, this suggests the old-order tenure in europe did not contribute to the differences reported here. taking the results shown in fig. at face value implies the between-group difference ( . standard deviations) is too large to be attributable to just simple genetic change with a predicted difference of . standard deviations, or . standard deviations if heritability is assumed to be as high as . . the best conclusion from comparing the data with the simple genetic model is that other social forces, e.g. family transmission, are mostly responsible for the difference between the amish and non-amish young men. homicide decline in european history much of quantitative genetics is about some measurable metric character in a population. a convenient and useful way to study many discrete characters is to treat them as threshold characters that only manifest when an underlying hidden quantitative trait exceeds some threshold. cleft palate, for example, is a failure of the palate to close at the midline during intrauterine development. there is an underlying metric trait in the model that is the strength of the mechanism leading to midline fusion. fusion fails for trait values below a threshold strength of this mechanism. the model further specifies what the incidence should be in relatives of varying degree. it predicts cleft palate risk in relatives of those affected quite closely (fraser, ). cloninger et al. ( b) proposed a threshold model for the prevalence of sociopathy and hysteria in humans. in their model both disorders reflect the same underlying heritable trait but the sociopathy liability threshold in females is higher than the male threshold. they consider hysteria to be a less deviant form of sociopathy in women whose liability is above the male, but below the female, threshold. sociopathy prevalence in the general population, the higher proportion of sociopathy among relatives of female sociopaths, and the clustering of both male and female sociopaths within families closely fit the predictions of the model (cloninger et al., a, b). eisner ( ) tabulated the historical decline in homicide rates, excluding war deaths, in several european countries from the fourteenth to the twentieth century. a typical models of selection https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core finding is that homicide rates fell from per , people in to per , in , a forty-fold decline. this striking social change seems ‘large’. a threshold model is considered to sharpen this intuition. this simple model posits that the propensity to murder is a normally distributed trait in the population. individuals with high propensity will murder while those with low propensity will not. following eisner the threshold for homicide is placed at / , . tables of the standard normal distribution show that this threshold corresponds to the right tail of the propensity distribution beyond . standard deviations. after years the rate declined to in , , corresponding to the tail beyond . standard deviations. if the whole distribution moved with no change in the variance, as expected with the simple fisher model, the shift was just . − . ≈ . standard deviation. on a stature scale in which the standard deviation is . cm this corresponds to a change in mean stature of . cm in years. this amount of change in / or generations amounts to just over / cm per generation. this is in the order of known changes in stature in european history. from this perspective the decline in homicide is not especially remarkable at all and plausibly reflects genetic change. this is not to claim that genetic change accounts for most of the decline in homicide. however, it does suggest that this model is an informative yardstick for measuring how substantially homicide rates declined. modifying the assumptions of this plausible quantitative genetics model yields some insight into its robustness. assume, for example, that murderers have a variable number of victims and that the average number of victims is two. since the average number of homicides per murderer is two, the thresholds are estimated by a decline from to . , rather than to , per , people. these numbers correspond to . and . standard deviations on the right of normal curves. the change is therefore about . standard deviation – the same result found in the simple model. the qualitative conclusion of unremarkable change in years remains the same. a perhaps more realistic elaboration of the model is that murderers murder other murderers. the homicide rate under these conditions reflects not the density, but the square of the density, of murderers assuming a mass action rate of random encounters between murderers. in this case the change in the square root of the homicide rates is looked at so that the tails of this distribution in and are respectively at . and . standard deviations to the right of the mean. this elaborated model predicts a shift in the distribution of just . standard deviation, even less than in the simple model. it is concluded that the simple quantitative genetic model is fairly robust to perturbations. frost and harpending ( ) examined english homicide and execution rates, in addition to death rates of murderers awaiting trial, during this period in more detail. they also concluded that genetic transmission could account for much of the social change. conclusions in this paper an index was constructed from responses to a personality questionnaire that distinguishes old-order amish from their non-amish neighbours. the associated genetic model posited that the differences between the two populations are a m. d. weight and h. harpending https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core consequence of selective ‘boiling-off’ of amish individuals over time. this process is equivalent to truncation selection from the viewpoint of the amish population since those who found the community least congenial left. this example illustrates that groups in particular demographic or occupational niches with reproductive assortment can undergo genetic change that significantly differentiates them from other groups. the data suggest that the simple model incompletely accounts for the observed group differences. even if the heritability of aq is high, differences accumulate under genetic transmission at about . standard deviations per generation. the difference between the group means is . standard deviations. if this difference were a consequence of purely genetic change such a difference would require generations, or generations with a more reasonable heritability estimate of . . historical evidence suggests that the pattern is no older than ten generations. the implication is that the cultural difference between the amish and their neighbours reflects mostly strong cultural transmission. unfortunately, previous cultural transmission models of amish defection proposed by markle and pasco ( ), ericksen et al. ( , ), meyers ( , ), wasao and donnermeyer ( ) and greksa ( ) give only plausibility arguments that are difficult to quantify and falsify. on the other hand, quantitative genetic theory is well established in agriculture and other areas of biology like biometrics and conservation biology. the present study illustrates the utility of quantitative genetic models as a useful yardstick for describing social traits and evaluating transmission models, suggesting that quantitative genetics deserves more attention and use in human evolutionary biology. the historical decline in european homicide rates since the fourteenth century was also considered. gregory clark ( ) described this decline as part of a larger heritable change in europe leading up to the industrial revolution. several variants of a simple model of selection against homicide indicate such genetic change is entirely plausible. acknowledgments the authors thank adrian bell, stephen beckerman, elizabeth cashdan, gregory cochran, james lee, alan rogers and ryan schacht for their comments and criticisms, which substantially improved this paper. references bailey, j. m. 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( ) the personality profile of excellent nurses in china: the pf. contemporary nurse: a journal for the australian nursing profession , – . m. d. weight and h. harpending https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at www.telegraph.co.uk/news/worldnews/asia/india/ /indias-dwindling-parsi-population-to-be-boosted-with-fertility- clinics.html www.telegraph.co.uk/news/worldnews/asia/india/ /indias-dwindling-parsi-population-to-be-boosted-with-fertility- clinics.html http://nitro.biosci.arizona.edu/zbook/newvolume/_ /newvol .html https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core 	some uses of models of quantitative genetic selection in social science 	introduction 	thought experiment 	the old-order amish 	assortative mating 	endogamy 	the pf 	table factors assessed in the f questionnaire 	fig. principal components analysis (pca) of pf score differences among groups of - to -year-old indiana men. the left panel portrays similarities (i.�e. correlations) among the sixteen traits listed in table� . the right panel portrays differences 	fig. principal components analysis (pca) of individual pf scores of - to -year-old indiana men. the left panel portrays similarities among traits, and the right panel among individuals along with the overall uk population. trait labels are given in 	a model of (self-)selection 	homicide decline in european history 	conclusions 	acknowledgments 	acknowledgements 	references the ggleam study: understanding glaucoma in the ohio amish international journal of environmental research and public health article the ggleam study: understanding glaucoma in the ohio amish andrea r. waksmunski , , yeunjoo e. song , tyler g. kinzy , reneé a. laux , jane sewell , denise fuzzell , sarada fuzzell , sherri miller , janey l. wiggs , louis r. pasquale , jonathan m. skarie , , jonathan l. haines , and jessica n. cooke bailey , ,* ���������� ������� citation: waksmunski, a.r.; song, y.e.; kinzy, t.g.; laux, r.a.; sewell, j.; fuzzell, d.; fuzzell, s.; miller, s.; wiggs, j.l.; pasquale, l.r.; et al. the ggleam study: understanding glaucoma in the ohio amish. int. j. environ. res. public health , , . https://doi.org/ . / ijerph academic editor: paul b. tchounwou received: december accepted: february published: february publisher’s note: mdpi stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. copyright: © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (https:// creativecommons.org/licenses/by/ . /). department of population and quantitative health sciences, case western reserve university, cleveland, oh , usa; axw @case.edu (a.r.w.); yeunjoo.song@case.edu (y.e.s.); tgk @case.edu (t.g.k.); ral @case.edu (r.a.l.); jls @case.edu (j.s.); mxf @case.edu (d.f.); slf @case.edu (s.f.); sdm @case.edu (s.m.); jskarie @gmail.com (j.m.s.); jlh @case.edu (j.l.h.) cleveland institute for computational biology, case western reserve university, cleveland, oh , usa department of ophthalmology, harvard medical school, massachusetts eye and ear infirmary, boston, ma , usa; janey_wiggs@meei.harvard.edu department of ophthalmology, icahn school of medicine at mount sinai, new york, ny , usa; louis.pasquale@gmail.com ohio eye associates, mansfield, oh , usa * correspondence: jnc @case.edu abstract: glaucoma leads to millions of cases of visual impairment and blindness around the world. its susceptibility is shaped by both environmental and genetic risk factors. although over risk loci have been identified for glaucoma, a large portion of its heritability is still unexplained. here we describe the foundation of the genetics of glaucoma evaluation in the amish (ggleam) study to investigate the genetic architecture of glaucoma in the ohio amish, which exhibits lower genetic and environmental heterogeneity compared to the general population. to date, we have enrolled amish individuals in our study from holmes county, ohio. as a part of our enrollment process, ggleam study participants ( glaucoma-affected and unaffected individuals) received comprehensive eye examinations and glaucoma evaluations. using the data from the anabaptist genealogy database, we found that of the ggleam study participants were related to one another through a large, multigenerational pedigree containing people. we plan to integrate the health and kinship data obtained for the ggleam study to interrogate glaucoma genetics and pathophysiology in this unique population. keywords: glaucoma; vision; endophenotype; family history; founder population; amish . introduction vision loss is a significant public health concern that is worsening with the increasing size of the elderly population [ ]. glaucoma is a leading cause of irreversible blindness with about million individuals affected around the world [ ]. this complex phenotype is considered a collection of disorders characterized by the progressive loss of peripheral vision resulting from degeneration of the optic nerve [ ]. while there are early-onset forms of glaucoma, the most common type of glaucoma, primary open-angle glaucoma (poag), manifests in adulthood and has a complex pattern of inheritance [ ]. poag is a multi- factorial condition with both genetic and environmental risk factors [ – ]. genome-wide association studies of large cohorts of unrelated individuals have identified loci associ- ated with poag risk as well as many loci associated with quantitative poag traits called endophenotypes like intraocular pressure (iop) and optic disc parameters [ – ]. however, genetic variation in these loci only accounts for less than % of poag heritability [ , ]. therefore, a substantial portion of poag heritability remains unexplained [ ]. int. j. environ. res. public health , , . https://doi.org/ . /ijerph https://www.mdpi.com/journal/ijerph https://www.mdpi.com/journal/ijerph https://www.mdpi.com https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://doi.org/ . /ijerph https://doi.org/ . /ijerph https://creativecommons.org/ https://creativecommons.org/licenses/by/ . / https://creativecommons.org/licenses/by/ . / https://doi.org/ . /ijerph https://www.mdpi.com/journal/ijerph https://www.mdpi.com/ - / / / ?type=check_update&version= int. j. environ. res. public health , , of previous studies have shown the utility of working with founder populations and population isolates to elucidate genetic variation for glaucoma [ – ] and other age- related ocular traits [ – ]. the amish comprise an isolated, founder population that is culturally and genetically segregated from the general population of european descent [ ]. they typically practice a conservative, uniform lifestyle that includes similar dietary habits, occupations, and physical activity as well as minimal smoking [ ]. nearly all present-day amish are descendants of a few hundred swiss–german anabaptists who emigrated to the united states to escape religious persecution in europe in the late eighteenth and early nineteenth centuries [ , ]. the resettlement of these individuals in north america resulted in a population bottleneck that has been sustained across generations because amish typically marry within their faith group and non-amish individuals rarely join the amish community [ ]. therefore, the amish are a valued population in genetics research due to their relatively homogeneous environments and their reduced genetic variation due to their founder event and consanguinity [ ]. our multidisciplinary team set out to establish the genetics of glaucoma evaluation in the amish (ggleam) study to understand the prevalence and risk factors of glaucoma in amish communities in ohio. this represents a previously unexplored area in genetics research, especially given that the incidence of common, age-related forms of glaucoma are unknown in the amish. furthermore, we hypothesize that studying a complex ocular disease like glaucoma in a genetically and environmentally homogeneous population, like the amish, will facilitate the discovery of novel loci associated with glaucoma and its endophenotypes and aid in our understanding of its pathophysiology. this work also highlights the importance of establishing a working relationship with study participants in biomedical research, especially those from special populations. . materials and methods . . study participants the participants in the genetics of glaucoma evaluation in the amish (ggleam) study are amish individuals living in and around holmes county, ohio. the holmes county, ohio amish community comprises one of the largest amish settlements in north america [ ]. it began in when amish settlers moved there from somerset county, pennsylvania [ ]. our initial contacts with this community were made over twenty years ago through advertising in the local amish papers and meeting with community lead- ers to establish a working relationship with the amish community in holmes county as previously described [ – ]. due to their lifestyle, amish abstain from using mod- ern technology in their homes [ , ]; therefore, study participants were recruited for this study using door-to-door methods and through advertisements in local newspapers. the recruitment for our ggleam study branched from the ongoing amish eye study, which recruited amish individuals from ohio, indiana, and pennsylvania to interrogate age-related macular degeneration (amd) genetics and identify possible biomarkers for amd from optical coherence tomography (oct) [ ]. in that study, some participants self-reported prior glaucoma diagnoses, and we found that all the self-reported glaucoma diagnoses were clinically confirmed (positive predictive value: %). these observations inspired us to increase our engagement with the amish in holmes county, ohio and launch the ggleam study to focus on glaucoma in this population. the ggleam study enrollment process was comprised of two visits with partici- pants. in the first visit, the study coordinator consented the study participants in their homes, and study participants completed health and family medical history questionnaires (supplementary materials), which were based on the questionnaires developed for the collaborative amish aging and memory project (nih grants ag and ag ) and the amish eye study (nih grant ey ). the second visit occurred at the ohio eye associates office in mansfield, ohio at which the participant received a comprehensive eye examination performed by a fellowship-trained md glaucoma specialist. during this visit, a blood sample was also drawn by the study coordinator for dna extraction and biomarker int. j. environ. res. public health , , of analysis. the examination included visual acuity assessment by snellen eye chart, auto- mated - humphrey visual field testing (zeiss, oberkochen, germany), stereo optic disc photography, optical coherence tomography assessment of the optic disc, retinal nerve fiber layer and ganglion cell complex (cirrus sd-oct, zeiss, oberkochen, germany), gonioscopy, central corneal thickness measurement by pachmate (dgh technology, inc., exton, pa, usa), assessment of corneal hysteresis via the ocular response analyzer (reichert technologies, buffalo, ny, usa), intraocular pressure (iop) measurement with goldman applanation tonometry, detailed slit lamp biomicroscopy, and fundus exam via indirect ophthalmoscopy following pupil dilation. glaucoma diagnoses were defined by current american academy of ophthalmology preferred practice guidelines [ , ]. briefly, glaucoma determination was based on iop measurements taking into account corneal thickness and hysteresis as well as optic nerve examination, visual field testing, and assessment of the retinal nerve fiber layer and ganglion cell layer complex. individuals were invited to participate in the study if they met one of the following criteria: (i) reported to have or were diagnosed with any type of glaucoma; (ii) were at least years old and did not have glaucoma; or (iii) had family members with glaucoma. individuals under without a prior diagnosis of glaucoma or family history of glaucoma were not invited to participate in the study. all study participants provided informed consent, and the study was conducted within the guidelines of the declaration of helsinki. the study protocol was approved by the institutional review board at case western reserve university (irb- - ). . . amish pedigree the amish are a culturally isolated population with extensive genealogical records dating back to their emigration to north america [ ]. community-based directories and research-based resources, described elsewhere, have been developed to curate these data [ ]. to determine the relatedness of the ggleam study participants, we queried the anabaptist genealogy database (agdb) [ ]. based on these pedigree data, we constructed an all-connecting path pedigree (acp) to depict all known familial relationships among study participants and their ancestors. the acp was drawn using the pedigraph software tool [ ]. kinship and inbreeding coefficients were calculated using kininbcoef software and genealogical information from the all-connecting path pedigree [ ]. . results . . ggleam study participants thus far, we have enrolled study participants from the amish community in and near holmes county, ohio. of these individuals, we have obtained phenotypic data from comprehensive eye exams and health questionnaires for individuals to date. a fellowship-trained md glaucoma specialist determined glaucoma type and severity based on the results of each individual’s comprehensive glaucoma examination and testing results. most of the glaucoma-affected study participants ( %) have primary open-angle glaucoma (poag) (table ). on average, individuals with glaucoma were older than individuals without glaucoma ( . years old and . years, respectively) (table ). . . family history of glaucoma family history is a well-established risk factor for glaucoma [ , – ]. individuals who have first-degree relatives with glaucoma have a -fold higher risk of developing glaucoma compared to individuals without glaucoma-affected first-degree relatives [ ]. we enrolled study participants based on their self-reported or clinically confirmed diagnosis of glau- coma as well as their self-reported family history of glaucoma. specifically, we asked study participants if they had a family history of glaucoma in first-degree relatives or extended family members. as a consequence of the interrelatedness and close-knit nature of this population, several of the study participants were from the same amish nuclear families. we found that about % of the ggleam study participants had first-degree int. j. environ. res. public health , , of relatives with glaucoma (table ). of the glaucoma-affected amish individuals in our study, . % had first-degree relatives affected by glaucoma, and . % had extended family members with glaucoma (table ). most of the unaffected individuals in this study reported that they did not have a family history of glaucoma (table ). table . features of genetics of glaucoma evaluation in the amish (ggleam) study participants. of the glaucoma-affected amish in this study, individuals have poag and have another form of glaucoma including person with chronic angle closure glaucoma and individuals with pigmentary glaucoma. age values represent the study participants’ ages at their eye exams. the age range for study participants with glaucoma was – , and the age range for unaffected individuals was – . affected unaffected n age mean ± sd . ± . . ± . sex male female table . family history of glaucoma in ggleam study participants. first-degree relatives include parents and siblings. extended family includes aunts, uncles, and cousins. family history of glaucoma affected (%) unaffected (%) all (%) first-degree relatives no ( . ) ( ) ( . ) yes ( . ) ( ) ( . ) extended family unknown ( . ) ( ) ( . ) no ( . ) ( ) ( . ) yes ( . ) ( ) ( . ) . . quantitative ocular measurements the majority of the ggleam study participants (n = ) underwent comprehensive eye exams that yielded various quantitative ocular measures for glaucoma-affected individuals and unaffected individuals (table ). this includes quantitative ocular mea- surements for poag-affected individuals (table ). sixteen of the glaucoma-affected study participants either received iop lowering medications (i.e., prostaglandin analogs, beta-blockers, alpha agonists, oral carbonic anhydrase inhibitors, topical carbonic anhy- drase inhibitors, or cholinergic agents) or had prior surgery (i.e., glaucoma filtering surgery, laser iridotomy, argon laser trabeculoplasty, selective laser trabeculoplasty, or minimally invasive glaucoma surgery). average iop was higher among individuals with glaucoma (i.e., average iop: mmhg) compared to individuals without glaucoma (average iop: mmhg) (table ). the average iop in study participants with poag was about mmhg (table ). the average vertical cup-to-disc ratio (vcdr) was over . times higher in glaucoma-affected individuals (od vcdr: . ± . , os vcdr: . ± . ) com- pared to unaffected individuals (od vcdr: . ± . ; os vcdr: . ± . ) (table ). the average vcdr in the right and left eyes of poag-affected individuals were . and . , respectively (table ). although the average refractive error for individuals without glaucoma was substantially lower than for study participants with glaucoma, the ranges of values observed for both groups overlapped (table ). the mean central corneal thickness (cct) was lower in glaucoma-affected individuals compared to unaffected individuals (table ). the average cct for poag-affected individuals was also lower than the aver- age cct observed in unaffected individuals (tables and ). average axial length was nearly the same in glaucoma-affected individuals compared to unaffected individuals (tables and ). int. j. environ. res. public health , , of table . quantitative eye measurements. measurements were obtained from eye exams performed at the ohio eye associates office. these data were obtained for glaucoma-affected study participants and unaffected individuals. iop: intraocular pressure. vcdr: vertical cup-to-disc ratio. od: oculus dexter (right eye). os: oculus sinister (left eye). refractive error: spherical equivalent from distance refraction. d: diopters. affected unaffected average ± sd (range) average ± sd (range) od iop (mmhg) . ± . ( – ) . ± . ( – ) os iop (mmhg) . ± . ( – ) . ± . ( – ) od vcdr . ± . ( . – . ) . ± . ( . – . ) os vcdr . ± . ( . – . ) . ± . ( . – . ) od refractive error (d) . ± . (− . , + . ) − . ± . (− . , + . ) os refractive error (d) . ± . (− . , + . ) − . ± . (− . , + . ) od cct (microns) ± ( – ) ± ( – ) os cct (microns) ± ( – ) ± ( – ) od axial length * (mm) . ± . ( . – . ) . ± . ( . – . ) os axial length * (mm) . ± . ( . – . ) . ± . ( . – . ) * these values were calculated based on measurements from glaucoma-affected individuals and controls. . . genealogy of ggleam study participants the amish represent a unique population in genetics research due to their recent founding event and the extensive genealogical records available for this community [ , , , ]. one of these resources includes the anabaptist genealogy database (agdb) [ , ]. we queried the agdb and found that of the ggleam study participants are re- lated to one another through a -person all-connecting path (acp) pedigree (figure ). one individual enrolled in this study only had parental information in the agdb and could not be connected to the pedigree. kinship coefficients were calculated among the connected ggleam study participants using kininbcoef [ ]. the average kinship coefficient among these study participants was . (sem: . ). the maximum kinship coefficient observed was . , and the minimum was . . the average inbreeding coefficient calculated by kininbcoef was . (sem: . ). int. j. environ. res. public health , , of table . quantitative eye measurements for poag-affected ggleam study participants. measure- ments were obtained from eye exams performed at the ohio eye associates office for poag- affected individuals. iop: intraocular pressure. vcdr: vertical cup-to-disc ratio. od: oculus dexter (right eye). os: oculus sinister (left eye). refractive error: spherical equivalent from distance refraction. d: diopters. poag average ± sd (range) od iop (mmhg) . ± . ( – ) os iop (mmhg) . ± . ( – ) od vcdr . ± . ( . – . ) os vcdr . ± . ( . – . ) od refractive error (d) . ± . (− . , + . ) os refractive error (d) . ± . (− . , + . ) od cct (microns) ± ( – ) os cct (microns) ± ( – ) od axial length * (mm) . ± . ( . – . ) os axial length * (mm) . ± . ( . – . ) * these values were calculated based on measurements from individuals with poag. int. j. environ. res. public health , , x for peer review of figure . all-connecting path pedigree of ggleam study participants. this multigenerational pedigree connects of the amish individuals enrolled in the ggleam study and includes individuals. genealogy data was obtained from the anabaptist genealogy database (agdb) [ ]. the ggleam study participants are highlighted in blue. men are represented by squares, and females are represented by circles. pedigree was drawn using the pedigraph software tool. figure . all-connecting path pedigree of ggleam study participants. this multigenerational pedigree connects of the amish individuals enrolled in the ggleam study and includes individuals. genealogy data was obtained from the anabaptist genealogy database (agdb) [ ]. the ggleam study participants are highlighted in blue. men are represented by squares, and females are represented by circles. pedigree was drawn using the pedigraph software tool. int. j. environ. res. public health , , of . discussion glaucoma is a leading cause of irreversible blindness, but its genetic architecture and disease etiology are not fully understood. we established the ggleam study to under- stand glaucoma risk in the ohio amish, which are a population isolate. while previous studies in the amish examined congenital glaucoma [ ] or described glaucoma as a clinical feature in a few individuals with a homozygous mutation in the samhd gene associated with cerebral vasculopathy and early onset stroke [ ], glaucoma risk and prevalence have not been extensively studied in the amish population. therefore, this study can provide the foundation for future work assessing glaucoma risk in this population, which could inform understanding of glaucoma risk in general. to date, we have enrolled amish individuals from in and around holmes county, ohio, and of these study participants have received comprehensive eye exams. moving forward, we plan to enroll additional ohio amish community members in our study and obtain data from glaucoma-specific eye exams for all of the ggleam study participants. because our study design includes obtaining glaucoma status and quantitative eye measurements of the ggleam study participants, we can study the genetic variation potentially associated with glaucoma and its endophenotypes. examining heritable en- dophenotypes such as iop and vcdr rather than disease status alone increases the sta- tistical power to detect associated loci for a heterogeneous phenotype like glaucoma [ ]. identifying genetic variants associated with these glaucoma-related traits may improve our understanding of glaucoma development and disease pathophysiology [ ]. additionally, studying a complex trait like glaucoma in an isolated, founder population like the amish may allow for the identification of novel genetic variants that have been indiscernible from previous genetics studies in large datasets of unrelated individuals due to low minor allele frequency (maf) [ ]. in traditional case-control genome-wide association stud- ies, the sample size needed to detect trait-associated variants increases with /maf [ ]. therefore, sample sizes in these population-based cohorts are in the tens of thousands. by comparison, association studies of rare variants in families require lower sample sizes if disease-associated variants are present since they are likely to be inherited by other family members [ ]. this aspect of family-based study designs is augmented in studies with pop- ulation isolates, which can have densely affected families with potentially causal variants inherited identical-by-descent from a small set of common ancestors [ , ]. the amish resettlement in north america and generations of intra-faith marriages has resulted in the accumulation of alleles that are rare in the general population of european descent. the all-connecting path pedigree we constructed for the ggleam study participants using data from the agdb [ ] confirmed that we are effectively working with one large family, which will greatly aid in future genetics studies of this cohort. furthermore, the av- erage kinship and inbreeding coefficients we calculated for these individuals were . and . , respectively. the estimated inbreeding coefficient for the amish population, in general, is . [ ]. the average kinship coefficient previously calculated for amish families in ohio and indiana was . [ ]. leveraging kinship information enables us to account for familial relationships among study participants, which are highly inter- connected due to generations of endogamy in the population [ ]. we can then utilize this information in family-based association tests between glaucoma affection status and snp genotype to identify novel glaucoma risk variants or in gene-set methods (i.e., kernel, burden, and collapsing methods) to identify rare variants for glaucoma [ , – ]. in addition to being genetically homogeneous as a result of their recent popula- tion bottleneck and generations of intra-faith marriages, the amish adhere to a con- servative lifestyle that is mostly consistent across amish communities, which are pre- dominantly in rural areas [ ]. therefore, environmental exposures, modifiable health behaviors, and lifestyle factors are more homogeneous in the ohio amish compared to the general population of european descent. these features of the amish popu- lation make them a valued population to investigate both genetic and environmental risk factors for glaucoma as well as the possible interactions among these risk factors. int. j. environ. res. public health , , of through health, physical activity, environmental exposure, and family history question- naires in the ggleam study (supplementary materials), we aim to investigate non-genetic factors pertaining to glaucoma risk in this population. we are especially interested in factors that have been inconsistently associated with glaucoma risk and development, including diet, physical activity, caffeine consumption, smoking, and alcohol consump- tion [ – ]. as a part of their cultural traditions, the amish generally do not use motorized vehicles; therefore, they have higher activity levels than non-amish individuals [ , ]. most amish community members consume a similar diet that consists of foods that are rich in carbohydrates and lipids as well as homegrown fruits and vegetables [ ]. they also typ- ically abstain from smoking and consuming alcoholic beverages [ , ]. all ggleam study participants who completed our questionnaires stated that they had never smoked, and only one individual self-reported alcohol consumption. therefore, our ggleam study is well-positioned to assess the contributions of risk factors like physical activity, diet, and caffeine consumption in a uniquely homogeneous population that generally prohibits smoking and alcohol consumption. increased knowledge of the effects and interplay of genetic and environmental risk factors may broaden our understanding of glaucoma patho- physiology, facilitate earlier disease detection based on more comprehensive risk profiles, and inspire the development of novel therapeutics to treat glaucoma. amish communities in north america have been engaged in genetics research since the s [ , ]. these early efforts led to better understanding of various mendelian genetic disorders [ ], and several studies have also shown the utility of studying com- plex traits in amish families due to their reduced heterogeneity in genetic variation and environmental exposures [ , , , , – , – ]. initial research efforts with amish communities involved collaboration among researchers and local amish liaisons who were familiar with the families in the community, could speak pennsylvania dutch, and under- stood the customs and values of the amish [ ]. some of these practices were continued as more research teams began working with the amish population, including the amish research program [ – ] and the amish eye study [ ]. the factors most valued by the amish include faith, family, and community [ , ]. their participation in research has partially been shaped by their altruistic nature and their belief that, by participating in these studies, they are helping others [ ]. to ensure that our study design and enrollment practices were respectful of these values, we engaged in door-to-door recruitment methods, performed study enrollments in the study participants’ homes, and used paper questionnaires to generate some of our study data. our study coordinators also built rapport with the ggleam study participants from engagement in prior research studies and years of community involvement [ , ]. with the growing field of genomics research in diverse and understudied populations, it is paramount that researchers form longitudinal partnerships with study participants, especially those from vulnerable populations [ , ]. . conclusions glaucoma significantly contributes to global cases of vision loss and blindness. while large population-based cohorts have successfully identified numerous loci contributing to glau- coma risk, most of the additive genetic variance for glaucoma is not attributable to known loci. we started the ggleam study to study glaucoma risk in the amish population by ascertaining individuals with and without glaucoma from in and around holmes county, ohio. to date, amish individuals have been enrolled in this study, and phenotypic data has been generated for of these individuals through health, lifestyle, environmental exposure, and family history questionnaires as well as comprehensive eye exams. we de- termined that these study participants are highly interrelated through a multigenerational pedigree and plan to incorporate this kinship information into our future genetics studies. our hope is that the health, phenotypic, and genetic data we are gathering, together with the extensive genealogical records for this special population, will be invaluable in expand- ing our understanding of the genetic epidemiology of glaucoma. int. j. environ. res. public health , , of supplementary materials: the following are available online at https://www.mdpi.com/ - / / / /s , supplementary item : ggleam study questionnaire for family history of eye disease, supplementary item : questionnaire for ggleam study participants’ physical activity and environmental exposure, and supplementary item : questionnaire for ggleam study participants’ health and activities. author contributions: conceptualization, j.n.c.b., j.m.s., j.l.h., j.l.w. and l.r.p.; methodology, j.n.c.b., j.m.s. and j.l.h.; formal analysis, a.r.w., y.e.s. and t.g.k.; data curation, a.r.w., y.e.s., t.g.k., j.s., d.f., s.f. and s.m.; writing—original draft preparation, a.r.w.; writing—review and editing, a.r.w., t.g.k., j.l.w., l.r.p., j.m.s., j.l.h. and j.n.c.b.; visualization, a.r.w.; supervision, j.n.c.b. and j.l.h.; project administration, r.a.l.; funding acquisition, a.r.w., j.l.w., l.r.p., j.m.s., j.l.h. and j.n.c.b. all authors have read and agreed to the published version of the manuscript. funding: this work was supported by the brightfocus foundation (g ) and the clinical and translational science collaborative of cleveland (kl tr ) from the national center for advancing translational sciences (ncats) component of the national institutes of health (nih). this work also received support from the following grants through the nih: ag and ey . a.r.w. received support from the postdoctoral nih visual sciences training program at case western reserve university (t ey - ). l.r.p. received support from ey . institutional review board statement: the study was conducted according to the guidelines of the declaration of helsinki and approved by the institutional review board of case western reserve university (irb- - ). informed consent statement: informed consent was obtained from all subjects involved in the study. data availability statement: deidentified data are, in principle, available to qualified investigators once institutional agreements have been reached. acknowledgments: we would like to thank the amish families in holmes county, ohio for their participation and contributions to this research study. we also appreciate the data contribution from the anabaptist genealogy database (agdb). this work made use of the high performance computing resource in the core facility for advanced research computing at case western reserve university. conflicts of interest: the authors declare no conflict of interest. references . world health organization. world report on vision; 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(p. ) while working on the identification of haplotype-tagging snps using chromosome data, zhang et al. stumbled across an interesting phenomenon that they dub “yin yang haplotypes.” these pairs of high-frequency haplotypes are composed of completely mismatched snp alleles; that is, they differ from each other at every snp position. to study these divergent haplotypes in a more systematic manner, zhang et al. developed an algorithm to identify regions containing yin yang haplotype pairs and used it to characterize two genomewide data sets. they found that %– % of the human genome is spanned by yin yang haplotypes and that these haplotypes make up a significant portion of the total haplotype diversity. higher coverage with yin yang haplotype was achieved after re- moval of low-frequency snps from the analysis, which led the authors to suspect that these haplotypes are an- cient. further supporting this idea is the high degree of conservation of yin yang haplotypes across populations, suggesting these patterns emerged prior to the african diaspora. although it might seem that evolution must have had a hand in the rise of opposing haplotype alleles, simulations indicate that yin yang haplotypes can occur under a neutral evolution model. g-actin gene mutations and progressive deafness, by zhu et al. (p. ) because mutations in several genes encoding actin-inter- acting proteins have been found to cause hearing loss, it is perhaps not surprising that zhu et al. report mutations in the gene for g-actin itself in four families with pro- gressive deafness. what is unexpected, though, is the fact that, when this protein is widely expressed and highly conserved, the only phenotype in the affected individuals is hearing loss. all four mutations are missense mutations at conserved residues, and they occur in three of the four subdomains of the g-actin protein. zhu et al. speculate that the mutations have mild effects on protein function and that the auditory hair cells may be more sensitive to mutations in this gene because they do not regenerate, so subtle mutations can have long-term effects. this is in contrast to other g-actin–expressing tissues in which cells are regenerated frequently, such as the intestinal epi- thelium. hair cells may also be more susceptible to these mutations than other g-actin–expressing cells because their function is dependent on precise control of their � by the american society of human genetics. all rights reserved. - / / - $ . ultrastructural organization, which is determined by the actin cytoskeleton. mutations and clinical correlations in xlrp, by sharon et al. (p. ) in an attempt to define genotype-phenotype correlations in x-linked retinitis pigmentosa (xlrp), sharon et al. screened rp and rpgr for mutations in a large sample of males with a diagnosis (or a suspected diagnosis) of xlrp or cone-rod degeneration. mutations were found in % of the people with a prior clinical diagnosis of xlrp, with the most common location being orf of rpgr, a region that was overlooked in initial screens of this gene. although there is a good deal of overlap in measures of ocular function, people with rp mutations have, on average, a significantly lower visual acuity than patients with rpgr mutations. subgroup analyses of the people with rpgr mutations indicate that those with orf mutations have milder disease than those with mutations in exons – . within this orf mutation group, the severity of the visual effects depended on the length of the wild-type amino acid sequence expressed. confirming a previous report, mutations in orf downstream of codon were often associated with cone-rod degeneration rather than a strict xlrp pheno- type, suggesting that these mutations are more deleterious to cones than to rods. these findings move us toward the ultimate goal of this research, which is to make mutation- based, long-term visual prognoses, but the heterogeneity in visual impairment within the mutation groups does not yet make this fully possible. mapping and cloning the gene for mast syndrome, by simpson et al. (p. ) in a recent journal editorial, crosby and proukakis ( : – ) proposed that a common underlying mech- anism in some hereditary spastic paraplegias (hsps) may be a defect in cellular transport. now, with the finding that mutations in acp are found in people with mast syndrome, simpson et al. believe they have found addi- tional evidence for this model. mast syndrome is a com- plicated form of autosomal recessive hsp that is found in old order amish. the disorder is progressive, and onset generally occurs in young adults, although milestone delays and subtle cognitive and motor difficulties are often reported in childhood. through use of a large amish pedi- gree, simpson et al. identified a single-nucleotide insertion in acp that leads to premature truncation of the en- coded protein. acp was originally identified as a cd - am. j. hum. genet. :i–ii, ii interacting protein that appears to regulate cd -depen- dent t cell activation (see zeitlmann et al. reference in simpson et al.). the protein is partitioned between the cytoplasm and the endosomal/trans-golgi network, and it has been proposed that it may be involved in intra- cellular trafficking and protein sorting. the hsps result from degeneration of the longest motor and sensory axons in the spinal cord. dr. crosby’s group has speculated that, because of their size, these axons are particularly susceptible to disruption of intracellular trafficking pro- cesses, which are vital for neuron survival. they propose that the acp gene product should be renamed “mas- pardin” for “mast syndrome, spastic paraplegia, autoso- mal recessive with dementia.” pten mutations upregulate akt in ldd, by zhou et al. (p. ) pten mutations cause a variety of disorders, including proteus syndrome, bannayan-riley-ruvalcaba syndrome, and cowden syndrome (cs). the explanation for pheno- typic differences in the expression of pten mutations and the relationship between these syndromes has not been fully uncovered. lhermitte-duclos disease (ldd) is another disorder associated with pten mutations, and it often occurs in association with features of cs, sug- gesting that these disorders share a common etiology. cs is characterized by multiple hamartomous lesions and typically involves neoplasms of internal organs, whereas ldd is a hamartomous overgrowth of hypertrophic gan- glion cells. to better understand the relationship between cs and ldd, zhou et al. gathered subjects with ldd, without regard to the presence or absence of other features in these individuals, and performed mutational analysis on pten. they discovered pten mutations in adult-onset cases, both with and without features of cs. on the basis of six matched germline dna sam- ples, at least these six were germline mutations. several points suggest that loss of pten is sufficient to cause ldd and that ldd is a component of cs. these include the high frequency of pten mutations, the complete or partial loss of pten staining in ∼ % of samples, and the identification of loss of heterozygosity or a second- hit somatic pten mutation in three cases. in contrast to the mutations in the adult-onset cases, all three child- hood ldd cases lacked pten mutations, both somatic and germline. in conjunction with three other reported patients with childhood ldd who never developed fea- tures of cs, this finding makes the interesting suggestion that adult-onset and childhood ldd may be distinct dis- orders, although this will require further study. kathryn garber deputy editor biomarkers of acute appendicitis: systematic review and cost–benefit trade-off analysis r e v i e w biomarkers of acute appendicitis: systematic review and cost–benefit trade-off analysis amish acharya • sheraz r. markar • melody ni • george b. hanna received: march / accepted: july / published online: august � the author(s) . this article is published with open access at springerlink.com abstract background acute appendicitis is the most common sur- gical emergency and can represent a challenging diagnosis, with a negative appendectomy rate as high as %. this review aimed to evaluate the clinical utility of individual biomarkers in the diagnosis of appendicitis and appraise the quality of these studies. methods a systematic review of the literature between january and september using of pubmed, ovidmedline, embase and google scholar was con- ducted. studies in which the diagnostic accuracy, statistical heterogeneity and predictive ability for severity of several biomarkers could be elicited were included. information regarding costs and process times was retrieved from the regional laboratory. european surgeons blinded to these reviews were independently asked to rank which charac- teristics of biomarkers were most important in acute appendicitis to inform a cost–benefit trade-off. sensitivity testing and the quadas- tool were used to assess the robustness of the analysis and study quality, respectively. results sixty-two studies met the inclusion criteria and were assessed. traditional biomarkers (such as white cell count) were found to have a moderate diagnostic accuracy ( . ) but lower costs in the diagnosis of acute appen- dicitis. conversely, novel markers (pro-calcitonin, il and urinary -hiaa) were found to have high process-related costs including analytical times, but improved diagnostic accuracy. quadas- analysis revealed significant poten- tial biases in the literature. conclusion when assessing biomarkers, an appreciation of the trade-offs between the costs and benefits of individual biomarkers is needed. further studies should seek to investigate new biomarkers and address concerns over bias, in order to improve the diagnosis of acute appendicitis. keywords acute appendicitis � biomarkers � cost–benefit trade-off acute appendicitis is the most common surgical emer- gency, with an annual incidence in the usa of . per , [ ]. cases are characterized by an acute inflam- matory process, but in approximately . % the appendix has perforated and become gangrenous or there is overt peritonitis, termed ‘complicated appendicitis’ [ ]. whilst in rare special circumstances management may differ, the mainstay of treatment for the majority of patients remains surgery either by an open or by laparoscopic approach. with , appendectomies performed in the usa during , at an average estimated cost of $ [ ], appendicitis represents a highly prevalent condition with significant expenditure associated with its treatment. despite the frequency of appendicitis, accurate diagno- sis remains difficult. the national surgical research col- laborative in the uk has estimated that the negative appendectomy rate is as high as . % [ ]. the use of ultrasound and computerized tomography (ct) has in some cases been shown to improve appendicitis diagnostic accuracy and reduce the number of negative appendec- tomies [ ], with the latter shown to decrease rates to less electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. & george b. hanna g.hanna@imperial.ac.uk division of surgery, department of surgery and cancer, imperial college london, th floor qeqm building, st mary’s hospital, south wharf road, london w ny, uk surg endosc ( ) : – doi . /s - - - and other interventional techniques http://dx.doi.org/ . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf than % [ ]. however, exposing patients to high levels of radiation is undesirable given the lifetime risk of cancer, along with the increase in costs associated with increased utilization of ct, representing negatives to this approach. whilst this radiation dosage is avoided by using ultrasound scanning, the technique is operator dependent, and in as many as % of cases the appendix fails to be visualized [ ]. several studies have previously examined a variety of biomarkers associated with appendicitis to more appropri- ately assign risk and allocate further diagnostic investiga- tion. these have the potential of providing noninvasive objective criteria to aid clinicians in the diagnosis of appendicitis and in some cases predict the severity of the condition, with no adverse effects upon the patient. in several studies, biomarkers have been shown to have potentially good diagnostic accuracy and reliability, but with variable financial and timing implications. the latter significantly limits the clinical effectiveness of a biomarker in the emergency setting. the ‘ideal’ diagnostic biomarker would therefore maximize clinical utility and minimize procedural cost including analytical time. the aim of this study was to evaluate specific characteristics of biomarkers that surgeons’ value and to critically assess the cost–benefit of both traditional and novel biomarkers in the diagnosis of acute appendicitis from published literature. materials and methods literature search strategy a literature search of pubmed, ovidmedline, embase and google scholar electronic databases was conducted from january , , up to and including september , , for studies regarding the use of urine or serum biomarkers in the diagnosis of appendicitis or the predic- tion of complicated appendicitis. search terms used included: appendicitis, serum, blood, urine, biomarkers, diagnosis, diagnostics, perforation, complicated and severity in various combinations, as well as the name of specific biomarkers previously identified. research titles were then screened for suitability, and full-text copies were retrieved. further potentially appro- priate papers were highlighted by assessing the reference lists and citations of the articles being screened. all studies that investigated the diagnostic ability of a single or mul- tiple biomarkers that could be tested in the urine or blood of patients were included. exclusion criteria involved studies with no available english translation, no full-text edition available, and those assessing the predictive ability of biomarkers for severity in which no diagnostic accuracy could be calculated. of those studies meeting inclusion criteria, the year of publication, population demographics, the number of patients enrolled and the stated specificity and sensitivity of the biomarker for diagnosis and severity were extracted. for studies that did not explicitly state the sensitivity and specificity of the biomarker, provided sufficient data were available, these were independently calculated. literature standard the quadas- tool was used to appraise the standard of the literature. it was implemented, as it has been previously described, to assess the quality and risk of bias of the included studies [ ]. the tool involves four domains: patient selection, index test, reference standard and the flow of subjects through the study. prompting questions are used to allow the reviewer to assess whether there is a risk of bias with respect to each of the four domains. it also allows the reviewer to gauge the applicability of the study to the review with respect to the first three domains. in this review, the reference standard is the histological exami- nation of the appendix. biomarker survey general surgeon members of the european association of endoscopic surgery (eaes) were asked to complete an anonymous survey regarding their opinions on the most desirable characteristics the ideal diagnostic biomarker of acute appendicitis would possess (table ). the surgeons were asked to rank each characteristic in the order of importance, including diagnostic benefits (high sensitivity, high specificity, reproducibility and predictive ability of perforation), process-related financial costs, time for result, ease of testing and patient acceptability. the average rank for each of the attributes, e.g., sensitivity, was then cal- culated, to identify which were the most desired charac- teristics. these ranks were used to inform the weightings for the cost–benefit trade-off, with greater importance placed upon higher ranked attributes. statistical methodology for each of the assessments of acute appendicitis and severity of appendicitis (perforation), paired sensitivity and specificity were calculated for diagnosis or severity, as appropriate, from each eligible study. a bivariate model for meta-analysis of statistical accuracy provides more accu- rate results than fixed-effect modeling. following the val- idated methodology of harbord et al. [ ], bivariate meta- analyses were therefore performed to generate pooled point estimates and % confidence intervals for the sensitivity and specificity of the biomarker under investigation with surg endosc ( ) : – histopathological confirmation of acute appendicitis, toge- ther with hierarchical summary receiver operating charac- teristic (roc) curves. the software used for this analysis was the custom-designed statistical package midas [ ]. areas under the hierarchical summary roc curves, and i statistics, were obtained directly from the midas output. see zhou and tu [ ] for an in-depth description of the statistical methods used. cost–benefit trade-off analysis to evaluate the biomarkers, we applied decision analysis methodology, employing multi-criteria decision analysis (mcda) [ ] to assess trade-offs between cost (both time and financial) and benefit amongst the biomarkers, in terms of their performance characteristics (table ). the list of performance characteristics was grouped into three areas, namely monetary costs, time to results and benefits, encompassing all the remaining characteristics that were neither costs nor time. through the literature review and expert survey, we determined the mean level of perfor- mance of the biomarkers on each of the characteristics. criteria on which all biomarkers had identical perfor- mance, such as patient acceptability, were removed. the performance level was converted to a score by assigning a value of to represent the worst performance (e.g., the highest unit price or worst sensitivity) and a value of to represent the highest performance (e.g., lowest unit price or highest sensitivity). we assumed linearity between per- formance and value, such that for any intermediate level the corresponding value was interpolated from the worst and best performances on that criterion (valued and , respectively). criteria weightings were derived from the rankings assigned by the european surgeons. the highest ranked criterion was given a weighting of , the second highest ranked criterion was given a weighting of , and so forth. the weightings were normalized so that they totaled , for each performance area. we applied a weighted average rule to combine the value scores across criteria as in: table definitions of the characteristics of biomarkers the consultants were asked to rank definitions outcome utilized sensitivity result of pooled sensitivity for diagnosis of acute appendicitis specificity results of pooled specificity for diagnosis of acute appendicitis predictive of perforation area under the curve of summary roc for diagnosis of perforated appendicitis cost cost of investigation from imperial college nhs trust east of testing level of invasiveness of testing acceptability the impression of patient acceptability time for result time from sample being taken to result being available for clinician interpretation as described by imperial college nhs trust reproducibility i statistic for heterogeneity: increasing value indicates less consistency table performance of various biomarkers with respect to the surgeon rankings biomarker sens. (%) spec. (%) ease of test predictive of perforation (%) cost (£) time for result (h) acceptability reproducibility wcc easy . good crp easy good bilirubin easy good pro-calcitonin easy . good il- easy . good -hiaa easy good surgeon rank acceptability considered ‘good’ as all can be done routinely. ease of testing all considered ‘easy’ as all are noninvasive wcc white cell count, crp c-reactive protein, il- interleukin , -hiaa urinary serotonin, sens sensitivity, spec specificity surg endosc ( ) : – value ¼ x k wkvaluek; where vk indicates the value of an option on the kth cri- terion and wk is the weighting assigned to that criterion. the overall value was therefore bounded between and : a biomarker that had the worst performance on all the criteria would have an overall value of , whereas the biomarker that had the best performance on all the criteria would have an overall value of . the more desirable the biomarker was, the higher this value was. two-way cost– benefit maps highlighted the trade-offs between different aspects of the biomarkers. sensitivity analyses examined the robustness of the results. trade-off analyses were per- formed using decision analytic software hiview (version . . . , educational copy). results literature search sixty-two full-text articles met the inclusion criteria and were appraised following the literature search (fig. ). forty-nine of these were used to assess the diagnostic accuracy of biomarkers. eight studies assessed urinary markers ( for urinary serotonin and for leucine-rich gly- coprotein). forty-three studies investigated serum biomarkers ( on white cell count, on c-reactive protein, on bilirubin, on serum amyloid a, on s a / protein, on calprotectin, on pro-calcitonin, on d-dimer, on interleukin , on interleukin , on leucine-rich glycoprotein, on fibrinogen, on liposaccharide binding protein and on high mobility group box protein- ). thirty- seven studies assessed whether biomarkers were predictive of severity ( on white cell count, on c-reactive peptide, on bilirubin, on pro-calcitonin, on interleukin and on urinary serotonin) [ , , , , , , – , – , – , , , , , , , – ]. the demographics of these studies are shown in appendixes and in esm. quadas- evaluation the results of the quadas- assessment of the studies are shown in fig. . fifty-nine percent of studies had an ‘un- clear’ or ‘high’ risk of bias with respect to patient selection due to constraining exclusion criteria. this limited the applicability of fifty-eight percent of the studies with respect to patient selection. forty-one percent and thirty- one percent of studies had an ‘unclear’ or ‘high’ risk of bias with respect to the index and reference standards, respec- tively. this was due to a lack of information regarding blinding, thresholds and the order in which they were assessed. only thirteen percent of studies had an ‘unclear’ or ‘high’ risk of bias with respect to the patient flow. fig. schematic to show the strategic literature search surg endosc ( ) : – biomarkers that were included in more than studies were taken forward for pooled analysis. pooled analysis for individual serum biomarkers in acute appendicitis white cell count the pooled sensitivity of white cell count for the diagnosis of acute appendicitis was . ( % ci . – . ; i = . %), and its pooled specificity was . ( % ci . – . ; i = . %). the area under the curve for the summary roc was . ± . . for the diagnosis of perforated appendicitis, the pooled sensitivity was . ( % ci . – . ; i = . %) and pooled specificity was . ( % ci . – . ; i = . %), giving an area under the curve of . ± . . c-reactive protein the pooled sensitivity of c-reactive protein for the diag- nosis of acute appendicitis was . ( % ci . – . ; i = . %), and its pooled specificity was . ( % ci . – . ; i = . %). the area under the curve for the summary roc was . ± . . for the diagnosis of perforated appendicitis, the pooled sensitivity was . ( % ci . – . ; i = . %) and pooled specificity was . ( % ci . – . ; i . %), giving an area under the curve of . ± . . bilirubin the pooled sensitivity of bilirubin for the diagnosis of acute appendicitis was . ( % ci . – . ; i = . %), and its pooled specificity was . ( % ci . – . ; i = . %). the area under the curve for the summary roc was . ± . . for the diagnosis of perforated appendicitis, the pooled sensitivity was . ( % c.i . – . ; i = . %) and pooled specificity was . ( % ci . – . ; i = . %), giving an area under the curve of . ± . . pro-calcitonin the pooled sensitivity of pro-calcitonin for the diagnosis of acute appendicitis was . ( % ci . – . ; i = . %), and its pooled specificity was . ( % ci . – . ; i = . %). the area under the curve for the summary roc was . ± . . for the diagnosis of perforated appendicitis, the pooled sensitivity was . ( % ci . – . ; i = %) and pooled specificity was . ( % ci . – . ; i = %), giving the area under the curve of . ± . . il- the pooled sensitivity of il- for the diagnosis of acute appendicitis was . ( % ci . – . ; i = . %), and its pooled specificity was . ( % ci . – . ; i = . %). the area under the curve for the summary roc was . ± . . for the diagnosis of perforated appendicitis, the pooled sensitivity was . ( % ci . – . ; i = . %) and pooled specificity was . ( % ci . – . ; i = %), giving an area under the curve of . ± . . pooled analysis for -hiaa from urine in acute appendicitis the pooled sensitivity of urinary -hiaa for the diagnosis of acute appendicitis was . ( % ci . – . ; i = . %), and its pooled specificity was . ( % ci . – . ; i = %). the area under the curve for the summary roc was . ± . . pooled analysis for severity was precluded as only one study met the inclusion criteria. fig. a graph displaying the percentage of studies with varying degree of bias for each of the four quadas- domains. b graph displaying the percentage of studies of varying applicability with respect to three of the four quadas- domains surg endosc ( ) : – biomarker survey six hundred and eighty-eight surgeon members of the eaes responded to the survey ( % of which were consultants, % registrar level and % other grades), giving a response rate of . %. diagnostic sensitivity was given the highest average rank by the surgeon consensus and was thus weighted as the most important biomarker characteristic. the results of the other parameters are listed in table . cost–benefit trade-off since all biomarkers had identical performances in terms of ‘ease of test’ and ‘acceptability,’ these two criteria were removed from the trade-off analysis. table displays the normalized weighted scores out of for each of the six biomarkers with respect to the costs, time for result and benefits (diagnostic sensitivity, specificity, prediction of perforation and reproducibility), as well as an overall performance score. figure a displays trade-offs between the benefits, as defined above, and the costs. white cell count and bilirubin performed best overall with the latter scoring marginally higher. when appraising the benefits in isolation, inter- leukin- performed the best. sensitivity analysis demon- strated how the performance of the biomarkers would change if the relative importance of the various charac- teristics, as determined by the survey, was altered. if less importance was placed upon the financial cost or the time for result than its relative benefits (such as sensitivity), then the surgeons’ preference would be shifted further in favor of novel markers such as il- (fig. b, c). the remaining biomarkers (c-reactive peptide, sero- tonin and pro-calcitonin) were inferior to those previously mentioned in a way that probabilistically dominated by the other three tests. discussion this study has highlighted the variable performance of biomarkers in the diagnosis of appendicitis, which reduces their potential to provide established objective criteria when used in isolation. this analysis has shown that whilst traditional markers including white cell count are associ- ated with low temporal and financial cost, their overall diagnostic accuracy is relatively poor. as such weighting the analysis in favor of diagnostic characteristics such as high sensitivity or specificity, as opposed to process-related performance, would favor the use of novel biomarkers. the low diagnostic accuracy of elevated wcc is likely due to the presence of the underlying generalized inflammatory process seen with acute appendicitis, but also a number of other inflammatory conditions [ ]. conversely, novel markers that are less commonly used clinically in the diagnosis of appendicitis such as interleukin- have been shown to have a higher diagnostic benefit, but are associ- ated with significant costs. the results of the literature search also highlight the expansion of work to look for novel diagnostic biomarkers, which to date remain only tested in isolated studies preventing meaningful analysis for clinical application [ ]. there was a ‘high’ or ‘unclear’ risk of bias in % of the studies with respect to patient selection. this was due to insufficient information regarding selection criteria. a number of studies assessing novel markers utilized healthy controls, or for example, with bilirubin, excluded patients in whom this could be caused by alternative pathology. this, however, leads to a selection bias when assessing the diagnostic ability of the biomarker with respect to sus- pected appendicitis and can spuriously improve the speci- ficity. there was also an ‘unclear’ bias with respect to the index tests, especially with novel biomarkers, as diagnostic thresholds were not stated. the majority of the studies showed good applicability, but the assessment of a restricted demographic, such as pediatric patients, limited the studies performance with respect to this domain. this study has highlighted the challenges associated with using single biomarkers in the diagnosis of appen- dicitis. radiological investigation, especially ct, has been shown to have far superior diagnostic ability, with a reported sensitivity and specificity of and %, respectively [ ]. however, the estimated radiation dose associated with a ct abdomen is msv, equating to an increase of . % in the cancer risk for a -year-old table normalized scores (out of ) for the six biomarkers with respect to financial cost, time, diagnostic benefit (composite of sensitivity, specificity, reproducibility and prediction of perforation) and overall performance wcc crp bilirubin pro-calcitonin il- -hiaa cost performance time performance diagnostic benefit . overall performance . . . . . . wcc white cell count, crp c-reactive protein, il- interleukin , -hiaa urinary serotonin surg endosc ( ) : – patient [ ]. furthermore, ct remains a relatively expen- sive modality that could not be practically used in all patients in many areas of the world. several studies have already suggested the use of diagnostic algorithms to ensure judicious use of radiology [ ] and have demon- strated the potential to halve the use of ct scanning without increasing the negative appendectomy rate. biomarkers could therefore be incorporated into these diagnostic algorithms in order to rationalize and more appropriately allocate further investigations. previous studies on biomarkers in appendicitis have focused solely upon their diagnostic accuracy. however, this study has highlighted the importance of considering clinical utility when assessing biomarkers. interleukin- had the overall highest overall beneficial characteristics; however, this neglects its -h process time and expensive cost per test, which would preclude it from actual clinical use. this is further highlighted by the sensitivity analysis, which demonstrated that factoring in the significance of costs, more traditional biomarkers such as wcc, will be preferred. this study has therefore highlights the potential importance of cost–benefit modeling to improve this decision-making process when considering regional or national allocation of resources for diagnostic investigations. in fact, no single biomarker had all the desired charac- teristics for the diagnosis of acute appendicitis. more commonly used biomarkers have less process-related costs due to the widespread availability of the testing, but are of relatively poor diagnostic accuracy when used in isolation. new proposed biomarkers whilst having high diagnostic value often require more complex assays, in which some circumstances require them to be sent to regional centers for analysis. however, a combination of biomarkers, as is used by some institutions clinically with white cell count and crp, may improve the diagnostic ability [ , ]. alternatively, the use of a biomarker in conjunction with a consistent clinical history and examination may improve diagnostic accuracy in a more feasible manner. this could be achieved by utilization of stratification scores such as the alvarado, which is a -point scoring system incor- porating the typical signs and symptoms seen with appendicitis. with a cutoff of , this diagnostic algorithm has been shown to have a reported specificity as high as % [ ]. however, the limitation of the utilization of these scoring systems is the subjective interpretation of b fig. a cost–benefit trade-off for the six biomarkers. benefits include a summation sensitivity, specificity, predictive ability and reproducibility. b sensitivity analysis revealing the effect of changing the current weighting (dashed line) placed upon financial cost and overall benefits. c sensitivity analysis revealing the effect of changing the current weighting (dashed line) placed upon time for result and overall benefits surg endosc ( ) : – clinical history and examination findings [ ]. further- more, a surgeon’s clinical impression has in some cases been shown to be of equivalent diagnostic value as these scoring systems, highlighting the value of clinical experi- ence and the limitations of the widespread utilization of scoring systems [ ]. in effect, therefore we have shown that clinically white cell count and bilirubin should be considered of greater use in the diagnosis of acute appen- dicitis when compared other biomarkers. however, given the limitations associated with current biomarkers, a high level of discrimination is required when interpreting these in practice, and the use of clinical impression in conjunc- tion with radiological investigations remains the mainstay of the diagnostic paradigm. the limitations of this study are primarily as a result of the studies included to inform the cost–benefit trade-off. patient selection varied, and a lack of details regarding exclusion criteria limited the applicability of the studies to a patient population. moreover, there was heterogeneity in the study designs, with a number of retrospective studies being included. many of these trials did not explicitly mention blinding of the investigators, which is another potential source for bias and limitation of this review. inherently with the use of novel biomarkers, no preexisting widely accepted threshold exists, leading many studies to assess various diagnostic cutoff values. without blinding the investigators to the results of the histology, this increases the scope for bias. furthermore, these studies often employed ‘healthy’ controls to formulate the testing thresholds; however, minimal details were provided as to the demographics of these controls, as well as leading to the aforementioned issues regarding specificity. a further limitation of this type of review is the potential for publi- cation bias. whilst this was mitigated by conducting a thorough multi-database search, the presence of language and publication bias still persists. the results are further limited by the fact that the weighting was based upon the results of an online survey which had a response rate of . % and represented only surgeons affiliated with the eaes. moreover, as the best overall marker changed with increasing the importance of sensitivity, the reliance upon the weighting system demonstrates how the conclusions would change depend- ing on the opinions of the surgeons. conclusion appendicitis continues to pose a diagnostic challenge to emergency physicians and surgeons. clinical impression remains a crucial tool in diagnosis, and treatment allocation in those with suspected appendicitis. as yet no biomarker has been shown to have sufficient diagnostic performance to be used in isolation clinically. this would suggest that further areas of research should focus upon the search for new novel diagnostic tests and the clinical utility of the tests, rather than repeat existing research into previously studied biomarkers. through this approach, the accuracy of diagnosis of appendicitis can be enhanced, reducing the number of negative appendectomies performed, implied adverse impact to patients and treatment costs to hospitals. funding mr. sheraz markar is funded by the national institute of health research (nihr). this research was supported by the national institute for health research (nihr) diagnostic evidence co-operative london at imperial college healthcare nhs trust. the views expressed are those of the authors and not necessarily those of the nhs, the nihr or the department of health. compliance with ethical standards disclosures amish acharya, sheraz r. markar, melody ni and george b. hanna have no conflicts of interest or financial ties to disclose. open access this article is distributed under the terms of the creative commons attribution . international license (http://creative commons.org/licenses/by/ . /), which permits unrestricted use, dis- tribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. references . d’souza n, nugent k ( ) appendicitis. am fam physician ( ): – . national surgical research collaborative ( ) multicentre observational study of performance variation in provision and outcome of emergency appendicectomy. br j surg ( ): – . nguyen nt, zainabadi k, mavandadi s, paya m, stevens cm, root j, wilseon se ( ) trends in utilization and outcomes of laparoscopic versus open appendectomy. am j surg ( ): – . jones k, pena aa, dunn el, nadalo l, mangram aj ( ) are negative appendectomies still acceptable? am j surg ( ): – . d’souza n, d’souza c, grant d, royston e, farouk m ( ) the value of ultrasonography in the diagnosis of 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cost--benefit trade-off analysis 	abstract 	background 	methods 	results 	conclusion 	materials and methods 	literature search strategy 	literature standard 	biomarker survey 	statistical methodology 	cost--benefit trade-off analysis 	results 	literature search 	quadas- evaluation 	pooled analysis for individual serum biomarkers in acute appendicitis 	white cell count 	c-reactive protein 	bilirubin 	pro-calcitonin 	il- 	pooled analysis for -hiaa from urine in acute appendicitis 	biomarker survey 	cost--benefit trade-off 	discussion 	conclusion 	funding 	references science journals — aaas s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e g en e t i c s department of medicine, albert einstein college of medicine, bronx, ny , usa. department of genetics, albert einstein college of medicine, bronx, ny , usa. institute of aging research, albert einstein college of medicine, bronx, ny , usa. department of human evolutionary biology, harvard university, cambridge, ma , usa. inserm u , pincus building, bicêtre hospital, paris sud university, le kremlin bicêtre, france. department of pathology univer- sity of vermont, south park drive, colchester, vt , usa. leonard davis school of gerontology, university of southern california, los angeles, ca , usa. university of maryland school of medicine, baltimore, md , usa. department of human biology, faculty of natural sciences, university of haifa, haifa, israel. de- partment of epidemiology and population health, albert einstein college of medi- cine, bronx, ny , usa. inserm u and department of pediatric endocrinology anddiabetes,bicêtrehospital,pôlei e,parissuduniversity, lekremlinbicêtre,france. department of epidemiology, university of washington school of public health, seattle, wa , usa. public health sciences division, fred hutchinson cancer research center, seattle, wa , usa. geriatrics research and education clinical center, veterans administration medical center, baltimore, md , usa. *corresponding author. email: gil.atzmon@einstein.yu.edu ben-avraham et al., sci. adv. ; :e june copyright © the authors, some rights reserved; exclusive licensee american association for the advancement of science. distributed under a creative commons attribution noncommercial license . (cc by-nc). the gh receptor exon deletion is a marker of male-specific exceptional longevity associated with increased gh sensitivity and taller stature danny ben-avraham, , , diddahally r. govindaraju, , temuri budagov, , delphine fradin, peter durda, bing liu, sandy ott, danielle gutman, lital sharvit, robert kaplan, pierre bougnères, , alex reiner, , alan r. shuldiner, , pinchas cohen, nir barzilai, , , gil atzmon , , , * h ttp ://a d va d o w n lo a d e d fro m although bothgrowthhormone (gh) and insulin-like growth factor (igf- ) signaling were shown toregulate life span in lower organisms, the role of gh signaling in human longevity remains unclear. because a gh receptor exon de- letion (d -ghr) appears to modulate gh sensitivity in humans, we hypothesized that this polymorphism could play a role in human longevity. we report a linear increased prevalence of d -ghr homozygosity with age in four independent cohorts of long-lived individuals: participants [ of the longevity genes project (lgp) ( % increase; p = . ), of the old order amish ( % increase; p = . ), of the cardiovascular health study ( . % increase; p = . ), and of the french long-lived study ( . % increase; p = . )]. in addition, mega analysis of males in all cohorts resulted in a significant positive trend with age ( % increase; p = . ), suggest- ing sexual dimorphism for gh action in longevity. further, on average, lgp d /d homozygotes were inch taller than the wild-type (wt) allele carriers (p = . ) and also showed lower serum igf- levels (p = . ). multivariate regression analysis indicated that the presence of d /d genotype adds approximately years to life span. the lgp d /d -ghr transformed lymphocytes exhibited superior growth and extracellular signal–regulated kinase activa- tion, to gh treatment relative to wt ghr lymphocytes (p < . ), indicating a gh dose response. the d -ghr variant is a common genetic polymorphism that modulates gh responsiveness throughout the life span and pos- itively affects male longevity. n ce s.sc o n a p ril , ie n ce m a g .o rg / introduction growth hormone (gh) and insulin-like growth factor (igf) play a cen- tral role in development, differentiation, growth, and metabolism among divergent taxa ( ). for instance, the absence of the igf- gene is associated with poor prenatal growth both in rodents and in humans ( ). the activities of igf- are regulated by six igf binding proteins (igfbp to igfbp ), which act as carrier and modulatory proteins for igf- and are produced in a diverse array of tissues via complex regulatory processes. among these six proteins, igfbp is complexed to more than % of the circulating igf- ( ). dwarf individuals appear to live longer among many species ( , ), suggesting a role for the gh/igf- axis in modulating aging and life span. a considerable body of in vitro experimental evidence also sug- gests an important role for the igf axis in human longevity and aging-related processes in a tissue-specific manner ( ). furthermore, several studies in selected human populations lend support on the role of thisaxisin healthandlife span. forinstance, we have previously iden- tified a cluster of functional mutations in the igf- receptor in cente- narians( ). we showedthat laron dwarfs, who are naturally short, have decreased prevalence of diabetes, cancer, and stroke, suggesting increased health span although life span in this small sample size cannot be determined accurately ( ). also, we previously established that cen- tenarians with lower levels of igf- had significantly longer survival ( ). clearly, individuals with severe gh deficiency have reduced life expect- ancy ( ), suggesting that some gh is necessary for survival. on the other hand, interventional gh therapy in humans is commonly used to reverse age-related morbidities; hence, the kind of deficiency that will be most beneficial for health span and longevity needs to be further established. gh is produced by the anterior pituitary gland and circulates in a pulsatile manner, with peaks during sleep. the production of gh is mediated by ghrelin and gh-releasing hormone and inhibited by so- matostatin and igf- feedback inhibition. the peaks of circulating gh are modulated by several factors, including age, health conditions, gender, and nutrition. gh production is decreased with age; however, it is never completely diminished ( ). that said, there is accumulating evidence that gh may play a crucial role in modulating aging. surpris- ingly, gh deficiency or diminished secretion has been linked to longev- ity phenotypes both in mice models and in humans with familial longevity ( , ). the gh receptor (ghr) gene is located on the short arm of chro- mosome (p -p . ) and has nine coding exons ( ). it consists of two common isoforms: (i) full-length ghr—flghr (nm_ ) and (ii) a shorter form with a deletion of exon in which exon is of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e h ttp ://a d o w n lo a d e d fro m spliced in frame to exon , resulting in the deletion of amino acids within the n-terminal domain of the receptor—d -ghr ( , ). the allele frequencies of these isoforms among human populations range from to % and to % for flghr and d -ghr ( – ), respectively. the effects of ghr isoforms on human health have provided mixed results. likewise, results on the efficacy of human gh (hgh) therapy to d -ghr human subjects are variable. following the initial observation of a positive association between growth response and hgh therapy among d -ghr carriers ( ), both positive ( , ) and no relationship ( , ) have been reported, resulting in two meta-analyses showing modest positive dominant effects of the d -ghr genotype for the re- sponse to gh in various etiologies of short stature ( , ). in two ge- nome wide association studies (gwas) based on single-nucleotide polymorphisms (snps) ( , ), the ghr locus showed association with final height. however, to our knowledge, the association of d -ghr with final height has not been examined possibly because individuals with d -ghr are expected to maintain normal gh action despite lower gh production, as hypothesized by bougnères ( ) and pantel et al. ( ). hence, it is reasonable to hypothesize that increased gh sensitivity can also alter igf- secretion ( , ) and therefore regulate longevity ( , ). given the potential role of the gh/igf axis in longevity, we hypoth- esize that low igf- levels will assure longevity of the d -ghr carriers. to address this hypothesis, we genotyped d -ghr locus in four human cohorts with long-lived participants, and we tested its association with longevity-related phenotypes and stature with a relatively common ghr variation. o n a p ril , d va n ce s.scie n ce m a g .o rg / results relationship between d -ghr and phenotypic traits d -ghr and longevity in ashkenazi males, but not in females, a marked difference in allele frequency for the exon deletion polymorphism (d -ghr) was found between centenarian and control, as well as offspring and control groups. whereas the male control group carried only % homozygote deletions, male offspring of centenarians and male centenarians carried and %, respectively. although these differences did not reach sta- tistically significant levels among the offspring (p = . ), they crossed the significance threshold in centenarians (p < . ) versus control (fig. ). note that the mean age difference between the centenarians ben-avraham et al., sci. adv. ; :e june and their offspring is about years. we applied age trend analysis to test therelationshipbetweenthed -ghrpolymorphisminpooledcontroland centenarian’s data. the results showed a significant positive trend (p = . ), implying an enrichment of this allele with advancing age (fig. a). we further validated these results in three independent cohorts—the old order amish (ooa), cardiovascular health study (chs), and the french long-lived study (flls). in the ooa, homozygosity for the d -ghr increased with age among males, from % at years of age to % at years of age (p = . ) (fig. b). in chs, these differ- ences showed similar trends; the homozygotes increased from . % at to years of age to . % at to years of age (p = . ; fig. c). in the french cohort, the frequency of d -ghr homozygotes increased from . % at < years of age to % at > years of age (p = . ) (fig. d). finally, we conducted a mega analysis with males of the four cohorts in which d -ghr homozygotes increased from . % at < years of age to % at > years of age (p = . ) (fig. e). these results demonstrate a consistent relationship between homozygosity for the d -ghr deletion allele and longevity among the cohorts studied. however, this observation was limited only to males; the frequency of d -ghr deletion homozygosity among females did not differ with age in any of the cohorts studied. d -ghr and height because d -ghr has been shown to be associated with shorter stature ( ), we investigated height among homozygotes for this deletion using a recessive model adjusted for gender, age, and group in our ashkenazi jew study cohorts (ajs). in the combined data set of aj subjects, d -ghr homozygotes across ages were taller compared to wild type (wt) and the heterozygotes by inch {( . inches versus . inches, p = . ; tables ),thisphenomenonmightbedrivenbytheeffectobservedamong centenarians [ . inches versus . inches, p = . ; table s (other val- idation cohorts did not have this phenotype)]}. relationships among d -ghr, igf- , and igfbps we further tested whether the d -ghr polymorphism was related to the levels of igf- and its igfbps. previously, suh et al. ( ) demon- strated high igf- levels and short stature among female offspring of centenarians and suggested a mechanism by which igf- insensitivity could occur in females. however, this finding was not replicated among males in the offspring of centenarians. in contrast, the frequency of the homozygote ghr deletion polymorphism increased in males in all co- horts, suggesting gender specificity. also, this increment was associated with low igf- (tables s and s under dominant model). d -ghr homozygote deletion carriers or those with heterozygote under domi- nant model showed significant low levels of igfbp compared with wt, in offspring (table s ) and in allelic or dominant model among all groups (table s ), and control and offspring combined (table s ). other igfbps did not reveal this relationship. d -ghr and ghbp we further tested for association of the d -ghr deletion with the levels of ghbp. all association tests revealed a significant inverse relationship between the carriers of the deletion and growth hormone binding pro- tein (ghbp) levels (fig. and tables s to s ). d -ghr and survival of the genotyped male subjects in the longevity cohort, were homozygous for the deletion (> % of total). the relationship between the d -ghr deletion genotypes and mortality assessed through kaplan-meier survival function suggests that the probability of survival control offspring centenarian d -g h r f o e g a t n e c r e p *p < . versus control fig. . percentage of d -ghr homozygosity among female (black) (f) and male (gray) (m) ashkenazi centenarians ( f and m), offspring ( f and m), and controls ( f and m). of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e ben-avraham et al., sci. adv. ; :e june o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m was significantly greater among the deletion carriers than the noncar- riers (p = . ; fig. ). the survivors were years older, on average, than the wt carriers, suggesting plausible beneficial effects of this poly- morphism on age and longevity. functional studies of d -ghr polymorphism to assessthe functionalimpactof the deletion, we determinedthe in vitro effects of gh stimulation ( ng/ml) on the rate of proliferation of transformed lymphocytes from male centenarians. in the basal state, lymphocytes from d -ghr homozygous subjects had lower prolifera- tion rates compared to wt homozygotes. however, in the presence of gh, we observedhigherproliferation ratesin d homozygotes(fig. a). in addition, we assessed the phosphorylation of extracellular signal– regulated kinase (erk) in transformed lymphocytes from male cente- narians. lymphocytes from d -ghr homozygotes displayed lower basal activation of erk compared to wt homozygotes. conversely, higher activation of erk was found in response to gh treatment ( ng/ml), compared to wt homozygotes (p < . ) (fig. b). no effect on the phosphorylation of signal transducers and activators of transcription (stat ) and akt was evident (fig. s ), suggesting a unique, pathway-specific effect of the d -ghr polymorphism on gh signaling e ≥ – – – – age bin (years) – – – – – d -g h r h o m o z y g o te ( % ) d -g h r h o m o z y g o te ( % ) age bin (years) a a b c d fig. . d -ghr age prevalence within and between cohorts. prevalence of d -ghr homozygotes in relation to age groups in: (a) ashkenazi jew (aj) (female and male of control and centenarian, n = ) combined and (a ) split by gender ( males and females), (b) ooa males, (c) white males of the chs, and (d) french white males and (e) mega analysis of the four cohorts ( males). in each of the cohorts, there is increase prevalence of the homozygote d -ghr (*p < . for the trend). d -ghr het and wt g h b p ( p m ) genotype offspring control centenarian fig. . distribution of ghbp (in pm) among the centenarians ( f and m), their offspring ( f and m), and controls ( f and m), all of whom are of ashkenazi (aj) descent, in recessive model [homozygous ghr deletion of exon (d -ghr) versus heterozygote (het) and wt combined], adjusted for gender and age (*p < . ). of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e with decreased constitutive activation yet enhanced gh-driven sig- naling. these results are compatible with reports on the growth of d -ghr children showing an increased responsiveness to gh only in studies that use high doses of gh. o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m discussion although numerous genes have been shown to influence longevity ( ), certain genes, including igf , appear to affect life span across diverse or- ganisms, ranging from worms to humans ( ). in humans, igf- is as- sociated with key biochemical pathways that regulate energy metabolism, immunity, inflammation, and imprinting and hence influence growth, development, and senescence from fetus to old age ( ). hence, it is rea- sonable to suggest that the igfs exert pleiotropic effects on a wide range of cellular, physiological, and morphological variables throughout the life span of individuals. for instance, igf- has been shown to prevent age- related myopathy through attenuation of myocyte death, repair muscle damage through regeneration of skeletal muscles in old animals, and up- regulate telomerase activity ( ). furthermore, as presented elsewhere, mutations in igf have been shown to bring about a series of correlated responses: decreased igf- levels, lower incidence of heart failure and mortality, and increased longevity in mice ( ). our results corrobo- rate similar studies conducted on other cohorts. for instance, an inverse relationship between reduced igf- levels and longevity has been reported on an italian centenarian cohort ( ) and in the leiden -plus study ( ). similarly, ghr deficiency is associated with an unchanged life span but with a reduced incidence of cancer and diabetes ( ). the phenotypic effects of ghr could be attributed to the direct activation of tyrosine ki- nase or indirect induction of igf- ( ), whose excess can be detrimental. in centenarians, most igf- regulation seems to respond to caloric and protein nutritional signals, not from gh. igf- is not lower in car- riers of d -ghr during childhood, adolescence, and adulthood despite several reports showing that the ghr genotype may influence circulat- ing igf- under basalconditions.people with d -ghr or fl-ghr alleles produce comparable amounts of circulating igf- ( , – ). that said, we suspect people with d -ghr alleles to have a decreased gh secretion. gh secretion by pituitary somatotropic cells is normally regu- lated by hypothalamic neuroendocrine mediators that are, at least par- tially, sensitive to feedback effects of igf- or metabolic substrates. a higher level of gh transduction signaling through the d -ghr would ben-avraham et al., sci. adv. ; :e june result in the following conditions: a greater gh impact on liver and the growth plate; and among other targets, increased igf- generation, less feedback exerted on pituitary gh production, lower circulating gh levels, less binding to the d -ghr–equipped cells, and reestablishment of a desirable level of gh action on relevant targets. it appears that deletion of the ghr gene exon might have origi- nated from complex genomic events taking place after the emergence of old world monkeys, followed by homologous recombination between two retro-elements in homo sapiens. thereafter, it spread throughout the human clades to be present now in approximately % of caucasian chromosomes. strawbridge et al. ( ) suggested thatd -ghr mayoffer some degree of protection against type diabetes mellitus (t dm) because the per- cent of individuals homozygous for the d -ghr allele was only % in t dm versus % in normal glucose tolerance (ngt). the diminu- tion of t dm prevalence may increase longevity. this protective effect of the d -ghr genotype is independent of igf- , because no significant differences of circulating igf- were observed across the three d -ghr genotypic groups. this is consistent with previous gwas and studies that have not found that the ghrgenotype contributesto the individual variation of circulating igf- ( ). the mode of gh secretion has not yet been studied in centenarians. it is possible that people exposed lifelong to lower gh secretion live longer, because the activity of gh is involved with many physiological . . . . s u r v iv a l age at recruitment (years) d -ghr het and wt d -ghr het and wt a g e a t r e c r u it m e n t (y e a r s) * *p < . versus d -ghr fig. . kaplan-maier survival curves of male aj centenarians (n = ) and control (n = ) of d -ghr in recessive model [homozygous ghr deletion of exon (d -ghr) versus heterozygote (het) and wt combined]. . . . sf d /d n = fl/fl n = growth a b au * gh ng/ml ** sf gh ng/ml fold change in perk to t- erk fl/fl n = d /d n = ** ** fig. . functional effect of d -ghr carriers. (a) in vitro effect of gh stimulation ( ng/ml) on proliferation of transformed lymphocytes from male aj centenarians. in the basal state [serum-free (sf)], lymphocytes from d -ghr homozygous subjects have reduced proliferation rates; however, in the presence of gh, there is enhanced respon- siveness and overall higher growth rates. this is compatible with reports in humans showing an increased responsiveness to gh only in studies that use high doses of gh. au, arbitrary units. (b) phosphorylation of erk in homozygous wt and d -ghr transformed lymphocytes from male aj centenarians. lymphocytes from d -ghr homozygotes displayed significantly lower perk levels under serum-free conditions but higher activation of erk in response to gh treatment ( ng/ml), compared to wt (fl/fl) carriers (**p < . ). of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m systems associated with longevity. in addition, we and others have re- ported an association between low igf- and longevity ( , , ). simi- larly, several others ( , – ) have shown numerically higher igf- levels in d -ghr carriers, which did not reach significant levels be- cause of absence of recessive model analysis or small sample size. further, people with d -ghr may have increased in number because of these ad- vantages, including low gh secretion for a given igf- level: if nutrition increases igf- in these people, then they will keep their gh relatively low through a more efficient feedback. we should remember that gh–igf- relationships are regulated to favor growth in young ages but fit other purposes in older ages, such as body composition, insulin secretion, and energy flux. finally, ghrko mice demonstrated longer life expectancy and lower igf- levels compared with wt ( ). follow- ing this line of thought, we suggest that people with d -ghr should maintain normal gh action despite less gh being produced. the inverse relationship between low igf- and increased d -ghr preva- lence (as indicated in this paper) supports the notion that d -ghr is favorable to longevity. the positive association between d -ghr homozygote frequencies and longevity in the three distinct populations with different demographic histories found in our study agrees with the earlier reports that reduced gh/igf signaling may be involved in modulating human longevity and could signify a broader phenomenon. the positive relationship between the d -ghr homozygote frequency and longevity, as well as height, suggests that decreased levels of ghr expression in homozygotes may have favorable effects on longevity. further, whereas in most re- ports d -ghr homozygote carriers are associated with short or no difference in stature, audi et al. ( ) demonstrated a tendency of the d -ghr homozygote carriers to be taller, and strawbridge et al. ( ) re- ported significant +-inches-taller normal glucose-tolerant d /d subjects in an additive model compared with the other genotypes. this general trend was further confirmed through cell culture studies, in which cells carrying ghr homozygote exon deletions displayed a sig- nificantly slower rate of growth and lower activation of erk at baseline. however, with gh treatment, d -ghr homozygous lymphocytes showed superior growth and erk activation, relative to homozygous wt lymphocytes. this phenomenon could be explained by the positive relationship of ghbp with longevity among ajs, further suggesting the plausibility of a consistent relationship between d -ghr variation and gh secretion from cellular to organism levels over the life course of in- dividuals. these observationssuggest thatigf- may exert positive plei- otropy ( ) on many traits associated with healthy longevity. our observation that igfbp is higher in the d -ghr subjects suggests that they are more insulin-sensitive ( ). our study provides the first consistent evidence linking the ghr to human longevity. although our study consists of four cohorts with a total of subjects, the sample size ranged from to ; hence, it is generally low relative to the sample sizes used in many association studies. mutations in gh-related genes may exert pleiotropic ( ) effects throughout the life span of individuals, which appears to be a universal feature of most if not all major longevity assurance genes. be- cause igfs are found across a wide range of organisms and perform similar functions among these, as suggested by waddington ( ), they could be viewed as “canalized” genes playing a vital role in the survival of organisms across taxa. although the ghr deletion appears to show age-, cohort-, and gender-specific effects, we hypothesize that a combi- nation of empirical and systems analyses would provide answers toward understanding the contextual effects of gh on longevity in relation to age, stage, and gender. ben-avraham et al., sci. adv. ; :e june in summary, our findings suggest that the d -ghr, as indicated by the distribution of homozygotes among the four populations examined, may be involved in modulating human longevity. these results may have implications in devising precision medicine strategies, such as gh-related interventional therapies in the elderly. materials and methods subjects and phenotyping the ashkenazi centenarian cohort (longevity genes project) the contemporary aj population worldwide is descended from a founder population (estimated to be several thousand) originating in the th century. to a large extent, thispopulation exhibits bothcultural and genetic homogeneity. for these reasons, the aj population has been successfully used in the discovery of many disease-associated genes ( ). the aj cohort in the present study, which consists of three groups: cen- tenarians, their offspring, and controls (see below), was recruited as pre- viously described ( – ). most of the participants in this cohort were born in or moved to the united states before world war ii. the ages of male aj centenarians with exceptional longevity were or above, as verified from their passports. offspring of the long-lived centenarians consisted of subjects ( . % male and . % female) with a mean age of . years (range, to years). the control group, used for the purpose of comparison with the offspring group, consisted of indi- viduals ( % male and % female) recruited from two different sources: (i) spouses of the offspring (n = ) and (ii) a group of inde- pendently recruited aj from the einstein aging study (n = ) ( ). in the control group, we excluded individuals if the parents lived to the age of > years. the mean age of the control group was years (range, to years). because there were no differences between any measured traits or ghr genotype frequencies between the two subgroups, they were combined and were treated as one control group. a detailed med- ical history questionnaire was administered, and a physical examination on the aj was performed as previously described ( – ). informed written consent was obtained from the participants in accordance with the policy of the committee on clinical investigations of the albert einstein college of medicine. independent (validation) cohorts in addition to the subjects from the aj cohort, dnas from the ooa, the chs, and the flls were genotyped. additional details of these cohorts are provided below. old order amish. the amish family calcification study (afcs) was initiated in at the university of maryland to identify the joint determinants of bone mineral density (bmd) and vascular calcification specific to the amish community of lancaster county, pa. the amish immigrated to the united states from western europe in the late s and represent a genetically homogeneous founder population. the afcs cohort consisted of males and females with an average age of years (range, to years). of these, white males with a mean age of . years (range, to years) and d -ghr genotypes were reported in this study (table s a). the afcs was approved by the uni- versity of maryland institutional review board (irb), and written informed consent was obtained from all research participants. cardiovascular health study. thisisaprospectivepopulation-based cohort study consisting of adult men and women who are years and older. they were recruited from four field centers: forsyth county, nc; sacramento county, ca; washington county, md; and pittsburgh, pa ( ). baseline examination for the original cohort, of whom or % self-identified their ethnicity as white, was performed over year, of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m beginning in may . of these, white males with a mean age of years (range, to years) and d -ghr genotypes were reported in this study (table s a). all clinical examinations/procedures were conducted under institutionally approved protocols for use of hu- man subjects. french long-lived study. the flls included french long- lived caucasian male participants from to years old and adult controls from to years old. of these, white males with a mean age of years (range, to years) and d -ghr genotypes were reported in this study (table s a). written informed consent was obtained from the study participants. phenotyping wide ranging phenotypic data encompassing physiological (lipids: cho- lesterol, triglyceride, and high-density lipoprotein and low-density lipo- protein cholesterol) ( ), and igf- –related biomarkers (igf- , igfbps, insulin, and ghbp) ( ), disease prevalence (myocardial in- farction, stroke, diabetes, or cancer), and anthropometric (height and weight) ( ) traits have been collected from the ashkenazi centenarians, their offspring, and age-matched (to the offspring) controls (table s b). genotyping genotyping of the d -ghr polymorphism was performed using two consecutive polymerase chain reactions(pcrs) using the same genomic dna sample, one with the g -g set of primers followed by another with the g -g set of primers (table s ). we used ng of genomic dna in a -ml pcr reaction mix consisting of -ml amplitaq gold pcr master mix (applied biosystems), . pm of each primer, and double-distilled water. the pcr was performed in a bio-rad instru- ment with the following conditions: °c for min, cycles of °c for s, °c for s, °c for s, and a final extension at °c for min. we then analyzed the pcr-amplified products by electropho- resis on a % agarose gel stained with ethidium bromide. the lengths of the pcr products for the g -g and g -g were and base pairs (bp), respectively. these bands were classified as follows: bp– bp = homozygote for wt, bp– bp = heterozygote, and bp– bp = homozygote for the deletion. analysis of transformed lymphocytes blood samples obtained from subjects were rapidly processed at the general clinical research center at the albert einstein college of med- icine for generating epstein-barr virus–transformed lymphocytes using the established methods. informed written consent was obtained from all the participants, in accordance with the policy of the committee on clinical investigations of the albert einstein college of medicine. to assess the ability of gh to activate signal transduction via the ghr, we incubated lymphocytes harboring mutation and those without the mutation, as described ( ). growth assays on these cells were recorded, followed by total cell protein extraction from cell lysates. these were further subjected to sds–polyacrylamide gel electrophoresis; immuno- blotting with total- and phospho-akt, stat and erk using antibo- dies from cell signaling; and densitometry. results are means ± sd. statistical significance was determined using unpaired t tests. statistical analysis data on serum triglycerides were ln-transformed for analysis and were back-transformed for presentation. four comparisons of phenotypic data were performed on the aj cohort: (i) within each group (that is, centenarian, offspring, and control—all of whom are of aj descent); ben-avraham et al., sci. adv. ; :e june (ii) between centenarians, offspring, and controls; (iii) between off- spring and controls; and (iv) between centenarians and controls. all analyses were adjusted for age and sex. we applied the most fre- quently used models in association studies: allelic (carriers of the de- letion versus none), dominant (wt versus heterozygote and deletion homozygote), additive (wt versus heterozygote versus deletion homo- zygote), and recessive (deletion homozygote versus wt and hetero- zygote) in each comparison (tables s to s ). analysis of genotype data genotypes were checked for mendelian consistency using the pedcheck software ( ) before analysis. mendelian errors were resolved or removed before analysis. allele frequencies were calculated by gene counting, and all the genotypes conformed to hardy-weinberg expectations. we eval- uated the association between snp genotype and phenotype (for exam- ple, igf levels or height) under the additive or dominant models using a variance component approach. we modeled the probability that the subject was a case or control as a function of the individual’s age, sex, and genotype, conditional on the correlations in phenotype among relative pairs. statistical analyses were performed using jmp . supplementary materials supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/ / /e /dc fig. s . phosphorylation of stat and akt in homozygous wt and d -ghr transformed lymphocytes from male aj centenarians. table s . analysis (allelic, dominant, additive, and recessive models) of various variables (means ± se), includes all ajs groups (n = ) and adjusted for gender, age, and group. table s . analysis (allelic, dominant, additive, and recessive) of various variables (means ± se), includes aj centenarian (c) and control (c) groups (n = ) and adjusted for gender, age, and group. table s . groups analysis of various variables (means ± se) adjusted for gender and age within ajs. table s . analysis (allelic, dominant, additive, and recessive models) of various variables (means ± se), includes aj offspring (o) and control (c) (n = ) and adjusted for gender, age, and group. table s a. genotyping efforts among the four cohorts (n = ): aj (n = , % female), ooa males, chs males, and flls males. table s b. crude measurements of various variables (means ± se) in the three ajs (n = ) study groups. table s . pcr procedure using primers. references and notes . m. barbieri, m. bonafè, c. franceschi, g. paolisso, insulin/igf-i-signaling pathway: an evolutionarily conserved mechanism of longevity from yeast to humans. am. j. physiol. , e –e ( ). . q. fu, x. yu, c. w. callaway, r. h. lane, r. a. mcknight, epigenetics: intrauterine growth 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:e june acknowledgments: we thank all the participants from the three cohorts used in this study. funding: this study was funded in part by grants from the nih [(p ag to n.b.), ( r ag to g.a.), and ( r ag and p ag to p.c.)], the nathan shock center of excellence for the biology of aging (p ag to n.b.), the glenn center for the biology of human aging (paul glenn foundation grant to n.b. and p.c.), and diabetes center (dk- to n.b.). d.f. and p.b. were supported by inserm with additional contribution from association de recherche sur le diabète and an institutional grant from novo nordisk france. chs was supported by contracts n -hc- through n -hc- , n -hc- , n hc- , n hc- , n -hc- , and n -hc- ; grant number u hl from the national heart, lung, and blood institute; u ag from the national institute on aging longevity consortium; and p ag from the national institute on aging, claude d. pepper older americans independence centers; and grant r hl- (to a.r.), with additional contribution from the national institute of neurological disorders and stroke. a full list of participating chs investigators and institutions can be found at http://chs-nhlbi.org. genotyping services for chs were provided by the center for inherited disease research (cidr). cidr is fully funded through a federal contract from the nih to the johns hopkins university (contract number n -hg- ). author contributions: d.b.-a. and d.r.g. performed data analysis and participated in drafting the manuscript. t.b., d.f., b.l., s.o., and l.s. were responsible for laboratory preparation and data acquisition. d.g. and a.r. performed data acquisition and helped with drafting the manuscript. bps initial recruitment and data acquisition. p.d. and r.k. helped with drafting the manuscript. p.b., a.r.s., p.c., and n.b. coordinated and designed the studies and helped with drafting the manuscript. g.a. coordinated and designed the study, performed data analysis, and participated in drafting the manuscript. all authors read and approved the final manuscript. competing interests: a.r.s. is an employee of regeneron pharmaceuticals. the other authors declare that they have no competing interests. data and materials availability: all data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. additional data related to this paper may be requested from the authors. the data and materials in this work can only be used in accordance with the filed university of maryland irb. submitted august accepted april published june . /sciadv. citation: d. ben-avraham, d. r. govindaraju, t. budagov, d. fradin, p. durda, b. liu, s. ott, d. gutman, l. sharvit, r. kaplan, p. bougnères, a. reiner, a. r. shuldiner, p. cohen, n. barzilai, g. atzmon, the gh receptor exon deletion is a marker of male-specific exceptional longevity associated with increased gh sensitivity and taller stature. sci. adv. , e ( ). o n of a p ril , http://chs-nhlbi.org http://advances.sciencemag.org/ with increased gh sensitivity and taller stature the gh receptor exon deletion is a marker of male-specific exceptional longevity associated and gil atzmon danielle gutman, lital sharvit, robert kaplan, pierre bougnères, alex reiner, alan r. shuldiner, pinchas cohen, nir barzilai danny ben-avraham, diddahally r. govindaraju, temuri budagov, delphine fradin, peter durda, bing liu, sandy ott, doi: . /sciadv. ( ), e . sci adv article tools http://advances.sciencemag.org/content/ / /e materials supplementary http://advances.sciencemag.org/content/suppl/ / / / . .e .dc references http://advances.sciencemag.org/content/ / /e #bibl this article cites articles, of which you can access for free permissions http://www.sciencemag.org/help/reprints-and-permissions terms of serviceuse of this article is subject to the is a registered trademark of aaas.science advancesyork avenue nw, washington, dc . the title (issn - ) is published by the american association for the advancement of science, newscience advances copyright © , the authors o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m http://advances.sciencemag.org/content/ / /e http://advances.sciencemag.org/content/suppl/ / / / . .e .dc http://advances.sciencemag.org/content/ / /e #bibl http://www.sciencemag.org/help/reprints-and-permissions http://www.sciencemag.org/about/terms-service http://advances.sciencemag.org/ thb .tmp a financial training program for usda/fsa borrowers: evolution and impacts robert l. parsons, gregory d. hanson, wesley n. musser, roland freund, and lehan power a financial training program designed by cooperative extension specialists was provided to over , usda/fsa borrowers from the northeast during the period - . key to the success of the workshops was an in-depth, user-friendly curriculum that evolved over time, eventually replacing satellite-feed instruction with pre-taped videos. cluster analysis classified nearly % of workshop participants as “low fkrance priority” or “low fhance knowledge.” farmers in these clusters received a relatively greater educational benefit from the program than those not in these clusters. impact analysis indicated that perceived annual gain in farm net worth from application of workshop tools ranged from approximately $ , to $ , . the training addressed the needs of prodncers typically isolated from cooperative extension because the workshop was the only extension program attended that year by nearly two-thirds of them. more than , farm operators, partners, and spouses in delaware, maryland, new york, and pennsylvania completed a financial training pro- gram developed in response to a mandate in the u.s. farm bill requiring certain farmers with u.s. department of agriculture farm service agency (fsa) operating andlor ownership loans to receive instruction in financial statements, budget- ing, record keeping, and financial management practices (federal register ; hanson (table )). in order to make the program more use- ful for fsa borrowers, the curriculum incorporated the fsa farm record system and accompanying financial statements format. because fsa is a lender of “last resort” to limited-resource produc- ers, cooperative extension has historically viewed fsa clientele as critically important to its outreach mission (hanson ). the purpose of this paper is to describe the evo- lution and evaluate the effectiveness of this finan- cial training program. analysis is based on partici- pant evaluations of the program and information about the participants collected during the work- shops. in addition to tabulations of these data, clus- ter analysis and logit models are used in the analy- sis, this analysis is combined with descriptions of the evolution of the program. initial curriculum and workshops robert l. parsons is an assistant professor with the dep~ment of com- munity development and applied economics at the university of ver- mont. gregory d. hansen is an associate professor with the department of agricultural economics and rural sociology at the pennsylvania state university, ‘ this program is unique in its adoption of the usdaffsa record and financial statement format. panly as a result of the dkect linkage to the fsa record system and its success in the northeast, this finance curricu- lum was selected for a nationwide program of training for more than fsa farm loan officers, county executive directors, and district directnrs frnm - . the financial training workshops initially used sat- ellite down-link presentations that would be coor- dinated locally by an on-site extension agent. par- ticipants followed the satellite presentations and did exercises in their own workbooks. with an educationally diverse audience in mind, the cur- riculum emphasized practical applications and had minimal narrative. this format facilitated in-class agricultural and resource economics review / (october ) - copyright northeastern agricultural and resource economics association parsons et al. a financial training program table . northeast finance and production training: farms, attendees, - basic cost cost workshop farmers, farms per farma per spouses, training enrolled pa/non-pa person curriculum partners year pa/non-pa ($) ($) pa/non-pa finance / / / / / / / / / / / / finance total / - / , / / , production (pa) / / ~ ~ ~ production total - m finance and production total – , $ $ , source: short course office, penn state college of agricultural sciences. ‘the basic charge does not include late fees and charges for more than two participants per farm. the $ tuition fee included $ for meats. the / reduction in tuition to $ in pennsylvania reflects lower costs due to economies of size. the / pennsylvania finance training was a make-up program, since / , pennsylvania has rotated finance and production training in alternating years. the production training is presently offered only in pennsylvania. presentations via satellite of topics such as produc- tion-based accrual income. major sections of the curriculum were “the balance sheet,” “the income statement,”” the cash flow budget,” “financial ratio analysis,” “farm home budgeting,” “strategic plan- ning,” and “fixing broken finances.” the work- book was prepared for a ninth-grade reading level to facilitate the participation of old order amish producers, who do not attend high school, and other farmers with limited educational achieve- ment. however, sufficient conceptual depth was included to challenge college-educated producers. a benefit of keeping the narrative to a minimum was that borrowers took ownership of the text by highlighting and writing notes pertaining to key finance concepts in the text. in order to promote attendance and minimize participant travel time, concurrent workshops were scheduled in approximately different accessible locations. workshop duration was five to six hours per day, for six days. agronomy, livestock, and farm business management agents were trained as workshop site leaders. in the first year of the pro- gram, an extension finance specialist presented the text material via satellite up-link from penn state. this approach assisted the site instructors, many of whom had limited finance background, as well as promoted uniform teaching, no one single special- ist could have delivered all of these lectures on site. the extension agents on-site coordinated training facility logistics and led text exercises, homework, and quizzes. the workshop began with a pretest, followed by text instruction on financial concepts and statements, numerical exercises, and quizzes after completion of each major topic. each partici- pant was required to complete a balance sheet, an accrual income statement, and a projected monthly cash flow for the coming year. grades were “pass,” “pass with additional fsa-led training re- quired,” and “fail,” and were based on attendance, effort on exercises, quizzes, and completion of own-farm homework. a panel of experts partici- pated in two live satellite question-and-answer ses- sions that permitted participants to call or fax ques- tions to penn state. evolution of instruction methods the official fsa evaluation indicated that % of the participants found the topics covered in / to be helpful to the farm business (table ). while coverage and suitability of the material were considered excellent by only ?z and t of the producers, respectively, approximately % found that the course level, course length, and amount of outside work were “appropriate.” the percentage of respondents who gave ratings of “poor,” “too easy,” or “too short” ranged from only o to . post-workshop discussions between the site leaders and the extension specialist leading the program revealed dissatisfaction with the rigid schedule of satellite up-links. satellite instruction required that each site meet at the same time/date and complete workshop exercises on a tight sched- ule. another problem was that signal reception was interrupted at several sites because of equip- ment failure. accordingly, time dedicated to down- october agricultural and resource economics review table . fsa/usda financial management training participants evaluations, - de, md, de, md, response to pa md, ny, pa ny, pa pa evaluation evaluation / / / / item item (%) (%) (%) (%) number of workshop participants comdeting evaluations . . , . . . . . . — top;cs c&ered in the class yes were helpful to me in my partially business no o coverage of the subject matter excellent was sufficient poor suitability of the instruction excellent materials was sufficient poor o the level of the course was too advanced appropriate too easy the length of the course was too long appropriate too short the amount of outside work too much was appropriate too little the instructor(s) was excellent good poor will you continue to take yes training courses in production maybe and financial management no topics if not required? would you recommend this yes instructor to other individuals? no comment no links was reduced from six four-hour sessions in the first year to two one-hour sessions in the third year. as with other distance education programs at penn state (peterson ) and in other states (hiel and herrington ), distance education via satellite up-links had proven to be too cumber- some, rigid, and expensive compared to pre-taped video presentations, and so instruction by satellite was finally discontinued altogether in – . this evolution was beneficial mostly where the local down-link facility was inconveniently located for participants, too small to accommodate all par- ticipants, and/or where local scheduling conflicts existed. cost savings generated from elimination of satellite instruction helped to reduce tuition from $ to $ in pennsylvania, and to $ in surrounding states. on-site extension agents, aided by pre-taped instructional videos, provided more of the instruction themselves. these changes permitt- ed site leaders to exercise more control over the pacing of materials. the site instructors, whose knowledge about the subject had increased during the first two years of the program, felt confident about their ability to assume more instructional re- sponsibility after the satellite program was discon- tinued. increased instructor experience and the use of videos and on-site instruction probably all con- tributed to enhanced instructor ratings over time (table , item ). beginning in / , participants were re- quired to complete a four-year farm plan that in- cluded projected yields, expenses, revenues, pro- jected capital expenditures, and family living expenses. in addition, the own-farm financial state- ments that participants were required to prepare were made more challenging. text workbooks and farm plan booklets were revised annually, not only to keep them current but also to add improve- parsons et al. a financial training program table . evaluation results from - fsa finance workshops (pa, ny, md, de) mean values for participants by year participant characteristics and evaftration items / / / . . , , . , . , . . . . . . . number of participant evaluations . . $ , . . $ . . . $ , dairy major farm enterprise (%) number of cows in dairy herd annual farm sales (including contract income) ($) age (yrs) years managing a farm . b . ’ . h $ , .za,b , ’ , . ’ $ , o. a . bc . b . b,” $ , , ’ completed high school (%) annual avg. farm profit past years ($) change in view of importance of financial management (scale to ) change in knowledge level of farm financial statements (scale to ) , ... . ” lff change in knowledge level of farm financial plans (scale to ) , . . budgeting, analysis, and planning tools from workshop will help your farm to survive (scale to ) . . . satisfaction with financial workshop (state to ) . $ . $ . $ financial skols learned in this workshop will likely increase your farm’s net worth per year ($) training-related increase in net worth as percent of sales (item divided by item ) . . . . . . participants not attending other extension workshops in past year (%) ‘statistically significant difference between – and – at p < . level, bstatistically significant difference between – and – at p < , level. ‘statistically significant difference between – and – at p < . level, ments suggested by site leaders and participants. for the same reasons, the instructional video tapes used in the third year were also remade, including being shortened by - %. pennsylvania and maryland extension specialists and agents were the instructors on these revised tapes. in / , farm production management was taught instead of finance. this one-year break in the finance curriculum permitted an in-depth revi- sion of its text and instructional format, to correct mistakes and clarify material. these changes in the instructional format resulted in improved evaltta- tions, the “coverage of subject matter” and “suit- ability of instruction material” receiving an “excel- lent” rating by a respective % and % of the participants (table ). although unfavorable ratings increased over time in three of the evaluation cat- egories (on the length of the course, the amount of work required outside the classroom, and the amount of interest in taking additional courses on the same subjects if not required to do so), the rest of the categories, including those pertaining to in- structors, continued to receive favorable ratings. an additional evaluation instrument was added in / . the purpose of this instrument was to provide information on participant characteristics, change in knowledge levels, and perceived poten- tial impact of the training on net worth accumula- tion (table ). three items ( – ) are producer as- sessments of their beginning and ending knowl- edge levels of financial topics, and four items ( – ) are self-assessments of workshop satisfaction and impacts. it is important to note that changes in knowledge levels and impacts of knowledge are october agricultural and resource economics review table . evaluation results by education level, - fsa workshops (pa, md, de) mean values for participants by education level participant characteristics and evaluation items primary high school college . . . . , . . . . . , . . . . percent of participants dairy major farm enterprise (%) number of cows in dairy herd annual farm sales (including contract income) ($) age (yrs) years managing a farm annual avg. farm profit past years ($) change in view of importance of financial management (scafe to ) change in knowledge level of farm financial statements (scale to ) change in knowledge level of farm financial plans (scale to ) budgeting, anatysis, and planning tools from workshop will help your farm to survive (scale to ) satisfaction with financial workshop (scale to ) financial skills learned in this workshop will likely increase your farm’s net worth per year ($) training-related increase in net worth as percent of sales (item divided by item ) participants not attending other extension workshops in past year (yc) . . a’b . ’j’ $ , ’ . a’h g,ja.b $ , ’ , . . . f+b $ . . . , ”’” , ’ $ , ’ . ’ . ’ $ , ” . . . . , a $ . . , , b’c , b $ , . b . b $ , c . . . . . lb $ . , ‘statistically significant difference between primary and high school education at p < . level. bstatistically significant difference between primary and college education at p < . level. “statistically significant difference between high school and college education at p < . level, difficult for both resident and extension educators to assess. however, the consistency of the self- assessment scores over the years supports the view that workshop participants experienced little diffi- culty answering the impact questions. data in table indicate that the typical partici- pant had managed a farm for about years, was about years old, and had annual farm sales of approximately $ , –$ , . the view that financial management was important and the knowledge levels of farm financial statements and farm financial plans all increased substantially af- ter taking the course (table , items – ). the rating of , - . (on a scale of to ) indicated that the participants believed that the tools learned at the workshop would help their farms to survive. participants estimated that implementing the work- shop farrn/household analysis and planning tools could increase farm net worth by an average of about $ , in a typical year. as shown by a rating of , . (on a scale of to ), the partici- pants expressed a high degree of overall satisfac- tion with the workshop, the information provided on this evaluation also suggests that the training addressed the needs of producers typically isolated from cooperative extension—the workshop was the only extension program attended that year by nearly two-thirds of them. workshop evaluations by education level and farm size using data from the evaluation instrument, evalu- ations were tabulated based on educational level (table ) and farm size of the participants. partici- parsons et al. a financial training program table , evaluation results from - fsa finance workshops by gross sales mean values for participants by farm sales sales less than sales $ , sales greater participant characteristics and evaluation item $ , to $ , than $ , . , . . . . . , . . . . , . percent of participants dairy major farm enterprise (%) number of cows in dairy herd annual farm sales (including contract income) ($) age (yrs) years managing a farm completed high school (%) annual avg. farm profit past years ($) change in view of importance of financial management (scale to ) change in knowledge level of farm financial statements (scale to ) change in knowledge level of farm financial plans (scale to ) budgeting, analysis, and planning tools from workshop will help your farm to survive (scale to ) satisfaction with financial workshop (scale to ) financial skills learned in this workshop . , a’b . b $ , ”b . b . b , $ , a’b . b . . b . . $ ”b . . ’ . c $ , ’” , c , c . $ , ’” . . . , . $ ” . . b . b’c $ , ”c . b’c . b” . $ , b’c , b . . b , . $lo, b will likely increase your farm’s net worth per year ($) . training-related increase in net worth as . ’j’ , w b,c percent of sales (item divided by item ) , participants not attending other extension . . . workshops in past year (%) ‘statistically significant difference between low sales and medium sales at p < . level. bstatistically significant difference between low sales and high sales at p < . level. ‘statistically significant difference between medium sales and high sales at p < . level pants in - who had completed at least high school reported approximately $ , gross rev- enue, however, those who had completed high school but not college were more specialized in dairy, had more cows per herd, and reported about $ , more in profit than those who had gone to college, the greatest change in views of the im- portance of financial management was shown by the lowest education group, which included the amish farmers. the change in knowledge vari- ables and satisfaction with the workshop tended to increase with education level, as expected, col- lege-educated participants attended more extension meetings, overall, the evaluations indicate that the training had similar impacts on knowledge levels for participants at all education levels, even though the workshop experience was more satisfactory for better-prepared college-educated participants. evaluations were also tabulated by the amount of gross sales reported by the participants into three groups—those reporting sales greater than $ , , $ , -$ , , and less than $ , (table ). the one with the largest gross sales reported less knowledge gain in terms of statements and plans than did the group with the least sales. the higher-sales group did find the workshop slightly more satisfactory and slightly more beneficial in terms of helping their business survive than did the other group, even though the difference was not statistically significant. the october agricultural and resource economics review table . evaluation characteristics from - fsa finance workshops by cluster cluster low finance low finance high finance priority knowledge knowledge cluster cluster cluster participant characteristics and evaluation items (n = ) (n = ) (n = ) . . . . . . . . . io. il. . . , . . , — percent of participants , dairy major farm enterprise (%) . number of cows in dairy herd . a’h annual farm sales ($) , ’” age (yrs) . years managing a farm (yrs) . completed high school (%) . annual avg. farm profit past years ($) , b change in view of importance of , ,sa.h financial management (scale to ) change in knowledge level of farm financial i . h statements (scale to ) change in knowledge level of farm financial . b plans (scale to ) budgeting, analysis, and planning tools from . ” workshop will help your farm survive (scale to ) satisfaction with financial workshop (scale to ) , financial skills learned in this workshop will likely ’ increase your farm’s net worth per year ($) training-related increase in net worth as percent of , d.h sales (items divided by ) participants not attending any other . extension workshops in past year (%) percent of amish participants . ‘statistically significant difference between cluster and cluster at p < . level. !statistically significant difference between cluster and cluster at p < . level. ‘statistically significant difference between cluster and cluster at p < . level. group with the highest gross sales also gave the potential impact of the workshop on annual growth in farm net worth the highest dollar value. how- ever, the group with the lowest amount of gross sales gave a higher rating than the other groups for the potential of the workshop to increase net worth as a percent of sales. the group with highest gross sales entered the workshop better prepared; their pre-workshop scores for items – were each about . larger than for the groups with the lowest in gross sales. the post-workshop scores for these items were only about . larger for the highest gross sales group than the group with the smallest sales. thus, a general conclusion is that the train- ing succeeded for all education and farm sales levels. . . . ’” , ’” , . . , ’ o&,. . ’ . c . ” , ’ . ’ . , . . @.c , i b“ , . , , h’c ( hs , ,ob,c ~,sb,c . .y bc . b . , cluster analysis of workshop participants tabulations by single variables were helpful in evaluating the success of the workshops, however, analysis for groups defined by several variables further refined the evaluation. cluster analysis was utilized to delineate groups in a multivariate frame- work for further analysis. variables used in the cluster analysis were pre-workshop beliefs partici- pants had on several topics: ( ) their view of the importance of financial management, ( ) their knowledge about farm financial statements, and ( ) their knowledge about farm financial plans. changes in these variables (items - , table ) were tabulated in tables and ). the approach of cluster analysis is based on the view that partici- parsons et al. pants with similar perspectives and knowledge lev- els could be characterized by a similar set of char- acteristics (bernhardt et al. ). it is assumed that participant knowledge can be analyzed in terms of clusters that broadly share similar charac- teristics as c =fl(bj, k=l, , . . ..k c =f (bj,l =l, ,. ... l c~=f~(bz), z=l, , . . ..z. where ci(i = , , . . . , m) represents the ith clus- ter and bj q = k,l, . . . z) is a set of characteristics associated with the ith cluster. these cluster pro- files are mutually exclusive. the fastclus pro- cedure in sas (sas/stat users guide ) was used to determine the number of clusters and to group the participants. the three clusters identified in the analysis can be described as “low finance priority,” “high fi- nance knowledge,” and “low finance knowl- edge” (table ).’ the participants identified with a low finance priority had an average score of . (scale of – ) on their initial view of the importance of financial management. however, this group had the largest change in their view of financial management. the low finance priority group also had a strong belief that the financial tools acquired in the workshop would help their farms to survive, with an average score of , on a scale of to . given their initial low priority for finance, it is not surprising that the change in fi- nance knowledge was substantial for the low fi- nance priority group< the members of the low finance knowl- edge cluster had the largest increase in knowledge of financial statements and planning and estimated that use of workshop concepts would raise annual net worth by . percent (item , table ). the sales level of this group indicates the presence pri- marily of small farmers. the high finance knowl- edge cluster scored the lowest on change in view that use of workshop tools would contribute to farm survival, and they also had the smallest in- crease in knowledge of financial statements and planning. most importantly, the post-workshop view of the importance of financial management converged between . and , for the three clus- ters, and knowledge of financial statements and plans ranged from . to . , suggesting that the workshop tended to make the ending finance knowledge and finance perspective similar for the three clusters. the cluster analysis isolated the par- ticipants with low finance knowledge and a nega- tive belief in the importance of finance. these two groups would be expected to gain less from the workshop than individuals with more knowledge a financial training program and/or more positive beliefs. the fact that their post-workshop knowledge and beliefs had become nearly as high as the group with higher knowledge initially indicated that the curriculum allowed these potentially problem participants to fully par- ticipate and become finance-literate. logit models (madalla ) were estimated for further comparison of each cluster to the other two clusters. these models considered characteristics of each cluster in a multivariate framework rather than in the univariate tabulations discussed above. each model has the same set of explanatory vari- ables, which are defined as being when the group of participants (cluster i) has the characteristics, and o when it does not: g [pi/(l – ‘i)] = po+ ~~x~ + ~~x~ + . . . + ( bxlcj+ ei, where pi = probability that the respondent belonged to the group (cluster) xl = xj= x = x = x = x = x = xs = x = cha~ge in the view of the importance of financial management (scale of to ) change in the level of knowledge of farm financial statements (scale of to ) change in the level of knowledge of farm financial plans (scale of to ) - years of farm management experience more than years of farm management experience moderately satisfied with workshop (score of on scale of to ) highly satisfied with workshop experience (score of on scale of to ) farm sales greater than $ , off-farm income of $ –$ xlo = off-farm income greater than $ xl ~ = workshop skills will increase net worth $ -$ , x = workshop skills will increase net worth more than $ x = typical profit $ -$ , xlq = typical profit greater than $ , x = did not attend any other extension workshops in past year x = amish farmer. the regression coefficients are in the appendix. given that the explanato~ variables are binary, odds-ratios were computed instead of marginal probabilities (hosmer and lemeshow ), these odds are used to analyze differences in character- istics among the clusters. the odds of an outcome being present when a predictor variable (x) is equal to one is defined as t(l)/[( – t( )], the october agricultural and resource economics review table . logistic regression odds ratio from - fsa finance workshops by cluster low f]nance low finance high fhrance priority knowledge knowledge regression variable cluster (n = ) cluster (n = ) cluster (n = ) . . . . . . . . . . , . . . . . . — intercept change in the view of the importance of financial management change in the knowledge level of farm financial statements change in the knowledge level of farm financial plans farm management experience: – yrs farm management experience: more than yrs moderately satisfied with workshop highly satisfied with workshop farm sales greater than $ , off-farm income: $ -$ off-farm income: more than $ workshop skills will increase farm net worth between $ –$ workshop skius will increase farm net worth by more than $ typical profit: $ -$ , typical profiti greater than $ , participants not attending any other extension workshops in past year amish producer . . . . *** . *** . *** . ** . *** . *** . . *** . *** . . . . . . . ** . . *** . ** . . *** . , . . . . . . , * . . , . . . . . , . . ” . , . . . . * . *logistic regression parameter estimate statistically significant at p < . level. **logistic regression parameter estimate statistically significant at p < (). level. ***logistic regression parameter estimate statistical y significant at p < . i level. odds ratio, denoted by w, is defined as the ratio of the odds for x = to the odds for x = o,given by * = a/et, where, a=m(l)/[( –m(l)] and b = t(o)/ [( - t(o)]. in simple terms, an odds ratio of two implies that when x = the outcome (event) is twice as likely, while an odds ratio of . would suggest the event is only half as likely to occur. computed odds ratios are in table . compared to the other two groups, the odds were higher that members of the low finance priority cluster would have a much larger change in perception of the importance of financial management, tend to have more than years of farm management ex- perience, be less satisfied with the workshop ex- perience, and view workshop skills as contributing strongly to net worth growth. for example, the odds were greater than . ( . ) that a member of the low finance priority group would have more than years of farm management experience. the odds were only about . that a member of the low finance priority group would end the workshop moderately or highly satisfied with the learning experience, which suggests that they were more likely to not be in these categories. members of the low finance knowledge group were more likely to have a large change in the knowledge level of financial statements and plans, respectively . and , . members of this cluster also tended to have less farm management experi- ence, more off-farm income, and to be less satis- fied with the workshop experience, members of both the low finance priority and low finance knowledge clusters tended to find the workshop less satisfying than the high finance knowledge cluster. thus, we surmise that the lower the finance knowledge and priority, the harder the producers had to work to master the finance concepts, and the parsons et al. a financial training program more unfamiliar the topic, the less satisfying the learning experience. note that the amish farmers were about twice as likely to be members of the low finance knowledge cluster. the odds were , -to- . that members of the high finance clus- ter would have – years of farm management experience, and that . -to-l.o members of this cluster would have farm sales greater than $ , . the odds were about . -to-l that a high finance knowledge member would estimate that workshop skills could increase farm net worth gains annually by $ , –$ , . thus, logit analysis of the clusters allowed us to identify and understand the differences in impacts of the work- shop and farm characteristics among the clusters, concluding comments the borrower training program addressed outreach education issues that are frequently critical to workshop success. in-depth workshops with sev- eral days scheduled for presentations, exercises, and homework are ideal for training on complex topics such as agricultural finance. that stated, the logistics of organizing concurrent workshops re- quires a sharp focus on information presentation efficacy. in our case, the more high-tech satellite up-link approach was not sufficiently flexible to accommodate the scheduling and workshop lead- ership needs of the typical county agent. the draw- back with using pre-taped videos, the alternative, was that careful editing and frequent updating of the tapes were required to accommodate changes made to curriculum text materials. a key finding of this study is that the finance workshops were very successful in terms of knowl- edge gains and potential impacts on net worth growth and farm survival for most participants. the cluster and logit analysis provided some more specific information for subsets of participants, it is noteworthy that the small farm and lower- educated participants benefited relatively more in terms of change in knowledge of financial state- ments and planning than their neighbors with more education and larger farms. obviously, writing the text at a lower level than most extension materials, emphasizing exercises, and repetition and review were elements of the curriculum that made it ac- cessible to these less-educated participants from smaller farms. however, a key challenge is to de- velop educational approaches that increase the sat- isfaction levels of these less prepared and moti- vated participants, in this case the low finance priority and low finance knowledge producers. clearly, these two clusters entered the workshop with more deficiencies than the high finance knowledge group. this uncomfortable learning challenge needs to be made as positive as possible without lowering the knowledge achievement stan- dards of the course, two other important impacts from the training experience were that agronomy and dairy science agent site leaders became more knowledgeable of and confident with agricultural finance concepts, to the point where several chose to present the mate- rials themselves rather than to use video-tape pre- sentations. in addition, cooperative extension was able to integrate clientele previously not reached by extension programs. finally, a challenge for cooperative extension is to cultivate ties with or- ganizations such as fsa/usda so that our strengths as educators can be employed with pro- ducers who otherwise would not take the time to master difficult concepts. the borrower training workshops will ultimately enhance usda and co- operative extension partnerships in working with minimum resource producers. references bernhardt, kevin j,, john c. allen, and glenn a. helmers, . “using cluster anafysis to classify farms for con- ventional/alternative systems research,” review ojagri- cultural economics - : - . federal register. . rules and regulations. washington, d.c. - (dec ): - . hanson, g.d. . “a distance learning approach to bor- rower training.” agricultural finance review : - . hanson, g.d, , “the agricultural finance legacy of the new deal.” the southern business and economic journal : - , hiel, edwin, and david herrirrgton. . “plausible uses and limitations of videoconferencing as a tool for achieving technology transfer.” journal of extension ( ): pages. (electronic publication). hosmer, d. w., and s, lemeshow. , applied logistic re- gression. new york: john wiley, madalla, g.s. . limited-dependent and qualitative vari- ables in econometrics. new york: cambridge university press, peterson, gary. . coordinator of agricultural short courses, penn state university. personal communication. august . sas/stat users guide. . version , fourth edition, vol- ume . cary, nc: sas institute, inc. october agricultural and resource economics review appendix table. logistic regression results from - fsa finance workshops by cluster parameter estimates low finance priority low finance high finance cluster knowledge knowledge regression variable (n = ) chrster (n = ) cluster (n = ) . . . . . . . . , , , . . , . . . intercept – . . *** - . – . *** , – , *w+change in the view of the importance of farm financial management change in the knowledge level of farm financial statements – . ”” . *** - . *** change in the knowledge level of farm financial plans - . , *** -j. *** farm management experience: – yrs . , - . – . . . farm management experience: more than yrs moderately satisfied with workshop – . ”” – . ”” - . - . . . - . - . - . . . - . . *** , *** . - . - . ” . highly satisfied with workshop farm sales greater than $ , off-farm income: $ -$ off-farm income: more than $ workshop skills will increase farm net worth between $ –$ workshop skills will increase farm net worth by more than $ typical profit: $ -$ , . . - , . . . - . – . * – . , . - . typical profit: greater than $ , participants not attending any other extension workshops in past year amish farmer - . , * . *parameter estimate statistically significant at p < , level, * *parameter estimate statistically significant at p < . level. * **parameter estimate statistically significant at p < . level. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ urgent notice for foundation dentists urgent notice for foundation dentists faculty supports faster cancer referrals in april the intercollegiate advisory committee for sedation in dentistry (iacsd) of the dental faculties of the royal colleges of surgeons and the royal college of anaesthetists published new standards for conscious sedation in the provision of dental care which includes updated recommendations on conscious sedation training for the whole dental team. the standards state that ‘patients have the right to expect a high quality service to meet their dental needs. this can only be achieved through robust, validated education and training of the entire dental team.’ as a result, an innovative course was established by health education england, london and the department of sedation & special care dentistry at guy’s and st thomas’ nhs foundation trust. the new course was based on the learning outcomes outlined in the standards and was run over eight days. the first two days were non-clinical and dedicated to increasing and updating theoretical knowledge and improving practical sedation skills. topics covered included relevant physiology and pharmacology, patient assessment and treatment planning, cannulation, administra- tion of midazolam, sedation monitoring and managing sedation related emergencies. the next six days were dedicated to treating patients requiring a wide range of dental treatment under intravenous sedation with midazolam. all the participants were encour- aged to bring a dental nurse to observe the sedation technique, patient management and intravenous conscious sedation training for the whole team course report procedural skills. the course provided the opportunity to treat patients with intra- venous sedation under closely supervised conditions. in addition there were oppor- tunities to experience the use of inhalation sedation and intranasal midazolam sedation and to observe the use of conscious sedation using a propofol infusion. attendees gained confidence in treatment planning patients for sedation and also took part in a seminar on cognitive behavioural therapy. the course was organised and supervised by emily sherwin, specialist in special care dentistry, and david craig, consultant in special care dentistry. having four attendees allowed for close supervision on the clinics with readily available support to assist with any challenging cases. the course involved continuous clinical assessment with both informal and formal feedback including workplace-based assessments and an end- of-course review. there was also an mcq examination to assess theoretical knowledge. the course is designed for ‘new starters’ in dental sedation wishing to progress to inde- pendent (unsupervised) practice but it would also be helpful for experienced dentists who wish to expand their skills. its clinical focus makes this course ideal for primary dental care dentists who are interested in adding conscious sedation to their skill set. by amish patel and emily sherwin . standards for conscious sedation in the provision of dental care. report of the intercollegiate advisory committee for sedation in dentistry. the dental faculties of the royal colleges of surgeons and the royal college of anaesthetists, . (l-r) sheena quille (dental nurse), amish patel (course member), emily sherwin (supervisor), chris vondee (course member), david craig (supervisor), parimal patel (course member), and lena vakil (course member) the faculty of general dental practice (uk) has expressed its support for a new standards document on cancer referrals from the national institute for health and care excellence (nice). in the uk, over people a day are diagnosed with oral cancer, and fgdp(uk) is asking dentists to take note in particular of the recommendation in the new document that ‘people with suspected cancer who are referred to a cancer service are given written information encouraging them to attend’. the faculty is recommending that dental practices make suitable resources, such as the nhs’s ‘patient information for urgent referrals’ leaflet, available for patients. to download the leaflet visit: https://www. myhealth.london.nhs.uk/healthy-london/ cancer/pan-london-suspected-cancer-referrals/ patient-leaflets. the british dental association (bda) is reminding all new graduates who have recently been allocated to a dental founda- tion training post in england or wales starting on september this year that they must apply to be included in the dental performers list before that date. the same applies to inclusion in the hscb dental list in northern ireland. the pcs and area teams/ health boards then have three months to finalise the inclusion of names in the list. for those in northern ireland applying to be on the dental list, full completion of the hs form and attendance at the hscb dental information session is a pre-requisite to a ds number being issued. young dentists and their trainers/educa- tional supervisors should also check with the deaneries and pcs/ats/hbs whether the process for inclusion is moving forward smoothly, so that any problems can be clarified in good time and the inclusion is confirmed by the end of november at the very latest. if trainers/supervisors or foundation dentists are becoming aware of any problems with this process, please get in touch with the bda as soon as possible. british dental journal | volume no. | august upfront © macmillan publishers limited, part of springer nature. all rights reserved. https://www.rcseng.ac.uk/fds/publications-clinical-guidelines/docs/standards-for-conscious-sedation-in-the-provision-of-dental-care- https://www.rcseng.ac.uk/fds/publications-clinical-guidelines/docs/standards-for-conscious-sedation-in-the-provision-of-dental-care- https://www.myhealth.london.nhs.uk/healthy-london/cancer/pan-london-suspected-cancer-referrals/patient-leaflets https://www.myhealth.london.nhs.uk/healthy-london/cancer/pan-london-suspected-cancer-referrals/patient-leaflets https://www.myhealth.london.nhs.uk/healthy-london/cancer/pan-london-suspected-cancer-referrals/patient-leaflets https://www.myhealth.london.nhs.uk/healthy-london/cancer/pan-london-suspected-cancer-referrals/patient-leaflets 	urgent notice for foundation dentists wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ policies that protect their business practices. it also offers lessons on how to bring about change, highlighting what can be achieved by a global move- ment linking international and domestic action, involving national coalitions, ngos, the media, and mobilisation of the public to create sustained pressure for change. it is possible that success might be achieved without global regulation but it is essential that the option is not ruled out. some elements of the food industry have adopted policies that are likely to be beneficial but there is still a very long way to go. the global public health community might usefully recall theodore roosevelt’s advice to ‘speak softly but carry a big stick’. the views expressed here are not neces- sarily those of the authors’ organisations. the authors report no competing interests. references full list of references appears as online supplementary data accompanying the online publication of this paper. iotf. overweight and obesity in the european union. briefing paper for the european commission platform on diet, physical activity and health, brussels, march . available at: http://europa.eu.int/comm/ health/ph_determinants/life_style/nutrition/ documents/iotf_en.pdf. lobstein t, baur l, uauy r. obesity in children and young people: a crisis in public health. obes rev ; suppl : – . who/fao. diet, nutrition and the prevention of chronic diseases. report of a joint who/fao expert consultation. who technical series, no. . geneva: world health organization, . yach d, mckee m, lopez a, et al. improving diet and physical activity: lessons from controlling tobacco smoking. bmj ; : – . research fellow, european centre on health of societies in transition, london school of hygiene and tropical medicine, london, uk childhood obesity coordinator, international obesity taskforce, london, uk professor of european public health, european centre on health of societies in transition, london school of hygiene and tropical medicine correspondence: cécile knai, research fellow, european centre on health of societies in transition, london school of hygiene and tropical medicine, london, uk e-mail: cecile. knai@lshtm.ac.uk doi: . /eurpub/cki curbing obesity: prevention and treatment caroline braet and myriam van winckel* this viewpoint is written by two middle- aged women who have been working with obese children and their families since � years, one as a psychologist and the other as a paediatrician. as we have been treating obesity in children, our focus has not been prevention. the thoughts that follow originate not only from what we have been studying and observing in our contacts with obese chil- dren and their parents, but also from what we have been learning as mothers of our now teenage children. obesity develops when a genetically sensitive person comes in a favourable environment. our genes are not changing rapidly, whereas obesity has emerged as a frequent and growing problem, from young age on, everywhere in the world. the united states is on top of the list, followed by those developing countries that are making the transition to more wealth. but also in europe the same evolution is observed. when reflecting on what has changed in our environment and how to curb this evolu- tion, one ends up with thinking politics. our thoughts, described below, are - fold. on a macro-level, politicians will have to invest in new rules and regula- tions as well as in large programs which will be the only way to streamline society with all its different and conflicting pres- sure groups. secondly, serving healthy food and facilitating a healthy life style should be mission statements in socialis- ing agencies like schools, sport clubs, and summer camps. finally, on a micro-level, all of us can model a healthy living attitude which can influence the people and especially the parents and their children in the near environment. we will give now some examples below. move more it is clear that an active lifestyle is as important as healthy eating in the prevention of obesity. together with modification in food consumption pat- terns sedentarism has increased dramati- cally. reasons to explain this evolution are obvious. when we were children, we could bike alone to school and back home from the age of or years. our parents did not worry to let us play outside. now, there are at least times more cars, which are also a lot faster and make roads unsafe, and children cannot be allowed to bike independently. in primary and in secondary school standard programmes offer only · min of sport per week. a lot of sport- ing facilities are available and affordable. unfortunately, our patients tell us far too often that they stopped attending sport clubs because they were not competitive. if we want our children to retain the joy of moving till adulthood, more recreative and less competitive sporting facilities are needed. epstein et al. suggests an exer- cise programme in which mainly aerobic exercises of moderate intensity, such as cycling, swimming, walking, jogging, rope-skipping or rowing, are planned for minimum min a day. schools seem to be the best place to take respon- sibility for organising these daily activ- ities for all children. walking and biking are important components of an active daily lifestyle. the amish people in north america who refuse to use electricity and motor cars have a low incidence of obesity ( % in men, % in women). an average amish man walks twice as much as an average european man. it is of course not realistic to ban modern comfort such as cars and elevators. if we want, however, to achieve the necessary lifestyle change we will need to create car free areas where people live enabling children to play outside, and we need reliable and timely public transport, safe bicycle lanes between home and school or work, shower facilities at work, and strong incentives to diminish the use of cars. reduce ‘screen’-time to maximum h a day research has clearly shown that obesity is inversely related to the time spent watch- ing television. television was introduced in our homes when we were years old. until p.m. only music and a fixed abstract black and white box and line pattern was shown. at p.m. h of chil- dren’s programme would start. it was far less difficult for our parents to restrict television viewing than it is for contem- porary parents. now several clock-round children’s programmes are available. in order to restrict television viewing time, parents need to set rules and control these rules. this is a time-consuming and frus- trating experience for parents, while in the mean time they have their own agenda and their own plans and duties. trying to streamline tv watching as well as playing pc games is part of an curbing childhood obesity http://europa.eu.int/comm/ educating process where more and more the children seem to ‘win’ and reinforce their parents by stopping with wining when tv is on. altering the so-called coercive cycles asks for a strict conse- quent approach, which is not in conflict with a democratic, authoritative educational approach by the parents. in our opinion messages like ‘reduce tv time’ are far more difficult to implement than explicit unequivocal rules (e.g. tv from p.m. until p.m. or ‘screen-time’ from p.m. until p.m.). can we stop the bad habit of eating in between? some children, from young age on, refuse healthy food like vegetables and fruit. when they are hungry they prefer unhealthy snacks. anxious parents do follow the whims of their offspring. others are simply not hungry during meals because they drink soft drinks or eat sweets in between. by consuming energy-dense snacks, it is not unusual that children eat more than they need. how to break through these bad habits? first of all, it is advisable to develop the habit of eating only at fixed hours. the national institute for food suggests the following scheme: three standard meals with balanced carbohydrates, pro- teins, and fat as well as two (or three) scheduled snacks. we also need clear messages with regard to healthy snacks (e.g. < kcal.) and beverages: drink water during all meals, restrict milk consumption to – ml/day, limit consumption of soft drinks to once weekly, and drink not more than one fruit juice a day. second, children are strongly advised to reduce the number of places where they eat. researchers assume that as one eats in more situations there are more and more environmental stimuli reminding of food, whereas reducing these situations extinguishes the remind- ing value of an environmental stimulus. it is also recommended to do nothing else but eating during the meal and to avoid other activities such as walking around, reading, watching television, or making telephone calls. for children it is helpful to have fewer supplies in the house stored in as few cupboards as possible. probably, it will be necessary to coach the shopping culture of many parents. some people suggest to provide food in the market with a green (healthy, not dense in cal- ories), red (dense in calories, ‘empty’ cal- ories, not containing vitamins or minerals), or orange (not to use without restrictions, but containing valuable nutrients) sticker. if only parents have healthy food in their house, discussions about what or how much the child can eat will be easily solved. as satter sug- gests ‘parents decide what and when their children eat, children decide how much they eat’. financing preventive programs in at-risk groups obesity ‘runs’ in families. overweight in childhood is strongly affected by parental fatness. these families will need more specific help. moreover, these families are more situated in neighbourhoods with low socioeconomic status, where families have more financial problems and often more stress and less free space. it can be helpful to run specific programmes together with these families where ideas are shared regarding prepar- ing healthy food, managing the costs of healthy food, finding sport clubs, orga- nising safe play grounds, and setting rules and having them observed. however, there is little incentive for counsellors to set up programmes if no means of financing them are provided. treating obese children is prevention of later obesity overweight and obesity in childhood have been shown to be predictive for obesity in later adult life. if the over- weight can reach stability during child- hood, we can prevent this evolution. indeed, some evidence exists that effec- tive management of childhood obesity is possible, with long lasting effects on lifestyle. the treatment that shows to be effective is family based and needs a multidisciplinary approach focusing on behavioural changes. reimbursement for these programmes will be necessary. however, meta-analyses on treatment programmes of childhood obesity indi- cated that few randomised controlled trials were available and long-term follow-ups were scarce. moreover, no evaluation of tailoring treatment to spe- cificcharacteristics ofthechildrenisavail- able. it seems extremely urgent that the evaluation of non-pharmacological treat- ment programmes is provided by policy- makers. afterthoughts. . .. research in developing and evaluating prevention programmes of obesity needs priority financing. the cost of this research will only be a fraction of what is now already spent in the treat- ment of adult obesity. drugs currently available, such as those causing fat malabsorption, are marginally effective and cost �$ a month for the patient. hospital costs for bariatric surgery in the us increased more than -fold between and , from $ mil- lion to $ million a year. in many countries, over % of the adult popula- tion is overweight and it is difficult to reduce excessive weight once it is established. obesity prevention should therefore focus on children in the first place. the kiel obesity prevention study is one of the few programmes demonstrat- ing positive outcomes in - to -year-old schoolchildren, by giving simple educa- tional messages to all children and their parents. these are (i) eat fruit and vege- tables each day, (ii) reduce the intake of high fat foods, (iii) keep active at least h a day, and (iv) decrease television viewing to < h a day. school meetings were combined with targeted programmes for families with overweight and or/ obese parents. however, effective preven- tion programmes are based on the knowledge of risk factors and causes. even if most risk factors for the develop- ment of obesity seem to be self evident, little research is available on what to base clear evidence based guidelines for effec- tive prevention. most of the ideas presented here ask an engagement of the parents of young chil- dren. during adolescence young people develop their own life style. a child is highly dependent on its parents’ attitudes and guidance, whereas adolescents are strongly influenced by their peers and idols. here, popular media people can use their influence in both directions and model a healthier life style. some of them are already strongly involved in different charity projects and others have set up big campaigns for people or animals all over the world who need help. because the obesity epidemic is one of the biggest problems of modern society, we must invite the media to think about their status and its influence on adolescents’ life style. the media, together with the academic world can be very influential on the policy to ask for action programmes or for european guidelines. perhaps, we hope, this will help develop a world-wide culture where healthy choices are cool and trendy. references epstein lh, myers md, raynor ha, et al. treatment of pediatric obesity. pediatrics ; : – . bassett dr, schneider pl, huntington ge. physical activity in an old order amish community. med sci sports exerc ; : – . available at: http//www.nutrition.gov. european journal of public health a checklist for curbing childhood obesity claude marcus* the insight that childhood obesity is a problem is spreading fast. most decision-makers involved in public health are aware of the fact that obesity early in life negatively affects self esteem and increases the risk of future diabetes and cardiovascular disease although the effect of obesity on cancer risk is less well known. thus, childhood obesity is one of our major threats to public health and will cause a reduced life expectancy in the future if we do not act now. the prevalence of obesity and over- weight has increased -fold or more in most western countries during the last years. it is generally accepted that the changes in society involving eating habits and physical activity are crucial. no other factors are of equal importance for the understanding of the childhood obesity epidemic. thus, it is quite simple. we have to reduce the intake of sweetened drinks, sweet, and fat snacks; go back to a more regular eating pattern without snacking between meals; and increase the physical activity. however, although simple, the realisation of such changes is so difficult that we have to realise that we have a lost generation of adolescents and adults with habits so firmly rooted that it is not possible to affect their behaviour within a democratic society. conse- quently we have to focus on younger chil- dren and start immediately when they are beginning school, i.e. at – years of age. measures taken to handle the situation are often too toothless. one reason seems to be the general but erroneous fear that tougher recommendations may lead to increased number of eating disorders especially among girls. there are no indi- cations that recommendations regarding good eating habits and physical activity increase the risk of eating disorders. another problem is that short-sighted economical considerations sometimes are more important than long-term efforts to reduce childhood obesity. the european union has difficulties handling the overproduction of butter and milk. in an attempt to both improve children’s eating habits and reduce the overproduction of milk products, school lunches milk is subsidised. however the benefits of this initiative are markedly hampered by the fact that whole milk is three times more subsidised than low fat milk and consequently an unfortunate intake of high animal fat among children is stimulated by the eu. the feeling of helplessness against the commercial and marketing powers of big companies such as coca cola and mc donald’s also seems to reduce the will- ingness to act. however, in my opinion, a consequent use of the school as an arena to influence children to adopt a healthy lifestyle should not be underestimated. furthermore, if schools have a firm attitude regarding sweet drinks and snacks it will be much easier for parents to have defined limits for their children as well. what is most important if we effec- tively want to prevent childhood obesity, is it to modify food intake or physical activity? if we consider that a normal -year-old child weighing kg has to walk for h or run km to burn the energy from an ice cream ( kcal), it is easy to realise that increasing physical activity without a concurrent change of eating habits will be almost without effect. this is problematic both for decision-makers and parents because directions regarding restricted eating and reduced snacking may be considered more repressive and more negative than increased physical activity. therefore most initiatives are aimed at physical activity. of course, despite this reasoning we must not forget that there is a group of both lean and overweight children today who live an extremely sedentary life who are in need of help to become more phy- sically active. my simple list of measures is not too expensive and many things can be launched without any long start-up time. � ban all sweets, sweetened drinks, ice cream, and energy-dense snacks from nurseries, schools, and after school care centres. inform parents about healthier alternatives if the children have packed lunch. � inform parents of their duty to ban all sweets, sweet breakfast cereals, swee- tened drinks, ice cream, and energy- dense snacks from daily family life and encourage them that they are good care-taking parents if they reduce irregular snacking to a minimum. � increase activity of daily living for chil- dren. teach them at a young age to take a walk instead of taking the bus. use consequently the stairs instead of an elevator together with children. � subsidise whole grain food and fresh fruit and vegetables. this might pro- vide a better and more functional incentive than extra tax on unhealthy foods. � reduce the subsidies on whole milk and increase them on low fat milk in schools � identify children with a pronounced sedentary behaviour and develop methods to increase their physical activity. � professor of pediatrics, karolinska institute, head of national childhood obesity centre, karolinska university hospital, stockholm, sweden correspondence: claude marcus, professor of pediatrics, karolinska institute and head of national childhood obesity centre, karolinska university hospital, stockholm, sweden e-mail: marcusclaude.marcus@ klinvet.ki.se doi: . /eurpub/cki satter e. how to get your kid to eat . . . but not too much. palo alto: bull publishing company, . müller mj, asbeck i, mast m, et al. prevention of obesity-more than an intention. concept and first results of the kiel obesity prevention study (kops). int j obes ; suppl :s – . � ghent university, belgium correspondence: caroline braet, department of developmental, personality and social psychology, ghent university, h. dunantlaan , ghent (b), belgium e-mail: caroline. braet@ugent.be doi: . /eurpub/cki curbing childhood obesity genome-wide and fine-mapping linkage studies of type diabetes and glucose traits in the old order amish evidence for a new diabetes locus on chromosome q and confirmation of a locus on chromosome q -q wen-chi hsueh, pamela l. st. jean, braxton d. mitchell, toni i. pollin, william c. knowler, margaret g. ehm, callum j. bell, hakan sakul, michael j. wagner, daniel k. burns, and alan r. shuldiner , we conducted a genome scan using a -cm map to search for genes linked to type diabetes in indi- viduals from a founder population, the old order amish. we then saturated two regions on chromosomes and showing promising linkage signals with additional markers to produce a � -cm map for fine mapping. analyses of both discrete traits (type diabetes and the composite trait of type diabetes and/or impaired glucose homeostasis [igh]), and quantitative traits (glucose levels during a -g oral glucose challenge, designated glucose – and hba c) were performed. we obtained significant evidence for linkage to type diabetes in a novel region on chromosome q (loga- rithm of odds [lod] for diabetes � . , p � . ). furthermore, we observed evidence for the existence of a diabetes-related locus on chromosome q -q (lod for type diabetes/igh � . , p � . ), a region shown to be linked to diabetes in several other studies. suggestive evidence for linkage to glucose traits was observed on three other regions: q -q (telomeric to that above with lod � . – . for glucose and ), p (lod � . – . for type diabetes and glucose – ), and p (lod � . , p � . for hba c and lod � . for glucose ). in conclusion, our findings provide evidence that type diabetes sus- ceptibility genes reside on chromosomes , , and . diabetes : – , t ype diabetes is a classic example of a complex disease; environmental variation, genetic influ- ences, and interactions among these factors all contribute to the risk of developing the disease ( – ). investigations targeting specific candidate genes have yielded relatively little insight into susceptibility genes for the common form of type diabetes ( ). more recently, researchers have turned to genome-wide ap- proaches for identifying genes linked to type diabetes and serum glucose levels ( , – ). promising linkage signals have been observed in several chromosomal re- gions in different study populations, with some of them overlapping ( ). to date, only one susceptibility gene (calpain- ) has been cloned through genome-wide ap- proaches ( ). the old order amish of lancaster county, pennsylva- nia, are a genetically well-defined caucasian founder pop- ulation who live in a relatively homogeneous environment and often have large sibships, thus potentially enhancing our ability to detect linkages to type diabetes and related traits ( ). the prevalence of type diabetes in the amish is � %, and the phenotypic characteristics of this disease in the amish are similar to common type diabetes in other populations ( ). here, we report the results of genome-wide linkage analyses that were carried out in extended families of the amish family diabetes study (afds). analyses were conducted on both discrete traits (type diabetes and/or impaired glucose homeostasis [igh]) and quantitatively distributed traits related to dia- betes (plasma glucose levels and hba c), and we used additional saturation markers placed in two chromosomal regions of particular interest. our findings provide evi- dence for susceptibility genes for type diabetes located on chromosomes q -q and p and in a novel region on chromosome q . from the department of genetics, southwest foundation for biomedical research, san antonio, texas; glaxosmithkline, research triangle park, north carolina; the department of medicine, university of maryland school of medicine, baltimore, maryland; the national institute of diabetes and digestive and kidney diseases, national institutes of health, phoenix, ari- zona; axys pharmaceuticals, la jolla, california; and the geriatrics re- search and education clinical center, baltimore veterans administration medical center, baltimore, maryland. address correspondence and reprint requests to alan r. shuldiner, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, w. redwood st., room , baltimore, md . e-mail: ashuldin@medicine.umaryland.edu. received for publication july and accepted in revised form november . w.-c.h. and p.l.s. contributed equally to this study. w.-c.h. is currently located at the university of california san francisco school of medicine, san francisco, california; c.j.b. at emergen, salt lake city, utah; and h.s. at pfizer, groton, connecticut. p.l.s., m.g.e., m.j.w., and d.k.b. are employed by and hold stock in glaxosmithkline; c.j.b holds stock in celera; and h.s. is employed by and holds stock in pfizer. additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. afds, amish family diabetes study; auc, area under the curve; igh, impaired glucose homeostasis; lod, logarithm of odds; lrt, likelihood ratio test; ogtt, oral glucose tolerance test; str, short tandem repeat. diabetes, vol. , february research design and methods subjects and phenotypes. the afds was initiated in with the goal of identifying susceptibility genes for type diabetes and related traits ( ). individuals with type diabetes were identified by door-to-door interviews with the help of liaisons from the amish community. individuals who reported diabetes onset between and years of age were invited to participate. all first- and second-degree family members of the probands who were � years of age were contacted and invited to participate in the study. if another diabetic individual was identified in the family (e.g., aunt or uncle), then the family was expanded further to include that person’s first- and second-degree relatives (� years of age). examinations were conducted at the amish diabetes research clinic in strasburg, pennsylvania, or in the subjects’ homes. a genome scan was performed on the initial set of individuals enrolled and examined in the afds between february and february . nearly all of these individuals share common ancestors insofar as the entire amish community of lancaster county (now numbering � , individuals) are descendents of a small number of amish families who emigrated to this area in the mid s. these subjects can be connected into a single -generation pedigree ( ). the study protocol was approved by the institutional review board at the university of maryland school of medicine, and informed consent was obtained from each study participant. a -h oral glucose tolerance test (ogtt) was administered to all subjects without a prior history of diabetes. after collection of a fasting blood sample, a -g oral glucose challenge was administered (trutol ; casco nerl diagnostics, baltimore, md), and additional blood samples were obtained at -min intervals for analysis of plasma glucose concentrations. glucose concentrations were assayed with a beckman glucose analyzer (beckman coulter, fullerton, ca) using the glucose oxidase method (interassay coeffi- cient of variation � . %). glucose area under the curve (auc) during the -h ogtt was calculated using the trapezoid method. hba c was measured by high-pressure liquid chromatography (interassay coefficient of variation � . % for low standard and . % for high standard). bmi was calculated as weight (kg) divided by height squared (m ). criteria for the diagnosis of diabetes were adapted from american diabe- tes association recommendations ( ). diabetes was defined by a single fasting venous plasma glucose level (� mmol/l), a -h ogtt venous plasma glucose level (� . mmol/l), or current treatment with insulin or oral hypoglycemic agents. diabetic subjects with an age at diagnosis � years were reclassified as diabetes status unknown to minimize heterogeneity due to inclusion of subjects with type diabetes. nondiabetic subjects were classified as having igh based on age-, sex-, and bmi-adjusted fasting and -h ogtt venous plasma glucose levels (fasting plasma glucose level between . and mmol/l or -h ogtt plasma glucose between . and . mmol/l). a combined phenotype, type diabetes/igh, consisted of subjects with type diabetes or igh as defined above. normoglycemia was defined as having fasting venous plasma glucose level � . mmol/l and a -h ogtt venous plasma glucose level � . mmol/l. due to missing data, diabetes and igh statuses could not be determined for and subjects, respectively. genotypes. we initially performed linkage analysis using highly polymor- phic microsatellite short tandem repeat (str) markers on autosomes and the x chromosome. these markers were part of the abi prism linkage mapping set (perkin-elmer) and have been described previously ( ). the mean marker heterozygosity was . , with a range from . to . . the marker order and sex-averaged distances between markers were estimated from our data by maximum likelihood methods using cri-map ( ). the average interval between markers was . cm, and the largest gap between markers was . cm, occurring on chromosome . the genetic maps calculated by cri-map using the amish data were on average . % longer than the corresponding map reported by marshfield (http://research.marshfieldclinic. org/genetics/mapmarkers/maps/indexmapframes.html). overall, there was excellent agreement with marker order between the amish map and the marshfield map, with only five occasions in which marker order was not concordant. in two regions where interesting linkage signals were observed in our initial scan, we typed additional str markers to increase the map density and information content. these included markers on chromosome between and cm (average intermarker distance . cm) and markers on chromosome between and cm (average intermarker distance . cm). these markers were integrated into the framework map by use of cri-map and by inferring genetic distance through the use of physical map information when the genetic distance could not be properly estimated (human genome project working draft at university of california, santa cruz, http:// genome.ucsc.edu/). marker allele frequencies used in all analyses were estimated from the entire set of subjects. to identify mendelian and/or pedigree errors, the -generation pedigree was broken down into smaller pedigrees that were generations in depth with marriage and inbreeding loops broken (d.m. nielsen, personal commu- nication). pedigree errors were identified and corrected by looking for subjects with systematic mendelian inconsistencies in the scan markers and by use of siberror ( ), a program that detects sibs, half-sibs, and monozy- gotic twins by investigating eight identity-by-state statuses using unlinked markers. mendelian inconsistencies were identified and genotypes involved in these discrepancies deleted via an algorithm based on unknown ( ), a program that detects, for each marker, the minimal set of subjects whose genotypes, when removed, result in eliminating said mendel errors (r. idury, personal communication). seven pedigree errors were identified and cor- rected. three sets of monozygotic twins were identified, and we removed one twin from each of the three sets before analysis. overall, the completeness of genotype calls for markers used in the linkage analysis was . � . % (means � sd) (range . -– . %). statistical analysis qualitative trait linkage analysis. the two qualitative traits, type diabetes and type diabetes/igh, were analyzed using two-point and multi- point nonparametric allele sharing methods as implemented in genehunter- plus ( ) with the exponential model and sall function. multipoint allele sharing was assessed at -cm intervals. the single -generation pedigree was subdivided into smaller pedigrees fitting the size constraints of the gene- hunter program. for the type diabetes phenotype, the single -generation pedigree was broken down into pedigrees with affected subjects and subjects who were either unaffected or of unknown diabetes status. for the type diabetes/igh phenotype, pedigrees were analyzed with affected subjects, unaffected subjects, and subjects with unknown type diabetes/igh status. separate analysis of pedigrees that are indeed related to one another introduces the risk of underestimating the degree with which any two individuals are related. this could result in the overestimation of the differ- ence between expected and observed allele sharing, possibly leading to inflated evidence of linkage. to address this issue, we simulated unlinked markers whose information content was equivalent to those in the observed linkage peaks. these unlinked markers were then dropped down through the entire -generation pedigree (d.m. nielsen, personal communication). this single large pedigree was divided into the smaller analysis pedigrees (see above), and these were then analyzed by genehunter-plus. we conducted , replicates and defined the probability that the observed logarithm of odds (lod) score is a false positive report as the proportion of replicates exceeding our observed lod score. the p values reported for the observed lod scores from the type diabetes and type diabetes/igh analyses were obtained from this simulation. conditional analyses were performed for the discrete trait, type diabetes/ igh, to test for interactions between genomic regions (the type diabetes trait was not tested due to its small sample size). to minimize the number of comparisons, we conditioned on regions with lod scores of � . (pointwise p � . [ ]) and examined the rest of the genome for further evidence of diabetes loci. using the genehunter npl score, two weighting schemes were applied; the first one tests a multiplicative model or epistatic model, and the second tests for heterogeneity among families ( ). to determine the empir- ical significance of lod scores from the conditional analyses, we randomly assigned weights to the families through permutation, keeping the overall number of families with a weight of for a given weighting scheme constant with the number observed with a weight of . these permutations were conducted , times. quantitative trait linkage analysis. linkage analyses were also per- formed on the quantitative traits, the plasma glucose levels obtained during a -g -h ogtt (fasting glucose levels and glucose levels obtained at -min intervals, noted as glucose , , , , , and ), glucose auc, and hba c. sample sizes used for each trait-specific analysis ranged from (for glucose auc) to (hba c), as described in table . to eliminate confound- ing influences of diabetes treatment, subjects taking medications for diabetes were excluded from these analyses. furthermore, to reduce skewness, extreme outliers were excluded from these analyses and hba c values were transformed by their natural logarithm. to reduce the computational com- plexity of these analyses, we divided the single large pedigree into separate smaller pedigrees, ranging in size from to individuals. even after splitting the large pedigree, the sample included a very large number of relative pairs, among these , pairs of siblings, , avuncular (aunt, uncle/niece, nephew) pairs, grandparent-grandchild pairs, and , first cousin pairs. these analyses were conducted using a pedigree-based likelihood method to partition the total phenotypic variation into effects due to covariates, effects of a specific locus (or linkage), and residual additive genetic effects (or herita- bility). statistical significance was assessed by computing the likelihood of the pedigree data under competing genetic models (linkage versus no linkage) and comparing the likelihoods using the likelihood ratio test (lrt), which w.-c. hsueh and associates diabetes, vol. , february yields a � statistic with (in this case) a single degree of freedom. p values obtained from the lrt were then converted to lod scores using the formula: lod � � /[ � ln( )]. linkage analysis for the quantitative traits was carried out using the solar software program ( ). use of the lrt to evaluate evidence for linkage using variance component methods can be problematic when the multivariate normality assumption is violated ( ). we therefore used simulation to empirically estimate the probability of obtaining false evidence for linkage. we derived the distribution of nominal lod scores under the null hypothesis of no linkage by simulating , unlinked markers, dropping them through the pedigrees, and conduct- ing linkage analysis with each of the markers for each of the glucose traits and hba c. the probability of obtaining a false positive result was defined as the proportion of replicates for which we obtained a specified lod score or higher. the p values obtained from the simulation study were then converted into lod scores as described above. all lod scores from quantitative trait locus analyses presented in this report were obtained from this simulation. results genome-wide linkage analysis. clinical characteristics of the study subjects are shown in table . the mean age was � years, and the mean bmi was . and . kg/m in men and women, respectively (p � . ). diabetes was present in . % of men and . % of women, and igh was present in . % of men and . % of women. the greater prevalence of type diabetes and igh in women was likely due to greater bmi. since participants were ascertained around family members with type diabetes, these prevalence rates do not reflect the preva- lences of type diabetes or igh in the general amish population, which are estimated to be and %, respec- tively ( ). the sibling relative risk(s) for type diabetes in this population was previously reported to be . ( % ci . – . ) ( ). the heritabilities for the glucose traits measured during the -h ogtt and hba c in family members of diabetic subjects not previously known to have diabetes are shown in table . the heritabilities for all glucose traits were significantly greater than zero (p � . ), with estimates ranging from . for glucose (fasting) and glucose to . for glucose . the heritability for the integrated measures, glucose auc and hba c, were . and . , respectively. results from the initial linkage analyses based on the -cm screening set of markers are summarized in table . these analyses revealed lod scores of � . for one or more traits in only three chromosomal regions. between and cm on chromosome , we observed linkage peaks for glucose with a lod of . and for glucose with a lod of . . at position – cm on chromosome , we observed lod � . for glucose at cm, lod � . for glucose at cm, and lod � . for glucose auc at cm. on chromosome at position cm, we observed linkage peaks for hba c (lod � . ) and glucose (lod � . ). lod scores of . – . for one or more traits were observed in three other chromosomal regions, all on chromosome : at position – cm (lod � . and . for glucose and glucose auc, respectively), at position cm (lod � . for glucose ), and at position cm (lod � . for hba c). eight more chromosomal regions (on chromosomes , , , – , and ) showed linkage signals, with lod between . (corresponding to a point- wise p � . ) (table ) and . . the complete genome scan results can be viewed at http://medschool.umaryland. edu/endocrinology/amish/amlinkindex.html or in an on- line appendix at http://diabetes.diabetesjournals.org. fine mapping on chromosomes and . based on results from the initial linkage analyses, we genotyped additional str markers on chromosomes and . on chromosome , we typed an additional markers within the - to -cm interval on our framework map (flanked by markers d s and d s ), and on chromosome , we genotyped an additional str markers falling within the region between and cm on our map (flanked by d s and d s ). results of the linkage analyses using the extended set of markers on chromosomes and are shown in fig. for the discrete traits, and glucose and (results for other traits can be viewed at http://medschool. umaryland.edu/endocrinology/amish/amlinkindex.html or http://diabetes.diabetesjournals.org). on chromosome p, evidence for linkage to the discrete type diabetes trait increased to . (p � . ), occurring at cm (nearest marker: d s ). results for quantitative trait locus analysis remained similar to those using framework markers only. the peak signal for glucose occurred at cm near d s (lod � . , p � . ). analyses table clinical characteristics of study population by sex* trait† n (m/f) male female h ‡ age (years) ( / ) . � . . � . na bmi (kg/m ) ( / ) . � . . � . ‡ . � . diabetes (%) ( / ) . . na igh (%) ( / ) . . na plasma glucose levels (mmol/l)§ glucose ( / ) . � . . � . . � . glucose ( / ) . � . . � . � . � . glucose ( / ) . � . . � . . � . glucose ( / ) . � . . � . � . � . glucose ( / ) . � . . � . ¶ . � . glucose ( / ) . � . . � . ¶ . � . glucose ( / ) . � . . � . � . � . glucose auc (mmol � l� � h) ( / ) . � . . � . � . � . hba c (%)§ ( / ) . � . . � . . � . *see reference for additional characteristics of the afds; †age, bmi, glucose traits, and hba c reported as mean � sd; §due to confounding influences of diabetes treatment on these traits, subjects with previously diagnosed diabetes were not included; ‡heritability estimates � se, all with p � . ; �p � . ; ¶p � . . na, not available. genome scan of type diabetes diabetes, vol. , february of three other quantitative traits also provided supportive evidence for linkage, including glucose (lod � . at cm near d s ), glucose (lod � . at cm near d s ), and glucose auc (lod � . at cm near d s ). since the analyses of glucose traits ex- cluded subjects with previously diagnosed diabetes, link- age of glucose traits and diabetes to the same region of chromosome p provide complimentary evidence that a gene influencing glucose homeostasis resides in this re- gion. in the region of q – , the lod score for type diabetes/igh increased to . (p � . ) at cm near d s . d s at . cm has the largest two-point lod score in this region (lod � . ). however, dropping this marker from the saturation map resulted in only a slight drop in the multipoint lod score at cm, from . to . . on chromosome , we also observed increased evi- dence supporting linkage using a denser genetic map. for the analysis of diabetes, the peak lod score increased to . (p � . ) at cm near d s . the information content at cm increased from . in the scan to . in the fine-mapping study. with an intermediate -cm map the lod score for type diabetes at cm was . . this -cm map did not contain d s , the marker with the largest two-point lod score (lod � . ) in this region. the peak lod score for glucose (lod � . , p � . ) occurred at the same position ( cm). again, since the analysis of glucose traits excluded subjects with previously diagnosed diabetes, linkage to both diabetes and glucose traits provide complimentary evidence for linkage in this region. similar to the analysis with the framework markers, the denser map showed a secondary peak for glucose (lod � . , p � . ) that coincided at virtually the same position (at cm near d s ) as the peak lod for two other traits, glucose (lod � . at cm), and glucose auc (lod � . at cm). conditional analyses for type diabetes/igh pheno- type. we examined the family-wise npl scores for the type diabetes/igh trait at three regions that had lod scores of � . (pointwise p � . ) for the conditional analyses. the three regions that were conditioned on were q near d s (lod � . at cm), q near d s (lod � . at cm), and q near d s (lod � . at cm). two regions showed increased evidence of linkage under a positive weighting scheme (epistatic model). at q -q (d s -d s , cm), the lod score for type diabetes/igh increased from . to . (p � . ) when we conditioned on the linkage results at the q region. after conditioning on linkage results at q , the lod scores at q near d s ( cm) increased from . to . (p � . ). under a negative weighting scheme (heterogeneity model), only one additional region was identified with a significant increase in lod score. when conditioning on linkage results at q , the lod score at q (d s , cm) increased from . to . (p � . ). the region at q is the same region showing evidence for linkage to the trait, diabetes (fig. ). conditioning on q did not significantly increase evidence for linkage elsewhere in the genome. discussion type diabetes has an important genetic basis in the amish, as it does in other populations. the sibling relative risk of type diabetes was estimated to be � . ( ), suggesting a familial aggregation of this disease in this population. furthermore, in nondiabetic family members of subjects with known diabetes, there are moderate yet table chromosomal regions showing evidence for linkage, with a lod � . (p � . ), to diabetes and related traits using framework str markers chromosome location (cm) trait peak lod score empirical p value lod- support interval (cm) flanking markers type diabetes . . – d s , d s glucose . . – d s , d s glucose . . – d s , d s glucose . . – d s , d s glucose auc . . – d s , d s glucose . . – d s , d s glucose . . – d s , d s hba c . . – d s , d s hba c . . – d s , d s type diabetes/igh . . – d s , d s glucose . . – d s , d s glucose . . – d s , d s glucose . . – d s , d s glucose . . – d s , d s glucose . . – d s , d s glucose . . – d s , d s glucose auc . . – d s , d s glucose . . – d s , d s glucose . . – d s type diabetes/igh . . – d s , d s glucose . . – d s , d s hba c . . – d s , d s glucose . . – d s , d s w.-c. hsueh and associates diabetes, vol. , february significant heritabilities for plasma glucose concentrations during an ogtt as well as for hba c, providing further evidence of genetic influence on type diabetes. despite the well-recognized genetic contribution to type diabe- tes, little is known about the specific genetic causes of diabetes or variation in glucose levels. most linkage stud- ies have examined diabetes or glucose levels during fast- ing and/or h after a glucose challenge. to our knowledge, our study is the first to investigate the genetic influence on plasma glucose levels during the course of a -h ogtt, allowing us to examine whether the variation in glucose levels during the ogtt have shared genetic influences. based on results from our genome scan, we identified several regions with suggestive evidence of linkage. al- though none of the linkage signals reached genome-wide significance at p � . , these studies provide evidence for linkage in the regions of p , q -q , and q . link- age signals for several glucose concentrations measured at different times during the -h ogtt (glucose , , and and glucose auc) were observed in a region on fig. . multipoint linkage analysis results of glu- cose and diabetes for chromosomes and in the fine-mapping study. genome scan of type diabetes diabetes, vol. , february chromosome p (between and cm). further- more, analysis of the discrete trait, diabetes, also provided evidence for linkage in the same region. on chromosome q -q , the peak lod score for type diabetes/igh was . (at cm). on chromosome q , we obtained much stronger evidence for linkage to diabetes, with a peak lod score of . . for the type diabetes/igh traits, the peak lod score was . on chromosome at virtually the same region. the peak signal for glucose also occurred at cm. linkage to a similar region on chromosome q for the dichotomous traits (type diabetes and type diabetes/igh), as well as glucose levels as quantitative traits (in which subjects with previ- ously diagnosed diabetes were not included), provides strong evidence for a novel type diabetes locus in this region. we also obtained evidence for linkage to glucose traits on chromosomes and . notably, evidence for linkage to hba c on chromosome at cm was quite strong (lod � . , p � . ) and deserving of further investigation. while evidence for linkage of more than one glucose trait in the same region was regarded as encour- aging, it should be noted that these traits were moderately correlated with each other. for example, the correla- tion coefficient was . between glucose and , . between glucose and , and . between glucose and . (for correlation coefficients between other glucose traits, see http://medschool.umaryland.edu/ endocrinology/amish/amlinkindex.html.) by conditioning on evidence for linkage previously detected in one or more regions of the initial genome scan, we may increase the power to strengthen linkages in other regions and to detect other diabetes-related loci. indeed, conditioning on the q region increased evidence of linkage to type diabetes/igh on both chromosomes and . these analyses extended evidence of linkage into the q -q region with a lod of . at cm. as seen in fig. , this region still overlaps with evidence of diabetes linkage reported in other populations. while conditioning on evidence of linkage at q , the lod score for type diabetes/igh on chromosome increased to . in the same region, providing further evidence for linkage to type diabetes (lod � . ). by contrast, no interaction occurred between q and q , suggesting that for the type diabetes/igh trait, the genetic effects from loci on q and q are independent of one another. our region of linkage on chromosome q showed a strong linkage signal for diabetes (lod � . ). this region contains a cluster of immune function genes, e.g., interleukin e (il e), interferon-stimulated gene transcription factor (isgf ), cytotoxic t-cell–associated serine esterase- (ctla ), t-cell antigen receptors 	 (tcra) and (tcrd), and the leukotriene b receptor (ltb r). although none of these genes have been previ- ously considered as candidate genes for type diabetes, there is an increasing body of evidence that the inflamma- tory process may be primarily involved in type diabetes pathogenesis ( , ). in addition, there are several genes in this region that may influence glucose and/or lipid metabolism through their effects on signal transduction or gene transcription, including protein kinase c � (prkcm), adenylate cyclase- (adcy ), ccaat/enhancer-binding protein � (cebpe), and the estrogen receptor- (esr ). we observed modest (lod � . – . ) evidence for linkage to four traits (diabetes and glucose , , and ) on chromosome p . although no other groups have reported evidence of linkage to diabetes on p, norman et al. ( ) observed a lod score of . for -h respiratory quotient between d s and d s (� cm on the amish map), and thompson et al. ( ) found linkage to acute insulin release at d s (� cm on the amish map). the ratio of carbohydrate to fat oxidation is a predictor of weight gain, and obesity is a known risk factor for type diabetes. similarly, decreased acute insulin release is an early indicator of glucose intolerance and type diabetes. this region is known to harbor the leptin receptor. linkage signals for diabetes to the region of chromo- some q -q have been reported in four other popula- tions (fig. ). in a genome scan conducted in the pima indians ( ), allele sharing of sib-pairs concordant (defined as onset before age years) and discordant (defined as nondiabetic at age years), revealed evidence for linkage of diabetes to d s on chromosome q (lod � . , at � cm on the amish map). moreover, when sib-pairs with age of diabetes onset of � years were analyzed separately in an affected sib-pair analysis, very strong evidence for linkage was detected near d s (lod � . , at � cm on the amish map). in utah caucasians ( ), linkage to diabetes was observed in the region between crp and apoa (� cm on the amish map) using both parametric analysis (lod � . ) and model- free affected sib-pair analysis (lod � . ). more recently, in a french study ( ), affected sib-pair analysis using lean (bmi � kg/m ) diabetic sib-pairs found a linkage peak near d s (at cm on the amish map) with an lod of . . in british affected sib-pairs, wiltshire et al. ( ) observed evidence for linkage to diabetes on q near d s , � cm on the amish map (lod � . after saturation markers were placed). it is often difficult to evaluate whether a putative gene underlying the linkage signal in one study (e.g., the amish) represents the same gene as that detected by linkage in other studies (e.g., pima indians and utah, french, and british caucasians). simulation studies have suggested fig. . regions showing linkage signals to diabetes and its related traits on chromosome q from five populations (traits shown in parentheses). *results from analysis conditioning on linkage signal to type diabetes/igh on chromosome q . dm, diabetes mellitus. w.-c. hsueh and associates diabetes, vol. , february that even under an ideal scenario (i.e., sufficient power and identical phenotype definition, ascertainment strategy, pedigree structure, genetic map, genetic and environment heterogeneity, and statistical approach), difference in sam- pling alone may cause the variation in location estimates of the linkage peaks to be as wide as cm ( – ). when we compared results from our linkage analysis with these four earlier studies showing linkage signals to diabetes on chromosome q ( , , , ), we found the genetic signals observed from these studies cluster in a -cm region on chromosome q -q (fig. ). it is thus possible that these linkages in several populations may represent the same gene. alternatively, there may be more than one type diabetes susceptibility gene in this region. chromosome q -q contains at least known genes, putative transcripts with homology to known genes, and as many as other potentially expressed sequences ( ). several of these genes are potentially associated with lipid or glucose metabolism, including lamin a/c (lmna) ( , ), phosphoprotein enriched in astrocytes (pea ) ( , ), potassium inwardly recti- fying channel, subfamily j, member (kcnj ) ( ), pre– b-cell leukemia transcription factor (pbx ) ( ), solute carrier family (thiamine transporter), member (slc a ) ( ), retinoid x receptor 
 (rxrg), insulin receptor–related receptor (insrr), and others. several other regions identified in our study with lod scores of � . have been reported by other researchers. these include chromosomes p -p ( ), q ( , ), q ( ), q . - q . ( ), q -q ( ), q . -q . ( , ), and p . -p . ( , ). by contrast, the link- ages we observed in the amish on chromosomes q -q , q . , p . - p . , q . , q . -q . , q . , q . , and p . -p appear to be novel. in summary, we obtained significant evidence for link- age to type diabetes with a novel locus on chromosome q , as well as suggestive linkage signals to its related traits on several other novel chromosomal regions. fur- thermore, we observed evidence for the existence of diabetes-related loci on chromosome q -q , a region previously linked to diabetes by several other groups. our findings provide the rationale for positional cloning of type diabetes susceptibility genes on chromosomes and . acknowledgments this study was supported in part by a research grant from glaxowellcome and axys pharmaceuticals, national insti- tutes of health grants dk and dk , and an american diabetes association research award to a.r.s. we thank wendy warren, mary ann drolet, denis massey, mary morrissey, janet reedy, and our amish liaisons for their energetic efforts in study subject recruit- ment and characterization; drs. alejandro schaffer and richa agarwala for assistance in pedigree construction; and dr. dahlia nielsen for supplying programs for simula- tion studies and for decomposing the -generation pedi- gree into subunits. we also thank don holt, derek traughber, darrin london, santhi sampath, bo zheng, keith tanner, and demian lewis for technical assistance. this study would not have been possible without the outstanding cooperation of the amish community. references . elston rc, namboodiri kk, nino hv, pollitzer ws: studies on blood and urine glucose in seminole indians: indications for segregation of a major gene. am j hum genet : – , . rich ss: mapping genes in diabetes: genetic epidemiological perspective. diabetes : – , . hanson rl, elston rc, pettitt dj, bennett ph, knowler wc: segregation analysis of non-insulin-dependent diabetes mellitus in pima indians: evi- dence for a major-gene effect. am j hum genet : – , . 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further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ obituary 	 the	journal	of	clinical	investigation      http://www.jci.org      volume        number        october  a tribute to victor a. mckusick victor  a.  mckusick,  md,  university  professor of medical genetics emeritus at  the  johns  hopkins  university  school  of  medicine, died of cancer on july  ,  ,  at the age of  . with james neel and arno  motulsky, victor was a founder of the dis- cipline of medical genetics. victor  had  only  one  earned  degree,  the  md, from johns hopkins in  . because  of world war ii, he was able to enter medi- cal  school  after  only  three  years  at  tufts  university and he referred to himself as a  college dropout. he became chief resident  of the osler medical service under a. mcge- hee  harvey,  perhaps  his  most  important  mentor ( ). in  , victor succeeded har- vey as the william osler professor of medi- cine  and  physician-in-chief  of  the  johns  hopkins hospital. initially, he pursued car- diology and championed a new technique,  phonocardiography. a few years after his  appointment as assistant professor in  ,  he published the book cardiovascular sound in health and disease,  which  established  his  clinical  and  academic  reputation.  when  he surprised his colleagues by deciding to  pursue  genetics,  some  suggested  he  was  committing academic suicide. but victor  was intrigued by genetics early in life. first,  he was one of monozygotic twins. second,  he was raised on a dairy farm in parkman,  maine. his first paper, unfortunately never  published,  concerned  the  inheritance  of  coat color in cattle. third, as an intern, he  cared  for  several  patients  with  polyposis  and pigmented macules on the lips; he col- laborated with harold jeghers in   on a  paper in the new england journal of medicine  that firmly established recessive inheritance  of  this  condition,  originally  described  by  j.l.a. peutz in  . additionally, as a cardi- ologist, he encountered patients with mar- fan syndrome. dilatation of the aortic root  and aortic dissection were reported a few  years earlier by helen tausig at hopkins,  among  others.  in  ,  victor  published  a seminal paper in circulation in which he  not only identified the causes of death of  dozens of patients with marfan syndrome  but established a new nosology, the heri- table  disorders  of  connective  tissue.  his   monograph on this topic described  seven quite distinct disorders that appeared  to be associated by primary defects in the  extracellular matrix. eventually, over    disorders would be thus classified. by  , he was a full professor and pur- sued the study of rare disorders full time  as director of the outpatient moore clinic.  he approached the old order amish first  as a physician and second as an investiga- tor and thereby identified a host of “new”  autosomal recessive syndromes made evi- dent by consanguinity. always the catalogu- er,  he  established  mendelian inheritance in man (mim) in  . victor recognized much  earlier than most the value of computers  and maintained this catalog on magnetic  tape,  starting  in  .  as  one  of  victor’s  trainees, i assisted him in the composition  of mim entries with a first-generation desk- top  computer  and  word  processing  pro- gram in  . eventually, mim morphed  into omim, the online version now main- tained by the national library of medicine  ( ). nearing  ,  entries, omim remains  a vital resource for clinicians and investiga- tors. victor was also a pioneer in mapping  human genes and an early practitioner of  linkage analysis in humans. a student of  his  established  the  first  autosomal  link- age, duffy blood group to chromosome  ,   in  . subsequently, victor helped estab- lish  the  human  gene  mapping  work- shops, was an early advocate of the human  genome project, and was the first president  of the human genome organisation. while  never self aggrandizing, he was secure in his  importance  to  academic  medicine,  and  a  number of personal reflections document  his contributions ( – ). any homage to victor must emphasize  his mentorship, for which he was greatly  respected  and  beloved.  he  counted    trainees. some of his earliest trainees are  long retired, but those still active and their  many “descendants” populate institutions  around  the  world.  one  enduring  legacy  began with his   paper on marfan syn- drome and continued when he introduced  me to a patient with that condition on my  first day at hopkins as an osler resident.  together  with  talented  colleagues,  we  implemented  treatments  that  improved  life  expectancy  to  near  normal  and,  in  ,  discovered  the  cause  of  this  syn- drome. more recently, the first incumbent  of the victor a. mckusick professorship in  medicine and genetics, hal dietz, discov- ered the basis of the pathogenesis of this  syndrome, which, if it leads to a cure, will  be an early triumph of translational medi- cine. this half-century saga gave victor tre- mendous satisfaction. his most powerful  — and unspoken — lessons were always the  value of persistence and hard work. virtu- ally no one was his equal. well into his  s,  he  would  be  viewing  posters  at  national image credit: keith weller, johns hopkins medicine. obituary 	 the	journal	of	clinical	investigation      http://www.jci.org      volume        number        october    meetings before breakfast, scribbling notes  for entry into omim later that day. when  i incorrectly introduced him as emeritus a  couple of years ago, he publicly noted that  he was still on the active faculty but only  working part time —   hours a day. victor was recognized with   honorary  degrees, election to the national academy  of sciences, the gairdner foundation inter- national award, the george m. kober medal  of the association of american physicians,  the national medal of science, the albert  lasker award for special achievement in  medical science, and the   japan prize  for medical genomics and genetics. victor  was well enough to attend the ceremony in  tokyo in april with dr. anne b. mckusick,  his wife of nearly   years. on his return,  he regaled us with tales of the pomp of the  ceremony and of dining with the emperor  and empress. at the end of the banquet, he  spontaneously took the empress’ hand to  introduce her to his twin, vincent, the for- mer chief justice of the supreme court of  maine. his eyes twinkled as he recounted  how the security detail swiftly intervened  in this breach of protocol. reed e. pyeritz division  of  medical  genetics,  depart- ment  of  medicine,  university  of  penn- sylvania  center  for  the  integration  of  genetic healthcare technologies, univer- sity  of  pennsylvania  school  of  medicine,  philadelphia, pennsylvania, usa. e-mail:   reed.pyeritz@uphs.upenn.edu.   . mckusick,  v.a.  .  abner  mcgehee  harvey,  - . proc. assoc. am. phys.  : – .   . mckusick-nathans institute of genetic medicine,  johns  hopkins  university,  and  national  center  for biotechnology information, national library  of  medicine.  .  omim  —  online  mendelian  inheritance in man [database]. http://www.ncbi. nlm.nih.gov/omim/.   . mckusick,  v.a.,  naggert,  j.,  nishina,  p.,  and   valle, d.  .   years of the annual ‘bar harbor  course’ ( – ): a pictorial history. clin. genet.  : – .   . mckusick, v.a.  . the anatomy of the human  genome: a neo-vesalian basis for medicine in the  st century. jama.  : – .   . francomano, c.a., mckusick, v.a., and biesecker,  l.g.  . medical genetic studies in the amish:  historical perspective. am. j. med. genet.  c: – .   . mckusick, v.a.  . a  -year tale of spots, maps,  and genes. annu. rev. genomics hum. genet.  : – .   . mckusick,  v.a.  .  mendelian  inheritance  in  man  and  its  online  version,  omim.  am. j. hum. genet.  : – . depth&#x ;resolved registration of transesophageal echo to x&#x ;ray fluoroscopy using an inverse geometry fluoroscopy system depth-resolved registration of transesophageal echo to x-ray fluoroscopy using an inverse geometry fluoroscopy system charles r. hatt department of biomedical engineering, university of wisconsin-madison, madison, wisconsin michael t. tomkowiak, david a. p. dunkerley, and jordan m. slagowski department of medical physics, university of wisconsin-madison, madison, wisconsin tobias funk triple ring technologies, inc., newark, california amish n. raval department of medicine, university of wisconsin-madison, madison, wisconsin michael a. speidela) departments of medical physics and medicine, university of wisconsin-madison, madison, wisconsin (received july ; revised september ; accepted for publication october ; published november ) purpose: image registration between standard x-ray fluoroscopy and transesophageal echocardiog- raphy (tee) has recently been proposed. scanning-beam digital x-ray (sbdx) is an inverse geometry fluoroscopy system designed for cardiac procedures. this study presents a method for d registration of sbdx and tee images based on the tomosynthesis and d tracking capabilities of sbdx. methods: the registration algorithm utilizes the stack of tomosynthetic planes produced by the sbdx system to estimate the physical d coordinates of salient key-points on the tee probe. the key-points are used to arrive at an initial estimate of the probe pose, which is then refined using a d/ d registration method adapted for inverse geometry fluoroscopy. a phantom study was conducted to evaluate probe pose estimation accuracy relative to the ground truth, as defined by a set of coregistered fiducial markers. this experiment was conducted with varying probe poses and levels of signal difference-to-noise ratio (sdnr). additional phantom and in vivo studies were performed to evaluate the correspondence of catheter tip positions in tee and x-ray images following registration of the two modalities. results: target registration error (tre) was used to characterize both pose estimation and registra- tion accuracy. in the study of pose estimation accuracy, successful pose estimates ( d tre < . mm) were obtained in % of cases when the sdnr was . or higher in seven out of eight poses. under these conditions, d tre was . ± . mm, and d (projection) tre was . ± . mm. probe localization error along the source-detector axis was . ± . mm. for the in vivo experiments, mean d tre ranged from . to . mm and mean d tre ranged from . to . mm. anatomy extracted from the echo images appeared well aligned when projected onto the sbdx images. conclusions: full dof image registration between sbdx and tee is feasible and accurate to within mm. future studies will focus on real-time implementation and application-specific analysis. c american association of physicists in medicine. [http://dx.doi.org/ . / . ] key words: inverse geometry, scanning-beam digital x-ray, image fusion, fluoroscopy, echocardiography . introduction catheter-based cardiac interventions allow for minimally inva- sive treatment of structural heart disease, reducing patient trauma and opening up treatment options for patients that are too sick and/or fragile to undergo surgery. while surgeons have the luxury of direct visualization of the treatment site, this comes at the cost of increased risk to the patient, greater morbidity, and longer recovery time. in contrast, interventional cardiologists employ imaging methods such as x-ray fluoros- copy (xrf) and echocardiography (echo) to visualize their devices, identify the anatomy they wish to treat and to avoid, and to monitor the success of the therapy. integration of these imaging methods is desirable for optimal clinical workflow and improved therapeutic success. image fusion between xrf and transesophageal echocar- diography (tee) has recently been proposed – and clinically implemented (echonavigator, philips healthcare). many structural heart interventions, such as transcatheter aortic valve replacement (tavr), left atrial appendage closure, and the mitral clip procedure, utilize both xrf and tee. these procedures may benefit from the enhanced guidance offered by combining information from both image modalities. for example, in tavr, a prosthetic valve is guided and deployed using xrf, but visualization of the anatomy is poor. if xrf/tee fusion is enabled, real-time anatomical information med. phys. ( ), december - / / ( )/ / /$ . © am. assoc. phys. med. http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://crossmark.crossref.org/dialog/?doi= . / . &domain=pdf&date_stamp= - - hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy from echo can be visualized continuously in the context of the devices without the need for nephrotoxic x-ray contrast. xrf/tee fusion is accomplished using d/ d registration techniques – or magnetic tracking sensors. , sensor-based methods require additional hardware and may be inaccurate due to electromagnetic field distortions in the catheterization lab. generally, d/ d registration techniques will estimate the d location and orientation (pose) of the tee probe by comparing the clinical xrf image to simulated xrf images of a d probe model (digitally reconstructed radiographs or drrs). the model pose is iteratively adjusted until the simi- larity between the clinical xrf and drr is maximized. after inferring the d position of the probe in the c-arm coordinate system, the d tee image data can be registered to xrf data. using a typical monoplane xrf c-arm system, the most challenging pose parameters to estimate are the so-called “out- of-plane” parameters, which include euler angle rotations about the detector axes (pitch and roll) and, in particular, trans- lations along the source-detector axis. this is because varying these parameters typically causes only subtle changes in the device appearance, which in turn do not strongly influence the similarity function maximized during pose estimation. performing the registration using two x-ray views can help resolve this issue but the increased radiation dose to the patient is a concern. scanning-beam digital x-ray (sbdx) is an inverse geom- etry x-ray fluoroscopy technology designed for dose reduc- tion and tomosynthesis-based d device tracking. the basic components of sbdx are a scanning x-ray tube, multihole collimator, high speed photon-counting detector, and a real- time reconstructor (fig. ). as the electron beam in the x-ray tube scans over an array of focal spot positions, small-field- of-view images of the patient are captured. after each frame, the detector data are reconstructed into a stack of full-field-of view tomosynthesis images ( planes× frames/s). the tomosynthesis images are a necessary precursor to the final live image display, termed the composite image. however, the plane stacks can also be exploited for frame-by-frame d localization of high-contrast devices. this principle has been previously applied to localize catheter tips and electrodes, fiducials, and coronary artery centerlines. in this paper, we present the first investigation of sbdx/tee image registration. the tomosynthesis capability of sbdx is used to obtain the position of the tee probe along the source-detector axis and an accurate initial estimate of the d probe pose. this is followed by refinement of the pose estimate using a d/ d registration procedure. a phantom study of d and d target registration error (tre) was conducted for a variety of probe orientations and image noise levels in order to quantify the performance of the new pose estimation algorithm. to demonstrate sbdx/tee image fusion in d and d visualizations, additional phantom and in vivo studies were conducted. in the d visualization, d tee data are registered and fused with d catheter tip positions localized from sbdx tomosynthesis imaging. . algorithm sbdx/tee registration is achieved by estimating the d pose of the tee probe based on its appearance in sbdx x- ray images. the pose estimation algorithm has two stages (secs. .b and .c). first, an initial estimate of the probe pose is obtained by performing tomosynthesis-based d local- ization of key-points on the probe. second, the initial pose is refined with a d/ d registration algorithm adapted for sbdx’s inverse geometry. given the d pose and a calibration relating the echo image volume to the tee probe, the echo f. . (a) sbdx imaging geometry, demonstrating shift-and-add tomosynthesis at multiple planes. (b) the central rays of the individual beamlets form a cone of rays originating from the center of the detector. the tomosynthesis image pixels are formed by subdividing the lateral shift between these rays. (c) the multiplane composite can be viewed as a “virtual projection” of the in-focus features of the tomosynthesis images. medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy t i. glossary of symbols. x,y,z coordinate axes of the sbdx system u,v integer column and row indices of a pixel in a reconstructed plane or composite image θx,θy,θz,tx,ty,tz rotation angles and translations corresponding to the x, y, and z axes i(u, v, z) tomosynthesis plane stack px,py virtual detector element pitch in the x and y directions dx,dy,sdd distance between center of the virtual detector and the virtual source point, along x, y, and z m denotes local coordinate system attached to the echo probe model v(x j, yj, z j) d point intensities defining the probe model in coordinate system m d(u, v) digitally reconstructed radiograph of the probe model sbdxtm transformation from probe model coordinates to sbdx coordinates cttecho transformation from echo image coordinates to ct coordinates used in echo calibration mtct transformation from echo calibration ct coordinates to probe model coordinates mtecho transformation from echo image coordinates to probe model coordinates tre target registration error image data may then be registered to xrf. details of sbdx image reconstruction, pose estimation, and visualization of the registered images are described in table i. .a. sbdx image reconstruction during sbdx imaging, an electron beam is raster-scanned over an array of focal spot positions. a multihole collimator defines a series of narrow overlapping x-ray beamlets directed at the detector. the detector captures a small image for each collimator hole illumination, and the images are transmitted to gpu-based hardware for real-time reconstruction. a two stage reconstruction process is executed for the detector images acquired in every / s scan frame. first, digital tomosynthesis is performed in parallel at a stack of planes spaced by mm. as described in ref. , an unfiltered backpro- jection technique is used (“shift-and-add” tomosynthesis). in the tomosynthesis images, in-plane objects appear sharp, and out-of-plane objects are progressively blurred as the plane-to- object distance increases. in the second stage, a d composite image is formed from the tomosynthesis stack in order to display all objects in focus simultaneously. the composite is generated by a plane selection algorithm, which, for each pixel position, selects the pixel value from the tomosynthesis plane with the highest local contrast and sharpness. field-of- view and frame rate are dictated by the number of focal spots scanned and the number of electron beam dwells per focal spot. in this work, scanning was performed with × holes, dwells per hole per scan frame, and scan frames/s. com- posite images and plane stacks were reconstructed at hz, and the isocenter plane reconstruction measured . cm wide. the source-to-detector distance (sdd) is fixed at mm. the coordinate system of the sbdx c-arm is defined such that x corresponds to the horizontal image direction, y the vertical image direction, and z is the distance along the source- detector axis. the (x,y,z) origin is located at the center of the focal spot array. the pixel coordinates of tomosynthetic and composite images are referred to by the integers u and v. the u- and v-axes are parallel to x- and y-axes, respectively. when a plane stack i(u,v,z) is described, z is assumed to take on the discrete values, in millimeters, corresponding to the plane positions. the sbdx/tee registration algorithm uses the d plane stack for initial probe pose estimation and the d compos- ite image for final pose refinement. since the sbdx image coordinate system is relevant to these tasks, a brief review is provided here. the pixel pitch in each tomosynthesis plane is defined by dividing the shift distance between adjacent back- projected images into a fixed number (m = ) of pixels. since the x-ray beamlets originate from a regularly spaced array of focal spot positions in the source and they all converge to a common point on the detector, the pixel centers for the stack of planes fall along a cone of rays originating from the center of the detector (see fig. ). that is, a ray corresponds to fixed (u,v) in the plane stack i(u,v,z). the composite image contains the in-focus pixel value for each of these rays. thus, the d composite can be viewed as an inverted “virtual projection” of the in-focus features in the patient volume, where the “virtual source” is at the center of the detector and the “virtual detector” is located at the source plane. the virtual detector pitch is the focal spot pitch ( . mm) divided by m = . for more details, we refer the reader to ref. . the use of this virtual projection model in d/ d registration is described in sec. .c. the coordinate system “m ” of the tee probe is defined such that the probe face from which the ultrasound volume emanates points in the positive z-direction (toward the sbdx detector), and the long-axis of the probe points in the negative y-direction of the sbdx system (toward patient inferior). the rotational pose parameters for the probe angle (θx, θy, and θz) correspond to sequential euler angle rotations about the sbdx coordinate system axes, in the order y → x → z. this corresponds to a rotation about the long-axis of the probe (“roll”), followed by a rotation about the short-axis of the probe (“pitch”), and then finally a rotation of the probe about the z-axis (“yaw”). figure demonstrates a tee probe model after it has been rotated and translated to a position in the sbdx coordinate system. .b. initial d pose estimation from tomosynthesis an initial estimate of the d position and orientation of the probe in the sbdx c-arm coordinate system is obtained from the tomosynthetic plane stack i(u,v,z) generated in a / s frame period. to obtain the position along the source- detector axis, the method exploits the fact that a device feature appears most in focus in the image plane closest to that feature and is progressively blurred as the plane-to-feature distance increases (fig. ). the z-location of the device feature is determined with finer precision than the plane-to-plane spac- ing by analyzing the distribution of feature sharpness versus medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy f. . the transformation sbdxtm maps d points in the local coordinate system of the probe model (m) to d points in the sbdx coordinate system. the z-coordinate of each plane. the method has three steps: (i) detection of probe key-points in the composite image, (ii) d localization of key-points using the tomosynthesis planes, and (iii) principal component analysis (pca) for orien- tation estimation (see fig. ). .b. . key-point detection first, the center pixel of the square transducer face of the tee probe is located in the composite image. this was done manually, although automatic techniques can also be applied. a segmentation of the tee probe is then generated by applying the frangi vesselness filter to the composite image followed by thresholding and dilation with a -pixels wide circular structuring element. the tee probe is typically the largest high-contrast object in the image. therefore, the largest connected component is found and all others are removed to produce the probe segmentation mask [fig. (c)]. to detect key-points within the mask, first the gradient magnitude of the composite image is computed following convolution with a gaussian kernel (σ = . pixel). next, a phase-symmetry filter is applied to enhance salient edges in the image while suppressing noisy edges. the result is multiplied by the mask to produce an edge image [fig. (e)]. a local maximum filter is applied where a pixel is set to if it is equal to the maximum value within a × window, and otherwise. the result specifies the initial key-point locations [fig. (f)]. .b. . d localization of key-points at each key-point position (uk,vk), the image gradient magnitude is sampled at all planes to create a -vector of edge strength values [fig. (g)]. the gradient magnitude in each plane of the stack i(u,v,z) is computed using the finite difference method after convolution with a d gaussian kernel (σ = . pixel). since the tomosynthetic blurring behavior is locally symmetric about the true object z-position, the vector of edge strength values can be viewed as a sampled version of a function with its centroid located at the true object position. local edge strengths about the object are obtained by applying a threshold [see fig. (g)]. denoting the original distribution of edge strengths as ck(z), the thresholded distribution is ĉk(z) = ck(z)− a if ck(z) > a otherwise , ( ) where a = . max(ck(z)). the z-position zk of a key- point (uk,vk) is then calculated as the center-of-mass of this distribution, zk =  i= ziĉk(zi)  i= ĉk(zi) . ( ) for each vector ĉk(z), the number of local peaks is found. vectors with more than one peak are removed from consid- eration, as they often result in unreliable d localization estimates. the localized key-point positions are converted from (u,v,z) to (x,y,z) coordinates using precalculated lookup tables. at this stage of the algorithm, most key-points belong to edges of the probe. however, some key-points have unrealistic z-coordinate values and should be labeled as outliers. to remove them, the median z-coordinate of all key- points is calculated, and any point that is greater than mm away from the median value is removed. (the distance mm was chosen based on the dimensions of the tee probe.) this mechanism is also designed to reject erroneous z-coordinates caused by overlapping objects, such as a catheter. .b. . initial d pose with the remaining set of d key-points, pca is used to determine a rough d pose. pca finds the directions of the highest variance in n -dimensional data. the first principal f. . top row: tomosynthesis images of a tee probe head reconstructed at different planes relative to the sbdx source. bottom row: the edge magnitudes grow weaker as the distance between the probe and the reconstructed image plane increases. the in-focus plane is indicated with the red rectangle. medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy f. . (a) original sbdx composite image. (b) composite image after applying the frangi filter. (c) the largest connected component of the filtered image is extracted and dilated to roughly segment the probe. (d) the gradient magnitude of a subwindow of the original image. (e) phase-symmetry filter applied to the gradient magnitude. (f) local maxima of the phase-symmetry image. (g) an example of the edge strength for each sbdx plane for a single d key-point. for the center-of-mass computation, only values greater than . of the maximal value are considered. (h) final d key-points, with principal direction computed via principal component analysis. component, therefore, defines the direction that the long-axis of the tee probe is aligned with, which in turn is used to determine the in-plane rotation of the probe (θz; yaw) and the out-of-plane pitch (θx). furthermore, the average z-location of the d key-points is used to estimate the central z-coordinate of the probe (tz) by finding the mean z-value of all key-points within mm of the center pixel (chosen based on the size of the tee probe). figure (h) demonstrates localized probe key-points in three dimensions along with the orientation vec- tor determined by pca. .c. pose refinement based on d/ d registration after the initial pose estimation step, the final estimation of all pose parameters is achieved through d/ d registra- tion. the tee probe is modeled as a point-cloud model (sec. .a. ), with its own coordinate system m . the pose parameters, applied to the probe model, refer to the three trans- lations (t x,t y,tz) and euler angle rotations (θx,θy,θz) about the axes (x,y,z) of the sbdx system. the full spatial transforma- tion of the tee probe is stored in the matrix sbdxtm, sbdxtm =  t x t y tz   cz −sz sz cz  ×  cx −sx sx cx   cy −sy sy cy  , ( ) where cj =cos(θ j) and s j =sin(θ j). as explained in sec. .a, the sbdx system geometry is different than the geometry of a standard c-arm imaging system. however, when considering the displayed composite image, the imaging geometry can be viewed as a single virtual inverted cone-beam projection, where the rays originate from the center of the detector and diverge in the direction of the x- ray tube. the matrix p defines the virtual projection geometry from the sdd, distance of virtual source to the center of the virtual detector in the x(dx) and y(d y) directions, and the virtual detector element spacing in the x(px) and y(py) directions, p =  /px dx /py d y − /sdd  . ( ) p was calibrated using a helix phantom (sec. .a. ). the value of sdd is mm, and the nominal virtual detector element spacing is . mm/ = . mm in both directions. with these definitions, the d/ d registration proceeds as follows: (i) given a vector of initial pose parameters, ϕ, generate a drr from a d model of the probe. (ii) compute the similarity between the drr and the sbdx composite image. (iii) using a nonlinear optimizer, repeat with different ϕ until the similarity is maximized. drrs were generated using a point splatting method, similar to wobbled splatting. using this method, a drr is generated by projecting point inten- sities, usually from a ct volume v (x,y,z), onto the image. each pixel in the drr image takes on a value equal to the sum of the values of the voxels that project onto it, d(ui,vi)=e −α  j∈si v(x j, yj,z j) , ( ) where si is the set of all voxel indices j such that the d point (x j,yj,z j) projects onto the d detector point (ui,vi), i.e., p ·sbdxtm · � x j,yj,z j �t = [ui,vi]t . the voxel intensities v medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy are normalized to a positive value range and the parameter α controls the contrast of the drr. to facilitate cross correlation calculations, α was set to achieve contrast approximately equal to that observed in an x-ray image. two similarity metrics were used for optimization: normal- ized cross correlation (ncc) and gradient cross correlation (gcc). normalized cross correlation is defined as ncc(i ,i )=  i  j � i � ui,vj � −µ �� i � ui,vj � −µ � σ σ , ( ) where µ is the image mean and σ is the image standard deviation. gcc is defined as gcc(i ,i )= . ·ncc(gx ,gx )+ . ·ncc(gy ,gy ), ( ) where gx is the image x-gradient and gy is the image y- gradient. d/ d registration consisted of three optimization stages: (i) optimization of the in-plane parameters (t x,t y, and θz) using ncc, (ii) all parameters except tz using ncc, and (iii) all parameters, including the drr contrast parameter α, using gcc. the nelder–mead optimizer was used at every registration stage. . methods .a. calibrations .a. . probe model in order to compute splat rendered drrs for d/ d regis- tration, a point-cloud model of the tee probe was generated from a cone-beam ct of the probe. this was done by manu- ally segmenting voxels belonging to the tee probe and then randomly sampling points within the segmented volume. the intensity associated with each point was obtained using linear interpolation from the ct volume. .a. . sbdx c-arm calibration the d/ d transformation matrix (p) describing the sbdx virtual projection geometry was calibrated using a precision manufactured phantom with steel beads arranged in a helical pattern. the helix phantom was placed at approximately the isocenter and imaged. to maximize snr, a -frame average was formed. the image was then manually thresholded to segment each fiducial, and the intensity centroid of each fidu- cial was calculated. an initial p matrix was generated using the nominal virtual projection geometry, and the helix model pose was manually initialized. next, the levenburg–marquardt algorithm was used to optimize helix model pose with fixed p. following convergence, the helix pose was fixed, and p was optimized. this was repeated until the fiducial registration error converged to a minimal value. .a. . echo calibration the spatial transform relating the echo image space to the tee probe model (m ), mtecho, was found using a wire phantom. the phantom consisted of a water-filled cylinder f. . illustration of tee probe model to echo image calibration. the probe model is registered to a ct image of the wire phantom. the wires from echo are then registered to the wires in the ct volume. this allows the spatial relationship between the tee probe model and the echo image volume to be established. containing metallic wires and an entrance port for the tee probe. a ct image was acquired of the entire setup while a simultaneous d echo of the metallic wires was recorded. using standard intensity-based registration, the echo image of the wires was computationally registered to the wires in the ct image to find cttecho (see fig. ). the probe model (generated from a previously acquired high-resolution ct) was similarly registered to the probe visible in the ct of the phantom to obtain mtct. these two transforms were combined to obtain mtecho, mtecho= mtct cttecho. ( ) to find the tre of the echo volume-to-probe registration, voxels from the echo volume, pecho, and the ct volume, pct, belonging to the wires were extracted by manually setting an intensity threshold for each image. for each wire voxel in echo, the distance to the nearest wire voxel in the ct image following registration was calculated. the tre defined as the rms distance over all of these distances was found to be . mm, treecho=  i � min(pct−cttecho·pi,echo) � n . ( ) .b. pose estimation accuracy a study was performed to compare the tee pose estimation results with a ground truth reference at eight different tee probe orientations (see fig. ), and a range of image signal-to- noise ratios. the ground truth was established by embedding the tee probe within a pvc cylinder covered with spherical steel ball bearings ( . and mm diameters). the probe was fixed in the cylinder using silicone rubber. a cone-beam ct of the entire fiducial/probe setup was used to establish the spatial relationship between the probe and fiducials. to measure the ground truth pose, the pose estimation algorithm was applied medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy f. . the eight different tee probe poses tested for the pose estimation experiments. the fiducials attached to the probe are used to estimate the ground truth pose. to the fiducials only. sbdx imaging of the probe/helix was performed at kv, mapeak ( % maximum tube current) in the × frames/s scan mode, with . cm acrylic in the x-ray beam (fig. ). sbdx image reconstructions were performed offline. five different levels of signal difference-to- noise ratio (sdnr) were generated by randomly sampling and averaging , , , , and frames. for each noise level and tee probe pose, this was repeated times, for a total of experiments. for all experiments, the sdnr was computed as sdnr= µprobe−µbackground σbackground . ( ) in order to measure tee probe and background signal statis- tics, roi masks were created by manually setting two intensity thresholds, one to segment out the probe and one to sample the background near the probe. σbackground was computed by sub- tracting two consecutive frames, finding the standard deviation of the difference image within the background, and dividing by√ . µprobe and µbackground were computed by finding the mean within their respective masks for one image frame. d and d tres were used to quantify pose estimation accuracy. for this experiment, the tre was based on a set of n = virtual points defined in the echo image space, randomly and uniformly distributed within a mm wide cubic volume. the virtual points p in echo space were trans- formed to the c-arm coordinate system using both the ground truth pose and the estimated pose, yielding point sets ptrue and pestimated, respectively. the tre d was then computed for each experiment as tre d= ∥ptrue−pestimated∥ n . ( ) the tre d was computed the same way, but only the x and y coordinates were used in the euclidean distance computation. tre d is the total target registration error, while tre d is representative of the error for points in echo in the plane paral- lel to the xrf detector. for this study, the overall registration error was based purely on pose estimation of the probe and did not include errors in registering the echo image space to the probe model (this additional error is considered in sec. .c). f. . the experimental setup for the phantom pose estimation experiment. left: the tee probe, embedded in the pvc cylinder with surrounding fiducials, was imaged between layers of acrylic to decrease the signal-to-noise. right: a zoomed out view of the experiment showing the sbdx c-arm. medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy .c. phantom and in vivo studies of sbdx/tee registration water tank phantom and in vivo experiments were con- ducted to evaluate two image fusion scenarios: echo-to-sbdx fusion, where features within the d echo image were pro- jected onto the d sbdx image, and sbdx-to-echo fusion, where the d locations of devices from the sbdx image space were transformed into the d echo image space. the first scenario maintains the conventional d x-ray display format while adding anatomical structures rendered/segmented from echo, whereas the second scenario enables the fusion of d sbdx catheter tracking results with the native display format of d echocardiography. .c. . phantom study for the phantom experiment, a cylindrical polyvinyl alco- hol (pva) phantom with a ventricle sized cylindrical cavity (height= mm, radius= mm) was fabricated. an injection catheter (myostar, biosense webster) with a metallic tip was guided through a plastic tube on the proximal side of the phantom, until it was positioned against the distal wall of the cavity. the proximal end of the catheter was attached to a trans- lation stage, and a mm/s catheter pullback was performed under simultaneous echo and sbdx imaging. the resulting trajectory of the catheter tip was a straight path mainly in the negative y-direction. sequences from two different c-arm angles ( ◦ lao, ◦ cc and ◦ lao, ◦ cc) were per- formed, resulting in different appearances of the tee probe. imaging was performed at kv, mapeak. background x-ray attenuation was provided by cm water, cm of wood, and cm of polyurethane plastic. to evaluate the d tre of sbdx-to-echo d image regis- tration, first tomosynthesis-based d tracking of the catheter tip in sbdx space was performed using the algorithm in ref. . the tip coordinate was then transformed to the echo image space using the tee probe pose estimate and the echo- volume-to-probe calibration. the transformed coordinate was then compared to the catheter tip location as manually identi- fied from the d echo images. for this task, the centroid of the reverberation artifact was located, which was presumed to correspond to the metal tip of the catheter (fig. ). the d tre was calculated for each frame using the following equation: tretip= � pecho−echotsbdx·psbdx � , echotsbdx= �sbdxtm mtecho �− . ( ) as in the pose estimation accuracy experiments, tre d was computed by considering only the x and y coordinates. .c. . in vivo study a kg healthy swine with cm anterior–posterior chest thickness was imaged in the × frames/s scan mode and kv, mapeak ( % maximum tube current) x- ray technique. procedures were approved by the local in- stitutional animal care and use committee. three image f. . method for catheter segmentation in the in vivo and water tank experiments. the catheter tip was found by determining the line that passed through the d reverberation artifact. sequences were performed under simultaneous sbdx and tee guidance. for the first two sequences, an injection cath- eter (myostar) with a metallic tip was guided into the left ventricle (lv). in sequence , the catheter tip was manipulated throughout the left ventricle to mimic navigation toward a target site. in sequence , the catheter was positioned at a single location against the left ventricular wall to mimic a catheter position confirmation task. in the latter case, the catheter only underwent cardiorespiratory motion. these two sequences were used to evaluate the registration accuracy for a discrete tip. the third sequence was used to evaluate the qualitative accuracy of anatomic echo-to-sbdx registration. specifically, a ventriculogram was acquired under simultaneous sbdx and echo imaging in order to compare a standard x-ray ven- triculogram with a proposed echo-based ventriculogram, in which the lv is segmented from the echo data and overlaid on the fluoroscopic image. additional tre measurements were obtained from the metallic markers of the pigtail injection catheter present in this sequence. since the sbdx and echo data were recorded simulta- neously on separate systems, temporal synchronization of im- age frames was necessary. to synchronize the images, each modality was first analyzed to determine the spatial axis with the largest variation of catheter motion. next, the d posi- tion of the catheter along that axis was recorded as a d signal. finally, the d motion “signals” from both modalities were compared and the time-shift that resulted in the highest normalized cross correlation was used to temporally align the image sequences. medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy tre was calculated in the same way as in the phantom study, with the exception of the ventriculogram sequence. for that sequence, the multiple metallic markers present on the pigtail catheter were indistinguishable in the echo image. therefore, a spline, secho, was fit to a set of manually segmented points on the catheter in the echo image, and the tre was the root mean square of the minimal distance between the markers registered from sbdx and the spline, trepigtail= � min(secho−echotsbdx·psbdx) � n . ( ) . results .a. pose estimation accuracy figures (a) and (b) show the average tre d and tre d for all successful registrations obtained in the pose estimation study. figure (c) shows the success rates, defined as the per- centage of registrations with a tre d less than mm. while this threshold is application dependent, mm was chosen because it represents a registration error that would result in suboptimal placement of a prosthetic valve during tavr (ref. ) or suboptimal catheter-based targeting of therapeutic injections. the eight poses tested are shown in fig. . the five sdnr levels tested were . ± . , . ± . , . ± . , . ± . , and . ± . . with the exception of pose , the success rate was . % for all experiments, with a mean tre d of . ± . mm and tre d of . ± . mm. pose , with a rotation of θy = ◦, rarely converged, an issue which is f. . mean tre d (top) and tre d (bottom) for varying levels of sdnr. addressed in sec. . considering all poses and sdnr levels, the probe localization z-error along the source-detector axis was . ± . mm. higher image sdnr tended to improve tre (fig. ), although for sdnr in the range of – the tre did not vary much. for experiments with probe orientations typically seen in clinical cases (tee probe roll < | ◦|, poses – and ), and with sdnr > . , the registration success rate was %, the tre d was . ± . mm, and the tre d was . ± . mm. for reference, the sdnrs in the in vivo study were – . .b. water tank phantom and in vivo studies table ii shows the tre results for the water tank phan- tom and in vivo studies. the full image registration pipeline f. . summary of pose estimation accuracy experiments for varying poses and increasing sdnr (via image averaging). bars indicate the mean value and error bars indicate one standard deviation. ten images were generated for each pose and sdnr level. (a) tre d. (b) tre d. (c) percentage of successful registrations, defined as tre d < . mm. for each sdnr/pose combination, n= . (truncated result in the top panel: pose , frame= . ± . mm.) medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy t ii. results for the water tank phantom and in vivo experiments. sequence frames tre d (mm) mean ± std tre d (mm) mean ± std sdnr water tank . ± . . ± . . water tank . ± . . ± . . in vivo . ± . . ± . . in vivo . ± . . ± . . in vivo . ± . . ± . . resulted in a mean d error of . mm over all experiments and image frames, with errors in individual frames ranging from . to . mm. the water tank experiments showed lower tre values than the in vivo experiments, which is likely due to the tighter control over experimental variables. potential causes of errors are outlined in sec. . figure demonstrates echo-to-sbdx registration and sbdx-to-echo registration for the catheter tip in the sec- ond in vivo sequence. in the echo-to-sbdx registration, the catheter tip segmented from echo is registered to the d sbdx image (blue circle). the tre d values in table ii represent the error in this registration. in the sbdx-to-echo registration, tomosynthesis-based catheter tip tracking is regis- tered to two planes of the echo image volume and displayed as red circles. the error in this process is characterized by tre d. figure demonstrates an echo-to-sbdx registration of the endocardial surface of the left ventricle. a d ventricular volume was manually segmented from an end-diastolic echo image volume and then registered to sbdx using the tee probe pose. the segmented d volume was then projected onto the sbdx image and the borders of the projected segmen- tation were displayed. for comparison, a contrast-enhanced ventriculogram was performed with sbdx. a good agreement exists between the visible borders of the x-ray contrast and the echo-based borders. . discussion xrf is generally considered the primary imaging modal- ity for guidance of devices in structural heart interventions, but soft tissue visualization is poor, the projection format creates ambiguity, and ionizing radiation dose is an ongo- ing source of concern. previous work has demonstrated the potential of sbdx to both reduce dose and provide d cath- eter tracking. , the registration of d echo with sbdx could address the remaining need for real-time soft tissue anatomy in a common visualization environment. to this end, we have developed and evaluated an algorithm for sbdx/tee registration. the sbdx/tee registration algorithm combines tomosynthesis-based d localization with a version of d/ d registration adapted for inverse geometry x-ray imaging. in the initial pose estimation stage, the algorithm was able to localize the correct z-position of the tee probe to within . mm on average. the ability of inverse geometry fluoroscopy to resolve depth in a single image frame is a unique advantage compared to standard fluoroscopy, which generally requires either biplane imaging or multiple acquisitions at different c-arm projection angles to localize the tee probe in three dimensions. the study of pose estimation accuracy found tre d < mm in individual images, for all experiments conducted at sdnr levels similar to those that were encountered in vivo. at lower sdnr levels, the pose corresponding to a primarily lateral view of the tee probe (pose , fig. ) resulted in poor registration convergence. visual inspection revealed this was due to an error in the final θy (roll) and θx (pitch) parameters. additional work is needed to address this issue, but we note that in tavr procedures performed at our own institution, the occurrence of this pose is extremely rare since the probe is almost always facing toward the x-ray detector while imaging the heart. future work should also validate pose estimation accuracy in the presence of over- lapping high-contrast objects in the field-of-view, such as a catheter. for the water tank and in vivo studies, a general increase in tre relative to the pose accuracy study was observed. this was expected because the targets were real catheter tips rather than virtual objects. under this scenario, additional sources of registration error included localization of the catheter tip in echo and sbdx, echo volume-to-tee probe calibration error (tre = . mm), and potential temporal synchronization er- rors. for example, the d localization of the catheter tip in the sbdx plane stack was expected to introduce approximately . mm error in the z-direction. f. . left: a sbdx composite image is shown, with the catheter tip location from echo overlaid onto the image, demonstrating tre d. right: two orthogonal slices from the d echo corresponding to the sbdx composite image on the left. the catheter tip, localized in sbdx, is transformed and overlaid onto the echo image. medical physics, vol. , no. , december hatt et al.: registration of transesophageal echo to inverse geometry fluoroscopy f. . left: two orthogonal slices through a d echo volumetric frame taken during the in vivo experiment, with a semiautomatically generated segmentation of the left ventricle. right: the corresponding sbdx composite image, with the left ventricle segmentation borders registered and projected onto the sbdx image. this study demonstrates two potential approaches to sbdx/echo visualization. in an echo-centric display, d echo images could be augmented with d representations of the catheter device derived from sbdx device localization. alter- natively, a live d fluoroscopic image could be combined with soft tissue anatomy segmented from simultaneous d echo. future work will investigate the utility of these approaches in different structural heart interventional tasks. note that in this initial study, sbdx/tee image fusion was implemented in . for real-time guidance experiments, implementation on gpu-based hardware will be required. additionally, future work should include automated procedures for initialization of the registration. . conclusions image registration between a low dose sbdx system and tee has been demonstrated. a novel degree-of-freedom localization algorithm was presented, and the registration feasibility and accuracy were evaluated in phantoms and in vivo. future technical work will focus on real-time implemen- tation and fully automatic registration initialization. acknowledgment partial financial support for this work was provided by nih grant no. r hl . a)author to whom correspondence should be addressed. electronic mail: speidel@wisc.edu g. gao, g. penney, y. ma, n. gogin, p. cathier, a. arujuna, g. morton, d. caulfield, j. gill, and c. a. rinaldi, “registration of d trans-esophageal echocardiography to x-ray fluoroscopy using image-based probe tracking,” med. image anal. , – ( ). p. lang, p. seslija, m. w. chu, d. bainbridge, g. m. guiraudon, d. l. jones, and t. m. peters, “us–fluoroscopy registration for transcatheter aortic valve implantation,” ieee trans. biomed. eng. , – ( ). c. hatt, m. speidel, and a. 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physics, vol. , no. , december http://dx.doi.org/ . / - / / /n http://dx.doi.org/ . / . http://dx.doi.org/ . /circinterventions. . http://dx.doi.org/ . /circinterventions. . http://dx.doi.org/ . / . a dual-motive model of scapegoating: displacing blame to reduce guilt or increase control zachary k. rothschild, mark j. landau, daniel sullivan, and lucas a. keefer university of kansas the authors present a model that specifies psychological motives underlying scapegoating, defined as attributing inordinate blame for a negative outcome to a target individual or group, (a) maintaining perceived personal moral value by minimizing feelings of guilt over one’s responsibility for a negative outcome and (b) maintaining perceived personal control by obtaining a clear explanation for a negative outcome that otherwise seems inexplicable. three studies supported hypotheses derived from this dual-motive model. framing a negative outcome (environmental destruction or climate change) as caused by one’s own harmful actions (value threat) or unknown sources (control threat) both increased scapegoating, and these effects occurred indirectly through feelings of guilt and perceived personal control, respectively (study ), and were differentially moderated by affirmations of moral value and personal control (study ). also, scapegoating in response to value threat versus control threat produced divergent, theoretically specified effects on self-perceptions and behavioral intentions (study ). keywords: dual-motive model, scapegoating, guilt, personal control, climate change scapegoating is the act of blaming and often punishing a person or a group for a negative outcome that is due, at least in large part, to other causes. infamous historical examples of scapegoating include the witch trials that occurred in europe and north america from the th to the th century, when members of christian institutions accused thousands of people (mostly women) of cor- rupting society’s moral integrity by practicing witchcraft, and the nazis’ attempted extermination of jews and other minority groups for their alleged responsibility for germany’s economic collapse. capetown, bosnia, and rwanda give additional testimony to the violent conflict that can result when one group is designated as the chief cause of major misfortunes. scapegoating continues to occur today in various forms. whether it is politicians blaming china for the worldwide eco- nomic recession (chen, ), americans blaming muslims for all incidents of terrorism (foley, ), or religious fundamentalists blaming homosexual individuals for the decline of traditional american values (eckholm, ), people seem all too eager to heap blame onto others for major misfortunes. it is therefore surprising that, after receiving a flurry of theoretical and empirical attention during the s and s (adorno, frenkel-brunswik, levinson, & sanford, ; allport, , / ; dollard, doob, miller, mowrer, & sears, ; hovland & sears, ), the topic of scapegoating has largely receded from focus in con- temporary social psychology. to fill this gap, we present a “dual-motive” model that attempts to provide an integrative and generative empirical framework for understanding when people are most likely to scapegoat and what psychological motives underlie this behavior at the level of the individual. we describe our model in more detail below, but briefly stated, it posits that scapegoating can serve two meaning- fully distinct motives: (a) maintaining perceived personal moral value by minimizing feelings of guilt over one’s responsibility for a negative outcome and (b) maintaining perceived personal control by obtaining a clear explanation for a seemingly inexplicable negative outcome that is otherwise difficult to explain or control. we assess this dual-motive model in three studies that test whether activating each motive influences scapegoating as a func- tion of theoretically specified predictors, mediating variables, and moderating variables. we also test whether the effect of activating each motive on scapegoating produces distinct downstream effects on self-perceptions and behavioral intentions. these studies test model— derived hypotheses in the context of people’s attitudes toward environmental destruction and global climate change— topics of contemporary societal concern. in the following two sections, we bring into relief two influential theoretical accounts of the motives behind scapegoating: one fo- cused on the motive to perceive the self as morally valuable, the other focused on the motive to perceive the self as having control over one’s environment. we then describe how our dual-motive model integrates both accounts in a way that permits the derivation of novel, testable hypotheses about the causes and consequences of scapegoating. scapegoating to maintain moral value gordon allport ( , / ) observed that the term scape- goat derives from the biblical story of the goat that ancient israelites used to carry their sins into the desert. interpreting this this article was published online first april , . zachary k. rothschild, mark j. landau, daniel sullivan, and lucas a. keefer, department of psychology, university of kansas. correspondence concerning this article should be addressed to zachary k. rothschild, department of psychology, university of kansas, jayhawk boulevard, room , lawrence, ks - . e-mail: zachary.rothschild@gmail.com journal of personality and social psychology © american psychological association , vol. , no. , – - / /$ . doi: . /a story and the psychology of blame displacement through the lens of psychoanalytic theory, allport argued that scapegoating can occur as a special case of defensive projection, that is, attributing to somebody else a thought or an impulse that is feared in oneself. in scapegoating, the individual or group seeks to symbolically purge their own (largely implicit) feelings of inferiority, guilt, and self-hatred by perceiving a target individual or an outgroup as immoral or dangerous, and by expelling, isolating, or otherwise punishing that scapegoated target. allport stressed that scapegoating is a flexible strategy for maintaining the perceived moral value of oneself or one’s group, by which he meant that people can relieve their negative self-views by projecting superficially unrelated negative characteristics onto scapegoats (e.g., suppressing the threatening awareness of one’s own sexual impulses by projecting greediness onto an outgroup). adorno et al. ( ) similarly proposed that people’s occasional desire to punish others who violate societal norms is driven by repressed hostility tracing back to superficially unrelated child- hood conflicts. however, this conception of scapegoating as a flexible strategy to maintain perceived moral value failed to garner empirical support (e.g., gollwitzer, ), and it fell into disfavor as psychoanalytic theory receded from the mainstream in the social sciences (glick, ). more recent theoretical accounts in this vein (e.g., t. douglas, ) posit that scapegoating is better understood as a strategy that people use to minimize feelings of guilt over their responsibility for a specific negative outcome by transferring blame for that outcome to another individual or group. consistent with this re- vised view is research showing that people externalize blame for negative outcomes that would otherwise incriminate themselves or their group, even when self-presentation concerns are minimized (campbell & sedikides, ; kelly & michela, ; mezulis, abramson, hyde, & hankin, ; sheppard, malone, & sweeny, ). however, to our knowledge, prior research has not provided a direct empirical test of whether people attribute inordinate blame for a negative outcome they experience to a target outgroup in order to minimize feelings of guilt connected to their potential responsibility for that outcome. one goal of the present research was to directly test this possibility. although an account of scapegoating emphasizing people’s motive to maintain perceived personal moral value offers a pro- vocative explanation for many instances of scapegoating, it would seem to have difficulty explaining those instances in which indi- viduals are unlikely to perceive themselves as responsible for the relevant negative outcome. for example, in , television evan- gelist pat robertson infamously declared that civil liberties orga- nizations, feminists, and other politically left-leaning groups were largely to blame for the september , , terrorist attacks on the united states, and yet it is unlikely that robertson felt implicit or explicit guilt over his personal responsibility for those attacks. such instances may be better explained by another theoretical account of scapegoating, described next. scapegoating to maintain perceived personal control classic (e.g., bandura, ) and contemporary (kay, whitson, gaucher, & galinsky, ) perspectives converge on the notion that people are fundamentally motivated to maintain the perception that they have effective control over their environment. people’s perceived personal control can be threatened when they encounter a significant negative outcome, such as a disease epidemic or an economic recession, that appears to be due to unknown or chaotic factors. focalizing responsibility for the negative outcome onto a scapegoat may serve as a strategy for restoring perceived control because scapegoats, in contrast to chaotic and impersonal forces, can be clearly identified, counteracted, and (at least) understood. note that this account of scapegoating is distinct from the previ- ously discussed account emphasizing moral value maintenance. if people are confronted with a hazardous or an otherwise threatening event or circumstance that lacks an easily comprehensible and controllable cause, they may be motivated to explain it in a way that restores perceived control even if they do not feel significant guilt over their responsibility for that negative outcome. theoretical support for this control-maintenance account comes from allport ( ), who proposed that scapegoating can help the individual maintain the perception that the external world is or- derly, stable, and predictable (rather than chaotic and dangerous). staub ( ) similarly proposed that genocide is largely fueled by the perpetrator group’s desire to find a scapegoat that can be seen as responsible for large-scale threatening outcomes that are other- wise difficult to explain. most recently, glick ( , ) pro- posed that individuals are attracted to ideologies that attribute widespread negative outcomes without clear causes to the actions of a target group because such ideologies provide a simple, cul- turally sanctioned explanation for why bad things happen, as well as a ready-made solution for restoring perceived control over the environment: punishing or eliminating the scapegoat. in addition to highlighting the control-restorative function of scapegoat ideologies, glick ( ) noted that whether or not scapegoating is an effective means of restoring perceived control following exposure to a seemingly inexplicable outcome depends on the scapegoated target’s perceived “viability.” a viable scape- goat is perceived as possessing both the ability and the malicious intent necessary to deliberately cause the threatening outcome that needs to be explained. in contrast, if a person or group appears patently incapable of having caused the outcome—for instance, if they appear too weak to have exerted the amount of influence necessary—then they will likely be perceived as a nonviable scapegoat and will therefore not restore perceived personal control. glick supports this analysis by pointing out that groups stereo- typed as competent and malicious, rather than as weak and vul- nerable, are most likely to be designated as scapegoats. for exam- ple, the nazis blamed the “worldwide jewish conspiracy” for causing germany’s collapse, citing the relative success of the jewish people in banking, industry, the media, and government. in support of this notion, bilwicz and krziminski ( ) found that perceptions of jewish control appeared to partially mediate the effect of deprivation on anti-semitism in a polish sample. although it might be expected that encountering powerful, malicious outgroups would reduce perceived control compared with weaker targets, it is possible that people prefer seeing viable scapegoats as responsible for a seemingly random negative out- come to leaving that outcome unexplained. even the most power- ful scapegoat target permits relatively greater control by presenting the individual or group with a clear agent against which to focus their efforts in coping with the negative outcome. glick’s ( ) analysis focuses on expressions of scapegoating at the level of group relations and ideologies, and therefore does a dual-motive model of scapegoating not emphasize the potential role of control motivation in driving scapegoating behavior at the individual level. recently, however, sullivan, landau, and rothschild ( ) demonstrated that control motivation lies behind the related individual-level tendency to attribute malevolent power to an enemy figure. these researchers theorized that people’s perception of control is threatened by their awareness that multiple sources of potential hazard are spread diffusely throughout their environment, because this implies that their well-being and even existence is subject to the influence of indifferent and indeterminate forces. to the extent that people perceive an enemy to be an influential source of misfortune in their life, they can perceive their environment as containing less ran- domly distributed risk and thus feel a greater sense of personal control. this analysis yields the hypothesis that people will imbue real or imagined enemies with undue power as a way of transferring ambient danger onto a more concrete and comprehensible adver- sary. consistent with this hypothesis, sullivan et al. ( ) showed that increasing the salience of diffuse potential hazards in the environment (e.g., natural disasters, airborne diseases) led partic- ipants to attribute increased responsibility to a personal enemy for a range of potential misfortunes unrelated to those salient hazards (e.g., lost computer files; suspicious bank account activity). a follow-up study showed that among participants who contem- plated diffuse chaotic hazards, those presented with an enemy figure capable of perpetrating a wide range of misfortunes per- ceived their environment as containing less randomly distributed risk and, consequently, perceived themselves as having more con- trol over their life. these findings provide indirect support for our claim that the motive to maintain perceived personal control lies behind scape- goating. nevertheless, it is important to recognize the conceptual differences between the processes behind scapegoating and en- emyship. theoretical perspectives on enemyship view it as a broad, distal strategy for maintaining perceived control by reduc- ing the perceived likelihood of encountering myriad prospective threats in the world (see m. douglas, ). consistent with this notion, research shows that people respond to salient chaotic hazards by projecting onto an enemy figure influence over mis- fortunes unrelated to the precipitating hazards (sullivan et al., ). in contrast, scapegoating is viewed as a more proximal strategy for maintaining perceived control in the face of a seem- ingly inexplicable negative outcome by blaming a plausible agent for that outcome. in light of these differences, we believe that the findings from sullivan et al.’s enemyship research are suggestive but do not bear directly on the possibility that increasing the salience of causal uncertainty surrounding a particular negative outcome will motivate people to attribute increased blame for that outcome to a viable scapegoat target, but not a nonviable scapegoat target. further- more, it remains to be tested whether this effect occurs indirectly through decreased perceptions of personal control over the relevant negative outcome, and whether it is moderated by situational varia- tions in perceived personal control. one goal of the present research was to directly test these possibilities. a dual-motive model of scapegoating the preceding literature review highlights two conceptually distinct motives that potentially lie behind scapegoating at the individual level: maintaining one’s perceived moral value by min- imizing guilt over one’s wrongdoing, and maintaining one’s per- ceived personal control by explaining a negative outcome in one’s environment that otherwise seems beyond one’s ability to under- stand or control. rather than attempt to determine which motive is paramount, reduce one to the other, or reduce both to a common source, we propose that each motive contributes independently to scapegoating. an advantage of this approach is that it allows us to propose separate “paths” to scapegoating. each path explains why scapegoating occurs in response to the salience of different con- struals of a negative outcome, how scapegoating is mediated and moderated by different intervening variables, and the different, and even divergent, consequences of scapegoating for self-perceptions and behavioral intentions. these two paths are integrated in our dual-motive model, which is graphically represented in figure and described next. the moral value maintenance path our model defines scapegoating along the moral value mainte- nance path as a response to a significant negative outcome con- figure . conceptual diagram of the dual-motive model of scapegoating. rothschild, landau, sullivan, and keefer strued as being at least partially the result of the harmful actions of oneself or one’s group. in this situation, individuals experience feelings of guilt over their harmful actions and a compensatory desire to minimize their guilt. these feelings of guilt partly stem from an elevated sense that the self or one’s group has control over, and is therefore responsible for, the negative outcome in question. if individuals are unable to restore perceived moral value by other means, they will attribute increased blame to an available scapegoat target that is perceived as capable of bearing responsi- bility for the outcome (i.e., a viable scapegoat), thus yielding their own perceived responsibility over the outcome. they will also report a corresponding desire to punish the scapegoat. the perceived control maintenance path our model defines scapegoating along the control maintenance path as a response to a significant negative outcome construed as caused by factors that are beyond one’s ability to explain or control. in this situation, individuals experience decreased percep- tions of personal control, defined as a general sense of personal efficacy, and a compensatory desire to restore perceived personal control. if individuals are unable to restore perceptions of general personal control by other means, they will attribute increased blame to an available scapegoat target seen as capable of providing a clear causal explanation for, and potential means of coping with, the relevant negative outcome. downstream consequences proposing independent motivational paths for scapegoating al- lows us to specify hypotheses about not only distinct predictors, mediators, and moderators, but also the divergent downstream consequences of scapegoating as a function of which path is involved. along the moral value maintenance path, blaming a viable scapegoat for the relevant negative outcome should reduce feelings of guilt. it should also result in lower perceived respon- sibility for the relevant negative outcome. the latter hypothesis is based on previous research and theory suggesting that feelings of guilt emerge in response to perceiving oneself as having causal control over, and thus responsibility for, a given negative outcome (e.g., mcgraw, ; tangney, ). insofar as feelings of guilt are predicated on feeling personal control over a particular nega- tive outcome, we expect that attributing blame to a viable scape- goat for that outcome will cause participants to report both reduced feelings of guilt and reduced perceptions of personal responsibil- ity. along the control maintenance path, attributing blame to a viable scapegoat for the relevant negative outcome should have the opposite consequence for perceptions of personal control defined more broadly as a general sense of personal efficacy. when people are exposed to a particular negative outcome that seems beyond their ability to comprehend and control, subsequent exposure to a scapegoat who can be viably blamed for that outcome should increase perceived personal control. this hypothesis is indirectly supported by evidence that exposure to an enemy figure after a reminder of diffuse hazards bolstered participants’ perceptions of general personal control (sullivan et al., ). in contrast to its hypothesized influence on perceived personal control over a spe- cific negative outcome, scapegoating along the control mainte- nance path is not hypothesized to influence feelings of personal guilt, because the salience of an uncontrollable negative outcome does not necessarily assign causal responsibility for that outcome to the self. our dual-motive model also specifies hypotheses related to the downstream consequences of scapegoating on behavioral inten- tions. prior research shows that the motivation to reduce feelings of guilt drives people to engage in behaviors aimed at repairing the harm they have caused (e.g., amodio, devine, & harmon-jones, ; ketelaar & au, ; nelissen, dijker, & de vries, ). therefore, along the moral value maintenance path, the presence of a viable scapegoat should, by reducing feelings of guilt, reduce individuals’ willingness to engage in behaviors aimed at amelio- rating the harm they have caused. in contrast, we do not expect scapegoating along the control maintenance path to have this effect on intentions to engage in reparative behaviors. overview of the present studies in the present studies, we tested hypotheses derived from our dual-motive model with the broad goal of demonstrating the ex- istence and independence of two motivational paths to scapegoat- ing. more specifically, we tested whether scapegoating can occur as a function of distinct, theoretically specified predictors, medi- ating variables, and moderating variables, and furthermore whether scapegoating along each path produces distinct down- stream consequences for self-perceptions and behavioral inten- tions. we tested our model in the context of people’s attitudes surrounding environmental destruction and catastrophic climate change, topics which we felt lent themselves equally to framings that threatened either perceived moral value or perceived personal control. in study , we tested the hypothesis that framing environmental destruction as a threat to either moral value or control would lead to scapegoating as a function of different mediating variables. specifically, we predicted that participants primed to view envi- ronmental destruction as due to their own negligent behavior (value threat condition) would attribute greater responsibility for environmental destruction to oil companies (and report a corre- sponding desire to punish oil companies) compared with partici- pants who were not primed with the hazardous consequences of environmental destruction (no threat condition), and that this effect would occur indirectly through feelings of personal guilt, but not through perceptions of personal control. we further predicted that participants primed to view environmental destruction as due to unknown causes (control threat condition) would attribute greater responsibility to, and desire stronger punishment of, oil companies compared with participants in the no-threat condition, and that this effect would occur indirectly through perceptions of personal control, but not through feelings of guilt. we further assessed the independence of the value- and control- maintenance paths in study by testing whether the effects of framing climate change in a value-threatening or control- threatening manner on scapegoating would be differentially mod- erated by distinct affirmation inductions. specifically, we pre- dicted that the effect of a value threat on scapegoating would be attenuated if participants had the opportunity to affirm their moral value in a domain superficially unrelated to climate change, whereas the effect of a control threat on scapegoating would be a dual-motive model of scapegoating attenuated if participants had the opportunity to affirm their per- ceived personal control in an unrelated domain. as a critical test of the proposed independence of these motivational paths, we hy- pothesized that the effects of value threat and control threat on scapegoating would not be attenuated, respectively, by affirmation of one’s personal control and moral value. to clarify, our empir- ical approach was to provide converging tests of our proposed model by measuring key hypothetical constructs in study and testing their hypothesized role in mediating scapegoating, and then experimentally manipulating those same constructs in study and testing their hypothesized role in moderating scapegoating. focusing on downstream consequences, in study we tested the hypothesis that participants in a value threat condition would report increased feelings of guilt and that this effect would be attenuated if participants were given the opportunity to blame a viable scapegoat (oil companies) for climate change, but not if they had the opportunity to blame a nonviable scapegoat (the amish) for climate change. we also predicted that, among participants in a control threat condition, those given a chance to blame a viable scapegoat, but not a nonviable scapegoat, would report bolstered perceptions of general personal control. finally, we hypothesized that participants under value threat and subsequently presented with a viable scapegoat would report less willingness to engage in behaviors meant to reduce their harmful impact on the environ- ment, and that this interactive effect would be mediated by de- creased feelings of guilt. study in recent years, there has been a growing popular concern about hazards resulting from human destruction of the natural environ- ment. people rarely assume personal responsibility for contributing to environmental destruction, however, and instead relegate blame to other groups such as governments or business organizations (lorenzoni & langford, ). in study , we used this real-life context of scapegoating to provide an initial test of our dual- motive model. specifically, we hypothesized that framing a neg- ative outcome in either a value-threatening or a control-threatening manner would increase scapegoating equally but that these effects would be differentially mediated by feelings of personal guilt and perceptions of personal control. we manipulated threat by framing the harmful consequences of environmental destruction as either the result of the participants’ own actions (value threat condition) or the result of unknown causes and thus beyond participants’ control (control threat con- dition). participants assigned to a third, no-threat condition were not reminded of the hazards of environmental destruction. after- wards, participants reported their feelings of personal guilt for their negative environmental impact and their perceptions of personal control over the harmful effects of environmental destruction. finally, participants indicated the extent to which they believed that oil companies should be blamed and punished for the harmful consequences of environmental destruction. we predicted that participants in both the value threat condition and the control threat condition would blame (and seek to punish) oil companies more than participants in the no-threat condition. we also predicted that the effect of threat on scapegoating would occur via distinct indirect effects according to condition: in the value threat condition, increased scapegoating should occur through increased feelings of personal guilt, but not through vari- ations in perceived control, whereas in the control threat condition, increased scapegoating should occur through decreased percep- tions of personal control, but not through variations in feelings of personal guilt. method one-hundred fourteen participants ( women; ages – ) completed a survey on a midwestern university campus in ex- change for candy. potential participants were approached by male and female experimenters and asked to complete a packet of opinion questionnaires dealing with current issues. only % of those asked declined to participate. participants were randomly assigned to conditions in a single-factor design with three levels: value threat versus control threat versus no threat. threat manipulation. the first questionnaire constituted the threat manipulation. in the value threat and control threat condi- tions, this questionnaire was described as an “environmental awareness survey.” in the value threat condition, the purported purpose of the survey was to assess people’s awareness of the lifestyle choices that contribute to environmental destruction. the instructions stated that threatening hazards result from environmental destruc- tion, which is caused by the lifestyle choices that some people make every day. participants were instructed to read seven state- ments describing environmentally destructive behaviors, which were purportedly proven to cause harmful environmental condi- tions, and to rate how true each statement was for them personally. in an effort to increase participants’ feelings of personal respon- sibility for environmental hazard, the seven statements refer to behaviors that we assumed would be common for participants in our sample (e.g., “i drive my car when i could walk, ride a bike, or take public transportation; i use the dryer when i could air-dry my clothing instead”). responses were made on a -point scale ( � not at all true for me, � completely true for me). support- ing our assumption that the items referred to common behaviors, a one-sample t test revealed that the grand mean of the composite scores averaging across the seven items (mgrand � . , sd � . ) was significantly higher than the scale’s midpoint ( ), t( ) � . , p � . . in the control threat condition, the purported purpose of the survey was to assess people’s beliefs about how much control they have over hazards caused by the unexplained destruction of the natural environment. the instructions stated that threatening haz- ards result from environmental destruction, which is caused by “unknown sources.” participants were instructed to indicate their agreement with seven statements regarding their personal control over hazardous environmental conditions, all of which concerned large-scale environmental hazards meant to be perceived as be- yond any individual’s control (e.g., “i have control over tornadoes and other natural disasters; i have control over whether my state suffers from drought or flooding”; � not at all true for me, � in this and the following studies, we originally performed our primary analyses including gender as a between-subjects variable. because we observed no significant main effects or interactions involving gender, we omit gender from our reporting of the results to simplify presentation. rothschild, landau, sullivan, and keefer completely true for me). although specific examples of hazardous environmental conditions were identified, the instructions empha- sized that the source of the environmental destruction purportedly causing these hazardous conditions is unexplained. supporting our assumption that these items referred to outcomes that participants would perceive to be beyond their personal control, a one-sample t test revealed that the grand mean of the composite scores aver- aging across the seven items (mgrand � . , sd � . ) was significantly lower than the scale’s midpoint ( ), t( ) � � . , p � . . participants in the no-threat condition responded to a “person- ality awareness survey” purportedly designed to assess the gap between university administrators’ awareness of student lifestyles and students’ actual behavior. participants indicated their agree- ment with seven statements referring to relatively benign aspects of daily life (e.g., “i like to stay busy and get bored easily”) using the same -point scale used in the other two conditions. guilt measure. after the threat manipulation, participants were presented with response items developed by ferguson ( ) for the purpose of measuring feelings of guilt over contributing to climate change, but here adapted to refer to one’s personal con- tribution to environmental harm more generally. specifically, par- ticipants were instructed to indicate their agreement with four statements referring to their guilt over contributing to environmen- tal harm (e.g., “i feel guilty for the negative impact my lifestyle has on the environment”). participants made their responses by mark- ing an x on a continuous line anchored on the left with “not at all” and on the right with “very much.” these responses were coded in centimeters from the left end of the line, such that higher scores indicated higher feelings of guilt (possible scores ranged from to ). responses across the four items were internally reliable (� � . ) and were averaged to create composite guilt scores (mgrand � . , sd � . ). personal control measure. because no existing measure could be found that specifically assessed perceptions of control over hazardous environmental conditions, this construct was mea- sured by asking participants to indicate their agreement with a single face-valid item: “there is nothing i can do to prevent worsening environmental conditions.” participants made their re- sponses using the same continuous scale used for the guilt measure (mgrand � . , sd � . ). responses on this item were reverse scored, such that higher scores indicated greater perceived per- sonal control. the guilt and control measures were counterbalanced in order of presentation, and preliminary analyses revealed no main effects or interactions involving presentation order; thus, this factor was omitted from subsequent analyses. scapegoating measure. finally, participants answered five questions assessing the extent to which they believed oil compa- nies should be blamed and punished for the harmful consequences of environmental destruction: “to what extent do you believe that: oil companies are responsible for the destruction of the environ- ment?; oil companies are to blame for the effects of environmental devastation?; oil companies are at fault for the effects of environ- mental damage?; oil companies are guilty of severely damaging the global environment?; oil companies should be punished for their contribution to the destruction of the natural environment?” responses were made on a -point scale ( � not at all, � very much) and were averaged to form composite scapegoating scores (� � . ). results guilt. first, we tested whether the threat manipulation influ- enced feelings of personal guilt in the hypothesized direction. submitting guilt scores to a one-way (value threat vs. control threat vs. no threat) analysis of variance (anova) revealed a significant omnibus effect, f( , ) � . , p � . , � � . . supporting predictions, pairwise comparisons (fisher’s least significant dif- ference test) revealed that participants in the value threat condition reported feeling more guilt over the negative impact that their lifestyle has on the environment (m � . , sd � . ) compared with participants in the control threat condition (m � . , sd � . ), f( , ) � . , p � . , and the no-threat condition (m � . , sd � . ), f( , ) � . , p � . . mean levels of guilt in the control threat and no-threat conditions were statis- tically equivalent (f � . , p � . ). personal control. a levene’s test for heterogeneity of vari- ance indicated that responses to our measure of perceived personal control violated the homogeneity of variance assumption, f( , ) � . , p � . . accordingly, we conducted welch’s alternative anova procedure on perceived personal control scores, which tomarken and serlin ( ) identify as the optimal procedure when the homogeneity of variance assumption is vio- lated. this analysis revealed a significant omnibus effect, f( , . ) � . , p � . . as predicted, post hoc contrasts showed that participants in the control threat condition reported feeling less control over worsening environmental conditions (m � . , sd � . ) compared with participants in the value threat condition (m � . , sd � . ), t( . ) � . , p � . , and no-threat condition (m � . , sd � . ), t( . ) � . , p � . . mean personal control scores in the value threat and no-threat conditions were statistically equivalent, t( . ) � . , p � . . scapegoating. submitting scapegoating scores to an anova revealed a significant omnibus effect, f( , ) � . , p � . , � � . . compared with participants in the no-threat condition (m � . , sd � . ), participants attributed more blame to, and reported greater desire to punish, oil companies for the harmful consequences of environmental destruction in both the value threat condition (m � . , sd � . ), f( , ) � . , p � . , and the control threat condition (m � . , sd � . ), across all three studies, we conducted a levene’s test for heterogene- ity of variance for all primary analyses. although this analysis yielded a significant f statistic for the one-way anova on personal control in study ( p � . ), all subsequent tests were nonsignificant (fs � . , ps � . ), indicating that the homogeneity of variance assumption was met for the remainder of our analyses. for all cases in which the assumption of homogeneity of variance was met, pairwise comparisons were tested with f tests using full-sample degrees of freedom and corresponding omnibus mse terms. however, because it is not appropriate to use an omnibus mse term for pairwise comparisons when the variance between groups is heterogeneous (howell, ), we performed pairwise comparisons on perceived personal control in study with t tests using the welch procedure and degrees of freedom estimated for each simple effect. a dual-motive model of scapegoating f( , ) � . , p � . . mean scapegoating scores did not significantly differ between the two threat conditions (f � . , p � . ). analysis of indirect effects. we then conducted separate indirect effect analyses to test our mediational hypothesis that the increased scapegoating produced by value threat and control threat occurs, respectively, through increased feelings of guilt and de- creased perceptions of personal control. using the bootstrapping procedure and corresponding spss macro of preacher and hayes ( ), we first regressed scapegoat- ing scores onto value threat condition (coded: value threat � /control threat � /no threat � ), with guilt scores and personal control scores entered as the proposed mediators and control threat condition (coded: value threat � /control threat � /no threat � ) entered as a covariate. five-thousand bootstrap resamples were performed. as predicted, the % confidence interval obtained for the indirect effect of value threat condition on scapegoating scores through the mediator of guilt did not contain zero [. , . ], whereas the confidence interval for the indirect effect of value threat through the mediator of perceived control did contain zero [�. , . ]. these results are consistent with our mediational hypothesis that the increase in scapegoating in the value threat condition (vs. the no-threat condition) occurs through a corre- sponding increase in feelings of guilt; and importantly that this effect does not occur through variations in perceptions of personal control. using the same bootstrapping procedure, we then regressed scapegoating scores onto dummy-coded control threat condition with guilt scores, and personal control scores entered as proposed mediators and dummy-coded value threat condition entered as a covariate. the % confidence interval for the indirect effect of control threat on scapegoating scores through the mediator of perceived personal control did not contain zero [. , . ], whereas the confidence interval for the indirect effect of control threat through the mediator of guilt did contain zero [�. , . ]. these results support our mediational hypothesis that the increase in scapegoating in the control threat condition (vs. the no-threat condition) occurs through the corresponding decrease in perceived personal control, but not though variations in feelings of guilt (see figure for a graphical depiction of the mediation model). discussion supporting hypotheses, the results of study show that, com- pared with participants who were not reminded of the hazards of environmental destruction, those primed to feel personally respon- sible for environmental destruction or uncertain of its cause attrib- uted more blame to oil companies and showed a greater desire to punish oil companies for the negative consequences wrought by such destruction. results were also consistent with our differential mediational hypothesis: the effect of value threat on scapegoating occurred indirectly through increased feelings of personal guilt, but not through perceptions of personal control, whereas the effect of control threat on scapegoating occurred indirectly through de- creased perceptions of personal control, but not through feelings of guilt. these results suggest that when individuals are confronted with a negative outcome framed as the result of their own harmful actions, they scapegoat in response to elevated feelings of personal guilt; when the cause of the harm is unknown, they scapegoat to compensate for decreased perceptions of personal control. these results support our broader theoretical claim that the desire to maintain a sense of personal moral value and the desire to maintain a sense of personal control constitute independent motivational paths to scapegoating. in the next study, we sought to complement the meditational approach used in study with a moderation approach that in- volved manipulating (rather than measuring) the constructs of perceived moral value and perceived personal control. specifi- cally, we tested whether the effect of value threat and control threat on scapegoating would be attenuated, respectively, by affirmations of moral value and personal control. in addition, in study we addressed one limitation of study , namely, that the general salience of negative environmental hazard differed as a function of threat condition. that is, beyond experiencing a value threat or a control threat, participants in both threat conditions were primed with the harmful consequences of environmental destruction, whereas those in the no-threat condition were not. this introduces the possibility that participants in both the value threat condition and the control threat condition showed higher scapegoating than participants in the no-threat condition simply because environmen- tal hazard was more salient in those threat conditions. this possi- figure . indirect effects of threat condition on scapegoating through feelings of personal guilt and perceived personal control (study ). all path coefficients represent standardized regression weights. the direct effect coefficients represent the effect of each threat variable on the dependent variable after controlling for the effect of the proposed mediators. total adjusted r for the model � . , f( , ) � . , p � . . total effect of value threat: � � . �. direct effect of value threat: � � . , ns. total effect of control threat: � � . �. direct effect of control threat: � � . �. � p � . . rothschild, landau, sullivan, and keefer bility seems unlikely given that we found evidence that these threat conditions increased scapegoating indirectly through distinct me- diating variables specified by our model. nevertheless, we at- tempted to rule out this possibility in study by exposing partic- ipants in all conditions to the same information about the perils of climate change and manipulating only the causal framing of this negative outcome. study study provides a further test of the existence of independent motivational paths to scapegoating by including experimental ma- nipulations designed to target the conceptual variables that we measured as mediators in study . specifically, in study we est whether the effects of value threat and control threat on scape- goating found in study are differentially moderated by affirma- tion inductions designed to restore perceptions of one’s moral value or personal control. if framing a negative outcome as due to individuals’ own harmful actions motivates them to compensate for the threat to their moral value by means of scapegoating, then providing an alternative means of restoring perceived moral value should attenuate this effect. analogously, if framing a negative outcome as due to unknown causes motivates individuals to restore perceived personal control by means of scapegoating, then provid- ing an alternative means of restoring perceived personal control should attenuate this particular effect. to test these hypotheses, we presented participants with what was purported to be a scientific article that, depending on condi- tion, framed catastrophic climate change as primarily caused by harmful actions on the part of participants’ ingroup (young amer- icans), unknown sources, or the harmful actions of an outgroup (middle-aged americans). including this third condition allowed us to expose all participants to the same information about cata- strophic climate change and thus control for possible effects of differential salience of environmental hazards. after the threat manipulation, participants were asked to com- plete a writing task that gave them the opportunity to affirm either their own moral value or their sense of personal control in a domain unrelated to climate change. participants then indicated the extent to which they blamed and desired to punish international corporations for the hazardous consequences of climate change. we changed the target scapegoat group from study (oil compa- nies) to study (international corporations) to improve the gen- eralizability of our hypothesized effects and ensure that they are not specific to oil companies as a targeted scapegoat. given the popularity of blaming international corporations for various neg- ative social, economic, and environmental outcomes (e.g., kortens, ), we felt that this group would readily serve as a viable scapegoat target for the present study. guided by our dual-motive model, we predicted that participants would show increased scapegoating of international corporations when the in- group (vs. an outgroup) was framed as responsible for hazardous climate change and that this effect would be attenuated by a moral value affirmation, but not by a personal control affirmation. fur- thermore, we predicted that participants would show increased scapegoating when hazardous climate change was attributed to unknown causes (compared with participants in the outgroup re- sponsible condition) and that this effect would be attenuated by a personal control affirmation, but not by a moral value affirmation. method sixty-one psychology undergraduates ( women) from a mid- western university participated in partial fulfillment of a course requirement. because in this and the following study, moral value was threatened through an article attributing responsibility for global climate change to people ages – , only participants within this age range were recruited. in a laboratory setting, participants were told that they would be taking part in two separate studies. the first study was described as an investigation of personality and knowledge about climate change, whereas the second study was described as an investigation of people’s judg- ments about different groups. in private cubicles, participants completed all the materials on computers. participants were ran- domly assigned to conditions in a (threat condition: value threat vs. control threat vs. no threat) � (affirmation: moral value vs. personal control) between-subjects factorial design, with scape- goating serving as the dependent variable of primary interest. threat manipulation. first, all participants read an article, ostensibly written by scientific experts, entitled “the causes and consequences of climate change.” the first section of this experimenter-fabricated article was identical across conditions and discussed the negative effects of climate change, including animal extinctions and an increasing threat of natural disasters. the sec- ond section, entitled “who’s to blame?” differed across condi- tions. in the value threat condition, this section identified young americans (ages – ) as the primary contributors to climate change. the article concluded by stating that “until young amer- icans start to acknowledge their responsibility for the damaging effects of climate change, things are likely to only get worse.” in the control threat condition, this section of the article stated that scientists are currently unable to determine what causes climate change. the article concluded “until we can identify who or what is responsible for the damaging effects of climate change, things are likely to only get worse.” in the no-threat condition, this section of the article was nearly identical to the corresponding section of the article in the value threat condition except that middle-aged americans (ages – ), rather than young ameri- cans, were identified as the primary contributors to climate change. affirmation manipulation. next, participants received a questionnaire purported to be a personality assessment. this ques- tionnaire constituted our affirmation manipulation. participants assigned to the moral value affirmation condition responded to the following writing prompt: “in a few sentences briefly describe something about yourself that makes you feel like a good and decent person.” participants in the control affirmation condition responded to the following writing prompt: “in a few sentences please briefly describe something in your life that you have com- plete control over.” underneath both prompts was a full page of blank lines. inspection of participants’ written responses revealed that no participant wrote about issues surrounding climate change. participants were then introduced to what was ostensibly a second, unrelated study. they were told that they would be asked to answer some questions about a group. to minimize suspicions about connections between the threat manipulation and our pri- mary dependent measure, we arranged it so that the computer chose a target group ostensibly at random from an assortment of a dual-motive model of scapegoating candidate groups. in actuality, all participants answered questions about international corporations. scapegoating measure. the degree to which participants blamed and sought to punish international corporations for their role in climate change was measured by having participants com- plete the same scapegoating measure used in study , with the exception that the five items referred to international corporations rather than oil companies (� � . ). results scapegoating. we predicted that participants primed to view climate change as due to their own group’s harmful actions (value threat condition) and those primed to view climate change as due to unknown causes (control threat condition) would show higher levels of scapegoating than participants primed to view climate change as due to an outgroup’s harmful actions (no-threat condi- tion). furthermore, we predicted that the effects of value threat and control threat would be attenuated, respectively, by affir- mations of moral value and personal control. to test these predictions, we submitted scapegoating scores to a (threat condition: value threat vs. control threat vs. no threat) � (affirmation: moral value vs. personal control) between- subjects anova. we obtained the predicted two-way interac- tion, f( , ) � . , p � . , � � . . supporting predictions, pairwise comparisons and the pattern of means depicted in figure revealed that, among participants who were not given the opportunity to affirm their moral value (i.e., who affirmed their personal control instead), those in the value threat condition exhibited higher levels of scapegoating (m � . , sd � . ) than participants in both the control threat condition (m � . , sd � . ), f( , ) � . , p � . , and the no-threat condition (m � . , sd � . ), f( , ) � . , p � . . also as predicted, among participants who were not given an opportunity to affirm their personal control (i.e., who affirmed their personal moral value), those in the control threat condition exhibited higher levels of scapegoating (m � . , sd � . ) than participants in both the value threat condition (m � . , sd � . ), f( , ) � . , p � . , and the no-threat condition (m � . , sd � . ), f( , ) � . , p � . . also supporting predictions, within the value threat condition, participants who affirmed their moral value following the threat induction exhibited lower levels of scapegoating than those who affirmed their personal control, f( , ) � . , p � . . sim- ilarly, within the control threat condition, participants who af- firmed personal control following the threat induction exhibited lower levels of scapegoating than participants who affirmed their moral value, f( , ) � . , p � . . affirmation condition had no effect on scapegoating level among participants in the no-threat condition (f � . , p � . ). discussion when confronted with their ingroup’s culpability for climate change, participants showed an increased tendency to scapegoat international corporations, and this effect was eliminated if they had an opportunity to affirm their moral value in an unrelated domain, but not if they had an opportunity to affirm their personal control. analogously, when participants were confronted with the causal indeterminacy of climate change, they showed an increased tendency to scapegoat international corporations, and this effect was eliminated if they had an opportunity to affirm their personal control, but not if they had an opportunity to affirm their moral value. these results support our broad theoretical claim that scape- goating can represent a strategy for restoring one’s perceived moral value or perceived personal control, depending on whether a particular negative outcome is framed in a way that threatens either of these self-perceptions. the fact that the salience of the negative impact of climate change was equivalent across condi- tions helps to rule out the possibility that the mere salience of harmful environmental conditions accounts for our primary scape- goating effects. in conjunction with the separate indirect effects reported in study , the threat-specific moderation effects obtained in study highlight the independence of our proposed motivational paths to scapegoating. simultaneously, they provide converging evidence to suggest that the effects of our threat conditions in study did in fact occur through elevated feelings of guilt or reduced perceptions of personal control. as discussed by spencer, zanna, and fong ( ), correlational indirect effects designs in which both pro- posed mediators and outcomes are measured rather than manipu- lated (as was the case for the present study ) do not yield conclusive evidence regarding mediation. however, by directly manipulating in study our key constructs (feelings related to moral value and perceptions of control) that were measured as mediators in study , we were able to establish more direct causal evidence to suggest that value threats and control threats increase scapegoating via increased guilt and reduced personal control, respectively. study in studies and , we examined the factors that influence scapegoating behavior. in study , we built on these studies by examining the downstream consequences of scapegoating. on the figure . scapegoating as a function of threat condition (climate change attributed to ingroup [value threat], unknown causes [control threat], or an outgroup [no threat]) and affirmation type (study ). higher scores indicate higher attributions of blame and greater desire to punish international corporations for the negative effects of climate change. scale ranges from to . error bars represent standard errors. rothschild, landau, sullivan, and keefer basis of our dual-motive model, we hypothesized that, depending on whether climate change was framed as threatening one’s moral value or perceived control, scapegoating should have differential effects on self-perceptions and behavioral intentions. focusing on self-perceptions, we hypothesized that when climate change was framed in a way that makes participants feel guilty for their own actions, providing them with the opportunity to attribute blame to a scapegoat would reduce feelings of personal guilt. also, in line with the findings reported by sullivan et al. ( ), we hypothe- sized that when climate change was framed in a causally indeter- minate way threatening perceived personal control in the world, the opportunity to scapegoat would bolster perceptions of general personal control over one’s life. we did not expect this causal indeterminacy framing of climate change to influence feelings of guilt, however, because there is no reason to expect that partici- pants would interpret this framing as implicating them personally in contributing to climate change. because our model portrays scapegoating along the control maintenance path as a strategy for maintaining general perceived personal control in the face of threatening outcomes that seem otherwise difficult to control, we sought to advance beyond study by measuring personal control in a way that captures a general sense of personal efficacy over one’s life. specifically, in study we assessed perceptions of general personal control, rather than perceptions of control or responsibility specifically in relation to the relevant negative outcome (in this case, climate change). nevertheless, we recognized the possibility that this measure might also be sensitive to variations in participants’ perceptions of their responsibility over climate change specifically, particularly in the value threat condition where participants are told they bear responsibility for climate change. as mentioned, past research and theory suggest that feelings of personal guilt are linked to per- ceived personal responsibility for a given negative outcome (e.g., tangney, ). thus, we allowed for the possibility that, in the value threat condition, the opportunity to blame a scapegoat would not only decrease feelings of personal guilt over climate change but also decrease participants’ perceptions of general personal control. to clarify, we believe that there is a useful distinction between perceived personal control, defined broadly as a global sense that one has efficacy over one’s life, and perceived personal control, defined more specifically as a sense of personal respon- sibility over a particular negative outcome. in study , we mea- sured the former type of perceived personal control because our primary goal was to test our model-derived hypothesis that blam- ing a scapegoat for an otherwise inexplicable negative outcome will bolster people’s general sense that they have efficacy over their lives. at the same time, we allow for the possibility that this measure captures specific perceptions of responsibility over cli- mate change; thus, insofar as value-threatened participants given the opportunity to scapegoat experience decreased guilt over cli- mate change, and decreased guilt corresponds to decreased respon- sibility perceptions, it is possible that these participants will also report decreased perceived personal control. we make this predic- tion tentatively because we believe that a proper test of decreased responsibility perceptions corresponding to decrease guilt should use a more direct measure of perceived personal responsibility over the relevant negative outcome, rather than perceptions of general personal control. focusing on behavioral intentions, we hypothesized that, insofar as increased feelings of guilt for illegitimate harm motivate repar- ative action (e.g., amodio et al., ; ketelaar & au, ; nelissen et al., ), participants made to feel responsible for climate change, but then presented with an opportunity to scape- goat, would be less willing to engage in environmental advocacy. we also predicted that this effect would occur indirectly through reduced feelings of personal guilt. to test these hypotheses, we again manipulated threat by fram- ing climate change as caused by either the harmful actions of participants’ ingroup (value threat) or unknown sources (control threat). because the focus of study was on differentiating be- tween the downstream consequences of scapegoating as a function of value threat versus control threat, rather than on scapegoating itself as an outcome, we did not include an additional no-threat comparison condition as in studies and . we manipulated opportunity to scapegoat by providing all par- ticipants with a target outgroup to blame for climate change, but varying whether or not that group represented a viable scapegoat- ing target. as discussed in the introduction, glick ( ) argued that a social group can viably serve as a scapegoat only when that group is perceived as sufficiently powerful and malevolent to have realistically contributed to the negative outcome or event requiring explanation (in this case, climate change). accordingly, we pre- sented participants with the opportunity to blame either oil com- panies (a viable scapegoat target) or the amish (a nonviable scapegoat target) for climate change. we subsequently assessed participants’ feelings of personal guilt for the negative effects of climate change, their perceptions of general personal control, and their willingness to take action to stop climate change. it is worth noting that glick’s ( ) notion of viability con- trasts with earlier accounts of scapegoating, which propose that weak and vulnerable groups, as opposed to powerful groups, tend to be scapegoated (e.g., allport, / ). in line with glick’s analysis of viability, we hypothesized that, insofar as the amish are perceived as lacking the power and influence necessary to be responsible for climate change, blaming the amish should fail as an effective means of either reducing guilt or reasserting control. alternatively, if earlier theories of scapegoating are correct, we would expect the amish to be readily scapegoated. past research has shown that individual differences can be important determinants of individuals’ attitudes toward climate change and related environmental conditions. for example, polit- ical orientation and general environmental concern have been found to be strong predictors of individuals’ belief in climate change and support for environmental advocacy (e.g., krosnick, holbrook, & visser, ; schuldt, konrath, schwarz, ). on the basis of these findings, we sought to advance beyond our first two studies by measuring and statistically controlling for these variables in study in order to isolate the unique effects of our experimental manipulations. method sixty-four undergraduates ( women; ages – ) from a midwestern university participated in partial fulfillment of a course requirement. the cover story was similar to that used in study . participants were randomly assigned to conditions in a (threat condition: value threat vs. control threat) � (scapegoat target: a dual-motive model of scapegoating viable vs. nonviable) between-subjects factorial design. effects on a number of dependent variables, discussed shortly, were exam- ined. individual differences. prior to any experimental manipula- tions, participants first responded to a single-item measure assess- ing their political orientation ( � very conservative, � very liberal; mgrand � . , sd � . ) and a single-item measure assessing their preexisting belief in the importance of environmen- tal responsibility (“how important is it for people to do everything they can to minimize their impact on the environment?”; � not at all important, � very important; mgrand � . , sd � . ). scores on these measures were used as covariates for all analyses. threat manipulation. participants assigned to the value threat condition read the same article used in study that framed climate change as due primarily to the harmful actions of young adults (the participants’ ingroup). participants assigned to the control threat condition read the article from study that framed climate change as due to unknown causes. scapegoat target manipulation. as in study , participants were then introduced to an ostensibly unrelated study in which they would answer questions about a group selected at random by the computer. unlike study , though, here participants were in fact randomly presented with one of two target outgroups. partic- ipants in the viable scapegoat condition were presented with oil companies, whereas participants in the nonviable scapegoat con- dition were presented with the amish. to test our assumption that participants would perceive oil companies as a more viable source of responsibility for climate change than the amish, we had participants rate their level of agreement with the statement “how much does this group contribute to climate change?” on a -point scale ( � not at all, � a very large amount). guilt. next, participants completed a series of filler surveys, embedded in which was a modified single-item measure of per- sonal guilt, selected from the larger guilt scale originally used by ferguson ( ) to assess guilt for contributing to global warming. participants were asked to rate their level of agreement with the statement “i feel guilty for my contributions to global warming” using a -point scale ( � very strongly disagree, � very strongly agree). a single-item measure of guilt was used in an attempt to shorten the study completion time, and this particular item was chosen because it is the most face-valid of the original scale items and had the strongest loading on the four-item com- posite in study . personal control. participants’ perceptions that they have control over their lives were measured by asking them to respond to the same single-item measure that sullivan et al. ( ) used to measure perceived personal control: “in general, how much con- trol do you feel you have over what happens in your life?” responses to this item were made along a -point scale ( � not at all, � very much). the guilt and personal control items were randomized in order of presentation. environmental advocacy measure. the final measure was a shortened version of ferguson’s ( ) environmental advocacy scale. participants responded to four items assessing their willing- ness to engage in behaviors intended to reduce the impact of global warming (e.g., “in order to reduce the impact of global warming, to what extent would you be willing to give a short presentation in an elementary school about global warming and how to reduce it?”). responses were made on a -point scale ( � not at all willing, � extremely willing) and were averaged to form com- posite environmental advocacy scores (� � . ). results scapegoating. we first tested our assumption that partici- pants would perceive oil companies as a more viable scapegoat target for climate change than the amish. we submitted ratings of the target group’s contribution to climate change to a (threat condition: value threat vs. control threat) � (scapegoat target: oil companies vs. amish) between-subjects anova. this analysis yielded the predicted main effect for target such that participants rated oil companies as contributing significantly more to climate change (m � . , sd � . ) compared with the amish (m � . , sd � . ), f( , ) � . , p � . . no other effects reached significance (fs � . , ps � . ). guilt. next, we tested our prediction that, when climate change was framed as the result of the harmful actions of their ingroup, participants would experience increased guilt, but this effect would be attenuated if participants additionally had the opportunity to attribute blame to a viable scapegoat, but not to a nonviable scapegoat. submitting guilt scores to a threat condi- tion � scapegoat target anova returned a significant interac- tion, f( , ) � . , p � . , � � . . supporting predictions, pairwise comparisons and the pattern of means depicted in figure revealed that, among participants given the opportunity to blame a nonviable scapegoat, those in the value threat (i.e., ingroup responsible) condition reported greater feelings of guilt (m � . , sd � . ) than those in the control threat (i.e., unknown cause) condition (m � . , sd � . ), f( , ) � . , p � . . however, participants in the value threat condition who were given an opportunity to blame a viable scapegoat reported feeling less guilt (m � . , sd � . ) than those in both the value threat/ nonviable scapegoat condition, f( , ) � . , p � . , and the control threat/viable scapegoat condition (m � . , sd � . ), f( , ) � . , p � . . figure . personal guilt as a function of threat condition and viability of scapegoat target (study ). higher scores indicate greater feelings of guilt for one’s own contributions to climate change. scale ranges from to . error bars represent standard errors. rothschild, landau, sullivan, and keefer unexpectedly, participants in the control threat condition who were exposed to a viable scapegoat reported more guilt than participants in the control threat/nonviable scapegoat condition, f( , ) � . , p � . . we return to this finding in the discussion section. personal control. next, we tested our prediction that, when climate change was framed as the result of unknown causes, exposure to a viable scapegoat would increase perceived personal control, whereas exposure to a nonviable scapegoat would not have this effect. submitting perceived personal control scores to the same two-way anova returned a significant interaction, f( , ) � . , p � . , � � . . supporting predictions, pairwise comparisons and the pattern of means depicted in figure re- vealed that, among participants exposed to a viable scapegoat, those in the control threat condition reported having more control over their lives (m � . , sd � . ) than participants in both the control threat/nonviable scapegoat condition (m � . , sd � . ), f( , ) � . , p � . , and the value threat/viable scapegoat condition, (m � . , sd � . ), f( , ) � . , p � . . we then tested our tentative prediction that participants in the value threat condition who were exposed to a viable scapegoat would report, in addition to less guilt (as reported above), less personal control over their lives compared with those exposed to a nonviable scapegoat. among participants in the value threat con- dition, perceived personal control was indeed lower after exposure to a viable scapegoat than after exposure to a nonviable scapegoat (m � . , sd � . ), f( , ) � . , p � . . environmental advocacy. we also tested the prediction that participants exposed to a value-threatening framing of climate change would be motivated to make reparative actions, unless they were first given the opportunity to attribute blame to a viable scapegoat. performing the same two-way anova on environ- mental advocacy scores yielded a significant interaction, f( , ) � . , p � . , � � . . pairwise comparisons and the pattern of means depicted in figure revealed that, as expected, participants in the value threat condition who were exposed to a viable scapegoat reported lower environmental advocacy scores (m � . , sd � . ) compared with participants in the value threat/nonviable scapegoat condition (m � . , sd � . ), f( , ) � . , p � . , and those in the control threat/viable scapegoat condition (m � . , sd � . ), f( , ) � . , p � . . also, participants in the value threat condition presented with a nonviable scapegoat reported higher environmental advocacy scores than participants in the control threat/nonviable scapegoat condition, (m � . , sd � . ), f( , ) � . , p � . . for participants in the control threat condition, mean environmental advocacy scores did not significantly differ between the two scape- goat target conditions (f � . , p � . ). indirect effect of threat condition � scapegoat target on environmental advocacy by guilt. using the bootstrapping procedure and corresponding spss macro of preacher and hayes ( ), we regressed environmental advocacy scores onto the interaction of threat condition (coded: value threat � /control threat � ) and scapegoat target (coded: nonviable � /viable � ) with self-reported guilt entered as the proposed mediator and our main effects as covariates. five-thousand bootstrap resamples were performed. the % confidence interval obtained for the indirect effects of the threat condition � scapegoat target inter- action on environmental advocacy scores through guilt did not contain zero [� . , �. ]. these results are consistent with our mediated moderation hypothesis that the decrease in self-reported intentions to participate in environmental advocacy among partic- ipants in the value threat/viable scapegoat condition is partially mediated by their corresponding decrease in feelings of guilt (see figure for a graphical depiction of the mediation model). discussion the presence of a viable scapegoat capable of bearing blame for climate change differentially affected participants’ self- perceptions and behavioral intentions as a function of the causal framing of climate change. when climate change was framed in a value-threatening manner as the result of the ingroup’s harmful actions, exposure to a viable scapegoat reduced participants’ feel- figure . perceived personal control as a function of threat condition and viability of scapegoat target (study ). higher scores indicate greater perceived control over one’s life in general. scale ranges from to . error bars represent standard errors. figure . environmental advocacy as a function threat condition and viability of scapegoat target (study ). higher scores indicate greater willingness to participate in environmental advocacy to help stop climate change. scale ranges from to . error bars represent standard errors. a dual-motive model of scapegoating ings of personal guilt. in contrast, when climate change was framed in a control-threatening manner as the result of unknown causes, exposure to a viable scapegoat bolstered participants’ perceptions of personal control. these findings lend further sup- port to our broad claim that scapegoating behavior can be driven by two distinct motives—to maintain feelings of moral value or perceptions of personal control— depending on the particular type of threat posed by a particular negative outcome. the findings of study also support glick’s ( ) assertion about the importance of a scapegoat target’s perceived viability and contradict earlier accounts of scapegoating, which suggested that groups perceived as weak and vulnerable are preferred for the purposes of displacing blame. we additionally found that value-threatened participants ex- posed to a viable (vs. a nonviable) scapegoat reported lower levels of perceived personal control in addition to lower levels of guilt. this suggests that our measure of general personal control, which we designed to specifically assess the control maintenance path of our model, also partly captured perceived causal responsibility for climate change. this is consistent with the idea that when a person is made to feel responsible for causing climate change, scapegoat- ing involves relinquishing specific feelings of agency connected to climate change in exchange for simultaneously reducing aversive feelings of guilt. this relinquishing of responsibility seems to have influenced broader perceptions of control among the value- threatened participants exposed to a scapegoat. insofar as our control measure may have partly reflected feelings of specific responsibility for climate change, the similar reduction in guilt and control in the value threat condition is consistent with previous research showing a positive association between guilt and personal responsibility for a specific negative outcome (e.g., tangney, ). nevertheless, correlational analyses yielded no significant association between participants’ personal feelings of guilt for climate change and perceptions of personal control across any of the conditions (rs � . , ps � . ). these null results may be due to the fact that whereas guilt was measured with an item that referred specifically to the context of climate change, perceived control was measured with an item designed to capture perceptions of general personal control beyond the climate change context. in support of this explanation, when in study we measured both guilt and control perceptions with specific reference to partici- pants’ responsibility for harmful environmental conditions, we indeed observed a significant positive correlation between guilt and personal control (r � . , p � . ). returning to study ’s results, one unexpected finding was that, within the control threat condition, participants exposed to oil companies reported significantly higher guilt for their personal contribution to climate change compared with participants exposed to the amish. one possible explanation for this effect is that participants who were initially made to feel uncertain about the cause of climate change may have come to associate some of their own behaviors (e.g., driving cars, using electricity) with the harm- ful actions of oil companies when given the chance to attribute blame to such companies. in turn, this may have led them to feel somewhat responsible for causing climate change and thus to experience increased feelings of personal guilt. it is also possible that participants may have felt guilty for failing to act when there was an identified scapegoat target to act against. study also tested the downstream consequences of scapegoat- ing on participants’ reported willingness to engage in reparative behaviors. consistent with our predictions, participants made to feel responsible for harmful climate change, and subsequently exposed to a nonviable scapegoat, exhibited an increase in their willingness to become environmental advocates (consistent with prior research; e.g., ferguson & branscombe, ). if, however, this value-threatening framing was followed by an opportunity to blame a viable scapegoat, participants reported significantly less willingness to engage in environmental advocacy. furthermore, results were consistent with our mediated moderation hypothesis suggesting that these effects are mediated by participants’ feelings of guilt about their own personal contribution to climate change. these results provide initial evidence that scapegoating can un- dermine individuals’ willingness to help stop a harmful outcome for which they are at least partially responsible. general discussion building on and integrating two influential theoretical accounts of scapegoating, we proposed a model that identifies two distinct motives behind scapegoating at the level of the individual: the desire to maintain feelings of personal moral value and the desire to maintain perceptions of personal control over one’s environ- ment. because each motive represents an independent path to scapegoating, the compensatory function served by scapegoating is dependent on whether the precipitating negative outcome primar- ily threatens the individual’s felt moral value or perceived personal control. specifically, although perceived moral culpability for a negative outcome motivates scapegoating to minimize elevated figure . indirect effect of threat condition � scapegoat target interaction on environmental advocacy through feelings of personal guilt (study ). all path coefficients represent standardized regression weights. the direct effect coefficient represents the effect of the interaction on the dependent variable after controlling for the main effects, the effect of the proposed mediator, political orientation, and preexisting feelings of environmental responsibility. total adjusted r for the model � . , f( , ) � . , p � . . total effect: � � �. �. direct effect: � � �. �. � p � . . rothschild, landau, sullivan, and keefer feelings of guilt, the perceived causal indeterminacy of a negative outcome motivates scapegoating to provide an explanation that restores perceived personal control. we provided empirical sup- port for this model in the present studies by testing whether scapegoating behavior is increased by theoretically specified threat inductions, is mediated and moderated by theoretically specified self-perceptions, and produces divergent downstream conse- quences on self-perceptions and behavioral intentions depending on which path is activated. study showed that participants primed to view the hazardous consequences of environmental destruction as the result of their own harmful actions (value threat), and participants primed to view the hazardous consequences of environmental destruction as the result of unknown causes (control threat), were more likely to blame and punish oil companies for the hazardous consequences of environmental destruction. moreover, results were consistent with the mediational hypothesis that scapegoating in response to a value-threatening framing of environmental destruction occurs in- directly through increased feelings of personal guilt, whereas scapegoating in response to a control-threatening framing of en- vironmental destruction occurs indirectly through decreased per- ceptions of personal control. study was designed to build on study by using experimental manipulations that targeted the same proposed mediating variables that were measured in the prior study. study conceptually rep- licated the basic scapegoating effect and further highlighted the independence of the two motivational paths to scapegoating by showing differential moderation effects: scapegoating of interna- tional corporations in response to a value-threatening framing of catastrophic climate change was eliminated by an affirmation of one’s own moral value, but not by an affirmation of personal control, whereas scapegoating in response to a control-threatening framing of climate change was eliminated by an affirmation of personal control, but not by an affirmation of moral value. complementing the findings from studies and , study illustrated the divergent effects of scapegoating along the value- and control-maintenance paths on participants’ self-perceptions and behavioral intentions. although the opportunity to scapegoat following a moral value threat effectively reduced feelings of personal guilt, the opportunity to scapegoat following a control threat effectively bolstered perceptions of personal control. in addition, some evidence was found for the hypothesized relation- ship between feelings of guilt and perceived control over a nega- tive outcome along the value-maintenance path to scapegoating. specifically, the fact that value-threatened participants exhibited lower perceived control after exposure to a viable scapegoat sup- ports our claim that scapegoating in response to a value threat represents an attempt to minimize guilt by transferring one’s own responsibility for a negative outcome to a scapegoat target. how- ever, the absence of a direct measure of perceived responsibility for climate change in this study, the lack of a significant correla- tion between guilt and control, and the unexpected increase in guilt among control-threatened participants exposed to a viable scape- goat suggests that any conclusions about the interplay between control and guilt in scapegoating processes remain tentative. finally, in line with predictions, participants in study who focused on their ingroup’s moral culpability for climate change, and who subsequently encountered a viable scapegoat, reported decreased behavioral intentions to engage in environmental advo- cacy. an indirect effects analysis yielded evidence consistent with our mediational hypothesis that this effect was partially mediated by decreased feelings of personal guilt for climate change. connections with past research by integrating the present studies with previous theory and research, we are able to see how the present findings relate to contentious issues in the literature and how they prompt new lines of research and inquiry. for instance, there has been some dis- agreement about whether scapegoating should be investigated at the level of the individual or the level of the collective. in line with allport ( , / ), the present studies approached scape- goating at the level of the individual’s motives. all three studies provide evidence that the individual’s tendency to blame and punish a (collective) scapegoat target for a negative outcome is motivated by the individual’s own concerns for personal value or control. however, social identity theorists argue that as a group- level phenomenon, scapegoating should be studied exclusively at the level of the group (e.g., billig, ; tajfel, ). consistent with this idea, some theorists have focused on the role scapegoat- ing plays in enhancing or maintaining ingroup cohesion in condi- tions of social instability (cantril, ; silverstein, ). of course, recognizing scapegoating as a group-level response to a shared misfortune does not imply an absence of individual- level motives, nor does a focus on individual-level motives nec- essarily ignore the shared social context in which scapegoating arises. although glick ( ) proposes that scapegoating is a collective process that manifests as a group-level ideology, he recognizes that individual-level motives can still play a role in determining how situational threats may lead individuals to show increased antagonism against a scapegoat target. the present re- search recognizes the role of group-level ideologies in providing “viable” scapegoat targets for collectively experienced negative events, but focuses on how individuals can be drawn to scapegoat ideologies by the perceived threats such events pose to individual- level needs for moral value and personal control. it thus bears conceptual similarity to recent work by kruglanski, chen, dechesne, fisman, and orehek ( ) and landau, rothschild, and sullivan ( ) on the psychology of terrorist and extremist behavior. these perspectives suggest that individuals who are threatened by the potential loss of personal significance may be attracted to extremist group-level ideologies that encourage them to aggress against scapegoated others. the present work is also consistent with work in the field of social identity theory, which suggests that threats to one’s ingroup can, under certain circum- stances, be experienced as threats to the self (hogg, ). all these approaches imply that individual- and group-level perspec- tives on scapegoating offer complementary rather than conflicting accounts of the phenomenon. another contentious issue in the previous literature concerns the exact process or mechanism through which scapegoating occurs. for example, allport ( , / ) claimed that scapegoating operates by way of defensive projection; others (e.g., wills, ) have argued that this is not the case. rather than taking a side in this debate, the present research focused instead on how different motivations can lead to scapegoating behavior. as such, the results from the present studies support allport’s claim that scapegoating can represent a strategy for minimizing guilt feelings and bolster- a dual-motive model of scapegoating ing self-esteem, but these results do not speak to the issue of whether scapegoating necessarily involves projection. it remains possible that scapegoating following a moral value threat operates by way of downward social comparison instead (wills, ). further research is required to determine whether scapegoating for moral value maintenance is best conceptualized via a hydraulic model of projecting one’s guilt onto a scapegoat target, or a nonhydraulic model of amplifying the scapegoat target’s guilt to maintain a relatively guiltless position. our results concerning the control maintenance function of scapegoating are also consistent with previous research on enemy- ship (sullivan et al., ). however, the present studies go beyond the findings of sullivan and colleagues by showing evi- dence consistent with the hypothesis that the tendency to focalize blame on a scapegoat target following a control-threatening fram- ing of a negative outcome is mediated by decreased perceptions of personal control over that particular event, and can be eliminated by alternate means of bolstering personal control. these findings provide more direct evidence for the role played by an individual’s underlying motivation to maintain perceptions of personal control when confronted with otherwise inexplicable negative outcomes. in the past, research on scapegoating has been hampered by theories that relied on either overly ambiguous and undifferenti- ated concepts, such as “frustration,” or a singular motive or pro- cess that was unable to explain scapegoating behavior across different contexts. in contrast, the present dual-motive model of scapegoating overcomes these limitations by presenting two clearly delineated motivational paths. by resisting the urge to reduce one motive to the other or seek a common distal motive, the present model allows us to see how the same behavioral phenom- enon can occur in response to different threats, be eliminated by different intervening variables, and lead to different downstream consequences. although we do not deny the possibility that other individual-level motives may influence the individual’s likelihood of engaging in scapegoating behavior, we believe that the present research provides a strong case that the motives to maintain perceived personal moral value and personal control underlie in- dependent routes to scapegoating. ultimately, the proposed dual- motive model encourages further empirical investigation of the individual-level and group-level motives behind scapegoating, and more generally illustrates the strengths of considering a multi- motive approach to understanding a variety of human behaviors. practical implications beyond its theoretical contributions, the present research offers important insights into a ubiquitous phenomenon shown to have serious real-world consequences. most importantly, the present studies offer potential avenues for reducing individuals’ scape- goating behavior. in particular, study found that participants’ tendency to scapegoat in response to a negative outcome framed as a threat to personal value or personal control threat was eliminated by a corresponding affirmation of personal value or personal control. these findings suggest that providing alternate means of bolstering threatened feelings of moral value or personal control in the face of aversive conditions or negative events may reduce the likelihood of scapegoating as a compensatory threat response, but only when these affirmations are appropriate to the threat. these findings highlight the fact that in order to determine the proper ameliorative action for scapegoating behavior, one must first iden- tify the type of precipitating threat posed by a given negative event. the results of study also have implications for how scape- goating can affect people’s willingness to resolve a negative out- come. in the context of devastating climate change and the haz- ardous effects of environmental destruction, the issue of how to motivate individuals to make different lifestyle choices is a topic of major concern. previous research has suggested that reminding participants’ of their ingroup’s responsibility for the harmful ef- fects of climate change increases feelings of guilt and motivates individuals to report a greater willingness to engage in proenvi- ronmental behaviors (ferguson & branscombe, ). however, in study , this effect was reversed when participants were able to blame a viable scapegoat, which reduced feelings of guilt and in turn their reported willingness to engage in environmental advo- cacy. these results suggest that although highlighting individuals’ moral culpability for environmental destruction can motivate mit- igating behavior, this strategy can backfire when a viable scape- goat is available. thus, the strategy of inducing guilt to motivate reparative behaviors may have the unintended effect of increasing scapegoating and reducing individuals’ willingness to take such corrective actions. references adorno, t. w., frenkel-brunswik, e., levinson, d. j., & sanford, r. n. 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( ). downward comparison principles in social psychol- ogy. psychological bulletin, , – . received july , revision received december , accepted december , � a dual-motive model of scapegoating ep journal - product research & review center 	skip to content 			 			 	 				 			 				 		 			 				 						 				 					ep journal 				 			 						 				 			 		 		 				 			 					 			 				main menu 				 							 		 			 			 		 				reviews 	blog 	about 	contact us 	privacy policy 			 		 			 		 			 		 			 					 				 		 	 		 		 			 best dry bags – keep your items safe from getting wet 						 				leave a comment			 			 / reviews / by 			 				william 			 		 		 		 				 			going on a trip and need to keep your items safe? then having one of the best dry bags will be a great choice. we know how annoying it can be to end up with all your items soaked in water. they end up all smelly and sometimes totally unusable. luckily, you can always prevent … best dry bags – keep your items safe from getting wet read more » 		 	 	 		 	 		 		 			 best waterproof jacket in – stay dry in adverse weather 						 				leave a comment			 			 / reviews / by 			 				william 			 		 		 		 				 			it doesn’t matter where you go – being safe is always crucial to getting a good time. and sometimes that means bringing the right jacket. if you’re going to a trip in a snowy mountain, a place with lots of humidity, or anywhere with cloudy weather – then you’ll want the best waterproof jacket to … best waterproof jacket in – stay dry in adverse weather read more » 		 	 	 		 	 		 		 			 best snorkel masks in – expert reviews! 						 				leave a comment			 			 / reviews / by 			 				william 			 		 		 		 				 			setting up a scuba diving trip demands a lot of time and money. just getting an oxygen tank and all the breathing equipment will take several hundred out of your bank account, and several hours of preparing. luckily, if you want to go scuba diving but don’t have the money – then going snorkeling can … best snorkel masks in – expert reviews! read more » 		 	 	 		 	 		 		 			 best sun hat in – editor choice 						 				leave a comment			 			 / reviews / by 			 				william 			 		 		 		 				 			engrossed in search of the essential companion in this scorching heat for your must-have summer plans? well, you’ve come to the right place! we comply with the fact to get your summer travels going on smoothly; hence, you require the best sun hat. it is undoubtedly the next most crucial thing to have in summer … best sun hat in – editor choice read more » 		 	 	 		 	 		 		 			 best beyblade in the world 						 				leave a comment			 			 / reviews / by 			 				william 			 		 		 		 				 			beyblade comes in different design and style in the market. the main thing with the best beyblade is its strength as well as its agility to move around. while speed, performance, and strength are significant to the device, the spin aesthetics with its customized features matter a lot. finding the best beyblade ever may be … best beyblade in the world read more » 		 	 	 		 	 		 		 			 best indoor outdoor thermometer – top pick’s and review 						 				leave a comment			 			 / reviews / by 			 				william 			 		 		 		 				 			so, you’re looking to buy an indoor outdoor thermometer. that’s a handy item to have around if you need to know the temperature all the time. unless you’re a weather geek, why else would you need to have an indoor outdoor thermometer? ok, so weather geeks are not the only creatures needing the best indoor … best indoor outdoor thermometer – top pick’s and review read more » 		 	 	 		 	 		 		 			 best firewood racks – review & buying guide 						 				leave a comment			 			 / reviews / by 			 				william 			 		 		 		 				 			despite many homeowners using electricity and gas to heat their homes, firewood remains a popular option. many people still prefer to use wood because it brings an authentic element to any home. however, one downside of firewood is that you will end up having a large pile of logs. that’s why you need a rack … best firewood racks – review & buying guide read more » 		 	 	 			 				 	 				 				 					search for: 					 				 				 			 		 		recent posts 			 					 best dry bags – keep your items safe from getting wet 									 	 					 best waterproof jacket in – stay dry in adverse weather 									 	 					 best snorkel masks in – expert reviews! 									 	 					 best sun hat in – editor choice 									 	 					 best beyblade in the world 									 		 	 	 	 		 			 			 	 		 			 									 						epjournal.net is a participant in the amazon services llc associates program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to amazon.com. amazon, the amazon logo, amazonsupply, and the amazonsupply logo are trademarks of amazon.com, inc. or its affiliates.					 				 				 			 		 	 			 		 			 			 			 	 [pdf] green tea catechin egcg inhibits ileal apical sodium bile acid transporter asbt. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /ajpgi. . corpus id: green tea catechin egcg inhibits ileal apical sodium bile acid transporter asbt. @article{annaba greentc, title={green tea catechin egcg inhibits ileal apical sodium bile acid transporter asbt.}, author={fadi annaba and p. kumar and a. dudeja and s. saksena and r. gill and w. alrefai}, journal={american journal of physiology. gastrointestinal and liver physiology}, year={ }, volume={ }, pages={ g - } } 	fadi annaba, p. kumar, + authors w. alrefai 	published 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology green tea catechins exhibit hypocholesterolemic effects probably via their inhibitory effects on intestinal bile acid absorption. ileal apical sodium-dependent bile acid transporter (asbt) is responsible for reabsorption of bile acids. the present studies were, therefore, designed to investigate the modulation of asbt function and membrane expression by green tea catechins in human embryonic kidney hek- cells stably transfected with asbt-v fusion protein and intestinal caco- monolayers… expand view on pubmed europepmc.org save to library create alert cite launch research feed share this paper citationsbackground citations methods citations view all figures and topics from this paper 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure view all figures & tables 	epigallocatechin gallate 	catechin 	plant extracts 	slc a gene 	bile fluid 	sodium 	plasma membrane 	detergents 	bile acids 	 -phosphatidylinositol -kinase 	bile duct carcinoma 	lipid raft 	renal tissue 	lipid metabolism disorders citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency identification of the catechin uptake transporter responsible for intestinal absorption of epigallocatechin gallate in mice 	shunsuke ishii, hidefumi kitazawa, + authors i. tamai 	chemistry, medicine 	scientific reports 	 	 	pdf save alert research feed cholestatic effect of epigallocatechin gallate in rats is mediated via decreased expression of mrp . 	p. hirsova, gabriela karlasova, + authors s. micuda 	chemistry, medicine 	toxicology 	 	 save alert research feed effect of epigallocatechin gallate on drug transport mediated by the proton-coupled folate transporter. 	maika kissei, t. itoh, tomoya narawa 	chemistry, medicine 	drug metabolism and pharmacokinetics 	 	 save alert research feed n-glycosylation is essential for ileal asbt function and protection against proteases. 	saminathan muthusamy, p. malhotra, + authors w. alrefai 	biology, medicine 	american journal of physiology. cell physiology 	 	 	pdf 	view excerpt, cites background save alert research feed luteolin and quercetin affect the cholesterol absorption mediated by epithelial cholesterol transporter niemann–pick c -like in caco- cells and rats 	mari nekohashi, m. ogawa, + authors shoko kobayashi 	biology, medicine 	plos one 	 	 	pdf 	view excerpt, cites background save alert research feed green tea polyphenol egcg alleviates metabolic abnormality and fatty liver by decreasing bile acid and lipid absorption in mice 	j. huang, s. feng, + authors c. yang 	chemistry, medicine 	molecular nutrition & food research 	 	 	 save alert research feed enteropathogenic escherichia coli inhibits ileal sodium-dependent bile acid transporter asbt. 	fadi annaba, zaheer sarwar, + authors w. alrefai 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	pdf 	view excerpts, cites methods save alert research feed the effects of (-)-epigallocatechin- -o-gallate (egcg), a green tea catechin, on blood cholesterol 	n. naumovski 	chemistry 	 	view excerpt, cites background save alert research feed anti-glycolipid disorder effect of epigallocatechin- -gallate on high-fat diet and stz-induced t dm in mice 	zhongkun ren, zhiyong yang, y. lu, r. zhang, h. yang 	chemistry, medicine 	molecular medicine reports 	 	 	pdf save alert research feed apical sodium-dependent bile acid transporter, drug target for bile acid related diseases and delivery target for prodrugs: current and future challenges. 	m. li, q. wang, + authors baohua gu 	chemistry, medicine 	pharmacology & therapeutics 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency modulation of ileal apical na+-dependent bile acid transporter asbt by protein kinase c. 	zaheer sarwar, fadi annaba, a. dwivedi, s. saksena, r. gill, w. alrefai 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	pdf 	view excerpts, references background save alert research feed green tea catechin enhances cholesterol α-hydroxylase gene expression in hepg cells 	mak-soon lee, ju-yeon park, h. freake, in-sook kwun, yangha kim 	medicine, biology 	british journal of nutrition 	 	 	pdf 	view excerpt, references background save alert research feed cholesterol modulates human intestinal sodium-dependent bile acid transporter. 	w. alrefai, zaheer sarwar, s. tyagi, s. saksena, p. dudeja, r. gill 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	pdf 	view excerpts, references methods and background save alert research feed modulation of ileal bile acid transporter (asbt) activity by depletion of plasma membrane cholesterol: association with lipid rafts. 	fadi annaba, zaheer sarwar, + authors w. alrefai 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	pdf 	view excerpts, references methods and background save alert research feed modulation of cholesterol metabolism by the green tea polyphenol (-)-epigallocatechin gallate in cultured human liver (hepg ) cells. 	c. bursill, p. roach 	chemistry, medicine 	journal of agricultural and food chemistry 	 	 	view excerpt, references results save alert research feed green tea as inhibitor of the intestinal absorption of lipids: potential mechanism for its lipid-lowering effect. 	s. koo, s. noh 	chemistry, medicine 	the journal of nutritional biochemistry 	 	 	highly influential 	pdf 	view excerpts, references background save alert research feed bioactive dietary polyphenolic compounds reduce nonheme iron transport across human intestinal cell monolayers. 	e. kim, soo-kyung ham, m. shigenaga, okhee han 	chemistry, medicine 	the journal of nutrition 	 	 	pdf 	view excerpt, references results save alert research feed effects of green tea catechins on membrane fluidity 	h. tsuchiya 	chemistry, medicine 	pharmacology 	 	 	view excerpt, references background save alert research feed hypocholesterolemic effects of chinese tea. 	t. t. yang, m. w. koo 	chemistry, medicine 	pharmacological research 	 	 	pdf 	view excerpts, references background and results save alert research feed inhibition of ileal bile acid transport and reduced atherosclerosis in apoe−/− mice by sc- published, jlr papers in press, june , . doi . /jlr.m -jlr 	b. g. bhat, s. rapp, + authors b. keller 	biology, medicine 	journal of lipid research 	 	 	pdf 	view excerpt, references methods save alert research feed ... ... related papers 	abstract 	figures and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue brief genetics report polymorphisms in the transcription factor -like (tcf l ) gene are associated with type diabetes in the amish replication and evidence for a role in both insulin secretion and insulin resistance coleen m. damcott, toni i. pollin, laurie j. reinhart, sandra h. ott, haiqing shen, kristi d. silver, braxton d. mitchell, and alan r. shuldiner , transcription factor -like (tcf l ) regulates genes involved in cell proliferation and differentiation. the tcf l gene is located on chromosome q in a region of replicated linkage to type diabetes. recently, a micro- satellite marker in intron (dg s ) and five corre- lated single nucleotide polymorphisms (snps) were identified in icelandic individuals that showed strong asso- ciation with type diabetes, which was replicated in danish and european-american cohorts. we genotyped four of the snps (rs , rs , rs , and rs ) in amish subjects with type diabetes (n � ), impaired glucose tolerance (igt; n � ), and normal glucose tolerance (ngt; n � ). we compared genotype frequen- cies in subjects with type diabetes with those with ngt and found marginal association for rs (p � . ; odds ratio [or] . ); comparison between ngt control subjects and the combined type diabetes/igt case group showed strong association with rs and rs (p � . – . ; or . – . ) and marginal association with rs and rs (p � . and p � . , respectively). in an expanded set of amish subjects without diabetes, we found no association with insulin and glucose levels during a -h oral glucose tolerance test. we also genotyped these snps in nondiabetic, non-amish sub- jects (n � ), in whom intravenous glucose tolerance tests were performed, and found an association between rs and rs and insulin sensitivity (p � . and p � . , respectively) and disposition index (p � . and p � . , respectively). these data provide replicating evidence that variants in tcf l increase the risk for type diabetes and novel evidence that the vari- ants likely influence both insulin secretion and insulin sensitivity. diabetes : – , t he transcription factor -like (tcf l ) gene is a member of the t-cell factor (tcf)/lymphoid- enhancing factor family of high mobility group box-containing transcription factors involved in the wnt signaling pathway. this pathway is a key compo- nent to the regulation of cell proliferation and differentia- tion. wnt signaling is initiated by the binding of wnts to their receptor complex, which results in the release of �-catenin from its degradation complex and translocation to the nucleus. in the nucleus, �-catenin heterodimerizes with the tcf/lymphoid-enhancing factor family of tran- scription factors to regulate the expression of wnt target genes ( , ). the tcf l gene spans . kb on chromosome q , a region of replicated linkage to type diabetes in mexican-american ( ) and icelandic ( ) cohorts. recently, grant et al. ( ) identified a microsatellite marker (dg s ) in intron of tcf l that showed strong association with type diabetes in icelandic, danish, and european-american populations. the authors then geno- typed the five single nucleotide polymorphisms (snps) from the ceph utah (ceu) hapmap samples with the strongest correlation to dg s (rs , rs , rs , rs , and rs ) and showed asso- ciation between all five snps and type diabetes in all three cohorts. although no evidence for linkage to type diabetes was detected on chromosome q in our ge- nome-wide scan in the amish (average marker density, . cm; logarithm of odds, . between markers d s and d s ) ( ), we genotyped four of the five snps in subjects enrolled in the amish family diabetes study (afds). the rs snp was not genotyped; however, according to the ceu hapmap ( ), rs is in high linkage disequilibrium (ld) with rs (r � . ). all snps conformed to hardy-weinberg expectations. fig- ure shows the tcf l gene structure, snps genotyped in the amish, and pairwise ld (r ) among the snps. from the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland; and the geriatric re- search and education clinical center, veterans administration medical cen- ter, baltimore, maryland. address correspondence and reprint requests to coleen m. damcott, phd, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood st., rm. , baltimore, md . e-mail: cdamcott@medicine.umaryland.edu. received for publication march and accepted in revised form june . additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. afds, amish family diabetes study; airg, acute insulin response to glucose; di, disposition index; igt, impaired glucose tolerance; ivgtt, intravenous glucose tolerance test; ld, linkage disequilibrium; ngt, normal glucose tolerance; ogtt, oral glucose tolerance test; snp, single nucleotide polymorphism; tcf, t-cell factor. doi: . /db - © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , september table summarizes the allele frequencies in subjects with type diabetes (n � ), impaired glucose tolerance (igt; n � ), and normal glucose tolerance (ngt; n � ) and the results of genotypic association analysis for each snp. for this case-control analysis, subjects were considered to have ngt if they were aged � years at the time of testing to minimize misclassification given the age-dependent penetrance of type diabetes. the compar- ison of genotype frequencies in subjects with type diabetes to those with ngt showed marginal significance for rs (p � . ; odds ratio [or] . ). to increase power, we combined the subjects with type diabetes and igt into one case group. in previous studies, we have shown that the prevalence of type diabetes is approxi- mately one-half that of the national health and nutrition examination survey caucasian population, despite similar bmi, while the prevalence of igt is the same or greater than that of the national health and nutrition examina- tion survey caucasian population ( ). it is likely that the high level of physical activity characteristic of the amish lifestyle protects genetically vulnerable individuals from progressing from igt to type diabetes. indeed, our previously reported genome-wide linkage analysis has revealed some of our strongest linkages to the combined trait igt/type diabetes ( ), and prospective studies of others ( ) have demonstrated the potent effect of lifestyle interventions on progression from igt to type diabetes. using this combined case group, we found that the geno- type frequencies of rs and rs differed sig- nificantly between type diabetic/igt subjects and ngt control subjects (p � . and p � . , respectively), with the minor allele being the at-risk allele for type diabetes/igt (or . and . , respectively). these two snps are in high ld (r � . ) (fig. ). the rs and rs snps showed marginal differences in geno- type frequency between the type diabetes/igt and ngt groups (p � . , or . and p � . , or � . , respectively). our results are similar to the findings by grant et al. ( ) in that the rs snp showed the strongest association with type diabetes in two (icelan- fig. . gene structure, location of polymorphic sites, and pairwise ld among snps in tcf l . the upper portion of the figure shows the gene structure and location of the polymorphisms genotyped in the amish and caucasian ivgtt subjects. two of the snps are located in intron and the other two snps are located in intron of the . -kb gene. the lower portion of the figure shows a schematic of the pairwise ld, calculated as r , among the snps in the amish. the dotted lines connect each snp name and position with the corresponding cell in the ld matrix. increasing level of ld is shown by darker grayscale. table allele frequencies and results of association analysis in amish subjects with type diabetes, igt, and ngt* for snps in tcf l minor allele frequency type diabetes vs. ngt† igt vs. ngt† type diabetes � igt vs. ngt† snp name chromosome location major/ minor allele diabetes (n � ) igt (n � ) ngt (n � ) p or p or p or rs t/c . . . . ‡ . . . . ‡ . rs c/t . . . . . . ‡ . . ‡ . rs c/g . . . . . . ‡ . . . rs g/t . . . . . . . . ‡ . *ngt control group restricted to subjects aged � years to avoid misclassification given the age-dependent penetrance of type diabetes. †p values are based on genotyped frequencies, and ors reflect the odds of disease associated with having two copies of the minor allele versus the odds of disease associated with having two copies of the major allele. the analysis models were adjusted for age, sex, and pedigree structure. reported p values were derived using the additive model and were not adjusted for multiple comparisons. ‡p � . . c.m. damcott and associates diabetes, vol. , september dic and dutch) of their three study populations and in the combined analysis of all three cohorts. in addition, the same alleles for all four snps that were associated with increased type diabetes risk in the cohorts studied by grant et al. ( ) were also associated with increased risk in the amish. we used haploview . ( ) to estimate four snp haplotype frequencies and perform haplotype analysis. none of the haplotypes provided stronger evidence for association with type diabetes/igt than rs (data not shown). in addition, we genotyped the snps in an expanded set of nondiabetic afds subjects (including subjects with ngt aged � years) and tested for association with insulin and glucose levels during a -h oral glucose tolerance test (ogtt), including ogtt-de- rived homeostatis model assessment of insulin resistance and insulin secretion measurements, but we found no association with any of these traits (table ). to shed light on a potential mechanism by which snps in tcf l contribute to type diabetes susceptibility, we used regression analysis to examine the effects of tcf l snp genotype on acute insulin response to glucose (airg), insulin sensitivity index (si), and disposition index (di) in a small sample of nondiabetic, non-amish caucasian sub- jects in whom intravenous glucose tolerance tests (ivgtts) were performed (n � ) (table ). subject characteristics are shown in the online appendix table (available at http://diabetes.diabetesjournals.org). after adjustment for age and sex, we found that rs and rs were significantly associated with si (p � . and p � . , respectively) and di (p � . and p � . , respectively), and the putative type diabetes risk alleles, identified in the amish case-control study and the study by grant et al. ( ), were associated with lower si and di. snp rs accounted for � % of the variance in age- and sex-adjusted si, although the effect size estimate has a wide confidence band attached to it because of the relatively small sample size. the power to detect an effect this large in our data was %. although the magnitude of the effects of rs and rs genotype on si (p � . and p � . , respectively) and di (p � . and p � . , respectively) were reduced after adjustment for bmi, the association remained significant with the exception of the marginal effect of rs on di. in addition, the risk allele of rs was associated with lower airg after adjustment for bmi (p � . ). the rs snp was also significantly associated with si (p � . ), but after correction for bmi, the effect was only marginal (p � . ). to reduce our reliance on distributional assump- tions, we repeated analysis of si, airg, and di using a table mean fasting glucose, fasting insulin, glucose and insulin area under the ogtt curve, insulin secretion index, and homeostatis model assessment of insulin resistance by genotype in nondiabetic amish subjects (n � ) for each tcf l snp mean trait value � se* p† rs tt ct cc n fasting glucose (mmol/l) . � . . � . . � . . ln(fasting insulin) (mmol/l) . � . . � . . � . . gauc . � . . � . . � . . iauc . � . . � . . � . . �is . � . . � . . � . . homa-ir . � . . � . . � . . rs tt ct cc n fasting glucose (mmol/l) . � . . � . . � . . ln(fasting insulin) (mmol/l) . � . . � . . � . . gauc . � . . � . . � . . iauc . � . . � . . � . . �is . � . . � . . � . . homa-ir . � . . � . . � . . rs gg cg cc n fasting glucose (mmol/l) . � . . � . . � . . ln(fasting insulin) (mmol/l) . � . . � . . � . . gauc . � . . � . . � . . iauc . � . . � . . � . . �is . � . . � . . � . . homa-ir . � . . � . . � . . rs gg gt tt n fasting glucose (mmol/l) . � . . � . . � . . ln(fasting insulin) (mmol/l) . � . . � . . � . . gauc . � . . � . . � . . iauc . � . . � . . � . . �is . � . . � . . � . . homa-ir . � . . � . . � . . *adjusted for age, sex, and family structure. †p values were derived using the additive model. gauc, glucose area under the ogtt curve; homa-ir, homeostatis model assessment of insulin resistance; iauc, insulin area under the ogtt curve; is, insulin secretion. tcf l polymorphisms and type diabetes diabetes, vol. , september nonparametric approach (spearman correlation) in which we assessed the additive effect of genotype on the ranked order of si, airg, and di (while adjusting for age and sex). in these analyses, each snp remained significantly associ- ated with si and di (table ). consistent with our results in the amish, the rs snp shows the strongest effects on insulin sensitivity in the non-amish ivgtt subjects. the t allele, which was associated with increased risk for type diabetes in the amish, is associated with lower si and impaired airg in the non-amish ivgtt subjects, suggesting both a reduc- tion in insulin sensitivity and a defect in insulin secretion from the �-cell. lower di in individuals with the t allele demonstrates a failure of the �-cells to fully compensate for the degree of insulin resistance, providing additional evidence for defects in both hallmarks of type diabetes. although the mechanism by which tcf l influences susceptibility to type diabetes is unclear, over target genes have been identified for the �-catenin/tcf complex ( ), including ccaat/enhancer-binding protein-� (cebpa) and peroxisome proliferator–activated recep- tor-	 (pparg), two important regulators of adipogenesis ( , ), and cell/tissue type-specific regulation of the pre- proglucagon (gcg) gene, a gene involved in glucose homeostasis and satiety ( ). grant et al. ( ) put forth the hypothesis that variants in tcf l influence type dia- betes susceptibility through altered transcriptional regula- tion of the insulinotropic hormone glucagon-like peptide- (glp ), a peptide encoded by gcg and expressed in the brain and gut. this hypothesis was driven by observations of intestine-specific roles for tcf l , including a role in the development of colon cancer ( – ) and the obser- vation that tcf l -null mice, which die shortly after birth, lack epithelial stem-cell compartments in the small intestine ( ). an alternative hypothesis is that variants in tcf l disrupt adipogenesis and/or adipocyte function by altering transcriptional regulation of cebpa and pparg, leading to deposition of triglycerides in peripheral tissues (i.e., liver and muscle) and resulting in insulin resistance. our results in the non-amish ivgtt subjects are consis- tent with one or both hypotheses in that we show defects in both insulin secretion and insulin sensitivity. in summary, we found evidence for association between snps in tcf l and the type diabetes/igt trait in the afds. these results replicate the report of association by grant et al. ( ) between these snps and type diabetes in three caucasian cohorts. this replication of the same snps with similar magnitudes of ors for the same risk alleles are characteristic of a true susceptibility gene and add tcf l to a growing list of bone fide type diabetes susceptibility genes that includes p a pparg, e k kcnj , and snps in capn . in addition, we found significant association with si, airg, and di in nondiabetic, non-amish caucasian subjects, suggesting for the first time a role for tcf l in regulating genes involved in insulin sensitivity and glucose-stimulated insulin release. studies in other populations, as well as functional analy- ses, will be required to further elucidate the role of variation in tcf l in the pathogenesis of type diabetes. research design and methods the afds was initiated in with the goal of identifying susceptibility genes for type diabetes in the old order amish in lancaster county, pennsylvania. details of the afds design, recruitment, phenotyping, and pedigree structure have been described previously ( ). briefly, probands with previously diagnosed type diabetes (onset age – years) and all willing first- and second-degree relatives of probands and spouses aged years were recruited. phenotypic characterization of participants included medical and family history, anthropometry, and a -h, -g ogtt with insulin levels. diabetes was defined by fasting plasma glucose level (� mmol/l), -h ogtt plasma glucose level (� . mmol/l), random plasma glucose level (� . mmol/l), the use of insulin or oral glucose–lowering agents, or a diagnosis of diabetes (with onset age � years) documented by a physician. igt was diagnosed based on ogtt plasma glucose levels ( -h ogtt plasma glucose level between . and . mmol/l). ngt was defined based on fasting plasma table mean si, airg, and di by genotype in non-amish caucasians for each tcf l snp regression model p value† regression model p value (bmi adjusted)† spearman rank correlation p value†mean trait value � se* rs tt ct cc n si ( � �min 
 pmol/l�� ) . � . . � . . � . . ‡ . ‡ . ‡ airg (pmol/l) . � . . � . . � . . . . di (si 
 airg) , � , � � . ‡ . . ‡ rs cc ct tt n si ( � �min 
 pmol/l�� ) . � . . � . . � . . ‡ . ‡ . ‡ airg (pmol/l) . � . . � . . � . . . ‡ . di (si 
 airg) , � , � � . ‡ . ‡ . ‡ rs gg cg cc n si ( � �min 
 pmol/l�� ) . � . . � . . � . . . . airg (pmol/l) . � . . � . . � . . . . di (si 
 airg) , � , � , � . . . rs gg gt tt n si ( � �min 
 pmol/l�� ) . � . . � . . � . . ‡ . . ‡ airg (pmol/l) . � . . � . . � . . . . di (si 
 airg) , � , � � . . . *adjusted for age and sex. †analyses were adjusted for age and sex, and p values were derived using the additive model. ‡p � . . c.m. damcott and associates diabetes, vol. , september glucose level (� . mmol/l) and -h ogtt plasma glucose level (� . mmol/l). for case-control association analysis, we genotyped the tcf l snps in subjects with type diabetes (n � ), igt (n � ), and ngt (n � ). ngt subjects included in this analysis were required to be aged � years. for association analysis of quantitative traits, a larger set of nondiabetic subjects (including the ngt and igt subjects described above and an additional ngt subjects aged � years) was studied. traits that were examined in this group included fasting glucose, fasting insulin, insulin and glucose area under the ogtt curve, insulin secretion index ([insulin at min � fasting insulin]/[glucose at min � fasting glucose]), and homeostatis model assessment of insulin resistance ([fasting insulin 
 fasting glucose]/ . ). we also genotyped the four tcf l snps in a set of nondiabetic, non-amish caucasian individuals in whom ivgtts were performed (n � ). subjects between the ages of and were recruited from the university of maryland baltimore campus and local workplace sites and underwent a standard -g, -h ogtt. if the ogtt demonstrated ngt, the subject underwent an ivgtt receiving at t � , glucose . g/kg i.v. and at t � min, insulin . units/kg i.v. airg and si were calculated using minimal model analysis. di was calculated as the product of si and airg. informed consent was obtained from all study subjects, and the study protocols were approved by the institutional review board at the university of maryland, baltimore. genotyping. all snps were genotyped using taqman snp genotyping assays (applied biosystems) according to the manufacturer’s protocol. all genotyping included % duplicate samples to determine mistyping rates, which were – %. statistical analysis. before analysis, genotypes were checked for mendelian consistency using the pedcheck software program ( ) in the extended amish pedigree. mendelian errors ( . % of genotypes) were resolved or removed before analysis. allele frequencies were calculated for each snp by gene counting, and observed genotypes were tested for fit to the expectations of hardy-weinberg using the � test. pairwise ld was computed between the snps, and haplotypes were inferred for each individual using haploview . ( ). in the amish, we evaluated the association between snp genotype and disease status (type diabetes versus ngt, igt versus ngt, and type diabetes/igt versus ngt) using a variance component approach in order to account for the relatedness among study subjects. using a liability threshold model, we modeled the probability that the subject was a case or control, as a function of the individual’s age, sex, and genotype, conditional on the correlations in phenotype among relative pairs. statistical testing was per- formed using the likelihood ratio test in which we compared the likelihood of the data under a model in which the genotype effect was estimated against the likelihood of a nested model, wherein the genotype effect was constrained to be zero. parameter estimates (i.e., �-coefficients) were obtained by maximum likelihood and ors by taking the inverse log of the �-coefficient. the or for the additive model was scaled to reflect the odds that a case was homozygous for the minor allele versus the odds that the case was homozygous for the major allele. variance components analysis was performed using solar ( ). quantitative trait means were estimated according to tcf l genotypes in the nondiabetic afds subjects (n � ). to account for the relatedness among family members, the measured genotype approach was used ( ) in which we estimated the likelihood of specific genetic models given the pedigree structure. parameter estimates were obtained by maximum likeli- hood methods, and the significance of association was tested by likelihood ratio tests. within each model, we simultaneously estimated the effects of age and sex. quantitative trait analyses were conducted using solar ( ). ivgtt quantitative traits were analyzed using multiple regression adjusted for age, sex, and with and without bmi as implemented in sas version (cary, nc). extreme outliers, defined as values greater than three sds above or below the mean, were set to missing (one si value and one airg value). values of di corresponding to a missing airg or si value were also set to missing (two di values). the snps were modeled as an additive effect, that is, quantitative predictor variables reflecting the number of “risk alleles” ( , , or ) as defined previously ( ). analyses of the ivgtt traits were repeated using the nonpara- metric spearman rank correlation test, in which we assessed the additive effect of genotype on the ranked order of si, airg, and di adjusted for age and sex. acknowledgments this work was supported by research grants r -dk (to a.s.), u -dk (to a.s.), k dk (to k.s.), and r dk (to k.s.); the university of maryland general clinical research center (gcrc) grant m rr ; the gcrcs program; the national center for re- search resources; the national institutes of health; and the baltimore veterans administration geriatric research and education clinical center. we gratefully acknowledge our amish liaisons and field- workers and the extraordinary cooperation and support of the amish community, without whom these studies would not be possible. in addition, we thank the volunteers who underwent ivgtts at the university of maryland, baltimore. references . nelson wj, nusse r: convergence of wnt, beta-catenin, and cadherin pathways (review). science : – , . prunier c, hocevar ba, howe ph: wnt signaling: physiology and pathology (review). growth factors : – , . duggirala r, blangero j, almasy l, dyer td, williams kl, leach rj, o’connell p, stern mp: linkage of type diabetes mellitus and of age at onset to a genetic location on chromosome q in mexican americans. am j hum genet : – , . reynisdottir i, thorleifsson g, benediktsson r, sigurdsson g, emilsson v, einarsdottir as, hjorleifsdottir ee, orlygsdottir gt, bjornsdottir gt, saemundsdottir j, halldorsson s, hrafnkelsdottir s, sigurjonsdottir sb, steinsdottir s, martin m, kochan jp, rhees bk, grant sf, frigge ml, kong a, gudnason v, stefansson k, gulcher jr: localization of a susceptibility gene 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september peer-reviewed oncology & cancer research journal | oncotarget 	 			 	 	 	 		 		 	 	online issn: - 	 		 			 			 			 			 			 			search: 			 			 		 	 oncotarget 	 									 					 	 	journal content 	 		home 	editorial board 	submission 	current issue 	advance online publications 	archive 	editorial policies 	publication ethics statements 	videos with outstanding authors 	oncotarget in the news 	search 	contact information 	special collections 	 	 	 		 			 				oncotarget (a primarily oncology-focused, peer-reviewed, open access, biweekly journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. 			 			 				its scope is unique. the term "oncotarget" encompasses all molecules, pathways, cellular functions, cell types, and even tissues that can be viewed as targets relevant to cancer as well as 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principal biologically-relevant down-regulated dlc family member in several cancers. 					 						wang d, qian x, rajaram m, durkin me, lowy dr. 					 					 					 						oncotarget. jul ; ( ): - . 					 					 					dynamic changes during the treatment of pancreatic cancer. 					 							wolff ra et al. 					 					 					 						oncotarget. ; ( ): - . 					 				 			 		 	 	 editors-in-chief 			 			 				 			andrei v. gudkov 				roswell park comprehensive cancer center, buffalo, ny 			 			 					andrei v. gudkov, phd, dsci, professor and garman family chair in cell stress biology, senior vice president for basic research of roswell park comprehensive cancer center, buffalo, ny 				 				 his academic degrees in experimental oncology and molecular biology were received in former ussr from national cancer center and moscow state university. he is co-founder and chief scientific officer of cleveland biolabs, inc and tartis, inc. his area of general research interest includes drug discovery, gene discovery, molecular targets for cancer treatment. 				 			 				 				 					mikhail v. blagosklonny 				 				roswell park comprehensive cancer center, buffalo, ny 			 			 					mikhail v. blagosklonny, m.d., ph.d., professor, roswell park comprehensive cancer center, buffalo, ny 				 				dr. blagosklonny is the author of over articles in peer-reviewed journals. he has served as associate editor of  					cancer res,  					cell death differ,  					cancer biol ther,  					autophagy,  					int j cancer,  					am j pathology,  					plos one and as editor-in-chief of  					cell cycle. his research interests range from molecular and cellular biology to clinical investigations. recently, he extended the study of signal transduction pathways from cancer to aging, revealing potential targets for slowing down aging and age-related diseases. 				 			 		 		editorial board 			 			 				cory abate-shen 				columbia university, new york, ny 			 			 					cory abate-shen, phd, professor, director of research, department of pathology, associate director, herbert irving comprehensive cancer center, columbia university, ny 				 			 		 			 			 				frederick alt 				harvard medical school, boston, ma 			 			 					frederick w. alt, ph.d., member of the national academy of sciences, professor, harvard medical school 				 				dr. alt is also investigator, howard hughes medical inst., charles a. janeway professor of pediatrics, hms, scientific director, cbri institute for biomedical research.fred alt received a phd from the department of biological sciences at stanford university. 			he is a howard hughes medical institute investigator, a member of the national academy of sciences and the american academy of sciences. 			he is the recipient of he is the recipient of the excellence in mentoring award from the american association of immunologists and the clowes memorial award from the american association of cancer research. 			editorial boards: 					mol. and cell. biology; 					advances in immunology; 					international immunology; 					j. exp. med.; 					current opinion in immunology; 					immunity (founding co-editor; -present); 					molecular medicine (contributing editor; -present); 					faculty of (co-head, immunology). honors and awards: fox award, stanford univ. ( ); hirschl award ( ); searle scholar; ( ) mallinckrodt scholar; ( ); nih merit award ( ); national academy of sciences ( ); american academy of microbiology ( ); american academy of arts and sciences ( ); associate (foreign) member, european molecular biology organization ( ); excellence in mentoring award,association of immunologists ( ); american association of cancer research b.h.a. clowes award ( ); rabi shai shacknai memorial prize in immunology & cancer research ( ); leukemia & lymphoma society de villiers international achievement award ( ), pasarow foundation prize in cancer research ( ); irvington institute scientific leadership in immunology award ( ); establishment of frederick w. alt award for new discoveries in immunology by the irvington institute ( ); national cancer institute alfred knudson award in cancer genetics( ). 				 			 		 			 			 				dario altieri 				wistar institute cancer center, philadelphia, pa 			 			 					dario altieri, md, robert and penny fox distinguished professor, director, the wistar institute cancer center, executive vice president, chief scientific officer, the wistar institute, philadelphia, pa. 				 			 		 			 			 				dafna bar-sagi 				nyu school of medicine, new york, ny 			 			 					dafna bar-sagi, ph.d., professor and chair, department of biochemistry new york university school of medicine, first avenue new york, ny 				 				 					after earning her undergraduate and master's degree in biochemistry from bar-ilan university in israel, dr. bar-sagi completed a phd in cell biology at the state university of new york, stony brook (suny). she received postdoctoral training and eventually served as senior staff investigator at cold spring harbor laboratory in long island. in , she joined the faculty of the department of molecular genetics and microbiology at suny and served as department chair from - . she relocated to new york university medical center in , where she is currently professor and chair of the department of biochemistry. dr. bar-sagi has published over peer-reviewed articles in leading scientific journals. in , she was appointed chair of the scientific advisory board of the pancreatic cancer action network. 				 			 		 			 			 				jiri bartek 				institute of cancer biology, copenhagen, denmark 			 			 					jiri bartek, md., ph.d., professor, head, department of cell cycle and cancer danish cancer society, deputy director, centre for genotoxic stress research cancer institute of cancer biology, copenhagen 				 				 					he received his m.d. degree from palacky university in olomouc, and his ph.d. degree in cell biology from the institute of molecular genetics in prague, czech republic. his main research interests include the molecular mechanisms of mammalian cell-cycle control and responses to dna damage, and the cancer-predisposing aberrations of these regulatory pathways. 				 				 					jiri bartek has a total of more than publications in peer reviewed journals (about - in nature, science and cell), with over . citations. he is currently member of the editorial boards of high-medium impact biomedical journals and has won a number of awards including: ( , ) czech medical association award, ( ) elected embo member, ( ) a. benzon prize, ( ) novo nordisk prize, ( ) g.j. mendel medal, ( ) danish cancer society prize 				 			 		 			 			 				stephen baylin 				johns hopkins university, baltimore, md 			 			 					stephen b. baylin, m.d., professor, deputy director, sidney kimmel comprehensive cancer center at johns hopkins university, virginia and dk ludwig professor of oncology, baltimore, md, usa 				 				dr. stephen b. baylin is professor of oncology and medicine, director of the cancer biology program at the oncology center, and the virginia and d.k. ludwig professor of cancer research at the johns hopkins university school of medicine, and the associate director for research at the sidney kimmel comprehensive cancer center at johns hopkins. 				dr. baylin has been a member of committees of the american cancer society and the national institutes of health, and his multiple honors include a research career development award from the national institutes of health. for the last years, dr. baylin has studied the role of epigenetic gene silencing in the initiation and progression of human cancer. 			 		 			 			 				joseph r. bertino 				cancer institute of new jersey, new brunswick, nj 			 			 					joseph r. bertino, m.d., professor of medicine and pharmacology, the cancer institute of new jersey, umdnj-robert wood johnson medical school 				 				he has been an american cancer society professor since . from to , dr. bertino served as director of the yale comprehensive cancer center, including director of the center and associate director for clinical research. dr. bertino has been internationally recognized for his role in finding curative treatments for leukemia and lymphoma. dr.bertino has received several awards and honors, including the rosenthal award from the american association of clinical research, the karnofsky award from the american society for clinical oncology, and the american cancer society medal of honor. he was the founding editor of the journal of clinical oncology. currently, he is the associate editor for cancer research and clinical cancer research and also the editor of the encyclopedia of cancer. dr. bertino served as president for the american society of clinical oncology in , and president of the american association for cancer research in - . dr. bertino is the author and co-author of more than scientific publications. 			 		 			 			 				mina j. bissell 				 lawrence berkeley national laboratory, berkeley, ca 			 			 					mina j. bissell, phd, member of the national academy of sciences, distinguished scientist, life sciences division, lawrence berkeley national laboratory, berkley, ca 				 				mina bissell has been recognized for her lifetime contributions to the fields of breast cancer research, the enhanced role of extracellular matrix (ecm) and the nucleus environment to gene expression in normal and malignant tissues. these works have ushered and have changed some central paradigms that have strengthened the importance of context in the development of cancer. 			 		 			 			 				bruce blazar 				university of minnesota, minneapolis, mn 			 			 					bruce r. blazar, m.d., professor, university of minnesota, minneapolis, mn 				 				bruce r. blazar is a regents professor of pediatrics, and chief of the pediatric blood and marrow transplantation program. dr. blazar is the director of the university of minnesota's clinical and translational science institute. 			 		 			 			 				kenneth cowan 				 university of nebraska medical center, omaha, ne 			 			 					kenneth cowan, m.d., ph.d., professor, university of nebraska medical center, omaha, ne, director, eppley institute, director, unmc eppley cancer center. 				 				dr. cowan has authored more than papers for scientific journals and has been appointed by president bush to a six-year term on the national cancer advisory board to help shape cancer policy. 			 		 			 			 				carlo m. croce 				the ohio state university, columbus, oh 			 			 					carlo m. croce, md, professor, member of the national academy of sciences, director of comprehensive cancer center, chair, molecular virology, immunology & medical genetics, the ohio state university, columbus, oh 				 			 		 			 			 				chi van dang 				 johns hopkins university, baltimore, md 			 			 					chi van dang, m.d., ph.d. professor, vice dean for research, the johns hopkins university school of medicine, baltimore, md 				 				dr. dang has written more than scientific papers. he is senior editor of cancer research and serves on the editorial boards of eight other scientific publications, including the journal of molecular medicine, current cancer therapy reviews, drug discovery today: disease mechanisms, journal of clinical investigations, neoplasia, clinical and translational science and the vietnamese medical journal. elected to the national academy of sciences' institute of medicine in , dr. dang has received the national institutes of health/national cancer institute merit award and numerous other accolades. 			 		 			 			 				ronald a. depinho 				the university of texas md anderson cancer center 			 			 					depinho, md, member of the national academy of sciences, past president, the university of texas md anderson cancer center, houston, tx, and professor, department of cancer biology, the university of texas md anderson cancer center, houston, tx. 				 				 			his recent awards includes albert szent-gyrgyi prize for progress in cancer research, ; helsinki medal, ; albert einstein college of medicine distinguished alumnus award, ; american cancer society edith a. pistorino research professorship, ; member, institute of medicine of the national academies, ; aacr-g.h.a. clowes memorial award, 			 			 		 			 			 				brian j. druker 				oregon health science univ., portland, or 			 			 					 brian j. druker, md, phd, member of the national academy of sciences, professor, oregon health & science university, director, ohsu knight cancer institute, oregon health & science university 				 				 			dr. druker is the director of ohsu knight cancer institute, jeld-wen chair of leukemia research, and professor of medicine. in he won the lasker clinical award and the meyenburg cancer research prize for his influential work in the development of sti , commonly known as gleevec, for the treatment of chronic myeloid leukemia. dr. druker is an investigator of howard hughes medical institute (hhmi), and was elected to the institute of medicine of national academies in , the american association of physician in , and the national academy of sciences in . 			 			 		 			 			 				wafik s. el-deiry 				fox chase cancer center, philadelphia, pa 			 			 					 						wafik s. el-deiry, md, phd, facp 						american cancer society research professor 						professor of medical oncology 						deputy cancer center director for translational research 						william wikoff smith endowed chair in cancer research 						co-leader, molecular therapeutics program 						fox chase cancer center, philadelphia, pa 					 				 			 		 			 			 				michael a. dyer 				 st. jude children's research hospital, memphis, tn 			 			 					michael a. dyer, phd, professor/member, st. jude faculty co-leader, developmental therapeutics for solid malignancies program 				 			 		 			 			 				andrew feinberg 				 johns hopkins university school of medicine, baltimore, md 			 			 					andrew feinberg, md, mph, king fahd professor of molecular medicine department of medicine, department of oncology and department of molecular biology & genetics, johns hopkins university school of medicine 				 			 		 			 			 				dean w. felsher 				 stanford university school of medicine, stanford, ca 			 			 					dean w. felsher, md, phd, stanford university school of medicine, stanford, ca 				 			 		 			 			 				antonio giordano 				 temple university, philadelphia, pa 			 			 					antonio giordano, md, ph.d, professor and director, sbarro institute for cancer research and molecular medicine, temple university, philadelphia, pa 				 				he has published over papers on his work in the fields of cell cycle, gene therapy and the genetics of cancer. giordano has been named a knight of the republic of italy for outstanding achievements in cancer research. in september of , giordano received the philip mazzei "the bridge" award from the american university of rome for his scientific and economic contributions to the united states and italy. in the past three years, dr. giordano's work has been profiled in philadelphia magazine, philadelphia business journal, and science. 			 		 			 			 				candace s johnson 				 roswell park comprehensive cancer center, buffalo, ny 			 			 					president & ceo of roswell park comprehensive cancer center, the wallace family chair in translational research, and professor of oncology. 				 			 		 			 			 				michael karin 				 university of california, los angeles, ca 			 			 					michael karin, ph.d, member of the national academy of sciences, professor of pharmacology at the university of california, san diego (ucsd; la jolla, ca) 				 				dr. karin has received numerous awards including the oppenheimer award for excellence in research from the endocrine society, the herman beerman lectureship from the society of investigative dermatology, c.e.r.i.e.s. research award for physiology or biology of the skin, the grossman lectureship form the american gastroenterology association and an american cancer society research professorship in . dr. karin was elected to the national academy of sciences in . dr. karin also serves on several advisory boards and was cofounder of signal pharmaceuticals (currently celgene). 			 		 			 			 				scott e. kern 				 johns hopkins university, baltimore, md 			 			 					 scott e. kern, m.d., professor of oncology, johns hopkins university, baltimore, md, co-director of the kimmel cancer center gastrointestinal cancer program, everett and marjorie kovler professor of pancreas cancer research. 				 				dr. kern is a molecular geneticist interested in the clonal mutations of cancer, especially pancreatic cancer. his laboratory identified the first human smad gene, smad , as well as the first cancer mutations in smad , brca , and other genes. current interests include pharmacogenomics, mitotic instability, and the clinical significance of cancer biomarkers. 			 		 			 			 				guido kroemer 				 research director, inserm, paris, france 			 			 					guido kroemer, professor, faculty of medicine of the university of paris descartes, director of the research team "apoptosis, cancer and immunity" of the french medical research council (inserm) 				 			 		 			 			 				 john s. lazo 				uva cancer center, charlottesville, va 			 			 					john s. lazo, phd, harrison distinguished professor, departments of pharmacology & chemistry, associate director for basic science, uva cancer center; adjunct professor virginia tech carilion research institute 				 			 		 			 			 				arnold levine 				institute for advanced study, princeton, nj 			 			 					arnold j. levine, ph.d. member of the national academy, professor, the simons center for systems biology in the school of natural sciences at the institute for advanced study, princeton, nj. 				 				levine was on the faculty of the biochemistry department of princeton university from to , when he became chair and professor in the department of microbiology at the state university of new york, stony brook, school of medicine. returning to princeton university in , he was named harry c. wiess professor in the life sciences in the department of molecular biology, a position he held until . he chaired the department between and . 			he was president and chief executive officer of the rockefeller university in new york city from to , as well as heilbrunn professor of cancer biology and laboratory head until joining the institute in . the recipient of many honors including: the medal for outstanding contributions to biomedical research from memorial sloan-kettering cancer center ( ); the keio medical science prize of the keio university medical science fund, japan ( ); 			the albany medical center prize in medicine and biomedical research ( ); and the award for basic research from the surgical society of oncologists ( ). 			levine is a member of the national academy of sciences and of the academy's institute of medicine; 			he is also the author or coauthor of over scientific papers, as well as a book, viruses ( ). 			he has served as board member or adviser to numerous scientific organizations and educational institutions, among them the n.j. biotechnology institute, the american cyanamid corporation, the suny health sciences center in brooklyn, albert einstein college of medicine, the weizmann institute, the huntsman cancer center of the university of utah, and the institute for cancer research in lausanne, switzerland. 			 		 			 			 				michael p. lisanti 				 university of salford, salford, uk 			 			 					professor lisanti, m.d., ph.d. serves as the director of the manchester breakthrough breast cancer research unit and holds the muriel edith rickman chair of breast oncology within the institute of cancer sciences. he is also professor of cancer biology and the new founding director of the manchester centre for cellular metabolism (mccm). 				 			 		 			 			 				gerry melino 				 university of rome, rome , italy 			 			 					 dr. melino is full professor of molecular biology at the university of rome “tor vergata” in italy. he also currently works as programme leader for the medical research council toxicology unit, in leicester, uk. 				 			 		 			 			 				harold l. moses 				vanderbilt-ingram cancer center, nashville, tn 			 			 					harold l. moses, phd, professor, vanderbilt-ingram cancer center, nashville, tn 				 			 		 			 			 				yusuke nakamura 				 university of chicago, il 			 			 					dr. nakamura received his md and phd from osaka university, and became head of the biochemistry department, at the cancer institute, japanese foundation for cancer research in . he was appointed as a professor at the institute of medical science, university of tokyo in and the department of medicine at the university of chicago in . from january to december , dr. nakamura was special advisor to the cabinet secretary general, office of medical innovation, cabinet secretariat, government of japan. dr. nakamura received the keio medical science prize and the tomizo yoshida award of the japanese cancer association. he is a member of the association of american physicians. 				 				dr. yusuke nakamura discovered the apc (familial adenomatous polyposis) tumor suppressor. dr. nakamura plays the leading role in the field of personalized medicine. he authored scientific articles. 			 		 			 			 				joseph nevins 				 duke university, durham, nc 			 			 					joseph nevins, phd, professor, duke university, durham, nc 				 			 		 			 			 				pier paolo pandolfi 				harvard medical school, boston, ma 			 			 					 						pier paolo pandolfi, phd, professor, harvard medical school, boston, ma 				 george c. reisman professor of medicine and professor of pathology, medicine, harvard medical school 				 chief, division of genetics, dept of medicine, beth israel deaconess medical center 				 director, cancer center, beth israel deaconess medical center 				 director, cancer research institute, beth israel deaconess medical center 					 				 			 		 			 			 				nickolas papadopoulos, 				 johns hopkins university, baltimore, md 			 			 					nickolas papadopoulos, ph.d. associate professor, department of oncology, director of translational genetics, ludwig center for cancer genetics & therapeutics, sidney kimmel comprehensive cancer center, the johns hopkins institutions, crb , room , orleans street, baltimore, md 				 			 		 			 			 				arthur b. pardee ( - ) 				harvard university, boston, ma 			 			 					arthur b. pardee, ph.d, member of the national academy of sciences, professor of biological chemistry and molecular pharmacology emeritus, harvard medical school 				 				 					 it is with sadness that we share the passing of our editorial board member, arthur pardee, who died february , at the age of . dr. pardee was a renowned cancer biologist who made several groundbreaking discoveries in the mid- th century that shaped modern molecular biology. we have been honored to have dr. pardee as a founding editor of oncotarget. 				 			 		 			 			 				jeffrey pollard 				albert einstein college of medicine, bronx, ny 			 			 					jeffrey pollard, phd, professor, albert einstein college of medicine, bronx, ny 				 			 		 			 			 				carol prives 				 columbia university, new york, ny 			 			 					carol prives, ph.d, member of the national academy of sciences, professor, columbia university, new york, ny 				 			 		 			 			 				edward v. prochownik 				 children's hospital of pittsburgh, pittsburgh, pa 			 			 					edward v. prochownik, md, phd, professor of molecular genetics and biochemistry, university of pittsburgh school of medicine, director of oncology research, children's hospital of pittsburgh, pa 				 			 		 			 			 				john c. reed 				 roche holding ag, basel, switzerland 			 			 					dr. john c. reed, m.d., ph.d. has been the head of roche pharma research & early development (pred) at roche holding ag since april , . he is an adjunct professor at the university of california san diego (ucsd) department of molecular pathology, university of florida, university of central florida, and in san diego state university's biology department. 				 			 		 			 			 				neal rosen 				memorial sloan-kettering cancer center, new york, ny 			 			 					neal rosen, m.d., ph.d. is a member in the department of medicine and in the molecular pharmacology and chemistry program at memorial sloan-kettering cancer center, where he serves as head of developmental therapeutics. he is also a professor of pharmacology, cell biology and medicine at cornell university medical school. 				 			 		 			 			 				andrew v. schally 				veterans affairs medical center, miami, fl 			 			 andrew v. schally won the nobel prize for medicine or physiology in , for his research into peptide hormone production in the brain. he is currently distinguished medical research scientist of the department of veterans affairs, head of the endocrine, polypeptide and cancer institute veterans affairs medical center, research service miami, fl and south florida va foundation for research and education, distinguished leonard miller professor of pathology, professor division of hematology/oncology and division of endocrinology department of medicine, miller school of medicine, university of miami, fl. 				dr. schally was one of a pair of scientists to first isolate several of the communicating chemical links between the brain and the pituitary gland and also determined their structure and succeeded in synthesizing them. schally’s discoveries have led to many practical clinical applications that are in wide use. he has written over , publications, more than , of them since receiving the nobel prize. european & south american universities: m.d.h.c., d.h.c., d.sc.h.c., d.nat. sc. h.c. 				lasker award 				nobel prize in physiology or medicine, 			 		 			 			 				gregg l. semenza 				johns hopkins university, baltimore, md 			 			 					gregg l. semenza, m.d., ph.d, member of the national academy of sciences, professor, johns hopkins university school of medicine, baltimore, md 				 			 		 			 			 				donald l. trump 				inova schar cancer institute, fairfax, va 			 			 					donald l. (skip) trump, md, facp, is ceo and executive director of inova schar cancer institute. prior to joining inova, dr. trump was president and ceo of roswell park comprehensive cancer center in buffalo, ny. 				 			 		 			 			 				alexander varshavsky 				california inst. of technology, pasadena, ca 			 			 					alexander varshavsky, ph.d, member of the national academy of sciences, professor, california institute of technology, pasadena, ca 				 				alexander varshavsky is a recipient of the albert lasker award for basic medical research, the wolf prize in medicine and the louisa gross horwitz prize from columbia university in for his research on ubiquitination. in he won the march of dimes prize in developmental biology and he won the $ million gotham prize for an original approach to killing cancer cells. 			 		 			 			 				bert vogelstein 				johns hopkins university, baltimore, md 			 			 					bert vogelstein, m.d., member of the national academy of sciences, professor, johns hopkins university, baltimore, md 				 				bert vogelstein is a howard hughes medical institute investigator. 			he has received the gairdner foundation international award, louisa gross horwitz prize from columbia university, prince of asturias award for technical and scientific research, and other awards for his research. 			 			 		 			 			 				peter k. vogt 				the scripps research institute, la jolla, ca 			 			 					peter k. vogt, ph.d, member of the national academy of sciences professor, the scripps research institute, la jolla, ca 				 				dr. vogt is a professor in the department of molecular and experimental medicine at the scripps research institute in la jolla, california. he has received many awards and honors, including the gregor johann mendel medal, charles s. mott prize, ernst jung prize for medicine, bristol meyers award, and icn international prize in virology. dr. vogt has been invited as a distinguished lecturer by more than twenty leading research institutions in the us, europe, and asia, among them the german cancer research center in heidelberg for the meyenburg foundation lecture, the princess takamatsu foundation, the alexander von humboldt foundation of the federal republic of germany and the agency for science, technology and research of singapore. he was also elected an honorary member of the japanese cancer association and received an honorary doctorate from the university of würzburg. dr. vogt is an elected member of many prestigious academies, including the american academy of arts and sciences, the national academy of sciences, the institute of medicine of the national academies, and the american academy of microbiology. he is the recipient of the th annual szent-györgyi prize for progress in cancer research. 			 		 			 			 				paul workman 				 the institute of cancer research, london, uk 			 			 					paul workman, the institute of cancer research, london, uk 				 			 		 			 			 				zeng yixin 				 cancer center and the state key laboratory of oncology in southern china, sun yat-sen university, guangzhou, china 			 			 					dr. zeng yixin, professor and director, cancer center and the state key laboratory of oncology in southern china, sun yat-sen university, guangzhou, china.  				 				he was elected as a member of the chinese academy of sciences in . besides, professor zeng also has many other academic positions including the vice president of the china anti-cancer association, president of the board of the international society on epstein-barr virus & related diseases ( - ), a member of the third world academy of sciences, and a member of the europe-asian academy of sciences 			 		 	 	 	see full editors list » 	 		 two members of oncotarget editorial board are nobel prize winners : andrew v. schally ( physiology or medicine ); gregg l. semenza ( physiology or medicine ) 			 				since , four members of oncotarget/oncoscience/gerotarget have won the breakthrough prize, the highest prize ever: bert vogelstein, michael n. hall, alexander varshavsky, stephen j. elledge 			 		 			 			 				 oncotarget 	 spotlight during the short history of oncotarget and oncoscience, a number of our members have also joined the national academy of sciences usa, and have won lasker, nobel and other prestigious awards. oncotarget is indexed/archived on pubmed and pmc. all volumes ( - ) of oncotarget are available on pubmed » volume ( ) issues ( - ) are now on pubmed » as stated by medline reviewers on / / , "this journal continues to play a major role in the publication of important basic science research papers. editorial practices are consistently high. ethical guidelines are consistently followed. this is an important research journal for the field." impact factor (if) web of science (clarivate analytics) 	year	if	total cites 	 	 . 	 	 	 . 	 	 	 . 	 	 	 . 	 	 	 . 	 	 	 . 	 web of science update » scopus/sjr ranking: -ongoing: q (highest rank). all years q in medicine and oncology (subject area). oncotarget ranks number for total cites ( - ) among all journals in oncology. news: (june ), scopus has released its latest ratings. oncotarget is in q (highest rank). furthermore, oncotarget ranks no. for total cites ( ) in oncology. andrew v. schally, a member of the editorial board of oncotarget, a nobel prize winner in physiology or medicine: “oncotarget is an outstanding and most important journal in the field of oncology and cancer research. oncotarget is performing an extremely useful function for those of us working not only in cancer research, but also on other important topics in the field of medicine. oncotarget deserves a strong support from investigators working in the area of oncology as well as from nih.” andrew v. schally has published papers in oncotarget https://link.springer.com/article/ . /s - - - the story behind oncotarget oncotarget podcast was named among top five open-access podcasts of , https://player.fm/podcasts/open-access oncotarget.org: blog posts on new & trending papers latest articles about new and trending papers published by oncotarget oncotarget.net: blog posts on community news & events latest articles about community news and events sponsored by oncotarget copyright © impact journals, llc impact journals is a registered trademark of impact journals, llc 	home 	editorial board 	current issue 	archive 	editorial policies 	ethics statements 	videos 	oncotarget in the news 	search 	contact 	special collections 	 cntnap and nrxn are mutated in autosomal-recessive pitt-hopkins-like mental retardation and determine the level of a common synaptic protein in drosophila article cntnap and nrxn are mutated in autosomal-recessive pitt-hopkins-like mental retardation and determine the level of a common synaptic protein in drosophila christiane zweier, , ,* eiko k. de jong, markus zweier, alfredo orrico, lilian b. ousager, amanda l. collins, emilia k. bijlsma, merel a.w. oortveld, arif b. ekici, andré reis, annette schenck, and anita rauch , heterozygous copy-number variants and snps of cntnap and nrxn , two distantly related members of the neurexin superfamily, have been repeatedly associated with a wide spectrum of neuropsychiatric disorders, such as developmental language disorders, autism spectrum disorders, epilepsy, and schizophrenia. we now identified homozygous and compound-heterozygous deletions and mutations via molecular karyotyping and mutational screening in cntnap and nrxn in four patients with severe mental retardation (mr) and variable features, such as autistic behavior, epilepsy, and breathing anomalies, phenotypically overlapping with pitt-hopkins syndrome. with a frequency of at least % in our cohort of patients, recessive defects in cntnap appear to significantly contribute to severe mr. whereas the established synaptic role of nrxn suggests that synaptic defects contribute to the associated neuropsychiatric disor- ders and to severe mr as reported here, evidence for a synaptic role of the cntnap -encoded protein caspr has so far been lacking. using drosophila as a model, we now show that, as known for fly nrx-i, the caspr ortholog nrx-iv might also localize to synapses. overexpression of either protein can reorganize synaptic morphology and induce increased density of active zones, the synaptic domains of neurotransmitter release. moreover, both nrx-i and nrx-iv determine the level of the presynaptic active-zone protein bruchpilot, indicating a possible common molecular mechanism in nrx-i and nrx-iv mutant conditions. we therefore propose that an analogous shared synaptic mechanism contributes to the similar clinical phenotypes resulting from defects in human nrxn and cntnap . introduction the etiology of severe mental retardation (mr) is heteroge- neous, and, despite a significant number of identified disease genes, the majority of cases, especially nonsyn- dromic cases, remain unsolved. many of the currently known mr-related genes are involved in neurogenesis and neuronal migration, and awareness of the implication of synaptic organization and plasticity in mr has only recently begun to rise. , in , haploinsufficiency of the basic helix-loop-helix (bhlh) transcription factor (tcf ) was identified as causative for pitt-hopkins syn- drome (pths [mim ]), a severe mr disorder with variable additional anomalies, such as breathing anomalies, epilepsy, and facial dysmorphism including a beaked nose and a wide mouth with a cupid’s-bow- shaped upper lip. , tcf belongs to the e-protein family of bhlh transcription factors, which bind as homo- and heterodimers to e-box consensus sequences in promoters of target genes. like other e-proteins, tcf shows a broad expression pattern and a high expression in the cns. , after the identification of the underlying gene in , approximately patients have been reported, , , – demonstrating the importance of a diagnostic test for the the american increased recognition and appreciation of a previously clinically underdiagnosed condition. because of a similar severe degree of mr, commonly associated seizures, and microcephaly, pths has evolved as an important differen- tial diagnosis to the two most common syndromic disor- ders in severe mr, rett (mim ) and angelman (mim ) syndromes. because only % of patients referred to us with suspected pths showed mutations in tcf (zweier et al. and unpublished data), the clinically relatively homogenous group of tcf -mutation-nega- tive patients, including two sibling pairs, represented a suit- able study cohort for searching for additional candidate genes for overlapping disorders. through molecular karyotyping and mutational anal- ysis, we indeed identified recessive defects in two genes, cntnap and neurexin i (nrxn ), in patients with a very similar severe mr disorder and variable additional symptoms, such as seizures and breathing anomalies, resembling pitt-hopkins syndrome. in light of the shared phenotype that characterizes our patients with recessive cntnap and nrxn defects, and on the basis of the theme of overlapping phenotypes being caused by genes that are linked with each other in molecular networks, we further aimed to address the hypothesis of a common institute of human genetics, friedrich alexander university erlangen-nuremberg, erlangen, germany; department of human genetics, nijme- gen centre for molecular life sciences, donders institute for brain, cognition and behaviour & radboud university nijmegen medical centre, ga nijmegen, the netherlands; unita operativa medicina molecolare, azienda ospedaliera universitaria senese, policlinico s. maria alle scotte, siena, italy; department of clinical genetics, odense university hospital, odense c, denmark; wessex clinical genetics service, princess anne hospital, southampton, so ya, uk; department of clinical genetics, leiden university medical centre, rc leiden, the netherlands; institute of medical genetics, university of zurich, zurich-schwerzenbach, switzerland *correspondence: czweier@humgenet.uni-erlangen.de doi . /j.ajhg. . . . ª by the american society of human genetics. all rights reserved. journal of human genetics , – , november , mailto:czweier@humgenet.uni-erlangen.de molecular pathogenesis. we therefore utilized the fruit fly drosophila melanogaster as a model and collected data that point to a common synaptic link between these two genes. subjects and methods patients our study group consisted of patients, including two sibling pairs, who were referred for tcf testing because of severe mr and variable additional features reminiscent of the pths spectrum, such as microcephaly, dysmorphic facial gestalt, or breathing anomalies. tcf mutational testing revealed normal results in all of these patients. ethics approval for this study was obtained from the ethics committee of the medical faculty, university of erlangen-nuremberg, and informed consent was obtained from parents or guardians of the patients. molecular karyotyping molecular karyotyping was performed in patients with the affymetrix k snp array and in patients with the affymetrix . snp array, in accordance with the supplier’s instructions. in the index patient of family , hybridization was performed with an affymetrix genechip mapping k snp array, and the second affected patient and both parents were analyzed with the affyme- trix genechip mapping k nsp snp array. copy-number data were analyzed with the nexus software (biodiscovery) and the affymetrix genotyping console . . software. molecular karyo- typing in patients and was performed with the affymetrix genechip mapping . array platform, and copy-number data were analyzed with the affymetrix software genotyping console . . . the identified copy-number variants (cnvs) were sub- mitted to the decipher database (patient a, ; patient , ; patient , ). mutational screening dna samples from patients, derived from peripheral-blood or lymphoblastoid cell lines, were screened for cntnap (nm_ ) and nrxn (nm ) mutations by unidirec- tional direct sequencing of the coding exons – of cntnap and the coding exons – of nrxn , including intronic flanking regions (abi bigdye terminator sequencing kit v. ; applied biosystems), with the use of an automated capillary sequencer (abi ; applied biosystems). mutations were confirmed with an independent pcr and bidirectional sequencing. primer pairs can be found in table s , available online. for splice-site predic- tion, the online tools nnsplice . and hsf v . were used. fish and mlpa fluorescence in situ hybridization (fish) analysis was performed in family with the directly cy -labeled bacterial artificial chro- mosome (bac) clone rp - l on metaphase spreads, in accor- dance with standard protocols. probes for all coding exons of cntnap were designed and mlpa reaction was performed in accordance with the guidelines of mrc-holland. the deletion in patient was confirmed with mlpa with the use of a probe within exon and a control probe within exon of nrxn . probe sequences are listed in table s . analysis of relationship the relationship of individuals within family was analyzed, with a four-generation family with known relationships used as back- the american journal of human genetics , – , novem ground, with the graphical representation of relationships (grr) software. for grr, we selected, from affymetrix k arrays, , randomly distributed autosomal snps with a minimal minor allele frequency of . in europeans. for each pair of indi- viduals, grr calculates over the , markers the identical- by-state (ibs) mean and standard deviation. the graphical plot of ibs mean versus ibs standard deviation facilitates distinguishing between relationships such as parents and offspring, siblings, half siblings, and cousins, as well as identical or unrelated individuals. additionally, snp genotypes around cntnap were analyzed in the family members. drosophila genes and lines drosophila orthologs of tcf , nrxn , and caspr (daughterless [cg ], nrx- [cg ], and nrx-iv [cg ]) were identified by the ensemble genome browser or by the reciprocal blast best-hit approach. two rnai lines, to nrx-iv and daughterless, respectively, were obtained from the vienna drosophila research center (vdrc) and gave consistent phenotypes. vdrc lines no. (nrx-iv) and no. (daughterless) were utilized for further analysis. rna interference was induced with the uas- gal system. the w line (vdrc no. ) was used as a control, representing the same genetic background as the rnai lines. flies were raised at �c for maximum efficiency of knock- down. the nrx-i overexpression line puast-nrx-i was obtained from wei xie from nanjing, china. gal driver lines and the inducible nrx-iv overexpression line p(ep)nrx-ivep were obtained from the bloomington stock center. quantitative real-time pcr rna extraction from l larvae of each genotype was per- formed with the rneasy lipid tissue kit (qiagen) in accordance with the supplier’s protocols. cdna synthesis was performed with iscript (biorad). quantitative real-time pcr was performed with the power sybr green pcr master mix on a fast real-time pcr system (applied biosystems), and results were normalized to the endogenous control actin. primer sequences can be found in table s . immunostaining and data acquisition we harvested – hr embryos and fixed them with . % pfa for – min. all primary antibodies—anti-elav (labels nuclei of all neurons), antibody c (sensory nervous system), antibody bp (axon tracts, central neuropile region), anti-fas ii (motor- and central pioneer axons), and antibody nc (anti-bruchpilot, synaptic active zones) (all from the developmental studies hybridoma bank [dhsb])—were used in a : dilution. late stage ( / ), nc- -labeled embryos were assigned to one of three phenotypic categories: strong peripheral staining, moderate stain- ing, and weak or residual staining, respectively. we performed statistical analysis of wild-type (wt) embryos (w ) and nrx-iv knockdown embryos from three independent experiments with a chi-square test and a fisher’s-exact test to obtain p values. the images for peripheral synaptic staining were obtained with a zeiss apotome. brains were dissected from l larvae and fixed for min in . % pfa. pictures of wt and mutant brains were acquired with the use of the same microscope settings. intensities of nc immu- nostainings were measured with image j within two fields at two standardized positions in each cns, one in the upper third and one in the lower third of the ventral nerve cord. the average of ber , these two values was normalized to the average of controls for comparison of results from independent experiments. a total of w brains from six independent experiments, elav-gal :: nrx-i brains from two independent experiments, double- elav-gal ::nrx-i brains from three independent experiments, elav-gal ::nrx-iv brains from two independent experiments, and double-elav-gal ::nrx-iv brains from four independent experiments were measured. p values were obtained with a wil- coxon test for two samples for comparison to the wt. type b neuromuscular junctions (nmjs) of muscle were analyzed after dissection of l larvae and fixation in . % pfa for min. costaining was performed with nc and dlg (both from dhsb) or hrp (jackson immuno research) antibodies in a dilution of : . nmj pictures were stacked in imagej and pro- cessed in adobe photoshop. numbers of active zones and branches were manually counted in an animated stack, and total synaptic area was determined by imagej. a total of wt nmjs, overexpression nrx-i nmjs, and overexpression nrx-iv nmjs from at least two independent experiments were counted. for the evaluation of branches, wt nmjs, overexpression nrx-i nmjs, and overexpression nrx-iv nmjs from three exper- iments were counted. we performed statistical evaluation with the wilcoxon test for two samples, comparing each of the genotypes to the wt. the antibody against nrxiv was obtained from chris- tian klämbt, münster, germany. secondary antibodies for all stainings were either alexa - or alexa -labeled antibodies against mouse or rabbit (molecular probes). all data were acquired blind to the evaluated phenotype. results identification of recessive deletions and mutations in cntnap and nrxn molecular karyotyping led to the identification of a homo- zygous deletion of exons – within the cntnap gene on chromosome q -q . in a sibling pair of european origin (p a and p b), formerly published as possible clinical cases of pitt-hopkins syndrome. this deletion was confirmed by fish analysis (figure s ) and mlpa (data not shown) and is predicted to be in frame but result in the loss of several functional domains (figure a, figure a, and figure s ). consanguinity of the parents had been denied, and no indication for consanguinity was found by analysis of relationship with the use of the information of . snps. however, when the snps within and around cntnap were analyzed, they showed homozygosity in both children, indicating an allele of common ancestry. by subsequent mutational screening of cntnap in a larger cohort of additional tcf - mutation-negative patients, we identified a third patient of european origin (p ) with compound heterozygosity for the splice mutation ivs - g>t and a partial in-frame deletion of exons – , identified by molecular karyotyping (figure b and figure a) and confirmed with mlpa. the splice-site mutation resulted in lack of recognition of the splice acceptor site by two splice-site-prediction programs and is therefore predicted to result in loss of exon , leading to a frame shift, and the deletion is predicted to result in the loss of two laminin g domains. the splice- the america site mutation was not found in control chromosomes, and no cntnap deletion was found in molecularly karyotyped control individuals. in both families, the parents were heterozygous carriers of one of the respective defects. in another european patient of our cohort who had a very similar phenotype (p ), we identified a heterozygous kb deletion within the nrxn gene on chromosome p . , spanning exons – , including the start codon. this deletion was inherited from the healthy mother, but no deletions affecting the coding region of nrxn were found in molecularly karyotyped healthy controls. subsequent sequencing of nrxn in this patient revealed a stop mutation in exon on the second allele, which was inherited from the healthy father (figure c and figure b). both mutations are predicted to result in loss of the so-called alpha-isoform of nrxn , one of two nrxn isoforms that are transcribed from alternative promoters. the presumably remaining shorter beta- isoform (figures b and c) appears not to be sufficient to ensure normal function, which is in accordance with findings in alpha-neurexin knockout mice. mutational screening of nrxn in our study cohort did not reveal any additional defects. clinical characterization as far as data are available, birth measurements of all patients (p a, p b, p , and p ) were normal. further growth development was also normal, apart from short stature in the siblings from family and additional microcephaly in one of them. all four patients with recessive defects in cntnap or nrxn showed severe mr with lack of speech or with speech limited to single words (p b), whereas motor milestones were normal or only mildly delayed, with a walking age of years in p . episodes of hyperbreathing occurred in all patients, and seizures with an age at onset between months and months were observed in p a, p b, and p . additional variable anomalies were cerebellar hypoplasia, autistic behavior, and stereotypic movements. apart from a wide mouth with thick lips in p a and p b and a wide mouth in p , no specific facial dysmorphisms were noted (figure d). parents of all patients were healthy, and the deceased sister of the father of p was said to have had epilepsy and mild mr. p a and p b have been described in detail by orrico et al., and an overview of clinical details of all patients is shown in table . lack of nrxn and cntnap expression in blood or fibroblasts (bakkaloglu et al. and data not shown) precluded functional studies on human material. analysis of cntnap and nrxn orthologs nrx-iv and nrx-i in drosophila although a synaptic role for nrxn is known, this has not yet been established for cntnap . however, the high simi- larity of clinical phenotypes caused by defects in the two genes suggested a potential common molecular contribu- tion. to address this, as well as a further possible connection n journal of human genetics , – , november , figure . pedigrees and results of molecular karyotyping (a) pedigree of family , with two affected children and homozygous deletion of cntnap affecting exons – . both parents are hetero- zygous carriers of the deletion. results are from molecular karyotyping with affymetrix k snp arrays and analysis with the genotyp- ing console . . software (affymetrix). the deletion-flanking snps in the k array of p a are snp_a- ( , , mb; ucsc human genome browser version [hg ]) and snp_a- ( , , mb; hg ), with a maximal deletion size of , , bp and a minimal size of , , bp (nexus software). (b) pedigree of p , with one affected child. the patient harbors an in-frame kb deletion affecting cntnap exons – and a splice- site mutation in the splice donor site of exon . results of molecular karyotyping data from the affymetrix . snp array were analyzed with the genotyping console . . software, showing a deletion from cn_ ( , , mb; hg ) to snp_a- ( , , mb; hg ). see figure s for snp copy-number profiles. the american journal of human genetics , – , november , with tcf , we utilized drosophila as a model organism. all three genes, tcf , nrxn , and cntnap , are highly conserved in evolution and have orthologs in drosophila. we initially hypothesized that the tcf ortholog daughterless might regulate nrx-i and nrx-iv as transcrip- tional targets. knockdown of daughterless to % of wt levels by the use of two different ubiquitous driver lines (promoter-gal lines that regulate inducible rnai alleles; see subjects and methods) resulted in pupal lethality, con- firming the importance of daughterless for fly development (c) pedigree of p , with a compound-heterozygous deletion of nrxn exons – and a stop mutation in exon . results of molecular karyotyping data from the affymetrix . snp array were analyzed with the genotyping console . . (affymetrix), showing a kb deletion between cn_ ( , , mb; hg ) and cn_ ( , , mb; hg ). (d) clinical pictures of p , with a compound-heterozygous deletion and mutation in cntnap and of patient , with a compound- heterozygous deletion and mutation in nrxn . apart from a wide mouth in patient , no specific dysmorphisms are noted. figure . structure of cntnap and nrxn (a) schematic drawing of the genomic structure of cntnap with color coding for domain-coding exons and localization of mutations and deletions. black bars represent deletions. abbreviations are as follows: sp, signal peptide; disc, discoidin-like domain; lamg, lam- inin-g domain; egf, epidermal growth factor-like domain; fib, fibrinogen-like domain; tm, transmembran region; pdzpb, pdz- domain-binding site ; f , family ; p , patient ; amish, homozygous mutation in the amish population, published by strauss et al. (b) schematic drawing of the genomic structure of a-nrxn and b-nrxn with color coding for domain-coding exons and localization of the mutation and deletion in patient , the deletion being represented by a black bar. abbreviations are as follows: sp, signal peptide; lamg, laminin-g domain; egf, epidermal growth factor-like domain; tm, transmembrane region; pdzpb, pdz-domain-binding site. (c) schematic drawing of the domain structure of neurexins, caspr , and caspr in humans and drosophila. in contrast to caspr, caspr contains a pdz-domain-binding site but lacks a pgy repeat region, rich in proline, glycine, and tyrosine residues. both neurexin i and caspr /nrx-iv contain pdz-domain-binding sites at their intracellular c terminus but differ in the presence of discoidin-like and fibrinogen-like domains and in the order of laminin-g domains. the american journal of human genetics , – , november , table . phenotype in patients with cntnap and nrxn mutations siblings patients p a p b p amish (n ¼ ) p mutations cntnap deletion of exons – , homozygous cntnap deletion of exons – , homozygous cntnap deletion of exons – þ ivs - g>t cntnap c. delg, homozygous nrxn deletion of exons – þ p.s x age yrs yrs yrs – yrs yrs sex f m f not reported f parents healthy healthy healthy not reported healthy birth weight length ofc g cm . cm not known g at term not reported g normal height <p <p normal p –p p –p weight p p –p p not reported p –p ofc <p p p p –p p mr severe severe severe all severe age of walking normal normal not known – mo yrs speech none single words none yes, but regression none age of seizure onset mo – mo – mo – mo none mri results cerebellar hypoplasia normal not known dysplasia in % normal hyperbreathing yes yes yes not reported yes stereotypies none not noted tooth grind., rep. hand movements yes autistic behaviour not noted not noted yes % yes developmental regression not noted not noted considered normal until months yes, with onset of seizures normal the first years constipation not noted not noted no not reported yes decreased deep tendon reflexes not known not known not known % ue: decreased le: normal other broad mouth, thick lips broad mouth, thick lips dry skin broad mouth, strabismus, protruding tongue, excessive drooling, abnormal sleep-wake cycles, hypermotoric behavior normal testinga fraxa, ube a, mecp , tcf fraxa,ube a, mecp ube a, cdkl , mecp , tcf array cgh:b ube a, mecp , zeb , tcf patients p a and p b are siblings from family , clinically described by orrico et al.; abbreviations are as follows: f, female; m, male; hom, homozygous; het, heterozygous; mr, mental retardation; ofc, occipito-frontal circumference; rep. hand movements, repetitive hand movements; tooth grind., tooth grinding; ue, upper extremities; le, lower extremities. a previous genetic testing with reported normal results. b array-based comparative-genome hybridization via an agilent k oligonucleotide array (agilent). and viability. however, expression of nrx-i and nrx-iv in l larvae of these genotypes was not significantly changed (figure s ). drosophila nrx-iv and nrx- both determine the level of the synaptic protein bruchpilot knockdown of nrx-iv, either ubiquitously with the use of actin-gal drivers or specifically in neurons with the use of an elav-gal driver, resulted in late embryonic lethality, with animals completing embryogenesis but failing to the american journal of human genetics , – , novem hatch. together with an only recently reported expression of nrx-iv in neurons, , this suggests a crucial role in this cell type. to evaluate the cause for lethality upon neuronal knockdown, we performed immunostainings on embryos with a series of neuronal markers, labeling all neuronal nuclei, the sensory nervous system, main central axon tracts, and motor- and central pioneer axons, respectively. none of these showed abnormal position or morphology (figure s and data not shown). however, we noted an overall diminished staining intensity of the presynaptic ber , figure . the presynaptic protein bruchpilot is misregulated in nrx-iv knockdown embryos and larval brains with neuronal over- expression of nrx-i and nrx-iv (a and b) for quantitative evaluation, embryos have been assigned to one of three categories of bruchpilot (nc ) intensities: strong, moderate, and weak peripheral staining. (a) images of strong and moderate central (ventral nerve cord, left panel) and peripheral (middle panel) presynaptic bruchpilot staining, representing the major fraction in wt and nrx-iv knockdown embryos, respectively (uas-nrx-ivrnai/þ; elav-gal /þ [elav::rnai nrx-iv]). note that peripheral and central synaptic staining of bruchpilot in the nrx-iv knockdown embryos is diminished. costaining with an anti-hrp marker, staining neuronal membranes, ensured the same focal plane in mutant and wt embryos. arrowheads point to a specific identifiable synapse (the muscle / synapse). bruchpilot staining of this synapse is depicted in insets in the middle panels. (b) quantitative analysis of nc (anti-bruchpilot, synaptic active zones) labeling. diagram shows mean with standard deviation. (c) representative pictures of bruchpilot staining in brains with neuronal overexpression of nrx-i or nrx-iv as a result of the following genotypes: wt, uas-nrx-i/ elav-gal ; elav-gal /þ (dbelav:: nrx-i) and elav-gal /þ; uas-nrx-iv/ elav-gal (dbelav:: nrx-iv). bruchpilot immunoreactivity is increased in both mutant conditions. (d) quantitative assessment of bruchpilot immunoreactivity in brains of genotypes shown in (c). the diagram shows the mean of normalized intensity with the standard error of the mean. p values in (b) and (d) are related to the wt, respectively. single asterisk, p < . ; double asterisk, p < . ; triple asterisk, p < . . protein bruchpilot (nc ) in nrx-iv knockdown embryos (figure a). quantification revealed that the fraction of embryos with weak or residual staining on peripheral motor synapses was significantly increased to % in nrx-iv knockdown embryos, compared to % in wt embryos, whereas the fraction of strongly stained embryos was decreased to % in nrx-iv knockdown embryos, compared to % in the wt (figure b). interestingly, bruchpilot is known to colocalize with nrx-i at presynaptic active zones, the domains of neuro- transmitter release, and reduced levels of bruchpilot immunoreactivity have been reported in larval brains of nrx-i mutants. we studied bruchpilot levels after the american neuronal overexpression of either nrx-i or nrx-iv and found a significant dosage-dependent increase of bruchpi- lot-staining intensity in larval brains. upon overexpres- sion with one copy of a panneuronal elav-gal driver, bruchpilot intensity was increased . - and . -fold, and introduction of a second copy (dbelav) resulted in a . - and . -fold increase (figures c and d). drosophila nrx-iv is present at synapses and can, like nrx-i, reorganize them for further analyses at the subsynaptic level, we utilized drosophila larval nmjs, giant synapses that share a series of features with central excitatory synapses in the journal of human genetics , – , november , figure . nrx-iv localizes to synapses and its overexpression reorganizes synapse architecture (a) presence of endogenous nrx-iv at type b neuromuscular junctions (nmjs) of muscle , overlapping with active zones stained with nc . white arrowheads point to nrx-iv, labeling overlapping active zones. (b) synaptic terminal with increased bouton number and increased number of synaptic branches upon nrx-i or nrx-iv overexpression, stained with an anti-hrp marker and the nc antibody. the enlarged section depicts the increased density of active zones. (c) increased bouton number per mm nmj area in uas-nrx-i/ elav-gal ; elav-gal /þ (dbelav:: nrx-i) and elav-gal /þ; uas-nrx-iv/ elav-gal (dbelav:: nrx-iv) versus control w animals (contr. ) and dbelav control animals (contr. ). (d) quantitative analysis of increased active-zone density (number per mm nmj area) in neuronal nrx-i and nrx-iv overexpression. (e) increased number of synaptic branches in nrx-i and nrx-iv overexpression. error bars indicate standard error of the mean. p values are related to the wt (control ). single asterisk, p < . ; double asterisk, p < . ; triple asterisk, p < . . mammalian brain and represent an established model for the study of synaptic development and plasticity. stain- ing of these synapses with a recently characterized specific antibody for nrx-iv detected the presence of nrx-iv at synaptic terminals. nrx-iv localizes in a pattern of subsy- naptic foci that overlap active zones (figure a), resem- bling the pattern previously reported for nrx-i. previous studies in nrx-i null mutants revealed a decreased number of synaptic boutons in nmjs, whereas overexpression of nrx-i resulted in an increased bouton number. our results, measuring total synaptic area in nrx-i-overexpression animals, show that this goes along with a morphological reorganization into smaller boutons, as illustrated by an increased number of boutons per mm nmj area (figures b and c). strikingly, overexpression the american journal of human genetics , – , novemb of nrx-iv is capable of inducing the same morphological changes (figures b and c). furthermore, overexpression of either nrx-i or nrx-iv resulted in a highly significant increase in density of active zones (figures b and d) and in a significant increase in branching of synaptic terminals (figures b and e). discussion recessive defects in cntnap and nrxn cause severe mr we report here on homozygous and compound-heterozy- gous deletions and mutations in nrxn and cntnap that cause severe mr with additional features such as er , epilepsy, autistic behavior, and breathing anomalies. heterozygous cnvs or snps in both genes have recently been extensively reported in association with autism-spec- trum disorder (asd) (auts , [mim ]), epilepsy, or schizophrenia. , – additionally, for cntnap , an asso- ciation with gilles de la tourette syndrome and obsessive compulsive disorder was reported to be due to a disrup- tion of the gene but was not confirmed in another family. furthermore, a homozygous stop mutation in cntnap in old order amish children was implicated in a distinct disorder, cdfe syndrome (mim ), which is characterized by cortical dysplasia and early onset, intractable focal epilepsy leading to language regression, and behavioral and mental deterioration; as well as periventricular leukomalacia and hepatomegaly in an additional patient. histological examination of temporal-lobe specimens of these patients showed evi- dence of abnormal neuronal migration and structure, as well as a possibly altered expression of kv . and nav . channels, therefore providing a possible explanation for the cortical dysplasia and epilepsy phenotypes. taken together, published data and the present study indicate that heterozygous defects or variants in both nrxn and cntnap can represent susceptibility factors for variable cognitive, neurological, and psychiatric disor- ders, whereas biallelic defects result in a fully penetrant, severe neurodevelopmental disease such as that observed in our patients, thus representing different ends of the clin- ical spectrum caused by either monoallelic or biallelic defects in these two genes. this is in accordance with a report by zahir et al., who described a patient with a heterozygous de novo nrxn deletion affecting the same exons as those in our patient but without detectable mutation on the second allele. this heterozygous deletion was associated with vertebral anomalies, behavioral prob- lems, and only mild cognitive abnormalities. of note, in our families, none of the heterozygous parents had a history of autism, epilepsy, or schizophrenia, and none of other family members are known to have these disorders. however, the deceased sister of the father of patient was said to have had epilepsy and mild mr. whether or not this relates to the mutation in the father’s family remains elusive. a common feature in the amish patients with a homozy- gous stop mutation and in our patients with homozygous deletions or compound-heterozygous deletion and muta- tion in cntnap is the early onset of seizures. in contrast, none of our patients showed regression of speech develop- ment. another difference is episodic hyperbreathing, present in our patients but not reported in the amish patients. cortical dysplasia, occurring in some of the amish patients, was not observed in our patients; however, the number of patients examined with mri is too low to allow any definite conclusions to be drawn. the pheno- typic differences between our patients and the reported amish patients may be explained by the loss of c-terminal transmembrane and cytoplasmic domains in the amish mutation and by the loss of n-terminal extracellular the american domains in our patients (figure and table ). however, clinical bias cannot be excluded. resemblance among the patients in this study who have recessive defects in cntnap or nrxn is high, with the exception of patient , who has nrxn defects but no seizures. it might even be speculated that the epilepsy, observed in patients with recessive cntnap defects but not in our patient with the compound-heterozygous nrxn defect, might be associated to the previously observed neuronal-migration anomalies, whereas the remaining common symptoms such as severe mr and autistic behavior might be caused by overlapping synaptic anomalies. all of the reported patients were originally referred for tcf analysis as a result of phenotypic resem- blance to pitt-hopkins syndrome with regard to facial aspects, the severity of mr, and breathing anomalies. nevertheless, phenotypical differences were noticeable. in contrast to patients with pitt-hopkins syndrome, who present an equally severe delay or lack of both motor and speech development, patients with cntnap or nrxn defects, also severely impaired in speech development, present with normal or only mildly to moderately delayed motor milestones. these findings are in line with the previously reported specific involvement of cntnap in language development. because of the similar phenotypes caused by defects in both nrxn and cntnap and the resemblance to pitt-hopkins syndrome, caused by haploinsufficiency of tcf , we investigated an as-yet-unappreciated common molecular basis contributing to these disorders, using the fruitfly drosophila as a model. the drosophila tcf ortho- log daughterless belongs to the achaete-scute complex, which encodes members of the bhlh class of transcrip- tional factors with a well-established proneural function. our initial hypothesis, that daughterless might regulate nrx-i and nrx-iv as transcriptional targets, could not be confirmed by our analysis of their expression levels in the daughterless knockdown condition (figure s ). however, given that the data were obtained from knock- down animals, not null animals, this excludes neither transcriptional regulation that is undetectable by our assay nor other nontranscriptional interactions. cntnap and nrxn drosophila orthologs nrx-iv and nrx-i might be involved in a common synaptic mechanism our identification of a fully penetrant and severe mr phenotype due to recessive defects in nrxn is in agree- ment with nrxn ’s important role in synaptic function, as previously suggested on the basis of its heterozygous alterations that are associated with neuropsychiatric disorders and work with animal models. nrxn belongs to the evolutionarily conserved family of neurexins, presynaptic transmembrane proteins. each of the three vertebrate neurexins has two promoters, generating longer alpha-neurexins and shorter beta-neurexins. in addition, extensive alternative splicing occurs, generating a large journal of human genetics , – , november , number of variants, which may mediate target recognition and synaptic specificity. alpha-neurexins also play a role in neurotransmitter release by coupling ca þ channels to synaptic-vesicle exocytosis. in contrast to the three neurexins in mammals, there is only one drosophila neurexin, termed nrx-i or dnrx. , previously, defects in several genes involved in synaptic pathways and complexes have been reported to be causative for or associated with mr or asd, among them neurexin interac- tors. the extracellular region of neurexins binds to post- synaptic neuroligins, a family of proteins associated with autism and mr, which in turn interact with the postsyn- aptic protein shank , also associated with asd. the binding of neurexins to neuroligins connects pre- and postsynaptic neurons and mediates signaling across the synapse, and the nrxn-nlgn-shank pathway is supposed to be crucial during synaptogenesis, given that mutations within this pathway lead to abnormal synapto- genesis and excess of inhibitory currents. the intracel- lular domains of neurexins interact with the synaptic vesicle protein synaptotagmin and with pdz-domain proteins such as cask, , in which x-linked recessive mutations were only recently identified to be causative for mr and brain malformations. in contrast, a synaptic role of cntnap has not yet been established. cntnap encodes caspr , a protein related to neurexins. however, on the basis of its additional motifs, different domain organization, and phylogenetic analyses, only a distant relationship was assumed. caspr regulates neuron-glia contact in vertebrates and has been shown to colocalize with shaker-like kþ chan- nels in the juxtaparanodal areas of ranvier nodes in myelinated axons of both the central and peripheral ner- vous system. , until recently, its fly ortholog nrx-iv was reported to be almost exclusively expressed in glial cells and to regulate glia-glia contact in insects via septate junctions that are important for maintaining an intact blood-brain barrier. a detected decrease in evoked neuro- transmitter release and an occasional failure to form synapses in nrx-iv null mutants was interpreted as a con- sequence secondary to glial dysfunction. however, recently, a neuronal nrx-iv isoform was also identified. , despite the knowledge of neuronal expression of vertebrate caspr , so far only a report on detection of the protein in fractionated rat synaptic plasma membranes pointed to a possible synaptic presence. our findings now support a synaptic localization of nrx-iv (figure ). previous studies at drosophila neuromuscular junctions had reported a decreased number of synaptic boutons in nrx-i null mutants and an increase upon overexpression of nrx-i. our observation that the latter goes in hand with an increased active-zone density and the finding that nrx-iv overexpression is causing the very same phenotypes (figure ) suggest a crucial and possibly common role for both proteins in the morphological orga- nization of synapses. moreover, published data for nrx-i mutants and our analysis of nrx-iv knockdown and the american journal of human genetics , – , novem overexpression conditions of nrx-i and nrx-iv suggest an important role of both proteins in bidirectional regula- tion of bruchpilot levels. bruchpilot is a presynaptic protein crucial for the structure of active zones, the subsynaptic domains of neurotransmitter release, and is present at most if not all synapses of drosophila. it shows high sequence and functional homology to the vertebrate family of elks/cast proteins, corresponding to erc and erc in humans with similar reported functions. the mechanism by which nrx-i and nrx-iv determine bruchpilot levels remains the subject of future study. given that all three contain c-terminal pdz-domain binding sites, which are necessary for the formation of large complexes with active-zone proteins, one reasonable hypothesis is that the three proteins might assemble into a synaptic complex or macromolecular network. in conclusion, we have identified here autosomal-reces- sive defects in cntnap and nrxn in patients with severe mr and variable additional features overlapping with pitt-hopkins syndrome. with a frequency of at least % in this cohort, mutations in cntnap in particular appear to significantly contribute to severe mr. using the fruitfly as a model organism, we observed that not only nrx-i but also nrx-iv, previously unrecognized as a synaptic protein in vertebrates and in drosophila, is present at synapses and regulates levels of the active-zone protein bruchpilot. it is therefore tempting to hypothesize that misregulation of the human bruchpilot ortholog might underlie a common synaptic pathomechanism contributing to the similar phenotypes observed in patients with defects in cntnap and nrxn . supplemental data supplemental data include four figures and three tables and can be found with this article online at http://www.cell.com/ajhg. acknowledgments we thank the patients and families for participation in this study and christine zeck-papp, michaela kirsch, and daniela schweitzer for excellent technical assistance. we are grateful to wei xie (genetics research center, southeast university medical school, nanjing, china) and to christian klämbt (institute for neurobi- ology, münster, germany) for providing the puast-nrx-i flies and the nrx-iv antibody, respectively. we thank h. brunner and h. van bokhoven for critical reading of the manuscript. this work was supported by a grant from the deutsche forschungsge- meinschaft (dfg) to c.z.; by a grant from the german mental retardation network (mrnet) funded by the german federal ministry of education and research (bmbf) as a part of the national genome research network (ngfnplus), to a.re., a.ra., and a.s.; and by a vidi grant from the netherlands organisation for scientific research (nwo) to a.s. received: july , revised: september , accepted: october , published online: november , ber , http://www.cell.com/ajhg web resources the urls for data presented herein are as follows: basic local alignment search tool (blast), http://blast.ncbi.nlm. 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( ). caspr , a new member of the neurexin superfamily, is local- ized at the juxtaparanodes of myelinated axons and associates with kþ channels. neuron , – . . wagh, d.a., rasse, t.m., asan, e., hofbauer, a., schwenkert, i., durrbeck, h., buchner, s., dabauvalle, m.c., schmidt, m., qin, g., et al. ( ). bruchpilot, a protein with homology to elks/cast, is required for structural integrity and function of synaptic active zones in drosophila. neuron , – . er , 	cntnap and nrxn are mutated in autosomal-recessive pitt-hopkins-like mental retardation and determine the level of a common synaptic protein in drosophila 	introduction 	subjects and methods 	patients 	molecular karyotyping 	mutational screening 	fish and mlpa 	analysis of relationship 	drosophila genes and lines 	quantitative real-time pcr 	immunostaining and data acquisition 	results 	identification of recessive deletions and mutations in cntnap and nrxn 	clinical characterization 	analysis of cntnap and nrxn orthologs nrx-iv and nrx-i in drosophila 	drosophila nrx-iv and nrx- both determine the level of the synaptic protein bruchpilot 	drosophila nrx-iv is present at synapses and can, like nrx-i, reorganize them 	discussion 	recessive defects in cntnap and nrxn cause severe mr 	cntnap and nrxn drosophila orthologs nrx-iv and nrx-i might be involved in a common synaptic mechanism 	acknowledgments 	web resources 	references the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context — vrije universiteit amsterdam 	 skip to main navigation 	 skip to search 	 skip to main content 	 help & faq 	 home 	 profiles 	 research units 	 research output 	 datasets 	 activities 	 prizes 	 press / media search by expertise, name or affiliation the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context m.d.n. popma, c.h. roessingh research output: contribution to journal › article › academic › peer-review downloads (pure) 	 overview 	 fingerprint abstract purpose – the paper aims to give an account to describe the way the south-south development programme is realized between taiwan and belize. design/methodology/approach – the paper is the result of ethnographic fieldwork research combined with a literature study. findings – the development of the newly industrialized country taiwan can be characterized by a drastic transformation from an impoverished agrarian society towards an affluent industrial economy. now, taiwan is aiming to help other developing countries to walk the same path by drawing lessons from their so-called “taiwan experience”. in order to transfer their experience, taiwan has established an overseas development organization, the international cooperation and development fund. this organization offers development assistance to countries of which taiwan receives political recognition. central and south america are the regions where most of taiwan's diplomatic ties exist. one of the recipients of taiwanese assistance in this area is belize, a small english speaking, central american country in the caribbean sea, with a multi-ethnic, densely populated, relatively poor population. research limitations/implications – future research might examine the ways in which these taiwanese farmers have integrated in belize and how their entrepreneurial activities contribute in this process. practical implications – after visiting taiwan's project sites in belize and interviewing government officials, taiwanese-belizeans, belizean farmers, and non-farmers, it was found that much of what the taiwanese brought was indeed considered beneficial, although incomplete. the links between the taiwanese development organization and the wider society, belize, were not clearly developed at the time, not least because of the problems in communicating with the taiwanese development workers(for instance due to language issues). it would be wrong to suggest, however, that taiwan's development project was only perceived as raising problems for it did have a positive impact. originality/value – although the first taiwanese project in belize was launched in it has until recently received little attention. this paper explores the social impact of this development project from the taiwanese in belize. © , emerald group publishing limited 	original language	english 	pages (from-to)	 - 	number of pages	 	journal	journal of enterprising communities: people and places in the global economy 	volume	 	issue number	 	dois		https://doi.org/ . / 	publication status	published - access to document 	 . / 	popmaroessinghfinal published version, kb 	roesschoonfinal published version, kb 	roesbrasfinal published version, . kb 	ijbg roesfinal published version, kb 	ara roesduijnberendsefinal published version, kb 	ijird roessmitsfinal published version, kb 	ijtp berendroesfinal published version, kb fingerprint dive into the research topics of 'the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context'. together they form a unique fingerprint. 	 taiwan business & economics 	 development projects business & economics 	 development organization business & economics 	 development assistance business & economics 	 farmers business & economics 	 international cooperation business & economics 	 central america business & economics 	 international development business & economics view full fingerprint cite this 	 apa 	 author 	 bibtex 	 harvard 	 standard 	 ris 	 vancouver popma, m. d. n., & roessingh, c. h. ( ). the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context. journal of enterprising communities: people and places in the global economy, ( ), - . https://doi.org/ . / popma, m.d.n. ; roessingh, c.h. / the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context. in: journal of enterprising communities: people and places in the global economy. ; vol. , no. . pp. - . @article{ d b bf a ba ee d ace , title = "the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context", abstract = "purpose – the paper aims to give an account to describe the way the south-south development programme is realized between taiwan and belize. design/methodology/approach – the paper is the result of ethnographic fieldwork research combined with a literature study. findings – the development of the newly industrialized country taiwan can be characterized by a drastic transformation from an impoverished agrarian society towards an affluent industrial economy. now, taiwan is aiming to help other developing countries to walk the same path by drawing lessons from their so-called “taiwan experience”. in order to transfer their experience, taiwan has established an overseas development organization, the international cooperation and development fund. this organization offers development assistance to countries of which taiwan receives political recognition. central and south america are the regions where most of taiwan's diplomatic ties exist. one of the recipients of taiwanese assistance in this area is belize, a small english speaking, central american country in the caribbean sea, with a multi-ethnic, densely populated, relatively poor population. research limitations/implications – future research might examine the ways in which these taiwanese farmers have integrated in belize and how their entrepreneurial activities contribute in this process. practical implications – after visiting taiwan's project sites in belize and interviewing government officials, taiwanese-belizeans, belizean farmers, and non-farmers, it was found that much of what the taiwanese brought was indeed considered beneficial, although incomplete. the links between the taiwanese development organization and the wider society, belize, were not clearly developed at the time, not least because of the problems in communicating with the taiwanese development workers(for instance due to language issues). it would be wrong to suggest, however, that taiwan's development project was only perceived as raising problems for it did have a positive impact. originality/value – although the first taiwanese project in belize was launched in it has until recently received little attention. this paper explores the social impact of this development project from the taiwanese in belize. {\textcopyright} , emerald group publishing limited", author = "m.d.n. popma and c.h. roessingh", year = " ", doi = " . / ", language = "english", volume = " ", pages = " -- ", journal = "journal of enterprising communities: people and places in the global economy", issn = " - ", publisher = "emerald group publishing ltd.", number = " ", } popma, mdn & roessingh, ch , 'the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context', journal of enterprising communities: people and places in the global economy, vol. , no. , pp. - . https://doi.org/ . / the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context. / popma, m.d.n.; roessingh, c.h. in: journal of enterprising communities: people and places in the global economy, vol. , no. , , p. - .research output: contribution to journal › article › academic › peer-review ty - jour t - the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context au - popma, m.d.n. au - roessingh, c.h. py - y - n - purpose – the paper aims to give an account to describe the way the south-south development programme is realized between taiwan and belize. design/methodology/approach – the paper is the result of ethnographic fieldwork research combined with a literature study. findings – the development of the newly industrialized country taiwan can be characterized by a drastic transformation from an impoverished agrarian society towards an affluent industrial economy. now, taiwan is aiming to help other developing countries to walk the same path by drawing lessons from their so-called “taiwan experience”. in order to transfer their experience, taiwan has established an overseas development organization, the international cooperation and development fund. this organization offers development assistance to countries of which taiwan receives political recognition. central and south america are the regions where most of taiwan's diplomatic ties exist. one of the recipients of taiwanese assistance in this area is belize, a small english speaking, central american country in the caribbean sea, with a multi-ethnic, densely populated, relatively poor population. research limitations/implications – future research might examine the ways in which these taiwanese farmers have integrated in belize and how their entrepreneurial activities contribute in this process. practical implications – after visiting taiwan's project sites in belize and interviewing government officials, taiwanese-belizeans, belizean farmers, and non-farmers, it was found that much of what the taiwanese brought was indeed considered beneficial, although incomplete. the links between the taiwanese development organization and the wider society, belize, were not clearly developed at the time, not least because of the problems in communicating with the taiwanese development workers(for instance due to language issues). it would be wrong to suggest, however, that taiwan's development project was only perceived as raising problems for it did have a positive impact. originality/value – although the first taiwanese project in belize was launched in it has until recently received little attention. this paper explores the social impact of this development project from the taiwanese in belize. © , emerald group publishing limited ab - purpose – the paper aims to give an account to describe the way the south-south development programme is realized between taiwan and belize. design/methodology/approach – the paper is the result of ethnographic fieldwork research combined with a literature study. findings – the development of the newly industrialized country taiwan can be characterized by a drastic transformation from an impoverished agrarian society towards an affluent industrial economy. now, taiwan is aiming to help other developing countries to walk the same path by drawing lessons from their so-called “taiwan experience”. in order to transfer their experience, taiwan has established an overseas development organization, the international cooperation and development fund. this organization offers development assistance to countries of which taiwan receives political recognition. central and south america are the regions where most of taiwan's diplomatic ties exist. one of the recipients of taiwanese assistance in this area is belize, a small english speaking, central american country in the caribbean sea, with a multi-ethnic, densely populated, relatively poor population. research limitations/implications – future research might examine the ways in which these taiwanese farmers have integrated in belize and how their entrepreneurial activities contribute in this process. practical implications – after visiting taiwan's project sites in belize and interviewing government officials, taiwanese-belizeans, belizean farmers, and non-farmers, it was found that much of what the taiwanese brought was indeed considered beneficial, although incomplete. the links between the taiwanese development organization and the wider society, belize, were not clearly developed at the time, not least because of the problems in communicating with the taiwanese development workers(for instance due to language issues). it would be wrong to suggest, however, that taiwan's development project was only perceived as raising problems for it did have a positive impact. originality/value – although the first taiwanese project in belize was launched in it has until recently received little attention. this paper explores the social impact of this development project from the taiwanese in belize. © , emerald group publishing limited u - . / do - . / m - article vl - sp - ep - jo - journal of enterprising communities: people and places in the global economy jf - journal of enterprising communities: people and places in the global economy sn - - is - er - popma mdn, roessingh ch. the overseas taiwanese in belize: an exploration of a south-south development project in a belizean context. journal of enterprising communities: people and places in the global economy. ; ( ): - . https://doi.org/ . / 	 	 	 	 	 powered by pure, scopus & elsevier fingerprint engine™ © elsevier b.v. we use cookies to help provide and enhance our service and tailor content. by continuing you agree to the use of cookies log in to pure vrije universiteit amsterdam data protection policy about web accessibility [pdf] the impact of molecular genetics on our understanding of the psychoses. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /bjp. . . corpus id: the impact of molecular genetics on our understanding of the psychoses. @article{mullan theio, title={the impact of molecular genetics on our understanding of the psychoses.}, author={m. mullan and r. murray}, journal={the british journal of psychiatry : the journal of mental science}, year={ }, volume={ }, pages={ - } } 	m. mullan, r. murray 	published 	psychology, medicine 	the british journal of psychiatry : the journal of mental science studies demonstrating the linkage to separate chromosomal locations of alzheimer's disease, manic depression, and schizophrenia require re-evaluation of our ideas of their genetic aetiology. this article reviews the findings, and explores the increasing contribution of the 'new genetics' to our understanding of the organic and functional psychoses.  view on pubmed cambridge.org save to library create alert cite launch research feed share this paper citations view all topics from this paper 	psychoses, substance-induced 	psychotic disorders 	bipolar disorder 	schizophrenia 	molecular genetics (discipline) 	alzheimer's disease citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency the potential and the pitfalls of molecular genetics for psychiatry: an overview 	r. murray, m. gill 	biology 	 	 save alert research feed genetics and psychiatry: an unheralded window on the environment. 	d. reiss, r. plomin, e. hetherington 	psychology, medicine 	the american journal of psychiatry 	 	 save alert research feed ethical implications of the new genetics for psychiatry 	a. pelosi, a. david 	psychology 	 	 save alert research feed new findings in psychiatric genetics: implications for social work practice. 	e. takahashi, j. turnbull 	psychology, medicine 	social work in health care 	 	 save alert research feed ethics, molecular genetics, and psychiatric disorders 	m. gill 	psychology 	 	 save alert research feed research strategies in 'slow' infections in psychiatry 	k. bechter, a. hodgkiss 	medicine 	history of psychiatry 	 	 save alert research feed schizophrenia: a neuropathological perspective. 	g. roberts 	psychology, medicine 	the british journal of psychiatry : the journal of mental science 	 	 save alert research feed brain dopamine receptor plasticity: testing a diathesis-stress hypothesis in an animal model 	s. cabib, a. oliverio, r. ventura, f. lucchese, s. puglisi‐allegra 	psychology, medicine 	psychopharmacology 	 	 save alert research feed lewy bodies in psychiatric patients 	d. peter birkett, a. desouky, l. han, m. kaufman 	psychology 	 	 save alert research feed families at risk for psychopathology: who becomes affected and why? 	r. rende, r. plomin 	psychology 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency molecular genetics and human disease. implications for modern psychiatric research and practice. 	m. baron, j. d. rainer 	medicine 	the british journal of psychiatry : the journal of mental science 	 	 save alert research feed family history studies: v. the genetics of mania. 	t. reich, p. clayton, g. winokur 	medicine 	the american journal of psychiatry 	 	 save alert research feed the continuum of psychosis and its implication for the structure of the gene. 	t. crow 	psychology, medicine 	the british journal of psychiatry : the journal of mental science 	 	 save alert research feed down's syndrome and alzheimer's disease: a review. 	c. oliver, a. holland 	psychology, medicine 	psychological medicine 	 	 save alert research feed partly dominant transmission of schizophrenia in iceland. 	j. karlsson 	psychology, medicine 	the british journal of psychiatry : the journal of mental science 	 	 save alert research feed bipolar affective disorders linked to dna markers on chromosome 	j. egeland, d. gerhard, + authors d. housman 	biology, medicine 	nature 	 	 save alert research feed localisation of a susceptibility locus for schizophrenia on chromosome . 	m. owen, d. craufurd, d. st clair 	biology, medicine 	the british journal of psychiatry : the journal of mental science 	 	 save alert research feed linkage between glucose- -phosphate dehydrogenase deficiency and manic-depressive psychosis. 	j. mendlewicz, p. linkowski, j. wilmotte 	psychology, medicine 	the british journal of psychiatry : the journal of mental science 	 	 save alert research feed the genetic defect causing familial alzheimer's disease maps on chromosome . 	p. st george-hyslop, r. tanzi, + authors d. drachman 	biology, medicine 	science 	 	 , save alert research feed a polymorphic dna marker genetically linked to huntington's disease 	j. gusella, n. wexler, + authors joseph b. martin 	biology, medicine 	nature 	 	 , save alert research feed ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue mennofolk: mennonite and amish folk traditions (review) mennofolk: mennonite and amish folk traditions (review) alan l. chan journal of american folklore, volume , number , winter , pp. - (review) published by american folklore society doi: for additional information about this article [ this content has been declared free to read by the pubisher during the covid- pandemic. ] https://doi.org/ . /jaf. . https://muse.jhu.edu/article/ https://doi.org/ . /jaf. . https://muse.jhu.edu/article/ 	 journal of american folklore	 	( ) ideology	and	gender	politics,	especially	in	its	 relationship	to	the	classical	jewish	languages	 and	the	majority	tongues	that	surrounded	and	 overlapped	its	territory. 	 although	katz	painstakingly	charts	the	his- tory	of	speakers	of	yiddish	and	provides	an	excel- lent	framework	for	understanding	how	the	lan- guage	 emerged	 and	 grew,	 he	 is	 rather	 parsimonious	with	the	details	of	how	the	lan- guage	itself	changed	over	time	and	distance.	he	 delves	into	this	subject	most	deeply	in	the	chap- ter	“genesis,”	in	which	he	shows	not	only	how	 historical	analysis	of	word	variants	and	grammar	 allows	for	a	rough	estimate	of	the	place	and	time	 of	the	language’s	origin	but	also	how	newly	ar- rived	jewish	immigrants	from	southern	europe	 and	the	near	east	to	germany	acclimated	to	the	 environment,	playfully	assigning	biblical	names	 to	the	european	territories	in	which	they	found	 themselves.	(ashkenaz,	the	name	given	to	ger- many	and	later	to	all	of	yiddishland,	comes	from	 the	book	of	jeremiah.)	then	again,	katz	is	clearly	 concerned	primarily	with	elucidating	the	social	 contexts	of	yiddish	rather	than	linguistic	nuts	 and	bolts.	as	a	professional	linguist,	katz	has	 covered	the	technical	details	of	yiddish	in	depth	 in	many	previous	publications. 	 the	final	chapter	of	the	book	addresses	the	 most	controversial	aspect	of	yiddish:	its	future.	 katz	treats	this	topic	with	admirable	clarity	and	 honesty,	and	he	states	unapologetically	what	yid- dish	enthusiasts	consider,	to	put	it	gently,	to	be	 a	bitter	pill	to	swallow:	the	linguistic	and	demo- graphic	evidence	suggests	that,	outside	of	aca- demia,	the	world	of	secular	yiddish	is	doomed	 to	die	a	natural	death,	albeit	one	tragically	has- tened	by	the	holocaust	and	stalin’s	purges.	the	 future	of	yiddish	lies	with	the	hasidic	sects	for	 whom	the	language	has	always	been	their	native	 tongue	and	an	important	literary	vehicle.	katz	 makes	the	claim	that	yiddish	as	a	living	language	 cannot	exist	without	its	speakers	maintaining	 intimate	contact	with	the	world	of	traditional	 jewish	scholarship	and	its	associated	classical	 languages—what	he	summarizes	as	the	“trilin- gualism	of	old	ashkenaz”	(p.	 )	—as	well	as	 retaining	a	privileged	place	in	the	home	and	in	 daily	life.	as	he	observes,	even	the	most	radical	 leftist	yiddish	writers	were	steeped	in	traditional	 learning	before	breaking	with	religion.	without	 the	classical	teaching,	katz	argues,	too	many	of	 the	nuanced	expressions	of	hebrew	or	aramaic	 derivation	lose	their	psychosocial	significance	 and	disappear	from	the	lexicon.	likewise,	with- out	pride	of	place	in	ordinary	communication,	 yiddish	will	gradually	cede	ground	to	the	host	 languages	that	surround	it	in	every	community.	 in	short,	yiddish	cannot	long	survive	outside	of	 a	jewish	community	that	largely	keeps	to	itself	 and	uses	yiddish	in	at	least	some	aspects	of	daily	 life.	katz	therefore	ends	with	a	call	for	linguists	 to	focus	seriously	upon	the	living	language	of	 the	hasidim,	even	as	masters	of	secular	yiddish	 literature	offer	a	few	last	pearls	of	their	craft	for	 us	to	appreciate. mennofolk: mennonite and amish folk tra- ditions. by	ervin	beck.	(scottsdale,	ny:	herald	 press,	 .	pp.	 ,	foreword,	preface,	 	pho- tographs	 and	 illustrations,	 notes,	 suggested	 readings,	credits.) alan l. chan lutheran theological seminary, hong kong the	forty-sixth	addition	to	the	studies	in	ana- baptist	 and	 mennonite	 history	 book	 series	 published	by	herald	press,	ervin	beck’s	men- nofolk: mennonite and amish folk traditions	 demonstrates	that	mennonites	and	amish	con- stitute	a	religious	faith	with	folk	traditions	that	 can	be	traced	to	the	anabaptists	in	europe	in	 the	sixteenth	century.	the	nine	chapters	cover	 diverse	traditional	genres	such	as	ethnic	slurs,	 origin	tales	and	beliefs,	trickster	tales,	urban	 legends,	 protest	 songs,	 material	 culture,	 and	 festival.	the	author	was	an	english	professor	at	 goshen	college	from	 	to	 	and	is	con- sidered	to	be	an	insider	of	the	mennonite	and	 amish	culture. 	 beck’s	 purpose	 for	 writing	 this	 book	 is	 to	 make	 both	 mennonites	 and	 interested	 non- mennonites	more	aware	of	the	group’s	cultural	 traditions.	these	traditions	have	been	learned	 by	word	of	mouth	or	customary	example	and	 have	been	transmitted	to	succeeding	generations	 of	mennonites.	they	involve	both	long-estab- lished	materials	and	creative	variants,	and	they	 express	feelings,	ideas,	and	values	that	are	im- portant	for	the	individuals	who	pass	them	on	in	 informal	performance	venues	and	also	for	the	 community	 that	 unselfconsciously	 sponsors	 them	(pp.	 – ).	the	study	does	not	only	focus	 on	narrative.	as	the	mennonites	and	amish	are	 famous	for	their	conscientious	objection	to	war,	 there	is	a	chapter	on	protest	songs.	glass	paint- ings	of	flowers,	birds,	and	butterflies	with	moral	 statements	are	a	common	genre	of	folk	art	in	the	 culture,	and	beck	provides	an	examination	of	 this	kind	of	cultural	production.	almost	every	 mennonite	home	has	a	family	record	book,	and	 genealogy	is	a	vigorous	form	of	historical	mem- ory	practiced	within	the	community,	including	 the	maintenance	of	detailed	birth,	marriage,	and	 death	records.	the	relief	sale	festival	is	a	folk	 festival,	not	a	fair,	organized	by	the	mennonites	 and	for	the	mennonites.	beyond	these	genres,	 this	is,	most	importantly,	a	book	of	countless	 tales.	it	shows	how	individual	stories	can	be	re- told	in	differing	versions	with	various	under- standings	and	interpretations,	and	it	also	ex- plores	humorous	narratives. 	 in	sum,	beck’s	mennofolk	is	an	interesting	 introduction	 to	 the	 mennonite	 folk	 culture	 through	stories	and	other	traditions.	the	lan- guage	used	in	the	book	is	plain	and	clear,	and	the	 concepts	conveyed	are	easy	to	grasp.	if	a	picture	 is	worth	a	thousand	words,	the	generous	inclu- sion	of	photographs	and	illustrations	in	the	book	 has	definitely	aided	my	understanding	of	the	 mennonites’	uniqueness	as	a	people.	finally,	this	 study	is	recommended	to	all	who	want	to	gain	a	 general	knowledge	of	mennonite	religious	and	 folk	traditions	from	an	insider’s	perspective. bodies: sex, violence, disease, and death in contemporary legend.	 by	 gillian	 bennett.	 (jackson:	university	of	mississippi	press,	 .	 pp.	x	+	 ,	preface,	key	texts,	references,	after- word,	index.) stiofán Ó cadhla university college cork, ireland when	we	consider	the	 s,	our	attention	can- not	help	but	be	drawn	to	urban	and	contempo- rary	legends.	this	decade	has	taken	on	the	con- notations	 of	 revolution,	 rock	 and	 roll,	 sex,	 hippies,	and	feminism,	all	jostling	in	the	final	 and	fateful	confrontation	of	tradition	and	mo- dernity.	here,	fairies	and	monsters	are	replaced	 by	aliens	and	hook-handed	killers,	and	myth	and	 folktale	are	replaced	by	news	and	history—but	 legend	continues	to	partake	of	both.	this	is,	per- haps,	legend’s	central	problematic.	in	her	mar- velously	accessible	but	scholarly	style,	gillian	 bennett	goes	straight	to	the	heart	of	this	prob- lematic,	“the	cultural	clash	of	discordant	catego- ries	and	concepts”	(p.	xv).	she	reminds	us	that	 one	of	the	key	facts	about	the	legend	is	that	it	is	 difficult	to	define.	legends	are	marked	by	their	 longevity,	geographical	spread,	style,	the	multi- plicity	of	audio	and	visual	media	through	which	 they	 are	 disseminated,	 and	 the	 recurrence	 of	 specific	details	or	motifs.	avoiding	the	carto- graphic	 pedantry	 (that	 is,	 the	 historical-geo- graphical	or	finnish	method)	of	definition	and	 delimitation,	bennett	points	out	that	legend	is	 not	a	scientific	term	and,	as	such,	it	has	no	real	 referent.	legend	can	be	superstition,	relic,	delu- sion,	and	curiosity,	or	it	can	be	cool,	new,	sexy,	 urban,	and	teenaged.	in	the	unfolding	reassess- ments	of	the	discipline,	legend	has	been	decon- structed	or	at	least	“declassified”—the	distinc- tion	 between	 reality	 and	 legend	 is	 no	 longer	 considered	to	be	clear-cut.	contemporary	leg- end,	itself	an	orphan	of	the	 s,	has	in	many	 ways	become	an	exemplar	of	the	contemporary	 life	of	the	discipline. 	 bennett’s	bodies,	therefore,	is	about	folklore	 as	much	as	it	is	about	contemporary	legend.	in	 her	encyclopedic	detail	and	analysis,	bennett	 draws	attention	away	from	the	supposed	nov- elty	 of	 the	 genre	 to	 broader	 generalizations	 about	the	discipline.	following	paul	klee’s	ap- proach	to	painting,	bennett	takes	“a	line	for	a	 walk,”	 exploring	 thematically	 the	 evolving	 shape	and	form	of	six	particular	legend	case	 studies	from	their	early	variants	to	their	con- temporary	inflections	(p.	xv).	here	the	shape- shifting	element	of	story	is	exemplified.	story	 is	information,	entertainment,	strategy,	news,	 gossip,	rumor,	warning,	lesson,	joke,	photocopy,	 graffiti,	fallacy,	or	political	commentary—in	 short,	a	palimpsest	of	life.	as	a	popular	poetics	 of	interpretation,	legends	may	be	better	under- stood	within	contemporary	discursive	para- digms	or	contexts.	they	are	a	kind	of	social,	 	 book	reviews	 edinburgh research explorer linkage disequilibrium mapping of the replicated type diabetes linkage signal on chromosome q citation for published version: prokopenko, i, zeggini, e, hanson, rl, mitchell, bd, rayner, nw, akan, p, baier, l, das, sk, elliott, ks, fu, m, frayling, tm, groves, cj, gwilliam, r, scott, lj, voight, bf, hattersley, at, hu, c, morris, ad, ng, m, palmer, cna, tello-ruiz, m, vaxillaire, m, wang, cr, stein, l, chan, j, jia, w, froguel, p, elbein, sc, deloukas, p, bogardus, c, shuldiner, ar & mccarthy, mi , 'linkage disequilibrium mapping of the replicated type diabetes linkage signal on chromosome q', diabetes , vol. , no. , pp. - . https://doi.org/ . /db - digital object identifier (doi): . /db - link: link to publication record in edinburgh research explorer document version: publisher's pdf, also known as version of record published in: diabetes publisher rights statement: readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. see http://creativecommons.org/licenses/by-nc-nd/ . / for details. general rights copyright for the publications made accessible via the edinburgh research explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. take down policy the university of edinburgh has made every reasonable effort to ensure that edinburgh research explorer content complies with uk legislation. if you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. download date: . apr. https://doi.org/ . /db - https://doi.org/ . /db - https://www.research.ed.ac.uk/portal/en/publications/linkage-disequilibrium-mapping-of-the-replicated-type- -diabetes-linkage-signal-on-chromosome- q(e fb -fb - a -adef- d bad).html linkage disequilibrium mapping of the replicated type diabetes linkage signal on chromosome q inga prokopenko, , eleftheria zeggini, , robert l. hanson, braxton d. mitchell, n. william rayner, , pelin akan, leslie baier, swapan k. das, , katherine s. elliott, mao fu, timothy m. frayling, christopher j. groves, , rhian gwilliam, laura j. scott, benjamin f. voight, , , , andrew t. hattersley, cheng hu, andrew d. morris, maggie ng, colin n.a. palmer, marcela tello-ruiz, martine vaxillaire, cong-rong wang, lincoln stein, , juliana chan, weiping jia, philippe froguel, , steven c. elbein, panos deloukas, clifton bogardus, alan r. shuldiner, and mark i. mccarthy, , , for the international type diabetes q consortium objective—linkage of the chromosome q – region to type diabetes has been demonstrated in multiple ethnic groups. we performed common variant fine-mapping across a -mb interval in a multiethnic sample to search for variants responsi- ble for this linkage signal. research design and methods—in all, , single nucleotide polymorphisms (snps) were successfully genotyped in , type diabetes case and control subjects from eight populations with evidence of q linkage. samples were ascer- tained using strategies designed to enhance power to detect variants causal for q linkage. after imputation, we estimate � % coverage of common variation across the region (r � . , europeans). association signals of interest were evaluated through in silico replication and de novo genotyping in � , case subjects and , control subjects. results—association mapping of the -mb region identified two strong signals, both of which were restricted to the subset of european-descent samples. the first mapped to the nos ap (capon) gene region (lead snp: rs , odds ratio . [ % ci . – . ], p � . � � , in case subjects and , control subjects); the second mapped within an extensive region of linkage disequilibrium that includes the ash l and pklr genes (lead snp: rs , odds ratio . [ . – . ], p � . � � , under a dominant model). however, there was no evidence for association at either signal on replication, and, across all data (� , subjects), there was no indication that these variants were causally related to type diabetes status. conclusions—detailed fine-mapping of the -mb region of replicated linkage has failed to identify common variant signals contributing to the observed signal. future studies should focus on identification of causal alleles of lower frequency and higher penetrance. diabetes : – , g enome-wide association (gwa) analysis has provided a powerful stimulus to the discovery of common variants influencing type diabetes risk, and, to date, � susceptibility loci have been identified with high levels of statistical confidence ( ). however, these known variants account for only a small proportion of the inherited component of disease risk (probably � %), and the molecular basis of the majority of the genetic predisposition to type diabetes has yet to be established ( ). the success of the gwa approach contrasts with the slow progress that characterized previous efforts to map susceptibility loci through genome-wide linkage ( ). how- ever, now that many of the common variants of largest effect have been identified (in european-descent popula- tions at least), there are cogent reasons to revisit regions previously identified through genome-wide linkage. first, variants within the genomic intervals representing repli- cated linkage signals can be considered to have raised prior odds for a susceptibility effect, and this information can be used to prioritize gwa signals (particularly those with only modest evidence of association) for targeted replication. second, genuine linkage signals are likely to be driven by causal variants—particularly low-frequency snps or copy number variants not captured by the com- from the wellcome trust centre for human genetics, university of oxford, oxford, u.k; the oxford centre for diabetes, endocrinology and metabo- lism, university of oxford, oxford, u.k.; the wellcome trust sanger institute, wellcome trust genome campus, hinxton, cambridge, u.k.; the phoenix epidemiology and clinical research branch, national institute of diabetes and digestive and kidney diseases, national institutes of health, phoenix, arizona; the school of medicine, university of maryland, balti- more, maryland; the endocrinology section, medical service, central arkansas veterans healthcare system, little rock, arkansas; the division of endocrinology and metabolism, department of internal medicine, col- lege of medicine, university of arkansas for medical sciences, little rock, arkansas; the institute of clinical and biomedical science, peninsula medical school, exeter, u.k.; the department of biostatistics and center for statistical genetics, university of michigan, ann arbor, michigan; the broad institute of harvard and massachusetts institute of technology, cambridge, massachusetts; the center for human genetic research, massachusetts general hospital, boston, massachusetts; the department of medicine, massachusetts general hospital, boston, massachusetts; the department of medicine, harvard medical school, boston, massa- chusetts; the shanghai diabetes institute, department of endocrinol- ogy & metabolism, shanghai jiaotong university no. people’s hospital, shanghai, china; the diabetes research group, biomedical research institute, university of dundee, dundee, u.k.; the department of medicine and therapeutics, chinese university of hong kong, shatin, hong kong, sar; the biomedical research institute, ninewells hospi- tal and medical school, dundee, u.k.; the cold spring harbor labora- tory, new york, new york; cnrs umr , institute of biology and lille university, pasteur institute, lille, france; informatics & biocomputing, ontario institute for cancer research, toronto, ontario, canada; genomic medicine, hammersmith hospital, imperial college london, london, u.k.; and the oxford national institute for health research biomedical research centre, churchill hospital, oxford, u.k. corresponding author: mark mccarthy, mark.mccarthy@drl.ox.ac.uk. received january and accepted april . i.p. and e.z. contributed equally to this work. published ahead of print at http://diabetes.diabetesjournals.org on april . doi: . /db - . © by the american diabetes association. readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. see http://creativecommons.org/licenses/by -nc-nd/ . / for details. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. brief report diabetes, vol. , july modity gwa arrays—with effect sizes larger than those currently detectable by gwa ( ). because alleles with these characteristics will have a more marked impact on individual disease predisposition than the common vari- ants found by gwa, identification of causal variants underpinning replicated linkage signals should accelerate efforts to obtain better predictors of disease ( ). for type diabetes, there appears to be only limited overlap between the regions identified by genome-wide linkage and those revealed by gwa ( ). although the discovery of tcf l was prompted by a search for causal variants within a region of replicated type diabetes linkage, neither the common variants in tcf l nor those in hhex and ide (a second nearby gwa signal) account for that linkage signal ( ). thus, the discovery of tcf l reflects either serendipity or the co-localization of com- mon and rare causal variants in the same locus—the former driving the association and the latter the linkage. similarly, whereas common variants in hnf a have been reported to explain the chromosome linkage signals seen in finns and ashkenazim ( , ), these associations have proved difficult to replicate ( ). chromosome q (in particular the -mb stretch adja- cent to the centromere) ranks alongside the regions on chromosomes and as among the strongest in terms of the replicated evidence for genome-wide linkage to type diabetes. linkage has been reported in samples of european (u.k., french, amish, utah), east asian (chi- nese, hong kong), and native american (pima) origin (summarized in supplementary table , which is available in the online-only appendix at http://diabetes.diabetes journals.org/cgi/content/full/db - /dc ; ref. ). the region concerned is gene rich and contains a dispropor- tionate share of excellent biological candidates ( ). the homologous region has also emerged as a diabetes sus- ceptibility locus from mapping efforts in several well- characterized rodent models ( – ). the international q consortium represents a coordi- nated effort by the groups with the strongest evidence for q linkage to identify variants causal for that signal. here, we report efforts to map causal variants using a custom linkage-disequilibrium (ld) mapping approach, predomi- nantly based around common snp variants, applied to a well-powered set of multiethnic samples. to improve power, ascertainment of type diabetes cases for this study aimed to enrich for q causal alleles through ) a focus on populations and samples that had shown q linkage; ) selection for positive family history; and ) for some samples, preferential recruitment on the basis of patterns of identity by descent sharing in the q region. for each set of case subjects, we selected a control sample of individuals from the same population. details of the recruitment have been reported previously ( ) and are summarized in the supplementary material available in the online appendix. in all, the case-control part of the study included , samples ( , case subjects, , control subjects) of european descent, ( case subjects, control subjects) of east asian origin, and ( case subjects, control subjects) who were native american (pima) (supplementary table ). we also included a small sample of individuals of african american origin ( case subjects, control subjects) and an additional pima individuals ( affected, nondia- betic after age years) from families who, after combination with the pima case-control set, were used for family-based association analyses. these samples were submitted to dense-map snp typing of the core region of interest (from . to . mb [build ]) using a series of , -plex beadarray designs (golden gate, illumina, san diego, ca). design of these arrays was contemporaneous with development of dbsnp and hapmap ( ). thus, whereas the first arrays were ld-agnostic and compiled using genomic localization as the primary consideration for inclusion, subsequent arrays used ld information from the ceu (european ancestry) and chb/jpt (asian ancestry) components of hapmap to guide snp selection and maximize coverage of the region. in all, we designed assays for , snps, of which , provided reliable data in all populations after passing through our extensive quality control (see the supplemen- tary material). we estimate that after imputation (using the appropriate set of hapmap data as a reference), coverage of the region (minor allele frequency � . ; r � . ) reaches � % in the european and � % in the east asian samples. coverage is harder to estimate (and impu- tation likely to be less valuable) in pima and african american samples, since reference data from these popu- lations are not available, although we estimate � % coverage in west africans based on yri data. genotyping quality was generally good, with over % of snps passing quality control in each population (see the supplementary material) and � . % of snps failing (p � � ) tests of within-sample hardy-weinberg equi- librium. significant departures from expectation in the distributions of test statistics observed in the amish and pima samples (as revealed by qq plots; see supplemen- tary material) likely reflect residual relatedness between subjects from those populations. we adjusted for this (and any population stratification effects) through genomic control methods ( ). association analyses treated each study as a separate stratum and used standard meta-analysis approaches to deliver estimates of joint effect size and statistical significance (see supplementary material). a series of nested meta-anal- yses were performed including ) european-descent samples only (“ -way”); ) non–african-descent samples only (“ -way”); and ) all samples (“ -way”). under an additive model with allele frequency of . , our sample provides � % power to detect per-allele odds ratios (ors) of � . (“ -way”) or � . (“ -way”) for � � � � . given that the region covers � % of the genome, we consider this a reasonable benchmark for “region- wide” significance (equivalent to consensus genome-wide thresholds of � � ). these power calculations are conservative: given the case ascertainment enrichment strategies used in this study, we would expect to detect variants with population-level effects in the . – . range. under reasonable assumptions (three independent alleles contributing to a linkage signal with a locus-specific sibling relative risk of � . ), we can expect the effect size of the variants we were seeking to detect (i.e., those responsible for the q linkage) to be substantially greater than this (e.g., allelic or . for a variant with risk allele frequency of %). our study was therefore well powered to detect putatively causal alleles within the european and/or combined datasets. across these analyses, none of the snps showed an association with type diabetes that withstood genome- wide correction (p � � � ) ( ). however, two clusters of snps showed association signals that ap- proached or exceeded “region-wide” significance thresh- olds. the first of these, involving rs and nearby i. prokopenko and associates diabetes, vol. , july snps, mapped to a . -kb interval (at � . mb) within the first intron of nos ap (nitric oxide synthase [neu- ronal] adaptor protein) with an estimated per allele or (in the -way, european-only analysis) of . ( % ci . – . , p � . � � , additive model, table , fig. ). rs lies � . kb from one of the snps (rs ) table association results for rs in the nos ap gene case subjects (n) control subjects (n) risk allele frequency in case subjects risk allele frequency in control subjects additive model or ( % ci) p u.k. . . . ( . – . ) . � � french . . . ( . – . ) . utah . . . ( . – . ) . � � amish* . . . ( . – . ) . meta-analysis: european descent populations , . ( . – . ) . � � shanghai . . . ( . – . ) . hong kong . . . ( . – . ) . pima* . . . ( . – . ) . meta-analysis: non-african populations , , . ( . – . ) . � � african american . . . ( . – . ) . meta-analysis: qc populations , , . ( . – . ) . � � replication samples independent wtccc sample , , . . . ( . – . ) . w c vs. bc , . . . ( . – . ) . ukt dgc , , . . . ( . – . ) . diabetes genetics initiative , , . . . ( . – . ) . fusion , , . . . ( . – . ) . meta-analysis: all replication samples , , . ( . – . ) . meta-analysis: all european descent populations , , . ( . – . ) . meta-analysis: all data , , . ( . – . ) . *p value for amish and pima was adjusted using estimated lambda for genomic control. wtccc, wellcome trust case control consortium. fig. . single-point type diabetes associations within the q region. this plot shows the “ -way” (european-descent samples only) meta-analysis using the additive model (cochran-armitage trend test). directly typed snps are shown in orange and imputed snps in blue. the pale blue track (and secondary y-axis) represents recombination rates (for hapmap ceu) across the region. blue diamonds represent the strongest association p value in the two regions taken forward for replication. in the case of the pklr/ash l region, the strongest association was seen for a dominant model (the equivalent additive model result is denoted with the red diamond). only a small subset of genes within the region is denoted on the gene track. chromosome q and type diabetes diabetes, vol. , july previously shown to influence cardiac repolarization and qt interval ( ): the two snps are in modest ld (r � . in hapmap ceu), and rs shows some evidence for association with type diabetes (p � . � � ) in the same -way meta-analysis. the second signal includes � snps in a -kb region of extensive ld at � . mb. this region includes the coding sequences of the genes encoding liver pyruvate kinase (pklr) and ash (absent, small, or homeotic)-like (drosophila) (ash l) among others. at the lead snp (rs ), the estimated or for the -way analysis was . ( . – . ) (p � . � � ) under the additive model. the effect size and significance were marginally greater ( . [ . – . ], p � . � � ), under a domi- nant model (table , fig. ). both signals were most prominent in the european samples, and there was no evidence that an equivalent association signal extended to the east asian, native american, or african american samples. though the asso- ciation p values for these two signals remained strong in the -way meta-analysis of all data ( . � � for rs and . � � for rs , tables and ), in each case they were driven by the larger european samples. analyses in the larger pima family-based associ- ation dataset also found no evidence of association (rs , p � . ; rs [r of one with rs in ceu and chb/jpt hapmap], p � . ). although neither signal was of sufficient effect size to be considered causal for the q linkage signal (the estimated sibling relative risk attributable to these loci in combination is only . ), we reasoned that these signals might nevertheless be pointers toward nearby causal variants (of lower frequency but higher pen- etrance) that were inadequately tagged by the snps we had typed. however, before proceeding to resequencing and fine-mapping, we first sought replication of our findings in independent datasets. mindful that our case ascertainment strategies may have led to inflated esti- mates of effect size compared with those evident in unselected case subjects, we recognized that large sample sizes would be required to test the observed associations. because the signals were clearest in european- descent samples, we focused replication on samples from northern europe. first, we used gwa data from the wellcome trust case control consortium ( , ). after removing overlap- ping case subjects, we examined , additional type diabetes case subjects and , control subjects with affymetrix k data (using imputation to test for associ- ation at the lead snps in each interval). no evidence of association was evident (rs , p � . ; rs , p � . ). similarly, analysis of gwa data from the diabetes genetics initiative ( ) and fusion ( ) studies provided no corroboration of either signal. furthermore, when analyzed jointly ( , case subjects, , control subjects), these three studies also failed to reveal any additional common variant signals of interest (p � � ) across the wider q region ( ) and no corroboration of any of the lesser signals evident in the q consortium analyses. tabl association results for rs in the pklr/ash l region case subjects (n) control subjects (n) risk allele frequency in case subjects risk allele frequency in control subjects additive model or ( % ci) p * u.k. . . . ( . – . ) . french . . . ( . – . ) . utah . . . ( . – . ) . amish* . . . ( . – . ) . � � meta-analysis: european descent populations , , . ( . – . ) . � � shanghai . . . ( . – . ) . hong kong . . . ( . – . ) . pima* . . . ( . – . ) . meta-analysis: non-african populations , , . ( . – . ) . � � african american . . . ( . – . ) . meta-analysis: qc populations , , . ( . – . ) . � � replication samples independent wtccc sample , , . . . ( . – . ) . w c vs. bc , . . . ( . – . ) . ukt dgc , , . . . ( . – . ) . diabetes genetics initiative , , . . . ( . – . ) . fusion , , . . . ( . – . ) . meta-analysis: all replication samples , , . ( . – . ) . meta-analysis: all european descent populations , , . ( . – . ) . meta-analysis: all data , , . ( . – . ) . *p value for amish and pima was adjusted using estimated lambda for gc. bc, british birth cohort; ukt dgc, united kingdom type diabetes genetics consortium; w c, warren cases; wtccc, wellcome trust case control consortium. i. prokopenko and associates diabetes, vol. , july finally, we genotyped the two lead snps (rs , rs ) using fluorogenic �-nuclease (taqman) as- says in , case subjects and , control subjects from the u.k. (the uk type diabetes genetics consortium and warren cases/ british birth cohort strata in tables and ). once again, there was no evidence of replication. taking into account all replication samples (� , case subjects, � , control subjects), there was no significant association with type diabetes (rs , or . [ % ci . – . ], p � . ; rs , or . [ . – . ], p � . [additive], . [ . – . ], p � . [dominant]). nominal significance was retained when these replication data were combined with the original q consortium case-control data (rs , p � . ; rs , p � . ), but these associations are unimpressive in either the region-wide or genome-wide context. even allowing for some heteroge- neity of effect size between the primary and replication datasets (due to ascertainment differences and the “win- ner’s curse”), there seems to be no substantive evidence that the association signals observed in the nos ap and the pklr/ash l region are genuinely associated with type diabetes. in summary, we have undertaken a detailed survey of common variants across the region of replicated q link- age, achieving coverage that exceeds that of available gwa data for the region. despite analysis of multiple ethnic groups in samples sufficiently powered (in the european-descent component at least) to have detected common variants causal for the linkage, we found no compelling signals. should we conclude therefore that the original evi- dence for q linkage was false? although this possibility cannot be discounted, it is worth considering that recent experience from gwa studies has shown that, for common susceptibility variants at least, effect sizes are modest and that none is of magnitude sufficient to generate a linkage signal detectable in achievable sam- ple sizes. efforts to explain the “missing heritability” for type diabetes (that is, the disparity between the predisposition attributable to the known loci and inde- pendent estimates of overall heritability and familiality) are now shifting toward the search for low-frequency, medium-penetrance alleles. alleles with these charac- teristics are likely to have escaped detection through the genome-wide approaches available so far, since they would be insufficiently penetrant to be detected with traditional linkage approaches applied to monogenic families and too infrequent to be reliably identified through gwa studies ( ). yet, low-frequency, medium- penetrance alleles could, particularly if several indepen- dent alleles map to the same locus, generate the kinds of linkage signals detectable in family-based studies (as is the case for nod /card and crohn’s, for example) ( ). detection of low-frequency susceptibility variants will require new approaches based around next-generation resequencing and large-scale fine-mapping. genome-wide resequencing of large case-control samples remains eco- nomically and logistically unfeasible, but targeted rese- quencing of selected regions is not, and the future plans of the q consortium include deep resequencing of the q region of interest, focusing at least initially on exons and conserved sequence. acknowledgments the principal funding for this study was provided as a supplement to niddk through r -dk and as a supplement to u -dk . other major support was provided by the national institutes of health (t - ag , r -dk , r -dk , k -dk , k - ca , r -dk , u -dk , and intramural funds); the university of maryland and arkansas general clinical research centers; the national center for re- search resources (m rr ); the department of vet- eran affairs and american diabetes association (u.s.); “ familles pour vaincre le diabète et l’obésité” and association française des diabétiques (france); diabetes u.k.; the hong kong research grants committee (cuhk / m; / c), chinese university of hong kong stra- tegic grant program (srp ) and hong kong innovation and technology support fund (its/ / ) (hong kong); and the national nature science foundation of china ( ), shanghai medical pioneer development project ( - - ; ) and shanghai science technol- ogy development foundation ( zb ) (china). we also recognize the funding support of the u.k. medical research council (g ), the oxford national insti- tute for health research biomedical research centre, the general clinical research centers program, the baltimore veterans administration geriatric research and educa- tion clinical center, and the wellcome trust (gr ). e.z. is a wellcome trust research career development fellow. for the birth cohort, venous blood collection was funded by the u.k. medical research council, and cell line production and dna extraction and processing was funded by the juvenile diabetes research foundation and the wellcome trust. no potential conflicts of interest relevant to this article were reported. we thank all the subjects participating in this study and those who contributed to collection of the clinical re- sources. in particular, we acknowledge members of the diabetes genetics initiative and the finland-u.s. investi- gation of niddm genetics (fusion) for sharing data from their studies. references . prokopenko i, mccarthy mi, lindgren cm. type diabetes: new genes, new understanding. trends genet ; 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: – . hugot jp, chamaillard m, zouali h, lesage s, cezard jp, belaiche j, almer s, tysk c, o’morain ca, gassull m, binder v, finkel y, cortot a, modigliani r, laurent-puig p, gower-rousseau c, macry j, colombel jf, sahbatou m, thomas g. association of nod leucine-rich repeat variants with suscep- tibility to crohn’s disease. nature ; : – i. prokopenko and associates diabetes, vol. , july mutations in kptn cause macrocephaly, neurodevelopmental delay, and seizures report mutations in kptn cause macrocephaly, neurodevelopmental delay, and seizures emma l. baple, , reza maroofian, , barry a. chioza, , maryam izadi, , harold e. cross, saeed al-turki, katy barwick, anna skrzypiec, robert pawlak, karin wagner, roselyn coblentz, tala zainy, michael a. patton, sahar mansour, phillip rich, britta qualmann, matt e. hurles, michael m. kessels, and andrew h. crosby ,* the proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. using linkage analysis and whole-exome sequencing on samples from families from the amish community of ohio, we have demonstrated that mutations in kptn, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and gfp-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis. extremes of brain growth have frequently been associated with impaired neurodevelopment and cognition. occipi- tofrontal circumference is an indirect measure of brain growth and the one most widely used in clinical practice in which macrocephaly (r sds above the mean) is indic- ative of brain overgrowth (megalencephaly) in the absence of hydrocephalus and cranial thickening. the differential diagnosis of macrocephaly relates to the underlying presence or absence of structural brain anomalies. the strong association between macrocephaly and neuro- developmental disability, autism, and other pervasive developmental disorders is well recognized. – where macrocephaly is associated with developmental disability, there appears to be a significantly increased risk of sei- zures. over recent years, family studies have begun to identify gene mutations that might cause inherited forms of developmental disability. these studies have shed important new light on the molecular and cellular pro- cesses that orchestrate the human neuronal circuitry and that might be dysfunctional in neurological disorders. the establishment of the incredibly intricate human neu- ral circuitry is critically dependent upon a complex and tightly regulated myriad of cellular processes and migra- tional cues. the actin cytoskeleton is known to play an important role in the formation, propagation, and steering of cell motility and migration during brain development. this in turn leads to the astonishing morphological intri- monogenic molecular genetics, university of exeter medical school, st. luke i, jena university hospital and friedrich schiller university jena, d- jena of arizona school of medicine, n. alvernon way, tucson, az , usa; ton, cambridge cb sa, uk; laboratory of neuronal plasticity & behaviour road, exeter ex ps, uk; windows of hope genetic study, holmes county, o healthcare nhs trust, london sw qt, uk; department of neuroradiolog these authors contributed equally to this work *correspondence: a.h.crosby@exeter.ac.uk http://dx.doi.org/ . /j.ajhg. . . . � the authors this is an open-access article distributed under the terms of the creative comm reproduction in any medium, provided the original author and source are cre the a . open access u cacy that neurons acquire during neuronal differentia- tion, which is required for the formation of the complex functional neuronal networks underlying human higher brain functions. mutations in genes encoding molecules important for normal function of the actin cytoskeleton have previously been implicated in inherited forms of developmental disability and brain development, – high- lighting the important role of the actin cytoskeleton in neuromorphogenesis. the studies described here derive from the analysis of blood samples obtained for dna extraction (informed consent was obtained from families from the anabaptist communities of ohio according to protocols approved by the institutional review board at the university of arizona and the wandsworth regional ethics committee). nine family members present in four nuclear families were affected by an inherited variable form of neurodevelop- mental delay. the most consistent features were global developmental delay, macrocephaly with frontal bossing, high levels of anxiety, and some features suggestive of a pervasive developmental disorder. additional features included craniosynostosis, recurrent pneumonia, and sple- nomegally. neuroimaging was performed in four cases, and no significant intracranial abnormalities were re- ported. a primary seizure disorder, involving absence or generalized tonic-clonic seizures, was described in three of the nine cases. dysmorphic features were subtle and ’s campus, magdalen road, exeter ex lu, uk; institute for biochemistry , germany; department of ophthalmology and vision science, university wellcome trust sanger institute, wellcome trust genome campus, hinx- , university of exeter medical school, hatherly laboratories, prince of wales h , usa; south west thames regional genetics service, st. george’s y, st. george’s hospital, london sw qt, uk ons attribution license, which permits unrestricted use, distribution, and dited. merican journal of human genetics , – , january , nder cc by license. mailto:a.h.crosby@exeter.ac.uk http://dx.doi.org/ . /j.ajhg. . . http://crossmark.crossref.org/dialog/?doi= . /j.ajhg. . . &domain=pdf http://creativecommons.org/licenses/by/ . / figure . family pedigree and gene mapping (a) pedigree diagram showing all four investigated nuclear families (families – ), which interlink into a single extended family. segregation of the two mutations identified (c. c>a [p.ser *] is denoted by x, and c. _ dup [p.met _gln dup] is denoted by dup) is shown (all genotypings were validated by dideoxy sequence analysis). (b) pictorial representation of the snp genotype data encompassing the chromosome homozygous (solid box) and compound- heterozygous (dashed box) regions in affected individuals. the locus containing the pathogenic variant is demarcated by snps rs and rs ( . mb; families and ). included frontal bossing, broad nasal tip, scaphocephaly, hooded eyelids with small, downslanting palpebral fis- sures, and a prominent chin (table s and figure s , avail- able online). in order to map the chromosomal location of the patho- genic variant, we genotyped samples from families and genome-wide by using illumina human cytosnp- beadchip arrays incorporating ~ , genetic markers. a single notable homozygous . mb region in q. . was found to be shared by all affected individ- uals in families and , although no notable homozygous regions were detected in affected members of families or (figures a and b). considered likely to harbor the path- ogenic variant in these families, the homozygous region identified in families and is delimited by recombinant snp markers rs and rs and contains genes. autozygosity across this interval was corroborated by microsatellite-marker analysis in all family members, which defined a haplotype cosegregating with the disease phenotype (data not shown). to identify the causative mutation, we undertook whole-exome sequence analysis of a single affected individual (ix: , figure a) to generate a profile of variants not present in publically available databases and rare sequence variants. coding regions were captured with a sureselect target enrichment system ( mb) and sequencing on a hiseq system (illumina) with bp paired-end reads. we obtained . gb of reads, of the american journal of human genetics , – , january , which % had a quality score r . approximately % of the reads were marked as duplicates by picard (v. . ) and were excluded before mapping to the human genome reference sequence (grch ). the genome analysis toolkit (gatk, v. . . ) was used to realign reads near potential indel sites and to recalibrate base qualities; single-nucleotide variants were called with gatk and samtools (v. . . ), whereas indels were called with gatk and dindel (v. . ). all variants were annotated with dbsnp ( ) and the genomes pilot study (may ) for minor allele frequency. the variant conse- quences on protein structure were predicted by the variant effect predictor (vep v. . ) with the use of ensembl (v. ). variants were filtered out if the read depth was < or > , , if the consensus quality was < , or if the snp quality was < . after filtering, only one likely delete- rious variant (g. g>t [nc_ . ] [c. c>a (nm_ . ); p.ser * (np_ . )]) in exon of kptn, encoding the amino acid protein kaptin, was identified within the critical region. the presence of the variant was confirmed by dideoxy sequencing, which also confirmed its cosegregation within families and (figure a). seven heterozygous carriers were identified in examined control chromosomes, indicating an allele frequency of approximately . in this commu- nity. the variant is also listed in the national heart, lung, and blood institute (nhlbi) exome sequencing project exome variant server, and one heterozygote has been reported in , european american chromosomes. we next investigated other amish families with chil- dren showing similar unexplained developmental delay associated with macrocephaly, leading to the detection of the c. c>a mutation in the heterozygous state in affected members of of such families (families and ). compared with this mutation’s occurrence in amish control studies, this frequency was higher than expected, prompting us to evaluate kptn for a second mutation that could be acting in conjunction with the c. c>a mutation in these families. subsequent dideoxy sequence analysis of all coding regions and associated splice junctions of kptn in these families revealed that all affected children were indeed compound heterozygous for the c. c>a variant, as well as an in-frame bp duplication (g. _ dup [nc_ . ] [c. _ dup (nm_ . ); p.met _gln dup (np_ . )]) in exon . both the c. c>a and the c. _ dup sequence mutations completely cosegregate with the disease phenotype, as would be expected of causative compound-heterozygous mutations (figure a, lodmax ¼ . simwalk ). the c. _ dup sequence duplication is not listed in genomic sequence databases, and one heterozygote was detected in amish control chromosomes. kptn encodes a largely uncharacterized protein (figure s ). our sequence analyses of kaptin identified no protein domains or homologous human proteins that could provide clues to the functional basis of the neurolog- ical deficits associated with its alteration. we therefore investigated the expression and localization of kaptin in neurons. in order to do so, we first cloned human kaptin from human embryonic kidney cell cdna obtained by rna isolation and rt-pcr as previously described. full-length kaptin (amino acids – ) was generated by pcr using primers bq ( -aagaattcatgatggggg aggcg- ) and bq ( -aaggatccttaagaggctg catt- ). the pcr product was digested with ecori and bamh and cloned in-frame into pegfp-c and subcl- oned into pcmv-tag . primary rat hippocampal cultures were prepared and cultured as previously described. , neurons were transfected with lipofectamine (invi- trogen) on days , , and in vitro, fixed in % parafor- maldehyde in pbs for min at room temperature and hr after transfection, and processed for immunofluores- cence microscopy. confocal imaging was performed with a zeiss axio observer equipped with apotome and zeiss plan-apochromat / . and / . objectives and an axiocam mrm ccd camera (zeiss). primary rat hippocampal neurons, identifiable by anti-map immu- nostaining (sigma, abcam), were transfected with wild- type flag-tagged kaptin (flag-kaptin) at div and imaged days later. wild-type flag-kaptin was observed to be local- ized at f-actin-rich foci in close proximity to the cell bodies and at growth cones (figures a and b, arrow heads). at later stages of development, when neurons established the a synapses (div ), wild-type flag-kaptin again colocalized with f-actin-rich sites. along dendrites, these sites appeared mainly to represent f-actin-rich postsynapses, given that almost all kaptin-enriched puncta were contacted by presynaptic structures containing bassoon (a marker for presynaptic active zones) (figure c). to be able to undertake immunolabeling experiments of endog- enous kaptin, we first characterized polyclonal rabbit anti- kaptin (sigma) in cos- cells. cells expressing wild-type flag-kaptin were highlighted by anti-kaptin immuno- labeling (figure s a). coimmunostaining of primary rat hippocampal neurons at div with anti-kaptin and anti-shank (neuromab) demonstrated that the dendritic accumulations of flag-kaptin at f-actin-rich puncta are indeed of physiological relevance and reflect the locali- zation of endogenous kaptin at postsynapses. the vast majority of anti-kaptin-immunolabeled puncta were not only enriched with f-actin but were additionally immuno- positive for shank , a postsynaptic scaffold protein inter- acting with f-actin binding proteins; , figure d). kaptin thus appears to be associated with dynamic actin cytoskeletal structures of neuronal cells. consistent with this, wild-type flag-kaptin accumulated at cos- cell lamellipodia (figure s b), the subcellular regions marked by dense arrays of dynamic actin filaments in mobile fibro- blasts. in neurons, the cortical actin cytoskeleton is known to be important for proper neuronal-network formation during development. our observations therefore suggest a role for kaptin in neuromorphogenesis. the c. c>a sequence variant is predicted to introduce a premature stop codon and result in loss of function as a result of degradation of the mutated transcript by mrna-surveillance mechanisms; however, because of a lack of patient material, we have been unable to confirm whether a truncated protein (lacking the c-terminal amino acids – ) is produced. in contrast, the in-frame c. _ dup mutation is likely to result in the insertion of six amino acids (met-trp-ser-val-leu-gln) into the full- length kaptin. in order to investigate the functional outcome of this mutation, we undertook in silico analysis of the secondary structural elements of wild-type and altered kaptin. this revealed that the n-terminal half of kaptin is likely to comprise a series of relatively densely organized b sheets, interspersed by only three a helices, and becomes more a-helical starting with a-helix (span- ning amino acids – ; figure a). sequence and pre- dicted structural conservation of both the n-terminal and c-terminal portions of kaptin are very high even between evolutionarily distant mammalian species (figure s ). strikingly, duplication of the six amino acids ( – ) is predicted to disrupt a-helix and result in its conversion into an extended b sheet (figure c) and is therefore likely to have a profound effect on kaptin function. in order to experimentally explore the functionality of any protein arising from translated mutant transcripts, we generated both disease-associated gfp-tagged mutants. we generated the p.met _gln dup altered kaptin merican journal of human genetics , – , january , figure . kaptin immunolocalization studies (a and b) flag-kaptin colocalized with f-actin-rich foci at the cell body and in growth cones (examples of both are marked by arrow heads in a) in div rat hippocampal neurons transfected at div . for clarity, the anti-map immunostaining was omitted from the merged images. scale bars represent mm. (c and d) endogenous kaptin immunostained together with the presynaptic marker bassoon at div (c) and with the postsynaptic marker shank at div (d). puncta enriched with anti-kaptin immunoreactivity (marked by arrow heads) were rich in f-actin, as shown by fluorescently labeled phalloidin. (c) furthermore, they were usually contacted by presynapses. the scale bar represents mm. (d) postsynapses marked by shank were largely positive for both f-actin and anti-kaptin immunolabeling (arrow heads). for clarity, the anti-f-actin staining was omitted from the merged image in (d). high-magnification images are shown. the scale bar represents . mm. (gfp-kaptinp.met _gln dup) by fusing an n-terminal portion carrying the duplication and a smai site introduced as a silent mutation (primers bq [ -aagaattcat gatgggggaggcg- ] and bq [ -tcacccggga gatgggcccgtcttgcaacacgctccacatctgcagg accgaccac- ]) with a c-terminal portion contain- ing a smai site also introduced by silent mutation (primers bq [ -atctcccgggtgattgtgttcag- ] and bq [ -aaggatccttaagaggctgcatt- ]). we generated the p.ser * altered kaptin (gfp- kaptin – ) by pcr using primers bq ( -aagaattc atgatgggggaggcg- ) and bq ( -aagtcgac ctagaggctgaacac- ). pcr products were cloned in- frame into pegfp-c . whereas wild-type gfp-tagged kaptin (gfp-kaptin) was found to localize at f-actin-rich lamellipo- dia of cos- cells, both altered forms of kaptin displayed no f-actin association but instead accumulated at irregular perinuclear sites (figures d– f). gfp-kaptin – showed a more pronounced tendency to form such accumulations; almost all cells were marked with little additional cyto- plasmic staining outside of these foci. alternatively, gfp- kaptinp.met _gln dup typically displayed fewer and slightly smaller accumulations; there were slightly higher levels of cytoplasmatic staining outside of these foci (fig- the american journal of human genetics , – , january , ures d– f). this indicates that both proteins are likely to benonfunctional, although wecannot exclude thepossibil- ity that misfolded altered protein might accumulate in neu- rons of affected individuals and lead to dominant-negative effects on other neuronal proteins or cell processes. finally, because kptn mutations result primarily in a form of neurodevelopmental disease, we also analyzed the behavior of the altered forms of kaptin in primary hippo- campal neurons during early development. whereas wild- typegfp-kaptin was againfoundtocolocalizewith dynamic f-actin in growth cones and foci at the cell body, both gfp- kaptin – and gfp-kaptinp.met _gln dup were found to accumulate in a manner reminiscent of the cos- studies in the cell body or at perinuclear sites (figures g– i). we investigated a number of families from the anabaptist communities of ohio and found that multiple individuals aged – years were affected by a syndrome in which the cardinal features include macrocephaly, global develop- mental delay, behavioral abnormalities, and seizures. our molecular studies determined that two distinct founder mutations affecting the same gene (kptn, encoding kaptin), both of which have become entrapped within the community, are responsible. compared with individuals found to be compound heterozygous for p.ser * figure . immunolocalization studies of altered kaptin (a–c) schematic representation of wild-type human kaptin (a), the truncated p.ser * (c. c>a; gfp-kaptin – ) (b), and the dupli- cation (gfp-kaptinp.met _gln dup) (c). on the left is a graphic overview of secondary structures; b sheets are shown as red boxes, and a-helices are shown as blue ellipsoids. on the right are amino acids and secondary-structure elements around amino acid . a-helix is predicted to be converted into a b sheet by the insertion of amino acids – , as shown in yellow. (d–f) localizations of gfp-kaptin, gfp-kaptin – , and gfp-kaptinp.met _gln dup (green in merges) in cos- cells counterstained with phalloidin (red in merged images). note that whereas wild-type kaptin was distributed in the cytosol and accumulated at f-actin-rich lamellipodia (d), both alterations showed accumulations at perinuclear regions (e and f). (legend continued on next page) the american journal of human genetics , – , january , and p.m _q dup, those individuals found to be homozygous for the p.ser * nonsense alteration ap- peared to be more severely affected given that they had a higher incidence of seizures and a greater degree of intel- lectual impairment. this might indicate that p.met _ gln dup retains a limited functionality in vivo, and perhaps consistent with this, we observed that p.met _gln dup showed a slightly lesser tendency than p.ser * to form perinuclear accumulations in our transfection studies. however, the sample cohort is currently too small for confidently determining any geno- type-phenotype correlation. kaptin is a largely uncharacterized protein originally iso- lated from human blood platelets but subsequently found to be expressed in fibroblasts and intestinal and sensory epithelia. a previous study of this molecule suggested a role at stereocilia tips, and so kptn was proposed as a candidate gene for hearing loss. however, the affected individuals described in this study have no evidence of sensorineural hearing deficits. during development, the actin cytoskeleton plays a pivotal role in neuronal cell morphology and migration, including the generation, pro- trusion, and steering of growth cones and the formation of postsynapse and dendritic spines. – our studies confirm kaptin expression in neuronal (map -positive) cells. given that kaptin was found to localize to f-actin-rich structures, it is conceivable that loss of kaptin function could either directly or indirectly lead to impairment of the neuronal actin cytoskeleton, required for dendritic arborization and/or spine formation, and result in the disease pheno- type described. support for this has been provided by studies of rab b, a small gtpase associated with the golgi apparatus; alterations in this protein lead to its downregulation and a concomitant reduction in dendritic arborization and synapse formation. this was previously associated with a disease phenotype comprising mental retardation, epilepsy, and macrocephaly, , features which overlap with those described here as arising from kaptin alterations. similarly, deficiencies of rho gtpases, which regulate the actin cytoskeleton by a growing variety of effector proteins, have been associated with intellectual disability and defects in spine structure. – several other actin-associated proteins, including drebrin a, cortactin, and abp , have also been found to decrease spine density or formation, – and a growing body of evidence sup- ports a role for the arp / complex and directly and indi- rectly associated proteins in postsynapse formation and proper development of neuronal morphology. , , , – our analyses reveal that wild-type kaptin is enriched in neuronal growth cones and at discrete cortical sites of neu- rons at early developmental stages. furthermore, wild-type kaptin was found to accumulate at postsynapses of neu- (g–i) transfection of div rat hippocampal neurons with wild-type a kaptin was found throughout the cell and showed accumulations at g in contrast, both alterations accumulated in areas of the cell body (h a green, map in red, and phalloidin in blue. scale bars represent m the american journal of human genetics , – , january , rons undergoing synaptogenesis (div ), as indicated by spatial correlation along dendrites of kaptin accumula- tions by phalloidin and synaptic-marker immunostaining. consistent with this, kaptin was also found to be present in the postsynapses of mature neurons. the sites demar- cated by kaptin localization represent areas of high f-actin content and high actin dynamics. an association between kaptin and dynamic f-actin was also indicated by the observed accumulations of flag-kaptin at the dynamic lamellipodia as opposed to the more static stress fibers in cos- cells. these observations are consistent with the suggestion of a lamellipodial localization of kaptin in chicken embryonic fibroblasts and with the original iso- lation of kaptin from blood cells with the use of f-actin columns. taken together, our studies indicate that both of the identified kptn mutations are likely to result in loss of function of kaptin, either by degradation of the mutant transcript via mrna-surveillance mechanisms (c. c>a) or by the production of mislocalized and/or nonfunc- tional protein products. these kptn mutations result in a distinctive clinical syndrome, and the presence of macrocephaly combined with global developmental delay should prompt the diagnostic analysis of kptn in affected individuals from anabaptist communities. the potential benefits of early diagnosis in this condition are indicated by the improvement in developmental markers in our study’s youngest two affected individuals, both of whom received early and intensive developmental interventions, although the lack of seizures in these individuals might also have been beneficial. finally, our identification of two kptn founder mutations within this anabaptist population parallels the situation seen for a number of other genes with multiple mutations that also commonly cause inherited diseases globally (e.g., gjb mutations in inherited hearing loss, and atm mutations in ataxia telangiectasia), indicating that kaptin developmental delay might be similarly widespread. supplemental data supplemental data include four figures and one table and can be found with this article online at http://www.cell.com/ajhg. acknowledgments first, we are very grateful to the amish families for partaking in this study and to the amish community for their continued support of the windows of hope project. we are grateful to olivia wenger for contributing clinical data. rabbit polyclonal anti- bassoon was a generous gift from e.d. gundelfinger (leibniz insti- tute for neurobiology, magdeburg). illumina cytosnp analysis and sanger sequencing were carried out at the medical biomics centre nd altered kaptin (cells were fixed and imaged at div ). wild-type rowth cones and f-actin-rich sites of the cell body (g; arrow heads). nd i; arrows). merged images show wild-type and altered kaptin in m. http://www.cell.com/ajhg (st. george’s university of london). the work was supported by the medical research council (g , g ), wellcome trust (wt ), newlife foundation, and research to prevent blindness. received: july , accepted: october , published: november , web resources the urls for data presented herein are as follows: genomes, http://browser. genomes.org/index.html ensembl genome browser, http://www.ensembl.org/index.html gatk, http://www.broadinstitute.org/gatk/about/citing-gatk ncbi, http://www.ncbi.nlm.nih.gov/ nhlbi exome sequencing project (esp) exome variant server, http://evs.gs.washington.edu/evs/ online mendelian inheritance in man (omim), http://www. omim.org polyphen- , http://genetics.bwh.harvard.edu/pph / provean, http://provean.jcvi.org/seq_submit.php pubmed, http://www.ncbi.nlm.nih.gov/pubmed/ samtools, http://samtools.sourceforge.net sift, http://sift.jcvi.org/ variant effect predictor (vep), http://useast.ensembl.org/info/ docs/tools/vep/index.html ucsc human genome browser, http://www.genome.ucsc.edu references . stevenson, r.e., schroer, r.j., skinner, c., fender, d., and simensen, r.j. 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( ). cop limits dendritic branching via cullin -dependent degradation of the actin- crosslinking btb-domain protein kelch. plos one , e . 	mutations in kptn cause macrocephaly, neurodevelopmental delay, and seizures 	supplemental data 	acknowledgments 	web resources 	references cells of origin in the embryonic nerve roots for nf -associated plexiform neurofibroma cancer cell article cells of origin in the embryonic nerve roots for nf -associated plexiform neurofibroma zhiguo chen, chiachi liu, amish j. patel, , chung-ping liao, yong wang, and lu q. le , , ,* department of dermatology cancer biology graduate program harold c. simmons cancer center utsw neurofibromatosis clinic university of texas southwestern medical center, dallas, tx - , usa *correspondence: lu.le@utsouthwestern.edu http://dx.doi.org/ . /j.ccell. . . summary neurofibromatosis type is a tumor-predisposing genetic disorder. plexiform neurofibromas are common nf tumors carrying a risk of malignant transformation, which is typically fatal. little is known about mech- anisms mediating initiation and identity of specific cell type that gives rise to neurofibromas. using cell-line- age tracing, we identify a population of gap + plp+ precursors in embryonic nerve roots as the cells of origin for these tumors and report a non-germline neurofibroma model for preclinical drug screening to iden- tify effective therapies. the identity of the tumor cell of origin and facility for isolation and expansion provides fertile ground for continued analysis to define factors critical for neurofibromagenesis. it also provides unique approaches to develop therapies to prevent neurofibroma formation in nf patients. introduction the tumor predisposition disorder neurofibromatosis type i (nf ) is among the most common human genetic diseases and is caused by mutation in the nf tumor suppressor gene, which encodes a gtpase activating protein (gap) that negatively regu- lates p -ras signaling (ballester et al., ; xu et al., ). nf patients have defects in neural crest-derived tissues, leading to a wide spectrum of clinical presentations, including develop- mental, pigment or neoplastic aberrations of the skin, nervous system, bones, endocrine organs, blood vessels, and the eyes (cichowski and jacks, ; ward and gutmann, ; zhu et al., ). while nf patients are predisposed to developing multiple tumor types (cichowski et al., ; jett and friedman, ; le and parada, ; shannon et al., ; vogel et al., ), the most common occurring are neurofibromas. neurofi- bromas are unique and complex tumors that contain proliferating schwann-like cells and other local supporting elements of the nerve fibers, including perineurial cells, fibroblasts, and blood significance in this study, we utilized a genetically engineered mouse mod broma. using cell-lineage tracing, we showed that the embryon nal nerve roots are the cells of origin for plexiform neurofibroma immediate progenitors in the initiation of tumors, consistent w primitive precursors and that most cells in an organ do not gene ular cancer (the cells of origin) will permit greater understandin for their treatment. can vessels, as well as infiltration of mast cells. neurofibromas are classified into different subtypes. however, for clinical and prog- nostic implications, many clinicians simply refer to these tumors as either dermal or plexiform. dermal neurofibromas are exclu- sively in the skin and occur in virtually all individuals with nf . they initially appear at puberty and increase in number with age. although similar to dermal neurofibromas at the cellular and ultrastructural levels, plexiform neurofibromas develop along a nerve plexus or involve multiple nerve bundles and are capable of forming large tumors. unlike their dermal counterpart, plexi- form neurofibromas are thought to be congenital and progres- sively enlarge throughout life. they carry a risk of malignant transformation that can metastasize widely and are often fatal. plexiform neurofibromas can occur anywhere along peripheral nerve plexus. in fact, deep-tissue neurofibromas occur in %– % of adult nf patients (tonsgard et al., ). the majority of internal plexiform neurofibromas manifest in the para-spinal re- gion associated with dorsal root ganglia (drg). their chance of malignant transformation is much higher compared with other el as a tool to identify the cell of origin for plexiform neurofi- ic gap + plp+ schwann cell precursors originate from spi- s. our studies point to the importance of stem cells and their ith the notion that these neoplasms originate in a subset of rate tumors. identifying which cell type gives rise to a partic- g of their pathogenesis and inform better design approaches cer cell , – , november , ª elsevier inc. mailto:lu.le@utsouthwestern.edu http://dx.doi.org/ . /j.ccell. . . http://crossmark.crossref.org/dialog/?doi= . /j.ccell. . . &domain=pdf cancer cell cells of origin for plexiform neurofibroma forms of plexiform neurofibromas and carries a poorer prognosis, in part because they are not evident clinically in the early stage. in addition, because of their location at the neural foramina of the vertebral column, they can impinge on the spinal cord and nerve roots, causing pain and neurological deficits. thus, these para- spinal neurofibromas represent a serious complication of nf . a large body of direct and indirect studies has provided evidence that nf gene deletion is the requisite initial step that precedes the cascade of interactions with other cell types in the microenvironment as well as additional cell autonomous modifications for neurofibromagenesis (joseph et al., ; le et al., ; wu et al., ; zheng et al., ; zhu et al., ). early speculation regarding the cells of origin for neurofi- bromas came from genetic studies examining the participation of different cell types including neural crest derivatives in the pathogenesis of many of the clinical presentations of nf , including neurofibroma. in human neurofibromas, schwann- like cells with biallelic nf mutations are the most consistently found cell type, leading to the argument that the tumors initiate in schwann cells or their earlier precursors. indeed, genetic mouse models have demonstrated that nf deletion in the schwann cell lineage is the genetic bottleneck for neurofibroma development (cichowski et al., ; joseph et al., ; vogel et al., ; wu et al., ; zheng et al., ; zhu et al., ). during the development of peripheral nerves, neural crest cells generate schwann cells in a process that parallels embryonic development. migrating neural crest stem cells emerge from the dorsal horns of the neural tube and move through immature connective tissue before the time of nerve formation and then differentiate into schwann cell precursors (scps). these scps then become immature schwann cells in the developing periph- eral nerves until early neonatal stages. the differentiation of immature schwann cells to form the non-myelinating and myeli- nating cells of mature nerves essentially occurs postnatally (jes- sen and mirsky, ; woodhoo and sommer, ). given that the majority of plexiform neurofibromas in human are congenital and generally detected at birth or in early infancy, they must arise from the early schwann cell lineage. indeed, genetic mouse models have demonstrated that nf deletion in the embryonic schwann cell lineage is the genetic bottleneck for neurofibroma development (cichowski et al., ; joseph et al., ; vogel et al., ; wu et al., ; zheng et al., ; zhu et al., ). however, the exact developmental stage and embryonic loca- tion of the schwann cell lineage that initiate neurofibroma forma- tion remain unknown. nf patients frequently have plexiform neurofibromas in the para-spinal region at the neural foramina associated with drg. in addition, existing mouse models of nf also develop neurofibromas at the drg regions. we wish to identify the cells of origin for these para-spinal plexiform neurofibromas. we reason that they reside in the vicinity of the embryonic drgs or at the nearby nerve roots. results loss of nf in embryonic drg/nerve root neurosphere cells gives rise to classic plexiform neurofibroma in vivo recent studies have established the value of in vitro sphere assay to identify cancer stem cells as well as neural stem/pro- cancer cell , – , november , ª elsevier inc genitor cells (le et al., ; williams et al., ). therefore, we examined whether nf -deficient embryonic drg/nerve root neurosphere cells (dnscs) could contain the elusive cells of origin of para-spinal neurofibromas. to obtain nf �/� dnscs, we dissected drgs/nerve roots from e . nf flox/flox; rosa (r r) mouse embryos (hall, ) and performed the neurosphere culture. these neurosphere cells have high self- renewal and proliferative potential (figures s a–s d available online). they were subsequently infected with adenovirus car- rying the cre recombinase (ad-cmv-cre) to ablate nf and generate nf �/�;lacz+ dnscs, which was verified by genomic analysis of nf (figure a). we next implanted nf �/�;lacz+ dnscs into the sciatic nerves where close proximity to the nerve environment could be achieved as previously established through an in vivo neuro- fibromagenesis assay (le et al., ). the transplanted nf �/�;lacz+ dnscs gave rise to plexiform neurofibroma within four months post implantation, whereas the transplanted nf flox/flox;r r controls showed no signs of tumor growth (figures ba– bd and table ). these tumors exhibited all histo- logical and immunohistochemical features of human plexiform neurofibromas, including disorganized spindle cells, abundant collagen matrix, heavy infiltration of mast cells and macro- phages, expression of the schwann cell marker s b, and gap (figure c; figure s e). to verify these tumors arise from transplanted drg/nerve root neurosphere cells rather than from the host cells, we took advantage of the fact that the transplant cells carried the lacz marker gene but the host did not. x-gal staining shown that these tumor cells were lacz+, which suggested that they arose from transplanted nf �/�;lacz+ dnscs. taken together, these data indicate that when placed in a favorable microenvironment (in proximity to a peripheral nerve), nf �/�;lacz+ dnscs can give rise to bona fide plexiform neurofibromas. as such, these results show that dnscs have the full potential to generate neurofibromas and indicate that the cells of origin for para-spinal neurofibromas may be in the embryonic drgs/nerve roots. cells of origin for para-spinal plexiform neurofibromas are inside the embryonic plp+ cells although we successfully induced neurofibroma formation by transplanting embryonic nf �/� dnscs to sciatic nerves, it is un- likely that all the embryonic dnscs could give rise to plexiform neurofibroma. in genetic mouse models, nf ablation in embry- onic schwann cell lineage (plp, krox , and dhh positive cells) was sufficient to induce para-spinal plexiform neurofibromas (le et al., ; wu et al., ; zhu et al., ), suggesting that all or at least part of the cells of origin might be inside these plp+, krox +, and dhh+ embryonic schwann cell lineage. therefore, we next sought to determine this subpopulation within the em- bryonic dnscs that could give rise to neurofibroma. to achieve this, we crossed plp promoter-driven, tamoxifen- inducible cre (plpcre-ert) (leone et al., ) transgenic mice into a nf flox/flox;r r-lacz (le et al., ) and flox-stop-flox r r-yfp (chen et al., ) background to generate plpcre-ert;nf flox/flox;r r-lacz-yfp mice. we selectively ablated the embryonic nf expression by administering mg tamoxifen orally at embryonic day . (e . ) to pregnant nf flox/flox;r r-lacz-yfp female mice that have been bred . figure . loss of nf in embryonic dnscs gives rise to classic plexiform neurofibroma in vivo (a) diagram of experimental design: isolation of drgs/nerve roots from e . nf flox/flox;r r-lacz embryos (a) and dnscs culture (b); dnscs were in- fected with ad-cre to ablate nf ; ad-gfp was used as a control (c); and nf �/�;lacz+ and nf flox/flox ;lacz� dnscs were implanted to right and left sciatic nerves of nude mice, respectively (d). (b) x-gal and h&e staining were performed on left (a and b) and right (c and d) sciatic nerve. (c) tumor on right sciatic nerve was stained with antibody against s b and gap . blue scale bars, mm. white and black scale bars, mm. see also figure s . table . the cells of origin for spinal plexiform neurofibroma are inside the embryonic plp+ dnscs genotype dnscs selected ablation of nf tumors/nude mice injected percentage p value nf f/f;lacz total ad-cre / . . total ad-gfp / plpcre-ert; nf f/f;lacz- yfp yfp+ / < . yfp� / plpcre-ert; nf f/f;lacz- yfp yfp+ ad-cre / . < . yfp� ad-cre / . krox -cre; nf f/f;lacz- yfp yfp+ / . < . yfp� / krox -cre; nf f/f;lacz- yfp yfp+ ad-cre / . . yfp� ad-cre / . dhh-cre; nf f/f;lacz- yfp total ad-cre / . yfp� ad-cre / cancer cell cells of origin for plexiform neurofibroma with plpcre-ert;nf flox/flox;r r-lacz-yfp male mice. we then dissected the drgs/nerve roots from embryos at e . and per- formed dnscs culture. in order to isolate the plp+ and plp� from these e . dnscs, we used fluorescence-activated cell sorting (facs) to obtain the yfp+ and yfp� dnscs which rep- resented plp+ and plp� dnscs, respectively. next, these plp+ and plp� dnscs were transplanted in proximity to the sciatic can nerve of nude mice. thirteen of ( %) mice transplanted with plp+ dnscs developed sciatic plexiform neurofibroma (table ). by contrast, plp� dnscs only led to neurofibroma in of ( %) mice (table ), which suggests that plp+ dnscs are more likely than plp� dnscs to contain the cells of origin and that neurofibroma development in the plp� dnscs im- planted mice is likely the result of contamination of plp+ dnscs during the sorting process as loss of nf is required for neurofi- broma development. therefore, given that the nf have been ab- lated in plp+ dnscs and not in plp� dnscs after the tamoxifen induction, we could not exclude the possibility that nf ablation in plp� dnscs would also give rise to neurofibroma. to address this issue, both plp+ and plp� dnscs were infected with ad- cmv-cre to generate the plp+;nf �/� and plp�;nf �/� dnscs. when transplanted to the sciatic nerve of nude mice, plp+; nf �/� dnscs maintained their higher capability to give rise to neurofibroma ( of , . %), whereas plp�;nf �/� dnscs could only give rise to plexiform neurofibroma in out of ( . %) mice (figure a and table ). strikingly, we also observed spontaneous transformation into malignant peripheral nerve sheath tumors (mpnst) arising from a fraction of these sciatic plexiform neurofibromas that originated from plp+; nf �/� dnscs (figures ai– al). this is consistent with human clinical observations that about % of plexiform neurofibromas undergo malignant transformation (evans et al., ). these data further indicate that the cells of origin might be within the embryonic plp+ dnscs population. however, since nf ablation in krox + and dhh+ embryonic schwann cell lineage could also give rise to para-spinal plexi- form neurofibromas, we next examined whether the cells of origin are also inside the embryonic krox + or dhh+ dnscs (wu et al., ; zhu et al., ). we utilized the same strategies as above using the krox -cre and dhh-cre transgenic mice. unexpectedly, we found that not only the krox +;nf �/� and total dnscs (contained dhh+;nf �/� dnscs) but also the cer cell , – , november , ª elsevier inc. figure . the cells of origin for para-spinal plexiform neurofibroma are inside the em- bryonic plp+ dnscs (a) facs was performed to obtain the yfp+ and yfp� dnscs from e . plpcre-ert;nf flox/flox; r r-lacz-yfp embryo, whose mother was administered with tamoxifen at e . (a and b). both yfp+ and yfp� dnscs were infected with ad-cre to ablate nf , then injected to right and left sciatic nerves of nude mice respectively (c). x-gal and h&e staining were performed on the left and right sciatic nerve (d–f). sections of right sciatic nerve were stained with antibody against s b (g) and gap (h). a fraction of these sciatic plexiform neurofibromas spontaneously trans- formed into mpnst, exhibiting both benign and malignant histologic characters (i–l). immunofluo- rescence staining of tumor on right sciatic nerve shows expression of yfp as well as gap and s b schwann cell markers (m–r). (b and c) a similar strategy to that in (a) was used for yfp+ and yfp� dnscs derived from e . embryos with genotype krox -cre;nf flox/flox; r r-lacz-yfp (b) and dhh-cre;nf flox/flox; r r-lacz-yfp (c). blue scale bars, mm. white and black scale bars, mm. cancer cell cells of origin for plexiform neurofibroma krox �;nf �/� and dhh�;nf �/� dnscs gave rise to neurofi- broma with high percentage (figures b and c and table ). these results demonstrate that plp+ dnscs encompass the cells of origin pool, whereas krox +;nf �/� and dhh+;nf �/� cancer cell , – , november , ª elsevier inc. dnscs might only account for partial population of the cells of origin. the plp+ cells of origin originate in the embryonic nerve roots to determine if the embryonic krox + and dhh+ cells are included within plp+ cell population, we administered tamox- ifen orally to mother mice at e . to activate plpcre-ert to induce the expression of yfp and genetically label the plp+ cells. we next performed immu- nofluorescence staining against krox and dhh and observed that the majority of the endogenous krox and dhh sig- nals colocalized with yfp (plp) in the nerve root, drg, and peripheral nerve (figure a), which indicated that most of the krox + and dhh+ cells were included in the plp+ cell population. we also found that plpcre+ (yfp+ cells) colo- calized with endogenous plp, suggest- ing that the plpcre+ population is inside the endogenous plp+ cell population (figure s a). we then took advantage of another marker, lacz, to compare the specific expression pattern of lacz among plp, krox , and dhh cre mice. specifically, for embryos with geno- type plpcre-ert;r r-lacz, we gave the mother mice a single dose of -hydroxytamoxifen, an active metabolic product of tamoxifen that can be cleared relatively quickly from serum figure . cells of origin for para-spinal plexiform neurofibromas are inside the em- bryonic plp+ nerve root cells (a) representative frozen sections from e . plpcre-ert;nf flox/flox;r r-lacz-yfp embryo, whose mother was administered with tamoxifen at e . , were stained for yfp, krox , dhh, and dapi. nr, nerve root; drg, dorsal root ganglia. (b–d) x-gal and h&e staining were performed on embryos with genotype plpcre-ert;r r-lacz (b, mother mouse was administered with -hy- droxytamoxifen at e . ), krox -cre;r r-lacz (c), and dhh-cre;r r-lacz (d) from e . to e . . arrow, area of enlarged view; arrow head, nerve roots; star mark, drg. (e) drg and dorsal nerve root were separated from e . embryos and cultured in neurosphere culture conditions. (f) nerve root derived neurospheres immuno- stained for nestin, gfap, s b, p , ki , and dapi. (g) immunostaining of nerve root derived neuro- sphere cells cultured in neurosphere medium (a, c, e, and g) or differentiation medium (b, d, f, and h) with antibodies against s b, gap , biii- tubulin, and olig . blue scale bars, mm. white and black scale bars, mm. see also figure s . cancer cell cells of origin for plexiform neurofibroma (robinson et al., ), to acutely induce the expression of lacz in plp+ cells at e . . on the other hand, krox -cre and dhh- cre are noninducible and, therefore, tamoxifen induction is not needed. we isolated the embryos at different time points and performed whole-mount x-gal staining. for the plpcre- ert;r r-lacz mice, the lacz (plp) positive staining began cancer cell , – , n to show up inside the nerve roots, drgs, and trigeminal ganglion (tg) at e . (figure b). histological analysis indicated that the lacz+ (plp+) cells were mainly inside the nerve roots and only few scattered lacz+ (plp+) cells could be seen in drg (figure b). from e . to e . , more lacz+ (plp+) cells appear in nerve roots, drgs, tgs, and eventually peripheral nerves (figure b). in contrast, in the krox -cre mice, the lacz+ (krox +) cells first appear in nerve roots at e . and stay in nerve roots until e . when some peripheral nerves started to express the lacz (krox ). however, there were very few lacz+ (krox +) cells inside the drg (figure c). similarly, in dhh-cre mice, we could only observe very few lacz+ (dhh+) cells in nerve roots after e . . from e . to e . , more lacz+ (dhh+) cells were detected only at nerve roots and some of the peripheral nerves (figure d), which were consistent with previous find- ings that dhh transcripts were expressed by scps both in the dorsal and ventral roots at e (parmantier et al., ). all of these data suggest that the plp+ cells first appear in embryonic nerve roots at least one day earlier and in a wider cell population than that of krox + and dhh+ cells and that plp+ dnscs represent a wider pool for the cells of origin. consistently, when we carefully separated out the e . ovember , ª elsevier inc. figure . phenotypic analysis identifies the plp+ cells of origin as embryonic schwann cell precursors (a) dnscs obtained from e . plpcre-ert; nf flox/flox;r r-yfp embryos, whose mother was administered with tamoxifen at e . , were stained for yfp, dapi, and lineage markers: nestin (a), p (b), blbp (c), gfap(d), s b (e), biii- tubulin (f), and gap (g–i). (b) formalin fixed paraffin embedded sections from e . plpcre-ert;nf flox/flox;r r-yfp em- bryos, whose mother was administered with tamoxifen at e . , were stained for yfp, dapi, and lineage markers: gap (a–c), nestin (d), p (e), blbp (f), biii-tubulin (g), gfap (h), and s b (i). drg, dorsal root ganglia; nr, nerve root; pn, peripheral nerve; sc, spinal cord. all scale bars, mm. see also figure s . cancer cell cells of origin for plexiform neurofibroma embryonic dorsal nerve roots from the drgs, we found that only the embryonic dorsal nerve roots could robustly give rise to neu- rospheres, but not the e . drgs (figure e). however, in adult mice, we found that the drgs can also give rise to neurospheres (figure s b). further immunofluorescence analyses demon- strate that these embryonic nerve roots generated neurosphere cells that express neural crest markers (nestin and p ), imma- ture schwann cell marker gfap, and proliferation marker ki- , but not the mature schwann cell marker s b (figure f). cancer cell , – , november , ª elsevier inc. under differentiation conditions, these neurosphere cells could differentiate into schwann cells and neurons, but not the oligodendrocytes (figure g). these data further narrow the candidate cells of origin to plp+ cells in the embryonic nerve roots. phenotypic analysis identifies the plp+ cells of origin as embryonic schwann cell precursors boundary cap (bc) cells are transient neural crest-derived population of stem cells located at the dorsal root entry zone and give rise to most schwann cells in the dorsal roots and drgs (coulpier et al., ; hjerling-leffler et al., ; topilko et al., ). therefore, bc cells might be the embryonic neurofibroma tu- mor cells of origin. however since krox is a marker of bc cells and both krox +; nf �/� and krox �;nf �/� dnscs could give rise to sciatic plexiform neurofi- broma, this indicates that bc cells are un- likely to be the cells of origin, but rather an even earlier cell that is plp+. to pheno- typically characterize these plp+ cells of origin, we gavaged mother mice with tamoxifen at e . , harvested drgs/ nerve roots from plpcre-ert;nf flox/flox; r r-yfp embryos at e . for neuro- sphere cell culture, and performed immunofluorescence ana- lyses. we found that the majority of neurosphere cells, either yfp+(plp+) or yfp�(plp�), expressed nestin and p , which confirms that they originate from the neural crest (figures aa and ab). we also observed the expression of embryonic schwann cell markers blbp and gfap in the minority of both yfp+(plp+) and yfp�(plp�) cells, but none expressed the immature and mature schwann cell marker s b (figures ac–a e). in addition, the majority of yfp+(plp+) cells did not figure . cell lineage tracing reveals plp+ precursors in the embryonic nerve roots as the cells of origin for plexiform neurofibromas (a) x-gal staining of whole spinal cord, drgs and partial peripheral nerves from plpcre-ert; nf flox/�;r r-lacz mouse, whose mother was administered with tamoxifen at e . . there is strong x-gal staining in the enlarged drgs (star mark), enlarged intercostal nerve (arrow head), and nerve root (arrow). (b) h&e and immunohistochemical staining for gap and s b in sections from these enlarged lacz+ drgs (star mark in a). (c) h&e staining of the drg and its associated enlarged intercostal nerve (ein) in (a) (arrow head). enlarged intercostal nerve was also stained for gap and s b. (d) representative h&e and immunohistochem- ical analyses of early neurofibroma in dorsal root and associated normal drg (arrow in a). drg, dorsal root ganglia; dr, dorsal root; sc, spinal cord; vr, ventral root. blue scale bars, mm. all other scale bars, mm. cancer cell cells of origin for plexiform neurofibroma express neuronal marker biii-tubulin (figure af). strikingly, every yfp+(plp+) cell also expressed gap , a schwann cell marker that is known to first appear at the precursor stage (fig- ures ag– ai), and sox , a marker for neural crest origin and future schwann cell lineage (figure s a) (kalcheim and rohrer, ). because these plp+gap + cells are also p positive but s b negative, they are at schwann cell precursor stage (jessen and mirsky, ). it is well known that cell culture in vitro may vary from that in vivo. therefore, we also character- ized these plp+ cells in e . plpcre-ert;nf flox/flox;r r-yfp embryos and observed that yfp+(plp+) cells were colocalized with gap mainly at nerve roots (figures ba– bc). similar to our in vitro data, they also expressed nestin, p , blbp, and sox (figures bd– bf; figure s b). they were also colocal- ized with biii-tubulin (figure bg). however, they did not express gfap and s b in vivo (figures bh and bi). these findings indicate that plp+gap + cells in the embryonic nerve roots are scps and that they may be the elusive cells of origin for para-spinal plexiform neurofibroma. cell lineage tracing confirms the plp+ precursors originate in the embryonic nerve roots as the cells of origin for plexiform neurofibromas tumor cells of origin are difficult to identify by characterizing cells within terminal stage tumors. a more definitive approach is to genetically label specific lineage early in development for cancer cell , – , n fate tracing prior to tumorigenesis. x-gal staining demonstrates that lacz+(plp+) cells are mainly located in nerve roots at e . when we gave -hydroxyta- moxifen to pregnant mother mice at e . (figure b). if these nf ablated lacz+(plp+) nerve root cells would give rise to neurofibroma, then neurofi- broma cells should carry the marker gene lacz. therefore, we crossed plpcre-ert;nf flox/flox;r r-lacz male mice with nf flox/�;r r-lacz female mice. we then administered -hydroxytamoxifen to pregnant mother mice for one dose at e . . at months of age, plpcre-ert;nf flox/�;r r-lacz progenies from these crosses began to show classic signs of illness: lethargy, weight loss, and paralyzed hind limbs. we observed the presence of plexiform neurofibromas with strong positive x-gal staining in close proximity to the cervical and thoracic drgs (figure a). because plpcre-ert mediates labeling solely at the time of tamoxifen administration at e . , x-gal positive staining of neurofibroma cells demonstrates unambiguously that the labeled neurofibromas were derived from e . plp+ precursors in the embryonic nerve roots. histopathological analysis of these tumors indicated that the tumors exhibited cardinal features of plexiform neurofi- bromas (figure b). strikingly, we also observed plexiform neurofibroma developed along the intercostal nerve whose associated drg appears histologically normal (figure c). in addition, we identified the earliest form of neurofibroma exhib- iting lacz positive staining: classic pathological characteristics of neurofibroma and expressing schwann cell marker s b and gap at the dorsal nerve roots near the drg (figure d). together, these findings point to the plp+;gap + scps in the embryonic nerve roots as the cells of origin for plexiform neurofibroma. ovember , ª elsevier inc. figure . therapeutic effect of mapk signaling inhibitor on dnsc-derived plexiform neurofibroma (a) diagram of experimental design for testing therapeutic effect of pd on plexiform neurofibroma. (b) immunostaining of erk / (total) and p-erk / (phosphorylated erk) in e . nf �/� dnscs derived sciatic plexiform neurofibroma (a). fold change of luminescence count measured during pd or vehicle treatment (b). after weeks of treatment, both pd and vehicle treatment group were divided into two subgroups: discontinued pd or continued pd and discontinued vehicle treatment or continued vehicle treatment subgroups for an additional weeks. tumors were then harvested for x-gal staining (c) and immunostaining for erk / and p-erk / (d). statistics were represented as the mean ± sem (*p < . ). blue scale bars, mm. all other scale bars, mm. see also figure s . cancer cell cells of origin for plexiform neurofibroma a robust plexiform neurofibroma model for preclinical drug screening the nf gene encodes for neurofibromin, a ras gtpase acti- vating protein (gap), and thus negatively regulates the ras signaling pathway (rubin and gutmann, ). this has promp- ted the use of specific map kinase pathway inhibitors to coun- teract nf loss-mediated tumor development (chang et al., ; jessen et al., ; staser et al., ) or to treat nf mutant animals in the effort to restore abnormal brain develop- ment and cognitive function (li et al., ; lush et al., ; wang et al., ). to determine if our non-germline plexiform neurofibroma model could be used as a rapid preclinical thera- peutic drug screening tool to identify effective therapies, we used a highly selective pharmacological inhibitor of mek, pd , which blocks activation and phosphorylation of erk / (chang et al., ), to test whether inhibition of the ras/raf/mek/erk signaling pathway can prevent neurofibroma progression in our plexiform neurofibroma model. in order to monitor the proliferation of tumor cells, we labeled these neurofibroma cells with luciferase by crossing the nf flox/flox;r r-lacz (le et al., ) mice with flox-stop-flox cancer cell , – , november , ª elsevier inc r r-luciferase mice to generate nf flox/flox;r r-lacz- luciferase mice. we then harvested e . dnscs and infected with ad-cmv-cre to generate nf �/�;r r-lacz-luciferase dnscs. we transplanted these nf �/�;r r-lacz-luciferase dnscs to sciatic nerves of nude mice to generate plexiform neu- rofibromas. mice were randomly assigned to pd treat- ment ( mg/kg/day, day/week) or vehicle as a control group (figure a). we also tested if the mapk pathway is activated in our neurofibromas and found that the erk pathway is highly acti- vated in the tumor tissues as shown by immunohistochemistry analysis (figure ba). we then started the treatment right after the transplantation. a striking reduction in luciferase signal was observed even after only one week of pd treatment (p < . ). continued treatment of pd further inhibited the proliferation of tumor cells. after weeks of pd treatment, tumor cell proliferation was almost completely blunt- ed as demonstrated by the diminishing luciferase signal. on the other hand, the luciferase signal in vehicle treatment group increased more than times after weeks of treatment (figure bb). these data suggested that the pd could effectively inhibit the proliferation of plexiform neurofibroma. . cancer cell cells of origin for plexiform neurofibroma however, it was not clear whether the inhibition effect of pd would be sustained after stopping the pd treatment. to answer this question, we divided the pd treatment group into two subgroups: discontinued pd treatment after weeks or continued pd treatment for an additional weeks in another group of mice. similar to this, we also divided the vehicle treatment group into two subgroups based on continuing or discontinuing vehicle treatment for another weeks. we again used the luciferase signal as a surrogate marker for tumor growth. we observed that continued treatment of pd further blunted the pro- liferation of neurofibroma; however, they regained the prolifera- tive capacity after pd withdrawal. eight weeks after transplantation of dnscs, we sacrificed all mice and performed x-gal staining of the sciatic nerves. vehicle treatment groups ( weeks or weeks) developed very large lacz+ neurofibromas, -week pd treatment dramatically reduced the growth of neurofibromas, while -week pd treatment almost completely prevented the proliferation of neurofibroma (fig- ure bc; figure s ). consistently, erk / phosphorylation was also reduced in both pd treatment groups compared with vehicle treatment groups (figure bd). all of these data showed the requirement of sustained ras/raf/mek/erk signaling for neurofibroma growth and that continuous pd treatment is effective in restraining this growth. thus, our non-germline plexiform neurofibroma model provides a powerful approach for preclinical therapeutic drug screening to identify effective therapies for human clinical trials to prevent neurofibroma formation in nf patients, where none exist today. discussion recent progress in cancer research points to the importance of stem cells and their immediate progenitors in the initiation and maintenance of many tumors, consistent with the notion that these neoplasms originate in a subset of primitive precursor cells and that most cells in an organ do not generate tumors (alcan- tara llaguno et al., ; barker et al., ; bonnet and dick, ; chen et al., a; goldstein et al., ; le et al., ; reya et al., ; tysnes, ). identifying which cell type gives rise to a particular cancer (the cells of origin) will permit greater understanding of their pathogenesis and inform better design approaches for their treating. in this study, we utilized a geneti- cally engineered mouse model as a tool to identify the cells of origin for plexiform neurofibroma. using cell lineage tracing, we demonstrated that the embryonic plp+;nf �/� scps, which originate from spinal nerve roots, give rise to plexiform neurofi- broma not only in para-spinal areas but also in distal peripheral nerve. we also report a non-germline model for plexiform neuro- fibroma that can be used broadly as preclinical models for ther- apeutic testing. a large body of direct and indirect studies has provided evi- dence that nf gene deletion is the requisite initial step that pre- cedes neurofibromagenesis (joseph et al., ; le et al., ; wu et al., ; zheng et al., ; zhu et al., ). cichowski et al. ( ) addressed this issue elegantly when they generated chimeric mice by injecting lacz+ nf �/� es cells into wild-type blastocysts. these mice developed microscopic neurofibromas derived from theinjected es cells,demonstratingthe requirement can of nf homozygosity for tumor formation (cichowski et al., ). however, the extent of chimerism in these mice occurs randomly and cannot be controlled genetically. as a result, it is difficult to establish the cells of origin for these tumors. conditional deletion of nf from embryonicneuralcreststemcells (ncscs) usingwnt- -cre transgenic mice leads to a transient increase in ncsc frequency and self-renewal, but the isolated ncscs fail to form tumors upon transplantation (joseph et al., ), suggesting that early ncsc derivatives may not contain potential cells of origin for plexiform neurofibroma. this observation indicates that later ncsc derivatives give rise to plexiform neurofibromas. consistently, ablation of nf in schwann cells using p a-cre; nf flox/� or dhh-cre;nf flox/flox mice, which deletes nf continu- ously from early scps through mature cells, efficiently induce neurofibromas development (wu et al., ; zheng et al., ). however, the cre recombinase transgenes used in these two studies compromise nf function at the early schwann cell stages of development (at e . ), and the cre recombinase activ- ity persists throughthe mature schwann cell stage. therefore, it is not possible to identify or exclude any particular schwann cell developmental stage as the critical one for nf loss-mediated tu- mor development. the interpretation of the data instead rests on correlations relating to detectable appearance of abnormal cells or structures. an alternative approach that can in theory provide a more precise evaluation of the evolution of nf -associated tumors is the application of inducible cre/loxp technology that al- lows temporal and spatial ablation of nf function in schwann cells.two differentstudies showthatacute lossof nf in bothem- bryo and adult can lead to plexiform neurofibroma formation (le et al., ; mayes et al., ). the fact that nf inactivation in adult mice also gives rise to neurofibroma, albeit at a much lower frequency (le et al., ), indicates that either ( ) mature schwann cells can, in rare cases, give rise to neurofibroma or ( ) that rare populations of progenitor cells persist within the adult peripheral nervous system. the notion that mature schwann cells have the capability to give rise to plexiform neurofibromas is less evident. nf patients rarely present with plexiform tumors for the first time as adults (ferner, ). as aforementioned, schwann cell development and terminal differentiation into myelinating cells mostly occurs postnatally. if mature schwann cells as a whole can give rise to neurofibromas when loss of heterozygosity (loh) occurs in adulthood, then it would be anticipated that plexiform tumors should develop widely in plpcre-ert mice induced postnatally since cre-mediated recombination remains efficient in mature schwann cells. it is not likely that the schwann cell lineage un- dergoes immature to mature transition en masse at tightly defined times from the ncsc stage, to the precursor stage, to the immature stage, and onward to more differentiated stages. instead, gradients of cells transition and migrate over extended periods with considerable overlap between the disappearance of one population and the emergence of another. therefore, re- maining compartments of precursor schwann cells may also persist into adulthood and retain the capacity to form plexiform neurofibromas within the peripheral nerves. indeed, given that embryonic nerve roots associate tightly with drgs and that the harvested drgs almost always contaminate with nerve roots, we carefully separated out the early embryonic dorsal nerve roots from the drgs for neurosphere culture. cer cell , – , november , ª elsevier inc. cancer cell cells of origin for plexiform neurofibroma unexpectedly, we found that only the embryonic dorsal nerve roots could robustly give rise to neurosphere but not the e . drgs. however, in adult mice, we found that the drgs can give rise to neurospheres and neurofibroma when nf was abla- ted and implanted into the sciatic nerves (data not shown). consistently, when we acutely labeled the plp+ precursor cells with lacz only in nerve roots at e . , we also observed plexi- form neurofibroma developed in the peripheral nerve distal to the drg. all of the data suggest that at least some of the cells of origin might migrate out along the nerve roots-drg-peripheral nerve and retain the capacity to form neurofibroma later in life. in the conditional knockout model using cre/loxp technology, the krox -cre;nf flox/flox mice (containing nf �/� schwann cells and nf +/+ microenvironment) do not develop neurofibromas. on the other hand, the krox -cre;nf flox/� mice (containing nf �/� schwann cells and nf +/� microenvironment) develop plexiform neurofibromas (zhu et al., ), demonstrating the essential role for the heterozygous environment in the development of neurofibroma. in another mouse model, a more robust dhh-cre driver causes plexiform neurofibroma formation in an nf -wild- type background (wu et al., ). in the current study, the plp+; nf �/� dnscs could also effectively give rise to plexiform neuro- fibromas when transplanted into the sciatic nerves of nude mice, which also have an nf -wild-type background. it is important to emphasize here that under these conditions, a large group of scps undergoes nf loh simultaneously and can therefore vie with the wild-type environment more efficiently than when only one or a very few isolated scps undergo loh. in nf patients that harbor germline heterozygous nf mutations, given the defined time of gestation and size of neural crest compartment, inactivation of the other normal nf in the schwann cell lineage must be rare and stochastic, such that single cells undergo completely inactivation of nf in a large pool of otherwise hetero- zygous nf mutant cells. in this setting, the increased secretion of scf or other factors from heterozygous cells, including mast cells, in the tumor microenvironment provides a permissive con- dition to foster tumor growth (yang et al., ). therefore, in the experimental setting, one possible advantage of having a large pool of precursors undergoing loh would be a rapid and dra- matic increase in cytokine and growth factor secretion that could be sufficient to elicit wild-type mast cells without the need for the hypersensitive heterozygous state of the latter. as such, in the physiologic scenario, one would envision a rare schwann cell undergoing loh that would normally disappear except for the response of the nf -heterozygous microenvironment that assists it to form a tumor. in another scenario, contribution of a multitude of cells that have undergone loh may override the otherwise requisite contributions from other factors, such as heterozygous mast cells or other cells in the microenvironment, to induce neurofibroma development. although the tumor microenvironment is critical for neurofi- broma formation (le et al., ; yang et al., ), intrinsic pro- liferative signals such as those in the mapk pathways are equally essential. therefore, both the tumor microenvironment and mapk pathways have been targeted in the preclinical setting to define clinically relevant pharmacodynamic variables and evaluating duration of drug effects on individual tumor (jessen et al., ; yang et al., ). the purpose of preclinical drug screening is to select the most effective and safe compound cancer cell , – , november , ª elsevier inc from a large library. our non-germline model for plexiform neuro- fibroma enables high efficiency of tumorigenesis and has a much shorter latency. therefore, it is a reliable and robust model for in vivo drug screening with a quantifiable and reproducible outcome measure (via bioluminescent scan) to select lead com- pounds for clinical assessment. in addition, the ability to isolate embryonic plp+ gap + cells for ex vivo expansion and nf ablation to generate classic sciatic plexiform neurofibromas al- lows flexible and speedy manipulation with multiple genetic tools to delineate additional pathways that are perturbed upon the loss of nf , which are essential for neurofibroma development. in conclusion, the identity of the tumor cells of origin and the facility for isolation and expansion for genetic alterations provide fertile ground for continued analysis to identify additional intrinsic and extrinsic factors within the tumor microenvironment that likely play essential roles in neurofibroma genesis. it is reason- able to speculate that lessons learned from investigating the mo- lecular interactions between the neurofibroma cells of origin and their microenvironment will provide us approaches to develop effective therapies to delay and possibly prevent neurofibroma formation in nf patients. experimental procedures mice animal care and use were approved by the institutional animal care and use committee at university of texas southwestern medical center. the nf flox/� and nf flox/flox mice have been described previously (zhu et al., ). for conditional ablation of nf , we used a tamoxifen-inducible cre line, the plpcre-ert transgenic mice (leone et al., ), and two non-induc- ible cre lines, krox -cre and dhh-cre (limpert et al., ; zhu et al., ). the rosa -yfp reporter mice were described previously (chen et al., ). the rosa -lacz and rosa -luciferase reporter mice were obtained from the jackson laboratories. tamoxifen and -hydroxytamoxifen induction tamoxifen (sigma-aldrich) and -hydroxytamoxifen was dissolved in a sunflower oil/ethanol mixture ( : ) at mg/ml and mg/ml, respectively. for embryonic induction, the pregnant mice were gavaged orally with mg of tamoxifen or . mg of -hydroxytamoxifen at e . or e . , respectively. cell culture and differentiation assays mouse embryos were removed from anesthetized . -day-old pregnant female mice. embryos were sacrificed. the spinal cord of each embryo was removed, and drgs/nerve roots were dissected from the vertebral column with the aid of stereomicroscope. fine scissors was used to cut and separate the nerve roots and drgs. drg and/or nerve root neurosphere culture was performed as previously described (le et al., ; patel et al., ; ratner et al., ). for cell proliferation assay, the single sphere cells were seeded to a -well plate coated with poly-d-lysine/laminin ( . cells per well) for monolayer culture. the cell numbers of wells were counted every hr for days. for differentiation assays, we seeded . cells per well of an -chamber slide coated with poly-d-lysine/laminin and cultured with fresh media containing b + % fetal bovine serum for – days. then we fixed the cells with % paraformaldehyde for min and performed immunostaining for lineage markers. fluorescence-activated cell sorting we harvested yfp+ and yfp� dnscs using facsaria instrument (bd biosci- ences) equipped with a nm solid-state laser. histology, immunostaining, and x-gal staining for hematoxylin and eosin (h&e) histology analysis, tissue specimens were harvested and fixed with % formalin in pbs for day and subsequently . cancer cell cells of origin for plexiform neurofibroma embedded in paraffin. sections ( mm thick) were stained with h&e per man- ufacturer’s protocol (statlab). the dilutions of primary antibodies used in immunohistochemistry and immunofluorescence studies were as follows: b iii-tubulin (rabbit, : , sigma-aldrich), blbp (rabbit, : , millipore), cd (rabbit, : , abcam), brdu (rat, : , abcam), cleaved caspase- (rabbit, : , cell signaling technology), dhh (rabbit, : , santa cruz biotechnology), total erk / (rabbit, : , cell signaling technology), phos- pho-erk / (rabbit, : , cell signaling technology), gap (rabbit, : , abcam), gfap (rabbit, : , dako), gfp (goat, : , rockland), krox (rabbit, : , covance research products), nestin (rabbit, : , abcam), p (rabbit, : , millipore), plp (rabbit, : , abcam), s b (rabbit, : , dako), sox (goat, : , santa cruz biotechnology), and vimentin (mouse, : , santa cruz biotechnology). for x-gal staining, after total body perfusion with % paraformaldehyde, tissues or embryos were harvested, equilibrated in % sucrose in pbs overnight at �c, and stained with x-gal at �c overnight. the tissues were then postfixed with % formalin, paraffin embedded, sectioned, and counter- stained with hematoxylin. transplantation experiments sciatic nerve implantation of dnscs was performed as previously reported (chen et al., b). briefly, mice were anesthetized by intraperitoneal injec- tion of ml of a mixture of ketamine ( mg/ml) and xylazine ( mg/ml) solu- tion. a skin incision was made above the femur. using iris scissors, a pocket was created within the quadriceps muscles to expose the sciatic nerve. the ml of l medium containing viable dnscs was then deposited into this pocket so that dnscs can be in contact with the sn. the quadriceps muscles were then closed with - vicryl suture, and the skin was closed with - prolene suture. mice were allowed to recover from anesthesia, then subject to mek inhibitor treatment and bioluminescent imaging. mek inhibitor treatment mek inhibitor pd were kindly provided by dr. kevin shannon. pd was dissolved in vehicle ( . % hydroxypropyl methylcellulose with . % tween ; sigma-aldrich) at a concentration of mg/ml. the solution was administered orally at the dosage of mg/kg (body weight) per mouse, once per day, for days/week. bioluminescent imaging bioluminescent imaging was performed as described previously (chau et al., ). briefly, mice were injected with ul mg/ml d-luciferin potassium salt (perkin elmer) in . % sterile nacl, and total flux was quantified using ivis lumina ii (caliper life sciences) weekly. statistics all data are displayed as the mean ± sem unless specified otherwise. a two-tailed t test and fisher exact test were applied as appropriate to evaluate statistical significance (p < . was deemed statistically significant). supplemental information supplemental information includes supplemental experimental procedures and four figures and can be found with this article online at http://dx.doi.org/ . /j.ccell. . . . author contributions l.q.l. contributed to conception and experimental designs, data collection and/or assembly, data analysis and interpretation, manuscript writing, and final approval of manuscript. z.c. contributed to experimental designs, data collection and/or assembly, data analysis and interpretation, and manuscript writing. c.l., a.j.p., c.-p.l., and y.w. contributed to data collection. acknowledgments we thank all members of the l.q.l. lab and dr. luis parada, dr. kristjan jessen, dr. rhona mirsky, and dr. wei mo for helpful suggestions and discus- sions. we also thank dr. ueli suter for the plpcre-ert mice. a.j.p. and c.-p.l. can are recipients of the young investigator awards from children’s tumor foun- dation. l.q.l. holds a career award for medical scientists from the burroughs wellcome fund. this work was supported by funding from the elisabeth reed wagner fund for research and clinical care in neurofibromatosis and cardio- thoracic surgery, the disease-oriented clinical scholar program, the national cancer institute of the nih grant number r ca , and u.s. department of defense grant number w xwh- - - to l.q.l. received: may , revised: july , accepted: september , published: october , references alcantara llaguno, s., chen, j., kwon, c.h., jackson, e.l., li, y., burns, d.k., alvarez-buylla, a., and parada, l.f. 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( ). plexiform and dermal neurofibromas and pigmentation are caused by nf loss in desert hedgehog-expressing cells. cancer cell , – . xu, g.f., o’connell, p., viskochil, d., cawthon, r., robertson, m., culver, m., dunn, d., stevens, j., gesteland, r., white, r., et al. ( ). the neurofibroma- tosis type gene encodes a protein related to gap. cell , – . yang, f.c., ingram, d.a., chen, s., zhu, y., yuan, j., li, x., yang, x., knowles, s., horn, w., li, y., et al. ( ). nf -dependent tumors require a microenviron- ment containing nf +/– and c-kit-dependent bone marrow. cell , – . zheng, h., chang, l., patel, n., yang, j., lowe, l., burns, d.k., and zhu, y. ( ). induction of abnormal proliferation by nonmyelinating schwann cells triggers neurofibroma formation. cancer cell , – . zhu, y., romero, m.i., ghosh, p., ye, z., charnay, p., rushing, e.j., marth, j.d., and parada, l.f. ( ). ablation of nf function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. genes dev. , – . zhu, y., ghosh, p., charnay, p., burns, d.k., and parada, l.f. ( ). neurofibromas in nf : schwann cell origin and role of tumor environment. science , – . . 	cells of origin in the embryonic nerve roots for nf -associated plexiform neurofibroma 	introduction 	results 	loss of nf in embryonic drg/nerve root neurosphere cells gives rise to classic plexiform neurofibroma in vivo 	cells of origin for para-spinal plexiform neurofibromas are inside the embryonic plp+ cells 	the plp+ cells of origin originate in the embryonic nerve roots 	phenotypic analysis identifies the plp+ cells of origin as embryonic schwann cell precursors 	cell lineage tracing confirms the plp+ precursors originate in the embryonic nerve roots as the cells of origin for plexifo ... 	a robust plexiform neurofibroma model for preclinical drug screening 	discussion 	experimental procedures 	mice 	tamoxifen and -hydroxytamoxifen induction 	cell culture and differentiation assays 	fluorescence-activated cell sorting 	histology, immunostaining, and x-gal staining 	transplantation experiments 	mek inhibitor treatment 	bioluminescent imaging 	statistics 	supplemental information 	acknowledgments 	references discovery of , rad-snps and development of a -snp array for monitoring river otters technical note discovery of , rad–snps and development of a -snp array for monitoring river otters jeffrey b. stetz • seth smith • michael a. sawaya • alan b. ramsey • stephen j. amish • michael k. schwartz • gordon luikart , received: july / accepted: june � springer science+business media dordrecht abstract many north american river otter (lontra canadensis) populations are threatened or recovering but are difficult to study because they occur at low densities, it is difficult to visually identify individuals, and they inhabit aquatic environments that accelerate degradation of bio- logical samples. single nucleotide polymorphisms (snps) can improve our ability to monitor demographic and genetic parameters of difficult to study species. we used restriction site associated dna (rad) sequencing to dis- cover , snps present in montana, usa, river otter populations, including , loci that were also variable in at least one other population range-wide. after applying careful filtering criteria meant to minimize ascertainment bias and identify high quality, highly heterozygous (ho = . – . ) snps, we developed and tested inde- pendent snp qpcr genotyping assays, including that performed well with diluted dna. the loci provided high power for population assignment tests with only misassignment ( . %) between closely neighboring pop- ulations. our snps showed high power to differentiate individuals and assign them to population of origin, as well as strong concordance of genotypes from high and diluted concentrations of dna, and between original rad and the snp qpcr array. keywords conservation genomics � rad � next generation sequencing � river otter � snp � population monitoring � noninvasive genetic tagging like other wide-ranging, elusive species, monitoring river otter populations remains a challenge for management agencies (melquist et al. ). individual otters are dif- ficult to distinguish visually and are known to travel con- siderable distances (melquist and hornocker ; newton ). radio-tagging studies, which require surgically implanting transmitters, are expensive and logistically difficult, and typically produce small samples sizes. for these and other reasons, studies aimed at estimating abundance, population growth rates, and connectivity have been difficult. interest in ensuring long-term population persistence in the face of harvest and habitat loss has led to the devel- opment of molecular tools, primarily microsatellite mark- ers, to monitor otter population dynamics (e.g., mowry et al. ). advancements in discovery and genotyping of single nucleotide polymorphisms (snps), with concurrent reduction in costs, present an opportunity to vastly improve our ability to monitor wildlife populations. relative to data available from the dryad digital repository: . /dryad. m r]. electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. & jeffrey b. stetz jeff.stetz@gmail.com sinopah wildlife research associates, n. higgins, suite , missoula, mt , usa fish and wildlife genomics group, division of biological sciences, university of montana, missoula, mt , usa mpg ranch, missoula, mt , usa national genomics center for wildlife and fish conservation, rocky mountain research station, united states forest service, missoula, mt , usa flathead lake biological station, university of montana, polson, mt , usa conservation genet resour doi . /s - - - http://orcid.org/ - - - http://dx.doi.org/ . /dryad. m r http://dx.doi.org/ . /dryad. m r http://dx.doi.org/ . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf microsatellites, snps are less prone to genotyping errors, easier to transfer and analyze consistently among labora- tories, genotyping samples is faster and cheaper, and snps can include neutral markers or those linked to regions under selection (morin et al. ; allendorf et al. ; helyar et al. ; fabbri et al. ). one of the strengths of using genetic methods for monitoring river otter populations is the ease of collecting fecal samples from shared latrine sites along water bodies. latrines are easy to locate and provide an opportunity to collect fecal material from multiple individuals at one site. likely because of degradation of dna due to moisture and ultraviolet light (vynne et al. ; stetz et al. ), these efforts have been largely unsuccessful due to poor genotyping success rates. for example, a study in mon- tana was able to obtain complete multilocus genotypes for just % of otter scat samples using microsatellites (newton ). as snps are considerably shorter than microsatellites, higher genotyping success rates (i.e., lower rates of allelic drop out and false amplifications) are likely, even with poor quality samples such as scat (morin and mccarthy ; fabbri et al. ; fitak et al. ). we therefore set out to develop a snp array for river otters that would improve our ability to assess and monitor otter populations in montana and across the species’ range. although our emphasis was on otter populations in montana, we used muscle tissue samples from a large geographic area to minimize issues of ascertainment bias and to ensure usefulness range-wide (fig. ; allendorf et al. ). sampling two populations that are somewhat close geographically (i.e., nb and qe) strengthened our test of marker power to assign individuals to population of origin. dna was extracted from tissue samples using the qia- gen dneasy protocol then quantified using the quant-it tm picogreen � dsdna assay to ensure dna concentrations [ ng/ll, needed for producing restriction-site-associated (rad) sequencing libraries (etter et al. ). rad libraries were sequenced on the illumina hiseq platform using base pair paired-end reads. following amish et al. ( ), we selected for infor- mative loci while applying strict data quality and assay design filters. samples were excluded from downstream analysis if [ % of their genotyped loci had\ reads or [fivefold read count difference between alleles. to facil- itate snp pcr genotyping assay design snp loci had to be located between – nucleotides from the end, and we allowed only snp per rad locus to avoid rad loci assembled from paralogs and to avoid physically linked snps. we then excluded rad loci where c samples had \ reads or [fivefold read count difference between alleles. we also required that observed heterozygosity (both range-wide and within montana) be . – . , that c sample from outside montana was heterozygous at each rad locus, and that each locus was successfully geno- typed in [ % of montana samples. these criteria produced candidate snps, from which we selected the with the highest expected heterozygosity for kasp-by-design fluidigm assays (lgc genomics � ). we tested these assays on samples from across otter range on a fluidigm microfluidic snp-chip. after excluding loci due to high linkage dis- equilibrium (p \ . ), we identified loci in hardy– weinberg proportions, with expected and observed heterozygosity [ . for montana samples, and with b instance where the initial rad genotype did not match the snp-chip genotype (table s ). within snp-chip geno- types, each sample was run at least three times, and there could be b instance of replicate genotypes not matching. we next required that each of the three possible genotypes from each locus was observed at least once on each of snp-chips. we set a call rate threshold of % for each snp-chip (i.e., % of individuals and replicates yielded quality genotypes), and mean genotype confidence had to be [ % for both chips. we then identified a subset of loci for use with low quantity dna samples by testing loci on samples where we reduced the original dna concentration (c ng/ll) by half (table s ). we excluded loci where c instance of normal and low concentration genotypes did not match, and where b instance of low concentration duplicate genotypes did not match. we used probability of identity statistics and population assignment tests to examine the snp panel’s power to answer questions of sample identity or population of origin. we used genalex . (peakall and smouse ) to cal- culate probability of identity, and geneclass . (piry et al. ; rannala and mountain ) to test how well indi- vidual otters assigned to populations (paetkau et al. ) using our two classes of snps. both sets of loci showed acceptable power to differentiate even closely related individuals within and among populations (fig. ). using loci produced misassignments ( . %) compared to just observed in the reduced set of loci ( . %). all putatively misassigned samples, however, originated in the closest neighboring population. for example, the indi- viduals from new brunswick that assigned to quebec had – % assignment scores to their native region, sug- gesting that these may have been related to recent migra- tion events. the snps we report here represent a new tool for monitoring demographic and genetic status and changes in river otter populations across north america. snps may be particularly well suited to studying otter populations given the observed increase in genotyping success of fecal conservation genet resour samples relative to microsatellite markers in other species (e.g., campbell and narum ; fabbri et al. ; fitak et al. ). further, this snp array may be a powerful tool to explore genetic structure and evolutionary potential of otter populations while taking advantage of noninvasive sampling techniques. such information is particularly valuable for reintroduction efforts and general questions on river otter ecology. necessary next steps include optimiz- ing sampling and preservation methods to maximize snp performance in spraints, and to directly compare perfor- mance of snps to microsatellites. acknowledgments we are grateful to mpg ranch for generously funding this research and to the university of montana for in-kind support, including laboratory facilities, equipment, and protocols. we thank the following for providing river otter tissue samples or source dna: e. bunting (cornell university), c. brown (rhode island division of fish and wildlife), c. bernier (vermont fish & wildlife department), j. cormier and l. elson (new brunswick department of environment), p. canac-marquis (quebec ministry of energy and natural resources), j. gude and n. anderson (montana department of fish, wildlife, and park), c. nelson (british columbia forests, lands and natural resource operations), j. harms (environment yukon), s. talbot (u.s. geological survey), j. hayden and z. lockyer (idaho department of fish and game), and k. pilgrim (u.s. forest service rocky mountain research station). fig. locations of tissue samples collected from north american river otter for snp development; population- specific snp statistics are reported within the figure conservation genet resour references allendorf fw, hohenlohe p, luikart g ( ) genomics and the future of conservation, invited review. nat rev genet : – amish sj, hohenlohe pa, painter s, leary rf, muhlfeld c, allendorf fw, luikart g ( ) rad sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic snp assays. mol ecol resour : – campbell nr, narum sr ( ) quantitative pcr assessment of microsatellite and snp genotyping with variable quality dna extracts. conserv genet. doi: . /s - - - etter pd, bassham s, hohenlohe pa, johnson ea, cresko wa ( ) snp discovery and genotyping for evolutionary genetics using rad sequencing. in: orgogozo v, rockman mv (eds) molecular methods for evolutionary genetics. humana press, new york, pp – fabbri e, caniglia r, mucci n, thomsen hp, krag k, pertoldi c, loeschcke v, randi e ( ) comparison of single nucleotide polymorphisms and microsatellites in non-invasive genetic monitoring of a wolf population. arch biol sci belgrade : – fitak rr, naidu a, thompson rw, culver m ( ) a new panel of snp markers for the individual identification of north american pumas. j fish wildl manag : – . doi: . / - jfwm- helyar sj, hemmer-hansen j, bekkevold d, taylor mi, ogden r, limborg mt, cariani a, maes ge, diopere e, carvalho gr, nielsen ee ( ) application of snps for population genetics of nonmodel organisms: new opportunities and challenges. mol ecol resour : – melquist w, hornocker m ( ) ecology of river otters in west central idaho. wildl monogr : – melquist we, polechla pj, toweill d ( ) river otter: lontra canadensis. in: feldhamer ga, thompson bc, chapman ja (eds) wild mammals of north america: biology, management, and conservation. johns hopkins university press, baltimore, pp – morin pa, mccarthy m ( ) highly accurate snp genotyping from historical and low-quality samples. mol ecol notes : – morin pa, luikart g, wayne rk ( ) snps in ecology, evolution and conservation. trends ecol evol : – mowry ra, gompper me, beringer j, eggert ls ( ) river otter population size estimation using noninvasive latrine surveys. j wildl manag : – newton de ( ) northern river otter population assessment and connectivity in western montana. thesis, university of montana, missoula, mt paetkau d, calvert w, stirling i, strobeck c ( ) microsatellite analysis of population structure in canadian polar bears. mol ecol : – peakall r, smouse pe ( ) genalex , genetic analysis in excel. population genetic software for teaching and research. mol ecol notes : – piry s, alapetite a, cornuet j-m, paetkau d, baudouin l, estoup a ( ) geneclass : a software for genetic assignment and first-generation migrant detection. j hered : – rannala b, mountain jl ( ) detecting immigration by using multilocus genotypes. proc natl acad sci : – stetz jb, seitz t, sawaya ma ( ) effects of exposure on genotyping success rates of hair samples from grizzly and american black bears. j fish wildl manag : – vynne c, baker mr, breuer zl, wasser sk ( ) factors influencing degradation of dna and hormones in maned wolf scat. anim conserv : conservation genet resour http://dx.doi.org/ . /s - - - http://dx.doi.org/ . / -jfwm- http://dx.doi.org/ . / -jfwm- 	discovery of , rad--snps and development of a -snp array for monitoring river otters 	abstract 	acknowledgments 	references [pdf] responsibilities of data monitoring committees | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / corpus id: responsibilities of data monitoring committees @article{bierer responsibilitiesod, title={responsibilities of data monitoring committees}, author={b. bierer and r. li and j. seltzer and l. sleeper and elizabeth frank and c. knirsch and carmen e. aldinger and r. levine and joe m massaro and a. shah and m. barnes and s. snapinn and j. wittes}, journal={therapeutic innovation & regulatory science}, year={ }, volume={ }, pages={ - } } 	b. bierer, r. li, + authors j. wittes 	published 	medicine 	therapeutic innovation & regulatory science background: a data monitoring committee (dmc) has special responsibilities for protecting the safety of clinical trial participants. [...] key method all members of the group have formed, served on, advised, or worked with dmcs. results: the group outlined the objectives and mechanics of running a dmc, including operational and practical considerations, membership characteristics, roles, members’ liability, and indemnification. further, it delineated the roles and responsibilities of each dmc member…expand view on sage mrctcenter.org save to library create alert cite launch research feed share this paper citations view all tables and topics from this paper 	 table 	 table 	 table 	 table 	 table 	 table 	 table view all figures & tables 	clinical trial independent data monitoring committee charter 	responsibility 	academia (organization) 	mechanics 	advocate (person) citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency data monitoring committees: promoting best practices to address emerging challenges 	t. fleming, d. demets, + authors s. ellenberg 	business, medicine 	clinical trials 	 	 	pdf save alert research feed reports to independent data monitoring committees 	k. buhr, matthew t. downs, janelle rhorer, robin bechhofer, j. wittes 	computer science, medicine 	therapeutic innovation & regulatory science 	 	 save alert research feed how to construct an optimal interim report: what the data monitoring committee does and doesn’t need to know 	j. neaton, b. grund, d. wentworth 	psychology, medicine 	clinical trials 	 	 save alert research feed issues for masked data monitoring 	o. d. williams, k. epnere 	business 	 save alert research feed references showing - of references liability issues for data monitoring committee members 	d. demets, t. fleming, + authors r. califf 	business, medicine 	clinical trials 	 	 	pdf 	view excerpts, references background and methods save alert research feed on independent data monitoring committees in oncology clinical trials. 	j. wittes, mark schactman 	medicine 	chinese clinical oncology 	 	 	view excerpts, references methods save alert research feed behind closed doors: the data monitoring board in randomized clinical trials. 	j. wittes 	medicine 	statistics in medicine 	 	 	view excerpts, references methods save alert research feed data monitoring in clinical trials 	d. demets, c. furberg, l. friedman 	medicine 	 	 	view excerpts, references methods and background save alert research feed data monitoring committees in clinical trials 	s. ellenberg, t. fleming, d. demets 	medicine 	 	 save alert research feed ethical and scientific implications of the globalization of clinical research. 	charla a andrews 	medicine 	the new england journal of medicine 	 	 save alert research feed and drug administration 	guidance for clinical trial sponsors: establishment and operation of clinical trial data monitoring committees. silver spring, md: us fda; 	 data monitoring committees in clinical trials: a practical perspective. 	patricia m. grambsch 	medicine 	 	 	view excerpts, references methods and background save alert research feed related papers 	abstract 	tables and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of rhoa signaling simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of rhoa signaling mathew j. wong, , crystal kantores, julijana ivanovska, amish jain, , , and robert p. jankov , , , physiology & experimental medicine program, hospital for sick children research institute, toronto, ontario, canada; heart and stroke richard lewar centre of excellence in cardiovascular research, university of toronto, toronto, ontario, canada; department of paediatrics, faculty of medicine, university of toronto, toronto, ontario, canada; and department of physiology, faculty of medicine, university of toronto, toronto, ontario, canada submitted august ; accepted in final form september wong mj, kantores c, ivanovska j, jain a, jankov rp. simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of rhoa signaling. am j physiol lung cell mol physiol : l –l , . first published september , ; doi: . /ajplung. . .— chronic neonatal pulmonary hypertension (pht) frequently results in early death. systemically administered rho-kinase (rock) inhibitors prevent and reverse chronic pht in neonatal rats, but at the cost of severe adverse effects, including systemic hypotension and growth restriction. simvastatin has pleiotropic inhibitory effects on iso- prenoid intermediates that may limit activity of rhoa, which signals upstream of rock. we therefore hypothesized that statin treatment would safely limit pulmonary vascular rhoa activity and prevent and reverse experimental chronic neonatal pht via downstream inhibitory effects on pathological rock activity. sprague-dawley rats in nor- moxia (room air) or moderate normobaric hypoxia ( % o ) received simvastatin ( mg·kg� ·day� ip) or vehicle from postnatal days – (prevention protocol) or from days – (rescue protocol). chronic hypoxia increased rhoa and rock activity in lung tissue. simva- statin reduced lung content of the isoprenoid intermediate farnesyl pyrophosphate and decreased rhoa/rock signaling in the hypoxia- exposed lung. preventive or rescue treatment of chronic hypoxia- exposed animals with simvastatin decreased pulmonary vascular re- sistance, right ventricular hypertrophy, and pulmonary arterial remod- eling. preventive simvastatin treatment improved weight gain, did not lower systemic blood pressure, and did not cause apparent toxic effects on skeletal muscle, liver or brain. rescue therapy with sim- vastatin improved exercise capacity. we conclude that simvastatin limits rhoa/rock activity in the chronic hypoxia-exposed lung, thus preventing or ameliorating hemodynamic and structural markers of chronic pht and improving long-term outcome, without causing adverse effects. chronic lung injury; rho-kinase; echocardiography; isoprenoid inter- mediates premature birth and associated chronic lung injury, known as bronchopulmonary dysplasia (bpd), is frequently associated with chronic pulmonary arterial hypertension (pht) ( ). chronic pht is characterized by pulmonary arterial remodel- ing, leading to increased pulmonary arterial pressure, increased pulmonary vascular resistance (pvr), and decreased arterial compliance ( , , ). the normal pulmonary circulation is a low-resistance, high-compliance system suited to accommo- date the entire cardiac output. increased thickening of pulmo- nary arteries from smooth muscle proliferation and extracellu- lar matrix deposition contributes to decreased pulmonary arte- rial compliance, increased resistance and increased right ventricle (rv) afterload ( ). the rv adapts by undergoing hypertrophy that may be followed by maladaptive chamber dilatation, which precedes rv failure and eventual death ( , ). newborns with chronic pht associated with severe bpd have a high mortality, with one study reporting a -year survival rate of % ( , ). moreover, survivors are at an increased risk for impaired neurological development ( ) and recurrence of pht later in life ( ). currently, there are no treatments of proven efficacy for chronic pht in neonates. previous work from our laboratory has implicated the ras- homolog gene family member a (rhoa)/rho-kinase (rock) signaling pathway in the pathogenesis of experimental chronic neonatal pht. rhoa/rock upregulation leads to increased pulmonary vascular tone and pulmonary arterial remodeling in chronic pht ( ). treatment with rock-specific inhibitors such as fasudil and y- has been shown to prevent or reverse chronic hypoxic- or bleomycin-induced pht in neo- natal rats ( , , , ). in pulmonary artery smooth muscle cells (smcs), vasoconstriction is regulated by phosphorylation of myosin light chain (mlc) through mlc kinase. dephos- phorylation by mlc phosphatase leads to vasorelaxation ( ). rock acts by phosphorylating the myosin phosphatase target subunit (mypt ), which inhibits mlc phosphatase activity, thus favoring a contractile state ( , ). rock mediates arterial remodeling via regulation of antiapoptotic and propro- liferative signaling in smcs and fibroblasts ( , , ). rhoa/rock signaling also negatively regulates endothelial nitric oxide synthase (enos) function ( , ). statins [ -hydroxy- -methylglutaryl (hmg)-coenzyme a (coa) reductase inhibitors] prevent conversion of hmg-coa to mevalonate, which is the rate-limiting step in cholesterol biosynthesis ( ). inhibition of mevalonate synthesis also de- creases the synthesis of isoprenoid intermediates (illustrated in fig. a): farnesyl-pyrophosphate (fpp) and geranylgeranyl- pyrophosphate (ggpp) ( ). the isoprenoids serve an essen- tial biological role through lipidation of proteins, a process also known as prenylation. of the isoprenoid intermediates, fpp interacts with the ras family of proteins while ggpp binds to the rho family ( ). an inactive reservoir of gdp-rhoa remains in the cytosol bound to rho-specific guanine nucleo- tide dissociation inhibitor (rhogdi) ( ). ggpp-mediated prenylation of rhoa is required for intracellular cytosolic trafficking and subsequent activation at the membrane ( , ). once localized to the membrane, rhoa is activated by guanine nucleotide exchange factors catalyzing the exchange of gdp for gtp. rhoa is inactivated by gtpase-activating proteins address for reprint requests and other correspondence: r. p. jankov, hospital for sick children, . peter gilgan centre for research and learning, bay st., toronto, on m g a , canada (e-mail: robert.jankov@sickkids.ca). am j physiol lung cell mol physiol : l –l , . first published september , ; doi: . /ajplung. . . - / copyright © the american physiological societyhttp://www.ajplung.org l downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . mailto:robert.jankov@sickkids.ca through hydrolysis of the phosphate bond ( ). ggpp- mediated prenylation of rhoa may also prevent membrane dissociation through the action of rhogdi, further enhanc- ing rhoa activation ( ). once active, rhoa binds to the rho-binding domain of rock, inducing an active confor- mational change ( ). previous work from our laboratory has confirmed the im- portance of upregulated rhoa activity and its downstream mediator rock in the pathogenesis of experimental chronic neonatal lung injury and chronic neonatal pht ( , ). work from other investigators has demonstrated pleiotropic inhibi- tion of rhoa activity by statins ( , , ). therefore, by limiting the synthesis of ggpp and prenylation of rhoa, statins may reduce pathological rhoa and rock activation ( ). the objective of this study was to examine the effects of simvastatin in the prevention or rescue of chronic hypoxic pht in neonatal rats. materials and methods materials. simvastatin was from cayman chemical (catalog no. , ann arbor, mi). oxygen-exposure chambers and auto- mated controllers, oxycycler model a xov, were from biospherix (parish, ny). tris-glycine precast gels (catalog no. xp box, well, – %) and pvdf membranes were from thermo scientific (rockford, il). phosphatase and protease inhibitors were from sigma life science (catalog no. p - ml, st. louis, mo) and calbio- chem (catalog no. - vl, san diego, ca), respectively. ac- ids, alcohols, organic solvents, paraformaldehyde, permount, and superfrost/plus microscope slides were from fisher scientific (whitby, ontario, canada). weigert’s resorcin-fuchsin stain was from rowley biochemical (catalog no. f- - , danvers, ma). luxol fast blue stain solutions were from electron microscopy sciences (catalog no. , hatfield, pa). elisa kits for alanine transaminase (alt; catalog no. ab ), creatine kinase (ck; catalog no. ab ), high-density lipoprotein (hdl), and low-density/very- low-density lipoprotein (ldl/vldl) cholesterol (catalog no. ab ) were purchased from abcam (toronto, ontario, canada). a free cholesterol assay kit (catalog no. ) was from cayman chemical. anti-rock i (catalog no. sc- ), anti-cleaved rock i (catalog no. sc- ), anti-glyceraldehyde- -phosphate dehydrogenase (gapdh; catalog no. sc- ) antibodies and protein a/g agarose beads (catalog no. sc- ) were from santa cruz biotechnology (santa cruz, ca). anti-rock ii (catalog no. ), anti-pan- mypt (catalog no. ), and anti-enos (catalog on. ) were from bd biosciences (san jose, ca). anti-phospho-threonine mypt (catalog no. - ) was from millipore (etobicoke, ontario, canada). anti-hif- � (catalog no. ) was from genetex (irvine, ca). anti-gtp-rhoa (catalog no. ) and anti-rhoa (catalog no. ) antibodies were from neweast bio- fig. . illustration of statin inhibition of cholesterol and iso- prenoid synthesis pathways. experimental design for preven- tion and rescue protocols. a: upstream inhibition of hmg-coa reductase enzyme limits bioavailability of diverging meval- onate products. reduction of the isoprenoid intermediate gera- nylgeranyl pyrophosphate (ggpp) limits membrane transloca- tion and activation of rhoa. b: overview of the time course and experimental design for prevention and rescue protocols. l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . sciences (king of prussia, pa). anti-�-smooth muscle actin antibody (catalog no. ma - ) was from thermo scientific. avidin-biotin- peroxidase complex immunohistochemistry kits, -diamino- - phenylindole aqueous mounting medium, and , -diaminobenzidine staining kits were from vector laboratories (burlingame, ca). goat anti-mouse immunoglobulin (igg)-peroxidase antibodies, and biotin- ylated goat anti-mouse igg (catalog no. ) and goat anti-rabbit igg (catalog no. ) were from cell signaling technologies (beverly, ma). in vivo experiments. all procedures involving animals were ap- proved by the animal care committee of the hospital for sick children research institute and conformed to the guidelines of the canadian council on animal care. timed-pregnant sprague-dawley rats were from taconic farms (germantown, ny). commencing on the day after birth [postnatal day (pnd) ], litters of sprague-dawley rat pups (equalized sex distribution) were chronically exposed to normobaric hypoxia ( % o ) or normoxia ( % o ) until pnd (prevention study) or until pnd (rescue study). concurrent with normoxia or hypoxia exposure, rat pups received daily intraperitoneal simvastatin ( mg/kg) or vehicle ( % dmso in pbs), from pnd to pnd (prevention study) or from pnd to pnd (rescue study). injury and treatment protocols are illustrated in fig. b. simvastatin was selected as it was the most frequently studied statin in the clinical and experimental pht literature during the conception of the study design, with a well-defined dose range in neonatal rats ( , , , , ). initial dose-response studies ( . , , , , and mg/kg) were conducted to determine the lowest effective dose in preventing pht, as guided by echocardiographic estimation of pvr. the highest dose ( mg/kg) adversely affected weight gain and the lowest dose ( . mg/kg) had a minimal effect on pvr (data not shown); therefore mg/kg was the dose chosen for all subsequent studies. each litter was maintained at n � pups, to control for nutritional effects. at the end of each - or -day exposure period, pups either were killed by pentobarbital overdose or were exsangui- nated after anesthesia. upon completion of the rescue protocol, a subgroup of animals was housed in room air until wk of age for exercise testing. simvastatin preparation and delivery. simvastatin was provided as a crystallized solid and stored in aliquots as a mg/ml solution in dmso stored at � °c. since simvastatin is not soluble in saline, a . mg/ml solution in % dmso in pbs was heated to °c in a temperature regulated shaker just prior to delivery to ensure solubility. injections were given intraperitoneally ( �l/g body wt) via a - gauge needle, once daily. vehicle controls received an equivalent volume of % dmso in pbs. two-dimensional echocardiography and pulse wave doppler ultrasound. two-dimensional echocardiography and pulsed wave doppler ultrasound was performed as a noninvasive method to assess pulmonary hemodynamics, using a vivid advantage (ge medical systems, milwaukee, wi) cardiovascular ultrasound machine with a small high frequency probe (i l), as previously described ( ). briefly, rat pups were anesthetized with % (vol/vol) isoflurane, and the animal was laid supine while spontaneously breathing – % (vol/vol) isoflurane through a modified face mask. a short axis view at the level of the aortic valve was obtained, and the pulmonary artery was identified using color flow doppler. the pulsed doppler gate was placed proximal to the pulmonary valve leaflets and aligned with an angle of insonation � °, maximizing laminar flow. the rv ejection time (rvet) and pulmonary arterial acceleration time (paat) were measured using the pulmonary doppler profile; rvet as the time from onset of systolic flow to completion of systolic flow, and paat from onset to peak pulmonary outflow velocity. a surrogate measure of pvr was calculated as a ratio of rvet to paat. to determine heart rate, measurements from three intersystolic intervals were av- eraged to account for beat-to-beat variability. analyses were under- taken in a blinded fashion using an offline analysis system (echopac, ge medical systems). right ventricular hypertrophy. measurement of rv hypertrophy using the fulton index (rv/lv�s) is a well-established marker of pht. the heart and lungs were separated, and the atria were removed inferior to the atrioventricular valves. the rv was separated from the left ventricle (lv) and septum. each component was freeze dried overnight and weighed separately, as previously described ( ). measurement of systemic blood pressure. systemic blood pressure was measured noninvasively using a tail cuff doppler device (le , harvard apparatus, holliston, ma), as previously de- scribed ( ). rats were anesthetized with % (vol/vol) isoflurane; the animal was laid supine while spontaneously breathing – % (vol/vol) isoflurane through a modified face mask. due to technical limitations of the device in smaller animals, measurements were carried out between pnd and pnd , as previously described ( ). exercise tolerance studies. adult rats ( wk of age) were trained daily on an exer- / animal treadmill (columbus instruments, co- lumbus, oh) over a -day period. the treadmill speed was initially set to m/min with a % uphill incline, and thereafter increased by increments of m/min every min up to a maximum speed of m/min. distance to fatigue was determined when the rat was unable to maintain pace with the treadmill belt for � s, despite encouragement to do so by tapping on the treadmill enclosure (electrical stimuli were not used). following the -day training period, the final testing regimen was as follows: speed of . m/min for min, followed by m/min for min, m/min for min, and m/min until fatigue. lowering of the hindquarters and a raised snout, resulting in an altered gait, generally precedes fatigue, which was confirmed by placing the animal on its back and observing a delayed (� s) righting reflex. in the case of a normal righting reflex, the test was repeated daily until fatigue was confirmed. distance covered until fatigue was recorded to the nearest tenth of a meter. tissue homogenate preparation. right lower lobes ( animals per group, equal sex distribution) were flash frozen, homogenized, and sonicated ( w for s) in ripa cell lysis buffer [ mm napo , . m nacl, . % (wt/vol) sodium dodecyl sulfate (sds), % (vol/vol) nonidet p- , % (vol/vol) sodium deoxycholate, mm edta, ph . ] containing protease (calbiochem) and phosphatase (sigma life science) inhibitors. left lung lobes ( animals per group, equal sex distribution) were flash frozen, homogenized in lysis buffer ( mm tris·hcl, ph , mm nacl, mm mgcl , mm edta, % triton x- ) containing protease and phosphatase inhib- itors for immunoprecipitation. the lung homogenates were left on ice for min before centrifugation ( , g for min). the supernatant was collected and protein concentration was measured by bradford assay. samples were stored at � °c until analysis. serum collection. following euthanasia of animals for tissue col- lection, the thoracic cavity was exposed and blood was drawn by direct lv cardiac puncture with a -gauge needle. blood was kept at room temperature for min to coagulate in microcentrifuge tubes. the tubes were spun down at , g for min. supernatant was collected by pasteur pipette and stored at � °c until assay. western blot analyses. lung homogenates from six animals per group (equal sex distribution) stored in ripa buffer containing protease and phosphatase inhibitors. tissue containing – �g of protein was boiled for min in sds sample buffer [ mm tris·hcl, % (wt/vol) sds, % (vol/vol) glycerol, mm 	-mercaptoethanol, ph . ] and separated under reducing conditions by sds polyacryl- amide gel electrophoresis for – h at – v depending on protein of interest. following electrophoresis, proteins were trans- ferred to pvdf membranes. all membranes were blocked with % bsa for h at room temperature, followed by incubation with primary antibody overnight at °c. blots were then washed with tris-buffered saline with tween and placed in secondary antibody for h at room temperature. following blotting, bands were imaged using an enhanced chemiluminescence kit (supersignal west dura chemiluminescent substrate, thermo scientific). dilutions of anti- bodies were as follows: : , for anti-rhoa ( kda), anti-cleaved l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . rock i ( kda), anti-p-thr mypt ( kda), anti-pan mypt ( kda), anti-hif- � ( kda), and anti-enos ( kda); : , for anti-rock i ( kda) and anti-rock ii ( kda); : , for anti-gapdh ( kda); : , for secondary antisera. blots were electronically captured using a microchemi chemilu- minescent camera (dnr bioimaging systems, jerusalem, israel) and quantified by digital densitometry of nonsaturated images with back- ground density removed using imagej software (national institutes of health, bethesda, md). quantitative analyses were conducted as follows: for each protein of interest, three gels were run comparing all samples from a group (normoxia vehicle, normoxia simvastatin, or hypoxia simvastatin) to all samples from the control group (hypoxia vehicle). membranes were stripped and then reblotted for the normal- izer protein (e.g., pan-mypt , rhoa, or gapdh) to account for differences in protein loading, and results expressed after normaliza- tion to the control band, as previously described ( ). representative images show contiguous lanes (the same lanes for the protein of interest and the normalizer) from each group that best represented the quantitative analysis. rhoa activity assay. the concentration of lung tissue homogenates were equalized to mg/ml in ml lysis buffer. anti-active rhoa monoclonal antibody ( �l) was added to the tube followed by �l of resuspended a/g agarose bead slurry. the tubes were incubated at °c for . h with gentle agitation following which beads were pelleted by centrifugation for min at , g at °c. the supernatant was removed and stored for measurement of non-gtp-bound rhoa. the beads were washed three times with . ml lysis buffer, centrifuging, aspirating, and discarding the supernatant each time. after the last wash, all supernatant was removed and discarded. the pellet was resuspended in �l reducing sds-page sample buffer. samples were boiled for min then centrifuged for s at , g. two western blots were run in parallel with samples from the pellet or the supernatant. the membranes were probed with anti-rhoa primary antibody. the proportion of active rhoa was determined by band densities of active rhoa normalized to total rhoa [gtp-rhoa/ (gtp-rhoa�non-gtp-rhoa)]. histological studies. two males and two females were randomly selected from each of the experimental groups and euthanized by pentobarbital sodium overdose. following the opening of the thoracic cavity and trachea cannulation, the pulmonary veins were divided. the pulmonary circulation was flushed with pbs and heparin, to clear the lungs of blood. the lungs were then perfusion fixed with parafor- maldehyde while air inflated at a constant pressure ( cmh o). following dehydration and clearance in xylene, tissues were embed- ded in paraffin, cut into -�m sections, mounted onto superfrost slides, allowed to air dry, and baked overnight at °c. for elastin staining, sections were dewaxed by immersion in xylene, rehydrated in ethanol, rinsed in several washes of distilled water, and then left overnight in weigert’s resorcin-fuchsin stain. for �-smooth muscle actin immunostaining, concentration of primary antibody for �-smooth muscle actin was : , and secondary goat anti-mouse antibody was : then counterstained with carazzi hematoxylin. slides were washed in tap water, dehydrated, and mounted with a coverslip using a % permount/ % xylene solution, as previously described ( , ). preparation of cardiac tissue. paraffin-embedded hearts, cut below the atrioventricular valves and oriented in the short axis, were cut into -�m sections and mounted onto superfrost slides, allowed to air dry, and baked overnight at °c. slides were dewaxed and rehydrated. sections were stained with hematoxylin, washed in distilled water, and partially dehydrated before counterstaining with eosin. slides were dehydrated and mounted with a coverslip using a % per- mount/ % xylene solution. whole sections were scanned by the imaging facility at the hospital for sick children research institute, using a dhistech pannoramic flash ii slide scanner and pannoramic viewer . . ( dhistech, budapest, hungary) soft- ware. preparation of brain tissue. paraffin-embedded left brain hemi- sphere oriented sagitally was cut into -�m sections just lateral to the ventricle and mounted onto superfrost slides, allowed to air dry, and baked overnight at °c. slides were dewaxed and rehydrated. sec- tions were stained with luxol fast blue solution (to identify myelin) overnight for h at °c, washed in distilled water, placed in lithium carbonate solution for s, and partially dehydrated before counter- staining with cresyl violet blue solution. slides were dehydrated and mounted with a coverslip using a % permount/ % xylene solution. whole sections were scanned as described above. measurement of percent medial wall area. percent medial wall area (% mwa) was used as a marker of pulmonary vascular remodeling. pulmonary arteries ( – �m external diameter) were identified on elastin-stained sections by the presence of an inner and outer elastic lamina and proximity to respiratory bronchioles. a minimum of arteries per animal from four unique sections were digitally captured (pixera penguin cl, san jose, ca) and analysis was performed in a blinded fashion. obliquely sectioned vessels where there was a greater than three times difference in perpendicular dimensions, were excluded. using the “quick selection” tool (adobe photo- shop cs , adobe systems, san jose, ca), the area of the inner lumen and the whole vessel were outlined and pixel count deter- mined. the following formula was used to determine the % mwa: [(whole vessel area � inner luminal area)/whole vessel area] , as previously described ( ). evaluation of arterial muscularization. degree of vessel muscular- ization was evaluated on sections stained for �-smooth muscle actin. pulmonary arteries ( – �m external diameter and proximity to respiratory bronchioles) were identified on �-smooth muscle actin- immunostained sections. a minimum of arteries per animal from four unique sections were digitally captured and evaluated in a blinded fashion. degree of muscularization was divided in to three categories: nonmuscular, partially muscular, or fully muscular based on the amount of brown stain surrounding each vessel (none, partially, or completely). category of muscularity was expressed as a fraction of the total number of vessels examined. lc-ms/ms. tissue samples weighing � mg were homogenized ( mg/ ml) in : -propanol- mm ammonium bicarbonate. the equivalent of mg ( �l of homogenate) was added to a . -ml plastic tube containing internal standards (sigma-aldrich, oakville, ontario, canada) and briefly vortexed. a standard curve was generated ( . – ng) for each analyte (mevalonic acid, fpp, and ggpp), treated in the same way as the tissue samples, but with �l of : -propanol- mm ammonium bicarbonate added instead of sample. each tube was processed as follows: �l of acetonitrile added, vortexed, chilled on ice, and then centrifuged for min at °c at , g. the supernatant was transferred to a clean tube and the pellet was reextracted with ml acetonitrile, vortexed, chilled on ice, and centrifuged for min at °c at , g. the supernatants were combined and taken to dryness under a gentle flow of nitrogen gas. the residues were reconstituted in �l of : methanol-ammonium hydroxide, transferred to . ml plastic tubes, and centrifuged for min at °c at , g. the supernatant was transferred to a -�l insert in a -ml autosampler vial and injected for lc-ms/ms analysis. samples were analyzed by lc-ms/ms using an agilent hplc with qtrap mass spectrometer (ab sciex, concord, ontario, canada) in negative ion mode. one microliter of sample extract was injected into a kinetex xb-c column ( . mm, . �m, phenomenex) at a flow rate of �l/min. mevalonic acid, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate were separated using a gradient of mm ammonium bicarbonate and . % triethylamine in water (mpa) and mm ammonium bicarbonate and . % triethyl- amine in % acetonitrile as follows: starting with % mpa, moving to % mpa from . to . min, then to % mpa from . to . min, % mpa was held from . to . min, after . min the system was returned to % mpa and allowed to equilibrate resulting in a total run time of min. the following mass transitions (precursor ion l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . to the product ion) were monitored for quantification in multiple reaction monitoring mode: mevalonic acid to , farnesyl pyro- phosphate to , and geranylgeranyl pyrophosphate to . data were analyzed and quantified using analyst software (ab sciex). elisa. serum alt, ck, ldl/vldl cholesterol and total free cholesterol were measured according to the manufacturer’s protocols. data presentation and statistical analysis. values are expressed as means � se. table values are expressed as means � sd. analyses were performed using sigma plot . (systat software, san jose, ca). statistical significance was determined by one-way anova or kruskal-wallis anova on ranks for data sets failing a normality test, followed by student-newman-keuls post hoc test where significant (p � . ) intergroup differences were found. fig. . simvastatin decreased farnesyl pyro- phosphate and rhoa/rock activity in the hypoxia-exposed lung. pups were exposed to chronic hypoxia ( % o ) or to normoxia ( % o ) from postnatal day until day (prevention) or day (rescue). pups were treated by daily intraperitoneal injections of simvastatin ( mg/kg) or % dmso in pbs (vehicle control) from birth to day (a, b, and d), or from postnatal days – (c and e). a: fpp lung isoprenoid interme- diates analyzed by lc-ms/ms. western blot analyses of gtp-rhoa normalized to total rhoa (b and c) and pthr -mypt nor- malized to pan-mypt (d and e). repre- sentative immunoblots are shown below each graph with noncontiguous gel lanes demarcated by black lines. values repre- sent means � se for n � – samples per group. *p � . , by -way anova on ranks (a) or -way anova (b–d), com- pared with all other groups. #p � . , by -way anova, compared with all other groups. l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . results lung fpp content and rhoa/rock activity were attenuated by simvastatin. statins inhibit mevalonate synthesis, which reduces isoprenoid intermediate content ( , ). as shown in fig. a, chronic exposure to hypoxia had no effect on lung fpp content, compared with normoxia-exposed controls. preven- tive treatment with simvastatin significantly reduced lung fpp content compared with hypoxia-exposed vehicle-treated pups (fig. a). rhoa activity was measured by lung gtp-rhoa content, as a ratio of total (gtp�non-gtp bound) rhoa. hypoxia-exposed, vehicle-treated pups had significantly ele- vated rhoa activity in the lung relative to normoxia controls at both pnds and (fig. , b and c). lungs of chronic hypoxia-exposed pups treated with simvastatin had signifi- cantly reduced gtp-rhoa content when given as either pre- ventive or as rescue therapy (figs. , b and c). lung rock activity was determined by pthr -mypt content, as a ratio of the pan-mypt protein. lungs of hypoxia-exposed, vehi- cle-treated pups had significantly increased rock activity compared with normoxia controls at both pnds and fig. . simvastatin prevented chronic hypoxia-induced pulmonary hypertension. pups were exposed to chronic hypoxia ( % o ) to normoxia ( % o ) from postnatal day until day . pups were treated by daily intraperitoneal injections of simvastatin ( mg/kg) or % dmso in pbs (vehicle control). a: pulmonary vascular resistance (pvr) index, as measured by the right ventricular ejection time (rvet)-to-pulmonary arterial acceleration time (paat) ratio (values represent means � se for n � – animals/group). b: right ventricle (rv)-to-left ventricle � septum (lv�s) dry weight ratios (fulton index) as a marker of right ventricular hypertrophy (values represent means � se; n � – animals/group). c: tiled low-power photomicrographs of hematoxylin and eosin-stained cardiac sections, oriented in the short axis below the atrioventricular valves (rv, right ventricular cavity; scale bar � , �m), are shown to demonstrate differences in rv free wall thickness between groups. *p � . , by -way anova on ranks, compared with all other groups. l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . (fig. , d and e). simvastatin significantly attenuated in- creased lung rock activity secondary to hypoxia, relative to the vehicle-treated animals, when given as either preventive or rescue therapy (fig. , d and e). simvastatin prevented chronic pht. similar to previous findings ( , ), exposure to hypoxia significantly increased pvr index (fig. a) and fulton index (fig. b) in vehicle- treated animals, compared with normoxia controls. animals exposed to hypoxia and treated with simvastatin had signifi- cantly reduced pvr index (fig. a) and fulton index (fig. b) compared with hypoxia-exposed vehicle-treated animals and had values comparable to normoxia controls. % mwa and degree of muscularization were used as measures of pulmonary arterial remodeling. as shown previously ( , , ), and in fig. a, pulmonary arteries of animals chronically exposed to hypoxia had greatly increased % mwa, which was accompa- nied by an increased proportion of fully muscularized arteries (fig. c). treatment with simvastatin reduced both % mwa (fig. a) and proportion of fully muscularized arteries (fig. c) relative to vehicle-treated controls. chronic hypoxia-induced pht was reversed and exercise capacity increased by rescue treatment with simvastatin. pre- vious studies have demonstrated that systemic or inhaled treat- ment with a rock inhibitor reversed pht induced by chronic fig. . simvastatin prevented chronic hypoxia-induced pulmonary arterial remodeling. pups were exposed to chronic hypoxia ( % o ) or to normoxia ( % o ) from postnatal day until day . pups were treated by daily intraperitoneal injections of simvastatin ( mg/kg) or % dmso in pbs (vehicle control). a: percentage arterial medial wall area (values represent means � se for n � animals/group) as a marker of pulmonary arterial remodeling. b: representative photomicrographs of elastin staining (dark brown inner and outer elastic laminae delineating the medial vascular wall; scale bar � �m) demonstrate differences between groups in medial wall thickening. c: degree of muscularization (values represent means � se for n � animals/group) as a marker of pulmonary arterial smooth muscle content. d: representative photomicrographs of �-smooth muscle actin staining (dark brown staining surrounding artery; scale bar � �m) demonstrate proliferation of pulmonary arterial smooth muscle. *p � . , by -way anova on ranks compared with all other groups. #p � . , by -way anova compared with normoxia vehicle group for proportion of partially muscularized arteries. †p � . , by -way anova, compared with all other groups for proportions of nonmuscularized and ‡p � . for fully muscularized arteries. l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . hypoxia in newborn rats ( , ). similarly, relative to vehicle- treated animals, sustained rescue treatment with simvastatin significantly decreased pvr (fig. a), rv hypertrophy (fig. b), % mwa (fig. c), and proportion of fully muscularized arteries (fig. d) compared with chronic hypoxia-exposed vehicle-treated animals. exercise capacity in chronic hypoxia, vehicle-treated rats was significantly reduced compared with air-exposed vehicle- or simvastatin-treated controls. female rats ran a significantly greater distance compared with male rats across all groups and therefore data were stratified by sex. l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . rescue treatment with simvastatin significantly increased dis- tance run in females and males compared with hypoxia-ex- posed, vehicle-treated controls (fig. e). preventive treatment with simvastatin improved hypoxia- induced growth restriction and acute treatment with simvasta- tin did not affect systemic blood pressure. we have previously reported significant adverse effects of systemic rock inhibi- tion, including worsened hypoxia-induced growth restriction and greatly decreased systemic blood pressure ( , ). as previously documented ( ), chronic exposure to hypoxia from birth lead to a significant growth restriction (fig. a). preventive (fig. a) treatment with simvastatin increased body weight at pnd compared with the hypoxia-ex- posed, vehicle-treated animals (fig. a). relative to nor- moxia-exposed controls, chronic hypoxia-exposed animals had significantly increased systolic blood pressure (fig. b). acute treatment with simvastatin had no effect on systolic blood pressure (fig. b). effects of hypoxia and preventive treatment with simvastatin on brain weight and periventricular myelin content. relative to normoxia-exposed vehicle-treated animals, chronic exposure to hypoxia significantly decreased brain weight (fig. c) and decreased periventricular myelin density (fig. d). treatment with simvastatin had no effects on brain weight in either normoxia- or hypoxia-exposed animals (fig. c). preventive treatment with simvastatin had no effect on periventricular myelin density in normoxia-exposed animals, while causing a significant increase in hypoxia-exposed animals (fig. d). preventive treatment with simvastatin did not increase se- rum markers of muscle or liver toxicity. statin therapy has been reported in adult humans to cause myopathy (increased ck) and liver injury (increased alt) ( ). as shown in table , neither chronic exposure to hypoxia nor treatment with simva- statin altered serum alt or ck. in addition, no changes in these parameters were observed at mg·kg� ·day� , the highest dose employed in preliminary studies (data not shown). preventive treatment with simvastatin had no effect on serum cholesterol. as shown in table , neither chronic exposure to hypoxia nor treatment with simvastatin had any effect on total serum cholesterol. to control for the possible effects of dmso vehicle on cholesterol levels ( ), serum cholesterol was also measured in normoxia- and hypoxia- exposed animals not injected with vehicle. there was no difference in serum cholesterol between noninjected and vehicle-treated groups (data not shown). as shown in table , chronic hypoxia significantly increased serum ldl cho- lesterol, which was unaffected by treatment with simvasta- tin. effects of hypoxia and preventive treatment with simvastatin on lung cleaved rock i, rock i, rock ii, hif- �, and enos contents. chronic exposure to hypoxia greatly decreased cleaved rock i (constitutively active form of rock) content in the lung compared with normoxia-exposed controls (fig. a). normoxia-exposed animals treated with simvastatin had decreased cleaved rock i content compared with vehicle- treated animals (fig. a). rock i content was significantly decreased by exposure to hypoxia, which was unaffected by treatment with simvastatin (fig. a). there were no differences in rock ii content between groups (fig. c). hif- � is known to be upregulated by hypoxia. accordingly, neonatal rat pups chronically exposed to hypoxia had significantly in- creased hif- � protein content in the lung compared with normoxia controls (fig. d). treatment with simvastatin had significantly reduced hif- � content in lungs exposed to chronic hypoxia (fig. d). enos expression has been de- scribed to be attenuated by increased rock activity ( ). as shown in fig. e, preventive treatment with simvastatin in- creased enos content in both normoxia- and hypoxia-exposed animals. discussion we have previously established a critical role for the rock signaling in the pathogenesis of chronic hypoxic pht in neonatal rats ( , , ). in the present study, we observed that simvastatin both prevented and reversed chronic pht, at least in part via modulation of rhoa and downstream rock activity. the novel aspect of this study was the examination of effects of simvastatin on chronic neonatal pht. despite favor- able results in adult experimental chronic pht ( , , , ), trials employing statins in human adults with chronic pht have thus far shown an apparent lack of efficacy ( ). the implications of these findings for translation of statins to the neonate remain open, given the potential for maturational differences in response to pharmacological agents that may affect the pulmonary vasculature. for example, selective sero- tonin reuptake inhibitors (ssris) impose a higher risk of pht in newborns when given to mothers in late pregnancy ( ), whereas studies in adults with chronic pht suggest that ssris may improve outcome ( ). a small observational study in children with chronic pht showed improvement in hemody- namic parameters with simvastatin therapy ( ). importantly, the potential utility of statins as a means of targeting rhoa/ rock activity in the newborn likely extends beyond pht. we have recently reported a contributory role for upregulated pulmonary rock signaling in experimental bpd-like lung fig. . simvastatin reversed chronic pht in juveniles and improved exercise tolerance in young adults. pups were exposed to chronic hypoxia ( % o ) or to normoxia ( % o ) from postnatal day until day . pups were treated by daily intraperitoneal injections of simvastatin ( mg/kg) or % dmso in pbs (vehicle control) from postnatal days to . a: pulmonary vascular resistance index (pvri), as measured by the right ventricular ejection time (rvet)-to-pulmonary arterial acceleration time (paat) ratio (values represent means � se for n � animals/group). b: right ventricle (rv)-to-left ventricle � septum (lv�s) dry weight ratios (fulton index) as a marker of right ventricular hypertrophy (values represent means � se n � – animals/group). c: percentage arterial medial wall area (values represent means � se for n � animals/group) as a marker of pulmonary arterial remodeling. d: degree of muscularization (values represent means � se for n � animals/group) as a marker of pulmonary arterial smooth muscle content. e: maximum exercise capacity measured in meters run (values represent means � se for n � – animals/group). *p � . , by -way anova on ranks, compared with all other groups. #p � . , by -way anova on ranks, compared with normoxia-exposed groups. †p � . , by -way anova, compared with all other groups, ‡p � . , by -way anova, compared with normoxia-exposed groups. §p � . (nonmuscularized arteries), �p � . (fully muscularized and partially muscularized arteries), by -way anova, compared with hypoxia vehicle group. **p � . , by -way anova, compared with hypoxia vehicle group of the same sex. ††p � . , by -way anova, compared with hypoxia vehicle group of the same sex. l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . injury ( ), a developmental lung disorder of prematurity characterized by inhibited pulmonary angiogenesis and alve- ologenesis. similarly, pulmonary hypoplasia and vascular re- modeling in fetal rats with nitrofen-induced congenital dia- phragmatic hernia (cdh), in which rhoa may play a patho- logical role ( ), was ameliorated by maternally administered simvastatin ( ). as there are currently no effective preventive therapies for developmental lung disorders such and bpd and l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . cdh, or to prevent or rescue chronic pht in neonates ( ), these findings point toward statins as a potentially useful therapy. we evaluated dose-response effects of simvastatin, establishing a dose that balanced efficacy and safety. this aspect is critical for potential translation since therapies de- signed to ameliorate injury to the lung and pulmonary vascu- lature could have unanticipated adverse effects on other sys- tems, particularly the developing brain. collectively, our find- ings provide compelling initial evidence for the translational potential of statin therapy for neonatal pulmonary disease. we observed that preventively administered simvastatin attenuated the activity of rhoa, normalized pvr, and reduced both rv and pulmonary arterial structural remodeling. in our rescue protocol, the reduction of rhoa/rock activity also reduced pvr and led to significant but incomplete reversal of cardiac and vascular remodeling. severity of pulmonary vas- cular changes secondary to chronic hypoxia peaks by days of exposure and is stable from days to ; therefore, effects of rescue therapies (given from days to ) reflect reversal rather than slowed progression of injury. rats exposed to chronic hypoxia receiving rescue treatment with simvastatin also had improved exercise capacity as young adults in keeping with an important and relevant functional improvement. the isoprenoid ggpp contributes to the pathogenesis of pht by prenylation-mediated intracellular trafficking and membrane localization, and subsequent activation of rhoa ( , , , ). in the present study, we observed an increase in fpp and in hif- � content in the lungs of chronic hypoxia- exposed rats. we also observed that simvastatin reduced lung content of fpp in hypoxia-exposed animals, with a trend toward reduction in normoxia controls. presumably, a reduc- tion of fpp also led to decreased ggpp as indicated by reduced rhoa activity; however, we were unable to reliably measure ggpp content in lung. it is known that hif- � activity is elevated in hypoxia, which in turn increases tran- scription of rhoa and/or rock ( ). treatment with simva- statin decreased pulmonary hif- � levels in chronic hypoxia- exposed animals but did not affect lung content of rhoa or of either rock isoform ( , ). therefore, the effect of simva- statin could be largely attributed to downregulation of rhoa activity via inhibition of isoprenoid intermediates, rather than by changes in protein content mediated by hif- �. in support of this observation, girgis and colleagues ( ) demonstrated that concurrent supplementation of mevalonate with simvasta- tin treatment negated any beneficial effects in experimental pht. the pathology of chronic hypoxic pht is characterized in neonatal animals by severe pulmonary vascular remodeling ( ). in previous studies, inhibition of rock signaling pre- vented vascular remodeling by inhibiting smc proliferation ( ). rescue therapy with rock inhibitor opposed the anti- apoptotic effect of rock (specifically by rock ii), therefore stimulating smc apoptosis and leading to reversal of remod- eling ( ). inhibited no-cgmp signaling plays a major role in the pathogenesis of chronic pht ( , ). rock inhibits enos activity and disrupts the stability of enos mrna, thus reducing no bioavailability ( , ). in the present study, we observed that simvastatin increased enos content, which fur- ther supports suppression of rock activity as a major mech- anism for simvastatin effects on chronic neonatal pht ( ). neonates are particularly susceptible to adverse and off- target effects of pharmacological agents ( ). documented ad- verse effects of statins in adult humans include hepatic injury and muscle pain. in rabbits, high-dose simvastatin ( mg·kg� ·day� ) increased serum lactate dehydrogenase, ck, and alt ( ). in the present study, we did not observe any significant increase in serum levels of alt or ck, even at doses of simvastatin as high as mg/kg. systemically admin- istered rock inhibitors restrict somatic growth, thus limiting translational potential to the neonate ( ). in the present study, simvastatin did not adversely affect somatic growth in nor- moxia-exposed animals and actually restored normal growth in chronic hypoxia-exposed animals. in addition, simvastatin did not affect systolic blood pressure, unlike systemic rock inhibitors ( ). we did not examine effects of exposure to hypoxia or of simvastatin therapy on rhoa/rock activity in systemic vessels, but we speculate that a lack of effect of simvastatin on systemic vascular tone reflects a greater hypox- ia-induced upregulation of rhoa/rock activity in the pulmo- nary, compared with the systemic, vasculature. the usual target of statin therapy is a reduction in serum cholesterol, which in the neonate may represent an adverse fig. . effects of chronic hypoxia from birth and preventive simvastatin treatment on somatic and brain growth, brain myelination, and systemic blood pressure. pups were exposed to chronic hypoxia ( % o ) or to normoxia ( % o ) from postnatal day until day . pups were treated by daily intraperitoneal injections of simvastatin ( mg/kg) or % dmso in pbs (vehicle control). a: body weight. values represent means � se for n � – animals per group. b: systemic blood pressure following therapy with simvastatin. values represent means � se for n � animals per group. c: brain weight. values represent means � se for n � samples per group. d: myelin pixel density. values represent means � se for n � samples per group. e: tiled low-power representative photomicrographs of luxol fast blue staining on sagittal brain sections (myelin � dark blue stain; scale bar � , �m). *p � . , by -way anova, compared with all other groups. #p � . , by -way anova, compared with normoxia controls. †p � . , by -way anova, compared with normoxia vehicle. ‡p � . , by -way anova, compared with normoxia controls. §p � . , by -way anova, compared with hypoxia vehicle group. table . effects of preventative treatment with simvastatin ( mg·kg� ·day� ) on serum markers of toxicity serum analyte normoxia vehicle normoxia simvastatin hypoxia vehicle hypoxia simvastatin alt, �mol/ml . � . . � . . � . . � . ck, �mol/ml . � . . � . . � . . � . total cholesterol, mg/dl . � . . � . . � . . � . ldl cholesterol, mg/dl . � . . � . . � . * . � . # values represent means � sd for n � animals per group. *p � . , by -way anova, compared to both normoxia groups. #p � . , by -way anova, compared to normoxia simvastatin group. l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . fig. . effects of preventive simvastatin on lung cleaved rock i, rock i, rock ii, hif- �, and enos content. pups were exposed to chronic hypoxia ( % o ) or to normoxia ( % o ) from postnatal day until day . pups were treated by daily intraperitoneal injections of simvastatin ( mg/kg) or % dmso in pbs (vehicle control). western blot analyses for cleaved rock i (a), rock i (b), rock ii (c), hif- � (d), and enos (e), all normalized to gapdh. representative immunoblots are shown with noncontiguous gel lanes demarcated by black lines. values represent means � se for n � – samples per group. *p � . , by -way anova on ranks, compared with all other groups. #p � . , by -way anova on ranks, compared with hypoxia simvastatin group. †p � . , by -way anova, compared with normoxia simvastatin group. ‡p � . , by -way anova, compared with all other groups. §p � . , by -way anova, compared with hypoxia vehicle group. l simvastatin prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . effect, given the reliance of the developing brain on cholesterol for normal development ( ). interestingly, neither simvastatin nor the dmso vehicle altered total serum cholesterol levels or ldl cholesterol in the newborn rat. this finding is likely explained by the fact that breastfeeding neonates rely less on endogenous synthesis of cholesterol to maintain serum levels, due to the presence of high levels of cholesterol in breast milk ( ). indeed, high doses of statins have not been found to alter brain cholesterol levels, which is required for myelin formation ( , , , ). lipid soluble statins such as simvastatin can cross the blood-brain barrier by lactonization. however, statins do not accumulate in the brain, being rapidly eliminated by the p-glycoprotein transporter ( ). furthermore, cyp a , the enzyme responsible for simvastatin metabolism and activation, is absent from the rodent brain ( , ) and has only been detected in the human brain at one-tenth the level of liver cyp a ( ). prematurely born humans have an increased risk for long-term neurodevelopmental disability. in models of newborn neurological injury and abnormal development, st- atins prevented injury through modulation of inflammation, leading to normalized morphology, white matter content, and brain weight ( , , ). while there were no reported adverse effects from simvastatin in a small observational study in children with pht, further study is needed to assess safety and efficacy in the newborn population despite our present reas- suring findings ( ). there are a number of limitations to this study. first, our chronic hypoxia model of pht does not mimic chronic pht secondary to developmental lung disorders, such as bpd, which is among the most common causes of chronic pht in neonates and infants ( ). therefore, effects of statin therapy should be studied in models mimicking inhibited pulmonary angiogenesis and arrested lung development, as observed in bpd ( ). second, use of simvastatin as a rescue therapy showed incomplete effects compared with prevention, which may have been overcome by prolongation of therapy. third, we did not conduct functional neurological examination to substantiate our morphological findings suggesting an im- provement in hypoxia-mediated disruption of myelination with simvastatin treatment. fourth, our focus was restricted to the activity of rhoa. many other gtpase proteins could be similarly affected by statin therapy, based on their dependence for prenylation-mediated activation. of note, the ras family of g proteins and other members of the rho family such as rac and cdc may warrant further investigation since they are known to be involved in cardiac hypertrophy, actin cytoskel- eton remodeling, and smc proliferation ( , ). lastly, eval- uation of sex differences in response to therapy is a relevant biological variable that should be evaluated in future studies. in summary, simvastatin-mediated inhibition of isoprenoid intermediates downregulates rhoa activity, leading to preven- tion and reversal of chronic pht. our findings suggest that simvastatin was well tolerated, lacked apparent adverse sys- temic or neurological effects, and therefore holds promise as a potentially translatable means of limiting pathological rock activity in the neonate. acknowledgments the authors thank denis reynaud and ashley st. pierre of the analytical facility for bioactive molecules, the hospital for sick children, toronto, canada, for assistance with lc- ms/ms analysis of lung tissue isoprenoid intermediates. grants this work was supported by operating funding from the canadian institutes of health research (cihr; mop- to r. p. jankov) and by infrastructure funding from the canada foundation for innovation (to r. p. jankov). a. jain was supported by a clinician-scientist training program award from the hospital for sick children research training centre and by a queen elizabeth ii/heart and stroke foundation of ontario graduate scholarship in science and technology. m. j. wong was supported by a graduate scholarship from the department of physiology, university of toronto and by a lorne phenix graduate award from the cardiovascular sciences collaborative program, faculty of medicine, university of toronto. disclosures no conflicts of interest, financial or otherwise, are declared by the author(s). author contributions m.j.w. and r.p.j. conceived and designed research; m.j.w., c.k., j.i., a.j., and r.p.j. performed experiments; m.j.w., a.j., and r.p.j. analyzed data; m.j.w. and r.p.j. interpreted results of experiments; m.j.w. and r.p.j. prepared figures; m.j.w. and r.p.j. drafted manuscript; m.j.w., c.k., j.i., a.j., and r.p.j. edited and revised manuscript; m.j.w., c.k., j.i., a.j., and r.p.j. approved final version of manuscript. references . adibhatla rm, hatcher jf. altered lipid metabolism in 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neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . the mobius airo mobile ct for image‐guided proton therapy: characterization & commissioning r a d i a t i o n o n c o l o g y p h y s i c s the mobius airo mobile ct for image-guided proton therapy: characterization & commissioning jasmine a. oliver | omar a. zeidan | sanford l. meeks | amish p. shah | jason pukala | patrick kelly | naren r. ramakrishna | twyla r. willoughby department of radiation oncology, uf health cancer center – orlando health, orlando, fl, usa author to whom correspondence should be addressed. twyla r. willoughby e-mail: twyla.willoughby@orlando health.com; telephone: . abstract purpose: the purpose of this study was to characterize the mobius airo mobile ct system for localization and image-guided proton therapy. this is the first known application of the airo for proton therapy. methods: five ct images of a catphan� phantom were acquired on the airo mobile ct system, varian edge radiosurgery system cone beam ct (cbct), philips bril- liance big bore slice ct simulator, and siemens somatom definition as slice ct simulator. doselab software v. . was utilized for image quality analysis. modulation transfer function, scaling discrepancy, geometric distortion, spatial resolution, overall uni- formity, minimum uniformity, contrast, high cnr, and maximum hu deviation were acquired. low cnr was acquired manually using the ctp module. localization accu- racy and ct dose index were measured and compared to reported values on each imag- ing device. for treatment delivery systems (edge and mevion), the localization accuracy of the d imaging systems were compared to d imaging systems on each system. results: the airo spatial resolution was . lp mm� compared with . lp mm� for the philips ct simulator, . lp mm� for the edge cbct, and . lp mm� for the siemens ct simulator. airo/siemens and airo/philips differ- ences exceeded % for scaling discrepancy ( . % and . %). the airo exhibited higher dose (> mgy) than the philips ct simulator. localization accu- racy (based on the mimi phantom) was . ° and . mm. localization accuracy (based on stereophan) demonstrated maximum airo-kv/kv shift differences of . mm in the x-direction, . mm in the y-direction, and . mm in the z-direction. conclusions: the localization accuracy of airo was determined to be within . ° and . mm despite its slightly lower image quality overall compared to other ct imaging systems at our institution. based on our study, the mobile airo ct system can be utilized accurately and reliably for image-guided proton therapy. p a c s . .bn, . .c-, . .n-, . .q- k e y w o r d s airo, igpt, mobile ct, proton therapy - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - this is an open access article under the terms of the creative commons attribution license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © the authors. journal of applied clinical medical physics published by wiley periodicals, inc. on behalf of american association of physicists in medicine. received: november | revised: january | accepted: february doi: . /acm . | wileyonlinelibrary.com/journal/jacmp j appl clin med phys ; : : – | introduction advances in in-room computed tomography (ct) scanners and cone- beam technology have led to the proliferation of ct localization for image guided radiation therapy (igrt). – these imagers provide improved treatment accuracy over conventional orthogonal imaging allowing for increased precision in radiation delivery. patient localiza- tion accuracy is particularly important in proton beam radiation ther- apy due to the sharp dose fall-off compared to conventional x-ray therapy. unfortunately, due to the size and geometry of proton ther- apy units, imaging has largely been limited to orthogonal kv/kv x-ray systems. recently, people have reported on the use of in- room ct scanners for proton beam radiation therapy localization. – some proton vendors are developing technologies for cbct (pro- teus�one, iba, belgium, hitachi, tokyo, japan). to date, stand- alone in-room ct scanners utilized have had large footprints and are either not amenable to or cumbersome to use in the more compact proton therapy centers, such as the s (mevion medical systems, littleton, ma, usa). for these reasons, a small-footprint mobile ct scanner, commonly used for image-guided surgery, could be of great value for d image-guided proton therapy (igpt). the airo mobile ct system (mobius imaging llc, shirley, ma, usa) is a large bore ( cm) helical slice ct scanner historically uti- lized for intra-operative imaging for spinal surgeries. our institution is the first to acquire and clinically implement the airo mobile ct system (airo) for igpt. the airo’s small footprint (w l h: . . . m) occupies . m of treatment floor space (in scan mode). when not in use, the airo is stored in the maze (fig. ) to avoid radiation-induced damage to its sensitive electronics. the airo’s motor-controlled castors provide effortless transport from the storage location (in the maze hallway) to the scanning location. the airo’s image quality characteristics have been reported by weir et al. for surgical applications. our study assessed the perfor- mance characteristics of the airo at our institution compared with igrt systems used for intensity-modulated radiation therapy (imrt) and ct simulators at our institution for radiotherapy applications. our current igpt workflow involves ct simulation using a philips big bore slice ct simulator with routine orthogonal kv/kv image pairs (flat- panel detectors) preceding each treatment fraction. the airo will be utilized for target localization and inter-fraction adaptive treatment assessment. to our knowledge, our facility is the first compact proton therapy system to use a mobile ct scanner for igpt. | materials and methods mevion medical systems has developed software, veritytm d, that uses ct images from any scanner to perform d image f i g . . mevion-airo room setup. (a) treatment room with couch in setup position. (b) treatment room with couch in imaging position. (c) birds-eye-view of the treatment room. the airo is shown in scan mode with cm max scan range and . cm image field-of-view. pa and left lateral kv imaging panels are also shown with associated track. oliver et al. | registrations for proton therapy. due to the ease of motion and small footprint, we have selected the airo ct scanner for igpt. the airo is sold commercially for surgical purposes by brainlab (munich, germany). mevion has developed an infrared camera tracking system to interface with the ct scanner to facilitate use of ct images for igpt. this system references the d image set to the in-room coordinates and planning ct via a reference frame, infrared camera, and ct scan. this reference frame attaches to the treatment couch and includes infrared markers to determine initial room coordinates as well as ceramic markers to reference the ct image to room coordinates. an additional infrared marker is rigidly attached to the gantry so that changes in the reference frame loca- tion in relation to the treatment room can be detected as the robotic couch moves between the initial treatment setup location and the ct imaging location. the airo unit was specifically adapted to use inside the proton vault by removing the integrated stand that supports the weight of surgical gurneys in order to avoid interference with the robotic couch (fig. ). .a | image quality characterization and comparison to simulators and igrt systems five ct images of a catphan� phantom (the phantom labora- tory, salem, ny, usa) were acquired on the airo mobile ct sys- tem, a varian edgetm radiosurgery system (edge) on board imager, a philips brilliance big bore slice ct simulator, and a sie- mens somatom definition as slice ct simulator. medial and lateral lasers were used to align the phantom prior to image acquisition. image acquisition was performed with various protocols. on the airo, images were acquired with soft, standard, and sharp recon- struction kernels with the pre-set clinical head protocol. the airo’s pre-set clinical protocols for head, thorax, abdomen, pelvis, shoulder are listed in table . the sharp, standard, and soft kernels are noise filtering gaussian smoothing kernels with r = . , . , and . , respectively. on the edge, images were acquired using the following pre-set clinical techniques: head, thorax, pelvis, pelvis obese, and image gently (table ). on the philips ct simulator, standard clinical proto- cols were used ( kvp, ma) with . mm, . mm, and . mm slice thickness. on the siemens ct simulator images were acquired with standard brain ( kv, mas), high res brain ( kv, mas), abdomen ( kv, mas), and thorax ( kv, mas) protocols with . mm, . mm, and . mm slice thickness. multimodality comparisons were made using the clinical head protocols on the airo, philips, and siemens ct simulators and the pelvis technique on the edge. the pelvis technique was used on the edge because it uses full ° gantry rotation and provides the high- est image quality. the edge head protocol involves a ° gantry rotation and is configured for low dose rather than image quality. doselab software v. . (mobius medical systems lp, houston, tx, usa) was used for image quality analysis. modules ctp , ctp , and ctp were analyzed. the following image quality measures were assessed: modulation transfer function (mtf), scaling discrepancy, geometric distortion, spatial resolution, overall unifor- mity, minimum uniformity, contrast, cnr, and maximum hu devia- tion (table ). the mean and standard deviation of five or more scans are reported. the low contrast module (ctp ) was assessed on the airo, philips, and siemens scans. low cnr was calculated using the . mm % supra-slice target. a region-of-interest (roi) was dropped onto the supra-slice target and another on a uniform region of the phantom. .b | localization accuracy a stereophan phantom (sun nuclear corporation, melbourne, fl, usa) was used to measure the localization accuracy of the mevion- airo system. the universal spacer insert and ct/mri insert were inserted into the cylinder cavity. the phantom was leveled with the precision leveling stand. treatment isocenter was placed at the bb target corresponding to the laser alignment marks on the outside of the phantom. the robotic couch was re-positioned to acquire the airo images following the mevion localization workflow. once the images were acquired, the robotic couch was moved back to the ini- tial treatment isocenter based on the robotic couch’s coordinates. the software then performed a ct registration correlating the refer- ence frame’s ceramic fiducials to its infrared markers (relative to treatment isocenter). the images were then manually registered to the d planning ct. successively, a kv/kv image pair was acquired and registered to the digitally reconstructed radiograph (drr). local- ization accuracy was defined as the difference between suggested shifts from the kv/kv pair versus the airo ct image set, where the kv/kv image was considered the gold standard. in addition, end-to- end tests were performed to illustrate both time and workflow for patient setup and target localization. additional localization accuracy tests were performed using the degree-of-freedom mimi phantom (standard imaging, inc. middleton, wi, usa). the default q-fix overlay couch top was used. with the proton gantry positioned at ° the reference frame was attached to the couch top and the couch was positioned to ° to capture the reference frame. known shifts were applied to the phantom sepa- rately including all translational directions (lateral, longitudinal, and vertical), all rotations (yaw, shift, and roll), and cumulatively (all t a b l e edge cbct & airo helical ct clinical pre-set protocols/ techniques. edge cbct techniques airo helical ct protocols techniques kvp mas protocols kvp mas head head thorax thorax pelvis pelvis pelvis obese abdomen image gently shoulder | oliver et al. degrees of freedom). a reference kv/kv image set was acquired pre- ceding the airo ct image. the couch was moved to the d imaging position and a d ct image was acquired. the image was sent to the mevion treatment console and verity, and then registered to the d planning ct. an initial manual rigid registration was performed followed by automated registration for fine tuning. suggested shifts were applied. the robotic couch was then re-positioned to standard imaging position and a d orthogonal kv x-ray pair was acquired. suggested shifts (kv/kv) were recorded. localization accuracy was defined as the difference between suggested shifts and known shifts. .c | measured imaging dose ct dose index for helical scans (ctdivol) was calculated using a -cm and -cm cylindrical polymethylmetacrylate phantom (cirs, computerized imaging reference systems, inc., norfolk, va, model & a) with mm pencil ionization chamber and electrome- ter (raysafe xi ct detector, unfors raysafe, inc., cleveland, oh, usa) on the airo and the philips ct simulator. the ion chamber was placed at the o’clock and the o’clock positions. three dose measurements were acquired at each position. measured dose was then compared to the vendor-reported ctdivol. acr requirements specify that dose must be within % of manufacturer-reported specifications. | results .a | image quality characterization and comparison to simulators and igrt systems .a. | airo reconstruction kernel effects on image quality the airo image quality results for various kernels are listed in table . the airo sharp kernel provided the best mtf ( . line pairs/mm). high cnr and spatial resolution ( . % difference) decreased from sharp to soft kernel. low cnr increased from sharp kernel to soft kernel ( . – . ). overall uniformity and mini- mum uniformity were comparable across reconstruction kernels (< . % difference). geometric distortion was equal for sharp and standard reconstruction kernels and < % difference between sharp/soft and standard/soft kernels. figure illustrates that the t a b l e image quality tests and calculation methods. image quality tests definition modulation transfer function (mtf)a the modulation of multiple rois in the phantom with various line bar patterns. as spatial frequency increases, roi’s modulation decreases. the modulation is normalized to its highest value and plotted. modulationa hu �hu hu þhu where hu s the th percentile ct number in the roi and hu m %ð Þ s the th percentile ct number in the roi. scaling discrepancya the maximum possible error in a measurement of distance. mð%Þ � %j j � l where m ð%Þ is the magnification of the image and l is the length of the image’s longest side. geometric distortiona verifies that correct distance measurement occurs in all regions of the ct image. overall uniformitya � max �min max þmin � � � % where max is the maximum th percentile ct number in all uniformity regions and min is the minimum th percentile ct number in all uniformity regions minimum uniformitya the lowest uniformity of all rois. uniformity is calculated as: � hu �hu hu þhu � � � % where hu is the th percentile of the pixel ct number in the roi and hu is the th percentile of the ct number in the roi. maximum hu deviationa the maximum deviation between mean hu value of an roi subtracted from its defined reference hu absolute value of all calculated hu deviation on an image. contrasta hu �hu hu þhu � � � % where hu is the mean ct number in the region with greater signal and hu is the mean ct number in the region with lesser signal. high contrast-to-noise ratioa highcontrast noise ¼ hu �hu hu þhu � % � �, ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðr Þþðr Þ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðhu Þ þðhu Þ p � � � % where r is the standard deviation of the high contrast roi, r is the standard deviation of background, hu is the mean ct number of the high contrast roi and hu is the mean ct number of background. low contrast-to-noise ratio lowcontrast noise ¼ huroi�hubj j rb where rb is the standard deviation of the background roi, huroi is the mean ct number inside the low contrast roi and hub is the mean ct number of the background roi. adefinitions derived from doselab v. . user manual. oliver et al. | mtf for the sharp kernel with small fov exceeded that of the soft kernel. .a. | comparison to simulators and igrt systems figure illustrates that the mtf and spatial resolution of the airo ( . lp/mm) was lower than the philips ct simulator ( . lp/mm), siemens ct simulator ( . lp/mm), and edge cbct ( . lp/mm). conversely, the philips ct simulator exhibited superior mtf and spatial resolution compared to all modalities (table and fig. ). the airo had comparable minimum and overall uniformity when com- pared to the philips ct simulator, siemens ct simulator or the edge (< . % difference, table ). the philips high cnr was superior to the airo with a . difference between the two values ( . % dif- ference). the siemens high cnr was superior to the airo with a . difference between the two values ( . % difference). philips maximum hu deviation was comparable to the airo ( . % differ- ence). siemens maximum hu deviation was larger than the airo’s maximum hu deviation ( . % difference). the edge demonstrated the smallest hu deviation ( . hu, . % difference from airo). airo/philips image quality differences were within % for over- all uniformity, minimum uniformity, and contrast and high cnr. airo/philips geometric distortion difference was . mm ( %). airo/siemens high cnr differences were . % and geometric distortion differences were %. airo/siemens differences exceeded % for scaling discrepancy ( . %). the airo low cnr was . for the standard reconstruction kernel. the philips and siemens ct simulator low cnr were . and . , respec- tively. refer to table for hu constancy values for the airo, phi- lips ct simulator, siemens ct simulator and edge cbct (module ctp ). .b | localization accuracy of mevion-airo system localization accuracy results using the stereophan phantom (based on two trials) demonstrated maximum airo-kv/kv shift differences of . mm in the x-direction, . mm in the y-direction, and . mm in the z-direction. (rotations were not included in the analysis because the target was a single bb inside of the phantom.) the mimi phantom was used to characterize the rotational corrections. the rms values were between . mm and . mm. localization accuracy (based on the mimi phantom) was within . °. registration (kv/kv) was within . ° and . mm from d/ d registrations. these results included robotic couch motion from the ct scanner imaging position to the standard kv imaging isocenter. t a b l e airo image quality results for varying reconstruction kernels. sharp standard soft scaling discrepancy (mm): . . . geometric distortion (mm): . . . spatial resolution ( % mtf, lp/mm): . . . overall uniformity (%): . . . minimum uniformity (%): . . . contrast (%): . . . high cnr: . . . low cnr: . . . maximum hu deviation (hu): . . . f i g . . airo normalized mtf for sharp, standard, and soft reconstruction kernels—calculated with doselab v . . f i g . . normalized mtf for philips ct simulator, airo, and edge cbct—calculated with doselab v . . t a b l e image quality results—airo, philips ct simulator, siemens ct simulator and edge cbct (doselab). philips airo siemens edgea scaling discrepancy (mm): . . . . geometric distortion (mm): . . . . spatial resolution ( % mtf, lp/mm): . . . . overall uniformity (%): . . . . minimum uniformity (%): . . . . contrast (%): . . . . high cnr: . . . . low cnr: . . . – maximum hu deviation (hu): . . . . aedge cbct images were acquired with the pelvis protocol. | oliver et al. the localization accuracy tests also included a workflow and time analysis to determine the optimal location of the ct scanner in the treatment room and to gauge the time required for a ct scan. the resultant workflow required one therapist to bring the ct scanner into the room while an additional therapist performed the initial patient setup. an additional – min were needed to acquire a ct scan. .c | ctdivol measured ctdivol for the airo, and philips ct simulator are illus- trated in table . the airo had the highest dose especially for the head protocol which gave . mgy more dose than the philips ct simulator. the standard helical airo head protocol ( kv, ma, mas (effective), . s rotation time, . mm slice collimation, . mm slice width, . pitch factor, soft kernel) gave . mgy ctdivol. measured ctdivol was . mgy (table ). the helical airo abdomen protocol ( kv, ma, mas (effec- tive), . s rotation time, . mm slice collimation, . mm slice width, . pitch factor, standard kernel) gave . mgy ctdivol. measured ctdivol was . mgy (table ). the philips clinical . mm head protocol ( kv, ma, mas (effective), . s rotation time, . mm slice thickness, . pitch factor) and clinical . mm body protocol ( kv, ma, mas (effective), . s rotation time, . mm slice thickness, . pitch factor) was used. ctdiw was also measured on the edge (pelvis protocol, table ). dose measurements were . mgy for head and . mgy for abdomen. in comparison, amer et al. reported cbdiw (cone beam ct dose index) values of . mgy, mgy and mgy for head, lung, and pelvis cbct protocols, respectively, on an elekta syn- ergy treatment machine. for the airo and the philips simulators, dose was within the acr requirements of % of manufacturer- specifications. | discussion this study describes the characterization and commissioning of the airo mobile ct system for igpt. our characterization of the airo’s image quality demonstrated that the spatial resolution of the airo was lower than our other simulators and igrt systems. weir et al. reported similar findings about the airo indicating its poor spatial resolution when compared to a siemens sensation slice ct scan- ner. weir et al. also reported ring artifacts which could not be elimi- nated. we found similar ring artifacts present when phantoms had widths at or near the width of the detector ( cm). ring artifacts were most noticeable at the phantom/air interface in cylindrical phantoms. these artifacts were not present in anthropomorphic phantoms, thus we conclude that ring artifacts should not appear on patient scans. image quality is protocol and reconstruction kernel dependent. we also found a large scaling discrepancy in comparison to the edge and simulators. we attribute this difference to the airo’s large bore. scaling discrepancy is calculated based on the image’s magnification and the length of the longest side of the image. one last potential disadvantage of the airo for daily igrt is the additional patient dose. we measured airo’s ctdivol in the range of . – . mgy. these doses are slightly higher than the values measured for the edge cbct ( . – . mgy), and may need to be taken into account if multiple ct datasets are acquired on a daily basis. although the airo’s image quality is worse than ct simulators and the edge cbct, the airo’s localization accuracy was within . mm and . ° measured using the mimi phantom. additionally, we demonstrated that the localization accuracy of the mevion- airo system using the stereophan phantom was within . mm when compared with orthogonal kv/kv pairs. this accuracy is com- parable to other commonly used igpt modalities. landry et al. reported . mm localization accuracy for a gantry-mounted cbct system and xu et al. ) reported the accuracy of the edge localization kv/kv pair to be within � . mm and � . °. it is also important to note that the airo’s localization accuracy included the motion of the robotic couch from treatment isocenter to the ct location. we suggest that ct scanner qa include addi- tional robotic couch tests to insure the airo’s localization accuracy is not adversely affected by robotic couch movement. based on this phantom study and initial commissioning, the airo can be used for accurate and reliable igpt. although other facilities have acquired mobile helical ct systems, such as the bodytom (neurologica corporation, danvers, ma, usa) and the aquilion lb (toshiba), our proton therapy facility is the first to acquire the airo mobile ct system for proton treatment local- ization. – we are also the first to use this technology for patient localization in a small-compact proton facility where space limitations exist. based on our study, the airo mobile ct system can be uti- lized for accurate and safe image-guided proton therapy. at the time of writing this manuscript, we have commissioned the system and begun clinical use of the system for treatment localization. our pre- liminary findings are that an additional – min of treatment time t a b l e measured ctdivol for airo helical ct and philips ct simulator for head and abdomen protocols. ctdivol (mgy) airo philips head . . abdomen . . t a b l e hu constancy (ctp ) for various materials in philips, edge, siemens and airo. philips (hu) edge (hu) airo (hu) siemens (hu) true (hu) acrylic . . . . air � . � . � . � . � polystyrene � . � . � . � . � ldpe � . � . � . � . � pmp � . � . � . � . � teflon . . . . ctp . . . . oliver et al. | are required to bring the scanner into the treatment room. future work will involve a time study for patient localization and evaluation scanning as well as characterization of the ct images as they relate to stopping power and ct number constancy for use in adaptive proton therapy. conclusions this work evaluated various image quality, localization accuracy, and dosimetric metrics of the airo and compared them to other com- mon modalities. based on our findings, we recommend that the airo mobile ct system can be applied clinically for safe and accu- rate image-guided proton therapy. conflict of interest there are no conflicts of interest. references . wen n, li h, song k, et al. characteristics of a novel treatment sys- tem for linear accelerator-based stereotactic radiosurgery. j appl clin med phys. ; : . . beltran c, pai panandiker a, krasin m, merchant t. daily image- guided localization for neuroblastoma. j appl clin med phys. ; : . . alvarado r, booth j, bromley r, gustafasson h. an investigation of image guidance dose for breast radiotherapy. j appl clin med phys. ; : . . fu l, perera h, ying x, yu y. importance of cbct setup verification for optical-guided frameless radiosurgery. j appl clin med phys. ; : . . sonier m, chu w, lalani n, erler d, cheung p, korol p. evaluation of kidney motion and target localization in abdominal sbrt patients. j appl clin med phys. ; : . . stanley d, papanikolaou n, gutierrez a. an evaluation of the stabil- ity of image-quality parameters of varian on-board imaging (obi) and epid imaging systems. j appl clin med phys. ; : . . unq n, wee l, hackett s, jones a, lim t, harper c. comparison of low-dose, half-rotation, cone-beam ct with electronic portal imaging device for registration of fiducial markers during prostate radiother- apy. j appl clin med phys. ; : . . yin f-f, wong j, balter j, et al. aapm therapy imaging committee task group , report no. ; . . li h, wu r, poenisch f, et al. clinical implementation of in room mobile ct for image guided proton therapy. med phys. ; : . . pham r, sun b, zhao t, et al. development of dose-based image guided proton therapy workflow. med phys. ; : . . moriya s, tachibana h, hotta k, et al. effectiveness of daily ct- based three-dimensional image guided and adaptive proton therapy. med phys. ; : . . zhao t, sun b, grantham k, et al. commissioning and initial experi- ence with the first clinical gantry-mounted proton therapy system. j appl clin med phys. ; : – . . weir vj, zhang j, bruner ap. dosimetric characterization and image quality evaluation of the airo mobile ct scanner. j xray sci tech- nol. ; : – . . doselab user manual version . : houston, tx: mobius medical sys- tems, lp; . . cody dd, pfeiffer d, mcnitt-gray mf, ruckdeschel tg, strauss kj, wilcox p. computed tomography: quality control manual. american college of radiology; . . amer a, marchant t, sykes j, czajka j, moore c. imaging doses from the elekta synergy x-ray cone beam ct system. br j radiol. ; : – . . lau skm, zhao x, carmona r, et al. frameless single-isocenter mod- ulated sterotactic radiosurgery for simultaneous treatment of multi- ple intracranial metastases. transl cancer res. ; : – . . landry g, nijhuis r, dedes g, et al. investigating ct to cbct image registration for head and neck proton therapy as a tool for daily dose recalculation. med phys. ; : – . . xu h, chetty i, wen n. analysis of systematic errors with d/ d image registration for target localization and treatment delivery in stereotactic radiosurgery. med phys. ; : . | oliver et al. diabetes care, volume , number , january o b s e rvat i o n s diabetes scre e n i n g practices among i n d i v i d u a l s a g e d ye a r s a nd o l d e r i n , the american diabetes associa- tion (ada) adopted new re c o m m e n d a- tions for screening the general popula- tion aged years for diabetes every years with an emphasis on those at high risk for undiagnosed diabetes ( ). few studies have examined the extent to which this screening has been adopted. this re p o rt describes the results of a telephone s u rvey of montana residents aged years to assess the diabetes screening prac- tices in this population. f rom october to december , the montana department of public health and human services conducted a random household telephone survey of montana residents aged years living in counties. respondents indicated whether they ever had been told by a physician that they had diabetes, the number of visits they made to a health care provider during the past year, their family history of dia- betes, whether they had ever been told they had high cholesterol and/or high blood pre s s u re, and their height and weight. respondents were asked the fol- lowing question to identify whether they had ever been screened for diabetes: “glu- cose or sugar is a substance found in your blood. have you ever had your blood glu- cose or sugar checked to see if you have diabetes?” when respondents re s p o n d e d “yes” to this question, they were asked to identify when screening was completed (“when was the last time your blood glu- cose or sugar level was measured by a health care professional?”). the re s p o n s e categories for this question included within the past year, within the past years, years ago, do not know/not sure, and refused to answer. pearson tests were used to assess associations between dia- betes screening and risk factors for dia- betes. logistical re g ression analyses were conducted to identify independent vari- ables associated with screening for diabetes during the past year. odds ratios ( % cis) were calculated. of the , respondents, ( . %) re p o rted that they had diagnosed diabetes. the remaining , respondents re p o rt e d that they did not have diagnosed diabetes and are included in the following analyses. of the respondents, % re p o rted a family h i s t o ry of diabetes, % re p o rted a bmi kg/m , % re p o rted having hyper- tension, and % re p o rted having high c h o l e s t e rol. excluding age, % of re s p o n- dents had one risk factor for diabetes, and % had two or more risk factors. of the , respondents without diagnosed dia- betes, % re p o rted that they had been s c reened for diabetes during the past year, % re p o rted screening from to years ago, and % re p o rted screening years ago or having never been scre e n e d . respondents who re p o rted being s c reened for diabetes during the past year w e re more likely to be age years and to have a family history of diabetes, two or m o re visits to a health care provider dur- ing the past year, hypertension, and high c h o l e s t e rol levels (table ). we found no association between recent screening and sex ( % men vs. % women), ameri- can indian ancestry ( % yes vs. % no), or bmi ( % kg/m vs. % kg/m ). respondents with three or m o re risk factors (e.g., aged years, american indian ancestry, family history of diabetes, hypertension, high choles- t e rol, or bmi kg/m ) were more likely to be screened for diabetes compared with respondents with only one risk factor ( vs. %, respectively). however, % of individuals with two risk factors for dia- betes and % of individuals with more than three risk factors had not been s c reened during the past years. based on logistical re g ression analysis, t h ree factors were associated with scre e n i n g for diabetes during the past year: two or m o re visits to a health care provider during the past year ( . [ % ci . – . ]), high cholesterol level ( . [ . – . ]), and family history of diabetes ( . [ . – . ]). respondents aged – years were less likely to re p o rt re c e n t s c reening than those aged years ( . [ . – . ] ) . a limitation of this assessment is that these data are self-re p o rted. pre v i o u s studies, however, have found that self- re p o rts of conditions such as diabetes and h y p e rtension are reliable ( , ). in addi- tion, the survey was conducted by tele- phone and does not re flect the experience of individuals in montana homes without t e l e p h o n e s . the findings suggest that diabetes s c reening is being adopted by physicians for individuals aged years at risk for diabetes and that the ada re c o m m e n d a- tions are being implemented in the general c o m m u n i t y. however, these data also indi- cate a need to develop strategies to encour- l e t t e r s table —characteristics of respondents aged years reporting screening for diabetes in montana in s c reening for diabetes past year – years years or never n age (years) – ( ) ( ) ( ) – ( ) ( ) ( ) ( )* ( ) ( ) family history of diabetes ye s ( )* ( ) ( ) n o ( ) ( ) ( ) visits to a health care provider during the past year ( )* ( ) ( ) ( ) ( ) ( ) h y p e rt e n s i o n ye s ( )* ( ) ( ) n o ( ) ( ) ( ) high cholestero l ye s ( )* ( ) ( ) n o ( ) ( ) ( ) data are n (%). *p . . diabetes care, volume , number , january letters age screening among all individuals at high risk for diabetes. todd s. harwell, mph jane g. smilie, ba janet m. mcdowall, rn, bsn steven d. helgerson, md, mph dorothy gohdes, md f rom the montana diabetes project, montana d e p a rtment of public health and human serv i c e s , helena, montana. a d d ress correspondence to todd s. harw e l l , mph, montana department of public health and human services, cogswell building, c- , p. o . box , helena, mt - . e-mail: t h a rw e l l @ s t a t e . m t . u s . a c k n o w l e d g m e n t s — this project was sup- p o rted through a cooperative agreement (u- / ccu- - ) with the centers for disease c o n t rol and prevention, division of diabetes translation, atlanta, georg i a . we thank linda priest and the staff mem- bers at northwest resource consultants for their work on the telephone surv e y. the contents of this letter are solely the responsibility of the authors and do not neces- sarily re p resent the official views of the centers for disease control and pre v e n t i o n . r e f e re n c e s . american diabetes association: screening for type diabetes. diabetes care (suppl. ): s –s , . kehoe r, wu sy, leske mc, chylack lt jr: comparing self-re p o rted and physician- re p o rted medical history. am j epidemiol : – , . jackson c, jatulis de, fortmann sp: the behavioral risk factor survey and the stan- f o rd five-city project survey: a comparison of cardiovascular risk behavior estimates. am j public health : – , h b a c is not recommended as a s c reening test for diabetes in cystic fibro s i s i n the june issue of diabetes care, h u n k e rt et al. ( ) recommend the use of h b a c for early detection of cystic fib ro- s i s – related diabetes (cfrd). their re c o m- mendation is based on the finding that mean hba c is slightly higher in cystic fib rosis (cf) patients requiring insulin t h e r a p y, compared with cf patients with i m p a i red or normal glucose tolerance. h o w e v e r, no data on the validity of this a p p roach for the diagnosis of asympto- matic diabetes in patients with cystic fib ro- sis are pre s e n t e d . our group has a long-standing intere s t in the early diagnosis of cfrd, comparing fasting blood glucose levels with oral glu- cose tolerance test results ( ). in our series, we have now cf patients with newly diagnosed diabetes based on a -h venous plasma glucose value mg/dl ( . mmol/l), and simultaneous determ i n a t i o n of hba c ( h i g h - p e rf o rmance liquid chro- matography method [pharmacia, erlan- gen, germany], normal range . – . %). only out of cf patients ( %) diag- nosed as diabetic according to the ameri- can diabetes association and world health o rganization criteria ( ) had an hba c value above the normal range (individual values . , . , . , and . %). in nine diabetic cf patients with normal hba c, values between . and . % were e n c o u n t e red (mean ± sd, . ± . %). the mean -h blood glucose value after inges- tion of oral glucose was not signific a n t l y d i ff e rent between diabetic cf patients with n o rmal hba c ( ± mg/dl, mean ± sd) and diabetic cf patients with elevated h b a c ( ± mg/dl, student’s t t e s t ) . these data clearly demonstrate that the d e t e rmination of hba c is not able to sub- stitute for the oral glucose tolerance test in the early diagnosis of cfrd. our fin d i n g s a re in agreement with several re p o rts in the l i t e r a t u re ( – ) as well as the consen- sus conference on cfrd ( ). in addition to its low sensitivity when used as a diagnos- tic tool for the detection of cfrd, the mea- s u rement of hba c has the disadvantage of considerable interassay variability and lack of standardization. there f o re, we stro n g l y advise against the use of glycosylated hemoglobin as a screening test for the early diagnosis of diabetes in patients with cystic fib ro s i s . reinhard w. holl, md christian buck, md christine babka, md anna wolf, md angelika thon, md f rom the department of pediatrics (r.w.h.), uni- versity of giessen, giessen; the department of pedi- atrics (c.bu., c.ba., a.w.), the university of ulm, ulm; and the medical school hannover (a.t. ) , h a n n o v e r, germ a n y. a d d ress correspondence to pd dr. reinhard w. holl, universitätskinderklinik giessen, feulgenstr. , d- giessen, germ a n y. r e f e re n c e s . h u n k e rt f, lietz t, stach b, kiess w: potential impact of hba c d e t e rm i n a t i o n on clinical decision making in patients with cystic fib ro s i s – related diabetes (let- ter). diabetes care : – , . holl rw, buck c, cario h, wolf a, thon a, heinze e, kohne e, debatin k-m: diag- nosis of diabetes in cystic fib rosis and tha- lassemia major. diabetes care : – , . the expert committee on the diagnosis and classification of diabetes mellitus: r e p o rt of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care : – , . lanng s, hansen a, thorsteinsson b, n e rup j, koch c: glucose tolerance in patients with cystic fib rosis: five year p rospective study. b m j : – , . deluca f, arrigo t, nibali sc, sferlazzas c, gigante a, dicesare e, cucinotta d: insulin secretion, glycosylated haemoglo- bin and islet cell antibodies in cystic fib ro- sis children and adolescents with diff e re n t d e g rees of glucose tolerance. h o rm metab r e s : – , . moran a, doherty l, wang x, thomas w: a b n o rmal glucose metabolism in cystic fib rosis. j pediatr : – , . moran a: highlights of the febru a ry consensus conference on cfrd. bonn, g e rm a n y, cystic fibrosis foundation, p ro gln p e roxisome p ro l i f e r a t o r- a c t i v a t e d r e c e p t o r- and o b e s i t y r istow et al. ( ) re p o rted an activating mutation in the peroxisome pro l i f- e r a t o r-activated re c e p t o r- g e n e ( p ro gln ppa r - ), which was pre s e n t in % ( of ) of obese and % ( of ) of nonobese german caucasians. these findings may have profound implica- tions, particularly if the presence of this variant and its association with obesity are c o n firmed in other populations. we perf o rmed polymerase chain re a c- t i o n – restriction fragment length polymor- phism analysis for the pro gln ppa r - variant as described ( ) on dna samples f rom several independent populations, including lean and obese caucasians fro m the baltimore, maryland, region; african- diabetes care, volume , number , january letters americans from jackson, mississippi, and forsyth county, north carolina; pima indians from arizona; and old ord e r amish from lancaster county, pennsylva- nia (table ). a pcr fragment corre s p o n d- ing to gastric insulinotropic peptide, which has two known restriction sites for h i n dii, was mixed with each sample as a positive control. among a total of sub- jects ( , alleles), the pro gln variant was not detected in a single subject. these findings were unexpected because there is substantial overlap of gene pools of caucasians from central e u rope and the baltimore and amish caucasians studied ( ). the germ a n caucasians studied by ristow et al. were said to be unrelated and re c ruited fro m the nord rh e i n - westfalen region, but they may be a genetic isolate whose gene pool does not re flect that of other caucasian populations. altern a t i v e l y, if the individ- uals carrying the mutation were re l a t e d , the true frequency of the pro g l n p pa r - variant may have been overe s t i- mated. the absence or very low fre- quency of this variant has also been doc- umented in danish ( ) and german ( ) populations. this study is the first, to our knowledge, to examine american popu- lations for this variant. in summary, the study by ristow et al. demonstrating that the pro g l n p pa r - variant is activating and can influence body weight in people is important. however, this variant appears to be absent or very r a re in the american populations studied. additional studies are re q u i red in other regions of europe and the u.s. to furt h e r d e fine the relevance of this intere s t i n g genetic variant to susceptibility to obesity. alan r. shuldiner, md william nguyen, bs w.h. linda kao, phd brock a. beamer, md ross e. andersen, phd richard pratley, md frederick l. brancati, md, phd f rom the department of medicine (a.r.s., w. n . ) , university of maryland school of medicine; the d e p a rtments of medicine (b.a.b., f.l.b.) and epi- demiology (w.h.l.k.), johns hopkins university school of medicine, baltimore, maryland; and the national institute of diabetes and digestive and kidney diseases (r.p.), national institutes of health, phoenix, arizona. a d d ress correspondence to alan r. shuldiner, md, professor and head, division of endocrinol- o g y, diabetes, and nutrition, department of medi- cine, university of maryland school of medicine, w. lombard st., room s- , baltimore, md . e-mail:ashuldin@medicine.umary l a n d . e d u . a c k n o w l e d g m e n t s — this study was sup- p o rted by nih r dk- , the american diabetes association, glaxowellcome, the b a l t i m o re geriatrics research and education clinical center of the baltimore ve t e r a n s administration medical center. r e f e re n c e s . ristow m, muller- wieland d, pfeiffer a, k rone w, kahn cr: obesity associated with a mutation in a genetic regulator of adipocyte diff e rentiation. n engl j med : – , . c a v a l l i - s f o rza ll, menozzi p, piazza a: t h e h i s t o ry of human genes. princeton univer- sity press, princeton, nj, . elk j, urhammer sa, sorensen ti, ander- sen t, auwerx j, pedersen o: homozygosity of the pro ala variant of the pero x i s o m e p roliferation-activated re c e p t o r- g a m m a ( p par-gamma ): divergent modulating e ffects on body mass index in obese and lean caucasian men. diabetologia : – , . mamann a, munzberg h, buttron p, busing b, hinney a, mayer h, siegfried w, hebe- brand j, greten h: missense variants in the human peroxisome pro l i f e r a t o r- a c t i v a t e d re c e p t o r-gamma gene in lean and obese subjects. eur j endocrinol : – , insulin secre t i o n , insulin sensitivity, and glucose e ffectiveness in nonobese individuals with va ry i n g d e g rees of glucose to l e r a n c e a lthough it is well known that insulin s e c retion, insulin sensitivity, and glu- cose effectiveness are impaired in type diabetic patients ( – ), little is known about the role of each of these fac- tors individually on the evolution of type diabetes. in this context, a major issue is that hyperglycemia per se impairs insulin s e c retion and insulin sensitivity and that obesity observed in type diabetic patients per se causes insulin resistance ( , ). to o v e rcome this problem, we studied u n t reated nonobese subjects classified as having normal glucose tolerance (ngt) (n = ; bmi . ± . kg/m [ r a n g e . – . ], mean ± sem), impaired glu- cose tolerance (igt) (n = ; bmi . ± . kg/m [ . – . ]), and type dia- betes (n = ; fetal bovine serum . ± . mmol/l [range . – . ], bmi . ± . k g / m [ . – . ]), based on the criteria of the world health organization ( ). they were normotensive and had norm a l renal, hepatic, and thyroid function. insulin sensitivity and glucose eff e c t i v e n e s s w e re estimated by the minimal model a p p roach ( – ). insulin secretion was e x p ressed as the area under the insulin c u rve between and min after an intra- venous glucose injection ( ). after the data w e re analyzed by one-way analysis of vari- ance, bonferroni correction was used to evaluate the diff e rences between any two of the three groups we studied ( ). no signifi- cant difference was observed in bmi among the three groups. compared with the subjects with ngt, the subjects with table —characteristics of subjects screened for pro gln ppa r - n ( a l l e l e s age ± sd f e m a l e bmi ± sd p o p u l a t i o n t y p e d ) ( y e a r s ) ( % ) ( k g / m ) c a u c a s i a n s b a l t i m o re longitudinal ( ) . ± . . . ± . study on aging johns hopkins we i g h t ( ) . ± . . . ± . management center amish, lancaster, pa ( ) . ± . . . ± . a f r i c a n - a m e r i c a n s a t h e ro s c l e rosis risk in ( ) . ± . . . ± . communities study pima indians a r i z o n a ( ) . ± . — . ± . all non-amish subjects were unrelated; amish subjects were not fir s t - d e g ree relatives of each other. diabetes care, volume , number , january letters igt had significantly lower insulin secre- tion ( , ± vs. , ± pmol l m i n , p = . ) and glucose eff e c- tiveness ( . ± . vs. . ± . m i n , p . ). insulin sensitivity index was lower in subjects with igt ( . ± . m i n pmol l) than in those with ngt ( . ± . min pmol l ) , but was not statistically significant (p = . ). in con- trast, disposition index calculated by the p roduct of insulin secretion and insulin sensitivity was significantly lower in sub- jects with igt ( , ± ) than in those with ngt ( , ± , p = . ). on the other hand, patients with type dia- betes had significantly lower insulin secre- tion ( ± pmol l m i n , p = . ) compared with subjects with igt. although no significant diff e rence was observed in insulin sensitivity index between subjects with type diabetes and igt ( . ± . vs. . ± . min pmol l, p = . ), disposition index was s i g n i ficantly diminished in type diabetic patients as compared with subjects with igt ( ± vs. , ± , p = . ). glucose effectiveness in type diabetic patients ( . ± . min ) was similar to that in subjects with igt ( . ± . m i n , p = . ) but was signific a n t l y lower than that in the subjects with ngt (p . ). from these results, the fol- lowing may be hypothesized: ) impair- ments in insulin secretion and disposition index and decreased glucose eff e c t i v e n e s s , but not insulin resistance, seem to consti- tute the basic characteristics of patients with igt or type diabetes in nonobese japanese populations. ) risk factors wors- ening to type diabetes in subjects with igt are associated with further impair- ments in insulin secretion and disposition index, but not associated with furt h e r derangement in glucose effectiveness in japanese populations. ataru taniguchi, md mitsuo fukushima, md masahiko sakai, md itaru nagata, md kentaro doi, md shoichiro nagasaka, md kumpei tokuyama, md yoshikatsu nakai, md f rom the first department of internal medicine ( a . t., m.s., i.n.), kansai-denryoku hospital, and the department of internal medicine, hoshida- minami hospital (m.f.), osaka; the second depart- ment of internal medicine (k.d.) and the college of medical technology (y.n.), kyoto university, kyoto; the department of internal medicine (s.n.), jichi medical college, tochigi; and the laboratory of biochemistry of exercise and nutrition (k.t. ) , tsukuba university, tsukuba, japan. a d d ress correspondence to ataru ta n i g u c h i , md, first department of internal medicine, kansai- d e n ryoku hospital, - - , fukushima-ku, fuku- shima, osaka city, osaka - , japan. e-mail: k @ k e p c o . c o . j p . r e f e re n c e s . b e rgman rn: to w a rd physiological under- standing of glucose tolerance: minimal- model approach. d i a b e t e s : – , . welch s, gebhart ssp, bergman rn, phillips ls: minimal model analysis of intravenous glucose tolerance test-derived insulin sensitivity in diabetic subjects. j c l i n endocrinol metab : – , . taniguchi a, nakai y, fukushima m, kawamura h, imura h, nagata i, tokuyama k: pathogenic factors re s p o n s i- ble for glucose tolerance in patients with niddm. d i a b e t e s : – , . nagasaka s, tokuyama k, kusaka i, hayashi h, rokkaku k, nakamura t, kawakami a, higashiyama m, ishikawa s, saito t: endogenous glucose pro d u c t i o n and glucose effectiveness in type diabetic subjects derived from stable-labeled mini- mal model approach. d i a b e t e s : – , . best jd, kahn se, ader m, watanabe rm, ni t-c, bergman rn: role of glucose eff e c- tiveness in the determination of glucose tol- erance. diabetes care : – , . rossetti l, giaccari a, defronzo ra: glu- cose toxicity. diabetes care : – , . taniguchi a, nakai y, doi k, fukuzawa h, fukushima m, kawamura h, to k u y a m a k, suzuki m, fujitani j, tanaka h, nagata i: insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis. m e t a b o l i s m : – , . world health organization: diabetes melli - tus: report of a who study gro u p . g e n e v a , world health org., (tech. rep. ser. , no. ) . winer bj: statistical principles in experimen - tal design. nd ed. new york, mcgraw-hill, , p. – late-onset tro g l i t a z o n e - i n d u c e d hepatic dysfunction r e c e n t l y, iwase et al. ( ) re p o rted a case of liver dysfunction occurring after months of troglitazone therapy. because it was thought before this re p o rt that the risk of liver dysfunction with tro g l i- tazone after months was negligible, we wish to re p o rt another patient who took t roglitazone intermittently and developed hepatic dysfunction after months. a -year-old white man with type diabetes, ischemic heart disease (post angioplasty and stent placement), hyper- tension, degenerative joint disease, benign p rostatic hypert ro p h y, dyslipidemia, and g a s t roesophageal re flux disease had tro g l i- tazone mg daily added to his re g i m e n of glimeperide mg daily and metform i n mg b.i.d. because of poor glycemic c o n t rol (hba c . % [normal – %]). the other medicines he used were aspirin and pravastatin. after months of triple oral therapy, his hba c level dropped to . %, and, after months, to . %. after months, the patient’s hba c began to rise: . % at months, . % at year, and . % at months. liver function tests were normal until months, when his aspartate amino- transferase (ast) was found to be (nor- mal – u/l) and alanine aminotrans- ferase (alt) (normal – u/l). the t roglitazone regimen was discontinued, and testing for hepatitis b and c, h e m a c h romatosis, autoimmune liver dis- ease, and gallbladder disease were nega- tive. two months after discontinuing t roglitazone, the patient’s ast and alt had decreased to and u/l, and, after months, had re t u rned to normal at and u/l, re s p e c t i v e l y. his ast and alt have remained normal since then and he has continued to take pravastatin, aspirin, m e t f o rmin, and glimeperide. when the patient was told to discon- tinue troglitazone, he admitted that he had been taking it only interm i t t e n t l y. he esti- mated that he took the drug regularly at first, but after the first months, he took the drug only once or twice weekly on aver- age. he gave the following three reasons for his lack of compliance: a lack of funds, a fear of liver disease, and a tendency to avoid taking drugs whenever possible. this case, like the case described by iwase et al., illustrates that the hepatic dysfunction caused by troglitazone can occur after months and further sup- p o rts the u.s. food and drug administra- t i o n ’s current recommendation that quar- terly liver function tests should be obtained when troglitazone utilization extends beyond year ( ). in this case, could the onset of hepatic dysfunction have been delayed because the diabetes care, volume , number , january letters d rug was being taken only interm i t t e n t l y after the first months, and the estimated total load presented to the liver would be equivalent to the exposure at months in a compliant patient? we doubt this, since t ro g l i t a z o n e ’s hepatic effects are thought to be idiosyncratic and there f o re the total e x p o s u re should be irre l e v a n t . david s.h. bell, mb fernando ovalle, md f rom the division of endocrinology and metabo- lism, department of medicine, school of medicine, university of alabama, birmingham, alabama. a d d ress correspondence to david s.h. bell, mb, th ave. s., birmingham, al . d.s.h.b. and f.o. have served on an advisory panel for sankyo parke-davis and have received con- sulting fees, re s e a rch grant support, and honoraria for speaking engagements from sankyo parke-davis. r e f e re n c e s . iwase m, yamaguchi m, yoshinari m, oka- mura c, hirahashi t, tsuji h, fujishima m: a japanese case of liver dysfunction after months of troglitazone tre a t m e n t . diabetes care : – , . parke-davis, division of wa rn e r- l a m b e rt : rezulin package insert. morris plains, nj, g l y b u r i d e - i n d u c e d hemolysis in m y e l o d y s p l a s t i c s y n d ro m e g lyburide, also known as gliben- clamide, is a widely used sulfonylure a to treat patients with type diabetes. hemolytic anemia is an extremely rare side e ffect of which there have been only a few re p o rts ( – ). we describe a patient with myelodysplastic syndrome who pre s e n t e d with glyburide-induced hemolysis. a -year-old man with a long history of type diabetes presented with left foot cellulitis of week’s duration. this patient was known to have slowly pro g re s s i v e pancytopenia for years, for which no work-ups had been perf o rmed. his med- ications included the following: glyburide, mg per day, which he had taken for m o re than year; buformine, mg per day; and boglibose, . mg per day. he was afebrile, and the physical examination was normal, except for localized cellulitis on his left foot, for which he was start e d on intravenous antibiotics. the laboratory studies revealed a white blood cell count of . / l , hemoglobin . g/dl, platelet count /l, reticulocyte count . %, mean cor- puscle volume fl, moderate anisocyto- sis, fasting plasma glucose mg/dl, h b a c . %, lactate dehydrogenase iu/l, total bilirubin . mg/dl, and hapto- globin mg/dl. red cell glucose- - phosphate dehydrogenase level was ade- quate. cold agglutinin test, ham’s test, and sugar water test were normal. both dire c t and indirect coombs’ tests were negative. red cell resistance to osmolarity was mildly low (parpart ’s method). urinalysis demonstrated no urobilinogen. endo- scopic studies did not reveal gastro i n t e s t i- nal bleeding. ultrasonography of the abdomen showed no splenomegaly. the result of bone marrow aspiration was equivocal. on the basis of pre s u m p t i v e glyburide-induced hemolysis, glyburide was discontinued and the patient was switched to subcutaneous insulin on the seventh day. there a f t e r, his hemoglobin level increased to . g/dl, re t i c u l o c y t e count decreased to . %, and anisocytosis d i s a p p e a red pro m p t l y. he was discharg e d with insulin therapy after month in the hospital, which is when the cellulitis re s o l v e d . t h ree months later, his hemoglobin level was . g/dl and his haptoglobin remained low. repeated bone marro w aspiration confirmed the diagnosis of myelodysplastic syndro m e . we conclude that this patient devel- oped glyburide-induced hemolysis super- imposed on red cell fragility secondary to an underlying bone marrow disord e r. t h e re have been several re p o rts of hemoly- sis caused by sulfonylureas, most of which have been considered immune-mediated ( , , ). our case points to the possibility that glyburide could cause hemolysis by a non–immune-medicated mechanism. it is i m p o rtant to be aware of this potential side e ffect of glyburide in light of this medica- t i o n ’s widespread prescription, even though such a side effect is rare . hiroshi noto, md kazuhisa tsukamoto, md satoshi kimura, md f rom the department of diabetes and metabolism, tokyo university hospital, tokyo, japan. a d d ress correspondence to hiroshi noto, md, d e p a rtment of diabetes and metabolism, tokyo uni- versity hospital, - - hongo, bunkyo-ku, to k y o - , japan. e-mail: noto-tky@umin.ac.jp. r e f e re n c e s . nataas ob, nesthus i: immune haemolytic anaemia induced by glibenclamide in selective iga defic i e n c y. b m j : – , . abbate sl, hoogwerf bj: hemolytic ane- mia associated with sulfonylurea use. d i a - betes care : – , . meloni g, meloni t: glyburide-induced acute haemolysis in a g pd-defic i e n t patient with niddm. br j haematol : – , . kopicky ja, packman ch: the mechanism of sulfonylurea-induced immune hemoly- sis: case re p o rt and review of the literature . am j hematol : – , e ffects of exposure at an altitude of , m on p e rf o rmance of glucose meters s elf-monitoring of blood glucose is m a n d a t o ry for type diabetic p a t i e n t s who participate in sports to adjust insulin dose and carbohydrate ingestion ( ). sports also include activities p e rf o rm e d at moderately high altitudes, such as hiking or skiing. capillary blood glucose monitors (bgms) have been shown to underestimate blood glucose values at an altitude of , m ( ) and at a simulated altitude of , m with t e m p e r a t u re and humidity kept constant ( ). the aim of the present study was to assess the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and p o can result in errors in blood glucose d e t e rmination ( ). two bgms, the lifescan one touch ii (ot) (ortho diagnostics, milpitas, ca) and the glucometer elite ii (ge) (bayer diagnostics, brussels, belgium), were tested during a study on the effects of acute exposure at an altitude of , m and exercise on blood pre s s u re and albu- min excretion rate in six type diabetic p a t i e n t s . all subjects (four men and two women) were free of disease-related com- plications and in good and stable glycemic control (ghb . ± . %). all subjects gave their informed and written consent to participate in the study pro t o- col. all subjects were investigated both at diabetes care, volume , number , january letters sea level and after ascent by car and cable car to the angelo mosso institute at col d’olen ( , m altitude), gressoney la trinité, italy. at sea level and at a moderately high altitude, bgm reliability at diff e rent blood glucose levels was tested, and blood glu- cose was assessed in fasting and re s t i n g conditions at : a.m.; at : a.m. b e f o re an in-field exercise test; and imme- d i a t e l y, min, and min after the exer- cise stopped. capillary glucose was simul- taneously assessed with the ot and the ge. both of these bgms measure capillary blood glucose through the glucose oxi- d a s e - p e roxidase reaction. bgms were cali- brated at the beginning of each test ses- sion. a venous blood sample was simulta- neously drawn from the contralateral antecubital vein in a sodium fluoride tube, centrifuged, and stored at °c. plasma glucose was assayed with the glucose oxi- dase method (go) within days. this last assessment was taken as a re f e re n c e method. statistical analysis compare d bgm capillary glucose values and go plasma glucose values for each blood col- lection time. measurement linearity was tested with pearson’s correlation coeff i- cient. the mean of the diff e rences between the bgm and go results re p resents the mean bias between the methods with accuracy expressed as percent error (pe): pe (%) = bmg – go % g o the level of statistical significance was c o n s i d e red to be p . . the ge and ot measurements had a good correlation with plasma glucose both at moderately high altitude and at sea level. p e a r s o n ’s correlation coefficients were . and . for the ge and . and . for the ot at sea level and at moderately high altitude, re s p e c t i v e l y. biases between plasma glucose and bgm measure m e n t s w e re as follows: for the ge, . ± . at sea level and . ± . at moderately high altitude; for the ot, . ± . at sea level and . ± . at moderately high alti- tude. at sea level, both the ge and the ot tended to underestimate glucose values (ns); at moderately high altitude, the ge tended to overestimate and the ot tended to underestimate glucose values (ns). mean pes between plasma glucose and bgm m e a s u rements were . (ot) and . (ge) at sea level and . (ot) and . (ge) at moderately high altitude. pe tended to be higher for both bgms at moderately high altitude (ns). figures and show the bias between the single measurements with both devices at sea level and at moderately high altitude, re s p e c t i v e l y. at moderately high altitude (fig. ), the tendency of the ge to o v e restimate was more evident for low ( mg/dl) and intermediate ( – mg/dl) blood glucose values, whereas the ot tended to underestimate mainly high blood glucose values (ns). in our study, bgm perf o rmance was similar and good at sea level. at a moder- ately high altitude, a tendency to overe s t i- mate blood glucose for the ge and to u n d e restimate for the ot was observ e d . the overestimation for the ge involved mainly low ( mg/dl) and interm e d i- ate ( – mg/dl) blood glucose val- ues. this could present a problem in the p resence of symptoms suggesting hypo- figure —relationship between plasma and capillary glucose at sea level. figure —relationship between plasma and capillary glucose at moderately high altitude. lifescan one touch ii glucometer elite ii lifescan one touch ii glucometer elite ii diabetes care, volume , number , january letters glycemia and normal blood glucose val- ues. the ot tended to undere s t i m a t e mainly high blood glucose values, although its perf o rmance with low to i n t e rmediate values was good. the pre s- ent study assessed the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and po can result in errors in blood glu- cose determination ( ). our results are consistent with previous studies ( , ). the decrease in po could alter the sec- ond phase of the chromogen reaction and u n d e restimate blood glucose values ( ); on the other hand, an increase in atmos- pheric pre s s u re could overestimate blood glucose values ( ). in our study, minimal o v e restimation by the ge at low interm e- diate blood glucose values at moderately high altitude cannot be explained by the a l t e red po . an increase in hematocrit, which is known to alter blood glucose m e a s u rements with bgms ( ), may also occur after prolonged exposure to high altitude or as a consequence of dehydra- tion. although our study did not deter- mine hematocrit, the exercise test was s h o rt, and the patients were instructed to drink according to their thirst during the , -m exposure; there f o re, dehydration was not likely to have occurred. in con- clusion, bgm perf o rmance is similar and good at sea level. at a moderately high altitude similar to that experienced during winter skiing or summer hiking, a ten- dency to overestimate low to norm a l blood glucose values for the ge and to u n d e restimate high blood glucose values for the ot was observed. the bias is not clinically meaningful for either bgm, both of which can be safely used by dia- betic patients during exposure to moder- ately high altitudes. some care in the eval- uation of low and intermediate blood glu- cose values measured with the ge is n e v e rtheless re c o m m e n d e d . oriana pecchio, md simona maule, md marco migliardi, md marina trento, bsc massimo veglio, md f rom the italian alpine club medical commission ( o . p.); the department of internal medicine (m.t. ) , university of turin; the s. giovanni battista hospital (s.m.); and the department of endocrinology (m.m., m . v.), mauriziano umberto i hospital, turin, italy. a d d ress correspondence to dr. m. veglio, via mancini , torino, italy. e-mail: veglio@ o n w. n e t . r e f e re n c e s . h o rton es: role and management of exer- cise in diabetes mellitus. diabetes care : – , . g i o rdano bp, trash w, hollenbaugh l, dube wp: perf o rmance of seven blood glucose testing systems at high altitude. diabetes educ : – , . gautier jf, bigard ax, douce p, duvallet a, cathelinau g: influence of simulated alti- tude on the perf o rmance of five blood glu- cose meters. diabetes care : – , . b a rnett c, ryan f, ballonoff l: effect of altitude on the self monitoring of blood glucose (smbg) (abstract). diabetes (suppl.): a, . piepmeier eh, hammett-stabler c, price me, kemper gb, davis mg: atmospheric p re s s u re effects on glucose monitoring devices (letter). diabetes care : – , . b a rreau pb, buttery je: effect of hematocrit concentration on blood glucose value d e t e rmined on glucometer ii. diabetes care : – , c o m m e n t s a n d r e s p o n s e s deterioration of glycemic contro l after long-te rm treatment wi t h troglitazone in nonobese type diabetic patients t roglitazone is an oral antidiabetic d rug used to treat type diabetic patients with insulin re s i s t a n c e . troglitazone improves overall insulin sen- sitivity in the liver and skeletal muscles, which are the largest consumers and metabolizers of glucose in the body ( – ). recent re p o rts showed that troglitazone is also effective in nonobese type diabetic patients whose hyperglycemia could not be controlled with sulfonylurea therapy ( , ). however, we aware that in some patients in whom adequate glycemic con- t rol is obtained during the first several months of troglitazone treatment, their glycemic control deteriorates several months later. we assume that two distinct g roups of type diabetic patients exist who respond diff e rently to long-term administration of troglitazone, one gro u p that maintains a steady response and another group that has a decre a s i n g response after certain periods. in this s t u d y, we re t rospectively examined patients with type diabetes who were t reated with troglitazone for months and whose hba c levels had improved by % with troglitazone by month . in of the patients ( %), hba c levels increased by . % after – months despite continuous tro g l i t a z o n e t reatment (group p). in contrast, the rest of the patients experienced steady glycemic c o n t rol with . % of hba c flu c t u a t i o n ( g roup g). during the first months, h b a c levels decreased from means ± sem . ± . to . ± . % in group p and fro m . ± . to . ± . % in group g, re s p e c- t i v e l y. no significant diff e rences were evi- dent between the two groups re g a rding the d e c rease in hba c during the first months (fig. ). from month onward, hba c l e v- els in group p climbed gradually by . % a month up to the baseline level at month , but hba c levels were stable in group g t h roughout the treatment period. a signifi- cant diff e rence in hba c levels was evident during months – (p . ) . among clinical characteristics, gro u p p had a significantly lower bmi ( . ± . vs. . ± . kg/m , p . ) and s i g n i ficantly lower fasting insulin levels ( . ± . vs. . ± . µu/ml, p . ). of the patients with a bmi of kg/m ( %), exhibited deteriora- tion of glycemic contro l . in this study, we re p o rt a group of patients who showed a renewed decline in glycemic control after long-term tre a t m e n t with troglitazone. these results seemed to suggest a secondary failure of tro g l i t a z o n e . our study demonstrates that this drug is indeed useful for a long-standing obese i n s u l i n - resistant diabetes but not for a nonobese type diabetes. yuko murase, md takanobu wakasugi, md kunimasa yagi, md hiroshi mabuchi, md f rom the department of internal medicine (y. m . , t. w.), fukui perfectural hospital; and the second d e p a rtment of internal medicine (k.y., h.m.), kanazawa university, ishikawa, japan. a d d ress correspondence to yuko murase, md, the second department of internal medicine, kanazawa university, - takara-machi, kanazawa, ishikawa - , japan. e-mail: diabe@med. k a n a z a w a - u . a c . j p . diabetes care, volume , number , january letters r e f e re n c e s . suter sl, nolan jj, wallace p, gumbiner b, olefsky jm: metabolic effects of new oral hypoglycemic agent cs- in niddm subjects. diabetes care : – , . o’rourke cm, davis ja, saltiel ar, corn i- celli ja: metabolic effects of troglitazone in the goto-kakizaki rat, a non-obese and n o rmolipidemic rodent model of nonin- sulin-dependent diabetes mellitus. m e t a b - o l i s m : – , . troglitazone study group: the metabolic e ffects of troglitazone in non-insulin dependent diabetes (abstract). d i a b e t e s (suppl. ): a, . mori k: the effect of troglitazone in combi- nation with sulfonylurea in non-insulin dependent diabetes mellitus (abstract). j japan diabetes soc (suppl. ): , . h o rton es, venable tc, whitehouse f, the troglitazone study group, ghazzi mn, whitcomb rw: troglitazone in combina- tion with sulfonylurea re s t o res glycemic c o n t rol in patients with type diabetes. diabetes care : – , figure —change from baseline in hba c. values are means ± sem. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ doi: . / am. j. hum. genet. : – , behavioral genetics ’ understanding the genetic basis of mood disorders: where do we stand? victor i. reus and nelson b. freimer center for neurobiology and psychiatry, department of psychiatry, university of california, san francisco, san francisco introduction as the predominant feature of the syndrome. in most current diagnostic systems, mood is viewed as carrying in this issue of the journal, sherman et al. ( ) de- greater heuristic and practical value than other signs and scribe the promise of genetic approaches for understand- symptoms that might also be present, such as anxiety, ing human behavior and point out a number of obstacles cognitive impairment, or alteration in vegetative func- to realization of this promise; these include the method- tions (american psychiatric association ). epidemi- ological challenge of identifying genes for complex traits ological studies have provided strong support for a ge- and the societal challenge of appropriately using the netic component in the etiology of these disorders information that will be gained if such genetic-mapping (tsuang and faraone ), particularly in comparison efforts are successful. genetic-mapping studies in hu- with other classes of mental illness. however, as noted mans rest on the premise that traits of interest can be above, our current categorical classification of psychiat- reduced to one or more discrete phenotypes and that ric disorders, although reliable, lacks demonstrated etio- these phenotypes result, at least in part, from particular logic validity. it is thus by no means certain that distur- alleles at susceptibility loci of reasonably large effect. as bance of mood represents the best indicator of common discussed in this review, abundant evidence suggests that genetic etiology for this class of conditions. for example, severe bipolar mood disorder (bp) fulfills this premise major depressive disorder (mdd), the most common better than other human behavioral traits (tsuang and mood disorder, is conceptualized as distinct from panic faraone ; escamilla et al. ). the diagnosis of disorder (pd), a common anxiety syndrome, although bp is highly reliable, and its delineation as a distinct two-thirds of patients with pd also will have a lifetime syndrome has proved to be clinically useful in predicting diagnosis of mdd (breier et al. ), and comorbidity course and response to treatment (goodwin and jami- between the two syndromes is common (kendler et al. son ). however, one must keep in mind that this ; coryell et al. ). psychiatric disorders are, diagnostic category, like all psychiatric classifications, is of course, not unique in this regard, and similar issues based on operational criteria (derived from a combina- regarding the etiologic validity of diagnostic constructs tion of epidemiological and clinical observations), rather also complicate genetic mapping of many medical disor- than on any anatomical or physiological evidence. this ders. fact differentiates psychiatric disorders from other etio- the list of mood disorders most commonly includes logically complex categories of disease, such as hyper- mdd, dysthymic disorder (a more chronic but generally tension or diabetes mellitus. in this review we discuss less severe depressive disorder), bp (in which episodes our current understanding of the genetic basis of bp and of major depression alternate with mania), cyclothymic other mood disorders and indicate how our body of disorder (characterized by many episodes of brief and/ knowledge has been influenced by different approaches or less severe depressions alternating with symptoms of to the definition of disease phenotypes. mild mania (hypomania)), and mood disorders due to a the term ‘‘mood disorders’’ encompasses a group of general medical condition or induced by substance conditions in which a disturbance of mood is recognized abuse. bp is divided further into bp-i (mdd and severe mania) and bp-ii (mdd and hypomania), and both of these categories also often are referred to as ‘‘manic- received april , ; accepted for publication april , . depressive illness.’’ the degree to which these diagnosesaddress for correspondence and reprints: dr. victor i. reus, center are related genetically or are distinct entities is currentlyfor neurobiology and psychiatry, department of psychiatry, univer- sity of california, san francisco, parnassus avenue, san fran- unknown (tsuang and faraone ; pauls et al. ; cisco, ca - . e-mail: vir@itsa.ucsf.edu spence et al. ). this uncertainty has impeded efforts this article represents the opinion of the authors and has not been to construct realistic models for linkage analysis of bp. peer reviewed. furthermore, although most (but not all) genetic-map-� by the american society of human genetics. all rights reserved. - / / - $ . ping studies treat mood disorders and thought disorders / a a$$ju - - : : ajhga uc-ajhg am. j. hum. genet. : – , (notably schizophrenia) as independent entities, a sub- were conducted § decades ago, using (a) phenotype- assignment methods that currently would not be widelystantial number of individuals are afflicted with schizo- affective disorder, in which disturbances in thought pro- accepted and (b) diagnostic categorizations that differ from those employed in almost all recent genetic-map-cesses are as prominent as the mood alteration (escamilla et al. ). ping studies. an example is the study by bertelsen et al. ( ), discussed above, which has had a substantial influence on current views regarding the genetic basis ofgenetic epidemiology of mood disorders mood disorders. the determination of phenotype in that investigation derived from an unstructured psychiatricuncertainty regarding the etiologic relationship be- tween bp and other psychiatric disorders has not been interview conducted by a single individual; in contrast, most current investigators would accept diagnostic-in-resolved by a large number of family, twin, and adoption studies aimed at evaluating the degree of familial aggre- terview data only if these were collected by standardized interviews. additionally, the findings of bertelsen et al.gation and heritability of various mood disorders. nev- ertheless, although family studies of bp, conducted over ( ) were based on diagnostic criteria different than those currently in use — for example, combining individ-a period of several decades, have utilized several differ- ent designs, including varying definitions of what consti- uals with hypomania and individuals with mania into the same category. reexamination of epidemiologicaltutes bp, they consistently have demonstrated familial aggregation of both severe and mild mood disorders. studies such as that by bertelsen et al. ( ) further emphasizes the special difficulty of psychiatric genetics;one of the most striking findings of these studies has been that bp shows far greater familial aggregation than there is inherent subjectivity in recording even individual behavioral features, let alone in determining categoricalother mood disorders. for example, weissman et al. ( ) observed a relative risk for bp of almost , diagnoses that change over time. this fact makes the delineation of affected status in current studies as prob-compared with a relative risk of Ç . for mdd. simi- larly, twin studies have indicated a higher heritability lematic as the proper interpretation of past data. it is not likely that we will attain a more refined ap-for bp, compared with that for other forms of mood disorder. for example, the classic twin study by bertel- proach for diagnostic classification of mood disorders until we begin to identify the genes that underlie riskson et al. ( ), utilizing the danish twin registry, indicated a concordance rate, for narrowly defined bp, for these disorders. from that point we may learn to make distinctions finer than are now possible betweenof . for mz twins, versus . for dz twins (as op- posed to . and . , respectively, for major depression). different classes of mood disorders and also may dis- cover that particular (and currently unexpected) pheno-although most epidemiological studies suggest that some cases of mdd probably are genetically related to typic features are etiologically related to one another. such use of genetics to redefine phenotypic categoriesbp, only inconclusive results have been obtained from efforts to identify particular mdd subtypes that are has, of course, been one of the most tangible results of positional cloning of genes responsible for relativelymost likely to reflect a shared genetic etiology with bp (goodwin and jamison ). simple disorders. such diagnostic refinement, however, will not be possible for mood disorders unless the cur-it is instructive to compare the evidence showing the high degree of heritability of narrowly defined bp to rently available classification schemes will permit us to first map the responsible genes. genes for several pheno-that for either of the traits used for illustrative purposes by sherman et al. in this issue of the journal — namely, typically complex (nonpsychiatric) disorders have now been mapped. success in mapping such traits usuallyschizophrenia and emotional stability; for example, the mz concordance rate of . for bp (bertelsen et al. has depended on the delineation of narrowly defined phenotypes that are most likely to share common genetic ) compares with an mz concordance rate of . for schizophrenia (results from several studies, pooled causation, as determined by examination of the findings of genetic-epidemiological studies (mcinnes and freimerby sherman et al.). the calculated heritability of bp has been estimated at . (tsuang and faraone ), ; escamilla et al. ). these examples could be used to guide our approach to genetic-mapping investi-compared with . – . for emotional stability (as com- piled by sherman et al.). gations of mood disorders; although the epidemiological studies of these syndromes are imperfect, they clearlysherman et al. point out that twin studies have a num- ber of inherent limitations and that their results there- suggest that severe bp (i.e., bp-i) should be considered such a narrowly defined phenotype.fore should be interpreted cautiously. such caution is particularly warranted for psychiatric disorders, because genetic mapping studies of mood disorderof the special complexities that we already have noted. relatively little attention has been paid to the fact that the effort to map genes for bp may have attracted more scrutiny from the scientific and general media thanthe majority of twin studies of psychiatric syndromes / a a$$ju - - : : ajhga uc-ajhg reus and freimer: behavioral genetics ’ has the search for genes responsible for any other human order amish community suggest possible sml inheri- tance only for bp-i, not for other mood disorders (paulstrait. remarkably, several cycles of misplaced excite- ment and exaggeration of failure were widely reported et al. ). these studies should not be interpreted as supporting sml inheritance of bp (craddock et al.before a single study had even attempted to screen the entire genome for linkage to bp. in fact, the initial series ; spence et al. ), but they clearly indicate that such a model is unlikely for broadly defined mood disor-of studies suggesting localization of bp genes on chro- mosomes (among the old order amish) and x ders. despite these observations, many (but not all) sub- sequent linkage studies have continued to focus on data(among non-ashkenazi israeli jews) were reported at a time when the available genetic markers permitted sets in which a high proportion of the genetic informa- tion derives from individuals with diagnoses other thanevaluation of linkage across only a tiny fraction of the entire genome (baron et al. ; egeland et al. ) bpi. efforts continue to attempt to identify forms of mood disorder that reflect a common genetic etiologyand when linkage had only been demonstrated for a handful of diseases, all with clear-cut mendelian trans- with bp; these remain unsuccessful (blacker et al. ). beginning in the early s, the availability of highlymission. these initial linkage studies were predicated on two broad assumptions: first, that relatively simple polymorphic microsatellite markers had a major impact on genetic-linkage studies for bp. several groups re-mendelian models could account for the transmission of an enormous range of mood-disorder phenotypes and, ported single suggestive localizations for bp genes, loci based on testing multiple markers in several locationssecond, that the power of linkage approaches was suffi- ciently great that bp genes could be identified even if but not, however, on complete screening of the genome. these studies utilized mainly cosmopolitan collectionsthe first assumption was incorrect. with the benefit of hindsight it is obvious that such preconceptions were of pedigrees from north america and europe. examples of these results include possible bp localizations on chro-naive, although it should be noted that the results of the studies, particularly that of the amish study, were mosomes q (straub et al. ), in the pericentro- meric region of (berrettini et al. ), on pwidely accepted until follow-up examination of the same pedigrees failed to support the original findings (mcin- (ewald et al. ), and on p (kelsoe et al. ). the results of berrettini et al. ( ) have attracted particu-nes and freimer ). it became clear that investigators had failed to account for uncertainty in the assignment lar attention, because they have been supported by an independent investigation using a different set of northof phenotypes; for example, the fall in the evidence for linkage on chromosome in the amish was due largely american pedigrees (stine et al. ). the results sup- porting bp linkage in the pericentromeric region of chro-to the development of mood disorder in two previously unaffected individuals whose marker genotypes in this mosome are confined largely to families in which bp appears to be inherited through the paternal lineage.chromosome region differed from those of the originally affected family members (kelsoe et al. ). addition- this intriguing observation has suggested the possibility of a parent-of-origin effect. it is unclear what mechanismally, the majority of the linkage information in both the amish and israeli studies derived from individuals with would account for such a finding; however, it may be noteworthy that this region contains one of the mostdiagnoses other than classic bp — for example, unipolar mdd (baron et al. , ; egeland et al. ; dramatic instances, in the genome, of suppression of male recombination relative to female recombinationkelsoe et al. ). as indicated above, it already had been known, from family studies, that the relative risk (silverman et al. ). several recently reported linkage studies of bp differfor bp was substantially greater than that for mdd (weissman et al. ) and, from twin studies, that the from previous ones in three important respects: ( ) link- age analysis is focused primarily on individuals withheritability of bp was substantially greater than that for mdd (bertelsen et al. ). severe mood disorder (bp-i only in ginns et al. ; mcinnes et al. ; and bp-i and bp-ii in blackwoodsubsequent to these initial ‘‘follow-up’’ linkage stud- ies, additional efforts have been made to evaluate the et al. ) ( ) the bulk of information for linkage is derived from affected, rather than from apparentlydegree to which the inheritance patterns of various forms of mood disorder are consistent with the models unaffected, individuals (blackwood et al. ; ginns et al. ; mcinnes et al. ). ( ) results can beused in most linkage studies — that is, the assumption that the action of a single major locus (sml) is involved. interpreted in the light of complete screening of the ge- nome in the relevant study populations (blackwood etspence et al. ( ), in the largest such study, have shown in a british columbian sample that, although al. ; ginns et al. ; mcinnes et al. ). what do these studies tell us? in the study by black-there is good evidence for sml inheritance of bp (in particular, of bp-i), the evidence argues against sml wood et al. ( ), an argument could be advanced for statistically significant linkage to chromosome p. thisinheritance for mdd and other psychiatric disorders. even more strikingly, segregation analyses of the old result was limited to a single extended scottish pedigree / a a$$ju - - : : ajhga uc-ajhg am. j. hum. genet. : – , and was not found in other pedigrees from the same prove spurious. in this respect the results of genome- screening studies of bp are roughly comparable to thosepopulation. additionally, the evidence for linkage de- pended on the inclusion of bp-ii individuals. ginns et recently observed for other complex traits, such as insu- lin-dependent diabetes mellitus and multiple sclerosisal. ( ), in a new analysis of the old order amish, identified regions on chromosomes , , and in (davies et al. ; hashimoto et al. ; ebers et al. ; haines et al. ; kuokkanen et al. ;which excessive allele sharing among affected individu- als suggest the possible location of loci for bp-i suscepti- sawcer et al. ). some commentators have suggested that the difficulties and uneven rate of progress inherentbility. none of these possible localizations, however, attain statistical significance. mcinnes et al. ( ), in in mapping genes for complex traits are somehow partic- ular to bp, perhaps because of the glib references thata study of two extended pedigrees from the genetically homogenous population of costa rica, identified possi- can be made to the euphoria of apparent success and the dysphoria of apparent failure (morell ; rischble localizations for bp-i in several chromosomal re- gions, notably p, p, and q. the q localization and botstein ). such publicity may have the unfor- tunate result of generating undue pessimism, among thewas supported by identification of extremely long haplo- types shared by most of the affected individuals in both scientific community and the general public, regarding the possibility of ultimately elucidating the genetic basisfamilies, including several alleles that are very rare in the general costa rican population (freimer et al. ). in of bp and other mood disorders. the costa rican study the linkage evidence also did not attain statistical significance. each of these reports, how- societal implications ever, identifies regions of the genome that require more intensive study. in two cases (blackwood et al. ; even if, in the near future, one or more susceptibility genes for bipolar disorder are identified, it will be diffi-freimer et al. ), the suggested linkage findings are accompanied by extended marker haplotypes in the ma- cult, as sherman et al. point out, to know how to utilize such information clinically. the societal stigma of bipo-jority of affected individuals within the relevant pedi- grees. these haplotypes indicate that the designated re- lar disorder and other psychiatric conditions, as well as their variable course, make it likely that the problemsgions are identical by descent (ibd) among affected individuals; since each of these studies draws on subjects already identified in the genetic testing of other common complex diseases, such as breast cancer and colon can-from genetically homogeneous populations, there is the possibility of testing whether these regions are shared cer, will be multiplied when genetic testing in psychiatric disorders becomes feasible. the opportunity exists,ibd by other (apparently unrelated) patients from the same populations. this opportunity also offers the hope however, to identify the salient issues and to plan for the day when genetic risk of psychiatric illness can bethat the region of ibd sharing can be narrowly deline- ated on the basis of the expectation that more distantly assessed reliably. for example, our group has surveyed individuals with mood disorder, their friends and rela-related affected individuals will share small regions ibd, as a result of recombination over several generations tives, and health-care providers, to explore the implica- tions of genetic testing in bipolar disorder (smith et al.that separate them from their common ancestor(s). ulti- mately it may be possible, by use of such isolated popu- ). we have observed that reproductive decisions and attitudes toward presymptomatic testing in childrenlations, to define, for bp loci, candidate regions suffi- ciently narrow to permit positional cloning of causative differ markedly between consumer groups and health- care providers and that they depend greatly on presumedgenes. it is unlikely that a single major susceptibility gene severity of illness and the availability of as yet undeter- mined preventive strategies that might ameliorate theexists that is responsible for a high proportion of bp cases in all populations. how, then, should we interpret severity or course of illness. individuals who are likely to pursue genetic testing may find it difficult to acceptthe fact that several genome-screening studies have high- lighted multiple possible chromosomal regions that the inherent uncertainty in the prediction of genetic risk and may misinterpret the information in such a way ascould contain bp-susceptibility genes? genetic-mapping studies of complex traits may be viewed as a highly to adversely affect their own and/or other family mem- ber’s life experiences. in our study, psychiatric physi-iterative process, in which initial genome-screening stud- ies serve to propose hypotheses to be tested through cians were much more likely to endorse termination of a pregnancy than were either patients and support-groupadditional investigation (mcinnes et al. ). ac- cording to this view, one does not expect the explosive members or medical students, if informed of a probable fetal inheritance of bipolar disorder. this difference inprogress toward gene identification that has character- ized the study of simple mendelian traits (jamison and interpretation of data was particularly evident in scenar- ios in which the likelihood of developing bp disordermcinnis ), and we can anticipate that many (if not most) of the currently suggested bp localizations will was high and in which the course of illness was severe. / a a$$ju - - : : ajhga uc-ajhg reus and freimer: behavioral genetics ’ cr, hostetter am, et al ( ) bipolar affective disorderssurprisingly, patients and support-group members ex- linked to dna markers on chromosome . nature :pressed greater interest in genetic testing than did psychi- – atrists, even when it was assumed that no prophylactic escamilla ma, freimer nb, reus vi ( ) the genetics oftreatment existed. the need for further study of attitudes bipolar disorder and schizophrenia. in: rosenberg rn,toward mood disorders is clear. it most likely will be at prusiner sb, dimauro s, barchi rl (eds) the molecular and least a few years before genes for bp are identified. this genetic basis of neurological disease, d ed. butterworth- provides us an opportunity to investigate systematically heinemann, boston, pp – the societal implications of the ability to determine ge- ewald h, mors o, flint t, koed k, eiberg h, kruse ta ( ) netic risk for this disorder — before the reality is thrust a possible locus for manic depressive illness on chromosome upon us. p . psychiatr genet : – freimer nb, reus vi, escamilla ma, mcinnes la, spesny m, leon p, service sk, et al ( ) genetic mapping using haplotype, association and linkage methods suggests a locusreferences for severe bipolar disorder (bpi) at q -q . nat genet : – american psychiatric association ( ) diagnostic and sta- tistical manual of mental disorders, th ed. american psychi- ginns ei, ott j, egeland ja, allen cr, fann cs, pauls dl, weissenbach j, et al ( ) a genome-wide search for chro-atric, washington baron m, freimer n, risch n, lerer b, alexander jr, straub mosomal loci linked to bipolar affective disorder in the old order amish. nat genet : – re, asokan s, et al ( ) diminished support for linkage between manic depressive illness and x-chromosome mark- goodwin fk, jamison kr ( ) manic-depressive illness. oxford university press, new yorkers in three israeli pedigrees. nat genet : – baron m, risch n, hamburger r, mandel b, kushner s, new- haines jl, ter-minassian m, bazyk a, gusella jf, kim dj, terwedow h, pericak-vance ma, et al ( ) a completeman m, drumer d, et al ( ) genetic linkage between x-chromosome markers and bipolar affective illness. nature genomic screen for multiple sclerosis underscores a role for the major histocompatibility complex. nat genet : – : – berrettini wh, ferraro tn, goldin lr, weeks de, detera- hashimoto l, habita c, beressi jp, delepine m, besse c,wadleigh s, nurnberger ji jr, gershon es ( ) chromo- some markers and manic-depressive illness: evidence for cambon-thomsen a, deschamps i, et al ( ) genetic mapping of a susceptibility locus for insulin-dependent dia-a susceptibility gene. proc natl acad sci usa : – betes mellitus on chromosome q. nature : – jamison kr, mcinnis mg ( ) genetic studies of manic-bertelsen a, harvald b, hauge m ( ) a danish twin study of manic-depressive disorders. br j psychiatry : – depressive illness. nat med : – kelsoe jr, ginns ei, egeland ja, gerhard ds, goldstein am,blacker d, faraone sv, rosen ae, guroff jj, adams p, weiss- man mm, gershon es ( ) unipolar relatives in bipolar bale sj, pauls dl, et al ( ) re-evaluation of the linkage relationship between chromosome p loci and the gene forpedigrees: a search for elusive indicators of underlying bipo- larity. am j med genet : – bipolar affective disorder in the old order amish. nature : – blackwood dh, he l, morris sw, mclean a, whitton c, thomson m, walker mt, et al ( ) a locus for bipolar kelsoe jr, sadovnick ad, kristbjarnarson h, bergesch p, mroczkowski-parker z, drennan m, rapaport mh, et alaffective disorder on chromosome p. nat genet : – ( ) possible locus for bipolar disorder near the dopamine transporter on chromosome . am j med genet : –breier a, charney ds, heninger gr ( ) major depression in patients with agoraphobia and panic disorder. arch gen kendler ks, heath ac, martin ng, eaves lj ( ) symp-psychiatry : – coryell w, endicott j, andreasen nc, keller mb, clayton pj, toms of anxiety and symptoms of depression: same genes, different environments? arch gen psychiatry : – hirschfeld rm, scheftner wa, et al ( ) depression and panic attacks: the significance of overlap as reflected in fol- kuokkanen s, sundvall m, terwilliger jd, tienari pj, wiks- trom j, holmdahl r, pettersson u, et al ( ) a putativelow-up and family study data. am j psychiatry : – vulnerability locus to multiple sclerosis maps to p -p in a region syntenic to the murine locus eae . nat genetcraddock n, vaneerdewegh p, reich t ( ) single major locus models for bipolar disorder are implausible. am j med : – mcinnes la, escamilla ma, service sk, reus vi, leon p, silvagenet : davies jl, kawaguchi y, bennett st, copeman jb, cordell s, rojas e, et al ( ) a complete genome screen for genes predisposing to severe bipolar disorder in two costa ricanhj, pritchard le, reed pw, et al ( ) a genome-wide search for human type diabetes susceptibility genes. na- pedigrees. proc natl acad sci usa : – mcinnes la, freimer nb ( ) mapping genes for psychiat-ture : – ebers gc, kukay k, bulman de, sadovnick ad, rice g, ric disorders and behavioral traits. curr opin genet dev : – anderson c, armstrong h, et al ( ) a full genome search in multiple sclerosis. nat genet : – morell v ( ) manic-depression findings spark polarized debate. science : – egeland ja, gerhard ds, pauls dl, sussex jn, kidd kk, allen / a a$$ju - - : : ajhga uc-ajhg am. j. hum. genet. : – , pauls dl, bailey jn, carter as, allen cr, egeland ja ( ) spence ma, flodman pl, sadovnik ad, ameli h, remick ra ( ) single major locus models for bipolar disorder arecomplex segregation analyses of old order amish families ascertained through bipolar i individuals. am j med genet implausible — response. am j med genet : – spence ma, flodman pl, sadovnick ad, bailey-wilson je, : – risch n, botstein d ( ) a manic depressive history. nat ameli h, remick ra ( ) bipolar disorder: evidence for a major locus. am j med genet : – genet : – sawcer s, jones hb, feakes r, gray j, smaldon n, chataway stine oc, xu j, koskela r, mcmahon fj, gschwend m, frid- dle c, clark cd, et al ( ) evidence for linkage of bipolarj, robertson n, et al ( ) a genome screen in multiple sclerosis reveals susceptibility loci on chromosome p and disorder to chromosome with a parent-of-origin effect. am j hum genet : – q . nat genet : – sherman sl, defries jc, gottesman ii, loehlin jc, meyer jm, straub re, lehner t, luo y, loth je, shao w, sharpe l, alexander jr, et al ( ) a possible vulnerability locuspelias mz, rice j, et al ( ) recent developments in human behavioral genetics: past accomplishments and future direc- for bipolar affective disorder on chromosome q . . nat genet : – tions. am j hum genet : – (in this issue) silverman ga, overhauser j, gerken s, aburomia r, o’con- tsuang mt, faraone sv ( ) the genetics of mood disor- ders. johns hopkins university press, baltimorenell p, krauter ks, detera-wadleigh sd, et al ( ) report of the fourth international workshop on human chromo- weissman mm, gershon es, kidd kk, prusoff ba, leck- man jf, dibble e, hamovit j, et al ( ) psychiatricsome mapping: boston, massachusetts, october – , . cytogenet cell genet : – disorders in the relatives of probands with affective dis- orders: the yale university – national institute of mentalsmith lb, sapers b, reus vi, freimer nb ( ) attitudes towards bipolar disorder and predictive genetic testing health collaborative study. arch gen psychiatry : – among patients and providers. j med genet : – / a a$$ju - - : : ajhga uc-ajhg wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top 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rhythm. in patients with af, only bvp improved lv diastolic function. these results indicate that bvp may be more beneficial than single-site lvp in patients with heart failure and af. groups #of patients exercise duratlon(min) met’s peak hr + spect, . * . . f . f % cl +spect, . * . . a . ’ * < % cl - spect, . f . ll.li . * so% cl -spect, . * . ’ . * . ” * < % cl poster session exercise testing: cardiac rehabilitation monday, march , , noonq:oo p.m. mccormick place, hall a presentation hour: :oo p.m.- :oo p.m. - predictors of negative exercise echocardiography in women with positive exercise electrocardiograms amish j. desai, amogh bhat, dallit bagha, mrudula guthikonda, ezra a. amsterdam university of california, davis medical center, sacramento, ca background: exercise (ex) electrocardiography (ecg) is the most widely used non- invawe test for evaluating symptoms suggestive of coronary artery disease (cad). in women. however, ex ecg has limited reliability due to an increased rate of false positive results. therefore, a stress-imaging study is frequently the initial test for evaluation of chest pal” in women. we have previously reported that certain exercise test variables predict a negative ex echocardlogram (echo). to further enhance this predictive value, we report additional exercise test variables predictive of negative ex echo. methods we analyzed the results of simultaneous ex echo and ex ecg in women with no known cardiac disease es part of the initial evaluation for chest pain suggestive of cad. all patients (pts) had a normal resting ecg and adequate exercise capacity by his- tory. all tests were symptom-limited utilizing a bruce protocol. a positive ex echo was defined es an ex-induced regional wall motion abnormality and a positive ex ecg was defined as ex-induced .o mm st segment depression - msec after the j point. results: the study group comprised consecutive women (mean age years [ - ) evaluated by simultaneous ex echo and ex ecg. ex echo was positive i” % ( / ) and negative in % ( / ). ex ecg was positive in % ( / ) and negative in % ( ). in % ( / ) pts with a positive ex ecg, ex echo was negative, suggesting a false positive ex ecg. in this group of pts, non-echo ex test data associated with a negative ex echo included: mets, double product z. . , st depression cl. mm, no ex-induced chest pain, st segment resolution <i minute into recovery, < leads with st depression. four or more of these factors were present in % ( / ) of the pts with a positive ex ecg and a negative ex echaconclusions: these results indicate that ( ) ex ecg was negative and concordant with ex echo in a majority of women and ( ) ~ % of positive ex ecg associated with negative ex echo were characterized by multiple indicators consistent with a false positive or low risk result. the evaluation of chest pain in women can be simple and cost effective utilizing ex ecg in those with a normal baseline egg and adequate exercise capacity. - abnormal peripheral thermoregulatory response to exercise substantially impairs exercise tolerance among patients with and without lschemia on myocardial perfusion single-photon emission computed tomography ehtasham a. qureshi, veeranna merla, pierre charouqi, umber burhan, amy . armenia. george reed, george diamond, alan rozanski, st. luke’s_roosevelt hospital center, new york, ny background: whereas spect myccardial ischemia predicts patients (pts) outcome, exercise (ex) duration generally adds substantial incremental information to event pre- diction. the pathophysiological factors mediating this association are unclear. paradoxi- cal vasoconstriction in finger blood flow during ex has recently been demonstrated, using peripheral arterial tonometry (pat), among cad patients. the relation between such vasoconstriction and ex duration is not known. methods: we employed pat to assess finger pulsewave amplitude (pwa) at rest and continuously during ex spect testing in cad pts (mean age * years. % males) divided into pts with ischemic and pts with non-ischemic spect studies. pwa patterns during ex were compared to previous normal limit values for ex pwa response; with values < % confi- dence intervals (cl) constituting abnormakty. resufts: abnormal finger blood flow response to ex was associated with a substantial reduction in ex duration and achieved ex mets (table). notably, this association was equally significant among patients with and without spect evidence of ischemia. conclusions: paradoxical reduction in finger blood flow during exercise is associated with significantly reduced exercise duration among cad patients. accordingly, this abnormal peripheral vascular response may rep- resent a potential, spect-independent, pathophysiological mechanism linking exercise duration to cardiac events. ‘pco. forz % cl vs. < % cl subgroups - using estimated functional capacity to optimize stress testing for diagnosis and prognosis of cardiovascular disease in women: the nhlbi-sponsored wise study leslee j. shaw, marian olson, sheryl kelsey, bernard r. chaitman. george sopko, c. noel bairey merz, wise study group, atlanta cardiovascular research institute. atlanta, ga, university of pittsburgh, pittsburgh, pa background: functional capacity, measured in mets, is an important component that affects the diagnostic and prognostic value of exercise stress testing. the aim of this study was to compare the prognostic value of predicted functional capacity based on the duke activity status index (dasi) compared with peak exercise mets methods: from the nhlbi-sponsored wise study, a total of wmen with chest pain (average age= years) underwent exercise testing and completed the -item dasi questionnaire. results: average exercw time was . i . minutes on the (modified) bruce or acip protocols and average dasi functional capacity was mets. . .year cardiovascular events (unstable angina, death, myocardial infarction, heart failure, or stroke) were reported in women. exertional lschemia occurred in % and % had a coronary stenosis+o%. in women with dasi-predicted mets < and > . , no inducible ischemia occurred more ( % vs. %), with % predicted maximum heart rate more ( % vs. %) often in functionally impaired women (p=o.o ); despite similar disease prevalence. in conclusion, among women referred for coronary angiography for suspected myocar- dial ischemia, marked functional impairment estimated by a simple estimate of functional capacity is associated with an adverse prognosis. use of the dasi prior to exercise stress testing may stratify candidates for exercise testing or pharmacologic stress. ll w . .year event rates % < . . - . . - . > . p value dascestimated mets . exercise mets . l-l - high treadmill workload in patients with exercise- induced st depression predicts a negative result on exercise echocardiography amish j. desai. amogh bhat, daljit bagha. mrudula guthikinda, ezra a. amsterdam, university of california, davis medical center, sacramento, ca background although exercise (ex) electrocardiography (ecg) is the most com- monly employed initial test to assess patients with symptoms suggestive of coronary artery disease (cad), it has limited diagnostic accuracy. therefore, patients (pts) with positive tests for myocardial ischemia are frequently referred for further evaluation by noninvasive stress imaging such as ex echocardiography (echo). a negative ex echo is considered evidence of absence of high risk cad and of low clinical risk. it has recently been shown that functional capacity is a strong predictor of prognosis. thus, in a group of pts with positive ex ecg but high treadmill workload, we investigated the results of sec- ondary evaluation by ex echo.methods we analyzed the results of consecutwe pts ( males, females; mean age yrs [ - ) referred for ex echo after a posi- tive ex ecg and a treadmill workload of mets. all pts had a normal resting ecg. exercise tests utilized a bruce protocol and were symptom-limited. a positwe ex ecg was defined as ex-induced .o mm st segment depression - msec after the j point and a positive ex echo was defined by an ex-induced regional wall motion abnor- mality. results: ex echo was negative in % ( / ) of pts and positive in % ( / ). conclusions: ex ecg performed to a high workload is highly predictive of a negative ex echo and thus low prognostic risk in pts referred because of positive ex ecg. pts with st depression on ex ecg, who achieve at least mets during treadmill ex. may not require additional noninvasive or invasive evaluation. - impact of obesity on inflammation and metabolic syndrome in coronary patients and effects of cardiac rehabilitation carl j. lavie, richard v. milani,ali morshedi, ochsner clinic foundation, new orleans, la background: obesity is epidemic in the us and represents a major risk factor for cad and type ii diabetes. limited data, however, exist on the eflects of obesity on such risk factors es inflammation and components of the metabolic syndrome (ms) es defined by atp ill in cad patients, and the effects of cardiac rehabilitation and exercise training programs &ret) in these patients. a comparison of periodontal disease among rural amish and non-amish adults j clin periodontol ; : - printed in denmark . all rights reserved copyright © murtksgaard clinical pencdontbioiy issn - a comparison of periodontal disease among rural amish and non-amish adults robert a. bagramian\ mahassen m. farghaly\ dennis lopatin\ maryfran sowers^ salam a. syed^ and julie l. pomerville^ ^school of dentistry, university of michigan; ^school of public health, university of michigan, ann arbor, michigan - bagramian ra, farghaly mm, lopatin d, sowers mf, syed sa and pomerville jl: a comparison of perio dont cd disease among rural amish and non-amish adults. j clin periodontol ; : - . © munksgaard, . abstract. periodontal disease can be more efficiently studied within a homogene- ous population where genetic influences and lifestyles are similar enough to negate their effect on the disease process. this study focuses on an amish population in southern michigan who isolate themselves from outside influences and their non-amish neighbors. a total of amish and non-amish were contacted resulting in amish and non-amish who were examined in their homes giving a participation rate of . %. ages ranged from to years. prevalence of periodontal disease tended to be higher among males and increased with age. there were slightly more amish females ( %) than non-amish ( %). means of periodontal conditions for amish were . mm for attachment loss, . mm for pocket depth, . for calculus, . for plaque and . for gingivitis. for non-amish, the means were . mm for attachment loss, . mm for pocket depth, . for calculus, . for plaque and . for gingivitis. it is of interest that the amish do not practice routine oral hygiene. only . % of amish reported brushing at least x a day compared to . % of non-amish. similarly, only . % of amish reported flossing at least x a week compared to . % of non-amish. key words: amish; periodontai disease; prevalence; epidemiology; attachment ioss. accepted for pubiication juiy the amish in this study isolate them- selves from the outside world by living without electricity, telephones, or auto- mobiles and marrying within their society. as reported previously (bagra- mian et al. ), the amish maintain a secluded lifestyle as a result of religious values established in the th century in switzerland. the extent of isolation and self-sufficiency of the amish com- munity is demonstrated by the existence, of amish schools, which children attend to th grade. only a few of the amish, %, in this study completed high school in public schools. households of or more comprising extended family mem- bers are common (mckusick , hos- tetler ). this secluded population provides a unique opportunity to inves- tigate the impact of a variety of factors on prevalence of periodontal disease. low levels of periodontal disease found among the amish in pilot studies (bagramian et al. ) formed the basis for the present intensive study. our pre- vious paper assessed dental examin- ations conducted on an amish farming community during the -year period - . the purpose of this paper is to report periodontal disease data col- lected for amish (â = ) and non- amish controls (â = ) over the entire -year period, - , assessments of behavior, attitudes toward oral hygiene, and knowledge of periodontal disease are examined along with various meas- ures of periodontal disease. iviethods amish farms are interspersed with non- amish farms in the area targeted for this study. a map of the amish community identified amish and non-amish farms. letters were mailed to all area residents informing them of our study and sol- iciting participation. the populations were approached in as similar a fashion as possible. for amish respondents, several steps were involved in gaining entry into individuals' homes to conduct the interview and exam. the st entry point was the religious leaders called bishops. each bishop has responsibility for apprixmately amish families and has ultimate authority over access to amish. after receiving a bishop's ap- proval, the team was able to contact individual families, where the nd entry point was the head of the household. once the household head agreed, the investigators were free to solicit partici- pation of individual family members. since amish participants were re- cruited door-to-door, non-amish were also recruited door-to-door. every non- amish house was approached to ensure the sample was as similar to the amish as possible. unhke the amish, these in- periodontal disease and amish dividuals could be accessed directly. sol- icitations were made during the evening as well as the daytime to avoid biasing the sample. a total of adult amish men and women over years of age were con- tacted and of these, were examined, giving a . % response rate. of the non-amish contacted, responded, a response rate of . %. all subjects were reimbursed for participation and no dental treatment was provided. con- ditions similar to the field were used to calibrate examiners to a reference periodontist, and x-statistics between each of the examiners were calculated for level of agreement on attachment loss and pocket depth. for attachment loss the xs ranged from . to . , and for pocket depth, they ranged from . to . , well within the limits in other studies and national surveys (brown et al. ). electricity was not available in homes, requiring examinations to be performed with a battery operated head light and portable dental chair. meas- urements were taken from standard sites of all existing teeth except rd mo- lars: distal-buccal, mid-buccal, mesio- buccal, disto-lingual, mid-lingual and mesio-lingual. the following indices were included: silness & loe ( ) plaque index, loe & silness ( ) gin- gival index, ramijord ( ) calculus index, and modified ramfjord ( ) pdi for pocket depth and attachment loss measurements. a hu-friedy no. periodontal probe was employed to measure loss of periodontal attachment and pocket depth to the nearest milli- meter. a -h, comprehensive questionnaire was administered by a trained inter- viewer. items regarding demographic characteristics, former health problems, current medications, allergies, tobacco use, frequency of physician visits, and family oral health history provided an overall picture of the respondents' gen- eral health. oral health attitudes and practices were probed through a series of questions on the respondent's oral health history, recent dental care, daily oral hygiene habits and satisfaction with dental services. the -page instrument was pre-tested on adult patients at the university of michigan school of den- tistry over a -week period. internal validity was checked through repetitive questions with altered wording or a re- verse response and modified. a knowledge score was computed based on questions that evaluated the respondent's ability to define peri- odontal disease. an index of oral hy- giene was also created from questions dealing with brushing, flossing, rinsing, and periodontal disease. severity of periodontal disease was examined to identify differences for amish compared to controls by ident- ifying the proportion of people with ^ site at various disease levels (kingman et al. ) and by assessing whole- mouth means. disease was delineated as having ^ tooth with mm of attachment loss or mm of pocket depth or a mean greater than . mm attachment loss or . mm pocket depth. logistic regression was applied to de- termine the relationship between ex- planatory variables: age, gender, amish or non-amish, oral health care; knowl- edge of periodontal disease, whether or not the person had mean plaque or mean gingivitis scores > or mean cal- culus scores > ; and presence or ab- sence of disease. each explanatory vari- able was entered into the model sequen- tially to determine the extent of any additional impact on the model ability to predict the probability of disease. in- teractions between explanatory vari- ables were also entered into the model. variables that did not show a significant dependent relationship with indepen- dent variables, as evidenced by the p- values for the wald / - statistic, were dropped from the model. this iterative process resulted in identification of ex- planatory variables that significantly re- late to periodontal disease. odds ratios were calculated. results amish respondents were younger ( % under ) than non-amish ( % were under ) and less educated; % had a th grade education compared to %) of non-amish. there were slightly more female amish ( %) than males ( %). gender for non-amish was almost evenly spht with %o female and % male. a larger proportion of non-amish had a full dentition at all age levels ex- cept for ages - years (tables , ). while %o of amish age - years had a full dentition, %o of non-amish at this age had all of their teeth. simi- larly, only %o of amish over years had a full dentition while %) of non- amish of this age had all of their teeth. the mean number of teeth present, however, differed little except for the age group - years, where the mean was . for amish and . for non- amish. mean attachment loss for amish was . mm while mean pocket depth was . mm. for non-amish, the means were slightly lower: . mm for attach- ment loss and . for pocket depth. mean calculus scores were low with an overall mean of . for amish and . for non-amish. plaque and gingivitis means showed somewhat higher levels but were still relatively low: . and . for amish and . and . , re- spectively, for non-amish. among amish, females had lower attachment loss levels than males at every age except the youngest. gingi- vitis and plaque showed similar age- and gender-related increases, while the change in pocket depth levels was not as dramatic. for amish, attachment loss rose from . mm at - years to . mm at age years and above (table ). for non-amish, attachment loss levels increased consistently with age from . mm at - years to . at age and above (table ). the distributions for oral hygiene practices and knowledge of periodontal disease showed significant differences between amish and non-amish respon- dents. given their secluded life style, the amish performed as expected, scoring lower than non-amish on the identifi- tabie , % distribution of numbers of teeth and mean number of teeth by age among amish age group (years) - - no. teeth - - + in= ) - - - + mean no. of teeth . . . . . . . . . . . . . . . . . . . . . . . . . bagramian et al. table . % distribution of numbers of teeth and mean number of teeth by age among non-amish no. teeth - - o : ^ , v . : : : • • : ; • . , + mean no teeth - ( v= ) . . . . . - . , , , . - . , , , - (tv= ) . . . . . + (/v= ) , , . . . table . mean periodontal disease measures by age among amish index plaque calculus gingivitis pocket depth attachment loss - ( y v = i i i ) . . . . . - (yv= ) , . , , , age group (years) - (a'= ) , , , , , - (a^= ) , . . , , + ( v= o) , , . , , total (a^= ) , . . , , table . mean periodontal disease measures by age among non-amish index plaque calculus gingivitis pocket depth attachment loss - ( v= ) . . . . . - (yv= ) , , , , , age group (years) - (a'= ) , , , , , - (/v= ) , . , , , + (yv= ) , , , , . total ( v= ) . . . . , table . % distribution for attachment loss of ^ mm by age for amish and non-amish attachment loss amish non-amish age (years) mm mm mm mm "a, n n n - - - + , , , , , , , , , , , , , , , , total , , , , cation and prevention of periodontal disease. % of the non-amish sample provided the correct response to knowledge questions while only % of the amish could. over -/^ of amish indi- cated incorrectly that cavities were in- dicative of periodontal disease and that widening of spaces between teeth was not an indicator. it is important to point out that this lack of knowledge is evi- dent in the reported oral hygiene prac- tices of amish, who show low behav- ioral scores when asked about actual practices. table shows that the proportion of persons with mm or more of attach- ment loss rose with age, as expected. a greater proportion of amish had mm of attachment loss at every age level. the overall difference in the proportion of respondents with ^ tooth at the mm level was % for amish and % for non-amish. logistic regression showed a high risk for attachment loss of mm for respon- dents who were amish, male, older, had calculus, and knew little about the signs of gum disease (/?< . , table ). for pocket depth of mm, the best-fitting model includes amish, older, male, having calculus, and having gingivitis as significant risk factors. the odds ratios presented in table show age as being the highest risk factor of mm of attach- ment loss. for pocket depth, the greatest risk is among those with gingivitis. behavior, plaque, and interactions between the independent variables did not reach statistical significance in the final model for attachment loss or pocket depth. however, when factors related to having gingivitis, calculus, or plaque were examined, interactions and behavior were important. the signifi- cant risk factors for plaque are being amish, older, having little knowledge about the signs of periodontal disease, and the interaction between amish and behavior (table ). significant factors related to having gingivitis are being amish, older, having little knowledge about the signs of periodontal disease, behavior, and the interactions between amish and knowledge of the signs of periodontal disease. no factors were significantly related to having calculus. when whole mouth means were examined, being amish, age, and gender (male) were significantly related to attachment loss. these variables con- stituted the entire set of independent variables for the final model. for mean pocket depth, having gingivitis, being amish, age, and male were significant risk factors. discussion the amish settlements in michigan present an exceptional opportunity to study demographic factors and behav- iors associated with periodontal disease. amish homes are interspersed among non-amish, thus reducing environmen- tal influences not related to genetics or life-style on the disease process. the amish and controls in this study were predominantly farmers. the examination of different meas- ures of periodontal disease in this study indicates that an assessment of severity when measured as the presence of ^ site at a specific disease level, yields more information than an assessment of whole-mouth means (burt ). only amish, gender, and age were significant- ly related to mean levels of attachment periodontal disease and amish table , odds ratios for periodontal disease amish gender age (years)* - - -t- plaque calculus gingivitis behavior disease knowledge attachment odds ratio . j . . . . . . . . loss ̂ mm p . . . . . . . . . . pocket d e p t h s odds ratio . . . . . . . . n / a n / a mm p . . . . . . . . n / a n/a reference group was respondents - years old. tabie , odds ratios for plaque and gingivitis amish gender age (years)* - - -f behavior knowledge score interactions: amish / behavior amish/knowledge plaque odds ratio . . . . . . . . . p . . . . . . . . . gingivitis odds ratio . . . . . . . . . p . . . . . . . . . reference group was respondents - years old. loss and pocket depth, whereas severity measured at ^ site at a specific level showed additional factors as being sig- nificant predictors. amish respondents reported lower education levels, scored lower on knowledge of signs of periodontal dis- ease, and reported less oral health care than non-amish. the difference in dis- ease levels, although statistically signifi- cant, is clinically small. overall, amish respondents had only . mm more attachment loss and . mm rnore pocket depth than non-amish. differ- ences in plaque, gingivitis, and calculus were also small. both measures of periodontal dis- ease, attachment loss of mm or pocket depth of mm, assessed among amish and non-amish respondents, demon- strated the expected relationships with age and gender. the odds of having attachment loss of mm were higher among persons with calculus and those who knew httle about the signs of peri- odontal disease. the highest odds ratio for pocket depth of ^ mm was for persons with gingivitis. this may be due to inflammation that occurs with gingi- vitis. a pocket may be measured deeper by the examiner if the area around the tooth is swollen due to gingivitis. calcu- lus showed the nd highest odds ratio for pocket depth. calculus is formed over a longer period of time than plaque and gingivitis; thus, it is not surprising that calculus was the only measure of the that was a strong predictor of attachment loss and pocket depth. the high odds ratio between having gingivitis or plaque and being amish reflect the low behavior score among the amish (table ). it is interesting to note that behavior was not statistically significant for attachment loss or pocket depth and was significant in the plaque and gingivitis models. levels of plaque and gingivitis in the mouth are affected by brushing or flossing, whereas the connection between oral hygiene and pocket depth or attachment loss is not as straightforward; these results are thus in line with current thought that con- siders plaque and gingivitis separate entities from the more advanced disease of attachment loss (williams ). oral health care and knowledge about the signs of periodonta] disease were significant risk factors for having plaque along with the interaction effect of oral health care and being amish. the gingivitis model was similar with being amish, male, high behavior score, high knowledge score, and the interac- tion between being amish and knowl- edge of periodontal disease as signifi- cant. the modest differetices between am- ish and non-amish oral health were in- teresting given the dramatic differences in oral hygiene practice and knowledge of periodontal disease. a lack of knowl- edge may well lead to a lack of dental care which is expected to lead to peri- odotital disease and missing teeth. this study of oral health indicates amish have slightly more disease than non- amish, which is in line with the reported lack of knowledge of periodontal dis- ease and poor oral hygiene practices among amish. acknowledgement this work was supported by the na- tional institute of dental research grant r deo - . zusammenfassung ein vergleich von parodontalerkrankungen zwischen erwachsenen der landlichen amisch- und nicht-amisch bevolkerung parodontalerkrankungen konnen besser in- nerhalb einer homogenen population unter- sucht werden, in der genetische einflusse und lebensgewohnheiten ahnlich genug sing, um keine auswirkung auf den erkrankungspro- zeb zu haben. diese studie konzentriert sich auf eine amisch-bevolkerung im stiden mi- chigans, die sich selbst auberen einflussen gegeniiber isoliert und ihren nicht-amisch nachbarn. insgesamt wurde mit amisch und nicht-amisch kontakt aufgenom- men. das ergebnis war, dab amisch und nicht-amisch bei sich zuhause unter- sucht wurden, wodurch sich eine teilnahme- rate von . % ergab. das alter lag zwischen und jahren. die pravalenz der par- odontalerkrankung tendierte dazu, bei man- nern hoher zu sein und im alter zuzunehmen. es gab etwas mehr weibliche amisch- ( %) als nicht-amisch ( %) -teilnehmerinnen. die mittelwerte fiir den parodontalen zu- stand ftir die amisch-bevolkerung waren . mm ftir attachmentverlust, . mm fiir taschentiefe, . fur zahnstein, . fiir pla- que und . fiir gingivitis. fiir die nicht- amisch-bevolkerung waren die mittelwerte . mm fiir attachmentverlust, . mm fur taschentiefe, . ftir zahnstein, . fiir pla- que und . fur gingivitis. es ist von inter- esse, dab die amisch keine regelmabige mundhygiene durchfuhren. nur , % der bagramian et al. amisch berichteten, dab sie wenigstens ein- mal pro tag putzen, im vergleich zu . % der nicht-amisch. auch berichteten nur . der amisch, dab sie zahnseide wenigstens einmal pro woche verwenden, im vergleich zu . % der nicht-amisch. r e s u m e , - • . '•;, •' ••• • • . • ; - . " " > ' ^ . comparaison de la maladie parodontale chez des adultes dans des populations rurales amish et non-amish l'etude de la maladie parodontale peut etre plus efficace si elle est faite au sein d'une population homogene ou les styles de vie et les influences genetiques sont suffisamment uniformes pour annuler ieur effet sur le pro- cessus pathologique. la presente etude est centree sur une population amish du sud du michigan qui s'isole des influences exterieu- res et de leurs voisins non-amish. on a pris contact avec au total amish et non- amish, ce qui a permis d'examiner amish et non-amish a ieur domicile, obtenant ainsi un taux de participation de . %. leur age allait de a ans. la prevalence de la maladie parodontale tendait a etre plus elevee chez les sujets du sexe masculin et augmentait avec l'age. ii y avait un peu plus de sujets du sexe feminin amish ( %) que non-amish ( %). les moyennes des parametres paro- dontaux chez les amish etaient , mm pour la perte d'attache. , mm pour la profon- deur des poches, , pour le tartre, , pour la plaque et . pour la gingivite. chez les non-amish. ces moyennes etaient . mm pour la perte d'attache, , mm pour la profondeur des poches, , pour le tartre, , pour la plaque et , pour la gingivite, ii est interessant de noter que les amish ne pratiquent pas de soins reguliers d'hygiene bucco-dentaire, chez les amish, . % seule- ment disaient se brosser les dents au moins une fois par jour, contre . 'mi chez les non- amish. de meme, . '/o des amish seulement disaient utiliser le fil dentaire au moins une fois par semaine. contre , 'm) des non- amish, references bagramian, r. a,, farghaly, m,, lopatin, d, e., sowers, m. f., syed, s. a. & pomervil- le, j, l. ( ) periodontal disease in an amish population. journal of clinical peri- odontology , - . bagramian, r. a., narendran, s. & khavari, a. m. ( ) oral health status, knowl- edge, and practices in an amish popula- tion. journal of public health dentistry , - , beck, j, d , lainson, p a., field, h. m. & hawkins, b. f. ( ) risk factors for vari- ous levels of periodontal disease and treat- ment needs in iowa. community dentistry and oral epidemiology , - . brown, j. l., oliver, r. c. & loe, h. ( ) evaluating periodontal status of us em- ployed adults. journal american dental association , - . brown, l. j., oliver, r. c. & loe, h. ( ) periodontal diseases in the u,s, in : prevalence, severity, extent, and role in tooth mortality. journal of periodontology , - , burt, b. a., ismail, a. i., morrison, e, c, & beltran, e, d ( ) risk factors for tooth loss over a -year period. journal of den- tal research , - . epidemiology and oral disease prevention program n ( ) oral health of united states adults: the national survey of oral health in us employed adults and seniors, - : regional findings. nih publi- cation no, - , epidemiology and oral disease prevention program n ( ) oral health of united states adults: the national survey of oral health in us employed adults and seniors, - : national findings, nih publi- cation no, - . hostetler, j. a. ( ) amish society, rd edi- tion, baltimore, md: john hopkins uni- versity press. ismail, a. i., morrison, e. c , burt. b. a,, caffesse, r, g, & kavanagh, m, t. ( ) natural history of periodontal disease in adults: fmdings from the tecumseh peri- odontal disease study, - . journal of dental research , - . kingman, a., morrison, e., loe, h. & smith, j. ( ) systematic errors in esti- mating prevalence and severity of peri- odontal disease. journal of periodontal re- search , - . loe, h., age, a., hans, b. & martyn, s. ( ) the natural history of periodontal disease in man. the rate of periodontal destruction before years of age. journal of periodontology , - . loe, h., anerud, a., boysen, h. & morrison, e. ( ) natural history of periodontal disease in man: rapid, moderate and no loss of attachment in sri lankan laborers to years of age. journal of clinical periodontology , - , loe, h. & silness, j. ( ) periodontal dis- ease in pregnancy (i). prevalence and se- verity. acta odontologica scandinavica , - , mckusick, v, a, ( ) the amish. endeavor , - . ramijord, s. p ( ) the periodontal dis- ease index (pdi). journal of peri- odontology >^, - , sillness, j. & loe, h. ( ), periodontal dis- ease in pregnancy (ii). correlation be- tween oral hygiene and periodontal con- dition. acta odontologica scandinavica , - , williams, ray c, ( ) periodontal disease. the new england journal of medicine , - . address: r. a. bagramian department of periodontics/prevention and geriatrics school of dentistry, university of michigan ann arbor, michigan - supported by national institue of dental research. roi de - usa news.fm full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iern expert review of neurotherapeutics issn: - (print) - (online) journal homepage: https://www.tandfonline.com/loi/iern new autism genetic risk factors identified to cite this article: ( ) new autism genetic risk factors identified, expert review of neurotherapeutics, : , - , doi: . / . . . to link to this article: https://doi.org/ . / . . . published online: jan . submit your article to this journal article views: view related articles https://www.tandfonline.com/action/journalinformation?journalcode=iern https://www.tandfonline.com/loi/iern https://www.tandfonline.com/action/showcitformats?doi= . / . . . https://doi.org/ . / . . . https://www.tandfonline.com/action/authorsubmission?journalcode=iern &show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iern &show=instructions https://www.tandfonline.com/doi/mlt/ . / . . . https://www.tandfonline.com/doi/mlt/ . / . . . news in brief – highlights from the latest news and research in neurotherapeutics . / . . . © future drugs ltd issn - www.future-drugs.com new autism genetic risk factors identified us researchers identify a hereditary and nonhereditary risk factor for autism in recent reports, two separate genetic defects have been shown to be associated with autism. one of the defects, caused by spontaneous deletion or duplication of a segment of chromosome , has been shown to be present in approxi- mately % of autistic children. three independent groups have also reported an association between the contactin- associated protein-like (cntnap ) mutation on chromosome and autism. published online in the new england journal of medicine, the autism consor- tium, a multicenter team, reported a -fold increased risk of developing autism in people who had deletions or duplications of a specific segment of chromosome , region p . . the mutations were found in autistic chil- dren but not their parents, suggesting that the mutation is spontaneous. the group used a high-resolution genomic copy-number variant analysis to examine chromosomes from families in which there was at least one autistic member as well as from people without the disorder. deletions or duplications in p . were found in of peo- ple from families with at least one autistic member, compared with only two of , people without the disorder. large instances of noninherited chro- mosomal abnormalities are extremely rare, as mark daly, lead author of the study commented. “finding precisely the same deletion in such a significant proportion of patients suggests that it is a very strong risk factor for autism.” the group is now attempting to iden- tify the specific gene or genes involved in the mutation. in three independent studies, published online in the american journal of human genetics, researchers have confirmed an association between cntnap , a gene previously identified in four amish chil- dren in , and risk of autism, in much larger samples. aravinda chakravarti, of the johns hopkins university school of medi- cine, who led one of the studies, explained their results. “we found a fac- tor that is probably present in every autistic kid. but while it may be neces- sary, it is not sufficient by itself to cause the disease.” “finding precisely the same deletion in such a significant proportion of patients suggests that it is a very strong risk factor for autism.” “cntnap is an excellent candidate gene for autism,” chakravarti com- mented. “it encodes a protein that’s known to mediate interactions between brain cells and that appears to enable a crucial aspect of brain-cell development. a gene variant that altered either of these activities could have significant impact.” in another of the studies, erik puffen- berger and colleagues built on their pre- vious finding in amish children. he commented. “this is a very exciting step for autism research. it also highlights the enormous potential of the ‘small science’ approach. our initial work used only four affected amish children. careful study of these four patients uncovered the association between cntnap and autistic behaviors. from that small beginning, cntnap has now been implicated as a significant risk factor for autism.” finally, matthew state and colleagues identified a rare mutation in the same gene that was associated with autism and appeared to be inherited. “this conver- gence of rare and common variants in autism is unusual, but reinforces the growing consensus among genetics researchers that both types of changes in dna sequence are going to be important contributors,” state commented. dietrich stephen, author of an accom- panying commentary, is extremely posi- tive about the findings and hopes that it may be possible to develop a diagnostic test for the cntnap mutation. “the field of genetics is replete with examples where researchers are unable to repro- duce results. here we have independent confirmation in multiple groups using large samples sizes,” he commented. “now that the results of the initial cntnap gene finding have been rep- licated, it strongly supports the notion that the ‘broken version’ of cntnap is recognized as a cause of autism in the general population,” he added. sources: weiss la, shen y, korn jm et al. association between microdeletion and microduplication at p . and autism. n. engl. j. med. ( ) (epub ahead of print); alarcón m, abrahams bs, stone jl et al. linkage, association, and gene-expression analyses identify cntnap as an autism-susceptibility gene. am. j. hum. genet. ( ), – ( ); arking de, cutler dj, brune cw et al. a common genetic variant in the neurexin superfamily member cntnap increases familial risk of autism. am. j. hum. genet. ( ), – ( ); bakkaloglu b, o’roak bj, louvi a et al. molecular cytogenetic analysis and resequencing of contactin associated protein-like in autism spectrum disorders. am. j. hum. genet. ( ), – ( ); stephan da. unraveling autism. am. j. hum. genet. ( ), – ( ). http://www.future-drugs.com k.rowland text box for reprint orders, please contact:
reprints@future-drugs.com expert rev. neurotherapeutics ( ), ( ) news in brief psychiatric comorbidity shown to be very common a recent study finds that the number of psychiatry outpatients who suffer from more than one psychiatric disorder is extremely high published in the february, , issue of psychological medicine, researchers from rhode island hospital (ri, usa) interviewed psychiatry outpatients to assess a wide range of psychiatric disorders. the majority of the patients tested were found to have two or more disorders, and more than one third of the patients suffered from at least three disor- ders. the average number of current diagnoses per patient was . . patients with a principal diagnosis of post-trau- matic stress disorder or bipolar disorder had the highest mean number of current comorbid disorders. for almost half of the outpatients, major depressive disorder was the reason for seeking treatment. about % of the patients suffered from social phobia; but % of these diagnoses came from initial treatment for another disorder. “…clinicians should assume that in outpatients presenting for the treatment of mood or anxiety problems, the patients have more than one diagnosis.” the lead author of the paper, mark zimmerman, director of outpatient psychiatry at rhode island hospital and associate professor of psychiatry and human behavior at brown medical school (ri, usa) commented, “for dis- orders like social phobia that are infre- quently diagnosed as the principle disor- der in clinical practice, it will be important for the next generation of treatment-efficacy studies to determine if treatment is effective when the disorder is a comorbid condition.” the authors also hope that these results will encourage clinicians to check for comorbidity in psychiatric outpatients, as zimmerman explained, “based on the results of this study, clinicians should assume that in outpatients presenting for the treatment of mood or anxiety prob- lems, the patients have more than one diagnosis.” source: zimmerman m, mcglinchey jb, chelminski i, young d. diagnostic co-morbidity in psychiatric out-patients presenting for treatment evaluated with a semi-structured diagnostic interview. psychol. med. ( ) (epub ahead of print). the us fda has recently granted cell- dex therapeutics, inc. (nj, usa) fast track designation for their cdx- investigational immunotherapy for the treatment of egfrviii-expressing glioblastoma multiforme (gbm). egfrviii, which is only found in cancerous tissue, is present in approxi- mately % of gbm patients. cdx- immunotherapy works by activat- ing a patient’s immune system against egfrviii, therefore selectively attack- ing the tumor cells. recent trials of the immunotherapy have shown highly promising results. fast track designation granted for cdx- for the treatment of glioblastoma multiforme drug: cdx- tradename: na manufacturer: celldex therapeutics, inc. indication: treatment of egfrviii- expressing glioblastoma multiforme in the activate phase ii study, patients with egfrviii-expressing gbm who were treated with cdx- had a median survival of months, compared with only . months in historical controls. the median time to progression was also longer, at months (p = . ) in cdx- - treated patients compared with only . months in historical controls. in addition, in patients whose gbm recurred following treatment, the tumors had lost egfrviii expression. “fast track status acknowledges cdx- ’s potential to fill an unmet need for glioblastoma patients…” in act ii, an extension study, a simi- lar patient population was treated with a combination of cdx- and chemo- therapy. although the study has not yet reached its median time to progression, preliminary progression-free survival and overall survival rates look similar to those found in the activate trial, and as such are highly promising. finally, in september , the first patient was randomized to the act iii phase ii/iii study of cdx- in com- bination with radiation therapy and temzolomide in newly diagnosed gbm. the study was designed to investigate the safety, anticancer activity and impact on survival of adding dcx- to the standard of care for gbm patients versus the standard of care alone. “fast track status acknowledges cdx- ’s potential to fill an unmet need for glioblastoma patients and gives it priority within the fda,” com- mented thomas davis, chief medical officer of celldex therapeutics. “con- firmation of the promising results we’ve already observed is a high priority at celldex, as it is within the brain cancer community in general.” source: www.celldextherapeutics.com news in brief www.future-drugs.com astrazeneca (ny, usa) recently sub- mitted two separate supplemental new drug applications (sndas) for the use of seroquel xr™ in the treatment of manic and depressive episodes associated with bipolar disorder. seroquel xr, which is currently approved in eight countries for the treatment of adults with schizophrenia, was reported at the th international forum on mood and anxiety disorders (budapest, hungary) in december to also be effective in the treat- ment of major depressive disorder and generalized anxiety disorder. the two snda submissions are both based on clinical studies of once daily treatment with seroquel xr compared with placebo. in the bipolar mania study patients receive seroquel xr once daily at doses of – mg/day. the primary end point is a change in baseline young mania rating scale (ymrs) total score at weeks. in the bipolar depression study patients receive mg/day seroquel xr once daily with a primary end point of a change from baseline in the mont- gomery asperg depression rating scale (madrs) total score after weeks. both studies have met their primary end points and further results are expected to be presented later in . sources: astrazeneca press releases www.astrazeneca.com/pressrelease/ .aspx; www.astrazeneca.com/pressrelease/ .aspx. astrazeneca seeks us fda approval for the use of seroquel xr™ for bipolar disorder drug: quetiapine fumarate tradename: seroquel xr™ manufacturer: astrazeneca current indication: treatment of schizophrenia in adults proposed indication: treatment of manic and depressive episodes associated with bipolar disorder positive effect of etanercept on alzheimer’s disease symptoms patient experiences rapid improvement in memory following treatment with the arthritis drug a recent case report, published online on january in the journal of neuroinflam- mation, has documented extremely rapid improvements in cognitive abilities in an alzheimer’s disease (ad) sufferer follow- ing spinal injection of the arthritis drug, etanercept (trade name enbrel®). etanercept is a tnf-α receptor fusion protein that binds to tnf-α and neutral- izes it. elevated levels of tnf-α are hypothesized to be involved in the patho- genesis of ad. therefore, researchers investigated the effect of treatment with etanercept on ad symptoms. the study reports the administration of etanercept into the spine of an -year old man with early stage ad. prior to injection a variety of cognitive tests were carried out on the man, who used to be a doctor. within min following the injection, the man could remember a variety of facts that he had previously been unable to recall, including the year, day of the week and the state in which he lived. he was also much calmer, more attentive and less frustrated following the injection. lead author of the study, edward tobinick, said the drug had “a very rapid effect that’s never been reported in a human being before”. he added, “it makes practical changes that are signifi- cant and perceptible, making a difference to his daily living.” “other experts in the field are also excited about the potential of this novel treatment…” sue griffin, the editor the journal of neuroinflammation who wrote an accom- panying commentary, said “it is unprece- dented to see cognitive and behavioral improvement in a patient with established dementia within minutes of therapeutic intervention. this gives all of us in ad research a tremendous new clue about new avenues of research.” she added, “even though this report predominantly discusses a single patient, it is of signifi- cant scientific interest because of the potential insight it may give into the proc- esses involved in the brain dysfunction of ad.” other experts in the field are also excited about the potential of this novel treat- ment, although they do stress that a great deal more research is required. rebecca wood, of the alzheimer’s research trust (cambridge, uk) commented, “this is promising and innovative research, but in the early stages and further work is needed before we can conclude etanercept could work as a treatment for ad. we need to investigate whether it is safe and works in a larger number of patients as well as moni- tor the long-term effects. scientists also need to check the benefits weren’t just due to the placebo effect and establish whether any benefit is just temporary or whether the disease itself is slowed.” sources: tobinick el, gross h. rapid cognitive improvement in alzheimer’s disease following perispinal etanercept administration. j. neuroinflammation ( ), ( ) (epub ahead of print). griffin ws. perispinal etanercept: potential as an alzheimer therapeutic. j. neuroinflammation ( ), ( ) (epub ahead of print). << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain %) /calrgbprofile (srgb iec - . ) /calcmykprofile (u.s. web coated \ swop\ v ) /srgbprofile (srgb iec - . ) /cannotembedfontpolicy /error /compatibilitylevel . /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel /emitdscwarnings false /endpage - /imagememory /lockdistillerparams false /maxsubsetpct /optimize true /opm /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true /preserveepsinfo true /preservehalftoneinfo false /preserveopicomments false /preserveoverprintsettings true 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settings require font embedding.) /jpn <feff e a d b a f ad e cf ea b cf b d ea d ec b e f f c b d b f f e e a d b a f c f f f f a e ee d a d e e a d b a b f d a f c e cb fbc f c fc > /fra <feff f f e f e f d e f e e e f c f e e c e f e c e d f e e e c a f f c f e e f c e c f c f e c e f f f e f c e> /deu <feff e e e c c e e a d c c e f e d f b d e e d e f e c c f e c d e c f fc b f a a c e e d f b d e b f e e e d f f d d e e f f e e e e c c e e e e f c e> /ptb <feff c a f e e f f d e f f d d f c e e f d d f f d c d e f d c f e f f d e f f d f f d f f c e f f e f e e f d e f f e e f f e e> /dan <feff e c c e c f d f b d e d f a c c f c f e e c d d b e e f a b c e d f b d e e b e e e d f f e f e e e c c e b e e e b e> /nld <feff b a e c c e e f d d f d e e d b e d e f c e f c f f b b e d f b c e e d f d e e d f d e e b e e e f e f e d f e e e f e a a e c c e d f e f e a a e e c f e e> /esp <feff f f e f d e f f e d f f c f e d e d e f e f d f e d f c e c f f d e f e f e f e f e f f e f e f e c e f e e e> /suo <feff e e e e c c f e c f d b a f a c a f e c f c f e b f b a b e b b e d b a f e f d a e d f a c d c c d d c c f c c e e e d e b c c e e f e e f e> /ita <feff d f a f e f d e f e e f c a f e d f e c e d d c f e f d e f f e f f e f e f e e d f a f e f e f c f f e e f f e> /nor <feff b e e c c e e c e f d f b d e d f c f c f e e f f b b c f b b e d f b d e b e e e d f f e f e e e e c c e e b b e e e e> /sve <feff e e e e e e c c e e e e e c c b d f b d e d f c c f e e f b f b c e d f b d e e b e f e d f f e c c e e e e c c e e b e e b c e b e e e> >> >> setdistillerparams << /hwresolution [ ] /pagesize [ . . ] >> setpagedevice no job name boundaries of social capital in entrepreneurship ivan light léo-paul dana our research begins with a theoretical critique of the social capital literature, and then focuses on old harbor, alaska. in this remote outpost, mainly populated by alutiiq people, all entrepreneurs self-identified as euro-americans or multi-ethnic, not alutiiq. although alutiiq people have abundant social capital, which they employed for economic purposes, they did not employ their social capital for commercial entrepreneurship. our findings suggest that social capital promotes entrepreneurship only when supportive cultural capital is in place. introduction the role of social capital in entrepreneurship has become an increasingly prominent topic in business literature, and the debate has become increasingly complex (casson & della giusta, ). early contributors to this literature were content to introduce social capital and then to portray its previously unacknowledged contribution to entrepreneur- ship (allen, , p. ; burt, ; light, ). on this conventional view, social capital involves relationships of trust and reciprocity that inhere in social networks (halpern, : chaps. – ; mcevily & marcus, ; mustafa & chen, ; ostrom, ; slotte-kock & coviello, , p. ). the contribution of social capital to entre- preneurship, understood broadly as self-employment in commercial business, is “the assets that may be mobilized” through networks, thanks to mutual trust and the norm of reciprocity (galbraith, rodriguez, & stiles, , p. ; nahapiet, , p. ). however, more recent approaches to social capital have complained that too much social capital squelches entrepreneurship whether because it protects mediocrities (light, a), reduces objectivity (locke, ), imposes mental conformity on whole groups (aldrich & kim, , p. ; dana & morris, ), or inhibits escape from failing allies and partners (adler & kwon, ). there is also concern that social capital can suppress please send correspondence to: ivan light, tel.: ; e-mail: light@soc.ucla.edu, and to léo-paul dana at lp.dana@supco-montpellier.fr. . thompson distinguished three forms of reciprocity: pooled reciprocity, sequential reciprocity, and recip- rocal interdependence ( : – ; see also etemad, wright, & dana, , p. ). what is essential here, however, is only the recognition that a trust-based capacity for any form of reciprocity conveys a huge advantage for economic action in general and entrepreneurship in particular. the asset flows along the network promote entrepreneurial success, both domestic (uzzi, ) and international (terjesen & elam, ). pte & - © baylor university january, doi: . /etap. entrepreneurship when a dominant group excludes subordinates “from the information, influence, and solidarity benefits” it enjoys (adler & kwon, p. ; also crow, , p. ). this debate has raised the question of when social capital is a catalyst, when an obstacle, and when unrelated to entrepreneurship. entering this debate, we explore both the pro and the con argument regarding social capital, concluding that both sides require specification. on the pro side, we address the frequent claim that resources born of social capital enable and enhance the entrepreneurship of groups endowed with social capital. this claim now approaches confirmation in the research literature, which has justly celebrated the contribution of social capital to entrepre- neurship. however, as we show, the research literature has relied overmuch on prevailing social contexts in which cultural capital supports entrepreneurship, thus concealing the supportive role of cultural capital just as one would conceal the contribution of oxygen to athletic performance if one conducted all research at sea level. we address a research location in which cultural capital does not support commercial entrepreneurship, and we identify a group with high social capital, alutiiq people, whose abundant social capital does not eventuate in commercial entrepreneurship. we infer that, beyond the boundary of supporting cultural capital, social capital ceases to encourage entrepreneurship. to this extent, then, the frequent claim that social capital supports entrepreneurship is apparently overstated, and its necessary symbiosis with cultural capital overlooked. on the negative side of this debate, we evaluate the frequently encountered claim that the social capital of powerful groups obstructs the entrepreneurship of less powerful groups (adler & kwon, , p. ; also crow, , p. ). again, that generalization usually holds true. that conceded, circumstances exist in which the social capital of the powerful does not prevent entrepreneurship by subordinates. the historical literature on middleman minorities makes that point. additionally, if subordinate groups bridge to dominant groups, then the social capital of the dominant group may support (rather than obstruct) the entrepreneurship of the subordinates. the empirical issue becomes how often the bridges mitigate the barrier posed to subordinate groups by the social capital of the powerful. methodological source of the problem under the usual circumstances of research, the people of interest participate in social networks that offer wide bridging access to neighboring networks (aldrich & kim, , p. ); and they approach economic life from a cultural standpoint congenial to com- mercial entrepreneurship. when these conventional conditions are in place, researchers reliably find that, as slotte-kock and coviello ( , p. ) summarize the literature, “networks have been shown to improve entrepreneurial effectiveness by providing access to resources.” if the world consisted only of such cases, the generalization would ring true. however, since the requisite support conditions are not always in place, this generalization may be generally but not universally true. we examined the issues of entrepreneurship theory and practice, published between and . of the articles in these issues, only two dealt with ethnic or racial minorities. this focus is quite understandable inasmuch as the entrepreneurship of ethnic and racial minorities is off the business mainstream and of restricted reader interest outside the social sciences. anthropology should not be a focal concern of the . for a comprehensive review of the voluminous social science literature on the subject of ethnic and immigrant minorities in self-employment, see light ( ). entrepreneurship theory and practice business literature, but we could learn about social capital’s boundary conditions by observing the experience in entrepreneurship of minorities in unconventional settings. after all, conventional circumstances are not universal circumstances. under some cir- cumstances, even given the availability of within-group (bonding) social capital, ethno- religious or ethno-racial groups are sharply distinct and unequal in respect to tangible economic resources, intergroup bridges are few or nonexistent, and cultural capital does not support entrepreneurship. under these circumstances, common in the world of indig- enous minorities, social capital does not produce entrepreneurship, which suggests that social capital’s beneficent effect is not what klyver, hindle and meyer ( , p. ) call a “simple universal.” indigenous communities of north america exactly these frustrating circumstances recurrently arise in indigenous communities across north america despite an abundance of social capital (anderson & giberson, ). if entrepreneurship depended only on bonding social capital, indigenous ameri- cans and canadians would be supremely entrepreneurial. however, indigenous north american communities exhibit now and have long exhibited below-average rates of self-employment (garsombke & garsombke, ; peredo, anderson, galbraith, honig, & dana, ). in , despite their abundant social capital, “american indians and alaska natives” in the united states operated . employer firms per , of population compared to employer firms per , in the total u.s. population (u.s. bureau of the census, economic census, ). exploring the reasons for this discrepancy, we identify boundary conditions that must be in place for social capital to support entre- preneurship. we propose that, in addition to their other economic handicaps, such as legal and administrative obstacles, already well addressed in the existing literature (anderson, dana, & dana, ; dana & anderson, , a), and which we neither discuss nor question, indigenous communities typically have three network-derived problems in developmental entrepreneurship: first, except for bonding social capital, which they have in abundance, they often lack business supportive cultural capital (frederick & henry, , pp. , ). they have abundant cultural capital, but their cultural capital does not necessarily support commer- cial entrepreneurship—although it does support other forms of economic activity, such as subsistence agriculture (wall & masayesva, ), hunting (povoroznyuk, ), and fishing (simeone, ). while food may be shared (meis-mason, dana, & alexander, ; van de velde, ; wenzel, ), there is not necessarily an economic transaction in such a context. true, in principle, any social capital “can be converted to other kinds of capital” such as economic, human, and cultural (adler & kwon, , p. ). that conversion need not occur, even though it can, and even when it does occur the conversion may require centuries to complete. second, lacking bridging ties to the powerful, indigenous communities are locked out of resource-swapping networks essential to introducing external resources of entrepre- neurship (jarillo, ; mcevily & zaheer, ). third, indigenous minorities exist outside the cultural orbit of encompassing market societies (dana & anderson, a, . this is common but not invariant; dana and anderson ( b) identify an indigenous culture of the canadian arctic in which commercial entrepreneurship is a growing trend. we thus note variations among peoples as well as change over time. january, p. ). in the indigenous cultural context, entrepreneurship is rarely a conscious desid- eratum or an appropriate occupation, especially for men; moreover, should indigenous communities generate any nascent entrepreneurs, they lack bridging access to external networks that control essential business resources. social capital as catalyst we acknowledge the advantages for entrepreneurship that social capital conveys in the developed world’s cultural orbit (halpern, , chaps. – ; mcevily & marcus, ; mustafa & chen, ; slotte-kock & coviello, , p. ). our negative case depends only on identification of the neglected boundaries outside of which social capital does not have this effect. social capital normally appears in the developed world in tandem with supportive cultural capital. under these circumstances, some readers and reviewers might assign to social capital all the credit for promoting entrepreneurship—at the risk of ignoring its indispensable partner. hence, claims of social capital’s advantageousness for entrepreneurship are overstated. the most sophisticated theoretical treatments of social capital warn against this mistake without, however, drawing out the methodological implications for research. adler and kwon ( , p. ) warn that “norms and beliefs figure in the analysis of social capital,” a caveat that suggests an intervening cultural variable needed to bring social capital to entrepreneurial fruition. similarly, edelman, bresnen, newell, scarborough, and swan ( , p. ) and lee and jones ( , p. ) introduce the concept of “cognitive social capital” in an attempt to introduce a cultural dimension into the concept. although this definition accesses a cognitive dimension in decision making, it conflates social capital and cultural capital, which bourdieu ( b, ) firmly distinguished. in the interest of reducing conceptual clutter, we prefer bourdieu’s ( a, b; weininger, ) older distinction between social capital and cultural capital. drori, honig, and ginsberg ( , pp. – ; also patel & conklin, ) have recently explicated bourdieu’s ( ) concept of habitus in respect to entrepreneurship. the habitus is a system of acquired dispositions that encompasses the social component of personality. bourdieu introduced the concept of habitus to explain the unproblematic reproduction of social worlds or, putting it negatively, failure to change. a unitary habitus confers a unitary economic direction to whole groups whereas cultural capital, its matrix, need not. to Özbilgin and tatli ( ), a habitus is “embodied history internalized as second nature” (p. ). de clercq and voronov ( ) state habitus is “practical sense of the game” (p. ). a group’s habitus orients participants to appropriate livelihoods, enabling whole groups to find their usual occupations in the usual way (light & gold, , p. ). as drori et al. ( , pp. – ) and their contributors rightly claim, a dual habitus can liberate entrepreneurship, but a unitary habitus tends to enslave groups to the repro- duction of traditional livelihoods (light, b). we subsume the static habitus within a broader concept of cultural capital that includes everything cultural that feeds into occupational choice and performance, includ- ing the habitus, but also externally introduced values, skills, attitudes, and beliefs that may . the habitus imposes “different definitions of the impossible, the possible, and the probable” that, in turn “cause one group to experience as natural or reasonable practices or aspirations which another group finds unthinkable or scandalous, and vice-versa” (bourdieu, , p. ). entrepreneurship theory and practice modify a habitus. for example, preferring to work outdoors is part of the vocational culture of farmers and lumberjacks. someone who prefers to work outdoors will be unhappy as a dentist. everyone has cultural capital. the cultural capital of bricklayers and entrepreneurs are not identical, but both have cultural capital. the cultural capital of bricklayers encourages laying bricks; that of entrepreneurs conduces to business owner- ship. bourdieu ( , p. ; b, ) distinguishes cultural capital from social capital, conceding the direct and unmediated contribution of social capital to economic action in general, but underscoring as well the independent contribution of cultural capital to entrepreneurship in particular or, for that matter, to any field. cultural capital directs social capital to approved vocational goals. max weber’s ( – ) celebrated protestant ethic was, we now realize, a specific form of cultural capital (light, a). obviously, the role of culture in entrepreneurship is not a new issue, but cultural capital still struggles to obtain recognition in the business academy for reasons that include, we believe, the methodological issue raised in this paper. theoretical awareness is present, but the prevailing methodology distorts the empirical results. “entre- preneurship is a value-driven activity” declare morris and schindehutte ( , p. ). similarly, adler and kwon ( , p. ) warn that entrepreneurship may be “seen as legitimate” in one culture, while another culture regards it as “opportunistic and self- seeking.” as dana ( , p. ) also explained, some cultures “do not value” entrepre- neurship. accordingly, we are not introducing a novel idea when asking whether or to what extent the cultural capital of any group endorses entrepreneurship as a legitimate much less desirable vocational choice. suppressive social capital now turning to the negative side of the social capital debate, we address the suppres- sion hypothesis. the suppression hypothesis proposes that the social capital of powerful groups locks subordinate groups out of entrepreneurship (adler & kwon, , p. ; also crow, , p. ). if so, social capital sometimes reduces entrepreneurship rather than invariantly strengthening it. sympathetic to the suppression hypothesis, which addresses a genuine barrier, we find that the hypothesis requires additional specification. first, clarifying an ambiguity in the existing literature, we distinguish malevolent suppression from inadvertent suppression. suppression is malevolent when powerful groups con- sciously exploit resource monopolies to lock less powerful groups out of entrepreneur- ship; suppression is inadvertent when the lock-out occurs simply out of the lopsided monopoly of resources that exists in a situation but without conscious intent. not every suppression is malevolent. additionally, two loopholes invalidate the simple and unmodified suppression hypothesis: ( ) we must specify the relative strength of social capital in the dominant and subordinate groups; ( ) we must specify the extent of bridging social capital between the dominant and subordinate group. first, the validity of the suppression hypothesis depends . additional citations might start with becker ( ), who noted that some societies consider entrepreneurship “unholy.” similarly, jones and sakong ( , pp. – ) explained that in korea, entrepreneurship “runs counter to the traditional value system.” min ( ) found that the perceived absence of key values reduces the probability of entrepreneurship within a culture group. enz, dollinger, and daily ( ) concluded that value orientation is an important explanatory variable in ethnic enterprise. dana ( ) found that alaska natives related differently to opportunities for entrepreneurship than did nonindigenous alaskans (p. ). dana ( , p. ) observed that the word trader in javanese language is synonymous with tramp (p. ). january, on the strength of the social capital of the dominant group vs. its strength in the subor- dinate. the suppression hypothesis works best when the dominant group has strong social capital, and subordinate groups have weak. however, if the social capital of the dominants is weak, and that of the subordinates strong, then the dominants will be unable to constrain the subordinates’ entrepreneurship so much as, given stronger social capital, they other- wise would have. in this case, dominants with weak social capital would not only enjoy less economic benefit from their own social capital, they would also prove less able to exclude the subordinates from it. even if the social capital of dominants is strong, if the social capital of the subordinates is stronger, then the dominants will prove less able to suppress the subordinates’ entrepreneurship than they would were the subordinates poorly endowed with social capital. this result would arise because the stronger social capital of the subordinates would, ceteris paribus, enable them to mount a stronger entrepreneurial challenge despite the resolute suppressive efforts of more powerful superordinates. for these reasons, the claim that dominants’ social capital always locks subordinates out of entrepreneurship may be generally true but cannot be universally true. the historical archive supports this deduction. the world’s middleman minorities were subject to intense hostility from host societies, as bonacich ( ; also pal, ) pointed out, including expropriation, riot, murder, and even genocide. all ethnic and immigrant minorities do not display elevated levels of entrepreneurship as do middleman minorities (portes & bach, ). but the jews of europe, the chinese of southeast asia, and the armenians of the levant, to name a prominent few, were for centuries extra- ordinarily entrepreneurial despite the most intense hostility of larger and more powerful groups (light & gold, , pp. – ). gradually, the entrepreneurship that began as a refuge became a habitus-linked competence. how was this transition possible? middle- man minorities had two overwhelming strengths: their cultural capital strongly endorsed business ownership as a vocation, and the hostility of more powerful hosts could not deflect them from this vocational goal. again, the middleman minorities mounted extraor- dinarily stronger social capital than what dominants mobilized against them. their enhanced social capital enabled the persecuted ethnic minorities to overcome the hostile social capital of the powerful, even establishing entrepreneurship as their main line of economic defense. the amish of pennsylvania, indiana, and ontario offer another empirical example of a weak minority with high entrepreneurship. although they lack education beyond grade eight, and cannot own automobiles, the amish have vast bonding (intragroup) social capital; and they isolate themselves from outside groups, who regard them with curiosity, if not derision; but the amish have nonetheless maintained extraordinarily high rates of self-employment for three centuries. here again, the cultural capital of the amish actually prescribes self-employment as a vocation; the amish do not need to acquire the motivation for self-employment from external sources. like the middleman minorities, the amish enjoy bonding social capital much stronger than what the non-amish majority enjoys (dana, ; kraybill & nolt, ). the same is true of sámi reindeer hunters of northern europe, who, like the amish are not middleman minorities. the sámis’ cultural capital prescribes entrepreneurship as the preferred occupation (dana & light, ). confronting the unsympathetic finnish welfare state, the sámi persist in extensive rein- deer entrepreneurship so their experience does not validate the suppression hypothesis either. that said, most excluded or marginalized ethno-religious groups are not middleman minorities with centuries of commercial entrepreneurship in their history. middle- man minorities are unique and exceptional (bonacich, ; dana, ). most ethnic minorities do not have cultural capital that resolutely supports and endorses commercial entrepreneurship theory and practice entrepreneurship nor bonding social capital that exceeds that of more powerful and possibly malevolent external groups (portes & bach, ). these were the key resources of middleman minorities. the suppression hypothesis succeeds quite well in explaining the frustration of entrepreneurship among numerous non-middleman minorities among whom we number the indigenous peoples of north america. since most ethno-religious minorities are not middleman minorities, the suppression hypothesis succeeds appreciably more often than it fails. an obvious example of success is african american entrepreneurs in the old south. unlike the chinese in mississippi (loewen, ), african americans of the old south were not middleman minorities. except in pockets of high density, african american entrepreneurship was persistently weak. the explanations offered support the suppression hypothesis (butler, ). the old south contained white people and an african american minority; white people were economically dominant, socially exclusive, and maliciously determined to frustrate the economic progress of the black people (davis, gardner, & gardner, , chaps. , , ). african americans had social capital, centered in the churches, but they lacked business skills, industrial infor- mation, financial capital, and bridging ties to the white people who had these resources of entrepreneurship, which they passed back and forth among themselves through bonding social capital. to the extent that the tangible economic resources in the old south were controlled by white people, and the black people were collectively irresolute regarding the vocational centrality of entrepreneurship, the social capital of the white people proved a serious obstacle to afro-american enterprise. the apparent exceptions in the old south, such as the brief business empire organized by the true reformers fraternal order, emerged precisely at those historical locations and moments when black people brought together a determination to mount entrepreneurship as well as the social capital to push forward that agenda (light, ). this discussion brings us at last to our second major criticism of the suppression hypothesis. the suppression hypothesis overlooks the possibility that, confronting an external and dominant group, well endowed with social capital, a subordinate minority may acquire social capital from that dominant group by bridging into it. in that case, bridging ties would open a flow of business resources from the dominant group to the subordinate group, ultimately enabling the subordinate group to mount an entrepreneurial response on the strength of externally introduced resources. among these imported resources might be the vocational legitimacy of entrepreneurship, a crucial form of cultural capital. that is, if a minority acquires this cultural capital from the dominant group, the minority acquires the desire to mount an entrepreneurial response, which is the indispensable first step toward actually doing it. to illustrate this discussion diagrammatically, figure introduces two schematic diagrams of simple social networks. in panel , the two networks abc and def are independent. they exchange no resources. each depends on its own cultural capital. if abc does not regard entrepreneurship as a valid occupational goal, whereas def do regard it that way, then abc will never acquire the cultural capital of entrepreneurship from def. the chances in entrepreneurship of both networks’ members depend entirely on the resources, including the habitus, their own network already controls. in panel , the discrete networks are bridged by the strong tie of c and e. as a result, a and b and d and f now enjoy potential exchange of resources across network boundaries. if now a and d, a and f, and b and d were also connected, everyone in panel would reach everyone else, and we could no longer speak of bridging between abd and def or even of two distinct networks. in effect, the two distinct networks would have merged into one. in this case, c and e would have lost their unique capabilities for bridging entrepreneurship between the groups, but the two networks would have gained maximum flexibility of resource january, exchange. bridging is clearly of great significance to groups whose tangible and cultural resources of entrepreneurship are weak, and the suppression hypothesis ignores the issue. but how important is this theoretical oversight? the answer depends on how com- mon are bridging relationships in start-up entrepreneurship. if bridging is very common between subordinate and dominant networks, then bridging usually reduces the ability of dominants’ social capital to stifle outsider entrepreneurship. in that case, the suppression hypothesis would rarely succeed. in the diagram’s lower panel, abc would import the entrepreneurship resources, including the cultural capital, that def enjoy. however, in point of fact, even in the developed world’s business system, most start-up entrepre- neurship occurs within social networks, and, therefore, on the basis of the resources the networks already control, not those they import. bridging is empirically unusual in any start-ups anywhere whether those who start up are ethno-racial minorities or majorities, and whether they live in manhattan or on kodiak island. the panel study of entrepre- neurial dynamics showed that even within advanced economies, “small world recruitment is the nearly invariant form of start-ups. . . . almost all start-ups assemble teams based on embedded ties from pre-existing relations within clusters” (aldrich & kim, , p. ). that is, in the business world of today in the advanced countries, almost all start-ups restrict team membership to known and trusted associates without bridging to outsiders. chen ( , p. ) and zain and ng ( ) argue that once firms are underway, their initially small world recruitment basis becomes increasingly diversified and instrumental, but chen acknowledges that start-ups emerge from homogeneous clusters. figure two social network panels panel : independent panel : bridged a d b c e f a d b c === e f entrepreneurship theory and practice the empirical infrequency of bridging relationships in start-up teams supports the suppression hypothesis. returning to figure , panel is much more common than panel in the start-up phase. that said, bridges are sometimes harder to build than in others, and bridging out of indigenous social networks is harder than bridging from one mainstream network to another in lower manhattan. caste systems institutionalize the most extreme restrictions on bridging relationships between the powerless and the powerful (anthonis- sen & blommaert, ). the whole point of caste is precisely to enforce the complete separation of groups through the suppression of bridging relations such as marriage and commensality. indeed, a litmus test for the existence of a caste system is social taboo upon eating with or marrying into people of a proscribed group such as the dalit of india or burakumin of japan. when caste barriers inhibit bridging relationships across groups or networks, members of poor groups cannot access resources of information, skill, and money that rich groups control (briggs, , p. ). if resource-poor, nascent entrepre- neurs cannot access resource-rich social networks, they probably cannot overcome their initial disadvantages (zain & ng, , p. ). old harbor, kodiak island, alaska to illustrate empirically the theoretical discussion thus far, and set the stage for our conclusion, we introduce the entrepreneurs of old harbor, kodiak island, alaska. why old harbor? to understand boundaries of social capital, we felt it useful to venture outside a conventional setting of mainstream society. usually, theories are developed in main- stream society, but we were looking for a location where we could interview people in a context other than mainstream. a conventional location might confirm what we know but with regards to building theory about boundaries of social capital, these boundaries might become more evident in a settlement isolated from conventional settings. several locations were considered, based on a list of remote communities where boundary conditions might become apparent. among these, many had not been the focus of academic research, and we ran the risk of encountering people not open to researchers. however, we recognized old harbor as having been the research site of a famous study by stanford’s professor haruni befu ( ), noting that residents of old harbor had not adopted american values. we contacted professor befu, and he kindly agreed to answer questions. it appeared that people in old harbor would welcome researchers. old harbor was settled by alitiiq (one people among many who are collectively grouped under the umbrella nomenclature, “alaska native”) people over , years ago. in , old harbor was home to mono-racial alaska natives, white, and persons of mixed heritage; given the small population, we set out to interview every entrepreneur willing to be recorded. our research plan followed the mentor model, wherein authoritative local individu- als facilitate the research process. we were first introduced to old harbor entrepreneurs by a well-respected leader who grew up there, professor gordon l. pullar of the depart- ment of alaska native and rural development at the university of alaska, fairbanks. thanks to his introductions, we interviewed alaska natives, non-indigenous alaskans, and people of mixed heritage. participants were selected by means of snowball sampling (goodman, ). . source: http://www.city-data.com/races/races-old-harbor-alaska.html, accessed february , . january, we did not enter old harbor equipped with hypotheses to test. first of all, as von bertalanffy ( ), morgan ( ), and berry ( ) have emphasized, hypotheses are value laden. given the cultural boundary, we were concerned that a hypothetico-deductive methodology might impose our beliefs and values. therefore, we adopted a holistic- inductive design to allow us to be open to whatever would emerge from the data (patton, ), and the theoretical ideas we propose are exactly that. our general objective was first to learn about entrepreneurship in old harbor, and then to theorize it. following eisenhardt ( ; also, glaser & straus, ), we read what was available, and we were opportunistic with regard to data collection methods. we photographed and digitally taped audiovisual recordings. we wrote down all impressions even those that did not seem very important at the time (eisenhardt, p. ), and we overlapped data collection and analysis including field notes. we found, as noted by mintzberg and waters ( , p. ) proposed, that empirical results often served as a stimulus for new thinking, and wide reading enabled us to synthesize literature and field research into a grounded theory. our interviewees included traditional subsistence hunters as well as commercial entrepreneurs and local leaders. respondents were encouraged to discuss each answer to every question in a manner with which they felt most comfortable. in some cases, respondents conferred with others in order to verify facts and context; this effort was encouraged as it provided insights into the attitudes and logic underlying a respondent’s answer, triangulated the information within the group, and also provided background information (bresnen, ). after each interview, we re-evaluated our interview protocol as we were inspired to modify questions and add new ones. the project took place between and with five separate field trips to alaska, and a total of days spent conducting interviews in the field. once all the inter- views were completed, responses to each question were compared. we also made use of triangulation (denzin, ). in total, persons were recorded on videotapes. following yin ( , ), we analyzed the cases, compared results with the litera- ture (eisenhardt, ), and sent our initial write-ups to participants to identify misinter- pretations. we then combined all our material and brain-stormed theory building. a first task was to reconstruct the history of old harbor from documents and interviews. long before free markets developed, indigenous alaskans were making a living off the land and sea. subsistence self-employment was the traditional livelihood in old harbor and across the region, and bonding social capital was central to alutiiqs livelihood. when we explained the concept of social capital, alutiiq respondents quickly understood its eco- nomic value. one respondent jokingly remarked, “even the walruses have social capital” around here. “when we’d harpoon one and try to pull it out of the sea, its mate would intertwine its ivory with our catch and we would pull, and it would pull and we had to be a strong team to beach one.” in alaska’s harsh climate, social capital was and remains essential for economic survival in the traditional economy. alutiiq men rarely hunt alone; they usually hunt in teams, usually of kinsmen, and they share the game (befu, , p. ; mishler & mason, ). however, alutiit (plural of alutiiq) hunt only with men deemed trustworthy in respect to skills, courage, and fidelity. every hunter’s life depends upon membership in a team, all of whose members possess these qualities. incompetent hunters would not return with game, and might themselves be killed by the game they stalked or cause the death of companions. this requirement meant that every alutiiq youth learned the same . the tapes were regrettably destroyed during the christchurch earthquake of . entrepreneurship theory and practice occupational lessons; few grew to maturity without the requisite skills, courage, and fidelity, but any who did could not hunt effectively. the hunters’ social networks excluded the unfit, which the community fed as a charitable duty. this economic strategy worked for many generations. the hunter networks reliably supported the alutiiq people. thanks to this economic arrangement, these indigenous alaskans survived and were content with their economic lot. happiness is not a simple function of money (anielski, ). however, though based in social capital, the kinship-based hunter networks did not transpose into commercial entrepreneurship because the alutiiq people were satisfied with the economic status quo, contentment observed elsewhere in alaska and in other traditional societies (dana & light, ). like the eighteenth century protestants, who preferred not to take chances on out- siders as business partners (lamoreaux, , p. ; light, a) alutiiqs economic conservatism was a defensible business decision. yes, it excluded entrepreneurial innovation; but, exclusion prudently guaranteed the survival of alutiiq society. why take chances with survival? in effect, these indigenous alaskans’ social capital reliably reproduced the society’s economic survival but suppressed economic innovation. the arrival of russian colonists in the nineteenth century, with their institutions, technologies, and market sector changed old harbor forever. nevertheless, in the early twenty-first century, traditional economic customs still remain strong in old harbor. men hunting today learned to hunt with their fathers and kinsmen in the traditional way. born in , paul kahutak (abalik) moved to old harbor when he was “about four.” “we all lived off the land . . . i started fishing when i was only years old.” his first cousin, george inga, sr., born at old harbor in , recalled that his father taught him to hunt, and he emphasized to us that the hunters wasted nothing. every sinew and particle of a slain animal had its ordained purpose. unlike the buffalo economy that, according to benson ( ) lacked environmental awareness, old harbor hunters assured us that their ancestors accepted responsible for stewardship of the natural environment and for the human community that depended on it. in fact, the hunters’ environmental ideas had been elaborated over generations into their animistic religion. missionaries introduced the idea of subjugating nature rather than sustaining nature, but christianity still had not extinguished the ancient beliefs and related spiritual obligations that long governed the indigenous alaskans’ economic theory and practice. possibly for this reason, kin groups still share the fish catch (mishler & mason, , p. ), although commercial catch is sold. traditional subsistence self-employment in fishing and hunting continues to this day, and is an important economic activity of alutiiq men. the division of subsistence of the alaska department of fish and game (fall & walker, ) found that virtually every household was involved in subsistence hunting and/or fishing at least part time; the same study found that old harbor had the largest per capita subsistence harvest of the six samples representing the indigenous alaskan communities across kodiak island. but old harbor is now part of the global economy. traditional fishing and hunting is a declining but still preponderant livelihood in terms of the share of the masculine population involved in it. nonetheless, commercial entrepreneurship exists side-by-side traditional fishing and hunting. the u.s. bureau of the census ( – ) identified self-employed persons in this town of households. we counted business licenses current in old harbor at this time. in the town’s civilian labor force of persons, nine ( . %) were unincorpo- rated self-employed. when we interviewed them, we learned that they identified with a culture other than that of alaska natives. again, we consulted the state of alaska commercial fishing permits held by old harbor residents in . of all the permits issued to individuals in old harbor with rights to fish in the waters surrounding kodiak january, island, all were held by euro-americans or persons of mixed ancestry (alaska commu- nity database, ). alutiiq people from old harbor have other interests than becoming commercial entrepreneurs in the fishing industry, operating independent fishing boats and selling their catch. all alaska native men from old harbor fish, and/or engage in other subsistence activity, but every participant with a commercial fishing license in was euro-american or of mixed ancestry. furthermore, euro-american alaskans and those of mixed ancestry also dominated the town’s new economy of tourism and restaurants. the following are examples. al cratty is a prominent fishing entrepreneur. the son of a bush pilot, cratty is a serial entrepreneur and commercial fisherman who also owns and operates the fishing vessel f/v ashlee christine c. he is also owner–manager of al’s charter service, providing transportation service to hunters and fishermen visiting kodiak island. additionally, cratty and his wife, jonetta, jointly operate a seafood-smoking facility and specialty processing company as well as bed and breakfast accommodations. he also serves the old harbor’s cape barnabas, inc., which is the only community quota entity that has purchased halibut quota shares. a military veteran of mixed ancestry, jeff peterson owns a boat called refuge rock. peterson has a license to operate kodiak combos that escort visitors to fish and hunt on the ocean. peterson’s business won the alaska federation of natives’ “small business of the year” award. peterson’s economic attitudes came from what alaskans call “the lower .” when interviewed, peterson proudly explained to us that he obtained no help from the government, no financing from any bank, nor assistance from any corpo- rations. although the peterson family has lived in old harbor for decades, peterson has swedish ancestors as well as alutiiq. he grew up on his father’s boat and owned his first vessel at the age of . in addition to operating this formal business in the modern sector, peterson provides food for his family from subsistence fishing and hunting; he explained that subsistence resource harvests have a traditional value not measured in dollars. another entrepreneur of mixed heritage and with a business license in old harbor is carl christiansen who owns and operates the ocean view lodge, a hotel. christiansen’s norwegian father was when he married a -year old indigenous woman from nearby eagle harbor, which is no longer inhabited. christiansen’s father was an entrepreneur in barth’s ( , ) sense. he was the first in the area to get an oil stove. carl’s parents adopted four indigenous children in addition to having children of their own, among them carl, who was born during the world war ii. asked whether he was more influenced by the norwegian cultural values of his father or by the indigenous alaskan cultural values of his mother, carl responded, “well, i guess the hard work is from my dad’s side.” carl is russian orthodox, as are many indigenous alaskans, but he told us that his protestant father transmitted the protestant work ethic, teaching as well that “welfare is no goal in life.” when carl received a welfare check in the mail, he recalled, his father demanded that he “take that check back to the post office and mail it back.” carl also told us about his three siblings and his fisherman son all of whom are entrepreneurs. carl explained to us that the price of salmon dropped; now, one even needs a quota to harvest halibut. undaunted, carl diversified his business, building the ocean view lodge for his son, carl jr., who manages the lodge and doubles as a guide. angie christiansen manages the firm, and meagan christiansen cooks for the business. carl opened a delicatessen in . carl’s son and three siblings are all commercial entrepreneurs in ocean fishing. one of carl’s sons is “captain” freddy christiansen, born in old harbor. in , captain freddy invested in sitkalidak lodge (old harbor’s first hotel) and later bought out his original business partner, captain rick berns. freddy then developed an ecotour- ism operation, first with the sitkalidak straits, a vessel that he has since replaced by entrepreneurship theory and practice several larger boats. he named his fishing boat, the tarissa jean c, after his eldest daughter. freddie no longer owns the sitkalidak lodge, which was acquired by outsiders who renamed it the kodiak sportsman’s lodge. ray and stella krumrey have a license for their mountain view guest house. business licenses have also been issued to several stores in old harbor, including: gwendolooks food store, a retail grocery; old harbor food store, selling specialty foods; and the retail grocer tidal wave. others to have business licenses in old harbor include the owners of the old harbor shuttle service and of larionoff’s gas station. if we ask why euro-americans and people of mixed ethnicity pioneered the tourist, hotel, commercial fishing, and restaurant businesses of old harbor, whereas alutiit clustered in other occupations, the answer can be framed in terms of social capital as retardant and as inadvertent suppression. ninety-five percent of old harbor residents identified themselves as mono-racial in heritage (the u.s. bureau of the census, – ). of these persons, % was mono-racial alaska natives, and one quarter was mono-racial euro-american. only % of old harbor residents identified themselves as of mixed ethnicity; e.g., part indigenous and part euro-american. the census estimate agrees with what we learned from the old harbor tribal village council who estimated that of old harbor residents two thirds belonged to the “native village of old harbor,” which is a federally recognized tribe with a total membership of . intermarriage is the key measure of assimilation. peach ( , p. ) declares that, “the united states has had an unparalleled, successful history of assimilating minorities,” but peach acknowledges that assimilation skipped some groups. in the absence of inter- marriage, ethno-racial groups remain culturally distinct. assimilation fails. the centuries- long shortage of multi-ethnic people reflects the continued ethno-cultural distinctness of euro-american and alutiiq people in old harbor. that situation resembles the separation of african american and white social worlds in the old south. in old harbor, euro- americans entered the alutiiq social world from the “lower ” and brought with them resources of money, education, business skills, and social capital that linked them to others in the lower . alutiit lacked the entrepreneurial resources of the euro-americans, except for bonding social capital, which they had in abundance. in effect, migrants monopolized the financial capital, human capital, and resource-connected social capital within old harbor and the bridging ties out of old harbor. the alutiiq hunters and fishermen did not lack bonding social capital; they had it in abundance, but they lacked access to mainstream social and financial capital. to some extent, persons of mixed ethnicity bridged the social capital of alutiit and euro-americans. some multi-ethnic persons became commercial entrepreneurs. however, there simply were few people of mixed ethnicity in old harbor, so bridges to the euro-american social networks were few. in this context, by excluding the alutiit socially, the social networks of the euro-americans implicitly (not malevo- lently) reduced alutiiq access to business ownership. conversely, newcomers lacked the requisite training and skills and, of course, the access to alutiiq social networks. de carolis, litzky, and eddleston ( , p. ) observe that “membership in a network or group can shape an individual’s consciousness” and exactly that appears to have happened to euro-americans and alutiit alike. in effect, the cultural capital of the euro-americans supported their entrepreneurship in the com- mercial sector, but it did not support their entry into subsistence hunting and fishing as a full-time occupation. newcomers wanted to become commercial entrepreneurs, rather than traditional alutiiq hunters and fishermen, while alutiiq people had the opposite aspiration. the cultural boundary thus defined the sphere within which euro-americans’ social capital was of economic value, and it directed them away from the subsistence economy of the indigenous people. january, the same holds true in reverse for the alutiiq people. when they did not value profit-maximizing entrepreneurship in the commercial sector, their abundant social capital did nothing to advance their chances in commercial entrepreneurship even though a diminished supply of ocean fish put some pressure on their ability to sustain the traditional economy (aarstad, haugland, & greve, , pp. – ), and even though % of old harbor families were below the poverty line in . if not commercial entrepreneurship, what did alutiiq people value? first, they valued their traditional way of life within which hunting and fishing in teams was an essential component as was sharing. unlike euro-americans, who viewed commercial entrepre- neurship as a secular business, and who could and did switch from one business to another as occasion demanded, alutiiq people viewed traditional hunting and fishing as the traditional way of life for people like them. changing an occupation and abandoning a way of life are changes of quite discrepant magnitudes. second, alutiiq hunters and gatherers regarded hunting and fishing as essential to a masculine identity. in their view, sanctified by many generations, real men hunt and fish in teams, undergoing danger and hardship in the process. the shared hardship strength- ened their bonding social capital. alutiiq people had reservations about the opportunistic pursuit of money income through business enterprise. this view is shared in many contexts of early economic development now (nida, ) and in the past (smelser, ). “entrepreneurship is for sissies” approximates their view. however, the alutiiq people accepted wage-earning in town as appropriate and acceptable for women. unlike their men, most alutiiq women worked part time or full time in old harbor offices, especially in schools and the numerous public agencies. for this reason, alutiiq house- holds can be understood as splitting their labor power between a traditional subsistence sector, dominated by men, and a modern cash sector, dominated by women. strictly speaking, this is a hybrid, not a traditional economy. empirically, we found that alutiit in old harbor underrepresented in commercial entrepreneurship. we found that these people have strong social capital, but there was no indication of social capital bridging to the euro-american or even to the small multi- ethnic population among whom alone there is commercial entrepreneurship. our results are novel, testable, and empirically reliable (eisenhardt, , p. ). summary our theoretical critique of the social capital literature has turned up conceptual problems that require specification on both sides of the debate. first, the business litera- ture has been content to address the contribution of social capital to entrepreneurship in cultural contexts that support entrepreneurship. this methodological limitation has obscured the supporting role of cultural capital. cultural habitus identifies occupations that are appropriate for group members. if a group’s cultural capital does not support and endorse the selection of entrepreneurship as a vocation, then the group’s strong social capital will not encourage entrepreneurship of group members. that negative contingency typically characterizes indigenous entrepreneurship in north america above and beyond the well-known legal and geographical barriers that also obstruct it. in the specific case of the alutiiq people of alaska, the strong social capital of indigenous people recreates among the men the traditional hunting and fishing economy, while bypassing commercial entrepreneurship. . source: http://factfinder.census.gov/servlet/acssafffacts? accessed february , . entrepreneurship theory and practice second, turning to the suppression hypothesis, we showed that the suppression hypothesis fails in the case of the world’s middleman minorities who have historically succeeded in entrepreneurship despite the vigorous opposition of dominants. that said, the suppression hypothesis works well when applied to ethno-racial and ethno-religious minorities who, like the alutiiq, are not middleman minorities. middleman minorities are exceptional and infrequent. we examined the possibility that bridging relationships with dominants might mitigate the suppression of subordinated minorities. from the literature, we learned that start-up bridging is empirically infrequent even in the business circles of the advanced world, and the obstacle is all the greater when dealing with entrepreneurship in caste or semi-caste societies such as the kodiak island. for this reason, we deem it highly significant that commercial entrepreneurship in old harbor is dominated by euro-americans and the descendants of multi-ethnic couples. the low intermarriage rate between alutiit and euro-americans testifies also to the social conditions that inhibited the creation of dual habitus people (drori et al., ) and therewith inhibited alutiiq entrepreneurship in the commercial sector. conclusion arguing against unbounded faith in the power of social capital to promote entre- preneurship, we concede its power to promote economic activity in general as well as its power to support entrepreneurship among groups already sharing the requisite cultural capital. social capital was essential to the economic life of the alutiiq men, but that economic life consisted of hunting and fishing, not commercial entrepreneurship. insist- ing on the contribution of cultural capital to social capital, we fully credit the vast body of evidence that demonstrates the power of bonding and bridging social capital to encourage entrepreneurship. our point of divergence from the existing consensus derives from starting on a remote alaskan island rather than in new york, london, or paris. we found alaskans abundantly endowed with social capital, and we found alaska natives tradition- ally self-employed in fishing, while commercial entrepreneurship in old harbor was dominated by outsiders and by people of mixed ancestry—who told us that they had origins from norway, russia, or sweden, while alutiit had other occupations of choice. the unusual sharpness of the ethnic separation in old harbor between the indigenous economic sector and the euro-american economic sector compelled us to ask how such a situation could have arisen. social conditions slowed assimilation (see befu, ), and therewith the trans- mission of the cultural capital of entrepreneurship, and the formation of bridging rela- tionships between the euro-american and the alutiiq people. as a result, nascent alutiiq entrepreneurs, if there were any, could not access entrepreneurial networks of the euro-americans. putting the issue that way implies that the social capital of the euro- americans implicitly (not malevolently) excluded the indigenous from entrepreneurship. the other part, equally valid, is that the cultural capital of the alutiiq people did not encourage them to turn their own abundant social capital into individual entrepreneurship, and collective entrepreneurship, often recommended for indigenous americans and canadians (anderson & giberson, , p. ) did not materialize either. instead, alutiiq people used their abundant social capital to catch fish and shoot game, an eco- nomic activity all right, but they did not focus on commercial entrepreneurship. on the strength of these results, we conclude that social capital’s apparent efficacy in promoting entrepreneurship actually depends on a number of understudied and usually ignored boundary conditions that must be in place for the social capital to have that effect. january, those conditions are standard in the developed societies, but they are by no means universal; venturing into a remote outpost in alaska, we found a context in which cultural conditions taken for granted in london, paris, or new york did not exist. as a result, social capital did not have the expected consequences for entrepreneurship on kodiak island that one would expect on the island of manhattan. our corrective to the prevailing view is summarized in table whose box depicts our research result, and whose other boxes synthesize our speculations regarding the general relationship between social capital, cultural capital, and entrepreneurship. on the basis of this alutiiq research, we also suppose that bonding social capital directly facilitates economic action in general but not entrepreneurship in particular. this is probably a universal relationship. however, to facilitate entrepreneurship specifically, social capital requires supportive cultural capital that directs the social capital toward a particular vocational goal, entrepreneurship. every culture does not value entrepreneur- ship, and social capital will not transpose into entrepreneurship where entrepreneurship is not valued. when research is conducted in cultural contexts that support entrepreneur- ship, the supporting role of cultural capital becomes invisible, and researchers wrongly conclude that social capital universally facilitates entrepreneurship. additionally, in contexts of separation between ethno-racial components of the popu- lation, such as existed in old harbor, bridging social capital is even more difficult to develop than in the more fluid social contexts in which research is usually conducted. it is hard there too, but it is even harder in outposts like old harbor. as a result, the existing entrepreneurs’ social networks are inadvertently closed to nascent entrepreneurs from the wrong side of the ethno-racial divide, frustrating their entrepreneurship. our results have implications for a broad range of situations involving entrepreneur- ship or the lack of it among propinquitous ethno-religious or ethno-racial 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( ). the impacts of network relationships on smes’ internationalization process. thunderbird international business review, , – . january, ivan light is a professor at the university of california, los angeles, ca, usa. léo-paul dana is a professor at groupe sup de co montpellier business school mrm, montpellier, france. the authors wish to thank professor gordon l. pullar and dixie masak dayo of the department of alaska native & rural development at the university of alaska fairbanks for assistance in old harbor and for introductions to its people, including alutiiq elders. also to dr. george p. charles, director of the national resource center for american indian, alaska native and native hawaiian elders, university of alaska, anchorage, who assisted us with several interviews, and to dr. sven haakanson, jr. for guidance. a special thank you to professor eric gedajlovic (sfu), professor benson honig (mcmaster university), and professor charlene zietsma (york university) for valuable feedback on an earlier version of this paper. entrepreneurship theory and practice the form of politics: aristotle and plato on friendship review the form of politics: aristotle and plato on friendship john von heyking (ed.) mcgill-queen’s university press, , xv + pp., isbn: - - - - contemporary political theory ( ) , s –s . https://doi.org/ . /s - - - ; published online september contemporary scholars have taken up the gauntlet thrown down at them by c. s. lewis who famously admonished that while ‘[t]o the ancients, friendship seemed the happiest and most fully human of all loves; the crown of life and the school of virtue. the modern world, in comparison, ignores it’ ( , p. ). indeed, lewis would hopefully look favourable upon contemporary attempts to make sense of friendship, whether in history (caine, ), in politics (smith, ), in international relations (koschut and oelsner, ), or in foreign policy analysis (berenskoetter and van hoef, ). while these works debate the essence of friendship, other works focus on the narratives of friendships between political actors (reid-henry, ; meacham, ). in the media as well, friendships between politicians can count upon heightened scrutiny, best illustrated by the attention newspapers give to the joint walks on the beach of french presidents and german chancellors. indeed, chancellor angela merkel and former french president nicolas sarkozy even earned the moniker merkozy in the press (van hoef, , p. ). ancient works on friendships can count upon renewed interest as well, and it is here where john von heyking’s work, based upon an elaborate reading of plato and aristotle, can be placed. because of the renewed interest in friendship, the first critical question must be whether von heyking has anything to add to previous interpretations (such as: smith pangle, ; vlastos, ). because friendship pervaded both greek personal and political culture, it is no wonder that the greeks made friendship the centre of their political theory. despite plato’s prominence, most works focusing on friendship draw upon aristotle’s three conceptualizations of friendship, rather than those of plato. friendship is addressed in several of plato’s works, most prominently in his symposium, lysis, and also in his phaedrus. plato’s account of friendship has been criticized for being cold-hearted and egocentric, most prominently by gregory vlastos who concluded that plato was not interested in other individuals for their own sake, but for his own needs and desires (a spiritual � macmillan publishers ltd. - contemporary political theory vol. , s , s –s www.palgrave.com/journals growth towards the forms) (vlastos, ). like frisbee c. c. sheffield, von heyking draws upon plato to provide a complete picture of political friendship, which is possible because plato, in contrast to aristotle, does not offer an account where friendship focuses solely on the other as an individual (sheffield, , p. ). von heyking’s work serves as a great introduction to aristotle and plato’s views on friendship, further cementing their seminal demarcations as the foundations upon which our conceptualizations of friendship must rest. to new students of friendship, the form of politics is a great introductory work to plato and aristotle, aided considerably by the fact that von heyking does not assume any prior knowledge of the reader, and therefore also consistently offers both the original greek and the english translations of important concepts side by side. students are either introduced or reacquainted with terms such as virtue–friendship (sunaisthe- sis: the joint perception of the good), utility, and pleasure–friendship. to the contemporary debate on friendship, von heyking offers a very important reconceptualization by bringing our attention to an oft-overlooked aspect of the ancient concept of political friendship: that of festivity. von heyking does this by strongly differentiating virtue–friendship from political friendship; the friendship of the city, of the community, and showing how the ideal polis debated by the athenian stranger in plato’s laws must culminate in the shared experience of festivity. the heart of the work, and its most praiseworthy ambition, lies in not only drawing our attention to the role of festivity, but also in trying to acquaint us with something that is so very far removed from our contemporary understanding, because: ‘‘[m]odern life offers few opportunities for such experiences of ‘withness’ in song and dance. we veer between the crowd-forming (rather than society- forming) ecstasies of popular music, and the rationalist contempt of jane austen’s mr. darcy, who refuses to join elizabeth bennett and her circle in dancing’’ (pp. – ). it is here that i have a minor gripe with the work. for although a number of illustrative examples of festivity are mentioned, such as alexandr onishenko’s impressionistic painting ‘‘jewish dance’’ and the maori haka dance (p. ), the book does not contain illustrations of these experiences that come close to festivity. the beautiful, but very safe cover, offers the familiar school of athens with plato and aristotle singled out. while an image would never convey the entirety and complexity of what a city or a political community engaging in festivity would entail, an illustration that comes partway is sorely lacking. this is noticeable too in the final chapter where the calgary stampede is dealt with in-depth as an illustration of a political community engaging in festivity. one or two striking images of festivity would have further strengthened the author’s insight. to an extent, the same goes for von heyking’s easy dismissal of robin dunbar in comparison with aristotle because the latter, ‘‘does not derive an algorithm to determine the optimal number of friends’’ (p. ). it seems to me that some of the review � macmillan publishers ltd. - contemporary political theory vol. , s , s –s s insights of dunbar’s extensive and ongoing quantitative analysis of friendship would complement, indeed even strengthen some of von heyking’s findings. for instance, dunbar’s research posits that humans bond through gossip: is there not some overlap with von heyking’s citizen’s that share in festivity? (sutcliffe et al., , p. ). does dunbar’s number (of average friends that a human can have) hold any further significance? it might, because this size of around : has been documented in a wide range of social, cultural, and historical circumstances, representing both top-down (community level) and bottom-up (egocentric personal network) perspectives […] examples include commu- nity size in traditional hunter–gatherers, average countywide village sizes in both the domesday book and eighteenth century england, company size in historical and modern armies, and parish sizes among the amish and huterites (sutcliffe et al., , p. ). it might very well be that the average of a close personal relationships does not only limit human relationships, but also restricts the amount of people that can participate in festivity, which then has important ramifications for this newly discovered concept. von heyking’s festivity begs to be further developed in debate with some of these other fundamental findings in the study of friendship. in sum, von heyking offers scholars a great introduction to plato and aristotle’s insights on political friendship, aptly differentiating between virtue–friendship on the one hand, and political friendship on the other. the most intriguing aspect of von heyking’s work is the idea of how the political community comes together and strengthens itself through festivity, an overlooked aspect of political friendship the author brilliantly brings back to the fore. the work is filled with both historical and contemporary examples of both forms of friendship, further adding to the book’s value as a reference work. if there is a weakness to be found, it is that some friendship scholars and their relevant insights should have been further called upon which, would have strengthened von heyking’s findings. that being said, there is no doubt that c. s. lewis too would look favourable upon the form of politics, and perhaps it even would have convinced him to add festivity as the fifth love. references berenskoetter, f. and van hoef, y. ( ) friendship and foreign policy, july. doi: . /acrefore/ . . . caine, b. (ed.) ( ) friendship: a history. london: routledge. koschut, s. and oelsner, a. (eds.) ( ) friendship and international relations. basingstoke: palgrave macmillan. lewis, c.s. ( ) the four loves. new york: harcourt, brace. meacham, j. ( ) franklin and winston: an intimate portrait of an epic friendship, st edn. new york: random house. review s � macmillan publishers ltd. - contemporary political theory vol. , s , s –s http://dx.doi.org/ . /acrefore/ . . http://dx.doi.org/ . /acrefore/ . . reid-henry, s. ( ) fidel and che: a revolutionary friendship, st u.s. edn. new york: walker and co. sheffield, f.c.c. ( ) viii—beyond eros: friendship in the phaedrus. proceedings of the aristotelian society ( ): – . doi: . /j. - . . .x. smith, g.m. ( ) friendship and the political: kierkegaard, nietzsche, schmitt. exeter: imprint academic. smith pangle, l. ( ) aristotle and the philosophy of friendship. paperback re-issue, digitally printed version. cambridge: cambridge university press. sutcliffe, a., dunbar, r., binder, j. and arrow, h. ( ) relationships and the social brain: integrating psychological and evolutionary perspectives. british journal of psychology ( ): – . doi: . /j. - . . .x. van hoef, y. ( ) friendship in world politics: assessing the personal relationships between kohl and mitterrand, and bush and gorbachev. amity: the journal of friendship studies ( ): – . vlastos, g. ( ) the individual as an object of love in plato. in: g. fine (ed.) plato . ethics, politics, religion, and the soul. oxford readings in philosophy. oxford: oxford university press, pp. – . yuri van hoef utrecht university, bs utrecht, netherlands y.vanhoef@uu.nl review � macmillan publishers ltd. - contemporary political theory vol. , s , s –s s http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /j. - . . .x 	the form of politics: aristotle and plato on friendship 	john von heyking (ed.) mcgill-queen’s university press, , xv + pp., isbn: - - - - 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ mh _ .. literature, but the last (hardy and tansey) is particularly welcome, constituting as it does one of the first serious attempts at a comprehensive historical survey of the development of medicine and medical science after the second world war. the authors have a huge task, for western medicine, in their period, became a global rather than european and american project. they also range very widely in subject matter, exploring scientific, social and economic issues. their account of the rise of the pharmaceutical industry is particularly useful. overall it might, however, be said that this section is the least well-organized thematically. this is not, i hasten to add, the authors’ fault— they have more ground to cover and they lack the benefit of the longer and deeper historiographical perspectives enjoyed by the other contributors. having said that, there is the occasional instance of repetition that a more careful editing might have eliminated. there can be no doubt that the second volume of the western medical tradition will be an essential addition to the reading list of every honours and master’s course in the history of medicine. the book is handsomely produced and also very reasonably priced, at least for the paperback edition, given the word count. the prospective reader may, however, be warned that it is not as entertaining a read as the volumes in the cambridge series nor, indeed, as its older companion textbook. this is partly because historians have, in the meantime, increasingly turned away from the sweeping grand narratives that gave the earlier texts, especially the cambridge ones, such rhetorical force. it is also because the very comprehensiveness of the western medical tradition to —its determination to cover all the major countries of europe, as well as north america—sometimes gets in the way of narrative clarity. overall, however, that is a price worth paying for what is a genuinely impressive scholarly and pedagogic achievement. malcolm nicolson, university of glasgow susan lindee, moments of truth in genetic medicine, baltimore, johns hopkins university press, , pp. xii, , £ . , $ . (hardback - - - ). historians of medical genetics have long been preoccupied with the field’s relationship to eugenics. that focus is certainly understandable given the manifest institutional, personal, and ideological entanglements of ‘‘reform eugenics’’ with medical genetics during the s and s, as well as continuing controversies concerning the eugenic content of such medical-genetic technologies as prenatal and pre-implantation genetic diagnosis. lindee notes that research in the field has resulted in more diagnostic tests than it has effective treatments for disease and indeed claims that selective abortion following prenatal diagnosis remains the ‘‘primary intervention’’ associated with genetic medicine (p. ). thus even she can not entirely escape the eugenics issue. nevertheless, the focus of her welcome new book is on aspects of the history of the field that have received much less attention from scholars, such as the central roles played by patient and parent advocacy groups in setting research agendas, financing studies, and providing critical information. moments of truth is not a systematic history of genetic medicine but an analysis of five key developments occurring between and —two decades during which human genetics was transformed from an institutional and intellectual backwater into a research frontier. each case study explores a different facet of the field. thus the routinization of newborn testing for phenylketonuria (pku) following the development of a blood test suitable for hospital-based mass screening is used to investigate the rise of public health genetics, and victor mckusick’s studies of the old order amish, the construction of human pedigrees and rise of mapping studies more generally. similarly, early research on human chromosomes is used to elucidate how standardization has transformed concepts and practices in genetic medicine, the development of the ‘‘twin method’’, a variety of issues in the book reviews https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core rise of human behaviour genetics, and the history of research on familial dysautonomia, the role of social organization and technology in both creating and eliminating a genetic disease. organizing the book around five quite disparate cases could have resulted in something of a hodgepodge. however, the studies are linked by several themes. thus running through the discussion of each case are reflections on the question of how nearly all disease came to be understood as genetic disease. lindee explores how this idea became crystallized during the s and s in texts, scientific and clinical practice, and public policy, and she considers what it meant and continues to mean for researchers, patients, and the public at large. in general, i found her arguments convincing, but i have a small quibble with the effort to fit the newborn screening case into this periodization. in the s and s, as lindee herself notes, pku was generally characterized as a treatable form of mental retardation, with genetics barely figuring in legislative and other debates surrounding screening, nor were many geneticists initially enthusiastic about efforts to mandate the test. it was only in the s that pku came to be commonly viewed as a success of genetic medicine, a reframing that in my view followed and served to validate the trend described in this book (a trend encapsulated by abby lippmann’s term ‘‘geneticization’’). a second theme uniting the individual cases concerns the varied types of work and workers involved in medical-genetic research. thus lindee argues that the production of scientific knowledge is a community project involving not just researchers, but also research subjects, patients and their families. she emphasizes that non-scientists have often functioned as active research collaborators, as crucial sources of knowledge and funds, and sometimes as validators of researchers’ claims. thus, in her account, scientific authority is more dispersed than it is often assumed to be and forms of labour not usually characterized as ‘‘scientific research’’ are shown to be integral to the enterprise and made visible. the resulting insight into the structure and organization of contemporary biomedicine is one of the chief contributions of this original and important new book. diane paul, umass boston stanley finger, doctor franklin's medicine, philadelphia, university of pennsylvania press, , pp. xiii, , illus., £ . , $ . (hardback - - - - - ). benjamin franklin and medicine was an excellent idea for a book, and stanley finger has executed the project admirably. as patient, advisor, author, publisher, inventor, and inquisitor, medicine permeated franklin’s life. franklin was a quintessential enlightenment figure, an optimist who considered that medicine (along with printing, the study of electricity, and the designing of spectacles and stoves) was among the best practical pursuits through which the human lot might be improved. franklin took on new medical interests throughout life thus making him a perfect subject for the historian. franklin’s changing concerns mirror innovations in eighteenth-century medicine and allow finger to tell a chronological tale weaving together medical developments and biography in a most unforced fashion. in the young printer in philadelphia published poor richard, an almanac full of maxims and medical advice. he promoted smallpox inoculation with characteristic relish and good sense—that is he never seemed to let any medical novelty blind him to its deficiencies. he experimented with electricity as a cure for palsies and his natural scepticism prevented his embracing it with the sort of enthusiasm that fired john wesley. he studied lead poisoning and music therapy. he conducted an investigation into mesmerism which he viewed with the same steely doubt that marked all he did. in his old age he suffered from gout and experimented unsuccessfully with cures for it. book reviews https://www.cambridge.org/core/terms. https://doi.org/ . /s x downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s x https://www.cambridge.org/core t:\t&f\us\wttm\wttm ( )\finals\wttm_a_ .dvi journal of travel & tourism marketing, : – , copyright © taylor & francis group, llc issn: - print / - online doi: . / . . a visitor-focused assessment of new product launch: the case of quilt gardens toursm in northern indiana’s amish country geunhee lee iis p. tussyadiah florian zach abstract. understanding the needs and wants of consumers in the process of new product devel- opment has been recognized as an essential aspect of preparing effective marketing strategies for the success of business. the new product development strategy has now moved into consumer-driven innovation (cdi), which not only asks consumers about their needs and wants but actually involves them in the product design, promotion, and even assessment processes. informed by the new concept of cdi, this study aims at identifying to what extent visitors as tourism product consumers and co-pro- ducers can be involved in a new product development process and reinvent the products by providing ideas and suggestions with their own creative insights. more specifically, using data collected from a trip diary and an online survey with respondents, this article examines visitor assessment on a newly launched product, quilt gardens toursm, in northern indiana’s amish country. the data were analyzed using geo-visualization of tourist spatiotemporal mobility, descriptive statistics, and qualita- tive analysis of visitors’ descriptions. the results show that the visitors are central role players in a new product development process, adding their creativity to the tour itinerary and design elements. several lessons and significance for future development of the tour are provided. keywords. consumer-driven innovation (cdi), new product development, visitor-focused assessment introduction in the recent highly competitive marketplace, new product development has been proven to be one of the vital factors to bring the growth and prosperity to most product and service providers (danneels, ; zirger & maidique, ). it is also emphasized that the new product development should be both timely geunhee lee (e-mail: ghlee@temple.edu), iis p. tussyadiah (e-mail: iist@temple.edu), and florian zach (e-mail: fzach@temple.edu) are affiliated with the school of tourism & hospitality management at temple university, north broad street, philadelphia, pa , usa. address correspondence to: geunhee lee at the above address. and responsive to consumer needs and wants (gruner & homburg, ; olson, walker, & ruekert, ) because it is consumers who judge the ultimate success of the new products and services (cooper & kleinschmidt, ; brown & eisenhardt, ). both academics and practitioners in market- ing have put a greater importance in under- standing consumers’ needs to provide effective journal of travel & tourism marketing and appropriate marketing strategies. howard ( ) argued that successful firms are more likely to be consumer-oriented than unsuc- cessful firms, thus marketing should serve as the basis for strategy decisions. furthermore, gruner and homburg ( ) pointed out that information on consumer needs and user expe- riences are viewed as resources companies depend upon to successfully develop new prod- ucts. consumers also have often been found to be the initial developers of what later became commercially important products and services (schreier & prügl, ). thus, it is stressed that companies should gain deeper understand- ing of the “voices of the consumers” in order to make their new product development success- ful. consequently, consumer research can be a useful tool to obtain consumers’ voices and very helpful to raise the odds of success in the market (van kleef, van trijp, & luning, ). in fact, it has been believed that consumer research could be conducted during any of the four stages of the new product develop- ment process: (a) opportunity identification, (b) development, (c) testing, and (d) launch (suh, ; urban and hauser, ). however, there are the often-heard arguments that asking con- sumers what they want and getting the “right answer” from them in the early stage of the new product development is hard because their solutions are often vague and they do not know what they really want without any experience of something that does not really exist (ulwick, ; von hippel, , ). therefore, consumers’ voices are now most widely and intensely adopted in the stages of development, testing, and launch (van kleef et al., ). in spite of increasing business literature on consumer research to gain their innova- tive insights in the new product development process, research about consumers’ impact on tourism products and services is still scant. thus, the goal of this study is to examine the extent to which visitors as tourism product consumers and co-producers can provide their creative insights in the new product develop- ment process, especially in the launch stage. this study shows what can be understood from the observation of the spatiotemporal movement patterns of visitors, how visitors evaluated the new tourism product, and the kind of improve- ment aspects visitors can indicate. theoretical background consumer-driven innovation given recent industry dynamics, the impor- tance of new product development has been magnificently emphasized for its ability to gain competitiveness, growth, and survival of orga- nizations (buxton, ; byrd & brown, ; cooper & kleinschmidt, ; leonard-barton & sinha, ). however, since new product development is proven to be a complex and dif- ficult task, thus easily plagued by high risks of failure (carlile, ; cooper & kleinschmidt), many researchers have tried to identify the fac- tors resulting in success or failure of the new product development process. after realizing that the old, sequential approach to developing new products is not sufficient for the success of business; consumers, of all the factors reported, have gathered an extensive attention as one of the important role players in the new prod- uct development process (cooper, ; harari, ; matthing, sanden, & edvardsson, ; takeuchi & nonaka, ). market managers are adopting market-driven strategies guided by the logic that all business strategy deci- sions should start with a clear understanding of consumers (cravens, ). the underlying premise of market-driven strategy is that the consumers that form the market should be the starting point in business strategy formulation. in the era of mass production, the main role player in new product development was product and service providers (i.e., manufactures, com- panies, and organizations). they were obsessed with making a brand new thing, and the creation from the obsession of making something totally unique is called invention. according to kotler ( ) and kotha ( ), however, current mar- ket segmentation have progressed into the era of mass customization as the substitute for dead mass production as consumers are emerging to be the new main role player in the new product development process. when invention is fully customized and gets accepted by a market, it can lee, tussyadiah, and zach be reproduced into an innovation. it character- izes the contemporary era of mass customiza- tion, and has been considered a newly focused tool that effectively drives organizational suc- cess. innovation is also heavily emphasized for its capability of providing competitive advan- tage for organizations. for example, byrd and brown ( ) describe innovation as an avenue that helps organizations grow and assert that the lack of innovation can stifle companies. buxton ( ) also advises that organizations might bet- ter strive for innovation with a thorough under- standing of consumer needs and their creative insights rather than focusing on the invention of the “brand new.” rothwell et al. ( ) emphasize the impor- tance of concentration on consumer needs for the success of innovation. they state that user needs must be precisely determined and met, and it is important that these needs are mon- itored throughout the course of the innova- tion since they very rarely remain completely static. many successful firms achieve this deep and imaginative understanding of user needs through the interaction with a representative sample of potential consumers throughout the development. cooper and kleinschmidt ( ) also indicate detailed market research as one of the success factors for new product develop- ment: consumer studies to pin down the exact consumer needs, requirements, and benefits by using techniques such as focus groups and con- sumer surveys. urban and von hippel ( ) suggest an accurate market research to under- stand the exact consumer needs and emphasize that such understanding is clearly an essential input to the new product development process. indeed, consumers have been shown to be the actual developers of most of the suc- cessful innovations and they also can con- tribute insights regarding solutions responsive to their needs (urban & von hippel, ). this explains why companies need to change themselves into market-oriented system in order to survive today’s fiercely competitive world. therefore, from merely asking consumers what they want and need, innovation strategy has moved into involving them in the process as the actual role players, suggesting that innova- tion should be driven by consumers’ insights (de marez & verleye, ; van kleef et al., ). according to toffler ( ), with his popular concept of “prosumerism” (i.e., a mix of both producers and consumers), consumers are increasingly participating in the process of conception, design, launch, and promotion of new products and services. this process is often called consumer-driven innovation (cdi, henceforth; harris, ; otsuka, ) and has been focused as the essential aspect for the suc- cess of business. cdi can be more effective and efficient for communication with consumers because organization strategy focuses on “by consumers” rather than “for consumers.” cdi mainly emphasizes on consumer involvement in the product development process while the cor- porate posture prior to cdi was just “to go out to consumers” (overby, ). in the modern organization, cdi typically includes consumer interaction at the early stage of new prod- uct development and gains consumers’ feed- back soon after the product launch (gruner & homburg, ). the ability of organizations to collect and then effectively exploit consumer information about demand and preferences is becoming a key aspect of competitive advantage (cox & mowatt, ). competitive advantage is also driven by constant innovation satisfying the quickly changing consumer fashions, trends, tastes, and patterns of demand (mowatt, ). for example, the intensity of consumer interac- tion is found to be a positive impact on the inno- vative product development process for keeping them on top of market competition (gruner & homburg). therefore, the roles that consumers play in the new product development process have been highly emphasized, especially in the recent market circumstances which emphasize the innovative ideas and products for the success of businesses (dahlsten, ; matthing et al., ). consumer-driven innovation in tourism despite being a popular topic in business lit- erature, there is very little discussion on cdi in tourism development. however, cdi deserves more attention in the tourism setting than in others because the market is getting extremely competitive across the world and tourist demand journal of travel & tourism marketing is rapidly changing due to the tremendous avail- ability of information. for a destination to meet the tourists’ quickly changing needs and wants and, therefore, to achieve the competitive advan- tage among others, the role of cdi through market-driven strategy in the tourism settings must be more explored than it has been. within the field of tourism, being market-driven can be translated as focusing on visitors’ needs and wants and obtaining visitors feedback to con- tinuously provide valuable tourism experience. particularly, as destinations are becoming more and more creative with the product offerings, it is of a paramount importance to understand the creative consumption of these new offerings and to gain visitors’ insights for a continuous co-creation of better products. as destination marketers face new chal- lenges from the rapid process of change in the field of tourism, developing new, innova- tive offerings can be an effective solution to create new markets and reinvent the old ones. in tourism literature, new product development is often discussed within the theme of cre- ativity. from their analysis on recent devel- opment in culture and tourism, richards and wilson ( ) use the terms “creative pro- duction,” referring to the innovative strategies of generating creative spaces within the des- tination; and “creative consumption,” referring to the new, different ways of experiencing tourism destinations. tourists are increasingly more creative and informed; they base their travel styles on values such as personal authen- ticity, altruism, whole process learning, and self- actualization (ray & anderson, ). these new tourists are creative individuals who are willing not just to accumulate tourism expe- riences but also to change them. the blurred boundary between tourism production and con- sumption often cause creative tourists to be co-producers of their own experiences, making them a part of the innovative process of creating new tourism products. thus, cdi is also consid- ered as an emerging chance to obtain and sustain a destination’s competitive advantage whereby destination marketing organizations (dmos) try to develop a valuable and engaging relationship with the tourists by giving them a central role in the innovation process. shaw, agarwal, and bull ( ) state that understanding tourism consumption and tourist experience is important to obtain their cre- ative consumption ideas, but these topics are somewhat neglected in tourism research because capturing the consumption and expe- rience of tourists is viewed as not an easy task. as such, tourism consumption and tourist experience have been operationally defined in many different ways. in an attempt to exam- ine tourist consumption and experience, some studies try to observe the actual movement patterns of tourists. haldrup ( ) asserts that research about tourists’ spatiotemporal movement is scant, in part, because tourist movement is so fundamentally obvious that its form and practice are taken for granted and often overlooked. however, lew and mckercher ( ) emphasize understanding tourists’ movements within a destination as the basic nature of their experience has impor- tant practical applications for destination man- agement, product development, and attraction marketing. based on what is observed in their move- ments, researchers try to model tourists’ move- ment and consumption patterns and draw their implications for product development (xia & arrowsmith, ). of the scant research deal- ing with tourists’ movements, some studies report tourists’ spatial implications of variations in attraction site visits (debbage, ; fennell, ; tideswell & faulkner, ). for exam- ple, debbage found that some tourists chose to follow the advised travel courses for much of their stay even though they stayed at the des- tinations for several days, while a substantial group of tourists seemed to be very adventurous and more likely to make their own travel routes. from the results, she concluded that tourists’ travel behavior or pattern were heterogeneous. other studies attempted to capture tourism con- sumption by interpreting tourist movement pat- terns (cooper, ; tussyadiah & fesenmaier, ; xia, ciesielski, & arrowsmith, ). from the spatiotemporal observation used in his study, cooper concludes that the tourists are prone to reduce uncertainty in their explo- ration of an area by visiting sites that are per- ceived to give the greatest reward for their effort. lee, tussyadiah, and zach he also points out that the number and the timing of tourist visits to each site are depen- dent upon the level of facility provision at that site and, thus, upon the position of that site on the hierarchy. this confirms that, early in the travel, tourists tend to visit sites which are perceived to give high recreational place utility. the tourists’ strategies continually min- imize the risk of disappointment at the expense of effort. based on the previous studies, it is argued that capturing tourists’ movements and descrip- tion of their experiences, including their com- ments and suggestions, can be considered as an effective approach to gain tourists’ feedback to assess a tour or other tourism programs, espe- cially in the process of new product develop- ment. therefore, this study attempts to provide a discussion on the importance of cdi in tourism settings through a case study. it examines how visitors experience a newly launched tourism product and, particularly, to what extent they add their creativity as co-producers of their own tourism experience through the analysis of their spatiotemporal movements and creative sugges- tions. this study also conceptualizes the insights provided by visitors for redesigning various aspects of the tour program to draw manage- rial implications for future development of the product. research context this article focuses on quilt gardens toursm, which is a new, innovative tourism product offered in northern indiana’s amish country. it launched in and engaged consumers (i.e., visitors in quilt gardens toursm) in the process of product development by asking them to participate in the surveys to collect their opinions while experiencing the product right after the product launch. the tour features quilt-themed gardens and hand-painted out- door murals, some of which are located at the same visit points, making a total of visit points. the unique gardens and murals are cre- ated based on patterns of traditional amish and contemporary quilts, offering both visitors and residents to experience a colorful and creative connection to the history and heritage of amish country. the tour resulted from a partnership between elkhart county convention & visitors bureau (eccvb) and local businesses and was designed to reinforce an existing product called heritage trailsm, a -mile trail connecting his- toric and scenic parts of amish country. the quilt gardens blossom from late may until first frost of , making this the time period for the tour. the tour comes with a marketer-suggested itinerary following the heritage trailsm. as an attempt to make the visitors follow the tour, eccvb provides an official tour map of the gar- dens and murals based on their order along the trail (see figure ). self-guiding quilt gardens toursm cds and cassette tapes are also provided to accompany visitors throughout the tour. the maps, cds, and cassette tapes can be obtained from the elkhart country visitor center mural (visit point no. ) or other stops along the trail. study method this article focuses on consumers’ feed- back for a new product at the introduction stage of product life cycle, using two differ- ent approaches of data collection: diaries and online survey. in order to fully understand how visitors experience the quilt gardens toursm, visitors were invited to participate in the study by filling out “quilt gardens toursm diary” as the first survey instrument during their trip. it was designed to accomplish two important goals: tracking visitors’ spatiotemporal move- ments and soliciting problem-solving design suggestions. the measures and scales for the diary were developed based on previous stud- ies focusing on visitor movements and expe- riences (see tussyadiah & fesenmaier, ; tussyadiah, fesenmaier, & yoo, ). to cap- ture the detailed information on various aspects of the visitor experience including the sequence of movement, the diary was designed to include three sections. part asks visitors to identify and evalu- ate at least gardens/murals according to the sequence of their visitations, consisting of journal of travel & tourism marketing figure . quilt gardens toursm map. visitors center mural ruthmere museum time was museum downtown elkhart commons linton’s enchanted gardens elkhart historical museum krider gardens varns & hoover mural das dutchman essenhaus menno-hof garden old bag factorymural old bag factorygarden ec -h fairgrounds goshen college mural mccormick creek downtown nappanee amish acres downtown wakarusa key bank mural american farmers market quilt mural heritage trail route quilt garden pages for each location. it starts with the name of gardens/murals and the time of their arrival, followed by the reason(s) of visiting the gardens and/or murals, their sensory expe- riences (i.e., color, sight, sound, smell, tex- ture, and taste) and emotional experiences (e.g., happy, sad, afraid, and calm) with the gardens and/or murals (in a -point likert scale rang- ing from for strongly disagree to for strongly agree), and ended with duration of stay at each specific location. this information was used to describe visitors’ spatiotemporal movements within the tour and to evaluate each individual garden/mural. in part , visitors were asked to list the three most and least interesting gardens/murals and the reasons for these selections. part also includes the future intention to participate in various activities related to the tour experience, consisting of seven measures with a -point likert scale ranging from for strongly dis- agree to for strongly agree. this information was used to evaluate the entire concept of the tour, to improve the quality of the individual venues within the tour, and to inquire of vis- itors’ involvement intention. the information from part was used to understand the market of this tour. the second approach of data collection was an online survey. it was targeted for tourists who already finished their trips and was designed to capture more general data on evaluations and suggestions on the overall tour (i.e., the visited gardens/murals, the three most and least inter- esting gardens/murals, the gardens/murals to visit or revisit in the future, and possible future engagement with the tour). both diaries and online survey were con- ducted from june to october . participants were solicited by eccvb through their website and by direct solicitation at the visitor center and point of interests throughout the area. the diary survey resulted in completed diaries with a total of garden/mural visitations while the online survey resulted in completed responses representing garden/mural visi- tations. all participants entered into a drawing to win a $ gift card, a $ gift card, or one of six $ gift cards. in addition, partic- ipants who visited at least gardens/murals and returned the completed diary received a free gift at the designated drop-off locations. the analysis was conducted using geo-visualization of tourist spatiotemporal mobility, descriptive statistics, and qualitative analysis of visitors’ descriptions. lee, tussyadiah, and zach results spatial sequences visitors’ movements throughout the tour were first mapped by the noted connections between the respective gardens. the results show various “subtours” created by the visi- tors. only . % of the visitors chose visitor center mural (visit point no. ) as the first point to start the tour; other visitors started at the first gardens they found upon entering the area. visitors who started the tour with visitor center mural stated that it was because of these reasons: “it was the first mural found upon entering the area” ( . %), “it was the closest to the accommodation” ( . %), “it was recom- mended by others” ( . %), and other reasons ( . %), including “wanted to start the tour with getting the information from the visitor center,” and “the starting point of the heritage trailsm.” interestingly, only a few of the visitors fol- lowed the trail as advertised/promoted in the brochures ( %). most visitors ( %) created their own itineraries. as presented in figure , barbara, who started her travel at the visitor center mural as suggested, was following the heritage trailsm until the fourth garden. however, she chose visit point no. as her next stop, toured the suggested route backward, and finally finished her tour at visit point no. . the routes she used when traveling to visit points no. and no. , which are illustrated with dotted arrows in figure , were not even on the heritage trailsm. the reason she stated for taking the different paths was “closeness to route taken”. in another case, darla started the tour at visit point no. and moved along different routes, which are also illustrated with dotted arrows, until she reached a part of the trail on the fifth stop. she finished her tour mov- ing along the advised routes backward. other specific reasons mentioned by the visitors who created their own routes instead of the suggested one are “[we] saw it as we drove by,” “[it’s] close to lodging,” and “[it’s convenience for] lunch time and shopping.” it appears that visitors’ movements from one garden/mural to another were driven mainly by “convenience, close proximity to the previous gardens/murals” ( . %). other rea- sons ( . %) included “following the trail/tour map,” “following the cassette tape/cd,” “close to other attractions to visit,” and “saw it while driving by.” only a few visitors stated that the reasons to visit a particular garden/mural after the previous ones are because “it was recom- mended at the previous garden/mural” ( . %), “it has similar plants as the previous garden” ( . %), “it has similar design patterns as the previous garden/mural” ( . %). these reasons indicate that while the movements of a few visitors were driven by the marketer’s instruc- tional material (e.g., map, trail, cd), most of the visitors moved along the area according to their perceived convenience and the proximity between a garden/mural and the others (see figure ). they, in the results, also suggest that market-advised routes are still perceived incon- venient or less convenient by the visitors to the destination. temporal sequences figure represents the temporal sequences of visitations. most respondents started the tour in the morning before : p.m. ( % of the first stops and % of the second stops). however, from the rd to the th stops, most of the vis- its occurred after : p.m. (about %). also, most of the overall gardens/murals visitations ( %) happened in the afternoon between : p.m. and : p.m., from short before noon until short before dusk. only a few recorded visits actually happened after : p.m. ( %). this is due to the nature of the attractions; the color- ful gardens and murals are best viewed with a sufficient amount of light. most visitors toured the quilt gardens/murals for day ( %) or days ( %). the shortest visit for a -day tour was for . hours spent in gardens/murals; the visitor was only driving by to see the quilt murals and spent to min- utes at the quilt gardens. the longest -day tour was for . hours, spent in gardens/murals; the visitor spent from to minutes at the quilt murals and to minutes at the quilt gardens. visitors stayed at each garden/mural from (i.e., just driving through) to minutes journal of travel & tourism marketing figure . marketer suggested tour and examples of generic visitors’ tours. figure . reasons of visit from one garden/mural to another. ( . hours); with an average length of stay of . minutes. visitor evaluations and involvement the participants were asked to evaluate each of the gardens and murals and the tour in gen- eral. table shows the evaluation scores for respective gardens and murals collected from the visitors. the scores range from for strongly disagree to for strongly agree. from the results, one can see that most of the visit points have similar scores on each evaluation category except three visit points: (a) varns & hoover mural (visit point no. ) has lowest scores on “crowded,” “noisy,” and “nice atmosphere”; (b) goshen college mural (visit point no. ) has lowest scores on “easy to find,” “attrac- tive,” “visually pleasing,” and “worth to time spent”; and (c) das dutchman essenhaus (visit lee, tussyadiah, and zach figure . aggregate temporal sequences of visitations (in number of visits). table . evaluation scores for each garden and mural (with for strongly disagree and for strongly agree) point name of easy to visually worth the nice number garden/mural find attractive pleasing crowded noisy time spent atmosphere visitors center mural . . . . . . . ruthmere museum . . . . . . . time was museum . . . . . . . mowntown elkhart commons . . . . . . . linton’s enchanted gardens . . . . . . . elkhart historical museum . . . . . . . krider gardens . . . . . . . varns & hoover mural . . . . . . . das dutchman essenhaus . . . . . . . menno-hof garden . . . . . . . old bag factory mural . . . . . . . old bag factory garden . . . . . . . ec -h fairgrounds . . . . . . . goshen college mural . . . . . . . mccormick creek . . . . . . . downtown nappanee . . . . . . . amish acres . . . . . . . downtown wakarusa . . . . . . . key bank mural . . . . . . . american farmers market . . . . . . . overall value . . . . . . . point no. ) has highest scores on “attractive,” “visually pleasing,” and “nice atmosphere.” it indicates that even though most of visit points are estimated equally by visitors, some of them have extremely high and low scores on partic- ular evaluation categories as compared to the others. also, the results show that their overall values on each evaluation category are mod- erately well-estimated (above . on positive aspects and below . on negative aspects). as a part of the evaluation process, visitors were asked to provide descriptions and comments journal of travel & tourism marketing about their experience with the tour. the com- ments collected from visitors provide thoughtful insights about their experiences, the gaps between their expectation and the provided tour, and rec- ommendations for solutions to their perceived problems. visitors were also asked to vote for their most and least interesting gardens/murals and state the reasons for their votes. based on the comments and votes, important aspects of the gardens and murals perceived by visitors as drivers of positive experience were identified; they are: design patterns, choice of plants and flowers, location (i.e., flat vs. ele- vated), visibility, and maintenance. some of the gardens/murals voted as the least interesting garden were identified as having bland or boring design, too repetitive in design, sparse flowers, having muted color, not well maintained, etc. interestingly, it appears many visitors enjoyed the gardens more than the murals. overall, most visitors enjoyed the tour and considered the new product as a wonderful idea and that eccvb should do it again next year. nearly all visitors stated that they have gardens/murals they would like to visit/revisit this season or in a more distant future. indeed, most visitors indicated that they agree to visit more gardens/murals in the future (average rate of . , with for strongly disagree and for strongly agree) and also stated that they would like to recommend the tour to their friends and relatives (average rate of . ). most impor- tantly, there are a number of visitors who stated that they agreed to participate in planting the future quilt gardens, attend seminars in quilting and gardening, and participate in a quilt gardens and murals photo contest organized by eccvb. these results show that visitors are more than willing to engage in the process of the tour development by active participation in various activities related to this tour. visitors’ ideas for product design besides the positive aspects of the tour, this study also found some problems faced by tourists as they experienced the attractions. the problems identified by tourists are cate- gorized into two groups: (a) attraction-related problems for each gardens and murals and (b) general problems for the overall tour product. respondents suggested the development solu- tions for each of the venues and for the overall tour setting. the problems and solutions for gardens and murals include: . visibility problem. visitors identified some difficulties enjoying the entire design pat- tern of a few gardens and capturing them in pictures, especially when the gardens are planted in relatively flat areas. visitors suggested the following solutions for this problem: – creating elevated platforms at each gar- den to allow visitors to view the full pattern and take pictures of the garden; – creating a pole with a large mirror to enable visitors to view the garden from a designated spot, as if from above; – planting gardens on the hill; – preparing only low growing plants for better viewing. . information and interpretation. some vis- itors wanted more background informa- tion and interpretation of all garden and mural designs to better understand the sig- nificance of the tour. they suggested the following solutions: – providing signs and descriptions accompanied by aerial pictures of every garden; – setting storytelling or interpretation boards for each quilt garden/mural design for easy understanding and more meaningful experience. . design problems. a few issues surround- ing the design of quilt gardens and murals were identified and several solutions were suggested as follows: – choosing plant colors that are not too distracting; – selecting color combinations of flow- ers that can distinguish the quilt pattern better; – preparing a variety of flowers or plants that display the same color. additionally, besides the comments about the gardens and murals itself, there were other important aspects of the tour highlighted by lee, tussyadiah, and zach visitors. some directional signs were identified as causing confusion because they gave visi- tors a wrong direction. more problematically, while the overall evaluation of the tour guide cd/cassette were described as very informa- tive, well-done, and interesting; there were also some negative comments about them, such as “confusing,” “not necessarily helpful,” or even “leading away from the tour.” these problems are considered to be urgent matters for the development of the tour in the following year. conclusion new product development has been empha- sized as one of the vital factors for the suc- cess and prosperity of organizations. from the market-driven strategies that all business should start with a clear understanding of consumers (cravens, ), involving the consumer as an important role player in new product develop- ment has been believed to be an efficient way to achieve a business success in the highly com- petitive market. within the field of tourism, since being market-driven can be interpreted as focusing on visitors’ needs and wants and obtaining their feedback to continuously pro- vide valuable tourism experience, this study examined to what extent visitors can be success- fully involved in the new product development process using a case study of quilt gardens toursm. this study is based on the logic that cdi should include consumer interaction at the early stage of new product development and obtain consumers’ feedback soon after the prod- uct launch (gruner & homburg, ). as xia and arrowsmith ( ) state, modeling tourists’ movement and consumption patterns through this study is found to be useful to draw the impli- cations for product development. the results also suggest that visitors, as the actual role players in innovation processes, have the poten- tial to be co-producers of tourism products by providing their own creative insights for the products (de marez & verleye, ; van kleef et al., ). as richards and wilson ( ) assert, this study illustrates how successful an invention (i.e., newly launched tourism product quilt gardens toursm) can be elaborated and reproduced into an innovation (i.e., a “creative production”) when it is fully customized with consumers’ creative insights (i.e., the “creative consumption”). by observing their spatial and temporal sequences of visitors’ movements as one aspect of their experience, it is shown that the move- ment pattern can better indicate the actual expe- rience regardless of tourism planners’ suggested itinerary or program. these movements are generic; yet, by doing so, tourists assemble var- ious versions of products customized to their needs and preferences. this can also be seen as an attempt at reducing uncertainty in their movements of an area by visiting sites per- ceived to reward the greatest benefit for their effort (cooper, ). these versions are valu- able insights for planners to improve the tourism offerings in the future. as a key person who actually experiences and enjoys the destination, a tourist could go beyond “just being a tourist” and provide suggestions that product planners were not able to come up with prior to the prod- uct launch. in addition, these findings demon- strate that, because of the inseparable nature of consumption and production of tourism prod- ucts, the process of soliciting consumer feed- back is comparable to a real-time integration of demand and supply, and therefore is a very use- ful approach for product design within a tourism setting. from the analyses, a series of suggestions for the improvement of the quilt gardens toursm were provided. some important design elements which should be considered in further devel- opment of this program are as the following. first, it is important to add more visibility to the quilt gardens so that visitors can enjoy the gardens more by capturing the entire quilt pat- terns. second, the coverage of plants and flowers (i.e., not too sparse and not too crowded) is also an important factor that can aid to the attractive- ness of the quilt gardens. third, visitors tend to prefer a good-sized mural with unique design patterns. fourth, quilt gardens/murals manage- ment should put a greater importance on the maintenance of the gardens/murals. well-kept and cared gardens and murals are perceived to be more attractive by visitors. last, the pro- vision of directional and informational aides will provide visitors with a more valuable and meaningful experience with the tour. journal of travel & tourism marketing the two different approaches of data collec- tion were also effective for the purpose of this study. by using tour diaries, researchers could obtain visitors’ opinions and ideas while they were on-site enjoying the tour. through tracking visitors’ movements, existing problems related with the itineraries and materials provided by product planners to guide visitors within the destination were identified. also, the online sur- vey was proven effective to gain visitors’ overall evaluation after experiencing the tour, targeting those who did not have a chance to participate in the diary survey. this study provides meaningful implications stemming from the identification of visitors as co-producers of the tourism experience. while information technology (e.g., online survey) has gathered attention from researchers as a genuine innovative communication tool with consumers for their interactive nature (kaplan & haenlein, ), the failure to collect a large number of online survey responses is one of the limitations of this study. additionally, due to the attributes of the diaries, the visi- tors were asked to write them simultaneously while they were moving around; the move- ment patterns were not fully completed in some of the diaries. high-technical digital equip- ment to understand tourists’ experience within the destination (e.g., by automatically tracking their movements, recording their narratives) has been used more commonly these days, espe- cially when observing tourists’ spatiotemporal experiences (o’connor, zerger, & itami, ; tussyadiah et al., ). thus, the application of high-technical devices is expected to enhance the accuracy of the observation results for fur- ther future research. furthermore, in order to compare how the product has been improved after the adoption of consumers’ feedback, a further research to look at how consumers’ sug- gestions affect the product positively in the redesigned tour is recommended to confirm the effectiveness of cdi. references brown, s. l., & eisenhardt, k. m. 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( ). a model of new product development: an empirical test. management science, ( ), – . submitted: january , final revision submitted: april , accepted: may , refereed anonymously wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ whole-genome association study identifies stk as a hypertension susceptibility gene ying wanga, jeffrey r. o’connella, patrick f. mcardlea, james b. wadeb, sarah e. dorffa, sanjiv j. shahc, xiaolian shia, lin pand, evadnie rampersauda, haiqing shena, james d. kime, arohan r. subramanyab, nanette i. steinlea, afshin parsaf, carole c. oberd, paul a. wellingb, aravinda chakravartig, alan b. wederh, richard s. cooperi, braxton d. mitchella, alan r. shuldinera, and yen-pei c. changa, adivision of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, md ; bdepartment of physiology, university of maryland school of medicine, baltimore, md ; csection of cardiology, department of medicine, university of chicago, chicago, il ; ddepartment of human genetics, university of chicago, chicago, il ; egeorgetown university school of medicine, washington, dc ; fdivision of nephrology, university of maryland school of medicine, baltimore, md ; gmckusick-nathans institute of genetic medicine, johns hopkins university school of medicine, baltimore, md ; hdepartment of internal medicine, university of michigan school of medicine, ann arbor, mi ; and idepartment of preventive medicine and epidemiology, loyola stritch school of medicine, maywood, il edited by gregg l. semenza, johns hopkins university school of medicine, baltimore, md, and approved november , (received for review august , ) hypertension places a major burden on individual and public health, but the genetic basis of this complex disorder is poorly understood. we conducted a genome-wide association study of systolic and diastolic blood pressure (sbp and dbp) in amish subjects and found strong association signals with common vari- ants in a serine/threonine kinase gene, stk . we confirmed this association in an independent amish and non-amish caucasian samples including the diabetes genetics initiative, framingham heart study, gennet, and hutterites (meta-analysis combining all studies: n � , , p < � ). the higher bp-associated alleles have frequencies > . and were associated with increases of . / . mm hg in sbp/dbp, respectively, in the amish subjects and with smaller but consistent effects across the non-amish studies. cell- based functional studies showed that stk interacts with wnk kinases and cation-chloride cotransporters, mutations in which cause monogenic forms of bp dysregulation. we demonstrate that in vivo, stk is expressed in the distal nephron, where it may interact with these proteins. although none of the associated snps alter protein structure, we identified and experimentally con- firmed a highly conserved intronic element with allele-specific in vitro transcription activity as a functional candidate for this asso- ciation. thus, variants in stk may influence bp by increasing stk expression and consequently altering renal na� excretion, thus unifying rare and common bp-regulating alleles in the same physiological pathway. blood pressure � essential hypertension � genome-wide association study � spak � stk hypertension is a leading cause of morbidity and mortalityworldwide, contributing to cardiovascular disease, stroke, and end-stage kidney disease ( ). the most common form, essential hypertension (eh), is widely believed to involve multiple genes with variant alleles. like other complex disorders, manifestation of eh in any single individual is likely dependent on a variety of genetic and environment factors, making identification of eh susceptibility genes in the general population a major challenge. studies on the genetic basis of rare monogenetic bp disorders, in contrast, have identified mutations in genes affecting a single physiological path- way, renal salt transport ( , ). although such studies highlight the importance of this pathway to bp regulation, the underlying genetic basis for eh remains poorly understood. to identify common eh susceptibility genes, we conducted a genome-wide association (gwa) analysis in the old order amish, a closed founder population who immigrated to the lancaster, pennsylvania area from switzerland in the early s. the mem- bers of this community are descendants from a small number of common founders and share a relatively homogeneous lifestyle, making this population ideal for identifying genes that underlie complex diseases. we analyzed genotype data (affymetrix gene- chip human mapping k platform) for systolic blood pressure (sbp) and diastolic blood pressure (dbp) measurements obtained in subjects of the amish family diabetes study (afds) (table ) ( ). this approach identified a novel hypertension susceptibility gene, stk , in which common variants are associated with bp levels. results gwas in afds subjects. in our initial gwa scan, we analyzed , autosomal snps that passed our genotype quality control procedure (median and mean inter-snp distances, . kb and kb, respectively). all association results are available upon request and those with p values � . are provided in supporting information (si) table s . our top signal, rs , is significant at the genome-wide level but is located in a gene desert kb away from known genes ( p . ; p � . � � with sbp). but many of the strongest signals from the gwa of sbp were with a cluster of snps located on chromosome q . (fig. ; p � . � � to . � � ) within the stk (serine threonine kinase ) gene, which encodes the spak (ste -related proline-alanine�rich ki- nase) protein. these snps also are associated with dbp (table s b), albeit at slightly lower levels of statistical significance. a growing body of evidence indicates that spak interacts with cation-chloride cotransporters as a part of an evolutionarily con- served signaling pathway important in controlling salt transport in osmotic cell volume regulation ( ). in addition to the known effects of spak on the na�-k�- cl� cotransporter (nkcc ), spak also phosphorylates the thiazide-sensitive na�-cl� cotransporter (ncc) and the loop diuretic-sensitive na�-k�- cl� cotransporter (nkcc ) ( ). both transporters play major roles in renal salt excretion, as underscored by the direct involvement of ncc and nkcc in rare autosomal recessive conditions of hypotension, hy- pokalemic metabolic alkalosis, and salt wasting, also known as gitel- man and bartter syndrome ( , ). spak is phosphorylated by wnk and wnk (lysine-deficient protein kinase and ) ( , ), and rare mutations in these genes lead to autosomal dominant pseudohypoal- author contributions: y.w., a.c., a.b.w., b.d.m., a.r. shuldiner, and y.-p.c.c. designed research; y.w., j.b.w., s.e.d., s.j.s., n.i.s., c.c.o., and y.-p.c.c. performed research; j.r.o., c.c.o., a.b.w., r.s.c., and a.r. shuldiner contributed new reagents/analytic tools; y.w., j.r.o., p.f.m., s.j.s., x.s., l.p., e.r., h.s., j.d.k., a.r. subramanya, a.p., c.c.o., p.a.w., a.c., b.d.m., and y.-p.c.c. analyzed data; and y.w., j.b.w., a.r. shuldiner, and y.-p.c.c. wrote the paper. the authors declare no conflicts of interest. this article is a pnas direct submission. to whom correspondence should be addressed. e-mail: cchang@medicine.umaryland.edu. this article contains supporting information online at www.pnas.org/cgi/content/full/ /dcsupplemental. © by the national academy of sciences of the usa – � pnas � january , � vol. � no. www.pnas.org�cgi�doi� . �pnas. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/content/full/ /dcsupplemental http://www.pnas.org/cgi/content/full/ /dcsupplemental dosteronism type ii, characterized by hyperkalemia, hypertension, and sensitivity to thiazide diuretics (gordon syndrome) ( ). our gwa finding, combined with the emerging role of spak in renal electrolyte transport, make stk a logical eh candidate gene. stk contains exons that span � kb. there are snps on the affymetrix k platform located within kb of stk . the associations of these snps with bp and their pairwise linkage disequilibrium (ld) relationships are shown in fig. . two groups of correlated snps (r � . within groups and r � . between groups) demonstrated strong evidence of association with sbp (p � . ). the average minor allele frequencies (mafs) of the groups were . and . . for simplicity, snps that are highly correlated with either rs or rs (r � . ) are referred to as belonging to ld bin and bin , respectively (fig. ). the association was highly significant when tested using an additive or dominant model (fig. a). the association remained significant when the analysis was restricted to subjects not taking antihyper- tensive medication (data not shown). replication in remaining afds subjects and an independently col- lected amish cohort. we genotyped tag snp from each ld bin, rs and rs , in the remaining afds subjects; the associations also were significant (p � . for rs [fig. b] and . for rs ). in addition, we selected a second amish replication set from the heredity and phenotype intervention (hapi) heart study ( ), an independently collected amish sample for which subjects were ascertained without considering diabetes status (table ). we genotyped rs and rs in this cohort and found strong evidence of association between rs and sbp (n � ; p � . ; fig. c). in this younger and less hypertensive cohort, the association was in the same direction as that observed in the afds, but was more significant when tested under the recessive model. after combining all sets of amish subjects to determine the effect size of this association, we estimated that having copy of the minor allele was associated with a -mmhg higher sbp and a -mmhg higher dbp (table ). replication in non-amish studies. seeking additional replication and extension of our findings in non-amish populations, we accessed publicly available gwa data from the framingham heart study (fhs) ( ) and the diabetes genetics initiative (dgi) ( ), with , and , subjects, respectively. based on the criteria that significance levels (p values) are � . and the associations are in table . characteristics of the amish study subjects initial screening sample, afds replication sample , afds replication sample , hapi number age, years . � . . � . . � . sex, % male % % % body mass index, kg/m . � . . � . . � . sbp, mm hg . � . . � . . � . dbp, mm hg . � . . � . . � . % t dm . % . % . % % on antihypertensive medication . % . % % fig. . whole genome association scan results for sbp in afds subjects. snps from each chromosome are represented by a different color and ordered by physical location. . . . . . . . . sbp dbp physical location (mb) a b exon , exon , exon - exon - exon bin bin -lo g ( p -v al ue ) fig. . (a) exonic structure, association between stk snps and sbp/dbp. (b) ld relationship among snps. the locations of associated snps in ld bins and are denoted by black and gray bars, respectively. pairwise ld relation- ships (r ) among snps are indicated by color (black � ; white � ; shades of gray � � r � ). t t t g g g t t t g g g t t t g g g m ea n s b p (m m h g) m ea n s b p (m m h g) m ea n s b p (m m h g) p= . , additive p= . , dominant n= n= n= n= n= n= n= n= n= p= . , dominant p= . , additive p= . , recessive p= . , additive afds, k gwa study subject (n= ) afds, replication set (n= ) hapi replication set (n= ) a b c fig. . rs genotype-specific mean sbp among afds subjects in the initial gwa scan (a), replication afds set (b), and hapi subjects (c). the number of subjects, mean, and sem are shown for each genotype group. genotype-specific means were calculated using sbp adjusted for age, sex, and diabetes status. signifi- cance levels of association to sbp are shown along with genetic models. wang et al. pnas � january , � vol. � no. � g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , the same direction as in our amish studies, the stk association was replicated with either sbp or dbp in both groups (table for ld bin and snps). in addition, studies with smaller sample sizes provided modest evidence of an association between stk and bp (table ). we observed the same trend and effect size in a sample from another founder population, the hutterites (n � ) ( ), but the difference did not reach significance level (ld bin and snps; p � . ). we found the same result when we genotyped and analyzed rs and rs in the gennet european- american subjects (n � ) ( ). meta-analysis combining association evidence from all studies. among the studies for which primary data were available (afds, hapi, gennet, and hutterites), stk snps were more signifi- cantly associated with sbp under the recessive model than under the additive model in both hapi (fig. c) and the hutterites (ld bin snps; p � . and . for recessive and additive models, respectively). for the sake of consistency, we performed meta- analysis using the additive model only, because test results based on the dominant and recessive models were unavailable for dgi and fhs. nevertheless, combining the associations from all studies (n � , ) provided compelling evidence of association (p � . � � for rs and . � � for rs ), with an estimated allelic effect size of mmhg sbp and mmhg dbp (table ). identification of a functional candidate for the gwas signal. the associated snps in ld bins and are located in a . -kb region (chr : , , – , , ) spanning introns – and contain- ing multiple intronic conserved elements (ces). we sequenced all exons (including exon–intron junctions) and ces, which, based on multispecies sequence alignment, are equally or more conserved evolutionarily compared with the coding exons (fig. a and table s ). although we noted no coding or splicing variants, we identified relatively common intronic polymorphisms (maf � . ) within ces: rs in ce , rs in ce , and a dinucleotide at deletion, rs , rs , in ce . among these, rs and rs were in complete ld with rs and rs , respectively, and were strongly associated with sbp in the amish (p � . with afds subjects). ce was not included in further analyses because it contained no common variant associated with bp. because neither of the ces harboring bp-associated snps demonstrated evidence of non-protein-coding rnas, we hypoth- esized that these ces might have a regulatory function and that the sequence variants in ce or ce may inf luence stk expression. we tested luciferase reporter gene constructs, each with minimal promoter and a different allele at the polymorphic sites of ce and ce , to determine whether these bp-associated variants altered luciferase expression. the constructs tested included ce :g and ce :c for alleles of rs and ce :del/t/a, ce :at/t/a, and ce :at/c/g for the common haplotypes of dinucleotide at deletion, rs , and rs in ce . the major allele- bearing constructs of both ce and ce demonstrated decreased luciferase activity compared with the control vector (fig. b). the presence of the g allele at rs in ce restored luciferase activity. in contrast, minor alleles of the other sites in either ce or ce had no impact on luciferase activity. to summarize, rs is a conserved nucleotide within a highly conserved sequence element (fig. s ). the less common g allele, associated with higher bp, enhances transcription in vitro, which is predicted to up-regulate expression of stk . the snps in ld bins and were correlated (rs and rs ; r � . in the amish and . in the hapmap ceu subjects), and their association with bp vanished when the geno- types of another snp from either bin were included as covariates in the analysis. our data suggest that these snps are most likely surrogates for the functional candidate, rs . some snps in ld bin (e.g., rs , rs ) were in complete ld in the amish (r � in the , amish subjects genotyped); thus, the association between sbp and rs was essentially the same as that between sbp and rs , with minor differences due to genotyping completeness. in contrast, rs and rs were partially correlated in the non-amish subjects (r � . in the gennet subjects genotyped). the functional snp rs provided a more significant association than the snps genotyped in our gwa effort (between rs and rs ; r � . ; p � . for rs , . for rs , and . for rs ), consistent with this snp being either a better surrogate for the functional variant or the functional variant itself. segment-specific renal expression of stk . the identification of stk as a susceptibility gene for eh raises the intriguing possi- table . effect sizes of ld bin snp rs and ld bin snp rs with bp in amish and in non-amish studies study maf sample size effect size (mm hg)* sbp p† dbp p† rs amish . , . [ . – . ] . . [ . – . ] . dgi‡ . , . [ . – . ] . . [� . – . ] . ¶ fhs§ . , . [� . – . ] . . [ . – . ] . gennet . . [� . – . ] . . [� . – . ] . hutterites‡ . . [� . – . ] . . [� . – . ] . combined , . [ . – . ] . e- . [ . – . ] . rs amish . , . [ . – . ] . . [ . – . ] . dgi‡ . , . [ . – . ] . . [� . – . ] . ¶ fhs§ . , . [� . – . ] . . [� . – . ] . gennet . . [� . – . ] . . [� . – . ] . hutterites‡ . . [� . – . ] . . [� . – . ] . combined , . [ . – . ] . e- . [ . – . ] . afds and hapi subjects were combined to estimate effect size. *effect sizes of sbp and dbp are the mm hg difference attributable to copy of the minor allele followed by % confidence intervals. †all p values are based on the additive model. ‡because rs and rs were not genotyped in dgi and the hutterites, data from rs and rs , which are in near-complete or complete ld with rs and rs , respectively, are presented. in both the hapmap ceu and amish subjects, r � . for rs and rs , r � for rs and rs . §effect sizes and significance levels are from examination . ¶only , dgi subjects were analyzed for dbp. � www.pnas.org�cgi�doi� . �pnas. wang et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=sf bility that spak controls bp by regulating renal ion transport. consistent with this idea, recent in vitro studies have demonstrated that spak binds to and phosphorylates nkcc and ncc, na�-dependent cation chloride cotransporters that mediate renal nacl reabsorption ( ). to explore whether spak has the capacity to regulate these transporters in vivo, we determined the renal expression pattern of spak through immunof luorescence micros- copy of rat kidney sections. we observed spak immunostaining at the site of nkcc expression, the thick ascending limb (tal) of the loop of henle (fig. a), as well as at the predominant site of ncc expression, the distal convoluted tubules (dcts) (fig. c). in addition, we observed robust spak expression in the cortical collecting ducts (ccds) (fig. a). the immunolocalization of spak to these nephron segments provides supporting evidence that higher transcriptional activity spak due to the presence of the g allele of rs may increase the activity of downstream nkcc and ncc, which would promote na� reabsorption, thereby increasing intravascular volume and bp. more studies are needed to define the role of spak in bp regulation, to establish whether rs alone explains the association signal detected in the . -kb region, and to determine whether the in vitro allele-specific transcriptional activity of ce is relevant to stk activity and renal na� excretion in vivo. discussion based on the gwa scanning set, the initial association in stk was no longer significant at the genome-wide level after bonferroni correction was applied, adjusting for the number of snps analyzed. but this approach is overly conservative, because , snps on the k arrays are highly correlated with or more snps ( ). false-positive findings due to multiple testing is unlikely here, because the association between stk and bp is consistent across independent amish studies and non-amish populations, and the effect size and statistical evidence for association in the combined sample is compelling. the associations in the amish and non-amish studies are all in the same direction. the studies differ in the genetic models that provided the most significant association (additive vs. recessive), possibly due to increased arterial stiffness in the studies with older subjects. thus, the relationship between stk geno- types and bp is recessive in the younger hapi subjects but additive or dominant in the older afds subjects. although the gwa approach has generated several successes for such traits as diabetes, coronary artery disease, and obesity ( , – ), no convincing susceptibility locus for eh has been identified to date. the initial strong gwa signal found in the amish may have been facilitated by the relative genetic and, more importantly, lifestyle homogeneity and somewhat greater ld in this founder population compared with the general population. in fact, the association between stk and bp was stronger when sodium intake was standardized (hapi study, data not shown). other than stk , several intriguing association signals were found elsewhere in the genome, mostly in intergenic regions (table s ). other than snps in stk , only association signal was located near a gene already implicated in bp regulation; rs is associated with dbp (p � . ) and is . kb telomeric to adra a, the alpha a adrenergic receptor ( , ). we were unable to confirm or refute any association to other known hyper- tension candidate genes, because most of these genes were poorly tagged by our genotyping array. in fact, only a fraction of the common variants in stk were analyzed by the affymetrix k array. it is very likely that there exist other common stk variants that are modestly associated with bp levels in the general popula- tion and rare variants that have a greater effect on bp levels, similar to the mutations in wnk , wnk , nkcc , and ncc. for example, a group of snps located in intron of stk also have been associated with bp in the afds, fhs, and dgi populations (p � . in or more studies; table s ). further analyses revealed that several stk snps associated with baseline bp also were associated with hypertension status in our amish subjects, the dgi, and the wellcome trust case control consortium (wtccc) (even with the different criteria used to define hypertension). among the afds subjects, snps in ld bins and were associated with hypertension at p � . (after adjusting for age and sex), and multiple snps in ld bin also were associated with hypertension in the dgi subjects (p � . ; data not shown). because a gwa of bp was not carried out in the wtccc , . k , . k , . k , . k , . k , . k , . k % % % % ce - ce - ce - ce - ce - ce - ce - pr om ote r o nly ce :g ce :c * ce :- -/t /a ce :a t/ c/ a ce :a t/ c/ g* . . . . . . . . hela hek r el at iv e lu ci fe ra se a ct iv ity a b fig. . (a) pairwise alignment of human-mouse (top) and human-opossum (bottom) genomic sequence of the bp-associated region in stk . percent sequence identity is shown on the right. exons are in blue; intronic ces are in red. the locations of ces are indicated by bars above the graph. (b) luciferase activity of ce and ce bearing different alleles. the alleles associated with higher bp are indicated by an asterisk (*). wang et al. pnas � january , � vol. � no. � g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st ( ), we did not include the wtccc in our effort to replicate the amish association with bp. nevertheless, several stk snps were associated with hypertension in the wtccc at p � . ; for example, rs , located within our associated region and � kb from the putative functional snp rs , was associated with hypertension status (p � . for the additive models and . for the general models) in the wtccc. this snp was in partial ld (r � . – . ) with the bin snps but not with the bin snps (r � . – . ) in both the amish and the hapmap ceu samples. all of the published studies with hypertension gwa results, including ours, recruited based on either diabetes or hypertension; thus, an unbiased estimate of hypertension risk in the general population associated with the stk genotype remains to be determined. besides renal electrolyte transport, spak has been linked to such functions as cytoskeleton rearrangement, cell differentiation, trans- formation, and proliferation ( ). in fact, in a subset of amish subjects with extensive metabolic syndrome-related phenotypes, the bp-associated stk snps were modestly associated with fasting glucose level, insulin response to glucose, and plasma triglyceride level (data not shown). furthermore, stk was found to be located in a genomic region in which multiple bp, obesity, and diabetes-related rodent quantitative trait loci have been mapped [information provided by mouse genome informatics (http:// www.informatics.jax.org) and the rat genome database (http:// rgd.mcw.edu/)]. whether or not stk is involved in other meta- bolic syndrome traits, after adjusting for its effect on bp, awaits further study. in addition, several snps within stk have dem- onstrated an association with autism ( ). although this association has not yet been replicated, it is intriguing, because several known and predicted phosphorylation targets of spak (e.g., nkcc and kcc ) are expressed in neuronal tissues ( ). a recent study based on the fhs subjects found that those heterozygous for rare variants in ncct, nkcc , and romk (frequency � . ) had a clinically significant reduction in bp and lower hypertension risk, demonstrating that at least some fraction of bp variation in the general population is due to rare, independent alleles ( ). it is noteworthy that our agnostic gwa study identified a gene that unifies in a single pathway the proteins involved in rare, monogenic forms of hypotension/hypertension, such as bartter’s syndrome and gordon’s syndrome, and that of common eh. stk has not been previously implicated as a susceptibility gene for eh, but its involvement in bp regulation is supported by our current understanding of this protein and its interaction partners. in summary, our data establish that snps within the stk gene are strongly associated with bp. the real value of this study is not the identification of snps associated with bp levels or eh susceptibility in the general populations; rather, our findings highlight the importance of identifying all common and rare variants in this gene that might inf luence bp regulation, as well as the need for additional biochemical and physiological studies of its role in bp-related pathways. for example, the stk genotype might be a predictor of eh patients who are more likely to respond to salt reduction or a specific type of diuretic as a measure to control bp. spak itself might be an excellent target for novel antihyper- tensive drug therapies. materials and methods study subjects and phenotypes afds. recruitment for the afds was initiated in with the goal of identifying susceptibility genes for type diabetes (t dm) and related traits. the study protocol was approved by the university of maryland school of medicine’s institutional review board, and informed consent was obtained from each study participant. details of the afds design, recruitment, and pedigree structure have been described previously ( ). from the entire afds, subjects ( with t dm, with impaired glucose tolerance, and with normal glucose tolerance) were genotyped using the affymetrix k set ( ). the remaining subjects (n � ) was subsequently genotyped to confirm the stk findings (table ). hapi heart study. this study was initiated in to measure the cardiovascular response to short-term interventions affecting cardiovascular health and to identify the genetic and environmental determinants of these responses ( ). before any intervention, baseline bp was obtained manually with the subject in the sitting position after he or she had been sitting quietly for min, and the average of of these measurements was used for analysis. hypertension medi- cation was discontinued in all hapi subjects before the start of the study. fhs. recruitment of men and women from the town of framingham, massa- chusetts began in with the purpose of investigating the causes of cardiovascular disease and related traits. fhs investigators have recently published results of a k gwa study based on , subjects ( pedi- grees), phenotypes, and the affymetrix genechip mapping k array ( , ). all data were available through dbgap. for replication of our stk signals, we used sbp and dbp from examinations – and the average of sbp/dbp from available examinations – . dgi. the dgi performed a gwa study in , patients with t dm and , controls to search for genetic variants that influence the risk for diabetes and related traits, such as blood glucose and lipid levels, obesity, and bp ( ). for the analysis of bp-related traits, cases and controls were combined. the numbers of subjects analyzed for sbp, dbp, and eh status were , , , , and , , respec- tively. the size of the dbp sample was considerably smaller than the other samples because subjects over age years were excluded from the analysis. for replication, p values after correction by genomic control were used, and regression coefficients (labeled as beta) were used to determine the direction of association. hutterites. the hutterites are a religious isolate that originated in the tyrolean alps in the s. in the s, approximately hutterites moved to what is now south dakota, and their descendants now live in � communal farms in the northern united states and canada. because of their communal lifestyles, hutterites share a relatively uniform environment. the subjects (none of whom take antihypertensive medication) in this study are descendants of ancestors and are related to one another in a -generation pedigree ( ). to test for the effects of each snp on sbp and dbp, the general -allele model test of association was used in the entire pedigree, with all inbreeding loops kept intact, as described previously ( ). gennet. details of the gennet subjects have been published previously ( , ). we analyzed european-american subjects who were not taking antihypertensive medication at the time of the study, collected from tecumseh, michigan. genotyping and genotype data quality control. for the initial genome-wide association scan conducted in the afds samples, genomic dna from leukocytes was genotyped using the affymetrix genechip mapping k array set. the genotyping a b c d fig. . immunolocalization of spak in kidney. (a) spak strongly localizes to the tal of the loop of henle and the ccds. (b) immunolocalization of aquaporin in the same section as in (a) as a segment-specific marker for ccds. (c) immunolocalization of spak in the dcts. (d) immunolocalization of ncc as a segment-specific marker for dcts. bar � . �m. � www.pnas.org�cgi�doi� . �pnas. wang et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , protocol and quality control procedures used to identify and remove poor-quality k data were described previously ( ). more details are given in si materials and table s . sequencing. the . -kb bp-associated region of stk contains exons (exons – ) and intronic ces. we defined noncoding ces as intronic elements with sequence identity as high as stk coding exons based on a lod score calculated using the phastcons program (phastcons conserved elements, -way verte- brate multiz alignment, provided by the ucsc genome browser) ( ). a total of samples were selected based on the rs genotype and residual bp levels, including homozygotes of the major allele with the lowest sbp residual, heterozygotes with the highest sbp residual, homozygotes of the minor allele with the highest sbp residual, subjects with the highest overall sbp residuals, and subjects with the lowest overall sbp residuals. pedigree structure was taken into account for sample selection, to minimize relatedness. exons and ces were amplified (primer sequences available on request) and sequenced using bigdye version . and an abi dna sequencer, and then analyzed using sequence analysis . software (all from applied biosystems). sequencher . (genecodes) was used to align individual sequences and identify variants. reporter gene assay. pcr products from individuals with known homozygous genotypes were cloned into the reporter vector (pgl . [luc /minp], promega) using an in-fusion . dry-down pcr cloning kit (bd biosciences). the following ces were cloned and tested: ce :g and ce :c for ce with different alleles of rs and ce :del/t/a, ce :at/t/a, and ce :at/c/g for ce with haplo- types involving a dinucleotide (at) deletion, rs (c/t), and rs (a/g) that we observed in the amish. haplotype del/t/g was not found in the amish population and was not tested. all recombinant plasmids were sequenced to confirm insert sequence and orientation. for each construct, hek- and hela cells (obtained from american type culture collection and cultured accord- ing to recommended condition) were cotransfected with �g of plasmid dna, . �g of pgl . (renilla luciferase), and �l of fugene hd reagent (roche), to normalize for transfection efficiency. after transfection and a -hour incuba- tion, cells were lysed and luciferase signals were assayed using the dual-luciferase reporter assay system (promega) and a td- / luminometer (turner designs). relative luciferase activity is given in arbitrary units corrected for renilla lucif- erase activity and normalized to the control. all luciferase experiments were performed in duplicate at least twice. statistical analysis. association analyses of bp-related traits were performed using the measured genotype approach that models variation in the trait of interest as a function of measured environmental covariates, measured geno- type, and a polygenic component to account for phenotypic correlation due to relatedness. a (n- )-degree-of-freedom t test was used to assess the significance of the measured genotype. age, age , sex, and t dm status were included as covariates, and the snp was coded using additive, dominant, and recessive modes. when analyzing the amish subjects, the polygenic component was mod- eled using the relationship matrix derived from the complete -generation pedigree structure, to properly control for the relatedness of all subjects in the study (data not shown). q-q plots comparing the observed gwa p values with those expected under the null are provided in fig. s . the genomic inflation factors (�) based on t-scores were . for sbp and . for dbp, indicating that systematic inflation of our association signals due to undetected genotyping error or hidden family relationship was highly unlikely. effect sizes in the amish studies were computed after the afds and hapi subjects were combined. age, age , sex, t dm status, and study designation (afds vs. hapi) were included as covariates, and genotype was coded based on an additive genetic model. pairwise r values between snps were calculated using haploview ( ). ld bin assignment was based on hapmap ceu genotypes, which provided an ld struc- ture of the stk region nearly identical to that observed in the amish. the bp association results for fhs and dgi were assessed from the websites referenced from their respective published studies ( , ). the directionality of the association was determined for each study from either the family-based association test (fbat) statistics or beta. study-specific adjustment for hyperten- sion medication was used when evaluating association signals from non-amish studies. a weighted z-score-based fixed-effects meta-analysis method was used to combine results across all studies, using the metal program (http:// www.sph.umich.edu/csg/abecasis/metal/index.html). in brief, for each snp, a ref- erence allele was identified, and a z-statistic summarizing the magnitude of the p value for association (under the additive model) and direction of effect was generated for each study. an overall z-statistic was computed as a weighted average of the individual statistics, and then a corresponding p value for that statistic was computed. the weights were proportional to the square root of the sample size in each study and scaled such that the squared weights summed to . immunolocalization of spak. kidneys from adult sprague-dawley rats were fixed by retrograde perfusion and embedded in paraffin. once sections ( �m) were picked up on cover slips, heat-induced target retrieval using a citrate buffer (ph ) was used to unmask epitopes. sections were then washed and incubated overnight with primary rabbit antibodies to spak (abgent, san diego, ca) at a concentration of �g/ml at °c, followed by secondary antibodies as described previously ( ). acknowledgments. this work was supported by national institutes of health (nih) research grants r dk – , r hl – , u hl – , r hl , u hl , and dk . we thank john shelton and kathy ryan for their expert genotyping and data support efforts. we also thank dr. martin larson for providing the fhs phenotype data for effect size estimate, dr. lei lu for assisting with genomic inflation, and dr. kirby d. smith for providing valuable discussions. we gratefully acknowledge the amish liaisons and fieldworkers, as well as the extraordinary cooperation and support of the amish community, without whom these studies would not be possible. . kearney pm, et al. 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( ) evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. genome res : – . . barrett jc, fry b, maller j, daly mj ( ) haploview: analysis and visualization of ld and haplotype maps. bioinformatics : – . . coleman ra, liu j, wade jb ( ) use of anti-fluorophore antibody to achieve high-sensitivity immunolocalizations of transporters and ion channels. j histochem cytochem : – . wang et al. pnas � january , � vol. � no. � g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=stxt http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=st http://www.pnas.org/cgi/data/ /dcsupplemental/supplemental_pdf#nameddest=sf http://tcn.sagepub.com journal of transcultural nursing doi: . / ; ; j transcult nurs carol savrin sharon m. weyer, victoria r. hustey, lesley rathbun, vickie l. armstrong, samantha reed anna, jeanne ronyak and a look into the amish culture: what should we learn? http://tcn.sagepub.com/cgi/content/abstract/ / / the online version of this article can be found at: published by: http://www.sagepublications.com on behalf of: transcultural nursing society can be found at:journal of transcultural nursing additional services and information for http://tcn.sagepub.com/cgi/alerts email alerts: http://tcn.sagepub.com/subscriptions subscriptions: http://www.sagepub.com/journalsreprints.navreprints: http://www.sagepub.com/journalspermissions.navpermissions: http://tcn.sagepub.com/cgi/content/refs/ / / citations by books editorial on january , http://tcn.sagepub.comdownloaded from http://www.tcns.org http://tcn.sagepub.com/cgi/alerts http://tcn.sagepub.com/subscriptions http://www.sagepub.com/journalsreprints.nav http://www.sagepub.com/journalspermissions.nav http://tcn.sagepub.com/cgi/content/refs/ / / http://tcn.sagepub.com . / articlejournal of transcultural nursing / april weyer et al. / a look into the amish culture a look into the amish culture: what should we learn? sharon m. weyer, msn, rn, np-c case western reserve university victoria r. hustey, msn, aprn, bc rainbow babies and children’s hospital, university hospitals of cleveland lesley rathbun, msn, rn, cnm vickie l. armstrong, msn, rn samantha reed anna, msn, rn jeanne ronyak, msn, rn carol savrin, nd, cpnp, fnp-c case western reserve university it is important to understand the amish culture in order to provide appropriate, acceptable, and accessible health care to this culturally diverse group. a case study pertaining to the care of a dying elderly amish woman living in a rural amish community is examined. this allows for exploration into the world of the amish community in greater detail. their overall beliefs, values, and behavior are discussed as well as how their lifestyle affects their health care decisions, access to health care, and reimbursement of services. nurse practitio- ners can offer culturally sensitive and appropriate health care to the amish population by recognizing important cul- tural values that have survived for more than three hundred years. keywords: amish culture; nurse practitioner; human caring; health care exploitation; culturally sensitive health care the amish community is a unique culture. as a group the members are religious, hard-working persons who value humility and focus their lives on maintaining family and com- munity values. the amish have many health care issues and needs. they strive to take care of their own; however, they will seek help from the outside world when necessary (banks & benchot, ). their culture has many differences from oth- ers living in the united states. it is vital for health care profes- sionals to respect these differences when dealing with amish individuals. nurse practitioners can play a vital role in caring for the amish community by providing education on disease prevention, health care maintenance, and avoidance of quack- ery medicine. the amish have continued to live simple lives with minimal technology in an ever-changing world around them. it is important to look into their culture and appreciate some of the aspects that have continued to thrive and grow despite these changes. the following is a case study that allows a glimpse into the amish world. case study on a cold, snowy, windy morning in january, a phone call was received from an amish family asking for assistance for their mother. mrs. t., a -year-old amish woman was diag- nosed with congestive heart failure (chf) months ago. prior to her symptoms of heart failure she had been healthy without chronic health problems, routine medications, and had never been hospitalized. mrs. t’s son stated she was not authors’ note: this case study occurred during a clinical rotation at a rural family practice office that served a large amish population. the authors de- veloped the article for a class assignment for a family nurse practitioner clini- cal course at frances payne bolton school of nursing at case western reserve university. journal of transcultural nursing, vol. no. , april - doi: . / © sage publications clinical practice department by books editorial on january , http://tcn.sagepub.comdownloaded from http://tcn.sagepub.com resting at night. she had been awake, anxious, and short of breath last night. mrs. t. had been taking digoxin (lanoxin) and furosemide (lasix) for the past months. a prescription was called to the pharmacy in town for alprazolam (xanax). the prescription was picked up and taken to the family. in this small, rural town with a fairly large amish population there is an understanding among shop owners and the amish, which allowed for the prescription to be charged to the family. we drove the miles to the home in a ford bronco in approximately to minutes. the horse and buggy ride to town would have taken half an hour for mrs. t and her family. mrs. t would have been most uncomfortable. along the way we passed an amish school, the children were sled riding down a large hill in front of their school. the roads to the home were narrow, winding, and composed of chip and seal. on arrival to the home, we pulled into the gravel drive and parked next to the hitching post. the driveways are usually circular so the horse and buggy do not have to back up. the house was a one-story, white ranch. there was no electric line going to the home. we entered the home through the back door; wooden steps led to the kitchen. the home was very neat, warm, and welcoming. a pie was cooling on the kitchen counter; the smell permeated the home. the hardwood floors were covered with rugs. decoration was sparse and what was present was of a religious nature. there were no electric plugs or light switches. the lights on the walls were kerosene lamps. mrs. t’s son and daughter greeted us and answered our questions about her condition. her activity had declined over the past weeks. they were providing assistance with all her activities of daily living. mrs. t’s daughter is the teacher at the school we passed. she has taken the past days off to help care for her mother. another woman from the amish church is covering for her at the school. mrs. t and her family had decided they wanted no heroic measures to prolong her life. they only wanted to provide comfort care. over the past days her food intake had decreased; she was taking intermittent sips of liquids. her uri- nation had decreased despite the furosemide. mrs. t was lying on the sofa in the living room. she was sitting at an degree angle, supported by three pillows. although she was frail looking, her white hair was neatly combed into a bun. she was wearing a crisp, white, cotton nightgown. her legs were covered with an afghan embroi- dered with the religious poem, “footsteps in the sand.” a small table was beside the couch; it contained a teacup and saucer. mrs. t was sleeping; her breathing was labored and irregu- lar with cheyne-stokes respirations. she aroused to verbal and tactile stimulation. despite being hard of hearing, she answered our questions appropriately. her pale skin was cool and dry with no rashes or lesions. her lips were moist and she displayed no sign of dehydration. her assessment was consis- tent with chf. crackles were auscultated bilaterally in her lungs an s gallop was audible. her bowel sounds were hypoactive. peripheral + pitting edema was noted in both lower extremities and her nail beds were slightly cyanotic. we explained the purpose and use of the new medication to the family and offered oxygen as an additional comfort mea- sure. at this point the family did not feel the oxygen was nec- essary. a return visit was planned in days and the family was encouraged to contact the office if they needed anything in the mean time. as we left the house, the family’s horse was wait- ing at the fence next to the driveway watching us leave, as if sensing something was going on. literature review there is a wide variety of literature pertaining to the amish community. some articles focus primarily on specific disease processes that are prevalent throughout the amish population; however, multiple articles were found detailing an overall view of the amish lifestyle, beliefs, culture, and views on childbirth and death. in addition, books have also been written about the amish community. it is first important to understand the background of the amish population. the amish are a branch of christianity that began in europe soon after the protestant reformation. a new group of believers around zurich, switzerland did not believe in government sponsorship of religion or infant bap- tism. they felt it was contrary to the teachings of christ in the new testament (kreps, donnermeyer, & kreps, ). it was believed that baptism should be reserved for consenting adults (o’neil, ). led by ulrich zwingli, the formal break came on january , , when the protestors symbol- ically rebaptized themselves (brewer & bonalumi, ). anabaptist means to be rebaptized and this was the original name given to the group. the group later split over differ- ences of anabaptist beliefs and laxity of practices (kreps et al., ). the separation from zwingli led to the formation of the mennonites, who are found throughout the world today, including pennsylvania (brewer & bonalumi, ). followers of a more conservative group led by jacob ammann, who separated from the mennonite church, came to be known as the amish (beachy, hershberger, davidhizar, & giger, ). many of the amish, today, consider themselves cousins of the mennonites (brewer & bonalumi, ). the first record of amish immigration to america was in , the settlements were in berks and lancaster counties of pennsylvania (kreps et al., ). there is no amish commu- nity remaining in europe, today. amish groups who remained in europe suffered through conditions of persecution and financial hardships during the th and th centuries. even- tually, the amish that stayed behind converted to local men- nonite and various protestant denominations (kreps et al., ). pennsylvania has the greatest number of settlements, but ohio has the largest population of amish. the largest set- journal of transcultural nursing / april by books editorial on january , http://tcn.sagepub.comdownloaded from http://tcn.sagepub.com tlement of amish is in a five-county area in ohio known as the holmes county and vicinity settlement area (kreps et al., ). the amish follow five basic tenets: adult baptism, separa- tion of church and state, ex-communication from the church for those who break moral law, living life in accordance to the teachings of christ, and refusal to bear arms, take oaths, or hold political office (andreoli & miller, ). historically, amish have been farmers. “they refer to the old testament passage from genesis when god commanded adam and eve to ‘dress’ and cultivate the garden” (kreps et al., , p. ). in some areas, about one in three amish breadwinners primarily make their living from agriculture (kreps et al., ). even in amish families where the bread- winner works in a factory, the woman of the house maintains a garden. the amish live in rural areas surrounded by farmland (brewer & bonalumi, ). the amish live by a code of conduct that forms their behavior, speech, and manner of dressing (banks & benchot, ). according to kreps et al. ( ), the ordnung (ott- nuing) is a set of rules and regulations for living the amish faith. it is largely oral, with only a few parts written down. it is what distinguishes between those in fellowship with the amish faith and all others. the amish are organized into church districts, each composed of several dozen families headed by a group of elders, including a bishop, two minis- ters, and one deacon. church leaders have no formal training or qualifications. they are nominated by the group based on their upholding of the amish beliefs. a piece of paper with a biblical quote is placed in a hymnbook. the nominees chose one of the hymnbooks. the one choosing the hymnbook with the piece of paper is considered chosen by god. each church district has its own diverse ordnung. “the amish are like the quilts they make, each patch may be different, but the amish are sewn together by their common history and religious val- ues” (kreps et al., ). two additional terms that are important in the amish com- munity are meidung and gelassenheit. meidung (mide-ung), or shunning, means cutting off fellowship with and avoiding former members who have been excommunicated, that is, told to leave the amish faith due to serious acts against the ordnung. only baptized adults are subject to the meidung. shunning is considered the last resort. fellowship can be restored, however, if the person sincerely repents (kreps et al., ). gelassenheit (gay las en hite) means yielding to god’s authority. this, also, helps guide amish behavior. it is the belief in living a simple life and giving thanks to god for their blessings. amish school children are taught the favorite motto of “joy”: j stands for jesus who comes first, o is for others that are second, and finally y stands for you that should come last (andreoli & miller, ). the amish avoid abu- sive, violent, and threatening words due to the belief that words can cause harm and disharmony (banks & benchot, ; kreps et al., ). aspects of the amish culture the amish are a diverse society with many traditions. they are changing all the time and have always changed to accommodate the challenges in the world around them. there are many unique characteristics that set the amish commu- nity apart from others living in the united states. first, they believe in living simply. one example of this is that electricity is not permitted in their homes and the use of electricity from public power lines is discouraged even outside the home. some compromises have been made, however. batteries and large diesel engines can be used to cool milk tanks and oper- ate workshops (kreps et al., ). also, if an ill family mem- ber needs to use electricity they have to get permission from the elders. another example of the simple living that the amish popu- lation follow is their dress. according to brewer and bonalumi ( ), their plain style of dress is to reject worldli- ness, vanity, and materialism. traditionally, men’s wear in the amish community includes button-front trousers with sus- penders and a black felt hat in the winter and a straw wide- brimmed hat in the summer. men shave until marriage and then grow a beard. the upper lip is always clean because church law forbids mustaches. women, who hand-make most of the clothing, wear solid colored dresses in blue, green, gray, purple, or wine along with a white organdy head cover- ing. a black bonnet is placed over the white covering when outdoors. black dresses are, also, worn for religious occa- sions. some communities use straight pins to fasten the bod- ice of their dresses and secure their head covering. the use of hooks and eyes versus snaps and buttons for construction of clothing varies by each community’s ordnung (beachy et al., ). the amish formal education system includes grades one through eight, taught by one or two amish teachers who are picked by the school board. the school board is made up of parents of the amish children. teachers do not have formal education beyond the eighth grade and are not required to have certification. they are selected by the school board based on their ability to uphold and teach amish values to the children. children attend school from september until the end of april, monday through friday. their only vacation days are usually thanksgiving and christmas. subjects con- sist of spelling, phonics, reading, vocabulary, english, geog- raphy, arithmetic, penmanship, and bible lessons. children are required to take both english and german (kreps et al., ). amish parents pay approximately $ , in tuition fees annually regardless of the number of children attending school (kreps et al., ). most amish parents refuse to send their children to high school in fear that non-amish chil- dren and advanced education will alter their thinking and weyer et al. / a look into the amish culture by books editorial on january , http://tcn.sagepub.comdownloaded from http://tcn.sagepub.com behavior in ways inconsistent with amish beliefs. some uni- versities offer vocational classes and workshops for the amish. some of the topics are farming, milk production, cul- tivation, buggy safety, and sawmill production (kreps et al., ). as mentioned previously, most amish are farmers. some, however, do work as laborers, carpenters, carriage makers, and other craftsmen. the amish prefer not to work for outsid- ers, but rather on family-owned farms and businesses. amish families can often be found selling homemade food or quilts at their homes or in nearby markets (brewer & bonalumi, ). children are often working full-time at home or in a nearby shop by age because school is completed by eighth grade (kreps et al., ). according to brewer and bonalumi ( ), the amish population speaks a mixture of german, english, and penn- sylvania dutch. children do not learn english until they attend school and speak pennsylvania dutch in the home. “in amish society, the pennsylvania dutch dialect is the lan- guage of work, family, friendship, play and intimacy” (kreps et al., , p. ). the amish also refer to the non-amish community as “english” because that is the language used by others around them (beachy et al., ). a characteristic that distinguishes the amish from many other denominations is that they conduct their church service in members’ homes. families in the community rotate ser- vices from one home to another, which means that or more people may attend at one time. services begin about : a.m. and last about three and one half hours. a meal, pre- pared by the host family, follows the service along with talk and visiting among the guests until afternoon chore time (kreps et al., ). meetings usually take place twice a month and scriptures from the bible are read. the amish dis- courage boasting of their bible learning and intimidation of others with bible quotes (o’neil, ). they also believe that “those who worship god, obey the church, provide for family and community will enjoy salvation” (o’neil, , p. ). the majority of the amish live in a household with six or more persons that often include extended family members. it is common for aunts, uncles, and cousins to live across the road or on the farm next door. a dawdy haus, which is right next to or often attached to the original house, is where elderly parents move when they “retire,” and the larger house is given to the youngest of their children. in amish society, the elderly are considered important sources of information for the youn- ger generation. they give advice on topics that include health and illness, household chores, cooking, gardening, farming, and predicting the weather (kreps et al., ). the amish do not believe in retirement homes; instead, they reside at home where they are cared for by their family (palmer, ). the average age of death in the amish population is approxi- mately years old (mitchell et al., ). the amish also believe in taking care of their young, therefore there are no day care centers. the amish community thinks they are responsible for the young, elderly, and the sick. the daily life of the amish is centered on the home. children, often born at home, play at home and attend a school that is within easy walking distance. many amish individuals work full-time in the home. recreational activi- ties take place at home or with nearby neighbors. church ser- vices and marriage ceremonies are held at the home. most meals are prepared and eaten at home. social gatherings and clothing making are also performed at home. finally, retire- ment occurs at home as well as end-of-life care and the funeral service (kreps et al., ). life almost always begins and ends in the amish home. members of the amish community do not practice divorce or birth control. therefore, large families are encouraged. the average amish family has six children (o’neil, ). they also tend to socialize with and marry their own. inter- marriages are very common among the amish and the major- ity of couples who marry are usually related to each other in some way (mckusick, ). this puts the amish population at risk for a high incidence of genetic diseases. recessive genetic disorders are increased among the amish (mckusick, ). finally, the amish view death as a natural part of life. they believe in eternal life. death is seen as “god’s will” and the amish often take their loved ones home to die rather than have death occur in a hospital setting (banks & benchot, ). when an individual in the amish community dies, friends and families gather at the home as soon as they receive word of the death. family members of the same gender dress the body for burial. men are dressed in a white shirt, vest, and pants and women are dressed in white with a white cape and apron. the coffin is wide at the head and narrows at the feet and is void of ornamentation (palmer, ). amish members of the community outside the family prepare the grave. the person is then buried in an amish cemetery (girod, ). the leading causes of death in the amish are heart disease and accidents and most men outlive the women (palmer, ). genetic diseases can also have a big impact on life expectancy and chronic health conditions. effects on health care decisions there are many factors that affect the health care of the amish population (see table ). the amish brought with them from the old country many home remedies and folk medicine. some amish still practice powwowing; also know as braucha or braucheri, which is an old world brand of faith healing. the belief holds that certain people inherit the power to heal by touch, or moving around a sick person, or through audible or silent incantations (adams & loverland, ). the amish are often preyed upon by quackery fraud and med- ical exploitation. journal of transcultural nursing / april by books editorial on january , http://tcn.sagepub.comdownloaded from http://tcn.sagepub.com modern health quacks are expert salesmen. they use sci- entific terms and many who target the amish quote bible scriptures. the amish cultural belief in rural living and rigor- ous physical labor as a means to good health makes “natural” treatments attractive. the quacks will use convincing testi- monials from amish community members to spread the word of their cure. many dishonest salesmen will discredit the medical establishment by claiming a massive conspiracy by licensed health care providers, drug companies, and medical associations. the budget, an amish newspaper written by correspondents from amish communities in north and south america is published in sugarcreek, ohio. it contains news, announcements of births, deaths, and marriages as well as a number of advertisements for questionable alternative health products and services (palmer, ). types of health fraud popular among the amish include chelation therapy, radon mines, tijuana clinics, medical devices, and herbal supplements. chelation therapy is a series of intravenous infusions containing disodium ethylene- diamine-tetra-acetic acid (edta) and is approved for hypercalcemia and heavy metal poisoning (sampson, ). some amish seek chelation therapy for atherosclerosis and neutralization of free radicals. chelation therapy can cause hypotension, shock, acute renal failure, and sudden cardiac death from hypocalcemia, however, and has been shown to have no effect on atherosclerosis or reduction in free radicals (sampson, ). also, many amish often visit abandoned mines and pay to purposely expose themselves to radon gas in the belief that it will cure arthritis, asthma, migraines, diabe- tes, and other ailments. radon gas is blamed for many lung cancer deaths (cannon, ). many clinics found in tijuana, mexico offer treatments appealing to the amish such as cof- fee enemas, goat blood therapy, and the healing power of cow embryos. some clinics also offer discounted surgeries includ- ing hip replacement and hysterectomy (barrett, ). finally, the amish use herbal remedies. often herbal tablets, garlic, and vitamins are their self-prescribed first-line treat- ment against illness and injury (mccollum, ). they believe strongly that vitamins and health foods are significant in maintaining one’s health and will often pay expensive herbal remedy prices (palmer, ). the functional ability to work, as well as the attitude of wanting to work, is important in the amish perception of health. significant disability, such as blindness or inability to walk, does not inhibit productivity. the amish primarily work as a team; therefore a person with a disability can per- form tasks that are contributory yet less physically demand- ing (hewner, ). these may include duties such as clean- ing vegetables or cutting scraps for rugs. disability, therefore, is minimized in large amish households. as mentioned ear- lier, the elderly live with their adult children, a practice that places less emphasis on individual function and more empha- sis on group function. independent function is much less important in amish culture than among the non-amish (hewner, ). the amish population will seek out westernized medical care and technology when necessary. the amish are not pro- hibited by church law from taking medication or seeking care from a doctor, however, as discussed earlier, they will often use other remedies first (girod, ). the amish define ill- ness as a failure to function in the work role instead of in terms of symptoms. for this reason, they may be more likely to seek care for acute traumatic conditions as opposed to chronic ill- nesses (palmer, ). when the amish do seek medical attention, they often have severe symptoms and increased risk of mortality secondary to delay in seeking care. the amish often do not practice preventative medicine, as well. this is partly due to a lack of education and understanding of micro- organisms and disease. many amish also believe that sins cause illness. if sins are the cause of illness, “they believe, then no amount of prevention or immunization will prevent illness, and there is no need to participate in preventive pro- grams” (adams & leverland, , p. ). this is an area that nurse practitioners can work closely with the amish and play a vital role in education. effects on access to care there are several factors that affect the amish’s access to health care. transportation is one factor. the horse and buggy is a distinct symbol of amish religious values and unique life- style. a medical appointment may be missed due to a horse that will not leave the barnyard (mcginn, ). the horse and buggy can usually travel about miles an hour, so the amish tend to live relatively close to their amish neighbors in weyer et al. / a look into the amish culture table reasons for medical exploitation of the amish education the amish only attend school through the eighth grade. they have a limited background in math and science. the amish do not use the internet or other electronic sources of information. insurance many amish do not have health insurance and mainstream medicine is too expensive. money the amish are cash paying customers who pay their bills in a timely manner. distrust many amish distrust mainstream medicine. some may have had a bad experience with doctors or hospitals. pacifism the amish are forbidden by their religious beliefs from suing. they rarely alert authorities. culture the amish have a strong cultural belief in folk medicine and home remedies. source: data from adams and loverland ( ). by books editorial on january , http://tcn.sagepub.comdownloaded from http://tcn.sagepub.com order to visit others in their community regularly and attend church services (kreps et al., ). this may make travel to health care facilities and hospitals a difficult task. many of the facilities are often located outside of their rural setting. traveling in bad weather is also another obstacle. transporta- tion by car or van from an “english” neighbor or friend is arranged for long distance travel or when traveling in larger groups. the majority of the amish do not have telephones in their homes, which affects access to immediate care particu- larly in times of emergency. the amish usually have to use a public phone or travel to a neighbor’s house to use a phone. effects on reimbursement or payment of services health care can be costly to the amish population, because they are self-pay clients who do not use commercial health insurance. the amish believe that they are responsible for their own group (dickinson, slesinger, & raftery, ). communities form their own type of insurance fund in which amish individuals contribute an initial amount and then again when someone has a need (brewer & bonalumi, ). this contribution is called “church aid” or “amish aid.” if an amish person incurs an unmanageable expense, the amish community will step forward and help with medical pay- ments or money for repairs (beachy et al., ; palmer, ). therefore, it is easy to see how medical payments can be trying on the amish community and how many often pro- long medical attention for what they believe may be minor ill- nesses or injuries. in many situations, however, delays in seeking treatment increase medical expenses (mccollum, ). implication for practice nurse practitioners, with physician collaboration, can play a vital role in the care of the amish. nurse practitioners specializing in family medicine and midwifery may provide an excellent source of care for this population because of the family and community needs in a rural setting. there are often fewer physicians in these settings, opening the door for nurse practitioners. nurse practitioners can remind their amish patients that there is no religious rule prohibiting immunizations against disease, and can use education to combat superstition and promote disease prevention (adams & leverland, ). education can be very vital in helping the amish avoid quackery medicine. nurse practitioners can play a big part through education in preventing the amish from getting caught up in scams and ineffective health treatments. acceptance of the amish for who they are will help in the development of a trusting relationship. according to watson (n.d., p. ; see also, mcgraw, ), developing and sustain- ing a helping-trusting, authentic, caring relationship is one of the necessary steps in providing human caring; she describes this as the essence of nursing practice. human caring is actualized in the moment based on the actions and choices made by both the one-caring and the one- being cared for . . . the goal in the relationship is the protec- tion, enhancement, and preservation of patient dignity, humanity, wholeness, and inner harmony. (mcgraw, , p. ) nurse practitioners should take the time to understand the different beliefs of the amish to provide the best care possible (adams & leverland, ). “they must be acutely aware of their own cultural biases, especially when those may be at odds with the amish orientation” (wiggins, , p. ). the best approach that the nurse practitioner can take with the amish is to talk simply and honestly with them. they are not impressed with increased education and are disinterested in individuals who act arrogantly or are bossy (beachy et al., ). nurse practitioners can reinforce an amish person’s health care practices, such as the use of creams and supple- ments, as long as it is not detrimental to his or her health (palmer, ). however, they may also want to offer the bio- medical perspective for consideration and develop a mutually agreed on plan of care. promoting mutual understanding and respect is always better than encouraging intolerance because intolerance leads to conflict (kreps et al., ). this article has discussed the amish culture and its rela- tionship to medical care. it has given us a look into a culture that continues to thrive with simple surroundings, despite the advances in technology and daily changes in the world. the amish have continued to maintain their faith, values, and strong sense of family and community. they have not allowed outside influences to penetrate their beliefs or alter their cul- ture. if we continue to look closely at the amish community, perhaps they can teach us some valuable lessons in life. as mrs. t was peacefully dying at home, surrounded by her friends and family, she was comfortable in her own surround- ings. she was not in a foreign place being cared for by strang- ers or being monitored by technology. she was dying in her home, with her family, in her community. it is easy for us to get caught up in our society with all the increased technology and medical advances, but perhaps we have lost some impor- tant things along the way. we may have lost sight of some valuable qualities that the amish can help us regain, if we let them. a quote from an amish man in the , summer issue of the small farm journal (as cited in kreps et al. ) sum- marizes some of what we can learn from the amish. we realize that not everyone is cut out to be one of the plain people. many have not the opportunity, but here is the chal- lenge. if you admire our faith—strengthen yours. if you admire our sense of commitment—deepen yours. if you admire our community spirit, build your own. if you admire journal of transcultural nursing / april by books editorial on january , http://tcn.sagepub.comdownloaded from http://tcn.sagepub.com the simple life—cut back. if you admire deep character and enduring values, live them yourself. (p. iv) references adams, c. e., & leverland, m. b. ( ). the effects of religious beliefs on the health care practices of the amish. nurse practitioner, ( ), , , . andreoli, e. m., & miller, j. s. ( ). aging in the amish community. nursing connections, ( ), - . banks, m. j., & benchot, r. j. ( ). unique aspects of nursing care for amish children. american journal of maternal child nursing, ( ), - . barrett, s. ( ). quackwatch. retrieved may , from http:// www.quackwatch.com/ beachy, a., hershberger, e., davidhizar, r., & giger, j. n. ( ). cultural implications for nursing care of the amish. journal of cultural diversity, ( ), - . brewer, j. a., & bonalumi, n. m. ( ). cultural diversity in the emergency department. journal of emergency nursing, ( ), - . cannon, s. ( ). plain prey. old mines yielding new bonanza: radioactive gas. retrieved april , , from http://www.kcstar.com/plain/stories/ mine .htm dickinson, n., slesinger, d. & raftery, p. ( ). a comparison of the per- ceived health needs of amish and non-amish families in cashton, wisc. wisconsin medical journal, ( ), - . girod, j. ( ). a sustainable medicine: lessons from the old order amish. journal of medical humanities, ( ), - . hewner, s. j. ( ). fertility, migration, and mortality in an old order amish community. american journal of human biology, , - . kreps, g. m., donnermeyer, j. f., & kreps, m. w. ( ). a quiet moment in time: a contemporary view of amish society. sugarcreek: carlisle press. mccollum, c. ( ). physicians’ perspectives on treating amish patients. wisconsin medical journal, ( ), - . mcginn, k. ( ). debbie laberge: caring for amish and mennonite fam- ilies. nurse practitioner forum, ( ), - . mcgraw, m. j. ( ). watson’s philosophy in nursing practice. in m. r. alligood & a. m. tomey (eds.), nursing theory: utilization and appli- cation (pp. - ). st. louis, mo: mosby. mckusick, v. a. ( ). the amish. endeavour, ( ), - . mitchell, b. d., hsueh, w., king, t. m., pollin, t. i., sorkin, j., agarwala, r., et al. ( ). heritability of life span in the old order amish. american journal of medical genetics, , - . o’neil, d. j. ( ). explaining the amish. international journal of social economics, ( ), - . palmer, c. v. ( ). the health beliefs and practices of an old order amish family. journal of the american academy of nurse practitioners, ( ), - . sampson, w. ( , fall/winter). the pharmacology of chelation therapy. scientific review of alternative medicine. retrieved june , from: http://www.quackwatch.com/ watson, j. (n.d.). watson’s caring theory. retrieved october , , from: http//www .uchsc.edu/son/caring/content/wct.asp wiggins, l. r. ( ). health and illness beliefs and practices among the old older amish. health values: achieving high level wellness, ( ), - . sharon m. weyer, msn, rn, np-c, is a research nurse in the de- partment of family medicine, research division at case western reserve university and a family nurse practitioner at the viola startzman free clinic in wooster, ohio. her research, teaching, and clinical interests include health promotion/disease prevention and access to health care for the uninsured. victoria r. hustey, msn, aprn, bc, is a registered nurse in the department of pediatric intensive care at university hospitals of cleveland, rainbow babies and children’s hospital, and pediatric clinical instructor at kent state univeristy, ashtabula. her clinical interests include family nurse practitioning. lesley rathbun, msn, rn, cnm, is an obstetrical staff nurse in the department of nursing at middlefield care center—amish birthing center. her clinical interests include midwifery and family practice. she is a master’s student in midwifery and family nurse practitioner at case western reserve university, frances payne bolton school of nursing. vickie l. armstrong, msn, rn, is a clinical nurse in the de- partment of medical/surgical nursing at university hospitals of cleveland. her clinical interests include geriatrics. she is currently a master’s student in the gerontological nurse practitioner pro- gram. she also serves on the clinical faculty for case western re- serve university, frances payne bolton school of nursing. samantha reed anna, msn, rn, is a staff nurse in the postanesthesia care unit at the cleveland clinic foundation. her teaching interests include undergraduate clinical education. she is currently a master’s student in the family nurse practitioner pro- gram at case western reserve university. she also serves on the clinical faculty at the frances payne bolton school of nursing. jeanne ronyak, msn, rn, is a clinical nurse in the family birth center at st. francis health center. her clinical interests include maternal and child health nursing and family nurse practitioning. carol savrin, nd, cpnp, fnp-c, is an assistant professor in the department of nursing at case western reserve university. her re- search interests include cardiovascular risk factors in adolescents. she is also lead faculty in the pediatric and family nurse practitioner programs at case western reserve university. weyer et al. / a look into the amish culture by books editorial on january , http://tcn.sagepub.comdownloaded from http://tcn.sagepub.com humanity and sustainability humanities , , - ; doi: . /h humanities issn - www.mdpi.com/journal/humanities editorial humanity and sustainability shu-kun lin mdpi ag, postfach, ch- basel, switzerland; e-mail: lin@mdpi.com; website: http://www.mdpi.org/lin/ received: september / accepted: september / published: september so far our open access publishing company mdpi (multidisciplinary digital publishing institute) has published mainly science, medicine and technology journals. to become a multidisciplinary publisher, we launched the journal sustainability [ ]. more recently, we started to run several social science journals, including societies [ ], religions [ ], administrative sciences [ ] and behavioral sciences [ ]. today we published the first paper [ ] of the inaugural issue of humanities (issn - ). this will be an international open access journal, publishing scholarly papers of high quality across all humanities disciplines. as a publisher, i would like to publish journals surrounding the topics of sustainability and i believe the humanities as a discipline of academic studies are very important. as a scientist, i believed science and technology will only benefit human beings. i was raised in a small village, living a very primitive life in a peasant family: no electricity, no machines, of course no tv and no refrigerator. now, the life of my children is completely different. even my own life has completely changed. i have witnessed very rapid changes: more and more machines are used to consume mineral resources and energy and to pollute the environment, in order to produce more and more powerful machines (we are also launching a journal titled machines, in which the relationship between man and machine should be an interesting topic.). machines are more and more like human individuals consuming resources themselves (we are launching a journal titled resources). what is the very human condition for us to preserve so that human beings’ existence is sustained? what features are the most important for a human to be labeled as rich, successful or highly respectable? regarding consumption and possession, is it material (for example, a man or a family or a country is the owner of isolated precious metals such as a large pile of gold bars, or the owner of many houses and cars) or alternatively information (for example someone has a lot of knowledge, or has watched many movies where the amount can be measured by number of bits, or know-how, etc.), or both? there are some other human traits which might find difficult to be quantitatively assessed by weights (for material) or bits (information); these characteristics of human beings as an intelligent animal are probably spirituality or morality, etc. it is said that humanities studies do not apply open access humanities , quantitative scientific methods. i wonder if degree of beauty, degree of honesty, degree of heroism, etc., can be defined or not. probably what is most important to the sustainability of human beings is virtues (heroism, altruism, honesty, frugality, etc.) and traditions. i watched the american movie witness several times [ ] from which i learnt that the amish communities are allowed to preserve their ordnung and live their own traditional life, tolerated by other communities and protected by the government. for another example, anson burlingame ( – ), an american, might be regarded as one of the most respectable figures in the history of the modern world: his great diplomatic work for the chinese qing empire (or manchu dynasty, – ) perhaps has prevented china from becoming divided colonies of western powers. while research, development and production of material objects in our industrial systems depend on resource exploitation, humanities studies and practice themselves may not necessarily need any significant amount of material resources. humanities studies deal almost exclusively with information. a typical intellectual (including scientists) of my previous generations in china must learn many humanities subjects, for example, the four arts of the chinese scholar qin (music), qi (strategic board game), shu (calligraphy) and hua (painting), and five confucian virtues ren (charity), yi (justice), li (courtesy), zhi (wisdom), xin (sincerity). a typical scholar in an eastern asian country (china, japan, korea, vietnam and perhaps other countries) in the early days learn and use wenyanwen (classical chinese or sometimes called literary chinese), similarly to how latin was studied and used by scholars in many western countries before. if we, the people, give up all humanities studies and practices, individuals will become more or less machines. humanities study and practice do not destroy our ecological system and the practices, if resumed or widely continued, are themselves the very sustainability of human tradition and identity. with rapid globalization and the increasingly widespread availability of the internet, the time is now ideal to publish the journal humanities in open access format. enjoy publishing with us. references and notes . sustainability journal homepage: http://www.mdpi.com/journal/sustainabilities/. . societies journal homepage: http://www.mdpi.com/journal/societies/. . religions journal homepage: http://www.mdpi.com/journal/religions/. . administrative sciences journal homepage: http://www.mdpi.com/journal/admsci/. . behavioral sciences journal homepage: http://www.mdpi.com/journal/behavsci/. . bednarik, r. the origins of human modernity. humanities , , - . . see: http://en.wikipedia.org/wiki/witness_( _film). © by the authors; licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution license (http://creativecommons.org/licenses/by/ . /). long-term exposure to particulate air pollution and brachial artery flow-mediated dilation in the old order amish research open access long-term exposure to particulate air pollution and brachial artery flow-mediated dilation in the old order amish shabnam salimi †, jeff d. yanosky *†, dina huang , jessica montressor-lopez , robert vogel , robert m. reed , braxton d. mitchell , and robin c. puett abstract background: atmospheric particulate matter (pm) has been associated with endothelial dysfunction, an early marker of cardiovascular risk. our aim was to extend this research to a genetically homogenous, geographically stable rural population using location-specific moving-average air pollution exposure estimates indexed to the date of endothelial function measurement. methods: we measured endothelial function using brachial artery flow-mediated dilation (fmd) in community- dwelling healthy amish participants. exposures to pm < . μm (pm . ) and pm < μm (pm ) were estimated at participants’ residential addresses using previously developed geographic information system-based spatio-temporal models and normalized. associations between pm exposures and fmd were evaluated using linear mixed-effects regression models, and polynomial distributed lag (pdl) models followed by bayesian model averaging (bma) were used to assess response to delayed effects occurring across multiple months. results: exposure to pm was consistently inversely associated with fmd, with the strongest (most negative) association for a -month moving average (− . ; % ci: − . , − . ). associations with pm . were also strongest for a -month moving average but were weaker than for pm (− . ; % ci: − . , − . ). associations of pm . and pm with fmd were somewhat stronger in men than in women, particularly for pm . conclusions: using location-specific moving-average air pollution exposure estimates, we have shown that - month moving-average estimates of pm . and pm exposure are associated with impaired endothelial function in a rural population. keywords: endothelial function, cardiovascular disease, air pollution, particulate matter © the author(s). open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. the creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/ . /) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * correspondence: jyanosky@phs.psu.edu †shabnam salimi and jeff d. yanosky are co-first author. department of public health sciences, college of medicine, the pennsylvania state university college of medicine, hope drive, hershey, pa , usa full list of author information is available at the end of the article salimi et al. environmental health ( ) : https://doi.org/ . /s - - -y http://crossmark.crossref.org/dialog/?doi= . /s - - -y&domain=pdf http://creativecommons.org/licenses/by/ . / http://creativecommons.org/publicdomain/zero/ . / mailto:jyanosky@phs.psu.edu introduction endothelial cells residing in the inner layer of blood ves- sels are a key determinant of vascular health. endothelial cell function includes vasoconstriction and vasodilation through nitric oxide release. endothelial damage results in aggregation of platelets and their adhesion to the vas- cular wall. these processes can lead to thromboses resulting in cardiovascular events (e.g., stoke, incident myocardial infarction, angina, coronary revasculariza- tion, cardiac arrest, cardiovascular (cvd)-associated death [ ];); such thrombotic conditions can be ad- dressed medically to prevent cardiovascular events in pa- tients with and without known cvd [ ]. endothelial dysfunction is widely recognized as an initial and revers- ible precursor in the progression of atherogenesis [ ]. through continued oxidation and inflammation, factors contributing to endothelial dysfunction lead to the pro- gression of atherosclerosis. brachial artery endothelial function is affected by sev- eral established risk factors for cvd, including hyper- tension, homocystinuria, oxidized low density lipoprotein (ldl) cholesterol, tobacco smoking, and oxi- dative stress [ , ]. further, brachial artery flow- mediated dilation (fmd), assessed non-invasively with ultrasound, is a broadly used assessment of endothelial function in adults [ , , , , ]. a substantial body of epidemiologic evidence has linked exposure to particulate matter (pm) air pollution to a wide array of adverse cardiovascular outcomes [ , , , , , , – , , ], including some effects con- sidered to be directly downstream of impaired endothe- lial function. for example, exposure to pm < . μm in aerodynamic diameter (pm . ) has been associated with cardiovascular risk factors including hypertension, sys- temic inflammation, oxidative stress, and established atherosclerosis [ , ]. though results from some earlier studies were inconsistent ([ ] and references therein), two recent epidemiologic studies that used pm air pollu- tion exposure modeling similar to that in the present analysis have reported associations between long-term pm . exposure and brachial artery fmd [ , ]. wilker et al. used a -year average of spatio-temporal pm . model predictions from the year , with m spatial resolution, as a surrogate for long-term exposure. krishnan et al. applied a hierarchical spatio-temporal universal kriging model of two-week average pm . levels, taking a -year average of model predictions for the year . neither of these analyses used moving av- erages specific to the date of fmd measurement, nor did they report on whether results were sensitive to the selected averaging period of year as compared to shorter or longer averaging times (with the exception that krishnan et al. evaluated and days prior to fmd measurement). also, neither of these analyses was performed in a primarily rural population. we sought to address these gaps using a genetically homogenous, geo- graphically stable population. we hypothesized that ele- vated pm exposures result in detrimental effects on endothelial function assessed by fmd. our objective was to determine whether and over what averaging period long-term exposure to pm . and pm < μm in aerodynamic diameter (pm ) were associated with fmd in a community-based sample of healthy indi- viduals from an amish community in lancaster county, pa. methods study design we performed a retrospective cohort study of partici- pants recruited into the heredity and phenotype inter- vention (hapi) heart study [ ]. the hapi heart study was conducted among the amish community in lancaster county, pa and designed to identify potential genetic and environmental risk factors of cvd [ ]. relevant to the present study, the lancaster amish are characterized by high levels of physical activity, homo- geneity of socioeconomic status and lifestyle, limited use of prescription medications, and relative geographic sta- bility. further details of the hapi heart study are avail- able elsewhere [ ]. briefly, the hapi heart study was a community-wide study of clinically healthy individuals aged years and older with the following major exclu- sions: currently pregnant or < months post-partum, blood pressure at the time of screening > / mmhg, and unable or unwilling to safely discontinue medications potentially affecting study outcomes (pre- scription medication use has been documented to be lower among this population than the general popula- tion [ ]). a total of participants were enrolled in the hapi study from to , of whom endothelial function was assessed on the exam day by fmd in participants (some of whom in family groups) in the present analysis. demographic, health behavior (e.g., smoking and eating habits), and family and medical history infor- mation was obtained through interviews by a study nurse and amish liaison in participant homes. body mass index (bmi) and fmd were measured at the amish research clinic in lancaster county, pa. all study participants provided written informed consent prior to data collection, and the hapi heart study protocol was approved by the institutional review board at the university of maryland. the protocol for the present air pollution ancillary study was ap- proved by the institutional review boards at the university of maryland and pennsylvania state university. salimi et al. environmental health ( ) : page of brachial artery fmd endothelial function was measured by brachial artery re- activity test (bart) to assess fmd using standardized procedures based on the international brachial artery task force and on expert guidelines [ , , ]. all partici- pants were fasting overnight ( – h) and were abstinent from food, caffeine, alcohol, and smoking. all medications (vasoactive and other), vitamins, and supplements were discontinued for days prior to the study. base brachial artery diameter (bad) and blood vel- ocity were measured in the left brachial artery above the antecubital fossa using mhz ultrasound (phillips hdi cv) with participants sitting supine for min prior to the measurement. then, using a standard sphygmo- manometer cuff above the antecubital fossa, inflation was applied by mmhg above systolic blood pressure for min to occlude blood flow to the brachial artery and induce ischemia. following the fast deflation of the cuff to induce post-ischemic hyperemia, the blood vel- ocity and brachial artery diameter were recorded. all images were measured in a blinded fashion by a trained technician and manually analyzed by a cardiolo- gist as a single reader of the records. percent fmd was computed as: ðmaxad−badbad Þ� % , where maxad is the maximum brachial artery diameter. shear stress and response to shear stress vascular endothelial cells are exposed to blood velocity- mediated shear stress. shear stress is the product of blood viscosity by shear rate: shear stress ¼ η v r η = blood viscosity; v = blood velocity; r = brachial ar- tery diameter. assuming blood viscosity is constant, an increase in blood velocity leads to an increase in wall shear stress, which then evokes endothelial cell-mediated nitric oxide release and vessel dilation to decrease the shear stress. in the context of endothelial dysfunction, however, dilation following increased blood flow is impaired and response to shear stress is low [ ]. after brachial artery occlusion, blood velocity increases which results in shear stress: shear stress � post occlusion blood velocity bad the vasodilation (maxad) following cuff deflation and hyperemia occurs as the response to the shear stress [ ]: response to shear stress � post occlusion blood velocity maxad the units of shear stress and response to shear stress were cm s− mm− . particulate air pollution exposure assessment residential addresses collected at the time of interview were geocoded using arcgis . software (esri, red- lands, california). long-term exposures to pm . and pm were estimated by applying previously-developed gis-based spatio-temporal generalized additive mixed models (gamms) of pm . and pm monthly-average mass concentrations, respectively [ , ], at the partici- pant’s geocoded residential addresses. these spatio- temporal models included ) monthly spatial smooth terms and ) smooth regression terms of a) gis-based covariates and b) time-varying meteorological covariates. the models were developed using pm . and pm mon- itoring data collected across the conterminous us from to ( and monitoring sites, respect- ively). exposure models were validated using cross- validation and had high predictive performance (cross- validation r ’s of . and . for pm . and pm , re- spectively). monthly exposure estimates specific to the residential location of each participant were averaged over a -month time period prior to the date of fmd measurement (the month prior to fmd measurement and the months before that) for use in our a priori analysis. also, in subsequent analyses, moving averages over time periods of , , , , , and months were generated. monthly exposure estimates were used to construct sets of polynomial distributed lag (pdl) basis coefficients [ ]. statistical analysis linear mixed-effect regression models were used to evaluate associations between pm . and pm exposure metrics with endothelial function measured by brachial artery fmd. analyses were conducted using sas version . (cary, north carolina) and mixed models analysis for pedigrees (mmap website [ , ]); mixed-effect models included a random effect for family structure to allow for the relatedness of participants within the amish community. statistical tests were -sided and p- values< . were considered statistically significant. in our regression models, we evaluated age, bmi, bad, sex, age by sex, smoking (current, ever, never), season of the year (four seasons based on calendar month), serum cholesterol, serum triglyceride, and hypertension as po- tential confounders. we repeated the above analyses ex- cluding bad from the model. we also performed the above analyses stratified by age (younger than vs. years and older) by sex. using interaction terms to assess effect modification, we evaluated: ) age by sex ) pm . or pm by age, ) pm . or pm by sex, and ) pm . or pm by sex and age < using interaction terms. we salimi et al. environmental health ( ) : page of did not evaluate pm . or pm by smoking interactions because none of our female participants were ever- smokers. because shear stress induces release of nitric oxide from endothelial cells resulting in response to shear stress, we evaluated associations of pm . and pm with shear stress and response to shear stress with adjustment for bad and the other covariates mentioned above. as a priori hypotheses, we first fit models using - month moving-averages of pm . and pm exposures. next, in subsequent exploratory analyses, we evaluated other averaging periods ( , , , , , months). the steps in our model development process were as follows: first, we fit crude models for a given averaging period for pm . and pm , then added potential confounders to these models, and next evaluated effect modification. to evaluate the assumption of a constant effect (i.e., one that does not taper off in time) of pm . or pm ex- posure over a given averaging period, we performed pdl models followed by bayesian model averaging (bma) [ ]. in exploratory analyses, we first identified the averaging period with the largest absolute value of effect among the fully adjusted moving-average models. we then fit pdl models from to th order using the number of months in the averaging period selected above as the lag period. we then used bma to calculate the probability-weighted average of the coefficients, given the data, from the resulting pdl models. all final models were performed using linear mixed-effects models to account for family structure among partici- pants (referred to as “polygenic mixed-effects models”). to compare the strength of associations of endothelial function measures between pm . and pm , we normal- ized the pm exposure variables by subtracting their re- spective mean and dividing by their respective standard deviation. all results presented are based on normalized exposures, except those in table s . results study population participant characteristics are presented in table . the mean age in our study population was . years (sd: . ) and . % were women. among men, . % were current smokers and . % were ever smokers; in con- trast none of the women reported smoking. few partici- pants reported a history of hypertension ( . %), high cholesterol ( . %), diabetes mellitus ( . %), or heart at- tack ( . %). fmd measures were approximately nor- mally distributed with a mean value of . % (sd: . ). mean pm . and pm -month moving averages were . μg m− (sd: . ; interquartile range (iqr): . ) and . μg m− (sd: . ; iqr: . ), respectively. distribu- tions of the pm . and pm exposure metrics and add- itional summary statistics are shown in fig. . associations of pm . and pm with fmd and shear stress measures table shows associations of pm . and pm exposure with fmd across all participants and for men and table characteristics of study participants clinical characteristic mean (sd) or n (%) across all men women number of participants ( %) ( . %) ( . %) age at examination (years) . ( . ) . ( . ) . ( . ) current smokers ( . %) ( . %) ( %) ever smokers (not currently smoking) ( . %) ( . %) ( %) never smokers ( . %) ( . %) ( %) body mass index (kg m− ) . ( . ) . ( . ) . ( . ) hypertension (diagnosed) ( . %) ( . %) ( . %) high cholesterol (self-report) ( . %) ( . %) ( . %) diabetes mellitus (self-report) ( . %) ( . %) ( . %) myocardial infarction (self-report) ( . %) ( . %) ( . %) vascular measures baseline brachial artery diameter (bad; mm) . ( . ) . ( . ) . ( . ) flow-mediated dilation (fmd; %) . ( . ) . ( . ) . ( . ) shear stress (cm s− mm− ) . ( . ) . ( . ) . ( . ) pre-occlusive blood velocity (cm s− ) . ( . ) . ( . ) . ( . ) post-occlusive blood velocity (cm s− ) . ( . ) . ( . ) . ( . ) response to shear stress (cm s− mm− ) ( . ) . ( . ) . ( . ) salimi et al. environmental health ( ) : page of fig. distributions and summary statistics of the -month moving average pm . and pm exposures, prior to normalization table associations of pm . and pm exposure metrics and fmd (%), across all participants and by sex, for increases in normalized pm . or pm exposure, in fully adjusted models particulate air pollution metrics (normalized) across all men women β se p-value % ci β se p-value % ci β se p-value % ci pm . -month moving-average* − . . . − . , − . − . . . − . , − . − . . . − . , . pm -month moving-average* − . . . − . , − . − . . . − . , − . − . . . − . , . *all models adjusted for age, sex, age by sex interaction, smoking (except in models for women only because there were no ever smokers), bmi, season, year, hypertension, and base brachial artery diameter salimi et al. environmental health ( ) : page of women separately with effect sizes for a normalized change in pm exposure (calculated by subtracting the mean and dividing by the sd). to afford comparisons with other studies, we also present these associations for a μg m− increment in pm . or pm exposure in table s . regression models were adjusted for age, bmi, and bad as linear variables, and sex, smoking (current, ever, never), season of the year (four seasons based on calendar month), and hypertension as categorical vari- ables. for neither pm . nor pm was there significant evidence for a pm by sex interaction (p > . ). pm . . associations for pm . were strongest (i.e., fur- thest from zero), among the averaging periods consid- ered, for a -month moving average. there was a significant inverse association between long-term expos- ure to pm . and fmd: for a one unit increase in nor- malized -month moving-average pm . , fmd decreased by . ( % ci: − . , − . ; p = . ) in our polygenic mixed-effects model. we repeated the analyses excluding bad from the model and the effect sizes were identical. in sex-stratified analyses, pm . was significantly asso- ciated with fmd in men (β = − . ; % ci: − . , − . ; p = . ), but not in women (β = − . ; % ci: − . , . ; p = . ) (table ). we also performed sex and age-stratified analyses: associations remained moder- ately larger in men than in women and were larger in older (≥ years) as compared to younger (< years) individuals, though none were statistically significant in these subgroups (table s ). pm . was not significantly associated with response to shear stress in the total population (p > . ), or in sub- analyses of men only or women only (p > . for both) (table ). pm . as for pm . , associations for pm were stron- gest (i.e., furthest from zero), among the averaging pe- riods considered, for a -month moving average. there was a significant inverse association between long-term exposure to pm and fmd. this association was stron- ger than that for pm . for the equivalent averaging period. for a one unit increase in normalized -month moving-average pm , fmd decreased by . ( % ci: − . , − . ; p = . ) in our polygenic mixed-effects model. there was no evidence of effect modification in associations between -month moving-average pm and fmd by age or sex (p > . for each). however, the association between fmd and pm was stronger and more significant in men (β = − . ; % ci: − . , − . ; p = . ) than in women (β = − . , % ci: − . , . ; p = . ) (table ). in age- and sex-stratified analyses, pm was more strongly associated with fmd in men than in women in both younger and older individuals. only in the sub- group of men younger than age years did the associ- ation between pm and fmd achieve statistical significance (β = − . ; %ci: − . , − . ; p = . ) (table s ). pm was marginally associated with response to shear stress (β = . ; % ci: − . , . ; p = . ) in the total study population, and in sex-stratified analyses the effect size was considerably larger in women compared to men, although in neither sex did the association achieve statistical significance (β = . ; p = . in women and β = . ; p = . in men) (table ). time course of effect of pm . and pm on fmd because associations of pm . and pm moving aver- ages were strongest for the time period months prior to the date of fmd measurement, a -month lag period was selected for pdl models. results from these pdl models and subsequent bma showed that . % and . % of the posterior probability corresponded to the zero-order model (equivalent to the moving-average) for pm . and pm , respectively (table s ). the next most influential model was the first-order, or linear decay model, which contributed only . and . % for pm . and pm , respectively. weighted coefficients from bma of these six models, for both pm . and pm did not in- dicate substantial non-linearity or even linear decay in the response to lagged exposures over the prior months (figure s ). discussion in our study population, significant inverse associations were observed between long-term residential pm . and pm levels and endothelial function measured by bra- chial artery fmd. though associations were suggestive of stronger pm . and pm effects in men than in women, interaction terms were non-significant. these trends persisted in age- and sex-stratified analyses, for table associations of pm . and pm exposure metrics and response to shear stress (cm s − mm− ), across all participants and by sex, for increases in normalized pm . or pm , in fully adjusted models particulate air pollution metrics (normalized) across all men women β se p-value % ci β se p-value % ci β se p-value % ci pm . -month moving-average* . . . − . , . − . . . − . , . . . . − . , . pm -month moving-average* . . . − . , . . . . − . , . . . . − . , . *all models adjusted for age, sex, age by sex interaction, smoking (except in models for women only because there were no ever smokers), bmi, season, year, hypertension, and base brachial artery diameter salimi et al. environmental health ( ) : page of which associations between pm . and pm and fmd were stronger in men than in women, for both those < years and greater. one explanation for stronger associations of pm . and pm with fmd in men as compared to women is that amish men have higher levels of physical ac- tivity than amish women, as we have previously shown using -day accelerometer counts [ ]. if this physical ac- tivity occurs largely outdoors, it is plausible that their per- sonal exposure would be more highly correlated with ambient pm levels. another explanation is that amish women are non-smokers and have less outdoor activity. finally, our results suggest greater effects of pm . and pm on fmd among older adults (> years), which may indicate increased susceptibility. however, because these interactions were not statistically significant, this conclusion requires replication in a larger sample size. in contrast to fmd, a stronger association between pm and response to shear stress was observed among women compared to men, suggesting greater nitric oxide bioavailability among women. for both pm . and pm , the data indicated selection of a -month averaging period was appropriate. earlier or later exposures to pm . or pm within the prior - month period did not substantially affect fmd re- sponses, as evidenced by the overwhelming dominance of the zero-order pdl models for both pm . and pm . results indicated stronger associations with pm com- pared to pm . for equivalent averaging periods, except in the women only group for which associations were non-significant (table ). one possible explanation is compositional differences in pm vs. pm . . another is that we measured endothelial function in the brachial ar- tery, which is a relatively large blood vessel, whereas pm . may exert the majority of its influence on smaller vasculature. finally, the spatial misalignment of the pm . and pm monitors may have contributed to ex- posure errors. the largest source of these errors appears primarily due to the apparent underestimation of pm levels at a pm monitor near to many of the participant residences; in contrast pm . monitors were farther away and reported higher levels. we note that pm . accounts for the majority of the mass of pm in most areas of the us [ ]. despite the limitation imposed by this spatial error, we believe the estimates of association for pm . and pm in our study are valid because consistent, sys- tematic overestimation of measured pm . levels is not ex- pected to change the rank-ordering of pm . exposure values; similarly, for underestimation of pm . however, dose-response interpretations based on this analysis should be viewed with caution. future research on pm ef- fects on endothelial function using personal exposure monitoring for pm . and pm to further clarify differ- ences in toxicity based on particle size fraction and/or composition is warranted. compared to previous studies, our results for pm . are consistent in direction but larger in magnitude of ef- fect (table s ). one explanation is that our exposure as- sessment approach better described gradients in exposure resulting in less misclassification. additionally, differences in time-activity patterns, with amish men spending more time outside than participants in other studies, may have affected our results. wilker et al. [ ] conducted an analysis of long-term pm . exposure and fmd among participants of the framingham offspring and third generation cohorts (n = ) and reported a smaller effect size compared to ours (over an approxi- mated μg m− increment: ( / . )*(− . ) = − . % in fmd vs. our result of − . % (table s )). in krishnan et al. [ ], an analysis among members of the multi- ethnic study of atherosclerosis and air pollution study (n = ), authors also reported an effect size for long-term pm . and fmd smaller than the present analysis (over an approximated μg m− increment: ( / . )*(− . ) = − . % in fmd vs. our result of − . %). both of these studies used average pm . levels over one calendar-year as surrogates of long-term exposure, and thus did not average over the spe- cific -months prior to fmd measurement (i.e., over a “moving-window”). as stated above, our analysis supports the use of a -month moving-average period, when avail- able, with regard to studies of health effects of pm . and pm on fmd. although the underlying biological mechanism of these effects is not currently well understood, it has been postulated that exposure to pm . may regulate endothe- lial function through altered expression or function of the enzyme nitric oxide synthase which results in re- duced bioavailability of endothelium-derived nitric oxide, a key component of vascular homeostasis [ ]. recent animal studies have elucidated the role of endo- thelial progenitor cells in this process [ ]. our results generally showed stronger, albeit not significantly, asso- ciations of pm exposure with response to shear stress in women than in men, which is consistent with greater resiliency of women to pm-induced endothelial dysfunc- tion, perhaps attributable to protective hormonal effects or absence of smoking in women. our study has several strengths. the homogeneity of the study population with regard to lifestyle and behav- ioral factors such as physical activity, diet, and formal education reduces the possibility that observed pm air pollution-fmd relationships are confounded by these factors. to our knowledge, this is the first study of air pollution and endothelial function in a genetically homogenous, rural population of clinically healthy indi- viduals. moreover, fmd was measured by a single well- trained sonographer and data was obtained by a single cardiologist using high quality-control standards. in addition, we used gis-based spatio-temporal exposure salimi et al. environmental health ( ) : page of models to predict time-varying (i.e., monthly) pm . and pm exposure estimates specific to each participant’s residential address and used this data to calculate expos- ure metrics based on the fmd exam date. limitations of our study include a lack of racial/ethnic variability such that our findings are not generalizable to other ethnicities. also, our exposure estimates were found to contain exposure error such that pm . esti- mates were sometimes slightly higher than pm esti- mates, limiting conclusions regarding the relative toxicity of pm . as compared to pm from these data. moreover, exposures to pm . and pm were based on monthly averages, preventing our analyses from address- ing acute exposures to pm . or pm . this study provides support for associations between long-term exposure to both pm . and pm with bra- chial artery fmd. this effect manifested maximally over the time course of approximately months for both pm . and pm . associations with fmd were stronger (i.e., more negative) for pm than for pm . . results were suggestive of stronger associations in men than women, though these interactions did not reach statis- tical significance. the findings bolster the existing evi- dence regarding the effects of pm air pollution on cvd risk and suggest long-term exposures to pm . and pm are plausible early risk factors of cardiovascular events. supplementary information supplementary information accompanies this paper at https://doi.org/ . /s - - -y. additional file : table s . polynomial distributed lag (pdl) model posterior probabilities obtained from bayesian model averaging (bma) for zero-order through th order models using lag periods, for pm . and pm . table s . associations of pm . and pm exposure metrics and fmd stratified by age (less than years vs. years and older) and sex for increases in normalized pm . and pm exposure in fully adjusted models. table s . associations of pm . and pm exposure metrics and fmd across all participants and by sex for a μg m− increment in pm . and pm exposure in fully adjusted models. figure s . effect esti- mates from the six pdl models (zero-order through th order) weighted using bma as discussed in main text, for: a) pm . and b) pm . the month prior to fmd measurement (lag zero) and months prior are shown, as well as the cumulative effect over all lag periods. abbreviations bma: bayesian model averaging; fmd: flow-mediated dilation; pm . : particulate matter < . μm; pm : particulate matter < μm acknowledgements the authors thank dr. joel schwartz for sharing sas code for the bma analysis and dr. duanping liao for sharing the sas code for the pdl models. authors’ contributions jdy performed data analyses and was the lead writer. ss contributed to the data collection and processing, performed data analyses, reviewed the manuscript, and participated in revisions. dh contributed to the analysis, reviewed the manuscript, and participated in revisions. jml contributed to the analysis, reviewed the manuscript, and participated in revisions. rv conceived of the study and reviewed the manuscript. rmr contributed to the analysis, reviewed the manuscript, and participated in revisions. bdm conceived of the study, coordinated study personnel, contributed to the analysis, reviewed the manuscript, and participated in revisions. rcp conceived of the study, coordinated study personnel, contributed to the analysis, reviewed the manuscript, and participated in revisions. the authors read and approved the final manuscript. funding this research was supported by nih grants u hl and p dk and by a university of maryland seed grant entitled “ambient air pollution and metabolic syndrome in the lancaster county amish”. also, ss was supported by nih grant number k ag - a . availability of data and materials supporting data is not available due to confidentiality constraints involved with human subject’s research. ethics approval and consent to participate informed consent was obtained from study participants and approval was obtained from the irb’s of both the university of maryland and the penn state college of medicine. consent for publication the authors consent to publication. competing interests the authors declare that they have no conflicts of interest. author details department of medicine, university of maryland school of medicine, baltimore, md, usa. department of public health sciences, college of medicine, the pennsylvania state university college of medicine, hope drive, hershey, pa , usa. department of epidemiology and biostatistics, university of maryland school of public health, college park, md, usa. maryland institute of applied environmental health, university of maryland school of public health, college park, md, usa. department of medicine, division of pulmonary and critical care medicine, university of maryland school of medicine, baltimore, md, usa. geriatrics research and education clinical center, baltimore veterans administration medical center, baltimore, md, usa. received: september accepted: march references . anderson jo, thundiyil jg, stolbach a. clearing the air: a review of the effects of particulate matter air pollution on human health. j med toxicol. ; : – . . bonetti po, lerman lo, lerman a. endothelial dysfunction: a marker of atherosclerotic risk. arterioscler thromb vasc biol. ; : – . . brook rd, brook jr, urch b, vincent r, rajagopalan s, silverman f. inhalation of fine particulate air pollution and ozone causes acute arterial vasoconstriction in healthy adults. circulation. ; : – . . brook rd, rajagopalan s, pope ca iii, brook jr, bhatnagar a, diez-roux av, holguin f, hong y, luepker rv, mittleman ma, peters a, siscovick d, smith sc jr, whitsel l, kaufman jd. particulate matter air pollution and cardiovascular disease: an update to the 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environmental health ( ) : page of http://edn.som/umaryland.edu/mmap/index.php http://edn.som/umaryland.edu/mmap/index.php https://doi.org/ . / . . n 	abstract 	background 	methods 	results 	conclusions 	introduction 	methods 	study design 	brachial artery fmd 	shear stress and response to shear stress 	particulate air pollution exposure assessment 	statistical analysis 	results 	study population 	associations of pm . and pm with fmd and shear stress measures 	time course of effect of pm . and pm on fmd 	discussion 	supplementary information 	abbreviations 	acknowledgements 	authors’ contributions 	funding 	availability of data and materials 	ethics approval and consent to participate 	consent for publication 	competing interests 	author details 	references 	publisher’s note pteridines € eur - euro £ gbp - pound $ usd - dollar en english deutsch × your purchase has been completed. your documents are now available to view. × changing the currency will empty your shopping cart. confirm cancel pteridines official journal of the international society of pteridinology issn: - first published: april , editor-in-chief: dietmar fuchs impact factor: . 	overview 	latest issue 	issues 	ranking 	submit 	editorial about this journal objective pteridines is an open acess international quarterly journal dealing with all aspects of pteridine research. pteridines are heterocyclic fused ring compounds involved in a wide range of biological functions from the color on butterfly wings to cofactors in enzyme catalysis to essential vitamins. of the pteridines, , , , -tetrahydrobiopterin is the necessary cofactor of several aromatic amino acid monoxygenases, the nitric oxide synthases and glyceryl ether monoxygenase (gemo). neopterin plays an essential role in the immune system and is an important biomarker in laboratory medicine for diseases such as hiv, cardiovascular disease, malignant tumors, among others. topics 	neopterin, dihydroneopterin, monapterin 	biopterin, tetrahydrobiopterin 	folates, antifolates, riboflavin 	phenylalanine, tyrosine, phenylketonuria, serotonin, adrenalin, noradrenalin, l-dopa, dopamine, related biogenic amines 	phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, nitric oxide synthases (inos), alkylglycerol monooxygenase (agmo), dihydropterin reductase, sepiapterin reductase 	homocysteine, mediators of inflammation, redox systems, iron article formats original articles, reviews, mini reviews, commentaries, opinions on controversial subjects > information on submission process your benefits your benefits: 	unique interdisciplinary forum devoted to conjugated and unconjugated pteridines 	basic and clinical approach to the topic combining the fields of chemistry, biochemistry, clinical chemistry, and laboratory medicine 	international, authoritative editors and reviewers boosting the prestige of published papers 	free language-correction services for authors from non-english speaking regions 	worldwide 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document × retrieving citations for document... laboratory diagnostic value of neopterin measurements in patients with covid- infection dietmar fuchs, magnus gisslen january , page range: - more cite open access pdf pdf abstract the new coronavirus sars-cov- was identified to be responsible for the covid- pandemic. there are striking differences in the response to infection, some people develop no or mild symptoms, while other outcomes are severe of even fatal. for those covid- patients who require hospitalization, prognostic markers could help clinicians to identify patients with a poor outcome early. the serum levels of the immune activation marker neopterin have already been shown to be of prognostic value in patients with sars-cov- and a similar pattern can be observed for sars-cov- . this comment discusses the biochemical circuits that support the clinical value of neopterin measurements in covid- patients. unable to retrieve citations for this document × retrieving citations for document... norepinephrine was superior in death risk reducing and hemodynamics compared to dopamine in treatment of patients with septic shock xudong lu, xianghua xu, yueying wu february , page range: - more cite open access pdf pdf abstract background to investigate the clinical effects of norepinephrine versus dopamine in treatment of septic shock by pooling the data form open published clinical trials. material and methods the clinical trials relevant to norepinephrine versus dopamine in treatment of septic shock were electronically searched in the databases of pubmed, embase, the cochrane library, web of science, google scholar and cnki. the original data related to the treatment effects such as death risk, oxygen metabolism and hemodynamics index were extracted from the included original studies. the death risk was pooled by the effect size of relative risk (rr), the oxygen metabolism and hemodynamics index were pooled by standard mean difference (smd) and the corresponding % confidence interval ( %ci). the publication bias was evaluated by begg's funnel plot and egger's line regression test. results thirteen clinical trials were included in the meta-analysis. the pooled results demonstrated the death risk was significantly decreased (rr= . , %ci: . to . , p= . ) in septic shock patients who received norepinephrine compared to those receiving dopamine. the hr (smd=− . , %ci: − . to − . , p< . ) and cardiac index (smd=− . , %ci: − . to − . , p< . ) were lower in norepinephrine group compared to dopamine group. the systemic vascular resistance index (smd= . , %ci: . to . , p< . ) in norepinephrine group was higher than those of dopamine group with statistical difference. the begg's funnel plot and egger's line regression test (t=− . , p= . ) showed no publication bias. conclusions based on the present evidence, norepinephrine was superior to dopamine in the aspects of death risk reducing and hemodynamics. unable to retrieve citations for this document × retrieving citations for document... clinical significance of serum homocysteine as a biomarker for early diagnosis of diabetic nephropathy in type diabetes mellitus patients bin ye, xiangying zhu, zhifu zeng, xiaozhen ji, meixia ji march , page range: - more cite open access pdf pdf abstract objective the aim of this study was to investigate the clinical significance of serum homocysteine (hcy) as a biomarker for early diagnosis of diabetic nephropathy (dn) in type diabetes mellitus (t dm) patients. methods fifty-five t dm patients with dn and t dm patients without dn were prospectively recruited from january to may in our hospital. the serum hcy was tested by electrochemiluminescence assay in dn and t dm groups and compared. the diagnostic efficacy of serum hcy as a biomarker for early diagnosis of dn was evaluated by calculating the diagnostic sensitivity, specificity and area under the roc curve (auc). results the serum levels of hcy were . ± . and . ± .  μmol/l for dn and t dm patients, respectively, with statistical difference ( t = . , p < . ). in the dn group, the serum hcy levels for patients with hyperfiltration, intermittent proteinuria, microalbuminuria, macroalbuminuria and uremic were . ± . , . ± . , . ± . , . ± . and . ± .  μmol/l, respectively, which indicated that serum hcy levels in dn were higher than those of t dm patients and correlated with patient’s renal damage. using the serum hcy level as the reference, the diagnostic sensitivity, specificity and auc were . ( . – . )%, . ( . – . )% and . ( . – . )%, respectively, with the cutoff value of . between dn and t dm. the serum hcy also had relatively good differential diagnostic efficacy between different dn stages with high sensitivity, specificity and auc. conclusion serum hcy was obviously elevated in dn compared to t md and correlated with the renal damage severity, which can be applied as a potential serological marker for early diagnosis of dn. unable to retrieve citations for this document × retrieving citations for document... the urinary biopterin in autism spectrum disorder aleksandra waligóra, aleksandra damasiewicz-bodzek, piotr gorczyca, sławomir waligóra, krystyna tyrpień-golder march , page range: - more cite open access pdf pdf abstract objective the aim of the study was to determine whether biopterin is present in significantly lower quantities in urine samples of patients with autism spectrum disorder (asd) compared to healthy individuals. methods the concentration of biopterin in urine samples was measured by elisa using commercially available kit. the study involved children aged – years with asd and healthy children aged – years. results significantly lower biopterin concentration was observed in autistic patients compared to the control group. however, no significant difference was observed between mild, moderate, and severe asd. conclusion one of the potential causes of decrease in urinary biopterin levels may be tetrahydrobiopterin (bh ) deficiency, which has extensive and serious health consequences for the nervous system. the results of measuring biopterin as a fully oxidized form of bh may suggest that biosynthesis or regeneration of bh may be decreased in children with asd. on the other hand, decreased urinary biopterin levels in children with asd may be due to bh overuse, a good regeneration process, and decreased urinary excretion; and abnormalities in bh metabolism appear to be related to the aetiology of asd or may be due to asd. volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue - 	 issue 	 issue volume ( ) 	 issue - 	 issue - volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue - 	 issue 	 issue volume ( ) 	 issue - 	 issue 	 issue special issue: th international symposium on pteridines and folates, antalya, turkey, may – , volume ( ) 	 issue - 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue - volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 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ablondi, harvard medical school, usa dr kate grayson (university of bristol, uk) dr laura mcveigh (university of new south wales, australia) dr morgan milton (rti international, usa) zandile nare (university of edinburgh, uk) associate editor qian (kevin) liu m.d. (tianjin lung cancer institute, tianjin medical university general hospital, tianjin, china) editorial office publisher de gruyter poland bogumiła zuga a str. - warsaw, poland t: + editorial contact jędrzej daszkiewicz, managing editor jedrzej.daszkiewicz@degruyter.com​ open access details 	online issn: - 	 type: journal 	 language: english 	 publisher: de gruyter 	 first published: april , 	 publication frequency: issue per year 	 audience: researchers in the fields of chemistry, biochemistry, clinical biochemistry, metabolic disorders, clinical chemistry, clinical immunology, immunology, laboratory medicine, biology, molecular biology, molecular medicine, nutrition, phytomedicine 	contact us 	customer service 	human resources 	press 	contacts for authors 	career 	how to join us 	current vacancies 	working at de gruyter 	open access 	articles 	books 	funding & support 	for authors 	publish your book 	publish your journal article 	abstracting & indexing 	for libraries & trade partners 	electronic journals 	ebooks 	databases & online reference 	metadata 	our partner publishers 	rights & permissons 	repository policy 	free access policy 	about de gruyter 	de gruyter foundation 	our locations 	help/faq 	privacy policy 	terms & conditions 	legal notice © walter de gruyter gmbh rev_iss_web_mec_ _ - .. genomic patterns of introgression in rainbow and westslope cutthroat trout illuminated by overlapping paired-end rad sequencing paul a. hohenlohe,* mitch d. day,* stephen j. amish,† michael r. miller,‡ nick kamps-hughes,‡ matthew c. boyer,§ clint c. muhlfeld,¶** fred w. allendorf,† eric a. johnson‡ and gordon luikart** *department of biological sciences, institute of bioinformatics and evolutionary studies, university of idaho, moscow, id - , usa, †fish and wildlife genomics group, division of biological sciences, university of montana, missoula, mt , usa, ‡institute of molecular biology, university of oregon, eugene, or , usa, §montana fish, wildlife and parks, kalispell, mt , usa, ¶u.s. geological survey, northern rocky mountain science center, glacier national park, west glacier, mt , usa, **flathead lake biological station, fish and wildlife genomics group, division of biological sciences, university of montana, polson, mt , usa abstract rapid and inexpensive methods for genomewide single nucleotide polymorphism (snp) discovery and genotyping are urgently needed for population management and conser- vation. in hybridized populations, genomic techniques that can identify and genotype thousands of species-diagnostic markers would allow precise estimates of population- and individual-level admixture as well as identification of ‘super invasive’ alleles, which show elevated rates of introgression above the genomewide background (likely due to natural selection). techniques like restriction-site-associated dna (rad) sequencing can discover and genotype large numbers of snps, but they have been limited by the length of continuous sequence data they produce with illumina short-read sequencing. we present a novel approach, overlapping paired-end rad sequencing, to generate rad contigs of > – bp. these contigs provide sufficient flanking sequence for design of high-throughput snp genotyping arrays and strict filtering to identify dupli- cate paralogous loci. we applied this approach in five populations of native westslope cutthroat trout that previously showed varying (low) levels of admixture from introduced rainbow trout (rbt). we produced rad contigs and used these data to filter and genotype previously identified species-diagnostic snp loci. our population-level and individual-level estimates of admixture were generally consistent with previous microsatellite-based estimates from the same individuals. however, we observed slightly lower admixture estimates from genomewide markers, which might result from natural selection against certain genome regions, different genomic locations for microsatellites vs. rad-derived snps and/or sampling error from the small number of microsatellite loci (n = ). we also identified candidate adaptive super invasive alleles from rbt that had excessively high admixture proportions in hybridized cutthroat trout populations. keywords: adaptive introgression, conservation genomics, hybridization, invasive species, natural selection, next-generation sequencing, salmonids, super invasive genes received july ; revision received december ; accepted december introduction hybridization between native and introduced taxa is an increasing concern for conservation and legal asse ssments of threatened species (allendorf et al. ). correspondence: paul a. hohenlohe, fax: ; e-mail: hohenlohe@uidaho.edu © john wiley & sons ltd molecular ecology ( ) , – doi: . /mec. hybridization can reduce fitness through outbreeding depression (muhlfeld et al. a), cause genomic extinc- tion (allendorf et al. ) and destroy important genetic and ecological adaptations (muhlfeld et al. b; kelly et al. ). the loci most responsible for the genetic effects of hybridization may be outliers in their degree of introgression because of natural selection in admixed populations (‘super invasive alleles’; gompert & buerkle ; fitzpatrick et al. ; teeter et al. ; miller et al. ). as a result, estimates of admixture averaged across loci at the individual or population level may miss important genetic factors in conservation and manage- ment of native taxa. current high-throughput sequencing techniques now allow genome scans for invasive alleles in natural populations of nonmodel species. anthropogenic hybridization is especially widespread in freshwater fishes due to decades of fish translocations and hatchery supplementation of wild populations. rain- bow trout (rbt, onchorhynchus mykiss) is the most widely translocated and problematic invasive fish worldwide (halverson ). rbt hybridize with cutthroat trout (o. clarkii), including the subspecies westslope cutthroat trout (wct, o. c. lewisi). wct is the most widely distrib- uted of extant cutthroat subspecies, and hybridization is the leading threat to persistence of genetically pure wct populations (shepard et al. ). management of wct populations would benefit from detection of hybridization and introgression at low levels and from the ability to precisely estimate individual-level admixture proportion. previous work has used microsat- ellites and other loci to assess levels of admixture from rbt into native wct populations (hitt et al. ; boyer et al. ; muhlfeld et al. a,c). muhlfeld et al. ( c) found that levels of rbt admixture were negatively related to distance from the source of rbt hybridization (abbot creek; see fig. ) and positively related to mean summer water temperature, suggesting potential for the existence of rbt alleles that are adaptive to warm water temperatures (perry et al. ; narum et al. ). how- ever, the low number of diagnostic markers available with microsatellites typically allows precise admixture estimates only at the population level, not at the individ- ual or genome-scan level. single nucleotide polymorphisms (snps) are ideal markers for hybridization assessment and monitoring because hundreds of snps can be rapidly, reliably and cheaply genotyped using new genotyping platforms (morin et al. ; seeb et al. , a; angeloni et al. ; twyford & ennos ). much recent effort has been committed to assembling a set of diagnostic snp loci for rbt and wct (finger et al. ; mcglauflin et al. ; harwood & phillips ; kalinowski et al. ; amish et al. ; campbell et al. ; pritchard et al. ). a high density of markers across the genome prom- ises individual-level estimates of admixture proportion, as well as detection of super invasive alleles. however, snp discovery in salmonid fish is especially challenging due to a recent genome duplication event, making it difficult to distinguish true snps from fixed sequence differences between homeologous duplicate chromo- somal regions (allendorf & danzmann ; everett et al. ; seeb et al. b) as well as more typical tandem-duplicated paralogous regions. one way to fil- ter out both paralogs and homeologs is to gather more sequence data around candidate snp markers to resolve between next-generation sequence reads that come from one locus vs. two different loci. we previously used restriction-site-associated dna (rad) sequencing (baird et al. ) to identify several thousand wct diagnostic snps (hohenlohe et al. ). those candidate diagnostic markers have shown a high rate of subsequent validation in microfluidic pcr-based genotyping assays (amish et al. ). however, primer fig. map of the north fork flathead river study area, show- ing the five admixed westslope cutthroat trout populations examined here plus the initial source of introduced rainbow trout individuals (abbot creek; see boyer et al. ; and muhlfeld et al. c for more information on these popula- tions). © john wiley & sons ltd genomic introgression and rad sequencing design for those genotyping assays required > bp of flanking sequence on each side of each snp, which we obtained from previously published sequence data, reducing the number of candidate markers for which assays could be designed (amish et al. ). in addi- tion, our ability to distinguish duplicate sequence based on the flanking sequence was limited to the bp single-end illumina read length in that study. the approach we present here can be used to simulta- neously identify and genotype snp markers, as well as gather substantial flanking sequence, in a single rad sequencing experiment. the amount of flanking sequence is more than sufficient for primer design and also allows better discrimination of paralogous loci. restriction-site-associated dna sequencing is one of a family of genomic approaches that provide sequence data adjacent to restriction enzyme recognition sites (davey et al. ). the primary difference between rad and related techniques is that rad incorporates a random shearing step in library preparation. as a result, while the forward reads are anchored at the restriction site, the reverse reads produced by paired- end illumina sequencing of rad libraries are staggered over a local genomic region (of several hundred base pairs). these staggered paired-end reads can be assem- bled into a ‘mini-contig’, a continuous stretch of geno- mic sequence that is longer than each individual read and potentially up to kb (baxter et al. ; etter et al. a; willing et al. ; etter & johnson ). here, we designed our rad libraries so that a substantial fraction of dna fragments would produce overlapping paired-end reads, allowing assembly of contigs contain- ing both the forward and reverse reads of each pair. these ‘rad contigs’ are anchored at one end by the restriction enzyme recognition site and contain several hundred base pairs of continuous genomic sequence data across dozens of individuals. the goals of this study were to: (i) assemble a large set of rad contigs from a sample of low-admixture wct populations; (ii) provide flanking sequence for finer fil- tering of candidate diagnostic snp markers between rbt and wct; (iii) genotype filtered diagnostic snps across five wct populations to assess the ability of rad sequencing compared with microsatellites to provide precise individual-level estimates of admixture; and (iv) identify outlier loci exhibiting the signature of super invasive alleles. methods study system we focus on wct populations in tributaries to the north fork of the flathead river in northwestern montana (fig. ). the north fork flathead river originates in canada and forms the western border of glacier national park before joining the main-stem flathead river, which flows into flathead lake. the presence of hybridization and rbt admixture was previously esti- mated in several populations using seven diagnostic mi- crosatellite loci (boyer et al. ; muhlfeld et al. c). here, we use five of these populations (meadow, nicola, dutch, lower hay and tepee) for which estimates of the mean population-level admixture based on microsatellite loci ranged from . % to . % (see boyer et al. ; muhlfeld et al. c for further infor- mation on these populations). we chose populations without f hybrids as identified in previous studies with the goal of using later-generation admixed popula- tions to detect specific loci with elevated levels of intro- gression. we used preserved dna samples, collected from to individuals in each population during to for the study by boyer et al. ( ), to allow individual-level comparisons between snp-based and microsatellite-based admixture estimates. we selected individuals across the range of admixture pro- portions previously estimated within each population. rad sequencing we prepared rad sequencing libraries for samples from the five wct populations described previously, following the protocol of etter et al. ( b). the rad protocol produces libraries of genomic fragments bounded on one end by a restriction enzyme cut site (therefore common across individuals), with the other end randomly sheared. typically, fragments in rad libraries are size selected simply to optimize the effi- ciency of the illumina sequencing process. here, we used the restriction enzyme sbfi and -nucleotide bar- coded adaptors differing from each other by at least three nucleotides to identify individuals. we modified the standard protocol to target dna fragments of – bp during gel size selection, so that the size of genomic dna inserts targeted the range – bp, to produce overlapping paired-end reads for a large proportion of sequenced fragments (fig. ). we sequenced the rad libraries in portions of two lanes (grouped with other rad sequencing experiments) on an illumina hiseq sequencer at the university of oregon, producing -bp paired-end reads. we processed the sequence data and grouped the read pairs from all individuals into rad loci using sev- eral modules from the stacks software package, version . (catchen et al. ). first, using the stacks program process_radtags.pl, we sorted read pairs by barcode, filtered for read quality and removed any pairs in which the forward read did not contain both a © john wiley & sons ltd p. a. hohenlohe et al. correct barcode and the remaining six bases of the sbfi recognition sequence. we then removed read pairs that represented pcr duplicates using the stacks program clone_filter. the random shearing step in rad sequenc- ing produces staggered paired-end reads as described previously, so that any set of read pairs that are identi- cal across both the forward and reverse reads are prob- ably pcr duplicates of a single original genomic dna fragment (davey et al. ). because genotyping depends on using read counts of alternative alleles in a statistical sampling model, pcr duplicates can be misleading because they do not represent independent samples from the genomic pool of dna. we identified rad loci by applying ustacks to the forward reads across all individuals. we enabled the deleveraging and removal algorithms to filter out highly repetitive, likely paralogous loci, and we used a maximum nucleotide distance between stacks of to achieve a balance between filtering paralogs and main- taining true alleles at a single locus approximately con- sistent with the expected number of rad loci (hohenlohe et al. ; miller et al. ). we created a catalog of rad tag loci using cstacks and matched indi- viduals against the catalog using sstacks. we populated and indexed a mysql database of loci using load_ radtags.pl and index_radtags.pl and then exported the data using export_sql.pl. finally, we grouped the for- ward and reverse reads from each individual corre- sponding to each rad locus using sort_read_pairs.pl. contig assembly we pooled many individuals for contig assembly to increase sequence coverage of read pairs at each rad locus (fig. b). however, we also wanted to limit levels of polymorphism that could complicate assembly. therefore, we pooled data from individuals from the three populations with the lowest level of admixture as estimated from previous microsatellite data (boyer et al. ): lower hay, nicola and tepee. we grouped the forward and reverse reads from all individuals in these populations into a separate file for each rad locus, using the stacks program sort_read_pairs.pl. we assem- bled the reads in each file separately to produce a set of rad contigs (fig. b), using both velvet (zerbino & bir- ney ) and cap (huang & madan ) assembly software. because cap performed better (see results), all further analyses mentioned below used the cap assemblies. because of our pooling strategy, the consen- sus sequences in this reference set of rad contigs rep- resent primarily wct with minimal rbt admixture. genotyping and admixture estimates we aligned the filtered read pairs for each individual from all five populations against the reference set of rad contigs (fig. c). (three individuals with very rad library preparation paired-end sequencing and identification of loci fragment size range rad contig assembly position along contig sequence depth alignment by individual and genotyping (a) (b) (c) (d) fig. schematic diagram of overlapping paired-end restriction- site-associated dna (rad) sequencing. (a) rad libraries are prepared according to etter et al. ( a,b), with the exception that a smaller size range of fragments are selected to obtain over- lapping reads. the green triangle indicates the restriction enzyme cut site, and fragments from only one side of the cut site are shown for three individuals (represented by different colours). (b) libraries are sequenced by illumina with paired- end reads. loci are identified with stacks software, using only the forward reads (solid lines) to cluster reads by locus. (c) both the forward and reverse reads from each locus are pooled across a set of individuals and assembled into a rad contig. the depth of sequencing coverage across overlapping paired-end rad con- tigs has a unique signature. (d) reads from each individual are separately aligned against the reference contig set and diploid single nucleotide polymorphism genotypes are called statisti- cally. the length of genotyped sequence data may vary across individuals, and in some cases genotype data may have a gap where paired ends did not overlap. © john wiley & sons ltd genomic introgression and rad sequencing low coverage were dropped: one each from meadow, nicola and tepee, leaving a total sample size of individuals.) we used the alignment software bowtie (langmead et al. ), allowing up to three nucleotide mismatches in the first bp of each read and up to mismatches over the total read. these parameters represent a compromise aimed at producing valid alignments to the reference, while minimizing bias against divergent rbt haplotypes. we chose them after aligning and genotyping a subset of the data across a wide range of parameter values, but we found that alignment parameters created only marginal differences in overall genotype calls (not shown). we retained only those read pairs that aligned uniquely to the reference contig set and that aligned in the expected orientation (i.e. the forward read aligns at position of the contig, matching the position of the restriction enzyme cut site and the reverse read aligns in the opposite direction along the same contig within a distance up to bp). we assigned diploid genotypes to each nucleotide position for each individual using the maximum-likeli- hood method of hohenlohe et al. ( ), modified by bounds on the per-nucleotide sequencing error rate of . < e < . and a significance level of a = . (custom software available at http://webpages.uidaho. edu/hohenlohe/software.html). these limits on e have the effect of being more likely to call a heterozygous genotype. while in de novo genotyping, these bounds on e would increase the frequency of false alleles; here, we are genotyping against previously identified wct and rbt alleles. this strategy and the relatively high signifi- cance threshold are also justified because of the quality filtering and removal of pcr duplicates described previ- ously, which increases confidence that each read repre- sents a true independent sample of genomic sequence. we used previously identified species-diagnostic snp loci to assess introgression from rbt into these wct populations. from the rad sequencing data in wct and rbt published by hohenlohe et al. ( ), we extracted all rad loci in which there was either one snp fixed between species and no other polymorphism in the -bp sequence ( loci), two fixed snps and no other polymorphism ( loci), or one fixed snp and one additional snp polymorphic within either spe- cies ( loci), for a total of diagnostic snps (see amish et al. for validation of some of these snp markers). we aligned both the wct and rbt alleles of these -bp sequences against the new reference set of rad contigs, using bowtie (langmead et al. ) and allowing up to two nucleotide mismatches. we retained only those diagnostic loci that aligned uniquely with up to two mismatches (for both the rbt and wct alleles) to the reference contig set. we then genotyped all individuals from the five admixed wct populations in the current study as wct, rbt or heterozygous at each of these loci for which genotype calls were made previously (any geno- type calls that did not match previously identified alleles at these snps were treated as missing data). as a final filtering step for paralogous loci, we removed loci for which these genotypes exhibited observed heterozygosity > . and fis < � . (hohenlohe et al. ). using all such diagnostic snps for which at least half of the individuals ( or more) were genotyped, we estimated proportion of admixture at the locus, individ- ual and population levels as the frequency of rbt alleles across diagnostic loci. we applied the heterogeneity test of long ( ) to test for super invasive alleles. this analysis tests whether the variance in admixture across loci exceeds that expected from random sampling as well as genetic drift across loci (other tests for admixture outliers do not account for drift and may suffer from a high false- positive rate, so our approach is a conservative test; fitzpatrick et al. ). because this method cannot handle allele frequencies of . , we used bayesian esti- mates of allele frequencies with an uninformative prior (fitzpatrick et al. ). we adjusted for differences in sample size of genotypes across loci, which affect the expected variance in allele frequency estimates, in equa- tion of long ( ). for each locus in each population, we calculated a p-value for the deviation from expected admixture and adjusted for false discovery rate at a level of a = . within each population (benjamini & hochberg ). we identified candidate super invasive alleles as those with significantly elevated admixture proportions in two or more populations. results rad sequencing and contig assembly after filtering for read quality and presence of a correct barcode and sbfi recognition site, we generated rad sequence read pairs across individu- als in five admixed wct populations. of these, % represented pcr duplicates and were removed, leaving unique read pairs. we identified a total of putative rad loci in stacks using the forward reads of each pair across all individuals. only of these loci represented eight or more read pairs across all individuals. we pooled the read pairs corresponding to these loci for individuals from three populations with the lowest previously estimated admixture proportions (lower hay, nicola and tepee). we conducted separate assemblies at each locus using both velvet (zerbino © john wiley & sons ltd p. a. hohenlohe et al. & birney ) and cap (huang & madan ). in velvet, we used fixed k-mer lengths of , , and bp as well as optimizing the k-mer length across these values independently at each locus. all of these assem- blies failed to connect overlapping paired-end reads at many loci, and the maximum contig length per locus was only ~ – bp (fig. s , supporting information). thus, in many cases, the contigs assembled were smaller than the read length of bp (after trimming the barcode) for the forward reads (fig. s , supporting infor- mation), meaning that sequences were broken into k-mers and unable to be reassembled. this difficulty in paired-end assembly of rad data has been observed elsewhere (davey et al. ), although that study had better success than we did in optimizing assembly parameters per locus. the general problem may be due to the unique signature of sequence coverage expected across contigs for overlapping paired-end rad data (fig. c; etter et al. a; fig. of davey et al. ). in contrast, the simpler algorithm of cap performed much better. while more computationally intensive, it is still feasible on a desktop computer because the locus identification from stacks significantly reduces the com- plexity of each individual assembly. of the loci, ( . %) assembled into single contigs, all but one containing both the overlapping forward and reverse reads. an additional loci assembled into two or more contigs, of which only the largest contig was anchored at the expected restriction enzyme recognition site. of these, all but contained both the forward and reverse reads. we combined these to produce our final reference set of rad contigs, which contained contigs from loci ( . %). fragment size selection to produce overlapping paired-end reads was remarkably successful, so that over % of loci pro- duced contigs spanning the forward and reverse reads. contig lengths ranged from to bp with most between and bp (fig. a), suggesting that longer fragments were carried through the gel-based size selection step. the mean number of read pairs con- tributing to each contig was . . contig length was positively related to the number of sequence pairs con- tributing to each assembly (fig. b), so our strategy of pooling individuals to increase coverage at this consen- sus assembly step appears sound. genotyping and admixture we aligned -bp rad sequences for previously identified snp loci (hohenlohe et al. ; amish et al. ) against the reference rad contig set. of these, ( . %) aligned uniquely to a single contig in the reference set with up to two mismatches for both the rbt and wct alleles. in addition, ( . %) aligned to multiple contigs with relatively few mismatches. these multiple contigs appear to represent genomic regions with duplicate sequence beyond the bp length of the previously identified rad sequence. figure s (sup- porting information) shows one such example in which sequences diverge relatively rapidly beyond the first bp, illustrating how longer reads and overlapping paired-end rad sequencing may provide powerful tools for distinguishing paralogous sequence from poly- morphism at homologous loci. we genotyped each individual at all nucleotide posi- tions aligned to the reference contig set using the maximum-likelihood statistical approach described pre- viously. of the uniquely aligned diagnostic snp loci, had diploid genotype calls for at least half the individuals sampled. two of these were probably paral- ogous loci, with elevated observed heterozygosity ( . and . ) and reduced fis (� . and � . , respec- tively), and these were removed from further analysis. the remaining loci had observed heterozygosity < . and fis > � . , suggesting a clear break between them and the two presumptive paralogous loci. we translated genotypes for the final list of loci into homozygous wct, heterozygous or homozygous rbt and assessed proportion of admixture as simply the fre- quency of rbt alleles. for all of the individuals genotyped here, we also had individual-level estimates of admixture proportion based on seven species-diagnostic microsatellite loci (boyer et al. ). our snp-based estimates were highly correlated with previous microsatellite-based estimates overall and within each population (table ), (a) (b) fig. (a) frequency histogram of consensus sequence lengths across contigs assembled by cap from overlapping paired- end restriction-site-associated dna (rad) sequencing in admixed westslope cutthroat trout populations. (b) relationship between sequencing depth at each locus (number of sequence pairs from pooled individuals) and rad contig length. © john wiley & sons ltd genomic introgression and rad sequencing although they tended to be slightly lower (fig. a). we detected evidence of introgression in all individuals for which no rbt alleles had been observed at the microsatellite loci. in these individuals, rbt alleles were detected at – loci, leading to individual admixture proportions ranging from . to . that were undetected in the microsatellite data. average popula- tion-level admixture proportions are also consistent with microsatellite-based estimates (pearson r = . ; p = . ; fig. b), in which dutch and meadow exhibited higher levels of admixture than the other three populations, although snp-based estimates were lower than microsatellite estimates for four of the five populations. comparing admixture proportions across snp loci reveals a positively skewed distribution within each population and overall, with many loci showing little or no admixture and a small set of outlier loci (fig. ). of the diagnostic snp loci genotyped, showed no rbt alleles in any of the five populations. however, loci exhibited admixture levels of . or greater across all five populations combined, up to a maximum of . (fig. f). these are candidate super invasive alleles: rbt alleles that may have spread rapidly or have higher probabilities of persistence in wct popula- tions. within each population, loci exhibited signifi- cantly elevated admixture proportions using the heterogeneity test of long ( ), corrected for false dis- covery rate (table ). three loci were significantly inva- sive in two or more populations, one of which was significant across all five populations (fig. ). we conducted a translated nucleotide blast search using the rad contig sequence for each of these three super invasive alleles. two of them aligned closely to annotated genes whose function is consistent with selec- tion in hybridized wct populations. the locus signifi- cantly admixed in all five populations (rad locus ) aligned significantly to the vertebrate gene latent transforming growth factor beta-binding protein (ltbp ), with the most significant hit in bos taurus (e-value = � ). the second locus, significantly admixed in the nicola and tepee populations (rad locus ), aligned to the vertebrate gene furry homolog-like (fryl), with the most significant hit in zebrafish (danio rerio, e-value = � ). it is worth noting that the blast alignments to these two annotated gene table correlation between previous microsatellite and current single nucleotide polymorphism (snp)-based estimates of individ- ual-level admixture proportions, and super invasive alleles exhibiting significantly elevated introgression with a false discovery rate corrected p-value population snp-microsatellite correlation # super invasive alleles fdr p-value thresholdr p-value meadow . < � . � nicola . < � . � dutch . . . � lower hay . < � . � tepee . < � . � all populations . < � . � (a) (b) fig. (a) individual-level admixture proportions estimated from seven diagnostic microsatellite loci (boyer et al. ) vs. current estimates from single nucleotide polymorphism loci across westslope cutthroat trout individuals from five populations. note that many of the points, particularly those with admixture proportions near . , lie on top of each other. (b) population-level admixture proportions esti- mated from the same two data sets, calculated using only the individuals genotyped by both boyer et al. ( ) and the current study. © john wiley & sons ltd p. a. hohenlohe et al. sequences began at nucleotide positions and , respectively, of the rad contigs so that the identifica- tion of these candidate genes would not have been pos- sible solely with single-end rad sequence data. discussion genomic tools hold remarkable promise for conserva- tion and management of many taxa. the ability to rap- idly identify and genotype large numbers of genetic markers allows improved estimates of demographic parameters (gene flow, effective population size, popu- lation-level admixture), as well as identification of out- lier loci (locally adapted genes, invasive alleles). overlapping paired-end rad sequencing offers advan- tages for rapid development of large numbers of candi- date snps that can be used in high-throughput genotyping assays, particularly in the case of large or repetitive genomes. in a specific application of this technique, here we assessed genomic patterns of introgression and were able to detect individuals with very low levels of admixture, precisely estimate individual- and population-level admixture and detect candidate super invasive alleles driven to high frequency by selection. below, we discuss some general aspects of the sequencing technique for conservation genomics and lessons from its application to the genomics of hybridization. overlapping paired-end rad for conservation genomics by assembling contigs of bp or more adjacent to rad loci, overlapping paired-end rad provides suffi- cient flanking sequence for snp assay design simulta- neous with snp discovery. the ability to generate sufficient flanking sequence has previously been a limi- tation of rad sequencing for converting rapid snp discovery to a set of high-throughput assays (ogden ; amish et al. ). our approach can rapidly provide a multitude of candidate snp markers for high-throughput assay development. here, we only analysed a few thousand diagnostic markers that had been previously identified. in general, the majority of contigs of – bp or longer would be expected to contain snps relevant for most population genomic or conservation applications. assembling rad contigs provides more continuous genomic sequence data for discriminating paralogous loci. this is a particular challenge in salmonids because of their ancestral genome duplication, which created homeologous duplicate sequence across the genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . admixture proportion # of s n p s i i i i i i ii ii ii iii iii iii (a) (b) (c) (d) (e) (f) fig. frequency histograms of admixture proportion across diagnostic single nucleotide polymorphism loci. (a) meadow. (b) nicola. (c) dutch. (d) lower hay. (e) tepee. (f) all five populations combined. arrows indicate super invasive alleles—loci with significantly elevated admixture proportion (a = . , corrected for false discovery rate) independently in two or more populations. (i) restriction-site-associated dna (rad) locus . (ii) rad locus . (iii) rad locus . © john wiley & sons ltd genomic introgression and rad sequencing (allendorf & danzmann ; everett et al. ; seeb et al. b). here we found examples of loci sharing very similar sequence over ~ bp, so that they were grouped together in previous analysis, but diverged beyond that length. as a result, we were able to further screen the candidate diagnostic snp loci we had previ- ously identified (hohenlohe et al. ; amish et al. ) by removing the % that aligned to multiple rad contigs. ongoing validation of the reduced set will determine the success rate of these refined candi- date markers. our approach to rad contig assembly produced a sin- gle contig with high average read depth for most of our rad loci. nonetheless, the assembly and validation of rad contigs can be challenging (davey et al. ). assemblies using the de bruijn graph technique of velvet (zerbino & birney ) produced consistently shorter contigs than a simpler (but more computationally intensive) assembly algorithm in cap (huang & madan ) (compare fig. and fig. s , supporting information). this contrasts with the results of etter et al. ( a,b), who had better success with velvet in assem- bling the reverse reads from nonoverlapping paired-end rad. willing et al. ( ) used nonoverlapping paired- end rad in guppies and assembled the reverse reads for . % of loci into a single contig with generally lower sequence coverage than used at the assembly step here. that study used the assembler locas, specifically designed by one of the authors for low-coverage data. davey et al. ( ) had poor results with locasopt and velvet in assembling paired-end rad data from helico- nius butterflies, but better results using the computation- ally intensive velvetoptimiser. in our trout data set, over % of loci produced a single contig of both forward and reverse reads with cap , and many of the remainder could be filtered out as paralogs. techniques like overlapping paired-end rad sequencing may allow new analytical power. compared with other markers like microsatellites, snps can be limiting in that they typically exhibit only two alleles in natural populations. more power to understand popula- tion genetic processes would come from using multi-allelic haplotypes instead of snps in analyses of high-throughput sequence data (gompert & buerkle ; buerkle et al. ). because of the relatively long contigs that can be generated (etter et al. a,b; will- ing et al. ) and because haplotype phase is known across read pairs and thus can be inferred along the length of rad contigs, paired-end rad offers the possi- bility of using haplotype- rather than snp-based analy- ses. genealogical relationships among multiple haplotypes are very useful for inferring demographic and evolutionary history (sunnucks ; beaumont & rannala ). assessing genome-wide patterns of introgression here we provide one of the first genomewide assess- ments of human-mediated introgressive hybridization in salmonid fishes (see also lamaze et al. ). our results confirm previous patterns of hybridization between introduced rbt and native wct in the north fork flat- head system (boyer et al. ; muhlfeld et al. c). population-level admixture estimates were generally consistent for diagnostic microsatellites and rad-based snp loci, suggesting that thousands of diagnostic loci are generally unnecessary for approximate estimates of pop- ulation-level admixture. however, one estimate did dif- fer: the estimate for dutch creek was over % higher using the microsatellite data (fig. b). this may be explained by selection against rbt alleles in chromo- somal regions near rad loci and/or sampling error from using only seven diagnostic microsatellite loci, especially for populations with low levels of introgression. given the variation in introgression we observed here among snp loci, the genomic location of those microsatellite loci could also be a major source of variation. overestimation of admixture (by using only a hand- ful of neutral loci) could cause populations to not be protected under conservation laws, such as the u.s. endangered species act (esa). for lahontan cutthroat trout, listed under the esa, % rbt admixture is the threshold for a population to be protected as if it were nonhybridized (pure native) lahontan. based on sam- pling theory for neutral loci, it is likely that – diagnostic loci would improve accuracy to levels approaching that of thousands of rad loci, if those diagnostic loci are widely distributed across the genome (amish et al. ). at the individual level, overlapping paired-end rad sequencing allowed detection of very low levels of rbt introgression. here, we detected rbt alleles in all samples analysed, over half of which did not exhibit rbt alleles at seven microsatellite loci (boyer et al. ). some of the assumed rbt-diagnostic alleles could actually exist in nonhybridized wct populations. additional rad sequencing of pure-native populations (e.g. isolated above barriers in the flathead river) could help identify assumed diagnostic rbt alleles that might exist in wct (e.g. due to maintenance of ancestral poly- morphism). genomewide marker coverage is an important advance for conservation and management because it allows pow- erful screening of individuals to prevent inadvertent release of hybridized individuals into populations (e.g. during assisted migration, broodstock development, translocation and reintroduction) and identification of markers for rapid screening for early detection of hybrid- ization. from a landscape genetics perspective, the ability © john wiley & sons ltd p. a. hohenlohe et al. to precisely estimate admixture would allow fine spatial mapping of hybridization and introgression patterns. this approach may be useful in monitoring and preventing the spread of invasive species and their alleles in many plant and animal species facing hybridization threats in nature (schwartz et al. ). dense coverage of markers across the genome allows for detection of candidate super invasive alleles—alleles of an invasive taxon that rise to much higher frequency (level of introgression) than the genomic background, analogous to outlier loci in genome scans for selection (luikart et al. ). here we detected several candidate super invasive alleles as evidenced by the distributions of admixture proportions among snps (in all popula- tions) containing a long tail of outlier loci. several of these loci were consistent as outliers across populations. further study is needed to confirm that these are indeed rbt alleles that have introgressed into these wct popu- lations. the haplotype information provided by longer overlapping paired-end rad (e.g. using -bp reads as provided by illumina miseq technology) may facilitate that analysis. further study would also be needed to identify the phenotypic and fitness consequences of these invasive alleles. blast searching revealed close sequence matches for two candidate invasive alleles to vertebrate genes (ltbp- and fryl). super invasive alleles may be under positive selection and increase fitness in hybrid- ized populations. alternatively, they may spread by having phenotypic effects on dispersal or through seg- regation distortion, despite reducing overall fitness from outbreeding depression (shine ). the ltbp family of proteins interacts with tgf-beta and has a wide range of developmental and physiological functions, including effects on fertility (mor�en et al. ; €okl€u & hesketh ; kosova et al. ), although the spe- cific relationship between ltbp- and tgf-beta is unclear (hirani et al. ). in rbt, the related protein ltbp- and other related proteins have been implicated in early ovarian development and early embryonic development (andersson & eggen ; lankford & weber ; gahr et al. ), suggesting the hypothesis that the rbt allele at this locus positively affects fecun- dity in admixed individuals. it is exciting that future research and additional studies like this one will help understand mechanisms driving super invasive alleles and genomewide introgression in natural populations. acknowledgements p.a.h. and m.d.d. received support from u.s. national insti- tutes of health/ncrr grant p rr (l. forney, pi). fwa and g.l. were partially supported by the u.s. national science foundation grants deb- . g.l. also received support from montana fish wildlife and parks and nsf grant deb- . this work was partially supported by bpa contract # . we thank j.j. giersch for providing the study area map, robb leary for helpful comments on the potential of some assumed diagnostic alleles for rbt to be present at low fre- quency in nonhybridized wct, and montana fish wildlife and parks and glacier national park for support and help in sam- pling. any use of trade, product or firm names is for descriptive purposes only and does not imply endorsement by the u.s. gov- ernment. this research was conducted in accordance with the animal welfare act and its subsequent amendments. references allendorf fw, danzmann rg ( ) secondary tetrasomic seg- regation of mdh-b and preferential pairing of homeologues in rainbow trout. genetics, , – . allendorf fw, leary rf, spruell p, wenburg jk ( ) the problems with hybrids: setting conservation guidelines. trends in ecology and evolution, , – . amish sj, hohenlohe pa, painter s et al. 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Érudit est un consortium interuniversitaire sans but lucratif composé de l’université de montréal, l’université laval et l’université du québec à montréal. il a pour mission la promotion et la valorisation de la recherche. https://www.erudit.org/fr/ document généré le avr. : ontario history in search of promised lands: a religious history of mennonites in ontario by samuel j. steiner julia rady-shaw volume , numéro , spring uri : https://id.erudit.org/iderudit/ ar doi : https://doi.org/ . / ar aller au sommaire du numéro Éditeur(s) the ontario historical society issn - (imprimé) - (numérique) découvrir la revue citer ce compte rendu rady-shaw, j. ( ). compte rendu de [in search of promised lands: a religious history of mennonites in ontario by samuel j. steiner]. ontario history, ( ), – . https://doi.org/ . / ar https://apropos.erudit.org/fr/usagers/politique-dutilisation/ https://www.erudit.org/fr/ https://www.erudit.org/fr/ https://www.erudit.org/fr/revues/onhistory/ https://id.erudit.org/iderudit/ ar https://doi.org/ . / ar https://www.erudit.org/fr/revues/onhistory/ -v -n -onhistory / https://www.erudit.org/fr/revues/onhistory/ ontario history samuel j. steiner’s the search of promised lands: a religious history of mennon- ites in ontario is a comprehensive account of the rich past of one of the province’s ear- ly settler groups. steiner’s archival record is voluminous, yet he weaves the evidence together into a rich, cogent, and accessi- ble history of the mennonites in ontario. drawing upon personal narratives, mu- nicipal records, the census, newspapers, and denominational records (to name a few), steiner’s book explores the nuances of mennonite origins in upper canada and follows those threads through to the near present. steiner incorporates the themes of religiousity, assimilation, conflict, and iden- tity politics throughout his book to trace the fragmentation and realignment that constantly occurred within the denomi- nation in order to sketch the spectrum of mennonite faith and practice in ontario. the promised lands of the title pro- vide a rich metaphor for the way early in search of promised lands a religious history of mennonites in ontario by samuel j. steiner kitchener, ontario: mennomedia, . pages. $ . hardback isbn: . $ . e-pub. (www.mennomedia.org ) mennonite, amish and quaker commu- nities sought a new life in north america upon which to build their faith. this prom- ise continued to animate settlement and emigration patterns as mennonites from pennsylvania traveled north to the niaga- ra region, the grand river valley, and the huron shores of present-day ontario, or when other mennonite communities im- migrated to canada in the twentieth cen- tury from places like the ussr, germany, asia, or south america. although not dissimilar from other immigration sto- about student health led to the creation of new sites through which administrators could exert their moral vision of the uni- versity and shape the student body” ( ). in doing so, gidney expands the understand- ing of the historical relationships between youth’s health, citizenship, and morality. her history of how university administra- tors tended the student body in the past also sheds light onto the ways universities are tending the student body today. she exposes the current conceptualization of universities as moral communities where students are seen as “immature youth, to be guided to full adulthood” ( ) and the university as their moral and intellec- tual guide. karissa patton university of saskatchewan book reviews ries, steiner’s history of the mennonites ably captures the way religion fundamen- tally and prominently directed the actions and reactions of this particular immigrant group to their new home. over the span of two centuries on this new frontier, mennonites adjusted and re- adjusted what constituted their “promised lands.” while people claimed allegiance to their mennonite identification, that identi- fication underwent internal change and was comprised of many variations. congrega- tions aligned and realigned their faith and faith practices in relation to conflicts, shift- ing religious trends, generational changes, and social changes. some groups held firm to conservative and fundamentalist practic- es, while others became much more liberal. steiner’s work pays great attention to the broader economic, political, and reli- gious climates. for example, in the early chapters on settlement in the thirteen colonies, steiner observes how the story of the mennonites, amish, and quakers was not atypical of the time. like the puritans or members of the church of england who settled in new england or virginia, respec- tively, the pacifist groups existed within this same immigration narrative. they all sought out a new opportunity in a new world where religion could factor prominently into daily life. examples from the twentieth century illuminate the way mennonite self-identifi- cation was a category in a constant state of flux. the great war bred greater acceptance of fundamentalist theology and cooperation as mennonites stood united in their pacifist stance to the war. after , the influx of mennonite settlers from the soviet union again reshaped broader patterns of belief and culture as new ideas emigrated with the new groups. the post-second world war moment and the evangelical zeal of the s and ’ s reshaped mennonite groups on the basis of practice, theology, and mis- sionary work as they attempted to “bring the church up to date” in the new world or- der. finally, later-twentieth century immi- gration of lowland german mennonites as well as groups from other parts of the world such as asia and south america forced the established mennonite churches in ontario to re-evaluate their core values as new inter- national ideas influenced traditions. why did some mennonites shift away from old order practices and others did not? this central question helps steiner to maintain cohesion in his encyclopaedic volume. he points to the effects of emigra- tion, of change over time, of the broader forces of secularization and modernization in society as just a few of the key reasons that division took place amongst the men- nonites. in one of the most interesting dis- cussions of the book, steiner explores the challenges of divorce and remarriage to the various orders. divorce posed a significant issue to involvement in the mennonite church; divorced individuals were denied membership. against the backdrop of the s and the rapid social change that oc- curred, this issue exemplified the way some orders like the mennonite brethren re-ex- amined their positions in order to reflect broader society and their choice to assimi- late more than their companion orders. steiner is meticulous in paying heed to the vast array of complicated themes and ideas that accompany the history of a de- nominational group over such a long period of time. debates such as the effect of secu- larization upon the religious community or the place of ecumenism certainly receive attention in the book; however, teasing out the implications of these weighty historical issues are sometimes of secondary concern. in a history as comprehensive as steiner’s this is not so much a criticism but a desire for a continued discussion. steiner’s book would be of interest to ontario history armchair historian and academic alike. his writing is accessible and his history sharp. his briskly-paced book would be of par- ticular use to students of ontario, religion, and the church. for teachers, in search of promised lands is an excellent example of archival history done well. it would help to augment discussions on the place of conflict in society, immigration in ontario, and how modernizing or changing society profound- ly impacted religious and cultural groups. most importantly, steiner’s concluding dis- cussion about the broad spectrum of men- nonite practices raises important questions about how secularization affected specific religious groups—an area of inquiry not yet fully enough explored in the historical record. for the mennonites, it has meant a history of debating the preservation of shared values, faith, and rituals and the mer- its of assimilation into broader society. julia rady-shaw university of toronto with the centennial of the first world war upon us, a large number of books have been published recently about canadian participation in that conflict. some cover old ground with fresh insight, while others venture into areas not previ- ously looked at in any detail. old enough to fight: canada’s boy soldiers in the first world war by dan black and john boile- au attempts the latter. of the roughly , men and women canada put into uniform, an estimated - , soldiers (or just over %) were boys younger than military regulations allowed. the authors note there is a literature on the history of boy soldiers in the commonwealth, but no detailed examination of the phenomena in canada. historians like desmond morton in when your number’s up: the canadian soldier in the first world war ( ) and old enough to fight canada’s boy soldiers in the first world war by dan black and john boileau toronto: james lorimer, . pages. $ . softcover. isbn - - - - . $ . hardback . $ . ebook . (www.lorimer.ca) tim cook in at the sharp end: canadians fighting the great war, - ( ) have noted the existence of recruits young- er than years of age, but this new book is the first detailed treatment of the topic. the question of how the canadian ex- peditionary force came to have underage soldiers is answered in the introduction and first chapter. the british and canadi- international journal of environmental research and public health article toxoplasma gondii serointensity and seropositivity: heritability and household-related associations in the old order amish allyson r. duffy , , jeffrey r. o’connell , , mary pavlovich , , kathleen a. ryan , , christopher a. lowry , , , , , melanie daue , , uttam k. raheja , lisa a. brenner , , , andré o. markon , cecile m. punzalan , aline dagdag , dolores e. hill , toni i. pollin , , andreas seyfang , maureen w. groer , braxton d. mitchell , , and teodor t. postolache , , , ,* mood and anxiety program, university of maryland school of medicine, baltimore, md , usa; aradford@health.usf.edu (a.r.d.); uttamraheja@gmail.com (u.k.r.); adagdag@som.umaryland.edu (a.d.) college of nursing, university of south florida, tampa, fl , usa; mgroer@health.usf.edu division of endocrinology, diabetes and nutrition, department of medicine, university of maryland school of medicine, baltimore, md , usa; joconnel@som.umaryland.edu (j.r.o.); pavlovichma@gmail.com (m.p.); kryan@som.umaryland.edu (k.a.r.); mdaue@som.umaryland.edu (m.d.); tpollin@medicine.umaryland.edu (t.i.p.); bmitchel@som.umaryland.edu (b.d.m.) program for personalized and genomic medicine, university of maryland school of medicine, baltimore, md , usa veterans health administration, rocky mountain mental illness research education and clinical center (mirecc), rocky mountain regional veterans affairs medical center (rmrvamc), aurora, co , usa; christopher.lowry@colorado.edu (c.a.l.); lisa.brenner@va.gov (l.a.b.) department of physical medicine and rehabilitation, university of colorado anschutz medical campus, aurora, co , usa military and veteran microbiome: consortium for research and education (mvm-core), aurora, co , usa department of integrative physiology and center for neuroscience, university of colorado boulder, boulder, co , usa center for neuroscience, university of colorado anschutz medical campus, aurora, co , usa us food and drug administration, college park, md , usa; andre.markon@fda.hhs.gov (a.o.m.); cecile.punzalan@fda.hhs.gov (c.m.p.) us department of agriculture, agricultural research service, beltsville agricultural research center, animal parasitic diseases laboratory, beltsville, md , usa; fithian @yahoo.com college of medicine, university of south florida, tampa, fl , usa; aseyfang@health.usf.edu geriatrics research education and clinical center (grecc), baltimore, md , usa mental illness research, education, and clinical center (mirecc), veterans integrated service network (visn ), va capitol health care network, baltimore, md , usa * correspondence: tpostola@som.umaryland.edu received: july ; accepted: september ; published: october ���������� ������� abstract: toxoplasma gondii (t. gondii) is an intracellular parasite infecting one third of the world’s population. latent t. gondii infection has been associated with mental illness, including schizophrenia and suicidal behavior. t. gondii igg antibody titers were measured via elisa. the heritability of t. gondii igg was estimated using a mixed model that included fixed effects for age and sex and random kinship effect. of old order amish participants, had positive titers ( . %). the heritability for t. gondii serointensity was estimated to be . (p = . × − and for seropositivity, it was estimated to be . (p = . × − ). shared household environmental effects (i.e., household effects) were also determined. household effects, modeled as a random variable, were assessed as the phenotypic covariance between any two individuals who had the same current address (i.e., contemporaneous household), and nuclear household (i.e., the phenotypic covariance between parents int. j. environ. res. public health , , ; doi: . /ijerph www.mdpi.com/journal/ijerph http://www.mdpi.com/journal/ijerph http://www.mdpi.com https://orcid.org/ - - - https://orcid.org/ - - - http://dx.doi.org/ . /ijerph http://www.mdpi.com/journal/ijerph https://www.mdpi.com/ - / / / ?type=check_update&version= int. j. environ. res. public health , , of and children only, not other siblings or spouses). household effects did not account for a significant proportion of variance in either t. gondii serointensity or t. gondii seropositivity. our results suggest a significant familial aggregation of t. gondii serointensity and seropositivity with significant heritability. the shared household does not contribute significantly to family aggregation with t. gondii, suggesting that there are possible unmeasured non-household shared and non-shared environmental factors that may play a significant role. furthermore, the small but significant heritability effects justify the exploration of genetic vulnerability to t. gondii exposure, infection, virulence, and neurotropism. keywords: environment; household; genetics; heritability; infection; mental illness; parasitic infection; toxoplasma gondii . introduction toxoplasma gondii is one of the most common obligate intracellular protozoan parasites, infecting one third of the world’s population [ ]. globally, the estimated seroprevalence of t. gondii infection ranges from % to %, with the most common form being a latent infection resulting in small cysts formed by intracellular organisms generally found in the brain as well as in skeletal and cardiac muscles [ , ]. a powerful humoral immune response is elicited in response to these cysts [ ]. any member of the feline family can be the definitive host of t. gondii, with oocysts only produced in the intestine of cats. intermediate hosts of the parasite include any warm-blooded mammal or bird. infection occurs through direct exposure to the oocysts (including cat litter, consumption of contaminated water or unwashed vegetables, or failing to adequately wash hands after soil-contact) or by ingestion of raw or undercooked meat containing t. gondii tissue cysts. once an intermediate host is infected, rapidly reproducing tachyzoites cause an acute infection, often presenting with flu-like symptoms [ ]. following acute infection, t. gondii persists in a slow-growing chronic form, in which bradyzoites are contained within intracellular cysts and may persist throughout the lifetime of the immunocompetent host, with occasional limited reactivation. chronic infection in rodents leads to behavioral changes, including nonspecific increases in exploratory behavior and specific attraction rather than aversion to a feline predator [ , ]. seropositivity of t. gondii previously has been associated with schizophrenia [ ], bipolar disorder [ – ], suicidal behavior [ – ], and possibly with depression [ – ] and personality disorders [ ]. risk-taking behaviors, delayed reaction time, and reduced neural processing speed have also been associated with positive t. gondii igg antibody titers as well as traits of impulsivity and aggression in healthy individuals and in patients with intermittent explosive disorder [ , ]. a recent systematic review and meta-analysis has confirmed previously reported links between t. gondii igg serointensity/seropositivity with traffic accidents and suicide attempts [ ]. although predictive associations increasingly confirmed links between t. gondii and brain and behavior, the direction of causality has not been demonstrated. for example, it is possible that impulsivity may represent a contributory cause of t. gondii infection (through less adequate washing of vegetables or inadequate cooking of meat) rather than a consequence of it. if infection occurs during pregnancy, consequences to the fetus are caused by vertical transmission and can be devastating. congenital toxoplasmosis can result in a wide array of clinical sequelae, the most severe being chorioretinitis, cerebral calcifications, hydrocephalus, pneumonia, and disseminated disease. it has been reported that primary infection during gestation was the only cause of congenital infection [ ]. spontaneous abortions and stillbirth may occur. while some babies can be asymptomatic at birth, delayed manifestations such as hearing and visual impairment, neurologic findings, and intellectual disability may develop years after birth [ ]. in a host that is immunocompromised or immunosuppressed, such as in a host with hiv infection, more symptomatic reactivation of dormant infections may occur. specifically, t. gondii bradyzoites int. j. environ. res. public health , , of convert back to tachyzoites [ ] with full invasive and spreading potential, leading to acute encephalitis. additionally, both primary infections and reactivations can lead to ocular lesions of toxoplasmosis, a cause of visual deficits [ ]. the old order amish of lancaster, pennsylvania, are a rural, primarily agricultural community, with a high seroprevalence of t. gondii. while exposure to t. gondii is required for infection, parasite factors, such as serotype, and host factors, both genetic and environmental, may play a role in susceptibility and disease course [ ]. in order to better understand the nature of infectivity of pathogens, it is important to consider resilience and vulnerability to infection. host genetic factors influence susceptibility to various infections, including mycobacterial infections [ , ] and malaria [ ]. heritability is useful for giving an approximate sense of the contributions of additive genetic effects while household analysis leads to identifying shared environmental effects. in addition, heritability is useful in suggesting whether or not there are genes to be identified and if the phenotype in question has an additive genetic component. a previous study found a high rate of chronic and recent t. gondii infections in fathers of congenitally infected children, suggesting that t. gondii infections cluster within families [ ]. yet, to our knowledge, there have been no prior attempts to investigate separate environmental household versus heritability contributions to the familial aggregation of t. gondii infection, and this is the first study to accomplish this aim. . materials and methods . . study population the study population consists of a total of old order amish individuals residing in lancaster county, pennsylvania, who participated in a community-wide genetic population-wide ascertainment study of cardiometabolic and other health issues (the amish wellness study). they represent approximately two-thirds of individuals who were targeted to participate ( . %). they lived in individual households, also approximately two-thirds of the households invited to participate ( . %). the number of individuals living in a household varied between one ( participants, households), two ( individuals, households) three ( individuals, households), four ( individuals, households), five ( individuals, households) and six ( participants in households). for this t. gondii study, we measured plasma igg antibody titers in these participants. this study was carried out in accordance with the recommendations of the university of maryland institutional review board (reference number hp- - ) with written informed consent from all subjects after a full explanation of the study and adequate time to ask questions. all subjects gave written informed consent in accordance with the declaration of helsinki. the protocol was approved by the institutional review board from the university of maryland (reference number hp- - ). the amish are ideal to examine heritability of t. gondii infection because (a) the large, well-characterized generation pedigree available through the anabaptist genealogy database (agdb) [ , ] provides greater power to partition the phenotypic variance into different sources of genetic and environmental variances, and (b) the relatively high t. gondii seroprevalence ( %) [ ] in this community provides substantial power to detect genetic effects. . . plasma collection the fasting blood draw was obtained by venipuncture either in the amish wellness study mobile clinic, the participant’s home, or at the amish research clinic. whole blood, collected in heparinized tubes, was centrifuged at ◦c at – rpm for min. plasma was separated and placed in eppendorf tubes for storage at − ◦c until assayed for t. gondii igg. all standard institutional biosecurity norms of the university of maryland school of medicine were respected and all personnel had training, retraining, and examination results set forth by biosafety procedures and research ethics norms listed by federal guidelines and maryland university institutional review board. int. j. environ. res. public health , , of . . laboratory analysis plasma t. gondii igg seropositivity and serointensity were measured by enzyme-linked immunosorbent assay (elisa) at the university of south florida college of nursing biobehavioral lab in tampa, florida, usa. the samples were analyzed by elisa that tests for levels of rh factor, which is the major tachyzoite antigen used in elisas (re , ibl international, männendorf, switzerland). all the assays used standards for validation. both a qualitative and a quantitative approach were used to define status. the qualitative approach uses a predetermined cutoff value as reported by the manufacturer of the kit. the optical density (od) of each sample was compared to the cutoff standard value (greater than iu/ml). concentrations of < iu/ml were defined as negative. when the elisa results indicated an equivocal concentration of t. gondii antibody (i.e., to iu/ml), we repeated the elisa (n = participants). when the second elisa remained in the equivocal range or showed a level less than iu/ml, the data were considered negative. if the second elisa in those with an equivocal antibody concentration on the first testing showed a concentration in the positive range (greater than iu/ml) the data were considered positive. for the quantitative analysis, the ods of the standards were plotted against their concentrations using graph pad prism software and a cubit spline method. the concentrations of the samples were then read from the standard curve. the serointensity was defined as the concentration of the igg antibodies, statistically analyzed as log titer. . . statistical analysis since t. gondii igg titer levels were skewed to the right, values were log transformed prior to analysis. heritability was defined as the proportion of the total trait variance attributable to the additive effect of genes and was estimated by modeling the degree of biological association. heritability was estimated using the maximum likelihood method with the solar software package [ ]. we additionally modeled household effects, also as a random variable, as the phenotypic covariance between any two individuals who had the same current address. we measured the contemporaneous (current) household effect, including parents, children, siblings, and spouses) as well as the nuclear household effect that measured the phenotypic covariance between parents and children only, not other siblings or spouses. . results the total number of participants who reported their occupation as farming was / ( . %). table . shows the mean characteristics of the study sample. table . demographics and heritability. all (n = ) men (n = , %) women (n = , %) age, mean (sd) . ( . ) . ( . ) . ( . ) bmi, mean (sd) . ( . ) . ( . ) . ( . ) t. gondii antibody titer, iu/ml, mean (sd) . ( . ) . ( . ) . ( . ) log (titer), mean (sd) . ( . ) . ( . ) . ( . ) seropositive, n/total (%) / ( . ) / ( . ) / ( . ) . . t. gondii igg antibody titers of the individuals with igg antibody titer measurements, the total number of seropositive individuals was ( . %) with more infected men ( . %) than women ( . %). the mean (sd) of igg antibody titer in the sample was . ( . ) iu/ml and was slightly lower in women ( . iu/ml) than in men ( . iu/ml). there was a positive association between age and transformed serointensity, with beta (se) and p-value of . ( . ), p < . . int. j. environ. res. public health , , of . . heritability the heritability for t. gondii serointensity was estimated to be . (p = . × − ). heritability for t. gondii seropositivity was estimated to be . (p = . × − ). the heritability of serointensity between mothers and offspring, h = . (p < . ) was higher than the heritability between fathers and offspring h = . (p = . ). . . household household effects did not account for a significant proportion of phenotypic variance in either t. gondii serointensity (p = . ) or for t. gondii seropositivity, regardless of whether heritability was included in the model (p > . for both). however, the nuclear household effect approached statistical significance (p = . ). . discussion to our knowledge, this is the first study to examine the heritability and household effects of t. gondii seropositivity and serointensity in any population. we found a moderate heritability of % for seropositivity and % for serointensity, and no significant effect of household (statistical trend for nuclear household, and no effect of contemporaneous household), suggesting that there were likely unmeasured non-household shared and non-shared environmental factors that may have played a significant role. studies of the old order amish provide an unparalleled opportunity to analyze family structure effects, including nuclear family and shared environmental effects. as suggested by the results of this study, the nuclear family is more important for serointensity than an individuals’ current household and is capable of capturing environmental developmental exposures to t. gondii oocysts and to factors that modulate non-th immune responses that may be very different in quality and impact of the adult exposure of the amish. while the current t. gondii igg seroprevalence in the united states is approximately %– % [ ], the seroprevalence in old order amish is much higher (boyer et al., ). specifically, in our study, we found a seroprevalence of %. the higher rate of seropositivity in the amish may be related to a higher exposure to food- and non-food-related risk factors [ ]. this raises the possibility that heritability of personality traits, such as impulsivity, may increase the risk of exposure to t. gondii (e.g., through food processing and hand hygiene). serointensity is indicative not only of the virulence of t. gondii, but also of reactivation from latent infection [ ]. serointensity is influenced by the duration since infection, meaning that the higher the antibody titer, the more recent the infection and the extent to which the tissue cysts have spread [ ]. for example, heritability estimates of hiv range from %– % [ ], and heritability of tuberculosis, from %– % [ ]. for malaria, another apicomplexan infection (as t. gondii), the heritability estimates for both maximum and overall parasite trophozoite density phenotypes for plasmodium falciparum (p. falciparum) and p. vivax in a karen population in thailand were significantly and relatively similar to the ones we are currently presenting (p. falciparum: % and p vivax: %– %) [ ]. similarly, the heritability of the number of the clinical episodes varied between % for p. falciparum and % for p vivax. in another study based on data from senegal, a significant heritability has been reported for asymptomatic infections with p. falciparum, but not for symptomatic infections. while, in general, estimations of heritability were similar across locations, there were also large differences between locations, suggesting geographic variation of heritability. for example, in dielmo and ndiop (senegal) heritability estimates for markers of asymptomatic infection with p. falciparum are reported as . % and . %, just a bit lower than for our current t. gondii study. meanwhile, gouye kouly (also in senegal) had a high level of heritability ( . %), higher than in our current study. similar to our current study the household effects were not significant [ ]. a somewhat divergent study in uganda has identified a significant . % heritability of plasmodium parasite density in children, but not adults, and a % household effect in adults, but not in children [ ]. in our study, the lack of household-related int. j. environ. res. public health , , of environmental effects could be due to homogeneity of risk factors in the household of the amish. it could also mean that non-household related environmental effects, for instance, occupational effects (contact with soil, animals, raw meat), could be more important than the household effect. of relevance, eating and hand/food hygiene habits are mainly acquired in early childhood rather than the current, i.e., contemporaneous, household. limitations of our study include not having a full history of shared household and the exposure time. additionally, this study was conducted in a highly specific population, and thus, the generalizability of the results might be limited. another limitation is the inability to distinguish between the effect of transmission of infection during pregnancy and true heritability, potentially spuriously inflating the heritability effect or even producing a falsely significant heritability. indeed, the heritability in serointensity between mothers and offspring, h = . (p < . ) was higher than heritability between fathers and offspring, h = . (p = . ). yet, the heritability between fathers and offspring was highly significant, arguing against a simple spurious association fully explained by an aftereffect of pathogen transmission of infection during pregnancy. we also did not calculate the heritability of the t. gondii clinical illness, rather than seropositivity. data from the better-studied apicomplexan-caused disease—malaria—shows that heritability estimates for outpatient and inpatient, i.e., more severe disease, were of % and % respectively, with a much stronger household effect ( % and %, respectively) than reported for asymptomatic infection [ – ]. finally, one-third of the invited population, in terms of both household number and person numbers, did not take part in the study. this could have affected the results in ways that are not fully known. we expect that it is possible that missing data could have inflated somewhat heritability estimates, as they appeared lower in males (as shown above), and males overrepresented females among those “missed” by recruitment. as strengths of the study, the high seropositivity and serointensity, and the accuracy of genealogical data in the amish provide unsurpassed advantages. our heritability results provide the supportive rationale for efforts to study genetic contributions to t. gondii seropositivity and serointensity. our next step is to further investigate genetic predisposition and resilience for t. gondii seropositivity and serointensity in the old order amish, including, sequentially, candidate gene, genome-wide association studies (gwas), and exome sequencing analyses. additionally, specific exploration of t. gondii serotype and type of infection (e.g., via oocyst vs. tissue cyst), and capturing seroconversion and exposure to risk factors using longitudinal designs, may further help understand, prevent, and manage t. gondii infection. it is possible that genetic effects resulting in a significant heritability may also lead to increased novelty seeking (exploration) [ , ] and impulsivity [ , ], which, in turn, may increase the risk of t. gondii infection through increased exposure and incomplete hand hygiene or non-hygienic food preparation, known risk factors for acquiring t. gondii. thus, increased heritability may be manifested not so much by vulnerability to infection when exposed to oocyst or tissue cyst, but through behavioral traits [ , , ], such as genetic effects on impulsivity and decision making that may lead to variation in individual exposure levels to t. gondii infection risk factors. to our knowledge, this is the first study of combined evaluation of, and reciprocal adjustment for, t. gondii heritability and household effects, in contrast to several such studies for malaria parasites. our study supports efforts to identify genetic, epigenetic, and environmental risk factors for t. gondii infection, in interaction, rather than in isolation, and might thus contribute to progress in identifying, and potentially reducing, vulnerabilities for t. gondii infection. this may lead to substantial preventative impact on negative child health outcomes (via reducing congenital toxoplasmosis), and negative adult health outcomes (perhaps by reduction in the rates of mental illness, such as schizophrenia [ ], and potentially lethal consequences attributed to behavioral dysregulation linked to t. gondii infection (such as suicidal behavior, and motor vehicle accidents). . conclusions our heritability results provide impetus for efforts to study genetic contributions to t. gondii seropositivity and serointensity. our next step is to further investigate genetic vulnerability and int. j. environ. res. public health , , of resilience for t. gondii seropositivity and serointensity in the old order amish, including, sequentially, candidate gene, gwas, and exome sequencing analysis, in interaction with reported environmental risk factors for t. gondii in the amish, currently being analyzed. author contributions: conceptualization, t.t.p., j.r.o., b.d.m.; methodology, b.d.m., j.r.o., a.r.d., a.s., t.t.p.; formal analysis, j.r.o., m.p., k.a.r., c.a.l., m.d., t.t.p.; investigation, t.t.p.; data curation, k.a.r.; writing—original draft preparation, a.r.d., j.r.o., t.t.p.; writing—review & editing, a.r.d., j.r.o., m.p., k.a.r., c.a.l., m.d., u.k.r., l.a.b., a.o.m., c.m.p., a.d., d.e.h., t.i.p., a.s., m.w.g., b.d.m., t.t.p.; supervision, t.t.p., b.d.m.; funding acquisition, t.t.p. all authors have read and approved the manuscript. funding: this study was funded by the university of maryland, joint institute for food safety and applied nutrition and the us food and drug administration (us fda) through the cooperative agreement fdu. (pi, t.t.p.) aditionally, this project was also supported in part by the national institutes of health , bethesda maryland nih through the pilot/exploratory grant p dk , and by the csr&d/veterans affairs administration via a merit award (grant number i cx - pi, t.t.p). the results and opinions presented here belong to the authors, and do not represent the official positions of the us fda, va, or nih acknowledgments: we thank the entire personnel of the university of maryland school of medicine amish research clinic, lancaster, pa, usa, in particular the amish liaisons, including naomi esh and hanna king, and the nurses of the amish research clinic, including donna trubiano, yvonne rohrer, theresa roomet, mary morrissey, nancy weitzel and susan shaub. we also thank dr. michael c. bazaco for his suggestions and comments and dr. anna spector for her comments on the final proofs of the manuscript. conflicts of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. references . flegr, j.; prandota, j.; sovickova, m.; israili, z.h. toxoplasmosis—a global threat. correlation of latent toxoplasmosis with specific disease burden in a set of countries. plos one , , e . 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[crossref] [pubmed] © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . /ajmg.c. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . / - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.biopsych. . . http://dx.doi.org/ . /pdb.top http://dx.doi.org/ . /j.diagmicrobio. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /s - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.smim. . . http://dx.doi.org/ . /journal.pone. http://dx.doi.org/ . /journal.pone. http://dx.doi.org/ . /ajtmh. . - http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /journal.pmed. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /s - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /s - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.biopsych. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /tp. . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /schbul/sbl http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.psychres. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	materials and methods 	study population 	plasma collection 	laboratory analysis 	statistical analysis 	results 	t. gondii igg antibody titers 	heritability 	household 	discussion 	conclusions 	references doi: . /s - ( ) -x iii-vs review the advanced semiconductor magazine vol - no - september/october biofunctional led roles as we delve further into the effects of light on human and animal biology, some regions of the visible spectrum are being found to have specific effects on animal and insect behaviour, even at fairly low intensities. therefore, light sources such as incandes- cent, fluorescent, or led lamps, must now be considered as more than just a light source, because of their potential control over important bio-functions, depending on the relative spectral intensities of the activating wavelengths. factors such as melatonin output and the related circadian rhythms (the natural . hour cycle of behaviour and metabol- ic changes exhibited by all plants and ani- mals) are mostly influenced by the light- dark cycle. however, it has been recently shown that melatonin output can alter cancer growth rates and colour related wavelengths are showing evidence of physiological activity that may affect both behaviour and/or health. it is already known that exposure to light is at least a partial cure for a seasonal form of depression (winter blues [not led related] or seasonal affective disorder), which is common in northern winter cli- mates (due to the long nights). exposure to bright light can cure this condition and can also assist night shift workers. in the latter case, minute exposures to bright lighting at the beginning, middle and end of the shifts is found to improve worker performance and well being.these effects are observed from either sunlight or artifi- cial lighting. however, beneficial wave- length-specific effects from led sources have been sought after and, to the poten- tial benefit of led demand, are now being discovered. red (white) & blue for health! in a recent report, mariana figueiro et al, from the lighting research center at rensselayer polytechnic institute, described the results of a pilot study where patients suffering from alzheimer’s disease (ad), with ambient fluorescent lighting levels of lux at the table top, were also exposed to rela- tively low intensity levels ( lux at the eye) of nm (blue) and/or red ( nm) light from led sources.the leds were arranged along the vertical sides of rectangular frames near to the patients to provide the required led intensities.when the patients received blue led exposure, their sleep behaviour became more normal, approaching day- time awake ness and – hours of sleep rather than the erratic sleep behaviour of short periods of sleep and awake ness common for ad patients. additional studies are planned with larg- er sample sizes and optimistically, light sources for this therapy are being designed.what will the blue leds do next? it is hoped that other led thera- pies can be developed, such as led exposure in the morning to help teenagers ward off their late morning awakenings. red led exposure appeared to cause little behaviour change for ad patients, but it has been shown to improve the rate of wound healing, a useful aid for the submariner. what other health related uses for leds lie in store for us? acne treatment and rosacea are both skin problems where the fda has given clearance for blue led use [iii-vs review, august page ]. led safety for the amish in the ever widening panorama of appli- cation categories for high brightness leds, one of the most novel must be the acceptance of led illumination for their transportation vehicles by the deeply reli- gious amish group. this historic sect of pennsylvanian farmers, part of the anabaptist group, (mennonites, quakers et al) have long believed that a simple and devout lifestyle will ease the way to heaven.the amish have existed on the meager livelihood of a simple farmer relying on horse power, oil lamps and a simple lifestyle with horse drawn bug- gies (see figure .) providing the essence of their transportation system, especially for religious duties.a characteristic that tends to restrict long distance travel and maintain their tightly knit, self-sufficient communities.thus, they have long eschewed many of the trappings of dr alan mills po box , mountain view, ca , usa tel/fax: + - - - / email :amills@inreach.com led’s in health and safetym a r k e t f o c u s horse power and oil lamps can be at risk in bad road conditions. but wind & solar powered leds could provide safer lighting ethically. pp - .qxd / / : page modern day civilization, including auto- mobiles, tractors, telephones, electricity, street lighting etc. present in several states and ontario, canada, the total population of the amish community in north america now exceeds one million and they are provid- ing unique market opportunities and novel outlets for high brightness leds. they may not constitute the largest mar- ket opportunities in the world, but they are leading to the development of advanced lighting concepts that have the potential to fulfill lighting needs in the less developed regions of the world, for other religious groups that do not accept all the modernities of the st century and for those that have to work in remote geographic locations. in this new application, the white led headlight units were found to be more effective than the high intensity dis- charge lamps (the hated blue tinted ones!), which draw six to eight amps per lamp at v. however, the high and directed lumi- nance efficiency of the led enables the use of (smaller) batteries that can be charged by solar or wind power and that provide about a fold increase in the time between charges.the new led headlight, which contains eight of the lat- est luxeon leds per unit (from lumileds, san jose, california), each modi- fied with individual optics, is made by sunline solar, a partially amish owned alternative energy business, located in gordonville, pa, deep in the pennsylvania dutch countryside. according to jerry stern from sunline, their white-led headlight design pro- vides wider beam coverage and about % extra viewing range, yet only draws . amps.as part of the total led solu- tion sunline also manufactures tail lamps [red leds] and yellow/amber running light/turn signals [amber leds], both of which have the ability to operate in the flashing mode.thus, sunline solar is the front runner (excuse the pun) in the buggy light-generation race. one up for the amish in transportation applications! the car manufacturers come second, with led-headlight introduction, not expected for three or four more years. the armed forces also have requirements for led headlights. but the present mil spec. design uses to small leds, yet having a performance comparable to the sunline product. the only negative factor for the led headlights appears to be cost. at about $ per unit, they are two to four times as expensive as the existing buggy headlights, but as usual, external factors such as longer bulb and battery life and the related safety issues are winning the day for the led system. the total cost for the full led buggy lighting system, including two to four marker or running lights and tail lamps, is about $ . the extended battery life, which improves from to hours, greatly increases safety margins by reducing the chances of lighting fail- ure —- at the same time increasing the life expectancy of the horse and the buggy passengers on dark country- nights. just think, the latest high bright- ness leds could become standard equipment on the new buggy —- back in your hp power unit and off you go! in spite of the new led-headlight advan- tages, life can be even more difficult for some of the stricter religious sects, because they still do not allow the use of headlights. however, in a meeting of the ancient and exceedingly modern, sunline has recently provided a different led solution for these travelers by devel- oping new tail lamp units that contain one forward facing white led per unit, providing some forward illumination and recognition, while still meeting their more stringent group regulations. other specialty led products in the ‘thanks to the government’ cate- gory, pennsylvania introduced a law that will soon require all milking houses in the state to have lighting, a service not normally demanded by amish cows. again, sunline came to the rescue with another led product.the dairy-farm special is based on an eight d-battery format and uses x mm white leds (this time from nichia). it provides ade- quate lighting, meets the legal and reli- gious requirement, lasts for hundreds of hours and from the state’s point of view, makes for happier, more produc- tive cows. stern also mentioned an additional new product soon to be released, a personal security light, suitable for most emer- gency applications.also based on dry batteries and x mm leds, this portable light will provide many hun- dreds of hours of useful light in disaster and other emergency situations, for an anticipated cost of $ retail. steve mellinger, also from sunline, men- tioned another recent and unique local application for their dry battery lights. in the amish communities, where midwives often have to assist in child birth by flashlight or candle light and the flash- light batteries are known to run down, one of these boxed led lights has been evaluated and requests from other mid- wives are pouring in. obviously, either in the d-battery or solar-rechargeable format, all of these led products developed in pennsylvania will have wider market appeal to mis- sionaries, residents and professionals in underdeveloped regions of the world, where electricity is not available or is an irregular commodity. but, the biggest hurdle to their market growth will probably be spreading their gospel around to all these remote locations! www.three-fives.com led’s in health and safety m a r k e t f o c u s $ for full led buggy lighting system a fine tailpiece for greater safety pp - .qxd / / : page wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ detection and prevention of wormhole attack in wireless sensor network using aomdv protocol procedia computer science ( ) – - © the authors. published by elsevier b.v. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). peer-review under responsibility of the organizing committee of icccv doi: . /j.procs. . . sciencedirect available online at www.sciencedirect.com th international conference on communication, computing and virtualization detection and prevention of wormhole attack in wireless sensor network using aomdv protocol parmar amisha ,v.b.vaghelab astudent, sankalchand patel college of engineering, visnagar- , india bprincipal, jashodaba polytechnic institute, sidhpur- , india abstract unique characteristics like limited bandwidth, limited battery power and dynamic topology makes wireless sensor network (wsn) vulnerable to many kinds of attacks. therefore interest in research of security in wsn has been increasing since last several years. infrastructure less and self-governing nature of wsn is challenging issue in terms of security. wormhole attack is one of the severe attack in wireless sensor network. in this paper, the techniques dealing with wormhole attack in wsn are surveyed and a method is proposed for detection and prevention of wormhole attack. aomdv (ad hoc on demand multipath distance vector) routing protocol is incorporated into these method which is based on rtt (round trip time) mechanism and other characteristics of wormhole attack. as compared to other solution shown in literature, proposed approach looks very promising. ns simulator is used to perform all simulation. © the authors. published by elsevier b.v. peer-review under responsibility of the organizing committee of icccv . keywords: wsn; wormhole attack; rtt; aomdv; malicious node. . introduction sensor nodes are used to perform communication in wireless sensor network. nodes in network here communicate directly with each other using wireless transceivers with no fixed infrastructure. sensor nodes are deployed in large number to monitor the environment or system by measurement of physical parameters such as pressure, characteristic of object temperature and their relative humidity or motion. each node of the sensor network consist of the three subsystems: the processing subsystem which performs local computations on the sensed data, the sensor subsystem which senses the environment and the communication subsystem which is responsible for message * corresponding author. e-mail address: amish.heartly@gmail.com © the authors. published by elsevier b.v. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). peer-review under responsibility of the organizing committee of icccv http://crossmark.crossref.org/dialog/?doi= . /j.procs. . . &domain=pdf parmar amish and v.b. vaghela / procedia computer science ( ) – interchange with neighbouring sensor nodes. cost and size on sensor nodes result in corresponding constraints on resources such as memory, energy, computational speed and communications bandwidth. the application scenarios for wsns are many including military surveillance, commercial, medical, manufacturing and home automation to name many but few [ ]. due to the broadcast nature of the transmission medium and fact that sensor nodes often operate in hostile environments wsns are vulnerable to variety of security attacks. according to the layers of the osi model classification of security attacks in wsns is done. the attacks which operate at the network layer are referred to as routing attacks. there are many types of attacks possible in network layer like selective forwarding, spoofed or replayed routing information, sybil attack, sinkhole attack, hello flood attack and wormhole attack. section ii describes about wormhole attack in detail. section iii describes related work proposed by various authors. section iv deliberates our proposed work for detection and prevention of wormhole attack. section v we present our results. in section vi we conclude. . wormhole attack this attack has one or more malicious node and a tunnel between them. the attacking nodes captures the packets from one location and transmits them to other distant located node which distributes them locally. the tunnel can be established in many ways e.g. in-band and out-of-band channel. this makes the tunnelled packet arrive either sooner or with a lesser number of the hopes compared to the packets transmitted over normal multi hop routes. routing mechanisms which rely on the knowledge about distance between nodes can get confuse because wormhole nodes fake a route that is shorter than the original one within the network [ ]. they can then launch a variety of attacks against the data traffic flow such as selective dropping, eavesdropping, replay attack, etc. wormhole can be formed using, first, in-band channel packet to another malicious node m using encapsulation even though there is one or more nodes between two malicious nodes, the nodes following m nodes believe that there is no node between m and m employ an physical channel between them by either dedicated wired link or long range wireless link shown in fig. . when malicious nodes form a wormhole they can disclose themselves or hide themselves in a routing path. the former is an exposed or open wormhole attack, while the latter is a hidden or close one. in fig , the destination d notice that the packet from the source s is transferred through the node a and b under hidden wormhole attack, while it believes that the packet is delivered via node a, m , m and b under exposed wormhole attack. . related work fig wormhole attack parmar amish and v.b. vaghela / procedia computer science ( ) – in this section we discuss some of the prevailing solutions to the wormhole attacks in wireless ad hoc networks and mobile ad hoc networks. one of the solution is s.gupta et al [ ] proposed a wormhole attack detection protocol using hound packet called whop for detecting wormhole attacks without using monitoring system or any special hardware. a hound packet is used by source node after route discovery process to detect wormhole attacks which counts hop difference between the neighbours of the one hop away nodes in the route. the destination node detects the wormhole based on the hop after the process, difference between neighbours of nodes exceeds the acceptance level. the technique called ‘packet leashes’ [ ] prevents packets from traveling farther than radio transmission range. the wormhole attack can be detected by an unchangeable and independent physical metric, such as time delay or geographical location. it disables wormhole attack by restricting the maximum distance of transmission, using either local information or tight time synchronization. an upper bound on packets lifetime is ensured by the temporal leash. when a node sends a packet to the destination, the sending packet includes the time at which it send the packet and the receiving node compares this value to the time at which it received the packet. the recipient of the packet is within a certain distance from the sender is ensured by geographical leash. the sending node location and its sending time is included in the sending packet. when they reach the receiving node computes the upper bound on the distance between the sender and its own. the drawback of temporal leashes is that they need highly synchronized clocks and of geographical leash is that, each node must know its own location and all nodes must have loosely synchronized clocks. chiu et al. [ ] introduce a delay analysis approach called delphi. it applies a multi-path approach and records the delay and hope counts in transmitting rreq and rrep through the paths. it calculates mean delay per hop of every possible route. the sender computes mean delay per hop of each route after collecting all response. in path with the wormhole attacks, the delay would be obviously longer than the normal path with the same hop count. hence, wormhole nodes could be avoided if the path with longer delays would not be selected to transmit the data packet. khalil et al. [ ] introduces liteworp in which they used notion of guard node. if one of the neighbour of guard node behaves maliciously it can detect the wormhole. the guard node is a common neighbour of two nodes to detect a legitimate link between them. however it is not always possible to find a guard node for a particular link in a sparse network. varsha et al. [ ] presented efficient method to detect a wormhole attack called modified wormhole detection aodv protocol (maodv). based on number of hops and delay of each node in different paths from source to destination wormhole attack is detected. it compares the delay per hop of every node in the normal path and a path that is under wormhole attack, finds that delay per hop of a path that is wormhole attack is larger in comparison of normal path. advantages of this method are that it requires no special hardware and it do not require positioning system and clock synchronization. shortcoming is that when all the paths are wormhole affected this method does not work well. . proposed mechanism to detect and prevent wormhole attack to discover multiple paths between the source and the destination in every route discovery ad-hoc on-demand multipath distance vector routing protocol (aomdv) is used which is an extension of the aodv protocol. in aomdv routing protocol the sender node checks in the route table whether a route is present or not for communication of any two nodes, if present it gives the routing information else it broadcasts the packet, if the route is not present then it broadcasts the rreq packet to its neighbours which in turn checks whether a route is present to the required destination or not. whenever the destination receives the rreq packet it sends rrep packet to the source along the same path through which the rreq packet has arrived. for all rreq packets arrived through other routes the rrep packets are sent along the same path. all the paths are stored in the routing table at source node. in this way the routes are established [ ]. the main idea in aomdv is during route discovery procedure to compute multiple paths for contending link failure. when aomdv builds multiple paths, it will select the main path for data transmission which is based on the time of routing establishment. only when the main path is down parmar amish and v.b. vaghela / procedia computer science ( ) – other paths can be effective and the earliest one will be regarded the best one. using aomdv protocol in this paper a technique is proposed to detect and prevent the wormhole attack in the network efficiently. details of the proposed algorithm is as follows. when the source node broadcasts a rreq packet note time and when the corresponding rrep packet is received by the source, again note the received time of the packet. if multiple rrep packets received, that means there is more than one route available to the destination node then note the corresponding times of each rrep packet. by using the above two values one can calculate the round trip time of the established route or routes [ ]. take round trip time of each route and divide it by respective hop count. calculate the average round trip time of all the routes with the help of this value say . the value obtained is threshold round trip time . after comparing the threshold value with each round trip time ,if the total round trip time is fewer than threshold round trip time and hop count of that particular route is equal to two than wormhole link is existing in that route else no wormhole link present in that route. since wormhole link spotted in that route, sender detects first neighbour node as wormhole node and sends dummy rreq packet through that route i and neighbour . at the destination end receiver receives dummy rreq packet from its neighbour and detects neighbour as wormhole node. routing entries for and are removed from the source node and broadcast to other nodes. thus wormhole affected link is jammed and is no more used. so, that from the next time onwards whenever a source node needs a route to that destination, first it checks in the routing table in the route established phase for a route and it will come to know that, the route is having wormhole link and it will not take that route instead it will take another route from the routing list of the source node which is free from wormhole link if available. benefit of using aomdv protocol in our proposed mechanism is that, it has less overhead and end to end delay. fig. shows the flow chart of proposed algorithm. parmar amish and v.b. vaghela / procedia computer science ( ) – algorithm: algorithm: . when sender broadcast route request packet it will note the time . . for each route reply received by the sender node, sender node notes time . . sender node calculates the round trip time for all routes using a formula = - . . calculate the threshold round trip time by using this formula = . take average of for i number of paths from step . . note this time as threshold round trip time for each route. [just to understand assume i= i.e. total three paths to the destination. then according to step = , = , = step will give the average of above values = which is threshold round trip time ] . if ( is less than and hop count on route i is equals to == true) then{ a. detect route i as a wormhole link b. sender detects first neighbour node as wormhole node c. sender sends dummy rreq through route i and neighbour d. receiver receives dummy rreq from its neighbour e. receiver detects its neighbour as wormhole node f. routing table entries for and are removed and also broadcasted to other nodes } else { there is no threat of wormhole attack and it is not detected } end if . simulation environment and results in this section the simulation results are shown for parameters like delivery rate, average end to end delay and average throughput of the packets at destination by comparing normal aomdv, wormhole affected aomdv and proposed aomdv protocols in a network. initially the readings of normal aomdv are noted based on above fig flow chart of proposed algorithm parmar amish and v.b. vaghela / procedia computer science ( ) – described parameters for , , and nodes respectively. then the wormhole nodes are added in normal conditions and results are noted again. lastly, proposed method is applied in the infectious network and results are compared for all the three scenarios. the wireless sensor network environment is formed using network simulator- . . the following table indicates the simulation parameters. table simulation parameters simulation area m x m routing protocol aomdv packet size bytes traffic rate cbr number of nodes , , , range of transmission m simulation time s mobility model fixed in all figures below on x-axis are the parameters and on y-axis are the routing protocols. fig. . shows that the values of average throughput are plotted against three routing protocols for network density (nodes). the difference in the value of throughput of protocol proposed aomdv and wormhole aomdv increases as the network density rises. thus in terms of throughput as a parameter the performance of network by given proposed algorithm increases for dense network. fig average throughput for , , and nodes in fig. . note that when wormhole nodes are kept in normal aomdv it increases the average end to end delay of the network. after applying proposed algorithm in this environment the average end to end delay value decreases. the values of delay obtained in proposed aomdv are even less than normal aomdv. the above values in the fig. . depicts that the average end to end parameter is improved for large density network. normal aomdv wormhole aomdv proposed aomdv nodes . . . nodes . . . nodes . . . nodes . . . t hr ou gh pu t ( bi ts /s ec ) parmar amish and v.b. vaghela / procedia computer science ( ) – fig average end to end delay for , , and nodes the results for packet delivery fraction in fig. . shows that in spite of variations the packet delivery fraction for the different network densities improves. here it can be seen easily by taking difference of wormhole aomdv and proposed aomdv. fig packet delivery fraction for , , and nodes . conclusion in this work, we have proposed and implemented a wormhole detection and prevention mechanism to detect and prevent the wormhole attacks. in our technique, no special hardware is required. all we have done is calculated the round trip time (rtt) of every route to calculate threshold rtt. according to simulation results of various parameters like average end to end delay, packet delivery fraction and average throughput it is proved that proposed mechanism performs better than wormhole affected aomdv. in future this proposed method can be implemented in mobile ad hoc network also. normal aomdv wormhole aomdv proposed aomdv nodes . . . nodes . . . nodes . . . nodes . . . se co nd s normal aomdv wormhole aomdv proposed aomdv nodes . . nodes . . . nodes . . . nodes . . . . . . . . . . . pa ck et d el iv er y fr ac tio n parmar amish and v.b. vaghela / procedia computer science ( ) – references . c. siva ram murthy and b. s. manoj, “ad hoc wireless networks-architecture and protocols,” prentice hall ptr, theodore s. rappaport, series editor. . s. gupta, s. kar and s. dharmaraja, "whop: wormhole attack detection protocol using hound packet". in the international conference on innovations technology, ieee, pp. - , april . . yih-chun hu, adrian perrig and david b. johnson, “wormhole attacks in wireless networks”, ieee journal on selected areas in communications, vol. , no. , pp. - ,february . . h.s. chiu and k.s. lui. delphi, “wormhole detection mechanism for ad hoc wireless networks”, st international symposium on wireless pervasive computing, pp. – , january . . umesh kumar chaurasia and mrs. varsha singh, “maodv: modified wormhole detection aodv protocol”, ieee, pp. - , . . khalil s. bagchi and n.b. shroff. liteworp, “a lightweight countermeasure for the wormhole attack in multihop wireless networks”, international conference on dependable systems and networks (icdsn), pp. - , . . s. r. biradar, koushik majumder, subir kumar sarkar, puttamadappa s.r.biradar, “performance evaluation and comparison of aodv and aomdv”, international journal on computer science and engineering (ijcse), vol. , no. , pp. - , . . v.karthik raju and k. vinay kumar. “a simple and efficient mechanism to detect and avoid wormhole attacke in mobile ad hoc networks”, international conference on computing sciences, pp. - , ieee . djs_dc_ _ _inthisissue-april .indd april in this issue of diabetes care edited by helaine e. resnick, phd, mph american diabetes association. economic costs of diabetes in the u.s. in . diabetes care ; : – hairston et al. comparison of bmi and physical activity between old order amish children and non- amish children. diabetes care ; : – $ billion spent on diabetes in updated estimates from the american diabetes association (p. ) put the economic bur- den of diabetes at $ billion in —a % increase over the last estimate, which was conducted using data for . this exhaustive economic analysis synthesized information from numerous national data sources administered by multiple federal agencies including the u.s. centers for disease control and prevention, the agency for healthcare research and quality, and the centers for medicare and medicaid services. the new analyses are the latest in a series that continues to document the increasing economic burden of diabetes in the u.s. cost estimates are provided for the diabetic population as a whole, as well as by race/ ethnicity, age-group, and insurance status. the new report shows that of the diabetes- associated costs, % were accounted for by direct medical expenditures and the balance by indirect costs such as lost productivity, unemployment due to diabetes-related disability, and premature mortality. among other key fi ndings are data showing that inpatient hospital care accounts for % of direct medical costs and that the medical expenditures of people with diabetes are about . times higher than what they would be if diabetes were absent. other fi ndings highlight the importance of the aging of the u.s. population on the economic burden of diabetes: % of diabetes-related health care costs are utilized by people aged and older. this suggests that as the baby boom generation continues to “age in,” the age- specifi c allocation of diabetes-related health care costs will continue to heavily favor older adults—a consideration that has obvious implications for the burden of diabetes-related morbidity and mortality in old age, as well as the medicare program itself. — helaine e. resnick, phd, mph slower weight gain may reduce diabetes prevalence in the amish community a new study examines whether childhood factors may contribute to the much lower preva- lence of diabetes in old order amish (ooa) adults in the u.s. (about half the rate) relative to the overall population of u.s. adults of european descent. since the number of years lived with obesity predicts diabetes risk, the authors hypothesized that deferment of weight gain may protect against diabetes. data from the study, published in this issue of diabetes care (p. ), indicate that ooa children are less likely to be overweight and are more physically active than non-amish children. the ooa community in pennsylvania adheres to strict life- style rules, including no use of electricity or cars. children often walk or scooter to school and do not watch television. to assess how this lifestyle may affect childhood obesity, the study examined anthropomorphic data from ooa children aged – years during the period – . it compared the children’s age- and sex-adjusted bmis with estimates from the national health and nutrition examination survey (nhanes). it also examined the correla- tion between physical activity (pa) and bmi. finally, the study compared ooa children’s pa levels with pa levels of non-amish children aged – years from maryland’s eastern shore (es), a nearby rural community. the study found an inverse correlation between pa and being overweight. ooa children spent more minutes per day in light pa and more minutes per day in moderately vigorous pa relative to es children. ooa children were also . times less likely to be overweight than both es and nhanes children. these data may lead to improved understanding of the relationship between weight gain over the life span and its relationship with risk of diabetes in adulthood. — helaine e. resnick, phd, mph care.diabetesjournals.org diabetes care, volume , april http://care.diabetesjournals.org/content/ / / http://care.diabetesjournals.org/content/ / / http://care.diabetesjournals.org/content/ / / http://care.diabetesjournals.org/content/ / / april the et al. timing and duration of obesity in relation to diabetes: fi ndings from an ethnically diverse, nationally representative sample. diabetes care ; : – adolescent obesity increases odds of diabetes in young adulthood the effect of obesity on diabetes risk over the life course is poorly understood. in this issue of diabetes care (p. ), a new study examines the infl uence of the timing and duration of obesity on diabetes in young adulthood among , individuals aged – years in the u.s. national longitudinal study of adolescent health. diabetes was defi ned as glycosyl- ated hemoglobin (a c) ≥ . % or self-reported diagnosis by a health care provider. during years of follow-up, . % of the cohort developed diabetes, including . % with undiagnosed diabetes (a c ≥ . % with no self-reported diagnosis). as young adults, , of , participants had been obese at least once during adolescence, most of whom had persistent obesity. in women, obesity prior to age years was associated with a . ( % ci . – . ) greater odds of diabetes in adulthood compared to obesity in later adolescence, even with adjustment for multiple factors including bmi, waist circumference, and age at menarche. persistent obesity in adolescence, rather than the specifi c timing of obesity, was related to a . ( % ci . – . ) greater odds of diabetes in men and a . ( % ci . – . ) greater odds in women compared to adults whose obesity started in adulthood. sex modifi ed the relationship between timing of obesity and odds of diabetes, but it had no impact on the effect of obesity duration and odds of developing diabetes. sensitivity analyses assessing the potential roles of selection bias and varied defi nitions of diabetes or obesity classifi ca- tions on the observed relationships showed consistent results. these fi ndings highlight the importance of preventing and reducing adolescent obesity as a diabetes prevention strategy and suggest that diabetes screening may be warranted for obese young adults. — elsa s. strotmeyer, phd, mph doi: . /dc -ti © by the american diabetes association. readers may use this article as long as the work is properly cited, the use is educational and not for profi t, and the work is not altered. see http://creativecommons.org/licenses/by-nc-nd/ . / for details. diabetes care, volume , april care.diabetesjournals.org http://care.diabetesjournals.org/content/ / / http://care.diabetesjournals.org/content/ / / << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma . ) /calrgbprofile (srgb iec - 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/$ . published by elsevier inc. doi: . /j.jacc. . . editorial comment he tell-tale heart now, optically mapped)* iguel valderrábano md, mish s. dave md, phd ouston, texas n edgar allan poe’s short story ( ) published in , a ameless narrator murders an old man, dismembers the ody, and then hides it under the floorboards. when the olice come to investigate, the murderer becomes quickly ormented by what he perceives is the sound of the dead an’s heartbeat and compels the officers to tear up the oorboards. as with the narrator in poe’s classic work of ction, countless researchers over the last century have been ompelled to seek out the source of the heartbeat. see page the sinoatrial node (san) has similarly obsessed anato- ists, physiologists, and cardiologists for more than ears since its original description by keith and flack in ( ). the fascination spans the spectrum from the olecular origins of pacemaker automaticity to the ana- omic and physiologic mechanisms of macroscopic propa- ation of the sinus node impulse to neighboring atrial tissue. t all levels, fascination has been accompanied by contro- ersy. even of the discoverers of the san is controversial, ir arthur keith being allegedly involved ( ) in the scien- ific scandal involving the fabrication of “piltdown man,” an volutionary missing link. recently, circulation research ublished a series of articles reviewing and revitalizing ontroversies around the san ( – ). the molecular mech- nisms of pacemaker activity generation have been disputed etween several proposed mechanisms, including various nward membrane currents (if, t- and l-type ca � cur- ents, the “membrane voltage clocks”) as well as intracellular a � cycling (the “calcium clocks” ( , , ), involving re- ease from the sarcoplasmic reticulum and possibly store- perated ca � channels [ ]). details of the generation of he pacemaker activity can be found elsewhere ( ). editorials published in the journal of the american college of cardiology reflect the iews of the authors and do not necessarily represent the views of jacc or the merican college of cardiology. from the division of cardiac electrophysiology, department of cardiology, c ethodist hospital, houston, texas. both authors have reported that they have no elationships to disclose. just as interesting, however, are the mechanisms of mpulse conduction from the san to the atria at the acroscopic level. successful propagation from the central acemaker cell (or group of cells) to atrial tissue is a remendous physiological challenge. normal propagation in ardiac tissue entails a delicate balance between depolarized ells (source) and the resting tissue ahead (sink) ( ). xcited cells serve as a source of electric charge for depo- arizing neighboring cells (sink). the relationship between ource and sink defines the safety factor of propagation. a arge volume of excited tissue (source) will easily propagate nto a small volume of quiescent tissue (sink). alternatively, f the sink is too large, propagation fails due to a source–sink ismatch. how does the san manage to take depolariza- ion from a small group of cells into the entire atrial tissue? ellular coupling holds the key to this challenge. in con- uction failure due to source–sink mismatch, propagation ails because cells close to the wave front fail to depolarize as he neighboring, well-coupled unexcited tissue downstream olds their membrane potential polarized. it is known that ropagation from a small group of cells (source) into a large roup of well-coupled cells (sink) has a low safety factor and s likely to fail. in this scenario, decreasing cellular cou- ling— e.g., via decreasing gap junction conductance— can aradoxically enhance propagation success, despite slowing onduction velocity ( ). it is intuitive that a similar echanism must play a role in propagating san conduc- ion. the slow conduction within the san supports un- oupling as a mechanism of slow-but-safe propagation. ow to successfully conduct from san to sites of initial trial activation remains challenging to explain. mapping studies ( , ) have shown that initial atrial ctivation sites can vary widely during sinus rhythm. in their study, meek and eyster ( ) used multiple string alvanometers and, by identifying locations with initial lectrical negativity under conditions of vagal stimulation or ocalized cooling, observed that initial atrial activation sites an vary. in , boineau et al. ( ) observed a trifocal rigin of the atrial waveform in the dog using multiple ipolar atrial electrode recordings. the subsequent oineau-schuessler san model proposed the existence of iscrete conduction pathways connecting the san with trial tissue to explain beat-to-beat divergent activation sites – ). in this issue of the journal, fedorov et al. ( ) add to the uthors’ extensive contribution over many years to the study f the san. in particular, the development of the technique f optical mapping has provided a modality by which to tudy the relationship between san anatomy and physiol- gy to begin to explain san function. applying this echnique, they recently found evidence for discrete exit athways connecting the san and atria in the dog ( ). hey were able to directly map the conduction inside the anine san and atrium, and identified or more discrete onduction pathways directed superiorly or inferiorly from t t a t p s e a r n h m p t m s p t a f w ( t i d a d f m c p t c r m t r valderrábano and dave jacc vol. , no. , the tell-tale heart october , : – he san. interestingly, they measured slowing of conduc- ion velocity in these pathways down to cm/s, and ttributed this to source–sink mismatch. they concluded hat the etiology of multifocal atrial activation is due to the ossibility of atrial excitation from any of multiple discrete an exit pathways. in the current report, they have xtended these findings to the human, for the first time pplying optical mapping techniques to obtain voltage ecordings of the coronary perfused intact human san and earby atria during normal sinus rhythm. they confirm the uman san is electrically insulated from nearby atrial yocardium with the exception of several discrete exit athways, noting a similar slowing of conduction within hese pathways as in the canine study. from the source–sink ismatch point of view, this insulation makes sense and eems necessary: if the atrial tissue were well coupled to the acemaker cells, propagation within the san would fail due o excessive current sink. controversies remain in the interpretation of the optical ction potential signals (which are a superposition of signals rom overlapping atrial myocardium and san layers) as ell as in the role of calcium dynamics in pacemaker activity ). notwithstanding such, understanding the mechanism hat overcomes the source–sink mismatch is likely to have mportant clinical implications. the authors suggest that ecreased conduction velocity is a result of the mismatch; nother explanation may be that cell– cell coupling is re- uced in these exit pathways so as to increase the safety actor for propagation, i.e., to overcome the source–sink ismatch. these questions and more remain; however, the urrent report continues a fruitful tradition of comparative hysiology research going back over years, and thanks o this work, we are no doubt closer to solving the ontroversies than ever before. eprint requests and correspondence: dr. miguel valderrábano, ethodist hospital, fannin street, suite , houston, exas . e-mail: mvalderrabano@tmhs.org. eferences . poe ea. the tell-tale heart. in: poe ea. the tell-tale heart and other writings. new york, ny: bantham dell random house; : – . k . keith a, flack m. the form and nature of the muscular connections between the primary divisions of the vertebrate heart. j anat physiol ; : – . . tobias pv. piltdown: an appraisal of the case against sir arthur keith. curr anthropol ; : – . . o’rourke b. be still, my beating heart: never! circ res ; : – . . efimov ir, fedorov vv, joung b, lin s. mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical map- ping. circ res ; : – . . difrancesco d. the role of the funny current in pacemaker activity. circ res ; : – . . lakatta eg, maltsev va, vinogradova tm. a coupled system of intracellular ca � clocks and surface membrane voltage clocks con- trols the timekeeping mechanism of the heart’s pacemaker. circ res ; : – . . christoffels vm, smits gj, kispert a, moorman afm. development of the pacemaker tissues of the heart. circ res ; : – . . maltsev va, lakatta eg. dynamic interactions of an intracellular ca � clock and membrane ion channel clock underlie robust initia- tion and regulation of cardiac pacemaker function. cardiovasc res ; : – . . maltsev va, lakatta eg. synergism of coupled subsarcolemmal ca � clocks and sarcolemmal voltage clocks confers robust and flexible pacemaker function in a novel pacemaker cell model. am j physiol heart circ physiol ; :h – . . ju y, chu y, chaulet h, et al. store-operated ca � influx and expression of trpc genes in mouse sinoatrial node. circ res ; : – . . fast vg, kléber ag. cardiac tissue geometry as a determinant of unidirectional conduction block: assessment of microscopic excitation spread by optical mapping in patterned cell cultures and in a computer model. cardiovasc res ; : – . . rohr s, kucera jp, fast vg, kléber ag. paradoxical improvement of impulse conduction in cardiac tissue by partial cellular uncoupling. science ; : – . . lewis t, oppenheimer a, oppenheimer b. the site of origin of the mammalian heart beat: the pacemaker in the dog. heart ;ii – . . meek wj, eyster jae. experiments on the origin and propagation of the impulse in the heart: iv. the effect of vagal stimulation and of cooling on the location of the pacemaker within the sino-auricular node. am j physiol ; : – . . boineau j, schuessler r, mooney c, et al. multicentric origin of the atrial depolarization wave: the pacemaker complex. relation to dy- namics of atrial conduction, p-wave changes and heart rate control. circulation ; : – . . boineau j, canavan t, schuessler r, cain m, corr p, cox j. demonstration of a widely distributed atrial pacemaker complex in the human heart. circulation ; : – . . schuessler rb. abnormal sinus node function in clinical arrhythmias. j cardiovasc electrophysiol ; : – . . fedorov vv, glukhov av, chang r, et al. optical mapping of the isolated coronary-perfused human sinus node. j am coll cardiol ; : – . . fedorov vv, schuessler rb, hemphill m, et al. structural and functional evidence for discrete exit pathways that connect the canine sinoatrial node and atria. circ res ; : – . ey words: bradycardia y exit pathway y optical mapping y sinus node. mailto:mvalderrabano@tmhs.org how liberal is (the liberal critique of) a liberal eugenetics? humana.mente journal of philosophical studies, , vol. , - how liberal is (the liberal critique of) a liberal eugenics? nathan van camp † nathan.vancamp@ua.ac.be abstract this article critically surveys the current bioethical and politico- philosophical debate about the ethical permissibility of a so-called ‘liberal eugenics’ and argues that neither the liberal argument for nor the liberal argument against human genetic enhancement is internally consistent as, ultimately, each ends up violating the very liberal principles it nonetheless pretends to defend. in particular, it will be shown that while the argument against a new eugenics necessarily entails a preemptive dehumanization of any potential enhanced form of life, the argument for it threatens to reduce any non-enhanced form of life to a “wrongful life” or a life not worth living. it will therefore be concluded that the specific stakes of this contentious issue cannot be grasped within a liberal conceptual framework. introduction recent progress in molecular biology and genetics has opened up the way for the deliberate manipulation of the human genome. although there are still numerous technical barriers that have to be overcome before human genetic modification will become a standard medical procedure “the question is no longer whether we will manipulate embryos, but when, where, and how” (stock, , p. ). the most direct benefit of genetic technologies will be in the prevention and healing of disease. but in addition to this obvious use, it will also be possible to employ these technologies for the purpose of human  this research was made possible by a generous scholarship granted by the flemish research foundation (fwo). † department of philosophy, university of antwerp, belgium. humana.mente – issue – may genetic enhancement. that is, if we can identify the gene(s) for phenotypic characteristics like height, strength, intelligence, and temperament, then it will be possible to use this knowledge to design human beings according to our personal preferences. in bioethical and politico-philosophical debates about genetic technologies, these developments are usually framed in terms of a return of eugenics. the central assumption guiding much of the literature on the subject is that if genetic technologies produce eugenic effects, then they are also morally unacceptable. most recently, however, some commentators have taken a different approach to this issue. they argue that there is nothing intrinsically wrong with the goals of eugenics as such and that its moral acceptability depends on the values and principles of the political ideology that regulates its implementation in society (agar, ; dworkin, ; harris, ). they reject as unjustified any comparison that might be drawn between the project of human genetic enhancement and earlier morally reproachable eugenic practices by arguing that the new eugenics will be firmly rooted in the core liberal principles of state neutrality and individual autonomy. liberal critics of human genetic enhancement, on the other hand, claim that this attempt to integrate the eugenic ideal into a liberal framework is bound to fail and that it will inevitably corrupt the central tenets of political liberalism to the point of its becoming something different altogether (fukuyama, ; habermas, ; sandel, ). in this article, it will be argued, however, that both liberal responses to the challenge of human genetic enhancement are internally inconsistent, as both are bound to lead to conspicuously illiberal conclusions. more specifically, it will be shown that while the liberal argument in favor of enhancement threatens to deprive all non-enhanced forms of human existence from any intrinsic value, the liberal argument against enhancement threatens to do exactly the same with regard to all future enhanced forms of human existence . the liberal eugenics it is notable that few advocates of the new eugenics are willing to call the practice they support by that name. john harris, for example, prefers to speak of “deliberate selection” (harris, , p. ) and gregory stock favors the term “human self-design” (stock, , p. ). these authors’ reluctance to use the term ‘eugenics’ obviously has much to do with the dark shadow that still how liberal is (the liberal critique of) a liberal eugenics? hangs over earlier attempts to make improvements to the biological foundations of human existence. many critics are indeed afraid that the emergence of a new eugenics will also prompt the return of some of the horrific acts committed in the field’s name, such as the atrocities committed by nazi eugenicists. this association is so strong that occasionally, when a practice is referred to as eugenic, it is in fact being described as morally reproachable (wilkinson, ; paul, ). apparently, the mere use of the label is enough to indicate that it refers to a field of practices that any reasonable person would find morally objectionable. conversely, those who argue in favor of a new eugenics are almost invariably accused of offering a thinly veiled justification of nazism. yet despite its inglorious history, the concept of eugenics continues to attract enthusiastic supporters. there appears to be something undeniably appealing in the essential idea of eugenics, something that prevents us from rejecting it in its entirety. who, after all, would not want to give his or her child the best possible genetic endowment? convinced that the potential benefits of genetic technologies in human reproduction are too valuable to renounce on the basis of past abuses, advocates of a new eugenics therefore argue that the main question is not whether the nazi eugenics program was abhorrent but whether the atrocities committed in the name of eugenics were not in fact the result of the underlying nazi ideology rather than something intrinsic to the field of eugenics itself. provided that the eugenic goal of ‘enhancing’ human beings still enjoys universal support and approval, and that the moral acceptability of eugenics depends on the values and principles of the political ideology regulating its implementation in society, then, they suggest, it might still be possible to devise a form of eugenics that is compatible with the basic tenets of contemporary liberal democracy. nicholas agar, one of today’s most vocal advocates of a new eugenics, has argued that the central principles of liberalism provide ample guidance for avoiding the moral pitfalls of earlier forms of eugenics: “[t]he addition of the word ‘liberal’ to ‘eugenics’ transforms an evil doctrine into a morally acceptable one” (agar, , p. ). in his view, the most important difference between the authoritarian eugenics of the past and the liberal eugenics he envisages is simply the degree of control that the state has over the reproductive choices of its citizens: “while old fashioned authoritarian eugenicists sought to produce citizens out of a centrally designed mould, the distinguishing mark of the new liberal eugenics is state neutrality” (ibid., p. humana.mente – issue – may ). state neutrality is, of course, central to any liberal democratic system which aims to protect the principle of value pluralism. in its original formulation, the principle of value pluralism was primarily intended to safeguard freedom of religion and expression, but liberal eugenicists believe that it is broad enough to cover the freedom to use genetic technologies in the field of reproduction (robertson, ). this means that governments must refrain from interfering not only with the more ordinary reproductive choices of its citizens but also with new reproductive choices made possible by genetic technologies. another important reason why liberal eugenicists are convinced that there is no need for moral panic in the face of a new eugenic era is that they think that there is no morally relevant difference between shaping humans by making modifications to their environment and shaping humans by making modifications to their genes (agar, , p. ). they argue, for example, that if parents are allowed and even encouraged to increase their children’s intelligence by providing them with the best possible education, then they should also be allowed to pursue the same goal through genetic technologies. there are two aspects to this claim. first, it allows liberal eugenicists to refute the common argument that genetic intervention is substantially more intrusive than any other influence we may have over the development of another human being. second, if there is no substantial difference between genetic intervention and other influences that parents have over the development of their children, then there is also no need to develop new ethical guidelines and legal regulations for genetic technologies, because the freedom to use such technologies is already protected by the existing right to reproductive freedom (harris, , p. ). yet, some critics have argued that there is indeed nothing morally suspect about human genetic modification as such but that one should nonetheless distinguish between genetic intervention for therapeutic purposes and intervention for enhancement (walters & palmer, , p. xviii; campbell et al, , p. ). there are two assumptions inherent to this argument. the first is that there is an objective difference between genetic interventions that aim at restoring the capacities of the body to their ‘normal’ state and interventions that aim at raising them above this state. the second assumption is that this distinction corresponds to the moral boundary between permissible and impermissible uses of genetic technologies. in other words, this argument holds that there is nothing morally wrong with using genetic technologies to how liberal is (the liberal critique of) a liberal eugenics? heal people, but that it is impermissible to use them to boost human capacities above what is normal, or for that matter, below what is normal (scully, ). while this argument appears to possess the merits of simplicity and fitness for practical application, both of its assumptions have met with severe criticism from liberal eugenicists. john harris, for one, thinks that “enhancements are not plausibly defined relative to normalcy, to normal species functioning, nor to species-typical functioning” (harris, , p. ). according to him, these notions “play no part in the definition of harm and therefore no part in the way the distinction between therapy and enhancement is drawn” (ibid., p. ). he gives a striking example to illustrate this. suppose it was possible to use genetic technologies to slow down or even halt the ageing process. if we would only allow genetic intervention to restore normal functioning, then we would have to forsake this clearly benevolent use of genetic technology because it would not simply restore our body to normal functioning but actually enhance it beyond its normal state. in other words, since it is perfectly normal for us to die of the diseases of old age, this intervention would go beyond the therapeutic use of genetic technologies and would therefore be morally unacceptable. as a libertarian consequentialist, harris believes that the moral imperatives either to provide therapy or enhancement derive from the fact that we value minimizing harm and maximizing benefits. what counts in deciding if it would be permissible to use genetic technologies is not the fact of whether an individual’s current state deviates from normal functioning, but the cost/benefit calculation regarding the body’s “possible functioning” (ibid., p. ). that is to say, the only pertinent questions are whether the harm the technologies aim to prevent is serious enough and whether the benefits they aim to produce are valuable enough to take the risks. . in defense of human nature some critical liberal observers have argued, however, that the proposed marriage between eugenics and liberalism will not so much redeem the former of its authoritarian drift as corrupt the central principles of the latter to the point of its becoming something different altogether. this argument can take a variety of different forms, but the basic assumption is that modifications to the human genome threaten to disrupt something that is valuable in itself. consequently, since our very understanding of human dignity and its legal reflection in human rights is founded upon the notion of human nature, then humana.mente – issue – may genetic modification could ultimately signal the end of the central principles of liberal democracy. in the future of human nature, jürgen habermas therefore argues that human nature should be legally protected against genetic enhancement. yet he founds this claim on a very specific understanding of what it essentially means to be human. what he seeks to protect are not the ‘species- typical’ characteristics and behaviors of homo sapiens, but “the conditions under which the practical self-understanding of modernity may be preserved” (habermas, , p. ). he argues that once we achieve a reflexive understanding of the necessary conditions for “our capacity to see ourselves as the authors of our own life histories” (ibid., p. ), we will realize that a liberal eugenics contradicts these conditions and should therefore be rejected. central to habermas’s argument is the notion that dignity is not a property one possesses simply by virtue of being human, but that it is the distinctive mode of being of a “communicatively structured form of life” (ibid., p. ). what he means by this is that we are only able to understand ourselves as free and autonomous agents worthy of respect in the context of a moral community that consists of equal members interacting with each other on the basis of norms and reasoning. thus, when habermas states that the danger of genetic technologies lies in their power to change human nature, he means that their free deployment threatens to undermine the very foundations of the moral community. to demonstrate why this is so, habermas invites us to consider that our lifeworld is still largely ‘aristotelian,’ in the sense that we tend to make automatic distinctions between “what is manufactured and what has come to be by nature” (ibid., p. ). this distinction is morally relevant insofar as it motivates us to adopt a particular mode of action when dealing with entities belonging to either one of these realms: while inert, inorganic entities are open to various forms of technical-instrumental intervention, self-regulated organic entities are not. according to habermas, this is due to the fact that we spontaneously feel ‘empathy’ for organisms which seem to possess a certain amount of subjectivity, no matter how minimal. we remain committed to this logic in the case of genetic interventions carried out on embryos for therapeutic purposes, firstly because our actions in this case are still guided by the natural processes of growth inherent to this prenatal form of life, but also because we imagine how the future person might give consent for any intervention that could prevent or cure a debilitating condition. in the case of genetic enhancement, however, a very different scenario emerges. here, prospective parents are not treating the embryo as another subject who will how liberal is (the liberal critique of) a liberal eugenics? come to be on an equal footing with them, but as an object they can simply dispose of if necessary. in other words, if the relationship between parent and child is reduced to that of producer and product, they will never be able to meet each other as equal members of the moral community. according to habermas, then, liberal eugenicists make the mistake of focusing solely on the freedom enacted in parental choice, while the proper question to ask is what consequences genetic intervention will have for the programmed person’s “capacity of being oneself” (ibid., p. ) on which one’s ethical self-understanding as a free and autonomous member of a liberal egalitarian society depends. it is true that if “we experience our freedom with reference to something which, by its very nature, is not at our disposal” (ibid., p. ). then the situation of the programmed person is not fundamentally different from that of an individual born “the natural way,” for neither have had any say in the genetic traits and characteristics they are endowed with. the crucial question to ask, however, is if it makes any difference whether these traits are the result of natural chance or of the deliberate intervention of a third person. liberal eugenicists tend to play down the impact of this intervention on the existential situation of an enhanced individual by suggesting that there is no substantial difference between improving a person by modifying her social environment and doing so by modifying her genes. in his view, however, while a genetically unenhanced person always retains the option of rejecting or reappraising her parents’ attempts to shape her personality through socialization, the enhanced person “who is at odds with genetically fixed intentions is barred from developing (…) an attitude towards her talents (and handicaps) which implies a revised self-understanding and allows for a productive response to the initial situation” (ibid., p. ). moreover, a liberal eugenics would not only deprive the genetically enhanced person of the spontaneous self-perception of being the singular author of her own life, but also create the child’s permanent and irreversible social dependence on the parent, which “is foreign to reciprocal and symmetrical relations of mutual recognition proper to a moral and legal community of free and equal persons” (ibid., p. ). . the new eugenics and the self-negation of liberalism it should be clear, then, that this debate mainly revolves around the question of whether the new eugenics concurs with or contradicts the central principles of humana.mente – issue – may liberalism. clearly, much depends on the actor that is given priority in this discussion: while liberal eugenicists tend to emphasize the parent, liberal critics believe that special consideration should be given to the prospective child. this difference of emphasis explains why the former group considers state neutrality in the domain of reproduction to be a sufficient guarantee of the liberality of the new eugenics. they argue that if the state remains neutral in this matter and does not intervene to enforce a particular conception of the good to be sought through genetic modification, then, by giving parents more control over which genetic traits their children will inherit, a liberal eugenics will actually strengthen the freedoms associated with reproduction. critics, on the other hand, point out that the main threat to the central tenets liberalism no longer comes from potential state intervention but from parents themselves. according to this group, it is not the freedom of parents that is at issue but the freedom of the children born to them. on the face of it, this way of framing the new eugenics debate may not be entirely satisfactory, for it gives the impression that liberal eugenicists believe there should be no moral or legal limitations whatsoever to the reproductive liberty of parents. this is obviously not the case. as with other individual liberties, reproductive choices tend to be judged for acceptability against john stuart mill’s principle of harm. as is well known, this principle broadly states that one is free to act as one chooses, as long as one’s actions do not cause harm to others. the problem in the specific case of genetic enhancement is, however, that the limit of individual freedom is not set by potential harm done to fellow citizens but to human beings who do not yet exist. one of the most influential approaches to this complicated issue was developed by derek parfit, and is known as the “nonidentity argument” (parfit, ). the example parfit gives is that of a -year-old girl who decides to have a child. intuitively, we would be inclined to believe that she is likely to harm her child because, by dint of having such a young mother, the child is likely to receive “a bad start in life” (ibid., p. ). furthermore, we would probably also believe that it would have been better for her child if the mother had waited longer to conceive, for then her child would have had better chances in life. parfit shows, however, that this is an inaccurate appraisal of the situation. if the girl had indeed waited longer to have a child, this child would have been the product of a different egg and a different sperm. it would, in other words, have been a different child. the further implication of this is that the child born to her at the age of has not been harmed, since the condition of this particular child should be compared how liberal is (the liberal critique of) a liberal eugenics? not to that of the hypothetical child born a couple of years later but to the condition of not being born at all. in other words, being born to a -year-old mother is no worse for a child than being born to, for example, a -year-old mother, because the alternative is not being born at all. one of the conclusions that has been drawn from this argument is that in reproductive freedom, the threshold of harm should be set at the point where the child would have been better off not being born. the underlying rationale is that all forms of life which fall short of this threshold constitute a “life not worth living” or a “wrongful life” (feinberg, ). obviously, the problem that some liberal eugenicists have with this argument is not that it would give prospective parents too little reproductive liberty, but that it would give them too much. indeed, very few are willing to accept the ultimate conclusion to which this argument seems to lead, namely that parents’ reproductive liberty should be so wide as to include even the freedom to endow their children with a physical or psychological disability. yet, according to the nonidentity argument, a child would usually not be harmed by such an anomalous reproductive choice, for very few cases are likely to arise in which a child would find herself in such terrible conditions that it would have been better for her not to be born at all. it would, for example, be very difficult to maintain that being born deaf is worse than not being born at all. in order to escape this conclusion, liberal eugenicists usually fall back on what is called the principle of procreative beneficence, which, in one version, states that parents “should select the child, of the possible children they could have, who is expected to have the best life, or at least as good a life as the others, based on the relevant available information” (savulescu, , p. ). broadly, this principle entails that parents are morally required to give their children the best possible genetic endowment. it is clear, however, that this principle is still much too formal to prevent parents from endowing their children with a disability how, then, do liberal eugenicists attempt to resolve this conflict between the principles of reproductive freedom and procreative beneficence in the case of selecting for disability? one solution could be, first, to define disability as a diseased state and subsequently argue that deliberately creating a disabled child constitutes a clear violation of medical deontology. this solution would not be wholly satisfying, though, because it would be necessary to reintroduce an objectivist notion of normality or normal functioning against which a given condition could be assessed. this is a solution that liberal eugenicists wish to humana.mente – issue – may avoid at all costs. john harris has therefore proposed defining disability as “a condition that someone has a strong rational preference not to be in and one that is moreover in some sense a harmed condition” (harris, , p. ). to determine whether a given condition is a harmed one, he suggests using what he calls the “emergency room test:” i have in mind the sort of condition for which if a patient presented with it unconscious in the emergency room of a hospital and the condition could be easily and immediately reversed, but not reversed unless the doctor acts without delay, a doctor would be negligent were she not to attempt reversal. (ibid.) according to harris, the main advantage of this conception of a harmed condition is that it is not defined in relation to the state of nonexistence, or to normal functioning, but “relative to possible alternatives” (ibid., p. ). suppose, he explains, that someone was brought into the hospital with her little finger severed at the first joint and it could be sewn on again. although it would obviously be absurd to maintain that the missing end joint of this person’s little finger meant that her life would be not worth living, there are nonetheless good moral reasons to maintain that the hospital staff would harm the patient by failing to reattach the finger. according to harris, the same holds true for all other injuries, diseases and disabilities. catherine mills has fiercely criticized this definition of disability, firstly because it neglects the simple fact that “some disabilities are neither irreversible nor removable” (mills, , p. ) and secondly because it uses the perspective of an “able-bodied person” (ibid.) as the standard against which to evaluate a given condition. yet, though this criticism may certainly hold true in the present, mills seems to ignore the fact that harris develops this argument in relation to genetic modification technologies of the future. what he actually suggests is that when we have the choice to have a child either with or without a disability, we have good moral reasons to choose the second option. another factor that critics have overlooked is that, as genetic science advances, it is likely not only to increase reproductive freedom and the responsibilities that come with it, but also to change the standards against which we seek to measure a harmed condition: it is normal now, for example, to be protected against tetanus; the continued provision of such protection is not merely permissive. if the aids pandemic continues unabated and the only prospect, or the best prospect, for stemming its advance is the use of gene therapy to insert genes coding for antibodies to aids, i cannot think that it would be coherent to regard making available such how liberal is (the liberal critique of) a liberal eugenics? therapy as permissive rather than mandatory (harris, , p. ; emphasis added). we cannot think of a stronger argument against deliberately endowing one’s children with a disability. what harris is saying here is that if parents have the power to prevent their child being born with a disease or disability, they should have not merely the freedom to use this power, but “the obligation to pursue human enhancement” (ibid., p. ) harris’s argument is not that the state should intervene to enforce this obligation —in his view, it is a moral obligation we have to our children—but we have no reason to assume that such demands will not be formulated as soon as these technologies become more widely available. if, upon closer examination, the liberal eugenicists’ argument for the freedom to intervene in the genetic make-up of future generations resembles an argument for the obligation to intervene, then it is seems that the critics are right to conclude that “liberal eugenics is a betrayal of liberal philosophy” (fox, , p. ). curiously, this is not how they themselves reach this conclusion. as we have showed, the danger that many see in a liberal eugenics is that it might change human nature. habermas has developed what is probably the most sophisticated version of this approach. his main point of critique is that being endowed with specific genetic traits and characteristics will deprive the programmed person of “an unobstructed future of his own” (habermas, , p. ). the idea is that a person who learns that some of her talents, skills and abilities were not given to her by “nature” but by means of the deliberate intervention of another person will find it impossible to understand herself as the singular author of her own life. interestingly, however, the underlying idea of this argument did not originate in the context of a discussion about the consequences of new genetic technologies. what actually prompted joel feinberg to write his seminal essay ‘the child’s right to an open future’ ( ) was a series of lawsuits in which members of the amish community challenged compulsory schooling laws in various states of the usa. as is widely known, the amish live an extremely secluded life, far removed from the complexity of the modern industrialized world. in wisconsin v. yoder the united states supreme court ruled in favor of an appeal made by the amish community, noting that by forcing amish children to attend state schools the state of wisconsin infringed on their constitutional religious rights. in his essay, feinberg disagrees with this decision by arguing that the amish way of life infringes on amish children’s humana.mente – issue – may right to an open future by prematurely closing off many of the other ways of life available in a free liberal society. it seems somewhat odd that habermas refers directly to feinberg’s essay in his argument against enhancement technologies (habermas, , p. ), for two reasons. firstly, by likening attempts to shape children by altering their social environments to attempts to shape them by altering their genetic profiles, he actually seems to be pursuing one of the strategies that liberal eugenicists employ to argue the opposite of what habermas himself intends. as explained above, if there are no substantial differences between genetic intervention and the other influences that parents have over the development of their children, then there is no reason to allow the latter while rejecting the former. secondly, at the core of habermas’s argument lies the contention that while the effects of “a pathogenic socialization process” can always be “revised by critical reappraisal” (ibid., p. ), this is impossible in cases of genetic intervention. if, as it appears, he actually disagrees with feinberg’s view on the intrusiveness of certain educational practices, why then does he claim to base his own argument on it? the most plausible explanation for this confusion seems to be that habermas wishes to retain the structure of feinberg’s reasoning but not its content. that is to say, he agrees with him insofar as we should be especially concerned about a child’s right to an open future, but disagrees with him insofar as he rejects the notion that the greatest threat to this right comes from a “pathological” socialization process. liberal eugenicists often liken the effects of socialization to those of genetic intervention in order to argue that the latter is no more problematic than practices that are now routinely accepted as part of normal parenting. habermas would be unlikely to disagree with the argument that parents’ reproductive freedom should also encompass genetic interventions. as soon as priority is given to the perspective of the ‘passive receiver,’ however, then a very different picture emerges. after all, whereas socialization occurs at a moment when a child is already able to respond to the actions of her educators, genetic intervention occurs before the child has even entered into existence and the resulting individual will therefore be unable to respond effectively to his or her producer’s intentions: (…) such an imposition fro m within the community, even if it is excluded from the relationships obtaining between morally acting persons, must nevertheless not be confused with an external or alien determination of the natural and mental constitution of a future person, prior to an entr y into the moral community (ibid., p. ). how liberal is (the liberal critique of) a liberal eugenics? most critics revert to the notion of human dignity in order to oppose these kinds of interventions. however, this road is not open to habermas because it entails giving full rights to unborn life and in his view, it is clear that the question of whether “the in vitro embryo were already ‘another,’ who possessed completely valid basic rights (…) can hardly be answered in the positive given the premises of an ethically neutral constitutional order” (ibid., p. ). if it is already extremely difficult—if not impossible—to reach consensus on the question of when life begins, then these problems are only likely to increase in the case of genetic intervention, for gene modification can be performed not only at the zygote and embryo stages, but also in sperm and egg cells. it seem quite reasonable to assume that few would be willing to accept the absurd consequences that would follow from giving sperm and egg cells full human rights. while the proposed dilemma is quite clear, however, the same cannot be said about habermas’s solution to it. when he contends that “legal protection might come to be expressed in a right to a genetic inheritance immune from artificial intervention” (ibid., p. ), then it remains far from evident who might be the beneficiary of this right. since he rejects the idea of giving such a right to prenatal forms of life, he seems to mean that it would be bestowed upon the adult enhanced person. but how could such a person ever exercise her right to a genetic endowment free from artificial intervention, given that this irreversible act would have taken place well before she was a position to do so? there is more to be said here, though. for what the debate between the advocates and opponents of a liberal eugenics makes evident is that the emergence of enhancement technologies is likely to be accompanied by a growing tendency to impose severe normative constraints on certain potential forms of life. this is clear enough in the argument of someone like habermas, who draws on a normative conception of human nature to argue against genetic enhancement. what has not been sufficiently emphasized thus far, however, is the fact that any attempt to give normative content to human nature may be mobilized politically to exclude those who deviate from this norm (mendieta, ). that is not to say that these authors’ conceptions of human nature could serve as grounds for excluding certain vulnerable groups, such as the disabled or the mentally ill, from the moral community. instead, it could be said that these definitions preemptively deny any genetically enhanced being that may be brought into existence in the future the status of human being. what else could habermas mean when he writes that “[t]his new type of relationship humana.mente – issue – may [between programming parent and programmed child, author’s remarks] offends our moral sensibility because it constitutes a foreign body in the legally institutionalized relations of recognition in modern societies” (habermas, , p. ; emphasis added)? this statement seems, moreover, to cast further doubt on the effectiveness of habermas’s call for a right to a genetic constitution free from genetic intervention. if an enhanced person is barred from establishing reciprocal relationships with ‘normal’ human beings, and thus from entering the moral community of equal citizens, on what grounds, then, may such a person appeal to this right in the first place? again we must ask who the bearers of this right would be if the only individuals to have an interest in it were denied legal subjectivity? this tendency is not absent from the discourse of liberal eugenics, however. quite the contrary, in fact. as explained above, many liberal eugenicists seek to avoid some of the more distressing consequences of the nonidentity argument by tempering the right to reproductive liberty with the principle of procreative beneficence. the ultimate result of this argument is that parents would have the obligation both to prevent their children from being born with a disability or with a disease and to boost their capacities to a maximum. we should not lose sight of the justification behind this line of reasoning, however. what liberal eugenicists reject is not the notion of ‘wrongful life’ as such, but only the criteria which are to be used to determine what forms of life are included in this category once genetic technologies become available. what they are actually arguing, therefore, is that while it may be true that it is currently better, for example, to be born deaf than not born at all, this may change once we have the power to choose between a deaf child and a hearing child. if it is true, on the other hand, that the emergence of genetic technologies will progressively raise the threshold of harm, then we are also about to witness a steady increase in the number of forms of human existence that will have to be categorized as wrongful life. it remains to be seen how far this category can be stretched but perhaps, in the not too distant future, human beings as we currently know them will all be judged as having a ‘life not worth living.’ conclusion human genetic modification is still in its infancy, but the issues discussed above suggest that liberal political and moral philosophy remains rather ill equipped to address this controversial field, in the sense that the two positions how liberal is (the liberal critique of) a liberal eugenics? appear to be conceptually inconsistent: ultimately, both lead to conspicuously illiberal conclusions. after all, as we have argued above, while the argument against a new eugenics necessarily entails a preemptive dehumanization of any enhanced form of life, the argument for it threatens to reduce any non- enhanced form of life to the status of wrongful life. the final analysis might conclude, then, that any kind of liberal response to the challenges of the new eugenics unwittingly produces a form of life devoid of any intrinsic value. this is not to say that this outcome is inevitable, but clearly we will need to rely on an alternate interpretative framework if we wish to gain a more precise understanding of this contentious issue. references agar, n. 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( ). redesigning humans: our inevitable genetic future. boston/new york: mariner books. walters, l., & palmer, j.g. ( ). the ethics of human gene therapy. new york: oxford university press. wilkinson, s. ( ). “eugenics talk” and the language of bioethics. journal of medical ethics, , – . 	introduction 	 . the liberal eugenics 	 . in defense of human nature 	 . the new eugenics and the self-negation of liberalism 	conclusion 	references reciprocal relationships: something for everyone reciprocal relationships: something for everyone nina tumosa narrative inquiry in bioethics, volume , number , spring , pp. - (article) published by johns hopkins university press doi: for additional information about this article [ access provided at apr : gmt from carnegie mellon university ] https://doi.org/ . /nib. . https://muse.jhu.edu/article/ https://doi.org/ . /nib. . https://muse.jhu.edu/article/ narrative inquiry in bioethics volume . ( ) – © by johns hopkins university press p roblems with maintaining a healthy working relationship are fodder for advice columnists and many day–time television program hosts. they have plenty of advice on how to recog- nize when a relationship is not working and what to do about that. even dr. phil has written a book on rescuing relationships (mcgraw, ), and has reminded us that, of course, both parties need to want to repair the relationship. establishing effective working relationships is not for the faint hearted. despite the plethora of advice out there on how to repair damaged relationships, the building and maintenance of good relationships is difficult. although numer- ous examples of common–sense advice on rela- tionship–building do exist, relationships still go bad and still need constant work. and this is true of all relationships, whether they be personal interactions, business partnerships, or research collaborations. when research collaborations are done right there are great rewards. from start to finish, a suc- cessful partnership must include: a project that is locally driven and supported by all partners; complementary skills and knowledge among the partners; project synergism that results in some- thing neither partner could do alone; recognized and consistent leadership; a dedicated core of workers; adequate resources for implementation and evaluation; plans for dissemination to other communities; and community–based change agents working to maintain the progress obtained during the collaboration (heaton, day, and britten, ). these partnership components are ideal but not always achievable. the struggles to final achieve- ment are illustrated in the narratives discussed commentary reciprocal relationships: something for everyone nina tumosa†* †u.s. department of health and human services *correspondence concerning this article should be addressed to nina tumosa, public health analyst, health resources and services administration, bureau of health workforce, division of medicine and dentistry, medical education and geriatrics branch, n a, fishers lane, rockville, md . email: ntumosa@hrsa.gov narrative symposium conflicts of interest. the author reports no conflicts of interest. abstract. reciprocal relationships based on mutual goals, respect and trust are key to maintaining working relationships and getting reliable research results. yet relationship building is not a concept taught in academia. these skills are often learned the hard way, with singular solutions found for case–by–case scenarios. several journeys to identify the components, barriers and rewards of reciprocal relationships are discussed. key words. reciprocal relationships; research collaboration; academic–community partnerships narrative inquiry in bioethics • volume • number • spring here that focus on community–academic partner- ships in research and public health. these narra- tives illustrate well that relationship building and maintenance is a continuous process. they take us through a journey of self–discovery, goal setting and readjustment, near misses, incremental successes, and ever renewing hope and perseverance. their discoveries provide us with a blueprint of essential components of the creation of successful commu- nity–academic partnerships in research and public health. and they remind us that, although we still have a long way to go, the journey is worth taking. essential components of reciprocal partnerships the authors are all enthusiastic, passionate, and most were inexperienced at forming community– academic partnerships at the start of the project. the same can be said for many of their commu- nity partners. but they are not naïve. they all approached difficult issues in healthcare disparity with a willingness to help and an understanding that the project was too big to be done alone. they all sought to establish common ground with their community–based partners. they sought out and spoke to persons of authority and listened to their needs and wants. they negotiated with commu- nity and academic advisors and granting agencies for time and money. they developed trust. they developed respect. they forged a bond between themselves and the community members. and then they went to work. one serious barrier to a reciprocal partnership is lack of confidence on the part of the community members. bravo et al. worked hard to blur the lines between researchers and promotoras by work- ing together to formulate research questions of interest to all in health prevention strategies in the hispanic community. however, all of the partners still struggle against the immediate relapse back into prior relationships once outside the research setting. this is a major barrier but recognition is the first step of overcoming that barrier. cultural differences are both a barrier and an opportunity. farrar spent years immersed in the amish community, willingly taking longer than originally anticipated to complete her research. but she is still surprised at the continuance of cultural misinterpretations on the part of healthcare pro- viders and realizes that she herself still needs to learn more about the amish culture. her journey, although off to a good start, will be a long one. faculty members have more research experience than graduate students but that does not make their journey smooth. saksena and mcmorrow found willing community partners who saw opportu- nity with enthusiastic academic partners, a new technology, external funding, and a community of congolese refugee women that would benefit from having its “voice” heard. despite these many positives, the loss of the original community leader led to an undervaluation of the contributions of the researchers. the researchers’ willingness to be flex- ible with their time and resources also appears to have reduced the respect from the new community leadership as evidenced by the fact that they were often made to wait until other work was completed or were asked to change dates and times of planned activities at the last minute. respect must be mutual and everyone’s contributions must be viewed as valuable. there are special problems that arise when researchers study their own community. raynor and penkin have the advantage of understanding both sides of the community–academic partner- ship. their dual perspectives brought a clarity to the discussion of priorities but it also introduced unanticipated struggles with ethical questions about privacy and trust. their description of how they recognized, addressed, and resolved the issues provides both academic and community partners with a sterling example of problem solving. not all community–academic partnerships are as clear–cut as originally imagined. pallai piloted a writing workshop that promotes the creation of patient narratives about illness, told by patients with assistance from medical students in writing them down. the plan was to help the medical com- munity by showing students how to better relate to their patients and their illnesses. however, this medical community possessed two communities: the student community and the patient community. the patient community unilaterally shared their community–academic partnerships in research and public health stories with the students. but the students failed to share their stories, and even denied ever being ill, which is highly unlikely. it is a rare person who has never suffered with at least a cold, flu, or stomach cramps. for fuller engagement the students will have to be asked what they would like to learn from these interactions. students have much to learn and give but often focus only on learning. with negotia- tion and relationship building they will learn that they can teach as well. every research project has multiple stages— including design, implementation, and evaluation of interventions—and each stage affects the oth- ers. the narrative by schuch reports on how the intervention phase of a study to increase access to healthcare by the hispanic immigrant community is affected when multiple partners are involved in the design stage. the interventions were developed by key informant interviews, focus groups, a pho- tovoice project, and community forums, as well as inclusion of community and academic partners. this led to the identification of multiple needs and goals. balance between research and service goals was difficult to maintain, leaving all sides anxious. communication was time consuming but critical to the project’s success. connecting participants to health and social services was key to the sustainabil- ity of the project. despite difficulties, the improve- ments in the health and wellbeing of underserved hispanic immigrants was worth the effort. when developing a community–academic part- nership your time is not always your own. when shirazi participated in a study on breast health in an afghan community, she knew the importance of the community having control over the produc- tion of knowledge and being engaged in all phases of research. community ownership of the entire program was key to its continuance following the termination of the study. what was unanticipated was the degree to which flexibility and patience were required. it took longer than anticipated to establish trust. academic timelines were jettisoned as community members led the pace of interactions and adoption of acceptable interventions. sometimes less is more. community–academic partnerships can happen unexpectedly when an overwhelming need is acknowledged for a project whose time has come. as part of an academic exercise salm ward et al. identified significant dis- parities in the infant mortality rate in milwaukee where african american babies die at a rate nearly three times that of white babies and proposed a hypothetical research intervention. the community members who heard the presentation agreed with the students’ assessment and asked that the study be done. the students’ lack of experience probably helped them to fully engage community members in all phases of the study, from start to finish with everyone claiming ownership. this was a success- ful partnership and a research study that had an important impact in milwaukee. community advisors have a special place in community–academic partnership heaven. they can assist academic researchers in identifying the community stakeholders who are often either voice- less or absent because they are not recognized as being a part of the community. often special effort is needed to ensure that all stakeholders are heard from. thomas et al. discuss a unique program in which disparate groups of stakeholders are brought together and engaged in personal storytelling designed to decrease stereotyping, promote under- standing and communication, and show each other why they care. this development of mutual trust empowers all the stakeholders, both academic–and community–based, to participate as equal partners in identifying and reducing health disparities and improving health outcomes for everyone. protection of personal information from research subjects is not a new topic. however, in terms of community–academic partnerships, it is a topic that bears discussion. collecting health data from com- munity members places the academic researcher in a position of power with the possession of health information that is sensitive. such is the case espe- cially when working with persons with a positive hiv status or a mental health issue. release of health status information can lead to social stigma- tization should it become widely known that they are participating in research. mason discusses the effect of stigma experienced by persons living with a positive hiv status or who are receiving mental health services has on their trust in their research partners. ethical dilemmas such as these negatively narrative inquiry in bioethics • volume • number • spring affect communication but, ironically, require more communication. exchange of information between academe and the community requires trust and openness. this reminder of social responsibilities is well placed. sometimes disadvantages can be advantages in disguise. normally developing a community–aca- demic research partnership over a large geographic divide would seem foolhardy. in the collaborative described by wilbricht the pros appear to outweigh the cons. synergism allowed for emic media mes- sages to be created and disseminated via local radio stations in a rural native american and an alaskan native community. distance dictated that many decisions were made locally, keeping control firmly in the community. the sincere interest and expertise of the researcher in the use of emic media provided expertise that allowed a project to be done that would not normally have been possible. the major cost was lack of control over the project, a price the researcher was gladly willing to pay for the ability to make a difference. some topics are so sensitive and potentially invasive that it takes extra measures to cement a community–academic partnership. this is the case with elk’s study of culturally–based attitudes towards palliative care in two rural communities, one african american, and the other white. not only is trust building important but so is the need to truly listen to what community members are saying. focus groups, advisory boards and local champions of palliative care all played a role in ensuring that conclusions drawn from past studies of palliative care preferences in other communities were used only as a guide to what questions to ask, not to what the answers would be. respectful listening and discussion led to improved cultural aware- ness and respect for cultural differences across the two different communities and with the academic researcher. in this case, allowing the community to direct the questions and conclusions ensured that results will be applied back to the community. with help from a researcher who provides research funds and expertise on research design, this community– academic partnership will go far. conclusions these narratives have shown that it is possible to develop community–academic partnerships that are truly win/win. each has shared a personal journey and has not hesitated to point out pitfalls, barriers, and challenges as well as successes. they talk of finding ways for all voices to be heard, the sometimes difficult road to mutual respect, and why equitable sharing of resources is important. other advice acknowledged in the narratives include the empowering local champions; being flexible and taking the time needed; asking what can be changed and respecting that some things cannot be changed; sharing the power; and leav- ing assumptions at the door. these are all strong lessons and well worth remembering. but are these lessons enough to support the maintenance of these partnerships? sustainability is always the holy grail of research and of funding. once these community–academic partnerships have accomplished their original goals, can the changes they have wrought be sus- tained and, perhaps equally important, can the relationships be maintained in order to extend the goals to others equally in need? a huge next step is to determine whether and how health care research that is successfully accomplished in one locality can be effectively translated to other settings. given the power of accomplishment that we have seen here in these community–academic partnerships that are done right, it is possible that such partnerships can indeed support new groups. and accomplish even more. dr. phil would be proud. references heaton, j., day, j., & britten, n. ( ). collaborative research and the co-production of knowledge for practice: an illustrative case study. implementation science, ( ). mcgraw, p. c. ( ) relationship rescue: a seven-step strategy for reconnecting with your partner. new york: hyperion books. a survey on application of non-orthogonal multiple access to different wireless networks electronics review a survey on application of non-orthogonal multiple access to different wireless networks asim anwar ,*,† , boon-chong seet ,†, muhammad amish hasan ,† and xue jun li ,† department of technology, the university of lahore, lahore , pakistan department of electrical and electronic engineering, auckland university of technology, auckland , new zealand; boon-chong.seet@aut.ac.nz (b.-c.s.); xuejun.li@aut.ac.nz (x.j.l.) department of electronics and electrical systems, the university of lahore, lahore , pakistan; amish.hassan@es.uol.edu.pk * correspondence: asim.anwar@tech.uol.edu.pk † these authors contributed equally to this work. received: september ; accepted: november ; published: november ����������������� abstract: the fifth generation ( g) wireless systems are anticipated to meet unprecedented capacity and latency requirements. in order to resolve these challenges in g, non-orthogonal multiple access (noma) is considered as a promising technique due to its ability to enhance spectrum efficiency and user access. as opposed to conventional orthogonal multiple access (oma) which relies on orthogonal resource sharing, noma has a potential of supporting a higher number of users by multiplexing different users in the same resource in a non-orthogonal manner. with advanced receiver techniques, such as successive interference cancellation (sic), the intra-user interference can be minimized at the noma receiver. to date, there are comprehensive surveys on noma, which describe the integration of noma with different communication technologies and discuss different noma classifications. however, the existing literature is scarce in reviewing state-of-the-art applications of noma from the perspective of its application to cellular networks (cns), device-to-device (d d) communications, and wireless sensor networks (wsns). therefore, the purpose of this survey is to fill this gap in knowledge. specifically, noma with its underlying concepts are elaborated in detail. in addition, detailed system model of different noma-based wireless networks is presented. furthermore, irrespective of the underlying spatial topology of the considered noma-based wireless network, general analytical expressions are presented to characterize the network performance. finally, some challenges related to noma design are highlighted and potential research directions are pointed out to address these issues. keywords: g; non-orthogonal multiple access; cellular networks; device-to-device communication; wireless sensor networks; outage probability; sum-rate maximization; interference . introduction the blistering growth in wireless devices, such as smart phones and tablets, coupled with rapid evolution in mobile internet technology, have massively transformed the use of traditional analog voice telephony to resource ravenous multimedia applications. subsequently, it is anticipated that there would be a thousand-fold increase in mobile data traffic by the year [ , ]. this explosive growth in mobile devices has ignited the demand for ubiquitous and seamless connectivity. driven by these requirements, the wireless communication technology has been entering into an era of hyper-connected society through the advent of upcoming fifth generation ( g) systems and services. particularly, the key stringent requirements of g systems which advocate its proposal can be stated as follows [ – ]: ( ) to support demands of massive and ubiquitous connectivity by providing at least connections/km , electronics , , ; doi: . /electronics www.mdpi.com/journal/electronics http://www.mdpi.com/journal/electronics http://www.mdpi.com https://orcid.org/ - - - http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /electronics http://www.mdpi.com/journal/electronics electronics , , of which is ten times superior than the connectivity density of current fourth generation ( g) systems; ( ) to meet the ultra low latency requirements of about ≤ ms; and ( ) to enhance the spectral efficiency by – times over current g systems. the last two decades have witnessed a paradigm shift in the development and use of wireless systems and technology. from an implementation perspective, the evolution of various wireless communications systems spans around using different multiple access schemes. more precisely, frequency division multiple access (fdma), time division multiple access (tdma), code division multiple access (cdma), and orthogonal frequency division multiple access (ofdma) have been utilized as the underlying multiple access (ma) schemes in first generation ( g), second generation ( g), third generation ( g), and fourth generation ( g) systems, respectively. from the working principle viewpoint, the aforementioned ma schemes belong to a class of orthogonal multiple access (oma) [ ]. an oma can be formally defined as a ma scheme in which radio resources (frequency/time/code/their combination) are orthogonally allocated to different users. in particular, multiple users access the channel in an orthogonal fashion i.e., the users are orthogonal in frequency/time/code domain. as such, the direct consequence of using oma is that there would be no interference among users’ message signals. as a result, the key benefit of using oma is that low-complexity and cost efficient-receivers are utilized to facilely decode the users’ message signals. however, the prime limitation of adopting oma is that it is severely limited in supporting a large number of users or connections. consequently, this is would result in spectral inefficiency and is particularly not feasible to support massive connectivity and internet of things (iot), which have been deemed as key features in g systems. further, the random nature of wireless channel induce impairments which perpetually destroy the orthogonality among users in oma systems [ ]. as a result, high-complexity receivers need to be invoked in order to rejuvenate the orthogonality among users. despite the fact that g systems are rapidly growing and are in wide use, due to the scarcity of available spectrum resources, the g technology and services are still inept to meet the stringent requirements of the upcoming g systems [ – ]. in order to meet the rigorous demands of g systems, the ingenious proposal of non-orthogonal multiple access (noma) has been admitted as a latest member of ma family. the key idea of noma is that multiple users simultaneously share the same radio resource i.e., the users access the channel in a non-orthogonal manner. this would enhance the spectrum efficiency at the expense of increased receiver complexity, which is ineluctably required to separate users’ messages. the superior spectral efficiency of noma over oma techniques can be illustrated with the aid of following example. consider a multiuser communication system in which there is a user with poor channel conditions that needs to send/receive a data with high priority. in order to maintain fairness among users, the conventional oma based system would allocate an exiguous dedicated orthogonal resource to this user. consequently, the spectral efficiency and overall system throughput would degrade. in contrast, noma would solve this problem by allowing multiple users to access the available radio resource simultaneously. this results in efficient spectrum utilization while concurrently maintaining the fairness among users [ ]. apart from improving spectral efficiency and fairness over conventional oma, noma has a strong potential to improve the cell-edge throughput, requires relaxed channel feedback, and to significantly reduce the overall latency [ ]. these properties make noma a suitable and promising ma candidate to support massive connectivity requirement in g systems [ , ]. the beauty and brilliance of the noma concept lies in a fact that it has excellent compatibility with other communication technologies. this allows noma to be readily integrated with the existing and future wireless systems. for instance, noma has proved to be concordant with the conventional oma techniques, such as tdma and ofdma [ ]. as a consequence, this remarkable feature offered by noma along with its potential to meet the demands of g systems has captured the attention from both academia and industry. particularly, noma has been adopted as an underlying ma scheme by various standardization bodies. for example, third generation partnership project ( gpp) has developed a standard long-term evolution advanced (lte-a) based on noma, where electronics , , of noma is termed as a multiuser superposition transmission (must). more precisely, adopting noma allows multiple users to simultaneously share the same subcarrier in ofdma without modifying the lte resource blocks [ ]. in addition, the latest advanced television systems committee (atsc) standard for digital television (tv) broadcasting is based on the noma principle, where multiple data streams are superimposed in a same resource. consequently, this would result in an enhanced spectral efficiency for the tv broadcasting system [ ]. moreover, apart from evaluating and analyzing basic principle of noma and proving its superiority over oma theoretically, industries like huawei technologies and nippon telegraph and telephone (ntt) docomo have performed field trials and prototype evaluations to verify the analytical performance gains of noma over oma. interesting readers are referred to see the references [ – ]. the aforementioned advantages of noma and its appreciation from standardization bodies inarguably imply that noma possesses an immense potential to meet the requirements of g and beyond g systems. to date, the existing state-of-the-art survey papers on noma can be broadly divided into two categories. the first category provides review of prior arts on noma from the perspective of different noma classifications such as power and code domain noma. the second category of survey papers review those works on noma which investigate the integration of noma with different communication technologies such as multiple-input multiple-output (mimo), cooperative communication, millimeter waves, full duplex (fd), and so on [ , , – ]. however, to the best of our knowledge, there is no current survey which attempts to review state-of-the-art on noma according to its application in cellular networks (cns), device-to-device (d d) communication, and wireless sensor networks (wsns). therefore, the goal of this survey is to exhaustively review the prior arts on noma from the perspective of its application to cns, d d communication, and wsns. in particular, apart from reviewing the state-of-the-art on noma-based cn, d d communications, and wsns, the main contributions of this survey are summarized as follows: • a comprehensive network model is provided for each of the considered noma-based cn, d d communication, and wsn. • in order to evaluate the performance of each considered noma-based network, outage probability expressions are presented. • in addition, the sum-rate maximization problem is formulated for each type of noma-based network, which could serve as a good reference point for beginners and practitioners working on noma-based cn, d d communication, or wsn. the rest of this paper is organized as follows. section provides the basic concepts of noma. the application of noma to cn, d d communication, and wsn has been comprehensively reviewed in section . the discussion of noma related research challenges and future directions is presented in section . finally, section concludes the survey. for quick reference, the organization of this survey is presented in figure . electronics , , of . introduction . basic concepts of noma . . the principal of superposition coding . . successive interference cancellation scheme . . noma scheme with sic . application of noma to wireless networks . . noma applied to cellular networks . . . noma in single cell single tier cellular networks . . . noma in single cell multi tier cellular networks . . . noma in multi cell single tier cellular networks . . . noma in multi cell multi tier cellular networks . . .a noma based scst cn system . . .b outage probability of noma based scst cns . . .c sum rate maximization problem in noma based scst cns . . .a noma based scmt cn system . . .b outage probability of noma based scmt cns . . .c sum rate maximization problem in noma based scmt cns . . .a noma based mcst cn system . . .b outage probability of noma based mcst cns . . .c sum rate maximization problem in noma based mcst cns . . .a noma based mcmt cn system . . .b outage probability of noma based mcmt cns . . .c sum rate maximization problem in noma based mcmt cns . . noma applied to device to device communications . . noma applied to wireless sensor networks . . . noma based group d d communications . . . outage in noma based group d d networks . . . sum rate maximization problem in noma based gd d networks . . . noma based wsns . . . outage in noma based wsns . . . sum rate maximization problem in noma based wsns . research challenges and future trends . conclusion . . alternate receiver for noma . . hybrid multiple access . . consideration of imperfect csi . . noma based data offloading in g networks . . noma aided full duplex networks figure . organization of this survey. . basic concepts of noma from conceptual and implementation perspectives, noma solutions can be classified into two broad categories, namely code domain and power domain noma [ ]. the fundamental working principle of code domain noma is that multiple users are allowed to share the same resources (time/frequency), which is very similar to the operation of classic cdma systems. however, code domain noma has maintained a vital difference with cdma systems because in contrast to cdma, it utilizes sparse spreading or non-orthogonal low cross-correlation sequences to realize multiplexing. some popular code domain noma solutions include sparse code multiple access (scma), low-density spreading cdma (lds-cdma), lds aided ofdm (lds-ofdm), must, and successive interference cancellation aided ma (sama). the readers are referred to see [ – ] and references therein to have more deep insights into the code domain noma. the other classification of noma is based on power domain multiplexing. in contrast to code domain noma, the multiple users are superimposed in a same resource (time/frequency/code) by allocating different power levels to multiple users. therefore, multiple users access the channel in a non-orthogonal fashion by applying the principle of superposition coding (sc). at the receiver side, advance multiuser detection (mud) techniques such as successive interference cancellation (sic) or dirty paper coding (dpc) are applied to decode the users’ message signals [ , ]. figure presents the classification of noma schemes. in this paper, the main focus is to provide a comprehensive review for the application of power domain noma to cns, d d communications, and wsns. therefore, unless otherwise stated, noma refers to power domain noma in the remainder of this paper. in addition, sc and sic techniques are the vital components of a noma system. therefore, the rest of this section discusses the principle of sc, the working of sic technique, and noma system with sic receiver. electronics , , of noma schemes major categories code domainpower domain scma low density spreading ofdm low density spreading cdma focus of this survey figure . classification of non-orthogonal multiple access (noma) schemes. . . the principle of superposition coding the kind of communication scenario in which a single source needs to communicate with many receivers arises in various wireless systems, such as base station (bs) communicating with cellular users, wifi access point sending information to multiple users, hotspot at airport or in shopping mall, military commander giving specific instructions to different units, and so on. the traditional technique to realize communication in these scenarios is to apply conventional oma, where orthogonal channels are allocated to each user by utilizing time/frequency/code domain. from an information-thoretic perspective, this approach is generally not optimal in achieving the sum-rate or capacity region of the gaussian broadcast channel (bc). as such, the non-orthogonal technique, known as sc, is considered as a promising scheme that has a potential for achieving the capacity of the gaussian bc. in addition, sc is capable of enhancing the spectral efficiency of the overall system [ – ]. the idea of sc was first proposed and investigated in the pioneer work of [ ], where sc is suggested as a method of simultaneously transmitting information from a single source to multiple receivers. the authors in [ ] proposed and discussed different strategies for implementing sc. in addition, with the aid of experimental results, the authors also present the set of rate-pairs which are capable of achieving superior spectral efficiency compared to the oma schemes. some common examples of sc include broadcasting tv transmission, a speaker/teacher delivering a lecture to people with different aptitudes, and radio transmission. specifically, sc is considered as a physical layer method to simultaneously transmit independent messages of multiple users. from the operational and implementation perspectives, sc technique can be regarded as a multi-layer modulation scheme in which each layer corresponds to the modulated and coded message signal of different users. the transmitter then superimposes independent users’ message signals by adding them in order to constitute a composite signal before transmitting to the channel [ ]. figure illustrates the basic transmitter implementing sc and the concept of sc based transmission system. electronics , , of user user user m s s sm s figure . transmitter implementing superposition coding (sc). in order to obtain deep insights for understanding the principle of sc, more rigorous treatment is required. consider a multiuser communication system with a single source and a total of m users. further, assume that the total transmission power of source is constrained by p watts and the message signal of user m, m = , , ..., m is denoted by sm. note that depending on the type of communication system, sm could be a baseband message signal or obtained as a result of using some particular modulation and coding scheme. however, for notational simplicity and ease of exposition, the discussion herein considers sm as a message signal regardless of its underlying construction method. by applying the sc principle, a source generates a superimposed composite signal, denoted as s, which is then simultaneously transmitted to all m users. the superimposed signal s at the source can be written as: s = m ∑ m= √ am psm, ( ) where am is the power allocation coefficient of user m and ∑mm= am = . by inspecting equation ( ), it can be observed that sc introduces intra-user interference, which needs to be removed by each user m before decoding its own message. as such, an efficient mud technique is required to suppress the intra-user interference in order to fully enjoy the benefits of sc. therefore, the next subsection discusses the working principle of sic, which is considered as a promising and relatively low-complexity mud scheme to be used at the users’ receiver side [ ]. . . successive interference cancellation scheme in order to decode the user m message from the superimposed signal s, the sic scheme is regarded as a rudimental approach [ ]. the basic structure of sic is presented in figure . the key operating principle of sic relies on exploiting the power differences among users participating in sc [ ]. the working of sic can be explained as follows. first, the users are ordered in descending order of their signal strengths. as such, consider that the users’ power allocation coefficients in equation ( ) are ordered as a ≥ ... ≥ am... ≥ am i.e., users m = and m = m are allocated with maximum and minimum powers, respectively. as a result, the users from , ..., m − are regarded as strong (higher order) users while the users from m + , ..., m are considered as weak (lower order) users for user m. next, the decoding is performed in a successive fashion, as depicted in figure . this essentially implies that user m first successively decodes the message signals of all the higher order users starting from user to m − . after decoding the higher order users, the user m decodes its own message by treating the lower order users’ messages as noise. considering the perfect sic operation (ideal cancellation electronics , , of of higher order users), the signal-to-interference-noise-ratio (sinr) at user m, denoted as Γm can be expressed as: Γm = pm ∑mi=m+ pi + σ , ( ) where pi is the signal strength or power (typically received power) of user i, and σ is the noise power. note that the summation term in equation ( ) is actually the intra-user interference from users m + , ..., m resulted due the application of sc. however, this intra-user interference is seen as a noise by user m because its allocated power level am p is larger than the power levels of all the weak users [ ]. this assumption simplifies the decoding operation and implementation of sic receiver but essentially increases the signal-to-noise-ratio (snr) level required for successful decoding. with the brief introduction to sc and sic, we are now in a position to discuss the concept of noma in next sub-section. hm(s +s +..+sn) hm(s +s +..+sn) s sm- sm hm(sm- +sm+..+sn) ue decoding s ue decoding ue decoding hm(sm+sm+ +..+sn) figure . block diagram of successive interference cancellation (sic) receiver. . . noma scheme with sic consider a single source which is simultaneously transmitting messages to total of m noma users. the users are assumed to be randomly distributed inside the coverage of source. further, denote by hm = gm +dαm as the channel gain (power) between user m and source. here, the variables gm and dm represent the fading (power) gain and distance between the source and user m, respectively, and α is the pathloss exponent. according to the noma principle, it allocates more power to users with poor channel conditions and vice-versa [ ]. as such, in order to apply noma technique, the users are first required to be ordered at the source. without loss of generality, consider that the users are sorted in ascending order of their channel gains as h ≤ ... ≤ hm. consequently, the power allocation coefficients are sorted as a ≥ ... ≥ am. the source then applies the principle of sc to generate a superimposed signal s as given electronics , , of by equation ( ), which is then sent to all the users simultaneously. at receiver of user m, the received signal, denoted by rm, can be expressed as [ ]: rm = m ∑ i= √ hm ai psi + nm, ( ) where nm represents the additive white gaussian noise (awgn) at user m receiver. at the receiver of user m, the decoding is performed in ascending order of the users’ channel gains. as a result, sic is applied to decode the message signals of all the higher order users , ..., m − . after removing the interference from higher order users, the user m decodes its own message by considering all the lower order users from m + , ..., m as noise. consequently, based on equation ( ), the sinr at user m can be expressed as: Γm = hm am p hm p ∑mi=m+ ai + σ ( ) = hm am ρ hm ρ ∑mi=m+ ai + ( ) where ρ = p σ is the transmit snr. . application of noma to wireless networks the previous section has provided an insight into the noma and its underlying basic concepts. with this background, the focus of this section is to present the application of noma to different types of wireless networks. more precisely, in the light of current literature, we will review noma applied to cn, d d communication, and wsn. our approach is to provide a basic structure and working of the considered noma-based wireless network in the context of existing literature, and then discuss useful analytical results which are important to evaluate the performance of noma-based wireless network. more precisely, we first review noma applied to the considered wireless network in the context of state-of-the-art prior works. second, the wireless network model based on noma is discussed. third, the performance of noma system under considered wireless network will be presented in terms of outage probability. finally, a sum-rate maximization problem for noma will also be formulated. . . noma applied to cellular networks from conceptual and operational viewpoints, a cellular network can be classified in the following four categories. ( ) single-cell single-tier (scst), ( ) single-cell multi-tier (scmt), ( ) multi-cell single tier (mcst), and ( ) multi-cell multi-tier (mcmt) cns. as such, the subsequent discussion comprehensively reviews the application of noma to the aforementioned classifications of cns. in addition, for illustration purposes, figures – present different types of noma-based cellular networks. electronics , , of rd ue ue ue ue m ue m- ue m figure . noma-based single-cell single-tier (scst) cellular network. rd sbs rd,k figure . noma-based single-cell multi-tier (scmt) cellular network. electronics , , of rd figure . noma-based multi-cell single tier (mcst) cellular network. rd sbs rd,k figure . noma-based multi-cell multi-tier (mcmt) cellular network. . . . noma in single-cell single-tier cellular networks the most common type of noma-based cns which are exhaustively investigated in current literature are scst cns. the initial works of [ , , ] proposed the concept of the noma technique with sic receiver and validated their proposal by performing system level simulations. the authors reported robust performance gain of %– % achieved by noma over oma in terms of overall cell-throughput. in addition, the work in [ ] also discussed the practical implementation issues related to the realization of noma such as power allocation, error propagation in sic, signaling electronics , , of overhead, and user mobility. the classical work of [ ] could be regarded as a pioneer paper which investigated the performance of single-cell downlink noma system with randomly deployed users. closed-form expressions for outage probability and ergodic sum-rate are derived to characterize the performance of the considered noma network. the authors in [ , ] integrated the lte specific practical considerations with noma related operations. more precisely, the problem of user scheduling was resolved using proportional fairness (pf) scheduler. in addition, the critical issue of users’ power allocation is addressed by applying dynamic transmit power allocation (tpa), fractional transmit power allocation (ftpa), and full-search power allocation (fspa) techniques. furthermore, hybrid automatic repeat request (harq) protocol was adopted for retransmissions when decoding error occurs. the impact of user grouping on noma performance is investigated in [ , , – ]. the authors in [ ] grouped users together whose channel gains differ significantly. this would facilitate users’ power allocation using tpa/fpa, resulting in superior user and cell throughput over conventional oma. in [ ], the authors investigated the impact of user pairing by considering fixed power allocation noma (f-noma) and cognitive radio inspired noma (cr-noma). the authors derived closed-form analytical expressions for achievable sum-rate in order to evaluate the performance of the proposed user pairing on the considered noma systems. the results demonstrate that after careful user grouping (pairing users with significant difference in channel qualities), f-noma outperforms conventional oma. this implies that when grouped users have similar channel conditions, there would be limited gain of noma over oma. on the other hand, in cr-noma scheme, the users with low and high channel gains are regarded as primary and secondary users, respectively. the secondary user is only permitted to transmit on the condition that it would not impact the transmission of primary user adversely. consequently, this setting would be sufficient to achieve significant difference in allocated powers of both users at the bs, which in turn facilitates the sic operation at users’ receivers. the problem of user pairing in uplink noma systems is studied in [ ] for two network settings under fixed power allocation policies. the first scenario assumes that both bs and users are equipped with single antenna. under this setting, a sub-optimum user pairing technique is proposed which is capable of achieving optimal performance with polynomial time complexity. in the second network setting, the bs is considered to have multiple antennas while users are equipped with single antenna. the results demonstrate that the optimal user pairing method achieves superior performance than the conventional random user pairing technique. based on the differences in channel conditions of the users, the authors in [ ] proposed user pairing and access theme algorithms. the results depict that the proposed schemes outperform the existing techniques in terms of capacity while ensuring the fairness among users. in [ ], the problem of user pairing is addressed by dividing the users in two groups. a closed-form power allocation strategy is proposed in order to solve the problem of power allocation for noma users. the authors presented the results in terms of sum-rate maximization of noma system and demonstrated that the proposed user pairing method is capable of achieving optimal performance. the authors in [ ] proposed an iterative algorithm in order to solve the joint optimization problem of user pairing and beamforming for noma systems. the results show that the proposed algorithm is capable of achieving superior rate fairness among noma users when compared to conventional random pairing and beamforming noma systems. in order to optimize the achievable rate, the problem of joint user pairing and power allocation is investigated in [ ] under the condition of meeting minimum rate requirements of each user. the authors first obtain the optimized solutions by considering simpler systems with two and four users, and then derived the closed-form global optimal solution for generalized noma system. the results demonstrate that the proposed optimal scheme outperforms the conventional oma as well as noma systems with random user pairing. an interesting problem of pairing mid-cell users who do not have significant difference in channel qualities is addressed in [ ]. in order to solve this issue, the authors proposed two user pairing schemes which have a potential of enhancing capacity for all the users while treating the problem of pairing mid-cell users. in [ ], the technique of pf is considered to achieve user pairing in the electronics , , of noma system. considering the two user noma system, the authors derived a closed-form optimal solution with pf objective in order to obtain user pairing and power allocation. based on adaptive pf technique, the authors in [ ] proposed an algorithm in order to obtain optimal user pairing, spectrum resource, and power allocation. the results show that the proposed algorithm achieves a comparable throughput performance to the global optimal search method and water-filling power allocation schemes, while also enhancing fairness among users. he et al. [ ] proposed a novel fast user pairing algorithm which pairs users based on pf metric under fixed power allocation scheme. the simulation results demonstrate that at the expense of negligible throughput loss, the proposed user pairing method performs considerably faster than the conventional exhaustive search technique. the authors in [ ] proposed an optimized scheme to jointly obtain user pairing and power allocation in order to maximize pf metric under the transmission power constraints. the simulation results illustrate a superior throughput performance and decreased complexity of the proposed scheme over current techniques in noma systems. in [ ], the authors proposed a sort-based method in order to obtain user pairing and power allocation in noma system. the results depict that the proposed sort-based technique has a significant potential to enhance the error probability and spectral efficiency of downlink noma systems. the authors in [ ] have investigated the problem of dynamic user clustering and power allocation by considering both uplink and downlink noma transmission scenarios. based on transmit power, users’ targeted rates, and sic constraints, the authors proposed an optimal user clustering and power allocation method which maximizes the overall system throughput. hsiung et al. [ ] proposed a packet level scheduling scheme for bs that dynamically switches between noma or oma. further, the proposed scheduling method also obtains user pairing and power allocation for the considered noma system. the results demonstrate that the proposed scheme outperforms the distance-based user pairing technique in terms of throughput. the issue of fairness for scst noma networks has been studied in [ – ]. the work of timotheou et al. [ ] can be regarded as a pioneer study investigating the issue of fairness for noma. by considering instantaneous and average channel state information (csi) at the transmitter, the authors proposed optimal power allocation for noma systems which guarantees fairness among downlink noma users. the authors in [ ] considered the asymmetric channel and studied the fairness comparison between noma and oma in terms of resource allocation. based on jain’s index, the authors proposed a fairness indicator metric which facilitates the users’ ability to select between noma and oma schemes, and thus constitute a hybrid noma-oma system. simulation results demonstrate that the proposed fairness metric improves the fairness among users in hybrid noma-oma system when compared to oma and noma transmission techniques. in [ ], the authors proposed a short term fairness scheme which aims to enhance the throughput–fairness trade-off for downlink noma systems. the results report an improvement of . % by the proposed scheme in user fairness over conventional techniques. the authors in [ ] suggested a new definition for user fairness which is based on the power distribution among different noma users. more precisely, under the proposed fairness index, the user’s fair rate is determined according to the amount of its allocated power i.e., the user with larger power should obtain a higher rate. in order to confirm the precision of the proposed fairness index, the authors carried out an asymptotic analysis by considering both low and high snr values. the problem of optimal resource allocation and energy efficiency for noma systems is investigated in [ – ] from the perspective of user fairness. in [ ], the authors proposed an optimal power allocation scheme which aims to maximize overall energy efficiency of the uplink noma system while also accounting fairness among users. based on an instantaneous csi, the authors in [ ] provide analytical expressions to obtain optimal power allocation in order to maximize the instantaneous sum-rate while maintaining the α-fairness among users. by considering statistical and perfect csi at the transmitter, the authors in [ ] proposed an optimal power allocation algorithm which aims to maximize the overall throughput of noma systems with α-fairness. considering the identical user fairness level, the numerical results demonstrate that the noma under the proposed optimal power allocation method significantly enhances the system performance over conventional electronics , , of oma technique. the authors in [ ] considers a two-user downlink noma system over fading channels and studied the trade-offs between system throughput and users’ fairness. based on full and partial csi at the transmitter, the authors derived closed-form analytical expressions to obtain optimal power allocation policies for the considered noma system. the optimal fairness policy for noma systems is proposed in [ ] which aims to maximize the achievable rate of the worst noma user. the authors derived a closed-form expression for the optimal fairness value and proposed an iterative algorithm in order to obtain the optimal solution with linear convergence. the trade-off between two conflicting metrics of sum-rate and fairness for multiple-input single-output (miso) noma systems is further investigated in [ ]. based on different network requirements and channel conditions, the authors proposed a flexible beamforming design which is capable of catering both sum-rate and user fairness. the aforementioned discussion reviews noma-related operations in the context of existing literature. apart from noma specific issues, there are two promising technologies, namely mimo and cooperative communications, which can be integrated with noma in order to further enhance the spectrum efficiency [ , ]. however, the main emphasis of this survey is to review the application of noma to different wireless networks and not to provide in depth survey on integrating different communication technologies to noma networks. therefore, for completeness of discussion, in what follows, we will briefly review some state-of-the-art works on mimo-noma and cooperative noma networks. the current literature e.g., see [ – ] explore the potentials of applying mimo technique to noma networks. in [ , ], the authors investigated the capacity gain achieved by mimo-noma over conventional mimo-oma systems. the authors derived closed-form analytical expressions for both sum channel and ergodic sum capacities to demonstrate the dominance of mimo-noma over mimo-oma. moreover, the authors in [ , ] performed system and link-level simulations to highlight the potentials of applying mimo to noma. in addition, the authors developed hardware test-bed for noma, where sic receiver was implemented in order to account the realistic hardware impairments. the results obtained after simulations and measurements demonstrate a % enhancement in cell throughput by noma over ofdma system. the authors in [ ] considered a downlink single user mimo-noma system and evaluated the performance of cell-edge users by using system level simulations. furthermore, a concept of signal alignment is applied to propose a general mimo framework for both uplink and downlink noma systems in [ ]. considering the randomly deployed users and interferers, the authors derived a closed-form analytical expressions by using techniques from stochastic geometry in order to evaluate the performance of the considered system. numerical results are presented in terms of outage probability which reveal the superior performance of mimo-noma over conventional mimo-oma system. the benefit of applying cooperation in noma is investigated in [ – ]. the work of ding et al. [ ], can be regarded as a pioneer work which proposed a cooperative downlink noma system. the entire transmission is divided in two phases, namely, direct and cooperative, transmissions. the operating principle of direct transmission phase exactly follows the working of conventional noma system. the consequence of direct phase is that every user would have decoded its message as well as the messages of all the higher order users. as a result, the users broadcast decoded messages of higher order users by utilizing time slots in cooperative transmission phase. in order to evaluate the performance, the authors derived closed-form expressions for outage probability and diversity order for the proposed scheme. the simulation results validate the accuracy of the derived analytical expressions and also demonstrate the superior performance of the proposed noma-based network over a conventional cooperative oma system. in [ ], the authors proposed a two-stage relay selection strategy for cooperative noma networks and provide closed-form analytical expressions for outage probability. the numerical results reveal that the cooperative noma network under the proposed relay selection method is capable of achieving lower outage probability and better diversity gain than the conventional max–min approach for relay selection. furthermore, the authors in [ ] considered a cooperative noma system based on amplify-and-forward (af) relay selection technique, and evaluated the performance of the considered electronics , , of system in terms of outage probability. the authors provided an approximation of the exact outage probability and investigated the accuracy of the approximation by studying its asymptotic behavior. the results demonstrate that proposed scheme outperforms the conventional cooperative oma system while maintaining the same diversity order. in addition, the authors in [ ] considered a cooperative noma network and proposed a novel two-stage relay selection scheme based on decode-and-forward (df) and af relaying methods. exact analytical expressions for outage probability are derived. further, the authors performed asymptotic analysis in order to obtain diversity gain of the proposed scheme. the numerical results reveal that the proposed two-stage df (af) relaying for noma outperforms the existing relay selection methods for cooperative noma as well as oma based networks. the authors in [ ] considered a cooperative noma system in which relay possesses a buffer. based on this relay feature, the authors proposed an adaptive transmission method which optimally selects the operational mode in every time slot. the results demonstrate that the proposed scheme significantly enhances the overall system throughput over conventional cooperative noma networks. in order to address the issues of high implementation complexity and power consumption of mimo-noma systems, the authors in [ ] proposed three node cooperative relaying system (crs) based on spatial modulation (sm) aided noma (crs-sm-noma) technique. closed-form analytical results for bit-error-rate (ber) are provided in order to characterize the performance of the considered system. the numerical results demonstrate the superiority of the proposed crs-sm-noma over crs-noma and sm-oma systems. the above discussion provides a brief and concise review for mimo-noma and cooperative noma networks. interested readers are referred to see [ – ] and [ – ] for more detailed treatment of mimo-noma and cooperative noma, respectively. a. noma-based scst cn system let us now shift our discussion on the working of scst noma networks in this subsection. in noma-based scst cn, there is a single bs communicating with multiple users by applying noma principle, as shown in figure . at the user’s receiver, sic technique is applied in order to decode the message signal by mitigating the multiuser interference [ ]. this arrangement constitutes a single-cell communication network, in which users are typically randomly distributed inside the coverage of bs. considering total of m noma users in a cell, the received signal at user m can be obtained by using equation ( ). consequently, the sinr available at user m for decoding can be expressed by equation ( ). b. outage probability of noma-based scst cns the user m is considered to be in outage when its fails to detect the message of user j, where ≤ j ≤ m. this essentially implies that whenever user m is unable to decode (meets the targeted rate) any of the higher order user or its own message, then it will suffer from complete outage. consequently, based on equation ( ), the sinr at user m to decode the message signal of j-th user, denoted by Γm→j, can be expressed as: Γm→j = hm aj ρ hm ρ ∑mi=j+ ai + . ( ) electronics , , of based on equation ( ), the outage probability at the m-th user in decoding j-th user message, denoted by pm→j under an scst noma-based cn can be expressed as follows: pm→j = pr ( Γm→j < ϕj ) = pr ( hm aj ρ hm ρ ∑mi=j+ ai + < ϕj ) = pr ( hm ρ ( aj − ϕj m ∑ i=j+ ai ) < ϕj ) = pr ( hm < τj ) , ( ) where ϕj = rj− , rj is the targeted rate of user j, ≤ j ≤ m, and τj = ϕj ρ ( aj−ϕj ∑mi=j+ ai ) . note that the result in equation ( ) can only be obtained if the following condition is satisfied: aj > ϕj m ∑ i=j+ ai. ( ) it can be observed that the outage probability of user m will always be one if condition in equation ( ) is not satisfied. this implies that careful allocation of rate and power would be required in order to keep the noma system operational. next, let us define τmaxm = max{τ , ..., τm}. the outage probability of the m-th user, denoted by pscstm can now be expressed as follows: pscstm = pr (hm < τ max m ) = fhm (τ max m ) ( ) where fhm (.) is the cumulative distribution function (cdf) of hm. it can be observed from equation ( ) that in order to obtain outage probability of the m-th user in scst noma cn, the cdf of channel gain hm would be required. recall that channel gain hm in equation ( ) is given as hm = gm +dα , where gm is the fading power. consider that gm has exponential distribution with unit mean ∼ exp ( ) i.e., assuming rayleigh fading [ ], then by applying order statistics [ ], the cdf fhm of channel gain can be expressed as follows [ , ]: fhm (x) = µm m−m ∑ q= ( m − m q ) (− )q m + q ( fĥ (x) )m+q , ( ) where µm = m!(m−m)!(m− )! , and fĥ (x) is the cdf of the unordered channel gain. next, consider that bs is located at the center of a disc with radius rd, where rd models the coverage of bs. further, since the users are randomly and uniformly distributed inside the coverage of bs and the fading is assumed to be rayleigh, then, the cdf of the unordered channel gain is given as follows [ ]: fĥ (x) = r d ∫ rd ( − exp (−( + rα) x)) rdr. ( ) note that finding the closed-form solution of equation ( ) for α > is very challenging. as such, the integral in equation ( ) can be approximated by gaussian–chebyshev quadrature in [ ] or by utilizing confluent hyper-geometric function in [ ]. now, based on equations ( ) and ( ), the outage probability of m-th user in scst noma cn can be expressed in closed-form as follows: electronics , , of pscstm = µm m−m ∑ q= ( m − m q ) (− )q m + q ( fĥ (τ max m ) )m+q . ( ) this completes the discussion of outage probability for scst noma cn and we now describe the problem of optimal power allocation in next subsection. c. sum-rate maximization problem in noma-based scst cn the fundamental objective of any wireless system is to maximize the overall sum-rate of the network. recall that noma operation is dependant on satisfying equation ( ) which would be met if rate and power allocations are done carefully. consequently, an optimal power and rate allocations would be required in order to maximize the aggregated sum-rate of the noma-based scst cn. in what follows, we will formulate and present a generic optimization problem that will maximize the overall sum-rate of the noma-based scst cn subject to constraints accounting for practical aspects of the system. let us define an objective function as, fscst = ∑mm= r̃m, where r̃m = log ( + Γm) is the achievable rate of user m. now, the constrained optimization problem can be formulated as follows: maxfscst ( ) subject to: c : rm ≤ r̃m c : m ∑ m= am p ≤ p c : ϕj m ∑ i=j+ ai ≤ aj, ∀ ≤ j ≤ m. the constraint c guarantees that the minimum rate requirements of all users are satisfied, which would also translate into minimum outage probability constraint for all the users. the condition c ensures that sum of powers allocated to all the users would ot exceed the total power budget p available at the bs. finally, the constraint c is added to appropriate users’ rate and power allocation in order to keep noma with sic operational. the above formulated problem is mixed-integer non-linear programming (minlp). the solution of this minlp problem has combinatorial nature. the given problem is hard to solve by utilizing standard optimization techniques. as such, one possible approach to solve this problem is to divide the problem into a sequence of linear programs and then develop an algorithm to obtain the optimal solution [ ]. another technique is to utilize standard optimization solvers such as cplex, matlab, and so on, to find the optimal solution numerically. note that based on underlying modulation used in noma, the generic optimization presented in equation ( ) problem can be be easily tailored for scst cn. . . . noma in single-cell multi-tier cellular networks in current literature, noma-based single-cell multi-tier (scmt) cns are relatively less explored. the problem of resource allocation for noma-based single cell heterogeneous (multi-tier) networks is investigated in [ ]. in the considered model, noma protocol is applied by only small-cell bss (sbss) to communicate with their users. in order to maximize the sum-rate of sbs users, the authors presented a resource allocation problem in terms of many-to-one matching game. the authors then proposed a novel distributed algorithm to obtain the solution of the formulated game. the results demonstrated the fast convergence of their proposed algorithm to a global optimal solution and sbs users achieve superior sum rate over oma under the proposed resource allocation strategy. however, the sbss are typically deployed in areas of higher user density, i.e., they usually follow user-centric electronics , , of deployments in which the locations of users and sbs are correlated. without considering this aspect, their modeling approach may not be accurate for modeling users’ locations in heterogeneous networks with sbss. further, the authors in [ ] considered a noma-based scmt cn with single mbs and multiple sbss, where sbss are communicating with small-cell users via noma protocol. considering the users’ fairness issue, the authors proposed an optimal joint spectrum allocation and power control algorithm in order to maximize the sum-rate of the small-cell users. the results demonstrate that under the proposed algorithm, noma-based scmt cn outperforms the conventional oma based scmt cn. furthermore, the problem of resource allocation in energy-cooperation enabled two-tier cns is investigated in [ ], where mbss and pico bss (sbss) apply the noma scheme to communicate with corresponding users. moreover, in contrast to conventional grid power, the bss in the considered system are also powered by renewable energy sources. in order to maximize the energy efficiency of such networks, the authors proposed an optimal algorithm to determine user association and power control while meeting the quality-of-service (qos) requirements. the results reveal that the noma in scmt cn outperforms the conventional oma in the considered network in terms of energy efficiency. the problem of optimal power allocation is considered in [ ] under imperfect csi in order to maximize the energy efficiency of the small-cells. based on the lagrangian method, closed-form optimal power allocation for each small-cell is derived. the results demonstrate that the considered scmt network under the proposed scheme outperforms the conventional algorithms. the issue of offloading mbs users in an scmt scenario under congested situations is investigated in [ ]. in order to serve the offloaded mbs users, fbs tier applies user pairing and then utilizes the noma protocol for serving the users. with the proposed offloading strategy, the authors have reported a significant improvement of . % and . % in outage probability for cell center and cell-edge users, respectively. furthermore, the authors considered a single mbs cell underlaid with femto bss (fbss) cells. in [ ], the authors considered a scmt scenario, where sbss (femto bss in [ ]) and d d tiers are underlaid with single mbs. noma transmission protocol is applied by sbs and d d tiers. the authors investigated the problem of congestion in the considered network and suggested to offload mbs load to sbss. further, when sbss are unable to pair the offloaded mbs users, the authors proposed to utilize the d d tier in order to support these unpaired offloaded mbs users. the results show that the proposed offloading scheme enormously decreases the outage probability and enhances the ergodic rate of offloaded mbs users. considering an ultra dense heterogeneous (scmt) network, the authors in [ ] proposed an optimal fair power allocation scheme which aims to maximize the overall energy efficiency of the considered network. the simulation results are presented in terms of energy efficiency for the considered network which demonstrate superior performance over conventional oma based scmt cns. the problems of energy and spectrum efficiencies are considered in [ ] for noma-based scmt cn. the constrained multi-objective optimization problem is formulated in order to maximize the overall energy and spectrum efficiency of the network. the authors proposed novel optimal algorithms to allocate power and sub-channels and reported superior performance of the considered noma-based cn over conventional oma. the authors investigated the problem of user scheduling and power optimization in[ ] and studied the trade-off between data rate and energy consumption for noma systems. by considering perfect and imperfect csi, the authors proposed user scheduling and power allocation schemes. under the proposed resource allocation methods, the results reveal significant enhancement in performance of noma-based scmt network for both perfect and imperfect csi scenarios. based on minimum user rate requirements, the authors in [ ] considered a noma-based scmt cn in which both mbs and sbs compete in order to maximize their throughputs. the power allocation required to optimize the mbs and sbs individual throughputs is modeled as a stackelberg game. in order to obtain the stackelberg equilibrium, the author proposed a distributed power allocation algorithm which has improved the spectrum efficiency of the considered noma-based scmt cn. furthermore, the authors in [ ] investigated the problem of subchannel and power allocation in order to maximize the energy efficiency for noma-based scmt cn. the optimal solution for subchannel and power allocation is derived in closed-form and the results show the fast electronics , , of convergence of the proposed optimal algorithm while significantly enhancing overall system energy efficiency. moreover, the authors in [ ] proposed an optimal joint subchannel and power allocation algorithm which is aimed to enhance the energy efficiency of mbs and sbs in a noma-based scmt cn. numerical results reveal that the proposed algorithm is capable of improving energy efficiency over the existing schemes. the problem of joint spectral and energy efficiency is investigated by the authors in [ ] for noma-based scmt cn. the results demonstrate that the proposed scheme achieve superior performance in terms of spectral and energy efficiency over conventional noma and oma. considering noma-based scmt cn, the authors in [ ] investigated the trade-off among four quantities, namely, sum-rate, fairness, energy efficiency, and harvested energy. the authors proposed an optimal algorithm in order to jointly allocate subcarrier and power which aims to obtain energy efficient resource allocation. furthermore, considering two scenarios of resource allocation based on subcarrier and intercell interference, the authors in [ ] proposed an optimal algorithm to maximize the sum-rate of the entire system. the results demonstrate that the system link performance is enhanced under the proposed scheme when the system sum-rate is kept low. the authors in [ ] studied the average rate, coverage probability, and quality of experience (qoe) for noma-based scmt cn. the results demonstrate that the considered network achieves better rate and qoe compared to the conventional oma. by considering the noma technique implemented by both mbs and sbs in scmt cn, the authors in [ ] proposed an optimal power allocation algorithm in order to maximize the overall system throughput. the results demonstrate that the considered network under the proposed power allocation algorithm significantly enhances the spectral efficiency and improves the outage performance over the conventional oma. the authors in [ ] considered a ratio fairness model and user scheduling scheme in order to increase the overall system capacity of the noma-based scmt cn. in addition, a user pairing method is also suggested to improve the working of existing sic. the results demonstrate that the proposed scheme achieves superior fairness performance compared to the existing noma fairness scheme and conventional oma. the authors in [ ] consider a noma-based scmt cn with self-organizing network (son) functionalities. in particular, the authors have proposed a distributed algorithm in which under situations of congestion, the users from congested tiers are offloaded to achieve load balancing while the resource allocation is obtained such that it would minimize the co-channel interference. the simulation results reveal that the improvement in data rate and user fairness has been observed by considering son functionality with noma-based scmt cn. a. noma-based scmt cn system a general noma-based k-tier single-cell cn is depicted in figure . it can be observed that there is a single macro-cell in which mbs is located at the center of a disc with radius rd, where rd models the coverage of mbs. without a loss of generality, it can be assumed that the mbs forms tier one. consequently, the bss from remaining k − tiers constitute small-cells, and are referred as sbss. further, it can be assumed that each k-th tier sbs is located at the center of a disc with radius rd,k, where rd,k represents the coverage of the k-th tier sbs and < k ≤ k. further, we call the users who fall inside coverage of any sbs as clustered users (cus), while those who do not fall inside coverage of any sbs are named as non-clustered users (ncus). note that at particular time instant, a ncu is always served by a mbs, whereas cu could be associated to mbs or sbs. as such, let us call the users who are served by mbs as mbs users (mus), while those served by sbs are termed as sbs users (sus). note that sus are always cus whereas mus can be further classified as non-clustered mus (ncmus) and clustered mus (cmus) [ ]. further, for analysis purposes, we consider a representative k-th tier small-cell in which n sus are randomly distributed. in addition, consider that there are total m mus which are distributed randomly inside coverage of mbs. moreover, assume that {hm}mm= and { hkn }n n= represent the channel gains (power) for m-th mu and n-th su in k-th tier, respectively. consequently, based on the noma principle, the mus and sus are considered to be ordered as h ≤ .... ≤ hm and electronics , , of hk ≤ .... ≤ h k n , respectively, where hm = gm dαm and hkn = gkn {dkn}α . here, gkn and d k n represent the fading power and distance between the n-th user and k-th tier sbs. as a result, power allocation coefficients for mus and sus in k-th tier are sorted as a ≥ ... ≥ am and ak ≥ ... ≥ a k n , respectively, where a k n is the power allocation coefficient of n-th su in tier k. further, consider that all bss are utilizing the same time and frequency resources. then, the received signal power at m-th mu, denoted as pscmtm can be expressed as follows: pscmtm =   hm am p︸ ︷︷ ︸ useful signal power + intra-user interference︷ ︸︸ ︷ hm m ∑ i= i =m ai p +σ , if m-th user is ncu hm am p︸ ︷︷ ︸ useful signal power + hm m ∑ i= i =m ai p ︸ ︷︷ ︸ intra-user interference +isbsm + σ , if m-th user is cu ( ) where isbsm is the total received interference (power) at m-th mu from sbss. similarly, the received power at the n-th su in tier k, denoted by pk,scmtn can be expressed as follows: pk,scmtn = h k n a k n pk︸ ︷︷ ︸ useful signal power + intra-user interference︷ ︸︸ ︷ hkn n ∑ i= i =n aki pk +i k,mbs n +i k,sbs n + σ , ( ) where ik,mbsn and i k,sbs n represent the total interference at n-th su in tier k from mbs and other sbss, respectively. note: the exact characterization of interference terms isbsm and i k,mbs n ,i k,sbs n in equations ( ) and ( ), respectively, and all the interference terms that will arise in subsequent discussions, require stochastic geometry based modeling of bss and user distributions, which is beyond the scope of this survey. interested readers are referred to see [ – ] for more detailed treatment of this topic. b. outage probability of noma-based scmt cn recall that ncu always connects to mbs whereas cu can be connected to mbs or sbs. as such, let us denote by ambs and −ambs as the probability that cu is associated to mbs and sbs, respectively. the probability of association for an arbitrary cu to connect to mbs can be expressed as follows: ambs = p ( r−αmbs p > r −α sbs pk ) = p ( rmbs < ( p pk ) α rsbs ) = ∫ ∞ [ − frsbs (( p pk ) α rmbs )] frmbs (rmbs) drmbs, ( ) where rmbs and rsbs represent the distance between a typical cu and the nearest mbs and sbs, respectively. it can be observed from equation ( ) that the association probability ambs depends on the distributions of random variables rmbs and rsbs. in current literature, one promising method to determine these distributions is to utilize a stochastic geometry framework, e.g., see the references [ , ]. therefore, after obtaining frsbs and frmbs and solving the integral in equation ( ) provides the association probability ambs. electronics , , of next, we focus our discussion on expressing the outage probability for typical mu and su in scmt cn. based on equation ( ), the received sinr at m-th mu after removing higher order users, denoted by Γscmtm , can be expressed as follows: Γscmtm =   hm am ρ hm ρ ∑mi=m+ ai+ , if m-th user is ncu hm am ρ hm ρ ∑mi=m+ ai+ρ̄i sbs m + , if m-th user is cu ( ) where ρ̄ = σ . now, at a particular time instant, mu can be of non-clustered or clustered type (but can’t be of both types). as such, let us define the following events in order to determine the outage probability of the m-th mu under scmt cn. ε = {tpical mu is ncu} ε = {tpical mu is cu} ε = {outage at tpical mu when it is ncu in scmt cn} ε = {outage at tpical mu when it is cu in scmt cn} based on the events ε − ε , the outage probability at the mth mu, denoted by pscmtm , can be expressed as follows: pscmtm = pε pε + pε pε . ( ) now following similar steps to deriving equation ( ), the probability pε can be expressed as follows: pε = p scst m . ( ) next, condition on interference isbsm and following similar steps to equation ( ), the probability pε can be written as: pε = pε |isbsm (a) = ∫ ∞ pscstm |τmaxm =τ̄maxm fisbsm (x) dx, ( ) where τ̄maxm = max { τ̄ , ..., τ̄j, ...., τ̄m } , τ̄j = ϕ̄j( +ρ̄isbsm ) ρ ( aj−ϕ̄j ∑mi=j+ ai ) , ϕ̄j = rscmtj − , ≤ j ≤ m, fisbsm (x) is the pdf of interference isbsm and (a) in equation ( ) is obtained by de-conditioning on fisbsm (x). finally, using equations ( ) and ( ) in equation ( ) derives the outage probability expression for m-th mu in scmt noma cn. next, recall that at some particular time instant, su can only be connected to sbs . as such, based on equation ( ), the sinr at typical k-th tier n-th su, denoted by Γk,scmtn , can be expressed as follows: Γk,scmtn = hkn a k n ρk hkn ρk ∑ n i=n+ a k i + ρ̄ ( ik,mbsn +i k,sbs n ) + , ( ) where ρk = pk σ . now following similar approach in deriving equation ( ), the outage probability the typical k-th tier n-th su, denoted by pk,scmtn , can be written as: pk,scmtn = ∫ ∞ ∫ ∞ pscstm |τmaxm =τ̄maxn,k fik,mbsn (x) fik,sbsn (y) dxdy, ( ) electronics , , of where τ̄maxn,k = max { τ̄ ,k, ..., τ̄j,k, ...., τ̄n,k } , τ̄j,k = ˇϕj,k( +ρ̄(x+y)) ρk ( akj −ϕ̌j k ∑ni=j+ a k i ) , ϕ̌j = rk,scmtj − , ≤ j ≤ n, rkj is the targeted rate for j-th su in tier k, fik,mbsn (x), and fik,sbsn (y) are the pdfs of interference ik,mbsn and ik,sbsn , respectively. this completes the discussion for outage probability in noma-based scmt cns. c. sum-rate maximization problem in noma-based scmt cn similar to scst cn case, let us consider a problem of maximizing overall sum-rate in scmt noma-based cn. as such, define an objective function as follows: fscmt = m ∑ m= r̃scmtm + k ∑ k= n ∑ n= r̃k,scmtn , ( ) where r̃scmtm and r̃ k,scmt n are the achievable rates of typical m-th mu and k-th tier n-th su in scmt cn. now, based on equation ( ), the optimization problem can be formulated as follows: maxfscmt ( ) subject to: c : rm ≤ r̃scmtm c : r k n ≤ r̃ k,scmt n c : m ∑ m= am p ≤ p c : n ∑ n= akn pk ≤ pk, ≤ k ≤ k c : ϕj m ∑ i=j+ ai ≤ aj, ≤ j ≤ m c : ϕ̌j k n ∑ i=j+ aki ≤ a k j , ≤ j ≤ n c : isbsm < hm am p, ≤ m ≤ m c : ik,mbsn +i k,sbs n < h k n a k n pk, ≤ n ≤ m, ≤ k ≤ k. the constraints c , c guarantee that the minimum rate requirements for mus and sus are met. further, the constraints c , c ensure that the allocated powers to mus and sus would not exceed the total transmitted powers available at mbs and sbs. the necessary conditions required to keep noma-sic operational are imposed by the constraints c , c . finally, the constraints c , c are imposed to ensure that the mus and sus are allocated with appropriate power levels such that the received powers at sus and mus should exceed corresponding interference levels. . . . noma in multi-cell single-tier cellular network much less attention has been paid in existing literature to investigating noma-based multi-cell single-tier (mcst) cns. the authors in [ ] analyze the uplink noma for large-scale cellular networks. the spatial locations of the bss are modeled by a homogeneous ppp. considering the locations of bss as a parent pp, the spatial topology of users is modeled by pcp, in which users are clustered around the locations of bss. this captures the correlation between the location of users and bs. in particular, they considered three scenarios of sic, namely perfect sic, imperfect sic, and imperfect worst case sic. in order to evaluate the performance of the considered network, the authors first derived a closed-form expression for intra-cluster interference. then, based on the interference results, an expression for rate coverage is provided under all three scenarios of sic. numerical simulations are electronics , , of conducted to validate the accuracy of the derived analytical results and compare the proposed network with ppp- and oma-based networks. the results show that the pcp-based network outperforms the oma-based network and offers more accurate analysis than the ppp-based approach, which provides optimistic results for noma. however, the considered system model is limited to a single tier case. the problem of power allocation for ofdm based noma systems is studied in [ ]. the considered cellular network is composed of hexagonal grid sites having three cells per site. the authors formulated an optimization problem to maximize the weighted sum rate of the entire system. due to the intractability of the presented optimization problem, it was solved in two steps. in the first step, the user selection across each subchannel and power assignment are performed by using greedy, and sub-optimal iterative power allocation algorithms, respectively. in the second step, using the iterative power allocation algorithm of the first part, the power assignment across the sub-channel is achieved. the simulation results demonstrate the superiority of their proposed scheme over existing ones in terms of achievable system throughput. however, a fundamental limitation of their system model is the assumption of hexagonal grids with a regular shape and size, which may not always hold true in real cell site deployments. a noma-based multi-cell network is analyzed in [ ]. the locations of bss are modeled by a homogeneous ppp, and a more realistic voronoi cell structure is considered. the authors provided close-form expressions for the coverage probability and achievable rate. numerical simulations are conducted to validate the accuracy of derived analytical results, and compare the achieved performance with that of an oma-based network. the results show that noma achieves higher coverage and achievable rate than oma under the considered network setting. however, there are two potential shortcomings: ( ) the performed analysis is limited to a case of only two users for single-tier networks; and ( ) modeling locations of mbss by ppp is not accurate because real deployments of mbss exhibit interaction between mbss locations, whereas ppp does not capture this property due to its completely spatial random property. the key challenges and benefits of applying noma in a mcst environment is discussed in [ ]. in addition, the authors have also comprehensively discussed the theory and working of noma in mcst and scst cases. a. noma-based mcst cn system figure illustrates the basic configuration of mcst cn utilizing noma protocol. similar to scst and scmt cns, it can be observed from the figure that each mbs is located at the center of a disc with radius rd modeling the coverage of mbs in mcst cn. the mbss are communicating with users by applying noma protocol. further, consider that there are total of m noma users distributed randomly in each macro-cell. for analysis purposes, we consider m-th user as typical mu in a representative macro-cell. the situation of m-th noma user in a representative macro-cell is similar to scst scenario except that there would be an additional interference from other mbss in the considered mcst cn. therefore, the received power at the m-th user in a representative macro-cell, denoted by pmcstm , can be written as follows: pmcstm = hm am p︸ ︷︷ ︸ useful signal power + hm m ∑ i= i =m ai p ︸ ︷︷ ︸ intra-user interference +imbsm + σ , ( ) where imbsm is the interference at the typical m-th mu in a representative macro-cell from other mbss in mcst cn. b. outage probability of noma-based mcst cn based on equation ( ), the sinr at m-th mu after the application of sic, denoted as Γmcstm , can be expressed as follows: electronics , , of Γmcstm = hm am ρ hm ρ ∑mi=m+ ai + ρ̄i mbs m + . ( ) next, following the similar approach as was used in obtaining equation ( ), the outage probability at m-th mu in mcst cn, denoted as pmcstm can de written as: pmcstm = ∫ ∞ pscstm |τmaxm =τ̂maxm fimbsm (x) dx, ( ) where τ̂maxm = max { τ̂ , ..., τ̂j, ...., τ̂m } , τ̂j = ϕ̂j( +ρ̄imbsm ) ρ ( aj−ϕ̂j ∑mi=j+ ai ) , ϕ̂j = rmcstj − , ≤ j ≤ m, and fimbsm (x) is the pdf of interference imbsm . c. sum-rate maximization problem in noma-based mcst cn let us consider an objective function fmcst for maximizing sum-rate of the representative macro-cell in mcst cn. note that this is identical of maximizing the overall sum-rate of the mcst cn in terms of average performance. the objective function fmcst can be now defined as: fmcst = m ∑ m= r̃mcstm = m ∑ m= log ( + Γmcstm ) = m ∑ m= log ( + hm am ρ hm ρ ∑mi=m+ ai + ρ̄i mbs m + ) . ( ) note that objective function fmcst in equation ( ) is different from fscst in equation ( ) due to the presence of interference term imbsm in the achievable rate of user m, ≤ m ≤ m in mcst cn. the optimization problem can now be formulated as follows: maxfmcst ( ) subject to: c : r mcst m ≤ r̃m c : m ∑ m= am p ≤ p c : ϕ̂j m ∑ i=j+ ai ≤ aj, ≤ j ≤ m c : imbsm < hm am p, ≤ m ≤ m. note that compared to optimization problem in equation ( ), there is an additional constraint c in equation ( ) which ensures that by virtue of appropriate power allocation at mbs, each mu would received a power level that could exceed the corresponding interference level. . . . noma in multi-cell multi-tier cellular networks the current literature is scarce in analyzing noma-based multi-cell multi-tier (mcmt) cns. in [ ], the authors consider the application of noma to k-tier multi-cell network. a comprehensive hybrid transmission framework is proposed under which mbss and sbss utilize massive mimo and electronics , , of noma technologies to enhance the overall spectral efficiency. under the considered k-tier network, the authors proposed a bs association policy for mbs and sbs users based on the biased average received power metric. the performance of the network is evaluated in terms of spectrum efficiency achieved by each tier, where the closed-form expressions are derived by using stochastic geometry tools. numerical results demonstrate that the proposed hybrid massive mimo for noma-based multi-tier network can achieve better spectrum efficiency then its oma counterpart. its potential shortcoming is the use of less realistic user distributions that do not capture the coupling between users’ and sbs locations. in addition, mbs locations are modeled by homogeneous ppp, which does not reflect the deployment trend of real mbss. the application of noma to multimedia broadcast/multicast service (mbms) is investigated for multi-cell k-tier network in [ ]. such a network aims to meet the high data rate demands of emerging applications by enjoying the efficient spectrum utilization of both noma and mbms techniques. the bs spatial topology of each tier is modeled by independent homogeneous ppp. based on noma and mbms, the authors proposed two transmission schemes and evaluated the performance of these schemes using stochastic geometry. an analytical framework is developed which is general enough to cover the cases of synchronous and asynchronous non-orthogonal mbms transmission. based on this framework, the authors derived closed-form expressions for coverage probability, sum rate and the number of users served. simulations are conducted to verify the analysis. it was shown that non-orthogonal mbms outperforms orthogonal mbms. in addition, synchronous transmission mode can achieve better performance than the asynchronous one. however, the users in each tier are modeled by independent homogeneous ppps. while this distribution is accurate for users who are located independent of bs locations, it does not capture the location coupling between users and sbs of multi-tier networks, where the latter is often deployed to enhance the service in areas of high congestion. in [ ], the authors considered a large-scale noma-based multi-tier cellular network in which sbss communicate with their users with the aid of noma protocol, whereas mbss are equipped with massive mimo capability. a tractable analytical framework is developed to analyze the performance of considered network. closed-form expressions for coverage probability are obtained for noma sbss users. in addition, a lower bound for achievable ergodic rate is derived for a user served by massive mimo enabled mbss. based on the coverage results, the energy efficiency of hybrid multi-tier networks is also investigated. numerical results reveal that the performance of sbss noma users is highly dependent on the choice of user targeted rates and power allocation coefficients. moreover, equipping mbss with massive mimo capability significantly enhances the network’s spectrum efficiency. while the developed model is analytically tractable, it suffers from two shortcomings: (i) noma is not utilized for mbss, and (ii) it uses homogeneous ppps to model mbss and sbss. the authors in [ ] considered a noma-based mcmt cn and investigated the system performance in terms of coverage and throughput under non-coordinated and coordinated joint noma transmission techniques. the authors derived a constraint on power allocation of users which mist be satisfied in order to enhance the coverage and throughput of the considered system. in addition, the authors have also showed the existence of optimal power allocation that would maximize the coverage and throughput of the system. the authors in [ ] considered a noma-based mcmt cn for ultra dense network deployments and proposed a network architecture which integrates ground–air–space radio access network and core networks by applying the concepts of virtualization. moreover, the authors also studied the impact of fog-computing coordination on resource allocation and macro-small cell coordination. simulation results demonstrate that utilizing network coordination would significantly enhance the system performance of both noma and oma based mcmt ultra dense cns. the authors in [ ] considered a noma-based mcmt cn where small-cells are deployed in a non-uniform manner. in order to evaluate the system performance, the authors derived closed-form expressions for coverage probability and achievable rate. numerical results are presented to validate the accuracy of the performed analysis and also demonstrate the superiority of noma over oma under the considered mcmt electronics , , of cn. furthermore, the authors in [ ] considered a noma-based multi-tier cn with cloud-based central station coordination functionality. by considering realistic power consumption of different cell types and wireless backhaul, the authors proposed a power allocation method for multiple cells from different tiers in the considered network. the authors reported that the overall energy efficiency of the network is hugely impacted by the power available at the cloud, cell types, and propagation environment. in [ ], the authors have proposed a framework to analyze ultra dense noma-based mcmt cn. the proposed framework can be modified and configured easily to accommodate various application scenarios. further, in order to highlight the effectiveness of their proposed framework, the authors have presented two case studies. a. noma-based mcmt cn system figure shows a mcmt cn in which bs from each tier implements noma in order to communicate with the cellular users. let us consider that there are total of k tiers, where without loss of generality, it is assumed that the tier one is comprised of mbss. it can be observed that each mbs and k-th tier sbs are located at the center of discs with radii rd and rk,d, < k ≤ k. further, similar to the scmt case, for analysis purposes, consider a representative macro-cell in which multiple small-cells are underlaid. note that in average terms, the performance of each macro-cell in mcmt cn is similar to the performance of representative macro-cell. as such, with this description and assumption for analysis, the representative macro-cell in the considered mcmt cn is similar to scmt case with exception of interference at a typical mu and su from other mbss and all mbss, respectively. consequently, all the underlying user classifications (cmus, ncmus, etc.) and definition/notations defined earlier in scmt cn system describing different variables (channel gains, power allocation, etc.) will remain same in the subsequent discussion. then, the received power at m-th mu in mcmt cn, denoted by pmcmtm , can be expressed as follows: pmcmtm =   hm am p︸ ︷︷ ︸ useful signal power + intra-user interference︷ ︸︸ ︷ hm m ∑ i= i =m ai p +imbsm,mcmt + σ , if m-th user is ncu hm am p︸ ︷︷ ︸ useful signal power + hm m ∑ i= i =m ai p ︸ ︷︷ ︸ intra-user interference +imbsm,mcmt +i sbs m,mcmt + σ , if m-th user is cu ( ) where imbsm,mcmt and i sbs m,mcmt represent the interference at typical m-th mu from other mbss and sbss, respectively. similarly, the received power at the typical k-th tier n-th su, denoted by pk,mcmtn , can be expressed as follows: pk,mcmtn = h k n a k n pk︸ ︷︷ ︸ useful signal power + intra-user interference︷ ︸︸ ︷ hkn n ∑ i= i =n aki pk +i k,mbs n,mcmt +i k,sbs n,mcmt + σ , ( ) where ik,mbsn,mcmt and i k,sbs n,mcmt are the interferences at typical k-th tier n-th su from mbss and other sbss, respectively. b. outage probability of noma-based mcmt cn based on equation ( ), the sinr at m-th mu after applying sic in mcmt cn, denoted by Γmcmtm , can be expressed as follows: electronics , , of Γmcmtm =   hm am ρ hm ρ ∑mi=m+ ai+ρ̄i mbs m,mcmt+ , if m-th user is ncu hm am ρ hm ρ ∑mi=m+ ai+ρ̄(i mbs m,mcmt+i sbs m,mcmt)+ , if m-th user is cu. ( ) now, similar to scmt cn, at a particular time instant, mu can be ncu or cu (but not both simultaneously). as such, define the following two events. ε = {outage at tpical mu when it is ncu in mcmt cn} ε = {outage at tpical mu when it is cu in mcmt cn} based on the events ε , ε , ε , ε , the outage probability at the mth mu in mcmt cn, denoted by pmcmtm , can be expressed as follows: pmcmtm = pε pε + pε pε . ( ) next, following similar steps to equation ( ), the probabilities pε and pε in equation ( ) can be expressed as follows: pε = ∫ ∞ pscstm |τmaxm =τ̃maxm fimbsm,mcmt (x) dx, ( ) where τ̃maxm = max { τ̃ , ..., τ̃j, ...., τ̃m } , τ̃j = ϕ̃j( +ρ̄imbsm,mcmt) ρ ( aj−ϕ̃j ∑mi=j+ ai ) , ϕ̃j = rmcmtj − , ≤ j ≤ m, and fimbsm,mcmt (x) is the pdf of interference imbsm,mcmt. similarly, pε can be written as: pε = ∫ ∞ ∫ ∞ pscstm |τmaxm =ϑmaxm fimbsm,mcmt (x) fik,sbsm,mcmt (y) dxdy, ( ) where ϑmaxm = max { ϑ , ..., ϑj, ...., ϑm } , ϑj = φj( +ρ̄(x+y)) ρ ( aj−φj ∑ni=j+ ai ) , φj = rmcmtj − , ≤ j ≤ n, rmcmtj is the targeted rate for j-th mu, fimbsm,mcmt (x) and fisbsm,mcmt (y) are the pdfs of interferences imbsm,mcmt and isbsm,mcmt, respectively. finally, using equations ( ) and ( ) in equation ( ) derives the result for outage probability of m-th mu in noma-based mcmt cn. next, based on equation ( ), the sinr at k-th tier n-th su after removing higher order users, denoted by Γk,mcmtn can be expressed as follows: Γk,mcmtn = hkn a k n ρk hkn ρk ∑ n i=n+ a k i + ρ̄ ( ik,mbsn,mcmt +i k,sbs n,mcmt ) + . ( ) now following similar approach in deriving equation ( ), the outage probability at the typical k-th tier n-th su, denoted by pk,mcmtn in mcmt cn, can be written as: pk,mcmtn = ∫ ∞ ∫ ∞ pscstm |τmaxm =ϑ̄maxn,k fik,mbsn,mcmt (x) fik,sbsn,mcmt (y) dxdy, ( ) where ϑ̄maxn,k = max { ϑ̄ ,k, ..., ϑ̄j,k, ...., ϑ̄n,k } , ϑ̄j,k = ¯φj,k( +ρ̄(x+y)) ρk ( akj −φ̄j k ∑ni=j+ a k i ) , φ̄j = rk,mcmtj − , ≤ j ≤ n, rk,mcmtj is the targeted rate for j-th su in tier k, fik,mbsn,mcmt (x) and fik,sbsn,mcmt (y) are the pdfs of electronics , , of interference ik,mbsn,mcmt and i k,sbs n,mcmt, respectively. this completes the discussion for outage probability in noma-based mcmt cns. c. sum-rate maximization problem in noma-based mcmt cn let us consider an objective function fmcmt which aims to maximize the sum-rate of the representative macro-cell. similar to scmt cn case, the objective function here can be defined as follows: fmcmt = m ∑ m= r̃mcmtm + k ∑ k= n ∑ n= r̃k,mcmtn , ( ) where r̃mcmtm and r̃ k,mcmt n are the achievable rates of typical m-th mu and k-th tier n-th su in mcmt cn. now, based on equation ( ), the optimization problem can be formulated as follows: maxfmcmt ( ) subject to: c : r mcmt m ≤ r̃ mcmt m c : r k,mcmt n ≤ r̃ k,mcmt n c : m ∑ m= am p ≤ p c : n ∑ n= akn pk ≤ pk, ≤ k ≤ k c : φj m ∑ i=j+ ai ≤ aj, ≤ j ≤ m c : φ̄j k n ∑ i=j+ aki ≤ a k j , ≤ j ≤ n c : imbsm,mcmt +i sbs m,mcmt < hm am p, ≤ m ≤ m c : ik,mbsn,mcmt +i k,sbs n,mcmt < h k n a k n pk, ≤ n ≤ m, ≤ k ≤ k, the constraints in optimization problem equation ( ) can be explained in a similar way as was described in optimization problem equation ( ) for scmt cn. . . noma applied to device-to-device communications in current literature, the application of noma to device-to-device (d d) communication is relatively less explored. particularly, the existing works pay little attention in investigating d d networks where d d nodes are communicating with the aid of noma transmission scheme. in [ ], the authors considered a cooperative noma system with full-duplex (fd) functionality. the bs applies noma protocol to serve strong and weak users. in order to enhance the outage performance of the weak noma user, the authors proposed to utilize fd at the strong user. under this setting, the users communicate with each other by utilizing oma based cooperative d d communication. furthermore, the authors in [ ] considered a cellular-network with underlay d d communications, where d d users can also operate in fd mode. in order to select between d d and fd modes, the authors proposed a novel selection criteria. based on stochastic geometry techniques, the performance of considered network is characterized by deriving closed-form outage probability expressions. the spatial locations of d d users are modeled by the poisson point process (ppp), which may not be an accurate distribution for modeling d d locations because it cannot capture the key characteristics electronics , , of of device clustering and spatial separation for d d communications [ ]. a mimo-noma-based multiuser downlink cellular network is considered in [ ]. moreover, d d communication in underlay mode is also invoked in order to further enhance the spectral efficiency of the considered network. as such, this configuration constitutes intra-beam interference as well as interference at d d users due to bs transmission. consequently, in order to mitigate both types of interference, the authors proposed two beamforming methods. further, the performance of both cellular and d d users is jointly investigated by formulating an optimization problem. however, a conventional oma-based paired d d communication is considered and the system model is limited to single-cell scenario only. the authors in [ ] considered a paired d d communication underlying a noma-based cellular network in which a single hybrid access point is used to receive from both d d and cellular users. in particular, they investigate the problem of resource allocation and propose a low complexity energy efficient algorithm for the d d pair while meeting the qos requirements of cellular users. the results demonstrated fast convergence of their proposed algorithm to an optimal solution while achieving superior energy efficiency over the existing schemes. a major limitation of their network model is that they considered only a single d d active pair, whereas in real situations, there can be more than one active d d pairs in the network, which requires joint optimization of energy efficiency of all active d d pairs. similarly, the problem of resource allocation in terms of power control and channel assignment for d d communication underlying a noma-based cellular network is investigated in [ ]. the authors derived the optimal conditions for cellular users under which power control on each subchannel is conducted. based on the derived conditions, they proposed an algorithm to maximize the sum rate of d d pairs while simultaneously satisfy the qos requirements of cellular users. however, the considered cellular network is restricted to the case of a single cell, thereby neglecting the impact of interference from multiple bss. in addition, noma is not utilized for d d communications, which may result in less efficient resource utilization. the concept of noma assisted d d relaying is proposed in [ ], where the transmission is composed of two phases. in the first phase, the bs transmits noma signal to near and far users. by exploiting the overhearing of this noma transmission at intermediate d d relay node, in the second phase, this relay node transmits superimposed overheard noma signal from first phase and its own signal for the d d receiver using noma. this helps to boost the performance of far user. although noma is used by the d d relay node, it is still communicating with a single d d receiver to form a paired d d communication. the work of [ ] can be regarded as a pioneer in investigating noma-based d d communications, where authors came up with a notion of the d d group. the key idea of group d d communication is that a d d transmitter (dt) acts like a bs and applies the noma transmission technique in order to communicate with multiple d d receivers (drs). the introduction of group d d communication would generate a network interference. as such, in order to realize a group d d network, an optimal resource allocation algorithm is proposed in order to efficiently perform interference management. furthermore, the work in [ ] can be regarded as the extension of [ ] in which a joint subchannel and power allocation algorithm is proposed in order to maximize the sum-rate of d d users. the results demonstrate that the proposed algorithm is able to achieve near optimal performance. however, again the system model is limited to a single-cell scenario and proper interference modeling and characterization would be required for multi-cell extension. the authors in [ ] considered a large-scale d d network and proposed a cooperative hybrid automatic repeat request (harq) scheme based on noma. the performance of d d users in the considered system is evaluated in terms of outage probability and throughput. the results reveal that the proposed cooperative harq assisted noma scheme outperforms both non-cooperative and oma based networks. however, the authors considered only two user noma in this work and also assumed that one noma user always exist in close proximity of the d d source. this would be a rather strong assumption which may not generally hold. a qos-based noma group d d communication system is proposed in [ ]. the authors introduced a notion of qos based power allocation. based on stochastic geometry techniques, the closed-form analytical expressions for outage probability are electronics , , of derived. the simulation results demonstrate that the proposed qos based noma group d d system achieves lower outage probability than the conventional oma-based paired d d communication. the performance of cooperative d d network with noma is investigated in [ ] where each d d link exhibits independent nakagami-m fading. in order to evaluate the system performance, closed-form expressions for outage probability and throughput are presented. in addition, numerical results reveal the accuracy of the derived analytical results. a cooperative d d system is considered in [ ] where bs communicates with all the users simultaneously by applying the noma protocol. the authors proposed two decoding schemes in order to study the impact of weak channel and different decoding strategies on the performance of the considered network. further, the authors presented the expressions for ergodic sum-rate, outage probability, and outage capacity to analyze the system performance. numerical results are also presented to highlight the superiority of the proposed schemes over conventional noma and oma. the authors in [ ] considered a noma system with underlay d d communication. in order to avoid the impact of interference on the performance of cellular user, the authors have proposed a strategy for d d users by which they can select to operate between interlay and underlay modes. further, a joint d d mode selection and resource allocation optimization problem is formulated which aims to maximize the sum-rate of the considered system. simulation results reveal that utilizing the proposed mode selection scheme for d d in the considered network significantly enhances the overall system sum-rate and d d access rate. the authors in [ ] consider a d d enabled noma network and proposed a novel framework in order to maximize the performance of the d d communications in terms of energy efficiency and sum-rate. as such, the optimization problem is formulated which jointly considers resource block assignment and power allocation based on sic decoding order of cellular users. the results show that the considered network under the proposed optimization algorithm achieves superior performance compared to the conventional oma system. the problem of resource allocation for noma-based d d network with underlay cn is investigated in [ ]. an optimization problem is formulated in order to optimize the total transmit power for all the users by jointly considering subchannel, user pairing, and power control. the authors reported that the minimization of total transmit power for the network has a high dependency on subchannel assignment and user pairing. the problem of joint d d group association and channel assignment is investigated in [ ] for uplink multi-cell noma systems. in order to utilize cellular channels at each bs by d d groups, the authors proposed sinr maximization based multi-objective power allocation solution with constraint of meeting qos requirements. in addition, the problems of d d group association and channel assignment are solved by applying principles of matching theory. simulation results reveal the effectiveness of proposed algorithms by achieving comparable sinr for each user and d d receiver to the joint group association, channel, and power allocations. the issue of security in the presence of eavesdropper for d d enabled noma-based cns are investigated in [ ]. in order to resolve the security problem in the considered network, the authors have formulated and solved a joint power optimization problem which targets to maximize the secrecy sum-rate of the entire network. in addition, closed form expressions for connection outage and secrecy outage probabilities are derived to characterize the performance. simulation results are provided to validate the accuracy of the derived analytical results as well as the superior performance of proposed algorithms in enhancing joint security of the network. the authors in [ ] considered an uplink d d communication based on noma and investigated the problem of joint sub-channel and power allocation in order to maximize the overall energy efficiency and throughput of the considered system. simulation results reveal that under the proposed optimal power allocation algorithm, the considered system achieves superior performance than the existing schemes in terms of energy efficiency and throughput. in order to enhance the data rate of d d communications, the authors in [ ] considered a d d and hybrid cn, where joint user pairing and power control optimization problem is formulated while taking into account the decoding threshold for cellular users. as such, an optimal power control algorithm is proposed to solve the optimization problem. the results show that the proposed algorithm achieves superior data rate when compared to the current techniques. electronics , , of . . . noma-based group d d communications a general group d d (gd d) network based on noma protocol is shown in figure . it can be observed that d d users are distributed randomly over the entire two-dimensional plane. further, it is assumed that the d d network is operating in an inband mode with underlay mcst cn. in addition, consider that dt, referred as group transmitter (gt), is communicating with multiple drs via noma protocol. note that at particular time instant, the selection of gt is performed by the bs. for analysis purposes, we consider a representative macro-cell in which m mus and multiple gts are randomly distributed, as shown in figure . further, let rd and rgt model the coverage of mbs and a gt, respectively. in addition, for ease of exposition, it is considered that each gt is serving l drs via noma protocol, which are assumed to be randomly distributed inside its coverage. without loss of generality, assume that channel gains of mus and drs (in coverage of representative gt) are ordered as h ≤ ... ≤ hm and h̃ ≤ ... ≤ h̃l, respectively, where h̃l = g̃l d̃αl . here, g̃l and d̃l represent the fading (power) gain and distance between l-th dr and representative gt, respectively, and ≤ l ≤ l. consequently, the power allocation coefficients for mus and drs are sorted as, a ≥ ... ≥ am and ã ≥ ... ≥ ãl, respectively. the received power at the m-th mu and l-th dr, denoted by pgd dm and pgd dl , can be expressed as follows: pgd dm = hm am p︸ ︷︷ ︸ useful signal power + intra-user interference︷ ︸︸ ︷ hm m ∑ i= i =m ai p +imbsm,gd d +i gt m,gd d + σ , ( ) pgd dl = h̃l ãl pgt︸ ︷︷ ︸ useful signal power + intra-user interference︷ ︸︸ ︷ h̃l l ∑ i= i =l ãi pgt +imbsl,gd d +i gt l,gd d + σ , ( ) where pgt is the transmission power of gt, imbsm,gd d, i gt m,gd d represent the interference at m-th mu from other mbss and gts, respectively, and imbsl,gd d, i gt l,gd d denote the interferences at l-th dr from mbss and other gts, respectively. electronics , , of drs gt rgd dr dr l dr l gt rgd dr dr l dr l gt rgd dr dr l dr l gt rgd dr dr l dr l figure . group device-to-device (d d) network with underlay cellular network. . . . outage in noma-based group d d network this subsection describes the outage probability at m-th mu and l-th dr in a representative macro-cell and gt coverage, respectively. based on equation ( ), the sinr after applying sic at m-th mu in noma-based gd d network, denoted as Γgd dm , can be written as follows: Γgd dm = hm am ρ hm ρ ∑mi= i =m ai + ρ̄ ( imbsm,gd d +i gt m,gd d ) + . ( ) next, based on equation ( ) and following similar steps to deriving equation ( ), the outage probability at m-th mu in noma-based gd d underlay cn, denoted as pgd dm , can be given as: pgd dm = ∫ ∞ ∫ ∞ pscstm |τmaxm =ϑ̃maxm fimbsm,gd d (x) fisbsm,gd d (y) dxdy, ( ) where ϑ̃maxm = max { ϑ̃ , ..., ϑ̃j, ...., ϑ̃l } , ϑ̃j = φ̃j( +ρ̄(x+y)) ρ ( aj−φ̃j ∑ni=j+ ai ) , φ̃j = rgd dj − , ≤ j ≤ l, rgd dj is the targeted rate for j-th mu, fimbsm,gd d (x) and fisbsm,gd d (y) are the pdfs of interferences imbsm,gd d and isbsm,gd d, respectively. similarly, based on equation ( ), sinr after applying sic at l-th dr in noma-based gd d network, denoted as Γgd dl , can be expressed as follows: Γgd dl = h̃l ãl ρl h̃l ρl ∑ l i=l+ ãl + ρ̄ ( imbsl,gd d +i sbs l,gd d ) + , ( ) where ρl = pgt σ . now based on equation ( ), the outage probability at l-th dr, denoted as pgd dl , can be expressed as: electronics , , of pgd dl = ∫ ∞ ∫ ∞ pscstm |τmaxm =ϑ̂maxl fimbsl,gd d (x) fisbsl,gd d (y) dxdy, ( ) where ϑ̂maxl = max { ϑ̂ , ..., ϑ̂j, ...., ϑ̂l } , ϑ̂j = φ̂j( +ρ̄(x+y)) ρ ( aj−φ̂j ∑ni=j+ ai ) , φ̂j = rgd dj − , ≤ j ≤ l, rgd dj is the targeted rate for j-th dr, fimbsl,gd d (x) and fisbsl,gd d (y) are the pdfs of interferences imbsl,gd d and i sbs l,gd d, respectively. this completes the discussion for outage probability in noma-based gd d networks. . . . sum-rate maximization problem in noma-based gd d networks similar to cn scenario, let us denote by fgd d, as the objective function in order to maximize the overall sum-rate of the noma-based gd d network. the function fgd d can be defined as follows: fgd d = m ∑ m= r̃gd dm + l ∑ l= r̃gd dl = m ∑ m= log ( + Γgd dm ) + l ∑ l= log ( + Γgd dl ) , ( ) where r̃gd dm and r̃ gd d l represent the achievable rates for m-th mu and l-th dr, respectively. based on definition of fgd d, the optimization problem can now be formulated as follows: maxfgd d ( ) subject to: c : r gd d m ≤ r̃ gd d m c : r k,mcmt n ≤ r̃ k,scmt n c : m ∑ m= am p ≤ p c : l ∑ l= al pl ≤ pl , c : φ̃j m ∑ i=j+ ai ≤ aj, ≤ j ≤ m c : φ̂j l ∑ i=j+ ai ≤ aj, ≤ j ≤ l c : imbsm,gd d +i gt m,gd d < hm am p, ≤ m ≤ m c : imbsl,gd d +i gt l,gd d < h̃l al pl , ≤ l ≤ l. the constraints c –c in equation ( ) follow similar explanation as the constraints described in optimization equation ( ) for mcmt cn. . . noma applied to wireless sensor networks the current literature is scarce in investigating the application of noma to wsns. the work in [ ] can be regarded as pioneer in investigating the noma-based wsns. by utilizing stochastic geometry tools, the authors proposed and modeled a noma-based ubiquitous wsn in which sensors and sink nodes are considered to be randomly distributed over the entire two-dimensional plane. further, the cross-technology (ct) nodes operating in the same frequency band are also considered to be co-located with the wsn. in order to evaluate the performance of the considered network, electronics , , of the authors derived closed-form expressions for outage probability, diversity, and throughput. in addition, computational complexity analysis is also presented in order to justify the proposal of noma for wsn. the simulation results demonstrate the superior performance of noma-based wsn over conventional wsn utilizing oma for transmission. the authors in [ ] considered a two-hop noma-based wsn with energy harvesting. based on two types of relying, the authors proposed a novel energy harvesting algorithm. in order to evaluate the performance, closed-form expressions for outage probability and ergodic rate are derived. numerical results demonstrate that the proposed scheme is able to achieve significantly lower outage probability and superior rate compared to the existing techniques. furthermore, noma-based wsn is considered in [ ] with time switching energy harvesting technique. closed-form analytical expressions for outage probability and achievable rate are derived. the monte carlo simulations are performed in order to validate the accuracy of the derived results. the application of noma-based wsn for smart agriculture system is investigated in [ ]. the authors considered a relay-aided uplink noma system and characterize the performance by deriving closed-form analytical expressions for outage probability and average sum-rate. the simulation results demonstrate that the proposed relay-aided noma-based scheme achieves superior performance over the conventional oma technique for wsn in agriculture. the authors in [ ] considered a wsn based on noma, where time-switching and power-splitting based relaying protocols are proposed in order to deploy energy harvesting. further, closed-form expression for outage probability and throughput are derived to characterize the network performance. in addition, an optimization problem is formulated for maximizing the data rate of the entire system. simulation results reveal that the performance of considered improves significantly under the two proposed algorithms. in [ ], the authors considered noma-based frequency hopping ad hoc network, where different users are grouped together to form noma clusters. the authors applied stochastic geometry tools to analyze the performance of considered network and derived closed-form expressions for coverage probability and average sum-rate. numerical results demonstrate the accuracy of the derived analytical expressions. in addition, the results also reveal that the by choosing appropriate cluster radius, number of frequency points, user power allocation, and cluster density could enhance the coverage probability of the considered network. . . . noma-based wsns consider a wsn as shown in figure , where sensors and sink nodes are randomly distributed across the entire two-dimensional plane. since, wsns generally operate in license a free band, therefore, there are other cross-technology (ct) nodes co-exist in the considered wsn, as depicted in the figure . further, assume that each sink node is located at the center of a disc with radius rsink, modeling the coverage of the sink node. moreover, the sink node is communicating with multiple sensor nodes by applying noma transmissions. in addition, let there are total of u sensor nodes distributed randomly inside the coverage of each sink node. furthermore, for analysis purposes, we consider a representative sink node. without loss of generality, the sensor nodes are ordered as ĥ ≤ ... ≤ ĥu , where ĥu = ĝu d̂αu , ≤ u ≤ u. here, ĝu and d̂u are the fading (power) and distance between the u-th sensor and representative sink node, respectively. consequently, under this network arrangement, there would be an interference at typical u-th sensor node in the coverage of representative sink node from noma transmissions of other sink and ct nodes. note that typically cns operate in a licensed spectrum and hence there would be no interference from cn to the sensors and sinks in a wsn. as a result, the received power at the u-th sensor, denoted by pwsnu , can be written as follows: pgd dm = ĥu au psink︸ ︷︷ ︸ useful signal power + intra-user interference︷ ︸︸ ︷ ĥu u ∑ i= i =u ai psink +isinku,wsn +i ct u,wsn + σ , ( ) electronics , , of where psink is the transmission power of sink node, isinku,wsn and i ct u,wsn represent the interference at the u-th sensor node from other sink nodes and ct nodes, respectively. rsink sink node sensor node ct node figure . noma-based wireless sensor network (wsn). . . . outage in noma-based wsns this subsection expresses the outage probability at the u-th sensor node in the considered noma-based wsn. after applying sic at the u-th sensor node, the sinr denoted as Γwsnu , can be written using equation ( ) as follows: Γwsnu = ĥu au ρu ĥu ρu ∑ui=u+ ai + ρ̄ ( isinku,wsn +i ct u,wsn ) + , ( ) where ρu = psink σ . next, following similar procedure to deriving equation ( ), the outage probability at u-th sensor node, denoted by pwsnu , can be expressed as follows: pwsnu = ∫ ∞ ∫ ∞ pscstm |τmaxm =ϑ̌maxu fisinku,wsn (x) fictu,wsn (y) dxdy, ( ) where ϑ̌maxu = max { ϑ̌ , ..., ϑ̌j, ...., ϑ̌u } , ϑ̌j = φ̌j( +ρ̄(x+y)) ρ ( aj−φ̌j ∑ui=j+ ai ) , φ̌j = rwsnj − , ≤ j ≤ u, rwsnj is the targeted rate for j-th sn, fisinku,wsn (x) and fictu,wsn (y) are the pdfs of interferences isinkl,wsn and i ct l,wsn, respectively. this completes the discussion for outage probability in noma-based wsns. . . . sum-rate maximization problem in noma-based wsns in order to maximize the sum-rate in noma-based wsn, let us consider an objective function, denoted by fwsn, and defined as: fwsn = u ∑ u= r̃wsnu = log ( + Γwsnu ) , ( ) electronics , , of where r̃wsnu is the achievable rate of u-th sensor node. now, based on fwsn in equation ( ), the optimization problem can be formulated as follows: maxfwsn ( ) subject to: c : r wsn u ≤ r̃ wsn u c : u ∑ u= au pu ≤ pu c : ϕ̌j u ∑ i=j+ ai ≤ aj, ≤ j ≤ u c : isinku,wsn +i ct u,wsn < ĥu au pu, ≤ u ≤ u. note that the constraints in problem equation ( ) can be explained in a similar manner as were described in equation ( ) for mcst cn. . research challenges and future trends the prior arts on noma have extensively investigated many noma specific issues and its integration with several communication technologies. however, there are still multiple unresolved issues which need to be addressed in order to further enhance the performance of noma systems. as such, this section discusses some key design challenges for noma and indicate potential research directions to solve theses issues. . . alternate receiver for noma in current literature, noma has been extensively investigated with the sic receiver. while carrying out theoretical analysis of noma systems with sic, it has been very commonly assumed that all the higher order users are perfectly removed at say m-th user receiver i.e., perfect cancellation is assumed. however, this assumption does not hold generally, rather in practical situations the cancellation is mostly imperfect (there is some residual interference left). therefore, there is a need to consider this imperfect cancellation aspect in theoretical analysis of noma-based wireless systems. in addition, the performance of sic technique is sensitive to power levels among users. this implies that appropriate rate and power allocation noma with sic would be required and failing to comply with this condition would always result in complete outage [ ]. furthermore, there is a problem of error propagation in sic. this means that whenever any of the higher-order user has been decoded incorrectly, the error will sequentially propagate to decoding of subsequent lower-order users [ ]. as a consequence, these limitations of sic receiver limit the noma functionality. therefore, in order to resolve the aforementioned sic related issues, either existing sic design could be improved or an alternate receiver for noma can be considered. the alternate receiver design here means to implement a receiver which does not utilize successive cancellation procedure for multiuser detection. one promising approach is to apply machine learning techniques in order to obtain predictions of noma users and then utilize these estimates to aid sic decoding. another potential solution is to consider parallel interference cancellation (pic) technique, message passing algorithm [ ], and deep learning methods to design an alternate receiver for noma. . . hybrid multiple access noma systems apply sc and are hence inherently interference limited due to the presence of intra-user interference. as such, the performance gain of noma over conventional oma is limited in the lower snr regime [ ]. consequently, the noma scheme in its pure form appears to be not very suitable for wireless systems that are operating at low snrs (iot, d d, m m communications, electronics , , of and so on). in order to resolve this problem, a hybrid ma based on combination of noma and oma could be designed, which is capable of switching between noma and oma operation modes under conditions of optimizing overall system throughput. . . consideration of imperfect csi the existing literature on noma commonly assumes a perfect csi order to either perform resource allocation at bs or suppress multiuser interference at the user receiver. however, this assumption is very strong as perfect csi is not possible to obtain in practical noma systems. consequently, real-time noma networks operate under channel estimation errors. as such, it is required to consider imperfect csi and channel estimation errors in theoretical analysis of noma systems. some attempts are made in existing literature in order to account the impact of channel estimation errors in theoretical analysis [ – ]. nevertheless, the upcoming g systems are envisioned to support a massive number of users, which in turn could increase inter-user interference, resulting in enhanced channel estimation errors. therefore, more sophisticated technique and algorithms are required in order to obtain accurate channel estimation in practical noma systems. . . noma-based data offloading in g networks the upcoming g wireless systems are anticipated to operate in a hyper-connected environment, where a massive number of connected devices will generate enormous amount of data on the backbone network. this poses a great challenge for network operators in order to store, process, and transmit this big data. a typical example of such a scenario could be a congested macro-cell. one promising solution to solve this problem is to offload data. for example, a mbs in a congested macro-cell can offload data to sbss or to wi-fi networks. however, utilizing conventional oma based offloading would result in increased latency, particularly under scenarios of huge data size and sources. consequently, this situation may not be feasible particularly for delay-sensitive applications. as such, by virtue of sc, noma has a potential to efficiently offload huge amounts of data and users simultaneously. therefore, it is required to design advance-offloading algorithms based on noma to resolve the problem of tackling big data in upcoming g wireless networks. . . noma-aided full duplex networks the fd duplex technique has a potential to enhance the spectral efficiency by simultaneously transmitting and receiving over the same frequency channel. in order to meet the ambitious demands of g wireless systems, the integration of noma with fd communication could be a promising solution to enhance overall system capacity. in current literature, some attempts are made to analyze noma-based fd networks, see [ – ] for quick reference. however, fd and noma systems are limited by self-interference and intra-user interference, respectively, which may degrade the system performance. consequently, an efficient low-complexity receiver design would be required to realize noma-aided fd networks. furthermore, advanced resource and power allocation algorithms would be required which could result in minimizing self-interference and intra-user interference and maximizing the overall sum-rate of the system. therefore, investigating noma-aided fd networks and its related issues could be a promising future research direction. . conclusions this survey has described the fundamental concept and potential benefits of noma protocol, which has been admitted as a new member of ma schemes. more precisely, the application of noma to cns, d d communications, and wsns has been reviewed and discussed in detail. moreover, outage probability expressions are presented and sum-rate maximization problems are formulated for each noma-based wireless network. the presented analytical expressions for a given noma-aided system (cn, d d, wsn) can be easily tailored based on the specific assumptions related to the spatial topology of the network. furthermore, this study also highlighted some of the key noma-related design issues electronics , , of along with some potential future research directions. it is strongly believed and anticipated that noma 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conference (vtc spring), seoul, korea, – may ; pp. – . . tan, y.; zhou, j.; qin, j. novel channel estimation for non-orthogonal multiple access systems. ieee signal process. lett. , , – . . struminsky, k.; kruglik, s.; vetrov, d.; oseledets, i. a new approach for sparse bayesian channel estimation in scma uplink systems. in proceedings of the ieee international conference on wireless communications & signal processing (wcsp), yangzhou, china, – october ; pp. – . https://doi.org/ . /vtcspring. . https://doi.org/ . /tvt. . https://doi.org/ . /access. . https://doi.org/ . /access. . https://doi.org/ . /tcomm. . https://doi.org/ . /access. . https://doi.org/ . /access. . https://doi.org/ . /hpsr. . https://doi.org/ . /healthcom. . https://doi.org/ . /access. . https://doi.org/ . /access. . electronics , , of . ding, z.; fan, p.; poor, h.v. on the coexistence between full-duplex and noma. ieee wirel. commun. lett. , , – . . sun, y.; ng, d.w.k.; ding, z.; schober, r. optimal joint power and subcarrier allocation for full-duplex multicarrier non-orthogonal multiple access systems. ieee trans. commun. , , – . . sun, y.; ng, d.w.k.; zhu, j.; schober, r. robust and secure resource allocation for full-duplex miso multicarrier noma systems. ieee trans. commun. , , – . . abbasi, o.; ebrahimi, a. secrecy analysis of a noma system with full duplex and half duplex relay. in proceedings of the ieee iran workshop on communication and information theory (iwcit), tehran, iran, – may ; pp. – . . lei, l.; lagunas, e.; chatzinotas, s.; ottersten, b. noma aided interference management for full-duplex self-backhauling hetnets. ieee commun. lett. , , – . c© by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	basic concepts of noma 	the principle of superposition coding 	successive interference cancellation scheme 	noma scheme with sic 	application of noma to wireless networks 	noma applied to cellular networks 	noma in single-cell single-tier cellular networks 	noma in single-cell multi-tier cellular networks 	noma in multi-cell single-tier cellular network 	noma in multi-cell multi-tier cellular networks 	noma applied to device-to-device communications 	noma-based group d d communications 	outage in noma-based group d d network 	sum-rate maximization problem in noma-based gd d networks 	noma applied to wireless sensor networks 	noma-based wsns 	outage in noma-based wsns 	sum-rate maximization problem in noma-based wsns 	research challenges and future trends 	alternate receiver for noma 	hybrid multiple access 	consideration of imperfect csi 	noma-based data offloading in g networks 	noma-aided full duplex networks 	conclusions 	references this is the accepted manuscript made available via chorus. the article has been published as: similarity of the signatures of the initial stages of phase separation in metastable and unstable polymer blends amish j. patel, timothy j. rappl, and nitash p. balsara phys. rev. lett. , — published january doi: . /physrevlett. . http://dx.doi.org/ . /physrevlett. . similarity of the signatures of the initial stages of phase separation in metastable and unstable polymer blends amish j. patel, timothy j. rappl, and nitash p. balsara , , ∗ department of chemical engineering, university of california, berkeley, california materials sciences division and environmental energy technologies division, lawrence berkeley national laboratory, university of california, berkeley, california time-resolved small angle neutron scattering was used to probe the initial stages of liquid-liquid phase separation in both, critical and off-critical binary polymer blends and the critical (qc) and most probable (qm) wave-vectors were identified for several quench depths. for the critical blend, the cahn-hilliard-cook theory provides a framework for analyzing the data and explains the observed decrease in qm with time. for the off-critical blend, qm is independent of quench time, regardless of whether the quench is metastable or unstable. pacs numbers: . .va, . .q-, . .nh, . .-g the standard phase diagram of binary liquid mixtures contains two curves: the binodal curve which is the lo- cus of the compositions of the coexisting phases and the spinodal curve which is the thermodynamic limit of the stability of the homogeneous phase. according to classi- cal treatments, the transformation of a homogeneous bi- nary liquid into a phase separated mixture after a quench from the single phase region of the phase diagram to the two-phase region occurs by two mechanisms: nucleation and growth, which occurs if the quench lies between the binodal and the spinodal curves (the metastable region), and spinodal decomposition if the quench lies within the spinodal curve (the unstable region) [ – ]. however, sub- sequent theoretical treatments that have examined the crossover from nucleation to spinodal decomposition sug- gest that the situation may be more complex [ , ]. in this letter, we present time-resolved small angle neu- tron scattering (sans) data from two polymer blends that are quenched from the one-phase to the two-phase region: a critical blend that is quenched directly into the unstable region and an off-critical blend quenched to various quench depths, both in the metastable and the unstable regions. the data were obtained during the ini- tial stages of phase separation, before coarsening sets in. sans results from both the blends have been presented in two separate papers [ , ]. the main purpose of this letter is to combine the data presented in these two pa- pers to illustrate that: ) for the off-critical blend, there are no qualitative differences between quenches in the metastable and unstable regions. ) quenches for the critical and off-critical blends display differences in the time evolution of the most probable wave-vector. this raises questions concerning the role of the spinodal in demarcating distinct phase separation mechanisms. both polymer blends are made up of high molecular weight liquid polyolefins: deuterated polymethylbutylene (dpmb) and hydrogenous polyethylbutylene (hpeb). the methods used to synthesize and characterize these nearly-monodisperse homopolymers are described in ref. [ ]. the weight-average molecular weights, mw, of the polymers that constitute the off-critical blend are kg/mol (dpmb) and kg/mol (hpeb). the radii of gyration, rg, of both chains are . ± . nm. the re- sults reported here are for a blend with dpmb volume fraction, φdpmb = . . for the critical blend, mw of the polymers are kg/mol (dpmb) and kg/mol (hpeb) and rg of both chains are . ± . nm. the critical composition, based on the flory-huggins theory [ , ], is φdpmb = . . all polymers are highly entan- gled, resulting in extremely slow phase separation, which can be tracked by time-resolved sans. the azimuthally averaged coherent scattering inten- sity, i, as a function of the magnitude of the scattering vector, q, was obtained by methods reported in ref. [ ]. static sans enabled the thermodynamic characteriza- tion of our system, while time-resolved sans enabled the study of the early stages of phase separation. we use the flory-huggins theory to quantify the thermodynamic properties of our blends. the t and p dependence of the flory-huggins interaction parameter, χ, for the system of interest and the phase diagrams of the blends used in this paper have been reported previously [ – ]. we define the quench depth, κ(t,p) = χ(t,p)/χb − , where χb is the value of χ at the binodal. in table , we list the final t and p of the quenches as well as the corresponding κ-values, for both critical and off-critical blends. table i: experimental conditions and quench depths t(◦c) p(kbar) κ t(◦c) p(kbar) κ critical blend off-critical blend . . . . . . . . . . . . . . . . off-critical blend . . . . . . . . . . . . . . in figure , we show the calculated binodal curves for both the critical and off-critical blends, as well as fig. : binodal curves for the critical (in red) as well as the off-critical (in blue) blend are shown along with the locations of the various quenches studied. inset shows the full phase diagram with both the binodal and the spinodal curves. the locations of the quenches that were investigated. [n = √ n n and φc is the critical composition; ni is the number of monomers per chain for each polymer, based on a . nm reference volume.] the locations of the quenches relative to the mean-field spinodal curves are shown in the inset of figure . typical data obtained from the off-critical blend are shown in fig. a, where we show the sans profiles dur- ing a quench from the one-phase region to . kbar and ◦c, which lies in the metastable region of the phase diagram. the arrow shows the location of the critical wave-vector, qc. the sans profiles are characterized by a rapid increase in i(t) for q-values smaller than qc as well as the appearance of a peak in i(q). in contrast, i(q > qc) does not change with time in the early stage of phase separation. we have argued that the size of the critical nucleus in the nucleation and growth regime is of order /qc [ ]. in fig. b, we show sans profiles for the off-critical blend during a quench to . kbar and ◦c, which lies in the unstable region of the phase diagram. there is no qualitative difference between the sans profiles resulting from quenches in the metastable and unstable regions (compare figures a and b). in the inset of fig. b, we show the sans profiles obtained from the critical blend during an unstable quench to . kbar and ◦c. the arrow shows the location of the critical wave-vector, qc, obtained by well-established methods for analyzing data during spinodal decomposition [ ]. data similar to those shown in figure were obtained from the critical and off-critical blends at various quench depths by controlling the final t and p . in fig. a, we show the position of the peak in i(q), qm, as a function of quench time for several quenches using the off-critical blend. there is no change (within experimental uncertainty) in qm as a function of quench time for quenches into the metastable as well as the un- stable regions. the kinetics of phase separation of the fig. : time-resolved sans intensity, i vs q measured for the off-critical blend during the (a) . kbar (metastable) quench, and the (b) . kbar (unstable) quench (shown at t = , , , , and min). inset shows i(q) for the critical blend during the . kbar quench. the arrows indicate the location of the critical wave-vector, qc. off-critical blend is thus governed by two characteristic wave vectors, qc and qp; we take qp to be the average value of qm for each of the quenches shown in fig. a. quenches of the critical blend into the unstable region of the phase diagram also result in scattering profiles with peaks. however, in this case, qm decreases monotonically with time as shown in fig. b. the cahn-hilliard-cook (chc) theory predicts the time evolution of the scattering profile of unstable sys- tems [ – ]: i(q,t) = it (q)+[i (q)−it (q)] exp[ r(q)t]. in ref. [ ], we showed that our measurements are in excellent agreement with the chc equation, enabling the determination of i (q), it (q), and r(q) for each of the quenches. this provides the basis for under- standing the time evolution of qm. at short times, i(q,t) ≈ i (q) + r(q)[i (q) − it (q)]t. hence, we ex- pect a peak in i(q) to emerge at the value of q, for which the initial rate of increase of i(q,t), denoted by j(q) = r(q)[i (q) − it (q)] is maximum. while it (q) has a pole at q = qc, r(qc) = and j(q) is a contin- fig. : sans peak position, qm, as a function of quench time for (a) off-critical and (b) critical blends. solid curves are single-parameter exponential fits to the data with τ = , , and min for κ = . , . , . and . respectively. the typical uncertainty in qm is also shown. inset shows the initial rate of increase in the scattering intensity, j(q), for the . kbar quench. the arrow indicates the location of qc. uous function of q. the pole at q = qc suggests the possibility of a maximum in j(q) in the vicinity of qc. this is indeed the case for all the quenches studied. we show j(q) for the . kbar quench in the inset of fig. b. there is good agreement between the location of the maximum in j(q) and qc, shown by an arrow. at long times, the exponent in the chc equation governs the behavior of i(q), resulting in a peak at a q-value, cor- responding to the maximum in r(q), which we call qp. thus qm(t = ) ≈ qc and qm(t →∞) = qp. the simplest function that captures the evolution of qm(t) from qc to qp is: qm(t) = qp + (qc − qp) exp(−t/τ). this functional form is used to fit the data from the critical blend using τ as the only adjustable parameter, and the fits are in good agreement with the data (fig. b). as was the case for the off-critical blend, phase separation kinetics of the critical blend are also governed by two character- istic scattering vectors, qc and qp. while the methodolo- gies for determining qc and qp in critical and off-critical blends are different, the physical significance of the wave- vectors is the same: /qc represents the length scale of the smallest structures that grow during phase separation while /qp represents the length scale that dominates the phase separated structure formed during the early stages. thus, the chc theory provides a framework for an- alyzing i(q,t) from critical blends, and explains the de- crease in qm. however, there is no framework for analyz- ing time-resolved scattering data from nucleating blends. while we do not have a theoretical basis for the time in- variance of qm for off-critical quenches, a possible expla- nation might be that qm represents the average distance between nucleating centers that does not change as the nuclei grow during the early stages of phase separation. in figures a and b, we plot qprg and qcrg as a function of the quench depth, κ, for the critical and off- critical blends. the dependences of both characteristic wave-vectors on quench depth are similar for the crit- ical and off-critical blend except for the fact that the off-critical data are shifted to the right along the κ-axis. fig. : dimensionless characteristic wave-vectors, (a) qm, and (b) qc as a function of the quench depth, (χ − χb)/χb for the critical (open squares) and off-critical (filled circles) blends. the insets show qm and qc as a function of an alterna- tive definition of quench depth (χ−χb)/χs. the dashed lines mark the position of the spinodal for the off-critical blend. typical uncertainties are also shown. the shift is larger in the qprg versus κ plot. in the in- sets of figures a and b, we show the same data plotted versus κs = (χ−χb)/χs, where χs is the value of χ at the spinodal. the distinction between critical and off-critical systems is significantly reduced when κs is used to define quench depth. the data in figures and indicate that the quali- tative features of the scattering profiles obtained during the initial stages of phase separation are independent of quench depth. while the spinodal may help in organiz- ing the data as we have shown in the insets of fig. , it is does not demarcate different mechanistic regimes. theoretical work of binder and stauffer [ ] showed that the characteristic length-scale of phase separation in mix- tures of low molecular weight compounds was unaffected by the presence of the spinodal. it was argued that the spinodal, a mean-field concept, is destroyed by concen- tration fluctuations. anticipation that the spinodal curve and mean-field behavior would be recovered in polymer blends was based on the scaling arguments of de gennes, who showed that the ginzburg number, gi, which quan- tifies the importance of fluctuations, follows a gi ∼ /n scaling law [ ]. more refined calculations by wang [ ] indicate that while this scaling law is correct in the large n limit, it is not obeyed for blends with n < . for gi to be significantly smaller than unity (say . ), the values of n required are of order for φ = . , corre- sponding to component mw ∼ kg/mol. while exper- iments on such large systems may one day be carried out, they represent a small portion of parameter space with virtually no practical significance. there are thus com- pelling reasons for eliminating the spinodal curve from the reported phase diagrams of binary mixtures that un- dergo liquid-liquid phase separation as we have done in figure . additionally, theoretical studies that improve upon the cahn-hilliard treatment by using a non-linear lo- cal free energy [ , ] report that there is no change in the mechanism of phase separation on crossing the spin- odal. novick-cohen uses a quartic free energy expression and reports that a parameter b, which is related to the higher derivatives of the free energy, governs the mecha- nism of phase separation [ ]. the non-linear theory pre- dicts a crossover inside the spinodal from a nucleation- like mechanism near the spinodal (b >> ) to clas- sical spinodal decomposition, deep within the spinodal (b << ). it can be shown that for a flory-huggins blend, b ≈ . √ (χs −χb)/|χ−χs|. for the critical blend, χb = χs =⇒ b = and thus classical spin- odal decomposition is observed. for the off-critical blend studied in this letter, the lowest value of b was . , i.e. the b << criterion was never reached and thus only the nucleation-like mechanism was observed. in conclusion, we have found similarities in the time- resolved scattering signatures of the initial stages of phase separation, with peaks observed in i(q) for all quenches and for both the critical and the off-critical polymer blends (fig. ). however, there are subtle dif- ferences, specifically in the evolution of qm with quench time (fig. ). for critical quenches, the chc theory ex- plains the observed decrease in qm with time. in contrast, for the off-critical blend, qm is independent of quench time, regardless of whether the quench is metastable or unstable. the difference between critical and off-critical quenches as well as the similarities between metastable and unstable off-critical quenches can be explained in the context of theories that incorporate non-linear effects into the cahn-hilliard analysis. however, a theoretical frame- work for describing i(q,t) for nucleating blends, akin to the chc theory for critical quenches, is still missing. we hope that the data in figures and will guide the de- velopment of such a framework. we acknowledge the national science foundation (nsf, grant no. cbet and dmr- ), and tyco electronics for financial support, the national institute of standards and technology, u.s. department of commerce, for providing the neutron research facilities used in this work (nsf, dmr- ), and boualem hammouda for his guidance. ∗ electronic address: nbalsara@berkeley.edu [ ] j. w. gibbs, the scientific papers of j. willard gibbs (dover, new york, ). 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[ ] z.-g. wang, j. chem. phys. , ( ). original article cntnap gene dosage variation is associated with schizophrenia and epilepsy ji friedman , , t vrijenhoek , , s markx , im janssen , wa van der vliet , bhw faas , nv knoers , w cahn , , rs kahn , , l edelmann , kl davis , jm silverman , hg brunner , a geurts van kessel , c wijmenga , , ra ophoff , and ja veltman departments of psychiatry and human genetics, mount sinai school of medicine, new york, ny, usa; department of human genetics, nijmegen centre for molecular life sciences, radboud university nijmegen medical centre, nijmegen, the netherlands; department of psychiatry, columbia university, new york, ny, usa; department of psychiatry, utrecht university, utrecht, the netherlands; rudolf magnus institute of neuroscience, utrecht university, utrecht, the netherlands; department of medical genetics, utrecht university, utrecht, the netherlands and department of genetics, university medical centre groningen, groningen, the netherlands a homozygous mutation of the cntnap gene has been associated with a syndrome of focal epilepsy, mental retardation, language regression and other neuropsychiatric problems in children of the old order amish community. here we report genomic rearrangements resulting in haploinsufficiency of the cntnap gene in association with epilepsy and schizophrenia. genomic deletions of varying sizes affecting the cntnap gene were identified in three non- related caucasian patients. in contrast, we did not observe any dosage variation for this gene in healthy controls. moreover, this genomic region has not been identified as showing large-scale copy number variation. our data thus confirm an association of cntnap to epilepsy outside the old order amish population and suggest that dosage alteration of this gene may lead to a complex phenotype of schizophrenia, epilepsy and cognitive impairment. molecular psychiatry ( ) , – ; doi: . /sj.mp. ; published online july keywords: epilepsy; schizophrenia; cntnap ; copy number variation introduction investigations of complex brain disorders such as schizophrenia (mim ) and epilepsy have demonstrated considerable heterogeneity of the ge- netic and pathophysiological components of these illnesses. moreover, the phenotypic manifestations of schizophrenia are numerous and variable, and may include delusions, disordered thought, hallucina- tions, negative symptoms, and cognitive and func- tional impairment. in addition, many distinct epilepsy syndromes do exist, which are defined by seizure characteristics, location and age of onset. these factors have complicated the identification of genes and their products that serve as vulnerability factors for these disorders. linkage and association studies have yielded several candidate genes for schizophrenia , and epilepsy, but despite these successes the genetic mechanisms underlying the comprehensive pathophysiology of these brain dis- orders remain poorly understood. technical developments in microarray-based gen- ome profiling have facilitated the routine genome- wide detection of genomic copy number variation (cnv) with a resolution of up to b kb. , as a result, subtle cnvs have been linked to the patho- genesis of a number of diseases, ranging from rare monogenic to complex disorders. – moreover, lim- ited evidence suggests a role for cnvs in the susceptibility to psychiatric disease such as schizo- phrenia. in the current study, we used both genome-wide and targeted genomic approaches to investigate cnvs in patients with epilepsy and schizophrenia. these approaches revealed the presence of unique cnvs on chromosome q – . in three unrelated patients with epilepsy and schizophrenia, all affecting the cntnap gene. cntnap variation has previously been associated with epilepsy, but not with schizo- phrenia. cntnap encodes caspr (contactin-asso- ciated protein ), a member of the neurexin superfamily, a group of transmembrane proteins that mediate cell–cell interactions in the nervous system. caspr is predominantly expressed in the nervous system in the juxtaparanodal region of axons of myelinated neurons and in oligodendrocytes. received april ; revised june ; accepted june ; published online july correspondence: dr ja veltman, department of human genetics, nijmegen center for molecular life sciences, radboud university nijmegen medical center, geert grooteplein , po box , hb nijmegen, the netherlands. e-mail: j.veltman@antrg.umcn.nl these authors contributed equally to this work. molecular psychiatry ( ) , – & nature publishing group all rights reserved - / $ . www.nature.com/mp http://dx.doi.org/ . /sj.mp. mailto:j.veltman@antrg.umcn.nl http://www.nature.com/mp caspr is essential for the localization of voltage- activated kþ channels in the juxtaparanodal region of axons, which may function to stabilize conduction and help to maintain the intermodal resting poten- tial. targeted disruption of caspr results in marked reduction in the accumulation of kþ channels at the juxtaparanodes in both central and peripheral nervous systems. this is the first study demonstrating an association between the cntnap gene and schizophrenia. moreover, this study is the first to demonstrate the presence of dosage variations at this gene, resulting in both epilepsy and schizophrenia. materials and methods patient material genomic dna from ethylenediaminetetraacetic acid venous blood was obtained from three independent sources. patient was brought to the diagnostics department at the radboud university nÿmegen medical center for routine karyotyping with a clinical presentation of severe mental retardation, epilepsy and language development problems among other findings (see table for details). patient , who presented with schizophrenia and epilepsy, was identified from a cohort of patients with syndro- mal schizophrenia (for example schizophrenia ac- companied by numerous minor physical anomalies, mental retardation, seizure disorder and other med- ical problems) recruited from pilgrim psychiatric center, w brentwood, ny, usa, and referred to the department of human genetics, radboud university nijmegen medical centre for array-based comparative genomic hybridization (array cgh) analysis. patient was identified from a cohort of schizophrenic patients recruited at the department of psychiatry, utrecht university, the netherlands for targeted single nucleotide polymorphism (snp) array analysis. these were analyzed together with healthy individuals of dutch origin as a control set. the cohorts were collected following institutional review board ap- proval and after obtaining signed informed consent. array-based comparative genomic hybridization array cgh was performed as described previously using a tiling-resolution bac array. all data were mapped on the national center for biotechnology information (ncbi) build of the ucsc genome browser hg (http://genome.ucsc.edu/). singe nucleotide polymorphism assay to assess genetic variation in the genomic region around cntnap , we developed a targeted snp assay for the illumina goldengate bead array (illumina inc., san diego, ca, usa). the assay consisted of haplotype-tagging snps in the cntnap locus (chromosome : – ; ncbi build of the ucsc genome browser hg (http://genome.ucsc.edu/); average spa- cing between two snps was kb). we performed a straightforward w test for association of individual snps with schizophrenia. we also checked for copy number changes by simultaneous measurement of both signal intensity variations (log r) and changes in allelic composition. multiplex ligation-dependent probe amplification to confirm the microarray analysis, samples were screened with multiplex ligation-dependent probe amplification (mlpa), essentially as described pre- viously. mlpa probes were designed for exons , , , , , (one probe for each exon) and intron of cntnap (three different probes), and for exon of pdia and exon of nos (one probe for each exon of both). sequences of test and control probes are in supplementary table s . probes were developed in accordance with a protocol provided by mrc-holland (amsterdam, the netherlands). ten probes were combined in one mlpa assay, in combination with three standard control probes. equal amounts of probe mix and hybridization buffer were added to – ng of dna from each sample, followed by heat denaturation and overnight incubation at c. ligation products were amplified by regular pcr with the use of a -fam-labeled primer set. amplification products were identified and quantified by capillary electrophoresis on an abi genetic analyzer (applied biosystems, foster city, ca, usa). data were normalized by dividing each probe’s signal strength by the average signal strength of the sample. this normalized peak pattern was divided by the average peak pattern of all samples in the same experiment. values for control wild-type peaks were centered at . . a copy number loss was defined as that below a fixed threshold value of . . fluorescent in situ hybridization analysis fluorescent in situ hybridization (fish) experiments were performed for de novo checking on metaphase spreads prepared from patient-derived lymphoblast cell lines using routine procedures. probe labeling, slide preparation and hybridization were carried out as described elsewhere, using probes listed in supplementary table s . sequence analysis to sequence all exons of the cntnap gene, we designed primers in the intronic region surrounding each exon using primer , and performed standard pcr reactions. primer sequences and conditions are in supplementary table s . results we identified a hemizygous deletion involving chro- mosome region q – q . in patient by routine chromosome analysis (data not shown). patient is a severely mentally retarded -year-old caucasian male born to healthy non-consanguineous parents. no other family members were reported to have mental retardation or epilepsy. at the age of , the cntnap deletions ji friedman et al molecular psychiatry http://genome.ucsc.edu/ http://genome.ucsc.edu/ patient suffered from seizures, first noticed by the parents and later confirmed by electroencephalogram (eeg). anticonvulsant medication (carbamazepine) was initiated and has remained necessary until today (table ). we confirmed this deletion by using both fish and mlpa (figure b). fish analysis of both parents failed to reveal any chromosome aberra- tions, indicating that the deletion occurred de novo in the patient. we also used tiling-resolution genome- wide array cgh to characterize the deletion in detail (figure a). by doing so, we found this deletion to be . mb in size and to encompass known genes, of which only ephb , ezh and cntnap are ex- pressed in the brain. we identified a second hemizygous deletion invol- ving cntnap in patient by tiling-resolution array cgh. this . mb deletion was identified at the q – q . locus and was found to encompass four genes: cul , ezh , pdia and cntnap (figure c). the microdeletion was confirmed by mlpa analysis (figure b). the patient is a -year-old caucasian female diagnosed with schizophrenia (age of on- set = ), epilepsy and severe cognitive impairment as indicated by a wais iii full-scale intelligence quotient (iq) = (table ). she completed years of mainstream education and was assessed to have a pre- morbid iq = (derived from wrat-r reading score), indicating a significant decrement in cognitive ability subsequent to the onset of schizophrenia. she also experienced a significant deterioration in communi- cative and self-care abilities requiring prolonged institutionalization. her mother and both of the table overview of clinical characteristics of the three patients with a deletion in chromosome q – q . patient patient patient deletion size mb . mb kb number of genes inheritance de novo unknown unknown; healthy sister does not show deletion patient details gender male female female year of birth ethnicity caucasian caucasian caucasian status at birth premature birth, severe respiratory problems, neonatal sepsis normal premature birth, dizygotic twin, severe respiratory problems family history schizophrenia — — aunt with bipolar disorder epilepsy — — father’s cousin with epilepsy physical illness — mother and two maternal aunts with chronic severe mental illness epilepsy age of first seizure type focal right frontotemporal epilepsia with secondary generalization general tonic-clonic seizure brainstem regulation disorder eeg slow theta/delta dysrythmic theta/delta not determined therapy carbamazepine dilantin none schizophrenia age of onset could not be determined type paranoid paranoid other features speech almost absent deteriorated normal cognitive abilities severe mental retardation progressive cognitive deterioration, iq = at ascertainment normal iq additional features severely delayed psychomotor development difficulties in communication autistic features mild cerebral atrophy abbreviations: eeg, electroencephalogram; iq, intelligence quotient. cntnap deletions ji friedman et al molecular psychiatry mother’s siblings suffered from unspecified chronic psychiatric illnesses requiring prolonged institutio- nalization. all three of this patient’s male siblings were free from psychiatric illness. at age , the patient experienced her first and only-documented seizure, characterized as generalized tonic-clonic. her eeg ( channel) demonstrated slow dysrythmia in theta and occasional delta, predominantly in the frontal area (table ). the above results prompted us to assess further genetic variation in the minimal deleted region using a targeted snp assay. we genotyped the cohort of patients with schizophrenia and a cohort of dutch control subjects for haplotype-tagging snps in the cntnap locus. the w test for association revealed no snp to be significantly (p < . ) associated with schizophrenia. however, a detailed copy number analysis of all data revealed a hemizygous deletion involving cntnap in one patient from the cohort of schizophrenic patients (patient ), represented by consecutive homo- zygous snps with an average log r-value of � . (figure a). subsequent mlpa analysis confirmed the deletion, which encompasses kb and affects only intron of the cntnap gene (figure b). the patient is a -year-old caucasian female diagnosed figure genomic deletions of the cntnap locus in chromosome in three patients. (a) microarray-based chromosome profiles for patients – . the profiles for patients and were obtained from tiling-path resolution genome-wide arrays containing human bac clones, indicated by small circles. these represent the log -transformed and normalized test- over-reference intensity ratios. the clones are ordered from pter – qter on the basis of physical-mapping positions obtained from ncbi build of the ucsc genome browser hg http://genome.ucsc.edu/. the normalized ratios were analyzed for loss and gain regions by a standard hidden markov model, as indicated by the red line. the profiles show the . mb deletion of chromosomal region q – q . in patient , and the . mb deletion of chromosomal region q – q . in patient . the profile for patient was obtained from an array that contained haplotype-tagging snps in the cntnap locus, indicated by small circles. in the upper profile, the circles represent the log -transformed total intensity ratio log r of both variants of an snp. in the lower profile, they represent the b-allele frequency of the snps. the kb deletion in cntnap is represented by a simultaneous decrease in log r and shift from heterozygous to homozygous state of the snps. (b) mlpa confirmation of the deletions in cntnap in all three patients. different probe sets were used to confirm the deletions, which varied in size. (c) schematic overview of the deletions in all three patients. for each patient, the deleted genomic region is shown in red. mlpa, multiplex ligation-dependent probe amplification; snp, single nucleotide polymorphism. cntnap deletions ji friedman et al molecular psychiatry with schizophrenia who suffered from epileptic seizures starting from age . her iq is reported as normal. family history revealed one aunt with bipolar disorder and a paternal cousin with epilepsy (table ). analysis of the healthy dizygotic twin sister of the patient did not reveal any deletion at this locus (figure b). no cnvs larger than kb have been reported in the q – q . chromosomal region, in which all deletions observed in the present study occurred (see the database of genomic variants; http://projects.t- cag.ca/variation/). in addition, we did not observe any deletions in our cohort of healthy controls tested on the targeted snp array, nor in an additional cohort of healthy individuals analyzed by mlpa. more- over, no cnv has been observed at this genomic locus in more than mentally retarded patients and unaffected parental samples tested on tiling-resolu- tion bac arrays in the diagnostics laboratory at the radboud university nijmegen medical centre. se- quence analysis of all exons of the cntnap gene revealed no additional mutations in these three patients. discussion recently, a syndrome characterized by focal epilepsy, macrocephaly, diminished deep tendon reflexes, mental retardation and other neuropsychiatric dis- turbances (designated as cortical dysplasia-focal epilepsy syndrome) was described in association with a homozygous mutation of the cntnap gene in old order amish children. the results of the present study demonstrate an association between dosage variation of the cntnap gene and epilepsy outside the old order amish community. sequence analysis of all exons of the cntnap gene in all three patients revealed no mutation in the remaining allele, indicating that haploinsufficiency of this gene leads to the disease. given that these deletions are not common events nor located in identified regions of high cnv, , these findings are unlikely to be coin- cidental. moreover, unlike the neuropsychiatric pre- sentation suggestive of a pervasive development disorder in the amish children with a homozygous mutation of cntnap , two out of three subjects in the present study presented with a diagnosis of schizophrenia. to our knowledge, this is the first report of an association between the cntnap gene and schizophrenia. however, it is noteworthy that a chromosomal translocation disrupting the cntnap locus seems to result in gilles de la tourette syndrome (mim ) and obsessive compulsive disorder, although the causality of this translocation was recently questioned. cntnap plays a role in the organization of myelinated axons, , and its disruption affects neuroblast migration. interest- ingly, brains of schizophrenic patients show evidence for myelin-related dysfunction and aberrant neuro- nal migration. , this may explain the observed association between cntnap disruptions and sus- ceptibility to schizophrenia. although the three patients in the present study showed moderate clinical overlap, they exhibited differences that may be related to the varying size of the deletion. the deletion in patient affected other genes in addition to cntnap . the more severe phenotype observed in this patient as com- pared to the other two (for example severe mental retardation, multiple epileptic seizures, severely delayed psychomotor development) may be the result of an additive effect of one or more of these other genes. because of the severe mental retardation and inability to communicate, a diagnosis of schizophre- nia could not be determined in this patient. similarly, the deletion in patient encompasses four additional genes, among which are the cullin- (cul ) and enhancer of zeste homolog (ezh ) genes, which are involved in protein ubiquitination and histone methylation, respectively. , although these genes have been reported to be only indirectly involved in brain function, , they might have also contributed to the phenotype of this patient. patient had the smallest deletion encompassing only intron of the cntnap gene, and presented with the least severe phenotype (normal iq, normal speech and higher levels of functioning), which still included epilepsy and schizophrenia. although no exons were affected in the cntnap deletion found in patient , regulatory motifs within this region may have affected the expression of the cntnap gene. alternatively, there are examples of enhancers that act over long distances, affecting other genes than those in which they reside. further functional analysis of this intronic cntnap deletion was not possible in this patient, owing to the restricted pattern of cntnap expression in the nervous system. our study and that of others indicate that rare point mutations and/or dosage variation at the cntnap gene can cause both neurological and psychiatric disorders. examples of other rare muta- tions of genes resulting in variable expression of neurologic and psychiatric disease include mutations in the kcnn gene as well as the syngr gene in schizophrenia, and the association of rare variants of the kcc gene with bipolar disorder. on the other hand, cnv of genes in the q region, which results in velo-cardio-facial syndrome, produces develop- mental neurologic abnormalities and is one of the most common known genetic risk factors for the development of schizophrenia. in conclusion, this study confirms the recently reported association between the cntnap gene and epilepsy in patients outside the old order amish community and demonstrates an association between cntnap and schizophrenia. our results highlight cntnap as a strong candidate gene for epilepsy and schizophrenia. the results further support the notion that cnv is a potentially important source of genetic susceptibility to both complex brain diseases. cntnap deletions ji friedman et al molecular psychiatry http://projects.tcag.ca/variation/ http://projects.tcag.ca/variation/ acknowledgments we thank r van’t slot, e strengman and d lugtenberg for their technical support. this work was supported by grants from the netherlands organization for health research and development (to jav, zonmw - - and - - ) and grant p mh - awarded by the nimh to kenneth davis. references andreasen nc. symptoms, signs, and diagnosis of schizophrenia. lancet ; : – . owen mj, craddock n, o’donovan mc. schizophrenia: genes at last? trends genet ; : – . lencz t, morgan t, athanasiou m, dain b, reed cr, kane jm et al. converging evidence for a pseudoautosomal cytokine receptor gene locus in schizophrenia. mol psychiatry ; : – . tan nc, mulley jc, berkovic sf. genetic association studies in epilepsy: ‘the truth is out there’. epilepsia ; : – . redon r, ishikawa s, fitch kr, feuk l, perry gh, andrews td et al. global 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www.nature.com/mp) cntnap deletions ji friedman et al molecular psychiatry http://www.nature.com/mp http://www.nature.com/mp 	cntnap gene dosage variation is associated with schizophrenia and epilepsy 	introduction 	materials and methods 	patient material 	array-based comparative genomic hybridization 	singe nucleotide polymorphism assay 	multiplex ligation-dependent probe amplification 	fluorescent in situ hybridization analysis 	sequence analysis 	results 	discussion 	acknowledgements 	note 	references rev_iss_web_men_ _ - .. special issue: sequence capture snp discovery in candidate adaptive genes using exon capture in a free-ranging alpine ungulate gretchen h. roffler,*† stephen j. amish,‡ seth smith,‡ ted cosart,‡ marty kardos,‡§ michael k. schwartz§¶ and gordon luikart‡** *alaska science center, u.s. geological survey, university drive, anchorage, ak , usa, †wildlife biology program, department of ecosystem sciences and conservation, college of forestry and conservation, university of montana, missoula, mt , usa, ‡fish and wildlife genomics group, division of biological sciences, university of montana, missoula, mt , usa, §evolutionary biology centre, uppsala university, se- uppsala, sweden, ¶us forest service rocky mountain research station, national genomics center for wildlife and fish conservation, e. beckwith ave., missoula, mt , usa, **flathead lake biological station, university of montana, polson, mt , usa abstract identification of genes underlying genomic signatures of natural selection is key to understanding adaptation to local conditions. we used targeted resequencing to identify snp markers in candidate adaptive genes associ- ated with known immunological, metabolic and growth functions in ovids and other ungulates. we selectively tar- geted exons in protein-coding and nearby and untranslated regions of chosen candidate genes. targeted sequences were taken from bighorn sheep (ovis canadensis) exon capture data and directly from the domestic sheep genome (ovis aries v. ; oviari ). the bighorn sheep sequences used in the dall’s sheep (ovis dalli dalli) exon cap- ture aligned to genes on the oviari genome with an average of exons each. we developed a microfluidic qpcr-based snp chip to genotype dall’s sheep from locations across their range and test for patterns of selec- tion. using multiple corroborating approaches (lositan and bayescan), we detected snp loci potentially under selection. we additionally identified candidate loci significantly associated with latitude, longitude, precipitation and temperature, suggesting local environmental adaptation. the three methods demonstrated consistent support for natural selection on nine genes with immune and disease-regulating functions (e.g. ovar-dra, apc, batf , mageb ), cell regulation signalling pathways (e.g. krit , pi k, orrc ), and respiratory health (cysltr ). char- acterizing adaptive allele distributions from novel genetic techniques will facilitate investigation of the influence of environmental variation on local adaptation of a northern alpine ungulate throughout its range. this research demonstrated the utility of exon capture for gene-targeted snp discovery and subsequent snp chip genotyping using low-quality samples in a nonmodel species. keywords: candidate genes, exon capture, next-generation sequencing, ovis dalli dalli, population genomics, snp chip received november ; revision received june ; accepted june introduction identifying patterns of adaptive variation in nonmodel species in natural environments has been challenging due to the difficulties of conducting experiments (e.g. controlled breeding) and obtaining direct measurements (e.g. common garden), especially for species with long generation intervals (holderegger & wagner ; scho- ville et al. ). the increased accessibility of high- throughput sequencing and genotyping methods has facilitated discovery and genotyping of snps in non- model organisms (brumfield et al. ; slate et al. ; stapley et al. ), including snps in functional or adaptive genes (cosart et al. ). these genomic tools facilitate the identification of genes with an adaptive function with higher certainty than with traditional genetic or observation-based methods (nielsen et al. ; eckert et al. ; hohenlohe et al. ), including candidate genes associated with fitness traits. candidate genes have a known function in a particular process, a metabolic pathway, or are related to a phenotype, and have been found to be under selection in previous stud- ies. therefore, candidate genes have a higher likelihood of being under selection than other genes, especially if correspondence: gretchen h. roffler, alaska department of fish and game, division of wildlife conservation, rd street, douglas, ak , usa, fax: - - ; e-mail: gretchen.roffler@alaska.gov © the authors. molecular ecology resources published by john wiley & sons ltd. this is an open access article under the terms of the creative commons attribution-noncommercial-noderivs license, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. molecular ecology resources ( ) , – doi: . / - . http://creativecommons.org/licenses/by-nc-nd/ . / their function relates to selection pressures that vary across the study area (luikart et al. ; nielsen ). these recent genomic developments are making broad- scale investigations of adaptive divergence in free-ran- ging species more possible than in the past (allendorf et al. ; ekblom & wolf ; ellegren ). exon capture (hodges et al. ) is an approach for discovering snps in wild species that are closely related to domestic species for which a sequenced genome is available and has been used to identify candidate genes associated with disease and other fitness traits in wild ungulates (cosart et al. ). exons are expressed por- tions of the genome that code for proteins and are there- fore generally well conserved. thus, it is possible to use the fully sequenced genomes of model species (e.g. domestic sheep; dalrymple et al. ; kijas et al. ; miller et al. ) as a reference to identify candidate snps in a related nonmodel species. this information in combination with a growing body of animal science studies on domestic sheep breeds (ibeagha-awemu et al. ; l€uhken ; heaton et al. ) can advance research on local adaptation by targeting candidate genes. candidate genes have been identified in wild sheep and other vertebrates related to immune function and parasite resistance and applied to studies of free- ranging sheep populations to test for areas of adaptive differentiation across a broad geographical scale (worley et al. ; johnston et al. ; luikart et al. ). fur- thermore, application of genomic data sets and land- scape genomic approaches has recently identified climate-mediated selection in sheep species (joost et al. ; pariset et al. ; lv et al. ) and provides evi- dence of the value of these methods to understand adap- tation to their environments. dall’s sheep have evolved in arctic and sub-arctic environments for > million years (kurten & anderson ; bunch et al. ) and are therefore expected to express specific adaptations to variability in environmen- tal factors that affect individual fitness. environmental extremes are typical in northern latitudes, thus it would be expected that selection on genes associated with speci- fic traits related to physiological limitations and pheno- logical timing would result. for example, exposure to cold temperatures and long winters would select genes associated with metabolic, thermogenic and circadian rhythm functions. the period of lambing in mountain sheep is correlated with plant phenology because it greatly affects the nutritional requirements of lactation which influences lamb survival (bunnell ; festa- bianchet ; rachlow & bowyer ). therefore, the relatively short summers in northern latitudes provide an abbreviated window of time during which ungulates must maximize their body condition and rear offspring. these conditions would select genes associated with signal pathways such as growth factors and lactation. finally, pathogen exposure levels and parasite abun- dance in northern ungulates tend to decrease with lati- tude (kutz et al. ; jenkins et al. ; mainguy et al. ). therefore, we would expect greater variation of genes associated with immune function at the southern end of the latitude gradient within the dall’s sheep sub- species range. our overarching objective was to develop a set of snps in candidate genes in a nonmodel organism and to extend this molecular approach into natural landscapes at a broad spatial scale to detect patterns of local adapta- tion. based on ecological knowledge of dall’s sheep, we developed hypotheses of factors limiting their fitness. we then selected genes with known functions related to these factors (immunological, metabolic, growth and environmental signalling functions) in ovids, domestic cattle or other well-studied taxa. we used exon capture to selectively target these genomic regions and then genotyped sheep at the range-wide scale. finally, we identified loci putatively under selection using two fst outlier approaches and correlation of candidate gene allele frequencies with environmental variables. materials and methods sample selection and extraction we collected samples throughout the dall’s sheep sub- species’ range in alaska and the yukon territory (fig. ). there are ~ dall’s sheep in this region (festa-bian- chet ) distributed across a latitudinal and ecological gradient from the southern, coastal influenced regions of alaska, to the arid brooks range in the arctic represent- ing the geographical limit of the subspecies. we attempted to include samples representing the major mountain ranges inhabited by dall’s sheep, as genetic structure has been demonstrated to coincide with these landscape fea- tures (worley et al. ; roffler et al. ). we obtained muscle or blood samples collected during – from hunter-killed rams or previous research efforts that had been preserved frozen or stored in tissue preservation buffer or % ethanol at room temperature (table ). genomic dna was extracted using gentra puregene blood and tissue kits following the recommended proto- cols (qiagen n.v., hilden, germany). the quality of the sample dna was evaluated at several points during the project. due to the age, collection method and/or preser- vation conditions, some samples produced low dna yields or showed high levels of inorganic contaminants. several different extraction protocols were tested, com- pared and optimized in an effort to get the highest qual- ity and concentration of dna possible (appendix s , supporting information). © the authors. molecular ecology resources published by john wiley & sons ltd. g. h. roffler et al. exon target sequences sequences for in-solution targeted re-sequencing and downstream snp discovery and assay design were pro- duced from several sources. forty-eight per cent of the dall’s sheep targeted exon sequences came from sequence reads from two bighorn sheep (ovis canadensis) from a previous study. the sequence reads were aligned to the domestic sheep (ovis aries) genome v (oviari ; archibald et al. ) with bwa (v . -r ) and genotyped using samtools v . . (li et al. ). the genotyped positions for one of the bighorn individuals were used to create sequences based on exon intervals in the oviari genome, as annotated by the collection of multiple spe- cies gene alignments to the oviari genome (obtained at the ucsc genome browser server at http://hgdown- load.cse.ucsc.edu/goldenpath/oviari /database/ xenorefgene.txt.gz). the final set of bighorn sheep sequences consisted of those that aligned to complete exons not only in the sheep genome, but also, in a second check for complete exon coverage, in the alignment of the same reads to the well-annotated cow genome btau v . (elsik et al. ; ncbi assembly accession no. gcf_ . ). the bighorn consensus sequences are available on request. the remaining % of targets were added from oviari genome (www.sheephapmap.org/ isgc_genseq.htm), including additional candidate gene exons and exons in chromosomal regions not enriched in the two bighorn sheep (fig. ). overall, a total of genomic regions including annotated genes were targeted in dall’s sheep samples using rna baits (sequences available upon request). exon capture one and a half micrograms of genomic dna in ll te-buffer was fragmented to a median size of bp using the covaris-s instrument following the kilometers western brooks gates of the arctic mackenzie lake clark kenai western chugach central alaska range yukon-tanana cassiar kluane-ruby wrangell-st.elias pelly-anvil eastern brooks western alaska range talkeetna coast mountains fig. map of sample locations and subspecies range (light grey) of dall’s sheep. © the authors. molecular ecology resources published by john wiley & sons ltd. using exon capture to discover snp in wild sheep http://hgdownload.cse.ucsc.edu/goldenpath/oviari /database/xenorefgene.txt.gz http://hgdownload.cse.ucsc.edu/goldenpath/oviari /database/xenorefgene.txt.gz http://hgdownload.cse.ucsc.edu/goldenpath/oviari /database/xenorefgene.txt.gz http://www.ncbi.nlm.nih.gov/nuccore/gcf_ . http://www.sheephapmap.org/isgc_genseq.htm http://www.sheephapmap.org/isgc_genseq.htm recommended settings. sheared samples were cleaned with agencourt ampure xp beads (beckman coulter, fullerton, ca, usa) using ll of resuspended beads and ll of dna. dna fragmentation was evaluated based on the distribution of fragment sizes using capillary electrophoresis on agilent dna chips, and the concentration of the dna was esti- mated by picogreen assay (invitrogen). individual sequencing libraries were prepared with lg of frag- mented dna using bioo scientific (austin, tx, usa) nextflex dna kits and barcodes following the stan- dard protocol. six in-solution hybridization captures of target dna using biotinylated rna baits from my- croarray v . . were carried out following the recom- mended protocol, with three samples multiplexed in each reaction. eighteen individual samples were sequenced in a sin- gle hiseq (illumina inc., san diego, ca, usa) lane producing approximately million bp single-end reads. the illuminaclip function with the truseq -se adapter sequences in trimmomatic v . (lohse et al. ) was used with the recommended settings to filter and trim the raw reads. the number of reads after trimming, the per-base and the per-sequence quality of the trimmed reads, were checked using fastqc v . . (www.bioinfor- matics.babraham.ac.uk/projects/fastqc). the quality fil- tered and trimmed sequence reads were aligned using bwa mapper software v . . (li & durbin ) to the domestic sheep genome oviari . coveragebed in bed- tools v . . (quinlan & hall ) was used to calculate the number of reads mapped to each targeted exon nucleotide. snp discovery samples were genotyped following the broad institute’s guidelines for nonmodel species (https://www.broad institute.org/) and a conservative set of hard filters to remove potentially false snps. using gatk’s recom- mended tools for genotyping (https://www.broadin stitute.org/gatk/guide), and gatk v . (mckenna et al. ), each sample was genotyped separately with the haplotypecaller, followed by a joint genotyping by gatk’s gentotypegvcfs. gatk’s variantfiltration tools, combined with an in-house script, were used to remove potentially false-positive snp calls. a snp genotyped locus was excluded if it showed one or more of the fol- lowing: (i) an allele was observed < times; (ii) the inbreeding coefficient at the locus was under � . , the upper bound of the lowest % of the inbreeding coef- ficients calculated for all loci under consideration (gatk’s inbreedingcoeff annotator: https://www.broadinstitute. org/gatk/gatkdocs/org_broadinstitute_gatk_tools_walk ers_annotator_inbreedingcoeff.php); (iii) more than two individual genotype calls had genotype qualities < ; (iv) more than two individual genotype calls had low coverage depth such that a homozygous alternate genotype call had coverage depth under five reads, or a heterozygous genotype call had coverage depth under eight reads; (v) more than two individual geno- types had coverage depth exceeding reads; (vi) more than individuals genotype calls were missing (i.e. the haplotypecaller did not find sufficient read data to call a genotype); (vii) more than of the individuals were genotyped as heterozygotes table summary information for sampled populations of dall’s sheep in alaska and the yukon territory, – (n = ). shown for each sample location are the coordinates of the sample area centroid, the number of samples included in the analyses, the expected (he) and observed (ho) heterozygosities, the proportion of polymorphic loci (pp) and inbreeding coefficient (fis) mountain range sample location n latitude longitude he ho pp fis alaska central alaska range . � . . . . . alaska lake clark npp . � . . . . � . alaska western alaska range . � . . . . . brooks eastern brooks range . � . . . . . brooks western brooks range . � . . . . . brooks gates of the arctic npp . � . . . . . kenai kenai . � . . . . � . talkeetna talkeetna . � . . . . . chugach western chugach . � . . . . . wrangell wrangell-st. elias npp . � . . . . . st. elias kluane-ruby ranges . � . . . . � . yukon cassiar . � . . . . � . yukon coast mountains . � . . . . . yukon pelly-anvil ranges . � . . . . . mackenzie mackenzie . � . . . . � . yukon-tanana uplands yukon-charley river np . � . . . . . © the authors. molecular ecology resources published by john wiley & sons ltd. g. h. roffler et al. http://www.bioinformatics.babraham.ac.uk/projects/fastqc http://www.bioinformatics.babraham.ac.uk/projects/fastqc https://www.broadinstitute.org/ https://www.broadinstitute.org/ https://www.broadinstitute.org/gatk/guide https://www.broadinstitute.org/gatk/guide https://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_gatk_tools_walkers_annotator_inbreedingcoeff.php https://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_gatk_tools_walkers_annotator_inbreedingcoeff.php https://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_gatk_tools_walkers_annotator_inbreedingcoeff.php (erroneous read mapping is suspected when substan- tially more than half of the individuals are genotyped as heterozygous); (viii) a snp locus was part of a clus- ter of at least three snp loci inside a bp window; or (ix) a snp locus was within bp of an insertion/ deletion call. consensus sequence generation and genotyping assay design we used our list of candidate genes to identify exons that could be used to test our hypotheses and contained vari- able snps for consensus sequence generation and later genotyping assay development. for each filtered snp locus, one sequence was generated for each individual, consisting of the filter-passed snp and its flanking posi- tions, base pairs upstream, base pairs down- stream. each position in the sequence had either a diploid genotype or an ‘n’ inserted at positions for which the individual had low coverage (≤ ) or low genotype quality (phred score ≤ ). these individual sequences were then aligned using mafft, v . (katoh & standley ). at each position, a nucleotide was included in the consensus genotype if it was observed at least eight times. a position was designated as having an unknown genotype (‘n’) if among the individuals, no genotype was observed in or more of the possible observations, more than of the possible observations were gapped by the alignment, or if the sum of nonobservations plus gaps was or more. consensus sequences were also designed for five snps within and near rxfp (appendix s , supporting information), a gene that has been inferred to be under strong selection in both wild bighorn (kardos et al. ) and domestic (kijas et al. ; johnston et al. ) sheep species, and putatively associated with variation in horn size in bighorn sheep (poissant et al. ). all consensus sequences were then aligned to the ncbi nucleotide collection (nt) database using blastn in blastn v . . (altschul et al. ). if a position was not already annotated, the top blastn hit was used to annotate the consensus sequence if the e-value for the alignment was < * � . consensus sequence quality and suitability for assay design were measured by calculating the num- ber of ns within bp and bp of all target snps. allele frequencies, observed heterozygosity, and expected heterozygosity were then calculated at the pop- ulation level for each locus. snps were also identified as either transitions or transversions. snps were preferen- tially targeted for assay design if they were in genes of interest (see above); had a consensus sequence with no ns within bp and fewer than ns within bp of the target snp; were transversions; or showed evidence of having allele frequencies that were highly variable across the range. specifically, if the frequency of the minor allele was < . in the eight samples that were sequenced coordinate (in millions) c hr om os om e x 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xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx sssss sss s ss sssss ssss ssss sss ssssssssssssss ss sssss ssss ss sss ssss ss sss s s s ssssss s s sss s ssssssss sssssssss s s sss s s s ss s sss s ss sss ss s s s s s s s ss ssssssssssssssssssssss ssss s s sssssssssssssssssssssssssssssssssssssss s ssssssssssssssssssssssssssssss sss ss sssssss ss s s s s sss ss sss s s ss s s ss ss s s ssss s s ss ssssssss s ss sssss s ssssss s ss ssss s ss s s ssssss s sss ss sss sssssssssssssssssssssssssssssssssssssssss s s s sssssssssssssssssssssssssssssssss s s s s s s sssss s ss s sss sssssss ss s ss s ss sss ss s s ssss s ss sss ss ss s s ssssssssssss s ss ssss sss ss ss ss sssss ss ssssssssssssssss s s ssssssssssssssssss sss s s s s s ss s ss s sss ssssssss sssssssssssssssssssssssssssssssssssssssssssss ssssssssssssssssssssssssssssss ss ss s s ss s s sss s sss sssssss sss ss sssss sssssssssss s s s s s s ssss sss sssssssss sssss ss s s s sss s ss s s s sss s s s s s ss s fig. on the ovis aries genome (oar_v . , genbank assession no. . ), the base pair coordinates of the targeted exon regions are marked with an ‘x’. coordinates of the snps that passed our filtering criteria are marked above their corresponding target with an ‘s’. © the authors. molecular ecology resources published by john wiley & sons ltd. using exon capture to discover snp in wild sheep http://www.ncbi.nlm.nih.gov/nuccore/ . from one source population, but > . in at least one of the other populations with exon capture genotypes, then the snp was preferentially targeted for assay design. additionally, loci that were fixed for different alleles in any combination of two populations were retained. con- sensus sequences for snps matching these criteria were submitted to fluidigm for snp type assay design using d assay design software (fluidigm inc., san francisco, ca, usa). this proprietary software takes into account primer and amplicon specificity and seeks to avoid plac- ing primers over snps, highly structured gc rich regions, and repeats (https://www.fluidigm.com/faq/ aa- ). because many of our samples were low quality, we relied on fluidigm snp type assays as this technol- ogy has proved to be the most sensitive and reliable for genotyping low dna concentration samples (campell & narum ). primers and sequence probes for snp assays tested are available upon request. genotyping assay evaluation all assays were run on the fluidigm ep genotyping sys- tem using . dynamic array ifcs under the manufac- turer recommended reaction conditions. prior to genotyping, all samples were quantitated using a nan- odrop spectrophotometer (thermo scientific, wal- tham, ma, usa). samples with concentrations below ng/ll were preamplified using the standard flu- idigm specific target amplification (sta) protocol. sta involves cycles of multiplex pcr using up to pairs of locus-specific (lsp) and sta primers provided with snp type assays. amplicons were then diluted and genotyped using the ep system. to test assays, samples from across the study area were each genotyped twice, including a subset of eight control samples that were used in the targeted rese- quencing experiment. prior to calculating call rate and concordance statistics, seven samples with abnormally low call rates (< %) were removed. this step was meant to help avoid a scenario where few, low-quality samples, controlled the majority of the variation in assay quality. several criteria were used to evaluate the performance of each snp assay for genotyping individual dall’s sheep samples. first, the plot of the snp alleles’ fluorescent intensity was checked for separation and clustering of each genotype class (heterozygotes and each homozy- gote) and assays with diffuse or unclear clusters were marked as potentially unusable. we then calculated the call rate for each assay across all replicates. each sample was run at least twice, so genotypes from each indepen- dent pcr reaction were compared for agreement. in addition, genotypes between the original exon sequenc- ing data and the snp assays were checked for concor- dance. finally, five populations (n from to ) were used to calculate hardy–weinberg proportions (hwp) and gametic disequilibrium (gd) between loci that passed initial genotype and clustering quality controls. descriptive statistics we tested for deviation from hwp and for gd using genepop (raymond & rousset ) across the sampling locations and snp loci using the markov chain monte carlo (mcmc) approximation of fisher’s exact test and a simulated exact test respectively. we ran dememorizations, batches and iterations, and applied a bonferroni-corrected alpha level of . . for each sampling location, we calculated observed and expected heterozygosities (ho and he) using genepop (table ). we calculated fis metrics for each genotyped individual within each population. fis values range from . to � . , with values less than � . indicating genotyping error (e.g. loci), or selection against homozygotes (waples ). identifying outlier loci we differentiated between neutral snp loci and those putatively under selection or linked to genes under selec- tion using two approaches. we first tested for departures from neutrality using an fst outlier approach based on simulation methods (beaumont & nichols ) imple- mented in lositan (ant~ao et al. ). we used replicates assuming an infinite alleles mutation model, with forced mean fst calculated for each pairwise com- parison, and tested for outliers at the and % confi- dence interval levels and used a false discovery rate (fdr) of ( . and) . . we also used bayescan ver. . (foll & gaggiotti ), a bayesian approach that directly estimates the posterior probability of a locus being under selection by comparing global and population-specific allele frequencies derived from fst coefficients. to iden- tify loci under selection using bayescan, we first per- formed pilot runs of iterations, followed by iterations on a sample size of and thinning interval of . we used a prior odds value of and identified outlier loci by comparing posterior probabili- ties and threshold values obtained from the fdr. we identified outlier loci as those with fdr q values < . and . , which approximately correspond to a values of . and . . lositan and bayescan have different assumptions and algorithms which may lead to differ- ences in the number of outlier loci detected. lositan assumes an island model (with equal population sizes, fst variances and migration rates), whereas bayescan does not make assumptions about fst variance equiva- lence and allows population and locus-specific effects in models. simulation studies have demonstrated a higher © the authors. molecular ecology resources published by john wiley & sons ltd. g. h. roffler et al. https://www.fluidigm.com/faq/aa- https://www.fluidigm.com/faq/aa- type i error of lositan vs. bayescan due to the failure to accommodate unequal fst variances (per�ez-figueroa et al. ; narum & hess ). type i error for balanc- ing selection was particularly high in this scenario (narum & hess ; lotterhos & whitlock ). environmental correlations we tested the hypothesis that loci under selection are correlated with particular environmental variables by implementing a sampling design distributed along envi- ronmental gradients (manel et al. ). we selected environmental and landscape variables (appendix s , table s , supporting information) to use in association tests with snp loci based on a priori hypotheses about ecological factors that affect dall’s sheep fitness. to test for associations between environmental characteristics and allele frequencies, we used an individual-based spa- tial analysis method (sambada; joost et al. ). sam- bada uses logistic regressions to test for associations between all possible pairs of environmental variables and allele frequencies at spatially referenced sample locations and thus estimates the probability of the pres- ence of a specific genetic marker given the sampling site environmental characteristics. models including and excluding the environmental variables are compared through examination of the significance of regression coefficients. model significance is then evaluated with likelihood ratio (g) and wald tests; if both tests indicate an environmental variable is more informative than a model with a constant only, the model is considered sig- nificant (joost et al. ). we recoded each allele from the snp loci as present (‘ ’) or absent (‘ ’), producing snp genotypes (i.e. , , for each of the snps). these genotypes were tested for associations with environmental variables (table s , supporting infor- mation), resulting in tests. we applied a bonfer- roni-corrected threshold corresponding to a = . and . to significant tests. results exon capture approximately million bp single-end reads were generated in the single hiseq (illumina inc.) sequen- cer lane. we obtained an average of approximately million high quality sequence reads from each of individuals distributed across the study area. two indi- viduals showed low read numbers (< ) and failed several fastqc per-base and per-sequence quality tests and were removed from the data set. after duplicate removal and realignment, individual bam files were used to calcu- late coverage for targeted exons using coveragebed. across these samples, a read depth of or greater was observed for % of nucleotides in targeted exons. genotyping assay design and evaluation the initial genotyping yielded potential snps. after applying our conservative snp filtration criteria necessary for consensus sequence generation, snps, each of which was the centre of a bp sequence, com- prised the initial set of potential snp assay sequences. of the sequences submitted to fluidigm, were suitable for snp type assay design. two hundred and sixty-one assays were selected from genes of interest and from across the genome and tested on a fluidigm ep . call rates varied from % to % across the tested assays; however, assay produced call rates > % (table ). duplicate genotype concordance varied from . % to %, with assays having genotypes that were concordant in > % of comparisons. concordance between exon capture and snp type genotypes ranged from % to %, with assays producing genotypes that matched those from exon capture in > % of cases. one hundred and eighty-eight assays were selected to be included in the final snp panel used to genotype all individuals. the majority of assays were chosen because they had high quality genotype clustering, high call rates and high levels of concordance. however, some loci were selected as a priori targets because they are located in a gene of interest. snp chip descriptive statistics after bonferroni correction, of tests for hwp were significant. seven loci deviated from hwp in > populations, thus were removed from further analyses. gametic disequilibrium was detected in of the tests after bonferroni correction, although gd was not observed for any snp pair in more than three populations. negative f is values (less than � . ) were observed for loci across seven populations; no more than two observations per population and no locus was homozygote deficient. the ho and he ranged between . – . and . – . , respectively (table ). outlier tests we detected a total of and fst outlier loci poten- tially under selection using lositan at the . and . thresholds, respectively (table ). of the outlier loci, were putatively under directional and under balanc- ing selection at the more conservative . threshold (table ). using bayescan, we identified a total of and outlier loci at the . and . thresholds, respec- tively, among which were possibly under directional © the authors. molecular ecology resources published by john wiley & sons ltd. using exon capture to discover snp in wild sheep (upper right corner fig. a), and nine under balancing selection (lower right corner fig. a). more outliers were detected with lositan than bayescan, and the majority ( of ) loci detected by lositan but not by bayescan were for balancing selection. the most robust support (demonstrated by coinciding results of the two methods) was for a subset of , and markers were identified as outliers (at the . and . thresholds, respectively), including nine for directional and six for balancing selec- tion at the most conservative threshold. the nine loci putatively under directional selection included loci in immune functions genes including ovar-dra (major histocompatibility complex, class ii, dr alpha), basic leucine zipper transcription factor (batf ), melanoma antigen family b (mageb ) and adenomatous polyposis coli (apc). other direc- tional selection candidate loci included synj (synapto- janin ; synaptic transmission) and cysltr (cysteinyl leukotriene receptor ; pulmonary inflammation media- tor). environmental correlations of models sambada computed ( environmental parameters snp genotypes) at the most conserva- tive . bonferroni-corrected confidence level, sig- nificant associations ( . % of the total) for both the likelihood ratio (g) or wald tests were identified in loci. of these, were significant for at least one fst out- lier test. applying the most conservative criteria (requir- ing both fst outlier tests to be significant at the . threshold), resulted in a subset of candidate loci with significant environmental associations (table ). all nine directional selection candidate loci had statistically sig- nificant environmental correlations, while the six balanc- ing selection candidate loci had only one. the majority of the environmental variables with significant associations were related to precipitation, temperature, position (lati- tude and longitude) and elevation (table ). discussion to facilitate studies of local adaptation, we identified snps in candidate adaptive genes using an exon capture approach. we detected adaptive variation of dall’s sheep and uncovered putatively adaptive loci cor- relations with environmental variables at a broad spatial scale. our results suggest that these genomics approaches can be used to identify adaptive variation across populations of dall’s sheep which could be useful to address wild sheep conservation by identifying adap- tation to local conditions. moreover, this work provides valuable insights into the general understanding of the genetic basis of adaptation in a free-ranging species. utility of a targeted approach our approach involved targeting a priori identified can- didate genes. research on adaptive differentiation of free-ranging species is greatly benefited using available information from closely related reference genomes. coding regions of the genome are generally well con- served, thus knowledge of genes related to fitness in model organisms may be translated into studies of multi- ple species in natural environments. this ‘bottom-up’ approach (sork et al. ; harrisson et al. ) is valu- able for gaining insight into an organism’s response to specific selective pressures. exon capture facilitated identification of putatively adaptive candidate genes (with known fitness trait asso- ciations in other species) at the range-wide scale in dall’s sheep. exons code for proteins that are involved in the table number of snp type assays tested by chromosome, mean call rate, duplicate genotype concordance and concor- dance of snp type assay genotype with exon capture genotypes chromosome n call rate duplicate genotype concordance snp assay and exon capture genotype concordance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m . . . x . . . total . . . © the authors. molecular ecology resources published by john wiley & sons ltd. g. h. roffler et al. table snp loci identified as potentially under selection for dall’s sheep using fst outlier and environmental correlation tests. for the fst outlier tests, loci with a % (*) and % (**) probability thresholds for balancing (b) and directional (d) selection are shown. snp that are significant for at least one outlier test and also have a genotype significantly correlated with environmental covariates for both the likelihood ratio (g) and wald tests at the bonferroni-corrected . alpha levels are shown. for environmental variables abbrevia- tions, see table s (supporting information) selected loci fst outlier detection environmental correlation gene functionlositan bayescan sambada chr _ b** il rb interleukin receptor, beta ; promotes the proliferation of t cells and nk cells chr _ d* lat , precip_wquart , anmeantemp , meantemp_cquart rxfp relaxin/insulin-like family peptide receptor ; associated with horn growth in sheep chr _ d* d** lat , lon , mintemp_month , antemprange , meantemp_cquart synj synaptojanin ; synaptic transmission and membrane trafficking chr _ b* precip_wquart , , annualprecip , alcam activated leucocyte cell adhesion molecule; interactions between nervous system cells, binding of t and b cells chr _ b** b* kiaa dna and calcium ion binding chr _ b* otol otolin chr _ b** tmprss transmembrane protease serine chr _ b* lpar lysophosphatidic acid receptor ; receptor for lysophosphatidic acid (lpa) chr _ d** d** min_temp , lat , precip_wetq , anmeantemp , anualprecip , meantemp_cquart , mintemp_month wars tryptophanyl-trna synthetase ; encodes mitochondrial tryptophanyl-trna synthetase chr _ b* lon acvr a activin a receptor, type iia; susceptibility to pregnancy-related disease chr _ b** bmpr bone morphogenetic protein receptor, type ii; endochondral bone formation and embryogenesis chr _ d* d** lat , , , precip_wquart , , meantemp_cquart , , annualprecip , , mintemp_month , , antemprange , , anmeantemp , precip_season rgs regulator of g-protein signalling ; gtpase activating proteins chr _ b* b** gabbr gamma-aminobutyric acid (gaba) b receptor, ; synaptic inhibition, slow wave sleep, muscle relaxation, and antinociception chr _ b** b** or a olfactory receptor, family , subfamily a, member chr _ b** b** olr oxidized low-density lipoprotein (lectin-like) receptor ; recognition, internalization and degradation of oxldl by vascular endothelial cells chr _ b* b** tspan tetraspanin ; regulates cell-surface receptor signal transduction © the authors. molecular ecology resources published by john wiley & sons ltd. using exon capture to discover snp in wild sheep table (continued) selected loci fst outlier detection environmental correlation gene functionlositan bayescan sambada chr _ d** d** lat , , anmeantemp , , meantemp_cquart , , precip_wquart , , lon , maxtemp_month , meantemp_wquart , mintemp_month , annualprecip , elv krit ovis aries krit , ankyrin repeat containing, transcript chr _ b** or g olfactory receptor, family , subfamily g, member chr _ d** d** lat , , precip_wquart , , meantemp_cquart , , annualprecip , , mintemp_month , , precip_season , , anmeantemp , , antemprange , precip_season , lon , elv lars leucyl-trna synthetase; infantile liver failure chr _ b* lon , , lat sin -family gtpase that may function in mitochondrial ribosome assembly, promotes growth and cell division chr _ b** kit type transmembrane receptor protein for mgf mast cell growth factor chr _ b* lat , , lon , , precip_season , a nmeantemp ip- chemokine (c-x-c motif) ligand ; antimicrobial gene encodes a chemokine of the cxc subfamily chr _ b** annualprecip , precip_wquart , precip_season rpgrip retinitis pigmentosa gtpase regulator interacting protein ; encodes a photoreceptor gene chr _ b* lon ptgdr prostaglandin d receptor (dp); respond to extracellular cues and activate intracellular signal transduction pathways chr _ b** b** esrrb oestrogen-related receptor beta; placental development chr _ d** d** lat , , lon , , elv , , maxtemp_month , , precip_wquart apc adenomatous polyposis coli; tumour suppressor chr _ d** d** lat , , precip_wquart , , annualprecip , , lon , maxtemp_month , elv , antemprange , meantemp_cquart , anmeantemp , mintemp_month orc origin recognition complex, subunit ; dna replication in eukaryotic cell chr _ b* b* phf phd finger protein ; transcription factor chr _ b** lon zhx zinc fingers and homeoboxes ; transcriptional repressor chr _ d* d** mintemp_month , , meantemp_cquart , , anmeantemp , , , antemprange , lat , , meantemp_wquart med mediator complex subunit ; regulated transcription of nearly all rna polymerase ii- dependent genes chr _ b* elv cpq carboxypeptidase q; hydrolysis of circulating peptide © the authors. molecular ecology resources published by john wiley & sons ltd. g. h. roffler et al. table (continued) selected loci fst outlier detection environmental correlation gene functionlositan bayescan sambada chr _ b* rxfp -intronic variant relaxin/insulin-like family peptide receptor ; associated with horn growth in sheep chr _ b* rxfp -intronic variant relaxin/insulin-like family peptide receptor ; associated with horn growth in sheep chr _ b* ssh slingshot protein phosphatase ; regulates actin filament dynamics chr _ d* per period circadian clock ; encode components of the circadian rhythms of locomotor activity, metabolism and behaviour chr _ b* irf interferon regulatory factor ; regulates epithelial cell proliferation chr _ b** tdrd tudor domain containing ; spermatogenesis chr _ d* precip_wquart , annualprecip , elv , lat , onsetgreen , endgreen , elv , precip_wquart , precip_wquart il ra interleukin- receptor subunit alpha; codes for proteins that inhibit the synthesis of proinflammatory cytokines chr _ b** lat , lon , precip_season abcc atp-binding cassette, subfamily c (cftr/ mrp), member ; transports molecules across extra- and intracellular membranes chr _ b* or m olfactory receptor m -like chr _ d** lat , lon , anmeantemp , meantemp_cquart , lat , lon , anmeantemp , meantemp_cquart , precip_season , mintemp_month ptcd pentatricopeptide repeat domain ; involved in mitochondrial rna maturation and respiratory chain function chr _ b** b* mrpl mitochondrial ribosomal protein l ; protein synthesis within the mitochondrion chr _ b** b** gabrb gamma-aminobutyric acid (gaba) a receptor, beta ; major inhibitory neurotransmitter, histamine receptor chr _ b* maxtemp_month gabra gamma-aminobutyric acid (gaba) a receptor, alpha ; mediates neuronal inhibition chr _ b* vrk vaccinia-related kinase ; regulate cell proliferation chr _ b** b* mapk mitogen-activated protein kinase ; cellular function chr _ d** d** lat , , precip_wquart , , meantemp_cquart , , annualprecip , , mintemp_month , , antemprange , , anmeantemp , , precip_season , ovar-dra major histocompatibility complex, class ii, dr alpha; immune function, presents peptides from extracellular proteins chr _ d* d** lat , , precip_wquart , , meantemp_cquart , , annualprecip , , mintemp_month , , antemprange , , anmeantemp , , precip_season , ovar-dra major histocompatibility complex, class ii, dr alpha; immune function, presents peptides from extracellular proteins © the authors. molecular ecology resources published by john wiley & sons ltd. using exon capture to discover snp in wild sheep production of phenotypes, and phenotype traits may influence the organisms’ fitness and therefore be affected by natural selection. consequently, we expected this approach might have a higher success rate at identifying adaptive variants than a broad-scale genomewide scan of genomic variation. in support of this exon-targeted approach, previous research using quantitative trait loci to identify relationships between fitness traits and candi- date genes in domestic cattle, goats and sheep has revealed most genetic polymorphisms associated with production (fatness, growth, milk production) are found in exons (ibeagha-awemu et al. ). indeed, our gene-targeted approach produced some- what higher proportions of candidate adaptive to puta- tively neutral genes than other next-generation genomics techniques. for example, lv et al. ( ) detected snps of from the ovinesnp beadchip with evi- dence of selection ( . %) across domestic sheep breeds. of these, were strong candidate genes for cli- mate-mediated selection ( . %). using alleles from microsatellite markers, joost et al. ( ) found five alle- les from four loci had significant correlations with envi- ronmental variables ( % of loci, . % of alleles) across breeds of domestic sheep distributed across europe and the middle east. the five alleles most likely to be under selection were all associated with precipitation and the number of wet days in a given month (joost et al. ), and two of the five had been previously associated with disease resistance. using genome scan approaches, limborg et al. ( ) identified % and narum et al. table (continued) selected loci fst outlier detection environmental correlation gene functionlositan bayescan sambada chr _ b** ifitm interferon-induced transmembrane protein; inhibits the entry of viruses to the host cell cytoplasm chr _ b** b** bola class i histocompatibility class i histocompatibility antigen; immune function chr _ d** d** mintemp_month , , meantemp_cquart , , antemprange , , anmeantemp , , precip_wquart , , mintemp_month , annualprecip , precip_season , lat , batf basic leucine zipper transcription factor, atf- like ; differentiation of lineage-specific cells in the immune system chr _ b** b** lon pi k family phosphatidylinositol -kinase regulator activity; positive regulation of development of blood vessels, t cell differentiation chr _ b* b* zp zona pellucida glycoprotein (sperm receptor); fertilization and preimplantation development chr _ b** b* supv l suppressor of var , -like; mitochondrial rna metabolism chrx_ d** d** lat , , anmeantemp , , meantemp_cquart , , meantemp_wquart , , mintemp_month , , maxtemp_month , cysltr cysteinyl leukotriene receptor ; contraction and proliferation of smooth muscle, oedema, eosinophil migration and damage to the mucus layer in the lung chrx_ d** d** lat , , lon , , anmeantemp , , precip_season , mageb melanoma antigen family b , may regulate various malignant phenotypes of cancer cells chrx_ d** lon , , lat , elv gata gata binding protein (globin transcription factor ); erythroid development; regulates switch of foetal to adult haemoglobin total ( . ) total ( . ) aa genotype, tt genotype, ta genotype, cc genotype, gg genotype, cg genotype. © the authors. molecular ecology resources published by john wiley & sons ltd. g. h. roffler et al. ( ) identified % of snps genotyped as putatively under selection. here, we identified loci of ( %) as putatively under selection; of these were detected as outliers with both fst-based approaches ( %). the relatively higher proportion of outliers detected in this study is potentially due to using a focused approach, specifically targeting candidate genes for which we had prior ovar dra apc batf mageb cysltr orrc krit mageb ovar dra apc batf orrc cysltr (a) (b) fig. (a) outlier loci detected at the % level with lositan software. the confidence area for candidate loci under positive selection is shown in red and for candidate loci potentially under balancing selection in yellow. (b) outlier loci identified by bayescan. x axis shows the posterior odds, and the y axis shows the fst index values. © the authors. molecular ecology resources published by john wiley & sons ltd. using exon capture to discover snp in wild sheep information (e.g. associated with specific fitness-related traits) and hypothesized would have a greater likelihood of being under selection. in contrast, the other studies referenced above used genome scan approaches to iden- tify outlier loci or alleles significantly correlated with environmental variables. a benefit to using genome scans is that it is not necessary to have a priori knowl- edge of loci associated with specific traits that are under selection and therefore may detect adaptive alleles with- out first identifying them (storz ). however, in com- parison with our targeted approach, which we were able to carry out due to an existing body of knowledge of domestic animal traits and their genetic associations, it may be necessary to assess a larger number of markers to detect putatively adaptive loci. although we began with a relatively small number of snp loci in contrast to other studies, we identified a comparable number of putatively adaptive loci. we expect that expanding our snp panel would also provide a higher number of candi- date outlier loci, loci with significant environmental cor- relations and a similar proportion of candidate adaptive vs. neutral loci. exon capture in nonmodel species bighorn sheep sequence reads from a previous exon cap- ture study proved to be an effective source of exon sequences to be used as baits in the dall sheep exon cap- ture. existing next-generation sequencing (ngs) data from a nonmodel species, along with their alignment to genome sequences from related model or domesticated and better-studied species like ovis aries and bos taurus, can be used to obtain gene sequence information on yet another nonmodel species. thus, we could leverage the closer relationship between the bighorn and the dall’s sheep, vs. ovis aries and the dall’s sheep, in the design of an exon capture on which to base the construction of our snp array. bighorn and the dall’s sheep are thought to have diverged . million years ago and are therefore expected to be more genetically similar than dall’s sheep and domestic sheep, who shared a most recent common ancestor . million years ago (bunch et al. ). this likely improved the overall success rate for our assay genotyping by increasing average read depth per target and allowing us to use higher ngs data quality thresh- olds for both the targeted resequencing genotyping, as well as the consensus sequence construction (cosart et al. ). our work joins a growing body of research that has capitalized on accumulated knowledge of domesti- cated species to select genes for use in studies of natural selection in wild populations (kardos et al. ; miller et al. ). the fluidigm platform is less commonly used in genomic research than other more high-throughput or massively parallel sequencing technologies that have become available (e.g. genotyping-by-sequencing, etc.). however, there are advantages to using this system including rapid turnaround time, straightforward data analysis, and a vetted methodology for dealing with low quality and low concentration samples. we found it to be an appropriate method for our research primarily because many of our samples were degraded and frag- mented (e.g. old or poorly preserved blood samples), and because using the fluidigm ep , only a small quan- tity of dna is required to obtain accurate and reliable genotypes (e.g. campbell & narum ; beja-pereira et al. ), particularly if samples are preamped prior to genotyping (kraus et al. ). specific target amplifica- tion primer pairs may be multiplexed at low dna con- centration ( nm per pair) and individual samples may be amplified with few cycles (~ ). an additional future goal of our research is to apply the snp chip to genotyp- ing of dall’s sheep samples collected noninvasively, specifically faecal pellets. faecal pellets may be collected broadly over large landscapes given access to dall’s sheep bedding sites (roffler et al. ). as is common with noninvasive samples, dna concentration is low, but this obstacle may be overcome using the fludigm snp chip developed in this research. an additional benefit of the fluidigm platform for our study was that it performed relatively efficiently and economically. a single laboratory technician could feasi- bly genotype samples at snps in day ( geno- types total), considering time estimates of approximately h of laboratory preparation, ~ h of pcr and an hour of data analysis. to the best of our knowledge, exon cap- ture has only been successfully performed once on low- quality noninvasive samples (perry et al. ), and rarely on museum samples (e.g. bi et al. ; mccor- mack et al. ; lim & braun ), and to apply this technology to our samples would require a large invest- ment in both time and finances. as these technologies are becoming increasingly available and applied to answer questions in molecular ecology, it is possible that in the near future exon capture could be effective with noninvasive samples from a broader array of taxa (beja- pereira et al. ). signatures of natural selection our results revealed signals of natural selection in dall’s sheep populations throughout their range. focusing on the results with the strongest support evidenced by mul- tiple significant outlier tests, we found candidate genes for immune function, signalling pathways for regulating cellular processes, tumour suppression, respiratory health, reproduction and olfactory receptors. we also demonstrate the relevance of environmental © the authors. molecular ecology resources published by john wiley & sons ltd. g. h. roffler et al. heterogeneity in locally adaptive genetic variation in can- didate genes. for all loci putatively under directional selection (identified by both outlier methods), tempera- ture variables were significantly associated with snp variants, with annual mean temperature (anmeantemp) most highly correlated, and also identified by principal component analyses as one of the environmental factors that explained most of the variability among regions (appendix s , supporting information). the most highly correlated precipitation variable was precipitation of the wettest quarter (precip_wquart), significantly associated with of the loci under putative directional selection. latitude was also identified as a potentially important driver for local adaptation, as it was also significantly associated with all candidate loci for directional selec- tion, whereas longitude and elevation were associated with . two of the candidate adaptive loci were in the ovar-dra gene located in the major histocompatibility complex (mhc) region. the mhc is made up of multi- ple genes and is generally highly polymorphic (at class i) across taxa. candidate loci within mhc have a demonstrated role in parasite resistance in sheep (buit- kamp et al. ; paterson et al. ). the ovar-dra is a class ii mhc gene and plays a central role in the ini- tiation of protective immune function in vertebrates. specifically, it encodes cell-surface proteins to bind and present peptides from extracellular proteins to t cells and invoke an immune response. this gene is well con- served and believed to have limited polymorphisms, although interestingly, comparatively high levels of diversity were described in domestic sheep breeds, potentially due to an independent evolutionary history (ballingall et al. ). in dall’s sheep, we found strong evidence of directional selection in ovar-dra, and strong balancing selection in the bola class i histo- compatibility antigen. balancing selection at mhc loci acting over long timescales and across multiple popula- tions can occur simultaneously with directional selec- tion at local populations. this pattern could be a result of different pathogens acting on different alleles at the mhc locus, or spatial inconsistency of pathogen expo- sure across a metapopulation. the candidate gene cysltr is a receptor for cys- teinyl leukotrienes which play a role in respiratory health by mediating inflammation, and eosinophil migration to the lung. in humans, variants of this gene are associated with occurrence of asthma and suscepti- bility to infection (sokolowska et al. ). we found strong evidence for directional selection in this gene and environmental correlations with all tested temperature variables and latitude. these results fit our hypothesis that genes related to immune function would vary across a latitudinal gradient, as pathogen exposure and abundance decline with latitude and decreasing temper- atures. wild sheep are believed to be immunologically na€ıve and therefore more susceptible to infectious dis- eases, especially in the case of respiratory diseases which have had catastrophic effects on bighorn sheep popula- tions in the u.s. rockies (foreyt et al. ; garde et al. ; jenkins et al. ; george et al. ). greater understanding of adaptive differentiation in disease resistance genes can aid in predictions of how dall’s sheep may respond to the spread of respiratory disease, which is expected to present an increasing threat to northern ungulates as a result of changing climates (kutz et al. ; jenkins et al. ). two other candidate genes related to immune functions with strong evidence for directional selection were mageb and apc. apc has a signalling pathway function which regulates stem cells and cancer tumour suppression, and mageb is expressed in tumours and may play a role in regulating malignant cancer cells. both demonstrated homozygous variants associated with latitude, longitude, temperature and precipitation variables, and apc was also signifi- cantly correlated with elevation. the relaxin/insulin-like family peptide receptor (rxfp ) was a lositan outlier for directional selection but not bayescan, providing weaker support of adaptive variation in this gene. however, the locus was signifi- cantly correlated with latitude, two precipitation vari- ables and annual mean temperature. two intronic variants of the rxfp gene were also identified as out- liers with lositan for balancing selection. this gene is associated with horn growth in sheep and has been demonstrated to be under strong selection in sheep spe- cies (kijas et al. ; johnston et al. ; kardos et al. ). in domestic sheep, selective breeding to remove horns resulted in a strong signal of selection (kijas et al. ). in free-ranging bighorn sheep, whole genome sequencing revealed a selective sweep at this gene, likely related to selection favouring rams with larger horns (kardos et al. ). our results provide additional evi- dence for selection on rxfp in a free-ranging and widely distributed sheep population. conclusions using a novel exon capture approach for gene-targeted snp discovery followed by qpcr-based snp chip geno- typing, we were able to extend population genomic approaches into natural landscapes and discover genes potentially under selection at the range-wide scale. we targeted genes with known functions, many of them in domestic sheep or cattle, and others from well-studied taxa. however, many traits are affected by multiple genes and interactions with the environment, and thus, we cannot account for unmeasured genetic or © the authors. molecular ecology resources published by john wiley & sons ltd. using exon capture to discover snp in wild sheep environmental effects. we recognize that while these results appear to be promising for future research, the possibility exists that the outlier loci detected may be simply linked to genomic regions experiencing natural selection and not under selection themselves. addition- ally, correlation of alleles with environmental variables does not imply causation. despite these limitations, our approach was valuable because it was founded on eco- logical knowledge of the organism, applied in a real life setting and can provide a step towards understand- ing the genetic basis of natural selection. further insights and increased certainty of selection could be gained by spatial and temporal replication of sample collections across environmental gradients in indepen- dent populations. detecting adaptive loci and natural selection in free- ranging populations is widely recognized as challenging, yet a critical endeavour for gaining insights into adaptive responses of species to changing environments (shafer et al. ). we provide an example of using targeted resequencing and snp genotyping in a nonmodel spe- cies to detect molecular patterns of natural selection and correlation of candidate adaptive genes with environ- mental variables. continued development and applica- tion of these methods will advance the accumulation of evidence for the genetic basis of adaptation. acknowledgements this work was funded by the national park service (nps) and the u.s. geological survey (usgs), and u.s. national science foundation grant . the nps (j. lawler, b. mangipane, k. rattenbury), the alaska department of fish and game (n. cassara, t. kavalok, t. lohuis, t. mcdonough, t. peltier, r. schwanke) and the yukon department of environment (t. hegel) provided samples and valuable insights. laboratory assistance was provided by y. horeaux and j. shabacker. l. adams, s. dobrowski and m. hebblewhite provided useful comments on early drafts of the manuscript. this work was supported by the u.s. national science foundation grant . g.r. was supported by the usgs and university of montana scholarships (bertha morton, les pengelly, and george and mildred cirica). g.l. was supported by nsf grants deb- and deb- . m.k. was supported by the national science foundation via the montana institute on ecosystems (eps- ) and the montana ecology of infec- tious diseases igert program (dge- ). any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the u.s. government. references allendorf fw, hohenlohe pa, luikart g ( ) genomics and the future of conservation genetics. nature reviews genetics, , – . altschul sf, gish w, miller w, myers ew, lipman dj ( ) basic local alignment search tool. journal of molecular biology, , – . ant~ao t, lopes a, lopes rj, beja-pereira a, luikart g ( ) lositan: a workbench to detect molecular adaptation based on a fst-outlier method. bmc bioinformatics, , . archibald al, cockett ne, dalrymple bp et al. 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( ) population genetic structure of north american thinhorn sheep ( ovis dalli ). molecular ecology, , – . worley k, carey j, veitch a, coltman dw ( ) detecting the signature of selection on immune genes in highly structured populations of wild sheep (ovis dalli). molecular ecology, , – . g.h.r., g.l. and m.k.s. designed the study. s.a. coordi- nated the exon capture, snp genotyping and provided analytical input. t.c. conducted bioinformatics analyses. s.s. conducted laboratory analyses. m.k. identified additional sheep genome targets for the capture. g.h.r. performed analyses, coordinated sample collection and led the writing of the manuscript with input from all co- authors. data accessibility snp data are available in dryad doi: doi: . / dryad.kk . supporting information additional supporting information may be found in the online version of this article: appendix s dna extraction, consensus sequence generation, and genotyping assay design. appendix s environmental and landscape association tests with snp loci. table s snp-type assays and primer sequences tested includ- ing alleles targeted by allele specific primer (asp ) and (asp ), sequences for allele specific primer (asp sequence) and (asp sequence), the sequences for locus specific primers (lsp sequence), and the sequences for specific target amplifica- tion primers (sta sequences) table s environmental and landscape variables tested for associations with snp loci fig. s principal component analysis (pca) plot of environmen- tal variables demonstrating the percentage of variation explained by each axis and correlations among variables. © the authors. molecular ecology resources published by john wiley & sons ltd. g. h. roffler et al. http://dx.doi.org/ . /dryad.kk http://dx.doi.org/ . /dryad.kk clopidogrel improves skin microcirculatory endothelial function in persons with heightened platelet aggregation clopidogrel improves skin microcirculatory endothelial function in persons with heightened platelet aggregation shabnam salimi, md, msc; joshua p. lewis, phd; laura m. yerges-armstrong, phd; braxton d. mitchell, phd; faisal saeed, md; jeffry r. o’connell, phd; james a. perry, phd; kathleen a. ryan, mph; alan r. shuldiner, md; afshin parsa, md, mph background-—platelet activation can lead to enhanced oxidative stress, inflammatory response, and endothelial dysfunction. to quantify the effects of platelet inhibition on endothelial function, we assessed platelet activity of healthy persons before and after clopidogrel administration and evaluated its effects on endothelial function. we hypothesized that clopidogrel, by attenuating platelet activity, would result in enhanced endothelial function. methods and results-—microcirculatory endothelial function was quantified by laser doppler flowmetry (ldf) mediated by thermal hyperemia (th) and postocclusive reactive hyperemia, respectively, in and relatively healthy and homogenous old order amish persons. ldf and platelet aggregation measures were obtained at baseline and after days of clopidogrel administration. our primary outcome was percentage change in post- versus preclopidogrel ldf measures. preclopidogrel th-ldf and platelet aggregation were higher in women than in men (p< . ). clopidogrel administration was associated with � -fold higher percentage change in th-ldf in participants with high versus low baseline platelet aggregation ( . � . % versus . � . %, p= . ). clopidogrel also increased absolute th-ldf measures in persons with high platelet aggregation ( � to � , p= . ), with a more prominent effect in women ( � to � , p= . ). there was no evidence that clopidogrel influenced postocclusive reactive hyperemia ldf measures. conclusions-—the administration of clopidogrel in healthy persons with high baseline platelet aggregation results in improved th- induced microcirculatory endothelial function. these data suggest that clopidogrel may have a beneficial effect on microcirculatory endothelial function, presumably through antiplatelet activity, and may confer additional vascular benefits. clinical trial registration-—url: https://www.clinicaltrials.gov. unique identifier: nct . (j am heart assoc. ; : e doi: . /jaha. . ) key words: clopidogrel • endothelial function • platelet aggregation • women p latelet activation can contribute to cardiovascular dis-ease through its role in thrombosis formation. this is mediated in part by the activation of the p y receptor (adenosine diphosphate [adp] receptor) and subsequent overexpression of glycoprotein iib/iiia, a platelet aggregator receptor. in addition to its role in thrombosis, platelet activation leads to an increase of circulating cd -l, resulting in release of inflammatory and oxidative stress mediators. – this causes endothelial dysfunction and attenuated nitric oxide (no) bioavailability, a sentinel event in the development and progression of both focal and systemic vascular disease and atherosclerotic-related morbidity. – clopidogrel, an adp (p y ) receptor antagonist, is activated by hepatic cytochrome p (cyp) isoenzymes in a biologically active thiol metabolite, significantly reducing adp-dependent platelet aggregation and thrombosis. – along with the antithrombotic benefits, interest has been emerging in studying the effect of clopidogrel on endothelial function in persons with – and without coronary artery disease. furthermore, studies in rat models have shown that clopidogrel can improve endothelium-mediated vascular response, presumably via no pathways. – in this study, we hypothesized that clopidogrel would result in enhanced microcirculatory endothelial function by primarily attenuating platelet activity. bearing in mind the significant variability in from the divisions of endocrinology, diabetes & nutrition (j.p.l., l.m.y-a., b.d.m., j.r.o’., j.a.p., k.a.r., a.r.s.) and nephrology (a.p., f.s.), department of medicine; department of epidemiology and public health (s.s., b.d.m., l.m.y-a.), university of maryland school of medicine, baltimore, md; geriatrics research and education clinical center (b.d.m.) and department of medicine, baltimore veterans administration medical center, baltimore, md (a.p.) correspondence to: shabnam salimi, md, msc, department of epidemiology and public health, university of maryland, school of medicine, baltimore, w baltimore st. mstf- , baltimore, md . e-mails: ssalimi@som. umaryland.edu, shabnam.salimi.m.d@gmail.com received april , ; accepted august , . ª the authors. published on behalf of the american heart association, inc., by wiley blackwell. this is an open access article under the terms of the creative commons attribution-noncommercial license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. doi: . /jaha. . journal of the american heart association original research d ow nloaded from http://ahajournals.org by on a pril , https://www.clinicaltrials.gov info:doi/ . /jaha. . http://creativecommons.org/licenses/by-nc/ . / baseline platelet aggregation and sex-specific variability in hypercoagulability state and endothelial function, we secon- darily hypothesized that sex and baseline platelet aggrega- tion level might affect the endothelial response to clopidogrel. considering that several methods exist for measuring microvascular endothelial function, we set out to measure the effect of clopidogrel on microcirculatory endothelial function and vascular response using complementary measures: laser doppler flowmetry (ldf) that was mediated by thermal hyperemia (th) as our primary measure and by postocclusive reactive hyperemia (porh) as a secondary measure. th-ldf has been shown to be largely mediated by the endothelial release of no and considered a valid proxy for endothelial function. , porh-ldf captures a complex microvascular response to induced transient ischemia in which the endothelium plays a pivotal role, primarily independent of no. the study was performed in a relatively healthy and homogenous population to evaluate whether clopidogrel, by inhibiting platelet activity, can have a beneficial effect on microcirculatory endothelial function in clinically healthy persons. materials and methods study population the pharmacogenomics of anti-platelet intervention (papi) study recruited relatively healthy old order amish persons from lancaster county, pennsylvania, to evaluate the genetic determinants of platelet aggregation before and after admin- istration of the antiplatelet drug clopidogrel. detailed charac- teristics of the papi study and its participants were published previously. briefly, all participants were aged ≥ years and were free of any known disease. participants were excluded if any of the following criteria were met: history of cardiovas- cular disease, severe hypertension (blood pressure > / mm hg or antihypertensive medication), diabetes melli- tus, anemia, cancer, thyroid malfunction, renal insufficiency, abnormal liver function tests, gastrointestinal bleeding, thrombocytosis or thrombocytopenia, current pregnancy or lactation, or continuation of any prescribed or over-the- counter medication that would interfere with the impact of clopidogrel. at least week prior to the initial study visit, participants discontinued the use of all medications, vitamins, and supplements. this study was approved by the university of maryland school of medicine institutional review board. in this investigation, we evaluated a subgroup of papi study participants who gave informed consent, and their ldf-based measures of endothelial function were recorded at baseline and after the clopidogrel intervention, which consisted of a -mg loading dose followed by days of daily dosing at mg. baseline demographic and cardiovascular-related clinical variables were obtained under standardized protocols and were published previously. , endothelial function measures laser doppler flowmetry ldf uses a laser light source and doppler shift effect to noninvasively measure microvascular blood flow on a relative blood-perfusion-unit unitless scale, allowing for reliable mea- surement of change in microcirculatory perfusion in response to various interventions. all measurements were performed by a single trained technician according to an established protocol using a perimed pf unit in a temperature- controlled room. thermal hyperemia our primary microcirculatory endothelial function outcome was based on skin th-ldf measures obtained from all study participants before and after clopidogrel administration. to obtain these measures, the ldf probe was placed on the volar aspect of the forearm and was heated to °c, causing dilatation of the skin vessels and increased flow. the ldf- based blood flow was recorded at baseline and then over minutes as the probe heated the skin. we then calculated the percentage change in flow from baseline to the observed peak flow over the -minute time interval, which constituted our final th-ldf measure. all results were abstracted by a single investigator who was blinded to the study participant status (f.s.). postocclusive hyperemia porh-ldf measurements were obtained on the same day as the th-ldf measures in participants who had th-ldf measured and who consented to a second ldf-based measure during the same study visit. after immobilizing the left arm, the ldf probe was placed over the volar aspect of the forearm skin � to cm proximal to the wrist to allow for the measure of baseline blood flow. next, a sphygmomanometer cuff located on the left arm above the antecubital fossa was inflated mm hg above systolic blood pressure for minutes to occlude arterial blood flow, resulting in ischemia-induced dilatation of resistance ves- sels. the percentage increase in flow from baseline was used as a proxy of endothelial function and vascular reactivity. platelet aggregometry ex vivo platelet aggregometry was measured before and after clopidogrel intervention in platelet-rich plasma ( doi: . /jaha. . journal of the american heart association clopidogrel improves endothelial function salimi et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , platelets/ll), which was isolated from the whole blood, as described previously. following stimulation of platelet-rich plasma samples with lg/ml of adp or collagen, platelet aggregation was quantified by optical aggregometry (pap e aggregometer; bio/data corp) using platelet-poor plasma as a reference, as described previously. to categorize our participants as high versus low platelet aggregators, the quantile distribution plot of standardized platelet aggregation after stimulation with adp or collagen was examined for each measure. based on the preclopidogrel platelet aggre- gation distribution, participants in the upper quartile of either platelet aggregation measures were classified as having high composite platelet aggregation (n= ) (figure a and b). clopidogrel active metabolite quantification clopidogrel active metabolite was quantified in the blood collected in an edta tube containing mmol/l (e)- -bromo- -methoxyacetophenone (mpb; sigma aldrich, st louis, missouri, usa) within hour after the last administration of clopidogrel using ultra–high-performance liquid chromatogra- phy–tandem mass spectrometry with an active metabolite calibration range of . to ng/ml, as described previ- ously. genotyping our group and others have previously shown that a common loss-of-function cyp c * variant (rs ) was associ- ated with differential clopidogrel active metabolite formation and variability in platelet aggregation response to clopidogrel. to account for potential genotype effect, we used our previously obtained rs genotype results within the parent papi study as a covariate. the mean genotype call rate for the papi study population was . %, and we observed > % concordance in a subset of blind duplicates. , statistical analysis our primary analysis was to compare percentage change in ldf measures of endothelial function before and after clopidogrel administration. percentage change in postclopi- dogrel th-ldf and porh-ldf from preclopidogrel mea- sures was calculated as follows: percentage change th=(thpostclopidogrel�thpreclopidogrel)/(thpreclopidogrel) %. similarly, percentage change porh=(porhpostclopidogrel� porhpreclopidogrel)/(porhpreclopidogrel) %. in addition, sec- ondary analyses of pre- and postclopidogrel absolute changes in the endothelial function measures were performed. to account for relatedness in the old order amish participants, the mixed models analysis for populations and pedigrees (mmap) program was used (http://edn.som.umaryland.edu/ mmap/index.php). mmap utilizes a regression-based approach that models variation of the trait of interest as a function of measured covariate (eg, age) and a polygenic component that accounts for phenotypic correlation due to relatedness. , in the current analysis, to reduce the effects of age and family relatedness, endothelial function and platelet aggregation were separately analyzed using a mixed model adjusting for age and family relatedness using mmap, and the residuals were then used for subsequent analyses. moreover, to eliminate any unit-related effect, standardized endothelial function and platelet aggregation measures were created by subtracting the mean and dividing by the observed standard deviation for outcomes and platelet aggregation as the main risk factor of interest and other conventional covariates. figure. distribution of ex vivo platelet aggregation stimulated by adding lg/ml of adenosine diphosphate (adp) or collagen. participants with values for either platelet aggregation measure within the upper quartile of distribution were consid- ered high aggregators. a, ex vivo platelet aggregation stimulated by adding lg/ml adp. b, ex vivo platelet aggregation stimulated by adding lg/ml collagen. doi: . /jaha. . journal of the american heart association clopidogrel improves endothelial function salimi et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , http://edn.som.umaryland.edu/mmap/index.php http://edn.som.umaryland.edu/mmap/index.php we assessed the mean differences of preclopidogrel platelet aggregations in persons with high and low platelet aggregation states. in addition, the mean of preclopidogrel th-ldf and porh-ldf by categorical cardiovascular-related factors were compared using the student t test. then, univariate and multivariate regression analyses were table . characteristics of the study participants total, n= women, n= men, n= p value* age, y . � . . � . . � . . sex, % men . — — — women . menopausal state, % yes — . — — bmi, kg/m . � . . � . . � . . < , % . . . . to < , % . . . ≥ , % . . . current smoker, % . . — sbp, mm hg . � . . � . . � . . dbp, mm hg . � . . � . . � . . crp, mg/dl, median (range) . ( . – . ) . ( . – . ) . ( . – . ) . total cholesterol, mg/dl . � . . � . . � . . ldl-c, mg/dl . � . . � . . � . . hdl-c, mg/dl . � . . � . . � . . tg, mg/dl . � . . � . . � . . pltagg-adp before clopidogrel . � . . � . . � . . after clopidogrel . � . . � . . � . . pltagg-coll before clopidogrel . � . . � . . � . . after clopidogrel . � . . � . . � . . high composite platelet aggregation, n (%) yes ( . ) ( . ) ( . ) . no ( . ) ( . ) cyp c * genotype allele . (n= ) . (n= ) . (n= ) . allele . (n= ) . (n= ) . (n= ) allele . (n= ) . (n= ) . (n= ) clopidogrel active metabolite . � . . � . . � . . th before clopidogrel � � � . th after clopidogrel � � � . porh before clopidogrel . � . . � . . � . . porh after clopidogrel . � . . � . . � . . data are shown as mean�sd except as noted. all p value are shown after adjustment for age and family relatedness. bmi indicates body mass index; crp, c-reactive protein; cyp c , cytochrome p c system; dbp, diastolic blood pressure; hdl-c, high-density lipoprotein cholesterol; ldl-c, low-density lipoprotein cholesterol; pltagg-adp , platelet aggregation on adding lg/ml adenosine diphosphate; pltagg-coll , platelet aggregation on adding lg/ml collagen; porh, postocclusive reactive hyperemia; sbp, systolic blood pressure; tg, triglyceride; th, thermal hyperemia. *sex-based p-value. doi: . /jaha. . journal of the american heart association clopidogrel improves endothelial function salimi et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , performed to assess the association among platelet aggrega- tion state (ie, high versus low), conventional cardiovascular risk factors, and percentage change in th-ldf or porh-ldf. furthermore, to calculate the absolute change in postclopi- dogrel endothelial function measures from function before clopidogrel, a paired t test was used in all study participants and based on stratification by sex. we then compared absolute change in th-ldf and porh-ldf in all participants with high platelet aggregation, with secondary stratification by sex. stata (statacorp) was used for statistical analysis of the residual data. a p value < . was considered statistically significance. for pre- and postclopidogrel microcirculation, endothelial function heritability (h ) was estimated while accounting for age and sex using the mmap program. results baseline characteristics and sex comparisons the characteristics of the study participants are shown in table . the mean age of the cohort was � years, and % of the participants were women, with no significant age discrepancy between the sexes (p= . ). the clinically healthy study population was slightly overweight (mean body mass index [in kg/m ] of . � . ), with elevated serum levels of low-density lipoprotein cholesterol ( . � . mg/dl) and high-density lipoprotein cholesterol ( . � . mg/dl). in old order amish culture, women tend to not use tobacco products, resulting in % of women reporting smoking, whereas . % of men reporting current smoking. women had higher body mass index than men ( . � . versus . � . , p= . ). in addition, women had lower low-density lipoprotein cholesterol ( . � . versus . � . mg/dl, p= . ) and higher high-density lipoprotein cholesterol serum concentrations ( . � . versus . � . mg/dl, p= . ). of the participants, . % (n= ) were classified as high composite platelet aggregators (based on adp and/or collagen stimulation), of which > . % (n= ) were women (table ). in both initial high and low composite platelet aggregations, there was significant change in platelet aggregation after administration of clopidogrel, with more prominent change in initial high composite platelet aggregation (table ). ex vivo preclopidogrel adp-stimulated platelet aggregation was significantly higher in women than in men (p= . ) (table ). the cyp c * genotype and related serum concentration of the clopidogrel active metabolite were equally distributed between men and women (table ). median c-reactive protein level was higher in participants with increased composite platelet aggregation compared with low composite platelet aggregation ( . versus . mg/dl, p= . ). th-mediated ldf of the participants with preclopidogrel th-ldf measure- ments, postclopidogrel measures could not be obtained in persons. in addition, th-ldf measures were excluded secondary to body motion artifacts resulting in unreliable measures. preclopidogrel th-ldf was higher in women than men ( � versus � , p= . ) (table ). of all examined cofactors, sex was the strongest determinant of preclopidogrel th-ldf endothelial function (p= . ) (table ). women with higher composite platelet aggregation had lower preclopidogrel th-ldf measures compared with those with lower platelet aggregation ( � versus � , respectively; p= . ). preclopidogrel th-ldf was not significantly different for pre- and postmenopausal women ( � versus � , p= . ). in univari- ate regression analysis, high composite platelet aggregation (b= . , p= . ) was significantly associated with percent- age change in th-ldf, and in our multivariate analysis, platelet aggregation (b= . , p= . ) was the sole risk factor that was significantly associated with percentage change in th-ldf. there were no significant associations between other conventional cardiovascular disease risk factors and preclopi- dogrel th-ldf measure (table ). postclopidogrel th-ldf was significantly higher in women than in men ( � versus � , p= . ). there was a significant percentage change in postclopidogrel th- ldf ( . � . versus . � . , p= . ) in participants with low versus high composite platelet aggregation, respec- tively. there was no overall significant absolute changes in postclopidogrel th-ldf from preclopidogrel th-ldf in the table . platelet aggregation response to clopidogrel in initial composite high and low platelet aggregation state high composite pltagg-adp or -coll low composite pltagg-adp or -coll preclopidogrel pltagg-adp . � . . � . postclopidogrel pltagg-adp . � . . � . p< . p= . preclopidogrel pltagg-coll . � . . � . postclopidogrel pltagg-coll . � . . � . p< . p= . pltagg-adp indicates platelet aggregation on adding lg/ml adenosine diphosphate; pltagg-coll , platelet aggregation on adding lg/ml collagen. doi: . /jaha. . journal of the american heart association clopidogrel improves endothelial function salimi et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , nonstratified analysis of composite platelet aggregation ( � versus � , p= . ). although there was no overall significant absolute change in postclopidogrel th-ldf from preclopidogrel th-ldf in women ( � versus � , p= . ) or men ( � versus � , p= . ) (table ), there was a significant absolute change in postclopidogrel th-ldf from preclopidogrel th-ldf in participants with high composite platelet aggregation ( � versus � , p= . ) (table ). moreover, premenopausal women with high com- posite aggregability showed significant absolute change in postclopidogrel from preclopidogrel th-ldf ( � ver- sus � , p= . , n= ). in women with high composite aggregability, percentage change in th-ldf was marginally higher in non-menopausal versus menopausal women ( . � . [n= ] versus . � . [n= ], p= . ). no association was observed between th and other cardiovascular disease risk factors (table ). porh-mediated ldf of the participants consented for the primary th-ldf measure, were available and agreed to participate in the supplementary porh-ldf measurement arm of the study. in addition, participants were excluded because of low- quality porh-ldf readings related to body motion artifacts, resulting in a total of participants with both pre- and postclopidogrel porh-ldf measurements. there was no significant difference between women and men in preclopi- dogrel porh-ldf (p= . ) (table ). in addition, preclopido- grel porh-ldf was not significantly different between premenopausal and menopausal women (p= . ). further- more, in both univariate and multivariate analyses, no association was observed between conventional cardiovascu- lar disease risk factors and preclopidogrel porh-ldf mea- sures, except for sex (table ). table . mean of preclopidogrel th-ldf and porh-ldf by categorical cardiovascular-related factors preclopidogrel th-ldf, mean�sd, p value preclopidogrel porh-ldf, mean�sd, p value high composite platelet aggregation no � . � . yes � , . . � . , . age, y < � . � . ≥ � , . . � . , . sex men � . � . women � , . . � . , . menopausal state no � . � . yes � , . . � . , . bmi, kg/m < � . � . ≥ � , . . � . , . current smoker no � . � . yes* � , . . � . , . sbp, mm hg < � . � . ≥ � , . . � . , . dbp, mm hg < � . � . ≥ � , . . � . , . crp, mg/dl < . � . � . ≥ . � , . . � . , . ldl-c, mg/dl < � . � . ≥ � , . . � . , . hdl-c, mg/dl ≥ for men or ≥ for women � . � . < for men or < for women � , . . � . , . tg, mg/dl < � . � . ≥ � , . . � . , . continued table . continued preclopidogrel th-ldf, mean�sd, p value preclopidogrel porh-ldf, mean�sd, p value cyp c * genotype† allele � . � . allele � . � . allele . � , . . � . , . bmi indicates body mass index; crp, c-reactive protein; cyp c , cytochrome p c system; dbp, diastolic blood pressure; hdl-c, high-density lipoprotein cholesterol; ldf, laser doppler flowmetry; ldl-c, low-density lipoprotein cholesterol; porh, postocclusive reactive hyperemia; sbp, systolic blood pressure; tg, triglyceride; th, thermal hyperemia. *smoking in men. †p value based on kruskal–wallis. doi: . /jaha. . journal of the american heart association clopidogrel improves endothelial function salimi et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , postclopidogrel porh-ldf was significantly higher in women than in men ( . � . versus . � . , p= . ) (table ). there was no association between composite platelet aggregation or conventional cardiovascu- lar risk factors and percentage change in porh-ldf (table ). there was no significant absolute change in postclopidogrel porh-ldf from preclopidogrel porh-ldf in women or men (table ), despite higher baseline composite platelet aggregation in women (p= . ) (table ). pretreat- ment platelet volume was measured but was not significantly associated with percentage change in th-ldf or porh-ldf (data not shown). table . univariate and multivariate regression models for association between various cardiovascular risk factors and percentage change in postclopidogrel th-ldf univariate models, percentage change th-ldf, b�sd, p value multivariate model, percentage change th-ldf, b�sd, p value high composite platelet aggregation yes . � . , . . � . , . age, y ≥ � . � . , . � . � . , . sex women . � . , . . . � . , . menopausal state yes . � . , . — bmi, kg/m ≥ � . � . , . � . � . , . current smoker* yes � . � . , . � . � . , . sbp, mm hg ≥ . � . , . � . � . , . dbp, mm hg ≥ . � . , . . � . , . crp, mg/dl ≥ . � . � . , . � . � . , . ldl-c, mg/dl > � . ( . ), . � . � . , . hdl-c, mg/dl < for men or < for women . � . , . . � . , . tg, mg/dl ≥ . � . , . . � . , . cyp c * genotype allele — . � . , . allele � . � . , . percentage change th-ldf was calculated as follows: preclopidogrel th-ldf� postclopidogrel th-ldf/preclopidogrel th-ldf) . bmi indicates body mass index; crp, c-reactive protein; cyp c , cytochrome p c ; dbp, diastolic blood pressure; hdl-c, high-density lipoprotein cholesterol; ldf, laser doppler flowmetry; ldl- c, low-density lipoprotein cholesterol; sbp, systolic blood pressure; tg, triglyceride; th, thermal hyperemia. *smoking in men. table . univariate and multivariate regression models for association between various cardiovascular risk factors and percentage change in postclopidogrel porh-ldf univariate models, percentage change porh-ldf, b�sd, p value multivariate model, percentage change porh-ldf, b�sd, p value high composite platelet aggregation yes � . � . , . � . � . , . age, y ≥ � . � . , . � . � . , . sex women . � . , . . � . , . menopausal state yes . � . , . — bmi, kg/m ≥ � . � . , . � . � . , . current smoker* yes � . � . , . . � . , . sbp, mm hg ≥ � . � . , . � . � . , . dbp, mm hg ≥ � . � . , . � . � . , . crp, mg/dl ≥ . . � . , . . � . , . ldl-c, mg/dl > . � . , . . � . , . hdl-c, mg/dl < for men or < for women � . � . , . � . � . , . tg, mg/dl ≥ � . � . , . � . � . , . cyp c * genotype allele . � . , . . � . , . allele . � . , . . � . , . percentage change porh-ldf was calculated as follows: preclopidogrel porh- ldf�postclopidogrel porh-ldf/preclopidogrel porh-ldf) . bmi indicates body mass index; crp, c-reactive protein; cyp c , cytochrome p c ; dbp, diastolic blood pressure; hdl-c, high-density lipoprotein cholesterol; ldf, laser doppler flowmetry; ldl-c, low-density lipoprotein cholesterol; porh, postocclusive reactive hyperemia; sbp, systolic blood pressure; tg, triglyceride. *smoking in men. doi: . /jaha. . journal of the american heart association clopidogrel improves endothelial function salimi et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , heritability of pre- and postclopidogrel th-ldf and porh-ldf measures th-ldf showed significant heritability when measured before clopidogrel administration (h . � . , p= . ), accounting for age and sex. heritability was slightly higher after the administration of clopidogrel (h . � . , p= . ). addi- tional adjustment for cyp c * attenuated postclopidogrel th-ldf heritability (h . � . , p= . ). the heritability estimate for porh-ldf was not significant. discussion a complex interplay exists between platelet function and the endothelium in which platelet activation can affect endothe- lial function and endothelial dysfunction may, in turn, influence platelet aggregation. given this multifaceted recip- rocal relationship, we evaluated the response of skin microcirculation endothelial function quantified by th-ldf and porh-ldf to clopidogrel administration in healthy persons. in addition, we assessed the effect of clopidogrel on microcirculatory endothelial function in persons with high versus low baseline composite platelet aggregation. we also assessed the association of microcirculatory endothelial function measures and conventional cardiovascular disease risk factors in persons without established coronary artery disease. finally, we calculated the estimated heritability of ldf measures of endothelial function before and after exposure to clopidogrel. we found that clopidogrel administration improves th- ldf–based measures of microcirculatory endothelial function in healthy persons with high composite platelet aggregability and that the endothelial response was more robust among women. we did not find a relationship between clopidogrel use and our porh-ldf measures. the discrepancy between porh-ldf and th-ldf results is consistent with the fact that they measure different biological components of endothelial function and vascular reactivity, which was the initial rationale for using both measures. porh- ldf captures a complex microvascular response to induced transient ischemia in which the endothelium plays a pivotal role, primarily independent of no and more dependent on other metabolic vasodilators, the myogenic response, and sensory nerves. in contrast, th-induced vasodilatation in skin microcirculation is contingent mainly on endothelial cell– dependent no bioavailability. , , previous in vivo studies found that clopidogrel can enhance the bioavailability of no release, , , and studies in animal models showed that clopidogrel may not confer its beneficial effect on endothelial function merely through p y receptors but possibly by interfering with the interaction between platelets and leuko- cytes, resulting in attenuated inflammation and no bioavail- ability. – these in vivo and animal-based studies implicating a beneficial role of clopidogrel in no bioavailability are consistent with our results showing improvement in th- ldf, which is mediated primarily by no. clinical studies have demonstrated that using antiplatelet therapy before percutaneous coronary intervention attenuates platelet acti- vation and endothelial dysfunction, supporting favorable outcomes in the patients with myocardial infarction. , in addition, studies have shown that antiplatelet therapy improves endothelial function by diminishing inflammation and oxidative stress biomarkers, thereby enhancing no bioavailability. – , in the current study, postclopidogrel th-ldf was increased in the participants with higher baseline platelet aggregation, and that finding is consistent with the hypothesis that the effect of clopidogrel results from modu- lation of high platelet activity. previous studies have suggested that platelet activation leads to the recruitment of inflammatory cells, releasing cytokines even in healthy women. , activated platelets release cd l, resulting in a local inflammatory response. in addition, activated platelets release platelet factor , which upregulates the expression of e-selectin on endothelial cells, resulting in further recruitment and adhesion of inflammatory cells to the endothelial cells. zhang et al showed that clopidogrel improved macrovascular endothelial function measured by flow-mediated dilation in a healthy chinese population. this study was performed in only participants and focused mainly on the macrovascular effect of clopidogrel and cyp c * polymorphism and did not stratify or report effect modification based on platelet function or sex. a previous study looking at the effect of table . paired change from post- to preclopidogrel th- and porh-mediated laser doppler flowmetry in the high composite platelet aggregation group total women men preclopidogrel th � � � * postclopidogrel th . � . � � * n= , p= . † n= , p= . † n= , p= . † preclopidogrel porh . � . . � . . � . postclopidogrel porh . � . . � . . � . n= , p= . † n= , p= . † n= , p= . † data are shown as mean�sd. porh indicates postocclusive reactive hyperemia; th, thermal hyperemia. *p< . for th difference between men and women, the analysis is of the residual data after adjustment for age and family relatedness. †p-value for difference between pre- and postclopidogrel th. doi: . /jaha. . journal of the american heart association clopidogrel improves endothelial function salimi et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , clopidogrel and/or clopidogrel with aspirin on porh-ldf brachial artery reactivity test did not find any effect ; however, this study had only older patients (mean age of years) with comorbidities and only female patients. moreover, the authors did not explore the influence of high platelet aggregability and did not measure microcirculatory response. in our larger study, we found a response in th-ldf measures based on baseline hyperaggregability, with a stronger effect on women. in contrast with the study by ostad et al, another study in relatively older participants showed long-term beneficial effect of clopidogrel on endothe- lial function in patients with coronary artery disease; however, this study used acetylcholine-mediated brachial vasodilation and not porh-ldf or th-ldf–based response. in another small study of participants with coronary artery disease, postclopidogrel endothelial function was measured based on a significantly different pulse wave amplitude system (no measure of blood flow), and it showed a favorable response. last, in a study of participants with established coronary artery disease and effect of clopidogrel, high platelet aggregability was associated with decreased endothelial function; however, the study did not have pre- and postclopidogrel measures. limited reports note that p y receptors reside on the vascular cells – but show no evidence that p y receptors are located on endothelial cells. moreover, in animal models, it has been shown that clopidogrel primarily mediates its effect on vascular endothe- lium by no pathways, presumably independent of p y receptors. – these findings, with ours, suggest that platelet aggregation is associated with decreased endothelial function and that clopidogrel can confer a beneficial effect on endothe- lial function, probably by a no-dependent pathway. we did not find an association between our ldf measures and conventional clinical cardiovascular risk factors except for sex. we believe that the primary reason for this lack of association pertains to the fact that our population was healthy, as opposed to most studies, which included and/or selected patients with chronic diseases. differences in blood pressure in a primarily normotensive population, for example, might not be associated with sufficient vascular disease to affect endothelial function significantly enough to be detectable in a study population with a modest sample size such as ours. in a distinct larger study of brachial flow-mediated dilation in > healthy participants, we similarly found no association between flow- mediated dilation and any established cardiovascular risk factors (shabnam salimi, md, msc, and afshin parsa, md, mph, unpublished data, ). in the current study, we also found that the endothelial function-related benefit of clopidogrel in healthy persons with high platelet aggregability was more robust in women. some studies reported increasing acute coronary syndrome in young women. – doughty et al reported that participants with younger age constituted > % of their patients with acute myocardial infarction, and > % of those participants were women. our sex-specific findings in younger women could also suggest a potential interaction between endothelial function and platelet aggregation or perhaps sex-specific hormones. moreover, we found, for the first time, significant heritability of baseline and postclopidogrel th-ldf endothelial function, suggesting a significant genetic component to microcirculatory-based endothelial function. strengths and limitations our study is the largest study to date aimed at testing the effect of clopidogrel on measures of microcirculatory endothelial function in a healthy population. the use of complementary ldf-based methods enabled us to more comprehensively test for dermal microcirculatory endothelial function and vascular reactivity. the use of a highly homoge- nous founder population allowed us to minimize potential confounders, and cyp c * genotype testing and our measures of platelet aggregability all allowed for careful analyses. nevertheless, stratification by sex and baseline composite platelet aggregation resulted in smaller subgroups, limiting overall power. the lack of a secondary control group with non–clopidogrel-mediated platelet inhibition did not allow us to rule out a non–platelet-mediated effect of clopidogrel on endothelial function. given that the positive effect of clopi- dogrel was noted only in those with high platelet aggregability, a non–platelet-mediated pathway seems less likely. finally, pre- and postclopidogrel serum proinflammatory cytokines measurements and oxidative stress markers could have shed more light on the findings. conclusion our findings demonstrated that clopidogrel can improve th- ldf, a proxy of no bioavailability, which may have vascular- related benefits in addition to its well-established antithrom- botic effects. the th-ldf response to clopidogrel appears to be most pronounced in premenopausal women with high baseline composite platelet aggregation. this sex-based difference suggests a possible interaction among sex- specific hormones, platelet function, and th-ldf. in addi- tion, we found a genetic component to th-ldf–based response, providing further impetus to study the genetic determinant of microcirculatory endothelial function. in summary, these findings implicate potential additional clinical benefits of clopidogrel on microcirculatory no- mediated endothelial cell response in persons with high platelet aggregability. doi: . /jaha. . journal of the american heart association clopidogrel improves endothelial function salimi et al o r ig in a l r e s e a r c h d ow nloaded from http://ahajournals.org by on a pril , acknowledgments we acknowledge the old order amish population and amish community and their cooperation, and the amish clinic staff in lancaster, pennsylvania. sources of funding this work was an ancillary to papi study which was conducted under an investigational new drug protocol (ind , ) and registered in clinicaltrials.gov (nct ), and supported by u hl , u hl , u gm , p dk , nih- k mcrcdp- k rr - and r and t ag . shabnam salimi was supported by nih training grant. disclosures none. references . davi g, patrono c. platelet activation and atherothrombosis. n engl j med. ; : – . . andre p, nannizzi-alaimo l, prasad sk, phillips dr. platelet-derived cd l: the switch-hitting player of cardiovascular disease. circulation. ; : – . . kr€otz f, sohn h-y, pohl u. 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pril , doi: . /j.bbadis. . . hal id: hal- https://hal.archives-ouvertes.fr/hal- submitted on jul hal is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. the documents may come from teaching and research institutions in france or abroad, or from public or private research centers. l’archive ouverte pluridisciplinaire hal, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. calcium-sensing receptor antagonism or lithium treatment ameliorates aminoglycoside-induced cell death in renal epithelial cells claire e. gibbons, david maldonado-pérez, amish n. shah, daniela riccardi, donald t. ward to cite this version: claire e. gibbons, david maldonado-pérez, amish n. shah, daniela riccardi, donald t. ward. calcium-sensing receptor antagonism or lithium treatment ameliorates aminoglycoside-induced cell death in renal epithelial cells. biochimica et biophysica acta - molecular basis of disease, elsevier, , ( ), pp. . � . /j.bbadis. . . �. �hal- � https://hal.archives-ouvertes.fr/hal- https://hal.archives-ouvertes.fr �������� ��	 ���
�� calcium-sensing receptor antagonism or lithium treatment ameliorates aminoglycoside-induced cell death in renal epithelial cells claire e. gibbons, david maldonado-pérez, amish n. shah, daniela riccardi, donald t. ward pii: s - ( ) -x doi: doi: . /j.bbadis. . . reference: bbadis to appear in: bba - molecular basis of disease received date: september revised date: january accepted date: january please cite this article as: claire e. gibbons, david maldonado-pérez, amish n. shah, daniela riccardi, donald t. ward, calcium-sensing receptor antagonism or lithium treatment ameliorates aminoglycoside-induced cell death in renal epithelial cells, bba - molecular basis of disease ( ), doi: . /j.bbadis. . . this is a pdf file of an unedited manuscript that has been accepted for publication. as a service to our customers we are providing this early version of the manuscript. the manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. http://dx.doi.org/ . /j.bbadis. . . http://dx.doi.org/ . /j.bbadis. . . a c c e p te d m a n u s c r ip t accepted manuscript calcium-sensing receptor antagonism or lithium treatment ameliorates aminoglycoside-induced cell death in renal epithelial cells *claire e gibbonsa, *david maldonado-pérezb,c, amish n shaha, daniela riccardib, donald t warda afaculty of life sciences, the university of manchester, manchester, uk bcardiff school of biosciences, cardiff university, cardiff, uk cmrc human reproductive sciences unit, edinburgh, uk * these authors contributed equally. key words: calcium-sensing receptor, aminoglycoside nephrotoxicity, lithium, proximal tubule, gentamicin. address correspondence to: dr. donald ward, faculty of life sciences, floor , core technology facility, the university of manchester, grafton street, manchester, m nt, united kingdom. tel: ; fax: ; email: d.ward@manchester.ac.uk a c c e p te d m a n u s c r ip t accepted manuscript summary the aminoglycoside antibiotic gentamicin elicits proximal tubular toxicity and cell death. in calcium-sensing receptor (car)-transfected hek- (car-hek) cells and car-expressing proximal tubule-derived opossum kidney (ok) cells, chronic gentamicin treatment elicits dose-dependent, caspase-mediated apoptotic cell death. here we investigated whether the renal cell toxicity of the car agonist gentamicin could be prevented by car antagonists or by lithium cotreatment which may interfere with receptor-mediated signalling. chronic treatment of ok and car-hek cells with low concentrations of gentamicin elicited cell death, an effect that was ameliorated by cotreatment with the car negative allosteric modulator (calcilytic) nps- . this calcilytic also attenuated car agonist-induced erk activation in these cells. in addition, mm licl, equivalent to its therapeutic plasma concentration, also inhibited gentamicin-induced toxicity in both cell types. this protective effect of lithium was not due to the disruption of phosphatidylinositol-mediated gentamicin uptake as the cellular entry of texas red- conjugated gentamicin into ok and car-hek cells was unchanged by lithium treatment. however, the protective effect of lithium was mimicked by glycogen synthase β inhibition. together, these data implicate car activation and a lithium- inhibitable signalling pathway in the induction of cell death by gentamicin in renal epithelial cells in culture. a c c e p te d m a n u s c r ip t accepted manuscript introduction the aminoglycoside antibiotics (agas) are an effective and economical treatment for life-threatening, gram-negative infections. however, the clinical usefulness of the agas is limited by their toxicity in the renal proximal tubule as well as their ototoxicity [ ]. we have shown previously that proximal tubule-derived opossum kidney (ok) cells express a calcium-sensing receptor (car)-like protein and elicit typical responses to car agonists [ ]. more recently we have shown that chronic exposure of ok cells to gentamicin, or indeed other car agonists such as spermine or poly-arginine, promotes cell death [ ]. in addition, the effect of gentamicin on cell-fate was also investigated using hek- cells and it was found that cells transfected with the car were considerably more susceptible to aga toxicity than non-transfected or empty vector-transfected controls. thus, given that car is expressed at the apical surface of the proximal tubule [ , ], where gentamicin-induced nephrotoxicity occurs, and where the acidic conditions enhance the potency of gentamicin as a car agonist [ ], these cell culture data suggest that the car could contribute to aga nephrotoxicity. however, in the absence of pharmacological inhibitors of the receptor it has not been previously possible to confirm an involvement of car in aga-mediated cellular toxicity. furthermore, the concentrations of gentamicin employed in the previous cell culture studies were at least an order of magnitude higher than those used clinically [ ] and therefore the contribution of car to aga toxicity in renal cell culture could not be demonstrated directly. therefore, here we began by assessing the contribution of the car to aga renal cell toxicity by testing whether a negative allosteric modulator of car, the calcilytic nps- , exerted a protective effect in a c c e p te d m a n u s c r ip t accepted manuscript renal-derived cells treated chronically with therapeutically relevant concentrations of gentamicin. despite being first described as a mood stabilizer in [ ], lithium currently remains an effective and inexpensive treatment for bipolar disorder [ ]. its therapeutic mechanism of action may result from its interference in phosphatidylinositol turnover [ ] given the inhibitory effect of lithium on inositol monophosphatase [ , ]. however, more recently the ability of lithium to inhibit glycogen synthase kinase (gsk)- β [ ] has been one of a number of mechanisms postulated to explain its therapeutic effect [ ]. in the context of aga nephrotoxicity, it has been reported that in rats injected with gentamicin for days, concurrent therapy with lithium decreased the gentamicin-induced formation of renal lysosomal myeloid bodies [ ], possibly via interference of lithium with phosphoinositide turnover. as a hydrophilic, polycationic drug, gentamicin binds negatively-charged phospholipid bilayers and as such this binding could contribute to the cellular uptake of gentamicin, at least in association with the binding of the drug to the megalin / cubilin complex [ , ]. therefore, we then tested whether lithium could ameliorate gentamicin-induced toxicity in ok and car- hek cells, and whether it did so by affecting drug uptake or not. a c c e p te d m a n u s c r ip t accepted manuscript materials and methods materials unless otherwise stated, items were obtained from sigma-aldrich (poole, dorset, uk) or from sources described previously [ ]. gsk- ß inhibitor xi ( -( -( - hydroxypropyl)- h-pyrrolo[ , -b]pyridin- -yl]- -pyrazin- -yl-pyrrole- , -dione) and gsk- inhibitor ix (( 'z, 'e)- - bromoindirubin- '-oxime) were purchased from calbiochem. cell culture opossum kidney (ok) cells (used within - passages of purchase from the american type culture collection, rockville, md) and hek- cells, stably transfected with human parathyroid car (car-hek) [ ], were a gift from dr. e.f. nemeth (nps pharmaceuticals, inc., salt lake city, utah, usa) and were cultured as described previously [ , ]. cell death counting cells ( - % confluency, mm dishes) were incubated in medium containing % serum supplemented with various treatments for up to days, or for up to days with an addition of % serum after days to maintain viability. adherent cells were mixed : (v/v) with . % trypan blue solution. viable cells (trypan blue-excluded) and non- viable cells (trypan-blue stained, including cells floating in the medium) were counted on a light microscope using a neauber’s counting slide. a c c e p te d m a n u s c r ip t accepted manuscript erk activation assay cells were grown to - % confluence in mm culture dishes and erk phosphorylation assayed as described previously [ , ]. briefly, cells were incubated at oc prior to lysis on ice in ripa buffer supplemented with protease and phosphatase inhibitors and proteins resolved by sds-page prior to immunoblotting using total erk and phospho-specific erk antibodies. flow cytometric dna analysis cells grown in culture flasks were treated as above and then resuspended in serum-free medium containing propidium iodide ( µg/ml). flow cytometric analysis was performed using a facs vantage flow cytometer equipped with an enterprise laser (innova technology; laser excitation at nm, mv). red fluorescence (dna- bound propidium) was detected at ± nm and acquired using logarithmic amplifiers. forward (fsc) and side (ssc) light scatter were also recorded to indicate cell size and granularity, respectively. a total of , cells were analyzed per sample and cell-fit software (becton dickinson) was used to evaluate the data. texas red gentamicin internalisation hek- cells, transfected with either pcdna . or egfp tagged car (car-egfp) [ ] vectors were incubated at °c in prewarmed hepes-buffered physiological saline containing texas red-conjugated gentamicin (trg, mg/ml; molecular probes, eugene, or) for min as described previously [ ]. after fixation, the relative levels of trg uptake were studied by confocal microscopy using an ultraview confocal optical scanner with a kr/ar laser (perkin elmer life sciences, cambridge, uk) mounted on an olympus ix inverted microscope. images were acquired with an ultrapix ccd a c c e p te d m a n u s c r ip t accepted manuscript digital camera and processed using perkin elmer ultraview software package. laser intensity, shutter speed and image capture speed were constant throughout the acquisition process of each experiment. quantification of trg internalisation was performed using a fluostar microplate based multi-detection reader (bmg labtech, durham, nc, usa). statistical analysis – unless otherwise stated, data are presented as means ± s.e. and statistical significance determined by one-way anova (tukey post-hoc test; p< . ). a c c e p te d m a n u s c r ip t accepted manuscript results we have shown previously that day exposure of proximal tubule-derived ok cells to µm gentamicin elicits cell death and that increased cell death was observed in car- hek cells following chronic exposure to µm gentamicin [ ]. however, the slow evolution of acute renal failure (depression of the gfr) does not normally manifest itself clinically before - days of treatment, underlying complications notwithstanding [ , , ]. in addition, the gentamicin concentrations used previously in our cell culture study were an order of magnitude greater than the target plasma concentration aimed for in antibiotic therapy [ ]. we therefore examined whether reproducing conditions resembling more closely those achieved therapeutically (i.e., more prolonged exposures and use of lower concentrations of the drug) we could still observe cytotoxicity. our results show that, indeed, chronic treatment for - days with - µm gentamicin elicited significant cell death in car-hek cells (figure a). similarly, ok cells exhibited significant cell death when treated with µm gentamicin (figure b). these dead cells included both floating cells and trypan blue-stained adherent cells. to demonstrate more conclusively a role for car in the development of aga- induced cellular injury, we used a car negative allosteric modulator. cotreatment with the calcilytic nps- resulted in a significant reduction in car-hek cell death in response to high concentrations of gentamicin (i.e., µm) and completely ablated the toxic effect of µm when the drugs were administered for days only (figure a). when ok cells were cotreated with nps- , a significant attenuation (- %, p< . ) of gentamicin ( µm, days) toxicity was observed (figure b). to confirm that nps- inhibits car-mediated responses in these cells, ok and car-hek cells were stimulated with either mm ca +o or µm gentamicin in the presence or absence a c c e p te d m a n u s c r ip t accepted manuscript of the calcilytic and then tested for extracellular-regulated kinase (erk) phosphorylation / activation, a well-established functional readout for car activation [ , ]. whilst both high ca +o concentration and gentamicin elicited erk activation, as demonstrated previously [ ], cotreatment with nps- blocked these responses in both cell types (figure c). there is evidence that lithium may interfere with gentamicin nephrotoxicity in rats [ ]. to determine whether lithium exerts its effect directly on renal cells, or via a more systemic effect, we studied gentamicin-induced cell death in both ok cells and car- hek cells in the presence or absence of the clinical target concentration of licl ( mm). indeed, licl cotreatment significantly inhibited the cytotoxicity of gentamicin in car- hek (figure a) and ok cells (figure b) as determined by trypan blue exclusion. these data were supported by flow cytometry experiments in which lithium cotreatment ameliorated gentamicin-induced cell death in both the car-hek cells (figure ) and ok cells (figure ). in the flow cytometry experiments, the dead cells were those exhibiting increased propidium iodide uptake (panel a), as well as decreased forward scatter (size) and increased side scatter (granularity; panel c) indicative of apoptosis [ ]. deterioration of proximal tubular function observed as a consequence of aga toxicity in patients has been traditionally ascribed to drug endocytosis and its sequestration into lysosomes, with formation of myeloid bodies and phospholipidosis [ ]. to examine whether the apparently protective effect of lithium occurs by inhibiting gentamicin internalisation, we employed a texas red-conjugated form of gentamicin (trg) [ ]. interestingly, lithium did not appear to reduce gentamicin cellular uptake. confocal images of car-egfp-transfected hek cells (figure a) and ok cells (figure b) pretreated with licl ( mm, min) and then incubated in trg (+ mm licl, min, oc) showed similar levels of trg uptake compared to that a c c e p te d m a n u s c r ip t accepted manuscript observed in non-lithium treated cells. these observations were reproduced using a quantitative fluorometric assay (ok cells, p> . ) (figure bii). finally, lithium is a known inhibitor for glycogen synthase kinase (gsk)- ß and inhibition of this kinase could contribute to the apparent cell-protective effect of lithium [ ]. therefore, we investigated whether gsk- ß inhibition is capable of attenuating gentamicin-induced cell death in ok and car-hek cells. indeed, the gsk- ß inhibitor xi inhibited ( µm) gentamicin-induced cell death in car-hek (figure a) and ok (figure b) cells, whilst the gsk- inhibitor ix also inhibited gentamicin toxicity in the ok cells (figure b). a c c e p te d m a n u s c r ip t accepted manuscript discussion here we provide pharmacological evidence that the car is capable of mediating aga- induced toxicity in cultured proximal tubule-derived cells and that such cellular toxicity can be ameliorated by lithium cotreatment. the car negative allosteric modulator (calcilytic) nps- has been shown previously to inhibit high [ca +]o-induced mineralisation in fetal rat calvarial cells [ ], and in car-hek cells to inhibit calcimimetic-induced actin polymerisation [ ] and cart phosphorylation [ ]. other calcilytics have been shown to stimulate pth secretion in rats and isolated bovine parathyroid cells [ - ] as well as inhibiting inositol phosphate metabolism and ca +i mobilisation in car-hek cells [ , , ]. here we demonstrate that nps- inhibits acute high [ca +]o- and gentamicin-induced erk activation and chronic gentamicin-induced cell death in ok and car-hek cells. initially, µm nps- was employed (figure a) as was used previously [ - ] however in later experiments (figures b & c) nm was found to be equally effective. the simplest explanation for these data is that nps- is acting by inhibiting car activity, presumably by decreasing receptor agonist sensitivity. this observation is supported by our previous studies in which we have demonstrated car expression and function in ok cells [ ] and shown [ ] that ok cell death can also be induced by the car agonists spermine [ ] and poly-arginine [ ]. furthermore, we have shown previously that using similar gentamicin concentrations, the drug elicits significant apoptotic cell death in car-hek cells but not in hek cells stably transfected with the empty vector alone [ ]. thus, it would be interesting to determine whether calcilytic cotreatment protects gentamicin- injected rats from proximal tubule injury. variations in basal cell death levels between experiments were observed. the a c c e p te d m a n u s c r ip t accepted manuscript precise amount of basal cell death recorded in any experiment depends on a variety of factors including the rate of growth of the cells, the depletion of nutrients, space and growth factors, as well as the method by which cells are collected and cell death quantified. in general however, we found both here and previously ( ) that the relative toxic effect of gentamicin tended to be quite consistent. with regards the gentamicin exposure conditions it should be noted that the drug was present in the media throughout the - day treatments whereas with clinical use, daily peaks and troughs in plasma gentamicin concentration would alter the pharmacodynamics of proximal tubular exposure to the drug. the possible consequence, if any, of such a difference is unclear and thus an in vivo study will be necessary to confirm the significance of the cell study. we also demonstrated a protective effect of lithium on gentamicin-induced cell death in the renal-derived cells. the effect was significant though partial in some experiments (figs & ), but complete in others (figs & ). whether higher concentrations of lithium would have consistently ablated gentamicin-induced apoptosis was not tested. the mm lithium treatment used here was chosen deliberately to correspond to the target therapeutic plasma concentration employed in bipolar disorders. since alternative theories of gentamicin nephrotoxicity involve aga endocytosis [ ], then one possible explanation for the apparent protective effect of lithium would be that it attenuates gentamicin cellular uptake by interfering with the turnover of negatively- charged inositol phospholipids with which gentamicin would bind on the membrane. however, at the gentamicin concentration tested, lithium failed to reduce the acute cellular uptake of texas red-conjugated gentamicin into the car-hek or ok cells, despite decreasing the chronic toxicity of the aga. therefore, since lithium can also interfere in the glycogen synthase kinase-mediated pathway, which is involved in the regulation of cell fate [ ], we tested the effect of gsk- ß inhibitors and found that they a c c e p te d m a n u s c r ip t accepted manuscript also inhibited gentamicin-induced car-hek and ok cell toxicity. our observations implicate gsk- ß signalling in the mediation of the car-mediated gentamicin toxicity seen here, possibly via the gsk / wnt / β-catenin pathway. whilst this does not prove that the protective effect of lithium occurs via gsk- ß inhibition, there at least appears to be good correlation between the protective effects of lithium and of the gsk- ß inhibitor xi. in the brain, the neuroprotective effect of lithium treatment has been associated with altered expression of pro- and anti-apoptotic proteins such as bax and bcl- respectively [ ] and this may play a role here. in any case, we propose that lithium should now be tested in rats to determine whether it may serve as an effective ameliorant for aga-induced nephrotoxicity. in this regard, lithium has already been shown to reduce the formation of lysosomal myeloid bodies in rat renal cortex following chronic gentamicin treatment [ ]. it should be noted that while we have shown that lithium fails to reduce total trg uptake into the cells while attenuating gentamicin toxicity, we cannot rule out the possibility that lithium may divert the gentamicin from one intracellular locale to another one in which the aga exerts reduced toxicity. in this regard, others have shown that porcine, proximal tubular-derived llc-pk cells traffic trg rapidly to the endoplasmic reticulum and then on to the cytosol and nucleus [ ] and thus lithium could act by interfering with such a process. previous studies have shown that aga-elicited cellular events include the impairment of glucose, protein and ion transport [ ] as well as numerous biochemical abnormalities [ , ]. since gentamicin elicits ok cell toxicity over a similar -day time course to the appearance of nephrotoxicity in humans and at concentrations as low as µm, we would propose that future cellular studies of gentamicin toxicity should use concentrations of drug much closer to the target plasma concentration in humans [ ]. at high concentrations, the agas exhibit non-cell specific cytotoxicity by a c c e p te d m a n u s c r ip t accepted manuscript interfering with protein translation and are indeed often employed as selection agents in mammalian expression systems. therefore, high gentamicin concentrations may induce cytotoxicity by means not related to the clinical nephrotoxic mechanism, especially given that gentamicin is not generally cytotoxic in other organs (except for its ototoxicity) during clinical therapy. in an elegant series of experiments, el mouedden et al demonstrated that rats receiving low dose gentamicin treatment, i.e. at low multiples of the therapeutic dose, exhibit proximal tubule cell apoptosis whereas higher doses of drug were required to elicit acute tubular necrosis [ ]. accordingly, low concentrations of gentamicin, as used in the current study, may elicit different toxic responses and by alternate cellular mechanisms than for higher doses and thus may have greater relevance in understanding the proximal tubular toxicity of the agas. the use of agas with lower nephrotoxic potential, such as amikacin, isepamacin or even the c gentamicin congener [ ] in addition to the use of single daily dosing will lead to a reduction in the prevalence of aga nephrotoxicity where employed. however due to its low cost, gentamicin therapy continues to be widely-used globally and thus it remains important to understand the molecular cause(s) of the proximal tubule injury particularly if an economical cotreatment adjunct such as lithium can be shown to be an effective ameliorant. in this regard, it will be necessary next to show that lithium actually ameliorates aga-induced nephrotoxicity in vivo, for example in rats. the physiological function(s) of the proximal tubular car may include the regulation of volume absorption, vitamin d α-hydroxylation and phosphate reabsorption [ , - ]. we propose therefore that during aga therapy the presence of luminal gentamicin may cause additional and excessive activation of the proximal tubular car causing sustained receptor-induced signalling leading to apoptotic cell death and that this should now be tested in an animal model. together, these data a c c e p te d m a n u s c r ip t accepted manuscript implicate the car and a lithium-inhibitable signalling pathway in the mediation of gentamicin toxicity in proximal tubular-derived ok cells and car-hek cells. a c c e p te d m a n u s c r ip t accepted manuscript acknowledgements this work was supported by a grant from the bbsrc (bbs/b/ ). dtw was a recipient of a kidney research uk career development fellowship (tf / ). the authors would like to thank dr. ed nemeth and nps pharmaceuticals, inc. (salt lake city, utah, usa) for supplying the calcimimetic and calcilytic reagents, mr mike jackson (faculty of life sciences, the university of manchester) for his advice and assistance with the facs analysis and tim stirling and laura tones for their technical assistance with the trypan blue cell counting. references [ ] r.a. santucci, j.n. krieger, gentamicin for the practicing urologist: review of efficacy, single daily dosing and "switch" therapy, j. urol. ( ) - . [ ] d.t. ward, s.j. mclarnon, d. riccardi, aminoglycosides increase intracellular calcium levels and erk activity in proximal tubular ok cells expressing the extracellular calcium-sensing receptor, j. am. soc. nephrol. ( ) - . [ ] d.t. ward, d. maldonado-perez, l. hollins, d. riccardi, aminoglycosides induce acute cell signaling and chronic cell death in renal cells that express the calcium- sensing receptor, j. am. soc. nephrol. ( ) - . 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[ ] e.i. christensen, h. birn, megalin and cubilin: synergistic endocytic receptors in renal proximal tubule, am. j. physiol. - renal physiol. ( ) f -f . [ ] s.k. moestrup, c. shyying, h. vorum, c. bregengard, s.e. bijorn, k. norris, j. gliemann, e.i. christensen, evidence that epithelial glycoprotein /megalin mediates uptake of polybasic drugs, j. clin. invest. ( ) - . [ ] j.e. garrett, i.v. capuano, l.g. hammerland, b.c.p. hung, e.m. brown, s.c. hebert, e.f. nemeth, f. fuller, molecular cloning and functional expression of human parathyroid calcium receptor cdnas, j. biol. chem. ( ) - . [ ] g.j. kaloyanides, aminoglycoside nephrotoxicity. in: r.w. schrier, c.w. gottschalk (eds.), diseases of the kidney, th ed., little brown and company, london, , pp. - . [ ] r.d. moore, c.r. smith, p.s. lietman, predicting aminoglycoside nephrotoxicity, jama ( ) - . [ ] o. kifor, r.j. macleod, r. diaz, m. bai, t. yamaguchi, t. yao, i. kifor, e.m. brown, regulation of map kinase by calcium-sensing receptor in bovine parathyroid and car-transfected hek cells, am. j. physiol. renal physiol. ( ) f - . [ ] u. trechsel, j.a. eisman, j.a. fischer, j.p. bonjour, h. fleisch, calcium- dependent, parathyroid hormone-independent regulation of , -dihydroxyvitamin d, am. j. physiol. ( ) e -e . a c c e p te d m a n u s c r ip t accepted manuscript [ ] r.a. giuliano, g.j. paulus, g.a. verpooten, v.m. pattyn, d.e. pollet, e.j. nouwen, g. laurent, m.b. carlier, p. maldague, p.m. tulkens, recovery of cortical phospholipidosis and necrosis after acute gentamicin loading in rats, kidney int. ( ) - . [ ] m. pap, g.m. cooper, role of glycogen synthase kinase- in the phosphatidylinositol -kinase/akt cell survival pathway, j. biol. chem. , ( ) - . [ ] m.m. dvorak, a. siddiqua, d.t. ward, d.h. carter, s.l. dallas, e.f. nemeth, d. riccardi, physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones, proc. natl. acad. sci. u s a ( ) - . [ ] s.l. davies, c.e. gibbons, t. vizard, d.t. ward, ca +-sensing receptor induces rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids, am. j. physiol. cell physiol. ( ) c - c . [ ] sl davies, a ozawa, wd mccormick, mm dvorak, dt.ward, protein kinase c- mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity, j. biol. chem. ( ) - . [ ] b.j. arey, r. seethala, z. ma, a. fura, j. morin, j. swartz, v. vyas, w. yang, j.k. dickson jr, j.h. feyen, a novel calcium-sensing receptor antagonist transiently stimulates parathyroid hormone secretion in vivo, endocrinology ( ) - . [ ] m. gowen, g.b. stroup, r.a. dodds, i.e. james, b.j. votta, b.r. smith, p.k. bhatnagar, a.m. lago, j.f. callahan, e.g. delmar, m.a. miller, e.f. nemeth, j. fox, antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats, j. clin. invest. ( ) - . [ ] e.f. nemeth, e.g. delmar, w.l. heaton, m.a. miller, l.d. lambert, r.l. conklin, m. gowen, j.g. gleason, p.k. bhatnagar, j. fox, calcilytic compounds: potent and selective ca + receptor antagonists that stimulate secretion of parathyroid hormone, j. pharmacol. exp. ther. , ( ) - . [ ] a. kessler, h. faure, c. petrel, d. rognan, m. cesario, m. ruat, p. dauban, r.h. dodd, n -benzoyl-n -[ -( -naphthyl)ethyl]-trans- , -diaminocyclohexanes: development of -chlorophenylcarboxamide (calhex ) as a new calcium sensing receptor ligand demonstrating potent calcilytic activity, j. med. chem. ( ) - . [ ] s.j. quinn, c.p. ye, r. diaz, o. kifor, m. bai, p. vassilev, e.m. brown, the ca +-sensing receptor: a target for polyamines, am. j. physiol. ( ) c - c . a c c e p te d m a n u s c r ip t accepted manuscript [ ] e.m. brown, c. katz, r. butters, o. kifor, polyarginine, polylysine, and protamine mimic the effects of high extracellular calcium concentrations on dispersed bovine parathyroid cells, j. bone miner. res. ( ) - . [ ] d.m. chuang, neuroprotective and neurotrophic actions of the mood stabilizer lithium: can it be used to treat neurodegenerative diseases? crit. rev. neurobiol. ( ) - . [ ] r.m. sandoval, b.a.molitoris, gentamicin traffics retrograde through the secretory pathway and is released in the cytosol via the endoplasmic reticulum. am. j. physiol. renal physiol. ( ) f -f . [ ] m.c. sassen, s.w. kim, t.h. kwon, m.a. knepper, r.t. miller, j. frokiaer, s. nielsen, dysregulation of renal sodium transporters in gentamicin-treated rats, kidney int. ( ) - . [ ] m. el mouedden, g. laurent, m-p mingeot-leclercq, h.s. taper, j. cumps, p.m. tulkens, apoptosis in renal proximal tubules of rats treated with low doses of aminoglycosides, antimicrob. agents chemother. ( ) – . [ ] r.m. sandoval, j.p. reilly, w. running, s.b. campos, j.r. santos, c.l. phillips, b.a. molitoris, a non-nephrotoxic gentamicin congener that retains antimicrobial efficacy, j. am. soc. nephrol. ( ) - . [ ] r. bland, e.a. walker, s.v. hughes, p.m. stewart, m. hewison, constitutive expression of -hydroxyvitamin d - alpha-hydroxylase in a transformed human proximal tubule cell line: evidence for direct regulation of vitamin d metabolism by calcium, endocrinology ( ) - . [ ] d.t. ward, d. riccardi, renal physiology of the extracellular calcium-sensing receptor, pflügers arch. – eur. j. physiol. ( ) - . [ ] a. maiti, n.c. hait, m.j. beckman, extracellular calcium sensing receptor activation induces vitamin d receptor levels in proximal kidney hk- g cells by a mechanism that requires phosphorylation of p alpha mapk, j biol chem. ( ) [epub ahead of print]. a c c e p te d m a n u s c r ip t accepted manuscript figures and legends figure . gentamicin induces cell death in car-hek and ok cells at low concentrations. car-hek cells were treated for days with , or µm gentamicin, with addition of fresh serum ( %) to each dish at day . cell death was then determined by trypan blue exclusion (panel a, n= - ) including both floating and adherent cells. alternatively, ok cells were treated for days with µm gentamicin, with addition of fresh serum ( %) to each dish at day and then cell death quantified as before (panel b, n= ). *** p< . vs control; +++ p< . vs all others by anova. figure . cotreatment with the calcilytic nps- attenuates gentamicin- induced cell death and car-induced erk activation in car-hek and ok cells. car-hek cells were treated for days with (panel a, n= ) or µm gentamicin (panel b, n= ) in the presence or absence of µm nps- ( . % dmso vehicle added to controls) and cell death determined by trypan blue exclusion. alternatively, ok cells were treated for days with or without µm gentamicin in the presence or absence of nm nps- , with addition of fresh serum ( %) to each dish at day and then cell death quantified as before (panel b, n= ). * p< . , ** p< . . , *** p< . vs control; + p< . , ++ p< . vs gentamicin by anova. panel c, car- hek (i) or ok cells (ii) were stimulated with either mm ca +o, or µm gentamicin in the presence or absence of nps- ( nm) for min at oc and then lysed in ripa buffer as described in methods. the phospho-erk content was then determined by immunoblotting using a phospho-specific anti-erk antiserum and a c c e p te d m a n u s c r ip t accepted manuscript the total levels of erk shown to be unchanged using an anti-erk polyclonal antibody. figure . cotreatment with lithium ameliorates gentamicin-induced cell death in car-hek and ok cells. car-hek cells were treated for days with µm gentamicin (panel a, n= - ) in the presence or absence of mm licl. cell death was then determined by trypan blue exclusion. alternatively ok cells received the same day treatments (panel b, n= - ). ** p< . vs all others, by anova. figure . flow cytometric analysis of lithium cotreatment on gentamicin-induced car-hek cell death. panel a, car-hek cells were treated for days in t culture flasks with µm gentamicin in the presence or absence of mm licl with subsequent cell death assessed by propidium iodide uptake as quantified by flow cytometry (facs vantage). panel b, a histogram shows the pooled cell death data (± s.e.m.) from three independent experiments (including replicates). * p< . gentamicin vs lithium & gentamicin by t-test. panel c, -dimensional plot showing the cell size and granularity profiles of the cells shown in panel a. in panels a & c, the data shown represent the median responses. gentamicin increased the proportion of smaller, more granular cells (marked by a polygon and consisting almost exclusively of pi-positive cells) and this effect was ameliorated by licl cotreatment. figure . flow cytometric analysis of lithium cotreatment on gentamicin-induced ok cell death. ok cells treated for days in t culture flasks with µm gentamicin in the presence or absence of mm licl were processed for cell death analysis by flow cytometry as described in the legend to figure . the gentamicin-induced increase in the proportion of pi-positive cells (panels a&b) and the proportion of smaller, more granular cells (panel c) was attenuated by licl cotreatment. ** p< . gentamicin vs a c c e p te d m a n u s c r ip t accepted manuscript lithium & gentamicin by t-test (n= ). figure . lithium cotreatment fails to prevent texas red-gentamicin uptake into car-hek and ok cells. panel a, hek cells, transfected stably with either empty vector (pcdna . /hygro) or human car, were preincubated for mins in the presence or absence of mm licl and then incubated for min in texas red-conjugated gentamicin (trg) ± licl. following cell washing to remove excess trg, microfluorescence imaging revealed equivalent gentamicin uptake in both sets of cells. panel b, an identical experiment was performed on ok cells. representative fluorescence images are presented (i) together with a histogram showing relative fluorescence unit (rfu) levels indicative of trg uptake (ii). image exposure times were identical for cell fluorescence within each experiment e.g. trg ± lithium. results are from three independent experiments performed at least in duplicate. figure . inhibitors of glycogen synthase kinase- attenuate gentamicin-induced cell death in ok and car-hek cells. panel a, car-hek cells were treated for days with µm gentamicin in the presence or absence of mm licl or nm gsk- ß inhibitor xi and then cell death was then determined by trypan blue exclusion. panel b, ok cells were treated as in a, but instead cotreated with either gsk- inhibitor xi or ix ( nm). *** p< . vs control; +p< . , ++p< . vs gentamicin, by anova; (n= ). a c c e p te d m a n u s c r ip t accepted manuscript a c c e p te d m a n u s c r ip t accepted manuscript a c c e p te d m a n u s c r ip t accepted manuscript a c c e p te d m a n u s c r ip t accepted manuscript a c c e p te d m a n u s c r ip t accepted manuscript a c c e p te d m a n u s c r ip t accepted manuscript a c c e p te d m a n u s c r ip t accepted manuscript wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ [pdf] amish burn ointment and burdock leaf dressings: assessments of antimicrobial and cytotoxic activities | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /bcr. b e a corpus id: amish burn ointment and burdock leaf dressings: assessments of antimicrobial and cytotoxic activities @article{rieman amishbo, title={amish burn ointment and burdock leaf dressings: assessments of antimicrobial and cytotoxic activities}, author={m. rieman and a. neely and s. boyce and william j kossenjans and p. durkee and jacquelyn m. zembrodt and barbara k. puthoff and r. kagan}, journal={journal of burn care & research}, year={ }, volume={ }, pages={e –e } } 	m. rieman, a. neely, + authors r. kagan 	published 	medicine 	journal of burn care & research amish burn wound ointment (abo) contains honey, lanolin, oils, glycerin, bees wax, and other natural additives. although there are many anecdotal reports that this ointment covered with a burdock leaf (bl) dressing promotes burn wound healing, little scientific testing of this treatment has occurred. the goal of this study was to evaluate in vitro some of the components of this treatment modality for antimicrobial and cytotoxic activities. the abo was tested for sterility using standard… expand view on wolters kluwer webcentral.uc.edu save to library create alert cite launch research feed share this paper citationsbackground citations view all topics from this paper 	ointments 	glycerin 	lanolin 	burn injury 	keratinocyte 	cell culture techniques 	evaluation procedure 	biological assay 	fibroblasts 	bees 	in vitro [publication type] 	freeze drying 	ploidies 	dry eye syndromes 	salve dosage form 	aloe vera leaf extract 	waxes citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency histopathological evaluation of burdock ( arctium lappa ) root hydroalcoholic extract on wound healing 	f. ghorat, m. azizkhani, shahriar naji, a. g. ranjbary, farzad doostishoar 	medicine 	 	 	pdf 	view excerpt, cites background save alert research feed honey-based salve and burdock leaf dressings as an alternative to surgical debridement of a traumatic wound eschar 	adam schell, jonathan copp, k. bogie, r. wetzel 	medicine 	advances in wound care 	 	pdf 	view excerpt, cites background save alert research feed amish culture and their utilization of burns and wounds ointment for the treatment of burns. 	krystal melich trinkle 	medicine 	holistic nursing practice 	 	 save alert research feed the management of paediatric burns with burns and wounds ointment and burdock leaves: a case series. 	shelby l. nathan, autumn d nanassy, b. burkey, w. davis, p. glat 	medicine 	journal of wound care 	 save alert research feed traditional and ethnobotanical dermatology practices in romania and other eastern european countries. 	m. gilca, g. s. tiplica, c. sălăvăstru 	geography, medicine 	clinics in dermatology 	 	 save alert research feed caring for the amish: what every anesthesiologist should know 	g. weller 	medicine 	anesthesia and analgesia 	 	 save alert research feed seventy-five years of amish studies, to : a critical review of scholarship trends (with an extensive bibliography) 	c. anderson 	history 	 	 	pdf save alert research feed cultural contexts of health and illness among the lancaster amish 	m. king 	sociology 	 	 	view excerpts, cites background save alert research feed references showing - of references sort byrelevance most influenced papers recency noncytotoxic combinations of topical antimicrobial agents for use with cultured skin substitutes. 	s. boyce, g. warden, i. holder 	biology, medicine 	antimicrobial agents and chemotherapy 	 	 	pdf save alert research feed cytotoxicity testing of topical antimicrobial agents on human keratinocytes and fibroblasts for cultured skin grafts. 	s. boyce, g. warden, i. holder 	medicine 	the journal of burn care & rehabilitation 	 	 	pdf save alert research feed a laboratory method for selection of topical antimicrobial agents to treat infected burn wounds 	p. nathan, e. law, d. murphy, b. macmillan 	medicine 	 	 save alert research feed quantitative assay for quality assurance of human cells for clinical transplantation 	s. boyce, b. a. anderson, horacio l. rodriguez-rilo 	chemistry, medicine 	cell transplantation 	 	 	pdf save alert research feed general practitioners' assessment of and interest in alternative medicine in canada. 	m. verhoef, l. sutherland 	medicine 	social science & medicine 	 	 save alert research feed the integration of bio-medicine and culturally based alternative medicine: implications for health care providers and patients 	b. lovell 	medicine 	global health promotion 	 	 save alert research feed moving lines and variable criteria: differences/connections between allopathic and alternative medicine 	fred m. frohock 	medicine 	 	 save alert research feed policy, the public, and priorities in alternative medicine research 	w. jonas 	sociology 	 	 save alert research feed mortality after treating severe burns with traditional amish home remedies: a case report, literature review and ethical discussion. 	s. a. kahn, r. demme, c. lentz 	medicine 	burns : journal of the international society for burn injuries 	 	 save alert research feed burn and wound dressing supplies 	newcomerstown (oh): holistic acres. catalog no 	 ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue apoc , coronary disease, and complexities of mendelian randomization cell metabolism previews of global metabolic control, affecting adi- pose tissue physiology and glucose use by the liver through a novel upr cell- nonautonomous mechanism. it remains to be shown whether long-term manipula- tion of this pathway influences other func- tional outputs of pomc neurons, but for the time being, the specificity and cell- type-restricted mode of action of xbp s in the hypothalamus can be considered a promising therapeutic candidate to treat prevalent metabolic diseases. acknowledgments the authors are supported by grants p - -f, fondecyt , conicyt-usa - , and act (c.h.); fondecyt (p.m.); and r ag (a.d.). references deng, y., wang, z.v., tao, c., gao, n., holland, w.l., ferdous, a., repa, j.j., liang, g., ye, j., lehrman, m.a., et al. ( ). j. clin. invest. , – . gao, q., and horvath, t.l. ( ). annu. rev. neurosci. , – . hetz, c. ( ). nat. rev. mol. cell biol. , – . hetz, c., and mollereau, b. ( ). nat. rev. neurosci. , – . hotamisligil, g.s. ( ). cell , – . cell metabolism , s ozcan, l., ergin, a.s., lu, a., chung, j., sarkar, s., nie, d., myers, m.g., jr., and ozcan, u. ( ). cell metab. , – . stanley, s., pinto, s., segal, j., pérez, c.a., viale, a., defalco, j., cai, x., heisler, l.k., and friedman, j.m. ( ). proc. natl. acad. sci. usa , – . taylor, r.c., and dillin, a. ( ). cell , – . taylor, r.c., berendzen, k.m., and dillin, a. ( ). nat. rev. mol. cell biol. , – . williams, k.w., liu, t., kong, x., fukuda, m., deng, y., berglund, e.d., deng, z., gao, y., liu, t., sohn, j.w., et al. ( ). cell metab. , this issue, – . apoc , coronary disease, and complexities of mendelian randomization jonathan c. cohen, ,* stefan stender, and helen h. hobbs , , ,* department of internal medicine department of molecular genetics howard hughes medical institute university of texas southwestern medical center, dallas, tx , usa *correspondence: jonathan.cohen@utsouthwestern.edu (j.c.c.), helen.hobbs@utsouthwestern.edu (h.h.h.) http://dx.doi.org/ . /j.cmet. . . two new studies report that triglyceride (tg)-lowering mutations in apoc reduce coronary heart disease (chd) (crosby et al., ; jørgensen et al., ). here, we explore limitations of using mendelian random- ization to evaluate chd risk, including potential confounding by the widespread use of statin therapy. the status of plasma tg levels as a risk factor for chd has been debated for de- cades. in most studies, plasma tg levels are associated with chd, but adjusting for confounding variables (e.g., smoking, insulin resistance, and diabetes) sub- stantially attenuates the association (di angelantonio et al., ). two recent studies take a genetic approach to untan- gle the gordian knot between tg levels and chd (crosby et al., ; jørgensen et al., ). instead of stratifying individuals based on plasma tg levels, the studies divide participants into two groups according to their apoc genotypes. plasma apoc and tg levels are highly corre- lated. stratifying by apoc genotype rather than plasma level of tg circum- vents confounding by factors that affect both plasma tg levels and chd, an approach referred to as mendelian randomization (katan, ). one study, led by sekar kathiresan, identified four rare variants in apoc that were associated with a % reduc- tion in plasma tg levels (crosby et al., ). the variants were then tested for association with chd in , individ- uals from different studies. mutation carriers had a % reduction in chd compared to noncarriers. the other study, led by anne tybjærg-hansen, used a similar strategy (jørgensen et al., ). they found three apoc variants that were associated with a % reduc- tion in plasma tg levels. in a cohort of , danes, carriers of these variants had a % reduction in chd. taken together, these findings provide compel- ling evidence that reducing apoc expression will reduce chd risk. the question remains as to whether the reduced chd risk in apoc variant carriers is due to lower plasma tg levels or to other associated factors, such as lower plasma levels of ldl cholesterol (ldl-c), apoc , or remnant lipoproteins, or to increased levels of hdl-c. reductions in ldl-c are consistently associated with reduced chd. figure a eptember , ª elsevier inc. mailto:jonathan.cohen@utsouthwestern.edu mailto:helen.hobbs@utsouthwestern.edu http://dx.doi.org/ . /j.cmet. . . http://crossmark.crossref.org/dialog/?doi= . /j.cmet. . . &domain=pdf figure . genetic and pharmacological reduction in ldl-c and coronary heart disease (a) reduction in chd risk associated with genetic variants (blue circles) and pharmacological agents (green circles) that lower plasma levels of ldl-c. genetic variations that reduce plasma ldl-c levels are associated with a greater reduction in chd compared to that seen in statin trials. the sources of the data shown in this figure are as follows: apob rs (pmid: ), ldlr rs (pmid: ), abcg rs (pmid: ), and pcsk (pmid: ). the red circle represents the chd reduction (� %) that is predicted for a loss-of-function mutation in apoc (r x) (crosby et al., ; pollin et al., ). woscops, the west of scotland coronary prevention study (pmid: ); care, cholesterol and recurrent events trial (pmid: ); hps, heart pro- tection study (pmid: ); s, the scandinavian simvastatin survival study (pmid: ). (b) effects of apoc loss-of-function variants on circulating lipid and lipoprotein levels and on chd. proven causal links are indicated by blue arrows. question marks indicate where causality has not been established. the red x indicates no causal relationship. idl, intermediate density lipoprotein; cr, chylomicron remnant. cell metabolism previews plots the reduction in chd as a function of the reduction in ldl-c in four studies in which subjects were treated for years with a cholesterol-lowering statin (green line). the blue line shows the reduction in chd in subjects with dna variations that lower ldl-c levels. for each per- centage reduction in ldl-c, the ldl-c- lowering variants produce a much greater reduction in chd than seen in the statin trials. presumably this reflects the fact cell metabolism , september , ª that dna variants lower ldl-c levels from birth, whereas statin treatment is initiated when atherosclerotic plaques have already developed. can the reduction in chd in the apoc mutant carriers be explained by a reduction in ldl-c levels? the effects of apoc inactivation on ldl-c levels remain inconclusive (pollin et al., ; tachmazidou et al., ). pollin et al. identified a nonsense mutation (r x) in elsevier inc. apoc that is common in the amish and is associated with a % reduction in plasma ldl-c levels (pollin et al., ). based on prior genetic studies, mutations that lower ldl-c by % should decrease chd by � % (fig- ure a), which is similar to the reduction observed in the two apoc studies ( % and %). the apoc carriers in the discovery cohort of the kathiresan study (crosby et al., ) had a % reduction in ldl-c level, which is similar to that observed in the amish, but corresponding data were provided for only a subset of the cohorts in the chd association study. in the tybjærg-hansen study (jørgensen et al., ), the mean plasma ldl-c level was only % lower in apoc carriers than in noncarriers. this modest reduction in ldl-c cannot account for the dramatic reduction in chd associated with the apoc variants. a factor that may mask the contribu- tion of plasma ldl-c levels to the reduc- tion in chd in apoc carriers is statin treatment. statins are more likely to be prescribed to individuals who have higher plasma ldl-c levels, multiple chd risk factors, or established chd. if apoc noncarriers have higher plasma ldl-c levels and/or more chd, they would be more likely to be treated with statins. this would lower their ldl levels and obscure differences in ldl-c levels between apoc carriers and noncarriers. could an excess of statin use among noncarriers mask the effect of apoc variants on ldl-c levels in these two studies? statin use was not described in the kathiresan study. in the tybjærg- hansen study, statin use was more prev- alent among noncarriers than carriers, although the difference did not reach statistical significance. given the large effects of ldl-c on chd risk and the as- sociation between apoc mutations and ldl-c levels, it is premature to conclude that the reduction in chd in apoc variant carriers is independent of plasma ldl-c levels. resolution of this issue will require additional studies in statin-naive individuals or in which pre-statin ldl-c levels are used in the analysis. alternatively, factors other than ldl-c levels may contribute to the reduction in chd. apoc has pleiotropic effects cell metabolism previews (figure b). it is possible that apoc itself promotes atherosclerosis (ginsberg and brown, ), or alternatively, that apoc retards clearance of atherogenic lipoprotein remnants. if the lower levels of tg in apoc carriers are atheroprotec- tive, studies using other variants that lower tg levels without affecting other chd risk factors should replicate the association. carriers of the apoc vari- ants also have higher plasma levels of hdl-c, which are inversely associated with chd. ironically, tybjærg-hansen and kathiresan performed the key genetic studies that showed plasma hdl-c levels are not causally related to chd risk (frikke-schmidt et al., ; voight et al., ). thus, it is unlikely that hdl-c is conferring the cardioprotective effect of the apoc mutations. the identification of other sequence variations that lower plasma tg levels without altering other risk factors would bolster the contention that tg lowering is causally linked to reduction in chd. apoc may be an excellent therapeutic target for patients with severe hypertri- glyceridemia. these patients are at risk of developing pancreatitis, a potentially life-threatening disorder, and the arma- mentarium of drugs to treat severe hy- pertriglyceridemia is extremely limited. the high circulating levels of apoc ( – mg/dl) may limit the efficacy of targeting apoc using antibody-based therapies, but strategies that target hepatic apoc mrna may prove efficacious. homozygotes for loss-of-function mu- tations can provide important clues as to the safety, as well as the efficacy, of therapies targeting a specific protein. neither study identified any individuals with total apoc deficiency. identifica- tion of such individuals would provide reassurance that extreme pharmacolog- ical inhibition of apoc would not have unforeseen detrimental effects. as these two studies attest, human genetic studies have fueled a resurgence of interest in lipid-modifying therapies for chd prevention. studies using a mendelian randomization approach hold the promise of identifying new thera- peutic agents for chd, but they must address problems associated with pleiot- ropy and account for effects of statin treatment on plasma ldl-c levels. short-term reductions in ldl-c levels due to statin therapy do not reflect lifetime exposure to this atherogenic lipoprotein. mendelian randomization, although a powerful approach, does not eliminate all confounding factors. cell metabolism , s references crosby, j., peloso, g.m., auer, p.l., crosslin, d.r., stitziel, n.o., lange, l.a., lu, y., tang, z.z., zhang, h., hindy, g., et al.; tg and hdl working group of the exome sequencing project, national heart, lung, and blood institute ( ). n. engl. j. med. , – . di angelantonio, e., sarwar, n., perry, p., kaptoge, s., ray, k.k., thompson, a., wood, a.m., lewing- ton, s., sattar, n., packard, c.j., et al.; emerging risk factors collaboration ( ). jama , – . frikke-schmidt, r., nordestgaard, b.g., stene, m.c., sethi, a.a., remaley, a.t., schnohr, p., grande, p., and tybjaerg-hansen, a. ( ). jama , – . ginsberg, h.n., and brown, w.v. ( ). arterios- cler. thromb. vasc. biol. , – . jørgensen, a.b., frikke-schmidt, r., nordest- gaard, b.g., and tybjærg-hansen, a. ( ). n. engl. j. med. , – . katan, m.b. ( ). lancet , – . pollin, t.i., damcott, c.m., shen, h., ott, s.h., shelton, j., horenstein, r.b., post, w., mcleni- than, j.c., bielak, l.f., peyser, p.a., et al. ( ). science , – . tachmazidou, i., dedoussis, g., southam, l., farmaki, a.e., ritchie, g.r., xifara, d.k., matchan, a., hatzikotoulas, k., rayner, n.w., chen, y., et al.; uk k consortium ( ). nat commun , . voight, b.f., peloso, g.m., orho-melander, m., frikke-schmidt, r., barbalic, m., jensen, m.k., hindy, g., hólm, h., ding, e.l., johnson, t., et al. ( ). lancet , – . stem cell lineage specification: you become what you eat clifford d.l. folmes ,* and andre terzic ,* center for regenerative medicine, mayo clinic, rochester, mn , usa *correspondence: folmes.clifford@mayo.edu (c.d.l.f.), terzic.andre@mayo.edu (a.t.) http://dx.doi.org/ . /j.cmet. . . nutrient availability and intermediate metabolism are increasingly recognized to govern stem cell behavior. oburoglu et al. ( ) now demonstrate that glutamine- and glucose-dependent nucleotide synthesis segre- gate erythroid versus myeloid differentiation during hematopoietic stem cell specification, implicating a metabolism-centric regulation of lineage choices. flexibility in energy metabolism enables cells to prioritize metabolic pathways in or- der to support stage-specific energetic de- mands (folmes et al., ). interrogation of stem cell metabolism has identified glycolysis as a key player in the mainte- nance of stemness through provision of energy and anabolic precursors (folmes et al., ), while oxidative metabolism al- lows for more efficient energy production to match energy-demanding processes of differentiating progeny (chung et al., ). furthermore, individual metabolic pathways underlying stem cell renewal versus lineage specification are starting eptember , ª elsevier inc. mailto:folmes.clifford@mayo.edu mailto:terzic.andre@mayo.edu http://dx.doi.org/ . /j.cmet. . . http://crossmark.crossref.org/dialog/?doi= . /j.cmet. . . &domain=pdf 	apoc , coronary disease, and complexities of mendelian randomization 	references a q duplication prenatally detected lable at sciencedirect taiwanese journal of obstetrics & gynecology ( ) e contents lists avai taiwanese journal of obstetrics & gynecology journal homepage: www.tjog-online.com research letter a q duplication prenatally detected rossella bruno, angelo valetto*, veronica bertini, cinzia cosini, benedetta toschi, caterina congregati, simona rossi, paolo simi cytogenetics and molecular genetic unit, azienda ospedaliera universitaria pisana, s. chiara hospital, pisa, italy a r t i c l e i n f o article history: accepted may this report is about an isolated de novo interstitial duplication of chromosome q detected in prenatal diagnosis. the duplication spans about . mb, and contains at least omim genes. as far as we know, this is the first case which has been detected prenatally; postnatal cases with a similar chromosomal anomaly are rare and the phenotype has been associated with a wide spectrum of clinical signs. this phenotypic variability may be due to the different extents of the duplicated regions, thus an accurate molecular definition of the chromosomal breakpoints is necessary to make better genotype-phenotype correlations [ ]. the mother was a -year-old healthy woman with two healthy children; amniocentesis was performed at the th week of gesta- tion because of her advanced age. ultrasound gynecological ex- amination did not show any fetal abnormalities except for a slight hypertelorism. conventional banding chromosome analysis on cultured amniotic fluid cells, with a resolution of about bands, revealed an apparent terminal duplication of chromosome q (fig. ), that was better characterized using molecular cytogenetic techniques. array-comparative genomic hybridization (array-cgh) using a k oligo platform (agilent technologies, santa clara, ca, usa) revealed that the duplication was not terminal but interstitial, from position , , bp ( q ) to position , , bp ( q ) (ncbi /hg map) (fig. ). the omim genes in the region can be seen in table and in fig. . fluorescent in situ hybridization, using the whole chromosome painting probe (cytocell, ltd cambridge, uk), excluded additional cryptic rearrangements; telvysion q (vysis, abbott s.p.a., milan, italy) probe for the q * corresponding author. molecular genetic unit, azienda ospedaliera uni- versitaria pisana, s. chiara hospital, via roma , pisa, italy. e-mail address: a.valetto@ao-pisa.toscana.it (a. valetto). http://dx.doi.org/ . /j.tjog. . . - /copyright © , taiwan association of obstetrics & gynecology. published subtelomeric regions showed two normal signals (fig. ), con- firming array-cgh data. parental karyotypes and array cgh profiles were normal. pregnancy was terminated at the st week of gestation. anal- ysis of the fetal anatomy showed minor facial dimorphisms with hypertelorism, a wide nasal base, a wide mouth and a thin upper lip (figs. e ), along with no specific signs such as microcalcinosis in the adrenal and hepatic parenchyma that can be associated with many other chromosomal anomalies, such as di george syndrome, edwards syndrome, patau syndrome, down syndrome, and mosaicism for partial trisomy of chromosome [ e ]. partial q duplication is a rare anomaly, and most of the pa- tients reported in literature have undergone postnatal analysis using classic cytogenetic techniques. the resolution of cytogenetic banding is about mb, thus a detailed analysis of breakpoints is missing. duplication q has been associated with a severe phenotype but the clinical consequences of this anomaly are far from being clarified, and extensive variability is present among the reported patients. manifestations of this anomaly include psycho- motor/mental retardation, growth retardation, and dysmorphic features such as facial asymmetry with hypertelorism, frontal bossing and temporal narrowness, a broad nasal bridge, epicanthal folds, wide mouth with a thin upper lip, micrognathia, webbed neck, low-set posteriorly angulated ears, and an abnormal hairline. moreover partial trisomy q is associated with polydactyly, long fingers, abnormal positioned feet, cerebellar hypoplasia, multiple cardiac anomalies, limb shortness, hyperlaxity, genital abnormal- ities, and accessory spleen [ , ]. the partial trisomy of q has been described in complex chromosomal rearrangements (table ). king et al [ ] reported a case of partial trisomy q -qter, detected by amniocentesis which was performed because of polyhydramios and ultrasound diagnosis of fetal anomalies. the chromosome complement of the cultured amniotic fluid cells was ,xx/ ,xx,- ,þder( ),t( ; ) (q . ; q . ) in a ratio of : ; the infant born showed the unique phenotypic features of mosaic partial trisomy q : frontal bossing, large mouth, brachyrhizomelia, and hexadactyly. babovic-vuksanovic et al [ ] presented a familial case of dup q -q . , whose clinical characteristics resembled ullrich- turner syndrome, indicating the presence of genes involved in skeletal development. kelly et al [ ] described an adult with dup by elsevier taiwan llc. all rights reserved. mailto:a.valetto@ao-pisa.toscana.it http://crossmark.crossref.org/dialog/?doi= . /j.tjog. . . &domain=pdf www.sciencedirect.com/science/journal/ http://www.tjog-online.com http://dx.doi.org/ . /j.tjog. . . http://dx.doi.org/ . /j.tjog. . . http://dx.doi.org/ . /j.tjog. . . fig. . cytogenetic analysis of the cultured amniotic fluid cells by q-banding. karyotype shows a duplication of the long arm of chromosome . r. bruno et al. / taiwanese journal of obstetrics & gynecology ( ) e q -q , suffering with epilepsy, sensorineural hearing loss, long fingers, and overlapping toes. in a study lukusa et al [ ] reported a case of a year-old girl, with pure q . ( . mb) duplication and a complex clinical presentation comprising main features of dup q syndrome and additionally striking distal arthrogryposis. most cases of partial trisomy for the distal region of q are due to adjacent- segregation of reciprocal translocation which were derived from either one of the parents [ e ]; some cases are due to a familial chromosome inversion [ , ], few are de novo events [ ]. this report shows the first case of a de novo isolated q duplication detected in a fetus without ultrasound abnormalities. the results of the fetal autopsy did not show the typical phenotypic fig. . array-comparative genomic hybridization profile of the duplicated region. array-com q -q (red dots represent duplicated oligos). alterations observed in cases diagnosed postnatally thus the cor- relation of genotype-phenotype is difficult, particularly during prenatal investigations. the lack of specific data concerning the prognosis of fetuses with dup q makes genetic counseling a difficult task. the q -qter portion is the region most commonly duplicated in almost all the dup q cases reported in literature. this region includes omim genes involved in several diseases, such as retinitis pigmentosa, ciliarydyskenesia, microcephaly, and bradyopsia (table ). so far, the duplication we have found is the largest described in prenatal diagnosis and it is likely that it was not compatible with life; if the pregnancy had not been interrupted the infant would have probably shown the major clinical signs of dup q syndrome immediately after birth. parative genomic hybridization showing the presence of a duplication of chromosome table omim genes located in the duplicated region q - . gene location phenotype carbonic anhydrase iv ca q . retinitis pigmentosa small patella syndrome sps q . small patella syndrome angiotensin i-converting enzyme ace q . renaltubulardysgenesis angiotensin i-converting enzyme, benign serum in crease alzheimer disease, susceptibility to microvascular complications of diabetes myocardial infarction, susceptibility to sars, progression of stroke, hemorrhagic ste -related kinase adaptor alpha strada q . polyhydramnios, megalencephaly, and symptomatic epilepsy growth hormone gh q . growth hormone deficiency, isolated, type ia growth hormone deficiency, isolated, type ib growth hormone deficiency, isolated, type ii kowarski syndrome cd b antigen cd b q . a gamma globulinemia sodium channel, voltage-gated, type iv, alpha subunit scn a q . hyperkalemic periodic paralysis, type hypokalemic periodic paralysis, type myasthenic syndrome, acetazolamide-responsive myotonia congenita, atypical, acetazolamide-responsive paramyotonia congenita polymerase, dna, gamma- polg q . progressive external ophthalmoplegia with mitochondrial dna deletions, autosomal dominant regulator of g protein signaling rgs q . bradyopsia family with sequence similarity member a fam a q . amelogenesis imperfecta and gingival fibromatosis syndrome sry-box sox q . acampomelic campomelic dysplasia campomelic dysplasia campomelic dysplasia with autosomal sex reversal component of oligomeric golgi complex cog q . congenital disorder of glycosylation, type iig dynein, axonemal, intermediate chain dnai q . ciliary dyskinesia, primary, , with or without situs inversus solute carrier family , member , regulator slc a r q . nephrolithiasis/osteoporosis, hypophosphatemic, solute carrier family (mitochondrial thiamine pyrophosphate carrier), member slc a q . microcephaly, amish type thiamine metabolism dysfunction syndrome (progressive polyneuropathy type) fig. . omim genes located in the duplicated region q - . fig. . fluorescence in situ hybridization performed using the probe whole chromo- some painting (cytocell). fig. . hypertelorism and a wide nasal base. r. bruno et al. / taiwanese journal of obstetrics & gynecology ( ) e fig. . apparently normal fetal anatomy. table duplication of q region as sole chromosome anomaly reported in literature and in our study. study duplicated q region babovic-vuksanovic et al [ ] q -q . kelly et al [ ] q -q lukusa et al [ ] from q . ( , mb) our study q -q ( . mb) r. bruno et al. / taiwanese journal of obstetrics & gynecology ( ) e conflict of interest the authors have no conflicts of interest relevant to this article. references [ ] lukusa t, fryns jp. pure de novo q . micro duplication characterized by micro-array cgh in a dysmorphic infant with growth retardation, develop- mental delay and distal arthrogryposis. genet couns ; : e . [ ] bertola g, giambona s, bianchi r, girola a, berra sa. di george syndrome: not always a pediatric diagnosis. recenti prog med ; : . [ ] simchen mj, toi a, bona m, alkazaleh f, ryan g, chitayat d. fetal hepatic calcifications: prenatal diagnosis and outcome. am j obstet gynecol ; : e . [ ] jay a, kilby md, roberts e, brackley k, platt c, mchugo j, et al. prenatal diagnosis of mosaicism for partial trisomy : a case report including fetal pathology. prenat diagn ; : e . [ ] bronshtein m, blazer s. prenatal diagnosis of liver calcifications. obstet gynecol ; : e . [ ] kelly bd, becker k, kermode v, stallings rl, murphy rp, green aj, et al. dysmorphic features and learning disability in an adult male with pure partial trisomy q -q due to a terminal duplication. am j med genet ; : e . [ ] cordier ag, braidy c, levaillant jm, brisset s, maurin ml, mas ae, et al. cor- relation between ultrasound and pathological examination in a prenatal diagnosis of cri du chat syndrome associated with partial trisomy q. prenat diagn ; : e . [ ] king pa, ghosh a, tang m. mosaic partial trisomy q . j med genet ; : e . [ ] babovic-vuksanovic d, westman ja, jalal sm, lindor nm. clinical character- istics associated with dup (q q . ) in a mosaic mother and two non- mosaic daughters. clin dysmorphol ; : e . [ ] ohdo s, madokoro h, sonoda t, ohba k. sibs lacking characteristic features of duplication of distal q. j med genet ; : e . [ ] cotter pd, stewart nl. partial trisomy q and monosomy p due to a familial translocation. ann genet ; : e . [ ] barros nu~nez p, rolon a, lizcano la, rivera h. pure trisomy q from a ; translocation. genet couns ; : e . [ ] greenberg f, stratton rf, lockhart lh, elder ff, dobyns wb, ledbetter dh. familial miller-dieker syndrome associated with pericentric inversion of chromosome . am j med genet ; : e . [ ] kingston hm, ledbetter dh, tomlin pi, gaunt kl. miller-dieker syndrome resulting from rearrangement of a familial chromosome inversion detected by fluorescence in situ hybridization. j med genet ; : e . http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref 	a q duplication prenatally detected 	conflict of interest 	references signatures of natural and unnatural selection: evidence signatures of natural and unnatural selection: evidence from an immune system gene in african buffalo lane-degraaf, k. e., amish, s. j., gardipee, f., jolles, a., luikart, g., & ezenwa, v. o. ( ). signatures of natural and unnatural selection: evidence from an immune system gene in african buffalo. conservation genetics, ( ), - . doi: . /s - - - . /s - - - springer version of record http://cdss.library.oregonstate.edu/sa-termsofuse http://survey.az .qualtrics.com/se/?sid=sv_ io d aayr vggx http://cdss.library.oregonstate.edu/sa-termsofuse tools and technology a method for improving the reliability of sound broadcast systems used in ecological research and management jonathon j. valente, department of forest ecosystems and society, oregon state university, corvallis, or , usa christa l. legrande, department of environmental and forest biology, state university of new york college of environmental science and forestry, syracuse, ny , usa vincent m. johnson, fisheries, wildlife, and environmental studies department, state university of new york cobleskill, cobleskill, ny , usa richard a. fischer, united states army engineer research and development center, environmental laboratory, vicksburg, ms , usa abstract automated sound broadcast systems have been used to address a variety of ecological questions, and show great potential as a management tool. such systems need to be reliable because treatments are often applied in the absence of a human observer and system failure can cause methodological ambiguity. during the breeding seasons of and , we used a sound broadcast system previously described by farrell and campomizzi ( ) in an experiment evaluating the use of post-breeding song in forest-bird habitat selection in southern indiana, usa. this system incorporates a portable compact disc (cd) player where the play button is permanently depressed using manual compression so that when a timer connects an electrical current to the unit, the cd player automatically starts. despite exhaustive efforts to find a reliable way to manually compress the play button on numerous cd player models, play button failure was the most significant source of broadcast system failure ( %) in . we attempted to resolve this problem in by removing the need for manual compression and soldering the play button contact poles on each cd players’ integrated circuit boards. though we did experience broadcast system failures during < % of treatment periods in , none of those were attributable to play button failure. by removing all possibility of failure from manual play button compression we improved our system reliability. thus, soldering the cd player play button on such broadcast systems represents a methodological improvement that can be used by researchers and managers interested in sound broadcast. � the wildlife society. key words bird song, cd player, momentary action pushbutton switch, play button depression mechanism, sound broadcast system. sound broadcast systems serve a variety of purposes in ecological research. they have been used to answer questions about perceived predation risk on reproductive performance (eggers et al. , zanette et al. ), movement ecology in fragmented landscapes (sieving et al. , , ; desrochers and hannon ; bélisle and desrochers ), habitat selection (doligez et al. , ward and schlossberg , nocera et al. , fletcher , betts et al. ), heterospecific interactions (diego-rasilla and luengo , fletcher , pupin et al. ), impacts of noise pollution (bee and swanson ), and to explore mechanisms driving species distribution patterns (fletcher ). additionally, audio broadcasts can be incorporated into survey protocols to improve detectability for some organisms (gibbs and melvin , conway and simon , kubel and yahner , ichikawa et al. ), and manipulation of species responses to conspecific attraction shows great promise as a manage- ment tool (ward and schlossberg , ahlering and faaborg ). in many instances, application of auditory experimental or management treatments is done most efficiently using automated systems that can be programmed to operate without a human present (ward and schlossberg , fletcher , ; betts et al. ; zanette et al. ). drawing correct and useful conclusions from such studies is contingent on broadcast treatments being applied correctly, making the reliability of automated systems a critical consideration. system failures lead to heterogeneity and ambiguity in treatment applications, and unreliable systems need to be visited with greater frequency, resulting in a loss of money and man hours. farrell and campomizzi ( ) presented a description of a sound broadcast system that incorporated a portable compact disc (cd) player on which the play button was permanently depressed so that when a timer switch connected an electrical current to the unit, the cd player automatically started. these authors describe using “a combination of adhesive received: september ; accepted: april published: august e-mail: jonathon.j.valente@gmail.com wildlife society bulletin ( ): – ; ; doi: . /wsb. valente et al. � improving sound broadcast systems tape, wooden dowels, and rubber stoppers to set the play button in the on position” (farrell and campomizzi : ). during field tests with comparable broadcast systems, we attempted to use similar tools for our play button depression mechanisms (pbdm), yet we were unable to develop a reliable method of securing the play buttons in the on position using compression. as such, pbdm failure was by far our greatest source of system failure, and we sought to develop a more reliable method of initiating playback for our experiments by bypassing the need for a pbdm on the cd player component. as part of a broader experiment evaluating the use of post-breeding song in breeding habitat selection by wood thrush (hylocichla mustelina) and kentucky warblers (oporornis formosus) in forest tracts of southern indiana, usa, we evaluated whether soldering the electrical play button connection would be a more reliable method of initiating playback in broadcast systems regulated by a timer than consistently applying pressure to the play button. study area our study area encompassed approximately , ha of land in the central hardwoods region of southern indiana, specifically within big oaks national wildlife refuge, naval surface warfare center crane, martin state forest, and the lost river tract of hoosier national forest. this region was dominated by corn and soybean agriculture and remnant tracts of temperate broadleaf and mixed forests. the mean annual rainfall in southern indiana was approximately cm (indiana state climate office ), and mean annual temperatures ranged from c in winter to c in summer (national climatic data center ). methods during the and breeding seasons, we deployed post-breeding song broadcast systems at point-count stations ( in and in ) previously unoccupied by target species (wood thrush and/or kentucky warblers). treatments were applied between june and august each year in order to experimentally test whether post-breeding song would attract future breeders. in ideal circumstances, treatment sites received between and hours of song broadcast ( hr/day) in intervals of – days based on the life of the batteries ( v a energy power absorbent glass mat battery; energy battery group, atlanta, ga). our study design incorporated paired control sites, though information about those is tangential to our focus and will not be discussed further. all broadcast systems contained of portable cd player models (insignia ns-p or insignia ns-p ; insignia products, richfield, mn). the play buttons on both cd player models utilize a momentary action pushbutton switch that, when depressed, connects power required to play the cd to contact poles on the integrated circuit board via a convex disk of flexible metal (fig. ). in , we rotated broadcast systems among treatment sites on a bi-weekly schedule, and each time a unit was placed at a treatment site, a trained technician used a combination of adhesive tape, rubber bands, metal pins, square keys, sticky tack, glue, and pieces of eraser to manually depress and secure the cd player play button. methods for permanently depressing the play buttons varied temporally as we attempted to identify a reliable solution, and varied among cd players because of subtle differences in model design. in summer of , we increased the number of broadcast systems to and did not rotate them among sites. to eliminate the need to apply manual pressure to cd player play buttons, we applied a thin layer of electrically conductive rosin soldering flux (radioshack corporation, fort worth, tx) to the contact poles and connected them with solder using a weler soldering iron (cooper hand tools, apex, nc) prior to system deployment (for detailed instructions, see supporting information, available online at www. onlinelibrary.wiley.com). this connection allows a constant electrical current to flow among contact poles to create a closed contact system, which is electrically identical to having the play button depressed at all times (eaton corporation ). both pbdms and soldering ensure that the poles are connected when the cd player receives power, but by soldering the connection we removed virtually all possibility of manual failure (e.g., play button deformation, pbdm dislodging, rubber bands stretching, adhesives failing, or human error in application). we then re-assembled the cd players to their original condition and incorporated them as components in the broadcast systems. all other components used in the treatments were identical for each year. we visited treatment sites once every or days to change the batteries and rotate the units ( only), and considered each period between battery changes (treatment period) as the experimental unit for our analyses. this is a logical temporal unit because a broadcast system operator would ideally be able to deploy a system and be confident that the components would continue working until the battery was scheduled to expire. when convenient, we made additional visits to treatment sites to verify that the units were working as intended. figure . a close-up image of the exposed upper integrated circuit board of a cd player (insignia ns-p ; insignia products, richfield, mn). when the contact poles (indicated by red arrow) associated with the play button are connected by an electrically conductive medium, the unit begins playing. wildlife society bulletin � ( ) http://www.onlinelibrary.wiley.com/ http://www.onlinelibrary.wiley.com/ if a unit was visited before the battery was scheduled to expire, and the unit was not playing appropriately, a technician thoroughly assessed all components, documented the reason for failure, and fixed the source of failure. when units were visited after the battery had likely expired, the technician connected a new, charged battery, and then assessed the status of the unit. any broadcast system malfunctions that could not be explained by a dead battery were also documented. if the playback unit or one of its components failed at least once during a treatment period, whereby bird song was not being broadcasted as intended, the treatment was considered a failure; whereas, if the unit continued broadcasting until the battery expired, it was considered a success. prior to analyses, we eliminated all failures resulting from human error (i.e., avoidable situations in which the broadcast system was not properly assembled by field personnel) and limit our discussion to treatment periods that were disrupted by component failure. results our sample size was treatment periods in , and we experienced treatment period failures, ( %) of which were specifically attributable to pbdm failures. all pbdm failures occurred when rubber bands stretched or snapped, depression components moved slightly so that pressure was no longer being applied in the correct area, or the cd player became warped. the only other failure occurred when the micro universal serial bus (usb) speaker charger stopped functioning in one unit. in , our sample size was treatment periods, and we experienced total failures, none of which were attributable to the cd player play button. all failures were due to malfunction of other broadcast system components, including speaker, speaker chargers, -v direct current (dc) power outlet y-adapter, and cd player power jacks. therefore, the soldering method had a % success rate while units were deployed in the field. discussion in , when all of our playback systems were activated using manual depression (pbdms), play button failure was by far the most significant source of treatment interruption. in , we were able to completely eliminate this problem by soldering the contact poles that are connected when the play button is depressed, resulting in a much more reliable broadcast system. we encountered primary issues that made manual play button depression challenging. first, each cd player model (we experimented with several additional models before selecting those we used in the field) has a unique design that requires a unique solution for depressing the play button, meaning there is no ubiquitous methodology. second, because the play button is designed to spring back after depression, applying too little pressure means the unit will not start playing as intended, and applying too much pressure can result in deformation of multiple pieces (e.g., the plastic play button, the convex metal disk, and the cd player shell itself) or physically prevent the cd from turning. in addition to the materials described above, we tried numerous other methods for securing cd player play buttons in the laboratory (including manual clamps, glue, rubber stoppers, and weighted pressure), but were never able to generate an infallible solution until we eliminated the need for manual pressure altogether. there are explanations for why we saw a greater number of electronic component failures in the second season. first, it is possible that by tampering with the internal circuitry on the cd players, we interfered with other internal mecha- nisms, causing the dc power jacks to eventually fail on of them. however, we believe that this scenario is very unlikely, given that every cd player that we disassembled, soldered, and put back together (n¼ ) worked as intended initially, and none of them failed until after they had been used in the field for several weeks. second, our study region received more than twice as much precipitation in the summer of (approx. . cm) as in the summer of (approx. . cm; indiana state climate office ). it seems likely that environmental moisture was responsible for many of the failures we endured with cd players and other electronic components in the second year, particularly given that others have identified moisture as a primary cause of failure in similar systems (farrell and campomizzi ). third, approximately % of the playback unit components (including of cd players) we used in had been previously used in the field in . as such, some may have simply failed from long, sustained periods of use in adverse environmental conditions (e.g., high heat and humidity). though we only used different cd player models in our field tests, our experiences suggest that our method can be utilized in most portable cd player models, eliminating the need to design a unique pbdm in each case. for instance, we employed our soldering method on a third model (memorex md sl; imation corporation, oakdale, mn) that utilizes a -pole momentary action pushbutton switch and on a fourth model (sony d-ej ; sony electronics, inc., san diego, ca) that utilizes a right-angle tactile switch. in each case, our method was successful at initiating playback, yet both of these latter models incorporated electronic volume buttons that are connected to the same integrated circuit board as the play button, rendering volume control unusable when the play button is activated (either by manual compression or electrical connection). we, therefore, chose to use the insignia models, which had a volume control dial not reliant on the integrated circuit board associated with the play button, and other experimenters should take this into consideration when designing their own systems. manipulating animal populations using broadcast systems for both experimental and management purposes holds great promise (nocera et al. , betts et al. , ahlering et al. , farrell et al. ), yet can also be expensive, time- consuming, and logistically challenging to implement. drawing correct and useful conclusions from manipulative experiments or management procedures that involve sound broadcast is contingent on broadcast treatments being applied correctly, often in the absence of a human observer. valente et al. � improving sound broadcast systems in such instances when a broadcast system fails, there is no way to determine when the failure occurred, making it impossible to quantify the number of broadcast hours for that treatment period and thereby adding another source of variability to the experiment. in the systems we used, for instance, failure of any component could result in between hours and hours of lost playback time, depending on when during the treatment period the component failed. as such, any improvement in system reliability will, in turn, improve our ability to generate and accurately interpret results, and reduce the number of man hours required to maintain such experiments. we suggest that our method of soldering the cd player play button is a reliable complement to broadcast systems used in ecological research and for management practices that involve using sound broadcast. acknowledgments we would like to thank m. betts, c. bochmann, k. mccune, b. slaby, r. snowden, j. suich, a. tucker, j. valente, c. winter, and the helpful staff from the best buy store in bloomington, indiana, for their assistance in designing reliable broadcast systems. we also thank s. andrews, j. robb, and b. walker for their logistical assistance, and s. destefano, k. mccune, and two anonymous reviewers for their help in improving this manuscript. this research was conducted under contract to the department of defense strategic environmental research and development program. the publication of this paper does not indicate endorsement by the department of defense (dod), nor should the contents be construed as reflecting the official policy or position of the dod. reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the dod. literature cited ahlering, m. a., d. arlt, m. g. betts, r. j. fletcher, j. j. nocera, and m. p. ward. . research needs and recommendations for the use of conspecific-attraction methods in the conservation of migratory songbirds. the condor : – . ahlering, m. a., and j. faaborg. . avian habitat management meets conspecific attraction: if you build it, will they come? the auk : – . 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white, m. c. allen, and m. clinchy. . perceived predation risk reduces the number of offspring songbirds produce per year. science : – . associate editor: destefano. supporting information additional supporting information may be found in the online version of this article at the publisher’s web-site. step-by-step instructions for soldering the electrical con- nection of a cd player play button that utilizes a tactile momentary action pushbutton switch. wildlife society bulletin � ( ) https://climate.agry.purdue.edu/climate/narrative.asp https://climate.agry.purdue.edu/climate/narrative.asp https://climate.agry.purdue.edu/climate/summary.asp https://climate.agry.purdue.edu/climate/summary.asp http://www.ncdc.noaa.gov/oa/climate/normals/usnormals.html http://www.ncdc.noaa.gov/oa/climate/normals/usnormals.html r e s e a r c h a r t i c l e signatures of natural and unnatural selection: evidence from an immune system gene in african buffalo k. e. lane-degraaf • s. j. amish • f. gardipee • a. jolles • g. luikart • v. o. ezenwa received: september / accepted: september / published online: october � springer science+business media dordrecht abstract pathogens often have negative effects on wildlife populations, and disease management strategies are important for mitigating opportunities for pathogen transmission. bovine tuberculosis (mycobacterium bovis; btb) is widespread among african buffalo (syncerus caffer) populations in southern africa, and strategies for managing this disease vary. in two high profile parks, kruger national park (knp) and hluhluwe-imfolozi park (hip), btb is either not actively managed (knp) or managed using a test-and-cull program (hip). exploiting this variation in management tactics, we investigated potential evolutionary consequences of btb and btb management on buffalo by examining genetic diversity at ifng, a locus which codes for interferon gamma, a sig- naling molecule vital in the immune response to btb. both heterozygosity and allelic richness were significantly and positively correlated with chromosomal distance from ifng in knp, suggesting that directional selection is act- ing on ifng among buffalo in this park. while we did not see the same reduction in genetic variation around ifng in hip, we found evidence of a recent bottleneck, which might have eroded this signature due to genome-wide reductions in diversity. in knp, alleles at ifng were in significant gametic disequilibrium at both short and long chromosomal distances, but no statistically significant gametic disequilibrium was associated with ifng in hip. when, we compared genetic diversity between culled and non-culled subsets of hip animals, we also found that individuals in the culled group had more rare alleles than those in the non-culled group, and that these rare alleles occurred at higher frequency. the observed excess of rare alleles in culled buffalo and the patterns of gametic dis- equilibrium in hip suggest that management may be eroding immunogenetic diversity, disrupting haplotype associations in this population. taken together, our results suggest that both infectious diseases and disease manage- ment strategies can influence host genetic diversity with important evolutionary consequences. keywords parasite-mediated selection � syncerus caffer � bovine tuberculosis � ifng � disease management � genetic diversity � gametic disequilibrium introduction parasites, including helminths, protozoa, bacteria, and viruses, can act as strong selective forces on host k. e. lane-degraaf and s. j. amish have contributed equally to this paper. electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. k. e. lane-degraaf (&) � v. o. ezenwa (&) odum school of ecology and department of infectious diseases, college of veterinary medicine, university of georgia, athens, ga , usa e-mail: lanedegraaf.kelly@gmail.com v. o. ezenwa e-mail: vezenwa@uga.edu s. j. amish � g. luikart fish & wildlife genomics group, division of biological sciences, university of montana, missoula, mt , usa f. gardipee u.s. fish and wildlife service, sacramento, ca , usa a. jolles college of veterinary medicine and department of integrative biology, oregon state university, corvallis, or , usa conserv genet ( ) : – doi . /s - - - http://dx.doi.org/ . /s - - - populations driving evolutionary change (smith et al. ; koskella et al. ; leung et al. ). selection by parasites may have particularly potent impacts on regions of the genome that are directly involved in immune defense. immune system genes often show much greater adaptive evolution than genes not directly involved in immune function, and this pattern is often interpreted as a signature of intense co-evolutionary interactions between hosts and parasites (mctaggart et al. ). parasite- mediated selection on immune genes can occur directly when parasites reduce individual survival or depress fecundity, increasing the frequency of resistant genotypes (altizer et al. ; thrall and burdon ), and also indirectly via management responses to infectious disease outbreaks (shim and galvani ). in the latter case, strategies used to control parasite spread, such as culling or vaccination, may accelerate the rate and intensity of observable selection or drive cryptic evolutionary change. parasites interact with the host immune system in dif- ferent ways, resulting in distinct forms of selection acting on immunity. although immune system genes are expected to experience strong selection as a group, the mode and degree of selection can be highly variable across loci. balancing selection was once viewed as a dominant form of selection acting on immune loci in wildlife populations in particular, due in part to a historical focus of research on major histocompatibility (mhc) genes (acevedo-white- house and cunningham ). however, more recent work on a broader set of immune function genes has expanded this view (e.g. downing et al. ; tonteri et al. ; tschirren et al. ; llewellyn et al. ). in the last five years, more than one thousand studies have described evidence of selection on immune genes in mammals (excluding humans); of these studies, almost half found evidence of directional selection, while less than a tenth found evidence of balancing selection, and fewer still found evidence of diversifying selection highlighting the level of variability in the mode of selection acting on immune genes. for example, in a study of microsatel- lites linked to immune genes in atlantic salmon (salmo salar), tonteri et al. ( ) found that genes for interleu- kin and calmodulin production, were under strong direc- tional selection. by contrast, a study of five different immune genes in the greater prairie-chicken (tympanachus cupido), including interleukin and transforming growth factor b , found evidence of directional selection at only one of five genes (inhibitor of apoptosis protein- ), while selection at the remaining loci was more consistent with balancing selection (bollmer et al. ). finally, in an analysis of the interleukin (il- ) locus, thought to be under selective pressure from helminth parasites in mam- mals, diversifying selection was found to play a role in maintaining genetic diversity at fifteen il- residues involved in receptor binding. this pattern of diversifying selection was maintained across multiple mammalian genomes, ranging from mice to primates, carnivores and ungulates (koyanagi et al. ). these case studies sug- gest that interactions between parasites and the host immune system result not only in distinct signatures of selection on different immune genes, but also that the form of selection can vary across species and populations. the environmental context of a particular host-parasite interaction can play a key role in determining the strength and form of selection acting on any immune gene. in recent years, disease management has emerged as an important factor potentially driving selection on immune loci (altizer et al. ; mccallum ). management strategies for controlling infectious diseases in natural populations are becoming increasingly common, and as such, understand- ing how management can shape the evolution of host immune defenses is critical (woodroffe ; cross et al. ; joseph et al. ). culling, in particular, is one disease management strategy used in wildlife that has enormous potential to drive selection on hosts, particularly when specific groups of individuals are targeted for removal (myerstud and bischof ; myerstud ; white et al. ). while a number of studies have docu- mented unintended ecological effects of culling (donnelly et al. ; woodroffe et al. , a, b), empirical evidence of evolutionary consequences are still very lim- ited (smith et al. ). however, culling has the potential to erode host evolutionary potential by facilitating the loss of genetic diversity via reductions in rare alleles (sackett et al. ), impeding the evolution of resistance (shim and galvani ), and even driving broad evolutionary changes in the ecological community if disease manage- ment for one species changes the demography and ecology of non-target species (chauvenet et al. ). in this study, we focus on bovine tuberculosis (btb) as a potential agent of selection on immune genes in a wild reservoir host population. btb is a chronic disease of wildlife and livestock, caused by mycobacterium bovis. the disease has a global distribution and accounts for significant economic losses worldwide (michel et al. ; goodchild et al. ). in south africa, african buffalo (syncerus caffer) serve as the primary wildlife reservoir of btb and are responsible for spillover into other wildlife populations (e.g. lions, cheetahs) and cattle (renwick et al. ; fitzgerald and kaneene ; de garine-wichatit- sky et al. ). as such, effective disease control in buffalo populations is of critical importance (de garine- wichatitsky et al. ). management strategies for con- trolling btb in south african buffalo range from passive surveillance to active test and cull programs (michel et al. ). for instance, in kruger national park (knp), where btb was first detected in the buffalo population between conserv genet ( ) : – and and the population is estimated to be between , and , individuals, btb management has focused on surveillance, population monitoring, and research (michel et al. ). by contrast, in hluhluwe- imfolozi park (hip) where the first btb positive buffalo was detected in and the buffalo population is con- siderably smaller (est. , individuals), a test and cull program was initiated in the mid- s (michel et al. ). current estimates of btb prevalence in both parks are between and % for buffalo herds in knp (cross et al. ), and – % for buffalo herds in hip (jolles et al. ). since negative effects of btb on survival and reproduction have been described for buffalo (jolles et al. ), it is possible that this disease could act as a direct selective force on buffalo populations. moreover, in hip where the btb control program culled approximately buffalo testing positive for btb (out of a total of , animals tested between and ; jolles et al., unpublished data), selective culling could impose addi- tional indirect selective pressure, particularly at loci involved in immune defense against the disease. thus, the african buffalo-btb system presents an ideal platform for studying potential direct and indirect effects of parasites on the evolution of immune genes in the wild. taking advantage of this unique study system, we tested for evidence of selection on the interferon gamma (ifng) gene in populations of african buffalo in knp and hip. the ifng locus codes for an immune signaling molecule of the same name that plays an important role in the response to m. bovis infection. ifnc is a key cytokine involved in t helper (th ) cell responsiveness that is triggered by intracellular pathogens, including m. bovis (waters et al. ). given the critical role of ifnc in pathogen defense generally, and the response to btb specifically, variability in the ifnc phenotype is likely to be associated with fitness in the face of infection. impor- tantly, btb diagnosis in buffalo relies on ifnc-based tests that measure the strength of an individual’s immune response to m. bovis antigen challenge (wood et al. ; ryan et al. ; cousins and florisson ), thus culling based on variation in this response could impose strong selection on the ifng locus. to explore the possibility that btb, btb-related cull- ing, or both could be driving selection at ifng, and to identify the form of selection that might be acting, we quantified genetic diversity (heterozygosity and allelic richness) and gametic (linkage) disequilibrium (gd) across neutral loci flanking ifng to examine how diversity and gd change with distance around a locus putatively under selection. first, we investigated patterns of genetic diver- sity and gd around ifng in the total population at both parks to evaluate how intracellular pathogens in general (and btb specifically) may be driving selection at immune genes, irrespective of management strategy. second, we explored whether culling had additional effects on ifng by examining whether the number and frequency of rare alleles at ifng and surrounding loci were directly impacted by culling. we predicted that parasite-mediated selection would result in a distinct signature of selection at ifng and nearby loci in both parks. specifically, we expected that if balancing selection is occurring, we would see higher levels of diversity at ifng relative to sur- rounding loci. by contrast, if directional selection is occurring, we would see lower levels of diversity at ifng compared to flanking loci. with respect to gd, we expected that directional selection might result in stronger patterns of gametic disequilibrium involving ifng versus flanking loci. finally, we also predicted that if disease management contributes to selection at ifng this would be evident as a stronger selection signature in the hip popu- lation where culling takes place. alternatively, culling might produce relatively cryptic genetic changes in this population, disrupting patterns genetic diversity and gd around ifng. methods sample collection we sampled buffalo at two sites, hluhluwe-imfolozi park (hip) and kruger national park (knp) in south africa. in hip, males and females were captured in the masinda section of the park as part a bovine tuberculosis control program in and . in knp, female buffalo were captured in the lower sabie and crocodile bridge regions in as part of a research study. captures in hip were carried out by park management using a helicopter and funnel system to drive herds into a capture corral. in knp, animals were darted from a helicopter by the south africa national parks veterinary wildlife services. whole blood from hip buffalo (n = ) was preserved on fta cards (whatman � inc, clifton, nj, usa), and dried cards were stored at room temperature for one year until dna extraction. in knp, tissue samples were collected from buffalo (n = ) and stored in ml tubes with silica gel at room temperature for up to months prior to dna extraction. in hip, all captured buffalo were tested for bovine tuberculosis using a tuberculin skin test. briefly, individual buffalo were injected with bovine tuberculin intra-der- mally, and a localized swelling response measured h later (ryan et al. ). animals were considered btb ? if the swelling response was greater than mm. skin test-positive buffalo were culled as part of the btb control program. conserv genet ( ) : – molecular methods we focused on the ifng gene which codes for ifnc, a protein critical in the immune response to a variety of intracellular pathogens, including m. bovis (bradley et al. , bream et al. , pollock et al. ). twelve flanking microsatellite loci were chosen based on their proximity to ifng, and range in distance from . (bms ) to . (kera) cm on either side of ifng (see table s ). studies of cattle and sheep suggest that all but one of these flanking loci are neutral, or functionally neutral, genes. bl has been associated with immunity and disease resistance in sheep and african buffalo (coltman et al. , , ezenwa et al. ); while the following six loci have been reported to be neutral: bms (kappes et al. ), rm (maddox et al. ), br (kappes et al. ), krt (maddox et al. ), ilsts (kappes et al. ), agla (kappes et al. ). we considered five additional loci to be functionally neutral based on their involvement or proximity to genes with functions not related to immunity or disease resis- tance, including: cssm , which codes for a retinoic acid receptor important in vitamin a absorption (barendse ); glycam , kera, and tex , which code for proteins important in lactation, corneal development, and chromosomal synapsis and meiotic recombination, respectively (groenen et al. ; tocyap et al. ; yang et al. ); and igf, which codes for insulin-growth factor, which modulates cell growth and is linked with increased tumor development (renehan et al. ). dna was extracted from tissue samples using the qia- gen blood & tissue kit (valencia, ca) following the manufacturer’s protocol; for fta cards, a modified protocol was used (lisette waits, pers. comm). multiplex pcrs were optimized, and ul reactions were performed in mjr ptc thermocyclers. each reaction contained: ul of template dna, . ul of qia multiplex mix (qiagen), and ul of pm forward and reverse primers. two different touch- down profiles with – cycles were used, one with an initial annealing temperature of �c stepping down to �c, and another starting at �c and stepping down to �c. fluo- rescently-labeled dna fragments were visualized on an abi xl automated capillary sequencer (applied biosys- tems). allele sizes were determined using the abi gs liz ladder (applied biosystems). chromatograms were analyzed and confirmed by two independent technicians using genemapper software v . (applied biosystems). locus descriptions we tested for the presence of null alleles at all loci using micro-checker v. . . (van oosterhout et al. ), and for deviations from hardy–weinberg proportions (hwp) using genepop v. . (raymond and rousset ). no null alleles were found at any locus. all loci were in hwp except for agla (p \ . in both populations; table ), which had a heterozygote deficit, so further analyses were run both with and without this locus. since inclusion of agla did not qualitatively change our results, we only report the results including all loci. we quantified the magnitude of deviation from hwp by computing fis in each population. we also calculated fst values to evaluate the degree of genetic differentiation between the two study populations. both fis and fst val- ues were calculated using fstat v. . . (goudet ). finally, mean population relatedness was calculated in genalex using the lynch & ritland estimator multiplied by two so that it scales from zero to one with full sibs sharing half their alleles (r = . ). indices of genetic diversity and gd to evaluate whether selection has affected the ifng locus, we calculated two measures of genetic diversity for ifng and the flanking loci. first, we calculated allelic richness (ar) at each locus using fstat v. . . (goudet ). next, we estimated the heterozygosity at each locus by calculating both observed and expected heterozygosity under hardy– weinberg proportions. observed and expected heterozy- gosities (ho and he, respectively) were calculated in arle- quin v. . (schneider et al. ), but subsequent analyses are limited to he as it more accurately reflects genetic vari- ation within the population as a whole (nei ). we calculated pairwise gametic disequilibrium, a mea- sure of non-independence between loci due to either prox- imity or function. gd was expressed as d , a derivative of d which is the deviation in the frequency of co-occurrence between multiple alleles (i.e. haplotypes) due to gametic disequilibrium (lewontin and kojima ). d is defined as d = d/dmax where dmax is the maximum value of d, given a set of allele frequencies, and d = is complete gametic disequilibrium (lewontin ). in addition to d , we also calculated gd as r , which is a measure of gd that is influenced by allele frequencies. however, results for r were not quantitatively distinct from d , and as such, we report only the results of d . d and r values were calcu- lated using powermarker (liu and muse ). the sig- nificance of pairwise d estimates were evaluated using genepop (v . ), and accepted at p b . . patterns of selection to test for a signature of selection within each study popu- lation, we examined the association between genetic diver- sity at each locus and its chromosomal distance (the absolute value in cm) from the putative gene under selection (ifng). conserv genet ( ) : – this use of multiple loci across the gene region helps control for genome-wide (e.g. demographic) variations and has been previously used to identify loci under selection and selective sweeps (ihle et al. ; makinen et al. ). we tested for correlations between distance and allelic richness and het- erozygosity using spearman rank tests. since differences in chromosomal distance between loci should reflect differ- ences in the rate of recombination and/or selection, we also tested for associations between statistically significant val- ues of pairwise d and the distance (cm) between locus pairs. linear regression tests were used to evaluate whether levels of gd decayed with increasing distance between loci. genetic diversity in hip we compared genetic diversity between the two study pop- ulations using wilcoxon paired sign rank tests. in hip, where overall genetic diversity was much reduced, we tested for evidence of potential bottlenecks using heterozygosity excess and deficiency tests in bottleneck (v. . ). we used a two- phase model of microsatellite evolution, a biologically appropriate model that captures the evolution of microsatel- lites (cornuet and luikart ; luikart and cornuet ). we parameterized the model by defining % of mutations as conforming to a stepwise mutation model (kimura and ohta ) and % to a multistep model, assuming a var- iance of for the geometric distribution of number of repeat units per multi-step mutation. mode shift tests were used to assess bottleneck strength (cornuet and luikart ). to identify potential effects of culling on genetic diversity in hip, we compared our measures of diversity (allelic richness and heterozygosity) between two subsets of the hip population: animals that were culled as a result of a positive tuberculin skin test (btb positive, n = ), and animals that were poor reactors on the skin test and were not culled (btb negative, n = ). comparisons between population subsets were done using wilcoxon paired signed rank tests. we also examined the occurrence of rare alleles in the culled and non-culled population subsets of hip to test if rare alleles are being removed disproportionately as a result of culling, potentially con- tributing to an overall erosion of genetic diversity in the park. to do this, for each population subset, we calculated the number and frequency of alleles at each locus with a frequency of less than . . we then tested for locus-specific differences in the number and frequency of these rare alleles in the two population subsets using wilcoxon paired signed rank tests. results locus descriptions thirteen microsatellite loci spanning ifng were geno- typed successfully in a total of individuals from two populations (knp: n = ; hip: n = ). locus-specific fis ranged from - . to . in hip and from - . to . in knp (table ). low and/or negative fis values found at of loci in hip and of loci in knp indicate that there is little to no cryptic subpopulation structuring occurring within these populations. pairwise table population structure statistics for hip and knp locus cm from ifng hwp fis fst ar ho he hip knp hip knp hip knp hip knp hip knp tex - . . . . . * . . . . . igf - . . . . . . * . . . . rm - . . . - . . . * . . . . . . br - . . . - . . . * . . . . . bms - . . . - . - . . . . . . ifng . . - . - . . * . . . . . bl ? . . . . - . . * . . . . . cssm ? . . . - . . . * . . . . . . krt ? . . . - . - . . * . . . . . . ilsts ? . . . - . - . . * . . . . . glycam ? . . . - . - . . * . . . . . . agla ? . . * . * . . . * . . . . . . kera ? . . . - . . . * . . . . . . ? and - signs denote chromosomal distance from ifng in centimorgans (cm). * denote significant (p b . ) deviations from hardy–weinberg proportions (hwp) and significant genetic structure (pairwise fst). also reported are fis values, allelic richness (ar), observed and expected heterozygosity (ho, he) conserv genet ( ) : – fst values between populations ranged from . to . among loci (table ). there was evidence of sig- nificant genetic differentiation between the two populations at all but one locus; bms was the only locus with a non-significant fst value (fst = . ; p = . ). this locus also had the lowest fis value in both populations (fis knp = - . ; fis hip = - . ; table ). mean relatedness among individuals in hip was . and . in knp. signature of selection: patterns of genetic diversity and gd overall, genetic diversity was lower at ifng compared to other loci in both populations. ar ranged from to . alleles per locus in hip and from to . in knp (table ). excluding bms for which we found no significant evidence of genetic differentiation between the two parks, ifng was the locus with the lowest ar value in both populations, with only alleles identified in hip and . alleles in knp. similarly, expected heterozygosity was also lowest at ifng in both populations ( . in hip, . in knp; table ). observed heterozygosity showed similar patterns as expected heterozygosity. by examining genetic variation across loci at increasing distances from ifng, we found evidence of a signature of selection in one population but not the other. in knp, both allelic richness and heterozygosity were significantly and positively correlated with distance from ifng (spearman rank correlation: ar: rho = . , p = . ; he: rho = . , p = . ; fig. a–b). although the distance pattern observed for allelic richness and heterozygosity in hip mirrored the pattern observed in knp, no significant associations were detected between either measure of genetic diversity and distance from ifng in the hip pop- ulation (ar: rho = . , p = . ; he: rho = . , p = . ; fig. a–b). given the reduced genetic diversity at bms relative to ifng and other surrounding loci in knp, we also examined whether the patterns we observed for ifng could have been driven by bms . to do this we re-ran the distance analyses (e.g., association tests) using bms as the target locus. we found no evidence of an association between chromosomal distance from bms and genetic diversity (ar: rho = . , p = . ; he: rho = . , p = . ). this supports the supposition that ifng, and not bms , is the putative target of selection in the region under analysis. fifteen pairs of loci were in significant gd in hip, with the average d = . ; by contrast, seven pairs of loci were in significant gd in knp, with the average d = . (table ). the two populations shared only four of significant locus pairs (table ), with two of these showing similar deviations in haplotype frequency across populations (ilsts -agla and krt -agla ), and two showing higher levels of d in hip (ilsts - glycam and rm -br ; table ). the median distance between loci with significant pairwise gd was . cm in hip and . cm in knp, with pairwise distances ranging from . to . cm (table ). in knp, the strongest deviation in haplotype frequencies was between ifng and bl (d = . ; . cm), while the longest significant gd observed was between ifng and gly- cam ( . cm). in hip, the strongest deviation in hap- lotype frequencies was between ilsts and glycam (d = . ; . cm), and there was no significant pair- wise gd at distances greater than . cm. when we tested for associations between gd and the chromosomal distance between loci, we found that gd declined signifi- cantly with pairwise distance in knp (n = , r = . , p = . ; fig. ), but not hip (n = , r = . , p = . ; fig. ). reduced genetic diversity in hip two tests—the mode shift and heterozygosity-excess tests—revealed evidence of a recent bottleneck in hip (mode shift: bimodal distribution; heterozygosity excess fig. patterns of a allelic richness and b expected heterozygosity at increasing chromosomal distance (cm) from ifng in knp and hip. hip is represented by the dashed line, and knp is represented by the solid line conserv genet ( ) : – test: p \ . ; see table s ). furthermore, a comparison of genetic diversity between the hip and knp populations indicated that diversity is reduced in hip compared to knp. both allelic richness and expected heterozygosity were significantly lower in hip compared to knp (wil- coxon paired signed rank test: ar, s = . , p = . ; he, s = . , p = . ). while the bottleneck in hip may account for the pattern of eroded genetic diversity in this population, btb man- agement (i.e. culling) could also contribute to the overall reduction in genetic diversity. to explore this possibility, we tested for differences in genetic diversity between culled (c) and non-culled (nc) groups of individuals from hip. although there was no difference between population subsets in either diversity index (wilcoxon signed rank test: ar: s = - . , p = . ; he: s = - . , p = . ), the culled group had no heterozygosity at ifng, possibly indicating strong selection acting on this locus. focusing on rare alleles, we found that individuals in the culled group had a significantly higher number of rare alleles at loci surrounding ifng (c = ; nc = ; s = . , p = . ; table ). moreover, for those rare alleles present in both the culled and non-culled groups, over % ( out of ) occurred at a higher frequency in the culled group (table ). rarity is influenced by the number of alleles per locus and therefore could vary between population subsets simply because of the greater number of alleles available to sample in larger populations. however, even when we considered only the seven rare alleles that were present in both population sub-groups, and that occurred at loci where all alleles were shared between groups, over % ( out of ) occurred at a higher fre- quency in the culled group (table ), suggesting that rare alleles were overrepresented in the culled subset of the population and that culling may be disproportionately eliminating these alleles. discussion our findings suggest that directional selection is acting on and around the ifng locus in the buffalo population of kruger national park. in the hluhluwe-imfolozi park population, reduced genetic diversity due to recent bottle- neck events may have masked any signature of directional selection driven by disease. however, we found evidence that disease management may be compounding the loss of genetic diversity in the ifng gene region. in particular, culling to reduce bovine tuberculosis (btb) may be selectively removing rare alleles from the hip buffalo population, prolonging genetic recovery from a recent reduction in population size. overall, our results suggest that disease can directly or indirectly drive selection at immune loci in wild populations, and that disease man- agement might result in unintended evolutionary table locus pairs with significant levels of gametic disequilibrium (gd) in hip and knp population locus locus cm d hip ilsts glycam . . glycam agla . . ilsts agla . . krt ilsts . . br bms . cssm krt . . krt glycam . . krt agla . . tex igf . . agla kera . . bl cssm . . rm br . . glycam kera . . ilsts kera . . krt kera . . knp ilsts glycam . . ilsts agla . . ifng bl . . krt agla . . rm br . . cssm agla . . ifng glycam . . chromosomal distance between loci is listed in centimorgans (cm) and the strength of gd is measured as d . the shortest distance between possible pairwise comparisons was . cm while the longest distance was . cm fig. relationship between pairwise gametic disequilibrium (gd) and chromosomal distance (cm) in knp and hip. hip is represented by the dashed line with open circles, and knp is represented by the solid line with filled circles conserv genet ( ) : – consequences by exacerbating underlying population demographic effects. evidence of selection in knp our conclusion that selection is acting on ifng in buffalo comes from several lines of evidence. by examining loci flanking ifng, we uncovered a significant, positive relationship between chromosomal distance from ifng and both allelic richness and heterozygosity in the knp population. the positive relationship between genetic diversity and chromosomal distance from ifng suggests that directional selection (e.g. a selective sweep) is occurring at this locus. in addition, we observed significant gametic disequilibrium (gd) between ifng and bl which is likely the effect of directional selection at ifng and genetic hitchhiking at bl . while there was a signif- icant association between pairwise chromosomal distance between loci and gd in knp, the level of gd between ifng and bl was higher than for any other pair of loci, irrespective of distance. this suggests that in this region of the chromosome, selection may be generating a non-ran- dom association between alleles at these two loci which is stronger than that expected based on distance alone. interestingly, despite this signature of directional selection, several low frequency alleles remain at ifng in the knp population (three alleles at \ % frequency), and the long distance gd observed between ifng and glycam ( . cm apart) suggests that haplotypes involving these low frequency alleles are being maintained in knp. in contrast to the genetic diversity-chromosomal dis- tance pattern we observed in knp, no such pattern was evident in hip. however, an overall reduction in genetic diversity in the ifng gene region in the hip population could have masked any distance effect. in the mid- s the hip buffalo population dropped to as few as individuals followed by rapid population growth thereafter (jolles ); this event and the elevated levels of relat- edness among hip individuals corroborates our evidence for a recent bottleneck in the population. bottlenecks and founder events can drastically reduce the amount of genetic diversity in populations (maruyama and fuerst ), table number and frequency of rare alleles in hip, parsed by culled (c, n = ) and non-culled (nc, n = ) population segments zeros represent alleles that are absent from the associated population segment s shared rare alleles loci na rare na rare alleles frequency total nc c total nc c t nc c ifng ifng allele . . bms na bl bl allele . . s . s bl allele . . s . s bl allele . . s . s br na cssm cssm allele . . . cssm allele . . krt krt allele . . rm rm allele . . s . s rm allele . . s . s rm allele . . rm allele . . s . s rm allele . . ilsts ilsts allele . . . igf na glycam glycam allele . . agla agla allele . . s . s agla allele . . agla allele . . s . s agla allele . . tex tex allele . . . tex allele . . . kera kera allele . . . kera allele . . s . s kera allele . . conserv genet ( ) : – making signatures of selection difficult to detect (frere et al. ). nevertheless, characteristics of individual loci hint that selection on ifng could be occurring in hip, as in knp. specifically, in both populations, heterozygosity at ifng was at or close to the lowest values recorded across all loci (knp = . , range: . – . ; hip = . , range: . – . , table ), a pattern suggestive of directional selection acting on ifng in both populations. evidence of directional selection acting on immune loci in response to bacterial pathogens, including m. bovis, has been reported in livestock, suggesting that disease, possibly btb, could be driving the pattern of selection we observed at ifng. for example, btb has been implicated as a potential force driving directional selection of the disease resistance gene, nramp , in african zebu cattle (kad- armideen et al. ). evidence of directional selection has also been found at the porcine tlr- gene in response to gram-negative bacterial pathogens (palermo et al. ). thus, it is possible that btb infection acts to reduce genetic diversity at the ifng locus in buffalo, resulting in the conservation of only those alleles important in mounting an effective immune response, as has been shown in for the prnp gene in cervids in north america in response to chronic wasting disease (robinson et al. ). management-driven selection in hip effects of disease management on genetic variation are poorly understood, rarely assessed, but potentially strong (allendorf and hard ). in hip, we exploited a rare opportunity to assess the role of disease management in driving indirect selection. although there was no clear signature of selection in this population, we did find evi- dence that disease management (i.e. culling) might affect diversity at immune system genes. specifically, we found that although there were no differences in allelic richness or heterozygosity between culled and non-culled segments of the hip population, the culled segment had a signifi- cantly greater number and frequency of rare alleles sug- gesting that these alleles are being lost from the population via culling. a comparison of gd patterns in the two pop- ulations further suggests that culling may have disrupted the pairwise distance-gd relationship in hip by eliminat- ing haplotypes that are being maintained between ifng and surrounding loci (e.g. glycam ) in knp. evidence that ifng haplotypes being maintained in knp have been lost from hip suggests that the rare allele loss observed in hip may have important fitness conse- quences. rare allele advantage can be an important com- ponent of a population’s ability to respond to infectious disease agents (spurgin and richardson ; lankau and strauss ). thus, the reduction in rare alleles in hip could have implications for population level responses to future infectious disease threats. given the small sample size we used for the rare alleles comparison ( vs. ), we recognize that these results need to be interpreted with caution. nevertheless, based on the strong preliminary patterns we report here and in light of work showing that rare alleles can confer fitness advantages, particularly with respect to response to pathogens (koskella and lively ; sommer ), the potential loss of rare alleles due to culling in the buffalo-btb system deserves further investigation. the bottleneck in the mid- s, and subsequent rapid growth of the hip population, likely resulted in the founder event and reduction in overall genetic diversity that we observed in the park. culling could be prolonging this bottleneck event by eliminating rare alleles from the pop- ulation and reducing heterozygosity at ifng. disease management in this population has had effects on the population ecology of buffalo, with rapid population declines and reduced population growth rates typically following culling events (jolles ). in addition to these ecological effects, our results suggest that culling is also affecting buffalo population genetics and long-term evo- lutionary potential. management strategies for controlling invasive and emerging diseases in wildlife will continue to be important due to the risk of disease spillover into live- stock, wildlife, and humans (michel et al. ; smith et al. ). for buffalo in hip, however, culling for dis- ease management may also be sustaining the effects of a historical population bottleneck. more generally, our find- ings suggest that there is real potential for unforeseen evolutionary consequences to arise from disease manage- ment in wildlife populations. there are several notable examples of unintended eco- logical consequences of culling (singleton et al. ; bowen ). one well-known example is badger culling in the uk, which was intended to limit spread of btb to cattle, but was instead linked to increases in transmission (donnelly et al. , ; pope et al. ; woodroffe et al. , a, b). far less is known about the evolu- tionary consequence of culling as a disease management strategy. sport hunting, which in some instances has the same characteristics as culling, has been shown to have a variety of unintended outcomes, including altering gene flow between populations and, most importantly, selec- tively removing individuals with desired traits (harris et al. ; allendorf and hard ). for example, hunter selection, in conjunction with deteriorating environmental conditions, led to a significant reduction in horn size among bighorn sheep rams in arizona (hedrick ). because btb-culling programs that use ifnc-based diag- nostic tests will selectively remove individuals that pro- duce high levels of ifnc in response to an antigen challenge (wood et al. ; ryan et al. ; cousins and conserv genet ( ) : – florisson ), an analogous potential consequence of btb-culling could be the selective removal of individuals capable of mounting strong immune responses to btb infection. this selection could lead to significant popula- tion biases in immune profiles. future research is needed to examine whether genetic differences in culled versus non- culled buffalo translate into observable phenotypic differ- ences in immune responses to btb. conclusions opportunities for disease transmission between wildlife, livestock, and humans are growing. however, the effects of disease and disease management on evolution in wildlife populations remain poorly understood. in particular, strat- egies used to control disease might unwittingly lead to cryptic evolutionary change (shim and galvani ). we found that in an african buffalo population where btb has been present for over years, and where there is no active disease management, there was reduced diversity at (and near) ifng, a locus involved in mounting an immune response to pathogens like tuberculosis. by contrast, in a population where disease-based culling occurs, reduced genetic diversity may be masking a signature of selection. furthermore, preliminary patterns show that management actions, and thus unnatural selection, could be removing rare alleles from the population, potentially driving cryptic evolutionary change. while disease is widely recognized as a potentially powerful force of selection, our results sug- gest that disease management strategies can also have important evolutionary consequences. acknowledgments we thank kwazulu-natal wildlife service, kwazulu-natal state veterinary service, and south africa national parks veterinary services for facilitating this research. in particular, d. cooper, p. buss, and m. hofmeyr provided invaluable assistance. we thank matt gruber for his assistance in the lab. animal protocols used in this study were approved by the university of georgia, the university of montana and oregon state university animal care and use committees. this work was supported by the national science foundation ecology of infectious diseases program (deb- / ef- , 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woodroffe r, donnelly ca, cox dr, gilks p, jenkins he, johnston wt, lefevre am, bourne fj, cheeseman cl, clifton-hadley rs, gettinby g, hewinson rg, mcinerney jp, mitchell ap, morrison wi, watkins gh ( b) bovine tuberculosis in cattle and badgers in localized culling areas. j wildl dis ( ): – yang f, eckardt s, leu na, mclaughlin kj, wang pj ( ) mouse tex is essential for double-strand break repair and chromo- somal synapse during male meiosis. j cell biol ( ): – conserv genet ( ) : – 	signatures of natural and unnatural selection: evidence from an immune system gene in african buffalo 	abstract 	introduction 	methods 	sample collection 	molecular methods 	locus descriptions 	indices of genetic diversity and gd 	patterns of selection 	genetic diversity in hip 	results 	locus descriptions 	signature of selection: patterns of genetic diversity and gd 	reduced genetic diversity in hip 	discussion 	evidence of selection in knp 	management-driven selection in hip 	conclusions 	acknowledgments 	references diabetes care, volume , number , january o b s e rvat i o n s diabetes scre e n i n g practices among i n d i v i d u a l s a g e d ye a r s a nd o l d e r i n , the american diabetes associa- tion (ada) adopted new re c o m m e n d a- tions for screening the general popula- tion aged years for diabetes every years with an emphasis on those at high risk for undiagnosed diabetes ( ). few studies have examined the extent to which this screening has been adopted. this re p o rt describes the results of a telephone s u rvey of montana residents aged years to assess the diabetes screening prac- tices in this population. f rom october to december , the montana department of public health and human services conducted a random household telephone survey of montana residents aged years living in counties. respondents indicated whether they ever had been told by a physician that they had diabetes, the number of visits they made to a health care provider during the past year, their family history of dia- betes, whether they had ever been told they had high cholesterol and/or high blood pre s s u re, and their height and weight. respondents were asked the fol- lowing question to identify whether they had ever been screened for diabetes: “glu- cose or sugar is a substance found in your blood. have you ever had your blood glu- cose or sugar checked to see if you have diabetes?” when respondents re s p o n d e d “yes” to this question, they were asked to identify when screening was completed (“when was the last time your blood glu- cose or sugar level was measured by a health care professional?”). the re s p o n s e categories for this question included within the past year, within the past years, years ago, do not know/not sure, and refused to answer. pearson tests were used to assess associations between dia- betes screening and risk factors for dia- betes. logistical re g ression analyses were conducted to identify independent vari- ables associated with screening for diabetes during the past year. odds ratios ( % cis) were calculated. of the , respondents, ( . %) re p o rted that they had diagnosed diabetes. the remaining , respondents re p o rt e d that they did not have diagnosed diabetes and are included in the following analyses. of the respondents, % re p o rted a family h i s t o ry of diabetes, % re p o rted a bmi kg/m , % re p o rted having hyper- tension, and % re p o rted having high c h o l e s t e rol. excluding age, % of re s p o n- dents had one risk factor for diabetes, and % had two or more risk factors. of the , respondents without diagnosed dia- betes, % re p o rted that they had been s c reened for diabetes during the past year, % re p o rted screening from to years ago, and % re p o rted screening years ago or having never been scre e n e d . respondents who re p o rted being s c reened for diabetes during the past year w e re more likely to be age years and to have a family history of diabetes, two or m o re visits to a health care provider dur- ing the past year, hypertension, and high c h o l e s t e rol levels (table ). we found no association between recent screening and sex ( % men vs. % women), ameri- can indian ancestry ( % yes vs. % no), or bmi ( % kg/m vs. % kg/m ). respondents with three or m o re risk factors (e.g., aged years, american indian ancestry, family history of diabetes, hypertension, high choles- t e rol, or bmi kg/m ) were more likely to be screened for diabetes compared with respondents with only one risk factor ( vs. %, respectively). however, % of individuals with two risk factors for dia- betes and % of individuals with more than three risk factors had not been s c reened during the past years. based on logistical re g ression analysis, t h ree factors were associated with scre e n i n g for diabetes during the past year: two or m o re visits to a health care provider during the past year ( . [ % ci . – . ]), high cholesterol level ( . [ . – . ]), and family history of diabetes ( . [ . – . ]). respondents aged – years were less likely to re p o rt re c e n t s c reening than those aged years ( . [ . – . ] ) . a limitation of this assessment is that these data are self-re p o rted. pre v i o u s studies, however, have found that self- re p o rts of conditions such as diabetes and h y p e rtension are reliable ( , ). in addi- tion, the survey was conducted by tele- phone and does not re flect the experience of individuals in montana homes without t e l e p h o n e s . the findings suggest that diabetes s c reening is being adopted by physicians for individuals aged years at risk for diabetes and that the ada re c o m m e n d a- tions are being implemented in the general c o m m u n i t y. however, these data also indi- cate a need to develop strategies to encour- l e t t e r s table —characteristics of respondents aged years reporting screening for diabetes in montana in s c reening for diabetes past year – years years or never n age (years) – ( ) ( ) ( ) – ( ) ( ) ( ) ( )* ( ) ( ) family history of diabetes ye s ( )* ( ) ( ) n o ( ) ( ) ( ) visits to a health care provider during the past year ( )* ( ) ( ) ( ) ( ) ( ) h y p e rt e n s i o n ye s ( )* ( ) ( ) n o ( ) ( ) ( ) high cholestero l ye s ( )* ( ) ( ) n o ( ) ( ) ( ) data are n (%). *p . . diabetes care, volume , number , january letters age screening among all individuals at high risk for diabetes. todd s. harwell, mph jane g. smilie, ba janet m. mcdowall, rn, bsn steven d. helgerson, md, mph dorothy gohdes, md f rom the montana diabetes project, montana d e p a rtment of public health and human serv i c e s , helena, montana. a d d ress correspondence to todd s. harw e l l , mph, montana department of public health and human services, cogswell building, c- , p. o . box , helena, mt - . e-mail: t h a rw e l l @ s t a t e . m t . u s . a c k n o w l e d g m e n t s — this project was sup- p o rted through a cooperative agreement (u- / ccu- - ) with the centers for disease c o n t rol and prevention, division of diabetes translation, atlanta, georg i a . we thank linda priest and the staff mem- bers at northwest resource consultants for their work on the telephone surv e y. the contents of this letter are solely the responsibility of the authors and do not neces- sarily re p resent the official views of the centers for disease control and pre v e n t i o n . r e f e re n c e s . american diabetes association: screening for type diabetes. diabetes care (suppl. ): s –s , . kehoe r, wu sy, leske mc, chylack lt jr: comparing self-re p o rted and physician- re p o rted medical history. am j epidemiol : – , . jackson c, jatulis de, fortmann sp: the behavioral risk factor survey and the stan- f o rd five-city project survey: a comparison of cardiovascular risk behavior estimates. am j public health : – , h b a c is not recommended as a s c reening test for diabetes in cystic fibro s i s i n the june issue of diabetes care, h u n k e rt et al. ( ) recommend the use of h b a c for early detection of cystic fib ro- s i s – related diabetes (cfrd). their re c o m- mendation is based on the finding that mean hba c is slightly higher in cystic fib rosis (cf) patients requiring insulin t h e r a p y, compared with cf patients with i m p a i red or normal glucose tolerance. h o w e v e r, no data on the validity of this a p p roach for the diagnosis of asympto- matic diabetes in patients with cystic fib ro- sis are pre s e n t e d . our group has a long-standing intere s t in the early diagnosis of cfrd, comparing fasting blood glucose levels with oral glu- cose tolerance test results ( ). in our series, we have now cf patients with newly diagnosed diabetes based on a -h venous plasma glucose value mg/dl ( . mmol/l), and simultaneous determ i n a t i o n of hba c ( h i g h - p e rf o rmance liquid chro- matography method [pharmacia, erlan- gen, germany], normal range . – . %). only out of cf patients ( %) diag- nosed as diabetic according to the ameri- can diabetes association and world health o rganization criteria ( ) had an hba c value above the normal range (individual values . , . , . , and . %). in nine diabetic cf patients with normal hba c, values between . and . % were e n c o u n t e red (mean ± sd, . ± . %). the mean -h blood glucose value after inges- tion of oral glucose was not signific a n t l y d i ff e rent between diabetic cf patients with n o rmal hba c ( ± mg/dl, mean ± sd) and diabetic cf patients with elevated h b a c ( ± mg/dl, student’s t t e s t ) . these data clearly demonstrate that the d e t e rmination of hba c is not able to sub- stitute for the oral glucose tolerance test in the early diagnosis of cfrd. our fin d i n g s a re in agreement with several re p o rts in the l i t e r a t u re ( – ) as well as the consen- sus conference on cfrd ( ). in addition to its low sensitivity when used as a diagnos- tic tool for the detection of cfrd, the mea- s u rement of hba c has the disadvantage of considerable interassay variability and lack of standardization. there f o re, we stro n g l y advise against the use of glycosylated hemoglobin as a screening test for the early diagnosis of diabetes in patients with cystic fib ro s i s . reinhard w. holl, md christian buck, md christine babka, md anna wolf, md angelika thon, md f rom the department of pediatrics (r.w.h.), uni- versity of giessen, giessen; the department of pedi- atrics (c.bu., c.ba., a.w.), the university of ulm, ulm; and the medical school hannover (a.t. ) , h a n n o v e r, germ a n y. a d d ress correspondence to pd dr. reinhard w. holl, universitätskinderklinik giessen, feulgenstr. , d- giessen, germ a n y. r e f e re n c e s . h u n k e rt f, lietz t, stach b, kiess w: potential impact of hba c d e t e rm i n a t i o n on clinical decision making in patients with cystic fib ro s i s – related diabetes (let- ter). diabetes care : – , . holl rw, buck c, cario h, wolf a, thon a, heinze e, kohne e, debatin k-m: diag- nosis of diabetes in cystic fib rosis and tha- lassemia major. diabetes care : – , . the expert committee on the diagnosis and classification of diabetes mellitus: r e p o rt of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care : – , . lanng s, hansen a, thorsteinsson b, n e rup j, koch c: glucose tolerance in patients with cystic fib rosis: five year p rospective study. b m j : – , . deluca f, arrigo t, nibali sc, sferlazzas c, gigante a, dicesare e, cucinotta d: insulin secretion, glycosylated haemoglo- bin and islet cell antibodies in cystic fib ro- sis children and adolescents with diff e re n t d e g rees of glucose tolerance. h o rm metab r e s : – , . moran a, doherty l, wang x, thomas w: a b n o rmal glucose metabolism in cystic fib rosis. j pediatr : – , . moran a: highlights of the febru a ry consensus conference on cfrd. bonn, g e rm a n y, cystic fibrosis foundation, p ro gln p e roxisome p ro l i f e r a t o r- a c t i v a t e d r e c e p t o r- and o b e s i t y r istow et al. ( ) re p o rted an activating mutation in the peroxisome pro l i f- e r a t o r-activated re c e p t o r- g e n e ( p ro gln ppa r - ), which was pre s e n t in % ( of ) of obese and % ( of ) of nonobese german caucasians. these findings may have profound implica- tions, particularly if the presence of this variant and its association with obesity are c o n firmed in other populations. we perf o rmed polymerase chain re a c- t i o n – restriction fragment length polymor- phism analysis for the pro gln ppa r - variant as described ( ) on dna samples f rom several independent populations, including lean and obese caucasians fro m the baltimore, maryland, region; african- diabetes care, volume , number , january letters americans from jackson, mississippi, and forsyth county, north carolina; pima indians from arizona; and old ord e r amish from lancaster county, pennsylva- nia (table ). a pcr fragment corre s p o n d- ing to gastric insulinotropic peptide, which has two known restriction sites for h i n dii, was mixed with each sample as a positive control. among a total of sub- jects ( , alleles), the pro gln variant was not detected in a single subject. these findings were unexpected because there is substantial overlap of gene pools of caucasians from central e u rope and the baltimore and amish caucasians studied ( ). the germ a n caucasians studied by ristow et al. were said to be unrelated and re c ruited fro m the nord rh e i n - westfalen region, but they may be a genetic isolate whose gene pool does not re flect that of other caucasian populations. altern a t i v e l y, if the individ- uals carrying the mutation were re l a t e d , the true frequency of the pro g l n p pa r - variant may have been overe s t i- mated. the absence or very low fre- quency of this variant has also been doc- umented in danish ( ) and german ( ) populations. this study is the first, to our knowledge, to examine american popu- lations for this variant. in summary, the study by ristow et al. demonstrating that the pro g l n p pa r - variant is activating and can influence body weight in people is important. however, this variant appears to be absent or very r a re in the american populations studied. additional studies are re q u i red in other regions of europe and the u.s. to furt h e r d e fine the relevance of this intere s t i n g genetic variant to susceptibility to obesity. alan r. shuldiner, md william nguyen, bs w.h. linda kao, phd brock a. beamer, md ross e. andersen, phd richard pratley, md frederick l. brancati, md, phd f rom the department of medicine (a.r.s., w. n . ) , university of maryland school of medicine; the d e p a rtments of medicine (b.a.b., f.l.b.) and epi- demiology (w.h.l.k.), johns hopkins university school of medicine, baltimore, maryland; and the national institute of diabetes and digestive and kidney diseases (r.p.), national institutes of health, phoenix, arizona. a d d ress correspondence to alan r. shuldiner, md, professor and head, division of endocrinol- o g y, diabetes, and nutrition, department of medi- cine, university of maryland school of medicine, w. lombard st., room s- , baltimore, md . e-mail:ashuldin@medicine.umary l a n d . e d u . a c k n o w l e d g m e n t s — this study was sup- p o rted by nih r dk- , the american diabetes association, glaxowellcome, the b a l t i m o re geriatrics research and education clinical center of the baltimore ve t e r a n s administration medical center. r e f e re n c e s . ristow m, muller- wieland d, pfeiffer a, k rone w, kahn cr: obesity associated with a mutation in a genetic regulator of adipocyte diff e rentiation. n engl j med : – , . c a v a l l i - s f o rza ll, menozzi p, piazza a: t h e h i s t o ry of human genes. princeton univer- sity press, princeton, nj, . elk j, urhammer sa, sorensen ti, ander- sen t, auwerx j, pedersen o: homozygosity of the pro ala variant of the pero x i s o m e p roliferation-activated re c e p t o r- g a m m a ( p par-gamma ): divergent modulating e ffects on body mass index in obese and lean caucasian men. diabetologia : – , . mamann a, munzberg h, buttron p, busing b, hinney a, mayer h, siegfried w, hebe- brand j, greten h: missense variants in the human peroxisome pro l i f e r a t o r- a c t i v a t e d re c e p t o r-gamma gene in lean and obese subjects. eur j endocrinol : – , insulin secre t i o n , insulin sensitivity, and glucose e ffectiveness in nonobese individuals with va ry i n g d e g rees of glucose to l e r a n c e a lthough it is well known that insulin s e c retion, insulin sensitivity, and glu- cose effectiveness are impaired in type diabetic patients ( – ), little is known about the role of each of these fac- tors individually on the evolution of type diabetes. in this context, a major issue is that hyperglycemia per se impairs insulin s e c retion and insulin sensitivity and that obesity observed in type diabetic patients per se causes insulin resistance ( , ). to o v e rcome this problem, we studied u n t reated nonobese subjects classified as having normal glucose tolerance (ngt) (n = ; bmi . ± . kg/m [ r a n g e . – . ], mean ± sem), impaired glu- cose tolerance (igt) (n = ; bmi . ± . kg/m [ . – . ]), and type dia- betes (n = ; fetal bovine serum . ± . mmol/l [range . – . ], bmi . ± . k g / m [ . – . ]), based on the criteria of the world health organization ( ). they were normotensive and had norm a l renal, hepatic, and thyroid function. insulin sensitivity and glucose eff e c t i v e n e s s w e re estimated by the minimal model a p p roach ( – ). insulin secretion was e x p ressed as the area under the insulin c u rve between and min after an intra- venous glucose injection ( ). after the data w e re analyzed by one-way analysis of vari- ance, bonferroni correction was used to evaluate the diff e rences between any two of the three groups we studied ( ). no signifi- cant difference was observed in bmi among the three groups. compared with the subjects with ngt, the subjects with table —characteristics of subjects screened for pro gln ppa r - n ( a l l e l e s age ± sd f e m a l e bmi ± sd p o p u l a t i o n t y p e d ) ( y e a r s ) ( % ) ( k g / m ) c a u c a s i a n s b a l t i m o re longitudinal ( ) . ± . . . ± . study on aging johns hopkins we i g h t ( ) . ± . . . ± . management center amish, lancaster, pa ( ) . ± . . . ± . a f r i c a n - a m e r i c a n s a t h e ro s c l e rosis risk in ( ) . ± . . . ± . communities study pima indians a r i z o n a ( ) . ± . — . ± . all non-amish subjects were unrelated; amish subjects were not fir s t - d e g ree relatives of each other. diabetes care, volume , number , january letters igt had significantly lower insulin secre- tion ( , ± vs. , ± pmol l m i n , p = . ) and glucose eff e c- tiveness ( . ± . vs. . ± . m i n , p . ). insulin sensitivity index was lower in subjects with igt ( . ± . m i n pmol l) than in those with ngt ( . ± . min pmol l ) , but was not statistically significant (p = . ). in con- trast, disposition index calculated by the p roduct of insulin secretion and insulin sensitivity was significantly lower in sub- jects with igt ( , ± ) than in those with ngt ( , ± , p = . ). on the other hand, patients with type dia- betes had significantly lower insulin secre- tion ( ± pmol l m i n , p = . ) compared with subjects with igt. although no significant diff e rence was observed in insulin sensitivity index between subjects with type diabetes and igt ( . ± . vs. . ± . min pmol l, p = . ), disposition index was s i g n i ficantly diminished in type diabetic patients as compared with subjects with igt ( ± vs. , ± , p = . ). glucose effectiveness in type diabetic patients ( . ± . min ) was similar to that in subjects with igt ( . ± . m i n , p = . ) but was signific a n t l y lower than that in the subjects with ngt (p . ). from these results, the fol- lowing may be hypothesized: ) impair- ments in insulin secretion and disposition index and decreased glucose eff e c t i v e n e s s , but not insulin resistance, seem to consti- tute the basic characteristics of patients with igt or type diabetes in nonobese japanese populations. ) risk factors wors- ening to type diabetes in subjects with igt are associated with further impair- ments in insulin secretion and disposition index, but not associated with furt h e r derangement in glucose effectiveness in japanese populations. ataru taniguchi, md mitsuo fukushima, md masahiko sakai, md itaru nagata, md kentaro doi, md shoichiro nagasaka, md kumpei tokuyama, md yoshikatsu nakai, md f rom the first department of internal medicine ( a . t., m.s., i.n.), kansai-denryoku hospital, and the department of internal medicine, hoshida- minami hospital (m.f.), osaka; the second depart- ment of internal medicine (k.d.) and the college of medical technology (y.n.), kyoto university, kyoto; the department of internal medicine (s.n.), jichi medical college, tochigi; and the laboratory of biochemistry of exercise and nutrition (k.t. ) , tsukuba university, tsukuba, japan. a d d ress correspondence to ataru ta n i g u c h i , md, first department of internal medicine, kansai- d e n ryoku hospital, - - , fukushima-ku, fuku- shima, osaka city, osaka - , japan. e-mail: k @ k e p c o . c o . j p . r e f e re n c e s . b e rgman rn: to w a rd physiological under- standing of glucose tolerance: minimal- model approach. d i a b e t e s : – , . welch s, gebhart ssp, bergman rn, phillips ls: minimal model analysis of intravenous glucose tolerance test-derived insulin sensitivity in diabetic subjects. j c l i n endocrinol metab : – , . taniguchi a, nakai y, fukushima m, kawamura h, imura h, nagata i, tokuyama k: pathogenic factors re s p o n s i- ble for glucose tolerance in patients with niddm. d i a b e t e s : – , . nagasaka s, tokuyama k, kusaka i, hayashi h, rokkaku k, nakamura t, kawakami a, higashiyama m, ishikawa s, saito t: endogenous glucose pro d u c t i o n and glucose effectiveness in type diabetic subjects derived from stable-labeled mini- mal model approach. d i a b e t e s : – , . best jd, kahn se, ader m, watanabe rm, ni t-c, bergman rn: role of glucose eff e c- tiveness in the determination of glucose tol- erance. diabetes care : – , . rossetti l, giaccari a, defronzo ra: glu- cose toxicity. diabetes care : – , . taniguchi a, nakai y, doi k, fukuzawa h, fukushima m, kawamura h, to k u y a m a k, suzuki m, fujitani j, tanaka h, nagata i: insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis. m e t a b o l i s m : – , . world health organization: diabetes melli - tus: report of a who study gro u p . g e n e v a , world health org., (tech. rep. ser. , no. ) . winer bj: statistical principles in experimen - tal design. nd ed. new york, mcgraw-hill, , p. – late-onset tro g l i t a z o n e - i n d u c e d hepatic dysfunction r e c e n t l y, iwase et al. ( ) re p o rted a case of liver dysfunction occurring after months of troglitazone therapy. because it was thought before this re p o rt that the risk of liver dysfunction with tro g l i- tazone after months was negligible, we wish to re p o rt another patient who took t roglitazone intermittently and developed hepatic dysfunction after months. a -year-old white man with type diabetes, ischemic heart disease (post angioplasty and stent placement), hyper- tension, degenerative joint disease, benign p rostatic hypert ro p h y, dyslipidemia, and g a s t roesophageal re flux disease had tro g l i- tazone mg daily added to his re g i m e n of glimeperide mg daily and metform i n mg b.i.d. because of poor glycemic c o n t rol (hba c . % [normal – %]). the other medicines he used were aspirin and pravastatin. after months of triple oral therapy, his hba c level dropped to . %, and, after months, to . %. after months, the patient’s hba c began to rise: . % at months, . % at year, and . % at months. liver function tests were normal until months, when his aspartate amino- transferase (ast) was found to be (nor- mal – u/l) and alanine aminotrans- ferase (alt) (normal – u/l). the t roglitazone regimen was discontinued, and testing for hepatitis b and c, h e m a c h romatosis, autoimmune liver dis- ease, and gallbladder disease were nega- tive. two months after discontinuing t roglitazone, the patient’s ast and alt had decreased to and u/l, and, after months, had re t u rned to normal at and u/l, re s p e c t i v e l y. his ast and alt have remained normal since then and he has continued to take pravastatin, aspirin, m e t f o rmin, and glimeperide. when the patient was told to discon- tinue troglitazone, he admitted that he had been taking it only interm i t t e n t l y. he esti- mated that he took the drug regularly at first, but after the first months, he took the drug only once or twice weekly on aver- age. he gave the following three reasons for his lack of compliance: a lack of funds, a fear of liver disease, and a tendency to avoid taking drugs whenever possible. this case, like the case described by iwase et al., illustrates that the hepatic dysfunction caused by troglitazone can occur after months and further sup- p o rts the u.s. food and drug administra- t i o n ’s current recommendation that quar- terly liver function tests should be obtained when troglitazone utilization extends beyond year ( ). in this case, could the onset of hepatic dysfunction have been delayed because the diabetes care, volume , number , january letters d rug was being taken only interm i t t e n t l y after the first months, and the estimated total load presented to the liver would be equivalent to the exposure at months in a compliant patient? we doubt this, since t ro g l i t a z o n e ’s hepatic effects are thought to be idiosyncratic and there f o re the total e x p o s u re should be irre l e v a n t . david s.h. bell, mb fernando ovalle, md f rom the division of endocrinology and metabo- lism, department of medicine, school of medicine, university of alabama, birmingham, alabama. a d d ress correspondence to david s.h. bell, mb, th ave. s., birmingham, al . d.s.h.b. and f.o. have served on an advisory panel for sankyo parke-davis and have received con- sulting fees, re s e a rch grant support, and honoraria for speaking engagements from sankyo parke-davis. r e f e re n c e s . iwase m, yamaguchi m, yoshinari m, oka- mura c, hirahashi t, tsuji h, fujishima m: a japanese case of liver dysfunction after months of troglitazone tre a t m e n t . diabetes care : – , . parke-davis, division of wa rn e r- l a m b e rt : rezulin package insert. morris plains, nj, g l y b u r i d e - i n d u c e d hemolysis in m y e l o d y s p l a s t i c s y n d ro m e g lyburide, also known as gliben- clamide, is a widely used sulfonylure a to treat patients with type diabetes. hemolytic anemia is an extremely rare side e ffect of which there have been only a few re p o rts ( – ). we describe a patient with myelodysplastic syndrome who pre s e n t e d with glyburide-induced hemolysis. a -year-old man with a long history of type diabetes presented with left foot cellulitis of week’s duration. this patient was known to have slowly pro g re s s i v e pancytopenia for years, for which no work-ups had been perf o rmed. his med- ications included the following: glyburide, mg per day, which he had taken for m o re than year; buformine, mg per day; and boglibose, . mg per day. he was afebrile, and the physical examination was normal, except for localized cellulitis on his left foot, for which he was start e d on intravenous antibiotics. the laboratory studies revealed a white blood cell count of . / l , hemoglobin . g/dl, platelet count /l, reticulocyte count . %, mean cor- puscle volume fl, moderate anisocyto- sis, fasting plasma glucose mg/dl, h b a c . %, lactate dehydrogenase iu/l, total bilirubin . mg/dl, and hapto- globin mg/dl. red cell glucose- - phosphate dehydrogenase level was ade- quate. cold agglutinin test, ham’s test, and sugar water test were normal. both dire c t and indirect coombs’ tests were negative. red cell resistance to osmolarity was mildly low (parpart ’s method). urinalysis demonstrated no urobilinogen. endo- scopic studies did not reveal gastro i n t e s t i- nal bleeding. ultrasonography of the abdomen showed no splenomegaly. the result of bone marrow aspiration was equivocal. on the basis of pre s u m p t i v e glyburide-induced hemolysis, glyburide was discontinued and the patient was switched to subcutaneous insulin on the seventh day. there a f t e r, his hemoglobin level increased to . g/dl, re t i c u l o c y t e count decreased to . %, and anisocytosis d i s a p p e a red pro m p t l y. he was discharg e d with insulin therapy after month in the hospital, which is when the cellulitis re s o l v e d . t h ree months later, his hemoglobin level was . g/dl and his haptoglobin remained low. repeated bone marro w aspiration confirmed the diagnosis of myelodysplastic syndro m e . we conclude that this patient devel- oped glyburide-induced hemolysis super- imposed on red cell fragility secondary to an underlying bone marrow disord e r. t h e re have been several re p o rts of hemoly- sis caused by sulfonylureas, most of which have been considered immune-mediated ( , , ). our case points to the possibility that glyburide could cause hemolysis by a non–immune-medicated mechanism. it is i m p o rtant to be aware of this potential side e ffect of glyburide in light of this medica- t i o n ’s widespread prescription, even though such a side effect is rare . hiroshi noto, md kazuhisa tsukamoto, md satoshi kimura, md f rom the department of diabetes and metabolism, tokyo university hospital, tokyo, japan. a d d ress correspondence to hiroshi noto, md, d e p a rtment of diabetes and metabolism, tokyo uni- versity hospital, - - hongo, bunkyo-ku, to k y o - , japan. e-mail: noto-tky@umin.ac.jp. r e f e re n c e s . nataas ob, nesthus i: immune haemolytic anaemia induced by glibenclamide in selective iga defic i e n c y. b m j : – , . abbate sl, hoogwerf bj: hemolytic ane- mia associated with sulfonylurea use. d i a - betes care : – , . meloni g, meloni t: glyburide-induced acute haemolysis in a g pd-defic i e n t patient with niddm. br j haematol : – , . kopicky ja, packman ch: the mechanism of sulfonylurea-induced immune hemoly- sis: case re p o rt and review of the literature . am j hematol : – , e ffects of exposure at an altitude of , m on p e rf o rmance of glucose meters s elf-monitoring of blood glucose is m a n d a t o ry for type diabetic p a t i e n t s who participate in sports to adjust insulin dose and carbohydrate ingestion ( ). sports also include activities p e rf o rm e d at moderately high altitudes, such as hiking or skiing. capillary blood glucose monitors (bgms) have been shown to underestimate blood glucose values at an altitude of , m ( ) and at a simulated altitude of , m with t e m p e r a t u re and humidity kept constant ( ). the aim of the present study was to assess the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and p o can result in errors in blood glucose d e t e rmination ( ). two bgms, the lifescan one touch ii (ot) (ortho diagnostics, milpitas, ca) and the glucometer elite ii (ge) (bayer diagnostics, brussels, belgium), were tested during a study on the effects of acute exposure at an altitude of , m and exercise on blood pre s s u re and albu- min excretion rate in six type diabetic p a t i e n t s . all subjects (four men and two women) were free of disease-related com- plications and in good and stable glycemic control (ghb . ± . %). all subjects gave their informed and written consent to participate in the study pro t o- col. all subjects were investigated both at diabetes care, volume , number , january letters sea level and after ascent by car and cable car to the angelo mosso institute at col d’olen ( , m altitude), gressoney la trinité, italy. at sea level and at a moderately high altitude, bgm reliability at diff e rent blood glucose levels was tested, and blood glu- cose was assessed in fasting and re s t i n g conditions at : a.m.; at : a.m. b e f o re an in-field exercise test; and imme- d i a t e l y, min, and min after the exer- cise stopped. capillary glucose was simul- taneously assessed with the ot and the ge. both of these bgms measure capillary blood glucose through the glucose oxi- d a s e - p e roxidase reaction. bgms were cali- brated at the beginning of each test ses- sion. a venous blood sample was simulta- neously drawn from the contralateral antecubital vein in a sodium fluoride tube, centrifuged, and stored at °c. plasma glucose was assayed with the glucose oxi- dase method (go) within days. this last assessment was taken as a re f e re n c e method. statistical analysis compare d bgm capillary glucose values and go plasma glucose values for each blood col- lection time. measurement linearity was tested with pearson’s correlation coeff i- cient. the mean of the diff e rences between the bgm and go results re p resents the mean bias between the methods with accuracy expressed as percent error (pe): pe (%) = bmg – go % g o the level of statistical significance was c o n s i d e red to be p . . the ge and ot measurements had a good correlation with plasma glucose both at moderately high altitude and at sea level. p e a r s o n ’s correlation coefficients were . and . for the ge and . and . for the ot at sea level and at moderately high altitude, re s p e c t i v e l y. biases between plasma glucose and bgm measure m e n t s w e re as follows: for the ge, . ± . at sea level and . ± . at moderately high altitude; for the ot, . ± . at sea level and . ± . at moderately high alti- tude. at sea level, both the ge and the ot tended to underestimate glucose values (ns); at moderately high altitude, the ge tended to overestimate and the ot tended to underestimate glucose values (ns). mean pes between plasma glucose and bgm m e a s u rements were . (ot) and . (ge) at sea level and . (ot) and . (ge) at moderately high altitude. pe tended to be higher for both bgms at moderately high altitude (ns). figures and show the bias between the single measurements with both devices at sea level and at moderately high altitude, re s p e c t i v e l y. at moderately high altitude (fig. ), the tendency of the ge to o v e restimate was more evident for low ( mg/dl) and intermediate ( – mg/dl) blood glucose values, whereas the ot tended to underestimate mainly high blood glucose values (ns). in our study, bgm perf o rmance was similar and good at sea level. at a moder- ately high altitude, a tendency to overe s t i- mate blood glucose for the ge and to u n d e restimate for the ot was observ e d . the overestimation for the ge involved mainly low ( mg/dl) and interm e d i- ate ( – mg/dl) blood glucose val- ues. this could present a problem in the p resence of symptoms suggesting hypo- figure —relationship between plasma and capillary glucose at sea level. figure —relationship between plasma and capillary glucose at moderately high altitude. lifescan one touch ii glucometer elite ii lifescan one touch ii glucometer elite ii diabetes care, volume , number , january letters glycemia and normal blood glucose val- ues. the ot tended to undere s t i m a t e mainly high blood glucose values, although its perf o rmance with low to i n t e rmediate values was good. the pre s- ent study assessed the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and po can result in errors in blood glu- cose determination ( ). our results are consistent with previous studies ( , ). the decrease in po could alter the sec- ond phase of the chromogen reaction and u n d e restimate blood glucose values ( ); on the other hand, an increase in atmos- pheric pre s s u re could overestimate blood glucose values ( ). in our study, minimal o v e restimation by the ge at low interm e- diate blood glucose values at moderately high altitude cannot be explained by the a l t e red po . an increase in hematocrit, which is known to alter blood glucose m e a s u rements with bgms ( ), may also occur after prolonged exposure to high altitude or as a consequence of dehydra- tion. although our study did not deter- mine hematocrit, the exercise test was s h o rt, and the patients were instructed to drink according to their thirst during the , -m exposure; there f o re, dehydration was not likely to have occurred. in con- clusion, bgm perf o rmance is similar and good at sea level. at a moderately high altitude similar to that experienced during winter skiing or summer hiking, a ten- dency to overestimate low to norm a l blood glucose values for the ge and to u n d e restimate high blood glucose values for the ot was observed. the bias is not clinically meaningful for either bgm, both of which can be safely used by dia- betic patients during exposure to moder- ately high altitudes. some care in the eval- uation of low and intermediate blood glu- cose values measured with the ge is n e v e rtheless re c o m m e n d e d . oriana pecchio, md simona maule, md marco migliardi, md marina trento, bsc massimo veglio, md f rom the italian alpine club medical commission ( o . p.); the department of internal medicine (m.t. ) , university of turin; the s. giovanni battista hospital (s.m.); and the department of endocrinology (m.m., m . v.), mauriziano umberto i hospital, turin, italy. a d d ress correspondence to dr. m. veglio, via mancini , torino, italy. e-mail: veglio@ o n w. n e t . r e f e re n c e s . h o rton es: role and management of exer- cise in diabetes mellitus. diabetes care : – , . g i o rdano bp, trash w, hollenbaugh l, dube wp: perf o rmance of seven blood glucose testing systems at high altitude. diabetes educ : – , . gautier jf, bigard ax, douce p, duvallet a, cathelinau g: influence of simulated alti- tude on the perf o rmance of five blood glu- cose meters. diabetes care : – , . b a rnett c, ryan f, ballonoff l: effect of altitude on the self monitoring of blood glucose (smbg) (abstract). diabetes (suppl.): a, . piepmeier eh, hammett-stabler c, price me, kemper gb, davis mg: atmospheric p re s s u re effects on glucose monitoring devices (letter). diabetes care : – , . b a rreau pb, buttery je: effect of hematocrit concentration on blood glucose value d e t e rmined on glucometer ii. diabetes care : – , c o m m e n t s a n d r e s p o n s e s deterioration of glycemic contro l after long-te rm treatment wi t h troglitazone in nonobese type diabetic patients t roglitazone is an oral antidiabetic d rug used to treat type diabetic patients with insulin re s i s t a n c e . troglitazone improves overall insulin sen- sitivity in the liver and skeletal muscles, which are the largest consumers and metabolizers of glucose in the body ( – ). recent re p o rts showed that troglitazone is also effective in nonobese type diabetic patients whose hyperglycemia could not be controlled with sulfonylurea therapy ( , ). however, we aware that in some patients in whom adequate glycemic con- t rol is obtained during the first several months of troglitazone treatment, their glycemic control deteriorates several months later. we assume that two distinct g roups of type diabetic patients exist who respond diff e rently to long-term administration of troglitazone, one gro u p that maintains a steady response and another group that has a decre a s i n g response after certain periods. in this s t u d y, we re t rospectively examined patients with type diabetes who were t reated with troglitazone for months and whose hba c levels had improved by % with troglitazone by month . in of the patients ( %), hba c levels increased by . % after – months despite continuous tro g l i t a z o n e t reatment (group p). in contrast, the rest of the patients experienced steady glycemic c o n t rol with . % of hba c flu c t u a t i o n ( g roup g). during the first months, h b a c levels decreased from means ± sem . ± . to . ± . % in group p and fro m . ± . to . ± . % in group g, re s p e c- t i v e l y. no significant diff e rences were evi- dent between the two groups re g a rding the d e c rease in hba c during the first months (fig. ). from month onward, hba c l e v- els in group p climbed gradually by . % a month up to the baseline level at month , but hba c levels were stable in group g t h roughout the treatment period. a signifi- cant diff e rence in hba c levels was evident during months – (p . ) . among clinical characteristics, gro u p p had a significantly lower bmi ( . ± . vs. . ± . kg/m , p . ) and s i g n i ficantly lower fasting insulin levels ( . ± . vs. . ± . µu/ml, p . ). of the patients with a bmi of kg/m ( %), exhibited deteriora- tion of glycemic contro l . in this study, we re p o rt a group of patients who showed a renewed decline in glycemic control after long-term tre a t m e n t with troglitazone. these results seemed to suggest a secondary failure of tro g l i t a z o n e . our study demonstrates that this drug is indeed useful for a long-standing obese i n s u l i n - resistant diabetes but not for a nonobese type diabetes. yuko murase, md takanobu wakasugi, md kunimasa yagi, md hiroshi mabuchi, md f rom the department of internal medicine (y. m . , t. w.), fukui perfectural hospital; and the second d e p a rtment of internal medicine (k.y., h.m.), kanazawa university, ishikawa, japan. a d d ress correspondence to yuko murase, md, the second department of internal medicine, kanazawa university, - takara-machi, kanazawa, ishikawa - , japan. e-mail: diabe@med. k a n a z a w a - u . a c . j p . diabetes care, volume , number , january letters r e f e re n c e s . suter sl, nolan jj, wallace p, gumbiner b, olefsky jm: metabolic effects of new oral hypoglycemic agent cs- in niddm subjects. diabetes care : – , . o’rourke cm, davis ja, saltiel ar, corn i- celli ja: metabolic effects of troglitazone in the goto-kakizaki rat, a non-obese and n o rmolipidemic rodent model of nonin- sulin-dependent diabetes mellitus. m e t a b - o l i s m : – , . troglitazone study group: the metabolic e ffects of troglitazone in non-insulin dependent diabetes (abstract). d i a b e t e s (suppl. ): a, . mori k: the effect of troglitazone in combi- nation with sulfonylurea in non-insulin dependent diabetes mellitus (abstract). j japan diabetes soc (suppl. ): , . h o rton es, venable tc, whitehouse f, the troglitazone study group, ghazzi mn, whitcomb rw: troglitazone in combina- tion with sulfonylurea re s t o res glycemic c o n t rol in patients with type diabetes. diabetes care : – , figure —change from baseline in hba c. values are means ± sem. rad capture (rapture): flexible and efficient sequence-based genotyping highlighted article | investigation rad capture (rapture): flexible and efficient sequence-based genotyping omar a. ali,* sean m. o’rourke,* stephen j. amish,† mariah h. meek,*,‡ gordon luikart,†,§ carson jeffres,** and michael r. miller*,**, *department of animal science, university of california, davis, california ; †division of biological sciences, university of montana, missoula, montana ; ‡department of natural resources, cornell university, ithaca, new york ; §flathead lake biological station, university of montana, polson, montana ; and **center for watershed science, university of california, davis, california abstract massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of dna in many individuals is cost-prohibitive and unnecessary for many studies. genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. here we develop a much improved restriction site-associated dna (rad) sequencing protocol and a new method called rapture (rad capture). the new rad protocol improves versatility by separating rad tag isolation and sequencing library preparation into two distinct steps. this protocol also recovers more unique (nonclonal) rad fragments, which improves both standard rad and rapture analysis. rapture then uses an in-solution capture of chosen rad tags to target sequencing reads to desired loci. rapture combines the benefits of both rad and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. our results demonstrate that rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. the methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science. keywords massively parallel sequencing; restriction-site associated dna (rad); sequence capture; genotyping; population genetics; rainbow trout massively parallel sequencing (mps) technologieshave revolutionized many aspects of agricultural, envi- ronmental, and biomedical science (shendure and ji ; poland and rife ; shokralla et al. ; koboldt et al. ). in population biology, mps enables de novo genome assembly for virtually any species (haussler et al. ; alkan et al. ) and subsequent characterization of within-species genetic variation through whole-genome resequencing (wheeler et al. ; consortium ). although mpsis widely used for whole-genome sequencing and resequencing, us- ing mps to discover and type genetic variation across entire genomes remains prohibitively expensive for many studies (luikart et al. ; sboner et al. ; shendure and aiden ). because sequencing large amounts of dna in many individ- ualscanbecost-prohibitive,researchersofteninterrogateasubset of the genome to reduce the cost per individual (baird et al. ). many genetic studies, such as those characterizing pop- ulation demography, performing genetic assignment, or describ- ing phylogenetic relationships often require information from a relatively small number of loci (from tens to hundreds). other studies—such as those using association mapping to identify loci that influence phenotypic variation or genome scans to describe differential adaptation between populations—typically require information from many more loci (from thousands to millions) (davey et al. ; narum et al. ). both the number of loci and the number of individuals analyzed contribute to the total cost of genetic analysis. the optimal genetic analysis strategy will vary dramatically by study. therefore, methods that facilitate copyright © by the genetics society of america doi: . /genetics. . manuscript received october , ; accepted for publication december , ; published early online december , . supporting information is available online at www.genetics.org/lookup/suppl/ doi: . /genetics. . /-/dc . corresponding author: department of animal science, university of california, shields ave., davis, ca . e-mail: micmiller@ucdavis.edu genetics, vol. , – february http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc mailto:micmiller@ucdavis.edu flexibility in the number of loci and individuals analyzed are needed for maximizing the efficiency of genetic analysis. sequence capture is one method to reduce genome com- plexity and thereby allow an increased number of individuals to be analyzed with mps. genome sequence information is used to design oligonucleotides that facilitate the isolation of desired genomic regions prior to sequencing (hodges et al. ; gnirke et al. ). capturing only genomic regions of interest prior to mps is more economical than sequencing the entire genome for many studies. in-solution capture has facili- tated extensive sequencing of target loci across an individual’s genome (gnirke et al. ). in addition, capture baits de- signed for one species can often be used in related species due to the conserved nature of functional sequence or a close phylogenetic relationship (cosart et al. ). although capture can generate high sequence depth at targeted loci, the method has drawbacks including a relatively high library preparation cost prior to capture and low multiplexing capacity during capture. restriction enzyme-based methods that limit sequencing to a subset of the genome offer an alternative approach to complexity reduction. examples of restriction site-based ge- nomiccomplexityreductionincluderestrictionsite-associated dna (rad) (miller et al. ; baird et al. ), reduced representation library sequencing (van tassell et al. ), and genotyping by sequencing (elshire et al. ). different individual restriction enzymes or enzyme combinations can be used to tailor the resolution of complexity reduction. when combined with barcoded adapters, these methods allow large numbers of individuals to be sequenced simultaneously in a single reaction (baird et al. ; hohenlohe et al. ; etter etal. ).furthermore,the per-individual costoflibrary prep- aration can be very low when samples are barcoded and multi- plexed early in library construction. reduced representation sequencing strategies are now being used extensively in conser- vation, ecological, evolutionary, and agricultural genetic studies (poland and rife ; davey et al. ; narum et al. ). however, these methods are much less flexible than sequence capture with respect to controlling the number and location of genomic loci represented after complexity reduction. current sequence-based genotyping technologies span a genomic resolution continuum from sequence capture and reduced representation methods to complete genome rese- quencing. each technique offers distinct benefits and limi- tations. whole-genome resequencing provides complete resolution but is cost-prohibitive and unnecessary for many studies involving a large number of individuals. restriction site-based methods offer rapid and inexpensive library prep- aration for large numbers of individuals but poor flexibility in the number and location of genomic loci analyzed. sequence capture provides great flexibility with respect to the number and location of genomic loci analyzed but is expensive when applied to large numbers of individuals. new methods that facilitate genotyping of hundreds to thousands of loci in a very large number of individuals would enable many studies that are not currently feasible. thus, we sought to develop a rapid, flexible, and cost-effective technology that is capable of ana- lyzing a very large number of individuals at hundreds to thousands of loci. here we develop a much improved rad sequencing pro- tocol and a new method called rapture (rad capture). the new rad protocol improves versatility by separating rad tag isolation and sequencing library preparation into two distinct steps. this protocol also recovers more unique (nonclonal) rad fragments, which improves both standard rad and rap- ture analysis. rapture then uses an in-solution capture of chosen rad tags to target sequencing reads to desired loci. rapture combines the benefits of both rad and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. our results demon- strate that rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. the methods pre- sented here should improve the efficiency of genetic analysis in many areas of biology. results new rad protocol outperforms the traditional protocol our initial goal was to investigate the potential of rapture as a flexible and efficient method for sequence-based genotyping. however, our initial rapture results contained very high pcr duplicate rates (e.g., . %; data not shown). these could be identified because we used paired-end sequencing and one end of each rad fragment is generated by a random shearing event (miller et al. ; andrews et al. ). upon further investigation, we determined that our rad libraries contained high clonality even before the capture step (see below). fur- thermore, although the traditional rad protocol (baird et al. ; miller et al. ) has worked well for us with high- quality dna samples, the protocol has been inconsistent when using low-quality and/or low-concentration dna samples, which are frequently encountered in conservation and ecolog- ical genetic studies. we reasoned that a new protocol that physically isolates rad tags from the rest of the genome prior to sequence library preparation would be more robust and reduce clonality (figure a). the new protocol employs bio- tinylated rad adaptors that purify the rad tags after ligation using streptavidin-coated magnetic beads (miller et al. ). the purified rad tags are then used as input to any commer- cially available library production kit (figure b). to directly compare the new and traditional rad proto- cols, we generated and analyzed data from rainbow trout individuals using both procedures. we normalized the se- quence data so the analysis of each protocol started with an equal number of reads. we separated the sequence reads according to individual barcode, aligned them, and produced summary statistics to evaluate the new protocol. specifically, wequantifiedtheaveragenumberofsequencedfragmentsper individual, average number of mapped fragments per indi- vidual,andaveragelocuscoveragepriortocloneremoval.the o. a. ali et al. new rad protocol produced similar numbers of sequenced fragments per individual (means of . for the new and . for the traditional) and slightly more mapped fragments per individual ( . for the new and . for the traditional) (table ). in addition, both rad procedures produced similar distributions of mapped frag- ments per individual (figure a), but the updated rad protocol yielded slightly more covered loci per number of sequenced fragments (figure b), as well as better mapping quality of fragments compared to the traditional protocol (table ). the dna used in this experiment was extracted from highly variable fin clips. more consistent samples or more effort in dna normalization before library preparation could decrease the variance among individuals. these results suggest that the updated rad protocol offers a modest im- provement over the traditional rad protocol even without clone removal. to determine if the updated rad protocol produces fewer pcr duplicates, we removed clonal sequences prior to deter- mining alignment statistics and locus coverage. strikingly, the traditional rad protocol produced high numbers of clones, which substantially reduced the number of unique mapped fragments per individual (figure c). with clone removal, the average locus coverage in the traditional protocol was reduced to . (a % loss of coverage) whereas in the new protocol coverage was . (a % loss) (table ). finally, the num- ber of fragments required for the traditional protocol to reach similar coverage levels of the updated protocol is substantially higher (figure d). our new rad protocol significantly im- proved the average number of mapped fragments, the cover- age per locus, and the number of loci covered per barcoded fragment. we conclude that the new rad protocol offers sub- stantial improvements over the traditional protocol. rapture produces high coverage from minimal reads per individual to test the rapture method, we designed and synthesized rna baits complementary to specific rainbow trout rad tags distributed across the chromosomes and performed rad capture. we produced rad sequencing libraries for each of three -well plates using the new protocol. each individual within a plate had a unique well barcode, and each plate had a unique plate barcode. this allowed the three rad libraries to be combined into a single library containing a total of individuals. we then performed a single capture reaction with the recommended bait concentration on the combined library (figure c). we sequenced both pre- and postcapture ver- sions of the combined library in a small fraction of an illumina lane ( %), which produced � million sequenced frag- ments for each of the pre- and postcapture libraries. this experimental design provides a direct comparison between rad and rapture and simulates the sequencing of thousands of individuals in an entire single lane. to evaluate the rapture results, we performed alignments, clone removal, and generated a number of summary statistics. as above, we quantified the average sequenced fragments per figure schematic overview of the new rad protocol and rad capture (rapture) method. (a) rad tag isolation. two wells are depicted in each of two different plates. genomic dna is digested with a restriction en- zyme and ligated to biotinylated well barcode adaptors (yellow and blue bars). (b) rad tag isolation and library preparation. dna from each well is pooled platewise, mechanically sheared, and incubated with streptavidin beads. following washing, dna is cleaved from the beads leaving the well barcodes. finally, a library preparation is performed where a unique plate barcode is added (red and purple bars). (c) rapture. multiple plate libraries are pooled, hybridized to biotinylated oligonucleotide baits cor- responding to the targeted rad tag loci, and captured to produce the final library enriched for the loci of interest. extreme multiplexed genotyping individual, the average mapped fragments per individual, and theaveragelocuscoverage.thedistributionofsequencereads perindividualwereconsistentbetweenprecapture(rad)and rapture (figure a). however, the average coverage at the captured loci was remarkably different between the rad and rapture protocols: rad produced a . average coverage whereas rapture produced average coverage (table ). strikingly, the number of sequenced fragments per individual required for or greater coverage of the captured loci is � , for rapture, while precapture (rad) failed to cover the captured loci to any extent (figure b). finally, as expected, the rapture procedure did not significantly enrich for nontargeted rad loci (figure c). we conclude that rap- ture effectively targets specific rad loci for high coverage with minimal sequencing. to simulate performing rapture with an increased number of individuals in a single capture reaction, we performed a second capture with the same libraries as above but used only one-fifth the recommended concentration of capture baits. the rad capture performed with a one-fifth capture bait concen- tration behaved identically to the original rapture data (figure , a–c). the amount of sequencing needed to gain specific coverage levels is shown in figure d. a high percentage of rapture loci are covered at or greater for . % of the individuals, whereas non-rapture loci were covered at or greater in , % of the individuals (figures , e and f). with both rapture trials producing identical results, we conclude that rapture can process a minimum of loci from individuals ( ) per capture reaction. rapture reveals population structure in fall river rainbow trout we next investigated the suitability of rapture sequence data for genetic analysis by discovering and genotyping snps using clone-removed alignments from the rapture one-fifth bait concentration experiment described above. we first deter- mined the distance from the restriction site for each snp discovered in the rapture loci. sequencing was done with -bp reads, but the first bases on the cut-site end of the sequenced fragment were removed because they contained the barcode and partial cut site. additionally, the shearing and size selection protocol used for these experiments produced fragments up to bases in length. therefore, positions beyond bases from the cut site should have lower coverage. as expected, most snps were discovered near the cut site due to higher sequencing depth generated on that end of the dna molecule (figure a). we discovered snps within the first bases following the cut site and snps between bases and (table ). the exact shearing, size selec- tion, and sequencing parameters could be adjusted in future experiments to influence the number of discovered snps. we then plotted the number of successfully genotyped individuals for each snp along the length of the rad frag- ments using different genotype posterior probability cutoffs. we found that snps within the first bases were successfully genotyped at each cutoff level used (figure , b–d). also, the number of snps genotyped within the first bases given a minimal number of reads (� , ) for each individual is extremely high (figure e). snps located after the first bases required more sequencing to approach saturation in the number of individuals with called genotypes (figure f). we conclude that rapture facilitates versatile and high-quality snp discovery and genotyping. to test the utility of rapture-generated genotypes for in- vestigating population structure, we calculated a covariance matrix from well-genotyped individuals and performed a principal component analysis (figure , a and b; see mate- rials and methods). the wild rainbow trout used in the study originated from the fall river watershed in shasta county, california. fin clips were collected from adult fish with un- known birth locations in the fall river system as well as from juvenile fish that were born at known upstream spawning locations (figure c). strikingly, the first principal compo- nent separated two distinct groups corresponding to individ- uals born in bear creek, ca and the spring-fed spawning locations (thousand springs, ca and spring creek, ca) (fig- ure a). furthermore, genetic differentiation is even appar- ent between spring-fed spawning sites as individuals from thousand springs and spring creek separate on the third component (figure b). thus, rapture facilitated the discov- ery of population structure on a fine spatial scale within a closed watershed. we conclude that rapture is a useful tool for characterizing population structure. discussion new rad protocol is superior to the traditional protocol pcrclonesareaseriousprobleminsequence-basedgenotyping because they produce incorrect genotype calls (hohenlohe et al. ; andrews et al. ). clones are easily detected with some rad sequencing protocols due to a random shearing table comparison of rad sequencing results from traditional rad and new rad protocols applied to the same individual dna extractions normalized sequenced fragments no. of individuals average sequenced fragments per individual average mapped fragments (no clone removal) average locus coverage (no clone removal) average mapped fragments (clones removed) average locus coverage (clones removed) traditional rad , , , , , ( . %a) . , ( . %a) . new rad , , , , , ( . %a) . , ( . %a) . a percentage of average sequenced fragments per individual. o. a. ali et al. step that produces a unique breakpoint in the dna fragment. because the rapture protocol used here relied on two pcr amplification steps (one during the rad library construction and another subsequent to the capture), clonality present in the initial rad libraries is exacerbated in the final postcapture li- brary. thus, we sought to minimize the level of clonality as much as possible in the rad libraries. one way to do this is to simply use more genomic dna; however, our samples are often finite and yield low dna concentrations due to small- sized or degraded tissue. therefore, we developed an improved rad sequencing procedure to maximize rad tag diversity. our redesigned rad protocol employs physical enrichment of rad tags rather than pcr-based enrichment. the new rad protocol outperforms the traditional protocol by yielding in- creased numbers of mapped fragments and better coverage per locus and requires fewer sequence data to achieve the same coverage. the physical separation of rad tags from other genomic fragments captures more unique (nonclonal) rad fragments than the older method of pcr enrichment. we have now used the new rad protocol on many diverse samples. the new protocol consistently produces higher concentration li- brariesusingthesameinputdnaandpcrcycles.furthermore, the new protocol is much more robust. for example, with low- concentrationand/orlow-qualitysamples, failed librarieswere fairly common when using the old protocol but are virtually nonexistent with the new protocol. a possible explanation for the relatively poor performance of the traditional protocol is that the pcr template contains a very high percentage of nonamplifiable dna fragments that have divergent “y” adapt- ers on both ends. figure comparison of rad sequencing results from traditional and new rad protocols on individuals. (a) histogram showing the number of individuals per bin of mapped fragments without clone removal. (b) scatter plot showing the relationship between the number of loci covered $ without clone removal and the number of sequenced fragments per individual. (c) histogram showing the number of individuals per bin of mapped fragments with clone removal. (d) scatter plot showing the relationship between the number loci covered $ with clone removal and the number of sequenced fragments per individual. extreme multiplexed genotyping the separation of rad tag isolation and sequencing library preparation into two distinct steps offers a significant benefit in addition to reduced clonality. new advancements in se- quencing library preparation reagents (such as hairpin loop adapters) were incompatible with the traditional rad pro- tocol due to the integrated steps of rad tag isolation and library preparation. however, the updated rad protocol produces barcoded, double-stranded, sheared dna that can be used as input material for any library preparation protocol on any sequencing platform that accepts fragmented dna. this new protocol should also be compatible with pcr-free library preparation kits that would completely remove pcr duplicates. in conclusion, the new physical rad tag isola- tion procedure generates higher quality data, is more cost- effective, and allows more flexibility for library production than the traditional protocol. figure comparisons of rad, rapture, and rapture with one- fifth bait concentration (rapture / ) sequencing results with clone removal on individuals. (a) histogram showing the number of individuals per bin of mapped fragments. (b) scatter plot show- ing the relationship between number of rapture loci covered $ and the number of se- quenced fragments per individual. (c) scatter plot showing the rela- tionship between the number of non-rapture loci covered $ and the number of sequenced fragments per individual. (d) scat- ter plot showing the relationship between number rapture loci covered at select levels and the number of sequenced fragments per individual for rapture / . (e) histogram showing the num- ber of rapture loci covered $ per bin individual for rapture / . (f) histogram showing number of non-rapture loci covered $ per bin individual for rapture / . o. a. ali et al. rapture combines the benefits of rad and sequence capture rad sequencing excels at sample multiplexing during library preparation because each plate of samples is rapidly com- binedintoasingletubeafterbarcodingandprocessedasasingle reaction.furthermore,thelibraryfromeachplatealsoreceivesa unique plate barcode, which allows processing multiple plate libraries in a single capture reaction. thus, the procedure that we present is scalable to many thousands ofsamples. the in-solution capture step in rapture uses a commercially available kit to selectively isolate the desired rad loci for sequencing. here we targeted loci,butanynumberofcapturebaitscouldbeused to target more or fewer rad loci. the mycroarray commercial product that we used offers up to , unique baits per kit. our results demonstrate the potential for rapture to ge- notype thousands of individuals in a single sequencing re- action. we analyzed rapture data obtained from � % of one illumina hiseq lane (� million reads) and still achieved . coverage across rapture loci in individuals. therefore, sequencing loci at � , , and cover- age could be achieved by multiplexing , , , and individuals per lane of sequencing, respectively. recent improvements in sequence outputs with new illumina ma- chines will allow even higher levels of multiplexing. further- more, even coverage as low as can provide sufficient data for many questions when using probabilistic genotyping ap- proaches (nielsen et al. ). experimental design considerations for rapture bait design for rapture can be obtained from a reference genome,priorraddata,orbyradsequencingofasubsample of individuals to be used for rapture. once candidate loci are identified, some number of loci are chosen to design a custom baitlibrarykitusedforsequencecapture.thisnumberisbased on the aims and budget of the study. rad libraries could be generated and baits designed adjacent to -bp (such as sbfi) or -bp (such as psti) restriction sites. either way, rad tags can be chosen to provide a random representation of the genome or designed with specific requirements depending on experimental needs. requirements for rad tags can be based on molecular constraints and/or genetic information from prior analysis such as linkage with respect to other rad tags and linkage with respect to phenotype, polymorphism, paralogy, position in the genome, or genetic maps (e.g., near genes), etc. if these factors are not considered, the quality and quan- tity of rapture sequence data may be diminished and po- tentially insufficient to answer the biological questions of interest. several other factors could produce low-quality data such as the molecular biology of sequence capture (suboptimal bait design), the designing of rapture baits that have paralogous (or highly similar) sequences repre- sented throughout the genome (off-target capture and se- quencing), and the total number of rad loci chosen or individuals sequenced (an inappropriate relationship be- tween the numbers of individuals, the numbers of loci, and amount of sequencing). therefore, rapture loci discovery and bait design is an important first step for a successful experiment. genetic population structure of fall river rainbow trout we demonstrated the successful use of the new rad protocol and rapture by detecting genetic population structure within a small geographic area (the fall river watershed of northern california) using a relatively small number of sequenced fragments per individual. we used rapture to generate se- quence at rad loci, thoroughly interrogating . , bases per individual. by knowing the hatching location of juvenile fish, we could infer origin of adult fish to bear creek or the spring spawning locations. we were very surprised to discover significant population structure in the rainbow trout from such a small watershed. the fall river flows � km from the uppermost spring and consists of two distinct sources of water: many individual spring inputs and a single rain/snow-melt stream (figure c). a dam just upstream of the pit river confluence blocks upstream fish passage, so the fall river trout population is self-sustaining and does not receive migrants from outside locations. bear creek is an ephemeral tributary of the fall river that fluctuates from zero to � cubic meters per sec- ond (cms) during the year, depending on precipitation events and snowpack. the major water source for the fall river is the multiple springs that discharge at a relatively constant rate of � cms. the large contribution of water from springs keeps the fall river within a constant temperature range and flow regime throughout the year, with the exception of high flow events from bear creek. the ephemeral and perennial differences between the spawning locations are likely respon- sible for producing the genetic differentiation between the two major distinct groups discovered here. table comparison of new rad, rapture, and rapture with one-fifth bait concentration (rapture / ) sequencing results normalized sequenced fragments no. of individuals average sequenced fragments per individual average mapped fragments (no clone removal) average mapped fragments (clones removed) average non-rapture locus coverage (clones removed) average rapture locus coverage (clones removed) rad , , , , ( . %a) , ( . %a) . . rapture , , , , ( . %a) , ( . %a) . . rapture / , , , , ( . %a) , ( . %a) . . a percentage of average sequenced fragments per individual. extreme multiplexed genotyping materials and methods genomic dna extractions we developed an economical high-throughput dna extrac- tion method using agencourt ampure xp beads and a liqui- dator manual -well pipettor (rainin). lifton’s buffer ( ml, mm edta, mm tris–hcl, ph . , % sds) was added to each well of a -well plate. fin clips measuring – mm were placed into each well. lifton’s buffer ( ml) containing . m dtt and . mg/ml proteinase k was added to each well. after mixing, the plate was sealed and incubated at � overnight to generate a crude dna lysate. to a new plate containing ml hybridization buffer ( . m nacl, % peg , . m dtt) and ml agencourt figure snp discovery using rapture with one-fifth bait con- centration data. (a) histogram showing the number of snps per bin of position in rapture locus. (b–d) scatter plots showing the relationship between the number of individuals genotyped and snp position using different posterior probability cutoffs. (e) scatter plot showing the relationship between the number of snps genotyped and the number of sequenced fragments per individual for snps in position – . (f) scatter plot showing the relationship between the number of snps genotyped and number of sequenced frag- ments per individual for snps in position – . o. a. ali et al. ampure xp beads (beckman coulter, a ), ml of the crude lysate was added. after thorough mixing, the plate was incubated for min and then placed on a magnet. the su- pernatant was aspirated and discarded. the plate was re- moved from the magnet, and ml freshly prepared % ethanol was used to resuspend the ampure beads. two ad- ditional % ethanol washes were performed. the beads were allowed to air-dry while on the magnet, and then a volume ( – ml) of low te ( mm tris–hcl, ph . , . mm edta) was used to elute the dna from the beads. traditional rad for each sample, genomic dna ( ng) was digested with . units of sbfi-hf [new england biolabs (neb) r l] at � for hr in a -ml reaction volume buffered with nebuffer (neb, b s). after heat inactivation at � for min, ml indexed sbfi/psti p rad adapter ( nm) was added to each sample (see supporting information, file s for sequences). to ligate the adaptors to the cleaved geno- mic dna, ml of ligation mix [ . ml water, . ml nebuffer , . ml ratp ( mm, fermentas r ), and . ml t dna ligase (neb, m m)] was added. ligations were performed at � for hr followed by incubation at � for min to inactivate the ligase. for each of the samples, ml was pooled and precipitated with agencourt ampure xp beads (beckman coulter, a ). the remaining sample was reserved for additional library preparation if desired. the pooled dna was resuspended in ml low te and sheared in a bioruptor ngs sonicator (diagenode). we used nine cycles of sec on/ sec off and evaluated the shearing efficiency with a fragment analyzer (advanced analytical technologies). additional shearing cycles were performed as necessary. the sheared dna was then concentrated to . ml using ampure xp beads. the concentrated dnawas used as template in the nebnext ultra dna library prep kit for illumina (neb e l; version . ) with the following modifications. instead of using the supplied illumina adaptor, we ligated a custom p adaptor onto the fragments. the indexed p was prepared by annealing an nebnextmultiplexoligoforillumina(neb,e l)totheoligo gatcggaagagcacacgtctgaactccagtcaciiiiiiatcaga aca*a (the asterisk represents a phosphorothioated dna base). we omitted the user enzyme step and used a universal p rad primer (aatgatacggcgaccaccgagatctacact ctttccctacacgac*g) and a universal p rad primer (caagcagaagacggcatacg*a) instead of the included nebnext oligos for the final amplification. new rad protocol for each sample, genomic dna ( ng) was digested with . units of sbfi-hf at � for hr in a -ml reaction volume buffered with nebuffer (note: more dna can and should be used when available. we have successfully used ng per sample with this exact protocol.) after heat inactivation at � for min, ml indexed sbfi/psti biotinylated rad adapter ( nm) was added to each sample (see file s for sequences). the new rad adapters feature -bp hamming barcodes (kozarewa and turner ). to ligate the adaptors to the cleaved genomic dna, ml of ligation mix ( . ml water, . ml nebuffer , . ml ratp, . ml t dna ligase) was added. ligations were performed at � for hr followed by incubation at � for min to inactivate the ligase. for each of the samples, ml was pooled and precipitated with ampure xp beads. the remaining sample was reserved for additional library preparation if desired. the pooled dna was resuspended in ml low te and sheared in a bioruptor ngs sonicator. we used nine cycles of sec on/ sec off and evaluated the shearing efficiency with a fragment analyzer. additional shearing cycles were performed as necessary. we used dynabeads m- streptavidin magnetic beads (life technologies, d) to physically isolate the rad- tagged dna fragments. a -ml aliquot of dynabeads was washed twice with ml of binding and wash buffer ( mm tris–hcl, ph . , mm edta, ph . , m nacl). the dynabeads were resuspended in a volume of binding and wash buffer equivalent to the sheared dna volume from above. the bead/dna mixture was incubated at room tem- perature for min with occasional mixing. the beads were washed twice by placing the tube on a magnetic rack, remov- ing the supernatant, and resuspending the beads in ml binding and wash buffer [ mm tris–hcl (ph . ), . mm edta, ph . , m nacl]. two additional washes were performed using � binding and wash buffer. two additional washes were performed using nebuffer . the beads were resuspended in ml nebuffer containing ml sbfi-hf. after incubation at � for hr, the supernatant containing the liberated dna was removed and precipitated with ampure xp beads. the dna was eluted in . ml of low te and used in nebnext ultra dna library prep kit for illumina with no modifications. sequence capture of rad tags for rapture baits were designed based on sequence from a previous exper- iment that identified , high-quality sbfi rad loci in table comparison of genotyping rate and position of snp in rapture locus average snps genotyped average individuals genotyped snp position no. of snps a a a no. of individuals a a a – bp . ( . %) . ( . %) . ( . %) . ( . %) . ( . %) . ( . %) – bp . ( . %) . ( . %) . ( . %) . ( . %) . ( . %) . ( . %) a posterior probability cutoff. extreme multiplexed genotyping http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc /files .xls http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc /files .xls http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc /files .xls rainbow trout (miller et al. ). a list of potential baits were chosen based on optimal gc content and minimal sequence similarity to other loci. from that list, loci were chosen for bait design such that all linkage groups (miller et al. ) had approximately equal coverage (see file s for sequences). we then ordered baits from mycroarray and used the mybaits protocol supplied with the capture probes. the only modification that we made was to use universal primers in the final library amplifica- tion because we combined several libraries, each made with unique barcoded primers. the universal primers had the following sequence: aatgatacggcgaccaccg agatctacactctttccctacacgac*g and caagca gaagacggcatacg*a (the asterisk represents a phosphor- othioated dna base). sequencing, alignments, and coverage analysis libraries were sequenced with paired-end -bp reads on an illumina hiseq . for each analysis, the sequencing li- braries were randomly subsampled to produce an equivalent number of reads for each library. the libraries were demulti- plexed by requiring reads to have a perfect barcode match as wellasaperfectpartialrestrictionsitematchforassignmentto an individual (see file s for barcodes). to demultiplex the traditional rad data, only the first reads were searched for a barcode and partial restriction site. in the new rad protocol, figure principal component analysis of rapture genotyping results from fall river rainbow trout. individuals are labeled based on birth location. individuals with known birth locations were collected as juveniles near spawning grounds. other individuals were collected as adults throughout the system below the spawning grounds. (a) scatter plot showing the first two principal components. (b) scatter plot showing the first and third principal components. (c) fall river map. o. a. ali et al. http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc /files .xls http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc /files .xls the barcode and partial restriction site can be on either read. therefore, both reads were searched during demultiplexing. rare cases in which a barcode and partial restriction site were present on both reads were discarded. reads were aligned to the rainbow trout reference genome assembly (berthelot et al. ) using the aln algorithm implemented in bwa (li and durbin ). of the , previously discovered rad loci (miller et al. ), , were present and represented only once in the reference ge- nome. a total of of the rapture baits were present and represented only once in the reference genome. there- fore, the rad analyses examined , loci and the rapture analyses examined loci. coverage statistics were obtained by analyzing these loci with samtools (li et al. ).the alignments werefiltered for proper pairs with samtools view, and pcr duplicates were removed with samtools rmdup, except in the first analysis comparing the new and traditional rad protocols. samtools flagstat was used to determine the number of fragments, number of align- ments, and number of unique alignments (clones removed). samtools depth was used to determine coverage per locus. snp discovery and population genetic analysis filtered bam files generated from the above analysis were used in angsd (korneliussen et al. ) for snp discovery, genotype posterior calculation, and population genetic anal- ysis. snp discovery was conducted on sites with a minimum base quality of and a minimum mapping quality of . sites were characterized by estimating their minor allele fre- quency using a uniform prior and the samtools genotype likelihood model (li ). sites were designated as poly- morphic if the snp p-value was # e- . individuals were gen- otyped at each site using posterior probability cutoffs of . , . , and . . we then parsed the output files to determine the position of each snp relative to the rad restriction site and the numbers of individuals genotyped for each snp. toperformtheprincipalcomponentanalysis,wesubsampled the bam files of each individual to the same number of mapped fragments ( , ), which left of the original individ- uals.anangsdgenotypeposterioroutputwasgeneratedwitha uniform prior and filtered by mapping quality ( ), base quality ( ), snp p-value ( e- ), minimum minor allele frequency ( . ), and minimum individuals ( ). this called genotype output was used to calculate a covariance matrix with ngstools ngscovar (fumagalli et al. ). principal component axes summarizing population genetic structure were derived from this covariance matrix by eigenvalue decomposition. data availability the raw sequence data from this study are available at the ncbi sequence read archive with identifier: srp . acknowledgments we thank the fall river conservancy; andrew braugh; california trout; s. d. bechtel, jr. foundation; springs ranch; steve mccanne; california department of fish and wildlife-heritage and wild trout program; members of the genetic diversity research group and the university of california, davis watershed science center, in particular, eric holmes, daniel prince, and ismail saglam for help with sample collection and data analysis; and iwanka kozarewa for hamming barcode sequences. g.l. and s.j.a. were supported by grants from the national science foundation (deb- ) and montana fish wildlife and parks. this work used the vincent j. coates genomics sequencing labora- tory at the university of california at berkeley, supported by nih s instrumentation grants s rr and s rr . literature cited alkan, c., s. sajjadian, and e. e. eichler, limitations of next- generation genome sequence assembly. nat. methods : – . andrews, k. r., p. a. hohenlohe, m. r. miller, b. k. hand, j. e. seeb et al., trade‐offs and utility of alternative radseq meth- ods: reply to puritz et al. mol. ecol. 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representation libraries. nat. methods : – . wheeler, d. a., m. srinivasan, m. egholm, y. shen, l. chen et al., the complete genome of an individual by massively par- allel dna sequencing. nature : – . communicating editor: j. shendure o. a. ali et al. genetics supporting information www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc rad capture (rapture): flexible and efficient sequence-based genotyping omar a. ali, sean m. o’rourke, stephen j. amish, mariah h. meek, gordon luikart, carson jeffres, and michael r. miller copyright © by the genetics society of america doi: . /genetics. . file s : rad protocol adapter sequences. (.xls, kb) available for download as a .xls file at: http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc /files .xls file s : bait sequences. (.xls, kb) available for download as a .xls file at http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc /files .xls file s : rainbow trout sample information. (.xls, kb) available for download as a .xls file at: http://www.genetics.org/lookup/suppl/doi: . /genetics. . /-/dc /files .xls 	combine.pdf 	files .pdf 	files .pdf 	files .pdf insulin resistance exacerbates genetic predisposition to nonalcoholic fatty liver disease in individuals without diabetes this is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the version of record. please cite this article as doi: . /hep . this article is protected by copyright. all rights reserved dr. lisa vanwagner (orcid id : - - - ) article type : original [hep - - ] insulin resistance exacerbates genetic predisposition to nafld in individuals without diabetes llilda barata (barata@wustl.edu), mary f. feitosa (mfeitosa@wustl.edu), lawrence f. bielak (lfbielak@umich.edu), brian halligan (halligan@med.umich.edu), abigail s. baldridge (abigail.baldridge@northwestern.edu), xiuqing guo (xguo@labiomed.org), laura m. yerges- armstrong (laura.m.yerges-armstrong@gsk.com), albert v. smith (albertvs@umich.edu), jie yao (jyao@labiomed.org), nicholette d. palmer (nallred@wakehealth.edu), lisa b. vanwagner , (lvw@northwestern.edu), j. jeffrey carr (j.jeffrey.carr@vumc.org), yii-der i. chen (ichen@labiomed.org), matthew allison (mallison@ucsd.edu), matthew j. budoff (mbudoff@labiomed.org), samuel k. handelman (samuelkh@med.umich.edu), sharon l.r. kardia (skardia@umich.edu), thomas h. mosley jr. (tmosley@umc.edu), kathleen ryan (kryan@som.umaryland.edu), tamara b. harris (harris @nia.nih.gov), lenore j. launer (launerl@nia.nih.gov), vilmundur gudnason , (v.gudnason@hjarta.is), jerome i. rotter (jrotter@labiomed.org), myriam fornage (myriam.fornage@uth.tmc.edu), laura j. rasmussen-torvik (ljrtorvik@northwestern.edu), ingrid borecki (iborecki @gmail.com), jeffrey r. o’connell (joconnel@som.umaryland.edu), patricia a. peyser (ppeyser@umich.edu), elizabeth k. speliotes* (espeliot@med.umich.edu), michael a. province* (mprovince@wustl.edu) a u th o r m a n u s c ri p t https://doi.org/ . /hep . https://doi.org/ . /hep . https://doi.org/ . /hep . mailto:halligan@med.umich.edu mailto:abigail.baldridge@northwestern.edu mailto:samuelkh@med.umich.edu this article is protected by copyright. all rights reserved division of statistical genomics, department of genetics, washington university school of medicine; st. louis, mo, usa department of epidemiology, school of public health, university of michigan, ann arbor, mi, usa division of gastroenterology, department of internal medicine, department of computational medicine and bioinformatics, university of michigan, ann arbor, michigan usa department of preventive medicine, northwestern university feinberg school of medicine, chicago, il usa the institute for translational genomics and population sciences, labiomed and the department of pediatrics, harbor-ucla medical center, torrance, ca, usa department of medicine, university of maryland school of medicine, baltimore, maryland usa department of biostatistics, school of public health, university of michigan, ann arbor, mi,usa department of biochemistry, wake forest school of medicine, winston-salem, nc, usa division of gastroenterology and hepatology, northwestern university feinberg school of medicine, chicago, il usa department of radiology, vanderbilt university school of medicine, nashville, tn, usa department of family medicine and public health, university of california, san diego, ca,usa division of cardiology, los angeles biomedical research institute, torrance, california. department of medicine, division of geriatrics, university of mississippi medical center, jackson, ms, usa laboratory of epidemiology and population sciences, national institute of aging, bethesda, md, usa icelandic heart association, kopavogur, iceland faculty of medicine, university of iceland, reykjavik, iceland university of texas health science center, houston, texas, usa * michael province and elizabeth k. speliotes equally supervised this work. keywords: gene-environment interactions, pnpla , hepatic steatosis, metabolic factors, gckr a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved corresponding authors: llilda barata phd, mph department of genetics division of statistical genomics washington university school of medicine south euclid ave st. louis, mo, usa barata@wustl.edu ( ) - elizabeth k. speliotes, md phd mph division of gastroenterology department of internal medicine university of michigan west medical center drive ann arbor, mi, usa espeliot@med.umich.edu ( ) - michael a. province, phd department of genetics division of statistical genomics washington university school of medicine south euclid ave st. louis, mo, usa mprovince@wustl.edu ( ) - abbreviations: nafld= nonalcoholic fatty liver disease; tg=triglycerides; ldl=low-density a u th o r m a n u s c ri p t mailto:barata@wustl.edu mailto:espeliot@med.umich.edu mailto:mprovince@wustl.edu this article is protected by copyright. all rights reserved lipoprotein cholesterol; hdl=high-density lipoprotein cholesterol; bmi=body mass index; whradjbmi= waist-to-hip ratio adjusted for body mass index; pnpla =patatin-like phospholipase domain-containing protein gene; gckr =glucokinase regulatory protein gene; ncan =neurocan gene; tm sf =transmembrane superfamily member gene; lyplal =lysophospholipase-like gene; ea=european ancestry; snp=single nucleotide polymorphism; aa=african ancestry; la=liver attenuation; homa-ir=homeostatic model of insulin resistance; ages=age, gene/environment susceptibility-reykjavik; amish=old order amish; cardia=coronary artery risk development in young adults; famhs=family heart study; fhs=framingham heart study; genoa=genetic epidemiology network of arteriopathy; mesa=multi-ethnic study of atherosclerosis; hu=hounsfield units; ivn=inverse normal transformation; laivn=inverse normal-transformed residuals of la; sd=standard deviation financial support: this work was performed under the auspices of the genetics of obesity-related liver disease consortium. funding for this study was made possible by the age, gene/environment susceptibility-reykjavik study (ages) funded by the national institute of health (nih) contracts n -ag- - and c, the national institute of aging intramural research program, hjartavernd (the icelandic heart association), and the althingi (the icelandic parliament). the study is approved by the icelandic national bioethics committee, vsn: - . the researchers are indebted to the participants for their willingness to participate in the study. the amish study gratefully acknowledges our amish liaisons, research volunteers, field workers and amish research clinic staff and the extraordinary cooperation and support of the a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved amish community without which these studies would not have been possible. the amish studies are supported by grants and contracts from nih, including u hl , u hl , u hl and p dk . funding for the coronary artery risk development in young adults study (cardia) is conducted and supported by the national heart, lung, and blood institute (nhlbi) in collaboration with the university of alabama at birmingham (hhsn c & hhsn c), northwestern university (hhsn c), university of minnesota (hhsn c), kaiser foundation research institute (hhsn c), johns hopkins university school of medicine (hhsn c) and vanderbilt university medical center (r hl ). cardia is also partially supported by the intramural research program of nia and an intra-agency agreement between nia and nhlbi (ag ). the national human genome research institute (nhgri) supported genotyping of cardia participants through grants u -hg- , u -hg- , and u -hg- . this manuscript has been reviewed by cardia for scientific content. exome chip genotyping and data analyses were funded in part by grants u -hg , r -hl and r -hl from nih to dr. myriam fornage. lisa b. vanwagner is supported by nih grant k hl . the family heart study (famhs) was supported by grant r -dk- from the national institute of diabetes and digestive and kidney diseases (niddk) and grant r hl from nhlbi. elizabeth k. speliotes and brian halligan are supported by nih grants r dk , r dk and the university of michigan department of internal medicine. data from the framingham heart study (fhs) came from the database of genotypes and phenotypes (dbgap). lawrence f. bielak and patricia a. peyser are supported, in part, by nih grants r dk and r dk . support for the genetic epidemiology network of arteriopathy (genoa) study was provided by nhlbi (hl , hl , hl , hl , and hl ) of the national institutes of health. the multi-ethnic study of atherosclerosis (mesa) and the mesa share project are conducted and supported by nhlbi in collaboration with mesa investigators. this research was supported by r hl , and mesa was supported by contracts n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , n -hc- from the national heart, lung, and blood institute, and by grants ul -tr- , ul - tr- , and ul -rr- from national center for research resources. the provision of exome chip genotyping data was supported in part by the nhlbi contract n -hl- , national center for advancing translational sciences, ctsi grant ul tr , and the national institute of diabetes and digestive and kidney disease diabetes research center a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved (drc) grant dk to the southern california diabetes endocrinology research center. funding support for mesa’s nafld dataset was provided by grant hl - a . the views expressed in this manuscript are those of the authors and do not necessarily represent the views of nhlbi, nia, niddk or nih. abstract the accumulation of excess fat in the liver (hepatic steatosis), in the absence of heavy alcohol consumption, causes nonalcoholic fatty liver disease (nafld), which has become a global epidemic. identifying metabolic risk factors that interact with the genetic risk of nafld is important for reducing disease burden. we tested whether serum glucose, insulin, insulin resistance, triglycerides, low density lipoprotein cholesterol, high density lipoprotein cholesterol, body mass index (bmi), and waist-to-hip ratio adjusted for bmi interact with genetic variants in or near the patatin-like phospholipase domain containing gene (pnpla ), the glucokinase regulatory protein gene (gckr), the neurocan gene (ncan/tm sf ), and the lysophospholipase-like gene (lyplal ) to exacerbate hepatic steatosis, estimated by liver attenuation (la). we performed association analyses in ten population-based cohorts separately and then meta-analyzed results in up to , individuals ( , of european a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved ancestry and , of african ancestry). we found that pnpla -rs significantly interacted with insulin, insulin resistance, bmi, glucose, and tg to increase hepatic steatosis in nondiabetic individuals carrying the g-allele. additionally, gckr-rs significantly interacted with insulin, insulin resistance and tg. conditional analyses, using the two largest european ancestry cohorts in the study, showed that insulin levels accounted for most of the interaction of pnpla -rs with bmi, glucose, and tg in nondiabetic individuals. insulin, pnpla-rs , and their interaction accounted for at least % of the variance in hepatic steatosis in these two cohorts. conclusion: our results suggest that insulin resistance, either directly or via the resultant elevated insulin levels, more than other metabolic traits, amplifies the pnpla rs -g genetic risk for hepatic steatosis. these results suggest that improving insulin resistance in nondiabetic individuals carrying pnpla -rs -g may preferentially decrease hepatic steatosis. nonalcoholic fatty liver disease (nafld) is a result of the excess accumulation of lipids in hepatocytes (hepatic steatosis) in the absence of heavy alcohol consumption( ). hepatic steatosis is also associated with the risk of developing dyslipidemia or dysglycemia( ), as well as cardiovascular disease, which is the number one cause of death in individuals with nafld( , ). hepatic steatosis may progress to advanced liver disease in the form of nonalcoholic steatohepatitis, fibrosis (cirrhosis), and cancer (hepatocellular carcinoma)( - ). in the u.s., the prevalence of hepatic steatosis in the adult population is between % to %; worldwide it is % to %( ). while the pathogenesis of nafld is not entirely understood, both genetic factors and metabolic traits increase the risk of hepatic steatosis. heritability of hepatic steatosis ranges from to % across all ancestries suggesting that specific genotypes may predispose individuals to nafld( ). previously, the genetics of obesity-related liver disease consortium conducted a genome-wide association study in , individuals of european ancestry (ea) with replication in histology-based samples ( ). this study identified that rs (pnpla ), a missense single nucleotide polymorphism (snp) first associated with hepatic fat content a decade ago ( ), the missense variant rs (ncan/tm sf ) and intronic variants rs (lyplal ) and rs (gckr) were significantly associated with hepatic steatosis( ). we and others have replicated the association of these common variants with hepatic steatosis in other populations and ethnicities ( - ), and the associations are consistent between those of ea and african a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved ancestry (aa) (direction of effect is similar)( ). further, the g allele for rs was associated with susceptibility to nonalcoholic steatohepatitis (or . , % ci: . - . , p≤ . e- ), nonalcoholic steatohepatitis severity (or . , % ci: . - . , p≤ . e- ) and fibrosis (or . , % ci: . - . , p≤ . e- ) in ea individuals( ). traits that predispose to metabolic syndrome, i.e. higher body mass index (bmi) ( ), dyslipidemia, hyperglycemia, and insulin resistance are associated with hepatic steatosis ( , , ). eighty to ninety percent of obese (bmi ≥ kg/m ) adults have hepatic steatosis( ), while - % of individuals with hepatic steatosis also have higher levels of triglyceride (tg) and low- density lipoprotein cholesterol (ldl), but lower levels of high-density lipoprotein cholesterol (hdl)( ). diabetes is also commonly associated with hepatic steatosis( ). how these modifiable metabolic traits interact with genetic variation to influence risk for hepatic steatosis is not known. in this cross-sectional study, we tested whether several metabolic traits interact with the four genetic variants previously associated with hepatic steatosis( ) to affect liver attenuation (la), a computed tomographic quantitative measure that is inversely related to histologically measured liver fat ( ). the metabolic traits tested were: insulin resistance (as homeostatic model of insulin resistance (homa-ir)), fasting insulin, fasting glucose, bmi, centralized fat deposition measured by waist-to-hip ratio adjusted for bmi (whradjbmi), fasting tg, fasting hdl and fasting ldl. we first carried out interaction analyses between each of these traits and each of the genetic variants in ten separate population-based cohorts from seven different studies. then we meta-analyzed results across cohorts in up to , individuals (ea, n= , and aa, n= , ). we then carried out conditional analyses in the two largest ea cohorts in the study to determine the driving metabolic factor. population and methods ethics statement the institutional review boards or equivalent committees of all participating studies approved this study. the principal investigator of each institution obtained written consent from participants. study description a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved the study was comprised of up to , individuals (ea, n= , and aa, n= , ); % of participants were female. the sample derived from seven population-based studies participating in the genetics of obesity-related liver disease consortium: age, gene/environment susceptibility-reykjavik (ages), old order amish (amish), coronary artery risk development in young adults (cardia), family heart study (famhs), framingham heart study (fhs), genetic epidemiology network of arteriopathy (genoa), and multi-ethnic study of atherosclerosis (mesa). in total, ten cohorts were included in the analysis, as three studies contributed two ethnic groups (aa, ea). each ethnic group was analyzed separately. cardia, mesa, and ages have unrelated individuals while fhs, amish, genoa, and famhs are family-based. detailed information about the characteristics and design of each study is provided in supplementary table . outcome variable and metabolic traits the outcome variable was la (liver attenuation), measured non-invasively with computed tomography in hounsfield units (hu) ( ). la is inversely proportional to liver fat, i.e. lower la values indicate a higher fat content in the liver (more hepatic steatosis)( ). the procedures followed by each cohort to measure la are described in supplementary table . individuals with active malignancies, focal lesions, or other incidental findings on computed tomography were excluded from the studies. metabolic traits of interest were harmonized across cohorts following standard clinical definitions. overall adiposity was characterized by bmi (kg/m ), and abdominal adiposity by waist-to-hip ratio adjusted for bmi (whradjbmi, cm). since waist-to-hip ratio is correlated with both bmi and visceral fat, we chose to use whradjbmi to have a measure that is independent of overall fatness (i.e. bmi), but does reflect visceral adiposity, and is easily measured in the clinic. fasting insulin (mu/l) and fasting glucose (mmol/l) were measured from plasma or serum using standard laboratory techniques detailed in supplementary table . when fasting glucose was measured from whole blood, it was converted to plasma glucose using a correction factor of . ( ). homa-ir was assessed using fasting glucose (mmol/l) x fasting insulin (mu/l) divided by . ( ). each cohort assayed fasting tg (mg/dl) and fasting hdl (mg/dl) using methods described in supplementary table . if fasting ldl (mg/dl) was assayed, it was used. otherwise, ldl was calculated using the friedewald formula, ldlf =(total cholesterol(mg/dl) - hdl(mg/dl) -tg(mg/dl)/ . ), only if tg < mg/dl ( ). a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved alcohol consumption, history of diabetes, and use of lipid lowering medications were acquired by questionnaire. total alcohol consumption, defined in drinks per week, was calculated from daily intake of beer, wine, and spirits. one drink was defined as a serving of grams of ethanol, the same as a oz. bottle or can of beer, oz. glass of wine, or . oz. shot of - proof spirits such as gin, vodka, or whiskey( ). heavy drinking was defined as ≥ drinks per week for women and ≥ drinks per week for men ( ). diabetes (type , type ) was defined as having fasting plasma glucose levels ≥ mmol/l ( mg/dl), or self-reporting the use of insulin or oral antidiabetic medications, or having a physician diagnosis of diabetes. the use of statins was assessed from medication questionnaires. genotyping and imputation four common variants were included in the analyses: rs - a missense variant in the patatin-like phospholipase domain containing gene (pnpla ); rs , an intronic variant within the glucokinase regulatory protein gene (gckr) that is in high linkage disequilibrium (r = . ) with rs , a likely functional missense variant in this gene; rs , a missense variant in the neurocan gene (ncan) that is in high linkage disequilibrium (r = . ) with rs , a likely functional missense variant in the transmembrane superfamily member gene (tm sf ); and rs , an intronic variant in the lysophospholipase-like gene (lyplal ). these variants were either directly genotyped (allele counts were coded , , or ), or dosages were imputed from hapmap ii or g. genotype calling algorithms and imputation methods are detailed in supplementary table . statistical analysis cohort-specific analyses cohorts performed analyses separately in each ancestry group (ea, aa). la and metabolic traits, used as continuous variables in all analyses, were adjusted for sex, age, principal component estimates of ancestry, and study-specific covariates using linear regression as detailed in supplementary table . la was also adjusted for alcohol consumption, a continuous variable (drinks/week), and for scan penetrance using phantom or spleen density. residuals from adjusted la and metabolic traits were transformed using inverse normal a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved transformation (ivn) to reduce the influence of outliers and to standardize the phenotypes across cohorts. inverse normal-transformed residuals of la, (laivn), and each metabolic trait (mtivn) were used to fit the interaction models. each cohort tested for statistical interactions between each variant and each metabolic trait using multivariable linear regression or mixed linear modeling. laivn was the dependent variable. the independent variables were each snp and mtivn, plus the interaction: laivn = α + β (snp) + β (mtivn) + β (snp x mtivn) + є. an additive model of inheritance was assumed. studies with family data (fhs, genoa, amish, and famhs) used linear mixed models to account for family relatedness among participants and computed robust standard errors. participants with diabetes (type and type ) were excluded from the insulin, glucose and homa-ir models, and those taking statins were excluded from the ldl model. as a secondary analysis, bmi was included as a covariate in the models to investigate whether the effect of the interaction between each snp and each metabolic trait on laivn occurred independent of overall adiposity. associations were carried out using mmap( ), r( ), and sas ( ) software. meta-analyses we conducted fixed-effects meta-analyses by ancestry and overall on the parameter estimates (β-coefficients and standard errors) for the main effects and interaction effects. we utilized the inverse variance weighting method implemented in metal ( ). using cochran’s q test ( ), we tested for heterogeneity of effects across all analyses. within ancestries, focusing on interactions, we found evidence of heterogeneity only for the interaction between tg and gckr in the ea cohorts. we did not find any heterogeneity for the interaction in the meta-analyses between the two ancestry groups (ea vs aa); thus, we report the combined ancestry meta- analyses. to determine the level of statistical significance while accounting for multiple testing, we applied a bonferroni correction that consisted of grouping correlated traits into three metabolic domains: insulin-glucose, adiposity, and lipids. the critical p-value α= . was divided by ( variants x metabolic domains) to obtain a corrected p-value. meta-analyses results and heterogeneity tests were considered significant if the two-tailed p-value was ≤ . e- . as a secondary analysis, to investigate whether the statistically significant interactions were consistent between genders, we fit the interaction models in men and women separately, and meta-analyzed results within gender. a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved conditional analyses in famhs and fhs to determine whether the interaction of bmi, glucose or tg with pnpla -rs was independent of insulin, we analyzed each trait’s interaction effect before and after including insulin in the model. the analyses were performed with ea individuals in famhs and replicated in fhs. we chose these two cohorts because they are the two largest cohorts in the study; together they represent more than / of our total sample. individuals with diabetes and/or missing information for the metabolic traits of interest were excluded resulting in a sample of , individuals in famhs and , in fhs. after adjusting la for phantom in both cohorts, and for field centers in famhs, la residuals were transformed using inverse normal transformation to approximate normality. la transformed residuals (laivn) were used as the dependent variable. using linear mixed models, we first regressed laivn on either bmi, glucose, or tg, and their interaction with pnpla -rs (supplementary text). we then added insulin to the models and its interaction with pnpla -rs and the metabolic trait (either bmi, glucose, or tg). insulin and tg were log-transformed due to the presence of influential outliers. models were adjusted for age, sex, and alcohol consumption (drinks/week), and for genotype batch effects in famhs. results from conditional analyses in each cohort were then meta-analyzed. the conditional models included principal components to adjust for population stratification. because the principal components were not associated with laivn in either cohort, and their inclusion in the conditional models did not change the inferences, we present the models without them. we also performed conditional analyses after excluding individuals from famhs (n= ), and fhs (n= ) who reported heavy alcohol use (≥ drinks per week for women, and ≥ drinks per week for men (supplementary tables - ) ( ). since the inferences were unchanged, to increase power, we included all individuals, and adjusted for alcohol as a covariate. additionally, we conducted the conditional analyses with log-transformed homa-ir instead of log-transformed insulin (supplementary tables - ). insulin and homa-ir provided similar inferences. because glucose explains significantly less of the variation in lainv, we focused on insulin over homa-ir since there was no added benefit of measuring glucose on variance explained by homa-ir than with just measuring insulin. illustration in famhs of the interaction between insulin and pnpla -rs in individuals without diabetes a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved to assess the interaction effect of insulin with pnpla -rs on hepatic steatosis prevalence in famhs, we plotted the percentage of individuals with la ≤ hu per pnpla - rs genotype by the lowest and highest quartile of insulin. individuals with diabetes and/or missing information for insulin were excluded and ancestries were combined to obtain a sample of n= , . la and insulin were not adjusted or transformed. the la cut point of ≤ hu, which corresponds to a liver/spleen ratio of . , has previously been shown to identify individuals with moderate to severe macrovesicular steatosis (≥ % of the liver parenchyma with fat) at histology with a high diagnostic accuracy ( ). in the literature, ≥ % liver fat suggests moderate to severe hepatic steatosis ( ). results demographics and clinical characteristics across the study cohorts are presented in table . the mean age ± standard deviation (sd) across cohorts ranged from . ± . to . ± . years old. all cohorts included more women than men. the mean±sd of la across cohorts ranged from . ± . hu to . ± . hu. mean±sd of fasting insulin levels in non- diabetics ranged from . ± . to . ± . mu/l and fasting blood glucose levels ranged from . ± . to . ± . mmol/l. the lowest mean±sd for homa-ir in non-diabetics was . ± . and the highest was . ± . . the mean±sd of bmi ranged from . ± . to . ± . kg/m . several cohorts reported mean fasting tg > mg/dl. mean±sd for fasting ldl cholesterol in non-statin users was borderline high in amish ( . ± . mg/dl) and ages ( . ± . mg/dl). across cohorts, the range of fasting hdl was within the recommended limit of ≥ mg/dl. heavy drinking varied among studies with genoa having the lowest percentage ( %) and cardia the highest ( %). pnpla -rs and gckr-rs interact with several metabolic traits we found significant interactions for pnpla -rs and gckr-rs with several metabolic traits in combined ancestries after adjusting for multiple comparisons (table , supplementary table ). pnpla -rs interacted with insulin (p= . e- ), homa-ir (p= . e- ), glucose (p= . e- ), bmi (p= . e- ) and tg (p= . e- ). as each of these metabolic traits increased, a decrease in laivn (i.e. higher fat content in the liver) became more pronounced in presence of the g allele at pnpla -rs as compared to the presence of the c allele. additionally, gckr-rs interacted with insulin (p= . e- ), homa-ir (p= . e- ), and tg (p= . e- ). as levels of insulin, homa-ir, and tg increased, a decrease in laivn (i.e. higher fat content in the liver) became more pronounced in a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved the presence of the t allele at gckr-rs , compared to the c allele. all interactions remained significant after adjusting for bmi (supplementary table ) suggesting that overall adiposity did not alter these effects. we did not find evidence of significant interactions between any of the four genetic variants and whradjbmi, ldl, or hdl. although the interaction between whradjbmi and pnpla did not reach the bonferroni significance level, it was borderline significant. this suggests that a larger sample size may be needed to detect an interaction. alternatively, the lack of statistical significance could be because whradjbmi does not represent overall fatness to the extent that bmi or other anthropometric measurements do. we also carried out meta-analyses in men and women separately to investigate possible gender differences focusing only on the statistically significant interactions with pnpla -rs and gckr-rs (supplementary table ). women made up % of our study sample. the interaction effects of insulin and homa-ir with pnpla -rs did not differ between men and women, and both reached statistical significance (women= p= . e- , men= . e- ; and women: p= . e- , men: p= . e- , respectively). for glucose, the interaction effect was slightly less in men than in women (beta smaller), and did not reach significance in men. these results suggest that gender did not alter the interactions between pnpla - rs and insulin/homair and the interaction effect of glucose was still present only in women in the present study. further, the interaction effects of bmi with pnpla -rs were similar between men and women, and reached significance in both (p= . e- and p= . e- , respectively). the interaction effect of tg with pnpla - rs did not reach statistical significance in either gender. moreover, the interaction effects of both insulin and homa-ir with gckr-rs reached significance only in women (p= . e- and p= . e- , respectively). similarly, the interaction of tg with gckr-rs was significant only in women (p= . e- ). stratifying by gender substantially reduced our sample size, and as a result power. conditional analyses suggest that insulin may mediate the interaction effect of bmi, tg and glucose on laivn in individuals without diabetes we observed that the interaction of insulin with pnpla -rs had a greater effect on laivn (hepatic steatosis defined by liver attenuation) than that of bmi, tg, or glucose. to determine if the interaction of bmi, tg, or glucose with pnpla -rs was independent of insulin, we carried out conditional analyses in famhs and fhs, and meta-analyzed results. we found that the interaction of bmi (p= . e- ), tg (p= . e- ), or glucose (p= . e- ) with pnpla - a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved rs was no longer statistically significant after including insulin as a main effect and interactor with pnpla -rs and the respective metabolic trait in the models (supplementary tables - ). in contrast, the interaction of insulin with pnpla -rs remained significant after controlling for bmi, tg, or glucose (pinsulin-bmi= . e- ; pinsulin-tg= . e- ; pinsulin-glucose= . e- ), although the effect sizes and p-values were attenuated. these results suggest that insulin may account for most of the interaction effect of bmi, glucose, and tg with pnpla -rs on laivn. previously, we reported that pnpla -rs explained . % of the variance in hepatic steatosis, estimated by la, in ea individuals ( ). in the present study, pnpla -rs , insulin and their interaction together explain as much as % of the variance in hepatic steatosis in the two largest ea cohorts excluding individuals with diagnosed diabetes. this suggests that insulin levels/insulin resistance may be a key contributor to nafld. excluding heavy drinkers from the conditional analyses did not change our inferences regarding pnpla -rs (supplementary table - ). we were not powered to carry out these analyses for gckr-rs . interaction effect of insulin with pnpla on hepatic steatosis prevalence in famhs we also assessed the interaction effect of insulin with pnpla -rs on hepatic steatosis prevalence in individuals without diabetes (figure ). in the lowest quartile of insulin levels (≤ . mu/l), the percentage of individuals with ≥ % liver fat (i.e. moderate to severe hepatic steatosis) was . %, . %, and . % for cc, cg and gg individuals, respectively. in the highest quartile of insulin levels (≥ . mu/l), the percentage of individuals with ≥ % liver fat was . %, . % and . % for cc, cg and gg individuals, respectively. the data show that as insulin levels increase the percentage of individuals with moderate to severe hepatic steatosis increases. however, among those with the gg genotype, this effect is magnified. the difference in the percentage of individuals with moderate to severe hepatic steatosis increases by percentage points between the lowest and highest insulin quartiles among those with gg genotype, and increases by percentage points among heterozygotes, while that difference increases only by percentage points among those with the cc genotype. these data suggest that insulin has a strong effect on exacerbating the accumulation of liver fat in individuals without diabetes who have or g- alleles at pnpla -rs . discussion in a sample of , ea and aa individuals, we found interactions between pnpla -rs and insulin, homa-ir, bmi, glucose, and tg on lainv (hepatic steatosis) after adjusting for a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved differences in age, sex, and alcohol consumption. we also found interactions between gckr- rs and insulin, homa-ir, and tg on lainv. conditional analyses in more than , ea individuals suggest that insulin, more than glucose, bmi, or tg drive the interaction with pnpla -rs to affect lainv in non-diabetics. we did not see significant interactions between pnpla -rs and bmi, tg or glucose once insulin was accounted for, whereas the reverse was not true. that is, there was still evidence for an interaction between pnpla - rs and insulin even after accounting for the other metabolic traits. these results persist after accounting for alcohol intake, gender and overall adiposity. we estimated in famhs and fhs that as much as % of the variance in hepatic steatosis is explained by pnlpa -rs , insulin and their interaction in non-diabetic ea individuals. in our previous study, pnpla - rs alone explained only . % of hepatic steatosis variance in ea individuals ( ). our findings suggest that non-diabetic individuals with pnpla -rs -g and high insulin levels may have a particularly high risk for hepatic steatosis. the pnpla gene encodes adiponutrin, an enzyme found on the membrane of lipid droplets within hepatocytes ( ). its function may be to break down tg stored in the droplets, helping regulate hepatic tg content ( , ). the missense polymorphism rs (c > g) in pnpla substitutes the amino acid isoleucine for methionine at residue (i m), changing the configuration of adiponutrin’s catalytic site, and rendering the enzyme inactive ( , ). the accumulation of the inactive enzyme on lipid droplets is associated with tg buildup in hepatocytes ( ). humans and mice carrying one or two copies of the i m mutation (rs cg or gg genotype) accumulate excess tg in lipid droplets, and show more pronounced hepatic steatosis and nafld than those without the mutation( , ). it is possible that having high insulin levels in addition to the pnpla -rs g allele may result in a strong synergistic effect that exacerbates the accumulation of fat in the liver of non- diabetic individuals, predisposing them to nafld. insulin resistance stimulates the hydrolysis of tg in adipose tissue releasing fatty acids in the bloodstream, which are taken up by the liver in an unregulated manner promoting the accumulation of tg in hepatocytes ( ). higher insulin levels also activate fatty acid synthesis in the liver further driving the formation and storage of tg ( ). in addition, insulin resistance elevates plasma glucose, which is sequestered by the liver, phosphorylated, and metabolized to make glycerol and acetyl-coa, the building blocks for the synthesis of tg ( , ). in this context, it is possible that increased lipid synthesis and fatty acid delivery to the liver may combine with the inability of hepatocytes to dispose of tg from a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved lipid droplets, due to the presence of pnpla -rs -g, and lead to increased hepatic steatosis. high insulin levels and pnpla -rs -g may also be involved in molecular feedback loops that increase hepatic steatosis. insulin resistance and increased insulin levels augment the activity of transcription factors such as srebp- c ( ). these transcription factors may promote tg synthesis in the liver and up-regulate the expression of pnpla i m by binding to its promoter in a positive feedback loop ( ). in this way, insulin and pnpla i m may synergize to promote hepatic steatosis. this conjecture is also consistent with the enhanced risk of steatosis and liver damage as evident by elevated liver enzymes and liver fat content seen with liver directed long-acting insulin analogues in type diabetics carrying the pnpla- variant ( ). when taken together, results show evidence that insulin and pnpla -rs interact to have an important role in hepatic steatosis, and as a result nafld. consequently, lowering the risk of hepatic steatosis and its liver complications in individuals with pnpla -rs -g may be achieved by reducing insulin resistance and concomitant high levels of insulin. one way to accomplish this could be through lifestyle changes that include increased exercise, weight loss, and better nutrition ( ). for example, decreasing exposure to carbohydrate rich diets, which adversely increase insulin levels, may mitigate risk ( , ). also, treatments that target insulin resistance may be of greater benefit for preventing or treating hepatic steatosis than drugs that simply lower glucose. for example, insulin sensitizing medications such as pioglitazone may be an option; it has already been shown to improve nafld, although at the expense of weight gain ( ). more studies are warranted to better understand the effect of the relationship between insulin levels and pnpla -rs -g on hepatic steatosis in different populations. we also observed significant interactions of pnpla -rs with bmi, glucose, and tg. our results support the findings of stender et al. who reported that high bmi augmented the effect of pnpla -rs -g on hepatic steatosis conferring susceptibility to nafld ( ). graff et al. also showed an interaction effect between pnpla -rs and visceral fat content, a measure of metabolic dysfunction ( ). however, we found that the effect of bmi in exacerbating hepatic steatosis in the presence of pnpla -rs -g is attenuated by controlling for insulin levels in the model. we made the same observation for glucose and tg suggesting that insulin/insulin resistance in the presence of pnpla -rs -g may confer most of the risk for hepatic steatosis on its own or through other metabolic intermediates. a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved studies have reported an association between ldl and hepatic steatosis ( , ). however, our study did not find an interaction between any of the genetic variants considered and ldl. this suggests that for individuals carrying pnpla -rs -g, reducing insulin levels or insulin resistance may have a greater effect on reducing the risk of hepatic steatosis than reducing ldl. in addition to pnpla , we found that gckr interacts with insulin resistance to increase susceptibility to hepatic steatosis. gckr encodes the glucokinase regulatory protein, which has an important role in glucose metabolism( ). the glucokinase regulatory protein binds to the glucose metabolizing enzyme, glucokinase, to inhibit its role in the uptake and storage of dietary glucose via stimulating de novo lipogenesis( ). the variant rs /rs in the glucokinase regulatory protein reduces its ability to inhibit glucokinase ( ). this results in an increased activity of glucokinase in the liver, which promotes de novo lipogenesis. when this mutation is combined with insulin resistance, it may amplify de novo lipogenesis to promote hepatic steatosis. we did not replicate the interaction between tm sf and bmi reported by stender et al. ( ); however, our results show a similar trend. the interaction was borderline non-significant in the combined ancestry meta-analyses (bint= - . , p= . e- ). some differences between stender et al. and this study may explain why we did not detect a statistically significant interaction. first, stender et al. used proton magnetic resonance spectrometry to measure steatosis, which is a more sensitive measure than computed tomography. second, they used the genotyped missense variant, rs ; we used the proxy, imputed variant, rs . the two variants are in high linkage disequilibrium (d’= . , r = . ). third, stender et al. combined the heterozygotes (ek), and homozygotes (kk), and compared them to those without the risk allele (ee). these three differences may have increased their power to see the weak effect they reported. our study has several limitations. it is a cross-sectional design that cannot prove temporal causality of insulin exposure on increasing hepatic steatosis. because we used population- based cohorts that lacked biopsy information, we do not know whether we included individuals with advanced stages of nafld such as nonalcoholic steatohepatitis, fibrosis, or cirrhosis. we also could not differentiate peripheral insulin resistance from hepatic insulin resistance with our data. moreover, even though in euglycemic individuals homa-ir was highly correlated to a single value of insulin (r = . ), we do not have direct measures of dynamic glucose regulation. therefore, functional studies are needed to gain more insight into the biological processes a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved driving our observations. finally, our study did not include the genetic variant mboat (rs ), which has been associated with hepatic fat accumulation ( ). in our prior association analyses ( ), we did not see an association between mboat and la (beta= - . , p= . ). because our inclusion criteria for variants was that they needed to be associated with la, and we could not substantiate the association of mboat in our sample, we excluded it. in conclusion, to our knowledge, this is the largest study examining the interaction between multiple metabolic traits and four genetic variants on hepatic steatosis in multiple cohorts representing two different ancestry groups. our findings suggest that insulin levels/insulin resistance more than other correlated metabolic traits including glucose, tg, and bmi interact with genetic variants in pnpla to promote hepatic steatosis. through conditional analyses, we show that insulin levels explain the interactions observed between pnpla -rs and bmi, as well as the interactions between pnpla -rs and glucose and tg, in almost , nondiabetic, ea individuals. our work suggests that improving insulin resistance and reducing insulin levels in pre-diabetic individuals carrying fatty liver promoting alleles at pnpla -rs may offer preferential benefit and mitigate their risk of developing nafld. although pnpla genotype information is not currently used to make clinical decisions, it may be helpful in the future not only to risk stratify individuals, but also to tailor their treatment. our work contributes to the understanding of the pathophysiology of nafld, and informs further interventional research to better diagnose and/or treat individuals with increased risk of nafld. references . kahali b, halligan b, speliotes ek. insights from genome-wide association analyses of nonalcoholic fatty liver disease. semin liver dis ; 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: - . . papandreou d, karabouta z, rousso i. are dietary cholesterol intake and serum cholesterol levels related to nonalcoholic fatty liver disease in obese children? cholesterol ; : . . raimondo a, rees mg, gloyn al. glucokinase regulatory protein: complexity at the crossroads of triglyceride and glucose metabolism. curr opin lipidol ; : - . . mancina rm, dongiovanni p, petta s, pingitore p, meroni m, rametta r, boren j, et al. the mboat -tmc variant rs increases risk of nonalcoholic fatty liver disease in individuals of european descent. gastroenterology ; : - e . a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved author names in bold designate shared co-first authorship. acknowledgment we thank the reviewers for their helpful suggestions, which have improved this paper. conflict of interest drr laura yerges-armstrong is a current employee stockholder for glaxosmithkline, however, the current work was conducted while at university of maryland school of medicine. dr. ingrid borecki owns stock in regeneron pharmaceuticals. dr. jeffrey r. o'connell was a consultant for regeneron pharmaceuticals for a period of time during this study. figure . shown is the percentage of non-diabetic individuals in famhs with ≥ % fat in the liver (moderate to severe hepatic steatosis) per pnpla -rs genotype in the lowest and highest quartile of insulin levels. as the level of insulin increases, the percentage of individuals with ≥ % fat in the liver increases more markedly with increasing copies of the g risk allele (non-parallel lines show interaction). among those with the gg genotype, the difference (∆) in the percentage of individuals with moderate to severe liver fat increases by percentage points between the lowest and highest insulin quartiles. in contrast, this difference is lower among those with the cg genotype ( %), and cc genotype ( %).a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved table . demographic and characteristics of study participants in each cohort by ancestry ages amish cardia famhs fhs mesa cardia famhs genoa mesa demographic european ancestry ( , ) african ancestry ( , ) n= , , , , , , , age . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . men ( , ) , ( %) ( %) ( %) , ( %) , ( %) ( %) ( %) ( %) ( %) ( %) women ( , ) , ( %) ( %) ( %) , ( %) , ( %) ( %) ( %) ( %) ( %) ( %) characteristics liver attenuation (hu) ¥ . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . insulin (mu/l) . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . homa-ir ょ . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . glucose (mmol/l) . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . bmi (kg/m ) . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . obese ゆ ( %) c((( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) whr (cm) § nval . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . tg (mg/dl) . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . ldl (mg/dl) . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . hdl (mg/dl) . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . alcohol (drinks/week) . ± . nval . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . heavy drinkers* ( . %) nval ( %) ( %) ( . %) ( %) ( %) ( %) ( %) statistics are presented as mean ± standard deviation (sd), or as n (%). the table includes individuals with liver attenuation and genetic information from each cohort that were included in analyses. la and metabolic traits were not adjusted for covariates. the sample size for each trait varied from n depending on the data available. summary statistics for fasting insulin, homa-ir and fasting glucose excludes diabetics; fasting ldl excludes statin users. ¥ raw liver attenuation measured in hounsfield units. ‡ calculated as [fasting insulin (mu/l) x fasting glucose (mmol/l)/ . ]; † defined as bmi ≥ kg/m ; § not adjusted for bmi; nval= not available in a u th o r m a n u s c ri p t this article is protected by copyright. all rights reserved cohort. *defined as ≥ drinks per week for women and ≥ drinks per week for men. the amish do not consume alcohol. units in the table are hu=hounsfield units; mu/l=milliunits per liter; mmol/l=millimoles per liter; kg/m = kilograms divided by height in meters squared; cm=centimeters; mg/dl=milligram per deciliter. table . meta-analyses results for interactions between four snps and inverse normal-transformed residuals of metabolic traits on laivn in combined ancestries. rs rs * rs * rs gene chr alleles (ref/o) ref af gene chr alleles (ref/o) ref af gene chr alleles (ref/o) ref af gene chr alleles (ref/o) ref af pnpla g/c . gckr t/c . ncan/ tm sf t/c . lyplal c/t . (snp x metabolic traits) metabolic traits βint se p-value n βint se p-value n βint se p-value n βint se p-value n insulin - . . . e- , - . . . e- , - . . . e- , - . . . e- , homa-ir - . . . e- , - . . . e- , - . . . e- , - . . . e- , glucose - . . . e- , - . . . e- , - . . . e- , - . . . e- , bmi - . . . e- , - . . . e- , - . . . e- , - . . . e- , whradjbmi - . . . e- , - . . . e- , - . . . e- , . . . e- , tg - . . . e- , - . . . e- , . . . e- , - . . . e- , ldl . . . e- , . . . e- , - . . . e- , . . . e- , hdl - . . . e- , . . . e- , . . . e- , - . . . e- , chr, chromosome; ref/o, reference/other allele (reference allele is the effect allele of each snp); ref af, reference allele frequency; βint, interaction effect size; se, standard error. p-values that reached significance threshold (p≤ . e- ) are in bold; n is the highest sample size in meta-analyses. *rs is in ld (r = . ) with rs , a functional missense variant in gckr; *rs is in ld (r = . ) with rs , a functional missense variant in tm sf . a u th o r m a n u s c ri p t hep _ _f .tif this	article	is	protected	by	copyright.	all	rights	reserved a u th o r m a n u s c ri p t 日喀则家具制造服务中心 	 	 	 search 	要闻动态 	新闻资讯 	公司简介 	行业资讯 	投资中国 	魅力中国 首页  >  要闻动态  >  广东要闻 日喀则家具制造服务中心_--- 来源：黑客技术     时间： - - : 日喀则家具制造服务中心 hqok,驻马店制鞋服务中心,上海卫浴经销部,镇江纺织业经销部,崇左纺织业有限公司 	 	 中新网 月 日电 据俄罗斯卫星网报道，美国国家航空航天局(nasa)发布消息指出，该国太空探索技术公司(spacex)的“龙”货运飞船 日将离开国际空间站，并将为地球带回最新科学实验成果以及有关太空生活的电影。 nasa称：“抵达约一个月后， 月 日‘龙’飞船将与国际空间站脱离，为地球带回实验样本、设备和有关所完成科学研究的数据。” 该飞船 月 日抵达国际空间站。据悉，在飞船离开空间站数小时后，航天器将在北京时间 日 时 分，于太平洋距加利福尼亚州海岸 公里处的海域着陆。 nasa表示，截止目前该飞船是唯一能将物资从国际空间站运回地球的航天器，乘飞船返回的有医疗、光纤制造、移动油箱等领域的实验成果。 除此之外，“龙”飞船还将带回一部有关在国际空间站生活的电影。 	 	 相关文章 	 端砚产自 	 z 	 晚托 	 聂耳拜师 	 乌木锭代码 	 开封养殖服务中心 	 求购不锈钢水箱 	 强尼戴普新片 	 一锤定音报名 	 古医书 关于本网 | 网站地图技术员qq | 联系我们技术员qq 版权所有：milvvw 新闻网 协办：新闻网 承办：新闻网 建议使用 × 分辨率 ie . 以上版本浏览器 	电脑版 	关于本网联系技术员qq 	联系技术员qq 版权所有：日喀则家具制造服务中心 homepage - brandeis center skip to content brandeis center independent, non-partisan institution for public interest advocacy, research and education search for: 	about	mission and values 	president 	founder and chairman 	board of directors 	academic advisory board 	legal advisory board 	what others are saying 	career opportunities 	best practices	communications 	anti discrimination policy 	freedom of speech 	academic freedom 	news	news releases 	in the news 	events 	publications	books 	research articles 	outside research and analysis 	testimony 	brandeis briefs 	resources	government documents 	quasi-government documents 	case materials 	non government organizations 	letters to universities 	united nations written submissions 	fact sheets and guides 	videos 	jigsaw initiative	jigsaw intake form 	about jigsaw 	jigsaw training 	pro bono credit 	apply to jigsaw 	law student chapters 	blog	litigation 	anti-semitism 	anti-israelism 	freedom of speech 	religious freedom 	contact 	donation 		about	mission and values 	president 	founder and chairman 	board of directors 	academic advisory board 	legal advisory board 	what others are saying 	career opportunities 	best practices	communications 	anti discrimination policy 	freedom of speech 	academic freedom 	news	news releases 	in the news 	events 	publications	books 	research articles 	outside research and analysis 	testimony 	brandeis briefs 	resources	government documents 	quasi-government documents 	case materials 	non government organizations 	letters to universities 	united nations written submissions 	fact sheets and guides 	videos 	jigsaw initiative	jigsaw intake form 	about jigsaw 	jigsaw training 	pro bono credit 	apply to jigsaw 	law student chapters 	blog	litigation 	anti-semitism 	anti-israelism 	freedom of speech 	religious freedom 	contact 	donation 						 						 						 						 						 						 the louis d. brandeis center, inc. 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fatemeh ostaresh; mojgan babanejad nilofar bazazzadegan; hossein najmabadi*; kimia kahrizi genetic research center university of social welfare and rehabilitation sciences, tehran, iran objectives: hearing loss (hl) is the most common sensory disorder, and affects in newborns. about % of hl is due to genetics and % of them are non-syndromic with a recessive pattern of inheritance. up to now, more than genes have been detected which are responsible for autosomal recessive non-syndromic hearing loss, (arnshl). in iran, hl is one of the most common disabilities due to consanguineous marriages. the aim was to investigate the prevalence of three new arhl genes (gjb , gjc , and slitrk ) reported in neighboring countries among iranian families with arnshl. methods: one hundred unrelated families with at least two affected siblings in consanguineous marriage, who were negative for gjb gene mutations, were selected. by using three str markers for each gene, homozygosity mapping was performed. results: two families showed linkage to gjb , six families were linked to gjc and only one family linked to slitrk . the samples of these families who showed linkage were sent for sanger sequencing to detect the causative mutations. however, after analyzing the sequencing results, no mutation could be detected in either of the families. molecular analysis for these nine families is underway in order to determine the pathogenic mutations using whole exome sequencing. discussion: these data demonstrate a very low prevalence of mutation in these three genes (gjb , gjc , and slitrk ) in the iranian population, since no mutation was detected in our study group of families. keywords: autosomal recessive non-syndromic hearing loss, homozygosity mapping, linkage analysis, iran submitted: june accepted: august introduction hearing loss (hl) is a common sensorineural defect that affects a person’s life by disrupting their ability to communicate. in industrialized countries, in infants is either born with profound hl or is affected during the pre-lingual period of their lives. in iran, hl is the second most common disorder after intellectual disability [ ]. hl can be classified by different systems. the first classification method is based on the time of hl appearance. if hl is manifested before a child speaks, it is called pre- lingual, while after a child speaks it is called post- lingual. the second method for classification of hl is whether the hl occurs along with other symptoms or disorders (syndromic), which accounts for - % of hl, or whether the disorder is only hl (non- syndromic), which accounts for - % of hl. deafness is also classified based on the degree of hl. for example if the severity of hl is more than db (decibels), it is considered as profound. the severity of hl ranges between - db in severe forms, - db in moderately severe, - db in moderate, - db in mild, - db in slight, and finally - db classified as normal hearing. the pattern of inheritance in hl can be different, which means that another classification can be its pattern of  all correspondence to: hossein najmabadi, email: < hnajm @yahoo.com > vol. , issue , autumn. inheritance. the most common pattern, which accounts for % of hl, is autosomal recessive. hl can also be dominant, which accounts for %. x- linked and mitochondrial pattern types only account for less than % of hl [ - ]. autosomal recessive non-syndromic hl (arnshl) is usually pre- lingual. in iran, due to its high percentage of consanguineous marriages, autosomal recessive disorders are more prevalent compared to other populations [ ]. arnshl is a genetically heterogeneous disorder in which loci have been mapped and genes have been identified to date (http://hereditaryhearingloss.org/). our previous study showed that mutations of the gjb gene do not contribute to the major genetic load of deafness in the iranian population (~ %). [ , ] recently, many countries are paying more attention to hl, and focusing their studies on the genes causing this disorder. in the past couple of years, some countries around iran have found mutations in three new hl genes (gjb , gjc , and slitrk ). the gjb gene (omim# ) located on chromosome p . , has exons compromising base pairs. the protein encoded by gjb is connexin protein . that is a component of a gap junction, and is composed of amino acids and is about . kda. gap junctions are specialized in cell-cell contacts between almost all eukaryotic cells which provide direct intracellular communication. gap junctions allow the passive diffusion of molecules up to kda, which includes nutrients, small metabolites, and ions. structurally, gap channels are composed of two hemichannels called connexons, which themselves are formed from six connexin molecules.[ ] gap junctions are regulated through post-translational modifications of the c- terminal cytoplasmic tail and phosphorylation modulates assembly and their physiological properties. they are continuously synthesized and degraded, allowing tissues to rapidly adapt to changing environmental conditions. connexins play a key role in many physiological processes including cardiac and smooth muscle contraction, regulation of neuronal excitability, epithelial electrolyte transport and keratinocyte differentiation. mutations in connexin genes are associated with human diseases including sensorineural deafness, a variety of skin disorders, peripheral neuropathy and cardiovascular disease. lopez et al. detected a deletion (c. del ) in gjb that causes a frameshift in .[ ] in , yang et al. studied taiwanese. among them, had non-syndromic hearing loss. they reported seven mutations in connexin . .[ ] in , kooshavar et al. studied iranian patients who were heterozygote for gjb mutation. they reported five variants and one of the variants, c. c>t, was not detected in the normal population [ ]. the gene gjc (omim# ) located on chromosome q . comprises two exons. the protein encoded by this gene is a member of the gap junction proteins called connexin . , and it is composed of amino acids.[ ] all cells in solid tissues are coupled by gap junctions, thus it is not surprising that mutations in connexin genes have been linked to a variety of human diseases, including cardiovascular anomalies, peripheral neuropathy, skin disorders, cataracts, and deafness. yang et al. reported a heterozygote transversion (c. a>t) in two taiwanese patients with arnshl in .[ ] in , hong et al. reported a novel missense mutation in hela cells (p.e d) in the gjc gene.[ ] afterwards, in , su et al. reported a novel missense mutation (p.l h) in hela cells that affects protein connexin . . [ ] slitrk gene (omim# ) is located on chromosome q . with two exons comprising base pairs. the proteins encoded by slitrk are integral membrane proteins with two n-terminal leucine rich repeat (lpr) domains. mostly, these proteins are expressed predominantly in neural tissues and have neurite-modulating activity. slitrk protein is composed of amino acids. mutations in this gene are associated with deafness and myopia [ ]. in , tekin et al. studied three consanguineous amish families that had sensorineural hl. they reported a nonsense mutation (c. c>t) in exon of slitrk . they also reported a mutation c. c>t in a consanguineous turkish family.[ ] morlet et al. studied closely related amish families in . they reported a homozygote nonsense mutation (c. c>t) which causes defects in cochlea of the inner ear and results in hl.[ ] table ( ) shows these genes and their locus, if available. iranian rehabilitation journal table . the candidate genes, their locations, the reported linked families in different countries. gene chromosome locus country mutation references gjb p . unknown taiwan iran p. r c (c. c>t) p. v m (c. g>a) p. v m (c. g>a) p. r c (c. c>t) p. r h (c. g>a) p. a w (c. c>t) p. c w (c. c>g) p. t m (c. c>t) yang et al. kooshavar et al. gjc q . unknown taiwan p. e d (c. a>t) yang et al. slitrk q . unknown turkey greece amish p.s x (c. c>a) p.r x (c. c>t) p. q x (c. c>t) tekin et al. morlet et al. these genes have either not been worked on iranian families, or they have been studied in small populations. our goal was to study these genes in a large sample size from different ethnic regions of iran, determining and comparing their prevalence with neighboring countries. methods patients-after providing a family history, all hearing-impaired family members underwent a clinical investigation, including a physical examination, and a pure-tone audiometry at - hz was performed for all individuals. one hundred unrelated patients with arnshl who were negative for the gjb mutation were selected for this study. these patients were from consanguineous families with at least two affected and one normal sibling. no clinical features, including mental retardation, that would indicate that the hl was part of a syndrome, were observed. in addition, no gross vestibular involvement was noted. the hl phenotype was severe to profound, and was not known to be caused by inflammatory middle ear disease or specific environmental factors. participating subjects were clinically evaluated by their medical history and physical examination. the consent form, as approved by the university of social welfare and rehabilitation sciences; tehran, iran, was signed by each family. homozygosity mapping - in this project, families were analyzed for allele segregation of three genes using three or four short tandem repeats (str) microsatellite markers flanking these genes. since the allelic frequency for these str markers were not available for the iranian population, we determined the heterogeneity for these markers in ten unrelated normal individuals from diverse ethnic groups. a minimum of two microsatellite markers per locus were genotyped in the parents, with at least two affected children and one healthy sibling in each family. the str markers that were either within the gene or closer to the gene, and the repeats which were tri or tetra rather than di repeats, were given priority over the rest of the markers. str amplification was carried out using the pcr method, and then the pcr products were analyzed on % polyacrylamide gel, followed by silver nitrate staining. the linked families were further investigated using sanger sequencing to confirm the mutation. primers for sequencing were selected using bioinformatics software, genome browser, primer , and oligoanalyzer. mutation analysis - complete dna sequencing was performed for each linked family using intronic primers flanking the exons and exon–intron boundaries by big dye terminators (applied biosystems genetic analyzer, foster city, ca). results in the present study, among the investigated families with arnshl, nine were linked to one of the three genes, gjb , gjc , or slitrk , after homozygosity mapping. unfortunately, no mutation was detected after sanger sequencing was performed. table ( ) shows the str markers selected for these genes from the genome browser. (http://genome.ucsc.edu/) vol. , issue , autumn. table . the panel of microsatellite markers for the homozygosity mapping of each of the candidate regions and the number of linked families to each gene. no. locus gene no. of linked families clinical features str markers unknown gjb severe to profound hl d s d s d s unknown gjc mild to moderate hl d s d s d s d s dfnmyp slitrk moderate to profound hl, myopia d s d s d s discussion hearing loss is very heterogeneous in different populations. here we investigated the prevalence of three different genes (gjb , gjc , and slitrk ) in arnshl in iran among families who were negative for gjb (dfnb ) mutations. nine families showed linkage to one of these genes. lopez et al. reported a deletion ( del ) in gjb in that causes a frameshift. [ ] in , yang et al. reported seven other mutations in gjb in a taiwanese population. [ ] recently, five variants were reported by kooshavar et al. in an iranian population, from which c. c>t was not detected in the normal population. they studied patients that were heterozygote for a gjb mutation, since about . % of arnshl is due to a mutation in this gene in iran. [ ] the families in our cohort were selected from different parts of iran, but no mutation was detected among them. yang et al. in , reported two taiwanese with c. a>t transversion in the gjc gene. [ ] in , hong et al. reported a novel missense in hela cells that stops the function of connexin. [ ] tekin et al. studied three amish families with consanguineous marriages who were affected with sensorineural hl along with high myopia in . they reported a mutation p.q x (c. c>t) in exon two of the slitrk gene. they also reported a mutation, p.s x (c. c>a), in a consanguineous turkish family. [ ] in , morlet et al. studied nine amish families. they reported a novel nonsense missense variant p.q x (c. c>t). all the patients were homozygote for this mutation. [ ] the linked family in our study group was only affected with hl without displaying any symptoms of myopia. the explanations for not detecting any mutation while the families were linked could be that the str markers are close to other genes that can cause hl, or it could be due to snps. they could be linked to the snp but not to the gene itself. finally, this method, homozygosity mapping, is an indirect method and the results are not very precise. the next step for detecting mutations for the families that have been linked to one of the three mentioned genes is whole exome sequencing (wes). the human genome comprises about × bases, with coding and noncoding sequences. about × base pairs ( %) of the genome are the coding sequences. it is estimated that % of the disease-causing mutations are located in the coding regions of the genome. since sequencing determines every nucleotide in a dna sequence, exome sequencing offers a look into the genome that large- scale studies of common variations, such as the genome-wide association study (gwas), cannot provide. gwas can only identify variations in dna that are common in the population, in at least one percent of people, but wes detects not only the ones known to vary, but it can also reveal rare mutations, mostly monogenic disorders. wes has been used for variant detection among both common and rare diseases, as well as for snp associations and pharmacogenetics. another advantage of using this technique is that wes is fast and it is not as costly as whole genome sequencing.[ ] if no mutation is detected after wes, this could mean that these three genes, gjb , gjc , and slitrk , have a very low prevalence in the iranian population. conclusion based on our findings, these three genes, gjb , gjc , and slitrk , have a very low prevalence in the iranian population. however, further studies are recommended on these genes, with a more precise and specific mutation detection system such as wes. acknowledgment we would like to thank our patients and their families for their collaboration in this research. iranian rehabilitation journal references . mahdieh, n., et al., genetic causes of nonsyndromic hearing loss in iran in comparison with other populations. j hum genet, . ( ): p. - . . cryns, k. and g. van camp, deafness genes and their diagnostic applications. audiol neurootol, . ( ): p. - . . hilgert, n., r.j. smith, and g. van camp, forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in dna diagnostics? mutat res, . ( - ): p. - . . morton, n.e., genetic epidemiology of hearing impairment. ann n y acad sci, . : p. - . . saadat, m., m. ansari-lari, and d.d. farhud, consanguineous marriage in iran. ann hum biol, . ( ): p. - . . najmabadi, h., et al., gjb mutations in iranians with autosomal recessive non-syndromic sensorineural hearing loss. hum mutat, . ( ): p. . . najmabadi, h., et al., gjb mutations: passage through iran. am j med genet a, . a( ): p. - . . plum, a., et al., connexin -deficiency in mice causes transient placental dysmorphogenesis but does not impair hearing and skin differentiation. dev biol, . ( ): p. - . . lopez-bigas, n., et al., a common frameshift mutation and other variants in gjb (connexin . ): analysis of hearing impairment families. hum mutat, . ( ): p. . . yang, j.j., et al., identification of mutations in members of the connexin gene family as a cause of nonsyndromic deafness in taiwan. audiol neurootol, . ( ): p. - . . kooshavar, d., et al., digenic inheritance in autosomal recessive non-syndromic hearing loss cases carrying gjb heterozygote mutations: assessment of gjb , gja , and gjc . int j pediatr otorhinolaryngol, . ( ): p. - . . altevogt, b.m., et al., connexin is uniquely distributed within myelinating glial cells of the central and peripheral nervous systems. j neurosci, . ( ): p. - . . hong, h.m., et al., a novel mutation in the connexin gene may contribute to nonsyndromic hearing loss. hum genet, . ( ): p. - . . su, c.c., et al., mechanism of two novel human gjc missense mutations in causing non-syndromic hearing loss. cell biochem biophys, . ( ): p. - . . aruga, j. and k. mikoshiba, identification and characterization of slitrk, a novel neuronal transmembrane protein family controlling neurite outgrowth. mol cell neurosci, . ( ): p. - . . tekin, m., et al., slitrk mutations cause myopia and deafness in humans and mice. j clin invest, . ( ): p. - . . morlet, t., et al., a homozygous slitrk nonsense mutation is associated with progressive auditory neuropathy in humans. laryngoscope, . ( ): p. e - . . rabbani, b., m. tekin, and n. mahdieh, the promise of whole-exome sequencing in medical genetics. j hum genet, . ( ): p. - . diabetes care, volume , number , may d iabetes is one of the most common chronic diseases in the u.s., having a prevalence of . % among people aged � years ( ). diabetes prevalence rates vary widely in different regions of the world. in some traditional communities, such as those of the mapuche indians and melanesians, diabetes is rare or absent, whereas high prevalence rates are observed in some arab, asian indian, chinese, and hispanic american populations ( ). in the u.s., diabetes is more common in african- americans, mexican americans, japanese americans, and native americans than in non-hispanic caucasians ( ). type diabetes accounts for % of all diabetes. although both insulin resistance and �-cell dysfunction are well docu- mented, the molecular basis of type dia- betes is poorly understood ( , ). it has long been considered a disorder resulting from both genetic and nongenetic influ- ences ( ). the current understanding of the genetic basis of diabetes is largely restricted to a few distinct monogenic forms of the disease with clear mendelian modes of inheritance ( , ); there has been limited progress in identifying specific genetic defects responsible for the most common form(s) of type diabetes, which is likely to be heterogeneous and polygenic. the amish family diabetes study was initiated in with the goal of identify- ing the genetic determinants of type dia- betes and related traits through positional cloning approaches. the amish, named after their original leader jacob ammann, immigrated from western europe (mainly switzerland) to the u.s. to escape religious persecution over a -year period begin- ning in ( ). the earliest immigrants settled in pennsylvania. later groups settled in ohio, indiana, and illinois. there are no longer any amish living in europe. approximately of these families settled in lancaster county, pennsylvania, and can be considered the founders of today’s lan- caster amish community ( ). today’s amish population in the lancaster area exceeds , ( ). all amish are ruralites, and most earn their living by farming. they are resistant to from the southwest foundation for biomedical research (w.-c.h., b.d.m.), san antonio, texas; axys phar- maceuticals, la jolla, california (r.a., h.s., c.j.b.); the university of maryland school of medicine (t.p., a.r.s.), baltimore, maryland; glaxo wellcome (m.g.e., m.j.w., p.l.s.j., d.k.b.), research triangle park, north carolina; the hagedorn research institute, gentofte, denmark (b.k.m.); and the national institute of diabetes and digestive and kidney diseases (w.c.k.), the national institutes of health, phoenix, arizona. address correspondence and reprint requests to alan r. shuldiner, md, division of endocrinology, dia- betes and nutrition, university of maryland school of medicine, w. lombard st., room s- , balti- more, md . e-mail: ashuldin@medicine.umaryland.edu. received for publication september and accepted in revised form december . d.k.b., m.g., p.l.s.j., and m.j.w. are employed by glaxo wellcome; w.-c.h. and a.r.s. have received funding and salary support from glaxo wellcome; and t.p. is employed by the division of endocrinology, diabetes and nutrition at the university of maryland, which has received research funds and salary support from glaxo wellcome. h.s. is employed by parke-davis pharmaceuticals, and b.m. is employed by and holds stock in novo nordisk a/s. c.j.b. is employed by and r.a. holds stock in axys pharmaceuticals. abbreviations: anova, analysis of variance; auc, area under the curve; dbp, diastolic blood pressure; ifg, impaired fasting glucose; igt, impaired glucose tolerance; lada, latent autoimmune diabetes of adults; nhanes iii, third national health and nutrition examination survey; ogtt, oral glucose tolerance test; ooa, old order amish; sbp, systolic blood pressure; str, subscapular-to-triceps ratio; whr, waist-to-hip ratio. a table elsewhere in this issue shows conventional and système international (si) units and conversion factors for many substances. diabetes in the old order amish characterization and heritability analysis of the amish family diabetes study o r i g i n a l a r t i c l e objective — the old order amish (ooa) are a genetically well-defined closed caucasian founder population. the amish family diabetes study was initiated to identify susceptibility genes for type diabetes. this article describes the genetic epidemiology of type diabetes and related traits in this unique population. research design and methods — the study cohort comprised amish probands with diabetes who were diagnosed between and years of age and their extended adult family members. we recruited adults who represented multigenerational families. phenotypic characterization included anthropometry, blood pressure, diabetes status, lipid profile, and leptin levels. results — the mean age of study participants was years, and the mean bmi was . kg/m . subjects with type diabetes were older, more obese, and had higher insulin levels. the prevalence of diabetes in the ooa was approximately half that of the caucasian individuals who participated in the third national health and nutrition examination survey ( % ci . – . ). the prevalence of diabetes in the siblings of the diabetic probands was . % compared with a prevalence of . % in spouses (λs = . , % ci . – . ). the heritability of diabetes-related quantitative traits was substantial ( – % for obesity-related traits, – % for glucose levels, and – % for insulin levels during the oral glucose tolerance test; p = . to � . ). conclusions — type diabetes in the amish has similar phenotypic features to that of the overall caucasian population, although the prevalence in the amish community is lower than that of the caucasian population. there is significant familial clustering of type diabetes and related traits. this unique family collection will be an excellent resource for investigating the genetic underpinnings of type diabetes. diabetes care : – , wen-chi hsueh, phd braxton d. mitchell, phd rami aburomia, ba toni pollin, ms hakan sakul, phd margaret gelder ehm, phd birgitte k. michelsen, msc michael j. wagner, phd pamela l. st. jean, phd william c. knowler, md, dr ph daniel k. burns, phd callum j. bell, phd alan r. shuldiner, md e p i d e m i o l o g y / h e a l t h s e r v i c e s / p s y c h o s o c i a l r e s e a r c h diabetes care, volume , number , may diabetes in the amish assimilation into the surrounding dominant culture and are widely known for their old- fashioned social and technological practices and characteristic dress. they do not pros- elytize and do not allow outsiders to marry into the sect. they represent both a religious and a genetic isolate ( ). there is a high degree of consanguinity in the old order amish (ooa). although first-cousin mar- riages are not permitted, on average, ooa married couples are more closely related than second cousins once removed but less related than second cousins ( ). other fea- tures of this population include low reloca- tion rates, a relatively high standard of living, large family sizes (average sibship size – ), and essentially complete genealo- gies dating back to the early s ( – generations). all of these characteristics facilitate the collection of large families and extended pedigrees for genetic studies. in this initial report, we describe the design of the amish family diabetes study, the genetic epidemiology of diabetes, and the heritability of diabetes-related traits in this unique founder population. research design and methods study recruitment subject recruitment for the amish family diabetes study began in february . the protocol was approved by the institu- tional review board of the university of maryland. informed consent, including permission to contact relatives, was obtained before participation. individuals with adult-onset diabetes were identified by door-to-door interviews and by word- of-mouth. probands were defined as indi- viduals with previously diagnosed diabetes with age at diagnosis between and years. the diabetic probands’ first- and sec- ond-degree family members aged � years were recruited. if another diabetic individual was identified in the family (e.g., an aunt or uncle), then the family was expanded further to include that person’s first- and second-degree relatives aged � years. the efficiency of this type of sequen- tial sampling strategy for genetic linkage studies has been previously described ( ). phenotypic characterization study subjects were examined either at the amish diabetes research clinic in stras- burg, pennsylvania, or at their homes. height and weight were measured using a stadiometer and calibrated scale with shoes removed and in light clothing. waist cir- cumference was measured at the level of the umbilicus, and hip circumference was measured at the widest protuberance across the pelvis. skinfold thickness was mea- sured with calipers in triplicate at sites, the subscapular and triceps. the ratios of waist-to-hip circumference (whr) and subscapular-to-triceps skinfold thickness (str) were calculated as indexes of abdom- inal and central adipose distributions. sys- tolic (first phase) and diastolic (fifth phase) blood pressure were obtained in duplicate by use of a standard sphygmomanometer with the subject sitting for at least min and was recorded to the nearest mmhg. after an overnight fast, an indwelling angiocatheter was placed in an antecubital vein. after acquisition of a fasting blood sample, a -g oral glucose tolerance test (ogtt) was administered. blood samples were then drawn for determination of glu- cose and insulin values at -min intervals for h during the ogtt. glucose concen- trations were assayed with a beckman glu- cose analyzer (beckman coulter, fullerton, ca) using the glucose oxidase method (interassay coefficient of variation = . %) ( ). insulin and leptin levels were deter- mined by radioimmunoassay (linco, st. louis, mo) (interassay coefficients of varia- tion = . and . %, respectively). the total glucose and insulin areas under the curve (aucs) during the -h ogtt were determined with the trapezoid method. hba c levels were measured by high- pressure liquid chromatography (interassay coefficient of variation = . % for low stan- dard and . % for high standard), and the fasting lipid profile (total cholesterol, hdl cholesterol, and triglyceride levels) was assayed by quest diagnostics (baltimore, md) (interassay coefficient of variation = . % for total cholesterol, . % for hdl cholesterol, and . % for triglycerides). levels of antibodies to gad, a marker of immune destruction of pancreatic �-cells, were measured by radioligand-binding assay ( ) in a subset of subjects ( subjects with diabetes). previously known diabetes was deter- mined by a self-report of diabetes and any of the following: ) a single fasting venous plasma glucose level � mmol/l or a -h ogtt venous plasma glucose level � . mmol/l; ) current treatment with insulin or oral hypoglycemic agents; or ) con- firmed diagnosis by a physician. newly defined diabetes was determined by the lack of diabetes history by self-report and positive ogtt results (either a fasting venous plasma glucose level � mmol/l or a -h ogtt venous plasma glucose level � . mmol/l). impaired glucose toler- ance (igt) was defined as a -h ogtt venous plasma glucose level � . mmol/l but � . mmol/l, and impaired fasting glucose (ifg) was defined as a fasting plasma glucose level � . mmol/l but � mmol/l. the definitions of igt and ifg excluded subjects with diabetes. statistical methods to obtain descriptive details of the collec- tion, clinical characteristics of study sub- jects were computed as unadjusted means ± sd. means were compared using analysis of variance (anova) (systat . ; spss, chicago); p values were adjusted for age and sex, and, where appropriate, bmi. to obtain unbiased estimates of the prevalence of diabetes and igt in the overall ooa population, we compared age-specific prevalence rates in spouses of ooa dia- betic probands and their family members. we compared diabetes and igt prevalence in the ooa with corresponding rates from the general u.s. caucasian population, as estimated from the third national health and nutrition examination sur vey (nhanes iii) ( ). using the indirect method of age adjustment ( ), we applied prevalence rates obtained from the nhanes iii to determine the expected number of diabetes cases in the ooa. direct comparisons of diabetes prevalence between ooa and the nhanes iii were then obtained by comparing the number of cases observed in the ooa with the num- ber expected if the ooa had the same age- specific prevalence rates as the nhanes iii. the confidence interval for the ratio of observed-to-expected was also calculated ( ). the λs was estimated by comparing the prevalence rates of type diabetes between siblings of diabetic probands and spouses of ooa diabetic probands and their family members, adjusting for age with the mantel-haenszel procedure ( ). the heritabilities of diabetes- and obe- sity-related traits were computed using standard quantitative genetic methods ( ). heritability was defined in the narrow sense as the proportion of total phenotypic vari- ance that could be attributed to the additive effects of genes. it was estimated as a func- tion of the covariance among all possible relationship pairs in the pedigree. we simul- taneously adjusted for the effects of age, age , and sex. parameter estimates were obtained using maximal likelihood proce- dures, as implemented in the solar soft- ware package (southwest foundation for biomedical research, san antonio, tx) ( ). although it is possible to combine all of the study participants into a single - generation pedigree ( ), it was not com- putationally feasible to perform heritability analysis with such a complex pedigree structure. therefore, we combined families with overlapping individuals so that the original families were reduced to pedi- grees ranging in size from to individ- uals. subjects with unknown diabetes status due to missing data (n = ) were excluded from all analyses, and subjects with diabetes (n = ) were excluded from heritability analysis of insulin. similarly, subjects cur- rently taking antihypertensive medications were excluded from heritability analysis of blood pressure. data on hba c, insulin, lep- tin, and triglycerides were transformed by their natural logarithms to normalize the data distributions. results — the amish family dia- betes study was well received by the ooa community, and the participation rate was excellent (� %). of those who did not participate, the most common reasons given were ) lived over miles from the clinic, ) personal reasons, and ) debili- tating illnesses that precluded their partic- ipation. by the end of march , adults aged � years from multigen- erational families were studied. the mean sibship size was . (range – ). the extended pedigrees formed by combining these families provided a very large number of relationship pairs for our genetic analyses (table ). overall, % of the study subjects were women. the overall frequency of diabetes in this study population ascertained through a diabetic proband was . %. diabetes was significantly more common in women ( . %) than men ( . %) (p = . ), although after removing probands from the sample, the prevalence in men and women was approximately similar ( . % in women, . % in men, p = . ). clinical characteristics of ooa subjects with dia- betes and igt and/or ifg were similar to those observed in other caucasian popula- tions (table ) ( , ). compared with euglycemic subjects, those with diabetes or igt and/or ifg were older, more obese, and had higher blood pressure and triglyc- eride levels. amish individuals with diabetes and impaired glucose tolerance also had higher insulin levels than euglycemic indi- viduals, suggesting insulin resistance. table compares the age-specific prevalence rates of diabetes and igt b e t w e e n s p o u s e s o f o o a d i a b e t i c probands and their family members and caucasians from nhanes iii ( ). for the sake of comparison, diabetes status was defined by world health organization cri- teria ( ) for both the ooa and nhanes iii. as in nhanes iii caucasians, the prevalence of diabetes increased with age in the ooa. diabetes prevalence was lower in the ooa across all age groups than in the general u.s. caucasian population. the overall prevalence of diabetes in the ooa was . % ( of subjects). a total of . cases would be expected among sub- jects aged – years if the ooa experi- enced the same prevalence rates as the nhanes iii. thus, the diabetes prevalence in the ooa was only . times that of the national rate for u.s. caucasians ( % ci . – . ). in contrast, the prevalence of igt in the ooa was . times that of n h a n e s i i i c a u c a s i a n s ( % c i . – . ). the mean bmi in all of the age groups was comparable with those of nhanes iii (data not shown) ( ). diabetes was diagnosed in a total of ooa individuals. characteristics of these diabetic subjects are summarized in table . of those individuals, % were newly diagnosed at our research clinic. approximately % of the diabetic subjects diabetes care, volume , number , may hsueh and associates table —number of pairwise relationships among examined subjects in the amish family diabetes study pairwise relationship n parent-offspring sib-sib , grandparent-grandchild avuncular , half-sib grand-avuncular great-grand–avuncular first cousins , first cousins, once removed , first cousins, twice removed , first cousins, three times removed second cousins , second cousins, once removed , third cousins , third cousins, once removed total , data are n from pedigrees consisting of subjects. table —clinical characteristics of study subjects by diabetes status variable euglycemic igt and/or ifg* diabetes n age . ± . . ± . † . ± . † sex (m/f) / / / bmi (kg/m ) . ± . . ± . ‡ . ± . ‡ leptin (ng/ml) . ± . . ± . ‡ . ± . † waist (cm) . ± . . ± . ‡ . ± . † whr . ± . . ± . . ± . ‡ str . ± . . ± . . ± . ‡ sbp (mmhg) . ± . . ± . † . ± . † dbp (mmhg) . ± . . ± . ‡ . ± . ‡ total cholesterol (mmol/l) . ± . . ± . . ± . ‡ hdl cholesterol (mmol/l) . ± . . ± . . ± . triglyceride (mmol/l) . ± . . ± . ‡ . ± . † glucose (mmol/l) fasting . ± . . ± . † . ± . † ogtt at h . ± . . ± . † . ± . † insulin (pmol/l) fasting ± ± ± † ogtt at h ± ± † ± † data are n unadjusted means ± sd. p values for bmi, leptin, waist, whr, and str were adjusted for age and sex; p values for all other traits were adjusted for age, sex, and bmi. of the study subjects, with unknown diabetes status were excluded. *of subjects, had igt, had ifg, and had both igt and ifg. †p � . between the euglycemic group and the igt/ifg or diabetic groups; ‡p � . between the eugly- cemic group and the igt/ifg or diabetic groups. dbp, diastolic blood pressure; sbp, systolic blood pressure. were diagnosed before the age of years. more than half of all previously known cases were on insulin ( . %), whereas only . % of previously known cases did not take any medications for diabetes. the prevalence of gad antibody pos- itivity in diabetic subjects whose age at diagnosis was � years was . %. this rate did not differ significantly from that in subjects with either normal glucose toler- ance ( . %) or igt ( . %). in contrast, the prevalence of gad antibody positivity was % in diabetic subjects with an age at diagnosis � years, which is significantly higher than that in nondiabetic subjects (p � . ). these results are in accor- dance with the hypothesis that a large pro- portion of diabetes cases with an age at diagnosis � years may have autoim- mune type diabetes or latent autoim- mune diabetes of adults (lada) ( ), and, therefore, we excluded subjects with age of diagnosis � years (n = ) from further analyses of type diabetes–related traits. to determine the magnitude of familial aggregation of type diabetes in the ooa, we compared prevalence rates of diabetes (age at diagnosis � years) between sib- lings of diabetic probands and the spouses of probands and their family members. the prevalence of type diabetes was signifi- cantly higher in the probands’ siblings aged � years ( of [ . %]) than in the spouse group ( of [ . %]). the sib- ling relative risk (λs) adjusted for age by the mantel-haenszel procedure was . ( % ci . – . ). in contrast, the prevalence of igt was similar between the groups ( . % among diabetic probands’ siblings vs. . % in the spouse group). heritability estimates for obesity, blood pressure, lipids, hba c, glucose, and insulin in the amish family diabetes study are shown in table . heritability of both bmi and leptin was %. heritability of the body composition measures were % for waist circumference, % for whr, and % for str (p � . for all). concen- trations of lipids were also highly heritable, with familiality accounting for , , and % of variation in triglycerides, hdl-cho- lesterol, and total cholesterol, respectively (p � . for all). familiality accounted for and % of the variation in systolic and diastolic blood pressure (p � . ), % of the variation in hba c values (p = . ), – % of the variation in glucose levels (p � . ), and – % of the variation in insulin levels during the ogtt (p = . for fasting insulin and p � . for insulin at min). among adult subjects, the majority of men made their living as farmers ( %) and laborers ( %), and the majority of women identified their occupations as housewives ( %), farmers’ wives ( %), or shopkeepers, teachers, or craft makers (e.g., quilting) ( %). of our respondents, % said they had no leisure physical activ- ity, and for those who did, such activities were mostly walking and playing ball (baseball, volleyball, or table tennis). approximately % of men reported they had ever smoked cigarettes, compared with only . % of women. among ever-smok- ing men, most started smoking cigarettes between ages and years, but then stopped smoking in their early s. less than % of all ooa subjects reported that they currently smoked. conclusions — the amish fam- ily diabetes study was designed to elucidate the genetic epidemiology of type diabetes in a genetically well-defined founder popu- lation. the study population included � % of the total adult ooa population in the lancaster area. participation rates for the study were high, resulting in the enroll- ment of large families. we were further able to reconstruct the complex pedigree struc- diabetes care, volume , number , may diabetes in the amish table —age-specific prevalence rates of diabetes and igt in the spouses of ooa diabetic probands, their family members, and the nhanes iii caucasian cohort, based on who criteria diabetes igt nhanes iii observed expected nhanes iii observed expected age-group (years) caucasians* in ooa in ooa† caucasians* in ooa in ooa‡ – na† . ( / ) na na . ( / ) na – . . ( / ) ( . / ) . . ( / ) ( . / ) – . . ( / ) ( . / ) . . ( / ) ( . / ) – . . ( / ) ( . / ) . . ( / ) ( . / ) � na . ( / ) na na . ( / ) na overall§ . . ( / ) . ( . / ) . . ( / ) . ( . / ) data are % (n). *data are from reference ; †ratio of observed-to-expected among those subjects aged – years was . ( % ci . – . ); ‡ratio of observed-to- expected among those subjects aged – years was . ( . – . ); §prevalence rate for subjects aged – years. na, not available. table —characteristics of subjects with diabetes diabetic characteristic n (%) newly diagnosed diabetes / ( . ) age at diagnosis � years / ( . ) mean hba c (%) . ± . previously diagnosed diabetes / ( . ) on insulin / ( . ) on oral agents / ( . ) on no medication / ( . ) diabetic cases with positive gad antibody* — age at diabetes diagnosis � years / ( . ) age at diabetesdiagnosis � years / ( . ) data are n (%) or means ± sd. *gad antibody was measured in a subset of subjects ( with diabetes). the prevalence rates of gad antibody positivity in euglycemic subjects and subjects with igt were . and . %, respectively. ture of the study population through exten- sive interviews with participating subjects and by record linkage with the extensive genealogical database previously compiled for the lancaster county ooa population ( ). the mean kinship coefficient in this population is � . , indicating that the average degree of relatedness between any two random individuals was less than that of first-cousins but greater than that of sec- ond-cousins ( ). the phenotypic characteristics of adult- onset diabetes in the ooa are similar to typ- ical type diabetes and, thus, atypical and/or monogenic forms of diabetes (e.g., maturity onset diabetes of the young, mater- nally-inherited diabetes and deafness, and type a syndrome of extreme insulin resis- tance) are uncommon in the amish. based on the association between age of diabetes diagnosis and gad antibody prevalence, it is likely that both types and diabetes exist in this population. to minimize the possi- bility that probands for our study had type diabetes, we required that age of diabetes onset in the proband be at least years. an unexpected result obtained from this study was the lower prevalence of diabetes observed among the ooa compared with the general u.s. caucasian population. the observed prevalence of diabetes among spouses of probands and their family mem- bers was only . as high as that expected if these individuals had experienced the same diabetes risk as in the general u.s. caucasian population. interestingly, the prevalence of igt in the ooa was similar to or slightly higher in spouse control subjects, as compared with the general u.s. cau- casian population. this raises the interesting hypothesis that the ooa may not be pro- tected against glucose intolerance, but fewer individuals with impaired glucose tolerance eventually develop overt diabetes. by virtue of their predominantly agrarian lifestyle, the ooa may be more physically active than the general u.s. caucasian population. one speculation is that this relatively active lifestyle provides partial protection against conversion from igt to type diabetes. several studies have reported beneficial effects of physical activity on insulin sensi- tivity and glucose tolerance ( , ). diabetes aggregates in families in the ooa as it does in other populations ( , ). in this study, the prevalence of dia- betes was . times as high in the siblings of diabetic probands than in the spouse control group, a finding similar to that observed in other caucasian populations ( – ). by contrast, there was no familial aggregation of igt in the ooa. these find- ings suggest that genes may be important determinants of progression of igt to dia- betes in the amish. to define type diabetes genes, there may be value in dissecting the type dia- betes phenotype into genetically less com- plex traits that may be more proximal to the underlying pathophysiology. we observed moderate heritabilities for many of these quantitatively distributed traits, including glucose, insulin, obesity, blood pressure, and lipid levels. the heritability estimates we obtained from the amish are in the range of those reported for other popula- tions (table ). the ooa ( , ) and other founder populations ( , ) have been used suc- cessfully to map genes for simple mendelian diseases, especially those characterized by recessive transmission. more recently, founder populations have been used to map genes for complex diseases ( , ), includ- ing diabetes and related quantitative traits ( , ). to understand further the genetic contribution to type diabetes, we have ini- tiated additional studies of the ooa, including a genome scan. because of their unique ancestral history, there may be advantages to trying to identify diabetes susceptibility genes in this population. first, because of the relatively small number of founders, it is possible that a complex genetic disease like type diabetes will have equally strong genetic determinants in this population, as compared with the general caucasian population, but will be attributed to a smaller number of genes, which should facilitate their identification and characteri- zation. second, diabetes susceptibility genes present in the amish are likely to be a sub- set of those that are relevant to the general caucasian population. third, the large fam- ily structure characteristic of the ooa and the large number of individuals for which quantitative trait information has been obtained lead to greater power because of the very large number of relative pairs that are present. fourth, uniformity of lifestyle may minimize potential confounding effects of variable phenotypic expression of sus- ceptibility genes, which would further enhance our ability to identify these genes. however, the use of founder populations to map genes for complex disease poses spe- cial challenges. for example, the complex pedigree structures can greatly complicate the estimation of allele-sharing among pedi- gree members. in addition, once linkage is observed, fine mapping may be difficult if linkage disequilibrium extends over large regions of the chromosome. furthermore, the genetic defects identified in these pop- ulations will need to be verified in other populations to evaluate their implication in public health. in summary, the ooa are a genetically and socioculturally well-defined founder population. type diabetes in the ooa is diabetes care, volume , number , may hsueh and associates table —heritability estimates of diabetes-related traits in the ooa h reported phenotype n h p by others references bmi . ± . � . . – . – , – , , ln (leptin) . ± . � . . – . – waist . ± . � . . whr . ± . . . – . , , str . ± . � . . – . , sbp (mmhg) . ± . � . . – . , , dbp (mmhg) . ± . � . . – . , , total cholesterol . ± . � . . – . – , hdl cholesterol . ± . � . . – . – , ln (triglycerides) . ± . � . . – . – , ln (hba c) . ± . . na na fasting glucose . ± . � . . – . , , glucose . ± . � . . – . , , glucose auc . ± . � . na na ln (fasting insulin) . ± . . . – . , , , ln (insulin ) . ± . � . . – . , insulin auc . ± . . na na data are n from pedigrees consisting of subjects. dbp, diastolic blood pressure; h , heritability estimates; ln, natural log transformed; na, not available; sbp, systolic blood pressure. phenotypically similar to that of the general caucasian population, although its preva- lence is lower. there is substantial familial aggregation of the disease, and diabetes- related quantitative traits are heritable. these results provide the rationale for a genome-wide search, which is currently in progress, for type diabetes susceptibility genes in this unique population. acknowledgments — this research was sup- ported by a grant from glaxo wellcome and sequana therapeutics. we thank wendy warren and our amish liaisons for their energetic efforts in study sub- ject recruitment, and drs. alejandro schaffer and richa agarwala for assistance in pedigree construction. this study would not have been possible without the outstanding cooperation of the amish community. references . harris mi, goldstein de, flegal km, little rr, cowie cc, wiedmeyer hm, eberhardt ms, byrd-holt dd: prevalence of diabetes, impaired fasting glucose, and impaired glu- cose tolerance in u.s. adults. diabetes 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measures in the lon- gitudinal quebec family study. genet epi- demiol : – , . sakul h, pratley r, cardon l, ravussin e, mott d, bogardus c: familiality of physical and metabolic characteristics that predict the development of non-insulin-dependent diabetes mellitus in pima indians. am j hum genet : – , . selby jv, newman b, quesenberry cp jr, fabsitz rr, carmelli d, meaney fj, sle- menda c: genetic and behavioral influ- ences on body fat distribution. int j obes : – , . watanabe rm, valle t, hauser er, ghosh s, eriksson j, kohtamaki k, ehnholm c, tuomilehto j, collins fs, bergman rn, boehnke m: familiarity of quantitative metabolic traits in finnish families with non-insulin-dependent diabetes mellitus. hum hered : – , . bier bj, stein de, morton dh, goodman si: gene structure and mutations of gly- taryl-coenzyme a dehydrogenase: impaired association of enzyme subunits that is due to an a v substitution causes glutaric acidema type i in the amish. am j hum genet : – , . pannain s, 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genes in utah caucasians. diabetes : – , diabetes care, volume , number , may hsueh and associates wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse 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following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ microsoft word - .docx oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ oncotarget, vol. , no. the herc ubiquitin ligase is essential for embryonic development and regulates motor coordination monica cubillos-rojas , taiane schneider , ouadah hadjebi , leonardo pedrazza , , jarbas rodrigues de oliveira , francina langa , jean-louis guénet , joan duran , josep maria de anta , soledad alcántara , rocio ruiz , , eva maría pérez-villegas , francisco j. aguilar-montilla , Ángel m. carrión , jose angel armengol , emma baple , andrew h. crosby , ramon bartrons , francesc ventura and jose luis rosa departament de ciències fisiològiques, idibell, campus de bellvitge, universitat de barcelona, l’hospitalet de llobregat, barcelona, spain laboratório de pesquisa em biofísica celular e inflamação, pontifícia universidade católica do rio grande do sul, porto alegre, rio grande do sul, brazil département de biologie du développement, institut pasteur, paris, france departament de patologia i terapèutica experimental, campus de bellvitge, universitat de barcelona, l’hospitalet de llobregat, barcelona, spain departamento de bioquímica y biología molecular, facultad de farmacia, universidad de sevilla, sevilla, spain departamento de fisiología, anatomía y biología celular, universidad pablo de olavide, sevilla, spain institute of biomedical and clinical science, university of exeter medical school, rild wellcome wolfson centre, exeter, uk correspondence to: jose luis rosa, email: joseluisrosa@ub.edu keywords: ubiquitin, p , angelman syndrome, purkinje cells, behavioural analysis, pathology section received: june , accepted: august , published: august , abstract a mutation in the herc gene has been linked to a severe neurodevelopmental disorder with similarities to the angelman syndrome. this gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p and is involved in important cellular processes such as dna repair, cell cycle, cancer, and iron metabolism. despite the critical role of herc in these physiological and pathological processes, little is known about its relevance in vivo. here, we described a mouse with targeted inactivation of the herc gene. homozygous mice were not viable. distinct from other ubiquitin ligases that interact with p , such as mdm or mdm , p depletion did not rescue the lethality of homozygous mice. the herc protein levels were reduced by approximately one-half in heterozygous mice. consequently, herc activities, including ubiquitin ligase and stimulation of p activity, were lower in heterozygous mice. a decrease in herc activities was also observed in human skin fibroblasts from individuals with an angelman-like syndrome that express an unstable mutant protein of herc . behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. this effect was not observed in p knockout mice, indicating that a mechanism independent of p activity is involved. morphological analysis showed the presence of herc in purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. in these animals, an increase of autophagosomes and lysosomes was observed. our findings establish a crucial role of herc in embryonic development and motor coordination. research paper: pathology oncotarget www.impactjournals.com/oncotarget introduction angelman syndrome (as) is a severe neurodevelopmental disorder that occurs in approximately one out of every , births. patients with as exhibit developmental delay, speech impairments, intellectual disability, epilepsy, abnormal electroencephalograms, puppet-like ataxic movements, prognathism, tongue protrusion, paroxysms of laughter, abnormal sleep patterns, hyperactivity, and a high prevalence of autism [ , ]. genetic studies revealed that as is associated with maternal deletions of chromosome q -q , paternal chromosome uniparental disomy, or rare imprinting defects that affect the transcription of genes within the q -q region. specific loss-of-function mutations in the maternally inherited ube a gene which resides within this chromosomal region have been identified in a subset of affected individuals [ ]. the ube a gene encodes an e ubiquitin ligase called ube a or e -associated protein (e ap). more recently, a mutation in the herc gene has been linked to neurodevelopmental delay and dysfunction in both as and autism-spectrum disorders among the old order amish [ , ]. molecular analysis associated a missense mutation in the herc gene (c. c>t, p.pro leu) with the disease phenotype. although the herc gene also resides in the q -q region, it seems that it is not imprinted [ ]. herc encodes an ubiquitin ligase that binds to ube a and stimulates its ubiquitin ligase activity [ ]. deregulation of the activity of ube a is well recognized as contributing to the development of as [ , ]. thus, disruption of herc function by this mutation is associated with a reduction in ube a activity resulting in neurodevelopmental delay with angelman-like features [ , ]. genetic variations in the herc gene are associated with eye pigmentation. although multiple genes contribute to eye colour in humans, most variation can be attributed to a strong interaction between herc and adjacent oca on chromosome [ ]. a distal regulatory element of the oca promoter is within intron of the herc gene and three different sequence variants of herc have been identified, such as predictors of eye colour in humans [ , ]. herc belongs to the herc gene family that encodes a group of proteins that contain multiple structural domains. all members have at least one copy of an n-terminal region showing homology to the cell cycle regulator rcc and a c-terminal hect (homologous to the e -ap carboxyl terminus) domain found in a number of e ubiquitin protein ligases. these two domains define the herc family (herc = hect + rcc ) [ ]. in humans, six members form the herc family. they are classified into two groups: large (herc - ) and small (herc - ) proteins. structurally, small herc proteins contain the two characteristic domains hect and rcc , whereas large herc proteins are giant proteins (approximately , amino acid residues) containing additional domains, including several rcc domains. functionally, the herc protein family regulates ubiquitination and isgylation processes associated with membrane trafficking, immune response, dna repair, cell stress response and cancer biology [ - ]. recently, several substrates of herc have been identified. herc targets ubiquitin-dependent proteasomal degradation to xeroderma pigmentosa a (xpa) during circadian control of nucleotide excision repair [ ] and the breast cancer suppressor brca during the cell cycle [ ]. these data, together with the interaction of herc with rnf [ ], indicate a regulatory role for herc in dna repair by nucleotide excision and by homologous recombination of dna double-strand breakage. more recently, other substrates, such as neurl , usp or fbxl , have been reported that also indicate the participation of herc in other important cellular processes such as centrosome architecture, β-adrenergic receptor recycling, ralb signaling, cancer cell migration, and iron metabolism [ - ]. herc may also interact with proteins in a manner independent of proteasomal degradation. the tumor suppressor p is a transcription factor that coordinates the cellular response to several kinds of stress through the regulation of a wide range of genes [ , ]. in response to stress, p transcriptional activation is dependent of its oligomerization state [ ]. thus, p mutations that impair its oligomerization have been associated with a rare hereditary cancer predisposition disorder called li-fraumeni syndrome [ , ]. herc interacts with p and modulates its transcriptional activity by regulating its oligomerization [ ]. rna interference experiments showed that herc knock-down inhibited p oligomerization affecting its transcriptional activity. under these conditions, up-regulation of cell growth and increased focus formation were observed, suggesting an important role of herc in proliferation [ ]. in agreement with these observations, an association of frameshift mutations of herc with gastric and colorectal carcinoma has been described [ ]. despite the critical role of herc in cellular processes regulated by p , little is known about its physiological relevance. the mutation of herc found among the old order amish with features similar to angelman syndrome also suggests an important role for herc in neurodevelopment [ , ]. to determine the physiological importance of herc , we decided to generate a mouse with targeted inactivation of the herc gene. oncotarget www.impactjournals.com/oncotarget results characterization of the herc mice to study the physiological role of herc , we generated a novel mutant allele at the herc locus by using a gene trapped embryonic stem (es) cell line from the sanger institute. these es cells, here called herc , contain a pgt lxr expression vector with a strong splice acceptor site integrated within intron of the mouse herc gene, which results in the expression of a truncated mrna. pcr experiments with genomic dna from the tails of mice generated with these es cells confirmed the integration of the trap between exon and (figure a). figure : generation of the herc mice. a. schematic representation of the herc wild-type allele (herc +) and herc allele and the designed primers to identify both alleles (left). the herc allele contains the pgt lxr vector that expresses the fusion of β-galactosidase and neomycin transferase within intron . the integration of the trap was determined by genotyping using the indicated primers (right), ko / ko for the wild-type allele and gal /gal for the allele. b. exon structure of herc + and herc (left). rt-pcr experiments with mrna from liver, spleen and kidney of herc + and herc mice was performed using the indicated primers. c. pcr products from b were sequenced. the trap was inserted after exon , for which the mutant protein contains the first amino acids of the herc and β-galactosidase protein. d. β-galactosidase expression in herc mice. the activity of β-galactosidase was determined in the testes, brain, heart and kidney of herc mice and detected by x-gal staining. e. scheme of herc protein and the expected product from the herc allele. the p l pathological mutation is indicated (*). rld: rcc -like domain. oncotarget www.impactjournals.com/oncotarget rt-pcr experiments with mrna from different tissues showed the formation of a truncated mrna of herc fused to β-galactosidase (β-geo) (figure b). sequencing analysis confirmed these results and revealed the fusion of β-galactosidase after amino acid residue of herc (figure c). β-galactosidase activity was determined in several tissues (figure d), confirming the expression of a fused transcript of the first amino acid residues of herc with β-galactosidase. because mouse herc protein has , amino acid residues, we can consider that the herc new mutant allele herc is functionally deleted (figure e). herc is an essential gene during embryonic development offspring born from the intercross heterozygous mice were genotyped by pcr. among these mice, ( %) were wild-type for herc , and ( %) were heterozygous for herc +/ (figure a). we could not figure : analysis of progeny from the herc +/ cross. a. analysis of offspring born from the intercross of herc +/ mice. ninety-one animals were genotyped by pcr of genomic dna isolated from mouse tails. the expected frequencies for herc +/+ and herc +/ were obtained; however, no homozygous mice (herc / ) were identified. b. analysis of embryonic lethality in herc / mice. embryos from herc +/ pregnant females at different stages were isolated and genotyped. the herc +/+ and herc +/ genotypes were identified, but not the herc / . the placentas without embryos could not be genotyped. c. analysis of herc protein levels during development. lysates from brains at different stages were analyzed by immunoblotting for herc and β-actin. e (embryonic day), p , p and p (post-natal day , and , respectively) and ad (adult animal). oncotarget www.impactjournals.com/oncotarget identify any viable homozygous herc / . these data suggested an embryonic lethality for null mice. to determine the time of embryonic lethality in herc / mice, genomic dna was isolated from embryos harvested at different stages (e . , e . and e . ) of pregnancy from herc +/ mice. we isolated placentas and observed ( %) without embryos (figure b). among the embryos, we identified both wild-type and heterozygous mice, but not homozygous mice. at day . and . in placentas without embryos, we observed some residuals. at day . , these residuals were completely resorbed. most likely, these residuals were rests of embryos homozygous for herc . these results indicate that the expression of at least a normal herc copy is essential for the completion of embryonic development before day . . genes important for development are usually highly expressed during embryonic stages. to determine the expression pattern of the herc protein during development, we analyzed the levels of the endogenous protein in samples from brains at embryonic day e , postnatal days p , p and p , and adult mice ( weeks). anti-herc antibodies detected a double band that decreased during development, with the highest levels in the embryonic stage and lower levels in the adult animal (figure c). similar expression profiles were observed for other members of the herc family, such as herc or herc (data not shown), suggesting an important role of the herc proteins during embryonic development. p inactivation did not rescue embryonic lethality of homozygous herc mice growth curves and survival rates were analyzed in herc +/ mice. the growth curve was not significantly altered in herc +/ mice during the time studied (figure a). the survival rate was also similar to wild-type mice during the period studied (figure b). figure : p inactivation did not rescue the lethality of herc / homozygous mice. graphs of growth rate a. and survival b. from herc +/+ and herc +/ mice. growth and survival were analyzed in male mice (n> ) at the indicated weeks. the survival for p -/- mice also was analyzed. c. the analysis of mice from a cross of double heterozygous herc +/ p +/- animals. the offspring was genotyped by pcr of genomic dna with the appropriate primers, indicating that the embryonic lethal phenotype of herc / embryos was not rescued by crossing with p -/- mice. oncotarget www.impactjournals.com/oncotarget figure : herc +/ mice show reduced levels of herc protein. a. herc protein levels were analyzed by immunoblotting using specific antibodies against herc in several tissues from week old mice. the levels of herc were quantified (n = ) and normalized with respect to β-actin levels. b. β-galactosidase expression in brain from herc mouse. the β-galactosidase activity was detected ubiquitously in all areas using x-gal staining. however, there were forebrain cortical and subcortical areas in which β-galactosidase labeling was the most intense (asterisks). c. the levels of herc were analyzed by immunoblotting in lysates of cerebellum, cerebral cortex and diencephalon from herc +/+ and herc +/ mice at p (post-natal day ). ba, basal amygdala. ca , pyramidal cell layer of the hippocampal cornu ammonis . den, dorsal endopiriform nucleus. dg, granular cell layer of hippocampal dentate gyrus. dm, dorsomedial nucleus of the hypothalamus. pir, piriform cortex. pv, paraventricular thalamic nucleus. rsg, retrosplenial granular cortex. vm, ventromedial nucleus of the hypothalamus. oncotarget www.impactjournals.com/oncotarget it had been previously demonstrated that knockout mice for the e ubiquitin ligases of p , such as mdm or mdm , are lethal in embryonic stages due to growth inhibition and apoptosis. interestingly, this lethality could be rescued by concomitant p depletion [ - ]. because herc is an e ubiquitin ligase that regulates p activity [ ], we asked whether p inactivation might also rescue the lethality of herc / homozygous mice. we crossed double heterozygous herc and p mice (herc +/ p +/- mice) to obtain double homozygous mice. we analyzed the genotype of the offspring by performing an adequate pcr assay on of genomic dna samples but did not observe any viable herc / mouse (figure c). these data suggest the non-involvement of p in the embryonic death caused by the depletion of herc . partial inactivation of the herc gene is sufficient to reduce herc protein levels and activity we analyzed protein levels of herc in herc +/ animals by immunoblotting. we observed that the levels of herc were decreased approximately % in the brain and kidney of herc +/ animals (figure a). the analysis of β-galactosidase activity in herc +/ mice allows the analysis of herc expression in more detail. ubiquitous expression of herc was observed in all tissues analyzed (figure d). in the brain, this expression was higher in the hippocampus (pyramidal cell layer and granular layer of dentate gyrus), hypothalamic nucleus (dorsomedial and ventromedial), amygdaloid nucleus (basal and medial), piriform cortex, dorsal endopiriform nucleus, entorhinal cortex, retrospenial cortex, paraventricular thalamic nucleus, and cerebellum (figure b and data not shown). figure : herc +/ mice show reduced activity of herc . a. usp , a substrate of ubiquitination of herc , was analyzed in lysates (cerebellum, cerebral cortex and diencephalon) from week old mice by immunoblotting. higher levels of usp were observed in all areas of herc +/ mice. levels of usp were quantified and normalized with respect to β-actin levels. b. herc +/ mice show reduced levels of p mrna. rt quantitative pcr analyses were performed in forebrain and cerebellum from herc +/+ and herc +/ mice to quantify p gene expression (n = ). the levels of expression were normalized with respect to gapdh gene expression. oncotarget www.impactjournals.com/oncotarget these observations were confirmed by immunoblotting. we dissected the cerebellum, cerebral cortex and diencephalon, detecting herc protein in all these areas (figure c). the levels of herc protein were reduced in herc +/ animals (figure c) with respect to control mice (herc +/+). these data show that the levels of herc protein are reduced almost % in herc +/ mice tissues and suggest that the herc activity must also decrease in the brain of heterozygous animals. two activities have been associated with herc protein; an e ubiquitin ligase activity which regulates protein levels of usp , brca or xpa [ , , ], and an activity as a stimulator of the p oligomerization that regulates the transcriptional activity of p [ ]. to analyze the activity of e ubiquitin ligase, we performed immunoblotting experiments in the mouse brain areas with antibodies against substrates ubiquitinated by herc . only the usp protein was detected by immunoblotting in these mouse samples (figure a). interestingly, in herc +/ mice, the levels of usp were higher than in control mice. to examine the activity stimulating p oligomerization and transcriptional activity, we analyzed the levels of p mrna by rt quantitative pcr analysis. a decrease in p mrna levels was observed in herc +/ mice (figure b). altogether, these data show that the partial inactivation of the herc gene in herc +/ mice is sufficient to reduce herc protein levels and activity. a homozygous mutation in human herc causes an angelman-like syndrome and reduces the activity of the herc protein herc has been implicated in a human disorder with some features similar to angelman syndrome. the substitution of proline by leucine at amino acid position in herc caused herc p l instability and almost total loss of the protein in homozygosis [ , ]. based on our data from heterozygous animals, we hypothesized that these patients would have lower levels of herc activity. to test this hypothesis, we analyzed the levels of usp protein from fibroblasts derived from an affected individual and a healthy control. we observed a high increase of usp levels in fibroblasts from a patient (figure a). to confirm that herc activity was diminished, we also analyzed the levels of p protein. we observed a decrease in the p protein levels in fibroblasts from this patient (figure a). however, the p and α-tubulin levels did not significantly change. the p mrna levels also confirmed the decrease in p transcriptional activity in fibroblasts from the patient (figure b). the level of p protein is regulated by the proteasome. in the presence of the proteasome inhibitor mg , we observed a similar stabilization of p protein levels in fibroblasts from the patient and the control (figure c). under these conditions, we also detected a decrease in p protein levels in fibroblasts from the patient (figure c), indicating a decrease in its p activity. these data show the low activity of herc in individuals carrying the herc p l mutation. in conclusion, the low herc activity found in herc +/ mice (figure ) and in individuals with the herc p l mutation (figure a- c) correlated with an increase in levels of the usp protein and a decrease in levels of the p protein. these data suggest that in conditions with low levels of herc protein, cells could contain more usp and less p . to confirm this point, human u os cells were depleted of herc using interference rna, and usp and p were analyzed by immunoblotting (figure d). herc knockdown increased levels of the usp protein and decreased levels of the p protein. table : behavioural tests of herc +/ mice cognitive task behavioural tests test values: mean ± error (n) p value herc +/+ herc +/ anxiety time outside of the dark box (s) ± ( ) ± ( ) . immobile time suspended by the tail (s) ± ( ) ± ( ) . learning and memory object recognition memory (di) stm . ± . ( ) . ± . ( ) . ltm . ± . ( ) . ± . ( ) . step through passive avoidance test (latency) stm . ± . ( ) . ± . ( ) . ltm . ± . ( ) . ± . ( ) . motor function open field (total activity) ± ( ) ± ( ) . rotarod (#falls) . ± . ( ) . ± . ( ) ** . forelimb grip strength (s) . ± . ( ) . ± . ( ) . stm: short term memory; ltm: long term memory; di: discrimination index; n: number of animals oncotarget www.impactjournals.com/oncotarget figure : a homozygous mutation in human herc that causes an angelman-like syndrome reduces the activity of the herc protein. a. fibroblasts derived from individuals with herc wild-type or herc with the mutation p l were analyzed by immunoblotting for the indicated antibodies. levels of usp and p proteins were quantified and normalized with respect to α-tubulin levels. b. levels of p mrna were analyzed by rt quantitative pcr analysis and normalized with respect to s gene expression. c. the levels of herc , p , p and α-tubulin proteins were analyzed in the presence or absence of the proteasome inhibitor mg . d. u os cells were transfected with non-targeting (nt) or herc sirnas and analyzed by immunoblotting against the indicated proteins. the levels of usp or p were quantified and normalized with respect to ran levels. oncotarget www.impactjournals.com/oncotarget herc regulates motor coordination individuals carrying the herc p l mutation present a severe developmental delay with an unstable gait [ , ]. we wondered whether herc +/ mice with a reduction of approximately % in herc protein could have some features found in herc p l patients. to examine this question, behavioural tests to measure different cognitive tasks were performed in herc +/ and wild-type mice. except for the rotarod test, no significant figure : impaired motor coordination in herc +/ mice. a.-b. the number of falls from the rotarod increases in herc +/ mice at months of age in comparison with control littermates. c. no difference was found in p +/- and p -/- mice in comparison with wt. (d-e) emg measurements of cmap amplitudes in the mg of control and herc +/ mice show normal neurotransmission efficacy in postnatal heterozygous mice. d. representative recordings during a train of stimuli at hz in a control and a herc +/ mouse. e. depression of cmap amplitudes (normalized to the first response) during a train of stimuli of ms at hz in control (n = ) and heterozygous mice (n = ). the train index f., corresponding to the depression at the end of the train, and ppf (pair -pulse facilitation) g., are similar between groups. oncotarget www.impactjournals.com/oncotarget differences between the two groups were found (table ; supplementary video). herc +/ mice displayed a statistically significant increase in rotarod falls compared to wild-type mice (herc +/+: . ± . , n = and herc +/ : . ± . , n = , p = . ) (figure a- b), suggesting a role for herc in motor coordination. because herc regulates p activity, we asked whether p inactivation could produce effects similar to herc +/ mice. we performed the rotarod test in p +/- and p -/- mice. no significant differences were observed compared to wild-type mice (figure c). these data indicate that the impaired motor coordination in herc +/ mice is specific and independent of p . next, we analyzed whether muscular function was affected in herc +/ mice. the electrical neuromuscular properties of the herc +/ mice were studied in vivo by performing electromyography (emg) on the medial gastrocnemius (mg) muscle using short train stimuli at hz (figure d). the depression at the end and the facilitation at the beginning of a train of stimuli were similar in mutant and wild-type mice when measured by normalization of the cmap (compound muscular action potentials) amplitude (p = . and p = . , respectively) (figure e- g). these results indicate a normal muscular function. the cerebellum regulates motor coordination, and alterations in its structure have been associated with impaired motor coordination. to examine whether the deficit of motor synchronization in herc +/ mice was caused by an alteration of cerebellar structure, we performed immunohistochemistry analysis of the specific marker of purkinje cells calbindin-d k (cabp) (figure ). cabp immunoreactive purkinje cell somata form a continuous cell layer in the cerebellar cortex of herc +/+ mice (figure a- c). in herc +/ mice, parasagittal zones devoid of immunoreactivity throughout the cerebellum indicative of purkinje cell loss were observed (arrows and arrowheads in figure d- f). these symmetrical purkinje cell-deprived bands -characterized by the presence of wide spaces lacking cell somata in the purkinje cells layer (asterisks in figure h) and dendritic debris through the molecular layer (mol in figure h)-, were distributed differently along a medio-lateral gradient (see comparison between figure d- f from a herc +/ mouse and figure a- c from a herc +/+ mouse). thus, vermal and paravermal purkinje cells were less affected than in the cerebellar hemispheres. in the vermis and paravermis, the loss of purkinje cells was distributed in narrow gaps (arrows in figure d and e), while at the hemispheres, the areas of purkinje cells loss reached a great extension (arrowheads in figure d and f), in which remain some surviving purkinje cells (small arrows in figure f). epifluorescence microscopy analysis showed that herc immunoreactivy in purkinje cells colocalize with cabp (figure ). in herc +/ mice, herc immunohistochemistry confirmed the loss of purkinje cells in narrow gaps at the vermis/paravermis or in a greater extension at the hemispheres (figure c- e). cabp immunohistochemistry also revealed the presence of pathological signs in herc +/ purkinje cells. rounded thickenings resembling axonal torpedoes were observed in purkinje cell axons, which contrast with the fine grained morphology of normal purkinje cells axonal plexuses (see arrows in the comparison between herc +/+ and herc +/ in figure a- c). phenotypic alterations of herc +/ purkinje cells were also observed in the . µm thick sections stained with toluidine blue (figure d- e). disappeared purkinje cells somata were substituted by glial golgi-epithelial cells (figure d: arrows, purkinje cells; small arrows, golgi-epithelial cells). high magnification allowed to detect degenerative dark accumulations within the cytoplasm of the soma (arrowheads in figure e) and the dendrites (arrows in figure e) of the purkinje cells. electron microscopy analysis of cerebella confirmed these degenerative signs in herc +/ mice (figure f- h and ). the cytoplasm of herc +/ purkinje cells contained a high number of lysosomes and electron-dense debris (figure f- h, asterisks), and autophagosomes with different degrees of evolution (figure g, arrows). damaged cisterns of golgi apparatus and numerous cisterns of the rough endoplasmic reticulum fused to the cytoplasmic face of the nuclear membrane were also observed (not shown). the difference between mice herc +/+ and herc +/ was even most evident in the principal purkinje cell dendrites (pcd in figure ). thus, numerous degenerative signs were present in herc +/ purkinje cells dendrite (figure b and e), while were almost absent in wild-type ones (figure a, c and d). these degenerative signs were found in and month-old animals (figure f- h), with a slight increase of these alterations in the older mice (not shown). these data show that the partial inactivation of herc in herc +/ mice causes purkinje cell loss, which explain motor incoordination detected here in rotarod test. the increase of autophagosomes and lysosomes observed in herc +/ purkinje cells (figure and ) led us to wonder whether herc may be involved in the regulation of autophagy. to this end, we have analyzed the adaptor-substrate p /sqstm by laser confocal microscopy. herc immunostaining colocalized with p (figure ). this colocalization, it was observed both in wild-type animals as in herc +/ animals (figure , arrows and arrowheads in the merged). in herc+/+ mice, p immunostaining showed a punctate labeling pattern that concentrated in purkinje cells somata (figure , arrows). this pattern was altered in herc +/ animals; thus, in addition to the somatic labeling, p immunostaining was also evident within the dendritic trees and the axonal torpedoes of purkinje cells (figure , arrows and arrowheads) indicating a dysregulation of autophagy in herc +/ purkinje cells. altogether, oncotarget www.impactjournals.com/oncotarget figure : purkinje cells loss in herc +/ mice. microphotographs of coronal sections through the cerebellar cortex of months old wild-type (wt, a-c, g) and months old herc +/ mice (+/ , d-f, h). calbindin immunohistochemistry shows as purkinje cell somata form a continuous cell layer in the herc +/+ (wt) cerebellum (a-c) however, parasagittal zones lacking of immunoreactivity throughout the herc +/ cerebellum indicative of purkinje cell loss are observed (arrows and arrowheads, in (d-f). these symmetrical purkinje cells deprived bands, characterized by the presence of wide spaces lacking purkinje cell somata (h, asterisks) and dendritic debris through the molecular layer (h, mol), distribute differently according a medio-lateral gradient. in the vermis and paravermal zones the immunonegative zones are sagittaly distributed in narrow gaps (d-e, arrows); while at the hemispheres the areas devoid of purkinje cells, also bilateral, reach a greater extension (d, f, arrowheads) in which remain some surviving purkinje cells (f, small arrows). g, illustrates the herc +/+ (wt) immunostaining of normal purkinje cells; note as dendritic trees fulfill the molecular layer (g, mol), while purkinje cells somata align in a continuous row. pcl, purkinje cells layer. bars = µm (a, d), µm (b-c, e-f), and µm (g-h). oncotarget www.impactjournals.com/oncotarget figure : herc is present in purkinje cells. microphotographs of coronal sections through the cerebellar cortex of months old herc +/+ (wt, a.-b.) and months old herc +/ mice (+/ , d.-e.). epifluorescence microscopy analysis shows that herc is expressed in all the adult purkinje cells colocalizating with the general marker of purkinje cell calbindin (cabp) (a-e) in the cerebellar cortex (a, cc,), and their axonal endings in the cerebellar nuclei (a, cn, arrow). herc +/ cerebellum displays parasagittal bands of purkinje cells loss in the vermis and paravermal zones (c., arrows; d, arrowheads), and areas of extensive purkinje cell loss in the cerebellar paraflocculus (asterisks). the arrows in e illustrate the co-expression of both proteins in the purkinje cell dendritic tree. bars = µm (c), µm (a), µm (d), µm (e), and µm (b). oncotarget www.impactjournals.com/oncotarget figure : purkinje cell degeneration in herc +/ mice. microphotographs of transmitted light a.-e. and electron microscopy f.-h. of parasagittal sections through the cerebellar cortex of months herc +/+ (wt, a), and (h) and months old herc +/ mice (+/ , b.-g.). calbindin immunohistochemistry reveals the presence of rounded thickenings resembling to axonal torpedoes in herc +/ purkinje cell axons (b-c, arrows), which contrast with the fine grained morphology of normal purkinje cells axonal plexuses (a, arrows). . µm thick sections illustrated purkinje cells (d, arrows) limiting a zone in which disappeared purkinje cells were substituted by glial golgi-epithelial cells (small arrows in d and e). high magnification allows detect degenerative dark accumulations within the cytoplasm of the soma (e, arrowhead) and the dendrites (e, arrows) of the purkinje cells. herc +/ purkinje cells cytoplasm possesses lysosomes, electron-dense debris (f-h, asterisks), and autophagosomes with different degrees of evolution (g, arrows). pcn, purkinje cell nucleus. bars = µm (a-d), µm (e), and µm (f-h). oncotarget www.impactjournals.com/oncotarget figure : ultrastructural analysis of herc +/ mice indicates accumulation of autophagosomes and lysosomes in purkinje cells. electron photomicrographs of parasagittal sections through the cerebellar vermis of months old herc +/+ (wt, a., c.- d.) and months old herc +/ mice (+/ , b., e.). an important difference in the presence of autophagic (arrowheads), and lysosomal (arrows) organelles can be observed between wild-type (a) and herc +/ (b) purkinje cells cytoplasm. the difference is even most evident in the principal purkinje cell dendrites. thus, numerous degenerative signs are present in herc +/ purkinje cells dendrite (e), while are almost absent in wild-type ones (c-d). pcd, purkinje cell dendrite. pcn, purkinje cell nucleus. bars = µm (a-c, e), and µm (d). oncotarget www.impactjournals.com/oncotarget these findings suggest that herc plays an active role in regulating the purkinje cells homeostasis, whose deregulation elicits alterations in motor coordination. discussion we have generated a new mutant allele of the herc ubiquitin ligase, herc , which has led us to identify herc as an essential gene for embryonic development. the herc gene encodes an unusual long polypeptide chain of almost , amino acid residues. the maintenance during evolution of this single and long polypeptide chain suggests an important physiological role for the giant herc protein. here, we showed that the inactivation of herc causes embryonic lethality before . days. at that time, we observed a high number of abnormal placentas, probably indicating resorption of unviable embryos. although we have not studied the cause of this embryonic lethality, a possibility could be defective implantation. the unviability of embryos when herc is inactivated would be indicative of the difficulty of finding human individuals homozygous for mutations of this gene. to our knowledge, only the herc p l mutation found in the amish community has been reported in humans [ ]. in these individuals, the herc protein is unstable and low levels are detected, although apparently high enough to avoid embryonic lethality. ubiquitin ligases that interact with p , such as mdm or mdm , had also been identified as essential during embryonic phases. the absence of mdm induces embryonic lethality in mice at the peri-implantation (e -e . ) stage of development, whereas mice deficient for mdm die in mid-gestation (e . -e . ) [ - ]. the lethality during these phases was rescued by a double knockout of the ubiquitin ligase and p [ - ]. because herc is also an ubiquitin ligase that interacts and regulates p activity [ ], we analyzed whether the double knockout of herc and p could also rescue the lethality. our results show that the double knockout did not rescue the embryonic lethality, indicating a p -independent role for herc in development. moreover, these data also confirm, at a genetic level, the different functions of mdm (or mdm ) and herc in p regulation. herc acts as an ubiquitin ligase tagging for degradation by the proteasome of substrates such as xpa, figure : p /sqstm in herc +/ mice. laser confocal microphotographs of coronal sections through the cerebellar cortex of the vermis of months herc +/+ (wt, a) and months old herc +/ (+/ , b) mice double labeled with herc and p antibodies. colocalizations of herc and p are indicated by arrows in dendrites and cell somata a., b., and by arrowheads in the axonal torpedoes of herc +/ purkinje cells (b). asterisks in b indicate the absence of purkinje cell bodies. bar = µm (a-b). oncotarget www.impactjournals.com/oncotarget brca , neurl , fbxl or usp [ , , - ]. herc may also positively regulate p activity through stimulation of its oligomerization [ ]. the analysis of levels of usp and p led us to analyze these two activities, respectively. these activities had been reported in culture cell but not in physiological conditions. the generation of herc +/ mice containing approximately one-half of the herc protein (figure ) led us to analyze the role of herc in vivo. herc +/ mice had higher levels of usp protein and lower levels of p mrna (figure ). these results show for the first time the inhibition of herc activity in physiological conditions. individuals from the amish community with a punctual mutation in herc , herc p l, suffer a developmental disorder with features similar to angelman syndrome [ , ]. these individuals produce an instable herc p l protein. skin fibroblasts obtained from these individuals also had higher levels of usp protein and lower levels of p , in agreement with a loss of herc activity. we showed how these results can be extrapolated to other human cells through interference rna experiments. herc knockdown caused an increase in the levels of usp protein and a decrease in the levels of p . in conclusion, we show how the loss of herc protein correlates with the loss of its activities, confirming the involvement of herc in cellular processes regulated by usp [ ] and p [ ]. the tumor suppressor gene p is mutated in approximately % of human sporadic cancers and in inherited cancer predispositions, such as li-fraumeni syndrome [ , ]. homozygous p -/- mice are highly prone to cancer, particularly t-cell lymphoma and sarcoma [ , ]. experiments with heterozygous p +/- mice indicate that a mere reduction in p levels may be sufficient to promote tumorigenesis [ ]. the p transcriptional activation is dependent of its oligomerization state [ ], and p mutations that impair its oligomerization have been associated with the li- fraumeni syndrome [ , ]. because herc interacts with p and modulates its transcriptional activity by regulating its oligomerization [ ] and herc +/ mice have less p activity (figure ), we would expect herc +/ mice to show an increased susceptibility to developing spontaneous tumors. however, we have not observed a greater number of tumors in herc mice (data not shown). in agreement with these data, an increased susceptibility to develop spontaneous tumors in individuals from the amish community with the herc p l punctual mutation [ , ] and lower p activity (figure ) has not been reported. the hypothesis that loss of transcription in p is the driving force selected during tumorigenesis needs to be reevaluated, as it is far from straightforward. for example, mice expressing p mutants transcriptionally defective for growth arrest, senescence and apoptosis, are not prone to cancer [ , ]. moreover, cells from mice deficient for the three p target genes, p , puma and noxa, are deficient in their ability to undergo p -mediated cell-cycle arrest, apoptosis, and senescence, although the animals remain tumor-free [ ]. our study identifies a new function for the herc ubiquitin ligase as a regulator of motor coordination through regulation of purkinje cells homeostasis. herc protein is expressed in purkinje cells (figure ). the decrease of its protein levels correlates with the loss of purkinje cells in the vermis and hemispheres of the cerebellum that would explain the motor incoordination detected in herc +/ mice with rotarod experiments (figures and ). the purkinje cell loss is bilateral and symmetrical as in other mutations characterized by the purkinje cells loss [ ], demonstrating the specificity of the degenerative process. this loss is not homogeneously distributed; thus, the loss of purkinje cells appears as discrete gaps in the continuous staining of the molecular layer of the vermis, while at the hemispheres, the loss reaches a great extension (figure ). despite their homogenous shapes and common morphological features, these results suggest some difference between purkinje cells and is in agreement with previous observations indicating that these neurons do not constitute a biochemically homogenous population [ , ]. pathological signs, such as varicose enlargements resembling axonal torpedoes and widespread accumulations of dense cytoplasmic material, were observed in purkinje cells of herc +/ mice. proliferation of golgi epithelial cells is also indicative of purkinje cell degeneration. electron microscopy analysis of herc +/ cerebella showed purkinje cells containing numerous degenerating signs in autophagosomes, lysosomes and golgi cisterns (figure and , and data not shown). these results resemble those obtained from a different mutant mouse called tambaleante [ , ]. in these animals, a progressive and specific loss of purkinje cell was observed with a very similar pattern of neurodegenerative signs. interestingly, the purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in herc [ ], the structural homolog of herc . in this regard, it seems that the functional alteration of one of them it is not compensated by the other. in agreement with this independent role of herc and herc , the essential role of herc for normal development and for neurotransmission at the mouse neuromuscular junction [ ] was not observed in herc +/ mice (figure ). thus, both ubiquitin ligases seem to have a crucial and independent role in purkinje cell physiology. this functional similarity of herc and herc was unexpected, because despite their structural homology, endogenous proteins do not interact between them, do not have common known interactors, have different subcellular locations and were involved in different pathways of cell signaling [ , , ]. however, oncotarget www.impactjournals.com/oncotarget an interaction between the first , amino acid residues of herc and herc has recently been detected using mass spectrometry analysis [ ], suggesting a possible interaction in some unknown circumstances. a functional role of cerebellar p protein in adult walking synchronization has been reported [ ]. because herc positively regulates p activity [ ] and herc +/ mice had less herc activity, it was plausible that the impaired motor synchronization in herc +/ mice was caused by a decrease in p activity. for this reason, we analyzed motor coordination in p knockout mice. we did not observe significant differences with rotarod experiments in these animals (figure ). our results are in agreement with other studies where no differences in motor synchronization were detected by tomasevic et al. [ ] in p knockout mice. these authors described a role for p in the recovery of neuromotor function after a traumatic brain injury but not before the injury [ ]. thus, the motor coordination regulated by herc seems to be independent of p activity. pioneering studies associated mutations at the mouse herc locus induced by ethylnitrosourea or ionizing radiation with a runty, jerky, sterile phenotype (rjs), also known as the juvenile development and fertility phenotype (jfd ), characterized by reduced size, jerky gait, fertility problems, including spermatocyte and oocyte abnormalities, defective maternal behaviour, and reduced lifespan with juvenile lethality [ - ]. more recently, a mutation in the herc gene has been linked to the neurodevelopmental delay and dysfunction seen in angelman syndrome and autism-spectrum disorders among the amish community [ , ]. now, our study demonstrates an important role of herc in the regulation of motor coordination through purkinje cells homeostasis that would explain some features observed in rjs/jdf mice and individuals with the herc p l mutation. it is important to note that, different to the above studies, the impaired motor synchronization is observed in mice with only a mutated allele of herc (herc +/ mice), indicating the relevance of herc activity in motor function coordination. studies of spontaneous mouse mutants have implicated autophagy in the death of purkinje neurons. lurcher mice with mutations in the delta glutamate receptor, pcd mice with loss of nna expression or tambaleante mice with mutation in the herc protein show an increase of autophagy associated with purkinje cell death [ , - ]. while conditional inactivation of autophagy genes such as atg or atg from purkinje cells in mice yields purkinje cell degeneration and death [ , ]. this dual role of the autophagy in purkinje cells degeneration seems to indicate that these neurons are very sensitive to the dysregulation of autophagy. although more studies will be necessary to establish the role precise of herc in autophagy, our results are in agreement with these previous observations in other mouse models, and reveal a dysregulation of autophagy in herc +/ purkinje cells. a recent study also suggests the involvement of herc in parkinson’s disease [ ], it would be interesting to analyze in future studies the herc role in the midbrain dopaminergic neurons of herc +/ mice. in summary, the generation of a mutant mouse of herc led us to identify the herc ubiquitin ligase as essential for embryonic development and an important regulator of motor coordination. these results may also explain some features observed among the old order amish with a homozygous missense mutation in herc . future studies will be necessary to identify additional partners involved in herc physiology and its role in human diseases. materials and methods animals the es cell line ar from the sanger institute containing a gene trap β-galactosidase/neomycin (β-geo- neo) cassette that has integrated between exons and in the herc gene was used. we choose this es cell line to study herc gene expression by β-galactosidase activity and to avoid the variability of the already existing herc mutations (deletions, point mutations, dna rearrangements) [ ]. es cells were injected into . day c bl/ j blastocysts, which were then implanted into pseudopregnant females. the resulting - % coat color chimeras were crossed with c bl/ mice to generate the heterozygous animals herc /herc (herc +/ ). p knockout mice b . s -trp tm tyj/j from jackson laboratories [ ] were kindly provided by dr. j. martin- caballero. all animal experiments were performed in accordance with guidelines approved by the ethical committee for animal experimentation of the university of barcelona. genotyping tail dna or embryo samples were purified using the nucleospin tissue kit (macherey- nagel) according to the manufacturer’s protocol. pcr amplification was performed using the primers: ko ’ggctgcccagtctcgccttg ’ and ko ’ctgtcacctctccgggagaac ’ for the amplification of the herc wild-type allele; gal ’tttccatatggggattggtg ’ and gal ’tgtctgttgtgcccagtcat ’ for the herc allele; and p ’acagcgtggtggtacct ’ and p ’tatactcagagccggcct p ’ ctatcaaggcatagcgttgg for the genotype of p . the pcr settings were °c for minutes, °c for minute, annealing at °c for minute and elongation at oncotarget www.impactjournals.com/oncotarget °c for minute, for cycles. rt-pcr to assess trap expression, total rna was isolated from mice tissues or cells using trisure reagent (bioline). two µg of total rna was reverse-transcribed using the cdna reverse transcription kit (applied biosystems) and random primers. to assess the trap insertion, pcr was carried out with primers: ko ’caggtctgaccacccggagg ’; ko ’gggagttctcgattttgtgc ’; and β-geo / ’agggttttcccagtcacgac ’. the pcr settings were °c for minutes, °c for minute, annealing at °c for seconds and elongation at °c for seconds, for cycles. additionally, cdna was sequenced using bigdye . on an abi xl genetic analyzer (applied biosystems). to analyze the expression of p , rt quantitative pcr was carried out using the abi prism ht fast-real time pcr system and commercially available taqman assays (applied biosystems): cdkn a (mouse p : mm _g ; human p : hs _ m ); gapdh (mouse gapdh: mm _g ; human gapdh: hs _m ); and s (hs _s ). the pcr data were captured and analyzed using the sequence detector software (sds version . , applied biosystems). histology and immunochemistry x-gal staining of β-galactosidase activity. mice were anesthetized and perfused via the left ventricle with % paraformaldehyde in a phosphate buffer ( . m). all tissues were dissected and fixed in the same solution for hours at ºc, and later were cryopreserved by immersion in % sucrose for h at ºc. whole tissues were then embedded into an % gelatin and % sucrose solution, frozen in liquid nitrogen and stored at - ºc. before staining for β-galactosidase activity, the tissues were sliced ( - μm), prepared on polylysinated slides, and permeabilized with pbs- . % triton. after washing in pbs, the slices were incubated in staining solution ( mm x-gal, mm k fe(cn) , mm k fe(cn) , mm mgcl in pbs) overnight while shaking in the dark at °c. after washing in pbs, the slices were stained with neutral red and mounted for microscopic observation. the protocol to study the cerebellar purkinje cells was reported previously [ ]. briefly, two and nine month old mice were deeply anaesthetised with pentobarbital ( mg/kg i.p.), and perfused transcardially with % paraformaldehyde in . m phosphate buffer (pb, ph . ). after dissection, the brains were post- fixed overnight in the same fixative and transferred to % sucrose in pb until they sank. sagittal and coronal sections of the cerebellum ( µm thick) were cut on a freezing microtome, and collected in pbs. the sections were incubated overnight with a polyclonal anti-calbindin d- k antibody ( : , ). after washing, the sections were incubated for one hour in a biotinylated secondary antibody ( : ) followed by incubation for one hour in the abc elite kit ( : ). a mixture of . % dab- . % nickel sulfate- . % hydrogen peroxide in pbs was used to reveal the immunoreaction. for double labeling analyses the sections were incubated overnight with the following primary antibodies mixtures: polyclonal anti-herc ( : )/monoclonal anti-calbindin d- k ( : ), and polyclonal anti-herc ( : )/monoclonal anti-p ( : ). after washing, the sections were incubated for one hour in a mixture of alexa fluor® donkey-anti- rabbit ( : ) and alexa fluor® donkey-anti-mouse ( : ). images were acquired in a zeiss axio-imager m microscope. laser confocal analyses were made on an olympus fluoview upright microscope. electron microscopy two and nine month old mice were deeply anaesthetized with pentobarbital ( mg/kg i.p.), and perfused transcardially with a mixture of % paraformaldehyde and % glutaraldehyde in , m phosphate buffer (pb, ph . ). thereafter, the brains were dissected out and immersed overnight in the same fixative. sagittal slices of the cerebella were cut and immersed in % oso in pb, stained in a block with ethanolic . % uranyl acetate, dehydrated with an increased gradient of ethanol, and embedded in durcupan (fluka®). semithin and ultrathin sections were obtained on a leica em uc ultramicrotome. semithin sections were stained with % toluidine blue. ultrathin sections were collected in copper grids ( and mesh) and observed without counterstaining in a zeiss libra em at kv (citius). behavioural tests anxiety to evaluate mice anxiety, two different protocols were used [ ]. tail suspension: mice were suspended above the floor by fixing the end of the tail to wire netting and immobility was scored by manual observation during a min test session. time outside the dark box: mice were placed in a rectangular arena ( x x cm ) with a dark box with a door. the time inside/outside the box was scored by manual observation during a min test session. oncotarget www.impactjournals.com/oncotarget learning and memory object recognition memory mice were tested as described previously [ ]. briefly, mice were placed in a rectangular arena ( x x cm ) and two identical objects were placed in the arena during the training phase. subsequently, the animal’s memory of one of the original objects was assessed by comparing the amount of time spent exploring the novel object as compared with that spent exploring the familiar one. the relative exploration of the novel object was expressed as a discrimination index [di (tnovel - tfamiliar)/ (tnovel + tfamiliar)]. step-through passive avoidance test the test was performed as described previously [ ]. briefly, in the habituation phase, the mice were handled and allowed to move freely for min in a chamber ( x x cm , manufactured by ugo basile). in the training phase, the mice were confined to the light compartment and then s later, the door separating the dark-light compartments was opened. once mice entered the dark compartment, the door closed automatically and the mice received an electrical stimulation ( . ma, s) delivered through the metal floor. in the retention tests, the latency to enter into a dark compartment (escape latency) is a measure of information learning or memory retention. to compare the results obtained in different experiments, the fold change in escape latency with respect to the latency obtained in the training session is calculated. motor function motor activity in the open field to evaluate locomotor and exploratory activity, mice were placed for minutes in an open field ( × × cm) (cybertec s.a.). this apparatus consisted of a walled platform containing infrared emitters and sensors (ir) coupled to an altimeter, and the movement sensor was connected to a computer that recorded the number of times the mouse interrupted the ir beams/min. fore limb grip strength to evaluate fore limb strength, mice were held above a horizontal wire and lowered to allow the fore limbs to grip the wire. the ability of the mice to remain attached by the fore limbs was scored during s. rotarod to habituate mice to the rotarod (ugo basile biological research apparatus), the animals were placed on the roller at a speed of rpm until they could remain on it for one minute without falling off. to assay motor coordination, animals were then tested at a rotational speed of rpm, accelerating to rpm in increments of rpm, and quantifying the number of falls at each increase in speed. electrophysiological analysis of the medial gastrocnemius (mg) muscle in mice compound muscular action potentials (cmaps) were recorded in anesthetized mice (tribromethanol %, . ml/ g body weight, i.p.) as described previously [ , ]. briefly, the recording needle electrode was placed into the medial part of the mg muscle and the reference electrode was situated at the base of the fifth phalanx. a ground electrode was placed at the base of the tail. stimulating needle electrodes were placed at the sciatic notch and the head of the fibula. stimulation protocols of supramaximal current pulses ( . ms duration, - ma amplitude) were applied as a short train of hz pulses generated by an isolated pulse stimulator (pulse train stimulator cibertec cs- ). the outputs recorded were differentially amplified (p ac amplifier astro-med, inc), digitally acquired at , samples/s (ced plus; cambridge electronic designed, cambridge, uk) and stored on a computer for later analysis. the analysis consisted of measuring the amplitude from the positive to the negative peak of the cmaps recorded during a train of stimuli, normalizing the amplitude to the first response. cell culture and transfection human fibroblasts and ethical statements were previously described [ ]. u os cells were obtained from atcc. cells were cultured at °c in dulbecco’s modified eagle’s medium (dmem) supplemented with % fetal bovine serum, units/ml penicillin, µg/ml streptomycin and mm glutamine. transfection of cells with sirnas (non targeting, nt: uagcgacuaaacacaucaa; herc , h : gacuguagccagauugaaa) purchased from genepharma was carried out using calcium phosphate. transfected cells were analyzed hours post-transfection. mg (z-leu-leu-leu-al) (sigma-aldrich) was added to the cells for hours to a final concentration of µm. antibodies used the following antibodies were used: anti-herc monoclonal (bd biosciences); anti-herc polyclonal [ ]; anti-p (c- ); anti-p (sqstm (d- ): sc- ); anti β-actin (santa cruz biotechnology, inc.); anti-calbindin d- k polyclonal (cb- a, swant); anti- calbindin d- k monoclonal (cb- , sigma); anti-p oncotarget www.impactjournals.com/oncotarget ab- (do- ) (neo markers); anti-usp (proteintech); anti-ran [ ]; anti α-tubulin (ab- , calbiochem); alexa fluor® donkey-anti-rabbit (a ), and alexa fluor® donkey-anti-mouse (a ) (invitrogen); horseradish peroxidase-conjugated secondary antibodies (invitrogen); biotin-conjugated secondary antibodies (vector); and the avidine-streptavidine elite kit (vector). lysate and immunoblot mice were euthanized by cervical dislocation. the organs were collected and frozen in liquid nitrogen and stored at - °c until analysis. tissues, human fibroblasts or u os cells were prepared in lysis buffer (consisting of mm trishcl, ph . , mm nacl, . % np , mm β-glycerophosphate, mm naf, mm sodium vanadate, mm phenyl-methylsulfonyl fluoride, μg/ml leupeptin, μg/ml aprotinin, μg/ ml pepstatin-a and benzamidine μg/ml) and the tissues were homogenized in a motor-driven polytron pt . the lysates were incubated on ice for minutes and centrifuged at , g for minutes at °c. total protein levels were measured by bca (pierce). equal amounts of supernatant proteins were analyzed using the tris-acetate page system [ ]. band intensities were analyzed using a gel documentation system (las- , fujifilm). proteins levels were normalized and expressed as a percentage of controls. statistical analysis the results are expressed as mean±sem. the data were analyzed by one-way analysis of variance (anova) or student’s t-test. for comparison of significance, tukey’s test was used as a post hoc test according to the statistical program graphpad prism. differences were considered significant at p values of less than . : *p < . , **p < . , and ***p < . . acknowledgments we would like to thank the sanger institute for the es cell line ar , j. martin-caballero for p -/- mice, and a. gimeno, e. adanero, a. angelo, e. castaño and b. torrejon for technical assistance. we also acknowledge s. sánchez-tena for valuable discussion throughout this study, and g. alvarez de toledo for the facilities to use the fluoview laser confocal scanning microscope. rr held a juan de la cierva contract jci- - from the mineco and vppi-us from the university of sevilla. ts and lp were supported by fellowships from the capes foundation (ministry of education from brazil) and from cnpq-programa ciências sem fronteiras (ministry of science, technology and innovation education of brazil), respectively. this article is based upon work from cost action (proteostasis bm ), supported by cost (european cooperation in science and technology). conflicts of interest all authors have not conflicts of interest to disclose. grant support this work was supported by grants from spanish ministerio de ciencia e innovación [bfu - to j.l.r. and bfu - p to f.v.] and brazilian scientific program “ciências sem fronteiras” from the ministry of science, technology and innovation education of brazil [ / - to j.l.r. and / - to j.r.o.]. editorial note this paper has been accepted based in part on peer- review conducted by another journal and the authors’ response and revisions as well as expedited peer-review in oncotarget. references . williams ca, beaudet al, clayton-smith j, knoll jh, kyllerman m, laan la, magenis re, moncla a, schinzel aa, summers ja, wagstaff j. angelman syndrome : updated consensus for diagnostic criteria. am j med genet a. ; : - 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. . ruiz r, tabares l. neurotransmitter release in motor nerve terminals of a mouse model of mild spinal muscular atrophy. j anat. ; : - . . rosa jl, casaroli-marano rp, buckler aj, vilaro s, barbacid m. p , a giant protein related to the chromosome condensation regulator rcc , stimulates guanine nucleotide exchange on arf and rab proteins. embo j. ; : - . . cubillos-rojas m, amair-pinedo f, tato i, bartrons r, ventura f, rosa jl. simultaneous electrophoretic analysis of proteins of very high and low molecular mass using tris- acetate polyacrylamide gels. electrophoresis. ; : - . pgen. .. a central role for grb in regulation of islet function in man inga prokopenko , , ., wenny poon ., reedik mägi , ., rashmi prasad b , s. albert salehi , peter almgren , peter osmark , nabila bouatia-naji , , , nils wierup , tove fall , alena stančáková , adam barker , vasiliki lagou , , clive osmond , weijia xie , jari lahti , , anne u. jackson , yu- ching cheng , jie liu , jeffrey r. o’connell , paul a. blomstedt , , joao fadista , sami alkayyali , tasnim dayeh , emma ahlqvist , jalal taneera , cecile lecoeur , , ashish kumar , , ola hansson , karin hansson , benjamin f. voight , hyun min kang , claire levy-marchal , , vincent vatin , , aarno palotie , , , , ann-christine syvänen , andrea mari , michael n. weedon , ruth j. f. loos , ken k. ong , peter nilsson , bo isomaa , , tiinamaija tuomi , , nicholas j. wareham , michael stumvoll , , elisabeth widen , timo a. lakka , , claudia langenberg , anke tönjes , , rainer rauramaa , , johanna kuusisto , timothy m. frayling , philippe froguel , , , mark walker , johan g. eriksson , , , charlotte ling , peter kovacs , , erik ingelsson , , mark i. mccarthy , , , alan r. shuldiner , , kristi d. silver , , markku laakso , leif groop , *, valeriya lyssenko , * oxford centre for diabetes, endocrinology and metabolism, university of oxford, oxford, united kingdom, wellcome trust centre for human genetics, university of oxford, oxford, united kingdom, department of genomics of common disease, school of public health, imperial college london, hammersmith hospital, london, united kingdom, department of clinical science, diabetes & endocrinology, lund university diabetes centre, malmö, sweden, estonian genome center, university of tartu, tartu, estonia, university of lille nord de france, lille, france, cnrs umr , institut pasteur de lille, lille, france, inserm u , paris cardiovascular research center parcc, paris, france, department of clinical science, neuroendocrine cell biology, lund university diabetes centre, malmö, sweden, department of medical sciences, molecular epidemiology and science for life laboratory, uppsala university, uppsala, sweden, department of medicine, university of eastern finland and kuopio university hospital, kuopio, finland, mrc epidemiology unit, institute of metabolic science, university of cambridge, cambridge, united kingdom, mrc lifecourse epidemiology unit, university of southampton, southampton, united kingdom, peninsula college of medicine and dentistry, university of exeter, exeter, united kingdom, institute of behavioural sciences, university of helsinki, helsinki, finland, folkhälsan research centre, helsinki, finland, department of biostatistics and center for statistical genetics, university of michigan, ann arbor, michigan, united states of america, division of endocrinology diabetes and nutrition, department of medicine, university of maryland school of medicine, baltimore, maryland, united states of america, department of chronic disease prevention, national institute for health and welfare, helsinki, finland, department of mathematics, åbo akademi university, turku, finland, epigenetics and diabetes unit, department of clinical sciences, lund university, crc, scania university hospital, malmö, sweden, swiss tropical and public health institute, university of basel, basel, switzerland, department of pharmacology and department of genetics, university of pennsylvania perelman school of medicine, philadelphia, pennsylvania, united states of america, inserm - institut de santé publique, paris, france, inserm cic ec , hôpital robert debré, paris, france, institute for molecular medicine finland (fimm), university of helsinki, helsinki, finland, wellcome trust sanger institute, wellcome trust genome campus, cambridge, united kingdom, department of medicine, helsinki university central hospital, helsinki, finland, program in medical and population genetics and genetics analysis platform, the broad institute of massachusetts institute of technology and harvard, cambridge, massachusettes, united states of america, molecular medicine, department of medical sciences, science for life laboratory, uppsala university, uppsala, sweden, cnr institute of biomedical engineering, padova, italy, department of clinical science, internal medicine, skåne university hospital malmö, malmö, sweden, department of social service and health care, jakobstad, finland, university of leipzig, department of medicine, leipzig, germany, university of leipzig, ifb adiposity diseases, leipzig, germany, institute of biomedicine/physiology, university of eastern finland, kuopio, finland, kuopio research institute of exercise medicine, kuopio, finland, department of clinical physiology and nuclear medicine, kuopio university hospital, kuopio, finland, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom, helsinki university, department of general practice and primary health care, helsinki, finland, helsinki university central hospital, unit of general practice, helsinki, finland, oxford nihr biomedical research centre, churchill hospital, oxford, united kindom, baltimore geriatric research, education and clinical center, baltimore, maryland, united states of america, steno diabetes center a/s, gentofte, denmark abstract variants in the growth factor receptor-bound protein (grb ) gene were in a gwas meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type diabetes (t d) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. grb is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. grb knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. together, these findings suggest that tissue-specific methylation and possibly imprinting of grb can influence glucose metabolism and contribute to t d pathogenesis. the data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father. citation: prokopenko i, poon w, mägi r, prasad b r, salehi sa, et al. ( ) a central role for grb in regulation of islet function in man. plos genet ( ): e . doi: . /journal.pgen. editor: scott m. williams, dartmouth college, united states of america received june , ; accepted january , ; published april , this is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. the work is made available under the creative commons cc public domain dedication. plos genetics | www.plosgenetics.org april | volume | issue | e http://crossmark.crossref.org/dialog/?doi= . /journal.pgen. &domain=pdf funding: studies at ludc in malmö (dgi, bps, ppp, mpp and human islets) were supported by grants from the swedish research council (sfo exodiab: dnr - ), linnaeus grant (ludc dnr - - ), project grants to vly, lg, nw, and cli, and financial support from the swedish research council (dnr - - , dnr - - , dnr - - , dnr - - , dnr - - , andis dnr - - ), the knut & alice wallenberg foundation (kaw . ), the torsten och ragnar söderbergs stiftelser (mt / ), the inga britt och arne lundberg’s research foundation (grant nr. ), the heart and lung foundation, the novo nordisk foundation, diabetes research & wellness, sydvästra skånes diabetesförening. the ppp-botnia study was supported by grants from the sigrid juselius foundation, the finnish diabetes research society, the signe and ane gyllenberg foundation, the swedish cultural foundation in finland, the ollqvist foundation, the foundation for life and health in finland, jakobstad hospital, the medical society of finland, the närpes research foundation and the vasa and närpes health centers. the mpp study was supported by a grant from the swedish heart and lung foundation, the e. lundström foundation and from the region skåne county council funds. the risc study was supported by european union grant qlg -ct- - and astrazeneca. the amish family diabetes study was supported by nih research grants r dk , r dk , u hl , the university of maryland general clinical research center (m rr ), hopkins bayview general clinical research center (m rr ), the mid-atlantic nutrition obesity research center (p dk ), the baltimore diabetes research and training center grant (p dk ), the baltimore veterans administration geriatric research and education clinical center, and by usda nifa nri competitive grant - - . the dr’s extra study was supported by grants to rr by the ministry of education and culture of finland ( ; - ), academy of finland ( ; ), kuopio university hospital, finnish diabetes association, finnish heart association, päivikki and sakari sohlberg foundation and by grants from european commission fp integrated project (exgenesis), lshm-ct- - , city of kuopio and social insurance institution of finland ( / / ). the metsim study was supported by grants from the academy of finland ( , ), diabetes research foundation, and sigrid juselius foundation. the ulsam project was supported by grants from the swedish research council, the swedish heart-lung foundation, the swedish foundation for strategic research, the royal swedish academy of sciences, swedish diabetes foundation, swedish society of medicine, and novo nordisk foundation. part of the genotyping in ulsam for this project was supported through the engage (european network for genetic and genomic epidemiology) consortium, which is funded through the european community’s seventh framework programme (health-f - - ). the snp & seq technology platform in uppsala was supported by science for life laboratory and the swedish research council for infrastructures (rfi). the sorbs work was supported by grants from the german research council (dfg - sfb ‘‘obesity mechanisms’’; a , b , c ), from the german diabetes association and from the dhfd (diabetes hilfs- und forschungsfonds deutschland). ifb adiposity diseases is supported by the federal ministry of education and research (bmbf), germany, fkz: eo . pk is funded by boehringer ingelheim foundation. rm acknowledges financial support from the european commission under a marie curie intra-european fellowship, efsd new horizons grant, and development fund of the university of tartu, in the frame of the centre of transitional genomics (grant sp gvareng) and by estonian government (grant #sf s ). the research of ip and vla was funded in part through the european community’s seventh framework programme (fp / - ), engage project, grant agreement health-f - - . helsinki birth cohort study (hbcs) has been supported by grants from the academy of finland (grant no. and to jge), the finnish diabetes research society, folkhälsan research foundation, novo nordisk foundation, finska läkaresällskapet, liv och hälsa, the wellcome trust (grant no. /z/ /z and /z/ /z), samfundet folkhälsan, finska läkaresällskapet, the signe and ane gyllenberg foundation, the univeristy of helsinki, ministry of education, ahokas foundation, emil aaltonen foundation, european science foundation (eurostress), juho vainio foundation, and wellcome trust (grant wt ). mim is a wellcome trust senior investigator, and an nihr senior investigator, and has also received funding from eu framework : engage consortium health-f - - , wellcome trust: , , , , medical research council: g and diabetes uk: rd / . the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. competing interests: i have read the journal’s policy and we have the following conflicts: vly declares that steno diabetes center is owned by novonordisk, pk is funded by boehringer ingelheim foundation, tf received payment for lectures from msd (merck), am is a consultant for eli lilly and poxel and has received grants from eli lilly and boehringer ingelheim, bi has received payment for lectures from boehringer ingelheim, msd, novo nordisk farma and astrazeneca, lg has been a consultant for and served on advisory boards for sanofi-aventis, gsk, novartis, merck, tethys bioscience and xoma, and received lecture fees from lilly and novartis. we confirm that these affiliations do not alter the adherence to all the plos genetics policies on sharing data and materials. * e-mail: leif.groop@med.lu.se (lg); vlly@steno.dk, valeriya.lyssenko@med.lu.se (vl) . these authors contributed equally to this work. introduction type diabetes (t d) is a multifactorial polygenic disease, in which genes interact with environmental and genetic factors to unmask the disease. to date, genome-wide association studies (gwas) have identified more than variants increasing risk of t d [ – ]. many of the identified variants seem to influence the capacity of b-cells to cope with increased insulin demands imposed by insulin resistance [ , ]. despite this, no gwas to date has explored the extent to which common genetic variants influence dynamic measures of insulin secretion and action. therefore, we have here performed the first large-scale meta-analysis for glucose- stimulated insulin secretion (gsis) during an oral glucose tolerance test (ogtt). association analyses included a gwas- based discovery stage and a replication stage using a custom- designed iselect cardiometabochip array containing , snps overlapping with the discovery gwas imputed from the hapmap reference panel in up to , non-diabetic individ- uals. we identified variants in the growth factor receptor-bound protein (grb ) gene (ncbi gene id: ) to be associated with impaired b-cell function at a genome-wide significant level (p, ). since grb has been shown to have parent-of- origin specific effects on expression in various tissues [ , ], we investigated the transmission patterns of the risk alleles and their effects on insulin and glucose levels during an ogtt, risk for t d, expression of grb and methylation status in human pancreatic islets, as well as evaluated the effects on islet function through disruption of grb in human pancreatic islets. results meta-analysis of gwas and cardiometabochip studies for insulin secretion and action during an ogtt we conducted a meta-analysis for dynamic measures of insulin response to glucose during an ogtt using gwas, cardiometa- bochip and de novo genotyping. the combined analysis in up to , non-diabetic individuals provided genome-wide significant association (p, ) with insulin secretion measured as correct- ed insulin response (cir) to glucose at min during an ogtt for the locus within the grb gene (lead snp rs located in intron ) (figure a, b), and at previously reported t d and glycemic trait variants, including mtnr b, hhex/ide/kif , cdkal , gipr/qpctl, c cd a (nlf ), gck and ank (table , table s a, figure s , s ). these associations remained virtually unchanged when cir was adjusted for insulin sensitivity (disposi- tion index) (table s a). in addition, nominal (p, . ) association with reduced insulin response to glucose (cir) during the ogtt was seen for the risk alleles in out of reported t d loci [ ], as well as for glucose and insulin loci out of reported being associated with these traits [ ]. notably, the risk alleles in of them (ankrd , grb , ppp r b, irs and arap (fasting glucose variant)) were associated with higher insulin response to glucose (p, . ) (table s b). in line with a previous report [ ], we confirmed the association of the index grb snp rs with fasting glucose levels (n = , , b = . , p = . ) (figure s ). however, we did not observe a significant effect of snp rs , previously grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e associated with glucose concentrations and risk for t d in an amish population [ ]. since grb is differentially expressed when transmitted from mothers and fathers, we explored whether the grb variant would have sex-specific effects on insulin and glucose levels. the sex- stratified analysis showed a greater insulin-reducing effect of the grb variant in women (cir: n = , ; b = . . ; p = . ) than in men (cir: n = , ; b = . . ; p = . ; sex heterogeneity p = . ) (figure c, d). complex pattern of genetic inheritance for variants in the grb gene: evidence for parent-of-origin effects given the complex parent-of-origin imprinting pattern de- scribed for grb , we next turned to families to explore in detail the inheritance patterns of identified variants and their potential effect on risk of t d and insulin/glucose levels. using a cohort of , parents-offspring trios with , individuals from finland and sweden, we first performed a transmission disequilibrium test, taking into account the parental phenotype being either t d or not, to increase power (parentdt) [ ]. we also used gene- dropping permutation to control for stratification and the depen- dence of related individuals [ ], or restricting the analysis to independent trios, including only the youngest affected offspring (n = , ; with t d) and oldest unaffected offspring (n = , ; unaffected). we observed an increased transmis- sion of the a-allele of rs from parents to diabetic offspring (p = . ) (table s a-i), particularly from diabetic fathers (p = . ) (table s a-v), and the g-allele was preferentially transmitted from non-diabetic parents to non-diabetic offspring (p = . ) (table s a-ii). in accordance with these findings, when simply counting transmission of the a- and g-alleles, we observed an increased transmission of the major a-allele of rs from a diabetic parent to a diabetic offspring (chi-square p = . ) (table s a-v). this effect was even stronger when we relaxed the definition of hyperglycemia to include ifg and igt in addition to t d (p = . ) (table s a-vi). we observed a similar pattern of an increased transmission of the t-allele of rs (ld with rs ; d = . , r = . ) to a hyperglycemic offspring (ifg/igt/t d) (p = . ) (table s a-iii). this latter association was confirmed using the family based association test (fbat) [ ] (p = . ), which can accommodate any type of genetic model and family construction. consistent with previous findings [ ], we confirmed the association of the snp rs with fasting glucose levels ( , nuclear families, p = . ) (table s a-vii). to explore whether grb rs would show a stronger effect on insulin secretion when inherited from either parent, we examined its effect on gsis in , non-diabetic individuals from parents-offspring trios from finland and sweden [ ] and usa [ ]. in these families, the maternally transmitted a-allele of rs was associated with reduced gsis (cir b = . , p = . ; ins adjbmi b = . , p = . ; ins b = . , p = . ; aucins b = . , p = . ; aucins/aucgluc b = . , p = . ) (figure a, b, table s b). no significant effect was observed for the paternally transmitted a-allele on gsis. surprisingly, the maternally transmitted a-allele was associated with reduced rather than elevated fasting glucose levels (b = . , p = . ). in contrast, the paternally transmitted a- allele was associated with elevated glucose levels (b = . , p = . ) (figure c, d, table s b). thereby, the a-allele of rs exerted virtually opposite effects on glucose metabolism if transmitted from the father than the mother. it is very likely that the association with risk or protection from t d would be missed or diluted in any traditional association study, which does not take the transmission pattern into account. in support of this, we did not observe any association between the snp rs and t d in , non-diabetic individuals, of whom , developed t d during a mean -year follow-up period (table s ) [ ]. also, in the recent diagram+ meta- analysis, none of the evaluated grb snps were associated with t d [ ]. a potential explanation for the paradoxical reduction in glucose levels despite reduced insulin secretion could be that the variant also enhances insulin sensitivity or reduces glucagon levels. in fact, the maternally transmitted a-allele was associated with enhanced, whereas the paternally transmitted a-allele was associated with decreased insulin sensitivity as measured by isi during the ogtt (p, . for difference between parental alleles). although we could not observe any significant effect of rs on fasting or hr glucagon levels in a finnish cohort with glucagon data available (table s b), we identified several grb snps from the same haplotype block which were in weak ld with rs and nominally (p, . ) associated with fasting and hr glucagon levels in the dgi gwas (table s ). unfortunately, there was no glucagon data available for the trios. effects of grb rs on gene expression grb protein was detected in human a-, b- and d-cells by immunofluorescence (figure a). we observed strong expression of grb mrna in total human islets, with no significant difference between islets from normoglycemic and hyperglycemic individuals (figure s a), or between carriers of different grb genotypes (figure s b). while we did not observe any correlation between the amount of grb and ins (insulin) mrna, nor between grb mrna and in vitro gsis, there was an inverse correlation between grb and gcg (glucagon) mrna in human pancreatic islets (all donors: rho = . , p = . ; normoglycemic: rho = . , p = . ; hyperglycemic: rho = . , p = . ), suggesting that higher grb expression is associated with lower glucagon (figure s c). author summary in this paper, we report the first large genome-wide association study in man for glucose-stimulated insulin secretion (gsis) indices during an oral glucose tolerance test. we identify seven genetic loci and provide effects on gsis for all previously reported glycemic traits and obesity genetic loci in a large-scale sample. we observe paradox- ical effects of genetic variants in the growth factor receptor-bound protein (grb ) gene yielding both reduced gsis and reduced fasting plasma glucose concentrations, specifically showing a parent-of-origin effect of grb on lower fasting plasma glucose and enhanced insulin sensitivity for maternal and elevated glucose and decreased insulin sensitivity for paternal transmissions of the risk allele. we also observe tissue- specific differences in dna methylation and allelic imbal- ance in expression of grb in human pancreatic islets. we further disrupt grb by shrna in human islets, showing reduction of both insulin and glucagon expression and secretion. in conclusion, we provide evidence for complex regulation of grb in human islets. our data suggest that tissue-specific methylation and imprinting of grb can influence glucose metabolism and contribute to t d pathogenesis. the data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father. grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e figure . gwas plots identifying grb rs . genome-wide quantile-quantile (q-q) (a) and manhattan (b) plots for cir in the meta- analysis. regional plots of identified grb genome-wide significant association for cir in men (c) and women (d). directly genotyped and/or imputed snps of grb are plotted with their meta-analysis p-values (as log values) as a function of genomic position (ncbi build ). in each grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e although there was no effect of rs on total grb mrna expression in human islets, we cannot exclude that the variant could influence splicing or methylation, especially as different transcriptional start sites (un , un a and un ) and tissue-specific expression have been described for the grb gene (figure b, s ) [ ]. we tested for allelic imbalance, i.e. deviation from the expected equal expression of both alleles. for this purpose, we used the snp rs (arg) located in exon as a reporter snp, as it is the nearest coding variant located kb from the index snp rs (d = , r = . ). this reporter snp indicated a clear allelic imbalance with a- to g-allele ratios ranging from % to % in pancreatic islets (table s ). we therefore examined whether the observed allelic imbalance could be related to specific transcripts arising from different promoters. transcripts containing exon un were monoallelically expressed from either the a- or g- allele, indicating imprinting of the promoter giving rise to the un transcripts (pun ) from one parent. until now, paternally expressed (i.e. maternally imprinted) un transcripts have only been observed in the brain [ ]. this differential imprinting was recurring in all tissues analyzed (figure s ). our findings extend this expression pattern to human islets. in contrast, transcripts containing exon un showed great variation (from % to %), but were mainly expressed from the other allele than those containing exon un (figure b, c), in line with the maternally expressed/paternally imprinted transcripts observed in placental trophoblasts [ ]. it can be hypothesized that these snps might regulate usage of alternative promoters and thereby influence the expression of grb . the opposite effects of maternally and paternally inherited rs allele could then be attributed to different effects of rs on the promoters pun and pun , e.g. especially if promoter preferences differ strongly between a- and b-cells. although allelic imbalance is an attractive model to explain differences in expression of different grb transcripts, as well as the observed differences in effects of the a-allele on risk of diabetes in the offspring when transmitted from father or from mother, the above data can only point at this possibility, as we did not have enough human islets for this kind of analysis. we therefore also tested for allelic imbalance using an alternative method, i.e. by comparing data from exome and rna sequencing. we found that another coding variant, snp rs , in the grb gene was associated with allelic imbalance in human pancreatic islet samples (p, . , fisher’s exact test). since grb is imprinted and methylated in humans and rodents in a tissue-specific fashion [ , , ], we studied whether grb would be methylated in human islets or in dna from human peripheral blood lymphocytes (pbl) and whether the degree of the dna methylation would correlate with gene expression in human islets. we found tissue-specific differences in dna methylation of grb in human islets compared to pbl and the degree of methylation in the region analyzed with the sequenom massarray epityper ranged from . % to . % (figure d). although we did not observe any significant effect of rs on the degree of dna methylation in human islets (figure s a), there was a nominal association between rs and dna methylation in pbl in the region analyzed using epityper (p = . ) (figure s b). moreover, we observed an inverse correlation between dna methylation at a cpg site located . kb downstream of rs and grb mrna expression (n = , rho = . , p = . ) (figure e) in human islets, particularly in islets from diabetic donors (n = , rho = . , p = . ), and at a cpg site , bp downstream from rs (n = , rho = . , p = . ) (figure s c), suggesting that decreased methylation in this region is associated with increased grb mrna. panel, the strongest associated snp is represented by a purple diamond (with meta-analysis p-value). estimated recombination rates (taken from hapmap) are plotted to reflect the local linkage disequilibrium structure around the associated snps and their correlated proxies (according to a blue to red scale from r = to , based on pairwise r values from hapmap ceu). gene annotations were taken from the university of california, santa cruz, genome browser, as implemented in locuszoom [ ]. doi: . /journal.pgen. .g table . snps associated with primary insulin secretion traits at genome-wide significance levels. snp nearest gene n alleles (effect/other) freq (effect allele) cir aucins/aucgluc effect se p effect se p identified in the present study rs grb , a/g . . . . . . . previously reported loci rs mtnr b , g/c . . . . . . . rs hhex/ide , c/t . . . . . . . rs cdkal , g/a . . . . . . . rs gipr , a/g . . . . . . . rs c cd a (nlf /vps c) , t/c . . . . . . . rs gck , t/g . . . . . . . rs ank , a/g . . . . . . . the results are from the meta-analyses of the discovery gwas, cardiometabochip and de novo genotyping. results are reported for the directly genotyped and imputed snps tested for association with insulin secretion measured as cir and aucins/aucgluc (trait abbreviations are listed in the methods ‘‘phenotype definition’’ section). freq denotes the allele frequency of the insulin secretion-reducing allele. n = sample size. since the index snp rs (a/g) from the discovery gwas was not present on the cardiometabochip platform, a variant (rs (c/t)) in strong ld with the former (r = . ) was used as a proxy snp for the meta-analyses. doi: . /journal.pgen. .t grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e expression of grb in human muscle and adipose tissue given that we observed differences in insulin sensitivity when the risk a-allele of snp rs was inherited from the mother compared to the father, and that grb is an inhibitor of insulin signaling, we also explored whether the snp would influence expression of the grb gene in human skeletal muscle and adipose tissue [ , ]. we observed that carriers of the a-allele had decreased grb mrna level in muscle (n = , b = . , p = . ) and adipose tissue (n = , , b = . , p = . ). effects of grb on islet function to gain insight into the mechanisms by which grb influences pancreatic b- and a-cell function, we disrupted grb expression in rat insulinoma ins- cells by sirna and in human islets by shrna achieved by lentiviral transfection. there was a clear reduction in gsis after sirna-disruption of grb in the ins- cell line lacking glucagon (figure a). in human pancreatic islets, decreased grb expression resulted in a reduction of both insulin and glucagon secretion and expression (figure b, c). in addition, grb knock-down was also associated with a decrease in forskolin- and k + -stimulated glucagon secretion (figure s a). grb has been reported to both increase [ ] and decrease [ ] apoptosis in islets, in addition to its effects on insulin signaling [ ]. disruption of grb was associated with a significant reduction in the number of viable human pancreatic islets, as assessed by an mts technique, supporting recent data in mice using shrna to disrupt grb in pancreatic islets [ ] (figure s c). discussion the present study provides mechanistic insights into the role of grb in the regulation of islet function and glucose metabolism in man. first, the a-allele of the grb rs variant was associated with different effects on insulin secretion, sensitivity and glucose concentrations, if transmitted from fathers or mothers. second, disruption of grb in human islets resulted in a reduction of both insulin and glucagon secretion, the latter of which can provide an additional explanation for the reduction in glucose levels despite reduced insulin levels. third, metabolic effects of grb on glucose homeostasis involved tissue-specific methylation and parental imprinting. grb is an adaptor protein, which interacts with a number of receptor tyrosine kinases and signaling molecules and serves as a down-regulator of insulin receptor activity [ ]. grb partici- pates in phosphorylation and activation of the mtorc protein, which is a central regulator of cellular metabolism, growth and survival [ ]. studies in mice have shown an abundant expression of grb in brain, fat, muscle and heart, and with the highest expression in pancreas [ ]. an interesting observation in the present study was that the common variant rs in the grb gene was associated with reduced gsis, enhanced insulin sensitivity and reduced glucose levels when inherited from the mother. on the contrary, the figure . parent-of-origin effect of grb rs on insulin secretion and glucose levels. (a) no significant effect for cir was observed from the paternally transmitted a-allele. (b) carriers of the maternally transmitted a-allele showed lower cir compared to the g-allele. (c) carriers of the paternally transmitted a-allele had elevated fasting plasma glucose levels, whereas (d) the maternally transmitted a-allele was associated with lower fasting plasma glucose levels. fin-swe = trios from finland and sweden, amish = amish family diabetes study, kuopio = kuopio offspring study. doi: . /journal.pgen. .g grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e paternally transmitted allele was associated with elevated glucose level and increased risk for t d. these findings might partially explain the modest or lack of effects seen in several studies for variants in the grb gene on t d risk [ ]. the effect on insulin sensitivity is indirectly supported by the view that carriers of the a-allele showed decreased expression of grb in skeletal muscle and adipose tissue. figure . grb expression in human islets. (a) immunostainings demonstrating that grb (green, panel a, e) is abundantly expressed in b- (panel b, d), a- (panel c, d) and d-cells (panel f, g) in human pancreatic islets. arrows indicate co-localization with insulin (panel d) and somatostatin (panel g). arrowheads indicate co-localization with glucagon (panel d). scale bar = mm. (b) schematic representation of the grb gene and snps investigated in the present study. grey boxes = untranslated exons. black boxes = translated exons. (c) examples of rt-pcr on islet cdna (top six rows) and pcr on genomic dna (gdna, bottom row) from two individuals heterozygous for the reporter snp rs . the first column states the forward primer location of each pcr and a forward primer in exon captures all transcripts. the peaks show the sanger sequencing trace across rs , which is underlined (a: green trace, g: black trace). percentages indicate the contribution from the paternal allele (p.a.) (g-allele in the first case, a-allele in the second case). the paternal genotype is identified assuming complete maternal imprinting of the un promoter, in line with previous findings [ ]. a sequence of heterozygous genomic dna (gdna) is shown on the bottom for comparison ( %- %). (d) to study if dna methylation of grb is tissue-specific, the degree of methylation was analyzed at cpg sites located , . kb downstream of rs in both human pancreatic islets of donors and pbl from trios using epityper. the exact position of each analyzed cpg site in relation to rs is given in the figure. data are presented as mean sem. * p, . for difference in methylation between human islets and pbl. (e) the grb mrna levels correlated negatively with the degree of methylation at the cpg site located , bp downstream of grb rs . doi: . /journal.pgen. .g grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e another potential explanation could be the effect of grb on glucagon secretion. unfortunately, due to lack of samples for glucagon measurements, we were not able to test this directly in the trios. to further explore whether grb influences glucagon secretion/expression and thereby mechanisms by which grb might influence glucose metabolism, we disrupted gbr in human pancreatic islets. while grb knock-down in the rat insulinoma cell line ins- lacking glucagon resulted in marked reduction of insulin, grb knock-down in human pancreatic islets resulted in reduction in both insulin and glucagon secretion and expression. the effect on glucagon seen in human islets is in line with data from mice, showing reduced glucagon levels after grb disruption using shrna [ ]. since rs is located in intron of the grb gene, one could envision an effect on gene expression. this could be a possible mechanism, as monoallelic and isoform-specific expres- sion of grb has been reported in fetal brain, skeletal muscle and in the placental trophoblasts [ , ]. unfortunately, we could not observe any effect of the snp on expression of the grb transcript in islets, nor could we examine parent-of-origin effects in the islets from human cadaver donors, as we lacked information on the parents. however, using two different strategies we could demonstrate allelic imbalance for snps in the grb gene, leaving this possibility open. in line with earlier findings, we also identified three transcripts arising from alternative gbr promoters, with the paternally expressed un isoform being imprinted from one parent in islets. however, there is sufficient amount of the maternally expressed un a isoform in islets to allow an effect of the maternally transmitted allele, which could contribute to the stronger effect of the maternal allele as described above. genetic imprinting is usually a consequence of increased dna methylation. in line with previous reports [ , ], we observed tissue-specific methylation of grb , showing highest degree of methylation in human pancreatic islets, which was associated with decreased expression of grb . although this relationship was not influenced by the snp in human pancreatic islets, there was a tendency for an effect of the snp on degree of methylation in pbl. our study has some limitations. despite large sample size for measurements of insulin secretion, we did neither have samples with measurements of glucagon in the trios, nor did we have parental information for islet donors. also, tissue limitations prevented us from directly exploring methylation patterns and imprinting in trios. finally, although this is the largest meta- analysis for insulin secretion to date, we discovered only one novel locus influencing gsis. while this could be a consequence of limited power, another plausible explanation is that many homeostatic mechanisms, as well as parent-of-origin specific effects, would dilute and neutralize such effects. in conclusion, our data demonstrate a complex genetic regulation of grb function in human islets with different effects of paternally and maternally transmitted alleles. together, these findings suggest that tissue-specific methylation and possibly imprinting of grb can influence glucose metabolism and contribute to t d pathogenesis. the data also emphasize the need in genetic studies to consider whether disease risk alleles are inherited from the mother or the father. methods ethics statement all studies were approved by local research ethics committees, and all participants gave informed consent. all procedures in human islets were approved by the ethics committees at the uppsala and lund universities and informed consent obtained by appropriate measures from donors or their relatives. figure . effects of disrupted grb through knock-down on islet function. (a) disrupted grb in ins- rat b-cells markedly reduced glucose-stimulated insulin secretion. (b) grb knock-down showed reduced glucose-stimulated insulin secretion at mm glucose and glucagon secretion at mm glucose in human pancreatic islets (ninsulin = , nglucagon = donors of human pancreatic islets; – measurements in each experiment for each donor). (c) grb knock- down resulted in a reduction of insulin and glucagon mrna expression (n = donors of human pancreatic islets; measurements in each experiment for each donor). * p, . ; ** p, . , *** p, . . error bars denote sem. doi: . /journal.pgen. .g grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e meta-analyses of genome-wide association and cardiometabochip studies association analyses of insulin secretion and action traits were performed within cohorts participating in the meta-analysis of glucose- and insulin-related traits consortium (magic) in a total of up to , individuals. in the discovery stage , we performed a meta-analysis of gwass (diabetes genetics initiative (dgi), amish family diabetes study, sorbs, helsinki birth cohort study (hbcs), french obese adults, and relationship between insulin sensitivity and cardiovascular disease study (risc)) for glucose- stimulated insulin secretion (gsis) during an oral glucose- tolerance test (ogtt) at time points (fasting, min, min) for primary traits measured as ( ) insulin response to glucose after the first min estimated as corrected insulin response (cir), and ( ) overall insulin response to glucose estimated as area under the curve (auc) for insulin over a total auc for glucose (aucins/aucgluc) in up to , non-diabetic individuals (table s a). as none of the traits gave genome-wide significant association, we selected the top independent signals from both primary and secondary traits (see ‘‘phenotype definition’’ below) after ld pruning (r , . ). signals prioritized for replication were ranked by the number of associations observed at primary traits and/or secondary traits, association p-value and number of times the signal was observed across the traits (more than ). we selected snps for replication genotyping and follow-up analyses, out of which loci were based on biological relevance: grb [ ] (rs , discovery p-value (cir) = . ), ucn (rs , discovery p-value (ins adjbmi) = . ) and inadl (rs , discovery p-value (aucins/aucgluc) = . ). replication stage a de novo genotyping was undertaken in five population- based studies (botnia-ppp, ulsam, metsim, bps and hague- nau; only gwas index snp rs in the latter three; max n = , ) (table s a, s a). replication stage b in silico was undertaken using an iselect cardiometabochip array (cm) (illumina, san diego, ca, usa) to genotype data in independent population-based studies (botnia-ppp, ulsam, ely, dr’s extra and metsim) including up to , individuals (table s a). the gwas/cm (stage and stage b) data including , snps were pooled together with the de novo genotyping results from stage a for non-overlapping individuals. in this meta-analysis, we defined all independent (r , . ) genome-wide significant (p- value, ) association signals for insulin secretion traits at genomic loci (table ). phenotype definitions the primary insulin secretion and action indices were: (i) corrected insulin response (cir) = ( insulin at min)/ (glucose at min (glucose at min– . )), and (ii) ratio of the area under the curve (auc) for auc insulin/auc glucose (aucins/aucgluc, mu/mmol) calculated using the trapezium rule [ ]. insulin sensitivity index (isi) = , /! (fasting plasma glucose (mg/dl) fasting insulin mean glucose during ogtt (mg/dl) mean insulin during ogtt). secondary insulin secre- tion and action indices during ogtt were: (i) disposition index (di) = cir isi; (ii) insulin at min (ins ); (iii) incremental insulin at min (increm ) = insulin min – fasting insulin; (iv) insulin response to glucose during the first min adjusted for bmi (ins adjbmi) = insulin at min/(glucose at min bmi); (v) area under the curve (auc) of insulin levels during ogtt (aucins, mu*min/l). individuals with missing data on any of the three time points included in the auc calculation were excluded. statistical analysis linear regression models were used for association of pheno- types (z-score residuals of insulin secretion and action traits) with genotypes coded additively. discovery (stage ) gwas analyses were carried out using a statistical tool that was able to account for genotype uncertainty, snptest [ ], or by using allele dosages in the linear regression model in mach qtl [ , ], probabel [ ], corrected for residual inflation of the test statistics using the genomic control method [ ]. the meta-analyses of effect sizes were performed with the fixed-effect inverse-variance method using gwama [ ]. the gc correction was applied only once to cohort-specific results before including them into the meta- analyses. sex-differentiated analyses were performed using gwama, with an assumed heterogeneity p-value of , . . effect sizes for glucose levels were estimated using a fixed-effect model using the metaphor package for r version . . (http:// www.r-project.org/). parent-of-origin effect analysis on insulin secretion and glucose levels the trios from finland and sweden, amish family diabetes study and kuopio offspring study (table s a, s a, b) consisted of a father, a mother and an offspring. genotype phase was determined using merlin and then analyzed using solar, which uses the kinship matrix to account for family. meta-analysis on insulin secretion and action and glucose levels during ogtt (as earlier described) was performed using a fixed-effect model. analyses were performed on ibm spss statistics . (ibm corp., chicago, il, usa), r version . . with the metaphor package (http://www.r-project.org/) and mmap (mmap: mixed models analysis for pedigrees and populations, http://edn.som. umaryland.edu/mmap/index.php). the transmission disequilibrium test (tdt) used to compare frequencies of transmission of the two alleles from heterozygote parents to an affected offspring was performed using plink (http://pngu.mgh.harvard.edu/purcell/plink/) [ ] (table s a). the deviations from mendelian transmissions were assessed and the power of the test was enhanced by incorporating information from phenotypically discordant parents (parentdt) [ ]. to confirm the association, another independent test was performed, which can accommodate any type of genetic model and family construction, i.e. the family based association test (fbat) [ ]. quantitative traits related to glucose metabolism and insulin secretion were assessed in non-diabetic individuals using qtdt and parent-of- origin effect tests. ibd estimates were calculated using merlin. permutations were performed using qfam (plink). ogtt values were natural log-transformed and adjusted for bmi. association of grb rs with glucagon levels the botnia prevalence, prediction and prevention of diabetes (botnia-ppp) study is a population-based study from the botnia region of western finland and has previously been described [ ]. for this study, we selected , non-diabetic individuals above the age of . linear regression analysis assuming an additive genetic risk model was performed to evaluate genotype-phenotype association. hyperglycemic individuals were identified based on previous diagnosis, fasting plasma glucose levels of . – . mmol/l and hr plasma glucose levels of . – . mmol/l (table s ). association of grb rs with grb mrna in human muscle a subgroup of men with igt at screening visit selected from the mpp study participated years later in more extensive grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e http://www.r-project.org/ http://www.r-project.org/ http://www.r-project.org/ http://edn.som.umaryland.edu/mmap/index.php http://edn.som.umaryland.edu/mmap/index.php http://pngu.mgh.harvard.edu/purcell/plink/ metabolic studies, including a new ogtt, a euglycemic-hyperin- sulinemic clamp combined with indirect calorimetry and infusion of [ - h]glucose [ , ]. the men were similar in age, but had varying degrees of glucose tolerance; were in the normal range, had ifg and/or igt, and had t d. t d patients were either treated with diet alone ( %) or with oral hypoglycemic agents, which were withheld the day before the test. microarray expression data were analyzed as previously described [ ]. association of grb rs with future risk for t d the malmö preventive project (mpp) is a large population- based prospective study from the city of malmö, sweden, and has previously been described [ ]. for this study, we selected , non-diabetic subjects, of whom , developed t d during a . year mean follow-up period. the odds ratio for risk of developing t d was calculated using logistic regression analysis assuming an additive genetic risk model. the analysis was adjusted for age, sex, bmi, participation period and an interaction term (participation period x sex) [ ]. ibm spss statistics . (ibm corp.) was used for the statistical analysis. human pancreatic islets islets from cadaver donors were provided by the nordic islet transplantation program (www.nordicislets.org) by the courtesy of prof. olle korsgren, uppsala university, sweden. the microarray experiments (human gene . st whole transcript) were performed on islets isolated from normoglycemic (mean sem; age . . yrs, bmi . . kg/m , hba c . . %) and hyperglycemic (age . . yrs, bmi . . kg/m , hba c . . %) islet donors. rna products were fragmented and hybridized to the genechip human hg u a array (affymetrix, santa clara, ca, usa) [ ]. statistical analyses of expression data were performed using two-tailed spearman’s t- test. immunocytochemistry immunocytochemistry with antibodies for grb (k ) (code sc- , santa cruz biotech. inc., ca, usa), insulin, glucagon and somatostatin was performed on human pancreatic sections as previously described [ ]. rna sequencing of human islets rna from human pancreatic islets donors ( with hba c , . % and donors with hba c . . %) was isolated and purified using mirneasy kit (qiagen, hilden, germany). as quality thresholds for rna samples, we demanded rin values . , s/ s ratio . . and the absorbance ratios / . . and / . . sample preparation for sequencing reactions was performed using trueseq sample prep kit (illumina). fragmentation was performed using inbuilt fragmentation in the sample prep kit to obtain fragments of approximately bp in length. sequencing was performed on the illumina hiseq platform. obtained sequenced reads were transformed into .qseq files using the illumina pipeline. alignment of the reads was performed using the tophat short read aligner (tophat.cbcb.um- d.edu). cufflinks (cufflinks.cbcb.umd.edu) was used for splice variant calling. identification of grb splice variants and measurement of allelic imbalance analytical rt-pcrs were performed on cdna from human islet, visceral fat, subcutaneous fat, liver and muscle in ml reactions using . ml amplitaq gold pcr master mix (applied biosystems, foster city, ca, usa) supplemented with . ml dmso and . mmol/l of forward and reverse primers. the following primers were used in various combinations: un fw: caaacgcctgcctgacgactg, un afw: gcccggga- cagtcttgagc, un fw: ggcgcacacgcagcgac, un fw: accacctacatcagagctgactgcc, bfw: cctgggct- accctctgcttc, fw: gcctgtactcggcctgcagc, fw: gcccctacagaccacgggct, fw: gctgtccccgttc- tcgacgc, rv: atgtgcacaggctgggagcg, rv: ctgg- ctgtcatgtctgct, rv: ctgctgagggattcggt, rv: ggatgcagtggtgcttga, the names referring to the target exon. pcr reactions were carried out with uc annealing temperature and over cycles. products were analyzed on % agarose. prior to sequencing, . ml pcr product was treated with . ml exosap-it (usb, cleveland, oh, usa) at uc for min followed by deactivation at uc for min. subsequently, ml was sequenced in both directions using bigdye . according to the manufacturer’s protocol (applied biosystems). the sequence reactions were purified and analyzed by gatc biotech ag (konstanz, germany). allelic imbalance measurements were performed by rt-pcr using the reverse primer rv and either of the forward primers un afw, un fw, un fw, un fw, bfw or fw in samples heterozygous for the common snp rs (grb exon ). the individual contribution from each allele was measured at the position of rs using sanger sequence traces and the software mutation surveyor (soft genetics, pa, usa). allelic imbalance in grb was also detected by a different method: after extensive quality and coverage filtering, we did a fisher exact test for comparing the ratio of reference/alternative alleles in the exome sequencing vs. rna-seq for each sample. exome sequencing was performed using the illumina exome sequencing protocols (truseq dna sample preparation kit v ). methylation studies sequenom’s massarray epityper protocol was applied to measure dna methylation (sequenom, san diego, ca, usa) in human islets of donors and peripheral blood lymphocytes (pbl) of diabetic offspring trios ( individuals). epidesigner was used for assay design at grb and the primer sequences were the following; forward: aggaagagaggggaaagggtgttaaattgtttatg, reverse: cagtaatacgactcactatagggagaaggctttttaaacccctca- aattcaaaaat. ng genomic dna was bisulfite-treated with the ez dna methylation kit (zymo research, orange, ca, usa). the spectra were analyzed and the methylation ratios were obtained by the epityper software v. . . . global methylation analyses were performed on dna extracted from pbl on the illumina infinium bead chip and the chips were scanned on an illumina iscan as per protocol (illumina). mg of dna was bisulfite-treated according to protocol (ez dna methylation kit, zymo research). for analysis, the genome studio methylation module of the genome studio genome browser was used, which facilitates integration of the snp and cpg location data (ncbi build ). methylation status was assessed after normalization to internal controls and background subtraction and expressed as b. the b values for the cpg sites were mapped to the gene and plotted to give an overview of methylation status for the region of interest (figure s d). disruption of grb for insulin and glucagon secretion analyses shrna-mediated knock-down of grb in human pancreatic islets. specific silencing of endogenous hgrb was achieved using a lentiviral-based shrna-silencing technique grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e www.nordicislets.org (santa cruz biotech. inc.). isolated human islets were incubated at . mmol/l glucose + polybree for min. thereafter, the medium was removed and the islets were washed before culture medium + lentiviral particles containing grb shrna ( ml/ml) was added, and the islets were cultured for h at mmol/l glucose. for comparison, a scramble (lentiviral particles without targeting any specific region) served as control. insulin and glucagon were measured using a radioimmunoassay kit (electro- box, stockhom, euro-diagnostica, malmö, sweden). sirna-mediated knock-down of grb in ins- / cells. for grb small interfering rna (sirna) experiments, – -nucleotide stealth-prevalidated sirna duplex designed for rat grb (santa cruz biotech. inc.) was used. ins- / cells were seeded in -well plates at a density of , cells in culture media without antibiotics and transfected with dharma- fect (dharmacon, lafayette, co, usa) according to the manufacturer’s instructions. assessment of b-cell viability human pancreatic b-cell viability assay was performed using a celltiter aqueous one solution cell proliferation assay reagent (promega, stockholm, sweden) according to the manu- facturer’s instructions. the actual performance is based on the spectrophotometric detection of a colored formazan product converted from a -( , -dimethylthiazol- -yl)- -( -carboxymethox- yphenyl)- -( -sulfophenyl)- h-tetrazolium (mts) compound by nadph or nadh via metabolically active cells. supporting information figure s regional plots for the top hits reaching genome- wide significance level. association with insulin secretion measured as corrected insulin response (cir) at min of ogtt for the novel genetic variant grb rs (a) and the previously reported t d and glycemic trait variants hhex/ide/kif (b), mtnr b (c), cdkal (d), c cd a (nlf ) (e), ank (f), gck (g) and gipr (h). (tiff) figure s genome-wide quantile-quantile (q-q) and manhat- tan plots for insulin secretion and action traits analyzed in the present study. corrected insulin response (cir) to glucose at min of ogtt (a, b), insulin sensitivity index (c, d), insulin response adjusted for insulin sensitivity (cir adj isi) (e, f), disposition index (g, h), insulin level at min of ogtt (i, j), incremental insulin at min of ogtt (k, l), insulin level at min of ogtt adjusted for bmi (m, n), area under the insulin curve (auc ins) (o, p) and ratio between area under the insulin and glucose curves (auc ins/auc gluc) (q, r). (tiff) figure s meta-analysis for association of grb rs with fasting plasma glucose in the participating cohorts. the insulin- reducing allele was associated with lower fasting plasma glucose levels in all individuals. (tiff) figure s grb mrna levels in human islets. (a) grb expression levels did not significantly differ between normoglyce- mic (hba c , . %) and hyperglycemic (hba c . %) islet donors (t-test, p = . ). (b) there was no significant difference in grb mrna expression levels between carriers of different grb genotypes (linear regression, pnormoglycemic = . ; phypergly- cemic = . ). (c) correlation between grb and gcg (glucagon), and grb and ins (insulin) mrna expression in islets from normoglycemic (hba c , %, n = ) and hyperglycemic donors (hba c . %, n = ). error bars denote se. (tiff) figure s schematic representation of the grb gene and its transcripts. the exon arrangements of grb splice variants were identified in human fat and islet samples by rt-pcr and sanger sequencing. the exon order at the gene level is shown in the top row. ucsc ids of matching transcripts are indicated to the right. (tiff) figure s allele-specific expression analysis of the grb - end in islet, muscle, liver and fat tissue. transcripts containing exon un are exclusively expressed from one allele (presumably paternal), whereas transcripts containing the upstream exons un or un a derive from both alleles to a varying degree. un a, un and un are mutually exclusive exons. a sequence of heterozy- gous genomic dna (gdna) is shown on top for comparison ( %- %). rs in exon was used as a reporter snp for the intronic index snp rs . (tiff) figure s pictorial representation of grb methylation status in human islets and peripheral blood lymphocytes. the impact of rs on dna methylation of three cpg sites analyzed using epityper in human islets (n = ) (a) and peripheral blood lymphocytes (pbl, n = ) (b). (c) the grb mrna levels correlated negatively with the degree of methylation at cpg sites located , bp downstream of rs of the grb gene in human pancreatic islets (n = ). (d) methylation status was tested for cpg sites using the illumina infinium k global methylation assay and is represented in the graph above the gene structure. the numbers on the y-axis on the right indicate the mean b values showing methylation status. the colored boxes represent the exons of the four isoforms annotated in the ncbi. arrows indicate position of the snps tested for association. distances between nearest cpg site tested and: (i) rs = . kb, (ii) rs = . kb and (iii) rs = . kb. hapmap does not show the linkage of the tested snps with any of the snps tested for methylation status. the cpg sites in the proximity were methylated (b. . ). grb is presented on a reverse strand. (tiff) figure s effect of grb disruption on islet function and cell survival in human pancreatic islets. (a) grb knock-down showed reduced glucagon secretion at mm glucose, particularly stronger for forskolin- and k + -stimulated glucagon secretion. reduced grb expression in human pancreatic islets had modest effects on insulin secretion, i.e. a slight increase in insulin levels at mm glucose. ninsulin = and nglucagon = donors of human pancreatic islets; up to measurements in each experiment for each donor. (b) effect of grb disruption on insulin and glucagon content ( non-diabetic donor, measurements in each experiment). (c) grb knock-down resulted in a reduction of the number of viable cells in human pancreatic islets. (d) effect of grb disruption on caspase- mrna expression ( non-diabetic donors, measurements in each experiment). * p, . , *** p, . . (tiff) table s (a) characteristics of cohorts and study details of analysis metrics and methods. (b) heritability estimates of parameters of insulin secretion and action indices used in the study. (c) spearman correlations between insulin secretion and action indices for the non-diabetic participants of the botnia-ppp study. (xls) grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e table s (a) association results of selected snps from the discovery gwas, the cardiometabochip and the de novo replication genotyping. (b) meta-analysis of gwas and cardio- metabochip studies: association results with parameters of insulin secretion and insulin action during ogtt for loci previously associated with type diabetes (t d), hour plasma glucose during ogtt ( hpg), fasting plasma glucose (fglu), fasting insulin (fins) and obesity. (xls) table s (a) assessing transmissions of grb rs and rs alleles in the trios from finland and sweden. (b) parent-of-origin effect of grb rs on parameters of insulin secretion and insulin action, and glucose levels during ogtt in the non-diabetic offspring from the trio cohorts. (xls) table s odds ratios for grb rs for risk of t d in the mpp study. (xls) table s (a) descriptive statistics for the botnia-ppp cohort. (b) association between grb rs and glucagon levels in the botnia-ppp cohort. (xls) table s grb snps associated with fasting and hr glucose levels in non-diabetic individuals from the dgi gwas study. (xls) table s measurements of allelic imbalance in the heterozy- gous carriers of grb rs in human islets. (xls) acknowledgments dgi, bps, ppp, mpp, human islets: human islets were provided by the nordic network for clinical islets transplantation by the courtesy of dr. olle korsgren, uppsala, sweden. microarrays were performed at sciblu genomics at lund university. we thank the patients for their participation and the botnia study group for clinically studying the patients. french obese adults: we thank jacques weill for recruitment of obesity families at jeanne de flandres hospital, marianne deweirder and frédéric allegaert for dna management, and stefan gaget for database management. amish family diabetes study: we thank our amish community and research volunteers for their long-standing partnership in research, and acknowledge the dedication of our amish liaisons, field workers and the amish research clinic staff, without which these studies would not have been possible. ulsam: genotyping was performed by the snp&seq technology platform in uppsala, sweden (?www.genotyping. se). we thank tomas axelsson, ann-christine wiman and caisa pöntinen for their excellent assistance with genotyping. sorbs: we thank all those who participated in the study. sincere thanks are given to knut krohn (microarray core facility of the interdisciplinary centre for clinical research, university of leipzig) for the genotyping support. hbcs: we thank all study participants and everyone involved in the helsinki birth cohort study. author contributions conceived and designed the experiments: ip wp rm rpb sas po nw td cli kds ml lg vly. performed the experiments: wp rm rpb sas po nw td kh. analyzed the data: ip wp rm rpb sas pa po nbn nw tf as ab vla co wx jla auj ycc jli jro pab jf sa td ea jt cle ak oh kh bfv hmk clm vv mnw rjl kko cla at rr jk cli pk kds vly. contributed reagents/materials/ analysis tools: sas pa po nw ap acs am pn bi tt njw ms ew tal tmf pf mw jge cli ei ars kds ml lg vly. wrote the paper: ip wp rm rpb po nw ab mim cli lg vly. references . diabetes genetics initiative of broad institute of harvard and mit and novartis institutes of biomedical research, lund university, saxena r, voight bf, lyssenko v, burtt np, et al. 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( ) locuszoom: regional visualization of genome-wide association scan results. bioinformatics : – . doi: . /bioinformatics/btq . grb , islet function, parent-of-origin, diabetes plos genetics | www.plosgenetics.org april | volume | issue | e http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id= &retmode=ref&cmd=prlinks http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id= &retmode=ref&cmd=prlinks http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id= &retmode=ref&cmd=prlinks http://mcb.asm.org/cgi/doi/ . /mcb. - http://mcb.asm.org/cgi/doi/ . /mcb. - http://www.ncbi.nlm.nih.gov/pmc/articles/pmc /pdf/bmj - .pdf http://www.ncbi.nlm.nih.gov/pmc/articles/pmc /pdf/bmj - .pdf hhe .indd fax + e-mail karger@karger.ch www.karger.com original paper hum hered ; : – doi: . / associations between genetic variants in the nos ap (capon) gene and cardiac repolarization in the old order amish wendy post a, b haiqing shen c coleen damcott c dan e. arking a, d w.h. linda kao b paul a. sack c kathleen a. ryan c aravinda chakravarti d braxton d. mitchell c alan r. shuldiner c, e a division of cardiology, department of medicine, johns hopkins university; b department of epidemiology, johns hopkins university bloomberg school of public health; c division of endocrinology, diabetes, and nutrition, department of medicine, university of maryland; d mckusick-nathans institute of genetic medicine, johns hopkins university school of medicine, and e geriatrics research and education clinical center, baltimore veterans administration medical center, baltimore, md. , usa val (rs , p = . and rs , p = . , additive models for both). snp rs explained . % of qt in- terval variability, with an average genetic effect of . ms. haplotypes that contained the minor allele for rs were associated with longer qt interval. conclusions: this study provides further evidence that nos ap variants influ- ence qt interval and further validates the utility of genome- wide association studies, a relatively new approach to gene discovery. copyright © s. karger ag, basel introduction: the electrocardiographic qt interval is a measure of cardiac repolarization which is moderately heritable [ , ] and predicts risk for cardiovascular events in the general population [ – ] . long qt syndrome is a rare heteroge- neous single gene disorder caused by mutations in one of a variety of ion channel genes, which can lead to fatal ar- rhythmias [ ] ; however, the majority of sudden arrhyth- mic deaths occur in people without mutations in these key words association analysis � candidate gene analysis � cardiovascular diseases � genetic polymorphisms � heritability � isolated population abstract background: through a genome-wide association study, we discovered an association of the electrocardiographic qt interval with polymorphisms in the nos ap (capon) gene. the purpose of the current study was to replicate this asso- ciation in the old order amish. methods: four nos ap snps were selected that captured all major haplotypes in the re- gion of interest ( � kb segment). genotyping was com- pleted in subjects from the heredity and phenotype in- tervention (hapi) heart study. association analyses were performed using a variance components methodology, ac- counting for relatedness of individuals. results: heritability of the qt interval was . . (p = . ! – ). all four snps were common with a high degree of correlation be- tween snps. two of the four snps (pairwise r = . ) were significantly associated with variation in adjusted qt inter- received: january , accepted after revision: april , published online: june , wendy post, md, ms division of cardiology, department of medicine, johns hopkins university blalock h, n. wolfe street baltimore, md (usa) tel. + , fax + , e-mail wpost@jhmi.edu © s. karger ag, basel – / / – $ . / accessible online at: www.karger.com/hhe http://dx.doi.org/ . % f nos ap (capon) gene and cardiac repolarization hum hered ; : – genes [ ] . indeed, determinants of the qt interval in the general population and the risk for sudden arrhythmic cardiac death are complex, and are determined by genet- ic background, the environment, and their interactions. we previously undertook a three-stage genome-wide association study, designed to identify genes or genomic regions that modulate the qt interval, by initially ge- notyping � , single nucleotide polymorphisms (snps) in subjects from each of the extremes of the qt interval distribution (total subjects) in the kora s study (‘cooperative research in the region of augs- burg’), a population based epidemiological study in augs- burg, germany [ ] . we identified and replicated an as- sociation between qt interval with snps in nitric oxide synthase (neuronal) adaptor protein ( nos ap also known as c apon) on chromosome , which is the car- boxy-terminal pdz ligand of neuronal nitric oxide syn- thase (nnos). the purpose of the current study was to evaluate, and replicate, the association between nos ap and the qt interval in a more genetically homogenous population, the old order amish, and to estimate the heritability of the qt interval. the old order amish of lancaster county, pennsylvania are a rural, closed, genetically iso- lated population who are descendents from a limited number of individuals who emigrated from europe in the ’s. they eschew modern technology, including many standard medical therapies, and live a relatively homoge- neous, active lifestyle [ ] . there are potential advantages to performing genetic association studies in the amish. genetic association studies in isolated populations are less likely to be inf luenced by spurious associations due to population substructure, and there are fewer non-ge- netic factors that could potentially confound associations between complex traits and genetic polymorphisms due to their relative environmental homogeneity. although the association between nos ap and the qt interval was replicated in our initial report, replica- tion in multiple populations helps to confirm that the association is valid in these populations that have the potential for different genetic backgrounds and environ- mental inf luences that have the potential for different genetic backgrounds and environmental inf luences that could negate or otherwise modify the main effect of the gene variant. moreover, if heterogeneity in associations is observed in different populations, assuming that the dif- ferences were not due to study design issues, our under- standing of the role of these genetic variations in different populations may be refined or additional insight into the potential interactions between genetic variations and other underlying risk factors in various populations may be gleaned [ ] . further validation of our genome-wide association study results would extend the association between nos ap and the qt interval to additional pop- ulations, and also strengthen the evidence for the poten- tial utility of genome-wide association studies, a relative- ly new approach to gene discovery. methods subjects/phenotyping the subjects in this study were amish men and women, en- rolled between and in the heredity and phenotype in- tervention (hapi) heart study, which was designed to identify genes that interact with the environment to inf luence cardiovas- cular risk. subjects were generally healthy and were initially re- cruited into the hapi heart study on the basis of their prior par- ticipation in one of our previous studies of bone mineral density and/or coronary calcification, although for practical consider- ations (e.g., to maintain rapport with the community and to more efficiently meet recruitment goals), recruitment guidelines were modified to allow other interested individuals in the community to participate. these subjects were initially identified through word of mouth, advertisements, amish-wide mailings, and refer- rals from local physicians. participating subjects were aged years and older, and their first and second degree relatives were encouraged to participate. subjects with severe hypertension (bp / mm hg), malignancy, kidney, liver or uncontrolled thy- roid disease were not eligible for the study. all subjects underwent a research examination at the amish research clinic in strasburg, pa. this examination included a -lead electrocardiogram (ecg) which was acquired using stan- dard methods with a ge marquette digital recording system. the qt measurements were calculated by the marquette computer system using ‘median beats’. there were median beats, one for each of the leads, which was developed using simultaneously sampled data, allowing for them to be time aligned synchronous- ly. a vector magnitude signal was formed by combining all - lead median beats, so-called ‘superbeat’. the qt interval was cal- culated from the superbeat, which measures the earliest onset of the qrs to the latest offset of the t wave across all leads. the analyses described in this report are based on subjects recruited into the hapi heart study with at least one genotype in the nos ap gene available. we excluded one subject with atri- al fibrillation and subjects with a qrs duration ms, as these conditions can alter repolarization (qt interval). our final analysis set included subjects. the research protocols were approved by the institutional re- view boards of the university of maryland and the johns hopkins university. all subjects signed informed consent, which included permission to perform genotyping. snp selection and genotyping four snps in nos ap (rs , rs , rs , rs ) were selected for genotyping based on our previous work in the kora study. in stage ii of that study, which consist- ed of individuals from each extreme of the qt interval dis- post /shen /damcott /arking /kao /sack / ryan /chakravarti /mitchell /shuldiner hum hered ; : – tribution (total of individuals), fine mapping had revealed that the � end of the gene, and in particular, a � -kb segment exhibiting strong linkage disequilibrium (ld), displayed the greatest association with qt interval [ ] . using the tagger tool in haploview (http://www.broad.mit.edu/mpg/haploview/) these four common snps capture all major haplotypes in this region ( % frequency). to estimate coverage, using an r of . in the hapmap ceu samples, we capture of the alleles with a mi- nor allele frequency . in this region. seventy-three percent of the captured alleles have an r . . genotyping was performed using taqman assays on demand or assays by design (applied biosystems) according to manufacturer’s protocols. statistical methods genotypes were checked for mendelian consistency using the pedcheck software program in the extended amish pedigree. al- lele frequencies were calculated for each snp by gene counting, and observed genotypes were tested for fit to the expectations of the hardy-weinberg law using the � test. pairwise ld (r ) was esti- mated by haploview. heritability and association analyses were performed using a variance components methodology to account for the relatedness of study subjects as implemented in the sequen- tial oligogenic linkage analysis routines (solar) program [ ] . we modeled variation in qt interval as a function of measured environmental covariates, additive genetic effects and a residual error component. the additive genetic effects were parameterized as a function of the kinship matrix. maximum likelihood methods were used to estimate the covariate and genetic effects simultane- ously. initially, we estimated the heritability of qt interval as the proportion of the total phenotypic variation that could be attribut- able to additive genetic effects. we then evaluated the association between nos ap genotype and qt interval while simultaneously adjusting for the effects of age, age , sex, and heart rate and additive genetic effects (heritability). statistical significance of the genotype effects were assessed using the likelihood ratio test, in which we compared the likelihood of the data under a model in which the genotype effect was estimated against the likelihood of a nested model in which the genotype effect was constrained to zero. anal- yses were performed assuming an additive genetic model. since some associations appeared to follow a dominant pattern, we re- peated the analyses assuming a dominant model. we evaluated gene-by-gender interactions for each snp using the log likelihood ratio test comparing the full model with a gene * gender term and a nested model without the gene * gender term. we estimated that a sample of size subjects provides % power to identify snps accounting for as little as % of the variation in qt interval, with an alpha level of . and minor allele frequency of . . haplotype frequencies and association were calculated based on the stochas- tic_em (sem) algorithm using thesias program [ ] . results the characteristics of the study population are de- scribed in table . the sample size of subjects includes individuals from sibships, with sibships ranging in size from to . additional relationship types were iden- tified by linking study subjects into larger pedigrees through their examined or unexamined parents. these individuals could be combined into multiplex pedigrees, ranging in size from to examined indi- viduals and representing sibpairs, parent-off- spring pairs, avuncular pairs and first-cousin pairs. the mean ( sd) age was years and % were women. qt was normally distributed (skewness = . and kurtosis = . ) with mean ( sd) of . . ms. heritability of the qt interval, adjusted for age, gender and heart rate, was estimated at . . (p = . ! – ). the proportion of the variance due to the covariates (age, age , gender, and heart rate) was . . all four snps were relatively common with minor allele fre- quencies between and % and satisfied hardywein- berg equilibrium. there was a high degree of correlation between snps (pairwise r ranging from . to . ) as displayed in figure . two of the four nos ap snps were significantly as- sociated with variation in adjusted qt interval, as shown in table (rs , p = . , additive model; p = . , dominant model; and rs , p = . , additive model; p = . , dominant model). of note, the r of pair- characteristics (mean sd) total men women (n = ) (n = ) (n = ) age, years . . . . . . mean qt interval, ms . . . . . . adjusted qt interval*, ms . . . . . . body mass index, kg/m . . . . . . systolic blood pressure, mm hg . . . . . . diastolic blood pressure, mm hg . . . . . . * adjusted for age, gender, heart rate and family structure. table . characteristics of the amish hapi heart study population nos ap (capon) gene and cardiac repolarization hum hered ; : – wise linkage disequilibrium between these two snps was . . there was a trend towards an association bet - ween rs and adjusted qt interval (p = . , dominant model). there were no associations between rs and the qt interval in single snp analyses. there were no significant gene-by-gender interactions. snp rs explained . % of the variability in the qt interval, and the average genetic effect, measured as the difference in the mean qt intervals between the two homozygote genotypes, was . ms. we also performed haplotype analyses using these snps. there were major haplotypes in this amish co- hort. one haplotype was comprised of all the major al- leles ( ) for these snps (haplotype frequency %) and the other of all the minor alleles ( ) (haplotype frequency %). there were three additional haplotypes ( , , ) that were each present in – % of the sample (note the order of the snps is the same as in table ). consistent with the single snp analyses, haplo- types that contained the minor allele for rs were associated with longer qt interval. discussion sudden unexpected cardiac death, generally attribut- ed to a lethal arrhythmia, accounts for nearly % of cor- onary heart disease mortality [ ] and has a familial com- ponent [ ] . abnormalities in cardiac repolarization, which can increase risk for arrhythmias, can manifest on a -lead electrocardiogram as an alteration in the qt interval. little is known about specific genes associated with risk for sudden cardiac death in the general popula- tion, other than a few candidate gene studies [ – ] . we found that the qt interval is a moderately heritable trait (h = . . ) in the old order amish. our herita- bility results are slightly higher than the heritability esti- mates in the framingham heart study (h = . ) [ ] and the family heart study (h = . ) [ ] . we have further validated the association between variants in the nos ap (capon) gene and the qt in- terval, which was recently discovered in a genome-wide association study [ ] . remarkably, we find very similar results in the old order amish, a genetically isolated population, and the results seen in the german kora study [ ] . in the amish, the proportion of the variability in the qt interval that was explained by snp rs was . % and the average genetic effect, measured as the difference in the mean qt intervals between the two ho- table . associations between nos ap snps and adjusted qt interval nos ap snp (n/n) maf adjusted qt interval* additive model p value* dominant model p value*nn nn nn rs (a/t) . . . . . . . . . rs (c/a) . . . . . . . . . rs (g/c) . . . . . . . . . rs (t/g) . . . . . . . . . n = major allele; n = minor allele. maf is minor allele frequency. * adjusted for age, gender, heart rate, and family structure. exon exon r s r s r s r s fig. . r plot of pairwise linkage disequilibrium between the four snps that cover a -kb region in the � portion of the nos ap gene. post /shen /damcott /arking /kao /sack / ryan /chakravarti /mitchell /shuldiner hum hered ; : – mozygote genotypes, was . ms. in kora s and kora f , two distinct samples from a population-based ger- man cohort, the proportion of the variability explained by the same allele of the same snp was . and . %, re- spectively, and the average genetic effects were . and . ms, respectively. the frequency of the minor allele was somewhat higher in the amish, versus % in the kora s and f populations, and % in the framing- ham heart study, in which association between nos ap and qt interval was also shown [ ] . a limitation of this study in the old order amish is the smaller sample size ( subjects versus , – , in the previous studies), leading to somewhat less power to detect associations and therefore less significant p values; however, the impor- tance of these results is ref lected in the remarkable con- sistency of the point estimates between this study and the others, suggesting that these findings are likely true pos- itives. we have added significantly to the previous study by performing these analyses in a genetically isolated popu- lation, in which spurious associations due to population substructure are less likely. we have also performed hap- lotype analyses, utilizing snps that were identified to tag the -kb region at the � region of the gene, which is likely to harbor the functional variant. since there was a high degree of ld between these snps in the old order amish, the haplotype analysis did not provide significant additional information, but did suggest that haplotypes containing the minor allele for rs are most as- sociated with longer qt intervals. most likely, the func- tional variant is in a non-coding region with regulatory function, since sequencing in kora did not identify any missense mutations in the exons [ ] . it is remarkable that when comparing the results across the various cohort studies, findings are relatively consis- tent even though each study used a different algorithm for measuring qt interval. at this time, there is no gold standard for population studies, and the association per- sists regardless of the qt measurement method uti- lized. in summary, we have further validated the discovery that the gene nos ap (capon) is associated with mod- ulation of cardiac repolarization as manifested on the surface electrocardiogram in a genetically isolated ho- mogeneous population. the trait is moderately heritable, and the functional variant is likely to reside in an area in the � region of the gene which contains a high degree of linkage disequilibrium. this validation supports the use of genome-wide association studies to identify genes or genomic regions that previously would not have been im- plicated to inf luence various disease traits. acknowledgements we gratefully acknowledge our amish liaisons and field work- ers and the extraordinary cooperation and support of the amish community without which these studies would not have been pos- sible. we thank dr. gordon tomaselli for his thoughtful com- ments on the manuscript. this work was supported by nih research grant u hl , the university of maryland general clinical research center, grant m rr and the johns hopkins university bayview general research center (m rr ), general clinical re- search centers program, national center for research resources (ncrr), nih, the baltimore veterans administration geriatric research and education clinical center (grecc) and the d.w. reynolds clinical cardiovascular research center, johns hop- kins university. dr. post is supported, in part, by the paul beeson physician faculty scholars in aging program. conflict of interest none. references carter n, snieder h, jeffery s, saumarez r, varma c, antoniades l, spector td: qt in- terval in twins. j hum hypertens ; : – . newton-cheh c, larson mg, corey dc, benjamin ej, herbert ag, levy d, d’agostino rb, o’donnell cj: qt interval is a heritable quantitative trait with evidence of linkage to chromosome in a genome-wide linkage analysis: the framingham heart study. heart rhythm ; : – . dekker jm, crow rs, hannan pj, schouten eg, folsom ar: heart rate-corrected qt in- terval prolongation predicts risk of coronary heart disease in black and white middle-aged men and women: the aric study. j am coll cardiol ; : – . okin pm, devereux rb, howard bv, fabsitz rr, lee et, welty tk: assessment of qt in- terval and qt dispersion for prediction of all-cause and cardiovascular mortality in american indians: the strong heart study. circulation ; : – . schouten eg, dekker jm, meppelink p, kok fj, vandenbroucke jp, pool j: qt interval prolongation predicts cardiovascular mor- tality in an apparently healthy population. circulation ; : – . priori sg, napolitano c: genetics of cardiac arrhythmias and sudden cardiac death. ann n y acad sci ; : – . myerburg rj, castellanos a: emerging para- digms of the epidemiology and demograph- ics of sudden cardiac arrest. heart rhythm ; : – . nos ap (capon) gene and cardiac repolarization hum hered ; : – arking de, pfeufer a, post w, kao wh, newton-cheh c, ikeda m, west k, kashuk c, akyol m, perz s, jalilzadeh s, illig t, gie- ger c, guo cy, larson mg, wichmann he, marban e, o’donnell cj, hirschhorn jn, kaab s, spooner pm, meitinger t, chakra- varti a: a common genetic variant in the nos regulator nos ap modulates cardiac repolarization. nat genet ; : – . sorkin j, post w, pollin ti, o’connell jr, mitchell bd, shuldiner ar: exploring the genetics of longevity in the old order amish. mech ageing dev ; : – . herbert a, gerry np, mcqueen mb: re- sponse to comments on ‘a common genetic variant is associated with adult and child- hood obesity’. science ; :e –e . almasy l, blangero j: multipoint quantita- tive-trait linkage analysis in general pedi- grees. am j hum genet ; : – . tregouet da, escolano s, tiret l, mallet a, golmard jl: a new algorithm for haplotype- based association analysis: the stochastic- em algorithm. ann hum genet ; : – . jouven x, desnos m, guerot c, ducimetiere p: predicting sudden death in the popula- tion: the paris prospective study i. circula- tion ; : – . sotoodehnia n, siscovick ds, vatta m, psaty bm, tracy rp, towbin ja, lemaitre rn, rea td, durda jp, chang jm, lumley ts, kuller lh, burke gl, heckbert sr: beta -adrener- gic receptor genetic variants and risk of sud- den cardiac death. circulation ; : – . splawski i, timothy kw, tateyama m, clan- cy ce, malhotra a, beggs ah, cappuccio fp, sagnella ga, kass rs, keating mt: variant of scn a sodium channel implicated in risk of cardiac arrhythmia. science ; : – . yang p, kanki h, drolet b, yang t, wei j, viswanathan pc, hohnloser sh, shimizu w, schwartz pj, stanton m, murray kt, nor- ris k, george al jr, roden dm: allelic vari- ants in long-qt disease genes in patients with drug-associated torsades de pointes. circulation ; : – . hong y, rautaharju pm, hopkins pn, ar- nett dk, djousse l, pankow js, sholinsky p, rao dc, province ma: familial aggregation of qt-interval variability in a general popu- lation: results from the nhlbi family heart study. clin genet ; : – . journal of mennonite studies for access to some consumer goods. the relationship between these two groups became so close that, when the shortage of farm land led old colony mennonites to emigrate to mexico in the s, the jewish population also departed from winkler ( , - ). werner attributes the post-war economic success of winkler to the creation of non-agricultural industries within the town, particularly a sewing factory and a recreational vehicle manufacturer (triple e). he excerpts heather robertson’s condemnation of the “hucksterism” of winkler’s annual sidewalk sale, known as old time value days. he himself is less critical of employer-employee relationships in winkler businesses. the tensions between school and business, for example, are left unexplored – specifically the connection between exploitation of a pool of uneducated (though skilled) labour and comparatively low high school graduation rates. this despite noting that “it was always a challenge” to see rural students graduate from garden valley col- legiate ( ). more effective is his brief explanation of the link between the absence of labour unions and the religious beliefs of mennonite business owners and workers alike in winkler. the production qualities of this book are excellent. interesting sidebars – ranging from quotations, letters to the editor, and newspaper articles, to poems – are interspersed with the text. numerous maps, photographs, and statistical tables are included, all with sources provided (too often a rarity in local histories). two appendices of early winkler families and of civic officials are included, as well as a bibliography, copious endnotes, and an index. werner, and the winkler heritage society, are to be congratulated on producing such an informative, well researched, and well written history. janis thiessen westgate mennonite collegiate reviews of religious studies and social science ray gingerich and ted grimsrud, eds., transforming the powers: peace, justice and the domination system. minneapolis: augsburg fortress press, . $ . (us). walter wink is well known for his work on “the powers,” which can be summed up in three statements: the powers are good, the powers book reviews are fallen, the powers must be redeemed. wink himself continues to develop his work (which draws on insights developed by hendrikus berkhof, g.b. caird, william stringfellow, john howard yoder, and so on), and others are also engaged in exploring and extending that work in various directions. transforming the powers: peace, justice and the domination system comes out of a conference at eastern men- nonite university, and includes essays, mostly by mennonite scholars, which seek to offer tribute to wink and to contribute to the task of testing and applying his insights in ever-broader spheres of life. transforming the powers is organized in three parts, including several contributions by wink. part , “worldviews and the powers,” focuses largely on discussions of wink’s notion of worldviews (funda- mental presuppositions about reality) in relation to the social sciences. in the second part, “understanding the powers,” the focus shifts to an attempt to gain clarity regarding the relationship of the powers to the difficult question of the existence and influence of evil in the world, and the distorting effects of fallen powers in areas such as epistemology, economics and politics. part , “engaging the powers,” includes essays that address questions of transformation, justice, nonresistance, and peacemaking. one of the interesting contributions made by this particular collec- tion of essays is that it nicely brings to view the fact that wink’s work can be taken in several different directions, both represented within the larger framework of anabaptist thought. that is, wink’s work can be taken up and extended further into the world of social science, as shown clearly by daniel liechty’s essay. liechty argues strongly that it is possible to arrive at a “social-scientific understanding of the biblical-theological category of the principalities and powers,” that “wink definitely designates as principalities and powers the same unseen forces toward which a social scientist would point.” in fact, argues liechty, “many paragraphs wink has written on what he calls the world domination system could have flowed from the pen of a phenomenological sociologist without any significant alterations.” this kind of an attempt to uncover common ground between biblical/theological notions and social-scientific view of the same phenonema is quite different from the direction that wink’s work is taken by scholars such as willard swartley. instead of seeking the kind of common ground in which liechty is interested, swartley believes wink is misleading in viewing the powers “primarily in the spirit- personality manifestations of structures and institutions.” swartley wants to rehabilitate an understanding of biblical teaching that pays closer attention to specific spiritual beings that act in this world. he is concerned that we are so steeped in the scientific worldview that we are prone to reduce all reality to the empirical. while he is not journal of mennonite studies explicit in saying so, swartley would undoubtedly see liechty’s essay as reductionist in exactly this way. in fact, swartley insists that “even god-talk points only to function of belief, not to an actual being,” which also holds true for talk of fallen powers and principalities. the divergent responses to wink’s work serve minimally to show the richness of his work, and the possibilities that lie within these insights. especially interesting to me are the essays which, in seeking to extend wink’s biblical/theological insights even further, take seri- ously spiritual realities, the life of the church, the life of jesus and the power of the spirit, and in doing so, engage the world within which these realities are manifested. paul doerksen mennonite brethren collegiate institute helmut isaak, menno simons and the new jerusalem. kitchener, on: pandora press, . pp. . paperback. $ . cdn., $ . u.s. this new book is important in that it seeks to alter the traditional portrait of menno simons. walter klaassen writes in the preface: “helmut isaak has moved the work of recovering menno simons ahead, in considerable measure. anyone working on menno from now on will need to engage this work.” the first chapter of the book, “the social and religious context for the emergence of anabaptism in the netherlands,” is in my view the best chapter in the book, describing the society in which menno lived and worked. most previous biographies of menno have not dealt in detail with the religious, social and economic world into which menno was born. the subsequent three chapters trace menno’s spiritual and theological development, dealing with: menno and münster: a vision of the new jerusalem; the heavenly jerusalem has descended upon this earth; and the eschatological anticipation of the new jerusalem. the concluding chapter summarizes isaak’s findings. of the pages, there are forty-two pages of notes and bibliography. mennonites who thought they knew their menno simons well, will be surprised to find that there are aspects to their spiritual leader they did not know before. having read and worked from the complete writings of menno simons (translated by leonard verduin and edited by j. c. wenger, published in ), i found, after reading this new book, that my portrait of menno did not quite fit the one painted by helmut isaak. isaak’s close reading of menno’s earlier and then his book reviews revised later works lead him to a more nuanced portrait of menno and a fresh understanding of his theology. this new portrait is different from the one readers of the complete writings and of bender’s biography have been familiar with. the new image brings menno closer to the fanatics of münster, the radical anabaptists from which he later sought to separate himself. historiographically, this book puts isaak into the so-called “polygenesis” camp of historians. the complete writings, according to isaak, do not always convey the exact meaning of menno’s words. for example, the english translation uses “polygamy” to translate the dutch “veelheyt der wijven” (plural- ity of wives?), but according to isaak, menno “is very reluctant to use this term in regard to münster” (p. , note ). or, verduin’s transla- tion of “dat uytwendige rijck christi op aerden” with “the visible kingdom of christ” is, according to isaak, “not correct.” isaak explains: “the difference between ‘external’ and ‘visible’ is very basic for the understanding of menno’s concept of the kingdom of god. münster was only external because it was motivated by selfish ambition, greed and lust. the truly spiritual kingdom of christ becomes visible reality through repentance, regeneration and new life” (p. , note ). while isaak deals primarily with menno’s original writings and does not rely much on the secondary literature, he does include second- ary works in his bibliography, and notes which agree with his view of menno and excludes those which don’t. for example, menno’s peace position might have received more attention in this book. scholars, including abraham friesen, have shown that menno was influenced especially by erasmus in this regard. there is, however, no reference to friesen’s work, nor to c. arnold snyder’s anabaptist history and theology which shows menno as an opponent of münster from the beginning. egil grislis’ insightful four articles in the journal of mennonite studies ( / , / , / , / ) on menno’s views of the incarnation, “good works,” the lords supper, and the apostle paul, are not referred to nor included in the book’s bibliography. had the above works been taken into account, menno simons would have retained more aspects of his traditional image. as it is, in isaak’s study menno appears as a sympathizer of the münsterites, whom he continued to call his “dear brothers and sisters.” only in his later works did he fight them because it was dangerous for menno and his followers to be identified with them. it is at this point that the book becomes contradictory in my view. on the one hand, menno is seen as a near-münsterite and on the other as a leader who seeks to follow scriptures in matters of a pure church, congregational discipline, and the eventual victory of the kingdom of god. the more traditional portrait of menno as one who grieved over those of his followers who were led astray by the münsterites, and then journal of mennonite studies wrote against the grave errors of his former followers, seems more convincing to me than the contradicting image which emerges in this book. isaak’s analysis of menno’s view of governments, while not all that novel, is an important part of this book’s theme, namely the kingdom of god (the new jerusalem) in menno’s thinking. it is known that menno did not follow the “schleitheim confession” ( ) of the swiss in regard to christians’ relationship to the state. he believed, at least at first, that the kingdom of god would transform society and rulers would play an important part in this transformation. in many of his writings he appealed to rulers and governments to repent and then govern as “christians.” in the end, however, menno was disappointed that rulers did not follow his advice, but he continued to believe in the possibility of the existence of pious christian rulers. at the end of the book (p. ) it comes as a bit of a surprise when isaak suggests that mennonites who established states within the state in th-century russia and in th-century latin american countries, may have realized, at least in part, menno’s vision of a “mennonite government.” i doubt that menno had such states as developed in russia and latin america in mind. were/are these “states” really christian? about fantasies to create a “mennostaat” in the s, see james urry’s article in journal of mennonite studies, vol. , . so mennonites and other believers will no doubt have to wait with menno simons for a real “new jerusalem” in the future! harry loewen kelowna, british columbia karl koop, editor and introduction, confessions of faith in the anabaptist tradition - . classics of the radical reformation . kitchener, on: pandora press (co-published with herald press), . pp. . paperback, $ . cdn, $ . u.s. in recent years we are learning that the earlier assumption was untrue that sixteenth-century anabaptists (and mennonites of the next generations) were non-creedal and more interested in ethics than in theology. relatively recent works that counter this stereotype include howard john loewen, one lord, one church, one hope, and one god, j. denny weaver, keeping salvation ethical, thomas finger, a contemporary anabaptist theology, gerald biesecker-mast, separation and the sword in anabaptist persuasion, and karl koop, book reviews anabaptist-mennonite confessions of faith. confessions now joins this last book as a companion volume, presenting the primary source material on which koop’s first book was based. confessions is a welcome addition to our growing resources for ana- baptist theology in english translation. the volume presents fourteen anabaptist and mennonite confessions of faith from four anabaptist streams. the time period of these confessions, to , bridges the era from the emergence of early anabaptism to the end of the first phase of anabaptist and mennonite doctrinal development. factors that brought an end to the confessional age of dutch mennonites in the s include early enlightenment influences, the rise of early pietism, and distaste for the divisions among mennonites brought about by strict confessionalism (p. ). selections for this volume from the swiss/south german anabaptist tradition include the swiss congregational order, the schleitheim brotherly union, jörg maler’s confession and the swiss brethren confession of hesse. from north german/dutch anabaptism are the kempen confession, the wismar articles and the concept of cologne. three documents come from the waterlander stream of anabaptism – the waterlander confession, the short confession and the thirteen articles. the final section has four confessions from the frisian, flem- ish and high german anabaptist tradition, namely the thirty-three articles, the jan cents confession, the dordrecht confession, and the continuation of this stream in a prussian confession. the book’s introduction provides an overview of scholarship on anabaptist confessions, along with brief discussions of the function, authority, and theological orientation of confessions. brief comments introduce each confession as well. reading these confessions side by side one after the other, impresses one as much with the variety of expression they contain as by any elements of a unifying theological tradition. putting these confessions in close proximity provides ample discussion fodder for both sides in the arguments about whether early anabaptist and mennonite theology is primarily orthodox christianity with a few additions or rather one or more new streams posing a contrast to inherited, standard orthodoxy. the introductions to the book and to the various individual confes- sions hint at an issue that needs further analysis. during the time frame of this book, mennonites produced a great many confessions, perhaps more than any other reformation group. these confessions frequently provoked divisions. but they were written with other goals in mind – discovering the identity of a group or providing a basis for unity among groups in conflict. what characteristics of anabaptists and mennonites produced the need for this number of confessions, and journal of mennonite studies what accounts for the two, apparently conflicting results of attaining unity and provoking divisions? what the introduction does not explain is that whether a confession provokes division or creates unity depends less on the character of the confession than on how it is used. to define identity by describing what a group believes or to develop a confession to discover what groups can agree on for unity purposes are primarily descriptive tasks. however, once the descriptive task is finished, an inevitable shift occurs. the moment it is declared, “this we believe,” the active impulse of the confession shifts from description to prescrip- tion and the confession begins to function as a norm that divides those inside from those outside the group. in other words, the one confession plays two roles, first as a uniting document and then as a potentially dividing document. this fine collection of documents invites extended historical analysis of the history that produced these confessions in light of the contrasting functions of description and prescription, or of uniting and dividing. a book designed for serious study needs an index. this book is no exception. a user misses being able quickly to locate theological themes throughout the confessions or to track the authorities quoted in the introductions. j. denny weaver bluffton university donald kraybill and james p. hurd, horse-and-buggy mennonites – hoofbeats of humility in a postmodern world. university park, pa: pennsylvania state university press, . hard cover, $ . us; paperback, $ . us. most people identify horse and buggy people as amish. in manitoba this summer, when a group of old order mennonites began a set- tlement, the press identified them as amish, because they assumed this was the identification that would communicate. in this volume, kraybill and hurd discuss the origin, faith, and history of old order mennonites, and as such, provide a much-needed corrective to popular misunderstandings about both old order mennonites and amish. old order mennonites in the united states began in , within the lancaster mennonite conference. the conference had been expe- riencing tensions over issues of modernization for some time. these tensions came to a head in the early s. the issue was not dress or use of technology, as one might have assumed since these are often the most visible signs of the old orders, but rather religious innovation book reviews and forms of worship.( ) as the division developed, other issues of modernity and relationship to the world were included, and gradually the two groups moved further and further apart. the long list of issues that divided the two groups primarily arose out of two major influences upon mennonites. one was evangelical- ism, and the innovations it promoted, like revival crusades, sunday schools, use of the english language in worship, evening church services, new hymnody, foreign missions, and higher education. all of these things seemed to be leading to pride, individualism, and “worldliness.” the other influence was the rapid increase in technol- ogy after the american civil war. new innovations like telephones and automobiles in the early twentieth century created new issues of how to relate to the “world.” on the issue of automobiles, the conviction not to use automobiles only developed gradually, with numerous members initially owning cars. the old orders eventually felt that owning a car was an expression of pride and individualism, and disruptive to community. the groffdale old order mennonite church initially consisted of about members, all living in lancaster county, pennsylvania. over the years the church has multiplied into many districts, each centred around a meetinghouse. (the term “church” is used for the people gathered, not the building.) because of the high cost of land in pennsylvania, the old order mennonites have moved into nine states, and number about , baptized members.( ) the total number of people in their communities, including children, is about , . they are scattered from new york in the east to iowa in the west, and from missouri and kentucky in the south to michigan in the north. they have been remarkably successful in passing on their faith from one generation to the next. groffdale old order mennonites are organized in about church districts. they meet in meetinghouses, and not in homes as do the amish. ministers from the districts meet twice a year to discuss issues related to the ordnung (principles and rules about life and faith), and thereby maintain a remarkable unity and similarity in all the church districts. they work together to make changes gradually, rationally, and in ways that will maintain community. they are in fel- lowship with old order mennonites in ontario and virginia, although neither of those groups is formally part of their conference. there are multiple names for this group of old order mennonites. they are also called horse-and-buggy mennonites, groffdale confer- ence mennonites, because their founding bishop, joseph o. wenger, was a preacher at the groffdale churchhouse in pennsylvania, wenger mennonites, because of their first bishop, and team mennonites, because they use the horse and buggy team. journal of mennonite studies none of the descendents of the groffdale old order mennonites have settled in canada. the old order mennonites in ontario began locally a few years earlier, in , when mennonites near waterloo divided between progressives and conservatives. their story is told very well in a recent publication by donald martin, old order mennon- ites in ontario: gelassenheit, discipleship, brotherhood. (kitchener, on: pandora press, ). after a very fine historical introduction to the groffdale old orders mennonites, kraybill and hurd discuss their faith and life under eight different topics. the topics range from “the fabric of faith and cul- ture” to “the rhythm of sacred ritual” to “pilgrims in a postmodern world.” the authors sensitively help readers see the older orders’ profound christian faith, their deep commitment to follow faithfully the teachings of the bible, their conviction that resources for renewal and change can be found within their own heritage, and their humble commitment to christian discipleship. the authors end their book with a very appropriate summary state- ment, “they [the old order mennonites] believe that true progress and deep satisfaction emerge when people yield to the collective wisdom of a redemptive community and that those who surrender to the precepts of providence, embedded in communal wisdom, will receive the bless- ings of contentment and fulfillment.”( ) john j. friesen canadian mennonite university format a j csian ournal of hemistrya j csian ournal of hemistry https://doi.org/ . /ajchem. . introduction biodiesel is future of fuel, since conventional feedstocks are limited and polluting. the feedstock for biodiesel synthesis is oil based on its availability is further classified as edible, non-edible and waste oil. the use of edible oil is criticized due to food security as well as forest land issue. various non-edible oils such as neem [ ], karanja [ ] and jatropha [ ] were explored for biodiesel synthesis. however, limited investigation on papaya oil as a feedstock for biodiesel was found in the research literature. india share % of papaya production and placed at th position worldwide [ , ]. an estimated papaya oil production is approximately . lakh tons per annum [ ]. in literature, conventional heat source was explored for esterification and transesterification of raw papaya oil [ ]. however, kinetic study of microwave-assisted transesterifi- cation of papaya oil is not yet reported in literature. microwave as a heat source has several advantage over conventional heat source in term of energy requirement and time required for conversion. lin et al. [ ] reported that energy required is kinetic and thermodynamic studies of microwave-assisted transesterification of papaya oil milap g. nayak ,*, and amish p. vyas , department of chemical engineering, vishwakarma government engineering college (affliated to gujarat technological university), chandkheda- , india department of chemistry, saffrony institute of technology (affliated to gujarat technological university), at & p.-linch, mehsana- , india *corresponding author: e-mail: milapnayak @gmail.com received: january ; accepted: march ; published online: june ; ajc- microwave-assisted transesterification due to drastic reduction in reaction time when compared with conventional method gain the research attention. pesent study explores the kinetic and thermodynamic study of microwave-assisted transesterification of non-edible papaya oil. the experiments were performed using pre-optimized process parameters having : molar ratio of methanol to oil, wt. % naoh catalyst. the experiments carried out in commercial microwave reactor with infrared temperature controller. kinetic study of microwave- assisted palm oil methyl ester (pome) conversion was carried out by varying temperature from to ºc with an increment of ºc. kinetic study revealed that microwave-assisted homogeneous alkali-catalyzed transesterification follows first order having lower activation energy . kj mol- . also, higher frequency factor min- and rate constant . min- at ºc revealed the non-thermal and thermal effect of the microwave. other thermodynamic properties such as ∆g, ∆h and ∆s were found out to be . kj/mol, . kj/ mol and - . kj/mol k, respectively, which show that reaction is non-spontaneous, endothermic and endergonic in nature. keywords: papaya oil, biodiesel, microwave, transesterification. asian journal of chemistry; vol. , no. ( ), - this is an open access journal, and articles are distributed under the terms of the attribution . international (cc by . ) license. this license lets others distribute, remix, tweak, and build upon your work, even commercially, as long as they credit the author for the original creation. you must give appropriate credit, provide a link to the license, and indicate if changes were made. times lower while time required for transesterification is times lower. on the other hand, patil et al. [ ] reported times lower energy consumption for given yield in transesteri- fication. susceptibility of heating under influence of micro- wave mainly depends upon dielectric constant and dielectric loss of material [ ]. methanol due to its high dielectric loss tangent and high reactivity is used for biodiesel synthesis. transesterification reaction involved the conversion of trigly- cerides into monoalkyl esters in presence of methanol. the overall reaction takes place in three steps: (i) conversion of triglycerides into diglycerides, (ii) diglycerides into monogly- cerides and fatty acid alkyl ester, (iii) finally monoglycerides to monoalkyl esters and glycerol. fig. presents overall trans- esterification reaction. present study investigates the kinetics and thermodynamic study of microwave-assisted transesteri- fication of papaya oil. experimental refined papaya oil was purchased from m/s katyani exports pvt, ltd., new delhi, india. all chemicals such as naoh, https://orcid.org/ - - - https://orcid.org/ - - - koh, and methanol were of ar grade and % pure. acid valve and saponification value of papaya oil were . and mg g- , respectively. the fatty acid composition of papaya oil were : oleic acid . %, palmitic acid . %, behenic acid . %, eicosenoic acid . %, arachidic acid . %, lignoceric acid . %, palmitoleic acid . %, linoleic acid . % and myristic acid . %. the degree of unsaturation of papaya oil is found to be . %. molecular weight of papaya oil based on fatty acid composition is g mol- . the physico-chemical properties of papaya oil such as density, viscosity and cloud point were found to be kg m- , . cst and − . ºc, respectively. general procedure: microwave-assisted transesterifi- cation of papaya oil was carried out in a ml glass flask with a water-cooled condenser at the top. it was charged with ml ( . g) of oil, : molar ratio of alcohol and wt. % of naoh of catalyst. the reaction temperature was controlled by infrared temperature sensor with a controller. the kinetic evaluation of biodiesel production via microwave-assisted transesterification from papaya oil was done at , , and ºc at a different time interval. the reaction mixture was stirred magnetically and refluxed for the required reaction time. the reaction was carried out at w and the reaction mixture was cooled and allowed to settle down into two layers. top layer mainly consists of papaya oil methyl ester and unreacted triglycerides (oil), while bottom aqueous layer mainly having glycerol and methanol. traces of methanol present in oil phase were removed by heating at ºc for several minutes. detection method: top layer biodiesel purity was confir- med by : test [ ]. the yield of palm oil methyl ester (pome) is calculated by eqn. : amount of pome produced yield of pome theoretical maximaum amount of pome produced = ( ) results and discussion rate constant and order of reaction: the transesterifi- cation reaction kinetics is represented by eqn. [ ]: n md[ca] k [ca] [meoh] dt − = × × ( ) as per stoichiometry, methanol requirement is : and during the kinetic study. during the experiment, the excess molar ratio : was used hence reaction kinetics is pseudo-order with respect to the concentration of methanol. it is expressed as: nd[ca] k [ca] dt − = × ( ) the initial concentration of triglycerides was . mol/ ml. as the reaction proceeds, the triglyceride converted into palm oil methyl ester (pome). fig. a presents the concen- tration of pome with time at , , and ºc. the amount of methyl ester formed was determined to calculate the moles of triglyceride unreacted during transesterification. the diffe- rential analysis was used to evaluate the rate of reaction and order of the reaction. fig. b shows the differential plot of ln d[-ca]/dt vs. ln [ca] for , , and ºc. a straight line is observed with the r values greater than . indicating that the good agreement with experimental data. the slope of straight line was used to find the order of the reaction while intercept on y-axis gave the value of ln k from which reaction rate constant was evaluated. the microwave- assisted transesterification of papaya oil followed first-order kinetics and the rate of the reaction varies linearly with a yield of methyl ester of palm oil. the order of reaction and rate constants are summarized in table- . m.w. naoh triglyceride methanol papaya oil methyl ester glycerol fig. . microwave-assisted transesterification of triglyceride of papaya oil into papaya oil methyl ester vol. , no. ( ) kinetic and thermodynamic studies of microwave-assisted transesterification of papaya oil activation energy: the arrhenius equation is used to relate the temperature dependency of rate constants. it is expressed by eqn. [ ]: k = ae –ea/rt ( ) where, a is frequency factor (min- ), which measures frequency of collision between reactants. ea is activation energy (j/mol) for product formation. r is the molar universal gas constant ( . j mol- k- ) and t is the absolute temperature (k). by taking the natural logarithm eqn. can be expressed as eqn : ln k = ln a – ea/rt ( ) the change in the yield of methyl ester of palm oil was studied with the change in temperature from to ºc and the results were used to plot the graph between k (rate constant) and temperature. the straight line with the r value of . obtained in the graph revealed that reaction follows the first order kinetics. the activation energy and frequency factor for the reaction was determined from the plot of ln k and /t. the activation energy and frequency factor found to be . kj/ mol and min- , respectively. the activation energy of oil depends upon the types of fatty acid as well as degree of unsaturation of fatty acid in the oil. oil with higher unsaturations is prone to other undesired side reactions. it lower down the reaction rate, hence it has higher activation energy. apart from nature of oil, other factors, affecting the activation energy are temperature, the source of heating, type of catalyst and catalyst amount. it was reported that, under influence of the nonthermal effect of the microwave, the active intermediate complex had a lower state of energy which reduced the activation energy [ ]. pre-exponential factor (a) in this study shows high frequency of collision in the microwave-assisted transesterification of papaya oil. it improved the possibility of molecular collision due to the dipolar rotation and ionic movement [ ]. thermodynamic parameters: eqn. presents the eyring-polanyi equation for determine the gibbs free energy (∆g) for known value of rate constant (k) [ ]. bk t gk exp h rt −∆ = ×     ( ) linear form eqn. can be expressed by eqn. as: bk t gln k ln h rt −∆ = +     ( ) relation of enthalpy and entropy change for gibbs free energy is written by ∆g = ∆h − t∆s. eqn. is written as: bkk h sln ln t rt r h −∆ ∆  = + +    ( ) where, k: rate constant (s- ); t: absolute temperature (k); r: universal gas constant . j mol- k- ; kb: boltzmann constant ( . × - j k- ); h: plancks constants ( . × - js). using eqn. and the values of slope and intercept of the erying plot (figure not shown) between /t and ln k/t, the values of ∆g, ∆h and ∆s can be calculated. the values of ∆h and ∆s found to be . kj/mol and - . kj/mol k, (a) (b) - . - . - . - . - . - . - . - . - . - . - . - . - . - . - . - . - . °c °c °c °c linear fit of °c ln(-dca/dt) linear fit of °c ln(-dca/dt) linear fit of °c ln(-dca/dt) linear fit of °c ln(-dca/dt) ln ca ln (- d c a /d t) time (min) p o m e y ie ld yield at °c yield at °c yield at °c yield at °c fig. . (a) pome yield with time at a different temperature, (b) plot of ln(-dca/dt) vs. ln ca for , , and °c table- order of reaction and rate constant for microwave assisted transesterification of papaya oil and anova of linear fit temp. ( °c) order of reaction (n) ln k k (min- ) r adj, r f value p value . - . . . . . × – . - . . . . . - . . . . . × – . - . . . . . × – nayak et al. asian j. chem. respectively. thus, at k, ∆g found to be . kj/mol. a positive value of ∆h shows that heat input is required to bring the reactants to the transition state to form the products. a negative value of ∆s indicates that the degree of ordered geometry/alignment of the transition state is better as compared to reactants in ground state. a positive value of ∆g indicates that the reaction is unspontaneous and endergonic in nature. table- summaries the comparison of kinetic and thermody- namic properties of present work with reported work in litera- ture. thermodynamic parameters such as ∆h and ∆s obtained in the microwave-assisted transesterification of papaya oil differs from soybean oil [ ], spirulina platensis algae [ ], leather tanning waste [ ] and, schleichera triguga oil [ ]. the variation in a kinetic and thermodynamic parameters is due to the variation in fatty acid composition, degree of unsatu- ration and difference in methodology adopted for biodiesel production. tlc analysis: thin layer chromatography (tlc) test was performed using silica gel fluorescent indicator f . the solvent hexane, diethyl ether and acetic acid with volume ratio : : was used for tlc. the spot observed at retention factor (rf) . , . and . correspond to the position of methyl esters, triglyceride and diglyceride, respectively. the spots at rf . and . represents monoglycerides and free fatty acid, respectively. the spot of triglyceride disappeared with time indicates the conversion of triglyceride into biodiesel. conclusion kinetic study of microwave-assisted transesterification of papaya oil was carried out by varying temperature from to ºc at an increment of ºc. the experiments were performed using pre-optimized process parameters having : molar ratio of methanol to oil, wt. % naoh catalyst. kinetic study revealed that microwave-assisted homogeneous alkali-catalyzed trans- table- summary of comparison of kinetic and thermodynamic properties of pome triglyceride source of heating reaction condition n k (min- ) a (min- ) ea (kj mol- ) ∆g (kj mol- ) ∆h (kj mol- ) ∆s (kj mol- k- ) ref. papaya oil . av u: % m, batch naoh wt %, : meoh:oil, °c . . . . - . present work waste cooking oil . av u: % m, batch cadg wt %, : meoh:oil, °c . . n.d. n.d. n.d. [ ] soybean oil . av c, batch cao/al o wt % : meoh:oil, °c . . n.d. n.d. n.d. [ ] soybean oil . av c, batch . wt % naoh, . wt % bentonite, - °c, . : meoh:oil . - . . . - . . - . [ ] sunflower oil . av c, batch wt % mgo-la o , meoh:oil : , °c, rpm, h, . . . - . . [ ] chinese tallow seed m, continuous naoh wt %, . : . : meoh:heane:oil(w/w) , °c . . × l mol- min- . . - . - . [ ] schleichera triguga oil . av us ba(oh) wt %, : methanol to oil molar ratio . . × l mol- min- . . - . . - . [ ] spirulina platensis algae biomass u % c h so wt % of biomass, : wt/volume ratio . . . . . - . [ ] soybean oil s ch ona °c, : meoh:oil, wt % naoh . . . . . . - . [ ] leather tanning waste u: % s non catalytic - °c, mpa, : meoh:oil . - . . . - . . - . [ ] av = acid value mg koh/g of oil, m = microwave, c = conventional, u = un-saturation, us = ultrasound, s = supercritical, n = order of reaction, k = rate constant, a = pre-exponential factor, ea = activation energy, n.d. = not determined, meoh = methanol, cadg = calcium diglyceride [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] vol. , no. ( ) kinetic and thermodynamic studies of microwave-assisted transesterification of papaya oil esterification follows first order having lower activation energy . kj/mol. also, higher frequency factor min- and rate constant . min- at ºc revealed the non-thermal and thermal effect of the microwave. other thermodynamic properties such as ∆g, ∆h and ∆s were found out to be . kj/mol, . kj/mol and − . kj/mol k, respectively, which showed that reaction is non-spontaneous, endothermic and endergonic in nature. conflict of interest the authors declare that there is no conflict of interests regarding the publication of this article. references . m.h. ali, m. mashud, m.r. rubel and r.h. ahmad, procedia eng., , ( ); https://doi.org/ . /j.proeng. . . . . y.c. sharma and b. singh, fuel, , ( ); https://doi.org/ . /j.fuel. . . . . a.k. tiwari, a. kumar and h. raheman, biomass bioenergy, , ( ); https://doi.org/ . /j.biombioe. . . . . faostat, faostat, http://www.fao.org/faostat/en/#data/qc; (accessed july , ). . c. reddy, all about papaya in india ( ); http://theindianvegan.blogspot.com/ / /all-about-papaya-in- india.html. (accessed july , ). . w.-j. lee, m.-h. lee and n.-w. su, j. sci. food agric., , ( ); https://doi.org/ . /jsfa. . . f.o.a. tolulope and a. adewole, j. fuels, , ( ); https://doi.org/ . / / . . y.-c. lin, s.-c. chen, c.-e. chen, p.-m. yang and s.-r. jhang, fuel, , ( ); https://doi.org/ . /j.fuel. . . . . p.d. patil, v.g. gude, a. mannarswamy, p. cooke, s. munson-mcgee, n. nirmalakhandan, p. lammers and s. deng, bioresour. technol., , ( ); https://doi.org/ . /j.biortech. . . . . a. mazubert, c. taylor, j. aubin and m. poux, bioresour. technol., , ( ); https://doi.org/ . /j.biortech. . . . . the - conversion test | quality testing; http://www.make-biodiesel.org/quality-testing/the- - -conversion- test.html. 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(jackson: university of mississippi press, . pp. x + , preface, key texts, references, after- word, index.) stiofán Ó cadhla university college cork, ireland when we consider the s, our attention can- not help but be drawn to urban and contempo- rary legends. this decade has taken on the con- notations of revolution, rock and roll, sex, hippies, and feminism, all jostling in the final and fateful confrontation of tradition and mo- dernity. here, fairies and monsters are replaced by aliens and hook-handed killers, and myth and folktale are replaced by news and history—but legend continues to partake of both. this is, per- haps, legend’s central problematic. in her mar- velously accessible but scholarly style, gillian bennett goes straight to the heart of this prob- lematic, “the cultural clash of discordant catego- ries and concepts” (p. xv). she reminds us that one of the key facts about the legend is that it is difficult to define. legends are marked by their longevity, geographical spread, style, the multi- plicity of audio and visual media through which they are disseminated, and the recurrence of specific details or motifs. avoiding the carto- graphic pedantry (that is, the historical-geo- graphical or finnish method) of definition and delimitation, bennett points out that legend is not a scientific term and, as such, it has no real referent. legend can be superstition, relic, delu- sion, and curiosity, or it can be cool, new, sexy, urban, and teenaged. in the unfolding reassess- ments of the discipline, legend has been decon- structed or at least “declassified”—the distinc- tion between reality and legend is no longer considered to be clear-cut. contemporary leg- end, itself an orphan of the s, has in many ways become an exemplar of the contemporary life of the discipline. bennett’s bodies, therefore, is about folklore as much as it is about contemporary legend. in her encyclopedic detail and analysis, bennett draws attention away from the supposed nov- elty of the genre to broader generalizations about the discipline. following paul klee’s ap- proach to painting, bennett takes “a line for a walk,” exploring thematically the evolving shape and form of six particular legend case studies from their early variants to their con- temporary inflections (p. xv). here the shape- shifting element of story is exemplified. story is information, entertainment, strategy, news, gossip, rumor, warning, lesson, joke, photocopy, graffiti, fallacy, or political commentary—in short, a palimpsest of life. as a popular poetics of interpretation, legends may be better under- stood within contemporary discursive para- digms or contexts. they are a kind of social, book reviews journal of american folklore ( ) psychological, and cultural diary full of scrib- bles, and yet they cannot be reduced to the encoded fears of society. defying national and generic boundaries in literature, journalism, theater, television, or the internet, legends appear both homeless and uni- versal, old and modern, urban and rural. at a, “the murdered son” (or “the killing of the prodigal son,” as bennett prefers to call it), has a life of four centuries. the bosom serpent complex can be traced back to twelfth-century ireland; indeed, the idea of reptiles infesting the body was orthodox medicine. ancient greek and roman literature and european medieval monk- ish culture are saturated with similar themes. one could be tempted to see the tale-type or motif indices themselves as inventories of uni- versal themes, veritable handbooks of humanity, rather than exclusively folkloric checklists. one might ask, if urban legends remained seamlessly hidden in the streams of discourse for so long, are there other unlisted, sleeping genres? bennett includes examples drawn from china, india, ireland, and greece as well as from biblical or apocryphal narratives. the protagonist of legend may be rock hudson, and the storyteller may be jackie collins. legends do not end hap- pily ever after but in death or madness. current contamination themes echo earlier epidemics of typhoid, cholera, plague, syphilis, and leprosy. what if aids mary is just a newer version of typhoid mary? how does the researcher avoid jaded or jaundiced reductionist analysis that ap- pears to debunk folklore, legend, or the gullibil- ity of humanity? according to bennett, the sto- ries themselves “are enough,” and folklorists should consider documenting the presentation and use of legends as more than a trivial pursuit (p. ). bennett’s interpretations here are as diverse as the symbolic, cultural, and psychological meanings implicit in the genre. the approaches to contemporary legend cobbled together in this book confront the ideas that legends are false or trivial stories; are told to discredit certain com- panies; are derived from private fear, anxiety, or distrust; are cautionary tales; are a psychocul- tural response; are a vernacular etiology; are symbolic or metaphorical truths; are a reflection of gendered psychology; are serious and danger- ous; and are a projection of a desolate view of the human condition. the preferred approaches to legend study used in the book involve sam- pling the cultural complex that involves the leg- end or including all related material associated with the particular case study, highlighting a specific example with local behavioral and cul- tural norms, or viewing legend as a sociopolitical language where the pathology or symptomatol- ogy of the body mirrors the sociological or eth- nographic analysis of the social body. legend might create mainstream opinion, but it also fol- lows it. for example, aids legends might absolve heterosexuals from culpability in the spread of the disease, or stories of street urchin syringe aggressors may relocate danger beyond the en- virons of the home. bennett’s analysis is more revealing and intriguing for its careful consider- ation of vernacular gender perceptions that shape and create the imaginative undercurrents in the ocean of stories that she presents. the legend is an emotionally powerful and challenging genre. gillian bennett’s innovative and questioning exploration of this topic re- minds us that folklore studies sometimes has a tendency to trivialize reality as mere urban leg- end. folklore studies was long a romantic hob- byhorse, and today it must insist on its right to explore the social and cultural fallout caused by the quarrying of “mere folklore” from the most grotesque nadir of human behavior. leg- end tends to echo life, and life echoes legend as well; as a result, a common rejoinder to ac- counts of legends should be, perhaps, that they are not in fact legend, but truth. types of the folktale in the arab world: a demographically oriented tale-type index. by hasan m. el-shamy. (bloomington: indiana university press, . pp. xxviii + , bibli- ography, register of tale types, list of changed tale-type numbers, register of motifs, index of authors and sources, register of countries, tale- type subject index, addendum.) david elton gay indiana university as hasan el-shamy notes, the classification scheme of aarne and thompson’s tale-type index “is seldom adequate for identifying folk cultural minority rights and the rights of the majority in the liberal state* jean-christophe merle abstract. traditional liberalism’s blindness to cultural concerns has often come under fire, while so-called “liberal multiculturalism” (taylor and kymlicka) has made it its business to take a good look at the place of culture within liberal law. according to them, cultural minorities should be recognized. in my opinion, however, their pro- posals, in fact, almost entirely preclude the possibility that cultural minorities would receive recognition within liberal society. in what follows, i explain my view of these matters and, above all, argue for a more vital understanding of cultural minorities. this will entail presenting a comprehensive view of minority rights within liberal society. . integration of differences without uniformity liberal society’s traditional neutrality toward individual cultures, as captured by the notion, “equal treatment of all people regardless of their cultural differences,” seems prima facie to be opposed to a more comprehensive recognition of cultural minorities which are often, at best, only tolerated. in this connection, i would like to recall the principle from which the demand for equal treatment was derived: “the right to treatment as an equal [i.e., as a fellow citizen].” according to dworkin ( , ), equal treatment would thus represent a restriction of the right to be treated as an equal, which for liberalism is the highest of all principles. liberal law is intended to guaran- tee the greatest possible freedom in equal measure to all citizens. i think in applying this principle, however, there are at least three levels to be distinguished. the first has been most often discussed. at this level, freedom ratio juris. vol. no. september ( – ) © blackwell publishers ltd , cowley road, oxford ox jf, uk and main street, malden, ma , usa. * translated from the german by michael mcgettigan and cara gendel ryan. i would like to thank dr. martin gessmann and dr. christoph horn for their helpful comments. i wish to thank the humboldt-stiftung for generously funding the research fellowship at georgetown university which made it possible for me to write this article. of conscience, which is all-important for minorities, and the right to be protected from violence, as well as other basic freedoms, should hold without restriction in liberal law. this is where the origin of traditional lockean tolerance makes itself manifest. guaranteeing these human rights to one individual it is by no means necessary to limit them for any other individual. at the second level, the liberal state has recently gained new ground. this is the level of basic institutions (and also of the “nightwatchman state”). the task of these institutions is to ensure the most fundamental forms of free- dom. right from the start, they have abstracted from all individual religious, ethnic and gender affiliations by treating all citizens equally. for example, all official posts have been open to anyone with the requisite skills or abilities. nevertheless, many of these institutions bear the stamp of the traditional cul- tural majority, and some of their regulations are either at odds with, or com- pletely alien to the cultural orientation of various minority groups. examples include: christian holidays, military service, uniforms for civil servants, and, not least, official languages. at the same time, however, the state can adapt its institutions to minority groups, for instance, waiving mandatory closing times for businesses; in canada, allowing sikhs who are members of the police force to don traditional ethnic clothing. other examples include: wearing turbans instead of helmets on motorcycles; exemption from military service for quakers and for the amish; reinforcement of federalist structures (and provisions for a democratic process of secession); classroom teaching in a minority language or the inclusion of a second national language in core curricula; and finally, new history textbooks and state symbols which do not identify the nation with opinions held by the majority. these are new solutions which do not affect the state’s central functions: for example, its system of defense, internal security, traffic safety, public order, and the school system. in this way, it is possible that the freedom of certain citizens is not needlessly restricted, while the freedom of others remains completely intact. but there are problems generated by a multicultural society which require expensive solutions, and sometimes so prohibitively expensive as to preclude implementation; for example, there is a definite limit to the number of languages a state can recognize as official. the justice of such solutions is obviously measured by the principle of proportionality. this principle can be seen at work in the relation between the percentage of different minorities making up the general population and how much consideration is given their claims within particular institutional frameworks. a third level, foreign to traditional liberalism and therefore requiring further justification, consists in the institutions of cooperation and utility distribu- tion, which do not only depend on the free market but also on public justice. yet rawls, the first to open up this area for liberalism, does not situate cultural concerns at this level. rather he envisions culture to be the locus of conceptions about the good, conceptions which are themselves highly jean-christophe merle © blackwell publishers ltd . controversial. on this account, all members of society are free to make use of the social primary goods available to them in order to realize their own concep- tions of the good. in this regard, it seems to me that rawls basically under- stands culture to be a sphere falling outside the jurisdiction of the law. i will suggest two possible strategies for opening up promising yet liberal options for minority rights. i will sketch out the first strategy by following samuel black’s case ( , – ) against rawls’ censure of the public subsidizing of cultural institutions. black argues that “if cultural goods are consigned to the market, then one can safely predict that the members of society will get less of those goods than they would be willing to pay for [...]; for an entrepreneur cannot realize a return commensurable with her investment during a single life- time” (black , – ). this argument is liberal in so far as it is neither perfectionistic nor paternalistic. black refers to dworkin’s auction criterion: cultural institutions (black’s examples are museums and libraries) corres- pond to the preferences of many present and future citizens who would be willing to pay for them (i.e., to invest basic goods for this purpose), but only under the condition that the state makes such participation possible. in this way, black emphasizes, all liberal states act, so to speak, as catalysts for cooperation. this is something more than traditional freedom of association. if this were not so, we would be making private autonomy absolute at the expense of public autonomy, as george sher ( ) has recently shown in his argument for liberal perfectionism. five aspects of this model for minority rights, which black unfortunately does not treat, are of interest with regard to the role of culture. first, the argument is not only valid for culture in the narrow sense of fine arts, theatre, etc., but also for many other projects which only have to do with culture in the broader sense. railroad lines, airports, highways, public parks, and the like, are all subsidized by the state for the same reason. these, however, have nothing to do with safeguarding basic freedoms, but rather with our liberal, modern way of life—and not with the amish way of life or the reservation life of native americans, as many of multiculturalism’s representatives have correctly reminded us. not only the welfare state, but also the minimalist liberal state is undisputably committed to supporting these aspects of liberal life which underpin the culture of the majority. nevertheless, this is, in fact, a matter of using public resources to implement specific conceptions of the good. as sher has recently demonstrated ( , ), if conceptions of the good really represent our convictions about what is good for us, then they have a much greater scope than rawls allows them. for example, they will encompass “health, security and freedom.” yet the state is to follow a neutral principle (larmore , ), a criterion for pro- portionality, so that it neither privileges nor discriminates against a par- ticular minority. kymlicka ( , ) rightly demands that a % minority “should win % of the time.” according to kymlicka’s example, this means cultural minority rights © blackwell publishers ltd . if % of the voters want an opera, and % want swimming pools, then the public funds are not to be spent only for swimming pools, but proportionally for the opera as well. second, public support benefits all cultural groups equally, whether they make up a majority or a minority, whether they are small or large, whether they are a traditional, well-organized community or a loosely associated, newly formed affiliation. this strategy can integrate differences without levelling them out to uniformity (see walzer , ). third, the model leaves completely open the question of just how comprehensive a minority is. michael walzer ( ) is not alone in describ- ing liberalism as the “art of separation” of individual spheres each regulated by its own criterion of justice (the spheres of the economy, work, education, leisure time, love, religion, recognition, political power, welfare, etc.). every individual’s freedom consists in being treated in every sphere as equal to all other individuals in any given sphere. in the liberal state, membership in a particular group can only result from free choice. seen in this way, minority rights amount to the right to voluntary associations (walzer , ). in general, each individual simultaneously belongs to several groups, minorities as well as majorities. the model described above offers sufficient freedom not only to large and highly comprehensive groups (groups which are actively present in most or all spheres), but to all possible groups within each sphere. this model is thus able to do justice to the complexity of modern society, i.e., to the multiplicity of small groups (walzer , ). all inter- ests are taken into account: they are not fused together into a higher commonality, as is the case for hegel, but rather integrated into a legal community which does not demand uniformity. the argument cannot be applied, however, where a particular cultural group loses it voluntary adherents but nevertheless continues to make the same claims for itself (raz , ) as if nothing had changed. fifth, this third level owes nothing to the liberal state, but calls only for its own just and proportional application. this model does not commit the state to the conception of either an active state or nightwatchman state, but solely to respecting minor- ity rights. yet the question arises, whether or not this third level should fall under the competence of the state, as is the case in all existing liberal states, where the responsibility is usually recognized in terms of subsidies or at least various forms of tax relief. the alternative to state responsibility would be to give the cultural realm completely over to private initiative, and the citizens’ financial involvement thus would be strictly on a voluntary basis. this seems incorrect to me for the following reason. the culture handed down from earlier generations is our common heritage which we enjoy together. it is almost a natural resource and, therefore, can be treated in the same way as the environment. fairness demands that we bequeath our common culture and the environment in an acceptable condition to all future generations. we should replace all the cultural resources we exhaust with equally valuable jean-christophe merle © blackwell publishers ltd . cultural resources. the question of what constitutes “equally valuable,” however, must be left open here. in matters of public support, the criterion should be set according to the percentage of the population represented by each group and not according to the social and economic influence a group has on the society at large. the german system of church taxes, for example, would appear doubly inappro- priate in the light of such a criterion. on the one hand, it privileges estab- lished religions while discriminating against less recognized religions as well as other cultural groups (pogge ). on the other hand, the state does not give the churches the same amount of support for each individual, but a sum based on the taxes each individual actually pays. the financial support the church receives is thus dictated by the income of its members. as opposed to the current church-tax model, a principle of distributive jus- tice should be introduced for matters concerning the public support of culture. a fourth level does not exist in the liberal legal community. rather, it has its place in society in the broadest sense and rests on the citizens’ free initiative. the liberal state cannot and should not want to change the fact that some groups have more members than others, or cohere better than others, or are culturally better equipped or socially and economically more successful than others. these considerations fall outside the sphere of law. . kymlicka’s view of culture as a basic good the second strategy, which i ascribe to kymlicka (kymlicka ), shares a common feature with the first. what i have described as the first point is also present in kymlicka’s account. but his strategy differs from the first in two other respects. it is well known that kymlicka does not proceed from a principle or criterion, but rather adopts a typology for his starting point. iris marion young ( , – ) has already perceptively criticized kymlicka’s typology for its omissions of intermediary steps and its lack of nuanced analyses, and i will not reiterate her objections here. instead, i will try to show that, as a consequence of his typological approach, kymlicka overlooks certain cultural groups. on the one hand, his categories consist in immigrant or “ethnic minorities” and, on the other hand, in aboriginals or “national minorities.” kymlicka’s model of the liberal state integrates the immigrant groups while according special rights to aboriginal people. his justification for this distinction rests partly on reasons of motivation. immigrants are pre- pared to integrate themselves as long as the liberal state makes the necessary adjustments on the second level, such as exemptions from mandatory busi- ness closing times, regulations concerning uniforms and the like. aboriginal people, according to kymlicka, do not want to be integrated, but rather desire affirmation of their special status as original inhabitants. interestingly, kymlicka has precious little to say about minority rights for immigrants or cultural minority rights © blackwell publishers ltd . for citizens who are not immigrants. in the case of immigrants, he only considers what i have identified as second-level concerns (exemption from laws regulating holidays, uniforms, military service, etc.), and not the further level of free cooperation. on this level, subsidies for cultural organ- izations and projects planned by immigrant minorities indeed constitute a minority right. they also play an important role in integrating minority groups into the liberal state without running the risk of compulsory uniformity. i will return to a discussion of this level. but first, kymlicka’s two nor- mative arguments need to be examined. his starting point is rawls’ theory, which he transforms in two ways. first, he transposes the cultural realm from the sphere where conceptions of the good arise to the sphere of resources, that is, to the sphere of social primary goods and, therefore, of cooperation. thus culture per se, conceptions of the good, must be supported by the state. second, kymlicka ( , ) makes membership in one culture, or more precisely, in one’s own cultural community, a social primary good. our own cultural community provides the background for our decisions (a “context of choice”). to this extent, such membership is to be supported. but this second alteration of rawls’ theory explicitly shows the one-sidedness which the first alteration concealed. to have a culture means to be educated as well as socialized. education requires both diachronic cooperation with a historical community’s tradition, and synchronic, highly differentiated cooperation with contemporaries. but a distinction must be made between the cognitive aspect of culture (trans- mission of practical and theoretical knowledge from one generation to the next) and the voluntary aspect of culture (the determination of actions and values by principles). by means of these two aspects, culture becomes individuated. we share the same cultural horizon in a cognitive sense with others. the voluntary side of individuation consists in how we determine ours lives on the basis of decisions, which may or may not coincide with the decisions others make. kymlicka also seems aware of the distinction between the cognitive and voluntary dimensions when he distinguishes the voluntary character from the cognitive structure of cultural communities (see kymlicka , – ). one can, of course, indiscriminately declare all of culture to be a mere resource, as kymlicka does. and it is entirely true that the cognitive element influences the voluntary; but unless one assumes a monocausal model, the reverse is also true. even such monocausality does not per se contradict liberalism; for a monocausal determination of voluntary actions does not in any way imply fatalism, so long as we as individuals do not understand how this form of determinism works. nevertheless, liberalism should avoid monocausal explanations. for liberalism, individual freedom is not a cogni- tive assumption, but rather a basic working hypothesis. this is precisely why the cognitive and voluntary dimensions must be carefully held apart. jean-christophe merle © blackwell publishers ltd . only in the first dimension can culture be understood as a social primary good; in the second dimension, culture consists in conceptions of the good. culture in general, and not a particular culture, is a social primary good. at the very latest, since kant wrote his idea for a universal history ( ), we have been aware that our humanity cannot be realized to its full extent without culture which is always social. and a particular culture is certainly a social good, but not a primary social good. in addition, kymlicka deals implicitly only with those comprehensive minorities actively present in all the different spheres. he does not distinguish among the spheres of individual, particular social goods. thus immigrants are only viewed from the perspective of their religion. for example, kymlicka portrays the inuit people as belonging to a comprehensive community which equally encompasses economic, political, social, linguistic and educational concerns. so much for kymlicka’s first step. the fact that we are fundamentally cultural animals certainly plays a part in how we arrive at our various conceptions about what the good is. but these conceptions can only be realized in a particular, concrete way. thus, if we are to follow kymlicka’s second step, we must also consider that par- ticular cultural communities are social goods which, for particular conceptions of the good, are nevertheless primary goods, i.e., goods which are uncon- ditionally valid for all possible conceptions of the good and are, therefore, the conditions for the good as such. it thus follows that particular cultural communities warrant unconditional protection. if, however, only culture in general is held to be a social primary good then it alone warrants protection against all forms of inhumanity. any demand for cultural integration, there- fore, is a human right. at least two important consequences follow from the claim that every particular cultural group represents a social primary good for its members. first of all, affiliations with a cultural minority will remain a good for the entire life of all the members of a given minority. taylor rejects viewing the language of a historical cultural community “as a collective resource which individuals might want to make use of” and which “is available for those who might choose it” (taylor , ). for his part, however, kymlicka recog- nizes the right of all individuals to leave their communities, but typically he neglects to pursue the point further. i will return to this issue after first laying out the second, more fundamental consequence. for rawls, social primary goods are valid for the entire society; on the level of the political community taken as a whole, they are distributed according to a principle of justice. in this case, if every particular group, and that means every cultural minority, is viewed as a social primary good, then ) every cultural group is simultaneously defined as a political group, and therefore ) different cul- tural groups should be considered as different political societies, that is, as states. this leads to a different notion of minority rights which i will now proceed to develop. cultural minority rights © blackwell publishers ltd . . integration or tolerance in the meaning of international law? on kymlicka’s account, immigrants are integrated. in contrast, the second minority category, the aboriginal community, is entitled to more than a right; they are entitled to recognition. it seems to me that the concept of recognition can be understood in terms of international law. at first, this may seem paradoxical given that kymlicka explicitly dis- cusses minorities, that is, internal groups. he works primarily with the examples given by the inuits and other native americans living in canada. he justifies aboriginal rights, which “entail special costs for other people, by restricting the rights and resources of non-aboriginal people” (kymlicka, , ), by arguing for equal opportunity. aboriginal rights are “a response (. . .) to unequal circumstances” ( , ). kymlicka compares the prospects of a young inuit girl with those of an anglo-canadian boy; the girl is socially and economically disadvantaged because she belongs to those who are “forced to try to execute their chosen life-styles in an alien culture, e.g., in their work, and, when the state superstructure is built, in the courts, schools, legislatures, etc.” ( , ). thus, for the aboriginal people “it is necessary to outbid non-aboriginal people just to ensure that their cultural structure survives, leaving them few resources to pursue the particular goals they’ve chosen from within that structure” ( , ). to this end, as is well known, different measures have been taken which are valid only for reser- vations: it is forbidden for aboriginals to sell land to non-aboriginals, and non-aboriginals are not allowed to settle on reservations. non-aboriginals can participate in local elections only after ten years have elapsed; they are denied the right to hunt and trap. the widows of aboriginals are not allowed to continue living on the reservation if they themselves are not aboriginals. and the rights of seasonal workers are restricted in still other ways. accord- ing to kymlicka, the justification for these special rights is the same as the justification for affirmative action ( , f.). leaving the controversial aspects of affirmative action aside, we may note that it aims at integrating members of minorities into common institutions. it is not the goal of any form of affirmative action to condemn minorities to live at the margins of society. before embarking on a critical examination of kymlicka’s arguments, i would like briefly to show that there is an alternative solution. the solution is classical but nevertheless effective. it consists in social measures as well as in a school system oriented toward integration, and it insures real, equal op- portunities for aboriginals in the “courts, schools, and legislature” (tomasi , – ). an appropriate system would also avoid the sad episode related by kymlicka ( , ): imitating a television series, a group of indonesian children jumped from a cliff to their death. a possible objection from taylor’s point of view ( , f.) would be that social and educational measures, and all redistribution models, overlook jean-christophe merle © blackwell publishers ltd . the special character of demands made within a politics of difference. but kymlicka justifies the special status of the aboriginal population by appeal- ing to the socio-economic inequality originating in the culture. in addition, social and educational measures also have an effect on cultural conditions. as joseph raz has argued, educational institutions could convey liberal culture in english and the local culture in the local language. in contrast, a school system with inuit as its only language will severely limit the cultural context—the social good in the above example of the young inuit girl. by the same token, a liberal state could also subsidize cultural and artistic asso- ciations which would help strengthen a sense of self among the aboriginal people. and not least, local enterprises would be supported as well. all these approaches could help safeguard cultural interests and thereby perhaps even augment aboriginal culture. donald kraybill and steven nolt ( ) show that other minorities, for example, the amish, even without such support, have been quite successful not only in maintaining their cultural tradition but also in increasing their economic productivity. the alternative i have been suggesting ensures that the inuit people will not suffer from disadvantages connected with their special status. the special status, however, involves disregarding the basic demand for coopera- tion, i.e., the principle of reciprocity (höffe ), and thereby infringes upon the majority’s rights and interests. also, kymlicka admits that “the special status of the aboriginal people” is an “imperfect” solution which is nonethe- less “acceptable” because compared to any other solution, it requires fewer “sacrifices” from the majority. i am of the opinion, however, that his solution would, in fact, impose greater sacrifices than the alternative i am proposing. first, although the infringement or restriction of the rights of the canadian majority in the northwest territories is obvious to kymlicka, he overlooks the fact that in the rest of canada the aboriginals are not in any way dis- advantaged. indeed, they profit from affirmative-action programs. and if they stay within the boundaries of their territory, they will also receive all the usual social services canada has to offer. this doubly one-sided support illustrates the tension described by david miller ( , ): the tension between the demand for a politics of difference and the demands for comprehensive equality put forward by affirmative action. it certainly mirrors canadian society’s bad conscience stemming from the past injustices it perpetrated on its own original inhabitants. kymlicka ( , , note ) admits that the special status cannot be founded on cor- rective justice (for, in this case, the solution would be, at best, only material, i.e., financial and social, and not institutional), though he emphasizes that only the least productive land was left to the aboriginal people. in canada, the same is also true with regard to the demands put forward by quebec’s independence movement; for example, the petition for representation in parliament regardless of plans for future secession, or the demand for a canadian passport for all quebecers after seccession without conceding the cultural minority rights © blackwell publishers ltd . same privilege in quebec, i.e., quebecer passports for all canadians after secession. second, a similar tension can be found within the aboriginal population. aboriginals are not allowed to sell property to non-aboriginals; this considerably infringes on their right to resettle elsewhere. also, in some cases, they are prevented from appealing to the canadian supreme court. non-aboriginal people are not allowed to remain in the territory after the death of their aboriginal spouses. children are only allowed to attend schools which teach in the local language, etc. these and many other similar restrictions are directed against those members of the aboriginal population who wish to leave their communities, even though this is one of their basic rights. perhaps one way to ameliorate this situation would be, for example, to offer options to buy land, which give priority to aboriginals. this would certainly be more liberal than a ban on non-aboriginal purchasing. tomasi ( , ) has detected in the aboriginals’ attitude toward their identity a contradiction between the desire to preserve their own culture and the desire to leave it for good. one reason for this is that the culture of many aboriginals is neither purely traditional nor that of the majority culture: it is a transitional culture. as i observed at the beginning, liberal minority rights no longer suffice for the preservation of cultural communities when their own members cease to cooperate actively. however painful this schism may be for those involved, the liberal state is not to “blame” for this process of modernization, and it is not in any way obliged to introduce corrective measures. a possible objection might be that the majority culture is just too tempting. but liberal society is not allowed to change this fact; for its task is to con- centrate on treating citizens, and not cultures, as equals. a classic example would be, as max weber discovered, how certain religions and the corres- ponding patterns of behavior they encourage strongly influence financial success. nevertheless, no one has ever seriously recommended granting catholics special rights denied to protestants. for this reason i have my doubts about rawls’ new method (as presented in rawls a, b). my position does not contradict multiculturalism, for while liberal multi- culturalism is not fundamentally against integration, it is certainly against compulsory integration (raz , ). liberal multiculturalism is a defense of liberal minority rights. if the demand for minority recognition is explained in terms of a special status based upon the model provided by international law, the demand will appear more coherent. tully ( ) documents the international legal con- tracts which forced the native american population to cede large portions of their territory and their sovereignty to the british colonial power. he explains how in the th century the white population, which had swelled to a majority, forgot the international status of these contracts. only recently have they been taken seriously by canadians. for tully, this is reason enough to demand a fundamental right for the aboriginal people, a right which does not need to jean-christophe merle © blackwell publishers ltd . be justified by the standards of the liberal majority in canada. it is remark- able that kymlicka sometimes also argues in terms of international law, for example, when he stresses that the special status of minorities is based upon a complete separation of two legal systems ( , chapter . , esp. ), or when he sees the ban on immigration to aboriginal territories being tied to the liberal state’s sovereignty in matters of immigration ( , ). all the same, kymlicka and tully do not argue for reinstating the sover- eignty of individual aboriginal groups. the status accorded these groups by both authors resembles more closely an enclave on the margins of and not within liberal society. this status is not even commensurate with a federal state which at least really does possess political authority and the responsi- bilities that go along with it. neither side is even interested in attaining offical status within a federal state. but this status invalidates tully’s prag- matic argument, according to which the presence of too many cultural groups makes the independence of each untenable and the special status for each necessary ( , f.). if the aboriginal population were to be granted federal state status, it would have to come to terms with the rights of present minorities and with the individual rights within its own minority commun- ity. moreover, it would have to secure the internal cooperation of its mem- bers, their social and economic development, as well as cooperation with the outside world. the organization of such foreign relations requires that mem- bers must be willing to cooperate on the home front so that the community would be compelled to face the problems surrounding individual rights— the right of free association, right to emigrate, and others as well. last but not least, this new state would be responsible for, and have to answer to international law, international organizations, and the community of nations. in grappling with these problems aboriginals would prove whether they really possessed, and really wanted to possess, the identity some have claimed on their behalf. instead of this, kymlicka has only “pluralism in the strong sense” to offer. as walzer correctly remarked, this is “possible only under tyrannical regimes” (walzer , ), as was the case in the turkish millet system. conclusion identity is not immutable, and it cannot be tied to a plot of land. it changes and requires continually renewed forces of subjective identification. rigor- ous preservation of an inherited “original” identity is possible, but seldom achieved. one success story in this respect is the amish community in liberal america. it has survived unchanged for hundreds of years, and it has only done so thanks to the extraordinary sense of belonging that the community has been able to instill in its members. it is interesting to note that the amish seldom make demands for special rights. they expand their property by purchasing land on the free market. they insist, however, on a completely cultural minority rights © blackwell publishers ltd . separate educational system which considerably impairs the ability to adapt to non-amish culture, especially among adults who want to leave the com- munity. because children are, in fact, different persons from their parents, the liberal state should not allow parents to have unlimited authority over them. some more recent minorities arising out of world-wide immigration enjoy strong solidarity as testified by the various chinatowns and little italies scattered across the globe. as long as they do not exercise violence up on their members, their existence—even as territorially settled groups—is com- pletely compatible with liberal society. but such groups do not hold as much fascination for us as those which draw upon the mythological, “original” authenticity we so often seek in native populations. while there is noth- ing wrong with a little rousseauian nostalgia, we must not translate such longing into law. nor should we partition off the liberal society of a disen- chanted world from regions basking in the aura of supposed authenticity and very real special rights. as walzer has written ( , ), no right “to cultural security” exists. “minority” means literally minority within society. on the one hand, kymlicka really only takes account of “national minor- ities” which are equipped with a special status partially based on consider- ations arising from international law. on the other hand, kymlicka looks to immigrants who have no wish to rely completely on the spectrum of minor- ity rights. the field of minority rights and multiculturalism is indeed much broader than kymlicka can account for. perhaps the identities of our liberal societies and their minorities change more rapidly and are more complex now than in previous times. this may be a cause for regret in that it undermines minority cohesion and thus multi- culturalism itself. for my part, i see in all this a welcome departure from the myth of the primacy of strong and rigid national identities (walzer , ), and the possibility of revitalizing minority cultures. it is unnecessary for the neutral liberal state either to strive for multiculturalism as a collective and individual identity or to fear it. but it makes such a plurality of identities pos- sible like no other form of legal community. georgetown university department of philosophy washington, d.c. usa references black, samuel. . revisionist liberalism and the decline of culture. ethics : – . dworkin, ronald. . taking rights seriously. london: duckworth. höffe, otfried. . political justice. oxford: polity press. kraybill, d.b., and s.m. nolt. . amish enterprise. baltimore: the johns hopkins university press. jean-christophe merle © blackwell publishers ltd . kymlicka, will. . liberalism, community, and culture. oxford: clarendon. ——— . . multicultural citizenship. oxford: clarendon. larmore, charles. . patterns of moral complexity. cambridge, ma: cambridge university press. miller, david. . on nationality. oxford: oxford university press. pogge, thomas. . group rights and ethnicity. in ethnicity and group rights. eds. i. shapiro and w. kymlicka. (nomos ). new york: new york university press. rawls, john. a. political liberalism. new york: columbia university press. ——— . b. law of people. in on human rights. eds. s. shute and s. hurley, – . new york: basic books. raz, joseph. . ethics in the public domain. essays in the morality of law and politics. oxford: clarendon. sher, george. . beyond neutrality: perfectionism and politics. cambridge: cambridge university press. taylor, charles. . multiculturalism and “the politics of recognition.” princeton: princeton university press. tomasi, j. . kymlicka, liberalism and respect for cultural minorities. ethics : – . tully, james. . strange multiplicity: constitutionalism in an age of diversity. cambridge: cambridge university press. walzer, michael. . pluralism: a political perspective. in harvard encyclopedia of american ethnic groups. ed. s. thernstrom. cambridge, ma: harvard university press. ——— . . spheres of justice: a defense of pluralism and equality. oxford: martin robertson press. ——— . . liberalism and the art of separation. political theory: . ——— . . the new tribalism. dissent: – . young, iris marion. . a multicultural continuum: a critique of will kymlicka’s ethnic-nation dichotomy. constellations / : – . cultural minority rights © blackwell publishers ltd . microsoft word - supplement_r _finalsubmission_ _clean.doc new loci associated with kidney function and chronic kidney disease supplement anna köttgen*, cristian pattaro*, carsten a. böger *, christian fuchsberger *, matthias olden, nicole l. glazer, afshin parsa, xiaoyi gao, qiong yang, albert v. smith, jeffrey r. o’connell, man li, helena schmidt, toshiko tanaka, aaron isaacs, shamika ketkar, shih-jen hwang, andrew d. johnson, abbas dehghan, alexander teumer, guillaume paré, elizabeth j. atkinson, tanja zeller, kurt lohman, marilyn c. cornelis, nicole m. probst-hensch, florian kronenberg, anke tönjes, caroline hayward, thor aspelund, gudny eiriksdottir, lenore launer, tamara b. harris, evadnie rampersaud, braxton d. mitchell, dan e. arking, eric boerwinkle, maksim struchalin, margherita cavalieri, andrew singleton, francesco giallauria, jeffery metter, ian de boer, talin haritunians, thomas lumley, david siscovick, bruce m. psaty, m. carola zillikens, ben a. oostra, mary feitosa, michael province, mariza de andrade, stephen t. turner, arne schillert, andreas ziegler, philipp s. wild, renate b. schnabel, sandra wilde, thomas f. muenzel, tennille s leak, thomas illig, norman klopp,christa meisinger, h.-erich wichmann, wolfgang koenig, lina zgaga, tatijana zemunik, ivana kolcic, cosetta minelli, frank b. hu, Åsa johansson, wilmar igl, ghazal zaboli, sarah h wild, alan f wright, harry campbell, david ellinghaus, stefan schreiber, yurii s aulchenko, janine f. felix, fernando rivadeneira, andre g uitterlinden, albert hofman, medea imboden, dorothea nitsch, anita brandstätter, barbara kollerits, lyudmyla kedenko, reedik mägi, michael stumvoll, peter kovacs, mladen boban, susan campbell, karlhans endlich, henry völzke, heyo k. kroemer, matthias nauck, uwe völker, ozren polasek, veronique vitart, sunita badola, alexander n. parker, paul m. ridker, sharon l. r. kardia, stefan blankenberg, yongmei liu, gary c. curhan, andre franke, thierry rochat, bernhard paulweber, inga prokopenko, wei wang, vilmundur gudnason, alan r. shuldiner, josef coresh, reinhold schmidt, luigi ferrucci, michael g. shlipak, cornelia m. van duijn, ingrid borecki, bernhard k. krämer, igor rudan, ulf gyllensten, james f. wilson, jacqueline c. witteman, peter p. pramstaller, rainer rettig, nick hastie, daniel i. chasman **,w. h. kao **, iris m. heid ** caroline s. fox ** *these co-authors contributed equally to this work **these senior authors jointly oversaw this work table of contents . supplementary note a. study-specific methods and full acknowledgements page . supplementary tables a. supplementary table a: study design and sample sizes page b. supplementary table b: genotyping and imputation platforms page c. supplementary table : genome-wide significant loci: snp imputation quality in stage discovery cohorts page d. supplementary table : genome-wide significant loci: snp association across renal traits in stage discovery and stage replication meta-analyses page e. supplementary table : additional gene biology of novel loci page f. supplementary table : effect sizes of association with egfrcrea across strata of diabetes and hypertension page g. supplementary table : expression associated snp analysis page h. supplementary table : additional snps associated with egfrcrea and ckd at an fdr of . page . reference list page . supplementary figures a. supplementary figure : quantile-quantile plots of observed vs. expected -log (p-values) from discovery analyses of egfrcrea (a), ckd (b), and egfrcys (c). page b. supplementary figure : locus-specific regional association plots for susceptibility loci for reduced renal function and ckd. page supplementary note – study-specific methods and full acknowledgements discovery cohorts age gene/environment susceptibility reykjavik study (ages): the ages-reykjavik study represents a sample drawn from the established prospective population-based cohort, the reykjavik study. between and , the ages-reykjavik study re-examined survivors of the original cohort. serum creatinine was measured at the icelandic heart association using the roche-hitachi p-module instrument with roche creatininase plus assay. the coefficient of variation (cv) for the creatinine assay was . %. covariates were obtained at examination. genotyping was performed at the molecular genetics section and laboratory of neurogenetics, nia, nih, using using the illumina cnv beadchip array on participants. standard protocols for working with illumina data were followed, with clustering score greater than . . amish studies (amish): old order amish individuals included in this study were participants of several ongoing studies of cardiovascular health carried out at the university of maryland. participants were relatively healthy volunteers from the old order amish community of lancaster county, pennsylvania and their family members. , study participants were enrolled within the - timeperiod. serum creatinine was measured using a modified kinetic jaffé reaction. cystatin c measured using a particle-enhanced immunonephelometric method bnii, (dade-behring). covariates were obtained at the index examination. atherosclerosis risk in communities study (aric): the aric study is an ongoing prospective population-based cohort study to investigate the etiology of atherosclerosis. the study started in - with the enrollment of , adults aged - years in four us communities; participants mostly self-identified as black or white. participants for the current study included those with measures of serum creatinine from visits , ( - ), or ( - ) for the analyses of egfrcrea and ckd, and from visit for the analyses of egfrcys. serum creatinine was measured using a modified kinetic jaffé reaction. ckd was defined as cumulative ckd prevalence for individuals with egfrcrea < ml/min/ . m at aric visits , , or . individuals were not counted as ckd cases if ckd at an earlier study exam was not apparent at a later study exam, unless there was also a ckd related hospitalization from continuously collected hospitalization discharge records. serum cystatin c was measured by a particle enhanced immunonephelometric assay (n latex cystatin c, dade behring). covariates were obtained at the visit from which egfr was used in analyses. austrian stroke prevention study (asps): the austrian stroke prevention study is a single center prospective follow-up study on the effects of vascular risk factors on brain structure and function in the normal elderly population of the city of graz, austria. the procedure of recruitment and diagnostic work-up of study participants has been described previously. , a total of european caucasian participants were randomly selected from the official community register stratified by gender and year age groups. individuals were excluded from the study if they had a history of neuropsychiatric disease, including previous stroke, transient ischemic attacks, and dementia, or an abnormal neurologic examination determined on the basis of a structured clinical interview and a physical and neurologic examination. since , blood was drawn from all study participants for dna extraction; covariates were obtained at the index examination. serum creatinine was measured. baltimore longitudinal study of aging (blsa): the baltimore longitudinal study of aging (blsa) is an observational study that began in to investigate normative aging in community dwelling adults who were healthy at study entry. participants are examined every one to four years depending on their age. currently there are approximately active participants enrolled in the study. serum and urinary creatinine was measured by an enzymatic method using the vitros analyzer (johnson & johnson co., rochester, ny). the modification of diet in renal disease (mdrd) study equation was used to calculate egfrcrea. the analysis was restricted to subjects with european ancestry. each analysis was further adjusted for the top two principal components derived from an eigenstrat analysis utilizing ~ , randomly selected snps from the k snp panel. cardiovascular health study (chs): the cardiovascular health study (chs) is a population- based longitudinal study of risk factors for cardiovascular disease and stroke in adults years of age or older, recruited at four field centers (forsyth county, nc; sacramento county, ca; washington county, md; pittsburgh, pa). individuals of predominantly european ancestry were recruited in - from random samples of medicare eligibility lists, followed by an additional african-americans recruited in - (total n= ). a total of persons were excluded from the gwas study sample due to the presence at study baseline of coronary heart disease, congestive heart failure, peripheral vascular disease, valvular heart disease, stroke or transient ischemic attack or lack of available dna. african american participants were excluded from this analysis since the other cohorts were predominantly of european ancestry. using a particle-enhanced immunonephelometric method, cystatin c was measured (bnii, dade-behring). covariates were obtained at baseline. erasmus rucphen family study (erf): the erasmus rucphen family (erf) study is comprised of a family-based cohort embedded in the genetic research in isolated populations (grip) program in the southwest of the netherlands. descriptions of erf’s design have been previously published. briefly, twenty-two families that had a minimum of five children baptized in the community church between and were identified with the help of detailed genealogical records. all living descendants of these couples, and their spouses, were invited to take part in the study. participants included in the current study total individuals for whom complete phenotypic and genotypic information was available. covariates were obtained during the baseline examination. family heart study (famhs): in , the family heart study began with the ascertainment of families, half due to excess of coronary heart disease (chd) or abnormal risk factors as compared with sex- and age-specific population, the other half randomly selected. these families, including about individuals, were sampled from four geographically diverse field centers: the framingham heart study, the utah family tree study, and two aric centers (minneapolis, and forsyth county, nc). after about years, a total of participants of european ancestry in extended pedigrees were invited for a second clinical exam. a two- stage design was used for the gwas conducted for this study. in the first stage, individuals chosen to be largely unrelated were selected, half from the highest quartile and half from the lowest quartile of sex- and age-adjusted coronary artery calcification values. the results presented here were derived using the first stage case-control sample. we did not observe association of egfrcrea with the first ten principal components derived from the gwas genotypes using eigenstrat. framingham heart study (fhs): in , the framingham heart study began when the original cohort was enrolled. beginning in , the offspring cohort was enrolled ( participants); the methodology and design has been described. , in , the third generation cohort was enrolled (n= ). participants for the current study include individuals from the original cohort who attended cohort exam ( to ) or exam ( to ) [n= ], as well as participants from the offspring cohort who attended the second exam ( - ) or the seventh exam ( - ) [n= ], and individuals from the third generation first examination (for egfr only). ckd was defined as cumulative ckd prevalence for individuals with egfr < ml/min/ . m at exam cycle (original cohort) and exam cycle (offspring cohort) for the earlier exams and exam (original cohort) and exam (offspring cohort) for the later exam cycles. using a particle-enhanced immunonephelometric method, cystatin c was measured (bnii, dade-behring). covariates were obtained at the index examination. we observed no association with ckd with the principal components estimated using eigenstrat. significant association between egfr and the principle components was observed, therefore principle components were included in the analysis for association between genotype and egfr. kora studies (kora f and kora f ): the kora surveys for genetic research have been described in detail previously and have been initiated as part of the monica (monitoring of trends of cardiovascular diseases) multi-center study. the third kora survey (kora s ) is a population-based sample from the general population of the south-german city of augsburg and surrounding counties, recruited / . a subsample consisting of individuals from this survey with -year follow-up (kora f ) information available was successfully genotyped..the fourth kora survey (kora s ) is a sample recruited - independent from kora s using the same platform with the same standard operating procedures and based on the same population. from the sample with a -year follow-up (kora f ), subjects were available for the gwa analysis. all participants had a german passport and were of european origin. serum creatinine in kora f and f was measured using a modified kinetic jaffé reaction. using a particle-enhanced immunonephelometric method, serum cystatin c was measured (bnii, dade-behring). korcula study (korcula): the korcula study (korcula) is a family-based, cross-sectional study on the dalmatian island of korcula. data for participants aged years and over were used for this analysis. fasting blood samples were collected and over health-related phenotypes and environmental exposures were measured in each individual. plasma creatinine was measured with the jaffé rate method. korcula, micros, nsphs, orcades, and vis used a polygenic linear model fitted to the residuals of log(egfrcrea) on age for estimating the inverse of the variance/covariance matrix, which accounts for the inter-individual relatedness and is based upon a genomic kinship matrix implemented in genabel. genome-wide association between snps and the residuals was assessed by means of an approximate score test statistic . for ckd, relatedness was accounted for by estimating the first three principal component of the genomic kinship matrix by multidimensional scaling. a genome-wide association scan was performed by means of a logistic regression model adjusted for age, sex, and the first three principal components. microisolates in south tyrol study (micros): the micros study is part of the genomic health care program 'gennova' and was carried out in three villages of the val venosta, south tyrol (italy), in - . it comprised members of the populations of stelvio, vallelunga and martello. a detailed description of the micros study is available elsewhere. information on the participant’s health status was collected through a standardized questionnaire. laboratory data were obtained from standard blood analyses. serum creatinine was measured by an enzymatic photometric assay using an advia clinical chemistry analyzer (siemens healthcare diagnostics gmbh, eschborn, germany) (pmid ) and cystatin c was measured on a bn-prospec analyzer (dade behring, marburg, germany). covariates were obtained during the interview phase. for study-specific statistical methods, see above page . northern swedish population health study (nsphs): the northern swedish population health study (nsphs) is a family-based study including a comprehensive health investigation and the collection of data on family structure, lifestyle, diet, medical history and of samples for laboratory analyses. , participants came from the northern part of the swedish mountain region (county of norrbotten, parish of karesuando). historic population accounts show that little migration or other dramatic population changes have occurred in this area over the last years. plasma creatinine was measured by an enzymatic photometric assay using an advia clinical chemistry analyzer (siemens healthcare diagnostics gmbh, eschborn, germany). for study-specific statistical methods, see above page . orkney complex disease study (orcades): the orkney complex disease study (orcades) is an ongoing family-based, cross-sectional study in the isolated scottish archipelago of orkney. genetic diversity in this population is decreased compared to mainland scotland, consistent with high levels of historical endogamy. participants were aged - years and came from a subgroup of ten islands. fasting blood samples were collected and over health-related phenotypes and environmental exposures were measured in each individual. plasma creatinine was measured by an enzymatic photometric assay using an advia clinical chemistry analyzer (siemens healthcare diagnostics gmbh, eschborn, germany). for study-specific statistical methods, see above page . rotterdam study i (rsi) and rotterdam study ii (rsii): the rs is a population-based cohort study aimed at assessing the occurrence of and risk factors for chronic diseases in the elderly. in brief, all inhabitants of ommoord, a district of rotterdam in the netherlands, who were years or older were invited and (rsi) agreed to participate ( % response rate). - the baseline visits took place between - . in , inhabitants who turned years of age or moved into the study district since the start of the study were invited (rsii) of whom participated ( % response rate). serum creatinine was assessed by a nonkinetic alkaline picrate (jaffé) method (kone autoanalyzer; kone corp, espoo, finland, and elan; merck, darmstadt, germany). covariates were obtained at baseline. diabetes was defined as use of antidiabetes medication or abnormal nonfasting glucose or an abnormal oral glucose tolerance test. a nonfasting or post load glucose level . mmol/l was considered abnormal. study of health in pomerania (ship): the study of health in pomerania (ship) is a longitudinal population-based cohort study conducted in west pomerania, the north-east area of germany. for the baseline examinations, a sample of eligible subjects aged to years was drawn from population registries. only individuals with german citizenship and main residency in the study area were included. baseline examinations were conducted between and . between and all participants were re-invited for an examination follow-up, in which subjects ( . % of eligible persons) took part. serum creatinine levels were determined according to the jaffè method. serum cystatin c levels were measured using the siemens n latex cystatin c assay, a particle-enhanced nephelometric immunoassay, on the bn prospec® system. the genetic data analysis workflow was created using the software inforsense. genetic data were stored using the database caché (intersystems). vis study (vis): the vis study (vis) is a family-based, cross-sectional study on the dalmatian island of vis. , data for participants aged years and over were used for this analysis. fasting blood samples were collected and over health-related phenotypes and environmental exposures were measured in each individual. serum creatinine was measured by an enzymatic photometric assay using an advia clinical chemistry analyzer (siemens healthcare diagnostics gmbh, eschborn, germany). for study-specific statistical methods, see above page . women’s genome health study (wghs): the women’s genome health study (wghs) is a prospective cohort of female north american health care professionals representing participants in the women’s health study (whs) who provided a blood sample at baseline and consent for blood-based analyses. participants in the whs were or older at enrollment and free of cardiovascular disease, cancer or other major chronic illness. serum creatinine was measured in the baseline blood sample. covariates were assessed at the index examination. the current data are derived from , wghs participants for whom whole genome genotype information was available at the time of analysis and self-reported european ancestry could be confirmed by multidimensional scaling analysis of ancestry informative markers in plink v. . . in silico replication cohorts aric: additional genotype data became available on self-reported white aric participants over the course of this study. these individuals were not part of the discovery samples, did not have a first-degree relationship with any individual in the discovery sample, nor would they have been classified as an outlier based on allele sharing measures applied in data cleaning of the discovery sample. family heart study: in the replication stage, family members with self-reported white ancestry are used for replication; and participants were genotyped. we used field centers in regression models to account for potential population stratification. genoa: the genoa study of the family blood pressure program (fbpp) was initiated between - to identify genetic determinants of hypertension in multiple ethnic groups. , in rochester, mn, the mayo clinic diagnostic index and medical record linkage system of the rochester epidemiology project were used to identify non-hispanic white (nhw) residents of olmsted county with a diagnosis of essential hypertension made before age . sibships in which either index hypertensive sibling was known to have impaired kidney function (e.g., serum creatinine . mg/dl) were not recruited, as impaired kidney function may cause secondary hypertension. a second stage of the fbpp was undertaken between - to identify genetic determinants of susceptibility to cardiac and renal complications of hypertension, consisting of whites in rochester ( . %), and siblings of rochester participants underwent an examination. serum creatinine was measured using a modified kinetic jaffé reaction. covariates were obtained at the second stage examination. gutenberg heart study: the gutenberg heart study began in as a community-based prospective study with participants ranging in age from to years. all participants have been drawn randomly from the local registry offices in the city of mainz and the district of mainz- bingen. the present analysis was based on an initial sample of , subjects successively enrolled into the ghs from april to april . genomic dna was isolated from all participants. serum creatinine was measured in heparin-plasma using the jaffe reaction method (abbott diagnostics; delkenheim, germany). genotyping, imputation, and the statistical analysis were performed as indicated in supplemental table . health abc: the health abc study is a community-based prospective cohort study that began in , consisting of men and women in the original cohort. participants of the cohort were recruited from medicare listings in pittsburgh, pennsylvania and memphis, tennessee. eligibility criteria included age – years, self-report of no difficulty walking one-quarter mile or climbing steps, or with activities of daily living, no history of active treatment for cancer in the prior years, and no plan to move out of the area. these analyses included only those health abc participants who reported their race/ethnicity as european-american. serum creatinine was measured using a modified kinetic jaffé reaction; serum cystatin c was measured using particle-enhanced immunonephelometric method (bnii, dade-behring). covariates were obtained at baseline. genomic dna was extracted from buffy coat collected using puregene® dna purification kit during the baseline exam. in , genotyping was performed by the center for inherited disease research (cidr). nurses health study/health professionals follow-up study (nhs/hpfs): the nhs was established in when , female registered nurses aged - years and residing in large u.s. states completed a mailed questionnaire on their medical history and lifestyle characteristics. every two years, follow-up questionnaires have been sent to update information on exposures and newly diagnosed illnesses. the hpfs was initiated in when , male u.s. health professionals aged - and residing in u.s. states answered a detailed questionnaire that included a comprehensive diet survey, and items on lifestyle practice and medical history. the cohort is followed through biennial mailed questionnaire. blood was collected from , nhs members between and and from , hpfs members between and . creatinine measures were performed on the samples from women and men with diabetes. diabetes was defined as initially self-reported diabetes subsequently confirmed by a validated supplementary questionnaire. , between and , a subset of these men and women were genotyped as part of a gwas of type diabetes. plasma creatinine was measured by a modified kinetic jaffé reaction. covariates were obtained from the questionnaire administered closest to the time creatinine was measured ( for nhs and for hpfs). standardized protocols for the nhs and hpfs genome-wide scans were developed as part of the geneva consortium. in the initial gwas, nhs (n= ) and hpfs (n= ) samples were genotyped approximately two months apart and underwent independent qc. popgen: using the popgen biobank, data on healthy control individuals from germany were obtained. as part of the gwas initiative of the german national genome research network (ngfn), genotyping was performed. serum creatinine was measured with an enzymatic in vitro assay (creaplus, cobas®, roche diagnostics, indianapolis, in). sorbs: all participants are part of a sample from an extensively phenotyped self-contained population from eastern germany, the sorbs. sampling comprised unrelated subjects as well as families; participants were available for the present study. serum creatinine was measured using a kinetic enzymatic method (roche inc). covariates were obtained at the index examination. adjustment for genomic control was used to control for increased genetic sharing due to long term isolation of this population. split: the split study (split) is a population-based, cross-sectional study in the dalmatian city of split. data for participants aged years and over were used for this analysis. fasting blood samples were collected and over health-related phenotypes and environmental exposures were measured in each individual. serum creatinine was measured using the photometric jaffé method. de novo genotyped replication cohorts genotyping methods of these four cohorts are described above (replication analysis). kora f /f : the subjects of kora f and kora f who had not been genotyped genome wide as described under “discovery cohorts” were used as replication samples including and subjects from kora f and f , respectively. covariates were obtained at the index examination. in kora f , the mean call rate was . %, the concordance among samples genotyped in duplicate across snps was . %. in kora f , the mean call rate was . %, the concordance among samples genotyped in duplicate across snps was %. sapaldia: the sapaldia study was originally designed to investigate the effects of air pollutants on respiratory health in a random sample of the adult population of switzerland. , the original study consisted of adults as described elsewhere. of the surviving participants, % (n = ) could be traced between and , and were re-contacted for sapaldia . in the second survey, there was additionally a collection of blood specimens for analysis of blood and dna markers. included in this study are subjects with blood and dna samples available (n= ) who had consented to the general blood marker and dna analyses. blood samples were processed and stored in a standardized fashion according to the sapaldia protocol. serum creatinine was measured using the jaffé reaction (roche) and calibrated to the roche enzymatic gold standard reference. dna was extracted as previously described. the mean call rate was . %, the concordance among samples genotyped in duplicate across snps was . %. saphir study: the "salzburg atherosclerosis prevention program in subjects at high individual risk" (saphir) is an observational study conducted in the years - involving healthy unrelated subjects: females from to years of age and males from to years of age. study participants were recruited by health screening programs in large companies in and around the city of salzburg as described recently. all individuals were of west-european origin. participants with established coronary artery, cerebrovascular or peripheral arterial disease, congestive heart failure, valvular heart disease, chronic alcohol (more than three drinks a day) or drug abuse, severe obesity (bmi> kg/m ) and pregnant women were excluded. serum creatinine (mg/dl) was measured using a modified kinetic jaffé reaction (crea , roche diagnostics gmbh, mannheim, germany). among the total of phenotyped participants, a total of had egfr and genotype information, and had ckd status and genotype data. however, there were too few cases with ckd (n= ) to warrant analysis of this trait. covariates were obtained at the index examination. the mean call rate was . %, the concordance among samples genotyped in duplicate across snps was . %. supplementary note – full acknowledgements discovery cohorts ages: we thank all participants in the study and the study staff for their invaluable contribution. the age, gene/environment susceptibility reykjavik study has been funded by nih contract n -ag- , the nia intramural research program, hjartavernd (the icelandic heart association), and the althingi (the icelandic parliament). amish: the amish studies are supported by grants and contracts from the nih including r ag (amish longevity study), r hl (amish calcification study), u gm - (papi study), u hl - (hapi study), u hl and nih k rr (university of maryland mcrdp), nih p dk (clinical nutrition research unit of maryland) and nih p dk drtc (baltimore diabetes research and training center), the university of maryland general clinical research center, grant m rr , the baltimore veterans administration medical center geriatrics research and education clinical center and the paul beeson physician faculty scholars in aging program. we thank our amish research volunteers for their long-standing partnership in research, and the research staff at the amish research clinic for their hard work and dedication. aric: the atherosclerosis risk in communities study is carried out as a collaborative study supported by national heart, lung, and blood institute contracts n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , n -hc- , r hl , r hl and r hl ; national human genome research institute contract u hg ; and national institutes of health contract hhsn c. the authors thank the staff and participants of the aric study for their important contributions. infrastructure was partly supported by grant number ul rr , a component of the national institutes of health and nih roadmap for medical research. asps: the research reported from the austrian stroke prevention study (asps) in this article was funded by the austrian science fond (fwf) grant number p -p and p . the medical university of graz supports the databank of the asps. the authors thank the staff and the participants of the asps for their valuable contributions. we thank birgit reinhart for her long-term administrative commitment and ing johann semmler and irmgard poelzl for the technical assistance at creating the dna-bank. blsa: the blsa was supported in part by the intramural research program of the nih, national institute on aging. a portion of that support was through a r&d contract with medstar research institute. charge consortium: we acknowledge the individual participating studies and investigators of the charge (cohorts for heart and aging research in genome epidemiology) consortium (ages, aric, chs, framingham heart study, rotterdam study). chs: the chs research reported in this article was supported by contract numbers n -hc- through n -hc- , n -hc- , n hc- , n hc- , n -hc- , n -hc- , grant numbers u hl and r hl , and r ag from the national heart, lung, and blood institute, with additional contribution from the national institute of neurological disorders and stroke. a full list of principal chs investigators and institutions can be found at http://www.chs-nhlbi.org/pi.htm. dna handling and genotyping was supported in part by national center for research resources grant m rr to the cedars-sinai general clinical research center genotyping core and national institute of diabetes and digestive and kidney diseases grant dk to the southern california diabetes endocrinology research center. erf: the erf study was supported by grants from the netherlands organisation for scientific research, erasmus mc and the centre for medical systems biology (cmsb). we are grateful to all study participants and their relatives, general practitioners and neurologists for their contributions and to p. veraart for her help in genealogy, j. vergeer for the supervision of the laboratory work and p. snijders for his help in data collection. family heart study: this research was conducted in part using data and resources from the nhlbi family heart study supported in part by nih grant r hl . framingham heart study: this research was conducted in part using data and resources from the framingham heart study of the national heart lung and blood institute of the national institutes of health and boston university school of medicine. the analyses reflect intellectual input and resource development from the framingham heart study investigators participating in the snp health association resource (share) project. this work was partially supported by the national heart, lung and blood institute's framingham heart study (contract no. n -hc- ) and its contract with affymetrix, inc for genotyping services (contract no. n -hl- - ). a portion of this research utilized the linux cluster for genetic analysis (linga-ii) funded by the robert dawson evans endowment of the department of medicine at boston university school of medicine and boston medical center. kora: the genetic epidemiological work was funded by the nih subcontract from the children’s hospital, boston, us, (h.e.w., i.m.h, prime grant r dk - a ), the german national genome research net ngfn and ngfnplus (h.e.w. gs ; wk project a , number gs ), the munich center of health sciences (h.e.w.), and by the else kröner-fresenius stiftung (p / //a / ; c.a.b., b.k.k.). the kidney parameter measurements in f were funded by the else kröner-fresenius stiftung (c.a.b., b.k.k.) and the regensburg university medical center, germany; in f by the university of ulm, germany (w.k.). genome wide genotyping costs in f and f were in part funded by the else kröner- fresenius-stiftung (c.a.b., b.k.k.). denovo genotyping in f and f were funded by the else kröner-fresenius-stiftung (c.a.b., b.k.k.). the kora research platform and the monica augsburg studies were initiated and financed by the helmholtz research center münchen for environmental health, by the german federal ministry of education and research and the state of bavaria. genotyping was performed in the genome analysis center (gac) of the helmholtz zentrum münchen. the linux platform for computations were funded by the university of regensburg for the department of epidemiology and preventive medicine at the regensburg university medical center. korcula: the korcula study in the croatian island of vis was supported through the grants from the medical research council uk to h.c., a.f.w. and i.r.; and ministry of science, education and sport of the republic of croatia to i.r. (number - - ). we would like to acknowledge the invaluable contributions of the recruitment team in korcula, the administrative teams in croatia and edinburgh (rosa bisset) and the people of korcula. micros: we owe a debt of gratitude to all participants. we thank the primary care practitioners raffaela stocker, stefan waldner, toni pizzecco, josef plangger, ugo marcadent and the personnel of the hospital of silandro (department of laboratory medicine) for their participation and collaboration in the research project. we thank dr. peter riegler (hemodialysis unit, hospital of merano) for the important discussions. in south tyrol, the study was supported by the ministry of health and department of educational assistance, university and research of the autonomous province of bolzano, the south tyrolean sparkasse foundation, and the european union framework program eurospan project (contract no. lshg-ct- - ). nsphs: the northern swedish population health study was supported by grants from the swedish natural sciences research council, the european union through the eurospan project (contract no. lshg-ct- - ), the foundation for strategic research (ssf) and the linneaus centre for bioinformatics (lcb). we are also grateful for the contribution of samples from the medical biobank in umeå and for the contribution of the district nurse svea hennix in the karesuando study. orcades: orcades was supported by the the chief scientist office of the scottish government, the royal society and the european union framework program eurospan project (contract no. lshg-ct- - ). dna extractions were performed at the wellcome trust clinical research facility in edinburgh. we would like to acknowledge the invaluable contributions of lorraine anderson, the research nurses in orkney, the administrative team in edinburgh and the people of orkney. rotterdam study: the rotterdam study is supported by the erasmus medical center and erasmus university rotterdam; the netherlands organization for scientific research; the netherlands organization for health research and development (zonmw); the research institute for diseases in the elderly; the netherlands heart foundation; the ministry of education, culture and science; the ministry of health welfare and sports; the european commission; and the municipality of rotterdam. the genome-wide association database of the rotterdam study was funded through the netherlands organization of scientific research nwo (nr. . . . , . . ) and the research institute for diseases in the elderly (ride). this study was supported by the netherlands genomics initiative (ngi)/netherlands organisation for scientific research (nwo) project nr. - - . we thank michael moorhouse, phd, department of bioinformatics, and pascal arp, bsc, mila jhamai, bsc, marijn verkerk, bsc, and sander bervoets, bsc, department of internal medicine, erasmus mc, rotterdam, the netherlands, for their help in creating the database. ship: ship is part of the community medicine research net of the university of greifswald, germany, which is funded by the federal ministry of education and research (grants no. zz , zz , and zz ), the ministry of cultural affairs as well as the social ministry of the federal state of mecklenburg-west pomerania. genome-wide data have been supported by the federal ministry of education and research (grant no. zik ) and a joint grant from siemens healthcare, erlangen, germany and the federal state of mecklenburg- west pomerania. the university of greifswald is a member of the ‘center of knowledge interchange’ program of the siemens ag. vis: the vis study in the croatian island of vis was supported through the grants from the medical research council uk to h.c., a.f.w. and i.r.; and ministry of science, education and sport of the republic of croatia to i.r. (number - - ) and the european union framework program eurospan project (contract no. lshg-ct- - ). we would like to acknowledge the invaluable contributions of the recruitment team (including those from the institute of anthropological research in zagreb) in vis, the administrative teams in croatia and edinburgh (rosa bisset) and the people of vis. wghs: the wghs is supported by hl and hl from the national heart, lung, and blood institute and ca from the national cancer institute, the donald w. reynolds foundation and the fondation leducq, with collaborative scientific support and funding for genotyping provided by amgen. replication cohorts genoa: this research was conducted in part using data and resources from the genetic epidemiology network of atherosclerosis (genoa) study. the analyses reflect intellectual input and resource development from the genoa study investigators participating in the snp health association resource (share) project. this work was partially supported by the national heart, lung and blood institute grant (hl- ) and its contract with mayo clinic college of medicine for genotyping services and statistical analyses. gutenberg heart study: the gutenberg heart study is funded through the government of rheinland-pfalz (“stiftung rheinland pfalz für innovation”, contract number az - / ), the research programs “wissen schafft zukunft” and “schwerpunkt vaskuläre prävention” of the johannes gutenberg-university of mainz and its contract with boehringer ingelheim and philips medical systems including an unrestricted grant for the gutenberg heart study. specifically, the research reported in this article was supported by the national genome network “ngfnplus” (contract number project a gs ) by the federal ministry of education and research, germany. health abc: this research was supported by nia contracts n ag , n ag , and n ag . the genome-wide association study was funded by nia grant r ag - a to wake forest university and genotyping services were provided by the center for inherited disease research (cidr). cidr is fully funded through a federal contract from the national institutes of health to the johns hopkins university, contract number hhsn c. this research was supported in part by the intramural research program of the nih, national institute on aging nhs/hpfs: the nhs/hpfs type diabetes gwas (u hg ) is a component of a collaborative project that includes other gwas (u hg , u hg , u hg , u hg , u hg , u hg , u hg , u hg , u hg , u hg , rfahg ; national institute of dental & craniofacial research: u de , u de ) funded as part of the gene environment-association studies (geneva) under the nih genes, environment and health initiative (gei). assistance with phenotype harmonization and genotype cleaning, as well as with general study coordination, was provided by the geneva coordinating center (u hg ). assistance with data cleaning was provided by the national center for biotechnology information. genotyping was performed at the broad institute of mit and harvard, with funding support from the nih gei (u hg ), and johns hopkins university center for inherited disease research, with support from the nih gei (u hg ) and the nih contract "high throughput genotyping for studying the genetic contributions to human disease”(hhsn c). additional funding for the current research was provided by the national cancer institute (p ca , p ca ), and the national institute of diabetes and digestive and kidney diseases (r dk , r dk ). m.c.c is a recipient of a canadian institutes of health research fellowship. we thank the staff and participants of the nhs and hpfs for their dedication and commitment. popgen: the popgen study was supported by the german ministry of education and research (bmbf) through the national genome research network (ngfn). it is currently funded by the ministry of science, commerce and transportation of the state of schleswig- holstein. the project has also received infrastructure support through the dfg excellence cluster “inflammation at interfaces”. sapaldia: the sapaldia cohort study is supported by the swiss national science foundation (grants - , co- , bo- , bo- , bo- , - . , - . , - . , - . ), the federal office for forest, environment and landscape, the federal office of public health, the federal office of roads and transport, the canton's government of aargau, basel-stadt, basel-land, geneva, luzern, ticino, zürich, the swiss lung league, the canton's lung league of basel stadt/basel landschaft, geneva, ticino and zürich. de novo genotyping was in part contributed to by the nhlbi intra-mural research program and the center for population studies, and by the else kröner-fresenius-stiftung (p / //a / ; c.a.b., b.k.k.). saphir: the saphir-study was partially supported by a grant from the kamillo eisner stiftung to b. paulweber and by grants from the "genomics of lipid-associated disorders – gold" of the "austrian genome research programme gen-au" to f. kronenberg. de novo genotyping was in part contributed to by the nhlbi intra-mural research program and the center for population studies, and by the else kröner-fresenius-stiftung (p / //a / ; c.a.b., b.k.k.). sorbs: this work was supported by grants from the interdisciplinary centre for clinical research at the university of leipzig (b to m.s., p.k. and a.t.; n to p.k.) and from the german diabetes association (to a.t. and p.k.). ms is supported by a grant from the dfg (kfo ). we would like to thank knut krohn from the microarray core facility of the interdisciplinary centre for clinical research (izkf), university of leipzig, germany, nigel william rayner from the wellcome trust centre for human genetics, university of oxford, uk and john broxholm from the bioinformatics core unit of the wellcome trust centre for human genetics for their excellent support. the research of inga prokopenko and reedik magi is funded in part through the european community's seventh framework programme (fp / - ), engage project, grant agreement health-f - - . split: the split study in the croatian city of split was supported through the grants from the medical research council uk to h.c., a.f.w. and i.r.; and ministry of science, education and sport of the republic of croatia to i.r. (number - - ) and the european union framework program eurospan project (contract no. lshg-ct- - ). we would like to acknowledge the invaluable contributions of the recruitment team from the croatian centre for global health, university of split, the administrative teams in croatia and edinburgh (rosa bisset) and the people of split. supplementary table a- study design and sample sizes study design overall sample size analyzed sample size egfrcrea / ckd/ egfrcys stage discovery ages population-based prospective , / , /na amish amish founder "healthy"population / na/ aric population-based / / asps community-based prospective longitudinal / /na blsa prospective population-based cohort / / na chs population-based / / erf cross-sectional population- based study with pedigree information / / na family heart study case-control / / na framingham heart study community-based family multi-generation / / kora f general population, unrelated, age-range - yrs / / kora f general population, unrelated, age - yrs / / korcula croatia cross-sectional population- based study with pedigree information / / na micros cross-sectional population- based study with pedigree information / / nsphs cross-sectional population- based study with pedigree information / / na orcades cross-sectional population- based study with pedigree information / / na rs population-based / /na rs population-based / / na ship population-based / / vis croatia cross-sectional population- based study with pedigree information / / na wghs population-based, women only / / na stage replication, in silico aric* population-based / / family heart study* family-based cohort / /na genoa community-based sibships / /na gutenberg heart study community-based prospective cohort / /na health abc population-based elderly cohort / / hpfs health professional –based cohort study , / /x nurses health study health professional –based cohort study , / /na popgen population-based biobank / /na sorbs self-contained population / /na split community-based cohort /na/na stage replication, de novo genotyped kora f * general population, unrelated, age-range - yrs / / kora f * general population, unrelated, age - yrs / / sapaldia random sample, switzerland / /na saphir health working cohort / /na * subjects independent from subjects contributing to discovery analyses supplementary table b - genotyping and imputation platforms array type genotype calling qc filters for genotyped snps used for imputation # snps used for imputati on imputation imputation backbone for phased ceu haplotypes (ncbi build) filtering of imputed genotypes data management and statistical analysis stage : discovery ages illumina cnv beadstudio call rate < %, maf < %, phwe < e- , mishap p < e- , a/t and g/c snps, mismatches between illumina, dbsnp and/or hapmap position , mach v . . hapmap release (build ) none plink, r amish affymetrix k brlmm call rate < %, maf < %, phwe < e- , snps not in hapmap , mach v . . hapmap release (build ) mapp, mixed model to account for relatedness aric affymetrix . birdseed call rate < %, maf< %, phwe< e- , mach v . . hapmap release (build ) none probabel, plink, r asps illumina human - quad beadchip® beadstudio call rate < %, phwe< e- , mach v . . hapmap release (build ) none probabel blsa illumina humanhap k beadstudio call rate < %, maf < %, phwe < e- , mach v . . hapmap release (build ) maf< %, r < . sas,merlin (fastassoc option),r chs illumina cnv beadstudio call rate < %, heterozygotes= , phwe< e- , snp not in hapmap , bimbam hapmap release a (build ) dosage variance < . r, robust variance option erf illumina, k illumina maf % , phwe e- , call rate % , mach v . . hapmap release (build ) none r, genabel , probabel, gc correction to account for relatedness family heart study illumina k beadstudio- gencall v . call rate < %, maf < %, phwe< e- , mach v . . hapmap release (build ) none sas, linear mixed effect and logistic models framingham heart study affymetrix k affymetrix k supplement al affymetrix phwe< e- , call rate< %, mishap p< e- , maf< . , mendelian errors> , snps not in hapmap or strandedness issues merging with hapmap , mach v . . hapmap release (build ) none r, linear mixed effect models and gee models, robust variance option to account for relatedness kora f affymetrix k brlmm per-chip call rate < %, maf < %, discrepancy for one of the snps common on both chips, gender checks , mach hapmap release , (build ) none mach qtl, probabel, r, visual basic kora f affymetrix . brlmm per-chip call rate < %, per-snp call rate < %, maf< %, gender checks , mach hapmap release , (build ) none probabel, r, visual basic korcula illumina, k beadstudio call rate < %, maf < %, phwe< e- , mach v . . hapmap release (build ) none r , probable micros illumina, k beadstudio call rate < %, maf < %, phwe< e- , mach v . . hapmap release (build ) none r, genabel, probabel; details in study-specific methods nsphs illumina, k beadstudio maf %, phwe e- , call rate % , mach v . . hapmap release (build ) none r, genabel, probabel; details in study-specific methods orcades illumina, k beadstudio maf %, phwe e- , call rate % , mach v . . hapmap release (build ) none r, genabel, probabel; details in study-specific methods rs-i, rs-ii illumina k beadstudio call rate < %, maf< %, phwe< e- , mach v . . hapmap release (build ) none linear and logistic regression using probabel, r ship affymetrix . birdseed v per-chip call rate < % , impute v . . hapmap release (build ) none snptest v . . , quicktest v . vis illumina, k beadstudio maf % , phwe e- , call rate % , mach v . . hapmap release (build ) none r, genabel, probabel; details in study-specific methods wghs illumina humanhap duo "+" beadstudio . call rate < % on snps; phwe < - , mach hapmap release (build ), snp positions updated to build none r, probabel, bash scripting array type genotype calling qc filters for genotyped snps used for imputation # snps used for imputati on imputation imputation backbone (ncbi build) filtering of imputed genotypes data management and statistical analysis stage : in silico replication aric* affymetrix . birdseed call rate < %, maf< %, phwe < e- , mach v . . hapmap release (build ) none probabel, plink, r family heart study* illumina human m genomestudio call rate < %, maf< %, phwe < e- , mach v . . hapmap release (build ) none sas, linear mixed effect models genoa affymetrix . birdseed v call rate < %, phwe < e- mach v . . hapmap release (build ) none plink; linear mixed models (r multic); gee models (r gee glm); robust variance option to account for relatedness gutenberg heart study affymetrix . birdseed v call rate < %, maf < %, phwe < e- , impute v . . hapmap release (build ) none r health abc illumina human m- duo illumina beadstudio call rate < %, hwe p< e- , maf< % , mach v . . hapmap release build none r hpfs affymetrix . birdseed call rate < %, phwe< e- , maf< %, > discordance/ replicates, significant plate associations , mach v . . hapmap release (build ) none probabel (r), sas . nurses health study affymetrix . birdseed call rate < %, phwe < e- , maf < %, > discordance/ replicates, significant plate associations , mach v . . hapmap release (build ) none probabel (r), sas . popgen affymetrix . birdseed v per-person-call rate < . , per-snp call rate < . , phwe< e- , maf < % , mach v . . hapmap release (build ) none plink, r sorbs k affymetrix, affymetrix . brlmm; birdseed call rate < %, phwe< e- , maf % , impute hapmap release (build ) proper- info> . , maf> %, hwe< - snptest, gc correction to account for relatedness split illumina cnv illumina gentrain algorithm call rate < %, phwe< e- na na na na r, genabel genotypin g platform quality control of genotyped snps # snps genotype d data management and statistical analysis stage : de novo genotyping replication kora f * sequenom, taqman mean call rate . %, concordance of duplicates . %, phwe > . na na na probabel (r) kora f * sequenom, taqman mean call rate . %, concordance of duplicates %, phwe > . na na na probabel (r) sapaldia sequenom, taqman mean call rate . %, concordance of duplicates . %, phwe > . na na na probabel (r) saphir sequenom, taqman mean call rate . %, concordance of duplicates . %, phwe > . na na na sas . . . * subjects independent from subjects contributing to discovery analyses supplementary table - genome-wide significant loci: snp imputation quality in stage discovery cohorts. study rs (ckd) rs rs rs rs rs rs rs rs rs rs ages . . . . . . . . . . . amish . . . . . . . . . . . aric . . . . . . . . . . . asps . . . . . . . . . . . blsa . . . . . . . . . . . chs . . . . . . . . . . . erf . . . . . . . . . . . famhs . . . . . . . . . . . fhs . . . . . . . . . . . kora f . . . . . . . . . . . kora f . . . . . . . . . . . korcula . . . . . . . . . . . micros . . . . . . . . . . . orcades . . . . . . . . . . . rs- . . . . . . . . . . . rs- . . . . . . . . . . . nhsph . . . . . . . . . . . ship . . . . . . . . . . . vis . . . . . . . . . . . wghs . . . . . . . . . . . median imputation quality . . . . . . . . . . . study rs rs rs rs rs rs rs rs rs rs ages . . . . . . . . . . amish . . . . . . . . . . aric . . . . . . . . . . asps . . . . . . . . . . blsa . . . . . . . . . . chs . . . . . . . . . . erf . . . . . . . . . . famhs . . . . . . . . . . fhs . . . . . . . . . . kora f . . . . . . . . . . kora f . . . . . . . . . . korcula . . . . . . . . . . micros . . . . . . . . . . orcades . . . . . . . . . . rs- . . . . . . . . . . rs- . . . . . . . . . . nhsph . . . . . . . . . . ship . . . . . . . . . . vis . . . . . . . . . . wghs . . . . . . . . . . median imputation quality . . . . . . . . . . study rs rs rs rs rs egfrcys: rs ages . . . . . . amish . . . . . . aric . . . . . . asps . . . . . . blsa . . . . . . chs . . . . . . erf . . . . . . famhs . . . . . . fhs . . . . . . kora f . . . . . . kora f . . . . . . korcula . . . . . . micros . . . . . . orcades . . . . . . rs- . . . . . . rs- . . . . . . nhsph . . . . . . ship . . . . . . vis . . . . . . wghs . . . . . . median imputation quality . . . . . . supplementary table - genome-wide significant loci: snp association across renal traits in stage discovery and stage replication meta-analyses. egfrcrea (n= ), ckd (n= ) or egfrcys (n= ) chr position (b ) genes nearby modeled allele# egfrcre a beta egfrcrea se p-value egfrcrea beta egfrcrea se -sided p- value renal function loci, association with egfrcrea discovery replication rs sypl ;atxn l ,cyb d ,psma ,amigo ,sort a - . . . e- - . . . e- rs anxa ;fam a,prune,b nipl,lass ,setdb c . . . e- . . . e- rs (ckd) sox c - . . . e- . . . e- rs gckr;ift ,fndc t . . . e- . . . e- rs nat ;nat b,alms g . . . e- . . . e- rs tfdp c . . . e- . . . e- rs dab ;c a - . . . e- - . . . e- rs slc a ;grk ,rgs ,lm an ,prr ,f ,pfn g - . . . e- - . . . e- rs vegfa g . . . e- . . . e- rs prkag a - . . . e- - . . . e- rs pip k b;fam a a - . . . e- - . . . e- rs rnaseh c;dkfzp e ,htatip,ovol g - . . . e- - . . . e- rs atxn t . . . e- . . . e- rs dach c . . . e- . . . e- rs ube q ;fbxo a - . . . e- - . . . e- rs slc a ;ccdc ,ecat c . . . e- . . . e- creatinine production loci, association with egfrcrea rs cps a - . . . e- - . . . e- rs slc a g - . . . e- - . . . e- rs tmem ;rsbn l,phtf c - . . . e- - . . . e- rs wdr t - . . . e- - . . . e- rs slc a ;jarid a,slc a c . . . e- . . . e- rs wdr c . . . e- . . . e- rs bcas ;tbx ,c orf c - . . . e- - . . . e- chr position (b ) genes nearby modeled allele# or ckd % ci p-value or ckd % ci -sided p-value renal function loci, association with ckd discovery replication rs sypl ;atxn l ,cyb d , psma ,amigo ,sort a . . - . . e- . . - . . e- rs anxa ;fam a,prune, bnipl,lass ,setdb c . . - . . e- . . - . . e- rs (ckd) sox c . . - . . e- . . - . . e- rs gckr;ift ,fndc t . . - . . e- . . - . . e- rs nat ;nat b,alms g . . - . . e- . . - . . e- rs tfdp c . . - . . e- . . - . . e- rs dab ;c a . . - . . e- . . - . . e- rs slc a ;grk ,rgs , lman ,prr ,f ,pfn g . . - . . e- . . - . . e- rs vegfa g . . - . . e- . . - . . e- rs prkag a . . - . . e- . . - . . e- rs pip k b;fam a a . . - . . e- . . - . . e- rs rnaseh c;dkfzp e , htatip,ovol g . . - . . e- . . - . . e- rs atxn t . . - . . e- . . - . . e- rs dach c . . - . . e- . . - . . e- rs ube q ;fbxo a . . - . . e- . . - . . e- rs slc a ;ccdc ,ecat c . . - . . e- . . - . . e- creatinine production loci, association with ckd rs cps a . . - . . e- . . - . . e- rs slc a g . . - . . e- . . - . . e- rs tmem ;rsbn l,phtf c . . - . . e- . . - . . e- rs wdr t . . - . . e- . . - . . e- rs slc a ;jarid a,slc a c . . - . . e- . . - . . e- rs wdr c . . - . . e- . . - . . e- rs bcas ;tbx ,c orf c . . - . . e- . . - . . e- chr position (b ) genes nearby modeled allele# egfrcys beta egfrcys se p-value egfrcys beta egfrcys se -sided p- value renal function loci, association with egfrcys discovery replication rs sypl ;atxn l ,cyb d , psma ,amigo ,sort a - . . . e- . . . e- rs anxa ;fam a,prune, bnipl,lass ,setdb c . . . e- . . . e- rs (ckd) sox c - . . . e- - . . . e- rs gckr;ift ,fndc t . . . e- . . . e- rs nat ;nat b,alms g . . . e- . . . e- rs tfdp c . . . e- - . . . e- rs dab ;c a - . . . e- - . . . e- rs slc a ;grk ,rgs , lman ,prr ,f ,pfn g - . . . e- - . . . e- rs vegfa g . . . e- . . . e- rs prkag a - . . . e- - . . . e- rs pip k b;fam a a - . . . e- - . . . e- rs rnaseh c;dkfzp e , htatip,ovol g - . . . e- - . . . e- rs atxn t . . . e- . . . e- rs dach c . . . e- . . . e- rs ube q ;fbxo a - . . . e- - . . . e- rs slc a ;ccdc ,ecat c . . . e- . . . e- creatinine production loci, association with egfrcys rs cps a . . . e- . . . e- rs slc a g - . . . e- - . . . e- rs tmem ;rsbn l,phtf c . . . e- . . . e- rs wdr t - . . . e- - . . . e- rs slc a ;jarid a,slc a c . . . e- . . . e- rs wdr c . . . e- . . . e- rs bcas ;tbx ,c orf c - . . . e- . . . e- #the minor allele based on sample size weighted mean allele frequency in the discovery cohorts is modeled. genes within kb were based on refseq genes (b ). the gene closest to the snp is listed first and bold if the snp is located within the gene. other genes in the region are listed after ";". betas for egfr refer to age-adjusted sex-specific residuals of natural log transformed egfr. p-values in discovery analyses are corrected for genomic control before and after meta-analysis. supplementary table - additional gene biology. this table lists additional genes in associated regions containing a gene highlighted in box , as well as genes in other associated regions. for novel regions associated with both egfrcrea and egfrcys, the gene(s) in closest physical proximity and/or in strong ld (r > . ) with the lead snp is presented. for novel regions association with egfrcrea only, the gene in closest physical proximity is presented. location gene function q lass lag homolog, ceramide synthase (lass ) is highly expressed in the kidney and may be involved in cell growth. a non-synonymous coding snp in lass , rs (e a), was in perfect ld with the lead snp in the region and of predicted damaging function. lass has been implicated in the synthesis of specific ceramides. ceramides and their product sphingolipids are important in genetic diseases of the kidney, and have a role in aging mechanisms. q , lass region anxa annexin a (anxa ) is expressed in kidney and functions as a calcium-sensitive protein. q , lass region setdb set domain, bifurcated (setdb ) is a key histone methyltransferase important in chromatin homeostasis and thus epigenetic regulation of gene expression. it is expressed in numerous tissues including the kidney (unigene). p . , gckr region fndc encodes fibronectin type iii domain containing gene. this gene shows low expression in the kidney, and is involved in cell adhesion activity. q cps encodes carbamoyl-phosphate synthetase . has isoforms and is involved in the hepatic urea cycle and in production of arginine, a precursor to creatine production that could potentially affect serum creatinine levels. moreover, cps is associated with hyperammonemia, which has been shown to decrease creatine synthesis. the associated snp causes a nonsynonymous amino acid change. q tfdp tfdp encodes e f dimerization partner (dp) . dimerization of dp proteins with e f proteins increases the transcription activity of e f. the role of tfdp has mainly been studied in the context of tumorigenesis via its known interaction with e f and with the tgf signaling pathway, and not in the context of renal disease. q , tfdp region atp b encodes atpase, na+/k+ transporting, beta polypeptide. esnp data points to this gene (table ). it contains a highly conserved actin nucleation and is involved in membrane growth and polarity. it is expressed in numerous tissues, including kidney (unigene). q slc a slc a encodes solute carrier family , member which functions as an organic cation transporter and mediates the uptake of a variety of organic cations, including creatinine in the basolateral membrane of renal tubular epithelial cells. neighboring genes are slc a and slc a , which have similar function. q . tmem encodes transmembrane protein . it is expressed in many tissues including the kidney and muscle. its biological function in unknown. q pip k b pip k b encodes phosphatidylinositol- -phosphate -kinase, type with a possible role in cell polarization. the lead snp we identified is an intronic snp in a region with high conservation across species. expression is complex, with multiple transcripts detected in a wide variety of tissues, including the kidney. no publications connecting this gene and kidney function. q . , pip k b region fam a encodes the protein family with sequence similarity a. no known association with kidney disease. p . wdr encodes wd repeat domain . members of this protein family are involved in a variety of cellular processes, including cell cycle progression, signal transduction, apoptosis, and gene regulation. specific biological function is unknown. p . slc a encodes solute carrier family (neurotransmitter transporter, betaine/gaba), member , which is expressed in the kidney. it is induced by hypertonicity and mediates chloride- and sodium- dependent transport of both betaine and the neurotransmitter gaba, and shows high homology to a known creatinine transporter encoded by slc a . q , atxn region brap in a monocyte differentiation model, brap (brca-associated protein) interacts directly with cdkn a. knockout of cdkn a ameliorates the development of chronic renal failure in a mouse model. q . wdr wd repeat-containing protein (wdr ) is a protein of unknown function. the snp rs is a coding snp in wdr with predicted damaging function (sift) and in ld with the lead snp we identified (r = . ). it is highly expressed in the kidney. q . ube q ubiquitin-conjugating enzyme e q family member is reported to catalyze the covalent attachment of ubiquitin to other proteins. ube q has a role in the cell cycle. it is expressed in the kidney. q . , ube q region fbxo encodes f-box protein , which contains an f box motif (omim). it constitutes one of four parts of the ubiquitin protein ligase complex with a role in ubiquitination (www.genecards.org) it is expressed in kidney. q bcas encodes breast carcinoma amplified sequence . involves a functional estrogen response element. it is ubiquitously expressed, and has been associated with human height, which could secondarily be associated with muscle mass. our identified snp was also in strong ld with the tbx gene. q . , slc a region ccdc coiled-coil domain containing (ccdc ) encodes a mitochondrial protein. it is expressed in the kidney (www.genecards.org). supplementary table : effect sizes of association with egfrcrea across strata of diabetes and hypertension. results are presented for the lead snp at known and novel loci related to renal function. diabetes no diabetes snpid locus chr pos a a freq beta se pval beta se pval pval diff rs anxa t c . - . . . e- - . . . e- . e- rs gckr t c . . . . e- . . . e- . e- rs alms a g . . . . e- - . . . e- . e- rs tfdp a c . - . . . e- - . . . e- . e- rs shroom a g . - . . . e- - . . . e- . e- rs dab a t . - . . . e- - . . . e- . e- rs slc a a g . . . . e- . . . e- . e- rs vegfa a g . - . . . e- - . . . e- . e- rs prkag a g . . . . e- - . . . e- . e- rs stc t c . - . . . e- - . . . e- . e- rs pip k b a c . - . . . e- - . . . e- . e- rs wdr t c . - . . . e- - . . . e- . e- rs atxn t c . . . . e- . . . e- . e- rs dach a c . - . . . e- - . . . e- . e- rs wdr a c . - . . . e- - . . . e- . e- rs ube q a g . - . . . e- - . . . e- . e- rs umod t g . . . . e- . . . e- . e- rs slc a t c . . . . e+ - . . . e- . e- hypertension no hypertension snpid locus chr pos a a freq beta se pval beta se pval pval diff rs anxa t c . - . . . e- - . . . e- . e- rs gckr t c . . . . e- . . . e- . e- rs alms a g . - . . . e- - . . . e- . e- rs tfdp a c . - . . . e- - . . . e- . e- rs shroom a g . - . . . e- - . . . e- . e- rs dab a t . - . . . e- - . . . e- . e- rs slc a a g . . . . e- . . . e- . e- rs vegfa a g . - . . . e- - . . . e- . e- rs prkag a g . - . . . e- - . . . e- . e- rs stc t c . - . . . e- - . . . e- . e- rs pip k b a c . - . . . e- - . . . e- . e- rs wdr t c . - . . . e- - . . . e- . e- rs atxn t c . . . . e- . . . e- . e- rs dach a c . - . . . e- - . . . e- . e- rs wdr a c . - . . . e- - . . . e- . e- rs ube q a g . - . . . e- - . . . e- . e- rs umod t g . . . . e- . . . e- . e- rs slc a t c . - . . . e- - . . . e- . e- p-value for difference are from a t-test for independent samples (b hat -b hat ~ n(b -b ,se ^ +se ^ )): under the null hypothesis, the difference between the effects in the two groups follows a normal distribution n( , ), with estimated as the sum of the squares of the standard errors. differences were considered significant if p< . * e- ( . / ) and are indicated in bold font. all discovery studies contributed to the meta-analyses of egfr among those without diabetes and without hypertension. the blsa study did not contribute to the meta-analysis among those with hypertension, and theamish, the blsa, and the family heart study did not contribute to the meta-analysis among those with diabetes. sample sizes for hypertension and diabetes are presented in table . supplementary table : expression associated snp analysis. snps significantly associated with egfrcrea, ckd or egfrcys in stage discovery analyses that are associated with gene expression in liver, lymphoblastoid cell lines (lcl) or lymphocytes. starred loci (*) were also detected in association with egfrcrea using the fdr method. tissue: liver p-value closest gene second closest gene snp e g f r c y s c k d e g f r c re a esnp ingene genes within kb name d is ta n c e (b p ) name d is ta n c e (b p ) expressed gene expressed probe r t o t o p s n p top snp n o te s rs . e- . e- . e- . e- sypl atxn l ; cyb d ; sypl ; psma ; amigo sypl atxn l sypl . rs rs . e- . e- . e- . e- fbxl med ; fbxl fbxl med perld * rs . e- . e- . e- . e- acvr b acvr b; xylb; endogl acvr b endogl hss * rs . e- . e- . e- . e- acvr b scn a; acvr b; endogl acvr b endogl xylb * rs . e- . e- . e- . e- shroom shroom shroom flj shroom . rs rs . e- . e- . e- . e- oip nusap ; oip ; chp oip nusap contig * rs . e- . e- . e- . e- alms alms alms nat alms . rs rs . e- . e- . e- . e- fbxl ; rpl ; cacnb ; stac fbxl stac crkrs * rs . e- . e- . e- . e- htatip; rnaseh c dkfzp e ; htatip; rela; rnaseh c rnaseh c htatip rnaseh c . rs rs . e- . e- . e- . e- pip k b fam a; pip k b fam a pip k b fam a . rs rs . e- . e- . e- . e- rsbn l; ptpn rsbn l ptpn bc . rs rs . e- . e- . e- . e- ube q ube q ; fbxo ube q fbxo ube q . rs rs . e- . e- . e- . e- senp igf bp ; senp senp igf bp hss rs . e- . e- . e- . e- tprkb; flj ; dusp ; nat b tprkb dusp tprkb . rs rs . e- . e- . e- . e- ccdc rhpn ; slc a ; c orf ; ccdc ccdc c orf slc a . rs rs . e- . e- . e- . e- zfp znf ; zfp ; znf ; znf ; znf zfp znf zfp rs . e- . e- . e- . e- mtch c qtnf ; mtch ; agbl ; ndufs mtch agbl cugbp rs . e- . e- . e- . e- slc a ; slc a slc a slc a slc a rs . e- . e- . e- . e- c orf ; chmp a; cdk ; dpep ; spata l; cpne chmp a dpep c orf rs . e- . e- . e- . e- ankrd ankrd ; rgs bp; nudt ankrd rgs bp snora rs . e- . e- . e- . e- stc stc nkx - c orf . rs rs . e- . e- . e- . e- hla-g hla-g hla-a hla-a rs . e- . e- . e- . e- c orf c orf ; tmem c orf tmem c orf tissue: lymphoblastoid cell lines (lcl) p-value closest gene second closest gene snp e g f r c y s c k d e g f r c re a esnp ingene genes within kb name d is ta n c e (b p ) name d is ta n c e (b p ) expressed gene expressed probe r t o t o p s n p top snp n o te s rs . e- . e- . e- . e- ptpn ptpn ptpn rsbn l ptpn _at . rs rs . e- . e- . e- . e- crkrs med ; crkrs crkrs med _at * rs . e- . e- . e- . e- mrpl ; slc a ap; rbks slc a ap mrpl _at rs . e- . e- . e- . e- tmem ; rsbn l; phtf tmem rsbn l ptpn _at . rs rs . e- . e- . e- . e- crkrs crkrs neurod _at * rs . e- . e- . e- . e- casp ela b; ela a; ctrc; casp ; dnajc casp ela b _s_at * rs . e- . e- . e- . e- crkrs crkrs crkrs med _at * rs . e- . e- . e- . e- fbxl fbxl fbxl med _at * rs . e- . e- . e- . e- fbxl fbxl ; rpl ; stac fbxl stac _at * rs . e- . e- . e- . e- fbxl fbxl ; stac fbxl stac _at * rs . e- . e- . e- . e- fbxl ; rpl ; cacnb ; stac fbxl stac _at * rs . e- . e- . e- . e- rsbn l tmem ; rsbn l rsbn l tmem ptpn _at . rs rs . e- . e- . e- . e- phtf phtf phtf magi ptpn _at . rs rs . e- . e- . e- . e- ppp r b; neurod ; stard ; crkrs neurod ppp r b _at * rs . e- . e- . e- . e- phtf phtf phtf tmem ptpn _at . rs rs . e- . e- . e- . e- med ; fbxl ; crkrs med crkrs _at * rs . e- . e- . e- . e- exdl exdl ; chp exdl chp _at * rs . e- . e- . e- . e- rbm ; ccdc ; rbm ; sptbn ; rbm b; ccs rbm rbm _at rs . e- . e- . e- . e- rsbn l tmem ; rsbn l rsbn l tmem ptpn _at . rs rs . e- . e- . e- . e- rsbn l tmem ; rsbn l rsbn l tmem ptpn _at . rs rs . e- . e- . e- . e- exdl ; chp exdl chp _at * rs . e- . e- . e- . e- c orf gckr; xab ; ccdc ; znf ; c orf c orf znf gpn _at . rs rs . e- . e- . e- . e- med med ; fbxl ; crkrs med fbxl _at * rs . e- . e- . e- . e- znf xab ; ccdc ; znf ; c orf znf c orf gpn _at . rs rs . e- . e- . e- . e- actn rbm ; ccdc ; bbs ; zdhhc ; ccs; ctsf; actn ; dpp actn ctsf _at rs . e- . e- . e- . e- actn rbm ; ccdc ; bbs ; zdhhc ; ccs; ctsf; actn ; dpp actn ctsf _at rs . e- . e- . e- . e- slc a ; slc a slc a slc a _s_at rs . e- . e- . e- . e- znf xab ; slc a ap; ccdc ; supt l; znf ; c orf znf ccdc gpn _at . rs rs . e- . e- . e- . e- dnajc ela b; agmat; casp ; dnajc dnajc casp _s_at * rs . e- . e- . e- . e- zfp znf ; zfp ; znf ; znf ; znf zfp znf _at rs . e- . e- . e- . e- znf znf znf _at rs . e- . e- . e- . e- rtf ltk; ndufaf ; rtf ; rpap ; itpka rtf itpka _at * rs . e- . e- . e- . e- rsbn l; ptpn rsbn l ptpn ptpn _at . rs rs . e- . e- . e- . e- rsbn l; ptpn rsbn l ptpn ptpn _at . rs rs . e- . e- . e- . e- sipa l sipa l znf _at rs . e- . e- . e- . e- magi ; phtf phtf magi ptpn _at . rs rs . e- . e- . e- . e- rapsn; psmc ; mybpc ; slc a ; spi slc a spi _at rs . e- . e- . e- . e- nup nup ptprj _at rs . e- . e- . e- . e- ela b ela b; ela a; ctrc; casp ; efhd ; dnajc ela b casp _s_at * rs . e- . e- . e- . e- ela b ela b; ela a; ctrc; casp ; efhd ; dnajc ela b casp _s_at * rs . e- . e- . e- . e- spi psmc ; mybpc ; slc a ; madd; spi spi mybpc _at rs . e- . e- . e- . e- spi psmc ; slc a ; madd; mybpc ; spi spi mybpc _at rs . e- . e- . e- . e- rtf ltk; ndufaf ; rtf ; itpka rtf itpka _at * rs . e- . e- . e- . e- mtch c qtnf ; mtch ; agbl ; ndufs mtch agbl _at rs . e- . e- . e- . e- mast ; pik r mast pik r _at rs . e- . e- . e- . e- gpbp l gpbp l ; nasp; ipp; tmem ; ccdc gpbp l ccdc _at rs . e- . e- . e- . e- gpbp l gpbp l ; nasp; ipp; tmem ; ccdc gpbp l tmem _at rs . e- . e- . e- . e- gpbp l ; nasp; ipp; tmem ; ccdc gpbp l tmem _at tissue: lymphocyte p-value closest gene second closest gene snp e g f r c y s c k d e g f r c re a esnp ingene genes within kb name d is ta n c e ( b p ) name d is ta n c e ( b p ) expressed gene expressed probe r t o t o p s n p top snp n o te s rs . e- . e- . e- . e- c orf ; spata l ; gatm gatm spata l spata l gi_ -s . rs rs . e- . e- . e- . e- spata l ; c orf ; slc a c orf spata l spata l gi_ -s . rs rs . e- . e- . e- . e- gckr gckr; ift ; fndc gckr fndc ift gi_ -s top sn p rs . e- . e- . e- . e- dab c ; dab dab c dab gi_ -s . rs rs . e- . e- . e- . e- c c ; dab c dab dab gi_ -s . rs rs . e- . e- . e- . e- c c ; dab c dab dab gi_ -s . rs rs . e- . e- . e- . e- slc a c orf ; slc a c orf slc a spata l gi_ -s . rs rs . e- . e- . e- . e- c c ; dab c dab dab gi_ -s . rs rs . e- . e- . e- . e- dab c ; dab dab c dab gi_ -s . rs rs . e- . e- . e- . e- spata l c orf ; spata l ; gatm spata l c orf spata l gi_ -s . rs rs . e- . e- . e- . e- fbxo nrg ; ube q ; fbxo fbxo nrg fbxo gi_ -i . rs rs . e- . e- . e- . e- alms alms alms egr alms gi_ -s . rs rs . e- . e- . e- . e- alms alms alms egr alms gi_ -s . rs rs . e- . e- . e- . e- alms alms alms egr alms gi_ -s . rs rs . e- . e- . e- . e- alms alms alms egr alms gi_ -s . rs rs . e- . e- . e- . e- alms alms alms nat alms gi_ -s . rs rs . e- . e- . e- . e- alms alms alms egr alms gi_ -s . rs rs . e- . e- . e- . e- alms alms alms nat alms gi_ -s . rs rs . e- . e- . e- . e- alms alms alms nat alms gi_ -s . rs rs . e- . e- . e- . e- nrg ; ube q ; fbxo ube q fbxo fbxo gi_ -i rs rs . e- . e- . e- . e- alms alms alms nat alms gi_ -s . rs rs . e- . e- . e- . e- casp ela b; agmat; ela a; casp ; dnajc casp dnajc gi_ -a * rs . e- . e- . e- . e- crkrs crkrs neurod gi_ -s * rs . e- . e- . e- . e- casp ela b; ela a; ctrc; casp ; dnajc casp ela b gi_ -a * rs . e- . e- . e- . e- atp b ; tfdp tfdp atp b gi_ -s rs . e- . e- . e- . e- spata l ; c orf ; slc a c orf spata l spata l gi_ -s . rs rs . e- . e- . e- . e- c orf c orf ; slc a ; pldn c orf slc a spata l gi_ -s . rs rs . e- . e- . e- . e- neurod ; crkrs crkrs neurod gi_ -s * rs . e- . e- . e- . e- map k ltbp ; map k ; rela; sipa ; sssca ; fam b; kcnk ; ehbp l map k kcnk gi_ -s rs . e- . e- . e- . e- jarid a jarid a; slc a jarid a slc a jarid a gi_ -s . rs rs . e- . e- . e- . e- inoc inoc exdl gi_ -a * rs . e- . e- . e- . e- acvr b acvr b; endogl acvr b endogl gi_ -s * rs . e- . e- . e- . e- c ; dab dab c dab gi_ -s . rs rs . e- . e- . e- . e- scn a; acvr b; endogl endogl acvr b gi_ -s * rs . e- . e- . e- . e- cyb d atxn l ; cyb d ; sypl ; gnai ; gpr ; amigo cyb d atxn l cyb d gi_ -s . rs rs . e- . e- . e- . e- scn a; acvr b; endogl endogl scn a gi_ -s * rs . e- . e- . e- . e- acvr b scn a; acvr b; endogl acvr b endogl gi_ -s * rs . e- . e- . e- . e- endogl scn a; acvr b; endogl endogl acvr b gi_ -s * rs . e- . e- . e- . e- med ; fbxl ; crkrs med crkrs gi_ -s * rs . e- . e- . e- . e- inhbc inhbe; gli ; arhgap ; mars; inhbc inhbc inhbe gi_ -s * rs . e- . e- . e- . e- arl arl arl ndufs hs. -s * rs . e- . e- . e- . e- nat ; alms nat alms alms gi_ -s . rs rs . e- . e- . e- . e- nat ; alms nat alms alms gi_ -s . rs rs . e- . e- . e- . e- map k ; rela; sipa ; kcnk ; ehbp l sipa rela hmm -s rs . e- . e- . e- . e- arl arl arl ndufs hs. -s * rs . e- . e- . e- . e- atp b ; tfdp tfdp atp b gi_ -s rs . e- . e- . e- . e- tprkb; dusp ; nat b nat b tprkb tprkb gi_ -s . rs rs . e- . e- . e- . e- tprkb; dusp ; nat b nat b tprkb tprkb gi_ -s . rs rs . e- . e- . e- . e- tprkb; nat ; nat b nat b nat nat gi_ -s . rs rs . e- . e- . e- . e- tprkb tprkb; flj ; dusp ; nat b tprkb nat b tprkb gi_ -s . rs rs . e- . e- . e- . e- alms alms alms nat alms gi_ -s . rs rs . e- . e- . e- . e- znf xab ; slc a ap; ccdc ; supt l; znf ; c orf znf ccdc gpn gi_ -s . rs rs . e- . e- . e- . e- actn rbm ; ccdc ; bbs ; zdhhc ; ccs; ctsf; actn ; dpp actn ctsf gi_ -s rs . e- . e- . e- . e- actn rbm ; ccdc ; bbs ; zdhhc ; ccs; ctsf; actn ; dpp actn ctsf gi_ -s rs . e- . e- . e- . e- znf xab ; slc a ap; ccdc ; supt l; znf ; c orf znf ccdc gi_ -s rs . e- . e- . e- . e- inoc inoc inoc chac gi_ -a * rs . e- . e- . e- . e- endogl scn a; acvr b; endogl endogl scn a gi_ -s * rs . e- . e- . e- . e- slc a slc a ; nfatc ; rbm b; slc a os; lypla ; prmt slc a lypla gi_ -s * rs . e- . e- . e- . e- kcnma kcnma kcnma dlg gi_ -s rs . e+ . e- . e- . e- hspc c orf ; higd a; kiaa ; cltb; hspc ; arl hspc higd a gi_ -s * rs . e- . e- . e- . e- rapsn; psmc ; mybpc ; slc a ; madd; spi spi slc a gi_ -s rs . e+ . e- . e- . e- cst ; cst l; cst cst cst gi_ -s rs . e+ . e- . e- . e- cst ; cst l; cst cst cst gi_ -s rs . e+ . e- . e- . e- cst ; cst ; cst cst cst gi_ -s supplementary table - additional snps associated with egfrcrea and ckd at an fdr of . . shown are snps with p-value > x e- in stage discovery analyses that are associated with egfrcrea and ckd at an fdr of . (p< . x e- ) trait snp^ chr pos beta se pval qval coded allele coded allele freq. esnp**, (r to fdr snp) beta cys pval egfrcys genes egfrcrea rs - . . . e- . c . rs ( ) - . . e- casp ; agmat, ctrc, ddi , dnajc , efhd , ela a, ela b egfrcrea rs - . . . e- . t . - . . e- ddx ; egfrcrea rs . . . e- . t . . . e- pard b; egfrcrea rs - . . . e- . t . rs ( . ) - . . e- arl ; egfrcrea rs - . . . e- . t . - . . e- egfrcrea rs . . . e- . c . . . e- rerg; egfrcrea rs - . . . e- . a . - . . e- itpk ; c orf , c orf , moap egfrcrea rs . . . e- . a . . . e- slc a ; snora a, snora b egfrcrea rs - . . . e- . a . rs ( ) - . . e- crkrs; med , neurod all snps within ^ bp of genomewide associations excluded from fdr analysis. ^best (smallest p-value) snp from each locus with at least one snp reaching fdr < . . **esnps were selected among those significant for the same trait listed in this table as the one with the highest r to the snp presented in this table. for ckd, the known gatm locus was additionally 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famhs: . ship: . fhs: . vis: . koraf : . wghs: . λ individual studies ages: na koraf : . amish: . korcula: na aric: . micros: . asps: na nsphs: na blsa: na orcades: na chs: . rs-i: na erf: na rs-ii: na famhs: na ship: . fhs: . vis: na koraf : . wghs: na egfrcrea, n= ckd, n= egfrcys, n= expected –log (p) expected –log (p) o bs er ve d –l og (p ) o bs er ve d –l og (p ) o bs er ve d –l og (p ) λmeta represents the genomic control parameters after discovery meta-analysis, and the λ for the individuals studies is reported next to each trait. the graphs present p-values corrected for inflation at the study-specific level before meta-analysis as well as after meta- analysis for the meta-analysis genomic control parameter. no correction was applied to data from studies with λ< .the graph for egfrcys is cut off; all snps with lower p-values are located at the cst locus on chromosome . na denotes phenotype unavailability. black: results from meta-analysis, orange: null hypothesis. suppl. figure : quantile-quantile plots of observed vs. expected -log (p-values) from discovery analyses of egfrcrea (a), ckd (b), and egfrcys (c). expected –log (p) regional association plots - susceptibility loci for reduced renal function and chronic kidney disease o b se rv e d ( -l o g p) supplementary figure -log p-values are plotted versus genomic position (build ). the lead snp in each region is labeled. other snps in each region are color-coded based on their ld to the lead snp (ld based on the hapmap ceu, see color legend). gene annotations are based on ucsc genome browser (refseq genes, b ) and arrows indicate direction of transcription. graphs were generated using the software snap (http://www.broadinstitute.org/mpg/snap/index.php). chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r tars ecm adamtsl mcl ensa golph l hormad ctss ctsk arnt setdb lass anxa fam a prune bnipl c orf cdc se mllt rp - i . sema c tnfaip l lysmd scnm tmod vps pip k apsmd znf pi kb rfx selenbp psmb pogz chromosome position (hg ) (kb) r e co m b in a tio n ra t e (cm /m b ) rs . . r mapre tmem agbl emilin khk cgref abhd preb c orf tcf slc a c orf cad slc a dnajc g trim ucn mpv gtf c eif b snx znf ppm g nrbp krtcap ift fndc gckr c orf znf ccdc gpn supt l slc a ap mrpl rbks bre chromosome , rs , gckr region chromosome , rs , lass region o b se rv e d ( -l o g p) o b se rv e d ( -l o g p) chromosome , rs , alms region chromosome , rs , tfdp region chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r rasa rnf grk atp b tfdp gk xrn atr o b se rv e d ( -l o g p) chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r smyd c orf cct fbxo egr alms nat nat b tprkb dusp flj stambp actg dguok tet bola o b se rv e d ( -l o g p) chromosome , rs , dab region chromosome , rs , slc a region chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r osmr rictor fyb c dab chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r hk uimc znf fgfr nsd rab prelid mxd lman rgs slc a pfn f grk prr dbn pdlim dok ddx flj tmed b galt fam a o b se rv e d ( -l o g p) o b se rv e d ( -l o g p) chromosome , rs , vegfa region chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r znf abcc dlk tjap c orf yipf polr c xpo polh gtpbp mad l bp c orf mrps a vegfa c orf mrpl tmem b capn slc a hsp ab slc b nfkbie tcte aars o b se rv e d ( -l o g p) chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r abcf csglca-t smarcd nub wdr crygn rheb prkag galntl galnt mll o b se rv e d ( -l o g p) chromosome , rs , prkag region chromosome , rs , pip k b region chromosome , rs , atxn region chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r pgm c orf pip k bfam a prkacg fxn tjp c orf o b se rv e d ( -l o g p) chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r cux fam a sh b atxn brap acad aldh mapkapk tmem erp c orf o b se rv e d ( -l o g p) chromosome , rs , dach region chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r sin a ptpn snupn imp snx cspg odf l ube q fbxo nrg c orf etfa isl scaper o b se rv e d ( -l o g p) chromosome position (hg ) (kb) r e co m b in a tio n ra te (cm /m b ) rs . . r dach chromosome , rs , ube q region o b se rv e d ( -l o g p) chromosome , rs , slc a region chromosome position (hg ) (kb) r e co m b in a tio n ra t e (cm /m b ) rs . . r znf dpy l pdcd ankrd rgs bp nudt tdrd slc a ccdc c orf rhpn gpatch wdr lrp slc a cebpa o b se rv e d ( -l o g p) 	supplement_r _finalsubmission_ _clean.doc [compatibility mode] 	supplement_r _finalsubmission_ _clean a 	supplement_r _finalsubmission_ _clean. 	qq_multipanel_ 	regional_plots_ _page 	regional_plots_ _page 	regional_plots_ _page 	regional_plots_ _page 	regional_plots_ _page 	regional_plots_ _page 	regional_plots_ _page slitrk mutations cause myopia and deafness in humans and mice mustafa tekin, … , jun aruga, andrew h. crosby j clin invest. ; ( ): - . https://doi.org/ . /jci . myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. in this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. our molecular investigation in families led to the identification of homozygous nonsense mutations (p.r x, p.s x, and p.q x) in slit and ntrk-like family, member (slitrk ), a leucine-rich repeat domain transmembrane protein. all mutant slitrk proteins displayed defective cell surface localization. high-resolution mri of wt and slitrk -deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. histological investigation of wt and slitrk -deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in slitrk -deficient animals. taken together, our results showed that slitrk plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness. research article ophthalmology find the latest version: https://jci.me/ /pdf http://www.jci.org http://www.jci.org/ / ?utm_campaign=cover-page&utm_medium=pdf&utm_source=content https://doi.org/ . /jci http://www.jci.org/tags/ ?utm_campaign=cover-page&utm_medium=pdf&utm_source=content http://www.jci.org/tags/ ?utm_campaign=cover-page&utm_medium=pdf&utm_source=content https://jci.me/ /pdf https://jci.me/ /pdf?utm_content=qrcode research article the journal of clinical investigation http://www.jci.org volume number may slitrk mutations cause myopia and deafness in humans and mice mustafa tekin, , barry a. chioza, yoshifumi matsumoto, oscar diaz-horta, harold e. cross, duygu duman, haris kokotas, heather l. moore-barton, kazuto sakoori, maya ota, yuri s. odaka, joseph foster ii, f. basak cengiz, suna tokgoz-yilmaz, oya tekeli, maria grigoriadou, michael b. petersen, , ajith sreekantan-nair, kay gurtz, xia-juan xia, arti pandya, michael a. patton, juan i. young, jun aruga, and andrew h. crosby john p. hussman institute for human genomics and dr. john t. macdonald department of human genetics, university of miami miller school of medicine, miami, florida, usa. division of pediatric genetics, ankara university school of medicine, ankara, turkey. centre for human genetics, st. george’s university of london, london, united kingdom. laboratory for behavioral and developmental disorders, riken brain science institute, saitama, japan. department of ophthalmology, university of arizona school of medicine, tucson, arizona, usa. department of genetics, institute of child health, ‘aghia sophia’ children’s hospital, athens, greece. south west thames regional genetics services, st. george’s university london, london, united kingdom. department of ophthalmology, ankara university school of medicine, ankara, turkey. department of clinical genetics, aalborg hospital, aalborg, denmark. windows of hope genetic study, geauga county, ohio, usa. department of human and molecular genetics, virginia commonwealth university, richmond, virginia, usa. myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. in this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. our molecular investigation in families led to the identification of homozygous nonsense mutations (p.r x, p.s x, and p.q x) in slit and ntrk-like family, member (slitrk ), a leucine-rich repeat domain transmembrane protein. all mutant slitrk proteins displayed defective cell surface localization. high-resolution mri of wt and slitrk -deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. histological investigation of wt and slitrk -deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in slitrk -deficient animals. taken together, our results showed that slitrk plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness. introduction myopia is the most common cause of visual impairment world- wide ( ). it affects more than % of adults in western europe and america, and its prevalence exceeds % in some urban areas of east asia ( – ). high myopia, defined as refractive error greater than – or more diopters, affects approximately %– % of the pop- ulation ( ) and is a significant risk factor for ocular comorbidity, including retinal detachment, macular degeneration, premature cataract, and glaucoma ( , ). data from several study types sug- gest a strong genetic component to myopia. the odds of a child becoming myopic rise with the number of myopic parents ( ), and even the nonmyopic offspring of myopic parents have been shown to have longer eyes than nonmyopic children of nonmyopic par- ents ( ). twin studies add further support, indicating heritabil- ity values between . and . ( – ). genome-wide association studies have identified putative gene loci on chromosomes q ( ) and q ( , ), as well as genetic variants in ctnnd , as being associated with myopia ( ). recently, a missense variant in leprel (encoding prolyl -hydroxylase [p h ]), a -oxogluta- rate–dependent dioxygenase that hydroxylates collagen molecules, was associated with an autosomal-recessive form of high myopia and early-onset cataracts in an israeli-bedouin kindred ( ). how- ever, although the magnitude of heritable factors in ocular refrac- tion has been convincingly established, few association studies have been replicated in independent studies or candidate genes validated in inherited human conditions involving myopia ( ). consequently, the genes and molecular mechanisms important for normal human refractive development remain to be discovered. hearing loss is another common sensory disorder, with a preva- lence of in , at birth that almost doubles by years of age ( , ). although the incidence of hearing loss in individuals with myopia is unknown, the prevalence of ocular abnormalities in deaf children is high ( ). it is known that greater than % of permanent childhood hearing impairment in the developed world has a genetic etiology ( ), and many syndromic forms of hearing loss have been described. nonsyndromic hearing loss is similarly genetically heterogeneous, with greater than loci and greater than genes identified ( ). more than genetic syndromes are associated with high myopia and more than with hearing loss (omim). several syndromes have previously been described that involve myopia as well as deafness, although all involve abnormalities in other sys- tems or involve other ocular abnormalities. a well-known example is stickler syndrome (omim ), in which patients present with midfacial underdevelopment, cleft palate, spondyloepiphyseal dysplasia, and/or precocious arthritis in addition to hearing loss and myopia ( ). mutations in genes encoding various collagen proteins (col a , col a , col a , col a , or col a ) cause stickler syndrome ( ). in donnai-barrow syndrome (omim ), a rare disorder also involving hearing loss and myopia due to mutations in lipoprotein receptor–related protein precursor authorship note: barry a. chioza, yoshifumi matsumoto, and oscar diaz-horta contributed equally to this work. conflict of interest: the authors have declared that no conflict of interest exists. citation for this article: j clin invest. ; ( ): – . doi: . /jci . research article the journal of clinical investigation http://www.jci.org volume number may (lrp ), affected individuals have ocular hypertelorism, enlarged fontanelle, agenesis of the corpus callosum, intellectual disability, and congenital diaphragmatic hernia and/or omphalocele ( ). we describe here the first syndrome of severe congenital myopia and sensorineural hearing loss in the absence of other systemic, ocular, or connective tissue manifestations. our findings dem- onstrated a major role of slit and ntrk-like family, member (slitrk ), in the molecular pathways important for normal human and mouse vision and hearing and shed light on biologi- cal processes that may be perturbed and subsequently result in high myopia and deafness. results slitrk mutations cause high myopia and sensorineural deafness in humans. we initially observed a previously unrecognized syn- drome characterized by high myopia and deafness in families: family was an old-order amish family with autosomal-reces- sive congenital myopia and prelingual sensorineural hearing loss (table , figure a, and supplemental figure ; supple- mental material available online with this article; doi: . / jci ds ). affected siblings have congenital high myopia (refraction error ranging from – . to – . diopters in affected individuals) corrected by glasses and prelingual-onset moderate to severe bilateral sensorineural hearing loss necessitating aids. the parents had low myopia (both – . diopters) of youth onset with normal hearing. family was a consanguineous turkish family with affected individuals (figure e). all affected indi- viduals had high myopia (refraction error ranging from – . to – . diopters) and prelingual-onset moderate to profound sen- sorineural hearing loss. the parents and unaffected siblings did not have hearing loss; the father and unaffected siblings had adult-onset myopia (less than – diopters). affected individuals in both families did not have delays in gross motor development; neither did they have balance problems, vertigo, dizziness, or spontaneous or positional nystagmus. tandem walking was nor- mal, and romberg test was negative. ct scans of temporal bones were normal in affected person in each family. clinical examina- tion confirmed the absence of additional findings, with normal facial appearance, neurological, connective tissue, and ocular manifestations. there was no retinitis pigmentosa in any affected subject. to map the chromosomal location of the disease gene in family , a genome-wide snp analysis was undertaken using samples from all affected family members (xi: , xi: , and xi: ; figure a). this identified a single homo- zygous region shared among all affected individuals of . mb of chromosome q . – q . (figure b), delimited by recombinant snp markers rs and rs , likely to correspond to the disease locus. no other notable regions of autozygosity were observed. autozygosity across this interval was corroborated by microsatellite marker analysis in all family members, which defined a haplotype that cosegregated with disease phenotype (supplemental figure ). the identif ied expanse of chromosome corre- sponded to a particularly gene-poor region contain- ing few coding sequences and putative functional transcripts. to identify the causative mutation, we sequenced all protein-coding refseq (http://www.ncbi. nlm.nih.gov/refseq/) genes (slitrk , omim ; slitrk , omim ; slitrk , omim ; gpc , omim ; the first exon of gpc , omim ) as well as numerous micrornas and other potentially functional mol- ecules located within the disease interval (loc ; mir ; mir ; loc ; mir hg, omim ; mir , omim ; mir a, omim ; mir a, omim ; mir a, omim ; mir b , omim ; mir a , omim ). this revealed a single novel sequence variant (nm_ . :c. c>t; np_ . :p.q x; chromo- somal variant nc_ . :g. c>t; figure d and supplemental figure ) in exon of slitrk . sanger sequence analysis of all family members indicated that all affected children were homozygous for this variant, while the parents and unaf- fected sibling were all heterozygous. as the variant creates a psii restriction endonuclease recognition site, this finding was also confirmed by restriction digestion of pcr products encompass- ing this base change (figure c). the variant was also detected in the rna transcript via sanger sequencing. it was not present in genomic databases (dbsnp , http://www.ncbi.nlm.nih.gov/ snp/; , genomes, http://www. genomes.org/) and was not detected in control individuals of european ancestry; only a single heterozygous carrier individual was detected in the analysis of amish controls. in parallel with the positional cloning effort in family , whole exome sequencing was performed in affected individuals in family (iv: and iv: ; figure e) in order to identify the caus- ative mutation. the exome sequencing experiment produced , , and , , reads for iv: and iv: , respectively. when measured at a minimum depth of ×, % of the target region was covered in both samples, with an average depth of × and × for iv: and iv: , respectively. likewise, when measured at × and × coverage, % and % (iv: ) and % and % (iv: ) of the intended target was covered, respectively. in terms of vari- ant calls, the burrows-wheeler alignment (bwa) predicted the presence of , variants in both samples, either homozygous or heterozygous. by using our filtering tool (see methods), only shared homozygous variant at position ch : g>t (hg ) was detected, corresponding to c. c>a (p.s x) in slitrk table clinical overview of patients patient (current age) myopia sensorineural hearing loss age at current age at current diagnosis severitya diagnosis severityb family (amish) xi: ( yr) yr – . congenitalc severe xi: ( yr) yr – . yr moderate xi: ( yr) yr – . yr severe family (turkish) iv: ( yr) infancy – . prelingual moderate iv: ( yr) early childhood – . prelingual severe iv: ( yr) early childhood – prelingual profound iv: ( yr) infancy – prelingual profound family (greek) ii: (young adult) na na prelingual severe ii: (young adult) na na prelingual severe adiopters in better eye. bbetter ear. cnewborn screen. research article the journal of clinical investigation http://www.jci.org volume number may (figure f). there were and sequence reads of the variant allele in iv: and iv: , respectively, and read of the reference. the identified variant was within a . -mb homozygous run between , , and , , bps on chromosome . it was absent in the dbsnp and nhlbi exome sequencing project databas- es (exome variant server; http://evs.gs.washington.edu/evs/). sanger sequencing confirmed the variant and showed its coseg- regation in the entire family and absence in turkish controls. to find additional mutations in slitrk , linkage to the slitrk locus was evaluated in multiplex families with autosomal- recessive deafness. affected individuals in these families had con- genital or prelingual-onset severe to profound sensorineural hear- ing loss. although myopia was present in some families, none was considered to have a syndromic form of deafness. when more than affected members had the same haplotype flanking slitrk , sanger sequencing of the entire gene was performed. this anal- ysis revealed a greek family with affected children having a homozygous c. c>t (p.r x) mutation (family ; figure , g and h). both parents were heterozygous for the mutation. this mutation was absent in greek controls. the p.r x and p.s x mutations were screened in american probands with sensorineural hearing loss who are not known to have a syndrome. this study identified no sequence variants. detected slitrk mutations do not cause mrna decay. since all detected mutations in slitrk were nonsense, they might trigger the nonsense-mediated mrna decay response, thereby leading to the absence of mrna for translation into the slitrk protein. serial quantitative pcr measurements of mrna levels of each mutant gene along with wt slitrk transfected into hek cells showed comparable stability of each mrna relative to the wt (supplemental figure ), which suggests that none of the identified mutations triggers the nonsense-mediated mrna response. slitrk mutations result in loss of function. slitrk belongs to the slitrk family of genes, which encode single-pass (type ) trans- membrane proteins with leucine-rich repeat (lrr) domains in the aminoterminal two-thirds of the molecule and a putative trans- membrane domain located toward the carboxyterminal end ( , ). rodent slitrk proteins are known to have synapse-inducing ( ) and neurite-modulating ( ) activity in cultured cells. we first studied the subcellular localization of wt human slitrk and all figure families and identified slitrk mutations. (a–d) family (amish). (a) pedigree diagram. (b) graphical representation of genotypes across chromosome . (c) psii restriction digestion of slitrk exon pcr product, confirming that all affected individuals were homozygous, and parents and unaffected sibling heterozygous, for the smaller digested c>t slitrk allele. dna marker is shown at far left; family members are as indicated. (d) electropherograms showing the identified mutation. (e and f) family (turkish). (e) pedigree diagram. individuals iv: , iv: , iv: , and iv: were homozygous and iv: , iv: , iii: , and iii: heterozygous for p.s x. (f) electropherograms showing the identified mutation. (g and h) family (greek). (g) pedigree diagram. individuals ii: and ii: were homozygous and i: and i: heterozygous for p.r x. (h) electropherograms showing the identified mutation. research article the journal of clinical investigation http://www.jci.org volume number may mutations. we constructed cag-slitrk -ires-egfp expression vectors and transfected them into nonneural (hek ) cells. wt slitrk and all mutants were detected in immunostaining of the permeabilized cells using an antibody recognizing an aminotermi- nal epitope of slitrk (figure ). however, in a nonpermeabilized condition, wt slitrk , but not the mutants, gave immunoposi- tive signals (figure ), which indicates that the mutations impaired cell surface expression. accordingly, when the transfectants were cocultured with dissociated rat hippocampal neurons, synapse- inducing activity was observed in human wt slitrk , but not in the slitrk variants (supplemental figure a). furthermore, human wt slitrk reduced both neurite number and length of the pc cells treated with nerve growth factor (ngf) in a manner comparable to mouse slitrk ( ), but the variants did not show significant effects on the neurite properties (supplemental figure b). taken together, these observations revealed dramatic effects of all nonsense mutations on slitrk function and suggest that a null mechanism most likely relates to these mutations. mouse slitrk ko presents with deafness and myopia. to verify the phenotypical consequence of the slitrk mutations obtained in humans, we undertook detailed phenotype analysis of slitrk ko (slitrk –/–) mice. we previously showed that both male and female slitrk ko mice grow without showing any external abnormalities and are fertile ( ). our previous experiments showed that in the developing inner ear, slitrk ko mice have pronounced reduction of cochlear innervation, despite normal gross morphology and general organization of the organ of corti ( ). more recently, we demonstrated functional deficits in adult slitrk ko mice, which have reduced startle response and impaired auditory brainstem responses consistent with mid–frequency range hearing loss ( ). this is consistent with the sensorineural hearing loss phenotype in human subjects with slitrk null mutations. since little is known about the eye phenotype of slitrk ko mice, we addressed the ocular manifestations in this study. we inves- tigated - to -month-old slitrk ko and wt adult male mice using high-resolution small animal mri scanning, as previously described ( ). this revealed a significant increase in axial length ( . %; p = . ), but not in lens thickness, of slitrk ko versus wt animals (figure ). this size difference in axial length was not apparent in newborn mice, as determined in serial sections of fresh- ly frozen eyes (figure ), which indicates that slitrk regulates eye growth after birth. it is known that axial length is the endophe- notype of myopia in animal models, with greater length being asso- ciated with more severe myopia ( ). consequently, slitrk ko mice appear to closely mimic the human phenotype involving myopia, in association with the previously documented deafness. figure slitrk q x, slitrk s x, and slitrk r x are defective in cell surface localization. wt slitrk and mutant slitrk expres- sion vectors were transfected into hek cells. total and cell sur- face proteins in the transfectants were detected by antibody against the aminoterminus of slitrk protein (red), whereas transfected cells were marked by gfp (green). only wt slitrk was detected at the cell surface. scale bar: μm. research article the journal of clinical investigation http://www.jci.org volume number may we next investigated the temporospatial expression pattern of slitrk during development. in the newborn, slitrk mrna was broadly detected in neural retina (figure , a and b, and supple- mental figure ). later, in p retina, expression was enhanced in the inner nuclear and outer plexiform layers (figure , c, d, g, and h). expression weakened as development proceeded, and slitrk mrna was detected in the inner nuclear layer and in a subset of cells in the ganglion cell layer of adult mice (figure , e and f). we next investigated the slitrk ko retina in postnatal development histologically. there were no overt abnormalities in gross layer organization in conven- tional histological staining (data not shown). however, when we examined the synapse markers ribeye, vglut , and vgat by immunof luorescence st aining in slitrk ko retina, it became clear that the ribeye signal in the outer plexiform layer was sig- nificantly lower than in wt retina ( %; p = . ) — and vglut in the outer plexiform layer and vgat in the inner plexiform layer tended to be lower — at p , when synap- togenesis was actively taking place (figure , a and b, and supplemen- tal figure ). ribeye expression was not detected at p in either slitrk ko or wt animals (figure a) and was detected equally in adult animals (supplemental figure ). in addition, anti-neurofilament staining for the retinal ganglion cell nerve fibers was reduced, albeit not significantly (fig- ure b and supplemental figure ). conversely, retinal cell type–specific markers (rhodopsin for rod photo- receptor cells, protein kinase c for bipolar cells, tyrosine hydroxylase for amacrine cells, thy for retinal gan- glion cells, and glutamine synthetase for müller glia cells) did not show clear genotype-dependent changes during immunostaining (supplemental figure ). collectively, these results were indicative of delayed synaptogenesis in slitrk ko retina. to our knowledge, this is the first indication of the involvement of slitrk in syn- aptogenesis in vivo. ribeye is the marker for “ribbon synapse,” which has been proposed to sustain high rates of exocytosis for relatively long periods ( ). interestingly, inner ear hair cells and retinal photoreceptor cells both possess this unique type of syn- apse, which indicates that slitrk may play a similar role in reti- nal and inner ear development. figure increased axial length of the eyes of slitrk ko mice. axial length (al) and lens thickness (lt) were measured in male - to -month-old wt (n = ) and slitrk ko (n = ) adult mice using mri. measure- ments of newborn (p ) wt (n = ) and slitrk ko (n = ) mice were made using serial sections of rapidly frozen fresh eyes. for each ani- mal, mean values of left and right eyes were first determined. mean values were then used for statistical analysis for genotype-depen- dent differences. bars indicate mean ± sem. a significant difference between wt and slitrk ko mice was observed in adult axial length. p values are based on -tailed t test. parameters used for the analysis are illustrated. figure postnatal slitrk expression in the murine eye. slitrk in situ hybridization (a, c, e, and g; purple stain) and hematoxylin and eosin staining (b, d, f, and h) in adjacent sections in p , p , and -week-old adult mice. (a–f) retinal layers. (g and h) horizontal sections through eye. (a and b) in the newborn, slitrk mrna was broadly detected in neural retina. (c and d) at p , expression became enhanced at the inner nuclear layer (in), whereas moderate expression continued in the ganglion cell layer (gc). images are higher-magnification views of boxed regions in g and h. (e and f) expression weakened as development proceeded, and expression was detected at the inner nuclear layer and in a subset of cells in the ganglion cell layer in adult mice. (g and h) in the tissues surrounding the eye, weak expression was detected in the hair root sheath (hs), lacrimal gland (lg), and olfactory epithelium (oe) at p . the gray line in the outer plexiform layer (op) and the layer containing outer and inner segments of photoreceptor cells (os) in adults (e, asterisk) and the brown staining in the lens (l) at p (g, asterisk) were not authentic in situ hybridization signals. parallel lines in h denote optic nerve. cr, cornea; em, extraocular muscle; ip, inner plexiform layer; n, retinal neuroblastic layer; on, outer nuclear layer; r, retina; s, sclera. scale bars: μm (a–f); mm (g and h). research article the journal of clinical investigation http://www.jci.org volume number may discussion although various heritability measures, twin studies, and genetic analyses have indicated a strong genetic component in myopia, lit- tle remains known about the specific genetic and molecular basis of this common eye disorder. in the current study, we showed that mutation of slitrk results in a human syndrome that includes high myopia and sensorineural deafness as the only clinical find- ings and confirmed a similar phenotype in slitrk mutant mice. slitrk gene family members are highly expressed in both human and murine central nervous systems ( , ) and are currently con- sidered emerging candidate genes for neuropsychiatric disorders ( ). the family consist of structurally related transmembrane proteins belonging to the lrr superfamily and are single-pass (type ) transmembrane proteins highly expressed in the central nervous system ( ). among the mouse slitrk family, slitrk has been shown to display strong expression in cochlea and retina in mouse embryos. specifically, in the developing cochlea, slitrk signals are evident in the primitive sensory neuroepithelium that gives rise to cochlear hair cells, and in the developing eye, expres- sion is observed in the retinal outer neuroblastic layer, eyelid, and extraocular orbital mesenchymal tissue ( , ). our present findings, along with previous studies investigat- ing the role of slitrk in inner ear development ( ), suggest that slitrk is involved in innervation of the developing murine eye and ear. we observed impaired synapse-inducing activity of slitrk mutants as well as delayed formation of synapses in the eyes of slitrk mutant mice. vglut and vgat, both markers of syn- aptic formation, were reduced in p retina of slitrk mutant mice, which indicates that slitrk may possess the ability to recruit or induce the formation of vglut - and vgat -positive neuronal structures ( ). consequently, while a general delay in neuronal maturation cannot be ruled out, the pathogenetic mechanism underlying the eye and ear phenotype in this syndrome may involve impairment of neural circuit formation. myopia and other refractive-error disorders are consequences of uncoordinated development and contributions of a number of ocular components to overall eye structure, in particular axial length, which is generally accepted to be the main determinant of refractive error. by investigating slitrk ko mice, we showed that the myopia was likely due to increased axial eye length, a feature not previously recognized in these animals, which indicates a key role for slitrk in this process. we detected no evidence for the occurrence of other eye disorders that typically accompany high myopia — such as cataract, glaucoma, and chorioretinal abnormali- ties — in either humans or mice; therefore, the slitrk ko mouse model represents a valuable tool for further investigation of the molecular and biological basis of the axial length endophenotype. numerous lines of evidence support the existence of an active emmetropization mechanism, which controls the location of the retina by remodeling the sclera so that images can be focused on the focal plane ( – ). alterations in axial elongation produced by the emmetropization mechanism may involve modulating normal postnatal growth, not ceasing or initiating growth, although much remains to be discovered. interestingly, in embryonic mice, cochlear slitrk is thought to regulate expression of neurotrophic factors ( ) that have been implicated in experience-dependent develop- ment of retinal function ( ), which suggests a possible role for slitrk in emmetropization via neurotrophin modulation. methods further information can be found in supplemental methods. sample preparation. dna was extracted from blood via standard meth- ods. affected individual from each family was prescreened and found to be negative for common causes of nonsyndromic deafness, including mutations in gjb (omim ), and for the m.a g mutation in mtrnr (omim ). rna was isolated from venous blood using the paxgene method (qiagen), and cdna synthesis from extracted rna was undertaken with primescript one step rt-pcr kit (version ; takara). genotyping and autozygosity mapping. autozygosity mapping was performed with illumina human cyto beadchip arrays incorporating approximately , genetic markers in family . to screen additional families for link- figure analysis of the synapse markers ribeye and vglut in slitrk ko mice. (a) the ribbon synapse marker ribeye and the glutamate-containing synaptic vesicle marker vglut were examined in p and p mouse eyes by immunofluorescence staining. layer abbreviations as in figure . (b) densitometric quantification of the immunopositive signals of immunostained images in a and supplemental figure . shown are results for ribeye, vglut , and the synaptic vesicle marker synaptophy- sin (syph) in the outer plexiform layer; the gaba-containing synaptic vesicle marker vgat and synaptophysin in the inner plexiform layer; and the neuronal marker neurofilament (nf) in the nerve fiber layer (nf). results are presented relative to wt levels (assigned as ). n = – p retinas from independent animals for each genotype. p values are based on -tailed t test. research article the journal of clinical investigation http://www.jci.org volume number may age to the slitrk locus, rs , rs , rs , and rs were genotyped with custom taqman probes in multiplex families with autosomal-recessive deafness. for each taqman reaction, ng dna was mixed with taqman universal pcr master mix ( ×; applied biosystems) and snp genotyping assay ( ×). pcr conditions were °c for min- utes, °c for minutes, cycles of °c for seconds, and °c for minute. data were analyzed with sds . software (applied biosystems). whole exome sequencing. genomic dna from affected individuals of family were used to sequence the whole exomes, as previously described ( ). the sureselect human all exon mb kit (agilent) was used for in- solution enrichment of coding exons and flanking intronic sequences fol- lowing the manufacturer’s standard protocol. adapter sequences for the illumina hiseq were ligated, and the enriched dna samples were sub- jected to standard sample preparation for the hiseq instrument (illu- mina). paired-end reads of bases in length were produced. the illumina casava version . pipeline was used to produce -bp sequence reads. bwa ( ) was used to align sequence reads to the human reference genome (hg ), and variants were called using the gatk software package ( , ). all variants were submitted to seattleseq (http://snp.gs.washington. edu/seattleseqannotation) for further characterization. the genomes management application (gemapp) of university of miami miller school of medicine (https://genomics.med.miami.edu/), was used to filter variants. the variants were filtered according to the inheri- tance model (autosomal-recessive with both homozygous and compound heterozygous), the variant function class including missense, nonsense, splice sites, in-frame and frame-shift indels, presence, and frequency at the dbsnp (http://www.ncbi.nlm.nih.gov/snp/) and nhlbi (http://evs. gs.washington.edu/evs/) databases (minor allele frequency of less than % was used). variants were also filtered for the absence in more than fami- lies samples in our internal database that includes more than exomes. gatk quality score (qual) was set to and genotype quality (gq) to . sanger sequencing. sanger sequencing was used to analyze refseq (www. ncbi.nlm.nih.gov/refseq/) genes as well as microrna and other poten- tially functional molecules within the disease interval in family , to con- firm the variant obtained through whole exome sequencing in family , to identify the variant in family , and to evaluate cosegregation in all fami- lies. pcr primers were designed using primer plus software (http://www. bioinformatics.nl/cgi-bin/primer plus/primer plus.cgi) encompassing all coding exons and associated intron-exon splice boundaries (slitrk primers shown in supplemental table ). pcr reactions were run in μl volume, applying a touch-down protocol and annealing temperatures between °c and °c. we visualized pcr products on agarose gels, cleaned over sephadex columns or with exosap-it (ge healthcare), and applied to bigdye reactions following the manufacturer’s recommenda- tions (applied biosystems). a dna sequencer (abi ) was used to detect the mutation. sequences were analyzed using mutation surveyor software (version . ; softgenetics). mrna nonsense-mediated decay. genomic dna from patients homozygous for c. c>t (p.r x), c. c>a (p.s x), or c. c>t (p.q x) and from a control individual was amplified by high-fidelity pcr using primer pairs shown in supplemental table . the resulting segments overlapping a unique ecori restriction site and comprising the ′ untranslated region, intron, and coding exon of slitrk were subcloned into a pcr . -topo vector (invitrogen). the inserts were removed with paci-ecori and ecori- noti and sequentially cloned downstream of the cmv promoter contained in an expression vector that also carries the ef- α–egfp expression cas- sette (pa vector; gift from z. zhou, university of pennsylvania, philadel- phia, pennsylvania, usa). hek cells were transfected with pa vectors containing wt slitrk and mutant slitrk minigenes to investigate the stability of mrna variants by quantitative real-time pcr using sybr green (applied biosystems) and primers shown in supplemental table after treatment with actinomycin-d, an inhibitor of rna synthesis. slitrk mrna level was normalized with s rrna, which is transcribed with an actinomycin-d–insensitive polymerase. post-pcr sanger sequencing confirmed correct splicing and transcription of mutant and wt slitrk . slitrk expression constructs. slitrk protein coding regions were pcr amplified from genomic dna of a healthy human subject or from cloned dna of the patients using primers ′-ccaccatgccgctgtg- gattcatc- ′ and ′-ctatgtttgctgctccaggac- ′ and cloned into pgemt easy plasmid vector (promega). the resultant noti fragments containing the entire protein coding region preceded by kozak consensus sequence were inserted into pef–ires–alkaline phosphatase expression vec- tor ( ) or pcag-ires-egfp vector ( ). slitrk ko mice. slitrk null allele (slitrk −) was generated by replac- ing its entire protein coding region by a loxp sequence ( ). slitrk ko (slitrk −/−) mice were generated using /ola-derived es cells and were backcrossed to c bl/ j mice for more than generations. both male and female slitrk ko mice grew without showing any external abnor- malities and were fertile ( ). mri analysis. mri analysis of adult mouse eyes was performed essentially as described previously ( ). mri images were acquired by subjecting anesthe- tized mice to mri scan using a vertical bore . -t bruker avance wb imaging spectrometer (bruker biospin). animals were anesthetized with intraperitoneal injection of ketamine ( mg/kg) and xylazine ( mg/kg) for induction, and . % isoflurane in air ( l/min flow rate) for maintenance. a contrasting agent ( mg/ml meglumine gadopentetate; fujipharma) was used as eyedrops to highlight the anterior chamber. mri images were obtained using the fisp- d protocol of paravision software (version . ; bruker biospin) with the following parameter values: effective te, . ms; tr, . ms; flip angle, °; average number, ; acquisition matrix, × × ; fov, . × . × . mm; total scan time, minutes, sec- onds. the resulted resolution was μm/pixel. for determination of axial length, the digitized d images were analyzed using osirix software (version . . ) ( ). first, the virtual horizontal plane through the optic nerve entry point was set. then, the virtual vertical plane through and the optical axis were obtained. axial lengths were manually determined in the images by a researcher blinded to genotype. means of left and right eye axial lengths were first calculated for each animal. the mean values for each animal were used to determine the mean and se for each genotype group (wt, n = ; slitrk ko, n = ). statistical analysis was performed using -tailed student’s t test. histological analysis. all histological analyses were performed after mice were deeply anesthetized and killed by ether inhalation. for axial length determination in newborn mice, heads were rapidly frozen on dry ice and serially sectioned in horizontal planes at a thickness of μm. after immersing in % paraformaldehyde plus . m sodium phosphate (ph . ) for minutes at room temperature (rt), the sections were stained with hematoxylin and eosin. for all sections, the digitized images were obtained by ndp slide scanner (hamamatsu photonics). images were manually aligned using free-d software ( ), and the axial length was calculated from the x, y, and z coordinates of the retinal internal surface of the optic nerve entry point and the anterior pole, which was determined as the center of the external surface of cornea with largest diameter among a series of sections. in situ hybridization analysis was performed both on fresh-frozen post- fixed tissue sections as above and on frozen sections of the tissue that was fixed with % paraformaldehyde immersion ( hour at rt) before freez- ing. the former sections (images in figure ) were better than the latter in terms of signal strength and morphology at low magnification. the pro- cedure for the in situ hybridization was previously described ( ). digitized images were color-range-selected and contrast-brightness-optimized using canvas x software (acd systems). research article the journal of clinical investigation http://www.jci.org volume number may hereditary hearing loss homepage (http://hereditaryhearingloss.org/). clinical evaluation of affected individuals included a thorough physical examination. all subjects underwent complete ophthalmologic examina- tions, including visual acuity test, anterior segment evaluation, intraocu- lar pressure measurement, and fundus examination as well as otoscopy, tandem walking, and romberg tests. all animal experiments were approved by the animal experiment com- mittee of the riken brain science institute (approval no. h - b ), and mice were maintained at the riken bsi research resource center. accession numbers. data were deposited in genbank (human slitrk , accession nos. nm_ and np_ ; mouse slitrk , accession nos. nm_ and np_ ). the targeted allele id is mgi: . acknowledgments this work was supported by nih grant r dc (to m. tekin), birth defects foundation (united kingdom)/newlife foundation for disabled children, riken bsi funds, strategic research program of riken, research to prevent blindness, and a mext (ministry of education, culture, sports, science, and tech- nology, japan) grant-in-aid for scientific research (a) ( ). we thank all families for participating in this study, tatiana v. tkatchenko for advice on mri analysis, hideto takahashi and jae- won ko for helpful advice on synapse induction assay, and research resource center riken staff for assistance in antibody generation and animal maintenance. illumina cytosnp analysis and sequenc- ing was carried out at the medical biomics centre (sgul). received for publication july , , and accepted in revised form january , . address correspondence to: mustafa tekin, dr. john t. macdon- ald foundation, department of human genetics, nw th avenue, brb- (m- ), miami, florida , usa. phone: . . ; fax: . . ; e-mail: mtekin@med.miami. edu. or to: andrew crosby, centre for genetics, st georges uni- versity london, london sw re. phone: . . . ; fax: . . ; e-mail: acrosby@sgul.ac.uk. or to: jun aruga, jun aruga laboratory, riken brain science institute, wako, saitama - , japan. phone: . . . ; fax: . . . ; e-mail: jaruga@brain.riken.jp. for immunofluorescence staining of retina, eyes at p , p , and adult stage were dissected out, fixed by % paraformaldehyde immersion for hour, cryoprotected in % sucrose plus pbs for hours, and frozen in oct compound (sakura finetechnical). adult eyes were freshly frozen, sec- tioned, and % paraformaldehyde immersion–fixed for minutes at rt. the sections ( μm) were rinsed with pbs and immersed in a blocking buffer consisting of % normal goat serum, . % triton x- , and pbs for hour at rt. the first antibody (rabbit anti-ribeye polyclonal, : , synaptic systems; guinea pig anti-vglut polyclonal, : , , millipore; rabbit anti-vgat polyclonal, : , synaptic systems; rat anti-synapto- physin monoclonal, : , sigma-aldrich; mouse anti-neurofilament monoclonal, : , nn , sigma-aldrich; mouse anti–βiii tubulin mono- clonal, : , , promega; rabbit anti-rhodopsin polyclonal, : , cosmo bio; rat anti-thy [cd . ] monoclonal, : , bd biosciences; mouse anti–protein kinase c α monoclonal, : , sigma-aldrich; rabbit anti– tyrosine hydroxylase polyclonal, : , chemicon; mouse anti–glutamine synthase monoclonal, : , chemicon) was diluted with blocking buffer and applied to the sections overnight at °c. after pbs wash ( minutes, ×), sections were incubated with secondary antibody (anti-rabbit igg and anti-guinea pig igg coupled with either alexa fluor or alexa fluor , : each, jackson immunoresearch labs) at rt for minutes, washed with pbs, and mounted with vectashield mounting medium with dapi (vector labs). confocal images were taken with an olympus fv microscope and quantitatively analyzed using imagej software. densito- metric values of immunofluorescence signals were determined in compara- ble regions of retina from right eyes. mean values of independent images were analyzed for each mouse and used to determine mean and se for each genotype group (wt, n = or ; slitrk ko, n = or ). statistics. parametric data were analyzed using -tailed student’s t test. data are presented as mean ± sem. differences were considered statisti- cally significant for p values less than . . study approval. human studies were approved by the ethics commit- tees of ankara university (ankara, turkey), south west london (london, united kingdom), and the institute of child health (athens, greece) and by the irbs at the university of miami (miami, florida, usa) and univer- sity of arizona (tucson, arizona, usa). informed consent was obtained from all participants, or, in the case of minors, from the parents. diagno- sis of sensorineural hearing loss was established via standard audiometry in a soundproofed room according to current clinical standards. hear- ing loss was classified according to the gendeaf guidelines from the . wojciechowski r. nature and nurture: the complex genetics of myopia and refractive error. clin 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. li h, durbin r. fast and accurate long-read align- ment with burrows-wheeler transform. bioinfor- matics. ; ( ): – . . mckenna a, et al. the genome analysis toolkit: a mapreduce framework for analyzing next-gen- eration dna sequencing data. genome res. ; ( ): – . . depristo ma, et al. a framework for variation dis- covery and genotyping using next-generation dna sequencing data. nat genet. ; ( ): – . . inoue t, ota m, ogawa m, mikoshiba k, aruga j. zic and zic regulate medial forebrain develop- ment through expansion of neuronal progenitors. j neurosci. ; ( ): – . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top 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cooperation with: fondazione eni enrico mattei (feem) suggested citation: crane, kristine ( ) : the city as an arena for the expression of multiple identities in the age of globalisation and migration, nota di lavoro, no. . , fondazione eni enrico mattei (feem), milano this version is available at: http://hdl.handle.net/ / standard-nutzungsbedingungen: die dokumente auf econstor dürfen zu eigenen wissenschaftlichen zwecken und zum privatgebrauch gespeichert und kopiert werden. sie dürfen die dokumente nicht für öffentliche oder kommerzielle zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. sofern die verfasser die dokumente unter open-content-lizenzen (insbesondere cc-lizenzen) zur verfügung gestellt haben sollten, gelten abweichend von diesen nutzungsbedingungen die in der dort genannten lizenz gewährten nutzungsrechte. terms of use: documents in econstor may be saved and copied for your personal and scholarly purposes. you are not to copy documents for public or commercial purposes, to exhibit the documents publicly, to make them publicly available on the internet, or to distribute or otherwise use the documents in public. if the documents have been made available under an open content licence (especially creative commons licences), you may exercise further usage rights as specified in the indicated licence. www.econstor.eu the city as an arena for the expression of multiple identities in the age of globalisation and migration kristine crane nota di lavoro . july know – knowledge, technology, human capital kristine crane, psychoanalytic institute for social research, rome, italy this paper can be downloaded without charge at: the fondazione eni enrico mattei note di lavoro series index: http://www.feem.it/web/activ/_wp.html social science research network electronic paper collection: http://papers.ssrn.com/abstract_id=xxxxxx the opinions expressed in this paper do not necessarily reflect the position of fondazione eni enrico mattei the special issue on economic growth and innovation in multicultural environments (engime) collects a selection of papers presented at the multidisciplinary workshops organised by the engime network. the engime workshops address the complex relationships between economic growth, innovation and diversity, in the attempt to define the conditions (policy, institutional, regulatory) under which european diversities can promote innovation and economic growth. this batch of papers has been presented at the second engime workshop: communication across cultures in multicultural cities. engime is financed by the european commission, fifth rtd framework programme, key action improving socio-economic knowledge base, and it is co-ordinated by fondazione eni enrico mattei (feem). further information is available at www.feem.it/engime. workshops • mapping diversity leuven, may - , • communication across cultures in multicultural cities the hague, november - , • social dynamics and conflicts in multicultural cities milan, march - , • governance and policies in multicultural cities rome, july • trust and social capital in multicultural cities athens, november • diversity as a source of growth milan, april partners of the engime network: • fondazione eni enrico mattei, milano, italy • psychoanalytic institute for social research, roma, italy • institute of historical, sociological and linguistic studies, university of ancona, italy • centre for economic learning and social evolution, university college london, uk • faculty of economics and applied economics, katholieke universiteit leuven, belgium • idea consult, bruxelles, belgium • maison de la recherche en science humaines, laboratoire d'analyse socio- anthropologique du risque, maison de la recherche en sciences humaines, université de caen, france • centre for economic research and environmental strategy, athens, greece • institute of higher european studies, the hague university of professional education, the netherlands the city as an arena for the expression of multiple identities in the age of globalisation and migration summary this paper discusses the concept of toleration as it has been manifested in modern society through two contrasting processes—the prevalence of group identity, and the prevalence of identity defined according to citizenship and individual rights. referring primarily to the work of political theorist michael walzer, the paper describes the historical development of toleration in the u.s., insofar as it is an immigrant nation which has passed through several phases of intolerance and toleration that continue to characterise the dynamics of american society, particularly in the city. special reference is made to the city of chicago, the largest city in the midwest, where immigrants from all over settled, establishing ethnic neighbourhoods. globalisation and migration have made diversity a defining feature of contemporary society, and cities in particular. the multiplication of identities is being experienced on an individual level as well, giving rise to the recognition of the increasingly ‘hybrid’ nature of social and personal identification. the paper concludes by calling into question the implications of this post-modern model on the conceptualisation of toleration as well as its manifestations. keywords: toleration, multiculturalism, migration, cultural pluralism, ethnic self-assertion address for correspondence: kristine m. crane psychoanalytic institute for social research passeggiata di ripetta, rome italy phone : + fax : + e-mail: kristinemcrane@hotmail.com, iprsit@iprs.it the city as an arena for the expression of multiple identities in the age of globalisation and migration kristine crane psychoanalytic institute for social research one of the most evident manifestations of the age of globalisation and migration is the multiplication of identities, embodied by the representation of many groups in the political space of cities. in this context, certain political concepts such as toleration are being re-defined. the centrality of toleration in contemporary political and social life is affirmed by political theorist michael walzer, who writes “toleration makes difference possible; difference makes toleration necessary.” in other words, a tolerant political climate allows for the existence of differences. at the same time, these differences make toleration a viable political concept. walzer examines toleration in modern society as it has existed within two paradoxical processes that confer identity. the first of these processes invokes the recognition of national identity that is defined by citizenship and individual rights, and prevails over citizens’ other cultural identities. this would be the case of u.s. citizens identifying themselves primarily according to their citizenship, with all other identities, such as jewishness or italian origins, as secondary. this process also posits the state’s intervention in protecting individual rights against group practices. for example, the legislation of democratic states in the west prohibits female genital mutilation, a “cultural tradition” practised in african and asian countries. immigrants from these countries are known to continue these practices within their ethnic groups in their host countries, and if caught, face penal charges. another example of a prohibited practice is polygamy, commonly used by mormons in the u.s., where the state justifies its attempt to stop the practice insofar as it enforces one law for all citizens. similiarly, the french state would be obligated to intervene on behalf of muslim girls who do not want to wear customary headdress in public schools, a special provision requested by the girls’ parents and granted only through a compromise with the secular state. the other process is characterised by the prevalence of group identity over the national one, exemplified by those immigrants who continue to practice female genital mutilation, and therefore violate the norms of the state in which they reside and in some cases, are citizens. some states also emphasise the maintenance of group identity insofar as it is considered fundamental to national identity. for example, the constitution of canada endorses multiculturalism, and the governance of migration refers to the creation of favourable conditions wherein different ethnic groups can express their cultures. canada’s mosaic multicultural model is manifested in the neighbourhoods of toronto, which are divided according to ethnic origin. ethnic constituents also provide for equal representation of these groups in political institutions and public decision-making. modern society has been characterised by the co-existence of these two processes of strong group identification and the protection of individuals’ rights. according to walzer, “the co-existence of strong groups and free individuals, with all of its difficulties, is an enduring feature of modernity.” in other words, cultural pluralism and common citizenship go hand in hand in strong democracies, and yet they are not on equal footing, at least for long. “it seems inevitable that individual rights will win out in the long run, for equal citizenship is the basic norm of both the nation-state and the immigrant society.” the discourse on citizenship is less complex since citizenship confers clearly-defined rights and protections. cultural pluralism, on the other hand, has various manifestations depending on its context, but toleration ought to be one of its basic conditions. toleration is itself characterised by a range of behaviour that includes resignation, indifference, acceptance, curiosity and enthusiasm. according to walzer, a successful regime of toleration does not depend on a particular standing on this continuum: co-existing with ‘otherness’ without necessarily understanding the ‘others’ themselves nor their practices, is considered to be an expression of toleration. the centrality of toleration has become increasingly imminent since globalisation and migration have made diversity a defining characteristic of contemporary society. highly-skilled immigrants are concentrated in the multi- walzer, michael. on toleration. yale university press: new haven, , p. xii. ibid, . koenig, matthias. “democratic governance in multicultural societies—social conditions for the implementation of international human rights through multicultural policies,” institute for sociology, university of marburg, germany. walzer, michael. on toleration. yale university press: new haven, , p. . ibid, - . nationals of global financial cities such as new york, los angeles and london. the silicon valley near los angeles has become synonymous with this type of immigration: thousands of mainly indian immigrants have special visas to work in the technology sector there, a pattern that has also developed in other industrialised countries such as the uk and germany. many of these immigrants end up remaining in their host countries, usually because of greater career opportunities. this brain drain phenomenon has been countered, however, by these specialised immigrants’ initiatives of developing the industries, educational institutions and general economic conditions of their home countries by utilising the trans-national knowledge networks that they have established abroad. this type of migration is still an exception, however. most migrants are doing the unskilled labour that the host societies’ decreasing population of native-borns, who are increasingly well-educated, will no longer do. in any case, both types of migrants, along with asylum seekers and refugees, constitute a broad spectrum of migrants that has given rise to an emergent ‘post-modern’ model of pluralism that transcends the limits of the opposing process of group identity and that defined by citizenship and individual rights. walzer writes: “in immigrant societies...people have begun to experience what we might think of as life without clear boundaries and without secure or singular identities.” the multiplication of identities thus implies a weakening of them, and an inevitable acceptance of the increasingly ‘hybrid’ nature of social and personal identification. some immigrants have begun to articulate their own multiple identities. albanian poet gëzim hajdari, exiled in italy and considered to be one of the best ‘migrant writers’ there, writes from the perspective of someone intimately familiar with the destructiveness of nationalism in the former yugoslavia. his poetry expresses a transcendence of national identity, an existence between borders and at the same time, as an enemy of those borders. this allows him to deflect persistent labels such as extracomunatario and even immigrant, as he reckons that fundamentally, all people are immigrants and foreigners. “man doesn’t understand this principle. i try to live by this. i am at home all over the world inasmuch as nowhere in the world.” hajdari also emphasises the importance of toleration: indeed, it was this concept that he chose to bring to an inter- cultural seminar called porto franco in tuscany dedicated to producing a dictionary. as defined by hajdari, toleration is the basis of a free society, whose importance he knew from having lived in a society that had been neither free nor tolerant. he describes the communist regime of enver hoxha, where as a young poet, he observed that any verse that did not praise the regime was not tolerated. in his first volume of poetry, the anthology of the rain (which had been censored), he describes this atmosphere on the eve of a national holiday: tomorrow we will all applaud and offer infinite smiles to the platform. tomorrow we must forget that which we have lost. yet the concept of ‘toleration,’ along with ‘globalisation’ (which i had contributed), became the most problematic at the seminar. how could that be, amongst a group of people purporting to be the enlightened interpreters and leaders of an evolving inter-cultural environment, in the heart of the most historically pluralistic regions of italy in one of the most pluralistic countries in europe? because toleration is easier to define than it is to live. as walzer writes, “toleration makes difference possible; difference makes toleration necessary.” diversity is nowadays something of a catch-word understood as having implicit value. but at the seminar, i found that any divergence from ‘acceptable diversity,’ was in short, not tolerated. we were a group from over countries, representing all the continents except australia, and each of us was expected to conform to prescribed cultural or ideological roles. the talented and striking young female poet from mali was heralded as the future see united nations report, “replacement migration: is it a solution to declining and ageing populations?” walzer, michael. on toleration. yale university press: new haven, , p. . see la letteratura italiana della migrazione: aspetti teorici e percorsi di lettura, edited by armando gnisci, giulia de martino, luciana menna, giulia perrozzi. rome: università degli studi di roma tre, . the italian term ‘extracomunitario’ was developed to define citizens from outside of the european community. after the european union was founded and all official documents replaced the term with the precise definition of “citizens of non-eu nations”, the term continued to be used in italy to describe immigrants in general, and has a somewhat derogatory connotation. see dizionario della diversità: le parole dell’immigrazione, del razzismo e della xenofobia. a cura di guido bolaffi, sandro gindro, tullio tentori. liberal libri: firenze, , p. . author’s interview with g. hajdari, october, . the seminar is porto franco. toscana. terra dei popoli e delle culture, region of tuscany, italy. see publication le culture della parola e della scrittura: tracce. parole di porto franco. edizioni polistampa: firenze, . hajdari, gëzim. antologia della pioggia, santarcangelo di romagna: faraeditore, . hope of young malian women and african women in general. as someone from the united states, anything other than an apologetic discourse about jim crow and an allegiance to seattle would have put me at odds with what was tolerated. hajdari had been cast as the exiled albanian, intimately involved with the future of his country and its people. off- putting, then, when he chose not to answer, at least directly, these heady questions: “where is albania going? what is the future of albania?” with lightness and candour in harmony with his art, he said that he felt part of no nation and all nations at the same time. the organisers of the seminar were clearly of their own political persuasions and cultural preferences, likely grounded in personal experience and ideological influence. and i am willing to bet that there is not a corner of the world without prejudice and stereotypes. for what it is worth, i certainly believe that italy—and this region—has represented an oasis of toleration for various groups of people throughout the ages. the experience at porto franco only goes to show how difficult diversity can be, and how ambiguous toleration. american law professor frank wu writes that true advocates of diversity are advocates of difference: “the supporters of diversity, for example, if they are to be true to the banner they fly, must at least acquiesce to the claim of the ku klux klan member who insists that he, too, must be presented in congress or in the boardroom. the born-again christian who asks why there are not more evangelicals like herself on the op-ed page or in front of the classroom has as strong an argument as anyone else.” (wu ) at my university in chicago, there was an engineering professor and known holocaust revisionist widely published on the subject. he maintains that the holocaust is largely a false tale propagated by jews. since this university has several jewish professors and students, there had been periodic efforts—mostly by students—to get this man fired, which as far as i know, have never been successful. another time, an african american student organisation had invited a member of the nation of islam to speak. during his speech, he made several offensive remarks about jews, of whom there were many in the audience. freedom of speech? tolerable discourse? questions of taste or toleration? in international society, toleration protects the principle of sovereignty, and diplomatic arrangements are the formal expression of this. limitations on this sovereignty and therefore on toleration, are manifested by international law and international human rights treaties. for example, a partial embargo was placed on south africa during the apartheid regime. nations’ collective condemnation and public pressure are more frequently invoked. the world did not tolerate, for example, that the nigerian woman, amina lawal, be stoned to death for having a child out of wed-lock, as the local islamic court had ruled. unforced but effective international pressure (even through petitions) was one of the means of at least getting her an appeals trial. the history of pluralism and toleration in u.s. society walzer is a jewish american and writes primarily about the situation in the u.s. regarding pluralism and toleration. he examines differences of culture, religion, and way-of-life in general since groups formed on these bases, differently from political groups, are inwardly focused and also require some kind of extended social space for the sake of assembly. the u.s. is of course an immigrant nation by definition, fundamentally distinct from the nation-state model, where there is implicit pressure to assimilate to the dominant nation and culture. in france, for example, muslim immigrant girls are discouraged from wearing traditional headdress in state schools since it conflicts with the norms of a nation-state in which cultural assimilation goes hand in hand with citizenship. by contrast, in immigrant societies, schools teach the state’s history and “civics”, which may inculcate a sense of civic religion as well as political or national identity, but not cultural identity, which is instead determined by individual choice. as walzer writes, “the state claims exclusive jurisdictional rights, regarding all its citizens as individuals rather than as members of groups. hence the objects of toleration, strictly speaking, are individual choices and performances: acts of adhesion, participation in rituals of membership and worship, enactments of cultural difference, and so on.” the historical context of cultural pluralism in the u.s. differs from that of europe, where it developed out of conquest and dynastic alliance. immigrants came to the u.s. for improved opportunities or mere survival. walzer writes, “whatever the pressures that had driven them to the new world, they had chosen to come, while others like themselves, in their own families, had chosen to remain.” the political naturalisation that took place when immigrants became u.s. citizens left a cultural void, given the wu, frank. “ a different world,” speakout.com. december , . see amnesty international web site: www.amnesty.org.uk/urgentappeal walzer, michael. on toleration. yale university press: new haven, , p. . ibid, . ibid, . ibid, . ibid, . diverse ethnicities that comprised the evolving society. the americanisation process was a response to this, the cultural naturalisation that corresponded to the melting pot model in which the various ethnicities were to assimilate into one common identity characterised by the dominant anglo-saxon culture. a play entitled “the melting pot” (by israel zangwill) was the biggest hit on broadway in i . in the climatic scene, the hero says: “america is god’s crucible, the great melting pot where all the races of europe are melting and re- forming! here you stand, good folk your groups, with your fifty languages and histories, and your fifty blood hatreds and rivalries... a fig for your feuds and vendettas! germans and frenchmen, irishmen and englishmen, jews and russians— into the crucible with you all! god is making the american.” so the u.s. aspired to transcending the ethnic divisiveness of the old continent. but the melting pot ideal and the americanisation process that it inspired had discriminatory consequences—as walzer says, brutal extremes but a benign centre, and timidity characterised immigrants’ behaviour until relatively recently. discrimination was directed at immigrants from southern and eastern europe, and there were various attempts to curb asian immigration. the national origins act in effectively gave preference to immigrants of anglo-saxon origin. but since immigration to the u.s. was in part voluntary, there was a certain acquiescence on behalf of immigrants to the americanisation process because it was seen as part of the ‘choice’ in having emigrated. people changed their family name to become more american, and they forbade their children to speak the languages of the old world. in private, they continued the cultural traditions of their ethnic groups, and this was expressed by the development of city neighbourhoods such as “little italy” or “china town”, where shops sold typical products from immigrants’ countries of origin, and restaurants were opened. but there were limits: unlike in toronto, people would not necessarily speak to each other in italian in italian shops, and the restaurants began catering to an “american” taste. the early opposition to the conformist views of the melting pot and the americanisation campaign was known as cultural pluralism. in , the philosopher horace kallen coined this term and defined it according to his ideal of u.s. society, which would function like a symphony orchestra in which each instrument was a distinctive group transplanted from the old world. kallen was a german jew and defended immigrants’ resistance to the assimilation that increased as a result of america’s involvement in world war i. in political terms, he envisioned the u.s. as being a canopy offering protection for a variety of descent-defined groups. a contemporary of kallen, randolph bourne, penned the essay “trans-national america,” ( ) in which he sustained the superiority of america’s cosmopolitanism in comparison to the more homogenous societies from which immigrants had come. bourne went one step further than kallen, however, in emphasising the dynamic mixing of immigrant cultures, what might be close to contemporary inter-culturalism, with its emphasis on cultural exchange and even contamination. a minority of american intellectuals in the s used the arguments of kallen and bourne in voicing their opposition to the intolerance of the americanisation process, but it was only in the s that their ideas would have a significant following. until then, religious toleration functioned better than ethnic toleration. although jews and catholics had suffered discrimination, and when john fitzgerald kennedy (jfk) became the u.s.’s first catholic president, it was a point of pride for both the irish and catholic communities, for the most part, the fact that religions came to resemble each other, thereby conforming to the melting pot myth, made them more tolerable. the amish is one exception. the group has roots in the anabaptist movement in europe that took place at the time of the reformation. in the u.s., the amish initially settled in pennsylvania and were part of william penn’s “holy experiment” of religious toleration. communities spread across the midwest, where there are sizeable communities today. the amish believe in separation from society: they hold their religious services at home, do not use electricity and travel by horse and buggy. a supreme court ruling exempted amish children from compulsory school attendance beyond the eighth grade, an innocuous compromise for both parties. walzer writes, “the arrangement is justified in part by the marginalisation of the amish, and in part by their embrace of marginalisation: their deep commitment not to live anywhere except on the margins of american society and not to seek any influence beyond them.” most religious groups have expressed their religious identity as secondary to the national one, whose importance is transmitted by civil religion. walzer writes, “so civil religion facilitates the toleration of partial differences—or it encourages us to think of difference as only partial. we are americans but also something else, and safe as something else insofar as we are americans.” wattenberg, ben. “melt. melting. melted,” united syndicated column, march , . walzer, michael. on toleration. yale university press: new haven, , p. . hollinger, david a. postethnic america: beyond multiculturalism. basic books: new york, , p. . ibid, . ibid, . walzer, michael. on toleration. yale university press: new haven, , p. . ibid, . ethnic self-assertion in the s, cultural pluralism gained a following in u.s. society for a number of reasons: the civil rights movement, resistance to the vietnam war, an end to immigration quotas and non-european immigrant influxes, namely from latin america and asia. there was an awakening in the conscience of many americans, a sense of returning to one’s roots, as the writer alex haley symbolised with his book roots, based on oral histories in search of his own family roots in west africa. other ethnic groups were also searching for the cultural origins which in part had been lost during the americanisation process. this socio-political context allowed for the expression of ethnic self-assertion by groups whose private resistance to the americanisation process had strengthened their solidarity and identity. african americans exemplify this, in having created a vibrant culture and literature based on their very resistance to the homogenising and discriminatory aspects of american culture. they also created the civil rights movement, which became a point of reference for national liberation movements throughout the world, and are themselves compared to the process of ethnic self-assertion. walzer writes: “ethnic self-assertion has been the functional equivalent of national liberation in other parts of the world.” the fundamental differences between the two lead us to some interesting observations about the nature of ethnic self-assertion. borders and their guards are among the first results of national liberation movements, but a similar outcome of the process of ethnic assertiveness would effectively give the state the power of choosing ethnic identities and establishing rigid distinctions among them. to ensure their political equality, the state would rely on a quota system that guarantees the representation of certain ethnicities in educational institutions and the professions. this quota system is commonly known as affirmative action, an issue that has come to the fore in the last decade. recognised as an attempt to make up for past discrimination, it has applied mainly to african-americans. it is controversial insofar as it is of questionable fairness and effectiveness. walzer writes that it is a mechanism that serves to enhance individuals’ wealth and progress and not necessarily that of the community; the privileged beneficiaries of the mechanism may indeed become role models for the group, but they rarely return to the group with the intention of improving its conditions. furthermore, there is an inevitable tension between quota systems and ethnic pluralism, since societies that are ethnically plural by definition ought to be culturally heterogeneous and economically and politically egalitarian. but quotas emphasise group uniformity instead of individual equality, meting out justice as a function of group patterns instead of individuals’ life chances. furthermore, privileging the members of subordinate groups may have long-term benefits, but it usually reinforces intolerance in the short term, breeding resentment between groups. the state should provide an adequate framework wherein groups may flourish. in this sense, the state encourages toleration, but it cannot guarantee that groups actually survive, which ultimately depends on the vitality of their centres and their ability to compete for political advantage, which in part derives from their ability to turn their group identity into a cause such as prohibition or suffrage. walzer writes that one of the functions of ethnic assertiveness is to build and sustain the reborn community by creating institutions, gaining control of resources, and providing educational and welfare resources. if groups are not sustained, pluralism may be a temporary phenomenon, particularly given the fact that the characteristic mobility in u.s. society—the lack of the territorially-defined ethnic identities inherent to national liberation movements—potentially weakens groups’ necessary vitality. the greatest difficulties arise for disadvantaged groups, whose conditions are likely to produce new forms of bigotry or else, political correctness. the fact that discrimination against ethnicities and lack of resources have gone hand in hand is another obstacle to pluralism and can feed into an atmosphere of intolerance. historian david hollinger warns against the flourishing of “ethno-racial particularisms” in light of the absence of more ambitious self-help programs for the poor. he cites glenn loury who argues that the merely “tenuous” commitment of the u.s. to provide for its poor creates an “ideological trap” for african american leaders and intellectuals who think that emphasising black victimisation provides the only secure basis for making claims for their disadvantaged peers. furthermore, historian of america’s poor walzer, michael. on toleration. yale university press: new haven, , p. . walzer, michael. the politics of ethnicity: dimensions of ethnicity, harvard university press: cambridge, , p. . ibid, . ibid, . ibid, . walzer, michael. on toleration. yale university press: new haven, , p. . walzer, michael. the politics of ethnicity: dimensions of ethnicity. harvard university press: cambridge, , p. . hollinger, david a. postethnic america: beyond multiculturalism. basic books: new york, . p. . jacqueline jones warns that for african americans to “identify one’s interests on the basis of skin colour is to continue to shoulder the burden of slavery.” this vicious cycle has direct implications for toleration. as walzer writes, “no regime of toleration will work for long in an immigrant, pluralist, modern, and post-modern society without some combination of these two: a defence of group differences and an attack upon class differences.” these dynamics are easily observable in the city of chicago, where the original immigrant neighbourhoods— clustering the chinese, italian, german and ukrainian communities—are still nestled amongst the skyscrapers and old industries. the city is now rimmed by flat stretches of american suburbia, lit up by the signs of commercial livelihood of the more recent immigrant communities: indian grocers and vietnamese beauty parlours along-side the kosher delis and synagogues from when jewish refugees from the second world war poured into the city. these are all middle-class areas, but delve deeper into the city, and the real poverty associated with one group in particular—the african-americans—becomes quickly apparent, as does the truism that poverty begets poverty. one july th evening, after fireworks along the lake side downtown, a friend and i were headed back to campus on the metro. caught up in our discussion, it was only several stops later when the train began emptying out, that my friend pointed out something that i had failed to notice: i was the only white person on the train. he is one of the ten children of haitian immigrants who have made their home in miami, florida. we had taken the train in the wrong direction and found ourselves in the heart of the south side of chicago, a reputably rough part of town whose bullet-poked and cocaine filled housing projects—the worst in the u.s.—have only recently been knocked down and their inhabitants re-located in subsidised housing throughout the city. nothing happened that july th evening—we simply hopped off the train and were lucky enough to get the last night train to campus. nor should something have necessarily happened, but we (or i, if only on the basis of appearance) were clearly outsiders, and territorial defence is a natural expression of ethnic assertion. once a group of university students had ventured into the same neighbourhood for a gospel concert and were being so attacked with glass bottles that a city bus driver stopped mid-road to pick up the students. another time i went to a campus meeting on reparations for slavery— financial compensation for the descendants of slaves. although i was interested in the issue, i was even more curious about the student group that had organised it. the group called itself fmo—for members only—and was made up of african american students, the same group, incidentally, that had invited the spokesman from the nation of islam to campus. as the american poet robert frost wrote, “good fences make good neighbours.” multicultural issues the re-birth of ethnic identity in the s paved the way for multiculturalism in the s, a movement that has been most diffuse in the schools and universities, as well as in local government and popular culture. i was in high school when the novelty of a multi-cultural club was created by a group of about twenty of the school’s better students and a handful of token minorities. the small numbers reflect the school’s minority population, as well as that of the state of iowa, which, along with the state of vermont, has the smallest non-white population in the u.s. iowa city is a university town priding itself on the international crowd that it draws, but foreign graduate students and visiting professors are not usually what comes to mind when one thinks of diversity in the u.s. for the most part, the real life situations of multiculturalism and the struggles it implicates were far removed from where it was safe to have discussions on the issue, often of a politically correct nature. the call for multicultural education reflects the recognition that american schools, however culturally neutral (but espousing of a ‘civic religion’) had catered to protestant and english history and excluded other voices. multiculturalism called for the recognition of cultures that had been considered marginal, and the teaching of these on equal footing with the canon of european studies. critics of multiculturalism have claimed that it reinforces pre-fixed identities instead of allowing for individual expression. this goes against the liberal tradition at school, which has aimed to inculcate in students the idea that while they have already been identified by their parents’ choices, they are entitled to make their own choices and are obligated to tolerate those of their peers. according to walzer, american liberalism is defined by this freedom and toleration, which, when manifested in an open and democratic political life, has created the conditions for the development of competition between ethnic groups for recognition and resources in a multicultural society. critics of multiculturalism also privilege identity defined by citizenship and its rights and protections over group identity. bharati mukherjee, an american writer of indian origin, writes that multiculturalism may hinder the expression of ibid, . walzer, michael. on toleration. yale university press: new haven, , p. . ibid, . walzer, michael. the politics of ethnicity: dimensions of ethnicity. harvard university press: cambridge, , p. . individual differences: “...the multiculturalist emphasis on race and ethnicity-based group identity leads to a lack of respect for individual differences within each group, and to vilification of those individuals who place the good of the nation above the interests of their particular racial or ethnic communities.” mukherjee is also critical of the multicultural model in canada, where she lived for several years. she writes, “for all its rhetoric about a cultural “mosaic,” canada refuses to renovate its national self-image to include its changing complexion. it is a new world country with old world concepts of a fixed, exclusivist national identity....the multicultural mosaic implies a contiguity of fixed, self-sufficient, utterly distinct cultures.” (ibid) mukherjee came to the u.s. as a creative writing student in her early twenties, intending to stay for two years and then go back to india to marry according to her religion, patrimony, caste and mother tongue. anything else was considered cultural dilution. instead, she immediately fell in love with and married a fellow student from canada, and they eventually settled in the u.s. she chose to have only citizenship in the u.s. because being two things did not make sense to her. for this reason, she has also rejected the hyphenated identities by which many americans increasingly define themselves. she writes: “i am an american, not an asian-american. my rejection of hyphenation has been called race treachery, but it is really a demand that america deliver the promises of its dreams to all citizens equally....rejecting hyphenation is my refusal to categorise the cultural landscape into a center and its peripheries; it is to demand that the american nation deliver the promises of its dream and its constitution to all its citizens equally.” similarly, washington-based columnist ben wattenberg says that it is the u.s. constitution and political power that have kept voting polls open to african americans in formerly segregated states, not racial data nor the affirmation of hyphenated identities. according to the census from , the million americans may identify themselves by more than racial and ethnic categories. wattenberg writes: “instead of slicing ourselves thin and thinner, we should say what most americans believe, that we are all americans, as diverse as we want to be, revelling in our unmeasured and immeasurable diversity, which nowadays should be none of the government’s diversity.” according to walzer, however, the use of hyphenated identities is merely the recognition that “american” is a political or national identity and cultural identity is expressed in the private sphere. this has always essentially been immigrants’ underwritten agreement with the u.s.—citizenship as a national, public identity that is separate from the private one, which, however, has only recently started to be assertively expressed in the public sphere as well. towards post-national identities the dualism of group identity and common citizenship that walzer says has characterised modernity is being challenged by the post-modern model of pluralism. the globalisation process may indeed breed cultural homogenisation as its critics contend. but the consequences of the process, namely migration, have created a world without clear boundaries and singular identities, multiplying difference. these dynamics have played out in cities, with their old and new immigrant neighbourhoods, multinationals (such as ikea, located in the peripheries of cities as far apart as chicago and rome), economic migrants in the regular labour market and illegal immigrants hiding out at the margins of society. this context has invoked a ‘post-national’ perspective in which citizenship and nationhood have been re-defined according to the conceptualisation of basic rights. yasemin soysal writes, “...what were previously defined as national rights become entitlements legitimised on the basis of personhood. the normative framework for, and legitimacy of this model derive from trans-national discourse and structure celebrating human rights as a world-level organising principle. post-national citizenship confers upon every person the right and duty of participation in the authority structure and public life of a polity, regardless of their historical or cultural ties to that community.” similarly, the ‘post-ethnic’ perspective challenges the primacy of ancestry in determining identity, the “prescribed affiliations on the basis of descent” and recognises the inherent multiplicity of individuals. this perspective posits resisting the rigidity of ascribed distinctions between people while recognising the psychological and political functions of groups. these perspectives have been expressed on a cultural level as well. the poet gëzim hajdari professes to live simultaneously between borders and as an enemy of those borders, feeling at home “all over the world inasmuch as nowhere mukherjee, bharati. “american dreamer,” in mother jones magazine (web site edition), . ibid. ibid. wattenberg, ben. “pigeonhole proliferation,” newspaper enterprise association, . walzer, michael. on toleration. yale university press: new haven, , p. . soysal, yasemin nuhoglu. limits of citizenship. migrants and postnational membership in europe. the university of chicago press: chicago, , p. . hollinger, david a. postethnic america. beyond multiculturalism. basic books: new york, , p. . in the world.” his sense of multiple identities is manifested by his curious decision to write poetry in the italian language and translate it into the albanian language. hajdari’s unique ability to negotiate cultures through his art has been widely recognised. migration literature specialist armando gnisci comments, “it is the very experience of being in exile, as testified by gëzim, that languages and nations can meet and exchange. from this position—not romantic or sentimental— the foreign writer speaks, teaching us all to be foreigners.” sceptics of the post-modern perspective and its implications for toleration point to its potential superficiality. according to walzer, the modernist dualism providing for individual and group identities would not be replaced by the post-modernist model, but the latter would be super-imposed over the former, resulting in dispersive and divisive identities. he writes: “the associations that these self-made and self-making individuals form are likely to be little more than temporary alliances that can be easily broken off when something more promising presents itself. won’t tolerance and intolerance in such a setting be replaced by mere personal liking and disliking? won’t the old public arguments and political conflicts about who to tolerate and how far to tolerate them be replaced by private melodramas? isn’t the post-modern project, considered without its necessary historical background, likely to produce increasingly shallow individuals and a radically diminished cultural life?” “modernity requires, i have argued, an enduring tension between individual and group, citizen and member. post- modernity requires a similarly enduring tension with modernity itself: between citizens and members on the one hand and the divided self, the cultural stranger, on the other. radical freedom is thin stuff unless it exists within a world that offers it significant resistance.” in other words, “toleration makes difference possible and difference makes toleration necessary”— but only if there is real difference does toleration have meaning. it follows that free individuals in post-modern society must realise that the objective of toleration was never “to abolish ‘us’ from ‘them’ but to ensure their continuing peaceful co- existence and interaction.” in conclusion, living and acknowledging our differences is also perhaps the key to sharing our common humanity at its most intimate. i was reminded of this with the film, september , a collection of short films by directors from several countries that evoked this tragedy as seen around the world. politics aside, it showed the september elevenths before “ - ”—the coup d’état in chile in , the massacre of men in sbrenica in (july), along with those september elevenths that persist in silent anonymity, such as the african boy’s daily search for the money to buy the medicine that will save his dying mother. sense of loss and pain are universal, but not abstract, notions. our ability to empathise with others begins with the deeply-rooted and often disturbing familiarity of what is right in front of us. author’s interview with g. hajdari, october, . author’s interview with a. gnisci, october, . walzer, michael. on toleration. yale university press: new haven, , p. . ibid, , . ibid, . ibid. note di lavoro della fondazione eni enrico mattei fondazione eni enrico mattei working paper series our working papers are available on the internet at the following addresses: http://www.feem.it/feem/pub/publications/wpapers/default.html http://papers.ssrn.com sust . k. tano, m.d. faminow, m. kamuanga and 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know . carole maignan, dino pinelli and gianmarco i.p. ottaviano: ict, clusters and regional cohesion: a summary of theoretical and empirical research know . giorgio bellettini and gianmarco i.p. ottaviano: special interests and technological change eta . ronnie schÖb: the double dividend hypothesis of environmental taxes: a survey clim . michael finus, ekko van ierland and robert dellink: stability of climate coalitions in a cartel formation game gg . michael finus and bianca rundshagen: how the rules of coalition formation affect stability of international environmental agreements siev . alberto petrucci: taxing land rent in an open economy clim . joseph e. aldy, scott barrett and robert n. stavins: thirteen plus one: a comparison of global climate policy architectures siev . edi defrancesco: the beginning of organic fish farming in italy siev . klaus conrad: price competition and product differentiation when consumers care for the environment siev . paulo a.l.d. nunes, luca rossetto, arianne de blaeij: monetary value assessment of clam fishing management practices in the venice lagoon: results from a stated choice exercise clim . zhongxiang zhang: open trade with the u.s. without compromising canada’s ability to comply with its kyoto target know . david frantz (lix): lorenzo market between diversity and mutation know . ercole sori (lix): mapping diversity in social history know . ljiljana deru simic (lxii): what is specific about art/cultural projects? know . natalya v. taranova (lxii):the role of the city in fostering intergroup communication in a multicultural environment: saint-petersburg’s case know . kristine crane (lxii): the city as an arena for the expression of multiple identities in the age of globalisation and migration (l) this paper was presented at the workshop “growth, environmental policies and sustainability” organised by the fondazione eni enrico mattei, venice, june , (li) this paper was presented at the fourth toulouse conference on environment and resource economics on “property rights, institutions and management of environmental and natural resources”, organised by fondazione eni enrico mattei, idei and inra and sponsored by mate, toulouse, may - , (lii) this paper was presented at the international conference on “economic valuation of environmental goods”, organised by fondazione eni enrico mattei in cooperation with corila, venice, may , (liii) this paper was circulated at the international conference on “climate policy – do we need a new approach?”, jointly organised by fondazione eni enrico mattei, stanford university and venice international university, isola di san servolo, venice, september - , (liv) this paper was presented at the seventh meeting of the coalition theory network organised by the fondazione eni enrico mattei and the core, université catholique de louvain, venice, italy, january - , (lv) this paper was presented at the first workshop of the concerted action on tradable emission permits (catep) organised by the fondazione eni enrico mattei, venice, italy, december - , (lvi) this paper was presented at the esf euresco conference on environmental policy in a global economy “the international dimension of environmental policy”, organised with the collaboration of the fondazione eni enrico mattei , acquafredda di maratea, october - , (lvii) this paper was presented at the first workshop of “cfewe – carbon flows between eastern and western europe”, organised by the fondazione eni enrico mattei and zentrum fur europaische integrationsforschung (zei), milan, july - , (lviii) this paper was presented at the workshop on “game practice and the environment”, jointly organised by università del piemonte orientale and fondazione eni enrico mattei, alessandria, april - , (lix) this paper was presented at the engime workshop on “mapping diversity”, leuven, may - , (lx) this paper was presented at the euroconference on “auctions and market design: theory, evidence and applications”, organised by the fondazione eni enrico mattei, milan, september - , (lxi) this paper was presented at the eighth meeting of the coalition theory network organised by the greqam, aix-en-provence, france, january - , (lxii) this paper was presented at the engime workshop on “communication across cultures in multicultural cities”, the hague, november - , series clim climate change modelling and policy (editor: marzio galeotti ) vol voluntary and international agreements (editor: carlo carraro) sust sustainability indicators and environmental valuation (editor: carlo carraro) nrm natural resources management (editor: carlo giupponi) know knowledge, technology, human capital (editor: dino pinelli) mgmt corporate sustainable management (editor: andrea marsanich) priv privatisation, regulation, antitrust (editor: bernardo bortolotti) eta economic theory and applications (editor: carlo carraro) series clim climate change modelling and policy (editor: marzio galeotti ) gg global governance (editor: carlo carraro) siev sustainability indicators and environmental valuation (editor: anna alberini) nrm natural resources management (editor: carlo giupponi) know knowledge, technology, human capital (editor: gianmarco ottaviano) iem international energy markets (editor: anil markandya) csrm corporate social responsibility and management (editor: sabina ratti) priv privatisation, regulation, antitrust (editor: bernardo bortolotti) eta economic theory and applications (editor: carlo carraro) ctn coalition theory network a common variant in the setd gene predicts serum lycopene concentrations nutrients article a common variant in the setd gene predicts serum lycopene concentrations christopher r. d’adamo ,*, antonietta d’urso , kathleen a. ryan , laura m. yerges-armstrong , richard d. semba , nanette i. steinle , braxton d. mitchell , alan r. shuldiner and patrick f. mcardle department of family & community medicine, university of maryland school of medicine, baltimore, md , usa the commonwealth medical college, scranton, pa , usa; adurso@tcmc.edu department of medicine, university of maryland school of medicine, baltimore, md , usa; kryan@medicine.umaryland.edu (k.a.r.); lyerges@medicine.umaryland.edu (l.m.y.-a.); nsteinle@medicine.umaryland.edu (n.i.s.); bmitchel@medicine.umaryland.edu (b.d.m.); ashuldin@medicine.umaryland.edu (a.r.s.); pmcardle@medicine.umaryland.edu (p.f.m.) wilmer eye institute, johns hopkins university school of medicine, baltimore, md , usa; rdsemba@jhmi.edu * correspondence: cdadamo@som.umaryland.edu; tel.: + - - ; fax: + - - received: december ; accepted: january ; published: february abstract: dietary intake and higher serum concentrations of lycopene have been associated with lower incidence of prostate cancer and other chronic diseases. identifying determinants of serum lycopene concentrations may thus have important public health implications. prior studies have suggested that serum lycopene concentrations are under partial genetic control. the goal of this research was to identify genetic predictors of serum lycopene concentrations using the genome-wide association study (gwas) approach among a sample of old order amish adults that consumed a controlled diet. linear regression models were utilized to evaluate associations between genetic variants and serum concentrations of lycopene. variant rs on chromosome , located in the intron region of the setd gene, was significantly associated with serum lycopene concentrations (p = . ˆ ´ ). our findings also provided nominal support for the association previously noted between scarb and serum lycopene concentrations, although with a different snp (rs ) in the region. this study identified a novel locus associated with serum lycopene concentrations and our results raise a number of intriguing possibilities regarding the nature of the relationship between setd and lycopene, both of which have been independently associated with prostate cancer. further investigation into this relationship might help provide greater mechanistic understanding of these associations. keywords: lycopene; carotenoids; genome-wide association study (gwas); setd ; scarb ; old order amish; prostate cancer . introduction the carotenoids are a group of over pigments that are synthesized by plants and microorganisms. lycopene is a red-pigmented carotenoid present in tomatoes, watermelon, papaya, and other fruits and vegetables. there is no endogenous production of lycopene or other carotenoids in animals and they are only obtained from the diet. higher intake and circulating concentrations of carotenoids have been associated with lower risk of cancer [ ], cardiovascular disease [ , ], metabolic syndrome [ ], and diseases of the eye [ ]. the protective effects of dietary carotenoids appear to be due in part to their antioxidant activity. lycopene has among the most potent antioxidant effects of the nutrients , , ; doi: . /nu www.mdpi.com/journal/nutrients http://www.mdpi.com/journal/nutrients http://www.mdpi.com http://www.mdpi.com/journal/nutrients nutrients , , of dietary carotenoids and is distinct from some other carotenoids because it does not form vitamin a [ ]. interventions utilizing lycopene-rich diets and lycopene supplementation have revealed reductions in inflammation, oxidative stress, markers of atherosclerosis, and increases in high density lipoprotein (hdl) [ , ]. meta-analysis of intervention trials has also revealed that lycopene supplementation reduces serum cholesterol and blood pressure [ ]. prostate cancer is the disease that has been studied most extensively in relation to lycopene. while the evidence is not conclusive, a number of large epidemiologic studies indicate that higher dietary intake and serum concentrations of lycopene are associated with lower risk of prostate cancer [ – ], particularly lethal prostate cancer [ ]. lycopene supplementation has also been shown to inhibit the progression of benign prostate hyperplasia [ ]. it has also been proposed that serum concentrations of lycopene and other carotenoids may be useful in the early detection of prostate cancer [ , ]. there are three chief proposed target pathways for lycopene’s involvement in prostate cancer: antioxidant and anti-inflammatory function, hormonal modulation, and epigenetic modification [ – ]. lycopene appears as if it may play a role in prostate cancer, but there have also been a number of large studies that have found no relationship between serum concentrations of lycopene and risk of prostate cancer [ , ]. thus, the relationship between both dietary intake and circulating concentrations of lycopene and prostate cancer is not fully understood and there has been a call for further study in this area [ ]. further confounding the relationship between lycopene and human disease is the relatively poor correlation between dietary intake of lycopene in isolation and its concentrations in serum [ ]. the absorption, bioavailability, and serum concentrations of lycopene vary substantially due to other accompanying dietary factors. for instance, consuming fat along with lycopene greatly increases circulating concentrations of this lipid-soluble nutrient [ ]. genetic variants may also play a role in how carotenoids are absorbed, transported, or metabolized and could help explain the discord between dietary intake and serum concentrations of lycopene. previous candidate gene studies have identified genetic variants associated with circulating lycopene concentrations. assessment of single nucleotide polymorphisms (snps) using candidate genes yielded variants in intestinal fatty acid binding protein, a cytoplasmic protein that transports long chain fatty acids [ ], as well as apo a-iv and apo b, proteins involved in lipoprotein metabolism [ ]. there are two previously reported genome-wide association studies (gwas) of lycopene. the first, a population based study in the tuscany chianti region of italy, did not identify any genome-wide significant associations with lycopene [ ]). in the other, three genetic loci were identified as associated with circulating lycopene concentrations in a multi-ethnic population of women [ ]. snps in the gene scarb , which encodes for a cholesterol membrane transporter, were genome-wide significant in the multi-ethnic meta-analysis, though the signal was driven by participants of hispanic and african heritage primarily. the other two associated loci in slt and dhrs , were identified in african-americans only. no genome-wide significant associations have been identified in populations of european descent. the main aim of this study was to identify novel genetic predictors of serum lycopene concentrations. we studied a population of old order amish adults living in lancaster county, pennsylvania in whom serum lycopene concentrations were measured at the conclusion of a controlled diet. the amish are a commonly studied population for these types of studies because, among other reasons, their relatively homogeneous genetics and lifestyles across the population increase the ability to detect genetic signals [ – ]. . experimental section . . study population the study sample was composed of caucasian participants from the heredity and phenotype intervention (hapi) heart study who completed a -day controlled diet and from whom frozen blood nutrients , , of samples were available for serum lycopene measurement. the hapi heart study is a family study and by design included individuals from the same nuclear family. moreover, many of the enrolled families were related to each other given the social structure of the old order amish community. the design of the hapi heart study, including study inclusion/exclusion criteria, demographics of the full study sample, etc., has been described previously [ ] and was registered on clinicaltrials.gov (nct ). of the old order amish adults recruited into the hapi heart study, were administered the controlled dietary intervention and subsequently provided a fasting blood sample at the conclusion of the -day diet. there were samples of insufficient quality to measure serum lycopene concentrations and one sample excluded from analysis due to extreme serum lycopene concentration ( , µg/dl) that was considered an outlier. there were nuclear families in our study sample, ranging from one participant to nine participants per family. the participant relatedness was as follows: parent-children, sibling, avuncular, first cousins, and six grandparents-grandchildren. the study was approved by the institutional review board of the university of maryland school of medicine and all participants provided written informed consent. . . controlled diet a controlled diet was prepared for participants by study staff. a registered dietitian visited several old order amish households to obtain diet histories and observe meals and foods that were in their homes. all meals in the controlled diet were designed to be representative of the typical diets of old order amish adults and were provided to the study participants by home delivery over a period of six days. study participants also abstained from both prescribed and over the counter medications and dietary supplements during this -day period. the full menus for the -day controlled diet that the participants in this study consumed are provided in the supplementary materials. the controlled diet contained an average of kilocalories per day; % from carbohydrate, % from protein, and % from fat. there was an average of mg of cholesterol per day in the diet. while designed to be representative of the typical diet of the old order amish, the controlled diet was higher in carbohydrate, total fat, and cholesterol than has generally been suggested in the dietary guidelines for americans of the united states department of agriculture. the diet contained approximately . mg of lycopene per day, coming primarily from tomatoes and tomato sauce. compliance with the controlled diet was assessed by comparing sodium, potassium, and creatinine levels from first morning urine samples obtained: ( ) prior to consuming the -day controlled diet that participants consumed in this study; ( ) on the final day of the -day controlled diet that the participants consumed in this study; and ( ) on the final day of a second isocaloric, -day controlled diet that was low in salt and consumed after the blood draw that was used to conduct the gwas in this study. the compliance data have been reported in detail previously [ ], but in brief, the excreted sodium, potassium, and creatinine levels that reflect varying salt content of the diets revealed high compliance with the controlled diet in this study. . . serum lycopene measurement frozen blood samples that had been obtained after a -h fast were assayed for serum lycopene concentrations at johns hopkins university. µl from each frozen blood sample were used for the reverse-phase high-pressure liquid chromatography (hplc) assessment of serum lycopene concentrations [ ]. there were batches run and the intra-assay and inter-assay coefficients of variation cvs for lycopene were . % and . %, respectively. . . genotyping a genome-wide association study assesses associations of phenotype with single nucleotide polymorphisms (snps) throughout the genome. study participants were genotyped using either the affymetrix k or affymetrix m snp chip v . by the genomics core laboratory at the university of maryland. genotyping calls were made using birdseed, which is part of the birdsuite nutrients , , of tools [ ]. a total of , snps were in common on both arrays and used for analysis. snps with a minor allele frequency (maf) ě %, a call rate exceeding % and conforming to the expectations of hardy-weinberg equilibrium (p > ´ ) were used for imputation with mach using the hapmap ceu reference sample [ ]. results were filtered using a maf ě % and an imputation quality score ě %, for a final analyzed snp count of , , . . . statistical methods descriptive statistics were performed to characterize the study sample and determine the mean concentrations of serum lycopene. for gwas analysis, we estimated the effect of genotype on lycopene levels, adjusting for the effects of age, sex, and body-mass index (bmi) using a general linear model. genotype was coded as the number of copies of the reference allele ( , , or ), thus corresponding to an additive genetic model. the gwas analyses were performed using the mmap software [ ], which accounts for family structure as a random effect. statistical analysis was performed using a variance component approach to account for relatedness among study participants. this approach has previously been shown to provide valid estimates of regression parameters [ ]. to account for the multiple snps tested, we considered associations at p < ˆ ´ to be statistically significant. at this genome-wide significance threshold, we estimated that our sample provided % power to detect snps accounting for %– % of trait variation. . results baseline characteristics of the study sample are provided in table . there were more men in the study than women ( men, women). participants were in their mid- s on average (mean = . years) with the men being slightly younger than the women. participants had a mean bmi of . kg/m and over half of both the men and the women could be classified as overweight (bmi ě kg/m ). mean lycopene values were . µg/dl (standard deviation = . µg/dl, standard error of mean = . µg/dl, range = ( . – . µg/dl)). the heritability was estimated to be . ˘ . . table . characteristics of old order amish study sample from lancaster county, pennsylvania after consuming a -day controlled diet. characteristic all (n = ) female (n = ) male (n = ) age (years) . ( . ) . ( . ) . ( . ) bmi (kg/m ) . ( . ) . ( . ) . ( . ) lycopene (µg/dl) . ( . ) . ( . ) . ( . ) retinol (µg/dl) . ( . ) . ( . ) . ( . ) lutein (µg/dl) . ( . ) . ( . ) . ( . ) zeaxanthin (µg/dl) . ( . ) . ( . ) . ( . ) β-cryptoxanthin (µg/dl) . ( . ) . ( . ) . ( . ) α-carotene (µg/dl) . ( . ) . ( . ) . ( . ) β-carotene (µg/dl) . ( . ) . ( . ) . ( . ) γ-tocopherol (µg/dl) . ( . ) . ( . ) . ( . ) α-tocopherol (µg/dl) . . ( . ) . ( . ) . ( . ) a manhattan plot summarizing results of the gwas is provided in figure . the top hits from the association analyses are presented in table . we detected genome-wide significant evidence for association of lycopene levels to a locus on chromosome q (lead snp = rs ; age, sex, and bmi-adjusted p = . ˆ ´ ). each copy of the a allele was associated with a . µg/dl decrease in serum lycopene, and this locus accounted for . % of the variation in lycopene levels. the minor allele at the locus (a) is common in the old order amish (maf = . ) as well as other populations (hapmap ceu = . , hapmap yri = . ). figure provides a regional association plot of the genome-wide significant association on chromosome . rs is in an intronic region of gene setd . gene setd encodes the enzyme histone h -k methyltransferase setd , one of the nutrients , , of histone methyltransferases (hmts) enzymes. the quantile-quantile plot provided in figure reveals little evidence for genomic inflation (lambda = . ), as the observed distribution of p-values for the genome-wide association tests is consistent with that expected under the null. nutrients  ,  ,     of    little evidence for genomic inflation (lambda =  . ), as the observed distribution of p‐values for the  genome‐wide association tests is consistent with that expected under the null.    figure  .  manhattan  plot  for  the  genome‐wide  association  study  (gwas)  of  serum  lycopene  concentrations  in  the old order amish study population  following a  ‐day controlled diet. the  x‐axis  represents  chromosomal  position  along  the  genome.  the  y‐axis  shows  the  p‐value  for  association  test  at  each  locus  on  the  log  scale.  the  colors  are  used  to  identify  the  different  chromosomes listed on the x‐axis.  table  . genome‐wide (p <   ×  − ) and sub genome‐wide (  ×  −  ≤ p <   ×  − ) associations with  serum lycopene concentrations.  snp  chromosome  position  gene maf coded allele beta (se)  p‐value genome‐wide significant  rs       setd   .   a  − .  ( . )  .  ×  − sub genome‐wide significant  rs       bc .   a  .  ( . )  .  ×  − rs       –  .   a  − .  ( . )  .  ×  − rs       –  .   c  − .  ( . )  .  ×  − rs       g pc   .   a  .  ( . )  .  ×  −   figure  . regional association plot of chromosome  q .  in the area of setd . variant rs   provides genome‐wide significant evidence of association with serum lycopene concentrations. the  x‐axis represents chromosomal position on chromosome   with the location of genes at the locus  figure . manhattan plot for the genome-wide association study (gwas) of serum lycopene concentrations in the old order amish study population following a -day controlled diet. the x-axis represents chromosomal position along the genome. the y-axis shows the p-value for association test at each locus on the log scale. the colors are used to identify the different chromosomes listed on the x-axis. nutrients  ,  ,     of    little evidence for genomic inflation (lambda =  . ), as the observed distribution of p‐values for the  genome‐wide association tests is consistent with that expected under the null.    figure  .  manhattan  plot  for  the  genome‐wide  association  study  (gwas)  of  serum  lycopene  concentrations  in  the old order amish study population  following a  ‐day controlled diet. the  x‐axis  represents  chromosomal  position  along  the  genome.  the  y‐axis  shows  the  p‐value  for  association  test  at  each  locus  on  the  log  scale.  the  colors  are  used  to  identify  the  different  chromosomes listed on the x‐axis.  table  . genome‐wide (p <   ×  − ) and sub genome‐wide (  ×  −  ≤ p <   ×  − ) associations with  serum lycopene concentrations.  snp  chromosome  position  gene maf coded allele beta (se)  p‐value genome‐wide significant  rs       setd   .   a  − .  ( . )  .  ×  − sub genome‐wide significant  rs       bc .   a  .  ( . )  .  ×  − rs       –  .   a  − .  ( . )  .  ×  − rs       –  .   c  − .  ( . )  .  ×  − rs       g pc   .   a  .  ( . )  .  ×  −   figure  . regional association plot of chromosome  q .  in the area of setd . variant rs   provides genome‐wide significant evidence of association with serum lycopene concentrations. the  x‐axis represents chromosomal position on chromosome   with the location of genes at the locus  figure . regional association plot of chromosome q . in the area of setd . variant rs provides genome-wide significant evidence of association with serum lycopene concentrations. the x-axis represents chromosomal position on chromosome with the location of genes at the locus annotated. the left y-axis shows the p-value for association tests at each locus (dot) on the log scale. the right y-axis provides recombination rates in centimorgans per megabase in the chromosomal region identifying recombination hotspots in the region (grey line). the diamond is the “top hit” (i.e., the strongest association). other snps in the region are represented by circles. the colors indicate linkage disequilibrium per the r map on top left. linkage disequilibrium associated with the top signal appears to span the entire region of gene setd . nutrients , , of table . genome-wide (p < ˆ ´ ) and sub genome-wide ( ˆ ´ ď p < ˆ ´ ) associations with serum lycopene concentrations. snp chromosome position gene maf coded allele beta (se) p-value genome-wide significant rs setd . a ´ . ( . ) . ˆ ´ sub genome-wide significant rs bc . a . ( . ) . ˆ ´ rs – . a ´ . ( . ) . ˆ ´ rs – . c ´ . ( . ) . ˆ ´ rs g pc . a . ( . ) . ˆ ´ nutrients  ,  ,     of    annotated. the left y‐axis shows the p‐value for association tests at each locus (dot) on the log scale.  the right y‐axis provides recombination rates in centimorgans per megabase in the chromosomal  region identifying recombination hotspots in the region (grey line). the diamond is the “top hit” (i.e.,  the strongest association). other snps in the region are represented by circles. the colors indicate  linkage disequilibrium per the r  map on top left. linkage disequilibrium associated with the top  signal appears to span the entire region of gene setd .    figure  . quantile‐quantile (qq) plot of gwas of serum lycopene concentrations. the axes plot the  observed (y‐axis) vs. theoretical (x‐axis) association p‐values on the log scale for all single nucleotide  polymorphisms (snps) with minor allele frequency (maf) greater than  %. the old order amish  are a closed  founder populations with  little admixture expected. the genomic control  lambda  is  estimated to be  . , indicating little bias due to population stratification.  we additionally identified three other loci for which associations were observed at p <   ×  −   that  are  provided  in  table  .  we  also  performed  look‐ups  for  snps  previously  reported  to  be  associated  with  serum  lycopene  levels  in a  multiethnic  gwas  [ ],  in  which associations were  reported  at  three  loci:  one  achieving  genome‐wide  association  to  three  snps  in  high  linkage  disequilibrium in scarb  (lead snp: rs ) in the meta‐analysis across three ethnic groups,  and the other two achieving genome‐wide significance  in the african‐american sample only (to  snps in slit  (lead snp: rs ) and dhrs  (lead snp: rs )). notably, the associated  snps in scarb  have a minor allele frequency (maf) of only  .  in european‐americans and the  associated snps in slit  and dhrs  were monomorphic in european‐americans. in the amish, the  maf of the three snps were even lower (maf =  . ), and there was no evidence for association  with  lycopene  levels  (p  =  . ).  although  not  a  replication,  we  did,  however,  detect  nominal  evidence for association of lycopene levels with a different snp in scarb  (rs ; maf =  . ;  p =  .  ×  − ).  . discussion  the key result from this study is the novel association observed between a common variant at  the setd  locus, rs , and serum lycopene concentrations in this genome‐wide association  figure . quantile-quantile (qq) plot of gwas of serum lycopene concentrations. the axes plot the observed (y-axis) vs. theoretical (x-axis) association p-values on the log scale for all single nucleotide polymorphisms (snps) with minor allele frequency (maf) greater than %. the old order amish are a closed founder populations with little admixture expected. the genomic control lambda is estimated to be . , indicating little bias due to population stratification. we additionally identified three other loci for which associations were observed at p < ˆ ´ that are provided in table . we also performed look-ups for snps previously reported to be associated with serum lycopene levels in a multiethnic gwas [ ], in which associations were reported at three loci: one achieving genome-wide association to three snps in high linkage disequilibrium in scarb (lead snp: rs ) in the meta-analysis across three ethnic groups, and the other two achieving genome-wide significance in the african-american sample only (to snps in slit (lead snp: rs ) and dhrs (lead snp: rs )). notably, the associated snps in scarb nutrients , , of have a minor allele frequency (maf) of only . in european-americans and the associated snps in slit and dhrs were monomorphic in european-americans. in the amish, the maf of the three snps were even lower (maf = . ), and there was no evidence for association with lycopene levels (p = . ). although not a replication, we did, however, detect nominal evidence for association of lycopene levels with a different snp in scarb (rs ; maf = . ; p = . ˆ ´ ). . discussion the key result from this study is the novel association observed between a common variant at the setd locus, rs , and serum lycopene concentrations in this genome-wide association study. this represents the first genome-wide significant genetic association of lycopene in a mixed-gender, caucasian population and the first study evaluating genetic determinants of lycopene concentrations among a sample that had consumed a controlled diet. we were unable to replicate an association previously noted between a snp in scarb with low maf in caucasians and serum lycopene concentrations, although we did observe a nominal association of lycopene levels with a different snp within this gene (rs ). interestingly, rs has previously been associated with lipoprotein-associated phospholipase a (lp-pla ) [ ]. both lp-pla and lycopene are primarily carried throughout circulation on low-density lipoprotein (ldl) [ , ]. our data do not provide replicative support for either slit or dhrs [ ], although this is not surprising as these prior associations were detected to a snp found only in african-americans and not in caucasian americans of european descent. our results raise a number of intriguing possibilities regarding the nature of the relationships previously noted between setd , lycopene, and prostate cancer. setd is proliferative and anti-apoptotic in prostate cancer cells and nuclear expression is upregulated in prostate cancer tissue [ ]. the activity of setd as a histone methyltransferase (hmt) may also play a role in prostate cancer. hmts have been shown to be upregulated in prostate cancer [ , ] and deregulation of hmts has also been associated with prostate cancer development and progression [ ]. it is also plausible that setd may be related to prostate cancer risk through its relationship to serum lycopene concentrations, as was identified in this study. the potential protective mechanisms of lycopene against prostate cancer include regulation of the antioxidant response element, exertion of effects on vegf signaling pathways, induction of cell cycle arrest, and mediation of apoptosis [ , ]. future studies containing prostate cancer endpoints would be necessary to confirm this relationship. there are several key strengths of the study. this was the first gwas aimed at identifying genetic predictors of serum lycopene concentrations that was conducted among a sample that had consumed a controlled diet. the controlled diet and consistent lycopene, fat, and cholesterol intake among the study participants enabled us to more closely isolate the genetic contributions to the variance in serum lycopene than would have been possible on a variable diet. a related strength was that adherence to the diet was also high, as verified by urinary excretion. the diet was informed by home visits of the study population performed by a registered dietitian, was designed to be culturally-appropriate based upon the foods and beverages present in the homes during these visits, and was delivered to the homes of the study participants to encourage adherence. conduct of this study in the old order amish population was also advantageous for several reasons. to our knowledge, this is the first study to estimate heritability of serum lycopene concentrations in humans, an analysis made possible by the relationship structure of the old order amish. the old order amish study sample also provided a population that was relatively homogenous with respect to genetics, environmental exposures, and lifestyle habits. this homogeneity, particularly with respect to genetics, provided increased power to detect genetic variants associated with lycopene concentrations. there were also several notable limitations to this study. the relatively small sample size (n = ) may have limited our ability to detect genome-wide significant associations between genetic variants and serum lycopene concentrations. furthermore, the relatively high inter-assay cv of . % for our serum lycopene measurements could have resulted in lower precision of our estimates. however, nutrients , , of despite the relatively small sample size and relatively high inter-assay cv, our study was able to identify a novel locus associated with serum lycopene concentrations. we attribute this success in part to the aforementioned advantages of studying an old order amish population as well as the controlled diet that the participants consumed prior to the fasted-state blood draw which enabled us to more closely isolate the genetic contributions to serum lycopene concentrations. a limitation of the controlled diet was its relatively short duration of six days. while the time to maximum concentration of lycopene after consumption is just six hours, lycopene has an elimination half-life of between five and nine days [ , ]. it is likely that the serum lycopene concentrations measured at the conclusion of the controlled diet were also influenced to some degree by variable dietary intake that occurred prior to the initiation of the controlled diet. however, the controlled diet was designed to be representative of the typical old order amish diet and to the authors’ knowledge, all previously published gwas of lycopene and carotenoid concentrations have been conducted among populations on uncontrolled diets. thus, we do not believe that this limitation of the controlled diet has a major influence on the findings of this study. finally, while the novel association noted between a variant in setd and serum lycopene concentrations, both of which have been associated with prostate cancer, may provide the rationale for further study into the specific mechanisms of this relationship, this study did not collect data on family history of prostate cancer, prostate specific antigen, or other markers of the disease and no direct inference can be made. in conclusion, this study provides the identification of a novel genetic association between rs , an intronic variant in setd , and serum lycopene concentrations. these findings provide further support that genetics may affect serum concentrations of lycopene. further studies are needed to clarify any potential relationships between setd , lycopene, and clinical endpoints such as prostate cancer. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . acknowledgments: the authors would like to thank the study participants for their partnership in research and the staff at the amish research clinic for their assistance. we would also like to acknowledge nga hong brereton, masters of science, registered dietitian, at johns hopkins university school of medicine for her work in designing and evaluating the nutrient content of the controlled diet in this study. author contributions: christopher r. d’adamo and patrick f. mcardle designed the research, patrick f. mcardle, and kathleen a. ryan conducted the research, patrick 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[crossref] [pubmed] © by the authors; licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons by attribution (cc-by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . /mjt. b e e d http://www.ncbi.nlm.nih.gov/pubmed/ http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /nar/gkq http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j. - . .tb .x http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /nature http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /erc- - http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /ijms http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /ajcn. . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /ajcn. . http://www.ncbi.nlm.nih.gov/pubmed/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . / 	introduction 	experimental section 	study population 	controlled diet 	serum lycopene measurement 	genotyping 	statistical methods 	results 	discussion � wiley-liss, inc. american journal of medical genetics part a a: – ( ) homozygosity for a novel splice site mutation in the cardiac myosin-binding protein c gene causes severe neonatal hypertrophic cardiomyopathy baozhong xin, erik puffenberger, john tumbush, j.r. bockoven, and heng wang , , * das deutsch center (ddc) clinic for special needs children, middlefield, ohio the clinic for special children, strasburg, pennsylvania the heart center, akron children’s hospital, akron, ohio department of pediatrics, rainbow babies & children’s hospital, cleveland, ohio department of molecular cardiology, cleveland clinic foundation, cleveland, ohio received may ; accepted july hypertrophic cardiomyopathy is typically inherited in an autosomal dominant pattern and has a variable age of onset and prognosis. mutations in the myosin-binding protein c (mybpc ) gene are one of the most frequent genetic causes of the disease. patients with mybpc mutations generally have a late onset and a relatively good prognosis. we report here more than old order amish children with severe neonatal hypertrophic cardiomyopathy caused by a novel homozygous splice site mutation in the mybpc gene. the affected children typically presented with signs and symptoms of congestive heart failure during the first weeks of life. echocardiography revealed hypertrophic non- obstructive cardiomyopathy. these children had a life span averaging – months. all patients died from heart failure before year of age unless they received a heart transplant. a genome-wide mapping study was performed in three patients. the disease related gene was localized to a . mb region on chromosome p . -p . . this homozygous block contained mybpc , a previously identified cardiomy- opathy related gene. we identified a novel homozygous mutation, c. þ t > g, in the splice-donor site of mybpc intron . the mutation resulted in skipping of the -bp exon , which led to a frame shift and premature stop codon in exon (p.asp glyfsx ). we have found a substantial incidence of this phenotype in old order amish communities. it is also concerning that many unidentified heterozygous individuals who are at risk for development of hypertrophic cardiomyopathy do not receive proper medical attention in the communities. � wiley-liss, inc. key words: hypertrophic cardiomyopathy; mybpc ; old order amish; mennonite how to cite this article: xin b, puffenberger e, tumbush j, bockoven jr, wang h. . homozygosity for a novel splice site mutation in the cardiac myosin-binding protein c gene causes severe neonatal hypertrophic cardiomyopathy. am j med genet part a a: – . introduction hypertrophic cardiomyopathy, clinically defined as thickening of the myocardial wall in the absence of other causes for left ventricular hypertrophy, affects one of every people [maron et al., ; zou et al., ]. the disease, generally being inherited in an autosomal dominant pattern, has a broad spectrum of clinical manifestations from a benign asymptomatic course to a malignant course with serious arrhythmias, heart failure, and sudden cardiac death. genetic causes of hyper- trophic cardiomyopathy are also fairly diverse with more than mutations identified in at least nine genes encoding sarcomeric proteins [ho and seidman, ; ashrafian and watkins, ]. one of most common genetic causes for hyper- trophic cardiomyopathy involves mutations in cardiac myosin-binding protein c (mybpc ) gene [charron et al., ; niimura et al., , ; erdmann et al., , ; konno et al., ; richard et al., ; van driest et al., ]. there are approximately this article contains supplementary material, which may be viewed at the american journal of medical genetics website at http:// www.interscience.wiley.com/jpages/ - /suppmat/index.html. *correspondence to: heng wang, m.d., ph.d., ddc clinic for special needs children, po box , madison road, middlefield, oh . e-mail: wang@ddcclinic.org doi . /ajmg.a. mutations identified so far as listed in the website (http://www.cardiogenomics.org) devel- oped by genomics of cardiovascular development [ ], adaptation, and remodeling program since the first disease-causing mutations were found in [bonne et al., ; watkins et al., ]. cardiac myosin-binding protein c is a sarcomeric protein belonging to the intracellular immunoglobulin super- family [einheber and fischman, ]. by binding to the myosin heavy chain and cytoskeleton protein titin, cardiac myosin-binding protein c contributes to the structural integrity of the sarcomere. the protein may also play a role in regulating cardiac contractility [flashman et al., ]. in general, patients with hypertrophic cardiomyopathy caused by mybpc gene mutations seem to have a more favorable clinical profile, characterized by a late onset and a relatively good prognosis [niimura et al., ]. the clinical expression of the mutations in the mybpc is often delayed until middle age or old age. homo- zygous mutation in the mybpc gene causing severe hypertrophic cardiomyopathy has not been reported to our knowledge, although two cases of severe hypertrophic cardiomyopathy caused by compound heterozygous mutations have been found recently [lekanne deprez et al., ]. here we describe a cohort of patients with severe hypertrophic cardiomyopathy presenting in the neonatal period, caused by homozygosity for a novel mutation in the mybpc gene. materials and methods subjects the study was approved by ddc clinic for special needs children (ddc clinic) institutional review board. all affected infants were old order amish, with of them from the geauga county settlement in ohio, one from the holmes county settlement in ohio, and two from a settlement in new york. the patients were clinically evaluated by at least one clinician in the list of authors and the diagnosis of hypertrophic cardiomyopathy was established based on family history, physical examination, and the characteristic clinical course of the disease. confirmation was made by both electrocardiogram and two-dimensional echocardiography reviewed by pediatric cardiologists. dna samples from three patients, their parents and siblings were acquired with informed consent. all three patients died before the study was completed. one of them expired before the study started, thus the dna sample preserved by another research laboratory was transferred to us per parent’s request. genotyping and mutation detection the dna isolation, genotyping, linkage analysis and mutation detection were performed as described previously [puffenberger et al., ; strauss et al., , ]. polymerase chain reaction (pcr) primers were designed to amplify each of the protein-coding exons and their flanking intronic sequences of mybpc . primer sequences are pro- vided in table of online supplementary material, which is published as supporting information on the ajmg web site (see the online table at http://www. interscience.wiley.com/jpages/ - /suppmat/ index.html). rna isolation and cdna amplification total rna was isolated from whole blood using qiaamp rna blood kit (qiagen, valencia, ca) according to the manufacturer’s protocol. the cdna was synthesized and amplified using primers f and r located in exons and . the primers were determined according to the mrna sequence nm_ . results clinical phenotype the children affected with hypertrophic cardio- myopathy were typically born after an uneventful pregnancy and delivery. they were usually full term with birth weight, length and occipitofrontal circum- ference all within normal ranges. there were no significant dysmorphic features noticed in any of those newborns at birth and afterwards. all patients passed the routine state newborn screening. a routine chromosomal analysis was performed on at least one newborn, which was reported as normal. approximately one third of the affected infants in this cohort presented with respiratory distress, an audible heart murmur or gallop rhythm soon after birth, which led to further evaluation before or soon after discharge from the hospital. the remaining two thirds of infants presented to the primary care physicians’ office during the first – weeks of life with poor feeding, excessive sweating during feed- ing, lethargy, difficulty with breathing, irritability and intermittent perioral cyanosis. abnormal find- ings from the initial physical examinations often included excessive sweating, poor perfusion with prolonged capillary refill time, tachypnea, sinus tachycardia, gallop rhythm and enlarged liver. chest x rays showed cardiomegaly. echocardiography revealed hypertrophic non-obstructive cardio- myopathy in the right or left ventricle or in both ventricles with ventricular dysfunction. mild to moderate ventricular dilation was observed in some patients. except for small ventricular defects discov- ered in several patients, a normal segmental anatomy without other significant structural heart defects was found in all the patients. the heart failure initially found in all our patients was progressive despite treatment with mybpc homozygosity causes severe cardiomyopathy american journal of medical genetics part a: doi . /ajmg.a beta-blockers, diuretics and inotropes. all patients died from heart failure before year of age unless they received a heart transplant. the life span of the affected children ranged from seven to days (average days with a median of days for infants with such information available to us). two patients who successfully received heart trans- plants remained fairly healthy except for some minor transplant related issues. genotyping and mapping we carried out a genome-wide mapping analysis using the affymetrix genechip mapping k snp arrays to identify the disease locus with three affected children from different families (fig. ). a large, shared block of homozygous snps was identified on chromosome p . -p . in the three affected individuals (see the online fig. s at http://www.interscience.wiley.com/jpages/ - /suppmat/index.html). the homozygous seg- ment contains contiguous snps and spans . mb. examination of the minimal shared region which was flanked by snps rs and rs in the affected individuals, revealed known or predicted genes based on both the ncbi and celera annota- tions, of which are characterized. among these genes, a known hypertrophic cardiomyopathy- related gene, mybpc , was selected for mutation analysis. mutation analysis genomic dna sequence analysis of the mybpc gene in one affected patient revealed a novel homozygous mutation in the consensus splice donor site of intron , c. þ t > g (fig. ). further sequencing analysis revealed that all three patients from the pedigrees (fig. ) were homozy- gous for the mutation, their parents were hetero- zygous, and no unaffected siblings (n ¼ ) were homozygous for the change (fig. ). rna analysis to further investigate the consequence of this mutation at the transcript level, lymphocyte rna from two heterozygous carriers was amplified by rt- pcr. amplification of mybpc cdna with primers f and r yielded two products: the expected -bp fragment and an abnormal shorter product (fig. ). direct sequencing of the pcr products after gel extraction revealed skipping of the -bp exon in the shorter fragment which led to a frame shift and premature stop codon in exon (p.asp glyfsx ). as a control, amplification of lymphocyte rna from two homozygous normal individuals only gave rise to the expected -bp product (fig. ). discussion in this study, we performed whole-genome linkage analysis and mutational analysis in three patients who suffered from severe unexplained hypertrophic cardiomyopathy from three consan- guineous families. we mapped the disease locus to a . mb region on chromosome p . -p . . we further identified a novel homozygous mutation, c. þ t > g, in a putative splice-donor site of the mybpc gene within this region that is associated with this severe condition. the c. þ t > g mutation in the mybpc gene has not been reported previously or been listed on the public database (http://www. cardiogenomics.org). due to the unavailability of rna or protein samples from cardiac tissue of the affected individuals at the current time, the con- sequence of the mutation was determined using lymphocyte rna from heterozygous carriers of the c. þ t > g mutation. it was demonstrated that the mutated allele produces an aberrant transcript with skipping of the associated exon . skipping of the -bp exon led to a frame shift. the aberrant mrna was predicted to encode a truncated protein fig. . pedigrees of the three families used in the mapping study and mutational analysis of hypertrophic cardiomyopathy. the probands are indicated with an arrow in each family. xin et al. american journal of medical genetics part a: doi . /ajmg.a (p.asp glyfsx ). as a consequence, amino acids of the conserved cooh terminus are deleted. we expect the same consequences of the splice donor site mutation in the myocardium. cardiac myosin-binding protein c is composed of domains referred to as c –c [carrier et al., ]. previous functional studies have demon- strated that the major myosin binding domain is located within the c consisting of the last amino acids [okagaki et al., ; alyonycheva et al., ]. the c. þ t > g mutation is predicted to produce a truncated protein with complete missing of c domain, which is required for the incorpo- ration of cardiac myosin-binding protein c into the a band, titin interaction and myosin binding [freiburg and gautel, ; gilbert et al., ]. it is speculated that homozygosity of the mutation reported in the present study acts as null alleles and leads to complete loss of function of cardiac myosin-binding protein c, which may explain the severity of the disease phenotype in these patients. indeed, all individuals affected with the disease present with signs and symptoms of heart failure during the neonatal period with an average life span of – months, and all patients die before year of age unless they receive a heart transplant. a recent report has described two lethal cases of neonatal hypertrophic cardiomyopathy caused by compound heterozygoity for mybpc mutations [lekanne deprez et al., ]. however, this is the first time, to our knowledge, that a homozygous mutation in the mybpc gene is reported causing a severe type of hypertrophic cardiomyopathy. it is noted that majority of children ( ) affected by this severe neonate hypertrophic cardiomyopathy in this study are from the geauga settlement of ohio and all of them were born during the last years. based on the number of amish births in this settlement during this interval, we estimate that a birth incidence of the disease is approximately in . by using the hardy-weinberg equilibrium and the incidence estimate, the heterozygous carrier frequency is calculated as approximately %. however, we are somewhat surprised with the prevalence of this severe type of hypertrophic cardiomyopathy beyond this settlement. during the study, we have been contacted by affected amish families or health professionals from delaware, illinois, indiana, kentucky, mississippi, new york, north carolina and pennsylvania with many similar cases as we reported here. the genotype of these affected children remains to be determined, but may be reasonably speculated as the same as reported here. in fact, we have been contacted by two mennonite families from different states, and each of them has one deceased child with hypertrophic cardiomyopathy. not unexpectedly, dna analysis in one of the mennonite couples with an affected infant has revealed that both parents carry the same single c. þ t > g mutation in the mybpc gene. thus, we speculate that this infant suffered from the same type of hypertrophic cardiomyopathy as well, thus the disease also affects the mennonite community. it has been illustrated that old order amish and old order mennonite populations have unique genetic heritages despite a common religious and geographic history [puffenberger, ]. the hypertrophic cardiomyopathy presented in this study might be one of a few diseases where fig. . identification of c. þ t > g mutation in the mybpc gene. partial sequence of the boundary region of exon and intron is shown and the three genotypes with respect to c. þ t > g mutation are presented (arrows). [color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] fig. . detection of the aberrant transcript as a consequence of c. þ t > g mutation. the mybpc cdna was amplified by rt-pcr from lymphocyte rna from homozygous normal individuals (n) and heterozygous carriers (c) using primers f and r located in exons and . samples from heterozygous carriers contain the normal -bp product and also a -bp product resulting from skipping of the -bp exon . m, dna molecular marker. mybpc homozygosity causes severe cardiomyopathy american journal of medical genetics part a: doi . /ajmg.a identical mutations segregate in both populations, as is the case for crigler-najjar syndrome and propionic acidemia in the amish and mennonites of lancaster county, pa. higher prevalence and larger geographic distribution of severe hyper- trophic cardiomyopathy might imply more distant common ancestors. in the past, many genetic disorders identified in the old order amish and mennonite communities have been autosomal recessive diseases related to the founder effect [mckusick, ; morton et al., ; puffenberger, ], and the parents of probands generally do not have any signs or symptoms of the disease. here, we are apparently dealing with disorder with a disease inherited in an autosomal dominant pattern with very severe phenotype in the homozygotes. although incom- plete penetrance often occurs in this autosomal dominant disorder, it remains concerning that many unidentified heterozygotes in the community are carrying a single c. þ t > g mutation and therefore might have, or have the potential to develop hypertrophic cardiomyopathy. indeed, we have noticed many reports of cardiac symptoms, such as chest pain, fatigue and palpitation from probands’ parents or relatives during the study. these individuals, presumably being heterozygotes of c. þ t > g mutation, have been one of our major concerns throughout the study. notably, a previously reported mutation c. þ g > c, very similar to the mutation c. þ t > g found in this study, has been documented as a cause of hyper- trophic cardiomyopathy in those heterozygous carriers [watkins et al., ]. the consequence of being a heterozygous carrier of c. þ t > g mutation during a lifetime is still to be determined, but we expect that certain individuals, particularly those middle age and older, might be affected to some degree. at least three direct family members of the affected infants have died from sudden cardiac death at middle age to our knowledge. it is disconcerting that many individuals with alarming symptoms do not receive proper medical attention in this amish community. while we are working on further understanding the pathology of both homo- zygosity and heterzygosity of this particular muta- tion, we believe that it is equally important to work with these individuals who are heterozygous carrier of c. þ t > g mutation to better define the clinical course of the disease. there is an urgent need to develop a practical strategy to deliver medical services to these individuals who often do not have health insurance. acknowledgments we thank the amish and mennonite families in this report for their support. this study would not have been possible without the invaluable assistance of family members. we are indebted to many pediatric cardiologists who provided outstanding and com- passionate care to the children affected by the disease, particularly dr. chandrakant r. patel of akron children’s hospital, dr. kenneth zahka of rainbow babies & children’s hospital, and dr. gerard j. boyle of children’s hospital of cleveland clinic among others. we are grateful for the help of dr. andrew crosby’s laboratory at st. george’s hospital medical school, london in the isolation of three dna samples. we appreciate mr. joe weaver for his genealogy consultation and dr. lawrence greksa of case western reserve university for his demographic consultation. the study was sup- ported in part by 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echocardiographic analysis of adults. am j med : – . mybpc homozygosity causes severe cardiomyopathy american journal of medical genetics part a: doi . /ajmg.a clinical and genetic validity of quantitative bipolarity bruce et al. translational psychiatry ( ) : https://doi.org/ . /s - - -z translational psychiatry a r t i c l e op e n a c c e s s clinical and genetic validity of quantitative bipolarity heather a. bruce , peter kochunov , braxton mitchell , kevin a. strauss , seth a. ament , laura m. rowland , xiaoming du , feven fisseha , thangavelu kavita , joshua chiappelli , krista wisner , hemalatha sampath , shuo chen , mark d. kvarta , chamindi seneviratne , teodor t. postolache , alfredo bellon , francis j. mcmahon , alan shuldiner and l. elliot hong abstract research has yet to provide a comprehensive understanding of the genetic basis of bipolar disorder (bp). in genetic studies, defining the phenotype by diagnosis may miss risk-allele carriers without bp. the authors aimed to test whether quantitatively detected subclinical symptoms of bipolarity identifies a heritable trait that infers risk for bp. the quantitative bipolarity scale (qbs) was administered to old order amish or mennonite individuals from multigenerational pedigrees; individuals had psychiatric diagnoses ( bp, major depressive disorders (mdd), psychotic disorders, other psychiatric disorders). familial aggregation of qbs was calculated using the variance components method to derive heritability and shared household effects. the qbs score was significantly higher in bp subjects ( . ± . ) compared to mdd ( . ± . ), other psychiatric diagnoses ( . ± . ), and no psychiatric diagnosis ( . ± . ) (all p < . ). qbs in the whole sample was significantly heritable (h = . ± . , p < . ) while the variance attributed to the shared household effect was not significant (p = . ). when subjects with psychiatric illness were removed, the qbs heritability was similar (h = . ± . , p < . ). these findings suggest that quantitative bipolarity as measured by qbs can separate bp from other psychiatric illnesses yet is significantly heritable with and without bp included in the pedigrees suggesting that the quantitative bipolarity describes a continuous heritable trait that is not driven by a discrete psychiatric diagnosis. bipolarity trait assessment may be used to supplement the diagnosis of bp in future genetic studies and could be especially useful for capturing subclinical genetic contributions to a bp phenotype. introduction bipolar disorder (bp) affects about % of the population, causing significant disability worldwide. bp is considered a highly heritable condition with an estimated – % heritability – . case-control genome-wide association studies (gwas) are beginning to illuminate the genetic risk for this complex polygenic disorder and have iden- tified a number of loci – though findings have been difficult to replicate. most genetic studies of bp define the study group by diagnosis. while this definition is impor- tant for clinical care of bp patients, whether this is the correct phenotype to use to search for genes conferring risks for bp is not clear. genetically susceptible indivi- duals without bp expression may be missed or even erroneously grouped into the control groups. our hypothesis is that bipolarity is not only expressed in bp but may also be a heritable subclinical trait that is present even in non-bipolar individuals, yet much more severe in bp and as such separates bp from other psychiatric diagnoses. the bipolar spectrum diagnostic scale (bsds) is a self- report scale designed to screen for bipolar spectrum © the author(s) openaccessthisarticleis licensedunderacreativecommonsattribution . internationallicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article’s creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article’s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. correspondence: heather a. bruce (hbruce@mprc.umaryland.edu) maryland psychiatric research center, department of psychiatry, university of maryland school of medicine, baltimore, md , usa department of medicine, university of maryland school of medicine, baltimore, md , usa full list of author information is available at the end of the article. () :,; () :,; () :,; () :, ; http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - x http://orcid.org/ - - - x http://orcid.org/ - - - x http://orcid.org/ - - - x http://orcid.org/ - - - x http://creativecommons.org/licenses/by/ . / mailto:hbruce@mprc.umaryland.edu disorders . bsds was shown to have a sensitivity of . and a specificity of . for bp in an outpatient clinic population , a range that was generally supported by other studies – . these data support the clinical validity of bsds to quantify clinical and subclinical bipolarity symptoms. the original bsds does not include all dsm- bp symptoms. we adopted much of the bsds content and format but modified items to cover symptoms in the dsm- bipolar i criteria, and also implemented a new severity rating for each item, henceforth called the quantitative bipolarity scale (qbs). to our knowledge the heritability of bsds or other quantitative bipolarity tools, such as the mood disorders questionnaire (mdq) , has never been evaluated. although qbs is designed to be consistent with dsm- , it is completed by patients, allowing independent evaluations of its validity using clinician assessed dsm- diagnoses. the ideal sample to test whether a quantitative bipo- larity measurement is a heritable endophenotype would be a population with bp and with a family structure which enables estimation of genetic vs non-genetic effects. pio- neered by egeland, there has been a long history of studying the genetics of bp through large pedigrees in the amish/mennonite population , . the old order amish and old order mennonite (ooa/m) population is a founder population whose large families and genealogical record keeping make the population a powerful resource for genetic and heritability analyses even in modest sample sizes. the prevalence of bp in the ooa/m appears similar to that in the general population though some pedigrees carry a heavier burden of mood dis- orders , . in addition, they share similar rural upbringing and school education. the high environmental homo- geneity reduces between-subject variations of uncon- trolled developmental and environmental factors theoretically yielding more precise estimates of genetic contributions. the low incidence of substance use dis- orders decreases another confounding factor commonly present in bp patients in the general population. there- fore, this study aims to develop a potentially novel phenotyping alternative to supplement traditional diagnosis-based genetic research in bp by taking advan- tage of the large family structures in the ooa/m, to test the hypothesis that qbs provides a heritable quantitative trait that separates bp from other psychiatric diagnoses. methods and materials subjects the study included members of ooa/m families [ male, female, age ( . ± . , mean ± s.d.)] from nuclear families in pennsylvania and maryland. the sample included sibling (sibship size ranged from to ), spouse pair, and parent-child pair relationships. sixty nuclear families had one individual participating (although they are related to other members in the extended pedigrees at second or third degree levels), families had two participants, families had three or more participants. as this is a founder population and marriages are kept within the community, most families are connected using genealogical records maintained by the ooa and the oom communities and digitalized in the nih anabaptist genealogy database (agdb) . the genealogical data were converted to the pedigree format by the solar-eclipse software (http://www.nitrc. org/projects/se_linux). exclusion criteria included major medical and neurological conditions and substance abuse in the past year. recruitment was based on the research domain criteria (rdoc) strategy and included all axis-i psychiatric illnesses, starting with identifying families with at least two cases of any psychiatric illnesses and then followed by recruiting members from the same household regardless of diagnosis. families without psychiatric ill- nesses were also recruited. note that this traditional definition of case and control families is a relative term in a population isolate where families are interrelated. for this study individuals without psychiatric illness were labeled as controls irrespective of their family status. figure gives an example of one pedigree. only indivi- duals that were directly interviewed were included in the analysis. the data included individuals with a lifetime diag- nosis of psychiatric disorders: bipolar disorders (n = , fig. an example pedigree. individuals whose diagnoses were estimated based on informant reports did not participate in the study. some members and birth orders were removed or altered to mask the family identity. mdd major depressive disorder, bp bipolar disorder bruce et al. translational psychiatry ( ) : page of http://www.nitrc.org/projects/se_linux http://www.nitrc.org/projects/se_linux including bipolar i and bipolar ii), major depressive disorder (mdd) (n = ), schizophrenia spectrum dis- orders (n = ), and other psychiatric disorders (n = , including: other specified depressive disorder , persistent depressive disorder , premenstrual dysphoric disorder , adjustment disorder , attention deficit hyperactivity dis- order , substance use disorder , social anxiety disorder , panic disorder , obsessive compulsive disorder , gen- eralized anxiety disorder , other specified anxiety dis- order ) and individuals with no lifetime psychiatric disorders. the schedule for clinical interview for dsm- (scid- ) was used to determine diagnoses by trained clinicians. each scid interview was reviewed in con- sensus meetings. for scid inter-rater reliability, a research team from another study interviewed of the current ooa/m participants . diagnoses for of subjects were in agreement while had minor dif- ferences (kappa = . ), supporting the reliability of the scid in this population. all study participants gave written informed consent as approved by the university of maryland irb. quantitative bipolarity rating scale (qbs) the original bsds contains items and a summary item in a format designed to extract the polarity of mood by self-report. we revised the bsds to reflect dsm- criteria for bipolar i disorder. for example, the dsm symptoms “inflated self-esteem or grandiosity” and “decreased need for sleep” were not represented in the original bsds. in the revision, we added items addressing those symptoms as well as items addressing racing thoughts and distractibility. the original bsds had one item “may be more talkative, outgoing, or sexual” which we separated into three items as they are represented by more than one symptom criterion in dsm- . also an item regarding increased substance use during periods of ele- vated mood was removed as this is not a specific criterion for bp in dsm- . furthermore, as we aimed to develop a quantitative phenotype, a severity scale was implemented for each item. subjects were asked to rate each item from to ( for “this description doesn’t really describe me at all”, for “this description fits me to some degree but not in most respects”, for “this description fits me fairly well”, and for “this description fits me very well or almost perfectly.”) the qbs analysis was based on the summed values from all individual items as well as a item asking how much the scale as a whole describes the individual. additional analysis was performed on predefined subscale scores. items through refer to depressive symptoms. items through refer to manic symptoms. items , , , and refer to mood fluctuation symptoms. hereafter these respective sums are referred to as depression sub- score, mania subscore, and mood fluctuation subscore. the quantitative bipolarity scale (qbs) is available online (www.mdbrain.org/qbs_instructions_and_scale.pdf). statistical analysis linear mixed-effects model fit by maximum likelihood estimation was used to compare qbs scores across diagnostic groups, where age and sex were fixed effects and familial relationships were random effects. this pro- cedure was repeated for qbs subscores. receiver oper- ating characteristic (roc) curve was performed to evaluate sensitivity and specificity of qbs. cutoff was based on the youden-index to determine the point for which sensitivity plus specificity is maximal . heritability estimates were obtained using the variance components method as implemented in the solar- eclipse software package (http://www.nitrc.org/projects/ se_linux). heritability (h ) is defined as the proportion of the total phenotypic variance that is explained by additive genetic factors in related individuals. the variance para- meters are estimated by comparing the observed pheno- typic covariance matrix with the covariance matrix predicted by kinship. inverse gaussian transformation was applied to ensure normality of the measurements. household effects were simultaneously estimated for shared environmental effects. siblings were grouped in the same household. significance of the heritability is tested by comparing the likelihood of the model in which additive genetic factors is constrained to zero with that of a model in which additive genetic factors is estimated. twice the difference between the loge likelihoods of these models yields a test statistic . age and sex were used as covariates when calculating the heritability of qbs scores. the familiality of qbs score was further evaluated by comparing family members (first or second degree rela- tives) of individuals with high qbs score (defined by qbs score cutoff > , which is the mean plus one sd of the entire group) to the remaining individuals on qbs scores. we further estimated the extent to which qbs score and bp diagnosis (coded as for bipolar diagnosis and for controls) were explained by shared genetic factors. the genetic correlation (ρg) of the two traits is modeled as a linear function of kinship coefficients that express relatedness among all pairs of individuals in the pedigree; the phenotypic variance–covariance matrix and its addi- tive genetic and random environmental components are then obtained. the significance of the components are then estimated directly by the likelihood ratio test , . if ρg is significantly different from zero then a significant proportion of the traits’ covariance is considered to be influenced by shared genetic factors . inverse gaussian transformation was applied to achieve normality of the qbs measure. to further explore whether there are latent structures of qbs not captured by the predefined depression, mania, bruce et al. translational psychiatry ( ) : page of http://www.mdbrain.org/qbs_instructions_and_scale.pdf http://www.nitrc.org/projects/se_linux http://www.nitrc.org/projects/se_linux and mood fluctuation subscales, factor analysis was per- formed using the whole sample. principal axis factor analyses were performed to identify the latent constructs in the data using oblique (promax) rotations. the optimal solution was based on a combination of common factor solution eigenvalues > . , factor structure using loadings > . , and percent of variance explained. results clinical validity age and sex ratio did not differ significantly across diagnostic groups (table ). the qbs score was sig- nificantly higher in the bp group compared to all other groups [f , = . , p < . ] (table ). post-hoc tests showed that there was a significant difference between the qbs score in subjects with bp ( . ± . , mean ± s.e.) compared to mdd ( . ± . ), other psychiatric diag- nosis ( . ± . ), and no psychiatric diagnosis ( . ± . ) (all p < . ). the difference was not significant when comparing bp and psychotic disorders ( . ± . , p = . ) although this may be due to the limited number of individuals with psychotic disorder in the sample (table ). the qbs score of bipolar patients was approximately double that of mdd, and individuals with mdd showed a two to three fold higher mean qbs score compared to individuals with other psychiatric illnesses. controls showed the lowest mean qbs score (fig. ). these char- acteristics support the specificity and sensitivity of this scale. we further compared patients with currently symptomatic bp ( . ± . ; n = ) to patients who have lifetime bp but are currently in full remission ( . ± . ; n = ), and found that their qbs scores were not sig- nificantly different (t = . , p = . ). this suggests that qbs captures the longitudinal trait aspect of bipolarity symptoms. there was no correlation between age and qbs score (r = − . , p = . ). the validity of qbs in terms of specificity and sensi- tivity was formally investigated with a roc curve ana- lysis. for bp vs controls, sensitivity and specificity was . and . ; for bp vs. all other psychiatric diagnoses, sensitivity and specificity was . and . ; for bp vs mdd sensitivity and specificity was . and . (fig. and table ). in terms of the subscales, mood fluctuation, depression, and mania subscores were all significantly different across the five diagnostic groups (table ). the key post-hoc tests were comparisons between bp and mdd. this analysis revealed that the mood fluctuation and mania subscores were significantly different between the two groups (p = . and p = × − respectively), but the depression subscore was not (p = . ). this supports the validity of qbs for identifying the converging symptoms (depression) and diverging symptoms (mania and mood table sample demographics, qbs (quantitative bipolarity scale) score and qbs subscores across diagnostic groups bipolar disorder major depressive disorder psychotic disorder other psychiatric illness control test statistic (f or x ) p-value n gender (male:female) : : : : : . . age . ± . . ± . ± . ± . . ± . . . qbs score . ± . * . ± . . ± . . ± . . ± . . × − mood fluctuation subscore . ± . * . ± . ± . ± . . ± . . × − depression subscore . ± . . ± . . ± . . ± . . ± . . × − mania subscore . ± . * . ± . . ± . . ± . . ± . . × − data are recorded as mean ± standard error. asterisk indicates measure with significant difference (p < . ) between bipolar disorder and major depression bipolar mdd psychosis other control q b s s co re diagnosis fig. qbs (quantitative bipolarity scale) score across diagnostic groups. mean qbs score is shown for each of five diagnostic groups [bipolar disorder (n = ), major depressive disorder (mdd) (n = ), psychotic disorder (n = ), other psychiatric diagnosis (n = ), controls (n = ). error bars represent standard error bruce et al. translational psychiatry ( ) : page of fluctuation) characterizing these two major mood disorders. factor analyses identified two factors that together accounted for % of the variance in the data. all depression items and mood fluctuation items loaded onto factor and all manic items loaded onto factor . genetic validity as measured by familial aggregation consistent with the literature, a diagnosis of bp was highly heritable in this sample (h = . ± . , p = . ). the qbs score also was significantly heritable (h = . ± . , p = × − ) (table ). repeating the qbs analysis with adjustment for shared environment (household effects), the heritability remained significant (h = . ± . , p = . ) and the proportion of phe- notypic variance attributable to household effects was not significant ( . ± . , p = . ) (table ). one possibi- lity is that the significant heritability of qbs was driven by the presence of bp cases. however qbs heritability remained significant even when removing all bipolar cases (h = . ± . , p = × − ). lastly, we removed all psychiatric illnesses and found that the heritability of the qbs score remained similarly significant (h = . ± . , p = × − ) while the phenotypic variance from shared environment in controls was zero (table ). it is unclear why the heritability of qbs in controls was greater than that in the subsample without bp, which was greater than that in the whole sample; however, standard methods indicate that these were not statistically significant differences. an alternative analysis of familiality showed that indi- viduals with a first or second degree relative with a high qbs score had a significantly higher qbs score ( . ± . , n = ) than individuals without such relatives ( . ± . , n = ) [t( ) = . , p = . )]. in further support of genetic validity, genetic correlation analyses demonstrated significant shared genetic variance between bp and qbs score (ρg = . , p = . ). exploring the heritability of the qbs subscores, we found that the mood fluctuation, depression, and mania . . . . . . . . . s en se tiv ity - specificity fig. receiver operating characteristic (roc) curves. lines show the roc curves for the quantitative bipolarity scale distinguishing bipolar disorder vs controls (red), non-bipolar psychiatric illness (green), and major depressive disorder (blue) table characteristics for each comparison in fig. comparison group sensitivity specificity auc se % c.i. p-value controls . . . . . – . × − other psychiatric illness . . . . . – . × − major depressive disorder . . . . . – . × − auc is area under the curve, se is standard error. all comparisons used a cutoff score of based on the youden index table heritability of qbs (quantitative bipolarity scale) score whole sample (n = ) whole sample without bipolar (n = ) non-psychiatric control subjects (n = ) unadjusted h . ( . ) . ( . ) . ( . ) p × − × − × − age and sex effect (r ) . . . adjusted h . ( . ) . ( . ) . ( . ) p(h ) . . × − household . ( . ) . ( . ) p(household) . . – age and sex effect (r ) . . . additive heritability estimates [h (se)] are shown with and without adjustment for shared environment. household is the proportion of the phenotypic variance attributed to shared environment (household effects). both models included age and sex as covariates. r is the phenotypic variance explained by the covariates age and sex (none was significant) bruce et al. translational psychiatry ( ) : page of subscores all had significant heritability (h = . ± . , p = × − ; h = . ± . , p = . ; h = . ± . , p = × − , respectively). in addition, all qbs subscores remained significantly heritable when the analysis was performed on controls only (h = . ± . , p = . ; h = . ± . , p = . ; h = . ± . , p = . , respectively). these subscore findings suggest that the significant heritability of qbs is not driven by any one facet of the scale. discussion we investigated whether bipolarity quantification can capture the genetic risk for bp beyond that obtained by categorical diagnoses. we found that a concise bipolarity quantification tool using a self-report format that covers symptoms in the current dsm- for bp is significantly heritable in pedigree samples from the ooa/m popula- tion, interestingly even in individuals without bp or psy- chiatric diagnosis. bp is a highly heritable condition. gwas of bp con- ducted outside the amish have identified ~ significant loci though many have not been replicated , , , which indicates that the strong heritability of bp may involve polygenicity and heterogeneity. the genetic loci that may be associated with qbs are yet to be determined. bp has been associated with q . region and also a number of single nucleotide polymorphisms including those in the odz (tenm ,encoding teneurin transmembrane pro- tein ), mad l (encoding mitotic arrest deficient-like ), and trank (encoding tetratricopeptide repeat and ankyrin repeat containing ) genes . it would be inter- esting to determine whether using the qbs phenotype can replicate some of these loci associated with bp or whether qbs identifies additional candidate loci. in addition to gwas studies, more recently whole-genome or -exome sequencing has been used in the search for bp related genes. as rare alleles can be enriched in founder populations, with better characterized phenotypes, the ooa/m may provide an important cohort for discovering risk variants for bp , . use of diagnosis as the primary phenotype in the search for genes conferring risk for bp has often gone unques- tioned though some have investigated temperamental traits as endophenotypes for bipolar disorder – . it is known that bp is prone to over- or under-diagnosis , which has led to the development of tools to screen for bp such as the mdq and bsds. a quantitative trait mea- suring bipolarity may be more informative than diagnosis for genetic studies . furthermore, as the onset of bp can occur later in age, future patients can be erroneously included as controls or with diagnoses of mdd that may convert to bp later, an issue particularly problematic in pedigree-based genetic search where candidate gene identification has been largely dependent upon comparing case vs non-case status within pedigrees. a tool that can identify subclinical bipolar symptoms and their genetic influence may address this important concern. the bivariate genetic correlation analysis here showed a sig- nificantly shared genetic correlation between qbs and bp, supporting that this quantitative bipolarity is at least partially tagging the genetic risk of bp. this measure of bipolarity scored much higher in bp and clearly separated bp from other psychiatric illnesses. however, the detection of subthreshold bipolarity may also be of value. subthreshold bipolarity has been exten- sively studied , and may indicate undiagnosed bipolar features in the general population or patients with mdd and may even predict the conversion of unipolar depression to bp . this view is consistent with our findings in which patients with mdd have a three times higher total bipolarity score, as well as higher mania, mood fluctuation, and depression subscores, compared to non-psychiatric controls. one of the limitations of this study is that the qbs findings were not evaluated in the general population. however, although ooa/m are culturally and envir- onmentally separated from other caucasians of european descent in north america, there is evidence that the clinical presentation of mood disorders is similar . the original version of the bsds has been applied to several populations leading to a range of values for sensitivity ( . – . ) and specificity ( . – . ) of bipolar vs non- bipolar patients depending on the cutoff score used – . using the qbs, the sensitivity and specificity were com- parable in ooa/m, which supports the generalizability of our findings. furthermore, research indicates that genetic findings in the ooa/m may be highly applicable to the larger population. for example, the contactin-associated pro- tein- (cntnap ) gene, first associated with autism in the ooa/m , has since been replicated in multiple stu- dies in the general population , . similarly, genetic stu- dies of non-psychiatric traits have identified associations and biological mechanisms that are replicable in the general population , . one of the novel findings of this study in the ooa/m cohort is that the heritability of qbs was substantial even in subsamples without psychiatric diagnosis, suggesting that the range of subtle bipolarity symptoms assessed by qbs is not specific to mental illness but rather that qbs may be indexing a heritable trait characterized along a continuous dimension from subclinical to overtly clinical symptoms. however, controls in families of a founder population with many cases of bp, may have inflated risk as they are genetically more closely related than discreet families in cohorts from the general population. there- fore, this particular conclusion must be re-tested by applying qbs to family or twin samples from the general bruce et al. translational psychiatry ( ) : page of population. however, even if this caveat of inflated risk is true, it may actually further support qbs as a marker of genetic risk for bp. in addition, a ooa/m cohort with more densely sampled nuclear families than ours may be helpful in a replication study. to the best of our knowledge, there has not been a tool designed to quantitatively assess mania vs depression vs mood fluctuation and then used to assess their relative heritability. the three quantitative subscales for mood fluctuation, depression, and mania within qbs were each found to be significantly heritable. our definitions of the subscores were based on the clinical description of each item. factor analysis confirmed the clear distinction between manic and depression items. it is unclear why the depression and the mood fluctuation items clustered together. we speculate that it may reflect fluctuations between normal and depressed moods occurring more commonly than fluctuations between normal and manic moods. factor analysis of qbs in a larger sample of bp may allow a clearer distinction. another notable limitation of this study is the lack of qbs administration over time, thus we did not address reliability. our study and other studies using the bsds address validity with measures of sensitivity and specifi- city, however, we do not know of any studies that address reliability. furthermore we did not evaluate the predictive power of qbs for risk of conversion to bp, which we plan to address in subsequent studies. some individuals in our study classified as controls were below the age of max- imum risk for bp and may go on to develop bp. however this may also be a strength of the qbs approach because a main purpose of this study is to investigate quantitative bipolarity with and without bp. another limitation of this study is the low number of individuals with a schizo- phrenia spectrum disorder ( % of the current sample, which is epidemiologically similar to the rate in the gen- eral population), given the data supporting a genetic overlap between schizophrenia and bp . an additional limitation is that only about % of the current sample have bp but their qbs scores are by definition higher and predominate in the distribution of the overall sample, which is a limitation of the current sample. future studies recruiting bp based samples are needed to further validate the questionnaire. heritability is only the first step of genetic validity; whe- ther qbs would assist in the search for genes conferring risk of developing bp remains to be seen. this study suggests that quantitative bipolarity as measured by the concise self- administered qbs task may be a useful phenotype in sup- plementing the diagnosis phenotype in bp genetic studies. acknowledgements the study was supported by national institute of health grants u mh , r eb , r da , r mh , p mh , and t mh . author details maryland psychiatric research center, department of psychiatry, university of maryland school of medicine, baltimore, md , usa. department of medicine, university of maryland school of medicine, baltimore, md , usa. clinic for special children, strasburg, pa , usa. department of psychiatry, university of maryland school of medicine, baltimore, md , usa. hershey medical center, department of psychiatry, penn state university school of medicine, hershey, pa , usa. human genetics branch, national institute of mental health intramural research program, bethesda, md , usa conflict of interest the authors declare that they have no conflict of interest. publisher’s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. received: october revised: march accepted: april references . bertelsen, a., harvald, b. & hauge, m. a danish twin study of manic-depressive disorders. br. j. psychiatry.: j. ment. sci. , 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and materials 	subjects 	quantitative bipolarity rating scale (qbs) 	statistical analysis 	results 	clinical validity 	genetic validity as measured by familial aggregation 	discussion 	acknowledgments this work is licensed under a creative commons attribution-noncommercial-noderivatives . international license. © the authors https://edm.bioscientifica.com/ � published�by�bioscientifica�ltd id: - ; july doi: . /edm- - catheter-related thrombosis in hyperinsulinism d yau and others central venous catheter-associated thrombosis in children with congenital hyperinsulinism daphne yau , maria salomon-estebanez , amish chinoy , john grainger , ross j craigie , raja padidela , mars skae , mark j dunne , philip g murray and indraneel banerjee departments of paediatric endocrinology, paediatric haematology, paediatric surgery, royal manchester children’s hospital, manchester, uk, and faculty of biology, medicine and health, university of manchester, manchester, uk summary congenital hyperinsulinism (chi) is an important cause of severe hypoglycaemia in infancy. to correct hypoglycaemia, high concentrations of dextrose are often required through a central venous catheter (cvc) with consequent risk of thrombosis. we describe a series of six cases of chi due to varying aetiologies from our centre requiring cvc for the management of hypoglycaemia, who developed thrombosis in association with cvc. we subsequently analysed the incidence and risk factors for cvc-associated thrombosis, as well as the outcomes of enoxaparin prophylaxis. the six cases�occurred�over�a� -year�period;�we�identified�an�additional� �patients�with�chi�who�required�cvc�insertion�during� this period (n =  �total),�and�a�separate�cohort�of�patients�with�chi�and�cvc�who�received�enoxaparin�prophylaxis�(n =  ).� the�incidence�of�cvc-associated�thrombosis�was� %�( / )�over�the�  years,�a�rate�of� . �thromboses/ �cvc�days.� there�was�no�difference�in�the�frequency�of�genetic�mutations�or�focal�chi�in�those�that�developed�thromboses.�however,� compound heterozygous/homozygous potassium atp channel mutations correlated with thrombosis (r  =  . ,�p =  . ).� no�difference�was�observed�in�cvc�duration,�high�concentration�dextrose�or�glucagon�infused�through�the�cvc.�in�patients� receiving enoxaparin prophylaxis, none developed thrombosis or bleeding complications. the characteristics of these patients�did�not�differ�significantly�from�those�with�thrombosis�not�on�prophylaxis.�we�therefore�conclude�that�cvc- associated�thrombosis�can�occur�in�a�significant�proportion�( %)�of�patients�with�chi,�particularly�in�severe�chi,�for�which� anticoagulant prophylaxis may be indicated. - - id: - correspondence should be addressed to d yau email daphne.yau@mft.nhs.uk learning points: •• cvc�insertion�is�one�of�the�most�significant�risk�factors�for�thrombosis�in�the�paediatric�population. •• risk factors for cvc-associated thrombosis include increased duration of cvc placement, malpositioning and infusion of blood products. •• to�our�knowledge,�this�is�the�first�study�to�evaluate�cvc-associated�thrombosis�in�patients�with�congenital� hyperinsulinism (chi). •• the�incidence�of�cvc-associated�thrombosis�development�is�significant�( %)�in�chi�patients�and�higher�compared� to other neonates with cvc. chi severity may be a risk factor for thrombosis development. •• although�effective�prophylaxis�for�cvc-associated�thrombosis�in�infancy�is�yet�to�be�established,�our�preliminary� experience�suggests�the�safety�and�efficacy�of�enoxoaparin�prophylaxis�in�this�population�and�requires�on-going� evaluation. downloaded from bioscientifica.com at / / : : am via free access https://creativecommons.org/licenses/by-nc-nd/ . / https://creativecommons.org/licenses/by-nc-nd/ . / https://creativecommons.org/licenses/by-nc-nd/ . / https://edm.bioscientifica.com/ https://doi.org/ . /edm- - mailto:daphne.yau@mft.nhs.uk d yau and others catheter-related thrombosis in hyperinsulinism doi: . /edm- - https://edm.bioscientifica.com/ id: - ; july background congenital hyperinsulinism (chi) is an important cause of persistent and severe hypoglycaemia in infancy due to excess insulin secretion ( , ). in cases where a genetic aetiology is identified, mutations in the atp-sensitive potassium (katp) channel genes, abcc and kcnj , are the most frequent. homozygous and compound heterozygous mutations in abcc /kcnj cause diffuse chi with severe and often refractory hypoglycaemia. a cardinal feature of chi is increased glucose demand, often requiring central venous catheter (cvc) insertion to achieve euglycaemia and prevent permanent neurologic injury. although cvcs are recognised as a significant risk factor for thrombosis in children ( , , , ), this has not been described in patients with chi. we describe six cases of cvc-associated thrombosis in chi patients, presented in chronologic order, and quantified the incidence and examined for potential risk factors. we additionally examined a separate cohort of patients with chi and cvc who were placed on enoxaparin prophylaxis. case presentation patient was a term male infant (birth weight . kg) who presented with a hypoglycaemic seizure at   days of life. chi was confirmed (glucose . mmol/l, insulin pmol/l, c-peptide pmol/l) and genetic testing revealed diffuse disease due to a homozygous mutation in abcc . glycaemic stability could only be achieved through high dextrose (up to %) and glucagon (up to . µg/kg/h) via cvc, delivering a maximal glucose infusion rate of mg/kg/min. his initial cvc, inserted in the right internal jugular vein (ijv), became dislodged after  days and ultrasound (us) later performed to assess for sites for intravenous access showed evidence of non- occlusive thrombus at this site. he had an extremely complicated treatment course. after his initial cvc, three further cvcs dislodged, and at the time of insertion of the fifth cvc, severe occlusion of the superior vena cava by thrombus was found with further thrombi identified in the inferior vena cava, common iliac and right common femoral veins on ultrasound scanning. treatment with low-molecular-weight heparin was commenced on advice from haematology specialists. other complications included several episodes of sepsis secondary to central line infections, necrolytic migratory erythema from glucagon infusion and raised transaminases from octreotide. pancreatectomy was performed at   months of age due to refractoriness to medical therapy but hypoglycaemia persisted. after the sixth cvc dislodged secondary to further thrombi, it was not possible to secure additional central venous access. temporary access was obtained via intraosseus and a peripheral venous cannula. however, he developed candida albicans sepsis complicated by disseminated intravascular coagulation and died. the presence of an inherited thrombophilia could not be ascertained. patient was born at   weeks with birth weight . kg and presented with jitteriness at h of age followed by hypoglycaemic seizures. chi was biochemically confirmed (glucose . mmol/l, insulin pmol/l, c-peptide pmol/l) and testing for mutations in abcc , kcnj and hnf a was negative. maximum dextrose and glucagon infusions of % and . µg/kg/h respectively were required to achieve euglycaemia. a peripherally inserted central catheter (picc) was placed at the referring hospital, and removed  days later after transfer to our centre when a tunnelled catheter was inserted into the right ijv. a routine echocardiogram performed  days after removal of the picc demonstrated a right atrial thrombus and the patient was promptly commenced on enoxaparin therapy. he was responsive to low-dose diazoxide and follow-up imaging demonstrated thrombus resolution with normal cardiac structure and function. patient presented with hypoglycaemic seizures at   days of life after uneventful term delivery. a hypoglycaemia screen demonstrated chi (glucose mmol/l, insulin pmol/l, c-peptide pmol/l), and he was found to have a paternal abcc mutation and f dopa pet ct scan demonstrated a focal lesion in the distal body of the pancreas. he required % dextrose, but not glucagon, delivered via a right ijv tunnelled catheter which was removed  days later due to infection and leakage. non-occlusive thrombus was noted at the site of the previous cvc, and he was treated with enoxaparin. the chi was successfully treated with focal lesionectomy. patient presented on day with hypoglycaemic seizures after emergency caesarean section for non- reassuring fetal heart rate at   weeks with birth weight . kg. she required % dextrose with glucagon infusion up to µg/kg/h and was unresponsive to diazoxide, octreotide and sirolimus. genetic testing revealed a homozygous abcc mutation. prior to subtotal pancreatectomy, a picc was inserted in the left femoral vein followed by a tunnelled cvc in the right ijv. malfunction and infection of the right ijv catheter was noted after   days, and the cvc was removed. downloaded from bioscientifica.com at / / : : am via free access https://doi.org/ . /edm- - https://edm.bioscientifica.com/ d yau and others id: - ; july doi: . /edm- - catheter-related thrombosis in hyperinsulinism https://edm.bioscientifica.com/ ultrasound scanning revealed thrombus in the right rij and therapeutic enoxaparin was commenced. patient was born at and -week gestation with a macrosomic birth weight of . kg. he required moderate respiratory support after birth and was noted on day to be hypoglycaemic due to chi (glucose . mmol/l, insulin and c-peptide both elevated). owing to the significant birth weight, mutation testing for abcc , kcnj , hnf a and beckwith-wiedemann were performed and negative. he initially required up to % dextrose and glucagon at µg/kg/h to achieve glycaemic stability. this was initially delivered via umbilical venous catheter and picc in both femoral veins. for long-term access, a tunnelled right ijv was inserted at the referring hospital, which dislodged after  days. a femoral line was placed for temporary access and when the patient was brought to theatre for further cvc insertion, swelling was noted at the previous cvc site and uss confirmed the presence of non-occlusive thrombus in the right ijv. treatment with enoxaparin was commenced and a trial of diazoxide, initially postponed due to respiratory issues and significant ventricular hypertrophy, was successful. patient presented at  days of life after an uneventful term delivery in cardiorespiratory collapse and was found to be severely hypoglycaemic due to chi (glucose . mmol/l, insulin pmol/l, c-peptide pmol/l). glucose stabilisation was achieved with . % dextrose and glucagon infusion at µg/kg/h via right femoral cvc. right leg swelling was noted  days after cvc insertion, which persisted despite cvc removal. ultrasound scanning demonstrated thrombus from the right femoral to the popliteal vein. the patient was responsive to diazoxide and intravenous support could be discontinued. genetic testing by targeted next-generation sequencing for known chi genes was negative. investigation doppler ultrasound demonstrated thrombosis in association with the cvc in patients , – as detailed above. thrombus was noted incidentally in patient in the right atrium on routine echocardiogram. treatment all six cases were treated with enoxaparin for   weeks or until thrombosis resolution by uss. doses were titrated to maintain an anti-factor xa level between . and . u/ml. with the exception of case , follow-up ultrasound demonstrated resolution and enoxaparin was discontinued. outcome and follow-up given the occurrence of these cases, we aimed to analyse the incidence and identify the potential risk factors. these six cases of cvc-associated thrombosis occurred from august to at a specialist centre for chi. all admitted cases of chi requiring cvc during this time period were retrospectively reviewed. chi was diagnosed according to well-established criteria ( , ). data were collected on chi characteristics, cvc features, dextrose and glucagon-containing fluids, enoxaparin dose for those on anticoagulation prophylaxis and bleeding events. venous thrombosis detection was based on clinical signs (i.e. swelling, erythema, warmth, reduced limb mobility, cvc occlusion) and confirmed by ultrasound. occlusive and table  characteristics of chi patients with and without thrombosis. thrombosis (n =  ) no thrombosis (n =  ) prophylaxis (n =  ) males, n (%) ( ) �( ) �( ) any mutation in abcc or kcnj , n (%) ( ) ( ) �( ) homozygous/compound heterozygous katp mutation, n (%) ( %) ( %) – catheter duration, days (range) �( – ) ( – ) ( – ) number requiring high dextrose, n (%) ( ) ( ) �( ) maximum high dextrose concentration, % (range) . ( – ) . ( – ) �( . – ) duration of high dextrose, days (range) �( – ) �( – ) ( – ) number requiring glucagon, n (%) ( ) �( ) �( ) maximum glucagon, micrograms/kg/h (range) ( – ) ( – ) ( – ) duration of glucagon, days (range) ( – ) �( – ) �( – ) thrombosis was associated with a marginally higher proportion of patients with compound heterozygous and homozygous katp mutations. there were no�significant�differences�in�chi�characteristics,�cvc�features,�high�concentration�dextrose�(defined�as�> %)�or�glucagon-containing�solutions�among�the� groups with and without thrombosis. the characteristics of the patients on enoxaparin prophylaxis were similar to those with thrombosis. downloaded from bioscientifica.com at / / : : am via free access https://doi.org/ . /edm- - https://edm.bioscientifica.com/ d yau and others catheter-related thrombosis in hyperinsulinism doi: . /edm- - https://edm.bioscientifica.com/ id: - ; july non-occlusive thromboses were included. fisher’s exact test and student’s t-test were used to test for significance in categorical and continuous variables, respectively, using spss. during this period, inpatients with chi required cvc including those who developed thrombosis, an incidence of % ( / ) over  years or . thromboses per catheter days. thrombosis occurred at a median of (range – ) days after cvc insertion at ( – ) days of life. patient, cvc and treatment-related factors were examined for associations with thrombosis development. there was no difference in the frequency of katp channel mutations in those with and without thrombosis (table  ). however, the frequency of homozygous and compound heterozygous katp channel mutations, associated with severe diffuse chi, tended to be higher but not statistically significant in those with thrombosis (table  ). in a stepwise backward logistic regression model, compound heterozygous and homozygous mutations correlated with thrombosis (r = . , p = . ), while sex, glucagon dose, cvc duration and focal chi were excluded. there was no association between thrombosis development and cvc characteristics (cvc type (i.e. tunnelled vs non-tunnelled), location or lumen number) or the maximum concentration of dextrose or glucagon infused via the cvc (table  ). the duration of cvc placement was marginally shorter in those with thrombosis reflecting earlier withdrawal following thrombosis detection (table  ). no patient had blood products infused via the cvc prior to thrombosis development, a previously identified risk factor in neonates ( ). following the recognition of cvc-related thrombosis in chi patients, seven patients with severe chi were commenced on enoxaparin prophylaxis. clinical parameters to consider prophylaxis were severe hypoglycaemia requiring high concentration (≥ %) dextrose infusion for the majority of fluid intake and/ or sustained glucagon infusion. baseline, cvc and treatment characteristics were similar for this group compared to those with thrombosis (table  ). none of these patients developed thrombosis or bleeding manifestations, suggesting the safety and efficacy of anticoagulation prophylaxis in our cohort of patients with severe chi. discussion we observed a clinically relevant incidence of cvc- associated thrombosis in patients with chi, % over  years or . events/ catheter days. the incidence is twice that reported in the neonatal literature ( . %) ( ) and is the first report in patients with chi. no direct association was identified with cvc characteristics, dextrose or glucagon dosage. thrombosis was associated with a marginally higher proportion of patients with compound heterozygous and homozygous katp mutations, suggesting a greater risk in patients with severe and persistent chi. in response to thrombotic events, we introduced anticoagulation prophylaxis with no development of thrombosis or bleeding complications. awareness of the heightened thrombosis risk and consideration of prophylaxis are therefore important in severe chi. declaration of interest the� authors� declare� that� there� is� no� conflict� of� interest� that� could� be� perceived as prejudicing the impartiality of the research reported. funding the study was supported by the northern congenital hyperinsulinism (norchi) charitable fund and by the manchester academic health sciences centre. patient consent written informed consent was obtained from the patients’ parents/ guardians for publication. for the deceased case described, however, it was not possible to contact the family. the study was approved by the local research ethics committee. author contribution statement i b conceived of the study. d y, m s e and i b designed the study; d y, m s e and a c collected the data; d y and i b analysed the data; d y drafted the manuscript, which was edited by all authors. j g provided guidance and input on the haematological aspects of the study. references banerjee i, avatapalle b, padidela r, stevens a, cosgrove ke, clayton pe & dunne mj. integrating genetic and imaging investigations into the clinical management of congenital hyperinsulinism. clinical endocrinology – . (https:// doi.org/ . /cen. ) stanley ca. perspective on the genetics and diagnosis of congenital hyperinsulinism disorders. journal of clinical endocrinology and metabolism – . (https://doi.org/ . / jc. - ) van ommen ch & sol jj. developmental hemostasis and management of central venous catheter thrombosis in neonates. seminars in thrombosis and hemostasis – . (https://doi. org/ . /s- - ) glaser dw, medeiros d, rollins n & buchanan gr. catheter-related thrombosis in children with cancer. journal of pediatrics – . (https://doi.org/ . /mpd. . ) downloaded from bioscientifica.com at / / : : am via free access https://doi.org/ . /edm- - https://edm.bioscientifica.com/ https://doi.org/ . /cen. https://doi.org/ . /cen. https://doi.org/ . /jc. - https://doi.org/ . /jc. - https://doi.org/ . /s- - https://doi.org/ . /s- - https://doi.org/ . /mpd. . d yau and others id: - ; july doi: . /edm- - catheter-related thrombosis in hyperinsulinism https://edm.bioscientifica.com/ massicotte mp, dix d, monagle p, adams m & andrew m. central venous catheter related thrombosis in children: analysis of the canadian registry of venous thromboembolic complications. journal of pediatrics – . (https://doi.org/ . / s - ( ) - ) smitherman ab, alexander t, connelly m, snavely ac, weston bw, liles ea & steiner mj. the incidence of catheter-associated venous thrombosis in noncritically ill children. hospital pediatrics – . (https://doi.org/ . /hpeds. - ) park ck, paes ba, nagel k, chan ak, murthy p & thrombosis and hemostasis in newborns (thin) group. neonatal central venous catheter thrombosis: diagnosis, management and outcome. blood coagulation and fibrinolysis – . (https://doi. org/ . /mbc. b e f b ) received in final form april accepted june downloaded from bioscientifica.com at / / : : am via free access https://doi.org/ . /edm- - https://edm.bioscientifica.com/ https://doi.org/ . /s - ( ) - https://doi.org/ . /s - ( ) - https://doi.org/ . /hpeds. - https://doi.org/ . /mbc. b e f b https://doi.org/ . /mbc. b e f b 	summary 	learning points: 	case presentation 	investigation 	treatment 	outcome and follow-up 	discussion 	declaration of interest 	funding 	patient consent 	author contribution statement 	references diagnosis and management of acute appendicitis. eaes consensus development conference consensus statement diagnosis and management of acute appendicitis. eaes consensus development conference ramon r. gorter , , • hasan h. eker • marguerite a. w. gorter-stam • gabor s. a. abis • amish acharya • marjolein ankersmit • stavros a. antoniou , • simone arolfo • benjamin babic • luigi boni • marlieke bruntink • dieuwertje a. van dam • barbara defoort • charlotte l. deijen • f. borja delacy • peter mnyh go • annelieke m. k. harmsen • rick s. van den helder • florin iordache • johannes c. f. ket • filip e. muysoms • m. mahir ozmen • michail papoulas • michael rhodes • jennifer straatman • mark tenhagen • victor turrado • andras vereczkei • ramon vilallonga • jort d. deelder • jaap bonjer received: august / accepted: september / published online: september � the author(s) . this article is published with open access at springerlink.com abstract unequivocal international guidelines regarding the diagnosis and management of patients with acute appendicitis are lacking. the aim of the consensus meeting of the eaes was to generate a european guideline based on best available evidence and expert opinions of a panel of eaes members. after a systematic review of the literature by an international group of surgical research fellows, an expert panel with extensive clinical experience in the management of appendicitis discussed statements and recommendations. statements and recommendations with more than % agreement by the experts were selected for a web survey and the consensus meeting of the eaes in bucharest in june . eaes members and attendees at the eaes meeting in bucharest could vote on these statements and recommendations. in the case of more than % agreement, the statement or recommendation was defined as supported by the scientific community. results from both the web survey and the consensus meeting in bucharest are presented as percentages. in total, statements and recommendations were selected for the electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. & ramon r. gorter rr.gorter@vumc.nl department of surgery, vu university medical centre, amsterdam, the netherlands department of surgery, red cross hospital, beverwijk, the netherlands department of surgery, spaarne gasthuis, haarlem, the netherlands department of surgery, st mary’s hospital, london, uk department of surgery, center for minimally invasive surgery, neuwerk hospital, mönchengladbach, germany department of surgery, university hospital of heraklion, heraklion, greece department of surgery, university of torino, torino, italy department of surgery, agaplesion markus krankenhaus, frankfurt am main, germany department of surgery, minimally invasive surgery research center, university of insubria, varese, italy department of surgery, maria middelares ghent, ghent, belgium department of surgery, hospital clinic of barcelona, barcelona, spain department of surgery, st. antonius hospital, nieuwegein, the netherlands department of surgery, noordwest clinics alkmaar, alkmaar, the netherlands department of surgery, university of medicine and pharmacy ‘‘carol davila’’, bucharest, romania medical library, vrije universiteit, amsterdam, the netherlands department of surgery, school of medicine, bahcesehir university, istanbul, turkey department of surgery, tel aviv sourasky medical centre, tel aviv, israel department of surgery, stepping hill hospital, stockport, uk surg endosc ( ) : – doi . /s - - - and other interventional techniques http://dx.doi.org/ . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf web survey and consensus meeting. more than mem- bers and attendees voted on them. in of statements and recommendations, more than % agreement was reached. all statements and recommendations are pre- sented in this paper. they comprise topics regarding the diagnostic work-up, treatment indications, procedural aspects and post-operative care. the consensus meeting produced statements and recommendations on the diagnostic work-up and management of appendicitis. the majority of the eaes members supported these statements. these consensus proceedings provide additional guidance to surgeons and surgical residents providing care to patients with appendicitis. keywords appendicitis � uncomplicated appendicitis � complicated appendicitis � appendectomy � laparoscopic appendectomy acute appendicitis is a common gastrointestinal disease affecting . – /per . individuals each year with the highest incidence in children and adolescents [ – ]. the variation of incidence is due to variations in ethnicity, sex, age, obesity and season of the year [ , – ]. based upon the entrenched idea that appendicitis is an irreversible pro- gressive disease eventually leading to perforation, removal of the appendix is the gold standard of treatment. the medical profession has gained much experience in manag- ing patients with acute appendicitis ever since fitz’s first report in [ ]. large heterogeneity exists, however, between existing intercontinental, european and national guidelines regarding diagnosing and managing acute appendicitis. for instance, in the netherlands, pre-operative imaging studies are promoted and considered mandatory in order to prevent negative appendectomies according to national guidelines, whereas in guidelines of other countries, it is not promoted nor considered mandatory [ ]. another example is the inconsistency regarding the management of an unexpected ‘‘normal appendix’’ during diagnostic laparoscopy [ , ]. this heterogeneity prompted the need for an european consensus development conference for the diagnosis and management of acute appendicitis. the european association of endoscopic surgery (eaes) initiated a consensus development conference meeting on the management of acute appendicitis for its meeting in bucharest. the aim of this consensus meeting was to develop practical guidelines based on the available evidence combined with the expertise of a selected panel of eaes surgeons. the findings are reported in this manuscript. materials and methods the coordinating team (hjb, rg, he and mgs) invited ten surgeons from nine european countries to serve as experts in this consensus development conference. an international research team of young surgical researchers across european countries was formed to evaluate and process the existing literature on the management of acute appendicitis. the coordinators generated a list of topics on acute appendicitis to be addressed (appendix ). an exploratory literature search was conducted in order to identify any additional topics of interest. all topics were approved by the experts and subsequently divided into three main parts: pre- operative care, operative care and after care. based upon the topics, research questions were formulated, reviewed and approved by the panel of experts. literature search and processing of the literature research questions were used as guidance to conduct lit- erature searches. the searches were conducted in cooper- ation with a medical information specialist of the vrije universiteit. searches were performed in the following databases: pubmed, web of science and the cochrane library from inception up to december . no limi- tation was used regarding year of publication. searches have been attached in appendix . all papers published in european languages, and all study types with the exception of case reports were included in the search. all articles were screened and reviewed by teams of two research fellows for eligibility, based on title and abstract. if eligible for inclusion, full text articles were obtained. if no full text was available, the article was excluded. in case of disagreement between the two research fellows, the coordinator dedicated to the topic acted as referee. full text articles were summarized, evaluated and discussed at research meetings to assess their eligibility for inclusion in the review process. all included studies were evaluated according to the grade system [ – ]. the grade system systematically evaluates the available literature and focuses on the level of evidence based upon the types of studies included. the level of evidence can be marked as high, moderate, low or very low. this could be either downgraded in case of significant bias or upgraded when multiple high-quality studies showed consistent results. department of surgery, hospital de la santa creu i sant pau, barcelona, spain department of surgery, medical school university of pécs, pecs, hungary department of surgery, university hospital vall hebrón, barcelona, spain department of pediatric surgery, vu university medical centre, p.o. box , dd amsterdam, the netherlands surg endosc ( ) : – the highest levels of evidence (systematic reviews) were assessed first. if the systematic review was of sufficient quality, it was used to answer the research question. if no systematic review of sufficient quality was found, ran- domized controlled trials (rcts) and cohort studies were evaluated. all selected studies were uploaded to a men- deley database that was accessible to all research fellows, coordinators and experts. after the literature search, an expert was assigned to every couple of researchers. this threesome was assigned research questions from the pre-operative care, operative care and after care. hereafter they were responsible for formulating a statement/conclusion and, if possible, a rec- ommendation on the assigned research questions. again, the quality of the evidence was evaluated according to the grade/sign system [ – ]. the strength of the rec- ommendation was based on the level of evidence and qualified as weak or strong. this was reflected in terms, using ‘‘recommend’’ in case of a strong recommendation and ‘‘suggest’’ in case of a weak recommendation. a face-to-face consensus meeting among the experts was held in amsterdam on the may to discuss the final statements and recommendations. the coordinating team all experts and members of the international research team attended the meeting. a modified delphi method was used. the delphi method is a structured process, commonly used to develop healthcare quality indicator and consists of four key components; iteration, controlled acquisition of feed- back, aggregation of responses and anonymity. as anon- ymity was not applicable in our situation, we used the term modified [ – ]. all statements and recommendations were shared with proposed levels of evidence with the entire group. after displaying the statements and recommenda- tions, the experts casted their votes of agreement or dis- agreement. refrain from voting was not allowed. no discussion was allowed between the experts at this point of time. in case of % consensus, the statement and rec- ommendation were accepted without further voting or dis- cussion. in case of lack of consensus, the research team responsible for the statement presented the underlying con- siderations. after discussion between the experts, a second voting round was conducted. the statement or recommen- dation was accepted in case of at least % consensus. those statements and recommendations with less than % consensus in the expert meeting were not included in the web survey or in the bucharest meeting. all finalized recommendations and statements with levels of evidence were entered into a web survey and distributed to all eaes members by e-mail. the web survey was open from may until july . the recommendations or statements as well as the levels of evidence were open to several voting options: ‘‘agree’’, ‘‘partly agree’’, ‘‘disagree’’ or ‘‘don’t know’’. the option ‘‘partly agree’’ meant that the voter agreed with the rec- ommendation, but did not agree with the strength of recommendation. all finalized recommendations and statements from the amsterdam meeting with levels of evidence were pre- sented at a plenary session of the rd annual meeting of the eaes on the june in bucharest. live voting was performed using a digital voting system. voting options were the same as the abovementioned. both results from the web survey and the bucharest meeting are presented in the ‘‘results’’ section. results the literature search yielded , articles. the title, abstract and full text were reviewed. in total, articles were selected and reviewed in detail to define state- ments and recommendations, which were subsequently discussed at the amsterdam meeting. (appendix ) during this meeting, the following statements and recommenda- tions were excluded: on incidence and prevalence of appendicitis (n = ), on the place of notes in acute appendicitis (n = ), on the learning curve of appendec- tomy (n = ), on day surgery for acute appendicitis (n = ) and on the skeletonizing technique of the meso- appendix (n = ). twenty-one statements were combined leaving a total of statement and recommendations; statements and recommendations for pre-operative care, statement and recommendations for operative care and statements and recommendations for aftercare (fig. ). of the articles, were excluded due to the fact that statements and recommendations were excluded or were combined, rendering articles (fig. ; appendix ). web survey in total, eaes members responded to the web survey; % were surgeons and % surgical residents. bucharest meeting the eaes congress in bucharest was attended by delegates. during the plenary consensus meeting, delegates voted. sixty-eight per cent were surgeons, % surgical residents and % scientists, physician assistants and others. surg endosc ( ) : – pre-operative care establishing the diagnosis of acute appendicitis remains challenging. the clinical presentation of acute appendicitis can vary from mild symptoms to signs of generalized peritonitis and sepsis. hence, the value of individual clin- ical variables to determine the likelihood of acute appen- dicitis in a patient is low [ , ]. biochemical testing is performed routinely in most patients. its value in con- firming acute appendicitis is debatable. a recent systematic review showed that elevated c-reactive protein levels render the highest diagnostic accuracy followed by increased numbers of leucocytes with an area under the curve of . [ % ci . – . ] and . [ % ci . – . ], respectively [ ]. the area under the curve represents the ability of a test to correctly classify patients. in case the score is between the . and . , it represents a fair test. both clinical and biochemical variables have been combined into clinical predicting rules (cpr) such as the alvarado score and paediatric appendicitis score (pas) [ , ]. this was done to increase the value of the indi- vidual variables. ohle et al. [ ] demonstrated that the alvarado score was good at ‘‘ruling-out’’ appendicitis with an overall sensitivity and specificity of and %, respectively. in children, however, it has been shown that the pas outperforms the alvarado score [ ]. to increase the predictive value of these two tests ebell et al. [ ] identified new cut-off values for the alvarado score and pas, which improved sensitivity and specificity rates. based upon the alvarado score, patients can now be cat- egorised into low risk (score \ ), intermediate ( – ) and high risk (c ). the use of such cprs appears useful to determine the likelihood of acute appendicitis. distin- guishing between low, intermediate and high risk provides guidance whether imaging studies are necessary. imaging studies in patients with a clinical suspicion of acute appendicitis can reduce the negative appendectomy rate, which has been reported to be as high as %. ultrasonography, abdominal computed tomography (ct) and magnetic resonance imaging (mri) are most com- monly used. ultrasonography is non-invasive, avoids radiation and is associated with a sensitivity rate between and % and a specificity rate between and %. the positive likelihood ratio of ultrasonography is high at values between and , while the negative likelihood ratio is moderate ( . – . ) [ – ]. ultrasonography is therefore reliable to confirm presence of appendicitis but unreliable to exclude appendicitis. furthermore, one should fig. flow diagram of the process prior to the eaes consensus meeting in bucharest surg endosc ( ) : – bear in mind that ultrasonography is highly operator dependent. inconclusive ultrasonography findings, mostly due to failure visualizing the appendix, mandate additional imaging studies. abdominal computed tomography (ct) for suspected appendicitis has sensitivity and specificity rates between – % and – %, respectively, and, therefore, is superior to ultrasonography. lower values of sensitivity and specificity can be explained by the use of enteral contrast [ , , – ]. however, the radiation exposure of abdominal ct is a concern particularly in children and during pregnancy. the estimated lifetime cancer-related mortality risk of developing a radiation-induced malig- nancy is approximately . % for a -year-old child and . % in a -year-old child if an abdominal ct is per- formed [ , ]. computed tomographies employing only a quarter of the standard radiation dose (low-dose cts) provide similar imaging results as standard cts and are, hence, an excellent alternative [ ]. regarding the administration of oral contrast, andersson et al. [ ] con- cluded in their meta-analysis that a ct scan without oral contrast was superior to cts with oral contrast in terms of sensitivity and specificity. therefore, low-dose cts with- out oral contrast are preferable in patients with suspected appendicitis [ ]. magnetic resonance imaging (mri) is used in pregnant patients and children with inconclusive findings at ultra- sonography [ ]. a recent meta-analysis on mri in patients with appendicitis, yielded a sensitivity rate of % [ % ci – %], a specificity rate of % [ % ci – %], a positive likelihood ratio of . [ % ci . – . ] and a negative likelihood ratio of . [ % ci . – . ] [ ]. these rates are comparable to those of ct imaging, although these findings should be interpreted with care as most studies have been performed in a selected group of patients. mri is associated with significant costs, and interpreting the images requires experience. therefore, at the present time, use of mri appears limited to pregnant women and children. the algorithm associated with the alvarado score (rec- ommendation ) is shown in fig. . in obese patients (definition depends on the reference study), the diagnostic accuracy of ultrasound is diminished due to an increase of the subcutaneous and intra-abdominal fat. anderson et al. [ ] demonstrated that the body mass index (bmi) does not alter the diagnostic accuracy of a ct scan. ct appears therefore more reliable than ultrasonog- raphy in obese patients with the exception of children and pregnancy. patients with appendicitis are classified as uncompli- cated or complicated appendicitis based upon pre-opera- tive, intra-operative and/or histopathological findings. in this report, uncomplicated appendicitis has been defined as an inflamed appendix without signs of gangrene, perfora- tion, intra-peritoneal purulent fluid, contained phlegmon or intra-abdominal abscess (iaa). complicated appendicitis applies to all patients with either a gangrenous inflamed appendix with or without perforation, intra-abdominal abscess, peri-appendicular contained phlegmon or purulent free fluid. classification is necessary as treatment strategies may differ. uncomplicated appendicitis appendectomy is still considered to be the gold stan- dard for uncomplicated appendicitis. two main approaches to remove an inflamed appendix are avail- able; the open approach (oa) or the laparoscopic approach (la). in , a large cochrane review on studies showed that la significantly reduced the rate of surgical site infection (ssi) (or . ; % ci . – . ) but significantly increased the risk of an intra-abdominal abscess (iaa) (or . ; % ci . – . ) compared to the open approach [ ]. it was stated that la was associated with fewer superficial wound infections, less post-operative pain, shorter hos- pital stay and earlier return to work, but the higher rate of iaa raised concerns [ ]. ever since, inconsistent results have been reported regarding the potential higher incidence of iaa after la [ – ]. benefits of la over oa reported in meta-analyses are: reduced incidence of ssi, post-operative and long-term bowel obstruction with better outcome in terms of shorter hospital stay, its diagnostic value, less pain, earlier return to work, ear- lier start of oral intake, improved scar and body satis- faction and fewer incisional hernias [ , , , – ]. disadvantages besides the possible higher incidence of iaa are longer operating time and possibly increased costs [ , ]. to reduce the surgical trauma even more, new treatment strategies have been introduced such as single-incision laparoscopic surgery (sils) first reported by pelosi et al. [ ]. since then, numerous studies (rcts and sr) have been published on the potential advantages and disadvan- tages of the sils technique. it can be concluded that sils is associated with comparable post-operative morbidity rates compared to conventional la [ – ]. the disad- vantage is the fact that sils is a more difficult technique as is reflected by the higher technical failure rate, longer operating time and conversion rate [ – ]. main advan- tages of sils would be less post-operative pain and better cosmetic outcomes, although inconsistent results have been reported [ , , , – ]. at the present time, evidence is lacking that sils is superior to conventional la [ , , ]. sils is, however, a safe and feasible alternative. surg endosc ( ) : – recently, initial non-operative management of appen- dicitis has been investigated in the adult population. five rcts reported an effectiveness of – % at -year fol- low-up [ – ]. meta-analyses of these studies revealed that non-operative treatment of acute appendicitis is less effective but could avoid surgery in – % of patients [ – ]. opponents of this strategy raise concerns such as recurrent appendicitis, missing an underlying malignancy and progression of uncomplicated into complicated appendicitis. due to the possible avoidance of surgery with an initial non-operative treatment strategy, morbidity was diminished [ , , ]. however, both rcts and meta- analyses showed significant heterogeneity of methodolog- ical quality, studies included and definitions of outcome parameters. until higher qualitative evidence has been obtained regarding the potential benefits of initial non-op- erative management of acute appendicitis and the potential long-term effects have been investigated appropriately, appendectomy remains the gold standard in acute uncom- plicated appendicitis. complicated appendicitis due to the heterogeneity of the definitions used in the lit- erature, it is difficult to draw firm conclusions regarding the treatment of complicated appendicitis. in , dimitriou published a retrospective cohort study on patients with complicated appendicitis (defined as perforated with an abscess or peritonitis). they showed that la reduced the incidence of ssi, number of reoperations and length of hospital stay as compared to oa with no difference in iaa rate [ ]. a rct encompassing patients with clinically and histopathologically confirmed complicated appendici- tis showed similar outcomes after oa and la [ ]. it should be noted, however, that the incidence of iaa after la for patients with complicated appendicitis was reported fig. algorithm. *the cut-off values are based upon the study by ebell et al. [ ]. **one could consider performing additional imaging studies in patients with high probability based upon the alvarado score in order to reduce the negative appendectomy rate. ***ultra- sound should be performed as a first level diagnostic imaging study, although in specific patient groups (such as the obese) an immediate ct scan might be considered. ****in case of an inconclusive result from the ultrasound, we recommend that additional imaging studies should be performed. either a ct or mri is preferred although it is recommended to perform an mri in children and pregnant patients. it is therefore obligated to rule out pregnancy before a ct scan is obtained in a woman of reproductive age suspected of appendicitis. *****in case all the imaging studies are inconclusive, patients should be observed and reassessed. diagnostic laparoscopy should be reserved for those patients with a continuous high index of suspicion after reassessment. ******in case of low probability based upon the alvarado score, other diagnoses should be excluded and the patient can be either discharged with good instruction (with an optional reassessment the next day) or admitted for observation if the clinical condition mandates this. in case appendicitis is excluded, patients should be treated for the set diagnosis according to the local protocols surg endosc ( ) : – to be higher in some studies. tuggle and colleagues reported that la in patients with complicated appendicitis was associated with an incidence of iaa of . versus . % in patients who underwent an open appendectomy [ ]. the incidence of small bowel obstructions after la is lower compared to oa (pooled odds ratio . [ % ci . – . ] with large heterogeneity regarding follow-up period) [ ]. in case of a contained phlegmon or abscess (peri-ap- pendicular mass), some authors opt for non-operative treatment while others advocate aggressive operative treatment. in , andersson et al. [ ] demonstrated that immediate surgical treatment of patient with an abscess or phlegmon was associated with higher morbidity compared to initial non-operative treatment (or . % ci . – . ). similis et al. showed in their meta-analysis of studies regarding this specific patient group that non-op- erative treatment was associated with fewer complications (ssi, iaa and bowel obstructions). it must be mentioned that this meta-analysis was subject to large heterogeneity [ ]. recent cohort studies draw opposite conclusions [ , ]. they opt for a more aggressive surgical approach at time of presentation in case of an appendicular mass or appendicular abscess, based upon the idea that there is a relative high failure rate for non-surgical treat- ment [ , ]. in our opinion, with this new evidence, a new systematic review should be performed. until then, initial non-operative treatment of an appendicular mass of appendicular abscess is the preferred treatment of choice. although not covered in this consensus guideline, the value of interval appendectomy after initial non-operative treat- ment of an appendicular mass is still subject of debate. some opt for an interval appendectomy based upon the chance of missing an underlying and untreated malignancy (incidence %) and the chance of developing recurrent appendicitis (incidence – %) [ – ]. both can be avoided with an interval appendectomy, although data are lacking on its benefits. specific patient groups obese patients abdominal surgery in obese patients is challenging for both the anaesthesiologist and surgeon due to higher inci- dence of respiratory dysfunction, difficult access to the abdominal cavity, blurred anatomical landmarks and reduced working space in the abdominal cavity. clarke et al. [ ] performed a subgroup analysis among patients ( la and oa) with a bmi higher than kg/ m and reported similar morbidity after la and oa [ ]. this was confirmed by a meta-analysis, although a reduced length of hospital stay was noted after la [ ]. more recently, two recent meta-analyses showed a reduction of mortality and morbidity rates after la [ , ]. pregnancy pregnancy induces anatomical and physiological changes that challenge the surgeon. the potential effects of carbon dioxide and increased abdominal pressure during la on the foetus remain unclear. loss of the foetus is most feared. in , walsh et al. [ ] published a systematic review of laparoscopic appendectomies in pregnant patients and noted foetal loss in approximately % of the patients, with the highest incidence in patients with complicated appendicitis. another review confirmed these findings and reported a nearly twofold increase of foetal loss in the la group [ ]. both reviews, however, are mainly dominated by one study and based on low-grade evidence (retro- spective studies with small numbers of patients) [ – ]. recently, a review suggested that based upon the little available evidence no recommendation can be made regarding the preferred approach in pregnant patients [ ]. more studies are necessary to ascertain the role of laparoscopic surgery during pregnancy. until more evi- dence comes available, the surgical approach should be at the surgeon’s discretion. based upon expert opinion, we recommend laparoscopy in case of sufficient experience. although not supported by the literature, we strongly advise a multi-disciplinary approach to the pregnant patient with appendicitis [ , , , , ]. children one meta-analysis included , children with both uncomplicated and complicated appendicitis [ ]. laparoscopic appendectomy in children with uncomplicated appendicitis la was associated with a significant reduction of hospital stay with similar morbidity compared to open surgery. in children with complicated appendicitis, la was associated with lower rates of morbidity, ssi, length of hospital admission and bowel obstruction. however, laparoscopic surgeries lasted longer and were followed by more intra-abdominal abscesses [ ]. in more recent prospective cohort studies in children below years of age, la was associated with fewer complications [ ]. non- operative treatment of acute non-complicated appendicitis appears more promising in children than in adults [ , ] elderly elderly patients have higher morbidity, reduced physio- logical reserves and impaired inflammatory responses, surg endosc ( ) : – which increases their peri-operative risks. all studies of laparoscopic appendectomy in elderly support the use of laparoscopic surgery [ – ]. one meta-analysis, com- prising more than , patients reported that la reduced post-operative mortality ( . ; % ci . – . ), post- operative complications ( . ; % ci . – . ) and length of hospital stay (- . ; % ci - . to - . ) compared to oa (tables , , , ) [ ]. timing determining the best moment to perform surgery in case of acute appendicitis is of crucial importance [ , ]. acute appendicitis has been considered to be an irre- versible progressive disease although recent studies have questioned this dogma [ , , ]. nowadays, the idea is endorsed that two types of appendicitis exist: uncom- plicated (non-perforating) and complicated (perforating) appendicitis. the aetiology and pathogenesis of acute appendicitis remain largely unknown. predicting a mild or fulminant course of appendicitis is not possible. delaying an appendectomy increases the risk of perfo- rated appendicitis, which is associated with higher inci- dence of short and long-term morbidity [ – ]. hence, it is recommended to perform appendectomy as soon as possible. although it should be noted that some studies have revealed that the clinical outcome was not affected by time to surgery (when surgery was performed within h after presentation at the emergency depart- ment) [ , ]. antibiotic prophylaxis antibiotic prophylaxis has been proven effective in pre- vention of superficial surgical site infections and intra-ab- dominal abscesses in patients with appendicitis [ – ]. prophylaxis should be commenced at the time of estab- lishing the diagnosis of acute appendicitis. the choice of antibiotics is dependent on the local microbiome and drug resistance pattern and is not influenced by age. technique open access to the abdominal cavity as well as closed access using the veress needle are accepted techniques to perform laparoscopy [ – ]. the debate on the pre- ferred technique continues. however, in children, the majority of surgeons employs open establishment of a pneumoperitoneum. the placement of the camera port and the work ports depend on the anatomy of the patient and preference of the operating surgeon. primary principle of trocar placement in laparoscopy is that a triangular working space should be pursued. intra-operative procedure increased employment of pre-operative radiologic testing (e.g. ultrasound, ct or mri) in cases of suspected appen- dicitis has significantly reduced the incidence of a normal appearing appendix encountered during surgery [ ]. macroscopic distinction between a normal appendix and appendicitis during surgery can be difficult [ , ]. the ‘‘gold standard’’ for defining appendicitis is histopathology. in some studies, histopathological assessment revealed abnormal findings in up to % of macroscopically normal appearing appendices [ , ]. therefore, it is recom- mended to perform an appendectomy in case of a normal appearing appendix during surgery for suspected appendicitis. several studies have investigated the safety of different methods of securing the appendicular stump [ , – ]. none of the different closure methods has a clear advan- tage in case of a healthy appendix base. stapler devices provide the most standardized and patent closure of the appendix base. suturing of the appendix base provides sufficient closure as well, but is technically more demanding than other techniques [ ]. in case of perfo- ration of the appendicular base, clips or endoloops do not provide secure closure and staple devices or laparoscopic suturing is required [ ]. reduction of bacterial load by meticulous suction of intra- peritoneal fluids is advised [ – ]. the right paracolic and pelvic area should be inspected to leave no fluid col- lections behind. irrigation of the intra-peritoneal space in case of perforated appendicitis seems to be contra-produc- tive leading to a higher number of abscesses [ , ]. it is believed that irrigation of the intra-peritoneal space leads to spreading of bacteria. routine use of drains does not reduce the incidence of abscesses [ , ]. necessity of a drain for special indications is left to the discretion of the surgeon. intra-operative unexpected findings when an appendicular mass is encountered during surgery, one should restrain from continuing the operation. con- tinuation of the operation can necessitate bowel resec- tion. antibiotic treatment of phlegmon and drainage of any abscess should be performed [ , , ]. surg endosc ( ) : – the extent of surgical resection in case of suspected malignancy depends on the location and size of the appendicular mass [ – ]. routine inclusion of the meso-appendix with the appendectomy is advised. definitive histological findings determine whether an additional resection after total appendectomy is table pre-operative care: statements web survey loe level of evidence x means present, box means not present table pre-operative care: statements eaes meeting loe level of evidence x means present, box means not present surg endosc ( ) : – table pre-operative care: recommendations eaes meeting loe level of evidence, sor strength of recommendation x means present, box means not present surg endosc ( ) : – indicated. in cases of small neuroendocrine tumours (net) or low-grade appendicular mucinous neoplasms (lamn), a total meso-appendicular resection can be sufficient. in cases of a net [ cm, lamn grade – or an adenocarcinoma of the appendix, a formal right hemicolectomy is indicated to provide an oncologically sufficient resection. it is advised to perform a total meso- appendicular resection at the primary operation and an table pre-operative care: recommendations web survey loe level of evidence, sor strength of recommendation x means present, box means not present surg endosc ( ) : – additional hemicolectomy at a later stage when indicated (tables , , , ) [ – ]. post-operative antibiotics the incidence of ssi after appendectomy has been reported to range from to % [ – ]. the severity of appendicitis strongly influences the risk of developing post-operative complications resulting in a substantially higher complication rate (up to – times) in patients with complicated appendicitis. in this specific group, post- operative administration of antibiotics significantly redu- ces the rate of ssi. in addition, to reduce bacteraemia and sepsis, these patients are uniformly treated with a course of post-operative antibiotics [ – , ]. in uncomplicated appendicitis, there is no evidence sup- porting routine administration of post-operative antibi- otics. therefore, only one pre-operative dose is advised [ – ]. advice on type of antibiotics depends on local microbiome and resistance patterns and therefore should be left up to the discretion of the surgeon [ , ]. available evidence on duration of treatment is limited and mainly focused on children. however, there is no firm evidence on the duration ( , , , days) and route of administration (usually intravenous administra- tion for h, then oral administration) [ , , , , ]. post-operative complications the incidence of post-operative complications ranges from . to . % [ – ]. complications include small bowel obstruction ( – . %.), ssi ( . – . %), iaa ( . – %), stump leakage and stump appendicitis [ – ]. literature suggests a higher rate of complica- tions in complicated appendicitis [ , , , ]. literature on stump leakage and stump appendicitis is limited, and no exact incidences have been reported in the literature, although it is assumed that it is more table operative care: statements eaes meeting loe level of evidence x means present, box means not present table operative care: statements web survey loe level of evidence x means present, box means not present surg endosc ( ) : – common in patients with complicated appendicitis and after oa [ ]. a recommendation to avoid stump leakage or stump appendicitis is to resect the appendix as a whole [ ]. therefore, the stump should be no longer than . cm and caecal taenia should be followed onto the appendix at removal to ensure complete resection. stump appendicitis is significantly more associated with perforation, as diagnosis is delayed by misled attention. this is caused by the assumption that the appendix as a whole is resected. prevention is cru- cial. in case of timely diagnosis, stump resection with laparoscopic or open approach is feasible. in case of perforation, extended bowel resection is usually required [ ]. in the initial management of iaa after appendectomy conservative measures (i.e. non-operative with antibiotics) are effective in most patients. however, in case of lack of improvement or deterioration, a more invasive strategy table operative care: recommendations eaes meeting loe level of evidence, sor strength of recommendation x means present, box means not present surg endosc ( ) : – should be applied (percutaneous drainage or surgical (la- paroscopic) drainage) [ – ]. post-operative care the use of prophylactic anti-emetics diminishes the incidence of post-operative nausea and vomiting. increasing the diet is best determined by the patient’s ability to tolerate oral intake. there is no evidence that a liberal diet causes complications in the post-operative period [ , ]. post-operative pain management should follow local protocol for pain management after abdominal surgery. post-operative analgesia with pca provides effective and safe pain relief in children and adults and is less time costly [ ]. recently positive results have been pub- lished regarding the pre-emptive incision site infiltration with a local anaesthetic. studies demonstrated that this decreases the total opioid consumption and lowers pain table operative care: recommendations web survey loe level of evidence, sor strength of recommendation x means present, box means not present surg endosc ( ) : – score experienced by patients in the first h after sur- gery [ – ]. pathology carcinoid is the most commonly found neoplasm in appendectomy specimens at an incidence between . and . % [ – ]. other unexpected findings can be encountered in . – . % of patients, including: divertic- ulitis ( . %), tuberculosis appendix . %, endometriosis ( . %), adenocarcinoma (\ %) and mucinous cystade- noma ( . – . %) [ – ]. treatment of unexpected findings ranges from no further surgical treatment, to right hemi colectomy and even hyperthermic intra-peritoneal chemotherapy (hipec) in some cases [ – ]. even though the incidence of unexpected findings seems low, the actual number of patients is significant and correct diagnosis is crucial for adequate treatment (tables , , , ) [ – ]. discussion this eaes consensus development conference regarding the diagnosis and management of acute appendicitis resulted in statements and recommendations based upon the available evidence. results from this meeting led to this paper, which can be used as a guideline for surgeons treating patients with appendicitis. local guidelines, national guidelines and guidelines from scientific commu- nities regarding appendicitis were available but showed great heterogeneity [ , , ]. with this consensus meeting, we managed to gather experts from different european nations to compare and debate management of patients with acute appendicitis. this led to a consensus meeting in which of the statements and the majority of the members of the eaes supported recommendations. the transfer of knowledge between the member countries, the opportunity to discuss views and above all, the creation of a widely supported paper appears valuable. our list of topics was created by the coordinating team and expert panel and was thought to cover the most important topics in the field of acute appendicitis. despite local differences, the general idea within the consensus group on the management of patients with acute appen- dicitis was comparable. in some cases, differences of treatment strategies between members of the expert panel were due to available surgical supplies and finances. this is reflected for instance on the statements and recommenda- tions regarding sils and mri. however, we want to emphasize that in defining statements we refrained from stating specific procedures. we rather stated the general principles to follow. in this way, the results from this table after care: statements eaes meeting loe level of evidence x means present, box means not present table after care: statements web survey loe level of evidence x means present, box means not present surg endosc ( ) : – consensus guideline can also be applied in areas with limited resources. the methodology of a consensus guideline is always subject to discussion. in the literature, there are several ways to conduct consensus conferences [ , – ]. however, not one was suited for our situation. it was therefore decided to modify the delphi method, as described in the method section, in order to systematically evaluate each statement and recommendation [ – ]. we decided to finalize only those statements and recommen- dation with % or more consensus, which is the arbitrary cut-off value we selected. the results of both the web- based survey and the live voting at the eaes conference in bucharest are presented independently rather than com- bined to rule out any bias. as expected, small differences were noted between the several voting rounds. although supported by the experts, some statements and recom- mendations were not supported by the scientific commu- nity in both the web survey as in the bucharest meeting. the topics that were not supported were on accuracy of mri compared to ct, the application of sils, extensive work-up in the elderly and treatment strategy for immune compromised patients and the open access to the peritoneal cavity. explanations for these discrepancies might be related to local habits, experience and financial situation. of more interest are the discrepancies noted between the outcome in the web survey and during the bucharest meeting. discrepancies were noted on the topic of mri application in children, the preferred approach in pregnant patients and the use of local anaesthetics prior to incision. this can again be explained by the fact that local habits, experience, composition of the voting public and financial situation might influence the outcome. the question was raised if the web survey alone would be sufficient to reach a consensus for future meetings. limiting a consensus meeting to only the web survey would limit the time as well as the costs involved. moreover, a higher percentage of surgeons participated in the web survey. in our opinion, however, the integration of an actual face-to-face meeting in the consensus methodology raises more awareness, provides an opportunity to discuss views and encourages the transfer of knowledge eventually leading to the creation of a widely supported paper. the literature review was ended in december . no studies after that were integrated for the consensus meeting as this was decided in our methodology. therefore, new studies might have been conducted on some topics. future research should be focused on the laparoscopic appendec- tomy in pregnant patients, elucidating the value of mri in specific patient groups, evaluating the outcomes of initial non-operative treatment for both uncomplicated and com- plicated appendicitis, specific patient groups and the need for interval appendectomy. we therefore propose that these statements are updated on a regular basis. table after care: recommendations eaes meeting loe level of evidence, sor strength of recommendation x means present, box means not present surg endosc ( ) : – although some limitations can be identified in our methodology, we have integrated a new systematic method for a consensus meeting. in our opinion, this is the way forward and we need to efflorescence this method. reproducibility, involving members of the scientific com- munity and applicability are key components of a con- sensus meeting. we believe that only after evaluation of the general opinion within the eaes such guidelines should be put into order. in conclusion, the consensus meeting of the eaes resulted in several statements and recommendations regarding the diagnosis and management of appendicitis based upon available evidence and expert opinion and was supported by the european surgical community. it provides guidance to surgeons and surgical residents facing patients with acute appendicitis. funding this project was supported by a grant from the eaes. compliance with ethical standards disclosures dr. antoniou received personal fees from the eaes (including journal and publication committee) and from the euro- pean hernia society. dr. muysoms received grants personal fees and non-financial support from medtronic and johnson & johnson. he received grants and personal fees from b. braun and dynamesh. he received personal fees from bard davol, cousin biotech, wl gore@ass and dansac outside the submitted work. prof. dr. bonjer received grants and personal fees from johnson & johnson, applied medical, medtronic and olympus. he received personal fees from cook. all outside the submitted work. drs. defoort, dr. go, prof. dr. ozmen, dr. rhodes, drs. gorter, dr. eker, drs gorter-stam, drs. abis, drs. acharya, drs. ankersmit, drs. arolfo, drs. babic, prof. dr. boni, drs. bruntink, drs. van dam, drs. deijen, drs. delacy, drs. harmsen, drs. van den helder, dr. iordache, drs. ket, drs. papoulas, drs. straatman, drs. tenhagen, drs. turrado, dr. ver- eczkei, prof. dr. vilallonga and drs. deelder have no conflict of interest or financial ties to disclose. open access this article is distributed under the terms of the creative commons attribution . international license (http://creative commons.org/licenses/by/ . /), which permits unrestricted use, distri- bution, and 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published version: richard, ma, huan, t, ligthart, s, gondalia, r, jhun, ma, brody, ja, irvin, mr, marioni, r, shen, j, tsai, p-c, montasser, me, jia, y, syme, c, salfati, el, boerwinkle, e, guan, w, mosley, th, bressler, j, morrison, ac, liu, c, mendelson, mm, uitterlinden, ag, van meurs, jb, franco, oh, zhang, g, li, y, stewart, jd, bis, jc, psaty, bm, chen, y-di, kardia, slr, zhao, w, turner, st, absher, d, aslibekyan, s, starr, jm, mcrae, af, hou, l, just, ac, schwartz, jd, vokonas, ps, menni, c, spector, td, shuldiner, a, damcott, cm, rotter, ji, palmas, w, liu, y, smith, ja, deary, ij & bios consortium , 'dna methylation analysis identifies loci for blood pressure regulation', american journal of human genetics, vol. , no. , pp. - . https://doi.org/ . /j.ajhg. . . digital object identifier (doi): . /j.ajhg. . . link: link to publication record in edinburgh research explorer document version: peer reviewed version published in: american journal of human genetics general rights copyright for the publications made accessible via the edinburgh research explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. take down policy the university of edinburgh has made every reasonable effort to ensure that edinburgh research explorer content complies with uk legislation. if you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. download date: . apr. https://doi.org/ . /j.ajhg. . . https://doi.org/ . /j.ajhg. . . https://www.research.ed.ac.uk/portal/en/publications/dna-methylation-analysis-identifies-loci-for-blood-pressure-regulation(f -b be- a -adf -cc b e b ).html dna methylation analysis identifies loci for blood pressure regulation melissa a. richard, , ,* tianxiao huan, , , symen ligthart, , rahul gondalia, min a. jhun, jennifer a. brody, marguerite r. irvin, riccardo marioni, , , jincheng shen, pei-chien tsai, may e. montasser, yucheng jia, catriona syme, elias l. salfati, eric boerwinkle, , weihua guan, thomas h. mosley jr, jan bressler, alanna c. morrison, chunyu liu, , , michael m. mendelson, , , andré g. uitterlinden, joyce b. van meurs, bios consortium, oscar h. franco, guosheng zhang, , , yun li, , james d. stewart, , joshua c. bis, bruce m. psaty, yii-der ida chen, sharon l.r. kardia, wei zhao, stephen t. turner, devin absher, stella aslibekyan, john m. starr, , allan f. mcrae, , lifang hou, allan c. just, joel d. schwartz, pantel s. vokonas, cristina menni, tim d. spector, alan shuldiner, , coleen m. damcott, jerome i. rotter, walter palmas, yongmei liu, tomáš paus, , , steve horvath, jeffrey r. o'connell, xiuqing guo, zdenka pausova, , themistocles l. assimes, nona sotoodehnia, jennifer a. smith, donna k. arnett, ian j. deary, , andrea a. baccarelli, jordana t. bell, eric whitsel, , abbas dehghan, , daniel levy, , , myriam fornage , , ,* brown foundation institute of molecular medicine, university of texas health science center, houston, tx , usa; population sciences branch, national heart, lung, and blood institute, national institutes of health, bethesda, md , usa; framingham heart study, framingham, ma , usa; department of epidemiology, erasmus university medical center, rotterdam , the netherlands; department of epidemiology, university of north carolina, chapel hill, nc , usa; department of epidemiology, school of public health, university of michigan, ann arbor, mi , usa; cardiovascular health research unit, department of medicine, university of washington, seattle, wa , usa; department of epidemiology, university of alabama at birmingham, birmingham, al , usa; centre for cognitive ageing and cognitive epidemiology, university of edinburgh, edinburgh eh jz, uk; medical genetics section, centre for genomic and experimental medicine, institute of genetics and molecular medicine, university of edinburgh, edinburgh eh xu, uk; queensland brain institute, the university of queensland, brisbane , australia; department of biostatistics, harvard t.h. chan school of public health, boston, ma , usa; department of twin research and genetic epidemiology, kings college london, se eh london, uk; division of endocrinology, diabetes, and nutrition, program for personalized and genomic medicine, university of maryland school of medicine, baltimore, md , usa; institute for translational genomics and population sciences, departments of pediatrics and medicine, los angeles biomedical research institute at harbor-ucla medical center, torrance, ca , usa; hospital for sick children, university of toronto, toronto, ontario, canada m g a ; department of medicine, stanford university school of medicine, stanford, ca , usa; human genetics center, school of public health, university of texas health science center, houston, tx , usa; human genome sequencing center, baylor college of medicine, houston, tx , usa; department of biostatistics, university of minnesota, minneapolis, mn , usa; department of medicine, university of mississippi medical center, jackson, ms , usa; department of biostatistics, boston university, boston, ma , usa; department of cardiology, boston children's hospital, boston, ma , usa; department of internal medicine, erasmus university medical center, rotterdam , the netherlands; department of genetics, university of north carolina, chapel hill, nc , usa; curriculum in bioinformatics and computational biology, university of north carolina, chapel hill, nc , usa; department of statistics, university of north carolina, chapel hill, nc , usa; department of biostatistics, university of north carolina, chapel hill, nc , usa; carolina population center, university of north carolina, chapel hill, nc , usa; cardiovascular health research unit, departments of medicine, epidemiology, and health services, university of washington, seattle, wa , usa; group health research unit, group health cooperative, seattle, wa , usa; division of nephrology and hypertension, mayo clinic, rochester, mn , usa; hudsonalpha institute for biotechnology, huntsville, al , usa; alzheimer scotland dementia research centre, university of edinburgh, edinburgh eh jz, uk; institute for molecular bioscience, the university of queensland, brisbane, qld , australia; department of preventive medicine, feinberg school of medicine, northwestern university, chicago, il , usa; department of preventive medicine, icahn school of medicine at mount sinai, new york, ny , usa; department of environmental health, harvard t.h. chan school of public health, boston, ma , usa; veterans affairs normative aging study, veterans affairs boston healthcare system, department of medicine, boston university school of medicine, boston, ma , usa; the regeneron genetics center, regeneron pharmaceuticals, tarrytown, ny , usa; department of medicine, columbia university, new york, ny , usa; wake forest school of medicine, winston-salem, nc , usa; departments of psychology and psychiatry, university of toronto, toronto, ontario, canada m s g ; rotman research institute, baycrest, toronto, ontario, canada m a e ; child mind institute, new york, ny , usa; department of human genetics, gonda research center, david geffen school of medicine, university of california los angeles, los angeles, ca , usa; departments of physiology and nutritional sciences, university of toronto, toronto, ontario, canada m s a ; cardiovascular health research unit, division of cardiology, university of washington, seattle, wa , usa; university of kentucky, college of public health, lexington, ky , usa; department of medicine, university of north carolina at chapel hill, chapel hill, nc , usa; department of biostatistics and epidemiology, mrc-phe centre for environment and health, school of public health, imperial college london, london w pg, uk; these authors contributed equally to this work. * correspondence: melissa.a.lee@uth.tmc.edu (m.a.r), myriam.fornage@uth.tmc.edu (m.f.) abstract genome-wide association studies have identified hundreds of genetic variants associated with blood pressure (bp), but sequence variation accounts for a small fraction of the phenotypic variance. epigenetic changes may alter the expression of genes involved in bp regulation and explain part of the missing heritability. we therefore conducted a two-stage meta-analysis of the cross-sectional associations of systolic and diastolic bp with blood-derived genome-wide dna methylation measured on the infinium humanmethylation beadchip in , individuals of european, african american, and hispanic ancestry. thirteen of discovery stage cytosine-phosphate-guanine (cpg) dinucleotides replicated after bonferroni correction (discovery: n= , , p value< . x - ; replication: n= , , p value< . x - ). the replicated methylation sites are heritable (h > %) and independent of known bp genetic variants, explaining an additional . % and . % of the interindividual variation in systolic and diastolic bp, respectively. bidirectional mendelian randomization among up to , individuals of european ancestry from four cohorts suggested that methylation at cg (taf b-ywhaq) influences bp, while bp influences methylation at cg (zmiz ), cg (cpt a), and cg (slc a ). gene expression analyses further identified six genes (tspan , slc a , unc b , cpt a, ptms, and lpcat ) with evidence of triangular associations between methylation, gene expression, and bp. additional integrative mendelian randomization analyses of gene expression and dna methylation suggested the expression of tspan is a putative mediator of association between dna methylation at cg and bp. these findings suggest that heritable dna methylation plays a role in regulating bp independently of previously known genetic variants. introduction elevated blood pressure (bp) confers a higher risk of heart disease, stroke, diabetes, dementia, renal failure, and pregnancy-related complications and is a leading risk factor for death worldwide. bp is a highly heritable trait and recent genetic studies have revealed part of its complex genetic architecture, – yet the genetic variants identified to date only account for a small fraction of its phenotypic variance. , , , complex phenotypes, such as bp, often result from the interplay between genetic and environmental influences. dna methylation, the covalent binding of a methyl group to the ’ carbon of cytosine-phosphate-guanine (cpg) dinucleotide sequences in the genome, plays a critical role in the regulation of gene expression and may reflect a link between genes, environment, and complex phenotypes such as bp. evidence is beginning to emerge that epigenetic modifications in genes relevant to bp may account for part of its regulation. variation in dna methylation may thus explain additional phenotypic variation in bp and provide new clues to the biological processes influencing its regulation. we conducted genome-wide dna methylation meta-analyses for systolic and diastolic bp with a discovery phase and independent replication among , individuals of european (ea), african american (aa), and hispanic ancestries in the cohorts for heart and aging research in genomic epidemiology (charge) consortium. dna methylation was measured in peripheral blood samples. we further sought to identify transcriptional changes for the replicated cpg sites and used mendelian randomization techniques to explore the causal relationship between dna methylation and bp. we report that the effect of dna methylation on bp is likely independent of previously known genetic variants, representing new insights into the biological mechanisms underlying bp regulation. material and methods study populations the discovery and replication studies were conducted in the framework of the cohorts for heart and aging research in genomic epidemiology (charge) consortium, which comprises multiple population- based cohort studies. cohorts participating at the discovery stage included , individuals of ea and aa ancestries in the atherosclerosis risk in communities (aric) study, cardiovascular health study (chs), framingham heart study (fhs), genetic epidemiology network of arteriopathy (genoa) study, genetics of lipid lowering drugs and diet network (goldn) study, lothian birth cohort (lbc ), normative aging study (nas), rotterdam study (rs), and twinsuk registry. cohorts participating at the replication stage consisted of , additional individuals of ea, aa, and hispanic ancestries in the amish complex disease research studies (amish), aric, the multi-ethnic study of atherosclerosis (mesa), rs, adults in the saguenay youth study (sys), and the women’s health initiative (whi). details for each cohort are provided in the supplementary material. all studies obtained written informed consent from participants and were approved by local institutional review boards and ethics committees. blood pressure measurements epigenome-wide association studies (ewas) were conducted for systolic and diastolic bp, in mmhg. in each cohort bp was measured in a sitting position after a period of rest and an average of sequential readings was used as the phenotype for each analysis. for most cohorts bp was measured concurrently at the time of tissue collection for dna methylation profiling, or in as close proximity as available for twinsuk ( . years) and sys adults ( . years). to adjust for the use of antihypertensive medication, we used the standard adjustment of adding mmhg and mmhg to measured systolic and diastolic bps, respectively, when the use of any antihypertensive medications were self-reported. dna methylation profiling dna methylation was measured on the infinium humanmethylation ( k) beadchip (illumina inc, san diego, ca) in all cohorts using whole blood samples, excepting that goldn measured dna methylation in cd + t-cells. to correct the beta value distributions of the two types of probes on the k array, each cohort normalized methylation beta values using bmiq, dasen, combat, swan, or quantile normalization. lbc did not normalize methylation beta values prior to analyses. cohort-level association analyses in each cohort, race-stratified linear mixed effect models were used to estimate associations adjusting for age, sex (in samples including men and women), blood cell counts, body mass index, smoking (current/former/never), and ancestry, as well as fixed and/or random effects for technical covariates to control for batch effects. surrogate variables were calculated and adjusted in the modeling for aric aas and fhs due to batch effects not controlled by other modeling techniques. the amish, fhs, goldn, and twinsuk accounted for sample relatedness in all analyses. study-specific modeling details can be found in the supplement. epigenome-wide meta-analyses effect estimates from all cohorts were combined using inverse variance fixed effects meta-analysis using gwama. we assessed heterogeneity of effect estimates between strata of races, sexes, and methylation tissue source among discovery cohorts using a degree of freedom chi-square test for effect differences between strata; no heterogeneous effects were observed so all cohorts were included in a single meta-analysis. meta-analyses were conducted separately for the discovery and replication cohorts to identify probes associated with bp. statistical significance was bonferroni-corrected for the epigenome-wide discovery meta-analysis (p value< . x - ) and the number of discovery cpg sites sought for replication in the second meta-analysis. an overall meta-analysis was additionally performed to combine effect estimates across all cohorts. significant cpg sites were annotated using information provided by illumina, including chromosome, position (grch /hg ), ucsc gene names, relationship to cpg islands, location in gene enhancer regions, and dnase i hypersensitivity sites (dhs). to assess the impact of antihypertensive medication use on our top findings, we additionally performed an overall meta-analysis among all individuals reporting no use of antihypertensive medications. for the top findings in the discovery meta-analysis, we compared effect and standard error estimates to those estimated in the non-medicated meta-analysis. percent variance explained percent variance explained was calculated in the aric aa and ea samples included in discovery and replication meta-analyses, as well as validated in a sample from the fhs third generation not included in the meta-analysis (n= , ). methylation profile scores for bp were calculated as the weighted sum of cpg sites significant for either bp trait in the replication and overall meta-analyses, with weights coming from the magnitude and direction of effects in the overall meta-analysis. selection of cpgs from meta-analyses including the prediction samples could overestimate percent variance explained, so additional meta-analyses were conducted excluding the aric samples to identify cpgs for their respective methylation profile scores. the probe sets based on exclusion of the aric samples and the probes identified in the primary replication and overall meta-analyses were used to generate methylation profile scores in the fhs sample. race- and cohort-stratified linear regression models were used to estimate the percent of age-, sex-, and bmi-adjusted systolic and diastolic bp variances explained by each methylation profile score; aric models were additionally adjusted for visit and study site, and aric aa and fhs models included surrogate variables. percent variance explained by the methylation profile scores is reported as the adjusted r from each model and compared to models without methylation profile scores (covariate-only models). we additionally assessed genetic risk scores derived using effect estimates from the uk biobank for previously reported independent variants (r < . ) and validated novel variants among the fhs third generation sample with available genetic data (n= , ). heritability the narrow-sense heritability estimate of a dna methylation trait (β score) (denoted as ℎ𝐶𝑝𝐺_𝑚𝑒𝑡ℎ𝑦 ) was the proportion of the additive polygenic genetic variance of the total phenotypic variance of a dna methylation trait: ℎ𝐶𝑝𝐺_𝑚𝑒𝑡ℎ𝑦 = σa /σ𝐶𝑝𝐺_𝑚𝑒𝑡ℎ𝑦 , where σa denotes the additive polygenic genetic variance and σ𝐶𝑝𝐺_𝑚𝑒𝑡ℎ𝑦 denotes the total phenotypic variance of a dna methylation trait. heritability estimation for all dna methylation traits was performed using the fhs-offspring participants (n= , ). functional tissue and gene set enrichment analyses functional dna elements regulated by methylation may be tissue specific, so the set of replicated cpgs was used to identify tissue and cell type specific signals using experimentally-derived functional element overlap analysis of regions from ewas (eforge). after pruning results for cpg sites within kb ( probes removed), we matched the top eleven ewas signals for overlap with dnase i hypersensitive sites using data from encode and roadmap epigenomics. one thousand matched sets were used with the k array as the background set. fdr correction was applied to the results. gene set enrichment analysis (gsea) was conducted on the results of the overall meta-analyses for systolic and diastolic bp. for each gene annotated to dna methylation measured on the k array, a composite ranking for bp was generated based on the cpg site with the minimum p value for either trait. all gene ontology biological process categories (c .bp.v . ) were assessed for enrichment at fdr q< . . methylation quantitative trait loci to determine methylation levels at cpg sites that may be influenced by nearby dna sequence, methylation quantitative trait loci (meqtl) analyses were performed for the replicated bp cpgs in ea individuals from aric (n= ), fhs (n= , ), and rs (n= ) and aa individuals from aric (n= , ) and genoa (n= ). residuals were obtained from regressing inverse-normal transformed methylation beta values on the first ten methylation principal components (pcs) and up to the first ten genetic pcs. the residuals were then regressed on genomes phase i imputed snps within kb of the probe (cpg position ± kb, grch /hg ). snps with low imputation quality (r < . ), low frequency variants (maf < . ), and snps present in only one cohort were removed from analyses. results for each probe were combined using race-stratified p-value-based meta-analysis weighted by sample size and direction of effects using metal. significant meqtls were determined using a bonferroni correction for all meqtls tested in each race (ea: . / , = . x - ; aa: . / , = . x - ). to maximize statistical power for identifying meqtls associated with bp, we then searched the largest genome-wide association studies (gwas) for bp in each race for suggestive association of meqtl regions with bp. to assess the association of snps reported by kato et al whose association may be mediated by dna methylation we additionally performed meqtl analyses for sentinel snps and additional gwas loci in high linkage disequilibrium (ld) with these regions. , , – we assessed the association of dna methylation within mb (cpg position +/- kb) of gwas snps among aric eas (n= ) using the previously described methodology. snps associated with methylation after bonferroni correction for the meqtls reported by kato et al (p value< . ) were then assessed for association with bp before and after adjustment for methylation at the cpg site. we additionally assessed the association of these cpg sites with bp in our overall meta-analysis. bidirectional mendelian randomization to assess the directional association of dna methylation and bp, we conducted bidirectional mendelian randomization (mr) using genomes imputed snps among ea individuals in aric, fhs, rs, and whi- empc (n= , ). forward mr was used to identify replicated cpg sites which may have an effect on bp. instrumental variables (ivs) for dna methylation were drawn from the meqtls estimated among eas and pruned for independence (r < . ). forward mr was conducted for the six sentinel cpg sites with at least three independent meqtls, which is the minimum number of ivs needed to perform multi- instrument mr. reverse mr was used to identify dna methylation at the sentinel cpg sites that may be caused by bp. the independent loci reported as associated with bp by the international consortium for blood pressure (icbp) were selected as ivs. the snp rs was not imputed in genomes and rs was used as a proxy when available (r = . in hapmap). each cohort estimated the associations of ivs with systolic bp, diastolic bp, and dna methylation at the respective cpg sites. cohort-level effect estimates for each iv were combined using inverse variance- weighted meta-analyses in metal. for each cpg in forward and reverse mr, causation was formally tested based on the inverse variance-weighted effects across all iv-bp and iv-cpg estimates using the r package mendelianrandomization. tests for causation with p value< . were considered significant. to ensure the validity of the inverse-variance weighted approach, the ivs were assessed for pleiotropy using the mr-egger test. inverse-variance weighted mr is invalid in the presence of pleiotropic effects of ivs, so egger regression estimates of causality were assessed only when pleiotropy was indicated at a particular cpg site. associations of dna methylation and gene expression association tests of bp-associated cpgs with transcripts that were located within +/- mb distance of the corresponding cpgs were performed in , fhs-offspring samples and rs samples whose dna methylation and gene expression data were both available. in fhs, linear mixed effect regression models were used with dna methylation β scores as the dependent variable, gene expression as independent variables, age, sex, and technical covariates as fixed effects, and family structure as a random effect. in rs, we first created residuals for both dna methylation and mrna expression after regressing out age, sex, blood cell counts (fixed effect) and technical covariates (random effect). we then examined the association between the residuals of dna methylation (independent variable) and mrna expression (dependent variable) using a linear regression model. estimates of the gene expression-methylation associations in rs and fhs were combined using sample size weighted fixed effects meta-analysis based on p-values and direction of effects using gwama. associations of gene expression and bp differential gene expression analysis of the transcripts assessed for association with dna methylation were performed for systolic bp, diastolic bp, and hypertension in , fhs offspring and rd-gen participants who were not receiving anti-hypertensive treatments. hypertension was defined as systolic bp ≥ mmhg or diastolic bp ≥ mmhg. see details in huan et al. two-step mendelian randomization for relationship of dna methylation, gene expression, and bp to identify gene transcription that functionally mediates the relationship of dna methylation and bp, we performed a two-step mr technique for genes with expression associated with both dna methylation and bp (fdr q value< . ). the first step was to establish a directional relationship between dna methylation and gene transcription. ivs for dna methylation were drawn from estimated meqtls pruned to be independent (r < . ). using whole blood eqtls estimated in the genotype-tissue expression (gtex) project, we verified the association of each iv with the implicated gene expression. in the second step, ivs for each implicated gene were selected from the gtex whole blood dataset in order to establish a directional relationship between gene expression and bp. the top eqtl also present in the icbp results was selected as the iv for each gene and assessed for association with systolic and diastolic bp in icbp published gwas results. genes with p value< . at both steps were considered to mediate a directional relationship of the respective cpg and bp; correction for multiple testing is not used as strong associations of ivs with an outcome would violate the assumptions of mendelian randomization. results cohort characteristics characteristics of the studies participating in discovery and replication meta-analyses are presented in table . each cohort included middle-aged and older adults with a wide range of bp values. mean systolic bp ranged from mmhg in goldn to mmhg among chs aas. mean diastolic bp ranged from mmhg in goldn to mmhg in the rs replication sample. prevalence of antihypertensive medication use varied with cohort age and health, with no use among the amish to more than % among the chs aa sample. identification of epigenome-wide cpg sites associated with blood pressure in the discovery stage, we conducted genome-wide associations of dna methylation with systolic and diastolic bp in nine cohort studies (n= , ). multiethnic meta-analyses identified methylation at cpg sites associated with bp after bonferroni correction for the number of dna methylation cpg sites measured on the illumina k array (p value< . x - ; table s , figures s and s ). replication of the discovery cpg sites was sought in multiethnic meta-analyses of an additional six cohort studies (n= , ). methylation at of the discovery cpg sites was associated with bp at p value< . in the replication meta-analysis ( . / ; table ). a schematic of the overall study design, including subsequent integrative analyses, is found in figure s . the top two cpg sites for both systolic and diastolic bp were at the phgdh locus, cg (systolic bp: coefficient= . % decrease in dna methylation per mmhg increase in bp, p value= . x - ; diastolic bp: coefficient= . % decrease in dna methylation per mmhg increase in bp, p value= . x - ), and the slc a locus, cg (systolic bp: coefficient= . % decrease in dna methylation per mmhg increase in bp, p value= . x - ; diastolic bp: coefficient= . % decrease in dna methylation per mmhg increase in bp, p value= . x - ). cg is located on chromosome p in the first intron of phgdh, which encodes a phosphoglycerate dehydrogenase that catalyzes the rate-limiting step of serine biosynthesis. located on chromosome q . , cg is in the first intron of slc a , which encodes a sodium-independent cysteine/glutamate antiporter. all replicated cpg sites demonstrated associations of decreased dna methylation with increases in bp (table s and figure s ). none of the replicated cpg sites cross-hybridize with sequence variation on the sex chromosomes and one cpg, slc a cg , is polymorphic. an additional cpg site in slc a , cg , was also associated with bp and not polymorphic, so we did not exclude cg from our results. narrow-sense heritability estimates of the replicated cpg sites are moderate to high (h = - %) relative to all epigenome-wide probes (average h = %; table ). four of the replicated cpg sites are in dnase i hypersensitivity sites and enhancer regions (table s ). in phgdh and slc a we identified two nearby cpg sites in each gene associated with bp. we regard cg as the sentinel cpg site in phgdh and cg as the sentinel cpg site in slc a due to the strength of association p value with bp. methylation levels at the two cpg sites in phgdh were strongly correlated (aa and ea ρ= . ), whereas the two cpg sites in slc a were only modestly correlated (aa: ρ = . , ea: ρ= . ; figure s ). heterogeneity (cochran’s q) that may be attributable to cell type or race was observed in the discovery panel for slc a cg (table s ), however, estimates in the replication panel for this cpg site were homogeneous with the same direction of effect and similar magnitude of association p value as in the discovery meta-analyses (table ). all other reported cpg sites showed homogeneous effects in discovery and replication meta-analyses. we additionally conducted an overall meta-analysis of the discovery and replication cohorts and identified cpg sites associated with bp after bonferroni correction (p value< . x - ; table s ). to assess the effects of antihypertensive medication use, we performed epigenome-wide meta-analyses among the , individuals reporting no concurrent use of antihypertensive medications in the discovery and replication samples. this combined sample free from antihypertensive medication use is of comparable size to the discovery meta-analysis. we did not identify a large difference in effect estimates among the discovery cpg sites that met our strict replication standards (figure s ). many replicated cpgs were also epigenome-wide significant in the non-medicated analysis and three cpg sites on chromosome p . were identified that were not significant in the discovery stage (table s ). these cpg sites map to the first intron of pfkfb , which encodes a glycolytic enzyme. percent variance explained a methylation profile score based on the replicated cpg sites explained an additional . % and . % of the interindividual variation in systolic and diastolic bp, respectively, beyond the traditional bp covariates of age, sex, and bmi in an additional sample set from the fhs (n= , , third generation cohort) not included in the discovery or replication meta-analyses (figure ). expanding the dna methylation risk score to include the cpg sites that were significant in the overall meta-analysis did not explain additional phenotypic variance in samples of either ancestry. up to bp-associated genetic variants explained minimal variance in the fhs third generation sample set (n= , ; pve= . % - . %). we elected to only report percent variances explained for methylation risk scores since our estimates are independent of the distally-located known genetic loci. functional tissue and gene set enrichment analyses tissues enriched for dnase i hypersensitive sites in regions of the replicated cpgs include blood cells, vascular tissues, brain tissues, and cardiac tissues (figure s ). gene set enrichment analysis (gsea) was conducted for intragenic cpg sites identified in the overall meta-analyses for systolic and diastolic bp. dna methylation associated with either bp trait mapped to genes involved in the transport of neutral amino acids (fdr q= . ; figure s ). the transport of neutral amino acids was also identified as significantly enriched in the individual meta-analyses for systolic and diastolic bp (fdr q< . ). forty- three biological processes reached fdr q< . including brain development, hematopoietic or lymphoid organ development, and the transport of amino acids and amines. methylation quantitative trait loci we assessed genetic determinants of dna methylation at the replicated cpg sites in , ea individuals and , aa individuals in aric, fhs, genoa, and rs. nine of the cpg sites demonstrated substantial evidence for methylation quantitative trait loci (meqtls) in both ancestries (ea p value< . x - , aa p value< . x - ), with evidence for weak meqtls at one additional cpg site in each ancestry (figure ). we confirmed our estimated ea meqtls in an independent ea dataset published by aries and found almost all estimated meqtls were significant or in linkage disequilibrium (r > . or d’= ) with aries meqtls. we assessed the association of ea meqtls with bp in genomes analysis by the international consortium for blood pressure (icbp) that is yet to be published. seven of the ten cpgs demonstrated nominal association with systolic or diastolic bp ( . >p value> . x - ; table s ). the strongest association with both systolic and diastolic bp was observed at rs for phgdh cg and cg (systolic p value= . ; diastolic p value= . ). though association of exposure snps can serve as an indication of causality, we chose to formally test causality using multi-instrument mendelian randomization, as follows, due to the complex genetic architecture of both dna methylation and bp. bidirectional mendelian randomization dna methylation can be the cause or consequence of complex phenotypes. to provide support for causal relationships between dna methylation and bp, we conducted bidirectional mendelian randomization among up to , ea individuals in aric, fhs, rs, and whi-empc. we used inverse- variance weighted tests to assess both forward causal roles of dna methylation on bp and reverse causation where bp influences dna methylation. for the six sentinel cpg sites with multiple genetic determinants, we were able to test forward causality using independent meqtls as the instrumental variables. the mean causal effect estimated across its seven independent meqtls suggests that methylation at cg (taf b-ywhaq) influences bp (causal effect estimate = . ( . ) change in systolic bp, p value= . , and . ( . ) change in diastolic bp, p value= . , per one-percent change in dna methylation; table ). there is also some evidence for reverse causation at cg (diastolic bp p value= . ), however, the causal p-values for both bp traits are smaller for, and thus favor, forward causation. we performed an additional mendelian randomization using bp effect estimates from icbp and confirmed a causal relationship of methylation at cg with bp (systolic bp p value= . ; table s ). we assessed reverse causation for sentinel cpg sites using independent gwas loci as instrumental variables to estimate the mean causal effect of bp on dna methylation. in the absence of pleiotropic effects, inverse-variance weighted tests suggest that dna methylation at cg (systolic bp p value= . , diastolic bp p value= . ) and slc a cg (systolic bp p value= . , diastolic bp p value= . ) is influenced by bp (table ). reverse causation at both cg and slc a cg is also supported by the lower-powered egger test for causality (table s ). additionally, tests for causality of the second cpg in slc a , cg support reverse causality at this locus (diastolic bp p value= . ; table s ). the significant reverse causal effect estimates are consistent in magnitude and direction with those estimated by our ewas. we additionally identified significant pleiotropic effects of the instrumental variables with methylation at cg and diastolic bp (p value= . ; table s ). pleiotropy overpowers the inverse-variance weighted test, and we did not identify a causal effect at cg using egger regression (p value= . ; table s ). there was a significant test result for forward causation at cg ; however, there also was evidence of pleiotropic effects among the forward instrumental variables and both the inverse-variance weighted and egger tests favored reverse causality (table s ). we also identified an effect of diastolic bp on dna methylation at cg using egger regression (p value= . ) in the presence of pleiotropic instrumental variables (table s ). using mendelian randomization we demonstrate that complex phenotypes can have an effect on dna methylation among top ewas signals and that forward causality can be assessed when instrumental variables are available. gene expression associations with replicated cpg sites and blood pressure traits in whole blood gene expression analyses, four of the replicated cpg sites were found to have one or more cis-located genes (tspan , slc a , unc b , cpt a, ptms, and lpcat ) where transcription levels are associated with both cpg methylation (fhs and rs, n= , ) and systolic bp, diastolic bp, or hypertension (fhs, n= , ; tables and s ). the direction of effects for all six gene transcripts was consistent with the negative associations of bp with dna methylation at each cpg (tables and ). the mrna expression of tspan showed the strongest associations with both cpg methylation and bp among all transcripts tested. methylation at cg , located in the first intron of tspan , was strongly associated with decreased expression of tspan in blood (p value= . x - ) and expression levels were associated with systolic bp (p value= . e- ), diastolic bp (p value= . x - ), and hypertension (p value= . x - ). we identified nominal triangular associations of gene expression levels with methylation at of the replicated cpg sites (p value< . ) and at least one bp trait (p value< . ) and present estimates of association and correlation in table s . these genes include ywhaq (cg and diastolic bp), ppif (cg and diastolic bp), and grlf (cg /cg and diastolic bp). two-step mendelian randomization for genes mediating the bp-dna methylation association we used two-step mendelian randomization to characterize causal mediation by gene transcripts significantly associated with methylation and bp. using expression data available from the genotype- tissue expression (gtex) project and bp gwas from icbp, we first sought to establish a directional relationship from dna methylation to gene expression, then a directional relationship from gene expression to bp. we showed that independent snps associated with methylation at cg are associated with expression of tspan and an additional independent variant associated with tspan expression in blood is associated with bp (table ). the instrumental variables used for the exposures in each step achieved the associations needed to establish causality without showing strong evidence of association with the outcome that would invalidate the causal test (generally, . >p value> x - ). using two-step mendelian randomization, we demonstrated that tspan expression in whole blood is influenced by methylation at cg and that tspan expression affects diastolic bp (figure ). taken together, the direction of causality and our epigenome-wide estimate of association suggest that diastolic bp may increase by mmhg for every . % decrease in dna methylation at cg . discussion in a two stage design of discovery and replication meta-analyses comprising , individuals, we identified dna methylation at cpg sites located in eight intragenic regions and three intergenic regions significantly associated with systolic or diastolic bp. these cpgs are heritable, which suggests dna methylation that is the cause or consequence of bp may have a transgenerational effect. we identified substantial cis-located genetic variation associated with methylation at many of these sites in both ea and aa populations, and these regions have moderate genetic associations with bp but are independent of the top gwas loci previously reported in either race. through cis gene expression analyses, we identified four cpgs significantly associated with one or more genes that may functionally connect dna methylation and bp. mendelian randomization techniques characterized the direction of association for six cpg sites with bp, including the mediation of a causal relationship of cg with bp through expression of tspan . through the analysis of dna methylation, we have identified genes that provide insight into the biological mechanisms underlying bp regulation and target genes possibly affected by bp-induced dna methylation. we identified expression of tspan to influence bp via dna methylation. tspan encodes the tetraspanin protein that is involved in signal transduction. tspan is highly expressed in vascular tissues and implicated in the contractile ability and differentiation of vascular smooth muscle cells. sequence variation mapped to tspan has previously been associated with large artery atherosclerosis- related stroke and migraine , and tspan suppresses inflammation in the central nervous system . we additionally identified dna methylation at cg to affect bp and the transcription of ywhaq has a suggestive triangular relationship. ywhaq encodes a - - theta protein involved in signal transduction by binding to phosphoserine-containing proteins. ywhaq has been implicated in phenotypes related to vascular response through transcriptional and dna methylation changes in human preeclamptic placental tissues, dna sequence variation associated with exercise heart rate response , and an effect on cardiomyocyte survival in animal models. we identified an effect of bp on dna methylation at four of the replicated cpg sites: zmiz cg , cpt a cg , and slc a cg /cg . previous epigenome-wide association studies have identified relationships of cpt a cg and slc a cg /cg with other metabolic phenotypes, particularly lipids and adiposity (table s ). an effect of triglycerides on methylation at cg has previously been identified, which supports our hypothesis that an underlying cardiometabolic disease process related to bp and lipids alters dna methylation within cpt a. transcriptional changes caused by dna methylation at these four cpg sites could affect the risk of downstream phenotypes. a recent gwas identified of sentinel snps to have methylation-mediated associations with bp using a mendelian randomization technique. in our overall meta-analyses, we assessed the association of the cpg sites reported by kato et al, but were not able to confirm the direct association of any of these cpg sites with bp (p value> . x - ; table s ). we further could not confirm the association of the sentinel snps with the cpg sites reported to mediate the associations with bp. however, we identified an association of rs (clcn ) with methylation at an upstream cpg, cg (draxin). this cpg site has a nominal association with bp (systolic bp p value= . , diastolic bp p value= . ) and adjustment for methylation at this locus attenuated the association of rs with systolic bp but not diastolic bp (table s ). diastolic bp was the primary phenotype identified by kato et al. differences between our findings and those reported by kato et al. may be due to differing linkage disequilibrium structure in the populations examined in the two studies. the strength of this study is our use of multiple data sources and analytic techniques to characterize functional relationships of dna methylation and bp. we used strict replication standards to identify cpg sites associated with bp and characterized gene transcriptional changes that could underlie these associations. however, these cpg sites were identified through cross-sectional analyses and we were only able to characterize the direction of association for six cpg sites. mendelian randomization requires exposure-predictive snps to be selected as instrumental variables. for three cpg sites we estimated no significant cis-meqtls (cg , cg , and cg ) and an additional two cpg sites had insufficient independent meqtls to assess forward causation using multiple instrumental variables (cg and cg ), so we lacked the ability to perform causal tests for select cpg sites. we additionally a priori chose to examine gene expression mb up- and downstream of each cpg site in whole blood, so more distal regulatory effects, and effects in different tissues, could have been missed. however, we did identify at least one biologically plausible gene to have a nominal triangular association with both methylation and bp for eleven of the replicated cpg sites, so it may be that larger sample sizes will uncover the functional and causal relationships of dna methylation and bp. despite these limitations, we discovered heritable cpg sites associated with bp among , individuals and showed that these cpgs explain additional phenotypic variance in an independent sample in which up to bp- associated genetic variants explained minimal trait variance. we characterized the methylation-bp relationship using gene expression analyses and mendelian randomization techniques to both understand which genes may regulate bp and how bp may affect gene transcription. in conclusion, our genome-wide analysis of dna methylation has uncovered loci influencing bp variation independently of underlying genetic variation. we additionally characterized functional and causal relationships connecting methylation at these loci with bp. in particular, we have identified tspan as a candidate gene for bp that is regulated by heritable dna methylation. tspan and other methylated regions point to vascular contractility and inflammatory processes that functionally and causally connect dna methylation and bp, and, thus, may represent new targets for the treatment and prevention of hypertension. additional studies are needed to provide further mechanistic insights into the environmental exposures and genetic variation that influence dna methylation and lead to high blood pressure. nonetheless, our findings suggest that information on dna methylation is likely to yield additional insights into the pathobiology of complex traits. supplemental data supplemental data include figures, tables, and additional material and methods, including funding acknowledgements. conflicts of interest o.h.f. works in erasmusage, a center for aging research across the life course funded by nestlé nutrition (nestec ltd.), metagenics inc., and axa; a.s. is the vice president and co-head of the regeneron genetics center, llc, a fully owned subsidiary of regeneron pharmaceuticals, inc; j.r.o’c. serves as a consultant for regeneron; all other authors declare no conflicts of interest. bios consortium members bastiaan t. heijmans, peter a. c. ’t hoen, joyce van meurs, aaron isaacs, rick jansen, lude franke, dorret i. boomsma, rené pool, jenny van dongen, jouke j. hottenga, marleen m. j. van greevenbroek, coen d. a. stehouwer, carla j. h. van der kallen, casper g. schalkwijk, cisca wijmenga, alexandra zhernakova, ettje f. tigchelaar, p. eline slagboom, marian beekman, joris deelen, diana van heemst, jan h. veldink, leonard h. van den berg, cornelia m. van duijn, albert hofman, andré g. uitterlinden, p. mila jhamai, michael verbiest, h. eka d. suchiman, marijn verkerk, ruud van der breggen, jeroen van rooij, nico lakenberg, hailiang mei, maarten van iterson, michiel van galen, jan bot, peter van ’t hof, patrick deelen, irene nooren, matthijs moed, martijn vermaat, dasha v. zhernakova, rené luijk, marc jan bonder, freerk van dijk, wibowo arindrarto, szymon m. kielbasa, morris a. swertz and erik w. van zwet acknowledgements we thank all participants for contributing to this study. see supplement for detailed study acknowledgements and funding. references . campbell, n.r., khalsa, t., world hypertension league executive:, lackland, d.t., niebylski, m.l., nilsson, p.m., redburn, k.a., orias, m., zhang, x.-h., international society of hypertension executive:, et al. 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( ). blood lipids influence dna methylation in circulating cells. genome biol. , . figure titles and legends figure percent variance explained by traditional covariates and methylation profile scores for systolic and diastolic bp. the plot presents adjusted r values from covariate-adjusted models including a methylation profile risk score based on methylation cpg sites identified to be associated with bp in the overall and replication meta-analyses. the number of cpg sites included in the methylation profile scores is indicated as n. percent variance explained for the cpg sites identified in the primary replication and overall meta-analyses was calculated among an independent sample from fhs. the two aric samples participating in the discovery and replication stages were excluded from meta-analyses used to identify cpgs for their respective methylation risk scores, which caused the sets of methylation sites to differ. abbreviations: aa, african american; aric, atherosclerosis risk in communities; bp, blood pressure; cpg, cytosine-phosphate-guanine; ea, european ancestry; fhs, framingham heart study. figure distribution of unpruned genomes imputed snps assessed for association with methylation relative to the cpg location (+/- kb). snp position relative to the replicated methylation cpg position (x= ) is plotted against –log of the p value for meqtl meta-analysis in each race. snps above the red line are significant after bonferroni correction for multiple testing (p value< . x - ). abbreviations: aa, african american; bp, base pair; cpg, cytosine-phosphate-guanine; ea, european ancestry; meqtl, methylation quantitative trait locus; snp, single nucleotide polymorphism. figure illustration of the relationship of methylation at cg with diastolic bp, mediated by expression of tspan . methylation at cg was identified as associated with diastolic bp in discovery and replication meta-analyses of genome-wide dna methylation (n= , ). expression of tspan was associated with methylation at cg in meta-analyses of fhs and rs and diastolic bp in fhs. the direction of arrows in the diagram are inferred from significant two-step mendelian randomization using data from the genotype-tissue expression project and international consortium for blood pressure, which suggests that methylation at cg influences bp through the expression of tspan . the epigenome-wide association of dna methylation and diastolic bp is interpreted given the evidence of causal direction and based on a . % change in dna methylation at cg . abbreviations: dbp, diastolic blood pressure. table characteristics of the discovery and replication cohorts age, years sbp, mmhg dbp, mmhg htn aht cohort race n cohort type tissue normalization mean sd mean sd mean sd % % discovery (n = , ) aric aa , unrelated blood bmiq . . . . . . . . chs aa unrelated blood swan . . . . . . . . chs ea unrelated blood swan . . . . . . . . fhs ea , family blood dasen . . . . . . . . genoa aa unrelated blood swan . . . . . . . . goldn ea family cd + t cells combat . . . . . . . . lbc ea unrelated blood - . . . . . . . . nas ea unrelated blood bmiq . . . . . . . . rs-iii ea unrelated blood dasen . . . . . . . . twinsuk ea twins blood bmiq . . . . . . . . replication (n = , ) amish ea family blood quantile . . . . . . . . aric ea , unrelated blood bmiq . . . . . . . . mesa aa unrelated blood quantile . . . . . . . . mesa ea unrelated blood quantile . . . . . . . . mesa hl unrelated blood quantile . . . . . . . . rs-iii ea unrelated blood dasen . . . . . . . . sys adults ea unrelated blood swan . . . . . . . . whi-baa aa unrelated blood combat . . . . . . . . whi-baa ea unrelated blood combat . . . . . . . . whi-baa hl unrelated blood combat . . . . . . . . whi-epmc aa unrelated blood bmiq . . . . . . . . whi-empc ea , unrelated blood bmiq . . . . . . . . whi-empc hl unrelated blood bmiq . . . . . . . . hypertension is defined as systolic bp ≥ mmhg or diastolic bp ≥ mmhg. antihypertensive treatment is defined as the self-reported use of any antihypertensive medication. whi-empc normalized dna methylation data using bmiq and plate-adjusted using combat. the discovery and replication samples from rs-iii do not include overlapping or related individuals. abbreviations: aa, african american; aht, antihypertensive treatment; bmiq, beta mixture quantile dilation; combat, combatting batch effects when combining batches of microarray data; dasen, background-adjusted (d) between-array (s) without dye bias correction (n); dbp, diastolic blood pressure; ea, european ancestry; hl, hispanic/latino; htn, hypertension; sbp, systolic blood pressure; sd, standard deviation; swan, subset-quantile within array normalization. table results of discovery, replication, and overall meta-analyses for cpg sites replicated for association with bp systolic bp diastolic bp discovery replication overall discovery replication overall cpg site chr position ucsc gene coeff p value coeff p value coeff p value coeff p value coeff p value coeff p value cg tspan - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg phgdh - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg phgdh - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg txnip - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg slc a - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg loc - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg zmiz - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg cpt a - . . x - - . x - . x - - . . x - - . . x - - . . x - - . . x - cg - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg slc a - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - cg slc a - . . x - - . . x - - . . x - - . . x - - . . x - - . . x - position is hg . coefficients give the percent change in dna methylation for every mmhg change in blood pressure. abbreviations: bp, blood pressure; chr, chromosome; coeff, coefficient; cpg, cytosine-phosphate-guanine; ucsc, university of california santa cruz. table narrow-sense heritability estimated in the fhs for cpg sites replicated for association with bp cpg site chr position gene cpg h ( % ci) cg tspan . ( . , . ) cg phgdh . ( . , . ) cg phgdh . ( . , . ) cg txnip . ( . , . ) cg . ( . , . ) cg slc a . ( . , . ) cg loc . ( . , . ) cg zmiz . ( . , . ) cg . ( . , . ) cg cpt a . ( . , . ) cg . ( . , . ) cg slc a . ( . , . ) cg slc a . ( . , . ) epigenome-wide average heritability is . . position is hg . abbreviations: chr, chromosome; cpg, cytosine-phosphate-guanine. table bidirectional mendelian randomization results showing the inverse-variance weighted effects of multiple snps used as instrumental variables in the association of dna methylation and bp forward mendelian randomization cpg  bp reverse mendelian randomization bp  cpg cpg trait iv snps, n mean estimate (se) p value iv snps, n mean estimate (se) p value cg sbp - . ( . ) - - . ( . ) . cg dbp - . ( . ) . - . ( . ) . cg sbp - . ( . ) . cg dbp - . ( . ) - cg sbp - . ( . ) . - . ( . ) . cg dbp - . ( . ) - - . ( . ) . cg sbp - - . ( . ) . cg dbp - - . ( . ) . cg sbp . ( . ) . - . ( . ) . cg dbp . ( . ) . - . ( . ) . cg sbp - - . ( . ) . cg dbp - - . ( . ) - cg sbp - . ( . ) . - . ( . ) . cg dbp ( . ) . - . ( . ) . cg sbp - . ( . ) . . ( . ) . cg dbp . ( . ) . . ( . ) . cg sbp - . ( . ) . cg dbp - - . ( . ) . cg sbp - . ( . ) . cg dbp - . ( . ) . cg sbp . ( . ) . . ( . ) . cg dbp - . ( . ) . . ( . ) . causal mean effect and standard error estimates for the sentinel cpgs are shown and causal p values have been omitted when ivs showed significant pleiotropic effects (p < . ). mean forward causal estimates are in percent dna methylation change per mmhg increase in bp; mean reverse causal estimates are mmhg change in bp per . percent change in dna methylation. abbreviations: bp, blood pressure; cpg, cytosine-phosphate-guanine; dbp, diastolic blood pressure; iv, instrumental variable; pos, position; sbp, systolic blood pressure; se, standard error; snp, single nucleotide polymorphism. table genes in a cis-region (+/- mb) of replicated cpg sites ) associated with dna methylation in meta-analyses of fhs and rs at fdr q value < . , and ) associated with bp traits with at least one fdr q value < . gene expression – dna methylation gene expression – blood pressure traits fhs rs meta-analysis cpg site chr gene coeff p value coeff p value z-score fdr q trait coeff t-test fdr q cg tspan - . . x - - . . - . . x - sbp . . . x - dbp . . . x - htn . . . x - - . . x - - . . - . . x - sbp . . . x - dbp . . . x - htn . . . x - cg slc a - . . x - na na - . . x - sbp . . . dbp . . . htn . . . cg unc b . . . . . . sbp - . - . . dbp - . - . . htn - . - . . cpt a - . . x - - . . - . . x - sbp . . . dbp . . . htn . . . cg ptms - . . - . . - . . x - sbp . . . dbp . . . htn . . . lpcat . . . . . . sbp - . - . . dbp - . - . . htn - . - . . abbreviations: chr, chromosome; coeff, coefficient; cpg, cytosine-phosphate-guanine; dbp diastolic blood pressure; fdr, false discovery rate; fhs, framingham heart study; htn, hypertension; rs, rotterdam study; sbp, systolic blood pressure. table two-step mendelian randomization results for genes with transcription significantly associated with dna methylation and bp step one: cpg  ge step two: ge  bp iv: meqtls gtex eqtl iv: eqtls icbp sbp icbp dbp cpg site gene snp pos p value p value snp pos p value p value p value cg tspan rs , , . x - . rs , , . x - . . rs , , . x - . rs , , . x - . rs , , . x - . rs , , . x - . cg slc a na na na na rs , , . x - . . cg cpt a na na na na rs , , . x - . . unc b na na na na rs , , . x - . . cg lpcat rs , , . x - . rs , , . x - . . rs , , . x - . ptms rs , , . x - . rs , , . x - . . rs , , . x - . position is hg . abbreviations: bp, blood pressure; cpg, cytosine-phosphate-guanine; dbp diastolic blood pressure; eqtl, expression quantitative trait locus; ge, gene expression; gtex, genotype-tissue expression project; icbp, international consortium for blood pressure genome-wide association studies; iv, instrumental variable; meqtl, methylation quantitative trait locus; pos, position; sbp, systolic blood pressure; snp, single nucleotide polymorphism. pone. .. uc irvine uc irvine previously published works title mitochondrial uptake of thiamin pyrophosphate: physiological and cell biological aspects permalink https://escholarship.org/uc/item/ n f tf journal plos one, ( ) issn - authors subramanian, veedamali s nabokina, svetlana m lin-moshier, yaping et al. publication date - - doi . /journal.pone. license https://creativecommons.org/licenses/by/ . / . peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ n f tf https://escholarship.org/uc/item/ n f tf#author https://creativecommons.org/licenses/https://creativecommons.org/licenses/by/ . // . https://escholarship.org http://www.cdlib.org/ mitochondrial uptake of thiamin pyrophosphate: physiological and cell biological aspects veedamali s. subramanian , , svetlana m. nabokina , , yaping lin-moshier , jonathan s. marchant , hamid m. said , * departments of medicine, physiology and biophysics, university of california irvine, irvine, california, united states of america, department of veterans affairs medical center, long beach, california, united states of america, department of pharmacology, university of minnesota medical school, minneapolis, minnesota, united states of america abstract mammalian cells obtain vitamin b (thiamin) from their surrounding environment and convert it to thiamin pyrophosphate (tpp) in the cytoplasm. most of tpp is then transported into the mitochondria via a carrier-mediated process that involves the mitochondrial thiamin pyrophosphate transporter (mtppt). knowledge about the physiological parameters of the mtpp-mediated uptake process, mtppt targeting and the impact of clinical mutations in mtppt in patients with amish lethal microcephaly and neuropathy and bilateral striatal necrosis are not fully elucidated, and thus, were addressed in this study using custom-made h-tpp as a substrate and mitochondria isolated from mouse liver and human-derived liver hepg cells. results showed h-tpp uptake by mouse liver mitochondria to be ph-independent, saturable (km = . . mm), and specific for tpp. mtppt protein was expressed in mouse liver and hepg cells, and confocal images showed a human (h)mtppt-gfp construct to be targeted to mitochondria of hepg cells. a serial truncation analysis revealed that all three modules of hmtppt protein cooperated (although at different levels of efficiency) in mitochondrial targeting rather than acting autonomously as independent targeting module. finally, the hmtppt clinical mutants (g s and g a) showed proper mitochondrial targeting but displayed significant inhibition in h-tpp uptake and a decrease in level of expression of the mtppt protein. these findings advance our knowledge of the physiology and cell biology of the mitochondrial tpp uptake process. the results also show that clinical mutations in the hmtppt system impair its functionality via affecting its level of expression with no effect on its targeting to mitochondria. citation: subramanian vs, nabokina sm, lin-moshier y, marchant js, said hm ( ) mitochondrial uptake of thiamin pyrophosphate: physiological and cell biological aspects. plos one ( ): e . doi: . /journal.pone. editor: james f. collins, university of florida, united states of america received june , ; accepted july , ; published august , copyright: � subramanian et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. funding: the study was supported by grants from the dept. of veterans affairs, and the nih [dk- , dk- to hms, gm- to jsm]. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. competing interests: the authors have declared that no competing interests exist. * e-mail: hmsaid@uci.edu introduction thiamin is indispensable for normal cellular functions due to its involvement as a co-factor (mainly in the form of thiamin pyrophosphate, tpp) in critical metabolic reactions related to oxidative energy metabolism, atp production, and reduction of cellular oxidative stress [ – ]; the vitamin also plays an important role in maintaining normal function/structure of the mitochondria [ ]. mammalian cells cannot synthesize thiamin endogenously, but obtain the vitamin from circulation via transport across the cell membrane; the latter process is mediated by the cell membrane thiamin transporters thtr- and thtr- (products of slc a and slc a genes, respectively) [ – ]. following internalization of free thiamin, the majority ( to %) of the vitamin is converted to thiamin pyrophosphate (tpp) via an enzymatic process that takes place exclusively in the cytoplasm [ , , ]. most of the generated tpp (, %) is then transported (compartmentalized) into mitochondria [ – ] for utilization in a variety of metabolic reactions (there is no tpp synthesis in the mitochondria; [ ]). uptake of tpp by mitochondria occurs via a carrier-mediated mechanism that involves the mitochondrial thiamin pyrophosphate transporter (mtppt; product of the slc a gene [ ]). previous studies that attempted at determining the physiological parameters/characteristics of the mitochondrial tpp (mtpp) uptake process have utilized either indirect enzymatic method to measure the level of the transported substrate (which as acknowledged by the authors is of limited accuracy [ ]), or a yeast complementation (growth) method [ ] that also does not allow accurate determination of transport kinetic parameters/characteristics. the mtppt ( amino acid residues) is a member of the mitochondrial carrier family (mcf) of transporters for which a large volume of targeting data has been collected. the mcf family members exhibit a three-fold tandem repeated domain (, amino acids in length), with each domain consisting of two transmembrane helices linked by a loop that contains part of the ‘signature sequence motif’ characteristic of this family of trans- porters (px[d/e]xx[k/r]x[k/r]… / residues.[d/ e]gxxxx[w/y/f][k/r]g) [ , ]. the proline residue of the signature motif enforces a sharp kink in the odd numbered helices (h , h & h ) and the charged residues contribute to a salt- bridge network critical for the transport mechanism of the carrier cycle [ , ]. prior experiments have established that members of the mcf are synthesized without a cleavable pre-sequence but rather contain internal signaling sequences within the three plos one | www.plosone.org august | volume | issue | e modules that contribute toward targeting to mitochondria [ – ]. however, nothing is known specifically about the mitochon- drial targeting determinants of the hmtppt transporter and whether the different modules contribute with equal efficiency to the targeting process. thus, we addressed these issues in the current investigation using a series of hmtppt truncated mutants. the slc a gene is clinically important as mutations in this gene cause amish congenital lethal microcephaly (an autosomal recessive disorder associated with retardation in brain develop- ment) [ , , ], and neuropathy and bilateral striatal necrosis [ ]. such mutations lead to drastic depletion in mitochondria tpp level [ , , , ]. little, however, is known about how such clinical mutations affect the physiology and cell biology of the mtppt system. our aims in this study were to establish the physiological parameters and characteristics of the mtpp uptake process using a direct and accurate radiolabeled tracer method for assessing transport of very low abundant substrates, as well as determine how the mtppt is targeted to the mitochondria. we also aimed at determining how clinical mutations in the mtppt found in patients with amish lethal microcephaly and neuropathy and bilateral striatal necrosis impact the function and cell biology of the mtppt system. thus, we used h-tpp as the substrate and mitochondria isolated from mouse liver and from human-derived liver hepg cells as models in our investigations. the results established the kinetic parameters of the mtpp uptake process and showed uptake is ph-independent and specific for tpp. truncation analysis showed that all three modules of hmtppt contribute in targeting the transporter to the mitochondria, and that the two clinical missense mutations in the hmtppt system impair functionality of the system most likely via reduction in transport protein level of expression. materials and methods materials a gfp vector, dsred-mito fluorescent protein plasmid and gfp polyclonal antibodies were from clontech (mountain view, ca). hepg cells were purchased from atcc (manassas, va). normal human liver total rna was obtained from biochain (nework, ca). dna oligonucleotides were from sigma genosys (woodlands, tx). h-thiamin pyrophosphate (specific activity , . ci/mmol) was from moravek biochemicals (brea, ca). isolation of mitochondria and uptake assays mitochondria were isolated from adult (, months old) mouse liver or from hepg cells (maintained in dulbecco’s modified eagle’s medium supplemented with % (v/v) fetal bovine serum and x appropriate antibiotics), as described previously [ , ]. use of animals was approved by the institutional animal care use committee (iacuc) of veterans affairs medical center at long beach, ca. the freshly prepared mitochondria were suspended in buffer [in mm] kcl, . edta, mgcl , mes, and hepes, ph . , to achieve a protein concentration of , – mg/ml and then immediately used for uptake studies using a rapid-filtration technique [ ]. briefly, mitochondria ( ml) were added to the uptake buffer ( ml) containing h-tpp ( . mm) and then subjected to the incubation at uc [uptake buffer was the same as suspension buffer but contained mm succinate (to maintain the function of mitochondria)]. the uptake reaction was terminated after min (unless otherwise stated) by the addition of ml of ice-cold stop solution [in mm: kcl, mannitol, and kh po , ph . ] followed by rapid filtration. the filter was washed two times with stop solution followed by measurement of radioactivity in a liquid scintillation counter. western blot analysis mitochondria isolated from mouse liver or hepg cells (, mg) were subjected to protein resolution in nupage – % bis-tris gradient minigels (invitrogen) followed by transfer onto immobilon polyvinylidene difluoride membrane (fisher scientific), and subsequent western blot analysis using specific antibodies. the primary antibody was rabbit polyclonal anti- mtppt antibody (abgent, ca) at mg/ml dilution. the secondary antibody was anti-rabbit irdye- antibody (li- cor bioscience, lincoln, ne) at : , dilutions. immunore- active bands were visualized using the odyssey infrared imaging system (li-cor bioscience) [ ]. mitochondria isolated from hmtppt-gfp, g s and g a stably expressing hepg cells ( mg of protein) were resolved as described above. membranes were then incubated with anti-gfp (clontech, ca) and anti-pyruvate dehydrogenase (pdh) antibod- ies (abcam, cambridge, ma). immunodetection was performed by incubating the membrane with secondary antibodies [irdye labeled anti-mouse or irdye labeled goat anti-rabbit ( : dilutions for both secondary antibodies), li-cor] for min at rt. signals were detected as described above. real-time pcr analysis total rna isolated from the mouse liver, human liver and hepg cells using trizol reagent was treated with dnase i and subjected to reverse transcription using iscript cdna synthesis kit (bio-rad, hercules, ca). the mrna expression level was quantified in a cfx real-time pcr system (bio-rad), using iq sybr green super mix (bio-rad) and primers specific for mouse mtppt (forward: -tccagattgaacgcctgtg - and reverse: -gacagctccgtagcctatggac - ) and acidic ribosomal phosphoprotein (arpo) (forward: - gctgaacatctcccccttctc- and reverse: - atatcctcatctgattcctcc- ). the hmtppt (forward: -agcatgagcgcctgtcgc- and reverse: - tgagctgggachtgtcctttcca- ) and human b-actin (forward: -agccagaccgtctccttgta- and reverse: - tagagagggcccaccacac- ). real-time pcr condi- tions were used as previously described [ ]. data were normalized to arpo or b-actin and calculated using a relative relationship method supplied by the icycler manufacturer (bio- rad) [ ]. generation of hmtppt full-length, truncated and mutated constructs the full-length gfp-hmtppt and hmtppt-gfp, and truncated constructs were generated by pcr amplification using the hmtppt specific primer combinations (table ) and conditions as described previously [ , ]. the gfp vectors and pcr products were digested with hind iii and sac ii, and the respective products were gel separated and ligated to generate in- frame fusion constructs with the gfp fluorescent protein fused to the nh /cooh-terminus of the construct. the quick change tm site-directed mutagenesis kit (stratagene, ca) was used to introduce insertions or deletions of nucleotides into the open reading frame (orf) of hmtppt (slc a ). overlapping primers containing the mutated nucleotides to the specified mutation sites (table ), and full-length hmtppt-gfp fused plasmid was used as a template for pcr based site-directed mutagenesis as described before [ ]. the nucleotide sequences of targeting of hmtppt to mitochondria plos one | www.plosone.org august | volume | issue | e full-length, truncated and mutated constructs were verified by sequencing (laragen, los angeles, ca). transient and stable transfections for transient transfection, hepg cells were grown on sterile glass-bottomed petri dishes (mattek corporation, ashland, ma) and transfected at % confluency ( mg plasmid dna) using ml of lipofectamine (invitrogen, ca). after – hrs, cells were analyzed by confocal microscopy. for stable transfection, hepg cells were selected using g ( . mg/ml) for – weeks. xenopus oocyte preparation and nuclear microinjection adult female xenopus laevis frogs were anesthetized by immersion in . % aqueous solution of -aminobenzoic acid ethyl ester (ms- ) for min, and after death by decapitation, whole ovaries were removed following procedures approved by university of minnesota iacuc. the epithelial layers of stage vi oocytes were removed and treated with collagenase ( . mg/ml for min) in dissociation solution (in mm: . nacl, . kcl, na hpo , and hepes, ph . ) to ensure complete defolliculation. for expression studies, , ng of plasmid cdna in nl of intracellular solution (in mm: kcl, hepes, mgcl , egta, and . cacl , ph . ) were injected using established methods [ , ]. injected oocytes were separated and maintained in barth’s solution for – hrs. western blotting of xenopus oocytes was performed as described [ ]. confocal imaging of hmtppt full-length, truncated and mutant constructs hepg cell monolayers grown on cover-slip petri-dishes were imaged using a nikon c- confocal scanner head attached to a nikon inverted phase contrast microscope. the fluorophores were excited using the -nm (gfp) or -nm laser lines and emitted fluorescence was monitored with a nm band pass (gfp) or at nm long-pass (red) filter as described before [ , ]. data presentation and statistical analysis data are presented as mean se of multiple uptake determinations and expressed in picomoles or femtomoles per milligram of protein per unit of time. student’s t-test was used in statistical analysis with statistical significance set at p, . . kinetic parameters of the saturable tpp uptake process [(i.e., maximal velocity (vmax) and the apparent michaelis-menten constant (km)] were calculated [ ]. uptake by the saturable component at certain tpp concentration was calculated by subtracting uptake by simple diffusion which was determined from the slope of the line between the point of origin and uptake at high pharmacological concentration of tpp ( mm) from total tpp uptake. results and discussion characteristics and kinetics of h-tpp uptake by freshly isolated mitochondria figure a shows the results of uptake of low physiological concentration of h-tpp ( . mm) by mouse liver mitochondria as a function of time. uptake was found to be linear [r = . ] for up to min of incubation (ph . ) and occurred at a rate of . . fmol (mg protein) . similarly, uptake of high concentration of tpp ( mm) was linear for up to min of incubation (data not shown). we chose a min incubation time to represent the initial-rate of uptake in all our subsequent studies. figure b depicts the results on the effect of buffer ph on tpp ( . mm) uptake by mouse liver mitochondria over the range of ph . – . . over the examined range, the rate of tpp uptake fluctuated slightly without clear preference to acidic, neutral or alkaline ph. considering this ph-independent nature, we performed subsequent studies at ph . . next, we examined the uptake of tpp as a function of substrate concentration ( . – mm tpp) in order to determine the kinetic parameters of the uptake process. the results (figure c) showed the initial-rate of uptake of h-tpp by mitochondria to be saturable. kinetic parameters of the saturable component were determined as described in ‘‘methods’’ and found to be of . . mm for the apparent km, and . . pmol (mg protein) ( min) for the vmax. table . combination of primers used to prepare the full length, truncated and mutated constructs of hmtppt by pcr. construct forward and reverse primers ( - ) positions (bp) fragment (bp) hmtppt[ – ]-gfp/gfp- hmtppt[ – ] cccaagcttatggttggctatgacccc; tccccgcgggcgctggctggctgtcct – hmtppt[ – ]-gfp cccaagcttatggttggctatgacccc; tccccgcgggaggatgccatggtactt – hmtppt[ – ]-gfp cccaagcttatggttggctatgacccc; tccccgcggggtccccacggcgtggcg – hmtppt[ – ]-gfp cccaagcttatgcaggcctctaggcagatt; tccccgcgggcgctggctggctgtcct – hmtppt[ – ]-gfp cccaagcttatggttggctatgacccc; tccccgcgggaggcccttgtatctccg – hmtppt[ – ]-gfp cccaagcttatggactgtgccaagcag; tccccgcgggcgctggctggctgtcct – hmtppt[ – ]-gfp cccaagcttatgcaggcctctaggcagatt; tccccgcgggaggcccttgtatctccg – hmtppt[ – ]-gfp cccaagcttatgtataggagcgaaggc; tccccgcgggcgctggctggctgtcct – hmtppt[ – ]-gfp cccaagcttatgctgacggagctggtc; tccccgcggtatggcccacttgtacag – hmtppt[ – ]-gfp cccaagcttatgctgacggagctggtc; tccccgcgggcgctggctggctgtcct – hmtppt[g s]-gfp cactttgtatgtagtggcctggctgcc; ggcagccaggccactacatacaaagtg hmtppt[g a]-gfp gttttctacaaagccttggctcccacc ggtgggagccaaggctttgtagaaaac restriction sites hind iii (boldface text) and sac ii (italic text) were added to the hmtppt primers to allow subsequent sub-cloning into the gfp vectors and mutant primers are in bold italic text. doi: . /journal.pone. .t targeting of hmtppt to mitochondria plos one | www.plosone.org august | volume | issue | e to determine specificity of the mitochondrial uptake process, we examined the effect of unlabeled tpp, its analogs/derivatives including free thiamin and thiamin monophosphate (tmp), as well as the structurally unrelated vitamin biotin (all at mm) on the initial rate of h-tpp ( . mm) uptake. the results showed that with the exception of unlabeled tpp, which caused a significantly (p, . ) inhibition in h-tpp uptake, none of the other compounds affected h-tpp uptake (figure d). expression of mtppt (slc a ) in mouse and human liver, and hepg cells the presence of mtppt mrna (product of slc a gene) was studied in mouse liver and in human-derived liver hepg cells by mean of rt-pcr using specific primers and expression was resolved in each of these preparations (figure a), similar expression of the mtppt mrna was observed in human liver (data not shown). we also observed expression of the mtppt protein in mouse liver and hepg cells studies using western blotting with anti-mtppt polyclonal antibodies (figure b). targeting of the hmtppt to mitochondria after initial characterization of the h-tpp transport process into native mouse liver mitochondria, we investigated the determinants that dictate the targeting of mtppt to mitochon- dria using cultured hepg cells. this system was chosen owing to experimental tractability overcoming the limitations in transfection and imaging in native mouse liver. hmtppt is predicted to harbor transmembrane domains with nh and cooh terminals oriented towards the cytosol (figure a). to visualize the targeting of hmtppt, nh and cooh terminal fusions of the full-length hmtppt cdna to the green fluorescent protein were constructed (gfp-hmtppt, hmtppt-gfp). these con- structs were transiently transfected into hepg cells together with a mitochondrial marker (dsred-mito) and the resulting fluores- cence distribution imaged using confocal microscopy. lateral (xy) images of transfected hepg cells showed that both hmtppt constructs targeted to the mitochondria regardless of the positioning of the fluorescent protein tag (figure b). similar mitochondrial targeting of hmtppt-gfp cdna was observed figure . characteristics of the tpp uptake process by the isolated mouse liver mitochondria. a) uptake as a function of time. mitochondria were incubated in uptake buffer, ph . , at uc. h-tpp ( . mm) was added to the incubation medium at the start of uptake. data are mean se of – separate uptake determinations. b) effect of incubation buffer ph on the initial-rate of h -tpp uptake. mitochondria were incubated in uptake buffer of varying ph at uc. h-tpp ( . mm) was added to the incubation buffer at the onset of a min of incubation time (i.e., initial-rate). data are mean se of – separate uptake determinations. c) uptake of h-tpp as a function of substrate concentration. mitochondria were incubated in uptake buffer, ph . , at uc in the presence of different concentrations of tpp. uptake was measured after min incubation (i.e., initial-rate). uptake by the carrier-mediated system was calculated as described in ‘‘methods’’. data are mean se of – separate uptake determinations. d) effect of structural analogs of tpp on the initial-rate of uptake. mitochondria were incubated in uptake buffer, ph . , at uc in the presence of h-tpp ( . mm) and unlabeled tpp, tmp, thiamin, and biotin ( mm for all). uptake was measured after min incubation (i.e., initial-rate). data are mean se of – separate uptake determinations. *p, . . doi: . /journal.pone. .g targeting of hmtppt to mitochondria plos one | www.plosone.org august | volume | issue | e when the construct was heterologously expressed in xenopus oocytes by nuclear microinjection (figure c). hmtppt-gfp expression was visible within a broad band of expression in the oocyte sub-cortex (figure c), with lateral (‘xy’) confocal sections revealing the clumps and vermiform structures diagnostic of mitochondrial morphology in the xenopus oocyte [ ]. figure d shows the hmtppt-gfp and dsred-mito co-expressing oocytes displays strong colocalization. single oocyte western blots revealed migration of the fluorescent protein-tagged fusion construct at , kda, consistent with expression of a , kda (untagged) transporter construct (compare with figure e). role of the multiple modules in targeting of the hmtppt to mitochondria to delimit regions of hmtppt important for mitochondrial targeting a series of truncated constructs were prepared (figures & ). on the basis of prior data implicating a key role for the signature motif in hmtppt targeting and translocation [ , ], we designed truncations to interrupt the signature motif in each hmtppt module, i.e. truncation between helices h and h , h and h , h and h . the results of these manipulations were as follows. first, disruption of the motif between h and h failed to impair mitochondrial targeting of the residual construct (hmtppt[ – ]-gfp) (figure ). truncation between h and h , which disrupted the third signature motif, also did not impact the targeting of the parent construct (hmtppt[ – ]- gfp, figure ) which co-localized with dsred-mito (data not shown) (figure ). the residual truncated sequences (hmtppt[ – ]-gfp and hmtppt[ – ]-gfp) when fused to gfp resulted in predominantly cytosolic fluorescence (figure ). therefore, the integrity of the first or the third signature motif proved non-essential for mitochondrial targeting of hmtppt. this conclusion was underscored by the observation that the double truncated mutant hmtppt[ – ]-gfp also localized to mitochondria (figure ). this construct comprised solely the second module of hmtppt (h –h ) flanked by an additional transmembrane helix at the nh (h ) and cooh terminus (h ). next, hmtppt was cleaved within the second signature motif (i.e. in half) resulting in two constructs (hmtppt[ – ]-gfp and hmtppt[ – ]-gfp, figure ). neither construct co-localize with dsred-mito (data not shown) suggesting that integrity of this region of hmtppt might be important for appropriate mito- chondrial targeting. while the second module appeared necessary for mitochondrial targeting, it was not sufficient by itself as this region alone (hmtppt[ – ]-gfp) failed to express in mitochondria. mitochondrial targeting was shown to be enhanced by flanking transmembrane regions at both the nh and cooh terminus (hmtppt[ – ]-gfp, see above), or nh terminus alone (hmtppt[ – ]-gfp) (figure ). these analyses reinforce the view that the modules of mcf proteins cooperate in targeting of these carriers to mitochondria [ ] rather than each act autonomously as independent targeting modules. additionally, our truncation data point to a non- equivalency in the modules, namely that the h -(h )-h region appears necessary for efficient targeting to the mitochondria. bioinformatic analysis suggests the second module (containing the h -(h )-h region) conforms to the consensus signature motif present in modules and of hmtppt, unique feature being a shorter length ( amino acids) compared with modules and ( – residues, respectively). our finding above showing a greater role for the second module of hmtppt in mitochondrial targeting is in contrast to some other observations with other members of the mcf which showed prominent role for other modules in the targeting event [ ]. effect of clinical mutations on physiology/cell biology of hmtppt two disease-linked mutations have been identified in slc a [ , , , ]. first identified was a g a (c. g.c) substi- tution [ , , ] associated with amish lethal microcephaly which is an inherited autosomal recessive disorder which markedly affects brain development and leads to alpha-ketoglutaric aciduria. the mutated residue comprises the terminal glycine within the mcf signature motif found between transmembrane helices h and h (px[d/e]xx[k/r]x[k/r]. / .[d/e]gxxxx[w/y/f][k/ r]g). this residue is highly conserved in mcf family members, and localizes about one and a half helix turns (between helices h and h ) below the substrate binding sites (figure a). the second disease-associated mutation in slc a is a g s allelic variant that is associated with neuropathy and bilateral striatal necrosis figure . expression of mtppt in the mouse liver and hepg cells at the mrna and protein levels. a) total rna was isolated and quantitative real-time pcr was performed as described in ‘‘methods’’. data (mean se) presented are from four independent mice normalized relative to arpo or multiple samples from hepg cells were normalized relative to b-actin. b) western blot analysis was performed with the use of isolated mouse liver or hepg cells mitochondria ( mg) and specific anti-mtppt polyclonal antibodies as described in ‘‘methods’’. data from a representative experiment involving two separate mitochondria preparations from two mice or hepg cells are shown. doi: . /journal.pone. .g targeting of hmtppt to mitochondria plos one | www.plosone.org august | volume | issue | e [ ]. this conserved glycine residue this time located at the start of h , within the ‘p-g level ’ region (figure a). we experimentally introduced point mutations into hmtppt to mimic both these clinical mutations (hmtppt[g s]-gfp and hmtppt[g a]-gfp). the hmtppt[g s]-gfp and hmtppt[g a]-gfp mutants were transfected into hepg cells and stable cell lines prepared for each mutant. after selection, stable hepg cells were co-transfected with dsred-mito. the confocal imaging revealed that both two missense hmtppt mutants exhibited mitochondrial expression in hepg cells (figure b). therefore these clinical phenotypes did not derive from mistargeting of hmtppt. next, we examined the effect of the impact of both clinical mutations on the functionality of transporter, by quantifying h- tpp uptake into mitochondria in stable hepg cell lines. the uptake results revealed a significant inhibition in h-tpp uptake (p, . for both mutants) in both hmtppt[g s]-gfp and hmtppt[g a]-gfp expressing cell lines when compared with those expressing the wild-type hmtppt-gfp construct or gfp alone (figure c). note, this is the first study utilizing the native tpp as a substrate to examine the functionality of clinical mutations in isolated mitochondria. the observed decrease in h- tpp accumulation was consistent with proposed structural roles of the mutated residues in hmtppt functionality. for g a, this residue lies in close proximity to one of the salt bridges (k to d ) in the cytosolic conformation of the carrier (‘c-state’) that may serve to close the base of the water-filled cavity of the transporter [ ]. the glycine residue has been proposed to act as a hinge residue (within p-g level , [ ]) serving to open/close the carrier on the matrix side during the carrier transport cycle. for g s also, this region has been implicated as a hinge region involved in conformational changes needed to translocate substrate [ ]. this region is found in the three-dimensional structure about one helical turn above substrate binding region toward the cytosolic face. consistent with prior findings (liposomes [ , ] vertebrate [ ] and yeast [ ]) our data demonstrated impaired h-tpp uptake functionality when substrate transport was assessed by hepg cells mitochondria (figure c). determi- nation of expression levels of the different hmtppt (wt) and mutant constructs were then assessed by western blot analysis with anti-gfp antibodies. densitometric analysis of several blots revealed a significant decrease in the protein expression of the clinical mutants compared with wild-type hmtppt (p, . for g s and p, . for g a, figure d). therefore, the observed loss of function phenotype likely results from both impaired protein expression as well as impaired transport figure . cellular distribution of hmtppt-gfp or gfp-hmtppt in hepg cells and x. oocytes. a) predicted transmembrane domains of hmtppt showing the location of three-fold tandem repeated domains and both nh and cooh-terminals oriented towards the cytosol. b) lateral (xy) confocal images of hmtppt-gfp or gfp-hmtppt co-transfected with dsred-mito (mitochondrial marker) and their overlay in hepg cells. c) resolution of hmtppt-gfp expression in a xenopus oocyte. top, axial image (‘xz’, blue) of a cortical band of hmtppt-gfp expression. individual lateral (‘xy’, red) images are displayed as image planes taken at successive mm intervals into the oocyte cortex. d) lateral (xy) confocal images of hmtppt- gfp co-transfected with dsred-mito and their overlay in xenopus oocyte. e) western blot was performed on two individual hmtppt-gfp expressing oocytes and probed with mouse anti-gfp ( : ) and goat anti-mouse antibodies ( : ). doi: . /journal.pone. .g targeting of hmtppt to mitochondria plos one | www.plosone.org august | volume | issue | e figure . systematic truncation of the hmtppt polypeptide and their expression pattern in hepg cells. sub cellular expression of indicated (five) hmtppt-gfp truncated constructs in hepg cells. lateral (xy) confocal images of different truncated constructs were imaged after – hrs of transient transfection. doi: . /journal.pone. .g figure . systematic truncation of the hmtppt polypeptide and their expression pattern in hepg cells. sub cellular expression of indicated (four) hmtppt-gfp truncated constructs in hepg cells. lateral (xy) confocal images of different truncated constructs were imaged after – hrs of transient transfection. doi: . /journal.pone. .g targeting of hmtppt to mitochondria plos one | www.plosone.org august | volume | issue | e capabilities first revealed by protein reconstitution experiments [ , ]. in summary, these studies have established the kinetic parameters and characteristics of the mitochondrial tpp uptake process using a sensitive radio-isotope approach. the results also showed that all three modules of hmtppt protein are essential for proper mitochondrial targeting, although they participate at a variable degree of efficiency. furthermore the results show that clinical mutations in the hmtppt system impair its functionality via affecting the level of expression of the transport protein with no impairment in its targeting to mitochondria. author contributions conceived and designed the experiments: vss smn jsm hms. performed the experiments: vss smn y-lm. analyzed the data: vss smn jsm hms. contributed reagents/materials/analysis tools: vss smn jsm hms. wrote the paper: vss smn jsm hms. references . berdanier cd ( ) advanced nutrition-micronutrients; new 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( ) functional and structural role of amino acid residues in the even-numbered transmembrane a-helices of the bovine mitochondiral oxoglutarate carrier. j mol biol : – . . kaplan rs, mayor ja, brauer d, kotaria r, walters de, et al. ( ) the yeast mitochondrial citrate transport protein. probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway. j biol chem : – . targeting of hmtppt to mitochondria plos one | www.plosone.org august | volume | issue | e proximal fibular resection improves knee biomechanics and enhances tibial stress fracture healing in patients with osteoarthritis with varus deformity: a prospective, randomized control analysis research open access proximal fibular resection improves knee biomechanics and enhances tibial stress fracture healing in patients with osteoarthritis with varus deformity: a prospective, randomized control analysis vikram indrajit shah , sachin upadhyay , *, kalpesh shah , ashish sheth , amish kshatriya and jayesh patil abstract background: the present study aimed to evaluate the functional outcome of single-stage total knee arthroplasty using long-stem tibial component with proximal fibular resection (pfr) for patients with knee osteoarthritis with varus deformity associated with tibial stress fracture. method: a cohort of patients with a mean age . ± . years who met the criteria were randomized to a study group and a control group. patients in the study group underwent single-stage total knee arthroplasty using long-stem tibial component with pfr. the control group received conventional treatment. all patients were followed at , , and month(s) after surgery. standard anteroposterior and lateral weight bearing knee x-rays were analyzed. western ontario and mc-master universities osteoarthritis index score (womac) and the visual analog scale (vas) score were used to assess the functional outcome. the level of significance was set at p < . levels. results: one patient in the study group was lost to follow-up, leaving patients for final assessment. the womac total score and mean vas score were significantly better in study group than in control group at final follow-up (p < . ). all fractures were successfully united in a mean time of . ± . weeks in study group. a total of patients in control group had delayed union, five had established nonunion and required further interventions. no complications relating to surgery was detected. conclusion: total knee arthroplasty with pfr for knee arthritis with varus deformity associated with tibial stress fractures restores limb alignment, improves biomechanics, enhances fracture healing and provides excellent functional outcome. keywords: total knee arthroplasty, stress fracture, womac, proximal fibular resection, vas © the author(s). open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. * correspondence: drsachinupadhyay@gmail.com department of orthopaedics, nscb medical college, jabalpur, mp, india joint replacement and minimal invasive surgery, shalby hospitals jabalpur, jabalpur, madhya pradesh, india full list of author information is available at the end of the article arthroplastyshah et al. arthroplasty ( ) : https://doi.org/ . /s - - -y http://crossmark.crossref.org/dialog/?doi= . /s - - -y&domain=pdf http://creativecommons.org/licenses/by/ . / mailto:drsachinupadhyay@gmail.com background stress fractures are considered to be multifactorial over- use injuries that are attributable to the repetitive sub- maximal stress, and were first reported in the metatarsals of prussian military soldiers in by breithaupt [ , ]. stress fractures are broadly classified into two types: an insufficiency fracture that results from normal stress or forces of low magnitude acting on ab- normal or compromised bone and a fatigue fracture that occurs as a consequence of increased and repetitive stress to normal bone [ – ]. tibial stress fractures are not an uncommon clinical entity but they rarely occur in elderly population with severe knee osteoarthritis koa [ – ]. the altered biomechanics, malalign- ment and abnormal stress on peri-articular bone second- ary to deformities in an arthritic knee all can result in stress fracture [ ]. however, surgical management of these conditions can be quite challenging, with the po- tential of high rates of complications and failure. key is- sues, such as residual varus alignment, failure to correct altered biomechanics, impaired bone fracture healing and delayed mobilization all lead to increased revision rates and poor functional outcomes. a procedure which addresses these factors seems to be the optimal treat- ment. in view of these critical concerns, the authors have advocated additional resection of proximal fibula in addition to total knee arthroplasty (tka) with modular stemmed tibial component as a single-stage surgical intervention for stress fracture associated with knee osteoarthritis. we believe that proximal fibular osteot- omy improves the functional outcome as it facilitates precise correction of deformities, improves the adverse biomechanics, decompresses the medial compartment more efficiently, and provides desirable biomechanical environment at fracture sites that enhances fracture union [ , ]. furthermore, to our knowledge, there has been no clinical study that has directly compared the outcomes of cohort of patients with proximal tibia stress fracture caused by severe arthrosis of the knee with varus deformity treated with tka with fibular oste- otomy with those without fibular osteotomy. the pur- pose of the present study was to present our experience with this technique and to prospectively compare out- comes of a cohort of koa patients with varus deformity associated with tibial stress fracture with and without fibular osteotomy. we hypothesized that the cohort of patients with and without proximal fibular resection would have different clinical outcomes. materials and methods we prospectively evaluated the effectiveness of proximal fibular resection in a cohort of patients who have under- gone unilateral tka for a diagnosis of koa with varus deformity associated with tibial stress fracture at our institute over a period of years from may to sep- tember . institutional ethics committee approval was obtained and all patients have consented to partici- pate in current research. patients of either sex with a diagnosis of koa with varus deformity associated with tibial stress fracture were eligible for inclusion in the study (fig. ). all stress fractures diagnosed by radiographic findings, including frank cortical break, periosteal reaction, endosteal callus, and horizontal or oblique patterns of sclerotic area [ ]. the exclusion criteria were: ( ) genu valgus or acute major trauma; ( ) preoperative evidence of infection (erythrocyte sedimentation rate and c-reactive protein); ( ) known history of cardiovascular diseases or cerebral vascular diseases; ( ) neuropathy; ( ) a history of patellar fracture, patellectomy, patello-femoral instability or prior unicondylar knee replacement or hto; ( ) hypersensi- tivity to nsaids or local anesthetic agents; ( ) preopera- tive abnormal hepatic or renal profile; ( ) history of peptic ulceration and upper gastrointestinal hemorrhage, cancer, hyperkalaemia; ( ) known history of coagulopa- thies, hematological or neuro-muscular disorders; ( ) known psychiatric diagnosis and/or any other circum- stances that would make participation not in the best interest of the cohort or could prevent the protocol- specified outcome evaluation. the patients were examined /screened for their sever- ity of arthritis (kellgren and lawrence system) and de- formity [ , ]. bone densitometry was not carried out, but all patients had radiological evidence of osteoporosis. among subjects, a cohort of patients with the mean age of . ± . years ( males and females) who met the criteria were randomly assigned by lottery to the study and control groups. study was designed to be a : case control study. patients in the study group underwent single-stage total knee arthroplasty using long-stem tibial component with proximal fibular resec- tion (pfr). the control group received conventional treatment (without fibular resection). the consort flow chart for the study is shown in fig. . clinico- demographic variables such as age, gender, grades of osteoarthritis, presenting symptoms, deformity (femoro- tibial angle) and comorbidities, if any, were recorded pre-operatively (table ). all operations were either per- formed or supervised by the senior author under spinal anesthesia. using longitudinal lateral incision, the fibula was exposed subperiosteally between the inter-muscular planes: peroneus muscle and soleus muscle. proximal fibular resection (pfr) was performed by removing a - to -cm length of fibula at a site to cm from the head of fibula and its end was sealed with bone wax. we preferred resection over osteotomy because of the possi- bility of osteotomized bone healing too rapidly. the joint was exposed through a standard midline incision with shah et al. arthroplasty ( ) : page of fig. an anteroposterior x-ray of the knee showing reduction degenerative changes, irregularity, diminution of medial joint space, osteophytes, varus deformity (arrow showing features suggestive of osteoarthritis) with stress fracture of proximal tibia (arrow showing stress fracture) fig. consort flow chart shah et al. arthroplasty ( ) : page of medial parapatellar arthrotomy. the anterior and poster- ior cruciate ligaments were resected. standard cuts and appropriate release were made and soft tissue balancing was done. patellar resurfacing was done in all cases. all had posterior stabilized metal backed pfc sigma fix bearing with stem extension prosthesis. all the compo- nents were cemented. the derotation fixation modality was not used in the cases where the bone strength and fitting of the stem of prosthesis were found to be satis- factory. in others, the fixation modality included lateral dynamic compression or locking plates so as to provide derotation stability (fig. a and b). good hemostasis was achieved before fascial closure. arthrotomy was closed in layers and staplers were used superficially. no drains were used in either group. a compression bandage was applied to the limb following closure. skin staples/su- tures were routinely removed days after the surgery. all surgeries were performed uneventfully without any intraoperative complications. outcome measurement all patients were followed , , and months post- surgery. the knee was evaluated pre- and postopera- tively against standard anteroposterior and lateral weight-bearing radiographs, the western ontario and mc-master universities osteoarthritis index score and the visual analog scale score of the knee joint. at each follow-up, lower extremity alignment was evaluated by measuring the femorotibial (fta) angle, residual varus component on weight bearing ap radiographs. postoper- atively and at each subsequent follow-up visit, average fracture healing time, pain scores, implant failure and other complications were studied. the union of the frac- ture was assessed both clinically and radiologically on ap and lateral radiographs. radiologically the fracture was believed to be united if union was present in at least three cortices of the tibia. absence of tenderness or pain at the fracture site and the ability to weight-bear were the clinical criteria to define fracture healing. table patient demographics and preoperative characteristics serial number characteristics control (n = ) treatment group (n = ) p-value . age (years) . ± . . ± . p = . t = . . sex female ( . %) male ( . %) female ( . %) male ( . %) χ = . p = . . severity of disease (kellgren and lawrence system) grade iv ( . %) grades iii ( . %) grade iv ( . %) grade iii ( . %) χ = . p = . . deformity (femorotibial angle)(in degree) (varus) . ± . . ± . p = . . flexion angle(in degree) . ± . . ± . p = . . co-morbidity (htn,ihd, dm) . % (n = ) . % (n = ) χ = . p = . fig. a: left knee x-rays. a) preoperative anteroposterior and lateral view showing features suggestive of osteoarthritis, with deformity with stress fracture of proximal tibia; b) recent follow-up anteroposterior and lateral view showing healed stress fracture with correction of deformity with modular stemmed knee prosthesis with implant (plate) in situ with proximal fibular resection. b: left knee x-rays. a) preoperative anteroposterior and lateral view showing features suggestive of osteoarthritis, with deformity with stress fracture proximal tibia; b) follow-up anteroposterior and lateral view showing healed stress fracture with correction of deformity with modular stemmed knee prosthesis with implant (plate) in situ with proximal fibular resection; c) recent follow-up anteroposterior and lateral view showing healed stress fracture with correction of deformity with modular stemmed knee prosthesis with implant (plate) in situ with proximal fibular resection shah et al. arthroplasty ( ) : page of postoperative physical therapy/rehabilitation schedule the aim of physical therapy during the early postoperative days was to achieve guarded and safe ambulation. all pa- tients received the same rehabilitation protocol. during immediate postoperative period, physical therapy (static quadriceps and ankle pump) was started as the effect of anesthesia weans off and patient felt comfortable. patient was allowed to engage in non-weight-bearing mobilization with walker and brace on day . patients were advised to wear brace in bed for week, assisted slr in brace with brace in situ from the third weeks; slr in high sitting from the sixth week, toe touch weight bearing for weeks, partial weight bearing for further weeks, high sitting from the sixth week. patients were allowed to have full weight bearing depending on radiological assessment at the th week. after six-week gait training, full weight bearing was encouraged (as tolerated). after the week, cane walking stick was encouraged (as per patients’ com- fort and confidence). twelve weeks after achieving inde- pendent weight bearing with cane, they were allowed to engage in staircase climbing. statistical analysis normally distributed data were expressed as mean ± standard deviation (sd) and range. during the critical analysis, numerically-coded categorical variables were cross-tabulated, and chi square or fisher’s exact test was applied as required. a fisher’s exact p-value was used in cases where the frequency was less than five. pearson’s chi square tests were used for other analyses. to test the difference between independent means, student t- test was used. differences were considered statistically significant at p < . . results sixty-two patients with a mean age of . ± . years (range – ) met the inclusion criteria for the current study. of the participants, ( %) were men and ( . %) were women (table ). follow-up lasted for . ± . month on average. one of patients in the study group was lost to follow-up, leaving patients who were followed for a minimum of months. complete vas and womac data were available in patients and were used in the final evaluation and ana- lysis. the two groups were similar in terms of their base- line parameters (p > . ) (table ). all fractures in both study group and control group healed at last follow-up. all fractures were successfully united in a mean time of . ± . weeks (range: – weeks) in study group (fig. ). however, ( . %) patients in control group fig. right knee x-rays. a) preoperative anteroposterior and lateral view showing features suggestive of osteoarthritis, with deformity with stress fracture proximal tibia; b) postoperative x-ray anteroposterior and lateral view showing correction of deformity with modular stemmed knee prosthesis with proximal fibular resection; c) recent follow-up anteroposterior and lateral view showing healed stress fracture with correction of deformity with modular stemmed knee prosthesis in situ with proximal fibular resection shah et al. arthroplasty ( ) : page of had delayed union ( . ± . weeks; range: – weeks) (fig. ). five ( . %) had established nonunion and required further interventions (fig. ) (table ). the mean tibio-femoral angle improved from . ± . ° varus to . ° valgus in study group while mean tibio- femoral angle in control group improved from . ± . ° to . ± . ° varus. eleven knees ( . %, / ) in the control group and only one knee ( . %, / ) in the treatment group showed persistent residual varus alignment ( . ± . ; range – degree) (table ). at the last follow-up, study group had significantly higher degree of flexion than the control group ( . ± . degree, vs. . ± . ) (p < . ). in both groups, all patients reported significantly less pain scores than base- line (p < . ) following total knee arthroplasty with long stem. the treatment group demonstrated significantly lower vas scores (p < . ) than the patients in the control group at the latest follow-up ( . ± . vs. . ± . ) (table ). the total womac scores, though bet- ter than baseline in both groups, the patients in the study group showed statistically significant improvement (p < . ) at the final follow-up ( . ± . vs. . ± . ) (table ). no infections were recorded in the present series of patients. there were no neurovascular complications. no revisions were performed during the course of follow-up. there was no evidence of prosthesis loosening, component migration and functional instabil- ity in any of the patients. discussion impaired bone fracture healing leading to delayed union or pseudarthrosis is a multi-factorial phenomenon and can exert a significant impact on a person’s personality (personal and professional productivity), lifestyle, and fig. left knee x-rays (delayed fracture healing) a) preoperative anteroposterior and lateral view showing features suggestive of osteoarthritis, with deformity with stress fracture proximal tibia; b) post operative x-ray anteroposterior and lateral view showing correction of deformity with modular stemmed knee prosthesis with intact fibula; c) week follow-up anteroposterior and lateral view showing healing of fracture in process with correction of deformity with modular stemmed knee prosthesis with intact fibula; d) week follow-up anteroposterior and lateral view showing healed stress fracture with correction of deformity with modular stemmed knee prosthesis with intact fibula; e) and f) recent follow-up anteroposterior and lateral view showing healed stress fracture with correction of deformity with modular stemmed knee prosthesis in situ with intact fibula shah et al. arthroplasty ( ) : page of ability to function—all of which compromise patients’ health-related quality of life, thus necessitating more ag- gressive approach [ , ]. a severely deformed arthritic knees will produce abnormal load on tibia [ ]. this re- petitive eccentric load/stress may lead to fatigue fracture of the proximal tibia [ ]. the incidence is expected to increase in the coming years, with an ageing population resulting in a greater number of tibial stress fracture as- sociated with koa, especially in indian context. correc- tion of deformity axis and fracture healing are the two key issues that need to be addressed. management can be either conservative or surgical. conservative treat- ment can lead to disuse muscle atrophy, joint stiffness, osteoporosis, malunion and it will not restore the mech- anical axis [ , ]. on other hand, surgical intervention aims to eliminate pain, correct the deformity axis, achieve fracture healing and improve function [ , – ]. opinions varied in the literature concerning the op- timal treatment for the tibial stress fracture associated with varus oa knee. most authors address this challen- ging problem by using modular stem prosthesis, which addresses both the deformity and symptoms of oa but has problems with durability and causes complications related to knee arthroplasty with a long tibial stem. frac- ture fixation with loose-fitting stem is likely to increase the incidence of delayed union, thereby increasing the chances of non-union. in our experience, supplementary fixation was not needed if a snugly-fitted stem was in place. loose stem and wide canal diameter warrant add- itional plate fixation to provide rotational stability. in fig. left knee x-rays (complication and intervention) a) preoperative anteroposterior and lateral view showing features suggestive of osteoarthritis, with deformity with stress fracture proximal tibia; b) and c) postoperative x-rays anteroposterior and lateral view showing correction of deformity with modular stemmed knee prosthesis with intact fibula; d) and e) follow-up anteroposterior view showing nonunion; f) recent follow-up anteroposterior view showing healed stress fracture with correction of deformity with modular stemmed knee prosthesis with implant (plate) in situ with resected fibula table fracture healing serial number study group (an = ) control group(n = ) p-value delayed union χ = . ; p = . time to union . ± . weeks . ± . weeks t = . ;p < . aone patient was excluded (lost to follow up) shah et al. arthroplasty ( ) : page of the present series, three patients in the study group and four patients in the control group required additional fixation with a plate to achieve rigid rotational stability. in the present prospective study, we found that pfr could significantly improve the functional outcome of the affected knee joint and minimize the risk of pseudar- throsis or delayed fracture union. there are several fac- tors that might contribute to our results. first, pfr technique was used during primary surgery so as to allow longitudinal pressure at the fracture site. second, a snugly-fitted stem and/or compression plate at the frac- ture site could provide rotational stability, thereby redu- cing the shearing forces. finally, more efficient decompression of medial compartment with no residual varus led to correction of biomechanical axis. (fig. ). the current study showed that significant number of patients [ ( . %)] in the control group had delayed fracture healing when compared to the study group (p < . ). in our control group, an intact fibula appears to be an important risk factor for delayed union. we be- lieve that, the fibula acts as an important lateral strut and may therefore prevent approximation of the frag- ments, and thereby delay healing. irigoyen dotti men- tioned the diminished pressure between the fragments, the influence of the interosseus membrane, and the per- sistence of a non-fractured fibula or a fibula that healed within the usual time were factors that led to delayed fracture union [ ]. furthermore, a gap at the fracture site is a critical factor that prolongs the healing time [ ]. delayed healing of a tibial stress fracture in the presence of an intact fibula pointed to resection of the fibula before non-union is established. sixteen percent of patients in the control group reported established pseudarthrosis and underwent internal fixation (plate osteosynthesis) along with bone grafting as a standard treatment (fig. ). in the present series, an altered strain (due to tibiofibular length discrepancy), decreased compression force on the stress fracture site, and inad- equate decompression of medial compartment owing to intact fibula led to delayed union and non-union in con- trol group while failure to correct extra-articular de- formity (s-shaped tibia) led to delayed union in one in the study group. furthermore, the lack of adequate compression or col- lapse at the fracture site and stem extension failure to bypass the fracture adequately could be a major factor in fracture healing and construct strength (fig. ). in the present study we found pfr could completely correct preoperative varus alignment in the cohort when compared with control group (p < . ). most patients in the control group had persistent residual varus align- ment. the most likely explanation for this finding is the intra-operative difficulty associated with obtaining neu- tral alignment owing to medial soft tissue contracture and increased tension [ ]. increased risk of revision was associated with malalignment, particularly in varus [ – ]. genu varum can be corrected by combination of larger soft tissue release, pie crusting and more com- plex bone cuts [ – ]. though clinical benefit has been shown, unfortunately, these techniques require specific psychomotor skills. furthermore, all these procedures results in more bleeding, more instability, thus poten- tially increasing joint trauma. the patients in the study group had significantly better range of motion than the control group at the last follow-up. this difference could be attributed to delay in rehabilitation or compromised rehabilitation in the control group. the delay in rehabilitation was due to inadequate pain relief owing to delayed union and repeated surgical intervention for established pseudarthrosis. during the course of study, authors noticed that post-surgical pain delayed early rehabili- tation and thus negatively affected patients’ satisfac- tion rate and functional outcomes [ ]. table residual varus alignment group tool residual varus angle ( . ± . ; range – degree) in (number of patients) p-value; χ value control (n = ) residual varus angle χ = . p < . treatment group (n = )* *one patient was excluded (lost to follow-up) (*n = ) table vas group tool pre-operative (baseline) month month month month control vas . ± . . ± . . ± . . ± . . ± . t–value . . . . p-values <. <. <. <. treatment group . ± . . ± . * . ± . * . ± . * . ± . * t-value . . . . p-value <. <. <. <. *one patient was excluded (lost to follow-up) (*n = ) shah et al. arthroplasty ( ) : page of in the current study, we started to use pfr as an add- itional procedure to decompress the medial compart- ment more efficiently. it was noticed that, during the surgery, in the pfr group, it was easier for us to restore the mechanical axis than in the control group. pfr re- leases soft tissue tension. currently, it is difficult to dis- cern underlying mechanism for the efficacy of pfr but it probably works by rebalancing or redistributing the load on the lateral and medial tibia plateau post surgery or due to non-uniform settlement theory [ , ]. authors articulated that corrective osteotomy alone, in the cases of an arthritic knee with extra-articular deformities, can facilitate the correction of malalign- ment. at times, pfr also facilitates the correction of alignment due to an associated extra-articular deformity by releasing tension. when extra-articular deformity as- sociated with proximal stress fracture exists, we usually combine corrective osteotomy with pfr to correct the adverse biomechanics both at the joint and at the frac- ture site. only corrective osteotomy in these cases would lead to delayed union or non-union with persistence of residual varus alignment. in the present series, four pa- tients in the study group were treated by corrective oste- otomy besides pfr and six patients in the control group table total womac score group tool pre-operative (baseline) month month month month control total womac . ± . . ± . . ± . . ± . . ± . t–value . . . . p-values <. <. <. <. treatment group . ± . . ± . * . ± . * . ± . * . ± . * t-value . . . . p-value <. <. <. <. *one patient was excluded (lost to follow-up) (*n = ) fig. schematic diagram showing the proposed hypothesis. the diagram showing that a) the intact fibula acts as a strut and produces distraction forces at the fracture site and hinders the efficient decompression of medial compartment; b) proximal fibular osteotomy facilitates precise correction of deformity, improves the adverse biomechanics, decompresses the medial compartment more efficiently, and provides desirable biomechanical environment at fracture sites that enhances fracture. shah et al. arthroplasty ( ) : page of received tibial osteotomy for correction of the extra- articular deformity. authors noticed that all these six cases in the control group showed features suggestive of delayed union and residual varus alignment. the intact fibula in these cases impeded the adequate collapse at the fracture site and correction of the alignment. au- thors recommend corrective osteotomy plus pfr in the cases of an arthritic knee associated with stress fracture and extra-articular deformity. during follow-up, all patients showed statistically sig- nificant improvement in their womac total scores (p < . ). mean vas scores were significantly lower than the preoperative data. treatment group showed sig- nificantly greater improvement than control group (p < . ). the answer probably lies in a combination of rea- sons: the control group had delayed fracture healing and non-union ( %) and persistent residual varus alignment. currently, we believe that due to the lack of bio- mechanical data, this supposition remains empirical. however, our findings suggest that the present tech- nique addresses all concerned issues. furthermore, authors believe that the present technique can be used as routine procedure for the correction of all varus deformities associated with stress fractures as it has the potential to correct significantly-deformed knee, improve the altered biomechanics and enhance the fracture healing without disturbing the soft tis- sues. this in turn could reduce the risk of delayed healing and/or ununited stress fractures, residual varus, and morbidity and thereby improve the func- tional outcome. in the cohort of the current study, no pfr-related complication was reported. there were some limitations to the current study. first, we analyzed only varus knees. therefore the find- ings of current study cannot be directly applied to valgus knees. further studies should investigate its efficacy in the cases of valgus knee. second, current study reported subjective outcome measures for patients with knee osteoarthritis and this may lead to biased evaluations. further research using biomechanical data is warranted. third, the small sample size in this research prevents the generalization of the finding and typically leads to type-ii errors. despite these limitations, to the best of our knowledge, this is the first pilot report which critic- ally analyzed the impact of proximal fibular resection on the severely varus-deformed arthritic knees associated with tibial stress fracture. these preliminary findings provide a rationale for future research using randomized controlled trials with larger sample sizes, and explor- ation into routine use of proximal fibular resection in patients with severely-deformed knee associated with stress fractures and its effect on biomechanical outcomes. conclusion the current study showed that modular stemmed tibial components with proximal fibular resection is suitable for the unusual and challenging problem of delayed and/ or ununited tibial stress fractures associated with an arthritic knee and it can significantly improve the func- tional outcomes in patients with koa. indeed, pfr rep- resents an intervention that is cost-effective, requires no sophisticated skills or armamentarium and produces minimal side effects. the present technique helps en- hance the fracture healing and achieves stable correction of severe varus deformity. abbreviations pfr: proximal fibular resection; koa: knee osteoarthritis; womac: western ontario and mc-master universities osteoarthritis index score; vas: visual analog scale; tka: total knee arthroplasty; hto: high tibial osteotomy; nsaids: nonsteroidal anti-inflammatory drugs; fta: femorotibial; ap: antero- posterior acknowledgements we are indebted to all the patients who participated in the study, nurses, paramedical staff involved in the study. we also acknowledge the contribution of entire research team. authors’ contributions vikram indrajit shah (resources; data curation; formal analysis). sachin upadhyay (conception; investigation; methodology; resources; supervision; validation; visualization; writing, review & editing). kalpesh shah (resources). ashish sheth (resources). amish kshatriya (resources). jayesh patil (resources). the author(s) read and approved the final manuscript. funding not applicable. availability of data and materials the data that support the findings of this study are available from [shalby hospitals india] but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. data are, however, available from the authors upon reasonable request and with permission of [shalby hospitals india]. ethics approval and consent to participate the study was approved by the scientific review committee and the institutional review board of the participating health service. written informed consent (about the surgical technique, risks and potential complications) was provided, according to the declaration of helsinki, and obtained from all participating patients. consent for publication informed consent was obtained from the patients for publication of their medical records for the providing evidence-based scientific literature for fur- ther research. competing interests the authors declare that they have no competing interests. author details department of knee and hip arthroplasty, shalby hospitals, ahmedabad, gujarat, india. department of orthopaedics, nscb medical college, jabalpur, mp, india. joint replacement and minimal invasive surgery, shalby hospitals jabalpur, jabalpur, madhya pradesh, india. shah et al. arthroplasty ( ) : page of received: february accepted: march references . boden bp, osbahr dc. high-risk stress fractures: evaluation and treatment. j am acad orthop surg. ; 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( ): . . teeny sm, et al. primary total knee arthroplasty in patients with severe varus deformity. clin orthop. ; : . . engh ga. the difficult knee: severe varus and valgus. clin orthop relat res. ; : . . verdonk, et al. soft tissue balancing in varus total knee arthroplasty: an algorithmic approach. knee surg sports traumatol arhtrosc. ; ( ): . . engh ga, ammeen d. results of total knee arthroplasty with medial epicondylar osteotomy to correct varus deformity. clin orthop relat res. ; : . . mullaji arun b, shetty gm. correction of varus deformity during tka with reduction osteotomy. clin orthop relat res. ; : . . shah vi, upadhyay s, shah k, sheth an, kshatriya a, saini d. multimodal cocktail injection relieves postoperative pain and improves early rehabilitation following total knee replacement: a prospective, blinded and randomized study. j recent adv pain. ; ( ): – . https://doi.org/ . /jp-journals- - . . wei xc, wang xh, li pc, et al. proximal fibular osteotomy, a new surgery for pain relief and improvement of joint function in human knee osteoarthritis: a short-term clinical study. poster #: . ors annual meeting at the disney’s coronado springs resort in orlando, florida, march – . . yang zy, chen w, li cx, wang j, shao dc, hou zy, gao sj, wang f, li jd, hao jd, chen bc, zhang yz. medial compartment decompression by fibular osteotomy to treat medial compartment knee osteoarthritis: a pilot study. orthopedics. ; 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ pii: s - ( ) - special article nufrifion vol. , no. , influences on diet, health behaviours and their outcome in select ethnocultural and religious groups bryna shatenstein, phd, pdt, and parviz ghadirian, phd from the department of nutrition, university of montrkal, and hdpital maisonneuve-rosemont, montr.kal, quebec, canada date accepted: may abstract diverse cultural components of behavior may have significant impacts on patterns of eating, drinking, and social interaction, irrespective of socioeconomic status. for example, the major world religions prescribe or proscribe specific dietary behaviors; some of these are rooted in historical or geographical origins as well as group folklore; and they have integral roles as expressions of religious piety and group cohesiveness. the literature is replete with ecological observations of between-country differences in disease trends, some of which have been associated with dietary practices. the study of distinct cultural and religious groups (especially migrants acculturating to new environments) and the extent to which they adhere to culturally-based dietary precepts, has advanced our knowledge of psychosocial influences on food habits, nutritional adequacy, and overall health. however, a relatively small proportion of culturally-based research studies conducted to date have explored cross-cultural, ethnic, or religious variables. this paper reviews some population-based differences in dietary habits and other behaviors by ethnocultural group or religious denomination; health consequences and suggestions for future research are discussed. nutrition ; : - . oelsevier science inc. key words: diet, culture, religion, ethnic groups, health behaviors introduction food behavior has social and cultural connotations resulting from acquired knowledge as well as carefully selected and main- tained traditions, - so that food has historically been intimately woven into the life fabric of a society. despite difficulties asso- ciated with precise and accurate assessment of dietary determi- nants and outcome,’ it is important to consider the symbolic value of foods when studying eating behaviors because foods reflect and are influenced by the cultural categories expressed by a society. - north american dietary studies have only recently begun to systematically explore dietary attributes of minority groups such as blacks, hispanics, or native americanss-‘ these data are becoming essential for addressing minority health issues. in both north america and europe, accelerated waves of migration have occurred in the last to years, with cultural groups from countries in africa, southeast asia, the near east, and the far east, among others, migrating to western countries. since , striking political changes in europe have seen many move from east to west. few dietary studies have been conducted among these groups in their new environments, although there is ample historical-as well as current-evidence of dietary acculturation among the children of migrants. is-is the proliferation and con- sumption of “ethnic” foods alongside the more traditional fare in most north american cities should be seen as clear testimony to the culinary influence of numerous cultures in western society today. the present overview aims to encourage health professionals to be more aware of cultural influences on their clients’ health, and to stimulate further research in the area of cross-cultural determi- nants of dietary and other health behaviors. concepts from two complementary disciplines-nutritional anthropology and nutri- tional epidemiology-will be explored, using selected historical and current research sources. a sampling of studies spanning the last two decades will illustrate some of the issues discussed and address implications for future research which seeks to diminish risk for chronic diseases. correspondence to: dr. bryna shatenstein, department of nutrition, university of montreal, unite de recherche en epidemiologic, rue st. urbain, montreal, quebec, canada, h w lt . nutrition : - , oelsevier science inc. printed in the usa. all rights reserved. elsevier - / /$ . pii so - ( ) - culture, religion, diet, and health food habits and health while the specific reasons for widespread and persistent social differences in health and disease are unknown, they are believed to result from differing socio-environmental factors between pop- ulations, as well as individual genetic and lifestyle differences within populations. cultural influences on behavior may affect dietary patterns and social relationships, independent of material conditions. indeed, a contributory nutritional etiology has been suggested for most chronic diseasesi for example, coronary heart disease has been related to a diet high in energy, total fat, and animal fat, contributing an excess of saturated fats and choles- terol.is hypertension may be influenced by an overconsumption of salt in susceptible individuals. i on the other hand, risk of osteo- porosis, particularly in white postmenopausal women, may be exacerbated by insufficient consumption of calcium-rich foods during childhood and adolescence. while diet-cancer relation- ships are less well-defined, excesses or insufficiencies of various nutrients have a purported role in the etiology of various cancers. however, while promising research consistently stresses the im- portance of fruit, vegetables, and dietary fiber in cancer preven- tion,*’ it is not yet known what constituent(s) in these foods are singly or synergistically responsible for the observed effects?*-* for some years, investigators have attested to the relationship between food habits and cardiovascular diseases in different na- tional groups; case-control and cohort studies on the epidemiology of circulatory diseases have shown that disease prevalence, inci- dence, and mortality may differ greatly from one population to another. it is thought that the differences observed are partially due to aspects of lifestyle (such as dietary habits), as well as other characteristics specific to the groups studied. - i environmental change favoring higher levels of consumption of saturated fat, along with decreased physical activity, may also have conse- quences for incidence rates in colorectal cancer or other can- cers. - immigrants’ mortality patterns are influenced both by their native country and adopted nation, where the social environment may contribute to host resistance. assessment of determinants of dietary and other health behaviors in cultural and religious groups can thus provide clues to the interactive consequences of social, environmental, and biological influences on disease prevalence and outcome. the study of waves of migration in populations has underscored the impact of secular change on the risk of develop- ing certain chronic illnesses . among japanese living in hawaii, their initially low colorectal cancer risk has increased in conjunc- tion with the population’s dietary transition from a typical japa- nese diet to a more western one .x a recent report on the effect of migration on blood pressure among the yi people, an ethnic group in china, examined differences in lifestyle attributable to migra- tion.a the authors found that migration had contributed to dietary changes among men, with a resultant weight gain and alteration of their dietary sodium/potassium ratio. these changes were associ- ated with increased blood pressure among the migrant men. such findings support the argument that environmental factors-often dietary in nature-are of major importance as causes of variation in disease rates. dietary and nutritional factors underlie many conditions that contribute to other health disparities between societies. while the ability to clarify these findings is still limited, because relatively few nutritional epidemiologic studies have compared distinct groups on similar attributes, such studies would increase our understanding of the importance and interaction of factors leading to heightened disease risk. moreover, to assure successful health education programs, it is important to have identified local cultural practices and beliefs. in hawaii, researchers have begun to draw on the traditional, culturally-accepted diet to redress the impact of westerners on native hawaiian society. these authors stress the potential of intervention programs that draw on traditional diet as a means for preventing diseases such as cancer. others see this application as a means of limiting obesity and other chronic diseases. a similar culturally-based approach has been explored to encourage pima indians in the southwestern united states to return to their traditional diet to help decrease the dramatically high incidence of noninsulin dependent diabetes. assessing dietary intake in heterogeneous populations in na- tional surveys poses many methodologic, statistical, and interpre- tive issues. z nonetheless, health professionals in any multicul- tural society must be aware of population demographics, cultural influences on food consumption patterns, and health care usage by ethnic groups, a subject of escalating importance in the western world. anthropological background a clarification of common, but sometimes confusing, concepts in social anthropology should emphasize the importance of cul- tural orientation and adherence to belief systems, and their col- lective influence on attitudes and behavior that may ultimately affect health interventions. a ‘world view’ (socially-shared values and concepts about the universe, and people’s place in it) is defined as the first universal component of ideology. as its public manifestation, culture encompasses a complex and dynamic set of social norms, values, beliefs, cosmology, knowledge, and experi- ence that describe the workings of the world to the culture. as the expression of public, standardized values of a community, culture mediates the experience of individuals, and it provides basic categories for the creation of a positive pattern in which ideas and values are tidily ordered. an ethnic group, on the other hand, implies a hereditary component. it has a common culture that is also firmly rooted in a communal familial, economic, and social structure; and it generally originates in a particular geo- graphic area. cultural authority, often mediated through peer pressure, induces assent via the assent of others. while individuals who identify with a common set of cultural attributes may be of different ethnic or religious origins, a common ethnic background (which generally implies a common religious affiliation) further underscores the impact of cultural authority on its adherents. religion has been considered as the second universal compo- nent of ideology. it has traditionally been firmly rooted in the ethical values of community life, functioning on personal and social levels to provide a stable niche in a predictable environ- ment; this ultimately provides its adherents with an understand- ing of the world and the relationships that exist between individ- uals individuals may choose to practice their faith communally or in solitude, and their form of worship may vary according to personal spiritual needs or by occasion. thus, religion should be viewed not only as a haphazard relation of a single individual to a supernatural power, but also as a manifestation of the relation- ship of the whole community-as a collective entity of individu- als-to a power that has the good of the community at heart. although it might appear that active religious practice has de- clined in recent years, religions still provide networks of social and emotional support to many millions, often comprising behav- ior-monitoring structures that advise and influence adherents on activities considered acceptable to the group. furthermore, the major world religions have experienced a resurgence in funda- mentalist practice that has had far-reaching effects in some sectors of western society. culture and diet since foods have symbolic potential as ethnic identifiers, di- etary patterns of members of a related group tend to be conser- vative; the specific identifying foods are likely to remain as the culture, religion, diet, and health sociodemographic health/ dietary food beliefs diet consumed fig. . determinants of food intake. last vestige of a cultural identity.d when ethnic groups are rela- tively closed to out-migration or intermaxriage with nonmembers, the transmission and reinforcement of the food system continue via social interactions. diet can thus be construed as a cultural construct influenced by multiple factors that operate alone or together, , ’ reflecting progressive lifelong development accord- ing to acquired experience. specific food habits result from the combined sources of influence exerted by attitudes, beliefs, and experience on the food practices of a group or community, as well as economic factors in conjunction with local market resources. historically, each culture has had its particular “superfoods.” within a given cultural context, they fostered a common identity via their prestige value, magical properties, and relationship to body image, sometimes performing a special role, particularly in stages of the lifecycle. there is evidence to show that cultural identity and food habits have long supported and reinforced one another. in most societies,fiodwuys (traditional foods and ways of eating) are passed on through the children of a society, so that each subsequent generation knows what is properly considered as “food.“ therefore, determinants of dietary action may be found on an individual and collective scale. these influences can entail (i) personal status and economic resources; ( ) social, professional, or familial milieu; and ( ) ethnic origin, age, and sex of the individual. these work synergistically to affect a range of behav- iors and lifestyle factors that may ultimately determine health practices and disease outcome. even in modem society, the foods people prefer and select from their environment are depen- dent on factors that interact in a complex and dynamic cultural, social, and economic milieu. sm such foods often reinforce both ethnic identity and religious beliefs, and they reflect their ac- ceptability and availability to the consumer.’ in summary, psy- chosocial, cultural, and situational factors interact to determine food purchasing habits, as well as methods of preparation and consumption of these foods. these factors may lead to marked geographic, cultural, and ethnic differences, even when the food distribution network is well-developed and the same foods are available country-wide. figure depicts these determinants of dietary behaviour. religion, health, and diet it has been observed that cultural-religious beliefs and prac- tices can have a significant impact on health risks. the following section thus draws on religion as the cultural focal point, because religions often have health and dietary “rules” that are clearly- defined to adherents in written (usually holy) texts. unfortunately, little data exist on the prevalence of observance of dietary rules within religious groups. numerous studies have described differ- ential overall mortality, and age- and sex-specific mortality, in religious or ethnic groupings. morbidity and mortality outcome have been related to country of birth and ethnicity, ,jg religious affiliation and migrant status, or socioeconomic status$l differ- ent morbidity rates have been observed for cardiovascular dis- eases, hypertension and stroke, some cancers, inflammatory bowel diseases, and general health status in religious groups. significant associations have also been found for morbidity and mortality by subjective religiosity and religious attendance. major religious denomination (catholic, jewish, protestant) has been linked with longevity, mortality due to all causes, all neoplasms and specific cancer sites, s. @- as well as diabetes, respiratory diseases, and circulatory diseases. , research on religion and well-being indicates that religiosity- the degree of adherence to a set of religious beliefs-may reduce stress, symptom prevalence, prolong survival, and contribute to, or even produce, a healthier lifestyle. - its absence may aggravate poor health in the elderly, while death may be postponed until after occasions of major importance to religious ill persons. s religiosity was found to be important for the maintenance of morale and coping attitudes among both healthy and sick aging individuals.‘ furthermore, hannay determined that active reli- gious allegiance was associated with significantly fewer symp- toms (particularly mental, but also social and physical) in a mixed, mostly christian community in glasgow. it must be remembered that religions evolved within existing cultures, whereby established environmental and magical food habits were simply incorporated into emerging faiths. while all religions serve many of the same functions, striving to make sense of the inexplicable and to decrease anxieties associated with the unknown, religious adherence can enhance a feeling of unity and social solidarity within a group, where practicing members par- ticipate in common activities in an atmosphere often charged with emotion. however, to unite a people, they must at times be defined, even to themselves. food prescriptions or avoidances as practiced by a (religious) group foster a sense of security within that community. for example, in praying for good weather and an abundant harvest, north american christian fundamentalist farm- ers, such as the amish, attempt to “enlist the cooperation” of the supernatural forces and ensure a successful outcome of planting. food taboos, common to all religions, illustrate the concept of pollution and reflect moral issues.*’ the “taboo” (from a polyne- sian language, meaning “restraint imposed by social usage or as a protective measure”) was originally intended as a sacred pro- hibition enacted upon certain objects (in this case, foods), which rendered them untouchable, outcast by the culture. we now inter- pret food taboos as prohibitions on foods that are forbidden because of tradition or convention. -* food taboos may have an adaptive value; production of milk or eggs has the potential to feed far more people than the flesh of one individual cow or hen.* in the hindu doctrine, the religious reason for holding cattle sacred derives from the belief that the cow was created by bruhma on the same day as the brahmin. however, the practical value of using cattle to provide manure for fertilizer or work pulling plows, and considering milk and ghee (clarified butter) to be holy, as products of a sacred creature, may have economic and nutritional value for the community. overall, food restrictions result from numerous influences, such as magical and religious beliefs, fear of contamination, health concepts, and social structure. in some cases, these influences have become intertwined with highly varied, culture-specific food superstitions. food taboos may have originated in cultural unfa- culture, religion, diet, and health miliarity with a food, or its opposite-the rejection of possible foods that stem from creatures too familiar to a group (not eating certain domesticated animals, for example). it is noteworthy that many of the foods prohibited by religions on a temporary or permanent basis, are of animal origin. devout hindus and bud- dhists eat no meat at all; judaism and islam forbid the consump- tion of pork; orthodox christianity restricts meat intake on fasting days, and the catholic church used to prohibit meat on fridays.i most religions advocate religious fasting, perceived as a vo- tive, penitential act that is practiced in order to gain spiritual merit. fasting is usually associated with particular religious observances: as a way to increase introspection, remember a mournful event in the history of the religion, pay homage to the hungry, and bring the individual to a plane closer to god. most instances of religious fasting are brief and, depending on the religion and occasion, may take one of two forms: ( ) total abstinence from food and drink, or ( ) abstaining from certain dietary items (usually of animal origin). fasts represent a depar- ture from secular time, when people merge with the myths and sacred history; fasting can also be seen as an expression of religious piety, shared identity, and the community bond of a shared past and present.i health consequences of intermittent, short-term religious fasting are unknown. however, fasting is medically contraindicated in pregnant women, those with renal problems, or those with a psychosis in remission; insufficient fluid intake will lead to electrolyte imbalance, and prolonged fasting causes reduction in circulating blood glucose concentrations, stim- ulating lipolysis, with the release of free fatty acids and ketogen- esisg s as a means of reinforcing ethnic identity and religious beliefs, food traditions are conducive to psychological well-being.” be- cause religion strives to make sense of the inexplicable, it is not surprising that guidelines, rules, standards of behavior, and activ- ities exist within religions to ward off sickness and death. historically, religious restrictions on particular foods were meant to prevent moral, psychological, and physical harm; the obser- vance of religious dietary regulations, preserved in texts holy to their respective groups, has been encouraged since early times. such behaviors and attitudes may have either beneficial or harmful effects. however, cultural mores are transmitted from one gen- eration to another; and, as a learned phenomenon, tradition may be differentially applied by individuals within the cultural sub- group. for example, some of the observances and taboos of the jewish dietary laws (kashruth) are very deeply rooted in the historical background of judaism, whereas others arose in the diverse environments where jewish communities had become established and whence their varied culinary habits evolved. s ~ diet, lifestyle, and health outcome among selected religious groups particular groups with clearly defined, unusual, or unique, practices (often related to religious aspects of their cultural pre- cepts) have been studied in order to better understand the nature of such habits in relation to health outcome. a sampling of studies published in the last years exploring associations between religion, diet, and health may be found in table i. among seventh-day adventists?’ mormons, or jews, z*s- some dietary and other lifestyle attributes related to religious beliefs, ethnicity, and birthplace (kark jd, personal communication, ) appear to be expressed in differential-generally advantageous- incidence and mortality rates for a range of diseases. other population groups follow religious dietary rules domi- nated by humoral concepts, such that particular foods are ordained or proscribed at critical periods in the life cycle.ss,ss- though these humoral concepts clearly have philosophical and social value, they may place young children, pubescent girls, and preg- nant and lactating women at nutritional risk during physiologically critical times. both islam and the baha’i faith have religious dietary rules; those of the former group resemble some aspects of the jewish dietary laws; the latter group’s dietary recommendations, while not obligatory to followers, resemble both the healthy eating principles of mormons and the reverence for life of the buddhists. however, we are unaware of published dietary studies among these groups. on the other hand, the buddhist doctrine stipulates that it is forbidden to kill living beings. ogata et al.iai have observed lower mortality from all causes in zen buddhist priests consuming traditional japanese diets with little western influence (fewer than % ate meat and fish on a daily basis), and having low rates of smoking but similar alcohol intakes, compared to other japanese men. in a study of diet and hemostatic factors associated with high risk of coronary heart disease, pan et a .i found that young buddhist vegetarians had significantly lower concentrations of plasma cholesterol, glucose, and uric acid; and they consumed % less fat, % more carbohydrate, and % less protein, compared to omnivores. the polyunsaturated to saturated fat (ps) ratios of these vegetarians were more than three times higher than those of the omnivorous group. perhaps the most widely-studied of modem religious groups, seventh-day adventists are noted for their health-promoting life- style. phillips et al. lo have examined the contribution of selection versus lifestyle on the risk of cancer and cardiovascular disease among american seventh-day adventists. though they were unable to clearly distinguish between these two hypotheses, it would appear that the adventists’ lifestyle afforded them the greater measure of protection from cancer and cardiovascular- disease mortality. in fact, these authors suggested that protective influences act even among converts who vigorously adopt church principles; consequently, in spite of their often lower socioeco- nomic status at conversion into the faith, they enjoy the protection afforded by adherence to the healthy living principles recom- mended by the church. these principles espouse lacto-ovo veg- etarianism, with little consumption of animal food, high intakes of vegetables and fruits, little caffeine, no alcohol, and no tobacco use. the impact of these recommendations has been carefully studied among adventists in california in relation to their degree of adherence to these religious dietary recommendations. findings indicate that intakes of meat and eggs are related to increased mortality from coronary heart disease, diabetes, and some neo- plasms; while consumption of leafy green vegetables has had the opposite impact in this population. i~i~ in another large cohort of american adventists followed since for determinants of coronary heart disease, frequent consumption of nuts and whole wheat bread has been observed to be protective.‘ while it was suggested that the unique fatty acid composition of the nuts and the high fiber content of both food groups might be chiefly responsible for this effect, it has also been speculated that these foods could merely be markers of other causal factors, although no evidence of confounding was identified. danish and japanese adventists have demonstrated lower risks for tobacco and alcohol- related cancers as well, not only due to their lesser use of these substances but also because of their low-meat, low-fat, and high- fiber dietary habits.ieevio in other adventist populations, low frequencies of consumption of broiled meat or fish (consequently contributing little benzo[cw]pyrene and nitrosomines to the diet) are likely to be responsible for their low risk for cancers of the digestive tract.lo .io in addition, their greater intakes of foods furnishing vitamins a and c may also protect against many of the major sites of cancer.io members of the church of jesus christ of latter-day saints (mormons) experience lower mortality from the “diseases of culture, religion, diet, and health table i. diet, lifestyle and health outcome in selected religious groups reference population/religious group country dietary characteristics studied other variables examined outcome jarvis, [ ] mormons phillips and kuzma, [io phillips et al., [io jensen, [ ] kahn et al., [lw ogata et al., do kuratsune et al., [ ] snowdon, [ ] fuchs et al., kapil et al., [ bitterman et al., [llo] fraser et al., llw pan et al., [lo shatenstein et al., a, b [ , seventh-day adventists seventh-day adventists seventh-day adventists seventh-day adventists zen buddhist priests seventh-day adventists seventh-day adventists old order amish jat hindus jews and non- jews seventh-day adventists buddhists ultra-orthodox jews canada japan united states denmark united states japan japan united states united states india israel united states taiwan canada “balanced diet”: whole grains, plant protein, fruits and vegetables, little meat low intakes of broiled foods; antioxidant vitamins lacto-ovo vegetarianism; low animal food intake lacto-ovo vegetarianism; fat, fiber lacto-ovo vegetarianism; animal food intake traditional japanese diet lacto-ovo vegetarianism; broiled fish consumption lacto-ovo vegetarianism; low animal products intake food and eating humoral theory (“hot” vs. “cold”; “light vs. “heavy”) fluid intake, olives and olive oil, cumin, pepper dietary determinants of chd risk factors buddhist vegetarian diet kosher diet cohesive community lifestyle, church life benzo[a]pyrenes, nitrosamines selection versus lifestyle smoking; alcohol intake health history; smoking; age at exposure to adventist church smoking, alcohol mutagenic pyrolyzates in foods mortality from major chronic diseases and all causes stress, obesity, physical activity, parity maternal beliefs on diet and childhood illnesses exposure to sun, chemical products, smoking habits lifestyle habits related to chd risk plasma lipids, apolipoproteins, glucose, uric acid jewish religious law; jewish lifestyle low mortality from “diseases of affluence” low cancers of digestive tract low mortality; low chd, cancer, diabetes low tobacco and alcohol- related cancers mortality higher with meat/egg intakes; lower with salad consumption low all-cause mortality overall reduced mortality low meat and egg intake, higher milk; low all-cause mortality and chronic disease mortality low hypertension, low stress, low circulatory disease mortality identification of local cultural practices for nutrition education differences in urologic cancers related to different dietary patterns low risk of coronary heart disease in frequent consumers of nuts and whole wheat bread low plasma cholesterol, glucose, uric acid, apolipoproteina- :b low animal protein, low relative fat intake, high p:s ratio chd, coronary heart disease. affluence” than the general population. adherents to this reli- gious philosophy follow a lifestyle prescribed by their church, in the belief that the human body is the temple of god and should be preserved in fit condition, by abstaining from caffeine, alcohol, and tobacco, while eating a balanced diet that emphasizes whole grains, plant proteins, fruits, and vegetables and discourages ex- cess intake of meat. a cohesive community lifestyle is advocated, stressing strong family ties and social activities revolving around church life. among the old order amish in rural ohio, fuchs et a . found differences in self-reports of behavioral risk factors as compared to non-amish rural ohio residents. lower rates of hypertension, smoking, alcohol consumption, and stress were re- ported in this amish community. some of these factors could contribute to their lower mortality due to circulatory and cardio- vascular diseases. although obesity was more prevalent (probably related to the great importance attached by the amish to food and eating, and possibly associated with high parity in the women, and decreased physical activity among middle-aged and older men, whose sons did much of the farm work), it appeared to be more tolerated or less stigmatized in amish culture. in the acre district in israel, a high incidence of urinary tract cancers has been observed among jews compared to non-jews: bitterman et al.l ° have attributed this to lower intakes of fluids, olives and olive oil, and the spices cumin and pepper by the jewish group. and, while ultra-orthodox jews in montreal are more likely to be culturally homogeneous than the various seventh-day adventist groups studied worldwide, an interesting parallel may be drawn between adventist converts who enthusiastically adopt church doctrine, and those non-traditional or secular jews who embrace fundamentalist judaism; many of the latter wholeheart- edly adopt dietary and lifestyle practices (eating a kosher, some- times vegetarian, diet, or giving up smoking) upon becoming religious, which may decrease their former levels of risk for some chronic diseases. most noteworthy is their low mean relative fat intake ( % of energy) and high p:s ratio ( . ).* , it has been observed that montreal jews overall have low all-cause mortality, principally due to their strikingly low rates of death due to circu- latory system diseases. the jewish motivation in health-related issues has a religious origin, where the bible refers to disease as a “manifestation of the anger of god,” who will heal if his commandments are heeded. jewish religious law (h&&&z) or- dains that anything potentially injurious to well-being must be avoided, and lifestyle must be conducive to good health. decades ago, it was postulated that low jewish mortality rates might result from adherence to “a traditional way of life, customs and rules of living.“ findings such as these have contributed to the highly contro- versial literature on associations between diet and the major chronic diseases of developed nations.gg however, recent dietary data and their determinants are largely unavailable for many minority ethnic groups in north america and europe or else- where, hampering our ability to estimate relative risks of diet and disease. cultural, ethnic, and religious dietary habits are in constant evolution from their place of origin, incorporating envi- ronmental adaptation and changes in societal mores, while form- ing an integral part of the individual’s self-perception. the pri- mary importance of family, community maintenance, and solidarity among certain religious groups, along with their philos- ophy and tradition, may well exert a salutary role. surveys of cultural or religious practices as determinants of health behaviors culture, religion, diet, and health and outcome appear to have strengthened this view, and certain types of community affiliation and cohesiveness seem to mediate life stressors. , for example, a recent study of mortality dif- ferences in two jewish populations appears to indicate that com- munity cohesiveness, socioeconomic status, and ethnic origin (possibly reflecting genetic differences as well) may play a major role in determining mortality patterns. implications for researchers and clinicians some investigators have undertaken specific evaluations of dietary attributes and other health behaviours according to estab- lished cultural practices, including religion and degree of religi- osity. , , - . , ’s however, comparative studies must be pur- sued in the changing multicultural environments of north america, europe, and elsewhere. this will provide valuable em- pirical information to add to our understanding of the interwoven influences of cultural attributes on health-related behaviors, and it will raise the potential for improving health through culture-based effective nutrition interventions. g, , while the suggestion of a differential impact on health related to active versus passive religious allegiance might provide some insight into this issue, others have indicated that the absence of a clearly defined notion of the perceived meanings of religion or religiosity, and inconsistencies in the way in which these variables are handled, have blocked progress.‘ the challenge lies in dis- tinguishing between membership and participation, defining ex- tent of observance or compliance, and separating the effects of religious injunctions from other variables (socioeconomic status, social support, ethnicity, dietary regimens, lifestyle, and others) that confound the issue. ,“ adherence to particular beliefs within a cultural context may be indicative of favorable or harmful health attributes. health professionals must seek to understand the contribution of these various religious or culturally-mediated behaviors and work to clarify their potential nutritional and overall health benefits. di- etary assessment should, therefore, integrate markers of the cul- tural determinants of food consumption and other behaviors into routine nutritional evaluation and health-assessment strategies. this approach will aid in evaluating dietary adequacy and related health practices within the context of the consumer’s sociocultural value system. it should result in improved counselling practices, in order to meet the ultimate goal of diminishing risk for chronic diseases. references . . . . . . thomassin l. l’kvolution socio-culturelle des comportements ali- mentaires. in: le comportement du consommateur dans l’kosysteme alimentaire. laval: colloque universitaire, : coates tj. eating-a psychological dilemma. j nutr ed ; 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levin js. is there a religious factor in health care utilisation? a review. sot sci med ; : . shovic ac. development of a samoan nutrition exchange list using curturally accepoted foods. j amer diet assoc ; : . levin js, vanderpool hy. is frequent religious attendance really conducive to better health?: toward an epidemiology of religion. sot sci med ; : (for an additional perspective see editorial comments on page .) prevalence and management of iron overload in pyruvate kinase deficiency: report from the pyruvate kinase deficiency natural history study | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /haematol. . corpus id: prevalence and management of iron overload in pyruvate kinase deficiency: report from the pyruvate kinase deficiency natural history study @article{beers prevalenceam, title={prevalence and management of iron overload in pyruvate kinase deficiency: report from the pyruvate kinase deficiency natural history study}, author={e. v. van beers and stephanie van straaten and d. morton and w. barcellini and s. eber and b. glader and h. yaish and s. chonat and j. kwiatkowski and jennifer a rothman and mukta sharma and e. neufeld and s. sheth and j. despotovic and n. kollmar and d. posp{\'i}{\vs}ilov{\'a} and c. knoll and k. kuo and y. pastore and a. thompson and p. e. newburger and y. ravindranath and w. wang and m. wlodarski and h. wang and s. holzhauer and v. breakey and m. verhovsek and j. kunz and melissa m mcnaull and m. rose and heather a. bradeen and kathryn addonizio and a. li and hasan al‐sayegh and w. london and r. grace}, journal={haematologica}, year={ }, volume={ }, pages={e - e } } 	e. v. van beers, stephanie van straaten, + authors r. grace 	published 	medicine 	haematologica pyruvate kinase (pk) deficiency is the most common red cell glycolytic enzyme defect causing hereditary non-spherocytic hemolytic anemia. current treatments are mainly supportive and include red cell transfusions and splenectomy.[ ][ ] regular red cell transfusions are known to result in iron  view on fsf haematologica.org save to library create alert cite launch research feed share this paper citationsbackground citations view all figures, tables, and topics from this paper 	 table 	 figure 	iron overload 	deficiency of pyruvate kinase 	lesch-nyhan syndrome 	beer paper mentions observational patient registry clinical trial pyruvate kinase deficiency natural history study the purpose of this study is to describe the range and incidence of symptoms, treatments, and complications related to pyruvate kinase deficiency (pkd). eligible patients are those of… expand 	conditions	congenital non-spherocytic hemolytic anemia, pyruvate kinase deficiency boston children's hospital december - may citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency comorbidities and complications in adults with pyruvate kinase deficiency 	a. boscoe, y. yan, + authors r. grace 	medicine 	european journal of haematology 	 	 save alert research feed management of pyruvate kinase deficiency in children and adults. 	r. grace, w. barcellini 	medicine 	blood 	 	 save alert research feed how we manage patients with pyruvate kinase deficiency 	r. grace, d. mark layton, w. barcellini 	medicine 	british journal of haematology 	 	 save alert research feed molecular heterogeneity of pyruvate kinase deficiency. 	p. bianchi, e. fermo 	medicine 	haematologica 	 	 	 save alert research feed the variable manifestations of disease in pyruvate kinase deficiency and their management. 	h. al-samkari, e. v. van beers, + authors r. grace 	medicine 	haematologica 	 	 	 save alert research feed a family affected by a life-threatening erythrocyte defect caused by pyruvate kinase deficiency with normal iron status: a case report 	karolina maciak, a. adamowicz-salach, j. poznański, m. góra, j. fronk, b. burzynska 	medicine 	frontiers in genetics 	 	 	view excerpts, cites background save alert research feed laboratory approach to investigation of anemia with a focus on pyruvate kinase deficiency 	a. agarwal, anton rets 	medicine 	international journal of laboratory hematology 	 	 save alert research feed development of the pyruvate kinase deficiency diary and pyruvate kinase deficiency impact assessment: disease‐specific assessments 	s. salek, a. boscoe, + authors michael storm 	medicine 	european journal of haematology 	 	 save alert research feed safety and efficacy of mitapivat in pyruvate kinase deficiency. 	r. grace, c. rose, + authors b. glader 	medicine 	the new england journal of medicine 	 	 	 save alert research feed iron overload in congenital haemolytic anaemias: role of hepcidin and cytokines and predictive value of ferritin and transferrin saturation 	w. barcellini, a. zaninoni, + authors m. cappellini 	medicine 	british journal of haematology 	 	 	 save alert research feed ... ... references showing - of references erythrocyte pyruvate kinase deficiency: status report 	r. grace, a. zanella, + authors b. glader 	medicine 	american journal of hematology 	 	 	 	view excerpt, references background save alert research feed clinical spectrum of pyruvate kinase deficiency: data from the pyruvate kinase deficiency natural history study. 	r. grace, p. bianchi, + authors w. barcellini 	medicine 	blood 	 	 	pdf 	view excerpts, references background save alert research feed iron chelation therapy in transfusion-dependent thalassemia patients: current strategies and future directions 	a. saliba, a. harb, a. taher 	medicine 	journal of blood medicine 	 	 	pdf 	view excerpt, references methods save alert research feed tailoring iron chelation by iron intake and serum ferritin: the prospective epic study of deferasirox in patients with transfusion-dependent anemias 	m. cappellini, j. porter, + authors a. kattamis 	medicine 	haematologica 	 	 	pdf save alert research feed liver iron concentrations and urinary hepcidin in beta-thalassemia. 	r. origa, r. galanello, + authors e. nemeth 	biology, medicine 	haematologica 	 	 	 	view excerpt, references results save alert research feed randomized phase ii trial of deferasirox (exjade, icl ), a once-daily, orally-administered iron chelator, in comparison to deferoxamine in thalassemia patients with transfusional iron overload. 	a. piga, r. galanello, + authors d. alberti 	medicine 	haematologica 	 	 	pdf save alert research feed the clinical spectrum of pyruvate kinase deficiency: data from the pyruvate kinase deficiency natural history study 	blood 	 iron-chelating therapy for transfusional iron overload. 	m. taccone-gallucci, s. manca-di-villahermosa, a. noce 	medicine 	the new england journal of medicine 	 	 	 	view excerpt, references methods save alert research feed related papers 	abstract 	figures, tables, and topics 	paper mentions 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue [pdf] management of lymphomas: consensus document by an indian expert group | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /s - - - corpus id: management of lymphomas: consensus document by an indian expert group @article{nair managementol, title={management of lymphomas: consensus document by an indian expert group}, author={r. nair and abhishek kakroo and a. bapna and a. gogia and a. vora and a. pathak and a. korula and a. chakrapani and d. doval and g. prakash and g. biswas and h. menon and m. bhattacharya and m. chandy and m. parihar and m. vamshi krishna and n. arora and nikhil gadhyalpatil and p. malhotra and p. narayanan and r. basu and sandip shah and s. bhave and s. bondarde and s. bhartiya and s. nityanand and s. gujral and t. k. tilak and vivek radhakrishnan}, journal={indian journal of hematology & blood transfusion}, year={ }, volume={ }, pages={ - } } 	r. nair, abhishek kakroo, + authors vivek radhakrishnan 	published 	medicine 	indian journal of hematology & blood transfusion the clinical course of lymphoma depends on the indolent or aggressive nature of the disease. hence, the optimal management of lymphoma needs a correct diagnosis and classification as b cell, t-cell or natural killer (nk)/t-cell as well as indolent or high-grade type lymphoma. the current consensus statement, developed by experts in the field across india, is intended to help healthcare professionals manage lymphomas in adults over  years of age. however, it should be noted that the… expand view on springer link.springer.com save to library create alert cite launch research feed share this paper citationsbackground citations view all figures, tables, and topics from this paper 	 table 	 figure 	 table 	 figure 	 table 	 figure 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table 	 table view all figures & tables 	lymphoma, follicular 	lymphoma 	chronic lymphocytic leukemia 	t-cell lymphoma 	natural killer 	lymphoid leukemia 	diffuse large b-cell lymphoma 	b-cell lymphomas 	leukemia, b-cell 	ki- + anaplastic large cell lymphoma 	chronic lymphocytic leukemia/small lymphocytic lymphoma 	reticulosarcoma citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency the usefulness of morphology in diagnosing various malignancies on bone marrow examination in adults- a single centre study 	nazia khalid, attika khalid, l. zafar, nadia arif, mehmood ul hasan 	medicine 	 	pdf save alert research feed image‐guided core needle biopsy as the first‐line diagnostic approach in lymphoproliferative disorders—a review of the current literature 	d. seviar, m. yousuff, z. chia, k. ramesar, j. newman, d. howlett 	medicine 	european journal of haematology 	 save alert research feed lymphoma in ‘india’ at the dawn of targeted therapies 	r. kapoor, r. kumar 	medicine 	indian journal of hematology and blood transfusion 	 	pdf save alert research feed przydatność badania pet/ct w diagnostyce chłoniaka hodgkina 	r. zając, a. pietrzak, w. cholewiński 	medicine 	 	pdf 	view excerpt save alert research feed lysophosphatidic acid promotes survival of t lymphoma cells by altering apoptosis and glucose metabolism 	v. gupta, pradip kumar jaiswara, pratishtha sonker, shiv govind rawat, rajan kumar tiwari, a. kumar 	chemistry, medicine 	apoptosis 	 	 	view excerpt, cites background save alert research feed references showing - of references sort byrelevance most influenced papers recency mantle cell lymphoma: evolving management strategies. 	e. campo, s. rule 	medicine 	blood 	 	 	pdf save alert research feed b cell non-hodgkin’s lymphoma: experience from a tertiary care cancer center 	g. prakash, a. sharma, v. raina, l. kumar, m. c. sharma, b. mohanti 	medicine 	annals of hematology 	 	 save alert research feed indian council of medical research consensus document for the management of non-hodgkin's lymphoma (high grade) 	d. doval, d. bhurani, + authors g. rath 	medicine 	indian journal of medical and paediatric oncology : official journal of indian society of medical & paediatric oncology 	 	 	 	view excerpts, references background save alert research feed mantle cell lymphoma: a north indian tertiary care centre experience 	c. das, a. gogia, l. kumar, a. sharma, m. sharma, s. mallick 	medicine 	asian pacific journal of cancer prevention : apjcp 	 	 	pdf save alert research feed esmo consensus conferences: guidelines on malignant lymphoma. part : marginal zone lymphoma, mantle cell lymphoma, peripheral t-cell lymphoma. 	m. dreyling, c. thieblemont, + authors e. zucca 	medicine 	annals of oncology : official journal of the european society for medical oncology 	 	 	pdf save alert research feed follicular lymphoma: an institutional analysis 	a. gogia, v. raina, l. kumar, a. sharma, m. sharma, s. mallick 	medicine 	asian pacific journal of cancer prevention : apjcp 	 	 	pdf save alert research feed guidelines for the investigation and management of mantle cell lymphoma 	p. mckay, m. leach, r. jackson, g. cook, s. rule 	medicine 	british journal of haematology 	 	 	pdf 	view excerpt save alert research feed relapse in common lymphoma subtypes: salvage treatment options for follicular lymphoma, diffuse large cell lymphoma and hodgkin disease 	b. seyfarth, a. josting, m. dreyling, n. schmitz 	medicine 	british journal of haematology 	 	 	 save alert research feed histopathological pattern of lymphomas and clinical presentation and outcomes of diffuse large b cell lymphoma: a multicenter registry based study from india 	rameshb. v. nimmagadda, r. digumarti, + authors a. pathak 	medicine 	indian journal of medical and paediatric oncology : official journal of indian society of medical & paediatric oncology 	 	 	 save alert research feed hodgkin lymphoma version . , nccn clinical practice guidelines in oncology. 	r. hoppe, r. advani, + authors hema m sundar 	medicine 	journal of the national comprehensive cancer network : jnccn 	 	 	pdf save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue tice of medicine and the life of the practitioner in the community. david loxterkamp is a family physician in a small coastal town in maine. in his th year in practice he chose to record his life with regular journal entries, which he then worked into this book. in doing so, he pulls the reader into his life and provides a very enjoy- able reading experience. in the process of examination he seems to have developed a fuller sense of self, home, family, and community, and we are privileged to share that journey. there is a chapter for each month of the year and for each of the four seasons, but the book is much more than an accounting of those days. by moving from the events of the day back into memory of earlier years and by anticipating the future, the author weaves a captivating narrative. the title indicates that the pages will reveal the daily life of a country doctor, and the book delivers on that promise. it reveals with hon- esty and candor the highs and lows and the growth of its author as physician, husband, father, and person of faith. it is a book about community and a deepening commionent to one community as home. dr. loxterkamp's gift with words ensures that the reader will experience the events and emotions of the days recorded. during the several days i was reading the book, i found myself experiencing elation, depres- sion, anxiety, and contenonent. at first i thought these emotions reflected the undulating circumstances of my own days, but i later concluded that was not so. i be- lieve the writing evokes in the reader the emotions and feelings of the author as he reacts to the circumstances of his life. i suspect also that each reader will react dif- ferently to the events described through memory of personal experience. a physician with community practice background will find this journal stimulates recall of the early years of establishing practice, home, and family. some who felt isolation and frustration in those years might find reassurance that their experiences were not unique. anyone who seeks to understand the practice of medi- cine in community would benefit from reading this work, as would those considering such a career. this book should be read by persons responsible for plan- ning, shaping, financing, or regulating community health care systems and organizations, for in doing so, they would develop an intimate understanding of the workings of such systems and the very personal needs they try to meet. such an understanding cannot be found in the collection of diagnostic classifications, lists of symptoms, reports of encounters and proce- dures, and similar tabulations used to describe or eval- uate a medical practice. dr. loxterkamp reveals his life to be one of inten- sity, commitment, and searching. he also shares his contenonents and satisfactions. one might wish him more sheer joy and happiness than what seems to come through in the narrative. but, he says of himself (page ): "there is always a tension between enjoying life and examining it." theodore j. phillips, md lopez island, wash patients are a virtue: practicing medicine in the pennsyl- vania amish country. by henry s. wentz. pp. morgan/gum, pa, masthofpress, . . . isbn - - - . have you wondered what it would be like to practice medicine from to among the amish in lan- caster county, pennsylvania? this fascinating period in medicine spanned the early years of antibiotics, the scourges of polio and rheumatic fever, the shift from solo to group practice, and the changes in health care financing from the $ fee-for-service office visit to medical insurance to managed care. dr. wentz chronicles these events, covering four decades, through short vignettes. he writes in a matter-of-fact, straightforward, conversational style that is most likely consistent with the way in which he practiced and cared for his patients. as he relates the satisfactions and frustrations of his practice, he inter- sperses reflections on lessons he learned, such as the value of nurses, the power of suggestion, the courage of patients facing adversity, and the importance of a supportive family and community for both patient and physician. perhaps his most interesting insights evolve from experiences with his amish patients that illustrate how their beliefs sustained them as they dealt with the challenges of life and death. he also describes the diffi- culties of getting the amish to accept such preventive measures as prenatal care, well-baby examinations, and immunizations. throughout these episodes dr. wentz conveys ac- ceptance of his patients, himself, and his times without undue moral judgment or philosophical speculation. nevertheless, the reader becomes aware of the unique problems associated with practicing alone out of an of- fice in one's home, the stresses on marriage and chil- dren, the interruptions of family gatherings by patients with emergencies, and the concerns of losing patients to a competing physician in the community. most of these stories are not particularly dramatic. their impact arises from the cumulative descriptions of a type of practice that will not be repeated. a physi- cian emerges who appeared to possess the qualities necessary to succeed during a unique time in medical history within a geographical location enriched by people with distinctive motivations and moral princi- ples. thus, this book might be of interest to those physicians who would like to know more about this way of life, to people living in the vicinity of lancaster county, and to lay persons who wonder about the daily activities of a busy general practitioner. vincent r. hunt, md brown university-memorial hospital of rhode island pav.'tucket book reviews o n a p ril b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://w w w .ja b fm .o rg / j a m b o a rd f a m p ra ct: first p u b lish e d a s . / - - - o n ja n u a ry . d o w n lo a d e d fro m http://www.jabfm.org/ the evolutionary history of membrane-integral pyrophosphatases supports na+ as the ancestral coupling ion in membrane bioenergetics stress conditions [ ]. the transporter contributes to virulence of pathogens such as staphylococcus aureus and helicobacter pylori. we utilize putp of escherichia coli as a model to explore structure and molecular mechanism of function of sssf proteins. here, we present a model of the helix bundle of putp obtained by molecular modeling constrained by experimentally determined intra- molecular distances and template restraints derived from the ten- helix core of the vsglt crystal structure [ ]. for this purpose, deer distance measurements between spin labels attached to helix ends were conducted and mean interspin distances were determined. fitting algorithm based on matrix geometry in combination with prediction of spin label conformations by a rotamer library approach [ ] resulted in an ensemble of helix bundle structures. the central structure of the ensemble showed a core structure with a fold similar to that of the vsglt template. furthermore, analysis of spin label motility and environmental polarity by cwepr yielded information on secondary structure elements and structural rearrangements of external loop (el) of putp upon sodium and/or l-proline binding. the results support the idea that el controls access to the sodium and/or l-proline binding site(s) similar as previously proposed for el of leut [ ]. references [ ] h. jung, febs lett. ( ) – . [ ] s. faham, a. watanabe, g.m. besserer, d. cascio, a. specht, b.a. hirayama, e.m. wright, j. abramson, science ( ) – . [ ] y. polyhach, e. pordignon, g. jeschke, phys. chem. chem. phys. ( ) – . [ ] d.p. claxton, m. quick, l. shi, f.d. de carvalho, h. weinstein, j.h. javitch, h.s. mchaourab, nat. struct. mol. biol. ( ) – . doi: . /j.bbabio. . . p mitochondrial carrier structure and diseases pierri ciro leonardo, palmieri ferdinando department of biosciences, biotechnology and pharmacological sciences, laboratory of biochemistry and molecular biology, university of bari, italy e-mail: ciroleopierri@gmail.com to date eleven disorders are known to be caused by defects of mito- chondrial carriers, a family of proteins that shuttle a variety of metabolites across the inner mitochondrial membrane. mutations of mitochondrial carrier genes are responsible for carnitine/acylcarnitine carrier deficien- cy, ornithine carrier deficiency (hhh syndrome), aspartate/glutamate isoform deficiency (global cerebral hypomyelination), aspartate/ glutamate isoform deficiency (ctln and niccd), amish microcephaly, early epileptic encephalopathy, congenital sideroblastic anemia, pic defi- ciency, adp/atp carrier isoform deficiency, neuropathy with bilateral striatal necrosis and adpeo (autosomal dominant progressive external ophthalmoplegia). structural, functional and bioinformatics studies have revealed the existence in mitochondrial carriers of a substrate-binding site in the internal carrier cavity, of two gates that close the cavity alternatively on the matrix or cytosolic side of the membrane, and of two sets of prolines and glycines in the six transmembrane a-helices located strategically between the substrate-binding site and the two gates. the key role played by these mitochondrial carrier areas is supported by the observation that they host most of the disease-causing missense mutations of the mitochondrial carriers. references [ ] e. pebay-peyroula, c. dahout-gonzalez, r. kahn, v. trézéguet, g.j. lauquin, g. brandolin, nature ( ) – . [ ] a.j. robinson, e.r. kunji, proc. natl. acad. sci. u. s. a. ( ) – . [ ] f. palmieri, biochim. biophys. acta ( ) – . [ ] f. palmieri, c.l. pierri, febs lett. ( ) – . [ ] f. palmieri, mol. aspects med. (in press). http://dx.doi.org/ . /j.mam. . . . doi: . /j.bbabio. . . p insights into the mechanism of the na+/ca + exchanger from atomistic molecular dynamics simulations fabrizio marinelli, josé d. faraldo-gómez theoretical molecular biophysics group, max planck institute of biophysics, max-von-laue strasse , frankfurt am main, germany e-mail: fabrizio.marinelli@biophys.mpg.de na+/ca + exchangers (ncx) and potassium-dependent na+/ ca + exchangers (nckx) are two related families of transporters involved in ca + signaling that function by extruding cytosolic ca + (and k+ for the potassium-dependent transporter) in exchange for extracellular na+ [ ]. previous studies have established that this exchange process is electrogenic and with a defined stoichiometry, and have identified specific acidic aminoacids believed to be crucial for ion binding and translocation [ – ]. recently the crystal structure of the ncx from methanococcus jannaschii was determined at . Å resolution [ ], revealing an intriguing transmembrane topology consisting of inverted structural repeats, and the presence of four putative ion binding sites formed by highly conserved residues. notwithstanding these groundbreaking insights, based on the struc- ture alone several ion occupancy states can be hypothesized that would be compatible with the experimental exchange stoichiometry. moreover, in the crystal the protein adopts a unique outward facing conformation, which does not immediately explain how ion binding to the protein facilitates the necessary outward-to-inward conforma- tional transition. here, we use extensive molecular simulations and molecular modeling to investigate the occupancy and specificity of the ion binding sites in ncx_mj, and the microscopic mechanism by which na+ and ca + are exchanged across the membrane. references [ ] v. frank, j. lytton, physiology ( ) – . [ ] k. kang, t.g. kinjo, r.t. szerencsei, p.p.m. schnetkamp, j. biol. chem. ( ) – . [ ] t.m. kang, d.w. hilgemann, nature ( ) – . [ ] j. liao, h. li, w. zeng, d.b. sauer, r. belmares, y. jiang, science ( ) – . doi: . /j.bbabio. . . p the evolutionary history of membrane-integral pyrophosphatases supports na+ as the ancestral coupling ion in membrane bioenergetics heidi h. luoto , alexander a. baykov , reijo lahti , anssi m. malinen department of biochemistry and food chemistry, university of turku, fin- turku, finland abstracts s http://dx.doi.org/ . /j.bbabio. . . http://dx.doi.org/doi: . /j.mam. . . http://dx.doi.org/doi: . /j.mam. . . http://dx.doi.org/ . /j.bbabio. . . http://dx.doi.org/ . /j.bbabio. . . belozersky institute of physico-chemical biology, lomonosov moscow state university, moscow , russia e-mail: hhluot@utu.fi membrane-integral pyrophosphatases (mppases) are primary h+- or na+-ion pumps directly energized by pyrophosphate, an abundant byproduct of anabolic reactions. mppases are widespread in all domains of life and provide the host necessary energy reserves, particularly during stress and low-energy conditions. the enzyme holds promise in biotechnology insofar as agricultural plants that overexpress mppase have salt- and drought-tolerant phenotypes. recent work has uncovered significant functional divergence among members of the mppase protein family. notably, mppases differ in pumping specificity and sensitivity to k+ ions [ , ]. all k+- independent mppases operate as h+-pumps, whereas most k+- dependent mppases are primary na+-pumps. however, several types of k+-dependent, h+-pumping mppases are known. one particular mechanism that allows a change in transport specificity from na+ to h+ is spatial repositioning of a glutamate residue that forms part of the cytoplasmic gate in the ion transport channel [ ]. the reconstructed evolutionary history of mppases suggests that the ancestral enzyme operated as a na+-pump and the transition to h+-pumping occurred in several independent enzyme lineages [ ]. these data lend support to the hypothesis of primordial na+-based membrane bioenergetics [ ]. na+- and h+-pumping mppases are structurally very similar [ ], supporting the concept, first proposed for a rotating atp-synthase/atpase, that switching between na+ and h+ transport specificities requires only subtle changes in structure [ ]. references [ ] g.a. belogurov, r. lahti, j. biol. chem. ( ) – . [ ] a.m. malinen, g.a. belogurov, a.a. baykov, r. lahti, biochemistry ( ) – . [ ] h.h. luoto, g.a. belogurov, a.a. baykov, r. lahti, a.m. malinen, j. biol. chem. ( ) – . [ ] a.y. mulkidjanian, m.y. galperin, k.s. makarova, y.i. wolf, e.v. koonin, biol. direct ( ) . [ ] s.m. lin, j.y. tsai, c.d. hsiao, y.t. huang, c.l. chiu, m.h. liu, j.y. tung, t.h. liu, r.l. pan, y.j. sun, nature ( ) – . doi: . /j.bbabio. . . p characterization and purification of the multi subunit type na+/h+ antiporter from alkaliphilic bacillus pseudofirmus of masato morino , , toshiharu suzuki , masahiro ito graduate school of life sciences, toyo university, oura-gun, gunma - japan department of pharmacology and systems therapeutics, mount sinai school of medicine, new york, ny , usa jst icorp atp, japan e-mail: masahiro.ito@toyo.jp mrp antiporters are monovalent cation/proton antiporters which exchange cytoplasmic na+, li+ and/or k+ ions for extracellular h+. they are widespread among bacteria and archaea. mrp antiporters have seven or six hydrophobic proteins that are encoded in the mrp operons, in contrast to most of bacterial na+/h+ antiporters which are single gene products. interestingly, the entire na+/h+ antiport activity requires all of these proteins, suggesting that mrp antiporters function as a hetero-oligomeric protein complex in the cytoplasmic membrane. purification and functional reconstitution of the mrp antiporter have not been reported. therefore, we purified and recon- stituted the mrp antiporter from alkaliphilic bacillus pseudofirmus of , because purification of target proteins and their complex with the native conformation is required for further functional and struc- tural research. the mrp antiporter expressed in major cation/h+ antiporter-defective escherichia coli strain knabc cells was purified by immobilized metal ion adsorption chromatography (imac). the purified mrp samples were reconstituted into artificial mem- brane vesicles (liposomes) with fof -atpase from bacillus sp. ps as the “power supply” to generate a proton motive force required for activation of the mrp antiporter. the na+/h+ antiport activity of the purified mrp antiporter was measured in the constructed proteoliposomes (protein-inserted liposomes). using talon resin, all of the mrp subunits could be purified from the e. coli membrane fraction expressing the mrp antiporter and seemed to be present as predominantly a mrpabcdefg complex dimer. apparent na+/h+ antiport activity was observed in the proteoliposomes into which purified mrp and fof -atpase were reconstituted. after elution of mrp proteins, they remained as mrp complexes and most of which were present as the mrpabcdefg complex dimer. this suggested that mrpabcdefg complexes detectable by bn-page are the active forms. it was also speculated that the mrp complex dimer is more stable than the complex monomer. this is the first report of the purification and functional reconstitution of a mrp antiporter. reference [ ] t.h. swartz, s. ikewada, o. ishikawa, m. ito, t.a. krulwich, the mrp system: a giant among monovalent cation/proton antiporters? extremophiles ( ) – . doi: . /j.bbabio. . . p crystal structure of the heterotrimeric egchead complex from yeast vacuolar atpase r.a. oot, l.s. huang, e.a. berry, s. wilkens department of biochemistry and molecular biology, suny upstate medical university, syracuse, ny, usa e-mail: ootr@upstate.edu the eukaryotic vacuolar atpase (v-atpase) is a rotary molecular motor and dedicated proton pump found on the endomembrane system of all eukaryotic cells and the plasma membrane of specialized cells in higher organisms [ ]. the enzyme is composed of a soluble catalytic subcomplex (v ) and a membrane integral complex (vo) involved in proton translocation. linking the soluble and membrane sectors are the stator subunits (e, g, c, h and ant) which absorb the torque generated during rotary catalysis. the unique mode of v-atpase regulation, known as reversible dissociation, involves the release of v -atpase from the membrane integral vo, and the activity of both domains is silenced [ ]. regulated release of v -atpase requires breaking of protein interactions mediated by three periph- eral stalks, each composed of a heterodimer of subunits e and g. two of the peripheral stalks (eg and eg ) connect the top of the v to the membrane bound a subunit while the third (eg ) is bound to subunit c, which is released from both v and vo during enzyme dissociation. we have previously characterized and quantified the affinities of some of these interactions and have found that the globular “head” domain of subunit c (chead) binds to one eg heterodimer with high affinity [ , ]. here, we present x-ray crystal structures of two conformations of the egchead complex from saccharomyces cerevisiae at . and . Å abstractss http://dx.doi.org/ . /j.bbabio. . . http://dx.doi.org/ . /j.bbabio. . . percutaneous mechanical assist for severe cardiogenic shock due to acute right ventricular failure case report percutaneous mechanical assist for severe cardiogenic shock due to acute right ventricular failure ryan kipp, md and amish n. raval,* md acute right ventricular failure can lead to severe cardiogenic shock and death. recov- ery may be achieved with early supportive measures. in many patients, intravenous fluid and inotropic resuscitation is inadequate to improve cardiac output. in these cases, percutaneous mechanical assist may provide a non-surgical bridge to recovery. herein, we describe a case series of patients with severe, refractory cardiogenic shock due to acute right ventricular failure who received a continuous flow percutaneous ventricular device primarily utilizing the right internal jugular vein for out flow cannula placement. vc wiley periodicals, inc. key words: heart failure; right ventricular function; shock; cardiogenic introduction cardiogenic shock due to severe, refractory right ventricular failure is associated with high mortality [ , ]. common causes of this condition include acute right ventricular myocardial infarction and acute pul- monary embolism. immediate treatment consists of reperfusion, intravenous fluid resuscitation, and intrave- nous inotropic support; however, despite these efforts, many patients will continue to have refractory shock and will eventually die. percutaneous mechanical ven- tricular assist devices have been widely adopted in the united states to support patients with cardiogenic shock due to left ventricular failure. these devices include intermittent aortic balloon counter-pulsation and continuous flow mechanical support with and with- out adjunctive extracorporeal membrane oxygenation for cardiogenic shock due to acute left ventricular fail- ure [ ]. there is less experience using such devices for the treatment of cardiogenic shock due to acute right ventricular failure. a modified use of the tandem heart (cardiac assist, pittsburg, pa) continuous flow device has been described for the treatment of refractory right ventricu- lar failure [ – ]. the typical configuration for left ven- tricular support is placement of the inflow cannula in the left atrium and the outflow cannula in an iliac artery. for right ventricular assist, the inflow cannula is placed in the right atrium and the outflow cannula in the pulmonary artery. in this configuration, the route of access is typically via the femoral veins. an alternative cannula configuration utilizing the right internal jugular vein for the outflow cannula and the right femoral vein for the inflow cannula has been previously reported [ , ]. herein, we report a series of seven patients where tandemheart percutaneous ventricular assist was employed for right ventricular support (prvad) in severe, refractory cardiogenic shock. in six of these, the right internal jugular vein was the access site chosen for placement of the outflow cannula. division of cardiovascular medicine, department of medicine, university of wisconsin school of medicine and public health, madison, wisconsin conflict of interest: nothing to report. *correspondence to: amish n. raval, md, division of cardiovas- cular medicine, department of medicine, csc h / , high- land ave, university of wisconsin school of medicine and public health, madison, wi . e-mail: anr@medicine.wisc.edu received january ; revision accepted february doi: . /ccd. published online month in wiley online library (wileyonlinelibrary.com) vc wiley periodicals, inc. catheterization and cardiovascular interventions : – ( ) case report all patients that required prvad support using the tandemheart device were identified between january and july . patients considered eligible for prvad support at our institution require (i) evidence of sustained and refractory cardiogenic shock (systolic blood pressure < mm hg, severely reduced cardiac output or clinical evidence of systemic under-perfu- sion) despite at least two inotropic/vasopressor agents, (ii) suitable volume resuscitation with evidence of increased central venous pressure (cvp), (iii) at least moderate right ventricular dysfunction on echocardiog- raphy, and (iv) no significant hypoxemia. in the event of significant hypoxemia, extracorporeal membrane ox- ygenation was employed. clinical history, echocardio- graphic, and hemodynamic data were utilized to determine whether right ventricular failure was a sig- nificant contributing factor to the patient’s persistent shock. in all patients, extracorporeal membrane oxy- genation was not felt to be necessary due to preserved oxygenation and predominant right ventricular failure contributing to cardiogenic shock, or previously placed surgical left ventricular assist device. medical records were reviewed and baseline demo- graphics, hemodynamics, and clinical outcomes were recorded. all post-prvad hemodynamics were recorded within hr after prvad placement. all percutaneous mechanical assist devices were placed in the cardiac catheterization laboratory. the tan- demheart device consists of a centrifugal pump, drive lines, a console, and two cannulae. during de- vice maintenance, heparin was administered intrave- nously to maintain an activated clotting time of � sec. if heparin-induced thrombocytopenia was suspected, argatroban was used to maintain an acti- vated partial thromboplastin time (aptt) of . times normal. all patients were sedated and intuba- ted during prvad placement and for the duration of its use to prevent cannula migration from inadvertent patient movement. patients were monitored in the cardiac intensive care unit by nurses trained in the use of the tandemheart. two prvad cannulae configurations were used: (i) femoral vein–femoral vein and (ii) femoral vein–inter- nal jugular vein for the inflow and outflow cannulae respectively (figs. and ). for (i) femoral vein–femo- ral vein insertion, a or fr introducer sheath was initially placed in each femoral vein. under fluoro- scopic guidance, a fr balloon tipped swan ganz catheter or fr multi-purpose catheter was advanced into the pulmonary artery via the femoral vein. follow- ing wire/catheter exchanges, a . cm � cm amplatz extra stiff wire was placed carefully in either the right or left pulmonary artery. a fr ( cm) cannula (outflow) was advanced over the amplatz wire and positioned in the pulmonary artery. another fr cannula was exchanged for the opposite venous sheath and positioned in the right atrium. the pump was primed, the cannulae were de-aired, connected, and secured, and the pump was activated. for (ii) femoral vein–internal jugular vein insertion, two fr cannulae were utilized. a fr introducer sheath was placed in the right internal jugular vein and a femoral vein. via the right internal jugular vein introducer, a fr multi- purpose catheter was used to advance a . cm � cm amplatz extra stiff wire into the pulmonary artery. subsequently, the introducer sheath was removed and a fr cannula (outflow) was advanced over the wire into the main pulmonary artery. via the femoral vein, another fr cannula (inflow) was advanced into the right atrium. approximately, – feet of sterile extension tubing was cut and a connec- tion adapter was used to connect to the outflow can- nula and bridge the distance from the internal jugular and femoral vein. notably, the use of extension tubing may not be required in patients of smaller stature, because the cannulae provided with the fig. . example of internal jugular vein—femoral vein prvad configuration. the inflow cannula is positioned in the right atrium via the right femoral vein and attached to the tandem heart drive unit. the outflow cannula is positioned in the pul- monary artery via the right internal jugular vein. the cannulae are connected to extension tubing placed using a tubing adapter. patients of smaller stature may not require the exten- sion tubing. [color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] kipp and raval catheterization and cardiovascular interventions doi . /ccd. published on behalf of the society for cardiovascular angiography and interventions (scai). http://wileyonlinelibrary.com tandemheart kit may be suitably long to directly con- nect with the connection adapter and pump. the pump was primed, the cannulae were de-aired, connected, and secured, and the pump was activated. all cannulae were secured at the access points using a horizontal drain/tube attachment device (hollister inc., libertyville, il). between the attachment device and the skin entry site, a silk suture was used to further stabilize the cannula and prevent migration. the can- nula sites were covered with an anti-microbial dressing and clear adhesive dressing. results between january and july , a total of seven patients with severe, refractory cardiogenic shock due to acute right ventricular heart failure, and adequate oxygenation underwent tandemheart me- chanical assist in a prvad configuration at our insti- tution. none of these patients acutely required extracorporeal oxygenation. mean age was . years and four of the seven were men. four patients ( %) had acute right ventricular myocardial infarction with delayed presentation; one ( %) patient had acute viral myocarditis with biventricular failure and refrac- tory cardiogenic shock despite placement of a surgical left ventricular assist device; one patient ( %) with heparin-induced thrombocytopenia had simultaneous proximal right coronary artery in-stent thrombosis and sub-massive pulmonary embolism; and one ( %) patient had massive pulmonary embolism (table i). all patients had refractory hypotension (systolic blood pressure < mm hg) and clinical signs of systemic mal-perfusion despite at least two intravenous inotropic/vasopressor agents, increased central venous fillings pressures, intra-aortic balloon pump in five patients, and surgical left ventricular assist device in one patient. the mean time from identification of right ventricular failure until prvad placement was . . hr. no patient had a history of prior car- diac disease, stroke, diabetes, or renal disease. device insertion the device was successfully placed in all seven patients without acute complication. six of the seven patients had outflow cannulae placed via the right in- ternal jugular vein plus femoral vein approach, whereas one patient had the standard femoral vein plus femoral vein configuration. none of the six patients with the outflow cannula placed via the right internal jugular vein required it to be repositioned, while one patient with bilateral femoral vein cannulae suffered repeated outflow cannula displacement into the enlarged right ventricle. in this case, the outflow cannula was re- positioned under fluoroscopic guidance twice and it was subsequently removed hr after initial placement (table ii). following placement, the average revolu- tions per minute from the device was , , with a mean output of . . l/min. hemodynamic and echocardiography outcomes hemodynamic data pre- and post-prvad place- ment was available for all patients (table iii). pre- prvad placement, the mean cvp was . . mm hg and the mean cardiac index (ci) was . . l/min/m . within hr of prvad place- ment, the mean cvp decreased to a mean of . mm hg and the ci increased to a mean of . . l/min/m across the entire cohort. there was no dif- ference in the pulmonary artery systolic pressure before and after prvad placement. echocardio- graphic data were available for all seven patients pre- rvad placement and for six of seven post-prvad placement. pre-prvad placement, six of seven patients had acute, severe rv systolic dysfunction by echocardiography, whereas it was moderately impaired in one of seven. following prvad placement, rv systolic function normalized or was only mildly reduced in three patients and remained severely reduced in three patients. three patients had severely reduced left ventricular ejection fraction at the time of prvad placement. the remaining four patients had normal left ventricular function. clinical outcomes duration of mechanical prvad therapy was between and hr (mean hr). three of seven patients fig. . chest x-ray of a patient with internal jugular vein— femoral vein prvad configuration. a, outflow cannula; b, inflow cannula. device assisted right ventricular support catheterization and cardiovascular interventions doi . /ccd. published on behalf of the society for cardiovascular angiography and interventions (scai). died during their hospitalization. two patients died from refractory multi-system organ failure, days and days following prvad placement. a third patient with a sur- gical lvad died from a hemorrhagic stroke, days fol- lowing prvad removal. the four surviving patients were discharged home (n¼ ) or temporary assisted living (n¼ ). five of seven patients had developed acute renal failure requiring temporary hemodialysis. no patients required surgical lvad or rvad placement after prvad deployment. the act ranged from to sec, and aptt ranged from to . significant bleeding requiring transfusions occurred around both the internal jugular and femoral vein cannulae in all patients. two patients required a large volume of blood transfusion (> units of packed red blood cells). one patient requiring large volume of blood transfusion had a surgical lvad placed prior to prvad placement. the second patient required repositioning of the prvad device resulting in a large volume of blood loss. excluding table i. patient characteristics subject age (years) sex indication for prvad treatment before prvad placement time from rv failure until prvad (hr) no. inotropes a no. vasopressor b inhaled nitric oxide iabp slvad f rv infarct no yes no m rv infarct & pulmonary embolism no yes no m rv infarct no no no f viral myocarditis yes yes yes m rv infarct no yes no m rv infarct no yes no f pulmonary embolism yes no no rv, right ventricle; iabp, intra-aortic balloon pump; slvad, surgical left ventricular assist device. a dopamine, dobutamine, epinephrine. b norepinephrine, vasopressin, phenylephrine. table ii. prvad cannula position and complications subject no. out flow cannula access cannula repositioned? duration of prvad (hours) units of prbc stroke renal failure requiring dialysis in-hospital death right ijv no yes, embolic no no right ijv no yes, hemorrhagic yes no right ijv no no yes yes right ijv no yes, hemorrhagic yes yes right ijv no no yes no left fv yes no no no right ijv no no yes yes ijv, internal jugular vein; fv, femoral vein; prbc, packed red blood cells. table iii. hemodynamic and echocardiographic data subject no. hemodynamic and echocardiographic data pre-prvad placement hemodynamic and echocardiographic data post-prvad placement lvef cvp ci pa systolic cvp ci pa systolic na na na (e) na . (e) . (e) . (e) . . na . na . (e) . na na . na . (e) . (e) lvef, left ventricular ejection fraction (%); cvp, central venous pressure (mm hg); pa systolic, pulmonary artery systolic pressure (mm hg); ci, cardiac index (l/min/m ); na, not available. “e” denotes hemodynamic data obtained from echocardiography. all other hemodynamic data obtained from direct measurement using a pulmonary artery catheter. kipp and raval catheterization and cardiovascular interventions doi . /ccd. published on behalf of the society for cardiovascular angiography and interventions (scai). the patient who received the surgical lvad, on aver- age approximately unit of packed red blood cells was transfused for every . . hr of device placement. additionally, approximately . . units of platelets and . . units of fresh frozen plasma were trans- fused on average per patient. the five patients who did not have massive bleeding required significantly fewer units of packed red blood cells averaging . . units per patient, or one unit of packed red blood cells per . . hr of device placement. discussion this series suggests that the tandemheart continu- ous flow percutaneous mechanical assist device can be used to treat extreme, refractory cardiogenic shock due to acute right ventricular failure, that right internal jug- ular vein access for the outflow cannula offers stable, long-term support and that severe bleeding despite attempting to target therapeutic range anticoagulation continues to be a major challenge in using this device for long intervals. all commercially available percutaneous mechanical assist devices in the united states were designed and optimized to provide hemodynamic support for the fail- ing left ventricle. the tandemheart, impella, cardio- help left ventricular support systems are fda approved for short-term left ventricular mechanical support. clinical trials are underway testing modifica- tions to these systems for right ventricular assist, but these devices are still considered investigational in the united states. previous reports have described off-label use of the tandemheart as a prvad for hemodynamic support post-myocardial infarction [ – ], post-massive pulmo- nary embolism [ ], treatment of severe pulmonary hypertension [ ], during rejection after orthotopic heart transplant [ , ], following surgical lvad place- ment [ , ], and post-pericardiotomy [ ]. our series was comprised of four patients in cardiogenic shock post-right ventricular infarction; one patient with acute viral myocarditis and biventricular failure with refrac- tory cardiogenic shock despite placement of a surgical left ventricular assist device; one patient with heparin- induced thrombocytopenia with simultaneous proximal right coronary artery in-stent thrombosis and sub- massive pulmonary embolism; and one patient with isolated massive pulmonary embolism. five out of seven patients had refractory shock despite inotropic agents and concomitant left ventricular assistance, whether with a surgically placed lvad or intra-aortic balloon pump. in addition to right ventricular dysfunc- tion, three of our seven patients also had severe left ventricular systolic dysfunction. prvad placement facilitated a marked improvement in hemodynamics and improved rv systolic function. all except one prvad devices placed in our series were via a femoral plus internal jugular vein position for the inflow and outflow cannulae, respectively. none of the outflow cannulae positioned by this tech- nique required repositioning suggesting this position combination may allow improved outflow cannula sta- bility versus the femoral–femoral position. in addition, fr side-by-side femoral vein–femoral vein cannulae position may cause partial obstruction of the inferior vena cava which may potentially impair right ventricu- lar filling or facilitate thrombosis. cannula migration into the enlarged right ventricle could cause acute he- modynamic collapse, right ventricular arrhythmia, or perforation. in our experience, the right internal jugular vein approach for the outflow cannula with cannula fixation at the skin entry site provided for a consistent and secure device position in all patients. in our experience, the tandemheart prvad required a significant amount of blood product transfu- sion due to persistent bleeding around cannula sites, gasterointestinal bleed, and hemolysis. large transfu- sion requirements were notable in two patients due to a need for cannula reposition or due to surgical lvad related bleeding and hemolysis. in addition, many of these patients also develop systemic coagulopathies due to shock liver, disseminated intravascular coagula- tion, and drug induced thrombocytopenia which can be responsible for excess bleeding. our maintenance tar- get activated clotting time was sec which is in the range recommended by the manufacturer for typical left ventricular assist. there is limited data in the literature describing the use of prvad support for refractory right ventricular failure and cardiogenic shock. while we found that uti- lizing the internal jugular vein for outflow cannula position was most stable, larger prospective studies are needed to address this question further. prospective studies are also needed to provide further guidance on patient selection for prvad placement and to address excess bleeding. conclusion continuous flow, percutaneous right ventricular assist device therapy is an option for patients with re- fractory cardiogenic shock due to right ventricular failure. the right femoral vein and right internal jug- ular vein for inflow–outflow cannula configuration offers stable cannulae position for prolonged prvad assist. bleeding remains a challenging problem for this therapy. device assisted right ventricular support catheterization and cardiovascular interventions doi . /ccd. published on behalf of the society for cardiovascular angiography and interventions (scai). acknowledgment sincere thanks to matthew c. tattersall md for his contribution 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university of groningen loci influencing blood pressure identified using a cardiovascular gene-centric array ganesh, santhi k.; tragante, vinicius; guo, wei; guo, yiran; lanktree, matthew b.; smith, erin n.; johnson, toby; castillo, berta almoguera; barnard, john; baumert, jens published in: human molecular genetics doi: . /hmg/dds important note: you are advised to consult the publisher's version (publisher's pdf) if you wish to cite from it. please check the document version below. document version publisher's pdf, also known as version of record publication date: link to publication in university of groningen/umcg research database citation for published version (apa): ganesh, s. k., tragante, v., guo, w., guo, y., lanktree, m. b., smith, e. n., johnson, t., castillo, b. a., barnard, j., baumert, j., chang, y-p. c., elbers, c. c., farrall, m., fischer, m. e., franceschini, n., gaunt, t. r., gho, j. m. i. h., gieger, c., gong, y., ... lifelines cohort study ( ). loci influencing 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https://doi.org/ . /hmg/dds loci influencing blood pressure identified using a cardiovascular gene-centric array santhi k. ganesh ,{, vinicius tragante , ,{, wei guo ,{, yiran guo , matthew b. lanktree , erin n. smith , toby johnson , , berta almoguera castillo , john barnard , jens baumert , yen-pei christy chang , , clara c. elbers , , martin farrall , mary e. fischer , nora franceschini , tom r. gaunt , johannes m.i.h. gho , christian gieger , yan gong , aaron isaacs , marcus e. kleber , , irene mateo leach , caitrin w. mcdonough , matthijs f.l. meijs , olle mellander , , cliona m. molony , ilja m. nolte , sandosh padmanabhan , tom s. price , ramakrishnan rajagopalan , jonathan shaffer , sonia shah , haiqing shen , nicole soranzo , peter j. van der most , erik p.a. van iperen , , jessic a. van setten , judith m. vonk , li zhang , amber l. beitelshees , gerald s. berenson , deepak l. bhatt , jolanda m.a. boer , eric boerwinkle , ben burkley , amber burt , aravinda chakravarti , wei 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(f.w.a.) human molecular genetics, , vol. , no. – doi: . /hmg/dds advance access published on january , at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/ division of cardiovascular medicine, university of michigan health system, ann arbor, mi, usa, department of cardiology, division heart and lungs, department of medical genetics and julius center for health sciences and primary care, university medical center utrecht, utrecht, the netherlands, department of epidemiology and biostatistics, school of medicine and department of molecular medicine, cleveland clinic lerner college of medicine, case western reserve university, cleveland, oh, usa, center for applied genomics, abramson research center, the children’s hospital of philadelphia, philadelphia, pa , usa, departments of medicine and biochemistry, schulich school of medicine and dentistry, university of western ontario, london, on, canada, department of pediatrics 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department of ophthalmology and visual sciences, university of wisconsin, madison, wi, usa, department of epidemiology, university of north carolina at chapel hill, chapel hill, nc, usa, mrc centre for causal analyses in translational epidemiology, school of social and community medicine, university of bristol, oakfield house, oakfield grove, bristol bs bn, uk, department of pharmacotherapy and translational research and center for pharmacogenomics and departments of pharmacotherapy and translational research and community health and family medicine, university of florida, gainesville, fl, usa, genetic epidemiology unit, department of epidemiology and departments of epidemiology and internal medicine, erasmus medical center, rotterdam, the netherlands, luric study nonprofit llc, freiburg, germany, mannheim institute of public health, social and preventive medicine, mannheim medical faculty, university of heidelberg, mannheim, germany, department of cardiology, department of epidemiology, division of nephrology, department of medicine, molecular genetics, medical biology section, department of pathology and medical biology, department of genetics, lifelines cohort study and interdisciplinary center psychopathology and emotion regulation, groningen university, university medical center groningen, groningen, the netherlands, hypertension and cardiovascular disease, department of clinical sciences, lund university, malmö, sweden, centre of emergency medicine, skåne university hospital, malmö, sweden, department of genetics, rosetta inpharmatics, seattle, wa, usa, bhf glasgow cardiovascular research centre, university of glasgow, university place, glasgow g ta, uk, mrc sgdp centre, institute of psychiatry, london, uk, department of medicine, division of medical genetics, university of washington, seattle, wa, usa, department of medicine, columbia university, new york, ny, usa, ucl genetics institute, department of genetics, evolution and environment, university college london, kathleen lonsdale building, gower place, london wc e bt, uk, human genetics and department of haematology, university of cambridge and nhs blood and transplant, cambridge and human genetics, wellcome trust sanger institute, hinxton, uk, durrer center for cardiogenetic research, department of clinical epidemiology, biostatistics and bioinformatics, department of vascular medicine and department of cardiology, amc, amsterdam, the netherlands, department of epidemiology, tulane university, canal street, suite , new orleans, la, usa, va boston healthcare system, brigham and women’s hospital, and harvard medical school, boston, ma, usa, national institute for public health and the environment (rivm), bilthoven, the netherlands, human genetics center and institute of molecular medicine and division of epidemiology, university of texas health science center, houston, tx, usa, the university of texas health science center at houston, houston, tx, usa, center for complex disease genomics, mckusick-nathans institute of genetic medicine and department of medicine, johns hopkins university school of medicine, baltimore, md, usa, merck research laboratories, po box , rahway, nj , usa, department of medicine and school of nursing, university of mississippi medical center, jackson, ms, usa, translational genomics research institute, phoenix, az, usa, division of cardiovascular sciences, national heart, lung, and blood institute, bethesda, md, usa, center for population studies, national heart, lung, and blood institute, framingham, ma, human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/ usa, department of community health and epidemiology, dalhousie university, canada, pathology and laboratory medicine institute, cleveland clinic, cleveland, oh, usa, medical college of georgia, augusta, ga, usa, division of public health sciences, fred hutchinson cancer research center, seattle, wa, usa, department of medicine, boston university school of medicine, boston, ma, usa, national heart, lung and blood institute’s framingham heart study, mt.wayte avenue suite # , framingham, ma, usa, department of preventive medicine, northwestern university feinberg school of medicine, chicago, il, usa, department of vascular medicine, university of amsterdam, amsterdam, the netherlands, institute for stroke and dementia research and neurologische klinik, klinikum grosshadern, ludwig-maximilians-universität, münchen, germany, broad institute of harvard and mit, cambridge, ma, usa, center for human genetic research, massachusetts general hospital, boston, ma, usa, department of psychiatry/emgo institute, vu university medical centre, amsterdam, the netherlands, division of cardiovascular medicine, university of florida college of medicine, gainesville, fl, usa, department of radiology, tufts medical center, department of medicine, brigham and women’s hospital, harvard medical school and division of genetics, brigham and women’s hospital, harvard medical school, boston, ma, usa, clinical institute of medical and chemical laboratory diagnostics, medical university of graz, austria, the scripps translational science institute and the scripps research institute, n. torrey pines ct. ste , la jolla, ca, usa, geriatric research and education clinical center, veterans administration medical center, baltimore, md, usa, cardiology group, frankfurt-sachsenhausen, division of endocrinology and department of internal medicine i—cardiology, university of ulm medical centre, ulm, germany, departments of medicine & psychiatry, columbia university, new york, ny, usa, the institute for translational medicine and therapeutics, school of medicine, university of pennsylvania, philadelphia, pa, usa, hannover unified biobank, hannover medical school, hannover, germany, synlab academy, mannheim, germany, scripps translational science institute and scripps health, n. torrey pines ct. ste , la jolla, ca, usa, cardiovascular institute, the perelman school of medicine at the university of pennsylvania, pa, usa, department of genetics and genomic sciences, mount sinai school of medicine, new york, ny, usa, molecular & cellular therapeutics, royal college of surgeons in ireland, st stephens green, dublin , ireland, department of cardiovascular sciences, university of leicester and leicester nihr biomedical research unit in cardiovascular disease, glenfield hospital, leicester le qp, uk received july , ; revised november , ; accepted december , blood pressure (bp) is a heritable determinant of risk for cardiovascular disease (cvd). to investigate gen- etic associations with systolic bp (sbp), diastolic bp (dbp), mean arterial pressure (map) and pulse pressure (pp), we genotyped ∼ single-nucleotide polymorphisms (snps) that capture variation in ∼ can- didate genes for cardiovascular phenotypes in individuals of european ancestry from cohort studies in the usa and europe. we identified novel associations between rs and sbp (chromosome p . , in an intron of hrh ) and between rs and dbp (chromosome q . in an intron of mdm ) and between rs and sbp (chromosome p in an intron of sox ), previously reported to be associated with map. we also confirmed previously known loci associated with sbp, dbp, map or pp (adrb , atp b , sh b /atxn , csk, cyp a , furin, hfe, lsp , mthfr, sox ) at array-wide significance (p < . ). we then replicated these associations in an independent set of individuals of european ancestry. the findings from expression qtl (eqtl) analysis showed associations of snps in the mdm region with mdm expression. we did not find any evidence of association of the two novel snps in mdm and hrh with sequelae of high bp including coronary artery disease (cad), left ventricular hypertrophy (lvh) or stroke. in summary, we identified two novel loci associated with bp and confirmed mul- tiple previously reported associations. our findings extend our understanding of genes involved in bp regu- lation, some of which may eventually provide new targets for therapeutic intervention. human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/ introduction blood pressure (bp) is a cardinal risk factor for cardiovascular disease (cvd). systolic bp (sbp) and diastolic (dbp) levels are associated with increased risk of atherosclerotic vascular disease and other cardiovascular causes of death ( ). much of the excess cvd risk imparted by bp elevation can be ame- liorated through interventions to decrease bp ( ). the identifi- cation of novel genes and pathways involved in bp regulation may highlight new ways of reducing bp and cvd risk asso- ciated with hypertension. the mean arterial pressure (map) and pulse pressure (pp, the difference between sbp and dbp) are single bp components associated with cvd risk ( – ). the latter is an indicator of conduit artery stiffness and is known to increase with age, as aortic elasticity decreases. to date, � common genetic variants associated with bp and hypertension have been reported, largely through genome-wide association studies (gwas), meta-analyses and admixture mapping approaches ( – ). the identification of common var- iants associated with bp may be enriched through further gene- centric approaches ( – ). accordingly, we tested the hypoth- esis that candidate gene analysis would identify known and novel associations with sbp, dbp, map and pp and would confirm previously reported associations. to further investigate and discover associations with bp, we genotyped approximate- ly single-nucleotide polymorphisms (snps) on a gene- centric array (itmat-broad_care [ibc] array, illumina san diego, ca, usa) that captures variation in � candidate genes for cardiovascular traits including bp ( ) in indi- viduals of european ancestry. we identified two novel bp-associated loci, at the candidate genes mdm and hrh and have validated these associations through in silico replica- tion analysis in a large set of independent samples. results discovery association analyses in the primary discovery meta-analysis, four bp traits were analyzed in individuals from cohorts, as described in table . we analyzed sbp, dbp, map and pp as continu- ous traits. cohort characteristics, including age, sex, bp values and the proportion of individuals treated with bp lowering med- ications, are provided in table . the details of the cohorts are provided in the supplementary material, table s and supple- mentary materials. association analyses were successfully carried out for up to snps, and summaries of the quality-control (qc) steps and numbers of snps removed at each step are provided in supplementary material, table s a. cohort-specific genomic control inflation factors, lgc, did not suggest the presence of inflation (supplementary material, table s ). meta-analysis quantile – quantile plots are shown in supplementary material, fig. s and p-values for association for all snps are provided in supplementary material, table s . we identified signifi- cant snp-trait associations with sbp, dbp, map and pp at different loci (p , . × ), including two novel loci near hrh and mdm for bp traits and one novel snp-trait associ- ation in the sox locus, a region previously described in asso- ciation with map. replication analyses replication testing was performed in additional indivi- duals, including individuals in seven cohorts with genome-wide snp genotypes imputed to hapmap (supplemen- tary material, table s b) and individuals genotyped on the same ibc chip used for the discovery analyses. through the joint analysis of snps considered relevant during the discovery phase combined with replication data, we identified robust asso- ciation of snp-trait associations at independent loci meeting our array-wide significance threshold of p , . × : six loci were associated with dbp (mthfr, mdm , hfe, sh b /atxn , csk, furin), nine loci were associated with sbp (mthfr, hrh , cyp a , lsp , sox , atp b , sh b /atxn , csk, furin), six loci were associated with map (mthfr, adrb , atp b , atxn , csk, furin) and one locus associated with pp (cyp a ). the association find- ings are summarized in table . we confirmed previously reported bp associations at loci, and identified two novel loci: rs associated with sbp (chromosome p , in an intron of hrh , p ¼ . × ) (fig. a); rs associated with dbp (chromosome q , in an intron of mdm , p ¼ . × ) (fig. b). we additionally found evidence of asso- ciation of rs (chromosome p in an intron of icam ) with dbp, in our discovery analysis, although it was not con- firmed in the replication analysis (p ¼ . × in discov- ery, p ¼ . × in the joint analysis). finally, one of the snp-trait associations we identified was novel in our analysis, with the association of rs with sbp (chromosome p , in an intron of sox , p ¼ . × ), whereas pre- viously only association with map had been reported. a second bonferroni correction of our results for testing four traits did not result in a change in the overall results, so we present the ori- ginal results here, as the four traits are highly correlated. full association results for snps in our discovery analysis with asso- ciation p , × are reported in supplementary material, table s . despite ascertainment biases in some of the discovery cohorts, due to inclusion or exclusion based upon bp or hyper- tension status (as noted in supplementary material, table s ), we show replication of the key findings. we comprehensively compared the results of our analysis with all published associa- tions at the time of this report ( – , ) (supplementary ma- terial, table s ). we reviewed previously reported loci for our bp traits of interest and found that were represented on our genotyping array, with one region containing a proxy snp (r ¼ . ) rather than the index snp previously reported (supplementary material, table s ). at a nominal association threshold (p ¼ . ), snps were associated with one or more bp traits in our study, and with a multiple testing correc- tion (p , . ), we observed snps with bp associations. sex interaction in a secondary sex-specific analysis of our discovery sample (supplementary material, table s ), we had no new signifi- cant associations. to follow-up possible sex differences in the two novel associations identified in our discovery efforts, we tested for interactions of rs (hrh ) and rs (mdm ) with sex and identified a modest sex- specific effect for all four continuous bp traits at rs human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/ (p-value for interaction was . for sbp; . for dbp; . for map and . for pp) (supplementary material, table s ), with the association observed in women but not in men (in females only sbp beta was . , se . , and in males only sbp beta was . , se . , p-value for inter- action of rs with sex was . ). table . clinical characteristics of discovery and replication cohorts age female/ male sbp dbp map pp bmi taking anti- hypertensive medication discovery cohorts amc-pas . + . / . + . . + . . + . . + . . + . . % amish . + . / . + . . + . . + . . + . . + . . % aric . + . / . + . + . + . . + . . + . . % bhs . + . / . + . . + . + . . + . + . . % cardia . + . / . + . . + . . + . . + . . + . . % cccs . + . / . + . . + . . + . . + . . ( . )/ . ( . ) . % cfs . + . / . + . . + . . + . . + . . + . . % chs . + . / . + . . + . . + . . + . . + . . % clear . + . / . + . . + . . + . . + . . + . . % epic_nl . + . / . + . . + . . + . . + . . + . n/a fhs . + . / . + . . + . . + . . + . . + . % girafh . + . / . + . . + . . + . . + . . + . . % gq . + . / . + . . + . . + . . + . . ( . )/ . ( . ) . % invest . + . / . + . . + . . + . . + . . ( . )/ . ( . ) . % luric . + . / . + . . + . . + . . + . . + . . % medal . + . / . + . . + . . + . . + . . + . . % mesa . + . / . + . . + . . + . . + . . + . . % monica/ kora f . + . / . + . . + . . + . . + . . + . . % monica/ kora s . + . / . + . . + . . + . . + . . + . . % nshs . + . / . + . . + . . + . . + . . + . n/a pear . + . / . + . . + . . + . . + . [ . ( . )/ . ( . )] % penncac . + . / + . . + . . + . . + . . + . n/a penncath . + . / + . . + . . + . . + . . + . . % smart . + . / . + . . + . . + . . + . . + . . % whi . + . / . + . . + . . + . . + . . ( . ) . % replication cohorts aibiii . ( . ) / . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . % ascot ( . ) / . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . % bright (controls) . ( . ) / ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . % bright (cases) ( . ) / . ( . ) . ( . ) ( . ) . ( . ) ( . ) . % bwhhs . ( . ) / . ( . ) . ( . ) ( ) . ( ) . ( . ) . % graphic . ( . ) / . ( . ) . ( . ) . ( . ) ( . ) . ( . ) . % lifelines . + . / . + . . + . . + . . + . . + . . % mdc . ( . ) / . ( . ) . ( . ) . ( . ) ( ) . ( . ) . % nbs . ( . ) / n/a n/a n/a n/a n/a n/a nesda . + . / . + . . + . . + . . + . . + . . % nordil ( ) / . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . % prevend . + . / . + . . + . . + . . + . . + . . % procardis . ( . ) / . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . % rotterdam study . ( . ) / . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . % trails clinical cohort . + . / . + . . + . . + . . + . . + . n/a trails population cohort . + . / . + . . + . . + . . + . . + . n/a wghs . + . / . + . . + . . + . . + . . + . . % whii . ( . ) / . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . % mean + standard deviation is given for each phenotype, except % were indicated. bp values shown are actual values without modification for medication treatment. human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/ table . loci associated with hypertension traits using discovery and replication data (betas and ses corresponding to the trait are highlighted in bold) discovery results replication results discovery + replication results gene snp chr bp a a caf beta se dbp map pp sbp dbp map pp sbp dbp map pp sbp dbp mthfr rs g a . . . . e . e . e . e . e . e . e . e . e e mdm rs g c . . . . e . . e hfe rs g c . . . . e . e . e atxn rs a g . . . . e . e . e . e . e . e . e . e . e csk rs t c . . . . e . e . e . e . e . e . e . e . e furin rs g c . . . . e . e . . e . e . e . e . e . e map mthfr rs g a . . . . e . e . e . e . e . e . e . e . e e adrb rs c t . . . . e . e . e atp b rs g a . . . . e . e . e . e . e . e atxn rs a g . . . . e . e . e . e . e . e . e . e . e csk rs t c . . . . e . e . e . e . e . e . e . e . e furin rs t c . . . . e . e . e . e . e . e . e . e . e pp cyp a rs c g . . . . e . e . e . . e . e sbp mthfr rs g a . . . . e . e . e . e . e . e . e . e . e e hrh rs g t . . . . e . . e cyp a rs c g . . . . e . e . e . . e . e lsp rs c t . . . . e . e . e sox rs t c . . . . e . e . e atp b rs g a . . . . e . e . e . e . e . e atxn rs a g . . . . e . e . e . e . e . e . e . e . e csk rs t c . . . . e . e . e . e . e . e . e . e . e furin rs t c . . . . e . e . e . e . e . e . e . e . e h u m a n m o le c u la r g e n e tic s, , v o l. , n o . at university of groningen on may , http://hmg.oxfordjournals.org/ downloaded from http://hmg.oxfordjournals.org/ conditional analyses for the loci described in table , except those containing fewer than three genome-wide significant snps, conditional analyses were conducted using the allele dosage of the snps within a kb (+ kb) window around the most signifi- cant snp per locus as a covariate in a subset of discovery cohorts (aric, cardia, chs and mesa). statistical models were identical to those used in the discovery analyses except for the additional snp covariates. no snps remained significant in these conditional models after correcting the total number of tests (snps) in the kb window, suggesting that only the strongest signal of association at each locus explained our findings. the complete results for these analyses are shown in supplementary material, table s . annotations of the identified loci annotations for the loci associated with bp traits in our study showed that the majority of the variants we identified were located within an intron of the corresponding gene (rs in mthfr; rs in mdm ; rs in cyp a ; rs in lsp ; rs in sox ; rs in atp b ; rs and rs in atxn ; rs and rs in furin; rs in hrh ). in aggregate, the associated snps accounted for . – . % of the pheno- typic variance in sbp, dbp, map and pp (supplementary material, table s ). these estimates are in line with other association studies in which , % of the overall phenotypic variance was explained by the common variants identified ( ). figure . regional association plots of the (a) hrh and (b) mdm loci are shown with negative log (p-value) on the y-axis and chromosomal position on the x-axis. human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/ expression qtl (eqtl) analysis showed that several of our associated snps (or proxy snps that were highly correlated with our lead snp of interest [r . . ]) were associated with the expression levels of nearby genes, as assessed by the micro- array analysis of rna expression in various tissues, including blood, liver, lymph tissues, lymphoblastoid cell lines (lcls) and peripheral blood t cells and monocytes, brain, adipose tissue and liver. these results are summarized in supplementary material, table s . testing for eqtl associations at our novel loci showed no eqtl associations in the hrh region, but we did observe significant eqtl associations in the mdm region. expression of mdm transcripts in lymph tissues, lcls and monocytes was associated with rs (p ¼ . × in lymph tissue) and rs (p ¼ . × in lcls and p ¼ . × in monocytes). both rs and rs are highly correlated with the lead snp we identified in our association analyses, rs in mdm (r . and . , respectively). additional eqtl associations were identified for rs with the hfe tran- script (chromosome p , p ¼ . × ), rs and rs with sh b and atxn transcripts ( q , p ¼ . × ); rs with the csk transcript ( q , p ¼ . × ). additionally, we queried the encode database (http:// www.regulomedb.org/). http://www.regulomedb.org/. we examined the annotations in the region of mdm snps (these snps include rs , rs , rs ). these snps show no new encode regulatory annotations. we also queried hrh (rs ) which did not have an eqtl association. this snp does show overlap via position weight matrix for the htf (her ) transcription factor and also via dna footprinting. this is potentially encouraging but no chip-seq data are available for htf, so this is not ne- cessarily strongly confirmed. the snps which do overlap with features are those with eqtl associations in hfe (rs ), sh b (rs ) and csk (rs ). pleiotropy evaluations we tested association of rs (chromosome p , hrh ) and rs (chromosome q , mdm ) with traits known to be associated with hypertension, including coronary artery disease (cad), left ventricular hypertrophy (lvh) and stroke. in these analyses, no additional trait associations with these snps were identified (supplementary material, table s ). discussion using a genotyping array that covers common genetic vari- ation in � candidate genes for several cardiovascular traits including bp, we identified robust associations at known and two novel loci associated with continuous bp traits. in our primary discovery experiment, we identified loci associated with our traits of interest in an analysis of individuals (p , . × ). through the joint ana- lysis of snps considered relevant during the discovery phase combined with replication data, in a total of indivi- duals, we identified robust association of snp-trait associa- tions at independent loci meeting our significance threshold of p , . × . associations at two of these loci were not previously known to be associated with bp. the association of sbp with variants in the hrh locus, and the association of dbp with variants in the mdm locus were novel and repli- cated in silico in an additional individuals. additional- ly, the sox locus contains a novel snp-trait association for sbp with rs ; previously an association with map was shown for this locus ( ). finally, one additional region, chromosome p . containing icam , was significant in our discovery experiment but was not replicated in the joint analysis of discovery and replication samples. only one of the replication cohorts had data available for this snp (uk, p ¼ . ). although still meeting the array-wide significance criterion in the combined analysis, we do not consider this association to be robust in the replication experiment. the histamine receptor h (hrh ) gene product is expressed in numerous tissues, such as smooth muscle and neurons. hrh is expressed in the nucleus tractus solitarii, where it has a role in regulating arterial pressure in rats ( ). congenic mapping in a rodent model linked hrh to autoimmune t-cell responses and vascular responses regulated by histamine after bordetella pertussis toxin sensitization ( ), and this locus appears to have a role in regulating blood brain barrier permeability ( ). in a mouse model of atherosclerosis, apolipoprotein e-null mice treated with a histamine h recep- tor selective antagonist developed % fewer aortic plaques compared with mice treated with a h receptor antagonist and higher levels of inflammatory markers within the plaques and higher numbers of inflammatory cells, despite equivalent plasma lipoprotein levels, suggesting that the h receptor enhances low density lipoprotein cholesterol perme- ability into the intimal space of the artery ( ). hrh was selected for the ibc array as a lower priority gene based on the presence in a protein analysis through evolutionary relationships (panther) inflammatory/immune response pathway. the hrh gene is located � kb from the atp b gene with no linkage disequilibrium (ld) detected in hapmap ceu between common variants in these genes. al- though atp b has not been directly implicated in bp or hypertension, it is one of the ca( +)-atpases, a family of plasma membrane pumps encoded by at least three additional genes: atp b on chromosome q ; atp b on xq and atp b on q . atp b contains a robust snp association for sbp, dbp and hypertension ( ). mice null for atp b are embryolethal ( ), but recently a mouse with conditional knockout for the atp b in vascular smooth muscle cells was generated and found to have significantly elevated bp ( ). in humans, no associations have been found between var- iants in the atp b gene and bp or other vascular traits. add- itionally, within the recombination interval containing hrh and the peak association signal, snps within the atg gene were associated but well below the statistical significance threshold for association, decreasing the likelihood that var- iants in atg are driving the signal in this region. mouse double minute homolog (mdm ) encodes a nuclear protein that is a critical regulator of p tumor sup- pressor protein by binding to this protein and inhibiting its ac- tivity, promoting cell viability and growth. mdm does not have a described vascular function, but it has a critical role in regulating p , a transcription factor which plays an human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/ important role in regulating target genes that induce cell cycle arrest, apoptosis and cell senescence ( ). mdm was also selected for the ibc array as a lower priority gene, based on the presence in a panther apoptosis pathway. mice null for p shows a variety of aging-related phenotypes ( ). mdm , another regulator of p , has been shown to have a functional role in aldosterone-induced vascular remodeling ( ), and mdm and mdm have been shown to have non- overlapping but a similar regulation of p pathways ( – ). a genome-wide linkage analysis for loci associated with pp has shown suggestive linkage on chromosome (bp qtl , lod ¼ . ) for blacks in the hypergen network ( ), with mdm overlapping with this linkage peak. an overlap with the mdm region has also been shown for quantitative trait loci (qtl) related to bp in rats (human mdm : http://rgd.mcw.edu/rgdweb/report/gene/main.html?id= ; rats mdm : http://rgd.mcw.edu/rgdweb/search/qtls. html?term=mdm [gene]&speciestype= ). the sex interaction we observed is of unclear significance since it was not confirmed in independent samples. mdm has also been associated with measures of cognitive performance in a gwas ( ). within the recombination interval containing mdm , correlated snps in pik c b were associated, so we cannot exclude that the asso- ciation signal may in part be due to variants in this gene. pik c b has no known function related to bp or vascular disease. the association of the sox locus with sbp expands our prior knowledge of associations in this region with associated bp traits, with this locus previously reported as associated with map ( ). map is derived from a calculation incorpor- ating sbp and therefore these traits are highly correlated. add- itionally, we replicated previous reports of snp-trait associations with bp, with acknowledgement that some of the cohorts in the published literature were genotyped on mul- tiple platforms and have therefore been included in not only our ibc analysis but also previous reports. the strengths of this study include the large size (n ¼ ) of the discovery meta-analysis with replication in an additional individuals and access to gene expression data from humans subjects. this meta-analysis has led to the identification of two novel signals not previously detected by prior association studies. another unreported locus was found in the discovery phase, e.g. icam , which could not be replicated due to the inadequate sample size in the replica- tion cohorts with high-quality genotypes for this snp. the non-replication may also be explained by gwas with less dense coverage used for the replication phase. in addition, future functional studies are needed to fully comprehend the underlying mechanisms responsible for the detected associa- tions. however, we did find that the mdm locus was related to expression of mdm transcripts in several tissues, and the lead snp identified in our study is in tight ld with the expression-associated snps, suggesting a transcriptional effect of the associated snp we identified. finally, it needs to be emphasized that the results were generated in european ancestry populations and that additional studies are needed in other ethnic groups. in summary, our study has identified two novel loci contain- ing the hrh and mdm genes associated with bp traits of clinical significance. the identification of these loci expands our understanding of the genetic determinants of bp. materials and methods study subjects the phenotype and genotype data of individuals of euro- pean ancestry, belonging to participating studies (supple- mentary material, table s ), were analyzed in the discovery phase, and additional individuals of european ancestry from additional studies were used in the replication phase. individuals of european ancestry, as confirmed by principal component analysis of genetic ancestry, were analyzed in this study. all individuals in these studies provided informed consent, and each study was approved by its own local ethics committee. more detailed information about each participating cohort is provided in the supplementary material. phenotype bp ascertainment in each study was performed according to the protocols described in the supplementary material. pp was defined as sbp minus dbp, and map was defined as / dbp plus / sbp. in the discovery analyses, each cohort provided regression models for its data, adjusted for age, age-squared, body mass index (bmi) and study-specific corrections for popu- lation substructure (based on principal component analysis). for individuals taking bp lowering medications, the bp values were adjusted by adding mmhg to the sbp and mmhg to the dbp in the discovery and replication cohorts. these adjust- ments were also implemented prior to the calculation of esti- mated off-treatment map and pp. genotyping and quality control a total of snps were genotyped and after filtering for an mismatches . % with hapmap, removing snps without an rsid in dbsnp and snps with more than two possible bases for a single snp, snps included in the discovery meta-analyses, all present in at least one of the three versions of the illumina humancvd beadchip (‘cardiochip’, itmat- broad_care [ibc] array, illumina san diego, ca, usa) ( ), which was used by all cohorts participating in the discov- ery analysis. for the ibc array, gene and specific snp information was assimilated from published studies systematically ana- lyzed up until may . an emphasis was placed on the sample size, data quality and strength of the described associa- tions. genes with known or putative association with pheno- types for sleep, lung and blood diseases were also nominated. several pathway-based tools were used to identify additional biologically plausible candidate genes: kyoto en- cyclopedia of genes and genomes; panther and biocarta. these tools were employed to collate additional genes from key pathways including lipid metabolism, thrombogenesis, circulation and gas exchange, insulin resistance, metabolism, and inflammation, oxidative stress and apoptosis. early access was provided to a number of unpublished mouse atherosclerosis expression qtl (eqtl) datasets. genes pre- dicted to be causal for the atherosclerotic lesion size in genetic crosses of mice with differing susceptibility to atherosclerosis were identified. early access was provided to a number of key findings from a number of cvd-related gwass. human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/ genotypes were called using beadstudio (illumina) and the data were processed using stringent qc filters, as summarized in supplementary material, tables s and s . variants with minor allele frequencies , % were excluded from the ana- lysis. further details on genotyping methods are provided in the supplementary material, tables s and s . qc measures were taken during the various steps of this work. individuals with , % call rate (completeness) across all snps were removed. snps with , % call rate (complete- ness) or snps causing heterozygous haploid genotype calls were removed across all remaining individuals. snps with p , × for the hardy – weinberg equilibrium test were also removed. in the nhlbi candidate gene association re- source (care) samples ( ), snps associated with chemistry plate effects were also removed. statistical association and meta-analysis initial association analyses were calculated within each cohort for males and females separately, adjusting for age, age- squared, bmi and study center when appropriate. in each cohort, except fhs, cfs and amish, association analysis was performed using plink ( ) using linear regression under an additive genetic model. the family structure was modeled using a linear mixed effects model implemented in r ( ) in fhs and cfs, and the mixed model analysis for pedigrees soft- ware program in the amish ( ). meta-analysis was conducted using the summary statistics contributed by each discovery study, using an inverse variance weighted, fixed-effects method. at the meta-analysis stage of analysis, snps with frequencies incompatible with hapmap frequencies were removed (defined as . % difference in the allele frequencies). two analysis groups independently performed the meta-analysis using different software packages: metal ( ) and mantel ( ); both applied a fixed-effects model weighted by inverse variance. the results from both the groups were compared and a concord- ance check was performed as a validation of the results (data not shown). genomic control ( ) was applied to each study result and then to the meta-analysis summary data to control effects possibly due to population stratification or cryptic relatedness. quantile – quantile plots are shown for each trait in supplementary material, fig. s . previous studies using the ibc array have used different significance thresholds from p , × to × [( ) and ( ), re- spectively]. the care ibc array studies ( ), which are included in this meta-analysis, determined that after account- ing for ld, the effective number of independent tests was � for europeans producing an experimental or ‘array- wide’ statistical threshold of p ¼ . × , respectively, to maintain an false-positive rate of % ( ) and thus, we have adopted these thresholds for this study. since we have ana- lyzed four traits, although highly correlated, we examined the effect of further bonferroni correction for four tests. for each associated locus, the ld patterns were examined and in- dependence between the loci identified in this study was veri- fied using snap ( ) (r , . ). for loci with multiple snps showing association with the traits, we also conducted conditional analyses to evaluate inde- pendent signals. at each locus with variants associated with bp traits, we added the most significant snp within the locus as a covariate in the association tests in each cohort in the nhlbi care consortium. then, we performed meta- analysis of cohort-specific conditional analysis results. this conditional analysis was performed for the snps within a kb region around the most significant snp. the p-values for snp association testing were then recorded, respectively, for associated locus for each trait. replication analysis independent snps with p , . × in the discovery analysis were carried forward for replication analysis using independent samples for each trait. the significance threshold for association in the replication phase was a bonferroni-corrected p-value based on an a ¼ . and the final number of independent (r , . ) snps tested, and we also combined the discovery and replication data in a meta-analysis, in which evidence of positive replication was defined by p , . × in the meta-analysis of combined discovery and replication samples. associated loci were tested for replication by carrying forward to replication testing the lead snp (minimum p-value) at each locus. interaction testing for the lead snp identified in novel genes, we tested for inter- actions with sex by first calculating the residual after adjusting for age, age-squared, bmi, sex and principle components, and then performed sex-specific linear regression on a snp for each cohort separately. this analysis was done using the plink g × e function. the sex-specific estimates were further combined by meta-analysis for men and women separ- ately, using metal ( ). the interaction between gene and sex was tested by comparing the regression coefficients in men and women. that is, we calculated a t statistic: t = (b̂m − b̂f)/ ��������� ŝ m + ŝ f √ , where b̂m, ŝ m, b̂f and ŝ f are the estimated regression coefficients and their standard errors from male and female meta-analysis, respectively. t follows the standard normal distribution. variance explained the proportion of the trait variance explained by the discov- ered associations was calculated by first obtaining the resi- duals after adjusting for age, age-squared, bmi and pcs and then performing linear regression on all identified asso- ciated variants together in a subset of the cohorts comprising the nhlbi care consortium cohorts (aric, chs, mesa, cardia, cfs). the variance explained was calculated by a standard analysis of variance. annotation eqtl analysis we identified alias rsids for significant index snps using snap ( ). further proxy snps in high ld (r ¼ . ) were identified with snap using multiple hapmap ceu builds. current and alias rsids were searched for primary snps and ld proxies against a collected database of expression snp (esnp) results including the following tissues: fresh lymphocytes ( ), fresh human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/ leukocytes ( ), leukocyte samples in individuals with celiac disease ( )), lcls derived from asthmatic children ( ), hapmap lcl from three populations ( ), a separate study on hapmap ceu lcl ( ), peripheral blood monocytes ( , ), omental and subcutaneous adipose ( , ), stomach ( ) and whole blood samples ( , ), endometrial carcinomas ( ), brain cortex ( , ), three large studies of brain regions includ- ing prefrontal cortex, visual cortex and cerebellum, respectively (emilsson, personal communication), liver ( , , ), osteo- blasts ( ), skin ( ) and additional fibroblast, t-cell and lcl sample datasets ( ). the collected esnp results met the criteria for statistical thresholds for association with gene transcript levels as described in the original papers. in each case, where an index or proxy snp was associated with a transcript, we further examined the strongest esnp for that transcript within that dataset (best esnp), and the ld between the best esnp and bp-selected esnps to assess the concordance of the bp as- sociation and expression signals. annotation of snps in the region was done with the snap web-based tool ( ). evaluation of pleiotropy of bp variants with cardiovascular disease we evaluated the effect of the novel loci identified in our study with traits known to be related to elevated hypertension including cad, lvh and stroke. the definitions of the cad, lvh and stroke phenotypes and association tests were carried out as described in the supplementary material. evidence of association for the additional traits was alpha of . adjusted by the two snps tested for association (p ¼ . ). supplementary material supplementary material is available at hmg online. acknowledgements we thank the national heart, lung and blood institute and the research institutions, study investigators and field staff for their support in creating the care program for biomedical re- search. we also thank the study participants, without whom this endeavor would not have been possible. dna samples were genotyped in part through the broad institute (n -hc- ). discovery cohort acknowledgements amc-pas: funding for pas was provided by ipse movet; bloodomics (lshm-ct- - ). amish: the amish studies were funded by the national insti- tutes of health (r hl , r ag , u hl , u gm with additional funding for cardiochip analysis provided by an american heart association scientist develop- ment grant ( n to h.s.). genotyping of cardiochip was carried out in the genomics core at the university of maryland, baltimore with support from the mid-atlantic nutrition and obesity research center (nih p dk ). aric: the atherosclerosis risk in communities study is carried out as a collaborative study supported by national heart, lung and blood institute contracts (hhsn c, hhsn c, hhsn c, hhsn - c, hsn c, hhsn c, hhsn c and hhsn c), r hl- , r hl and r hl ; national human genome research institute contract u hg and na- tional institutes of health contract hhsn c. atherosclerotic risk in communities: university of north carolina at chapel hill (n -hc- , n -hc- ), baylor medical college (n -hc- ), university of mississippi medical center(n -hc- ), university of minnesota (n -hc- ), johns hopkins university (n -hc- ), university of texas, houston (n -hc- ). the authors thank the staff and participants of the aric study for their important contributions. infrastructure was partly sup- ported by grant number ul rr , a component of the national institutes of health and nih roadmap for medical research. bhs: ens, ssm and njs are supported in part by nih/ ncrr grant number ul rr . the bhs was sup- ported by grants hd- and hd- from the nation- al institute of child health and human development, and ag- from the national institute on aging. cardia: coronary artery risk in young adults: univer- sity of alabama at birmingham (n -hc- , n - hc- ), university of minnesota (n -hc- ), north- western university (n -hc- ), kaiser foundation research institute (n -hc- ), tufts-new england medical center (n -hc- ), wake forest university (n -hc- ), harbor-ucla research and education institute (n -hc- ), university of california, irvine (n -hc- , n -hc- ). cccs: cccs was supported by grant p hl from the national heart, lung and blood institute, national insti- tutes of health, us department of health and human ser- vices. the authors thank the charisma investigators for phenotype and sample collection. cfs: cleveland family study was supported by funding from the national heart, lung and blood institute, national institutes of health, us department of health and human ser- vices and case western reserve university (r -hl- , m -rr- ). chs: cardiovascular health study: university of washing- ton (n -hc- , n -hc- , u -hl- ),wake forest university (n -hc- ), johns hopkins university (n -hc- , n -hc- ), university of pittsburgh (n -hc- ), university of california, davis (n -hc- ), university of california, irvine (n -hc- ), new england medical center (n -hc- ), university of vermont (n -hc- ), georgetown university (n - hc- ), university of wisconsin (n -hc- ). clear: clear support (gpj) came from r hl , the northwest institute of genetic medicine and the state of washington life sciences discovery fund. the clear investigators sincerely thank the participants for their efforts. epic-nl: the epic-nl study was funded by ‘europe against cancer’ program of the european commission (sanco), dutch ministry of public health, welfare and sports (vws), netherlands cancer registry (nkr), lk re- search funds, dutch prevention funds, dutch cancer society; zonmw the netherlands organization for health human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/lookup/suppl/doi: . /hmg/dds /-/dc http://hmg.oxfordjournals.org/ research and development, world cancer research fund (the netherlands). genotyping of the ibc-chip was funded by iop genomics grant ige from nl agency. fhs: the framingham heart study research included in this study is funded by nih grant/contract n -hc- , r -hl- , r -hl- , r -ag- and by the nih intramural research program. invest: the international verapamil sr trandolapril (invest) genetic substudy was funded by nih grants hl , hl , hl , gm and rr , a grant from abbott pharmaceuticals and the florida opportun- ity fund and nih/ncrr clinical and translational science award (ctsa) to university of florida ul tr . invest and pharmacogenomics evaluation of antihyperten- sive responses (pear) studies thank the participants and the investigators who made this collection possible. luric: luric thanks their participants and researchers and acknowledges that it has received funding through the sixth framework program (integrated project bloodomics, grant lshm-ct- - ) and seventh framework program (inte- grated project atheroremo, grant agreement number ) of the european union. medal: medal was supported by merck & co (white- house station, nj, usa). the authors thank amarjot kaur and the current or former employees of merck research laboratories who contributed to the conduct and analysis of the medal data. mesa: multi-ethnic study of atherosclerosis: support for mesa is provided by contracts n -hc- through n - hc- and ctsa ul -rr- . funding for genotyp- ing was provided by nhlbi contract n -hl- - and n -hc- . monica/kora: the monica/kora augsburg studies were financed by the helmholtz zentrum münchen, german research center for environmental health, neuherberg, germany and supported by grants from the german federal ministry of education and research (bmbf). part of this work was financed by the german national genome research network (ngfnplus, project number gs ), by the german research foundation (th- / - and th- / - ), by the european foundation for the study of diabetes and through additional funds from the helmholtz zentrum münchen, the german diabetes center and the university of ulm. furthermore, the research was supported within the munich center of health sciences (mc health) as part of the ludwig maximilians university innovative. nshs : nova scotia health survey (nshs ) was supported by grants hl- , hl- , hl- , hl- , hl- , hl- and hl- from the nhlbi; by the national health and welfare of canada, ottawa, ontario; by the nova scotia department of health, halifax and by the heart and stroke foundation of new bruns- wick, saint john. pear: pear was supported by the nih pharmacogenetics research network grant u -gm ; and ctsa grants ul -tr (university of florida), ul -tr (emory university) and ul -tr (mayo clinic); and funds from the mayo foundation. penncac: the university of pennsylvania coronary artery calcification study (penncac) gratefully acknowledges in- ternal funding from the university of pennsylvania and the participation of the study subject and is indebted to the inves- tigators on these teams. penncath: the university of pennsylvania catheterization study program (penncath) gratefully acknowledges internal funding from the university of pennsylvania and the participa- tion of the study subject and is indebted to the investigators on these teams. procardis: this work was supported by the british heart foundation; by the european community sixth frame- work program (grant number lshm-ct- - ) and by astrazeneca ab. r.c., m.f. is supported by the british heart foundation center for research excellence. smart: folkert w. asselbergs is supported by a clinical fellowship from the netherlands organization for health re- search and development (zonmw grant ). m.f.l.m. was financially supported by eugeneheart, grant number lshm-ct- - . the authors acknowledge all mr technicians, research nurses and medical students involved in smart heart for valuable support. whi: the women’s health initiative (whi) program is funded by the national heart, lung and blood institute, na- tional institutes of health, us department of health and human services through contracts hhsn c, hhsn c, hhsn c, hhsn - c, hhsn c and hhsn - c. replication cohort acknowledgements this work was supported by the british heart foundation (grant number pg/ / / to p.b.m.) for humancvd beadchip genotyping for the aibiii, ascot, bright, mdc and nordil cohorts; by the wellcome trust (grant number /z/ /z to t.j.) and in part by a vip award from the wellcome trust to queen mary university of london in the / academic year. aibiii: the allied irish bank workers iii study was sup- ported by the higher education authority (ireland), program for research in third-level institutions cycle , program for human genomics. we thank the allied irish bank and their employees for facilitating the study. ascot: this work was supported by pfizer, new york, ny, usa, for the ascot study and the collection of the ascot dna repository; by servier research group, paris, france; and by leo laboratories, copenhagen, denmark. we thank all ascot trial participants, physicians, nurses and practices in the participating countries for their important contribution to the study. in particular, we thank clare muckian and david toomey for their help in dna extraction, storage and handling. bright: "this work was supported by the medical re- search council of great britain (grant number g d) and by the british heart foundation (grant number pg/ / ). a.f.d. was supported by the british heart foundation (grant numbers rg/ / / , sp/ / / ) and by the european union ingenious hypercare consortium: inte- grated genomics, clinical research, and care in hypertension (grant number lshm-c - - ). the bright study is extremely grateful to all the patients who participated in the study and the bright nursing team. we would also human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/ like to thank the barts genome center staff for their assistance with this project. this work forms part of the research themes contributing to the translational research portfolio for barts and the london cardiovascular biomedical research unit, which is supported and funded by the national institute for health research. bwhhs: the british women’s heart and health study is supported by funding from the british heart foundation and the department of health policy research program (england). we thank the bwhhs data collection team, general practi- tioners who helped with recruitment of participants and the par- ticipants. we thank all of the participants and the general practitioners, research nurses and data management staff who supported data collection and preparation. the bwhhs is coor- dinated by shah ebrahim (pi), debbie lawlor and juan-pablo casas, with genotyping funded by the bhf (pg/ / / , pi tom gaunt).the bright study was supported by the medical research council of great britain (g d) and the british heart foundation. (pg/ / ). graphic: this work was supported by the british heart foundation (grant numbers rg/ , pg/ / / ) for recruitment and genotyping of the graphic cohort. n.j.s. was supported by a british heart foundation chair of cardiology (grant number ch/ / . this study is part of the research portfolio supported by the leicester national institute for health research biomedical research unit in cardiovascular disease. lifelines: the lifelines cohort study, and generation and management of gwas genotype data for the lifelines cohort study, is supported by the netherlands organization of scientific research nwo (grant . . . ), the economic structure enhancing fund (fes) of the dutch gov- ernment, the ministry of economic affairs, the ministry of education, culture and science, the ministry for health, welfare and sports, the northern netherlands collaboration of provinces (snn), the province of groningen, university medical center groningen, the university of groningen, dutch kidney foundation and dutch diabetes research foun- dation. we thank behrooz alizadeh, annemieke boesjes, marcel bruinenberg, noortje festen, ilja nolte, lude franke, mitra valimohammadi for their help in creating the gwas data- base, and rob bieringa, joost keers, rené oostergo, rosalie visser and judith vonk for their work related to data collec- tion and validation. the authors are grateful to the study par- ticipants, the staff from the lifelines cohort study and medical biobank northern netherlands, and the participating general practitioners and pharmacists. lifelines scientific protocol preparation: rudolf de boer, hans hillege, melanie van der klauw, gerjan navis, hans ormel, dirkje postma, judith rosmalen, joris slaets, ronald stolk, bruce wolffen- buttel; lifelines gwas working group: behrooz alizadeh, marike boezen, marcel bruinenberg, noortje festen, lude franke, pim van der harst, gerjan navis, dirkje postma, harold snieder, cisca wijmenga, bruce wolffenbuttel. mdc: this work was supported by the swedish medical research council; by the swedish heart and lung founda- tion; by the medical faculty of lund university, malmo university hospital; by the albert p_ahlsson research founda- tion; by the crafoord foundation; by the ernhold lundstroms research foundation, the region skane; by the hulda and conrad mossfelt foundation; by the king gustaf v and queen victoria foundation; by the lennart hanssons memor- ial fund and by the marianne and marcus wallenberg foun- dation. nbs: this work was supported by the wellcome trust (grant number /c/ /z); by the national institute for health research (grant number rp-pg- - ) program grant to nhsbt, and a national institute for health research grant to the cambridge comprehensive biomedical research center; and by the british heart foundation (grant number pg/ / / ) for humancvd beadchip genotyping for the ukbs-cc cohort. we acknowledge use of dna from the uk blood services collection of common controls (ukbs-cc collection), funded by the wellcome trust and by the national institute for health research. the collection was established as part of the wellcome trust case control consortium (wtccc ). nesda: the infrastructure for the nesda study is funded through the geestkracht program of the dutch scientific organ- ization (zon-mw, grant number - - ) and matching funds from participating universities and mental health care organizations. genotyping in nesda was funded by the genetic association information network of the foundation for the us national institutes of health. statistical analyses were carried out on the genetic cluster computer (http:// www.geneticcluster.org), which is financially supported by the netherlands scientific organization (nwo - - ) along with a supplement from the dutch brain foundation. nordil: this work was supported by the british heart foundation (grant number ch/ to a.f.d., rg/ / / to a.f.d., s.p. and c.d.) and a special project, for genotyping of the swedish extremes from the nordil and mdc cohorts; and by pharmacia. we thank professor thomas hedner (department of clinical pharmacology, sahl- grenska academy, gotheburg, sweden) and professor sverre kjeldsen (ullevaal university hospital, university of oslo, oslo, norway), who are investigators of the nordil study. professor kjeldsen is also an investigator of the ascot trial. prevend: prevend genetics is supported by the dutch kidney foundation (grant e ), the national institutes of health (grant lm ), the netherlands organization for health research and development (nwo veni grant . . ) and the dutch inter university cardiology institute netherlands. procardis: this work was supported by the british heart foundation; by the european community sixth framework program (grant number lshm-ct- - ) and by astrazeneca ab. r.c., m.f. is supported by the british heart foundation center for research excellence. rotterdam study: the contributions of inhabitants, general practitioners and pharmacists of the ommoord district to the rotterdam study are gratefully acknowledged. the rotterdam study is supported by the erasmus medical center rotterdam, the erasmus university rotterdam, the netherlands organiza- tion for scientific research (nwo), the netherlands organ- ization for health research and development (zonmw), the research institute for diseases in the elderly (ride), the ministry of education, culture and science, the ministry of health, welfare and sports, the european commission (dg xii), and the municipality of rotterdam. human molecular genetics, , vol. , no. at u niversity of g roningen on m ay , http://hm g.oxfordjournals.org/ d ow nloaded from http://hmg.oxfordjournals.org/ trails: trails (tracking adolescents’ individual lives survey) is a collaborative project involving various depart- ments of the university medical center and university of gro- ningen, the erasmus university medical center rotterdam, the university of utrecht, the radboud medical center nij- megen, and the parnassia bavo group, all in the netherlands. trails has been financially supported by grants from the netherlands organization for scientific research nwo (medical research council program grant gb-mw - - ; zonmw brainpower grant - - ; zonmw risk behavior and dependence grants - - - and - - - ; zonmw culture and health grant - - ; social sciences council medium-sized investment grants gb-magw - - and gb-magw - - ; social sciences council project grants gb-magw - - , gb-magw - - , and gb-magw - - ; nwo large-sized investment grant . . . ; nwo longitu- dinal survey and panel funding - - ); the sophia foun- dation for medical research (projects and ), the dutch ministry of justice (wodc), the european science foundation (eurostress project fp- ), the dutch biobanking and bio- molecular resources research infrastructure (bbmri-nl cp ) and the participating universities. we are grateful to all adolescents, their parents and teachers who participated in this re- search and to everyone who worked on this project and made it possible. statistical analyses were carried out on the genetic cluster computer (http://www.geneticcluster.org), which is fi- nancially supported by the netherlands scientific organization (nwo - - ) along with a supplement from the dutch brain foundation. wghs: women’s genome health study: the wghs is funded by the donald w. reynolds foundation (las vegas, nv), the fondation leducq (paris, france), the national heart, lung and blood institute (nhlbi; hl ) and the national cancer institute (nci; ca ). funding for genotyping and collaborative scientific support was provided by amgen. whii: this work was supported by the british heart foun- dation (grant numbers pg/ / / , rg/ / , sp/ / / ), senior fellowship to a.d.h. (grant number fs/ / ), chair for s.e.h.; by the national heart lung and blood institute (grant number hl ) for m.kivimaki’s and m.kumari’s contributions to this work; by the medical research council (grant number g ) population health scientist fellowship to m.v.h.; by the health and safety executive; by the department of health; by the nation- al institute on aging in the usa (grant number ag ); by the agency for health care policy research (grant number hs ); by the john d. and catherine t. macarthur foun- dation research networks on successful midlife development and socioeconomic status and health. conflict of interest statement. c.m.m. and s.c. are employees of merck pharmaceuticals, usa. authors’ contributions conceived and designed the experiment, alphabetically f.a., p.deb., s.g., b.k., d.l., x.z. performed the experiments: individual cohorts conducted genotyping and cohort-level ana- lyses, except for the nhlbi care program samples were genotyped under a contract with the broad/mit. analyzed the data: f.a., p.deb., s.g., w.g., b.k., d.l., v.t., x.z. con- tributed reagents/materials/analysis tools: all authors. wrote the paper: f.a., p.deb., s.g., w.g., b.k., d.l., v.t., x.z., with contributions from all authors. funding the grants and contracts that have supported care are listed at http://www.nhlbi.nih.gov/resources/geneticsgenomics/programs/ care.htm. the funders had no role in study design, data collection and analysis, decision to publish or preparation of the manu- script. references . lewington, s., clarke, r., qizilbash, n., peto, r. and collins, r. 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microbiome in asthma: the gut–lung axis franco frati , cristina salvatori , cristoforo incorvaia , alessandro bellucci , giuseppe di cara , francesco marcucci and susanna esposito ,* pediatric clinic, department of surgical and biomedical sciences, università degli studi di perugia, perugia, italy; francofrati @gmail.com (f.f.); crisalva_@libero.it (c.s.); alessandro.bell @gmail.com (a.b.); giuseppe.dicara@unipg.it (g.d.c.); fmarcucci @yahoo.it (f.m.) cardiac/pulmonary rehabilitation, asst g. pini/cto, milano, italy; cristoforo.incorvaia@gmail.com * correspondence: susanna.esposito@unimi.it; tel.: + - - - ; fax: + - - - received: october ; accepted: december ; published: december ���������� ������� abstract: asthma is one of the most common chronic respiratory diseases worldwide. it affects all ages but frequently begins in childhood. initiation and exacerbations may depend on individual susceptibility, viral infections, allergen exposure, tobacco smoke exposure, and outdoor air pollution. the aim of this review was to analyze the role of the gut–lung axis in asthma development, considering all asthma phenotypes, and to evaluate whether microbe-based therapies may be used for asthma prevention. several studies have confirmed the role of microbiota in the regulation of immune function and the development of atopy and asthma. these clinical conditions have apparent roots in an insufficiency of early life exposure to the diverse environmental microbiota necessary to ensure colonization of the gastrointestinal and/or respiratory tracts. commensal microbes are necessary for the induction of a balanced, tolerogenic immune system. the identification of commensal bacteria in both the gastroenteric and respiratory tracts could be an innovative and important issue. in conclusion, the function of microbiota in healthy immune response is generally acknowledged, and gut dysbacteriosis might result in chronic inflammatory respiratory disorders, particularly asthma. further investigations are needed to improve our understanding of the role of the microbiome in inflammation and its influence on important risk factors for asthma, including tobacco smoke and host genetic features. keywords: asthma; atopy; gut microbiota; immunity; lung microbiota; microbiome . introduction asthma is one of the most common chronic respiratory diseases worldwide. it affects all ages but frequently begins in childhood. it is a chronic inflammatory disorder of the lower airways that is characterized by wheezing, shortness of breath, chest tightness, and cough, which may vary over time in their occurrence, frequency and severity [ ]. the symptoms are associated with variable expiratory airflow impairment, i.e., breathing difficulty with prolonged expiration due to bronchoconstriction (airway narrowing), airway wall thickening, and increased mucous production. epidemiological studies have estimated that , deaths can be linked to this disease each year, and more than million people have asthma-related symptoms [ ]. a new approach was recently developed using a standardized unit, called “disability-adjusted life year” (daly). daly assesses the years of healthy life lost because of a disease, combining information about morbidity and mortality in terms of healthy years lost. worldwide, asthma accounts for nearly % of all dalys lost, resulting in a particularly high morbidity [ ]. int. j. mol. sci. , , ; doi: . /ijms www.mdpi.com/journal/ijms http://www.mdpi.com/journal/ijms http://www.mdpi.com https://orcid.org/ - - - http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /ijms http://www.mdpi.com/journal/ijms int. j. mol. sci. , , of asthma is a complex disease that includes multiple phenotypes with diverging clinical and pathophysiological characteristics [ , ]. asthma initiation and exacerbation may depend on individual susceptibility, viral infections, allergen exposure, tobacco smoke exposure, and outdoor air pollution [ , ]. if one considers all allergic inflammation triggers, the role of environmental allergen exposure is very relevant. the most common allergens involved in asthma development and exacerbation are dust mites, grass, tree pollen, animal epithelia, fungi, and mold [ , ]. the incidence of allergic diseases has increased dramatically over the last five decades, with large variations in the prevalence of asthma in different countries. although asthma symptoms are generally more common in some high-income countries, several low- and middle-income countries also show high levels of asthma prevalence [ ]. among children, asthma is usually more severe in low- and middle-income than high-income countries [ ]. the decreasing number of infections in western countries, and more recently in developing countries, seems to be at the origin of the increasing incidence of both autoimmune and allergic diseases [ ]. the underlying mechanisms of this event are multiple and complex, involving various regulatory t cell subsets and toll-like receptors (tlrs). these mechanisms could derive, to some extent, from changes in the microbiota caused by changes in lifestyle. the “hygiene hypothesis” was the first to suggest a link between microbes and allergy [ , ]. recently, the original concept of the hygiene hypothesis was expanded to include the increase of antibiotic use and vaccinations, as other lifestyle changes have reduced childhood infections and altered the microbiota [ ]. moreover, other important perinatal and early postnatal factors include caesarean birth and milk formula feeding [ ]. another significant issue is the change in the modern diet based on high levels of fat and low levels of fiber, which has profound consequences for the composition of the intestinal microbiome [ – ]. interestingly, an important study by stein et al. underlined the importance of living in a healthy environment for asthmatic patients, especially near dairy farms [ ]. the study evaluated amish and hutterite children; levels of allergens and endotoxins were measured, and an assessment of the microbiome composition of indoor dust samples was performed. these u.s. farming populations have a similar lifestyle but different agricultural practices. in fact, the amish use traditional farming, while hutterites make use of mechanized techniques. albeit comparable in genetic heritage and standard of living, it was found that in the amish the prevalence of asthma and allergies is four and six times lower than in hutterites, respectively. this is associated with the occurrence in amish dwellings of average endotoxin levels approximately seven times higher. analyzing dust samples from amish and hutterite houses, differences in microbial presence were detected. in addition, in two groups of amish and hutterite children, significant differences were observed concerning innate immune cells in quantitative and functional terms, as well as in phenotypes [ ]. the conclusion was that the amish environment provides protection against asthma by engaging and shaping the innate immune response. the aim of this review was to analyze the role of the gut–lung axis in asthma development considering all asthma phenotypes and to evaluate whether microbe-based therapies may be used for asthma prevention. . microbiome and atopy atopy can be defined as a genetic disposition to develop an allergic reaction and produce elevated levels of ige upon exposure to an environmental antigen and especially one inhaled or ingested [ ]. the chronic inflammation of atopic disease is caused by an enhanced t-helper (th )-type immune response against common, non-pathogenic, environmental antigens (i.e., allergens) in susceptible individuals because of their genetic background [ , ]. th cells play an important role in immune system regulation: th and th cells in particular direct immune response and coordinate other cells (i.e., b cells, eosinophils, mast cells, or neutrophils). th cells produce various cytokines, such as interleukin (il)- , il- , il- , il- , and il- . furthermore, il- stimulates b cells to produce eosinophils and ige antibodies, which in turn enhance mast cells to release histamine, serotonin, and int. j. mol. sci. , , of leukotrienes to cause bronchoconstriction, contributing to allergic responses. the differentiation of naive t cells into il- -secreting t cells is one of the hallmarks of allergy [ , ]. a potential role of the gut microbiota in atopy is based on the observation of germ-free mice, which are born and raised in a sterile environment. they are more susceptible to anaphylaxis induced by the administration of oral antigens when compared to mice that are not germ-free, demonstrating how difficult it is to achieve oral tolerance in animals with altered microbiota [ , ]. to induce and maintain oral tolerance, the role of regulatory t cells (tregs) is fundamental. tregs are a subpopulation of t cells that modulate immune system activity, maintain tolerance to self-antigens, and prevent autoimmune disease development. in recent years, it has also become clear that the induction of tregs is influenced by symbiotic microbes and may therefore provide a possible link between our environment and the susceptibility to allergic disorders [ ]. several studies in the last years have confirmed a role for the microbiota in regulating immune function. for example, bacteroides fragilis modulates the th type / (th /th ) balance [ ], and segmented filamentous bacteria directly stimulate th cell differentiation [ ], whereas clostridium spp. induce treg production [ ]. furthermore, the microbiome produces several mediators, such as lipopolysaccharides (lps), peptidoglycans, short-chain fatty acids (scfas), and gaseous molecules, which influence host physiology depending upon the dose, developmental time period, and tissue type [ – ]. furthermore, the administration of lps to germ-free animals was sufficient to restore oral tolerance [ ]. for example, clostridium spp. are producers of propionic acid (ppa) following the fermentation of complex carbohydrate fibers that are implicated in the modulation of cell signaling, immune function (generation of tregs), and neurotransmitter synthesis and release [ , ]. another important metabolite that depends on microbiota metabolism is tryptophan, which can regulate serotonin production and induce many significant effects on brain function, contributing to psychiatric diseases [ – ]. the innate immune response is based on a number of defensive systems providing nonspecific reactions to environmental stimuli. such responses include both cellular and soluble factors. in addition to their role as a mechanical barrier, the airway epithelium and mucosal stratum guarantees innate immunity provided by immune cells such as dendritic cells, various types of innate lymphoid cells (ilcs), as well as different types of leukocytes, including neutrophils, eosinophils, and macrophages [ ]. different ligand motifs are recognized by receptors such as retinoic acid inducible gene i (rig-i)-like receptors, tlrs, and nucleotide-binding oligomerization domain (nod)-like receptors. these receptors are important in facilitating the timely responses that result in the ensuing adaptive immunity [ ]. the host defense also includes several noncellular secreted elements with antimicrobial action, comprising defensins, lactoferrin, interferons, cathelicidin (ll ), and leukocyte protease inhibitor (slpi) [ ]. . microbiome and asthma asthma has been extensively studied concerning the innate and adaptive immune response. the continuous exposure of the respiratory system to airborne agents in patients with a genetic predisposition offers repeated opportunities for contact with mucosal immunity of inhaled material from upper airways or from the digestive tract, which may contain microbes and bile salts and also provide immune stimuli [ ]. the epithelial mucosa and the dendritic cells, which are in continuous contact with the airway lumen, and antimicrobial peptides produced by immune cells play an important role in the response to environmental agents [ ]. the epithelium controls the local immune activities of iga antibodies, defensins, and lysozymes, which are also regulated by the production of il- , il- , and thymic stromal lymphopoietin (tslp), that in turn stimulate a th type inflammation, which is known to favor the development of asthma. the relevance of th responses driven by the epithelium was supported in a recent study investigating the effect of treatment with an anti-tslp antibody, which reduced allergen-induced bronchoconstriction and diminished the markers of th -related airway inflammation, including the level of exhaled nitric oxide and the number of eosinophils in the sputum [ ]. epithelial cells also produce other regulatory cytokines, such as il- int. j. mol. sci. , , of and transforming growth factor (tgf)-beta. this makes it apparent that the airway epithelium has an important role in orchestrating both the innate and adaptive immunity that is involved in the development of asthma. airway mucus, in addition to the known mucociliary clearance, has other key properties of importance in asthma [ ]. mucus contains both aqueous and non-aqueous constituents. mucins (mucs) are large glycoproteins, predominant types in human airways being muc ac and muc b. experimental data from mice suggest a critical role in protection of airways for muc b, but not for muc ac. in fact, the lack of muc b resulted in lung inflammation, weakened immune homeostasis, and infections from several bacterial species [ ]. proteins with antimicrobial activity are naturally occurring in airway mucus and may exert their action once microbes are trapped in mucus but incompletely eliminated by mucociliary clearance. this is true for cystic fibrosis (cf), wherein repeated chronic infection from pseudomonas aeruginosa is commonly observed. as opposed to the p. aeruginosa phenotype that causes acute infections, in chronic p. aeruginosa infection in cf, a noninvasive phenotype with high resistance to eradication prevails. it was recently suggested that in cf the mucus, as well as the high levels of neutrophil elastase, may favor the growth of p. aeruginosa aggregates resistant to antibiotics [ ]. a further mucous–microbial relation concerns bacteriophages, which are viruses infecting bacterial species. bacteriophages have the ability to bind to mucin glycans, thus reducing bacterial adherence to epithelial cells of the airways [ ]. this adherence has an important role because it may be a form of protection from bacterial infections that are non-host-derived. an important link between the innate and adaptive immunity is represented by dendritic cells (dcs), which influence the response to virus infection and the development of allergic inflammation [ ]. dcs present to the immune system fragments of microbes that promote a number of regulatory and adaptive responses, including th , th , and th pathways. the role of the lung dcs in asthma was recently studied [ ]. dcs may activate several immune cell types, such as t cells and ilcs, the latter comprising a group of cells occurring at barrier surfaces, which are able to provide environmental signals that also target commensal bacteria. the human gut in otherwise healthy subjects is colonized by bacteria and contains more than bacterial species [ ]. the interaction of the human gut and its microbiome has been conditioned by dietary and environmental variations, leading to the selection of a high variety of bacteria interacting both for defense and nutritional advantages. the microbiome is present in both the placenta and meconium, suggesting that the fetus is exposed to bacteria in the prenatal period [ ]. the time and mode of childbirth, maternal age, diet, hospitalization, body mass index, smoking status, socioeconomic status, breastfeeding, and antibiotic use all influence the development of the infant microbiome. long-term stability for many microbiome species begins at approximately two years of age [ – ]. for several years, the respiratory tract was considered germ-free, but this belief, which was due to difficulties in bacterial culturing, was recently negated by metagenomic data analysis that revealed the presence of a microbiome at this site also in healthy neonates. although the gut microbiome contributes to the generation of tregs and probably influences susceptibility to oral allergens, asthma is thought to originate from inhalation, making the lung (and its microbiome) more relevant for asthma initiation. the differing composition of the lung microbiome between asthmatic and healthy people suggests that bacteria may contribute to the initiation of asthma, also indicating a possible important role in influencing the immune responses for microbiota residing in other sites, such as the gut [ ]. this has led to the concept of the “gut–lung axis”. stiemsma et al. studied a population of children diagnosed with asthma at preschool age in whom they found evidence of gut bacterial dysbiosis [ ]. in particular, a reduction of lachnospira in favor of clostridium spp. was potentially linked to asthma. the individual opposing shift in the abundance of lachnospira and clostridium neonatale in the first three months of life suggests that these specific gut bacteria play a role in protecting or promoting development of a preschool age asthmatic phenotype, in addition to the previously identified roles in other atopic disorders. this bacterial dysbiosis was confirmed int. j. mol. sci. , , of in other studies of the same group of authors, in which they showed the relative abundance of the bacterial genera lachnospira and the decrease of veillonella, faecalibacterium, and rothia in children at risk of asthma [ ]. this reduction was accompanied by decreased levels of faecal acetate and dysregulation of enterohepatic metabolites. inoculation of germ-free mice with these four bacteria ameliorated airway inflammation [ ]. clostridium spp. are implicated in the increased risk of asthma in several other studies [ – ]. to underscore the role of mediators produced by the microbiota, another study highlighted the protective effect of a factor called a that is expressed by lung epithelial cells. asthmatic patients showed reduced levels of a in epithelial cells, which might make these patients more susceptible to allergic asthma due to failed lps tolerance induction [ ]. the first days of life seem to be the early life “critical window” during which microbial dysbiosis is very influential in promoting the development of ige-mediated hypersensitivities in humans [ ]. from these findings, we can assume that external agents, such as antibiotic or probiotic administration, can modulate the immune response. in experimental models of murine allergic airway disease, noverr et al. described how antibiotic treatment in immunocompetent hosts followed by oral administration of candida albicans induced susceptibility to allergic airway disease [ , ]. however, oral administrations of mycobacterium vaccae [ ], helicobacter pylori [ ], as well as conventional probiotic strains [ , ] reduced symptoms of allergic airway disease in mice. one study showed that tregs produced in the periphery (but not in the thymus) are known as induced tregs (itreg) and are principally stimulated in the mesenteric lymph nodes (mln), peyer’s patches, and lamina propria (lp) of the small and large intestines [ ]. mice deficient in itregs spontaneously develop th type pathologies characterized by higher percentages of cd + t cells producing il- , il- , and il- cytokines in the mln; il- in the lp of the large intestine; and il- and il- in the lp of the small intestine. an enhanced th immune response and the production of cytokines, such as il- , il- , and il- , contribute to the induction of atopic diseases [ ]. figure shows differences in the lung bacterial microbiota between asthmatic and healthy people, showing that the respiratory tract of an asthmatic patient had an increase in il- , il- , and il- and these differences, together with other environmental factors in individuals with a specific genetic background, may have an impact on the gut microbiome. int. j. mol. sci. , , of int. j. mol. sci. , , x for peer review of figure . differences in the lung bacterial microbiota between asthmatic and healthy children. in the upper section, the names and the image of pathogens that can influence asthma development are represented. in the lower section, on the left it is shown the respiratory tract of an asthmatic child with an increase in interleukin (il)- , il- , and il- ; on the right is shown the respiratory tract of a healthy child with normal values of il- , il- , and il- . early viral infections, mainly due to rhinovirus or respiratory syncytial virus, are also linked to the development of asthma [ , ]. moreover, it has been reported that colonization at one month of age with streptococcus pneumoniae, moraxella catarrhalis, or haemophilus influenzae is associated with an increased risk of subsequent asthma development and enhanced th -associated immunity [ ]. the leading hypotheses to date have been limited to the role of the bacterial and viral microbiota in affecting allergic responses [ – ]. recently, it has been shown that nasal microbiome composition differs in subjects with exacerbated asthma, nonexacerbated asthma, and healthy controls, and nasal taxa could be further investigated as possible biomarkers of asthma activity [ ]. furthermore, it has been observed that one-year-old children with an immature microbial composition have an increased risk of asthma at five years of age [ ]. conversely, adequate maturation of the gut microbiome in this period may protect these pre-disposed children from asthma development [ ]. however, in addition to the complex bacterial communities that have been detected in farm dust, fungal loads have also been reported to be high [ ]. within the context of allergies, fungi are typically considered within the framework of allergens or inducers of allergic inflammation [ ]. nevertheless, many factors complicate the analysis of dysbiosis in subjects with figure . differences in the lung bacterial microbiota between asthmatic and healthy children. in the upper section, the names and the image of pathogens that can influence asthma development are represented. in the lower section, on the left it is shown the respiratory tract of an asthmatic child with an increase in interleukin (il)- , il- , and il- ; on the right is shown the respiratory tract of a healthy child with normal values of il- , il- , and il- . early viral infections, mainly due to rhinovirus or respiratory syncytial virus, are also linked to the development of asthma [ , ]. moreover, it has been reported that colonization at one month of age with streptococcus pneumoniae, moraxella catarrhalis, or haemophilus influenzae is associated with an increased risk of subsequent asthma development and enhanced th -associated immunity [ ]. the leading hypotheses to date have been limited to the role of the bacterial and viral microbiota in affecting allergic responses [ – ]. recently, it has been shown that nasal microbiome composition differs in subjects with exacerbated asthma, nonexacerbated asthma, and healthy controls, and nasal taxa could be further investigated as possible biomarkers of asthma activity [ ]. furthermore, it has been observed that one-year-old children with an immature microbial composition have an increased risk of asthma at five years of age [ ]. conversely, adequate maturation of the gut microbiome in this period may protect these pre-disposed children from asthma development [ ]. however, in addition to the complex bacterial communities that have been detected in farm dust, fungal loads have also been reported to be high [ ]. within the context of allergies, fungi are typically considered within int. j. mol. sci. , , of the framework of allergens or inducers of allergic inflammation [ ]. nevertheless, many factors complicate the analysis of dysbiosis in subjects with food allergy [ ]. comparisons between studies are difficult because of considerable heterogeneity in study design, sample size, age at fecal collection, methods of analysis of gut microbiome, and geographic location. fungi could also be key regulators of inflammation and immune homeostasis [ , ], opening the avenue of beneficial roles of fungi in shaping immune responsiveness. indeed, a recent report by wheeler et al. reported that disruption of the “mycobiome” with an antifungal drug can predispose mice to both colitis and allergic airway inflammation [ ]. interestingly, using advanced multiplex quantitative imaging methods, a recent study has identified an extensive and persistent phosphorylated-stat signature in group ilcs, and intestinal epithelial cells that are induced by il- and il- in mice that lack cd + t cells [ ]. by contrast, in immune-competent mice, phosphorylated-stat activation is induced only transiently by microbial colonization at weaning. this early signature is extinguished as cd + t cell immunity develops in response to the expanding commensal burden. physiologically, the persistent il- production from group ilcs that occurs in the absence of adaptive cd + t cell activity results in impaired host lipid metabolism by decreasing lipid transporter expression in the small bowel. these findings provide new insights into how innate and adaptive lymphocytes operate sequentially and in distinct ways during normal development to establish steady-state commensalism and tissue metabolic homeostasis [ ]. germain’s group showed that ilc s possess properties considered characteristic of adaptive t lymphocytes, namely local activation and distant effector function, but in response to alarm cytokines instead of specific antigens [ , ]. on the other hand, other authors showed the distinct role of individual commensal bacteria in maintaining immune functions during/following dysbiosis induced by antibiotic therapy, thereby shaping host immunity and opening novel therapeutical avenues in conditions of perturbed microbiota composition [ ]. . conclusions recent studies have demonstrated a role for the gut microbiome in influencing remote organs and mucosal and hematopoietic immune functions [ ]. the underlying inflammation in atopic asthma seemed related to the composition of microbiota and appeared associated with the severity of airway obstruction [ ]. treatment with inhaled corticosteroids was associated with changes in the airway inflammatory response to microbiota [ , ]. the interaction of different mucosal barriers, including the gut–lung cross-talk, is likely to be mediated by locally resident microbes and circulating immune cells, but further studies are needed to fully understand this issue. at present, the available treatments for the main non-communicable lung diseases are aimed only at reducing symptoms but are unable to effectively prevent and/or cure the diseases. recent clinical and basic studies to date have identified possible therapeutics that can target innate immunity and the microbiota in asthma [ ]. studies have examined gut microbiota maturation over the first year of life in infants at high risk for asthma, and whether it is modifiable by early-life lactobacillus supplementation [ ]. results showed that early-life gut microbial development is distinct, but plastic, offering a novel strategy for early-life preventive interventions. considering the data already collected on the gut–lung axis, manipulation of the airway and gut microbiome, particularly in early life, might be a strategy to prevent asthma initiation and exacerbation. a better understanding of microbiome-driven pathophysiology and inflammation, in conjunction with the interaction of major risk factors for asthma development, such as host genetics and tobacco smoke, would aid in optimizing current treatments and in managing this chronic lung condition. furthermore, by improving our understanding of the role of the microbiome in these diseases, novel therapeutic strategies of modifying the microbiome through diet, probiotics, or faecal or selected bacterial transfers may be developed. the effects of these therapies on the overall microbiome and consequently on disease severity/progression remain largely unknown and need to be properly understood to realize the full impact of these treatments. int. j. mol. sci. , , of author contributions: f.f. and c.s. wrote the first draft of the manuscript; c.i., a.b., and g.d.c. participated in the literature collection and evaluation; f.m. and s.e. supervised and critically revised the manuscript. all the authors have read and approved the final manuscript. funding: this study was funded by the world association of infectious diseases and immunological 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[crossref] © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . /nature http://dx.doi.org/ . /j.coi. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /science.aam http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /fmicb. . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /jem. http://dx.doi.org/ . /journal.pone. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.jaci. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	microbiome and atopy 	microbiome and asthma 	conclusions 	references microsoft word - ijesb roessingh.doc int. j. entrepreneurship and small business, vol. , no. , copyright © inderscience enterprises ltd. ‘we are growing belize’: modernisation and organisational change in the mennonite settlement of spanish lookout, belize carel roessingh* and kees boersma department of culture, organization and management, faculty of social sciences, vrije universiteit amsterdam, de boelelaan , room z- , hv amsterdam, the netherlands fax: + e-mail: ch.roessingh@fsw.vu.nl e-mail: fk.boersma@fsw.vu.nl *corresponding author abstract: this article addresses the entrepreneurial and organisational activities of a specific mennonite group in belize called the kleine gemeinde community of spanish lookout. building upon christian beliefs, agricultural skills and a strong working ethos, this group was able to build up a stable, local economic network. the authors suggest that their collective resistance against other social groups and their day-to-day strictness lead to processes of ‘selective modernity’. as we make clear in this chapter, the kleine gemeinde mennonites identity contains elements of ethnicity and partial exclusion based upon religious motives. the relative successful economic progression of this group is a sign of both their working ethos inspired by their religious background, and their will to progress and expand. keywords: religious organisation; selective modernity; family networks; mennonite community. reference to this paper should be made as follows: roessingh, c. and boersma, k. ( ) ‘‘we are growing belize’: modernisation and organisational change in the mennonite settlement of spanish lookout, belize’, int. j. entrepreneurship and small business, vol. , no. , pp. – . biographical notes: carel roessingh studied cultural anthropology and received his phd from the university of utrecht. his phd research was on the belizean garifuna. his central research topic now is religious entrepreneurs, focusing on the organisational activities of the mennonites in belize and central america. he works as an associate professor at the vrije universiteit amsterdam, faculty of social sciences, department of culture, organisation and management. his publications appeared amongst others in journals such as journal of mennonite studies, journal of developmental entrepreneurship, international journal of entrepreneurship & small business, international journal of business and globalisation and belizean studies. kees boersma is an associate professor at the vu university amsterdam in the group of culture, organisation and management. his research interest is in ‘science and technology studies’, ‘business history’ and ‘organisational culture and power’. he has published widely on r&d history, organisational learning, enterprise-wide systems, and organisational culture and higher education. his c. roessingh and k. boersma publications appeared amongst others in journals such as enterprise and society, history and technology, human relations, business process management journal and journal of strategic information systems. he is a teacher in the courses organisational politics, organisational and management theory and technology and culture. introduction the mennonites settlements are in the middle of a process of selective modernity which is leading to a visible role on the belizean economic and entrepreneurial market. from its early beginnings, the mennonites enclosed significant differentiations concerning religious principles, ideas and opinions (urry, ). the mennonites originate from the anabaptist movement of the protestant reformation in europe during the first half of the th century (everitt, ; redekop ). the term ‘anabaptist’ stands for ‘re-baptiser’, which means that this religious group believed that adults should be baptised based on their choice to follow christ. in contrast to the then popular approach that children should be baptised soon after birth, the anabaptists thought that the basis of faith must be a conscious rational decision. the anabaptists were convinced that a clear distinction was needed between church and government. therefore, they rejected the authority of a civil/religious government, demanding to baptise children, swear oaths and join military service, because they felt that the scripture suggested a different approach to a life of faith (ryman, ). hostile reactions from the more established churches, to these practices led to persecution. in the th and th century, the mennonite migrated from western europe to areas like northern america and russia where they could live in a relatively isolated manner and where they mainly lived as farmers (dyck, ; loewen ; redekop, ; scott ). later, in the th century, the mennonites moved on to countries in latin america like paraguay and bolivia for several reasons (dana and dana, ; hedberg, ). in march , eight years before british honduras received the right to an internal self-government, a group of kleine gemeinde mennonites arrived to settle at the banks of the belize river at the present site of spanish lookout [higdon, ( ), p. ; quiring ( ); sawatzky ( )]. the first years of the mennonites in belize were difficult and full of new experiences. the description koop, a prominent mennonite member with interest in history, gives in his book the pioneer years in belize provides a dramatic picture: “after a weary and tiring journey, they arrived on the southern bank of the belize river at the present side of spanish lookout’s southern edge. neither bridge or ferry awaited them. on the northern bank of the river was a dark and forbidding jungle with its strange noises and smells. underneath the dense bush, giant snakes and jaguars made their home. the apprehensive settlers may had thoughts akin to those of the children of israel in numbers : : ‘why did the lord bring us to this land to allow us to fall by the sword (tropical diseases)? our wives and children will be taken as plunder. would it not have been better for us to return to egypt?’” [koop, ( ), p.vii] literature tells us that a mennonite is first of all to be a member of a community, a tiny part of an integrated whole. in small settlements like in belize, these christians labour ‘we are growing belize’ and worship together and have little contact with people outside their settlement. this, they believe, is according to god’s plan. one of the mennonites’ strongest basic beliefs is their separation from society. referring to the bible, they state that they are to be ‘strangers and pilgrims’ in the world. this implies that all activities should result in better service to their god. also, the aspect of gelassenheit is reflected in the selective participation in processes of modernity. the mennonite internal system is organised around congregation, which are the basic social and religious unit beyond the extended family. communities or congregations with common rules and discipline “participate in the same conference, which is an organisational unit held together by a biannual meeting of ordained leaders” [kraybill and bowman, ( ), p. ]. in an analysis of the agricultural system of the kleine gemeinde mennonites in spanish lookout in belize, hall ( , p. ) mentions some cultural factors, which explain the economic success of this community and its organisations. these factors are group cohesion, homogeneity, pervasive religion, a high degree of social organisation, a self-sufficient economy, and a low threshold for migration. ever since their arrival in , all mennonite settlements in belize locate themselves on the edge of or even outside the society. for example: inside their settlement the mennonite speak a language called ‘low german’. with outsiders they speak english or spanish. language is an instrument to exclude oneself from society. nevertheless, more than years after their arrival in belize the kleine gemeinde mennonites of spanish lookout are economically one of the most successful communities in belize. the kleine gemeinde community has evolved from an isolated group of families dependent on subsistence agriculture and logging, into a more complex economy with commercial agriculture and agribusiness as its primary engines of growth (higdon, ). peculiar in this case is that an ethnic/religious group, which, culturally, acts in a rather isolated way inside the multi-ethnic society of belize, would be capable of establishing such a strong economic position in this country. in this chapter, we want to address the relationship between the mennonite’s way of being careful with cultural and organisational changes and their perspective on modernity. in connection, viewing this religious group as an organisation, our story also deals with (collective) identity. it is the mennonite struggle to remain a socially isolated group, but at the same time to be successful entrepreneurs. literature on amish-mennonite groups in america provides us with examples of a selective adaptation and a dynamic process of isolation and strictness, which enables individual mennonites to retain their ethnic and religious identity while simultaneously adapting to economic pressure (kraybill, ). according to weber ( ), who wrote about the mennonites as a protestant ‘sect’, members of these communities practice a strict avoidance of ‘the world’. an alternative concept, proposed in this chapter, to explain the dynamics of the belizean mennonites is ‘selective modernity’ in relation to their entrepreneurship, which would possess community and settlement dynamics departing from a differential appropriation of western influence and values. in fact, the mennonites are commonly regarded as the economic motor of belize. this is especially true for the mennonites of spanish lookout and blue creek. this observation has served as the starting point for our fieldwork, data collection and analyses. throughout this chapter, we will interpret the significance of the success of the kleine gemeinde mennonites entrepreneurial organisation and organisational changes in c. roessingh and k. boersma spanish lookout and address their struggle to hold foot as a pure religious community by creating a specific position in a multi-ethnic post-colonial context. selective modernity in relation to mennonite entrepreneurship: a theoretical investigation in social-cultural debates, the process of modernisation, including capitalism, industrialism and the growth of rational organisations and institutions, appears to be inevitable. these aspects of modernity influence personal life and give rise to questions of identity. “modernity is a post-traditional order, in which the question, ‘how shall i live?’ has to be answered in day-to-day decisions about how to behave, what to wear, what to eat – and many other things - as well as interpreted within the temporal unfolding of self-identity” [giddens, ( ), p. ]. however, these individual choices can also constitute a form of collective identity. this type of identity building can make the power of entrepreneurial and organisational instruments into a form of collective resistance against other social groups [castells, ( ), pp. – ]. in the case of the mennonite settlements in belize, the collective identity is one of the basic elements of their economic existence. trust and recognition inside the mennonite settlement and more specific the church community are fundamental aspects to create a collective identity. “the cohesive mennonite community (the village) is designed to care for its members. there is mutuality of recognition; regular and mutual affirmation and certification. the old colony mennonite village becomes a hallowed place” [driedger, ( ), p. ]. driedger points out that trust and recognition inside the mennonite settlement and more specific the church community are based on fundamental elements out of the past which are continuous transplanted from one location to another. generally speaking, all mennonite communities want to be ‘pure’ congregations, in the sense of being members of a blameless conduct. to develop this line of thought, and following miller, we “…would also expect that local congregations that reassert distinctive historical features will out-perform less distinctive congregations within the same denomination, even if they do not become independent of the parent church” [miller, ( ), p. ]. this is in accordance with simmel’s investigation on social conflicts, which states that conflict is admitted to cause or modify interest groups, unifications, and organisations. however, conflict can be a way of achieving some kind of internal unity and understanding of identity (simmel, ). such conflicts may be caused by external tensions, but also by internal fragmentation or segregation. to avoid such self-defeating processes, restrictions and enforcement mechanisms on for example smoking, drinking, eating and other potentially private activities are compelled within strict religious communities to keep members in line (iannaccone, ). in fact, these processes can be seen as normative, cultural control mechanisms (kunda, ), which are based on questions about ‘purity’. in the first place, ‘purity’ is a question of pure religious identity. this religious identity is connected with the discourse about the concepts of ‘church’ and ‘sect’. the question raised here is whether a specific religious group should be seen as part of a church or be marked as a sect. “the ideal-typical ‘sect’ might be defined as a religious organization with a highly committed, voluntary, and converted membership; a separatist orientation; an exclusive social structure; a spirit of regeneration; and an attitude of ethical austerity and demanding asceticism. the ideal-typical ‘church’ would have its ‘we are growing belize’ own complex list of attributes: birth-based membership; inclusiveness and universalism; hierarchical structures; an adaptive, compromising stance vis-a-vis the larger society; and so forth” (iannaccone, ). we want to stress that these definitions fail, in a sense, to analyse communities or even settlements like the belizean mennonites, because theoretical literature and empirical studies about communities and settlements like the mennonites provides a more complex picture and offer insight into a non-ideal, mixed-type case. for the mennonite tradition-oriented communities, the primary social unit is not the individual but a redemptive church community. therefore, instead of individual rights and personal achievements, they are committed to obedience, self-denial, and the authority of the church (kraybill and bowman, ). in practice, the church leaders become some kind of ‘guardians’ of the internal religious values. the bishop, for instance, has the power to excommunicate members of the congregation (roessingh and plasil, ). in the second place, ‘purity’ is also a matter of social identity and organisational continuity. in the case of the mennonites, the organisational trust is in the hands of the church and the individual members are closely connected to each other by means of community and family ties. it is not only this social system which guarantees organisational continuity, but also the commitment of the individuals to the collective history and ethnic identity. and this commitment especially creates a basis for their organisational change and entrepreneurial power. “behind the stories of mennonite businesses and businesspeople lie tales of individual and collective struggles: the struggle to reconcile the accumulation of personal wealth with responsibilities to the collective good; the struggle to reconcile the autonomy and self-interests of the individual with a traditional submission to group authority; the struggle with individualism and commitment” [redekop et al., ( ), p. ]. generally speaking, religious organisational commitment has specific features including a minimal professional staff and mostly volunteer workers, with a low financial compensation and a community structure, which limits the need for professional workers and which constitutes a source of credibility (miller, ). one can also see that in communities where much of the church financing and administration is in the hands of laymen, these laymen constitute an instrumental elite. the lay elite exerts power over the communities instrumental activities like building infrastructures and houses [etzioni, ( ), p. ]. lower participants can be highly integrated into the organisational collectivity – potential informal leaders are recruited for formal organisational positions and can become full-time leaders (roessingh and smits, a). in other words, the participants in the community offer their time, their organisational animation and financial commitment. this is not to say that the lay elite is shaped according to arbitrary decisions. it is the church, whose leadership is organised around three roles: bishop, minister and deacon, that plays a key role in the selection and appointment of trustworthy people. these people then become representative servants of god for the community (roessingh and plasil, ). this is in line with weber’s idea of the concept of calling: “it follows that work in a calling is also affected by this aim, and hence this-worldly work stands in service to the community as a whole” [weber, ( ), p. ]. it is not only service to the local community, but also to other ‘sister’ communities which may be located at other places in the country and even abroad. these communities are highly inter-connected by means of social, religious and business networks. in other words, their social, religious and business networks reaffirm the mennonite collective identity. their social networks help the mennonites to form joint business c. roessingh and k. boersma ventures, long-time business relationships based on trustworthiness, because they provide useful information about the individual’s reliability and creditability (roessingh and smits, b). networks have been founded to make important contributions to entrepreneurial firms (shaw ; granovetter ; arnoldus ; dana and dana ). they provide entrepreneurs with accurate information and tactics. in this chapter, networks are seen as connections between mennonite agricultural related firms and business entrepreneurs. these networks are part of the social environment of the mennonite communities. it is likely that the solidarity between the firms in this network is identical to the solidarity in the social networks of the mennonites. a kleine gemeinde mennonite history the kleine gemeinde separated from the molotschna church community, which was founded in south russia in . in , a young minister by the name of klaas reimer opposed to the contributions of the mennonites in russia to the russian government in the war against napoleon (plett, ). “finding little response in the church, he began meeting separately with like-minded members in , and by they were organised as a separate group. the others mockingly called this conservative minority group the kleine gemeinde (small church), a name which the group itself soon accepted fully as indeed indicating the true nature of the faithful church in a hostile world” [dyck, ( ), ]. in , the kleine gemeinde migrated to manitoba, canada as a response to the pressure of the russian government to gain more influence on the mennonite schools and the fear that young mennonite men would have to fulfil military service. in , the kleine gemeinde left canada for chihuahua and durango in mexico to avoid the total integration of their school system with a larger canadian educational programme (reimer, ). this integration of their educational system into a ‘worldly’ one was seen as a direct threat for the mennonites social and religious identity and formed a major reason to leave this country. the disappointment of meeting yet another unwilling government confronted the mennonites in mexico with a dilemma. because the mexican government proclaimed its intention to incorporate the mennonites into a national social security system, some of the mennonites made the decision to move on. ten years after their migration to mexico the kleine gemeinde and some other mennonite groups moved away from mexico to british honduras (driedger, ; sawatzky, ). this move, however, was also caused by internal tensions between members of the community and church leaders [redekop, ( ), pp. – ]. among other things, this behaviour is the reason that the mennonites are also known as ‘the roaming people’. the kleine gemeinde mennonites migration was the result of an agreement made on the th december between the government of british honduras and a delegation of the quellen colony in chihuahua and durango in northern mexico. in this agreement, the government of british honduras granted to the mennonites among other privileges (british honduras gazette, ): “- the right to run their own churches and schools, with their own teachers, in their own german language, according to their own religion; - the privilege of affirming with the simple ‘yes’ or ‘no’ instead of making oaths in or out of the courts; ‘we are growing belize’ - the right to administer and invest the estates of their own people, especially those of widows and orphans, in their own ‘trust system’, called the ‘waisenamt’, according to their own rules and regulations; - exemption from any social security or compulsory system of insurance”. in turn the kleine gemeinde mennonites agreed among other things to (british honduras gazette, ): “- bring into british honduras capital investment in cash and kind amounting to five hundred thousand dollar more or less british honduras currency; - produce food not only for themselves but also for local consumption and for export market”. both parties, the mennonites and the government of british honduras, had their reasons for this agreement. when we look at the history of this migration process it is obvious that the basic motives of the mennonites have always been ideological and religious. the colonial government of british honduras did have more practical reasons to stimulate the migration plans of the mennonites. around british honduras had a total population of . which increased to . in [bolland, ( ), p. ]. in the late s, the situation in british honduras was very complicated. local political leaders, like the former prime minister of belize george price, were overtly demanding autonomy within the british colonial empire. next to this internal claim, the ever-lasting dispute over the territorial rights between belize and guatemala was intensified. the inside situation of british honduras was very fragile and the country was depending on the outside support of great britain on all levels. “one problem facing the independence movement was a profound economic dependence on colonial authorities for capital, imported food supplies and manufactured goods. the colony had a small population, and only a few farmers engaged in subsistence agriculture” [hidgon, ( ), p. ]. the government therefore acknowledged that skilled farmers were required to make the rough tropical forest suitable for cultivation and stock-breeding and to establish a commercial agricultural sector in the country. george price, who was the member for natural resources of the government of british honduras in , stated that: “one reason for encouraging them, was that the mennonites are good farmers and since belize had a small population and an excess of land, i thought it would be a good idea to introduce modern techniques of agriculture in our traditional ways.” [shaw, ( ), p. ]. however, the choice of the government was criticised because they preferred white european mennonites over black west indians (daily clarion, ). nevertheless local newspapers and political leaders agreed that the need of agriculture products for the local market was evident. a belize billboard commentator wrote: “one community of the mennonites will, we understand, begin their settlement on land purchased by them for some half million dollar. this illustrates two important points. first that the mennonites are no paupers who are likely to become charges of the state, and secondly it is a sign of the high value placed on agricultural land by the mennonites. this is an important demonstration in a country where the people place little value on agriculture. the mennonites obviously mean to make agriculture a paying business in british honduras.” (belize billboard, ) c. roessingh and k. boersma the context of the mennonite settlements in belize: die stillen im lande? the country that they entered at the time was british honduras. on september , this former british colony has become independent. this date marked the formal end of a process of independence that took years. in , british honduras received the right to an internal self-government and in the name of the country was changed into belize. nowadays, with an area of , km and approximately , inhabitants, belize is one of the smallest and most under-populated countries in central america (belizean government, ). the country has a multi-ethnic population consisting of, among others, mestizos, creoles, garifuna, maya’s, east indian and chinese (roessingh, ). according to the census of , . % of belize’s inhabitants are mennonites – which comes down to , people on a total in of , inhabitants. however, the census indicates that there are , religious mennonites ( . %) in this multi-religious country (central statistical office, ). the difference between the percentages of ethnic and religious mennonites is at least something that requires an explanation. in practice, mennonite identity turns out to be a dual concept. the ethnic identity of the mennonites is based on a combination of shared assumptions on life values, life style and ideologies. the mennonites share their common descent from western europe, especially the netherlands, switzerland and germany. this specific background is shown in the different kinds of denomination in which the lineage of the background is reflected. the old order mennonites, whose name does not refer to a specific group, have their roots in switzerland and south germany. the old colony mennonites and the kleine gemeinde, who live in belize, have their origin in the netherlands and the northern parts of germany [dyck, ( ), pp. – ]. the mennonites share socially relevant cultural characteristics, like their anabaptist background, their cultural and social repertoires, and their ‘white’ phenotypic features. the mennonites have a common coding system of attitudes and behaviour, which can be found in concepts like the principle of ‘gelassenheit’, or the submission to the will of god (which results in values like obedience, humility and simplicity), and the system of the ‘ordnung’, that contains common rules and discipline. in an article he wrote on the interaction between the mestizo and maya population in western belize with the mennonite community of spanish lookout, jantzen ( ) notes that this interaction was not only based on work relationships and business transactions but was also rooted in faith. some mestizo and maya people turned to the anabaptist religion of the mennonites. this way their religious identity became a mennonite one but this does not mean that they have been accepted as ethnic mennonites. their roots are not in western europe, their features are not like the ethnic mennonites and they do not speak low german, the internal language. although the low german language is vanishing in the daily conversation of the people in spanish lookout and blue creek (roessingh and schoonderwoerd ; roessingh and smits a), it is still the language of their religion. the dual concept of mennonite identity is based on ethnicity and partial exclusion. even in this mosaic of cultural and ethnic diversity it is rather striking to come across mennonites. men from the old colony community wear blue denim overalls or jeans with plain coloured shirts, black shoes and caps or straw cowboy hats. the women of this community are expected to wear bonnets or a shawl on their heads and dresses, which is a sign of simplicity and modesty, possibly with an apron. clothes and artefacts, are ‘we are growing belize’ important outward signs of internal attitudes, which can be interpreted as a sign of symbolic capital (bourdieu, ). as a result of this strong symbolic capital belizeans often consider the mennonite settlements as one of a kind. but in practice there is no uniformity. inside the different settlements in belize, strong controversial opinions exist of the way one should interpret the bible. the consequence is the possibility of fragmentation between and inside the mennonite settlements. there are some mennonite settlements which are rather isolated with a strong inward focus. they are very careful in picking out people they interact with and with whom they trade. however, other mennonite settlements have more interactions with other people of the society. in practice, there is no uniformity (roessingh, ). first of all, there are mennonites settlements with different numbers of inhabitants in belize as shown in the table below. table mennonite settlements in belize inhabitants less than – – , , – , , – , settlements pine hill, pilgrimage valley, richmond hill lower barton creek, upper barton creek, springfield blue creek, indian creek spanish lookout shipyard, little belize this differentiation and lack of uniformity does not mean that there is no basic connection between the settlements. the fact that there are differences is related with the notion of (collective) identity, social conflicts, and the will to be a pure community, as we discussed before. in practice, individual families have relatives in other settlements. basically because of ideological differences, economic problems or shortage of ground, families have migrated to other places. because of this most mennonite settlements are interrelated on some level. in spite of these kinship relationships, there are three main streams to which the different church communities in belize belong, namely the conservative amish mennonite communities, the traditional old colony or altkolonier mennonites and the progressive church communities like the kleine gemeinde and the evangelical mennonite mission church (emmc). the distinction between these three main streams is based on the level of restrictions on the use of technological innovation in the agricultural machinery. “a case in point, which has become the crux of serious controversy among the altkolonier, is the use of modern rubber-tired tractor. the proscription against them has its roots back in when altkolonier leaders in manitoba and saskatchewan [both in canada] agreed to ban the automobile forever and to arrest technological innovation in agricultural machinery at the then existing level. this meant that the farm tractor, which by then was fairly common, would be tolerated, but pneumatic tires are banned because, so the argument goes, they make the tractor equivalent to an automobile” [sawatzky, ( ), p. ]. in practice, this means that the conservative mennonites, who ban any form of modernisation, work with horsepower and if necessary, hire a tractor with driver. the traditional mennonites use tractors but have a ban on pneumatic tires. when they to go to the market to sell their furniture they rent a car and driver. the progressive mennonites do not have a ban on technological innovation, they use automobiles and all kind of modern machinery in their farm and farm related businesses. c. roessingh and k. boersma . the conservative settlements richmond hill used to be a very small conservative settlement in the orange walk district in the northern part of belize. a group of old colony mennonites from alberta, canada established this settlement in . the inhabitants “soon began to break up, however, and by the early s some members of this group had bought land in shipyard, some had returned to canada and some had moved with other dissatisfied belizean mennonites to bolivia and paraguay” [everitt, ( ), p. ]. richmond hill is a good example of the way these people seek for land in countries where they are able to live according to their own rules. but when the circumstances do not live up to their religious prescriptions or farming abilities, they abandon their homestead and move on. another very small mennonite settlement is pilgrimage valley in the cayo district. the census indicates that there are now only inhabitants in this settlement (central statistical office, ). it was founded in by a mixed group of ten amish and old order families from pennsylvania, ohio, arkansas and ontario [sawatzky, ( ), p. ]. although this settlement never really got to the level of expansion, the interesting thing about these mennonites is the fact that they had a ‘different kind of background’. beside that they were old order and not old colony; there was also the influence of the amish which makes this group special. one thing is clear; this settlement never came to the point where it was possible to create a stable existence. “consequently, several families moved to the more remote settlements at barton creek or they returned to north america” [everitt, ( ), p. ]. the question arises what role was played by this conservative group in the religious notions of the other settlements in this area, especially that of the upper barton creek settlement. the mennonites of upper and lower barton creek, springfield in the cayo district and pine hill in the toledo district are also conservative in their lifestyle. “their reasons for migrating to this quite isolated area are many and varied, but they are basically conservative and dislike association with other mennonites in belize, mexico and canada who have become too worldly “ [everitt, ( ), p. ]. a deacon in springfield reconfirmed that he did not recognise the kleine gemeinde of spanish lookout as mennonites because they were too worldly in the eyes of the springfield community members. in , the settlement of upper barton creek was established. three men and their families were very important in the process of founding this settlement. two of them came from pilgrimage valley and one from spanish lookout (schneider, ). in , lower barton creek followed. mennonite families from all over belize composed both settlements. springfield and pine hill are settlements, which were founded because of the land shortage in the barton creek area. like the lower barton creek and pine hill settlements, the upper barton creek and springfield mennonites are connected by family ties. this connection is: “a result of the population growth of upper barton creek and in-migration of lower barton creek families and proof of its stability as a unique cultural entity and agricultural success. the decision to seek new land for a daughter community [namely springfield, which was established in ] came about gradually after the influx of additional families. quite simply, there is nowhere for upper barton creek to expand the occasional small parcel notwithstanding. with so many youth coming onto age and young couples in need of land, it became necessary to purchase another large tract, preferably a minimum of one thousand acres” (nippert, ). one of the striking features of these mennonites is their beard. this may be a very strange point to discuss. ‘we are growing belize’ but one of the characteristics of the mennonites is their appearance. most of the mennonites in belize do not have beards, except the barton creek and springfield mennonites. following kraybill and bowman ( ), the mennonites do not wear beards with which they distinguish themselves from anabaptist, like the amish and the brethren. a springfield bishop explained that their beard originated in russia to distinguish from the russian soldiers and did not indicate, as suggested by the researchers, any influence of the amish. . the traditional settlements little belize, which is a traditional old colony community, is located in the corozal district in the northern part of the country and originated out of the shipyard settlement in the orange walk district. in fact there is a kind of connection between shipyard, little belize and indian creek, which is also situated in the orange creek district. in , a group of old colony mennonites established shipyard. this group lived according to their traditional lifestyle. in the course of time migrants from other places, like spanish lookout and blue creek, who disagreed with what they interpreted as the increasing worldliness and modernisation, settled in shipyard (roessingh and plasil, ). because of land shortage, but also because of the fear for non-traditional incursions upon their culture, many of the more ‘conservative’ traditional mennonites from shipyard began to move to a new established settlement, called little belize, around (everitt, ). the expansion of little belize has been remarkable. together with shipyard, little belize is the largest mennonite settlement in the country. at the end of the eighties another settlement was started for the mushrooming younger generation of shipyard, called indian creek. all three settlements are the offspring from the old colony mennonites and characterised by their tradition-based lifestyle which is symbolised through their horse and buggy transportation, their ban on rubber wheels for tractors and the power of the church leader (roessingh and plasil, ). shipyard, little belize and indian creek are old colony mennonite based settlements where the influence of modernity is integrated bit by bit. nonetheless the old colony mennonites protect their religious values and try to live according to their traditional farming systems. shipyard has for some years now, in contrast to for instance springfield, been confronted with an internal religious schism. the progressive emmc, which has its headquarters in manitoba (canada) and a church in blue creek have slowly started to enter the shipyard settlement. the old colony mennonites of shipyard do not accept people who are member of this church. they avoid them because they are excommunicated from the old colony church. most old colony mennonites are embedded in transnational networks. these networks are mainly based on ideological religious exchange and family visits. for the shipyard mennonites these networks includes brother communities in canada, usa, mexico and bolivia. because of a lifestyle which is also based on interaction with people from outside the settlement and a more differentiated agricultural related entrepreneurial system, the old colony mennonites of shipyard are more visible on the belizean marked then for instance the springfield mennonites. interesting is what role the internal religious differentiation will have on the possibilities for social and economic changes inside the settlement in the future. c. roessingh and k. boersma . the progressive settlements together with spanish lookout, the blue creek settlement in the orange walk district is regarded as more progressive mennonite settlements in belize. the shipyard and blue creek mennonites are from the same background in mexico. originally, it was the intention of these old colony mennonites to establish one settlement, blue creek, in . but from the beginning there were problems between two sections in this settlement. although both sections of this old colony church community lived according to their traditional lifestyle there were disagreements about the way one should use agricultural systems and machinery in this tropic area. the more ‘conservative’ traditional mennonites decided to start a new settlement and founded shipyard. nonetheless more traditional orientated kleine gemeinde mennonites from spanish lookout migrated not only to shipyard but also to blue creek, were they founded their own kleine gemeinde church. increasingly modern technology began to play an important role in the blue creek settlement (roessingh and smits, a, b). economically this settlement prospered. in the course of time the basic structures of the old colony church vanished and were replaced by the emmc, which came over from canada (roessingh and smits, a). the spanish lookout settlement, in the cayo district, is also known as a progressive mennonite settlement. like blue creek, the emmc has also settled itself in spanish lookout. the emmc is seen as an even more progressive church than the kleine gemeinde church. in practice, this means that there is some disturbance in the settlement because the inner relations and structures are at stake (roessingh and schoonderwoerd, ). nonetheless, the kleine gemeinde church is still dominant and the most influential in this settlement (roessingh and mol, ). in the introduction, we mentioned that the kleine gemeinde mennonites of spanish lookout are regarded as one of the economically most successful church communities of belize. of course there is always the question of how to decline successful. for the conservative mennonites it will mean something different from what the kleine gemeinde mennonites will attribute to it. the best way to show the differences and the different position of a conservative and a progressive settlement might be to show the contrast of the road in-use. . the contrast between two settlements the inhabitants specific religious backgrounds have consequences for the features of the settlements. one of the most eye-catching features for an outsider for instance is the construction of the roads to the settlements. the differences in the quality of the road in-use, as part of the new infrastructure to be built and in the use of cars and farm tractors are signs of different religious identities. the way a settlement like spanish lookout, in which the progressive kleine gemeinde mennonites are living, gives a totally different picture than the settlements of the upper barton creek and the springfield settlement of the conservative amish mennonites do. visitors to spanish lookout have three roads to enter the settlement. two of them are bumpy, dusty and not paved. the third road is paved all the way, which was constructed and paid for by the spanish lookout mennonite road committee. one road, which is the short cut, passes a hand-operated ferry. after a couple of kilometres, the entrance to spanish lookout is amazing. a beautiful valley with the most well paved road of belize, connecting the southern part of the village with the northern end. the whole sight gives ‘we are growing belize’ an impression of an area, which one associates, with the midwest of northern america. the wooden houses are in a good state and nice looking. around the houses one can see short cleaned gardens, large sheds for farming machinery, big chicken-farms, fields with corn, red kidney beans and sorghum. on the paved road tractors, pickups and four-wheel driven land rovers are passing by. the road to upper barton creek and springfield is dusty, bumpy and unpaved. but once one enters the village the road has become a cart track. these conservative amish mennonites use horse drawn wagons. the small farms lie scattered between the hillsides of the maya mountains. the houses are small and modest. the fields are more like horticultural land. in contrast to spanish lookout, barton creek and springfield look tranquil. the kleine gemeinde mennonites of spanish lookout are progressive in their acceptance of technology. they own tractors, trucks, caterpillars, automobiles and they use electricity. mennonites from places like upper barton creek and springfield on the other hand are not allowed to use machinery or electricity. horsepower drives the sawmill in springfield, which is owned by the people of the settlement. a struggle to stay pure: entrepreneurial behaviour and organisational power of spanish lookout ‘we are growing belize’ is the slogan on a sign of reimers feed mill beside the main road through spanish lookout. this sign is a symbol of the entrepreneurship of the mennonites of spanish lookout. we’re growing belize. it claims an image of self-confidence and even organisational power. however, when the kleine gemeinde mennonites came to belize they were far from self-confidential. they entered a world, which was very different from canada and mexico. in the introduction, koop ( ) describes that the first settlers had a hard time. there was much uncertainty about the future and their position in this new homeland. from the moment the mennonites settled in belize, some families moved back to mexico or even canada. internal fragmentation caused removal to other settlements in the country. the kleine gemeinde mennonites had ‘some advantages’ compared to some of the other church communities who migrated to belize. most of the church communities in belize migrated around from the southern part of russia (the present day ukraine) to canada and the united states. from this group some started to move to mexico around . the kleine gemeinde settled in mexico around . the fact that the kleine gemeinde mennonites stayed in canada for a longer period reflected in some internal changes. this original conservative church community started to adopt “new farm methods and produced new products as dictated by their perspective physical environments and regional markets and required by their aim to secure a familiar standard of living and obtain the resources to pass the farm onto the next generation” [loewen, ( ), p. ]. it seems that the kleine gemeinde mennonites are farmers with the capacity to use their internal organisation to adapt and transform outside circumstances into a level that enables the church community and the household economic unit (the family) to exist (penner et al., ). of course changes do take place in spanish lookout, but intrinsic moral rules are deeply rooted in the way the mennonites accept these changes. aspects and elements out of their past are used and integrated in a conscientious way to keep c. roessingh and k. boersma structure in their system. these aspects and elements are based on the link between the family and kinship networks, the church as a moral hub, the collective way of farming and the accurate type of entrepreneurship. all these aspects are mutually connected with their religious identity in the weberian sense. an aspect, which requires some special attention because it is related to the collective way of farming and everything farming is about, is the subject of land and land resources. although belize is the most under-populated country in central america, there are rules and limitations in land resources. the government keeps this matter under its administration and jurisdiction. in practice, this means that farmers do not have free access to land. little belize, indian creek and springfield are settlements which, for one reason, are established because of the need of farming land. the mennonites in spanish lookout have also been in need for land. since , their land expansion has been progressive. the internal system of land tenure for the members of the kleine gemeinde church is based on a communal property. this means that a farmer is a kind of ‘partial’ owner. he can make his own farming plan and produce the crops or keep the animals he chooses. but nonetheless he is depending on the cooperative with whom he trades to approve of the amount of production. the farmer is entitled to bequeath or to rent his property to his children. otherwise he has to sell his property to one of the other church members. the organisational power of this system is based on the control of the community lay elite on the resources and the ability to distribute land to the church members without losing land and production. poultry has always been one of the elements of the kleine gemeinde mennonite agriculture business, next to other farming activities. “an early emphasis on poultry has led to one of the most visible and economically important aspects of spanish lookout agriculture. from modest beginnings, the complex of hatchery, feed mills, and broiler-killing plant has grown to dominate the colony and has greatly expanded these products availability in belizean markets. nearly every colonist [kleine gemeinde mennonite] is somehow involved in selling eggs, raising broilers, or preparing or marketing poultry products” [jantzen, ( ), p. ]. this is in line with our empirical findings, which show a stable growth in chicken production of the spanish lookout community. the growth of the broilers is especially important, because it indicates the entrepreneurial and organisational power of the kleine gemeinde mennonites in the belizean chicken industry. in other words, the mennonites provide belize with chicken meat, which is one of the main sources of protein in the country. “from very modest beginnings in the early s these businesses have grown to a dominant position in their respective areas in the economy of belize” [snider, ( ), p. ]. from the beginning, the chicken industry was a niche in the market the kleine gemeinde mennonites entered. as a consequence, the belizean inhabitants became independent of expensive, imported chicken meat. “this broiler business is conductive to the corn raising business on spanish lookout. the corn farmers were able to feed a portion of the crop to their broilers or sell it to farmers who had broilers operations. this double advantage was a real boost for the hard-pressed frontier farmers” [koop, ( ), p. ]. this entrepreneurial behaviour was exactly the reason why the belizean government was willing to welcome this group: to enrich the country with farming relating business activities. besides this, it is a sign of their organisational power, which is reconfirmed by the way they organise their church community and settlement internally. the organisational committee chart at the end of this chapter, which shows the intentional organisational routines, is an indication of the rationalisation of settlements organisational processes. the different committees are headed by lay-members of the ‘we are growing belize’ spanish lookout settlement – the so-called instrumental elite. in particular, it is the land chairman and, more specifically the zoning committee members who, besides building barns for hogs, cattle and other agricultural purposes, are responsible for the chicken industry infrastructure like buildings and chicken coops. this infrastructure is part of the settlement as a whole. however, it is the individual farmer and the broiler cooperative ‘quality poultry products’, which are responsible respectively for the chicken production on a local level and the distribution countrywide. the farmer trading center, also headed by a mennonite cooperative committee like the quality poultry products, plays a major role in the financial funding and guarantees continuity in process. the process runs as follows. the individual farmer grows the chickens and sells them to the quality poultry products cooperative. this cooperative has three functions. first of all it has a processing function in the sense that it prepares the chickens for the market. secondly, the cooperative has a responsibility to its shareholders. it contributes the profits and members are able to make a loan, with poultry related innovations. thirdly, it functions as an intermediary actor between the individual farmer and the farmer trading center that operates as an internal settlement bank. the only people who are allowed to participate in this internal financial system are members of the two cooperatives. this implies that those chicken farmers who are not a member of the cooperatives are excluded from internal advantages. “the community’s cooperatives are an important source of capital for member households. the farmers trading center (ftc) is the main purchasing cooperative and de facto bank within the community. the ftc takes in cash deposits, issues bank checks and administers a ‘coupon’ system for internal colony [mennonite members] use. the ftc grants loans for all types of purposes including home and barn construction, equipment purchases, and most importantly, land purchases” [higdon, ( ), ]. next to this internal organisation of capital advantages, the kleine gemeinde church also has a credit and loan system for their members called the ‘hilfsverein’ or ‘help association’. adult male members of the church make an annual contribution, the ‘auflagen’. this contribution which is a percentage of the members total financial assets is deposited in a general fund. by making this contribution adult males are able to get a loan against a profitable and low interest rate. the entrepreneurial behaviour and organisational power as sketched above are based on an internal network of members of the kleine gemeinde mennonite community. this network is based on religious, organisational and family ties. it does not only function as an enabling structure for entrepreneurial behaviour and change, but also contains elements of exclusion. this system, in short, is a cultural instrument of normative control to keep the church community pure. conclusions the case of the kleine gemeinde mennonites offers us an insight in the specific religious and ethnic background of this group and the relation to the process of organisational change in general. the story contains elements of migration history, frontier experiences, adaptation to local contexts and entrepreneurial behaviour. by showing their collectivity, networks of acquaintance, power of distinctions and mentality of preservation, our chapter comes about struggles for purity. the spanish lookout kleine gemeinde mennonites success is built upon stable self-defeating structures of internal solidarity. these structures, as we have shown, are based upon institutional aspects c. roessingh and k. boersma of the church and the intrinsic mentality of family ties connected by a strong, local household-based network-economy. we can reconfirm driedger’s observation ( ) that trust and recognition inside the mennonite settlement are fundamental aspects of the creation of a collective identity and suggest that this aspect is also connected with their ability of organisational change. trust and recognition inside spanish lookout are fundamental aspects for the creation of collective identity. the organisation of the chicken industry, which we take as a major example of their entrepreneurial expansion, helps to explain this collective identity. rooted in the settlement, this industry does not only connect agribusinesses, but at the same time different mennonite families. in other words, it is the solidarity between agribusiness firms in this network that mirrors the solidarity in the social network of the spanish lookout mennonites. the mennonites are often called ‘die stillen im lande’. this characterisation is not only used by the mennonites themselves but also by outsiders. the phrase ‘die stillen im lande is taken from the bible (ps. : : “gegen die stillen im lande”) and symbolises the ‘good’ people and their ‘gelassenheit’ in a world of wickedness and nonbelievers. these pure and humble people are quiet and are the ones who work on the land, which was created and given to them by god. however, in our case we have shown that these people can be quite successful. this contrast raises the following question: to what extent the kleine gemeinde mennonites of spanish lookout are symbols of ‘the quiet ones in the country’? when they entered british honduras in the mennonites were confronted with an environment which they had to conquer. by working hard and leaning on their organisational skills they were able to get a grip on their surroundings. the instrument to get control over the existential circumstances was and still is their strategic and accurate way of using their entrepreneurial skills (roessingh and plasil, ). it is not so much the amount of land that is owned by the kleine gemeinde mennonites of spanish lookout per se that stands for their success, but the way it is cultivated. these aspects of land and the cultivation of it, however, are only one side of the coin. it is their ability to transplant traditional cultural elements into a new context that is the basis of both their stability and flexibility. in line with this, driedger ( ) points out that trust and recognition inside the mennonite settlement are based on the continuous transplantation of fundamental elements of the past. according to us this element is a very strong reliable mechanism within spanish lookout, which is used by die ‘stillen im lande’ and forms a starting point to adapt their new frontier environment. right from the start the kleine gemeinde mennonites used the farming knowledge of the indigenous people to broaden their own perspectives on cultivating newly obtained land. however, in this phase of the process the kleine gemeinde mennonite ideology was centred around the concept of selective modernity in relation to their entrepreneurial processes. the duality between the need of purity and the need to progress was illustrated by the road in-use contrast between a conservative and a progressive settlement. inside the settlement this duality is best illustrated by the expansion of the broiler industry and the austere way in which the kleine gemeinde families maintain their household system. in belize this austere image is related to the mennonite identity, in other words, it is their symbolic capital. to conclude, the remark ‘we are growing belize’ is a sign of both their working ethos inspired by their religious background, and their will to progress and expand. ‘we are growing belize’ references arnoldus, d. 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[ ] t.m. kang, d.w. hilgemann, nature ( ) – . [ ] j. liao, h. li, w. zeng, d.b. sauer, r. belmares, y. jiang, science ( ) – . doi: . /j.bbabio. . . p the evolutionary history of membrane-integral pyrophosphatases supports na+ as the ancestral coupling ion in membrane bioenergetics heidi h. luoto , alexander a. baykov , reijo lahti , anssi m. malinen department of biochemistry and food chemistry, university of turku, fin- turku, finland abstracts s http://dx.doi.org/ . /j.bbabio. . . http://dx.doi.org/doi: . /j.mam. . . http://dx.doi.org/doi: . /j.mam. . . http://dx.doi.org/ . /j.bbabio. . . http://dx.doi.org/ . /j.bbabio. . . e med oral patol oral cir bucal. jul ; ( ):e - . ellis-van creveld syndrome journal section: special patients publication types: case report ellis-van creveld syndrome. case report and literature review daniela alves-pereira , leonardo berini-aytés , cosme gay-escoda dds. fellow of the master degree program in oral surgery and implantology. school of dentistry of the university of barce- lona (spain) dds, md, phd. dean. professor of oral and maxillofacial surgery. professor of the master degree program in oral surgery and implantology. school of dentistry of the university of barcelona (spain). researcher of the ub-idibell institute dds, md, phd. chairman and professor of oral and maxillofacial surgery. director of the master degree program in oral surgery and implantology. school of dentistry of the university of barcelona. researcher of the ub-idibell institute. oral and maxillofacial surgeon of the teknon medical center, barcelona (spain) correspondence: centro médico teknon c/ vilana - barcelona. spain cgay@ub.edu received: / / accepted: / / alves-pereira d, berini-aytés l, gay-escoda c. ellis-van creveld syn- drome. case report and literature review. med oral patol oral cir bucal. jul ; ( ):e - . http://www.medicinaoral.com/medoralfree /v i /medoralv i p .pdf abstract ellis-van creveld syndrome is a genetic disorder that was first described by richard ellis and simon van creveld in . the four principal characteristics are chondrodysplasia, polydactyly, ectodermal dysplasia and congenital heart defects. the orofacial manifestations include multiple gingivolabial musculofibrous fraenula, dental anoma- lies, hypodontia and malocclusion. the disease can be diagnosed at any age, even during pregnancy. the differ- entiation should be made between jeune syndrome and other orofaciodigital syndromes. key words: ellis-van creveld syndrome, chondrodysplasia, orofacial anomalies. article number: http://www.medicinaoral.com/ © medicina oral s. l. c.i.f. b - pissn - - eissn: - email: medicina@medicinaoral.com indexed in: -sci expanded -journal citation reports -index medicus / medline / pubmed -embase, excerpta medica -scopus -indice médico español introduction ellis-van creveld syndrome (evc) or chondroecto- dermal dysplasia is a rare autosomal recessive disease resulting from a genetic defect located in chromosome p ( - ). it was first described in by richard el- lis and simon van creveld ( ). the exact prevalence of this illness is unknown. around cases are described in the literature ( ). the principal features of this syndrome are chondroec- todermal dysplasia, polydactyly and congenital heart defects. the patients have small stature, short limbs, fine sparse hair and hypoplastic fingernails. oral manifesta- tions include multiple musculofibrous frenula, dental transposition, conical teeth, hypoplasia of the enamel, hypodontia and malocclusion. the teeth can erupt and exfoliate prematurely ( - ). this review of the clinical and radiographic features of evc syndrome provides the dentist with the possibil- ity of making an early diagnosis and treatment, and to establish a differential diagnosis with other clinically similar entities. we describe a clinical case of a patient who visited the odontological clinic of the university of barcelona pre- senting the typical clinical features of evc syndrome. e med oral patol oral cir bucal. jul ; ( ):e - . ellis-van creveld syndrome case report the patient attended the pathology and periodontal surgery unit of the department of the oral surgery of the university of barcelona, spain, referred by the orthodontic service. the objective was to assess the periodontal condition and to control the plaque index in preparation for orthodontic treatment to align and level the teeth for subsequent prosthetic rehabilitation. the patient, a -year-old woman with no toxic habits or known allergies. no family history of interest, and a personal pathological history of having been diagnosed at birth with evc syndrome without heart involvement. at months of age the polydactyly had been surgically corrected (fig. a). fig. . a) the presence of a surgically operated sixth finger is perceived on both hands and the hypoplastic fingernails are observed. b) frontal photographs. facial symmetry, fine and lip seal. fig. . view of occlusion and dental arcades. a) frontal view of the mouth. multiple musculofibrous frenula, diverse morphological anomalies of the teeth and malocclusion are observed. b) occlusal image of the upper dental arcade. c) occlusal view of the lower dental arcade showing agenesis of the lower incisors. fig. . the orthopantomography reveals extruded upper third molars, agenesis of the lower third molars and the incisors and an anteroinferior radiopacity due to the presence of a bone graft fixation screw. a b c e med oral patol oral cir bucal. jul ; ( ):e - . ellis-van creveld syndrome the patient is generally healthy (fig. b) although with very short stature ( . m). the hands are deformed and the presence of a sixth finger can be perceived (fig. a). at orofacial level, no anomalies are detected on exami- nation of the temporomandibular joint. regarding soft tissues, the appearance of gingivolabial fibrous bands or multiple muscular frenula is highlighted. mid-line deviation presents, also malocclusion with posterior crossbite in the second and third quadrants, and extru- sion of teeth . , . , . and upper third molars. dental morphologic anomalies of number (hypodontia with agenesis of the incisors and the lower third molars), of size (microdontia of the canines and premolares) and in shape (conical canines and incisors) (fig. and ) are observed. the patient wears no prosthesis and presents good oral hygiene. the orthopantomography (fig. ) shows extruded upper third molars, altered dental mor- phology, dental agenesis and a radiopacity in the anter- oinferior sector due to a fixation screw from a previous bone graft made for later implant rehabilitation. no parental consanguinity nor anomalies in the analytic tests were present. no genetic study had been made. discussion evc syndrome is a genetic disorder with autosomal re- cessive transmission most often described in families with a history of consanguinity ( ). in our case this has not been verified. the gene responsible for this syn- drome was identified in the short arm of the chromo- some with five different mutations ( ). it is described in all the ethnic groups, however it is most prevalent in the amish population of lancaster in pennsylvania, u.s.a. ( , ). the syndrome can be diagnosed during the prenatal pe- riod, starting from the th week of gestation, by ultra- sonography, or later by clinical examination after birth ( ). chondrodystrophia is the most common clinical feature, affecting the tubular bones producing a serious ossifica- tion defect ( , ). in consequence, the distal extremities of the limbs are short and the patients small in stature ( ). aldegheri ( ) recommends osteogenic distraction to lengthen the limbs. baujat et al. ( ) have demonstrated that growth hormone treatment with these patients is not effective. however, it is important to highlight that there is one case published in the literature in which a favorable result in growth is described following hor- monal treatment ( ). the thorax is usually narrow with pectus excavatum, lumbar lordosis and genu valgum, the hair is sparse and fine. the polydactyly, wide hands and feet, sausage- shaped fingers, and dysplastic fingernails are also typi- cal features of evc syndrome ( ). congenital heart malformations are described in a to % of patients affected by this syndrome. defects of the mitral and tricuspid valves, patent ductus arteriosus, ven- tricular septal defects and atrial septal defects are some of the malformations described as the principal cause of decreased life-expectancy in these patients ( , , ). we coincide with all published studies in regard to the oral features presented in these patients. we highlight the malocclusion, the hypodontia, the appearance of conical teeth, hypoplasia of the enamel and multiple musculofibrous frenula as constant clinical features ( , - , , ). however, additional clinical findings affect- ing other organs (lungs, kidneys, liver, pancreas and central nervous system) may occasionally be observed, ( , , ) although these were not diagnosed in our case. genitourinary anomalies such as agenesis and renal dysplasia, ureterectasia and nephrocalcinosis usually present in % of cases ( ). exceptionally, hematologi- cal anomalies have been reported. in the literature, one case with dyserythropoiesis is described and another with associated perinatal myeloblastic leukemia ( , ). digoy et al. ( ) reported a unique case of a patient with evc syndrome presenting congenital stridor due to the appearance of a cyst in the upper airway that impeded autonomous breathing on displacing the larynx. other infrequent features include: strabismus, epi- and hypospadias, cryptorchidism and malformations in the thoracic or lung walls ( ). the cognitive and motor development of patients af- fected by evc syndrome are normal ( , ). evc syndrome requires multidisciplinary therapeutic planning. the odontologist plays a fundamental role in the control of the oral and dental manifestations. a combination of orthodontics-surgery-prosthetics is es- sential to correct the craniofacial morphology and den- tal defects, aiming to achieve satisfactory functional and aesthetic results ( ). dental treatment should be made under low antibiotic prophylaxis due to the high incidence of heart pathology in these patients ( , ). the prognosis for this syndrome is related to the dif- ficulty in breathing during the first months of life which derives from potential heart problems and a narrow tho- rax ( ). the differential diagnosis includes jeune syndrome and orofaciodigital syndromes ( , , , ). jeune syndrome is a rare, potentially lethal, autosomal recessive disease; characterized by thoracic dystrophy, short limbs, small stature, polydactyly and generalized bony dysplasia. there are anomalies in pigmentation of the retina, renal involvement and hypoplastic lungs ( , ). the orofaciodigital syndromes result from dominant sex-linked inheritance, they are limited to women and clinically characterized by multiple gingivolabial frenu- la, hypoplasia of the nasal cartilages, moderate mental retardation, fissured tongue and in a third of the cases ankyloglossia ( , ). e med oral patol oral cir bucal. jul ; ( ):e - . ellis-van creveld syndrome references . arya l, mendiratta v, sharma rc, solanki rs. ellis-van creveld syndrome: a report of two cases. pediatr dermatol. ; : - . . tompson sw, ruiz-perez vl, blair hj, barton s, navarro v, robson jl, et al. sequencing evc and evc identifies mutations in two-thirds of ellis-van creveld syndrome patients. hum genet. ; : - . . baujat g, le merrer m. ellis-van creveld syndrome. orphanet j rare dis. ; : . . winter gb, geddes m. oral manifestations of chondroectodermal dysplasia (ellis-van creveld syndrome). report of a case. br dent j. ; : - . . ghosh s, setty s, sivakumar a, pai km. report of a new syn- drome: focus on differential diagnosis and review of ellis-van creveld, curry-hall, acrofacial dysostosis, and orofacial digital syndromes. oral surg oral med oral pathol oral radiol endod. ; : - . . katsouras cs, thomadakis c, michalis lk. cardiac ellis-van creveld syndrome. int j cardiol. ; : - . . sergi c, voigtländer t, zoubaa s, hentze s, meyberg-solomeyer g, troeger j, et al. ellis-van creveld syndrome: a generalized dys- plasia of enchondral ossification. pediatr radiol. ; : - . . aldegheri r. distraction osteogenesis for lengthening of the tibia in patients who have limb-length discrepancy or short stature. j bone joint surg am. ; : - . . versteegh fg, buma sa, costin g, de jong wc, hennekam rc, evc working party. growth hormone analysis and treatment in el- lis-van creveld syndrome. am j med genet a. ; a: - . . mintz sm, siegel ma, seider pj. an overview of oral frena and their association with multiple syndromic and nonsyndromic conditions. oral surg oral med oral pathol oral radiol endod. ; : - . . kolenc-fusé fj. tooth agenesis: in search of mutations be- hind failed dental development. med oral patol oral cir bucal. ; : - ; - . . black d, reutter j, johnson m, fair j, woosley j, gerber d. liver transplantation in ellis-van creveld syndrome: a case report. pediatr transplant. ; : - . . digoy gp, greenberg m, magit a. congenital stridor secondary to an upper airway cyst in a patient with ellis-van creveld syndrome. int j pediatr otorhinolaryngol. ; : - . . scurlock d, ostler d, nguyen a, wahed a. ellis-van crev- eld syndrome and dyserythropoiesis. arch pathol lab med. ; : - . . poveda-roda r, jiménez y, carbonell e, gavaldá c, margaix- muñoz mm, sarrión-pérez g. bacteremia originating in the oral cavity. a review. med oral patol oral cir bucal. ; :e - . a m e r i c a n e d u c a t i o n a l r e s e a r c h j o u r n a l fall , vol. , no. , pp. - liberal democracy, equal educational opportunity, and the challenge of multiculturalism k e n n e t h r. h o w e university o f colorado a t b o u l d e r l i b e r a l p o l i t i c a l t h e o r y i n g e n e r a l , a s w e l l a s l i b e r a l e d u c a t i o n a l t h e o r y in p a r t i c u l a r , h a s b e e n l a r g e l y s i l e n t o n the c h a l l e n g e p o s e d b y m u l t i c u l - t u r a l i s m . this l a c u n a r e s u l t s f r o m the t e n d e n c y to c o n f l a t e " c u l t u r a l " a n d " p o l i t i c a l " c o m m u n i t i e s a n d to c o n c e i v e o f e q u a l i t y e x c l u s i v e l y in t e r m s o f the latter. the r e s u l t is t h a t e q u a l i t y o f e d u c a t i o n a l o p p o r t u n i t y is p o t e n t i a l l y r e n d e r e d a s h a m f o r c u l t u r a l m i n o r i t i e s i n s o f a r a s t h e y a r e r e q u i r e d to c o n f r o n t e d u c a t i o n a l i d e a l s a n d p r a c t i c e s t h a t a r e ' ' c u l t u r a l l y e n c u m b e r e d " in a w a y t h a t reflects o n l y the v a l u e s a n d i n t e r e s t s o f the d o m i n a n t social g r o u p . this a r t i c l e a r g u e s t h a t ' ' p r o g r e s s i v e " liberal e d u - c a t i o n a l t h e o r y c a n s a t i s f a c t o r i l y r e s p o n d to the c h a l l e n g e p o s e d b y m u l - t i c u l t u r a l e d u c a t i o n w h e n c o n c e p t s s u c h a s " f r e e d o m " a n d " o p p o r t u n i - t y " a r e p r o p e r l y a n a l y z e d a n d w h e n the d e m a n d to p r o m o t e s e l f - r e s p e c t a m o n g c i t i z e n s is t a k e n s e r i o u s l y . kenneth howe is a n a s s o c i a t e p r o f e s s o r i n t h e s c h o o l o f e d u c a t i o n at t h e u n i - v e r s i t y o f c o l o r a d o , b o u l d e r - . h e s p e c i a l i z e s i n p h i l o s o p h y o f e d u c a t i o n . this work may be downloaded only. it may not be copied or used for any purpose other than scholarship. if you wish to make copies or use it for a non-scholarly purpose, please contact aera directly. h o w e i n , t h e l a n d m a r k b r o w n v. t h e b o a r d o f e d u c a t i o n d e c i s i o n t h r u s t t h e c o n c e p t o f e q u a l e d u c a t i o n a l o p p o r t u n i t y c e n t e r stage, a n d it h a s re- t a i n e d a c e n t r a l r o l e i n e d u c a t i o n a l p o l i c y a n d r e s e a r c h e v e r s i n c e . d e s p i t e its p r o m i n e n c e , h o w e v e r - - o r b e c a u s e o f i t - - t h e c o n c e p t has b e c o m e m o r e a n d m o r e e l u s i v e , m o r e a n d m o r e " a w i t c h e s ' b r e w o f e q u i v o c a t i o n a n d v a g u e n e s s . "' c o n t r o v e r s i e s h a v e u n f o l d e d r e g a r d i n g b o t h t h e g e n e r a l m e a n i n g o f e q u a l e d u c a t i o n a l o p p o r t u n i t y a n d its m e a n i n g i n t h e c o n t e x t o f s p e c i f i c p o l i c i e s . u n f o r t u n a t e l y , t h e s e t w o l e v e l s o f a n a l y s i s h a v e r a r e l y b e e n j o i n e d . at t h e g e n e r a l level, t h e d e b a t e has b e e n l a r g e l y p h i l o s o p h i c a l a n d has f o c u s e d o n i s s u e s s u c h as h o w c o m p e t i n g t h e o r i e s o f j u s t i c e e n t a i l c o m p e t i n g c o n - c e p t i o n s o f e q u a l e d u c a t i o n a l o p p o r t u n i t y ; w h e t h e r t h e c r i t e r i o n o f e q u a l - i t y s h o u l d b e e q u a l i t y o f a c c e s s o r e q u a l i t y o f results; a n d w h e t h e r t h e c o n - flicts t h a t p u t a t i v e l y e x i s t b e t w e e n t h e p r i n c i p l e o f e q u a l i t y a n d p r i n c i p l e s s u c h as m e r i t a n d f a m i l y a u t o n o m y c a n b e r e s o l v e d . a l t h o u g h t h e s e d e - b a t e s are u s e f u l for a r t i c u l a t i n g a n d e s t a b l i s h i n g g e n e r a l ideals, t h e g u i d a n c e t h e y c a n p r o v i d e w i t h r e s p e c t to s p e c i f i c p o l i c i e s is q u i t e l i m i t e d . at t h e s p e c i f i c level, t h e p r o b l e m is j u s t t h e r e v e r s e . d e b a t e s s u r r o u n d - i n g d e s e g r e g a t i o n , t r a c k i n g , b i l i n g u a l e d u c a t i o n , e d u c a t i o n o f t h e h a n d i - c a p p e d , a n d g e n d e r e q u i t y , for i n s t a n c e , t y p i c a l l y fail t o e x h i b i t c a r e f u l at- t e n t i o n t o t h e m o r e f u n d a m e n t a l p o l i t i c a l a n d p h i l o s o p h i c a l a s s u m p t i o n s a n d p r i n c i p l e s that i m p l i c i t l y u n d e r l i e t h e m . i n s t e a d , " e q u a l e d u c a t i o n a l o p - p o r t u n i t y " is e m p l o y e d as if its m e a n i n g w e r e w h o l l y t r a n s p a r e n t , a n d t h e d e b a t e s p r o c e e d a l m o s t e x c l u s i v e l y i n t e r m s o f t h e legal r e a s o n i n g o f b r o w n a n d v a r i o u s a p p l i c a b l e f e d e r a l laws. i gratefully acknowledge the support of the national academy of education spencer foundation fellowship program in the preparation of this manuscript. also thank katharine dougherty, dan liston, and ernie house for their helpful comments. an abbreviated version of this paper was presented at the american educational research association annual meeting in chicago in april, . tparaphrase of j.r lucas, in michael levin, "equality of opportunity," the philosophical q u a r t e r l y , no. ( ): - . regarding conflicts among liberal principles, see especially james fishkin, justice, equal opportunity, a n d the f a m i l y (new haven: yale university press, ). regarding the controversy about access versus results as the criterion for equality of educational op- portunity, see, for example, james coleman, "the concept of equality of educational op- portunity," h a r v a r d e d u c a t i o n a l review , no. ( ); - ; nicholas burbules and ann sherman, "equal educational opportunity: ideal or ideology," proceedings o f the philosophy o f e d u c a t i o n society ( ): - ; nicholas burbules, brian lord, and ann sherman, "equity, equal opportunity, and education," e d u c a t i o n a l evaluation a n d policy analysis, , no. ( ); ; christopher jencks, "whom must we treat equally for educa- tional opportunity to be equal?" ethics , no. ( ): - ; and my "in defense of outcomes-based conceptions of equal educational opportunity," e d u c a t i o n a l theory , no. ( ): - . these include titles iv, vi, vii, and ix of the civil rights act; the bilingual education act of ; sec. of the rehabilitation act; the equal educational opportunities act; and the education for all handicapped children act of . e q u a l e d u c a t i o n a l o p p o r t u n i t y a n d m u l t i c u l t u r a l i s m m y p r i m a r y t a s k i n t h i s a r t i c l e w i l l b e t o j o i n t h e s e t w o l e v e l s o f a n a l y s i s w i t h a n e y e t o w a r d p r o v i d i n g a p h i l o s o p h i c a l l y g r o u n d e d e x a m i n a t i o n o f t h e c o n c e p t o f e q u a l e d u c a t i o n a l o p p o r t u n i t y as it a p p l i e s t o m u l t i c u l t u r a l e d u c a t i o n . i n m y v i e w , b e c a u s e t h e c o n c e p t o f e q u a l e d u c a t i o n a l o p p o r - t u n i t y h a s " m a n y f a c e s , " its p r e c i s e m e a n i n g a n d i m p l i c a t i o n s d e p e n d o n t h e p o l i c y q u e s t i o n at h a n d . a c c o r d i n g l y , v a r i o u s i s s u e s t h a t r a i s e q u e s t i o n s r e g a r d i n g e q u a l i t y o f e d u c a t i o n a l o p p o r t u n i t y a r e b e s t e x a m i n e d o n e at a t i m e b y r e n d e r i n g a g e n e r a l p h i l o s o p h i c a l f r a m e w o r k s e n s i t i v e t o e t h i c a l p r i n c i p l e s a n d e m p i r i c a l c o n s i d e r a t i o n s t h a t a r e p e c u l i a r t o s u c h i s s u e s . e m p l o y i n g t h i s m e t h o d , i w i l l p r o c e e d b y first p r o v i d i n g a g e n e r a l p h i l o - s o p h i c a l c h a r a c t e r i z a t i o n o f t h e c o n c e p t o f e q u a l e d u c a t i o n o p p o r t u n i t y a n d t h e n s k e t c h i n g o u t t h e f e a t u r e s o f t h e p a r t i c u l a r " f a c e " it a s s u m e s w i t h r e s p e c t t o m u l t i c u l t u r a l e d u c a t i o n . a g e n e r a l c h a r a c t e r i z a t i o n o f e q u a l e d u c a t i o n a l o p p o r t u n i t y q u e s t i o n s a b o u t t h e a i m s o f s c h o o l i n g a n d n a t u r e o f t h e c u r r i c u l u m c a n o n l y b e a n s w e r e d f r o m w i t h i n a p o l i t i c a l t h e o r y t h a t a d u m b r a t e s t h e m o r e g e n e r a l s o c i o p o l i t i c a l f u n c t i o n s o f p u b l i c e d u c a t i o n . i w i l l s e t a s i d e b r o a d e r q u e s t i o n s o f p o l i t i c a l t h e o r y a n d p r e s u p p o s e a l i b e r a l d e m o c r a t i c t h e o r y - t h e k i n d o f p o l i t i c a l t h e o r y i n w h i c h t h e p r i n c i p l e o f e q u a l e d u c a t i o n a l o p - p o r t u n i t y f i n d s its h o m e . m y t a s k in t h e r e m a i n d e r o f t h i s a r t i c l e w i l l b e t o s h o w h o w t h e l i b e r a l d e m o c r a t i c t r a d i t i o n i n g e n e r a l a n d t h e p r i n c i p l e o f e q u a l e d u c a t i o n a l o p p o r t u n i t y i n p a r t i c u l a r a r e r o b u s t e n o u g h t o a c c o m - m o d a t e t h e p e c u l i a r c h a l l e n g e p o s e d b y m u l t i c u l t u r a l e d u c a t i o n . t h e p r i n c i p l e o f e q u a l e d u c a t i o n a l o p p o r t u n i t y s e r v e s t o j u s t i f y d e - m a n d i n g o f p u b l i c e d u c a t i o n s o m e t h i n g s h o r t o f f u l l e q u a l i t y : it d e m a n d s o n l y e q u a l i t y o f o p p o r t u n i t i e s , w h i c h it is t h e n t h e r e s p o n s i b i l i t y o f s c h o o l c h i l d r e n o r t h e i r p a r e n t s t o a c t o n . i n t h i s w a y , t h e c o n c e p t o f f r e e d o m is b u i l t i n t o t h e c o n c e p t o f e q u a l e d u c a t i o n a l o p p o r t u n i t y . as o n o r a o ' n e i l r e m a r k s , " t h e c o n c e p t o f e q u a l e d u c a t i o n a l o p p o r t u n i t y c a n n o t b e r i d o f its l i b e r t a r i a n b i r t h m a r k , e v e n a f t e r r a d i c a l s u r g e r y . '' a t l e a s t s i n c e c o l e m a n ' s r e f l e c t i o n s , a c o n t r o v e r s y h a s e x i s t e d r e g a r d - i n g w h e t h e r t h e c r i t e r i o n o f e q u a l i t y o f e d u c a t i o n a l o p p o r t u n i t y s h o u l d b e this method approximates "reflective equilibrium," which draws on john rawls, a theory of justice (cambridge, mass.: harvard university press, ), and also rawls's "kan- tian constructivism in moral theory," journal of philosophy , no. ( ): - . amy gutmann acknowledges the influence of rawls on the method of analysis she employs in democratic education (princeton, n.j.: princeton university press, ). sin this context, by "liberal" i mean a general tradition in political theory. within this tradition, there are liberals and conservatives in the more popular sense that would distinguish, for example, ted kennedy from william f. buckley. onora o'neill, "opportunities, equalities, and education," theory and decision , no. ( ): - . i, for one, have attempted to perform such surgery. see my "in defense of outcomes-based conceptions of equal educational opportunity." :coleman, "the concept of equality of educational opportunity." h o w e e q u a l i t y o f a c c e s s o r e q u a l i t y o f r e s u l t s ; a n d b o t h c r i t e r i a h a v e p r o v e n p r o b - l e m a t i c . a l t h o u g h g u a r a n t e e i n g e q u a l i t y o f a c c e s s is a n a d v a n c e o v e r s u c h p r a c t i c e s as d e j u r e s e g r e g a t i o n , it c a n b e q u i t e h o l l o w i f it m e r e l y a m o u n t s t o r e m o v i n g f o r m a l b a r r i e r s t o t h e c h o i c e s s t u d e n t s a n d t h e i r p a r e n t s m i g h t m a k e , as d e f a c t o s e g r e g a t i o n a p t l y i l l u s t r a t e s . o n t h e o t h e r h a n d , g u a r a n t e e - i n g e q u a l i t y o f r e s u l t s s e e m s t o d e m a n d t o o m u c h , b o t h in t e r m s o f t h e c a p a b i l i t i e s o f s c h o o l s a n d in t e r m s o f h o w it t h r e a t e n s t o b l o c k t h e f r e e d o m t h a t s t u d e n t s a n d t h e i r p a r e n t s m i g h t o t h e r w i s e w i s h t o e x e r c i s e . t h i s w a y o f f r a m i n g t h e p r o b l e m s e e m s t o l e a v e o p e n t w o w a y s o f r e s p o n d i n g : a b a n d o n i n g f r e e d o m a n d c h o i c e , o n t h e g r o u n d s t h a t t h e y a r e i d e o l o g i c a l s h a m s t h a t m e r e l y s e r v e t o j u s t i f y v a s t i n e q u a l i t y , o r a b a n d o n - i n g c q u a l i t y o f r e s u l t s , o n t h e g r o u n d s t h a t f r e e d o m is a c h e r i s h e d v a l u e t h a t o u g h t n o t t o b e s a c r i f i c e d a n d t h a t r e s u l t s c a n n o t b e e q u a l i z e d in a n y c a s e . t h e r e is a t h i r d r e s p o n s e , h o w e v e r , t h a t m e r i t s c a r e f u l e x a m i n a t i o n : a b a n d o n i n g t h e q u e s t f o r t i d y s o l u t i o n s t o c l a s h e s a m o n g f u n d a m e n t a l p r i n - c i p l e s , o n t h e g r o u n d s t h a t u n c e r t a i n t y , t e n t a t i v e n e s s , a n d t e n s i o n s a m o n g p o l i t i c a l p r i n c i p l e s a r e p e r m a n e n t f e a t u r e s o f t h e p r o j e c t o f d e m o c r a c y . i w i l l b r i e f l y d e s c r i b e a n d d e f e n d t h i s t h i r d a p p r o a c h , w h i c h , b o r r o w i n g f r o m w a l z c r , i s h a l l c a l l " i n t e r p r e t i v e . ''~ t h e i n t e r p r e t i v e a p p r o a c h h a s t w o m e t h o d o l o g i c a l f e a t u r e s t h a t e x i s t in t e n s i o n . f i r s t , s o c i a l c r i t i c i s m is c o n s t r u e d as i m m a n e n t , w h i c h is t o s a y it m u s t g a i n a f o o t h o l d i n t h e v o c a b u l a r y a n d a c c e p t e d p r i n c i p l e s o f a g i v e n p o l i t i c a l c o m m u n i t y if it is t o h a v e a n y t h i n g t o s a y t o t h e m e m b e r s o f s u c h a c o m m u n i t y a n d t o h a v e a n y c h a n c e o f c o n s t r u c t i v e l y i n f l u e n c i n g t h e m . i n t h i s v e i n , w a l z e r e n c o u r a g e s s o c i a l c r i t i c s " t o i n t e r p r e t t o o n e ' s f e l l o w c i t i z e n s t h e w o r l d o f m e a n i n g s t h a t w e s h a r e " a n d w a r n s a g a i n s t a b s t r a c t p h i l o s o p h i z i n g : justice and equality can c o n c e i v a b l y be w o r k e d out as p h i l o s o p h i c a l artifacts, but a just or an egalitarian society cannot be. if such a society isn't alrcady h e r e - - h i d d e n , as it were, in our concepts and categories - - w e will n e v e r k n o w it c o n c r e t e l y o r realize it in f a c t . g i v e n t h e p r o m i n e n c e o f t h e p r i n c i p l e o f e q u a l e d u c a t i o n a l o p p o r t u n i t y in t h e c o n v e r s a t i o n a b o u t a j u s t s y s t e m o f e d u c a t i o n , t h e i m m a n e n t f e a t u r e o f t h e i n t e r p r e t i v e a p p r o a c h a r g u e s a g a i n s t a b a n d o n i n g it is as m e r e i d e o - l o g i c a l s h a m o r as h o p e l e s s l y m u d d l e d . r a t h e r , t h e a i m s h o u l d b c t o d e v i s e p h i l o s o p h i c a l l y d e f e n s i b l e i n t e r p r e t a t i o n s t h a t h a v e s o m e c h a n c e o f w i n - n i n g b r o a d a c c e p t a n c e . w o r k i n g i n t h e o t h e r d i r e c t i o n , h o w e v e r , is t h e s e c o n d f e a t u r e o f t h e i n t e r p r e t i v e a p p r o a c h : c o n c e p t u a l r e v i s i o n i s m . t h i s f e a t u r e r e q u i r e s t h a t ~michael walzer, interpretatiotl and social criticism (cambridge, mass.: harvard university press, t ). ';michael walzer, spheres of justice: a defense of pluralism and equality (new york: basic books, ), xiv. equal educational opportunity a n d multiculturalism political a r g u m e n t b e p r o g r e s s i v e a n d d y n a m i c , a n d n o t m e r e l y a lexico- g r a p h i c a l o r historical a c c o u n t o f w h a t political p r i n c i p l e s m e a n o r h a v e m e a n t . i n s t e a d , political a r g u m e n t m u s t i n v e s t i g a t e s h a r e d p r i n c i p l e s a n d their i m p l i c a t i o n s , p o i n t t o conflicts a n d i n c o n s i s t e n c i e s , a n d r e s p o n d to c h a n g i n g c i r c u m s t a n c e s a n d k n o w l e d g e . this feature o f the i n t e r p r e t i v e ap- p r o a c h a r g u e s in f a v o r o f r e v i s i n g t h e c o n c e p t o f e q u a l e d u c a t i o n a l o p p o r - t u n i t y as n e c e s s a r y s o that it b e s t a c c o m m o d a t e s c o m p e t i n g p r i n c i p l e s a n d t h e i r i m p l i c a t i o n s - - t h e p r i n c i p l e s o f e q u a l i t y a n d f r e e d o m in p a r t i c u l a r - - in light o f c u r r e n t c i r c u m s t a n c e s . t h e a p p r o a c h just d e s c r i b e d has b e e n a d o p t e d in o n e s h a p e o r a n o t h e r b y v a r i o u s t h i n k e r s , m o s t n o t a b l y g u t m a n n . ° in particular, g u t m a n n b e g i n s w i t h the a s s u m p t i o n that e q u a l i t y o f e d u c a t i o n a l o p p o r t u n i t y is a s e r v i c e a b l e p r i n c i p l e , t h e n e n t e r t a i n s a n d rejects s e v e r a l c o n c e p t i o n s , a n d finally r e a c h e s t h e s o m e w h a t c o u n t e r i n t u i t i v e c o n c l u s i o n that e q u a l i t y o f e d u c a t i o n a l o p p o r t u n i t y r e q u i r e s equalizing c e r t a i n e d u c a t i o n a l results ( n a m e l y , t h o s e that g o i n t o t h e " d e m o c r a t i c threshold"). it will b e suffi- c i e n t f o r p r e s e n t p u r p o s e s to set t h e intricacies o f s u c h a r g u m e n t s aside a n d to n o t e t h r e e p i v o t a l issues that a n y a d e q u a t e i n t e r p r e t a t i o n o f equali- ty o f e d u c a t i o n a l o p p o r t u n i t y m u s t a c c o m m o d a t e . . freedom a n d opportunities worth wanting. t h e c o n c e p t o f free- d o m has d i f f e r e n t senses. t h e w e a k e s t s e n s e r e q u i r e s o n l y v o l u n t a r i n e s s a n d i n t e n t - - a kind o f f r e e d o m p o s s e s s e d e v e n b y y o u n g children. a s t r o n g e r s e n s e r e q u i r e s t h e s e f e a t u r e s plus t h e ability to i d e n t i f y a n d w e i g h alter- n a t i v e s a n d t h e i r c o n s e q u e n c e s a n d to c h o o s e t h e o n e j u d g e d b e s t f r o m a m o n g t h e m - - a k i n d o f f r e e d o m a t t r i b u t e d t o n o r m a l adults. it s h o u l d require n o a r g u m e n t to establish that these senses o f f r e e d o m are n o t equally w o r t h w a n t i n g . t h e first s e n s e is s i m p l y t o o w e a k ; in o r d e r t o b e free in e v e n a m i n i m a l s e n s e o f b e i n g in c o n t r o l o f o n e ' s life, the s e c o n d s e n s e is r e q u i r e d . a n e c e s s a r y c o n d i t i o n o f f r e e d o m sufficiently w o r t h w a n t i n g , t h e n , is t h e ability t o d e l i b e r a t e e f f e c t i v e l y , b u t this is clearly n o t a sufficient c o n d i - tion. for, to m a k e u s e o f t h e ability to d e l i b e r a t e effectively, a n i n d i v i d u a l m g u t m a n n , d e m o c r a t i c e d u c a t i o n . ~ g u t m a n n denies that s h e r e q u i r e s i n p u t s o r o u t c o m e s to be equalized. i think s h e is s i m p l y m i s t a k e n a b o u t this. for a l t h o u g h s h e clearly denies that a l l educational o u t c o m e s m u s t he equalized, in the e n d she n o n e t h e l e s s h o l d s that s o m e m u s t be, namely, t h o s e that are r e q u i r e d b y the " d e m o c r a t i c t h r e s h o l d . " t a still s t r o n g e r kind r e q u i r e s the features o f the first t w o kinds p l u s the ability to reflect a b o u t o n e ' s basic value c o m m i t m e n t s and w a y o f l i f e - - a kind o f f r e e d o m a t t r i b u t e d to especially reflective adults. as it t u r n s out, the s e c o n d level is all that s c h o o l s s h o u l d be required (or permitted) to foster, because the third level entails having q u e s t i o n e d o n e ' s m o s t f u n d a m e n t a l c o m m i t m e n t s (e.g., o n e ' s religious c o m m i t m e n t s ) to qualify as free. a l t h o u g h this is w h a t p h i l o s o p h i c a l t y p e s strive for, it is i n a p p r o p r i a t e to d e m a n d this o f the p o p u l a - tion in general. see, for example, stephen macedo, l i b e r a l v i r t u e s (new york: o x f o r d univer- sity press, ). h o w e m u s t also have the o p p o r t u n i t y to exercise it. the o p p o r t u n i t y to exercise it, in turn, requires ( ) that information necessary for deliberation is available a n d ( ) that social c o n d i t i o n s d o n o t i m p o s e a b u r d e n for acting o n the results o f deliberation that is d i s p r o p o r t i o n a t e to the b u r d e n o f o t h e r deliberators. as an illustration o f condition ( ), consider dennett's distinction between " b a r e " a n d "real" o p p o r t u n i t i e s . ~ he gives the e x a m p l e o f a g r o u p o f prisoners w h o have their p r i s o n d o o r s u n l o c k e d b y the prison guards while t h e y are asleep a n d l o c k e d again b e f o r e t h e y awaken. a c c o r d i n g to den- nett, b e c a u s e the prisoners d o n o t have the i n f o r m a t i o n t h e y n e e d to deliberate, t h e y have o n l y a " b a r e " o p p o r t u n i t y to escape. as an illustra- tion o f c o n d i t i o n ( ), imagine a family that displays its disapproval o f u.s. military i n v o l v e m e n t in the persian gulf and receives threats to its safety a n d p r o p e r t y as a result. here, a l t h o u g h the requisite i n f o r m a t i o n for deliberation is available, acting o n the results o f deliberation entails a b u r d e n that is d i s p r o p o r t i o n a t e to the b u r d e n o f those w h o wish to express their s u p p o r t for military i n v o l v e m e n t . t h e principle o f f r e e d o m o f e x p r e s s i o n in this case is blunted, and t h e r e f o r e resembles o n l y a " b a r e " o p p o r t u n i t y . t h e p o i n t is that neither the prisoners n o r the dissenting family e n j o y kinds o f o p p o r t u n i t i e s w o r t h wanting, a n d similar examples are easy to find in e d u c a t i o n . for example, imagine a ninth-grade s t u d e n t w h o is being " c o u n s e l e d " into a vocational track and w h o , along with his or her parents, lacks k n o w l e d g e a b o u t the c o n s e q u e n c e s o f such a decision. also imagine that the family's cultural m a k e u p leads it to be intimidated b y a n d deferen- tial to school authorities. first, the k n o w l e d g e required for effective delibera- tion is missing. s e c o n d , the family is p r e s s u r e d b y social c o n d i t i o n s that are implicitly hostile to m a k i n g a different decision. in general, s o m e t h i n g m o r e is r e q u i r e d in the n a m e o f equalizing educational o p p o r t u n i t y than equalizing these kinds o f " b a r e " opportunities. . e q u a l e d u c a t i o n a l o p p o r t u n i t y a s enabling. education is, n o doubt, valuable in its o w n right, but it also is e n a b l i n g in the sense that it serves ( h o w e v e r imperfectly) as the g a t e w a y for obtaining o t h e r societal g o o d s , such as desirable e m p l o y m e n t , a d e q u a t e i n c o m e , and political p o w e r . for this reason, equal e d u c a t i o n a l o p p o r t u n i t y is related to equal o p p o r t u n i t y m o r e generally b e c a u s e it serves as an i m p o r t a n t link in w h a t might be t e r m e d an o p p o r t u n i t y chain. accordingly, the strength o f the educational link d e t e r m i n e s the overall strength o f the o p p o r t u n i t y chain in the sense that the array o f o p p o r t u n i t i e s o p e n to an individual is (again, imperfectly) d e t e r m i n e d b y the quality o f his o r her education. t h e o p p o r t u n i t y chain is c o m p l i c a t e d b y the fact that e d u c a t i o n a l op- p o r t u n i t y itself has this same chain-like character. that is, taking advantage * daniel dennett, e l b o w r o o m : the varieties o f free will worth w a n t i n g (cambridge, mass: mit press, ). equal educational opportunity a n d multiculturalism o f early educational o p p o r t u n i t i e s is related to h a v i n g later ones. for ex- ample, c h i l d r e n w h o fail to learn to read early o n h a v e their curricular op- tions progressively n a r r o w e d as t h e y p r o c e e d t h r o u g h the k - curriculum, as c o m p a r e d to their counterparts w h o d o learn to read. consequently, their e d u c a t i o n a l o p p o r t u n i t i e s will be likewise n a r r o w e d s u c h that t h e y will be incapable o f e n j o y i n g equality o f educational o p p o r t u n i t y a n d equality o f o p p o r t u n i t y m o r e generally as t h e y a p p r o a c h a d u l t h o o d . several lessons m a y be d r a w n f r o m this observation, first, w h a t at o n e p o i n t in time serves as an e d u c a t i o n a l e n d (like reading) later serves as a m e a n s to o t h e r e n d s (like reading t e x t b o o k s for content). thus, certain educational ends (or results) must be a c c o m p l i s h e d in o r d e r for certain o t h e r e d u c a t i o n a l o p p o r t u n i t i e s to exist. s e c o n d , a n d as a c o n s e q u e n c e o f this, the c o n c e p t o f equal educational o p p o r t u n i t y needs to be c o n c e i v e d in terms o f educational careers rather than specific e p i s o d e s w i t h i n such careers, lest educational o p p o r t u n i t i e s b e c o m e m e r e l y " b a r e " a n d n o t w o r t h wanting. w o r k i n g o u t the details o f this claim outstrips the aims o f this article. by w a y o f a brief illustration, h o w e v e r , c o n s i d e r h o w far a free adult literacy p r o g r a m goes t o w a r d equalizing e d u c a t i o n a l o p p o r t u n i t y . t h e a r g u m e n t that s u c h p r o g r a m s p r o m o t e equality o f educational o p p o r t u n i t y gains its force b y isolating particular c h o i c e s f r o m the b r o a d e r social s c h e m e that d e t e r m i n e s the s c o p e a n d kinds o f o p p o r t u n i t i e s that individuals possess, a n d b y glossing o v e r the fact that adults w h o are free to u n d e r t a k e o r pass u p literacy p r o g r a m s suffer f r o m a restricted range o f opportunities. it seems quite reasonable to suggest that the n e e d for adult literacy p r o g r a m s signals a failure o f earlier e d u c a t i o n , a failure to p r o d u c e earlier results r e q u i r e d to e x p a n d the s c o p e o f adult o p p o r t u n i t y - - e d u c a t i o n a l a n d otherwise. it also seems quite reasonable to suggest that to be p u t in the p o s i t i o n o f be- ing an adult h a v i n g the c h o i c e o f w h e t h e r to b e c o m e literate h a r d l y seems a c h o i c e w o r t h wanting. ( c o m p a r e a c o m p e n s a t o r y p r o g r a m like h a v i n g the c h o i c e o f w h e t h e r to receive free medical t r e a t m e n t for w o r k - r e l a t e d lung disease.) . equal educational opportunity a n d children. children raise a v e r y special p r o b l e m w i t h respect to the c o n c e p t o f equal e d u c a t i o n a l o p p o r - tunity: because children (especially y o u n g ones) lack the capacity for effec- tive deliberation, this capacity m u s t be instilled in t h e m b e f o r e q u e s t i o n s regarding the o t h e r t w o requisites for f r e e d o m a n d o p p o r t u n i t y w o r t h w a n t i n g - - a d e q u a t e information a n d social s u p p o r t - - e v e n arise. up to a cer- tain age, then, children c a n n o t possess f r e e d o m a n d opportunities genuinely w o r t h wanting. thus, it is u p to s o m e o n e e l s e - - s c h o o l s , parents, o r b o t h - - to act o n c h i l d r e n ' s b e h a l f to ensure that t h e y o n e d a y are able to possess i am currently developing a more elaborate analysis and defense of this point in a paper tentatively entitled "equal educational opportunity as educational opportunities worth wanting." h o w e t h e s e t h i n g s . i n o t h e r w o r d s , p a t e r n a l i s t i c i n t e r f e r e n c e i n c h i l d r e n ' s f r e e d o m (in t h e w e a k s e n s e ) is j u s t i f i e d i n t h e n a m e o f p r e p a r i n g t h e m to e n j o y f r e e d o m (in t h e s t r o n g s e n s e ) later o n i n life. i n v o k i n g p a t e r n a l i s t i c i n t e r f e r e n c e i n t h e n a m e o f c h i l d r e n ' s b e s t e d u c a - t i o n a l i n t e r e s t s raises a n u m b e r o f p o t e n t i a l (as w e l l as real) c o n f l i c t s - - b e t w e e n s c h o o l s a n d c h i l d r e n , p a r e n t s a n d c h i l d r e n , p a r e n t s a n d p a r e n t s , s c h o o l s a n d p a r e n t s , a n d so f o r t h - - i n s o f a r as w h a t e d u c a t i o n a l o p p o r t u n i - ties are i n d e e d w o r t h w a n t i n g o f t e n c a n b e ( a n d is) i n d i s p u t e . t h i s is a large a n d c o m p l e x issue, a n d m u c h o f it lies b e y o n d t h e s c o p e o f this arti- cle. i n m y s u b s e q u e n t d i s c u s s i o n , i will l i m i t m y s e l f to t h e c o n f l i c t b e t w e e n s c h o o l s a n d c u l t u r e , l a r g e l y i g n o r i n g t h e o t h e r k i n d s o f c o n f l i c t s t h a t c a n arise. i n s u m m a r y , w h e n a p p l i e d t o c h i l d r e n , ~ a d e f e n s i b l e i n t e r p r e t a t i o n o f t h e p r i n c i p l e o f e q u a l e d u c a t i o n a l o p p o r t u n i t y is r e q u i r e d to take i n t o a c c o u n t t h e o b s e r v a t i o n s t h a t ( ) e d u c a t i o n s h o u l d b e e n a b l i n g , ( ) t h e c o n - c e p t o f e q u a l e d u c a t i o n a l o p p o r t u n i t y is b e s t a p p l i e d to e d u c a t i o n a l c a r e e r s r a t h e r t h a n i s o l a t e d i n c i d e n t s , a n d ( ) c h i l d r e n are n o t i n a p o s i t i o n t o e x e r - cise f r e e d o m a n d o p p o r t u n i t i e s ( w o r t h w a n t i n g ) u n t i l t h e y g a i n t h e capaci- ty to d e l i b e r a t e e f f e c t i v e l y . e q u a l e d u c a t i o n a l o p p o r t u n i t y a n d m u l t i c u l t u r a l e d u c a t i o n will k y m l i c k a has o b s e r v e d t h a t l i b e r a l d e m o c r a t i c t h e o r y is s u r p r i s i n g l y s i l e n t o n issues o f m u l t i c u l t u r a l i s m . he a t t r i b u t e s this s i l e n c e to a t e n d e n - c y a m o n g l i b e r a l t h e o r i s t s to i g n o r e " c u l t u r a l " c o m m u n i t i e s i n f a v o r o f t h e " p o l i t i c a l " c o m m u n i t y a n d to c o n c e i v e o f e q u a l i t y e x c l u s i v e l y i n t e r m s o f t h e latter. t h e u p s h o t is t h a t e q u a l i t y is p o t e n t i a l l y r e n d e r e d a s h a m for c u l t u r a l m i n o r i t i e s . for, a c c o r d i n g to k y m l i c k a , it only makes sense to invite people to participate in politics (or for people to accept that invitation) if they will be treated as equals . . . . and that is incompatible with defining people in terms of roles they did n o t shape or endorse. ~ k y m l i c k a c o n t e n d s t h a t a l t h o u g h t h e failure to g r a p p l e w i t h m u l t i c u l - t u r a l i s m is a s i g n i f i c a n t l a c u n a i n l i b e r a l t h e o r y , it is n o t a fatal flaw. i n his ~si include the conditional because some thinkers believe that children's lack of autonomy and the associated authority to represent their own interests requires a different moral perspective and vocabulary. onora o'neill, "children's rights and children's lives," e t h i c s ( ): - , for example, is willing to (proposes to) forgo rights language in the case of children. a similar move could be made with respect to equal educational op portunity, namely, it could be iudged as having no defensible application to children. for a response, see my "in defense of outcomes-based conceptions of equal education opportunity." i~"will kymlicka, l i b e r a l i s m , c o m m u n i t y , a n d c u l t u r e (new york: oxford university press, ). *rlbid., . equal educational opportunity a n d multiculturalism v i e w , r e s p e c t f o r c u l t u r a l i d e n t i t y is i m p l i c i t in liberal t h e o r i s t s ' (particular- ly j o h n r a w l s ' s ) c o m m i t m e n t t o s e l f - r e s p e c t as a " p r i m a r y g o o d " t h a t m u s t b e p r o t e c t e d b y liberal d e m o c r a t i c r e g i m e s . it is s i m p l y i n c u m b e n t u p o n liberal d e m o c r a t s t o w o r k o u t this i m p l i c i t c o m m i t m e n t . liberal e d u c a t i o n a l t h e o r y , like liberal p o l i t i c a l t h e o r y m o r e g e n e r a l l y , h a s also b e e n l a r g e l y silent o n t h e p r o b l e m o f m u l t i c u l t u r a l i s m - - a n d f o r a c l o s e l y r e l a t e d r e a s o n . liberal e d u c a t i o n a l t h e o r i s t s h a v e l a r g e l y c o n f i n e d t h e m s e l v e s t o t h e q u e s t i o n o f w h a t k i n d o f i n d i v i d u a l is s u i t a b l e t o t a k e his o r h e r p l a c e as a c i t i z e n in a liberal d e m o c r a t i c political c o m m u n i t y , a n d t h e a n s w e r has b e e n w h a t i shall r e f e r t o as t h e liberal educational ideal. this ideal m a y b e i d e n t i f i e d w i t h t h e g o a l o f e n g e n d e r i n g in s t u d e n t s a c a p a c i t y f o r t h e k i n d o f e f f e c t i v e d e l i b e r a t i o n d e s c r i b e d earlier as w e l l as a c o m m i t m e n t t o liberal p r i n c i p l e s s u c h as n o n d i s c r i m i n a t i o n , n o n r e p r e s - s i o n , a n d t o l e r a n c e , m r e a c h i n g this g o a l results in a d u l t c i t i z e n s w h o c a n e v a l u a t e a n d c h o s e t h e i r o w n life p l a n s a n d e f f e c t i v e l y p a r t i c i p a t e in d e m o - c r a t i c politics. t h e liberal e d u c a t i o n a l ideal is i n t i m a t e l y r e l a t e d t o t h e p r i n c i p l e o f e q u a l e d u c a t i o n a l o p p o r t u n i t y b e c a u s e it s u p p l i e s t h e a n s w e r t o t h e q u e s - t i o n o f w h a t e d u c a t i o n a l o p p o r t u n i t i e s are i n d e e d w o r t h w a n t i n g a n d t h u s w h a t e d u c a t i o n a l o p p o r t u n i t i e s ( o r results °) a r e t o b e e q u a l i z e d a m o n g s c h o o l c h i l d r e n . c o n s e q u e n t l y , t h e liberal ideal is culturally encumbered, w h i c h is t o s a y t h a t it rules c e r t a i n g o a l s f o r e d u c a t i o n in, s u c h as e f f e c t i v e d e l i b e r a t i o n , a n d c e r t a i n o t h e r s out, s u c h as i n d o c t r i n a t i n g c h i l d r e n w i t h a p a r t i c u l a r r e l i g i o u s faith. i n t h e c o n t e x t o f m u l t i c u l t u r a l e d u c a t i o n , t h e liberal e d u c a t i o n a l ideal t h u s faces m e a s u r i n g u p t o a c h a l l e n g e t h a t m a y b e c h a r a c t e r i z e d in t e r m s o f a slightly m o d i f i e d v e r s i o n o f k y m l i c k a ' s earlier o b s e r v a t i o n : it only makes sense to invite people to participate in schooling (or for people to accept that invitation) if they will be treated as equals. and that is incompatible with defining people in terms o f roles they did n o t shape or endorse. t h i s c h a l l e n g e t o t h e liberal e d u c a t i o n a l ideal h a s b o t h p o l i t i c a l a n d e m p i r i c a l d i m e n s i o n s . t h e political d i m e n s i o n is that n o t all g r o u p s e n d o r s e this ideal as a c e n t r a l a i m o f e d u c a t i o n . t h e a m i s h , t o t a k e a w e l l - w o r n ex- a m p l e , r e j e c t t h e liberal e d u c a t i o n a l ideal in f a v o r o f e d u c a t i n g their c h i l d r e n f o r w o r k , p i e t y , a n d a s t r o n g s e n s e o f c o m m u n i t y . christian f u n d a m e n t a l i s t s r e j e c t t h e liberal e d u c a t i o n a l ideal as e x e m p l i f y i n g " s e c u l a r h u m a n i s m . " t o t a k e a less e x t r e m e e x a m p l e , political c o n s e r v a t i v e s s e e k t o p l a c e t r a d i t i o n a l lurawls, a theory o f justice. ~ggutmann, democratic education. °see note . h o w e values a n d associated political a n d e c o n o m i c practices b e y o n d criticism in such a w a y as to c i r c u m s c r i b e significantly the liberal educational ideal. t h e empirical d i m e n s i o n o f the challenge is that p s y c h o l o g i c a l a n d social barriers exist to educational o p p o r t u n i t i e s e v e n w h e n explicitly ar- ticulated political ones d o not. minorities are often stigmatized in ways that can d e s t r o y self-respect a n d m o t i v a t i o n o r result in " d i s i d e n t i f y i n g " with schooling. related to the latter, so-called "caste-like" minorities exhibit " o p p o s i t i o n a l cultures" that r e n d e r t h e m ill-equipped to, i n d e e d resistant to, take advantage o f the present o p p o r t u n i t y structure, e v e n w h e n they d o n o t explicitly reject the liberal educational ideal. some n o d o u b t think these kinds o f p r o b l e m s are fatal for the liberal e d u c a t i o n a l ideal, as well as for the allied principle o f equal educational op- p o r t u n i t y . a l t h o u g h the p r o b l e m s are i n d e e d s e r i o u s - - a n d are p r o b l e m s that have received t o o little a t t e n t i o n - - t h e liberal educational ideal a n d the principle o f equal educational o p p o r t u n i t y are, if suitably interpreted, capable o f a c c o m m o d a t i n g these p r o b l e m s . s h o w i n g h o w will be m y task in the r e m a i n d e r o f this article. i will begin by, first, distinguishing conser- vative a n d progressive positions w i t h i n the liberal tradition, next, refining the liberal educational ideal, and, finally, examining equality o f educational o p p o r t u n i t y w i t h respect to three kinds o f cultural minorities. e.d. hirsch is a g o o d representative o f a c o n s e r v a t i v e in the liberal d e m o c r a t i c tradition vis-a-vis multicultural education. he makes free use o f the principles a n d r h e t o r i c o f the liberal d e m o c r a t i c tradition, c o n t e n d - ing that it is o n l y b y acquiring cultural literacy that the " d i s a d v a n t a g e d (pri- marily african americans a n d hispanics) can participate in d e m o c r a c y a n d e n j o y equality o f o p p o r t u n i t y . hirsch's s o l u t i o n to multiculturalism is thus to eliminate it b y using public education to p r o m o t e a u n i f o r m cultural liter- acy. a c c o r d i n g to hirsch, this n o t o n l y benefits the disadvantaged, but it is also r e q u i r e d to p r e s e r v e d e m o c r a c y . hirsch is w r o n g o n b o t h c o u n t s because his p r o p o s a l is b o t h naive and a betrayal o f the liberal democratic tradition. it is naive to think that it w o u l d benefit p e o p l e to strip t h e m o f their identities a n d that t h e y w o u l d so easily a b a n d o n s o m e t h i n g so i m p o r t a n t to them; it is a betrayal o f the liberal d e m o c r a t i c tradition to think that s o m e t h i n g so i m p o r t a n t to p e o p l e ' s iden- tities s h o u l d be eliminated rather than a c c o m m o d a t e d . c o n t r a hirsch, there is n o t h i n g in the liberal d e m o c r a t i c tradition that c o m p e l s it to strip individuals o f their cultural heritage in service o f the e~claude m. steele, "race and the schooling of black americans," atlantic monthly , no. ( ): - . -'-'john ogbu and maria matute-bianchi, "understanding sociocultural factors: knowledge, identity, and school adjustment," in beyond language: social a n d cultural factors in schooling language minority students (los angeles: evaluation, dissemination and assessment center, california state university, ), - . ~e.d. hirsch, cultural literacy (new york: vintage books, ). e q u a l e d u c a t i o n a l o p p o r t u n i t y a n d m u l t i c u l t u r a l i s m p o l i t i c a l - e c o n o m i c c o m m u n i t y . t h i n k e r s i n t h e t r a d i t i o n h a v e l o n g a p - p r e c i a t e d t h e s o c i o s t r u c t u r a l n a t u r e o f l i b e r a l c o n c e p t s s u c h as f r e e d o m a n d o p p o r t u n i t y . f o r i n s t a n c e , d e w e y o b s e r v e s r e g a r d i n g f r e e d o m , all c o n d u c t is interaction b e t w e e n e l e m e n t s o f h u m a n nature and the e n v i r o n m e n t , natural a n d s o c i a l . . , f r e e d o m is f o u n d in the in- t e r a c t i o n w h i c h maintains an e n v i r o n m e n t in w h i c h h u m a n desire and c h o i c e c o u n t for s o m e t h i n g . . . . r e g a r d i n g o p p o r t u n i t e s , d e w e y o b s e r v e s , the resistance a n d the c o o p e r a t i o n of o t h e r s is the central fact in the furtherance o r failure o f o u r schemes. c o n n e c t i o n s w i t h our fellows furnish b o t h the o p p o r t u n i t i e s for action and the instrumen- talities b y w h i c h w e take a d v a n t a g e o f o p p o r t u n i t y . b y i n c o r p o r a t i n g s o c i o c u l t u r a l f a c t o r s i n t o t h e i n t e r p r e t a t i o n o f f r e e d o m a n d o p p o r t u n i t y , t h i n k e r s l i k e d e w e y , p r o g r e s s i v e s in t h e l i b e r a l d e m o c r a t i c t r a d i t i o n i n g e n e r a l , h o l d a v i e w t h a t is p o t e n t i a l l y m u c h m o r e r e s p o n s i v e t o c u l t u r a l d i f f e r e n c e s t h a n a c o n s e r v a t i v e v i e w s u c h as h i r s c h ' s . h o w e v e r , b y e n d o r s i n g t h e p r o m o t i o n o f t h e c u l t u r a l l y e n c u m b e r e d l i b e r a l e d u c a - t i o n a l i d e a l t h r o u g h p u b l i c e d u c a t i o n i n o r d e r t o c r e a t e c i t i z e n s s u i t e d f o r a l i b e r a l d e m o c r a t i c p o l i t i c a l - e c o n o m i c c o m m u n i t y , p r o g r e s s i v e s i n t h e l i b e r a l d e m o c r a t i c t r a d i t i o n a p p e a r i n t h e e n d t o b e s u b j e c t t o t h e s a m e c r i t i c i s m t h a t m a y b e l e v e l e d a g a i n s t c o n s e r v a t i v e s : t h e y d i s m i s s c u l t u r a l c o m m u n i t i e s i n f a v o r o f t h e p o l i t i c a l - e c o n o m i c c o m m u n i t y a n d , in t h e p r o - c e s s , p r o m o t e a k i n d o f p u b l i c e d u c a t i o n i n w h i c h c u l t u r a l m i n o r i t i e s a r e r e q u i r e d t o g i v e u p t h e i r c u l t u r a l i d e n t i t i e s i f t h e y a r e t o s u c c e e d . t h e r e is n o t i d y , c o m p l e t e l y u n p r o b l e m a t i c r e s p o n s e t o t h i s o b j e c t i o n . h o w e v e r , it h a s m o r e o r l e s s f o r c e d e p e n d i n g o n ( ) h o w f u l l y s p e c i f i e d , h o w c u l t u r a l l y e n c u m b e r e d , t h e l i b e r a l e d u c a t i o n a l i d e a l is, a n d ( ) w h a t c u l t u r a l m i n o r i t i e s a r e at i s s u e . t h e l i b e r a l e d u c a t i o n a l i d e a l s h o u l d b e m o d e r a t e l y s p e c i f i e d - - s p e c i f i e d j u s t e n o u g h s o t h a t its r e a l i z a t i o n e n a b l e s p e o p l e s t o c o n t r o l t h e i r o w n lives, a n d n o m o r e . w h e n s p e c i f i e d t o t h i s d e g r e e , it m a y , a g a i n , b e i d e n t i f i e d w i t h t h e c a p a c i t y f o r e f f e c t i v e d e l i b e r a t i o n i n c o n j u n c t i o n w i t h a c o m m i t - m e n t t o p r i n c i p l e s s u c h as n o n d i s c r i m i n a t i o n , n o n r e p r e s s i o n , a n d t o l e r a n c e . b y c o n t r a s t t o t h i s c o n c e p t i o n , h i r s c h ' s c o n c e p t i o n is t o o s p e c i f i c i n a c o n - s e r v a t i v e d i r e c t i o n b e c a u s e it t o o h e a v i l y i n c o r p o r a t e s a p a r t i c u l a r c u l t u r a l h e r i t a g e . c a n t h i s m o d e r a t e c o n c e p t i o n o f t h e l i b e r a l e d u c a t i o n a l i d e a l - - t h i s c o n - c e p t i o n o f w h a t e d u c a t i o n a l o p p o r t u n i t i e s a r e s u f f i c i e n t l y w o r t h w a n t i n g 'john dewey, human nature and conduct (new york: random house, ), . slbid., . h o w e to be equalized a m o n g s c h o o l c h i l d r e n - - p r o m o t e a n d be c o n s i s t e n t with equality o f educational o p p o r t u n i t y vis-a-vis cultural minorities? t o a n s w e r this question, i will consider, in turn, three kinds o f cultural minorities identified b y o g b u a n d matute-bianchi: a u t o n o m o u s , immigrant, a n d caste- like. an e x a m p l e o f an a u t o n o m o u s m i n o r i t y is the amish. as n o t e d before, the amish reject the liberal educational ideal as a threat to their w a y o f life. in response, the amish are simply p e r m i t t e d to establish their o w n , largely u n r e g u l a t e d s y s t e m o f formal education. as a practical matter, t h e y are t o o small in n u m b e r to p o s e a significant threat to a liberal d e m o c r a t i c society. f u r t h e r m o r e , t h e y are separatists w h o n e i t h e r appeal to t h e state t o p r o t e c t c o m m u n i t y m e m b e r s ' welfare n o r a t t e m p t to influence public education. regarding equality o f educational o p p o r t u n i t y , permitting the amish to establish their o w n s c h o o l s is t a n t a m o u n t to allowing t h e m to d e t e r m i n e their o w n view o f educational o p p o r t u n i t i e s w o r t h w a n t i n g in d e f e r e n c e to their cultural (particularly religious) sensibilities a n d to a b a n d o n i n g the quest for equality o f o p p o r t u n i t y . amish children are p r o v i d e d an equal educational o p p o r t u n i t y o n l y in the a t t e n u a t e d sense that t h e y are p r o v i d e d with the o p p o r t u n i t y to at- t e n d public schools, w h i c h their parents turn d o w n o n their behalf. o n the o t h e r hand, the liberal educational ideal is n o t a threat insofar as the amish d o n o t have to give up their cultural c o m m u n i t y and identity to b e c o m e a part o f the p o l i t i c a l - e c o n o m i c c o m m u n i t y , for t h e y simply d o n o t participate significantly in the latter. thus, as a result o f o b s e r v i n g the principle o f equality at o n e level, namely, equal respect for cultural identi- ty, amish children forgo equality at a n o t h e r level, namely, equality o f educa- tional o p p o r t u n i t y . an e x a m p l e o f an immigrant m i n o r i t y is the american chinese. ac- c o r d i n g to o b g u a n d matute-bianchi, g r o u p s like the american chinese, w h o enter the u.s. voluntarily, exhibit an "alternation m o d e l o f b e h a v i o r , " w h e r e b y t h e y neither are assimilated into n o r reject the mainstream u.s. culture reflected in the public schools. instead, they m o v e back and forth b e t w e e n cultures as circumstances dictate. given this response, g r o u p s such as the american chinese also seem to have n o t h i n g to fear f r o m thc liberal educational ideal. i n d e e d , in o n e i m p o r t a n t situation in w h i c h t h e y were e x c l u d e d f r o m e n j o y i n g the fruits o f u.s. public e d u c a t i o n , t h e y a d v a n c e d ~'"throughout will be s p e a k i n g in t e r m s o f a g g r e g a t e t e n d e n c i e s . i d o not m e a n to sug g e s t ( n o r d o t h e a u t h o r s i cite) t h a t i m p o r t a n t d i f f . ' f e n c e s d o n o t e x i s t w i t h i n the c a t e g o r i e s o f m i n o r i t i e s h a v e c h o s e n . i n d e e d , e d u c a t o r s s h o u l d in g e n e r a l a v o i d a s s u m i n g that in- d i v i d u a l s can be d e s c r i b e d as t h u s a n d so s i m p l y b e c a u s e t h e y fall i n t o o n e o r a n o t h e r g e n e r a l c u l t u r a l c a t e g o r y . -ogbu a n d m a t u t e - b i a n c h i , ~ ' u n d e r s t a n d i n g s o c i o c u l t u r a l f a c t o r s . " -'~ibid. e q u a l e d u c a t i o n a l o p p o r t u n i t y a n d m u l t i c u l t u r a l i s m their claims for equality o f educational o p p o r t u n i t y in the u.s. legal s y s t e m in the celebrated l a u v. n i c h o l s case. caste-like minorities include g r o u p s like african americans a n d mex- ican americans. unlike a u t o n o m o u s minorities such as the amish, t h e y par- ticipate significantly in (and are s u b j u g a t e d by) the d o m i n a n t political- e c o n o m i c c o m m u n i t y ; unlike immigrant minorities s u c h as the a m e r i c a n chinese, t h e y b e c a m e a part o f the political-economic c o m m u n i t y involun- tarily. because o f their peculiar circumstances, rather t h e n a d o p t i n g the separatist strategy o f the amish o r the alternation strategy o f the a m e r i c a n chinese, caste-like minorities have a d o p t e d an o p p o s i t i o n a l strategy to p r e s e r v e their cultural identity. ° such a strategy typically entails p o o r s c h o o l p e r f o r m a n c e because w h a t is i n v o l v e d in d o i n g well in s c h o o l re- quires a c c e p t i n g the values o f the d o m i n a n t culture to w h i c h casteqike minorities are in o p p o s i t i o n . o f the three kinds o f minorities c o n s i d e r e d , caste-like minorities p o s e the greatest p r o b l e m s for the principle o f equal e d u c a t i o n a l o p p o r t u n i t y . unlike the amish, t h e y a t t e n d public s c h o o l s a n d d o n o t e s c h e w the de- m a n d for equality o f educational o p p o r t u n i t y ; unlike the american chinese, t h e y fare p o o r l y there a n d are unwilling or unable to a d o p t the p r e d o m i - nant n o r m s . t h e crucial q u e s t i o n for the p r e s e n t discussion is w h e t h e r this state o f affairs is the inevitable o u t c o m e o f e n d o r s i n g the liberal educational ideal as a central aim o f e d u c a t i o n a n d e n d o r s i n g the principle o f equal e d u c a t i o n a l o p p o r t u n i t y as the sine q u a n o n o f a just educational system. t h e r e are g o o d reasons to believe that the a n s w e r to these q u e s t i o n s s h o u l d be " n o . " caste-like minorities seem to be reacting m o r e against false p r o m i s e s a n d h a v i n g their identities d e f i n e d for t h e m than against the prin- ciple o f equality o f educational o p p o r t u n i t y and the liberal educational ideal. i n d e e d , t h e y have e m p l o y e d the c o n c e p t o f equality o f educational o p p o r - t u n i t y as a basic principle in the c o u r s e o f p u r s u i n g their grievances w i t h i n the u.s. legal system, b r o w n b e i n g the m o s t c e l e b r a t e d example. further- m o r e , o p p o s i t i o n a l b e h a v i o r is m o s t likely w r o u g h t b y despair a n d frustra- tion a n d is p r o b a b l y as m u c h a reaction against b e i n g relegated to a l o w l y p o s i t i o n in the class structure as to p r e s e r v i n g cultural identity; it is n o t a deliberate strategy a n d is o f t e n self-defeating. it results f r o m the p e r c e p - tion (often accurate) that unless o n e is a m e m b e r o f the d o m i n a n t culture, d o i n g well in s c h o o l will n o t result in the benefits that are p r o m i s e d , a n d f r o m the p e r c e p t i o n (again, o f t e n accurate) that o n e m u s t allow o n e ' s b e h a v i o r a n d identity to be d e f i n e d in terms o f the d o m i n a n t culture (e.g., b y " a c t i n g w h i t e " ) in o r d e r to d o well. zglau v. n i c h o l s u.s. ( ). ~°paul willis, l e a r n i n g to l a b o r (new york: c o l u m b i a university press, ), argues for the s a m e k i n d of oppositional culture w i t h respect to w o r k i n g class b o y s in england. 'consider willis's " l a d s " in l e a r n i n g to labor. howe a l t h o u g h w e have a clear obligation to respect the cultural identities o f a u t o n o m o u s a n d immigrant m i n o r i t i e s - - a n d c o u l d d o m u c h better in this r e g a r d - - t h e obligation is especially d e m a n d i n g for caste-like minorities b e c a u s e o f the peculiar w a y t h e y b e c a m e a part o f and the peculiar roles t h e y o c c u p y w i t h i n the p o l i t i c a l - e c o n o m i c c o m m u n i t y . f u r t h e r m o r e , because o f their peculiar history a n d position within the political-economic c o m m u n i t y , caste-like minorities are the m o s t o f t e n criticized b y certain quarters for failing to take advantage o f the opportunities that are o f f e r e d to them. ~ but requiring p e o p l e to sacrifice their identities in o r d e r to suc- c e e d is n o t a kind o f o p p o r t u n i t y w o r t h wanting; n o r is w o r k i n g h a r d a n d d o i n g well p u r s u i n g the false p r o m i s e that e d u c a t i o n is enabling. the con- clusion that i reach f r o m the a b o v e o b s e r v a t i o n s is that the principle o f equal educational o p p o r t u n i t y is being b e t r a y e d vis-a-vis caste-like minorities, n o t that it is bankrupt. c o n c l u s i o n i set o u t in this article to s h o w that the liberal tradition with its central em- phasis o n equality is r o b u s t e n o u g h to a c c o m m o d a t e the challenge p o s e d b y multicultural e d u c a t i o n . given that the principle o f equal educational o p p o r t u n i t y is i n d e e d n o t b a n k r u p t , but is being betrayed, i will c o n c l u d e b y offering a few brief suggestions regarding h o w it might be better realized. in general, institutional and c l a s s r o o m practices n e e d to i n c o r p o r a t e a m u c h greater a p p r e c i a t i o n o f the n e e d for g e n u i n e participation a n d self- definition. at the institutional level, the participation o f parents o f cultural m i n o r i t y students s h o u l d be enlisted a n d s h o u l d be s u p p o r t e d w h e n it oc- curs. at the c l a s s r o o m level, t e a c h i n g m e t h o d s such as c o o p e r a t i v e learn- ing strategies s h o u l d b e c o m e c o m m o n p l a c e , a n d teachers s h o u l d d e v e l o p a m u c h greater sensitivity to cultural (including linguistic) differences a n d h o w to deal with them. these are quite familiar suggestions a n d i shall leave their further specification to empirical investigations. what needs to be e m p h a s i z e d here is that, although the classroom a n d institutional practices i suggest are quite familiar, m y u n d e r l y i n g justifica- tion for a d v o c a t i n g t h e m p r o b a b l y is n o t (and w o u l d likely influence the w a y in w h i c h t h e y are carried out). specifically, i have in m i n d e m p l o y i n g these practices so as to truly negotiate the curriculum. for example, i d o n o t a d v o c a t e c o o p e r a t i v e learning as b u t a n o t h e r educational t e c h n o l o g y for achieving the same old educational objectives. instead, cooperative learn- ing s h o u l d be p r a c t i c e d so as to e x e m p l i f y a n d p r o v i d e practice in d e m o - cratic give-and-take. a n d i d o n o t a d v o c a t e parental participation so that s c h o o l s m a y garner s u p p o r t at h o m e for carrying o u t their o w n p r e s e n t ~ patrick mcquillan, d i s n e y l a n d in the j u n g l e : the m y t h o f e d u c a t i o n a l o p p o r t u n i - ty in a n u r b a n a m e r i c a n h i g h school (forthcoming doctoral dissertation, brown university). ~see, for example, b e y o n d l a n g u a g e . equal educational opportunity a n d multiculturalism agenda. although the precise f o r m such participation s h o u l d take is an o p e n question, t w o things are clear. first, s t u d e n t s w h o s e p a r e n t s d o n o t partici- pate s h o u l d n o t be penalized, for there are a variety o f reasons that might a c c o u n t for parental non-participation, a n d c h i l d r e n s h o u l d n o t be held responsible for their parent's behavior in a n y case. second, schools o f c h o i c e b a s e d o n m a r k e t principles are a v e r y b a d idea. again, c h i l d r e n s h o u l d n o t be held hostage by their p a r e n t s ' j u d g m e n t a n d behavior. in addition, the market t o o heavily favors those w h o possess the c a p i t a l - - e c o n o m i c as well as c u l t u r a l - - a n d it d o d g e s the d e m o c r a t i c p r o c e s s o f deliberation a n d ne- gotiation about w h a t kind o f individuals a n d society public education s h o u l d foster b y supplanting it with " v o t i n g with o n e ' s feet" o n the basis o f unex- a m i n e d a n d self-serving " p r e f e r e n c e s . '' i balk at c h o i c e a n d separatist s c h e m e s in general b e c a u s e i believe it w o u l d be vastly b e t t e r to create a public s c h o o l s y s t e m in w h i c h s u c h s c h e m e s w e r e n o t necessary. h o w e v e r , i c o n c e d e that certain n o n - m a r k e t based s c h e m e s might be justified if w a r r a n t e d b y circumstances. in par- ticular, w h e r e g r o u p s are h a r m e d b y participation in culturally h e t e r o g e n o u s s c h o o l s that are d o m i n a t e d b y mainstream culture, it might be acceptable for t h e m to separate themselves voluntarily so as to p r o t e c t their cultural identities a n d to p r e s e r v e a sense o f p u r p o s e , b e l o n g i n g , a n d self-respect. for example, schools exclusively for black males might be justified o n these g r o u n d s . (note that such s c h o o l s are n o t simply the flip side o f the practice o f de j u r e segregation that was struck d o w n in b r o w n insofar as the latter was j u d g e d harmful b e c a u s e it segregated blacks involuntarily a n d implied racial inferiority.) t h e principle o f equal e d u c a t i o n a l o p p o r t u n i t y can o n l y be realized for cultural minorities b y r e n d e r i n g educational o p p o r t u n i t i e s w o r t h want- ing, a n d r e n d e r i n g educational o p p o r t u n i t i e s w o r t h w a n t i n g requires that minorities n o t be required to give up their identities in o r d e r to e n j o y them. for minorities w h o can live w i t h the liberal e d u c a t i o n a l i d e a l - - a n d i think m o s t c a n - - s o m e "cultural e l b o w r o o m '' must be p r o v i d e d w i t h i n the area it circumscribes. t o reiterate m y earlier paraphrase o f kymlicka: it only makes sense to invite people to participate in schooling (or for people to accept that invitation) if they will be treated as equals. and that is incompatible with defining people in terms of roles they did not shape or endorse. r e n d e r i n g educational o p p o r t u n i t i e s w o r t h w a n t i n g also requires en- suring that success in s c h o o l is truly enabling. that the d e v e l o p m e n t o f h a v e in m i n d p a r t i c u l a r l y t h e a r g u m e n t s b y j o h n c h u b b a n d t e r r y m o e , politics, markets, a n d america's schools ( w a s h i n g t o n , d.c.: b r o o k i n g s institution, ). t h e r e s p o n s e is o n e o f t h e s e v e r a l p r o v i d e d b y g u t m a n n in d e m o c r a t i c education. sthe c o n c e p t o f " e l b o w r o o m " is b o r r o w e d f r o m d e n n e t t ' s b o o k b y t h a t n a m e . t t o w e o p p o s i t i o n a l cultures a n d t h e failure o f e d u c a t i o n t o b e e n a b l i n g is deter- m i n e d b y the p o l i t i c a l - e c o n o m i c o r d e r o u t s i d e o f s c h o o l i n g is cause f o r ex- t r e m e p e s s i m i s m , p e s s i m i s m that is e x a c e r b a t e d b y t h e f r e q u e n t o b s e r v a - t i o n that s c h o o l i n g s i m p l y reflects a n d r e p r o d u c e s t h e p o l i t i c a l - e c o n o m i c o r d e r . a n y p r o p o s a l s f o r e d u c a t i o n a l r e f o r m that i g n o r e these o b s e r v a t i o n s are d o o m e d to e i t h e r s i m p l y fail o r f u r t h e r e n s c o n c e t h e status q u o . o n the o t h e r h a n d , calling a t t e n t i o n to the influence o f b r o a d political- e c o n o m i c i n f l u e n c e s is a t w o - e d g e d s w o r d . t o o o f t e n , p o i n t i n g t o s u c h influences serves to justify d o i n g nothing, o n the g r o u n d s that b r o a d political- e c o n o m i c i n f l u e n c e s are b e y o n d the p o w e r a n d p u r v i e w o f e d u c a t o r s q u a e d u c a t o r s a n d that s c h o o l s s h o u l d stay o u t o f politics, but, s c h o o l s are in- h e r e n t l y political, if o n l y b y default, a n d e d u c a t o r s c a n a n d s h o u l d take t h e political lead in r e s h a p i n g p u b l i c s c h o o l i n g s o that it b e c o m e s an im- p o r t a n t l o c u s for p r o g r e s s i v e social c h a n g e that f u n c t i o n s to p r e p a r e a l l c h i l d r e n to p a r t i c i p a t e in w h a t g u t m a n n calls " c o n s c i o u s social r e p r o d u c - tion. ''-~c' public s c h o o l i n g c a n b e r e s h a p e d in this w a y , h o w e v e r , o n l y if e d u c a t o r s p r o m o t e w h a t the p r i n c i p l e o f e q u a l e d u c a t i o n a l o p p o r t u n i t y re- q u i r e s by, first, c o n s t r u i n g it in a w a y that is c o n s i s t e n t w i t h p h i l o s o p h i c a l - ly d e f e n s i b l e i n t e r p r e t a t i o n s o f the c o n c e p t s o f f r e e d o m a n d o p p o r t u n i t y and, s e c o n d , a c c o m m o d a t i n g w h a t t w e n t i e t h - c e n t u r y social s c i e n c e has r e v e a l e d r e g a r d i n g t h e i n f l u e n c e s that s h a p e o p p o r t u n i t y structures. received august , revision received january , accepted march , ~'gutmann, democratic education. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" 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(typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ book review enough: staying human in an engineered age b mckibben, henry holt company, new york, , hardback, pp . isbn bill mckibben’s book, enough, is about cloning, genetic enhancement, and nanotech- nology. his thesis is that these things are bad—nay, they are downright evil. in vivid and readable prose, mckibben explains what will soon be possible with these technologies and provides a dystopian portrait of the future. his alarmist tone is effective. despite having read several similar works and remaining unmoved, mckibben’s images struck home with me. i began to feel, in my gut, anxiety about the genetically engineered future. mckibben is no stranger to raising the dangers of new technology. his the end of nature is widely acclaimed to have alerted an unsuspecting public about the danger of global warming. alarmism was appropriate for the end of nature, since little was written on global warming prior to . but despite mckibben’s effective writing, his alarmism seems out of place for his current topic, since the potential harms of cloning and genetic engineering have been discussed in the academic and popular press for some time. remember, it has been years since the appearance of paul ramsey’s fabricated man, and years since huxley’s brave new world. mckibben’s most prominent argument against human genetic engineering is that it will cause psychological harm to the people produced from the use of the technology. mckibben claims that people with the knowledge that they resulted from engi- neered embryos will undergo a crisis in personal responsibility. so, he argues, engi- neered people will not know whether to take credit for their achievements, (they were designed for great things, after all). likewise, engineered people will tend to feel guilty for their failures (since their genes provided the aptitude needed to succeed, their failures must result from a culpable lack of will). mckibben makes these points with a series of hypothetical examples. here he is imagining the psychological state of a genetically engineered daughter: ‘‘and what can she take pride in? her good grades? she may have worked hard, but she’ll always know she was specced for good grades. her kindness to others? well, yes, it’s good to be kind—but perhaps it’s not much of an accomplishment once the various genes with some link to sociability have been catalogued and manipulated’’ (pp – ). the problem with this argument is that the potential psychological harm seems to arise because of genetic determinism, rather than because such individuals were intentionally designed. but, if knowledge about genetic determinism causes psychological harm, one would expect it to have been a problem even in the pre-engineered age. thus, sally could worry that it is her genes that make her a virtuoso pianist (she herself deserves no praise), even if her parents did not purchase a particular set of genes for her. the fact that we do not now suffer any great psychological harm from the knowledge that we are genetically deter- mined suggests that ‘‘engineered’’ people who are genetically determined will not suffer any harm either. at least, mckibben does not provide any reasons for thinking so. mckibben misses the opportunity to explain why the intentional engineering of offspring has additional harms, harms not associated with genetic determinism per se. at least, he does not do this in any systematic way, though he has certainly opened the door to an analysis. it is obvious that the inten- tional design of offspring has ethical implica- tions for those doing the designing. the ethically relevant distinction here is between being causally responsible for a state of the world, and merely lamenting (or celebrating) a state of the world that one has had no hand in bringing about. so, parents who have a child engineered are morally responsible for the health of the offspring in a way in which parents who use the more usual way of determining the genotype of offspring are not responsible. but perhaps genetic engineering also has implications for the moral psychol- ogy of the engineered offspring. perhaps any guilt felt by parents will seep over to affect the children. perhaps (as mckibben does suggest) the expectations of the parents will put too much pressure on the children. perhaps engineered children will not feel responsible for their actions because they were programmed (although i have already suggested that this is not materially different from worries about genetic determinism resulting from natural reproduction). perhaps there are more direct sources of harm in simply knowing that one was designed. mckibben’s writing leaves us with the feeling that this is the case, but it is difficult to translate the feeling into an articulate and appropriately weighty set of worries. mckibben’s book also covers a number of familiar issues. he predicts that class divi- sions will widen as the rich purchase genetic enhancements for their children—class divi- sions will be written into our biology. he is concerned about an arms race: since the technology will improve every several years, one can expect that younger people will be smarter, faster, and better than those just three or four years older. how will our employment system handle this: if the newer model is always better, why keep older models around? these concerns are familiar, but they are also particularly vivid in mckibben’s telling. mckibben unifies these worries under the theme of meaning. past technological advances have stripped away the ‘‘contexts’’ (for example, church, village, family, nature) that have given meaning to the lives of previous generations of humans. currently, our self-image allows us to find meaning only in autonomy and individuality. however, according to mckibben, because of biotechnology ‘‘we stand on the edge of dis- appearing even as individuals’’ (p , italics in original). mckibben’s point is that we will no longer be able to view ourselves as making free choices which warrant praise or blame— we will instead see ourselves as automatons. this is a potent concern, but it is also one which we have been struggling with since the recognition that physical determinism seems to be incompatible with free will. i am not sure that reflection on genetic engineering does anything more that put a new face on an old monster. understandably, mckibben’s discussion is at its weakest when discussing nanotechnol- ogy and its current state of development. likewise, his description of the harms of nanotechnology is somewhat inchoate. (michael crichton’s novel prey might be a better primer on these topics.) however, nanotechnology may be too new an idea to discuss with much specificity. at the very least, mckibben lets us know who is making news in the area and provides citations for further research. mckibben provides a knowledgeable and nuanced treatment of preimplantation diagnosis (pp – ). he advocates screening for genetic diseases, and though he acknowledges that there is only a murky line between screening for disease and screening for merely suboptimal mental and physical traits, he suggests that we can implement this technology without falling prey to a brave new world of genetic enhancement. a less familiar theme in mckibben’s work is his discussion of our resources for resisting technological change. he offers three case studies: the present-day amish, china’s self- imposed moratorium on sea trade in , and japan’s outlawing of weapons that used gunpowder in the early s. in each case, a newer technology was successfully resisted because of the ruling group’s concern about how the technology would affect the fabric of society. these examples provide much fuel for reflection (as do other examples drawn from environmental studies). in particular, one wonders how long new technologies can successfully be suppressed. one also wonders about the forces that propel societies to implement new technologies even when they tend to erode the cultural status quo. for example, why do societies seem to more strongly resist ideological reforms (such as those prompted by feminism or gay rights) than changes from technological advances, since both have the potential to erode the cultural status quo? is it merely the profit motive that makes technological change so difficult to resist? mckibben’s book does not break new philosophical ground. nonetheless, it is chock full of news items, rich hypothetical scenar- ios, interesting analogies, and provocative quotes from experts and quacks alike. it may be the best place that i know of to begin to philosophise about biotechnology. f chessa j med ethics ; :e (http://www.jmedethics.com/cgi/content/full/ / /e ) of www.jmedethics.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm e .b m j.co m / j m e d e th ics: first p u b lish e d a s . /jm e . . o n n o ve m b e r . d o w n lo a d e d fro m http://jme.bmj.com/ microsoft word - brian k. hand current zoology ( ): – , received sep. , ; accepted dec. , .  corresponding author. e-mail: hohenlohe@uidaho.edu © current zoology genomics and introgression: discovery and mapping of thousands of species-diagnostic snps using rad sequencing brian k. hand , tyler d. hether , ryan p. kovach , , clint c. muhlfeld , stephen j. amish , matthew c. boyer , sean m. o’rourke , michael r. miller , winsor h. lowe , paul a. hohenlohe *, gordon luikart , flathead lake biological station, fish and wildlife genomics group, university of montana, polson, mt , usa institute for bioinformatics and evolutionary studies, department of biological sciences, university of idaho, moscow, id , usa u. s. geological survey, northern rocky mountain science center, glacier national park, west glacier, montana , usa fish and wildlife genomics group, division of biological sciences, the university of montana, missoula, montana , usa montana fish, wildlife and parks, kalispell, montana , usa department of animal science, university of california, davis, one shields avenue, davis, ca , usa university of montana, division of biological sciences, missoula, montana , usa abstract invasive hybridization and introgression pose a serious threat to the persistence of many native species. understand- ing the effects of hybridization on native populations (e.g., fitness consequences) requires numerous species-diagnostic loci dis- tributed genome-wide. here we used rad sequencing to discover thousands of single-nucleotide polymorphisms (snps) that are diagnostic between rainbow trout (rbt, oncorhynchus mykiss), the world’s most widely introduced fish, and native westslope cutthroat trout (wct, o. clarkii lewisi) in the northern rocky mountains, usa. we advanced previous work that identified , species-diagnostic loci by using longer sequence reads ( bp vs. bp) and a larger set of individuals (n = ). we sequenced rad libraries for individuals from diverse sampling sources, including native populations of wct and hatchery broodstocks of wct and rbt. we also took advantage of a newly released reference genome assembly for rbt to align our rad loci. in total, we discovered , putatively diagnostic snps, , of which we mapped to anchored chromosome locations on the rbt genome. a small portion of previously discovered putative diagnostic loci ( of , ) were no longer diagnostic (i.e., fixed between species) based on our wider survey of non-hybridized rbt and wct individuals. our study suggests that rad loci mapped to a draft genome assembly could provide the marker density required to identify genes and chromosomal regions in- fluencing selection in admixed populations of conservation concern and evolutionary interest [current zoology ( ): – , ]. keywords conservation genetics, hybridization, invasive species, next generation sequencing, salmonid fish, snp discovery species invasions and resulting hybridization be- tween native and non-native species provide exciting evolutionary experiments where natural selection on novel gene combinations can be studied in ecological time and in different environments (fitzpatrick et al., ; rieseberg, ). hybridization between native and invasive species is one of the most serious threats to global biodiversity, and is a major conservation concern (allendorf et al., ). the rate of hybridization for numerous taxa is increasing with habitat degradation, species translocations, and climate change (kelly et al., ; muhlfeld et al., ). in many cases, natural selection and dispersal by hybrids play key roles in the spread of hybridization in nature (e.g., rhymer and simberloff, ; kovach et al., ). the genetic basis for mechanisms influencing introgression can now be assessed by discovering and genotyping thousands of genetic markers, thereby illuminating the genomic basis of hybridization, fitness, adaptive capacity, and disper- sal (allendorf et al., ; twyford and ennos, ). here we report on the development and mapping of thousands of diagnostic loci between rainbow trout (rbt, oncorhynchus mykiss) and westslope cutthroat trout (wct, o. clarkii lewisi). the rbt is the most widely introduced cold-water fish in the world (halver- son, ), and is one of the most problematic exo- hand bk et al.: genomics and introgression tic invasive species (lowe et al., ). wct and all other extant cutthroat trout taxa are threatened with genomic extinction due to introgressive hybridization with rbt (allendorf et al., ; trotter, ), and introgressive hybridization is considered the primary threat to the persistence of cutthroat trout (e.g., shepard et al., ). for example, known non-hybridized wct populations occupy only about % of their historic range primarily due to hybridization with introduced rbt (shepard et al., ). consequently, wct have twice been petitioned for listing under the u.s. endan- gered species act (usfws, ; shepard et al., ). next generation sequencing (ngs), particularly re- striction-site-associated dna (rad) sequencing, has enabled the discovery and genotyping of thousands of single-nucleotide polymorphisms (snps) distributed across the genome of non-model plants and animals (baird et al., ; narum et al., ). the increased density of loci and genome coverage offered by rad sequencing has enormous promise to help researchers understand the mechanisms driving hybridization and its effects on species fitness, persistence, and adaptive potential (allendorf et al., ), including in the con- text of climate change (muhlfeld et al., ). we previously identified a set of , diagnostic lo- ci using relatively short-read ( bp) rad sequencing in individuals ( wct and rbt) and de novo assembly of loci (hohenlohe et al., ; amish et al., ). we further filtered those loci using a set of rad contigs assembled for wct to assess patterns of intro- gression in populations across a large river drainage (hohenlohe et al., ). here we extend this work by applying paired-end rad sequencing with longer reads ( bp) to a larger set of individuals from pure rbt and wct populations, and take advantage of a newly released reference genome assembly for rbt (berthelot et al., ). our objectives were: (i) to discover a more reliable and expanded set of diagnostic snp loci for rbt and wct; (ii) to physically map these snps to the rbt genome; and (iii) to validate previously identified diagnostic loci against this broader population sample and compare admixture estimates produced by each approach. we show that the combination of an expanded set of populations, longer reads, and alignment to the refer- ence genome increases the number of diagnostic snps detected. an increased number of species diagnostic markers aids in the discovery of putative super-invasive alleles and development of low-cost diagnostic snp panels (e.g., the – most highly informative di- agnostic snp markers). mapping to the rbt genome offers the additional advantage of enabling diagnostic snp panels that are evenly distributed across chromo- somes, thus increasing statistical power to detect ge- nomic regions of reduced or elevated introgression, and to identify candidate adaptive loci within them. for the purposes of conservation and management, a mapped, well-chosen set of diagnostic loci is crucial for early detection and intervention in populations at the early stages of introgression, for choosing only non-hybri- dized individuals for translocations, and for prioritizing conservation and recovery programs (allendorf et al., ). materials and methods . wct and rbt source populations it can be difficult to find the non-hybridized popula- tions required for discovery of species-diagnostic mark- ers. for wct we focused on three stream populations (south creek, addition creek, danaher creek) of non-hybridized wct located in the south fork flathead river drainage in northwestern montana (fig. ). we also included samples from the washoe park hatchery in anaconda, montana, which is the source of the mixed origin wct m broodstock widely used as a source for reintroductions for conservation programs and in- troductions for recreational fisheries. the wct m broodstock is composed of a diverse set of pure stream populations, including south creek and addition creek, throughout the south fork flathead river drai- nage, and two populations in the clark fork river drai- nage (fig. ). these populations have all been tested repeatedly for rbt admixture, and rbt introgression has never been detected (montana fish, wildlife and parks, unpublished data). we included individual samples from each of south creek, danaher creek, and addition creek and samples from the washoe park hatchery, for a total of wct samples. we used individual samples of rbt from diverse populations including three hatchery stocks (the ennis and arlee hatcheries in montana, and the rock creek hatchery in oregon). samples from the ennis hatchery included a mix of known broodstock sources: mcco- naughy, nebraska; eagle lake, california; fish lake, utah; erwin hatchery, tennessee; shasta, california; arlee hatchery, montana. six additional individual samples from the arlee hatchery were also included. the arlee hatchery rbt strain was the principle source of fish stocked in the flathead drainage until the prac- tice ended in . the arlee strain began in with current zoology vol. no. fig. study area for westslope cutthroat trout (wct; oncorhynchus clarkii lewisi) in northwest usa black lines delineate the clark fork river drainage (left) and the south fork flathead river drainage (right). individuals from three wild populations (silver dots; south creek, addition creek and da- naher creek) were sampled, in addition to the inclusion of individuals from the m broodstock, which represents a mixture of multiple source populations (red dots and including south creek and addition creek). the arrow at bottom right denotes the danaher creek popula- tion from which individuals have never been used in the mo broodstock. the crossing of two major strains (the donaldson trout strain and the missouri strain; stephanie espinoza, personal communication). historically, the arlee hat- chery broodstrock has included coastal strains of rbt with some previous contributions from inland redband rainbow trout oncorhynchus mykiss gairdneri from the kootenai drainage. finally, we included four individual samples from a previous study using clonal lines from the rock creek hatchery on the north umpqua river in southwestern oregon, the clearwater river in north central idaho, and the swanson river in south central alaska (described in miller et al., ). . rad sequencing, alignment and genotyping we prepared libraries for rad sequencing from ge- nomic dna from wct and rbt individuals according to standard protocols using the restriction enzyme sbfi and unique bp barcodes for each sample (miller et al., ). we multiplexed these samples, along with rad libraries (at equal concentration) for an additional other salmonid samples for another study at equal concentration, in sequencing lanes on an illumina hiseq machine. we conducted initial processing of the sequence data using several modules from the stacks software package, version . (catchen et al., ). we used process_radtags from stacks to sort read pairs by barcode and remove any pairs in which the forward read did not contain both a correct barcode and the re- maining six bases of the sbfi recognition sequence. paired end reads from the same individual were only used to identify pcr duplicates. the random shearing step in traditional rad sequencing produces staggered paired-end reads, so that any set of read pairs that are identical across both the forward and reverse reads are likely pcr duplicates of a single original genomic dna fragment (davey et al., ). we therefore removed all read pairs that represented pcr duplicates using the stacks program clone_filter. no further quality filtering was performed at this stage. we aligned filtered read pairs from each individual to the recently published rbt reference genome (berthe- lot et al., ). we used the alignment software bow- tie v . . (langmead and salzberg, ), using end- to-end alignment without allowing gaps, and allowing up to an average of one high-quality nucleotide mis- match per bp. we retained only those read pairs that aligned uniquely to a single genomic location according to these criteria. all further analysis considered only forward reads (i.e., sequence within the first bp of each restriction enzyme cut site) to provide a set of snp loci that could be efficiently and reliably genotyped using further sin- gle-end rad sequencing. we assigned diploid geno- types at each nucleotide position in each individual us- ing the bounded maximum-likelihood method described by catchen et al. ( ), with a minimum phred quali- ty-score threshold of at each nucleotide, the upper bound of the sequencing error rate set to . , and a likelihood ratio significance level of α = . , and we calculated fst across all loci between the two species (custom software is available at http://webpages.uidaho. edu/hohenlohe/software.html). the upper bound on the sequencing error rate effectively makes the genotyping more sensitive to rare alleles and more likely to call a heterozygous genotype when two alleles are observed, making our analysis generally conservative for detect- ing loci that are fixed within each species. . identifying diagnostic and polymorphic loci we applied multiple filters to identify snps that were fixed for different alleles in rbt and wct (i.e., spe- cies-diagnostic). for each putatively diagnostic locus, we required genotypes from at least wct individu- als from each of the four populations and at least rbt individuals. one hybrid individual from addition creek was present in our database. however, given the fact that all previous genetic testing indicated that this hand bk et al.: genomics and introgression population is composed of non-hybridized wct, we suspected that this sample was mislabeled and from another population. we did not include this individual in further analysis, as it substantially reduced (in the thou- sands) the number of diagnostic snps when included in the dataset. previous work identified , putatively diagnostic snps (hohenlohe et al., ; amish et al., ) using bp reads, the same restriction enzyme, and de novo assembly of loci. to validate this prior set of diagnostic snps against the new data, we first aligned these prior rad loci to the rbt reference genome using bowtie (langmead and salzberg, ), providing chromosome and base pair positions for previous snps that exactly corresponded to the current dataset. we rejected any of the prior diagnostic snps that were genotyped in fewer than of individuals in the new dataset, or that showed a minor allele frequency (maf) greater than . in either species (effectively removing all snps with more than one copy of the “wrong” allele observed in either wct or rbt). we also tested our newly discovered diagnostic snps against raw data from hohenlohe et al. ( ). from the individuals used in that previous study, we removed individuals (jocko river and abbot creek samples) from populations where hybridization has occurred (hitt et al., ; corsi et al., ). we aligned the sin- gle-end rad data from that study to the rbt reference genome assembly using bowtie with parameters iden- tical to those described above. we removed all reads that did not align uniquely to the genome, and we called diploid genotypes as described above. finally, we re- moved any snps in the new set of diagnostic loci that were found to share a single allele (e.g., shared ancestral polymorphisms) between wct and rbt if at least one individual was genotyped for each species. lastly, we compared admixture proportions of in- dividuals and population admixture in hybridized populations that were previously analyzed with , snps (hohenlohe et al., ) and microsatellite loci (boyer et al., ). we aligned the raw paired-end rad data (described by hohenlohe et al., ) to the rbt genome using bowtie and called genotypes for the forward reads, using identical parameters as de- scribed above. we extracted the genotypes for our new set of diagnostic snps and calculated individual-level admixture as the proportion of rbt alleles across all loci. this compares results from not only the new set of snps to those used previously, but also the approach of aligning sequence reads to the rbt genome vs. using wct-based rad contigs (hohenlohe et al., ). results . discovery of species-diagnostic snps rad sequencing of the wct and rbt samples yielded a total of . million sequence read pairs. we removed pcr duplicates and aligned the remaining . million read pairs against the recently published rbt reference genome assembly (berthelot et al., ). on average, . % (sd = . %) of read pairs aligned uniquely to a single genomic location for each individu- al. this proportion was slightly higher ( . %) for the rbt individuals compared to the wct individuals ( . %). we then estimated diploid genotypes per indi- vidual at each nucleotide position in the first bp of each rad locus (i.e., using only the forward reads), with an average of , rad loci genotyped per in- dividual. after filtering and alignment, the mean se- quencing coverage at successfully genotyped rad loci per individual was . x (range . – . x; sd = . x). filtering based on the criteria outlined above for number of individuals genotyped per locus, and apply- ing a minor allele frequency threshold of . across the whole dataset, produced a set of , snps, each genotyped at a mean of . individuals (sd = . ). across this set of snp loci, fst = . between the two species. of these snps, , were fixed for al- ternative alleles between the two species. a subset of these snps ( , out of , , or . %, that oc- curred in the first bp of each rad locus) was vali- dated against the additional wct and rbt population samples from hohenlohe et al. ( ). we found of these loci to be polymorphic within one or both species; these loci were removed, leaving a final set of , diagnostic snp loci. data for these diagnostic loci is available on the dryad database (http://datadryad.org). . mapping and validation the newly discovered set of diagnostic snps were fairly evenly spaced across the rbt genome. the rbt assembly comprises mb ( %) on anchored and or- dered chromosomes, along which position and orienta- tion of contigs is known; mb ( %) on anchored chromosomes, on which local ordering and orientation of contigs is not known; and mb ( %) on unanc- hored scaffolds not assigned to a chromosome (berthe- lot et al., ). we found that putatively diagnostic snps were distributed in similar proportions on anc- hored and oriented chromosomes ( . %; fig. ), over- represented on anchored and globally ordered chromo- somes ( %; fig. ), and underrepresented on unanc- hored regions ( %; table ). current zoology vol. no. fig. chromosomal positions of species-diagnostic single-nucleotide polymorphisms (snps) on anchored and ordered chromosomes in the recent assembly of the rainbow trout oncorhynchus mykiss genome (berthelot et al., ). red lines delimit the known length of each chromosome (berthelot et al., ). black dots are newly discovered diagnostic loci ( snps), khaki- colored triangles are snps shared between the new set of diagnostic loci and a previously published set (hohenlohe et al., ). green asterisks represent snps from hohenlohe et al. ( ) that were mapped to chromosomal regions, but were not found in the new set of diagnostic loci. fig. chromosomal positions of species-diagnostic single-nucleotide polymorphisms (snps) on globally ordered but not oriented chromosome sequences (where the order of two adjacent contiguous sequences might be ambiguous), in the recent assembly of the rainbow trout oncorhynchus mykiss genome (berthelot et al., ) red lines delimit the known length of each chromosome (berthelot et al., ). black dots are new putatively diagnostic loci ( snps), khaki- colored triangles are snps shared between the new set of diagnostic loci and a previously published set (hohenlohe et al., ). green asterisks represent snps from hohenlohe et al. ( ) that were mapped to chromosomal regions, but were not found in the new set of diagnostic loci. hand bk et al.: genomics and introgression table discovery, validation, and mapping of two sets of species-diagnostic snps: those from previous work (hohenlohe et al., ; amish et al., ), and those from the current study description anchored, ordered chromosomes anchored chromosomes unanchored scaffolds totals previous data map to rbt genome genotyped in new dataset confirmed diagnostic polymorphic current study confirmed diagnostic polymorphic the rainbow trout (rbt; oncorhynchus mykiss) reference genome (berthelot et al., ) is split into three groups: i) anchored and ordered chro- mosomes, ii) anchored chromosomes, which are globally ordered but where local order and orientation is unknown; and iii) unanchored scaffolds (not associated with any chromosome). polymorphic sites refer to snps identified in a single dataset as diagnostic, but rejected here because of polymorphism detected within at least one of the species in the other dataset. shown are numbers of snps in each category. we further screened and validated previously identi- fied loci within the rbt genome, and compared the utility of aligning against the rbt reference genome to that of our previous set of rad contigs from wct (hohenlohe et al., ). from hohenlohe et al. ( ), contig lengths ranged from to bp with most between and bp. a total of , out of , snp loci ( %) aligned uniquely to a single rad con- tig from hohenlohe et al. ( ). we were able to map , ( %) of these snps, of which , were geno- typed in a sufficient number (> ) of individuals to al- low validation against the expanded set of populations. of these, , were confirmed to be diagnostic and the remaining were polymorphic in either wct or rbt. in our final test, we compared individual and popula- tion-level admixture proportions previously estimated with rad-contig alignment (hohenlohe et al., ) and microsatellite loci (boyer et al., ) to rates of admixture proportion calculated using alignment to the rbt genome and an increased number of snps ( , vs. , ). the old and new estimates of individual ad- mixture proportion were strongly correlated for snp loci (r = . ; fig. , right panel). the new snp loci were more consistent with population level estimates from microsatellite loci, despite individual estimates being more highly variable for microsatellite loci (fig. , left panel). thus both the old and new snp loci give a more precise estimate, but the new set of snp loci ap- peared to eliminate some bias that was present in the previous set of snp loci. discussion . discovery of species-diagnostic snps we validated a previously-discovered set of , species-diagnostic snps between rbt and wct (ho- fig. individual-level and population-mean admixture proportions (marked by +) estimated from a previous set of di- agnostic loci (left panel; boyer et al. ) vs. current estimates from , snp loci, and (right panel) using , snps (hohenlohe et al., ) vs. current estimates estimates are for the same westslope cutthroat trout individuals from five populations in the north fork flathead river, usa as described pre- viously (hohenlohe et al., ). populations are meadow (black), nicola (green), dutch (blue), lower hay (purple), and tepee (orange). the dashed line shows the expectation of equality between the two estimates. current zoology vol. no. henlohe et al., ), and discovered thousands ( , ) of new diagnostic snps. in total, we have now identi- fied , snps that can be used to describe genome- wide patterns of introgressive hybridization between these species (figs. and ). the new discovery of thousands of snps is due to several factors. first, we used longer sequence reads ( bp) compared to our previous discovery of diagnostic loci ( bp). second, we aligned sequence data against the newly available rbt reference genome rather than conducting a de no- vo assembly of loci, which has been shown to improve clustering of reads to form loci (catchen et al., ). third, we used paired-end sequencing, allowing us to improve sequence alignment to the reference genome and to filter out pcr duplicates. lastly, we used geno- typing parameters that were sensitive to loci with a strong excess of heterozygotes and thus more conserva- tive in identifying diagnostic loci. all of these consider- ations are particularly acute in salmonid fish taxa, in which a recent ancestral genome duplication led to abun- dant paralogous sequence variants (psv) that plague development of genetic markers (seeb et al., ; berthelot et al., ). it is worth noting that although we have identified and removed psv from our data set, this also means that we are under-representing dupli- cated genomic regions. how this may influence inter- pretation of genome-wide patterns of admixture remains an ongoing, and poorly understood, problem in popula- tion genomics. the available reference genome for rbt is still a work in progress, with the large majority of contigs or scaffolds either assigned to chromosomes but not local- ly ordered (or oriented; %), or not assigned to chro- mosomes at all ( %). nonetheless, this resource ap- peared to improve snp discovery when compared to the previous set of rad contig loci assembled from wct (hohenlohe et al., ). even unanchored contigs pro- vided a valuable reference for alignment, which served to further filter reads by quality and to better assign reads to loci in comparison to de novo clustering. ac- cordingly, a higher proportion of diagnostic snps from the previous work aligned uniquely to the reference genome compared to the previous set of rad contigs ( % vs. %, respectively). the large number of species-diagnostic snps be- tween rbt and wct identified here was slightly higher than the number of diagnostic loci discovered for other hybridizing species in three recent studies (stölting et al., ; li et al., ; pujolar et al., ). it should be noted, however, that direct (quantitative) compari- sons between the studies is difficult because of several confounding factors influencing the snp discovery process (e.g., methods, time since divergence, effective population size, genome size, sample size, etc.). we found , diagnostic snps out of a total of , with an overall fst = . , using samples ( in wct and in rbt) and bp reads. work on euro- pean eel anguilla anguilla and american eel a. rostrata, with mean fst ~ . – . for microsatellite loci, identified , diagnostic loci (fst > . ) using the european eel draft genome and individuals per spe- cies with rad bp sequencing (mank and avise, ; wirth and bernatchez, ; pujolar et al., ). in two hybridizing tree species (populus alba and p. tremula), with fst ~ . between species for snp loci, a total of , fixed snps were found using the popu- lus reference genome, samples from each species, and bp rad sequencing reads (stölting et al., ). fi- nally, in hybridizing florida bass micropterus florida- nus and largemouth bass m. salmoides, with fst ~ . for microsatellite loci, identified , putatively diag- nostic snps using samples ( of each species and f hybrids) to conduct rna sequencing and de novo transcriptome assembly with bp single-end reads (barthel et al. ; li et al., ). these studies all demonstrate the power of genomic techniques to effi- ciently discover thousands of species-diagnostic snps in non-model taxa, allowing for the direct study of ge- nomic patterns of introgressive hybridization. . genomic studies of introgression ample coverage over large genomic regions is cru- cial for early detection of introgression when levels of hybridization are low and hard to detect with a limited number of microsatellite loci. the updated set of mapp- ed snps discovered here will be used to develop a spe- cies-diagnostic snp chip for testing thousands of indi- viduals across the flathead river drainage, and to more accurately detect expansion of rbt introgression and inform wct conservation strategies range-wide (hitt et al., ; boyer et al., ; muhlfeld et al., ; muhlfeld et al., ). this panel will also be used to assess the genetic status of individual fish, improving our ability to detect even small amounts of nonnative genetic admixture (e.g., super-invasive alleles). in this study, we used samples from three non-hybri- dized wct populations in one drainage as well as a hatchery strain developed from a wide range of wct populations. we used a smaller number of rbt indi- viduals (though more than in hohenlohe et al. ). because introduced rbt come mainly from a few hat- hand bk et al.: genomics and introgression chery sources, we expected that a smaller sample size would be sufficient to capture variation in rbt ance- stral polymorphic loci. however, this approach runs the risk of missing ancestral polymorphism in rbt, which could lead to underestimates of true admixture propor- tion in hybridized populations if putative diagnostic loci scored as pure wct actually represent introgression from rbt. this did not appear to be the case, however, as our overall admixture estimates closely matched pre- vious microsatellite-based estimates and were consis- tently higher than a previous diagnostic snp panel (fig. ). the increased estimates compared to the previous snp set may be explained by higher alignment success in rbt individuals to the rbt genome, although this difference was slight (see above). instead, the agree- ment of our current estimates with the microsatellite estimates suggests the possibility that our current set of diagnostic snps has removed some bias toward wct that was present in the previous snp panel because of alignment to wct-based rad contigs (hohenlohe et al., ). when the source of invasive populations is not well known, a sample size of at least individuals would likely allow for a high probability of detecting even rare alleles (> % of detecting an allele with a minor allele frequency of . , or % with maf = . ) that repre- sent ancestral polymorphism (allendorf et al., ). the degree of shared ancestral polymorphism is af- fected by genetic connectivity, local effective popula- tion sizes and genetic drift, so these geographic and demographic factors should also be considered in sam- pling design for diagnostic markers. increasing the nu- mber of diagnostic markers in a comprehensive snp panel may reduce the variance in admixture estimates caused by undetected ancestral polymorphism when sample sizes are low. these diagnostic snps will help identify mechani- sms influencing the spread of adaptive introgression into non-hybridized wct populations. adaptive intro- gression has important evolutionary consequences, but has been studied in detail only in a handful of systems, and will be greatly aided by the dense, genome-wide marker coverage we report here (hedrick, ). grea- ter genomic coverage and density of markers is essential for identifying genomic regions under natural selection in admixed populations of rbt and wct (hohenlohe et al., ) and other cases of invasive hybridization (fitzpatrick et al., ). genome-typing a large panel of diagnostic loci across hybridized populations or through time can also help improve understanding of how environmental variation or change influences rates of introgression at neutral and adaptive loci (e.g., fitz- patrick et al., ; muhlfeld et al., ). this study illustrates the major leap forward that ngs allows in generating dense, genome-wide snp coverage crucial to studying hybridization, one of the most serious threats to global biodiversity. acknowledgements thanks to morten limborg for a help- ful review of the manuscript. this work was supported by nsf (deb- ), bonneville power administration ( ), and used the vincent j. coates genomics se- quencing laboratory at uc berkeley, supported by nih (s instrumentation grants s rr and s rr ). bkh was supported in part by funding from the department of the interior northwest climate science center. bkh and gl were supported by nasa grant number nnx ab g. tdh and pah received support for data analysis from nih grant p gm . any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the u.s. government. references allendorf fw, hohenlohe pa, luikart g, . genomics and the future of conservation genetics. nature reviews genetics : – . allendorf fw, leary rf, hitt np, knudsen kl, boyer mc et al., . cutthroat trout hybridization and the u.s. endangered species act: one 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cutthroat trout as threatened throughout its range. federal register . – . wirth t, bernatchez l, . decline of atlantic eels: a fatal synergy? proceedings of the royal society of london b : – . v p -koenekoop pagemaker molecular vision ; : <http://www.molvis.org/molvis/v /p > received march | accepted july | published july correspondence to: dr. robert koenekoop, ophthalmology depart- ment, montreal children’s hospital, tupper, montreal, que- bec h h p , canada; phone: ( ) - ext. ; fax: ( ) - ; email: rkoeoph@mch.mcgill.ca leber congenital amaurosis (lca) is the earliest form of retinitis pigmentosa (rp) and is characterized by autosomal recessive inheritance, visual impairment at birth, nystagmus, and an abolished electroretinogram (erg). finding the cause of lca commands considerable interest because it is a com- mon cause of retinal blindness in children [ - ], and its gene identification will allow study of the molecular developmen- tal biology of the human eye. genetic heterogeneity of lca has been suspected since waardenburg’s report [ ] of normal children born to two affected parents, and rare reports of auto- somal dominant inheritance [ ]. patients with lca might maintain visual function, despite progressive retinal changes supporting the hypothesis that some forms of lca are an apla- sia and not a degeneration with impaired development of rod and cone photoreceptors [ , ]. other pathological findings ranging from diffuse retinal atrophy [ ], to ganglion cell ab- normalities [ ] absent rods [ ], or cones [ ], immature [ ], or normal rods and cones [ ], also support the aplasia hy- pothesis. recently, the genetic heterogeneity of this disease has been confirmed, when mutations in three genes were found to be associated with the lca phenotype: the guanylate cyclase gene [ ], rpe [ ] and crx [ ]. also a recent linkage study reported a new lca locus on chromosome q , but the gene remains to be identified [ ]. of these genes, guanylate cy- clase is located in p . , the first locus for lca described [ , ]. until now, only lca patients of maghrebian origin from north africa have been linked to p . [ ]. how- ever, in the same report, it was demonstrated that from eight families linked to p . , only four show mutations in retgc. another candidate gene, pigment epithelium-derived factor (pedf), also localizes to p . [ ]. pedf is a serine pro- tease inhibitor (serpin) expressed in fetal retinal pigment epi- thelial cells. in vitro studies [ ] show, that in pedf-treated retinoblastoma cells (which represent primitive photorecep- tor cells), neurite like projections develop, and neuron spe- cific enolase including neurofilament nf- are expressed, unlike untreated cells. based on linkage data and a compel- ling physiological profile, pedf is still, an obvious and in- triguing candidate gene for photoreceptor degenerations, par- ticularly lca. methods patients with lca from around the world ( multiplex, simplex) entered the study. criteria of inclusion were: ( ) se- vere visual impairment at birth or shortly after, ( ) nystag- mus, ( ) a diminished erg, and ( ) other systemic diseases were also allowed. exclusions were zellwegers disease, abetalipoproteinemia, infantile phytanic acid storage disease, and neuronal ceroid lipofuscinosis. most patients have been © molecular vision four polymorphic variations in the pedf gene identified during the mutation screening of patients with leber congenital amauro- sis robert koenekoop, ana luisa pina, malgali loyer, joe davidson, johane robitaille, irene maumenee , joyce tombran-tink mcgill ocular genetics laboratory, montreal children’s hospital research institute, montreal, canada; johns hopkins center for hereditary eye diseases, baltimore, md; laboratory of retinal cell and molecular biology, national eye institute, national institutes of health, bethesda, md purpose: leber congenital amaurosis (lca) has been mapped to chromosome p . . from the candidate genes mapped to this region, thus far, only retinal guanylate cyclase (retgc), has been found to have pathogenic lca mutations, in families from north african origin. however, early reports, demonstrated eight lca families linked to p . , but only four of them showed mutations in retgc. mapped in proximity to this locus is the candidate gene pigment epithelium derived factor (pedf), a factor implicated in photoreceptor differentiation and neuronal survival. our purpose in this study was to identify mutations and polymorphisms in the pedf gene in lca patients of diverse ethnic origin. methods: automated genotyping with four p . markers flanking the pedf gene was performed to assess homozy- gosity and pcr-sscp combined with direct sequencing was used to detect mutations in the pedf gene in lca patients. results: homozygosity of markers d s and d s was found and four new intragenic basepair alterations were discovered: a met thr polymorphism in exon (t c), a thr thr polymorphism in exon (t c), a g to a transition in intron (nine base pairs upstream from splice acceptor site), and a tyr tyr polymorphism in exon (c t) were detected. conclusions: we report the discovery of four new polymorphic alterations in the pedf gene in lca patients and exclude by rflp analysis the pedf gene as a common cause of leber congenital amaurosis. these single nucleotide polymor- phisms will aid in future linkage analysis of complex multifactorial diseases involving retinal and rpe dysfunctions. previously reported [ , ]. pcr-sscp was performed to de- tect mutations, and automated genotyping was done with microsatellite markers to detect homozygosity. for all pcr- sscp, our µl amplification mixture contained ng of genomic dna, mm tris-hcl (ph . ), mm kcl, . mm mgcl , . % (w/v) gelatin, . µm of each dntp (dctp, dgtp, dttp), . µm datp, . u taq polymerase (gibco-brl, burlington on), ng/ µl of each intronic primer (described in table ) and α- s-datp ( . mci/ml, nen, boston ma) as a tracer. denaturation at °c for s, was followed by annealing at primer specific temperatures (see table ) for s, and elongation at °c for s in a dna thermocycler (perkin-elmer, oakville on) for a total of cycles. pcr products were run on % polyacrylamide gels with % or % glycerol. all abnormal sscp variants were further analyzed by direct sequencing using dideoxy termina- tion kits and α- s-datp (sequenase pcr product sequenc- ing kit, us biochemicals, cleveland oh). every change in the dna was then confirmed by a restriction diagnostic di- gest. nonradioactive pcrs were performed with the same conditions as before, including . mm dntp. for restriction digestion of exon , units of maeiii (boehringer mannheim, indianapolis in) were used and the digest products were run on a % polyacrylamide gel. automated genotyping using fluorescein labelled microsatellite markers selected from the whitehead genome data base flanking the pedf gene were run on the dna sequencer (applied biosystems, foster city ca). allele analysis was performed using the genescan and genotyper (applied biosystems) pro- grams. normal controls consisted of children (equal sex ratio) from the province of quebec ( chromosomes). al- lele frequencies are noted per chromosome. results we established homozygosity for p alleles in one greek lca proband for markers d s and d s but not d s or d s (data not shown). we also found four new intragenic base pair alterations, namely a met thr poly- morphism in exon (t c), a thr thr polymorphism in exon (t c), a g to a transition in intron (nine base pair upstream from splice acceptor site), and a tyr tyr poly- morphism in exon (c t). alterations in restriction sites are listed in table . normal controls were checked for the presence of each base pair change by restriction digestion. the © molecular visionmolecular vision ; : <http://www.molvis.org/molvis/v /p > primer sequences fragment length t-ann ---------------------------- --------------- ----- exon '-atcacagaatccaaattgagtgcag bp °c '-tcttttctgctccctggagtgcccc exon '-gaaggggtagcggggcagtgcagtg bp °c '-gttccctgtcctgtttcccgtgctg exon '-gacagtccctgtgcatctctgcgag bp °c '-tcagccacgtttacgcagaggccac exon '-tgagtatagtgtctgtgttctggga bp °c '-aagacccccccagcctgcagcatgg exon '-gctctcaaagacgggatgcttgtcg bp °c '-agggctacagagtaagactccatcg exon '-cagcatggcgccactgtctttctgg bp °c '-taccctgttttgcttctctatcctc exon '-ggaaggcagctcctggctgtgtctg bp °c '-cacagtggaaggcccaaagaccctg exon '-atcccagcttgcttgcaaagggatc bp °c '-tgaaaccttacaggggcagccttcg table . intronic primer sequences are shown with location, pcr fragment length, and the annealing temperature (t-ann) figure . example of a restriction diagnostic digest for the tyr tyr polymorphism. the more frequent human allele has maeiii sites that generate bands of , and bp (a bp fragment cannot be seen) as shown in the first and third lanes from left to right. a ho- mozygote for the tyr tyr polymorphism (with one maeiii site destroyed) would have bands of , and bp. a heterozygote has bands at , , , and bp (as shown in the second and fourth lanes on the gel image). an example from a greek lca pedi- gree shows that one affected sib is heterozygous for the polymor- phism (second lane), the other has two copies of the common allele (third lane). lanes in the image correspond to each of the family members of the pedigree (lane one, the father; lane two, affected sib number ; lane three, affected sib number , and lane four, the mother). the low molecular weight band in each lane is compatible with primer dimer formation. exon polymorphism enzyme frequency frequency in lca patients in normals ---- ------------ --------- --------------- ------------ met thr bsssi / ( %) / ( %) t c created thr thr bsteii / ( %) / ( %) t c created g+ a bstui / ( %) / ( . %) intronic destroyed tyr tyr maeiii / ( . %) / ( %) c t destroyed table . four new polymorphisms with their respective exons, created or destroyed restriction enzyme sites and relative frequencies. frequency of each polymorphism is shown in table . pedi- gree analysis with the new rflps excluded pedf as the caus- ative gene in three of five families. one family is shown in figure . the families excluded using the same polymorphism (data not shown) were from the middle east, another was an amish family from northern germany, and one family was from greece. linkage exclusion of the p . lca locus in the large amish family has also been reported by others [ ]. discussion early reports, on the molecular basis of lca, demonstrated its mapping to p . and its genetic heterogeneity [ , ]. shortly after, perrault et al. [ ], described lca , caused by mutations in the retgc gene. they analyzed eight families linked to p and demonstrated that only four of them have mutations in the retgc gene. thus, the possibility exists that more than one gene in the same region can cause lca. in this report we explored the possibility that pedf, a gene that may promote photoreceptor differentiation and neuronal survival, can cause lca in families of diverse ethnic origin. our results here excluded the pedf gene as a common cause for leber congenital amaurosis [ ]. however, pedf remains an important candidate gene for inherited retinal dis- eases that map to chromosome p [ ] such as, autosomal dominant retinitis pigmentosa [ - ], autosomal dominant cone-rod dystrophy [ , ], and central areolar choroidal dys- trophy [ ]. for this group of diseases, our four newly de- scribed single nucleotide polymorphisms will aid in future link- age analyses. evidence that pedf may have an effect on the differentiation of rods and cones is demonstrated by experi- ments that show differentiation of retinoblastoma cells into neuronal-like cells [ ]. this suggests that pedf may be a factor involved in a programmed series of events that includes both proliferation and commitment to a final differentiated neuronal phenotype [ ]. further exploration towards the elu- cidation of the role of pedf in normal and pathological reti- nal development remains to be done. acknowledgements this study was supported by the e. mildred kanigsberg es- tate fund and an frsq award ( - ) to rk and a re- tinitis pigmentosa foundation fellowship to alp. references . leber t. uber retinitis pigmentosa und angeborene amaurose. archiv für ophthalmologie ; : - . . franceschetti a. l’importance diagnostique de l’erg dans les degenerescences tapeto-retiniennes avec retrecissement du champ visuel et hemeralopie. confin neurol ; : - . . schappert-kimmijster j, henkes he, van den bosch j. amaurosis congenita (leber). arch ophthalmol ; : . . 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on chromosome q . hum genet : - . . camuzat a, dollfus h, rozet jm, gerber s, bonneau d, bonnemaison m, briard ml, dufier jl, ghazi i, leowski c, et al. a gene for leber’s congenital amaurosis maps to chromo- some p. hum mol genet ; : - . . camuzat a, rozet jm, dollfus h, gerber s, perrault i, weissenbach j, munnich a, kaplan j. evidence of genetic het- erogeneity of leber’s congenital amaurosis (lca) and map- ping of lca to chromosome p . hum genet ; : - . . tombran-tink j, pawar h, swaroop a, rodriguez i, chader gj. localization of the gene for pigment epithelium-derived factor (pedf) to chromosome p . and expression in cultured human retinoblastoma cells. genomics ; : - . © molecular visionmolecular vision ; : <http://www.molvis.org/molvis/v /p > © molecular visionmolecular vision ; : <http://www.molvis.org/molvis/v /p > . steele fr, chader gj, johnson lv, tombran-tink j. pigment epithelium-derived factor: neurotrophic activity and identifica- tion as a member of the serine protease inhibitor gene family. proc natl acad sci u s a ; : - . . schroeder r, mets mb, maumenee ih. leber’s congenital amau- rosis. retrospective review of cases and a new fundus find- ing in two cases. arch ophthalmol ; : - . . robitaille jm, palincsar lk, zhu d, maumenee-hussels ie. link- age analysis in an amish family with leber’s congenital amau- rosis. invest ophthalmol vis sci ; :s . . davidson jk, tink j, loyer m, traboulsi e, maumenee i, koenekoop rk. pigment epithelium derived factor as a candi- date gene for leber’s congenital amaurosis. invest ophthalmol vis sci ; :s . . joshi ar, mullen l, small kw. the retina: genetic studies of several retinopathies located on the short arm of chromosome . curr opin neurol ; : - . . greenberg j, goliath r, beighton p, ramesar r. a new locus for autosomal dominant retinitis pigmentosa on the short arm of cromosome . hum mol genet ; : - . . goliath r, tombran-tink j, rodriquez ir, chader g, ramesar r, greenberg j. the gene for pedf, a retinal 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september of , the french government sent some roma (gypsies) from bulgaria and romania back to their home countries. it was alleged that they engaged extensively in begging and petty theft (pick-pocketing) (economist ). some european union (eu) commissioners complained bitterly that this action violated the roma’s rights to travel freely within the eu, and most commentators outside of france agreed with these commissioners (see, e.g., goldston ). no one seemed to question the proposition that eu citizens ought to have the right to locate anywhere within the eu. in this article, i call this presumption into question. more generally, i challenge the now widely held presumption, which underlies debates like these over rights, that there exists some set of universal human rights of which free movement is a member. my argument builds on a theory of constitutional rights, which sees them being defined by citizens seeking to maximize their expected utility under the constitution. the theory is briefly reviewed in section ii. a key feature of constitutional rights, as i define them, is that they are relative to the preferences and expectations of the citizens writing the constitution, which in turn are likely to depend on the history and culture of the community. this makes constitutional rights quite different from the universal human rights many claim exist. an example of why some communities will choose different sets of rights is given in section iii. in section iv, i examine the bill of rights in the u.s. constitution (u. s. const.) and show that most of these rights have characteristics which make it likely that they would have been chosen – at the time the us constitution was ratified – under the conditions postulated by my theory. in sections v and vi, i critically examine the un’s universal declaration of human rights (un general assembly ) and the eu’s charter of fundamental rights (european * professor emeritus, universtiy of vienna, hohenstaufengasse , vienna, austria. dennis.mueller@ univie.ac.at . the more familiar and historic term is “gypsy,” although this is regarded by some people as pejorative. i use the term most often seen in recent newspaper articles. romani is still another option. kyklos, vol. – november – no. , – © blackwell publishing ltd., garsington road, oxford ox dq, uk and main street, malden, ma , usa union ). as their names imply, each of these would seem to be a candidate for the definitive set of universal human rights. i argue otherwise. i also show that all lists of universal and fundamental rights such as these are inherently relative. in section vii, i take up the issue of whether a right to free movement within the eu is necessarily a good thing. in addition to the issue of the roma, i look at the riviera, and access to universities and health care. in each case i show how free movement can produce negative externalities and result in outcomes that are not pareto optimal. these sub-optimal outcomes are particularly problematic, because the actions causing them are protected as rights, thus making it extremely difficult to eliminate the externalities. the final section draws some conclusions. chief among these is that the definition of any enforceable consti- tutional right should be made by weighing the benefits to the actor protected by the right against any costs this action may impose on others. universal human rights, as they are usually propounded, generally fail this test by ignoring the external costs of the protected actions on others. many who claim the existence of human rights use them to make moral arguments. humans are said to possess a right to a decent standard of living and this, it is argued, creates a moral obligation for the rich to give to the poor, or for rich countries to make transfers to poor ones. no moral claims are made for the kinds of constitutional rights described in this article. for example, article vi of the u.s. bill of rights provides individuals accused of a crime with a right to a trial by jury. such a right can be justified using the theory presented here. it cannot be argued from this, however, that it is immoral for a country to allow judges to determine guilt or innocence instead of citizen juries. although i make no moral claims, my theory can be regarded as normative in that it assumes that individuals define certain rights as part of a constitution designed to advance their collective interests. if one thinks that this is what constitutions and, more generally, political institutions ought to do, then the theory provides a framework for making normative judgments about constitutions. ii. a theory of constitutional rights i have developed my theory of constitutional rights elsewhere, and so only briefly describe its main features here (mueller, , , ch. ). as in buchanan and tullock ( ) a polity’s constitution is assumed to be written by the citizens themselves under conditions of uncertainty about their future positions under the constitution. when choosing a voting rule for a particular category of decisions, the constitution writers weigh the future utility gains of the winners, if an issue passes, against the utility losses to the losers. the larger the losses to the losers . although buchanan and tullock did not discuss rights in their pioneering treatise on constitutions, buchanan ( ) has more recently stressed their importance for a liberal democracy. dennis c. mueller © blackwell publishing ltd. are relative to the gains to the winners, the higher is the required majority that maximizes the expected utility of a participant at the constitutional convention. in the extreme case of huge expected losses, if one is on the losing side, relative to the gains to the winners, the constitution framers will protect future citizens from these huge losses by requiring unanimous agreement. bans on religious practices could be an example of a category of decisions which might be assigned the unanimity rule in the constitution, if participants at the constitu- tional stage envisaged great utility gains for people who are allowed to practice their religions and small external costs on the rest of the community from such practices. unanimous agreement to ban a religious practice could only be achieved if members of the religion in question could somehow be convinced, or bribed, to vote for the ban. if, however, the constitution framers were correct about the relative gains and losses from such a ban, it is unlikely that members of a religious group would be convinced to abandon one of their religious practices, or that an offered bribe would be large enough. realizing this, the constitution writers can avoid long and fruitless debates over bans of religious practices by defining a right to practice one’s religion in the constitution, instead of requiring unanimity for bans of religious practices. thus, under my theory constitutional rights arise as low transaction cost substitutes for the unanimity rule, and are defined only for collective decisions where the costs from being on the losing side of a vote are thought to be extremely high relative to any gains for the winners. in developing their theory of the optimal voting rule, buchanan and tullock ( , pp. – ) justified the assumption of uncertainty over future positions by pointing to the long-run nature of constitutional choices. thus, to the extent that the uncertainty at the constitutional stage which produces unanimous agreement is real, their theory of the optimal voting rule, and by implication my theory of constitutional rights, might be regarded as positive theories with predictive value. in section iv, i argue that most of the rights defined in the u.s. bill of rights can be defended by this theory. on the other hand, buchanan and tullock ( , pp. , ) clearly state that theirs is a normative theory. such an interpretation might be further advanced by assuming that the uncertainty at the constitutional stage arises because individuals consciously step behind a veil of ignorance of the kind rawls ( ) used to derive a theory of justice. rawls, however, wished to offer his account of justice as an alternative to utilitarianism, and thus assumed a veil of ignorance so thick that utility information could not penetrate. the implicit veil in the theory of buchanan and tullock is not so thick, as individuals at the constitutional stage can envisage decision-making and external costs, presum- ably measured subjectively in units of utility, associated with future collective decisions. this makes both their theory and mine utilitarian in the sense that they rely on utility information. neither theory is utilitarian, however, in the classic rights, religion, riviera, and roma © blackwell publishing ltd. sense of assuming that the goal of society is to maximize a social welfare function containing individual utilities. three features of constitutional rights under this theory need to be noted. first, explicit rights will be defined only for actions capable of generating sufficiently strong negative externalities to elicit efforts by some members of the community to restrict them. even actions that provide considerable benefits for the actor need not be protected if they are not expected to be challenged. second, there is an inherent tension between constitutional rights and the normative principles justifying majoritarian democracy. when explicit rights and the simple majority rule both exist in a constitution to deal with situations where individual interests conflict, these situations should differ dramatically in the perceived losses imposed on the different sides from curtailing the action. the simple majority rule is optimal for resolving a negative externality, when an individual at the constitutional stage expects the utility gain from undertaking the action to equal the loss the externality causes. rights are defined precisely where the simple majority rule is not optimal, because the expected gains and losses from a ban are dramatically different, and the constitution framers wish to preclude its use. because rights will be defined only when significant losses are expected for those prevented from acting relative to the losses imposed on others, disputes over rights are likely to be emotionally charged, as they pit a perhaps substantial majority that feels harmed by the action against an intense minority that benefits from it. third, constitutional rights in this theory are inherently relative, and thus differ from definitions, which see rights as absolute in some meaningful sense. in a community where everyone is a member of the same religion, it may not occur to anyone that someone would ever challenge practicing this religion, and the constitution may, therefore, not explicitly protect religious freedom. actions explicitly protected as constitutional rights should vary across communities depending upon their particular histories, the preferences (values) of their citi- zens, and their expectations of the future benefits and costs from the actions. the next section provides an illustration. iii. the relative nature of constitutional rights: an example habeas corpus protection is found in many liberal constitutions. it can be justified by my theory of constitutional rights under the assumption that the loss in utility to someone imprisoned is very large, and the costs on the rest of the community from letting this person go free if she has not been charged and convicted of a crime are rather modest. now consider the country libertystan. its constitution contains strong habeas corpus protection. ten years ago, donald faith founded a religious group dedicated to the worship of ba’al of tyre, an dennis c. mueller © blackwell publishing ltd. ancient phoenician god. the group seeks to replace the democratic institutions of libertystan with a theocracy devoted to the worship of ba’al. seven years ago, three baalists were convicted of blowing up a bus and killing people. no evidence could be found implicating other members of the sect, which then numbered . five and a half years ago, a bomb went off in the capital, killing people. police suspected four baalists, but could not gather enough evidence to convict them. a year later, the same four baalists were charged and convicted of blowing up a train killing people. once again, no evidence was found implicating other members of the sect, which now numbered over . three years ago baalist suicide bombers entered libertystan’s parliament and killed people, including over half of the members of parliament. as these events unfold, libertystanians may begin to rethink constitutional habeas corpus protection. waiting until a baalist commits a crime and then arresting him or her has proved rather costly. indeed, for suicide bombers, the threat of capture is inoperative. if the baalists and other libertystanians stepped behind a veil of ignorance, they might unanimously agree to allow preventive incarceration, when the state perceives a grave threat to the community from a particular group. even a right as seemingly basic as habeas corpus protection may not be chosen by a community that is maximizing its expected utility if it anticipates that some members may pose significant threats to its welfare. here we might consider the dilemma faced by the united states with respect to the people imprisoned at guantanamo. that the united states mistreated some prisoners in violation of its own rules seems indisputable. similarly, it seems to have imprisoned some people who did not pose a threat to the country. but, unless we assume that the cia and american military are totally incompetent, some prisoners are bent on attacking the united states should they be released. freeing all prisoners and waiting to see which ones carry out attacks is a risky strategy. iv. the bill of rights as discussed above, uncertainty at the constitutional stage might be thought of as real due to the long-run nature of constitutional choices, or artificially imposed by persons who adopt a kind of veil of ignorance as proposed by rawls ( ). it is reasonable to assume that individuals are more likely to adopt a rawlsian frame of mind when making the kind of basic choices for a community that are involved when writing a constitution, and that agreement on the constitution is reached for this reason. the theory might thus be used to predict what sorts of actions will receive rights protection in constitutions. in this section, i illustrate that the bill of . let b be the loss inutility to a baalist from being imprisoned, l the gain to a non-baalist from the reduced threat of death, and a the fraction of the population that is baalist. then the incarceration of baalists would be unanimously approved from behind a veil of ignorance if ab < ( -a)l, which is equivalent to a(b + l) < l. with sufficiently small a, and l being large relative to b, this condition will be satisfied. rights, religion, riviera, and roma © blackwell publishing ltd. rights appended to the u.s. constitution seems, at the time of the ratification at least, to have satisfied the utilitarian calculations underlying my theory. the first amendment congress shall make no law respecting an establishment of religion, or prohibiting the free exercise thereof; or abridging the freedom of speech, or of the press; or the right of the people peaceably to assemble, and to petition the government for a redress of grievances. (us const. amend i) many colonists migrated to america to escape religious persecution, and recognized that religious practices often come under attack. if americans at the end of the th century felt that the loss to someone prevented from practicing their religion would be very large relative to any externality this action caused, the conditions for defining a religious freedom article were met. free speech, free press and freedom of assembly are essential requisites for a healthy democracy. free speech can cause injury to others. article i of the bill of rights fits my three criteria for a constitutional right to a tee. the second amendment a well regulated militia, being necessary to the security of a free state, the right of the people to keep and bear arms, shall not be infringed. (us const. amend ii) section of article i of the main constitution limits congress’s power to support an army to two years. the gains from having each citizen armed and ready to join a militia would, at the time of the constitution’s ratification, have been large relative to any negative external effects from an armed citizenry. article ii illustrates the relative nature of constitutional rights. at the end of the th century, the united states was sparsely populated, and many citizens owned guns for hunting and protection. these conditions do not characterize the us at the beginning of the st century, and article ii today cannot be defended as a constitutional right. the third amendment no soldier shall, in time of peace be quartered in any house, without the consent of the owner, nor in time of war, but in a manner prescribed by law. (us const. amend iii) article iii is the first to reflect the injuries that the colonists suffered under british rule, and their desire to be protected from similar injuries under the new government. it and the articles that follow reflect the heavy weight at the founding placed on individual freedom, and the founders great suspicion of the state. this suspicion is perhaps most clearly evidenced in the fifth amendment, which forbids both trying a person for the same crime twice, and compelling him dennis c. mueller © blackwell publishing ltd. to testify against himself. to reconcile this amendment with my theory, one must assume that imprisonment imposes tremendous costs, and that allowing a person suspected of a crime to go free would produce relatively modest costs for the community. only under these assumptions can one justify denying the prosecu- tion the possibility to cross-examine someone accused of a crime, or retrying someone should new evidence be found that conclusively shows that this person was guilty. similar utility calculations can justify the sixth amendment, which introduces habeas corpus protection. a careful review of each of the first eight articles of the bill of rights would, i believe, reveal that most fit easily into my theory, when the constitution was ratified, under plausible assumptions about perceived costs and benefits from protecting certain individual freedoms. the ninth and tenth amendments are rather different from the first eight, and do not fit my theory as nicely. articles ii and iii and perhaps some additional elements in the bill of rights would not be included in a new constitution written today. the fact that they are still in force can be attributed to the difficulty of amending the u.s. constitution created by the procedures specified in the original document. thomas jefferson ( ) felt that each generation should be allowed to write its own constitution and not be forced “to remain under the regimen of their barbarous ancestors,” and i am inclined to agree. ideally, a constitution should contain provisions for periodic reviews, so that its provisions reflect the preferences and environment of the current generation. v. the universal declartion of human rights on december , , the general assembly of the united nations adopted and proclaimed the universal declaration of human rights (un general assembly ). as its name implies, the declaration presumes that a set of human rights exists, and that they are universal. scholars making similar claims often cite the un declaration to justify their positions (e.g., pogge ). this in itself is rather strange, since on most issues, the un general assembly would not generally be regarded as a moral authority. its members today include zimbabwe, myanmar (burma) and cuba, and in included stalin’s soviet union. given the frequency with which it is cited, however, the declaration deserves a closer look in light of the arguments put forward here. the declaration contains articles, many of which have multiple parts. several involve economic entitlements, and thus raise the question of who will fulfill them in poor countries. others are vague. for example, article pro- claims a right to “rest and leisure.” i shall, however, focus only on one article that is not vague. part of article asserts, “parents have a prior right to choose the . for further discussion, see mueller ( , - , - ). rights, religion, riviera, and roma © blackwell publishing ltd. kind of education that shall be given to their children.” by “prior” i assume it means prior to the state. such a right was effectively granted to the parents of amish children by the us supreme court. wisconsin had a law requiring school attendance through age . the amish removed their children after the th grade to protect the amish way of life. chief justice berger’s majority opinion defended the right of amish parents to withdraw their children from secondary schools with the following argument. broadly speaking, the old order amish religion pervades and determines the entire mode of life of its adherents. their conduct is regulated in great detail by the ordnung, or rules, of the church community . . . amish objection to formal education beyond the eighth grade is firmly grounded in these central religious concepts. they object to the high school and higher education generally, because the values they teach are in marked variance with amish values and the amish way of life; they view secondary education as an impermissible exposure of their children to a “worldly” influence in conflict with their beliefs. the high school tends to emphasize intellectual and scientific accomplishments, self-distinction, competitiveness, worldly success, and social life with other students. amish society emphasizes informal “learning through doing;” a life of “goodness,” rather than a life of intellect; wisdom, rather than technical knowledge; community welfare, rather than competition; and separation from rather than integration with, contemporary worldly society. (justia: wisconsin vs. yoder ) in finding for the amish community, the supreme court chose to protect parents’ freedom to determine their children’s life plans over the children’s freedom to choose their own life plans. should an amish girl at age wish to follow a life plan that includes intellectual and scientific accomplishments, self-distinction, competitiveness, worldly success, and a social life with people outside of the amish society, this option would be largely precluded by the parent’s decision to deny her the education necessary to follow this path. under my theory, a constitution would protect the right of parents to choose their children’s education, including no education, if the benefits to the parents were very great and the costs to the rest of the community quite small. the benefits to the amish parents are the psychological satisfaction of knowing their children are highly likely to follow the amish way of life. the law exists, as berger later concedes, because wisconsin citizens benefit from their fellow citizens having good educations. the largest external costs of amish parents withdrawing their children from school, however, fall on the children them- selves. their life choices are severely constrained by their parents. a liberal community which weighed the benefits to both parents and children might well decide not to grant parents the right to deny their children an education. if human rights exist and are universal in the sense that everyone possesses the same set of rights, then everyone should agree about these rights once they are explained. why would anyone deny the existence of a right that he or she possesses? i, and i am sure others, would not grant parents the right to deny their . see the dissent of justice william o. douglas in wisconsin v. yoder at findlaw (http:// caselaw.lp.findlaw.com/cgi-bin/getcase.pl?court=us&vol= &invol= , accessed . . ). dennis c. mueller © blackwell publishing ltd. children an education. unanimous acceptance of other articles of the declaration also seems highly unlikely. if the rights contained in the declaration are not universally recognized as rights, how can we be certain that they are indeed universal in the sense that we all possess the same rights? vi. the charter of fundamental rights the eu’s charter of fundamental rights (european union ) came into full effect with the lisbon treaty on december , (european union ). although the charter does not claim to be a set of universal rights, the word “fundamental” in the title suggests that the drafters had something like the un’s declaration in mind. indeed, many rights in the charter have similar wording to those in the declaration. while the un declaration has only articles, the charter has . many of the charter’s articles are also vague with respect to the nature of the protection offered. some imply economic entitlements (health care, education) without specifying how they are to be fulfilled. others are totally vacuous. many would be difficult to rationalize under the kind of utilitarian calculus underlying my theory of constitutional rights. some illustrations follow. article . political parties at union level contribute to expressing the political will of the citizens of the union. (european union ) in what sense is this a right protecting a specific freedom or action? article . the union recognizes and respects the rights of the elderly to lead a life of dignity and independence and to participate in social and cultural life. (european union ) does this article seek to prevent the union from passing a law prohibiting the elderly from participating in social and cultural life? alternatively, is it implying that the community has an obligation to facilitate such participation by the elderly by providing free transportation to social events, free tickets and the like? if the first is meant, then the article would seem to rest on a rather dubious premise that the union is likely to try to prevent the elderly from participating in its social and cultural life. if the second is implied, then the article contains an undefined and seemingly open-ended financial commitment to the elderly. article contains similar ambiguities with respect to “persons with disabilities.” when i drive into italy, i anticipate being subject to italy’s laws and rules of the road. now imagine an article in the eu constitution that would read as follows: article s. drivers have, in accordance with community law and national laws and practices, the right to drive at any speed that they choose. rights, religion, riviera, and roma © blackwell publishing ltd. what would such an article imply? at first glance it seems to create a right to drive at any speed across the entire union. a closer reading reveals that when i drive in italy i am subject to its rules of the road. but that is the case already. the article has changed nothing and as such is totally vacuous. consider now article of the charter. article . workers and employers, or their respective organizations, have, in accordance with community law and national laws and practices, the right to negotiate and conclude collective agreements at the appropriate levels and, in cases of conflicts of interest, to take collective action to defend their interests, including strike action. (european union ) a literal interpretation of article , like that of article s, implies that workers and employers are subject to national laws and practices. the article changes nothing. why then did the charter’s drafters write such a vacuous article? an alternative interpretation is that the drafters intended to enshrine certain workers’ rights, including the right to strike, in the eu constitution. if this was the intent, then article is no longer vacuous, but rather pernicious. an action should be protected as a right only when the loss to a person prevented from acting is very large relative to any negative externalities caused by the action. the most powerful unions in europe today are public sector unions. strikes by their members, air traffic controllers, railroad workers, fire- fighters, and teachers, often impose heavy costs on the community for seemingly modest benefits for the strikers – higher wages, shorter hours, or early pensions. the relative utility payoffs to justify a right are reversed; the actors’ gains are modest and the costs imposed on others are large. indeed, other rights in the charter, such as article , which guarantees that “the family shall enjoy legal, economic and social protection” (european union ) would seem to preclude strikes by workers who provide essential services. thus, under either interpre- tation of article , it has no place in the eu’s constitution. several articles are so vague that the rights proclaimed are largely meaning- less. article , for example, ensures “a high level of human health protection.” articles and ensure “high levels” of environmental and consumer protec- tion. presumably ensuring high consumer protection would involve a vigorous competition policy. but is the loss to a consumer from a cartel always very large relative to the benefits to the rest of the community, including cartel members? what about workers’ cartels (unions), farmers? the eu should have a strong competition policy, but this should be governed by ordinary legislation and not treated as an outgrowth of some “consumer rights.” the rights in the charter to high levels of health care, education, environmen- tal protection and the like would seem to require state funding for these activities. yet, article . explicitly precludes the eu from providing such funding. article . this charter does not establish any new power, or task for the community or the union, or modify powers and tasks defined by the treaties. (european union ) dennis c. mueller © blackwell publishing ltd. but where then do the resources come from to provide these high levels of public services? enough examples have been presented to indicate that many articles in the charter cannot be defended using the theory of constitutional rights presented above nor, i would argue, by any other criterion for constitutional rights. thus, as a list of fundamental human rights with some claim to universality, the charter, like the un declaration, is sorely lacking. one might argue that the charter is not meant to be a set of rights defensible in court, but merely a statement of european values. europeans value the elderly, children, families, consumers, the environment, and so on. this interpretation runs into the difficulty that many rights in the charter, such as freedom of speech and assembly, are enforceable in court. moreover, in some cases the european commission is using them to shape policies like those dealing with the free movement of individuals. vii. the right to free movement the problematic nature of defining rights without considering the externalities caused by the protected actions is illustrated by the rights of eu citizens to free movement. . the riviera article . of the lisbon treaty states that “the union shall offer its citizens an area of freedom . . . in which the free movement of persons is insured . . .” (european union ) this right in restated in the charter of fundamental rights. article . . every citizen of the union has the right to move and reside freely within the territory of the member states. (european union ) these rights presumably allow all eu citizens to buy property and live anywhere in the eu. the riviera is an attractive place to live. over time we can expect eu citizens from less desirable parts of europe to migrate to the riviera. two possible consequences follow. ( ) the stock of accommodation on the riviera expands to meet demand, and the expanding population causes overcrowding on the beaches, traffic congestion, air and water pollution, and the other ill-effects of overpopulation. under this option, migration can be expected to continue until the quality of life on the riviera sinks, due to overcrowding, to the levels in the least desirable parts of europe. ( ) the stock of accommodation is constrained . for further discussion of the externalities associated with migration, see mueller ( , ch. ). rights, religion, riviera, and roma © blackwell publishing ltd. to avoid overcrowding. the price of housing rises due to excess demand. since there are more wealthy europeans than wealthy french, the french tend to lose out in bidding for housing, and the riviera comes to be populated by a cross- section of wealthy europeans. southern california is an example of the first scenario. blessed with a mod- erate climate on the pacific ocean, it has drawn migrants from all parts of the us. today, traffic congestion is so bad that the average commuter spends hours a year (the equivalent of -hour days) just driving to and from work. water is in short supply, and air pollution can be severe (diamond ). venice serves as an example of the second scenario. much of the housing in the canal area is now devoted to hotels or owned by people from other parts of europe. the first scenario is clearly not optimal. both it and the second scenario could be avoided by restricting housing on the riviera to french citizens. but this would violate the eu’s right of free movement. from a european perspective, one might welcome the second scenario. why should the french have a greater right to live on the riviera than other europeans, or the italians have a greater right to live in venice? if many french and italians think they should have such rights, however, resentment of the eu is likely to rise over time. . universities in its effort to advance european integration and citizen mobility, the eu com- mission requires that all eu universities have the same entrance requirements and charge the same fees to students from other eu countries as apply for domestic students. to see the potential difficulty of this policy, consider what the consequences would be if it were implemented in the united states. public universities in a few states, such as california, michigan, and wisconsin, are among the best in the world. in most other states this is not the case. every state university charges tuition to its residents that is far below the costs of educating a student. non-residents are charged much higher tuition. if the supreme court were to rule that this discrimination was unconstitutional, the immediate conse- quence would be a flood of applications for admission to berkeley, ucla, michigan, wisconsin and the other top public universities. similar to the riviera example, two consequences are possible. budgets remain fixed and enrollments expand to accommodate both in- and out-of-state applicants, and the quality of education at top-ranked universities sinks. alternatively, enrollments are kept steady to maintain quality, and taxpayers in states with high-quality universities find themselves increasingly subsidizing the education of students from other states. mobility across universities in europe would be unproblematic, if it were balanced. greek students learn spanish and study in spain, spanish students study in denmark, and danes in hungary. the flow of students across the eu is dennis c. mueller © blackwell publishing ltd. not balanced, however. english is the most common second language in non- english speaking countries, and some british universities are among the best in the world. more greeks travel to britain for an education than british traveling to greece. rising student numbers and budget cuts in recent years have threat- ened the quality and even the very survival of many british universities. in response, the british government announced at the end of tuition increases of up to percent, a change that precipitated large and often violent protests. the fees british students must pay for their education could be kept lower, if students from other eu countries were charged higher fees. these students presently make up only five percent of university enrollment in the uk, but their enrollments are increasing at a faster rate than for uk students (higher educa- tion statistics agency). at some point in time, the wrath of british students may be re-channeled toward the eu. imbalances are also possible between french-speaking belgium and france, dutch-speaking belgium and the netherlands, and germany and austria. german universities have entrance exams. austrian universities have tradition- ally not had them. austria limited german enrollments by requiring germans to prove that they had passed the entry requirements of a german university. the eu ruled that this discriminated against the german students, violating their rights to free mobility within the eu. as a consequence, german enrollments at austrian universities have increased greatly, producing a steep decline in their quality. at the university of vienna, for example, enrollment surged by percent, while its position in the qs world university rankings fell from th in to rd in (qs topuniversities). its london times ranking fell from th in to th in (studium.at). . healthcare every resident of britain has access to its free, universal healthcare. someone with an independent income, say a retiree, who lives in an eu country with poor healthcare might easily improve his welfare by moving to britain and taking advantage of its free healthcare. in july of , the commission issued a directive to facilitate “medical tourism” and in january of the eu parlia- ment passed legislation that endorsed and modified this directive (health tourism, international medical travel journal). should large numbers of euro- peans choose to migrate in search of better medical care, the same two possible scenarios will arise with respect to health care as for university education: quality . a report by the university of college union, released in december , predicted that as many as a third of british universities might close due to inadequate funding. this report did not take into account the recent tuition hikes, however. see academica group http://www.academicagroup.ca/top /stories/ (accessed . . ). rights, religion, riviera, and roma © blackwell publishing ltd. deteriorates in countries with high quality health systems, or their taxpayers increasingly subsidize the medical costs of other europeans. . roma many roma are educated, hold good jobs and lead lives that do not differ significantly from their non-roma neighbors. some, however, are poorly edu- cated, unemployed, and live in what can only be called encampments that are overcrowded and unsightly. these roma often resort to begging or petty crime to earn a living. it is the migration and expulsion of roma such as these, which caused a furor in france in , and still earlier in italy. to justify a right for the roma to migrate, under my theory two conditions must be met: the benefits to the roma from migrating must be great, and the costs imposed on other citizens from their migration must be small. that the roma benefit from moving from romania and bulgaria to richer parts of the eu is obvious. they would not move if it were not the case. it is also obvious that some citizens in the recipient countries sense that they have been made worse off. are the gains to the roma sufficiently large to offset the costs on the others? i leave it to the reader to answer this question. . discussion in a world of high mobility, people migrate from locations with poor amenities to places with attractive amenities. attractive amenities created by nature tend to be limited in supply, and free migration either leads to overcrowding and a deterioration of the amenities’ quality, or policies to avoid overcrowding, which result in the displacement of local beneficiaries by migrants. when the amenities are manmade, such as universities and hospitals, supply can be increased, but at a cost. if the citizens in a country with high-quality institutions wish to maintain their quality, their taxes will rise to subsidize the consumption of migrants. if they fail to raise taxes, quality will deteriorate. the eu’s right to free movement resembles other rights many deem to be “fundamental” or “universal.” an action or, in the case of entitlements, a transfer, which is perceived to have great benefits for the actor/recipient, is justified by claiming it is a fundamental (basic) human right. any external costs created by the action are ignored, or assumed to be negligible. when these costs are not negligible, a constitutional right cannot be justified. migration almost tautologically benefits the migrant, and often does not impose significant costs on the host country. a roma with an mba who takes a job in paris benefits herself and probably the citizens of paris and france as well. but all forms of migration are not so anodyne. when migration imposes signifi- dennis c. mueller © blackwell publishing ltd. cant costs on the recipient community, that community will naturally object to it. in ignoring the possible costs of migration or, as in the case of universities, thwarting efforts to avoid these, the eu commission risks alienating citizens in countries adversely affected by its forced integration policies. just as free speech and habeas corpus rights must optimally have exceptions and boundaries, so too must free movement rights. in its zeal to promote eu integration, the commis- sion is not only producing outcomes that are not pareto optimal; in the long run, it may be undermining citizen support for the union itself. viii. conclusions this article makes two claims, one general and one specific. the general claim is that any theory of rights which purports to delineate the rights citizens have and provide guidance as to what rights should go into a constitution should take into account both the benefits to the actor protected by a right and the potential costs of the action on others. the specific claim is that the right to free mobility embedded in eu treaties and the charter of fundamental rights cannot be justified as an unqualified right. the first claim is controversial and so, by implication, is the second. the philosopher richard brandt has made the following claim: virtually all philosophers now agree that human beings – and possibly the higher animals – have moral rights in some sense, both special rights against individuals to whom they stand in a special relation (such as a creditor’s right to collect from a debtor), and general rights, against everybody or against the government, just in virtue of their human nature. (brandt , ) brandt goes on to observe that “some philosophers also think, however, that anyone who is a utilitarian ought not to share this view” (ibid.). perhaps this explains why i favor a form of utilitarian theory of constitutional rights, and eschew the idea that people possess rights simply because they are humans, or are granted rights by nature. the main advantage of my utilitarian theory is that it gives some criteria for judging whether a particular claimed right would or should find its way into a constitution. slavery is often invoked in attacks on utilitarianism by its critics, because utilities could be assigned to slaves and slave owners to make slavery optimal under a utilitarian calculus. i argue just the opposite. interpersonal comparisons are always somewhat subjective, but most people would probably agree that the loss of utility to someone forced into slavery would far outweigh any utility gain to the slave owner. the problem with human or natural rights claims is that one has no benchmark for comparison. slaves and slave owners are both humans. the right to own property usually makes its way onto lists of . should progress continue in the measurement of happiness levels, an objective basis for making interpersonal comparisons may yet become available. see frey ( ). rights, religion, riviera, and roma © blackwell publishing ltd. human rights. slaves have traditionally been viewed as forms of property. how does one decide whether a human right to be free trumps a human right to own property without making utility comparisons? today it seems obvious that freedom trumps property, but this was not always so. a philosopher as astute as aristotle claimed that slavery was a “natural condition.” thomas jefferson and james madison each owned slaves, and they were not obviously lacking in ethical convictions. almost any action can be defended as a human or natural right, if one does not have to examine the likely gains and losses from it. this point is nicely illustrated by amartya sen’s “liberal paradox.” (sen , , ; see also mueller , ch. ). in his original illustration of the paradox, sen used the example of reading lady chatterley’s lover to create preferences for two people which could lead to an outcome that is not pareto optimal if each person exercises the liberal right to read or not read the book. such a right might be defended as part of a free-speech right and could easily meet the conditions set forth for a constitutional right by my theory. in subse- quent discussions of the liberal paradox, however, sen has used the examples of choosing the color of tiles in one’s bathroom, or whether one sleeps on one’s stomach or back. the protection of such actions by a constitutional right is difficult to justify using my theory. a law banning red tiles in bathrooms would be unlikely to cause a great loss of utility to those wishing to install red tiles, and it is highly doubtful that anyone would propose such a law. these additional examples serve to illustrate sen’s main argument, that almost any action might cause an externality and lead to an outcome which is not pareto optimal, but they also illustrate the disadvantage of defining rights without regard to the magni- tudes of their utility consequences, as advocates of human and natural rights generally do. thus, somewhat ironically, this broad latitude for defining human or natural rights makes these rights inherently relative. unlike the constitutional rights defined by my theory, however, which are relative to the preferences and per- ceptions of the citizens in the community, definitions of human or natural rights are relative to the values of the person or committee propounding them. this elastic nature of the human rights concept is illustrated by comparing the eu’s charter of fundamental rights written in , and its predecessor, the con- vention for the protection of human rights and fundamental freedoms written in . the convention is much shorter than the charter and the rights in it are easier to defend using utilitarian criteria. with the passage of time, more and more actions or entitlements benefitting some group of individuals are thought of, and the list of fundamental human rights grows. a new list of fundamental human rights written years from now will most certainly be longer than today’s charter. i have given several examples of negative externalities that can arise from free mobility. the undesirable effects of mobility can arise in any country, of course, dennis c. mueller © blackwell publishing ltd. and southern california is a parallel case to the riviera. there is, however, an important difference between the ill effects of mobility in the us and the eu. the us’s federalist institutions are defined by a constitution, which most ameri- cans accept as legitimate. this constitution protects the rights of states to adopt policies, which avoid some of the costs of high mobility, such as higher univer- sity tuition for out-of-state students. the eu has no constitution, and when it tried to adopt one, the voters of france and the netherlands rejected it. thus, eu political institutions do not possess the same degree of legitimacy as those in the us in the eyes of many eu citizens. the charter of fundamental rights came into force without eu citizens contributing to either its drafting or its adoption, and by and large without their knowledge of its content. the directives regarding mobility within the eu, which are imposing unwanted costs on citizens in some countries, are issued by unelected administrators in the eu commission. this democratic deficit in the eu, combined with the commission’s zeal to foster integration through enforcing rights to free movement, may eventually pose a threat to the union itself, as more and more eu citizens become aware of the costs they bear from these policies. references academica group (n.d.). http://www.academicagroup.ca/top /stories/ (accessed . . ). brandt, richard b. ( ). morality, utilitarianism, and rights, cambridge: cambridge university press. buchanan, james m. ( ). direct democracy, classical liberalism, and constitutional strategy, kyklos, : – . buchanan, james m. and gordon tullock ( ). the calculus of consent, ann arbor (mich.): university of michigan press. diamond, jared ( ). collapse, london: allen lane. economist, ( ). hard traveling. sept , , . european union ( ). treaty of lisbon amending the treaty on european union and the treaty establishing the european community, december , /c / . european union ( ). charter of fundamental rights of the european union, december , official journal of the european communities, december , /c / . findlaw (n.d.). http://caselaw.lp.findlaw.com/cgi-bin/getcase.pl?court=us&vol= &invol= (accessed . . ). frey, bruno ( ). happiness: a revolution in economics, cambridge (mass.): mit press. goldston, james a. ( ). roma and the e.u. international herald tribune, sep. http:// www.nytimes.com/ / / /opinion/ iht-edgoldston.html. (accessed . . ). health tourism (n.d.). eu directive on cross border healthcare boosts health tourism http:// treatmentabroad.blogspot.com/ / /eu-directive-on-cross-border-healthcare.html (accessed . . ). higher education statistics agency (n.d.). press release – students in higher education institu- tions / , http://www.hesa.ac.uk/index.php?option=com_content&task=view&id= & itemid= (accessed . . ). international medical travel journal (n.d.). european parliament passes eu cross-border healthcare directive european parliament passes eu cross-border healthcare directive, http://www.imtj.com/ news/?entryid = (accessed . . ). rights, religion, riviera, and roma © blackwell publishing ltd. jefferson, thomas ( , [ ]). letter to samuel kercheval, reprinted in a. koch and w. peden, eds., the life and selected writings of thomas jefferson, new york: modern library, – . justia us supreme court center (n.d.). wisconsin v. yoder ( ), http://supreme.justia.com/us/ / /case.html (accessed . . ). mueller, dennis c. ( ). constitutional rights, journal of law, economics, and organization, : – . mueller, dennis c. ( ). constitutional democracy, oxford: oxford university press. mueller, dennis c. ( ). public choice iii, cambridge: cambridge university press. pogge, thomas w. ( ). moral universalism and global economic justice, politics, philosophy & economics, : – . qs topuniversities (n.d.). world university rankings® / http://www.topuniversities.com/ university-rankings/world-university-rankings/home (accessed . . ). rawls, john ( ). a theory of justice, cambridge (mass.): belknap. sen, amartya k. ( ). the impossibility of a paretian liberal, journal of political economy, : – . sen, amartya k. ( ). minimal liberty, econometrica, : – . sen, amartya k. ( ). rights, formulation and consequences, analyse & kritik, : – . studium.at (n.d.). http://www.studium.at/ -uni-ranking-platzierte-uni-wien-auf-platz- (accessed . . ). u.s. const. amend. i u.s. const. amend. ii u.s. const. amend. iii summary the recent conflict between the french government and the eu over roma motivates a more general examination of the eu’s free mobility policies. the article describes three additional situations, access to land in desirable locations, access to universities, and access to health care, in which the eu’s policies with respect to mobility can lead to pareto sub-optimal outcomes. more generally, the article challenges the now widely held presumption, which underlies debates like these over rights, that there exists some set of universal human rights of which free movement is a member. a utilitarian theory of constitutional rights is offered instead. the article demonstrates that some constitutional rights, as those contained in the u.s. bill of rights, do fit the theory rather well. the un’s universal declaration of human rights, and the eu’s charter of fundamental rights are used as examples of universal human rights, which are difficult to defend under the theory proposed here, or under any other theory. dennis c. mueller © blackwell publishing ltd. science journals — aaas s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e h e a l t h a n d m e d i c i n e division of cardiology, department of medicine, northwestern university feinberg school of medicine, chicago, il , usa. feinberg cardiovascular research insti- tute, northwestern university feinberg school of medicine, chicago, il , usa. department of preventive medicine, northwestern university feinberg school of med- icine, chicago, il , usa. center for human development and aging, new jersey medical school, newark, nj , usa. school of kinesiology, university of british columbia, vancouver, british columbia, canada. indiana hemophilia and thrombosis center, indianapolis, in , usa. tohoku university graduate school of medicine, miyagi, japan. *these authors contributed equally to this work. †corresponding author. email: d-vaughan@northwestern.edu khan et al., sci. adv. ; :eaao november copyright © the authors, some rights reserved; exclusive licensee american association for the advancement of science. no claim to original u.s. government works. distributed under a creative commons attribution noncommercial license . (cc by-nc). d o w n lo a d e a null mutation in serpine protects against biological aging in humans sadiya s. khan, , , * sanjiv j. shah, , * ekaterina klyachko, , abigail s. baldridge, mesut eren, aaron t. place, abraham aviv, eli puterman, donald m. lloyd-jones, , meadow heiman, toshio miyata, sweta gupta, amy d. shapiro, douglas e. vaughan , † plasminogen activatorinhibitor– (pai- ) has beenshown to be akey component of the senescence-related secretome and a direct mediator of cellular senescence. in murine models of accelerated aging, genetic deficiency and targeted inhibition of pai- protect against aging-like pathology and prolong life span. however, the role of pai- in human longevity remains unclear. we hypothesized that a rare loss-of-function mutation in serpine (c. _ dupta), which encodes pai- , could play a role in longevity and metabolism in humans. we studied members of the berne amish community, which included carriers of the null serpine mutation. heterozygosity was associated with sig- nificantly longer leukocyte telomere length, lower fasting insulin levels, and lower prevalence of diabetes mellitus. in the extended amish kindred, carriers of the null serpine allele had a longer life span. our study indicates a causal effect of pai- on human longevity, which may be mediated by alterations in metabolism. our findings demonstrate the utility of studying loss-of-function mutations in populations with geographic and genetic isolation and shed light on a novel therapeutic target for aging. d fr o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / o m introduction age is the primary risk factor for most of the chronic diseases, including type diabetes mellitus (dm), metabolic syndrome, and cardiovascular diseases (cvd) ( ). the prevalence of age-related diseases has increased concordantly with the growing proportion of elderly individuals in the population. aging remains one of the most challenging biological pro- cesses to unravel, with coordinated and interrelated molecular and cellular changes ( ). humans exhibit clear differential trajectories of age-related decline on a cellular level with telomere attrition across var- ious somatic tissues and on a physiological level across multiple organ systems ( ). in addition to telomere length, lópez-otín and colleagues ( ) proposed several molecular drivers of aging, including genomic in- stability, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell ex- haustion, and altered intercellular communication. despite knowledge of these potential molecular causes of aging, no targeted interventions currently exist to delay the aging process and to promote healthy lon- gevity ( , ). in the united states, cardiometabolic disease influences life span as a leading cause of death and disability in adult men and women ( ). car- diometabolic disease is associated with a shorter leukocyte telomere length (ltl) ( , ). telomere shortening, which results from replication of somatic cells in vitro and in vivo, may cause replicative senescence. senescent cells and tissuesexhibit adistinctive pattern of protein expres- sion, including increased plasminogen activator inhibitor– (pai- ) as a part of the senescence-associated secretory phenotype (sasp) ( , ). pai- , which is encoded by the serpine gene, is the primary inhibitor of endogenous plasminogen activators and is synthesized in the liver and fat tissue. in addition to its role in regulating fibrinolysis, pai- also contributes directly to cellular senescence in vitro ( ). genetic absence or pharmacologic inhibition of pai- in murine models of accelerated aging provides protection from aging-like pathology, prevents telo- mere shortening, and prolongs life span ( ). cross-sectional human studies have demonstrated an association of plasma levels of pai- with insulin resistance ( , ). mendelian randomization analyses from large genome-wide association studies (gwas) provide an additional supportive evidence fora casual effect of pai- on insulin resistance and coronary heart disease ( ). the role of the sasp, in general, and specifically pai- in longevity in humans is uncertain. we have previously reported the identification of a rare frameshift mutation (c. _ dupta) in the serpine gene in the old order amish (ooa), living in relative geographic and ge- netic isolation in the vicinity of berne, indiana; this mutation results in a lifelong reduction in pai- levels ( , ). therefore, we tested the as- sociation of carrier status for the null serpine mutation with ltl as the prespecified primary end point in the only known cohort with a serpine null mutation. the secondary endpointsof the study includ- ed fasting insulin level, prevalence of dm, and life span. the findings of this study were extended by assessing the novel aging scores derived in the ooa cohort in a u.s. population–based cohort study, the coronary artery risk development in young adults study (cardia). results baseline characteristics of the study participants the clinical characteristics of the study participants by serpine geno- type status are shown in table and table s . a total of participants was enrolled. forty-three participants were identified as carriers of the null serpine mutation, and seven participants were identified as homozygous for the null serpine mutation with an overall minor (null) allele frequency of % in the study population. the observed serpine genotype frequencies were in hardy-weinberg equilibrium. heterozygous carriers of the null serpine mutation had % lower of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m mean circulating plasma pai- levels ( . ± . ng/ml versus . ± . ng/ml; p < . ) compared with unaffected participants. homo- zygous individuals for the null serpine mutation had no evidence of detectable pai- antigen in the plasma, consistent with the loss-of- function mutation. unaffectedamishparticipantshadlowerbody mass index, fasting glucose, and triglyceride levels than the cardia partici- pants sampled from the u.s. urban communities (table ). rates of clinically overt disease with hypertension and type dm were similar in both unaffected amish participants and the cardia participants. association of null serpine heterozygosity with telomere length and life span overall, the mean ltl was highly correlated with chronological age (r = − . , p < . ) and % shorter per decade of age (p < . ) in unaffected amish participants. carriers of the serpine mutation had a % longer mean ltl, the prespecified primary end point, after adjustment for age, sex, and familial relatedness compared with noncar- riers (p = . ) (fig. ). interaction between age and carrier status of the serpine null mutation in association with ltl was not signifi- cant. secondary analyses, including participants who were homozygous for the serpine null mutation (n = ), demonstrated similar results (p = . ; table s ). the overall heritability (h ) estimate of ltl was . (p < . ), suggesting that the additive effects of genetic vari- ation, including the serpine mutation, accounted for % of the total variation. khan et al., sci. adv. ; :eaao november vital status was available on individuals identified as directly re- lated to our study participants. genotype status for serpine was as- certained by direct genotyping or obligate ascertainment from ancestral data in deceased individuals with known dates of birth and death (n = ). the median survival in serpine null carriers compared with un- affected individuals was longer [ ( to ) versus ( to ); p = . ], as shown in fig. . association of null serpine heterozygosity and metabolic traits carriers of the serpine mutation had similar mean body mass index compared with noncarriers (p = . ). the prevalence of type dm, one of the prespecified secondary end points, was significantly lower in carriers of the serpine null mutation compared to noncarriers, as shown in table ( % versus %; p = . ). fasting insulin levels were % lower in serpine heterozygotes (p = . ) after adjustment for age, sex, and familial relatedness in comparison to unaffected partici- pants. secondary analysis including amish participants (n = ) who were homozygous for the null serpine mutation demonstrated simi- lar results (p = . ). sensitivity analysis performed with exclusion of amish participants with diabetes did not alter the results. in addition, we observed a significant positive correlation between pai- and fasting insulin levels in the amish participants (r = . , p < . ) and the cardia participants (r = . , p < . ). association of null serpine heterozygosity and cardiovascular parameters tissue doppler e′ velocity was slightly higher in carriers than controls but did not achieve significance ( . ± . versus . ± . ; p = not table . clinical characteristics of the berne amish kindred by serpine genotype status. continuous values are listed as mean ± sd unless otherwise specified. hdl, high-density lipoprotein; ldl, low-density lipoprotein. serpine +/+ (n = ) serpine +/− (n = ) p* age (years) ± ± . female, n (%) ( ) ( ) . hypertension, n (%) ( ) ( ) . systolic blood pressure (mmhg) ± ± . diastolic blood pressure (mmhg) ± ± . obesity, n (%) ( ) ( ) . body mass index (kg/m ) . ± . . ± . . diabetes, n (%) ( ) ( ) < . fasting insulin (uiu/ml)† . ( . – . ) . ( . – . ) . total cholesterol (mg/dl) ± ± . hdl cholesterol (mg/dl) ± ± . triglyceride (mg/dl)† ( – ) ( – ) . ldl cholesterol (mg/dl) ± ± . serum creatinine (mg/dl) . ± . . ± . . ever smoked, n (%) ( ) ( ) . *polygenic model adjusted for carrier status and family structure in solar. †non-normally distributed, reported as median ( th to th percentile). table . comparison of clinical characteristics of the berne amish kindred serpine +/+ and cardia. serpine +/+ (n = ) cardia (n = ) p age (years) ± ± . female, n (%) ( ) ( ) . hypertension, n (%) ( ) ( ) . systolic blood pressure (mmhg) ± ± < . diastolic blood pressure (mmhg) ± ± . obesity, n (%) ( ) ( ) < . body mass index (kg/m ) ± ± < . diabetes, n (%) ( ) ( ) . fasting glucose (mg/dl)* ( – ) ( – ) < . total cholesterol (mg/dl) ± ± . hdl cholesterol (mg/dl) ± ± . triglyceride (mg/dl)* ( – ) ( – ) < . ldl cholesterol (mg/dl) ± ± . serum creatinine (mg/dl) . ± . . ± . < . ever smoked, n (%) ( ) ( ) . *non-normally distributed, reported as median ( th to th percentile). of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m significant). brachial pulse pressure [ ( to ) versus ( to )] and carotid intima-media thickness (cimt) [ . ( . to . ) versus . ( . to . )] were lower in carriers than controls, but differences were not significant. several cardiovascular measures that correlated significantly with chronological age in unaffected individuals (|r| > . , p < . ; fig. ) were identified. a composite score of cardiovascular aging that represents vascular and myocardial health (e′ velocity, brachial pulse pressure, and carotid imt) was . units lower, on average, in carriers of the serpine mutation than noncarriers, but this difference did not achieve statistical significance (p = . ). a second composite score rep- resentative of cardiometabolic aging that included cardiovascular khan et al., sci. adv. ; :eaao november parameters and fasting insulin was . units lower, on average, in car- riers (p = . ). composite score , which was representative of com- prehensive biological aging, including score parameters and relative ltl, was . units lower, on average, in carriers (p = . ). these three composite scores of aging were assessed in the cardia cohort to determine their associations with cardiovascular outcomes. each score was significantly associated with the -year outcomes of cardiovascular morbidity and mortality (tables s and s ). similar results were obtained when restricting the analysis to white participants in cardia. in addition, plasma pai- levels in the cardia partici- pants correlated significantly with all three composite scores (r = . , . , and . for composite scores , , and , respectively; p < . ). founder identification in family structure pedigree a simplified diagram of the extended berne amish pedigree high- lighting individuals with heterozygosity and homozygosity for the null serpine gene isshown in fig. s . we identifiedeither individuali- or i- as highly likely to have introduced a single copy of the mutated allele into the population six generations ago. this husband and wife couple is a common ancestor to all individuals newly and previously identified in the study as heterozygous or homozygous for the null serpine allele. although the genetically restricted nature of this cohort has resulted in a relatively high prevalence of the serpine null allele, the mean kinship coefficient among carriers of the null serpine allele was %. the oldest known homozygous individual for the serpine mutation was born in as a result of the first marriage to occur between two heterozygotes. although these individuals provide a unique oppor- tunity to examine the biology of pai- in humans, the rarity of this con- dition in the world and their young age limit comparisons at this time, but baseline characteristics are described (table s ). discussion the central findings of our study are that heterozygosity for the null serpine gene encoding pai- , which is associated with a lifelong re- duction in pai- , is associated with longer ltl, a healthier metabolic fig. . association of serpine genotype status and leukocyte telomere length as a function of age in the berne amish kindred. (a and b) ltl in serpine null allele carriers and noncarriers in the berne amish kindred as quantified by (a) polymerase chain reaction (pcr) and (b) southern blot. relative ltl is shown in (a), and mean terminal restriction fragment (trf) length is shown in (b) as a function of age stratified by serpine mutation status. p value represents difference in mean ltl and trf by serpine mutation status (carriers versus noncarriers) after adjustment for age, sex, and family structure in sequential oligogenic linkage analysis routines (solar) (p = . and p = . , respectively). every -year increase in age of study participant was associated with a . lower relative ltl (p < . ) and a –base pair lower mean trf (p < . ). fig. . age at death in the extended berne amish kindred by genotype sta- tus for serpine . on the basis of ancestral data obtained from the extended pedi- gree and extensive fieldwork, we identified individuals with known dates of birth and death. genotype status for serpine was ascertained by direct genotyping or ob- ligate ascertainment in family members. the mean ± sd age at death was delayed by years in serpine null carriers compared with unaffected individuals ( ± ver- sus ± ; p = . by wilcoxon rank sum test). of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m profile with lower prevalence of diabetes, and a longer life span. the berne amish kindred provide an unprecedented opportunity to study the biological effects of a private loss-of-function mutation with a large effect on circulating pai- on longevity in humans ( , ). we confirm previous reports that suggest that ltl is inversely correlated with chronological age and is substantially heritable ( ). in epidemiologic studies, ltl has been validated as a potent predictor of diabetes, cvd, and all-cause mortality ( , , – ). in addition, inherited defi- ciencies in telomere maintenance in mice and humans manifest with an accelerated aging phenotype, supporting the critical role of telomere khan et al., sci. adv. ; :eaao november attrition in the pathophysiology of aging ( , ). these perspectives informed both our study design and the selection of ltl as the prespe- cified primary end point. although age-adjusted ltl is significantly longer in the carriers of the null serpine allele, the age-related phe- nomenon of ltl attrition across the cohort is the same. this supports previous data that absolute ltl may be largely determined at birth, and individuals with a longer ltl may shift the onset of insulin resistance and cvd to an older age, promoting longer life span ( – ). the current study builds upon the available cellular and animal ev- idence supporting the role of pai- , the product of serpine , as an fig. . association of serpine genotype status and cardiovascular measures of aging as a function of age in the berne amish kindred. (a to c) components of the aging composite scores, including brachial pulse pressure (a), e′ velocity (b), and carotid imt (c) as a function age in amish participants by genotype status for serpine null allele. (d to f) composite scores of cardiovascular, cardiometabolic, and comprehensive biological aging inserpine null allele carriers and noncarriers in the berne amish kindred. several cardiovascular measures that correlated strongly with chronological age with absolute correlation coefficients (|r|) greater than . (p < . ) were identified. pulse pressure [pp = . + . (age in years)] and carotid imt [ . + . (age in years)] increased with age. e′ velocity [e′ = . − . (age in years)] decreased with age, as expected. these measures of vascular structure andstiffness and myocardial health were then standardized to have mean = and sd = (z scores) and were integrated into composite score (cardiovascular age composed of brachial pulse pressure, e′ velocity, and carotid imt), composite score (cardiometabolic age composed of score plus fasting insulin), and composite score (comprehensive biological age composed of score and ltl). z scores were coded so that higher values corresponded to older levels of the measures of biological aging; that is, the z scores for ltl and e′ velocity, which decline with age, were reverse-coded so that higher z scores corresponded to lower levels [**p value represents difference in components and composite scores of aging by serpine mutation status (carriers versus noncarriers) after adjustment for age, sex, and family structure in solar]. of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m important contributor to aging ( ). pai- expression is increased in senescent cells and tissues and is a fundamental component of the sasp ( ). there is a compelling evidence that senescent cells accumulate in the tissues and contribute to the aging process ( , ). in addition to contributing to the molecular fingerprint of senescence, pai- is nec- essary and sufficient for the induction of replicative senescence in vitro and is a critical downstream target of the tumor suppressor p ( , ). the contribution of pai- to cellular senescence is broadly relevant in the organism as a whole, and age-dependent increases in plasma pai- levels have been identified in wild-type mice as they age, murine models of accelerated aging (klotho and bubr h/h), and humans ( , , ). partial pai- deficiency in klotho-deficient animals extended median survival nearly threefold with preserved telomere length and substantial protection against aging-like pathology in vari- ous organs ( ). both genetic and pharmacological inhibition of pai- protected against the development of hypertension and arteriosclerosis and was associated with longer telomere length in mice treated with long-term nitric oxide synthase inhibition ( ). these preclinical data are consistent withourfindingsof a longer ltl and a greater life span in carriers of the null serpine allele in humans. metabolism plays a fundamental role in the biology of aging, and insulin and insulin-like growth factor (igf ) are widely endorsed as critical contributors to senescence and aging in several experimental models (for example, flies, worms, and mammals) ( ). potential anti- aging interventions have focused on caloric restriction and drugs with metabolic effects, including metformin and resveratrol, all of which mechanistically intersect in reducing pai- expression ( – ). con- versely, pai- production is enhanced by insulin, free fatty acids, and glucose ( – ). in addition, pai- impairs the proteolytic degradation of igf-binding protein– (igfbp ) and igf , both of which are capa- ble of initiating cellular senescence ( , ). in observational human studies, pai- levels were higher in obesity and insulin resistance and independently predicted the future development of type dm ( , ). in the only gwas of circulating pai- in , participants, a genotype-phenotype analysis identified a significant association be- tween single-nucleotide polymorphisms (snps) that regulate pai- and type dm ( ). further, mendelian randomization analyses from large gwas studies support a casual role for pai- and hyperglycemia ( ). here, we demonstrate that serpine null carriers have lower fasting insulin levels and lower prevalence of type dm, and in a diverse u.s. population–based study (cardia), we demonstrate that circulating pai- levels are strongly correlated with fasting insulin levels. our findings from the only known pai- –deficient kindred provide the first example of a private gene mutation on age-dependent molec- ular and metabolic changes in humans. whole-genome sequencing and gwas for healthy aging and life span have had limited success in iden- tifying associated genetic loci, supporting the value of rare genetic var- iants (such as the one described here) in understanding biological mechanisms that regulate aging and life span ( – ). the genetic mechanism of pai- deficiency in the berne amish kindred is ex- plained by the presence of a dinucleotide insertion (ta) within the coding sequence of the pai- gene (exon ), resulting in a frameshift mutation and formation of a premature stop codon and the synthesis of a truncated, nonfunctional pai- protein. this frameshift mutation has not been identified in the generalpopulation based on the sequence data publicly available for review from , unrelated individuals in the exome aggregation consortium (exac). ten distinct loss-of-function mutations, including three frameshift mutations, are reported in the exac database in serpine and have exceedingly rare mutated allele khan et al., sci. adv. ; :eaao november prevalence ranging from . to . ( ). further, in gwas, snps account for no more than . % of the total variation in plasma pai- levels beyond that of age and sex ( ). therefore, discov- ery and validation of serpine snps in gwas of longevity may be difficult, given the limited contribution of these variants in the general population to circulating pai- levels, and highlight the strength of our study with a large effect size on circulating pai- levels as a result of a loss-of-function mutation. additional longitudinal studies in this cohort will help inform potential links between the null serpine mutation, metabolism, and life span. although there is a possibility that the serpine muta- tion segregates with other mutations that may contribute to the pheno- type observed, the qualitative and quantitative effects we observed with serpine heterozygosity in humans are fully concordant with the pre- dicted effects observed in preclinical experimental models ( , ). we acknowledge that the generalizability of the findings identified here may be limited, given that this cohort carries a rare private mutation, is func- tionally isolated from the “modern world” in several ways, and appears to be relatively healthier when compared to a general population sample as represented by cardia. average plasma pai- levels even in un- affected amish participants without the null serpine allele were % lower than participants enrolled in u.s. population–based cohorts such as cardia and the framingham heart study ( ). use of small-molecule selective inhibitors of human pai- or other components of the sasp may be of interest in preventing or treating age-related morbidities. orally active pai- inhibitors have already completed extensive preclinical evaluation and are currently in early- phase clinical testing in humans in japan ( , ). the absence of bleeding complications in heterozygotes for the serpine mutation in over years of clinical observation suggests that therapeutic agents that selectively reduce but incompletely inhibit pai- activity may be safe from a hemostatic perspective ( ). our study has several limitations. first, our results are based on an observational study and include limited assessments of ltl, metabolic parameters and disease, and measures of cardiovascular structure and function. given the current lack of consensus regarding the quantitative assessment of biological age, we developed novel composite scores of aging that integrate age-dependent physiological measures of cardio- vascular health including vascular structure and stiffness, subclinical atherosclerosis, myocardial relaxation, and ltl ( , ). although we demonstrate that our composite scores are significantly associated with the -year cardiovascular outcomes in cardia, events are limited, and these quantitative assessments should be replicated in additional population-based cohorts with longer follow-up times. second, although the aging phenotype of homozygous individuals is certainly of interest, these individuals are extremely rare in the kindred and relatively young (all less than years old), limiting any meaningful comparisons at the present time. therefore, we focused on the potential impact of serpine heterozygosity and ltl in this study. third, all of the het- erozygous participants share a common ancestor in generation i from the late s, and this lends a possibility that other unidentified genetic or epigenetic factors contribute to the present findings. to mitigate this risk, we adjusted our analyses for relatedness in solar ( ). in conclusion, our data indicate that pai- is associated with a number of parameters that may reflect biological aging, including ltl, metabolism, and life span. overall, our findings are the first to identify the physiological association of a null mutation in pai- with ltl and life span in humans and suggest that pai- , a component of the senescence-related secretome, may influence the aging process. of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e future studies will provide the opportunity to investigate the contribu- tion of pai- to individual telomere attrition over time, the develop- ment of incident diabetes and other age-related diseases, and perhaps ultimately differences in health and life span in humans. o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m materials and methods amish study participants this cross-sectional observational study was conducted in the ooa—a founder population who originally settled in berne, indiana, which is characterized by a homogeneous diet and lifestyle—and included par- ticipants aged years and older. the institutional review board at the indiana hemophilia and thrombosis center and the northwestern university approved the study protocol. a total of individuals was invited to participate in the study, and individuals were enrolled in the study. all study participants provided written informed consent and were genotyped for the c. _ dupta frameshift mutation in serpine . for the primary analyses, participants homozygous for the null serpine allele were excluded because of small numbers (n = ) and their relatively young age ( to years). the primary end point of telomere length and the secondary end points of fasting insulin, di- abetes status, and life span were prospectively selected. molecular markers and laboratory testing genotyping for serpine null mutation status was performed at the university of michigan with a preidentified primer for the presence of the c. _ dupta frameshift mutation in the pai- (serpine ) gene. genomic dna isolated from peripheral blood leukocytes (dneasy blood and tissue kit, qiagen) was used to measure relative ltl by quantitative pcr and trf length by southern blot by blinded technicians using previously described methods ( – ). quantitative pcr samples were run in triplicate, and southern blot samples were run in duplicate. the inter-assay coefficient of variation (cv) was . % for relative ltl and . % for mean trf. plasma samples preserved in citrate were assayed for human pai- total antigen with the molecular innovations pai- elisa (molecular innovations) using biotek synergy ht gen software (biotek). all samples were run in duplicate, and the mean of two measurements was used. the intra-assay cv was . %, and the inter-assay cv was . %. metabolites (glucose and insulin) and cholesterol (total cholesterol, hdl cholesterol, ldl cholesterol, and triglyceride) were measured in morning fasting blood samples according to a standardized protocol in the main laboratory at the northwestern memorial hospital. cardiovascular assessment all study participants underwent a comprehensive two-dimensional echocardiographic examination with doppler and tissue doppler imag- ing using a commercially available ultrasound system with harmonic imaging (vivid or vivid i, ge healthcare) and a . -mhz transducer (ge healthcare) by blinded sonographers. cardiac structure and function were quantified as recommended by the american society of echocardiography (ase) ( ). deidentified images were copied in raw format and analyzed offline using ge echopac software version . . (ge healthcare). all measurements were made by a single experienced research sonographer and verified by an experienced inves- tigator with expertise in echocardiography (both blinded to clinical data and genotype status) according to the guidelines of the ase. carotid imaging was performed by an experienced sonographer blinded to genotype status using a commercially available ultrasound khan et al., sci. adv. ; :eaao november machine (vivid or vivid i, ge healthcare) and an -mhz transducer (ge healthcare). deidentified images were copied in raw format for offline analysis and analyzed using ge echopac software version . . (ge healthcare). all measurements were performed by a single experienced research sonographer and verified by an experienced inves- tigator (both blinded to clinical data and genotype status). imt mea- surements were performed using ge’s semiautomated edge detection method according to the ase recommendations ( ). genealogical information and life span detailed genealogic data spanning three generations were obtained from each participant. the pedigree was supplemented by an extensive fieldwork to fill in available birth and death dates and was used to adjust for family relatedness by calculation of kinship matrix for each of our enrolled participants in the solar polygenic models. we additionally used ancestral data from the extended pedigree for life-span analysis. we restricted our life-span analysis to individuals known to have died at the age of ≥ years to minimize the potential bias from premature cause of death, including infection, accidental trauma, and childbirth, as previously described in similar analyses ( ). validation of composite aging scores we sought to validate the biological aging composite scores in a gen- eralized u.s. population using the cardia study. the cardia study is a multicenter, community-based longitudinal cohort study designed to investigate the risk factors for the development of cvd. in – , black and white men and women aged to years were recruited from four urban sites across the united states (birmingham, al; chicago, il; minneapolis, mn; and oakland, ca). recruitment was balanced within each center by sex, age, race, and education. the cardia participants have been followed for more than years with collection of detailed demographic and clinical data. all participants provided written informed consent at each examination, and institu- tional review boards from each field center and the coordinating center approved the study annually. detailed descriptions of the study design and conduct had been previously published ( ). for this analysis, all participants who attended the year- examination and had available data for components of composite scores and were included (n = ). a subset of participants who also had data for composite score (n = ) were also examined separately. statistical analysis we compared baseline demographics between heterozygous and un- affected participants with adjustment for family structure. we used polygenic models adjusted for age, sex, and family structure to deter- mine the association of heterozygosity in serpine with ltl as the primary end point, as measured by quantitative pcr and southern blot. we estimated heritability (h ) defined as the proportion of total varia- tion explained by additive genetic effects for ltl. we also investigated the association of serpine status with parameters of metabolism, in- cluding fasting insulin and the prevalence of type dm and life span. composite scores of aging were created by the summation of standard- ized z scores among aging-related characteristics that had the highest values for pearson correlation with chronological age (|r| > . ). composite score included log-transformed brachial pulse pressure, tissue doppler e′ velocity (a marker of left ventricular relaxation), and log-transformed average carotid imt. composite score included components in score and fasting insulin, and composite score in- cluded components of score and ltl. the three composite aging of http://advances.sciencemag.org/ s c i e n c e a d v a n c e s | r e s e a r c h a r t i c l e d o w n lo scores were validated in a generalized u.s. population using the cardia study. supplemental descriptive analysis was performed including the homozygous participants (n = ) with limited power given their small sample size and young age. polygenic analyses were conducted using solar(version . . ; southwest foundation for biomedicalresearch). all other analyses were conducted using sas software version . (sas institute). two-sided p values < . were considered to be statistically significant. supplementary materials supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/ / /eaao /dc supplementary materials and methods table s . clinical characteristics of homozygous participants for the null serpine mutation. table s . association of null serpine mutant allele status with pai- , telomere length, and fasting insulin: secondary analyses. table s . association of cardiometabolic aging composite scores with -year cvd morbidity and mortality in the cardia cohort (n = ). table s . association of biological aging composite scores including telomere length with -year cvd morbidity and mortality in the cardia cohort (n = ). fig. s . abbreviated pedigree of the berne amish kindred. o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / a d e d fro m references and notes . m. kaeberlein, p. s. rabinovitch, g. m. martin, healthy aging: the ultimate preventative medicine. science , – ( ). . l. fontana, l. partridge, v. d. longo, extending healthy life span—from yeast to humans. science , – ( ). . l. daniali, a. benetos, e. susser, j. d. kark, c. labat, m. kimura, k. k. desai, m. granick, a. aviv, telomeres shorten at equivalent rates in somatic tissues of adults. nat. commun. , ( ). . c. lópez-otín, m. a. blasco, l. partridge, m. serrano, g. kroemer, the hallmarks of aging. cell , – ( ). . v. d. longo, a. antebi, a. bartke, n. barzilai, h. m. brown-borg, c. caruso, t. j. curiel, r. de cabo, c. franceschi, d. gems, d. k. ingram, t. e. johnson, b. k. kennedy, c. kenyon, s. klein, j. j. kopchick, g. lepperdinger, f. madeo, m. g. mirisola, j. r. mitchell, g. passarino, k. l. rudolph, j. m. sedivy, g. s. shadel, d. a. sinclair, s. r. spindler, y. suh, j. vijg, m. vinciguerra, l. fontana, interventions to slow aging in humans: are we ready? aging cell , – ( ). . c. lópez-otín, l. galluzzi, j. m. p. freije, f. madeo, g. kroemer, metabolic control of longevity. cell , – ( ). . k. d. kochanek, j. xu, s. l. murphy, a. m. minino, h.-c. kung, deaths: preliminary data for . natl. vital stat. rep. , – ( ). . m. j. j. d’mello, s. a. ross, m. briel, s. s. anand, h. gerstein, g. paré, association between shortened leukocyte telomere length and cardiometabolic outcomes: systematic review and meta-analysis. circ. cardiovasc. genet. , – ( ). . p. c. haycock, e. e. heydon, s. kaptoge, a. s. butterworth, a. thompson, p. willeit, leucocyte telomere length and risk of cardiovascular disease: systematic review and meta-analysis. bmj , g ( ). . t. tchkonia, y. zhu, j. van deursen, j. campisi, j. l. kirkland, cellular senescence and the senescent secretory phenotype: therapeutic opportunities. j. clin. invest. , – ( ). . s. 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f hl to s.s.k.; r hl and hl to s.j.s.; and r hd , r hl , and r hl to a.a.). the cardia is conducted and supported by the national heart, lung, and blood institute (nhlbi) in collaboration with the university of alabama at birmingham (hhsn c and hhsn c), northwestern university (hhsn c), university of minnesota (hhsn c), kaiser foundation research institute (hhsn c), and johns hopkins university school of medicine (hhsn c). cardia is also supported, in part, by the intramural research program of the national institute on aging (nia) and an intra-agency agreement between nia and nhlbi (ag ). this manuscript has been reviewed by cardia for scientific content. author contributions: d.e.v., s.s.k., and s.j.s. provided substantial contribution to the conception and design of the study, data acquisition and analysis, and drafting and critical revision of the manuscript. a.s.b. contributed to data analysis and critical manuscript revisions. e.k., m.e., a.t.p., d.m.l.-j., e.p., a.a., t.m., and m.h. contributed to data acquisition and critical revision of the manuscript. s.g. and a.d.s. contributed substantially to the conception and design of the study, data acquisition, and critical revision of the manuscript. all coauthors approved the final version of the manuscript to be published and agreed to be accountable for the accuracy and integrity of all aspects of the work. d.e.v. claims responsibility for all figures in the manuscript and the supplementary materials. competing interests: the authors declare that they have no competing interests. data and materials availability: all data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. additional data related to this paper may be requested from the authors. submitted june accepted october published november . /sciadv.aao citation: s. s. khan, s. j. shah, e. klyachko, a. s. baldridge, m. eren, a. t. place, a. aviv, e. puterman, d. m. lloyd-jones, m. heiman, t. miyata, s. gupta, a. d. shapiro, d. e. vaughan, a null mutation in serpine protects against biological aging in humans. sci. adv. , eaao ( ). of http://exac.broadinstitute.org http://advances.sciencemag.org/ protects against biological aging in humansserpine a null mutation in puterman, donald m. lloyd-jones, meadow heiman, toshio miyata, sweta gupta, amy d. shapiro and douglas e. vaughan sadiya s. khan, sanjiv j. shah, ekaterina klyachko, abigail s. baldridge, mesut eren, aaron t. place, abraham aviv, eli doi: . /sciadv.aao ( ), eaao . sci adv article tools http://advances.sciencemag.org/content/ / /eaao materials supplementary http://advances.sciencemag.org/content/suppl/ / / / . .eaao .dc references http://advances.sciencemag.org/content/ / /eaao #bibl this article cites articles, of which you can access for free permissions http://www.sciencemag.org/help/reprints-and-permissions terms of serviceuse of this article is subject to the is a registered trademark of aaas.science advancesyork avenue nw, washington, dc . the title (issn - ) is published by the american association for the advancement of science, newscience advances license . (cc by-nc). science. no claim to original u.s. government works. distributed under a creative commons attribution noncommercial copyright © the authors, some rights reserved; exclusive licensee american association for the advancement of o n a p ril , h ttp ://a d va n ce s.scie n ce m a g .o rg / d o w n lo a d e d fro m http://advances.sciencemag.org/content/ / /eaao http://advances.sciencemag.org/content/suppl/ / / / . .eaao .dc http://advances.sciencemag.org/content/ / /eaao #bibl http://www.sciencemag.org/help/reprints-and-permissions http://www.sciencemag.org/about/terms-service http://advances.sciencemag.org/ biomed centralbmc genomics ss open accemethodology article identification of disease causing loci using an array-based genotyping approach on pooled dna david w craig† , matthew j huentelman† , diane hu-lince , victoria l zismann , michael c kruer , anne m lee , erik g puffenberger , john m pearson and dietrich a stephan* address: neurogenomics division, translational genomics research institute (tgen) phoenix, arizona , usa and clinic for special children, strasburg, pa , usa email: david w craig - dcraig@tgen.org; matthew j huentelman - mhuentelman@tgen.org; diane hu-lince - dhlince@tgen.org; victoria l zismann - vzismann@tgen.org; michael c kruer - mkruer@tgen.org; anne m lee - alee@tgen.org; erik g puffenberger - epuffenberger@clinicforspecialchildren.org; john m pearson - jpearson@tgen.org; dietrich a stephan* - dstephan@tgen.org * corresponding author †equal contributors abstract background: pooling genomic dna samples within clinical classes of disease followed by genotyping on whole-genome snp microarrays, allows for rapid and inexpensive genome-wide association studies. key to the success of these studies is the accuracy of the allelic frequency calculations, the ability to identify false-positives arising from assay variability and the ability to better resolve association signals through analysis of neighbouring snps. results: we report the accuracy of allelic frequency measurements on pooled genomic dna samples by comparing these measurements to the known allelic frequencies as determined by individual genotyping. we describe modifications to the calculation of k-correction factors from relative allele signal (ras) values that remove biases and result in more accurate allelic frequency predictions. our results show that the least accurate snps, those most likely to give false-positives in an association study, are identifiable by comparing their frequencies to both those from a known database of individual genotypes and those of the pooled replicates. in a disease with a previously identified genetic mutation, we demonstrate that one can identify the disease locus through the comparison of the predicted allelic frequencies in case and control pools. furthermore, we demonstrate improved resolution of association signals using the mean of individual test-statistics for consecutive snps windowed across the genome. a database of k-correction factors for predicting allelic frequencies for each snp, derived from several thousand individually genotyped samples, is provided. lastly, a perl script for calculating ras values for the affymetrix platform is provided. conclusion: our results illustrate that pooling of dna samples is an effective initial strategy to identify a genetic locus. however, it is important to eliminate inaccurate snps prior to analysis by comparing them to a database of individually genotyped samples as well as by comparing them to replicates of the pool. lastly, detection of association signals can be improved by incorporating data from neighbouring snps. published: september bmc genomics , : doi: . / - - - received: may accepted: september this article is available from: http://www.biomedcentral.com/ - / / © craig et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. page of (page number not for citation purposes) http://www.biomedcentral.com/ - / / http://creativecommons.org/licenses/by/ . http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ http://www.biomedcentral.com/info/about/charter/ bmc genomics , : http://www.biomedcentral.com/ - / / introduction the ability to genotype hundreds of thousands of single nucleotide polymorphisms (snps) across the genome and to perform association analysis between cases and controls provides, for the first time, a discovery-based approach for determining the underpinnings of complex human genetic disorders. technologies from affymetrix (microarray-based genechip® mapping arrays), illumina (beadarray™), and sequenom (massarray™) are now available with sufficient density to detect linkage disequi- librium between informative snps and nearby disease- causing nucleotide variants through non-hypothesis based whole-genome association scans for certain popula- tions [ - ]. several practical issues make whole-genome association studies by utilizing individual genotyping difficult to implement [ ]. power estimates predict that somewhere on the order of a thousand cases and control subjects must be genotyped to detect allelic differences of < % between the cohorts, as well as to detect rare alleles which may be causative in only a subset of the cohort [ ]. addi- tionally, population stratification and allelic imbalance may identify snps that have statistically significant allelic frequency differences yet have no relation to the disease [ , , ]. whole-genome association studies are now tech- nologically possible, though the cost is several million dollars if samples are individually genotyped. here we describe the validation of pooling genomic dna samples as a rapid pre-screening to detect disease-causing loci for a few thousand dollars on snp genotyping microarrays. it is possible to identify snps that have significant differ- ences in allelic frequencies between two populations while saving a significant amount in resources by pooling genomic dna and then snp genotyping on a single microarray, or preferably on a series of replicated arrays. indeed, a limited number of studies have been conducted that demonstrate the possibility to predict accurately the allelic frequencies of a snp from a pooled sample on a microarray, and, in fact identify quantitative trait loci [ - ]. typically, these studies have validated the pooled allelic frequencies by later individually genotyping between ten to a few hundred snps. the most elegant val- idations of pooling have used indirect approaches, such as spiking a single individual of known genotype into a pooled group with unkown genotypes [ ]. one cannot realistically expect all probes on a microarray to function equally, especially considering that the objec- tive of these platforms is to identify allelic differences of %, %, and %. indeed, as platforms move to , + snps, the ability to select preferentially the best performing snps, such as was done in the design of the affymetrix k genechip®, will likely be compromised. as a result, our prediction is that many snps will be unre- liable for pooling, and thus may be more likely to lead to false positives. in a pooling study, limiting false positives that are a result of the assay, rather than the underlying population, will be a major factor in being able to realis- tically identify snps that can predict disease status. in this study, we investigated the reliability of snp allelic fre- quency measurements as determined from pooling genomic dna samples on snp mapping arrays. we fur- ther demonstrate our ability to identify poorly predictive snps prior to analysis. results we compared the predicted allelic frequencies from pools of genomic dna to the known allelic frequencies deter- mined by individual genotyping in order to establish the accuracy of pooling. the goal was to compare allelic fre- quencies for all the snps on a microarray, since not all snps will be equally accurate for the prediction of fre- quencies. inaccurate snps are expected to be problematic as microarrays progress to probe hundreds of thousands of snps, whereby snps are chosen primarily for their physical position in the genome and not for their repro- ducibility. indeed, in order to identify , snps for the affymetrix k genechip® mapping array nearly , snps were screened by affymetrix for reliability in the assay [ ]. individual genotyping of snps for samples allelic frequencies for , snps on samples were determined by individually genotyping on the k gene- chip®. these samples were genotyped over a one-year period; therefore, some samples were genotyped on ver- sion . of this platform and others on version . . only snps genotyped on both platforms were utilized for this study. accuracy of snp calls was approximately . %, as determined by inheritance errors in family pedigrees, in line with the accuracy reported by affymetrix ( . %) [ ]. we found no significant decrease in accuracy between the two versions of the k genechip®. the aver- age percentage of snps called across all samples was . %. only individuals with a call rate above % were included in the present study. for example, , snps were called for all individuals and , snps were called for > % of individuals. in our experience using this plat- form on over , samples we determined that the call rate is highly dependent on dna quality and that high quality genomic dna yields a call rate of – %. the samples used in this study have been collected over sev- eral years with variable dna quality. it is to be expected expect that large-scale whole-genome association studies will also be forced to utilize dna of less than optimal quality since hundreds to thousands of individuals are needed. thus the genomic dna used in this study will likely be representative of what could be expected in a page of (page number not for citation purposes) bmc genomics , : http://www.biomedcentral.com/ - / / whole-genome association study on a disease where sev- eral-thousand individuals are needed. construction of pools pools were created in triplicate from the individually gen- otyped samples. the individuals were from the old order amish and old order mennonite populations of south- eastern pennsylvania [ ]. pool consisted of individ- uals, pool consisted of a different individuals, and pool consisted of patients who died of a form of sud- den infant death syndrome known as siddt and had a known region of identity-by-descent (shared a pre- defined allele on all six chromosomes across the case cohort) [ ]. this region was defined on the k microar- ray by consecutive autozygous snps, of which were informative. all dna was quantitated using picogreen reagent (molecular probes, eugene, oregon) to ensure equal amounts were contributed to the pool from each individual. these three pools were then genotyped in rep- licates of three on the k genechip®. in all, microar- rays were used for the pooled genotyping compared to microarrays for the individual genotyping. calculation of allelic frequencies from pooled samples the predicted allelic frequencies from pooled genotype samples were calculated for each snp using a k-correction factor based on their derivation from over , individ- uals genotyped on the k genechip®. the training set consisted of , individuals that were genotyped in our lab within the past year. all had call rates above % with an average call rate of %. none of the , individuals used for calculation of k-correction factors were included in the pooled genomic dna. the purpose of the k-correction factor is to allow for cal- culation of a predicted allelic frequency from peak heights, or in this case fluorescence signal, whereby p = a/ (a+kb) [ ]. k-correction factors have recently become well established and have been used successfully in primer extension assays whereby measurements in snp allelic frequencies on pooled genomic dna have been taken by hplc, mass spec, and by fluorescence in taq- man assays [ - ]. for the k mapping array assay, p is the predicted allelic frequency of the a allele, a is the flu- orescent signal intensity measure of the a allele, and b is the fluorescent signal intensity measure of the b allele. the k-correction factor can be calculated for a given snp using a heterozygote who is ab, effectively % a and % b. conveniently, output of the affymetrix genechip software for the affymetrix k mapping array includes relative allele signal (ras) values which have been previ- ously used to determine k-correction values (see figure ) [ ]. generally, ras = a/(a+b). here, a refers to the median match/mismatched differences of the major allele and b for the minor allele (affymetrix technical manual). example of ras statistics for three snps based on genotyp-ing of individuals with an average call rate of all snps greater than %figure example of ras statistics for three snps based on genotyp- ing of individuals with an average call rate of all snps greater than %. these example snps illustrate how snp call reliability can vary both between snps and within the same snp, as measured by ras and ras values. blue spheres are bb individuals, orange triangles are aa individu- als, and green squares are ab individuals, grey stars are "not called". page of (page number not for citation purposes) bmc genomics , : http://www.biomedcentral.com/ - / / there are two ras values, ras (sense) and ras (anti- sense) since both sense and antisense directions are probed. whereas k-correction factors based on the affymetrix k genechip® mapping array have been previously calcu- lated directly using only heterozygous ras values [ ], we suggest that this can be improved upon since the ras val- ues are generally not or for homozygotes (see figure ). indeed, we observed significant deviation for many snps, which could potentially add significant bias (see discussion) [ ]. thus for each snp, we normalized ras values, referred to as nras, using the individuals from the training set that were aa (normalized to ) and bb (nor- malized to zero). without this normalization, predicted frequencies will be systematically biased as the pooled samples approach homozygosity. thus nrasx = (rasx- aaave)/(bbave) where aaave is the average rasx score for individuals aa in the training set, and bbave is the rasx score for individuals bb in the training set. the value of x refers to whether the calculation is for ras or ras , and nras values are calculated for both ras and ras . thus, two predictions of allelic frequency are obtained: one from ras and one from ras . each ras variable has dis- tinct variability, and as shown in figure (b), ras may be very precise with low variance, while ras may exhibit high variance, and vice versa. averaging the two ras val- ues will mask the ras value with lower variance. because of this independent variability, we do not recommend averaging ras and ras for all snps as was suggested in other pooling studies [ , , ]. rather, we recommend treating the two ras values as separate experiments, and preferably removing ras values with the greatest variance prior to analysis. values making up each of the ras and ras mean values are provided for homozygous aa, homozygous bb, and heterozygous individuals on a website based on our , person database which is being made available to the pub- lic as part of this publication. these k-correction factors derived from ras and ras values using this training dataset are available at [ ] and as supplementary material. comparison of allelic frequencies: pooling vs. individual genotyping for the , snps on the affymetrix k genechip® we found a median difference in allelic frequency between individually genotyped samples and pooled samples of . %, a mean difference of . %, and a standard devia- tion of . %. figure shows a histogram for all the snps and their difference between the predicted frequency from the pools and the individual genotypes data. other stud- ies have reported slightly lower differences between pooled and individually genotyped methods for determining allelic frequencies ( – %) [ , , ]. many reasons are likely for this difference: we used dna that was collected over ten years and was of varying quality; we also compared all the snps on the microarray rather than selecting only a few snps for comparison. realistically, the greater difference seen in this study may be more rep- resentative of large association studies, in which thou- sands of genomic samples of varying quality are pooled. (a) allele frequency differences between individual and pooled genotypesfigure (a) allele frequency differences between individual and pooled genotypes. histogram representing the total number of snps at each allele frequency difference between individ- ual and pooled samples. (b) accuracy of predicted snp fre- quencies increases for those snps that perform well on mapping k individual assays and decreases for poorly per- forming snps. the mean and median absolute difference between the predicted allelic frequency and individually gen- otyped allelic frequencies are shown vs. the binned perform- ance of snps on individual assays. performance is ranked by the frequency of calls in a set of , individually genotyped samples. page of (page number not for citation purposes) bmc genomics , : http://www.biomedcentral.com/ - / / identification of assay false positives while the differences in frequencies between pooled and individual genotyped samples show that calculating fre- quencies from pooled samples is highly accurate, it is per- haps of greater importance that we are able to predict those snps that are unreliable and largely inaccurate. assays genotyping , snps will likely not have the ability to be as selective and thus are likely to provide a large number of snps that do not reliably quantitate allelic frequencies from pooled genomic samples. as shown in table , we found that the snps most likely to give a "nocall" in individual genotyped samples more often gave unreliable predictions of allelic frequencies in pooled samples. furthermore, as shown in figure b, those snps that are the worst % (in terms of % called for individual genotypes) also gave rise to higher allelic frequency differences. we found that rarely called snps are also likely to be called inaccurately (table and figure c). in this case, constructing k-correction factors and predicting allelic fre- quencies will be unreliable for these snps, even if pooled replicates show low variability. snps with the highest var- iance in pool replicates were also unreliable. as a practical measure, we found that applying both filters for too many "nocalls" in a training set and having a high variance in pooled replicates was more effective than either measure alone. we could identify / rd of the worst performing snps (greater than % difference), by removing the worst performing % of snps based on variance in pool replicates and removing the worst performing % of snps based on excessive "no calls" when individually geno- typed. consequentially, the removal of those snps that are either poorly called in a training set of individually genotyped samples or highly variable across pooled repli- cates significantly decreases the number of false positives. the number of snps removed should maintain a balance between retaining dense snp coverage and excluding those snps more likely to give false positives. ultimately, removing potential false positives will be a compromise between the coverage of the snp microarray and the genetic diversity of the population. it is of interest to note that allelic frequencies calculations were more accurate as snps approached homozygosity. for example, for those snps with allelic frequencies from % to % and from % to % the mean difference was . % vs. a mean difference of . % for snps with allelic frequencies between % and %. this finding may be due to inaccuracies in the assays as snps approach %, since the variance for heterozygotes is higher than the variance measured for homozygotes. identification of a disease locus from genotyping of pooled samples in order to assess whether it is feasible to use pooled gen- otyping to identify the genetic locus for a disease, we created case and control pools for the disease sudden infant death with dysgenesis of the testes (siddt) and a pool of amish control individuals. a test for proportions was employed to detect statistical differences between cases and controls. this test-statistic is more often used in pooled studies since frequency data are generally not whole-integers [ ]. shown in equation is the calculation for the test statistic (t) where fcase is the allele frequency for the case group and fcontrol is the allele frequency for the control group. table : inaccurate snps with the largest difference between snp allele frequencies when genotyped individually vs. calculated from pooled dna can be partially predicted. nearly % of the snps found to be the most inaccurate snps were also either (a) one the worst performing snps in individual genotyping or (b) had the largest variability between replicates in the pool. all snps (a) worst performing snps criteria: nocalls on person database (b) worst performing snps criteria: pool variability from replicates snps found in (a) or (b) inaccuracy (predicted vs. genotyped) most inaccurate snps (individual genotyped vs. pooled) . % . % . % . % most inaccurate snps (individual genotyped vs. pooled) . % . % . % . % remaining snps . % . % . % . % t f f f f f f case control case control case case = − + = var( ) var( ) , var( ) ( − ( )f n case case ) page of (page number not for citation purposes) bmc genomics , : http://www.biomedcentral.com/ - / / the distribution follows an approximate χ distribution with one degree of freedom. the snp with the highest sig- nificance, rs , had a p-value of . and was in the siddt locus at chromosome q . however, it is expected that the snp with the lowest p-value will not always be at the correct disease locus. even strong single snp association signals will likely be obscured in the noise when , + snps are probed. thus we employed a moving window whereby the mean test-statis- tic of several consecutive snps was calculated at each snp position across the genome. the objective of the moving window was to leverage the fact that neighbouring snps will likely be in linkage disequilibrium, whereby one snp is at least partially predictive of the neighbouring snp. the number of snps contained in the moving window was varied between and . shown in table is the rank of the q region for varying window sizes. it is of inter- est to assess sensitivity of this windowing approach to snps within the region. thus, we consecutively removed the top three snps contributing to the overall association signal. removing the first two snps has little effect on detecting the association signal. the q region remains the most significant for window sizes of four and greater even when these top two snps are removed. in compari- son, all three top snps has a marginal effect, lowering the rank of the region from highest to within the top ten. to compute the statistical significance of averaged test-sta- tistics, we used a permutation test. with this approach the consecutive order of snps was randomized in four hun- dred separate bootstrapped datasets. p-value statistics were calculated from the distribution of these datasets. shown in table , the siddt locus ( q . -q . ) was generally revealed as the most significant region of associ- ation for window sizes between and snps. it will not always be the case that snps are in linkage dis- equilibrium and a windowing-based approach will be effective. the permutation statistics can be used to test this scenario in order to see if the frequency of a given mean window test-statistic is indeed significant. the old order amish and mennonite populations used in this study identification of the siddt locus from pooled genomic dna by calculating the mean test-statistic for a rolling window of con-secutive snpsfigure identification of the siddt locus from pooled genomic dna by calculating the mean test-statistic for a rolling window of con- secutive snps. the moving window was determined across the genome and the p-value was calculated from a distribution of bootstraps of the original dataset. mean window sizes of , , , , , and are shown and the siddt locus is high- lighted in yellow. the siddt disease locus is the top region for window sizes of , , , , and . page of (page number not for citation purposes) bmc genomics , : http://www.biomedcentral.com/ - / / arise from a population founded in approximately the six- teenth century with expectedly larger regions of identity by descent. the amish and mennonites are not one large isolated population. it is more accurate to say that both these populations derive from the swiss anabaptists (circa ). these groups are socially and genetically unique even though both came from the same geographical region. thus undoubtedly some stratification exists between our two cohorts and it is encouraging that the correct region was easily identifiable despite any stratifica- tion [ ]. based on previous research in this population, the k mapping array was anticipated to be of sufficient density whereby many of the snps would be in relative linkage disequilibrium throughout this regional population. indeed, the permutation statistics of moving windows support this notion as the q region shows a p-value of < e- for window sizes of snps and greater, far lower than would be expected with ~ , snp measure- ments. other methods have been developed that reduce noise using haplotype data from snps in linkage disequi- librium [ ]. in the absence of this haplotype data, which may often be the case, it is encouraging that the very straightforward statistical approach described here is effective at identifying the correct locus. discussion our results show that ( ) pooling genomic samples is highly accurate; ( ) unreliable snps most likely to give false-positives can be largely identified and removed prior to association analysis; and ( ) a moving window of aver- aged test-statistics can be used to detect association sig- nals. additionally, we have described modifications as to how allelic frequencies are calculated from ras values of pooled samples that remove systematic biases. pioneering work on pooling studies by other research groups has shown that the average relative allele signal (rasave) can be effectively used to derive k-correction fac- tors by k = rasave/( -(rasave), and as such, can be used to accurately predict allelic frequencies [ , , ]. pooling studies are intended to be screening approaches. ras val- ues are highly convenient since they are generated by the affymetrix gdas software on the k platform and fairly intuitive to understand. we suggest significant improve- ments to this innovative approach that will remove biases; allow for continued use of ras values; and result in more accurate predictions. these improvements focus on lower- ing the number of false positives due to added variance or systematic biases, since the utility of pooling-based approaches will be based on how one can detect associa- tion signals given a high number of false positives. first, ras and ras should not be averaged since they are separate probe sets with distinct variances. one may unnecessarily propagate unwanted variance by averaging. for example in figure b, it is clearly visible that ras is highly predictable of the particular snp allele whereas ras is highly inaccurate. in this case, averaging ras and ras will produce a rasave value that is less accurate than ras alone. we suggest instead that these values be treated as separate measures, each with their distinct vari- ance. in the case of ras values with a large variance, these values should not be used due to the increased chance of a false positive. table : identification of disease locus using a moving window. snps were ranked by test statistics and sorted by physical position. the average was calculated for a moving window of consecutive snps across the genome. the region q . was already known to contain the mutation leading to the siddt. the rank of region q . for a various window sizes in shown in the second column. in the rd, th, and th columns, the top , , and snps were removed from the q . regions to probe sensitivity of window size. # snps averaged in moving window q . rank region (all snps) q . region rank (exclude top snp in region) q . region rank (exclude top snps in region) q . region rank (exclude top snps in region) page of (page number not for citation purposes) bmc genomics , : http://www.biomedcentral.com/ - / / second, we highly recommend that ras values for each snp be normalized prior to calculation of allelic frequencies. when these values are not normalized prior to calculating a predicted allelic frequency a significant bias is introduced since the ras values, as produced by the affymetrix gdas software, generally are not . or . for homozygous bb and aa respective alleles. indeed, on a training set of individuals we found that % of snps who were called aa had a ras value less than . and % of snps called bb had a ras value greater than . . this bias can be seen in an example calculation using k-correction factors derived from a typical ras value directly obtained from the gdas software. for example, the average ras for a given snp of an aa individual may be . , the average ras for a heterozygous individual may be . , and the average ras for a bb individual may be . . when one uses the approach outlined by butcher, et al, the k-correction factor is . , whereby the ras value of the average heterozygote is divided by one minus this value [ ]. in a pooled sample, the same snp is expected to have a ras value of . if it is completely homozygous for aa. however, using the k-correction approach on non- normalized ras values, one would predict an allelic fre- quency of %, whereas the actual frequency is %, a bias of %. these biases would be most pronounced as pools approach dominance by one allele type, as would often be the case for a snp highly associated to a disease. while ras values are readily obtainable from the affyme- trix software for the k genechip® arrays, they are not provided for the k or k. this is partly due to the fact that ras values are no longer used to make a snp call. we have developed a simple perl script which generates ras values, still useful in pooling, for the k and k affymetrix genechip® platform from chp files. this tool is available on our website [ ]. while one may use these ras values to find obvious differences in cases and con- trols, for many snps allelic frequencies are not linearly dependent on the ras values; thus, one should calculate allelic frequencies when possible to reduce uneven biases between different snps. additionally, we are making public on the same site both normalized and non-normalized k-correction factors derived from over , genotyped individuals for the k version . snp genotyping platform. other research groups have created central repositories for k-corrections using non-normalized ras values and we will work with these teams to contribute these values to this valuable cen- tralized resource [ ]. conclusion prior to the investment of large resources into individual genotyping thousands of individuals, one may first con- sider pooling samples at a low cost to rapidly ascertain gross population stratification concerns and potentially identify the regions of the genome with the strongest asso- ciation to the trait. the sheer number of snps interrogated will lead to a high number of false positives, due to both actual variation in genotype frequencies of the underlying groups and to technical variance. we demonstrate that technical variance can be detected a priori for each snp using training sets from large numbers of individual microarrays or by replicates of pooled samples. we further show that despite the issues of population stratification, admixture, and subgroups that are difficult to detect when pooling, the cost savings make pooling a first step that we suggest should logically precede the investment of mil- lions of dollars. we describe here a method by which k and k affymetrix snp array data can be parsed into ras scores and pooled inbalances accurately assessed in an outbred population. methods k genechip® mapping array genotyping k snp genotyping was performed as detailed by affymetrix on the k genechip® mapping . and . arrays [ ]. in short, ng of genomic dna was digested with units of xba i (new england biolabs, beverly, ma) for hours at °c. adaptor xba (p/n , affymetrix, santa clara, ca) was then ligated onto the digested ends with t dna ligase for hours at °c. after dilution with water, samples were subjected to pcr using primers specific to the adaptor sequence (p/n , affymetrix) with the following amplification parameters: °c for minutes initial denaturation, °c seconds, °c seconds, °c seconds for a total of cycles, followed by °c for minutes final extension. pcr products were then purified and frag- mented using . units of dnase i at °c for min- utes. the fragmented dna was then end-labeled with biotin using units of terminal deoxynucleotidyl trans- ferase at °c for hours. labeled dna was then hybrid- ized onto the k mapping array at °c for – hours at rpm. the hybridized array was washed, stained, and scanned according to the manufacturer's instructions. the chp_ _ras.pl script processes one or more chp text files from affymetrix k and k snp chips, calculates ras and ras scores and outputs them in an excel spreadsheet. testing shows that for k chips, chp_ _ras.pl produces the same scores as those produced by affymetrix' gdas software. chp_ _ras.pl is distributed as part of tgen-array, a collection of perl scripts and mod- ules that provide parsing and object-oriented interfaces to common microarray files. the script can be downloaded at the tgen bioinformatics website [ ]. page of (page number not for citation purposes) bmc genomics , : http://www.biomedcentral.com/ - / / authors' contributions dwc and mjh performed snp genotyping, participated in the concept of the paper, and drafted the manuscript. dhl, vlz, mjh, and aml conducted pooling and snp genotyping. dwc and jmp performed statistical analysis of the snp data. das participated in study design, coordi- nation, and manuscript drafting. all of the authors have read and approved the final manuscript. additional material acknowledgements we thank the old order amish families who participated in the research and the old order amish community for their willingness to participate in research studies. references . matsuzaki h, dong s, loi h, di x, liu g, hubbell e, law j, berntsen t, chadha m, hui h, yang g, kennedy gc, webster ta, cawley s, walsh e, jones kw, fodor sp, mei r: genotyping over , snps on a pair of olignucleotide arrays. nature methods , : - . . fan jb, oliphant a, shen r, kermani bg, garcia f, gunderson kl, hansen m, steemers f, butler sl, deloukas p, galver l, hunt s, mcbride c, bibikova m, rubano t, chen j, wickham e, doucet d, chang w, campbell d, zhang b, kruglyak s, bentley d, haas j, rigault p, zhou l, stuelpnagel j, chee ms: highly parallel snp genotyping. cold spring harb symp quant biol , : - . . marnellos g: high-throughput snp analysis for genetic associ- ation studies. curr opin drug discov devel , : - . . cardon lr, bell ji: association study designs for complex diseases. nat rev genet , : - . . risch n, teng j: the relative power of family-based and case- control designs for linkage disequilibrium studies of complex human diseases i. dna pooling. genome 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[http://www.tgen.org/neurogenomics/data]. . puffenberger eg: genetic heritage of the old order mennon- ites of southeastern pennsylvania. am j med genet c semin med genet , : - . . hinds da, seymour ab, durham lk, banerjee p, ballinger dg, milos pm, cox dr, thompson jf, frazer ka: application of pooled gen- otyping to scan candidate regions for association with hdl cholesterol levels. hum genomics , : - . . website : . [http://bioinformatics.tgen.org/software/tgen-array/]. additional file calculated k-correction factors for pooling on affymetrix k genechip mapping array based on , person database. click here for file [http://www.biomedcentral.com/content/supplementary/ - - - -s .zip] additional file the chp_ _ras.pl script processes one or more chp text files from affyme- trix k, k, and k ea snp chips, calculates ras and ras scores and outputs them in an excel spreadsheet. testing shows that for k chips, chp_ _ras.pl produces the same scores as those produced by affymetrix' gdas software. gdas does not calculate ras values for k chips. it should be noted that snps on k chips do not necessarily contain even numbers of sense and antisense probes and in fact only about % have sense and antisense probes. the remaining snps have a – or – probe bias towards either sense or antisense. this is important because part of the ras calculation involves taking the median of the "successful" probes and median may not be the best approach if only probes exist in one direction and some may have failed and been dis- carded. chp_ _ras.pl is distributed as part of tgen-array, a collection of perl scripts and modules that provide parsing and object-oriented inter- faces to common microarray files. the tgen-array site contains online documentation for all modules and scripts in the distribution including pages that show the source code so the code and algorithms may be inspected. click here for file [http://www.biomedcentral.com/content/supplementary/ - - - -s .xls] page of (page number not for citation purposes) http://www.biomedcentral.com/content/supplementary/ - - - -s .zip http://www.biomedcentral.com/content/supplementary/ - - - -s .xls http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= 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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.tgen.org/neurogenomics/data http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://bioinformatics.tgen.org/software/tgen-array/ 	abstract 	background 	results 	conclusion 	introduction 	results 	individual genotyping of snps for samples 	construction of pools 	calculation of allelic frequencies from pooled samples 	comparison of allelic frequencies: pooling vs. individual genotyping 	table 	identification of assay false positives 	identification of a disease locus from genotyping of pooled samples 	table 	discussion 	conclusion 	methods 	 k genechip® mapping array genotyping 	authors' contributions 	additional material 	acknowledgements 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . 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and environment university of michigan e. university, ann arbor, michigan - , usa stephen kaplan* departments of psychology and computer science and engineering university of michigan e. university, ann arbor, michigan - , usa abstract / the patterns of living, planning, and resource utilization as practiced in western countries for decades are not sustainable; change of some sort is essential. but what changes are appropriate, and, perhaps more important, will people accept them? the small experiment framework pro- vides a strategy for meeting the challenge of change. by en- couraging participation, limiting the scale of initial change, and incorporating some aspects of the familiar, many of the diffi- culties that make change so problematic can be mitigated or even eliminated. an exploration, from a psychological per- spective, of the characteristic difficulties surrounding potential change provides the context for a discussion of the compo- nents of the small experiment and an analysis of how these elements address these characteristic challenges. a compari- son to adaptive management is drawn, and several concrete examples illustrate how the strategy has been used success- fully to address a variety of environmental problems. the adoption of more environmentally appropriate patterns of living necessarily involves changing the cur- rent way of doing things, at both a personal and societal level. it is far less clear, however, exactly how best to foster potentially beneficial changes. there are not only questions of what to change but, of equal impor- tance, of what not to change, as some tempting inno- vations are fraught with hidden costs. in the recent past environmental organizations have often considered legislation, frequently at a national level, as the method of choice for achieving needed change. laws are far- reaching, often creating mandates on a large scale. though understandably an attractive approach, expe- rience has shown that legislating solutions to complex problems entails many risks; even the best intended legislation can have unanticipated costs so severe as to make the very problem addressed worse rather than better (sieber ). questions have also been raised about the efficiency and effectiveness of working at large scales to promote and encourage change. estava and prakash ( ) note that the popular phrase “think globally, act locally” suggests that actions taken at the local level can make as much (if not more) of a difference when compared to larger scale action. in addition to praising local action, the authors argue for thinking locally rather than glo- bally, pointing out that it is easier to “think wisely about what one knows well” (estava and prakash , p. ). drawing on what one “knows well” may facilitate taking action toward the attainment of environmental sustainability. if one takes seriously the concept of thinking locally, and acting locally, one must also take seriously the local human resources that are necessarily part of the solu- tion to a problem. all too often the public is a ne- glected, or largely untapped, resource. finding ways to utilize this resource might go a long way toward solving our environmental problems and achieving sustainabil- ity. before we begin our analysis, a note on terminology might be appropriate. one difficulty in discussing the need for change and the types of change necessary is the strong feelings some have about the terms sustain- able and environment. some argue that sustainability is an inadequate goal; given the damage done to so many of the world’s ecosystems, mere sustainability is not enough (hawken , mcdonough and braungart ). there is certainly merit to this position; none- theless current human practices are, as a whole, still far from sustainable (meadows ). the goal of sustain- ability is in the long run not sufficient, but in the short term it is clearly a step that is necessary and by no means trivial. thus, given the importance of incremen- talism (johnson , ) and the motivating power of attainable subgoals and “small wins” (weick ), we have chosen to use the term sustainability. at the same time, the approach we propose is equally appro- key words: sustainability; participation; experiment; local scale; adaptive management; environmental problem solving *author to whom correspondence should be addressed. doi: . /s environmental management vol. , no. , pp. – © springer-verlag new york inc. priate to the steps beyond sustainability, as framed by such farsighted visions as “the next industrial revolu- tion” (mcdonough and braungart ), “natural cap- italism” (hawken and others ), and the “global green deal” (hertsgaard ). in this article we propose an approach for achieving environmental sustainability that makes public partici- pation an essential and constructive part of the process. based on the “small experiment” concept (de young and kaplan , s. kaplan , r. kaplan ), this approach provides a way to test ideas without the risk of making a far-reaching or radical departure from the status quo. as a way to structure the process of solving environmental problems, the small experiment con- cept can facilitate the creation, evaluation, and the ultimate acceptance of potential solutions. by relying heavily on public participation, such an approach fos- ters familiarity with and a sense of ownership of solu- tions developed, both of which can aid the adoption process. public participation can also lead to explora- tions of possible change that can be both more exten- sive and more economical than would otherwise be possible. and finally, by calling on human talent and ingenuity, small experiments can provide not only effi- cient solutions but ones that are satisfying as well. the article first addresses the issue of why attempts to change the status quo are sometimes difficult for people. we then describe the small experiment as a possible approach to change. this is followed by a comparison of the small experiment approach to the adaptive management concept. we end with specific examples to illustrate how the small experiment strat- egy has been used successfully to address a variety of environmental problems. the challenges of change: a psychological perspective change suggests that something is going to be mod- ified, to be made different from its current incarnation. people have an inclination to be threatened by change (grabiner and miller , kaplan ). the thought of changing jobs, visiting a new city, or implementing new technology is frequently viewed with some appre- hension. this fear of change is especially acute during times when many changes are occurring at once (gra- biner and miller ). what is it that makes change so difficult? why is it that a species as mobile, creative, and adaptable as humans has such difficulty with change? more pragmatically, how can solutions to pressing en- vironmental problems be reached given this all too pervasive inclination? the first step in dealing with people’s resistance to change is to face the challenge of finding the “right” change; that is, a change that has a good chance of producing the desired effect and of minimizing unde- sirable side effects. then one can proceed in good conscience (and with better chance of success) to the second step, the challenge of getting the proposed change adopted. finding the “right” change the process of identifying a change that will im- prove a situation or correct a problem would, from a psychological perspective, be considered an instance of problem solving. if an individual or group stops to ponder a problem, the solution is presumably not ob- vious, because if it were, it would no longer be a prob- lem. thus, not knowing what to do next is the charac- teristic state of mind that leads to problem solving. not knowing what to do next is generally due to a deficiency in one of three areas: pertinent knowledge, a source of variation, or a means of evaluation (posner ). let us examine each in turn. solving problems typically requires knowledge. in the modern world experts are often counted on to have the knowledge essential for solving problems in their respective areas. examining the sorts of knowledge ex- perts possess is instructive. experts are trained to know what to focus on, what factors to attend to. this may be cash flow or water table level or form, line, color and texture, depending on the particular expertise in- volved. they also, because of their vast experience, have a rich repertoire of possibilities, of what might be done in a given situation and of what the consequences of a given decision might be. these are significant areas of knowledge and are often essential in solving a problem. it is important to remember, however, that there are other realms of knowledge that can also be useful. in the planning and management process, for example, knowledge of local tradition, local history, and local circumstances can play a central role (ostrom ). these sorts of knowledge tend not to be available from the individuals traditionally regarded as experts. not infrequently a problem may resist solution not because of a lack of knowledge but because a crucial kind of variation is missing. the variation that is needed, interestingly enough, is not in generating pos- sible solutions, but in ways of looking at the problem that needs to be solved. research on problem solving indicates that the most common difficulty when prob- lem-solving fails is that the usual ways of looking at the problem have not worked (posner , bardwell ). the way one thinks of a problem (or, more technically, the way one represents the problem) turns out to be the single most important determinant of k. n. irvine and s. kaplan whether the problem is solvable (posner ). the remedy for this might appear to be obvious: simply change the way of looking at the problem. this, how- ever, is easier said than done. not surprisingly, the way one perceives a problem is typically experienced as the right and natural way to understand the problem. in fact, it often seems to be the only reasonable way one could think about it. thus, although some alteration in the way the problem is conceptualized is often essential, such variation typically is not easily achieved. the third hazard in negotiating the problem solving process is evaluation (anderson ). there are two sorts of situations in which evaluation is essential. some- times, when an impasse is reached, a solution is found by relaxing constraints (e.g., time, budget, goals). one becomes willing to entertain solutions that do not meet all of one’s criteria. a solution obtained in this fashion may, of course, be unworkable; therefore, it is essential that the proposed solution receive an appropriate eval- uation. at the other end of the spectrum, it is some- times possible to think of a large number of solutions (a not uncommon situation in the context of planning and management). the difficulty then is not one of coming up with a solution but of deciding which solu- tion, from among many, to implement. here, too, eval- uation is essential. in both cases evaluation is exceed- ingly difficult due to the impossibility of predicting in advance how a proposed solution will work out. facilitating adoption once one or more possible “right” changes have been identified, there is the challenge of getting the proposed change or changes adopted. the reason this is so challenging can be stated simply, humans are much more comfortable with the known than the un- known (zajonc , tversky and kahneman , kaplan ). although this is seemingly an inconse- quential statement, it provides an important observa- tion about human nature. the “known,” whether it is a particular environment, person, or issue, is something about which one has some understanding. understand- ing and comprehension are critical for knowing what to do. being familiar with something is a way to achieve this understanding, whether that “something” is the surrounding physical or social environment or a more abstract concern (e.g., sustainability, ecosystem man- agement). it appears, therefore, that humans have good reason for preferring the familiar. (at the same time people are motivated to make modest exploratory forays from the stable base of the known. the approach we describe in the next major section takes advantage of this motivation). to understand why the bias toward the familiar is so powerful, it is useful to think about a specific instance of change. computer software (e.g., word processing, data management), which is constantly being up- graded, provides a pertinent example. while often use- ful, the modifications may create anxiety and perhaps some resistance to the change. actions that with the current version were routine, took little effort, and provided support for accomplishing a goal (e.g., writ- ing a report, organizing data), may suddenly take longer and require considerably more attention. there may be uncertainty as to whether one will be able to meet deadlines; one may also experience irritation at the prospect of the time needed to learn the new version. although perhaps a trivial example of the power of the familiar, this example illustrates the benefits famil- iarity brings. being familiar with a physical place (e.g., a city, a park, a neighborhood), an issue (e.g., forest management, water policy), or a relationship (e.g., be- tween selective harvesting and wildlife biodiversity) al- lows one to recognize where one is, make decisions, predict, and evaluate possible next steps of action (craik , kaplan ). familiarity, in essence, as the above example illustrates, helps a person function more effectively in the world. the ability to function in one’s surroundings, in turn, leads to feeling competent in one’s abilities. feeling as though one is good at and able to accomplish what one is doing is a powerful motivational force for humans (white , de young ). being able to function effectively is not the only reason to maintain the status quo. if people are resis- tant to change that in the long run is likely to work out, one would expect them to be even more hesitant where there is uncertainty about the alternative outcome (maccrimmon and taylor , slovik ). this con- cern may well be realistic. using a new, alternative approach does not guarantee that the goal of a more sustainable relationship with the natural environment will be met. the potential for unintended conse- quences does, unfortunately, exist (sieber ). cou- pled with these may be the fear that something valuable could be lost (e.g., an endangered species, a “way of life”) in the process of attaining sustainability. given the time and energy involved in learning a new way of doing things (whether it be managing forests or main- taining viable populations of endangered species), in the face of uncertain results the tendency to be skepti- cal of new approaches is hardly surprising. even if one knows it may not be the best alternative, there is indeed a strong inclination to stick with the current way of doing things in the face of possible future surprises. coping with change small experiments: a possible strategy perhaps if the process of change were less confusing and less overwhelming the resistance to adopting envi- ronmentally appropriate patterns of living would be minimized. in working to overcome this resistance to change, it would be particularly helpful if an approach could be found that enhanced familiarity with possible new alternatives. one way to lessen resistance and increase familiarity would be to introduce change in incremantal steps; small steps would maintain a certain level of the famil- iar while still allowing for exploration of alternatives. taking small steps would also make it possible to keep track of what effect each step had, an essential compo- nent for evaluating what one has done and deciding what step to take next. last, an approach that encour- ages participation would be useful. participation helps build familiarity and ownership of the process and in- corporates the local knowledge and local talent that all too often go to waste. the above description may sound similar to a con- cept known as “muddling through” (or simply “mud- dling”). there is indeed a similarity both to this con- cept and to “adaptive management” (holling , walters ). we discuss the former in this section because it provides pertinent background to the small experiment notion. the comparison to an adaptive management approach is discussed later in the article after the small experiment concept is presented more fully. “muddling through” is a phrase introduced by lindblom ( ) to reflect the incremental, even grop- ing, character of decision-making in large organizations (e.g., bureaucracies). lindblom suggests that decision- making is actually quite a slow process, characterized by taking modest steps forward rather than grand leaps of faith. johnson ( , ) subsequently applied this concept to environmental management. the small ex- periment concept is akin to muddling in that they both approach change through taking small, manageable steps. because steps are small, both techniques avoid big, potentially damaging mistakes. the scale of imple- mentation, however, differs; small experiments focus on change at the local level while most discussions of muddling center on change at a larger scale (e.g., a large organization or an entire country). both techniques address the human bias toward the familiar by tying proposed changes to results of past changes, but small experiments allow many solutions to be explored simultaneously. traditional muddling, by contrast, tends to focus on instituting only one policy at a time. this may be due to another difference between the approaches, namely, the role of citizen participa- tion. in muddling, the decision-making and implemen- tation process are typically carried out by experts. citi- zen input, when it occurs, may be sought primarily at the beginning of the process, when advice is garnered from those most likely to be impacted by a policy, or near the end, when the possibility of change is minimal. with small experiments, special emphasis is placed on providing creative opportunities for citizen involve- ment, permitting far more person power for parallel experiments. a frequent criticism of muddling is its inability to respond quickly. here the possibility of many parallel explorations is a major advantage of small experiments. trying out several alternatives at the same time makes it easier to incorporate modifications in response to changes in the environment or the process. the possi- bility of multiple alternatives being explored at once, however, requires some way to assess their effectiveness. the luxury of just “getting an intutive feel for how things worked out” is unlikely to be affordable or useful when many efforts are being implemented simulta- neously. the small experiment concept, thus, has an explicit focus on evaluation. the small experiment concept undoubtedly many activities that would appropri- ately be called small experiments are already under way. one might argue they are going on all the time. sometimes called pilot projects, small studies, field tests, or prototyping, they are often simply a local re- sponse to local problems—innovative programs that arise out of frustration with the status quo. if small experiments are indeed a way in which to try out new and different approaches to the way people– environ- ment relationships are managed, how can we make these efforts more helpful to the present state of affairs? how, also, might small experiments be made a more common approach to change? some insight into these issues can be gained by examining the components of the “ideal” small experiment. purpose or goal. small experiments have a specific purpose. this could be in the form of a question to explore (e.g., what would happen if. . .), a particular value to maintain (e.g., small-town character), or goal (e.g., improved water quality). identifying a purpose provides the motivation for exploring ways of changing how things have always been done. a purpose also focuses attention on the ultimate outcome rather than on any one specific solution. the process thus becomes an exploratory one; the proposed changes are a possi- ble solution rather than the solution. it is often easier to try something new if one knows that if it does not work or brings unintended consequences, one has not k. n. irvine and s. kaplan made a long-term commitment to it. viewing the pro- cess as exploratory also means that some method of evaluating it is required. having an articulated purpose provides clarity about what would constitute success or failure and facilitates the evaluation of the experi- ment’s effectiveness. information gathering and evaluation. formal or infor- mal tracking of what happens both during and after implementing an “exploration” is a vital component of a small experiment for two reasons. first, it allows for feedback to participants. this feedback lets people know how things are going in general (e.g., are popu- lation numbers of a certain endangered species increas- ing) and provides information to individuals active in the effort about how their particular contribution has helped. second, information gathering provides fodder for the process of determining what worked and what did not work. such evaluation helps assess what steps, if any, to take next as well as identifies findings that are appropriate to share with others. scale and scope. experiments need not be large and all-encompassing to be useful. what makes an experi- ment small, however, will vary from one circumstance to the next. “smallness,” therefore, can have a variety of referents. it may refer to the scale of the experiment or the elaborateness of the measurement, or the size of the sample. taking a local scale approach, for example, may help avoid many of the potential pitfalls often associated with larger scale projects, such as the lack of funds and potential for details to overwhelm the pro- cess. smaller-scale approaches often result in greater participation, with the scope often being dictated and managed by the people who are actually engaged in the experiment rather than by external considerations (e.g., source of funding). given the uncertainty associ- ated with change and the eternal question of whether the experiment will have the hoped-for results, perhaps it is safer to err on the side of too small rather than too big. this way any possible negative effects can be more easily remedied with minimal impact on the commu- nity. communication and dissemination of results. letting others know about a particular strategy taken and the results of the effort is also an important aspect of the small experiment approach to change. communicating the findings of a small experiment to a larger audience (people not directly involved in the project as well as people who live elsewhere) is essential if others are to profit and learn from the efforts. the strategy under- taken becomes, via dissemination, an available alterna- tive that others can try in their own setting. sharing information is one way to expand the level of knowl- edge of different approaches to sustainability. present- ing the information in concrete and vivid language with a suggestion that more information is available a little farther down the road can enhance familiarity (kear- ney ). meeting the challenges of change: small experiments and participation small experiments can in principle be carried out without the benefit of participation. it is our conten- tion, however, that there are often considerable bene- fits to involving local citizens in the process. in this section we consider three interrelated issues concern- ing local participation. first we examine the way such participation can facilitate finding a promising solu- tion. then we turn to the issue of how it can aid adoption once such a solution is identified. but these benefits are for naught if local citizens are unwilling to participate. thus, we conclude this section with a dis- cussion of the basis for believing that participation will be experienced as a positively valued activity. the impact of participation on finding the “right” change. the kind of knowledge that experts have is usually well represented in efforts to solve environmental problems. other realms of knowledge, however, are often over- looked. these include the history and idiosyncrasies of the setting, the special resources that might be avail- able, and what has been tried in the past. these kinds of knowledge are commonly held by local people. a vivid example of the different ways of looking at the same issue comes from the green revolution. clarkson ( ) describes a prototypic confrontation between an agricultural expert and an indigenous farmer of a less-developed country. based on maps and theory, the expert suggests that a certain crop be grown in a certain field. the farmer refuses, referring to ghosts traditionally associated with the field. although this may seem to be a straightforward conflict between reason and superstition, clarkson (a geographer) points out that the local is likely to be in possession of long-term, detailed information about the productivity of that particular field— despite the information on the generalized map and despite the local’s failure to de- scribe this information in a way understandable to the expert. thus, when local people are central to the problem-solving process a wider array of knowledge is available. participation in the planning and carrying out of small experiments on the part of locals reduces the danger of neglecting information that may be vital to finding a solution to a problem. the solution of difficult problems often depends on new and different ways of looking at the problem. this in turn requires diversity in how a problem is concep- tualized. incorporating local people into the process coping with change also addresses this need for diversity. people from the local community who are participating in the process are unlikely to have the same backgrounds, training, and perspectives the experts possess. thus, they are likely to bring a variety of perspectives to the process. and, to the extent that citizens come from different backgrounds and have different work identities, they are likely to differ not only from experts but from each other as well, another useful source of diversity. evaluating a potential solution prior to widespread implementation is another facet of the problem solving process that can be aided through participation. it is often difficult to adequately evaluate proposed solu- tions without actually trying them out. in fact, in many situations there is no substitute for trying out an inno- vation on a small scale first. peters and waterman ( ) describe how this approach to evaluation has become institutionalized in some of the country’s most highly regarded companies (e.g., ibm, m). local par- ticipation provides more people power for trying things out on a small scale. when a sufficiently large group of people is interested, it becomes possible to test several alternative solutions at the same time. in this way the problem-solving process can be moved along at a more rapid and informed pace and the implementation of sweeping changes without the benefit of prior small scale evaluation can be avoided. the impact of participation on adoption. people are often resistant and even disturbed by proposed change. one way to view this phenomenon is in terms of a lack of clarity. it is important to distinguish a lack of clarity from uncertainty. one can know a great deal about something and still face uncertainly (e.g., a surgeon facing surgery where the probability of a successful outcome is . ). people routinely make decisions where the outcome is uncertain. unclarity, by contrast, entails not understanding the pertinent issues, not knowing what is important in the situation, and not knowing how to evaluate what is going on. thus, not surpris- ingly, unclarity often inclines people to leave the situ- ation or to avoid thinking about it (kaplan , ). considerable evidence suggests that people react with discomfort and avoidance in the face of things they are unclear about (bruner and postman , maslow and diaz-guerrero , fortune ). because potential change is a likely source of unclarity, their negative reaction is hardly surprising. there are three key factors that reduce unclarity, namely, familiarity, understanding, and concreteness (s. kaplan , r. kaplan and others ). any activity that facilitates these three factors should reduce people’s almost reflexive resistance to proposed changes. the small experiment is an ideal vehicle for achieving this. by giving people the opportunity to try something out, their familiarity is, by definition, in- creased. the proposal becomes more concrete as they implement a small-scale version of it in the real world. it becomes more understandable as they participate in carrying it out and observe its workings. some might worry that those participating in a small experiment will come to favor the proposed change independent of its merits. there are two reasons why this is unlikely to be the case. first, if the experiment is going badly the participants are not likely to have the confidence and enthusiasm necessary to convince oth- ers of its value. second, persuading others is far more difficult when the results of the experiment are not favorable. the problem of adoption lies in the human tendency to resist change even when there is good reason to believe that it is needed and beneficial. a small experiment is a procedure that differentially fa- vors the acceptance of workable proposals. on the face of it, it might seem that the beneficial increase in clarity will only be experienced by those who have been directly engaged in a small experiment. if this were the case, it would be an important draw- back, as the individuals who have participated are likely to constitute only a small fraction of the total popula- tion impacted. but a small experiment readily becomes a case study, or, more informally, a story about what other people did. such shared experiences of having been involved in a small experiment have shown them- selves to be a powerful source of information that makes it easier for people to take appropriate action themselves (monroe and kaplan ). stories of this kind are more likely to be effective when people hear several of them rather than a single instance. this is not surprising because multiple examples are necessary to build a concept one can be comfortable with and con- fident of (hebb , posner , rosch ). for- tunately small experiments are generally inexpensive, so that the launching of several in parallel may well be feasible. further, such multiple experiments provide a sounder basis for action as well as a more effective means of building the understanding that facilitates adoption. will people actually get involved? these benefits of par- ticipation are, of course, contingent on a sufficient number of individuals being willing to participate. though this might seem to be a serious limitation of the approach, there are, in fact, quite a few reasons for believing that people find such opportunities both at- tractive and satisfying. despite a strong desire for settings that foster under- standing, humans, at the same time, have a natural inclination toward exploring and learning. opportuni- k. n. irvine and s. kaplan ties for exploration have, in fact, been shown to be highly preferred (kaplan and kaplan ). the small experiment provides an opportunity for meeting these two potentially conflicting needs. in small doses, and in settings that are relatively familiar, exploring new ap- proaches can be quite engaging and rewarding. explo- ration is, after all, a process of expanding one’s knowl- edge of the world. small experiments allow discovery to occur while involving small enough increments of change to permit a feeling of familiarity and under- standing to be preserved. second, because there are often fewer people and resources available for small-scale projects, involvement tends to be active rather than passive. thus a small experiment might involve developing and testing dif- ferent ways to reduce runoff as opposed to the more common citizen role of voting on a plan that was de- veloped by an outside expert. the relative scarcity of resources and people in an “understaffed” context has been shown to increase the level of participation as well (gump and barker ). fortunately, people tend to respond positively to opportunities to “do something” (kaplan and kaplan ), so the kinds of opportuni- ties provided by small-scale projects draw on human motivations and inclinations (e.g., to learn, to be in- volved) that may be difficult to express under other circumstances. as the name implies, a small experiment is some- thing that takes place at a modest scale. there are many advantages of such an arrangement. overall familiarity is maintained; mistakes, should they occur, are less costly. participants understand what they can do and how their actions contribute to the process as a whole. having this kind of an understanding of a situation has many advantages. it helps overcome the all-too-preva- lent belief that individuals cannot make a difference, makes involvement enjoyable, and frequently leads to a sense of ownership and confidence in the process. an important by-product of participation is the fact that people find it satisfying (de young – , ). in further support of this observation, wandersman ( a) found that in contrast to the those who did not participate, individuals in the participation condition “felt significantly more creative, responsible, helpful, and significantly less alienated and anonymous” (wan- dersman b, p. ). concerning the meaning of “small” and “experiment”. the expression “small experiment” refers to a way of learning from experience. it is an expression of the belief that there are many ways in which an experiment, a trying out of something, can be made less expensive and more manageable. it is an expression of the belief that an experiment need not be the intimidating task that it so often turns out to be. given the many contexts in which the small experiment concept can be applied, it is impossible to define the “small” aspect of a small experiment with any exactness. in essence, it refers to any modification from the usual approach that reduces the cost, the effort, the duration, or the number of people affected relative to the usual way of doing things. thus, the word small in small experiment plays a similar role to the word appropriate in appropriate technology; it is a reaction to the tendency of technol- ogy to get out of hand, to foster ever more elaborate and expensive solutions (schumacher ). there are two key consequences of such downsizing. first, the reality of current practice is that innovations in programs, policies, arrangements, and solutions are often put into effect on a large scale without the benefit of trying them first (schumacher , peters and wa- terman ). untold harm has been done by appar- ently good ideas that not merely failed the test of time but did so on an unconscionably large scale. thus, the focus on smallness is, to no small degree, an expression of frustration with current approaches. a second benefit of taking smaller scale action con- cerns the notion of “experiment.” the very idea of research has become so intimidating that it is not cur- rently considered by individuals who are ideally posi- tioned to learn from something that is about to happen anyway. there are so many new programs tried, so many opportunities to learn from experience that would be far more informative if they were thought of as experiments. not as official, traditional, experi- ments, but as approximations, as modest efforts, as ways of preserving essential components of research (i.e., purpose, evaluation, communication) without bringing with them the expensive baggage. the alternative to runaway technology in the realm of trying things out needs a name, a philosophy, and a legitimacy. it is our hope and intention to contribute to the small but grow- ing literature that attempts to address these needs. (it must be acknowledged that what one learns by experi- menting at a smaller scale does not guarantee that it will behave similarly at a larger scale. but it is nonethe- less vastly safer than not testing it at all.) small experiments and adaptive management— a comparison not surprisingly, the idea of employing a more ex- perimental approach to guiding environmental deci- sion-making is not without precedent. in particular, the adaptive management approach has received consider- able attention for its utilization of this concept (holling , walters , walters and holling , johnson a). it is useful to examine this approach both for its coping with change similarities and differences with respect to small exper- iments and for how it has fared in applied contexts. grounded in industrial operation theory (everett and ebert ), adaptive management was first ap- plied to natural resource management in the s (holling ). central to the concept is the issue of uncertainty (e.g., about the resource, about the effect of management efforts). unlike conventional manage- ment practices, which, for example, often attempt to make precise predictions and presume certainty, adap- tive management accepts as given the reality of incom- plete knowledge. it thus focuses on building opportu- nities to learn into the design and implementation of policies (holling , walters , walters and holling ). in this way, adaptive management em- phasizes learning by doing (walters and holling ), treating management actions and policies as hypothe- ses, designing and implementing them so as to gener- ate “critical information about the resource being man- aged” (johnson a, p. ). the approach typically includes several steps (holling , lessard , lee, , johnson, b). the assessment phase seeks to identify specific goals for the natural resource being managed (e.g., desired future condition of an aquatic ecosystem). these emerge from a collection of current knowledge about the resource with input from various stakehold- ers. the second phase identifies and assesses the signif- icance of gaps in knowledge and generates alternative management actions. the actual implementation of management actions is treated as an experiment. infor- mation is collected about the resource, impacts are monitored, and management actions are evaluated as to whether they are meeting management goals for the resource. the final step includes utilizing findings to revise policy as necessary. it is instructional to look at where adaptive manage- ment and the small experiment concepts overlap and where they diverge. both approaches attempt to bring a more scientific approach to the solution of practical problems. the emphasis on goal identification provides both concepts with a benchmark against which to de- termine whether efforts are addressing identified needs. information collected during the process of im- plementation provides the basis for this continuous evaluation in both adaptive management and small experiments. both approaches identify the importance of sharing information and findings. the audience and intent, however, differ. adaptive management focuses primar- ily on communicating findings to those immediately engaged in the process, with the intention of informing the implementation of management actions and poli- cies. small experiments emphasize dissemination of information to a wider audience. in addition to those involved in the project, it also targets individuals in the vicinity but not directly involved with the project at hand, as well as individuals further away geographically, working on similar or different projects altogether. in essence, although both approaches create a feedback loop so that a current activity can inform future deci- sion-making, the small experiments concept extends the dissemination beyond the immediate project at hand. a second, and perhaps more significant difference is that of participation. both approaches highlight the importance of participation, but the extent of involve- ment and who specifically is involved differ. in adaptive management participation occurs primarily during the goal identification phase, when relevant stakeholders are gathered to pool existing data about the resources and identify goals for the resource. stakeholders are often identified by the managing organization (e.g., forest service) and usually consist of individuals with relevant expertise and knowledge (e.g., biologists; pink- erton , shindler and cheek ). there is less involvement at later stages of adaptive management and few opportunities for the average citizen. the small experiment expressly relies on participation through- out the process by experts as well as nonexperts. in fact, the distinction between experts and nonexperts is pur- posefully deemphasized in the small experiment con- cept, viewing the resource user (e.g., individuals using resource for fishing, walking) as an important source of creativity and of valuable local information. a third difference is the scale of implementation. much of the current literature discusses the application of adaptive management to the management of ecosys- tems (lessard , gunderson ). ecosystems, or eco-regions, typically cross boundaries (e.g., political, ecosystem type, ownership), presenting biological, eco- nomic, and social complexity on a large scale. thus, adaptive management has tended to operate at and be perceived as a tool for larger scale management efforts. the small experiment concept suggests the value of applying an experimental approach to small-scale ef- forts as well (e.g., neighborhood water use campaign). in light of the above discussion about the small experiment and adaptive management concepts, it may be useful to focus on what can be learned about and, perhaps more important, applied to efforts to create environmentally appropriate patterns of management and living. adaptive management has been described as a tool for organizational decision-making, particu- larly for resource managers who are responsible for developing and implementing strategies and policies k. n. irvine and s. kaplan for natural areas (johnson b). the implementa- tion and use of the adaptive management concept have, however, met with limited success (mclain and lee , walters , gunderson , lee ). describing the concept as “scientifically sound” yet “so- cially challenged,” johnson ( a) identifies a need for greater participation from nonexperts. drawing from the conceptualization of small experiments, a role for nonexperts should exist beyond merely providing additional information about the resource; such partic- ipants can also contribute new perspectives and ap- proaches (gunderson ). participation by a broader array of stakeholders also has the added advantage of gaining greater acceptance for identified goals. even though the involvement might require a change in the approach taken, the ultimate goal may be reached rather than stymied by public indifference or outright opposition. adaptive management puts emphasis on a systematic identification and collection of environmental data, and the use of technology for the development of multiple management strategies (e.g., computer mod- eling). this focus can make the process costly and slow (walters and others , lee ). the small exper- iment concept takes a more informal, organic ap- proach to information collection and solution genera- tion. this could lead to missed information. yet both concepts have their advantages, and in many situations the two approaches can be complimentary. there are, however, situations where speed, diversity of input, and acceptability to the public are sufficiently important that the small experiment approach might be prefera- ble. in general, adaptive management and small experi- ments appear to share considerable common ground. both allow for the implementation of potentially risky strategies precisely because they are treated as experi- ments to be monitored and evaluated before adoption. at the same time, the adaptive management approach might benefit from adopting the broader concept of participation that is so central to the small experiment approach. can modest projects make a difference? is it realistic to think that such small-scale efforts can accomplish anything of substance in pursuit of the challenging goal of sustainability? a number of exam- ples indicate not only that this is possible but that it is an approach rich in potential. examples are drawn from different cultures, include national and interna- tional projects, and cut across several environmental issues. innovative efforts undertaken in davis, califor- nia; the amish community; curitiba, brazil; a small town in wisconsin; and ann arbor, michigan, demon- strate both the usefulness and the flexibility of the small experiment concept. energy efficient buildings—davis, ca the first example comes from attempts to reduce energy consumption. in the mid- s, in response to a national goal of energy conservation and conversion to renewable resources, davis, california, developed a lo- cal-level solution based on local needs, constraints, and resources rather than relying on national legislation to solve the problem. the city designed its own building code around local climate conditions in hopes of in- creasing energy efficiency of new buildings. the idea arose out of research done at the university of califor- nia, davis campus and was shaped by the comments and criticisms of many different segments of the com- munity, such as environmental groups, energy groups, consumer advocates, builders, and developers. evalua- tion was done primarily by those opposing the code to see if it really worked. one builder found that the inside temperature of unoccupied buildings, built un- der the new code, was °f cooler than the outside temperature on hot summer days, thus reducing the need for air conditioning and therefore energy use (brunner ). city leaders in davis have received numerous requests for information about their local energy policies. builders now accept the new code as efficient and cost-effective, and the code has been adopted in nearby counties (brunner ). the city of davis provides a nice example of decisions and policies that were devised, explored, and implemented at the local level, in contrast to legislation passed at the na- tional level, which would not have involved as many local groups and might not have adequately addressed local issues. farming technology usage—amish culture in the context of environmentally sustainable pat- terns of behavior, decisions need to be made both about new practices intended to increase sustainability and about proposed innovations that may turn out to reduce sustainability and thus should be modified or rejected. if sustainability is a serious goal, then it is a consideration with respect to any innovation, not merely those intended to have an effect on the envi- ronment. the next example illustrates the use of the small experiment concept to purposefully evaluate, and ultimately reject, a new technological innovation. the amish maintain a lifestyle that in many respects is remarkably sustainable. their practice is to examine innovation for consequences that may be quite far re- coping with change moved from the purposes that made the innovation attractive in the first place. the scale of these “experi- ments” remains small; the church units within amish communities are inherently small and often only a few families “try out” the new technology (huntington per- sonal communication). the effects of an innovation— a tractor, for example—are watched with keen interest and observations are communicated throughout the community both formally (e.g., at specific membership meetings) and informally (e.g., conversations with com- munity members). decisions about innovations are made against a backdrop of stability and community stemming from common, identifiable values shared by all in the community. the amish wish to maintain their separateness from the “english,” to maintain equal sta- tus among all members of their own community, and to maintain strong, healthy, families and communities (huntington ). thus, the amish will question em- bracing the use of tractors, despite the increase in productivity a tractor could bring to their farming, because observation and communication would indi- cate that the use could ultimately upset the balance within the community and the separateness of the com- munity from the larger society. public transportation and waste management— curitiba, brazil curitiba, brazil, provides an inspiring example of an attempt both to preserve key values and to make im- provements with respect to both sustainability and the quality of life. throughout efforts to modernize the city, the use of high-tech (and expensive) practices, believed to be essential to a modern city, was ques- tioned; well-thought-out alternatives were explored, evaluated, and if determined to be appropriate, imple- mented. a particularly striking example was the deci- sion to emphasize public transportation rather than individual car use, and to implement this decision using buses rather than a subway system. the city achieved remarkably high utilization levels through the use of such elements as dedicated bus lanes, subway-like ter- minals, and a public-private partnership for operating the buses. interestingly, another factor favoring the choice of a bus system was the fact that the city was already familiar with the operation and management of buses. thus, the thorny issue of how to provide trans- portation routes for the city’s citizens was accomplished at a fraction of the cost of a subway system, drew on existing skills and knowledge, and had minimal impact on the environment. this is only one of the many experiments occurring at the same time in this fast- developing city. each is targeted to a specific problem and places emphasis on appropriate technology, in achieving an environmentally and humanly satisfactory solution. as a result, the city has kept its pollution and crime rates lower and its educational level higher than is often the case with rapidly developing cities (rabin- ovitch and leitman ). curitiba has also created a waste management sys- tem that does not center around mechanized trash- separation plants—a recommended necessity for cities generating more than , tons of solid waste a day (rabinovitch and leitman ). the city has instead implemented labor-intensive programs that encourage citizen participation. through both a separation pro- gram, in which citizens voluntarily separate their waste, and a purchase program, which allows families from neighborhoods difficult to service to exchange bags of trash for bus tokens, food, and school notebooks, cu- ritiba has managed to clean up the city, reduce waste going to landfills, provide employment, and conserve resources (rabinovitch and leitman ). despite many important differences, curitiba perhaps shows a parallel to the amish example in their ability to say “no” to conventional wisdom and to the use of expensive high technology. recycling—wisconsin the last two examples focus on efforts made by individuals not normally viewed as agents of change. the first is of a wisconsin woman nearing retirement who started a recycling company in (sinclair and others ), well before recycling was a household word, let alone a household behavior. her goal—to reduce the amount of waste destined for the land- fill—was an outgrowth of her well-water being polluted due to leakage from a nearby landfill, and her realiza- tion that landfills were not the only solution to dealing with solid waste. as of the company had recycled , tons of refuse, saving the county $ , in dumping fees (sinclair and others ). the idea of recycling was initially greeted with skepticism. however, according to the company’s owner, this attitude has changed dramatically; people now bring items that are washed, flattened, and ready for recycling. thus, in this case, an individual brought change to the traditional practice of sending all household solid waste to a land- fill. water quality and lawn care—ann arbor, mi the issue of water quality provides the venue for the final example. officials responsible for watershed pro- tection are increasingly concerned about the impact of lawn chemicals on the quality of groundwater. due to the vast commercial investment in providing such lawn care and the cultural attachment to the traditional k. n. irvine and s. kaplan lawn, addressing this problem is likely to be challeng- ing. perhaps children, who will ultimately pay the price for chemically green lawns, could be an appropriate vehicle for this message. an experiment at the scale of a local neighborhood provides an indication of how such an approach might work (romaker ). a group of preschool students in ann arbor, michigan, went door-to-door, explaining to their neighbors the implications of and their concerns with lawn chemicals. they asked individuals to sign a pledge agreeing to stop applying chemical lawn sprays. about % of the peo- ple with whom the children talked agreed to sign the pledge and received a “safe lawn” sign to place on the lawn to let others know of their commitment (romaker ). results of the children’s efforts were written up in the local newspaper. there are many problems implicit in moving from current practice to sustainability. the small experiment approach is presented as a framework for addressing the challenges of building a sustainable relationship between people and the natural environment. the ap- proach can be thought of in terms of three themes: local emphasis, experimental stance, and information sharing (see figure ). the examples cited above all incorporate, to a greater or lesser degree, the three themes and provide insight into how they can be uti- lized. local emphasis can occur in a variety of ways. for instance, in the examples cited above, note the differ- ent scales at which efforts to change the status quo can be successful— city, community, small town, and neigh- borhood. in each instance, an issue (e.g., water quality, energy conservation, community) was addressed in an exploratory manner, at a scale smaller than the issue might usually be treated, with an emphasis on learning about consequences before extensive resources were committed and great disruption occurred. participa- tion by individual citizens, a key concept in the small experiment approach, goes beyond providing input on other people’s decisions. citizens can have a role in shaping policy, as in the case of davis’s energy policy, or in the amish’s decisions regarding use of technology. citizens can actively participate in alleviating problems, as in curitiba, or could develop a program to reduce the use of toxic chemicals on lawns, as in ann arbor. in each case, local knowledge is utilized in meaningful ways. the examples also illustrate the numerous ways in which the purpose or goal, central to the experimental stance, can be formulated. for example, the amish emphasize maintenance of community-oriented values, the city of curitiba is working toward solutions of civic and environmental problems in the context of citizen participation, whereas in the other examples conserva- tion of resources, such as water quality or energy, is the focus. how information is gathered and evaluated also varies, from the informal networks between neighbors, as with the amish and the preschool children, to more formal venues such as the testing done by opponents of the davis housing code system. information sharing was also present in all cases. findings and results were communicated and dissemi- nated via word-of-mouth and informal networks of col- leagues (as between davis city officials and other cities) and more formal channels, such as biannual meetings of the community (as done in the amish setting) or newspaper articles. it is hopeful to see that many noteworthy efforts have been and are being made to address the issue of envi- ronmental sustainability. at the same time, the efforts cited, though promising, could benefit from a more vivid awareness of the importance of the local emphasis and experimental stance themes, which might have increased their effectiveness. additionally, the scope of information sharing was limited. the dissemination that did occur was due as much to chance factors as to conscious intent. if the small experiment is to realize its full potential, more systematic means of making this rich source of information available need to be em- ployed. by encouraging participation in the explora- tion of alternative solutions, and by making participants aware of the usefulness of the various features of a small experiment, many worthy goals would be accom- plished. knowledge about what works would grow rap- idly. vast quantities of human talent and ingenuity now underutilized or wasted would be engaged in stimulat- ing and worthwhile effort. the incorporation of local knowledge and skills would increase the number and diversity of alternatives worthy of consideration. addi- tionally, familiarity with new alternatives and openness to making appropriate changes is likely to grow as the level of participation increases. finally, there is reason figure . small experiment themes. coping with change to believe that both life satisfaction and commitment to environmental concerns would benefit as well. acknowledgments we would like to thank trudy huntington for her help in exploring the connection between the small experiment concept and the amish approach to living. we are also indebted to jodi asarch, gordon bradley, rachel kaplan, steve lippman, elizabeth mccance, bill nichols, and ali phillips for their input. the feed- back from the three reviewers, richard e. saunier, d. scott slocombe, and robert staib, was most helpful and 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the most frequent organic aciduria detected in tandem mass spectrometry–based neonatal screening programs. the phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. mcc is a heteromeric mitochondrial enzyme composed of biotin-containing α subunits and smaller β subunits. here, we report cloning of mcca and mccb cdnas and the organization of their structural genes. we show that a series of mcc-deficient probands defines two complementation groups, cg and , resulting from mutations in mccb and mcca, respectively. we identify five mcca and nine mccb mutant alleles and show that missense mutations in each result in loss of function. article find the latest version: https://jci.me/ /pdf http://www.jci.org http://www.jci.org/ / ?utm_campaign=cover-page&utm_medium=pdf&utm_source=content https://doi.org/ . /jci http://www.jci.org/tags/ ?utm_campaign=cover-page&utm_medium=pdf&utm_source=content https://jci.me/ /pdf https://jci.me/ /pdf?utm_content=qrcode introduction mcc (ec . . . ) is a biotin-dependent carboxylase that catalyzes the fourth step in the leucine catabolic path- way. isolated, biotin-resistant mcc deficiency (also known as methylcrotonylglycinuria [mim ]) is inherited as an autosomal recessive trait ( ). the clinical phenotype is highly variable: some patients present in the neonatal period with seizures and muscular hypoto- nia ( , ); others are asymptomatic women identified only by detection of abnormal metabolites in the neona- tal screening samples of their healthy babies ( ). there is a characteristic organic aciduria with massive excretion of -hydroxyisovaleric acid and -methylcrotonylglycine, usually in combination with a severe secondary carnitine deficiency. mcc activity in extracts of cultured fibrob- lasts of patients is usually less than % of control. no correlation between the level of residual enzyme activity and clinical presentation has been observed. tandem mass spectrometry (ms/ms), recently intro- duced for newborn screening, provides for the first time to our knowledge a way to detect a large variety of organ- ic acidurias including mcc deficiency ( ). surprisingly, mcc deficiency appears to be the most frequent organ- ic aciduria detected in ms/ms screening programs in north america ( , , ), europe ( ), and australia ( ), with an overall frequency of approximately in , . mcc is a member of the family of biotin-dependent carboxylases, a group of enzymes with diverse meta- bolic functions but common structural features ( , ). members of this family have three structurally con- served functional domains: the biotin carboxyl carrier domain, which carries the biotin prosthetic group; the biotin carboxylation domain, which catalyzes the car- boxylation of biotin; and the carboxyltransferase domain, which catalyzes the transfer of a carboxyl group from carboxybiotin to the organic substrate spe- cific for each carboxylase ( , ). in addition to mcc, there are three other biotin-dependent carboxylases in humans: propionyl-coa carboxylase (pcc), pyruvate carboxylase (pc), and acetyl-coa carboxylase (acc) ( , ). the genes for all human carboxylases except mcc have been cloned and characterized ( – ). mcc carboxylates -methylcrotonyl-coa at the - carbon to form -methylglutaconyl-coa (figure ) ( ). the reaction uses atp and bicarbonate and is reversible. bovine mcc has an approximate size of kda and appears to comprise six heterodimers (αβ) ( ). like pcc, mcc has a larger α subunit, which covalently binds biotin, and a smaller β subunit ( ). mcc is predominantly localized to the inner mem- brane of mitochondria and is known to be highly expressed in kidney and liver ( ). cdnas encoding both subunits of mcc recently have been cloned in ara- bidopsis thaliana and other plants ( , ). here we report cloning of human mcca and mccb cdnas, confirmation of their identity by biochemical and molecular studies, and identification of mutations in mcc-deficient patients. the journal of clinical investigation | february | volume | number the molecular basis of human -methylcrotonyl-coa carboxylase deficiency matthias r. baumgartner, , shlomo almashanu, , terttu suormala, cassandra obie, , robert n. cole, seymour packman, e. regula baumgartner, and david valle , mckusick-nathans institute of genetic medicine, johns hopkins university, baltimore, maryland, usa metabolic unit, children’s hospital, university of basel, basel, switzerland howard hughes medical institute, and department of biological chemistry, johns hopkins university, baltimore, maryland, usa department of pediatrics, university of california, san francisco, california, usa address correspondence to: david valle, pctb, johns hopkins university, n. wolfe street, baltimore, maryland , usa. phone: ( ) - ; fax: ( ) - ; e-mail: dvalle@jhmi.edu. received for publication december , , and accepted in revised form january , . isolated biotin-resistant -methylcrotonyl-coa carboxylase (mcc) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry–based neonatal screening programs. the phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. mcc is a heteromeric mitochondrial enzyme composed of biotin-containing α subunits and smaller β subunits. here, we report cloning of mcca and mccb cdnas and the organiza- tion of their structural genes. we show that a series of mcc-deficient probands defines two complementation groups, cg and , resulting from mutations in mccb and mcca, respective- ly. we identify five mcca and nine mccb mutant alleles and show that missense mutations in each result in loss of function. j. clin. invest. : – ( ). the journal of clinical investigation | february | volume | number methods patients, complementation analysis, and mcc assay this study includes mcc-deficient probands, all of whom had the diagnostic pattern of organic acid excre- tion and less than % mcc activity in extracts of cul- tured skin fibroblasts. clinical and biochemical data of ten patients have been reported in the literature: ( ), ( ), ( ), ( ), ( ), ( ), ( ), ( ), ( ), and ( ). fibroblasts or dna of the remaining patients were referred by b.t. poll-the (patient ), h.g. koch (patient ), j. smeitink (patient ), u. von döbeln (patient ), r.d. de kre- mer dodelson (patient ), and d.h. morton (patient ). skin fibroblasts were cultured in eagle’s minimal essential medium (life technologies inc., rockville, maryland, usa) supplemented with mmol/l l-gluta- mine and % fcs. complementation analysis was modified from that described by wolf et al. ( ). briefly, we produced heterokaryons by a - or -second treatment with % peg harvested the cells days later and measured mcc activity as described previously ( , ). mcc activity in cultures of mixed but unfused cells was subtracted as blank and self fusions were included as a negative control. expression of biotin-containing proteins in fibroblasts we harvested cultured fibroblasts by trypsinization, disrupted the washed cells by homogenization, har- vested the mitochondria by differential centrifugation ( ), suspended them in sds-buffer ( mm tris- acetate, % sds [wt/vol], mm dtt, . % [wt/vol] bromphenol blue), and dissolved the proteins by boil- ing for minutes. the cellular proteins were separated by sds-page, and the biotin-containing proteins were detected with an avidin alkaline phosphatase conjugate (avidin-ap, : , ; bio-rad laboratories ag, glat- tbrugg, switzerland). fibroblasts of an unaffected control, a patient with isolated pc deficiency, and a patient with isolated pccα deficiency were used to confirm the identity of the protein bands. cdna cloning, sequence analysis, and chromosome mapping we obtained two mouse (ai and ai ) and two human (aa and aa ) mcca est clones, and one human mccb (ai ) est clone from the image consortium (genome systems inc., st. louis, missouri, usa) and sequenced both strands with an abi automated sequencer. to design primers corresponding to the ′ utr of our putative mcca, we used the sequence of the mcca est clone aa (not available commercially). we used primers dv (sense, ′-gacgcagctgcctctg tac) and dv (sense, ′-tggccgggctccagggacatg), complementary to the ′ utr, and dv (antisense, ′-aactgctctttatgagacccc), complementary to the ′ utr, to amplify full-length human mcca from a human retina cdna library ( ); and primers dv (sense, ′-aggacctgagctcagcttcc) and dv (sense, ′-tcggtgcccgccgccatg), complementary to the ′ utr, and dv (antisense, ′-actg- taacagcctcatgttcg), complementary to the ′ utr, to amplify full-length human mccb from the same human retina cdna library ( ). we gel-purified the pcr products and sequenced them directly. the sequence alignments were prepared with megalign (dnastar inc., madison, wisconsin, usa). we mapped hsmcca using the genebridge radia- tion hybrid panel (research genetics inc., huntsville, alabama, usa) using primers dv (sense, ′- tttgtcgtctcagactcgatg) and dv (antisense, ′-agtcagaaaaataaggccaacc) corresponding to ′ flanking intronic sequence of exon and ′ flanking intronic sequence of exon . isolation and mass spectrometry analysis of biotin- containing proteins enrichment for biotin-containing proteins. we homogenized . g of flash frozen male mouse kidney in . ml buffer a ( mm tris-hcl [ph . ], mm dtt, mm edta, . % (vol/vol) triton x- , % (vol/vol) glycerol, µm dmsf, tablet cocktail protease inhibitor (boehringer-mannheim) and centrifuged the homogenate at , g for minutes at °c. peg was added to the supernatant to a final concentration of % (wt/vol), and the mixture was centrifuged at , g for minutes at °c. the pellet was resus- pended in ml of buffer a, approximately × pre- washed m streptavidin dynabeads (dynal inc., lake success, new york, usa) were added, and the slur- ry mixed by rotating for hour at °c. we washed the beads five times with buffer b ( . m kcl in buffer a), resuspended them in µl of xi protein loading buffer, boiled for minutes, and placed the solution on ice. we loaded µl in each lane of a % polyacry- lamide gel and stained the separated proteins with coomassie brilliant blue r . s-carboxymethylation and proteolytic digestion. we per- formed in gel digestion of the proteins using the coomassie blue–stained sds-polyacrylamide gels according to williams et al. ( ) with the following modifications. cystines were modified by car- boxymethylation as described elsewhere ( ). after rehydrating gel pieces with ng/µl tpck-treated trypsin in % acetic acid, excess trypsin solution was removed and the gel piece was covered with gel vol- umes of mm nh hco (ph ), at °c for hours. the resulting tryptic peptides were extracted from the gel piece with % acetonitrile in . % tfa and concentrated by drying. mass spectrometry analysis. tryptic peptides were resus- pended in % acetic acid and loaded into a fused silica capillary column ( µm inner diameter) packed with cm of µm c reverse-phase resin (ymc inc., atlanta, georgia, usa) as described elsewhere ( ). a -minute, . – % methanol gradient in % acetic acid was applied to the column at f low rates of – nl/min. eluting peptides were electrosprayed directly into a finnigan lcq atmospheric pressure ionization quadrupole ion trap mass spectrometer (thermoquest corp., san jose, california, usa). pos- itive-ion mass spectra were obtained at using xcal- ibur software (thermoquest corp.). peptides were fragmented by a % collision energy using a two- atomic-mass-unit isolation width. fragmentation data were screened against the protein.nrdb.z data- base from the frederick biomedical supercomputing center (ftp://ftp.ncifcrf.gov/pub/nonredun/) using the sequest browser ( ) (thermoquest corp.). mutation analysis by rt-pcr and genomic pcr we extracted rna and genomic dna from cultured skin fibroblasts and/or blood using the puregene rna and dna isolation kits (gentra systems, minneapolis, minnesota, usa) and performed rt-pcr using – µg fibroblast rna and a cdna cycle kit (invitrogen corp., carlsbad, california, usa) following the manu- facturers’ recommendations. we generated first-strand cdna with primers dv (antisense, ′-gacc- caaatgcatgattctcc), complementary to sequence in the mcca ′ utr region, and dv (antisense, ′-ggtagaaaagtacaa tgcacag), complementary to sequence in the mccb ′ utr region. we then ampli- fied first-strand mcca cdna with primers dv and dv to generate a , -bp fragment (– to + , where + is the a of the initiation methionine codon); and we amplified first-strand mccb cdna with primers dv and dv to generate a , - bp fragment (– to + ). in some instances, when the amount of amplified product was inadequate, we went through a second round of pcr with nested primers dv and dv . we gel-purified the pcr products and sequenced them directly. to confirm mutations identified in rt-pcr products, we amplified a genomic fragment containing the corre- sponding exon using flanking intronic primers and sequenced the pcr product directly. in the compound heterozygous patients in whom we identified only one of two alleles in rt-pcr products, we amplified and sequenced all exons and flanking intronic sequences. all pcr reactions ( µl) contained primers ( ng each), × standard pcr buffer (life technologies inc.), dntps ( µm), and taq polymerase ( . u; life tech- nologies inc.). the sequences of all primers are available upon request. to survey a control population for the identified mis- sense mutations, we amplified the relevant exon from genomic dna and performed allele-specific oligonu- cleotide analysis as described previously ( ). construction of wild-type and mutant human mcca/b expression vectors we ta cloned the full-length human mcca (– to + ) and mccb (– to + ) cdnas into pcr blunt ii topo (invitrogen corp.). to introduce the mcca mutations r s, a v, and l p, we har- vested an -bp acci fragment from rt-pcr–ampli- fied patient cdna and subcloned this fragment into the pmcca-topo construct. we then transferred the wild-type and mutant mcca constructs into a mam- malian expression vector (ptracer-cmv ; invitrogen- corp.) at the ecor i site. this vector contains a green fluorescent protein (gfp) gene fused to the zeocin resistance gene. similarly, to introduce the mccb mis- sense mutation e q, we harvested a -bp econ i/bste ii fragment from rt-pcr amplified patient cdna, subcloned this fragment into the pmccb- topo construct and then transferred the wild-type and mutant constructs into ptracer-cmv . to intro- duce the mccb missense mutations s l, v m, r c, p r and r c, we harvested a -bp bste ii/sfi i fragment from rt-pcr–amplified patient cdna and subcloned this fragment directly into mccb-ptracer-cmv . we sequenced all constructs in both directions to validate the sequences. transfections we transformed primary fibroblasts from proband (homozygous for mcca q fs(+ ), from proband (homozygous for mccb s l), and from a con- trol as described previously ( ). for expression stud- ies, we electroporated the indicated constructs into transformed cells as described elsewhere ( ). we har- vested the cells after hours and measured mcc and pcc activity radioisotopically as described previously ( ). our standard mcc assay enables us to reliably detect activity as low as – pmol/min/mg protein. all the journal of clinical investigation | february | volume | number figure the mcc-catalyzed reaction and its position in the leucine catabol- ic pathway. the dashed arrow indicates the metabolites that accu- mulate due to deficiency of mcc. the journal of clinical investigation | february | volume | number transfections were in duplicates. transformed fibrob- lasts from an unaffected individual were used as con- trol. transfection efficiency was assessed by coexpress- ing gfp in the same construct. results genetic complementation and assignment to mcca or mccb as an initial step in defining the molecular basis of mcc deficiency, we performed biochemical and somatic cell genetic studies with fibroblasts from mcc-deficient probands. using restoration of mcc activity in peg-induced heterokaryons of fibroblasts as an end point, we defined two complementation groups (cgs), one comprising eight probands (mcc- cg , - ) and the other, six probands (mcc- cg , - ) (data available upon request). given that mcc is composed of αβ heteromers, we antici- pated that the two cgs likely corresponded to muta- tions in genes encoding the α and β subunits of mcc (encoded by mcca and mccb, respectively). to investigate the complementation phenotype at the protein level, we examined the expression of the mccα subunit using the covalently bound biotin as a tag in fibroblasts of five of six cg and in all of the eight cg probands (figure ). in cg , mccα was not detected in four of five probands, whereas in the remaining cg cell line (proband ), the mccα band was at least as intense as in controls (figure ). in all the cg cell extracts, mccα was reduced but present. these results suggested that mcc-cg is caused by mutations in mcca and, by exclusion, mcc-cg , by mutations in mccb. identification of mammalian candidate mcca and mccb cdnas database search. we used the amino acid sequences of a. thaliana mcca and mccb ( , ) and the tblastn algorithm to probe the public est data- bases to identify murine and human cdnas encoding candidate mccas and mccbs. we used the murine candidates to assemble full-length mouse putative mcca cdna (genbank accession number: mmmc- ca: af ) and the human candidate ests to design primers corresponding to the predicted ′ and ′ utr of the putative human mcca and mccb cdnas. using these primer pairs, we amplified a sin- gle fragment of the predicted size for both mcca and mccb from a human retina cdna library ( ). gen- bank accession numbers: hsmcca: af . hsm- ccb: af . we used additional ests to extend the ′ and ′ utr sequences. the sequence of the human mcca candidate has bp of ′ utr, a , -bp orf, and bp of ′ utr extending to a polyadenylation signal (aauaaa). the murine can- didate mcca cdna has bp of ′ utr, a , -bp orf, and bp of ′ utr extending to a probable polyadenylation signal (auaaa). the sequence of the amplified human mccb candidate has bp of ′ utr, a , -bp orf, and bp of ′ utr. the candidate cdnas predict a human mccα of amino acids with a calculated molecular mass of kda, a mouse mccα of amino acids with a calcu- lated molecular mass of kda, and a human mccβ of amino acids with a calculated molecular mass of kda. human mccα has % and % identity to mccα of mouse and a. thaliana, respectively (figure a). human mccβ has % identity to mccβ of a. thaliana (figure b). similar to pcc, the mccα subunit contains an nh -terminal biotin carboxylation domain and a cooh-terminal biotin carrier domain ( ). the biotin carrier domain is centered on the motif amkm, which is found in most biotinylated proteins (figure a) ( ). biotin is covalently attached to the ε-amino group of lysine ; the ε-biotinyl lysine amide is termed biocytin (figure a). as with other biotin- dependent carboxylases, there is a conserved (a)pm motif residues nh -terminal and a hydrophobic residue (f ) - residues cooh-terminal of bio- cytin ( ). the biotin carboxylation domain is located in the nh -terminal two-thirds of mccα and is linked to the biotin carrier domain by a less-conserved (only % identity to a. thaliana residues - ) “hinge” region of residues - . additionally, there is per- fect conservation of residues (figure a, asterisks) that are highly conserved among all biotin-dependent figure expression of the biotin-containing mccα subunit in fibroblasts. proteins in mitochondrial enriched fractions from cultured fibrob- lasts were separated by sds-page, and the biotin-containing sub- units of mcc, pcc, and pc were detected with an avidin alkaline phosphatase conjugate. most, but not all, cg -probands lack the mccα subunit, while it is detectable in all cg probands. the journal of clinical investigation | february | volume | number figure sequence alignment of human mccα and mccβ with orthologs from mouse and a. thaliana. amino acids identical to the human sequence are highlighted. missense mutations identified in mcc-defi- cient patients are indicated above with the substituted amino acid. potential cleavage sites for the nh -terminal mitochondrial leader sequences are indicated by vertical arrowheads. (a) sequence align- ment of human, mouse, and a. thaliana mccα. the predicted atp- binding site in the nh -terminal biotin carboxylation domain and the predicted cooh-terminal biotin carboxyl carrier domain are indicated by solid and dashed over- lines, respectively. the arrow indi- cates the lysine residue that links covalently to biotin (biocytin). the residues marked with an asterisk within the biotin carboxylation domain are thought to play an important role in catalysis ( , ). (b) sequence alignment of human and a. thaliana mccβ. the putative -methylcrotonyl-coa binding domain is indicated by a solid over-line. the journal of clinical investigation | february | volume | number figure coomassie-stained sds-page gel of biotin-containing proteins puri- fied from a mouse kidney extract with streptavidin dynabeads. we excised the proteins from the gel, digested with trypsin, and analyzed the tryptic peptides using maldi-tof and liquid chromatography coupled to esi-ms/ms. we analyzed all the indicated bands includ- ing those that we identified as mccα and β. carboxylases and thought to play a role in catalysis ( , , ). consistent with this prediction, the putative carboxylation domain contains the conserved sequence ggggkgmriv at positions - (figure a), which is part of the atp binding pocket in the biotin carboxylation domain of escherichia coli acc ( ) and is similar to a consensus p-loop atp-binding site [g′xgk(ts)] ( ). the β subunit has a putative coa binding motif (figure b) ( , ). mccα and β have candidate nh -terminal mito- chondrial targeting sequences with multiple arginine residues and a paucity of acidic residues ( ). possible cleavage sites are indicated by vertical arrowheads in figure . because the sequence of mitochondrial lead- ers is not highly conserved, we favor the more cooh- terminal cleavage site in mccα just before a highly conserved region (figure a). isolation and mass spectrometry analysis of biotin-contain- ing proteins. we also used a biochemical strategy to identify mccα and β. we enriched biotin-containing proteins in a mouse kidney extract using streptavidin dynabeads and separated these by sds-page (figure ). using maldi-tof and electrospray ionization mass spectrometry (esi-ms/ms), we analyzed tryptic fragments of these proteins to confirm the identity expected on the basis of the size of acc, pc, and pccβ. the analysis of the expected pccα identified, in addition to fragments derived from pccα, unique fragments corresponding to the conceptual transla- tion of the putative murine mcca cdna (figure ). we identified peptides with % identity to the putative murine mccα subunit covering amino acids or % of the putative full-length murine mccα. the protein comigrating with the -kda marker (fig- ure ) contained tryptic fragments with sequences cor- responding to the conceptual translation of the puta- tive human mccb cdna. we identified peptides with % identity to the putative human mccβ sub- unit covering amino acids or % of the putative full-length mccβ. these sequence results strongly supported the identity of the mammalian mcca and mccb cdnas. organization of human mcca and mccb structural genes during the course of determining the structural organization of these genes by long-range pcr and direct sequencing, we identified sequences correspon- ding to mcca and mccb in the human high through- out genome sequence database (genbank accession numbers ac and ac , respectively). these clones contain exons encoding the complete mcca and mccb cdnas and provide all exon/intron boundaries including the flanking intronic sequences with one exception ( ′ flanking intronic sequence of mcca exon ). mcca has exons and mccb exons. given that the draft sequence has gaps, our information on the size of some introns is incomplete. because the mcca gene was not mapped in the uni- gene database, we searched the flanking sequence in the bac clone containing mcca for other mapped genes. we identified the unigene cluster hs. represented by several ests present in the mcca genomic clone and localized to q -q (d s - d s ). to confirm this localization, we used the genebridge radiation hybrid panel to regionally localize mcca . cr from the wi- marker cor- responding to the same region on chromosome . similarly, we identified unigene cluster hs. representing several est clones covering the ′ end of the mccb cdna. this cluster maps to chromosome q -q . (d s -d s ). patients with isolated mcc deficiency have mutations in mcca or mccb to confirm the identity of mcca and mccb, we sur- veyed these genes for mutations in our collection of unrelated mcc-deficient probands. we grouped the probands according to their cgs and used rt-pcr to amplify the corresponding mrna from their cultured skin fibroblasts. we sequenced the entire orf in each proband and confirmed all mutations by direct sequencing of pcr-amplified genomic dna. in two additional probands ( and ) (ref. ) from the amish/mennonite population in lancaster county, pennsylvania, we searched for mutations by direct sequencing of pcr-amplified genomic dna. we iden- tified five mcca mutant alleles in cg cell lines accounting for ten of possible mutant mcca genes (table ). the mutations include three uncomplicated missense mutations (figure a), one missense mutation that alters splicing (figure a), and one -bp insertion. in cg cell lines, we identified nine mccb mutant alle- les accounting for of possible mutant genes (table ). the mutations include uncomplicated missense mutations (figure b), one missense mutation that alters splicing (figure b), one splice site mutation, and one -bp insertion. in spite of sequencing all exons and flanking intronic sequences (with the exception of the alleles has a low frequency in this population. we did not screen for alleles for which we did not have the appropriate control population (vietnamese, mccb- r q, -p r; turkish, mccb-s l, -v m). discussion using a combination of homology probing and mass spectrometry we cloned human mcca and mccb full length cdnas. the conceptual translation of mcca shows the expected nh -terminal biotin carboxylation domain and the cooh-terminal biotin carboxyl carrier domain (figure a), separated by a less conserved “hinge” region ( , ). presumably because of differ- ences in substrate specificities, carboxyltransferase domains are less conserved among biotin-dependent carboxylases. mcc catalyzes the carboxylation of methylcrotonyl-coa ( ). the acceptor binding site is thought to be on mccβ ( , ). consistent with this, human mccβ shares high identity with a. thaliana mccβ ( %) and only % identity with human pccβ ( ) that supports the role of the β subunit in determin- ing substrate specificity of these enzymes (figure b). our series of mcca- and mccb-deficient ′ flanking intronic sequence of mcca exon ), we were not able to identify a second allele in two cg and four cg probands. in five of these probands, the one allele identi- fied appeared to be homozygous in the rt-pcr product, but was clearly heterozygous in genomic dna, sug- gesting that the steady level of mrna from the second allele was not detectable as would be the case for a promoter mutation or an intra- genic deletion or insertion missed by genomic pcr. these results strong- ly support the identification of the mcca and mccb genes and their assignment to mcc deficiency in cg and cg , respectively. expression of mcca and mccb alleles as a final test of the identity of our candidate human mcca and mccb cdnas, we subcloned them into a mammalian expression vector (ptracer-cmv ), electroporated the recombinant constructs into a sv t transformed reference cg or cg cell line, and measured mcc activity hours later ( ). as a ref- erence, we also measured pcc activ- ity ( ). wild-type mcca and mccb alleles restored mcc activity to % and % of untransfected control fibroblasts, respectively (table ). transfection efficiency, assessed by scoring a subset of cells in each transfection for the presence of the coexpressed gfp, ranged from to % in these experiments. similarly, to test the functional consequences of the missense mutations, we expressed three mcca and six mccb missense alleles. mccb-r c and -v m had activity of and pmol/min/mg protein, respectively, or about % of the activity produced by the wild-type allele, whereas the remaining four mccb alleles and the three mcca alleles produced no detectable activity (table ). these results confirm the deleterious func- tional consequences of the tested missense mutations. population frequency of selected mcca and mccb alleles additionally, we used allele-specific oligonucleotide analysis ( ) to survey a north american control popu- lation of individuals for three mcca alleles (r s, a v, and l p) and two mccb alleles (e q and r c). aside from one individual heterozygous for r s, we did not identify any of these alleles in this collection of control chromosomes (data not shown). these results indicate that each of these mutant the journal of clinical investigation | february | volume | number figure mcca and b missense mutations that alter splicing. (a) mcca d h. the g→c trans- version of the last bp of exon results in the missense mutation d h. the ′ base of an exon also contributes to donor splice site recognition and, as shown in the lower panel, rt-pcr of mcca cdna in this patient with primers corresponding to the ′ and ′ utr resulted in a product smaller than in wild-type. sequence analysis of this product showed that exon ( bp) is skipped, which shifts the reading frame. thus, the deleterious consequences of this mis- sense mutation appear to be entirely due to the splicing defect. (b) mccb i v. as shown in the upper panel, the a→g transition in exon results in the replacement of isoleucine by valine, a conservative change. however, the mutation also activates a cryptic splice donor. use of this new donor splice site deletes the last bp of exon from the mature transcript. as shown in the lower panel, direct sequencing of the rt-pcr product shows that virtually all the transcript present uses the new, more ′ splice donor. the second allele of this compound heterozygous patient does not produce detectable rna. wt, wild-type; mut, mutant. the journal of clinical investigation | february | volume | number probands is characterized by the fact that almost every proband had a unique genotype with no prevalent mutant allele for either gene. in agreement with this observation, using allele-specific oligonucleotide analy- sis to screen north american controls ( chro- mosomes), we found only a single heterozygote from one allele (mcca-r s) and no carriers for the others tested (mcca-a v, -l p; mccb-e q, -r c). for mcca, we assume functional significance for the frameshift mutation q fs(+ ) and the missense mutation d h, which alters splicing (figure a), because both result in truncated proteins lacking func- tionally important domains. the mcca mutations r s, a v, and l p all change conserved residues (figure a), and the corresponding alleles con- fer no detectable mcc activity when expressed in the cg reference cell line (table ). in contrast to the other four cg probands tested, who had no detectable mccα, we detected normal amounts of mccα protein in the proband homozygous for r s (figure ). this result is consistent with the pre- diction based on the structure of the biotin carboxyla- tion domain of e. coli acc that the residue correspon- ding to mccα r is part of a positively charged pocket for bicarbonate binding ( , ). for mccb, we assume functional significance for the frameshift mutation s fs(+ ), the splice site muta- tion in ac- g→a and the missense mutation i v, which alters splicing (figure b). the remaining six mccb missense mutations all change conserved residues and were the only coding alterations we found in sequencing the full-length orf (figure b). in expression studies, we showed that the mccb-r q, -p r, -s l, and -e q alleles had no detectable mcc activity, whereas mccb-v m and -r c had some residual activity, about % of the experimental control value (table ), when expressed in cg -deficient reference cell lines. we identified v m in two com- pound heterozygous turkish probands (table ), the only patients in our collection with residual mcc activity in fibroblasts. although % residual activity is at the detec- tion limit of the standard mcc assay we used for the expression studies ( ), these results are in accordance with the residual activity detect- ed in fibroblasts of these patients with a modi- fied mcc assay of increased sensitivity ( , ). mccα was reduced, but clearly present, in all cg cell lines in our biochemical detection of the biotin-containing α subunit (figure ). this suggests that the mccα subunit is less stable when the β subunit is absent or defective. interestingly, we detected the mccβ s fs(+ ), a t insertion, as one allele in a mild- ly affected swiss compound heterozygote ( ) and in an asymptomatic mennonite homozy- gote from lancaster county, pennsylvania (table ). the ancestors of the lancaster county amish/men- nonite population originated from switzerland ( ) and may have brought this allele with them. haplotype analysis will be necessary to confirm a founder muta- tion. moreover, the amish proband is homozygous for a different mccb allele, e q. thus, despite the small size and common origins of the amish/mennonite population in this region, there is allelic heterogeneity for mcc deficiency. combining our results and the published clinical reports ( – , – ), we were not able to discern a phenotype-genotype correlation. probands and , homozygous for truncating mutations in mcca or mccb (tables and ), have, in one case, no symp- toms and, in the other, a mild phenotype with late onset and no residual damage ( ). by contrast, probands , , and , homozygous for mis- sense mutations mcca-r s, mccb-s l, and - e q, have a severe phenotype with early-onset, major neurological involvement and, in one case, a fatal outcome (table ) ( , , ). proband , an adult amish patient homozygous for the same mccb-e q, has only mild symptoms ( ). further- more, the two turkish patients with mccb-v m that have some residual mcc activity both have a severe phenotype. taken together, these results sug- gest that factors other than the genotype at the table mccb mutant alleles no. allele exon nucleotide clinical ethnic proband change phenotypea origin r q g→a mild vietnamese p r c→g mild vietnamese s fs(+ ) inst mild swiss/mennoniteb c, d i v a→ge mild dutch c r c c→t mild dutch in ac- g→a in ac- g→a mild dutch s l c→t severe turkish d e q g→c severe/mild turkish/amish d, d v m g→a severe turkish c, c amild: late onset, good recovery after acute attack, no or mild developmental delay; severe: onset in infancy, severe neurological involvement with severe developmental delay. bdetect- ed by newborn screening. conly one allele identified. dhomozygous. eactivates cryptic splice donor (figure b). table mcca mutant alleles no. allele exon nucleotide clinical ethnic proband change phenotypea origin r s a→c severe german b q fs(+ ) insg mild swedish/amer. c, b a v c→t mild american c d h g→cd severe turkish b l p t→c severe argentine b amild: late onset, good recovery after acute attack, no or mild developmental delay; severe: onset in infancy, severe neurological involvement with severe developmental delay. bhomozygous. conly one allele identified. dalters splicing (figure a). the journal of clinical investigation | february | volume | number mcca and mccb loci (modifying genes, environ- mental variables) must have a major influence on the phenotype of mcc deficiency. since the widespread introduction of ms/ms to new- born screening, many new patients with mcc defi- ciency have been detected. surprisingly, mcc deficien- cy appears to be the most frequent organic aciduria in these screening programs ( , – ) with an overall fre- quency of approximately in , . studies of these prospectively identified individuals should provide insight into the factors that determine the phenotypic severity of mcc deficiency. note added in proof. we have recently learned that the molecular basis of mcc deficiency has also been identi- fied by gallardo et al. ( , am. j. hum. genet. : ). acknowledgments we thank a. kohlschütter, u. von döbeln, s. berry, j. smeitink, r.d. de kremer dodelson, w. lehnert, u. wiesmann, u. wendel, w.j. kleijer, b. steinmann, b.t. poll-the, d.h. morton, and h.g koch for referring fibroblasts of their patients, and j. nathans for provid- ing the human retina cdna library and s. muscelli for assistance in preparing the manuscript. m.r. baum- gartner, t. suormala, and e.r. baumgartner are sup- ported by grants from the swiss national science foun- dation ( - . for t. suormala and e.r. baumgartner). r.n. cole was supported by the ameri- can health foundation. d. valle is an investigator in the howard hughes medical institute. . sweetman, l., and williams, j.c. . branched chain organic acidurias. in the metabolic and molecular bases of inherited disease. th edition. c.r. scriver, a.l. beaudet, w.s. sly, and d. valle, editors. mcgraw-hill. new york, new york, usa. – . . bannwart, c., wermuth, b., baumgartner, r., suormala, t., and weis- mann, u.n. . isolated biotin-resistant deficiency of -methyl- crotonyl-coa carboxylase presenting as a clinically severe form in a newborn with fatal outcome. j. inherit. metab. dis. : – . . lehnert, w., niederhoff, h., suormala, t., and baumgartner, e.r. . isolated biotin-resistant -methylcrotonyl-coa carboxylase deficiency: long-term outcome in a case with neonatal onset. eur. j. pediatr. : – . . gibson, k.m., bennett, m.j., naylor, e.w., and morton, d.h. . - methylcrotonyl-coenzyme a carboxylase deficiency in amish/men- nonite adults identified by detection of increased acylcarnitines in blood spots of their children. j. pediatr. : – . . levy, h.l. . newborn screening by tandem mass spectrometry: a new era. clin. chem. : – . . naylor, e.w., and chace, d.h. . automated tandem mass spec- trometry for mass newborn screening for disorders in fatty acid, organic acid and amino acid metabolism. j. child neurol. (suppl. ):s –s . . smith, w.e., et al. . evaluation of elevated hydroxyisovaleryl- carnitine in the newborn screen by tandem mass spectrometry. am. j. hum. genet. : . (abstr.) . roscher, a.a., liebl, b., fingerhut, r., and olgemöller, b. . prospective study of ms-ms newborn screening in bavaria, germany. j. inherit. metab. dis. : . (abstr.) . wilcken, b., wiley, v., and carpenter, k. . two years of routine newborn screening by tandem mass spectrometry (msms) in new south wales, australia. j. inherit. metab. dis. : . (abstr.) . samols, d., et al. . evolutionary conservation among biotin enzymes. j. biol. chem. : – . . wolf, b. . disorders of biotin metabolism. in the metabolic and molecular bases of inherited disease. th edition. c.r. scriver, a.l. beaudet, w.s. sly, and d. valle, editors. mcgraw-hill. new york, new york, usa. – . . jitrapakdee, s., and wallace, j.c. . structure, function and regu- lation of pyruvate carboxylase. biochem. j. : – . . lamhonwah, a.-m., et al. . isolation of cdna clones coding for the a and b chains of human propionyl-coa carboxylase: chromoso- mal assignments and dna polymorphisms associated with pcca and pccb genes. proc. natl. acad. sci. usa. : – . . freytag, s.o., and collier, k.j. . molecular cloning of a cdna for human pyruvate carboxylase. j. biol. chem. : – . . abu-elheiga, l., jayakumar, a., baldini, a., chirala, s.s., and wakil, s.j. . human acetyl-coa carboxylase: characterization, molecular cloning and evidence for two isoforms. proc. natl. acad. sci. usa. : – . . lau, e.p., cochran, b.c., and fall, r.r. . isolation of -methyl- crotonyl-coenzyme a carboxylase from bovine kidney. arch. biochem. biophys. : – . . weaver, l.m., et al. . molecular cloning of the biotinylated sub- unit of -methylcrotonyl-coenzyme a carboxylase of arabidopsis thaliana. plant physiol. : – . . mckean, a.l., et al. . molecular characterization of the non- biotin-containing subunit of -methylcrotonyl-coa carboxylase. j. biol. chem. : – . . mourmans, j., et al. . isolated (biotin-resistant) -methyl- crotonyl-coa carboxylase deficiency: four sibs devoid of pathology. j. inherit. metab. dis. : – . . gitzelmann, r., et al. . isolated (biotin-resistant) -methyl- crotonyl-coa carboxylase deficiency presenting at age months with sopor, hypoglycaemia and ketoacidosis. j. inherit. metab. dis. (suppl. ): – . . beemer, f.a., et al. . isolated biotin-resistant -methylcrotonyl- coa carboxylase deficiency in two sibs. eur. j. pediatr. : – . . wiesmann, u.n., suormala, t., pfenninger, j., and baumgartner, e.r. . partial -methylcrotonyl-coa carboxylase deficiency in an infant with fatal outcome due to progressive respiratory failure. eur. j. pediatr. : – . . tsai, m.y., johnson, d.d., sweetman, l., and berry, s.a. . two siblings with biotin-resistant -methylcrotonyl-coenzyme a car- boxylase deficiency. j. pediatr. : – . . steen, c., et al. . metabolic stroke in isolated -methylcrotonyl- coa carboxylase deficiency. eur. j. pediatr. : – . . jurecki, e., and packman, s. . nutritional therapy for beta- methylcrotonylglycinuria. metabolic currents. : – . . wolf, b., willard, h.f., and rosenberg le. . kinetic analysis of genetic complementation in heterokaryons of propionyl coa car- boxylase-deficient human fibroblasts. am. j. hum. genet. : – . . suormala, t., wick, h., bonjour, j.-p., and baumgartner, e.r. . rapid differential diagnosis of carboxylase deficiencies and evalua- tion for biotin responsiveness in a single blood sample. clin. chim. acta. : – . . old, s.e., and devivo, d.c. . pyruvate dehydrogenase complex deficiency: biochemical and immunoblot analysis of cultured skin fibroblasts. ann. neurol. : – . . nathans, j., piantanida, t.p., eddy, r.l., shows, t.b., and hogness, d.s. . molecular genetics of inherited variation in human color vision. science. : – . table expressiona of mcca and mccb alleles allele enzyme activityb (pmol/min/mg protein) experiment experiment mcc pcc mcc pcc mcca wild type r s a v l p vector mccb wild type s l – – e q – – v m – – r q – – p r – – r c – – vector control fibroblasts atransient transfection in sv t-transformed reference cg or cg cell lines. bnumbers represent average of duplicates. the journal of clinical investigation | february | volume | number . williams, k., lopresti, m., and stone, k. . internal proteins sequencing of sds-page-separated proteins: optimization of an in gel digest protocol. in techniques in protein chemistry. d.r. marshak, editor. academic press. new york, new york, usa. – . . smith, b.j. . chemical cleavage of proteins. methods mol. biol. : – . . kennedy, r.t., and jorgenson, j.w. . preparation and evaluation of packed capillary liquid chromatography columns with inner diam- eters from to µm. anal. chem. : – . . eng, j.k., mccormak, a.l., ashley, l., and yates, j.r.i. . an approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. j. am. soc. mass spectrom. : – . . braverman, n., et al. . human pex encodes the peroxisomal pts receptor and is responsible for rhizomelic chondrodysplasia punctata. nat. genet. : – . . leon-del-rio, a., and gravel, r.a. . sequence requirements for the biotinylation of carboxyl-terminal fragments of human propi- onyl-coa carboxylase alpha subunit expression in escherichia coli. j. biol. chem. : – . . waldrop, g.l., rayment, i., and holden, h.m. . three-dimen- sional structure of the biotin carboxylase subunit of acetyl-coa car- boxylase. biochemistry. : – . . thoden, j.b., blanchard, c.z., holden, h.m., and waldrop, g.l. . movement of biotin carboxylase b-domain as a result of atp bind- ing. j. biol. chem. : – . . saraste, m., sibbald, p.r., and wittinghofer, a. . the p-loop: a common motif in atp- and gtp-binding proteins. trends biochem. sci. : – . . lópez-casillas, f., et al. . structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme a carboxylase. proc. natl. acad. sci. usa. : – . . hendrick, j.p., hodges, p.e., and rosenberg, l.e. . survey of amino-terminal proteolytic cleavage sites in mitochondrial precur- sor proteins: leader peptides cleaved by two matrix proteases share a three-amino acid motif. proc. natl. acad. sci. usa. : – . . nolt, s.m. . a history of the amish. good books. intercourse, penn- sylvania, usa. pp. microsoft word - huang_pmid_ uc irvine uc irvine previously published works title electronic health record solutions to reduce central line-associated bloodstream infections by enhancing documentation of central line insertion practices, line days, and daily line necessity. permalink https://escholarship.org/uc/item/ vb c cx journal american journal of infection control, ( ) issn - authors quan, kathleen a cousins, sarah m porter, darlene d et al. publication date - - doi . /j.ajic. . . peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ vb c cx https://escholarship.org/uc/item/ vb c cx#author https://escholarship.org http://www.cdlib.org/ 	 electronic health record solutions to reduce central line- associated bloodstream infections by enhancing documentation of central line insertion practices, line days, and daily line necessity kathleen a. quan msn, rn, cic, cphq a, sarah m. cousins bs b,*, darlene d. porter bsn, rn, phn c, margaret o’brien bsn, rn d, scott rudkin md c, brian lambertson bsn, rn c, dennis hoang pharmd c, amish a. dangodara md e, susan s. huang md, mph a,b a epidemiology and infection prevention, university of california irvine health, orange, ca b division of infectious diseases and health policy research institute, university of california irvine school of medicine, irvine, ca c health affairs information services, university of california irvine health, orange, ca d nursing informatics, university of california irvine health, orange, ca e university of california irvine hospitalist program informatics, orange, ca key words: central venous catheter checklist background: central line-associated bloodstream infections (clabsis) continue to cause preventable morbidity and mortality, but methods for tracking and ensuring consistency of clabsi-prevention activities remain underdeveloped. methods: we created an integrated electronic health record solution to prompt sterile central venous catheter (cvc) insertion, cvc tracking, and timely line removal. the system embedded central line insertion practices (clip) elements in inserter procedure notes, captured line days and new lines, matching each with its clip form and feeding back compliance, and enforced daily documentation of line necessity in physician progress notes. we examined changes in clip compliance and form submission, number of new line insertions captured, and necessary documentation. results: standard reporting of clip compliance, which measures compliance per clip form received, artificially inflated clip compliance relative to compliance measured using cvc placements as the denominator; for example, % per clip form versus % per cvc placement. this system established a higher threshold for clip compliance using this denominator. identification of cvcs increased %, resulting in a decrease in clabsi rates. the system also facilitated full compliance with daily documentation of line necessity. conclusions: integrated electronic health records systems can help realize the full benefit of clabsi prevention strategies by promoting, tracking, and raising the standard for best practices behavior. major national efforts have resulted in a large reduction in central line-associated bloodstream infections (clabsi). nevertheless, clabsi continue to cause preventable morbidity and mortality in , inpatients annually in acute care hospitals. successful strategies to reduce clabsi have included ensuring sterile processes for central venous catheter (cvc) insertion, establishing maintenance and access protocols, and promoting line removal. these activities are considered gold standard care for cvcs. , however, methods for tracking and ensuring consistency of these activities are not well developed. 	 one method to ensure appropriate insertion processes to prevent clabsi is the use of checklists. the central line insertion practices (clip) checklist form was developed by the centers for diseases control and prevention to confirm appropriate hand hygiene, skin preparation with a chlorhexidine product, and maximal sterile barrier precautions. the states of california and new hampshire have mandated public reporting of compliance with this form. despite the benefits of the clip form, inserters (or an observer) often fail to submit it, making comprehensive assessment of insertion techniques difficult. another method of preventing clabsi is to monitor and feed- back clabsi rates at the unit or hospital level to highlight good performance and encourage poor performers to improve. monitoring requires the accurate capture of the number of central line days as the denominator for rates. often a manual or periodic sampling technique is used in place of a true count of central line days, which may contribute to inaccuracy in reported rates. a third way to reduce clabsi is to ensure prompt line removal. to this end, california law requires that physicians document the necessity for continuing a cvc each day. as with the clip form, daily documentation of line necessity requires initiative on the part of the attending physician to complete the form. given the intensity of medical care frequently required in patients needing central lines, this documentation is often not prioritized and adherence with daily documentation of necessity is poor. techniques that rely on clinician initiative to complete cvc- related infection prevention documentation have limited success. when good intentions fail, electronic tools can be very helpful in prompting best practice behaviors. we describe the successful use of electronic health record (ehr) solutions to improve clip documentation, capture cvc line days, and ensure documentation of daily necessity assessment. methods clip documentation in response to the california mandate to ensure clip form documentation, we created a progression of documentation tools leading up to our ultimate ehr solution. for rapid deployment, we initially created a paper-based form that had to be printed, completed, and faxed by the submitter (eg, inserter or observer) to the hospital’s epidemiology and infection prevention (eip) program, and then manually entered into a database by eip staff. to improve compliance, we temporarily placed an electronic version of the clip form within the nursing electronic documentation system to be completed online by nurses after witnessing or assisting with a cvc insertion. during this second phase of electronic nursing documentation we no longer accepted paper clip forms. our permanent ehr solution created an electronic procedure note with embedded clip form elements for documenting cvc insertion. the form provided a series of check boxes (fig a) that generate a narrative procedure note using templated language that could be edited by the physician before being finalized (fig b). this allowed physicians to complete the note rapidly and, if necessary, remotely. to finalize the note, inserters were required to report adherence with clip elements using additional check boxes. inserters could select a single check box to confirm that all elements of maximal sterile precautions were employed or select from a series of individual elements. 	 to evaluate the fraction of cvc insertions for which clip forms were completed, we identified the total number of clip forms received divided by the number of cvc insertions based on electronic nursing documentation indicating the new presence of a line. adherence with clip elements within the form was calculated ways: by assessing the fraction of forms with adherence to all clip components among forms submitted and by assessing the fraction of forms with adherence to all clip components among lines placed. capture of line days and identification of new lines comprehensive capture of cvc line days is essential for calculation of clabsi rates, defined as the number of clabsi events divided by the number of device days. we calculated both the total number of line days (eg, lines in patient for day counts as line days) and the national healthcare safety network (nhsn) device patient days (eg, lines in patient for day counts as line day). when not otherwise specified, the nhsn definition of device day as day per patient who has or more lines was used. before the development of our ehr solution, we captured line days by requiring each unit to maintain daily paper records of all patients who had cvcs. we then moved to an interim electronic system in which nurses documented the number of patients with any cvcs for every shift. this system did not track each cvc individually, so device-specific dwell times could not be calculated. in our permanent ehr solution, we developed comprehensive electronic nursing documentation for individual cvcs. daily e-flowsheets were modified to collect cvc insertion site (eg, internal jugular, subclavian, antecubital, and femoral), left or right side, type of cvc (eg, peripherally inserted central catheter, tunneled/ nontunneled single or multilumen catheter, tunneled/nontunneled hemodialysis line, and vascular access port), and assessment of skin integrity as part of the nursing assessment conducted during each shift. for newly placed cvcs, inserter name and unit of insertion were also collected, facilitating clinician-specific follow-up for missing clip forms. an automated report matched nursing cvc documentation of new lines and electronically submitted clip forms. cvc insertions without associated electronic clip forms were reported to the inserting unit medical director, nurse manager, and inserting clinician, as well as the eip program. cvc data were linked between shifts, allowing for device- specific cvc information and calculation of dwell times. this allowed us to calculate device days based on the total number of lines or based on the nhsn patient days definition. a cvc’s device days were counted from the first to last calendar day that had nursing documentation for a specific cvc and attributed to the first documenting unit of the day. we then calculated unit-specific or patient- specific total monthly nhsn patient device days (maximum device day per patient per day) and device use ratio (number of device days divided by number of patient days) as required for mandated re- porting. in addition to use for mandated reporting, the dwell time for each line was cascaded from nursing e- flowsheets to the physician e-progress note to ensure physician attention was drawn to long dwell times. daily documentation of line necessity before our ehr solution, physicians were expected to self- initiate documentation of cvc necessity each day in accordance with california law. to facilitate this, intensive care units used a 	 variety of mechanisms. in certain units, the unit clerk stamped a template for documenting cvc necessity on the patient chart for the primary team physicians to complete each day. others incorporated documentation of daily necessity into templated paper progress notes in patient charts. no facilitating interventions were implemented for non-intensive care units. in our ehr solution, electronic physician progress notes (e- progress note) replaced paper progress notes. within the e-progress note for a given patient, each existing cvc from the electronic nursing flowsheet documentation was automatically cascaded into the e-progress note with an indication of the cvc type, side, and site, and device-specific dwell time. next to each listed cvc were templated options (device is needed/not needed) to confirm the necessity of each cvc (fig ). any physician documenting an e-progress note would see cascaded cvc line data and have the option to document daily necessity. however, only notes written by the primary service (self- identification as primary or consulting service is required when an e-progress note is initiated) had the requirement that these fields be completed before the note could be finalized. we calculated the distribution of dwell times before and after implementation of our e-progress notes. these phases of implementation are summarized in table . 	 fig . (a) input elements for the electronic physician procedure note documenting insertion of a central venous catheter. central line insertion practices (clip) elements are embedded into the form (bottom right) and inserters were required to report adherence or nonadherence with clip elements via check boxes of the individual clip elements. for ease of reporting, a single box was available to confirm that all elements of maximal sterile precautions were used. (b) the narrative procedure note that was automatically generated using the elements inputted into the electronic physician procedure form shown in figure a. following completion of the procedure form, documentation of the procedure was generated with templated language. this note could be generated at the bedside or remotely and could then be edited by the physician before being finalized. 	 fig . the elements of daily documentation of necessity that were found in all physician note templates in the electronic health record. data on each central line were cascaded into the note from the daily nursing documentation record, including body location and line type. dwell time was automatically calculated. physician notes created by the primary team were required to indicate whether each line was still needed before the daily progress note could be finalized, ensuring compliance with daily documentation of necessity. all created physician notes required the user to indicate whether they represented the primary or consulting team. table phases of increased electronic capture of central line insertion practices (clip) form compliance and central venous catheter (cvc) line days phase : paper documentation ( -october ) • paper nursing flowsheets indicate new cvcs • observer (nurse)-submitted paper clip form • unknown total number of cvc insertions • unable to facilitate coordinated feedback phase : nonintegrated e-documentation (november -october ) • electronic nursing capture of cvc days • electronic clip form available in nursing documentation for observer (nurse) submission • manual process for matching newly-inserted cvcs with clip forms • unable to facilitate coordinated feedback phase : integrated e-documentation (november -present) • electronic health record-based e-nursing flowsheet capture of individual lines* • electronic health record-based inserter e-procedure note with imbedded clip components • automated process to match new cvc with clip form • automated process to coordinate feedback *the ability to track individual lines in the phase integrated e-documentation allowed us to also track clip documentation, daily documentation of necessity, and dwell time for each individual line. results clip documentation following the state mandate for clip form documentation for each cvc placement and before the design and implementation of our present ehr solution, physicians did not routinely include clip form components in their procedure notes for cvc insertion and compliance with the clip 	 form was near zero. the institution of a paper form to be completed and faxed by physicians or observers marginally improved compliance. the subsequent interim solution shifted the responsibility to nurse observers using an electronic nursing clip form as part of nursing documentation and in- creased clip form submission to % (table ). although nurses accepted the interim partnership to complete clip forms, it was uniformly believed that the clip form should be completed by the inserter of the cvc. following the implementation of the permanent ehr solution based on integrated data from electronic nursing documentation and physician procedure notes, clip form submission significantly increased from %- % (χ p < . ) (table ). to verify that physician self- assessment would continue to document compliance appropriately, we evaluated compliance using both physician self- assessment and nurse observer assessment during a -month study period. during this evaluation, we found no differences in documentation of compliance. under both electronic documentation systems, compliance differed based on whether percent compliance was calculated using the total number of cvc insertions (including those missing clip forms as noncompliant) or counting only cvc insertions for which clip forms were submitted (table ). periods for which no data are reported were transition times during which new ehr implementation phases were being rolled out. before the implementation of our interim electronic documentation system, our -month average clabsi rate per , line days was . , before the beginning of our permanent system it was . , and as of december , it was . . capture of line days and identification of new lines our ehr solution for cvc documentation in daily nursing flowsheets increased identification of cvcs from approximately , - , line days per month ( % increase) (table ). validation of our electronic system confirmed that capture of line days is improved using this system. annual device use per , patient days increased from . - . . the tracking system also allowed for more accurate capture of line days by allowing de-duplication of lines in a single patient. the ehr solution also allowed automatic calculation of dwell times. the number of lines was , in and , in . the mean dwell time was . ± . days (sd) in and . ± . days (sd) in . for both years, the median dwell time was days (interquartile range, - days). daily documentation of line necessity nonelectronic efforts to prompt documentation of daily necessity of cvcs, such as physician self-initiation and the stamped template, were largely unsuccessful. only on the few intensive care units where physicians routinely used a standardized, fixed- template paper progress note was the form generally completed. in contrast, the e-progress note led to near % documentation of daily necessity. the only reason that daily necessity would not have been documented was if the primary service did not write a progress note for a particular day. 	 table central line insertion practices (clip) form submission and compliance over phases of electronic health record implementation* % clip-compliant implementa tion phase paper documentati on submis sion of clip forms clip- compliant forms (denominat or = forms submitted) cvc insertions (denomina tor = cvcs inserted) , mo unknown ( , / , ) unknown nonintegrated electronic documentation november -october , mo ( , / , ) ( , / , ) ( , / , ) integrated electronic documentation january - december , mo ( , / , ) ( , / , ) ( , / , ) january - december , mo ( , / , ) ( , / , ) ( , / , ) note. values are presented as % (n/total sample). *includes clip-compliance for inpatient and outpatient cvc insertions. table average central venous catheter (cvc) device days and average device utilization per month* average national healthca re safety network device inpatient days average number of total line days avera ge dur ability to track cvc- specific cvc documentation implementation phase per mont h per mont h per mont h dwell times paper ( ) , unknown . ( , / , ) no nonintegrated electronic (november -october ) , unknown . ( , / , ) no integrated electronic nursing flowsheet ( - ) , , . ( , / , ) yes *national healthcare safety network device inpatient days and number of total line days 	 are calculated for inpatients only. discussion we show that integrated ehr solutions can be instrumental in improving physician adherence to mandatory clip form documentation, consistent capture of central line days for the purpose of determining clabsi rates, and daily documentation of cvc necessity. our ehr solutions achieved success for main reasons: they were imbedded into usual care, required completion, and gave feed- back. the system increased the accuracy of nhsn cvc line day capture, which decreased clabsi rates. most importantly, it changed and improved our metric for insertion practices compliance. our ehr system was built within electronic daily nursing and physician documentation to streamline workflow. the capture of line days was built into the electronic nursing flowsheet assessments, thereby being incorporated into a daily practice activity. by creating an electronic physician procedure note for cvc insertion, we incentivized use by ensuring a process that was more rapid, consistent, and comprehensive than a handwritten paper note. we further incentivized use by creating a process where physicians could easily document a procedure at a time and location separate from the patient’s bedside paper chart. by embedding the clip elements into this preferred documentation platform, we were able to enforce documentation. similarly, by creating an e-progress note that replaced a paper-based documentation system, we were able to cascade data to allow rapid assessment of the cvcs present in a patient and highlight the dwell time for a physician while they were documenting their daily note. this provided both a ready opportunity for easy documentation using check boxes and drop-down menu selections for the most common reasons for persistent need of a cvc. the system achieved a high rate of use by forcing documentation of clabsi-prevention activities. the procedure note could not be submitted without clip elements nor could the e- progress note be submitted without daily documentation of necessity for each cvc for a primary service note. this improved physician adherence to completion of clip forms. the ehr solutions were also successful because of feedback. the clip elements in the procedure note reminded physicians of the proper protocols for decreasing infections. the system gave an error message if doctors or nurses tried to submit forms without answering required elements. the system also automatically calculated dwell time to alert physicians to long-dwelling cvcs that might require heightened attentiveness to skin integrity or removal. finally, the capture of line days from nursing flowsheets allowed our eip program to calculate and feedback accurate clabsi rates to clinical areas. our % increase in the capture of line days increased the denominator in our clabsi rate calculation, which resulted in a % decrease in our calculated clabsi rate following de-duplication of lines in a single patient. finally, our ehr solution allowed us to make a very important change to our metric for clip compliance. the current standard for measuring clip compliance is to calculate the percentage of all clip forms submitted on which use of all clip elements is documented. whereas we previously calculated clip compliance only for cvc insertions for which we had a clip form according to that standard, we were now able to determine compliance for all cvc insertions. this allowed us to more accurately determine whether cvc insertions were compliant with required insertion practices and to associate changes in insertion practices with clabsi rates. this study is limited by use of data from a single medical center, which may not be generalizable to 	 other hospitals. our system itself is limited in that it could only be used by organizations that use an ehr, and not all ehrs have the capacity to allow for the addition of these features. however, ehr capabilities are rapidly increasing across all us hospitals due to meaningful use requirements. although we saw a reduction in the clabsi rate during the implementation of our ehr solution, we took a multimodal this was component—to address clabsi. in analyzing the results of implementing our system, we are limited by the lack of data for the total number of cvc insertions during phase , before the implementation of our electronic system. this means we cannot compare the percentage of cvc insertions for which clip forms were submitted or the clip-compliance for all cvc insertions (not just those with submitted clip forms) between and - . our ehr solution represents a novel way to address challenges to physician documentation when their processes and practices are not necessarily uniform or standardized on a day-to-day basis. using this system, we improved documentation of clip, increased accuracy in calculation of clabsi rates, and ensured documentation of line necessity, all steps toward decreasing central line infections. 	 references . siempos ii, kopterides p, tsangaris i, dimopoulou i, armaganidis ae. impact of catheter-related bloodstream infections on the mortality of critically ill patients: a meta-analysis. crit care med ; : - . . magill ss, edwards jr, bamberg w, beldavs zg, dumyati g, kainer ma, et al. multistate point-prevalence survey of health care–associated infections. n engl j med ; : - . . marschall j, mermel la, classen d, arias km, podgorny k, anderson dj, et al. strategies to prevent central line– associated bloodstream infections in acute care hospitals. infect control hosp epidemiol ; (s ):s - . . o’grady np, alexander m, dellinger ep, gerberding jl, heard so, maki dg, et al. guidelines for the prevention of intravascular catheter–related infections. clin infect dis ; : - . . pronovost p, needham d, berenholtz s, sinopoli d, chu h, cosgrove s, et al. an intervention to decrease catheter- related bloodstream infections in the icu. n engl j med ; : - . . centers for disease control and prevention. national healthcare safety network. central line insertion practices adherence monitoring cdc . rev , v . . available from: http://www.cdc.gov/nhsn/forms/ . _clip_blank.pdf. accessed july , . . california health and safety code § . (b). available from: http:// www.leginfo.ca.gov/cgi- bin/displaycode?section=hsc&group= - &file= . - . . accessed december , . . n.h. rev. stat. ann. § : . available from: http://www.gencourt.state.nh.us/ rsa/html/xi/ / - .htm. accessed december , . . gastmeier p, schwab f, sohr d, behnke m, geffers c. reproducibility of the surveillance effect to decrease nosocomial infection rates. infect control hosp epidemiol ; : - . . california department of public health. new regulatory requirements for compliance with senate bill — joining cdph group, clip reporting (afl - ). available from: http://www.cdph.ca.gov/certlic/facilities/ documents/lnc-afl- - .pdf. published . accessed july , . lack of association between stk and hypertension in the chinese population original article lack of association between stk and hypertension in the chinese population j xu , , , l-d ji , , , l-n zhang , c-z dong , l-j fei , s hua , j-y tsai and y-p zhang , genome-wide association study (gwas) has identified serine/threonine kinase (stk ) as a candidate gene for hypertension. a replication study provided supporting evidence that stk functional polymorphism rs was associated with hypertension. recently, another study also showed rs within the stk was associated with blood pressure responses. however, these studies were all conducted in caucasians. thus, we carried out a case–control study to test whether stk is a common candidate gene for hypertension, and to examine the interaction of genetic factors and non-genetic risk factors in the chinese population. thousand twenty four hypertensive cases and controls were genotyped for five polymorphisms. four single-nucleotide polymorphisms (snps) are located within stk , and rs , the snp that showed the strongest signal is located in a gene desert. results indicated that none of these snps was associated with hypertension in the chinese population. logistic regression analysis found body mass index (bmi) and triglyceride level were higher in the hypertension group when compared with the control group. multifactor dimensionality reduction analysis indicated that the interaction between bmi and rs may have an impact on hypertension. taken together, the present study found no evidence that stk was associated with hypertension in the chinese population. instead, non-genetic risk factors such as bmi have an important role in chinese hypertensive subjects, and the ‘missing inheritability’ requires further investigation. journal of human hypertension ( ) , – ; doi: . /jhh. . ; published online november keywords: genome-wide association study; serine/threonine kinase ; polymorphism; interaction introduction genome-wide association study (gwas) has identified serine/ threonine kinase (stk ) as a candidate gene for hypertension in the american old order amish. recently, a replication study in two sweden cohorts showed that the stk functional polymorphism rs a g was associated with blood pressure (bp) and hypertension. consequently, stk becomes a very important ‘susceptibility gene’ for hypertension. stk encodes a serine/threonine kinase known as a ste - related proline–alanine-rich kinase (spak). when phosphorylated by the wnk (wnk lysine-deficient protein kinase ) and wnk (wnk lysine-deficient protein kinase ), spak can bind and phosphorylate two naþ-dependent cation chloride cotranspor- ters, including furosemide-sensitive naþ/kþ/ cl� channel (nkcc ) and thiazide-sensitive naþ/cl� channel (ncc). – this pathway has a critical role in salt homeostasis in renal physiology and is strongly implicated in bp regulation and hypertension development. recently, donner and colleagues selected single-nucleotide polymorphisms (snps) from five gwass and studied their association with bp responses to four different antihypertensive drug monotherapies in finnish population. the result indicated only rs (within the stk gene) was significantly associated with bp responses. however, these studies were all conducted in caucasians, and whether it is a common candidate gene for hypertension in other populations is not clear. therefore, we carried out a case–control study and an interaction analysis to verify whether stk is associated with hypertension in the chinese population. methods study population the participants were chosen from our established community-based epidemiology study of common diseases. with informed consent, we collected health records. subsequently, participants who fulfilled the following criteria were put into our database: – years old, han chinese, living in ningbo city (east side of china) for at least three generations and do not have a migration history. thousand twenty four essential hypertension cases and controls were chosen from this database. the hypertensive subjects and control subjects were matched for age and gender. in addition, only subjects who do not have cardiovascular disease or other major chronic illnesses according to their health records were included in the control group. bp and clinical parameters bp measurements were conducted in the morning. after the participant had been in the sitting position for min, three bp measurements were obtained at -min intervals using standard mercury sphygmomanometer, and the average of last two measurements was taken as the bp for that participant. hypertension in this study is defined as a sitting systolic bp (sbp) x mm hg and/or a diastolic bp (dbp) x mmhg, or self-reported use of antihypertension medication. patients with secondary hypertension were excluded. normal bp is defined with sbp p mm hg and dbp p mm hg. state key laboratory of genetic resources and evolution, kunming institute of zoology, chinese academy of sciences, kunming, china; department of public health, school of medicine, ningbo university, ningbo, china; institute of biochemistry, school of medicine, ningbo university, ningbo, china; center for cardiovascular genetics, brown foundation institute of molecular medicine, university of texas health science center at houston, houston, tx, usa and laboratory for conservation and utilization of bio- resource, yunnan university, kunming, china. correspondence: professor y-p zhang, state key laboratory of genetic resources and evolution, kunming institute of zoology, chinese academy of sciences, no. jiaochang donglu, kunming , china. e-mail: zhangyp@mail.kiz.ac.cn these authors contributed equally to this work. received september ; accepted september ; published online november journal of human hypertension ( ) , – & macmillan publishers limited all rights reserved - / www.nature.com/jhh http://dx.doi.org/ . /jhh. . mailto:zhangyp@mail.kiz.ac.cn http://www.nature.com/jhh blood samples were collected with informed consent. subsequently, total cholesterol (tc), high-density lipoprotein (hdl), and triglyceride (tg) were measured from these blood samples. simultaneously, clinical infor- mation including body mass index (bmi), smoking habit and alcohol abuse were also obtained. the protocol of this study was reviewed and approved by the ethics committees of kunming institute of zoology, chinese academy of sciences and ningbo university. snp genotyping genomic dna was obtained from the whole blood by standard phenol/ chloroform extraction. genotyping was performed by the genomelab snpstream genotyping system (beckman coulter, fullerton, ca, usa) according to the protocols provided by the manufacturer. of the five candidate snps, rs was replaced by a nearby snp (rs , d ¼ , r ¼ in chb) due to unavailability of appropriate primers (www.autoprimer.com). the primers for snpstream were listed in supplementary table . rs was genotyped by pcr-restriction fragment length polymorphism using mismatched primers (forward primer ’-ctggcatttgtattactttc- ’, reverse primer ’-tgaacaggtccca tactc- ’), and the pcr products were digested by the restriction enzyme spei (new england biolabs, beverly, ma, usa). statistics continuous variables are presented as the mean±s.d. and analyzed by t-test between the two groups. statistical analysis of allele and genotype frequencies between case and control groups and among different sex groups were performed by w -test. effect of confounding variables were identified by logistic regression (spss . , spss inc., chicago, il, usa). hardy–weinberg equilibrium was determined by the software pedstats v . . (http://www.sph.umich.edu/csg/abecasis/). multifactor dimensionality reduction (mdr) was used to identify and characterize interactions among the snps and the non-genetic factors, including bmi, serum hdl, tc and tg level, as well as frequency of smoking and alcohol abuse. the software used for mdr is distributed in a java platform with a graphical user interface and is freely available (http://www.epistasis.org/mdr.html). all tests were two-sided and p-values o . were considered statistically significant. for w -test, the p-values were adjusted for the total number of tested snps using the bonferroni correction method (a ¼ . / ¼ . ). results the baseline characteristics of the participants are summarized in table . in this study, hypertensive participants and controls, who have all been long-term residents of ningbo city, were analyzed. the male to female ratio was equal in both groups, and mean age of hypertensive participants and controls were similar, demonstrating that the experimental and control groups were well-matched and are appropriate for the following analyses. bmi, serum tg and tc level in the hypertensive group were significantly greater than the control group (po . ). furthermore, significantly more subjects drank alcohol regularly in hypertensive group than those in control group (po . ). however, hdl level and smoking habit were similar in both groups. as shown in supplementary figures s –s , the results of snpstream and pcr-restriction fragment length polymorphism were clear and reliable. table shows the genotypes and minor allele frequency of each snp. the genotyping success rate was . %, and all five snps did not deviate from hardy–weinberg equilibrium (p . ). in addition, with the prevalence, odds ratio, and minor allele frequency in this study, the genetic power calculator (available online http://pngu.mgh.harvard.edu/bpur- cell/gpc/) indicated that the sample size is big enough to do case– control analysis with % power. according to the w -test p-values and odds ratios, none of the snps was shown to associate with hypertension, even after stratification by gender (table ). considering the effect of confounding variables, we further carried out logistic regression analysis with genetic and non- genetic factors. the result indicated that only bmi and tg were associated with hypertension (po . ), and none of the snps was positive (table ). finally, mdr was used to analyze the interaction among different snps and non-genetic risk factors for hypertension. after input the five snps together with information of tg, tc, hdl, bmi, smoking and alcohol drinking, the software output the best model for bmi with / cross-validation consistency, and ‘bmi, rs ’ with / cross-validation consistency (table ). discussion stk encodes spak, and the wnk-spak-nkcc /ncc pathway is involved in salt renal reabsorption and bp homeostasis. therefore, it is not surprising to see that snps within stk were identified to associate with hypertension through gwas. to avoid the possibility of false-positive results, fava et al. conducted a replication study in two independent sweden cohorts. their results indicated that the functional rs a g polymorphism was associated with higher sbp and dbp values in the malmö diet and cancer (n ¼ ), but not in the malmö preventive project (n ¼ ). after stratification for sex, the results remained statistically significant only in women. table . baseline characters of the investigated participants variables case control p-value number na sex (male) (%) . . na age (years) . ± . . ± . . tg (mm) . ± . . ± . o . hdl (mm) . ± . . ± . . tc (mm) . ± . . ± . o . bmi (kg m� ) . ± . . ± . o . smoking (%) . . . alcohol drinking (%) . . o . abbreviations: bmi, body mass index; hdl, high-density lipoprotein; tc, total cholesterol; tg, triglyceride. table . association statistics for five snps snp group maf p-value or % ci rs case . . . . – . control . male . . . . – . female . . . . – . rs case . . . . – . control . male . . . . – . female . . . . – . rs case . . . . – . control . male . . . . – . female . . . . – . rs case . . . . – . control . male . . . . – . female . . . . – . rs case . . . . – . control . male . . . . – . female . . . . – . abbreviations: ci, confidence interval; maf, minor allele frequency; or, odds ratio; snp, single-nucleotide polymorphism. stk and hypertension j xu et al & macmillan publishers limited journal of human hypertension ( ) – www.autoprimer.com http://www.sph.umich.edu/csg/abecasis/ http://www.epistasis.org/mdr.html http://pngu.mgh.harvard.edu/~purcell/gpc/ http://pngu.mgh.harvard.edu/~purcell/gpc/ the population studied in the initial gwas from wang et al. is american old order amish, which is a closed founder population emigrated from switzerland in the early s. the study populations in articles of fava et al. and donner et al. are from swede and finland. these populations are all caucasians and close in geographical location. when we checked the details of the positive snps (rs , rs and rs ) in wang’s gwas, we found that the minor allele frequency differs greatly between europeans and asians. to determine whether stk is a common candidate gene for hypertension, we set to find out whether it is associated with hypertension in the chinese population, as china has now become a nation with rapidly increasing hypertension prevalence. we first studied two snps (rs and rs ) within stk , which were putative functional snps based on a multispecies sequence alignment. however, neither snp was significantly asso- ciated with hypertension in the chinese population. as the result was quite different from fava’s replication study, we additionally genotyped two tag snps (rs and rs ) within stk . these two snps belong to ld bin and bin . both snps were significant with sbp in amish (po . ), marginally significant in population from diabetes genetics initiative (p ¼ . for rs and . for rs ), but not significant in populations from framingham heart study from, gennet and hutterites. however, in the present study, these two tag snps were still not associated with hypertension. we further genotyped rs as it has the minimum p-value in wang’s gwas. this snp rs is not a stk polymorphism, but a snp that is located in a gene desert kb away from known genes. in our study, the p-value for rs is . , and the odds ratio is . ( % confidence interval: . – . ). even after stratification with gender, none of the five snps showed association with hypertension. hereto, five genome-wide significant snps genotyped in this study did not show a positive association with hypertension in the chinese population. similarly, cunnington et al. also did not find any association between the three snps (rs , rs , rs ) within stk and sbp or dbp from a cohort of british caucasians. furthermore, in fava’s study, after exclusion of individuals from the malmö preventive project cohort, who also participated in the malmö diet and cancer study, g allele of rs was no longer associated with hypertension. thus, it is still questionable whether stk can be accepted as a common susceptibility gene for hypertension. pickering and colleagues have initially suggested that hypertension and bp are complex traits. before the era of gwas, epidemiological studies have found dozens of risk factors, such as smoking, alcohol abuse, excessive salt intake, obesity, mental stress and others to associate with high bp. , although no positive snp was found within this study, we found interesting interactions between genetic factors and non-genetic risk factors. high bmi and serum tg level were already believed to be risk factors for hypertension, , and were also confirmed by logistic regression in current study. the mdr analysis further demonstrated that these important risk factors interacted with the genetic factor. thus, the present interaction analysis gave a little more information than the single genetic study. the participants in recent gwass are mainly from well-organized cohorts (global bpgen and cohorts for heart and aging research in genomic epidemiology), which means the epidemiology data are readily available. – with the development of statistic methods for evaluation of gene–environment interaction, more missing inherit- ability will be found. , all in all, studies from wang et al., fava et al. and donner et al. have demonstrated that stk is associated with hypertension or bp regulation in three different caucasian populations, , , but this result could not be replicated in the chinese population. in addition, in our interaction analysis, a significant interaction was found between genetic and non-genetic factors, demonstrating that gwas alone is not a sufficient indicator for hypertension. to decipher causal factors leading to the development and the pathogenesis of hypertension, future work will require analysis of both genetic (epigenetic) and non-genetic (environmental) factors. what is known about the topic � genome-wide association study has identified stk as a candidate gene for hypertension. � a replication study in two sweden cohorts provided supporting evidence that stk functional polymorphism rs was associated with hypertension. another study conducted in finnish population also showed rs within the stk was associated with blood pressure responses. what this study adds � none of the snps within the stk was associated with hypertension in the chinese population. � logistic regression showed that only body mass index (bmi) and tg were associated with hypertension. � mdr analysis indicated that the interaction among bmi and rs may have an impact on hypertension. � the present study found no evidence that stk was associated with hypertension in the chinese population. instead, interaction analysis indicated that non-genetic risk factors such as bmi have an important role for chinese hypertensive subjects, and the ‘missing inheritability’ requires further investigation. table . logistic regression for genetic and non-genetic factors variables b p-value exp (b) % ci gender � . . . . – . age . . . . – . rs . . . . – . rs . . . . – . rs . . . . – . rs . . . . – . rs . . . . – . bmi . . . . – . tg . . . . – . hdl . . . . – . tc . . . . – . smoking . . . . – . alcohol . . . . – . constant � . . . abbreviations: b, b-regression coefficient; ci, confidence interval. table . mdr analysis of gene-environment interaction best model testing accuracy testing sensitivity testing odds ratio testing w cross-validation consistency bmi . . . ( % ci: . – . ) . (p ¼ . ) / bmi, rs . . . ( % ci: . – . ) . (p ¼ . ) / bmi, tc, rs . . . ( % ci: . – . ) . (po . ) / abbreviations: bmi, body mass index; ci, confidence interval; mdr, multifactor dimensionality reduction; tc, total cholesterol. stk and hypertension j xu et al journal of human hypertension ( ) – & macmillan publishers limited conflict of interest the authors declare no conflict of interest. acknowledgements we acknowledge dr ali j marian for helpful suggestions to this study. this study was supported by zhejiang natural science foundation (y ), ningbo natural science foundation ( a , a ) and china postdoctoral science foundation ( ). references wang y, o’connell jr, mcardle pf, wade jb, dorff se, shah sj et al. whole- genome association study identifies stk as a hypertension susceptibility gene. proc natl acad sci usa ; ( ): – . fava c, danese e, montagnana m, sjogren m, almgren p, engstrom g et al. serine/ threonine kinase is a candidate gene for primary hypertension especially in women: results from two cohort studies in swedes. j hypertens ; ( ): – . vitari ac, deak m, morrice na, alessi dr. the wnk and wnk protein kinases that are mutated in gordon’s hypertension syndrome phosphorylate and activate spak and osr protein kinases. biochem j ; (pt ): – . moriguchi t, urushiyama s, hisamoto n, iemura s, uchida s, natsume t et al. wnk regulates phosphorylation of cation-chloride-coupled cotransporters via the ste -related kinases, spak and osr . j biol chem ; ( ): – . delpire e, gagnon kb. spak and osr : ste kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells. biochem j ; ( ): – . donner km, hiltunen tp, hannila-handelberg t, suonsyrja t, kontula k. stk variation predicts the ambulatory blood pressure response to losartan in hyper- tensive men. hypertens res ; ( ): – . bell pa, chaturvedi s, gelfand ca, huang cy, kochersperger m, kopla r et al. snpstream uht: ultra-high throughput snp genotyping for pharmacogenomics and drug discovery. biotechniques ; (suppl: – ): – . wigginton je, cutler dj, abecasis gr. a note on exact tests of hardy-weinberg equilibrium. am j hum genet ; ( ): – . motsinger aa, ritchie md. the effect of reduction in cross-validation intervals on the performance of multifactor dimensionality reduction. genet epidemiol ; ( ): – . purcell s, cherny ss, sham pc. genetic power calculator: design of linkage and association genetic mapping studies of complex traits. bioinformatics ; ( ): – . cunnington ms, kay c, avery pj, mayosi bm, koref ms, keavney b. stk poly- morphisms and blood pressure: an association study in british caucasians and assessment of cis-acting influences on gene expression. bmc med genet ; : . pickering gw, keen h, rose g, smith a. the nature of essential hypertension. lancet ; ( ): – . horan mj, lenfant c. epidemiology of blood pressure and predictors of hyper- tension. hypertension ; ( suppl): i –i . gavras i, manolis a, gavras h. genetic epidemiology of essential hypertension. j hum hypertens ; ( ): – . filipovsky j, simon j, chrastek j, rosolova h, haman p, petrikova v. changes of blood pressure and lipid pattern during a physical training course in hypertensive subjects. cardiology ; ( ): – . bhan v, yan rt, leiter la, fitchett dh, langer a, lonn e et al. relation between obesity and the attainment of optimal blood pressure and lipid targets in high vascular risk outpatients. am j cardiol ; ( ): – . kato n, takeuchi f, tabara y, kelly tn, go mj, sim x et al. meta-analysis of genome-wide association studies identifies common variants associated with blood pressure variation in east asians. nat genet ; ( ): – . levy d, ehret gb, rice k, verwoert gc, launer lj, dehghan a et al. genome-wide association study of blood pressure and hypertension. nat genet ; ( ): – . newton-cheh c, johnson t, gateva v, tobin md, bochud m, coin l et al. genome- wide association study identifies eight loci associated with blood pressure. nat genet ; ( ): – . mukherjee b, ahn j, gruber sb, chatterjee n. testing gene-environment inter- action in large-scale case-control association studies: possible choices and com- parisons. am j epidemiol ; ( ): – . kazma r, babron mc, genin e. genetic association and gene-environment inter- action: a new method for overcoming the lack of exposure information in con- trols. am j epidemiol ; ( ): – . supplementary information accompanies the paper on the journal of human hypertension website (http://www.nature.com/jhh) stk and hypertension j xu et al & macmillan publishers limited journal of human hypertension ( ) – http://www.nature.com/jhh 	lack of association between stk and hypertension in the chinese population 	introduction 	methods 	study population 	bp and clinical parameters 	snp genotyping 	statistics 	results 	discussion 	acknowledgements 	note 	references case reports and large, low set ears with a well formed helix. the lenses and fundi were normal. systemic examination showed marked failure to thrive, hypotonia, mental retardation, unilateral undescended testis, and small penis (fig ). both arms were normal in length. there was loose skin over the dorsal aspect of the meta- carpophalangeal joints and an increase in fine palmar creases. dermatoglyphic examination showed bilateral simian creases, two arches, one radial loop, and two ulnar loops on each hand. his palms appeared to be longer than usual because of syndactyly of the skin between all fingers. all joints were hyperextensible. his mental age was around months. laboratory studies routine haematological, biochemical, and urinary studies were normal. his bone age was within nor- mal limits. fifty metaphases, obtained by standard leucocyte culture techniques and stained with giemsa, were analysed. twelve of the metaphases had chromo- somes. each of these cells lacked one chromosome : ,xy, - (fig ). in other metaphases the chromosomal complement showed a normal ,xy pattem. the chromosomal pattern of the mother was normal. discussion despite the fact that banding techniques have been in use for almost a decade now, only one case of monosomy without mosaicism has been de- scribed. our patient is the first case of mosaicism for monosomy , the majority of cells ( of ; %) having a normal ,xy karyotype. patients with partial deletion of g group chromo- somes have two different clinical syndromes: g, (antimongolism) and g (involving chromosome ). the patient of decicco et al with monosomy did not correspond to either of these. in patients with ring chromosome , as reported by hunter et al,s mental retardation, microcephaly, growth failure, and hypotonia were found, as in other deletion syndromes. in our patient, minor anoma- lies such as flat occiput, epicanthus, full eyebrows, dental malocclusion, and cutaneous syndactyly were also present, as in r( ) patients. more cases will have to be documented in detail, however, before a specific monosomy syndrome can be delineated. m s moghe, z m patel, j j peter, and l m ambani unit of medical genetics, institute for research in reproduction, jehangir merwaniji street, parel, bombay , india references al-aish ms, de la cruz f, goldsmith la, volpe j, mella g, robinson jc. autosomal monosomy in man. n engl j med ; : - . dhadial rk. monosomy for g-autosome. arc/h dis child ; : . warren rj, rimoin dl, summitt rl. identification by fluorescent microscopy of abnormal chromosomes, associated with g deletion syndrome. am j hum genet ; : - . decicco f, steele mw, pan s, parck sc. monosomy of chromosome no . a case report. j pediatr ; : - . hunter agw, ray m, wang hs, thompson dr. phenotypic correlations in patients with ring chromosome . clin gentet ; : - . requests for reprints to professor l m ambani, unit of medical genetics, institute for research in reproduction, jehangir merwanji street, parel, bombay , india. variable expression in pfeiffer syndrome summary a female infant with pfeiffer syn- drome (acrocephalosyndactyly v) is presented. her mother has no limb malformations, but has craniofacial features which strongly suggest that she is also affected, although more mildly. this family indicates that wide intrafamilial variation of pfeiffer syndrome is possible and suggests that without detailed investigation mildly affected subjects can remain undiagnosed, which may lead to erroneous genetic counselling. in , pfeiffer' described a family in which eight subjects in three generations had a syndrome con- sisting of craniosynostosis, broad thumbs and big toes, and partial soft tissue syndactyly of the hands and feet. vertical transmission of the trait, and the fact that males and females were equally affected, supported an autosomal dominant mode of in- heritance. because of the close phenotypic similarity to the apert syndrome, pfeiffer reported this family as having a mild form of that syndrome. however, in pedigree studies, no transition from one type to the other was observed. the syndromes are recog- nised by most investigators as separate entities, although recent reports have cast doubt on this conclusion. we report a girl with typical pfeiffer syndrome whose mother has abnormalities limited to the cranium and face, suggesting she is mildly affected. received for publication april o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n f e b ru a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ case reports case report the proband was examined by us when she was years months old. she was the product of an uneventful pregnancy and the second child of non- consanguineous parents who were years old at her birth. at birth, anomalies of cranium, face, and extremities were noted. because of coronal and sagittal suture synostosis, she was operated on at months of age and again two and a half years later. physical examination (fig ) showed acrocephaly, high forehead, hypertelorism, proptosis, malar hypoplasia, flattened face, short philtrum, open mouth, high palate, relative prognathism, and short neck. her thumbs were broad and there was almost right angle deviation of the distal phalanx, rigidity of the second finger without flexion creases, and hyperconvex nails (fig ). both halluces were broad, there was soft tissue syndactyly between the second and third toes, and the nails were hyperconvex (fig ). her weight was on the th centile and ......... *"x,.,., fig feetofprobandshowing broadhalluces:d hegh on the t cetl. raioraphcevaluation' triangularshaped distal phalanges, and bilateral~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~msl~ 'tarsal wer wide andt dyslasic. t'' : s-n ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.... :... .. ...he. rm t r f hrad e high f e a tacro verticachyephan cranium, left e of al stturas s, phlage of the seon fingers..thee-irst .xeita-,' brahyephli ca ium, lef exera srb sm s fig face ofproband. fig hand ofproband showing the typical shape of the thumb in pfeiffer syndrome. fig mother ofproband. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n f e b ru a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ case reports proptosis, malar hypoplasia, and relative prog- nathism. on physical and radiological examination, her hands and feet were normal. metacarpophalan- geal pattern profile (mcpp) analysis on photographs of the hands and feet of the mother by dr victor escobar (university of louisville) did not support the hypothesis that she was affected. the proband's older brother and her mother's half sister were examined by us and found normal. no information could be obtained about the proband's grandparents. chromosomal analysis using con- ventional and g banding techniques was normal: ,xx. discussion the association of facial anomalies and cranio- synostosis, with or without anomalies of the hands and feet, is common to the acrocephalosyndactylies and crouzon syndrome. blank was one of the first to classify the acrocephalosyndactyly (acs) syn- dromes by dividing them into two groups: typical (apert syndrome) and several atypical forms, each the result of different mutations. this theory was based on the observation that only one type had been detected in the same family. pfeiffer' in his original paper believed that this syndrome could represent a mild form of the apert syndrome. however, cohen disagreed since no transition from one form to another in a single family has been observed. on the other hand, jackson et a reported, in a large amish kindred, patients with acs; an additional relatives were also reliably reported to be affected. they observed considerable phenotypic variation, and with the exception of the apert syndrome, all forms of acs were represented, including one patient with polysyndactyly of the feet. these observations suggest that there is con- siderable variation in the acs syndromes. escobar and bixler recently described a family in which both pfeiffer and apert syndromes were present, while seven other subjects had an unusual head shape and face but without abnormalities of the hands or feet, resembling the crouzon syndrome. these authors suggested that the classification of acs syndromes should be reviewed to determine whether apert, pfeiffer, saethre-chotzen, and crouzon syndromes are not, in reality, the same disorder. despite the lack of confirmation by mcpp analysis that the proband's mother is affected, we believe that her craniofacial features are sufficiently demonstrative to support our hypothesis. in the family reported, wide variation in expression appears to exist in pfeiffer syndrome, suggesting that relatives of affected subjects should be carefully examined for minor stigmata of this disorder before providing genetic counselling. we are very grateful to dr william nyham of the department of pediatrics, school of medicine, university of california, san diego, and to dr l stefan levin of the department of otolaryngology, johns hopkins hospital, baltimore, for their editorial advice. jose maria sanchez and teresa ciacci de negrotti fundacion de genetica humana, salta / , buenos aires , argentina references pfeiffer ra. dominant erbliche akrocephalosyndactylie. z kinderheilkd ; : - . cohen mm. an etiologic and nosology overview of craniosynostosis syndromes. birth defects ; , no : - . jackson ce, weiss l, reynolds wa, forman tr, peterson ja. craniosynostosis, midfacial hypoplasia and foot abnormalities: an autosomal dominant phenotype in a large amish kindred. j pediatr ; : - . escobar v, bixler d. on the classification of the acro- cephalosyndactyly syndromes. clin genet ; : - . escobar v, bixler d. the acrocephalosyndactyly syn- dromes: a metacarpophalangeal pattern profile analysis. clin genet ; : - . blank ce. apert's syndrome (a type of acrocephalo- syndactyly): observations on a british series of thirty- nine cases. ann hunt genet ; : - . requests for reprints to dr j m sanchez, fundacion de genetica humana, salta / , buenos aires , argentina. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n f e b ru a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ n-glycosylation is essential for ileal asbt function and protection against proteases call for papers cell signaling: proteins, pathways and mechanisms n-glycosylation is essential for ileal asbt function and protection against proteases saminathan muthusamy, pooja malhotra, mobashir hosameddin, amish k. dudeja, sujata borthakur, seema saksena, ravinder k. gill, pradeep k. dudeja, , and waddah a. alrefai , jesse brown veterans affairs medical center, chicago, illinois; and division of gastroenterology and hepatology, department of medicine, university of illinois at chicago, chicago, illinois submitted january ; accepted in final form april muthusamy s, malhotra p, hosameddin m, dudeja ak, borthakur s, saksena s, gill rk, dudeja pk, alrefai wa. n-glycosylation is essential for ileal asbt function and protection against proteases. am j physiol cell physiol : c –c , . first published april , ; doi: . /ajpcell. . .—the bile acid transporter asbt is a glycoprotein responsible for active absorption of bile acids. inhibiting asbt function and bile acid absorption is an attractive approach to lower plasma cholesterol and improve glucose imbalance in diabetic patients. deglycosylation of asbt was shown to decrease its function. however, the exact roles of n-glycosylation of asbt, and how it affects its function, is not known. current studies investigated the roles of n-glycosylation in asbt protein stability and protection against proteases utilizing hek- cells stably transfected with asbt-v fusion protein. asbt-v protein was detected as two bands with molecular mass of � and � kda. inhibition of glycosylation by tunicamycin sig- nificantly decreased asbt activity and shifted asbt bands to � kda, representing a deglycosylated protein. treatment of total cellular lysates with pngase f or endo h glycosidases showed that the upper -kda band represents a fully mature n-acetylglucosamine-rich glycoprotein and the lower -kda band represents a mannose-rich core glycoprotein. studies with the glycosylation deficient asbt mutant (n q) showed that the n-glycosylation is not essential for asbt targeting to plasma membrane. however, mature glycosylation significantly increased the half-life and protected asbt protein from digestion with trypsin. incubating the cells with high glucose ( mm) for h increased mature glycosylated asbt along with an increase in its function. these results unravel novel roles for n-glycosylation of asbt and suggest that high levels of glucose alter the composition of the glycan and may contribute to the increase in asbt function in diabetes mellitus. bile acid absorption; n-glycans; bile acids; intestinal transport pro- teins apical sodium-dependent bile acid transporter (asbt) is a plasma membrane protein expressed in the distal ileum and mediates the na�-dependent transport of bile acids across the apical membrane of intestinal epithelial cells ( ). asbt func- tion represents the first and rate-limiting step of reabsorption of bile acids that are then transported back to the liver via the portal blood where they influence cholesterol metabolism and insulin signaling, as well as lipid and glucose metabolism ( ). the essential role of asbt in controlling enterohepatic circu- lation of bile acids is supported by the fact that surgical removal of the distal ileum and genetic deletion of asbt in mice leads to bile acid malabsorption and the depletion of their circulating pool between the liver and intestine ( , ). on the other hand, a decrease in asbt expression was shown in disorders with lipid abnormalities such as familial type iv hypertriglyceridemia ( ). further, asbt function and expres- sion are increased in diseases that are associated with bile acid overload in the liver like necrotizing enterocolitis (nec) and progressive familial intrahepatic cholestasis (pfic) ( , ). therefore, much attention has been focused on investigating asbt regulation and defining molecular targets for modulat- ing its function and expression. the transcriptional regulation of asbt has been extensively investigated. asbt mrna expression and promoter activity is decreased by proinflammatory cytokines and by cholesterol ( , ). also, bile acids through fxr-shp axis suppress asbt expression by reducing the binding of rar/rxr activating complex to asbt promoter ( ). on the other hand, the activation of glucocorticoid receptor increases asbt expres- sion and promoter activity ( ). besides the transcriptional regulation, recent studies also demonstrated posttranscriptional modulations of asbt. for example, the activation of pkc �-dependent pathway decreases asbt level on the plasma membrane ( ). also, asbt function is dependent on lipid raft domains of the plasma membrane and the depletion of plasma membrane cholesterol decreases asbt function ( ). n-glycosylation represents one of the most important post- translational modifications of plasma membrane proteins. the attachment of glycans to membrane proteins is known to influence many functional aspects including targeting to plasma membrane ( , , , , ). further, changes in the pattern of glycosylation of plasma membrane proteins occur in diseases with hyperglycemia affecting their function and con- tributing to the associated pathophysiology as shown for the urea transporter ut-a in the kidney ( ). asbt has been previously shown to be n-glycosylated on the asparagine residue n in the first extracellular loop of the protein ( ). deglycosylation led to a decrease in asbt function without affecting its targeting to plasma membrane ( ); however, the exact mechanism by which the deglycosylation of asbt alters its function remains elusive. also, bile acid absorption was increased in diabetic patients and animal models of diabetes mellitus, and whether hyperglycemia affects asbt function and n-glycosylation is not known ( , , ). address for reprint requests and other correspondence: w. a. alrefai, jesse brown va medical center, medical research service ( / ), s. damen ave., chicago, il (e-mail: walrefai@uic.edu). am j physiol cell physiol : c –c , . first published april , ; doi: . /ajpcell. . . http://www.ajpcell.orgc downloaded from journals.physiology.org/journal/ajpcell at carnegie mellon univ ( . . . ) on april , . mailto:walrefai@uic.edu the current studies were focused at investigating the mech- anisms by which glycosylation affects asbt function and surface expression in hek cells stably transfected with asbt-v fusion protein. our studies provide novel molecular insights into the roles of n-glycosylation in increasing the half-life of asbt protein on the plasma membrane and pro- tecting asbt against digestion by proteases. further, these data suggest that hyperglycemia-induced changes in the gly- cosylation may underlie the increase in asbt function and bile acid absorption associated with diabetes mellitus. materials and methods materials. taurocholic acid and cycloheximide were purchased from sigma (st. louis, mo); [ h]taurocholic acid (tca) was pur- chased from perkin-elmer (boston, ma). anti-v -hrp was pur- chased from invitrogen (carlsbad, ca). anti-gapdh-hrp antibody was purchased from santa cruz biotechnology (paso robles, ca). cell culture medium was purchased from attc (manassas, va), and the transfection reagents were purchased from invitrogen. agarose-bound lectins were purchased from vector laboratories (burlingame, ca). cell culture and stable cell lines. we have previously established human embryonic kidney (hek)- cells that are stably transfected with human asbt-v fusion protein (designated as bt cells) ( , ) as a suitable model to investigate posttranslational regulation of asbt. cells were cultured in mem supplemented with % fbs and were plated at a density of � /well in -well falcon plates. bt cells reached – % confluence after – days in culture and were utilized for experiments. the medium was supplemented with �g/ml of geneticin in case of the stably transfected cells. transient transfections in hek- cells were carried out using lipofectamine (invitrogen) as previously described ( ). western blotting. cells were lysed in ripa buffer (sigma) supple- mented with freshly added protease inhibitor cocktail (roche, bran- ford, ct). protein concentration was measured by the method of bradford using protein assay kit (bio-rad, hercules, ca). proteins ( �g) were then solubilized in laemmli sample buffer ( % sds, % glycerol, �m dtt, mm tris, ph . , . % bromophe- nol blue) and separated on – % gradient sds-page gels (bio- rad). separated proteins were then transferred to nitrocellulose mem- brane, and the blots were incubated with the anti-v hrp-conjugated antibody diluted in the blocking solution for h at room temperature. blots were washed extensively with pbs containing . % tween- , and then the bands were visualized by enhanced chemiluminescence ecl kit according to the manufacturer’s instructions (amersham, arlington heights, il). cell surface biotinylation. hek- cells stably or transiently transfected were washed with ice-cold pbs and incubated with mg/ml of ez-link sulfo-nhs-biotin (pierce, rockford, il) dissolved in borate buffer ph . for h. incubating with mm glycine in ice-cold pbs for min quenched the reaction; cells were then lysed in ripa buffer and insoluble debris was cleared by centrifugation ( ). biotinylated proteins were separated by neutravidin agarose beads (pierce), washed with ripa buffer, and eluted by boiling in x sds buffer (bio-rad). the eluted proteins were analyzed by western blotting. to validate biotinylation of surface proteins, the same blot was stripped and probed with anti-gapdh-hrp antibodies. lectin pull-down assay. lectin-pull downs were performed in total cell lysates prepared with ripa buffer. four hundred micrograms of total cell lysates was added to �l of agarose bound lectins (vector laboratories) in a total volume of �l ripa buffer and mixed for overnight at �c. the beads were washed three times in ripa buffer; boiled in x sds buffer for min to elute bound proteins, and the resulting eluent was analyzed by western blotting with anti-v -hrp antibodies. treatment with glycosylation inhibitors and glycosidases. tunica- mycin (sigma, st. louis, mo) at a concentration of . �g/ml was used to inhibit n-glycosylation in stably transfected hek- cells for h. cell lysates were digested with endoglycosidase h (endo h) for h at °c, (new england biolabs, ipswich, ma) to remove high mannose glycans (core-glycosylated). peptide-n-glycosidase f (pngase f) (new england biolabs) was used to remove n-linked glycans from both complex and high mannose glycoproteins for h at °c. digested proteins were analyzed by western blotting with anti-v -hrp antibodies. taurocholic uptake assay. hek- cells stably transfected with hasbt were used for uptake experiments. cells were seeded in -well plates, and uptake was carried out – days postplating. uptake was carried out as described previously ( ). to determine the sodium dependency of the uptake, na� in the buffer was isosmoti- cally replaced with choline chloride. uptake rates were measured as picomoles per milligram of protein per min. statistical analysis. results were expressed as means � se of – experiments performed on separate occasions. student’s t-test was utilized for statistical analysis. p � . was considered statistically significant. results deglycosylation decreases asbt protein levels on plasma membrane. since asbt has previously been shown to be a glycoprotein, we aimed at investigating how n-glycosylation affected asbt function and membrane expression. we first examined the pattern of asbt glycosylation in hek- cells stably transfected with asbt-v fusion protein (designated as bt cells). as shown in fig. a, asbt-v fusion protein was detected as two bands with molecular masses of about kda and kda as previously reported in cos- cells ( ). cell surface biotinylation presented in fig. a demonstrated that the two asbt bands were detected on the plasma membrane fractions. also, fig. a shows that the higher molecular weight band was more enriched on the plasma membrane compared with the lower molecular weight band. treatment of bt cells with the n-glycosylation inhibitor tunicamycin ( . �g/ml, h) blocked asbt glycosylation and shifted both asbt bands to a lower band with a molecular mass of about kda (fig. a). previous studies have shown that the asparagine residue n located on the extracellular nh terminus of asbt is the only site responsible for asbt glycosylation. we have gener- ated hek- cells that express asbt-v fusion protein in which the n residue was mutated to glutamine (n q). as shown in fig. b, the n q mutant was detected as a -kda immunoreactive band similar to the band that was detected after treatment with tunicamycin. these data show that n-gly- cosylation of asbt generates two different glycosylated pro- teins. densitometric analysis presented in fig. c shows that in response to tunicamycin treatment, total cellular expression of asbt is significantly lower than that of the wild-type asbt (considering the sum of densities of higher and lower molec- ular weight bands). the observed reduction mainly occurs because of the loss of the higher molecular weight asbt protein. similar to the decrease in the surface levels, asbt function is also significantly decreased � % in cells treated with tunicamycin as depicted in fig. . n-glycosylation is not essential for asbt trafficking to plasma membrane. we next investigated whether n-glycosyl- ation is important for asbt trafficking by evaluating the rate by which asbt is targeted to the plasma membranes as c posttranslational modulation of ileal bile acid transport ajp-cell physiol • doi: . /ajpcell. . • www.ajpcell.org downloaded from journals.physiology.org/journal/ajpcell at carnegie mellon univ ( . . . ) on april , . previously described ( ). hek- cells were transiently transfected with wild-type or n q asbt mutant and then surface expression of the newly synthesized asbt protein from transfected plasmid was assessed by cell surface biotiny- lation at different time points ( , , and h) posttransfection. as shown in fig. a, the two bands of asbt were detected at the surface after h from the start of transfection. data depicted in fig. a show that the asbt mutant is also detected on the plasma membrane by h posttransfection, and its level was increased by time. densitometric analysis of the levels of surface asbt over time, shown in fig. b, demonstrates that the rate of trafficking of wild-type asbt glycoprotein is similar to that of the nonglycosylated n q asbt mutant. these data suggest that glycosylation is not essential for the trafficking of asbt to the plasma membrane. n-glycosylation profile of asbt glycoprotein. as the two immunoreactive bands of wild-type asbt represent different glycoproteins, we next examined the nature of carbohydrates that contribute to each of these two glycoproteins of asbt. total protein lysates from bt cells were treated with pngase f and endo h glycosidases. the endo h recognizes high mannose carbohydrate chains, whereas pngase f cuts the first n-acetylglucosamine of the glycan that is attached to aspara- gine residues on the protein. as expected, treatment with pngase f shifted the two asbt bands to the lower band of � kda similar to treatment with tunicamycin (fig. ). however, treatment with endo h shifted only the low molec- ular weight band to kda, whereas, the high molecular weight band appears to be insensitive to endo h treatment. these data clearly suggest that the lower band of wild type asbt is mannose rich core glycosylated protein, whereas the higher band is the mature n-acetylglucosamine rich glycosy- lated asbt protein. to further confirm the nature of carbohydrates associated with asbt glycoprotein, total cellular proteins from bt cells or from hek- cells stably expressing n q asbt mutant were subjected to pull-down assays with different types of lectins that interact with different carbohydrate chains associ- ated with glycoproteins. as shown in fig. , the agarose bound cona lectin selectively enriched the mannose rich core-glyco- sylated asbt, whereas the agarose bound wheat germ agglu- tinin (wga) selectively pulled down the highly complex glycosylated asbt protein (upper band). the nonglycosylated asbt-n q mutant did not interact with the lectins, indicat- ing the specificity of the assays. these results support the notion that asbt glycosylation occurs as a core mannose rich or mature complex forms. a gapdh ub lb asbt dg total cellular asbt tunicamycin [ g/ml] . cell surface asbt tunicamycin [ g/ml] . s kda s kda s kda b gapdh ub lbasbt dg asbt (wt) asbt (n q) s kda s kda c a s b t p ro te in /g a p d h ( % o f c on tr ol ) * . tunicamycin [µg/ml] fig. . deglycosylation decreases asbt protein levels on the plasma membrane. a: hek- cells stably expressing wild-type asbt-v fusion protein were treated with tunicamycin ( . �g/ml) or vehicle for h followed by cell surface biotinylation. the labeled cell surface proteins were precipitated with streptavidin beads and resolved by sds-page followed by immunoblotting with anti-v antibody. ub, upper band (� kda); lb, lower band (� kda); dg, deglycosylated band (� kda). the specificity of the cell surface labeling was confirmed by absence of gapdh on the cell surface. b: hek- cells stably expressing glycosylation deficient mutant asbt (n q)-v total protein was extracted and separated by sds-page followed by western blotting with anti-v antibody. c: densitometric analysis for asbt relative expression in total cell lysates normalized to the levels of gapdh. values are expressed as % of control and represent means � se of – measurements. *p 	 . compared with control. c posttranslational modulation of ileal bile acid transport ajp-cell physiol • doi: . /ajpcell. . • www.ajpcell.org downloaded from journals.physiology.org/journal/ajpcell at carnegie mellon univ ( . . . ) on april , . n-glycosylation of asbt increases protein stability. since the level of the mature glycosylated asbt was higher than that of the mutant, we next examined whether n-glycosylation affects the stability of asbt. for these studies, protein de novo synthesis in bt cells and hek- cells stably transfected with asbt mutant was blocked by treatment with cyclohexi- mide ( . mg/ml) for different periods of time. at the end of the incubation, cell surface proteins labeled by biotin were precipitated by streptavidin beads. as shown in fig. a, the mature glycosylated asbt glycoprotein appears to have lon- ger half-life compared with that of the core glycosylated asbt when investigated with the total cell protein lysates and plasma membrane biotinylated fractions (mature glycosylated: � . h; core glycosylated: � . h; and nonglycosylated: . � . h). interestingly, the half-life of asbt mutant was com- parable to the half-life of core glycosylated asbt. densito- metric analysis presented in fig. b showed that mature n-glycosylation significantly increased the half-life of asbt protein by � – fold on the plasma membrane consistent with the increase in its function. n-glycosylation of asbt protects against proteases. glyco- sylation is known to protect plasma proteins against digestion by external proteases ( ). we then examined whether n- glycosylated asbt is more resistant to cleavage by proteases. total protein lysates from bt cells and hek- cells stably expressing asbt n q mutant were treated with trypsin ( �m) for min and the proteolytic products were examined by western blotting. figure a shows that the trypsin treatment caused a decrease in the level of asbt mutant protein more than the decrease observed in wild-type asbt protein. the numeric representation of the data shown in fig. b clearly demonstrates that the digestion by trypsin was significantly increased in nonglycosylated (n q mutant) compared with n-glycosylated asbt. these data indicate that n-glycosyla- tion protects asbt against the proteolytic effects of proteases such as trypsin. high glucose increases asbt glycosylation and function. recent studies in animal models and humans demonstrated that diabetes mellitus and hyperglycemia modulate n-glycosylation by increased glucose flux through the hexosamine pathway ( , , ). to examine whether high glucose in the cell culture media alters asbt n-glycosylation levels, bt cells were incubated with medium containing mm concentration of n a + -d e p e n d e n t h -t c u p ta k e (% o f c o n tr o l) * . tunicamycin [ g/ml] fig. . tunicamycin decreases asbt function. hek- cells stably expressing wild-type asbt-v fusion protein were treated with tunicamy- cin ( . �g/ml) or vehicle (control) for h and asbt function was then assessed as described in materials and methods. asbt function was determined as na� dependent [ h]taurocholate ([ h]tc) uptake and ex- pressed as % of control. data are expressed as means � se of – experiments. *p 	 . compared with control. a wt n q cell surface asbt post transfection (h) ub lb s kda s kda s kda a s b t s ur fa ce e xp re ss io n (% o f m ax im al e xp re ss io n) post transfection (h) b fig. . n-glycosylation is not essential for asbt trafficking to plasma membrane. a: transiently transfected cells were biotinylated at various time points posttransfection, and cell surface proteins were precipitated with strepta- vidin beads followed by western blotting with anti-v antibody. ub, upper band; lb, lower band. b: densitometric analysis for the surface expression of wild-type asbt (up triangles, ub; down triangles, lb) and asbt-n q (circles) at various times posttransfection. values are expressed as % of maximal expression and represent means � se of experiments. control pngasef endo h asbt s kda s kda fig. . asbt n-glycosylation profile. total cell lysates extracted from wild- type asbt-v fusion protein were treated with peptide-n-glycosidase f (pngase f) and endoglycosidase h (endo h), and then samples were solubi- lized in laemmli sample buffer and resolved by sds-page followed by western blotting with anti-v antibody. c posttranslational modulation of ileal bile acid transport ajp-cell physiol • doi: . /ajpcell. . • www.ajpcell.org downloaded from journals.physiology.org/journal/ajpcell at carnegie mellon univ ( . . . ) on april , . glucose (normal) or mm concentration of glucose (high) for h. figure shows that asbt activity, judged by the na�-dependent [ h]taurocholate uptake, was significantly in- creased when cells where incubated with mm compared with mm glucose. western blot analysis presented in fig. , a and b, showed that the expression of the fully mature glycosylated asbt protein was significantly increased in re- sponse to incubation with mm glucose. these findings suggest that high levels of glucose increase asbt glycosyla- tion, leading to an increase in the expression of the fully mature glycoprotein and an increase in asbt function. discussion ileal asbt activity has previously been shown to decrease in response to deglycosylation ( ). however, the mechanisms by which glycosylation affects asbt function remains un- clear. data presented in the current study for the first time demonstrate that n-glycosylation is essential for asbt protein stability on the plasma membrane and for the protection against digestion with proteases. we investigated asbt glycosylation utilizing human em- bryonic kidney (hek)- cells stably transfected with asbt-v fusion protein that has been previously established by us as an excellent model to examine posttranslational regulation of asbt ( ). asbt-v protein in bt cells was detected by western blotting as two bands. in the current studies, the nature of the asbt bands was scrutinized by two complementary approaches: digestion with glycosidases and lectin pull-down assay. the peptide-n-glycosidase f (pngase f) cleaves the bond between the asparagine residue on the glycoprotein and the first n-acetylglucosamine (glcnac) sugar molecule in the attached polysaccharide releasing the full glycan chain from all types of the n-glycosylated proteins. pngase f treatment to n q wt cona cona wga pull down n q wtinput asbt supernatant asbt s kda s kda s kda s kda fig. . lectin pull-down assays for wild-type asbt and glycosylation defi- cient n q mutant asbt protein samples. equal amounts protein from total cellular lysate were incubated with agarose-bound lectins ( °c) overnight along with protease inhibitor cocktail. after washing with ripa buffer, bound samples were eluted by boiling with x laemmli buffer and used for western blotting with anti-v antibody. top panel shows the pull-down fractions and bottom panel shows unbound proteins in the supernatant. a total cellular asbt post cycloheximide (h) wt n q cell surface asbt post cycloheximide (h) ub lb s kda s kda s kda b a s b t s ur fa ce e xp re ss io n ( % o f c on tr ol ) post cycloheximide (h) fig. . n-glycosylation increases asbt protein stability. a: hek- cells stably expressing wild-type asbt or glycosylation deficient n q mutant asbt were treated with . mg/ml of cycloheximide for different periods of time and then cell surface proteins were biotinylated. the labeled cell surface proteins were precipitated with streptavidin beads and resolved by sds-page followed by western blotting with anti-v antibodies (ub, upper band; lb, lower band). b: densitometric analysis of surface levels of wild-type asbt and glycosylation deficient mutant asbt-n q (up triangles, ub; down triangles, lb; circles, n q mutant). values are expressed as % control (values at h). c posttranslational modulation of ileal bile acid transport ajp-cell physiol • doi: . /ajpcell. . • www.ajpcell.org downloaded from journals.physiology.org/journal/ajpcell at carnegie mellon univ ( . . . ) on april , . protein lysates of bt cells shifted both the high and low molecular weight bands of asbt to a lower molecular weight band (� kda), indicating that both the bands represent glycosylated asbt. this pattern was similar when the cells were incubated with the glycosylation inhibitor tunicamycin. the observation that the band generated by treatment with pngase f and tunicamycin is a nonglycosylated asbt was further confirmed by showing that asbt band generated by the treatment has the same molecular weight as the glycosyl- ation deficient n q-asbt mutant. the other glycosidase used was endoglycosidase h (endo h) that selectively cleaves the oligomannose-type of glycans attached to glycosylated proteins. the low molecular weight, but not the high molecular weight, glycosylated asbt was sensitive to endo h and was cleaved to a nonglycosylated asbt. collectively, these data suggested that the low molecular weight asbt band represents a glycoprotein with a mannose rich glycan, whereas, the high molecular weight band represents a fully mature glycosylated protein. it is worth noting that dawson et al. ( ) previously demonstrated the presence of glycosylated asbt protein in brush-border membrane vesicles prepared from mouse intes- tine that was not sensitive to digestion by endo h indicating a mature glycosylated protein. this previous observation implies that asbt protein expressed on the apical membrane of native intestinal epithelial cells is a mature glycosylated protein. therefore, investigating the functional aspects of the mature glycosylated asbt in bt cells is relevant to the native intestinal epithelial cells. the complex structure of the glycan attached to the high molecular weight band of asbt was further confirmed by the ability of wheat germ agglutinin (wga) lectin, which binds to glycoproteins rich with glcnac and sialic acid, to pull down the upper asbt band. notably, wga lectin did not com- pletely pull down the upper asbt band suggesting the pres- ence of multiple types of complex glycan species forming the mature glycosylated asbt protein. incubation with lectin cona that binds to -linked mannose glycan, pulled down only the lower asbt band further supporting the notion that the low molecular weight asbt band represents mannose rich asbt glycoprotein. n-glycosylation plays important roles in protein folding and trafficking from the endoplasmic reticulum (er) to the golgi apparatus as well as targeting of proteins to the apical mem- brane of epithelial cells ( ). in this regard, a polysaccharide chain is usually attached to a newly synthesized plasma mem- brane protein in the er that is then transported to the golgi apparatus for further processing ( ). some proteins, however, traffic through the golgi and post-golgi without any process- ing and reach the plasma membrane as mannose-rich glyco- proteins ( ). our data showed that the core glycosylated, mannose rich asbt glycoprotein (the low molecular weight band) is expressed on the plasma membrane indicating that a pool of asbt protein could be targeted to the plasma mem- brane through the golgi apparatus without any additional processing. both the two glycosylated asbt proteins and the glycosylation-deficient asbt mutant (n q) were targeted to the plasma membrane with similar efficiencies after the start of transient transfection of their corresponding expression vector in hek- cells. our data show that glycosylation is not essential for asbt targeting. these data are consistent with the previous studies of zhang et al. ( ) showing comparable a trypsin [ μm] asbt (wt) asbt (n q) trypsin [ μm] control control s kda s kda b a s b t e x p re s s io n (% o f c o n tr o l) * trypsin [μm] asbt (wt) asbt (n q) fig. . n-glycosylation protects against proteases. a: equal amount of protein from total cell lysates prepared from wild-type asbt or glycosylation defi- cient mutant asbt-n q were digested with �m trypsin for min and then reaction was stopped with trypsin inhibitor. samples were then solubilized in laemmli buffer and used for western blotting with anti-v antibody. b: densitometric analysis of wild-type asbt and glycosylation deficient mutant asbt-n q is presented. data are presented as % of respective controls and are shown as means � se of experiments. *p 	 . compared with control. n a + -d e p e n d e n t h -t c u p ta k e (% o f c o n tr o l) * glucose [mm] fig. . high glucose increases asbt function. a: hek- cells stably expressing wild-type asbt were incubated with media containing or mm glucose for h, and then asbt function was assessed as described in materials and methods. asbt function was determined as na� dependent [ h]tc uptake and expressed as % of control ( mm glucose). values are shown as means � se of experiments. *p 	 . compared with control. c posttranslational modulation of ileal bile acid transport ajp-cell physiol • doi: . /ajpcell. . • www.ajpcell.org downloaded from journals.physiology.org/journal/ajpcell at carnegie mellon univ ( . . . ) on april , . levels of surface membrane expression of wild-type asbt and the glycosylation-deficient asbt. our results and the previous findings indicate that glycosylation is not essential for asbt targeting to the plasma membrane. protein glycosylation was shown to increase the half-life of plasma membrane transporters like the potassium channel kv . and the receptor potassium channel trpp as well as receptor potential melastatin b* protein ( , ). our studies showed that the half-life of the mature glycosylated asbt is significantly higher than the half-life of the core glycosylated and the deglycosylated forms of asbt, thus providing novel evidence showing that complex glycosylation of asbt in- creases its protein stability on the plasma membrane. asbt protein on the plasma membrane of ileal epithelial cells is exposed to a milieu that is rich with digestive enzymes including proteases. indeed, glycosylation was shown to be one of the important mechanisms protecting intestinal epithelial proteins from degradation by digestive enzymes ( ). this was shown to be the case for the h�/k�-atpase proton pump and intestinal slc a chloride transporter ( , ). similarly, our data showed that mature glycosylated asbt is resistant to digestion by trypsin suggesting that glycosylation is essential for the protection of the asbt protein from digestion by luminal proteases. changes in the n-glycans attached to proteins were altered in diseases such as diabetes mellitus ( ). hyperglycemia associated with diabetes mellitus may lead to an increase in the flux of glucose through the hexosamine pathway resulting in an increase in the production of udp-glcnac and hence affecting protein glycosylation ( , , ). for example, studies showed that the carbohydrate structure of the n-glycan attached to renal urea transporter ut-a was altered in diabetes mellitus causing an increase in the total level of the protein ( ). our data demonstrated a significant increase in asbt function in bt cells in response to prolonged incubation ( h) with high levels of glucose. this increase in the function is not due to transcriptional regulation since an exogenous promoter in bt cells drives asbt expression. indeed, the level of the mature glycosylated, but not the core glycosylated, asbt protein was significantly increased by exposure to glucose. these data suggest that high levels of glucose enhanced asbt glycosyl- ation leading to an increase in asbt protein levels, likely via promoting its protein stability. in conclusion, our data for the first time show that asbt half-life on the plasma membrane is significantly increased by mature n-glycosylation. also, n-glycosylation of asbt pro- vides protection against digestion with luminal proteases that may have access to the brush-border membrane of intestinal epithelial cells. interestingly, high levels of glucose increased the glycosylation of asbt and enhanced its function. in this regard, we have previously shown that asbt mrna and protein expression were significantly elevated in rat model of diabetes mellitus ( ). our current studies provide novel evi- dence for a posttranslational mechanism that contributes to an increase in asbt protein levels in response to hyperglycemia associated with diabetes mellitus. indeed, elucidating the mechanisms by which asbt glycosylation is altered by high glucose may unravel novel pathways that could be targeted to inhibit bile acid absorption in diabetes mellitus. future studies are needed that focus on investigating the nature of the changes in the n-glycans attached to asbt in diabetic patients and in animal models of diabetes mellitus. acknowledgments we are grateful to dr. k. ramaswamy for critical reading and discussion of the manuscript. grants these studies were supported by the department of veterans affairs (w. a. alrefai, p. k. dudeja) and by national institute of diabetes and digestive and kidney diseases grants dk- (w. a. alrefai), dk- , dk- , dk- (p. k. dudeja), dk- (r. k. gill), and dk- (s. sak- sena). disclosures no conflicts of interest, financial or otherwise, are declared by the author(s). author contributions author contributions: s.m., s.s., r.k.g., p.k.d., and w.a.a. conception and design of research; s.m., p.m., m.h., a.k.d., and s.b. performed experiments; s.m., p.m., m.h., a.k.d., s.b., s.s., r.k.g., p.k.d., and w.a.a. analyzed data; s.m., p.m., m.h., a.k.d., s.b., s.s., r.k.g., p.k.d., and w.a.a. interpreted results of experiments; s.m., r.k.g., and w.a.a. prepared figures; s.m., r.k.g., and w.a.a. drafted manuscript; s.m., s.s., a glucose [mm] asbt gapdh ub lb s kda s kda s kda b h ig h ly g ly c o s y la te d a s b t /g a p d h (% o f c o n tr o l) * glucose [mm] fig. . high glucose increases asbt glycosylation. a: hek cells stably expressing wild-type asbt were incubated with media containing or mm glucose for h. total cellular proteins were extracted and equal amounts of protein were resolved by sds-page, and western blotting was performed with anti-v antibody. b: densitometric analysis of the mature glycosylated for asbt in mm glucose treated cells and expressed as % of control ( mm glucose). *p 	 . compared with control. c posttranslational modulation of ileal bile acid transport ajp-cell physiol • doi: . /ajpcell. . • www.ajpcell.org downloaded from journals.physiology.org/journal/ajpcell at carnegie mellon univ ( . . . ) on april , . r.k.g., p.k.d., and w.a.a. edited and revised manuscript; s.m., s.s., r.k.g., p.k.d., and w.a.a. 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bile acid transport ajp-cell physiol • doi: . /ajpcell. . • www.ajpcell.org downloaded from journals.physiology.org/journal/ajpcell at carnegie mellon univ ( . . . ) on april , . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ neurology .. neuroimages pseudo-foster-kennedy syndrome with optic nerve compression by the gyrus rectus a -year-old woman presented with headaches and left eye visual loss. examination revealed acuity / od and finger counting os, a left afferent pupillary defect, papilledema od, and optic atrophy os. left atrophy was unexplained until orbital mri revealed left nerve compression by the gyrus rectus (figure, a), displaced by an intraventricular central neurocytoma (figure, b). foster-kennedy syndrome is characterized by optic atrophy on one side due to direct optic nerve mass lesion compression with contralateral papilledema. this case is termed pseudo-foster-kennedy with indirect compressive optic neuropathy due to brain displacement from a tumor distant from the optic nerve. ninad desai, mbbs, raymund l. yong, md, amish doshi, md, janet c. rucker, md from nyu langone medical center (n.d., j.c.r.); and mount sinai medical center (r.l.y., a.d.), new york, ny. author contributions: ninad desai: manuscript writing and editing. raymund yong: conception of idea, manuscript writing and editing. amish doshi: manuscript writing and editing. janet rucker: conception of idea, manuscript writing and editing. study funding: no targeted funding reported. disclosure: the authors report no disclosures relevant to the manuscript. go to neurology.org for full disclosures. correspondence to dr. rucker: janet.rucker@nyumc.edu . klingele tg, gado mh, burde rm, coxe ws. compression of the anterior visual system by the gyrus rectus. j neurosurg ; : – . figure optic nerve compression by gyrus rectus and intraventricular mass causing displacement of gyrus rectus coronal t -weighted orbital and brain mri reveals left lateral ventriculomegaly with downward displacement of the gyrus rectus, resulting in left optic nerve compression (a), due to a left intraventricular mass (b). © american academy of neurology ª american academy of neurology. unauthorized reproduction of this article is prohibited. http://neurology.org/lookup/doi/ . /wnl. mailto:janet.rucker@nyumc.edu doi . /wnl. ; ; neurology ninad desai, raymund l. yong, amish doshi, et al. pseudo-foster-kennedy syndrome with optic nerve compression by the gyrus rectus this information is current as of july , services updated information & http://n.neurology.org/content/ / / .full including high resolution figures, can be found at: references http://n.neurology.org/content/ / / .full#ref-list- this article cites articles, of which you can access for free at: subspecialty collections http://n.neurology.org/cgi/collection/primary_brain_tumor primary brain tumor http://n.neurology.org/cgi/collection/optic_nerve optic nerve following collection(s): this article, 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http://n.neurology.org/subscribers/advertise an initial brainstem attack sharing chronic lympho- cytic inflammation with pontine perivascular enhancement responsive to steroids (clippers) fea- tures. after steroid weaning, mog-seropositive lon- gitudinally extensive transverse myelitis (letm) involving the conus appeared, but in absence of brainstem lesions. a diagnosis of clippers is dif- ficult in this clinical picture. brainstem punctate and curvilinear enhancements, a characteristic radiologic finding of clippers, may conceal several diseases such as glioma, primary cns lymphoma, lymphomatoid granulomatosis, primary cns vasculitis, and multiple sclerosis. except for gli- oma, all of these diseases initially respond to high doses of steroids, and some could have a relapsing-remitting course in absence of immunosuppressive therapy. however, unlike clippers, the brainstem is not sys- tematically affected at each relapse and lesion distribu- tion does not remain concentrated in the pons. – the symmonds et al. case highlights a possible pathophysiologic connection between clippers and demyelinating diseases. recently, a postmortem analysis performed in a patient sharing all clippers features revealed the classical perivascular lymphohis- tiocytic infiltrates but also perivenular demyelinating lesions (as seen in acute disseminated encephalomye- litis). since perivenular demyelinating lesions were found in only one clippers patient, it is unlikely that clippers is a primary demyelinating disease. however, as suggested by the authors, clippers may induce immunization against mog antigen. author response: mkael symmonds, m. isabel leite, ursula g. schulz, oxford, uk: we agree with the important points that drs. taieb and lab- auge raised in response to our recent report. our patient presented with clinical and radiologic features consistent with clippers, even though the subse- quent episode of letm involving the conus had not been previously reported as part of this disease spec- trum. while clippers can relapse, typically with recurrence of brainstem inflammatory features, this does not form key diagnostic criteria and the diagno- sis could be consistent with a single episode. as discussed, the differential diagnosis of clippers is wide and many of these alternatives will clearly define themselves in time. the histo- pathologic findings of demyelination in an isolated case at postmortem provides interesting additional support for the hypothesis that clippers may not be a distinct entity, but rather have a range of underlying etiologies. our case raises the possibility that anti-mog antibodies may be causal in some clippers cases, although we cannot exclude the possibility that clippers itself causes immuniza- tion against mog epitopes. © american academy of neurology . symmonds m, waters pj, küker w, et al. anti-mog anti- bodies with longitudinally extensive transverse myelitis pre- ceded by clippers. neurology ; : – . . jones jl, dean af, antoun n, et al. “radiologically com- patible clippers” may conceal a number of pathologies. brain ; :e . . limousin n, praline j, motica o, et al. brain biopsy is required in steroid-resistant patients with chronic lympho- cytic inflammation with pontine perivascular enhancement responsive to steroids (clippers). j neurooncol ; : – . . ferreira rm, machado g, souza as, et al. clippers-like mri findings in a patient with multiple sclerosis. j neurol sci ; : – . . moreira i, cruto c, correia c, et al. chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (clippers): postmortem findings. j neuropathol exp neurol ; : – . correction pseudo-foster-kennedy syndrome with optic nerve compression by the gyrus rectus in the neuroimage “pseudo-foster-kennedy syndrome with optic nerve compression by the gyrus rectus” by n. desai et al. (neurology® ; : ), there is an error in the correspondence address. the note at the bottom should read “corre- spondence to dr. rucker: janet.rucker@nyumc.org.” the authors regret the error. author disclosures are available upon request (journal@neurology.org). neurology october , ª american academy of neurology. unauthorized reproduction of this article is prohibited. mailto:janet.rucker@nyumc.org mailto:journal@neurology.org biomed centralbmc pediatrics ss open accecorrespondence care for amish and mennonite children with cystic fibrosis: a case series jonathan f henderson and ran d anbar* address: department of pediatrics, state university of new york upstate medical university, syracuse, ny, usa email: jonathan f henderson - hendersonj@upstate.edu; ran d anbar* - anbarr@upstate.edu * corresponding author abstract background: published articles have described a lack of willingness to allow preventative measures, as well as other types of modern therapies, as an obstacle to providing medical care for amish and mennonite populations. methods: we present data regarding the amish and mennonite patients at the suny upstate medical university pediatric cystic fibrosis center and three representative case reports. results: families of patients from these communities receiving care at our center have accepted preventive therapy, acute medical interventions including home intravenous antibiotic administration, and some immunizations for their children with cystic fibrosis, which have improved the health of our patients. some have even participated in clinical research trials. health care education for both the child and family is warranted and extensive. significant cystic fibrosis center personnel time and fundraising are needed in order to address medical bills incurred by uninsured amish and mennonite patients. conclusion: amish and mennonite families seeking care for cystic fibrosis may choose to utilize modern medical therapies for their children, with resultant significant improvement in outcome. background while the amish and mennonite communities offer the opportunity for study of closed populations with an increased incidence of certain genetic disease and defects, including cystic fibrosis (cf) [ - ], no published data have dealt with treatment and socioeconomic impact of cf on amish and mennonite families. in the united states, the population of the amish in was reported as approximately , while the men- nonite population was approximately , [ ]. the incidence of cf among these populations has been diffi- cult to estimate because of the closed nature of their com- munities. for example, in one ohio amish isolate the incidence of cf was / live births, while in another isolate there was no occurrence of cf among live births [ ]. the incidence of cf in the general united states population has been approximately / live births, however this incidence is falling, perhaps as a result of preconception and prenatal screening offered to the gen- eral population in order to identify carriers of cystic fibro- sis [ ]. as a majority of amish and mennonites may approve of cf carrier testing that can impact whether cf carriers marry each other [ ], it is possible that these pop- published: january bmc pediatrics , : doi: . / - - - received: august accepted: january this article is available from: http://www.biomedcentral.com/ - / / © henderson and anbar; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ - / / http://creativecommons.org/licenses/by/ . http://www.biomedcentral.com/ http://www.biomedcentral.com/info/about/charter/ bmc pediatrics , : http://www.biomedcentral.com/ - / / ulations also are experiencing a decline in the incidence of cf. however, the extent of genetic testing and counseling services available to these communities is unclear [ ]. the mennonites separated from the "old order" amish in as a result of adoption of new practices by the men- nonites, however, many of the central tenants of each group remain similar [ ]. both the amish and mennon- ites believe that good health is a gift from god, resulting from hard work and strict obedience to the teachings of the bible. the ability to work defines a "healthy" individ- ual [ - ]. conversely, illness is generally believed to be "god's will," while death is considered a natural part of life and a new beginning, rather than an end or punish- ment [ ]. the amish and mennonites believe in the strict separation of church and state, which historically has extended to refraining from use of government funds, including medicaid and social security [ , ]. further, typically they do not purchase commercial insurance [ ]. thus, barriers to provision of modern medical care include reimbursement issues, as well as the amish repu- diation of worldly conveniences such as telephones, elec- tricity, and automobiles [ ]. in most instances of illness, the amish rely on folk reme- dies and herbal medications, among other types of "alter- native" care [ ]. patients coming to modern medical facilities typically do so with chronic illness of many years, only after symptoms have become severe and herbal remedies have not proven beneficial [ ]. such will- ingness has been attributed to a lack of trained profession- als within their own communities to deal with severe illness [ , ]. contrary to the implications of the literature, we present data regarding the amish and mennonite patients at the suny upstate medical university pediatric cf center ( % of our cf patients) along with three representative case reports in order to alert physicians that families of these patients can be receptive to modern medical ther- apy, including preventive measures, which can greatly benefit the patients. table shows the proportion of our amish and mennonite patients who have accepted the recommended standard therapies for cf at our center. nine of the patients have undergone genotype testing and found to be homozygous for the Δf cf mutation. five of the patients have participated in clinical research trials through our center. due to the small nature of the amish and mennonite communities, we were concerned that members of their communities could identify the patients in the case reports. further, we were concerned by the potential impact of the current article on such communities [ ]. therefore, following consultation with the suny upstate institutional review board (irb), the families of the patients described in the case reports and their commu- nity elders reviewed and approved this manuscript prior to its submission for publication, in order to minimize risk of group harm, and harm to the families involved in each of the three presented cases. approval of the manu- script also was obtained from the suny upstate irb. case reports patient a – amish patient a was four-years-old when he began receiving care at our center. he had multiple siblings, including an older, much healthier sister who had cf. at the start of therapy, the patient's parents considered the long-term prognosis of their son when deliberating what treatments should be used. would the disease be painful? what help, if any, would medications and other medical technology provide that herbal therapies had not? is treatment futile in children with cf? following lengthy conversations involving not only the patient's parents, but also the bishop and elders in his community, it was felt to be in patient a's best interest to begin care with standard therapy for cf at our center (table ). a gas-powered generator was used to power the vest. patient a, as well as all of our amish and mennonite patients, qualified for pharmaceutical companies' patient assistance programs for many of his medications. those medications not covered under individual patient assist- ance programs were secured from pharmaceutical com- pany representatives in the form of samples. our hospital established a fee reduction program to help offset the cost of outpatient visits and inpatient hospital admissions for these families, all of whom qualified based on their income level. notably, all of the aforementioned assist- ance was secured by our cf center social worker, who was table : rate of standard cf therapy use by amish and mennonite patients at our center chest physiotherapy manual percussion % high frequency chest wall oscillator (vest) % nebulized mucolytic therapy with rhdnase % multivitamins fortified with vitamins a, d, e, and k % pancreatic enzymes % nutritional supplementation % antibiotic therapy % page of (page number not for citation purposes) bmc pediatrics , : http://www.biomedcentral.com/ - / / vital in communicating with the parents and helping them complete required paperwork. while standard therapies appeared to slow progression of the patient's lung disease, when his status worsened it was recommended by our center physician that intravenous antibiotic therapy be instituted. the family consulted with their elders who recommended that such therapy be with- held because of its cost, and as use of such therapy would only prolong the dying process in a patient with a termi- nal disease. during discussions with the family, our center physician stated that he believed the patient still would have a reasonable quality of life for several months or even a few years with use of intravenous antibiotics. further, withholding of antibiotic therapy at the time of the discussions would result in a much longer dying proc- ess than if the patient lived a longer life with concomitant lung disease progression prior to withholding of aggres- sive therapy. after a number of discussions at our center, the patient's parents allowed the introduction of intrave- nous therapy. they stated that if the medications did not seem to help their son's symptoms, it would be "god's will." at such a point they would not want to give him fur- ther intravenous therapy. home intravenous therapy was instituted because the family could not afford to pay in- patient hospital charges. several courses of intravenous antibiotic therapy were associated with a significant improvement of the patient's respiratory condition. however, after two years, he failed to respond to two intravenous antibiotic courses. at that time, the decision was made to withhold further intrave- nous therapy. the patient was provided supplemental oxygen at home that was generated by a concentrator, as well as on-going vest therapy. at the invitation of the patient and his family, a physician, respiratory therapist, and social worker from our center made home visits to check on the patient as he deteriorated. as he grew sicker, the patient was prescribed oral narcotics to be used as necessary for discomfort. four months after withholding intravenous therapy, following eight years of treatment at our center, patient a died. according to his family, as has become custom within this community, all of his supplies, including the vest and medicines, were given to another child in the community with cf; in this case, his sister. the strong bond between the family and providers at our center has led to amish from other com- munities in new york to seek care at our center at this family's advice. patient b – mennonite at the age of two months, patient b was seen at another center for failure to thrive, emesis, bloody stools, rash and bruising as a result of a vitamin k deficiency. follow- ing an episode of significant cough and wheezing, he was diagnosed with cf. later, it was found that patient b also had biliary cirrhosis. after receiving care elsewhere for four years, the patient came to our center because his family became discon- tented with the medical care they had been receiving. the parents stated they were unhappy because they were not provided with accurate information regarding the patient's health condition, need for testing, and treatment options. reportedly, the family was told that the patient would require hospitalization every few months for his entire life, as well as a liver transplant. the parents report- edly felt much pressure from staff members of the other institution to obtain state aid because of the high on- going and projected costs of his medical care. moreover, herbal remedies that patient b had used since he first became ill were discouraged by his physician, even though the family felt his caregivers did little to under- stand the need for these remedies. after transferring to our center, the parents of patient b were willing to try therapies they had heard worked well in others with cf in their community, including standard therapies offered at our center (table ). according to these parents, their readiness to try such therapies, includ- ing those that they may have previously rejected, was the result of the willingness of our center staff to discuss the potential benefit and harm of standard as well as alterna- tive therapies. for example, our cf center physician was open to use of herbal therapies for patient b, although he informed the family that no studies have demonstrated the effectiveness of treatment of cf. notably, the deacon and some of the bishops of patient b's mennonite com- munity actively discouraged use of the herbs because their use was thought to represent witchcraft. this created sig- nificant discomfort between the family and their bishop. the parents felt that while some of their community were supportive of herbs and would be willing to help fund this therapy, they felt uncomfortable asking for financial resources from other community members. ultimately, without pressure from our center, the family decided to obtain insurance coverage through medicaid. four months after institution of our standard cf therapies and nebulized tobramycin (tobi®) in treatment of the patient's airway colonization with pseudomonas aerugi- nosa, the patient's pulmonary crackles cleared, and his hemoglobin saturation in room air rose from % to %. his body mass index over the same time interval increased from . kg/m ( th percentile for age) to . kg/m ( th percentile for age). helping others within their community with cf was an important aspect of patient b's overall care, according to page of (page number not for citation purposes) bmc pediatrics , : http://www.biomedcentral.com/ - / / his parents. therefore, they consented to his inclusion in the cf foundation national patient registry, which tracks demographics of cf patients throughout the united states. moreover, because of the significant history of bil- iary disease in their child, the parents also consented to his enrollment in a gene modifier study of patients with cf liver disease, for which participation consisted of pro- vision of a single blood specimen. patient c – amish patient c was diagnosed with cf following postnatal test- ing done as a result of a sibling with the disease. she showed few symptoms of cf until she was two-years-old, at which time she was brought by her parents to a hospital in michigan because she was "pale". while she did not have any significant respiratory disease prior to this admission, her condition warranted the start of treatment for cf including rhdnase, and a bronchodilator, which largely was covered under michigan's trust fund for chil- dren with special needs (formerly the crippled chil- dren's fund). shortly thereafter, the patient and her family relocated to another state, where coverage for her expensive therapies was less available. more importantly to the family, they "didn't like the doctor" taking care of their children in the new cf center because they felt he did little to understand the beliefs of their culture, and why certain therapies are accepted while others are not. after talking to others in their community, the parents of patient c were told to come to our center because of the work we have done with the amish. a generator was installed by the family for the use of a nebulizer and vest. medications needing refrigeration have been kept on ice. three years ago, the family was contacted by an individual who sought to, "help amish children with cf." the family was presented with a plan to help cover the costs of treat- ment and were told that it "did not involve government assistance." ultimately, this plan turned out to be medic- aid through the state of new york, which caused distress for this family, when they learned about this at a subse- quent visit to our center. the social worker at our center worked to amend this problem with the state, and assured the elders and others within their community that the family in fact did not knowingly apply for state or govern- mental aid. the family of patient c was advised by the social worker to refrain from applying for any sort of "help" from others outside our facility without first con- tacting our center. the parents consented to have patient c participate in two studies, including one involving growth hormone. the latter study provided reimbursement for the family's transportation costs to our center and there were no charges associated with the center visits for the duration of the study. the family also agreed to allow immuniza- tion for influenza virus on a yearly basis. this family explained that even though amish patients often have refused immunizations of any kind, such preventative care currently is being left to the family's discretion as atti- tudes towards preventative care among the amish have shifted significantly over the past decade. discussion publications have described a lack of willingness to allow preventative measures as an obstacle to providing modern medical care for amish and mennonite populations [ - ]. based on the experiences reported by our patients, this information may have led some within the medical community to assume that amish and mennonite fami- lies are unwilling to allow preventative care, as well as other types of modern therapies, which has resulted in provision of suboptimal care for patients with cf. nonetheless, it is evident that amish and mennonite fam- ilies can be open to effective, modern therapy for this dis- ease. after extensive exploration of the beliefs and expectations of families from these communities, they have accepted preventive therapy, acute medical interven- tions including home intravenous antibiotic administra- tion, and some immunizations for their children with cf. some even have participated in clinical research trials. significant cf center personnel time and fundraising are needed in order to address medical bills incurred by unin- sured amish and mennonite patients with chronic disease such as cf. several options are available. churches within the community often have fundraisers, through selling amish and mennonite foods, quilts and furniture. "amish aid" is a type of medical insurance governed by business- men within the amish church to help pay for hospital bills [ ]. such funds are used by members of the church community, but often fall short of covering costs. with the availability of programs such as our institution's fee reduction program to offset costs, caring for amish patients becomes more manageable, and helps these fam- ilies to seek care. health care education for both the child and family is war- ranted and extensive. by openly discussing the rationale for state-of-the-art and alternative treatments, side effects and outcomes, amish and mennonite families can embrace state-of-the-art medical therapies, with signifi- cant positive results. participation of amish and mennon- ite patients in some of our clinical research trials also has been very helpful in that participating patients are reim- bursed for their transportation costs, and clinic charges are minimized for the duration of studies sponsored by pharmaceutical companies. further, participation in such page of (page number not for citation purposes) bmc pediatrics , : http://www.biomedcentral.com/ - / / trials allows for more frequent visits at the cf center for the enrolled patients as well as siblings with cf, which leads to provision of more timely and thus improved health care. as presented in this report, some members of each group also are willing to allow certain preventative measures. a recent study by yoder and dworkin [ ], in which ques- tionnaires were mailed to all households in an illinois amish community, showed that the majority of the com- munity vaccinates all ( %) or some ( %) of their chil- dren, with only a small minority objecting due to concerns about vaccine safety, and an even smaller cohort objecting due to religious reasons. thus, amish families often are willing to utilize vaccination as a form of pre- ventative care. this report illustrates that health care geared to the cul- tural needs of patients and their families can lead to an improved outcome. it has been proposed that cultural sensitivity improves establishment of rapport and thus promotes cooperation and adherence to therapy [ ]. such sensitivity should include assessment of the patients' preferences and beliefs, and adjustment of health care delivery accordingly [ ]. for example, in some cultures more emphasis is placed on collective rather than individ- ual decision making, communication patterns may differ (e.g., there may be a relative emphasis on non-verbal com- munication), and views may differ regarding physicians, suffering, and the afterlife [ ]. conclusion it appears essential for the health care team to understand, consider, and incorporate current beliefs of amish and mennonite communities into the development of effec- tive programs for treatment of their members with cf. similar efforts should be undertaken whenever health care providers encounter patients from different cultures or religions. abbreviations cf: cystic fibrosis competing interests the authors declare that they have no competing interests. authors' contributions jh wrote the manuscript, and it was edited by ra, who was the attending physician for the described patients. both of us approve the submission of this version of the manuscript, and take full responsibility for it. acknowledgements this study was not funded. written consent was obtained from the families of the described patients for publication of this report. references . miller sr, schwartz rh: attitudes toward genetic testing of amish, mennonite, and hutterite families with cf. am j public health , : - . . tsui lc, barker d, braman jc, knowlton r, schumm jw, eiberg h, mohr j, kennedy d, plavsic n, zsiga m, markiewicz d, akots g, brown v, helms c, gravius t, parker c, rediker k, donis-keller h: cf locus defined by a genetically linked polymorphic dna marker. science , : - . . morton dh, morton cs, strauss ka, robinson dl, puffenberger eg, hendrickson c, kelly ri: pediatric medicine and the genetic dis- orders of the amish and mennonite people of pennsylvania. am j med genet c sem med genet , c: - . . francomano ca, mckusick va, biesecker lg: medical genetic studies in the amish: historical perspective. am j med genet c sem med genet , c: - . . kraybill db, bowman cf: on the background of heaven: old order hutterites, mennonites, amish, and brethren. balti- more, maryland: johns hopkins university press; . . klinger kw: cystic fibrosis in the ohio amish: gene frequency and founder effect. hum genet , : - . . hale je, parad rb, comeau am: newborn screening showing decreasing incidence of cystic fibrosis. n engl j med , : - . . brensinger jd, laxova r: the amish: perceptions of genetic dis- orders and services. j genet counseling , : - . . "mennonite beliefs and practices." britannica online. britannica [http://www.britannica.com/eb/article- /mennonite]. . weyer sm, hustey vr, rathbun l, armstrong vl, anna sr, ronyak j, savrin c: a look into the amish culture: what should we learn? j transcult nurs , : - . . brewer ja, bonalumi nm: health care beliefs and practices among the pennsylvania amish. j emerg nurs , : - . . banks mj, benchot rj: unique aspects of nursing care for amish children. the american journal of maternal/child nursing , : - . . shapiro ht: national bioethics advisory commission: ethical and policy issues in research involving human participants. bethesda, maryland . . yoder js, dworkin ms: vaccination usage among an old-order amish community in illinois. pediatr infect dis j , : - . . donate-bartfield e, lausten l: why practice culturally sensitive care? integrating ethics and behavioral science. j dent educ , : - . . searight hr, gafford j: cultural diversity at the end of life: issues and guidelines for family physicians. am fam physician , : - . pre-publication history the pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/ - / / /prepub page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.britannica.com/eb/article- /mennonite http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ - / / /prepub 	abstract 	background 	methods 	results 	conclusion 	background 	case reports 	patient a – amish 	patient b – mennonite 	patient c – amish 	discussion 	conclusion 	abbreviations 	competing interests 	authors' contributions 	acknowledgements 	references 	pre-publication history doi: . / am. j. hum. genet. :i–ii, i this month in the journal this month in the journal, we present a review article by karl pfeifer on the mechanisms of genomic imprinting (p. ). this article outlines current knowledge on the imprinting process, with emphasis on both the human p . region, which is associated with beckwith-wie- demann syndrome and wilms tumor, and the q - q region, which is associated with the prader-willi and angelman syndromes. dr. pfeifer explains in detail how a cell might use a methylation imprint as both a positive and a negative signal for transcription. also included in this issue are two invited editorials. in the first (p. ), stephen mount cautions that, even as mapping of the human genome nears completion, researchers are still limited in their ability to predict gene structure. especially as gene annotation becomes more automated, researchers will need to be aware of the ex- ceptions to the rules for genetic-structure predictions. in the second invited editorial (p. ), rando allikmets discusses the role of the abc transporter abcr in ret- inal disease. abcr mutations lead to a clinically het- erogeneous array of phenotypes, including stargardt disease, autosomal recessive retinitis pigmentosa, and autosomal recessive cone-rod dystrophy. dr. allikmets outlines current knowledge on the role of abcr in a variety of retinal disorders and highlights the work of both rivera et al. (p. ), who find that mutations in abcr are the major cause of autosomal recessive cone- rod dystrophy, and maugeri et al. (p. ), who provide further evidence that sequence variation in abcr is as- sociated with age-related macular degeneration. tnnt gene mutation in nemaline myopathy, by johnston et al. (p. ) amish nemaline myopathy is an autosomal recessive disorder that is very common in the old order amish, with an incidence of ∼ / . this muscular disorder presents as tremors in infants. progressive muscle atro- phy and proximal contractures develop and eventually lead to respiratory insufficiency and death at an early age. mutations in the genes for three sarcomeric pro- teins—nebulin, a-tropomyosin, and a-actin—have been associated with nemaline myopathy. to identify the gene involved in the unique amish form of nemaline myopa- thy, johnston et al. performed a genome scan and iden- tified, on chromosome , a -cm region of interest that included the gene for the sarcomeric protein troponin t. sequencing of this gene revealed a truncating mutation, e x, in an affected individual. although it is not clear � by the american society of human genetics. all rights reserved. - / / - $ . whether this mutation would result in nonsense-medi- ated decay, protein instability, or defective protein ac- tivity, loss of the c terminus of the protein would re- move the sites of interaction with other components of the troponin complex and would disrupt the coupling of excitation and contraction in muscle fibers. linkage disequilibrium in the nf gene region, by eisenbarth et al. (p. ) linkage disequilibrium (ld) is not evenly distributed throughout the human genome, but the reason for this lack of uniformity is unclear. since the extent of ld has a major impact on genetic-association studies, a greater understanding of the ld distribution would allow more- efficient gene mapping. eisenbarth et al. have examined ld in the region of the nf gene on chromosome q . , an area that shows variation in the extent of ld. they have been able to correlate differences in ld with the dna composition in this region. more specif- ically, the recombination fraction begins to increase pre- cisely at an isochore-transition boundary. because iso- chore class is defined by gc content, the change in recombination is associated with a change in the general dna composition; a higher recombination fraction is associated with a higher gc content. as the authors point out, if this correlation between ld and isochore structure is a general phenomenon, it will have an impact on the marker density required for genetic-association studies. markers could be concentrated in the regions with lower ld (high-gc content), thereby increasing the efficiency of these studies. apoe haplotype variation, by fullerton et al. (p. ) all populations studied to date are polymorphic at the apoe locus, which encodes apolipoprotein e. the three common alleles of apoe—e , e , and e —vary in their associated risk for both cardiovascular and alzheimer disease. individuals possessing an e allele have an in- creased risk of both diseases, whereas e is protective. more-complete examination of the alleles of apoe has demonstrated variation within each common isoform, and these data led fullerton et al. to study further var- iation of apoe. through careful haplotype analysis, these authors discovered considerable variation in the population-specific distribution of apoe haplotypes. this variation provides a potential explanation for the interpopulation differences in association of apoe with disease risk. on the basis of the haplotypes, reduced- median networks were created to study the history of am. j. hum. genet. :i–ii, ii variation at the apoe locus. it appears that e is the ancestral allele but that e has risen in frequency during the past , years. fullerton et al. hypothesize that this change in allele frequency, as well as a reduction in apoe variation, result from the rise of an advantageous mutation that is present in the e and e alleles. admixture-generated linkage disequilibrium, by wilson and goldstein (p. ) the study by wilson and goldstein provides an impor- tant bit of information on the relationship between ld and admixture. ultimately, this provides data on the number of markers necessary for genomewide associa- tion studies, an important unanswered question. it has been believed for quite some time that admixture creates ld, and this study is the first to conclusively prove this fact. using the lemba population from south africa, which is a bantu-semitic hybrid population, these au- thors report elevated ld at large genetic distances, as well as increased ld at most genetic distances. this is in contrast to the situation in the putative parental pop- ulations, bantu and ashkenazi jews, where ld extends only – cm. analysis of allele-frequency differentials between the parent populations shows that, in the lemba, there is a positive correlation between the dif- ferentials and ld, and this demonstrates that ld is gen- erated by admixture. because ld measurements in dif- ferent populations and different genetic regions have shown considerable variability, researchers should no longer generalize ld across the genome and between populations. population-specific measurements of ld, such as this one, will allow more-efficient use of markers in genomewide association studies, and they will also help us to understand better the factors, such as admix- ture, that affect ld variation. report (mutational hotspots in mtdna), by stoneking (p. ) in attempt to resolve the controversy concerning the or- igin of the hypervariable sites in human mtdna, mark stoneking has analyzed the evolutionary rates for new mutations at these sites. although the hypervariable sites have generally been hypothesized to be mutational hot- spots, an alternative theory has been proposed—that is, that they may have resulted from ancient mutations that were moved to various mtdna lineages via recombi- nation. stoneking has found that new mtdna mutations occur preferentially at the hypervariable sites, support- ing the hypothesis that the sites are mutational hotspots. however, the molecular basis for the increased mutation rate at these sites is unknown. kathryn beauregard editorial fellow pre-pubertal bipolar disorder: origins and current status of the controversy duffy et al. int j bipolar disord ( ) : https://doi.org/ . /s - - - r e v i e w pre-pubertal bipolar disorder: origins and current status of the controversy a. duffy , *, g. carlson , b. dubicka and m. h. j. hillegers abstract background: evidence from epidemiological, clinical and high-risk studies has established that the peak period of risk for onset of bipolar disorder spans late adolescence and early adulthood. however, the proposal of the existence of a pre-pubertal form of bipolar disorder manifesting in early childhood created substantial debate. in this narrative review, the literature and contributing factors pertaining to the controversy surrounding the proposed pre-pubertal bipolar disorder subtype are discussed. the resolution of the debate and lessons learned are highlighted. main body: in the mid s us researchers proposed that chronic irritability and explosive temper in pre-pubertal children with pre-existing adhd and/or other learning and developmental disorders might represent a variant of mania. a number of factors contributed to this proposal including severely ill children with no diagnostic home given changes in the adhd dsm diagnostic criteria and over-reliance on symptoms and structured interviews rather than on a clinical assessment incorporating developmental history, social context and clinical course. prospective studies of children at high familial risk did not support the proposed pre-pubertal bipolar phenotype; but rather provided convergent evidence that bipolar disorder onset in adolescence and early adulthood not uncommonly preceded by sleep and internalizing symptoms and most often debuting as depression in adolescence (after puberty). epidemio- logical studies of population and hospital discharge data provided evidence that the pre-pubertal bipolar phenotype was largely a us driven phenomenon. conclusions: psychiatric diagnosis is particularly challenging given the current lack of objective biomarkers. how- ever, validity and utility of clinical diagnoses can be strengthened if all available predictive information is used to formulate a diagnosis. as in other areas of medicine, critical information required to make a valid diagnosis includes developmental history, clinical course, family history and treatment response—weighed against the known trajecto- ries of classical disorders. moreover, given that psychiatric disorders are in evolution over childhood and adolescence and symptoms, in of themselves, are often non-specific, a thorough clinical assessment incorporating collateral his- tory and psychosocial context is paramount. such an approach might have avoided or at least brought a more timely resolution to the debate on pre-pubertal mania. keywords: bipolar disorder, pre-pubertal bipolar disorder, high-risk, epidemiology, cross-national, debate, controversy, diagnosis © the author(s) . this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article’s creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article’s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. background as international clinician researchers who share a focus on bipolar and related mood disorders in children and adolescents, we have come together to update the status of the controversy surrounding the diagnosis of bipo- lar disorder in pre-adolescent children. the aim of this paper is to succinctly summarize what factors led to the proposal of a pre-pubertal or very early onset subtype of bipolar disorder and discuss key evidence that has helped to inform the debate. in the concluding remarks, we provide our collective thoughts more broadly on the lessons learned and future priorities for understanding open access *correspondence: anne.duffy@queensu.ca; anne.duffy@all-souls.ox.ac.uk department of psychiatry, university oxford, oxford, uk full list of author information is available at the end of the article http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf page of duffy et al. int j bipolar disord ( ) : psychopathology and the developmental trajectories of mental illness in young people. origins of the controversy authors are required to disclose conflicts of interest. however, given the degree of discordance and debate, it would be helpful to also have a disclosure whereby authors or reviewers state what they fundamentally believe bipolar disorder is—especially in young chil- dren. in the mid to late s a pre-pubertal bipolar subtype was proposed characterized by chronic irritabil- ity and explosive temper (taken as a manic equivalent) in the context of neurodevelopmental and externalizing behavior problems. “mania” in these chronically ill chil- dren began between and   years of age with estimated “episode” duration of + years on average at the time of diagnosis; moderate to severe interference in functioning (cgas under ) and a high rate of comorbid externaliz- ing disorders and learning problems (wozniak et al. ; geller et  al. ; tillman et  al. ) were also charac- teristic. the proposal raised the question as to whether the definition of bipolar disorder should be restricted to the presence of discrete mood episodes that repre- sent a clear departure from a person’s prior functional and clinical state or be broadened to include extremes of mood regardless of the duration or change from base- line. there was the additional question around whether a pediatric clinical subtype of an illness should have at least some continuity with the adult form of the illness. further complicating the effort to validate the proposed pre-pubertal bipolar subtype was the fact that the crite- ria and structured interviews that were supposed to yield consistent samples didn’t (duffy et al. a). thus, peo- ple reading articles from various research groups might assume that the samples being described reflected the reader’s view of what bipolar disorder is—which is not necessarily the case. a case in point is a recently published genetic study on the association of genetic and environmental risks for attention deficit hyperactivity disorder (adhd) with hypomanic symptoms in youth (hosang et al. ). the findings hinge on two questionnaires that were given to assess hypomania—the child mania rating scale-short form (henry et al. ) and the mood disorders ques- tionnaire (wagner et  al. ). these instruments have been said to distinguish bipolar disorder from adhd. however, a closer look at these studies reveals that the children identified as having bipolar disorder might be among those children now re-conceptualized as hav- ing disruptive mood dysregulation disorder (dmdd) or affect lability related to some other problem or condition. for instance, there is a developing body of literature that recognizes the fact that adhd in childhood is indeed sometimes complicated by significant affective lability (shaw et  al. ; faraone et  al. ) and that this may be mistaken for bipolar disorder (pataki and carlson ). questionnaires cannot magically solve this prob- lem. a different interpretation of the purported genetic association between adhd and bipolar disorder would argue that there is a genetic association because in these children they are two forms of the same condition. if one is to make any sense out of a particular article, then, it is important to know who, exactly, comprises the sample. carlson and klein ( ) have examined reasons why there have been such divergent views regarding the pre- pubertal or very early onset bipolar disorder diagnosis: i. adhd is confused with mania, that is  many chil- dren with adhd have a problem with low frus- tration tolerance, currently labelled irritability or mood dysregulation, was ignored. if mood lability is considered to be unique to bipolar disorder, then a group of children that some might diagnose with adhd will be given a bipolar diagnosis by others. this goes for their families too, since such traits may well be familial (saudino ). ii. a poor definition of distinctive episodes for mania increased the emphasis on symptoms to make the diagnosis. episodes were defined by symptoms of “at least a week” but that definition did not require an offset of symptoms thus leading to “manic” epi- sodes in children that were typically years in length (geller et al. ). iii. irritability, a cardinal symptom of the proposed pre-pubertal mania criteria, was never defined. it was initially present as part of depression in dsm iii (“dysphoric mood is characterized by symptoms such as the following: depressed, sad, blue, hope- less, low, down in the dumps, irritable” p. ) but was eliminated from adult depression criteria in dsm iii-r. it was kept as a symptom of child depression—but not without some disagreement (stringaris et  al. ). dsm iii-r recognized that adjustment disorder and oppositional defiant dis- order could present with irritability (p. )—again not defined. the term irritability is never used in conjunction with adhd, but starting in dsm-iii, under associated features, references to “increased mood lability, low frustration tolerance, temper out- bursts, low self-esteem” can be found. therefore, in children, irritable mood with symptoms of adhd could easily be construed as a “mixed episode” (wagner et al. ). iv. the absence of developmentally informed criteria in dsm iii-iv added to the confusion. for instance, the symptoms of adhd are defined: six (or more) page of duffy et al. int j bipolar disord ( ) : … symptoms of hyperactivity impulsivity [that are] inconsistent with developmental level. unfortu- nately, we don’t have developmentally informed information about manic symptoms e.g. we don’t know what constitutes” developmentally appropri- ate euphoria”. little children can be silly in ways that would look inappropriate in an adult. lit- tle children can say things that in an adult would sound grandiose (carlson and meyer ). v. there has been no consistent approach to man- age information from multiple informants. this is especially problematic in child psychiatry because diagnoses differ depending on whether everyone’s positive symptoms are counted even if inconsist- ent versus counting only those where there is agreement between informants. rather than try- ing to understand what the discrepancy tells us, the approach has been to count one or the other informant or combine everyone’s positive symp- toms. such diagnostic approaches can be highly susceptible to confirmatory bias. for example, disparate teacher information in the longitu- dinal assessment of manic symptoms (lams) study (horwitz et  al. ; findling et  al. ) was ignored because it didn’t agree with interview diagnoses or parent ratings—instead it was par- ents’ information that counted toward a diagno- sis. if teachers observe no evidence of irritability, hyperactivity, rapid speech etc. where parents rate the child as severely ill, the discrepancy needs to be understood. mania does not just occur at home. vi. equating all structured or semi-structured inter- views even though they actually collect different information (galanter et al. ). vii. double counting symptoms so that someone meets criteria for two disorders with one set of symptoms or thinking one can avoid the problem simply by not counting the overlapping symptoms (wagner et al. ). even with the unintended downstream effects of dsm criteria and shifts in diagnostic approaches, had the con- cept of bipolar disorder in very young children not met a clinical need it might not have become an accepted or popular diagnosis. there was and continues to be a need to reach an understanding as to how to conceptualize and treat children with explosive, aggressive behavior who do not fit typical cases of adhd, conduct disorder, pervasive developmental disorder or other conditions in which aggression plays a role (carlson and klein ). further, a recent systematic review suggests that pre- pubertal mania preferentially responds to antipsychot- ics (often with concurrent stimulant medication) rather than to lithium or anticonvulsants (duffy et  al. a). recently, explosive behavior has been subsumed under the umbrella of chronic irritability with leibenluft and the nimh developing an extensive study of irritabil- ity in children. originally called “severe mood dysregu- lation” the condition was defined by chronic irritability with rages and hyperarousal and impairing symptoms of either in at least two settings (leibenluft et al. ). chronic irritability is no longer felt to be a harbinger or alternative form of bipolar disorder. one definition of irritability is “proneness to anger compared with peers at the same developmental level” and it cuts across diag- noses (stringaris et  al. ). however, this has its own short-comings as irritability severity is determined in most studies only by the frequency of temper loss. the frequency and intensity, duration and context of the resulting explosive behavior (carlson and klein ) has not been adequately operationalized. to accurately reflect the behaviors that cause impairment, it will be necessary for measures to address the resulting verbal and physical aggression—what the child does when irri- tated. otherwise, there will be no way to meaningfully distinguish unpleasant, grouchy children from those with significant explosive temper and aggression—those same children previously at the centre of the pre-pubertal bipolar diagnostic controversy. longitudinal high‑risk studies bipolar disorder is highly heritable (mcguffin et al. ) and relatives of bipolar patients are at an increased risk of developing bipolar and related mood disorders. while specific estimates vary between families and studies, con- vergent evidence supports that first degree relatives of an adult with bipolar disorder have an – -fold risk of developing bipolar disorder and a – -fold risk of devel- oping unipolar disorders compared to the general popu- lation or low-risk controls (duffy et  al. ). the risk is even higher for children with both parents affected (gottesman et  al. ). therefore, children of bipolar parents are an important high-risk group that can inform the onset and early course of bipolar disorder. moreover, children at confirmed familial risk would be the most likely to manifest early onset subtypes thus shedding light on the controversy surrounding the validity of the proposed pre-pubertal bipolar phenotype. the question therefore is does the proposed pre-pubertal bipolar phe- notype occur in children at confirmed familial high-risk? there have been several published studies, narrative reviews and meta-analyses informing the prevalence and age of onset of psychopathology in children of variably identified and diagnosed samples of bipolar parents pub- lished over the last few decades (duffy et al. , b; delbello and geller ; lau et  al. ; lapalme et  al. page of duffy et al. int j bipolar disord ( ) : ; ellersgaard et  al. ). several of these studies have repeatedly assessed high-risk children through the peak period of risk from childhood into early adulthood (mesman et  al. ; duffy et  al. b; axelson et  al. ; preisig et al. ; egeland et al. ). while meth- ods of identifying and assessing parents and children vary (duffy et al. ), all have reported an increased risk for mood disorders in general, with depressive disorders more prominent than bipolar disorder. for example, the dutch and swiss studies reported lifetime rates of mood disorder of % and %, and lifetime rates of bipolar spectrum disorder of % and % respectively, for off- spring observed into early adulthood (mean age of and  years). the bios study reported a lifetime rate of mood disorder of % and bipolar spectrum disorder of % by age . in the dutch, amish and canadian stud- ies, onset of clinically significant manic symptoms began in adolescence. the dutch and canadian studies, reported that bipo- lar disorder debuted as a depressive disorder in the vast majority of cases ( % and %, respectively) with onset in mid-adolescence ( and   years) and the first diag- nosable activated episode (i.e. hypomanic or manic) fol- lowing years later (   years on average) (mesman et  al. ; duffy et al. , b). further, the dutch study found that in over one-third of offspring who developed bipolar disorder the preceding depressive disorder was recurrent (not chronic) in nature (mesman et  al. ). similarly, the canadian study found that the first five mood episodes of diagnosed bipolar disorder in high- risk offspring were predominantly depressive (not manic) (duffy et al. b). the age of onset for bipolar disorder was .   years (range . – ) in the canadian cohort and .   years (range – ) in the dutch cohort. simi- larly, the amish high-risk study reported a median age of onset of mania of   years (range –   years) (egeland et  al. ). the bios study reported a younger mean age of onset of mania or hypomania at . ± .   years (axelson et  al. ). while no high-risk children met diagnostic criteria for bipolar disorder prior to age in the amish, dutch or canadian studies ( total sub- jects) (mesman et  al. ; duffy et  al. b; egeland et  al. ), the bios study reported that of the children manifest the first manic episode prior to age (axelson et al. ). factors that might explain this dis- crepancy include differences in the bios parent sample (i.e. high rates of comorbidity including substance abuse, lower familial ses, assortative mating and ascertainment bias) (duffy et  al. ), as well as cross-national differ- ences in diagnostic conceptualization of psychopathology in young children discussed below (mesman et  al. ; dubicka et al. ). longitudinal high-risk studies have not only been help- ful in characterizing the onset and early course of bipo- lar disorder, but also in characterizing early childhood functioning and clinical antecedents. the dutch study reported normal or good overall level of social func- tioning in high-risk offspring compared to the general population from ages –   years (reichart et  al. ). the onset of mood disorders in this cohort was associ- ated with lower scores in family and adaptive function- ing compared to the general dutch population, but with small effect sizes ( . % and . % respectively). the canadian study reported normative or gifted childhood functioning (social and academic) in the offspring of lithium responders (proxy for classical manic depressive illness) compared to offspring of healthy controls, and somewhat lower early childhood functioning on aver- age in the offspring of lithium-non-responders (proxy for heterogeneous bipolar subtype) (duffy et  al. ). at last assessment, the global assessment of functioning (gaf) scores were stable in well or remitted offspring of lithium responders (median gaf ), but had declined from baseline in the well or remitted offspring of lithium non-responders (median gaf ). further, there were comparable rates of adhd and disruptive behaviour disorders (dbd) to the general population and low-risk comparison groups in the dutch, amish and canadian high-risk offspring studies. the bios study reported sig- nificantly higher rates of adhd and dbd in high-risk offspring compared to controls; however this difference diminished when adjusting for confounds related to the general level of family psychopathology and dysfunction (birmaher et al. ). based on longitudinal observations into adulthood, there have been recent efforts to model the developmen- tal trajectory of emergent bipolar disorder in children at confirmed familial risk (duffy et  al. , b) and to advance individualized risk prediction (hafeman et  al. ). based on the canadian data, as a general model of the developmental trajectory of bipolar disorder sup- ported a progressive sequence of psychopathology; shift- ing from sleep and anxiety symptoms and disorders in childhood to minor depressive and adjustment disorders (internalizing symptoms related to stressors) in early ado- lescence (around or after puberty) to major depressive disorder and hypomanic symptoms in mid-adolescence and to bipolar disorder in late adolescence and early adulthood (duffy et al. , b). the transition from depression to bipolar disorder was more likely if depres- sion was recurrent and/or included psychotic symptoms within the episode. also, substance abuse onset earlier in high-risk offspring and in close proximity to the onset of depressive disorders (duffy et  al. , ). based on page of duffy et al. int j bipolar disord ( ) : bios data, baseline (childhood) parent-reported anxiety and depressive symptoms and child-reported mood labil- ity together with more proximal (to the first manic epi- sode) mood lability and hypomanic symptoms predicted new-onset bipolar spectrum disorders. in the bios, canadian and swiss studies, earlier onset of parental bd was associated with a higher risk of developing bipolar disorder (duffy et al. b; preisig et al. ; hafeman et al. ). in summary, several independent longitudinal pro- spective studies of children of parents diagnosed with bipolar disorder according to dsm or icd criteria, have provided important and mostly convergent insight into the onset and early course of bipolar disorder that do not align with the proposed pre-pubertal bipolar subtype hypothesis. key findings in high-risk children include that (i) premorbid social and cognitive/academic functioning is comparable to the general population; (ii) childhood internalizing symptoms and disorders and sleep problems but not neurodevelopmental, cognitive or externalizing disorders predict for onset of mood dis- order in adolescence and early adulthood; (iii); bipolar disorder typically debuts with depressive episodes with a recurrent illness course starting in adolescence; (iv) psychotic symptoms in depressive episodes predict for conversion to bipolar disorder; (v) clinically significant sub-threshold manic symptoms (often episodic) have a variable age of onset (typically not pre-adolescent), and predict for subsequent onset of bipolar disorder in emer- gent adulthood. finally, and relevant to the bipolar con- troversy, the so-called pre-pubertal bipolar phenotype has not been observed in children of parents with a con- firmed bipolar diagnosis in the vast majority of studies and is therefore  consistent with a different illness trajec- tory (fig.  ). it is important to note that substantial differences in the quality of remission, nature and rates of comorbid disorders, as well as global functioning in subgroups of high-risk children have been reported. for example, in the canadian study there were significant differences in the quality of early childhood functioning (academic and social), nature of non-mood comorbidity (i.e. sequential vs concurrent), quality of mood disorder remission, and spectrum of end stage disorders in the offspring of par- ents with a classical bipolar disorder responsive to long- term lithium compared to the offspring of parents with a less typical lithium-non-responsive psychotic subtype of bipolar disorder (duffy et  al. , b). therefore, heterogeneity of our current diagnoses, including bipo- lar disorder, require more detailed attention if we hope to understand stable developmental trajectories, map reliable biomarkers and develop specific effective treat- ments. this notwithstanding, there was no evidence of a pre-pubertal form of bipolar disorder across these high- risk subtypes. cross‑national differences the debate around the diagnosis of bipolar disorder in pre-pubertal children ignited in the s with the pub- lication of data from a us tertiary care centre suggest- ing that up to % of children with adhd also exhibited “mania” (wozniak et  al. ; biederman et  al. ). these reports caused significant debate as findings were not replicated in several centres outside the us. for example, using a nationwide psychiatric case register in denmark covering a background population of . mil- lion inhabitants, children ( boys, girls) were identified has having the diagnosis of manic-depressive psychosis between and before the age of (thomsen et  al. ). in addition, no cases of mania were found in a british clinic survey of children aged and under (harrington and myatt ) and only one child met diagnostic criteria for both icd– hypo- mania and dsm–iv bipolar disorder nos in a uk sam- ple of young people with adhd ( –   years, mean age . ) (hassan et  al. ). a recent meta-analysis re-examined prevalence rates of paediatric bipolar disor- der in epidemiological samples (van meter et  al. ). the authors concluded that there was no difference in rates of paediatric bipolar disorder in the us compared to other countries based on data from structured inter- views in epidemiological populations. however, the stud- ies included mostly adolescent not pre-pubertal subjects and there was evidence of significant heterogeneity likely associated with bipolar subtype (i, i or ii, spectrum), age, sample size, diagnostic criteria and structured interviews scoring externalizing behaviour as a bipolar equivalent (parry et al. a). therefore, questions arose regarding cross-national or other factors influencing clinician diagnosis of pre- pubertal bipolar disorder, previously recognised in con- ditions such as schizophrenia (gupta ) and adhd (prendergast et al. ). a us–uk cross-national study anxiety and sleep problems developmental disorders and adhd mood dysregulation (chronic) mood dysregulation and depressive disorders depressive disorder and hypomanic symptoms (episodic) bipolar disorder early childhood adolescence early adulthood classical bipolar disorder dmdd fig. developmental trajectory of bipolar disorder vs severe mood dysregulation or disruptive mood disregulation disorder page of duffy et al. int j bipolar disord ( ) : investigated potential diagnostic biases as a source for the discrepancy in prevalence rates (dubicka et al. ). five vignettes were presented to uk and us clinicians, four representing complex scenarios where the diagnosis of mania was thought to be controversial and one a ‘classi- cal’ case of mania in an adolescent where it was expected there would be good agreement. as predicted, overall us clinicians were significantly more likely to diagnose mania than uk clinicians in the younger complex cases, but there was good agreement on classical mania in the adolescent case. uk clinicians were significantly more likely to diagnose pervasive developmental and adjust- ment disorders, while us clinicians were more likely to diagnose mania and additional comorbid disorders. in a recent dutch-us cross-national comparison study of psychopathology in offspring (aged –   years) of parents with bipolar disorder, the inter-site reliability was measured using us audiotapes of a semi-structured psychiatric interview. specifically, selected audiotapes from the bios study assessments were blindly rated by interviewers from pittsburgh and interviewers from the netherlands (mesman et  al. ). this study indi- cated good agreement in assessing the narrow definition of bipolar disorder (bd i and ii), but cross-national dif- ferences in assessing co-morbid externalizing disorders. given that the bios sample were younger this might have impacted the rate of co-morbidity and age of onset of bipolar disorder. however reducing this possibility, the danish high risk and resilience study—via , a nation- wide population-based cohort study of -year-old (age range . – .   years) high-risk children (ellersgaard et  al. ), reported a higher prevalence of anxiety dis- orders, stress and adjustment disorders compared with controls and no cases of prepubertal bipolar disorder. another cross-national study of mania in adults reported that uk psychiatrists were less likely to endorse symp- toms on the young mania scale than were clinicians from the us or india (mackin et  al. ). these studies pro- vided evidence that with the same information the inter- pretation and thus diagnosis varied across clinicians from different countries. using outpatient treatment data, moreno and col- leagues showed a substantial increase ( -fold) in the rate of paediatric bipolar disorder diagnosis in the us between and (moreno et  al. )—a trend that has not been replicated in the same way in other countries. for example, james and colleagues reported a . times higher hospital discharge rate between the us and england for bipolar disorder in children and ado- lescents, reduced to a . -fold difference after adjusting for length of stay (james et al. ). the largest disparity in discharge rates occurred in the pre-adolescent period and by age   years the rate of discharge in the us was greater than the maximum discharge rate in england, which occurred at age . the study of hospital discharge rates for bipolar disorder was expanded to include other countries compared to the us (clacey et  al. ). for those under , the discharge rates for bipolar disorder per , population were: us . , australia . , new zealand . , germany . and england . . the most marked difference was in - to -year-olds with per , population rates: us , new zealand . , aus- tralia . , germany . and england . . interestingly the trend in discharge rates in the us showed a sharp peak in –  year olds, while in the other countries there was a gradual rise in discharge rates starting from early adolescence (clacey et al. ). cross-national differences in clinical practice guide- lines and diagnostic criteria reflect the pre-pubertal bipolar diagnostic controversy (parry et  al. b). in a review of published articles, among articles with us authorship, the majority ( %) were found to support the validity of a pre-pubertal form of mania. of articles by non-us authors, most ( %) supported the tradi- tional view that mania and therefore bipolar disorder is rare before mid-adolescence. dsm is based on symptom counting, whereas icd, more commonly used outside the us, allows for a pattern recognition approach to diagno- sis and has generally avoided arbitrary cut-offs and pre- cise requirements regarding symptom counts. the icd approach is intended to reflect the way clinicians make diagnoses (reed et al. ). in addition, icd- requires more than one manic episode for a diagnosis of bipo- lar disorder, whereas the dsm-iv-tr and the dsm- require only one such episode. to avoid over-diagnosis in children and adolescents, icd- states that the diag- nosis of bipolar disorder requires the presence of mania with unequivocal euphoria (not just irritability) and an episodic course. similarly, the uk national institute for health and care excellence (nice) (national collaborating centre for mental health ) clinical practice guideline rec- ommends conservative use of the diagnosis of bipolar i disorder in children and adolescents and cautions against making bipolar ii disorder diagnoses in this age group. it also states that irritability should not be used as a core diagnostic criterion. whereas some us centres have maintained that pre-pubertal bipolar disorder is char- acterised by non-episodic, chronic, ultra-rapid cycling, mixed/irritable states, in the uk, and perhaps else- where, such cases are more likely to be conceptualised as oppositional defiant disorder (odd), conduct disorder and/or adhd with emotional dysregulation (sobanski et  al. ). the icd- odd category includes a “with chronic irritability and anger” qualifier to characterize presentations with prevailing, persistent irritable mood page of duffy et al. int j bipolar disord ( ) : or anger. this presentation is recognized to significantly increase the risk for subsequent depression and anxi- ety. the icd- conceptualization of this presentation as a form of odd diverges from the dsm- approach of introducing a new disorder “disruptive mood dysregula- tion disorder” highlighting international differences in the approach to this constellation of symptoms. unique aspects of the us health system may have been important in the greater acceptance of pre-pubertal mania  (bipolar disorder) as a valid diagnosis. it has been argued that the us health system often drives clinicians to engage in “diagnostic upcoding” and managed care has been anecdotally reported as providing more funding for a diagnosis like bipolar disorder, than for difficulties such as parent–child relational problems (parry et  al. b). it has been argued that the expansion of bipolar disorder has been created in order to market new drugs into the more profitable realm of everyday emotional problems, rather than limiting them to classical forms of bipo- lar disorder, and in so doing, medicalizing personal and social difficulties (moncrieff ). finally, there are cross-national differences in the gen- eral acceptance of diagnoses in children and a proclivity to not diagnose children with a mental disorder is more active outside than within the us (national collaborat- ing centre for mental health ). in some countries, there is a policy movement towards a focus on mental well-being (which remains a nebulous concept) and away from mental illness. for example, the netherlands is cur- rently in the middle of a substantial decentralization and transformation of the dutch youth care system including youth mental health (sobanski et  al. ). this transi- tion tasks dutch municipalities with the coordination of most services in the social domain and promotes de- medicalization—a concept that arises from philosophical and ideological driven frameworks, rather than robust scientific justification. this would potentially remove consideration of mental disorders as discrete entities and substitute existential experiences as the target for men- tal health care. how this approach will further or hinder access to best evidence-based care for those young peo- ple who require it, or how this approach will better assist us with developing necessary and effective treatments remains to be proven. conclusions several different forces have contributed to the pro- posal that chronic aggression and explosive temper in very young children with comorbid adhd and associ- ated problems in social and academic functioning repre- sented a pre-pubertal form of bipolar disorder. although based on findings from clinical, high-risk and epidemio- logical studies, the pendulum is now swinging away from that point of view, we still have no consensus regarding the specificity of irritability or explosive behavior across different conditions, which was at the core of the pre- pubertal  mania  controversy. disruptive mood dysregu- lation disorder, proposed as an alternative diagnosis for these children, is problematic in that explosive temper is defined by frequency rather than severity of outbursts and it is difficult to define “mood between outbursts” (gal- anter et al. ). moreover, it is not at all clear whether adding a comorbid diagnosis is more helpful than con- sidering explosive temper and mood lability as subtype of adhd denoted by a diagnostic specifier—coming full circle  as it were  and supported by  convergent evidence that childhood adhd is associated with psychotic and neurodevelopmental disorders (hamshere et  al. a; b; maccabe and murray ; murray et al. ). nev- ertheless, this journey has been helpful in underscoring the importance of a comprehensive clinical assessment, which as in other areas of medicine should include col- lecting  collateral history from multiple sources, distin- guishing illness episodes from baseline functioning, and assessing symptoms  in the context of the developmen- tal history, family history, clinical course, treatment response and psychosocial context (duffy et al. a). longitudinal prospective studies of children at con- firmed familial high-risk of developing bipolar disorder have not supported the validity of the pre-pubertal bipo- lar phenotype proposed by geller et  al. ( ), wozniak et al. ( ) and others. this is important because bipolar disorder is highly heritable (mcguffin et al. ; bienv- enu et  al. ) and thus children of affected parents would be the most likely group expected to manifest an early-onset prepubertal form of bipolar disorder—if it existed. this assumption is underscored by the fact that the amish and canadian high-risk families were selected initially for genetic studies owing to the high penetrance of bipolar illness across multiple generations. the validity of the proposed pre-pubertal form of bipo- lar disorder has been a focus of significant debate and controversy stretching over two decades. without reli- able and objective biomarkers to validate clinical diag- noses differences of conceptualization and opinion are more likely and difficult to resolve. the corollary of the pre-pubertal mania controversy is the recognition that, while there are many instances of clinical uncertainty especially on the basis of a cross-sectional structured (symptom focused) assessments  of children with emerg- ing clinical pictures, to improve our diagnoses we should rely upon what we know about prototypical develop- mental trajectories of severe mental illnesses. combin- ing developmental course, with the context of symptoms and family history of mental illness will help our assess- ment as to whether something is (i) diagnosable as a page of duffy et al. int j bipolar disord ( ) : separate  comorbid illness, (ii) part of a developing or evolving illness  trajectory or (iii) a normative  develop- mental variant  or reaction to a specific psychosocial stressor or context and as such not diagnosable. further, any proposed early-onset  subtype should have proven association and continuity with the prototypical adult form—otherwise there is no basis for a relationship. diagnosis is a cornerstone of medical practice. yet, there is growing recognition that the current practice of basing diagnoses on symptom checklists is highly prob- lematic (duffy et  al. a). as stated by kendler “since dsm iii, our field has moved toward a reification of the dsm that implicitly assumes that psychiatric disorders are just the dsm criteria. that is, we have taken the index of something for the thing itself” (kendler ). such an approach is insufficient for accurately defining an illness with precision, differentiating it from other illnesses and mapping the clinical illness to underlying pathophysiol- ogy in order to develop specific targeted treatments and refine prognostic predictions. ultimately this debate profoundly matters because diagnoses have significant meaning and consequences for the individual and their family and for scientific advancement. acknowledgements we thank dr. stanley kutcher for his constructive comments on this manuscript. authors’ contributions all authors contributed to the planning, writing and revisions of this manu- script. all authors read and approved the final manuscript. funding anne duffy was a visiting fellow at all souls college, oxford university when writing this manuscript. availability of data all studies reviewed are published and available. ethics approval and consent to participate all studies cited in this manuscript are published and have stated that they had ethics approval and obtained proper informed content from participants. consent for publication all authors met criteria for authorship and read and approved the final version of this manuscript. competing interests the authors declare that they have no competing interests. author details queen’s university, kingston, canada. department of psychiatry, university oxford, oxford, uk. renaissance school of medicine, stonybrook university, stony brook, ny, usa. faculty of biology, medicine and health, university of manchester, manchester, uk. department of child and adolescent psychiatry and psychology, erasmus medical center-sophia children’s hospital, rotterdam, the netherlands. received: january accepted: march references axelson d, goldstein b, goldstein t, monk k, yu h, hickey mb, et al. diagnostic precursors to bipolar disorder in offspring of parents with bipolar disor- der: a longitudinal study. am j psychiatry. ; 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( ): – . van meter a, moreira alr, youngstrom e. updated meta-analysis of epidemio- logic studies of pediatric bipolar disorder. j clin psychiatry. . https :// doi.org/ . /jcp. r . https://doi.org/ . /jcp. r https://doi.org/ . /jcp. r page of duffy et al. int j bipolar disord ( ) : wagner kd, hirschfeld rm, emslie gj, findling rl, gracious bl, reed ml. validation of the mood disorder questionnaire for bipolar disorders in adolescents. j clin psychiatry. ; ( ): – . wozniak j, biederman j, kiely k, ablon js, faraone sv, mundy e, et al. mania-like symptoms suggestive of childhood-onset bipolar disorder in clinically referred children. j am acad child adolesc psychiatry. ; ( ): – . publisher’s note springer nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 	pre-pubertal bipolar disorder: origins and current status of the controversy 	abstract 	background: 	main body: 	conclusions: 	background 	origins of the controversy 	longitudinal high-risk studies 	cross-national differences 	conclusions 	acknowledgements 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ article adiponectin receptor genes: mutation screening in syndromes of insulin resistance and association studies for type diabetes and metabolic traits in uk populations s. c. collins & j. luan & a. j. thompson & a. daly & r. k. semple & s. o’rahilly & n. j. wareham & i. barroso received: july /accepted: october / published online: january # springer-verlag abstract aims/hypothesis adiponectin is an adipokine with insulin- sensitising and anti-atherogenic properties. several reports suggest that genetic variants in the adiponectin gene are associated with circulating levels of adiponectin, insulin sensitivity and type diabetes risk. recently two receptors for adiponectin have been cloned. genetic studies have yielded conflicting results on the role of these genes and type diabetes predisposition. in this study we aimed to evaluate the potential role of genetic variation in these genes in syndromes of severe insulin resistance, type diabetes and in related metabolic traits in uk europid populations. materials and methods exons and splice junctions of the adiponectin receptor and genes (adipor ; adipor ) were sequenced in patients from our severe insulin resistance cohort (n= ). subsequently, polymorphisms were tested for association with type diabetes in population-based type diabetes case–control studies (n= , ) and with quantitative traits in a population-based longitudinal study (n= , ). results no missense or nonsense mutations in adipor and adipor were detected in the cohort of patients with severe insulin resistance. none of the polymorphisms (allele frequency . – . %) tested was associated with type diabetes in the case–control study. similarly, none of the polymorphisms was associated with fasting plasma insulin, fasting and -h post-load plasma glucose, -min insulin increment or bmi. conclusions/interpretation genetic variation in adipor and adipor is not a major cause of extreme insulin resistance in humans, nor does it contribute in a significant manner to type diabetes risk and related traits in uk europid populations. keywords adipor . adipor . association studies . insulin resistance . polymorphisms . type diabetes abbreviations adipor adiponectin receptor adipor adiponectin receptor ld linkage disequilibrium sir severe insulin resistance (cohort) snp single nucleotide polymorphism introduction adiponectin, encoded by the gene adipoq (also known as -kda adipocyte complement-related protein, diabetologia ( ) : – doi . /s - - - electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorised users. s. c. collins : a. j. thompson : a. daly : i. barroso (*) metabolic disease group, the wellcome trust sanger institute, the wellcome trust genome campus, hinxton, cambridgeshire cb sa, uk e-mail: ib @sanger.ac.uk j. luan : n. j. wareham mrc epidemiology unit, elsie widdowson laboratory, fulbourn road, cambridge, uk r. k. semple : s. o’rahilly department of clinical biochemistry, university of cambridge, cambridge, uk r. k. semple : s. o’rahilly department of medicine, university of cambridge, cambridge, uk http://dx.doi.org/ . /s - - - acrp , apm- , apm , acdc, and gelatin-binding protein- or gbp ), is an adipokine with insulin- sensitising [ , ] and anti-atherogenic actions [ ]. its levels correlate strongly with insulin sensitivity in humans and animal models, and increasing levels of plasma adiponectin produce a sensitising effect to the biological action of insulin [ ]. several genetic reports have detected association between adiponectin gene variants and obesity, insulin resistance, type diabetes, and adiponectin levels [ – ]. recently, two adiponectin receptors were identified, adiponectin receptor (adipor ), cloned from a human skeletal muscle expression library, and adiponectin receptor (adipor ), identified using computational tools by yamauchi et al. [ ]. in mice, adipor is expressed ubiquitously, with higher levels in skeletal muscle, and has a higher affinity for the globular form of adiponectin. adipor , on the other hand, is most abundant in the liver and preferentially binds the full-length form of adiponectin [ ]. in contrast, both human adipor ( aa) and adipor ( aa) were predominantly expressed in skel- etal muscle [ , ]. in mexican americans, glucose-tolerant individuals with a family history of type diabetes were reported to exhibit significantly lower levels of mrna for adipor and adipor in skeletal muscle than subjects without a family history of diabetes. mrna levels of both receptors were also reported to positively correlate with glucose disposal [ ]. it is possible therefore that lower expression or altered function of the receptors would predispose to increased insulin resistance and type diabetes. in fact, common variants in the adipor gene were recently tested for association in a case–control study with white and african american individuals, but no association was reported [ ]. two additional studies have evaluated the role of adiponectin receptor variants and risk of type diabetes. in the old order amish population, two intronic variants in adipor were reported to associate with risk of type diabetes, while in adipor an extended haplotype block was associated with increased risk of disease [ ]. in contrast, in a japanese population no associations between polymorphisms in adiponectin re- ceptor genes and risk of type diabetes were detected [ ]. more recently, studies in french and finnish populations reported no association between adipor single nucleotide polymorphisms (snps) and type diabetes [ , ], although evidence for association between rs in adipor and type diabetes in a french population has been suggested [ ]. in light of these studies, and the potential role of these receptors in insulin action and diabetes, we sought to identify and investigate the effects of genetic variants in these genes in uk populations. subjects and methods participants severe insulin resistance cohort a cohort of human patients with severe insulin resistance (sir cohort) was collected at the university of cambridge, uk. the inclusion criteria for this cohort were: ( ) fasting insulin > pmol/l or exogenous insulin requirement > u/day; ( ) acanthosis nigricans; and ( ) bmi < kg/m . in the present study, patients from this cohort were screened for mutations in exons and splice junctions of adipor and adipor genes. cambridge local research ethics committee approval was obtained, and informed consent was received from all individuals before participation. cambridgeshire case–control study the cambridgeshire case–control study has been described previously [ ]. briefly, this population-based case–control study consists of type diabetes patients and matched control subjects. dna was available from cases and control subjects for this study. the cases were a random sample of europid men and women with type diabetes, aged to years, from a population-based diabetes register in a geographically defined region in cambridgeshire, uk. the presence of type diabetes in these participants was defined as onset of diabetes after the age of years without use of insulin therapy in the first year after diagnosis. the control participants were individually matched to each of the diabetic subjects by age, sex and geographical location, but not by bmi. potential control subjects with hba c levels greater than % were excluded, as this subgroup could have contained a higher proportion of individuals with previously undiagnosed diabetes. ethical approval for the study was granted by the cambridge local research ethics committee. epic-norfolk participants this is a nested case–control study within the epic-norfolk prospective cohort study; both the case–control and full cohort study [ , ] have been described in detail previously. briefly, the case–control study consists of incident type diabetes cases and two sets of control subjects, matched on age, sex, time in study and family physician, with the second set additionally matched for bmi. a case was defined by a physician’s diagnosis of type diabetes, with no insulin prescribed within the first year after diagnosis, and/or hba c > % at baseline or the follow-up health check. controls were selected from those in the cohort who had not reported diabetes, cancer, stroke or myocardial infarction at baseline, and who had not developed diabetes by the time of selection. potential control subjects with measured hba c levels > % were excluded. dna was available for this diabetologia ( ) : – analysis from cases and control subjects. ethical approval for the study was granted by the norwich local research ethics committee. ely study this is a population-based cohort study of the aetiology and pathogenesis of type diabetes and related metabolic disorders in the uk [ ]. it uses an ethnically homogeneous europid population, in which phenotypic data were recorded at the outset and after . years. the cohort was recruited from a population sampling frame with a high response rate ( %), making it representative of the general population for this area in eastern england. this analysis included , men and women, aged – years and without diagnosed diabetes, who attended the study clinic for a health check between and . of these, , were attending a follow-up health check, while the remaining were newly recruited in from the original population sampling frame. participants attending the health check underwent standard anthropometric measurements and a -g oral glucose tolerance test. plasma glucose was measured using the hexokinase method. plasma insulin was measured by two-site immunometric assays with either i or alkaline phosphatase labels. cross-reactivity with intact proinsulin was less than . % and interassay cvs were less than %. ethical approval for the study was granted by the cambridge local research ethics committee. pcr and sequencing genomic dna from patients was randomly preamplified in a genomiphi reaction (ge healthcare uk, chalfont st. giles, uk) prior to amplification with gene-specific primers. primers were designed using primer software [ ] to cover all coding exons and splice junctions. pcr primers and expected product sizes are described in electronic supplementary material (esm) table . follow- ing pcr, performed using standard conditions, products were purified using exonuclease i and shrimp alkaline phosphatase (usb corporation, cleveland, oh, usa), and bi-directional sequencing was performed using a dna sequencing kit (big dye terminator . ; applied biosys- tems, foster city, ca, usa). sequencing reactions were run on abi capillary machines (applied biosystems) and sequences were analysed using mutation surveyor version. . (softgenetics llc, state college, pa, usa). genotyping snp selection all snps with a minor allele frequency greater than % in our sir cohort were selected for genotyping. to increase coverage in areas not re-sequenced, a number of dbsnps were selected in an attempt to eliminate gaps, between genotyped snps, of greater than . – kb on average (additional snps were selected prior to hapmap phase i data release). snp choice was based on the following criteria: ( ) all putative non-synonymous snps in dbsnp were selected irrespective of whether or not there was frequency or validation information for the snp; ( ) snps with frequency information were selected if their minor allele frequency was greater than or equal to %; ( ) for snps with no frequency information the choice was based on whether the snp was a double-hit snp, had been validated by-cluster or by-sub- mitter; and finally ( ) some snps with no validation information were included to try and eliminate gaps of greater than . – kb between snps selected for genotyping. genotyping and quality control samples were arrayed on -well plates with three replicates and one water control per plate. for the case–control populations, cases and control samples were randomly distributed across each - well plate, with approximately the same number of cases and controls per plate. genotyping of samples was performed in -well plates at the wellcome trust sanger institute, cambridge, using an adaptation of the homoge- nous massextend protocol for the massarray system (sequenom, san diego, ca, usa) [ ]. assay results for case–control are described in esm table . the following criteria were used to pass assays resulting from genotyping: ( ) call rates had to be greater than or equal to % (in one case a call rate of % was accepted); ( ) concordance rates between duplicate samples had to be greater than or equal to %; ( ) minor allele frequency had to be greater than or equal to % in the genotyped populations; ( ) agreement with hardy–weinberg equilibrium was tested separately in cases and controls using a χ goodness-of-fit test, and if p< . in controls, the assay was failed, while if p< . in cases, the assay was flagged but included in primary analysis. assays that failed quality control were excluded from further analysis. in total we analysed results from nine snps in adipor and snps from adipor . statistical analysis all analyses used sas . (sas institute, cary, nc, usa) or stata . (stata corporation, college station, tx, usa) statistical programs, unless otherwise stated. all used genotypes were in hardy–weinberg equilibrium. the pair- wise linkage disequilibrium (ld) coefficient for the con- trols (r ) was calculated for genotyped snps and is represented in fig. . for each snp, two primary models were used to assess association with diabetes and quanti- tative traits, the linear trend (additive model) on df and the general model on df. since the results from these analyses were not materially different, we present only the results diabetologia ( ) : – diabetologia ( ) : – from the linear trend test. tests for association were performed by logistic regression combined in the two case–control populations adjusting for age, sex and popu- lation. between-study heterogeneity was tested by log- likelihood ratio tests. quantitative trait analysis was undertaken in the ely study population. association between fasting plasma insulin, fasting and -h post-load plasma glucose, -min insulin increment ( -min insulin minus fasting insulin over -min glucose in an ogtt), bmi and genotype was tested in a multiple regression model, adjusted for age and sex. results to evaluate whether genetic variation in adipor and adipor contributed to severe insulin resistance in humans, we sequenced exons and splice junctions in both genes in individuals from our sir cohort. these individuals were unrelated and had a variety of syndromes of severe insulin resistance [ ]. we identified and polymorphisms in adipor and adipor respectively, none of which altered the protein sequence of either gene (esm table ). this suggests that no functional mutations were identified in these genes. we next tested whether common variants at these genes impacted on type diabetes predisposition or related quantitative traits in uk europid populations. we selected all variants with a minor allele frequency greater than % in the sir cohort and supplemented our snp selection with additional variants from the dbsnp database plus snps with significant association results in other published studies. thirteen polymorphisms from adipor and polymorphisms from adipor were selected for genotyping in two type diabetes case–control studies (n= , ) and one metabolic quantitative trait study (n= , ) (fig. ). those snps that passed our genotyping quality control criteria (described in methods) were used to investigate the degree of ld in control individuals across adipor and adipor . in total nine snps in adipor and snps in adipor were included in the analysis. ld was measured by r statistic and is depicted in fig. . under both general and linear trend models no evidence was found for statistically significant associations between snps and disease risk (table ). in adipor , snps rs , rs and rs were removed from the quantitative trait analysis as they were out of hardy–weinberg equilibrium (p< . ). for the remaining snps there was also no evidence for asso- ciation of the snps tested with bmi, fasting and -h glucose levels, fasting insulin or -min insulin incremental response (esm table ). in adipor a few snps showed nominally significant association with bmi and -h glucose levels. however, these results are likely to be chance findings given the number of statistical tests performed (esm table ). discussion the current study, including sequencing of patients with syndromes of severe insulin resistance and genotyping of both population-based type diabetes case–control studies (n= , ) and a metabolic quantitative trait study (n= , ), suggests that adipor and adipor genetic variants are unlikely to be major risk factors for type diabetes and insulin resistance in uk europid populations. although sequencing of adipor and adipor genes in a cohort of patients with syndromes of severe insulin resistance (n= ) led to the identification of poly- morphisms, including novel rare variants, none altered the protein sequence. given that this group of patients comprises a heterogeneous cohort representative of a variety of syndromes of extreme insulin resistance, the lack of variants affecting the protein sequence suggests that func- tional mutations in the genes adipor and adipor are not major causes of extreme insulin resistance in humans. for adipor , our data in case–control studies are consistent with previous reports showing that snps in this gene are not associated with type diabetes risk in europid [ , , ], african [ ] or japanese populations [ ]. this is in contrast to evidence from the old order amish, where an association of rs (and rs which is in perfect ld with it) and rs with type diabetes risk has been reported [ ]. since the amish represent an isolated population, it is possible that variants in adipor play a role in type diabetes predisposition among them, which is not apparent in more heterogeneous populations. however, given that both reported associated snps are present in all populations, it is unlikely that either is the true causal variant, although they could be detecting, through ld, the effect of an untested snp. alternatively, given the relative small sample sizes used and lack of adjustment for multiple testing, the authors may have reported a false– positive association. for adipor the data are less consistent. while no evidence of association between adipor snps and type diabetes was present in a japanese population [ ], an association has been suggested between snp rs and type diabetes risk in french populations [ ]. our data, fig. genomic structure and pair-wise marker ld in adipor (a) and adipor (b). the location of snps identified in this study and/or genotyped is represented along the gene (bold type: snps selected for genotyping, and failed or monomorphic snps). exons are represented in boxes (black for coding and open for untranslated). introns and flanking sequences appear as lines. the pair-wise marker ld measured by r statistics is shown below the genomic structures and indicated by the shade of grey blocks (white to black) and the r value. a, b, isoforms a and b in adipor � diabetologia ( ) : – t a b le a d ip o r an d a d ip o r s n p as so ci at io n re su lt s w it h ty p e d ia b et es g en e s n p a ll el e c as es c o n tr o ls o d d s ra ti o p v al u e tr en d p v al u e h o m o g en ei ty / (% ) (% ) (% ) (% ) (% ) (% ) ( % c i) a d ip o r rs g /a ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs a /g ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs c /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . iv s + c /g ( . ) ( . ) ( ) ( . ) ( . ) ( . ) . ( . , . ) . . rs c /t ( . ) ( . ) ( ) ( . ) ( . ) ( . ) . ( . , . ) . . rs c /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs g /a ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs c /g ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs c /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . a d ip o r rs a /c ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs a /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs a /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs a /g ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs g /c ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . * rs c /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . i c /a ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs c /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs g /a ( . ) ( . ) ( . ) ( . ) ( . ) ( ) . ( . , . ) . . rs c /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs c /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs a /t ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs t /c ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs t /c ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . rs g /a ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) . ( . , . ) . . , h o m o zy g o u s fo r al le le , , h et er o zy g o u s, , h o m o zy g o u s fo r al le le . g en o ty p e co u n ts ar e sh o w n w it h fr eq u en cy (% ), o d d s ra ti o s ar e sh o w n p er al le le w it h si g n if ic an ce ca lc u la te d fo r th e li n ea r tr en d (p v al u e tr en d ). h o m o g en ei ty in re su lt s fr o m as so ci at io n b et w ee n e p ic an d c am b ri d g e c as e– c o n tr o l p o p u la ti o n s w as te st ed (p v al u e h o m o g en ei ty ) * p v al u es ≤ . diabetologia ( ) : – and those from the old order amish [ ], do not support this finding. notably, in the french population, meta- analysis of rs , including , individuals with type diabetes and , controls, demonstrated allelic associ- ation of nominal significance only (p= . ), while the most significant result was under a recessive model (p= . ). our study has only % power to detect such small recessive effects (odds ratio . ) with an allele frequency of . , and this could explain our discrepant result. further large scale studies of this snp in additional populations will be required to elucidate its role in conferring risk of disease. a haplotype was also reported to increase risk of diabetes in the old order amish [ ]. however, when we performed haplotype analyses in both adipor and adipor , we were unable to detect any significant associations with diabetes risk (data not shown), and this discrepancy might also be accounted for by differences in the populations studied. with regard to quantitative metabolic traits, to date two studies have reported nominally significant results with insulin sensitivity and body size [ , ]. the first study showed nominally significant associations between two tightly linked snps (rs and an intronic – snp) in adipor and decreased insulin sensitivity and increased hba c levels [ ]. we did not test association with hba c, but have not replicated any association with insulin sensitivity (including with snp rs ) as assessed by fasting insulin measurements. recently kantartzis and colleagues reported that the association between rs and insulin sensitivity is observed only in more obese, but not in lean individuals [ ]. this dependence on the degree of adiposity could explain some of the discrepant results observed for this snp, if there are substantial differences in mean bmi between populations tested. to explore this hypothesis, we performed snp×bmi interaction tests (using bmi both as a contin- uous trait and splitting the population above and below the median) on all adipor snps tested. our data provided no evidence for such an interaction (data not shown). we also specifically tested for association between rs , and measures of insulin and glucose in participants with bmi above and below . again we found no statistically significant difference between the two groups (p> . ). therefore, it is unlikely that bmi differences between populations are at the root of our discrepant results. of note, our data cannot exclude possible small effects of this snp on insulin sensitivity, in particular if this effect is only observed in subjects with higher bmi. the second study suggested there was evidence of association between two markers (rs and rs ) and bmi, but this evidence came from men only. furthermore, three other markers (rs , rs and rs ) were reported to associate with fasting and -h insulin levels, particularly in men at baseline [ ]. we did not test rs , but did test rs (d′=r = with rs ) and found no evidence for snp×sex interaction on any of the quantitative traits we analysed. our data also did not replicate the sex effect of snp rs on insulin measurements. although we did not test snp rs , previously published data was conflicting. thus while the c allele was suggested to be associated with higher -h insulin levels in men (p= . ), in women the t allele was associated with higher levels (p= . ) [ ]. given that neither we, nor others [ ], have found evidence for sex×snp interaction effects on measures of insulin sensitiv- ity, we suggest that further confirmatory studies are required to test this hypothesis. until recently, remaining uncertainty regarding the identity of the true physiological receptors for adiponectin [ ] had hampered interpretation of the functional rele- vance of polymorphisms in adipor and adipor with respect to adiponectin’s insulin sensitising effects. howev- er, a recent yeast two-hybrid screen identified an adipor interacting molecule, appl , thought to mediate many of the effects of adiponectin [ ]. this molecule was shown to interact with both adipor and adipor in an adipo- nectin-sensitive manner, and was shown to mediate many of adiponectin’s insulin-sensitising effects. this suggests that adipor and adipor could be therapeutic targets for drug development and should renew interest in association studies, such as those we present here, testing polymorphisms in adipor and adipor for effects on type diabetes risk and metabolic traits. in summary, sequencing of adipor and adipor genes in a cohort of patients with syndromes of severe insulin resistance (n= ) suggests that functional muta- tions in these genes are not a major cause of extreme insulin resistance in humans. furthermore, testing of common genetic variants (n= ) did not find evidence for associa- tion of these genes with type diabetes risk (n= , ) or with five additional quantitative metabolic traits (n= , ). these data suggest that adipor and adipor variants are unlikely to be major risk factors for type diabetes and insulin resistance in uk europid populations, although more detailed analyses of gene variants may be required to exclude a potential minor role of these genes in insulin resistance and glucose homeostasis. acknowledgements we thank s. bumpstead for genotyping sup- port. i. barroso is funded by the wellcome trust. s. o’rahilly and i. barroso acknowledge support from eu fp funding (contract no. lshm-ct- - ). the epic-norfolk study is funded by mrc uk and cancer research uk. the mrc ely study is funded by the mrc and wellcome trust (to n. j. wareham). we are grateful to all patients, volunteers, referring physicians and staff of the sir, cambridgeshire case–control, epic-norfolk, and mrc ely studies. duality of interest the authors declare that no conflicts of interest exist. diabetologia ( ) : – references . berg ah, combs tp, du x, brownlee m, scherer pe ( ) the adipocyte-secreted protein acrp enhances hepatic insulin action. nat med : – . yamauchi t, kamon j, minokoshi y et al ( ) adiponectin stimulates glucose utilization and fatty-acid oxidation by activat- ing amp-activated protein kinase. nat med : – . ouchi n, kihara s, arita y et al ( ) adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class a scavenger receptor expression in human monocyte-derived macro- phages. circulation : – . yamauchi t, kamon j, waki h et al ( ) the fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. nat med : – . hara k, boutin p, mori y et al ( ) genetic variation in the gene encoding adiponectin is associated with an increased risk of type diabetes in the japanese population. diabetes : – . menzaghi c, ercolino t, di paola r et al ( ) a haplotype at the adiponectin locus is associated with obesity and other features of the insulin resistance syndrome. diabetes : – . vasseur f, helbecque n, dina c et al ( ) single-nucleotide polymorphism haplotypes in the both proximal promoter and exon of the apm gene modulate adipocyte-secreted adiponectin hormone levels and contribute to the genetic risk for type diabetes in french caucasians. hum mol genet : – . gibson f, froguel p ( ) genetics of the apm locus and its contribution to type diabetes susceptibility in french cauca- sians. diabetes : – . qi l, li t, rimm e et al ( ) the + polymorphism of the apm gene, plasma adiponectin concentration, and cardiovascu- lar risk in diabetic men. diabetes : – . yamauchi t, kamon j, ito y et al ( ) cloning of adiponectin receptors that mediate antidiabetic metabolic effects. nature : – . civitarese ae, jenkinson cp, richardson d et al ( ) adiponectin receptors gene expression and insulin sensitivity in non-diabetic mexican americans with or without a family history of type diabetes. diabetologia : – . wang h, zhang h, jia y et al ( ) adiponectin receptor gene (adipor ) as a candidate for type diabetes and insulin resistance. diabetes : – . damcott cm, ott sh, pollin ti et al ( ) genetic variation in adiponectin receptor and adiponectin receptor is associated with type diabetes in the old order amish. diabetes : – . hara k, horikoshi m, kitazato h et al ( ) absence of an association between the polymorphisms in the genes encoding adiponectin receptors and type diabetes. diabetologia : – . vaxillaire m, dechaume a, vasseur-delannoy v et al ( ) genetic analysis of adipor and adipor candidate poly- morphisms for type diabetes in the caucasian population. diabetes : – . siitonen n, pulkkinen l, mager u et al ( ) association of sequence variations in the gene encoding adiponectin receptor (adipor ) with body size and insulin levels. the finnish diabetes prevention study. diabetologia : – . barroso i, luan j, middelberg rp et al ( ) candidate gene association study in type diabetes indicates a role for genes involved in beta-cell function as well as insulin action. plos biol :e . harding ah, day ne, khaw kt et al ( ) dietary fat and the risk of clinical type diabetes: the european prospective investigation of cancer—norfolk study. am j epidemiol : – . day n, oakes s, luben r et al ( ) epic-norfolk: study design and characteristics of the cohort. european prospective investigation of cancer. br j cancer (suppl ): – . williams dr, wareham nj, brown dc et al ( ) undiagnosed glucose intolerance in the community: the isle of ely diabetes project. diabet med : – . rozen s, skaletsky h ( ) primer on the www for general users and for biologist programmers. methods mol biol : – . whittaker p, bumpstead s, downes k, ghori j, deloukas p ( ) snp analysis by maldi-tof mass spectrometry. in: celis j, carter n, simons k, small jv, hunter t (eds) cell biology: a laboratory handbook, rd edn. academic, san diego . barroso i, gurnell m, crowley ve et al ( ) dominant negative mutations in human ppargamma associated with severe insulin resistance, diabetes mellitus and hypertension. nature : – . stefan n, machicao f, staiger h et al ( ) polymorphisms in the gene encoding adiponectin receptor are associated with insulin resistance and high liver fat. diabetologia : – . kantartzis k, fritsche a, machicao f, haring hu, stefan n ( ) the - g/a polymorphism of the adiponectin receptor gene is associated with insulin sensitivity dependent on adiposity. diabetes care : . hug c, wang j, ahmad ns, bogan js, tsao ts, lodish hf ( ) t-cadherin is a receptor for hexameric and high-molecular- weight forms of acrp /adiponectin. proc natl acad sci usa : – . mao x, kikani ck, riojas ra et al ( ) appl binds to adiponectin receptors and mediates adiponectin signalling and function. nat cell biol : – diabetologia ( ) : – 	adiponectin... 	abstract 	abstract 	abstract 	abstract 	abstract 	introduction 	subjects and methods 	participants 	pcr and sequencing 	genotyping 	statistical analysis 	results 	discussion 	references << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma . ) /calrgbprofile (srgb iec - . ) /calcmykprofile (iso coated) /srgbprofile (srgb iec - . ) /cannotembedfontpolicy /error /compatibilitylevel . /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /perceptual /detectblends true /detectcurves . /colorconversionstrategy /srgb /dothumbnails true /embedallfonts true /embedopentype 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cartilage hair hypoplasia: characteristics and orthopaedic manifestations patrick riley jr. • dennis s. weiner , , , • bonnie leighley , • david jonah • d. holmes morton • kevin a. strauss • michael b. bober , • martin s. dicintio received: july / accepted: february / published online: march � the author(s) . this article is published with open access at springerlink.com abstract purpose cartilage hair hypoplasia (chh) is a rare metaphyseal chondrodysplasia characterized by short sta- ture and short limbs, found primarily in amish and finnish populations. cartilage hair hypoplasia is caused by muta- tions in the rmrp gene located on chromosome p . . the disorder has several characteristic orthopaedic manifestations, including joint laxity, limited elbow ex- tension, ankle varus, and genu varum. immunodeficiency is of concern in most cases. although patients exhibit orthopaedic problems, the orthopaedic literature on chh patients is scant at best. the objective of this study was to characterize the orthopaedic manifestations of chh based on the authors’ unique access to the largest collection of chh patients ever reported. methods the authors examined charts and/or radiographs in cases of chh. we analyzed the orthopaedic manifestations to better characterize and further understand the orthopaedic surgeon’s role in this disorder. in addition to describing the clinical characteristics, we report on our surgical experience in caring for chh patients. results genu varum, with or without knee pain, is the most common reason a patient with chh will seek orthopaedic consultation. of the cases reviewed, pa- tients had undergone surgery, most commonly to correct genu varum. conclusion this paper characterizes the orthopaedic manifestations of chh. characterizing this condition in the orthopaedic literature will likely assist orthopaedic sur- geons in establishing a correct diagnosis and appreciating the orthopaedic manifestations. it is important that the accompanying medical conditions are appreciated and evaluated. keywords pathoanatomy � mckusick-type metaphyseal chondrodysplasia � cartilage hair hypoplasia introduction mckusick et al. [ ], in an extensive study of the old order amish community in eastern pennsylvania, clearly eluci- dated in cases a distinct type of short-limb dwarfism termed cartilage hair hypoplasia, also known as mckusick metaphyseal chondrodysplasia. mckusick presented evi- dence that this disorder was inherited as an autosomal re- cessive with a frequency of – per thousand live births in the old order amish [ ]. cartilage hair hypoplasia (chh) has been reported in many countries and many peoples outside the amish electronic supplementary material the online version of this article (doi: . /s - - -z) contains supplementary material, which is available to authorized users. & dennis s. weiner mdicintio@chmca.org department of pediatric orthopaedic surgery, akron children’s hospital, akron, oh , usa akron children’s hospital, northeast ohio medical university, akron, oh , usa regional skeletal dysplasia clinic, akron children’s hospital, akron, oh , usa little people’s research fund, baltimore, md , usa clinic for special children, strasburg, pa , usa skeletal dysplasia program, alfred i. dupont hospital for children, wilmington, de , usa locust street, ste. , akron, oh - , usa j child orthop ( ) : – doi . /s - - -z http://dx.doi.org/ . /s - - -z http://crossmark.crossref.org/dialog/?doi= . /s - - -z&amp;domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - -z&amp;domain=pdf community [ – ]. the largest population of cases outside the amish community is found in finland, where mäkitie in described the incidence as roughly : , live births [ ]. mäkitie and colleagues, in several excellent publications, have described several associations of chh beyond the presence of chondrodysplasia [ , – ]. chh is associated with a broad spectrum of mild-to-moderate, cell-mediated immunodysfunction, including occasionally severe combined immune deficiency, neutropenia, lym- phopenia, disordered erythrogenesis, and a predisposition to lymphoma [ – , – ]. recurring infections are commonly seen in % of all patients, likely linked to the immunodeficiency. the severity is reflected in a report by rider et al. [ ] in which % of their patients ( ) had recurrent infections, with two requiring bone marrow transplantation [ – ]. individuals affected with chh can have marked impairment of t-lymphocyte function due to an intrinsic defect in cell proliferation. beta cells and fi- broblasts also show defective proliferation. morbidity and mortality to varicella is clearly increased in chh, but there is no apparent increase in susceptibility to severe or fatal infections with other viruses. morbidity and mortality are directly related to immune dysfunction. the presence of infections, particularly in the first years of life in chil- dren, is increased in frequency and hematologic malig- nancy is increased in adults. mäkitie et al. [ ] in reported an increased incidence of non-hodgkin lymphoma and an excess risk of basal cell carcinoma in chh. others have reported similar findings, most commonly non- hodgkin lymphoma [ , , , , – ]. anemia is also commonly encountered and significantly correlated with the severity of immunodeficiency and growth failure. it has been clearly documented that chh is caused by mutations in the rmrp (rna component of mitochondrial rna processing endoribonuclease) gene located on chro- mosome p –p and more recently narrowed to chro- mosome p . [ , , , – ]. the rmrp a?g mutation is the most frequent ancestral mutation seen in finnish and old order amish people. patients with chh are classically short at birth and the shortening may increase in the first years of life. a very weak or absent pubertal growth spurt has been reported by mäkitie. typically, the patients present with fine, sparse, light- colored, ‘‘silky’’ short hair that breaks easily. mckusick et al. [ , ] and coupe and lowry [ ] examined the hair grossly, microscopically, and in diameter. the hair has been demonstrated to be diminished in caliber and lacks a pigmented core, a finding also observed by van der burgt et al. [ ]. the hands are short and ‘‘pudgy’’ with marked excessive joint laxity, particularly of the metacarpal, pha- langeal, and interphalangeal joints [ , ]. the elbows typically lack full extension. bowing of the lower extremities and disproportionate length of the fibula com- pared to the shortened tibia is consistent, although un- proven as the cause of the ankle varus, resulting in ankle varus from relative fibular overgrowth (fig. ). mäkitie et al. reported that the median adult height was . cm (range . – . cm) for males and . cm (range . – . cm) for females [ ]. the radius and ulna are commonly shorter than the humerus and the femur shorter than the tibia, with the tibia being more severely involved than the fibula. several nonskeletal issues are of considerable impor- tance in association with chh. in the mckusick series, a few patients had evidence of aganglionic colon, two pa- tients died of varicella, and three others had had virulent infections with varicella. intestinal malabsorption and hirschsprung disease has been reported in association with chh [ , ]. mäkitie et al. [ ] estimated the incidence of these two disorders as occurring in % of the patients. prominence of the upper sternal region has been mentioned in the literature but is of no functional significance, but can be useful in diagnosis. typically, the ossification centers of the epiphyses are radiographically normal. metaphyseal abnormalities re- flecting the chondrodysplasia are manifested by flaring, cupping, marginal serration, fragmentation, and scalloping of the metaphyses of the tubular bones, most particularly seen at the knee [ , , , – ] (fig. a, b). the hips are typically spared these findings. there are irregular cystic radiolucencies in the metaphyses with extension into the diaphyses. abnormal epiphyseal shape fig. frontal view demonstrating bowing of right femur and tibia in patient with cartilage hair hypoplasia j child orthop ( ) : – occurs, seemingly as a consequence of the deformed metaphyseal region. there is marked shortening of the metacarpals, metatarsals, and phalanges, with metaphy- seal cupping and ‘‘cone-shaped’’ epiphyses. costochon- dral junctions have reflected mild flaring of the lower ribcage, slightly anterior angulated sternum, and lumbar lordosis. there is commonly delay in appearance of the proximal femoral epiphyses but absence of the proximal femoral metaphyseal changes that are typical at the knee. the entire metaphyseal region is widened and flared at the knee and ankle. the skull and spine are normal. ray and dorst [ ] also reported mild-to-mod- erate c and c subluxation. mäkitie et al. [ ], how- ever, reported in that none of patients had cervical subluxation. lachman [ ] mentioned that several patients showed forward subluxation of c and c in flexion (lateral c-spine x-rays taken in neutral flexion and extension). mäkitie et al. demonstrated that the growth retardation in chh correlates well with the severity of the metaphy- seal changes [ , ]. ray and dorst [ ] reported that one-third of elbows showed lateral subluxation or dislo- cation of the radial head. histologic examination was performed in one of mckusick’s cases and demonstrated paucity of cartilage cells and deficiency in columnar or- ganization that was interpreted as cartilage hypoplasia. we could find no infrastructural histopathologic information in the literature to date. prenatal diagnosis has been reported [ , ]. a summary of the orthopaedic abnormalities that are typical in this disorder comprises significant joint laxity, particularly of the hands and feet, limited elbow extension, bowing of the lower leg and thigh, increased lumbar lor- dosis, varus deformity of the ankle due to fibular over- growth, occasional atlantoaxial radiographic hypermobility, and scoliosis that rarely requires orthopae- dic treatment. material: data a unique opportunity was afforded the authors to access data on a large number of patients through clinical ex- aminations, records, and radiographs of the largest accu- mulation of chh cases so far encountered in north america. approval from our institutional review board was obtained. information was obtained from the files of the senior author (d.s.w.) on patients with chh com- prising males and females: old order amish and non-amish patients. documentation of diagnosis was obtained by clinical examination ( patients), radio- graphic examination ( patients), or genetic diagnosis ( patients). complete physical examination was available on patients and the remainder was included based on ra- diographic assessment and/or genetic documentation. fig. a anteroposterior radiograph of the lower extremity in cartilage hair hypoplasia demonstrating bowing. b illustration depicting the osseous and cartilaginous deformities of lower extremities in cartilage hair hypoplasia j child orthop ( ) : – for the purpose of this paper, we have arbitrarily di- vided the orthopaedic characteristics of chh into typical (found in over % of cases), frequent ( % or more), occasional (\ %), and rare. radiographic abnormalities associated with chh were similarly separated by frequency. results the age distribution of the patients at initial visit is shown in fig. . the clinical findings seen in our population of patients are recorded in table in order of their fre- quency. likewise, in table , radiographic features en- countered are presented in accordance with their relative frequency. in our series, % of the cases where radio- graphs were reviewed ( / ) showed coxa vara radiographically. several surgical procedures were performed on patients with angular and/or rotational deformity. in this series of patients, roughly % had undergone surgical realignment of the lower extremities. the ages at surgery ranged from to years, with an average of . years and mean of . years. we did not encounter in our patients any cases that required cervical surgical stabilization ( cases with lateral view of c-spine in neutral flexion and extension). discussion it is interesting that over two-thirds of the patients in mckusick’s study derive ancestry from one john miller who was married to catherine hochstetler. over % of the patients trace their ancestry to jacob hochstetler, a relative of catherine. eighty percent of parents of the affected patients studied by mckusick were descended from either jacob or catherine hochstetler, who immi- grated to the united states in the mid- s. amish mi- gration has resulted in many cases seen in other states, particularly in ohio at present [ , ]. the orthopaedic manifestations that have clinical sig- nificance are related to the metaphyseal chondrodysplasia resulting in bowing, angulation, and rotational deformities. femoral bowing and coxa vara are occasionally severe enough to warrant surgical intervention by osteotomy and realignment. in the lower leg, the shortening and bowing of the tibia and the ankle varus appears directly related to the relative overgrowth of the fibula. it would appear that the loss of elbow extension, although typically seen, is most likely related to radial head subluxation or dislocation [ ]. this is probably the best explanation, inasmuch as the other joints generally demonstrate increased laxity. although typically there is lack of elbow extension, we have not encountered a single patient who had any issues of consequence with that loss of motion. the most common mutation in the rmrp gene located on chromosome p . is identical in the old order amish population and the finnish population. although c - subluxation has been described, we have not, in our series, fig. patient age distribution at initial visit (m months, yrs years, m male, f female) table frequency of clinical findings in chh clinical manifestation cases/patients reviewed % occurrence frequency short-limbed disproportionate dwarfism / typical varus deformity of femur / typical short pudgy hands with marked joint laxity / typical varus deformity of tibia / frequent diminished elbow extension / frequent ankle varus deformity / frequent table frequency of radiographic findings in chh radiographic manifestation cases/patients reviewed % occurrence frequency disproportionate short- limb dwarfism / typical metaphyseal abnormalities of femur and tibia / typical shortening of the metacarpals and phalanges / typical genu varum / frequent coxa vara / occasional j child orthop ( ) : – encountered any cases that have required stabilization nor have we seen any cases of scoliosis that required ortho- paedic management. coxa vara is likely due to a combination of relative discrepancy in growth of the upper femoral physis to trochanteric growth (slower proximal femoral growth plate in relation to trochanter and neck) combined with bowing and shortening of the femur, although this is admittedly speculative (fig. ). the severity of the bowing of the fe- mur and tibia appears to correlate with the extent of metaphyseal radiographic changes. the metaphyseal alterations in the magnified illustration (fig. ) show a convoluted pattern of the bony metaphysis and junctural physis. fibular overgrowth and joint laxity and genu varum coexisted but causation could not be established in this review. computer reconstructions of the cartilaginous distal femoral condyles demonstrate a weight-bearing point of contact on the medial tibial plateau while the lateral portion of the knee shows an opening gap. online resource depicts a rotating d computer model of fig. . the convoluted pattern of the bony metaphysis likely reflects irregular cartilaginous invaginations into the ossi- fication zone of the metaphysis from a disordered maturation process within the physis (fig. a, b). online resource depicts a rotating d computer model of fig. . likewise, even in the face of excessive laxity of the joints of the hand and concomitant shortness, we did not encounter any clinically significant complaints. inasmuch as a large number of patients were old order amish ( patients), the usual types of employment probably correlate with the lack of complaints (blacksmith, farmer, lumber work, rough and finish carpenter). conclusion the spectrum of orthopaedic manifestations typically and frequently seen in chh has been elucidated in this paper based on this study of the largest group of patients so far reported in the north american literature. thirty-four fig. anteroposterior radiograph demonstrating coxa vara and femoral bowing fig. illustration of osseous and cartilaginous abnormalities in frontal view j child orthop ( ) : – percent of patients with the condition had undergone surgical realignment osteotomies of the long bones of the lower extremities. acknowledgments the authors wish to express their appreciation to richard pauli, m.d., ph.d., for providing access to non-amish patients’ information and to the women’s board of akron children’s hospital for their research funding support. this study was supported in part by a grant from the women’s board of akron children’s hospital. conflict of interest the authors declare no conflict of interest. open access this article is distributed under the terms of the creative commons attribution license which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. references . mckusick va, eldridge r, hostetler ja, ruangwit u, egeland ja ( ) dwarfism in the amish. i. cartilage-hair hypoplasia. bull johns hopkins hosp : – . mckusick va ( ) medical genetic studies of the amish: se- lected papers, assembled with commentary. johns hopkins university press, 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ds, phillips op, shulman lp ( ) car- tilage-hair hypoplasia syndrome: implications for prenatal diag- nosis. fetal diagn ther : – . alanay y, krakow d, rimoin dl, lachman rs ( ) angu- lated femurs and the skeletal dysplasias: experience of the in- ternational skeletal dysplasia registry ( – ). am j med genet a a: – j child orthop ( ) : – 	cartilage hair hypoplasia: characteristics and orthopaedic manifestations 	abstract 	purpose 	methods 	results 	conclusion 	introduction 	material: data 	results 	discussion 	conclusion 	acknowledgments 	references challenges and opportunities for north american hardwood manufacturers to adopt customization strategies in an era of increased competition review challenges and opportunities for north american hardwood manufacturers to adopt customization strategies in an era of increased competition david l. nicholls ,* and matthew s. bumgardner research forest products technologist, pacific northwest research station, sitka, ak , usa research forest products technologist, northern research station, delaware, oh , usa; mbumgardner@fs.fed.us * correspondence: dlnicholls@fs.fed.us; tel.: + - - received: march ; accepted: april ; published: april ���������� ������� abstract: much of the north american wood products industry was severely impacted by the recession of – . in addition, many sectors within this industry face intense global competition. against this backdrop, we examine economic opportunities for hardwood manufacturers to achieve greater competitive advantage via product customization, through a literature review and synthesis. we also discuss several related themes including agility, lean manufacturing, and clustering. we found that, in globally competitive environments, hardwood producers must be agile to adapt to economic conditions and dynamic customer demand. we discuss how some sectors of the hardwood industry have effectively exhibited customized production, and subsequently fared relatively well in the current economy. we conclude the synthesis by evaluating the importance of supply chains to achieving customization for hardwood producers. in the future, supply chains will need to be configured to rapidly respond to changing consumer demands, and pressure to provide more services will likely extend further back up the supply chain to hardwood sawmills. it is expected that sustainability practices, including green supply chain management, will impact operational and economic performance of hardwood firms as well. keywords: customization; competitive advantage; lean; agile; supply chain; hardwood products . introduction . . the hardwood value chain: from forest to consumer competition among firms is dynamic and is closely related to innovation and the search for strategic differences, according to porter [ ]. competition is also pervasive, whether it involves companies contesting markets, countries coping with globalization, or organizations responding to societal needs. directly related to competition is the creation of customer value, or delivering a desired product at an acceptable price. although value may not have a consistent universal meaning, it is clear that the process of delivering value to consumers is becoming the imperative for many organizations—in the wood products sector and elsewhere. therefore, the importance of understanding competition and value creation is often a prerequisite for business success. it is within this context that the present review synthesizes literature pertaining to the increasing role that customization plays in the north american hardwood industry, especially in secondary sectors such as furniture and cabinets. hardwood forests are noted for their ecological diversity, containing numerous commercial species. forest economics plays an overriding role in aspects of forest regeneration, silviculture, harvest, transportation, manufacture, distribution, product use, and ultimately recycling. the hardwood species forests , , ; doi: . /f www.mdpi.com/journal/forests http://www.mdpi.com/journal/forests http://www.mdpi.com http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /f \num [minimum-integer-digits = ]{ }\num [minimum-integer-digits = ]{ } http://www.mdpi.com/journal/forests forests , , of mix preferred by consumers is dynamic, requiring manufacturers to stay abreast of not only current forest and economic conditions but also consumer preferences. although the focus of this paper relates to the competitiveness of the north american secondary hardwood industry, an overarching theme is the role of the complete forest value chain. this includes all “upstream” value-chain entities, including forest landowners, managers, harvesters, and manufacturers of primary products such as lumber and veneer. these upstream entities are especially integral, as forest management and sawmill decisions can influence the appearance characteristics and value of primary hardwood products used as inputs to secondary manufacturing [ , ]. in addition, “downstream” customers, and ultimately consumers, are increasingly important to the hardwood supply chain. the north american hardwood industry faces new challenges in dynamic international markets. this sector now must compete with global supplies of raw materials and global manufacturers for u.s. consumers. the recent loss of manufacturing infrastructure for some components of the north american industry has brought high levels of change and uncertainty. of greatest importance and concern is the ability to competitively manufacture and maintain supply chains, while providing stability to other sectors of the overall forest industry [ ]. in addition, repercussions of the housing crisis are still being felt today across many wood product sectors, which has cast new emphasis on the way firms do business and interact with others in their supply chains. for example, the size of the average u.s. secondary manufacturer had not fully returned to pre-recession levels by , but the trend was upward and secondary manufacturers were requiring increasing services from their hardwood lumber suppliers [ ]. thus, we emphasize current economic events in our synthesis as well as the markets served, which are increasingly global in nature. . . mass customization—a paradigm shift for the wood products industry for wood products industries to realize competitive advantage, they must identify competitive strengths at all points in the value chain. this was especially evident during the recent u.s. housing crisis. panwar et al. [ ] recount the relevant macroeconomic factors and resulting dislocations felt by domestic wood products firms during the recession, identifying specific actions that could help maintain competitive advantage throughout the forest sector supply chain during periods of economic crisis. firms that successfully adapted to decreased demands, fluctuating prices and exchange rates, and greater competition were better able to survive the recession. they also identified strategies for maintaining coherent and intact supply chains during times of financial crisis. optimum strategies for maintaining and enhancing competitive advantage are constantly evolving—what worked well in the most recent business cycle may not be as effective in the future, especially during economic downturns. brown and blackmon [ ] introduced the concept of strategic resonance and strategic dissonance to compete within highly volatile markets. they discuss two ways that firms can gain competitive advantage. a market-led view suggests that the key is to identify external opportunities in both new and current markets (including niche markets). by contrast, a resource-based view argues that firms should mobilize resources that are likely to maximize returns in their chosen markets. here, competitive advantage is realized by the valuable firm-level resources that competitors do not have access to. competitive strategies must then be based on approaches that consider both market requirements and manufacturing capabilities under dynamic and unpredictable business environments. new production paradigms, such as mass customization, can increase this flexibility for secondary hardwood manufacturing firms, and this has become a frontier issue for the industry. it has been said that mass customization is a complete paradigm shift from traditional manufacturing [ ]. in north america, europe, and other regions with high labor and land costs such as singapore and hong kong, much research has looked at mass customization, lean manufacturing, strategic supply chains, and other strategies for maintaining or enhancing manufacturing competitiveness [ ]. in the u.s. wood products industry, a paradigm shift has been called for in sectors especially affected by globalization. one such sector is the wood household forests , , of furniture industry [ , ], where customized production and strategic supply chain alliances are key elements of competitiveness. similarly, dugan [ ] has called for several “new rules” for the u.s. furniture industry, including agility, niche marketing, supply chain development, and lean production. azouzi et al. [ ] claim that competitive priorities for the furniture enterprise of the future are short lead times, high product quality, product variety and innovativeness, and a focus on profit margin. a key component of a paradigm shift is the desire of consumers for customized products [ ]. thus, by leveraging this demand with development of customized production, the u.s.-based industry can be more competitive. shortening the lead times associated with customization is a key component of competitiveness, and as will be discussed in this synthesis, can be enhanced through factors such as lean manufacturing, supply chain agility, and local sourcing/purchasing of goods. mass customization has been defined as production of individually customized products using flexible and responsive manufacturing systems at a cost and speed near that of mass-produced items [ , ]. the potential advantage lies in realizing price premiums for products customized to customers’ specific demands, while at the same time maintaining the cost associated with large-scale production [ ]. gilmore and pine [ ] posit that consumers are increasingly moving away from a mindset of settling for standardization (and thus forgoing their individuality) simply to achieve a lower price, thus creating “customer sacrifice gaps”. this is a frontier provision where producers in higher-cost locations can compete, by moving away from price and towards provision of customized goods [ ]. previous studies have shown that consumers are willing to pay more for customized products [ ]. successful implementation of mass customization in the hardwood products industry has broad-reaching economic ramifications throughout the forest value chain. this includes not only manufacturing, but also product sourcing, procurement, and even harvesting and silvicultural methods, which can influence wood quality and/or product quality. for example, alignment between forest owners, logging contractors, and wood products manufacturers was identified as a key element of entrepreneurial success, which included the presence of strong networks [ ]. customization is therefore on the “frontier” of research and practice for the hardwood industry and will be the focus of this review and synthesis. we discuss some of the most central, state-of-the-art, and recent research involving customization and related concepts and how they are being applied in the north american hardwood industry. the end result, if this frontier can be successfully expanded, is more efficient production of the market goods derived from hardwood forests. . methods and objective in this review and synthesis, the objective was to discuss the literature regarding mass customization, including the related concepts of agility, lean manufacturing, economic clustering, and supply chain management with the intent to find linkages to competitiveness for the north american hardwood industry. while the entire hardwood supply chain is considered, the focus was on secondary manufacturers. we utilize a combination of articles that specifically address the forest products industry, as well as more general articles. the primary source of articles was a search carried out using google scholar in the summer of based on the following key words: customization, competitiveness, competitive advantage, lean, clusters, agile, and supply chain. the qualifying words of wood products and hardwood also were used to focus on articles specific to the secondary wood products industry. initially, of the articles discovered in this fashion were pre-screened as potentially relevant to the synthesis and sought for analysis. in addition, other articles were found through cross-referencing the articles identified in the google scholar search, or included via knowledge of the authors. this led to the inclusion of other relevant articles not identified through the primary search. the body of research in these fields is quite expansive; for example, mishra et al. [ ] provide a recent review of articles related to manufacturing flexibility alone, while bhamu and sangwan [ ] recently reviewed articles related to lean manufacturing. a goal of this work therefore was not to provide an exhaustive list but rather to weave a subset of these papers together, combining them with forests , , of work specific to forest products, to provide a practical discussion. a total of articles ultimately were referenced in this synthesis, with over % being published since . while the synthesis did not consider a specific time frame, preference was given to more recent studies since the focus was on the “frontier” of the field. the following sections discuss customization and its relationships with the related concepts outlined above. linkages between these concepts and competitiveness are discussed. the paper concludes with a discussion of the frontier of customization research with regard to the hardwood industry supply chain, focusing on the secondary wood products industry. . discussion . . agility and customization agility has been said to go “hand in hand” with mass customization [ ] while other authors have claimed it is a related but different construct [ ]. for example, um [ ] claims that agility is constrained by a parallel focus on economies of scale in many industries where efficiency is the focus, but that supply chain agility is requisite in high-level customization environments such as production of innovative or fashion products with short lifecycles. stump and badurdeen [ ] refer to agility as responsive manufacturing in unpredictable environments, without specific reference to customizing for individual customers. however, in their extensive review of mass customization research, fogliatto et al. [ ] found that a common feature of the literature is a blurring of mass customization, lean, and agile concepts. they conclude that both lean and agile concepts are relevant to implementation of mass customization and they call for more research aimed at elucidating these linkages. according to azouzi et al. [ ], agility is “ . . . the ability to respond to, and create new windows of opportunities in a turbulent market environment driven by individualizing customer requirements cost effectively”. similarly, um [ ] claims that agility refers to a firm’s capability to “ . . . transform market uncertainty into opportunities through rapid response, especially in high-level customization environments”. routroy and shankar [ ] evaluated agile supply chains, finding that they can be effective at functioning in challenging markets such as those characterized by uncertain demand, new and/or innovative products, and dynamic customer expectations. they developed a methodology known as “performance value analysis” to evaluate agile supply chains along three different time horizons for apparel supply chains, finding that significant performance improvements in supply chains could be realized in as little as two years. agility has been said to be enabled by the introduction of new technology, which is driven by market interaction and product design [ , ]. to these authors, a given process best contributes to the agility of a manufacturing enterprise when it possesses several capabilities, including flexibility, responsiveness, and autonomy. the result is the “furniture enterprise of the future” where the technology employed matches the customization strategy of the firm. the furniture enterprise of the future seems consistent in many ways with the paradigm shift described by schuler and buehlmann [ ] and the new rules for the industry described by dugan [ ]. a note should be made on the distinction between flexibility and agility, even though these terms often are used interchangeably in the literature to refer to responsiveness to changes in the business environment. for example, azouzi et al. [ ] claim that flexibility is a subset of agility, referring specifically to the ability for a process to outlive the products for which it was originally built. regardless of the definitional refinement, agility and flexibility are important in light of an increasingly global and customized economy, and the plurality of research on flexibility has focused on its relationship to environmental uncertainty [ ]. in the wood products industry, the importance of upstream suppliers to think strategically about the competitiveness of their downstream customers in the midst of uncertainty over globalization (i.e., the loss of the furniture industry as a market for hardwood lumber) has also been discussed [ ]. forests , , of comparative studies of the u.s. furniture and cabinet industries have shown that agility and customization strategies are associated with competitiveness. for example, lihra et al. [ ] found that, when studying these industries in north america and europe, the cabinet industry scored highest for implementation of mass customization while the household furniture industry scored lowest. the upholstered and office furniture sectors scored between the other two. cumbo et al. [ ] likewise found that the cabinet and upholstered furniture industries were implementing lean manufacturing at a higher rate than other sectors. and, lihra et al. [ ] found that the cabinet, upholstery, and office furniture sectors were following an assemble to order production model (more customized), while household furniture was following a ship to order model (less customized). this is consistent with the findings of luppold and bumgardner [ ] that the u.s. cabinet industry carried less inventory, offered more customized products, and competed less on price than did the u.s. wood household furniture industry. these factors were enabling the cabinet industry to be more competitive than the wood household furniture industry. import market shares are a useful way to analyze the competitive positions of different u.s. wood products industries [ ]. figure shows estimated market share statistics for three sectors for the latest year data were available. it can be seen that the wood kitchen cabinet industry (naics ) has been less affected by globalization to date than the nonupholstered wood household furniture (naics ) and upholstered furniture (naics ) sectors—a finding consistent with the comparative studies discussed above. in turn, the upholstered furniture sector (which has been the subject of several lean manufacturing studies) has been less affected by globalization than the wood household furniture sector. this notion of relative industry competitiveness will be revisited in the clustering section that describes how sectors of the furniture industry that customize products (similar to the cabinet industry) have realized success in the united states. figure . estimated import market shares in the wood household furniture, upholstered furniture, and kitchen cabinet sectors in the united states. import market shares in the united states calculated as: imports/consumption, where: consumption = value of product shipments (production) + imports − exports (data sources: [ – ]). . . lean manufacturing and customization—the skinny on wood value chains lean refers to far more than just dieting. lean manufacturing is generally described as a production system that uses fewer resources than mass production systems (for example less labor, equipment, and space), while achieving superior results. in fact, lean producers often aim for forests , , of perfection [ ], leading to declining costs, almost zero defects and inventories, and greater product variety [ ]. others have defined lean production as “an integrated socio-technical system whose main objective is to eliminate waste by concurrently reducing or minimizing supplier, customer, and internal variability” [ ], although variance reduction also is associated with related concepts such as six-sigma. returning to the cabinet industry discussion from the previous section, less inventory is often needed using lean manufacturing as a competitive strategy. however, mass customization has been said to be incompatible with lean manufacturing [ , ]. for example, mass customization follows a “design-sell-make” model where the final product is not manufactured until the end-consumer order is received. in lean manufacturing, items are made to meet the needs of immediate customers (i.e., intermediate to the end-consumer in the supply chain) and not the end-consumers themselves. olhager and prajogo [ ] explored potential differences between “make-to-order” and “make-to-stock” firms in australia. after considering firms, they found that make-to-order firms benefit significantly from supplier integration, whereas make-to-stock firms benefit significantly from lean practices. these findings indicate that the difference between these models is important when considering potential manufacturing and supply chain improvements. nag et al. [ ] consider the influence of inventory management on the competitive position of manufacturing firms. they identify three stages of inventory—raw material, work-in-process, and finished goods—the management of which can influence a firm’s competitive position. the authors follow porter’s “five forces model”, analyzing factors driving supply chain strategies, including: industry intensity of rivalry, threat of new entrants, threat of substitutes, bargaining power of suppliers, and bargaining power of buyers. they found that this framework can influence whether an industry shows low versus high levels of raw material inventories and finished goods inventories. inventory management also can greatly influence how supply chains are configured. stavrulaki and davis [ ] identify four different supply chain structures: make to stock, assemble to order, built to order, and design to order, where various structures are best suited to different combinations of products and demand characteristics. a key finding of this study was that high-volume products with little uncertainty about their demand should be matched with lean supply chains enabled by efficient processes. conversely, low-volume, high-uncertainty products should be matched with agile supply chains enabled by flexible processes. in the wood products industry, cumbo et al. [ ] found that implementing lean manufacturing might not be applied evenly across the facility. in particular, the rough mill, where lumber is converted to parts for later assembly, was less likely to be lean than other parts of the facility. ray et al. [ ] found that primary wood products manufacturers are inherently leaner than secondary manufacturers and suggest that not all classical lean metrics are entirely relevant to wood products manufacturers. haartveit et al. [ ] explored supply chain management in the forest products industry of western canada; successful implementation of supply chain improvements were found to focus on efficiencies through increasing throughput and reducing inventories. however, it was found to be challenging to apply supply chain management techniques to commodity-based supply chains such as wood products producers. this was primarily due to uncertainty in raw material supply and relatively long lead times in production. other authors have claimed that lean manufacturing can help enable mass customization. according to bhamu and sangwan [ ], “the goal of lean manufacturing is to be highly responsive to customer demand by reducing waste”. according to these authors, product variety and the integration of product development with supply chain and operations management are some of the goals for which lean manufacturing is implemented. modern definitions of lean production state in part that it provides companies with the agility to face market demands and environmental changes [ ]. several of the definitions of lean manufacturing found in the literature reference the provision of product variety that can be achieved through lean production [ ]. the advent of computer optimization has made concepts such as dynamic prioritization strategies feasible. in such systems, emphasis is placed on different part sizes as part needs are met and different forests , , of lumber grades and sizes are used [ ]. however, cumbo et al. [ ] found that yield of parts was the primary performance metric even though maximizing yield did not necessarily mean high-demand parts were being produced. pirraglia et al. [ ] also suggest that customer-driven inventory practices could result in yield losses (i.e., disposal of clear wood) in wood products companies. however, movement toward lean principles was associated with several customer satisfaction variables, suggesting multiple potential benefits to lean manufacturing beyond cost reduction. czabke et al. [ ] found similarly that, even though most benefits were manufacturing-related, implementation of lean thinking allowed manufacturers to get closer to their customers and could even pave the way for implementation of mass customization programs. in the case study presented by czabke et al. [ ], this was believed by the company to have led to competitive advantage. ariss and zhang [ ] discussed the notion of “flexible process capability” and found evidence that it was possible for firms, through flexible technology and employee involvement, to achieve high product variety while maintaining low process variety, essentially achieving mass customization. buehlmann et al. [ ] discuss the role that the digitization of manufacturing likely will play in the future of the hardwood industry and in helping to enable lean and agile manufacturing. in the wood products industry, implementation of lean manufacturing has been associated with shorter lead times, with an average -day shorter lead time for lean companies [ ] as well as greener production [ ]. time to market is an important component of mass customization [ ], so lean practices seemingly can help here. stump and badurdeen [ ] discuss “job shop lean” as a system where certain lean practices, such as value stream mapping, are applied to small firm customized environments. given the small size of many firms in the secondary wood products industry [ ], this concept seems well-suited to the industry, although literature to date on actual application is limited. others have noted that implementing lean is more difficult in smaller firms for a variety of reasons, including use of the wrong lean tools, use of a single tool to fix all problems, lack of understanding, and overall lack of financial and management resources [ ]. one potential compromise that has been found between lean manufacturing and customization, is determining an optimal lot size. yao and carlson [ ] found that, for a large upholstered furniture company with many possible product design combinations, a small lot size of units per batch was the key to balanced and synchronized production. coupled with an on-line, real-time data capture system that tracks status and location of materials in each batch, this was a way to link agility with leanness and realize competitiveness. other authors have noted the importance of using information systems to collaborate with consumers to obtain their design and preferences for customized products [ , ] and in monitoring supply chain performance measures [ ]. however, hunter et al. [ ] noted that batch and queue operations are outdated and based on a system of material movement through distinct departments within a shop where each worker is assigned to a single process or workstation. they instead argue for a system based on manufacturing cells, where each cell contains multiple functions and has the ability to produce a wide variety of products at low cost (i.e., economy of scope) and permits rapid design changes for upholstered furniture customers. manufacturing has been defined by hunter et al. [ ] as “ . . . the creation of wealth by adding value to raw materials”. this notion is at the core of searching for successful competitive strategies for the forest products industry. however, while much research has been conducted on the product side of mass customization, much less has been published concerning the manufacturing costs [ ]. in the forest products literature, product costing was addressed by andersch et al. [ ], who found that percent of the companies surveyed relied on traditional accounting systems that failed to account for many of the costs associated with lean and/or mass customization activities (product diversity, operational complexity, etc.). additional economic analysis of cost drivers would therefore seem to be an important area for future research. forests , , of . . economic clustering and customization clusters can be defined as “ . . . geographic concentrations of interconnected companies and institutions in a particular field” [ ]. clusters may be thought of in two dimensions as they can extend downstream to customers, and also laterally to manufacturers of complementary or related products. clustering can also serve as a means for small companies to realize some of the economies of scale usually reserved only for large companies [ ]. clusters of forest products companies can exhibit competitiveness and there are several examples of success [ , ]. walcott [ ], and kodzi and gazo [ ] discuss that the history of furniture manufacturing in the united states has followed a regional clustering pattern over time, with new formations in regions with lower-cost factors of production. the extension to global manufacturing also follows this pattern, with movement to taiwan and later china [ ]. more recently vietnam has emerged as a low-cost manufacturer of household furniture for the global market and it also has benefitted from u.s. duties on some chinese wood furniture [ ]. one example of a paradigm shift in the u.s. industry is amish furniture manufacturing in ohio and elsewhere. unlike the other regional u.s. furniture clusters described above where costs ultimately drove change, the ohio amish cluster formed through a sense of embeddedness in the community [ ] where important motivation for firms is to remain a viable family-based shop and continue living in the area [ ]. in this cluster, competitiveness arises by other means based largely on a customization model. the clustering aspects that enable customization include specialization among the many small shops and well-defined supply chains that allow flexibility among several shops [ ]. for example, brookfield [ ] found that firms operating within clusters outsource inputs to a greater degree than do non-clustered firms, in essence forming a “system of network production”. a good example of how such a production system can work is in the aggregate productivity of the cluster. even though the firms are quite small on average, their input productivity (wood use per employee) was found to be quite similar to the broader u.s. furniture industry when factoring in the firms specializing in component manufacturing [ ]. buehlmann and schuler [ ] make a connection between local markets, mass customization, and competitiveness; the customer/producer interface and short lead times necessary for mass customization give local and regional producers inherent advantages over importers, citing the u.s. architectural millwork industry as an example. similar observations have been discussed for the furniture industry (especially upholstery) in the united states by walcott [ ], who added logistics considerations as a key factor to regional competitiveness. others also have noted the potential value of buyer-supplier proximity in developing lean supply chains [ ]. maskell [ ] describes a successful wood furniture cluster in denmark where competitiveness was attributed to the ease of communication and exchange of knowledge arising from the close proximity of producers. the amish furniture cluster model follows what stump and badurdeen [ ] refer to as low-level mass customization, whereby consumers are involved in the assembly decisions for their products but design and fabrication are more modular. multiple choices in species, finish, and hardware are offered at the retail level and consumer orders are then placed with local manufacturers [ ]. there are now even options for the type of wood to be used, i.e., whether or not to use character-marked or naturally blemished woods, with a price differential. this practice is consistent with the findings of brinberg et al. [ ] and bumgardner et al. [ ] that the best opportunities for greater use of character-marked wood might be realized by manufacturers and consumers working together directly to design the product. increased use of character-marked wood has long been sought as a means to more efficiently use harvested timber [ ], but proven difficult to implement in traditional (i.e., non-customized) furniture marketing systems [ ]. in essence, consumers are participating in the design stage of furniture manufacture in the system described above [ ]. jensen et al. [ ] report a similar situation in the swedish construction industry, where modularization was found to be a bridge between mass production of standardized prefabricated components and being able to meet customers’ increasing requirements for customization in multi-story forests , , of timber housing. in the system described, jensen et al. [ ] explain how the design phase of the construction project was replaced by a configuration phase. hock soon and udin [ ] found similarly that organizations could enhance manufacturing flexibility by creating supply and logistics networks that were highly responsive to customers. christensen et al. [ ] also evaluated the role of customers in supply chain regimes, comparing build-to-order and just-in-time management. they found that build-to-order strategies can positively influence market performance through a greater supply chain knowledge with customers (i.e., a downstream application). conversely, just-in-time strategies affect upstream application of knowledge with suppliers. in fact, this is a similar model followed in the u.s. cabinet industry, which as described previously, has been more competitive than the wood household furniture industry [ , ]. consumers can choose from a variety of styles, species, and finishes, and the final product is then transported to where it is needed [ ], in this case, cabinets delivered to the point of installation. in such systems, lean manufacturing concepts fit well in that single pull signals for standardized components are used in downstream assembly [ ]. jiao et al. [ ] describe a similar concept in terms of “reusability” of design and process capabilities thereby creating underlying product architecture from which customized products can then be derived. . . pulling everything together with supply chains—at the frontier of customization a basic hardwood product value chain includes key elements from stump to consumer, including trees, sawmills, distributers, secondary manufacturers, and ultimately, end users (figure ). value chains help visualize the flow of manufacturing from raw material to final product by showing the steps where value is added [ ]. it has been said that: “the value chain has become the focal point of mass customization competition” [ ]. indeed, um [ ] states that the ultimate aim of supply chain management is to reduce costs and improve customer service, which at the base level are the two primary tenets of mass customization. maintaining consistent, reliable, and economical supply chains that are also manageable is one of the leading challenges for hardwood manufacturers. this is in part due to a multitude of product parts, manufacturing methods, harvesting stages, and transportation networks, as well as the design and fashion aspects associated with dynamic consumer tastes for many hardwood products. in recent years, global competition also has greatly changed the way that hardwood producers approach supply chain decision making. figure . basic value chain for north american hardwood product manufacturing (adapted from cohen and kozak [ ]). forests , , of li et al. [ ] conceptualized aspects of supply chain management, and collected data from firms and tested the effects on organizational performance. they found that higher levels of supply chain management practice can lead to enhanced competitive advantage and improved organizational performance. the five supply chain aspects were: strategic supplier partnerships (e.g., long-term relationships needed between organization/manufacturer and suppliers), customer relationships (e.g., practices to enhance long-term relationships with customers), level of information sharing (e.g., critical/proprietary information communicated to supply chain partners), the quality of information sharing (e.g., accuracy, timeliness, and credibility of information), and postponement (e.g., moving operations or activities to later points in the supply chain). kipserska-moron and swierczek [ ] introduce the concept of postponement with respect to modern supply chains. using an international survey approach, they found that dynamic supply chains can undergo reconfigurations, and that this is in response to increased demand for customized products. they also found that a “full postponement strategy” is possible but depends on the level of product customization as well as a firms’ manufacturing and logistical capabilities. supply chains also are becoming longer and more complex due to globalization [ , ]. this can further complicate the already complex nature of hardwood supply chains [ , ]. therefore, logistics management and supply chain tuning become key to remaining flexible in a high customization environment [ ]. bozarth et al. [ ] modelled the complexity of international supply chains and provided empirical tests from plants in seven countries. they considered both detail complexity (the distinct number of parts that make up a supply chain) and dynamic complexity (the unpredictability of a system’s response to given inputs). they found that several forms of complexity—including upstream complexity, internal manufacturing complexity, and downstream complexity—can all negatively impact plant performance. further, individual firms must assess the optimal degree of complexity. bhamu and sangwan [ ] found that simultaneous adoption of leanness by both the manufacturer and the supply chain is critical to overall lean manufacturing implementation. similarly, miller et al. [ ] found that lean concepts could successfully be applied to both manufacturing processes and supply chain optimization within a small furniture company. in recent research, um [ ] found that, among highly customized manufacturers (approximately percent of the sample were wood products companies), supply chain agility was not associated with business performance directly; actually it was negatively related. the author cited the costs associated with developing supply chain agility, including set-up and investment costs [ ] as well as the operational costs of using small batches [ ] to respond to customer requirement, as contributing to this negative direct relationship. however, improved customer service and product differentiation capability were both positively associated with supply chain agility, and both also led to higher business performance. in this study [ ], the supply chain agility construct was comprised of items, including the ability to rapidly: reduce the product development cycle time, reduce manufacturing lead time, increase the level of product customization, improve the level of customer service, improve delivery reliability, improve responsiveness to changing market needs, and reduce delivery lead time. so how can supply chain agility be achieved? in a high customization environment, the key is in variety management [ ]. interestingly, the elements of variety management bring us back to previously discussed topics. internal to the firm, variety management includes modularity [ , , ], cellular manufacturing [ , ], and postponement of customer involvement in customization decisions (sometimes called the decoupling point) to latter stages of production. examples of this strategy can be seen in the amish furniture model described previously [ ] and the u.s. upholstery industry, where imported components can be received as unfinished “white wood” and customized finishing and upholstery features are added at regional hubs closer to consumers [ , ]. research based on a cross-section of australian manufacturers found that firms in the lumber and wood products category (sic ) and the furniture and fixtures category (sic ) were not appreciably different than several other broad manufacturing categories on a measure of supply chain flexibility, although some of the lumber and wood products firms were among the highest scorers [ ]. forests , , of external to the firm, factors such as close customer relationships and collaboration with upstream and downstream partners are crucial to variety management [ ]. numerous studies have pointed to the importance of strong linkages between producers and customers as an integral part of supply chains. lefaix-durand et al. [ ] examined the construct of “relationship value” and its potential to help suppliers create value in their customer relationships, which in turn can improve competitive advantage. they studied a specific example of the canadian wood products industry supplying u.s. customers. they also developed a framework for differentiating customer relationships as “questionable”, “supportive”, “promising”, or “strategic”. these findings could have implications for not only wood products producers, but other business sectors as well. parhizkar et al. [ ] compared competitive factors for small- and medium-sized forest products firms. they found that export firms differed with regard to transportation methods, marketing activities, and production profiles compared to non-exporting hardwood lumber manufacturers. exporting firms were also significantly larger in production and employment. they point to the importance of firms developing customer relationships in order to seek new markets in a global economy. more complex supply chains are also possible. collin et al. [ ] suggest that alternative supply chain configurations can be customized to consumer demands. in their example (i.e., the telecommunications industry), a leading supplier deployed different supply chains to meet different customer demand chains. however, they point out that when supply chain design is too specialized, product backlogs may result, along with the potential for “inventory creep” and product obsolescence. although these outcomes are less likely in the secondary hardwood industry, the principle of matching supply chains to unique customer demands could be considered for specialized products. um’s [ ] research confirmed the predicted relationship that variety management, partnerships with suppliers, and closeness to customers were positively related to supply chain agility and flexibility. the implication is that supply chains can be managed (internally and externally) in high customization environments such that even though more complicated, the net benefits of customization can be achieved. greening of the supply chain bildsten et al. [ ] posit that purchasing strategy is central to lean management. they discuss the contrasts between market-driven purchasing and value-driven purchasing. the former is characterized by buying in large quantities (and subsequently large inventory costs), mostly based on price, with frequent switching of suppliers over time. value-driven purchasing, on the other hand, is based on close collaboration with a sole supplier who understands the technical aspects of the buyer’s products, leading to just-in-time delivery, zero defects, and customized products. in a case study, bildsten et al. [ ] show that value-driven purchasing of kitchen cabinets by an industrialized housing construction company could possibly lead to a reduction in the costs, complexity, and time associated with installation of bulk-purchased cabinets in the company’s modularized production process. hughes et al. [ ] considered modern supply chain management techniques and supply chain modeling, reviewing over papers. however, only three papers addressed demand-driven modeling approaches to value/supply chains in the wood pellets sector. related to purchasing strategy is the notion of “lean and green” supply chains. hardwood products firms can incorporate multiple objectives into their supply and value chains. a leading option on the “frontier” is the combination of agile supply chains with those that are focused on sustainability. carvalho et al. [ ] evaluated supply chains that had both “lean” and “green” paradigms. their work is distinguished from past research in that “lean and green” aspects are considered jointly within proposed models. a set of lean and green management practices are proposed to enhance the operational, economic, and environmental performance of supply chains. although lean and green paradigms share common goals, the primary distinction is that lean paradigms seek to minimize waste while green paradigms seek to minimize environmental impacts. forests , , of chavez et al. [ ] studied “green” supply chains in the context of comparing antecedent factors (for example, customer demands and pressures) with outcome factors (for example, operational performance and customer satisfaction). surveying manufacturers, the authors found that customer pressure can lead to operational improvements such as flexibility, delivery, product quality, and cost. of these factors, product quality and delivery aspects are most strongly associated with customer satisfaction. although customer-centric considerations are important, this study noted that other stakeholders—for example, regulatory stakeholders and community stakeholders—are also able to exert influence on firms with regard to environmental performance. cohen and kozak [ ] also considered the environmental performance of small- to medium-sized wood products firms in terms of their value chains. they furthered their focus on secondary (value-added) manufacturers. their research highlighted influential points in the value chain that could be leveraged to create environmental behavior changes. it is clear that the natures of various types of supply chains are changing quickly, with factors like globalization, green supply chains, and agility in supply chain design all influencing the ultimate goal of businesses—profitably serving customers. this has important ramifications for hardwood lumber firms that are competing in this ever-changing business environment. for example, espinoza et al. [ ] found that hardwood sawmills and distributors were providing more product-related services in recent years in response to increasing customer demands, including quicker delivery, just-in-time orders, certified products, and more lumber sorting for color and width. the numerous certification programs that currently exist for forest management and manufacturing processes can provide opportunities for companies to leverage green-compliant performance in their product promotion. . conclusions today’s economy calls for increasing customization of products to meet consumers’ increasing demands for product variety. in locations worldwide where cost factors of production are high and a cost leadership strategy is difficult, this trend provides considerable opportunity for competitiveness. a synthesis of the literature regarding customization and several related concepts reveals that a move toward customization by companies requires a combination of technology investment, training, and management insight to be successful. furthermore, technology investments and strategy development must match within a given company to move towards successful customized production. a key link is the supply chain, where companies can leverage both internal and external factors to enhance their competitiveness through agility and customization. such agility and lean principles are critical to shortening lead times for consumers to receive their customized products, and the advantages of producing and sourcing locally also can be leveraged by domestic manufacturers. to date in the united states, the wood kitchen cabinet industry generally has followed the mass customization model more closely than the wood household furniture industry, and subsequently performed better in terms of maintaining domestic market share. although lean implementation can be difficult for smaller firms, many of the supply chain efficiencies necessary for successful customization can be found in economic clusters. in such clusters, specialization develops among many small shops and well-defined supply chains, a result that allows for flexibility among the shops. proximity also can enhance communication and knowledge exchange among producers and customers. a good example is the amish furniture manufacturing clusters located in ohio and elsewhere, as well as clusters in parts of europe. once models of modularized customization are developed, consumers can configure their desired products and manufacturers can then build to order. this model can open the door for new beneficial product innovations, such as use of character-marked wood in products. implementation of this practice has long been sought by researchers and practitioners alike, and has started to become reality in some customized furniture lines where consumers can choose this product feature if desired. a related trend appears to be the “greening” of the supply chain. green supply chain management has been found to influence not only environmental performance but also operational performance and forests , , of economic performance [ ]. the most important link in the supply chain—consumers—will be the ultimate determinant of how widely sustainability measures are accepted. a culture of customization will require changes throughout the hardwood supply chain. at each value-added step, firms will need to determine what their customers need and then profitably deliver products and services that meet those needs. recent research has shown that this is already beginning to occur with hardwood sawmills, which have increased the product services they are offering to customers as downstream manufacturers seek to become leaner and more customized. this synthesis has shown that there is no one strategy to universally adopt, but elements of competitiveness are apparent in several existing models and research findings that can be adapted by individual firms to help them be more competitive. author contributions: bumgardner and nicholls jointly conceived of this synthesis paper. bumgardner researched and wrote sections on competition, lean management, and clusters. nicholls researched and wrote sections on supply chain management and greening the supply chain. both authors collaborated on locating key reference papers, writing the introduction, and writing the conclusions. conflicts of interest: the authors have no conflicts of interest to report. references . porter, m.e. location, competition, and economic development: local clusters in a global economy. econ. dev. q. , , – . 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[crossref] © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . / http://dx.doi.org/ . /j.jom. . . http://dx.doi.org/ . /j.omega. . . http://dx.doi.org/ . /j.ijpe. . . http://dx.doi.org/ . / - - . . http://dx.doi.org/ . /j.ejor. . . http://dx.doi.org/ . /ajibm. . http://dx.doi.org/ . /j.jom. . . http://dx.doi.org/ . / http://dx.doi.org/ . /ijbpscm. . http://dx.doi.org/ . /bse. http://dx.doi.org/ . /biores. . . - http://dx.doi.org/ . / http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	the hardwood value chain: from forest to consumer 	mass customization—a paradigm shift for the wood products industry 	methods and objective 	discussion 	agility and customization 	lean manufacturing and customization—the skinny on wood value chains 	economic clustering and customization 	pulling everything together with supply chains—at the frontier of customization 	conclusions 	references rb- - .pmd indian pediatrics volume __august , multicystic dysplastic kidney: a retrospective study *sathish sharada, mahalingam vijayakumar, prahlad nageswaran, sudha ekambaram and amish udani from the departments of *pediatrics and pediatric nephrology, mehta children’s hospital, chetpet, chennai, india. rrrrr eeeee sssss eeeee aaaaa rrrrr ccccc hhhhh b b b b b rrrrr iiiii eeeee fffff objective: to report the renal structural and functional anomalies in children with multicystic dysplastic kidneys. methods: retrospective descriptive analysis of children with multicystic dysplastic kidney seen in a pediatric nephrology unit over a period of years. results: antenatal diagnosis of multicystic dysplastic kidney was made in ( . %) patients. on follow up of children for more than months, ( %) had involution, [ %] had non-regression, and ( %) were nephrectomized. vesico-ureteric reflux (n= ; %) was the commonest renal abnormality. the serum creatinine values were higher (p= . ) in children with contralateral reflux. sub-nephrotic proteinuria was noted in ( %) and was significantly associated with complete involution (p=< . ). none of the patients developed hypertension and ( . %) had renal failure. conclusion: close nephrological follow-up is needed in children with multicystic dysplasia of kidneys. keywords: hyperfiltration injury, multicystic dysplastic kidney, proteinuria. correspondence to: dr m vijayakumar, consultant pediatric nephrologist, mehta children’s hospital, no. (e) mc nichols road, rd lane, chetpet, chennai , tamilnadu, india. doctormvk@gmail.com. received: july , ; initial review: july , ; accepted: may , . b etter and advanced antenatal ultrasound screening has led to an increased diagnosis of fetal renal abnormalities. multicystic dysplastic kidney (mcdk) is the most common form of cystic renal dysplasia with an incidence of : live births [ ]. the natural course of unilateral mcdk is either complete or partial involution which begins early in fetal life and progresses throughout the postnatal life. the renal function depends on the function- ing contralateral kidney [ ]. thus close nephrological follow-up becomes essential. this retrospective analysis of the records of children with mcdk was done to describe the follow-up of the clinical parameters and associated renal and urological abnormalities. methods records in pediatric nephrology services at mehta children’s hospital, chennai between the years - were reviewed. children with antenatally and/or postnatally detected mcdk were included. mcdk was defined as presence of multiple non communicating cysts of various sizes detected sonographically with no evidence of functioning renal parenchyma by dimercaptosuccinic acid renal scan (dmsa) [ ]. children with other cystic renal diseases and those with less than months of follow-up were excluded. initial assessment was done by demographic data collection, blood pressure measurement along with investigations, including blood urea, serum creatinine and electrolytes, urinalysis, spot urine protein-creatinine ratio (upcr), ultrasound abdomen, dmsa renal scan and micturating cysto-urethrogram (mcu). -hours urine protein analysis was done, if considered necessary. during follow-up (every months), blood pressure measurements, renal function tests and ultrasonography were done. mcu and dmsa scans were repeated, if necessary. hypertension was defined as blood pressure more than th centile for age, gender and height [ ]. proteinuria was considered when spot upcr was > . mg/mg or -hours urinary protein was > mg/ . m /day. urinary tract infection (uti) was defined on the basis of urine culture done during febrile episodes associated with urinary symptoms. compensatory hypertrophy of the contralateral kidney was defined as renal length of > sd of normal kidneys for age [ ]. modified schwartz’ formula was used to calculate estimated glomerular filtration rate (egfr). complete involution was defined as disappearance of the mcdk by usg abdomen [ ]. statistical analysis was done using spss software. involution rate was calculated using kaplan meier survival analysis. chi-squared test, fisher exact test and student t-test were applied to find out the association between variables. results of the children who attended the out-patient department from to , children with unilateral mcdk were enrolled; their characteristics are described in table i. indian pediatrics volume __august , sharada, et al. multicystic dysplastic kidney the characteristics of children with vur is depicted in table ii. resolution of reflux was documented only in children who had unilateral low grade contralateral reflux with no associated complications. surgical intervention for vur (ureteric reimplantation) was performed in one child with high grade contralateral reflux which was associated with recurrent uti, proteinuria and multiple scars on dmsa. the mean (sd) serum creatinine values were higher in children with contralateral vur than in those without vur [ . ( . ) vs. . ( . ); p= . ]. the median egfr was ml/min/ . m (range: - ml/min/ . m ]. the median egfr was ml/min/ . m [range: - ml/min/ . m ] in children with proteinuria and ml/min/ . m (range: - ml/min/ . m ] in those who underwent nephrectomy. other associated ureteric anomalies were ipsilateral ectopic ureter and ipsilateral ureterocele. six of the children diagnosed antenatally showed compensatory hypertrophy of the contralateral kidney at the initial postnatal scan. two children diagnosed postnatally showed scars on dmsa during their initial visit at and years of age. a total of children were followed-up for more than months with a mean follow up period of . months (range - months). their characteristics are described in table i. twenty-one ( . %) had involution of mcdk. the mean period of involution was months with a minimum period of months. the estimated median ( % ci) time during follow-up for complete involution of mcdk was ( - ) months by kaplan meier survival analysis. all children had compensatory hypertrophy of the contralateral kidney. the probability of complete involution at , and years were %, % and %, respectively. the probability of children without complete involution at year follow-up was %. the mean follow-up of those who did not have involution of mcdk was months. four children underwent nephrectomy. two among them had increasing size of mcdk of whom one had hypertension which resolved after nephrectomy. one child with ipsilateral ectopic ureter and another with ipsilateral high grade reflux underwent nephro- ureterectomy. both had scars on contralateral kidney by dmsa of which the child with ectopic ureter presented at years of age and the other child diagnosed antenatally, developed scars on follow-up. eight children developed at-least one episode of culture positive uti with children having contralateral grade iv vur and proteinuria. none of them had recurrent uti, renal failure or scars by dmsa. none of the children showed acute kidney injury during their infection episodes. among children with proteinuria, had undergone nephrectomy and had vur. there was significant association between proteinuria and complete involution of mcdk (p< . ). all of them had compensatory hypertrophy of the contralateral kidney. there was no significant association between grades of reflux and proteinuria. there was no significant difference in the table i characteristics of children with unilateral multicystic dysplastic kidney patient characteristics number (%) at baseline (n= ) males ( . ) right sided ( . ) antenatal diagnosis ( . ) urological abnormalities ( . ) vur ( . ) ipsilateral / contralateral / unilateral / bilateral / ureterocele ( . ) ectopic ureter ( . ) at follow up (n= ) ipsilateral complete regression ( . ) partial regression ( . ) increase in size ( . ) no change ( . ) nephrectomy ( . ) contralateral compensatory hypertrophy ( ) renal failure ( . ) sub-nephrotic proteinuria ( ) table ii characteristics of children with multicystic kidney disease and associated vesicoureteric reflux (n= ) characteristics number unilateral vur ipsilateral (grade iv) contralateral grade iii grade ii/i each bilateral vur ipsilateral grade iii grade i or ii each contralateral grade iii or ii each indian pediatrics volume __august , sharada, et al. multicystic dysplastic kidney egfr between proteinuric and non-proteinuric children. two children with postnatal diagnosis had renal failure. both had high grade reflux into contralateral kidney with multiple scars on dmsa. the child who underwent ureteric reimplantation showed decreased kidney size on follow-up. discussion mcdk is increasingly being recognized due to routine antenatal ultrasonographic screening [ ]. our retrospective analysis showed that antenatal diagnosis was made in majority of children. associated renal anomalies were seen in one-third of patients, and contralateral vur was seen in one-fourth. the proteinuria in completely involuted mcdk was probably due to hyperfiltration. the usual course in any single kidney status is progressive hyperfiltration, glomerulo- sclerosis and decrease in renal function [ ]. hence, these children need to be under close monitoring. singh, et al. [ ] described predominant postnatal diagnosis of mcdk. rabelo, et al. [ ], suggested a median time of months for the mcdk to become undetectable by usg, which is similar to our study. the overall involution rate in our study was lesser than that reported by kessler, et al. [ ]. we found significant difference in the involution rates at the -year follow-up between mcdk with an initial size of ≥ cm versus mcdk with an initial size of ≤ cm, similar to the finding of study by hayes, et al. [ ]. the rate of associated abnormalities of the contralateral kidney has been reported to be - % [ ]. according to mansoor, et al. [ ], children with contralateral anomalies are at risk for developing decreased renal function. hence, early diagnosis and treatment of contralateral anomalies to prevent renal injury is necessary. seeman, et al. [ ] suggested that the main risk factor for developing hypertension is contralateral kidney damage. in our study, even though the contralateral renal anomalies were high, no child developed persistent hypertension. in conclusion, long term nephrological follow up is needed in children with mcdk. as renal functions depend on the contralateral functioning kidney, its anomalies need to be detected early and managed appropriately. contributors: ss, se, mvk: formulated the manuscript; mvk, what this study adds? • associated anomalies in the contralateral kidney are common in multicystic dysplastic kidneys. np: were in-charge of the patients; mvk: will act as a guarantor. funding: none; competing interests: none stated. references . wiesel a, queisser-luft a, clementi m, bianca s, stoll c. euroscan study group. antenatal detection of congenital renal malformations by fetal ultrasonographic exami- nation: an analysis of , births in european countries. eur j med genet. ; : - . . mansoor o, chandar j, rodriguez m, abibtol cl, seeherunvong w, freundlih m. long-term risk of chronic kidney disease in unilateral multicystic dysplastic kidney. pediatr nephrol. ; : - . . stuck kj, koff sa, silver tm. ultrasonic features of multicystic dysplastic kidney: expanded diagnostic criteria. radiology. ; : - . . national high blood pressure education program working group on high blood pressure in children and adolescents: fourth report on the diagnosis, evaluation, and treatment of high blood pressure in children and adolescents. pediatrics. ; : - . . konus ol, ozdemir a, akkaya a, erbas g, celik h, isiks. normal liver, spleen, and kidney dimensions in neonates, infants, and children: evaluation with sonography. am j roentgenol. ; : - . . menster m, mahan j, koff s. multicystic dysplastic kidney. pediatr nephrol. ; : - . . brenner b, lawler e, mackenzie h. the hyperfiltration theory: a paradigm shift in nephrology. kidney int. ; : - . . singh jk, kanojia rp, narasimhan kl. multicystic dysplastic kidney–a need for conservative and long term approach. indian j pediatr. ; : - . . rabelo ea, oliveira ea, silva gs, pezzuti il, tatsuo es. predictive factors of ultrasonographic involution of antenatally detected multicystic dysplastic kidney. bju int. ; : - . . kessler oj, ziv n, livne pm, merlob p. involution rate of multicystic renal dysplasia. pediatrics. ; :e . . hayes wn, watson ar, trent and anglia mcdk study group. unilateral multicystic dysplastic kidney: does initial size matter? pediatr nephrol. ; : - . . kuwertz-broeking e, brinkmann oa, von lengerke hj, sciuk j, fruend s, bulla m, et al. unilateral multicystic dysplastic kidney: experience in children. bju int. ; : - . . seeman t, john u, bláhová k, vondrichová h, janda j, misselwitz j. ambulatory blood pressure monitoring in children with unilateral multicystic dysplastic kidney. eur j pediatr. ; : - . r the american political science bn w i eii ww social structure and political participation: developmental relationships, i norman h. nie, g. bingham powell, jr., and kenneth pretcitt culture and political development: herder's suggestive insights f. m. barnard politics, ideology, and belief systems giovanni sartori who pays for defense? bruce m. russelt political matrix and political representation: prolegomenon to a new departure from an old problem kenneth pretcitt and heinz eulau wilbur d. mills: a study in congressional influence john f. manley citizen demands and the soviet political system james h . oliver the electoral polities of gaulliets in the fourth french republic: ideology or constituency interest? howard rosenthal majority decision-making with partial unidimensionality richard g. niemi presidential elections: an explanation of voting defection richard ip. boyd research notes campaign strategy and party loyalty: the electoral rele- vance of candidate decision-making in the con- gressional elections robert a. schoenberger the desired political entropy henri theil communications to the editor book reviews and notes announcements richard f. fenno (ed.) vol. lxiii june, no. published quarterly by the american political science association htt p s: // d o i.o rg / . /s d o w n lo ad ed f ro m h tt p s: // w w w .c am b ri d g e. o rg /c o re . c ar n eg ie m el lo n u n iv er si ty , o n a p r a t : : , s u b je ct t o t h e c am b ri d g e c o re t er m s o f u se , a va ila b le a t h tt p s: // w w w .c am b ri d g e. o rg /c o re /t er m s. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms race and poverty, both of longstanding concern to the editors of commentary, have been the focus of some of the most important articles we have published. now reissued as commentary reports, many of them are available for use in college classrooms and by adult discussion groups. dating from the start of and coming up to the present moment, they serve as a remarkable record of shifting thought and calls for action in the stormy era that has followed national confrontation of these problems. we invite you to send for examination copies of all eighteen of these commentary reports or to select one of the four subject groupings in which they are listed below. your letterhead is the ticket of admission, write to howard gladstone.fcommentary reports, east street, new york city ^the reports are priced at fifty cents each, but your invoice will be cancelled whenever you or your bookstore place an order for twenty-five or more copies* of any one report. poverty in the city daniel p . moynihan: the professors and the poor david danzig and john. feild: the betrayal of the american city daniel p. moynihan: the president and the negro — the moment lost john slawson: mutual aid and the negro herbert j. gans: the failure of urban renewal charles e. silberman: up from apathy — the woodlawn experiment integration a n d education • maurice j. goldbloom: the new york school crisis midge decter: the negroes & the new york schools richard schickel: p.s. negroes a n d j e w s earl raab: the black revolution and the jewish question nathan glazer: negroes & jews — the new challenge to pluralism norman podhoretz: my negro problem — and ours the changing face of civil rights bayard rustin: the lessons of the long hot summer bayard rustin: "black power" and coalition politics david danzig: in defense of "black power" bayard rustin: the watts "manifesto" & the mccone report bayard rustin: from protest to politics — the future of the civil rights movements david danzig: the meaning of negro strategy *orders for over twenty-five copies merit a % discount please mention t h e american political science review when writing to advertisers h tt p s: // d o i.o rg / . /s d o w n lo ad ed f ro m h tt p s: // w w w .c am b ri d g e. o rg /c o re . c ar n eg ie m el lo n u n iv er si ty , o n a p r a t : : , s u b je ct t o t h e c am b ri d g e c o re t er m s o f u se , a va ila b le a t h tt p s: // w w w .c am b ri d g e. o rg /c o re /t er m s. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms new books from the university of chicago press the fiscal revolution in america herbert stein between and there was a revolution in american economic policy. the principle of balancing the budget gave way to the doctrine of managing government expenditures and taxes to achieve prosperity. noted economist herbert stein tells the story of this revolution in terms of the leading participants in it and'offers his conclusion that no single party or school of thought was alone responsible. studies in business and society series. lc: - pages, $ . the rules of the game in paris nathan leites translated by derek coltman a longtime resident of france, with a profound and often uncomfortably acute understand- ing of what makes people tick, nathan leites probes the nuances of the french character. with wit and urbanity, he relates their political tendencies to their behavior and attitudes in other spheres of life. 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b r y c e a. l a w r e n c e l o w e l l w o o d r o w w i l s o n s i m e o n e . b a l d w i n a l b e r t b u s h n e l l h a r t w . w . w i l l o u g h b y j o h n b a s s e t t m o o r e e r n s t f r e u n d j e s s e m a c y m u n r o e s m i t h h e n r y j o n e s f o r d p a u l s. r e i n s c h l e o s. r o w e w i l l i a m a. d u n n i n g h a r r y a. g a r f i e l d j a m e s w . g a r n e r c h a r l e s e . m e r r i a m c h a r l e s a. b e a r d former presidents w i l l i a m b . m u n r o j e s s e s. r e e v e s j o h n a. f a i r l i e b e n j a m i n f . s h a m b a u g h e d w a r d s. c o r w i n w i l l i a m f . w i l l o u g h b y i s i d o r l o e b w a l t e r j . s h e p a r d f r a n c i s w . c o k e r a r t h u r n . h o l c o m b e t h o m a s r e e d p o w e l l c l a r e n c e a. d y k s t r a c h a r l e s g r o v e h a i n e s r o b e r t c. b r o o k s f r e d e r i c a. o g g w i l l i a m a n d e r s o n r o b e r t e . c u s h m a n l e o n a r d d . w h i t e j o h n m . g a u s w a l t e r f . d o d d a r t h u r w . m a c m a h o n h e n r y r . s p e n c e r q u i n c y w r i g h t j a m e s k . p o l l o c k p e t e r h . o d e g a r d l u t h e r h . g u l i c k p e n d l e t o n h e r r i n g r a l p h j . b u n c h e c h a r l e s m c k i n l e y h a r o l d d . l a s s w e l l e . e . s c h a t t s c h n e i d e r v. o. k e y , j r . r . t a y l o r c o l e c a r l b . s w i s h e r e m m e t t e s. r e d f o r d c h a r l e s s. h y n e m a n c a r l j . f r i e d r i c h c. h e r m a n p r i t c h e t t d a v i d b . t r u m a n g a b r i e l a. a l m o n d r o b e r t a . d a h l m e r l e f a i n s o d c h tt p s: // d o i.o rg / . /s d o w n lo ad ed f ro m h tt p s: // w w w .c am b ri d g e. o rg /c o re . c ar n eg ie m el lo n u n iv er si ty , o n a p r a t : : , s u b je ct t o t h e c am b ri d g e c o re t er m s o f u se , a va ila b le a t h tt p s: // w w w .c am b ri d g e. o rg /c o re /t er m 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o re . c ar n eg ie m el lo n u n iv er si ty , o n a p r a t : : , s u b je ct t o t h e c am b ri d g e c o re t er m s o f u se , a va ila b le a t h tt p s: // w w w .c am b ri d g e. o rg /c o re /t er m s. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms t h e e c o n o m i c a p p r o a c h t o political s c i e n c e publ^ct choice the latest intellectual revolution ' in political science is economic analysis of political problems. public choice was established for the purpose of accelerating work in this new and exciting field of investigation. in the less than three years of its existence, it has already published articles by james s. coleman, duncan black, james buchanan, vincent ostrom, gordon tullock, otto davis, kenneth arrow, william mitchell, kenneth boulding, ferdinand levy, oliver williamson, and . many others. public choice is essential reading for political scientists interested in keeping abreast of the latest developments in their field. public choice is published twice a year by the center for study of public choice, virginia poly- technic institute, blacksburg, virginia . subscription price (includes membership in the public choice society) is s . for the paperback edition and $ . for the hardback. please mention t h e american political science review when writing to advertisers h tt p s: // d o i.o rg / . /s d o w n lo ad ed f ro m h tt p s: // w w w .c am b ri d g e. o rg /c o re . c ar n eg ie m el lo n u n iv er si ty , o n a p r a t : : , s u b je ct t o t h e c am b ri d g e c o re t er m s o f u se , a va ila b le a t h tt p s: // w w w .c am b ri d g e. o rg /c o re /t er m s. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms comments on the communicable disease issue by the guest editors neal d. goldstein, phd, mbi ; deborah kahal, md, mph, facp . drexel university dornsife school of public health, department of epidemiology and biostatistics, philadelphia, pa; william j. holloway community program, christiana care health system, newark, de . william j. holloway community program, christiana care health system, newark, de the focus on infectious diseases in public health ebbs and flows. students of the field are taught that developed countries have undergone an epidemiological transition whereby morbidity and mortality from non-infectious diseases has eclipsed that of infectious causes. nevertheless, infectious etiologies of disease remain a substantial threat to the public’s health. the last decade has seen a re-emergence of vaccine preventable diseases, including pertussis and measles; an increase in sexually transmitted infections (stis) most notably syphilis among gay, bisexual, and other men who have sex with men ; a worsening hepatitis c infection (hcv) crisis in the setting of the opioid epidemic ; and the ongoing struggle to control the hiv epidemic well into its fourth decade. to that end, we have solicited a wide range of experts in infectious disease epidemiology, public health practice, and clinical medicine to contribute insightful, and at times, provocative discussions of important infectious diseases within delaware. globally, nationally, and locally, healthcare systems face many challenges related to infectious diseases, from creating the infrastructure and microenvironments to support responsible antibiotic prescribing practices to immunization advocacy (and rapid response to complications stemming from a lack of adequate vaccination coverage) to ongoing efforts to test and treat for stis. this issue of the delaware journal of public health tackles these broad issues. certainly, we are all cognizant of the on-going debate surrounding vaccinations in our country. with the recent measles outbreak in washington and elsewhere, public health is in the challenging position of advocating for vaccination while responding to disease outbreaks from those who choose to abstain from this remarkable preventive intervention. james talbot and paul hess review the state of immunization in delaware and how the delaware division of public health (dph) has responded to recent vaccine preventable disease outbreaks. a pertussis outbreak in the amish community in delaware, as highlighted in the article by paula eggers et al., provides a local example of the repercussions of low vaccination rates. margot savoy enriches the immunization discussion with a closer examination of human papillomavirus vaccination and a call to action for us all to work collaboratively toward improved vaccination acceptance rates. a common theme throughout these articles is an emphasis on the importance of public health practitioners closely engaging with communities that have lower vaccination rates. hcv presents a multi-faceted story of incredible therapeutic success and implementation challenges. there is much work still to tackle as we strive to meet the four goals of the national viral hepatitis action plan in : . prevention of new hepatitis infections, . reduction of death and improvement in health of those living with hepatitis, . reduction of viral hepatitis health disparities, and . effective implementation of viral hepatitis activities. continuing with the engagement of the community by public health professionals, william mazur describes caring for patients with hcv who are incarcerated in delaware’s correctional facilities. with recent advances in hcv therapeutics, treating individuals in an institutionalized setting represents an opportune time to improve the patient’s health and provide treatment as prevention of forward hcv transmission subsequently reduces the community hcv burden. stephen eppes highlights the unique opportunities surrounding hcv amongst women of childbearing potential and their children, while navin vij shares his profoundly personal story with hcv. while eppes and vij offer different perspectives, both share compelling arguments in support of universal hcv screening for all pregnant women and adults, respectively. we have also included several articles that focus on the programmatic management of infectious diseases in delaware. vital statewide efforts to improve antimicrobial stewardship are eloquently outlined by marci drees et al. the ebrighthealth choosing wisely initiative emphasizes the importance of sustainable multidisciplinary collaboration in working toward appropriate antibiotic utilization in upper respiratory tract infections in inpatient and ambulatory settings. joanna suder tackles the sometimes difficult task of public health in the prevention, isolation, and quarantine of individuals who have tuberculosis, focusing on the medicolegal implications. this issue would be incomplete without including several articles focusing on hiv, both the successes and ongoing opportunities for improvement. nguyen tran and seth welles detail how understanding the epidemiology of hiv among sexual and gender minority communities, who have been particularly affected by hiv, is used to inform public health policy and action. as they discuss, the epidemiology of hiv has drastically changed since the disease was first recognized in the early s. in large part due to the increase in hiv screening, reduction in stigma, and the development and roll-out of antiretroviral therapy (art), hiv has transitioned from a death sentence to a chronic infection. consequently, we are seeing an aging population with hiv, the focus of brianne olivieri-mui’s article which considers a medicare-eligible hiv population and implications for art access in a nursing home setting. in closing, we hope you enjoy this issue and give pause to consider both the remarkable achievements of public health in reducing infectious disease sequalae and serious challenges that lay ahead. lastly, we wish to express our sincere gratitude to the invited contributors who have devoted considerable time and energy to making this issue of the djph a success. references . 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( ). the epidemiologic transition: a theory of the epidemiology of population change. . the milbank quarterly, ( ), – . pubmedhttps://doi.org/ . /j. - . . .x . adams, d. a., thomas, k. r., jajosky, r. a., foster, l., baroi, g., sharp, p., . . . anderson, w. j. ( , august ). summary of notifiable infectious diseases and conditions - united states, . mmwr. morbidity and mortality weekly report, ( ), – . pubmedhttps://doi.org/ . /mmwr.mm a . an, q., wejnert, c., bernstein, k., & paz-bailey, g., & the nhbs study group. ( , july ). syphilis screening and diagnosis among men who have sex with men, - , u.s. cities. journal of acquired immune deficiency syndromes, (suppl ), s –s . pubmedhttps://doi.org/ . /qai. https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://doi.org/ . /j. - . . .x https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://doi.org/ . /mmwr.mm a https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://doi.org/ . /qai. . valdiserri, r., khalsa, j., dan, c., holmberg, s., zibbell, j., holtzman, d., . . . compton, w. ( , may). confronting the emerging epidemic of hcv infection among young injection drug users. american journal of public health, ( ), – . pubmedhttps://doi.org/ . /ajph. . . the lancet. ( , july ). the global hiv/aids epidemic-progress and challenges. lancet, ( ), . pubmed https://doi.org/ . /s - ( ) - . centers for disease control and prevention. measles cases and outbreaks. https://www.cdc.gov/measles/cases-outbreaks.html. accessed march , . . centers for disease control and prevention. viral hepatitis action plan - . https://www.cdc.gov/hepatitis/hhs-actionplan.htm. accessed march , . https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://doi.org/ . /ajph. . https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract https://doi.org/ . /s - ( ) - 	comments on the communicable disease issue by the guest editors 	references in defence of state directed enhancement in defence of state directed enhancement tim fowler abstract this article considers the ways in which a liberal society ought to view the potential to cognitively or physically enhance children. at present, the dominant approach in the literature is to leave this decision to parents. i suggest that the parental choice approach is often inadequate and fails to account properly for the interests of children and wider society in enhancement decisions. instead i suggest that the state should play a greater role in determining when, and how, to enhance. to make this case, i compare the case of enhancement with existing practices of education, an area in which the state already plays a significant role. finally i suggest that some concerns with a statist approach are not as serious as has been argued. introduction one of the most significant debates in contemporary bioethics concerns the permissi- bility of human enhancement. recent developments suggest that humans will soon have the potential to enhance significantly many physical and cognitive capacities in young children. this article begins with the assumption that at least some enhancements are permissible. it addresses the separate question of who should decide whether to enhance a child and in what ways. a dominant view within the literature suggests that parents should control enhancement choices. i term this the ‘parental choice’ view of enhancement control (pce). pce is the core claim of the ‘liberal eugenics’ project defended most forcefully by nicholas agar, who writes that: on the liberal approach to human improvement, the state would not presume to make any eugenic choices. rather, it would foster the development of a wide range of technologies of enhancement ensuring that prospective parents were fully informed about what kinds of people these technologies would make. parents’ particular conceptions of the good life would guide them in their selection of enhancements for their children. on agar’s view, there are two important restrictions on parental freedom. one is that parental decisions cannot reduce the total amount of ‘real freedom’ that their children possess. following amartya sen, real freedom is defined in terms of a capabilities approach. agar writes that ‘a person whose collection of alternative functionings is greater than another’s is freer than that other person.’ the second restriction is that enhancement decisions cannot directly contribute to existing unjust prejudices in society. for instance, agar argues that parents cannot choose to have white children if this is likely to increase problems faced by racial minorities. agar suggests that pce can be justified without appeal to any contested notion of what kinds of lives are valuable or worth pursuing. he writes that ‘the distinguishing bs_bs_banner journal of applied philosophy,vol. , no. , doi: . /japp. © society for applied philosophy, , john wiley & sons ltd, garsington road, oxford, ox dq, uk and main street, malden, ma , usa. mark of the new liberal eugenics is state neutrality about the good life’. pce is thus consistent with a version of the principle of liberal neutrality on which state actions are freestanding from disputed questions of the good. this view is associated with theorists such as john rawls and charles larmore. agar’s view has received some important critical scrutiny, most notably by robert sparrow who writes that: liberal eugenicists confront the horns of a dilemma. they may allow parents the freedom to act on their own assumptions about these matters [conceptions of the good], with the consequences that the options available to children will be significantly determined by their parents and that future distributions of life plans will be strongly influenced by existing ideas about the nature of the good life. alternatively, they may require the state to protect the future wellbeing of children against their parents, at the cost of placing controversial decisions by the state at the heart of the ‘new’ eugenics. however, sparrow argues that despite the problems with pce, it is still the most appropriate way for a liberal society to make enhancement choices given the greater problems with the statist view. i argue that sparrow is wrong to think this, and, instead, i defend a statist alternative. further, i suggest that state regulation of enhancement can permissibly be premised on achieving an ideal of what constitutes a good and flourishing person. my account is thus perfectionist in the sense defined by joseph chan: ‘perfec- tionism is the view that the state should promote valuable conceptions of the good life’. following the most influential liberal perfectionist, joseph raz, i assume that one important component of the good life is personal autonomy. for raz, autonomous agents must meet three conditions ‘they must possess certain mental capacities, they must have an adequate range of valuable options, and they must enjoy independence from coercion and manipulation’. the practical implication of this view is that enhancement choices will be subject to much stricter regulation than those envisioned by pce. i also argue that it implies a duty for the state to fund some enhancements. finally, i suggest that in some cases the state ought to require that children receive enhancement even if this goes against their parents’ wishes. . education, enhancement and upbringing to make the case for a statist control of enhancement, i will compare the ethical issues raised by enhancement with those raised by education. i assume that given the moral and functional similarities between education and enhancement, principles which apply to one area should be assumed to have some salience in the other unless some good reason can be found to show why this should not be so. this analogy is suggestive in the light of the debate between pce and the statist alternative. in particular, it is significant that liberal democracies mandate that children attend school, provide a direct provision of education and operate a system in which the state controls the content of the curriculum. i will suggest that the reasons which justify these policies justify a similar series of policies in the case of enhancement. tim fowler © society for applied philosophy, . problems with parental choice as noted above, i will highlight serious concerns with pce by comparing this policy with a similar set of principles that apply to the upbringing of children. i will refer to these principles as the parental choice view of upbringing (pcu). according to pcu, parents should be able to make all significant choices on behalf of their children until such time as the children are able to make decisions for themselves. as with pce, this view does not allow parents to reduce the real freedom of their children, nor does it allow them to act in such a way that they directly contributes to the creation of social problems. pcu would lead to a wide diversity of institutions catering to children’s upbringing. this would include expensive private schooling and many different schools run by faith organisations. pcu would also lead to many children being schooled at home. under pcu, these institutions would be generally free of state regulation. in this section i will briefly outline these issues and show why dealing with each will often require state intervention, and why a similar concern might arise with pce. . . equality of opportunity a serious concern with the world created by pcu is that children of wealthy parents would enjoy a superior education compared to less fortunate children, leading to them having better employment opportunities. many might see this situation as unfair, since it implies that many children are disadvantaged due to factors outside of their control. this worry can be captured by the principle of fair equality of opportunity (feo) as defended by john rawls. this holds that considerations such as a student’s race, class or gender should have no bearing on their employment prospects.the only relevant issues for competition over valuable jobs and social offices ought to be an individual’s natural talent. several authors have offered problems with rawls’ account. one prominent objec- tion to feo is that it seems vulnerable to the levelling down objection. developed by derek parfit, this holds that in any case where equality is seen as intrinsically valuable there is some reason to favour worlds in which no one benefits relative to a more unequal one. in the case of feo, it appears that a state committed to equalising opportunities has a powerful reason to restrict the opportunities of those who are more fortunate in their natural talents. this could be achieved by sabotaging their schooling to ensure they received poor quality teaching. crucially, there would be some reason to favour these policies even if restricting the opportunities of the fortunate made no difference at all to the life chances of the unfortunate. thus, the state would have achieved equality only by creating a dead-weight loss. parfit suggests that this objection can be rebutted by abandoning egalitarian principles in favour of the priority view, which holds that it is morally valuable to give priority to the interests of the worst off, but that benefits to better off people are always valuable. this prioritarian view yields a plausible view of education policy explored by gina schouten. while the debate between egalitarianism and prioritarianism is significant for the best distribution of educational advantages (or enhancements), in one respect these latter cases are different from more standard advantages such as money or health. this is because educational advantages are, in large part, positional goods and gain their value via their place in a competitive ranking. the value of a degree from a prestigious university is dependent on the number of other students with similar or better degrees. in defence of state directed enhancement © society for applied philosophy, harry brighouse and adam swift suggest that this aspect of educational advantage provides a powerful reason to create equality of education rather than merely give some priority to the worst off. this is because a world in which the worst off get a reasonably good education (by current standards) but wealthy individuals get one that is signifi- cantly better in all respects is still one in which the worst off will predictably lose out. agar is sensitive to this concern, and suggests that the state should try and restrict enhancement choices made merely to garner positional advantage. however, he does not think this ought to restrict the enhancement of intelligence because it is both a positional good and an intrinsic benefit. he writes, ‘those who want to use enhancement tech- nologies to boost the iqs of their children do prepare them for some winner-takes-all competitions, but they can also point to many competitions that do not reward only the winners. the brightest may get the best jobs but those who are slightly less intelligent also get good jobs.’ while this point is important, it underplays the importance of the positional goods argument. the point is that where a good is positional, even if it is also an intrinsic benefit, benefiting one person harms the interests of others. a better response to goods which are both positional and intrinsically valuable is to increase them in an equal manner, allowing all to gain the benefit and none to lose position. another important feature of the education case is societal benefits that may be garnered from equality. in brief, it seems plausible to think that a society in which each knows her position in society is determined by her own efforts would be much more conducive to relations of mutual respect than one in which a favoured class has advan- tages merely because of birth. one could imagine a highly troubling society in which enhanced children went on to become wealthier and spent this money on their own children, who would then themselves tend to go on to be successful. while this society might have formal equality of opportunity between the enhanced and non-enhanced, in the sense that rawls terms ‘careers open to talents’, it would surely be a deeply problematic society for those with egalitarian or democratic sensibilities. taken together, the fact of positional goods and these social concerns are sufficient to show that equality of opportunity does have value in societies like ours, even if the levelling down objection shows us that we have no intrinsic reason to prefer reducing the opportunities of the more fortunate. this kind of consideration tells against agar’s view that the slightly less enhanced still get good jobs, just not the best, since it implies a class of careers forever out of bounds to those from some social backgrounds. these alternative accounts of educational fairness have been explored in the enhance- ment debate by oliver feeney who defends a principle of strict equality of opportunity. one worry with this view, raised by colin farrelly, is that an egalitarian approach to enhancement would imply either a very expensive and probably unfeasible program of funding free enhancement for all or banning most uses of enhancement. instead, farrelly suggests that the state should permit enhancement but use taxation to funnel the additional revenue gained from higher productivity into benefits for the worst off. in part, this debate rests on how far idealised political theorising ought to stray from feasibility constraints. one could hold the view that in the ideal world enhancement should be distributed in an egalitarian fashion, but in our world only a range of less egalitarian distributions are possible. however, worries about feasibility cannot ever justify pce as agar and others describe it if the general arguments for equality of opportunity are sound. this is because there are many cases in which the state can tim fowler © society for applied philosophy, feasibly (and relatively cheaply) create a fairer distribution than that created by pce. this would be true if treatments were both difficult to conceal and difficult to perform (thus decreasing enhancements done by non-sanctioned providers). it is also true whenever the state can plausibly fund a program of free enhancement financed by general taxation. therefore, concern with equality of opportunity justifies offering free education to poorer children. it also justifies a series of actions to mitigate the effects of parental wealth on life chances. since enhancement is both positional and has important social connotations these similar arguments justify state provision of useful enhancements to poorer children funded by taxation, and the banning of enhancements which cannot be provided to all whenever this is feasible. . . parental control and manipulation another concern about the world created by pcu is that parents will have extensive control over the lives of their children. in a world organised along pce lines, parental control would extend to selecting elements of their child’s genome and deciding whether they will possess biomechanical enhancements. while it is unobjectionable to claim that parents should have significant influence over their children’s lives, there must come a point at which this control becomes too extensive and children’s lives become dominated by their parent’s wishes and concerns. in the case of education, worries about parental influence are generally addressed by a principle requiring that children be raised to be autonomous. eammon callan suggests that there is consensus on some version of this principle amongst liberal treatments of education. however, there has been significant disagreement over the justification and strength of this principle. one important distinction is between those who defend a child’s autonomy as intrinsically valuable and those who defend it on instrumental grounds. the intrinsic view holds that children’s autonomy is a valuable feature of their lives, and should be protected for this reason alone. a strong version of this argument suggests that only choices made autonomously are ever valuable, and hence some measure of autonomy is a prerequisite of having a flourishing life. in contrast, an instrumental defence of autonomy does not rely on a close connection between autonomy and flourishing. instead, the core claim of this view is that being autonomous increases the chances of children ending up in a life in which they will flourish. this account of education is deployed by theorists such as brighouse, arneson and shapiro. this instrumental defence of autonomy is the one adopted by agar in his support of pce. he writes that ‘expanding a person’s real freedom expands the range of functionings available to him. this means that those with real freedom are more likely to be able to successfully pursue their distinctive life plans.’ therefore, on this view, one important reason to be concerned about parental influ- ence is that a child who rejects their parents’ plans may be ill suited to the life-plan they eventually endorse. this reason provides the justification for the requirement that parents do not reduce their children’s real freedom. agar writes that: the difference between selecting a life plan and raising its probability is a significant one. were prospective parents to be able to choose their child’s life plan then they could justify equipping her with the specific set of capabilities in defence of state directed enhancement © society for applied philosophy, required for that plan. if they can only raise the probability that a life plan will be chosen, they must make provision for the eventuality of their child’s rejecting their choice. this is to say: they must respect their child’s autonomy. at its core, agar’s defence of children’s autonomy rests on an empirical claim about enhancement technology, which he suggests will only be able to raise the probability of children selecting a given way of life. parents will never be able to choose for their children. one problem with this response is that agar underestimates the potential for parents to select values for children because he concentrates solely on the efficacy of genetic interventions. while it may well be true that such technology will never be able to determine someone’s character and life choices, it is far from the only means open to parents to shape their children’s characters. future technologies might provide all manner of biomedical interventions applied after birth, and of course parents already possess substantial control over their children’s lives. further, agar’s arguments provide a faulty philosophical foundation for the impor- tance of autonomy. after all, complete manipulation is at least conceptually possible and agar’s view appears to give us no reason to object to it. nor does it offer reasons to object to successful instances of manipulation that might have gone awry at an earlier stage so long as the (now heavily manipulated, possibly down to the genetic level) child does not reject the view into which they have been manipulated. this implication of agar’s view seems perverse. intuitively, a case where a child has been so successfully manipulated that they do not even consider the possibility of rejecting the way of life into which they are inculcated, and whose life is set from birth or soon after, is a case of lost autonomy. this worry can be rebutted by embracing an intrinsic view of the importance of autonomy. in razian terms, children designed by their parents to follow a certain path (and later educated such that they would pursue it) would not be the author of their own lives. what this case suggests is that autonomy requires a certain kind of independence: ‘you are sovereign as against others not because you get to decide about the things that matter to you most, but because nobody else gets to tell you what purposes to pursue, you would be their subject if they did’. therefore, there is strong reason to reject the purely instrumental defence of autonomy, though of course it is important to recognise the instrumental benefits of having more possible options. this has important implications for debating the limits of parental choice. on my view, one highly significant goal of enhancement and educa- tion should be to promote a child’s prospective autonomy by developing the relevant capacities and opening up an adequate range of genuinely valuable alternatives. in education, this goal would justify a number of actions that would go beyond pcu. for instance, the state would be justified in controlling the curriculum to ensure that all children had a chance to learn skills valuable in the labour market. this would often (though not always) be true even if parents objected. it is perhaps more difficult to see how enhancement technology might promote autonomy in ways that go beyond opening up options, but there are some plausible interventions that might be possible. for instance, most accounts of autonomy note the importance of critical thinking, which might be promoted by increases in intelligence. in addition to critical reasoning, autonomy requires a number of other personal character- istics. for instance, being autonomous requires a degree of courage to challenge others’ views and perhaps a certain curiosity to motivate self-investigation of issues. all of these tim fowler © society for applied philosophy, characteristics are ones which have a plausible genetic influence, and hence ones which might be promoted via genetic intervention or enhancement, and might form the basis of a program of enhancement which goes beyond pcu in order to both safeguard and promote children’s autonomy. . . missed opportunities the third problem with pcu is that many children will not receive an adequate education, even though their parents could have provided it. on the pcu view, parents have no legal obligation to ensure their children receive education. of course, in many cases we assume that parents have a strong desire to promote the interests of their children, and thus that they would make adequate provision for their children even without a state mandate. however, our experience suggests that this would not always be the case. in some cases, parents might decide not to educate their children. a much-discussed example amongst educational theorists is the supreme court case of wisconsin v.yoder (henceforth yoder). in this case, amish parents wished to withdraw their children from state education, a choice eventually approved by the court.this decision was widely criticised, often on the grounds that the children’s interests had been unduly set back. while this kind of freedom might be a feature of some versions of pcu, agar and others might suggest that no analogous worry is present in pce. after all, agar specifically considers cases such as yoder and argues that the parents’ choices were misguided and violated the real freedom restriction. however, i believe agar mischaracterises the case, and that it is not true that the real freedom restriction is sufficient to block parental choices. recall that the real freedom restriction holds that: . . . it does not require that parents use enhancement technologies to expand their children’s real freedom, or to have children who have greater real freedom than those they would otherwise have had had. instead, it demands only that they do not, in their pursuit of their eugenic visions, reduce their children’s real freedom, or have children with less real freedom than those they would other- wise have had. i believe that this restriction is under specified, and that it can block the amish request only on the basis of assumptions about the relevant alternatives which are as yet undefended. to show this, suppose (very crudely) that there are only options available to the children in question, each of which has a corresponding score of ‘real freedom’. these choices are: i) life in the amish community: ; ii) no education at all: ; iii) attending high school: . agar’s arguments rely on the fact that moving children off a path in which they are going to high school towards the amish path will involve a reduction of their real freedom. however, it is also true (merely by stipulation in this example, though i believe it to be plausible claim) that moving children into an amish upbringing would increase their real freedom relative to a world in which they received no education whatsoever. in defence of state directed enhancement © society for applied philosophy, what this example shows is that to understand the ‘real freedom’ restriction, we must have an understanding of the baseline of analysis. if we assume that the baseline is having no education, then the amish parents are increasing the real freedom of their children; they are simply not increasing it by as much as they could do by sending them to school. the suggestion that parents must send their children to high school requires assuming that this option is the baseline. it is natural to take the high school path as the baseline in the yoder case since other laws required sending the children to school, but these laws and policies have independent justification and cannot be defended merely in terms of the real freedom restriction. the relevance of this discussion to pce is that it shows that many of agar’s arguments depend on the claim that the relevant baseline is one in which children receive no enhancement. this baseline underlies the claim of advocates of the pce who argue that parents should be free to enhance their children so long as they do not reduce the amount of real freedom the children would have relative to the amount they would have had without enhancement. in many circumstances, this may well be the most plausible baseline. at present, enhancement technologies are expensive and of limited effective- ness in many cases.this makes a policy of state mandated enhancement highly expensive and ill judged. however, there is no reason to think that this will be true indefinitely. imagine a society in which many enhancements were widely available and relatively inexpensive. the result is that most individuals do have some enhancements, and these enhancements increase their real freedom in various ways by opening up ways of living that would otherwise be unavailable to them. it might still be true in this society that many would prefer not to enhance, and their decision would be protected by pce, since not enhancing does not, obviously, reduce a person’s real freedom relative to the amount they would have without enhancement. the problem for agar is that this choice seems analogous to that of the amish in yoder which he wished to condemn. of course, there are many distinctions one could draw between these cases. we might suggest that enhancement is more personal than educa- tion, or appeal to some version of the unnatural quality of enhancement relative to education. however, suggesting a dis-analogy between the two cases would appeal to similar arguments used by opponents of enhancement, which advocates of pce wish to disavow. if one accepts the basic premise that enhancement and education are morally similar, then under certain circumstances the decision not to enhance looks precisely like the decision not to educate. those same arguments which justify a state mandate to educate would apply in this case, and a state mandate goes beyond the requirements of pce. . . harm to wider society a final problem with pce is that in many cases the state has good reason to wish to overrule parental choices in order to advance legitimate social goals, for instance creating a tolerant society. there are difficult issues involved in determining precisely how biotechnology might make individuals more tolerant, but in this section i merely high- light that liberal eugenics faces a moral problem in that it appears to rule out the promotion of tolerance and other morally valuable qualities. agar considers the issue of tolerance because of the danger posed to his project by various racial and sexual prejudices. the problem is that, in current societies, many tim fowler © society for applied philosophy, parents would face a temptation to engineer their children such that they conform to dominant sexual preferences or are part of a privileged race or gender. agar believes that using biotechnology to alter a child’s race, gender or sexual preference for this goal can be permissibly restricted even by a liberal state committed to parental freedom. his reason is that allowing parents to make these choices would make the situation of prejudice worse, and hence harm existing members of disadvantaged groups as well as future children who are not engineered to avoid being a member of such groups. similarly, the state has a legitimate interest in there not being a gender imbalance, which might create serious societal problems. however, even if accurate, agar has only shown why enhancement technologies might avoid becoming part of the problem of intoler- ance. this is a deeply limited aim because, as i will go on to discuss, these technologies may well be capable of providing solutions to prejudice via the promotion of an attitude of tolerance. the timid nature of agar’s proposals is not merely incidental to his view, but stems from deep features of pce. the reason for this is that many individuals who grow up with intolerant attitudes do so because of the influence of their parents. therefore, to the extent that the promotion of tolerance might be served by genetic enhancements, we have no reason to believe that the children whom society would most like to become more tolerant will become so via parental choice. this problem of liberal eugenics is highlighted via the experiences of liberal societies of promoting tolerance through education. it is well documented that children become more tolerant if they attend schools that are mixed in terms of ethnic and social background. many parents would see this as a good reason to send their children to a mixed school, and would certainly not resist doing so on the basis that it was mixed. however, the research cited above suggests that, in the standard case, the children of these tolerant parents would already be in an environment conducive to raising toler- ance. the children we would be most concerned about are children raised by more intolerant parents, but these are precisely the parents whom we would expect to resist the use of schools to promote tolerance. in important past cases it has thus only been possible to achieve the goal of toleration by overruling parental objections. . the statist alternative the above discussion highlighted several problems with pce, and suggested remedies which might be taken to ameliorate these difficulties. taken together, these suggestions form the basis for an attractive alternative set of principles for the governance of enhancement that is decidedly more reliant upon state authority than writers like agar, sparrow and habermas believe is justified. i) funded. many kinds of enhancement technology may well need to be state funded and free at the point of use to citizens in order to address issues of equality of opportunity. ii) banned. a perfectionist account will ban many more types of enhancement than pce. enhancements will be banned if they i) threaten equality of opportunity by giving advantages to wealthy children that cannot be compensated for by other means, or ii) threaten children’s autonomy by dramatically raising the probability of in defence of state directed enhancement © society for applied philosophy, them pursuing a way of life favoured by their parents, or iii) if they would tend to increase the chance of children pursuing ways of life which are less valuable than those they otherwise would do. iii) mandatory. a perfectionist account holds that in some cases enhancements might be mandatory; parents would be required to allow their children to have certain enhancements just as the state requires inoculations or requires school attendance. this specification of the statist alternative is necessarily vague. importantly, it does not specify either the level of funding, or the costs that should be applied to people who either perform enhancements which are banned or fail to perform mandatory ones. this level of specificity is not needed to defend the view at this stage, and specifying these issues will raise a number of difficult problems of technology, availability of state funds and the possibility of enforcement. however, it is important to note two possibilities which i do not defend. first, i do not suggest that all possible useful enhancements should be funded by the state regardless of the costs. new technologies provide one way of boosting cognitive capacities, but there are other ways of doing so. moreover the education of children is only one role of government and it is one which competes with others for funding. there can clearly be cases when a possible enhancement is valuable, but too expensive. in such cases, my argument from equality of opportunity suggests restricting this technology until it is available to more than simply the wealthy. second, i do not claim that parents who fail to perform mandatory enhancements or perform illegal enhancement should thereby lose all rights over themselves or their children. of course, making something mandatory implies imposing costs on those who do not do so, but individuals retain important rights. the statist view need not, and indeed must not, imply such obviously objection- able actions as forcibly aborting unenhanced foetuses. . concerns with a state based approach above i sketched the outline of a statist account of enhancement control. in this section i address two of the most serious concerns that have been raised against this view in the literature by sparrow and others. these concerns are: ) the state intrusion problem and ) the disrespect of parents problem. i will suggest that both of these problems can be addressed. . . the state intrusion problem the possibility of greater state involvement in enhancement is discussed by sparrow, who considers this route as one of the ways to respond to the problems present in the current project of liberal eugenics. however, sparrow eventually argues that agar and other liberal eugenicists should reject the statist approach and that while there are problem with the current account of liberal eugenics the statist/perfectionist account i am suggesting has even more serious troubles. he writes that: if . . . we insist that the state should guard the wellbeing of future citizens against their parents, then liberal eugenics does not look all that different from tim fowler © society for applied philosophy, a more traditional eugenics. it will rely on the regulative authority of the state, and do so in the service of non-neutral assumptions about the relative merits of different life plans. however, the statist can respond to this problem. although sparrow is correct that, in one respect, a state-driven liberal eugenics will be similar to more traditional eugenics, he is wrong to suggest that this is a deep problem for this account. although a liberal eugenics program of the kind i advocate has one feature in common with objectionable eugenics programs in the past — namely, the promotion of a non-neutral account of human flourishing — it also has many distinguishing features that render it unobjec- tionable. specifically, my view promotes an account of human flourishing tied to toler- ance and personal autonomy, whereas, for example, the nazi program of propaganda tried to promote a view of human flourishing based on militaristic ideals and the intrinsic superiority of a master race. to illustrate, consider the following example. the state freedonia is a liberal democ- racy, but in the past was ruled by a deeply objectionable fascist party which ran an education system based on mandatory attendance at schools teaching a deeply racist view of the world. freedonia currently has an education policy relying entirely on parental choice. all children are home-schooled by their parents, who are free to make whatever choices about the curriculum they like. given that freedonia is a pluralistic society, this educational arrangement implies a very diverse range of early years’ experiences for children. however, this policy comes under criticism on the basis that it is failing many children. many citizens believe, quite rightly, that some parents’ choices about education are based on faulty values or beliefs. as such, concerned citizens complain that many children are being inculcated into a belief system which is deeply detrimental to those children’s long term interests. further, they complain that many parents are simply mistaken about basic aspects of history and science. they therefore propose to create a national curriculum, through which the state will create guidelines on appropriate beliefs and values for children, and a state-run school system which mandates that all children attend a state-run institution to learn the curriculum. now imagine that a citizen of freedonia objects to the proposal to create a state-run school system on the basis that this proposal does not look very different from the education policies of the previous fascist state. after all, he argues, both would involve the state in the education of children and both would involve the state teaching children some views which were different from those held by their parents. however, while there may be legitimate concerns with the specifics of the school curriculum (or with the very idea of school curricula), to suggest that the mere fact that the proposed education system is state run makes it equivalent to the earlier fascist system, is obviously misguided.while it would be true that both standard liberal education systems and those of fascist states rely on non-neutral opinions about science, history and values, this does not seem a particularly salient objection to the proposal in freedonia or to current practice in existing liberal democracies. we can imagine that the older fascist education system was run to promote a militaristic worldview, encouraging boys to see themselves as soldiers and girls as providers of future soldiers. further, science lessons in this state might well be based on a deeply misguided racist view of biology. in contrast, the liberal state school system will emphasise the importance of personal choice, as well as a respect in defence of state directed enhancement © society for applied philosophy, for diversity, and teach mundane (and true) scientific principles. these differences in educational content seem to vastly outweigh the importance of the similarities of the form of the system. while this comparison is obviously somewhat crude, the problems i have raised with pce are precisely those which might be raised with a policy of home-schooling all children, an extreme version of pcu. current liberal societies reject this policy in favour of a statist view. there are many possible problems with the statist account, and perhaps good reason to prefer one with more parental choice, but the mere fact of state involve- ment does not seem a particularly serious worry and certainly not one that evokes valid comparisons to the profoundly pernicious eugenics efforts of some past states. . . the disrespect of parents problem another worry with a state based view of enhancement control can be called the disrespect of parents problem. the idea is that it disregards any role for parents in the formation of their children. while it is true that many of the problems i have outlined for pce stem from its over-reliance on parental choice, it is not true that this implies the statist alternative has no room for parents. this is because there is a justification for parental involvement in children’s upbringing and decisions about enhancement that is compatible with the perfectionist account. this justification begins by recognising that the relationship with parents is the core of most children’s experiences, and is a rela- tionship which provides unique and highly beneficial support to children as they develop. a perfectionist account which promoted capacities like intelligence or sporting prowess, but ignored this relationship, would thus be extremely deficient on its own terms. the most prominent elaboration of the value of families is provided by brighouse and swift, who investigate the ‘relationship goods’ created and sustained by families. these goods include the loving attention of an adult carer, taken to be essential to children’s emotional development, a sense of continuity with the past, and security provided by a single stable carer. children’s interest in these goods is powerful, and as such according to brighouse and swift the state has reason to foster and protect family units. signifi- cantly for the argument of this article, brighouse and swift also believe that children’s interests in these goods justifies allowing parents to shape the values of their child, since doing so will better enable the parent-child relationship to produce the valuable familial goods they describe. they write that: . . . it is hard for us to imagine that two people can maintain intimacy without having some distinctive enthusiasms and interests in common. these shared enthusiasms provide for the interactions which to a considerable extent consti- tute the intimacy of the relationship and allow for the relationship to be pursued in a way that is not constantly self-conscious. from this it follows that it is in children’s interests to allow parents to shape their values and pursuits, even if these pursuits are not intrinsically better than the alternatives. in the case of genetic choices, or other enhancements, this gives a reason to allow parents to make many different choices about which specific capacities to enhance. for instance, suppose (probably wrongly) that it were only possible to enhance either a set of sporting capacities or a gift in music. also suppose that music and sports are each things that could lead to the realization of genuine and important human goods. it may be true that tim fowler © society for applied philosophy, in some impartial sense there is no reason to prefer one enhancement to the other. however, the fact that sports would allow a series of shared interest with a parent may provide a decisive reason to think it is (likely) to be more valuable to a specific child. since a parent will obviously know their own life-plans, and the likely life-plans of the child, much better than anyone else, there are thus powerful perfectionist reasons to allow a large degree of parental choice. the difference between parental choices on my account, as opposed to the standard pc view, is that they are limited by a requirement to make actually valuable choices about enhancements, and not to infringe their child’s autonomy understood in a fuller sense than agar allows. conclusion there is, undoubtedly, a serious worry with the idea that the state ought to have a role in choosing what sorts of people there should be, right down to their genetic code. one aspect of this worry is the horrendous history of past eugenics projects. against this, i have argued that it is not the mere fact of state involvement which was problematic, but rather the content of those states’ policies. modern states already play an expansive role in the raising of children, and do so without being thought of as the inheritors of any terrible instances of state indoctrination of pernicious beliefs. another aspect of the worry is the thought that the state might impose one view about what is best on all others. julian savulescu writes that: ‘as rational people we should all form our own ideas about what is best. but to know what is the good life and impose this on others is at best overconfidence-at worst arrogance.’ this kind of concern might be taken to be a problem for the perfectionist and statist view i have defended, but in fact it constitutes an endorsement of it. properly understood, the belief that rational people should form their own idea of what is best requires the promotion of autonomy and, if liberals really believe that the imposition of ideals on others is objectionable arrogance, then we have reason to want to raise a tolerant citizenry. designing policies to promote tolerance, autonomy and opportunity above parental objections is not illiberal, but is in fact the outcome of a deep commitment to liberal values. therefore, i concur with agar that ‘the addition of the word “liberal” to “eugenics” transforms an evil doctrine into a morally acceptable one’ but disagree with him over which liberal values are key to this transformation. , tim fowler, department of sociology, politics and international studies, university of bristol, priory rd, bristol bs tu, uk. tim.fowler@bristol.ac.uk notes note that the article is concerned solely with enhancements made to existing children. this excludes some technologies which might be thought to ‘enhance’ unborn children such as sperm selection. it does this to avoid philosophically difficult issues of personal identity, since enhancement via sperm selection might be thought to create an entirely different person. however, on many plausible responses to the non-identity problem, the arguments of this article would suggest some reasons to be sceptical of allowing parents to control pre-birth enhancements as well. to be more precise, i assume that some enhancements beyond those plausibly described as merely treatments are permissible. in defence of state directed enhancement © society for applied philosophy, mailto:tim.fowler@bristol,ac.uk in addition to agar, robert sparrow correctly attributes some version of the pc view to writers such as alan buchanan, norman daniels and julian savulescu.the pc view is also held by jürgen habermas who writes: ‘it virtually goes without saying that decisions regarding the genetic composition of children should not be submitted to any regulation by the state, but rather should be left to the parents’ (j. habermas, the future of human nature (cambridge: polity, ), p. ). nicholas agar, liberal eugenics: in defence of human enhancement (oxford: blackwell, ), . amartya k. sen, commodities and capabilities (oxford: oxford university press, ). agar op. cit., p. . agar op. cit., p. . there is a wide array of anti-perfectionist views, many of which appeal to importantly different accounts of political neutrality. given the target of this article, i confine myself primarily to agar’s view and the anti-perfectionist arguments consistent with this (and which would support pce if accurate). john rawls, political liberalism, exp. edn. (newyork: columbia university press, ); charles e. larmore, the morals of modernity, modern european philosophy (cambridge: cambridge university press, ). robert sparrow, ‘liberalism and eugenics’, australasian journal of philosophy , ( ): – , at p. . joseph chan, ‘legitimacy, unanimity and perfectionism’, philosophy and public affairs , ( ): – , at p. . joseph raz, the morality of freedom (oxford: clarendon press, ). ibid., p. . john rawls, a theory of justice, rev. edn. (cambridge, ma: belknap press of harvard university press, ), pp. – . see richard j. arneson, ‘against rawlsian equality of opportunity’, philosophical studies , ( ): – ; and matthew clayton, ‘rawls and natural aristocracy’, croatian journal of philosophy , ( ): – . derek parfit, ‘equality and priority’, ratio , ( ): – at p. . gina schouten, ‘fair educational opportunity and the distribution of natural ability: toward a prioritarian principle of educational justice’, journal of philosophy of education , ( ): – . harry brighouse & adam swift, ‘equality, priority, and positional goods’, ethics , ( ): – . adam swift, how not to be a hypocrite: school choice for the morally perplexed parent (london: routledge, ). agar op. cit., p. . rawls op. cit., p. . this argument does not suggest that improving the productivity of some can never be justified if it undermines fair equality of opportunity, only that there is some cost to the losers even if their jobs are adequately paid because of the range of careers no longer available to them. oliver feeney, ‘equality of whom? a genetic perspective on equality (of opportunity)’, res publica , ( ): – . one possible objection to this view is that parents might be permitted to advantage their children in ways that are less than optimal from a social perspective; for instance, reading bedtime stories might create inequalities. i respond to this thought below. eamonn callan, creating citizens: political education and liberal democracy, oxford political theory (oxford: clarendon press, ). see harry brighouse, on education (oxford: routledge, ); and richard arneson & ian shapiro, ‘democratic authority and religious freedom: a critique of wisconsin v.yoder’ in i. shapiro (ed.) democracy’s place (ithaca, ny: cornell university press, ). agar op. cit., p. . ibid., p. . arthur ripstein, force and freedom: kant’s legal and political philosophy (cambridge, ma: harvard univer- sity press, ), p. . wisconsin v. yoder, u.s. ( ). agar op. cit., p. . this kind of parity underlies agar’s principle that ‘if we are permitted to produce certain traits by modifying our children’s environments then we are also permitted to produce them by modifying their genomes’: agar op. cit., p. . tim fowler © society for applied philosophy, a more difficult case is engineering people to have a certain quality for purely instrumental reasons. for instance, one might engineer children to be heterosexual simply because there will be more possible partners for them. i leave this issue aside. a useful set of data is offered in amy gutmann, ‘civic education and social diversity’, ethics , ( ): – . (sometimes via the court system as in brown v. board of education, u.s. ( ).) sparrow op. cit., p. . harry brighouse & adam swift, ‘parents’ rights and the value of the family’, ethics , ( ): – , at p. . julian savulescu, ‘deaf lesbians, “designer disability”, and the future of medicine’, british medical journal ( ): – , at p. . agar op. cit., p. . i’d like to thank members of c.e.l.p.a at the university of warwick, and participants at a workshop at the university of leeds. in particular, i’d like to thank matthew clayton, kimberley brownlee and matt matravers. in defence of state directed enhancement © society for applied philosophy, biomed centralcases journal ss open accecase report unilateral hemothorax in a year old south indian male due to a giant arteriovenous hemodialysis fistula: a case report shihas salim*, prasanthi ganeshram, amish dilip patel, anita a kumar, divya vemuri, vijay jeyachandran, deepan rajamanickam and ghanshyam palamaner subash shantha address: department of general medicine, sri ramachandra university, chennai, india email: shihas salim* - shihas.salim@gmail.com; prasanthi ganeshram - preshg@gmail.com; amish dilip patel - myshtikal@gmail.com; anita a kumar - anitakumar @hotmail.com; divya vemuri - dvem @yahoo.com; vijay jeyachandran - vijayjeyachandran@yahoo.co.in; deepan rajamanickam - deepan_rm@yahoo.co.in; ghanshyam palamaner subash shantha - drpssghanshyam@yahoo.co.in * corresponding author abstract in a patient undergoing regular hemodialysis through an arteriovenous fistula access, pleural effusion is a known long term complication. however, a unilateral hemothorax is relatively uncommon. here we report a year old male, end-stage renal disease patient, on maintenance hemodialysis, who presented with a giant brachiocephalic av fistula in his left arm and progressive breathlessness. radiological imaging revealed a left sided pleural effusion. ultrasound guided aspiration revealed a hemorrhagic pleural fluid. a doppler study of the fistula revealed a high velocity blood flow through the fistula, thereby establishing the cause of the unilateral hemothorax. ligation of the fistula resulted in complete resolution of the hemothorax. the other possible causes for hemothorax in a dialysis patient are also discussed in this case report. introduction av (arteriovenous) fistulas are recognized as the preferred access method for hemodialysis in patients with end stage renal disease. however, they are associated with a separate subset of complications, in addition to those that can potentially occur with hemodialysis. pleural effusion, with or without hemorrhage, is one among these compli- cations. high flow through the arteriovenous fistula is rec- ognized as an uncommon cause of unilateral hemothorax in such patients. here we report a patient with a giant av fistula and a same sided hemorrhagic pleural effusion. case presentation a year old male, a known hypertensive on treatment for the past years, was diagnosed to have chronic renal failure years ago. consequently he progressed to esrd and hemodialysis was initiated in december for the same, with the creation of a brachiocephalic arteriov- enous fistula over the left arm. he has been undergoing regular thrice weekly hemodialysis since then. he is a school teacher by occupation. he had no past history of diabetes mellitus, hypothyroidism, coronary artery dis- ease, or hepatic disease. no past history of tuberculosis or tb contact. he is married and has two sons. he was a non- smoker and a non-alcoholic. over the past year, the artificial av fistula enlarged in size to attain its current dimensions (figure ). on rd june , he presented to the emergency department of our tertiary care hospital with complaints of progressive published: october cases journal , : doi: . / - - - received: september accepted: october this article is available from: http://www.casesjournal.com/content/ / / © salim et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.casesjournal.com/content/ / / http://creativecommons.org/licenses/by/ . http://www.biomedcentral.com/ http://www.biomedcentral.com/info/about/charter/ cases journal , : http://www.casesjournal.com/content/ / / breathlessness over the past days. there was no history of associated chest pain, palpitations, syncope, cough, expectoration, or orthopnea. on examination, he had pallor and bilateral pitting pedal edema. he was afebrile, with a pulse rate of /min, bp of / mmhg, respiratory rate of /min. a giant arte- riovenous fistula was seen extending from the cubital fossa of the left arm up to the left supraclavicular region (figure ). respiratory system examination revealed stony dullness over the left mammary, infra-axillary and sub- scapular areas on percussion. on auscultation, there was decreased intensity of breath sounds over the above men- tioned areas. normal vesicular breath sounds were heard over all other areas. other systems examination was unre- markable. his blood investigations showed hemoglobin of . g/dl, bun – mg/dl, creatinine- . mg/dl, with other param- eters being within normal limits. his coagulation profile was slightly deranged with a raised inr (table ). chest x-ray revealed a massive pleural effusion on the left side (figure ). usg guided pleural aspiration revealed a hem- orrhagic fluid. pleural fluid analysis showed no wbcs, rbc – /mm (normocytic %, crenated %), sugar – mgs/dl, protein – . g/dl. doppler study of the av fistula showed a high peak velocity of cms/sec, with no evidence of thrombosis or stenosis. in order to drain the hemorrhagic pleural effusion, an intercostal drain was placed. patient was managed with units packed red cell transfusion, oxygen and other supportive measures. with this presentation the possible cause for the left hemothorax was suspected to be due to the av fis- tula. hence after the patient was stabilized, the giant av fistula was ligated on the th day after admission. follow- ing ligation of the fistula, the patient's left sided pleural effusion decreased. the chest drain was removed after giant arteriovenous fistulafigure giant arteriovenous fistula. photograph of the patient's upper body, with the left arm showing a giant arteriovenous fistula extending from the cubital fossa of the left arm up to the left supraclavicular area. table : laboratory investigations parameters values hb . g/dl tc cells/mm platelet . lakhs/mm inr . pt . sec (control- . sec) ptt . sec (control- . sec) bleeding time min seconds clotting time min seconds d-dimer < ng/ml bun mg/dl creatinine . mg/dl pleural fluid analysis: wbc nil rbc /mm (normocytic %, crenated %) sugar mgs/dl protein . g/dl pcr for mycobacterium tuberculosis negative cytology for malignant cells negative page of (page number not for citation purposes) cases journal , : http://www.casesjournal.com/content/ / / days and patient was discharged after more days. on fol- low up weeks later, the patient did not have a recurrence of pleural effusion (figure ). for further hemodialysis, the creation of a new fistula on his right arm has been planned. discussion the incidence of pleural effusion in hospitalized patients receiving long-term hemodialysis is . %; of them % have unilateral effusion [ ]. unilateral hemorrhagic pleu- ral effusion can occur secondary to uremia, an emboli arising from the arteriovenous fistula, anticoagulation therapy, subclavian/brachiocephalic vein stenosis/throm- bosis, a complication of central venous catheterization, or excessive flow through the avf access. [ - ] uremia causes uremic pleuritis. this evolves into a fibri- nous pleuritis which, if untreated, progresses to become a fibrothorax. the pleural effusion takes the form of exu- dates, which at times is hemorrhagic, and often unilateral. it usually disappears with dialysis and adequate renal replacement therapy, or develops into a fibrothorax. an ultrasonogram may depict septae, fibrinous bands, and debris [ - ]. considering the clinical presentation of our patient – a giant av fistula, acute onset massive pleural effusion, ultrasound showing no evidence of septae and adequate control of uremic status, the possibility of ure- mia being the cause seems unlikely. in patients on dialysis, hemothorax may occur due to coagulopathy. this can arise secondary to platelet dys- function of uremia and/or anticoagulant use [ , , , ]. although the coagulation profile in this individual was altered (inr – . ), it is more likely that this alteration contributed to the extent of hemorrhage as opposed to it being the primary cause. hemothorax could arise as a complication of central venous catheterization. this is usually secondary to mal- positioning or malfunctioning of the vascular catheter [ , ]. in our patient, a central venous catheter was not used. as a result of the increased venous pressure due to the high venous flow through the arteriovenous fistula, sten- osis and/or thrombosis of the brachiocephalic and/or subclavian veins can occur. a stenosis of the brachio- cephalic vein, in association with high venous flow rates, considerably increase venous pressures in the intercostal and bronchial veins of the left side of the chest. conse- quently, local hemodynamics would be altered such that pleural fluid resorption would not occur normally, and excess pleural fluid formation would occur. this leads to unilateral pleural effusion over the same side as the vein stenosis/thrombosis [ ]. doppler study in our patient did not reveal any evidence of stenosis or thrombosis in the brachiocephalic or subclavian vessels. x-ray chest on admissionfigure x-ray chest on admission. x-ray of the chest (pa-view), showing a left sided massive pleural effusion. this x-ray was taken on admission. x-ray chest -weeks after ligation of the fistulafigure x-ray chest -weeks after ligation of the fistula. x-ray of the chest (pa-view), taken -weeks after ligation of the fis- tula, showing resolution of the pleural effusion with no recur- rence. page of (page number not for citation purposes) cases journal , : http://www.casesjournal.com/content/ / / the other possibility of pulmonary embolism due to a thrombus arising from the av fistula also seemed unlikely, as there was no evidence of thrombosis in the fis- tula and the patient's d-dimer levels were within normal limits. pulmonary embolism usually produces minor pleural effusions unlike the massive effusion in our patient [ , ]. the possibility of the hemothorax occurring secondary to tuberculosis or other infective etiology seems improbable as the patient improved significantly following ligation of the fistula, and had no evidence of recurrence even after weeks. during this entire period he was not given antibi- otics or antituberculous therapy. a tuberculous hemotho- rax resolving without antituberculous therapy is not possible. a malignant etiology for hemothorax is again unlikely in view of rapid resolution and absence of recurrence of the hemothorax. in addition, the pleural fluid showed no evi- dence of malignant cells. there have been reports of hemothorax occurring as a consequence of excessive flow in an avf access [ ]. as a result of the av fistula, local hemodynamics is altered and the high venous pressure could impede pleural fluid resorption, eventually leading to a same sided pleural effusion with or without hemothorax. this condition usu- ally resolves with ligation of the arteriovenous fistula. [ ]. in our patient, this could have been the most likely mechanism, as the patient presented with a high flow giant av fistula, and once the av fistula was ligated there was dramatic resolution of the pleural effusion. in addi- tion, on follow-up there was no evidence of recurrence. conclusion high flow state of an arteriovenous fistula access must be thought of as a differential diagnosis for ipsilateral hemot- horax, in a patient on hemodialysis through the same. dramatic improvement results with ligation of the fistula. patient's perspective people used to give me odd stares because of the snake- like structure over my arm. when i was admitted, the sight of blood coming out of my chest when the tube was inserted really scared me. i feel a lot more content now that this fistula has been removed. i feel like i have been cured of this problem. abbreviations av: arteriovenous; avf: arteriovenous fistula; esrd: end stage renal disease; tb: tuberculosis; hb: hemoglobin; tc: total count; rbc: red blood cells; wbc: white blood cells; bun: blood urea nitrogen; pt: prothrombin time; ptt: partial thromboplastin time; inr: international normal- ized ratio; usg: ultrasonogram; pcr: polymerase chain reaction. competing interests the authors declare that they have no competing interests. authors' contributions ss, pg, adp, aak, dv, vj, dr, gpss were involved in the patient care, acquisition of data, analysis and interpreta- tion of data, review of literature, drafting and revising the manuscript. consent written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor – in – chief of this journal. references . bakirci t, sasak g, ozturk s, akcay s, sezer s, haberal m: pleural effusion in long-term hemodialysis patients. transplantation proceedings , ( ): - . . kim yookyung, shim sung shine, shin jung hee, choi gyu bock, lee kyung soo, chin-a yi, yu-whan oh: variable pulmonary mani- festations in hemodialysis patients. j korean radiol soc , : - . . schwartz e: thoracic manifestations of chronic renal disease. contemporary diagn radiol , : - . . galen ma, steinberg sm, lowrie eg, lazarus jm, hampers cl, merrill jp: hemorrhagic pleural effusion in patients undergoing chronic hemodialysis. ann intern med , ( ): - . . nasiłowski j, krenke r, wewnetrznych klinika chorób, pneumon- ologii i, alergologii am, warszawie w: hemothorax with high number of eosinophils following warfarin overdose. pneumo- nol alergol pol , ( – ): - . . bensman a, grimfeld a, vasmant d, montagne jp: massive haemot- horax during haemodialysis in a child. rev fr mal respir , ( ): - . . cheah fk, sheppard mn, hansell dm: computed tomography of diffuse pulmonary haemorrhage, with pathological correla- tion. clin radiol , : - . . gavelli g, zompatori m: thoracic complications in uremic patients and in patients undergoing dialytic treatment: state of the art. eur j radiol , : - . . wright robert s, quinones-baldrich william j, anders alpha j, dano- vitch gabriel m: pleural effusion associated with ipsilateral breast and arm edema as a complication of subclavian vein catheterization and arteriovenous fistula formation for hemodialysis. chest , : - . . varan b, karakayali h, kutsal a, ozdemir n: spontaneous hemot- horax in a hemodialysis patient. pediatric nephrology , ( ): - . page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= 	abstract 	introduction 	case presentation 	discussion 	conclusion 	patient's perspective 	abbreviations 	competing interests 	authors' contributions 	consent 	references software for constructing and verifying pedigrees within large genealogies and an application to the old order amish of lancaster county richa agarwala, , leslie g. biesecker, katherine a. hopkins, clair a. francomano, and alejandro a. schaffer , laboratory for genetic disease research (lgdr)/national human genome research institute (nhgri)/national institutes of health (nih), bethesda, maryland usa; (under contract to r.o.w. sciences, inc., rockville, maryland); immunogenetics laboratory, division of medical genetics, department of medicine, johns hopkins university, school of medicine, baltimore, maryland usa; medical genetics branch (mgb)/nhgri/nih, bethesda, maryland usa, and center for medical genetics and department of medicine, johns hopkins university, school of medicine, baltimore, maryland usa; this paper describes pedhunter, a software package that facilitates creation and verification of pedigrees within large genealogies. a frequent problem in medical genetics is to connect distant relatives with a pedigree. pedhunter uses methods from graph theory to solve two versions of the pedigree connection problem for genealogies as well as other pedigree analysis problems. the pedigrees are produced by pedhunter as files in linkage format ready for linkage analysis. pedhunter uses a relational database of genealogy data, with tables in specified format, for all calculations. the functionality and utility of pedhunter are illustrated by examples using the amish genealogy database (agdb), which was created for the old order amish community of lancaster county, pennsylvania. many genetic studies require linkage analysis, a key step toward positional cloning of disease suscepti- bility genes. linkage analysis requires describing pedigrees for a set of people exhibiting a specific trait and verifying relationships in pedigrees (poly- meropoulos et al. ; khoury et al. ). if a pedigree is implicitly part of a large genealogy, it is possible to use computer science techniques to find it. there may be many potential (sub)pedigrees that connect the individuals with the trait, and it is de- sirable to find a (sub)pedigree that is optimal in a rigorous mathematical sense. this paper describes software tools, called pedhunter, to keep genealogi- cal data in a relational database and to analyze the data systematically. pedhunter solves two distinct formulations of the optimal pedigree connection problem, as well as other problems related to pedi- gree construction, verification, and analysis. closed populations are a rich source of material for medical genetics studies. for example, closed populations provide powerful data sets to map re- cessive disease genes by homozygosity mapping (kruglyak et al. ). some closed populations have compiled genealogies because of their inter- ests, and these genealogies enhance the utility of the populations for genetic studies. the old order amish of lancaster county in pennsylvania is one such population. most ancestors of the amish in the united states migrated in the early eighteenth cen- tury from germany and switzerland. they settled in berks county, pennsylvania, and gradually radiated to other parts of pennsylvania, ohio, indiana, illi- nois, and iowa. some settled in lancaster county, in about . information about amish pedigrees of lancaster county is available in a book called the fisher family history (fisher family history ), also known as the ‘‘fisher book,’’ which traces the lineage of the amish in magnificent detail. in spite of the rich information in this book, it is not easy to use in medical genetics for a number of reasons. ( ) the book ( version) has ∼ pages of informa- corresponding author. e-mail schaffer@helix.nih.gov; fax ( ) - . research : – © by cold spring harbor laboratory press issn - / $ . ; www.genome.org genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com tion in small font. ( ) the organizers grouped infor- mation about extended families in the book. this ordering is useful for amish readers interested in their proximal genealogy but is problematic for the construction of extended pedigrees needed for ge- netic analysis. ( ) many people have the same first and last name. ( ) there are no pedigree diagrams; only text. ( ) some information about a person may appear in several places because that person has his/ her own record and also appears in the records of parents and children. data are sometimes inconsis- tent across multiple entries. because of these limitations, tracing family re- lationships for genetic studies requires laborious searching. this paper describes a database called amish genealogy database (agdb) for the geneal- ogy of the old order amish of lancaster county, pennsylvania. agdb contains information from the version of fisher family history that has been corrected for errors and contains additional infor- mation compiled subsequent to . keeping the data in agdb and querying the data with pedhunter facilitates usage of the genealogy by medical geneti- cists. beyond the old order amish, pedhunter is a general tool that can be used to analyze any gene- alogy. the use of pedhunter requires only a database with information for that population. for reasons of confidentiality no phenotype information is stored in the database. phenotype information known only by the user may be helpful in posing geneti- cally interesting queries. tools from computer science this section summarizes relevant data storage and data analysis tools from the domain of computer science and suggests how they can be used to make a genealogy database. the data in agdb are stored in a relational database (date ). pedhunter is de- signed to use relational databases developed in sybase. sybase provides an implementation of structured query language (sql) useful for answer- ing simple queries and an interface with the c pro- gramming language useful for implementing more complex queries. data organization each record in the fisher book contains information about a nuclear family. for example (information in this example of a family record is fictitious, but the syntax is accurate), . noah karp b jan. , , d may , , farmer, m dec. , , mary green ( ) b sep. , , d . children: victor ( ), sarah ( ), amy. m , annie green ( ) b aug. , . children: henry. . victor karp ( ) b may , , m june , , mildred sweeney. . john green b dec. , , m sarah fisher. children: annie ( ), mary ( ). the data were obtained as an ascii text file with some typesetting commands inserted. using standard methods from the theory of formal lan- guages these family records were parsed and col- lated into a record of the information for each per- son. each record in the fisher book contains the fol- lowing pieces of information relevant to genealo- gies: ● name of family head; e.g., noah karp, victor karp, and john green in the above three examples, re- spectively. ● a number called the fisher number or family number. for the above examples, the fisher num- bers are , , and , respectively. ● if the parents of a family head are known, the record contains the fisher number for that family; e.g., record refers to record , victor’s par- ents. ● list of spouses. for each spouse, the record gives the name and attempts to give a fisher number in following order: ( ) fisher number for the family in which spouse is the family head; ( ) fisher number for the family in which spouse is a child. for example, in record noah is married first to mary and then to annie green; the fisher number following those names refers to the family in which they were children. ● list of children of each spouse. this list contains names of children and attempts to give a fisher number for each child in the following order: ( ) fisher number for the family in which the child is the family head, e.g., for victor karp in record ; ( ) fisher number for the family in which the child is a spouse, e.g, for both annie and mary in record . data inconsistencies the analysis of data with a relational database re- quires ( ) resolving or deleting inconsistencies in the data, ( ) designing the database, ( ) creating the agarwala et al. genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com tables, and ( ) writing programs to analyze the da- tabase. of these four steps, the only task that is spe- cific to a population is deleting or resolving incon- sistencies. the other three tasks are greatly facili- tated by using a commercial sql implementation. finding inconsistencies is feasible if the raw geneal- ogy data have redundant and conflicting informa- tion. because of the structure of the records in the fisher book, the detectable inconsistencies include parent–child relationships and husband–wife rela- tionships. some inconsistencies are caused by infor- mation that is present in one record but absent in another; it is assumed that the information is cor- rect in this case. other inconsistencies are appar- ently caused by typographical errors in fisher num- bers, in which case it may be straightforward to decide which numbers are (in)correct. a dispropor- tionate number of the errors occur in the data en- tered after , presumably because those data have not been published and hence have not been proofread by large numbers of amish. in addition, the number of recorded births, deaths, and mar- riages per year dropped precipitously after , suggesting that much recent information is missing. efforts have been initiated with the amish commu- nity to correct remaining errors and update records. database design each person in the database is assigned a unique integer, called the program id. there are two cen- tral tables in the database—the person table and the relationship table. the person table has information exclusive to the individuals, and the relationship table keeps information relating the individuals by containing an entry for each couple and a list of their children, if any, in terms of their program id. please see appendix for details of database de- sign. analyzing the database after the database is created, the next step is to use analytic tools to extract useful information. the analytic tools in pedhunter rely heavily on graph theory (even ). in the appendix, the notion of a graph is introduced along with related terminol- ogy. results and examples this section describes the queries available in pedhunter and shows the utility of pedhunter with a usage of agdb on a published pedigree. pedhunter supports functions for finding relatives of individu- als (father, mother, siblings, half-siblings, uncles and aunts, children, first cousins, ancestors) as well as functions for testing if two individuals are related according to a specified relationship. the more complicated queries supported by pedhunter are as follows: ● lca(s): [lowest common ancestors for s] this query finds the most recent common ancestors of the given set of people s; the set of most recent common ancestors of s is the set of people a such that ( ) everyone in a is an ancestor of everyone in s, and ( ) no one in a has a descendant who is also an ancestor of everyone in s. ● asp(s): [all shortest paths pedigree for s] if there is at least one common ancestor for the given set of people s, then for each lowest common ances- tor a, asp(s) finds the pedigree containing all shortest paths between a and each person in s. pedhunter contains an implementation of dijk- stra’s algorithm (dijkstra ) for finding all shortest paths. pedhunter takes a few seconds for creating modest size pedigrees (e.g., connecting – individuals resulting in a pedigree of ∼ individuals). this is a significant improvement over the tens of hours typically required to create such pedigrees by manually searching the fisher book. ● minped(s): [minimal pedigree for s] if there is at least one common ancestor for the given set of people s, then for each lowest common ancestor a, minped(s) finds a pedigree connecting a and each person in s such that ( ) the pedigree has the minimum number of vertices, and ( ) every path from a to each person in s is the shortest path. the problem in finding a minimal pedigree for s is the steiner tree problem, with asp(s) being the given graph and s being the set of vertices to be connected. pedhunter uses a ‘‘branch and bound’’ algorithm for finding a minimal pedigree (de- scribed in the appendix). ● pedigree length(x,n): prints a pedigree contain- ing all descendants of x who are at most n gen- erations apart from x. for example, pedigree- length(x, ) lists all children and grandchildren of x. ● kinship(x,y): computes the kinship coefficient between x and y where kinship(x,y) is the prob- ability that a randomly selected allele of x and a randomly selected allele of y are identical by de- scent. pedhunter uses the algorithm in weir ( , pp. – ). software for constructing pedigrees genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com ● inbreeding(x): computes the inbreeding coeffi- cient for x where inbreeding(x) is the probability that the two alleles of x are identical by descent. because x inherits a randomly selected allele from mother and a randomly selected allele from fa- ther, inbreeding(x) = kinship(x’s mother, x’s father). pedhunter uses the algorithm in weir ( , pp. – ). ● print(x): print all information about person x. all pedigrees are produced in linkage format (terwilliger and ott ). the pedhunter output pedigree files can be read into some versions of pedi- gree/genotype maintenance programs such as cyrillic . (chapman ) and pedigree/draw . (mamelka et al. ). two of the drawings here were produced by feeding pedhunter output into pedigree/draw . . the all shortest paths pedigree may contain many connections between pairs of individuals, and it can be quite large. this pedigree may be too com- plicated for linkage analysis with current tools and may be impossible to draw comprehensibly (e.g., the drawing in mckusick et al. ). these two problems justify the desire for a minimal pedigree that is a subpedigree of the all shortest paths pedi- gree. a minimal pedigree can be used to explain most likely paths of inheritance of the mutant trait gene. a minimal pedigree can be used as a starting point for the geneticist to reinstate more individuals that are in the all shortest paths pedigree in an it- erative linkage analysis. to verify a given pedigree (p), one must verify all paths in p. a path is considered to be verified if the parent–child relationships represented by the edges on the path are consistent with the informa- tion in the database. the paths in p can be verified as follows: . find the set of people s in p who do not have a child in p and have at least one parent in p. . find asp(s). paths in p that are present in asp(s) are verified. if all the paths in p are verified, the verification is complete. . for each ‘‘long’’ path p from u to v that is not yet verified, find asp(r) where the elements of r are u and v. if p is a path in asp(r), p is verified. if all the paths in p are verified, verification is complete. . for each edge u → v in p that is not yet verified, do the query is child(v,u), which returns true if v is a child of u and false otherwise. if it returns false, the edge is in error. [it can be corrected by using query features father, mother, ancestor, chil- dren, as appropriate for the pedigree. for in- stance, if confidence about v is high, u can be corrected by using father(v) and mother(v).] it is not practical to have a query feature for verifying an entire pedigree at once because it is difficult to determine what to do when there are multiple related errors in the pedigree. in many cases, the corrections would depend on knowledge not present in the pedigree database. the utility of agdb and pedhunter are demon- strated in an example concerning a genetic disorder currently under study. mckusick ( ) published a pedigree that connects the children of six couples. field work and consultation with the authors of the original study yielded the names of the persons, so they could be accessed in agdb. three of the couples are present in the fisher book and agdb and will be used as a test example. part of the pub- lished pedigree that connects the three couples that are in agdb is shown in graph representation in figure and in pedigree representation in figure . the pedigree in figure is designated mkped. let s be the set with elements , , , , , , rep- resenting the couples that are in agdb. using lca(s), it turns out that there are five least common ances- tors for the three couples. figure shows the all shortest paths pedigree (aspped) for the least com- mon ancestor that has the maximum number of vertices in common with mkped. a minimal pedi- figure graph for the partial mckusick pedigree. agarwala et al. genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com gree with the same least common ancestor is shown in figure . mkped (fig. ) can also be used to illustrate pedigree verification. the paths in mkped that are verified by aspped (fig. ) are as follows: ( ) path from to ; ( ) path from to ; ( ) path from to ; ( ) path from to and ; and ( ) path from to . according to aspped and are siblings, whereas in mkped and are siblings and is a child of . this discrepancy is due to an error in mkped—person should not be present in mkped. the following relationships are verified using is child/siblings query features: ( ) is the father of ; ( ) is the father of ; ( ) is the mother of and ; ( ) is the mother of ; and ( ) and are siblings. discussion much attention has been paid toward database management and analysis [e.g., gdb (fasman et al. ), genbank (benson et al. ), swiss-prot (bairoch and apweiler )] of genomes as well as to making extensive family trees [e.g., gedcom ( ), ged html ( )] but there is no tool suf- ficient for a medical geneticist who has a large genealogy as well as genetic information for some of the individuals. existing software packages [e.g., cyrillic (chapman ), dgene (lange et al. ), pedigree/draw (mamelka at al. ), pedraw/wpedraw (curtis ), phenodb (cheung et al. ), schesis (round )] man- age genetic information, store pedigrees, and draw pedigrees but do not automatically extract or verify pedigrees from a genealogy. pedsys (dyke ) supports pedigree extraction but not based on the type of optimality criteria implemented in pedhunter. pedhunter provides tools to organize ge- nealogical information in a database and a query facility to access the database. this functionality can create and verify pedigrees according to criteria specified by the user. advantages of keeping data in a relational database include the following: ● simplicity: the concept of a relation provides a self-contained means of representing all types of data relationships using simple tables. ● flexibility and restructuring: there is complete flexibility in database design. relations can be combined or broken down in other relations with ease. ● accessibility and protection: keeping data in a rela- tional database provides variable and ready access figure all shortest paths pedigree (aspped). figure partial mckusick pedigree (mkped). software for constructing pedigrees genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com to all data. data can be protected and can be used by multiple users. ● update: updating the information in a database is simple because one can verify whether informa- tion is consistent. it is important to keep data current for future use. ● optimization: a good database design does not du- plicate information, which reduces data storage and redundancy. ● easy storage: physical data storage and logical da- tabase structure are independent. there is no worry about where and how the files are stored and they can be accessed using the relational tables. the usage of pedhunter should be extensible to other existing genealogies besides the fisher book: ( ) there are other genealogy books of the old or- der amish that overlap with the fisher book; ( ) there are genealogy books of other amish commu- nities from the midwestern united states; ( ) there are genealogy books of other closed north american communities, such as hutterites and mennonites; ( ) there are genealogies loosely compiled for sev- eral island populations and for parts of finland. ef- forts have been initiated to obtain some of these genealogies, to obtain permission to computerize them, and to get the raw data into a computer- readable text file. to use pedhunter on a genealogy, one need only create a database with two tables— person table and child parent table, as described earlier and use the utility program provided to cre- ate generation table. pedhunter is freely avail- able to scientists studying populations with written genealogies. interested scien- tists should send an electronic mail request to richa@helix. nih.gov or to schaffer@helix. nih.gov. agdb is intended to be available to scientists con- ducting national cancer in- stitute internal review board (irb)-approved research on the old order amish. access to agdb is carefully con- trolled and limited by irb protocol. in the initial phase, the irb approved usage by five groups that have proto- cols approved by their irbs to study the amish. each user group is represented by at least one coinvestigator on the protocol for agdb. more user groups meeting these criteria can be added. the hunt for disease susceptibility genes has be- come increasingly automated. one step that has been lacking automated tools is that of connecting distant relatives with the same phenotype into a pedigree for linkage analysis. when the desired pedigree is implicitly part of a large, existing gene- alogy, computer science techniques can be adapted to extract the pedigree or to verify a putative pedi- gree systematically. pedhunter provides this automa- tion. the utility of pedhunter is demonstrated by using it to query a database, agdb, of a large old order amish genealogy. these tools were used to verify and correct parts of a previously published pedigree. the combination of agdb and pedhunter are immediately useful in ongoing genetic studies of this population (e.g., stone et al. ). conversations with geneticists who use the fisher book suggest that they are content when they can find any pedigree to connect a specified set of individuals. there is no reason to believe that the first pedigree found is the best. in contrast, the all shortest paths and steiner criteria that pedhunter of- fers are mathematically rigorous and genetically sensible. methods agdb was created in sybase sql server release . .x of the sybase database management soft- figure minimal (steiner) pedigree. agarwala et al. genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com ware. the functions are implemented using trans- act-sql and c version of open client db-library. pedhunter was developed on unix system v release . running under sunos . . using sun work- shop compiler c . but it is compatible with other unix computers. all of the pedhunter functions are available as executable files that can be used from the command line prompt; agdb is available to sci- entists conducting irb-approved research on the old order amish. the automatic compilation of the fisher book genealogy into agdb was approved by the national cancer institute irb at nih. acknowledgments this research was performed within the division of intramu- ral research (nhgri). we thank dr. robert nussbaum for frequent and active encouragement, for helpful guidance about the conventions of genetics research, and for sugges- tions on this manuscript. we also thank katie beiler and our other amish colleagues and friends in lancaster county. we are grateful to gunther birznieks and ed whitley for assis- tance with sybase. we thank dr. cyril chapman, kevin crawford, and dr. paul mamelka for their help with cyrillic and pedigree/draw. we thank carol machan for providing the raw data files for agdb. the research of k.a.h. was sup- ported by a grant (nih p hg ) from the national cen- ter for human genome research. the publication costs of this article were defrayed in part by payment of page charges. this article must therefore be hereby marked ‘‘advertisement’’ in accordance with usc section solely to indicate this fact. references bairoch, a. and r. apweiler. . the swiss-prot protein sequence data bank and its supplement trembl. nucleic acids res. : – . benson, d.a., m.s. boguski, d.j. lipman, and j. ostell. . genbank. nucleic acids res. : – . chapman, c.j. . a visual interface to computer programs for linkage analysis. am. j. med. genet. : – . cheung, k.-h., p. nadkarni, s. silverstein, j.r. kidd, a.j. pakstis, p. miller, and k.k. kidd. . phenodb: an integrated client/server database for linkage and population genetics. comp. biomed. res. : – . curtis, d. . a program to draw pedigrees using linkage or linksys data files. ann. hum. genet. : – . date, c.j. . an introduction to database systems (vol. ). addison-wesley, new york, ny. dijkstra, e.w. . a note on two problems in connection with graphs. num. math. : – . dyke, b. . pedsys: a pedigree data management system. population genetics laboartory, southwest foundation for biomedical research, san antonio, tx. even, s. . graph algorithms. computer science press, rockville, md. fasman, k.h., s.i. letovsky, r.w. cottingham, and d.t. kingsbury. . the gdb(tm) human genome database. nucleic acids res. : – . fisher family history. . first ed. ( ) compiled by john m. fisher family, published by amos l. fisher. second ed. ( ) edited by janice a. egeland, published by moore clinic, johns hopkins hospital, baltimore, md. third ed. ( ) revised and updated by a committee of fisher descendants (ed. katie beiler) eby’s quality printing, pa. gedcom. . gedcom coordinator- t, family history department, salt lake city, ut . e-mail: gedcom@gedcom.org. ged html: a gedcom to html translator. . eugene w. stark, port jefferson, ny . e-mail: stark@cs.sunysb.edu. url: http://www.gendex.com/ged html. hwang, f.k. and d.s. richards. . steiner tree problems. networks : – . khoury, m.j., b.h. cohen, e.l. diamond, g.a. chase, and v.a. mckusick. . inbreeding and prereproductive mortality in the old order amish. iii. direct and indirect effects of inbreeding. am. j. epidemiol. : – . kruglyak, l., m.j. daly, and e.s. lander. . rapid multipoint linkage analysis of recessive traits in nuclear families, including homozygosity mapping. am. j. hum. genet. : – . lange, k. and r.c. elston. . extensions to pedigree analysis. i. likelihood calculations for simple and complex pedigrees. hum. hered. : – . lange, k., d. weeks, and m. boehnke. . programs for pedigree analysis: mendel, fisher, and dgene. genet. epidemiol. : – . mamelka, p.m., b. dyke, and j.w. maccluer. , nd ed. pedigree/draw for the apple macintosh, population genetics laboratory technical report no. . southwest foundation for biomed. research, san antonio, tx. mckusick, v.a. . the william allan memorial award lecture: genetic nosology: three approaches. am. j. hum. genet. : – . mckusick, v.a., j.a. egeland, r. etheridge, and d.e. krusen. . dwarfism in the amish. i. the ellis-van creveld syndrome. bull. johns hopkins hosp. : – . papdimitriou, c.h. and k. steiglitz. . combinatorial optimization: algorithms and complexity. prentice-hall, englewood cliffs, nj. software for constructing pedigrees genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com polymeropoulos, m.h., s.e. ide, m. wright, j. goodship, j. weissenbach, r.e. pyeritz, e.o. da silva, r.i. ortiz de luna, and c.a. francomano. . the gene for the ellis-van creveld syndrome is located on chromosome p . genomics : – . round, a.p. . schesis: computation in a multipoint linkage analysis program. prog. clin. biol. res. : – . stone, d., r. agarwala, a.a. schäffer, j.l. weber, d. vaske, t. oda, s.c. chandrasekharappa, c.a. francomano, and l.g. biesecker. . genetic and physical mapping of the mckusick-kaufman syndrome. hum. mol. genet. : – . terwilliger, j.d. and j. ott. . handbook of human genetic linkage. the johns hopkins university press, baltimore, md. weir, b.s. . genetic data analysis ii. sinauer associates, sunderland, ma. zelikovsky, a. . a series of approximation algorithms for the acyclic directed steiner tree problem. algorithmica : – . received september , ; accepted in revised form february , . appendix this appendix describes the database design, graphs and related terminology, and method used by pedhunter to solve the minimal pedigree, or steiner pedigree problem. database design each person in agdb is assigned a unique integer, called the program id. utility programs were writ- ten to translate from a fisher id to the program id of the first person in that record, and back from a program id to the fisher id of one record where that person is listed. there are two central tables in the database—the person table and the relationship table. the person table (person table) has informa- tion exclusive to the individuals. the columns of this table are program id, name, birth date, death date, address, gender, special status, number of spouses, and other information. the special status field contains information about whether a person is a twin, triplet, foster child, or adopted child. the relationship table (child parent table) keeps in- formation relating the individuals. it contains an entry for each couple and a list of their children, if any, in terms of their program id specified in per- son table. the columns of child parent table are program id of father, program id of mother, marriage date, and program id of each child. the last field is a list of program ids of each child, if the couple has any. as each row/column combina- tion can have only one value, the list is kept as a string with special delimiters between children. pedhunter contains a utility program for creat- ing generation table, which contains the maxi- mum generation level for each person relative to other persons in the database. the two columns of this table are program id of the individual and maximum generation level. the generation table is used in computations including inbreeding and kinship coefficients. graphs and related terminology a graph is a mathematical representation of objects and the relationship between pairs of objects. some standard graph terminology is defined below and illustrated by references to the graph in figure . the set of objects represented in a graph are called its vertices. if two objects are related, they are joined by an edge. for convenience, the following notation is used: ● v(g) represents the set of vertices for graph g. ● e(g) represents the set of edges for graph g. ● e is u–v if edge e joins vertices u and v and the order of u and v is not important. such an edge is called an undirected edge. ● e is u → v if edge e joins vertices u and v and the edge is ordered from u to v. such an edge is called a directed edge. ● v is in the set v(g) if v is a vertex in graph g. ● e is in the set e(g) if e is an edge in graph g. a graph is a directed graph if its edges are di- rected edges; otherwise, it is an undirected graph. a pedigree can be represented by a graph that has a vertex for each person and an edge for each child– parent pair among the persons in the pedigree. sometimes it is desirable to make the graph directed by directing each edge from the vertex for parent to the vertex for child. for example, the pedigree (fig. ) that is used later as an example, can be repre- sented as in figure . it is useful to view a pedigree as a graph because it traces the passage of genetic information over generations and provides a conve- nient mathematical representation. a graph repre- senting a pedigree is called a pedigree graph. for the pedigree graph in figure , the set of vertices v(g) is { , ,. . ., }, and there are directed edges, includ- ing → and → . a path from vertex u to vertex v in g is an alter- agarwala et al. genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com nating sequence of vertices and edges of g, begin- ning with u and ending with v, such that no vertex is repeated and every edge joins the vertices imme- diately preceding it and following it. a path p con- necting u to v is denoted p = u, u , u , . . ., un, v, where u , u , . . ., un is the sequence of other vertices on the path. for example, → → → is a di- rected path in the graph of figure . the undirected sequence — — — — — — — is a path in the undirected version of this graph, but not in the directed version. the two paths are denoted as p = , , , , and p = , , , , , , , , respectively. a directed path p from u to v in a pedigree graph traces one way that v receives genetic material from u. the vertices on p are also a (partial) list of ances- tors of v. absence of a directed path from u to v indicates that u is not an ancestor of v. hence, there is no way that v can receive genetic material from u; for example, in figure , person does not receive genetic material directly from person , although they have a common ancestor. a pedigree graph g connects a set of individuals s if there is a vertex in v(g) for each individual in s, and in the undirected version of the graph, there is a path between each pair of vertices representing people in s. the pedigree of figure is connected. geneticists usually use the term pedigree to mean a set of parent–child relationships that induce a pedi- gree graph whose undirected version is connected. therefore, any formal definition of pedigree construc- tion should require that the output pedigree induce a connected pedigree graph. however, this require- ment does not specify the output pedigree uniquely, and different specifications of pedigree construction are useful for different purposes. a cycle is a path that begins and ends at the same vertex. if a graph has no cycles, it is called acyclic. any directed pedigree graph is acyclic, as the edges are directed from parent to child, and it is biologi- cally impossible to have a cycle in this setting. the term loop is not used here because formally a loop in a pedigree is a cycle in a more complicated, undi- rected representation called a marriage graph (lange and elston ). for example, a first-cousin mar- riage with offspring leads to a loop in the undirected marriage graph but not to a cycle in the directed pedigree graph. the length of a path is the number of edges in the sequence defining the path; for example the length of path p = u , u , u , . . ., un is n. a path from vertex u to vertex v is a shortest path if it has mini- mum length of any path from u to v. the length of a shortest path from u to v is also called the distance d(u,v) from u to v. a shortest path from u to v makes the fewest assumptions about how an allele could have passed from u to v. by the principle of parsimony, a hy- pothesis that a disease-causing allele passed from u to v requires consideration of all shortest paths from u to v. this observation motivates the asp pedigree construction query. if g and h are graphs, then h is a subgraph of g if and only if v(h) is a subset of v(g) and e(h) is a subset of e(g). given a pedigree graph g that connects a set of people s, it is sometimes useful to find a subgraph of g that still connects s. one such situation is when the pedigree graph has too many edges to be drawn comprehensibly. a subgraph of g that contains minimum number of vertices and connects s is called a minimal subgraph, and the problem of find- ing a minimal connected subgraph is called a steiner tree problem in the computer science literature. therefore, a minimal pedigree subgraph that con- nects specified individuals is called a steiner pedigree. pedhunter finds steiner pedigrees with the query called minped. steiner pedigrees are useful for draw- ing and for linkage analysis when the pedigree graph has too many edges. pedhunter’s method to construct steiner pedigrees and some background from the computer science literature are explained in the next section. steiner pedigree steiner tree problems are widely studied due to ap- plications such as designing road networks and vlsi chips. a survey of steiner tree problems can be found in hwang and richards ( ). many steiner tree problems are quite intractable computationally, but pedigree graphs have special features that may make the problem easier. pedigree graphs are acy- clic, and the vertices that must be included typically represent children at the bottom generation. such vertices that have no outgoing edges are called ter- minals. in a recent paper zelikovsky ( ) studied the steiner tree problem for all directed acyclic graphs with the constraint that the vertices to be connected are terminals. this problem has applica- tions outside genetics. zelikovsky showed that this version of the steiner tree problem is intractable in a formal sense and gave an approximation algorithm for the problem. an approximation algorithm finds a subgraph that contains all of the prescribed vertices whose size is bounded relative to the size of the minimal subgraph, where the size of a graph is the number of vertices in the graph. however, both the performance ratio and the computation time of this software for constructing pedigrees genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com approximation algorithm grow exponentially with the length of the longest path in the graph. instead of zelikovsky’s approximation algo- rithm, pedhunter uses an algorithm that finds an exact smallest subgraph (subpedigree) as measured by the number of vertices (individuals). the ap- proximation algorithm may be inadequate for the subsequent linkage analysis. the exact algorithm applies a general technique called branch and bound, which is commonly used to explore exponential-size search spaces in intractable search problems (papdimitriou and steiglitz ). branch and bound works well in problems where one can quickly get to a good solution. the weight of the known solution can be used to eliminate many partial solutions whose weight is larger (for a minimization problem such as steiner tree). when a partial solution is eliminated, all full solutions con- taining that partial solution are implicitly elimi- nated also. branch refers to steps that expand the current partial solution by branching in the search space; bound refers to using a known solution as a bound. branch and bound algorithms start by finding an initial solution, keep track of the current best solution, and probe other solutions until it is pos- sible to determine that the current solution is at least as good as any other solution. branch and bound algorithms typically use retracing or back- tracking, which means that part of the current (par- tial solution) is removed, and the algorithms pro- ceed to consider other solutions that do not contain the removed part. to describe a specific branch and bound algorithm one must describe ( ) what makes one solution better than another? ( ) how to find an initial solution? ( ) how to probe all solutions? for the minimal pedigree problem, the algorithm solves the three basic questions as follows. comparing solutions the number of edges in the solution is the measure for comparing solutions and the solution with fewer number of edges is better. the number of edges in a solution is called the cost of the solution. the prob- lem statement asks to minimize the number of ver- tices, but any connected graph with no cycles that has e edges, must have exactly e+ vertices (even ). therefore, minimizing number of edges is equivalent to minimizing number of vertices. the algorithm transforms the initial steiner problem where all edges have weight to a related problem where edges may have larger weight. in the weighted version, the cost of a solution is the sum of weights of edges the solution contains. initial solution the steps for finding an initial solution are as fol- lows: . start with the specified set of individuals as the vertices in the solution. . for each vertex v in the solution, except the root, that is not connected to a parent, choose a parent p that is present in the input graph. if the parent chosen is not in the solution, add a vertex for p in the solution. add the edge p → v to the solution. . repeat step until there is only one vertex in the solution that is not connected to a parent. that vertex is the root of the pedigree. probing all solutions every edge in the input graph g is either in the cur- rent solution or it is out. the edges are numbered , . . .,|e(g)| such that edge u → v is preceded in the order by all edges that are incoming into u. such an order is possible for pedigree graphs because they are acyclic (even ). consider the edges in order e = , , , . . . . add edge e to the current solution unless it ( ) creates an undirected cycle or ( ) creates a (partial) solution with higher cost than the current best. when the current partial solution is a full solution (detectable by testing whether number of edges equals number of vertices minus ), compare its cost to the current best. there are four cases that need to be specified further. . if the current partial solution is a full solution and it has lower cost than the previous best, re- place the best solution and retrace decisions back to the last edge added in. omit that edge and proceed to look for better solutions. . if the addition of e creates an undirected cycle, then omit e and proceed to add more edges. . if the addition of e creates a partial solution with higher cost than the current best full solution, then as in case , retrace back to the last edge added in. omit that edge and proceed forward. . if the last edge was considered and the solution is not full, then as in case , backtrack to the last edge added in, omit it and proceed. all solutions have been considered when retracing returns all the way past edge . agarwala et al. genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com the worst-case computation time of the algo- rithm is exponential in the number of edges in the input graph. the computation time is lowered sub- stantially by reducing the size of the graph to be considered in the branch and bound algorithm. the graph is reduced by three transformations that are applied repeatedly in any order until none of them can be applied: . replace paths consisting of k + vertices with one incoming edge and one outgoing edge with a single edge of cost k. this is valid because in the original graph, either all k edges must be in- cluded or excluded from a solution. . delete unselected terminals with one incoming edge. this is valid because they cannot serve to connect the selected terminals. . delete edge u → v if there is another path from u to v. this is valid because the input graph is an all shortest paths pedigree. therefore, all paths from u to v have the same cost. the vertices on the multiple-edge path cannot lead to a worse solu- tion than using the single edge u → v and might lead to a better solution. the first transformation introduces edges whose weight is > . the third transformation can apply only after the first transformation has created edges of weight > because the cost of all paths from u to v is the same. the cost of a solution in the transformed graph is the sum of costs of all edges in the solution. the transformation is reversed to pro- duce the output steiner pedigree. software for constructing pedigrees genome research cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/ http://www.cshlpress.com . /gr. . . access the most recent version at doi: : - genome res. richa agarwala, leslie g. biesecker, katherine a. hopkins, et al. county lancaster genealogies and an application to the old order amish of software for constructing and verifying pedigrees within large license service email alerting click here.top right corner of the article or receive free email alerts when new articles cite this article - sign up in the box at the https://genome.cshlp.org/subscriptions go to: genome research to subscribe to cold spring harbor laboratory press cold spring harbor laboratory press on april , - published by genome.cshlp.orgdownloaded from http://genome.cshlp.org/lookup/doi/ . /gr. . . http://genome.cshlp.org/cgi/alerts/ctalert?alerttype=citedby&addalert=cited_by&savealert=no&cited_by_criteria_resid=protocols; . /gr. . . &return_type=article&return_url=http://genome.cshlp.org/content/ . /gr. . . .full.pdf https://genome.cshlp.org/subscriptions http://genome.cshlp.org/ http://www.cshlpress.com wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ 'howard hughes medical institute and department of physiologyand biophysics, the university of iowa college ojmedicine, iowa city, iowa , usa fondationjean- dausset-ceph, paris, france git~&thon, evry, france university of texas, howard hughes medical institute, biopolymer facility, dallas, texas , usa jinserm u , paris, france ~ e n r y ford hospital, division of clinical and molecular genetics, c.f.p. , detroit, michigan , usa correspondence should be addressed to k.p.c. and j.s.b. +thefirst three authors contributed equally to this work. p-sarcoglycan: characterization and role in limb-girdle muscular dystrophy linked to q leland e. liml*, franck ~ u c l o s ' " , odile broux *, nathalie our^^, yoshihide sunadal, valerie allamand , jon ~ e ~ e r l , isabelle ~ i c h a r d ~ , carolyn moomaw , clive slaughter , fernando m.s. tome , michel fardeau , charles e. jackson , jacques s. ~ e c k m a n n ~ > ~ & kevin p. campbell p-sarcoglycan, a kda dystrophin-associated glycoprotein, is an integral component of the dystrophin-glycoprotein complex. we have cloned human p-sarcoglycan cdlva and mapped the p-sarcoglycan gene to chromosome q . pericentromeric markers and an intragenic polymorphic ca repeat cosegregated perfectly with autosomal recessive limb- girdle muscular dystrophy in several amish families. a thr-to-arg missense mutation was identified within the p-sarcoglycan gene that leads to a dramatically reduced expression of p-sarcoglycan in the sarcolemma and a concomitant loss of adhalin and dag, which may represent a disruption of a functional subcomplex within the dystrophin- glycoprotein complex. thus, the p-sarcoglycan gene is the fifth locus identified (lgmd e) that is involved in autosomal recessive limb-girdle muscular dystrophy. the dystrophin-glycoprotein complex (dgc) - is a large oligomeric complex of sarcolemmal proteins and glycoproteins, consisting of dystrophin, a large f-actin binding intracellular syntrophin, a kda intracellular proteins, ; adhalin, a kda transmem- brane glycoproteinlo; a kda transmembrane glyco- protein doublet (p-dystroglycan and a b) , ,"; a kda transmembrane glycoprotein; a kda trans- membrane protein; and a-dystroglycan, a large extra- cellular laminin-binding gly~oprotein"-'~. together, the dystrophin-glycoprotein complex acts as a struc- tural link between the cytoskeleton and the extracellu- lar matrix, and is believed to confer stability to the sarcolemma and protect muscle cells from contrac- tion-induced damage and necrosis . the dgc has been implicated in several forms of muscular dystrophy. in duchenne muscular dystrophy (dmd), mutations in the dystrophin gene cause the complete absence of dystrophin and a dramatic reduc- tion of its associated glycoproteins at the sarcolemma resulting in severe muscular dystrophy , . in the milder becker muscular dystrophy, mutations in dystrophin result in the ~ r o d u c t i o n of a dvsfunctional vrotein . more recently, some cases of severe childhood autoso- ma recessive muscular dystrophy (scarmd or lgmd d) were shown to be caused by missense and null mutations in the adhalin genel >l , which result in the reduction or absence of adhalin at the sarcolem- ma . non-fukuyama congenital muscular dystrophy (cmd) has recently been linked close to the merosin locus on chromosome q , , which is likely to be responsible for this disease. thus, in these muscular dystrophies, mutations in one component of the dgc cause the disruption of the complex and consequently lead to the dystrophic process. the limb-girdle muscular dystrophies (lgmds) represent a clinically heterogeneous group of diseases which are characterized by progressive weakness of the pelvic and shoulder girdle m u ~ c l e s ~ ~ , ~ ~ . these disor- ders may be inherited as an autosomal dominant or recessive trait, the latter being more common with an estimated prevalence of i in , indi~idua .s~~. several genes have been implicated in the aetiology of these disorders. the autosomal dominant form, lgmdla, was mapped to q -q (ref. ), while four genes involved in the autosomal recessive forms were mapped to chromosomes ~ -p (lgmd b) , q (lgmd c) , , . (lgmd a) , and q -q . (lgmd~d)". the genes responsible for lgmd d and lgmd a have been identified: adhalin and muscle-specific calpain ( c a n p ~ ) ~ ~ , respectively. cases of autosomal recessive limb-girdle muscular dystrophy among members of the old order of amish of northern and-southern indiana were described by iackson and carev and tackson and strehler . most of the families of these communities are interrelated by multiple consanguineous links and common ancestry which can be traced to the th and th century in the canton of bern. switzerland . in view of the high u degree of consanguinity and the similar clinical pre- sentation of all amish lgmd patients, the demonstra- tion of genetic heterogeneity within this community was unexpected . although families from northern indiana were shown to carry the same r q calpain mutation , involvement of this locus was excluded in nature genetics volume november article ( u ~ o u t o ( u o t t t u t o m a o t o o c c % ~ ~ m ~ t a t c t o l t ~ d s u z t b z s o l l r t x ~ v s d u o q ~ t c c m c c t c ~ t m ~ t ~ ~ t t t c m u t c v i b p l x x s t v g o r r n z n l v i w-cmcuocctattgttttt---twda- t a n n q p i v i q q o t @ x l s v z n w t t c t a t t h c m g - x o o c a t ~ t t t t i ~ c c ( w w l c t - w x t ~ i t s d i ~ u q ~ ~ ~ p r t _ p ? a t c t t a t t ~ g s t a t g a m c t c a t ~ g ~ t c a t t t o c c m g t o ( u m ~ t i l ~ s t d x z t b e ~ b l p s g v x s ttommtt-ocitctxt~ttacugcmtoctxuot~tttlut l u v q x a s t l r i t s p a t s d l n at-ttmtgoocgtgctattotocot-tomogtgtattuttatg- i x v d g r a i v r o n l g v r i m g k xutiomtttuutgoat tmtato(ugttluooco umuotatutccta t i l t b m o o n m l l x a z n i i i l mtwtctgtgatgtuouccacccocctacccaottcctcuot-cwg w g s v n v s t t r l p s s s s g d q l tmitqotolctgffitacdctacuoctct~totmoctgitw(ucoctctt~ o s o d y v r x x l c u c a d g t l i x s q t o c u g t m c c m c u o m c a t o o o c t d c c m a t c t ~ c c c c t g t ~ c t v q v t s q n m g c q i s d n p c g n t ulttiiljiuimcccu(uggtcmcuuntttatatcttb)rctt cttttttatou b stop touutcattgtttttauwtttgtdltmctututtattttmtooca~ fig. human p-sarcoglycan cdna and translated amino acid sequence. human p-sarcoglycan cdna is shown with the deduced amino acid sequence below. the predicted transmem- brane domain is indicated by the shaded box. three consensus sites for n-linked glycosylation are indicated by asterisks, *. the intracellular consensus phosphorylation site by protein kinase c or casein kinase ii is indicated by a #. peptide sequence frag- ments identified in the amino acid sequence are underlined. the amino acid that is mutated in the amish limb-girdle muscular dystrophy patients is circled. amish families from southern indiana, as none of the examined patients from these cohorts carried this mutation, even in a heterozygous state (data not shown). furthermore, the role of known lgmd loci, as well as several other candidate regions involved in other neuromuscular disorders, were all excluded . these results thus implied the existence of yet another locus, lgmd e, involved in autosomal recessive limb- girdle muscular dystrophy. here we report the cloning and characterization of human p-sarcoglycan, a kda component of the dys- trophin-glycoprotein complex, and demonstrate its involvement in lgmd e. we show that p-sarcoglycan colocalizes with the dgc at the sarcolemma and is expressed ubiquitously, but predominantly in muscle. we report the primary localization of this gene to chromosome q , the identification of an intragenic polymorphic ca repeat, and linkage of lgmd e to the intragenic p-sarcoglycan microsatellite in southern indiana amish lgmd families. we have also identified a missense mutation in the p-sarcoglycan sequence, which is present in a homozygous state in all patients, and is apparently responsible for the muscular dystro- phy in all chromosome -linked amish lgmd families screened. by immunofluorescence of affected individ- uals, we demonstrate a dramatic reduction in the level of the p-sarcoglycan protein at the sarcolemma, as well as a concomitant reduction of adhalin and the kda dag, thereby disrupting the integrity of the dgc. together, these data demonstrate that a defect in - sarcoglycan is responsible for lgmd e. because of this protein's tight association with other components of what has been called the sarcoglycan complex , which includes adhalin, this kda protein, the kda dag, and the kda dap, the name 'p-sarcogly- can' has been proposed. this name was mutually agreed upon by us and the authors of an accompany- ing manuscript which describes similar results . under this nomenclature, a-sarcoglycan becomes syn- onymous with adhalin. p-sarcoglycan cdna a n d primary structure the dystrophin-glycoprotein complex (dgc) was identified in based on the ability of dystrophin to be retained on a wheat-germ agglutinin when p-dystroglycan, a kda dag, was cloned ', the translated peptide sequence was compared with peptide sequence fragments obtained from the kda band of the purified dgc. of these fragments, only one was found in the primary structure of p-dystrogly- can. this suggested the presence of another protein of similar molecular weight, consistent with the observed kda doublet in the dgc , . to further investigate this protein, we searched the genbank database of expressed sequence tags (dbest) with the unidentified peptide sequences using the tblastn search pro- gram . several ests encoding peptide sequence frag- ments of the p-sarcoglycan protein were identified and isolated from a normalized human infant brain cdna library constructed by dr. bento soares et ale '. clones and , from which ests were generated, were received from the image consortium at the lawrence livermore national laboratories. the larger of these clones, , was fully sequenced on both strands. in addition, we isolated clones from a xzapii human skeletal muscle cdna library using the - bp insert of clone as a probe. sequence analysis revealed a single open reading frame that encodes a protein with a predicted molecular weight of , da (fig. ). several peptide fragments obtained from sequencing of the kda doublet were found in the primary structure of the protein. there was no other significant homology with any other known protein or functional protein domains in the database. addition- ally, our sequence data provided evidence of alternate polyadenylation. two distinct poly(at) tails were iden- tified, one that is about bases downstream of the stop codon in brain, and one that is approximately kb downstream in skeletal muscle (data not shown). hydropathy analysis of the amino acid sequence revealed a single transmembrane domain and n d func- tional signal sequence at the n terminus, despite the presence of eight hydrophobic alanine residues follow- ing the initiator methionine. thus, the small n-termi- nal domain of the protein is predicted to be intracellular, whereas the large c terminus is extracel- lular. this membrane topology is consistent with the location of the three putative n-linked glycosylation nature genetics volume november article a s . . . . - . . . . . . . . . . - . . . . . ~ ~ . . . . . . . c * . . , - m. ' . . . . - . . .- - ~. - . . . . - - . . . . . . . . . . . ' probe - . . ' -: . . . .- ~ . . - . . . . . . . . . . . . . . . , "' . . . . . . f .) .. " full-length probe adult fetal fig. expression of -sarcoglycan in adult and fetal tissues. rna blots containing pg of poly(a+) rna from the indicated human adult and fetal tissues were hybridized as follows. molec- ular size in kb is indicated on the left. a, northern blots hybridized with a -bp ' segment of the p-sarcoglycan cdna. b , northern blots hybridized with the -bp insert of p- sarcoglycan clone . sites. all of which are c-terminal to the transmem- brane domain. there are also five extracellular cysteine residues which may form disulfide bonds. there is also o n e potential intracellular consensus site for phospho- rylation by protein kinase c o r casein kinase i at ser . t h e predicted membrane organization is similar to that of p-dystroglycan a n d adhalin, both of which have single transmembrane domains a n d large extra- cellular a n d short intracellular domains' ,' '. t i s s u e d i s t r i b u t i o n of p - s a r c o g l y c a n to determine the tissue-specific expression of p-sarco- glycan, we performed rna hybridization analysis. human adult a n d fetal multiple tissue northern blots were probed in two different ways: once with a pcr fragment encompassing bases - of the p-sarco- glycan coding region, and a second time with the b p insert of clone described above (fig. ). t h e predominant transcript is approximately . kb in length; however, there are also weaker signals of . a n d . kb. seauence data demonstrating evidence of " alternate polyadenylation can account for the smallest and largest transcript. p-sarcoglycan rna is present in all tissues, a n d is particularly enriched in skeletal a n d cardiac muscle. this pattern of expression is different from adhalin, which is expressed only in muscle tis- ~ u e ~ ~ ~ ' ~ , but is similar to dystroglycan, which is ubiqui- tously e x p r e ~ s e d ' ~ . interestingly, the northern blot results are different between t h e two probes. when probed with a -bp pcr fragment corresponding to nucleotides - , the signals in the fetal liver a n d adult pancreas lanes are weak o r absent (fig. a). however, when probed with the larger clone, which contains all the coding region and nearly b p of ' untranslated region, these signals are significantly stronger, particularly in the adult pancreas (fig. b). this suggests that the ' e n d of the coding region is alternatively spliced among different tissues. ed a glutathione-s-transferase (gst) fusion protein (fp-i) containing residues c-terminal to the trans- membrane domain (fig. ). anti-fp-i polyclonal antibodies. ~ r o d u c e d in rabbits. were used to detect b- , , sarcoglycan in isolated membranes a n d purified dgc. t h e antibody specifically recognizes a kda protein i n both crude sarcolemlna and purified dgc, but does not recognize gst alone (fig. b). sheep polyclonal antibodies produced against a peptide fragment (residues - ) of the p-sarcoglycan protein also rec- ognized a kda protein in the purified dgc (data not shown). identification of p-sarcoglycan during the purification of the dgc demonstrates that p-sarcogly- can is an integral component of the dgc. to determine the subcellular localization of b-sarco- glycan by immunofluorescence, serial transverse cryosections of control human biopsied skeletal nius- cle were immunostained with anti-fp-i antibody pre- absorbed with gst, as well as with antibodies against other components of the dgc including dystrophin, p-dystroglycan, syntrophin, adhalin, and kda dag. as shown in fig. , the anti-fp-i antibody labelled throughout the entire sarcolemma a n d showed colo- calization of p-sarcoglycan with other components of the dgc. however, immunofluorescence from d m d muscle biopsy samples demonstrated a loss of the p- sarcoglycan protein at the sarcolemma along with all other components of the d g c (data not shown). l o c a l i z a t i o n to c h r o m o s o m e q primers were designed to amplift a fragment of the h u m a n p-sarcoglycan gene from a panel of hunian- rodent somatic cell hybrids containing various combi- nations of h u m a n chromosomes. restriction digests of the amplified product with taqi specifically cleaved the human allele, a n d allowed assignment of the p-sarco- glycan gene to chromosome . to f i ~ r t h e r narrow the gst i i i fp-i fig. lmmunoblot analysis of p-sarcoglycan. a, schematic model of human p-sarcoglycan cdna. the transmembrane domain is shaded; three sites of potential n-linked glycosylation sites are indicated. a schematic representation of fusion protein i (fp-i) is shown below. b , nitrocellulose transfer of sds-page gel stained with anti-fp-l antibody. membrane contains the fol- lowina: kci-washed microsomes (mic), crude rabbit skeletal muscie sarcolemma (csm). duriied 'dvstro~hin-a~vcoorotein l o c a l i z a t i o n of p - s a r c o g l y c a n p r o t e i n complex (dgc), f ~ s i o n ' ~ r o t i i n i (fp-i), anh glutathige-~-trans- ferase (gst). anti-gst antibodies were removed prior to stain- to confirm that the 'ioned represents the ing. molecular weight in kda is indicated on the left. arrow on kda dystrophin-associated glycoprotein, we construct- right indicates position of p-sarcoglycan protein. nature genetics volume november article dystrophin d-dystroglycan syntrophin sarcoglycan gene between nucleotides and of search using the highly' informative microsatellit& fig. lmmunohistochemical analysis of p-sarcoglycan in normal and amish lgmd e muscle. skeletal muscle biopsy samples were stained with antibodies against dgc components as described in the methods. dys, anti-dystrophin; p-dg, anti p- dystroglycan; dag, anti- kda dystrophin-associated glyco- protein. control, normal subject; patient, amish lgmd e patient. bar, lim. chromosonlal region, the same process was used t o analyse dna isolated from human-rodent somatic cell hybrids containing various fragments of cl~romosome (ref. ). p-sarcoglycan fragments could be amplified only from hybrids containing the region p -q . , which overlaus the centromere. we isolated two cosmids spanning approximately kb of the human p-sarcoglycan gene by screening a human chromosome cosmid library. the smaller cosmid, which contained a . kb insert, was used as a probe for fluorescence i n s i t u hybridization (fish), and resulted in the specific labelling of the centromere o n the long arm of chromosome , corresponding to band q (data not shown). we searched for polymorphic microsatellites within the p-sarcoglycan gene. southern blots of restriction fragments of the c o s ~ n i d s and genomic pcr fragments were probed with oligonucleotides encoding a dinu- cleotide (ca) repeat and several tetranucleotide repeats. only hybridization with the ca repeat oligonucleotide was detected. sequencing subsequent- ly located a novel ca repeat within a n intron of the p- fig. recombinant haplotypes in w - <\i-- lgmd e families. the ordered marker loci m , r c r u have arbitrarily been represented as =. : - - ; q ; s s z $ d ; equidistant. loci bracketing the smallest ., x i " - - , .$ - z a g ~~~~~~ interval defined by recombination events ( ua~i-a -a : z = = --- --- are noted in larger letters. the intragenic ,.::::: d-sarcoalvcan microsatellite is underlined. ..w.v -- s o l ~ d squares, affected lndlvlduals, open :;;;el, = - : : : - : = - l- squares, healthy carrlers the numbers .,,,,,,, -; i --; : indicate the family and the individual. a , \ * r a --ct . ...*~- - -i- chromosomes segregating with the dis- i::::::: i-?<--= - = - ease allele or the normal allele are coded n a l b l l ~ --*---- as solid or open circles, respectively. thin horizontal lines represent the recombina- tion interval. uninformative markers are coded by a line in place of a circle, and nongenotyped markers are left blank. the last two recombinants define the critical interval for the location of the morbid locus. a, previously described amish lgmd fami- lies. b, additional southern indiana amish families. in autosomal recessive disorders, affected individu- als from consanguineous families often show homozy- gosity by descent at the region surrounding a disease locus . haplotypes were ~nanually constructed for the chromosome markers assuming a minimal number " of recombinations. we identified a unique carrier hap- lotype segregating within all the southern indiana amish population (data not shown), suggestive of a unique founder effect, though different from the o n e found in the northern indiana and pennsylvania amish lgmd a families . six affected and one unaf- fected offspring showed informative crossovers (fig. ). this identified d s and . ' as newr flanking loci which define a region of approximately cm, based o n analyses of ceph reference families". five additional southern indiana lgmd families also showed linkage to this new locus, thereby increas- ing the number of informative meioses. a maximum lod score of . at = . was obtained with marker d s (table in). genotyping of these families with new microsatellite markers allowed a further narrow- ing of the lgmd e interval, flanked by markers d s and d s (fig. b ) . ho~nozygosity map- ping and reconstitution of historical crossing over events suggested that the lgmd e interval is flanked by markers d s and d s (data not shown). based o n physical maps for chromosome (ref. and d. cox. versonal communication^, ceph yacs , l spanning this region were used to localize the p-sarco- glycan gene inside the lgmd e interval, between markers d s a n d d s . genotyping of the intragenic microsatellite in lgmd e families yielded a lod score of . at = . (table l b ) . this lower lod score, as compared t o d s , is d u e t o a lower observed heterozygosity and hence lower inforrnativity of the intragenic marker. identification of a mutation in amish patients \ve performed northern blot analysis o n total rna isolated from skeletal muscle biopsies of two affected siblings in these families to determine whether p- sarcoglycan mrna size or abundance were affected. the major muscle p-sarcoglyca~~ transcript ( . kb) was present at normal levels and size in both affected sibs (one of which is shown in fig. ) c o ~ n p a r e d to an nature genetics volume november article -- table ~ i n k a ~ e of lgmdpe families to chromosome markers ), and laminin a chain (not shown) were present at comparable levels with control muscle. however, the z at recombination ( ) of immunostaining of -sarcoglycan was greatly onelodsupport decreased, with- a concomitant-reduction of adhalin marker o.oo o.o . o. ° . (') and the kda dag. the apparent differences in mus- a -m . . . . . ( . ) , ~ . cle cell size between control and ~ a t i e n t samples is due -m . . . . . ( . ) - to cell hypertrophy associated with dystrophic changes. -- . . . . . ( . ) . - . d i s c u s s i o n limb-girdle muscular dystrophy in the amish of indi- ana was first described over years ago ', but only recently has the nature of the disease been closely stud- ied. the lgmd in the northern indiana amish popu- lation is caused by mutations in calpain , a cysteine proteinase that niay be involved in muscle protein pro- cessing or signal t r a n s d u ~ t i o n ' ~ . however, analysis of southern indiana amish revealed no linkage to this locus '. the genetic heterogeneity of lgmd in this population was unexpected, because of evidence of common ancestry and multiple consanguineous mat- ings within these populations. the human cdna of p-sarcoglycan, a component of the dystrophin-glycoprotein complex, has now been cloned. the identity of this clone is confirmed by the identification of peptide sequences in the primary c t r mutation -- - - - - - a, maximum pairwise lod scores and their corresponding recombination fractions with one-lod support intervals obtained in ten southern indiana families between the lgmd e locus and chromosome markers. b, the intragenic microsatellite. c, the ti r mutation. marker loci are listed according to their order on the regional map of chromosome . unrelated control. this strongly suggested that the causative mutation was most likely to involve a small deletion, insertion, or base substitution. fragments of the p-sarcoglycan cdna were ampli- fied following reverse-transcription from total rna prepared from biceps brachii muscle biopsies of these two affected sibs. sequencing the rt-pcr products structure, recognition of a kda protein in purified dgc by a p-sarcoglycan-specific antibody, and sar- colemrnal colocalization of this protein with other components of the dgc. the predicted molecular weight of this translated product is , da. experi- ments with endoglycosidase f indicate that the dis- crepancy between the predicted and observed revealed a single c to g transversion at nucleotide in both patients in a homozygous state (fig. ). the codon change is aca to aga and results in a thr-to- arg substitution at residue (t r). segregation of this mutation was assessed in this family and in other amish lgmd e families by sequencing and 'touchdown' pcr". results showed perfect cosegregation of this missense mutation with the disease in all southern indiana amish families test- molecular weights is primarily due to the glycosylation of this ~ r o t e i n (data not shown). the p-sarcoglycan gene has been mapped to chro- mosome q , the same region to which we have linked lgmd in the southern indiana amish. in agreement with the common haplotype displayed by these families around the disease locus, a mutation common to all southern indiana amish lgmd e patients tested has been found. this suggests that a unique founder effect may account for lgmd e in this population, as has already been observed in the lgmd a families of the northern indiana and penn- ed, as expected from the common haplotype at this locus (fig. ). to exclude the possibility that this mis- sense mutation might be a polymorphism, unre- lated chromosomes taken from the ceph reference families were tested; none showed this mutation, nor did any northern indiana lgmd a amish patients (data not shown). linkage analysis with the mutation was performed (table l c ) . a maximum lod score of . at = . a go" "' ,.c‘ b * control + s a i i c * . g t " l c i , c a . s r c * . . * r t , . * patlent between the mutation and the disease in these amish families was obtained. in addition, assuming complete linkage disequilibrium, the calculated lod scores are significantly increased (data not shown), further con- firming that the mutation in p-sarcoglycan is responsi- ble for the disease in these patients. fig. : northern blot and sequence analyses of -sarcoglycan gene in family a . a, an r n a blot containing pg of human skeletal muscle total r n a per lane from an unrelated control and an affected member of family a hybridized with a human p- sarcoglycan cdna probe, representing nucleotides to , and exposed overnight. b, direct nucleotide sequence determi- nation of reverse-transcribed total r n a representing nucleotides to performed on the same individuals described above and on another affected member of the family a . arrowheads indicate the position of the mutation. both affected members of family a showed the same mutation. p-sarcoglycan deficiency in lgmd e m u s c l e skeletal muscle biopsy specimens from the two patients described above were examined by immuno- fluorescence to test the effects of the t r mutation on p-sarcoglycan expression. serial frozen sections were stained with antibodies against p-sarcoglycan (anti-fp-i) or other dgc components as described above. dystrophin, p-dystroglycan, syntrophin (fig. nature genetics volume november article explain the long survivability a n d slow disease progres- sion of affected individuals. in the future. it will be interesting to determine whether patients with null mutations exhibit anv svlnvtoms related to the trunca- , , . tion of the p-sarcoglycan protein, particularly in the nonmuscle tissues in which the protein is also known to be expressed (fig. ). a feature colnmon t o lgmd d and lgmd e is a selective absence of p-sarcoglycan, adhalin, a n d the kda dag at the sarcolemma. t h e close association of these proteins had previously been s ~ g g e s t e d ~ ~ , ~ ~ , ~ ~ , a n d recent immunoprecipitation experiments have further demonstrated the existence of a subcomplex composed of these proteins within the dgc ( u n p u b - lished data). the loss of all cotnponents of this sub- complex in both lgmd d and lgmd e indicates that mutations in o n e component can affect the stabili- ty o r targeting of other components, further support- ing the hypothesis that the missense mutation found in the southern indiana amish is the cause of lgmd, since this mutation affects the function of not just p- sarcoglycan, but adhalin and kda dag as well. this also suggests yet another mechanism by which lgmd could occur, through a mutation in the kda dag gene. the precise function of p-sarcoglycan and its sub- complex as a whole is currently unknown. in duclienne muscular dystrophy, disruption of the dgc causes the loss of a functional link between the extra- cellular matrix a n d the cytoskeleton, and may result in sarcolemmal instability a n d greater susceptibility to stress-induced damage. it is likely that the b-sarcogly- fig. illustration of the segregation of the thr to arg substitution in the southern indi- canladhalinl kda ~ a g complex plays a similar h e ana lgmd e amish population. a, schematic representation of 'touchdown' pcr in maintaining the structural and functional integrity amplification. products of -bp and -bp are produced using primer r cou- of the cell membrane. in the bio . cardiotnvo~athic - . , . pled with primer m l and primer t coupled with primer m , respectively. primer hamster, a deficiency of this subcomplex is apparently sequences are given in the methods. b, family a pedigree with affected and unaf- fected individuals indicated by closed and open symbols, respectively. c, duplex for the a n d c a r d i o m ~ o - deposit of "touchdown" pcr amplification products. d, chromosome haplotypes pathic changes in this dystrophin and a- segregating within the family. disease-bearing chromosomes are boxed. ca t repre- dystroglycan are not tightly associated with the sents the intragenic microsatellite. sarcolemma, indicating that disruption of the sarcogly- sylvania amish. t h e perfect cosegregation of the muta- tion with the disease trait within the lgmd e amish population a n d lack of the mutation in over a hundred control c h r o m o s o n ~ e s is strong evidence to support the hypothesis that this base change is responsible for the disease. these genetic data, together with o u r bio- chemical results, demonstrate the role of p-sarcoglycan i n the aetiology of this disorder. as yet, however, the functional importance of the region in which the mutation occurs has not been defmed. bonnemann et al. have cloned the cdna of the same protein, and their results are reported in a manuscript published concurrently with this . they have identi- fied a compound heterozygote with mutations result- ing in significant truncations of p-sarcoglycan. this patient's clinical presentation is much niore severe than that of the amish lgmd e patients, indicating that the ~ mutations in this patient produce a much more deleterious effect. this pattern of severity, com- pared to the milder clinical course of the amish patients, is similar to that seen in the spectrum of adhalin mutations that cause l g m d ~ d i ' s ~ ~ . thus, the presence of a ~nissense rather than a nonsense muta- tion in the southern indiana amish population could can cotnplex can lead to the d e ~ t ~ b i l i z a t i o n of the dys- troglycan complex as well " beca~tse n o mutations have been found in the adhalin cdna o r mrna of the cardiomyopathic hamster ', mutations in p-sarcogly- can may be responsible for the loss of this cotnplex and the resulting phenotype. it is also possible, liowe\ler, that p-sarcoglycan alone serves an important function- al role in nonmuscle tissues as well as in muscle, as suggested by the presence of p-sarcoglycan transcript in all tissues studied. because the pattern of adhalin expression is different from that of p-sarcoglycan, p- sarcoglycan may function in a manner independent of the other components of the complex. in nonmuscle tissues, p-sarcoglycan [nay be part of another subcom- plex. it will be of interest to determine the structure a n d cellular localization of p-sarcoglycan in these tis- sues a n d to identify its associated proteins. t h e pres- ence of p-sarcoglycan in brain is particularly interesting because of the involvement of dystroglycan in synapse f o r m a t i ~ n ~ ~ , ~ ~ , though its involvement in this o r in other processes in the nervous system remains to be determined. the genetic a n d clinical heterogeneity of the limb- girdle muscular dystrophies has broad implications for the diagnosis a n d management of persons afflicted nature genetics volume novernber artzcle with this disease. because defects in several distinct proteins can cause phenotypically similar clinical and biochemical manifestations, a definitive diagnosis can only be achieved through genetic testing of all candi- date genes. at the current stage of neuromuscular dis- ease research and therapy, such measures can only provide information on the precise aetiology of lgmd, but this information will be important in directing proper therapy in the future. methods peptide sequencing and isolation of ests. the kda band of the purified dystrophin-glycoprotein complex was partially sequenced, and several peptide fragments were obtained and are indicated in fig. . peptide sequences were used to search the genbank database of expressed sequence tags (dbest) using the tblastn search program . several overlapping ests were identified that represented portions of the p-sarco- glycan cdna. human p-sarwglycan cdna cloning a n d characterization a n d mrna analysis. two of the clones, and , from which ests were generated, were obtained from the image consortium at lawrence livermore national laboratories. the larger of these clones, , was fully sequenced o n both strands using an applied biosystems, inc. automated sequencer. this clone was determined to contain the full cod- ing region of the p-sarcoglycan cdna as well as about bp of ' untranslated region and a poly(a+) tail. clones were iso- lated from a xzapii human skeletal muscle cdna library (stratagene) using the bp insert of image clone as a probe. primary structure and site detection analyses were performed using pcigene software (intelligenetics). this sequence data is available from the genbank database, acces- sion number u . clontech adult and fetal human multiple tissue northern blots containing pg of poly(a+) rna per lane were probed with a -bp pcr-amplified probe that represents nucleotides to of the p-sarcoglycan sequence, and with the -bp insert d o n e . fusion protein construct. a -bp region of the p-sarcogly- can cdna downstream of the predicted transmembrane domain was amplified by pcr using the following primers: sense, '-gccgggatccgtgattcgcattggaccaaa- '; antisense, '-gcgcgaattcctttgttgtcccttgct- gaa- '. following restriction digest with bamhi and ecori, the product was subcloned into pgex tk and introduced into e. coli dh a cells. ml overnight cultures were diluted : and induced with iptg to promote fusion protein (fp-i) production. fusion proteins were purified o n a glutathione- agarose column . antibodies. anti-p-sarcoglycan antibodies were generated by intramuscular and subcutaneous injection of new zealand white rabbits with pg of purified fp-i in an emulsion of freund's complete adjuvant. rabbits were boosted two weeks later with a subcutaneous injection of pg of fp-i in pbs ( mm sodium phosphate, p h . , . % nac ). rabbits were bled two weeks following boost and the serum was tested for the presence of anti-fp-i antibodies. the serum was cleared of anti-gst antibodies with a glutathione column and anti-fp-i antibodies were affinity-purified using immobilon-p strips containing pg of fp-i. monoclonal antibodies via against dystrophin, and ivd against adhalin were previously ~ h a r a c t e r i z e d ~ , ~ ~ . monoclonal antibody d against -dystro- glycan was kindly provided by louise anderson. an affinity- purified rabbit antibody against kda dag (d. jung, unpublished results) was also used in this study. monoclonal antibody against human laminin a chain was purchased from chemicon. western blot and immunofluorescence. kc -washed micro- somes, crude rabbit skeletal muscle sarcolemma, and purified dgc were prepared as previously d e ~ c r i b e d ~ . ~ ~ . proteins were resolved on a % % sds polyacrylamide and trans- ferred to nitrocellulose by electroblottings . blots were incubat- ed overnight in a : dilution of affinity-purified anti-fp-i antibody in blotto ( % nonfat dried milk in tbs [ mm tris- hcl, mm nac , ph . ]), then incubated with a horserad- ish peroxidase-conjugated goat anti-rabbit igg secondary antibody (boehringer mannheim) for hr. antibody staining was detected with h in tbs with -chloro- -naphthol as a substrate. for immunofluorescence, p m transverse cryosec- tions were prepared from control and amish lgmd muscle. the following procedures were performed at room tempera- ture. sections were treated with ab blocking solutions (vecter), blocked with % bsa in pbs for min, and then incubated with a : dilution of affinity-purified anti-fp-i antibody for min. antibodies against the following components of the dgc were also tested: dystrophin, laminin a chain, p-dystro- glycan, syntrophin, adhalin, and kda dag. after extensive washing with pbs, sections were incubated with biotinylated secondary antibodies ( : ) for min, washed with pbs, then incubated with fitc-conjugated streptavidin ( : ) for min. after rinse with pbs, sections were mounted with fitc-guard (testog) and observed under a zeiss axioplan flu- orescence microscope. photographs were taken under identical conditions with the same exposure time. p-sarcoglycan gene localization t o chromosome q . primers corresponding to human p-sarcoglycan cdna nucleotides - (sense) and - (anti-sense) were used in pcr using dna from a panel of human-rodent somatic cell hybrids (bios corporation) containing various combinations of human chromosomes. subsequent restriction digest of the pcr products by taqi was used to distinguish between the human and rodent alleles. use of a somatic cell hybrid panel containing various regions of chromosome (ref. ) further narrowed the location of the gene using the same approach described above. a chromosome cosmid library (kindly provided by jeffrey murray) was screened with a p-labelled pcr product repre- senting nucleotides - . two cosmids with inserts of . kb and kb were obtained. csc -purified dna from the smaller cosmid was used for fluorescence in situ hybridization mapping which was performed by genome systems. families. six previously described lgmd amish families from southern indiana ( individuals, affected) were analysed in the linkage search. subsequently, dna from additional southern indiana families were included in this study ( indi- viduals, affected). au of these kindreds show multiple con- sanguineous links (data not shown). in these families, patients were clinically reevaluated after exclusion of the cal- pain locus. au patients presented with proximal symmetrical weakness and atrophy of limb and trunk muscles. clinical onset was at . years of age (range to years), loss of walk- ing at years (range to years), with marked intrafamilial variability. calf hypertrophy was similar to that observed in bmd and lgmd d patients, but different from reunion island lgmd patients and lgmd a patients from the northern indiana amish community. genotyping and linkage analysis. markers were selected from the microsatellite panel described or from chlc maps '. dna ( ng) was used as templates in a pl polymerase chain reaction as described . southern blots of restriction fragments of cosmids were probed with (ca), and tetranucleotide oligonucleotide repeats labelled with y- p atp. the positive fragment was subcloned and sequenced. the identified intra- genic polymorphic ca repeat was amplified using the follow- ing primers: sense ( '-tatcttctaatgtcttctgtctat- ') nature genetics volume novernber article and antisense ( '-gaaacaagaataacatgccattt- '). pcr conditions for this marker were - denaturation at oc for min, annealing at "c for min, and extension at oc for min, for cycles. primer sequences, pcr conditions, and other information concerning the highly polymorphic microsatellites used in this study can be obtained from the genome database, john hopkins university. two-point and multipoint linkage analyses were carried out using the link- age software package, version . (ref. ) assuming fully pen- etrant autosomal recessive inheritance with a gene frequency of . . rna isolation and reverse-transcription pcr. total rna was extracted from - mg of skeletal muscle from one control and two amish lgmd e patients from family a (ref. ) using rnazol (tel-test) according to manufacturer specifica- tions. rna samples were run on % formaldehyde/l. % agarose gels and transferred to hybond n+ membrane (amer- sham). membranes were then hybridized with the pcr labelled cdna as described above. total rna ( pg) was used for reverse transcription with a specific primer representing nucleotides - (antisense) in the p-sarcoglycan ' cdna untranslated region, in a reaction mixture containing mm mgc , mm dntp, mm kci, mm tris ph . , u rnasin, pmol specific primer, u amv reverse transcriptase, and incubated for min at "c. pcr on the reverse-transcribed product was performed using the same ' primer and one of two ' primers (representing nucleotides - and - respectively) to cover the p-sarco- glycan cdna coding sequence. the rt-pcr amplification products were analysed by agarose gel electrophoresis and by direct sequencing. touchdown pcr. dna ( ng) was subjected to touchdown p c r ~ * in a p reaction mix containing mm tris-hc , ph . , mm kc , . mm mgcl,, . % triton x- , mm of each dntp, ng of each primer, and u taq polymerase (perkin elmer). after min denaturation at "c, amplifica- tion cycles were carried out as follows: s denaturation at "c followed by s annealing steps starting at 'c with a decrease of "c every two cycles until "c. twenty-five addi- tional cycles of amplification consisting of s at oc and s at oc, were performed. primer pairs r iml and t m were designed to yield, respectively, a -bp product from individuals carrying the mutation, and a -bp product from individuals not carrying the mutation. primers sequences were: r : '-gtttttcagcaagggacaag- '; t : '-gtttttcagcaagggacaac- '; m l : '-tattttgag- tcctcgggtca- '; m : '-cttttcactccacttgg- caa- '. pcr products were mixed in a :l volume ratio and run in a single lane on % agarose gels stained with ethidium bromide. acknowledgments we thank m.b. soares and g. lennon of the image consortium forproviding the clonesfrom the normalized infant brain library; j. murray for the chromosome specific cosmid library and critical comments on this manuscript; j. chamberlain for thegift of the microsatellite repeatprobes; l. anderson for the p- dystroglycan antibody; d. jung for the dag antibody; s.a. cullen forperforming the muscle biopsies; c. leveille, j. lee, s. cutshall, d. venzke, and d. tischfield for their expert technical assistance; v shefield for helpful consultation; l. eberly and d. moser of the university of iowa dna core for their sequencing assistance; n. chiannilkulchai, d. fugman, f. quetier, j. tischfield, and all members from our respective labs for all their help. we are particularly indebted to all patients and their families. this research was supported by grants from the muscular dystrophy association, l'association fran~aise contre les myopathies (afm), and the groupement de recherche europken sur le gknome. l.e.l. is supported by a grant from the iowa afiliate of the american heart association. va. is funded by a grantfrom afm. k.j?c. is an investigator of the howard hughes medical institute. received july; accepted october . nature genetics volume november article . campbell, k.p. & kahl, s.d. association of dystrophin and an integral membrane glycoprotein. nature , - ( ). . emasti, j.m., ohlendieck, k., kahl, s.d., gaver, m.g. &campbell, k.p. deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. nature . - . 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(current protocols, brooklyn, new york, ). . jorgensen, a.o. et a/. identification of novel proteins unique to either transverse tubules (ts ) or the sarcolemma (sl ) in rabbit skeletal muscle. j. cellbiol. . - ( ). . ohlendieck, k., e~asti, j.m.. snook, j.b. &campbell. k.p. dystrophin- glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma. j. ceilbiol. , - ( ). . laemmli, u.k. cleavage of structural proteins during the assembly of the head of bacteriophaget . nature , - ( ). . towbin, h., staehelin, t. &gordon, j. electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. proc. natn. acad. sci. u.s.a. , - ( ). . fardeau, m. et a/. juvenile limb-girdle muscular dystrophy: clinical. histopathological, and genetics data on a small community living in the reunion island. brain (in the press). . murray, j.c. et a/. a comprehensive human linkage map with centimorgan density. science , - ( ). . fougerousse f. et al. mapping of a chromosome region involved in limb-girdle muscular dystrophy. hum. molec. genet. . - ( ). . lathrop g.m.. lalwel j.-m.. julier, c. & ott, j. multilocus linkage analysis in humans: detection of linkage and estimation of recombination. am. j. hum. genet. , - ( ). nature genetics volume novernber sodium nitrite augments lung s-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats research article sodium nitrite augments lung s-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats robert p. jankov, , , , kathrine l. daniel, shira iny, crystal kantores, julijana ivanovska, nadya ben fadel, and amish jain , molecular biomedicine program, children’s hospital of eastern ontario research institute, ottawa, ontario, canada; faculty of medicine, department of pediatrics, university of ottawa, ottawa, ontario, canada; faculty of medicine, department of cellular and molecular medicine, university of ottawa, ottawa, ontario, canada; translational medicine program, hospital for sick children research institute, toronto, ontario, canada; department of physiology, university of toronto, toronto, ontario, canada; and lunenfeld-tanenbaum research institute, mount sinai hospital, toronto, ontario, canada submitted april ; accepted in final form august jankov rp, daniel kl, iny s, kantores c, ivanovska j, ben fadel n, jain a. sodium nitrite augments lung s-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats. am j physiol lung cell mol physiol : l –l , . first pub- lished august , ; doi: . /ajplung. . .—deficient nitric oxide (no) signaling plays a critical role in the pathogenesis of chronic neonatal pulmonary hypertension (pht). physiological no signaling is regulated by s-nitrosothiols (snos), which act both as a reservoir for no and as a reversible modulator of protein function. we have previously reported that therapy with inhaled no (ino) in- creased peroxynitrite-mediated nitration in the juvenile rat lung, although having minimal reversing effects on vascular remodeling. we hypothesized that sodium nitrite (nano ) would be superior to ino in enhancing lung snos, thereby contributing to reversal of chronic hypoxic pht. rat pups were exposed to air or hypoxia ( % o ) from postnatal days to . dose-response prevention studies were conducted from days – to determine the optimal dose of nano . animals then received rescue therapy with daily subcutane- ous nano ( mg/kg), vehicle, or were continuously exposed to ino ( ppm) from days – . chronic pht secondary to hypoxia was both prevented and reversed by treatment with nano . rescue nano increased lung no and sno contents to a greater extent than ino, without causing nitration. seven lung sno proteins upregulated by treatment with nano were identified by multiplex tandem mass tag spectrometry, one of which was leukotriene a hydrolase (lta h). rescue therapy with a lta h inhibitor, sc a ( mg·kg� ·day� sc), partially reversed chronic hypoxic pht. we conclude that nano was superior to ino in increasing tissue no and sno generation and reversing chronic pht, in part via upregulated sno-lta h. cofilin; leukotriene; newborn; nitration; nitric oxide introduction chronic pulmonary hypertension (pht), characterized by increased pulmonary vascular resistance (pvr), increased pul- monary arterial pressure, and arterial wall remodeling due to smooth muscle hyperplasia ( ) is commonly observed in newborns and infants with bronchopulmonary dysplasia and other developmental lung disorders. chronic pht heralds a greatly increased risk of death and severe morbidity, and no therapies yet exist that are proven to modify the disease course ( ). abundant evidence implicates deficient nitric oxide (no) signaling as central to the pathogenesis of chronic neonatal pht ( , , , ). in addition to endothelial no synthase (nos)-derived no stimulating signaling pathways leading to vascular smooth muscle relaxation, physiological no signaling is regulated by reversible s-nitrosylation of cysteine thiols ( ), producing s-nitrosothiols (snos) ( ). s-nitrosylation modifies protein structure, alters interactions with binding partners, or leads to changes in subcellular localization and degradation, causing a reversible alteration in protein function ( , , , , ). exogenous (inhaled) no (ino) is a commonly utilized no-based therapy in the neonate. unfortunately, ino has not proven effective at reversing or slowing progression of chronic pht in human infants ( , ) and has been of variable efficacy in reversing established chronic pht in neonatal rats ( , ). a biochemical obstacle to effective no-based therapy relates to the high potential for no to be diverted to produce reactive nitrogen species such as peroxynitrite ( ); particularly in the lung, which is directly exposed. reactive nitrogen species cause irreversible oxidation and tyrosine nitration ( ), which permanently alters protein function ( , , ) and directly contributes to pulmonary vascular disease in neonatal rats ( , , , ). nitrite anion (no �) is now recognized to contribute impor- tantly to local no bioavailability and to sno formation by acting as a stable, nonreactive endocrine pool of no, formed from circulating and tissue-bound no � reductases ( , ). unlike adults, in whom the majority of circulating no � is derived from dietary nitrate, sources of nutrition in neonates are poor in no � ( ), resulting in lower plasma no � levels ( ) and a likely greater dependence upon nos to generate no. inhaled ethyl (alkyl) nitrite, a volatile gas that acts as a no � donor, was more effective than ino in preventing hyper- oxic lung injury in neonatal rats ( ) and was highly efficacious as an acute pulmonary vasodilator in human neonates ( ). these observations support no �-based therapy as a promising strategy. systemic or inhaled sodium nitrite (nano ) is an address for reprint requests and other correspondence: r. p. jankov, children’s hospital of eastern ontario, smyth rd., ottawa, on, k h l , canada (e-mail: rjankov@cheo.on.ca). am j physiol lung cell mol physiol : l –l , . first published august , ; doi: . /ajplung. . . - / copyright © the american physiological society http://www.ajplung.orgl downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . http://doi.org/ . /ajplung. . mailto:rjankov@cheo.on.ca alternative no �-based therapy shown to be strongly protective in adult experimental models of chronic pht ( , , , , ). however, no previous studies, to our knowledge, have examined effects of nano on chronic pht in the neonatal or juvenile animal. we hypothesized that therapy with nano will be superior to ino in enhancing no bioavailability and s-nitrosylation, thus allowing for rescue of chronic hypoxic pht. herein, we report that systemically administered nano both prevented and reversed chronic hypoxic pht and in- creased lung no and sno contents to a far greater extent than ino. furthermore, unlike ino, nano did not increase lung nitration. we identified several lung sno proteins upregulated by nano therapy, including sno leukotriene (lt) a hy- drolase (lta h). rescue effects of nano were partially replicated by an lta h inhibitor, sc a, suggesting increased sno-lta h as one mechanism by which nano reversed chronic pht. materials and methods materials. sodium nitrite (nano ; cat. no. s- ) and sc a (cat. no. ) were purchased from sigma-aldrich (oakville, can- ada) and tocris biosciences (bristol, uk), respectively. s-nitrosylated biotin switch (cat. no. ) and nitrate/nitrite detection (cat. no. ) kits, peroxynitrite solution (cat. no. ), and c solid phase extraction cartridges (cat. no. ) were from cayman chemical (ann arbor, mi). oxygen and no exposure chambers and automated controllers, oxycycler models a xov and at n, were from biospherix (parish, ny). no -ppm cylinders (balance n ) were from linde canada (mississauga, canada). iodo-tandem mass tag (tmt) labeling kits (cat. nos. and ), immobilized anti-tmt antibody resin (cat. no. ), and tmt elution buffer (cat. no. ) were from thermo fisher (waltham, ma). mass spectrometry grade trypsin/lys-c mix (cat. no. v- ) was from promega (madison, wi). precast tris-glycine – % gradient gels (mini-protean tgx stain-free), chemiluminescent reagent (clar- ity or clarity max western ecl substrate), imaging system (chemi- doc touch), and analysis software (image laboratory . ) were from bio-rad (mississauga, canada). protease inhibitors were from cal- biochem (cat. no. - -vl, san diego, ca). acids, alcohols, organic solvents, paraformaldehyde, permount, and superfrost/plus microscope slides were from thermo fisher. weigert’s resorcin- fuchsin stain was from rowley biochemical (cat. no. f- - , dan- vers, ma). avidin-biotin-peroxidase complex (cat. no. pk- ) and , -diaminobenzidine staining kits (cat. no. pk- ) were from vector laboratories (burlingame, ca). an antigen retriever device was from aptum biologics ltd. (antigen retriever , southam- pton, united kingdom). ihc-tek antibody diluent (cat. no. iw- ) and epitope retrieval solution (cat. no. iw- ) were from ihc world (woodstock, md). anti-lta h (cat. no. nbp- - ) was from novus biologicals (littleton, co). anti-nitrotyrosine (cat. no. - ) was from emd millipore (billerica, ma). anti-cofilin- (cat. no. ) and goat anti-rabbit igg-peroxidase (cat. no. ) anti- bodies were from cell signaling technologies (beverly, ma). anti- phospho-serine myosin phosphatase target (mypt)- (cat. no. sc- ) and goat anti-rabbit igg-biotin (cat. no. sc- ) secondary antibody were from santa cruz biotechnology (santa cruz, ca). anti-pan-mypt- was from bd biosciences (cat. no. ; mis- sissauga, canada). colorimetric competitive elisa kits for biotin (cat. no. k ) and -nitrotyrosine (cat. no. k- ) were from biovision (milpitas, ca). elisa kits for ltb (cat. no. adi- - ) were from enzo life sciences (farmingdale, ny). unless specified, all other chemicals and reagents were from sigma-aldrich. in vivo experiments. all procedures involving animals were ap- proved by the animal care committee at the university of ottawa and conformed to the guidelines of the canadian council on animal care. a total of timed-pregnant sprague-dawley rats were pur- chased from taconic farms (germantown, ny). experiments were conducted in multiples of four litters ( litters in hypoxia and litters in normoxia, one vehicle-treated, and one drug-treated). except for samples used for mass spectrometry (n � per group, performed twice), sample size estimates were based on previous work and set a priori [n � for cardiac end points (pulmonary hemodynamics and fulton index) and n � for all other end points] incorporating animals from two to three litters per experimental group with equal- ized sex distribution. analysis of all end points was conducted in a blinded fashion. commencing on the day after birth [considered postnatal day (pnd) ], litters of sprague-dawley rat pups were chronically exposed to normobaric hypoxia ( % o ) or normoxia ( % o ) until pnd . automated controllers (biospherix) main- tained o concentrations to within . % of set point. the room in which chambers were housed had -h light/dark cycles. chamber temperature was maintained at � °c, humidity at � %, and food and water were available ad libitum. rat pups received daily subcutaneous nano ( , , , or mg/kg in . % saline vehicle; �l per g body wt) or vehicle alone from pnds – to determine the dose of nano that maximally prevented chronic pht without significantly (� %) increasing blood methemoglobin (methb). for rescue treatment studies, rats received daily subcutaneous nano ( mg/kg), which was determined to be the dose that best met the above criteria, or sc a (lta h inhibitor) at a dose ( mg· kg� ·day� sc) previously reported by our group to be effective in neonatal rats ( ) or vehicle ( . % saline or % dmso in pbs, respectively) from pnd to pnd . separate animals were exposed to chronic hypoxia with or without ino ppm. effects of rescue treatment reflect “reversal” of chronic pht (rather than limit- ing further progression) as pht markers have previously been deter- mined in this model to remain stable between pnds and ( ). each litter was maintained at n � pups throughout the exposure period to control for nutritional effects. at the end of the exposure/ treatment period, pups were either killed by pentobarbital overdose or were exsanguinated after inhalational or ketamine-induced anesthesia. two-dimensional echocardiography and pulsed wave doppler ultrasound. two-dimensional echocardiography and pulsed wave doppler ultrasound was performed as a noninvasive method to assess pulmonary hemodynamics as previously described ( ), using a philips affiniti cardiac ultrasound system with a small high frequency linear probe (l - io, philips healthcare, richmond hill, ontario, canada). the right ventricular ejection time (rvet) and pulmonary arterial acceleration time (paat) were measured using the pulmonary doppler profile obtained from the parasternal short axis; rvet is the time from onset of systolic flow to completion of systolic flow and paat is from onset to peak pulmonary outflow velocity. a previously validated ( ) index of pvr was calculated using the average ratio of rvet/paat from three systolic traces. blood methb. at the end of each treatment period, pups were anesthetized as described above and the jugular vein isolated and severed. blood was collected in a capillary tube and immediately analyzed using an abl flex co-ox blood gas analyzer (radi- ometer america, brea, ca). right ventricular hypertrophy. measurement of right ventricular hypertrophy (rvh) using the fulton index (right ventricle/left ven- tricle � septum) is a well-established marker of pht. the heart and lungs were separated, and the atria were removed inferior to the atrio-ventricular valves. the right ventricle was separated from the left ventricle and septum, freeze-dried, and weighed separately. histological studies. three males and three females (one from each of three litters per group) were euthanized by sodium pentobarbital overdose. following the opening of the thoracic cavity and tracheal cannulation, the pulmonary veins were divided. the pulmonary cir- l sodium nitrite prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . culation was flushed with pbs and heparin ( u/ml) to clear the lungs of blood, following which the lungs were perfusion-fixed with para- formaldehyde while air inflated at constant pressure ( cm of h o). following dehydration and clearance in xylene, whole left lungs were embedded in paraffin, cut into -�m sections, mounted, air-dried, and baked overnight at °c. for elastin staining, sections were dewaxed by immersion in xylene, rehydrated in ethanol, rinsed in several washes of distilled water, and then left overnight in weigert’s resor- cin-fuchsin (hart’s elastin) stain. slides were then washed with distilled water and counterstained with . % (vol/vol) tartrazine in acetic acid followed by dehydration and mounting. percentage medial wall area (%mwa) was measured on elastin-stained sections as a marker of pulmonary vascular remodeling. pulmonary arteries ( – -�m external diameter) were identified by the presence of an inner and outer elastic lamina and proximity to respiratory bronchioles. a minimum of arteries per animal from unique left lung sections were digitally captured and analyzed. obliquely sectioned vessels where there was a greater than three times difference in perpendicular dimensions were excluded. as previously described ( ), using the “quick selection” tool (adobe photoshop cs , adobe systems inc., san jose, ca) the area of the inner lumen and the whole vessel was outlined and pixel count determined. the following formula was used to determine the percent medial wall area: [(whole vessel area�inner luminal area)/whole vessel area] � . for -nitrotyrosine immuno- staining, slides were incubated with anti-nitrotyrosine ( : ) in antibody diluent overnight at °c, followed by biotin-conjugated secondary antibody ( : , ) for h at room temperature, then washed, dehydrated, counterstained with hematoxylin, and mounted. measurement of no oxidation products. nitrate/nitrite oxidation (nox) was measured in lung tissue homogenized in pbs and depro- teinated by ultrafiltration (amicon ultra, emd millipore, burlington, ma). nitrate in deproteinated samples was enzymatically converted to nitrite (cayman nitrite/nitrate detection kit) and quantified, along with nitrite standards, using a sievers i no analyzer (zysense, weddington, nc), according to the supplier’s instructions. values were normalized to wet tissue weight. quantification and identification of sno proteins. sno protein modifications, because of their labile nature, can be difficult to study employing traditional methods used for detecting phosphorylated proteins. for this reason, several “switch” assays have been developed to label sno-modified cysteines with stable markers ( , ). total snos were quantified in lung tissue homogenates using a biotin switch assay and competitive biotin elisa, both according to the manufacturer’s instructions. specific sno proteins were identified from lung tissue homogenates (two normoxia-exposed vehicle- treated, two hypoxia-exposed vehicle-treated, and two hypoxia-ex- posed nano -treated) labeled with sixplex-tmt and affinity column purified according to the manufacturer’s instructions. samples were quantified by multiplex liquid chromatography/tmt spectrometry (tmt-lc/ms/ms; q exactive hybrid quadrupole-orbitrap mass spectrometer, thermofisher scientific) by the sparc biocentre (hospital for sick children, toronto), following previously reported methods ( ). reporter ions observed in the m mass spectrum ( – ), generated from higher-energy collisional dissociation fragmenta- tion, were used for relative quantification between samples. mass spec- trometry data were searched using peaks software ( ) version . (bioinformatics solutions, waterloo, canada) and the reporter ions quan- tified using proteome discoverer software (version . ; thermofisher scientific). seven sno proteins were identified (table ) where content was altered (significance score � ) by nano treatment with results that were consistent between two separate sets of samples and tmt-lc/ ms/ms runs. two of the seven candidates (lta h and cofilin- ) were confirmed by western blot, despite lc/ms/ms identifying one unique peptide with relatively low sequence coverage ( and %, respectively; table ). western blot analyses. lung lysates from three males and three females per group (representing litters) were tmt-labeled accord- ing to the manufacturer’s instructions, purified by agarose bead immunoprecipitation, and matched with unlabelled, unpurified lysates both of which were separated under reducing conditions by sds- page. following electrophoresis, proteins were transferred to pvdf membranes. all membranes were blocked with % skim milk for h at room temperature, followed by incubation with primary antibody overnight at °c. blots were then washed with tris-buffered saline- tween and placed in secondary antibody for h at room temper- ature. dilutions of primary antisera were : , for lta h ( kda) and secondary antiserum and : , for cofilin- ( kda), phospho-serine and pan-mypt- ( kda). bands were quanti- fied by digital densitometry of nonsaturated images with background density removed. bands were normalized to corresponding pan- protein content, each corrected for total protein/lane quantified by stain-free protein imaging (bio-rad). in preliminary experiments, we confirmed that stain-free protein imaging was equivalent to gapdh as a marker of protein loading (data not shown). raw values are expressed as a multiple or fraction of the normoxia-exposed, vehicle- treated group, which was assigned a value of . elisa. lung lysates from three males and three females per group (representing litters) were purified and analyzed according to the manufacturer’s instructions. values were normalized to protein con- tent ( -nitrotyrosine) or wet tissue weight (ltb ). data presentation and statistical analysis. data are expressed as means � se after any outliers (� sd from mean value) were removed. analyses were performed using sigma plot (systat software, san jose, ca). statistical significance (p 	 . ) was determined by t-test or one-way anova followed by tukey post hoc test where significant intergroup differences were found or by kruskal-wallis one-way anova for nonparametric data. table . candidate sno proteins identified by tmt-lc/ms/ms accession no. │ uniprot id (protein name) unique peptides, n sequence coverage, % known functions p │lkha (leukotriene a hydrolase) epoxy hydrolase catalyzing biosynthesis of proinflammatory leukotriene b . also possesses amino-peptidase activity. p │cof (cofilin- ) depolymerizes f-actin. regulates cell morphology and cytoskeletal organization. b bmy (histone h ) nucleosomal protein. q │ptpa (serine/threonine-protein phosphatase a activator) regulation of apoptosis. q │myo d (unconventional myosin-id) cytoskeletal protein. regulation of cell motility. q jlu │shan (sh and multiple ankyrin repeat domains protein ) cytoskeletal protein. regulation of cell motility. o │prdx (peroxiredoxin ) thiol-specific peroxidase enzyme/antioxidant. sno, s-nitrosothiols; tmt-lc/ms/ms, multiplex liquid chromatography/tmt spectrometry. l sodium nitrite prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . results systemic nano prevented and reversed chronic hypoxic pht. dose response prevention studies are shown in fig. . nano at doses of and mg·kg� ·day� prevented chronic pht as evidenced by significantly (p 	 . ) de- creased pvr (fig. a) and rvh (fig. b). blood methb was significantly greater (p 	 . ; mean value . %) at mg/kg when compared with mg/kg (mean value . %). rescue effects of nano mg·kg� ·day� are shown in fig. . rescue nano normalized pvr (fig. a) and almost completely normalized rvh (fig. b) and %mwa (fig. c). in contrast, and as previously reported ( ), ino had no significant reversing effect on increased rvh (fulton index . � . ; p 	 . vs. nano -treated, n � animals per group) or %mwa ( . � . %; p 	 . vs. nano - treated, n � animals per group) in chronic hypoxia-exposed animals. methb values in animals receiving rescue nano were all 	 % (data not shown). rescue treatment with nano increased lung nox, sno protein content, and cgmp activity, without causing increased nitration. lung nitrate/nitrite (nox) was measured as a marker of total no content. nano significantly increased (p 	 . ) lung nox (fig. a) and sno protein (fig. b) contents in both normoxia- and hypoxia-exposed animals when com- pared with animals treated with vehicle- or hypoxia-exposed animals that were treated with ino. lung phospho-serine /pan-mypt- ratio was used as a marker of cgmp-medi- ated protein kinase g signaling (pkg), as previously described ( ). as shown in fig. c, rescue treatment with both nano and ino significantly increased lung cgmp-pkg activity in chronic hypoxia-exposed animals. chronic exposure to hyp- oxia or treatment with nano had no effect on total lung -nitrotyrosine content (fig. d). in contrast, and as previously reported ( ), lung -nitrotyrosine was greatly increased by rescue treatment with ino (p 	 . vs. all other groups; fig. d). representative images of -nitrotyrosine immunohisto- chemistry are shown in fig. e. rescue treatment with nano increased lung contents of sno-lta h and sno-cofilin- . as shown in fig. , a and b, sno-lta h and sno-cofilin- were significantly increased by rescue treatment with nano in hypoxia-exposed animals. there were no significant differences in pan-lta h (p � . , by anova; data not shown) or pan-cofilin- (p � . , by anova; data not shown) between groups. increased sno-lta h in nano -treated hypoxia-exposed animals was associated with a nonsignificant (p � . ) trend toward decreased lta h activity, as indicated by lung ltb content, when compared with hypoxia-exposed vehicle-treated animals (fig. d). rescue treatment with sc a, a lta h inhibitor, par- tially reversed chronic hypoxic pht. treatment with sc a partially but significantly (p 	 . ), decreased pvr (fig. a) rvh (fig. b) and %mwa (fig. c) in chronic hypoxia-exposed animals. these effects were accom- panied by significantly decreased lung ltb content ( . � . pg per lung wet wt in hypoxia-exposed vehicle-treated animals versus . � . in hypoxia-exposed, sc a- treated animals, n � per group; p 	 . , by t-test). fig. . dose-response studies. from postnatal days – , rat pups were exposed to normobaric hypoxia ( % o ) or normoxia ( % o ). beginning on day , pups received daily subcutaneous injections of sodium nitrite ( , , , or mg/kg) or . % saline vehicle. a: pulmonary vascular resistance (pvr) index. ratio of right ventricular ejection time (rvet) to pulmonary arterial acceleration time (paat), n � animals per group. b: right ventric- ular hypertrophy (rvh) assessed by right ventricle (rv)/left ventricle (lv) � septum (s) weight ratio (fulton index), n � animals per group. c: blood percentage methemoglobin levels, n � animals per group. bars represent means � se; *p 	 . , by anova, vs. vehicle-treated group. #p 	 . , by anova, vs. all other groups. l sodium nitrite prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . discussion we present the first data, to our knowledge, to demonstrate therapeutic efficacy of nano and superiority to ino in reversal of chronic pht in the juvenile animal. nano has many properties that are favorable for clinical translation, including stability, low cost, and ability to be administered systemically or by inhalation. as confirmed in this study, nano leads to upregulation of physiological no signaling [increased snos ( , , )] without an apparent concurrent propensity to cause nitration ( ). in humans, an injectable form of nano is marketed as therapy for cyanide poisoning ( ), and nebulized nano was safe and well tolerated in healthy adults ( ) and acutely reduced pvr in pulmonary hypertensive patients with -thalassemia ( ). in rats chronically exposed to hypoxia from birth, we report that increased pvr, rvh, and pulmonary arterial wall remod- eling were almost completely reversed by rescue treatment with systemic nano , in contrast to ino, which had no significant reversing effect ( ). furthermore, rescue nano increased lung nox and total sno protein contents to a much greater extent than ino, without causing nitration. in addition to avoidance of nitration, increased bioavailability of no and enhanced cgmp-pkg signaling, a likely mechanism behind the efficacy of nano was reversible and specific modification in protein function that arises from s-nitrosylation ( ). inter- estingly, we observed that rescue ino greatly increased cgmp-pkg activity, which was surprising in light of the lack of reversing effect on markers of chronic pht. possible ex- planations are that enhanced nitration may have negated any benefits of enhanced cgmp signaling and/or that increased s-nitrosylation is of greater importance to reversal of pulmo- nary arterial remodeling in this model. as there is currently no knowledge on the functional role/s of specific sno proteins in neonatal cardiopulmonary disease, fig. . rescue sodium nitrite reversed chronic pulmonary hypertension. from postnatal days – , rat pups were exposed to normobaric hypoxia ( % o ; hypoxia) or normoxia ( % o ). from days – , pups received daily subcutaneous injections of sodium nitrite (nano ) mg/kg or . % saline vehicle or were continuously exposed to ppm inhaled nitric oxide (ino). a: pulmonary vascular resistance (pvr) index. ratio of right ventricular ejection time (rvet) to pulmonary arterial acceleration time (paat), n � animals per group. b: right ventricular hypertrophy (rvh) assessed by right ventricle (rv)/left ventricle (lv) � septum (s) weight ratio (fulton index), n � animals per group. c: pulmonary arterial medial wall area as a marker of vascular remodeling, n � animals per group. bars represent means � se; *p 	 . , by anova, vs. all other groups. d: high-power photomicrographs demonstrating elastin-stained pulmonary arteries. bar length � �m. l sodium nitrite prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . fig. . rescue sodium nitrite increased lung nitric oxide oxidation (nox), s-nitrosylated (sno) proteins, and cgmp-pkg activity, without increasing nitration. from postnatal days – , rat pups were exposed to normobaric hypoxia ( % o ; hypoxia) or normoxia ( % o ). from days – , pups received daily subcutaneous injections of sodium nitrite (nano ) mg/kg or . % saline vehicle or were continuously exposed to ppm inhaled nitric oxide (ino). a: lung nox products as a marker of tissue nitric oxide abundance, n � animals/group. b: total lung sno proteins, n � animals per group. c: western blot analyses of lung phospho-serine /pan-myosin phosphatase target (mypt)- ratio as a marker of cyclic guanosine monophosphate-protein kinase g signaling, n � animals per group. representative immunoblots show two contiguous lanes for each group. d: lung -nitrotyrosine content, n � animals per group. bars represent means � se; *p 	 . , by anova, vs. all other groups. #p 	 . , by anova, vs. normoxia groups. †p 	 . , by anova, vs. vehicle-treated groups. e: representative medium-power photomicrographs of lung -nitrotyrosine immunoreactivity, as a marker of nitric oxide-derived reactive nitrogen species-mediated nitration. bar length � �m. inset: positive control section pretreated with mm peroxynitrite. l sodium nitrite prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . we undertook tmt-lc/ms/ms and identified seven lung sno proteins that were consistently upregulated by treatment with nano . despite there being relatively low sequence coverage for most peptides identified by lc/ms/ms, changes in two of these proteins, lta h and cofilin- , were able to be confirmed by western blot analyses. s-nitrosylation is the covalent mod- ification of cysteine thiols by addition of a nitrosyl group (by either no or no �). as a result, cysteine-rich proteins contrib- ute importantly to physiological no signaling by acting as a circulating and tissue reservoir for no ( , , , , ). in addition, s-nitrosylation possesses the essential criteria for a signaling modification (akin to phosphorylation), including a rapid reaction, specificity for particular cysteine residues, and enzymatic removal [by s-nitrosoglutathione reductase and the thioredoxin reductase system ( )]. the functional implications for the majority of the � , sno proteins identified to date remain obscure. we have previously reported that lta h inhibition pre- vented vascular remodeling and chronic pht in bleomycin- exposed neonatal rats ( ). in the present study, we observed that rescue nano significantly decreased lung ltb content (product of lta h epoxy hydrolase activity) and that rescue therapy with sc a partially reversed chronic hypoxic pht. a potential role for s-nitrosylation in modulation of lta h activity has not, to our knowledge, been previously described. in addition to production of ltb , which is known to stimulate inflammation, production of matrix proteins, and to increase smooth muscle contractility and airway/vascular remodeling in the lung ( , ), lta h is also known to act as an aminopeptidase, products of which may also contribute to fig. . rescue treatment with nano increased lung contents of sno-leukotrienea hydrolase (lta h) and sno-cofilin- . from postnatal days – , rat pups were exposed to normobaric hypoxia ( % o ; hypoxia) or normoxia ( % o ). from days – , pups received daily subcutaneous injections of sodium nitrite (nano ) mg/kg or . % saline vehicle. western blot analyses of lung sno-lta h (a) and sno-cofilin- (b). representative immunoblots for sno and pan-proteins (c). unlabeled control � purified normoxia-exposed, vehicle-treated sample not labeled with tmt. lung leukotriene b , as a marker of lta h activity, quantified by elisa (d). bars represent means � se for n � – samples per group. *p 	 . , by kruskal-wallis one-way anova, compared with all vehicle-treated groups. #p 	 . , by kruskal-wallis one-way anova, compared with all other groups. h, heavy chain; l, light chain; sno, s-nitrosothiols; tmt, tandem mass tag. l sodium nitrite prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . lung inflammation and injury separate from the leukotriene pathway ( ). our present data provide indirect evidence for inhibitory effects of s-nitrosylation on lta h activity. the identity of specific cysteine residues on lta h that are prone to s-nitrosylation and whether inhibition of hydrolase or ami- nopeptidase pathways predominated in reversal of chronic pht awaits further study. cofilin is an intracellular actin-modulating protein that binds and depolymerizes filamentous f-actin. cofilin activity is in- creased in hypertrophic cardiomyocytes ( ) and in chronic hypoxia-exposed murine lung ( ) and is activated down- stream of upregulated rhoa and rho-kinase activity [via enhanced lim kinase-mediated phosphorylation ( )]. patho- logical rho-kinase activity is a critical mediator of chronic neonatal pht in the present model ( , , ), in bleomycin- exposed neonatal rats ( , ) and in pulmonary hypertensive fetal sheep ( , ). s-nitrosylation is known to modulate the activity of cofilin- in endothelial cells ( ). in the present study, we observed that lung sno-cofilin- was decreased by chronic exposure to hypoxia and restored by rescue nano . as most sno protein modifications lead to inhibition of protein function, we speculate that smooth muscle cofilin- activity was reduced by enhanced s-nitrosylation, potentially also con- tributing to reversal of vascular remodeling. there are several limitations to this study. first, it is likely that multiple sno protein modifications contributed to nano -mediated reversal of chronic pht; however, we chose to explore lta h as a candidate because of amenability to pharmacological inhibition and the known effects of the leu- kotriene pathway in mediating pht ( ). of the other candi- date sno proteins identified, all may contribute to the patho- genesis of chronic pht (modulators of actin depolymerization, oxidative stress, cell survival) and warrant further exploration. second, although we were able to measure total and specific lung snos in whole tissue, we were unable to localize them in situ. commercially available sno-nitrosocysteine antibodies are available; however, we were unable to obtain any mean- ingful data employing several of these antibodies. third, we fig. . rescue treatment with sc a, a leukotrienea hydrolase (lta h) inhibitor, partially reversed chronic hypoxic pulmonary hypertension (pht). from postnatal days – , rat pups were exposed to normobaric hypoxia ( % o ) or normoxia ( % o ). from days – , pups received daily subcutaneous injections of sc a mg/kg or % dmso in pbs (vehicle). a :pulmonary vascular resistance (pvr) index. ratio of right ventricular ejection time (rvet) to pulmonary arterial acceleration time (paat), n � animals per group. b: right ventricular hypertrophy (rvh) assessed by right ventricle (rv)/left ventricle (lv) � septum (s) weight ratio (fulton index), n � animals per group. c: arterial medial wall area as a marker of vascular remodeling, n � animals per group. bars represent means � se; *p 	 . , by anova, vs. all other groups. #p 	 . , by anova, vs. normoxia groups. d: high-power photomicrographs demonstrating elastin-stained pulmonary arteries. bar length � �m. l sodium nitrite prevents and reverses chronic neonatal pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . did not explore the relative contributions of no-cgmp versus sno signaling in the therapeutic effects of nano . this could be explored in future studies employing a smooth-muscle- specific soluble guanylate cyclase knockout model, either in vitro or in vivo. finally, although pulmonary-selective (inha- lation) therapy with nano has the greater translational po- tential, we chose to limit the present study to systemic therapy as proof of concept of efficacy and superiority to ino. in conclusion, we provide the first preclinical evidence, to our knowledge, for efficacy of nano in the immature animal. ongoing and future studies will examine the utility of the inhalational route of therapy and efficacy in alternative neona- tal models that replicate bronchopulmonary dysplasia and other developmental lung disorders. acknowledgments we thank dr. jonathan krieger (sparc biocentre, hospital for sick chil- dren, toronto, canada) for valuable assistance with multiplex liquid chromatog- raphy/tmt spectrometry s-nitrosothiol protein measurement and analysis. grants this work was supported by operating funding from the physicians’ services incorporated foundation and the heart and stroke foundation of canada and by infrastructure funding from the canada foundation for inno- vation (all to r. jankov). disclosures no conflicts of interest, financial or otherwise, are declared by the authors. author contributions r.p.j. conceived and designed research; r.p.j., k.l.d., s.i., c.k., j.i., n.b.f., and a.j. performed experiments; r.p.j., k.l.d., s.i., n.b.f., and a.j. analyzed data; r.p.j. and a.j. interpreted results of experiments; r.p.j. prepared figures; r.p.j. and a.j. drafted manuscript; r.p.j., k.l.d., c.k., j.i., n.b.f., and a.j. edited and revised manuscript; r.p.j., k.l.d., s.i., c.k., j.i., n.b.f., and a.j. approved final version of manuscript. references . angelo m, singel dj, stamler js. an s-nitrosothiol (sno) synthase function of hemoglobin that utilizes nitrite as a substrate. proc natl acad sci usa : – , . doi: . /pnas. . . arnelle dr, stamler js. no�, no, and no- donation by s-nitrosothi- ols: implications for regulation of physiological functions by s-nitrosyla- tion and acceleration of disulfide formation. arch 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for disease control and prevention, clifton road ne, mailstop a- , atlanta, ga (e-mail: vid @cdc.gov; ) author affiliations: division of viral diseases, centers for disease control and prevention, atlanta, georgia (paul a. gastañaduy and gregory s. wallace); division of nutrition, physical activity, and obesity, centers for disease control and prevention, atlanta, georgia (prabasaj paul), centre for the mathematical modelling of infectious diseases, london school of hygiene and tropical medicine, london, united kingdom (sebastian funk), ohio department of health, columbus, ohio (lilith tatham, nicholas fisher, jeremy budd, brian fowler, sietske de fijter, and mary diorio); department of ecology and evolutionary biology, princeton university, princeton, new jersey (bryan grenfell). funding information: this work was supported by the uk medical research council (fellowship mr/k / to sebastian funk); and by the bill and melinda gates foundation (grant opp ), the rapidd program of the science and technology directorate, u.s. department of homeland security, and the fogarty international center, national institutes of health (bryan grenfell). conflicts of interest: none declared. running head: impact of control measures during a measles outbreak. abbreviations: r, effective reproduction number; mmr, measles-mumps-rubella; rt, instantaneous reproduction number; rc, case reproduction number; r , basic reproduction number; vc, vaccine coverage; ve, vaccine effectiveness; su, proportion of unvaccinated individuals that are susceptible. or ig in al u ne di te d m an us cr ip t published by oxford university press on behalf of the johns hopkins bloomberg school of public health . this work is written by (a) us government employee(s) and is in the public domain in the us. downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april mailto:vid @cdc.gov abstract we quantified measles transmissibility during a measles outbreak in ohio in to evaluate the impact of public health responses. case incidence and the serial interval (time between symptom onset in primary and secondary cases) were used to assess trends in the effective reproduction number r (average number of secondary cases generated per case). a mathematical model was parameterized by early r values to determine outbreak size and duration if containment measures had not been initiated, and the impact of vaccination. as containment started, we found a fourfold decline in r (~ to ) over weeks, and maintenance of r< as control measures continued. under a conservative scenario, the model estimated , cases ( % confidence interval [ci]: , , , ) over days ( % ci: , ) without control efforts, and cases ( % ci: , , ) over days ( % ci: , ) when including vaccination; , fewer cases ( % ci: , , , ) and fewer outbreak days ( % ci: , ) were attributed to vaccination. vaccination may not account entirely for transmission reductions, suggesting changes in community behavior (social distancing) and other control efforts (isolation, quarantining) are important. our findings highlight the benefits of measles outbreak response and of understanding behavior change dynamics. keywords: measles; outbreak response; transmissibility; reproduction number; united states or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april measles is a highly contagious viral disease that can lead to serious complications and death. even with repeated importations of measles into the united states, most introductions do not result in additional transmission, outbreaks are generally small ( ), and endemic measles transmission has been declared eliminated since ( ). the success of the u.s. measles elimination program is credited to high measles-mumps-rubella (mmr) vaccine coverage, as well as the rapid implementation of control measures once cases are reported ( ). yet, the relative contributions of baseline coverage and of various simultaneous control measures (vaccination, isolation, quarantine) in preventing large outbreaks is not fully understood ( ). because, outbreak responses by health agencies are labor intensive and costly ( ), efforts to measure the effectiveness of these interventions are important. on march , the return of two unvaccinated amish men to ohio from the philippines, where they were unknowingly infected with measles, led to the largest outbreak of measles in the us in over two decades ( , ). the outbreak provided an opportunity to measure the impact of public health responses in mitigating measles transmission in an under-immunized community. we aimed to quantify measles transmissibility during the outbreak, and to evaluate the effect of public health responses in limiting the size and duration of the outbreak. methods transmissibility was measured by estimating the effective reproduction number, r, the average number of cases generated by a single infectious individual ( ). the goal of outbreak response is to reduce r below the threshold value of ; transmission wanes when r is maintained at < , bringing an outbreak under control. using a ready-to-use tool ( ), r was estimated from case incidence time series data and the distribution of the serial interval (the time between the onset of symptoms in primary and secondary cases) ( ). we tracked r over time during the outbreak and correlated changes in transmissibility to control efforts. two distinct time-varying estimates of r were measured: the instantaneous reproduction number (rt), and the case reproduction number (rc) ( ). rt measures the expected transmissibility at or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april calendar time, t, based on the ratio of the number of new infections in that time step, to the total infectiousness of previous cases. thus, rt can be estimated in real-time, as new cases are identified.( ) rc measures the actual transmissibility of cases with symptom onset at calendar time, t, and is calculated once all secondary cases are detected and thus it is retrospective ( , ). to maintain precision, rt and rc were calculated for each day of the outbreak over a -day time window ending on that day.( ) details of these methods are available elsewhere ( , ). the estimation procedures were applied to outbreak notification data (incidence by the date of rash onset) using a serial interval for measles derived from household studies (gamma distribution with mean of . days and a standard deviation of . days) ( ). to evaluate the probable size and duration of the outbreak if control measures had not been introduced, we simulated potential outbreak trajectories using a continuous time stochastic susceptible-exposed- infectious-recovered compartmental model; stochastic variability was incorporated using the adaptive tau-leaping algorithm ( ). the model tracks classes of persons: ) susceptible to infection and disease (s), ) exposed but not yet infectious and asymptomatic (e), ) infectious with symptoms (i), and ) recovered and immune (r) (figure ). all persons in the s class can be infected at a rate λ(t), the force of infection, and move into the e class. exposed individuals then become infectious, and progress from the e class into the i class at a rate σ. finally, persons recover (i.e., become immune) and move from the i class into the r class at a rate γ. static model inputs are detailed in table . λ(t) is proportional to β, the per capita rate at which two individuals come into sufficient contact to lead to infection per unit time, and to the number of infectious individuals at time t (it). the rate β itself can be written as a combination of three parameters: the basic reproduction number r (the average number of cases generated by a single infectious individual if the entire population is susceptible), the duration of infectiousness, and the population size. r may vary considerably for the same disease in different populations ( ). however, r in a particular population can or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april be estimated from r and the ≥ -dose mmr vaccine coverage (vc), as follows: r=sr , where s is the proportion of the population that is susceptible, s=vc( -ve)+su( -vc), where ve is the median vaccine effectiveness of dose of mmr ( %) ( ), and su is the proportion of unvaccinated individuals that are susceptible (i.e., not immune through previous exposure), so that r =r/[vc( -ve)+su( -vc)]. immunization levels in the amish community were unknown. thus, we modeled two distinct scenarios, using a lower and upper bound in vc. a lower bound in vc of % was derived from a coverage assessment in a subset of affected amish families ( households with measles cases selected by convenience sampling, totaling individuals) ( ); these data were obtained through review of vaccination cards and of the ohio immunization registry (impactsiis) ( ). an upper bound in vc of % was obtained from a recent study showing that this was proportion of amish children in holmes county, ohio that were reported to have received ≥ doses of any vaccine ( ). the estimate of su was based on a measles attack rate of . % among exposed unvaccinated family members, part of a household transmission study conducted during the outbreak. lower and upper bounds in vc were then used to calculate the corresponding r and β parameters to model a range of possible scenarios. reassuringly, our r estimates ranged between - (table ), consistent with prior estimates for measles in various settings (ranging between - ) ( ). of note, in any given population with a particular contact pattern, the transmissibility potential of measles would be described by a single r value; the true r for this community is likely somewhere in between the range estimated. the σ and γ parameters are inversely proportional, respectively, to the pre-infectious period (the time period between infection and onset of infectiousness), and to the duration of infectiousness. we assumed random mixing and a finite population size of , persons – the estimated amish population in the affected settlement during ( ). due to natural (and maternal) immunity, as well or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april as vaccine coverage prior to the outbreak, a proportion of the population was assumed to be recovered at the outset, bypassing the s class, although they were allowed make contacts and thus were accounted for in the calculation of the incidence. to assess the impact of the vaccination campaign, we queried impactsiis for the number of doses of mmr given at local health departments in affected counties during the time vaccine clinics were offered (april -july , ). unvaccinated individuals who received mmr were removed from the s class and added to the r class based on day of vaccine receipt; during broad vaccination campaigns, the vaccine may reach unvaccinated individuals before or around the time of exposure ( , ), and when administered within hours after exposure, mmr vaccine can protect or modify the clinical course of measles ( , ). five hundred iterations of the model were run for each of the two scenarios, in the absence and presence of the vaccination campaign. the median size and duration of predicted outbreak trajectories, and the corresponding daily changes in rc, are presented and compared to what was observed. we modeled transmission in the affected amish community only, without potential spill over to the general (non- amish) population, where immunity levels are high and almost no measles spread was seen ( ). to evaluate the appropriateness of using early estimates of r to inform the susceptible-exposed- infectious-recovered model, we compared the expected (as predicted by the model) and the observed number of cases during the first days of the outbreak, prior to initiation of the control measures. sensitivity analyses were conducted to examine ( ) the choice of time window width used to estimate rt, ( ) the use of symptom onset instead of rash onset to estimate r, ( ) the impact of advancing or delaying the vaccination campaign by week (to evaluate delays in vaccine protection [immunologic response] and outbreak response), ( ) a range of measles vaccine effectiveness ( . % to . %) at baseline ( ), ( ) a measles vaccine effectiveness of . % for campaign doses ( ), and ( ) a shorter infectious period of days ( ). or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april analyses were performed in r . . . since the investigation was part of a public health response, it was not considered by the centers for disease and control to be research, and was designated as exempt from human subject policy. results the outbreak affected one of the largest amish communities in north america, located in the holmes county, ohio area ( ). a total of confirmed measles cases were reported over days. vigorous containment efforts were instituted by local health departments to limit measles spread, including the delivery of mmr doses to , unvaccinated individuals ( ). the epidemic curve and the estimated rt and rc are shown in figure . rt increased from an initial value of . ( % confidence interval (ci): . - . ) at the end of the third week to a maximum value of . ( % ci: . - . ) in the mid-fifth week, then varied between . and . from the mid-sixth week to the mid-seventh week. thereafter, as the vaccination campaign started to get under way, estimates declined over two weeks, with rt falling below by the mid-ninth week. as the campaign continued, rt remained below during the next months, except at the very end of the epidemic, when it increased from a minimum value of . ( % ci: . - . ) to . ( % ci: . - . ). this late increase in rt occurred after introduction of measles into a single, unimmunized family consisting of six individuals, four of which developed measles. similar patterns in transmissibility were observed with rc, i.e., high initial values with variability, a steady decrease as control measures started, and maintenance below thereafter. the distribution of the expected number of cases (as determined by the model) during the first days of the outbreak, prior to initiation of control measures, captured well the number of observed cases; the observed and projected daily case incidence tracked each other well in the early stages of the outbreak (figure b), and the number of observed cases consistently fell between the th and th percentiles of the predicted data, for the range of scenarios that were evaluated. or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april the range of possible outbreak sizes and durations projected by the model are shown in table . under the first scenario, assuming an initial vc of %, the model estimated approximately , measles cases presenting over days if no control efforts had been introduced, and approximately , measles cases presenting over days when including the vaccination campaign; the model attributes ~ , fewer cases to vaccination efforts. under the second scenario, assuming an initial vc of %, the model estimated approximately , measles cases presenting over days if no control efforts had been introduced, and approximately measles cases presenting over days when including the vaccination campaign; ~ , fewer cases and fewer outbreak days were attributed to vaccination efforts. results based on anecdotal immunization levels in the community of - % reported by local health departments, are presented in figure . assuming an initial vc of %, in the absence of containment measures, the model predicts an outbreak with a median of approximately , cases. when the vaccination campaign is included, the model predicts a smaller outbreak, with a median of approximately , cases (figure a); , fewer cases are attributed to vaccination efforts. when comparing model predictions that include the vaccination campaign with what was observed (figure b), an excess ~ , cases were projected by the model, which may be accounted by other factors (e.g., changes in community behavior including social distancing, and other control efforts such as isolation and quarantining). a comparison of the changes in the observed and projected daily estimates of rc are shown in figure . the observed rc trajectory (which represents the effect of the vaccination campaign, other control efforts, and changes in community behavior) indicated a rapid decline in measles transmissibility over time. by contrast, declines in transmissibility were slower from a depletion of susceptible individuals from infection plus the vaccination campaign (model with the vaccination campaign), and from a depletion of susceptible individuals from infection alone (model without the vaccination campaign). or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april sensitivity analyses found that varying several of the assumptions in this evaluation resulted in little change on the overall patterns of measles transmissibility; these include the choice of time window used to estimate r (web figure ), the use of daily counts of onset of symptoms (instead of onset of rash) to estimate r (web figure ), an evaluation of a range of measles vaccine effectiveness at baseline (web table ), an evaluation of the effect of vaccination assuming an effectiveness of . % for campaign doses (web table ), and an evaluation of delays in vaccine protection (immunologic response) and outbreak response (web table ). discussion through monitoring of measles communicability during this outbreak, we show that containment efforts likely contributed to reducing measles spread, and demonstrate that launching comprehensive and timely public health responses can help avert large outbreaks from occurring in under-immunized populations. these findings corroborate previous results of an individual-based model, also showing the potential of measles spread to other north american amish communities in the absence of outbreak responses ( ). as containment measures started to get underway, we found a ~ -fold reduction in transmissibility (rt declined from . to ) over weeks, and subsequent maintenance of rt below unity as control measures continued. based on the observed epidemic curve, in the absence of vaccination or behavioral changes, cases could have continued to double approximately every days in the early stages of the outbreak (web appendix), and assuming a conservative scenario (initial vaccination coverage of %), the number of affected individuals might have increased to ~ , , i.e., > times the number of cases observed ( cases). outbreaks of this magnitude have not been seen in the us since elimination was declared.( ) based on hospitalization rates for measles in this community ( ), and post-elimination measles case-fatality ratios ( ), such an outbreak could have resulted in ~ hospitalizations and ~ deaths ( hospitalizations and no deaths were reported during the outbreak). or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april evidence supporting measles outbreak response immunization is increasing. a review of vaccination during outbreaks in middle- and low-income countries noted an impact in ( %) of papers ( ), and updated who guidelines recommend this strategy in countries with measles mortality reduction goals ( ). fewer studies have evaluated the benefits of vaccination during outbreaks in elimination settings ( , - ), where background immunization coverage is high and outbreaks occur in under-vaccinated subpopulations. in such evaluations, it is often challenging to account for a depletion of susceptible persons from infection, or for other aspects related to outbreak control and community behavior – isolation of cases, quarantining of susceptible contacts, and self-imposed social distancing, e.g., limited attendance to church gatherings or social events because of measles awareness. our results suggest that the vaccination campaign could not have accounted entirely for the observed decrease in transmissibility (figure ). by immediately reducing the number of susceptible contacts each ill individual makes, these other factors seem to play an important role. based on an initial coverage of ~ %, we show that ~ , fewer cases might be attributed to community engagement and behavioral changes. yet, any factor that affects the force of infection during an outbreak, including spatial and social heterogeneity in mixing, unevenness in mmr coverage, or varying effects of control interventions in different areas (e.g., targeting primarily those exposed), could also have curbed transmission. data on each of the components of outbreak response (particularly isolation and quarantining) are needed to disentangle their relative effectiveness ( ), and quantifying and modeling the dynamics of behavior change in response to epidemics is a particular challenge and priority. our evaluation highlighted a few interesting aspects of measles transmissibility and outbreak control. first, considerable variability in transmission was evident early during the outbreak; estimates of rc varied between . and . before interventions began. this variability may be an artifact of the estimation method resulting from an initial low number of incident cases ( ), or a reflection of differences in the contact rates of the first several case-patients. the latter could be an observation bias – or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april if the first few cases generate many other cases, the outbreak gets past the chance of initial extinction and can spread beyond the initial cluster. second, cases continued to occur for ~ months after r fell below , indicating that sustained transmission can occur when r is near unity, and that both elimination efforts and outbreak control measures should aim to reduce r as close to as possible. a similar effect has been observed in a previous outbreak of influenza ( ) and could be the effect of the interaction with the behavioral or public health response, where cases cause small clusters of secondary cases that are largely contained but might seed other sub-outbreaks elsewhere ( ). third, measures to control an exceedingly contagious disease like measles are likely more successful when the susceptible population is embedded in a general population with high mmr vaccine uptake. the measles containment strategies ( ) implemented during this outbreak could serve as a guide on how to halt propagation of the disease in non-immunized subpopulations in elimination settings. in the region of the americas, for example, despite -dose measles vaccine coverage being maintained at ≥ % since ( ), and a declaration of measles elimination in ( ), recent outbreaks reported in ecuador, canada ( ), the us ( , ), and brazil ( ), indicate that coverage is not homogenous. these outbreaks ranged considerably in size and duration ( to , confirmed measles cases, weeks to . years long), and were characterized by varying containment efforts. in the us, reports of measles cases are expected within hours of confirmation ( ), triggering the implementation of enhanced surveillance, and of measures to limit spread. key elements to curtail transmission include isolation of cases until no longer infectious, vaccination of susceptible contacts, and quarantining of susceptible contacts who cannot be vaccinated ( ). our analysis has several limitations. first, often all causes of heterogeneity cannot be accounted for in models. measles was reported in nine ohio counties ( ), and homogenous mixing does not account for more complex spatial patterns of spread in this community, or for preferential mixing by age. however, at least part of the heterogeneity in contact rates is captured by the initial r values, which we then use to or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april get the projected model estimates. similarly, any underlying heterogeneity in immunity was unknown, yet, a broad range of coverage scenarios were modeled, and an impact of controls measures was evident under a conservative scenario. of note, the model also assumes a homogenous effect of vaccination efforts, which may underestimate their impact. in addition, final outbreak sizes and the impact of the interventions depend on the estimated population at risk, and we did not measure the impact of the response in limiting spread to other amish communities, including outside ohio ( ). because of these caveats, the trajectories we present should not be viewed as exact projections, but characterize probable trends in transmissibility and in the potential for control. second, case under-ascertainment might have occurred, however, enhanced surveillance, widespread knowledge of the outbreak, and established relationships with the community likely improved case identification ( ). importantly, estimates of r would not be affected as long as surveillance does not change considerably during the outbreak ( , ), and sudden decreases or increases in case reporting were unlikely. third, we chose an infectious period of days based on outbreak control guidelines ( , , ), which may be closer to maximum duration of infectiousness, and long for models assuming this period is the mean of an exponential distribution. however, the number of cases prevented by vaccination using a shorter infectious period ( ) was still significant, and our base model tracked the initial outbreak trajectory better (web table ). fourth, we did not account for imperfect or delayed vaccine protection from campaign doses. however, sensitivity analyses using a lower effectiveness ( ), and delaying vaccine protection by week, did not have a substantial impact on our findings. finally, this is an evaluation of a single outbreak and our findings may not be generalizable to all communities where importations occur, and the assumption of homogenous mixing may not be applicable to other measles outbreaks in post-elimination settings. the findings in this report demonstrate the substantial public health impact of rapid measles containment efforts in an unvaccinated community in an elimination setting. our results reinforce who’s measles elimination strategy, that includes outbreak preparedness as one of the core components to achieve or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april elimination targets in of the who regions by ( ). measles elimination is a fragile state ( ) and the data provided here may serve as an impetus for local and international health organizations to allocate resources to build and maintain capacity for measles outbreak readiness, including in countries where measles incidence is sufficiently low or elimination has been achieved. the single best means of measles containment, however, is maintaining high initial levels of vaccination coverage across the population. or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april affiliations and acknowledgments affiliations: division of viral diseases, centers for disease control and prevention, atlanta, georgia (paul a. gastañaduy and gregory s. wallace); division of nutrition, physical activity, and obesity, centers for disease control and prevention, atlanta, georgia (prabasaj paul), centre for the mathematical modelling of infectious diseases, london school of hygiene and tropical medicine, london, united kingdom (sebastian funk), ohio department of health, columbus, ohio (lilith tatham, nicholas fisher, jeremy budd, brian fowler, sietske de fijter, and mary diorio); department of ecology and evolutionary biology, princeton university, princeton, new jersey (bryan grenfell). funding information: this work was supported by the uk medical research council (fellowship mr/k / to sebastian funk); and by the bill and melinda gates foundation (grant opp ), the rapidd program of the science and technology directorate, u.s. department of homeland security, and the fogarty international center, national institutes of health (bryan grenfell). conflicts of interest: none declared. acknowledgments: we are indebted to the personnel who led case investigations and vaccination clinics at the following departments of health in jurisdictions affected by the outbreak: ashland county city health department, coshocton county health department, holmes county health department, knox county health department, richland county public health, and wayne county health department. disclaimer: the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical 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(accessed july ). or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april http://www.paho.org/hq/index.php?option=com_content&view=article&id= :region-americas-declared-free-measles&itemid= &lang=en http://www.paho.org/hq/index.php?option=com_content&view=article&id= :region-americas-declared-free-measles&itemid= &lang=en http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/ vol /acs-dcc- /assets/pdf/meas-roug-eng.pdf http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/ vol /acs-dcc- /assets/pdf/meas-roug-eng.pdf http://www.health.gov.au/internet/main/publishing.nsf/content/bd ad fd bfd ca bf d c /$file/measles-song-final-april .pdf http://www.health.gov.au/internet/main/publishing.nsf/content/bd ad fd bfd ca bf d c /$file/measles-song-final-april .pdf https://www.cdc.gov/measles/downloads/expert-panel-elimination-measles.pdf https://www.cdc.gov/measles/downloads/expert-panel-elimination-measles.pdf figure . schematic representation of disease states, flow between states, and parameters controlling flow in the measles model. the model represents a constant (closed) population in which individuals are either susceptible (s) or recovered (r) to measles infection and disease, and into which a measles introduction occurs (i.e., a number of infectious individuals (i) is introduced). persons in the susceptible pool become exposed at the force of infection λ(t), and then progress through the exposed pre-infectious (e) and the infectious (i) stages, before arriving in the removed compartment (r), where individuals are immune. the symbols σ and γ denote the rates at which individuals progress into the i and r compartments, respectively. the model tracks, each day, the number of individuals in each of the compartments, and incorporates stochasticity using the adaptive tau-leaping algorithm.( ) the effect of the vaccination campaign is represented by θ, the number of unvaccinated individuals who received a dose of mmr during containment efforts. unvaccinated individuals are removed from the s compartment and added to r compartment (bypassing e and i) based on the day mmr was administered. figure . a) the daily epidemiologic curve; the daily total numbers of confirmed outbreak-associated measles case-patients in ohio in according to day of rash onset are shown (n= ); for three case- patients, the rash onset date could not be determined, and the illness onset date + days (the median number of days between illness onset and rash for all other cases) is shown; one lab-confirmed case- patient did not develop rash and the date of illness onset is shown. b) daily estimates of the instantaneous reproduction number rt over sliding -day windows; the black line shows the median estimate, the gray areas the % confidence intervals, and the horizontal dashed line the threshold value rt= ; c) daily estimates of the case reproduction number (rc) over sliding -day windows; the red circle shows the mean estimate, the bars the % confidence intervals, and the horizontal dashed line the threshold value rc= . as expected, estimates of rc were ahead of the estimates of rt, with the highest estimate of rc occurring around the end of the third week, about one serial interval ( - days) before the peak in rt; the peak in rt in the mid-fifth week indicates increased transmissibility among cases with rash onset one generation before.( ) superimposed in all figures is the cumulative number of daily doses of mmr vaccine given at local health department vaccination clinics during the outbreak. figure . projected and observed daily measles case incidence assuming an initial vaccination coverage of %. one hundred iterations of the modeled epidemic curves are presented; dashed lines show the median daily measles case incidence of all iterations. panel a compares model trajectories with and without the vaccination campaign. panel b compares the model trajectories including the vaccination campaign to the observed epidemic curve (that included other control measures). note that the scale of the axes differ. cumulative case incidence was as follows: model without vaccination, , ( % confidence interval [ci] , , , ) cases over ( % ci: , ) days; model with vaccination campaign, , ( % ci: , , , ) cases over ( % ci: , ); observed with control interventions, cases over days. figure . panel a shows the observed and projected daily estimates of the case reproduction number (rc) over sliding -day windows; the black and blue circles show the median rc of model trajectories without and with the vaccination campaign, respectively, assuming an initial vaccination coverage of %. the red circles show the observed mean rc estimate (that included other control measures, changes in community behavior), the red bars represent the % confidence intervals, and the horizontal dashed line indicates the threshold value rc= . observed and projected rc estimates were derived from the likelihood-based estimation procedure and directly from the models, respectively. panel b shows the proportion of decline in rc attributable to changes in community behavior (social distancing) and other control efforts (isolation, quarantining) during the outbreak; data presented are or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april from the initiation of containment efforts, to the time when projected estimates of rc fall below . the reduction in transmissibility that may be ascribed to these factors varied between ~ - % during the outbreak. or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april table . fixed input parameters for each model scenario parameter symbol formula value references transmission probability β r /nd scenario a : . * − per day na transmission probability β r /nd scenario b : . * − per day na basic reproduction number r r/s scenario : . na basic reproduction number r r/s scenario : . na r of cases prior to initiation of control measures r na . estimates likelihood-based estimation procedure( , ) proportion of the population that is susceptible at outset s vc ( −ve c )+su d ( −vc) scenario : . na proportion of the population that is susceptible at outset s vc ( −ve c )+su d ( −vc) scenario : . na population size n s + e + i + r , young center for anabaptist and pietist studies, elizabethtown college( ) average pre- infectious or latent period na na days cdc measles surveillance manual and acip recommendations( , ) average duration of infectiousness d na days cdc measles surveillance manual and acip recommendations( , ) rate at which individuals become infectious σ /average pre- infectious or latent period . per day na recovery rate γ /average duration of infectiousness . per day na na, not applicable; vc, ≥ -dose mmr coverage; ve, median vaccine effectiveness of dose of mmr; su, proportion of unvaccinated individuals that are susceptible; cdc, centers for disease control and prevention; acip, advisory committee on immunization practices a assuming ≥ -dose mmr coverage of % to calculate s (lower bound) – from a coverage assessment in a subset of affected amish families( ) b assuming ≥ -dose mmr coverage of % to calculate s (upper bound) –from the literature( ) c ve= %( ) d unvax.s= . % – from household transmission study or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april table . model predictions of measles outbreak sizes and durations in an amish community in ohio in , with and without the vaccination campaign a , and based on two initial levels (lower and upper bounds) of mmr coverage prior to initiation of containment efforts assumed mmr coverage b no. of measles case-patients duration of outbreak (days) absolute reduction vaccination campaign included no yes no yes no. % ci no. % ci duration (days) % ci duration (days) % ci no. % ci duration (days) % ci % (lower) , , , , , , , , , , , , , , − − , % (upper) , , , , , , , , , , , , , a county health department clinics offering vaccination were held from day to day of the outbreak; first doses of mmr were delivered to , unvaccinated individuals b ≥ -dose mmr coverage c values are the medians and % confidence intervals (ci) generated from model simulations or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april s e r i λ(t) σ γ θ or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april , , , , , d o se s o f a n  m m r  v a ccin e c a se  r e p ro d u ct io n  n u m b e r or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april or ig in al u ne di te d m an us cr ip t downloaded from https://academic.oup.com/aje/advance-article-abstract/doi/ . /aje/kwy / by london school of hygiene & tropical medicine user on april 	manu_aje_ . . 	figure 	figure_ 	figure_ _a_b 	figure_ _a_b a day in the life: matt ruark error_outline javascript disabled you have to enable javascript in your browser's settings in order to use the ereader. or try downloading the content offline download journal of the college of physicians and surgeons pakistan , vol. ( ): - introduction ellis-van creveld syndrome is a rare form of mesenchymal-ectodermal dysplasia. it has an autosomal recessive inheritance caused by evc gene located at p . evc gene has also been described as head on head mutation at same location. this is a rare condition with high family clusters reported in an old order amish community of lancaster county, pennsylvania, usa. around cases have been reported in literature presenting with features of chondroectodermal dysplasia. the classical tetrad includes chondro- dystrophy, polydactyly, ectodermal dysplasia and cardiac anomalies. other clinical findings frequently reported with the syndrome include oral (absence of mucobuccal fold, short upper lip, serrated lower alveolar ridge), dental (neonatal teeth, partial anodontia, enamel defects, malocclusion) and genitourinary anomalies. uncommon findings reported in literature include cor triatrium, double orifice mitral valve, syndactyly, airway cysts, lung anomalies, strabismus, retinitis pigmentosa and dyserhythropoiesis. early deaths have been reported during infancy associated with respiratory problems mainly due to severe chest narrowness or congenital cardiac lesions. this report presents two cases of this rare dysplasia with some unusual features. case report case was a -year-old boy who presented with decreased vision in both eyes for the last months. he belonged to consanguineous parents, having intrauterine growth restriction in antenatal period and presently short for his age. no other siblings had any similar complaints. he was disproportionately short ( . cm with us/ls: . ), having irregular dentition, abnormal shaped teeth, absent mucobuccal fold, hypoplastic dysplastic nails, narrow chest, bilateral postaxial polysyndactyly both hands, inability to form fist, genu valgum deformity, and hypospadias (figures , ). he also had a bulging pre-cordium with grade / pansystolic murmur at left sternal border. skeletal survey revealed rhizomelic limb shortening, bony ulnar polysyndactyly, fused carpel bones, small iliac crests and dome shaped upper end of tibia. orthopantogram showed absent permanent dentition. echocardiography showed complete atrioventricular septal defect with abnormal tricuspid valve. ultra- sonography revealed no structural abnormality of urinary tract. eye evaluation revealed bilateral posteriosuperior lens subluxation. developmental profile showed fine and motor age of months, cognition of months and language of months using portage developmental assessment materials. case was an -year-old boy presenting with recurrent chest infections and failure to thrive since years of age. he belonged to related parents with no adverse prenatal, natal or postnatal history. he was the only sibling with history of two neonatal deaths of unknown cause. he was a developmentally delayed, dispro- portionately short boy ( cm with us/ls: . ), with absent maxillary central incisors, serrated alveolar ridge, abnormal shaped teeth, absent mucobuccal fold, dystrophic nails, narrow chest, post axial polydactyly, genu valgus deformity and wide gap between st and nd toe (figure ). he had a bulging precordium with right ventricular heave and grade / ejection systolic murmur in the left upper sternal border. abstract ellis-van creveld syndrome is a rare form of mesenchymal - ectodermal dysplasia. it is an autosomal recessive disorder characterized by disproportionate short stature, postaxial polydactyly, ectodermal dysplasia and congenital heart defect. this case report presents two cases with classical clinical findings along with some unusual features including rhizomelic limb shortening, global developmental delay and bilateral lens subluxation not reported previously. key words: skeletal dysplasia. short stature. cardiac defect. ellis-van creveld syndrome. mesenchymal-ectodermal dysplasia. lens subluxation. department of paediatric medicine, unit- , children’s hospital and institute of child health, lahore. correspondence: dr. ahmad usaid qureshi, -a, gor- , faisal town, lahore. e-mail: qureshiahmad@yahoo.com received january , ; accepted august , . disproportionate short stature with multisystem involvement – ellis-van creveld syndrome ahmad usaid qureshi, yasser masood and agha shabbir ali case report skeletal survey revealed rhizomelic limb shortening, funnel chest, bony ulnar polydactyly, small iliac crests and dome shaped upper end of tibia. orthopentogram confirmed absence of maxillary incisors and malformed permanent dentition. echocardiography revealed septum primum atrial septal defect with moderate pulmonary hypertension. developmental profile found gross and fine motor skills at months, cognition at months and language at months using childhood adaptive behaviour scale for children. discussion the condition was first described by richard ellis and simon van creveld in . in the general population, the incidence is one in , live births and much lower in the asian population. only around cases have been reported from the indian subcontinent. parental consanguinity among the reported patients is as high as %. the reported cases had the classical tetrad with certain unusual features. the chondrodystrophy results in disproportionate short stature and mesomelic limb shortening. both reported cases had rhizomelic and acromelic limb shortening sparing the forearms. post- axial polydactyly was present in both children. case also had polysynmetacarpalism. ectodermal dysplasia involving dysplastic nails and dental anomalies were present in both cases. case also had serrated upper alveolar ridge while previous reports showed mostly mandibular ridge serration. cardiac anomalies in both cases were atrioventricular canal defect. narrow chest and genu valgum deformity commonly observed in this syndrome were present in both cases. genitourinary abnormalities are occasionally found including hypoplastic penis, renal agenesis and megaureter. hypospadias found in case was also among rare findings described with the syndrome previously. mental retardation documented in a few cases was secondary to cns structural anomalies. both cases had global delay in all fields of development despite the absence of any cns anomaly on neuroimaging. mild mental retardation has been documented in one pedigree with allelic deletions. significant developmental delay has not been described with this syndrome as well. motor development comparatively more affected due to skeletal abnormalities. case had bilateral lens subluxation which is not previously reported with the syndrome. identification of the obvious dysmorphic features of this syndrome as early as possible is essential to prevent early deaths due to chest narrowness or congenital cardiac defects. planning appropriate interventions for dental anomalies, cardiac defects and infective endocarditis prophylaxis is only possible with early identification. there has been a reported case of ellis van creveld syndrome where dental procedure was done without infective endocarditis prophylaxis as the cardiac lesion was never diagnosed. in case , the atrial septal defect was left unnoticed and the patient had started to develop pulmonary hypertension. growth hormone therapy may improve height. limited data does not allow any conclusions but the few patients studied have shown improved growth velocity with hormone therapy. early detection of a constellation of features suggestive of ellis-van creveld syndrome is essential for establishing accurate diagnosis and early management as the condition involves multiple systems with little variation. references . tompson sw, ruiz-perez vl, blair hj, barton s, navarro v, robson jl, et al. sequencing evc and evc identifies mutations in two thirds of ellis-van creveld syndrome patients. hum genet ; : - . epub sep . . kurian k, shanmugam s, harsh vardah t, gupta s. chondroectodermal dysplasia (ellis-van creveld syndrome): a report of three cases with review of literature. indian j dent res ; : - . . cahuana a, palma c, gonzáles w, geán e. oral manifestations in ellis-van creveld syndrome: report of five cases. pediatr dent ; : - . journal of the college of physicians and surgeons pakistan , vol. ( ): - ahmad usaid qureshi, yasser masood and agha shabbir ali figure : case having disproportionate short stature, rhizomelic limb shortening, narrow chest, genu valgus, nail hypoplasia, hypospadias and post axial bony polysyndactyly. figure : case showing serrated alveolar ridge and abnormal dentition. figure : case showing absent mucobaccal fold. . chakraborty pp, bandyopadhyay d, mandal sk, subhasis rc. a rare variant of ellis-van creveld syndrome. singapore med j ; : - . . khan i, ahmed sa, kiren m. ellis-van creveld syndrome: a case report. j pak assoc derma ; : - . . temtamy sa, aglan ms, valencia m, cocchi g, pacheco m, ashour am, et al. long interspersed nuclear element- (line )- mediated deletion of evc, evc , c orf , and stk b in ellis- van creveld syndrome with borderline intelligence. hum mutat ; : - . . versteegh fg, buma sa, costin g, de jong wc, hennekam rcm; evc working party. growth hormone analysis and treatment in ellis-van creveld syndrome. am j med genet ; a: - . journal of the college of physicians and surgeons pakistan , vol. ( ): - ellis van creveld syndrome l l l l l ol l l l l [pdf] amish enterprise: the collective power of ethnic entrepreneurship | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /gber. . corpus id: amish enterprise: the collective power of ethnic entrepreneurship @article{kraybill amishet, title={amish enterprise: the collective power of ethnic entrepreneurship}, author={donald b. kraybill and s. nolt and erik j. wesner}, journal={global business and economics review}, year={ }, volume={ }, pages={ - } } 	donald b. kraybill, s. nolt, erik j. wesner 	published 	economics 	global business and economics review this paper examines how amish communities build and sustain enterprises that produce and/or sell goods to both ethnic and non-ethnic markets. based on qualitative research including interviews with amish entrepreneurs in communities in the usa, the authors develop a transformative model of ethnic community entrepreneurship. the analytical model conceptualises the dynamic interaction between three forces/agents – cultural constraints, cultural resources, entrepreneurs – and shows how they… expand view via publisher users.etown.edu save to library create alert cite launch research feed share this paper citationshighly influential citations background citations view all figures from this paper 	 figure citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency navigating ethnic entrepreneurship in religion and culture meld 	s. ojo 	sociology 	 	 save alert research feed the confluence of religion and ethnic entrepreneurship in the informal economy 	s. ojo 	sociology 	 	 save alert research feed diaspora entrepreneurship : a 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research feed sources of enterprise success in amish communities 	donald b. kraybill, s. nolt, erik j. wesner 	sociology 	 	 	pdf save alert research feed ... ... related papers 	abstract 	figures 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue books: chasing the sun: the new science of sunlight and how it shapes our bodies and minds chasing the sun: the new science of sunlight and how it shapes our bodies and minds linda geddes wellcome collection, , pb, pp, £ . , - let there be light the sun has had bad press. as gps we correctly declaim grim warnings to our patients about skin cancer. we rightly warn of the sharp rise in malignant melanomas, the risks of sunbathing on holidays, and frequenting those tanning salons that benight our high streets. on the other hand, we are aware of the sun’s benefits in the production of vitamin d. my results inbox daily has at least one patient with low vitamin d, and we seem to be testing it for a range of conditions, physical and psychological, with a corresponding increase in prescribed vitamin d supplementation. the health benefits of sunlight have been known for centuries. hippocrates built a solarium at his treatment centre on the greek island of kos, and a fellow physician, aretaeus of cappadocia, recommended sunlight for ‘lethargics.’ more recently, florence nightingale would position patients close to windows, arguing that ‘second only to their need of fresh air is their need for light … not only light but direct sun-light’. in the early th century, niels finsen developed light therapy for the treatment of skin tuberculosis, and robert koch showed that the bacterium responsible for tb could be killed by sunlight. there followed a fashion for heliotherapy, until the rise of critical voices such as those of the british surgeon john lockhart-mummery, who, in his book nothing new under the sun, dismissed sunlight therapy as ‘pseudo-magic’. linda geddes argues that we are missing the point in this age-old binary discussion, and instead we should focus on the integral role of the sun in the development of our circadian rhythms. this starts in utero, but, as all new parents are well aware, circadian rhythms do not fully develop until months after birth, and they continue to evolve throughout life. in adolescence the biological rhythms are shifted later, making it harder for teenagers to fall asleep at night and get up in time for school. adults generally have well-developed rhythms, with defined changes in body temperature throughout the day and night. in order to illustrate the importance of circadian rhythms for our health, geddes travelled far and wide. she starts with the amish, an american people whose rhythms are completely dictated by the sun. she describes a yard-sale at . am: ‘already a man with a chin-curtain beard, and the distinctive amish uniform of straw hat, plain shirt and braces, is firing up a barbecue, and the smell of smoke and grilled chicken intermingle with the sweet smell of desserts’. living their lives in the outdoors, the amish are exposed to far greater levels of natural light than the rest of us, and geddes suggests that this may help account for their lower levels of depression. in complete contrast, geddes describes her trip to a conference in las vegas in when, suitably, the international agency for research on cancer added night-shift work to the list of ‘probable’ human carcinogens. she describes her experience of vegas as lurching ‘like a confused moth, through underground malls and vast casino floors, my sense of time becoming ever more distorted’. she explains that exposure to bright light at night ‘forces the body to feel alert when it should be sleeping, setting off a cascade of damaging effects’. as an example of how we can all re-engage with our circadian rhythms, geddes travels to the german spa town of bad kissingen, bavaria, historically a place of health and healing, which has now refashioned itself along the lines of ‘chronobiology’. guided by the ‘chronobiologist’ thomas kantermann, the town starts schools later in the day, holds classes outdoors, and encourages businesses to offer flexitime for the larks and owls in their workforce. most controversially, bad kissingen has proposed unilaterally abandoning daylight saving time, which curtails teenagers’ sleep even further, and may well have an adverse effect on their exam results. geddes ends her travels where she began, at stonehenge. there she describes the -year-old understanding we have had of the sun and its importance for the ‘circularity of our biology’. justifiably, after writing a book full of complex biomedical theories intertwined with delightful history and travel writing, we learn that she has graduated from a mere visitor to a guest of the cotswold order of druids. rhodri evans, gp, newport, south wales. email: evans.rhodri@gmail.com doi: https://doi.org/ . /bjgp x * * * * * did he save lives? a surgeon’s story david sellu sweetcroft publishing, , pb, pp, £ . , - a miscarriage of justice one ordinary evening in february an experienced consultant colorectal surgeon saw his last patient of the day, a -year old retired builder who had developed abdominal pain shortly following an elective knee replacement. three years later on a cold november day david sellu was sentenced to prison for and a half years for unlawfully killing john hughes. did he save lives? charts the events between these two dates that would lead to the conviction of a surgeon with a previously unblemished record of gross negligence manslaughter. sellu’s methodical, sparse yet descriptive prose depicting the day-to-day of prison life belies the quiet horror of a life stripped of freedom, autonomy, and dignity. born in a rural village in sierra leone to illiterate farmers it was not in sellu’s destiny to become an eminent surgeon in england. life & times books british journal of general practice, february doi: . /j.ajhg. . . article phosphodiesterase b gene variants are associated with serum tsh levels and thyroid function lisette arnaud-lopez, , gianluca usala, , graziano ceresini, braxton d. mitchell, maria grazia pilia, maria grazia piras, natascia sestu, andrea maschio, fabio busonero, giuseppe albai, mariano dei, sandra lai, antonella mulas, laura crisponi, toshiko tanaka, stefania bandinelli, jack m. guralnik, angela loi, lenuta balaci, gabriella sole, alessia prinzis, stefano mariotti, alan r. shuldiner, , antonio cao, david schlessinger, manuela uda, gonçalo r. abecasis, ramaiah nagaraja, serena sanna, and silvia naitza ,* thyroid-stimulating hormone (tsh) controls thyroid growth and hormone secretion through binding to its g protein-coupled receptor (tshr) and production of cyclic amp (camp). serum tsh is a sensitive indicator of thyroid function, and overt abnormalities in thyroid function lead to common endocrine disorders affecting ~ % of individuals over a life span. by genotyping , snps in , sar- dinians, we identified a strong association (p ¼ . � ) between alleles of rs and circulating tsh levels; each additional copy of the minor a allele was associated with an increase of . miu/ml in tsh. the single-nucleotide polymorphism (snp) is located in intron of pde b, encoding a high-affinity camp-specific phosphodiesterase. the association was replicated in , individuals, including additional sardinians and two genetically distant cohorts from tuscany and the old order amish (overall p value ¼ . � ). in addition to association of tsh levels with snps in pde b, our genome scan provided evidence for association with pde a and several biologically interesting candidates in a focused analysis of genes. in particular, we found evidence for association of tsh levels with snps in the thrb (rs , p ¼ . � ), gnaq (rs , p ¼ . � ), tg (rs , p ¼ . � ), pou f (rs , p ¼ . � ), pde d (rs , p ¼ . � ), and tshr (rs , p ¼ . � ) loci. overall, the results suggest a primary effect of pde b variants on camp levels in the thyroid. this would affect production of t and t and feedback to alter tsh release by the pituitary. pde b may thus provide a candidate target for the treatment of thyroid dysfunction. introduction the thyroid controls several metabolic pathways through the synthesis and release of thyroxine (t ) and its active derivative triiodothyroxine (t ), which binds to nuclear receptors to regulate gene expression and impact growth, development, and metabolism. consistent with its criti- cal role in muscle, bone, central nervous system, and heart physiology, abnormal thyroid function can lead to hyper- thyroidism, hypothyroidism, and related childhood neu- ropsychological abnormalities including severe cretinism. the key regulator of thyroid function is thyroid-stimu- lating hormone (tsh). secreted by the pituitary, tsh inter- acts with the tsh receptor (tshr) on thyroid cells to upre- gulate cyclic amp (camp) and induce ca þ release and activation of phosphoinositol. these effectors then lead to the expression of downstream gene targets, culminating with pinocytosis of thyroglobulin and the release of t and t , enhanced iodide uptake, and, on a longer time scale, growth and differentiation of thyroid follicular cells. tsh levels are themselves controlled in several ways. tsh production is promoted by thyrotropin-releasing hor- the american journal of human genetics , – , june mone (trh) and inhibited by somatostatin, both produced by the hypothalamus. , in addition, negative-feedback control on hypothalamic release of trh and pituitary re- lease of tsh is exerted by the blood levels of thyroid hormone. when the levels of t and t are low, the pro- duction of trh and tsh increases, and conversely, when t and t levels are high, trh and tsh production decreases. serum tsh concentrations are a sensitive indicator of thyroid function. high and low tsh levels reflect hypo- and hyperfunction of the thyroid gland, respectively. how- ever, even within the normal range, tsh is a sensitive measure of thyroid function, and normal (euthyroid) indi- viduals show narrow individual variation, suggesting that the thyroid-hormone axis is tightly regulated. tsh levels are genetically regulated and ~ % heritable in several populations; however, specific gene variants that influence tsh levels are not known. – to identify specific possible genetic factors affecting tsh levels and thyroid function, we studied a sardinian cohort in whom the founder-population structure can simplify genetic analyses of complex traits and diseases. this has laboratory of genetics, national institute on aging, baltimore, md , usa; istituto di neurogenetica e neurofarmacologia, consiglio nazionale delle ricerche, cagliari, italy; department of internal medicine, section of geriatrics, university of parma, parma, italy; division of endo- crinology, diabetes and nutrition, university of maryland school of medicine, baltimore, md , usa; clinical research branch, national institute on aging, baltimore, md , usa; geriatric rehabilitation unit, azienda sanitaria firenze (asf), florence, italy; laboratory of epidemiology, demography and biometry, national institute on aging, bethesda, md , usa; endocrinology, department of medical sciences ‘‘m. aresu,’’ univer- sity of cagliari, cagliari, italy; geriatric research and education clinical center, veterans administration medical center, baltimore, md , usa; center for statistical genetics, department of biostatistics, university of michigan, ann arbor, mi , usa these authors contributed equally to this work. *correspondence: silvia.naitza@inn.cnr.it doi . /j.ajhg. . . . ª by the american society of human genetics. all rights reserved. mailto:silvia.naitza@inn.cnr.it table . top snps associated with tsh levels from the gwa in sardinia affymetrixcode chr position dbsnp allele (þ/�) freq (þ) effecta seb h c p value snp_a- rs a/g . . . . % . � snp_a- rs g/a . . . . % . � snp_a- rs g/t . . . . % . � snp_a- rs a/c . . . . % . � snp_a- rs c/t . . . . % . � snp_a- rs t/c . . . . % . � snp_a- rs a/g . . . . % . � snp_a- rs a/c . . . . % . � snp_a- rs g/a . . . . % . � snp_a- rs t/a . . . . % . � snp_a- rs t/c . . . . % . � snp_a- rs a/g . . . . % . � snp_a- rs c/g . . . . % . � snp_a- rs t/c . . . . % . � snp_a- rs a/g . . . . % . � snp_a- rs g/a . . . . % . � snp_a- rs g/c . . . . % . � snp_a- rs a/t . . . . % . � snp_a- rs t/a . . . . % . � snp_a- rs a/t . . . . % . � snp_a- rs c/g . . . . % . � snp_a- rs a/g . . . . % . � snp_a- rs g/a . . . . % . � snp_a- rs c/t . . . . % . � snp_a- rs c/t . . . . % . � the table summarizes the top association signals observed in the gwa scan in the sardinian cohort. chromosome assignments and physical position refer to the ncbi build map. alleles are ordered such that the first allele (þ) is associated with increased tsh levels. snps in bold fall in the pde b gene region. a the effect size is measured in standard-deviation units and is estimated as the b coefficient of the regression model when the normalized trait is used (e.g., an effect size of . implies that each additional copy of the allele being evaluated increases trait values by . standard deviation). b se represents the standard error of the effect. c h represents the amount of phenotypic variability explained by the marker and, thus, under an additive model, the amount of the heritability of the trait explained by the marker. recently facilitated the finding of genes associated with susceptibility to asthma, obesity-related traits, uric acid, lipid levels, height, and severity of b-thalasse- mia. here we report analyses that point to common var- iants in pde b [mim ], a gene that encodes a high-affinity camp-specific phosphodiesterase (pde), as genetic modulators of tsh levels. the association was rep- the am licated in another group of sardinians and in samples of italians from tuscany and of old order amish from penn- sylvania. , furthermore, our sample provides evidence for association with single-nucleotide polymorphisms (snps) in the pde a gene [mim ], as well as thy- roid-hormone receptor, b (thrb) [mim ] (p ¼ . � ) and g protein, q polypeptide (gnaq) (p ¼ . � ) figure . results of genome-wide association scan for tsh levels for each marker, the �log of the p value resulting from an association test that evaluates its additive effect on the phenotype is plot- ted. the position of pde b is annotated. erican journal of human genetics , – , june figure . association with tsh levels and linkage-disequilibrium patterns in the region surrounding pde b (a) the top panel summarizes association between the snps and tsh levels in each individual (�log of the p value). the snp showing strongest association (rs ) is highlighted and indicated with a red square. other snps are colored according to their degree of disequilibrium with rs , ranging from high (red) to intermediate (green) to low (blue). r values of rs with rs and rs (red dots) are r ¼ . and r ¼ . , respectively. the transcript for all genes in the region is indicated in the next panel, with an arrow indicating transcript direction. (b) the two panels summarize the patterns of disequilibrium in sardinia and in the ceph from utah (ceu; utah residents with ancestry from northern and western europe) hapmap populations. r values are colored as in (a) with gold, which schematically draws the ld plot according to the physical position of markers, resulting in a line divided according to marker-marker distance. the gray bar marks the region of association and facilitates comparisons between the panels. the american journal of human genetics , – , june [mim ], among genes in a focused analysis of candidates for involvement in the dynamic regulation of thyroid function. overall, our results suggest that pde b, by affecting camp concentrations in the thyroid, may alter thyroid-hormone levels in the serum and affect tsh re- lease from the pituitary. pde b variants thus modulate thyroid physiology and may affect the course of thyroid disease. material and methods sample description we recruited and phenotyped , individuals, males and fe- males, ages – yr, from a cluster of four towns in the lanusei valley of sardinia. during physical examination, a blood sample was collected from each individual and divided into two aliquots. one aliquot was used for dna extraction and the other to charac- terize several blood phenotypes, including evaluation of serum tsh levels. tsh was measured with the siemens tsh assay (immu- lite ) according to the manufacturer’s instruction. the method is a solid-phase, chemiluminescent, competitive analog immunoassay and has analytical sensitivity of . miu/ml and upper limit of miu/ml of tsh. this method is a third-generation tsh assay and has a sensitivity and detection range comparable to the methods used to evaluate tsh levels in the other cohorts studied (see below). thyroid ultrasound examination was also performed on all the individuals enrolled in the study, with a portable real-time instru- ment using a . -mhz linear transducer. subjects were examined in the supine position, with neck hyperextended, by transverse and longitudinal scans to evaluate overall thyroid size and echo- texture. thyroid volume was calculated for each lobe according to the ellipsoid formula (length breadth width . ) (nor- mal range . . ml to . ml). goiter was scored when the total thyroid volume was above the mean thyroid volume. re- duction of thyroid volume associated with diffuse alteration of echotexture indicative of chronic thyropathies was also detected. presence, structure, size, and vascularization of nodules were de- termined by ultrasound and color-doppler sonography. records of self-reported thyroid disease status (i.e., autoimmune thyroid- itis, thyroid cancer, partial or total thyrectomy) and hormone- replacement therapy were available for all subjects. each participant signed an informed consent form. all study methods have been approved by the local ethics committee. gwas genotyping during the study, we genotyped , individuals selected from the whole sample to represent the largest available families, re- gardless of their phenotypic values. specifically, , were geno- typed with the k affymetrix mapping array set and , with the k mapping array set, with individuals genotyped with both arrays. this genotyping strategy allowed us to examine the majority of our cohort in a cost-effective manner because geno- types for the snps that passed quality-control checks could be propagated through the pedigree via imputation. , tsh mea- surements were available for , individuals among the , the am genotyped. a total of , snps passed initial quality-control checks and were tested for association with tsh levels with an additive model. further details of the strategy for imputation and data analysis are given in the statistical analysis section be- low. an additional , individuals were genotyped with the k mapping array set but were not included in the genome- wide association scan (gwas) because no close relatives were typed with the k mapping array set. these individuals were, however, included in the fine-mapping analysis. replication and fine mapping we designed a parallele custom chip (from affymetrix) to repli- cate and refine the regions associated with tsh levels as well as other traits studied in the sardinia project. , specifically, for the locus associated with tsh levels, we included markers that were chosen as snp tags of markers falling in the coding re- gions of the pde b gene and that also passed reliability criteria in the design phase. snp rs showed the strongest associa- tion with tsh levels in an initial analysis performed on a subset of phenotyped individuals and was thus included in the custom chip (instead of rs , which ranked second in the initial analy- sis). genotyping was performed in sardinian individuals selected for replication or for fine-mapping efforts. in particular, for replica- tion, we genotyped and analyzed , individuals from the sardi- nia cohort (stage ), who were unrelated (kinship coefficient ¼ ) to the individuals in stage . for fine mapping, we genotyped a subset of individuals from stages and including couples (mothers and fathers) already typed with the k mapping array set and individ- uals representing children from the larger sibships. inchianti the inchianti study is an ongoing epidemiological study in two italian towns in the chianti area (tuscany). a detailed description of the study design and data-collection methods has been pub- lished. , plasma tsh levels were measured with commercial kits (vitros tsh reagent, ortho-clinical diagnostics, johnson & johnson medical s.p.a section) by chemiluminescent assay. the assay has an analytical sensitivity of . miu/ml. the intra-assay coefficients of variation (cvs) were . % to . % over the range . – . miu/ml. snp rs was genotyped in this cohort by taqman single snp genotyping assays (applied biosystems). old order amish the old order amish (ooa) study participants reported here were from ongoing studies of cardiovascular disease and longevity; none were ascertained on the basis of thyroid disease. , a total of , individuals from these two studies had serum tsh mea- sured by quest diagnostics (nichols institute) with a standardized third-generation assay and were previously genotyped for rs with the k affymetrix mapping array set. statistical analysis to evaluate the additive effect of each marker, we fitted a simple regression model and used a variance-component approach to account for correlation between different observed phenotypes within each family. gender, age, and age squared were included (c) the bottom panel summarizes the results of the fine mapping in the pde b gene. the association results are for all snps in the sardinia sample genotyped with either the k or k mapping array set or the parallele custom chip. the top three markers, all in intron , are highlighted. the transcripts for all the pde b isoforms are indicated at the bottom. erican journal of human genetics , – , june as covariates in all analyses, and the trait was normalized with quantile normalization to avoid inflation of type i error rates. for individuals genotyped with a sparse map, we used a modified version of the lander-green algorithm , to estimate ibd shar- ing at the location of the snps being tested and identify stretches of haplotype shared with close relatives who were genotyped at higher density and probabilistically infer missing genotypes. in brief, in the statistical approach to estimate each genotype, we first calculated the likelihood of the observed genotype data. then, we assigned each missing genotype to a specific value and updated the likelihood pedigree. the ratio of the two likelihoods gave a posterior probability for the proposed genotype conditional on all available data. furthermore, instead of assigning the most likely genotype, we estimated an expected genotype score, repre- senting the expected number of copies of a reference allele (a frac- tional number between and ), which allowed us to partially account for uncertainty in genotype assignment. the genotype scores were then used in a variance-component-based association test as described. because of computational constraints, we di- vided large pedigrees into subunits with ‘‘bit-complexity’’ of or less (typically, – individuals) before analysis. false-discov- ery rates (fdrs) were calculated with r’s p.adjust() procedure via the method of benjamini and hochberg. in sardinia stage replication and in inchianti samples, asso- ciation with tsh was tested as in the sardinia gwa sample, ex- cept that no imputation was required. association analyses to test for additive effects in the old order amish sample were carried out under a variance-component framework implemented in solar; these analyses modeled the effect of genotype on tsh levels while adjusting for the effects of age and sex and accounting for a background polygenic contribution to variation in tsh levels occurring because of the relationships existing among the exam- ined individuals. the choice of solar was based on the pres- ence of larger pedigrees in the ooa families and the lack of a re- quirement for genotype imputation in these samples. for fine mapping in the sardinia sample, we merged all avail- able genotypes from all the platforms ( k mapping array set, k mapping array set, parallele custom chip) for a total of , nonoverlapping individuals. because fewer markers were analyzed than in the gwas, larger pedigrees were divided into units of bit-complexity of or less before analysis. missing geno- types for individuals typed with only one platform were inferred with the same approach used in the gwa analysis. we then eval- uated the additive effect of each marker by using allele dosages as above. results genome-wide association scan for tsh levels in sardinians genome-wide association analyses were carried out in , volunteers characterized for circulating tsh levels (see material and methods). , analyses revealed three snps with genome-wide significant association at a single chromosome locus (p < � , see table and figure ). these snps and three other snps among our top sig- nals lie in intron or upstream of the pde b gene (table and figure ). their locations and linkage-disequilibrium (ld) relationships in the sardinia and ceu hapmap pop- ulations are illustrated in figure . , a quantile-quantile the american journal of human genetics , – , jun (qq) plot (figure ) shows that most of the deviation from the null hypothesis is accounted for by markers falling in the pde b gene. after those markers were removed, the corresponding qq plot (blue line) suggests that there are nevertheless likely to be some additional true signals (see below). the three snps that showed the strongest ge- nome-wide significant association (rs , p ¼ . � ; rs , p ¼ . � ; rs , p ¼ . � ) are all in strong linkage disequilibrium (figure , average pairwise r > . ). among these, the snp showing strongest association, rs , explains . % of the var- iance of tsh levels. each copy of the minor a allele is asso- ciated with an average increase of . miu/ml in tsh levels. replication of the association of rs /pde b in other populations to confirm the association of rs with tsh levels, we analyzed two additional cohorts, one comprising , tuscan subjects from the genetically distinct in- chianti study population , and the second , indi- viduals from the founder population of the old order amish. , we also analyzed a replication cohort of , individuals enrolled in the sardinia study but unrelated to the first group (designated here as stage analysis). the features of these cohorts are given in table , and the results of association are summarized in table . analysis revealed that snp rs or its surrogate rs was associ- ated with the tsh trait in all the cohorts tested, with the same direction of effect as observed in our original sample (table ). notably, the effect size in the replication samples figure . quantile-quantile plot of snps associated with tsh level in the sardinia study red dots represent all the , snps analyzed in the gwas. blue dots represent all analyzed snps not located within the pde b gene. the gray area corresponds to the % confidence region from a null distribution of p values generated from simula- tions. e table . characteristics of samples used in genome-wide and follow-up analyses population study n (% female) mean age (sd) mean tsh (sd) % under treatment % recorded disease status sardinia ( . %) . ( . ) . ( . ) . % % follow-up samples sardinia stage ( . %) . ( . ) . ( . ) . % % inchianti ( . %) . ( . ) . ( . ) . % % old order amish ( . %) . ( . ) . ( . ) . % na the table shows the mean and standard deviation (sd) for age and tsh values for each study group, as well as the percentage of individuals under thyroid- hormone therapy and of individuals with clear reports of disease status (i.e., thyroid cancer, surgery, and autoimmune thyroiditis). na, not assessed. was about half that observed in the initial gwas ( . ver- sus . ). this may be due to less accurate estimates of ef- fect size as a result of reduction in sample size and conse- quent decrease of statistical power, or it may reflect the ‘‘winner’s curse’’ phenomenon, where effect-size estimates for markers that reach statistical significance may be in- flated. the combined p value from stage analyses in the three cohorts was highly significant (p ¼ . � ), leading to a cumulative-association p value of . � . table also summarizes the distribution of the tsh levels for each genotype class for the snp rs and shows that in all the samples, individuals carrying at least one copy of the minor a allele have higher circulating levels of tsh than those homozygous for the major g al- lele, showing a trend for an additive model. in the amish sample, the mean tsh value for individuals with the a/g genotype appears slightly higher than that observed for the a/a subjects. this could result from the different fre- quency of the rare a allele and the lower frequency of the a/a genotype in the amish compared with the other cohorts; this lower frequency would yield less accurate mean-value estimates for this genotype class compared to values in the other cohorts. we also repeated association analyses in all cohorts after stratifying subjects into two groups, one group including individuals with tsh levels in the normal range ( . – . miu/ml), with no reported history of thyroid disease, and not taking thyroid medications (group , table ), and the am the other group including the remaining subjects (group ). the results confirmed the association of rs in both the unaffected and the thyroid-affected individuals (table ). because of the reduction in sample size, the p value was lower in the two subsets, but the effect size of the associated a allele was larger in group individuals (in all but the ooa cohort), suggesting a potential involve- ment of pde b activity in the course of thyroid disease. further analysis in a larger subgroup of thyroid-affected sardinian subjects, including all the individuals from group as well as individuals with nodules and goiters (formerly in group ), showed stronger association when compared to the remaining subjects, again confirming a larger effect size of the a allele in thyroid-affected indi- viduals (see table s available online). despite the consis- tently larger effect in thyroid-affected individuals in sev- eral studies, this difference does not reach statistical significance (cochran’s q statistic p ¼ . , i ¼ %); consequently, larger studies will be necessary to test this hypothesis further. association analysis of rs in males and females did not suggest gender-specific effects (table s ). signals for other loci that did not reach ge- nome-wide significance (table ) did not replicate strongly in other cohorts and have in general not been followed up further. for two snps, rs on chromosome (in the abo blood group gene [mim ], with borderline bon- ferroni p value) and snp rs on chromosome (in pde a, a gene that belongs to the same enzyme family table . summary of association results for snp rs in all cohorts population study n a/a a/g g/g freq(a) effecta (se) p value sardiniab . ( . ) . ( . ) . ( . ) . . ( . ) . � stage sardinia stage c . ( . ) . ( . ) . ( . ) . . ( . ) . � inchianti . ( . ) . ( . ) . ( . ) . . ( . ) . old order amish . ( . ) . ( . ) . ( . ) . . ( . ) . � meta-analysis stage . � overall . � the table summarizes association results for rs in all cohorts. for each study, genotype medians (irq range) of unadjusted tsh values are reported in miu/ml; effect sizes (se) and p values are given relative to the normalized trait. a the effect size is measured in standard-deviation units and is estimated as the b coefficient of the regression model when the normalized trait is used (e.g., an effect size of . implies that each additional copy of the allele being evaluated increases trait values by . standard deviation). b sardinia genotype medians are relative only to individuals directly genotyped with the k mapping array set. c results are for rs , in strong ld (r ¼ . ) with rs , which was not genotyped (see material and methods). erican journal of human genetics , – , june table . results of association analysis of rs in healthy and thyroid-affected individuals group group population study n effecta (se) p value n effecta (se) p value sardinia . ( . ) . � . ( . ) . � stage sardinia stage b . ( . ) . � . ( . ) . inchiantic . ( . ) . . ( . ) . old order amish d . ( . ) . � . ( . ) . meta-analysis meta-analysis stage . � . � overall . � . � we compared association results in two subgroups. group identifies healthy subjects and consists of individuals with tsh levels in the normal range of . – miu/dl and with no history of thyroid disease (thyroid medications, thyroid cancer, surgery, or autoimmune thyroiditis). group consists of the remaining subjects. a the effect size refers to the minor allele frequency (maf) allele; it is measured in standard-deviation units and is estimated as the b coefficient of the regression model when the normalized trait is used (e.g., an effect size of . implies that each additional copy of the allele increases trait values by . standard deviation). b results are for rs , in strong ld (r ¼ . ) with rs , which was not genotyped (see materials and methods). c thryoid autoimmunity and thyroid cancer information were not reported in the inchianti database. old older amish database did not contain any in- formation regarding thyroid disease, so only out-of-range tsh values and history of taking thyroid-hormone therapy were considered to define group . d old older amish database did not contain information regarding thyroid disease, so only out-of-range tsh values and history of taking thyroid-hormone therapy were considered to define group . as pde b), some further testing was performed. snp rs did not replicate in the amish or inchianti co- horts, whereas rs was replicated in inchianti, with a one-sided p value of . , but was not supported in the amish samples (data not shown). thus, its involve- ment in the variability of tsh remains suggestive, requir- ing study in additional cohorts. fine mapping in the pde b locus to identify possible coding and/or functional variants in- volved in the regulation of pde b activity in the context of tsh variation, we genotyped additional snps falling in the pde b gene or in the neighboring kb upstream and downstream regions in , sardinians. we analyzed all the available markers ( from the gwas and from the custom chip) in the individuals genotyped with at least one platform and for whom tsh values were available (n ¼ , ). as shown in figure c and table s , the strongest association signals all pointed to intron , common to all five pde b isoforms, in which ten significant snps were lo- cated. the top three snps confirmed the previous top three markers detected in the gwas, though with a shifted rank order (rs , p ¼ . � ; rs , p ¼ . � ; rs , p ¼ . � ). several of the snps show linkage disequilibrium (r > . ) with snps in neigh- boring regions (figures a and b), suggesting that the functional variant(s) may lie outside of intron . however, it seems likely that they will reside in a noncoding se- quence, because sequence analysis of all the pde b exons in subjects homozygous for the rare and for the com- mon alleles of the top two associated snps, rs and rs , did not reveal any coding variants (data not shown). in addition to intron , we also found evidence of association for two new snps within intron of the american journal of human genetics , – , june pde b . the functional relevance of these variants re- mains to be elucidated. further studies, including large- scale resequencing and molecular analyses, will be neces- sary to verify the effects of the associated variants on the regulation of the tsh levels. association analysis of candidate genes with tsh levels our gwa analysis implicated pde b as the only gene with a significant effect on variation of tsh. because pde b var- iants contribute only . % of tsh variation, which repre- sents a small part of the total genetic component of the trait (~ %), we expected other genes to contribute to tsh levels, albeit with smaller effects. to begin to look for such genes, we carried out in the sardinia cohort a sub- analysis focusing on candidate genes, where a less strin- gent significance threshold would increase power. we se- lected candidate genes on the basis of their reported function in camp metabolism, tsh signaling, and the reg- ulation of thyroid function. no linkage and association studies showing a correlation with tsh levels had previ- ously been performed for any of these candidates. when markers falling in the candidates and already genotyped in the gwas were tested for their association with tsh levels, as expected none reached genome-wide significant association (table ). however, substantive evidence of as- sociation was detected for some genes involved in the bio- logical activity and negative feedback of thyroid hormone (thrb), in signaling of tsh (tshr [mim ] and gnaq), , in thyroid-hormone production (tg [mim ]), and in phosphodiesterases involved in the ca- tabolism of camp (pde d [mim ]) , (see discus- sion for more details). among the various pde genes, pde d and pde b [mim ] were included in the table . tag snps that show strongest association with tsh in previously identified candidate genes in the tsh signaling pathways and thyroid function gene # affymetrix snps # hapmap snps coverage ( . ) coverage ( . ) best p value snp allele (þ/�) freq (þ) effecta h thrb . . . x � rs g/a . . . % gnaq . . . x � rs g/a . . . % tg . . . x � rs a/c . . . % pou f . . . x � rs g/a . . . % pde d . . . x � rs g/a . . . % tshr . . . x � rs g/a . . . % gnao . . . rs t/c . . . % dio . . . rs g/a . . . % pde b . . . rs g/a . . . % gnai . . . rs c/t . . . % gnas . . . rs a/t . . . % ttf . . . rs g/a . . . % tpo . . . rs g/a . . . % dio . . . rs c/t . . . % tsh-a . . . rs c/t . . . % pax . . . rs t/c . . . % prop . . . rs t/c . . . % trhr . . . rs t/c . . . % crebbp . . . rs c/t . . . % creb . . . rs a/g . . . % ctla . . . rs g/a . . . % tsh-b . . . rs g/c . . . % thra . . . rs c/t . . . % prkar b . . na na na na na na the first column indicates the name of a previously identified candidate gene. the second column indicates the number of snps in our affymetrix arrays that are within kb of the gene. the next column indicates the number of hapmap snps within kb of the gene and the proportion of these that are covered at r ¼ . or r ¼ . by snps in the affymetrix arrays. the remaining columns indicate the snp that showed strongest association in our analysis, the p value, the tested allele and its frequency, and the estimated additive effect in standard-deviation units. association was tested either with intragenic snps or with the best available tag for intragenic snps (r > . ). shown in bold are candidate genes with snps reaching p values % . . a the effect size refers to the maf allele; it is measured in standard-deviation units and is estimated as the b coefficient of the regression model when the normalized trait is used (e.g., an effect size of . implies that each additional copy of the allele increases trait values by . standard deviation). focused analysis on the basis of their camp specificity and reported expression in thyroid tissues. we have calculated the corresponding fdrs by taking into account all the snps tested in the candidate-gene analysis. notably, by this criterion, the q value for snps falling in the thrb and gnaq genes reached the significant threshold of . (q ¼ . and q ¼ . , respectively), suggesting a possible involvement in the variability of tsh levels. discussion the pde b gene, encoding a high-affinity camp phospho- diesterase, is inferred to modulate circulating tsh levels in three independent populations, with a combined p value of . � . of the five characterized pde b iso- forms, the major isoform pde b and minor isoforms pde b and pde b are abundantly expressed in the thy- roid. , because pde b is undetectable in the pituitary, we infer it to act primarily in the thyroid to catalyze the hy- drolysis and inactivation of camp after tsh signaling. pde b could then influence tsh levels by feedback from an effect on the generation of t and t in the thyroid, the am in line with reports that both thyroglobulin endocytosis and thyroid-hormone secretion are stimulated by tsh via a camp-dependent pathway. although no signals other than pde b reached genome- wide significance (table ), several candidate genes showed suggestive evidence of association. these included two other camp-specific phosphodiesterases, pde d, , and, at a much lower level of significance, pde b. it is relevant that the camp-specific pde family has also been sug- gested to be involved in modulating the camp signal after tsh stimulation of thyrocytes. furthermore, some evi- dence for association was also detected in our gwas in pde a, a phosphodiesterase with dual specificity for cy- clic nucleotides and stimulated by camp. analyses using differential inhibition or transfection of the range of phos- phodiesterases and their variants in thyrocytes should help to determine their contribution to camp levels. as for other genes previously implicated in tsh homeo- stasis, the thyroid-hormone receptor, b (thrb) and several g protein genes—in particular, gnaq, previously re- ported to be required for tsh-induced thyroid-hormone synthesis and release—implicated in the signal transduc- tion of hormone receptors, also showed suggestive erican journal of human genetics , – , june association signals (table ). finally, some evidence of as- sociation was also noticed in two thyroid-specific genes, thyroglobulin (tg) and thyroid-stimulating hormone receptor (tshr), and in pou class homeobox (pou f [mim ]), a gene expressed in the pituitary and impor- tant for the regulation of a number of pituitary hormones. we infer that much of the tsh-level variance that subtly affects normal and possibly pathological states seems to be exerted at the level of camp degradation. it is possible that genomic pde b mutations could be responsible for certain thyroid diseases—for example, for increased serum tsh occasionally observed in individuals with no evidence of thyroid autoimmunity or loss-of-function mutations in the thyroid-hormone- or tsh-receptor genes. because pde b has the highest affinity for camp of any known phosphodiesterase, the enzyme would be active even at low concentrations of camp, and a change in its level or activity could have an especially marked effect on tsh signaling. consistent with this notion, a mutation in pde b that leads to elevated camp levels has now been identified in a case of adrenal hyperplasia in cushing syn- drome (aimah [mim ]). increased camp-degrad- ing pde b activity has also been detected in autonomous thyroid adenomas, where it may represent a compensatory mechanism opposing the constitutive activation of the camp pathway. other reports have noted pde b upregu- lation in alzheimer’s disease (ad [mim ]) brain and pituitary adenomas, as well as involvement in a model of modified insulin secretion. pde family members have increasingly been implicated in the pathogenesis of a number of other diseases, includ- ing cardiovascular disorders, renal failure, adrenocortical hyperplasia, , and a wide variety of inflammatory pa- thologies. selective isoform-specific pde inhibitors are already employed to treat several diseases, and thus pde b may provide a pharmaceutical target for certain thyroid pathologies. supplemental data five tables are available at http://www.ajhg.org/. acknowledgments we thank the many individuals who generously participated in this study, monsignore piseddu (bishop of ogliastra), the mayors and citizens of the sardinian towns (lanusei, ilbono, arzana, and elini), and the head of the public health unit asl for their volun- teerism and cooperation. the team also thanks the physicians an- gelo scuteri, marco orrù, maria cristina spada, danilo fois, liana ferreli, marcello argiolas, francesco loi, and pietro figus; nurses paola loi, monica lai, and anna cau, who carried out participant physical exams; and monica balloi for the recruitment of volun- teers. we are grateful for the provision of inchianti data and sam- ples for analyses and for helpful discussion and critical comments from their leadership for this manuscript. we also wish to thank the amish community for their cooperation and partnership in re- search and the personnel at the amish research clinic for their ex- the american journal of human genetics , – , june traordinary efforts. this work was supported by the intramural re- search program of the national institute on aging (nia), national institutes of health (nih). the sardinia (‘‘progenia’’) team was supported by contract no -ag- – from the nia; the efforts of g.r.a. and s.s. were supported in part by contract -ma- from the nia to the university of michigan and by re- search grant hg and hl from the nih (to g.r.a.). s.n. was partially supported by a grant from the fondazione banco di sardegna. the inchianti study was supported in part by the italian ministry of health (ics . /rs . ) and u.s. nia (con- tracts n -ag- , n -ag- , md , and md ). the work with the amish was supported by re- search grants u hl and r ag and by the univer- sity of maryland general clinical research center, grant m rr ; the johns hopkins university general clinical research center, grant m rr ; general clinical research centers program, national center for research resources (ncrr), nih; and the baltimore veterans administration geriatric research and education clinical center (grecc). received: march , revised: april , accepted: april , published online: may , web resources the url for data presented herein is as follows: online mendelian inheritance in man (omim), http://www.ncbi. nlm.nih.gov/omim/ references . kopp, p. 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( ). therapeutic benefit of pde inhibitors in inflammatory diseases. curr. opin. investig. drugs , – . 	phosphodiesterase b gene variants are associated with serum tsh levels and thyroid function 	introduction 	material and methods 	sample description 	gwas genotyping 	replication and fine mapping 	inchianti 	old order amish 	statistical analysis 	results 	genome-wide association scan for tsh levels in sardinians 	replication of the association of rs /pde b in other populations 	fine mapping in the pde b locus 	association analysis of candidate genes with tsh levels 	discussion 	acknowledgments 	web resources 	references [pdf] evidence for several independent genetic variants affecting lipoprotein (a) cholesterol levels. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /hmg/ddu corpus id: evidence for several independent genetic variants affecting lipoprotein (a) cholesterol levels. @article{lu evidencefs, title={evidence for several independent genetic variants affecting lipoprotein (a) cholesterol levels.}, author={w. lu and yu-ching cheng and k. chen and hong wang and g. gerhard and c. still and xin chu and r. yang and a. parihar and j. o'connell and t. pollin and e. {\'a}ngles-cano and m. quon and b. mitchell and a. shuldiner and m. fu}, journal={human molecular genetics}, year={ }, volume={ }, pages={ - } } 	w. lu, yu-ching cheng, + authors m. fu 	published 	biology, medicine 	human molecular genetics lipoprotein (a) [lp(a)] is an independent risk factor for atherosclerosis-related events that is under strong genetic control (heritability = . - . ). however, causal mutations and functional validation of biological pathways modulating lp(a) metabolism are lacking. we performed a genome-wide association scan to identify genetic variants associated with lp(a)-cholesterol levels in the old order amish. we confirmed a previously known locus on chromosome q - and found lp(a) levels also to… expand view on pubmed academic.oup.com save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all figures, tables, and topics from this paper 	 table 	 figure 	 figure 	 table 	 figure 	 table 	 table 	 figure 	 figure view all figures & tables 	cholesterol 	lipoprotein (a) 	nitroprusside 	atherosclerosis 	chromosome q 	lpa gene 	kremen gene 	null value 	mental association citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency a genome-wide association meta-analysis on lipoprotein (a) concentrations adjusted for apolipoprotein (a) isoforms[s] 	salome mack, s. coassin, + authors f. kronenberg 	biology, medicine 	journal of lipid research 	 	 	pdf 	view excerpt, cites methods save alert research feed genome-wide association study highlights apoh as a novel locus for lipoprotein(a) levels—brief report 	mary hoekstra, h. chen, + authors j. engert 	biology, medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 	 save alert research feed deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of european and african ancestries 	s. zekavat, s. ruotsalainen, + authors s. zoellner 	medicine, biology 	nature communications 	 	 	pdf save alert research feed lipoprotein(a) concentration and risks of cardiovascular disease and diabetes. 	d. gudbjartsson, g. thorgeirsson, + authors k. stefánsson 	medicine 	journal of the american college of cardiology 	 	 	 save alert research feed deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of european and african ancestries 	s. zekavat, s. ruotsalainen, + authors p. natarajan 	biology, medicine 	 	 	pdf save alert research feed a novel but frequent variant in lpa kiv- is associated with a pronounced lp(a) and cardiovascular risk reduction 	s. coassin, gertraud erhart, + authors f. kronenberg 	medicine 	european heart journal 	 	 	pdf save alert research feed a novel variant associated with hdl-c levels by modifying daglb expression levels: an annotation-based genome-wide association study 	d. zhou, dandan zhang, + authors m. lai 	biology, medicine 	european journal of human genetics 	 	 	pdf save alert research feed effect of two lipoprotein (a)-associated genetic variants on plasminogen levels and fibrinolysis 	hong wang, chan e hong, + authors m. fu 	biology, medicine 	g : genes, genomes, genetics 	 	 	pdf 	view excerpts, cites methods, results and background save alert research feed lipoprotein (a): impact by ethnicity and environmental and medical conditions 	b. enkhmaa, e. anuurad, l. berglund 	medicine 	journal of lipid research 	 	 	pdf save alert research feed lipoprotein(a) concentration, genetic variants, apo(a) isoform size, and cellular cholesterol efflux in patients with elevated lp(a) and coronary heart disease submitted or not to lipoprotein apheresis: an italian case-control multicenter study on lp(a). 	c. stefanutti, l. pisciotta, + authors g. watts 	medicine 	journal of clinical lipidology 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency genetic variants associated with lp(a) lipoprotein level and coronary disease. 	r. clarke, j. peden, + authors m. farrall 	medicine 	the new england journal of medicine 	 	 	pdf 	view excerpts, references results save alert research feed multiple qtl influence the serum lp(a) concentration: a genome-wide linkage screen in the procardis study 	s. barlera, c. specchia, + authors h. watkins 	biology, medicine 	european journal of human genetics 	 	 	pdf 	view excerpts, references background save alert research feed lipoprotein(a): resurrected by genetics 	f. kronenberg, g. utermann 	biology, medicine 	journal of internal medicine 	 	 	highly influential 	view excerpts, references background save alert research feed genome-wide linkage analysis for identifying quantitative trait loci involved in the regulation of lipoprotein a (lpa) levels 	s. lópez, a. buil, + authors j. soria 	biology, medicine 	european journal of human genetics 	 	 	pdf 	view excerpts, references background save alert research feed characterization of the genetic elements controlling lipoprotein(a) concentrations in mexican americans. evidence for at least three controlling elements linked to lpa, the locus encoding apolipoprotein(a). 	d. l. rainwater, c. kammerer, j. vandeberg, j. hixson 	biology, medicine 	atherosclerosis 	 	 save alert research feed the apolipoprotein (a) gene: a transcribed hypervariable locus controlling plasma lipoprotein (a) concentration 	h. kraft, s. köchl, h. menzel, c. sandholzer, g. utermann 	biology, medicine 	human genetics 	 	 	view excerpts, references background save alert research feed meta analysis of candidate gene variants outside the lpa locus with lp(a) plasma levels in , participants of six white european cohorts. 	d. zabaneh, m. kumari, + authors s. humphries 	biology, medicine 	atherosclerosis 	 	 	 save alert research feed apolipoprotein(a) kringle iv repeat number predicts risk for coronary heart disease. 	h. kraft, a. lingenhel, + authors g. utermann 	medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 	view excerpt, references background save alert research feed a polymorphism in the protease-like domain of apolipoprotein(a) is associated with severe coronary artery disease 	may m. luke, j. kane, + authors s. ellis 	biology, medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 	pdf 	view excerpt, references background save alert research feed two rare variants explain association with acute myocardial infarction in an extended genomic region including the apolipoprotein(a) gene 	w. koch, j. mueller, + authors a. kastrati 	biology, medicine 	annals of human genetics 	 	 	view excerpt, references results save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - 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) core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core articles the role of godly magistrates in the church: melanchthon as luther's interpreter and collaborator james m. estes madame guyon and experiential theology in america patricia a. ward theology as entertainment: oral debate in american religion e. brooks holifield french politics and alfred loisy's modernism harvey h i l l the shift from character to personality in mainline protestant thought, - heather a. warren books book reviews and notes kiley, mark, et al., eds., prayer from alexander to constantine: a critical anthology william r mcdonald sharer, peter, judeophobia: attitudes toward the jews in the ancient world paula fredriksen early church fathers: book collection on cd-rom james j . o'donnell hultgren, a r l a n d j . , a n d steven a . h a g g m a r k , eds., the earliest christian heretics: readings from their opponents gary r. brower salisbury, j o y c e e., perpetua's passion: the death and memory of a young roman woman a m y g. oden perkins, judith, the suffering self: pain and narrative representation in the early christian era dennis e. trout elliott, t. g., the christianity of constantine the great charles odahl b u r r u s , virginia, the making of a heretic: gender, authority, and the priscillianist controversy richard valantasis macmullen, ramsay, christianity and paganism in the fourth to eighth centuries averil c a m e r o n urbainczyk, theresa, socrates of constantinople: historian of church and state richard lim amory, patrick, people and identity in ostrogothic italy, - michael m a a s power, kim, veiled desire: augustine on women maureen a. tilley o'rourke boyle, marjorie, divine domesticity: augustine of thagaste to teresa of avila merry wiesner-hanks l e m a i t r e , j e a n - l o u p , michel dmitriev, a n d pierre g o n n e a u , eds., moines et monastères dans les sociétés de rite grec et latin: Études publiées avec le concours de la fondation singer-polignac david paul hester core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core potts, cassandra, monastic revival and regional identity in early normandy charles b. paris epstein, marc michael, dreams of subversion in medieval jewish art and literature kalman p. bland vauchez, andre, and jean birrell, trans., sainthood in the later middle ages maiju lehmijoki-gardner voolstra, sjouke, menno simons: his image and message william c. ringenberg maag, karin, ed., the reformation in eastern and central europe gregory j. miller o'reilly, terence, from ignatius loyola to john of the cross: spirituality and literature in sixteenth-century spain carlos m. n. eire chatellier, louis, and brian pearce, trans., the religion of the poor: rural missions in europe and the formation of modern catholicism, c. - kathleen m. comerford boersma, owe, and auke j. jelsma, eds., unity in multiformity: the minutes of the coetus of london, , and the consistory minutes of the italian church of london, - peter iver kaufman schutte, anne jacobson, transc, transl., ed., cecelia ferrazzi: autobiography of an aspiring saint charmarie j. blaisdell wiesner-hanks, merry, ed., joan skocir, and merry wiesner-hanks, trans., convents confront the reformation: catholic and protestant nuns in germany marygrace peters, o.p. parnham, david, sir henry vane, theologian: a study in seventeenth-century religious and political discourse jameela lares coffey, john, politics, religion and the british revolutions: the mind of samuel rutherford dewey d. wallace jr. swatland, andrew, the house of lords in the reign of charles ii gary s. de krey hinds, hilary, god's englishwomen: seventeenth-century radical sectarian writing and feminist criticism paul wesley chilcote herrick, james a., the radical rhetoric of the english deists: the discourse of skepticism, - charles wallace jr. kaufman, peter iver, prayer, despair, and drama: elizabethan introspection norman jones butler, jon, and harry s. stout, eds., religion in american history: a reader mark a. noll stout, harry s., and d. g. hart, eds., new directions in american religious history robert bruce mullin gilpin, w. clark, a preface to theology e. brooks holifield shriver, george h., ed., dictionary of heresy trials in american christianity david r. bains faull, katherine m., trans., moravian women's memoirs: their related lives, - catherine a. brekus helmstadter, richard, ed., freedom and religion in the nineteenth century charles d. cashdollar compton, todd, in sacred loneliness: the plural wives of joseph smith gerald e. jones vogel, dan, ed., early mormon documents. vol. thomas g. alexander weaver, j. denny, keeping salvation ethical: mennonite and amish atonement theology in the late nineteenth century steven m. nolt howard, victor b., the evangelical war against slavery and caste: the life and times of john g. fee marty g. bell evans, christopher hodge, social gospel liberalism and the ministry of ernest fremont tittle: a theology for the middle class merrill m. hawkins jr. fahey, david m., temperance and racism: john bull, johnny reb, and the good templars william l. fox core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core denton james a., rocky mountain radical: myron w. reed, christian socialist david b. mccarthy emmons, terrence, alleged sex and threatened violence: doctor russel, bishop vladimir, and the russians in san francisco, - marks. still klejment, anne, and nancy l. roberts, eds., american catholic pacifism: the influence of dorothy day and the catholic worker movement maryj. oates knotts, alice g., fellowship of love: methodist women changing american racial attitudes, - linda m. martin chaves, mark, ordaining women: culture and conflict in religious organizations randi jones walker miller, keith graber, wise as serpents, innocent as doves: american mennonites engage washington richard g. kyle blodgett, jan, protestant evangelical literary culture and contemporary society james g. moseley dorgan, howard, in the hands of a happy god: the "no hellers" of central appalachia samuel s. hill gilbert, james, redeeming culture: american religion in an age of science michael s. hamilton miller, donald e., reinventing american protestantism: christianity in the new millennium jackson w. carroll brown, michael r, the channeling zone: american spirituality in an anxious age kelly jarrett hanegraaff, wouter, ]., new age religion and western culture: esotericism in the mirror of secular thought evelyn a. kirkley jeffrey, david lyle, people of the book: christian identity and literary culture wesley a. kort terray, laszlo g., and erik w. gritsch, trans., he could not do otherwise: bishop laps ordass, - tandy mcconnell burgess, john, the east german church and the end of communism johns. conway xing, jun, baptized in the fire of revolution: the american social gospel and the ymca in china: - jane harris munro, doug, and andrew thornley, eds., the covenant makers: islander missionaries in the pacific robert a. schneider wiles, maurice, archetypal heresy: arianism through the centuries harry o. maier prokurat, michael, alexander golitzin, and michael d. peterson, historical dictionary of the orthodox church john l. boojamra zwi werblowsky, r. ]., and geoffrey wigoder, eds., the oxford dictionary of the jewish religion shaye j. d. cohen books received society notes core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core sharing the load: amish healthcare financing healthcare article sharing the load: amish healthcare financing kristyn rohrer and lauren dundes ,* department of sociology, kutztown university of pennsylvania, kutztown, pa , usa; krohr @live.kutztown.edu department of sociology, mcdaniel college, westminster, md , usa * correspondence: ldundes@mcdaniel.edu; tel.: + - - - academic editor: sampath parthasarathy received: september ; accepted: december ; published: december abstract: when settling healthcare bills, the old order amish of lancaster county, pennsylvania rely on an ethos of mutual aid, independent of the government. consonant with this philosophy, many amish do not participate in or receive benefits from social security or medicare. they are also exempted from the affordable care act of . this study expands the limited documentation of amish hospital aid, an amish health insurance program that covers major medical costs. interview data from amish adults in lancaster county depict how this aid program supplements traditional congregational alms coverage of medical expenses. the interview data delineate the structure of the program, its operation, and how it encourages cost containment and community interdependence. the manner in which the amish collaborate to pay for medical expenses provides a thought-provoking paradigm for managing health care costs. keywords: amish; obamacare; healthcare; insurance; exemptions; amish hospital aid; alms; community . introduction . . purpose of study this study amplifies the limited documentation of amish hospital aid (established in ) that helps amish members pay for health care. published reports of their unique system can also supplement understanding of the amish whose evolving code of rules and conduct (the ordnung), varies by subgroup and lacks systematic or explicit written rules. specific information about the structure of amish hospital aid and its role in how amish pay for healthcare can inform discussions of how to innovate mainstream us healthcare. . . background since , members of religious groups that conscientiously oppose social security benefits may apply for an exemption from the self-employment tax (according to the medicare segment of the social security act). the exemption applies to old order amish, and other religious groups that conscientiously object to insurance, if the sect has been in existence since december [ ]. amish are part of the “plain” community, a term that distinguishes amish and other anabaptists from outsiders who are called english, fancy people, or white folk. amish people, whose origins date back to the anabaptist movement that began in zurich, switzerland in , now live in the united states and ontario, canada. (the last amish church in europe closed in [ ]). since the amish first began to immigrate to north america in the s, their population grew to , by and to , in u.s. states [ , ]. members of exempted religious groups—including the amish—also must have a reasonable means of caring for their own elderly or dependent members, obviating the need for retirement communities or nursing homes, when each family takes care of its own [ , ]. healthcare , , ; doi: . /healthcare www.mdpi.com/journal/healthcare http://www.mdpi.com/journal/healthcare http://www.mdpi.com http://www.mdpi.com/journal/healthcare healthcare , , of amish commonly believe that commercial insurance plans undermine the religious duty of community accountability [ ]. their sense of community is strengthened by the belief that most modern technology brings a worldliness that detracts from their lifestyle. amish are increasingly likely to use telephones (even cell phones), while televisions, bicycles, gas-powered tractors, and owning vehicles are still forbidden [ ]. the exemptions allow self-employed amish to avoid paying the combined employee and employer social security tax for religious reasons (irs form : application for exemption from social security and medicare taxes and waiver of benefits is for followers of established religious groups that are “conscientiously opposed to accepting benefits of any private or public insurance that makes payments for the cost of medical care; or provides services for medical care”). amish who work for amish-owned employers were granted the same exemption in [ , ]. amish employed by businesses that are not owned and operated by amish, however, depend on a different exception—one for individuals who are part of healthcare-sharing ministries [ ]. the government describes a health care sharing ministry as “a tax-exempt organization whose members share a common set of ethical or religious beliefs and share medical expenses in accordance with those beliefs”. in addition, the ministry must reject all types of insurance, including social security and medicare, and must have been in existence and sharing medical expenses continuously since december [ ]. these exceptions do not extend to any other taxes, such as real estate taxes, state and federal income taxes, county taxes, and sales tax. the affordable care act of (obamacare) similarly allows exemptions from the requirement to obtain health care insurance for certain religious groups. to compensate for their lack of commercial insurance, the amish turn to their own community [ , ]. their way of managing medical costs includes amish hospital aid, an insurance program (that predates obamacare). this aid program—the focus of this study—is relatively unknown, but received more notice after the passage of the affordable care act in . access to those who administer and use amish hospital aid was facilitated by a mennonite member of the lancaster county community (referred to as “male relative” in this paper). . . amish vs. mennonite mennonites and amish (as well as hutterites) share the same roots as participants of the anabaptist movement, occurring shortly after the protestant reformation. the anabaptists split into different subgroups before settling in north america, though many of them settled within close proximity to one another. in lancaster county, there are few amish subgroups. it is almost exclusively old order amish who have remained together under the same doctrine of faith. this contrasts with mennonite groups and amish communities in other areas, which have experienced multiple religious splits [ ]. there are two major groups of mennonites: plain mennonites and assimilated mennonites. plain mennonites or old order mennonites are often confused with the amish, as they share many of the same religious beliefs and cultural ideologies. there are numerous different divisions of plain mennonites in lancaster county. some plain mennonites use horse and buggy for transportation (team mennonites), while others such as the black-bumper mennonites drive all-black cars (i.e., the chrome, bumpers, etc. are painted black). there are general differences in the guidelines and style of clothing, head coverings, and buggies within the plain community, distinguishing the amish from the plain mennonites. mennonites are also permitted to ride bicycles, while the amish are not. on the other hand, unlike the amish, plain mennonites believe other religious groups (including the amish and assimilated mennonites) are not “saved”. assimilated mennonites are essentially indistinguishable within mainstream society. they are allowed to wear contemporary clothing, use electricity and new technology, attain higher education, and do not live in community settings (as do the amish and plain mennonites). healthcare , , of . methods . . access to the amish the ability to interview members of the lancaster county amish community required special arrangements and stipulations given their reluctance to associate with and trust outsiders, known as “english”. this term refers to the language of outsiders that contrasts with the variant of german (called pennsylvania dutch) which amish speak among themselves (“dutch” is likely derived from the word deutsch meaning “german”, not dutch). since amish learn english in school, the interviews could be conducted in english. the first author received assistance from an assimilated mennonite male relative who has a -year career in business and is well known in the amish community for his integrity. his heritage also provided valuable insight into the amish culture (e.g., his mennonite grandfather in retirement refused to accept social security income beyond what he had paid into the system) the relative granted permission to include all of the information identifying him in this paper. the relative offered to approach and help interview contacts in the old order amish community of lancaster county, pa, usa (the largest amish community comprising about , persons) [ ]. he was able to advise the authors on factors critical to developing a rapport with amish interviewees, including norms for appropriate dress (ankle-length skirts for women), the prohibition on taking pictures of the amish (although photos of their homes are not forbidden), and scheduling norms (e.g., working around the wedding season in mid-october through mid-december, and the inability to schedule interviews more than a few days or hours ahead of time) [ ]. . . obtaining interviews after receiving irb approval from mcdaniel college on october , and in accordance with the rules of the declaration of helsinki of , the first author (accompanied by her male relative) interviewed individuals after they signed an informed consent form. the sample consisted of seven men and four women from nine separate households who had experienced major medical expenses or were heavily involved in the amish hospital aid program, including those in administrative positions. the relative specifically recruited interviewees to provide a variety of perspectives, both in administering and receiving amish hospital aid. a majority of the interviewees ( / ) had previously established a relationship with the male relative that facilitated interviewee recruitment, while the remaining respondents were referred to the male relative by initial respondents because of their knowledge of amish hospital aid (either due to having had major medical expenses or administrative involvement in the program). one respondent not previously acquainted with the male relative—an amish hospital aid administrator—sought additional clarification about the purpose of the study before he consented to the interview. the response rate among those approached was %. the individuals were told that the purpose of the study was to collect and publish data documenting how the amish manage health care needs within their own system (given their exemption to coverage mandated by obamacare). ten of the interviewees were members of the amish hospital aid plan at the time of the interviews, although one had only joined amish hospital aid subsequent to a major medical expense. as is normal among the amish, all of the respondents received a formal education only through th or th grade. the first author and her male relative conducted the interviews in the kitchen of interviewees’ homes or in their office/workspaces between – november . interview questions concerned their involvement in the program, participation in conventional medical care, and the operation of the amish hospital aid program. interviews lasted between – min and were documented by the first author with handwritten notes. healthcare , , of . results . . traditional ways of paying for health care although much of the information shared during interviews has not been previously documented, existing literature (when available) corroborated the findings. while the focus in the interviews was amish hospital aid (a type of amish insurance), respondents also discussed traditional ways their community helps with unmanageable healthcare costs that at times operate in tandem with amish hospital aid. one source of handling medical costs that exceed an individual’s ability to pay out of pocket is voluntary donations to congregations, called alms, which are in line with a tradition of sharing burdens in the amish community [ ]. alms are tithes or offerings donated to the congregation by its members. as with tithes in other christian churches, members are encouraged to give % of their annual income to the amish church district. church deacons, who are in charge of both disciplinary and financial matters in the congregation, visit members in need of medical assistance to see how they are faring and then distribute the alms as they see fit. in the case of more serious injuries, when an individual’s congregation cannot afford to pay the medical bills solely with their own alms funds, amish congregations may use community collections. community collections are a form of alms that are gathered from the alms of other amish congregations in the area. these funds can be requested at the discretion of a deacon. community collections were used when a man was paralyzed from the waist down after a diving accident. occasional auctions of donated food, furniture, quilts, and livestock can raise as much as three hundred thousand dollars in one evening to help supplement alms and/or community collection coverage of healthcare bills [ , ]. in extremely rare cases, the government has covered medical costs (e.g., when an uninsured driver hit a buggy). . . implementation of amish hospital aid amish hospital aid covers only major medical needs. in serious cases (normally when hospitalization is necessary), those who participate in the program contact the treasurer in charge of their district once they know the costs incurred or to be incurred. those requiring care typically pay the health care provider used, and amish hospital aid then reimburses them. members who are unable to pay upfront allow the board to make arrangements with the hospital or care facility, in order for the board to pay the provider directly. the first % of the bill is expected to be paid by the individual, while the other % is covered by the amish hospital aid plan. those who are unable to pay the first % often rely on alms money from their congregation. . . how amish hospital aid manages medical costs when ill, amish seek treatment at their local hospital and are billed the same as non-amish. the individual then requests help from the congregation and/or amish hospital aid for any unaffordable medical expenses (e.g., maternity complications, surgeries, head injuries, physical therapy, and geriatric care). the amish hospital aid board also works closely with bill negotiators at different hospitals and facilities, just like commercial or governmental insurance companies, to negotiate discounts for individuals with specific needs [ ]. incentives to provide discounts include the promptness with which bills are normally paid (within days), less paperwork, as well as assurance that the facility will not be sued (since doctors are seen as fallible but autonomous individuals doing their best) [ ]. typically, participants of amish hospital aid receive a discount slightly above medicare rates, although each medical provider has its own particular discount. not all care facilities offer a discount for members of the amish community, however. hospitals sometimes refuse to consider lower rates beyond existing negotiated rates with government or commercial insurance companies [ ]. on the other hand, health care facilities like the clinic for special children provide pediatric care, especially for genetic disorders and syndromes (in strasburg, pennsylvania) for amish and old order mennonites, who may travel a great distance to reach the facility in order to receive healthcare , , of state-of-the-art care and save money on treatment. the clinic offers substantial reductions in health care costs by such means as lowering the price of testing, gauging when expensive treatment is warranted, and sometimes by devising treatments that prevent costly disability [ , ]. (a short video ( : ) about the clinic is available in a link in a wall street journal article [ ].) . . restrictions in amish hospital aid plan coverage the amish hospital aid plan includes limitations in its coverage, namely because it covers only major medical costs (hence the word “hospital” in its name). this discourages routine and preventive medical services—particularly by family doctors—and is a source of discontent for some interviewees. in addition, amish hospital aid does not cover physical disability costs, such as those for cerebral palsy. another amish-run organization, disability relief aid, covers costs for necessary items such as wheelchairs, ramp installations, and special bathroom installations, in addition to supplying an annual check to help with personal care costs. as with alms, disability relief aid is funded by community donations. neither amish hospital aid nor congregational alms funding cover health care needs that result from prohibited activities within the amish community. one interviewee mentioned an incident that occurred with a teenage boy in her congregation who was injured in a snowmobiling accident. the use of motor vehicles (e.g., cars, tractors, snow mobiles) is strictly forbidden in old order amish culture. even though the boy was not yet baptized into the amish congregation (and therefore still under the aegis of his parents), the deacon would not provide alms money to help pay for his hospital care. the boy’s parents also participated in the amish hospital aid plan, which likewise did not provide financial support, even though since he was under , he was covered under his parents’ plan. . . extent of participation in amish hospital aid while members of the amish community are not required to participate in amish hospital aid and may just rely on alms, a growing number of amish want the extra security provided by amish hospital aid. interviewees cited an estimated – participants in lancaster county or a participation rate of approximately %. the amish national steering committee (the plain community’s liaison to the national government) had new work as a result of obamacare. in , the committee instructed each head of household in the amish community to sign forms from health and human services (hhs) to extend their exemption to obamacare. hhs provided each deacon with the forms to be distributed to members of their church congregations. members gathered at the usual sunday service, with men sitting in groups of ten at round tables. one man explained the meaning of the forms to the others before they all signed the forms. in response to obamacare, participation in amish hospital aid increased by % from to . . . hierarchy of amish hospital aid administration the amish hospital aid plan is run by an all-male board (consisting of a chairman, vice chairman, and four treasurers). each treasurer is in charge of the funds for approximately congregations. the leaders appoint a committee man for each congregation to act as a liaison between the members and the administration. the hospital aid committee (including an estimated committee men and the board members) meets annually to discuss the program. the entire committee participates in voting, with board members holding six-year terms with no limit on reelection. current committee men are candidates for members of the board, by recommendation. those with amish hospital aid typically contact their treasurer once they receive their medical bill. in some cases, the treasurer actually contacts whoever receives the bill. since news travels fast within the amish community, if someone has been injured or hospitalized, members of the congregation will know, including the individual’s treasurer. healthcare , , of all members of the committee are men, since there are no women in administrative positions in the amish hospital aid program, as is consistent with organizations in the amish community. however, women are allowed and often encouraged to have their own personal businesses (selling quilts, fabrics, baked goods, etc.), and are sometimes in charge of the family finances instead of their husbands. . . role of unpaid administrators the amish system of paying health care bills has existed very informally, driven partly by the notable fact that administrators are not paid for their time. “remarkably, common to all these aid programs is their ability to address major needs without bureaucratic red tape, paid employees, underwriters, offices, computers, threat of lawsuits, or profits” [ ] (p. ). this system contrasts with administrative expenses among us private insurers who spend % of their budget on costs like claims processing, marketing, and general overhead [ ]. administrative costs at us hospitals are even higher, at % of total hospital expenditures [ ]. all respondents in this study were aware of and supported a lack of paid administrators. they offered comments such as, “if it ain’t broke, don’t fix it”. . . cost of amish hospital aid members pay a flat rate per person on a monthly basis. the monthly cost of $ per individual is exactly the same as what was reported in a wall street journal article seven years prior to data collection for this study [ ]. a married couple pays $ per month; however, all of their children until the age of would also be covered by the $ payment. one interviewee who complained about the high monthly cost (mentioned above) speculated that the “english” (i.e., non-amish people) “probably don’t pay nearly that much”. this interviewee was very surprised to hear that the costs for non-amish who use conventional insurance are in fact significantly higher than what he was paying for amish hospital aid (although the type of coverage provided by conventional insurance varies). the funds are payable to the committee men on the first day of every month. members tend to be very punctual with their payments and are contacted (sometimes by phone) if their check does not arrive by the due date. the committee man then transfers the funds collected to the district’s treasurer who then transfers them to the bank for later dispersal, as needed. . . complementary care used to minimize conventional care respondents were asked about ways they stayed healthy and minimized the need for health care. they mentioned attention to good nutrition and exercise, including their vigorous farm work. in addition, unmarried amish youth often participate in sports such as softball and volleyball. they frequently take natural vitamins and probiotics in order to minimize health problems, such as vitamin c for a bad cough. home remedies are typically used for less serious ailments (such as colds, minor burns, and infections, etc.), and replace visits to a family doctor. remedies mentioned included a combination of herbs and whiskey (known as tincture) for a sore throat, burdock for burns, and charcoal for infections, many of which can be found in an often-mentioned book, be your own doctor [ ] (a local health food store miller’s natural foods located in bird-in-hand, pa supplies many amish families in the lancaster county area with natural vitamins and supplies for home remedies). it is common for individuals to follow their family’s conventions in their inclinations to go to a medical doctor. nonetheless, there is a lot of variability in opinion: one respondent stated that he would go to the doctor if he had a cold he could not shake, while his wife said that for similar problems, she prefers home remedies. the continued used of complementary medicine in the amish community, however, does not preclude mainstream medical treatment (perhaps in part an acknowledgement of the limits of complementary care which is not always beneficial). healthcare , , of . . willingness to seek conventional medical care amish should not be confused with other groups loathe to seek modern health care for themselves or their children (e.g., [ ]). chiropractic care (including for infants) and phenology are especially popular [ ]. despite stereotypes that the amish are luddites—perhaps assumed because of their use of the horse and buggy—amish are generally very willing to take advantage of the most cutting-edge technology to help remedy their children’s maladies, no matter the cost to them personally [ ]. they avoid only technology that they believe detracts from their relationship with god, or family and community life. many interviewees expressed a willingness to benefit from modern health care, without moral (if not financial) reservations. many indicated that they would seek conventional care: “if something happens” or “whenever we need it”. additional related comments included: “if it’s a doctor thing, it’s a doctor thing” and “if something looks fishy, check it out”. some amish only visit their family doctor for an annual check-up, while others wait until an injury occurs. most amish praised the health care they had received, especially doctors who were good listeners and willing to “cater to the plain community” by not necessarily thinking like a medical doctor (e.g., allowing amish families to practice home remedies). one woman whose young daughter had a genetic disorder that required her to be in and out of the hospital for the first few months of her life spoke highly of the extra care that her doctors from the above-referenced clinic for special children provided to the family, including monthly visits directly to their family farm to check on her daughter. the amish approach to seeking conventional medical care also takes religion into account: “amish people are more willing to stop interventions earlier and resist invasive therapies than the general population because, while they long for healing, they also have a profound respect for god’s will. this means taking modest steps toward healing sick bodies, giving preference to natural remedies, setting common-sense limits, and believing that in the end their bodies are in god’s hands” [ ] (p. ). . . why respondents support amish hospital aid interviewees referred to amish independence in explaining the need for having their own system for health care payments. comments included: “the amish community is not in favor of government hand-outs”, “amish prefer to take care of their own”, “obamacare requires too much government involvement”, “the less government the better”. one respondent opined that “obamacare commandeers freedom of choice” and contrasts with the ability of members of the amish hospital aid plan to choose whichever medical facility best suits their needs. respondents also mentioned the benefits of assisting others and helping others while helping themselves: “we appreciate the privilege to take care of our own people”. their belief that a communal approach to covering health costs is morally right is consistent with their strong commitment to community members looking after one another. . . divisions in the amish community the amish hospital aid program is not without challenges, however, as more conservative amish (who tend to reside in the southern areas of lancaster county) are less likely to participate in the program, reflecting a well-known north–south divide in lancaster county (roughly represented by route ). one southern respondent who objected to amish hospital aid explained that “plainer amish don’t use it” and perceived it as, “the rich helping the rich”. northern lancaster county amish (e.g., those living in and around intercourse) tend to be more affluent than their more conservative southern neighbors living in towns like quarryville. relative to much more conservative amish in other parts of pennsylvania, however, the difference between northern and southern lancaster county amish is much less prominent. healthcare , , of some of the more conservative amish see the amish hospital aid plan as inappropriately progressive and institutionalized. many believe it detracts from neighbors helping each other completely voluntarily (with its set monthly fees, etc.) and view it as taking away from donations to the alms fund, a belief that remains to be substantiated. these individuals are more apt to approve only of the traditional amish alms support (akin to church tithing) for members’ medical needs [ ]. part of the appeal of alms is the reliance on voluntary donations, which bears no resemblance to standard health insurance. nonetheless, the extent of participation in amish hospital aid is growing in southern settlements of lancaster county. some northern amish prefer the amish hospital aid plan because alms are sometimes considered “poor money”, making wealthier amish feel guilty if they receive financial support from the congregation that is traditionally meant for those who are less financially stable. the question of wealth is complicated for amish who have assets in land but few liquid assets, such as cash or things they could sell quickly to pay a medical bill. one respondent frowned on wealthier amish whom he believed would be exploiting alms if they have significant funds tied up in land. he gave a hypothetical example of someone who owned a couple of farms who had a bad accident. he claimed that the congregation would not expect an individual to sell one of their farms to make the payments, so the deacon would provide alms money. he added, “i’d be a hypocrite for having two farms and taking money from the church”. this hypothetical example has similarities with the publicized case of jesse martin, an old order mennonite farmer from denver, pennsylvania who received national news coverage in because of his struggles to pay for healthcare. because martin did not sell any part of his two valuable farms, he was unable to pay bills totaling hundreds of thousands of dollars for nine of his children who had serious genetic disorders including hirschsprung disease and maple syrup urine disease [ ]. in cases where there are many high medical bills, even a combination of personal resources, alms, and amish hospital aid can be insufficient [ ]. nevertheless, these cases are the exception. according to a health survey of lancaster county old order amish (response rate: %), their system is adequate: only % of amish surveyed had delayed care in the past months (most commonly because of the expense), and only % (vs. % of county residents) rated their health as fair or poor [ ]. . . limitations this research relies on a small but select sample of individuals in a limited geographical area who were able and willing to discuss the administration of amish hospital aid and its advantages and shortfalls. although the sample was biased based on the male relative’s business connections, individuals were selected for their knowledge and insight on the topic, as well as a commitment to providing new in-depth and candid information about the amish. it is important, however, to acknowledge that the small sample size limits the generalizability of these findings, despite the thoughtfulness of respondents’ answers. data from a random sample would likely have revealed different reactions to amish hospital aid, although a random sample would have yielded a significantly lower response rate from those outside of the social network of the community shared by amish and mennonites. in addition, given that the data provided were confidential, but not anonymous, some interviewees may have limited what information they disclosed during the interview. nevertheless, because the main goal of the study was to understand the structure of amish hospital aid, interviewees may have felt more comfortable than if the research were designed to assess the program’s popularity or effectiveness. . discussion this paper describes a unique community-based approach to healthcare financing. the amish have achieved and sustained a large measure of self-sufficiency in their own system for managing costs that reflects the spirit of mutualism. the amish approach provides a stark contrast to the healthcare , , of current mainstream healthcare environment, where there is significant federal government control over healthcare decision-making and a pressing need to curtail spending. the amish also provide a small-scale example of healthcare rationing by implementing a program that covers only major medical needs. by limiting coverage, they have devised a manageable system bolstered by a strong sense of personal and group responsibility. because members of amish hospital aid can, if necessary, seek help to pay their % share of a bill through alms, they must be cognizant of their standing in the community, since coverage of an individual’s share is subject to review. this facet of the system reinforces amish inter-connectedness, as they face healthcare challenges collectively, mindful that judicious use of health care resources—including preventative measures—benefit the community. in the amish system, individuals are more likely to understand that it is impossible to cover all medical desiderata. in contrast, in the mainstream healthcare system, there is no way for patients to see that spending money on one individual perforce reduces the amount that can be spent on others. amish insight into this equation helps to provide an incentive on an individual level to limit care or costs that are otherwise very abstract. collaborative efforts by the amish to manage healthcare costs could inspire new ways of thinking about containing costs while building community. . conclusions the amish norm of reciprocal assistance without government interference is the basis for a system of paying for healthcare that builds on existing resources while limiting coverage. this article elucidates the unique way that a sample of old order amish of lancaster county manage healthcare financing by providing never-before reported information about the organization and administration of amish hospital aid. this information was obtained through the assistance of a trusted mennonite community liaison, imperative to elicit the level of candor needed to learn the kinds of details contained in the interview data presented in this article. although the sample size is small, interviewees include persons very familiar with the inner workings of amish hospital aid. documenting the unorthodox manner in which the amish community collaborates in managing healthcare costs could inform innovative alternatives to mainstream healthcare financing. acknowledgments: the authors are grateful to the department of sociology, mcdaniel college for covering the costs to publish in open access. author contributions: kristyn rohrer and lauren dundes conceived and designed the study; kristyn rohrer collected and transcribed the data; kristyn rohrer and lauren dundes analyzed the data; lauren dundes and kristyn rohrer wrote the paper. conflicts of interest: the authors declare no conflict of interest. references . social security administration. available online: 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[crossref] [pubmed] © by the authors; licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc-by) license (http://creativecommons.org/licenses/by/ . /). http://lancasteronline.com/news/local/cell-phones-computers-more-and-more-part-of-lancaster-county/article_ d c-def -- e -- e -- a bcf .html http://lancasteronline.com/news/local/cell-phones-computers-more-and-more-part-of-lancaster-county/article_ d c-def -- e -- e -- a bcf .html http://lancasteronline.com/news/local/cell-phones-computers-more-and-more-part-of-lancaster-county/article_ d c-def -- e -- e -- a bcf .html https://www.ssa.gov/pubs/en- - .pdf http://www.npr.org/templates/story/story.php?storyid= http://www.npr.org/templates/story/story.php?storyid= https://www.healthcare.gov/exemptions-tool/#/results/ /details/healthcare-sharing-ministry https://www.healthcare.gov/exemptions-tool/#/results/ /details/healthcare-sharing-ministry http://dx.doi.org/ . / http://www.ncbi.nlm.nih.gov/pubmed/ https://mosaicscience.com/story/amish-healthcare-medicine http://www.readingeagle.com/ap/article/small-lancaster-county-clinic-treating-amish-breaking-ground-on-genetics http://www.readingeagle.com/ap/article/small-lancaster-county-clinic-treating-amish-breaking-ground-on-genetics http://dx.doi.org/ . /ajph. . http://www.ncbi.nlm.nih.gov/pubmed/ http://www.wsj.com/articles/sb http://www.wsj.com/articles/sb https://cdn.americanprogress.org/wp-content/uploads/issues/ / /pdf/papercuts_final.pdf https://cdn.americanprogress.org/wp-content/uploads/issues/ / /pdf/papercuts_final.pdf http://dx.doi.org/ . /hlthaff. . http://www.ncbi.nlm.nih.gov/pubmed/ http://www.nytimes.com/ / / /us/ faith.html?_r= http://dx.doi.org/ . /s - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	purpose of study 	background 	amish vs. mennonite 	methods 	access to the amish 	obtaining interviews 	results 	traditional ways of paying for health care 	implementation of amish hospital aid 	how amish hospital aid manages medical costs 	restrictions in amish hospital aid plan coverage 	extent of participation in amish hospital aid 	hierarchy of amish hospital aid administration 	role of unpaid administrators 	cost of amish hospital aid 	complementary care used to minimize conventional care 	willingness to seek conventional medical care 	why respondents support amish hospital aid 	divisions in the amish community 	limitations 	discussion 	conclusions the abcg g r variant, serum plant sterol levels, and cardiovascular disease risk in the old order amish | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /atvbaha. . corpus id: the abcg g r variant, serum plant sterol levels, and cardiovascular disease risk in the old order amish @article{horenstein theag, title={the abcg g r variant, serum plant sterol levels, and cardiovascular disease risk in the old order amish}, author={r. horenstein and b. mitchell and w. post and d. l{\"u}tjohann and k. von bergmann and k. ryan and m. terrin and a. shuldiner and n. steinle}, journal={arteriosclerosis, thrombosis, and vascular biology}, year={ }, volume={ }, pages={ – } } 	r. horenstein, b. mitchell, + authors n. steinle 	published 	biology, medicine 	arteriosclerosis, thrombosis, and vascular biology objective—to determine whether long-term exposure to moderate elevations in plasma plant sterol levels increases risk for atherosclerosis. methods and results—in old order amish participants aged to years, with (n= ) and without (n= ) copy of the abcg g r variant, we compared mean plasma levels of plant sterols and cholesterol precursors and carotid intima-media wall thickness. carriers of a single r allele had increased plant sterol levels (eg, %– % higher plasma levels of… expand view on wolters kluwer ahajournals.org save to library create alert cite launch research feed share this paper citationsbackground citations methods citations view all figures, tables, and topics from this paper 	 table 	 figure 	 table 	 figure 	 figure view all figures & tables 	phytosterols 	cardiovascular diseases 	lipoproteins 	lanosterol 	campesterol 	triglycerides 	stigmasterol 	atherosclerosis 	sitosterols 	lathosterol citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency the abcg / cholesterol transporter and myocardial infarction versus gallstone disease. 	s. stender, r. frikke-schmidt, b. nordestgaard, a. tybjærg‐hansen 	medicine 	journal of the american college of cardiology 	 	 	pdf 	view excerpt, cites background save 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... ... references showing - of references sort byrelevance most influenced papers recency no association between plasma levels of plant sterols and atherosclerosis in mice and men 	k. r. wilund, l. yu, + authors h. hobbs 	biology, medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 	 save alert research feed relationships of serum plant sterols (phytosterols) and cholesterol in hypercholesterolemic subjects, and familial aggregation of phytosterols, cholesterol, and premature coronary heart disease in hyperphytosterolemic probands and their first-degree relatives. 	c. glueck, j. speirs, t. tracy, p. streicher, e. illig, j. vandegrift 	biology, medicine 	metabolism: clinical and experimental 	 	 save alert research feed noncholesterol sterols and cholesterol lowering by long-term simvastatin treatment in coronary patients: relation to basal serum cholestanol. 	t. miettinen, t. strandberg, h. gylling 	biology, medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 	 save alert research feed independent association of serum squalene and noncholesterol sterols with coronary artery disease in postmenopausal women. 	r. rajaratnam, h. gylling, t. miettinen 	medicine 	journal of the american college of cardiology 	 	 	pdf save alert research feed disruption of cholesterol homeostasis by plant sterols. 	chendong yang, l. yu, w. li, fang xu, j. cohen, h. hobbs 	biology, medicine 	the journal of clinical investigation 	 	 	pdf save alert research feed familial combined hyperlipidemia is associated with alterations in the cholesterol synthesis pathway. 	t. v. van himbergen, s. otokozawa, + authors a. stalenhoef 	chemistry, medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 	pdf save alert research feed identification of a gene, abcg , important in the regulation of dietary cholesterol absorption 	mi-hye lee, k. lu, + authors s. patel 	biology, medicine 	nature genetics 	 	 	 save alert research feed new insights into the molecular actions of plant sterols and stanols in cholesterol metabolism. 	l. calpe-berdiel, j. c. escolà-gil, f. blanco-vaca 	biology, medicine 	atherosclerosis 	 	 save alert research feed serum sterols during stanol ester feeding in a mildly hypercholesterolemic population. 	h. gylling, p. puska, e. vartiainen, t. miettinen 	chemistry, medicine 	journal of lipid research 	 	 	pdf save alert research feed serum cholesterol and cholesterol and lipoprotein metabolism in hypercholesterolaemic niddm patients before and during sitostanol ester-margarine treatment 	h. gylling, t. a. miettinen 	chemistry, medicine 	diabetologia 	 	 	pdf save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization 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time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ microsoft word - _file _ .docx ifnγγγγ influences epithelial anti-viral responses via histone methylation of the rig-i promoter c. mirella spalluto , , akul singhania * , doriana cellura , christopher h. woelk , tilman sanchez-elsner , karl j. staples , , tom m.a. wilkinson , , . clinical & experimental sciences, university of southampton faculty of medicine, southampton general hospital, southampton, uk; southampton nihr respiratory biomedical research unit, southampton general hospital, southampton, uk; wessex investigational sciences hub, university of southampton faculty of medicine, southampton general hospital, southampton, uk. *current address: the francis crick institute, london, uk; running title ifnγ priming mediates rig-i epigenetic regulation corresponding author dr c. mirella spalluto phd, clinical & experimental sciences, university of southampton, faculty of medicine, mailpoint , wessex investigational sciences hub, southampton general hospital, southampton so yd page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society united kingdom tel: + . fax: + e-mail: c.spalluto@soton.ac.uk author contribution conceived and designed experiments c.m.s, k.j.s., t.s-e., t.m.a.w.; performed experiments c.m.s., d.c.; microarray analysis a.s.; interpretation of the data c.m.s, a.s., k.j.s., t.s-e.,t.m.a.w., c.h.w.; wrote the paper c.m.s, t.m.a.w.; all authors contributed to the final editing of the manuscript. word count for the abstract (max ): word count for the text (max ): conflict of interest: the authors have declared that no competing interests exists. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society abstract the asthmatic lung is prone to respiratory viral infections that exacerbate the symptoms of the underlying disease. recent work has suggested that a deficient th response in early life may lead to these aberrant anti-viral responses. we investigated whether the inflammatory environment of the airway epithelium could modulate anti-viral gene expression via epigenetic mechanisms, in order to study the development of a long-term dysregulation of innate responses, which are a hallmark of asthma. we primed aaleb, a human bronchial epithelial cell line, with ifnγ and il and subsequently infected cells with respiratory syncytial virus (rsv) and innate anti-viral genes expression and their epigenetic markers were analysed. priming epithelial cells with ifnγ reduced rsv viral load. microarray analysis identified that ifnγ-priming enhanced rig-i mrna expression and this expression correlated with epigenetic changes at the rig-i promoter that influenced its transcription. using chromatin immunoprecipitation, we observed a reduction of trimethylated histone lysine (h k me ) at the rig-i promoter. addition of inhibitor bix- to this model indicated an involvement of lysine methyltransferase g a in rig-i epigenetic regulation. these data suggest that prior exposure to ifnγ may leave an epigenetic mark upon the chromatin that enhances airway cells’ ability to better resist infection possibly via epigenetic upregulation of rig-i. these observations provide further evidence for a crucial role of ifnγ in the development of mature anti-viral responses within a model of respiratory infection. further clinical validation is required to determine if this effect in early life leads to changes in anti-viral responses associated with asthma. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society key words: ifnγ, cytokine priming, rig-i, epigenetic regulation of innate immune response, asthma. clinical relevance the airway inflammatory milieu at or before birth, may have important effects on the development of anti-viral innate immunity at the level of the epithelium. as rapid maturation of adaptive immunity from a tolerant (th ) to an anti-infective (th ) state occurs in the neonatal period, the presence of a th milieu appears key to the development of effective anti-viral responses. introduction the pulmonary innate immune system matures rapidly in early life ( ), but the age of the first viral infection and hence the state of maturity of the immune system at the time are critical factors predisposing to both severe infection and to the risk of long term airways disease ( - ). respiratory syncytial virus (rsv) is the most common cause of severe respiratory infection in new-born babies and each year leads to over million hospitalizations and around , deaths worldwide each year ( ). some infants have an inherent predisposition to severe rsv infection and to a tendency to post-infectious wheezing which is associated with increased pro-inflammatory cytokine production ( ). previous studies have investigated the role of cytokines in the development of asthma early in life, particularly the th cytokine, il . however, there are conflicting reports of the role of il in asthma development. il can drive normal human pediatric pbecs toward an asthmatic phenotype in vitro ( ), contrastingly, williams et al. ( ) have described deficient il production in babies that later developed atopic disease. furthermore, early life bacterial and viral infection increase il responses ( ). gern et al. ( ) examined peripheral page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society blood responses to activation by phytohemagglutinin (pha) in children and found that infants who wheezed with rsv infection had reduced pha-induced il at birth and babies with detectable pha-induced ifnγ were less likely to wheeze in the first year. studies in murine models showed that production of ifnγ during rsv infection determines the outcome of subsequent rsv reinfections ( ) and also mice infected for the first time as neonates have low ifnγ response compared to mice infected as adults. upon reinfection ifnγ deficient mice developed enhanced lung response ( , ). work from copenhaver et al. ( ) showed that cord blood ifnγ responses were inversely related to the frequency of subsequent viral respiratory infections. juntti et al. ( ) measured also cytokines responses to lps stimulation in cord blood and found that ifnγ responses were lower among the children hospitalised for rsv infection. in their key message juntti et al. states that infants with a severe rsv infection differ from healthy children in their innate immunity cytokine profile before the infection. taken together these data suggest that early life lung exposure to interferons may influence antiviral immunity in the longer term more than exposure to il . recently the copsac study linked asthma development with the number of viral respiratory tract infections episodes in the first year of life, but not with a particular viral trigger ( ). airway epithelial cells are not only a physical barrier between the human body and the external environment ( ), but also a key site in the innate immune response, being the first line of defence against lung pathogens. activated epithelial cells are able to produce antiviral and pro-inflammatory cytokines that enhance the innate immune response and activate adaptive immunity, in the lung one key function of ifnγ is to upregulate antiviral gene transcription ( - ). page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society environmental exposures in early life are also crucial in influencing how immune responses are programmed pre- and postnatally ( ). a study into the role of the environment in to the development of asthma and allergy was recently designed comparing amish and hutterite children. amish community follow traditional farming practices whereas hutterites live on large, highly industrialized, communal farms. taking advantage of their similar genetic ancestries and lifestyles the study showed that there was and times low incidence of asthma and allergic sensitization in amish children and endotoxin levels in amish house dust was . times as high suggesting one more that the amish environment has a protective effect against asthma modulating innate immune response ( ). von mutius and vercelli ( ) describes this ifnγ centred model and cite “farm living” as an example of the environment characterised by intense microbial exposure, in which mothers live during pregnancy, that is protective against asthma and allergies for the children later in life. these combined exposures, which occur at a crucial time for programming immune responses, upregulate regulatory t (treg) cell function and ifnγ production at birth, which in turn enhance innate immune responses (through increased expression of pattern-recognition receptors), and dampen th cell dependent allergic inflammation in early childhood ( ). the ability to produce high levels of ifnγ at birth may ensure effective responses to respiratory viral infections in early life, thereby counteracting the contribution of these infections to increased asthma susceptibility ( ). the ifnγ priming in our cellular model mimic the increased ifnγ upregulation following microbial exposure and help us to identify the effects of ifnγ priming in the antiviral response early in life. we investigated how ifnγ is able to modulate the innate immune response to rsv by the airway epithelium. we used an unbiased approach to identify modulation in gene page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society transcription induced by ifnγ-primed epithelium and further investigated the mechanisms by which ifnγ-priming may modulate the epithelial response to viral infection. in addition to transcription factors, gene expression can also be modulated in the longer term via epigenetic mechanisms. post-transcriptional modifications of nucleosomal histones in the chromatin, including methylation, leading to changes in gene transcription that affect the cellular phenotype and reflect the nature and timing of the environmental stimulus ( ). we therefore investigated the potential epigenetic mechanisms employed by ifnγ to regulate anti-viral gene transcription. some of the results of these studies have been previously reported in the form of abstracts: ( , ). page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society methods cell culture and rsv infection aaleb human bronchial epithelial cells ( ) were cultured in begm and used at a passage number between and and plated in well plates at approximately x cells per well. primary bronchial epithelial cells (pbecs) isolated from bronchial brushes from healthy controls undergoing bronchoscopy. subjects gave written informed consent and the study was approved by the south central—southampton b national research ethics service (nres) committee ( /sc/ ). pbecs were kept in culture in flasks coated with collagen (purecol, nutacon, netherlands) using bronchial epithelial growth medium (bebm plus singlequots of growth supplements) (lonza, slough, uk). cells at passage or were then plated in collagen coated well plates at x cells per well for subsequent experiments. pbecs and aaleb cells prepared in well plate and maintained for h in starvation media (bebm supplemented with its x (insulin, transferrin, selenium, life biotechnology, thermo fisher scientific waltham, ma usa) and . % bsa (sigma dorset, england). where required, h before viral infection, il or ifnγ (both ng/ml, r&d systems, abingdon, uk) were added to media. before rsv infection, cells were washed twice with dmem (sigma, dorset, england) supplemented with mm glutamine (sigma) before rsv subtype a memphis (amsbio abingdon, uk) ( ) at moi was added to the wells required in a volume of µl for h at o c with gentle agitation. then cells were washed twice with dmem mm glutamine and cultured for h in starvation media. cells were harvested and stored in trifast (peqgold peqlab, southampton, uk) at - o c prior to rna extraction. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society rna extraction rna extraction was performed as per manufacturer’s instructions (see online protocol). microarray analysis global gene expression was assessed using the affymetrix genechip u plus . (see online protocol). geo accession number: gse (reviewer access link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=utmbyqyqbnmrxix&acc=gse ). epigenetic inhibitors aaleb cells, were plated in well plates at about x cells per well can cultured for h and then exposed to a range of epigenetic inhibitors for hours in starvation media (table e ). ifnγ was then added at ng/ml for h and cells were collected in trifast (peqgold peqlab, southampton, uk) and rna extraction was performed with standard protocol as stated earlier. rt-qpcr (see online protocol and table e for taqman gene expression assays) chromatin immunoprecipitation (chip) chip was performed essentially as previously described ( ) (see online protocol). antibodies and pcr primers in tables e and e respectively. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society production of cell lysates and protein quantitation for western blot experiments see online protocol. statistical analysis data were analysed with software graphpad prism software version using one-way anova (holm-sidak's multiple comparisons test) for gene expression analysis and two-tailed wilcoxon paired t-test for analysis of pull down isolated chromatin from chip experiments. number of samples between and as indicated in the legend. error bars represent mean with sem. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society results viral shedding is reduced in the presence of ifnγγγγ. in an attempt to investigate the effects of the local cytokine environment on the airway epithelium we first investigated the effects of th (ifnγ) and th (il ) cytokines in modulating epithelial responses to viral infection. aaleb cells, a cell line derived from human airways epithelial cells, ( ) previously used by tesfaigzi’s group ( ) to study the link between p and ifnγ were used for these sets of experiments. aaleb cells, were pre- treated with ifnγ or il ( ), then cells were infected with rsv (figure a). rsv efficiently replicated within the aaleb cells as measured by rt-qpcr (figure b). ifnγ pre-treatment reduced rsv replication compared to un-primed cells but il had no statistically significant effect on viral replication (p= . ) (figure c). ifnγγγγ-modulated genes are involved in the viral innate immune response. unbiased gene expression analysis using microarrays was then performed to identify innate immune genes that may be responsible for the priming effect of ifnγ on the respiratory epithelium. gene expression changes were determined for the following conditions compared to the untreated control: ifnγ treated, rsv infected, ifnγ pre-treated and rsv infected. (figure a). as expected, several genes involved in inflammation, interferon response, and antigen presentation were upregulated following rsv infection in ifnγ-primed cells. as a consequence of ifnγ priming, amongst the top genes differentially expressed were the interferon stimulated genes, isgs: mx , ifit and ifit , known to inhibit viral replication. we focused on the antiviral receptor rig-i (also known as ddx ) because of its key role in page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society the detection and eradication of replicating viral genomes ( ). rig-i is known to be upregulated in hela cells stimulated by ifnγ ( ) and similarly in intestinal cells ( ). furthermore, other groups have previously highlighted the robust link between rig-i and viral load. kato et al. demonstrated that rig-i -/- mice are more susceptible to viral infections compared to control mice ( ). in addition, sirna-mediated knockout of rig-i expression in human bronchial epithelial tissue showed reduced innate cytokine responses and rhinovirus replication ( ). similarly, foronjy et al. stated that silencing rig-i in a epithelium had a significant impact on rsv infection ( ). overall the work already published clearly shows an association between rig-i expression and modulation in viral infection. furthermore, genetic association studies have already linked polymorphisms in this gene to viral respiratory illness ( ) and also to asthma exacerbations ( ). ifnγ upregulated the expression of rig-i compared to both control cells and rsv infected cells (figure b). a further increase in rig-i expression by ifnγ-primed rsv- infected cells, was also observed and confirmed by rt-qpcr (figure ). in addition to ifnγ effects on cells we investigated the effects of il and no significant changes in rig-i expression were seen when were primed with il with or without exposure to rsv. we further investigated the relevance of this observation of increased rig-i expression in response to ifnγ in primary human bronchial epithelial cells (pbecs). similarly to aaleb, ifnγ priming caused upregulation of rig-i expression in pbecs h after ifnγ treatment (figure a). intriguingly, in the pbecs this effect of ifnγ on rig-i expression was still manifest up to days after ifnγ was removed from the culture medium (figure b). furthermore ifnγ reduced viral load in ifnγ-primed rsv-infected pbecs compared to rsv control cells and il -primed rsv-infected pbecs (figure c). a further increase in rig-i expression by ifnγ-primed rsv-infected pbecs, was also observed and confirmed by rt- page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society qpcr (figure d). we didn’t reach significance in pbecs due to a small number of repeats (n= ) when we measured viral load and rig-i following priming and rsv infection but these data indicated a long term effect of ifnγ priming in pbecs and suggest epigenetic regulation of rig-i expression may play a role alongside any temporary transcriptional regulation. due to their almost unlimited availability and similarity of rig-i responses in pbecs following ifnγ priming, we opted to continue analysis of the epigenetic control of gene regulation in aaleb cells. epigenetic analysis requires great amounts of chromatin and an immortalised cell line like aaleb would be an ideal model to use for this project. effects of ifnγγγγ-priming on epigenetic regulation of rig-i expression we investigated if the increase of rig-i transcription induced by ifnγ−priming correlated with epigenetic changes at the rig-i promoter and hence focused on changes in the levels of histone methylation associated with increased rig-i transcription. in association with an increase in rig-i mrna detected at h following ifnγ priming, we saw a significant reduction of the methylation of the lysine residue on histone (h k me ) (figure ), but not of lysine (h k ) or lysine (h k ) at the rig-i promoter (figure b), in aaleb cells using chromatin immunoprecipitation (chip). trimethylation of h k is associated with transcriptional repression and hence its reduction following ifnγ priming is consistent with the observed increase in rig-i expression. in addition we investigated histone acetylation of the lysine residue (h k ac) as this also has a functional link to gene expression ( ). in our cellular model h k ac was not altered by ifnγ, indicating that h k ac may not be involved in ifnγ-regulated rig-i expression. in addition, rnapolii, an essential element in the transcription complex, recruited to the rig-i promoter was detected at the promoter as expected but it did not change significantly with priming (figure c). page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society functional effects of epigenetic markers on rig-i expression to further analyse the epigenetic factors influenced by ifnγ priming and to investigate their functional consequences we employed small molecule inhibitors of histone demethylases and methyltransferases (table e ) to identify the role of histone methylation in rig-i transcription (figure ). first we employed the methyltransferase inhibitors wdr - and dznep. wdr - inhibits methylation of lysine on histone h (h k me ) ( ), whilst dznep inhibits trimethylation of lysine on histone h (h k me ) ( ). when cells where pre-treated either with wdr - or dznep with or without ifnγ priming, there was no effect on rig-i transcription compared to the untreated cells (data not shown) further confirming that h k me and h k me were not involved in rig-i modulation by ifnγ. analysis of histone demethylase function on rig-i epigenetic regulation as h k methylation appeared to be involved in these ifnγ-mediated epigenetic changes of the rig-i promoter, we next explored if the histone-modifying enzymes involved in the regulation of h k tri- and di-methylation were ifnγ-modulated. unlike monomethylated h k which promotes transcription, di- and tri-methylation of h k are capable of preventing activation of gene expression ( ). several enzymatic complexes are required to modulate methylation that ultimately regulates transcription and ensures that epigenetic information is carried over through cellular replication ( ). based on our findings that ifnγ priming was associated with a reduction of tri-methylation of h k at the rig-i promoter we investigated two possible options: a) ifnγ removes methyl residues from h k me by recruiting demethylases, b) ifnγ inhibits the methyltransferases that add methyl groups to h k me and h k me . to investigate which mechanism is used by ifnγ to modulate rig-i page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society transcription we used jib- a pan inhibitor of the activity of the jumonji family of histone demethylases ( ) and bix- (diazepin-quinazolin-amine derivative) that inactivates the lysine methyltransferase g a involved in h k me methylation ( ). additionally, for testing if lysine-specific demethylase (lsd ) was involved in mono- and di- demethylation of h k , we used pargyline and tranylcypromine as inhibitors ( , ) pargyline and tranylcypromine treatment did not cause any significant increase in rig-i (data not shown) possibly indicating that lysine demethylation of mono and di methylation of h k is not involved in ifnγ-mediated rig-i regulation. following treatment with µm jib- , contrary to expectations, we observed a significant increase of rig-i transcription in ifnγ-primed cells compared to cells treated with jib- alone. the effects of this compound are dose dependent and the concentration we used ( um) was aimed at inhibiting jmjd c (kdm c) the enzyme responsible for demethylating h k me (figure a). we further analysed the effects of jib- on the rig-i promoter by chip. intriguingly, no differences in h k me were detected at the rig-i promoter in ifnγ primed cells following jib- treatment (not shown). this result suggests that jib- may be acting on rig-i transcription through other regulatory mechanisms that do not involve h k me . similarly, no changes were observed for h k me , h k me , h k ac and rnapolii in the presence of jib- (data not shown). the inhibitor jib- without ifnγ priming, promotes an increase in transcription; this indicates that some of its effects are independent of ifnγ (figure a). due to the purpose of this investigation, aimed a characterise regulation ifnγ-mediated, jib- was not investigated any further. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society involvement of lysine methyltransferase g a in rig-i epigenetic regulation we next studied whether ifnγ modulates rig-i via inhibition of methylation, possibly interfering with lysine methyltransferase g a that uses mono-methylated h k (h k me ) as substrate to add a second methyl group (h k me ). the activity of g a leads to an increase of di-methylated h k that is then the substrate for h k trimethylation, a transcription repressive marker. inhibiting g a with bix- should therefore results in an increase of transcription. treatment of aalebs with bix- alone did not lead to any change in rig-i expression at either the rna or protein level. as expected, addition of bix- to ifnγ primed cells led to a significant increase in rig-i expression at both the rna and protein level compared to ifnγ alone (figure b and c). this suggests the involvement of lysine methyltransferase g a in rig-i epigenetic regulation by ifnγ. chip analysis revealed that the levels of h k me , did not change after bix- treatment alone compared to untreated and with or without ifnγ pre-treatment (figure e a). we observed no correlation between rig-i expression and the levels of h k me after bix- treatment (figure e b). taken together these results suggest that these epigenetic inhibitors may stabilise the ifnγ-induced reduction in h k me leading to enhanced rig-i transcription. to demonstrate that the effects seen on rig-i were specific, we measured the gene expression levels of a housekeeping gene glyceraldehyde -phosphate dehydrogenase (gapdh) and showed that its expression (figure e a) and levels of h k me or me (figure e c and e e) were not affected by ifnγ priming or bix- treatment. the toll like receptor (tlr ) gene was expressed in aalebs at barely detectable levels, but was also not affected by ifnγ priming or bix- treatment (figure e b), tlr gene shows at the promoter high levels of repressive epigenetic marks h k me and me compared to page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society gapdh consistent with its transcription levels (figure e d and e f). this indicates that the effects of the methyltransferase inhibitor bix- are gene specific and here we see an effect of this inhibitor on ifnγ regulated genes. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society discussion we have demonstrated that ifnγ priming of epithelial cells reduces viral load and the enhanced rig-i mrna expression following ifnγ priming correlated with epigenetic changes at the rig-i promoter linked with increased transcription. global gene expression analysis using microarrays following the exposure of epithelial cells to ifnγ and rsv identified induction of many epithelial expressed anti-viral genes including the cytosolic viral sensor rig-i known for its role in the detection and eradication of replicating viral genomes ( ). rig-i upregulation was also confirmed using rt-qpcr. ifnγ priming caused a reduction of transcriptionally repressive histone methylation, h k me , at the rig-i promoter. furthermore, use of the inhibitors jib- and bix- suggested that more than one mechanism may be involved in this ifnγ-mediated epigenetic regulation of rig-i. the specific mechanism of ifnγ priming still needs to be elucidated but taken together these results suggest that exposure to ifnγ can prime the anti-viral response of the airway epithelium by epigenetic changes to key pathways. increasingly strong evidence suggests that the environment is capable of inducing innate immune memory and that epigenetic changes, including histone methylation, play an important role in the development and persistence of this memory ( ). ooi et al. ( ) showed that mechanisms of epigenetic regulation involved in allergic airway inflammation were key in the development of a chronic asthmatic phenotype. early life has already been shown to be a critical stage where allergens, poor air quality and repeated viral infections impacting on pre-existing genetic susceptibility can impact on the evolution of asthma ( , ). in contrast, exposure to farming environments and endotoxins for instance can lead to higher levels of ifnγ pre- and post-natally and may be protective against an asthma page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society phenotype ( , - ). studies in mice have shown that ifnγ is required for viral clearance ( , ) but there is also data showing that ifnγ can prevent replication ( ). our data do not provide an indication as to which of these mechanisms predominate in our model. il has been linked to airway inflammation and remodelling but it seems to be a response to rsv rather than the cause of a severe response during viral infection ( , ). our cellular model data support these conclusions in that the addition of il did not affect viral load or rig-i expression, an rna sensor known to have a pivotal role in the antiviral immune response ( ). these data suggest that the “th hypothesis”, an upregulated th and a downregulated th response drive the development of the disease ( ), may be re-read as “a lack of a th and not a th predominant immune milieu per se that drives disease development”. ifnγ regulates downstream genes through phosphorylation and activation of stat homodimers which activate gamma activating sequence (gas) at the promoter of immune related genes and increases transcription by recruiting interferon regulatory factors (irfs) ( ). rig-i gene is under the regulation of stat ( ) and in our model we show that the regulation of rig-i by ifnγ is further influenced at the epigenetic level by a reduction of the repressive marker h k me leaving a signature on the histones. interestingly, robertson et al. ( ) showed that % of stat promoters do not contain h k me and similarly we did not find involvement of h k me and h k me in the regulation of rig-i transcription suggesting that the polycomb/trithorax enzymatic complex ( ) are not modulating rig-i regulation by ifnγ. this trimethylation of lysine on histone is associated with heterochromatin and inactive genes, although there are exceptions where methylation on lysine contributes to the regulation of euchromatic genes ( - ). in our cellular model, where rig-i is expressed page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society even in unstimulated cells, the presence of h k me at low levels is permissive of transcription and its further reduction following ifnγ priming is linked with an increase of rig-i gene expression ( ). in an attempt to understand how ifnγ induces these epigenetic changes at the rig-i promoter we used small molecule epigenetic inhibitors. the lysine methyltransferase inhibitor (bix- ) and the pan inhibitor of jumonji histone demethylases (jib- ) upregulated rig-i expression in the presence of ifnγ, but neither of them indicate what the epigenetic changes caused by ifnγ priming are. jib- induced a trend towards upregulation of rig-i expression in aaleb cells in the absence of ifnγ. this observation may suggest a direct correlation between jmjd c/kdm c (the enzyme responsible for demethylating h k me ), which is inhibited by jib- , and rig-i expression. whilst in the presence of ifnγ, the effect of jib- on rig-i expression was additive, the primary aim of this investigation was to identify regulatory mechanisms that were mediated purely by ifnγ. we therefore concentrated on bix- , which, in contrast to jib- , had significant effects on rig-i expression only in ifnγ primed cells. bix- alone did not cause a reduction in the tri-methylation levels of h k (h k me ) at the rig-i promoter. tri- methylation of h k was reduced when cells were primed with ifnγ, but this was not further reduced in the presence of bix- . this could indicate that these inhibitors may have other unidentified ways to potentiate the mechanisms used by ifnγ to regulate rig-i. additionally we investigated whether ifnγ modulates rig-i by interfering with lysine methyltransferase g a ( ) that uses mono-methylated h k (h k me ) as substrate to add a second methyl group (h k me ). the activity of g a physiologically leads to an increase of h k me a substrate for h k me known to be a transcriptional repressor marker. when we consider that bix- inhibits g a involved in h k me methylation, we would expect a reduction in the level of h k me in cells exposed to bix- although page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society in our experiments this reduction did not reach significance. the removal of h k me did not increase rig-i transcription on its own and the levels of h k me described earlier remained high, which may mask any effects produced by bix- . thus, even though we have inhibited demethylases and methyltransferases known to be associated with h k methylation we saw no effect on baseline methylation. however, ifnγ reduced h k me of the rig-i promoter and increased gene transcription in the presence of both inhibitors. therefore, ifnγ may modulate h k methylation and rig-i transcription via a different mechanism and further work is required to elucidate this. it would also be informative to analyse other areas of the rig-i promoter to have a clearer picture of the histone profile and possibly where the effects of the epigenetic inhibitors would be more evident. we may have not investigated the histone markers directly regulated by ifnγ and what we have shown here is a downstream effect and not the cause of change in transcription levels. these findings do not identify the mechanisms associated with ifnγ regulation, although the results strongly suggest that the cytokine environment may epigenetically modulate genes involved in the viral response and drive innate future responses. there are limitations in this investigation that may have restrict our findings. the use of aaleb cells as an epithelial model instead of primary airways epithelial cells may have given us an incomplete or an imperfect picture of the epigenetic profile of rig-i promoter. we also do not take into account the effects that other cytokines may have in conjunction with ifnγ on modulating gene expression. additionally we analyse the effects of ifnγ priming after only after hours and although it is enough to show an effect on rig-i promoter, it is possible that longer time points may have been more informative. the innate immune response and the th / th predisposition in the lung changes along the life course and these variations are evident in the interaction between host and page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society environment and correlate to susceptibility to disease ( , ). the inflammatory environment of naive epithelial cells may epigenetically modulate innate immune responses altering the levels of histone methylation and acetylation and hence potentially may lead to long term impacts on anti-viral immunity. the presence of a th and th milieu appear key to be involved in the development of innate immune responses. the effect of the cytokine milieu on the anti-viral responses rely on the environmental influences that modulate cytokine production, genetic background and on the timing of viral infections. further investigation using chip-seq analysis may identify other genes that are regulated in a similar fashion by cytokines and clarify mechanisms that once understood may provide a way to learn how to better modulate the innate system against viral infections and virus induced asthma. we describe a potentially important regulatory mechanism that need to be explored further but may explain the ability of the airway immune environment to modulate gene transcription early in life to prime immune responses. the airway inflammatory milieu, driven by the maturing immune response to the environment, may have important effects on the development of epithelial anti-viral innate immunity and the consequent development of its long term dysregulation, a key feature of asthma. page of ajrcmb articles in press. published on -may- as . /rcmb. - 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. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure legends figure detection of rsv replication in aaleb cells. (a) experimental design of ifnγ priming and viral infection on aaleb cells. aalebs, after being plated, were primed with ng/ml ifnγ (rsvifnγ ), ng/ml il (rsvil ), or untreated (rsv) for h before being exposed to rsv for h. (b) viral replication measured by rt-qpcr in aaleb cells exposed to rsv subtype a memphis at moi for h (rsv), with no virus quantified in uv–treated rsv cells (uv) and in the untreated cells control (un). n= , (c) ifnγ priming significantly reduced rsv replication. virus replication was detected with custom taqman assays (life technologies) specific for the n gene of the rsv genome. values were normalized against hprt and calculated using Δct method. n= figure (a) heatmap depicting the log expression values for genes with top peak fold changes in untreated aaleb cells (unt), rsv infected (rsv), primed with ifnγ (ifnγ) and primed and infected (rsv+ifnγ). hierarchical clustering was performed using pearson correlation metric to calculate distances and clustered using complete linkage. the genes expression values were averaged and scaled across every row to indicate the number of standard deviations above (red) or below the mean (blue), denoted as the row z-score. (b) log expression values for rig-i mrna with top peak fold changes in untreated cells (unt), rsv infected (rsv), primed with ifnγ (ifnγ) and primed and infected (rsvifnγ) n= . figure detection of rig-i mrna in aaleb cells measured by rt-qpcr after cytokine priming and rsv infection. aaleb cells were untreated (unt), primed with ng/ml il (il ) or ng/ml ifnγ (fnγ ) for h. aaleb cells were untreated (rsv), primed with ng/ml il (rsvil ) or ng/ml ifnγ (rsvifnγ ) for h before being exposed to rsv subtype a memphis at moi for h. values were normalized against hprt and calculated using Δct method. n= . figure (a) detection of rig-i mrna measured by rt-qpcr in pbecs exposed ifnγ priming ( ng/ml) for h, n= . (b) detection of rig-i mrna measured by rt-qpcr in pbecs being exposed to ifnγ ( ng/ml) for h and after being washed twice in pbs left in culture media for additional days, n= . ifnγ primed (ifnγ), or untreated (unt). values were normalized page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society against hprt gene and calculated using Δct method. (c) pbecs cells after being plated, were primed with ng/ml ifnγ (rsvifnγ ), ng/ml il (rsvil ), or untreated (rsv) for h before being exposed to rsv subtype a memphis at moi for h. virus replication was detected with custom taqman assays (life technologies) specific for the n gene of the rsv genome. n= (d) detection of rig-i mrna in pbecs measured by rt-qpcr after cytokine priming and rsv infection. values were normalized against hprt and calculated using Δct method. n= figure analysis of methylation and acetylation levels at lysines residues on histone at the promoter of rig-i gene on aaleb cells by chromatin immune precipitation (chip). aalebs primed with ifnγ (ifnγ) or left untreated (unt). (a) analysis of methylation levels at lysines residue on histone (h k me ). antibodies against trimethylated lysine on histone (h k me ) were used, n= . other antibodies against the following modifications were used: (b) trimethylated lysine on histone (h k me ), trimethylated lysine on histone (h k me ) n= , (c) acetylation on lysine on histone (h k ac) and against rna polymerase ii (rnapolii), n= . the pull down chromatin is amplified by qpcr with custom taqman assays specific for rig-i promoter sequence. percentage of input is calculated as × [ct(input) – ct(ip)], after adjusting the mean input ct value for / starting material (fraction of input chromatin reserved). the levels of h k me , and h k me do not change significantly. ifnγ causes a not significant reduction of h k ac and an increase of rnapolii. figure schematic representation of experimental conditions used to prime aaleb cells with ifnγ alongside the chemical compounds used to inhibit histone demethylases and methyltransferases. figure detection of rig-i mrna measured by rt-qpcr in aaleb cells exposed to epigenetic inhibitors. (a) aalebs exposed to µm jib- for h before ifnγ priming ( ng/ml) for h. jib- exposure and ifnγ priming (jib +ifnγ), ifnγ primed (ifnγ), exposed to jib- only (jib) or untreated (unt), n= . (b) aalebs were exposed to µm bix- for h before ifnγ priming ( ng/ml) for h. bix- exposure and ifnγ priming (bix +ifnγ), page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society ifnγ primed (ifnγ), exposed to bix- only (bix) or untreated (unt). both the inhibitors upregulate rig-i expression when cells were also ifnγ primed. values were normalized against hprt and calculated using Δct method. n= . (c) detection of rig-i protein levels measured by western blot following ifnγ priming and bix- treatment. representative image of independent experiments. tubulin served as loading control, ug of lysate. page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure (a) (b) (c) r s v n g e n e ( c t) r s v r s v il r s v if n page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure (a) (b) page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure (a)(a)(a)(a) (b(b(b(b)))) r ig -i m r n a ( c t) u n t if n r ig -i m r n a ( c t) u n t if n ((((cccc)))) (d(d(d(d)))) page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society � � ��������� � � � � �� � � � � � 	 � � � �� � � � � � 	 � � � �� � � � � � 	 � � � �� � � � � � 	 � � �$�$�$�$����� �%�%�%�%����� �&�&�&�&����� page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure h h h h h h h h ---- h h h h cellscellscellscells platedplatedplatedplated mrnamrnamrnamrna chipchipchipchip +/+/+/+/----ifnifnifnifnγγγγ dznepdznepdznepdznep wdr wdr wdr wdr ---- jibjibjibjib---- bixbixbixbix---- pargylinepargylinepargylinepargyline tranylcyprominetranylcyprominetranylcyprominetranylcypromine ---- h h h h page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure (a)(a)(a)(a) (b)(b)(b)(b) r ig -i m r n a ( c t) u n t b ix if n b ix + if n page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society u n t b ix u n t + i f n g b ix + if n g rig-i tubulin (c) page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society ifnγγγγ influences epithelial anti-viral responses via histone methylation of the rig-i promoter c. mirella spalluto , , akul singhania , doriana cellura , christopher h. woelk , tilman sanchez-elsner , karl j. staples , , tom m.a. wilkinson , , online supplement extended material and methods rna extraction per ml of trifast . ml of chloroform were added and the sample were centrifuged at , × g for minutes at °c. the aqueous phase was precipitated at - o c for minutes in the presence of µg of rnase-free glycogen as a carrier with . ml of % isopropanol. the rna was centrifuged at , × g for minutes at °c and the pellet washed twice in cold % ethanol. the rna used for microarray gene expression analysis was further purified with rneasy mini kit (qiagen, manchester, uk). as standard quality measurements rna was analysed with eukaryote total rna nano series ii assay kit on bioanalyzer (agilent technologies, cheshire, uk). rin values were above for every sample. microarray analysis global gene expression was assessed using the affymetrix genechip u plus . . labeling and array hybridisations were performed according to standard protocols (source bioscience, page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society berlin, germany) in aaleb cells subjected to the following conditions: ( ) untreated ( ) ifnγ treated, ( ) rsv infected and ( ) ifnγ treated and rsv infected. gene expression analysis was done in duplicate for every condition and rna for each condition was the result of three independent experiments pooled together. raw microarray gene expression data was normalized using gene chip robust multi-array analysis (gcrma) ( ) and subjected to quality control procedures. fold changes were calculated by averaging replicates and subtracting the log expression values from control. only genes with actual fold changes greater than or less than - were considered for functional enrichment analysis. direct protein interaction networks (pins) were generated using metacore™ (genego, st. joseph, mi, usa) and visualized in cytoscape (version . . ) ( ) and pathways significantly over- represented for differentially expressed genes in a hypergeometic test (fdr-corrected p- value < . ) were identified using toppgene ( ). rt-qpcr total extracted rna concentration was determined using a spectrophotometer (nanodrop ; thermo fisher scientific waltham, ma usa). ng of rna were reverse transcribed using the high capacity cdna reverse transcription kit (life technologies) with random examers primers carried out accordingly to the manufacturer’s protocols. rna expression was measured using taqman gene expression assays and rsv replication was detected with custom taqman assays as listed in table e . for rt-qpcr, we employed taqman® universal pcr master mix, no amperase® ung in a ht fast real-time pcr system machine (both from life technologies, thermo fisher scientific). the values were normalized against hypoxanthine-guanine page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society phosphoribosyltransferase (hprt ) from the same manufacturer using comparative ct method ( -delta ct ). chromatin immunoprecipitation (chip) cells were plated in cm petri dishes at concentration of . x . - h later cells were treated for h with the relevant inhibitor and then with ifnγ for further h. cells were crosslinked with % final concentration of formaldehyde for minutes. crosslinking was stopped by the addition of glycine for minutes to a final concentration of mm. cells washed three times with pbs, scraped and collected, and then resuspended in lysis buffer ( mm tris–hcl ph . , mm nacl, mm edta, % (v/v) triton-x- , . % (w/v) sds) with proteases inhibitors (roche, basel, ( - ) switzerland). the lysate was then sonicated with soniprep (mse, london, uk) with pulses of seconds each. the sample was centrifugated and the supernatant resuspended in lysis buffer with proteinases inhibitors and aliquoted. every chromatin aliquot was immunoprecipitated with the chosen antibody (table e ) and allowed to form complexes overnight at o c with agitation. one chromatin aliquot was used as negative control to which no antibody was added and µl of chromatin was also reserved as input. after the overnight antibody incubation, the immune complexes were incubated with protein g-agarose beads (ge healthcare) blocked with salmon sperm dna and bsa (life technologies). the protein g agarose/antibody/protein/dna complexes were washed three times with each of the following buffer: lysis buffer; ip buffer ( mm tris–hcl ph . , mm nacl, mm edta, % (v/v) triton-x- , . % (w/v) sds); ip buffer ( mm tris–hcl ph . , page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society mm licl, . % (v/v)np- , . % (w/v) sodium deoxycholate, mm edta); and two washes with te (  mm tris ph . ,  mm edta) to purify precipitated dna, complexes were incubated with dnase-free rnase (sigma) for min at o c, then . % sds and proteinase k (sigma) were added to remove proteins and incubated h at o c. after enzyme treatment, chromatin was incubated at o c for h to reverse the cross-link. dna was purified by phenol/chloroform extraction, precipitated with ethanol, and resuspended in te. recovered dna was amplified with custom taqman assays (life technologies) specific for the promoter of the genes analysed. primers and assay id in table e : duplicates qpcrs were performed using an abi ht fast real-time system (life technologies). production of cell lysates and protein quantitation for western blot experiments cells were lysed with % sds + x protease inhibitor cocktail (merck, kenilworth, new jersey, usa). ug of total protein lysate was loaded onto precast polyacrylamide gel (thermo fisher scientific ) in reducing conditions and proteins transferred onto hybond c extra nitrocellulose membrane (ge healthcare, buckinghamshire, uk). rig-i was detected with anti-ddx (ab abcam, cambridge, uk) : and tubulin (sc- santa cruz biotechnology, dallas, texas) : . detection was performed using horseradish peroxidase–conjugated secondary antibody goat anti-mouse igg-hrp (sc- santa cruz) : in % non-fat milk/tbs-t and e enhanced chemiluminescence (ecl)- based system (super signal west dura kit, thermo fisher scientific). images were acquired using a xograph x photographic film processor (xograph healthcare ltd, gloucestershire, uk). page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society references: . chen j, bardes ee, aronow bj, jegga ag. toppgene suite for gene list enrichment analysis and candidate gene prioritization. nucleic acids res ; (web server issue):w - . . doerks t, copley rr, schultz j, ponting cp, bork p. systematic identification of novel protein domain families associated with nuclear functions. genome research ; ( ): - . . wu z, irizarry r, gentlemen r. a model-based background adjustment for oligonucleotide expression arrays. j am statist assoc . . purvis tl, hearn t, spalluto c, knorz vj, hanley kp, sanchez-elsner t, hanley na, wilson di. transcriptional regulation of the alstrom syndrome gene alms by members of the rfx family and sp . gene ; ( - ): - . page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society supplemental tables and figures table e epigenetic inhibitors inhibitor concentration used company -deazaneplanocin a . µm r&d systems jib- µm r&d systems tranylcypromine µm enzo life science bix µm cambridge bioscience pargyline . mm cambridge bioscience wdr - µm cambridge bioscience page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society table e taqman gene expression assays (applied biosystems): gene name assay id primers sequence (where available) rsv (perkins et al. ) ait tb forward: catccagcaaatacaccatcca reverse: ttctgcacatcataattaggagtatcaa probe: cggagcacaggagat rig-i hs _m tlr hs _s gapdh hs _g hprt hs _m table e antibodies (abcam) used in chip experiments antibody code h k me ab h k me ab h k me mabcam h k me ab h k ac ab rna polymerase ii ab page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society table e taqman gene assays for chip (applied biosystems): gene name assay id primers sequence (where available) rig-i ajhsnwi tlr forward primer cgacctgatctttgtagttggaa reverse primer ggcaggaagtgtagtcacaaagac probe tccagggctggctg gapdh forward primer cctaattatcaggtccaggctacag reverse primer cgggaggcggcttga probe ctgcaggacatcgtg page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society supplementary figure legends figure e analysis of methylation levels at lysines residues on histone at the promoter of rig-i gene on aaleb cells by chromatin immune precipitation (chip). cells primed with ifnγ (ifnγ), untreated (unt), exposed to bix- (bix) and primed with ifnγ following exposure to bix- (bix+ifnγ). (a) antibodies against trimethylated lysine on histone (h k me ) n= and (b) dimethylated lysine on histone (h k me ) n= were used. the pull down chromatin was amplified by qpcr with custom taqman assays specific for rig-i promoter sequence. percentage of input was calculated as × [ct(input) – ct(ip)], after adjusting the mean input ct value for / starting material (fraction of input chromatin reserved). figure e detection of gapdh (a) and tlr (b) mrna measured by rt-qpcr in aaleb cells exposed to µm bix for h before ifnγ priming ( ng/ml) for h. cells primed with ifnγ (ifnγ), untreated (unt), exposed to bix- (bix) and primed with ifnγ after exposure to bix- (bix+ifnγ). analysis of methylation levels at lysines residues on histone at the promoter of gapdh and tlr genes on aaleb cells by chromatin immune precipitation (chip). antibodies against trimethylated lysine on histone (h k me ) (c and d) and dimethylated lysine on histone (h k me ) (e and f) were used. the pull down chromatin was amplified by qpcr with custom taqman assays specific for gapdh and tlr promoter sequence. percentage of input was calculated as × [ct(input) – ct(ip)], after adjusting the mean input ct value for / starting material (fraction of input chromatin reserved). n= page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure e (a)(a)(a)(a) (b)(b)(b)(b) page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society figure e (a)(a)(a)(a) (b)(b)(b)(b) (c)(c)(c)(c) (d)(d)(d)(d) (e)(e)(e)(e) (f)(f)(f)(f) gapdh h k me u n t b ix if n b ix + if n . . . . . . tlr h k me % in p u t u n t b ix if n b ix +i f n gapdh h k me u n t b ix if n b ix +i f n - . . . . tlr h k me u n t b ix if n b ix +i f n . . . . . page of ajrcmb articles in press. published on -may- as . /rcmb. - oc copyright © by the american thoracic society eur psychiatry ( ) , - © elsevier, paris letter to the editor linkage studies of major affective disorders: the impact of the extension of pedigrees ac bruni ' , mp montesi ' \ r manfredi , a di c e l l o , g gei , d fragiacomo , p de f a z i o , m p u c a \ a amati , jf foncin 'unit of neurology, ussl ; -smid-sud research center. lamezia terme; 'psychiatric care unit, uss , lainezia terme; icd-fidia abano terme; ussl , smid-sud center, via dei campioni, lamezia terine; 'chair of psychiatry, univer- sity of reggio calabria, school of medicine at catanzaro, italy; ecole pratique des hautes etudes, la salpetriere, paris, france strongly discordant results have been obtained so far in linkage studies of major affective disorders. egeland el al ( ) reported preliminary evi- dence suggesting a linkage between a major locus for bipolar disorder and two markers on chromo- some . however, linkage with the same markers was excluded (kelsoe el al, ) after the pedigree had been extended and new subjects developing the illness were assessed. both studies were conducted with great accuracy as regards ascertainment of phenotype, laboratory techniques and statistical analysis. the authors sug- gested that the initial results may have been obtained by chance or, alternatively, that a genetic heter- ogeneity of the disease exists in the amish popu- lation. whatever the cause of these discrepant findings, the only way to overcome such problems is by way of an extensive epidemiological-genetic study. the direct experience of our group is enlightening in this sense. bruni et al described in a family with a bipo- lar disorder segregating in an apparently dominant manner (fig ): four generations of affected people, no generation skipped, male to male transmission, and an affected father, married twice, who had generated affected children from both wives. sub- sequently, both affected and unaffected lines were extended vertically as well as horizontally, through a genealogical method called the blanket method (foncin et al, , ). data on birth, marriage and death were collected from municipal archives dating back to , and from parish archives dat- ing back to the th century. the kindred, as recon- stituted, contained not only blood relatives but also relatives by marriage and tended to be a descrip- tion of an intermarrying population. the extension of the pedigree (fig ), with the presence of affected people in branches apparently not at risk raises doubts as to the initial evidence, generating new hypotheses of transmission related to peculiar features of the kindred, such as genetic isolation, inbreeding, apparently assortative mating. we suggest that pedigrees in which major affec- tive disorders are segregating should be submitted to a systematic investigation through this epidemi- ological genetic method, in order to clarify fun- damental issues in the psychiatric field: model(s) of transmission, effects due to environment and/or genotype, comorbidity, assortative matings and phenocopies. i . , . . ~< jt tjprjro o '! ''' ̂ 's' ' ill : n, . . ii i? nor nt i'ik . original pedigree. subjects are identified by their pedi- gree number, black symbols = major affective disorders; s = suicide or attempted suicide. ac bruni et al d- o o- t̂ > o •a d - t - o d d- - ^l -ti -o o-ro d- i-n-<ji-ro - • a fig . a lateral extension of the original pedigree. the numbers identify the subjects reported in figure . black symbols = major affective disorders. acknowledgment supported by smid-sud center and cnr grant no . . . references bruni ac, montesi mp, manfrcdi r, gei g, ermio c, caruso g ( ) un esteso ceppo con probabile psicosi maniaco depressiva. rivisla di psychialria , , - egeland ja, gerhard ds, pauls dl, sussex jn, kidd kk, allen cr, hostetter am, housmann d ( ) bipolar affective disorders linked to dna markers on chromosome . nature , - foncin jf, salmon d, bruni ac ( ) extended kindreds as a model for research on alzheimer's dis- ease. in: genetics and alzheimer's disease. (sinet pm, lamour y, christen y, eds) springer-verlag, berlin heidelberg, - foncin j f , salmon d, bruni ac ( ) notes on the methods for the study of kindreds with familial al- zheimer's disease. in: familial alzheimer's disease. molecular genetics and clinical perspectives. (miner gd, richter rw, blass j p , valentine jl, winters- miner la, eds) marcel dekker, inc new york and basel, - kelsoe jr, ginns ei, egeland ja, gerhard ds, gold- stein am, sherri jb, pauls dl, long rt, kidd kk, conte g, housman de, paul sm ( ) re-evaluation of the linkage relationship between chromosome l i p loci and the gene for bipolar affective disorder in the old order amish. nature , - am. j. hum. genet. : – , calpainopathy—a survey of mutations and polymorphisms i. richard, c. roudaut, a. saenz, r. pogue, j. e. m. a. grimbergen, l. v. b. anderson, c. beley, a-m cobo, c. de diego, b. eymard, p. gallano, h. b. ginjaar, a. lasa, c. pollitt, h. topaloglu, j. a. urtizberea, m. de visser, a. van der kooi, k. bushby, e. bakker, a. lopez de munain, m. fardeau, and j. s. beckmann ura cnrs, généthon, evry, france; department of neurology and experimental unit, hospital ntra. sra. aranzazu, san sebastian, spain; school of biochemistry and genetics, university of newcastle upon tyne, newcastle upon tyne; mgc-department of human and clinical genetics, leiden university medical center, leiden; institut de myologie, inserm u. , hopital salpétrière, paris; hospital sant pau, unitat genetica molecular, barcelona; medical biology department, hacettepe university, ankara; and amsterdam medical center, amsterdam summary limb-girdle muscular dystrophy type a (lgmd a) is an autosomal recessive disorder characterized mainly by symmetrical and selective atrophy of the proximal limb muscles. it derives from defects in the human capn gene, which encodes the skeletal muscle–specific member of the calpain family. this report represents a compi- lation of the mutations and variants identified so far in this gene. to date, distinct pathogenic calpain mu- tations have been identified ( nonsense mutations, deletions/insertions, splice-site mutations, and mis- sense mutations), of which have not been described previously, together with polymorphisms and non- classified variants. the mutations are distributed along the entire length of the capn gene. thus far, most mutations identified represent private variants, although particular mutations have been found more frequently. knowledge of the mutation spectrum occurring in the capn gene may contribute significantly to structure/ function and pathogenesis studies. it may also help in the design of efficient mutation-screening strategies for calpainopathies. introduction limb-girdle muscular dystrophy type a (lgmd a [ mim ]) is an autosomal recessive disorder char- acterized by symmetrical and selective atrophy of the pelvic, scapular, and trunk muscles; elevated serum cre- received january , ; accepted for publication april , ; electronically published may , . address for correspondence and reprints: dr. jacques s. beckmann, généthon, rue de l’internationale, evry, france. e-mail: beckmann@genethon.fr q by the american society of human genetics. all rights reserved. - / / - $ . atine kinase; and a necrotic regeneration pattern on mus- cular biopsies. classically, calf hypertrophy is rare and always discrete, and there is no mental, cardiac, or facial disturbance. in the vast majority of cases, the symptoms arise in childhood, progress gradually, and, – years after onset, patients are frequently unable to walk and are confined to a wheelchair (beckmann and fardeau ). a limb-girdle muscle involvement can also be recognized in a number of muscular disorders—in par- ticular, in the other progressive muscular dystrophies that also have been regrouped under the term “lgmd ”; the latter comprise the four sarcoglycano- pathies (lgmd c–lgmd f; (roberds et al. ; bönnemann et al. ; lim et al. ; noguchi et al. ; nigro et al. ), which are characterized by a disruption of the sarcoglycan complex (campbell ), and three additional lgmd entities (lgmd b, lgmd g, and lgmd h [bashir et al. ; moreira et al. ; weiler et al. ]), which present no al- terations in the sarcoglycan complex. the causative gene for lgmd b recently has been identified as coding for dysferlin (bashir et al. ; liu et al. ). the gene responsible for lgmd a had been local- ized, by linkage and linkage-disequilibrium analyses, to chromosome q . - . , within a -cm region al- most entirely covered by a . -mb yac (beckmann et al. ; fougerousse et al. ; allamand et al. ). characterization of genes from this region led eventually to the identification of the lgmd a gene that encodes calpain , a member of the calpain family (chiannilk- ulchai et al. ; richard et al. ). the latter re- groups nonlysosomal calcium-dependent cysteine pro- teases, whose precise functional roles remain elusive. the human calpain gene (capn ) covers a genomic region of ∼ kb (richard et al. ). it is expressed predominantly in the skeletal muscle tissue, as a . -kb transcript, driving the translation of a -kd protein containing a proteolytic domain, a ca -activated do- main, and two other domains with no known homology richard et al.: calpain mutations and polymorphisms table primer pair used for detection of mutations, by allele-specific pcr mutation primer(s)a ( ′r ′) touchdown pcr uppera lower temperature range ( c) formamide concentration (%) da ataagatgactgcctgccaac ( ) ttcctgtgagtgaggtctcg – tagatgactgcctgccac ( ) g r ctacgaagctctgaaaggtg ( ) ggctttcttcatgatcttgt – ctacgaagctctgaaaggta ( ) ivs gra ctctggttactgctctacaa ( ) agcacgaaaagcaaagataaa – ctctggttactgctctacag ( ) r w gccctgatgcagaagaacc ( ) ccaggagctctgtgggtca – gccctgatgcagaagaact ( ) r q gccctgatgcagaagaaccg ( ) ccaggagctctgtgggtca – gccctgatgcagaagaacca ( ) r w agggggaattcatcctccg ( ) ttcaacctctgggagtgggcc – agggggaattcatcctctg ( ) r q agggggaattcatcctccg ( ) ttcaacctctgggagtgggcc – agggggaattcatcctcca ( ) s g aagaatggggttgatttggag gcatttcgcatctcgtagct ( ) – gcatttcgcatctcgtagcc ( ) r q aagaatggggttgatttggaga tgcgtcgttgactgcatttc ( ) – tgcgtcgttgactgcatttt ( ) r q aagaatggggttgatttggaga atgtgtttgtctgcgtacc ( ) – atgtgtttgtctgcgtact ( ) crt caccagagcaaaccgtccac cagggcttgttttgcctttg ( ) – ) cagggcttgttttgccttta ( ) dca caaggacctgaagacacacg ( ) accctgtatgttgccttgg – cacaaggacctgaagacacg ( ) agrtcatct catctgctgcttcgttag ( ) ggagattatcaggtgagatgcc – tgctgcttcgtttcatct ( ) a for each mutation, two sequences are used—one corresponding to the normal allele ( ) and the other corresponding to the mutated allele ( ). (sorimachi et al. ). in addition, capn has three unique sequences—ns, is , and is . additional tran- scripts resulting from alternative splicing and alternative promoter use have been characterized (ma et al. a, b; herasse et al., in press; f. fougerousse, personal communication). since the discovery of the molecular defect in lgmd a, we have collectively undertaken studies to determine the nature, number, and distribution of capn mutations and to assess potential genotype-phe- notype correlations. to date, distinct mutations have been identified, of which have not been described previously. subjects and methods description of families and preparation of samples from patients this report summarizes studies, performed in five lab- oratories (généthon [france], hospital ntra. sra. ar- anzazu [spain], hospital sant pau [spain], the dna di- agnostic laboratory [netherlands], and university of newcastle upon tyne [united kingdom), of families from diverse ethnic or geographic origins (including bra- zil, bulgaria, canada, france, greece, israel, italy, japan, lebanon, the netherlands, poland, portugal, russia, spain, switzerland, turkey, united kingdom, united states, and vietnam). families with lgmd that were from of the u.s. amish community, from réunion is- land, and basque country were distinguished from, re- spectively, families in the remaining united states, met- ropolitan french (i.e., continental france), and spanish families, since each of them represents a particular ge- netic isolate with a predominant founder effect. all pa- tients presented in this report fulfill the clinical criteria of a classic lgmd phenotype—namely, autosomal re- cessive inheritance, progression of muscle weakness in a limb-girdle distribution (with sparing of facial mus- cles), and a clearly dystrophic muscle biopsy (fardeau et al. a, b). calpain –deficient patients were also ascertained by the newcastle group, by demon- stration of both reduced -kd-band labeling and pre- served sarcoglycans and dystrophin on western blots of skeletal-muscle biopsies (anderson et al. ). table mutations in the capn gene exon/intron and origin no. of families (no. of homozygous patients) positiona (bp) mutationb position in amino acidc mutation reference : spain gtcratcd v i hospital sant pau turkey ( ) – dgcatc – – dgcatc dinçer et al. ( ), richard et al. ( ) réunion island dg dg généthon spain da da hospital sant pau italy ccgrctgd p l généthon france – inst – inst généthon france gacraac d n généthon lebanon ( ) tctrttt s f richard et al. ( ) france ( ) – -bp change – dfpiqfvwk généthon : brazil ( ) cgartgad r x richard et al. ( ) bulgaria agargga r g généthon : united states dc dc richard et al. ( ) france ( ) tgcrcgc c r généthon polish – insc – insc mgc leiden vietnam cagrtag q x hospital ntra. sra. aranzazu france atcrctc i l généthon : france ctgrcag l q richard et al. ( ) france ccarcta p l généthon france da da richard et al. ( , ) greece university of newcastle italy ( ) richard et al. ( ) netherlands mgc leiden russia ( ) généthon turkey ( ) dinçer et al. ( ) uk university of newcastle réunion island acgratgd t m généthon united kingdom ctgrccg ! l p university of newcastle bulgaria – -bp change – dfwsal généthon germany häffner et al. ( ) iv s : b as q u e , -b p ch an ge iv s d g én ét h o n : n et h er la n d s g g t r a g t g s m g c l ei d en f ra n ce t c c r c c c s p r ic h ar d et al . ( ) u n it ed st at es – -b p ch an ge – d sy e a l k g r ic h ar d et al . ( ) u n it ed k in gd o m g a a r a a a e k u n iv er si ty o f n ew ca st le b as q u e g g g r a g g g r u rt as u n et al . ( ) sp ai n ( ) h o sp it al sa n t p au sp ai n g a g r a a g e k h o sp it al n at r. sr a. a ra n za zu f ra n ce a c a r a t a t i g én ét h o n f ra n ce g g g r g a g g e r ic h ar d et al . ( ) l eb an o n ( ) d t d t r ic h ar d et al . ( ) f ra n ce – d g a a – d k g én ét h o n iv s : g er m an y g t r a t ) iv s g r a h äf fn er et al . ( ) : f ra n ce d a d a g én ét h o n f ra n ce d g d g r ic h ar d et al . ( ) iv s : r éu n io n is la n d ( ) a g r a a ) iv s g r a r ic h ar d et al . ( ), p en is so n -b es n ie r et al . ( ) : l eb an o n c c g r c t g d p l r ic h ar d et al . ( ) f ra n ce c a c r c a g h q r ic h ar d et al . ( ) t u rk ey ( ) t a c r a a c y n d in çe r et al . ( ) : f ra n ce ( ) g t g r g g g v g r ic h ar d et al . ( ) f ra n ce t g g r t a g w x r ic h ar d et al . ( ) ja p an ( ) t g g r t g c w c k aw ai et al . ( ) iv s : f ra n ce a r g ) iv s a r g g én ét h o n : s p ai n – in st – in st h o sp it al n at r. sr a. a ra n za zu f ra n ce c g c r t g c d r c h o sp it al n at r. sr a. a ra n za zu is ra el ( ) c g g r t g g d r w r ic h ar d et al . ( ) sp ai n – d g g g g – d g g g g h o sp it al n at r. sr a. a ra n za zu f ra n ce g g t r g a t g d g én ét h o n n et h er la n d s g g a r a g a d g r m g c l ei d en n et h er la n d s c g c r t g c d r c m g c l ei d en sp ai n h o sp it al n at r. sr a. a ra n za zu f ra n ce ( ) c g c r g g c r g g én ét h o n f ra n ce c g c r c a c d r h g én ét h o n u n it ed st at es g én ét h o n (c o n ti n u ed ) table (continued) exon/intron and origin no. of families (no. of homozygous patients) positiona (bp) mutationb position in amino acidc mutation reference : united kingdom dc dc university of newcastle spain agcrggc s g hospital natr. sra. aranzazu united kingdom university of newcastle basque cagrgag q e urtasun et al. ( ) basque cggrtggd r w urtasun et al. ( ) réunion island cggrcagd r q généthon united states cggrtggd r w richard et al. ( ) france richard et al. ( ) france ( ) cggrcagd r q généthon turkey dinçer et al. ( ) france cggrtggd r w généthon spain hospiatl sant pau italy gggragg g r richard et al. ( ) netherlands attract i t mgc leiden : france tacrtaa y x généthon turkey ( ) dinçer et al. ( ), richard et al. ( ) france cggrcagd r q généthon switzerland ( ) gggrtgg g w richard et al. ( ) france ( ) cggrtggd r w richard et al. ( ) réunion island ( ) cggrcagd r q richard et al. ( ) france généthon europe / dtg – dtg hospital natr. sra. aranzazu : spain / daaag – daaag hospital sant pau japan ( ) / insa – insa kawai et al. ( ) : italy tcgrttgd s l richard et al. ( ) europe hospital natr. sra. aranzazu france da da généthon france dag dag généthon réunion island ( ) ggcrggtd crt richard and beckmann ( ) netherlands cagrccg q p mgc leiden : bulgaria / datag – datag généthon france da da généthon united kingdom da da university of newcastle iv s : sp ai n g t r a t ) iv s g r t h o sp it al n at r. sr a. a ra n za zu u n it ed k in gd o m a g r a t ) iv s g r t u n iv er si ty o f n ew ca st le : r éu n io n is la n d / d a c d c a r ic h ar d et al . ( ) u n it ed k in gd o m c g t r c c t r p u n iv er si ty o f n ew ca st le t u rk ey ( ) g c g r g t g d a v d in çe r et al . ( ), r ic h ar d et al . ( ) f ra n ce ( ) g én ét h o n f ra n ce g a t r c a t d h h o sp it al n at r. sr a. a ra n za zu g re ec e g a t r g c t d g u n iv er si ty o f n ew ca st le iv s ex o n : sp ai n ( )/ -b p ch an ge ) v (i v s )/ d h o sp it al n at r. sr a. a ra n za zu iv s : f ra n ce a g r g g ) iv s a g r g g g én ét h o n c an ad a ( ) g én ét h o n : f ra n ce t t c r t c c f s g én ét h o n r éu n io n is la n d a g c r g g c s g r ic h ar d et al . ( ), p en is so n -b es n ie r et al . ( ) b as q u e c g a r c a a d r q u rt as u n et al . ( ) sp ai n ( ) h o sp it al sa n t p au , h o sp it al n tr a. sr a. a ra n za zu t u rk ey ( ) d in çe r et al . ( ), r ic h ar d et al . ( ) iv s : sp ai n g t r g a ) iv s t r a h o sp it al n at r. sr a. a ra n za zu : u n it ed st at es (a m is h ) ( ) c g g r c a g d r q r ic h ar d et al . ( ) b ra zi l ( ) r ic h ar d et al . ( ) f ra n ce r ic h ar d et al . ( ) b ra zi l – d a g a c – d a g a c r ic h ar d et al . ( ) f ra n ce – a a a c a r t – a a a c a r t g én ét h o n n et h er la n d s – in sa – in sa m g c l ei d en b ra zi l – a g r t c a t c t a g r t c a t c t r ic h ar d et al . ( ) r éu n io n is la n d t c a t c t r ic h ar d et al . ( ) b as q u e ( ) u rt as u n et al . ( ) sp ai n ( ) h o sp it al sa n t p au , h o sp it al n at r. sr a. a ra n za zu u n it ed st at es r ic h ar d et al . ( ) a n u m b er ed o n th e b as is o f th e cd n a (e m b l ac ce ss io n n u m b er ), st ar ti n g fr o m a t g . b m u ta te d n u cl eo ti d es ar e u n d er li n ed . c n u m b er ed o n th e b as is o f th e p ro te in se q u en ce s, st ar ti n g fr o m th e fi rs t m et h io n in e re si d u e. d m o d ifi ed c p g si te . am. j. hum. genet. : – , table nature and frequency of the polymorphisms identified in the capn gene location name position (bp) event cpg? frequencya promoter trc trc no nd exon t t accract no !. exon t t actracc no . exon c c tgcrtgt yes . exon e k gagraag yes . exon f f ttcrttt no . exon q q cagrcaa no !. exon a t gcaraca no . intron ivs crg crg no . exon i i atcratt yes !. exon l l ctcrgtc no !. intron da da no . a based on study of independent chromosomes; nd not determined. altogether, families were selected on the basis of identification of variation in the capn gene. several series of control chromosomes were examined, including those of the parents of patients, those of ceph individ- uals, and/or those of control individuals from matched geographic origins. genomic dna was prepared from either peripheral-blood lymphocytes or cultures of lym- phoblastoid cell lines. identification of dna sequence variants there was no uniform strategy for variant identifi- cation, since different protocols were used in each of the centers involved. these protocols included the following: . illegitimate transcription analyses of cdna—these were performed as in the study by richard et al. ( ), by use of total cellular rna extracted from lympho- blastoid cell lines, followed by a final characterization by dna sequencing. . heteroduplex analysis—ten microliters of the pcr product were denatured at ˚c for min and were allowed to renature at room temperature for > h. the product was electrophoresed on hydrolink gel prepared according to the manufacturer’s recommendations. for pcr products of ∼ bp, the conditions of migration are an overnight (∼ h) run at v. after the gel was stained by ethidium bromide, the products are visualized under uv light. identification of the variant sequences was performed by dna sequencing. . sscp—five microliters of the pcr products were denatured at c for min and cooled on ice, with ml of loading buffer ( % formamide and . % brom- ophenol blue). the samples were separated on . -mm- thick % acrylamide % glycerol gels in . # . gels were run at room temperature,tris-borate edta at w for h. the samples were transferred to nylon filters and hybridized with the primers used in the pcr, according to the ecl protocol (amersham), as described by vignal et al. ( ). identification of the variant se- quences was performed by dna sequencing. . direct sequencing—one hundred nanograms of dna were amplified under buffer and cycle conditions described by richard et al. ( ). each exon was am- plified by specific primers, chosen in introns. the prod- ucts of pcr were directly sequenced, with the same primers, by dye-dideoxy sequencing, after purification through either microcon devices (amicon) or polyacryl- amide gel biogel p- (bio-rad). . allele-specific pcr—oligonucleotides for muta- tions da, g r, ivs gra, r w, r q, r w, r q, s g, r q, r q, crt, dca, and agrtcatct were designed such that their ′ bases were at the point of the mutation (table ). one hundred nanograms of dna were amplified by touchdown pcr in a -ml volume containing mm tris-hcl, ph . , mm kcl, . mm mgcl , . % triton x- , mm of each dntp, ng of each primer, and units of taq polymerase (perkin-elmer). it was necessary to add various amounts of formamide, to increase the specificity of some pcrs. after -min denaturation at c, thermocycling was performed as follows: c for s and then -s annealing steps starting c higher than the annealing temperature and decreasing c every two cycles until the annealing tem- perature was reached. these steps were followed by cycles at c for s and s at the annealing tem- perature. after pcr, the products were electrophoresed on a % horizontal agarose gel stained with ethidium bromide. haplotype analysis short tandem repeats covering a region of cm at the lgmd a locus at q . (markers d s , d s , d s , d s , and d s ) were richard et al.: calpain mutations and polymorphisms figure distribution of mutations along the capn transcript and protein. the mutations are represented, according to their type, along the schematic representation of the capn gene and protein. the number of independent events is given either inside or above the symbol of the mutation. the exons are indicated by unblackened boxes (with the exon numbers below them). the four domains (i–iv) and the muscle-specific sequences (ns, is , and is ) of calpain also are shown. table unclassified variants exon/intron origin no. of families position (bp) event amino acid position putative effect spain, netherlands tgcrtgta c splicing? switzerland ( ) ctgrctaa l splicing? ivs japan ( ) – dc ) splicing? turkey cacrgac h hrd united kingdom gcargaa a are note.—data are as defined in the footnotes totable . a modified cpg site. used to assign haplotypes, as described in a previous publication (richard et al. ). results altogether, variants have been detected in the capn gene (table ). ninety-seven mutations were identified after screening of the capn gene in a total of families from different ethnic or geographic origins. ninety-six mutations were characterized in our laboratories, and the additional one (ivs gra) was previously reported as having occurred in a german family (häffner et al. ). in most families, both mu- tations were identified, with the exception of families for which only one allele each has been uncovered. twelve polymorphisms were uncovered in the course of these studies (table ). five additional variants (table ), found only in patients, were not demonstrated to be causative mutations and therefore could not be classified as belonging to either of the two formerly described groups. mutations identified a summary of all the capn mutations is presented intable and figure . the names of the mutations fol- low published guidelines (antonarakis ). the mu- tations include single-base-pair substitutions—which resulted in a change of amino acid in cases, in pre- mature stop codons in cases, and in splicing defects in cases. twenty-three ( %) of these point mu- tations affect a cpg dinucleotide. an additional mu- tations were small insertions and/or deletions. therefore, ∼ % ( / ) of the capn mutations are likely to result in a truncated protein and, therefore, are likely to be inactivating. for the missense mutations, the follow- ing arguments are used to infer their pathogenic nature: (i) the fact that they were not encountered in control populations (in general, > independent chromo- somes were examined) and (ii) the observation that all but one of these mutations involve changes of amino acids that are strictly conserved among the human, mouse, rat, bovine, and porcine capn sequences, if not among all the calpains (table ). furthermore, some mutations were demonstrated to induce consequences with regard to three biochemical characteristics of capn —namely, titin-binding ability, autolysis capac- ity, and fodrinolysis capacity (ono et al. ). interestingly, one of the splicing mutation ( crt) changes the third base of a codon to a synonymous co- don yet leads to both the creation of a novel internal splice site and the subsequent loss of the end of the exon and, eventually, to a premature in-phase stop codon (ri- chard and beckmann ). demonstration of the path- ogenic nature of this sequence variant required the ex- amination of the transcription product. the seven remaining splice mutations are located within ′ or ′ splice sites. ivs gra was reported to induce skipping of exon (häffner et al. ), and ivs agraa leads to the use of a cryptic splice site upstream in the intron. the consequences, at the rna level, of the other splice-site mutations were not ex- am. j. hum. genet. : – , table conservation of amino acids at locations of missense mutations and nonclassified variants of the capn protein mutation amino acids in capn ofaa other calpainsb calpain homologue(s)bhuman mouse rat porcine bovine chicken mammalian nonmammalian v i v v v v ) v not relevantc not relevantc not relevantc p l p p p a ) s not relevantc not relevantc not relevantc d n d d d d ) d d d d s f s s s s ) s s, a a, s s, a r g r r r r ) r r r n, r, s c r c c c c ) c s, c s, n, c c, s, n i l i i i i ) i i i, v i l q l l l l ) l l l l p l p p p p ) p p p p t m t t t t ) t t, i t, v t l p l l l l ) l l l l g s g g g g ) g g g g s p s s s s ) s s, c c, s c, s e k e e e e ) e e e, a e, q g r g g g g ) g g g g, l e k e e e e ) e e e d, e t i t t t t ) t t t t g e g g g g ) g g g g, t p l p p p p p p not relevantc not relevantc not relevantc h q h h h h h h h h h, y y n y y y y y y y y y v g v v v v v v i i, m i, v w c w w w w w w w w w, l r c r r r r ) r n, t, r, s e, q, n, s, t e, d, n r w r r r r ) r r r, p l, r, p, q g d g g g g ) g g g, n m, g, q g r g g g g ) g g g g r h or g or c r r r r ) r r, l r, i y, i, r s g s s s s ) s s, t s, t t q e q q q q ) q q q q r w or q r r r r ) r r r, i, t l, k, r r q or w r r r r ) r r, q r r r w r r r r ) r r, k r, k r g r g g g g ) g g g g i t i i i i ) i i i i r q r r r r ) r r r r g w g g g g ) g g, a g, a, v g, s r q or w r r r r ) r r r r s l s s s s ) s not relevantc not relevantc not relevantc q p q q q k ) ) not relevantc not relevantc not relevantc r p r r r r ) r r, k r, k, n k a v a a a a ) a d, n, s, h n, a, d, h d d h or g d d d d ) d d, e d, q, t d f s f f f f ) f f, y f, y, i y s g s s s s ) s s, a, t f, s, g, a s r q r r r r ) r r r, d r r q r r r r ) r r r r h d h h h y ) n d, e, n, q, k d, e q a e a a a a ) a q, a, c, s, l, i e, a, k, s q a indicative of conservation among all known calpain proteins; an ellipsis ()) denotes that no information is available. b in each cell, amino acids are listed according to frequency in published sequences. c with regard to amino acids of the three specific parts of calpain : ns, is or is . amined, because cells were not available from these pa- tients. nevertheless, present knowledge of splicing mech- anisms could be used to infer putative consequences, in accordance with the exon-definition model (robberson et al. ). ivs arg generates a new dinucleotide, ag, that could potentially be used as a splicing anchor (smith et al. ); use of the latter would result in a frameshift mutation, by addition of bases richard et al.: calpain mutations and polymorphisms table haplotype analysis of recurrent mutations mutation (cpg?) and origin haplotypea d s d s d s d s d s g e: france ( )n france ( )n r q (yes): france ( )n / / / france ( )n / / / turkey ( )n r w (yes): france ( )n / france ( )n r q (yes): réunion island ( )n france ( )n ivs agrgg: france ( )n / / / canada ( )n r q (yes): spain ( )n spain ( )n / / turkey ( )n r q (yes): amish ( ), france ( )n n / brazil ( )n / a only haplotypes corresponding to recurrent mutations are presented. markers are listed ac- cording to their linear map order. the disease locus is located between markers d s and d s . in situations in which the haplotype could not be reconstructed, the two alleles have been given, separated by a virgule (/). for all markers except d s , the numbers represent allele size (in bp); in the case of marker d s , the numbers represent allele designations as given in the ceph database. in the mrna. ivs grt, ivs grt, and ivs agrgg may lead to either single-exon skip- ping or cryptic–splice-site utilization. most of the deletion/insertion mutations caused a frameshift, leading to a secondary premature stop co- don. four of the mutations (dfpiqfvw, dfwsal, dsyealkg, and dk ), however, were in-frame de- letions that would theoretically result only in limited amino acid deletions. two mutations were slightly more complex and resulted in the combination of small insertions and deletions. aaacart consists of a -bp deletion of nucleotides – of the cod- ing region, coupled with a -bp insertion, and agrtcatct consists of a -bp deletion coupled with a -bp insertion. the net result in both cases is a shift in the reading frame, followed by a premature stop codon. the first major genomic deletion in the calpain gene was identified in a basque patient. reverse transcrip- tion–pcr of the illegitimate transcription products showed that exons – were missing. since no sequence alteration was identified in the splice sites of exons – , a genomic deletion was suspected. two pcr prim- ers—one located in exon and the other located in exon —that, in normal genomic dna, were separated by ∼ kb, led to the amplification of a fragment of ∼ . kb in dna from this patient, whereas no amplification was obtained in dna from control individuals. se- quencing of the corresponding pcr product enabled us to identify the mutation event as a , -bp deletion spanning exons – . the ′ breakpoint is in the right arm of the ′r ′-oriented alu-jb repeat in intron , and the ′ breakpoint maps to the left arm of the alu-sg repeat in intron , which is oriented in the same direc- tion. the deletion therefore seems to result from an un- equal recombination event between two adjacent alu elements. polymorphisms to date, polymorphisms have been characterized in the capn gene (table ). they were excluded from being lgmd a mutations, because of their occurrence in normal healthy populations and/or occurrence along the same chromosome that has a mutation. the range of frequencies of these polymorphisms varies between ! . % and % (table ). none of these polymor- phisms is in linkage disequilibrium with a particular mu- tation. three of the variants are due to crt transitions. three polymorphisms map in noncoding regions. seven am. j. hum. genet. : – , table detection of variants by modification of restriction enzymes mutation enzymatic site(s)a normal allele variant allele dgcatc hhai, hinpi, sfani alui dg alwni, nspbii haeii, eco iii da ava i p l bcni, ncii t t ddei seci d n avaii s f mnli i l alwi, bstyi banii, bsp i t m maeii s p avaii, sau i dk mboi dg ncii, bcni avaib p l mspi, hpaii h q stui a t hphi w x nlaiv, seci w c nlaiv, seci eaei, gdiii ivs crg tth ii l l psti inst tth i r c nspii, haeiii r w rsai q e sfani r w cfr i r q cfr i nspbii r w avaii g r maei ddei r w bcni, ncii r q bcni, ncii da alwni datag bbvi, fnu hi da bglii a v hinpi, hhai hgiai f s xmni s g alui aaacart tth ii bsphi, plei a e nsii bsphi a enzymatic sites corresponding to enzymes that cut too frequently to be used in an analysis are not given. b present only on genomic dna, not on cdna. figure evolution curves for lgmd a patients. functional stages are graded as i–vii, according to the gardner-medwin and walton ( ) scale. only the curves for patients for whom two points of the disease evolution are known are represented, whereas the cal- culation of means and duration takes into account all available data. exonic polymorphisms do not lead to an alteration of the encoded amino acid, and the majority of them in- volve the third base of a codon. the last two polymor- phisms, e k and a t, which are present, respec- tively, in exons and , lead to an amino acid substitution. it should be noted, however, that the minor variants correspond to an amino acid that can also be found at the equivalent positions in other members of the calpain family: for e , a lysine is present at the equivalent position in the human ncl protein, and, for a , a threonine is present at the corresponding po- sition in rat, mouse, and porcine calpain , as well as in the human, mouse, rat, and chicken calpain pro- teins. this suggests that these changes may not have major deleterious effects on the protein’s function(s). unclassified variants also detected were five changes that could not be clas- sified as either a polymorphism or a deleterious muta- tion, even though they were observed only in patients (table ). one intronic change, leading to a deletion of a c in intron at position of exon , was ob- served in a homozygous state in a japanese patient who was homozygous for a chromosome haplotype. two additional changes are single-base substitutions involv- ing the third base of a codon: a tgcrtgt transversion at position in exon (c rt [c c]) and a ctgrcta transversion at position in exon richard et al.: calpain mutations and polymorphisms figure location of missense mutations and in-frame deletions along the capn protein (g ra [l l]). c rt (c ) was observed in three unrelated lgmd families of different geographic origins but not in control individuals. we are con- fident that two of these families are authentic lgmd a families, since one mutant allele already has been char- acterized. no additional changes were identified in the corresponding patients’ dna, despite exhaustive screen- ing. g ra (l ) was encountered only in a swiss family in which the segregation is consistent with the chromosome location of the disease locus. all these changes may represent neutral polymor- phisms; however, it is also possible that they represent authentic lgmd a mutations that affect the structure of the transcript. unfortunately, no biopsies or cell lines were available for the corresponding patients. hence, we could not examine whether these changes have con- sequences at the rna level. we can, however, attempt to predict their putative manifestations on the transcrip- tion products. ivs dc may affect the branch site, since it is located at the corresponding consensus posi- tion. c rt and g ra create potential splicing sites, with scores of % and %, respectively (shapiro and senapathy ). the last two unclassified variants, h d and a e, lead to a change in the amino acid composition; but the presence, at the corresponding po- sition, of the variant amino acid in other members of the calpain family is a strong argument in favor of their representing rare innocuous polymorphisms (table ). recurrent mutations or founder effect twenty-nine mutations were each found in more than one family; however, it is necessary to include a correction for founder effect, since several mutations am. j. hum. genet. : – , have been amplified in specific populations. this is particularly true for the amish community, in which the r q mutation is encountered in a homozygous state in all families with lgmd a that were tested. a predominant mutation is also observed in two other populations with high consanguinity. ivs gra and agrtcatct are present on % ( / ) and % ( / ) of the carrier chro- mosomes in families from réunion island and in the basque isolate, respectively. haplotype analyses by microsatellite markers that flank the lgmd a locus confirmed that the mutations derived from the same ancestral mutational event (allamand et al. ; ri- chard et al. ; urtasun et al. ). the same analysis allowed us also to infer a relationship be- tween families, even in the absence of any known ge- nealogical or geographic link. in particular, the da mutation observed in patients from eight dif- ferent countries was found, each time the information was available, to be associated with the same lgmd a marker haplotype. this is also the case for (i) y x and a v, which are found in turkish and metropolitan french families; (ii) r q, which is present in both amish and metropolitan french families; and (iii) agrtcatct, which is present in north american, brazilian, réunion island, and spanish families. in other cases, haplotype data allowed us to rule out the possibility of a com- mon founder effect and suggested, instead, that the mutations represent events that have occurred several times independently. overall, if only the clearly in- dependent events are considered, the number of re- current mutations collapses to seven, representing independent events (table ); among these mutations, five correspond to crt transitions involving cpg sites. thus, altogether, independent mutations were observed. distribution of capn mutations mutations are relatively evenly distributed over all exons of the calpain gene, with the exception of exons , , , , and , all of which are among the smallest exons and in which no mutations have been found so far (fig. ). these exons are to be contrasted with exon , which carries an excess of mutations ( ). whenp ! . the nature of the mutations is considered, exons , , and show an excess of missense mutations ( ,p ! . . , and . , respectively), and, when allowance is made for the small numbers, exons , , and may show an excess of deletions or insertions. interestingly, a mutation cluster spanning a region of amino acids in exon accounts for % ( / ) of the independent missense events. this may be due to the elevated number of cpg sites at this location. no differences in the dis- tribution of mutations were observed between the four calpain domains. rapid screening for particular mutations in some cases, rapid testing for the presence of a par- ticular mutation in a patient’s dna may be important, especially in the case of recurrent or population-specific mutations. direct sequencing may be the method of choice, but alternatives allowing the analysis of a great number of individuals exist. some capn mutations can be tested rapidly because they cause a change in a restriction-enzyme site (table ). short deletions could be tested directly by electrophoresis of the corresponding pcr products. in addition, we have developed allele- specific pcr assays (table ) for the mutations present in families from the amish community, from réunion island, and from the basque country. this has allowed us to test rapidly the presence of the corresponding mu- tations in new patients with these origins. we also have done this for the frequently observed mutation da, which is always associated with the same haplotype, allowing to test candidate carriers presenting either this particular haplotype or a related haplotype. phenotypic characteristics of patients with lgmd a clinical information was available for of the pa- tients presented here. for many of them, clinical char- acteristics have been reported elsewhere, and a typical formula has been defined, first in patients from réunion island (fardeau et al. b). this formula is charac- terized mainly by a symmetrical, very selective atrophic involvement of limb-girdle and trunk muscles, with the gluteus maximus and thigh adductors being most af- fected (fardeau et al. a, b). the same pattern of muscle involvement was also reported for / of the examined metropolitan french patients, with occasion- ally minor variations around this pattern. the same holds for the majority of lgmd a patients of turkish or basque origin (dinçer et al. ; topaloglu et al. ; beckmann and fardeau ; urtasun et al. ). overall, there was a marked heterogeneity of severity. the mean age at onset is . years (range – years old; fig. ), and the mean age at loss of walking ability is . years (range – years; fig. ). no sex difference was evident in either age at onset or evolution. in an attempt to draw correlations between the clinical severity and the nature of gene mutations, evolution curves associating age at onset and functional stages were separated and compared according to the nature of the mutations (null/null, null/missense, and missense/ missense) (fig. ). the age at onset in patients carrying two null mutations is quite homogeneous and occurs generally at age years. in contrast, age at onset in patients who either had two missense mutations or were richard et al.: calpain mutations and polymorphisms compound heterozygotes for one missense mutation and one null mutation is much more variable and can occur as early as age . years or as late as age years. furthermore, patients carrying two null mutations lost walking ability at age ! years, whereas patients with either one missense and one null mutation or two mis- sense mutations had broader variability in the evolution curves. these data further confirm and extend prior ob- servations regarding the relative severity of null muta- tions versus missense mutations. discussion in this report, we have presented all the mutations and polymorphisms identified so far in capn , the gene involved in lgmd a. a total of variants have been identified in the calpain gene: mutations, poly- morphisms, and variants that could not be classified as either mutation or polymorphism. until one assesses the impact of the latter on mrna processing and sta- bility, it may be difficult to ascertain the neutrality or nonpathogenicity of these variants. the precedent dem- onstration of the pathogenic character of the crt mutation, which does not change the nature of the en- coded amino acid (richard and beckmann ), con- firms that one needs to interpret with caution the ap- parently “neutral” mutations. the mutations reported thus far, of which are newly described, include nonsense, deletion/inser- tion, splice-site, and missense mutations. if we consider the additional, unclassified ivs dc, c rt, and g ra variants as causative muta- tions, then the number of mutations characterized thus far would total . the mutations are distributed along the length of the capn gene, with a slight mutational “hot spot” in exon . if we consider missense mutations alone, exon shows a notable excess of mutations. this exon constitutes a good primary target for screening of mild calpainopathies, in light of the degree of disease severity of carriers of either two missense mutations or a missense mutation associated with a null mutation. most mutations ( / [ %]) represent private var- iants, although particular mutations were found more frequently. among these particular mutations, ( %) are associated with a founder effect, ( %) are really recurrent, and no information was obtained for the re- maining mutations. capn mutations were identified in families originating from countries, further demonstrating the worldwide distribution of the disease. for families, only one mutant allele was identified, despite the fact that all coding regions in these patients have been examined. these observations therefore sug- gest that mutations may occasionally lie in an essential noncoding regions, such as the promoter region or an intron. we also have described the first large genomic deletion of the calpain gene, which is due to an unequal recombination between two intragenic alu elements. the methodologies usually used for mutation screening are not oriented toward identification of such altera- tions. it would be interesting, given the relatively high alu-sequences content of the calpain region, to verify what fraction of the unidentified mutations can be explained by such events. for this purpose, it may be useful to resort to use of additional procedures, such as southern blot. diagnostic applications after the initial confirmation of capn as the lgmd a gene, the primary clinical motivation for per- forming a systematic mutation analysis was to provide accurate and unambiguous lgmd a diagnosis for pa- tients with progressive muscular dystrophies. in partic- ular, the identification of mutations in a family allowed the latter to be classified as belonging to the lgmd a group. this is of importance in light of (i) the genetic heterogeneity of lgmd (which has at least eight caus- ative genes, lgmd a–lgmd h) and (ii) the difficulty in clinically distinguishing one entity from another. even if it is now possible, with the help of antisarcoglycans antibodies, to test for defects in the corresponding pro- teins and to distinguish sarcoglycanopathies from the other progressive muscular dystrophies, unambiguous diagnosis still rests on the identification of the underlying mutations. hence, genetic and immunochemical analy- ses provide important means by which to refine and simplify diagnosis. the wide spectrum of capn mutations, the relative large size of the capn gene, and, finally, the fact that most mutations seem to be private and relatively evenly distributed over most calpain exons create significant practical problems for diagnosis of calpainopathies. be- cause of all these characteristics, no single mutation- detection method appears to be ideal, and the challenge of mutation identification remains important. the as- sessment of the calpain protein in muscle biopsies, by either western blot or immunohistochemistry, may be- come an additional tool with which to investigate cal- painopathies, although it should be remembered that the autoproteolytic nature of calpain could render this diagnostic test difficult to interpret (anderson et al. ; beckmann and fardeau ). nevertheless, the potential of this diagnostic strategy, which has been fol- lowed by the newcastle team, is clearly demonstrated in the present study. since the realization of an objective molecular-genetic diagnosis still presents a formidable challenge, it could be helpful, whenever possible, before a direct mutation search is performed, to further classify the family—or, at least, to exclude particular loci—by linkage analysis. am. j. hum. genet. : – , to streamline the genetic analyses, we have developed a fluorescent-marker kit, using a set of markers brack- eting the lgmd a–lgmd f disease loci (richard et al., in press). in an additional step, haplotype analyses can be used to infer a common ancestral origin and thus to point to a specific mutation(s). the geographic origins of the patients is another element that can direct the mutation screening. we also have reported herein the polymorphisms iden- tified in the study of our patient’s cohort, since it is essential, in a diagnostic test, to distinguish between them and the morbid dna-sequence anomalies and, thereby, to prevent erroneous diagnosis. this is of par- ticular importance in the detection of mutations in exons and and in intron , which contain polymor- phisms that can reach %– % heterozygosity in the population examined. the inclusion of appropriate con- trols in the mutation analysis of this exon will help us to distinguish between these neutral polymorphisms and causative mutations. lgmd phenotype the availability of molecular diagnosis has enabled precise symptomatology, by identification of clues al- lowing the recognition of specific clinical features of cal- painopathies, compared with other progressive muscular dystrophies caused by defects in structural proteins. mo- lecular classification of patients as belonging within the different lgmd forms helps us to validate the specific topography and characteristics of muscle involvement (for a precise description of lgmd a patients, see (far- deau et al. a, b; dinçer et al. ; topaloglu et al. ; beckmann and fardeau ; urtasun et al. ). to sum up, the typical formula with regard to calpain –deficient patients includes, in the early stage of the disease, a predominant wasting of muscles from the posterior compartments of the limbs, clearly visible by both clinical examination and computed-tomography (ct) scan, whereas in sarcoglycanopathies there is a marked quadriceps femoris involvement (eymard et al. ). the differences, in the distribution of muscle wasting, between the different forms renders ct-scan analysis an important element in a clinical diagnosis. an additional sign that may help us to distinguish calpain- opathies from sarcoglycanopathies is the fact that, in the former, calf hypertrophy is less frequent, whereas ma- croglossia is never seen. both the characterization of mutations and the study of phenotype/genotype correlations should allow us to elucidate the molecular basis for the expression of dis- parate phenotypes among calpain –deficient patients. no evident correlations between the mutations and the clinical manifestations of lgmd a has been estab- lished, either between the families or even within the families, although, in general, null mutations result in clinical consequences that are more severe than those that result from missense mutations. furthermore, the study of this large cohort of calpain –deficient patients has failed to show that the patient’s gender has any influence on either age at onset or disease evolution. locus-specific database given both the widespread geographic distribution of this disease and the growing number of reported cases, it seems important, in order to help investigators and clinicians in their diagnostic process, to maintain and update a capn -mutation database. with this goal in mind, we are in the process of constructing a locus- specific database website in accordance with hugo guidelines for content and structure of mutations da- tabases, and we encourage others to join us and to par- ticipate in a collective effort to regroup all capn mu- tations in a publicly available database. meanwhile, some of these data are already accessible in the leiden muscular dystrophy pages. acknowledgments we would like to thank the lgmd a families. we are grate- ful to all of those clinicians who have collected samples, in particular drs. o. f. brouwer (lumc, leiden), j. m. bur- gunder (neurologie inselpital, bern), p. dinçer (hacettepe uni- versity, ankara), and c. e. jackson (henri ford hospital, de- troit) and profs. g. lefranc (university of montpellier, france), c. legum (ichilov hospital, tel aviv), and l. merlini (istituto rizzoli, bologna). this study was supported by grants from the association française contre les myopathies, by marato tv grant / , and by fis grant / - , from the spanish ministry of health. a.s. is a predoctoral fellow from the basque ministry of health. electronic-database information online mendelian inheritance in man (omim), http:// www .ncbi.nlm.nih.gov: (for lgmd a [mim ]) leiden muscular dystrophy pages, http://www.dmd.nl references allamand v, broux o, richard i, fougerousse f, chiannilk- ulchai n, bourg n, brenguier l, et al ( ) preferential localization of the limb-girdle muscular dystrophy type a gene in the proximal part of a -cm q . -q . interval. am j hum genet : – anderson lvb, davison k, moss ja, richard i, fardeau m, tomé fms, hübner 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for health? jonathan flint and guy goodwin attempts to map the genes for psychotic illnesses have been fraught with problems. studying why some family members do not become ill might prove to be a more successful strategy. address: university department of psychiatry, warneford hospital, oxford ox jx, uk. current biology , :r –r http://biomednet.com/elecref/ r © elsevier science ltd issn - one of the few well accepted pieces of evidence we have about the biology of psychiatric illnesses is that there is a genetic component to (almost) all of them, but very little is yet known about the number of genes, where they are on the genome or what they are doing to produce illness. ten years ago, a breakthrough was announced with a report in nature [ ] that a gene, or genes, on chromosome was associated with the development of manic depres- sive psychosis. manic depressive psychosis, also known as bipolar affective disorder, is a condition characterized by debilitating fluctuations in mood. in mania, patients experience elation or euphoria, which may spill into psy- chotic grandeur. there is a reduced need for sleep, boundless optimism and, too often, hopelessly flawed judgement in the sexual and financial spheres. in depres- sive episodes, patients experience profound sadness and a depressed affect of almost painful intensity. action and thinking are slowed and there is significant impairment of concentration and memory. in depression, there may be persistent thoughts of suicide and as many as % of patients who require hospitalisation eventually do kill themselves. it was with heavy hearts that those of us committed to a scientific psychiatry read, a couple of years later, that the claim of a breakthrough in manic depressive psychosis was wrong [ ]. it was overturned by the incidence of only a few new cases in apparently unaffected members of the original families. this, and other false positive findings in the field, led to a greater caution among the research com- munity in their approach to the declaration of positive results. there was also a wider pessimism. a curious con- sensus was formed by scientists who thought all psychiatry unscientific and an anti-psychiatry lobby who still regard all efforts to be scientific about psychiatry as inhumane. the burden of disability that mood disorder imposes guarantees, however, the importance of the clinical problem. cautious enthusiasm for mapping genes for manic depressive psychosis has been sustained, and a number of groups have reported linkages to mood disorders (see table ). to put the table in perspective, the standard measure of the likelihood that a specific chromosomal posi- tion is linked to a disorder given a set of segregation data, the ‘log of the odds ratio’ or lod score, is reckoned to be significant if it exceeds . . to find significance is not to establish confidence that a linkage is real, however. such studies are simply too small to achieve that, so convincing evidence for linkage to any one chromosome is still lacking. the authors of the largest survey — markers on subjects — admitted that no chromosomal area met criteria for significant or even suggestive linkage [ ]. three years ago risch and botstein [ ], commenting on a series of linkage reports for manic depressive psychosis in nature genetics, asked if the field had advanced and concluded that “compared to other fields to which genet- ics has been applied, one would have to argue not”. they suggested that the genetics of manic depressive psychosis was much more complex than anyone had anticipated. genetics can be complex for a number of reasons. in disorders caused by mutations in a single gene, the situa- tion can be bad enough; consider the difficulties in under- standing the inheritance of fragile x syndrome [ , ], or the complex series of different phenotypes that arise from mutations of fibroblast growth factor receptors [ ]. when we deal with disorders that are likely to be due to mutations in more than one gene, the ways in which com- plexity can arise are legion: there may be different genes in different populations, different combinations of genes producing different phenotypes, variable interactions between genes and environment and so on. how complex is the genetics of manic depressive psychosis likely to be? the answer appears to be that it is very complex indeed. consider first how the genetic analyses are done. the basic methodology of genetic linkage and association studies is to ask what affected indi- viduals have in common, genetically speaking. thus, if all affected individuals share some segment of chromosome and this is rare or non-existent in unaffected individuals, we have evidence for a gene predisposing to the disorder somewhere in the shared segment of chromosome . perhaps the sole uncontested result from linkage studies of manic depressive psychosis is that the disorder is not, at least commonly, the result of a mutation in a single gene. assume that mutations in any ten of a hundred different genes can give rise to the disorder. in outbred popula- tions, if we picked affected individuals, the chance that they all have at least one common mutated gene is very low. our genetic test is therefore not going to have much power to detect an effect, unless we study many thousands of individuals. one way to simplify things is to study inbred populations, or those where all affected indi- viduals are genetically related (inhabitants of small islands tend to attract the interest of geneticists for this reason). in a population where all affected individuals are descended from a single progenitor, then all will have the same set of susceptibility genes. this is one reason why the old order amish community in pennsylvania — who inhabit a virtual island bounded by their culture — were selected for one of the largest genome screens undertaken for any disorder. they have large extended families with well-documented genealo- gies dating back to the progenitors who arrived from europe in the th century. all affected pedigrees trace back to a very small number of individuals with the disor- der (perhaps even a single affected founder). despite such remarkably propitious circumstances for a gene hunt, the results have been disappointing: no single chromosome region stands out as a convincing place to look for the genes that predispose to this psychiatric illness. this is why risch and botstein [ ] concluded that the genetics of manic depressive psychosis are complex. there is now a new twist to the amish story. hitherto, no one has thought to look at the absence of illness as a genetically determined trait in high risk families. why not see what the well relatives have in common, geneti- cally speaking? you might think, from the arguments given above, that such a strategy would only make things more complicated. if finding evidence of genetic suscep- tibility for manic depressive psychosis is complex, then surely finding evidence of genetic predisposition to health will be equally complex? yet the results indicate otherwise [ ]. ginns et al. [ ] have recently reported evidence of linkage to good mental health at a locus on chromosome p, with a p value in favour of linkage of . × – (lod score of . ), and a locus on chromosome q, with a p value of . × – (lod score of . ). in comparison, the most encouraging result of their search for loci linked to manic depressive psychosis, in the same population, was one at chromosome p with a lod score of . . of course, the size of the lod score is not everything: an earlier report [ ] of apparent linkage to xq reported a lod of . that was never replicated. but the new result is certainly more encouraging than most. furthermore, the use of simulated data to obtain empirical p values and an analysis relatively free of assumptions about the mode of inheritance gives the result a certain authority. but why should ‘wellness’ be easier to study than disease? in families with manic depressive psychosis members, wellness means either the absence of manic depressive psychosis predisposing genes, or the presence of protec- tive alleles. assuming that the latter is the case, then one reason why wellness loci have been found might be because they are rarer than disease loci. in other words, for the family members studied, health is just a cover-up of the effects of disease genes. this is not to say that disease is more common than health within these families: there may be fewer wellness loci, but each has a larger effect, and it is this larger effect that has made them easier to find, relative to disease loci. this would also explain why, when the investigators increased the age-at-risk cut-off for defining the wellness phenotype from to , the number of mentally healthy members decreased, while the p values in favour of linkage increased. there are certainly plenty of examples of protective alleles in other conditions: a classic finding in mouse genetics is that a mutation may lose or radically alter its phenotype when transferred onto a different genetic background [ ] and similar moderating effects have been reported for a few complex human disorders ([ ] for example). the dis- covery of protective alleles might make it easier to find the disease genes. by allowing for the effect of the protective alleles the genetics of manic depressive psychosis can be simplified, a little at least. theoretically, the linkage analy- ses will have more power to find effects of disease loci. studying wellness rather than ill health has other advantages too. although linkage of genes for particular receptors or enzymes to the illness may offer clues to develop treatments, it might be more fruitful to investi- gate what neurobiological processes keep individuals well in high-risk families. attempts to mimic or stimulate the molecular actions of the products of such genes might lead dispatch r table candidate genetic linkages to mood disorders. location lod reference year xq . [ ] p . [ ] q . [ ] . [ ] q . [ ] p/ q . [ ] . – . [ ] p . [ ] p . [ ] r current biology, vol no to protective treatments that can transform high risk individuals into low risk individuals. a similar strategy is already being pursued in relation to apolipoprotein e and susceptibility to alzheimer’s dementia. at the very least, wellness alleles might prove to be predictive of differen- tial responsiveness to the variety of treatments we already have. too often, claims for the molecular genetic approach to human disease are couched in revolutionary terms: there is much reference to new dawns and fresh horizons. to improve the modest benefits of existing treatment would actually represent a major public health gain. let us look forward to it being achieved, along with more breakthroughs, of course. references . egeland ja, gerhard ds, pauls dl, sussex jn, kidd kk, allen cr, hostetter am, housman de: bipolar affective disorders linked to dna markers on chromosome . nature , : - . . kelsoe jr, ginns ei, egeland ja, gerhard ds, goldstein am, bale sj, pauls dl, long rt, kidd kk, conte g, et al.: re-evaluation of the linkage relationship between chromosome p loci and the gene for bipolar affective disorder in the old order amish. nature , : - . . group ngib: genomic survey of bipolar illness in the nimh genetics initiative pedigrees. am j med genet , : - . . risch n, botstein d: a manic depressive history. nature genet , : - . . sherman sl, jacobs pa, morton ne, froster-iskenius u, howard- peebles pn, nielsen kb, partington mw, sutherland gr, turner g, watson m: further segregation analysis of the fragile x syndrome with special reference to transmitting males. hum genet , : - . . oberle i, rousseau f, heitz d, kretz c, devys d, hanauer a, boue j, bertheas mf, mandel jl: instability of a base pair dna segment and abnormal methylation in fragile x syndrome. science , : - . . wilkie aom: craniosynostosis: genes and mechanisms. hum mol genet , : - . . ginns ei, stjean p, philibert ra, galdzicka m, damschroder-williams p, thiel b, long rt, ingraham lj, dalwaldi h, murray ma, et al.: a genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the old order amish. proc nat acad sci usa , : - . . baron m, risch n, hamburger r, mandel b, kushner s, newman m, drumer d, belmaker rh: genetic linkage between x chromosome markers and bipolar affective illness. nature , : - . . crawley jn, belknap jk, collins a, crabbe jc, frankel w, henderson n, hitzemann rj, maxson sc, miner ll, silva aj, et al.: behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. psychopharm , : - . . todd ja, bell ji, mcdevitt ho: hla-dq-beta gene contributes to susceptibility and resistance to insulin dependent diabetes mellitus. nature , : - . . straub re, lehner t, luo y, loth je, shao w, sharpe l, alexander jr, das k, simon r, fieve rr, et al.: a possible vulnerability locus for bipolar affective-disorder on chromosome q . . nature genet , : - . . aita vm, liu jj, knowles ja, terwilliger jd, baltazar r, grunn a, loth je, kanyas k, lerer b, endicott j, et al.: a comprehensive linkage analysis of chromosome q supports prior evidence for a putative bipolar affective disorder locus. am j hum genet , : - . . craddock n, owen m, burge s, kurain b, thomas p, mcguffin p: familial co-segregation of major affective disorder and darier’s disease (keratosis follicularis). br j psychiatry , : - . . berrettini wh, ferraro tn, goldin lr, weeks de, detera-wadleigh s, nurnberger, ji: chromosome dna markers and manic depressive illness. evidence for a susceptibility locus. proc natl acad sci usa , : - . . stine oc, xu j, koskela r, mcmahon fj, gschwend m, friddle c, clark cd, mcinnes mg, simpson sg, breschel ts, et al.: evidence for linkage of bipolar disorder to chromosome with a parent- of-origin effect. am j hum genet , : - . . ewald h, mors o, flint t, koed k, eiberg h, kruse ta: a possible locus for manic-depressive illness on chromosome p . psychiatr genet , : - . . blackwood dhr, he l, morris sw, mclean a, whitton c, thomson m, walker mt, woodburn k, shar-cm, wright af, et al.: a locus for bipolar affective disorder on chromosome p. nat genet , : - . 	a genetic basis for health? 	references 	table 	table effect of phenobarbitone on amplitude-integrated electroencephalography in neonates with hypoxic-ischemic encephalopathy during hypothermia original paper neonatology ; : – effect of phenobarbitone on amplitude-integrated electroencephalography in neonates with hypoxic-ischemic encephalopathy during hypothermia poorva deshpande a, b amish jain a, b patrick j. mcnamara a, c adivision of neonatology, hospital for sick children, toronto, on, canada; bdepartment of pediatrics, mount sinai hospital, university of toronto, toronto, on, canada; cdepartment of physiology, university of toronto, toronto, on, canada received: january , accepted: september , published online: january , poorva deshpande department of paediatrics, staff neonatologist room - , mount sinai hospital, university avenue toronto, on m g x (canada) poorva.deshpande @ sinaihealth.ca © the author(s) published by s. karger ag, basel karger@karger.com www.karger.com/neo doi: . / keywords hypoxic-ischemic encephalopathy · amplitude-integrated electroencephalography · phenobarbitone · hypothermia · neonates abstract background: phenobarbitone induces suppression of cere- bral electrical activity on amplitude-integrated electroen- cephalography (aeeg) in neonates with hypoxic-ischemic encephalopathy (hie); however, its effect during therapeutic hypothermia (th) has not been well characterized. objec- tive: to evaluate the effect of phenobarbitone on aeeg in neonates with hie undergoing th. methods: thirty-five neo- nates born at ≥ weeks gestational age (ga), who received phenobarbitone as first-line antiepileptic drug during th for ≥ sarnat stage ii hie with aeeg recordings were retrospec- tively studied. background pattern, upper and lower margin voltages were characterized for a -min period before and – min after phenobarbitone administration. primary outcome was presence of severely abnormal aeeg pattern after phenobarbitone administration. results: mean (±sd) ga and median birth weight were . ± . weeks and . ( . – . ) kg, respectively. phenobarbitone ( – mg/kg), administered at median age . h, was associated with background pattern worsening in / ( . %) cases. se- vere background patterns were more prevalent in post- ver- sus pre-phenobarbitone tracings ( / [ %] vs. / [ %]; p = . ). presence of severe pattern versus either continuous normal voltage or discontinuous normal voltage pattern post-phenobarbitone, ( / [ %] vs. / [ %]; p = . ) was associated with death or moderate-to-severe injury on mri brain. median time to trace recovery, when measurable, was h ( min– h). conclusions: phenobar- bitone induces significant suppression on aeeg in infants with hie undergoing th. development of severe aeeg back- ground patterns after phenobarbitone may unmask a popu- lation at greater risk of abnormal outcome. © the author(s) published by s. karger ag, basel background amplitude-integrated eeg (aeeg) is commonly em- ployed in neonatal intensive care units (nicus) to mon- itor background cortical activity, seizure management, this is an open access article licensed under the creative commons attribution-noncommercial- . international license (cc by-nc) (http://www.karger.com/services/openaccesslicense), applicable to the online version of the article only. usage and distribution for com- mercial purposes requires written permission. deshpande/jain/mcnamaraneonatology ; : – doi: . / and aid prognostication in infants with hypoxic-ischemic encephalopathy (hie) [ – ]. previously, presence of a severely abnormal aeeg background pattern at < h of age was considered to be a strong predictor for adverse neurodevelopmental outcome [ , ]. therapeutic hypo- thermia (th), however, has shown to delay the overall recovery of aeeg in hie patients, with recent studies demonstrating persistence of aeeg abnormalities ≥ h of age and time to normalization of trace being better pre- dictors of adverse outcomes [ – ]. antiepileptic drugs (aed), such as phenobarbitone being the commonest first-line agent, are frequently used for seizure management in infants with moderate and se- vere hie and are well known to induce aeeg suppression [ – ]. in clinical practice, trends in aeeg characteris- tics are often used as an ancillary tool to refine prognosis. hence, accurate documentation of the degree and/or du- ration of drug-induced aeeg suppression may help clini- cians differentiate these “iatrogenic” effects from hie-re- lated suppression and potentially avoid misinterpreta- tions. although the suppressive effects of phenobarbitone on neonatal aeeg have been described under normothermic conditions, its impact and clinical relevance in the pres- ence of th remains unknown [ – ]. therefore, the primary aim of this study was to characterize the effect of phenobarbitone on aeeg background pattern in infants with moderate and severe hie undergoing th. second- ary aims were to assess the effect of phenobarbitone on aeeg voltage, investigate the time to trace recovery and to study the association between aeeg suppression fol- lowing phenobarbitone and severity of hie. we hypoth- esized that during hypothermia, phenobarbitone causes suppression of aeeg background in > % of tracings. methods study design this retrospective cohort study was conducted at the nicu of the hospital for sick children, toronto, over a -year period when aeeg recordings were archived and phenobarbitone was the first-line aed used. the study was approved by the institu- tional research ethics board and parental consent requirement was waived. inclusion and exclusion criteria all infants with sarnat stage ii (moderate) or iii (severe) hie, born at gestational age ≥ weeks, who received treatment with intravenous phenobarbitone for clinical or electrical sei- zures while undergoing th, and had aeeg recorded during phe- nobarbitone administration were considered for inclusion. only the first episode of seizure treatment after admission to our unit was considered for analysis. infants who may have had a prior phenobarbitone dose in the community before admission were included. infants who received aeds other than phenobarbi- tone, including lorazepam as first treatment after admission, and those who had seizures after completion of rewarming were excluded. tracings with impedance > ohms or where time of phenobarbitone administration was not marked were also ex- cluded. study setting our center is an outborn quaternary nicu, where infants with suspected hie are referred from community hospitals after initial stabilization. when appropriate, th is commenced pre-transfer either by the community physicians or the neonatal transport team or in the nicu after admission, in all cases according to our stan- dardized guideline. typically, th is commenced at age < h and continued for h. all infants undergoing th also receive aeeg monitoring for the duration of treatment, starting soon after ad- mission until completion of rewarming or death/withdrawal of life sustaining treatment, whichever occurred earlier. during the study period, aeeg was recorded using single-channel olympic cfm monitor (natus medical incorporated, san carlos, ca, usa) with electrodes at p -p location or brm brainz monitor (brainz instruments, new zealand) with electrodes at c -c and p -p locations, using hydrogel electrodes, and archived electron- ically on our hospital’s server. phenobarbitone, given intravenous- ly at a dose of , , or mg/kg over min was our first-line aed and the timing was marked on the aeeg monitor by the bed- side nurse. the decision to treat, whether for a clinical and/or elec- trical seizure, as well as the choice of dose of phenobarbitone, was at the attending physician’s discretion. conventional -h eeg was obtained at some point during the infant’s admission period. for table . national institute of child health and human development (nichd) scoring system for classifying brain injury on mri for infants with hie score injury pattern observed on brain mri no injury seen a minimal cerebral lesions without involvement of bgt, alic, or plic, and no watershed area infarction b more extensive cerebral lesions but without bgt, plic, or alic involvement or watershed area infarction a any bgt, alic, or plic involvement or watershed area infarction, but without any other cerebral lesions b any bgt, alic, or plic involvement or watershed area infarction, and additional cerebral lesions cerebral hemispheric devastation hie, hypoxic-ischemic encephalopathy; bgt, basal ganglia or thalamus; alic, anterior limb of the internal capsule; plic, posterior limb of the internal capsule. phenobarbitone and aeeg in hypothermia neonatology ; : – doi: . / the study period, full montage continuous eeg monitoring was undertaken only for patients with intractable seizures needing midazolam infusion, in consultation with our institute’s pediatric neurology team. brain mri was performed between days and after birth for all surviving infants treated with th for hie and reported by our institute’s pediatric neuroradiologists. data collection patients who underwent th for hie were identified from our unit’s computerized database, and their health records were re- viewed to determine eligibility. clinical data were collected for de- mographics and perinatal history, sarnat stage of hie, timing of th initiation and rewarming, presence of liver dysfunction, phe- nobarbitone dose and age at administration, concomitant use of opioid sedatives and outcomes (mortality and results of brain mri, whenever available). only the first episode of phenobarbitone ad- ministration after admission to the nicu and initiation of aeeg monitoring was included for analysis. assessment of aeeg tracings for a duration of min prior to (baseline) and be- tween and min after phenobarbitone administration were reviewed and categorized by one of the investigators (pd and aj), who had > years of clinical experience in interpreting neona- tal aeeg. for consistency between the aeeg devices, only the p -p traces were used for trace analysis. for each time period, the worst background pattern and lowest upper margin voltages (umv) and lower margin voltages (lmv) were recorded. the umv and lmv were determined visually by drawing a line across the uppermost and lowermost dense part of the tracing, respec- (n = ) admissions with hie for th (n = ) infants included (n = ) unknown time of phenobarbitone administration (n = ) received other aeds prior to phenobarbitone (n = ) jet ventilation artifact (n = ) no aeeg monitoring during phenobarbitone treatment (n = ) seizures after rewarming (n = ) impedance> ohms (n = ) flat trace prior to phenobarbitone (n = ) seizures (n = ) infants included for background pattern comparison pre and post phenobarbitone (n = ) umv and lmv not interpretable (n = ) background pattern not interpretable (n = ) infants included for umv and lmv comparison pre and post phenobarbitone fig. . eligibility and analysis criteria. hie, hypoxic-ischemic encephalopathy; th, therapeutic hypothermia; aeeg, amplitude-integrated electroencephalography; aeds, anti-epileptic drugs; ft, flat trace; lmv, lower mar- gin voltage; umv, upper margin voltage. deshpande/jain/mcnamaraneonatology ; : – doi: . / tively. whenever background pattern changed after phenobarbi- tone, tracings were further reviewed either until it recovered to baseline status or until completion of rewarming, whichever oc- curred first. for the former, time to trace recovery was calculated. definitions background pattern was categorized according to the classifi- cation previously described by hellström-westas [ ]. continu- ous normal voltage (cnv) was considered a normal background, discontinuous normal voltage (dnv) as moderately abnormal, and burst suppression (bs), continuous low voltage (clv), or flat trace (ft) as severely abnormal. umv was categorized as > µv, – µv, – µv, or < µv, and lmv as > µv, – µv, or < µv. a “clinically significant” change after phenobarbitone was de- fined a priori based on (i) background pattern: cnv to any other pattern or dnv to bs/clv/ft or bs/clv to ft or (ii) changes in umv: – µv to < µv or > µv to < µv or (iii) changes in lmv: > µv to < µv or – µv to < µv. brain mri findings were scored from to , as per the national institute of child health and human development system (table  ) [ ]. for this study, adverse outcome was defined as death or moderate-to-severe in- jury on mri (score ≥ a). furthermore, liver dysfunction was de- fined as alanine transferase > units/l and/or aspartate transfer- ase > units/l as per our laboratory normal values. study outcomes presence of new-onset, severely abnormal background patterns af- ter phenobarbitone was considered the primary outcome. secondary outcomes included: (i) changes in umv and lmv after phenobarbi- tone compared to baseline, (ii) time to trace recovery, where applicable, (iii) association between pre- and post-phenobarbitone aeeg charac- teristics and death or moderate-to-severe injury on brain mri. statistical analysis data are described as frequency (percentage), mean (standard deviation), or median (range), as appropriate. the frequency of aeeg patterns pre- and post-phenobarbitone as well as their as- sociation with adverse outcome of death or moderate-to-severe mri injury was analyzed using fisher’s exact test. time to trace recovery was described in hours and compared between infants with sarnat stage ii versus stage iii hie, with or without liver dys- function and phenobarbitone dose < mg/kg versus mg/kg using wilcoxon signed-rank test. furthermore, interobserver reli- ability was tested on tracings ( pre- and post-phenobarbi- tone) from randomly selected subjects using cohen’s kappa sta- tistic, which was . and . , for categorization of background pattern and lmv, respectively. table . perinatal and clinical characteristics of the study cohort (n = ) female gender ( %) gestational age, weeks . ± . birth weight, g , ( , , , ) known sentinel eventα ( %) history of fetal distressβ ( %) caesarean delivery ( %) intubation at birth ( %) chest compressions ( %) apgar score at min ( , ) cord ph . ± . base deficit − . ± . persistent pulmonary hypertension ( %) hypotension – requiring treatment ( %) prior phenobarbitone before admission ( %) clinical seizures only ( %) dose of phenobarbitone for the episode studied , mg/kg ( %) , mg/kg ( %) , mg/kg ( %) age at phenobarbitone administration, h . ( . , . ) liver dysfunction ( %) severe injury on mri ( %) mortality ( %) data are presented as percentage, mean ± sd, or median (range) as appropriate. α  include placental abruption, prolonged labor, and shoulder dystocia. β defined as documentation of fetal heart decelerations, tachycardia, or abnormal variability. γ  rest were either electrical or electroclinical. cnv dnv bs clv ft ft clv bs dnv cnv pre- phenobarbitone background pattern bs dnv cnv clv ft fig. . changes in aeeg background pattern before and after phe- nobarbitone in infants. background pattern could not be deter- mined in infants due to prolonged seizure activity prior to phe- nobarbitone administration. aeeg, amplitude-integrated electro- encephalography; cnv, continuous normal voltage; dnv, discontinuous voltage; bs, burst suppression; clv, continuous low voltage; ft, flat trace. phenobarbitone and aeeg in hypothermia neonatology ; : – doi: . / results a total of infants with hie, with sarnat stage iii and with stage ii, satisfied the inclusion criteria (fig.  ). the cohort characteristics are listed in table  . two infants demonstrated ft even before phenobarbi- tone, and were not included in further analysis. four tracings had pre-seizure recording duration < min, where background pattern could not be ascertained, in- cluding , where lmv and umv could not be ascer- tained. in comparison to baseline, post-phenobarbi- tone tracings demonstrated higher frequency of severe- ly abnormal patterns and umv and lmv below pre-defined thresholds (table  ). none of the traces demonstrated cnv pattern after phenobarbitone ad- ministration (table  ). a clinically significant change table . characteristics of background aeeg traces at baseline and post-phenobarbitone aeeg characteristics baseline n (%) post-phenobarbitone n (%) p value background patternα severely abnormal trace (bs/clv/ft)β / ( ) / ( ) . continuous / / . discontinuous / ( ) / ( ) . upper margin < µvγ / ( ) / ( ) . upper margin < µvγ / ( ) / ( ) . lower margin < µvγ / ( ) / ( ) . lower margin < µvγ / ( ) / ( ) . aeeg, amplitude-integrated electroencephalography; bs, burst suppression; clv, continuous low voltage; ft, flat trace. valies in italics indicate p < . . α pre-phenobarbitone background pattern could not be ascertained for infants due to presence of seizures at onset of aeeg recording. β excluding infants with baseline flat trace. γ pre-phenobarbitone upper and lower margin voltage could not be classified for infant due to seizures at onset of aeeg recording. dnv upper margin – µv cf m , µ v phenobarbitone burst suppression pattern upper margin – µv lower margin – µv ed : / / : : wed lower margin – µv burst suppression pattern upper margin – µv fig. . example of an aeeg tracing illustrating effect of phenobar- bitone administration to an infant with hie and seizures while receiving therapeutic hypothermia. the left side of the tracing shows a discontinuous background pattern (umv between and μv and lmv at – μv) with repetitive seizures. the dotted line in the middle part of the tracing indicates intravenous administra- tion of phenobarbitone at mg/kg over min. following this, the background changed to a bs pattern with a drop of both umv and lmv to – μv and – μv respectively. aeeg, amplitude- integrated electroencephalography; hie, hypoxic-ischemic en- cephalopathy; umv, upper margin voltage; lmv, lower margin voltage; bs, burst suppression; dnv, discontinuous normal volt- age. deshpande/jain/mcnamaraneonatology ; : – doi: . / in background pattern was seen in / ( %) (fig.  , ) and in umv and/or lmv in / ( %) infants. there was no difference within the subgroups of infants with clinical and electrical seizures with respect to clin- ically significant change in the background ( / vs. / , p = ) or any clinically significant change ( / vs. / , p = . ). fourteen of the traces recovered to baseline status prior to exposure to any further aed. the median (range) time to trace recovery was ( . – ) hours. four traces remained suppressed until the end of aeeg recording and infant was given another aed before trace recov- ery. time to trace recovery did not differ between infants with sarnat stage ii versus iii hie ( . [ – ] vs. [ . – ] hours; p = . ), with versus without liver dys- function ( . [ – ] vs. . [ . – ] hours; p = . ), or phenobarbitone dose < mg/kg versus mg/kg ( . [ . – ] vs. . [ – ] hours; p = . ). for infants, where the study episode was the only exposure to pheno- barbitone and therefore not proceeded by any aed in the community, median (range) time to trace recovery was ( – ) hours. death or moderate-to-severe mri injury occurred more frequently in those with severely abnormal post- phenobarbitone background pattern versus cnv/dnv ( / [ %] vs. / [ %]; p = . ). no association was seen between adverse outcome and pre-phenobarbitone severely abnormal background pattern versus cnv/ dnv ( / [ %] vs. / [ %]; p = . ). discussion in this study, we found that phenobarbitone treatment for seizures in neonates with hie undergoing th is char- acterized by important changes in various clinically rel- evant aeeg parameters including background pattern and voltage margins. we also provide data on time to trace recovery, where applicable, that clinicians may be able to apply while interpreting aeeg in the context of hie and th. furthermore, we found an association be- tween aeeg patterns – min post-treatment but not pre-treatment, with adverse outcome of death or moder- ate-to-severe brain injury. although the suppressive effect of aeds on neonatal aeeg have previously been described, to the best of our knowledge there is no previous report on the effect of phenobarbitone on neonatal aeeg in the setting of th studied in the clinical context [ , ]. shany et al. [ ] described the suppressive effects of commonly used aeds from aeeg tracings in neonates, who received treatment for seizures. while % of patients had a diag- nosis of hie, none received th. each repeat aed expo- sure was considered as a separate episode. the authors reported worsening in background pattern, umv, and lmv in up to , , and % tracings, respectively. overall, mean (range) time to trace recovery was . ( . – ) hours and . h for phenobarbitone. data spe- cifically from the hie subgroup or clinical outcomes were not evaluated. although there are differences in the pop- ulation and aeds used by shany et al. [ ], we noted a comparatively higher frequency of post-treatment sup- pressed tracings and slightly longer median time to trace recovery; however, trace recovery was noted in only half of the traces in our cohort. the mechanism behind suppression after phenobarbi- tone in the setting of hie and th may likely be because of the following reasons: first, the severity of suppression may relate to the severity of brain injury; specifically, it is plausible that treatment with aeds may unmask a sub- population with increased risk of brain injury. this hy- pothesis is supported by our finding of an association of adverse clinical outcome and abnormal tracing only after but not before phenobarbitone administration. second, it may be related to seizure burden, which could not be ad- dressed in our study. although it may be plausible that severe hie or a high seizure burden may increase the sen- sitivity of the brain to the suppressive effect of phenobar- bitone, our findings do not explain the mechanism be- hind these hypotheses. finally, although we do not have data on plasma phenobarbitone levels, pharmacokinetic studies have demonstrated no change in phenobarbitone clearance in hypothermia versus normothermia [ , ]. in fact, a study using simulated pharmacodynamic mod- eling found phenobarbitone treatment to be associated with reduced rate of transition in aeeg pattern from cnv to dnv [ ]. phenobarbitone clearance may also be impacted by liver dysfunction; however, a lack of as- sociation between hepatic dysfunction and trace suppres- sion in our study also suggests a likely lack of role of dif- fering pharmacokinetic profile. our observations, how- ever, are based on a small sample size and need further validation. strengths of our study include strict eligibility criteria, well-defined population, and minimizing the confound- ing influence of multiple drugs and escalating dosage. limitations include retrospective design and small sample size, which prevented us from accounting for the indepen- dent effect of confounders such as severity of hie, seizure burden, dose of phenobarbitone, and concomitant use of phenobarbitone and aeeg in hypothermia neonatology ; : – doi: . / opioids. second, we were unable to correlate degree of aeeg suppression with plasma phenobarbitone levels due to limited and variable testing. furthermore, though we did not use automated quantification of aeeg upper and lower margins, the categorization of margins we used makes the results clinically applicable. finally, although we found an association between severity of aeeg sup- pression and adverse short-term outcomes, long-term neurodevelopmental outcome data are lacking. in conclusion, a significant number of infants who re- ceive phenobarbitone for seizures while undergoing th for moderate-to-severe hie may demonstrate clinically significant suppression in aeeg characteristics after treatment, including new-onset severely abnormal back- ground patterns. presence of severely abnormal aeeg patterns – min post-phenobarbitone may be a better indicator of risk of adverse outcome of death or moder- ate-to-severe injury on mri brain than aeeg pattern pri- or to treatment. a larger prospective study that includes information on drug levels may provide more mechanis- tic insights. clinicians should consider the potential sup- pressive effect of phenobarbitone on aeeg during deci- sion-making and prognostication. statement of ethics this research was approved by the institutional research eth- ics board of the hospital for sick children (reb# ). parental consent was not required due to the retrospective nature of the study. conflict of interest statement the authors have no conflicts to declare. funding sources no financial support. author contributions p.d. conceptualized the research idea, and p.d. and p.m. de- vised the methodology. a.j. provided feedback to refine to meth- odology and analysis. p.d. and a.j. carried out amplitude-integrat- ed encephalography trace analysis as well as clinical data collec- tion. p.m. provided guidance and supervision during trace analysis. p.d. drafted the manuscript, and a.j. and p.m. reviewed the man- uscript and provided critical feedback. references bjerre i, hellström-westas l, rosén i, sven- ningsen n. monitoring of cerebral function after severe asphyxia in infancy. arch dis child. dec; 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( ): – . https://www.karger.com/article/fulltext/ ?ref= #ref https://www.karger.com/article/fulltext/ ?ref= #ref https://www.karger.com/article/fulltext/ ?ref= #ref https://www.karger.com/article/fulltext/ ?ref= #ref https://www.karger.com/article/fulltext/ ?ref= #ref https://www.karger.com/article/fulltext/ ?ref= #ref 	starttablebody 	starttablebody 	starttablebody vol. , no. journal of clinical microbiology, aug. , p. - - / / - $ . / copyright (o , american society for microbiology lipopolysaccharide gel profiles of haemophilus influenza type b are not stable epidemiologic markers robert w. tolan, jr., robert s. munson, jr., and dan m. granoff* edward mallinckrodt department of pediatrics, washington university school of medicine, and division of infectious diseases, st. louis children's hospital, st. louis, missouri received february /accepted april sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lipopolysaccharide (lps) was performed to assess the usefulness of this technique for the epidemiologic analysis of haemophilus influenza type b isolates. lps samples were prepared from isolates which had been passaged either in vitro or in infant rats. preparations from paired isolates from a number of epidemiologically related clinical situations also were examined. the gel patterns of lps prepared on different occasions from an individual isolate were stable. however, the lps gel patterns changed in of ( %) of the passaged isolates, and differences in gel patterns also were observed among epidemiologically related isolates. the variability in lps electrophoretic patterns of individual isolates indicated that this technique is not useful for the epidemiologic analysis of h. influenzae type b disease. haemophilus influenza type b is the most common cause of bacterial meningitis in children in the united states. outer membrane protein (omp) profiles ( , , , ), serum bactericidal assays with cross-adsorbed antisera ( ), and biotype analyses ( ) have been used to distinguish among type b isolates in an effort to assess the heterogeneity of these organisms as well as to provide important epidemio- logic information on the transmission of this organism. in the united states, more than % of the type b isolates from children with bacteremic disease are biotype i ( ), and approximately % have a common omp subtype, h ( ). therefore, in many situations the application of these tech- niques provides only limited information to aid in distin- guishing among isolates of h. influenza type b. recently, inzana and pichichero ( , ) demonstrated that the lipopolysaccharide (lps) of h. influenza type b also exhibits strain heterogeneity as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds- page). this technique was then applied to evaluate the relatedness of isolates obtained from patients and carriers in an epidemic ( ) and to investigate endemic disease in an apache population ( ). more recently, it has been applied to quantitate lps production by antibody-sensitive and -resistant strains of h. influenza type b ( ) and to analyze cell wall alterations in a transformed type b strain ( , ). one requirement of an epidemiologic tool is that the char- acteristic being measured be a stable feature of an isolate and not subject to frequent variation. thus, the present study was undertaken to ascertain the stability of the lps gel profiles after a variety of experimental manipulations of the organism. we also compared the lps profiles of isolates obtained from patients with those of isolates from asympto- matic contacts in closely related clinical situations. materials and methods bacteria. bacterial isolates were collected, serologically typed for capsular antigen, and stored as described previ- ously ( ). the clinical and epidemiologic characteristics of selected bacterial isolates used are summarized in table . * corresponding author. isolates were subcultured from frozen stocks onto chocolate agar and incubated at °c in % c overnight. a single, representative colony was selected and streaked onto a fresh chocolate agar plate and incubated at °c in % c overnight; colonies were then scraped from the chocolate agar and suspended in dulbecco phosphate-buffered saline ( ) for lps sample preparation. in some experiments, bac- teria were grown to mid-log phase in broth cultures and harvested as described previously ( ). rapid isolation micromethod, whole-cell lysates, and pro- teinase k digestion. two rapid techniques for lps extraction were developed recently ( , ). the rapid isolation micromethod of inzana uses a simplified hot phenol-water extraction followed by ethanol precipitation (avoiding the ultracentrifugation steps in the classic purification procedure [ ]) ( ), and the other technique uses sds solubilization followed by proteinase k digestion ( ). in previous studies of both h. influenza type b and other bacteria, these extraction procedures yielded preparations with gel profiles similar to those of preparations made by conventional pro- cedures ( , , ) and were more convenient than the conventional methods for examining the large numbers of isolates encountered in epidemiologic studies ( , ). re- cently, kimura and hansen ( ) showed that h. influenza type b lps prepared by these two methods yields identical sds-page profiles. in selected experiments, the lps was purified by using the hot phenol-water extraction ethanol precipitation procedure described previously ( ), with log- phase broth-grown cells as the source of the lps. however, the proteinase k procedure of hitchcock and brown ( ) was found to be easier and was used in most of our experiments. there were several minor modifications, and the procedure was as follows. cells were harvested with sterile cotton swabs and suspended in ml of cold dulbecco phosphate-buffered saline (ph . ) to an optical density of . + . at a wavelength of nm. portions (either . or . ml) of these suspensions were centrifuged for . min in an eppendorf microfuge (model ; brinkmann instruments, inc., westbury, n.y.), and the supernatants were discarded. the pellets were solubilized in ,ul of solubilizing buffer containing % sds (bdh, poole, england), % -mercaptoethanol (eastman kodak co., o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://jcm .a sm .o rg / d o w n lo a d e d fro m http://jcm.asm.org/ tolan et al. table . clinical and epidemiologic characteristics of h. influenza type b and salmonella isolates used designation site diagnosis locale date notes and reference(s) omp source durst csfb meningitis cleveland, , , , l ohio eagan csf meningitis boston, , , , , , l porter mass. anderson minn a csf meningitis minneapolis, isolate from day-care center h janet minn. ( ); , gilsdorf blood facial st. lous, h cellulitis mo. csf meningitis st. louis, h mo. csf meningitis st. louis, , l mo. csf meningitis madison, unknown patient ( ); l bruce wis. edmonson a csf meningitis buffalo, mo. / / case ( ) h dean rising a blood cellulitis buffalo, mo. / / case ( ) h dean rising b throat buffalo, mo. / / community contact ( ) h e throat buffalo, mo. / / healthy father of a child h with meningitis (case [ ]) e throat buffalo, mo. / / healthy mother of a child h with meningitis (case [ ]) w blood cellulitis buffalo, mo. / / case ( ) h blood meningitis minneapolis, unknown h michael minn. osterholm g rc mutant of salmonella mary jane typhimurium lt ; , osborn g a re rough mutant of s. mary jane typhimurium g ; osborn atcc salmonella typhimurium a omp subtypes per barenkamp et al. ( ). b csf, cerebrospinal fluid. rochester, n.y.), % glycerol, . m tris chloride (ph . ), and . % bromphenol blue. after heating at °c for min, ,ug of proteinase k (sigma chemical co., st. louis, mo.) in ji.l of solubilizing buffer was added to each lysate, and the samples were incubated at °c for h. samples were applied immediately to polyacrylamide gels or were frozen for later use. frozen samples were reheated at °c for min before application to the gel. quantitation of the lps in the samples added to the wells was not practical because of the small amount of lps present. thus, the sample volumes added to the gels were varied to optimize visualization in subsequent gels. approximately ,ug of lps was added to each well, based on comparison with staining intensities of known amounts of purified lps standards ( ). sds-page. lps preparations were subjected to sds- page by using the system of laemmli ( ) with modifica- tions described by hitchcock and brown ( ) and inzana and pichichero ( , ). stacking gels ( . %) and separating gels ( %) were cast in laemmli buffers containing . % sds, . m edta (tetrasodium salt), and m urea. the electrophoresis was performed at room temperature with equipment from hoefer scientific instruments, san francisco, calif., at a constant current until the dye front migrated off the gel ( to h). the running buffer was that of laemmli ( ) with the addition of . m edta (tetrasodium salt). the gels were fixed for at least h in ml of % (vol/vol) -propanol in % (vol/vol) acetic acid before staining. the sds-page of sarcosyl-insoluble omp preparations was performed as described before ( ). silver staining. gels were stained by a modification of the method of tsai and frasch ( ) as modified by hitchcock and brown ( ). after fixation, the gels were oxidized for min in a mixture of . g of periodic acid- ml of distilled water- ml of ethanol:acetic acid:distilled water ( : : , vol/vol/vol). after four -min washes in ml of distilled water, the gels were stained for min in a solution prepared by mixing ml of % (wt/vol) agno , ml of . n naoh, . ml of to % nh h, and ml of distilled water. after four -min washes in ml of distilled water, the gels were developed in ml of distilled water contain- ing . ml of . % formaldehyde and . g of citric acid. the gels were then washed for h in ml of distilled water containing ml of % (vol/vol) acetic acid, rinsed, and stored in distilled water. in vivo and in vitro passage. the procedure for serial in vivo passage of the isolates through infant rats has been described before ( ). in addition, clinical isolates were carried on chocolate agar. isolated colonies from of the isolates were picked and replated times, and the remain- ing isolates were passaged times. results stability of lps mobilities. the lps was purified by the modified proteinase k method on one or more occasions (mean, . ; range to ) from isolates. these prepara- tions were electrophoresed on multiple sds gels (mean, . ; range to ). for each isolate, the gel profiles of the lps samples were reproducible in all cases. lps mobilities of clinical isolates. an outbreak of four cases of h. influenzae type b disease occurred among j. clin. microbiol. o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://jcm .a sm .o rg / d o w n lo a d e d fro m http://jcm.asm.org/ h. influenzae type b lps children in a close-knit amish community in rural missouri ( ). isolates from the patients and carriers in the community had the same omp subtypes (fig. , lanes , , and to ) and identical alloenzyme patterns ( ) characteristic of elec- trophoretic type . (a total of enzymes was tested, and there was an average of three to four variants at each of polymorphic loci [ ].) thus, the isolates were judged to be a single clone. the lps from the three invasive isolates (from cases , , and ) obtained from the amish outbreak was examined by the proteinase k purification method of hitchcock and brown ( ). the lps from cases and , separated by months, showed identical mobility (data not shown). the lps from case , which occurred months after case , had a different mobility. in several gels, the major fraction of the staining material from isolate w from case (fig. , lane ) migrated with a slightly lower apparent molecular weight than that of isolate a from case (fig. , lane ). h. influenzae type b throat isolates e and e were available from the parents of another amish child (case ) with meningitis. the isolate from the child was not available, so we compared the electrophoretic mobilities of lps preparations from the two parental throat isolates with those of preparations from the invasive isolates from cases , , and . the lps from isolate e from one parent (fig. , lane ) appeared to be identical to the lps from isolate a from case (fig. , lane ), whereas the lps from the other throat isolate ( e) appeared to have a slightly lower molecular weight (fig. , lane ). six isolates from the outbreak of h. influenza type b k k r ; fig. . sds-polyacrylamide gradient slab gel ( to %) of sarcosyl-insoluble omp from h. influenza type b isolates. isolates representing three common subtypes ( ) as well as isolates which exhibited changes or differences in electrophoretic mobility of lps after passage or in epidemiologically related situations are shown. lanes: and , isolate eagan (omp subtype ); and , isolate durst (omp subtype ); and , isolate (omp subtype ); and , isolate durst before and after four rat passages; and , isolate before and after four rat passages; and , isolate a, a blood isolate from patient (case [ ]), before and after in vitro passages; and , isolate w, blood isolate of patient (case [ ]), before and after in vitro passages; , isolate a; and , isolates e and e, respectively, throat isolates (taken the same day) from the parents of a child with meningitis (case [ ]) in the amish community (an isolate from the child was unavailable). the method for preparation of sarcosyl-insoluble preparations and gradient gels has been described previously ( ). molecular weight standards, a-lactalbumin ( . x ); soybean trypsin inhibitor ( . x ); carbonic anhydrase ( x ); ovalbumin ( x ); bovine serum albumin ( x ); phosphor- ylase b ( x ) ( ). fig. . sds-polyacrylamide slab gel ( %) of lps from protein- ase k digests of whole-cell lysates of isolates of h. influenza type b and salmonella typhimurium. profiles of h. influenza type b isolates exhibiting representative changes in the electrophoretic mobilities of lps are shown in lanes to , corresponding to fig. , lanes to , in which the omp profiles of the h. influenza type b strains are shown. the controls are as follows: lanes and , strain g , an rc mutant of s. typhimurium lt ( , ); lanes and , strain g a, an re rough mutant of s. typhimurium g ( ); lanes and , strain atcc , smooth s. typhimurium. approximately ,ug of lps was added to each lane. the preparation of the gels and the lps samples, as well as the staining of the gels, is described in the text. disease in an isolated amish community ( ) were passaged times in vitro. changes in sds-page mobility were observed in a (fig. , lanes and ), w (fig. , lanes and ), and a (data not shown). compared with the prepassage isolates, isolates a and a both produced lps with a lower apparent molecular weight after passage, and isolate w produced lps with a higher apparent molecular weight. again, the omp profiles of these three isolates remained unchanged after passage in vitro ( a, fig. , lanes and ; w, fig. , lanes and ; and a, data not shown). lps mobilities before and after in vivo passage. four isolates (durst, eagan, , and ) were passaged four times each in infant rats. the electrophoretic mobilities of the lps prepared from proteinase k-digested whole-cell lysates of two of these strains (eagan and ) remained unchanged after passage in rats (data not shown). however, the lps mobilities of the two remaining isolates were different after passage (durst, fig. , lanes and ; , fig. , lanes and ). to examine further these changes, lps was prepared from the isolates obtained after each of the four rat passages. after one passage, isolate durst produced lps with a greater apparent molecular weight (slower elec- trophoretic mobility) than that of the lps from before passage (data not shown). the mobilities of lps produced after subsequent passages remained unchanged. in contrast, isolate produced lps of unchanged mobility over three passages. after passage , the lps had a lower apparent molecular weight (faster electrophoretic mobility; data not shown). the omp profiles of these isolates before and after passage were unchanged (durst, fig. , lanes and ; , fig. , lanes and ). lps mobilities before and after in vitro passage. isolates durst, eagan, minn a, and were passaged times vol. , o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://jcm .a sm .o rg / d o w n lo a d e d fro m http://jcm.asm.org/ tolan et al. each in vitro. in contrast to the changes observed when isolates from the amish were passaged in vitro (see above), no changes were observed in the mobilities of the lps produced from these isolates after in vitro passage. discussion the electrophoretic profiles of lps prepared from epide- miologically related and laboratory isolates of h. influenzae type b were compared on silver-stained sds-page gels. lps was prepared with log-phase broth-grown cells by the method of inzana ( ) and by the proteinase k method of hitchcock and brown ( ) with cells scraped from plates. lps samples prepared from a single isolate yielded repro- ducible gel profiles, but we observed different mobilities for lps samples from epidemiologically related isolates and changes in lps mobility in both laboratory and epidemio- logically related strains after passage in vivo or in vitro. although we were unable to resolve the h. influenza type b lps patterns into several distinct bands as previously de- scribed ( - , , ), changes in the lps profiles of individual isolates were apparent in gels of samples prepared by both the methods of hitchcock and brown ( ) (fig. ) and inzana ( ) (data not shown). these changes were obvious and indicative of as yet undefined changes in the lps from these strains. an increase in the resolution of the gels would only enhance the differences observed and would not alter the conclusions of our study. although multiple lps preparations from a single isolate yielded stable gel patterns, we observed that the electropho- retic mobility patterns of lps from several isolates were unstable after repeated passage in vivo or in vitro. four isolates were passaged in infant rats; two produced lps with altered mobilities. three of six isolates from a recent out- break of h. influenza type b disease in an amish commu- nity produced lps with altered mobilities after passage in vitro. shifts to lower apparent molecular weights as well as shifts to higher apparent molecular weights were observed. a direct comparison of lps patterns from isolates from different patients and carriers in the amish community indicated that variation in lps structure among isolates also occurred after transmission among humans. the outbreak in the amish community occurred during a -month period. therefore, it might be argued that more than one h. influ- enzae type b strain was involved. however, the community is located in rural missouri, and the children have little or no contact with non-amish children. all of the h. influenza type b isolates from the community had identical antibiotic susceptibility patterns, omp profiles, and alloenzyme pat- terns. furthermore, differences in lps mobilities were ob- served in these isolates after passage in vitro. finally, throat isolates e and e cultured from the parents of patient on the same day differed in lps mobility (fig. , lanes and ); it is highly unlikely that these two house- hold contacts, who earlier in the outbreak had had negative throat cultures, became colonized by different h. influenza type b strains. lps mobility on sds-page has been used in previous studies to investigate the transmission of h. influenza type b in an epidemic ( ) and to examine the spread of h. influenzae type b infection on an apache reservation ( ). in the former study, isolates from two patients with onset of disease weeks apart and who attended the same day-care center differed in lps subtype but exhibited an identical omp profile ( ). the researchers concluded that lps subtype is more useful for distinguishing between these two isolates than are omp profiles, implying that case was caused by the introduction of a new h. influenza type b strain into the day-care center. similar conclusions were reached concerning isolates from patients and carriers in the latter study ( ). however, our results indicate that isolates producing lps of altered electrophoretic mobility occur frequently after in vivo or in vitro passage in the laboratory and may be seen in epidemiologically related situations. while this manuscript was in preparation, kimura and hansen ( ) independently reported similar instability in h. influenza type b lps electrophoretic mobility. thus, their data and ours indicate that lps electrophoretic gel mobility is an unstable strain marker and that this technique is not a useful epidemiologic tool to study the transmission of h. influenza type b infection. the chemical basis and the genetic mechanisms responsible for these changes remain to be elucidated. acknowledgments this work was supported in part by public health service grant -roi-ai from the national institute of allergy and infec- tious diseases. r.w.t. was supported by biomedical research support grant -so rr - from the national institutes of health. we thank susan grass and kathleen mccollough for excellent technical assistance and paul gulig, eric hansen, penny hitchcock, and thomas inzana for helpful suggestions. literature cited . anderson, p., a. flesher, s. shaw, a. l. harding, and d. h. smith. . phenotypic and genetic variation in the suscepti- bility of haemophilus influenza type b to antibodies to somatic antigens. j. clin. invest. : - . . anderson, p., r. b. johnston, jr., and d. h. smith. . human serum activities against haemophilus influenza, type b. j. clin. invest. : - . . barenkamp, s. j., d. m. granoff, and r. s. munson, jr. . outer-membrane protein subtypes of haemophilus influenza type b and spread of disease in day-care centers. j. infect. dis. : - . . barenkamp, s. j., r. s. munson, jr., and d. m. granoff. . subtyping isolates of haemophilus influenza type b 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population: studies of the effects of genetic factors, immunization, and rifampin prophy- laxis on the course of an outbreak. pediatrics : - . . granoff, d. m., and g. a. nankervis. . infectious arthritis in the neonate caused by haemophilus influenza. am. j. dis. child. : - . . granoff, d. m., and g. a. nankervis. . cellulitis due to haemophilus influenzae type b. am. j. dis. child. j. clin. microbiol. o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://jcm .a sm .o rg / d o w n lo a d e d fro m http://jcm.asm.org/ h. influenzae type b lps : - . . hampton, c. m., s. j. barenkamp, and w. m. granoff. . comparison of outer membrane protein subtypes of haemoph- ilus influenza type b isolates from healthy children in the general population and from diseased patients. j. clin. micro- biol. : - . . hitchcock, p. j., and t. m. brown. . morphological heter- ogeneity among salmonella lipopolysaccharide chemotypes in silver-stained polyàcrylamide gels. j. bacteriol. : - . . inzana, t. j. . electrophoretic heterogeneity and interstrain variation of the lipopolysaccharide of haemophilus influenza. j. infect. dis. : - . . inzana, t. j., and p. anderson. . serum factor-dependent resistance of haemophilus influenza type b to antibody to lipopolysaccharide. j. infect. dis. : - . . inzana, t. j., and m. e. pichichero. . lipopolysaccharide subtypes of haemophilus influenza type b from an outbreak of invasive disease. j. clin. microbiol. : - . . johnson, k. g., and m. b. perry. . improved techniques for the preparation of bacterial lipopolysaccharides. can. j. micro- biol. : - . . kilian, m., i. s rensen, and w. frederiksen. . biochemical characteristics of recent isolates from haemophilus influ- enzae meningitis. j. clin. microbiol. : - . . kimura, a., and e. j. hansen. . antigenic and phenotypic variations of haemophilus influenza type b lipopolysaccharide and their relationship to virulence. infect. immun. : - . . laemmli, u. k. . cleavage of structural proteins during the assembly of the head of bacteriophage t . nature (london) : - . . loeb, m. r., and d. h. smith. . outer membrane protein composition in disease isolates of haemophilus influenza: pathogenic and epidemiological implications. infect. immun. : - . . losonsky, g. a., m. santosham, v. m. sehgal, a. zwahlen, and e. r. moxon. . haemophilus influenza disease in the white mountain apaches: molecular epidemiology of a high risk population. pediatr. infect. dis. : - . . munson, r. s., jr., j. l. shenep, s. j. barenkamp, and d. m. granoff. . purification and comparison of outer membrane protein p from haemophilus influenza type b isolates. j. clin. invest. : - . . musser, j. m., d. m. granoff, p. e. pattison, and r. k. slander. . a population genetic framework of the study of invasive diseases caused by serotype b strains of haemophilus influenza. proc. natl. acad. sci. usa : - . . osborn, m. j., s. m. rosen, l. rothfield, and b. l. horecker. . biosynthesis of bacterial lipopolysaccharide. i. enzy- matic incorporation of galactose in a mutant strain of salmo- nella. proc. natl. acad. sci. usa : - . . shenep, j. l., r. s. munson, jr., s. j. barenkamp, and d. m. granoff. . further studies of the role of noncapsular anti- body in protection against experimental haemophilus influ- enzae type b bacteremia. infect. immun. : - . . shenep, j. l., r. s. munson, jr., and d. m. granoif. . human antibody responses to lipopolysaccharide after menin- gitis due to haemophilus influenza type b. j. infect. dis. : - . . tsat, c.-m., and c. e. frasch. . a sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. anal. biochem. : - . . westphal, o., and k. jann. . bacterial lipopolysaccharides. extraction with phenol-water and further applications of the procedure. methods carbohydr. chem. : - . . zwahlen, a., l. g. rubin, c. j. connelly, t. j. inzana, and e. r. moxon. . alteration of the cell wall of haemophilus influenza type b by transformation with cloned dna: associ- ation with attenuated virulence. j. infect. dis. : - . . zwahlen, a., j. a. winkelstein, and e. r. moxon. . surface determinants of haemophilus influenza pathogenicity: com- parative virulence of capsular transformants in normal and complement-depleted rats. j. infect. dis. : - . vol. , o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://jcm .a sm .o rg / d o w n lo a d e d fro m http://jcm.asm.org/ case reports inheritance was dominant and therefore unlike the present family. moynahan' reported male sibs with mental subnormality, epilepsy (onset at to years), and alopecia which involved the scalp only. the hair later regrew. the mode of inheritance in this family is difficult to ascertain as the boy's father only grew hair at the age of years and the boy's mother's sister was bald until the age of years. the regrowth of the hair, the restriction of the baldness to the scalp, and the epilepsy differentiate moynahan syndrome from the one described here. a similar condition in two sibs whose parents were con- sanguineous was reported by perniola et al. deafness occurred in both. there are at least three other syndromes in which hair loss is associated with mental retardation. menkes disease can be excluded as the hair is kinky and not totally absent. prognosis for life is poor and seizures are frequent. inheritance is x linked recessive. in monilethrix the hair is normal at birth but is then lost within the first few months. red pustular lesions on the scalp may be present. alopecia is seldom total and through the pustules brittle hairs which break easily emerge. the nails and teeth are abnormal and mental retardation is only occasionally a feature. in the hair-brain syndrome (amish brittle hair syndrome) those affected had short stature and relatively mild mental retardation. all the affected had hair but it was brittle and broke easily. total alopecia was not a feature and inheritance was recessive. in the condition described in this paper, the alopecia was total and involved all areas of normal hair growth. it probably represents a distinct entity. m baraitser, c carter, and e m brett* clinical genetics unit and *department of neurology, the hospital for sick children, great ormond street, london wcin jh. references moynahan ej. familial congenital alopecia, mental retardation with unusual electroencephalograms. proc r soc med ; : - . shokeir mhk. universal permanent alopecia, psycho- motor epilepsy, pyorrhoea and mental subnormality. clin genet ; : - . perniola t, krajewska g, carnevale f, lospalluti m. congenital alopecia, psychomotor retardation, convul- sions in two sibs of a consanguineous marriage. j inher met dis ; : - . requests for reprints to dr m baraitser, clinical genetics unit, the hospital for sick children, great ormond street, london wcin jh. a female infant with features of mohr and majewski syndromes: variable expression, a genetic compound, or a distinct entity? summary a female child, the offspring of a consanguineous mating, had a cleft palate, tongue tumours, hypoplastic tibiae, and poly- syndactyly. the relationship to the mohr and majewski syndromes is discussed. variable expression is a common problem en- countered by those who seek to define syndromes. the best method to determine the extent of the phenotypic expression of a gene defect is to study the variability within sibships, but in rare disorders this is not always possible. it is then necessary to depend on somewhat arbitrary definitions. the mohr and majewski syndromes each behave as a probable autosomal recessive disorder with a relatively distinct phenotype. temtamy and mc- kusick reported two subjects whose clinical features fulfilled the definition of both syndromes. we report a female infant, the second child of consanguineous parents, in whom combined features of both syndromes were seen. case report the patient was born on . . at weeks' gestation weighing g. she was the second child of first cousin pakistani moslem parents. the mother and father were and years old, respectively. a sib was normal and there was no relevant family history. the child (fig ) was noted to have low set ears, micrognathia, and shallow orbits causing mild proptosis. there was a high arched palate with a posterior cleft and fleshy tumours were present on the underside of the tongue. bilateral postaxial polysyndactyly was noted in the hands with poly- syndactyly, severe talipes equinovarus, and distal shortening of both lower limbs. at months her weight of - kg was well below the rd centile. she fed poorly and had frequent chest infections. her development was slightly delayed. radiological examination revealed severe bilateral tibial dysplasia (fig ), whereas the chest x-ray did not show significant rib shortening. there was a normal ,xx karyotype. received for publication july . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n f e b ru a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ case reports fig (a) facial appearance ofproband. note flat nasal bridge andprominent eyes. (b) sublingual fleshy tumours. (c) postaxial polydactyly of hands. (d) pre- and postaxial polydactyly and severe talipes affecting both feet. fig x-ray appearance of limbs. note hypoplasia of tibia. ,:::.s. . discussion the similarities between the orofacial digital syn- drome type (mohr syndrome) and the short rib polydactyly syndrome type (majewski syndrome) are shown in the table. it is apparent that mid-facial flattening, hyper- telorism, tongue tumours, and multiple frenulae may occur in both syndromes. the majewski syndrome is distinguishable by the presence of short ribs, probably a major factor in the invariable neonatal death, and hypoplasia of the tibiae. in mohr syndrome, survival is the rule and the tibiae are not hypoplastic. possible exceptions have occurred. spranger et a gave a brief report of a patient with features of majewski syndrome whose sib might have been similarly affected and survived for minutes. rimoin and edgerton reported a family in which a brother and sister had the typical features of mohr syndrome, whereas a third sib who ;ift i.w o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n f e b ru a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ rable comparison of clinical features in the present patient with those of alohr and majewski syndromes. majewski syndrome mohr syndrome present patient oral manifestations median cleft, or pseudo-cleft of pseudo-cleft tongue tumours, tongue tumours, exaggerated upper lip exaggerated multiple frenulae multiple frenulae deafness ? + ? nose broad and flat broad and flat broad and flat eyes hypertelorism hypertelorism hypertelorism hand postaxial polydactyly pre- and postaxial polydactyly postaxial polydactyly foot postaxial polydactyly pre-axial polydactyly postaxial polydactyly tibia hypoplastic normal hypoplastic thorax narrow normal normal perinatal death + inheritance ar ar ?ar (parental consanguinity) was stillborn had the digital malformations and a cleft lip and palate, identical to those seen in the surviving sibs. x-rays of the lower limbs of the surviving sibs showed the proximal metaphysis of the tibiae and fibulae to be wide and misshapen. the proximal epiphysis of the tibiae and the distal epiphysis of the fibulae were flattened. the x-rays of the sib who died were not available. temtamy and mckusick reported two patients who combined the features of the two syndromes. in the first, there was a median pseudo-cleft of the upper lip, multiple frenulae, a normal tongue with a single yellow nodule on the lateral border, poly- dactyly, and syndactyly; the infant died at weeks following an aspiration. this was probably related to a hypoplastic epiglottis thought to be character- istic of the majewski syndrome. x-rays of the tibiae were not shown. the second case had a small thorax, but survived despite respiratory problems in infancy. the limbs were short, particularly the tibiae which had rounded upper metaphyses. the facial features were compatible with either syndrome. the present case represents, perhaps, the best illustration to date of a patient falling midway between the two conditions. the normal thorax and prolonged survival are against the diagnosis of majewski syndrome, while the severe hypoplasia of the tibiae is against the diagnosis of mohr syndrome. the child may be regarded as either a mild example of majewski syndrome or an unusually severe example of mohr syndrome. the general tendency of these syndromes to 'breed true' is some- what against an explanation that suggests the two syndromes are not distinct. a third possibility is that the child represents a compound heterozygote for separate recessive alleles. the consanguinity in the parents is more in favour of homozygosity for a single abnormal allele within the family. a fourth possibility is that this child is homozygous for a distinct recessive gene defect, possibly disturb- ing a similar developmental pathway to that involved in the other syndromes. the consanguinity of the parents provides support for this possibility, though in view of the high level of inbreeding in the population from which this family originates, cousin marriage in the parents is of less significance. in each of these four explanations, it is implied that children with the features described in the present report have a recessive condition and that the parents face a i in risk of recurrence. m baraitser*, j burnt, and j fixsen* *the hospitalfor sick children, great ormond street, londont wcin jh; and tmrc clinical genetics unit, institute of child health, guilford street, lonidon wcin jeh. references gorlin rj, pindborg jj, cohen mm. syndrotnes of the head and neck. nd ed. new york: mcgraw-hill, : - . black il, fitzsimmons j, fitzsimmons e, thomas aj. parental consanguinity and the majewski syndrome. j med genet ; : - . temtamy s, mckusick v. the genetics of hand malforma- tions. birth defects ;xiv:no . spranger j, grimm b, weller m, et al. short rib-poly- dactyly (srp) syndromes, types majewski and saldino- noonan. z kinderheilkd ; : - . rimoin dl, edgerton mt. genetic and clinical hetero- geneity in the oral-facial-digital syndromes. j pediatr ; : . requests for reprints to dr m baraitser, clinical genetics unit, the hospital for sick children, great ormond street, london wcin jh. addendum since the submission of this report for publication, a second affected child has been born to these parents. the infant has the same clinical and radio- logical features, details of which will be provided in a future report. this development adds weight to the view that this syndrome results from a distinct autosomal recessive gene defect. case reports o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n f e b ru a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ we really do have the same goals: the push and pull of one community–academic partnership to support congolese refugee women we really do have the same goals: the push and pull of one community–academic partnership to support congolese refugee women jyotika saksena, shannon mcmorrow narrative inquiry in bioethics, volume , number , spring , pp. - (article) published by johns hopkins university press doi: for additional information about this article [ access provided at apr : gmt from carnegie mellon university ] https://doi.org/ . /nib. . https://muse.jhu.edu/article/ https://doi.org/ . /nib. . https://muse.jhu.edu/article/ community–academic partnerships in research and public health caregiver and older adults in every story. an aspect of the study that i did not anticipate, and fuels the research partnership we now have, is the lack of understanding of the amish culture from western healthcare providers. every story had an aspect of cultural miscommunications, financial or trans- portation based barriers that was not appreciated. this information made me realize that there is still so much i don’t understand. during this period i became pregnant, my parents retired and my personal caregiving role began to shift. normally you might think that these personal events might complicate the research rela- tionship. however, because of the unique oppor- tunity partnership with a community can have, my research partners are more than my research participants. we have more than an exchange of data and forms. in alignment with a key principle of participatory research, a sense of mutual growth pervades our research and our personal relation- ship. several of the older adults who were being cared for during data collection passed on during the study, and i was grateful to be included in their funerals. my son enjoys the friendship of other amish children born during the same time period, and i have new insight into the changing role of parent and child, as my own parent’s age. i did not anticipate how much engaging in this partnership would affect my own life. i am humbled to be in this research relationship and i am grateful that i can do this work with this community. acknowledgements. the fran and earl ziegler college of nursing, university of oklahoma, and the amish and mennonite community members that assisted in this work. funding. the research discussed in this article was funded by the d.w. reynolds center for geriatric nursing excellence at the university of oklahoma, fran and earl ziegler college of nursing and the jonas foundation. the opinions expressed in the article are those of the author.  we really do have the same goals: the push and pull of one community– academic partnership to support congolese refugee women jyotika saksena and shannon mcmorrow w e never expected a mundane conver- sation about university curriculum to blossom into a community based par- ticipatory research endeavor. however, looking back, it seems natural that an international relations faculty member who had worked with a refugee organization for years and a public health faculty member who used photovoice research with vul- nerable populations found common ground. the main purpose of our project was to assess the needs of congolese refugees coming into indianapolis in order to improve the ability of refugee resettlement agencies and public policy makers to provide effec- tive and, culturally appropriate services. photovoice methodology was intentionally selected to give the women voice through the photographs and subsequent storytelling sessions, thus making them active participants in the study and providing a firsthand view of needs in their immediate environ- ments through photos. our initial conversation led to envisioning a photovoice project with refugees, acquiring internal funding, securing an external grant associated with state public health funds, and utilizing photovoice to understand perceptions and experiences of health and integration among congolese refugee women living in indianapolis. our community partner was a non–profit orga- nization dedicated to refugee resettlement. from the outset, we strived to engage them in all facets of the project including choosing the specific refugee subpopulation, formulating research questions, recruitment, and implementation. we approached our partner with the idea of using photovoice to better understand integration and health of refugees and asked two questions: ) is this something of interest and utility to you? ) if so, which population of refugees do you feel would be most helpful to conduct photovoice with? their answer was yes and people fleeing the democratic republic of congo, narrative inquiry in bioethics • volume • number • spring iraq and syria were prioritized by the resettlement agency due to those being new refugee groups in the state. we agreed the focus would be on women pri- marily due to alignment with an existing women’s program at the agency. our partner was particularly excited about the use of photovoice methodology to engage and give voice to refugee women, particularly the newer groups like the syrians, iraqi and congolese that they had limited experience in serving. one curve ball that occurred was restriction of the study popu- lation to congolese refugees only due to the funding agency’s application of the u.s. office of manage- ment and budget (omb) definition of ethnic and racial groups. the omb considers iraqi and syrian women as caucasian and therefore do not count as minorities and had to be excluded from the study. this is a case where the omb definitions were not helpful in serving the greater needs of refugees or refugee resettlement agency and all parties involved were dismayed about the shift in our project. initially, external funding was secured in close collaboration with the former executive director. during the course of the project, that initial execu- tive director left the agency, so it was inherited by the next executive director. later, we worked closely with two assigned personnel to flesh out the details of the project including working with our univer- sity’s institutional review board (irb). since we applied for the funding together, the amount was split between us, with the majority going to our community partner. the funding was limited in that it did not pay for our time as researchers, but simply for materials and supplies to conduct the project. this becomes relevant because we engaged in conducting the research in addition to our regular teaching and service responsibilities as opposed to buying out any time for the project. one of several positive aspects of this partner- ship was generous investment of the community partner in terms of human resources, space, and transportation resources. they assigned their sole medical case manager to work as an interpreter for the photovoice project. this was written into the budget, but in the end she invested more time than she was compensated for. she was an invaluable resource as an insider of the community, ability to speak several languages and as a critical bridge in helping us recruit and retain the participants. it was also wonderful that we were able to use the space of the community partner to hold initial interviews. additionally, the partner used their connections to find another, third partner to donate community space close to the homes of most of our participants where we held all photovoice sessions. finally, the partner provided their bus to transport participants to and from photovoice meetings. this was instrumental for maximizing participation since the women did not have to rely on public transportation. while we agreed in principle to the common goal of serving the refugee population, we faced multiple challenges. we differed throughout the project about the level of priority and significance of the project for the community partner and the women, lack of clarity in the point person with the partner institution, and appropriate use of funding. though the new leadership appeared to be on board with the project, we felt the commitment to and context of the project was not sufficiently conveyed to staff assigned to work with us on the ground. we endeavored to communicate what we perceived as the relevance of the project for the day–to–day work of the agency, but we often missed the mark. sometimes, we felt perceived as stereotypical, leisurely academics conducting research. other times, it appeared our partner personnel felt grudg- ingly required to help us out. they would not hesitate to ask us to wait if they had other work and sometimes changed appointment times and days at the last minute. our perception was that the partner was not taking the project as seriously as us, viewing it as our research rather than a joint project. at different points, we were told that the project was taking too much time and commit- ment. this was disappointing to us because we thought the participatory process through which the agency had agreed the project was beneficial and had originally selected the priority population was evidence of their commitment. on the other hand, it seemed our community partner felt that community–academic partnerships in research and public health we did not sufficiently understand the day to day pressures of a non profit organization. while the project was important to them, serving their clients on a daily basis and dealing with emergency situa- tions was clearly, understandably, their priority. a project that would help their clients in the future, therefore, could wait. there were multiple challenges throughout the project that served as “ah ha!” moments and lessons learned for future work with partners. one such challenge was lack of a central point person from the partner agency. it was originally the “baby” of the first executive director who left for another organization before the project got off the ground. while the new director was interested, there sud- denly were multiple things to manage such as adapting to the new position, and therefore, atten- tion was clearly diverted. the previous executive director had committed to be the main point person, but the new director could not focus on the project in the same way. we were assigned two different personnel to work with us, an intern familiar with photovoice and their medical case manager who was also an interpreter. the intern lived in another city and was available just two days per week and the health navigator had to divide the responsibil- ity between what she saw as her “real job” that she was paid for at the organization and the research project, which felt like extra work for her. this meant that she was often unavailable and did not hesitate to back out if she had other commitments. though the agency received funding to support part of her salary while assisting with the project, the intense time period of work for the study occurred on top of the regular workload and she justifiably, felt overworked. while we kept ourselves flexible outside of our regular university responsibilities and schedule, it became frustrating to constantly negotiate her availability with her or her supervi- sor. it was not clear who our point of contact was in the organization. we felt that partnership implied a commitment from our community partner but our sense was that they were doing us a favor by partnering with us. this became apparent from the way the assigned personnel dealt with us as well. there was friction at different points due to their assumption that we did not know how to interact with refugees and unnecessary negotiations about resources like access to the bus, availability of drivers, or delivery of food without checking with the leadership struc- ture. in frustration and trying to stick to a tighter timeline, we sometimes went to the leadership or executive director to get what we needed, which in turn led to more friction. we addressed this through conversations with the agency leadership and felt encouraged by a change and more amiable interactions during the remainder of the project. the assigned representatives from the resettlement agency became much more responsive and respect- ful of our requests regarding the project. one major lesson learned was that we need to better communicate in advance about how both parties plan to use grant money. our understanding was the agency would hire an additional interpreter or personnel. the community partner, like most nonprofits, wanted to use the grant money as an additional resource to support the organization. as a result, no new personnel were hired to assist with the project. existing employees had to handle both their assigned work and additional work of the proj- ect. therefore, for the duration of the two months that the project lasted, they were constantly torn in different directions. the organization did not want to pay them overtime, so there were restrictions on how many hours they could work. the employees wanted, and we felt they deserved, time off or extra pay to do the extra work for the photovoice proj- ect. when they got neither, there was resentment towards us and the organization. we learned to keep in mind that for nonprofits, funding is always an issue and therefore, they are always going to be strategic about spending money. this means that it is up to us to ensure that we have a clear idea of how much time and commitment our project will require and convey it to our partner institution. we did not know how much time the project might take and therefore did not sufficiently communicate to our community partner how much time and resources the project might require. this led to the partner being overwhelmed to some extent by the obliga- tion of the project. narrative inquiry in bioethics • volume • number • spring another key lesson learned was that prior to commencing the project, we needed to better com- municate with the partner to identify one desig- nated and committed staff person to be in charge of the project, to be our main point of contact, and responsible for all aspects of execution of the project. we spent a lot of time seeking input from various supervisor level staff and the executive director to negotiate the availability of the personnel, as well as the use of funding. at several times the personnel assigned to us did not know how to set their priori- ties. it also left us confused at times and questioning the commitment of the agency to the project. a final lesson we will take with us for future partnerships is the need to build in more flexibility to project timelines to deal with unexpected situa- tions, particularly when working with vulnerable populations such as refugees. for the women we were trying to engage, time was of the essence due to the urgency of trying to maintain consistent jobs. this means that the longer we took to implement our project, the more chance there was of losing par- ticipants. due to many factors such as our inexperi- ence in working with a refugee resettlement agency on a project of this nature, an intensive and lengthy irb review, and the unusual pressures faced by our partner organization, the timeline for our project dragged on longer than anticipated. luckily, due to the extraordinary assistance from our interpreter/ research team member and unusual flexibility in our schedules we retained most participants. however, it is clear that in the future we need to build in the longer timeline as an expectation. overall, during the course of the project our part- ner organization experienced extraordinary change with sudden change in top leadership, engagement in a lawsuit against the state government, and planning an office move; all of which had impact on the project. in the end, we would absolutely do the project again with the same partner. we proudly worked together to share some of the photos and narratives at a local world refugee day event last summer. the friction that occurred during imple- mentation faded away into satisfaction and pride in the shared accomplishment of giving voice to con- golese refugee women in our community. moving forward, we foresee reuniting with our partner to apply our lessons learned about how to better work together as the united states moves into times of great uncertainty and potential threat to refugees. acknowledgements. we are grateful for the cour- age and commitment of our participants as well as thankful for the strong collaboration with our community partner agency. we are also grateful to the creek community center for providing us space to conduct our meetings. funding. university of indianapolis, interdisciplin- ary grant and indiana minority health coalition state master research plan grant, / .  dual relationships in specialty care: reflections from the field lewis raynor and amy penkin introduction the creation of the oregon health & science uni- versity (ohsu) transgender health program (thp) was a grassroots effort involving transgender and gender nonconforming (tgnc) community mem- bers, local organizations serving the tgnc commu- nity, clinicians, administrators, and researchers. the thp, which launched in january , offers com- prehensive, affirming, and competent healthcare to tgnc individuals across their lifespan. in the thp had over referrals for tgnc patients and in that number grew to over referrals. amy penkin is a cisgender, lgbtq community member and licensed clinical social worker who was hired as the thp program coordinator in . her duties include, but are not limited to, workforce education, assisting patients with healthcare naviga- tion, tgnc policy development, clinical alignment of departments offering gender affirming care, and community engagement to ensure program devel- opment and services align with community needs. during the first year amy also helped establish a thp volunteer program to ensure the program tilburg university in the shadow of christ? on the use of the word "victim" for those affected by crime van dijk, j.j.m. published in: criminal justice ethics publication date: link to publication citation for published version (apa): van dijk, j. j. m. 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https://research.tilburguniversity.edu/en/publications/ cc b - e- c a- bf -d c cd a e winter/spring  when pressed, english speakers are inclined to sup- pose that the word “victim” is derived from the latin verb vincere, which means to conquer. in this mis- taken interpretation “victim” refers to the opposite of the victor: he who lost the battle (the loser). in reality, “victim” is not a derivative of the verb vincere but of the unrelated latin word for sacrificial object, victima.  “victim” is, for example, used in latin versions of the bible to denote a sacrificial animal. the victim is someone or something slaughtered and offered as a sacrifice to the gods. in june i heard george fletcher speaking about the etymology of “victim” at a seminar on victimol- ogy and human rights held at the hebrew university in jerusalem. he discussed the original meaning of the word and the universality of the victim label as a name for those affected by crime in the languages of christianity, judaism, and islam. in modern hebrew, the word used for victim is korban, originally meaning both the sacrifice and the sacrificial animal. in arabic, the word for victims of crime or terrorism,  udhiya, also refers to both the sacrifice and the sacrificial ani- mal. the same root concept is used in the name of the important islamic holiday of the sacrifice. fletcher also presented a tentative explanation about how the victima concept was adopted into the abrahamic lan- guages. this explanation is discussed in more detail in his recent study, the  grammar  of  criminal  law, in his chapter on language. in the following essay i will comment on fletcher’s explanation for the use of the word “victim” and compare it with my own hypoth- esis elaborated in a public lecture at the university of tilburg, the netherlands, around the same time. in a final section, i will briefly discuss the implications of these etymological analyses for a better understand- ing of the role of the victim in criminal procedure, an- other topic raised in fletcher’s book. as fletcher rightly points out, victima is used to de- note those harmed by crime not only in english but in all romance languages, such as french and ital- ian (victime  and vittima,  respectively). the sacrificial connotations of “victim” may be hidden for most na- tive speakers of english, french, or italian, but this is not the case in germanic languages. in german, for instance, the victim is called das opfer, meaning the sacrifice; and in swedish, brottsoffer, the sacrifice of the crime. in icelandic the word used for victim is fo- ernarlamb, meaning the sacrificial lamb. in dutch the word is slachtoffer. the dutch term means, literally, the butchered, sacrificial object. the identification of crime victims with sacrificial objects is not limited to english, romance, or germanic languages. in rus- sian (zherta), hungarian (aldozot), and modern greek (tema), to name just a few, crime victims are also re- ferred to as sacrificial objects. a brief etymological excursion therefore confirms that the word used for in the shadow of christ ? on the use of the word “victim” for those affected by crime jan van dijk jan  van  dijk  is  professor  of  victimology  and  research  director  at  the  international  victimology  institute  (in- tervict), tilburg university, the netherlands. i am not a victim simply because other people say i am. other people cannot make you a victim, only you can do that . . . i want to be taken seriously and for the events of my case not to be swept under the doormat. — natasha kampusch introductory remarks in the shadow of christ?  /  criminal justice ethics those affected by crime refers in all european lan- guages to animals killed in a religious ritual as part of the worship of god. “victim” is replete with deep religious meaning in all these languages. the similar adoption of the victim label across all these different languages is the more puzzling since it seems to be a characteristic of modern versions of these languages. in classical greek, latin, and he- brew, the wider use of the victim label for those af- fected by crime is non-existent. in the ancient world, nobody called a crime victim a sacrificial object. neither is the label common among other language groups. in chinese, the word for sacrificial animal (jipi) is definitely not used for those persons affected by crime. the latter are described by the characters for “person,” “harm,” and “in a passive sense”; in other words, they are neutrally described as the per- son-receiving-harm (bei hai ren). likewise in japanese, a person against whom a crime has been perpetrated is called a receiving-harm-person. in this factual ter- minology, all connotation of sacrifice is absent. why then do modern languages of the western and arabic world unanimously call those affected by crime sacrificial persons rather than opt for a more neutral, legal term such as “wronged persons”? why was a term with such cruel as well as holy connota- tions chosen to refer to ordinary persons harmed, in- jured, or wronged by ordinary crimes? i agree with fletcher that the use of the term is puzzling—myste- rious even—and that this linguistic phenomenon has attracted surprisingly little attention from scholars. also, my interest in the topic goes beyond etymologi- cal curiosity. a better understanding of the reasons for the adoption of this word might offer new insights into the prevailing attitudes toward crime victims in western culture, including prevalent arguments in the philosophies of criminal justice. jan van dijk  /  a talmudic interpretation i agree with fletcher that the victim label is not applied in many languages to those affected by crime simply because such persons can somehow be regarded as sacrificial persons in a literal sense. clearly, they are not animals and they have not been offered to the gods. they have been killed or hurt for selfish reasons of the perpetrators. but how then to explain the extensive linguistic adoption of the victima label in a figurative sense? fletcher, who asserts that many of our legal sen- sibilities have their roots in biblical wisdom, starts his inquiry with a reflection on the double meaning of guilt in the torah. the book of leviticus provides detailed instructions for the proper execution of sacrifices in the temple to atone for both sins and crimes of guilt. through the sacrifice of a korban, believers can achieve atonement. the instructions include those for the day of atonement, when a goat is to be ritually killed by the high priest and another goat is to be driven into the desert laden with the community’s sins. this ritual is the etymological source of the socio-psychological phenomenon of scapegoating (redirecting social ag- gression against an outsider). after a brief elaboration on the meaning of guilt and sin in old hebrew, fletcher returns to the linguistic phe- nomenon at issue, namely, that “the word for ‘victim’ in a criminal trial is the same as for the ‘victim’ in the first five chapters of leviticus” [ ]. it is important to note that fletcher looks at the adoption of the victim label in the context of a criminal trial. this means that he looks for the possible rhetorical functions of the label within a legal argument. he tries to understand how the label makes sense in the discourse of criminal justice. it is far from obvious, however, that the label was adopted into the criminal justice context and thereafter entered into colloquial usage. the sequence of its adoption might have been the other way round (a colloquial term that subsequently entered legal terminology). i will return to this subject shortly, but first i will present fletcher’s own—and by his own admission, specula- tive—thoughts on these matters. according to fletcher, the text of the torah offers two possible explanations for the adoption of the term “victim,” or the korban label, in a criminal trial. in the first explanation, the label refers to the story of abraham’s aborted sacrifice of his only son, isaac. in genesis, god instructs abraham to bind and subse- quently kill his beloved only son. in fletcher’s view, the story expresses symbolically the belief that human sacrifices are a matter for divine sovereignty, strictly forbidden to human calculation. only god is allowed to designate persons as sacrificial objects. therefore, someone who takes the life of another person usurps a function reserved for divine sovereignty. such a perpetrator commits blasphemy, the ultimate crime. fletcher theorizes that those affected by crime are metaphorically called victims in criminal trials in order to underline the horrible guilt of their offenders. winter/spring  fletcher is himself not fully convinced by this first explanation. he therefore develops a second explana- tion, more or less along the same lines, but this time based on leviticus. according to the instructions in leviticus, the sacrificial goat must be absolutely pure in order to be an appropiate carrier of the sins of the israelite community. the scapegoat, in other words, needs to be without blemish. christian theology re- produces this jewish imagery. the figure of christ is presented as the fundamentally innocent “lamb of god” who absolves humankind of its sins. according to fletcher’s second interpretation, the person affected by crime is called an intrinsically innocent victim in order to underline the guilt of the offender. the use of the victim label is a rhetorical gesture to portray one party as intrinsically innocent and the other as intrin- sically bad. fletcher follows up this point with a brief discussion about the dichotomous logic of criminal justice: the blame for a crime is typically, and entirely, laid upon one of the two parties, regardless of any pre- cipitative behavior practiced by the person believed to be trespassed against. the victim label suits this type of legal reasoning very well. in the shadow of christ?  /  supporting evidence support for fletcher’s first interpretation can be found in conventional theological wisdom that the prohibition of human sacrifice is at the core of the old testament, the new testament, and the qur’an. the god of the abrahamic religions rejects human sacrifice and in this light a person who kills a fellow human being contravenes the shared core values of the three religions. support for fletcher’s second interpretation can be found in a classic of critical criminology, nils chris- tie’s article on the “ideal victim.” in this essay, the norwegian criminologist argues that conventional criminal justice maintains an idealized and totally unrealistic vision of the victim as a frail, innocent old lady in order to portray the offender as a totally evil person. in his eyes, victimologists who argue for more victims’ rights contribute to a tendency within criminal law toward offender bashing. other writ- ers have repeated this critique—for example, dutch criminal law professor ybo buruma, who argues that the growing role of the victim in criminal procedure “demonizes” the offender. on these grounds, chris- tie and others have argued to replace criminal trials with supposedly more humane restorative justice ap- proaches outside criminal justice. fletcher’s inter- pretation of the use of the victim label adds fuel to this fundamental criticism of victim-friendly reforms of criminal justice. in fletcher’s view, victim-friendly reforms may result in more repressive criminal poli- cies as unintended side effects. furthermore, the very use of the word “victim” in the context of criminal law is actually meant to produce such effects. in other words, fletcher’s analysis confirms the worst fears of the critics of victimology. finally, some indirect support for fletcher’s theo- ries can be found in the main etymological diction- ary of the dutch language, het woordenboek der ned- erlandsche  taal. this dictionary includes one of the oldest known sentences in which slachtoffer is used to denote someone affected by a crime: “murderer! see there: your victim.” (“moordenaar! daar ligt uw slachtoffer.”) in this citation from the nineteenth cen- tury, the term “victim” is clearly used to shame and accuse the offender. it presents a useful illustration of the reasoning that, according to fletcher, has per- suaded so many languages to adopt “victim” in its current, figurative sense. conflicting evidence in my criticism of fletcher’s hypothetical explana- tions, i want first to point out some logical problems. according to fletcher, a murdered person is suppos- edly called a victim in order to make the statement that the offender has usurped the role of god and/or is, in contrast to the innocent victim, full of guilt. my main objection is that the word “victim” in a bibli- cal context suggests quite different associations from those imagined by fletcher. in the old testament, the word “victim” is used first and foremost in connection with the sacrifices in the temple and not the story of abraham. in fact, as fletcher himself observes, isaac is not called a korban or victima in either the hebrew or latin versions of the old testament. i can add to this criminal justice ethics that in the qur’an the saved son of abraham is not called an udhiya either. furthermore, for jewish and christian readers of the old testament, those who kill the sacrificial animals are priests. this association puts offenders in a strangely favorable light. in the biblical context, calling the killed person or animal a victim does not incriminate the killer as a bad person. the use of this label for the affected party, on the con- trary, portrays the actor as someone who acted in the interest of the community and out of respect for god. to make someone or something a victim is a deeply religious and morally outstanding act. a sacrificer is the antagonist of a criminal. in my opinion, fletcher’s interpretations, clever as they are, ignore the immedi- ate and dominant associations of the term “victim” in biblical language and presuppose an implausible theological and legal sophistication on the part of or- dinary users of the word. these objections are in my view confirmed by the linguistic fact that, to my knowledge, there is no place in any version of the torah, old testament, or qur’an where the word “victim” is used to identify a person affected by a crime. if the victim label has such ob- vious, indirect associations with blasphemy or the guilt of the offender in a biblical context as fletch- er’s hypothesis assumes, one would expect at least some translators to have used this word for those af- fected by homicide or other crimes. in contrast, such persons are uniformally described in neutral terms in these religious texts—commonly as “the beaten ones.” fletcher observes this complication himself: “the linkage does not appear in the bible, which nev- er mentions the victim korban as a victim of crime” [ ]. this fact seriously undermines the validity of his argument that the biblical context suggests such associations to the extent that they have entered col- loquial language. i argue instead that these founding religious documents promote an altogether different set of associations that almost preclude the use of the victim label in the broader sense. another fact-based argument against fletcher’s hypothesis can be directed against his underlying assumption that the adoption of the victim label for those affected by crime occurred primarily in the con- text of criminal justice. in my view, it is one of the strik- ing characteristics of the broader, figurative meaning of the word “victim” that it does not, according to the dictionaries consulted, derive from legal language. it is clearly a word that first appeared in colloquial lan- guage. to my knowledge, the word “victim” does not feature as a technical legal term in the common law, the napoleonic, or the germanic systems of law. in fact, it is only because of the victims movement in the past three decades that “victim” has incrementally entered legal discourse. in the common law countries, the use of phrases such as “victim impact statement” or “victim restitution or compensation” are recent in- novations. in other european legislation, “victim” is rarely if ever used. the other party of a crime is usu- ally described in procedural law as the person who claims civil damages. if “victim” in a broader figurative sense originates from colloquial language, one could still argue that ordinary people have chosen the label for those af- fected by crime in order to express their angry feel- ings about the guilt of the offender. however, euro- pean dictionaries reveal that the broader figurative meaning is not specifically used for those affected by crime but for those affected by any event that causes serious harm, including disasters and accidents. in some languages the use of the term “crime victims” seems to be a relatively late phenomenon. any link between the use of “victim” in a figurative sense and the wish to stress the culpability of the actor is simply absent in colloquial language. those victimized by so-called acts of god for which nobody is to blame are called victims as well. this linguistic fact alone puts fletcher’s hypothesis on very shaky ground. jan van dijk  /  a phenomenon of late christianity fletcher acknowledges in his book that his interpre- tations do not by themselves clarify how exactly the broader—supposedly accusatory—use of the word “victim” for those affected by crime found its way into so many languages. he writes, “why and how this association between the two senses of victim- hood came into being remains a mystery” [ ]. in his final comment on the topic, he observes: “the as- sociation probably developed in the early christian period when jesus came to be called korban of the crucifixion, and was probably understood to be a kor- ban or victim in both senses. he was at once the sac- rificial lamb to redeem humankind from the fall and the victim of the crime of torture and biased prose- cution” [ ].  if the roots of the broader meaning of the concept lie in christianity, fletcher’s hypothesis winter/spring  must be revisited against the background of the story of the gospel. if those affected by crime are called vic- tims because of their resemblance to jesus christ, the hypothesis that this is done to indirectly accuse the perpetrators becomes far-fetched. this would imply that christian and jewish speakers started to refer to those affected by crime as victims in order to put the perpetrators of crimes in the role of the jewish accus- ers of jesus! in my view the accusatory hypothesis simply loses any sense if the victim label is supposed to have christian roots, most notably in the context of judaism. in the shadow of christ?  /  etymological explorations i will now turn to etymological evidence about the origins of the victim label contained in dictionaries. in the latin version of the bible, the vulgate, sacrificial animals are described as victima. in the oldest trans- lations of the bible in english, sacrificial animals are not called victims but sacrifices (from the latin sacer  facere, meaning to make sacred). the word “victim” was not used for the simple reason that at the time of these early translations “victim” did not exist in the english language. according to the oxford english  dictionary ( ), “victim” appeared for the first time in the english language in the so-called rhemian bi- ble, written in the sixteenth century by scholars at the english college in rheims. it was initially used only as a name for sacrificial animals. in english, the first recorded time “victim” seems to have been used for a human person was in . in that year it was used as an honorary name for jesus christ, the crucified, in a translation of the new testament. christ was called the expiatory victim: the person who through his vic- timhood redeemed mankind. the oxford english dic- tionary also documents the first use of this figurative speech for victims of disasters and crimes as occur- ring in . the broader use of the word surfaced for the first time in colloquial (that is, non religious and nonlegal) english not more than two centuries ago. according to the preeminent dictionary of the french language, le  grand  robert, the oldest docu- mented figurative use of “victim” for a human per- son is also a reference to jesus christ. it appears in a play by corneille, published in , in which cor- neille calls jesus christ victime volontaire. the word “victim” was used in other french texts to refer to scapegoats, but was not used for those affected by crime or disaster before the eighteenth century. as in english, the figurative concept of a victim emerges first as name for jesus christ and acquires its broad- er use much later. finally, in dutch, one of the old- est documented citations of slachtoffer in a figurative sense for a human being is from . in that year, the religious author, gnapeus, published the follow- ing sentence: “the blood of our lord jesus (who had become a victim on behalf of us poor sinners) that cleanses us from our sins.” the early appearance of the word “victim” as a descriptor for jesus christ in the dutch language suggests that this usage may have stemmed from protestant theology. according to andré lascaris, a contemporary dutch catholic theologian, john calvin replaced the softer catholic imagery of god’s lamb, who voluntarily sacrificed himself to redeem mankind, with more blatantly sacrificial imagery in his major publication institutio  christianae  religionis  ( ), written in classical lat- in. here calvin explains that god’s only son had to die to atone for human sinfulness. he writes, “in sum, the scripture consistently tells us that the arrival of god’s son among humans and also the command thereto from the lord, served no other purpose than to make him a victima in order to reconcile us with the lord.” calvin’s sacrificial account of the crucifixion is interpreted by andré lascaris as a psychologically inspired response to the growing uncertainties of the times, spawning the collective need for a fresh scapegoating story. in the same period, catholic theo- logians such as thomas aquinas had also started to stress the sacrificial aspects of the crucifixion. how- ever, calvin may have been inclined to come up with more severe sacrificial imagery to buttress his criti- cism of the corrupt practices in the catholic church. by emphasizing the harsher aspects of the fate of je- sus, he perhaps wanted indirectly to attack the prac- tices of easy redemption in the catholic church of his time. calvin’s use of the latin word victima for christ in is at any rate one of the oldest instances of the use of the figurative victim label known in european languages. criminal justice ethics thus in english, french, dutch, and academic latin, the first use of “victim” was reserved for jesus christ and appeared in the sixteenth or seventeenth century. its broader use as a word for those persons affected by crime appears much later in these languages—in the late seventeenth or early eighteenth century. why did it take christian cultures another two centuries to assign the victim label to other suffering human beings, including victims of crime? the very fact that “victim” was initially reserved as a special characterization of jesus christ would probably have impeded its ready adoption as a name for human beings. its delayed adoption as a colloquial name for those affected by disaster, accident, or crime seems to have been de- pendent on a series of cultural transformations. the first important transformation is probably the gradual humanization of the figure of christ. the first steps in this direction were the personal portraits of christ (and mary) in italian renaissance art. furthermore, millions of ordinary people must have been exposed to gruelling depictions of the suffering of christ in passion plays and other forms of christian-realist art. incrementally, the image of christ as a suffering human being would have been impressed upon the collective western mind. a much later, and telling, example of the identification of the public with the passion of christ is the painting by pablo picasso, ecce homo, which shows a self-portrait of the painter superimposed on the face of the crucified christ. fletcher himself alludes to the double meaning of defining christ as victim. christ was, according to fletcher, “at once the sacrificial lamb to redeem humankind from the fall and the victim of the crime of torture and biased prosecution” [ ]. he was, in other words, both a mythical figure and a scapegoat. however, this second image of christ as a human vic- timized by torture and biased prosecution presupposes a rudimentary understanding of the phenomenon of scapegoating. according to rené girard, the psycho- logical mechanism of scapegoating was not widely understood before the seventeenth century. it was only thereafter that persons killed in sacrificial stories, including the story of the crucifixion, were recognized as persecuted scapegoats and could become a symbol for other suffering people. although causality is difficult to prove, the adop- tion of the word “victim” to describe persons affected by disasters and crimes was probably the result of the gradual humanization of images of the crucified christ and the growing awareness of the psychological reality behind sacrificial stories. these developments have gradually dissociated “victim” from its original christian connotations of holiness. only then could those affected by crime or disaster be seen as victims, in the sense of those suffering innocently from bad luck or from other people’s criminal behavior. label- ling those wronged by crime as victims appears to be a modern development of christian ideology. to sum up, the use of the victim label for victims of crime is preceded by its adoption as a special name for the crucified christ in christian theological writ- ings in the sixteenth and seventeenth centuries. in these texts a more overtly sacrificial interpretation of the life and death of jesus is presented. the subse- quent development of the victim label in colloquial language as a term for victims of crimes and disasters has been catalyzed by the incremental humanization of the stories of the passion of christ and a growing understanding of the psychological mechanisms of scapegoating. through ordinary people’s identifica- tion with the deep suffering of christ, the concept of victimhood has found its way into the popular cultures and languages of the west. the idea of victimhood has developed out of christian theology and permeated everyday language. although the appearance and use of the concept in individual languages needs further detailed analysis, the general history of the phenom- enon has been established here. what remains unresolved is the development of the victim concept in modern hebrew and arabic. without the christian imagery of the crucifixion, it is hard to understand how a word for sacrificial animals can be- come a colloquial term for humans affected by crime in the cultural sphere of world religions that categorically reject human sacrifice. were it to be confirmed that korban in its broader sense did not appear in modern hebrew before the seventeenth or eighteenth century, it is possible that it found its way into the language due to the influence of one or more european languages. such adoption, however, remains curious, considering the jewish denial of the story of the gospel. the use of the arabic word udhiya for victims of crime is more puzzling still, considering that islam, unlike judaism, acknowledges the historicity and religious significance of jesus christ as a prophet but emphatically denies his crucifixion. the image of the suffering christ at the cross is totally absent from islamic theology and it is hard to imagine how such an image can have led jan van dijk  /  the passion of christ winter/spring  here i will briefly elaborate on the possible impli- cations of the victim label for the role of victims in criminal procedure, including its implications for the provision of special rights or services for crime vic- tims. as explained, the current victim label originates from popularized christian theological imagery. its primary association with deep suffering elicits the emotional response of compassion. various authors have characterized compassion as the core value of christianity. girard goes so far as to call the chris- tian god the “god of victims.” in light of the chris- tian core value of compassion for the vulnerable and weak, one would expect the use of the victim label for persons hurt by crime to have led to an outpour- ing of compassion for those so labelled. in one of his later books, girard heralds victim care as the quin- tessential operationalization of christian morality. victim care, girard asserts, is practical christianity at its best. he reminds us that from the medieval period onward, christian churches have spearheaded the build-up of extensive provisions of care and protec- tion for the vulnerable and weak, including prison- ers. this assertion is historically well-documented. but this history of charity is remarkable in that it has never included the care of victims of crime. parallel to the gradual establishment of the fledgling welfare state, the position of crime victims in criminal pro- cedure has become more and more marginal. this incremental weakening of the position of victims within criminal procedure induced early victimolo- gists such as stephan schafer to speak of the golden age of the victim in medieval europe. everywhere in modern christian europe the procedural rights of victims have been incrementally reduced, in contrast to the permanency of these rights in legal systems that apply versions of islamic sharia law. even more striking, in no western country has any church-based organization played a prominent role in the victim- friendly reforms of welfare and criminal policies in the past three decades. those affected by crime are in my view called vic- tims because their suffering resembles that of the fig- ure of christ. surprisingly, however, this honorary label has not brought any favors for those so labeled. on the contrary, crime victims have for centuries been marginalized in criminal procedure and ex- cluded from the modern welfare state and voluntary work. such neglect of the interests of crime victims in countries with christian cultural roots is paradoxical. it invites a question as to what theological arguments or other forces within christian institutions have hin- dered christianity from extending christian compas- sion to the victims of crime? in the shadow of christ?  /  implications of the victim label for those persons so labelled the countervailing command of forgiveness as noted, christian identification with the passion of christ elicits an emotional response of compassion. this identification could also easily elicit a strong emotional response of anger and vengefulness. in christian theology, however, the latter response is reined in by the moral command to forgive. at the time of his crucifixion jesus stated about his tormen- tors: “forgive them, lord, because they do not know what they do.” with these famous words, jesus practiced what he had preached all his life. in his ser- mon on the mount he preached forgiveness, symbol- ized by the turning of the other cheek to one’s ag- gressor. in the old testament “vengeance is mine” is still the warrior’s cry of the god of israel. in the new testament it has come to mean the opposite, for vengeance is claimed as the exclusive prerogative of the lord. vengeance is mine becomes a command- ment to believers to abstain from retaliatory action. the new testament is replete with exhortations to forgive those who sin against one—when necessary, even seventy times seventy. in the torah and old testament, forgiveness is highly valued but it is dependent on the payment of fair compensation to the victim. in the new tes- tament, victims are expected to offer it uncondition- ally. in the qur’an, the retributive principle of an to the broader use of udhiya in arabic. possibly, other stories and images of sacrifice within the islamic tra- dition have somehow inspired this modern use of the term. for the moment, the etymology of the arabic victim label remains a mystery. we will return to this puzzling issue in the concluding paragraph. criminal justice ethics eye for an eye is fully recognized, though forgiveness is recommended as an admirable option. where for- giveness in islam is welcomed as a form of charity that may bring victims the reward of atonement, it is nothing less than an absolute moral duty for chris- tians. the refusal to forgive is, in fact, the only unfor- givable sin. unsurprisingly, wrath, the denial of for- giveness, is one of the seven deadly sins in catholic morality. in dante’s divina  commedia, special places in hell are reserved for the envious and the wrathful. they have to suffer in the fourth circle. even deeper down in hell, especially gruelling places are reserved for those who have actually murdered their attack- ers by way of retaliation. and before descending to this most horrible part of hell, dante encounters the furies, the avenging goddesses of ancient greece. there is no mercy for the merciless in dante’s hell. calling those affected by crime “victims” after the figure of christ implies that those so labelled ought to refrain from retaliation. for example, lascaris ap- peals to victims of crime to follow the example of jesus christ literally and to forgive their offenders. imitating jesus, christians should recognize their of- fenders as victims like themselves and therefore of- fer them unconditional forgiveness. in this purest of evangelical views, victims and offenders should em- brace each other as fellow victims of a higher order. stitutions toward victims of crime. because victims of crime may very well be angry and vengeful, sup- porting them is a morally hazardous undertaking. by reaching out to victims of crime, one may become an accomplice to unchristian thoughts and perhaps even activities. christian churches have for centuries collected alms for prisoners but never for victims. in recent times, they have stayed away from initiatives to provide specialized services to victims. by way of an alternative, following the call of norwegian criti- cal criminologist nils christie to design alternatives for victim-centred criminal justice, they have actively supported reconciliation between victims and offend- ers. as george pavlich observes: “the now pervasive values of restoration, healing, reintegration, forgive- ness and compassion within restorative governmen- talities often derive from theological roots. church- based restorative justice initiatives seemed to align particularly well with community mediation-panels and victim-offender reconciliation programs.” strik- ing examples of this are the many projects for restora- tive justice initiated and maintained by the mennonite churches in the u.s. and canada, theological cousins of the amish. in other words, if christian churches presently reach out to crime victims, it is not usually for the sake of the victims but with a view to reconcil- ing them with their offenders. the “ideal victim” for those advocating victims’ rights in criminal justice may be a social construct that serves an ideological agenda of punitiveness. but the alternative images of victims put forward by christie and other advocates of restorative justice should not be taken at their face value either. the “ideal victim” of restorative justice is someone who, according to chris- tian belief, carries his suffering gracefully and offers his attackers unconditional forgiveness. such forgiveness serves the interests of both community and offender but not necessarily the interests of the victims them- selves. in many cases, the expectation of forgiveness is unrealistic considering the seriousness of the crimes and the negative or insincere attitudes of the offenders. in such cases the command of forgiveness puts a heavy additional burden on the victim. for many victims, the example of jesus christ that lascaris would urge them to follow is psychologically out of reach in the early stages of coping—sometimes even permanently so. many crime victims may see forgiveness as an impossible demand and feel guilty about their failure to live up to it. even worse, their social environment, sensing their failure to forgive, may forsake the soli- darity and support that they need and deserve. in this bleak scenario, the labelling of those affected by crime as victims in a christian sense sets them up as poten- jan van dijk  /  calling those affected by crime “victims”  after the figure of christ implies that those  so labelled ought to refrain from retaliation. in october , members of the devoutly christian amish community in the u.s. experienced the horrific shooting of a group of school children by a mentally disturbed man. days after, they were seen embrac- ing the offender’s widow and offering forgiveness on national tv. when questioned about the foundations of these extraordinary acts of forgiveness, an amish spokesperson said they were simply following the ex- ample set by christ as described in the gospel. as a cultural symbol for crime victims, jesus christ does not just stand for innocent suffering. his image also stands for meekness and mandatory forgiveness, or, in the german words of the amish, for gelassenheit in the face of misfortune. christianity offers charity to victims on the condition that they sincerely forgive their offenders. this theological position, it seems, underpins the ambiguous policies of christian in- winter/spring  tial sinners. the label may then be an impediment to succesfully coping with their pain and suffering. in the case of victims of sexual abuse, for example, the com- mand of forgiveness in amish communities is known to exacerbate existing feelings of shame and self-blame among abused women and girls. the emblematic christian forgiveness practiced by the amish also sheds light on the curious mar- ginalization of the crime victim in western law. the expectation that victims must forgive their offenders explains such marginalization. a forgiving victim has no role to play in criminal proceedings other than to make a plea for mercy toward the offender, a plea that the prosecution may deem unhelpful. if the victim is not forgiving, his participation in the proceedings would contravene christian morality. in both cases exclusion of the victim from criminal proceedings seems justifiable from a christian perspective. for fear of acting revengefully, members of the amish communities are reluctant to take part in criminal tri- als and sometimes refrain from reporting criminal in- cidents to the police or to testify. the christian value of forgiveness fundamentally denies the moral right of victims to be angry and seek gratification for their emotional need to see their victimization avenged. it therefore presents a strong intuitive justification for reducing to an absolute minimum the victim’s rights in criminal procedure. in the shadow of christ?  /  the victim as a secondary scapegoat, or why the label fits so well as discussed above, the crucifixion of christ can be understood as an instance of scapegoating in a theo- logical sense (collective redemption through the self- sacrifice of christ) and as scapegoating in a more mundane, psychological sense (an instance of anger and hatred within the community vented at an in- nocent outsider). in both interpretations, the figure of christ fulfills the typical role of the sacrificial ob- ject who brings peace to the community through his meek suffering. in the view of girard, primitive com- munities restore a broken social peace through ritu- ally sacrificing people, such as foreigners or orphans, who are not only incapable of defending themselves but also incapacitated from arranging any form of re- taliation. victims in sacrificial rites are supposed to plead guilty to alleged sins and forsake any thought of retaliation. the story of the crucifixion places the figure of christ in exactly this position of chosen de- fenselessness. in his polemical tirade against chris- tianity, nietzsche portrays christ’s behavior at the cross in the following terms: “he doesn’t resist, he doesn’t put up a fight, he doesn’t lift a finger to avoid the worst, in fact, he provokes it. . . . and then he is praying, suffering, he loves together with or in the ones hurting him.” in the context of christian culture, victims are chal- lenged to follow in the steps of christ at the cross. they are expected to play a healing role in the after- math of the crime. the label of victim invites them to render a service to the community by relinquishing their natural right to seek vengeance. by labelling them as victims, those affected by crime are forced into a passive role. they are prohibited from engaging in retaliatory action and, until recently, were expected to remain silent in court about their suffering and anger. like victims of scapegoating rituals, they are incapaci- tated from arranging any form of retaliation. crime victims, then, are victims in a double sense: they have been damaged first by the offender and second by society’s institutional response to their vic- timization—a response that restricts their freedom to arrange a revenge. the label of victim, with its dual connotations of suffering and non-retaliation, seems then particularly appropriate for those affected by crime. by calling them victims, we acknowledge their suffering while at the same time restraining their vengefulness. the application of the victima label to those harmed by crime is a linguistic stroke of genius. this may well explain why all european languages have opted for this loaded label for those affected by crime. this speculative interpretation also explains why the concept did not, contrary to fletcher’s as- sumption, originally emerge as a legal term in crimi- nal trials. the victim label has been adopted to try to exclude victims from criminal trials or to minimize their role. in this view, the function of the victim label is diametrically opposed to the accusatory role as- sumed by fletcher and others. it is an oxymoron if someone called “a victim” speaks up assertively or vindictively in a criminal trial. these final thoughts on the implications of the victim label suggest that the increasing resistance to crime victims being defined as such is etymologically well-grounded. critics rightly reject the implied con- notations of the victim label of passivity, shame, and helplessness. they have sound reasons to demand criminal justice ethics to be called survivors or wronged parties. by reject- ing the victim label they can enhance their chances of being taken seriously, being adequately assisted in coping, and getting access to justice and voice. in the final analysis, the opposition of many legal schol- ars to stronger procedural rights for crime victims in criminal justice law seems grounded in insufficiently understood or articulated religious beliefs. in my view, the hidden assumptions about victims should be critically re-examined in order to arrive at less bi- ased opinions on the proper place of the wronged in criminal proceedings. these conclusions about the secondary meaning of the victim label, that of self-effacing healer, also suggest a new line of inquiry regarding the puzzling adoption of the victim label in hebrew and arabic. as noted, the story of abraham and isaac/ishmael seems an unlikely basis for calling crime victims kor- ban or udhiya. unlike crime victims, the sons of abra- ham did not suffer but were saved from victimization by divine intervention. however, the label’s connota- tion of a passive, self-effacing healer may apply neatly to victims of crime in a jewish and islamic theological setting. in both religions, the story of abraham cent- ers round the readiness of the protagonist to bring the highest sacrifice. in fact, the islamic holiday of the sacrifice celebrates the human capacity to make sacrifices in a moral sense. it seems worth explor- ing whether, in modern hebrew and arabic, the vic- tims of crime are perhaps called victims not so much because they are sufferers resembling christ but be- cause the label usefully invites them to sacrifice their right of revenge in the interest of communal peace. sharia law may fully acknowledge the talionic prin- ciple, but islamic clergymen play an important role in persuading victims to be moderate in requesting blood money or to engage in other acts of mercy or reconciliation. by calling the affected party  udhiya, the muslim community perhaps hopes to persuade that person or his family to sacrifice their right to seek revenge and to adopt a more reconciliatory stance. if this interpretation is correct, those affected by crime are called victims in these three religions partially for the very same reason—namely, the wish to instill in them a spirit of sacrifice in spite of their recognized suffering. with this final hypothesis, we may have identified common ground in the foundations of christian, jewish, and islamic criminal law. on this basis, legal scholars of all three religions could per- haps assist each other in finding the right balance be- tween legitimate retributive impulses of the wronged party and the need to treat offenders humanely. jan van dijk  /  notes natasha kampusch is a young woman who was kidnapped and kept locked in a cellar in vienna for eight years. from the film “natasha a year on,” shown on channel orf, as report- ed in the telegraph, august , <http://www.telegraph. co.uk/news/main.jhtml?xml=/news/ / / /wnata- sha .xml>. the equivalent term in arabic isthe equivalent term in arabic is qurbani, which means both the sacrifice and the sacrificial animal. the feast of the sacri- fice is known as the feast of the korban in turkish and many other languages of the islamic world. george p. fletcher, the grammar of criminal law: american,  comparative,  and  international, vol. : foundations (new york: oxford university press, ). [bracketed page numbers in[bracketed page numbers in the text refer to this volume.] j. j. m. van dijk, “the mark of abel: reflections on the so-j. j. m. van dijk, “the mark of abel: reflections on the so- cial labeling of victims of crime,” (inaugural lecture, pieter van vollenhoven chair in victimology and human security, tilburg university, tilburg, the netherlands, november , ). hyam maccoby,hyam maccoby, the sacred executioner (new york: thames and hudson, ). rené girard,rené girard, the  scapegoat, trans. yvonne freccero (balti- more: johns hopkins press, ); girard, job: the victim of his  people, trans. yvonne freccero (stanford: stanford university press, ). abdullah yusef ali,abdullah yusef ali, the  holy  quran,  text,  translation  and  commentary (st. brentwood, md: amana corp., ). nils christie, “the ideal victim,” innils christie, “the ideal victim,” in from  crime  policy  to  victim policy, ed. ezzat fattah (hampshire, uk: palgrave mac- millan, ). ybo buruma,ybo buruma, de aandacht van de strafrechter (arnhem, nl: gouda quint, ). the title of this inaugural lecture given atthe title of this inaugural lecture given at the university of nijmegen can be translated as, “the atten- tion of the criminal judge.” ezzat fattah, “from a handful of dollars to tea and sym-ezzat fattah, “from a handful of dollars to tea and sym- pathy: the sad story of victim support,” in caring for crime  victims, ed. j. j. m. van dijk, ron g. h. van kaam, and joanne wemmers (monsey, ny: criminal justice press, ); r. elias, “which victim movement? the politics of victim policy,” in victims  of  crime;  problems,  policies  and  programs, ed. robert c. davis, arthur j. lurigio, and susan a. herman (newbury park, ca: sage, ). woordenboek  der  nederlandsche  taal  (den haag: nijhoff, ), available at: http://wnt.inl.nl/. one could also argue that fletcher’s interpretation is rath-one could also argue that fletcher’s interpretation is rath- er far-fetched. is a label for the killed person chosen in order winter/spring  to label indirectly the other party? would it not be rhetorically more economical just to introduce terms labeling the perpetra- tor as a blasphemous or a very guilty person? is not stigmatiz- ing the offenders as evil persons exactly what accusers have always been doing? in the qur’an, ishmael is calledin the qur’an, ishmael is called zabiyullah (the sacrifice re- quested by god) ( , xix, , see annotation abdullah yusef ali, the  holy  quran,  text,  translation  and  commentary (st. brentwood, md: amana corp., ). according to theaccording to the oxford  english  dictionary  (oxford: ox- ford university press, ), victima/victim is related to the germanic concept of weihen, to consecrate. the word “victim” seems therefore to be etymologically related to the german word for christmas: weihnacht. the verb weihen is, accord- ing to the oed, etymologically related to the sanskrit word vinakti, which means setting apart or singling out. etymologi- cally, “victim” refers to the sacred scapegoat. in this context, reference should also be made to the etymology of the words “sacred” and “sacrifice” (sacer and facere). both words are de- rived from the latin word, sacer, which means both the holy and the accursed (chris fleming, rené  girard:  violence  and  mimesis [cambridge: polity press, ], ). the verb sac- rare, like vinakti in sanskrit, means to set apart. at a deeper etymological level, then, “victim” refers to those sacrificed, meaning those who achieved holiness by having been killed as scapegoats. paul rockpaul rock,  constructing  victim’s  rights:  the  home  office,  new labour, and victims (oxford, uk: oxford university press, ). le grand robert de la langue francaise: dictionaire alphabeti- que et analogue de la langue francaise (paris: le robert, ). woordenboek  der  nederlandsche  taal  (den haag: nijhoff, ). andré lascaris,andré lascaris, het soevereine slachtoffer: een theologisch es- say over geweld en onderdrukking (baarn, nl: ten have, ). the dutch title can be translated as, the  sovereign  victim:  a  theological essay on violence and repression. translation from dutch into english by the author. in histranslation from dutch into english by the author. in his dutch translation of the latin text lascaris inserts victima be- tween brackets after the dutch word slachtoffer, indicating that calvin used this latin term. lascaris used an edition of john calvin’s institutio  christianae  religionis of edited by a. tholuck and published in london in . rené girard,rené girard, things hidden since the foundation of the world, trans. stephen bann and michael metteer (stanford: stanford university press, ). for example, friedrich nietzsche,for example, friedrich nietzsche, werke  in  zwei  bänden (münchen: carl hanser verlag, ). strident criticism of christian compassion is most notable in his two last publicati- ons ecce homo and the anti-christ. girard,girard, things  hidden  since  the  foundation  of  the  world. the book’s last chapter dealing with the god of the gospel is called “god of the victims.” rené girard,rené girard, i see satan fall like lightning (new york: or- bis books, ). chapter , in which girard praises chris- tian care for actual victims, is called “victim care.” in the shadow of christ?  /  abram de swaanabram de swaan, in care of the state, health care, education  and welfare in europe and the usa in the modern era (new york: oxford university press, ). stephan schafer,stephan schafer, the  victim  and  his  criminal, (new york: random house, ). for the united kingdom, see rockfor the united kingdom, see rock,  constructing  victim’s  rights. in the netherlands, the main impetus for the establish- ment of provisions for victim support has come from an or- ganization promoting voluntary work based on the traditions of humanism, called humanitas. an inquiry into the link between the story of the cruci-an inquiry into the link between the story of the cruci- fixion and the command to forgive lies outside the scope of this article. as discussed by girard and many others, a reli- gion that re-enacts the killing of a scapegoat, while identify- ing with the scapegoat rather than the killers, runs the risk of promoting an endless series of vendettas against presumed killers. the command of forgiveness is therefore not to be seen as accidental but as the essential countervailing power of the passion of christ within christianity (see also chris fleming, rené  girard, violence  and  mimesis [cambridge: polity press, ]). without the meekness of christ, christianity would have a strong paranoid streak. luke : .luke : . matt. : .matt. : . deut. : .deut. : . rom. : .rom. : . matt. : .matt. : . andré lascaris,andré lascaris, het soevereine slachtoffer: een theologisch es- say over geweld en onderdrukking (baarn, nl: ten have, ). the dutch title can be translated as, the  sovereign  victim:  a  theological essay on violence and repression. “tooth for tooth, and wounds equal for wounds. but if“tooth for tooth, and wounds equal for wounds. but if anyone remits the retaliation by way of charity, it is an act of atonement for himself.” from abdullah yusef ali, the  holy  quran, text, translation and commentary , verser (st. brent- wood, md: amana corp., ). matt. : , : .matt. : , : . lascaris,lascaris, het soevereine slachtoffer, page - . cited in d. b. kraybill, s. m. nolt, and d. l. weaver-�erch-cited in d. b. kraybill, s. m. nolt, and d. l. weaver-�erch- er, amish grace: how forgiveness transcended tragedy (san fran- cisco: wiley, ), - . pierre allard and wayne northey, “christianity: the re-pierre allard and wayne northey, “christianity: the re- discovery of restorative justice,” in the  spiritual  roots  of  re- storative  justice, ed. michael hadley (albany, ny: state uni- versity of new york press, ). george pavlich,george pavlich, governing  paradoxes  of  restorative  justice (london: glasshouse press, ), . annalise acorn,annalise acorn, compulsory  compassion:  a  critique  of  re- storative  justice (vancouver: university of british columbia press, ). van dijk, “the mark of abelvan dijk, “the mark of abel.” kraybill, nolt, and weaver-�ercher,kraybill, nolt, and weaver-�ercher, amish grace, - . criminal justice ethics kraybill, nolt, and weaver-�ercher,kraybill, nolt, and weaver-�ercher, amish grace, - . girard,girard, things hidden since the foundation of the world. nietzsche,nietzsche, werke in zwei bänden, . according to rolf kleber, one of the founders of the in-according to rolf kleber, one of the founders of the in- stitute for psycho-trauma in the netherlands, both male and female victims increasingly refuse to call themselves victims because of the term’s association with powerlessness. the vic- tims’ movement is confronted with increasing numbers of vic- tims who reject stereotypical victim labels and try to present themselves with personalized labels revolving around their newly found strength. (rolf kleber, in i. van teeseling, het  oog van de storm, slachtoffers in actie (in the eye of the tempest;  victims in action) (amsterdam: veen), . see also basia spa- lek: “if the stereotype of victim as ‘passive’ and ‘helpless’ is perpetuated in dominant representations of victimhood, du- ring a time when individual strength is valued in society, then both males and females may increasingly refuse to situate themselves in terms of victimhood. they may even reject ser- vices, despite the harms experienced, due to their distaste for the label ‘victim’ and the kind of stereotypes it elicits” spalek,  crime victims: theory, policy and practice (new york: palgrave, ), . in the u.s., victims of crime are increasingly called survi- vors (for example, survivors of rape, sexual abuse, or human trafficking) and organizations for victim support are called survivor agencies. in israel, activists have proposed to replace the word korban by the more neutral term nifga. in turkey, the jan van dijk  /  word korban is increasingly perceived as old-fashioned in the more secular western part of the country.   during the holiday of the sacrifice, animals are killed as sacrifice to allah, with the strict obligation not to waste any surplus of meat but distribute it among the needy (see ali, the  holy quran). it cannot be ruled out that the killing of goats or camels during this important religious feast forms part of the explanation for the adoption of the udhiya concept for victims of crime, but considering the cruelty of the actual slaughtering this does not seem very likely. m. rahami, “islamic restorative traditions and their re- flections in the post revolutionary criminal justice system of iran,” european journal of crime, criminal law and criminal jus- tice,  , no. ( ): - . the reader is reminded that the words korban  and udhiya  (qurbani) share the double meaning of sacrifice and sacrificial animal in hebrew and arabic. leslie sebba, third  parties:  victims  and  the  criminal  justice  system  (columbus, oh: ohio state university press, ); m. groenhuijsen, “victims’ rights in the criminal justice sy- stem: a call for a more comprehensive implementation the- ory,” in caring for crime victims, ed. van dijk, van kaam, and wemmers, - ; e. erez, “integrating a victim perspective in criminal justice through victim impact statements,” in in- tegrating a victim perspective within criminal justice, ed. adam crawford and jo goodey (aldershot, uk: ashgate, ), - . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ ellis-van creveld syndrome: mutations uncovered in lebanese families case report ellis-van creveld syndrome: mutations uncovered in lebanese families maria valencia, lara tabet, nadine yazbeck, alia araj, victor l. ruiz-perez, , khalil charaffedine, farah fares, rebecca badra, and chantal farra , instituto de investigaciones biomédicas, consejo superior de cient́ıficas, universidad autónoma de madrid, madrid, spain department of pathology and laboratory medicine, american university of beirut medical center, p.o. box - riad el solh, beirut , lebanon department of pediatrics and adolescent medicine, american university of beirut medical center, p.o. box - riad el solh, beirut , lebanon centro de investigación biomédica en red de enfermedades raras (ciberer), instituto de salud carlos iii (isciii), madrid, spain correspondence should be addressed to chantal farra; cf @aub.edu.lb received march ; accepted april academic editor: philip d. cotter copyright © maria valencia et al. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. background. ellis-van creveld (evc) syndrome is a rare, autosomal recessive disorder characterized by short stature, short limbs, growth retardation, polydactyly, and ectodermal defects with cardiac anomalies occurring in around % of cases. evc syndrome has been linked to mutations in evc and evc genes. case presentation. we report evc syndrome in two unrelated lebanese families both having homozygous mutations in the evc gene, c. c>t (p.(arg *)) and c. del (p.(leu *)) in exons and , respectively, with the latter being reported for the first time. conclusion. although evc has been largely described in the medical literature, clinical features of this syndrome vary. while more research is required to explore other genes involved in evc, early diagnosis and therapeutic care are important to achieve a better quality of life. . introduction ellis-van creveld (evc) syndrome, also known as chondroec- todermal and mesoectodermal dysplasia, was first described in by elis and creveld, as an autosomal recessive disorder characterized by short stature and ribs, polydactyly, and ectodermal defects [ – ]. more than half of evc patients manifest congenital heart defects [ ]. while relatively rare, with a prevalence of . / , live birth and only around cases reported worldwide [ , , ], it is mainly reported in highly consanguineous populations such as the amish population [ ]. the syndrome can be diagnosed either by ultrasonography starting from th week of gestation or through clinical examination right after birth [ ]. diagnosis after birth is based on clinical observation of features and symptoms described above. it is also supported by an x-ray of the skeleton, chest radiography, ecg, and echocardiography [ ]. evc is related to a group of diseases with alteration of cilia (ciliopathies). such abnormalities are caused by mutations in the evc genes (evc and evc ) found on chromosome p [ , ] in around two-thirds of the cases. in this paper, we report evc syndrome in two unrelated lebanese families both having homozygous mutations in the evc gene, nm . : c. c>t p.(arg *) and nm . : c. del (p.(leu *)) taat (p.(leu *)) in exons and , respectively. while the first one has been recently reported in a chinese patient, the latter is a newly described mutation. informed consents were obtained from the patients’ guardians. genetic analysis was approved by the institutional review board at the instituto de investigaciones biomédicas. patients were clinically assessed by an experienced clinical geneticist. the pedigrees of families affected are shown in figures and . hindawi publishing corporation case reports in genetics volume , article id , pages http://dx.doi.org/ . / / http://dx.doi.org/ . / / case reports in genetics v iv iii i i figure : pedigree of family . ii i figure : pedigree of family . . case presentations . . family . a two-year-old girl born to healthy, young consanguineous parents was referred to our genetics clinic at the american university of beirut medical center (aubmc) for short stature. the patient had a trial septal defect for which she underwent surgical repair. there was no family history of similar problems and the pregnancy and delivery were reportedly uneventful. upon physical examination, both height and weight were on the th percentiles according to cdc growth charts (ht = cm;< th percentile; wt = kg;< th percentile). upper and lower segment ratio was / (figures (a) and (b)). radiology results showed shortening of the paired long bones and slight elongation of the thorax with relatively short ribs, early ossification of femoral heads, and irregular acetabular roofs. the patient had bone deformities and polydactyly with short fingers. laboratory work-up yielded a normal xx karyotype but a high thyroid hormone level (tsh = . 𝜇iu/ml) with free t within normal range. gene sequencing showed a nm . : c. c>t (p.(arg *)) stop codon muta- tion in exon of evc . . . family . a one-year-old boy born to first degree cousins was referred to our genetics clinic for assessment because of short stature and polydactyly. he was the second child of first cousin lebanese parents. the patient was delivered at term via c-section to a - year-old mother (g p ). his birth weight was grams (< %), with a length of cm (< %) and a head circumfer- ence of . cm (< ). an echo done at the th month of gestation showed a bell-shaped chest. no otherwise prenatal complications were reported. an echocardiogram done at birth showed patent foramen ovale (pfo). upon physical examination the patient’s stature was below the th percentile (height: cm < th%; wt: cm < th%). radiology results showed shortening of the limbs and ribs. he had a brother who passed away at day of age and who was born with cleft lip and palate and bilateral polydactyly. one of his paternal cousins, a -year-old male with normal karyotype ( , xy), was reported to have polydactyly, short stature, and dysplastic nails and teeth. he was labelled as having dwarfism without proper follow-up or accurate assessment. upon examination, he had widely spaced conical shaped teeth and a height of cm (figure ; figures (a) and (b)). dna analyses for evc and evc genes on both cousins revealed a nm . : c. del taat in exon of evc . . discussion evc is a rare disorder involving several embryonic tissues and resulting in polymorphic symptoms. although largely case reports in genetics (a) (b) figure : clinical photos of patient from family depicting short upper and lower limbs and polydactyly. (a) (b) figure : clinical photos of patient from family showing conical shaped teeth and polydactyly. described in the medical literature, the clinical features of this syndrome differ between patients [ , ] with chondrodys- trophia being the most commonly reported and the main reason behind short stature [ , , , ]. both families are consanguineous and presented with the classical findings of evc syndrome. so far, around mutations in either evc or evc genes have been reported in the literature with the majority causing premature termi- nation codons [ , , ]. in both families, mutations were detected in evc gene. in the first family, a homozygous nonsense mutation c. c>t (p.(arg *)) was found. this mutation was previously reported in a -year-old chinese female [ ] who was a compound heterozygous for another mutation (ivs - a>g). both our patient and the chinese patient had similar clinical presentation [ ]. in our second family, we detected a homozygous deletion of nucleotides nm . : c. del taat in exon of evc . this deletion generates a frameshift that runs into a premature stop codon immediately. to our knowledge, this mutation is reported for the first time. amongst arabs and middle eastern populations, muta- tion panels for recessive disorders in general differ from one country to another and in between religious denominations [ , ] mainly because of the wide migratory movements that occurred over the centuries resulting in a great variability of ethnicity and origins that constitute these populations. lebanon is a small country with several ethnic and demographic groups originating in part, from european crusader christians and arabian muslims, resulting in a het- erogeneous background of our families [ ]. consanguineous marriages in lebanon are relatively common ( . %) leading to a high prevalence of autosomal recessive disorders [ , ]. despite this fact the need for genetic services in lebanon is still not widely established. indeed, lack of compliance for genetic referrals and testing is still witnessed mainly due to economic burdens since these tests do not benefit from third party coverage and also because of social taboos that are still associated with inherited diseases. for this reason, a substantial number of patients with genetic disorders may be misdiagnosed or not followed up properly. reporting rare case reports in genetics cases from our population will raise further awareness on the occurrence of these disorders and will increase the chance for a proper genetic assessment. this could also eventually contribute to unravel other genes that might be involved in evc and to improve the quality of care for these patients. consent the patients have given their consent for the case reports to be published. conflict of interests the authors declare that there is no conflict of interests regarding the publication of this paper. references [ ] r. kamal, p. dahiya, s. kaur, r. bhardwaj, and k. chaudhary, “ellis-van creveld syndrome: a rare clinical entity,” journal of oral and maxillofacial pathology, vol. , no. , pp. – , . 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[ ] b. barbour and p. salameh, “consanguinity in lebanon: prevalence, distribution and determinants,” journal of biosocial science, vol. , no. , pp. – , . identification of a sporozoite-specific antigen from toxoplasma gondii | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /ge- . corpus id: identification of a sporozoite-specific antigen from toxoplasma gondii @inproceedings{hill identificationoa, title={identification of a sporozoite-specific antigen from toxoplasma gondii}, author={d. hill and c. coss and j. p. dubey and k. wroblewski and m. sautter and tiffany hosten and c. munoz-zanzi and e. mui and s. withers and k. boyer and gretchen l hermes and jessica j. coyne and f. jagdis and a. burnett and patrick mcleod and h. morton and d. robinson and r. mcleod}, booktitle={the journal of parasitology}, year={ } } 	d. hill, c. coss, + authors r. mcleod 	published in the journal of parasitology 	biology, medicine abstract reduction of risk for human and food animal infection with toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. existing serological assays can determine previous exposure to the parasite, but not the route of infection. we have used difference gel electrophoresis, in combination with tandem mass spectroscopy and western blot, to identify a… expand view on bioone europepmc.org save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all topics from this paper 	toxoplasma gondii 	sporozoites 	toxoplasmosis 	oocysts 	embryonic development 	gel electrophoresis (lab technique) 	documented 	differential in-gel electrophoresis 	western blotting 	animal 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medicine 	parasitology international 	 	 	 save alert research feed serum metabolic profiling of oocyst-induced toxoplasma gondii acute and chronic infections in mice using mass-spectrometry 	chun-xue zhou, w. cong, xiao-qing chen, s. he, h. elsheikha, x. zhu 	biology, medicine 	front. microbiol. 	 	 	pdf 	view excerpts, cites background save alert research feed proteomic analysis of fractionated toxoplasma oocysts reveals clues to their environmental resistance 	h. fritz, paul w. bowyer, m. bogyo, p. conrad, j. boothroyd 	biology, medicine 	plos one 	 	 	pdf 	view excerpts, cites background save alert research feed diagnosis of toxoplasmosis and typing of toxoplasma gondii 	q. liu, zedong wang, s. huang, x. zhu 	biology, medicine 	parasites & vectors 	 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency identification of stage-specific sporozoite antigens of toxoplasma gondii by monoclonal antibodies. 	l. kasper, m. s. bradley, e. pfefferkorn 	biology, medicine 	journal of immunology 	 	 	view excerpt, references background save alert research feed identification of stage-specific antigens of toxoplasma gondii. 	l. kasper 	biology, medicine 	infection and immunity 	 	 	pdf 	view excerpt, references background save alert research feed toxoplasma gondii: from animals to humans. 	a. tenter, a. heckeroth, l. weiss 	biology, medicine 	international journal for parasitology 	 	 , 	 save alert research feed biology and epidemiology of toxoplasma gondii in man and animals 	d. hill, sreekumar chirukandoth, j. dubey 	medicine, biology 	animal health research reviews 	 	 	view excerpt, references background save alert research feed the development and biology of bradyzoites of toxoplasma gondii. 	l. weiss, k. kim 	biology, medicine 	frontiers in bioscience : a journal and virtual library 	 	 	pdf 	view excerpt, references background save alert research feed high prevalence and abundant atypical genotypes of toxoplasma gondii isolated from lambs destined for human consumption in the usa. 	j. dubey, n. sundar, + authors c. su 	biology, medicine 	international journal for parasitology 	 	 	view excerpt, references background save alert research feed strain and stage specific variation in toxoplasma gondii antigens. 	p. appleford, j. smith 	biology, medicine 	international journal for parasitology 	 	 	view excerpt, references background save alert research feed antibody responses measured by various serologic tests in pigs orally inoculated with low numbers of toxoplasma gondii oocysts. 	j. dubey, c. andrews, p. lind, o. kwok, p. thulliez, j. lunney 	biology, medicine 	american journal of veterinary research 	 	 	view excerpt, references methods save alert research feed specific detection of neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction 	d. hill, s. liddell, m. jenkins, j. dubey 	biology, medicine 	the journal of parasitology 	 	 	view excerpt save alert research feed multicenter evaluation of strategies for serodiagnosis of primary infection with toxoplasma gondii 	a. roberts, k. hedman, + authors e. petersen 	biology, medicine 	european journal of clinical microbiology and infectious diseases 	 	 	view excerpt, references background save alert research feed ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue fontographer . genetic influences on premature parturition in an australian twin sample susan a treloar , george a macones , laura e mitchell and nicholas g martin queensland institute of medical research and joint genetics program, the university of queensland, brisbane, australia the university of pennsylvania health system the children’s hospital of philadelphia, philadelphia, pa, usa we investigated possible genetic influences on women’s liability to preterm birth, using data from a large sample of australian female twin pairs. in a – questionnaire survey, both members of parous twin pairs ( monozygotic and dizygotic) reported on whether deliveries had been more than two weeks preterm. tetrachoric twin pair correlations for first birth were rmz = . ± . and rdz = – . ± . , and for any birth were rmz = . ± . and rdz = . ± . . best-fitting models to data contained only additive genetic influences and individual environmental effects. heritability was % for preterm delivery in first pregnancy, and % for preterm delivery in any pregnancy. in the former case, however, we could not reject a model without genetic influences. although our data did not allow for differentiation of the varying aetiologies of premature parturition, results from this exploratory analysis suggest that further investigation of genetic influences on specific reasons for preterm birth is warranted. twin research ( ) , – . keywords: preterm birth, pregnancy outcome, twins, genetics the risk of preterm birth has been found to be higher for women who were themselves born prematurely, suggesting a repeating pattern across generations. the relevance of genetic influences on prematurity had been highlighted by an earlier study using old order amish records in lancaster county, pennsyl- vania, which suggested that prematurity was mostly related to the maternal genotype. in order to explore the possibility of genetic influences on a woman’s liability to preterm births we have examined self-reported, retrospective data on premature birth provided by a large number of australian, female twin pairs. these twins are members of the australian national health and medical research council twin registry, and partic- ipated in a health and lifestyle survey conducted between and . – responses to this survey were obtained from both members of female monozygotic (mz) and dizygotic (dz) twin pairs aged and older, for a pairwise response rate of %. the study questionnaire included questions on their menstrual cycles and reproductive events, including specific obstetric factors relating to each delivery. – premature delivery (preterm birth) was defined in the questionnaire as delivery ‘over weeks early’, rather than the standard definition of curtailment of pregnancy before the th week of gestation. we did not ask for number of weeks gestation. we investi- gated parous twins’ independent reports of pre- mature delivery of first baby, and also reports of whether any birth was premature using the > week criterion. premature births may have been sponta- neous or induced, vaginal deliveries or performed by caesarean section. data were also available on these items, but analyses are not presented here. twin pair matrices of tetrachoric correlations, and corresponding asymptotic covariance matrices, were computed separately for mz and dz twin pairs, using the windows version of prelis . . these correlations apply appropriately to dichotomous categorical data where the underlying distribution is assumed to be continuous and normally distributed. genetic models were fitted by the method of asymp- totic least squares to estimate the contributions of additive genetic, shared and non-shared environ- mental effects, using mx. we proceeded by system- atically testing the significance of dropping parame- ters in turn. in addition to the likelihood ratio ø test (lr), the akaike information criterion (aic, meas- ured as ø - df) was used as an additional indicator of fit. on the grounds of parsimony the model with the least number of parameters that offered a fit not significantly worse than the full model was chosen. data analysis methods are described more fully elsewhere. correspondence: susan a treloar, phd, queensland institute of medical research, po royal brisbane hospital, queensland , australia. tel: ; fax: ; e-mail: suet@qimr.edu.au received november ; accepted november twin research ( ) , – y © macmillan publishers ltd all rights reserved – / $ . www.nature.com/tr both members of parous twin pairs ( mz pairs and dz pairs) answered the question concerning prematurity of their first and any sub- sequent birth. ages of parous twins ranged from to years, and mean age was . ± . years. mean ages of mz twins ( . years) and dz ( . ) twins did not differ significantly. the prevalence of premature first birth in the sample was . % of individuals in mz pairs and higher at . % in the dz twins, giving an overall prevalence of . %. premature parturition relating to any birth using the > week criterion was reported by . % of individ- uals in mz pairs and . % of twins in dz pairs. the percentage of nulliparae was the same ( %) for both mz and dz individuals. non-parametric one-way median score tests showed that parous mz and dz twins did not differ significantly on median total pregnancies. however, they did differ on median number of full-term (vs aborted) pregnancies (p < . ) and of offspring (p < . ), the latter median being higher for dz twins ( ) than for mz twins ( ). this suggested a higher prevalence of multiple births in the dz twins. the median number of preterm babies reported by dz twins was significantly higher than for mz twins (p < . ), consistent also with a higher rate of multiple births. the ratio of mz to dz twin pair correlations ( > : ) shown in table suggests that genetic influ- ences may be operating on premature parturition. for both prematurity variables, using the aic as the indicator, best-fitting most parsimonious models contained only additive genetic variation (a) and individual environmental effects (e). for having had a premature delivery for any birth, the best fitting model (aic = – . ) included a heritability of %. a model including non-additive genetic influ- ences resulted in a better fit than a model containing shared environment (c), but was not a significant improvement on the ae model. a ce model contain- ing no additive genetic variation was rejected on significance grounds. for premature delivery, the ae model was again the model of choice (aic = – . ) with a heritability of %; however, in this case a model containing no genetic influences could not be rejected on significance grounds. evidence suggests that in australia the impact of changes in obstetric care that might influence pre- mature labour were minimal up to . we would not expect the impact of obstetric practices to differ for individuals on the basis of their zygosity. increased prevalence of multiple births to dz twins may have influenced pairwise concordance for pre- term birth, but any effect would have been too small to detect in this sample. data were retrospective and collected in – , before the widespread availa- bility of assisted reproductive technologies, which are increasing the prevalence of non-monozygotic multiple births, and hence may influence prevalence of premature delivery. we cannot unequivocally separate out the differ- ent aetiologies for preterm birth in our data. the aetiologies of spontaneous premature membrane rupture or preterm labour may well differ, and those of indicated preterm births relating to foetal or maternal morbidity clearly may vary even further. retrospectivity may have influenced accuracy of recall of preterm delivery. data on length of gestation or date of last menstrual period (lmp) were not available. furthermore, genetic heterogeneity may be impli- cated in the predisposing conditions. for example, the prevalence of pre-eclampsia, which may be associated with indicated premature delivery, is higher for first than subsequent births. if the aetiol- ogy is foetal in origin rather than maternal, which may well be the case, the effect may be to reduce the (maternal) twin pair correlations. this might suggest that the heritability we have observed is occurring in the spontaneous rather than the indi- cated preterm deliveries, but to test this hypothesis would require a very large sample indeed. it would be fascinating to know whether, for example, there is a genetic contribution to factors such as cervical incompetence per se. nevertheless, our data do suggest that genetic influences may be important in influencing preterm birth, and further investigation of this hypothesis may well be warranted. acknowledgements we thank the national health and medical research council of australia for support (no. , no. ) and the twins for their co-operation. references porter t, fraser a, hunter c, ward r, varner m: the risk of preterm birth across generations. obstet gynecol ; : – . khoury m, cohen b: genetic heterogeneity of prematurity and intrauterine growth retardation: clues from the old order amish. am j obstet gynecol ; : – . table tetrachoric twin pair correlations for prematurity of first delivery and of any delivery mz pairs (n= ) dz pairs (n= ) premature by twin pair twin pair > weeks correlation se correlation se first birth . . – . . any birth . . – . . twin research genetic influences on preterm birth in australian twin mothers sa treloar et al y do k-a, treloar sa, pandeya n, purdie d, green a, heath ac, martin ng: predictive factors of age at menopause in a large australian twin study. hum biol ; : – . heath a, cloninger c, martin n: testing a model for the genetic structure of personality: a comparison of the person- ality systems of cloninger and eysenck. j pers soc psychol ; : – . treloar s, martin n, heath a: longitudinal genetic analysis of menstrual flow, pain and limitation in a sample of australian twins. behav genet ; : – . treloar s, martin n, dennerstein l, raphael b, heath a: pathways to hysterectomy: insights from longitudinal twin research. am j obstet gynecol ; : – . jöreskog k, sörbom d: prelis . for windows. scientific software international inc: chicago, . neale m: mx: statistical modeling. department of psychiatry: mcv, richmond, va, . neale m, cardon l: methodology for genetic studies of twins and families nato asi series. kluwer academic publishers: dordrecht, . heath ac, martin ng, eaves lj, loesch d: evidence for polygenic epistatic interactions in man? genet ; : – . stanley f: perinatal outcome in western australia, to . perinatal mortality and birthweight. med j aust ; : – . thornton j, macdonald a: twin mothers, pregnancy hyper- tension and pre-eclampsia. br j obstet gynaecol ; : – . twin research genetic influences on preterm birth in australian twin mothers y sa treloar et al wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no 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http://europepmc.org/abstract/med/ dmso- -causes-and-consequences-of-obesity--epigenetics-or-hypokines © graham et al. this work is published by dove medical press limited, and licensed under creative commons attribution – non commercial (unported, v . ) license. the full terms of the license are available at http://creativecommons.org/licenses/by-nc/ . /. non-commercial uses of the work are permitted without any further permission from dove medical press limited, provided the work is properly attributed. permissions beyond the scope of the license are administered by dove medical press limited. information on how to request permission may be found at: http://www.dovepress.com/permissions.php diabetes, metabolic syndrome and obesity: targets and therapy : – diabetes, metabolic syndrome and obesity: targets and therapy dovepress submit your manuscript | www.dovepress.com dovepress l e t t e r open access to scientific and medical research open access full text article http://dx.doi.org/ . /dmso.s causes and consequences of obesity: epigenetics or hypokinesis? michael r graham julien s baker bruce davies llantarnam research academy, cwmbran, torfaen, uk; exercise science research laboratory, institute of clinical exercise and health science, school of science, university of the west of scotland, hamilton, uk; science department, university of south wales, newport, uk correspondence: julien s baker exercise science research laboratory, institute of clinical exercise and health science, school of science, university of the west of scotland, hamilton, lanarkshire, scotland ml ojb, uk email jsbaker@uws.ac.uk dear editor epigenetics can be defined as the study of heritable changes that affect gene function without modification of the deoxyribonucleic acid (dna) sequence. the transfer of epigenetic marks through generations is not well understood, and their transmission is in dispute. epigenetic marks are tissue-specific and include dna methylation and histone modifications that mediate biological processes, such as imprinting (figure ). many imprinted genes are regulators of gene expression controlling growth. imprinting disorders often feature obesity as one of their characteristics. genomic imprinting determines expression of alleles, creating an equilibrium between the expression of the parental alleles influencing growth and metabolism. the identification of susceptible loci, and finding the causal genes and variants in each locus for obesity may allow molecular and physiological investigations. however, the knowledge of the mechanisms through which these contribute to susceptibility to obesity will not necessarily provide the requisite management strategies. geneticists have long since identified the obesity gene (fto) and a low-fat gene (apoa ), and that variations in the adiponectin gene (single nucleotide polymor- phism [snp] g allele) can lead to hypoadiponectinemia, which then results in insulin resistance, the metabolic syndrome, increased atherosclerosis, morbidity, and ultimately, premature mortality. using exome sequencing, a low-frequency coding variant has been identified in the sypl gene associated with morbid obesity. this gene may be involved in the development of excess body fat. hypokinesis, in conjunction with excess caloric intake over expenditure, leads to obesity, which can then result in the metabolic syndrome, type diabetes mellitus, cardiovascular disease (cvd), coronary heart disease, and cerebrovascular accident, or “stroke”. – obesity is also believed to predispose to certain types of cancer, such as breast and colon cancer. however, the assertion that the complete absence of fat cells (adipocytes) is meta- bolically advantageous is a fallacy. adipocytes play an important role in health by their hormonal role in the regulation of energy intake and fat storage, and function as an endocrine gland (figure ). , adipocytes secrete approximately adipokines with diverse functional roles. the average human phenotype contains billion fat cells, weighing approximately . kg. to establish the dynamics within the stable population of adipocytes in d ia b e te s, m e ta b o lic s yn d ro m e a n d o b e si ty : t a rg e ts a n d t h e ra p y d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -a p r- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / http://www.dovepress.com/permissions.php http://creativecommons.org/licenses/by-nc/ . / http://www.dovepress.com/permissions.php www.dovepress.com www.dovepress.com www.dovepress.com http://dx.doi.org/ . /dmso.s mailto:jsbaker@uws.ac.uk diabetes, metabolic syndrome and obesity: targets and therapy : submit your manuscript | www.dovepress.com dovepress dovepress graham et al adults, adipocyte turnover can be measured by analyzing the integration of c derived from nuclear bomb tests in genomic dna. if excess weight is gained as an adult, fat cells increase approximately fourfold in size before dividing and increasing the absolute number of fat cells present. however, even after marked weight loss, the number of fat cells stays constant in adulthood in lean and obese individuals, indicating that the number of adipocytes is set during childhood and adoles- cence. approximately % of fat cells die and are replaced annually. the expansion of the fat cell population and the final number of adipocytes in the adult body appears to occur at an earlier age in obese people. fatter people experience a period of rapid adipoctye production around the age of years and reach their adult number of fat cells when they are approximately . years old. lean people, however, recruit fat cells most rapidly at approximately years old, with their fat cell population reaching its adult size at approximately . years. obese people have as many as twice the number of adipocytes as lean people. scientif ic evidence supports the concept that gene expression in our physically active ancestors reduced adverse physiological effects that may occur with a seden- tary lifestyle. to prevent the development of these adverse physiological effects, children who experience appropriate nutrition and regular exercise during their growing years display healthy patterns of physical maturation consistent with their genetic potential. however, a genetic explanation cannot explain how humans have become so fat over such a short time frame. heredity can only explain % of the population variance in obesity. genetics are unlikely to account fully for the prevalence of obesity – it must be due to the declining rates of physical activity and an increase in the consumption of energy-dense foods. , the differences in phenotypes and physiology between two geographically separate but genetically similar pima indian tribes suggests the importance of the environmental effect, especially physical activity, on the genome. the mexican pima indians were found to expend , – , kj day- more in physical activity than did their obese arizonian pima equivalents, to have had a diet lower in fat and higher in fiber content, and to weigh kg less. as a consequence, the chromosome epigenetic mechanisms organism development (conception–death); epigenetic factors: environmental effects: diet, chemicals, medicines, toxins, physical activity, aging dna ← ← histone modification (gene regulation) dna ← methylation (gene regulation) adverse effect on metabolism obesity? metabolic syndrome? type diabetes mellitus? cardiovascular/cerebrovascular disease? ← ← ← ← ← ← figure epigenetic mechanisms. notes: dna methylation: addition of a methyl group to dna nucleotides, cytosine or adenine, from dietary sources can activate or repress gene response. methylation modifications that regulate gene expression can cause genomic imprinting. histone modification: dna winds around histone proteins which can activate or repress gene response. histone is the main protein component of chromatin (complex of macromolecules comprising dna, protein, and rna). abbreviation: dna, deoxyribonucleic acid. d ia b e te s, m e ta b o lic s yn d ro m e a n d o b e si ty : t a rg e ts a n d t h e ra p y d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -a p r- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com diabetes, metabolic syndrome and obesity: targets and therapy : submit your manuscript | www.dovepress.com dovepress dovepress epigenetics or hypokinesis? mexican pima indians did not have the diabetes prevalence of the arizonian pima indians. children of pregnant women exposed to the dutch famine of were more susceptible to diabetes, obesity, cvd, renal disease, and other health problems. the children of the women who were pregnant during the famine were smaller, and when these children grew up and had children, those children were also smaller than average. this suggested that the famine experienced by the mothers caused epigenetic changes that were passed down to the next generation. genetic predisposition is the primary cause of most of the rare diseases that affect one in people. , every human possesses approximately three million sequence variants of which approximately can predispose to pathology, in adverse environmental circumstances. could lifestyle assessments and avoidance of potential risks have beneficial effects on a person’s health despite genetic variants? this question can only truly be answered if we are able to sequence individual genomes on demand and then, only if it is cost effective. the general public should be concerned with prevention rather than the management of obesity and its sequelae. despite considerable financial investment, the pharmaceu- tical industry has provided only palliation of obesity rather than cure. physical activity should be the main agenda of the department of health and the responsibilities for its effec- tive prescription and assessments placed in the hands of appropriate health professionals. more consideration should be given to effective exercise prescription rather than the premature introduction of prescription medicines, such as the latest insulin analog or glucagon-like peptide- agonist (glp- agonist), to type diabetics, which appears to be only palliating the disease state and elevating the public purse’s annual budget. in the uk in the anti-obesity drugs’ bill was £ , . and in it was £ million, with increased prescribing of drugs such as orlistat and sibutramine. in it was £ million. the annual drugs bill in the uk is currently around £ billion, which equates to just approximately % of national health service (nhs) expenditure, having risen around . % a year between and . when the nhs was launched in it had a budget of £ million (circa £ billion at today’s value). for it is predicted to be £ . billion. the obesity epidemic that we are experiencing is a global phenomenon. in more than . billion adults over the age of years, were overweight and million were obese. by it has been predicted that more than . billion endocrine effects (hyperinsulinism, insulin resistance, hypercortisolism, increased erythropoietin secretion) cardiovascular and rheologic effects, sleep apnea renal effects activation of the sympathetic nervous system metabolic effects (dyslipidemia, carbohydrate intolerance) increased coagulation/decreased fibrinolysis (↑ pai- ) hematologic effects ↑ nfκb ↑ pai- ↑ tnfα ↑ angiotensin↓ adiponectin ↑ resistin obesity adipocytes ↑ leptin figure the endocrine function of an adipocyte. abbreviations: nfκb, nuclear factor kappa-light-chain-enhancer of activated b cells; pai- , plasminogen activator inhibitor- ; tnf, tumor necrosis factor. d ia b e te s, m e ta b o lic s yn d ro m e a n d o b e si ty : t a rg e ts a n d t h e ra p y d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -a p r- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com diabetes, metabolic syndrome and obesity: targets and therapy : submit your manuscript | www.dovepress.com dovepress dovepress graham et al adults over the age of years, will be overweight and at least million will be obese. with global projections, more than . billion will be overweight and . billion will be obese individuals by . watson and crick discovered the structure of dna in , and sanger identified the amino acid sequence of insulin in , and nucleic acid sequencing becoming a major target of early molecular scientists. however, we are no nearer to eradicating the most basic of diseases, associated with obesity. have we really had a single or series of genetic mutations to account for a . % and a % increase in obesity in males and females respectively, in a mere third of a century? sequencing the first human genome took years and was completed in at a cost of £ billion. today we can sequence a complete human genome in two days at a cost of approximately £ , . . bulk sequencing all – , human protein coding genes, the exome, now costs less than £ . targeting the screening of a specific panel of genes known to be associated with a particular phenotype, as opposed to screening mutations, would appear necessary in the belief that eating five pieces of fruit and vegetables a day will keep us slim, healthy, and disease free. scientists are required to identify variants in genes, known to be pathogenic in an effort to decide on preventative, curative or palliative action. the investment continues as the government’s decision to gene sequence , whole genomes at a cost to the public purse of £ million hoping to identify the reason for the current obesity pandemic, pursuant to the work of the wellcome trust’s sanger institute initiating the human genome project. , however, will such an investment stimu- late the general population to cease eating refined processed foods and commence exercising? the fact there is so much money available for such a resource is surprising. should not that money be available to employ more exercise scientists, who can advise these obese phenotypes that eating the correct diet and performing even limited amounts of exercise can bring about positive transgenerational epigenetic changes to the major cvd risk factors. , currently the vast sums of money spent in researching pharmacotherapy alone appear to be having very little effect. a greater investment should be applied to promoting exer- cise in conjunction with exome sequencing and epigenetic research. human growth hormone (hgh) which controls the body’s ability to metabolize carbohydrate, fat, and protein and deposit any excess fat in the appropriate site, commences a downward spiral, after full adult growth has been attained. it decreases by % per decade from the age of years. exercising for more than – minutes at an oxygen uptake (vo max) of %, stimulates pituitary release of hgh by five- to tenfold. protein consumption stimulates pituitary release of hgh. carbohydrate and fat ingestion inhibit pituitary release of hgh. also obesity causes a blunted response to hgh com- pounding the adverse situation. physical activity can ultimately prevent many problems associated with obesity. there is a lower cancer incidence rates among the adult amish population, compared with non-amish population, due to increased physical activity. physical activity differences can be identified in childhood in the amish compared with non-amish population and accounts for amish children being approximately . times less likely to be overweight compared to non-amish children. the average american would aspire to walk approximately , steps per day to maintain a healthy body mass. in one study the average number of steps walked per day was , for amish men versus , for amish women, who have a very low incidence of obesity. the old order amish refrain from driving cars, using electrical appliances, and employing other modern conveniences. labor-intensive farming is still the preferred occupation. conclusion obesity is a consequence of a high saturated fat, a refined carbohydrate diet, and an increased input over expenditure, combined with reduced exercise. proactive research scientists are working to identify explanations, at the molecular level, relating exercise to health improving epigenetic changes. prevention must always be better than cure. when a large segment of the population adopts modest improvements in health behaviors, the overall disease burden in the population is likely to be reduced more dramatically than if a modest segment of the population adopts large changes. disclosure the authors repor t no conflicts of interest in this communication. references . bird a. perceptions of epigenetics. nature. ; ( ): – . . chong s, youngson na, whitelaw e. heritable germline epimutation is not the same as transgenerational epigenetic inheritance. nat genet. ; ( ): – ; author 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environmental factors and disease: the man made environment. br med j (clin res ed). ; ( ): – . d ia b e te s, m e ta b o lic s yn d ro m e a n d o b e si ty : t a rg e ts a n d t h e ra p y d o w n lo a d e d f ro m h tt p s: // w w w .d o ve p re ss .c o m / b y . . . o n -a p r- f o r p e rs o n a l u se o n ly . powered by tcpdf (www.tcpdf.org) / www.dovepress.com www.dovepress.com www.dovepress.com http://www.dailymail.co.uk/health/article- /slimming-drugs-cost-nhs- m.html http://www.dailymail.co.uk/health/article- /slimming-drugs-cost-nhs- m.html http://www.dailymail.co.uk/health/article- /slimming-drugs-cost-nhs- m.html http://www.nhs.uk/nhsengland/thenhs/about/pages/overview.aspx http://www.nhs.uk/nhsengland/thenhs/about/pages/overview.aspx diabetes, metabolic syndrome and obesity: targets and therapy publish your work in this journal submit your manuscript here: http://www.dovepress.com/diabetes-metabolic-syndrome-and-obesity-targets-and-therapy-journal diabetes, metabolic 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defined by segregation analysis in centre d’etude polymorphisme humain (ceph) families m. p. martin & r. m. single & m. j. wilson & j. trowsdale & m. carrington published online: december # springer-verlag erratum to: immunogenetics doi . /s - - -y in the original version of the manuscript, we failed to discuss and cite a key study closely related to ours (norman et al. ). this study described and analyzed data from of the families used in our study. thus, our study extends that of norman et al. by including additional families. we view our manuscript as a source of reference material as opposed to the analytical synthesis provided by the norman et al. study. we sincerely apologize for our omission and wish to correct the manuscript as follows: . norman et al. should be included as one of the citations for the sentence below: human populations can vary remarkably in the frequencies of kir genes and haplotypes, possibly a reflection of selection pressures conferred by population-specific diseases (cook et al. ; crum et al. ; denis et al. ; frassati et al. ; gendzekhadze et al. ; jiang et al. ; kulkarni et al. b; middleton et al. ; norman et al. ; norman et al. ; rajalingam et al. ; toneva et al. ; velickovic et al. ; whang et al. ; witt et al. ; yawata et al. ). . a statement referring to the families used in the study should have read as follows: in order to more fully characterize the kir locus in caucasians, we typed for the presence/absence status of kir genes (martin and carrington ) in members of ceph families of european descent and determined haplo- types based on segregation analysis (figure ). these families included one amish family, ten french families, and forty six families from utah. kir data from of the utah families used in the present study were previously reported by norman et al. . our study includes an additional families and thus is an extension of this previous work. norman pj, cook ma, carey bs, carrington cvf, verity dh, hameed k, ramdath dd, chandanayingyong d, leppert m, stephens haf, vaughan rw ( ) snp haplotypes and allele frequencies show evidence for disrup- tive and balancing selection in the human leukocyte receptor complex. immunogenetics : – doi: . /s - - - immunogenetics ( ) : doi . /s - - - the online version of the original article can be found at http://dx.doi. org/ . /s - - -y m. p. martin : m. carrington (*) cancer and inflammation program, laboratory of experimental immunology, saic-frederick, inc, nci-frederick, frederick, md , usa e-mail: carringt@ncifcrf.gov r. m. single department of mathematics and statistics, university of vermont, burlington, vt , usa m. j. wilson csl limited, bio institute, parkville, victoria , australia j. trowsdale immunology division, department of pathology, university of cambridge, tennis court road, cambridge cb qp, uk http://dx.doi.org/ . /s - - -y http://dx.doi.org/ . /s - - -y 	kir haplotypes defined by segregation analysis in centre d’etude 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e e> >> >> setdistillerparams << /hwresolution [ ] /pagesize [ . . ] >> setpagedevice microsoft word - .aiso - david c. wyld et al. (eds) : csen, aiso, ncwc, sipr - pp. – , . © cs & it-cscp doi : . /csit. . hierarchical deep learning architecture for k objects classification atul laxman katole, krishna prasad yellapragada, amish kumar bedi, sehaj singh kalra and mynepalli siva chaitanya samsung r&d institute india - bangalore, bagmane constellation business park, doddanekundi circle, bangalore, india abstract evolution of visual object recognition architectures based on convolutional neural networks & convolutional deep belief networks paradigms has revolutionized artificial vision science. these architectures extract & learn the real world hierarchical visual features utilizing supervised & unsupervised learning approaches respectively. both the approaches yet cannot scale up realistically to provide recognition for a very large number of objects as high as k. we propose a two level hierarchical deep learning architecture inspired by divide & conquer principle that decomposes the large scale recognition architecture into root & leaf level model architectures. each of the root & leaf level models is trained exclusively to provide superior results than possible by any -level deep learning architecture prevalent today. the proposed architecture classifies objects in two steps. in the first step the root level model classifies the object in a high level category. in the second step, the leaf level recognition model for the recognized high level category is selected among all the leaf models. this leaf level model is presented with the same input object image which classifies it in a specific category. also we propose a blend of leaf level models trained with either supervised or unsupervised learning approaches. unsupervised learning is suitable whenever labelled data is scarce for the specific leaf level models. currently the training of leaf level models is in progress; where we have trained out of the total leaf level models as of now. we have trained the leaf models with the best case top- error rate of . % on the validation data set for the particular leaf models. also we demonstrate that the validation error of the leaf level models saturates towards the above mentioned accuracy as the number of epochs are increased to more than sixty. the top- error rate for the entire two-level architecture needs to be computed in conjunction with the error rates of root & all the leaf models. the realization of this two level visual recognition architecture will greatly enhance the accuracy of the large scale object recognition scenarios demanded by the use cases as diverse as drone vision, augmented reality, retail, image search & retrieval, robotic navigation, targeted advertisements etc. keywords convolutional neural network [cnn], convolutional deep belief network [cdbn], supervised & unsupervised training computer science & information technology (cs & it) . introduction deep learning based vision architectures learn to extract & represent visual features with model architectures that are composed of layers of non-linear transformations stacked on top of each other [ ]. they learn high level abstractions from low level features extracted from images utilizing supervised or unsupervised learning algorithms. recent advances in training cnns with gradient descent based backpropagation algorithm have shown very accurate results due to inclusion of rectified linear units as nonlinear transformation [ ]. also extension of unsupervised learning algorithms that train deep belief networks towards training convolutional networks have exhibited promise to scale it to realistic image sizes [ ]. both the supervised and unsupervised learning approaches have matured and have provided architectures that can successfully classify objects in & categories respectively. yet both the approaches cannot be scaled realistically to classify objects from k categories. the need for large scale object recognition is ever relevant today with the explosion of the number of individual objects that are supposed to be comprehended by artificial vision based solutions. this requirement is more pronounced in use case scenarios as drone vision, augmented reality, retail, image search & retrieval, industrial robotic navigation, targeted advertisements etc. the large scale object recognition will enable the recognition engines to cater to wider spectrum of object categories. also the mission critical use cases demand higher level of accuracy simultaneously with the large scale of objects to be recognized. in this paper, we propose a two level hierarchical deep learning architecture that achieves compelling results to classify objects in k categories. to the best of our knowledge the proposed method is the first attempt to classify k objects utilizing a two level hierarchical deep learning architecture. also a blend of supervised & unsupervised learning based leaf level models is proposed to overcome labelled data scarcity problem. the proposed architecture provides us with the dual benefit in the form of providing the solution for large scale object recognition and at the same time achieving this challenge with greater accuracy than being possible with a -level deep learning architecture. . related works we have not come across any work that uses -level hierarchical deep learning architecture to classify k objects in images. but object recognition on this large scale using shallow architectures utilizing svms is discussed in [ ]. this effort presents a study of large scale categorization with more than k image classes using multi-scale spatial pyramids (spm) [ ] on bag of visual words (bow) [ ] for feature extraction & support vector machines (svm) for classification. this work creates ten different datasets derived from imagenet each with to , categories. based on these datasets it outlines the influence on classification accuracy due to different factors like number of labels in a dataset, density of the dataset and the hierarchy of labels in a dataset. the methods are proposed which provide extended information to the classifier on the relationship between different labels by defining a hierarchical cost. this cost is calculated as the height of the lowest common ancestor in wordnet. classifiers trained on loss function using the hierarchical cost can learn to differentiate and predict between similar computer science & information technology (cs & it) categories when compared to those trained on - loss. the error rate for the classification of entire k categories is not conclusively stated in this work. . problem statement supervised learning based deep visual recognition cnn architectures are composed of multiple convolutional stages stacked on top of each other to learn hierarchical visual features [ ] as captured in figure . regularization approaches such as stochastic pooling, dropout, data augmentation have been utilized to enhance the recognition accuracy. recently the faster convergence of these architectures is attributed to the inclusion of rectified linear units [relu] nonlinearity into each of the layer with weights. the state of the art top error rate reported is . % for classification into k categories [ ] that utilizes the above mentioned generic architectural elements in layers with weights. unsupervised learning based architecture model as convolutional dbn learns the visual feature hierarchy by greedily training layer after layer. these architectures have reported accuracy of . % for classifying objects [ ]. both the architectures are not yet scaled for classification of k objects. we conjecture that scaling a single architecture is not realistic as the computations will get intractable if we utilize deeper architectures. figure . learning hierarchy of visual features in cnn architecture computer science & information technology (cs & it) . proposed method we employ divide & conquer principle to decompose the k classification into root & leaf level distinct challenges. the proposed architecture classifies objects in two steps as captured below: . root level model architecture: in the first step the root i.e. the first level in architectural hierarchy recognizes high level categories. this very deep vision architectural model with weight layers [ ] is trained using stochastic gradient descent [ ]. the architectural elements are captured in the table . . leaf level model architecture: in the second step, the leaf level recognition model for the recognized high level category is selected among all the leaf models. this leaf level model is presented with the same input object image which classifies it in a specific category. the leaf level architecture in the architectural hierarchy recognizes specific objects or finer categories. this model is trained using stochastic gradient descent [ ]. the architectural elements are captured in the table . cdbn based leaf level models can be trained with unsupervised learning approach in case of scarce labelled images [ ]. this will deliver a blend of leaf models trained with supervised & unsupervised approaches. in all a root level model & leaf level models need to be trained. we use imagenet k dataset [ ], which is compiled from synsets of the fall- release of imagenet. each leaf node has at least labelled images which amount to m images in total. figure . two levels – hierarchical deep learning archtecture . supervised training in vision the low level features [e.g. pixels, edge-lets, etc.] are assembled to form high level abstractions [e.g. edges, motifs] and these higher level abstractions are in turn assembled to form further higher level abstractions [e.g. object parts, objects] and so on. substantial number of the recognition models in our two-level hierarchical architecture is trained utilizing supervised training. the algorithms utilized for this method are referred to as error-back propagation computer science & information technology (cs & it) algorithm. these algorithms require significantly high number of labelled training images per object category in its data set. . . cnn based architecture cnn is a biologically inspired architecture where multiple trainable convolutional stages are stacked on the top of each other. each cnn layer learns feature extractors in the visual feature hierarchy and attempts to mimic the human visual system feature hierarchy manifested in different areas of human brain as v & v [ ]. eventually the fully connected layers act as a feature classifier & learn to classify the extracted features by cnn layers into different categories or objects. the fully connected layers can be likened to the v area of the brain which classifies the hierarchical features as generated by area v . the root level & the leaf level cnn models in our architecture are trained with supervised gradient descent based backpropagation method. in this learning method, the cross entropy objective function is minimized with the error correction learning rule/mechanism. this mechanism computes the gradients for the weight updates of the hidden layers by recursively computing the local error gradient in terms of the error gradients of the next connected layer of neurons. by correcting the synaptic weights for all the free parameters in the network, eventually the actual response of the network is moved closer to the desired response in statistical sense. table . cnn architecture layers [l] with architectural elements for root level visual recognition architecture layer no. type input size #kernels convolutional x x x x max pool x x x convolutional x x x x convolutional x x x x max pool x x x convolutional x x x x convolutional x x x x max pool x x x convolutional x x x x convolutional x x x x convolutional x x x x max pool x x x convolutional x x x x convolutional x x x x convolutional x x x x max pool x x x convolutional x x x x full-connect full-connect full-connect -softmax . . architectural elements architectural elements for the proposed architecture are: computer science & information technology (cs & it) • enhanced discriminative function: we have chosen deeper architectures & smaller kernels for the root & leaf models as they make the objective function more discriminative. this can be interpreted as making the training procedure more difficult by making it to choose the feature extractors from higher dimensional feature space. • relu non-linearity: we have utilized relu nonlinearities as against sigmoidal i.e. non- saturating nonlinearities in each layer as it reduces the training time by converging upon the weights faster [ ]. • pooling: the output of convolutional-relu combination is fed to a pooling layer after alternative convolutional layers. the output of the pooling layer is invariant to the small changes in location of the features in the object. the pooling method used is either max- pooling or stochastic pooling. max pooling method averages the output over the neighborhood of the neurons where-in the poling neighborhoods can be overlapping or non-overlapping. in majority of the leaf models we have used max pooling approach with overlapping neighborhoods. alternatively we have also used stochastic pooling method when training for few models. in stochastic pooling the output activations of the pooling region are randomly picked from the activations within each pooling region, following multinomial distribution. this distribution is computed from the neuron activation within the given region [ ]. this approach is hyper parameter free. the cnn architecture for stochastic pooling technique is captured in table . • dropout: with this method the output of each neuron in fully connected layer is set to zero with probability . . this ensures that the network samples a different architecture when a new training example is presented to it. besides this method enforces the neurons to learn more robust features as it cannot rely on the existence of the neighboring neurons. table . cnn architecture layers [l] with architectural elements for leaf level visual recognition architecture with max pooling strategy layer no. type input size #kernels convolutional x x x x max pool x x x convolutional x x x x convolutional x x x x max pool x x x convolutional x x x x convolutional x x x x max pool x x x convolutional x x x x convolutional x x x x max pool x x x convolutional x x x x full-connect full-connect full-connect -softmax computer science & information technology (cs & it) table . cnn architecture layers [l] with architectural elements for leaf level visual recognition architecture with stochastic pooling strategy layer no. type input size #kernels convolutional x x x x stochastic pool x x x convolutional x x x x convolutional x x x x stochastic pool x x x convolutional x x x x convolutional x x x x stochastic pool x x x convolutional x x x x convolutional x x x x stochastic pool x x x convolutional x x x x full-connect full-connect full-connect -softmax . . training process we modified libccv open source cnn implementation to realize the proposed architecture which is trained on nvidia gtx™ titan gpus. the root & leaf level models are trained using stochastic gradient descent [ ]. the leaf models are trained as batches of models per gpu on the two gpu systems simultaneously. the first leaf models were initialized and trained from scratch for epochs with learning rate of . , and momentum of . . the rest of the leaf models are initialized from the trained leaf models and trained with learning rate as . . the root model has been trained for epochs with learning rate of . , after having been initialized from a similar model trained on imagenet k dataset. it takes days for a batch of leaf models to train for epochs. currently the root model and / leaf models have been trained in weeks. full realization of this architecture is in progress and is estimated to conclude by second week of september’ . unsupervised training statistical mechanics has inspired the concept of unsupervised training fundamentally. specifically statistical mechanics forms the study of macroscopic equilibrium properties of large system of elements starting from the motion of atoms and electrons. the enormous degree of freedom as necessitated by statistical mechanics foundation makes the use of probabilistic methods to be the most suitable candidate for modelling features that compose the training data sets [ ]. computer science & information technology (cs & it) . . cdbn based architecture the networks trained with statistical mechanics fundamentals model the underlying training dataset utilizing boltzmann distribution. to obviate the painfully slow training time as required to train the boltzmann machines, multiple variants of the same have been proposed where the restricted boltzmann machine [rbm] is the one that has provided the best possible modelling capabilities in minimal time. the resulting stacks of rbm layers are greedily trained layer by layer [ ] resulting in the deep belief networks [dbn] that successfully provides the solution to image [ - ], speech recognition [ ] and document retrieval problem domains. dbn can be described as multilayer generative models that learn hierarchy of non-linear feature detectors. the lower layer learns lower level features which feeds into the higher level and help them learn complex features. the resulting network maximizes the probability that the training data is generated by the network. but dbn has its own limitations when scaling to realistic image sizes [ ]. first difficulty is to be computationally tractable with increasing image sizes. the second difficulty is faced with lack of translational invariance when modelling images. to scale dbn for modelling realistic size images the powerful concept of convolutional dbn [cdbn] had been introduced. cdbn learns feature detectors that are translation invariant i.e. the feature detectors can detect the features that can be located at any location in an image. we perform the block gibbs sampling using conditional distribution as suggested in [ ] to learn the convolutional weights connecting the visible and hidden layers where v and h are activations of neurons in visible & hidden layers respectively. also bj are hidden unit biases and ci are visible unit biases. w forms the weight matrix connecting the visible and hidden layer. the gibbs sampling is conducted utilizing ( ) & ( ). the weights such learnt give us the layers for convolutional rbms [crbm]. the crbms can be stacked on top of each other to form cdbn. we had probabilistic max pooling layers after convolutional layers [ ]. mlp is introduced at the top to complete the architecture. this concept is captured in figure . we train the first two layers in the leaf architecture with unsupervised learning. later we abandon unsupervised learning and use the learnt weights in the first two layers to initialize the weights in cnn architecture. the cnn architecture weights are then fine-tuned using backpropagation method. the architecture used for training with unsupervised learning mechanism is same as captured in table . also a two-level hierarchical deep learning architecture can be constructed entirely with cdbn as depicted in figure . computer science & information technology (cs & it) figure . convolutional dbn constructed by stacking crbms which are learnt one by one. . . training process we have used contrastive divergence cd- mechanism to train the first two layers of the architecture as specified for unsupervised learning. the updates to the hidden units in the positive phase of cd- step were done with sampling rather than using the real valued probabilities. also we had used mini-batch size of when training. we had monitored the accuracy of the training utilizing – . reconstruction error: it refers to the squared error between the original data and the reconstructed data. while it does not guarantee accurate training, but during the course of training it should generally decrease. also, any large amount of increase suggests the training is going wrong. . printing learned weights: the learned weighs needs to be eventually visualized as oriented, localized edge filters. printing weights during training helps identify whether the weights are “approaching” that filter-like shape. when the ratio of variance of reconstructed data to variance of input image exceeds , we decrease the learning rate by factor of . and reset the values of weights and hidden bias updates to ensure that weights don’t explode. the initial momentum was chosen to be . which is increased finally to . . the initial learning rate is chosen to be . . computer science & information technology (cs & it) figure . proposed level hierarchical deep learning architecture constructed entirely utilizing cdbns for classification/recognition of k+ objects . two-level hierarchy creation the -level hierarchy design for classification of k objects categories requires decision making on the following parameters – . number of leaf models to be trained and . number of output nodes in each leaf model. to decide these parameters, we first build a hierarchy tree out of the synsets (classes) in imagenet k dataset (as described in section . ). then using a set of thumb-rules (described in section . ), we try to split and organize all the classes into leaf models, each holding a maximum of classes . . building hierarchical tree using the wordnet is a relationship, all the synsets of imagenet k dataset are organized into a hierarchical tree. the wordnet isa relationship is a file that lists the parent-child relationships between synsets in imagenet. for example a line “n n ” in the relationship file refers to the parent synset to be n (cow) and child synset as n (heifer). however the isa file can relate a single child to multiple parents, i.e. heifer is also the child of another category n (young mammal). as the depth of a synset in imagenet hierarchy has no relationship to its semantic label, we focused on building the deepest branch for a synset. we utilized a simplified method that exploits the relationship between synset id and depth; the deeper a category nxxx the larger its number xxx. hence we used the parent category of heifer as cow, instead of young mammal. computer science & information technology (cs & it) the algorithm htree as depicted in figure a & b is used to generate the hierarchy tree. in this paper, the results from the ninth ( th) iteration are used as base tree. a sample illustration is captured in figure . . . thumb-rules for building hierarchical tree from the hierarchy tree, it is evident that the dataset is skewed towards the categories like flora, animal, fungus, natural objects, instruments etc. that are at levels closer to the tree root i.e. % of the synsets fall under % of the branches. figure a. pseudo-code for hierarchy tree generation (htree) algorithm figure b. pseudo-code for hierarchy tree generation (htree) algorithm computer science & information technology (cs & it) figure . hierarchy tree generated by htree algorithm at iterations , & for imagenet k. the categories at depth- are part of leaf- model. taking into account the number of models to be trained and the time & resources required for fine-tuning each model, the below thumb-rules were decided to finalize the solution parameters: . the ideal hierarchy will have leaf models each capable of classifying categories . the synsets for root and leaf level models have to be decided such that the hierarchy tree is as flat as possible . the total number of synsets in a leaf model should be less than and . if leaf level models have more than sub-categories under it, the remaining subcategories will be split or merged with another leaf. . . final solution parameters the final solution parameters are as follows, leaf models and with each leaf model classifying ~ synsets. . results we formulate the root & leaf models into -level hierarchy. in all a root level model & leaf level models need to be trained. each leaf level model recognizes categories that range from to in numbers. we use imagenet k dataset [ ], which is compiled from synsets of the fall- release of imagenet. each leaf node has at least labelled images which amount to m images in total. the top error rates for out of leaf level models have been computed. the graph in fig. plots the top errors of leaf models vis-à-vis the training epochs. we observe that when the leaf models are trained with higher number of training epochs the top error decreases. the top error rate for the complete k objects classification can be computed upon training of all the models as required by the -level hierarchical deep learning architecture. computer science & information technology (cs & it) figure . graph captures the decrease in top- error rate with increase in increase in number of epochs when training with supervised training method training for classification of k objects with the proposed -level hierarchical architecture is in progress and is estimated to be completed by mid of september’ . in this architecture, the root model & the / leaf models are based on cnn architecture and trained with supervised gradient descent. utilizing unsupervised learning we have trained a leaf model leaf- that consists of man-made artifacts with categories. the model for leaf- is cdbn based architecture as described in section . we have trained the first layer of this cdbn architecture with contrastive divergence (cd- ) algorithm. later on the first layer weights are utilized to initialize the weights for leaf- model in supervised setting. the same are then fine-tuned with back-propagation utilizing supervised learning. the feature extractors or kernels learnt with supervised & unsupervised learning are captured in fig. . we intend to compute the top error rates for categories using the algorithm as captured in the fig. . figures – depict the top- classification results using this -level hierarchical deep learning architecture. from top-left, the first image is the original image used for testing. the remaining images represent the top- categories predicted by the hierarchical model, in descending order of their confidence. . conclusions the top- error rate for the entire two-level architecture is required to be computed in conjunction with the error rates of root & leaf models. the realizations of this two level visual recognition architecture will greatly simply the object recognition scenario for large scale object recognition problem. at the same time the proposed two level hierarchical deep learning architecture will help enhance the accuracy of the complex object recognition scenarios significantly that otherwise would not be possible with just -level architecture. computer science & information technology (cs & it) figure . : the left image depicts the first layer numbers of * filter weights learned by leaf cd- . the right image depicts the same first layer weights after fine the trade of with the proposed model. the total size of the -level recognition models including the root & leaf models amounts to approximately gb. this size might put constraints towards on low-end devices. the same size is not a constraint when executed with high end device or cloud based recognition where ram size is higher. besides we can always split the hierarchical model between device & clou the novel device-cloud collaboration architectures. the proposed k architecture will soon be available for classifying large scale objects. this breakthrough will help enable applications as divers targeted advertisements, augmented reality, retail, robotic navigation, video surveillance, search & information retrieval from multimedia content etc. the hierarchical recognition models can be deployed and commercialized in various devices like smart phones, tv, home gateways and vr head set for various b b and b c use cases. figure . algorithm to compute top error rates for k categories as evaluated by computer science & information technology (cs & it) the first layer numbers of * filter weights learned by leaf . the right image depicts the same first layer weights after fine-tuning with back propagation. the trade of with the proposed -level architecture is the size of the hierarchical recognition level recognition models including the root & leaf models amounts to approximately gb. this size might put constraints towards executing the entire architecture end devices. the same size is not a constraint when executed with high end device or cloud based recognition where ram size is higher. besides we can always split the hierarchical model between device & cloud which paves the way for object recognition utilizing cloud collaboration architectures. the proposed k architecture will soon be available for classifying large scale objects. this breakthrough will help enable applications as diverse as drone vision, industrial robotic vision augmented reality, retail, robotic navigation, video surveillance, search & information retrieval from multimedia content etc. the hierarchical recognition models can be mmercialized in various devices like smart phones, tv, home gateways and vr head set for various b b and b c use cases. figure . algorithm to compute top error rates for k categories as evaluated by -level hierarchical deep learning algorithm the first layer numbers of * filter weights learned by leaf- utilizing tuning with back propagation. level architecture is the size of the hierarchical recognition level recognition models including the root & leaf models amounts executing the entire architecture end devices. the same size is not a constraint when executed with high end device or cloud based recognition where ram size is higher. besides we can always split the -level d which paves the way for object recognition utilizing the proposed k architecture will soon be available for classifying large scale objects. this industrial robotic vision, augmented reality, retail, robotic navigation, video surveillance, search & information retrieval from multimedia content etc. the hierarchical recognition models can be mmercialized in various devices like smart phones, tv, home gateways and level hierarchical computer science & information technology (cs & it) figure . the test image belongs to wilson's warbler. the predicted categories in order of confidence are a) yellow warbler b) wilsons warbler c) yellow hammer d) wren, warbler e) audubon's warbler figure . the test image belongs to fruit kumquat. the predicted categories in order of confidence are a) apricot b) loquat c) fuyu persimmon d) golder delicious e) kumquat figure . the test image belongs to racing boat. the top- predicted categories are a) racing boat b) racing shell c) outrigger canoe d) gig e) rowing boat computer science & information technology (cs & it) figure . the test image belongs to category oak. the top- predicted categories are a) live oak b) shade tree c) camphor d) spanish oak acknowledgements we take this opportunity to express gratitude and deep regards to our mentor dr. shankar m venkatesan for his guidance and constant encouragement. without his support it would not have been possible to materialize this paper. references [ ] yann lecun, koray kavukvuoglu and clément farabet, “convolutional networks and applications in vision,” in proc. international symposium on circuits and systems (iscas' ), ieee, [ ] a. krizhevsky ilya sutskever g. hinton. 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[ ] s. lazebnik, c. schmid and j. ponce, "beyond bags of features: spatial pyramid matching for recognizing natural scene categories", in ieee computer society conf. computer vision and pattern recognition, . authors atul laxman katole atul laxman katole has completed his m.e in jan in signal processing from indian institute of science, bangalore and is currently working at samsung r&d institute india bangalore. his technical areas of interest include artificial intelligence, deep learning, object recognition, speech recognition, application development and algorithms. he has seeded & established teams in the domain of deep learning with applications to image & speech recognition. krishna prasad yellapragada krishna prasad yellapragada is currently institute india-bangalore. his interests include deep learning, machine learning & content centric networks. amish kumar bedi amish kumar bedi has completed his b.tech in computer science from iit roorkee, batch. he is working in samsung r&d institute india his technical areas of interest include deep learning/machine learning and artificial intelligence. sehaj singh kalra sehaj sing kalra has completed his b.tech in com . he is working in samsung r&d institute india interest lies in machine learning and its applications in various domains, specifically speech and image. mynepalli siva chaitanya mynepalli siva chaitanya has completed his b.tech in electrical engineering from iit bombay, batch of . he is working in samsung r&d institute india since june . his area of interest includes neural networks and artificial intelligence. computer science & information technology (cs & it) c. r. dance, j. willamowski, l. fan, c. bray and g. csurka, "visual categorization with bags of keypoints", in eccv international workshop on statistical learning in computer vision, . ebnik, c. schmid and j. ponce, "beyond bags of features: spatial pyramid matching for recognizing natural scene categories", in ieee computer society conf. computer vision and pattern completed his m.e in jan in signal processing from indian institute of science, bangalore and is currently working at samsung r&d institute india- bangalore. his technical areas of interest include artificial intelligence, deep learning, ition, speech recognition, application development and algorithms. he has seeded & established teams in the domain of deep learning with applications to krishna prasad yellapragada is currently working as a technical lead at samsung r&d bangalore. his interests include deep learning, machine learning & amish kumar bedi has completed his b.tech in computer science from iit roorkee, batch. he is working in samsung r&d institute india- bangalore' since july . his technical areas of interest include deep learning/machine learning and artificial sehaj sing kalra has completed his b.tech in computer science from iit delhi, batch of . he is working in samsung r&d institute india - bangalore since june . his interest lies in machine learning and its applications in various domains, specifically mynepalli siva chaitanya has completed his b.tech in electrical engineering from iit bombay, batch of . he is working in samsung r&d institute india - bangalore since june . his area of interest includes neural networks and artificial c. r. dance, j. willamowski, l. fan, c. bray and g. csurka, "visual categorization with bags of keypoints", in eccv international workshop on statistical learning in computer vision, . ebnik, c. schmid and j. ponce, "beyond bags of features: spatial pyramid matching for recognizing natural scene categories", in ieee computer society conf. computer vision and pattern [pdf] vaccine-preventable diseases, immunizations, and the epidemic intelligence service. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /aje/kwr corpus id: vaccine-preventable diseases, immunizations, and the epidemic intelligence service. @article{hinman vaccinepreventabledi, title={vaccine-preventable diseases, immunizations, and the epidemic intelligence service.}, author={a. hinman and w. orenstein and a. schuchat}, journal={american journal of epidemiology}, year={ }, volume={ suppl}, pages={ s - } } 	a. hinman, w. orenstein, a. schuchat 	published 	medicine 	american journal of epidemiology during - , vaccine-preventable diseases were the topic of approximately % of all epidemic-assistance investigations by the centers for disease control and prevention. both in the united states and abroad, current and former epidemic intelligence service officers have played a critical role in describing the epidemiology of vaccine-preventable diseases, contributing to development of immunization policies, participating in the implementation of immunization programs, and establishing… expand view on pubmed academic.oup.com save to library create alert cite launch research feed share this paper citationsbackground citations view all tables and topics from this paper 	 table 	 table 	 table 	vaccination 	immunization programs 	secondary immunization 	adverse event 	contribution 	policy citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency vaccine-preventable diseases: from paediatric to adult targets. 	s. esposito, p. durando, s. bosis, f. ansaldi, c. tagliabue, g. icardi 	medicine 	european journal of internal medicine 	 	 save alert research feed focusing on the implementation of st century vaccines for adults. 	p. bonanni, g. bonaccorsi, + authors a. bechini 	medicine 	vaccine 	 	 	 save alert research feed the effects of vaccinations on public health 	caitlin reveal 	medicine 	 save alert research feed adolescents and vaccines in the western world. 	n. principi, s. esposito 	medicine 	vaccine 	 	 save alert research feed a decade of the national health mission : full immunization coverage and vaccine preventable diseases in india 	sowmya thota, dnyaneshee dudhal 	 	pdf save alert research feed editorial: epidemic-assistance investigations by the centers for disease control and prevention: the first years. 	t. o'brien 	medicine 	american journal of epidemiology 	 	pdf save alert research feed diphtheria outbreak in yemen: the impact of conflict on a fragile health system 	f. dureab, maysoon al-sakkaf, + authors a. jahn 	medicine 	conflict and health 	 	 	 	view excerpt, cites background save alert research feed frequency and erroneous usage of temporary medical exemptions and knowledge of immunization guidelines among some miami-dade county florida providers. 	alazandria r. cruze, g. zhang, l. thomas, j. alonso, lydia sandoval 	medicine 	vaccine 	 	 save alert research feed cd + subcapsular macrophage role in antigen adjuvant activity 	christina lisk, rachel yuen, jeff kuniholm, danielle antos, m. reiser, lee m. wetzler 	frontiers in immunology 	 	 save alert research feed tetanus vaccination is associated with differential dna-methylation: reduces the risk of asthma in adolescence. 	vimala devi janjanam, n. mukherjee, + authors w. karmaus 	biology, medicine 	vaccine 	 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency a statewide mass measles immunization program. 	e. byrne, b. rosenstein, a. jaworski, r. a. jaworski 	medicine 	jama 	 	 save alert research feed epidemic assistance by the centers for disease control and prevention: role of the epidemic intelligence service, – 	s. thacker, d. stroup, d. j. sencer 	medicine 	american journal of epidemiology 	 	 	pdf save alert research feed prevention of rotavirus gastroenteritis among infants and children. recommendations of the advisory committee on immunization practices (acip). 	u. parashar, j. alexander, r. glass 	medicine 	mmwr. recommendations and reports : morbidity and mortality weekly report. recommendations and reports 	 	 , 	pdf save alert research feed historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states. 	s. roush, t. murphy 	medicine 	jama 	 	 	pdf save alert research feed clinical epidemiology of sporadic measles in a highly immunized population. 	w. schaffner, a. schluederberg, e. byrne 	medicine 	the new england journal of medicine 	 	 save alert research feed intussusception among recipients of rotavirus vaccine--united states, - . 	medicine 	mmwr. morbidity and mortality weekly report 	 	 	pdf save alert research feed epidemic measles in a divided city. 	p. landrigan 	medicine 	jama 	 	 	pdf save alert research feed prevention of secondary transmission of pertussis in households with early use of erythromycin. 	m. a. sprauer, s. cochi, + authors p. a. patriarca 	medicine 	american journal of diseases of children 	 	 save alert research feed high attack rates and case fatality during a measles outbreak in groups with religious exemption to vaccination. 	d. v. rodgers, j. gindler, w. l. atkinson, l. markowitz 	medicine 	the pediatric infectious disease journal 	 	 save alert research feed immunogenicity of tetanus-diphtheria toxoids (td) among ukrainian adults: implications for diphtheria control in the newly independent states of the former soviet union. 	r. sutter, i. r. hardy, + authors m. wharton 	medicine 	the journal of infectious diseases 	 	 	pdf save alert research feed ... ... related papers 	abstract 	tables and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue manic depressive illness and tyrosine hydroxylase gene: linkage heterogeneity and association | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /nbdi. . corpus id: manic depressive illness and tyrosine hydroxylase gene: linkage heterogeneity and association @article{malafosse manicdi, title={manic depressive illness and tyrosine hydroxylase gene: linkage heterogeneity and association}, author={a. malafosse and m. leboyer and t. d'amato and s. amad{\'e}o and j. mallet}, journal={neurobiology of disease}, year={ }, volume={ }, pages={ - } } 	a. malafosse, m. leboyer, + authors j. mallet 	published 	psychology, medicine 	neurobiology of disease several studies have implicated the tyrosine hydroxylase (th) locus within the p region in susceptibility to manic depressive illness (mdi). this possibility was further investigated by both parametric (lod score) and nonparametric (affected-pedigree-member and a case-control study) methods of analysis in french mdi families and in a sample of unrelated subjects. both types of analyses corroborate the implication of this locus, and positive lod scores were obtained in two families… expand view on elsevier doi.org save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all topics from this paper 	bipolar disorder 	th gene 	depressive disorder 	 p 	genetic heterogeneity 	mixed function oxygenases 	scientific publication citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency linkage of mood disorders with d , d and th genes: a multicenter study. 	a. serretti, f. macciardi, + authors j. mendlewicz 	psychology, medicine 	journal of affective disorders 	 	 save alert research feed analysis and metaanalysis of two polymorphisms within the tyrosine hydroxylase gene in bipolar and unipolar affective disorders. 	r. furlong, j. rubinsztein, + authors d. rubinsztein 	medicine 	american journal of medical genetics 	 	 save alert research feed multicentre italian family‐based association study on tyrosine hydroxylase, catechol‐o‐methyl transferase and wolfram syndrome polymorphisms in mood disorders 	a. serretti, c. cusin, + authors g. nappi 	psychology, medicine 	psychiatric genetics 	 	 	view excerpts, cites background save alert research feed dopamine d receptor and tyrosine hydroxylase genes in bipolar disorder: evidence for a role of drd 	p. muglia, a. petronis, e. mundo, s. lander, t. cate, j. l. kennedy 	biology, medicine 	molecular psychiatry 	 	 	pdf save alert research feed no evidence of an association between tyrosine hydroxylase gene polymorphisms and suicide victims haruki hattori 	h. nushida, k. maeda 	 	pdf 	view excerpt, cites background save alert research feed no evidence of an association between tyrosine hydroxylase gene polymorphisms and suicide victims. 	h. hattori, o. shirakawa, n. nishiguchi, h. nushida, y. ueno, k. maeda 	medicine 	the kobe journal of medical sciences 	 	 	view excerpt, cites background save alert research feed genetic associations with clinical characteristics in bipolar affective disorder and recurrent unipolar depressive disorder. 	l. ho, r. furlong, j. rubinsztein, c. walsh, e. paykel, d. rubinsztein 	medicine 	american journal of medical genetics 	 	 save alert research feed searching high and low: a review of the genetics of bipolar disorder. 	j. potash, j. depaulo 	psychology, medicine 	bipolar disorders 	 	 save alert research feed post-genomic era and gene discovery for psychiatric diseases: there is a new art of the trade? 	r. meloni, n. f. biguet, j. mallet 	biology, medicine 	molecular neurobiology 	 	 save alert research feed chapter . sychiatric genetics current perspective 	d. levinson 	psychology 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency association and haplotype analysis at the tyrosine hydroxylase locus in a combined german‐british sample of manic depressive patients and controls 	j. körner, m. rietschel, + authors p. propping 	biology, medicine 	psychiatric genetics 	 	 save alert research feed absence of linkage between chromosome p markers and manic-depressive illness in a belgian pedigree. 	j. mendlewicz, m. leboyer, + authors j. mallet 	biology, medicine 	the american journal of psychiatry 	 	 save alert research feed linkage between tyrosine hydroxylase gene and affective disorder cannot be excluded in two of six pedigrees. 	l. lim, h. gurling, d. curtis, j. brynjólfsson, h. petursson, m. gill 	biology, medicine 	american journal of medical genetics 	 	 save alert research feed rflp alleles at the tyrosine hydroxylase locus: no association found to affective disorders 	j. körner, j. fritze, p. propping 	biology, medicine 	psychiatry research 	 	 save alert research feed chromosome dna markers and manic-depressive illness: evidence for a susceptibility gene. 	w. berrettini, t. ferraro, + authors e. gershon 	biology, medicine 	proceedings of the national academy of sciences of the united states of america 	 	 	pdf save alert research feed molecular genetic evidence for heterogeneity in manic depression 	s. hodgkinson, r. sherrington, + authors j. brynjólfsson 	biology, medicine 	nature 	 	 save alert research feed close linkage of c-harvey-ras- and the insulin gene to affective disorder is ruled out in three north american pedigrees 	s. detera-wadleigh, w. berrettini, l. goldin, d. boorman, stewart anderson, e. gershon 	biology, medicine 	nature 	 	 save alert research feed a possible vulnerability locus for bipolar affective disorder on chromosome q . 	r. straub, t. lehner, + authors m. baron 	biology, medicine 	nature genetics 	 	 save alert research feed tyrosine hydroxylase gene not linked to manic-depression in seven of eight pedigrees. 	w. byerley, r. plaetke, + authors m. leppert 	biology, medicine 	human heredity 	 	 save alert research feed approaches to the genetics of affective disorders. 	n. craddock, p. mcguffin 	psychology, medicine 	annals of medicine 	 	 	view excerpt, references background save alert research feed ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue moyamoya disease in a member of the roma gypsy community | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / corpus id: moyamoya disease in a member of the roma gypsy community @article{bertora moyamoyadi, title={moyamoya disease in a member of the roma gypsy community}, author={p. bertora and c. lovati and p. gambaro and a. vicenzi and s. rosa and m. osio and f. resta and c. mariani}, journal={european neurology}, year={ }, volume={ }, pages={ - } } 	p. bertora, c. lovati, + authors c. mariani 	published 	medicine 	european neurology the age of of cerebral haemorrhage (not further investigated), and a patient’s son – a heterozygote twin – had died soon after birth for unknown causes. a brain ct scan performed at admission showed several hypodense areas within the cerebral parenchyma in both hemispheres. brain mri confirmed the presence of multiple t -hypointense and t -hyperintense lesions lodear sir, moyamoya disease is a clinical entity in which occlusion of intracranial large vessels, in particular carotid… expand view on pubmed karger.com save to library create alert cite launch research feed share this paper citations view all figures and topics from this paper 	 figure 	moyamoya disease 	seizures 	intracranial hemorrhages 	hemorrhage 	representational oligonucleotide microarray analysis 	basal ganglia 	blood vessel 	hemiplegia 	autosomal dominant inheritance 	parenchyma 	community 	penetrance 	statistical cluster 	cessation of life 	partial 	carotid arteries citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency moyamoya disease: case report and literature review 	p. janda, jonathan g bellew, v. veerappan 	medicine 	the journal of the american osteopathic association 	 	 	pdf save alert research feed a case report of moyamoya disease from nonendemic region of upper part of brahmaputra valley of north eastern india 	d. das 	medicine 	 save alert research feed moyamoya disease presenting with ischemic stroke in association with diabetic ketoacidosis 	h. habib 	medicine 	 	 	view excerpt save alert research feed etiology and pathogenesis of moyamoya disease: an update on disease prevalence 	s. huang, zhen-ni guo, ming-chao shi, y. yang, m. rao 	medicine 	international journal of stroke : official journal of the international stroke society 	 	 save alert research feed review of the scientific literature on the health of the roma and sinti in italy. 	l. monasta, a. erenbourg, s. restaino, v. lutje, l. ronfani 	medicine 	ethnicity & disease 	 	 save alert research feed moyamoya disease-two case reports 	g. kundu, benzamin, + authors s. akter 	medicine 	 	 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency familial occurrence of moyamoya disease: a clinical study 	h. seol, k. wang, seungki kim, yong-seung hwang, k. kim, byung-kyu cho 	medicine 	child's nervous system 	 	 save alert research feed clinical features of probable moyamoya disease in japan 	k. ikezaki, t. inamura, t. kawano, m. fukui 	medicine 	clinical neurology and neurosurgery 	 	 save alert research feed primary and acquired forms of moyamoya syndrome. a review and three case reports. 	n. gadoth, m. hirsch 	medicine 	israel journal of medical sciences 	 	 save alert research feed neuromuscular disorders in the gypsy ethnic group. a short review. 	c. navarro, s. teijeira 	medicine, psychology 	acta myologica : myopathies and cardiomyopathies : official journal of the mediterranean society of myology 	 	 save alert research feed inheritance pattern of familial moyamoya disease: autosomal dominant mode and genomic imprinting 	y. mineharu, k. takenaka, + authors a. koizumi 	medicine 	journal of neurology, neurosurgery & psychiatry 	 	 	 save alert research feed human leukocyte antigen in patients with moyamoya disease. 	m. aoyagi, k. ogami, y. matsushima, m. shikata, m. yamamoto, k. yamamoto 	medicine 	stroke 	 	 save alert research feed high-resolution turbo magnetic resonance angiography for diagnosis of moyamoya disease 	i. yamada, t. nakagawa, y. matsushima, h. shibuya 	medicine 	stroke 	 	 	 save alert research feed high-resolution turbo magnetic resonance angiography for diagnosis of moyamoya disease 	ichiroyamada, tsuneakinakagawa, yoshiharumatsushima, hitoshishibuya 	medicine 	 	 save alert research feed hereditary motor and sensory neuropathy – lom (hmsnl): refined genetic mapping in romani (gypsy) families from several european countries 	d. chandler, d. angelicheva, l. heather, r. gooding, l. kalaydjieva 	biology, medicine 	neuromuscular disorders 	 	 	pdf save alert research feed a newly discovered founder population: the roma/gypsies. 	l. kalaydjieva, b. morar, raphaelle chaix, h. tang 	biology, medicine 	bioessays : news and reviews in molecular, cellular and developmental biology 	 	 	pdf save alert research feed ... ... related papers 	abstract 	figures and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue coverage error embedded in self-selected internet-based samples: a case study of northern indiana | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / corpus id: coverage error embedded in self-selected internet-based samples: a case study of northern indiana @article{hwang coverageee, title={coverage error embedded in self-selected internet-based samples: a case study of northern indiana}, author={yeong-hyeon hwang and d. r. fesenmaier}, journal={journal of travel research}, year={ }, volume={ }, pages={ - } } 	yeong-hyeon hwang, d. r. fesenmaier 	published 	computer science 	journal of travel research the internet is rapidly evolving as a new tool for conducting tourism research. given known advantages, steps to examine the possible drawbacks need to be taken to verify internet survey methods. this article focuses, in particular, on coverage error of self-selected e-samples. based on the response to a question regarding willingness to provide contact information on the internet, the respondents of two conventional tourism surveys were assigned to likely, somewhat likely, and not likely… expand view on sage save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations view all figures, tables, and topics from this paper 	 figure 	 table 	 table 	 table 	 table 	 table view all figures & tables 	internet 	behavior 	embedded system 	sampling (signal processing) 	demography citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency contrasting online and onsite sampling methods: results of visitor responses to a destination survey 	p. benckendorff, g. moscardo, l. murphy 	geography 	 	view excerpts, cites background save alert research feed online versus paper: format effects in tourism surveys 	s. dolnicar 	 	 	view excerpts, cites background save alert research feed can online surveys substitute traditional modes? an error-based comparison of online and on-site tourism destination surveys. 	n. kim, xiaojuan yu, z. schwartz 	economics 	 	 save alert research feed format effects in tourism surveys 	s. dolnicar 	 	 	pdf 	view excerpts, cites background save alert research feed online versus paper 	s. dolnicar, c. laesser, katrina matus 	psychology 	 	 	 	view excerpts, cites background save alert research feed non‐response bias in internet‐based advertising conversion studies 	s. park, d. fesenmaier 	economics 	 	 	 	view excerpts, cites background save alert research feed intentions to use social media in organizing and taking vacation trips 	eduardo parra-lópez, j. bulchand-gidumal, desiderio gutiérrez-taño, ricardo díaz-armas 	psychology, computer science 	comput. hum. behav. 	 	 	view excerpt, cites background save alert research feed hospitality and travel 	a. wilson, h. murphy, j. c. fierro 	business 	 	 	view excerpt, cites methods save alert research feed small tourism accomodation distribution patterns in canada 	d. gilbert, s. hudson 	business 	 save alert research feed a systematic comparison of first-time and repeat visitors via a two-phase online survey 	xiang li, chia-kuen cheng, hyoung-gon kim, j. petrick 	psychology 	 	 	pdf 	view excerpts, cites background save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency e-surveying for tourism research: legitimate tool or a researcher’s fantasy? 	s. litvin, goh hwai kar 	sociology 	 	 	view excerpts, references background save alert research feed internet-based evaluation of tourism web site effectiveness: methodological issues and survey results 	patrick t. tierney 	economics 	 	 	view excerpts, references background save alert research feed using e-mail to survey internet users in the united states: methodology and assessment 	k. sheehan, m. g. hoy 	computer science 	j. comput. mediat. commun. 	 	 	view excerpts, references background save alert research feed sampling for internet surveys. an examination of respondent selection for internet research 	n. bradley 	computer science 	 	 	highly influential 	view excerpts, references background and results save alert research feed an assessment of the generalizability of internet surveys 	s. j. best, b. s. krueger, clark hubbard, a. smith 	computer science 	 	 	view excerpts, references background save alert research feed virtual subjects: using the internet as an alternative source of subjects and research environment 	m. smith, b. leigh 	psychology 	 	 	highly influential 	pdf 	view excerpts, references background save alert research feed expanding the functional information search model 	c. vogt, d. fesenmaier 	business 	 	 	view excerpt, references background save alert research feed an exploratory study of the perceived benefits of electronic bulletin board use and their impact on other communication activities 	michaela james 	computer science 	 	 	pdf 	view excerpt, references background save alert research feed methodological variables in web-based research that may affect results: sample type, monetary incentives, and personal information 	k. o'neil, steven d. penrod 	psychology, medicine 	behavior research methods, instruments, & computers : a journal of the psychonomic society, inc 	 	 save alert research feed research methodology: taming the cyber frontier 	b. k. kaye, t. johnson 	computer science 	 	 	highly influential 	view excerpts, references background save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue book reviews journal of medical genetics, , , - medical genetic studies of the amish. selected papers by v. a. mckusick. (pp. x + ; figures + tables. £ - .) baltimore and london: johns hopkins university press. . to read this edited collection of papers is both a fascinating experience and, for one already familiar with the amish, a pleasurable reminder of the persistence and success of their remarkable way of life in th century america. a visitor to the moore clinic at johns hopkins hospital in the late s might have been excused if he had thought that the amish formed a majority of the baltimore popula- tion, but this book shows clearly how worthwhile the intensive study of this group has proved in both medical and population genetics. the papers are reproduced unchanged, but their value is greatly increased by the addition of notes on subsequent developments. they are grouped into those dealing with general and population genetics, studies of previously recognised mendelian syn- dromes, and (the largest section) 'new recessively inherited entities, many of which would have been difficult, if not impossible, to recognise outside the setting of a defined, closed, and well documented community as provided by the amish'. among these delineations of new disorders, that of 'cartilage hair hypoplasia' still stands out for its clarity and its masterly combination of clinical and genetic information. the value of the studies in this book extends far beyond the demonstration that the disorders in question are recessively inherited. the range of clinical expression produced by what clearly must be a single gene is something that is difficult to document in the more heterogeneous patients seen in most populations. the studies of autosomal recessive limb-girdle muscular dystrophy and of the 'troyer syndrome' provide excellent examples of this. another particularly impressive feature of the amish studies is the detail and accuracy of the genealogical and demographic work which underlies them, much of it due to the attachment of amish people themselves to this type of documentation. it is of interest that a number of the main contributors to the book are themselves of amish origin, and it is fortunate that the amish antipathy to further education has not prevented the emergence of those individuals to transmit the unique aspects of amish culture to the world in general. anyone working in medical genetics to whom the amish are unfamiliar should read this book without delay; it will be a revelation as to the value of studying genetic isolates. those who do know of the amish will not need encouragement to refresh their memory of the subject and gain new ideas. it should also serve as a stimulus for people to study their own equivalents of the amish. though few of us have access to such an ideal population, there are many minority groups which would repay much closer investigation, where studies of genetic diseases have not been attempted. many of these groups, less robust than the amish, may soon disappear for ever if the opportunity is not taken soon. p. s. harper british medical bulletin: the hla system vol. , no. , september . scientific editor, w. f. bodmer. (figures + tables. £ o .) london: the british council. . medical journals are now replete with articles on hla and, though weary voices have been heard to exclaim 'not hla again', we should take heart. cytogenetics had similarly inauspicious beginnings when, years ago, pictures of 'squashed spiders' began to appear in medical journals. there are many aspects of hla which are relatively simple, though others seem comprehensible only to experienced travellers in the exotic lands of lymphomania. the latest issue of the british medical bulletin provides an excellent overview of the hla system and will do much to enlighten the weary. it is a convenient introductory package for this fascinating genetic system. i recommend reading 'evolution and function of the hla system' at an early stage as, in it, central issues relating to the system are dealt with succinctly and comprehensibly. the other articles can be divided into several categories. firstly, the 'technology' of hla: serology, cellular typing, the chemistry of hla antigens, and the 'new' hla- drw locus are considered in articles. secondly, the role of hla as part of the major histocom- patibility system (mhs) is discussed in articles devoted to complement genetics and to the immune response. thirdly, the somewhat variable importance of hla in human organ transplantation is described objectively. the fourth category, that of the associa- tion between hla and various diseases, takes up a large part of the bulletin in a total of papers o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ outstanding animal studies in allergy ii. from atopic barrier and microbiome to allergen-specific immunotherapy | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /aci. corpus id: outstanding animal studies in allergy ii. from atopic barrier and microbiome to allergen-specific immunotherapy @article{jensenjarolim outstandingas, title={outstanding animal studies in allergy ii. from atopic barrier and microbiome to allergen-specific immunotherapy}, author={e. jensen-jarolim and i. pali-sch{\"o}ll and f. roth-walter}, journal={current opinion in allergy and clinical immunology}, year={ }, volume={ }, pages={ - } } 	e. jensen-jarolim, i. pali-schöll, f. roth-walter 	published 	medicine 	current opinion in allergy and clinical immunology purpose of review animal studies published within the past months were assessed, focusing on innate and specific immunomodulation, providing knowledge of high translational relevance for human atopic and allergic diseases. recent findings allergic companion animals represent alternative models, but most studies were done in mice. atopic dermatitis mouse models were refined by the utilization of cytokines like il- and relevant skin allergens or enzymes. a novel il- reporter mouse allows… expand view on wolters kluwer europepmc.org save to library create alert cite launch research feed share this paper citationsbackground citations view all topics from this paper 	allergens 	atopy 	drug allergy 	immunotherapy 	dermatitis 	intestinal microbiome 	poly iclc 	cholecalciferol 	hypersensitivity 	sensitization (observable entity) 	food allergy 	sublingual location 	skin barrier 	dermatitis, atopic 	probiotics 	poly a paper mentions blog post april , microbiome digest - bik's picks april citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency recent developments and advances in atopic dermatitis and food allergy. 	k. sugita, c. akdis 	medicine 	allergology international : official journal of the japanese society of allergology 	 	 	 save alert research feed microcrystalline tyrosine and aluminum as adjuvants in allergen-specific immunotherapy protect from ige-mediated reactivity in mouse models and act independently of inflammasome and tlr signaling 	deborah s leuthard, a. duda, + authors p. johansen 	chemistry, medicine 	the journal of immunology 	 	 	pdf save alert research feed contributions of innate lymphocytes to allergic responses 	juan m. inclan-rico, john j. ponessa, m. siracusa 	medicine 	current opinion in allergy and clinical immunology 	 	 	 save alert research feed role of the microbiome in allergic disease development 	andrea c. aguilera, isabelle a. dagher, k. kloepfer 	medicine 	current allergy and asthma reports 	 	 	 save alert research feed clinical efficacy of sublingual immunotherapy is associated with restoration of steady-state serum lipocalin after slit: a pilot study 	f. roth-walter, rené schmutz, + authors e. jensen-jarolim 	medicine 	the world allergy organization journal 	 	 	view excerpt, cites background save alert research feed comparing immediate‐type food allergy in humans and companion animals—revealing unmet needs 	i. pali-schöll, m. d. de lucía, h. jackson, j. janda, r. mueller, e. jensen-jarolim 	medicine 	allergy 	 	 	 	view excerpt, cites background save alert research feed tamanu oil potentiated novel sericin emulgel of levocetirizine: repurposing for topical delivery against dncb-induced atopic dermatitis, qbd based development and in vivo evaluation 	ravi raj pal, poonam parashar, indu r. singh, s. saraf 	medicine 	journal of microencapsulation 	 	 save alert research feed strategic outlook toward : japan's research for allergy and immunology - secondary publication. 	t. adachi, keigo kainuma, + authors m. tamari 	political science, medicine 	allergology international : official journal of the japanese society of allergology 	 	 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency the future of immunotherapy for canine atopic dermatitis: a review. 	d. deboer 	medicine 	veterinary dermatology 	 	 	pdf save alert research feed a distinct microbiota composition is associated with protection from food allergy in an oral mouse immunization model 	s. diesner, cornelia bergmayr, + authors e. untersmayr 	biology, medicine 	clinical immunology 	 	 	pdf save alert research feed house dust mite-specific sublingual immunotherapy prevents the development of allergic inflammation in a mouse model of experimental asthma 	s. hagner, c. rask, + authors h. garn 	medicine 	international archives of allergy and immunology 	 	 	 save alert research feed specific immunotherapy in combination with clostridium butyricum inhibits allergic inflammation in the mouse intestine 	y. shi, l. xu, + authors p. yang 	biology, medicine 	scientific reports 	 	 	pdf save alert research feed transplantation of human skin microbiota in models of atopic dermatitis. 	ian a. myles, k. williams, + authors s. k. datta 	biology, medicine 	jci insight 	 	 	 save alert research feed vitamin d improves the effects of low dose der p allergoid treatment in der p sensitized balb/c mice 	c. petrarca, e. clemente, + authors m. di gioacchino 	medicine 	clinical and molecular allergy 	 	 	 save alert research feed use of a canine model of atopic dermatitis to investigate the efficacy of a ccr antagonist in allergen-induced skin inflammation in a randomized study. 	c. murray, k. ahrens, + authors r. marsella 	medicine 	the journal of investigative dermatology 	 	 	 save alert research feed epicutaneous allergic sensitization by cooperation between allergen protease activity and mechanical skin barrier damage in mice. 	s. shimura, t. takai, + authors s. ikeda 	chemistry, medicine 	the journal of investigative dermatology 	 	 	 save alert research feed biodegradable antigen-associated plg nanoparticles tolerize th -mediated allergic airway inflammation pre- and postsensitization 	charles b. smarr, woon teck yap, + authors s. miller 	medicine 	proceedings of the national academy of sciences 	 	 	pdf save alert research feed longitudinal evaluation of the skin microbiome and association with microenvironment and treatment in canine atopic dermatitis. 	c. bradley, d. morris, + authors e. grice 	medicine 	the journal of investigative dermatology 	 	 	pdf save alert research feed ... ... related papers 	abstract 	topics 	paper mentions 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators blog posts, news articles and tweet counts and ids sourced by altmetric.com terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue original article variation within the gene encoding the upstream stimulatory factor does not influence susceptibility to type diabetes in samples from populations with replicated evidence of linkage to chromosome q eleftheria zeggini, , coleen m. damcott, robert l. hanson, mohammad a. karim, , n. william rayner, , christopher j. groves, leslie j. baier, terri c. hale, , andrew t. hattersley, graham a. hitman, sarah e. hunt, william c. knowler, braxton d. mitchell, maggie c.y. ng, , jeffrey r. o’connell, toni i. pollin, martine vaxillaire, mark walker, xiaoqin wang, , pamela whittaker, xiang kunsun, weiping jia, juliana c. n. chan, philippe froguel, , panos deloukas, alan r. shuldiner, steven c. elbein, , and mark i. mccarthy, , for the international type diabetes q consortium* the gene encoding the transcription factor upstream stim- ulatory factor (usf) influences susceptibility to familial combined hyperlipidemia (fchl) and triglyceride levels. phenotypic overlap between fchl and type diabetes makes usf a compelling positional candidate for the widely replicated type diabetes linkage signal on chromo- some q. we typed variants in the f r/usf region ( per kb), including those previously implicated in fchl- susceptibility (or proxies thereof) in , samples pref- erentially enriched for q linkage. we also examined glucose- and lipid-related continuous traits in an overlap- ping set of , subjects of european descent. there was no convincing evidence for association with type diabetes in any of seven case-control comparisons, individually or combined. family-based association analyses in pima subjects were similarly negative. at rs (the variant most strongly associated with fchl), the combined odds ratio, per copy of the rarer a-allele, was . ( % ci . – . , p � . ). in utah subjects, rs was significantly associated (p � . ) with triglyceride lev- els, but direction of this association was opposite to pre- vious reports, and there was no corroboration in three other samples. these data exclude usf as a major con- tributor to type diabetes susceptibility and the basis for the chromosome q linkage. they reveal only limited evi- dence for replication of usf effects on continuous meta- bolic traits. diabetes : – , p ositional cloning within regions previously high- lighted by genome-wide linkage analysis repre- sents one of the dominant strategies for identification of sequence variants influencing individual risk of type diabetes. a region of chromosome q (from to mb on the ncbi assembly) has emerged as among the strongest signals ( ) with linkage to type diabetes demonstrated in diverse populations of european ( – ), east-asian ( , ), native-american ( ), and african-american origin (s.c.e., unpublished observa- tions). moreover, signals for several diabetes-related traits have also been mapped to this region ( – ). the inter- national type diabetes q consortium represents a collaborative effort between many of the groups with q linkage signals to identify the variant(s) responsible. in addition to dense-map indirect linkage disequilibrium (ld) mapping of the region, the most compelling positional candidates are being targeted for detailed analysis. the gene encoding upstream stimulatory factor (usf) is one such candidate. usf maps in the middle of the region of interest (� mb), close to the peak of linkage in several of the type diabetes-linked populations from the oxford centre for diabetes, endocrinology and metabolism, university of oxford, oxford, u.k.; the wellcome trust centre for human genetics, university of oxford, oxford, u.k.; the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland; the phoenix epidemiology and clinical research section, national institute of diabetes and digestive and kidney diseases, phoenix, arizona; the endocrinology section, medical service, central arkansas veterans health- care system, little rock, arkansas; the division of endocrinology and metabolism, department of internal medicine, college of medicine, university of arkansas for medical sciences, little rock, arkansas; the institute of clinical and biomedical science, peninsula medical school, exeter, u.k.; the centre for diabetes and metabolic medicine, bart’s and the london queen mary’s school of medicine and dentistry, london, u.k.; the wellcome trust sanger institute, hinxton, u.k.; the department of medicine, university of chicago, chicago, illinois; the department of medicine and therapeutics, chinese university of hong kong, shatin, hong kong; the institut de biologie de lille, lille, france; the department of medicine, university of newcastle, newcastle, u.k.; the department of endocrinology and metabo- lism, shanghai diabetes institute, shanghai jiaotong university no. people’s hospital, shanghai, china; and the faculty of life sciences, imperial college, london, u.k. address correspondence and reprint requests to mark mccarthy, robert turner professor of diabetes, oxford centre for diabetes, endocrinology and metabolism, university of oxford, churchill hospital, old road, headington, oxford ox lj, u.k. e-mail: mark.mccarthy@drl.ox.ac.uk. received for publication january and accepted in revised form june . *a list of additional members of the consortium can be found in the acknowledgments. additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. di, disposition index; fchl, familial combined hyperlipidemia; fsigt, frequently sampled intravenous glucose tolerance test; ld, linkage disequilib- rium; maf, minor allele frequency; snp, single nucleotide polymorphism; usf, upstream stimulatory factor. doi: . /db - © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , september ( , , , ). the gene encodes a ubiquitously expressed member of the basic helix-loop-helix leucine zipper tran- scription family, which, either as a homodimer or through heterodimerization with the related usf protein, binds the e-box motif found in the promoters of many genes involved in glucose and lipid metabolism ( , ). although the relative functional importance of usf and usf (and of sterol regulatory element– binding protein , which interacts with the same motif) remains unclear ( , ); considerable evidence implicates the usfs in the regula- tion of key metabolic genes including liver pyruvate kinase ( ), fatty acid synthase ( , ), glucokinase ( ), and pyruvate dehydrogenase complex component x ( ). the biological plausibility of usf as a positional candidate for type diabetes is enhanced by evidence implicating the gene in susceptibility to familial combined hyperlipidemia (fchl) ( , ). in finnish pedigrees seg- regating fchl, in which prior evidence for q linkage had been reported ( ), several single nucleotide polymor- phisms (snps) within usf (and to a lesser extent, within the neighboring f r [or jam] gene) were associated with fchl and triglyceride levels. though no single caus- ative variant was identified, the strongest associations involved the common allele at two highly correlated snps, rs (within the � untranslated region) and rs (within intron ). these findings were recently replicated in families from utah ( ). given the strong phenotypic overlap between fchl and type diabetes (which is characterized by an fchl-like dyslipidemia) and growing suggestions that the �-cell dysfunction and insulin resistance of type diabetes may, in fact, be secondary to disturbances of lipid metabolism, usf emerges as a strong candidate vis-à-vis the q linkage signal. there have been only limited studies addressing the role of usf variation and glucose metabolism to date. in young men from the european atherosclerosis research study, putt et al. ( ) tested three intronic usf snps (one of which, rs , was in complete ld with the snps displaying the strongest fchl association signal in finns) reporting weak haplotypic associations with glu- cose levels during an oral glucose tolerance test. a recent study in hong kong chinese reported associations of usf snps (including rs ) with type diabetes and metabolic syndrome in one case-control analysis (using family cases showing q linkage), but these were not replicated in a second chinese sample ( ). most recently, a study of eight usf snps (including rs and rs ) reported no association with type diabetes in , french case-control samples ( ). the present study has made use of dense-map, large- scale genotyping data from the consortium’s mapping efforts on chromosome q to evaluate the influence of usf variation on type diabetes risk and related meta- bolic traits, making explicit use of populations and sam- ples selected because of their prior evidence for linkage to the region. research design and methods we used three complementary approaches to the analysis of usf variation: ) a large multiethnic case-control analysis in seven q-linked populations, ) family-based association analyses in an enlarged pima dataset, and ) analysis table characteristics of the samples studied n male (%) ethnicity age at diagnosis or age at study (years)* bmi (kg/m ) u.k. sibpair probands european descent . � . . � . control subjects european descent — — french diabetic case subjects european descent . � . . � . control subjects european descent . � . * . � . shanghai sibpair probands east asian . � . . � . chinese control subjects east asian . � . * . � . hong kong sibpair probands east asian . � . . � . control subjects east asian . � . * . � . utah case subjects european descent . � . . � . control subjects european descent . � . * . � . intermediate trait european descent . � . * . � . amish familial case subjects european descent . � . . � . control subjects european descent . � . * . � . other samples european descent . � . * . � . pima early-onset case subjects native american . � . . � . elderly control subjects native american . � . * . � . other family members native american . � . . � . . � . * arkansas intermediate trait european descent . � . * . � . data as means � sd. age at diagnosis was taken for case subjects only. *age at study was taken for control subjects or other nondiabetic subjects. usf and type diabetes diabetes, vol. , september of diabetes-related quantitative traits in amish, utah, and arkansas subjects of european origin. multiethnic case-control study. we examined , case-control samples from seven populations with evidence for q linkage (table and online appendix [available at http://diabetes.diabetesjournals.org]). for five of the populations, cases included probands from some (u.k.) or all (french, shanghai, hong kong, and utah) pedigrees from the relevant genome scans ( – , , ), allowing us to exploit the increase in power when cases are selected for membership of a multiplex sibship ( ). in french and utah samples, additional cases were recruited from other sources (online appendix). in these five samples, cases were unrelated, and ethnically matched locally recruited control subjects were used (online appendix). all of the amish and a quarter of the pima case-control samples were recruited from the pedigrees typed in the relevant linkage studies ( , ). the pima case-control sample compared case (age of diagnosis � years) with control subjects (nondiabetic after age years). no case or control subject was a first-degree relative of another individual in the sample. the amish case-control set consisted of type diabetic case and control subjects (normal glucose tolerance beyond the age of years). given sustained endogamy within amish communities, ascertainment of unrelated case and control subjects was not feasible. thus, analyses in the amish take account of the correlations among related individuals (see below). differences in clinical parameters of case and control subjects (table ) are consistent with recognized ethnic and geographic variation in anthropometry and the clinical presentation of type diabetes (for example, the early onset of diabetes in pima indians and the relatively low adiposity of chinese subjects). note that the hong kong and french samples reported here represent a subset of those separately analyzed for usf variation at selected snps by ng et al. ( ) ( case and control subjects in common) and gibson et al. ( ) ( case and control subjects in common). family-based association study. a further pima samples from the original linkage pedigrees were typed and, with individuals from the case-control sample, analyzed using family-based association methods. these pima samples include type diabetic subjects (diagnosed before age years), nondiabetic siblings (aged � years), and parents (to reconstruct family relationships). quantitative trait study: amish. in addition to case-control samples, the q consortium genotyped a further amish individuals from the same pedi- grees, including with impaired glucose tolerance and/or impaired fasting glucose and “linking” individuals to allow pedigree reconstruction. fasting levels of (serum) total and hdl cholesterol and triglycerides (quest diagnos- tics, baltimore, md) were available for of individuals, including type diabetic case subjects, subjects with normal glucose tolerance, subjects with impaired glucose homeostasis, and of the linking subjects ( with normal and with unknown glucose tolerance). utah and arkansas. to examine the impact of usf snps on insulin sensitivity (si), insulin secretion (airg), and �-cell function (disposition index [di]), we analyzed two samples. the first consisted of nondiabetic members of utah families with evidence of chromosome q linkage ( , ). the second sample included unrelated arkansas caucasians with normal glucose tolerance tests, ascertained primarily for age – years. character- istics of the latter population, not previously reported, are summarized in table . all utah subjects and of those from arkansas underwent a tolbut- amide-modified frequently sampled intravenous glucose tolerance test (fsigt) ( , ). subjects on lipid-lowering medications were excluded from analysis. tolbutamide became unavailable during the arkansas study, and the remaining were studied with an insulin-modified fsigt. after baseline sampling, the fsigt was initiated with a . g/m % dextrose bolus over min; a further samples were collected over h. at min, subjects were given tolbutamide ( mg/m ) or insulin ( . units/kg). if the glucose between and min differed by � . mmol/l or showed a clear upward or downward trend, sampling was continued to min. in addition, we analyzed trait data from those members of the utah case-control population (see above) with bmi, cholesterol, and triglyceride measures ( case and control subjects). subjects from this group on lipid-lowering medications were not excluded from analyses. for utah samples, insulin was measured with a radioimmunoassay with appreciable proinsulin crossreactivity. for arkansas samples, insulin was measured by the general clinical research center core laboratory using an insulin-specific immunochemilluminometric assay (mlt assay, wales, u.k.). plasma glucose was measured by a glucose oxidase assay, and blood lipids were measured using standard clinical assays by labcorp (burlington, nc). ethical information. all samples were ascertained with written informed consent under protocols approved by respective local ethics committees or institutional review boards. use of samples for q consortium purposes was, where necessary, supplemented by further specific approval. the oxfordshire local research ethics committee assessed all ethical documentation and provided specific approval for each sample genotyped in the u.k. snps and genotyping. q consortium samples were typed for snps covering usf and f r (fig. ). these included snps typed as part of the consortium’s effort to cover the q region of interest at kb density and snps specifically chosen given the fchl association data ( ). all snps were typed using the illumina golden gate assay ( ) at the wellcome trust sanger institute, as part of two -plex assays. the utah and arkansas intermediate trait samples were independently genotyped at the university of arkansas using pyrosequencing (psq- ; biotage, uppsala, sweden). using genotypes gathered in utah subjects for usf snps selected from prior data ( ), six tag snps (rs , rs , rs , rs , rs , and rs ) were defined using pairwise ld methods (ldselect; r � . ) ( ). further details of primer sequences and assay conditions are available from the authors. data quality control. consortium genotyping data from the illumina plat- form were subjected to extensive quality control, which required snps to pass criteria for call rates, illumina quality score, and duplicate error rates. we also tested for departures from hardy-weinberg equilibrium and expected allele frequencies, for excessive within-sample plate-to-plate variation, and, in pima and amish samples, for mendelian inconsistency. as a result, snps rs and were excluded from analysis in all populations; rs from pima, french, shanghai, and u.k. populations; rs from french, shanghai, and u.k. populations; and rs from the hong kong popula- tion. for snps passing this quality control pipeline, we estimate, on the basis of � , cryptic duplicate genotypes, an overall genotyping error rate of � . %. statistical methods multiethnic case-control study. for the case-control samples, between- group differences in genotype frequency were evaluated on a population- specific basis using standard contingency table methods with the additive model as default. exact p values were calculated, where necessary, using stata se version and statxact (cytel, cambridge, ma). in the amish sample, population structure results in substantial relatedness within (and between) case and control subjects; failure to allow for this will underestimate the variance and upwardly bias the test statistic. we therefore repeated case-control comparisons in the amish by modeling a residual familial effect as a random effect using solar. specifically, we used the measured genotype approach to assess the effect of genotype on diabetes status while simulta- neously estimating the residual correlations in phenotype among related individuals ( ). single-point data from the seven case-control samples were combined using the mantel-haenszel fixed-effects method (stata se version and statxact). for these analyses, we first established the homogeneity of odds ratios (ors) across combined strata before generating combined ors under dominant, additive, and recessive models. the mantel-haenszel method assumes independence of case and control data. to account for departures from this assumption in the amish, this analysis was repeated using an inverse variance-based method ( ). the logarithm of the or (logor) (under the additive model) and its variance were calculated for each population by logistic regression and the study-wide common logor estimated as the weighted sum of the study-specific values, with weights taken as the inverse of the variance estimates. in the amish, the corrected value of the logor variance was taken as the p value for the measured genotype approach using the wald statistic. pairwise measures of ld were calculated, and haplotype analyses were performed using haplotype trend regression ( ) implemented in helixtree (goldenhelix, bozeman, mt). family-based association analysis. analysis of the extended pima data set (n � ) was conducted in two ways. first, case-control comparisons were made across the full resource (using a cutoff for age at diagnosis of years) using binomial generalized estimating equations to control for family membership. second, we conducted an explicit family-based association analysis using the method of abecasis ( ) to partition the association into between- and within-family components. quantitative trait analyses. in the amish, full pedigree structure was explicitly specified and analyses conducted in a regression framework with trait measures as the dependent variable. in utah and arkansas datasets, si was estimated from glucose and insulin measures during the fsigt using the minmod (utah) or minmod millennium (arkansas) programs ( , ). airg was calculated as the mean insulin response above basal from to min following the glucose bolus and di as si � airg. genotypic effects on si, airg, di, and lipids were tested using mixed-effect general linear models in spss version . primary tests were conducted under an additive model unless homozygosity for the rare allele was observed in � cases, in which case a recessive model for the common allele was tested. skewed variables (si, airg, di, bmi, triglycerides, and free fatty acid levels) were ln-transformed to normality before analysis. all models included age (and bmi, as appropriate) e. zeggini and associates diabetes, vol. , september as covariates and sex and genotype as fixed factors. additionally, diagnosis and pedigree membership were included in analysis of family members from the utah study and protocol type (tolbutamide or insulin) as a fixed factor for arkansas subjects. the significance of associations detected was examined by comparison of marginal means using the least significant difference test. we considered p � . to be significant throughout without correction for multiple testing. results multiethnic case-control study. following quality con- trol, between and (depending on the population) of the original snps (covering a -kb range) were avail- able for analysis. within the immediate usf region (� kb), complete data were available from nine snps. these include three of four snps showing association in the finnish fchl study ( ) (rs , rs , and rs but not rs for which assay design failed). using deep resequencing and genotype data from seattle snps (http://pga.mbt.washington.edu/), these typed snps discriminate over % of all usf haplotypes observed in chromosomes of european origin (snps with minor allele frequency [maf] � %) and tag � % of all snps with maf � % (the exception being rs and its associated haplotype, both with a frequency of %) with a r � . . the untyped fchl-associated snp (rs ) is in almost complete ld with rs in samples of european descent ( , ). ld plots for the seven samples are shown in online appendix table . single-point results for each of the seven case-control populations are summarized in fig. and detailed in online appendix table . the amish case-control data shown were derived using the measured genotype ap- proach to allow for family structure ( ). there was no suggestion of association with rs in any popula- tion. in the u.k. case-control analysis, nominally signifi- cant (p � . ) associations were seen for several variants, including a pool of highly correlated snps within f r and rs and rs within usf but none was replicated in the other samples (see fig. ). in the hong kong sample, there was nominal evidence of an association with type diabetes at rs (p � . , additive model); again, this was not substantiated in other samples. finally, haplotype analyses of the nine core usf fig. . single-point case-control association analyses for the usf gene region. the central part of the figure shows the regional gene structure (for usf , f r, and arhgap ), the snps typed, and those associated with fchl or triglycerides (pajukanta et al. [ ]), as well as the snps attempted in this study (n � ) and those successfully typed in at least one population (n � ). snps rs and are shown in parentheses, as they failed quality control in all populations. the three asterisked snps failed only in some populations. the right panel displays the case-control association p values for the four samples from european descent. for the amish, these are corrected for family structure by the measured genotype method. the left panel displays the case-control association p values for the east-asian and pima samples. the dotted vertical lines define p � . (�log p � . ). usf and type diabetes diabetes, vol. , september snps (from rs to rs ) showed no associa- tion with type diabetes in any of the populations studied (online appendix table ). analyses combining data from these seven case-control comparisons are summarized in table (additive model only). at rs , no evidence for association was observed under the additive (table ), dominant (for the rarer allele a: or . [ % ci . – . ], p � . ), or recessive ( . [ . – . ], p � . ) models. nominally significant associations under the additive model were seen for a cluster of highly correlated variants in f r and for snps and within usf (generating ors between . and . ). with the exception of rs (model recessive for g, p � . ), combined analyses under the dominant and recessive models were not significant (data not shown). family-based association study. the same set of snps were also typed in the full pima dataset (n � ). case-control analysis (allowing for family structure) re- vealed no significant associations (online appendix table ). the most significant p value was seen at rs (maf . in control subjects, . in cases, p � . ). at rs , the equivalent allele frequencies were . (unaffected) and . (affected) (p � . ). the explicit test of within-family association was again nonsignificant for all snps (including rs , p � . ). quantitative trait study. in the full set of amish subjects ( with trait measures), there was no evidence of association with lipid or adiposity traits. for example, at rs , there was no hint of association with lipid (triglycerides p � . ; hdl cholesterol p � . ) or anthropometric (bmi p � . ; waist-to-hip ratio p � . ; leptin p � . ) measures. the utah and arkansas fsigt subjects were genotyped separately for six tag snps. no associations with si, airg, di, or free fatty acid levels were seen among the nondiabetic utah family members. however, in this sam- ple, several snps (rs , rs , and rs ) showed associations with cholesterol levels (p � . , p � . , and p � . , respectively) when corrected for age, sex, bmi, and family membership (table ). further- more, snps rs and were associated with triglyceride levels (p � . and p � . , respectively). crucially, however, it is the rare allele (a) at rs that is associated with raised triglycerides (in contrast with other reports [ , ]). in the second fsigt sample (arkansas), we found no significant associations with mea- sures of si, �-cell function, glycemia, or lipids for any snp. finally, we tested each of these six snps in the utah case-control subjects with bmi, cholesterol, and triglycer- ide measures ( case and control subjects) after adjusting for bmi (in the lipid analyses), sex, and diabetes status. in contrast to the younger nondiabetic utah family members, we found no association of any usf snp with cholesterol or triglyceride concentrations. discussion the main finding is that analysis of � , subjects (using case-control and family-based association methods) found no reproducible evidence that variation within the usf gene (or its neighbor f r) is associated with type diabetes. though several nominally significant associa- tions were found in the case-control study, these were not observed in the family-based association analysis. in any event, the variants displaying these associations have low prior odds for functional involvement (since none was strongly associated with fchl) ( ), and the estimated effect sizes are modest, particularly in the context of the q consortium’s ongoing ld mapping efforts across the re- gion, which have already identified many variants display- ing far stronger replicated associations with type table single snp analyses for association with type diabetes for all seven samples combined additive additive (inverse variance) rs no. ncbi position alleles ( , ) or ( % ci)* p* or ( % ci)† p† c,t . ( . – . ) . . ( . – . ) . a,g . ( . – . ) . . ( . – . ) . c,t . ( . – . ) . . ( . – . ) . a,g . ( . – . ) . . ( . – . ) . c,g . ( . – . ) . . ( . – . ) . c,t . ( . – . ) . . ( . – . ) . a,g . ( . – . ) . . ( . – . ) . c,t . ( . – . ) . . ( . – . ) . c,g . ( . – . ) . . ( . – . ) . g,t . ( . – . ) . . ( . – . ) . a,g . ( . – . ) . . ( . – . ) . c,t . ( . – . ) . . ( . – . ) . c,g . ( . – . ) . . ( . – . ) . a,g . ( . – . ) . . ( . – . ) . a,g . ( . – . ) . . ( . – . ) . c,g . ( . – . ) . . ( . – . ) . c,t . ( . – . ) . . ( . – . ) . a,g . ( . – . ) . . ( . – . ) . c,g . ( . – . ) . . ( . – . ) . a,t . ( . – . ) . . ( . – . ) . allele designations ( , ) are in line with illumina convention (i.e., alphabetical for base name; see third column). *p values based on assumptions of independence. where appropriate (any cell with fewer than six observations), exact methods were used. or and % cis are calculated per copy of allele (with reference to genotype ) by modification of the method by liu and agresti ( ). †p values estimated using inverse-variance method to take into account of lack of independence in the amish sample. or is calculated per copy of allele . e. zeggini and associates diabetes, vol. , september diabetes ( ). crucially, the direction of these weak asso- ciations (favoring haplotypes carrying the more rare allele at rs ) is opposite to that reported in previous studies ( – ). while we cannot formally exclude the possibility that these nominal associations reflect true susceptibility effects (perhaps involving ld with other untyped variants), it is clear that previously reported type diabetes susceptibility effects involving rs and other usf variants ( , ) are not substantiated in this larger, more informative dataset in which cases were selected to be enriched for evidence of linkage to the usf region. in any study of this type, it is vital to be explicit about the extent to which the variants analyzed allow exclusion of effects at other untyped variants. in terms of capturing common variation in the usf region, the snps typed provide excellent coverage, as shown by their high density (close to one per kilobase in the usf coding region), their documented capacity to tag hidden variation (at least in europeans and east asians), and by comparison to variants typed in previous studies ( , , ). recent evi- dence ( ) suggests that rs has the best credentials as a functional etiological variant within usf . though we were unable to generate a working assay for this snp, it is clear (from genotypes in finns [ ], french [ ], and caucasians [http://pga.mbt.washington.edu/]) that rs and rs are in almost complete ld (r exceeding . ) in europeans. ld patterns in the region suggest the same is likely to be true in the other popula- tions studied. thus, failure to type this snp directly will have had minimal adverse effect on power. as with any indirect association study using common tag snps, we cannot exclude a role for rare variation within the gene (mafs � %) ( ). in terms of effect size, our study focused on populations with documented q linkage and preferentially sampled cases from multiplex families contributing to that linkage signal. both maneuvers substantially boost power to de- tect variation contributing to the linkage signal ( , ). taking these factors into account, we estimate that the current sample has (for 	 � , maf of %, multipli- cative model), % power for a variant with an allelic or of . and % for an or of . . given the relative power of linkage and association approaches when the etiologi- cal variants are typed ( ), we can demonstrate, using reasonable assumptions, over % power to detect a variant contributing to at least % of the q linkage signal. while the conclusions regarding usf variation and type diabetes seem clear, interpretation of the findings with respect to the other traits (especially lipid parame- ters) is less straightforward. in one sample ( nondia- betic relatives of type diabetic subjects from utah), we did detect statistically strong associations with certain usf snps (including rs ). however, the associa- tion between rs and triglycerides lies in the oppo- site direction to that described in finns ( ) and a different set of utah pedigrees ( ), and none of these associations could be replicated in the other samples (combined n � , ) or in a recent study of usf variation in french subjects ( ). the possibility should also be considered that usf variants having modest effects on lipid param- eters in other populations may be relatively less penetrant in the amish, owing to the generally higher degree of physical activity in this population. while the analysis of lipid traits was not a major aim of our study, our data certainly suggest that the trait associations evident in several previous studies ( – ) are not reproducible in all other samples. despite the strong biological candidacy of usf and its location within the well-replicated peak of linkage on chromosome q, we conclude, with confidence engen- dered by the large sample size, comprehensive capture of common variation within and around the gene (and the detailed analytical plan) that common variation within usf has no discernible impact on type diabetes sus- ceptibility and that usf makes no detectable contribu- tion to the replicated linkage signal on chromosome q. table metabolic traits by usf genotype in utah family members snp (rs) allele , model (with respect to allele ) , (n) trait values for / , (or combined group) (n) trait values for , (or combined group) , (n) trait values for , p value cholesterol (mmol/l) c,g general . � . . � . . � . . dominant . � . . � . — . recessive — . � . . � . . a,g general . � . . � . . � . . dominant . � . . � . — . c, t general . � . . � . . � . . dominant . � . . � . — . triglycerides (mmol/l) a,g general . ( . – . ) . ( . – . ) . ( . – . ) . a,g general . ( . – . ) . ( . – . ) . ( . – . ) . recessive — . ( . – . ) . ( . – . ) . data are marginal means � se or means ( % ci). marginal means, adjusted for age, ln(bmi), sex, and pedigree membership are shown by genotype under general (three genotypes compared), dominant, and recessive models (with respect to allele as designated in the second column). all values are in mmol/l. to convert to mass units, multiply cholesterol by . and triglycerides by . . p values are shown for comparison of marginal means using the least significant difference test with df under general and df under dominant or recessive models. triglycerides, which were ln-transformed for analysis, were converted back to a linear scale. only statistically significant findings at p � . are shown. usf and type diabetes diabetes, vol. , september acknowledgments the principal funding was provided as a supplement to national institute of diabetes and digestive and kidney diseases award u -dk . other major support has been provided by the national institutes of health (t - ag , r -dk , k -dk , k -ca , r - dk , and intramural funds), the university of maryland and arkansas general clinical research cen- ters, the national center for research resources (m rr ), the department of veteran affairs, and the american diabetes association (u.s.); “ familles pour vaincre le diabète et l’obésité” and the association fran- çaise des diabétiques (france); diabetes uk (u.k.); the hong kong research grants committee (cuhk / m; / c), the chinese university of hong kong strategic grant program (srp ) and the hong kong innovation and technology support fund (its/ / ) (hong kong); and the national nature science foundation of china ( ), the shanghai medical pioneer development project ( - - ; ), and the shanghai science tech- nology development foundation ( zb ) (china). additional funding and support is detailed in the online appendix. we thank all the subjects participating in this study and the involvement of the following additional members of the q consortium: nancy cox (chicago); clifton bogar- dus, michal prochazka, and peter kovacs (phoenix); lon cardon, steven wiltshire, simon fiddy, and richard mott (oxford); christian dina (lille); mao fu and mona sabra (baltimore); winston chu (little rock); alan herbert, josee dupuis, james meigs, and adrienne cupples (bos- ton); and rudy leibel and wendy chung (new york). references . mccarthy mi: growing evidence for diabetes susceptibility genes from genome scan data (review). curr diab rep : – , . elbein sc, hoffman md, teng k, leppert mf, hasstedt sj: a genome-wide search for type diabetes susceptibility genes in utah caucasians. diabetes : – , . vionnet n, hani eh, dupont s, gallina s, francke s, dotte s, de matos f, durand e, lepêtre f, lecouer c, gallina p, zekiri l, dina c, froguel p: genomewide search for type diabetes-susceptibility genes in french whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome q -qter and independent replication of a type -diabetes locus on chromosome 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a, saarela j, koistinen ha, pajukanta p, taskinen m-r, peltonen l: usf and dyslipidemias: converg- ing evidence for a functional intronic variant. hum mol genet : – , . zeggini e, rayner w, morris a, hattersley a, walker m, hitman g, deloukas p, cardon l, mccarthy mi: hapmap sample size and tagging snp performance: an evaluation in large-scale empirical and simulated data sets. nat genet : – , . fingerlin tl, boehnke m, abecasis gr: increasing the power and effi- ciency of disease-marker case-control association studies through use of allele-sharing information. am j hum genet : – , . risch n, merikangas k: the future of genetic studies of complex human diseases. science : – , usf and type diabetes diabetes, vol. , september wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . 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elbein, maggie c.y. ng, ravindranath duggirala, rector arya, giuseppina imperatore, adebowale adeyemo, toni i. pollin, wen-chi hsueh, juliana c.n. chan, charles rotimi, robert l. hanson, sandra j. hasstedt, johanna k. wolford, and the american diabetes association gennid study group* dyslipidemia is a major risk factor for coronary heart disease, which is the predominant cause of mortality in individuals with type diabetes. to date, nine linkage studies for quantitative lipid traits have been performed in families ascertained for type diabetes, individually yield- ing linkage results that were largely nonoverlapping. dis- crepancies in linkage findings are not uncommon and are typically due to limited sample size and heterogeneity. to address these issues and increase the power to detect linkage, we performed a meta-analysis of all published genome scans for quantitative lipid traits conducted in families ascertained for type diabetes. statistically sig- nificant evidence (i.e., p < . ) for linkage was ob- served for total cholesterol on q . -q . ( . – cm; p � . ), p . -p ( . – . cm; p � . ), p -q . ( . – . cm; p � . ), and q . - q . ( . – . cm; p � . ), as well as ldl on p . -p (p � . ). suggestive evidence (i.e., p < . ) for linkage was also observed for ldl on p - q . , triglycerides on p -q . ( . – . cm), tri- glyceride/hdl on p -q . and p -q . , and ldl/ hdl on q . -q . ( . – . cm) and p -q . . linkage for lipid traits has been previously observed on both chromosomes and in several unrelated studies and, together with the results of this meta-analysis, pro- vide compelling evidence that these regions harbor impor- tant determinants of lipid levels in individuals with type diabetes. diabetes : – , c oronary heart disease (chd) is the leading cause of death in individuals with type diabe- tes ( ). the high prevalence of chd among these individuals is largely due to dyslipidemia, which is influenced by both environmental and genetic factors ( ). over genome scans have been performed to identify loci for lipid levels, including nine linkage studies of quantitative lipid traits performed in ethnically diverse populations of families ascertained for type diabetes ( – ). common chromosomal regions have been identi- fied in some of these studies; for example, linkage on chromosome was observed for triglyceride levels in families with northern-european ancestry ( ) and total cholesterol levels in pima indians from arizona and afri- can americans from the genetics of niddm (gennid) study ( , ). similarly, linkage on chromosome was observed for ldl cholesterol levels in an old order amish population ( ) and for triglyceride/hdl levels in cauca- sian families from the gennid study ( ). however, most of the regions observed in these genome scans have not been overlapping, including linkage on q and p for plasma triglycerides and hdl cholesterol, respectively, in mexican-american families ( , ); chromosome for hdl cholesterol in africans ( ); chromosome for triglycerides in a chinese population from hong kong ( ); and chromo- some for triglyceride/hdl cholesterol in hispanics from the gennid study ( ). while some of these analyses yielded logarithm of odds (lod) � . , the majority of lod scores reported in these studies were well below the threshold normally considered statistically significant. while disparities in linkage findings for lipid traits in families ascertained for type diabetes and lack of statis- tically significant evidence for linkage can result from both locus heterogeneity and utilization of different study de- signs, in most cases, they are most commonly attributable to inadequately sized study samples. in the nine genome scans for lipid traits conducted in families with type diabetes, sample sizes ranged from to individuals, and most of these studies had low power to detect statisti- from the diabetes and obesity research unit, translational genomics research institute, phoenix, arizona; the endocrinology section, university of arkansas, little rock, arkansas; the department of medicine and thera- peutics, chinese university of hong kong, shatin, hong kong; the depart- ment of genetics, southwest foundation for biomedical research, san antonio, texas; the division of clinical epidemiology, university of texas health sciences center, san antonio, texas; the division of diabetes translation, centers for disease control and prevention, atlanta, georgia; the national human genome center, howard university, washington, dc; the division of endocrinology, diabetes, and nutrition, university of maryland, baltimore, maryland; the department of medicine, university of california san francisco, san francisco, california; the diabetes and arthritis epide- miology section, national institute of diabetes and digestive and kidney diseases, phoenix, arizona; and the human genetics department, univer- sity of utah, salt lake city, utah. address correspondence and reprint requests to alka malhotra, diabetes and obesity research unit, genetic basis of human disease, translational genomics research institute, n. th st., phoenix, az . e-mail: amalhotra@tgen.org. received for publication july and accepted in revised form december . *a complete list of the american diabetes association gennid study group members can be found in the acknowledgments. aadm, africa-america diabetes mellitus; afds, amish family diabetes study; chd, coronary heart disease; gennid, genetics of niddm; gsma, genome scan meta-analysis; hkfds, hong kong family diabetes study; lod, logarithm of odds; safads, san antonio family diabetes study. doi: . /db - © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , march cally significant evidence for linkage. the goal of this study, therefore, was to overcome many of the limitations associated with detecting statistically significant evidence of linkage for a complex trait and augment the power to detect statistically significant evidence of linkage for lipid traits in families with type diabetes. to this end, we combined lod scores from each of the nine genome scans using a meta-analysis approach. this study is the first meta-analysis of linkage results for quantitative lipid traits measured in families ascertained for type diabetes and represents the largest genetic linkage study of lipid traits conducted to date in a population with diabetes. research design and methods characteristics of participants from the nine populations used in the present study are shown in table . linkage results for quantitative lipid traits (triglycerides, total cholesterol, ldl, hdl, triglyceride/hdl, and ldl/hdl) were used from the following populations with families ascertained for type diabetes: african-american families, caucasian families, and hispanic families from the gennid study ( ); chinese nuclear families from the hong kong family diabetes study (hkfds) ( ); ( siblings), ( siblings), and ( siblings) pima indian families for total cholesterol, hdl cholesterol, and triglycerides, respectively, from the gila river indian community in arizona ( ); individuals from families as part of the san antonio family diabetes study (safads) ( , ); families ( individuals) with northern-european ancestry ( ); sibling pairs from the africa-america diabetes mellitus (aadm) study ( ); and old order amish pedigrees collected as part of the amish family diabetes study (afds) ( ). extensive details of the study populations and methods used in each study have been previously described ( – ). statistical analysis. the genome scan meta-analysis (gsma) program ( , ) was used to assess evidence for linkage utilizing results from analyses of triglycerides, ldl, total cholesterol, hdl, ldl/hdl, and triglyceride/hdl. bins were created using marshfield map distances. of the nine genome scans, only two used marker maps with marshfield map distances; for the remaining studies, distances were converted to marshfield map distances by inserting the marker names in the marshfield database (http://research.marshfieldclinic. org/genetics/). in total, bins of � cm (range . – . ) spanning the genome were created. following this, the maximum lod score for a bin in each study was identified and were used to assign ranks to each bin in each separate study. ranks were then summed across studies and used to estimate the probability of observing a bins summed rank by chance, i.e., the propor- tion of replicates with summed rank equal to or greater than that observed when combining the results of all studies ( ). we have described this method in detail elsewhere ( ). because sample sizes varied considerably among the studies evaluated here, both unweighted and weighted gsma analyses were performed. the data were weighted according to the number of individuals genotyped in the respective study populations, such that weight � [�n(genotyped individu- als)]/[mean of �n(genotyped individuals) over all studies] where n � , , , , , , , , and (or or depending on the trait being analyzed) for the afds, safads, aadm, hkfds, gennid (cauca- sians), gennid (hispanics), gennid (african americans), caucasians with northern-european ancestry, and pima indian studies, respectively ( – ). statistically significant evidence for linkage, equivalent to lander and kruglyak’s ( ) definition of one false positive per meta-analyses, was assessed using a bonferroni correction ( . / ), giving p � . , whereas suggestive evidence for linkage, defined as one false positive per meta- analysis, corresponded to p � . . we, however, did not correct for the six lipid traits because they were correlated. results nine genome scans for lipid traits have been performed in families ascertained for type diabetes. characteristics of the participants from each study are shown in table . these populations were drawn from diverse ethnic back- grounds including american-indian, old order amish, african, african-american, and chinese study samples, as well as two groups of unrelated caucasian and hispanic individuals. we performed a meta-analysis for the follow- ing quantitative lipid traits: ldl (seven studies), hdl (nine studies), triglycerides (nine studies), total cholesterol t a b l e c h aracteristics o f p articip an ts fro m th e stu d ies u sed in th e g s m a s tu d y a f d s s a f a d s a a d m h k f d s g e n n id n o rth ern -e u ro p ean an cestry p im a in d ian s e th n ic b ack gro u n d o ld o rd er a m ish m ex ican a m erican s a frican s c h in ese c au casian s a frican a m erican s h isp an ics c au casian a m erican in d ian n m ale (% ) . . . . . . . . . f em ale (% ) . . . . . . . . . w ith d iab etes (% ) . . . . a ge (years) . � . . � . . � . � . � . . � . . � . . � . . � . s tu d y d esign e x ten d ed p ed igrees e x ten d ed p ed igrees s ib -p airs n u clear fam ilies n u clear fam ilies n u clear fam ilies n u clear fam ilies e x ten d ed p ed igrees s ib -p airs p ro gram u sed s o l a r s o l a r m erlin m erlin g h g h g h s o l a r s a s m ax l o d * . ( ) . ( ) . ( ) . ( ) . ( ) . ( ) . ( ) . ( ) . ( ) m ax l o d trait l d l t g h d l t g t g /h d l t c t g /h d l t g t c d ata are m ean s � s d u n less o th erw ise in d icated . *h igh est l o d sco re o b served ; th e ch ro m o so m e n u m b er is given in p aran th esis. t c , to tal ch o lestero l; t g , triglycerid e. a. malhotra and associates diabetes, vol. , march (eight studies), triglyceride/hdl (five studies), and ldl/ hdl (five studies). the number of studies included in the meta-analyses varied according to the availability of trait data from the original genome scans for a given population (table ). we observed statistically significant evidence (i.e., p � . ) for linkage in four bins for total cholesterol on q . -q . ( . – cm), p . -p ( . – . cm), p -q . ( . – . cm), and q . -q . ( . – . cm), as well as one bin for ldl on p . - p (table ). to evaluate the distribution of p values across the genome, summed ranks were plotted against the bin number for total cholesterol and ldl, which were the only traits showing statistically significant evidence for linkage (figs. and , respectively). in addition, eight regions also showed suggestive evidence (i.e., p � . ) of linkage for different traits: p -q . and q . -q . for ldl, p -q . ( . – . cm) and p -q . for triglyceride/hdl, p -q . for triglyc- erides, and q . – q ( . – . ), q -q . ( . – . cm), and p -q . for ldl/hdl. however, in the analysis of hdl, no bins met the criteria for statistically significant or suggestive evidence for linkage, as described in research design and methods. discussion in this study, we report the results from the first meta- analysis of linkage data undertaken to date to identify loci underlying quantitative lipid traits in families ascertained for type diabetes. we have found statistically significant evidence of linkage for total cholesterol on q . -q . and chromosome and for ldl on p . -p . sugges- tive evidence for linkage was also observed on p - q . and q . -q . for ldl, p -q . for triglycerides, p -q . and p -q . for triglycer- ide/hdl, and q . – q , q -q . , and p - q . for ldl/hdl. these results demonstrate that combining data from unrelated studies conducted in fam- ilies ascertained for type diabetes can successfully address important limitations commonly associated with linkage analysis, such as relatively modest sample size, and increase the power to detect statistically significant evidence for linkage. the strongest evidence of linkage for lipid traits identi- fied in this study was found on chromosomes and . linkage for lipid traits has been previously observed in both of these regions in several studies. for example, linkage on chromosome for triglyceride/hdl ( ) was found in randomly ascertained families and for triglyceride levels in two unrelated studies of families ascertained for obesity and one study of families ascertained for type diabetes ( , , ). a statistically significant p value iden- tified in this meta-analysis, combined with multiple obser- vations of linkage in unrelated studies, supports the presence of a gene(s) in this region with important effects on triglyceride levels. it is worth noting that only nominal evidence for linkage was observed for this locus in the individual studies, indicating that meta-analysis may be a powerful tool for identifying susceptibility loci for com- plex traits such as lipid levels. while the q . -q . locus is a strong candidate for genetic control of triglyceride levels, the most consistent and compelling evidence of linkage for lipid traits was observed on chromosome . linkage for various lipid traits to chromosome has been observed in severalt a b l e l o d sc o re s an d p va lu es m ee ti n g cr it er ia fo r si gn ifi ca n t o r su gg es ti ve ev id en ce fo r li n k ag e (i .e ., p � . o r p � . , re sp ec ti ve ly ) c yt o ge n et ic lo ca ti o n cm lo ca ti o n (m b in p ar an th es is ) t ra it a f d s s a f a d s a a d m h k f d s g e n n id c au ca si an g e n n id a fr ic an a m er ic an g e n n id h is p an ic n o rt h er n - e u ro p ea n an ce st ry p im a in d ia n g s m a (l o d )* g s m a (p )† q . -q . . – ( . – . ) t c . — ‡ . . . . . . . . . § p . -p . – . ( – . ) t c . — . . . . . . . . § p -q . . – . ( . – . ) t c . — . . . . . . . . . § q . -q . . – . ( . – . ) t c . — . . . . . . . . . § p . -p . – . ( – . ) l d l . — . . . . . — . . § p -q . . – . ( . – . ) l d l . — . . . . . — . . � q . -q . . – . ( . – . ) l d l . — . . . . . — . . � p -q . . – . ( . – . ) t g . . . . . . . . . . . � p -q . . – . ( . – . ) t g /h d l — — — . . . . . — . . � p -q . . – . ( . – . ) t g /h d l — — — . . . . . — . . � q . – q . – . ( . – . ) l d l /h d l — — — . . . . . — . . � q - q . . – . ( . – . ) l d l /h d l — — — . . . . . — . . � p -q . . – . ( . – . ) l d l /h d l — — — . . . . — . . � *l o d sc o re fo r w ei gh te d an al ys is . †p va lu e fo r w ei gh te d an al ys is . ‡r es u lt s w er e n o t av ai la b le . §s ig n ifi ca n t ev id en ce (b o n fe rr o n i co rr ec ti o n : p � . ) . �s u gg es ti ve ev id en ce (b o n fe rr o n i co rr ec ti o n : p � . ) . t h e cy to ge n et ic lo ca ti o n , l o d sc o re s fo r in d iv id u al st u d ie s, g s m a l o d sc o re s, an d g s m a p va lu es ar e gi ve n fo r th e re gi o n s id en ti fi ed . n o d if fe re n ce s b et w ee n w ei gh te d an d u n w ei gh te d an al ys es w er e fo u n d , an d re su lt s fo r w ei gh te d an al ys es ar e sh o w n h er e. t c , to ta l ch o le st er o l; t g , tr ig ly ce ri d e. meta-analysis of lipid level genome scans diabetes, vol. , march studies including total cholesterol in pima indians ( ) and african americans ( ), triglycerides in non-hispanic cau- casians ( ), and ldl in old order amish ( ) and africans ( ); notably, these five studies involved families ascer- tained for type diabetes. in addition, linkage to chromo- some was also observed for ldl cholesterol in randomly ascertained american indians ( ) and cauca- sians ( ) and a combination of randomly and obesity- ascertained families ( ), as well as for ldl particle size, in randomly ascertained mexican-american families ( ) fig. . summed ranks in relation to bin number in the analysis of lipid traits. summed ranks are plotted against the gsma bin number. there are a total of bins. vertical lines divide the bins according to chromosome. a: total cholesterol levels: three bins on chromosome and one bin on chromosome showed significant evidence as indicated. b: ldl levels: one bin on chromosome showing significant evidence for linkage. a. malhotra and associates diabetes, vol. , march and african americans ascertained for hypertension ( ). together, these findings are consistent with chromosome being an important locus for regulation of lipid levels among individuals from diverse ethnic backgrounds. fur- ther, the identification of this locus in five of the nine published genome scans conducted in families ascertained for type diabetes supports the idea that studies of such populations, in whom effects of genetic variants affecting lipid levels may occur earlier or more severely, may facilitate the discovery of genes with critical roles in lipid metabolism in individuals with diabetes and in the general population. the identification of chromosome both in a single bin identified for multiple traits (total cholesterol, ldl, triglyc- eride/hdl, and ldl/hdl) and across multiple bins (total cholesterol) suggests possible pleiotropic effects of a gene fig. . lod score plots for chromosome (a) and chromosome (b). horizontal lines indicate the lod score of the meta-analysis for a given bin that extends . cm for chromosome and . cm for chromosome , with cm interval for each bin given above the line. meta-analysis of lipid level genome scans diabetes, vol. , march in this region and/or multiple genes affecting a single trait. this effect is not unique to our study, as multiple peaks have also been observed in other studies. heijmans et al. ( ) identified peaks on each arm of chromosome , and a broad linkage peak extending from the p to the q arm was observed in an african-american population ( ). there are many genes located in this region that encode proteins with direct effects on lipid metabolism, including hormone-sensitive lipase (lipe at q . -q . ), apoli- poprotein e (apoe at q . ), and ldl receptor (ldlr at p . ). it is therefore possible that variants in multiple genes within this region interact with one another to exert effects of varying magnitude on lipid levels. in addition, the low proportion of variance of cholesterol levels explained by these genes, e.g., the apoe locus contributed as low as % of the variance in one study ( ), might suggest the role of genes on other chromosomes, including cholesteryl ester transfer protein (cetp) and apolipoprotein b (apob). while we have found statistically significant evidence for linkage for different quantitative lipid traits using meta-analysis, we recognize that utilization of ethnically diverse populations may yield results that are difficult to interpret, in light of potential effects of locus heterogene- ity. however, low heterogeneity was observed for total cholesterol in bins corresponding to q . -q . , p . - p , p -q . , and q . -q . (results not shown). the fact that common regions of linkage were identified in this analysis, despite differences in ethnicity in the study samples, is consistent with common genetic influences across the different populations. in summary, we observed statistically significant evi- dence for linkage for total and ldl cholesterol on chro- mosome and for total cholesterol on chromosome in a meta-analysis of genome scan results from families ascertained for type diabetes. these findings, combined with previous reports of linkage on chromosomes and , provide compelling evidence that these regions harbor genes with important effects on lipid levels. acknowledgments the gennid study was supported by the american dia- betes association (ada). we thank the investigators and staff who contributed to the gennid repository, as well as the individuals who participated in the gennid study. genetic material collected by, and families characterized by, the ada gennid study group, which includes eric boerwinkle, phd, university of texas health science center; john buse, md, phd, university of north carolina; ralph defronzo, md, university of texas health science center; david ehrmann, md, university of chicago; steven c. elbein, md, university of utah/university of arkansas; wilfred fujimoto, md, and steven e. kahn, mb, chb, university of washington; craig l. hanis, phd, university of texas health science center; richard a. mulivor, phd, and jeanne c. beck, phd, coriell cell repositories; jill norris, phd, university of colorado school of medicine; m. alan permutt, md, and philip behn, md, wa university school of medicine; leslie raffel, md, cedars-sinai medical center; and david c. robbins, md, medlantic research institute. the safads project was supported by grants from the national institute of diabetes and digestive and kidney diseases (niddk): dk , dk , and dk . we warmly thank the safads families for their enthusiasm and cooperation. the study performed in families from northern-european ancestry was supported by national institutes of health (nih) grant dk and the re- search service of the department of veterans affairs. the hkfds study was supported by the hong kong research grants committee (cuhk / m; / c), the chinese university of hong kong strategic grant program (srp ), and the hong kong innovation and technology support fund (its/ / ). support for the aadm study was provided by nih grant t tw - s from the office of research on minority health. aadm was also supported in part by the national center for research resources, the national human genome research insti- tute, and by the niddk grant dk- . genotyping services were provided by the center for inherited disease research (cidr) (http://www.cidr.jhmi.edu). cidr is fully funded through a federal contract from the nih to johns hopkins university, contract number n -hg- . this research was supported in part by the intramural research program of the niddk. we also thank investigators and staff of the afds based at the university of maryland, including alan r. shuldiner, md, braxton d. mitchell, and associated faculty, including technical and clinical staff, and especially the outstanding cooperation of the amish community, without which the afds would not have been possible. we thank the aadm investigators and are deeply grateful to aadm partici- pants, their families, and physicians. references . haffner sm, lehto s, ronnemaa t, pyorala k, laakso m: mortality from coronary heart disease in subjects with type diabetes and in nondiabetic subjects with and without prior myocardial infarction. n engl j med : – , . kannel wb, castelli wp, gordon t, mcnamara pm: serum cholesterol, lipoproteins, and the risk of coronary heart disease: the framingham study. ann intern med : – , . malhotra a, wolford jk: analysis of quantitative lipid traits in the genetics of niddm (gennid) study. diabetes : – , . ng mc, so wy, lam vk, cockram cs, bell gi, cox nj, chan jc: genome-wide scan for 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are associated with decreased risk for cardiovascular disease. precision medicine aims to capitalize on recent findings that rare variants such as apoc r x (rs ) are associated with risk of disease, but it is unclear how population-based associations can be best translated in clinical settings at the individual-patient level. methods: to explore the potential usefulness of screening for genetic predictors of cardiovascular disease, we surveyed biovu, the vanderbilt university medical center’s biorepository linked to de-identified electronic health records (ehrs), for apoc x mutations among adult european american patients (> and > years of age for men and women, respectively) with the lowest percentile of tg levels. the initial search identified patients with the lowest tg levels in the biorepository; among these, patients with sufficient dna and the lowest tg levels were chosen for illumina exomechip genotyping. results: a total of two patients were identified as heterozygotes of apoc r x for a minor allele frequency (maf) of . % in this patient population. both heterozygous patients had only a single mention of tg in the ehr ( and mg/dl, respectively), and one patient had evidence of previous cardiovascular disease. conclusions: in this patient population, we identified two patients who were carriers of the apoc x null variant, but only one lacked evidence of disease in the ehr highlighting the challenges of inclusion of functional or previously associated genetic variation in clinical risk assessment. keywords: precision medicine, triglycerides, biobank, electronic health records, apoc background personalized or precision medicine is meant to distin- guish tailored treatment from trial and error. the con- cept of precision medicine is not new and has been in practice arguably since the dawn of modern medicine [ ]. health care providers have long collected detailed data on patients, ranging from basic personal histories to technical laboratory assays and diagnostic procedures, to provide specific diagnoses and treatments. these tools ordered in the precision medicine setting are constantly evolving, for example, the evolution of myocardial in- farction diagnosis [ ], resulting in high resolution and, in some cases, highly predictive individualized data. today’s concept of precision medicine has evolved to specifically include the genetic profile of a patient in the prevention, diagnosis, and treatment of disease [ , ]. previous proxies for genetic profiles such as sex, race/ ethnicity, family history, and response to therapy are now being augmented by clinical laboratory improve- ment amendments-certified genotyping and sequencing * correspondence: dana.crawford@case.edu department of population and quantitative health sciences, institute for computational biology, case western reserve university, cornell road, wolstein research building, suite - , cleveland, oh , usa full list of author information is available at the end of the article © the author(s). open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/ . /) applies to the data made available in this article, unless otherwise stated. crawford et al. bmc medical genomics , (suppl ): https://doi.org/ . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf mailto:dana.crawford@case.edu http://creativecommons.org/licenses/by/ . / http://creativecommons.org/publicdomain/zero/ . / at both the targeted and whole genome level. indeed, technological advances in high-throughput genomics coupled with their rapid decreases in costs have made generating the data almost trivial, and the emergence of electronic health records (ehrs) in part through the hitech act [ , ] make it possible to effectively deliver personalized medicine to the patient. a major challenge in the delivery of personalized medicine is not the collection of data, but the interpret- ation of the data. large-scale population sequencing studies have demonstrated that potentially functional variants exist in all dna samples sequenced, but the biological and statistical data lag in filtering the potential from truly functional, and even less data are available to direct the course of clinical action based on these geno- types. as such, major areas of active research focus on methods to translate genomic discoveries into clinical applications [ ]. this broad area of investigation in- cludes research on who to test, what to test, how to test, what to report, how to report, and how to measure its effectiveness, to name a few. to begin to address some of these gaps in research, we have undertaken a pilot study of genotyping for a loss of function variant, apoc r x (rs ), in a targeted population of european american adults (men > and women > year of age) with very low triglyceride levels in biovu, the vanderbilt university medical center (vumc)‘s biorepository linked to de-identified ehrs. triglyceride levels (tg) are a com- mon biomarker measured in the clinic, and patients with extreme triglyceride (tg) levels may be flagged for further evaluation for cardiovascular disease risk assess- ment (tg ≥ mg/dl). recent studies have identified a loss-of-function variant in apoc (r x or rs ) associated with low triglyceride levels and diminished post-prandial lipemia in heterozygous carriers [ ] and im- proved clearance of plasma tgs after a fatty meal in homozygous carriers [ ]. although rare in the general population [ ], we hypothesized that the loss-of-function allele would be at an increased frequency in this extreme population. based on previous reports, we also hypothe- sized that evidence of cardiovascular disease would be absent in ehrs of these apoc x carriers with very low tg levels. methods study population the study population presented here is from biovu, the vumc’s biorepository linked to de-identified ehrs. biovu operations [ ] and ethical oversight [ ] have been previously described. briefly, dna is extracted from discarded blood drawn for routine clinical care at vanderbilt outpatient clinics in nashville, tennessee and surrounding areas. the dna samples are linked to a de-identified version of the patient’s ehr. the data in this study were de-identified in accordance with provi- sions of title , code of federal regulations, part ( cfr ); therefore, this study was considered non-human subjects research by the vanderbilt university internal review board. phenotyping the de-identified ehr contains both structured (inter- national classification of diseases, ninth revision, clinical modification (icd- -cm) billing codes; current procedural terminology (cpt) codes; problems lists; labs) and unstruc- tured (clinical free text) data accessible for electronic phe- notyping. we extracted all available labs for triglyceride levels in september for european american adults (> years of age of men and > years of age for women) and examined individuals whose median tg levels constituted the lowest % of biovu at the time of data extraction. a total of individuals were identified. manual review of the clinical text for random patients with low tg levels prior to selection for genotyping failed to identify obvious documented diagnoses or notes that may have led to very low tg levels in these patients. from these individuals, were selected for illumina humanexome beadchip genotyping based on dna quality and quantity, and preference was given to individuals with more than one triglyceride level reflecting consistently low tg levels. the de-identified ehrs for the patients genotyped on the illumina humanexome beadchip were re-examined in july for evidence of myocardial infarction, revascu- larization, and other heart disease. myocardial infarction was defined by mention of icd- -cm codes or .* or a problem list mention of “mi” or “myocardial infarc- tion.” revascularization was defined by cptcodes (table ). other heart disease was defined as a problem list mention of “coronary artery disease,” “cad,” “coronary heart dis- ease,” or “chd.” genotyping and statistical methods vanderbilt technologies for advanced genomics (vant- age) genotyped biovu samples and eight international hapmap reference samples using the illumina humanex- ome beadchip (v . for samples and v . for samples). as recommended by other research groups with exome beadchip experience [ ], genotypes for these samples were called as part of larger, ongoing exome bead- chip projects in biovu genotyped by vantage. apoc r x (rs /exm ) was directly assayed by the exome beadchip, and we extracted these called genotypes for further analysis. we also extracted genotypes for snps previously asso- ciated with incident myocardial infarction in european americans [ ]. of the previously associated snps, crawford et al. bmc medical genomics , (suppl ): page of were directly assayed by the exome beadchip (additional file : table s ). we then calculated both unweighted and weighted genetic risk scores (grs) based on the genotypes of these snps. unweighted grs were calculated by counting the number of risk alleles per individual. weighted grs were calculated based on counts of risk alleles multiplied or weighted by odds ratios recently reported for european american cases of coronary artery disease in comparison with con- trols [ ]. results as detailed in the methods section, european ameri- can men > and women > years of age had the lowest tg levels in biovu in . slightly less than half ( %) of the patient sample was female, and the average birth decade was the s. approximately half of the patients ( . %) with the lowest tg levels had more than one tg available in the ehr. for those with more than one tg level, the median values were calculated. the overall median tg level in this lowest % was mg/dl. the genotyped study population characteristics are given in table . like the patients considered, the patients genotyped were majority male born in the s and s. the mean body mass index was . kg/m , which is considered within the normal range. the first mention of tg in the clinical record was on average . mg/dl. the median of all tg levels available for this patient population was mg/dl, ranging from . to mg/dl (fig. ). among the patients with low tg levels in biovu genotyped here, . %, . %, and . % had evidence in the ehr of a myocardial infarction, revascularization, and other heart disease, respectively. only six out of patients ( . %) had evidence of all three. the unweighted and weighted grs for patients with events (myocardial in- farction or revascularization) or evidence for other heart disease were higher compared with patients without events or evidence for other heart disease, although these differences were not statistically significant (table ). we next examined the frequency of apoc r x (rs ) in this patient sample of extremely low tg levels. among patients successfully genotyped for this variant, two heterozygotes were identified for a sam- ple allele frequency of . %. the allele frequency for the loss-of-function allele (t) in this sample of very low tg levels is . to . fold higher compared with allele frequency estimates for american adults drawn from the general population [ , ] and three to fold lower in frequency compared with isolated populations [ , , ] (table ). previous studies in outbred [ ] and isolated [ , ] populations suggest that the x mutation arose once on a single haplotype background. we therefore exam- ined the diplotypes spanning apoa /c /a /a gene cluster assayed by the illumina humanexome beachip for evidence of a single haplotype background contain- ing the x mutation. of the snps assayed (spanning table current procedural terminology codes used to define revascularization among european american patients in biovu with very low triglyceride levels cpt code description – ; coronary artery bypass grafting with venous grafting only ( – or more grafts) coronary artery bypass (old code) – ; – coronary artery bypass grafting with venous and arterial grafting ( – or more vein grafts); billed in conjunction with – ( – cannot be billed alone). coronary artery bypass (old code) – coronary artery bypass grafting with arterial grafting only ( – or more grafts) – transcatheter placement of an intracoronary stent, percutaneous, with or without other therapeutic intervention, any method (single vessel and each additional vessel) ; percutaneous transluminal coronary balloon angioplasty (single vessel and each additional vessel) ; percutaneous transluminal coronary atherectomy, by mechanical or other method, with or without balloon angioplasty (single vessel and each additional vessel) table study population characteristics female, % . decade of birth, % . . . . . . mean (±sd) body mass index (kg/m ) . (± . ) mean (±sd) first tg level (mg/dl) . (± . ) study population characteristics (sex, decade of birth, average body mass index, and average first mention of triglyceride levels in patient’s electronic health record) are given for the genotyped patients with the lowest % triglyceride levels in biovu among european american men and women > and > years of age, respectively abbreviations: sd standard deviation, tg triglyceride crawford et al. bmc medical genomics , (suppl ): page of chr : – , , ), seven snps overlapped with variants used to infer haplotypes in non-hispanic whites of the third national health and nutrition exam- ination survey (nhanes iii): rs , rs , rs , rs , rs , rs , and rs . we found that one x carrier was homozygous at all variants in this region that passed quality control except for x, and that this variant-containing haplotype background was similar to x backgrounds in nhanes iii (g-c-c-c-a-t-t). in contrast, the second x carrier was heterozygous at eight sites including rs . interestingly, one of these eight heterozygous sites in- cluded rs (ivs + g > a), another apoc rare variant associated with decreased levels of tg [ , , ]. a query for rs in the patients with very low triglycerides revealed an additional carrier for this variant. both carriers of rs are, with the inclusion of this rare variant, heterozygous at more than one site, and one carrier has missing data ( / sites) at this gene cluster; therefore, his or her haplotypes could not be unambiguously inferred with confidence. much like r x, the frequency of rs ( . %) is higher in this patient population of low tg levels compared with outbred population estimates [ , ]. a third rare apoc variant (rs ) associated with low tg levels [ ] failed genotyping in this patient population. as expected when stratified by apoc r x genotype (fig. ), the mean tg level in carriers ( mg/dl; . standard deviation) was lower compared with the non-carriers ( . mg/dl; . standard deviation). however, the difference was not statistically significant (p = . ) in this sample of adults with very low tg levels. similarly, the mean tg level in ivs + g > a carriers was . mg/dl ( . standard deviation) for first-mentioned tg level. previous observational studies have suggested that tg levels are associated with risk of cardiovascular disease [ ]. more recently apoc x carrier status among other apoc mutations was associated with lower risk of coronary heart disease [ ]. of the two apoc x carriers, one had no evidence in the ehr of myocardial infarction, revascularization, or other heart disease. one apoc x carrier had evidence in the ehr of all three. a more detailed review of the de-identified ehrs of the fig. distribution of low triglyceride levels among european american adults in biovu. a total of european american adults (men > years and women > years) with at least one triglyceride level in the lowest % of biovu were genotyped on the illumina humanexome beadchip for apoc r x. the frequency (expressed as percent in the study population) is given on the y-axis and the median triglyceride levels (in mg/dl) are given on the x-axis table genetic risk scores, unweighted and weighted, by case status among european american patients with very low triglyceride levels all patients with very low tg levels (n = ) patients with no evidence of mi, revascularization, or other heart disease (n = ) patients with evidence of mi (n = ) patients with evidence of revascularization (n = ) patients with evidence of other heart disease (n = ) patients with evidence of mi, revascularization, and other heart disease (n = ) unweighted grs . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) weighted grs . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) we calculated unweighted and weighted genetic risk scores (grs) based on snps and previous association estimates in european americans with and without coronary artery disease (additional file : table s ). unweighted grs were calculated as counts of risk alleles per patient, and weighted grs were calculated using the pooled odds ratios from cardiogram [ ]. shown are the means (standard deviations) for unweighted and weighted grs for the total study population as well as by cases status. although unweighted and weighted grs were higher among all patient groups with events or other heart conditions compared with patients lacking evidence of events or other heart disease, these differences were not statistically significant (unpaired t-tests; p > . ) crawford et al. bmc medical genomics , (suppl ): page of two apoc x carriers was performed to identify other possible cardiovascular disease risk factors. the fe- male x carrier was born in the s, and her ehr contained a medical history significant for remote breast cancer treated with surgery and radiation, controlled hypertension, and overweight (bmi ~ kg/m ). this fe- male x carrier had never smoked cigarettes, and there was no evidence of coronary artery disease or myocar- dial infarction in her ehr. a single assessment of lipids was available for this female x carrier: low-density lipoprotein cholesterol (ldl-c) mg/dl, high density lipoprotein cholesterol (hdl-c) mg/dl, and tg mg/dl. the female x carrier had unweighted and weighted grs of and . , respectively. the second apoc x carrier was a male born in the s. the male x carrier had a past medical history of uncon- trolled hypertension, was overweight (bmi kg/m ), and was a prior smoker. the male carrier had an exten- sive history of cardiac disease including atrial fibrillation, coronary artery disease with prior coronary artery bypass grafting, myocardial infarction, and ischemic cardiomy- opathy (ejection fraction %) with heart failure. the table apoc r x frequency, by population population sample size carriers identified (overall allele frequency) allele frequency in european-descent populations pubmed id or website european american adults with very low triglyceride levels ( . %) . % - (present study) pennsylvania amish ( . %) . % greek isolate ( . %) . % greek isolate ( . %) . % americans regardless of health status , ( . %) . % exome aggregation consortium (exac) , ( . %) . % (http://exac.broadinstitute.org/ accessed may ) national heart, lung, and blood institute (nhlbi) grand opportunity (go) exome sequencing project (esp) ( . %) . % (http://evs.gs.washington.edu/evs/ accessed june ) ohio and indiana amish (−) – genomes project (−) – (http://useast.ensembl.org/index.html accessed june ) european americans from baltimore (−) – fig. mean triglyceride levels by apoc r x genotype. a total of european american adults (men > years and women > years) with at least one triglyceride level in the lowest % of biovu were genotyped on the illumina humanexome beadchip for apoc r x. two samples failed genotyping. the means for the first mentioned triglyceride level (y-axis) were calculated for the non-carriers (cc genotype) and carriers (ct genotype) at apoc r x (x-axis). although the mean triglyceride level in carriers ( mg/dl; . standard deviation) was lower compared with the non-carriers ( . mg/dl; . standard deviation), the difference between the two is not statistically significant (two-sided t-test assuming unequal variances; p = . ) crawford et al. bmc medical genomics , (suppl ): page of male x carrier was not treated with statins due to re- ported intolerance, and a single measurement of lipids was available: ldl-c mg/dl, hdl-c mg/dl, and tg mg/dl. the male x carrier had unweighted and weighted grs of and . , respectively. neither apoc x carrier has died as of . the male x carrier was also a carrier for ivs + g > a, the only potentially compound heterozygote described in the literature to date. the other ivs + g > a carrier was a female born in the s. the female ivs + g > a carrier was normal weight (bmi kg/m ) with two men- tions of tg levels ( and mg/dl) in the ehr. this female ivs + g > a carrier has no evidence of myocar- dial infarction, revascularization, or other heart disease in the ehr. discussion we evaluated adult european american patients with very low tg levels extracted from ehrs for the presence of the loss-of-function allele ( x) for apoc rs . overall, we identified two carrier patients, and as hypothe- sized, the resulting allele frequency of apoc x was higher in this extreme patient population compared with the general population [ ]. neither carrier patient had the lowest tg levels among this patient population. a review of the ehr revealed only one of the two apoc x carriers was free of myocardial infarction, revascularization, and other heart disease. coincidentally, the apoc x carrier with evidence of cardiovascular disease was also a carrier of ivs + g > a, representing to our knowledge po- tentially the first compound heterozygote for these muta- tions in the literature [ ]. based on the current literature [ ], we would expect that the addition of these genomic data to the ehr would assist a physician in the assessment of the car- riers’ risk of future cardiovascular disease. apoc r x was originally identified in a genome-wide association study (gwas) of tg levels in the pennsylvania amish where a variant in linkage disequilibrium with r x was significantly associated with decreased tg levels [ ]. follow-up sequencing revealed the loss of function mu- tation in apoc likely responsible for the gwas find- ings [ ], and subsequent studies in both isolated [ , ] and outbred [ , ] populations have confirmed the strong association between lower tg levels and x car- riers. more recently, prospective epidemiologic studies have demonstrated that x carriers have lower rates of cardiovascular disease compared with non-carriers [ ]. interestingly, one of the two apoc x carriers iden- tified here has evidence in the ehr of a myocardial in- farction, revascularization, and other heart disease. also, apart from sharing low tg levels, the two x carriers had different cardiovascular risk profiles. while the stat- istical evidence for the association between lower tg levels and lower risk of coronary heart disease is strong at the population level, these data highlight the difficulty in translating a genetic association finding in the clinic for risk prediction at the patient level as envisioned for precision medicine. these data also highlight the genetic and environmental heterogeneity that drives cardiovas- cular disease risk. thus, the addition of apoc r x in a clinical setting may contribute to the patient’s risk as- sessment for cardiovascular disease, but it is not absolute and must be considered with other genetic and environ- mental risk factors [ ]. we specifically targeted adult european americans with low tg levels for apoc r x genotyping. the loss-of-function variant is common in isolated popula- tions such as the pennsylvania amish [ ] and greek isolates [ , ], but a study in nhanes confirmed the mutation is rare in the general population [ ]. the fre- quency of x also varies by race/ethnicity. in nhanes [ ] and in other studies [ ], x is exceedingly rare in african americans or african-descent populations com- pared with european-descent populations. in the present study of european americans with low tg levels, we ob- served that the frequency of x was higher in this patient population compared with the general population, an ob- servation consistent with the known genetic epidemiology of this loss-of-function variant. although the carriers identified in this study had lower mean tg levels com- pared with non-carriers, we did not observe a statisti- cally significant association most likely due to the fact that all patients genotyped already had low tg levels. also, we only identified two carriers resulting in low statistical power. the strategy of genotyping or targeting individuals with extreme phenotypes has been a popular and successful strategy in genetic epidemiology for gene discovery for many years [ ]. indeed, in the field of cardiovascular genetics, sequencing individuals with extreme ldl-c, hdl-c, and tg levels in multiple populations has identi- fied several genetic variants and potential drug targets such as pcsk [ ]. an analogous strategy could be im- plemented in a clinical setting to augment the ehr with specific genotypes for an individual patient’s risk assess- ment. for example, if a patient presents with an extreme lipid level, a panel of known functional variants (missense and loss-of-function) could be ordered for genotyping and added to the ehr to inform risk assessment for future cardiovascular events in that patient. a major advantage of this targeted approach is that the genetic assays would be ordered only on a fraction of the patients (e.g., patients with extreme labs) making it cost-effective compared with offering the panel to all patients regardless of lab results. there are major disadvantages to a targeted approach to augmenting the ehr with genomic data. for most human traits and diseases, the known functional or crawford et al. bmc medical genomics , (suppl ): page of strongly associated variants were discovered in european- descent populations [ , ]. as such, diverse populations such as african americans and hispanics may not benefit from a european-centric genotyping panel. and, even among european-descent populations the catalog of genotype-phenotype associations is far from complete or strongly predictive of clinical events [ ]. as technology improves to sort functional from neutral variants [ , ], all patients may benefit from whole genome sequencing. conclusions further work is needed in developing appropriate tools for ehr integration and delivery of clinical decision support [ ] for this and other clinically relevant genetic variants as envisioned in an era of precision medicine. additional file additional file : table s . genetic variants previously associated with risk of coronary artery disease in european americans. (docx kb) abbreviations bmi: body mass index; cad: coronary artery disease; chd: coronary heart disease; cpt: current procedural terminology; ehr: electronic health record; grs: genetic risk score; gwas: genome-wide association study; hdl-c: high density lipoprotein cholesterol; icd- -cm: international classification of diseases, ninth revision, clinical modification; ldl-c: low-density lipoprotein cholesterol; maf: minor allele frequency; nhanes: national health and nutrition examination survey; tg: triglycerides; vantage: vanderbilt technologies for advanced genomics; vumc: vanderbilt university medical center funding the cost of publication was funded by case western reserve university’ institute for computational biology. the dataset (s) used for the analyses described were obtained from vanderbilt university medical center’s biovu which is supported by institutional funding and by the vanderbilt ctsa grant funded by the national center for research resources, grant ul rr – , which is now at the national center for advancing translational sciences, grant ul tr – . availability of data and materials the data that support the findings of this study are available from vanderbilt university medical center but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. data are however available from the authors upon reasonable request and with permission of vanderbilt university medical center. about this supplement this article has been published as part of bmc medical genomics volume supplement , : selected articles from the th translational bioinformatics conference (tbc ): medical genomics. the full contents of the supplement are available online at https://bmcmedgenomics.biomedcentral.com/ articles/supplements/volume- -supplement- . authors’ contributions dcc designed the study. ked, ef-e, and qsw collected and prepared the data. dcc and nar performed analyses. dcc drafted the manuscript. dcc, nar, and qsw were major contributors in revising the manuscript critically for all important intellectual content. all authors gave approval to the final version of the manuscript and agreed to be accountable to all aspects of the work. ethics approval and consent to participate the data in this study were de-identified in accordance with provisions of title , code of federal regulations, part ( cfr ); therefore, this study was considered non-human subjects research by the vanderbilt university internal review board. consent for publication not applicable. competing interests the authors declare that they have no competing interests. publisher’s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. author details department of population and quantitative health sciences, institute for computational biology, case western reserve university, cornell road, wolstein research building, suite - , cleveland, oh , usa. cancer biology, vanderbilt university school of medicine, nashville, tn, usa. vanderbilt institute for clinical and translational research, vanderbilt university medical center, nashville, tn, usa. departments of medicine and pharmacology, vanderbilt university medical center, nashville, tn, usa. published: september references . murray jf. personalized medicine: been there, done that, always needs work! am j respir crit care med. ; 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( ): – . https://doi.org/ . /ajmg.c. . pmc . shirts bh, salama js, aronson sj, chung wk, gray sw, hindorff la, et al. cser and emerge: current and potential state of the display of genetic information in the electronic health record. j am med inform assoc. ; ( ): – . https://doi.org/ . /jamia/ocv . pmc crawford et al. bmc medical genomics , (suppl ): page of https://doi.org/ . /journal.pone. https://doi.org/ . /journal.pone. https://doi.org/ . /ng. https://doi.org/ . /nejmoa https://doi.org/ . /nejmoa https://doi.org/ . /ncomms https://doi.org/ . /ncomms https://doi.org/ . /hmg/ddw https://doi.org/ . /ncomms https://doi.org/ . /circgenetics. . https://doi.org/ . /cir. b e https://doi.org/ . /cir. b e https://doi.org/ . /nejmoa https://doi.org/ . /nrg https://doi.org/ . /ng https://doi.org/ . /ng https://doi.org/ . /nrg https://doi.org/ . / a. pmc https://doi.org/ . /mol. https://doi.org/ . /jama. . https://doi.org/ . /jama. . https://doi.org/ . /ajmg.c. https://doi.org/ . /jamia/ocv 	abstract 	background 	methods 	results 	conclusions 	background 	methods 	study population 	phenotyping 	genotyping and statistical methods 	results 	discussion 	conclusions 	additional file 	abbreviations 	funding 	availability of data and materials 	about this supplement 	authors’ contributions 	ethics approval and consent to participate 	consent for publication 	competing interests 	publisher’s note 	author details 	references summary of indiana farm fatalities involving individuals years and older— – safety article summary of indiana farm fatalities involving individuals years and older— – sean a. tormoehlen * and william e. field department of agricultural and biological engineering, purdue university, west lafayette, in , usa; field@purdue.edu * correspondence: stormoeh@purdue.edu received: may ; accepted: june ; published: june ���������� ������� abstract: agriculture has historically been one of the most hazardous of all occupations, with a variety of potential safety risks to workers and even higher risks documented for older agricultural workers. this study was undertaken to document and summarize indiana farm work-related fatalities involving persons years and older over the year period from to . data were mined from the purdue university agricultural safety and health program’s fatality database that dates back to the s. a total of fatalities involving persons years and older was documented. the average age of the victims was . years old, and an overwhelming majority of the cases involved males ( . %). the average number of deaths per year has remained fairly consistent, though it has occasionally been erratic, with an unanticipated increase in the number of documented fatalities over the period – . there appeared to be a direct positive correlation between the level or intensity of agricultural production in a county and the frequency of fatalities. the type of fatal injury most commonly reported was crush/run-over, with cases ( %). the most common agent or source of injury involved was tractors, with cases ( . %). another noted contributing factor was the high frequency of incidents in which the victim was reported to be working alone at the time of death. findings will be used to develop evidence-based injury prevention strategies, including the development of agricultural safety training materials and methods more relevant to older farmers. a special emphasis should be placed on reducing the risk of tractor and farm machinery overturns, especially when older, non-roll over protection structure (rops) equipped tractors are being operated. keywords: agriculture; aging; farmer; fatal injury; farm fatalities . introduction the purpose of this summary was to identify the primary contributing factors of indiana farm work-related fatalities involving agricultural producers years of age and older. it was anticipated that findings would provide a detailed look at documented characteristics and trends of fatalities over the year period from to , with an analysis of selected causative factors. the research was undertaken in response to the recent upward trend in the number of annual fatalities documented within this age group. review of literature the average age of u.s. farm operators has been on the rise for the past years, with the average going from . in , to . in , and to . in [ – ]. the three highest age categories for principal operator— to years old, to years old, and years and older—showed an increase of . %, . %, and . % from to and . %, . %, and . % from to , respectively [ , , ]. safety , , ; doi: . /safety www.mdpi.com/journal/safety http://www.mdpi.com/journal/safety http://www.mdpi.com http://www.mdpi.com/ - x/ / / ?type=check_update&version= http://dx.doi.org/ . /safety http://www.mdpi.com/journal/safety safety , , of farming is well documented as a dangerous profession for all ages. the census of fatal occupational injuries (cfoi) reported in that farmers, ranchers, and other agricultural managers of all ages had a fatal injury rate of . per , full-time equivalent workers, as compared to the all-worker fatal injury rate of . [ ]. the injury rate has been reported to be even higher for older farmers, with a fatality rate of . / , reported for farmers years and older between and [ ]. the increased risk of injury due to aging has been well documented. all of the senses and functions of the body go through changes with age. mobility issues, such as those caused by arthritis, increase the risk of injury with age [ , ]. the risk of eye injuries and diseases that impair vision increases with age [ ]. age-related hearing impairments are common and are considered the second most common handicapping condition in the united states [ ]. cognitive changes also take place, with reaction times slowed in comparison to younger adults [ ]. older farmers, however, have generally been shown to have a lower risk of obtaining a non-fatal injury as compared to younger farmers [ – ]. in a study of farmers years and older, marcum et al. ( ) found that an incremental increase of years for a farmer reduced their non-fatal injury risk by % [ ]. salminen ( ) conducted a literature review that looked at the risk of occupational injuries for younger workers across industries, including agriculture [ ]. he found that older farmers who had sustained injuries were more likely to die from their injuries. summaries of farm-related occupational fatalities have often reported large representations of older farmers [ , – ]. voaklander et al. ( ), for example, found that . % of all farm fatalities during – in canada were years or older and that the fatality rate per , increased with age [ ]. gorucu et al. ( ) found that % of pennsylvania farm fatality cases from to consisted of farmers years and older, even though this age group only accounted for . % of the farm household populations [ ]. another study by cheng and field ( ) found that about half of the fatality cases in indiana since have been farmers years or older [ ]. it has also been reported that common aging related changes in the body, such as arthritis, hearing and vision impairment, and mobility or medication usage, could increase the risk of sustaining specific types of farm-related work injuries [ , , , ]. in the study of how arthritis, mobility, and farm tasks affected injuries among older farmers, heaton et al. ( ) found that farmers with mobility issues were twice as likely to be injured by a farm-related task [ ]. even considering the higher risk of potential injuries, there seems to be limited attention given to agricultural safety material that is designed and oriented for older farmers. an example would be tree cutting and chainsaw safety materials developed and disseminated by organizations such as extension programs. the majority of the publications reviewed did not mention any potential issues to worker safety due to age-related changes in the body or other challenges more specific for the older farmer population involved in chainsaw operations [ – ]. a review of the images contained in these publications and related audio–visual material found that most depicted younger workers. nationally, the documentation of farm-related fatalities, has, in general, also been problematic for several reasons including: the lack of federal or state reporting requirements for farms with less than hired workers [ , ]; the lack of a central site for documenting and storing farm fatality data; and the nature of current reporting protocols that may not document a fatality when the injuries to the victim resulted in death sometime after the injury occurred. this lack in reporting requirements and inadequacies in the classification process contributes to the historical failure of the bureau of labor statistics’ (bls) records of national farming fatalities to include fatalities that occur on smaller farms relying primarily on family labor or with few employees [ ]. the bls data were also known to combine their data of farming fatalities with fatalities that may not be occupational-related, such as operating all-terrain vehicles (atvs) on farm land, or unrelated occupations, such as fishing, forestry, and hunting [ ]. another source of federal fatality data was the census of fatal occupational injuries (cfoi), which gathers descriptive data and counts of fatalities nationally [ ]. a concern regarding the completeness of farm fatalities reported through the cfoi is that the individual was required to safety , , of have been employed at the time of the incident, generally excluding children and unpaid family labor, such as retired family workers [ ]. historically, the cfoi has significantly under reported indiana farm-related fatalities. . methods the data used were drawn from the purdue university farm fatality database and previously published in annual summaries by the purdue agricultural safety and health program (puashp). a total of reported farm work-related fatalities have been recorded by puashp in indiana since . a variety of sources have been used to document these incidents, including death certificates, news clippings, web searches, obituaries, and post-incident interviews with family members, extension educators, first responders, expert witnesses in litigation, and others associated with the incidents. the general information included the date, county, age, sex, type of incident, and a short narrative of the incident. for this study, only data of confirmed fatalities of farmers years and older that occurred during the period from to were extracted for review. the definition of an older worker used, as being workers over the age of , was taken from current published studies, including the national institute for occupational safety and health (niosh), that found that farm workers, aged years and older, were at higher risk of injury and death [ ]. no follow-up investigations of individual cases were conducted by the authors other than the review of original sources. the coding sheet used for classifying the data was adapted from previously published coding sheets and the farm and agricultural injury code (faic) [ , – ]. the coding sheet helped to categorize the fatality cases to allow for summarization by reported type of fatal injury and the contributing agents. faic codes were not used due to a lack of sufficient data for the majority of reported cases [ ]. limitations the primary limitation of the summary relates to the comprehensiveness of the data due to the data collection and documentation methods. as noted, since there is no official centralized site where these data are gathered, puashp used a variety of sources to identify cases and related information. for more recent years, the data were compared with the indiana department of labor’s annual summary of work-related fatalities to identify any differences. these differences were generally due to the different criteria used by the u.s. department of labor to fulfill federal reporting requirements, such as excluding children under who are often not identified as being involved in work-related activities. the use of on-line reports or media “clippings” can also be problematic, since these reports are not always reliable due to the abilities of the individual doing the reporting [ , , ]. though the data are not recognized as comprehensive, they remain the best possible representation of indiana farm-related fatalities. the introduction of the internet in recent years has increased access to information on fatality cases. this new flow of data, however, may distort the overall data set because it may mean that the increase in number of recent cases may be due to better reporting versus an actual increase in fatality cases as compared to past years. these additional cases during the latter part of the period studied could affect any projected trends. online sources also appear to increase the risk of multiple reporting of the same incidents, with widely differing accounts that suggest multiple incidents when, in fact, all were reporting on the same incident [ , ]. . results the total number of fatalities documented and used in this summary for the year period was , making up . % of all reported cases over the time period. safety , , of . . age the average age of the older farmers was . , while the average age of all documented fatalities during the same period was . years old. the average age of current farm owner/operators in indiana is . years old [ ]. the oldest victim reported was years old. the number of fatalities decreased rapidly with age, as seen in figure . there were peaks at , , and , which offset the downward trend. when the ages were put into -year groups of – years, – years, and years and older, as seen in table , there was a fairly similar age distribution. the high percentage of victims over the age of was not anticipated. , , x of . . age the average age of the older farmers was . , while the average age of all documented fatalities during the same period was . years old. the average age of current farm owner/operators in indiana is . years old [ ]. the oldest victim reported was years old. the number of fatalities decreased rapidly with age, as seen in figure . there were peaks at , , and , which offset the downward trend. when the ages were put into -year groups of – years, – years, and years and older, as seen in table , there was a fairly similar age distribution. the high percentage of victims over the age of was not anticipated. figure . age distribution of fatalities for ages – . table . breakdown of ages by group. category frequency percent – years old . – years old . years and older . total - . . year the distribution of fatalities per year is shown in figure . the data shows variation over the year period with a positive trend that is statistically significant over time for farmers years and older, which is inconsistent with the negative trend for farm fatalities for indiana farmers of all ages. the years with the highest number of fatalities were with , with , and a tie between and with each. the years with the lowest number of fatalities were with four, with six, and a tie between and with seven each. the past five years, to , have shown a strong positive trend, with some of the highest number of fatalities in years. figure . age distribution of fatalities for ages – . table . breakdown of ages by group. category frequency percent – years old . – years old . years and older . total - . . year the distribution of fatalities per year is shown in figure . the data shows variation over the year period with a positive trend that is statistically significant over time for farmers years and older, which is inconsistent with the negative trend for farm fatalities for indiana farmers of all ages. the years with the highest number of fatalities were with , with , and a tie between and with each. the years with the lowest number of fatalities were with four, with six, and a tie between and with seven each. the past five years, to , have shown a strong positive trend, with some of the highest number of fatalities in years. safety , , of , , x of figure . number of fatalities per year. . . month the distribution of fatalities per month, when known, as seen in figure , showed a bimodal pattern. the two highest peaks were may with fatalities ( . %) and september with fatalities ( . %) reflecting the two periods of the year with the most intense agricultural production activities. the month with the lowest number of reported cases was march with cases ( . %), and the second lowest was february with cases ( . %). this monthly distribution also confirms other studies, indicating a distribution based upon seasonal work [ , , ]. figure . number of fatalities per month. figure . number of fatalities per year. . . month the distribution of fatalities per month, when known, as seen in figure , showed a bimodal pattern. the two highest peaks were may with fatalities ( . %) and september with fatalities ( . %) reflecting the two periods of the year with the most intense agricultural production activities. the month with the lowest number of reported cases was march with cases ( . %), and the second lowest was february with cases ( . %). this monthly distribution also confirms other studies, indicating a distribution based upon seasonal work [ , , ]. , , x of figure . number of fatalities per year. . . month the distribution of fatalities per month, when known, as seen in figure , showed a bimodal pattern. the two highest peaks were may with fatalities ( . %) and september with fatalities ( . %) reflecting the two periods of the year with the most intense agricultural production activities. the month with the lowest number of reported cases was march with cases ( . %), and the second lowest was february with cases ( . %). this monthly distribution also confirms other studies, indicating a distribution based upon seasonal work [ , , ]. figure . number of fatalities per month. figure . number of fatalities per month. . . gender of the documented fatalities, ( . %) were males, and ( . %) were females in the same age range. due to the recent upward trend in the number of female farmer fatalities, the data were safety , , of analyzed to see if there were any trends for older female agriculture workers. the dataset is small, with only deaths over the year period, so it was not possible to accurately infer trends, but the trend line from the available data shows that the number of female fatalities has been steady over the year period. the number of female deaths per year (figure ) shows an increase of deaths during and [ ]. the small increase in the number of female fatalities in recent years may be due to an increase in number of principal female farmers, increasing % from to [ , ]. however, the frequency of older females in fatal farm work incidents has historically been minimal and remains so. , , x of . . gender of the documented fatalities, ( . %) were males, and ( . %) were females in the same age range. due to the recent upward trend in the number of female farmer fatalities, the data were analyzed to see if there were any trends for older female agriculture workers. the dataset is small, with only deaths over the year period, so it was not possible to accurately infer trends, but the trend line from the available data shows that the number of female fatalities has been steady over the year period. the number of female deaths per year (figure ) shows an increase of deaths during and [ ]. the small increase in the number of female fatalities in recent years may be due to an increase in number of principal female farmers, increasing % from to [ , ]. however, the frequency of older females in fatal farm work incidents has historically been minimal and remains so. figure . female fatalities per year. the types of fatalities that affected older females are shown in table . the most common type of fatality was roadway related, with almost half of the cases ( . %). tractors, including tractor rollovers, were the third highest with three cases ( %). fourteen of the documented fatalities involved the operation of or exposure to farm machinery and tractors. table . breakdown of reported agent of fatal injury of female cases. category frequency percent roadway . farm machinery-related . tractors . smothering and asphyxiation . total - . . county fatalities were identified in almost all of indiana’s counties during the year period. only scott, union, vermillion, and warrick counties did not experience a documented farm fatality over the age of . the top five counties with the highest number of fatalities were allen with ( . %); harrison with ( . %); lawrence with ( . %); dubois with ( . %); and morgan with ( . %). allen county has a large number of amish/old order farm families, with the th largest amish community in the united states as of [ ]. historically amish/old order communities have represented a disproportionate share of farm-related deaths in indiana [ ]. there are other counties with a high density of amish/old order members, such as lagrange and elkhart, but these counties did not represent a disproportionate number of older fatalities [ ]. due to the tendency for fatalities trendline n = figure . female fatalities per year. the types of fatalities that affected older females are shown in table . the most common type of fatality was roadway related, with almost half of the cases ( . %). tractors, including tractor rollovers, were the third highest with three cases ( %). fourteen of the documented fatalities involved the operation of or exposure to farm machinery and tractors. table . breakdown of reported agent of fatal injury of female cases. category frequency percent roadway . farm machinery-related . tractors . smothering and asphyxiation . total - . . county fatalities were identified in almost all of indiana’s counties during the year period. only scott, union, vermillion, and warrick counties did not experience a documented farm fatality over the age of . the top five counties with the highest number of fatalities were allen with ( . %); harrison with ( . %); lawrence with ( . %); dubois with ( . %); and morgan with ( . %). allen county has a large number of amish/old order farm families, with the th largest amish community in the united states as of [ ]. historically amish/old order communities have represented a disproportionate share of farm-related deaths in indiana [ ]. there are other counties with a high density of amish/old order members, such as lagrange and elkhart, but these counties did not represent a disproportionate number of older fatalities [ ]. due to the tendency for amish/old order farmers to retire from many more hazardous farm work activities at an earlier age due to their large families, the risk to older amish/old order farmers may be less than might be anticipated [ ]. in addition, the intensity of agriculture is an important part of the economy of those counties reporting safety , , of high numbers of farm fatalities, including allen county, which had the state’s third highest total gross domestic agricultural product impact and the top producer of soybeans in the state for [ ]. the county is home to farms, ranking second in the state [ ]. the population of the county was , in , reflecting a high mix between urban and agricultural use of land increasing the risk of roadway crashes involving farm equipment [ ]. the other four counties, although not as highly ranked in the number of fatalities, have strong agricultural-based economies. an example of this would be the strong animal production that takes place in lawrence county, a leading producer of beef cattle, and dubois county, first in value of sales for turkeys for – [ ]. there does appear to be a positive correlation between the level of agricultural production, especially livestock production, and the larger number of farm fatalities involving older farmers. the distribution of fatal cases of farmers years and older by county is shown in figure , with each heart representing a farm work fatality that occurred during the period of to . a geographic distribution of all documented indiana farm fatalities from to can be found in a study by cheng and field ( ) [ ]. the geographic distribution of incidents was divided in half between the northern and southern parts of the state, with the dividing line running along u.s. . the two regions were then compared, since the southern portion tends to have rougher terrain, greater proportions of timber land, and older and smaller farms due to the state being settled from the south to the north, which can be seen in figure . the northern part of the state had fatalities, and the southern portion had fatalities, indicating no significant difference. , , x of amish/old order farmers to retire from many more hazardous farm work activities at an earlier age due to their large families, the risk to older amish/old order farmers may be less than might be anticipated [ ]. in addition, the intensity of agriculture is an important part of the economy of those counties reporting high numbers of farm fatalities, including allen county, which had the state’s third highest total gross domestic agricultural product impact and the top producer of soybeans in the state for [ ]. the county is home to farms, ranking second in the state [ ]. the population of the county was , in , reflecting a high mix between urban and agricultural use of land increasing the risk of roadway crashes involving farm equipment [ ]. the other four counties, although not as highly ranked in the number of fatalities, have strong agricultural-based economies. an example of this would be the strong animal production that takes place in lawrence county, a leading producer of beef cattle, and dubois county, first in value of sales for turkeys for – [ ]. there does appear to be a positive correlation between the level of agricultural production, especially livestock production, and the larger number of farm fatalities involving older farmers. the distribution of fatal cases of farmers years and older by county is shown in figure , with each heart representing a farm work fatality that occurred during the period of to . a geographic distribution of all documented indiana farm fatalities from to can be found in a study by cheng and field ( ) [ ]. the geographic distribution of incidents was divided in half between the northern and southern parts of the state, with the dividing line running along u.s. . the two regions were then compared, since the southern portion tends to have rougher terrain, greater proportions of timber land, and older and smaller farms due to the state being settled from the south to the north, which can be seen in figure . the northern part of the state had fatalities, and the southern portion had fatalities, indicating no significant difference. figure . distribution of fatalities per county. figure . distribution of fatalities per county. safety , , of , , x of figure . map of indiana’s agriculture-cultivated areas during [ ]. reproduced from indianamap, the name of the publisher: indiana geographic information council. . . reported type of fatal injury in the majority of cases, no official or medically determined cause of death was available. the reported, generally unofficial, types of fatal injuries were divided into ten categories based upon the terminology used in the reporting source. the distribution by type is shown in table . the most common fatalities by far were caused by crushing/run-over injuries, accounting for over half of all cases ( %). the unknown category was high because some documented cases only reported the fatality but did not include any background information on type of injury, cause of death, or other causative factors. there was no attempt to pursue additional information on the reported cause of death, and access to official death certificates was limited to only the first decade of the study period. the “other” categories included: a work-related heart attack, fatal electrocutions, an unintentional on-farm shooting, strikes by lightning, heat, and bleeding out (with cause not identified). table . breakdown of reported type of fatal injuries. category frequency percent crushed/runover figure . map of indiana’s agriculture-cultivated areas during [ ]. reproduced from indianamap, the name of the publisher: indiana geographic information council. . . reported type of fatal injury in the majority of cases, no official or medically determined cause of death was available. the reported, generally unofficial, types of fatal injuries were divided into ten categories based upon the terminology used in the reporting source. the distribution by type is shown in table . the most common fatalities by far were caused by crushing/run-over injuries, accounting for over half of all cases ( %). the unknown category was high because some documented cases only reported the fatality but did not include any background information on type of injury, cause of death, or other causative factors. safety , , of table . breakdown of reported type of fatal injuries. category frequency percent crushed/runover trauma from impact . asphyxiation . entanglement/caught in machinery . burns . head injury . other . drowning . chemical exposure . unkown . total - there was no attempt to pursue additional information on the reported cause of death, and access to official death certificates was limited to only the first decade of the study period. the “other” categories included: a work-related heart attack, fatal electrocutions, an unintentional on-farm shooting, strikes by lightning, heat, and bleeding out (with cause not identified). . . type of contributing agent the contributing agents involved were divided into ten different categories: tractors, farm machinery-related, roadway, smothering and asphyxiation, falls, cutting and trimming trees, fires/burns/explosions, livestock, other, and unknown. table provides a distribution of the categories of agents when known. the most common agent involved, by far, was tractors, with incidents ( . %); farm machinery-related was the second highest, with incidents ( . %). case information did not provide sufficient information to determine whether or not tractors involved were equipped with roll over protection structure (rops) or that the rops were in use at the time of incident. the five incidents in the other category included two cases of round bales rolling onto the victim, an unintentional shooting, a heart attack while attempting to remove a dead pig, and collapsing due to unknown issues, such as heat stress or a heart condition. table . breakdown of type of contributing agent. category frequency percent tractors . farm machinery-related . roadway cutting and trimming trees smothering and asphyxiation . falls . livestock . fires, burns, and explosions . unknown . other . total incidents were broken down further to better understand the primary causes of tractor-related fatalities, as seen in table . the most common incident type largely involved tractor rollovers, with cases ( . %), or . % of all documented incidents. the second most common incident type was tractor runovers, with cases ( . %), or . % of all cases. the third most common was pinned against, between, or underneath a tractor, with cases ( . %), or . % of all cases. safety , , of table . breakdown of fatal tractor-related incidents. category frequency percent percentage of all cases tractor rollover . . tractor runover . . pinned against, between, or underneath the tractor . . fall from a tractor . . unspecified tractor incident . . total . the next breakdown made was the machinery-related incidents, as shown in table . the most common incident type was “pinned against, between, or underneath an object or equipment,” with cases ( . %). the second most common was “equipment rollover,” with cases ( . %), and the third most common was “equipment runover,” with cases ( . %). table . breakdown of fatal machinery-related incidents. category frequency percent percentage of all cases pinned against, between, or underneath an object or equipment . . equipment rollover . . equipment runover . . entanglement in pto driveline . . struck by flying, falling, or thrown material . . fall from equipment . . entanglement in other, non pto components . . atv incident during farm-related work . . equipment incident, unspecified . . total . the breakdown of roadway incidents is shown in table . the most common fatality type was “roadway collisions involving farm tractors or equipment,” with cases ( . %), and the second highest was “farm truck incident during farm work,” with six cases ( . %). based upon indiana fatal motor vehicle crash data, the number of farmers involved in fatal work-related crashes, especially those involving farm trucks, is most likely under reported [ ]. table . breakdown of fatal roadway incidents. category frequency percent percentage of all cases roadway collision involving farm tractors or equipment . . farm truck incident during farm work . . roadway collision at railroad crossing . . roadway incident, unspecified . . total . the breakdown of incidents involving cutting and trimming trees is shown in table . all of the cases fell under the category of struck by a tree or tree limb ( cases), or . % of all cases. no fatality cases were reported relating to injuries sustained during the operation of chainsaws. safety , , of table . breakdown of fatal cutting and trimming tree incidents. category frequency percent percentage of all cases struck by a tree or tree limb . chainsaw total . the smothering and asphyxiation categories of fatality cases are shown in table . the most common type of incidents was “entrapped and suffocated by grain, feed, or other loose material,” with cases ( . %). the second most common type was “drowning in pond, lagoon, or stream,” with five cases ( . %). in some cases, it appears that the drownings were associated with being trapped beneath an overturned tractor or mowing equipment. table . breakdown of fatal smothering and asphyxiation incidents. category frequency percent percentage of all cases entrapped and suffocated by grain, feed, or other loose material . . drowning in pond, lagoon, or stream . asphyxiation or poisoning from gases . total . the breakdown of the category of falls is shown in table . the most common cases were “falls from farm structures,” with nine cases ( . %), and the second were “fall from ladder,” with five cases ( . %). data on falls that occurred in the home, though identified in some reports, were not considered work-related and therefore not included in the data set. considering the age of the population being studied, the number of non-work fatal falls could have been significant [ , ]. table . breakdown of fatal fall incidents. category frequency percent percentage of all cases fall from farm structure . fall from ladder . . fall incident, unspecified . . total . the breakdown of the livestock fatality type is shown in table . the most common type of incident involved bulls, with six cases ( . %), and the second involved cows, with five cases ( . %). the exotic livestock cases involved a wildebeest and a buffalo. table . breakdown of fatal livestock incidents. category frequency percent percentage of all cases bull . . cow . . horse . . exotic livestock . . total . the breakdown of the “fires, burns, and explosions” fatality categories is shown in table . the most common type of incident involved explosions, with five cases ( . %). the second most common type of incident was tied between “caught on fire with accelerant”, “electrocutions”, and “other fire-related incidents” with three cases each. safety , , of table . breakdown of fatal fire, burn, and explosion incidents. category frequency percent percentage of all cases explosion . . caught on fire with accelerant . . electrocution . . other fire-related incident . . farm structure fire . . smoke inhalation . . total . . discussion the data showed that the number of fatalities involving older farmers has been on the rise from to , with – having the highest reported number. this increase does not reflect the general decrease in the frequency of older fatal farm incidents over the same time period [ ]. the increase could be the result of several factors, including the increasing age of current farmers, farmers putting off retirement, older non-farmers becoming beginning farmers as part of their retirement, or due to improvements in reporting. the average age of all farm operators increased from . in , to . in , and to . in [ – ]. a survey by amshoff and reed ( ) of male farmers years or older found that % stated they were not retired from farm work, even though the average age of the sample was years [ ]. in addition, the number of principal operators of farms increased for the age groups of to years old, to years old, and years and older from to by . %, . %, and . %, respectively [ ]. the frequency of fatalities involving older farmers is clearly increasing, while the distribution of fatal incidents for all ages appears to be on a slight but continuous, decline. the increase in the number of small farms owned and operated by retired people cannot be ignored as a contributing factor. there was a . % increase in the number of farms owned by operators years and older from to [ , ]. in some of these cases involving new and beginning farmers, the lack of experience and safety training in the use of agricultural tractors and equipment may have played a significant role in the fatality. it is also important to mention that new methods in reporting, such as the internet, may have caused the increase in the number of recent cases. the distribution of the victims’ age showed a decrease around age up into the s. this may be explained by the fact that there is a smaller population of farmers still active at that age. the large number of fatalities in this age range, however, follows the literature that farmers work longer than the general workforce, with fatalities reported over , and the oldest case being years of age [ , ]. it appears that this trend parallels the % increase in the total number of farmers who were years or older from to in indiana [ ]. this is a concern because farm-related injuries occurring to older farmers are more likely to be fatal or result in long term or permanent disability. as noted, it is well documented that certain age changes, such as arthritis or hearing and vision impairments, contribute to increased risk of sustaining both non-fatal and fatal injuries. regarding type of contributing injury agents, tractors were identified as an area of concern, representing . % of the reported fatality cases. this finding aligns with other reports for the most common type of contributing agent for all ages [ , , – ]. the second highest type of fatality was farm machinery-related, with fatalities that accounted for . % of the deaths. the third and fourth highest fatality types were roadway and the cutting and trimming of trees, with and fatalities, respectively. tractor rollover incidents were clearly the most significant event, accounting for . % of cases, with most frequently reported fatal injuries of broken or crushed pelvis, broken bones, loss of blood, collapsed lungs, and chemical or other burns. other similar studies have reported that tractor rollovers, or overturns, have also been historically the most common type of fatal incident [ , , , – ]. it has also been reported that farm operators years and older are more likely to be operating a tractor without a rops [ ]. safety , , of occupational farm-related injuries and fatalities have a huge cost, estimated to be $ . billion in due to medical costs and reduced productivity [ ]. tractor overturns alone were projected to have a social cost of $ . billion over years [ ]. the problem gets even more complex when coupled with a large percentage of farmers and household members, . %, not having any health insurance in [ ]. with respect to older farmers, the length of recovery and rehabilitation costs associated with serious injuries can become unmanageable. . conclusions this study identified the most prevalent types of contributing injury agents and reported types of fatal injuries, as well as other demographic factors of indiana farmers years and older. the data are important to help guide the development and delivery methods of agricultural safety material to create a safer worker environment by illustrating what risks are most prevalent for older farm workers. the summary suggests that agricultural safety material and practices should be focused towards farm worker incidents relating to tractors, farm machinery, roadways, cutting and trimming trees, and smothering and asphyxiation risks. educational materials should clearly address how the aging process contributes to the increased risk of injury and measures needed to reduce the impact of age-related functional limitations. the most significant risk that should be addressed with this population is the potential for tractor overturn and the well-recognized hazard of operating older tractors without rops. the findings from this study should be an important tool to be given to stakeholders, including farm families and extension educators, so they can be better informed about the health and safety of the older farmers in their families and communities. findings should assist in initiating a discussion on ways to enhance the safety of these workers while still allowing them to continue contributing to the farm operation. future research is needed to better understand how this population perceives risks and to better understand the values and behaviors of older farmers, compared to other age groups, that motivate them to remain involved in performing hazardous farm-related tasks. work is also needed to develop and field test injury prevention strategies that are effective at modifying behaviors and attitudes that contribute to a higher risk of farm work injuries among this population. author contributions: s.a.t. and w.e.f. conceived and designed the experiments, which were conducted by s.a.t. s.a.t. 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[crossref] . usda-ers. health insurance coverage. . available online: https://www.ers.usda.gov/topics/farm- economy/farm-household-well-being/health-insurance-coverage/ (accessed on april ). © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /ajim. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.jsr. . . http://ashca.org/wp-content/uploads/ / /ashca- -ag-safety-fact-sheet- .pdf http://ashca.org/wp-content/uploads/ / /ashca- -ag-safety-fact-sheet- .pdf http://dx.doi.org/ . / . https://www.ers.usda.gov/topics/farm-economy/farm-household-well-being/health-insurance-coverage/ https://www.ers.usda.gov/topics/farm-economy/farm-household-well-being/health-insurance-coverage/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	methods 	results 	age 	year 	month 	gender 	county 	reported type of fatal injury 	type of contributing agent 	discussion 	conclusions 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ cloning the differences between two complex genomes cloning the differences between two complex genomes author(s): nikolai lisitsyn, natalya lisitsyn, michael wigler source: science, new series, vol. , no. (feb. , ), pp. - published by: american association for the advancement of science stable url: http://www.jstor.org/stable/ accessed: / / : your use of the jstor archive indicates your acceptance of jstor's terms and conditions of use, available at http://www.jstor.org/page/info/about/policies/terms.jsp. jstor's terms and conditions of use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the jstor archive only for your personal, non-commercial use. please contact the publisher regarding any further use of this work. publisher contact information may be obtained at http://www.jstor.org/action/showpublisher?publishercode=aaas. each copy of any part of a jstor transmission must contain the same copyright notice that appears on the screen or printed page of such transmission. jstor is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. we use information technology and tools to increase productivity and facilitate new forms of scholarship. for more information about jstor, please contact support@jstor.org. american association for the advancement of science is collaborating with jstor to digitize, preserve and extend access to science. http://www.jstor.org http://www.jstor.org/stable/ ?origin=jstor-pdf http://www.jstor.org/page/info/about/policies/terms.jsp http://www.jstor.org/action/showpublisher?publishercode=aaas * research articlemne ... .s . . .. . . .. ... .... cloning the differences between two complex genomes nikolai lisitsyn, natalya lisitsyn, michael wigler the analysis of the differences between two complex genomes holds promise for the discovery of infectious agents and probes useful for genetic studies. a system was de- veloped in which subtractive and kinetic enrichment was used to purify restriction endo- nuclease fragments present in one population of dna fragments but not in another. application of this method to dna populations of reduced complexity ("representations") resulted in the isolation of probes to viral genomes present as single copies in human dna, and probes that detect polymorphisms between two individuals. in principle, this system, called representational difference analysis (rda), may also be used for isolating probes linked to sites of genomic rearrangements, whether occurring spontaneously and resulting in genetic disorders or cancer, or programmed during differentiation and development. genetic alterations underlie various bio- logical processes. programmed gene rear- rangements occur in many contexts, in- cluding the generation of diversity in the immune system ( ), mating type switching in yeasts ( ), and antigenic variation in microbial organisms ( ). spontaneous loss- es or rearrangements of genetic material in somatic cells occur frequently during the development of cancers and leukemias. similar events occurring in the germ line can result in inherited disorders. even in- fectious disease can be viewed as the alter- ation of the genetic content of the infected organism. various time-consuming meth- ods have been applied to determining the nature of the genetic changes that occur in the above situations. a single method for defining the differences between two dna populations could, in principle, be applied to each of the above problems and lead to the discovery of the genetic basis of many types of biological phenomena. we present here a general method (representational difference analysis, or rda) for finding small differences between the sequences of two dna populations. the method builds upon subtractive hybridization techniques that have been used in the past to find probes for large sequence differences be- tween two genomes. in lamar and palmer applied a subtractive hybridization technique to clone probes for the y chromosome ( ). they used an excess of sheared dna from a female to drive hybridization of sau a-cleaved dna from a male. the y chromosome-specific dna was free to self-anneal and was subse- quently cloned after ligation into an accep- tor site of a plasmid. a similar method was applied by kunkel et al. ( ) and then by nussbaum et al. ( ) to clone probes for the the authors are at cold spring harbor laboratory, p.o. box , cold spring harbor, ny . duchenne muscular dystrophy and the chor- oidermia loci, respectively. in both cases, large deletions on the x chromosome made it possible to use subtractive hybridization techniques with dna from affected males as "driver" and dna from normals as "tester" ( ). in general, however, subtractive hybrid- ization techniques do not achieve sufficient enrichment of the sequences that occur only in tester (the "target") partly because of the high complexity of the human genome, which prevents effectively complete hybrid- ization ( ). even when subtractive steps are reiterated, "target" sequences are enriched only to times ( ). this enrichment is insufficient for more common situations in which the magnitude of enrichment required is . a second means for dna difference enrichment has been proposed on the basis of the second-order kinetics of self-reasso- ciation ( ). in theory, this method can be applied after an initial enrichment of target sequences has been achieved by subtrac- tion. if a population of dna fragments containing a target subpopulation enriched n times relative to unenriched fragments in tester is melted and reannealed so that only a small proportion of double-stranded tester dna forms, double-stranded target dna would be present n times relative to the other sequences present as duplex dna ( ). we call this "kinetic" enrichment. to apply this method, it is necessary to use some kind of dna amplification to purify the small quantities of double-stranded dna that form away from all driver and all unreannealed tester sequences. we achieved this by ligating oligonucleotide adaptors to tester so that only double- stranded tester molecules were amplified during the polymerase chain reaction (pcr) ( ). subsequent reiterations of the method led to exponentially increasing en- richments of target. in rda, we lowered the dna complex- ity of both tester and driver genomes by preparing a representative portion of each genome (a "representation"). the differ- ence analysis of the two representations is based on simultaneous combination of sub- tractive and kinetic steps. the lowered complexity of the representations allowed us to achieve greater completeness during subtractive enrichment and, hence, a more effective kinetic enrichment. we now dem- onstrate the cloning of probes for single copy sequences present in (or absent from) one of the two genomes, and the cloning of probes for binary polymorphisms between two individuals. we discuss the applica- tions of this approach to the discovery of probes for pathogenic organisms, for other- wise anonymous loci that have suffered genetic rearrangements, and for polymor- phisms located near the genes affected by inherited disorders. subtractive and kinetic enrichments of pcr amplicons. we began by making rep- resentations of dna populations that we call "amplicons"; dna cleaved with rela- tively infrequent cutting restriction endo- nucleases was ligated to oligonucleotide adaptors, and amplified by pcr. the result was similar to a size fractionation, since after rounds of amplification only low molecular size fragments, below kb, were effectively amplified. the advantage of this method for representation compared to a physical fractionation is that only small amounts of starting material (less than ,ug) are required. only a subset of the whole genome was represented. however, when different restriction endonucleases were chosen, sets of amplicons that scan the genome could be made. in the following examples, we made amplicons of mamma- lian dna after it underwent cleavage with bam hi, bgl ii, and hind iii ( ). we estimate the complexity of the resulting amplicons to be times, times, and times less than the complexity of the start- ing genomic dna, respectively ( ). once tester and driver amplicons were made, their adaptors were removed by cleavage, and only tester fragments were ligated to new adaptors at their ' ends. we then combined subtractive and kinetic steps into a single operation, the hybridiza- tion-amplification step (fig. ) ( ). we abandoned all use of physical separation techniques, such as biotinylation with sub- sequent biotin-avidin chromatography ( , ) or hydroxylapatite ( ), to enrich for double-stranded tester because these meth- ods are not completely reliable. instead, dna amplification was used for selective enrichment of double-stranded tester. after melting and reannealing in the presence of excess of driver amplicon, dna molecules were treated with taq dna polymerase at science * vol. * february elevated temperatures in the presence of all four deoxynucleotide triphosphates. only seif-reannealed tester molecules had ' adaptors at each end of the duplex dna and thus could be filled in at both ' ends. therefore only self-reannealed tester could subsequently be amplified by pcr at an exponential rate. driver dna acted as a competitive inhibitor for the self-reanneal- ing of those tester dna fragments common to driver. target dna, which occurs only in tester, was thus enriched relative to other tester dna after amplification. the pcr products were then either cloned, otherwise analyzed, or further enriched. in the last case, the pcr products were cleaved, and yet new pcr adaptors were added to their ' ends ( ). this material was then diluted ( i ) with fresh driver amplicon and the hybridization-amplifica- tion step was repeated. the first round of rda was mainly subtractive. subsequent rounds had a great- ly increased kinetic component equal to the enrichment achieved on the previous step. for example, if target is equimolar with respect to tester (that is, single copy), and if driver amplicon is taken in n times excess to tester amplicon, target may be enriched e times after the first round, where e < n ( ). after the second round, target would be enriched yet another e times because of repetition of the subtractive component, multiplied by another e times because of the kinetic component. after the third round, total enrichment would be at least the square of that. if e is , at the end of the second round, target would be enriched by about i , and at the end of the third round by more than . acquisition (or loss) of new sequences. in the first set of stringent tests of our protocols, we added, as single copies, ade- novirus or bacteriophage a dna (or both) to a human dna to create a model tester, and used the same dna without viral dna as driver. the viral dna's were the target and we can view this model as testing the efficacy of the procedure in either of two situations: the acquisition of a pathogenic genome at a single copy of genomic dna per infected cell; or the homozygous loss of dna by spontaneous mutation. we pre- pared bgl ii amplicons from human dna with adenovirus and x dna's as targets (fig. a) or hind iii amplicons with a dna as target (fig. b). if bgl ii amplicons were taken, small a and adenovirus frag- ments were the major difference products even after two rounds (fig. a, lane a). this represents an enrichment of more than x times from the starting material, and a probable enrichment of about x times from amplicons. there is a strong bias during amplification against fragments larg- er than . kbp, so that the larger bgl ii fragments of target (fig. a, lanes c and d) are not enriched. the enrichment from hind iii ampli- cons was not as effective. the a hind iii fragment was greatly enriched after the third round (fig. b, lane b), but other sequences were still present. nevertheless, after the fourth round the expected target fragment was purified to near homogeneity (fig. b, lane c). we attribute this out- come to the greater sequence complexity of the hind iii amplicon. in fact, we found that without some simplification of driver complexity, our method failed. presumably, when the complexity of the driver was too high, subtractive and kinetic enrichments were diminished and competing processes dominated. probes for polymorphic loci. in the experiments described above, the driver and tester were identical except for the tester amplicon driver amplicon a b (inexcess) ab cde ab cd efg abcdefg m~~~~~~~~~~~~~~~~~~ - = _ am c ugate to - - dephosphorylated - - | o _ ligate to _- ~~~~~~~~- - - adaptor strands - c c - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ~ vj mix, melt, anneal =_ ~~~~~~~~~~.............. -rrrrrrrrfttm xrrrrrtttrfm - fig. (left). schematic protocol for representational difference analysis, illustrating ds-tester hybrids ds-driver the hybridization and amplification steps after the preparation of amplicons. the ss-tester ss-dnrver representation stage is not illustrated. the details are described in ( ). fig. (above). representational difference analysis with viral dna's added as targets. . . + fill in the ends high molecular weight dna ( ,ug) purified from the lymphoid cell line drl was used for preparation of driver amplicons and .lg of the same dna, containing =_ = r ....... _trn equimolar amounts of target [ pg of adenovirus- dna or pg of x phage dna =_ ....... v (or both), both from new england biolabs] was taken for preparation of tester o add primer, pcr amplify amplicons ( ). difference products were prepared as described in ( ). (a) add primer, pcr amplify agarose gel electrophoresis of difference products of bgl ii amplicons, obtained after _______ gthe second (lane a) and third (lane b) hybridization-amplification steps. x phage (lane c) and adenovirus (lane d) amplicons were prepared as described ( ) taking to unear amplification ng of purified viral dna ligated to adaptors (primer set ; see table ). hae ilil __________ _______, digest of x rf dna is in lane e. sizes (bp) are indicated to the right. arrows on the left indicate major difference products. (b) agarose gel electrophoresis (b, ) and exponential amplification no amplification no amplification southern blot (b, ) of difference products of hind ilil amplicons. difference products ( . ag) obtained after two, three, and four hybridization-amplification steps are digest ss-dna wath mung b shown in lanes a to c. the x phage hind ilil amplicons were prepared as described nudease, pcr amplify ( ) taking to ng of purified viral dna ligated to adaptor set (lane d). the difference product after four hybridization-amplification steps was digested with hind difference product enrched in target ill (lane e). hind iil-digested x phage dna (lane f) and hae iil-digested +x dna (lane g, sizes indicated to the right) are as shown. these dna's were separated on digestwithrestrictio endonudease a percent agarose gel, transferred at reduced pressure with the lkb clone and analyze vacugene vacuum blotting system (pharmacia lkb biotechnology) to genescreen plus membranes (du pont) and hybridized to p-labeled a phage hind ilil amplicon under conditions recommended by the membrane supplier. arrow on left indicates the target x fragment. the slight upward mobility shift of this fragment in lanes b, c, and d relative to lanes e and f is due to the presence of adaptors. science * vol. * february a c bam hi bgl hind iii r , ,, h i ab cd ef g hi j klim no p qr a b c d e f g h i j k i m : : - - - : - _ - - -~ - - - .i y - b fig. . representational difference analysis of dna's from two individuals. (a) agarose gel electrophoresis of bam hi (lanes a to d), bgl ii (lanes e to h), and hind ilil (lanes i to ) difference products. tester dna amplicons (lanes a, e, and i) and difference products, after the first (lanes b, f, and j), second (lanes c, g, and k) and third (lanes d, h, and ) dna hybridization-amplification steps, were prepared as described ( , ). hae ilil x dna size markers (lane m) are indicated in base pairs. driver and tester amplicons were prepared from human lymphoblastoid cell cultures gm and gm , respectively (amish pedigree , human genetic mutant cell reposi- tory, camden, nj). (b) autoradiogram of conventional southern blot hybridized to bam hi parf probe (table ) cloned from the bam hi difference product. bam hi-digested dna's ( ,ug) isolated from human lymphoblastoid cell cultures gm , gm (tester), gm (driver), cl. w gm , gm , gm , and gm (lanes a to g) from amish pedigree and pug k-' v k) each of bgl li and hind ilil digests of gm dna (lanes h and i) were separated on a percent agarose gel, vacuum-transferred, and hybridized to the radioactive probe. sizes (kbp) are indicated to the right. the family tree is vertically aligned above the autorad. filled squares or circles indicate a b c d a f g h i individuals with the bipolar affective disorder. (c) autoradiogram of bam hi parf probe ..........j s-g; . . >e.-i ehybridization to a blot containing transferred bam hi amplicons ( i?g each) of human dna's . . isolated from three lymphoblastoid cell cultures (lanes a to c), two fibroblast cell lines (lanes d and _ e), five human placentas (lanes f to j), and seven amish pedigree lymphoblastoid cell cultures * -l . gm , gm , gm , gm , gm , gm , and gm (lanes k to q). the . l l bam hi difference product ( ng) after three hybridization amplification steps was placed in lane r. sizes (in base pairs) are indicated to the right. experimental perturbation introduced by the presence of exogenous dna in tester. in order to assess the method for potential applications, we compared tester and driver amplicons from different individuals and expected to obtain a subset of restriction endonuclease fragments that were polymor- phic between them. for example, if one of the two bam hi sites flanking a short bam hi fragment in tester was absent in both alleles from driver, leading to only large and poorly amplified bam hi fragments in driv- er, the short bam hi fragment of tester would be present in its bam hi amplicon but absent in the bam hi amplicon of the driver. we were able to study two sisters from an amish family with a well-established pedi- gree. amplicons were prepared after cleav- age with bam hi, bgl ii, or hind iii. difference products between amplicons were obtained ( , ) and size-fractionated by gel electrophoresis (fig. a). a discrete but complex pattem of bands was observed in each case. after three successive hybridiza- tions and amplifications (fig. a, lanes d, h, and ), difference products were cloned into plasmids. for each difference product we picked three probes for blot hybridization analysis, and found that all of them detected polymorphisms within the amish family. we analyzed bam hi difference products in the greatest detail (table ). of random- ly picked clones, different classes of clones remained after removing redundan- cies, and the inserts from nine of these were used as probes in dna (southern) blots of tester, driver, and five other members of the family. all probes detected two alleles, dis- tinguished by a large and a small dna fragment (table , allele size). the small allele was always present in the tester (fig. b, lane b, and table , sample b) and always absent in the driver (fig. b, lane c, and table , sample a). the probes detect- ed either one of these bands in presumed homozygotes (fig. b, lanes c and e), or both bands in presumed heterozygous indi- viduals (fig. b, lanes a, b, d, f, and g). moreover, the blot hybridization pattem for each probe was consistent with a mendelian pattem of inheritance. from these experi- ments we concluded that our method yields collections of probes for restriction endonu- clease fragment polymorphisms between two individuals. each of the bam hi probes derived from the above experiment was also used in blot hybridizations to amplicons from the family and ten other unrelated human dna's extracted from cell lines or placentas (fig. c and table ). such blots detect the presence or absence of small-size bam hi fragments in the tested dna, and thus are more readily performed than conventional southem blots to total genomic dna. we found that blotting amplicons was in com- plete concordance with blotting of total genomic dna, as described above, in out of cases (nine probes times seven individuals). the results of the amplicon blots indicate that probes capable of detect- ing polymorphisms within the amish fam- ily tend to detect polymorphisms in the human population at large. we call such polymorphisms parf's, for polymorphic amplifiable restriction endonuclease frag- ments. science * vol. * february , " i.research article table . sequences of primers used for representational difference analysis. primer set (r series) was used for preparing amplicon representations, and sets (j series) and (n series) were used for odd and even hybridization-amplifications, respectively, the oligo computer program (national biosciences) was used to check the oligonucleotide design for the absence of strong secondary structure. primer name sequence set r bgl '-agcactctccagcctctcaccgca- ' r bgl '-gatctgcggtga- ' j bgl '-accgacgtcgactatccatgaaca- ' j bgl '-gatctgttcatg- ' n bgl '-aggcaactgtgctatccgagggaa- ' n bgl '-gatcttccctcg- ' r bam '-agcactctccagcctctcaccgag- ' r bam '-gatcctcggtga- ' j bam '-accgacgtcgactatccatgaacg- ' j bam '-gatccgttcatg- n bam '-aggcaactgtgctatccgagggag- ' n bam- '-gatcctccctcg- ' r hind same as r bgl (see above) r hind '-agcttgcggtga- ' j hind same as j bgl (see above) j hind '-agcttgttcatg- ' n hind '-aggcagctgtggtatcgagggaga- ' n hind '-agcttctccctc- ' table . screening for presence of bam hi parf's in human dna samples. probe dna sample* allele size (kbp)t no. percent t a b c d e f g h i j k l m n o p q large small . - + - + + + + + + + + + + + + + + . , . ? . - + - - + + . . - + + + + + + + + + +- + - - + . . . - + + - + + + + + + - + - -+ + + . . - + - - + + + + + + . . - + + + - + + + - + f + + - + + + . . . - + + + - + + + + + + - - - + + + nd . . - + - + + + + +- + + - + + - + - > . , - ++ --- + + + -+ - + + + - , . . - + ----+ - + - nd . . - + + + + + + + + + + - + + + . . - + + + - + + + + + + - + - + + + nd . *bam hi amplicons were prepared from dna from seven amish pedigree lymphoblastoid cell cultures, gm (driver), gm (tester), gm , gm , gm , gm , gm (columns a to g), five different placentas (columns h to l), three lymphoblastoid cell lines established from the biopsies of leukemic patients (columns m, n, and ), and two fibroblast cell cultures, drl and drl , established from the biopsies of dmd patients (columns p and ), transferred to genescreen membrane, and hybridized to the indicated probes. the plus sign means that the small bam hi parf allele was present in the sample (that is, the probe hybridized to a band of the correct size in the amplicon); the minus sign means that the small allele was not detected (see, for example, fig. c). tthe sizes of the alleles hybridizing to parf's are indicated, where known. tthe percentage of clones in the bam hi difference product (obtained after three hybridization-amplification steps) that hybridized to the indicated clone. ?two different small alleles were found in the human population. iitwo different large alleles were found in the human population. nd, not determined. the probes for parf's are not equally abundant in the difference product. to ob- tain a measure of this uneveness, we hybrid- ized each cloned bam hi parf to a grid of individual randomly picked clones from the difference product of the two siblings, and its frequency in the collection was de- termined (see percentage value in table ). of randomly picked elements, only distinct polymorphic probes were present, and at very different frequencies. we esti- mate that there should be of the order of bam hi parfps between two individ- uals. the uneven distribution of parf's in our difference products probably reflects the specific conditions used in our protocol, which was designed for the detection of a small number of differences between two nearly identical genomes. we found that, where probes for polymorphic loci were de- liberately sought, more representative differ- ence products could be generated by dimin- ishing the number of rounds of hybridization and amplification, increasing the complexity of the representation or decreasing the total number of pcr cycles. applications to pathogen discovery. in principle, rda can be used to isolate probes for pathogens when dna from infected tissue is compared to dna from uninfected tissue from the same individual. we have demonstrated this in the model case of the acquisition of a large ( to kbp) viral genome even with a single copy per cell. probes for smaller viral genomes should also be detectable, but might require several ap- plications of this procedure with several different restriction endonucleases in order to find fragments from the viral dna that are readily amplified. as it is now described, rda cannot reasonably be expected to de- tect probes for pathogens that are present at less than one copy per ten cells in infected tissue. the kinetic enrichment component would select against target sequences consid- erably when they are present at concentra- tions lower than other tester sequences. however, procedures for "normalization" ( ), that equalize the concentrations of all tester sequences, could be applied prior to subtractive and kinetic enrichment. application of rda to the discovery of pathogens requires precise matching of the polymorphisms from the infected and unin- fected dna sources. tester and driver dna can derive from the same individual, if the individual is not a genetic mosaic. these dna's cannot derive from unrelated individuals, as the abundant polymorphic differences in their dna's would obscure the detection of the pathogen. however, the uninfected dna source (driver) could, in principle, come from an identical twin, or be the pooled dna from the parents of the infected individual, because virtually every dna restriction fragment found in the genomic dna of the infected individ- ual can be expected to be present in at least one parental dna ( ). pooled parental drivers might be sought if the pathogen is suspected to be ubiquitous in the infected individual. applications to detecting genetic ab- normalities in cancer. rda should enable the discovery of genomic alterations occur- ring in cancer cells. these could be of two distinct types: those that result in loss of restriction endonuclease fragments, such as might occur from deletions or gene conver- sions extending over heterozygous polymor- phisms, and those that produce new restric- tion endonuclease fragments, such as might result from genomic rearrangements. in the former case, rda could be applied without modifications with dna from cancer cells as driver and normal dna as tester. un- fortunately, the presence of normal stroma in a cancer biopsy would certainly interfere with the detection of loss of genetic infor- mation in the cancer cell. hence, either cultures of cancer cells, xenographs of tu- mors, or highly purified cancer cells ob- tained by physical separation would be needed as the source for tester. science * vol. * february fig. . schematic repre- a b sentation of restriction en- donuclease fragments of a b a b c d genomes that have suf- < < ? fered rearrangements. (a) , reciprocal translocation. d (b) deletion. restriction < d endonuclease sites a, b, c b d c, and d are as shown > before and after rearrange- c ments. below is depicted the mobility of the new (*) and unrearranged frag- d ments during gel electro- phoresis. we assume that *ad ab the normal chromosomes of the involved pair are cd cd present. ab *ad *bc bc tumor or normal tumor or normal spontaneous spontaneous mutant mutant these restraints do not apply to the detection of genomic rearrangements. ge- nomic rearrangements, including transloca- tions, insertions, inversions, and deletions, may result in the creation of new restriction endonuclease fragments "bridging" the site of the rearrangement (fig. ). some of these bridging fragments may be amplifiable by pcr (fragment bc in fig. a). such bridg- ing fragments would be discoverable by rda when dna from the tumor is used for preparation of tester amplicons and dna from normal tissue of the same individual is used for preparation of driver amplicons. the different-sized restriction endonucle- ase fragments created by genomic rearrange- ments can potentially be exploited another way. fractionated size classes from tumor dna digests may sometimes contain se- quences that are not present in comparable size classes from normal dna (fig. ). with the former as tester and the latter as driver, we can prepare amplicons after cleavage with a second restriction endonuclease and compare these in order to clone amplifiable restriction endonuclease fragments in prox- imity to the point of genetic rearrangement. the presence of normal cells among the tumor cells should not obscure the detection of probes for the rearrangement. application to genetic analysis. when rda is applied to different individuals, and amplicons are used as the representation, probes for a special type of polymorphism that we call parf's result. other types of representations can yield different types of polymorphisms. in general, rda may be useful for generating new sets of polymor- phisms not only for species that have not previously undergone extensive molecular genetic characterization, but even for such well-studied species as humans and mice. since parf's are most often binary, they may be especially useful for creating a panel of probes that can be used with a standard- ized format for genetic typing. rda may find special uses when applied to pairwise comparisons of amplicons ob- tained from dna's of individuals from spe- cific groups. straightforward application of rda could yield probes for parf's in the dna of all individuals from a founder group affected by some autosomal dominant inherited disorder (the tester), but absent in the pooled dna of all individuals from a normal group (the driver). conversely, rda could yield probes for parf's present in the dna of a normal individual (the tester), but absent in the dna of all individuals from a founder group affected by a recessive inherited disorder (the driver). combined with methods for coincidence cloning ( ), such applications might ac- celerate the discovery of probes for rare parf's in linkage disequilibrium with the dominant locus, or the absence of common parf's in linkage disequilibrium with the recessive locus ( ). many genetic diseases of progeny occur as a consequence of spontaneous germline genomic rearrangements in the gamete of one of the parents. the genome of the affected individual will in all probability have acquired restriction endonuclease fragments that are not present in the somat- ic cells of either parent. this situation is formally analogous to genetic rearrange- ments occurring in cancer cells (fig. ). in these special cases, rda might be applied directly to the isolation of probes close to the sites of genetic abnormalities by taking dna from the individual as tester and pooled dna's from parents as driver. programmed rearrangements in somatic cells may also be discovered by rda. such events, for example, occur during the gen- eration of diversity of the immune system ( ), and have also been postulated during development of the nervous system ( ). in principle, our method could be applied to the discovery of probes that detect these events. finally, rda may have special applications for the study of organisms that can be bred. multiple backcrossing of a mutant strain to a polymorphic strain could lead to the identification of clonable restric- tion endonuclease polymorphic fragments tightly linked to the mutant locus. cautions. the data obtained with our protocol were highly reproducible; the same bands and even their relative intensities were obtained in multiple independent runs. application of any new technique requires awareness of possible sources of artifact. one clear source can be pcr itself, because of the ease with which previous pcr products can contaminate reactions. moreover, pcr products present after sub- traction and enrichment do not necessarily reflect effective enrichment of target and may result from the stochastic nature of the process itself ( ). therefore each candidate difference product must be tested for its presence or absence in tester and driver amplicons. another source of artifact can be anticipated during tissue sampling. nor- mal flora contaminating a specimen of tester will be readily enriched during differ- ence analysis if that flora are not also present in driver. other sources of artifact can be readily imagined. for example, we do not know to what extent we may find programmed genetic rearrangements specif- ic for certain tissues or organs. we also do not know the extent of genetic mosaicism in somatic tissues, the result of spontaneous genetic events occurring during early devel- opment. even so, application of rda should provide an economical route to solv- ing many types of genetic problems. references and notes . . bernard, n. hozumi, s. tonegawa, cell , ( ). . j. n. strathern, e. spatola, c. mcgill, j. b. hicks, proc. natl. acad. sci. u.s.a. , ( ); d. h. beach, nature , ( ). . e. segal, e. billyard, m. so, s. storzbach, t. f. meyer, cell , ( ); l. h. t. van der ploeg, c. a. cornelissen, p. a. m. michels, p. borst, ibid. , ( ). . e. e. lamar and e. palmer, ibid. , ( ). . l. m. kunkel, a. p. monaco, w. middlesworth, h. d. ochs, s. a. latt, proc. natl. acad. sci. u.s.a. , ( ). . r. l. nussbaum, j. g. lesko, r. a. lewis, s. a. ledbetter, d. h. ledbetter, ibid. , ( ). . throughout this article, we use "target" to refer to sequences present in one population, the "tester," but absent in another, the "driver." . in general, a single cycle of subtraction can be expected to yield enrichments of target of the order of fnwhere nis the molar excess of driver to tester and f is the fraction of driver that reanneals. . i. wieland, g. bolger, g. asouline, m. wigler, proc. natl. acad. sci. u.s.a. , ( ). . to visualize this, consider viral sequences pres- science * vol. * february ........ .......... ... .. ... .... .... ... .... .............. ...... ........ ..... r esearch a rticle . . .... .. . ... . . . .. ..... .... ...... ... .......... . . ... .... . .......... . .. ...... ...... ... ...... ... . . . . . . . . ........ . .. .... .... .. ..... ........ ... ..... .... . ... ... . ... ....... ent in excess (ten times more) relative to single- copy -globin sequences. at early stages of self- reannealing, when . percent of the viral se- quences are reannealed, only . percent of the j -globin sequences will be reannealed. the ratio of the viral sequences to the p-globin sequences in the double-stranded dna will then be percent of to . percent of (one hundred times more). . r. k. saiki, et al. science , ( ). . for preparation of amplicons both tester and driver dna samples were digested with restriction endonuclease (new england biolabs) and ,ug of each dna digest was mixed with . nmol of -bp and of -bp unphosphorylated oligonucle- otides (table , primer set ) in ,ul of t dna ligase buffer (new england biolabs). oligonucle- otides were annealed by cooling the mixture gradually from ? to ?c for hour and then ligated to human dna fragments by overnight incubation with u of t dna ligase at ?c. after ligation, both tester and driver dna samples were amplified. each of ten tubes taken for prep- aration of driver amplicons and two tubes used for preparation of tester amplicons contained (in ,ul) mm tris-hci, ph . at ?c, mm mgci , mm (nh ) s , mm p-mercaptoethanol, bovine serum albumin ( pg/mi), pm (each) datp, dgtp, dctp, and dttp, ,um -bp primer, and ng of dna with ligated adaptors. the tubes were incubated for minutes at ?c in a thermal cycler (perkin-elmer cetus), u of taq polymerase (amplitaq, perkin-elmer cetus) was added, the reactions were overlaid with mineral oil and incubated for minutes to fill in ' protruding ends of ligated adaptors, and amplified for cycles (each cycle including minute incubation at ?c and minutes at ?c, with the last cycle followed by an extension at ?c for minutes). after amplification both driver and tester amplicons were digested with the same restriction endonuclease ( u/,ig) to cleave away adaptors. for removal of adaptors, ,ug of tester amplicon dna digest was subject- ed to electrophoresis through percent nusieve agarose (low melting point, fmc bioproducts), and dna fragments ( to bp) were recov- ered after melting of the agarose slice and qia- gen-tip o chromatography (quiagen inc.). . these estimates in degree of simplification were calculated from data on the mean restriction endo- nuclease fragment lengths and the proportion of fragments of < kbp [d. t. bishop, j. a. williamson, m. h. skolnick, am. j. hum. genet. , ( )]. our calculations of the number of fragments with an amplifiable size are not based on complete informa- tion. we do not know, for example, the exact math- ematical dependence of amplification efficiency upon fragment length, nor the proportion of frag- ments of any length that amplify poorly. moreover, the occurrence of restriction endonuclease cleav- age sites is not truly random. . in preparation for the hybridization and amplifica- tion step, fragments of tester amplicons were ligated to new adaptors (table , primer set , and fig. ). the tester amplicon ( . ,ug) ligated to adaptors and the driver amplicon ( ,g) dna's were mixed, ethanol-precipitated, dissolved in pi of x ee buffer ( ), overlaid with pl of mineral oil (perkin-elmer cetus), and denatured by heat; pi of m naci solution was added and dna was hybridized for hours at ?c. at the end of hybridization, part ( percent) of the resulting dna was incubated with u of taq polymerase ( minutes, ?c) in pi of pcr mixture without primer to fill in ends of the rean- nealed tester, and then amplified for cycles ( minute at ?c, minutes at ?c, and held for minutes more for the last round) after addition of the same -bp oligonucleotide to which the tester was ligated. single-stranded dna mole- cules present after amplification were degraded by a -minute incubation with u of mung bean nuclease (new england biolabs) in a volume pi, diluted ( : ) in mm tris-hci (ph . ), and heated ( ?c, minutes) to inactivate the en- zyme. a portion ( i) of the solution was ampli- fied for to cycles under the same conditions as before the mung bean nuclease treatment. amplified dna ( to pg) was digested with the original restriction endonuclease, and ng of the digest was ligated to the third adaptor (table , primer set ). this dna ( to ng) was mixed with pg of driver amplicon and the hybridization and amplification procedures were repeated as in the first cycle. a sample ( ng) of the digest obtained after the second hybridiza- tion-amplification step (table , primer set ) was then ligated to the second set of adaptors, and to pg of this material together with pg of driver amplicon was taken for the third round of hybridization, with a final amplification after mung bean nuclease digestion for to cycles. when a fourth hybridization-amplification step was performed, pg of material from the third round was ligated to the third adaptors (table , primer set ) and mixed with pg of driver amplicon before proceeding. . d. straus and f. m. ausubel, proc. natl. acad. sci. u.s.a. , ( ). . pcr adaptors were alternated between rounds of hybridization and amplification to avoid the accu- mulation of pcr products that might interfere with subsequent amplifications. the adaptors are de- signed to reconstruct the original restriction endo- nuclease cleavage sites that defined the tester fragments, and are therefore removable by cleav- age with that same enzyme. . if enriched tester is insufficiently diluted, further enrichment due to the subtractive component will be hampered because driver amplicon sequences may not then be in excess over amplicon se- quences from tester. further enrichment due to the kinetic component will also be diminished because of the excessive self-reannealing of non-target tester. if enriched tester is diluted too much, so little target will reanneal that it may no longer be detect- able by pcr. the protocols described should serve as a practical guide. . s. r. patanjali, s. parimoo, s. m. weissman, proc. nati. acad. sci. u.s.a. , ( ). . homologous germline recombination between two polymorphic restriction endonuclease sites would create a new restriction fragment length polymorphism present only in the progeny, but this should be a rare event. . a. j. brookes and d. j. porteous, nucleic acid. res. , ( ). . we do not have enough experience with rda to predict what would happen if dna's pooled from individuals of one group were used as tester and dna's pooled from individuals of another group were used as driver. we expect the difference product would yield probes to parfs common to most of the first but absent in all of the second group. . h. yokota and m. oishi, proc. natl. acad. sci. u.s.a. , ( ); j. j. chun, d. g. schatz, m. a. oettinger, r. jaenisch, d. baltimore, cell , ( ); m. matsuoka et al., science , ( ). . we thank b. stillman, t. grodzicker, j. watson, and r. axel for constructive criticisms of the manuscript; k. kidd for useful discussions, s. teplin for preparative synthesis of oligonucleo- tides, l. rodgers for growth of human cell cul- tures; t. c. caskey for dna purified from the lymphoid dr cell line ; j. duffy and m. ockler for preparation of figures; and p. bird for secre- tarial assistance. supported by grants from the national cancer institute, and the american can- cer society. m.w. is an american cancer society research professor. the first author is on leave from the institute of molecular genetics, russian academy of sciences, kurchatov square, mos- cow, russia. part of this work was con- ceived there. october ; accepted january science * vol. * february 	article contents 	p. 	p. 	p. 	p. 	p. 	p. 	issue table of contents 	science, new series, vol. , no. (feb. , ), pp. - 	front matter [pp. - ] 	editorial: evolution of atmospheres [p. ] 	letters 	asteroid or volcano: have the volcanists been heard? [p. ] 	basic research and society [pp. - ] 	correction: a li-fraumeni syndrome p mutation [p. ] 	corrections and clarifications: drawing a bead on superdense data storage [p. ] 	corrections and clarifications: top hhs lawyer seeks to block nih [p. ] 	sciencescope [p. ] 	news and comment 	hughes' tough stand on industry ties [pp. - ] 	a shot in the arm for tb research [p. ] 	nasa stakes its reputation on fix for hubble telescope [pp. - ] 	scripps-sandoz deal comes under fire [p. ] 	shalala backs reorganization [p. ] 	research news 	will future computers be all wet? [pp. - ] 	chemists work their way from pores to riches [p. ] 	unexpected intelligence turns up in a cellular gel [pp. - ] 	mathematicians gather to play the numbers game [pp. - ] 	cell communication failure leads to immune disorder [pp. - ] 	random samples [p. ] 	special section: evolution of atmospheres 	[introduction] [p. ] 	news 	is the geological past a key to the (near) future? [pp. - ] 	searching for clues to ancient carbon dioxide [pp. - ] 	an `outrageous hypothesis' for mars: episodic oceans [pp. - ] 	earth scientists look nasa's gift horse in the mouth [pp. - ] 	atmospheric evolution of the terrestrial planets [pp. - ] 	earth's early atmosphere [pp. - ] 	the ice record of greenhouse gases [pp. - ] 	the global carbon dioxide budget [pp. - ] 	perspectives 	the pluses of subtraction [pp. - ] 	rna polymerase marching backward [pp. - ] 	research article 	cloning the differences between two complex genomes [pp. - ] 	reports 	existence of a flat phase in red cell membrane skeletons [pp. - ] 	photoinduced polymerization of solid c$_{ }$ films [pp. - ] 	electrochemical processing of conducting polymer fibers [pp. - ] 	colicin e binding to membranes: time-resolved studies of spin-labeled mutants [pp. - ] 	the secretory granule matrix: a fast-acting smart polymer [pp. - ] 	structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase [pp. - ] 	deletion of irf- , mapping to chromosome q . , in human leukemia and preleukemic myelodysplasia [pp. - ] 	anti-oncogenic and oncogenic potentials of interferon regulatory factors- and - [pp. - ] 	"infectious" transplantation tolerance [pp. - ] 	carboxyl methylation of ras-related proteins during signal transduction in neutrophils [pp. - ] 	altered prevalence of gating modes in neurotransmitter inhibition of n-type calcium channels [pp. - ] 	shutdown of class switch recombination by deletion of a switch region control element [pp. - ] 	an adenovirus vector for gene transfer into neurons and glia in the brain [pp. - ] 	cd ligand gene defects responsible for x-linked hyper-igm syndrome [pp. - ] 	book reviews 	review: shifts and overthrows [pp. - ] 	review: aec and critics [pp. - ] 	books received [p. ] 	back matter [pp. - ] wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ virtual mentor virtual mentor american medical association journal of ethics november , volume , number : - . clinical case political discussions in the exam room jack p. freer, md dr. buccarelli is already behind schedule when he encounters his next patient, mr. van ware. mr. van ware is coming in for a follow-up appointment after a lingering, viral uri that finally resolved. he is politically engaged and has been following the affordable care act legislation closely. his own insurance premiums have risen, and he asks what dr. buccarelli thinks of the individual mandate for health insurance. “you think it’s fair that a young, healthy guy like me should be shouldering the bill for chronic care for the elderly?” mr. van ware asks. dr. buccarelli replies in a general way that the current health system has its flaws and he is just happy that legislators are attempting to address the problems. unsatisfied, mr. van ware repeats his question about young people subsidizing older people’s expensive end- of-life care. after a couple back-and-forths, in which dr. buccarelli politely explains that the aca is , pages long and very complicated and that it will be years before the regulations are ironed out, mr. van ware says, “seriously, doctor. you must know more about this new act than i do. do you think the mandate that all of us, healthy or not, have to buy insurance is constitutional? isn’t it socialism?” dr. buccarelli considers all the answers he can offer mr. van ware (who seems to have forgotten that, young healthy man that he is, he has had a serious uri for several weeks). dr. buccarelli feels his clinician’s role conflicting with his educator’s role and, meantime, his waiting room continues to fill. commentary dr. buccarelli’s predicament is familiar to physicians who have spent any time in ambulatory clinical care. falling behind schedule is annoying to patients and aggravating to doctors. mr. van ware, with his resolving uri, ought to be an easy opportunity to catch up a little. instead, patients often surprise us with unexpected symptoms or (as in this case) an urgent need to talk about current events. educating patients is an important part of practicing medicine. aside from clinically relevant teaching that concerns the patient’s own condition, physicians have a broader role to share their unique perspective and knowledge with the public. certainly, physicians have an inside track about health policy and health care reform. and yet, mr. van ware may not be asking for dr. buccarelli’s opinion as much as he is grandstanding and debating an issue dear to his heart. he may be simply www.virtualmentor.org virtual mentor, november —vol broadcasting his opinion to a captive audience (an opinion that is unlikely to change, no matter what dr. buccarelli says). having attempted a polite diversion, dr. buccarelli can either address the issue or unequivocally notify the patient that the discussion is over. giving mr. van ware the benefit of the doubt, it may be that he is truly interested in his physician’s views. in that case, dr. buccarelli should have a few tight, informed comments to make about this important public policy issue directly related to medical practice. health insurance is a gamble, a bet. in a sense, you are betting that you will get sick or injured. if you “lose” the bet, you lose your wager (the insurance premium). if you “win” the bet, your payoff is that the cost of your medical care (or a very large part of it) is covered. even young, healthy mr. van ware can get hit by a dump truck running a stop sign later today and wind up being the recipient of several thousand dollars’ worth of medical care before the month is out. groups like the amish recognize health insurance as gambling and reject it. interestingly, the aca contains a religious exemption [ ]. although not specified in the law, many feel that anabaptists (amish, mennonites, hutterites) and muslims might qualify under this provision [ ]. emergency medical care is already provided to the uninsured, and the costs are shared by others in society. when mr. van ware gets hit by that dump truck, he will be taken to a hospital and treated, regardless of whether he has insurance. the emergency medical treatment and active labor act, emtala, passed in as part of the consolidated omnibus budget reconciliation act (cobra), requires hospitals to provide emergency care regardless of insurance coverage or ability to pay [ ]. if mr. van ware were given the choice to opt out of insurance coverage altogether, society would still share the cost of his care. fortunately, we do not live in a society that allows uninsured people to die for lack of treatment. so, whether mr. van ware realizes it or not, young, healthy, intact people already pay for the care of the old, sick, and injured. costs are shifted to the government through taxes as well as higher insurance premiums across the board. universal coverage is more efficient and economical. in , more than $ billion in medical care to the uninsured was uncompensated [ ]. the majority of this care was provided by hospitals. these losses are offset by government in the form of disproportionate share hospital (dsh) payments. hospital care to the uninsured is fundamentally inefficient; it is emergency care, late in the course of an illness rather than preventive care earlier, when it is more effective. however one calculates the economic costs of caring for the uninsured (due to uncompensated, late, or forgone care), it is more than the cost of insuring them. the current health care system is financially unsustainable. the american health care system has grown in unbridled fashion for years. gaining control over this sprawling system is the first step in slowing and reversing cost escalation. analysts may disagree about the best way to gain control. while not perfect, the aca is expected to make a significant difference [ ]. virtual mentor, november —vol www.virtualmentor.org while physicians may want to have some prepared sound bites in response to common questions, they ought to have a deeper knowledge of some of the underlying issues. clearly this issue is extraordinarily complex and the details can be mind-numbing. still, there are some valuable resources that can be accessed easily through the internet. the henry j. kaiser family foundation has prepared a number of excellent analyses of health care reform and financing issues [ , ], the federal government has a useful website on the subject [ ], and healthcareandyou.org is an authoritative site sponsored by several organizations, including ama, aarp, aafp, and acp [ ]. finally, there is another issue that could arise in this situation. when people display strong opinions about social and political issues, there is often the potential for the discussion to become heated and personal. the doctor’s office is no exception. in this setting, however, there is a real concern about professionalism and the patient- doctor relationship. physicians must continually be aware of their place in relation to the patient. ordinarily, the patient is not the doctor’s buddy. in fact, preexisting friendships put considerable strain on a clinical relationship. when professional relationships get blurred with personal ties, both can suffer. the friendship can be strained when the patient is unhappy with the doctor’s decision. equally problematic is the way in which professional decision making can be compromised with a patient-friend. while not as striking as when caring for family members, the medical decisions made for friends can be similarly distorted. when political discussions take a bad turn in a friendship, people can just drift away, but what about a patient-doctor relationship? suppose you hear what sounds like racial bias (or even a blatant racial slur) in a political diatribe [ ]. does it affect your attitude toward that patient and can you objectively provide care for that person any longer? what does it say about how someone views you when he or she feels comfortable saying hateful things to you? of course, a patient who resists more subtle suggestions that political debates or speeches are off limits, may need to be told directly that the professional relationship cannot continue without a limit on that behavior. when both parties understand their roles, many aspects of the relationship can be presumed and go unsaid. in some situations, however, the terms must be explicitly restated. when that happens, honesty, transparency, and clarity are required to keep things on track. references . exemptions from individual responsibility requirements. patient protection and affordable care act, sec (b)( )(a). http://docs.house.gov/ energycommerce/ppacacon.pdf. accessed october , . . national committee for amish religious freedom. amish faq general information. http://www.holycrosslivonia.org/amish/amishfaq.htm. accessed october , . www.virtualmentor.org virtual mentor, november —vol . centers for medicare and medicaid services. emtala. https://www.cms.gov/emtala/. accessed october , . . hadley j, holahan j. the cost of care for the uninsured: what do we spend, who pays, and what would full coverage add to medical spending? the kaiser commission on medicaid and the uninsured. http://www.kff.org/uninsured/upload/the-cost-of-care-for-the-uninsured- what-do-we-spend-who-pays-and-what-would-full-coverage-add-to- medical-spending.pdf. accessed october , . . orszag pr, emanuel ej. health care reform and cost control. n engl j med. ; : - . . henry j. kaiser family foundation web site. http://www.kff.org. accessed october , . . henry j. kaiser family foundation. health reform source. http://healthreform.kff.org. accessed october , . . us department of health and human services. healthcare.gov website. http://www.healthcare.gov. accessed october , . . health care and you. what the affordable care act means for you. http://www.healthcareandyou.org. accessed october , . . capozzi jd. rhodes r. coping with racism in a patient. j bone joint surg. ( ); ( ): - . http://www.jbjs.org/article.aspx?volume= &page= . accessed october , . jack p. freer, md, is a professor of medicine, clinical professor of social and preventive medicine, and chief of the division of palliative medicine at the university at buffalo (suny) in new york. dr. freer is certified in hospice and palliative medicine, and his primary area of academic interest is communication skills. related in vm physician involvement with politics—obligation or avocation? november campaign posters in the clinic, january physicians and political advocacy, october health reform and the future of medical practice, november the people and events in this case are fictional. resemblance to real events or to names of people, living or dead, is entirely coincidental. the viewpoints expressed on this site are those of the authors and do not necessarily reflect the views and policies of the ama. copyright american medical association. all rights reserved. virtual mentor, november —vol www.virtualmentor.org http://virtualmentor.ama-assn.org/ / /ccas - .html http://virtualmentor.ama-assn.org/ / /ccas - .html http://virtualmentor.ama-assn.org/ / /ccas - .html http://virtualmentor.ama-assn.org/ / /msoc - .html wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ debate: no bipolar disorder in prepubertal children at high familial risk debate: no bipolar disorder in prepubertal children at high familial risk manon h.j. hillegers department of child and adolescent psychiatry/psychology, erasmus mc-sophia children’s hospital, rotterdam, the netherlands how do we classify school-aged children with severe emotional dysregulation, attention deficits, and irritabil- ity? over the last two decades, a huge increase in pedi- atric bipolar disorder (pbd) diagnoses has been observed, especially in the united states. from a histori- cal perspective, a shift toward an earlier age of onset of bipolar disorder (bd) is observed. this is also confirmed in a recent study in which a birth cohort effect toward an earlier age of onset has also been observed in european multicenter clinical observational studies. however, no prepubertal onset was found (scott, etain, azorin, & bel- livier, ). while the diagnostic criteria of bipolar dis- order during adulthood are generally accepted, the term pbd is inconsistently used applying to either prepuber- tal children or under the age of . here, pbd is referred to as prepubertal (or preadolescent) bipolar disorder. the rise in pbd prevalence rates and the related treat- ment regimen has an impact on a whole generation of severely affected young children. indeed, bipolar disor- der is a chronic, severe mood disorder which often requires lifelong treatment with second generation antipsychotics or mood stabilizers which are associated with a broad range of serious side effects. in europe, we also see such emotional dysregulated children with a broad range of explosive disruptive behavior. however, classification within the bipolar disorder spectrum is rare. these children are rather diagnosed within the dis- ruptive behavior adhd/odd/cd spectrum and treated accordingly. why are these prepubertal children diagnosed with mania and how does this fit with findings from bipolar offspring studies? bipolar disorder in adolescents has been well described and there are convergent findings indicating that mania presents with similar symptoms as in adulthood. in addition, we know that experiencing (hypo)manic symptoms is a common adolescent phe- nomenon that infrequently predicts mental health care use (tijssen et al., ). context and multi-informants are therefore crucial in the interpretation of these typical adolescent experiences. interestingly, adolescent bd patients often have a high familial load for bipolar disor- ders. since a first-degree family member with bd is the strongest risk factor for bipolar disorder, longitudinal offspring studies are ideal to study the development of bd. children at the highest genetic and environmental risk (offspring of bd parents) constitute, therefore, a population in which childhood mania, if valid, should present more often and clear as compared to the general population. worldwide, all six longitudinal bd offspring studies from different countries (switzerland, canada, usa amish, usa pittsburgh, australia, and the netherlands) show a mean age of onset of the narrow- defined bd type i after the age of years. however, cross-national variation is an issue with bd-nos, as studies show more variation when bd-nos is included as a result of different methods, interpreta- tion of criteria, and prevalence rates across countries. this was also the case in our study comparing categori- cal and dimensional psychopathology from the pittsburg bios study and the dutch bipolar offspring study (mes- man et al., ). bd-nos prevalence rate in the pitts- burgh sample was high ( . %), but was not assessed in dutch offspring because of unclear criteria and could therefore not be compared directly. however, both off- spring studies used the achenbach child behavior checklist (cbcl) and the k-sads-pl, a semistructured psychiatric interview. after controlling for age, rates of bipolar i ( . % pittsburgh, . % dutch) and bipolar ii ( . % and . ) were similar. by contrast, other disorders were significantly more common in the pittsburgh sam- ple, as was the prevalence of comorbidity in the bipolar offspring. in general, the pittsburg offspring was more severely affected compared to the dutch offspring based on the psychiatric interview. surprisingly, however, par- ent scores on the cbcl did not differ significantly between samples, whereas they should have as the symptom rates by interview were different. also, in the pittsburgh sample, fewer index-parents had bipolar i (vs. bipolar ii or nos disorder), they had an earlier age of onset, higher comorbid substance abuse rates, and lower rates of employment. in addition, the prevalence of psychopathology in the co-parent was higher in the pittsburgh sample and recruitment by advertisement was common, while recruitment in the dutch sample was through a patient advocacy group. these differences illustrate the importance of the psychopathology load of both the parents, the level of family functioning, and stress and effect of the recruitment strategy in under- standing rates of offspring psychopathology and com- paring cohorts. recently, results were published from the danish high risk and resilience study - via , a nationwide population-based cohort study of seven-year-old (age range . – . years) children, who were born and living in denmark, with no, one, or two biologi- cal parents diagnosed with schizophrenia spectrum or bipolar disorder. they were identified using the danish civil registration system and the danish psychiatric central research register (ellersgaard et al., ). the bd offspring showed a higher prevalence of anxiety disorders, stress, and adjust- ment disorders – compared with controls. no bipolar © the authors. child and adolescent mental health published by john wiley & sons ltd on behalf of association for child and adolescent mental health. this is an open access article under the terms of the creative commons attribution-noncommercial-noderivs license, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. child and adolescent mental health , no. , , pp. – doi: . /camh. https://orcid.org/ - - - x https://orcid.org/ - - - x https://orcid.org/ - - - x http://creativecommons.org/licenses/by-nc-nd/ . / disorder was diagnosed in these offspring with a mean age of seven. again, in this study recruitment and severity of the bipolar disorder are relevant. the bd parents in the danish study were recruited through the registers and likely to be more heteroge- neous in terms of severity of the disorders, which may affect the levels of psychopathology in the offspring. the research and clinical field of child and adolescent psychiatry would benefit from more uniform methods of assessing symptoms and determining bipolar disorder diagnoses. keeping in mind that questionnaires are not reliable, multiple informants are essential, and a clinical interpretation is more important than the sum of symp- toms which needs to define an episode with a clear “change from the usual state”. longitudinal studies in high-risk offspring and across diagnostic domains and countries, with a focus on harmonizing parental charac- teristics, recruitment procedures, and clear bd criteria are needed to completely understand the international perspective on pediatric bipolar disorder (maciejewski, hillegers, & penninx, ). acknowledgements the author has declared that she has no competing or potential conflicts of interest. ethical information no ethical information was required for this commentary. correspondence manon hillegers, department of child and adolescent psychiatry/psychology, erasmus mc-sophia children’s hospital, wytemaweg rotterdam cb, the netherlands; email: m.hillegers@erasmusmc.nl references ellersgaard, d., jessica plessen, k., richardt jepsen, j., soe- borg spang, k., hemager, n., klee burton, b., . . . & elgaard thorup, a.a. ( ). psychopathology in -year-old children with familial high risk of developing schizophrenia spectrum psychosis or bipolar disorder - the danish high risk and resilience study - via , a population-based cohort study. world psychiatry, , – . maciejewski, d., hillegers, m., & penninx, b. ( ). offspring of parents with mood disorders: time for more transgenerational research, screening and preventive intervention for this high- risk population. currunt opinion psychiatry, , – . mesman, e., birmaher, b.b., goldstein, b.i., goldstein, t., derks, e.m., vleeschouwer, m., . . . & hillegers, m.h. ( ). categorical and dimensional psychopathology in dutch and us offspring of parents with bipolar disorder: a preliminary cross-national comparison. journal of affective disorders, , – . scott, j., etain, b., azorin, j.m., & bellivier, f. ( ). sec- ular trends in the age at onset of bipolar i disorder - support for birth cohort effects from interational, multi- centre clinical observational studies. european psychiatry, , – . tijssen, m.j., van os, j., wittchen, h.u., lieb, r., beesdo, k., mengelers, r., . . . & wichers, m. ( ). evidence that bipolar disorder is the poor outcome fraction of a common develop- mental phenotype: an -year cohort study in young people. psychological medicine, , – . accepted for publication: december © the authors. child and adolescent mental health published by john wiley & sons ltd on behalf of association for child and adolescent mental health. manon hillegers child adolesc ment health ; ( ): – mailto:m.hillegers@erasmusmc.nl wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ jmed genet ; : - syndrome of the month pure hereditary spastic paraplegia evan reid the hereditary spastic paraplegias are a group of neurological conditions which are character- ised by the presence of progressive spasticity, predominantly affecting the legs. they may be subclassified into pure and complicated forms based on the presence of additional neurological or non-neurological features. (ymed genet ; : - ) keywords: pure hereditary spastic paraplegia; differen- tial diagnosis; molecular genetics pure hereditary spastic paraplegia (phsp) the first clear description of phsp was by strumpell in , who reported a family in which two brothers were affected by a late onset spastic paraplegia. autosomal dominant inheritance was likely, since their mother was "a little lame". subsequent reports have described autosomal dominant, autosomal recessive, and x linked recessive patterns of inheritance.' - autosomal dominant inherit- ance accounts for approximately - % of families, with autosomal recessive inheritance being responsible for most of the remainder. x linked recessive inheritance is very rare. prevalence pure hereditary spastic paraplegia is the most common form of hereditary spastic paraplegia (hsp). ' a rigorously conducted epidemiologi- cal study in the cantabria region of spain sug- gested a prevalence for phsp of . per . department of medical genetics, university of cambridge, box , addenbrooke's nhs trust, cambridge cb qq, uk e reid clinical features the principal clinical feature of phsp is, by definition, the presence of a progressive spastic paraplegia. often this is of insidious onset and the abnormal gait is frequently noticed by rela- tives before the affected person becomes aware of it. age at onset may range from early childhood, with delayed motor milestones and "clumsiness", to adult life. age at onset is not consistent between families, although it is consistent within some families. there is also considerable variation in disease severity. affected subjects may range from entirely asymptomatic ( - % of cases) to chair- bound ( - % of cases). although severity tends to increase with disease duration, consid- erable variation is present even when people who have been affected for the same length of time are compared. urinary symptoms are common, and % of patients may be affected by urinary frequency, urgency, or hesitancy. - anal sphincter disturbances have been reported but are rare. erectile impotence may occur, although its frequency is unknown. physical examination findings are typical of a spastic paraplegia. however, the degree of hypertonicity is often out of proportion to weakness, and is frequently the most disabling feature. weakness and hypertonicity are usu- ally restricted to the legs, although there have been occasional reports of mild upper limb weakness, and upper limb hyperreflexia is common. pes cavus is present in - % of cases. mild upper limb incoordination may occur and mild distal amyotrophy is well recognised, though rare. unexpected physical signs may be found; ankle jerks are absent and babinski reflexes plantar in a small proportion of patients. additional neurological abnor- malities, indicating involvement of systems other than the motor tracts, are frequent. a significant proportion of patients ( - %) have diminished vibration sense, and a small number have diminished joint position sense. subclinical sensory abnormalities affecting these and other modalities may be present in the majority of cases.' attempts have been made to subclassify autosomal dominant phsp on the basis of clinical features. harding found a bimodal distribution of age of onset and used this to divide families into type i, with age of onset predominantly below years, and type ii, with age of onset predominantly over years. subjects from type ii families had a disorder which progressed more rapidly and was more commonly associated with somatosensory and urinary sphincter disturbances than subjects from type i families. muscular weakness predominated in type ii families, while hyper- tonicity was more marked in type i families. however, there was considerable overlap be- tween these two groups and subsequent studies have not consistently supported the concept of two distinct types of phsp. no consistent clinical differences are apparent between fami- lies with different inheritance patterns. pathology pathological reports for phsp are scant and have usually described patients with long o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ standing disease. the principal pathological finding is of axonal degeneration involving the terminal ends of the longest fibres of the corti- cospinal tracts and dorsal columns. the spinocerebellar tracts are involved to a lesser degree. the cell bodies of the degenerating fibres are apparently normal; the process has been described as "dying back" of the nerve fibre endings. there is no evidence of primary demyelination and no abnormality of peri- pheral nerves, dorsal roots, or dorsal root gan- glia has been reported.' differential diagnosis and investigations the diagnosis of phsp in a family where sev- eral members have typical clinical features is relatively straightforward. standard neurological investigations should be used selectively to exclude alternative diagnoses. magnetic resonance imaging may show spinal cord atrophy, particularly in the cervical region." peripheral nerve conduction studies are almost always normal. cervical soma- tosensory evoked potentials and central motor conduction studies are abnormal in the major- ity of cases, although unfortunately do not pro- vide a means for early detection of affected family members."- interestingly, brain stem auditory evoked potentials and visual evoked p.otentials are abnormal in a minority of cases. other genetic conditions to be considered in the differential diagnosis include dopa respon- sive dystonia, which should actively be ex- cluded in families where age of onset is early and sensory signs are absent, particularly if there is marked diurnal variation of spasticity. dramatic and sustained improvement of symp- toms occurs with small doses of l-dopa.' the clinical picture should allow exclusion of the spinocerebellar syndromes and machado- joseph disease, in which ataxia is a much more prominent feature. adult onset forms of adrenoleucodystrophy, krabbe's leucodystro- phy, and metachromatic leucodystrophy may rarely present with spastic paraplegia, and can be detected with appropriate biochemical tests. atypical friedreich's ataxia should also be considered, since a proportion of patients homozygous for the frataxin gene triplet repeat expansion have retained or exaggerated lower limb reflexes and upgoing plantar responses. the differential diagnosis is more extensive in sporadic cases of spastic paraplegia and, in addition to the above, includes structural spinal cord lesions, intracranial parasagittal space occupying lesions, multiple sclerosis, myelopa- thies associated with deficiencies of vitamin b or e, and abetalipoproteinaemia. infective conditions including tertiary syphilis, htlv infection causing tropical spastic paraparesis, and the myelopathy associated with the ac- quired immune deficiency syndrome should be considered, although the clinical picture would be not entirely typical of phsp." diagnosis in at risk family members criteria for the diagnosis of phsp in at risk family members have been proposed, although they are perhaps most useful for standardisa- tion in a research setting (table ). the table suggested diagnostic criteria forfamily members from families with phsp. ' alternative diagnoses should first be excluded and the family history should be consistent with autosomal dominant, autosomal recessive, or x linked recessive inheritance. these criteria are perhaps most useful for standardisation in a research setting and are open to criticism. many definitely affected subjects lack grade hyperreflexia. the criteria for the definitely unaffected category are insufficiently rigorous in view of the wide range in age of onset which may occur in some families status criteria definitely affected progressive gait disturbance + frank corticospinal tract involvement of lower limbs, including grade hyperreflexia and extensor plantar reflexes probably affected subjects lacking history of progressive gait disturbance, or asymptomatic subjects with signs of spastic paraparesis, examined only once and so not proven to have a progressive gait disturbance + frank corticospinal tract involvement of lower limbs, including grade hyperreflexia and extensor plantar reflexes. serial examinations may allow recategorisation as definitely affected possibly affected asymptomatic + normal gait + questionably abnormal corticospinal tract signs, eg mild hyperreflexia, non-sustained clonus, but downward plantar reflexes definitely unaffected asymptomatic + normal neurological examination + age greater than maximal age of symptoms in family probably unaffected asymptomatic + normal neurological examination + age younger than maximal age of symptoms in family insidious onset of the condition can lead to diagnostic uncertainty. clues from the history can be helpful. patients may note poor athletic ability at school, tripping more often than nor- mal, and the presence of clonus at the knee or ankle joint. relatives may have made com- ments about an at risk subject having the "family walk". many affected people rapidly wear out shoes, particularly at the toe, and footwear should be examined. physical examination findings in at risk relatives must be carefully interpreted. anxiety may cause mildly increased muscle tone, hyperreflexia, and non- sustained clonus and these findings should be viewed with caution in subjects who lack one of the "hard" physical signs of sustained clonus (d five beats), bilateral upgoing plantar re- sponses, or progressive gait disturbance. if doubt remains, serial examinations should help. management no therapy is available which slows disease progression. treatment is aimed at maximising functional ability and preventing complications such as contractures. referral to a neurological physiotherapist, for an appropriate exercise programme, is essential. some patients in- crease functional activity with antispastic drugs such as the gaba agonist baclofen, or newer alternatives."' anecdotal reports in small num- bers of patients have described a useful functional response to continuous intrathecal baclofen, although there have been no good clinical trials of this treatment." intramuscu- lar botulinum toxin may improve function for carefully selected patients, although its use has not been fully evaluated in phsp. ortho- reid o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ pure hereditary spastic paraplegia paedic surgery, usually release of contractures and tenotomies, may rarely have a role, but requires very careful assessment of the likely consequences of the procedure on gait. genetic counselling the often late onset of phsp and the variabil- ity of age of onset within families showing dominant inheritance means that it may be impossible to reassure clinically normal young adults who are at % prior risk of having inherited the disease gene. the data of harding on type i families indicate that the clinically normal offspring of an affected parent have, at the age of , a % chance of having the abnormal gene, and at the age of , a % chance of having inherited the abnormal gene (table ). it should be emphasised that these data are only applicable to families in which the age of onset for the condition is predominantly under years. the risk figures are almost cer- tainly overestimates, since they are derived from an age at onset of symptoms cumulative frequency curve. the degree of reduction which can be made when an at risk patient has no signs is unknown. predictive testing may be feasible for at risk subjects from large families where definite linkage is established. the frequent occurrence of asymptomatic subjects means that every effort should be made to examine both parents of sibs with apparently recessive phsp. if both parents are normal, recessive inheritance is most likely. non-penetrance (only a few well documented cases exist ) or gonadal mosaicism in a parent are alternative possibilities. if both parents of affected sibs cannot be examined, empirical figures derived by harding (and only applica- ble to type i families) indicate a / chance that one parent was affected. the risks to the offspring of the affected sibs are therefore / . no empirical figures are available for the risks to offspring of apparently sporadic cases, where in addition to non-penetrance in a parent a new dominant mutation is a possibility. although x linked inheritance cannot be excluded in many families, it would appear to be rare and only a few convincing pedigrees have been described." those obligate carrier females examined have been almost always normal neurologically and none has had clear cut signs of spastic paraplegia." however, because x linked pedigrees are so sparse and because examination findings in carrier fe- males have not always been reported, it is diffi- cult to be certain that carrier females never have definite signs of spastic paraplegia." complicated hsp complicated hsp consists of a large number of rare conditions, which tend to be inherited in an autosomal recessive fashion. they have been well reviewed by bundey ' and by harding,' whose proposed classification is given in table . molecular genetics ofhsp three autosomal dominant genes causing phsp have been mapped, on chromosomes p, q, and q (to regions of cm, cm, and cm respectively). - linkage results have been published in complete form for families. - strong evidence of linkage to one of the three known loci has been found in approximately half of these families, with weaker evidence of linkage being found in a proportion of the remainder. of those families showing evidence of linkage, most are linked to chromosome , - with small numbers showing linkage to chromosomes (three families) and (one family). linkage to all three loci has been excluded in a number of families, strongly suggesting the existence of at least one further locus. anticipation has been postulated to occur for a proportion of the families where the disease gene maps to chromosome and to chromosome , suggesting the intriguing possibility that the underlying ge- netic abnormality might involve a trinucleotide repeat expansion, as has been described for several other neurodegenerative conditions. there may be some correlation between genetic locus and clinical phenotype, although assessment of this is hampered by scanty clini- cal details in mapping reports. all three of the families showing linkage to the chromosome locus had a very early mean age at onset, under years old. on the other hand, families showing linkage to chromosome have consid- erable inter- and intrafamilial variation in age of onset, corresponding to both types i and ii of harding, and further weakening the argument that these types of phsp are distinct. in none of these families was the mean age of onset as early as the chromosome linked families. the single family showing linkage to chromosome had a unimodal age of onset (mean years) and was characterised by the relative severity of the condition, with approximately % of its members being chairbound. a gene for autosomal recessive phsp has been mapped to the pericentric region of chro- mosome in three consanguineous and one non-consanguineous tunisian families. again there is evidence of locus heterogeneity, with linkage to chromosome markers being excluded in a fifth consanguineous tunisian family. the clinical picture was homogeneous in all five families, with an early age of onset (between and years), loss of vibration sense and proprioception, and bladder sphinc- ter disturbance in all affected members. while x linked spastic paraplegia is rare, it is important because its molecular pathology is more completely understood than that of the other forms. mutations in the gene encoding the neural cell adhesion molecule li (li- cam) at xq are responsible for a compli- cated form of spastic paraplegia, in which paraplegia is accompanied by mental retarda- tion and absence of the extensor pollicis longus muscle. different mutations in the same gene are responsible for the masa syndrome (men- tal retardation, aphasia, shuffling gait, ad- ducted thumbs) and x linked hydrocephalus. the same li-cam mutation may result in either an x linked hydrocephalus phenotype or a masa phenotype. li-cam is a cell surface glycoprotein which is expressed on the table risks ofhaving disease gene for clinically normal offspring of affected subjects, based on cumulative age of onset curve for autosomal dominant phsp for use with families in which age of onset is predominantly under years. modified from harding' risk of having age (y) disease gene (%) o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ reid table classification of complicated spastic paraplegias, updated and modifiedfrom harding,' with additional references - type clinicalfeatures inheritance pattern with amyotrophy resembling peroneal muscular atrophy of small hand muscles troyer syndrome charlevoix-saguenay syndrome resembling amyotrophic lateral sclerosis sjogren-larsson syndrome masa syndrome with cerebellar signs kjellin syndrome with optic atrophy with choreoathetosis/ dystonia mast syndrome with sensory neuropathy with disordered skin pigmentation with hyperekplexia mild paraparesis accompanied by ad features resembling charcot-marie-tooth disease mild paraparesis with ad moderate/severe amyotrophy of small hand muscles early onset with motor and speech ar delay. severe spasticity and distal amyotrophy of all limbs. pseudobulbar palsy, dysarthria, and emotional lability. described in amish early onset. dysarthria, nystagmus, ar cerebellar ataxia. amyotrophy in all limbs. spasticity prominent in legs. defective vertical pursuit eye movements. occurs in an isolated area of quebec ar/' congenital ichthyosis, severe ar. mental retardation and usually dehy non-progressive spastic paraplegia mental retardation, aphasia, xlf spasticity, and adducted thumbs. gene allelic with x linked hydrocephalus cerebellar dysarthria, mild upper ar/b limb ataxia and spastic paraplegia. distal wasting may occur mental retardation, progressive ar spastic paraplegia from s, distal neurogenic atrophy of limbs, dysarthria, central retinal degeneration rare. associated with a variety of arba additional features in different family reports additional features may occur in ar/b some families. consider dopa responsive dystonia as an alternative diagnosis! dementia, dysarthria, athetosis, and spastic paraplegia. described in amish population sensory neuropathy often affects ar/! all modalities and may lead to trophic ulceration and mutilation. trophic ulcers may have early onset two separate conditions reported. ar/ first with generalised depigmentation at birth, becoming patchily pigmented. second with hypopigmented skin below knee a single family described in which ad. hyperekplexia and spastic the paraplegia cosegregate 'ad caused by mutations in fatty aldehyde ydrogenase gene r. caused by mutations in li-cam . 'ad 'ad 'ad 'ad 'ad .caused by mutations in gene coding for alsubunit of glycine receptor ar=autosomal recessive, ad=autosomal dominant, xlr=x linked recessive. axons of postmitotic neurones and which is involved in neuronal migration and neurite extension. it is highly expressed in the devel- oping corticospinal tracts of rats and the congenital onset of the three conditions comprising the "li-cam" syndrome is con- sistent with the molecule's proposed role in neuronal development. pelizaeus-merzbacher disease is a dysmyeli- nating disorder of the central nervous system, characterised by congenital hypotonia, nystag- mus, slow psychomotor deterioration, and pro- gressive pyramidal, dystonic, and cerebellar signs. mutations in the proteolipid protein (plp) gene, which maps to xq -q , have been found in families with this condition, in a family with phsp and in families with compli- cated forms of spastic paraplegia." ' the plp gene codes for two myelin proteins, plp and dm . these proteins are thought to play a major role in oligodendrocyte maturation and, later in development, in myelin sheet compaction. it has been suggested that muta- tions or duplications which affect the role ofthe plp gene in oligodendrocyte maturation give rise to the more severe pelizaeus-merzbacher phenotype, while those which affect only myelin compaction give rise to the milder spas- tic paraplegia phenotypes, although this issue has not yet been resolved. it is possible that further x chromosome phsp genes exist. in a second x linked phsp family, the responsible gene mapped tightly to the plp gene region. however, no mutation in either the coding sequence or intron/exon boundaries of the plp gene was found, raising the possibility that the disease causing mutation may have been in a non-coding portion of the plp gene, or that phsp may also be caused by a second gene in the same region. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ pure hereditary spastic paraplegia thus, the x linked hereditary spastic para- plegias indicate that mutations in the same gene may be responsible for both pure and complicated forms of hsp, as well as other neurological conditions. in addition, they sug- gest that cell adhesion molecule genes and myelin genes are likely candidates for other forms of spastic paraplegia. conclusion knowledge of the genetics of the hereditary spastic paraplegias is advancing rapidly. even- tually, an understanding of the molecular pathology underlying these conditions may allow better genetic counselling for at risk fam- ily members, rational development of more effective treatments, and insight into the func- tion of the spinal cord in health and disease. i am grateful to dr charles ffrench-constant and dr andrew green for their helpful comments on the manuscript and to the mrc for their support of my work on phsp. harding ae. the hereditary ataxias and related disorders. edinburgh: churchill livingstone, . strumpell a. beitrage zur pathologie des ruckenmarks. arch psychiatr nervenkr ; : - . roe p. hereditary spastic paraplegia. j neurol neurosurg psychiatry ; : - . holmes gl, shaywitz ba. strumpell's pure hereditary spastic paraplegia: case study and review of the literature. j neurol neurosurg psychiatry ; : - . harding ae. hereditary "pure" spastic paraplegia: a clinical and genetic study of families. j neurol neurosurg psychiatry ; : - . boustany rmn, fleischnick e, alper ca, et al. the autosomal dominant form of "pure" familial spastic paraplegia: clinical findings and linkage analysis of a large pedigree. neurology ; : - . scheltens p, bruyn rpm, hazenberg gj. a dutch family with autosomal dominant pure spastic paraparesis (striim- pell's disease). acta neurol scand ; : - . polo jm, calleja j, combarros 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botulinum toxin in the management of the lower limb in cerebral palsy. dev med child neurol ; : - . bundey s. genetics and neurology. edinburgh: churchill liv- ingstone, . de laurenzi v, rogers cg, hamrock dj, et al. sjogren- larsson syndrome is caused by mutations in the fatty alde- hyde dehydrogenase gene. nat genet ; : - . jouet m, rosenthal a, armstrong g, et al. x-linked spastic paraplegia (spg ), masa syndrome and x-linked hydro- cephalus result from mutations in the li gene. nat genet ; : - . elmslie fv, hutchings sm, spencer v, et al. analysis of glra in hereditary and sporadic hyperekplexia: a novel mutation in a family cosegregating for hyperekplexia and spastic paraparesis. _ med genet ; : - . hazan j, fontaine b, bruyn rpm, et al. linkage of a new locus for autosomal dominant familial spastic paraplegia to chromosome p. hum mol genet ; : - . hentati a, pericak-vance ma, lennon f, et al. linkage of a locus for autosomal dominant familial spastic paraplegia to chromosome p 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of the immu- noglobulin superfamily.] cell biol ; : - . joosten eaj, gribnau aam. immunocytochemical localiza- tion of cell adhesion molecule li in developing rat pyrami- dal tract. neurosci lett ; : - . saugier-veber p, munnich a, bonneau d, et al. x-linked spastic paraplegia and pelizaeus-merzbacher disease are allelic disorders at the proteolipid protein locus. nlat genet ; : - . griffiths i, montague p, dickinson p. the proteolipid protein gene. neuropathol appl neurobiol ; : - . cambi f, tang xm, cordray ms, et al. refined genetic mapping and proteolipid protein mutation analysis in x-linked pure hereditary spastic paraplegia. neurology ; : - . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ pnas correction .. corrections evolution correction for “the abo blood group is a trans-species poly- morphism in primates,” by laure ségurel, emma e. thompson, timothée flutre, jessica lovstad, aarti venkat, susan w. margulis, jill moyse, steve ross, kathryn gamble, guy sella, carole ober, and molly przeworski, which appeared in issue , november , , of proc natl acad sci usa ( : – ; first published october , ; . /pnas. ). the authors note that fig. appeared incorrectly. some abo polymorphism statuses have been corrected. the corrected fig- ure and its legend appear below. www.pnas.org/cgi/doi/ . /pnas. fig. . the phylogenetic distribution of abo phenotypes and genotypes. shown is a phylogenetic tree of primate species, with a summary of phenotypic/ genotypic information given in the first column, and the genetic basis for the a versus b phenotype provided in the second column (functionally important codons at positions and are in uppercase letters). see dataset s for the source of information about phenotypes/genotypes. only species with available divergence times are represented here ( of ). the phylogenetic tree is drawn to scale, with divergence times (on the x axis) in millions of years taken from ref. . owm, old world monkeys; nwm, new world monkeys. under a model of convergent evolution, these data suggest that a is the ancestral allele, and a turnover (e.g., a neutral substitution) occurred on the branch leading to old world monkeys. if instead, b were ancestral, all old world monkeys would have had to serendipitously converge from atg to ttg to encode a leucine, whereas all new world monkeys and hominoids would have had to converge to the ctg codon. www.pnas.org pnas | april , | vol. | no. | – c o r r ec ti o n s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , www.pnas.org/cgi/doi/ . /pnas. evolution; earth, atmospheric, and planetary sciences correction for “mass extinction of lizards and snakes at the cretaceous–paleogene boundary,” by nicholas r. longrich, bhart-anjan s. bhullar, and jacques a. gauthier, which appeared in issue , december , , of proc natl acad sci usa ( : – ; first published december , ; . / pnas. ). the authors note the following: “the genus name lamiasaurus, which we proposed for a new lizard from the late cretaceous of wyoming, is preoccupied by lamiasaurus watson ( ), a tapinocephalid therapsid from the permian of africa. we therefore propose the name lamiasaura for the wyoming lizard; its type species is lamiasaura ferox, also proposed in our paper. fur- thermore, holotypes were figured for this and other newly pro- posed species but not explicitly identified in the text. we designate types and provide diagnoses as follows. cemeterius monstrosus, holotype: united states national museum . diagnosis: large stem varanoid characterized by a deep, massive jaw, teeth short, unserrated, robust, and labiolingually expanded. cerberophis robustus, holotype: university of california museum of paleon- tology . diagnosis: medium-sized (∼ m) alethinophidian, trunk vertebrae with broad, flat ventral surface, hypertrophied synapophyses, large, massive prezygapophyses with rudimentary prezygapophyseal processes and anterior ridges; neural arch with dorsolateral ridges, moderately tall neural spine. lamiasaura ferox, holotype: university of wyoming a, left dentary with four teeth. diagnosis: dentary straight, tapered in lateral view; teeth widely spaced, crowns weakly recurved, crowns with bottleneck constriction between the base and apex, low mesial and distal cusps, and ridged lingual surface. lonchisaurus trichurus, holotype: american museum of natural history . diagnosis: dentary long, low, and weakly bowed in lateral view; tooth crowns robust, weakly recurved, with weakly pointed crowns; tooth bases wider la- bially than lingually, tooth replacement reduced, coronoid overlaps dentary laterally. obamadon gracilis, holotype: university of cal- ifornia museum of paleontology . diagnosis: small poly- glyphanodontian characterized by the following combination of characters: dentary slender, symphysis weakly developed, tooth im- plantation subpleurodont, teeth lack basal expansion, tooth crowns with a tall central cusp separated from accessory cusps by deep lin- gual grooves. pariguana lancensis, holotype: american museum of natural history . diagnosis: small iguanid; teeth tall, slender, with tapering crowns and weak accessory cusps; coronoid extended onto lateral surface of jaw below last tooth, meckelian groove con- stricted suddenly ahead of anterior inferior alveolar foramen. so- cognathus brachyodon, holotype: yale peabody museum (princeton university collection) . diagnosis: socognathus with posterior teeth having strongly swollen, weakly tricuspid crowns.” “this correction formally validates the taxa proposed in our paper; thus, those taxa should be attributed to this note and accordingly dated as march , .” “we thank christian kammerer and christopher taylor for bringing these two issues to our attention.” . watson dms ( ) the deinocephalia, an order of mammal-like reptiles. proceedings of the zoological society of london – . www.pnas.org/cgi/doi/ . /pnas. neuroscience, psychological and cognitive sciences correction for “sensory adaptation as optimal resource allocation,” by sergei gepshtein, luis a. lesmes, and thomas d. albright, which appeared in issue , march , , of proc natl acad sci usa ( : – ; first published february , ; . /pnas. ). the authors note that, due to a printer’s error, some text ap- peared incorrectly. on page , right column, third full paragraph, line “in fig. a” should instead appear as “in fig. b”. on page , right column, second full paragraph, line “in fig. a” should instead appear as “in fig. a”. on page , left column, fourth full paragraph, lines – “in fig. a, sensitivity changes are plotted for two speeds (fig. a, upper) and for the entire domain of the sensitivity function (fig. a, lower)” should instead appear as “in fig. b, sensitivity changes are plotted for two speeds (fig. b, upper) and for the entire domain of the sensitivity function (fig. b, lower)”. on page , left column, first full paragraph, line “cortical visual area middle temporal (mt)” should instead appear as “middle temporal (mt) cortical visual area”. on page , right column, second full paragraph, lines – “as illustrated in fig. a for experiment and fig. a for ex- periment ” should instead appear as “as illustrated in fig. a for experiment and fig. a for experiment ”. www.pnas.org/cgi/doi/ . /pnas. chemistry correction for “enhanced surface hydrophobicity by coupling of surface polarity and topography,” by nicolas giovambattista, pablo g. debenedetti and peter j. rossky, which appeared in issue , september , , of proc natl acad sci usa ( : – ; first published august , ; . / pnas. ). the authors note the following: “recalculations confirm some of the conclusions reported in our paper but do not confirm others. specifically, we confirm that (i) polar surfaces can be hydrophobic, (ii) capillary evaporation can occur when water is confined between “polar hydrophobic” nanoscale surfaces, and (iii) inversion of surface polarity can alter the surface hydro- phobicity (i.e., water contact angle). however, because of the sensitivity of the results to the value of the ewald wave vector cutoff parameter mmax for the model surface studied, the re- ported observation that adding polarity to an apolar silica-based surface can enhance hydrophobicity beyond that of the original apolar surface is not confirmed. “we thank zhonghan hu for bringing to our attention dis- crepancies between his computer simulation results and some of our calculations reported in the above-cited pnas paper; richard c. remsing and john d. weeks for generously sharing their results with us; and sapna sarupria, amish patel, and sumit sharma for useful discussions. additional details regarding the recalculations are available from the authors upon request.” www.pnas.org/cgi/doi/ . /pnas. | www.pnas.org d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , www.pnas.org/cgi/doi/ . /pnas. www.pnas.org/cgi/doi/ . /pnas. www.pnas.org/cgi/doi/ . /pnas. what the world's religions teach, applied to vaccines and immune globulins r w j m a a r r a a k r b v a i c p h vaccine ( ) – contents lists available at sciverse sciencedirect vaccine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e eview hat the world’s religions teach, applied to vaccines and immune globulins ohn d. grabenstein ∗, erck vaccines, sumneytown pike, wp -b , west point, pa , usa r t i c l e i n f o rticle history: eceived october eceived in revised form december ccepted february vailable online february eywords: eligion eliefs accines ntibodies mmune globulins a b s t r a c t for millennia, humans have sought and found purpose, solace, values, understanding, and fellowship in religious practices. buddhist nuns performed variolation against smallpox over years ago. since jenner developed vaccination against smallpox in , some people have objected to and declined vaccination, citing various religious reasons. this paper reviews the scriptural, canonical basis for such interpretations, as well as passages that support immunization. populous faith traditions are considered, including hinduism, buddhism, jainism, judaism, christianity, and islam. subjects of concern such as blood components, pharmaceutical excipients of porcine or bovine origin, rubella strain ra / , and cell-culture media with remote fetal origins are evaluated against the religious concerns identified. the review identified more than reports or evaluations of vaccine-preventable infectious-disease outbreaks that occurred within religious communities or that spread from them to broader communities. in multiple cases, ostensibly religious reasons to decline immunization actually reflected concerns about vaccine safety or personal beliefs among a social network of people organized around a faith community, rather than theologically based objections per se. themes favoring vaccine acceptance included transfor- mation of vaccine excipients from their starting material, extensive dilution of components of concern, the medicinal purpose of immunization (in contrast to diet), and lack of alternatives. other important features included imperatives to preserve health and duty to community (e.g., parent to child, among neighbors). concern that ‘the body is a temple not to be defiled’ is contrasted with other teaching and quality-control requirements in manufacturing vaccines and immune globulins. health professionals who counsel hesitant patients or parents can ask about the basis for concern and how the individual applies religious understanding to decision-making about medical products, explain facts about content and processes, and suggest further dialog with informed religious leaders. key considerations for observant believers for each populous religion are described. © elsevier ltd. all rights reserved. ontents . background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . populous religious groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hinduism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . buddhism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . jainism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . judaism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . christianity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . islam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vaccine components and processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . bacteria, viruses, cell substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . wi- and mrc- cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ∗ tel.: + ; fax: + . e-mail address: john grabenstein@merck.com jdg is an employee of merck & co., inc., whitehouse station, nj. this work represents racticing roman catholic his entire life, and has explored religious aspects of immuniza - x/$ – see front matter © elsevier ltd. all rights reserved. ttp://dx.doi.org/ . /j.vaccine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . the author’s opinions and not those of merck & co. the author has been a tion since the s. dx.doi.org/ . /j.vaccine. . . http://www.sciencedirect.com/science/journal/ x http://www.elsevier.com/locate/vaccine mailto:john_grabenstein@merck.com dx.doi.org/ . /j.vaccine. . . j.d. grabenstein / vaccine ( ) – . . . rubella virus strain ra / . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . porcine excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . bovine excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . misunderstandings of vaccine production or content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pathogen route of exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . personal note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . appendix a. supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i c c b c d s d m a g o m b p r o k d o s v o c r s i c s i w t r h s c [ m p c m o n a n references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . background people conducting immunization programs may encounter ndividuals who hesitate, question, or decline some or all vac- ines or immune globulins based on religious beliefs or related ultural reasons. such matters are intensely personal and may e disconcerting for health professionals not comfortable dis- ussing religious issues or who usually make immunization ecisions based on matters more closely aligned to quantitative ciences. the word religion derives from the latin religio or religionem, escribing respect for the sacred or reverence for god or gods [ , ]. ultiple definitions of religion have been proposed, but religions re fundamentally sets of beliefs about god or spirituality held by roups of people. like all groups, religious groups develop their wn systems of culture. and yet, as we will see, behaviors of like- inded individuals are not necessarily related to the theological asis of their religions. “religious” differs from “theological,” in art, as social differs from scholarly. religious concerns about immunization have a long history, eaching back to those who rejected edward jenner’s mode f smallpox vaccination as contrary to god’s will [ ]. in the united ingdom, the anti-vaccination league formed in in lon- on to oppose compulsory vaccination acts [ – ]. similar events ccurred in the netherlands and elsewhere [ ]. in the united states, everal boston clergymen and devout physicians formed the anti- accination society in [ , , – ]. in contemporary cases, such bjections involve blood products, porcine or bovine pharmaceuti- al excipients, or the remote fetal origins of cell-culture media and ubella strain ra / . in contrast, it is also worth remembering that ome of the earliest descriptions of variolation to prevent smallpox nvolved the proponency of buddhist religious women [ ]. individual rights are deeply embedded in many cultures. with ontagious diseases, though, vaccine and immune globulin deci- ions may affect more than an individual’s health. this occurs f a parent chooses to withhold immunization from a child or here vaccine-exempting people increase the infectious risk of heir neighbors. numerous examples of vaccine-preventable outbreaks among eligious schools, congregations, and communities illustrate ow clusters of vulnerable people can enable epidemics, even preading beyond those foci to neighboring, well-immunized ommunities [ – ]. published examples include diphtheria , ], haemophilus influenzae type b [ , ], hepatitis a [ , ], easles [ – ], mumps [ – ], pertussis [ , , , , , ], oliomyelitis [ , , , – ], and rubella [ , – , ]. tetanus ases have also resulted [ , , ]. these infections occurred in ultiple countries (including transmission across borders and ceans) and among a range of cultural traditions and socioeco- omic situations, leading directly to hospitalizations, disabilities, nd deaths. in several analyses, the risk of measles or pertussis was – times higher among people claiming exemption to immu- ization, compared with the general population [ , , ]. this . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . elevated risk applies regardless of the faith tradition involved. the infectious risk has nothing to do with religious denomination or righteousness of the objection. to paraphrase the book of genesis (chapter , verse ), vaccine recipients are their brother’s keepers, as contributors to herd protection. this review is intended to provide a factual and contextual basis for discussions about religious concerns about vaccines and immune globulins, as well as the role of religion in promoting immunization. the perspective taken here is that of religious insti- tutions and authorities, as they would teach their doctrines to believers. it is important to note that there may be differences between what individual believers profess and what their canon- ical texts teach. indeed, different sects within a faith tradition can interpret the same scriptural passages differently. vaccines did not exist when the torah, bible, qur’ān, or major sanskrit texts were originally written. subsequent interpretations are fundamental to how contemporary believers approach immunization. this review is not intended to criticize or argue against any religious beliefs, but rather to objectively describe the basis from which the beliefs arise, as well as various religious positions that may enable or even expect immunization to be conducted. the goal is a clearer understanding of the nature of some motivations for or objections to immunization, how broadly or narrowly the objections tend to be applied, and to help dispel misunderstanding. philosophical objections to immunization are beyond the scope of this article. respectful consideration of religious beliefs within a clinical set- ting is important because medicine and religion come together to frame and enlighten choices made by patients as well as health pro- fessionals [ , , , , ]. scientists and clinicians confront moral and ethical choices daily and often observe a religious faith that helps guide their own personal conduct. indeed, the religious beliefs of countless historical and contemporary researchers and clinicians have been a source of motivation to help relieve human suffering by means of immunization. . methods to identify professional and lay documents related to the acceptability or unacceptability of vaccines and immune globu- lins based on religious beliefs, pubmed and google databases were searched using the search terms [outbreak and religion], [vaccine and religion], and [vaccine and “name of specific religious group”], specifying each of the world’s religions estimated to have at least million adherents: bahá’í faith, buddhism, christianity, confu- cianism, daoism, hinduism, islam, jainism, judaism, shinto, and sikhism. also searched were populous denominations within the christian tradition: roman catholicism, eastern orthodox and ori- ental orthodox churches, amish, anglican, baptist, church of christ (scientist), church of jesus christ of latter-day saints (including “mormon”), congregational, dutch reformed congregations, epis- copalian, jehovah’s witnesses, lutheran, methodist, pentecostal, presbyterian, and seventh-day adventist. j.d. grabenstein / vaccine table notable scriptural passages. a. hindu texts: bhagavad gı̄tā . . shikshapatri śloka and b. sayings of the buddha: sermon at benares. dhammapada x: and xv: . sāmaññaphala-sutta. sigālovāda-sutta, advice to sigāla. bodhicharyavatara of santideva iii c. hebrew bible: genesis : , leviticus : – , : – , : , and : , deuteronomy : , : – , : – , and : , and proverbs : – d. christian new testament:passages cited to support immunization: mark : – , luke : – , luke : – , corinthians : , timothy : , james : , and john : . passages cited in declining immunization: matthew : – and : , mark : [note similarities with luke : – and matthew : – ] and : , and corinthians : – ; : – . consider also (c), with regard to old testament e. jehovah’s witnesses: genesis : – , leviticus : – , and acts of the apostles : – f. qur’ān: : , : , : , : , : , : . f t c g s e i c c s t i t d g a w a o f c i ( t s f i v e i i h b a b t a s ull text of these passages appears in the supplemental material. hese selected scriptural passages should be interpreted in context with text pre- eding and following them. all documents identified via pubmed were assessed. for the oogle searches, at least the top entries for each individual earch were evaluated, more when the search results delivered rel- vant documents. after each search, reference lists were scanned to dentify other relevant documents. religious reference books were onsulted [ , ], as well as key scriptural texts (e.g., hebrew bible, hristian new testament, qur’ān, table ). . results . . populous religious groups discussion of the major religious groups appears below, equenced by the founding dates of these traditions. the chris- ian denominations are listed alphabetically. this review did not dentify any canonical doctrine that has led to religious objection o vaccines or immune globulins for bahá’í faith, confucianism, aoism, shinto, or sikhism. most ostensible objections to immunization attributable to reli- ious belief fell into three categories: (a) violation of prohibitions gainst taking life, (b) violation of dietary laws, or (c) interference ith natural order by not letting events take their course. each is ddressed further below. . . . hinduism various denominations of hinduism share a fundamental set f common beliefs, but philosophies and practices vary across dif- erent hindu denominations. with no single founder, hinduism onsiders itself sanatan dharma (the eternal tradition) and traces ts roots to the revelations in the vedic sacred texts of ancient india at least years before our common era (bce) to bce) [ , ]. here are four major branches of hinduism: shaiva, vaishnava, hakta, and smarta. the vedic sacred texts were transmitted orally or many centuries before being committed to writing [ , , ]. mportant hindu texts include the shrutis and the smritis (e.g., edas, mahābhārata, rāmāyana, bhagavad gı̄tā). in hinduism, the thics and metaphorical meanings of the texts, as revealed by spir- tually elevated gurus, may often be emphasized more than literal nterpretations. vaccination is widely accepted in predominantly indu countries. hindus advocate non-violence (ahimsa) and respect for life, ecause divinity is believed to permeate all beings, including plants nd non-human animals [ , , , ]. the degree to which hindu elievers apply the principle of non-violence varies. hindu scrip- ures support the use of violence in self-defense and do not equate himsa with pacifism [ ]. some reason that even vegetation must ubmit for human survival and that humans unknowingly destroy ( ) – life forms on a regular basis through daily activities, as mohandas gandhi acknowledged (table a). some hindus embrace vegetarianism to respect higher forms of life (table a); some eat meat only on certain days. food habits vary across communities and regions. observant hindus who do eat meat often abstain from beef. the cow in hindu society is tradi- tionally identified as a caretaking and maternal figure. verses of the rig-veda refer to the cow as devi (goddess), but hindus do not wor- ship cows, but rather venerate (deeply respect) them. this review did not identify contemporary hindu concerns with trace bovine components of some vaccines. . . . buddhism buddhism involves traditions, beliefs, and practices based on teachings attributed to siddhartha gautama, commonly known as the buddha (awakened or enlightened one). the buddha taught in the eastern part of what is now india between the th and th cen- turies bce, perhaps – bce. he is recognized by buddhists as an enlightened teacher who shared his insights (table b) to help end ignorance, craving, and suffering, and attain nirvana (nibbāna, freedom from suffering) [ , , – ]. major buddhist sects include theravada, mahayana, vajrayana, and zen. buddhism has no central text commonly referred to by each tradition, nor a central author- ity empowered to pronounce on doctrine or ethics. vaccination is widely accepted in predominantly buddhist countries. a key precept within buddhism generally prohibits killing, either humans or animals [ , , , ]. some canonical passages seem to accept meat consumption, whereas certain mahayana sutras (texts) denounce eating meat [ ]. in the modern buddhist world, attitudes toward vegetarianism vary by location. this review did not identify contemporary buddhist concerns with trace bovine components of some vaccines. buddhism does not oppose treatment of an existing illness by use of non-animal derived medicines, because treatment is an act of mercy [ – ]. antibiotics kill microorganisms, yet antibi- otics are accepted because they help people get closer to reaching enlightenment. serious diseases separate the body from the mind. preventing disease means preventing disharmony within the body. the nepalese lama zöpa rinpoche describes a prayer of the heal- ing buddha, to prevent diseases not yet experienced [ ]. he also describes logyönma (or loma gyönma), “a female healing bud- dha in leaf-wearing aspect,” known as an opponent to epidemic diseases [ , ]. the first written account of variolation describes a buddhist nun (bhikkhuni) practicing around – ce [ ]. she ground scabs taken from a person infected with smallpox (variola) into a powder, and then blew it into the nostrils of a non-immune per- son to induce immunity. continuing this tradition, the th dalai lama participated in poliovirus immunization programs personally [ ]. . . . jainism jainism arose in india between the th and th centuries bce, based on the teachings of nataputta vardhamana (also called mahavira), who prescribed a path of non-violence toward all liv- ing beings [ , , , ]. their scriptures are known as the jaina sutras. in the practice of ahimsa, expectations are less strict for lay persons than for monastics. jains recognize a hierarchy of life forms, such that mobile beings are accorded more protection than immobile ones [ ]. jains are vegetarians or vegans [ , ]. they avoid eating root vegetables in general, as cutting the root from a plant kills it, unlike other parts of the plant (e.g., leaves, fruits, seeds). although jains acknowledge that plants must be destroyed for the sake of food, they accept such violence only inasmuch as it is indispensable for human survival [ , , ]. cine t c t n [ b p t w r n n s i c ( j o fi w a h t t a v b p t j n ( a b w s a l [ h m s a p e f v a h h t p o j o t f j.d. grabenstein / vac jains may drink boiled water, cook food, use paper or soap, and ake necessary antibiotics, but perhaps with some regret. when onsidering vaccination, jains may benefit from an explanation of he seriousness of the diseases to be prevented, to explain the ratio- ale for killing microorganisms in the course of vaccine production , ]. jains agree with hindus that violence in self-defense can e justified [ , ]. jains filter water, to remove any small insects that may be resent. observant jains drink primarily water that has been fil- ered and boiled. boiling kills the multitude of tiny beings in the ater, but this is considered preferable to allowing the beings to eproduce in the water and later die, which would result in a greater umber of deaths. as one jain writer explained: “. . . we should ot cause violence to creatures; but we cannot live without water; o minimizing sins, we should use water. . . . meaningless use is mproper” [ ]. . . . judaism judaism is based on the relationship between god and the hildren of israel. judaism considers itself the religion of jacob alternately yisrael or israel), grandson of abraham and father of udah [ , ]. major western branches or denominations include rthodox, conservative, reform, and reconstructionist. the first ve books (torah) of the hebrew bible date to around bce, ith an evolution of ancient judaism that reached its present form round bce. the documentary basis of judaic teaching is the ebrew bible (tanakh or miqra), expounded in later texts such as he talmud and the shulchan aruch [ , ]. judaism traditionally expects certain actions of its believers o maintain health. pikuakh nefesh, acting to save one’s own or nother’s life, is a primary value, a positive commandment (mitz- ah aseh) [ – ]. judaic principles emphasize the community enefits of disease prevention in a manner superior to individual reference, based on scriptures such as leviticus : (table c) hat counsel not to stand idly by while a neighbor is in trouble. ewish scholars applied this directive to encourage smallpox vacci- ation in previous eras. rabbi and physician mosheh ben maimon also called maimonides or rambam) expounded: “anyone who is ble to save a life, but fails to do so, violates ‘you shall not stand idly y the blood of your neighbor”’ [ , , ]. indeed, in settings here vaccination services were intermittently available, several cholars stated it is permissible to set aside sabbath restrictions on ctivity to allow vaccination [ , , , , , , ]. simi- arly, there are exemptions from fasting if one is ill. parental responsibilities are detailed in a number of jewish texts , , ], based in proverbs : – (table c). the talmud as long encouraged parents to teach their children to swim, as a eans of preventing drowning in some unknown, but foreseeable cenario. scholars have taken this as a metaphor for vaccination gainst a future infection [ , , ]. maimonides wrote about revention: “one must avoid those things which have a deleterious ffect on the body, and accustom oneself to things which heal and ortify it” [ ]. another metaphor related to community responsibility is ele- ated to the status of a paradigm: the admonition to erect a railing round one’s roof, when it was often used as a porch, to prevent arm to others who may later walk there from an anticipatable azard (deuteronomy : , table c) [ , , , , , ]. his paradigm has been applied as a proactive call for communal rotection: vaccinating oneself and one’s family to reduce the risk f transmission of infectious diseases to neighbors and bystanders. within halacha (jewish law), the kashrut is the collection of ewish dietary laws, followed more closely by branches such as rthodox than by other branches. food considered fit for consump- ion is termed “kosher” in english, with most dietary laws derived rom the books of leviticus and deuteronomy (table c). ( ) – among these dietary laws are prohibitions on consuming ani- mals considered impure (e.g., pork, shellfish). products of impure or improperly slaughtered animals are also non-kosher (treif). ani- mal gelatin, for example, may be avoided as food; nonetheless, kosher gelatin (from cows or fish prepared to be kosher) may be an alternative food. in distinction to dietary laws, jewish medical issues are judged based on concepts of medical law contained in halachic codes. the propriety of using vaccines or immune globulins within judaism would be evaluated from a therapeutic or disease-prevention per- spective. multiple jewish authorities agree that limitations on medications with porcine components are only an issue with oral administration (for those who observe kosher rules), not prod- ucts given by injection [ , ]. thus, the teachings to avoid pork products do not apply to injectable medications, in contrast to food- stuffs. permissibility of oral administration of medications with non- kosher ingredients, if necessary to preserve life, is provided in the talmud [ ]. in the case of oral medications, the transformation (ponim chadashos) of “primary” pork components into processed materials would make them more acceptable. oral medication con- taining small amounts of material derived from non-kosher animals devoid of its taste could be kosher under some circumstances. according to a principle known as bitul b‘shishim, a small amount of non-kosher food mixed with a much greater quantity of kosher food may be acceptable if the non-kosher item loses its taste or is diluted beyond a : ratio [ ]. additional conditions (e.g., inten- tion, gentile source) need to be considered before this ruling can be made. rabbi abraham nanzig, writing in london in in the era of smallpox outbreaks, described the halachic basis for exposing a child to variola virus (variolation) to induce immunity against smallpox: “one who undergoes this treatment while still healthy, god will not consider it a sin. rather, it is an act of eager religious devotion, and reflects the commandment to ‘be particularly careful of your well-being”’ (deuteronomy : , table c) [ , ]. in the s, distinguished rabbi yisroel lipshutz described edward jen- ner as a “righteous gentile,” for his efforts in developing smallpox vaccination [ , ]. jewish communities (often ultraorthodox, those who adhere meticulously to jewish law and tend to be more isolated from others) in several countries have experienced measles and mumps outbreaks associated with declining vaccination [ , , , , , , , ]. the transnational social networks between such communities have allowed outbreaks to spread from one country to another [ ]. based on this review, contemporary jewish vaccine decliners are more likely to cite concerns about vac- cine safety than to invoke a specific religious doctrine that has not been considered by acknowledged jewish scholars. those scholars have rejected arguments that medical interventions interfere with divine providence [ , , ]. the orthodox hasidic jews who constitute most of the residents of the village of kiryas joel in orange county, new york, volunteered for several pivotal vaccine trials. these included trials for hepatitis a vaccine and mumps vaccine [ – ]. . . . christianity christians are followers of jesus, whom they consider the christ (i.e., messiah, anointed one). christians believe that jesus, descended from abraham through isaac, is the son of god proph- esied in the hebrew bible [ , ]. christianity began as a jewish sect around ce. today, the largest groups within christianity are the roman catholic church, the eastern orthodox and oriental ortho- dox churches, and the denominations of protestantism [ , ]. the life and teachings of jesus are presented in four canonical gospels (“good news”) and other writings appended to the hebrew cine b b t n o t o c l t s t c v p t w t k ( g c t b a d d d m a a o i i i n p r e o a t s e m i f f r w p h t k d j.d. grabenstein / vac ible (old testament) in the form of a new testament. various ranches of christianity define separate lists of books of the bible hat each considers canonical [ , ]. . . . . multiple christian denominations. most christian denomi- ations have no scriptural or canonical objection to use of vaccines r immune globulins per se, based on this review (table c and d). hese include roman catholicism, eastern orthodox and oriental rthodox churches, amish, anglican, baptist, the church of jesus hrist of latter-day saints (lds), congregational, episcopalian, utheran, methodist (including african methodist episcopal), pen- ecostal, presbyterian, and seventh-day adventist church. exceptions appear in following sections. roman catholicism and ome other christian denominations have expressed concern about he aborted fetal origins of the principal formulation of rubella vac- ine and some cell lines used to manufacture certain types of viral accines, discussed in later sections. the second half of table d rovides scriptural passages interpreted by a minority as contrary o vaccination. within a christian creation-fall-redemption-restoration frame- ork, immunization advocacy can form a basis for christian service o humanity. this is consistent with themes of being one’s brother’s eeper (genesis : , table c), loving your neighbor as yourself james : , table d), and acting kindly to strangers, as did the ood samaritan (luke : – , table d). . . . . amish and related communities. the amish, sometimes alled old-order amish or amish mennonites, are a group of chris- ian fellowships among mennonite churches. amish fellowships egan with a schism within a group of anabaptists in switzerland in ce. related groups in canada and the northern us are known s hutterites. immunization is not prohibited by amish or hutterite religious octrine, but vaccine acceptance varies from district to district. istricts that typically decline immunization reflect a social tra- ition within these religious communities, related to modernity, ore than a theological objection. low immunization rates in mish communities have been attributed variously to limited ccess to care, limited disease understanding, higher priority to ther activities, and concerns about vaccine safety, with variabil- ty among various communities [ , – ]. they tend to define llness in terms of failure to function in a work role, more than n terms of symptoms [ ]. within amish and related commu- ities, multiple haemophilus influenzae type b, measles, pertussis, oliomyelitis, rubella, and tetanus cases and outbreaks have been eported [ , , , , , , – , , , – , ]. district lead- rs have been more accepting of immunization at times of local utbreaks. . . . . church of christ, scientist. spiritual healing of disease is central tenet for members of the first church of christ, scien- ist, founded in ce in boston by mary baker eddy. christian cientists frequently decline some or all medical help for dis- ase. individual believers often forego immunization, and church embers have lobbied governments for religious exemptions from mmunization. eddy called believers to unmask the devil’s lies, one mani- estation of which is disease. disease, in this construct, is not undamentally real, but rather something that can be dispelled, to eveal the perfection of god’s creation. “sickness is part of the error hich truth casts out” [ ]. from this arose the christian science rinciple that disease is cured or prevented by prayer that affirms uman perfection as god’s child and denies the reality of disease. his principle is featured in eddy’s canon, science and health with ey to the scriptures [ ]. christian science “practitioners” (who o not practice medicine) aid believers in focused prayer. ( ) – in a interview with the new york herald, eddy said [ ]: “at a time of contagious disease, christian scientists endeavor to rise in consciousness to the true sense of the omnipotence of life, truth, and love, and this great fact in christian science realized will stop a contagion.” later, she said: “rather than quarrel over vaccination, i recommend, if the law demand, that an individual submit to this process, that he obey the law, and then appeal to the gospel to save him from bad physical results” [ ]. outbreaks of diphtheria, measles, and poliomyelitis have been reported among followers of christian science [ , , – , , , ], including repeat measles outbreaks at principia college and affiliated k- schools between and [ ]. three measles deaths and hundreds of cases occurred during those outbreaks. the church has a policy for members to report communicable diseases to health authorities, but members have limited ability to do so. first, their practitioners and nurses are not trained in disease recognition. second, members are taught that disease is healed by convincing oneself of its unreality. as a result, several outbreaks have been recognized only after many people were infected [ , ]. in such cases, christian science parents were more willing to accept immunization after outbreaks were recognized by health authorities. . . . . dutch reformed congregations. members of certain tra- ditional reformed (bevindelijk gereformeerden) christian denom- inations in the netherlands, founded in the s ce, have a tradition of declining immunization that dates back to con- cerns about adverse events after smallpox vaccination from onward [ , , , ]. these communities were the epi- centers of paralytic poliomyelitis, measles, congenital rubella syndrome, and mumps outbreaks between and [ , , , , , – , – , , ]. members of these denominations have familial and cultural ties to associated christian communities in other countries (e.g., canada, united states), where immunization rates may also be low. these ties have resulted in international transmission of vaccine- preventable diseases (e.g., measles, poliomyelitis, rubella) with multiple outbreaks in locations otherwise free of circulating disease [ , , , , – , ]. the contemporary basis for the objection of some members of these churches includes choosing to forego immunization rather than making a person less dependent on god [ , , , ]. for a subset, avoiding interference with divine providence before infec- tion may be paramount; another subset described immunization as a gift from god to be used with gratitude [ , ]. arguments against immunization have been refuted by other members of the traditional reformed community [ ], for example by pointing out that using agricultural practices or raising dikes, to prevent flood- ing, could also be construed as contrary to divine intent, yet are common practices [ ]. recent increases in immunization rates in dutch communities suggest that objections to immunization may be declining [ ]. . . . . jehovah’s witnesses. the jehovah’s witnesses is a christian denomination tracing its roots to the late s ce. the watch tower bible and tract society is its organizing body [ – ]. since , the watch tower society has instructed its followers to refuse transfusions of whole blood and certain blood components (e.g., red blood cells, white blood cells, platelets, whole plasma), which they consider a violation of god’s law. this interpretation derives from several scriptural passages (table e) [ – ]. their blood doctrine has undergone multiple changes since , princi- pally in , , and [ – ]. by abstaining from blood, witnesses express their faith that only the shed blood of jesus can redeem them and save their life. in this view, those who respect life as a gift from god do not try to sustain cine l m a t t a m w c h v f b w i d a c t b a t g t t f c n s a n e [ a b a o s i o b i c ( m g i o p t b b a s j.d. grabenstein / vac ife by taking in blood, even in an emergency [ , ]. while albu- in, antimicrobial immune globulins, rho(d) immune globulin, nd coagulation factors viii and ix have been declared acceptable o believers since [ , ], witnesses today are taught that he use of various blood fractions are “not absolutely prohibited” nd are a matter of personal choice [ , , – , – ]. ore recently permissible products include those derived from hite blood cells (e.g., interferons, interleukins), cryoprecipitate, ryosupernatant, erythropoietin, and preparations derived from emoglobin [ , , ]. it is unclear what proportion of jeho- ah’s witnesses offered such therapeutic products accept them. the watch tower society distributes worksheets and pre- ormatted power-of-attorney advance directives, on which mem- ers can specify which allowable fractions and treatments they ould personally accept, if any [ , , , , , , ]. mportant questions have been raised regarding how much free- om and what degree of information about risks, benefits, and lternatives are available to individual jehovah’s witnesses when onsidering these documents [ , , – , – ]. some jehovah’s witnesses dissent from the blood-product doc- rine, including the associated jehovah’s witnesses for reform on lood [ , ]. they see no biblical or ethical wrongness with ccepting transfusion of donor blood or with donating blood for ransfusion. this group functions with anonymity, because congre- ations have ostracized or expelled those who ignore or criticize heir doctrine [ , , – ]. the watch tower society denounced vaccination from the s through the s, citing scriptural passages such as those in able e [ – , , , ]. the group banned their members rom vaccination around this time, under penalty of excommuni- ation [ , ]. the society revised this doctrine in the december , , issue of the watchtower, saying that those passages did ot apply to vaccination [ ]. in , the society took a neutral tand, neither endorsing nor prohibiting vaccination. in the s, wake! magazine began acknowledging the clinical value of vacci- ation. a contemporary watchtower web page acknowledges the fficacy of vaccination in preventing hepatitis a and hepatitis b ]. . . . . churches that rely on faith healing. in addition to discussion bove, several small christian denominations or churches hold core eliefs that focus on healing through faith alone (table d), with ctive avoidance of medical care (e.g., faith tabernacle, church f the first born, faith assembly, end time ministries) [ ]. everal vaccine-preventable outbreaks (and associated deaths) nvolved faith healing to the exclusion or neglect of immunization r treatment after infection [ , , , , , – ]. these out- reaks involved both adults who choose not to have themselves mmunized and parents who withheld routine vaccines from their hildren. . . . islam islam professes beliefs articulated by their holy book, the qur’ān the recitation), and through the teachings and example of muham- ad ( – ce). muhammad, the last messenger of allāh (the od, in arabic), descends from abraham (ibrahim) through his son shmael [ , ]. the two major islamic sects are sunni and shı̄‘āh. the qur’ān and tradition forbid consumption of several animals utright (e.g., “the flesh of swine,” table f), while other animals are ermitted (halal) or forbidden (haram) based on conditions of how hey died or were slaughtered. gelatin made from porcine skin or ones is forbidden as food. gelatin made from other halal animals, eef or fish for example, is acceptable as food. opinions or rulings on interpretation of the qur’ān are issued s fatwas by islamic scholars (mujtahids), with varying degrees of trictness. but fatwas are not always widely held to be authoritative, ( ) – in part because of varying degrees of expertise and also because the relationship for each muslim is directly with god. according to the qur’ān, a person is not guilty of sin in a situation where the lack of a halal alternative creates an undesired necessity to consume that which is otherwise haram (qur’ān : ). this is the basis for the “law of necessity” in islamic jurisprudence: “that which is necessary makes the forbidden permissible” in exceptional circumstances (table f). opposition to immunization programs among selected mus- lim communities has occurred during poliovirus immunization programs in nigeria, pakistan, and afghanistan [ ]. the oppo- sition within northern nigeria, notably in the state of kano, was particularly long-lasting and an impediment to the global eradi- cation effort [ , – ]. detailed consideration of the nigerian situation revealed that what was described as ostensibly religious objections and assertions that vaccines spread the hiv virus or were vehicles for sterilization programs masked deeper struggles related to political power, inadequate health services, and a controver- sial clinical trial of an investigational antibiotic [ , , ]. while the boycott was centered within islamic social networks, most of the objections raised related to social issues, rather than theo- logical issues. eventually, the nigerian government sent religious representatives to south africa, indonesia, and india to observe quality-control tests of poliovirus vaccines to be used in their areas and then sourced the vaccine from manufacturers they trusted [ , ]. in contrast, multiple imams and other islamic leaders issued clear statements and fatwas describing how immunization is con- sistent with islamic principles [ , , ]. in the nigerian case, engagement of the organization of the islamic conference (includ- ing african countries) and the th annual conference of the international fiqh council (a global forum of islamic lawyers, scholars and philosophers to address the practice of islam in con- temporary life) provided assurances to nigerian leaders [ ]. earlier, in , the islamic organization for medical sciences, a well-regarded set of jurisprudents and medical experts con- ducted a seminar in kuwait on “the judicially prohibited and impure substances in foodstuff and drugs” [ ]. participants included the muftis (experts in islamic law) of egypt, tunisia, oman, and lebanon, the secretary general of the islamic fiqh academy in jeddah, and many other accomplished islamic scholars. citing the accepted principle of transformation (fundamental change, as from wine to vinegar) within islam, they concluded that “the gelatin formed as a result of the transformation of the bones, skin, and tendons of a judicially impure animal is pure, and it is judicially permissible to eat it” (see also section . . ) [ ]. the full docu- ment also addressed issues related to medication capsules, alcohol, pig fat, and porcine insulin. omar kasule, professor of islamic medicine at the institute of medicine university of brunei darussalam noted that polio immu- nization is obligatory (wajib) when disease risk is high and the vaccine shown to have benefits far outweighing its risks [ , ]. muslims will be interested in issues of vaccine safety, professor kasule explained, because immunization to prevent disease should not lead to side effects of the same magnitude as the disease. he based this judgment on the purpose of the law to protect life, the principle of preventing harm (izalat aldharar), and the principle of the public interest (maslahat al-ummah). he noted that the qur’ān uses the concept of wiqaya in multiple situations to refer to tak- ing preventive action (e.g., against hell-fire, punishment, greed, bad acts, harm, heat) and concludes that prevention is one of the laws of allāh, with obvious application to medicine. muslims may apply additional scrutiny to vaccines required for pilgrims to the annual hajj in mecca, when purity takes on extra significance [ – ]. another guiding principle comes from the prophetic statement of muhammad: “god has not made cine t [ m t d i a w s o t n r t r p t v c m o l f l s [ v t f z v w s b s c s e v t p g c b s o w u d w f j.d. grabenstein / vac hings that are unlawful for you to consume to be your medicine” ]. . . . . nation of islam. the nation of islam is a us-based move- ent that aims to improve the condition of african-americans in he us [ ]. its religious practices have some similarities and some ifferences, compared with traditional islam. in , the min- ster of health of the nation of islam advised believers to avoid ll immunizations, based on concern about viral contamination ith pathogens that cause “aids, ebola, hanta, chronic fatigue yndrome, gulf-war syndrome, ‘mad cow’ disease, etc.” [ ]. no bjective evidence to substantiate these claims has been offered. hat statement was framed as “until further notice,” although it o longer appears on the nation of islam website. the basis was ooted in safety and distrust-of-government concerns, rather than heological grounds. . . vaccine components and processes . . . bacteria, viruses, cell substrates the hindu, buddhist, and jain religions have long prioritized especting all forms of life, in the form of ahimsa [ ]. the jains in articular extend this respect even to the bacteria or viruses con- ained in a vaccine, as well as the culture-media cells used to grow iruses or produce recombinant proteins. the google searches identified a posting contending that jains annot take vaccines because microbes are killed in the process of anufacturing the vaccines. but this would seem to be a misreading f the jain approach to regretting the loss of microbial (one-sensed) ife, yet taking actions necessary to sustain life (e.g., ingesting life orms along with food, boiling water) [ , , ]. mohandas gandhi observed: “the very fact of his [humanity’s] iving—eating, drinking and moving about—necessarily involves ome himsa, destruction of life, be it ever so minute” (table a) ]. . . . wi- and mrc- cell lines unlike bacteria, viruses do not replicate on their own. to make iral vaccines, large numbers of viruses must be grown in cell cul- ures specific to each virus. some licensed viral vaccines (i.e., some ormulations of hepatitis a, poliovirus, rabies, rubella, and varicella- oster viruses or combination vaccines containing such component iruses) are produced by growing viruses that infect humans in i- or mrc- cell cultures [ , ]. wi- and mrc- repre- ent two commonly used lineages of human diploid cell cultures, atches of immature cells with twice as many chromosomes as perm or egg cells. embryonic diploid cells are valuable in vac- ine manufacture, because each aliquot of these cells can propagate everal dozen times before senescence. each of these cell lines started with cells harvested from a delib- rately aborted fetus [ , ]. the cell lines are used to grow the iruses, then discarded and not included in vaccine formulations. hese cell lines cannot form a human being. the wi- line was developed at the wistar institute in philadel- hia in , with lung cells from a female fetus of months estation aborted in sweden, whose parents felt they had too many hildren [ , , – ]. similarly, british scientists funded y the medical research council developed the mrc- line in eptember with fetal lung fibroblasts “taken from a -week- ld male fetus removed for psychiatric reasons from a -year-old oman. . .” [ , , ]. these cell lines, still in use today, grad- ally replaced primary cultures of monkey, duck, rabbit, chicken, og, or mouse tissue, an approach vulnerable to contamination ith viruses and bacteria [ , , ]. vaccine manufacturers have few options for viral culture media, or reasons of microbiology and safety [ , , , – ]. it is ( ) – not possible to simply replace one cell line with another, because various viruses grow abundantly only in some kinds of cell lines. wi- and mrc- lines are well described and understood, with experience accumulated via hundreds of millions of vaccinations, important for safety-assessment reasons. the fetal origins of wi- and mrc- cell lines pose an ethical or moral problem for people who disapprove of abortion. criti- cally, the two abortions were not conducted for the purpose of harvesting the cells that were transformed into these cell lines [ , , – ]. this lack of intention is a key element in break- ing the complicity link that could otherwise make use of the vaccines unacceptable. no additional abortions are needed to sus- tain vaccine manufacture. the cell lines are not the final product, and no human cells are present in the final vaccine formulations. in the late s—early s, teams of ethicists at the national catholic bioethics center and then at the vatican’s pontifical academy for life and elsewhere considered the virology, epidemi- ology, and theology of the matter in detail [ , , – ]. their considerations included both cooperation with evil and the principle of double effect. in this case, the cooperation related to those involved with the specific abortions in the s. the prin- ciple of double effect applied insofar as using implicated vaccines today could appear to endorse or acquiesce to the acceptability of additional abortions in our current time. these teams concluded that the association between implicated vaccines and abortion was noncomplicit, and that using these vaccines is not contrary to a principled opposition to abortion. these centers reasoned that, because the abortions that enabled the production of these vaccines are in the past and (critically) the abortions were not undertaken with the intent of producing the cell lines, being immunized does not involve any sharing in immoral intention or action of others. in short, they are morally separate actions. in , this position was elevated to the status of official roman catholic teaching [ ]. the bioethicist teams agreed that use of a vaccine in the present does not involve sharing in the action of those who carried out the abortion in the past [ , – ]. further, they found that parents have a moral obligation to provide for the life and health of their children by means of immunization [ , – ]. the situation with vaccines differs morally from ongoing harvest of fetal tissue for pharmaceutical manufacturing or research, which could be used to justify future abortions [ ]. still, these ethicists concluded that alternate vaccines should be used if available. they also recommended that parents and cli- nicians should speak out against abortion by asking governments and vaccine manufacturers to stop using cell lines that have links to aborted fetuses [ , ]. . . . rubella virus strain ra / in , the wistar institute developed the ra / strain of rubella virus. the rubella virus isolate “was recovered from the explanted [kidney] tissue of a fetus obtained at therapeutic abor- tion from a mother who had been infected with rubella virus” [ , – ]. the scientific literature of that era indicates that the abortion was not conducted with the motive of isolating the virus, but rather because the mother was infected with rubella virus and risked major birth defects [ , , ]. after the ra / strain was isolated, it has been propagated serially in human diploid cells. the ra / strain produced superior antibody responses and was better tolerated, compared with other rubella vaccine strains available in the s [ , ]. no further abortions are neces- sary to sustain the manufacture of additional batches of rubella ra / -strain vaccine. use of the ra / rubella virus strain was also considered by the national catholic bioethics center and the pontifical academy for life. using the same logic, they reasoned that because the one abortion that yielded the viral isolate was not undertaken with the cine i n a s m c o i p r p i g d t m c a p v e a v w t p p p a n t m d i f ( ( ( p m t e t t c o b s u o a with both hbv and hpv, a person could forego the vaccines, lead a life fully compliant with religious belief, and still be infected. many j.d. grabenstein / vac ntent to retrieve the virus and because no additional abortions are eeded to obtain more virus, being immunized is morally accept- ble and also associated with parental duty [ , – ]. the ame provisions for preferring alternatives and petitioning govern- ents and manufacturers also apply. some find it meaningful that rubella vaccination prevents many ases of fetal death and congenital rubella syndrome that would therwise occur if women were infected with rubella virus dur- ng pregnancy. immunized women exposed to the virus during regnancy are no longer confronted with the question (what some eligions might consider temptation) of whether to terminate their regnancies on that basis. . . . porcine excipients all vaccines require the use of excipients (inactive ingredients) n manufacturing. some of these products, such as hydrolyzed elatin or trypsin, may have a porcine (pork) origin. hydrolyzed gelatin is a mixture of peptides and proteins pro- uced by partial hydrolysis of collagen (connecting fibers and issues) typically extracted from skin, bones, or other components, ost often from pigs or cattle. hydrolyzed refers here to the pro- ess of breaking down collagen molecules into chains of amino cids (polypeptides) by acidic or alkaline treatment, followed by urification [ , , ]. gelatin hydrolysates are added to some accine formulations to help stabilize and preserve active ingredi- nts during freeze-drying and storage; hydrolyzed gelatin may also ct as a solvent [ , , ]. the enzyme trypsin may be used in producing some viral accines, to resuspend cells adhering to the cell-culture dish all during the process of harvesting cells [ , , ]. trypsin ypically is removed from the product physically before further rocessing. like hydrolyzed gelatin, trypsin is often derived from orcine or bovine sources. some jews, muslims, and others have expressed concern about orcine-origin components, derived from faith-based concerns bout consumption of pork in their diet, despite the injectable ature of most vaccines. injectable medications are not subject o kosher rules [ , ]. permissibility of oral administration of edications with such ingredients, if necessary to preserve life, is escribed in earlier sections. scholars of judaism and islam have ssued various rulings or waivers that allow use of such vaccines, or several reasons [ , – , , , ]: ) the components of concern (e.g., hydrolyzed gelatin) have been sufficiently transformed from original pork origins, ) the minute quantities per dose administered (e.g., hydrolyzed gelatin, trypsin) invoke exceptions based on dilution, or ) the vaccine is intended for important medicinal purposes and not a matter of ingestion, to which dietary rules apply. other important considerations include the necessity of the roduct to save life and the lack of alternatives. different scholars ay evaluate and weigh these criteria differently. for muslims, sharı̄‘ā law includes the principle of transforma- ion (istihaalah) in which unclean products can be made clean by xtensive processing, transforming the original product into some- hing new (e.g., from wine to vinegar). under certain circumstances, his can make it permissible for observant muslims to receive vac- ines, even if the vaccines contain porcine excipients. this principle f transformation was invoked by the conference convened y the islamic organization for medical sciences [ , ]. the cholars explicitly concluded that transformation of pork prod- cts into gelatin alters them sufficiently to make it permissible for bservant muslims to receive vaccines containing porcine gelatin nd certain other medicines, including those formulated in gelatin ( ) – capsules [ ]. even so, alternative products without components of concern may be preferred, if available. in , the european council of fatwa and research issued a fatwa finding the permissibility of using oral poliovirus vaccine pro- duced with porcine-origin trypsin [ ]. their decision centered on lack of similarity between pork and purified trypsin, physical removal during processing, dilution of any residual, necessity, and lack of alternative. . . . bovine excipients bovine serum or albumin may be added to some cell cultures as a source of nutrition (in the form of albumin, amino acids or pep- tides, and growth factors); albumin can act as a protein stabilizer [ , ]. hydrolyzed gelatin or trypsin (see porcine excipients, above) may alternately derive from a bovine source. this review identified no explicit religious objection to bovine components. . . . misunderstandings of vaccine production or content google searches revealed multiple postings (data not shown), both by members of the public and by those who describe them- selves as health professionals, that misstate the actual contents of vaccines and immune globulins. these searches revealed erroneous assertions that all vaccines are grown in chicken eggs, that vaccines are blood products, or that vaccines are contaminated with alcohol, toxins, or heavy metals. some sites incorrectly asserted that most vaccines are genetically modified, claiming that such products are forbidden in both judaism and christianity based on leviticus : and matthew : (table c and d). objections of certain catholic officials in the philippines in the mid- s that tetanus toxoid immunization for adult women actually contained contraceptives or abortifacients were based on misunderstanding [ – ]. similar confusion disrupted immu- nization programs in kenya, méxico, nicaragua, and tanzania [ , ]. several websites objected to immunization on the basis that god created humans in his own image and that the body is a temple not to be defiled (table d). interpretations of the first letter of paul to the corinthians contrast with mark’s gospel in this regard. even so, vaccines are no less pure than various other commonly used medications and are subject to extensive quality-control and audit procedures by the manufacturers and by multiple government reg- ulators [ , ]. . . . pathogen route of exposure many religions traditionally have been proponents of sexual propriety [ , ]. this review identified several objections to hepatitis b immunization or to human papillomavirus (hpv) immunization, centered on the sexual route of exposure that can be associated with the corresponding pathogens. these objections to immuniza- tion were not theologically based per se, but rather arose indirectly as religious beliefs (usually of parents) affected views of accept- able sexual practices or timing. in the case of hepatitis b virus (hbv), sexual activity is only one of many risk factors for infection, including mother-to-child transmission. for hpv, several studies have shown that immunization does not increase or accelerate a woman’s likelihood of sexual behavior [ – ]. the proportion of never-married teenaged females in the us who had been sexually active at least once fell from % in to % in – [ ]. religions have rites that allow for atonement or forgiveness of sins, but the many diseases caused by hbv and hpv (including multi- ple cancers) remain among the most difficult infectious diseases to attempt to cure. j.d. grabenstein / vaccine table summary of key points from perspectives of selected religions. jainism, buddhism, hinduism (linked via ahimsa) © respect for all life, favoring nonviolence [ , , ] © recognize the need to sustain human life, with regretful acceptance of cooking food, boiling water, using antibiotics and vaccines [ , , , , , , ] judaism © consider the imperative for pikuakh nefesh, acting to save one’s own or another’s life [ , , , ] © consider the duty to protect one’s children and one’s neighbors; do not stand idly by [ – ] © dietary kosher limitations on medications with porcine components apply to oral administration, but not to injection [ , ]. even so, consider the importance of medicine in preserving life christianity © vaccines with remote fetal implications are morally acceptable (with a duty to protect children), unless alternative products are available [ , , – ] © jehovah’s witnesses may accept certain blood derivatives, including immune globulins, interferons, coagulation factors, erythropoietin, and others [ , , , , ] © concern that ‘the body is a temple not to be defiled’ contrasts with other scripture passages (table d) and modern quality-control requirements for vaccines and immune globulins [ , ] islam © consider the law to protect life, the principle of preventing harm (izalat aldharar), and the principle of the public interest (maslahat al-ummah) [ , ] © transforming haram components may generate halal products (e.g., wine to vinegar) [ , ] © extensive dilution of components of concern may result in minute quantities per dose [ , , ] © vaccines are intended for important medicinal purpose, not diet [ , , , ] © vaccines help protect others [ , – , ] t o a i m m h t t i r i m p ( n l t a f a a g t t a e cal considerations of clinical issues [ , , , , , , ]. when © consider the law of necessity, whether alternative vaccines are available [ ] . discussion this review is intended to explain pivotal aspects of religious eaching that have been applied for and against the acceptability f vaccines and immune globulins. as various examples described bove show, the scriptural, canonical passages cited here are not nterpreted uniformly by each believer within a faith tradition. the ultiple sects, denominations, and branches within each of the ajor religions demonstrates the multiple ways various passages ave been applied [ , , ]. this review identified multiple religious doctrines or impera- ives that call for preservation of life, caring for others, and duty o community (e.g., parent to child, neighbors to each other). even n cases where vaccine components could be objectionable, this eview found several themes favoring vaccine acceptance, includ- ng transformation of components of concern from their starting aterial, extensive dilution of such components, the medical pur- ose of immunization (in contrast to diet), and lack of alternatives see table ). this review revealed few canonical bases for declining immu- ization, with christian scientists a notable exception. along these ines, it would seem that the instances of personal objections hat are properly theological in nature (defined here as system- tic and rational exploration of the nature of god) are relatively ew, and that the preponderance might more accurately be defined s philosophical (i.e., a more general consideration of existence nd reason) or simply personal choice [ ]. for several religious roups, declination of immunization is more traditional or social han an essential religious precept [ , ]. the bulk of the objec- ions identified in the searches for this review reflected concerns bout vaccine safety, not matters of theology, as did an analysis of xemptions for school-aged children [ ]. for christian scientists ( ) – who believe “man is incapable of sin, sickness, and death” [ ], vaccines would be superfluous. one question-and-answer webpage started with this question: “i need to get a religious exemption or medical exemption for my children. we are moving to hi [hawaii] and these are the only two exemptions they offer. anyone know how to get around vaccinat- ing my children?” [ ]. how can we understand the intent of this writer? understanding people’s actual motives is important when discussing immunization. clinicians counseling people reluctant to be immunized may wish to probe for understanding of vaccine contents and provide factual information. from a netherlands perspective, ruijs et al. suggest discussing vaccine decision-making processes (e.g., criteria used, consequences), rather than medical information or an author- itarian stance [ ]. collaboration between public-health leaders and (religious) community leaders historically has helped resolve objections and enabled immunization programs to continue. reli- gious communities are a powerful social force, as shown in this review and in other studies [ , ]. the accumulation of susceptibles within a community creates vulnerability to infection [ , , , , , , ]. a community can afford to have a small number of conscientious objectors to immunization. but each unimmunized person adds to the vulnerability of the group. if geographic clusters within a city neigh- borhood, among preschoolers, or within a suburb, a rural town, an island, a parish, or some other focal area are immunized at only % or % levels, herd protection does not occur and outbreaks can develop. an increasing collection of vulnerable people is like an increasing collection of kindling wood. introduce a spark and fire can spring forth. one contagious person among a cluster of vul- nerable people can ignite an outbreak involving many, including those unable to respond to vaccination. one of the limitations of this review is that information about beliefs of less populous religions or denominations were not explic- itly sought. on the other hand, the many searches and traces through reference lists frequently led to documents describing other religious traditions or denominations. none of those docu- ments featured a canonical objection to immunization not already described above. but review of the medical literature identified multiple outbreaks of vaccine-preventable diseases among them [ , , , , , , , ]. outbreaks rooted in personal or philo- sophical beliefs are not referenced here, but are numerous. the outbreak reports cited in this review are likely not an exhaustive list of all religious-centered outbreaks, for several rea- sons: some publications may not have been identified (especially those not written in english or relevantly coded in pubmed), some publications about outbreaks related to personal-belief exemptions may not have specified a religious basis for those beliefs, and some relevant outbreaks (or individual cases) may not have been pub- lished. one element of acceptability for some believers is whether vac- cines of concern have any alternatives [ , , , , – ]. alternatives can be determined by comparing ingredients and culture media described in product prescribing information. con- trary to several web pages, measles vaccine is not a prophylactic alternative to measles-mumps-rubella (mmr) vaccine, insofar as selecting measles vaccine alone would be a decision to reject pro- tection against mumps and rubella. manufacturers attentive to global acceptability will endeavor to replace or avoid components of concern whenever possible. if we are to serve our patients’ needs in all their humanity, we should help them gain access to reasoned ethical and theologi- dealing with vaccines, the implications of a personal infectious- disease decision reach beyond the self, to affect neighbors [ , , – , , , , , , , ]. my decision to immunize cine o y s h o i a v c s m s w d m p a e a a f j r j.d. grabenstein / vac r not immunize my family members changes the likelihood that ou or your family will contract a contagious disease, and vice versa. . personal note the coming together of public health and religion is not a colli- ion; rather it involves repeated intersections. we can advance both ealthcare and our own condition by discussing them openly more ften. i remain open to finding and reading doctrinal teachings not dentified in my searches to date. cknowledgements the assistance of numerous religious scholars, believers, and accine experts who critiqued drafts of this review is greatly appre- iated, including: ibrahim abuammar, brant biehn, joye l. bramble, hehla hussain, chester j. kitchen, barbara j. kuter, mark loeb, anal morsy, paul a. offit, diane c. peterson, samir shaikh, neel heth, benoît soubeyrand, walter l. straus, rita swan, deborah l. exler, charles (skip) wolfe, robert m. wolfe, and karie young- ahl; from the associated jehovah’s witnesses for reform on blood, arvin shilmer; from the interfaith center of greater philadel- hia, abby stamelman hocky, rev. nicole diroff, bijan etermad, nd suketu 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[ ] omer sb, enger ks, moulton lh, halsey na, stokley s, salmon da. geographic clustering of nonmedical exemptions to school immunization requirements and associations with geographic clustering of pertussis. am j epidemiol ; : – . http://omarkasule- .tripod.com/id .html http://www.eimjm.com/vol -no /vol -no -h .htm http://www.muslimpopulation.com/america/cuba/how cuba is tapping into the.php http://www.muslimpopulation.com/america/cuba/how cuba is tapping into the.php http://www.wikipedia.com/ http://www.mosque .org/minofhealth/declarationofexem.doc http://www.ncbenter.org/vaticanresponse.pdf http://www.ncbenter.org/vaticanresponse.pdf http://www.vatican.va/roman_curia/congregations/cfaith/documents/rc_con_cfaith_doc_ _dignitas-personae_en.html http://www.vatican.va/roman_curia/congregations/cfaith/documents/rc_con_cfaith_doc_ _dignitas-personae_en.html http://www.who.int/immunization_standards/vaccine_quality/vmc/en/index.html http://www.who.int/immunization_standards/vaccine_quality/vmc/en/index.html http://islamqa.info/en/ref/ http://www.sciencedirect.com/science/article/pii/ http://www.sciencedirect.com/science/article/pii/ http://www.path.org/vaccineresources/files/combatting_antivac_rumors_unicef.pdf http://www.path.org/vaccineresources/files/combatting_antivac_rumors_unicef.pdf http://www.cdc.gov/nchs/data/series/sr_ /sr _ .pdf http://www.mothering.com/community/t/ /what-religions-dont-vaccinate 	what the world's religions teach, applied to vaccines and immune globulins 	 background 	 methods 	 results 	 . populous religious groups 	 . . hinduism 	 . . buddhism 	 . . jainism 	 . . judaism 	 . . christianity 	 . . . multiple christian denominations 	 . . . amish and related communities 	 . . . church of christ, scientist 	 . . . dutch reformed congregations 	 . . . jehovah's witnesses 	 . . . churches that rely on faith healing 	 . . islam 	 . . . nation of islam 	 . vaccine components and processes 	 . . bacteria, viruses, cell substrates 	 . . wi- and mrc- cell lines 	 . . rubella virus strain ra / 	 . . porcine excipients 	 . . bovine excipients 	 . . misunderstandings of vaccine production or content 	 . . pathogen route of exposure 	 discussion 	 personal note 	acknowledgements 	appendix a supplementary data 	appendix a supplementary data wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ pii: s - ( ) - b-sarcoglycan: genomic analysis and identification of a novel missense mutation in the lgmd e amish isolate f. duclos a, o. broux b, n. bourg b, v. straub a, g.l. feldman c, y. sunada a, l.e. lim a, f. piccolo d, s. cutshall a, f. gary b, f. quetier b, j.-c. kaplan d, c.e. jacksonc, j.s. beckmann b , e, k.p. campbell a ,* ahoward hughes medical institute, department of physiology and biophysics and department of neurology, university of iowa college of medicine, iowa city, ia , usa bcnrs ura , france, généthon, evry, france chenry ford hospital, department of medical genetics, detroit, mi , usa dhopital cochin maternités, inserm u , paris, france efondation jean dausset-ceph, paris, france received august ; revised version received october ; accepted november abstract the sarcoglycan complex is involved in the etiology of four autosomal recessive limb-girdle muscular dystrophies (lgmd c–f). a missense mutation (t r) in the b-sarcoglycan gene on chromosome q has been shown to cause a mild form of lgmd e in families from a southern indiana amish community sharing a common haplotype. we now report that two sibs from another amish family with mild lgmd e are compound heterozygotes for chromosome q markers. in order to characterize the genetic defect in this new family, we determined the genomic organization of the b-sarcoglycan gene. a second missense mutation (r c) has now been identified in this lgmd e amish family. this mutation is also present in the homozygous state in another family of probable amish ancestry. finally, analysis of all the components of the dystrophin-glycoprotein complex was performed for the first time on a biopsy from a patient homozygous for the b-sarcoglycan mutation (t r). interestingly, in addition to the loss of the entire sarcoglycan complex, we detected a reduction of a-dystroglycan which suggests a role for the sarcoglycan complex in stabilizing a-dystroglycan at the sarcolemma.  elsevier science b.v. keywords: limb-girdle muscular dystrophy; b-sarcoglycan; sarcoglycan complex; a-dystroglycan . introduction in the dystrophin-glycoprotein complex (dgc) [ – ], b- sarcoglycan, a kda glycoprotein, is closely associated with a-, g-, and d-sarcoglycan [ – ]. this subcomplex can be distinguished from a-, and b-dystroglycan [ ], the syntrophins [ , ] and from dystrophin [ ] by differential solubilization [ ] or by immunoprecipitation [ ]. together, the dgc confers a structural link between laminin in the extracellular matrix, via a-dystroglycan, and the cytoskele- ton, via dystrophin, and is believed to protect muscle cells from contraction-induced damage [ , ]. the dgc has been implicated in several forms of mus- cular dystrophy. in duchenne muscular dystrophy (dmd), mutations in the dystrophin gene result in a severe dys- trophic phenotype [ , ], whereas in becker muscular dys- trophy, dystrophin mutations result in a dysfunctional dystrophin and are associated with a milder phenotype [ ]. severe and mild forms of congenital muscular dystro- phy have recently been characterized by mutations in the laminin a -chain gene [ – ]. limb-girdle muscular dystrophies (lgmds) represent a clinically and genetically heterogeneous group of diseases which are characterized by progressive weakness of the pelvic and shoulder girdle muscles [ – ]. the genes neuromuscular disorders ( ) – - / /$ .  elsevier science b.v. all rights reserved p i i s - ( ) - * corresponding author. tel.:+ ; fax: + ; e-mail: kevin-campbell@uiowa.edu these authors contributed equally to this work. responsible for lgmd c–f have been identified and encode for g-sarcoglycan [ ], a-sarcoglycan [ ], b-sarco- glycan [ , ], and d-sarcoglycan [ , ], respectively. in lgmd c–f caused by sarcoglycan mutations, a mutation in one of the sarcoglycan proteins leads to the concomitant loss or reduction of the other sarcoglycans at the sarco- lemma [ , – ]. to facilitate the analysis of lgmd e patients and to characterize the b-sarcoglycan gene, we performed an extensive analysis of kb of genomic sequences overlap- ping the b-sarcoglycan gene on chromosome q . using various predictive algorithms, we analyzed the entire geno- mic sequence and detected two putative exons that could be part of tissue specific isoforms of b-sarcoglycan. we have also found several additional polyadenylation sites that may account for the two transcripts of . and . kb which have not been yet characterized. a second lgmd e mutation was identified in an amish family with two affected sibs carrying the common haplotype [ ] at the heterozygous state. this second mutation was also present in the homo- zygous state in patients belonging to another family with probable amish origins. in addition, we screened for a- sarcoglycan deficiency cases diagnosed with myopathy, primarily characterized with normal immunohistochemical findings for dystrophin. we identified sporadic cases with these criteria and screened them for b-sarcoglycan mutations. a novel missense mutation i f was identified in the heterozygous state in the b-sarcoglycan gene of one of these patients. of particular interest, we demonstrated with immunofluorescence analysis, a reduction in the expression level of both the sarcoglycan complex and of a-dystrogly- can at the sarcolemma of one individual carrying a homo- zygous b-sarcoglycan t r mutation. . materials and methods . . subcloning and sequencing of cosmid cos dna of cosmid cos (cloning vector, supercos ) was sonically sheared and subcloned into a m vector. insert dna was amplified by pcr under standard conditions using ml of overnight culture in a ml total volume containing the m forward and m reverse primers. after a min hot start at °c, units of taq polymerase (perkin-elmer) were added, followed by cycles of s denaturation at °c, s annealing at °c, and s extension at °c. the pcr amplification products were analyzed by agarose gel electrophoresis and only those dis- playing an insert size higher than bp were sequenced on one strand of dna for sizes , bp or on both dna strands for size . bp. . . . ecori/noti restriction map of cos restriction fragments of cos obtained upon ecori and noti digestion were separated by agarose gel electrophor- esis and capillary transferred to (amersham) nylon mem- brane and hybridized with t and t primers for mapping. . . . sequence analysis the sequences were analyzed and alignments performed using xbap software of the staden package (version . ). gaps between sequence contigs were filled by walk- ing with internal primers and by sequencing both ends of several ecori-ecori, noti-noti and ecori-noti restriction fragments. the total consensus sequence was scanned using the following software programs: grail a and xgrail a (version . b); fexh, hexon, fgeneh, tssg, tssw of the bcm gene finder package (version . . . ); and promoter scan program (version . ). accession number embl# y . . . immunofluorescence analysis amish patients were ascertained through the henry ford hospital, at the department of medical genetics, detroit, mi, usa. skeletal muscle tissue from lgmd patients were obtained from diagnostic biopsies. human control muscle was received from patients undergoing orthopedic surgery not related to neuromuscular disorders. antibody staining on mm transverse cryosections was performed at room temperature. sections were treated with ab blocking solu- tions (vector), blocked with % bovine serum albumin (bsa) in phosphate-buffered saline (pbs) for min, and then incubated with a dilution of the primary antibody for min. antibodies against the following components of the dgc were tested: dystrophin, laminin a -chain, a-, b-, g-, d-sarcoglycan, and a- and b-dystroglycan. sections were then treated and analyzed as previously described [ ]. monoclonal antibodies via against dystrophin and ivd against a-sarcoglycan were previously characterized [ , ]. monoclonal antibody b against b-sarcoglycan and b against g-sarcoglycan were produced in collaboration with dr. louise anderson (muscular dystrophy research labs, newcastle general hospital, newcastle upon tyne, ne be, uk). monoclonal antibody d against b-dys- troglycan was kindly provided by dr. louise anderson. an affinity-purified d-sarcoglycan antibody (rabbit ) was produced against the n-terminal sequence (mmpqe- qythhrstmpgm) of human d-sarcoglycan. this syn- thetic peptide (research genetics) with an additional cysteine at the n terminus was conjugated with the n-term- inal cysteine of keyhole limpet hemocyanin (pierce) using m-maleimidobenzoic acid-n-hydroxysuccinimide ester (pierce), mixed with freund’s adjuvant (sigma) and injected into rabbit . polyclonal antibody against the peptide was affinity purified from crude sera using bsa- conjugated peptide as described previously [ ]. antibodies against different components of the dystrophin-glycoprotein complex were produced in a goat (goat ) using the pur- ified rabbit skeletal muscle dgc [ ] as previously described [ ]. a specific antibody against a-dystroglycan was affi- f. duclos et al. / neuromuscular disorders ( ) – nity-purified from goat antiserum using immobilon p (millipore) strips of a-dystroglycan fusion protein-d [ ]. monoclonal anti-human laminin a chain antibody (clone h ) was purchased from chemicon. . . mutation screening of b-sarcoglycan gene for b-sarcoglycan cdna fragments were amplified fol- lowing reverse-transcription from total rna prepared from biceps brachii muscle biopsies of patients [ ]. primer sequences used for these experiments are reported in table . the rt-pcr products were directly sequenced and mutations confirmed by allele specific oligonucleotide hybridization or restriction digest using hinf due to the r c mutation leading to the loss of a restriction site for this enzyme. exons - of the b-sarcoglycan gene including flanking intronic sequences were amplified from patients’ dna and sequenced. the absence of variants in control chromosomes from the normal population was tested on dna from the ceph reference families. . results . . b-sarcoglycan gene: exon-intron structure and genomic sequence to facilitate the mutation search and the identification of regulatory domains in the b-sarcoglycan gene, we have determined its genomic sequence and structure. we had table primers used for rt-pcr and genomic analysis of b-sarcoglycan gene/fragment sequences of primers length (bp) annealing t°c exon – bf : gga.cag.tcg.ggc.ggg.ga (rtpcr) br : ctt. tgt.tgt.ccc.ttg ctg.a exon – bf : agt.tcc.aat.ggt.cct.gta.aag.a (rtpcr) br : tta.ggc.tct.ctg.aga.aga.tt exon bf : agt.tcc.aat.ggt.cct.gta.aag.a br : aat.aaa.gtc.tat.gat.ttc.act.ata exon bf : ggc.cta.cca.aca.gcg.gac br : aat.atc.tct.ttc.caa.taa.tca.act.ag exon bf : ttc.agg.aat.ttg.ttt.gca.gtc.t br : act.cta.gag.aat.aat.tct.ctc exon bf : tct.gat.aac.aat.tct.cac.att.aat br : tat.gga.ttt.atg.tac.cca.aga.a exon bf : cct.cat.gga.ggt.aaa.taa.atc br : aag.tca.agt.caa.gat.ata.aa primers designed to amplify b-sarcoglycan cdna or b-sarcoglycan exons are indicated with the annealing temperature used for pcr amplification of the corresponding fragment. positions are indicated according to the sequence reported for b-sarcoglycan cdna (genbank u ). table analyses of the human b-sarcoglycan genomic sequence splice acceptor score (%)a exon (size, bp) exon~position splice donor score (%)a intron (size, bp) power of prediction algorithmsc grail a fexh hexon exon-intron organization of the b-sarcoglycan gene corresponding to the cdna ( ) – aacaggtctgt . ( ) med good – tatttcatttttagcaaag . ( ) – taatagtgagt . ( ) exc exc exc gtgttcttttcagataac . ( ) – agcctgtaagt . ( ) exc med med tgttttttttgaagattgt . ( ) – aaagggtatga . ( ) good – – ttttaaattttcagattac . ( ) – aggcggtgagt . ( ) exc exc exc tattctcctaataggaaaa . ( ) – exc good – additional predicted exonsb actgtattgtttagggaat . a ( ) – tacaggtgcgt . – – good good tttcctttttccaggcatt . b ( ) – caagggtgagt . – exc exc exc ~ position corresponds to the first nucleotide on the sense strand of the sequenced cosmid cos (accession # y ). aa score expressing adherence to the consensus was calculated for each size according to shapiro and senapathy [ ]. bonly those exons showing at least one excellent or two good scores are listed. cmed, good, exc and – refer to mediocre, good, excellent and no detected scores, respectively. the values given by bcm were arbitrarily translated into qualitative scores as follows, exc ≥ ; . . good . ; med ≤ . . the scores listed for grail a correspond to the best score obtained using either grail a and xgrail a (version . b). f. duclos et al. / neuromuscular disorders ( ) – previously isolated a cosmid (cos ) for the b-sarcoglycan gene [ ]. the cos insert (nucleotides – ; acces- sion embl# y ) was entirely sequenced and it will be referred to hereafter as the sense strand. the consensus sequence has been determined with an average × coverage value (minimal local value ×). the genomic organization of the b-sarcoglycan gene was determined from comparison of the published cdna sequences [ , ] with the genomic sequence using several prediction algorithms. a comparison of the relative power of the latter demonstrated the benefits that can be obtained in the absence of prior exon informa- tion by the use of all existing algorithms: individually, none of these programs were able to identify all six exons. since the ′ stretch of the cdna published by bönnemann et al. [ ] extended upstream of that of lim et al. [ ], the former was taken into account. furthermore, local discrepancies in the translated parts were easily ascertained by genomic sequencing ( × coverage in these regions). the human b-sarcoglycan gene (table and fig. ) is organized into exons, which range in size from to bp, separated by introns (from to bp). all donor and acceptor splicing sequences display the gt/ag consen- sus. the translation start codon is the atg at position , and the stop codon at position is followed by several putative polyadenylation sites (fig. ) which could account for several transcript isoforms. the possible biological roles of these isoforms either in regulating the stability or addres- sing of the mrna [ ] remain to be elucidated. analysis of the public domain expressed sequence tag (est) databases and of the northern blot expression patterns [ ] bring further credence to the biological relevance of these alter- native polyadenylation sites. indeed, we reported the pre- fig. . exon-intron structure of the b-sarcoglycan gene. ecori restriction map of cos dna was determined. size in bases of ecori fragment is indicated at the top of the cosmid dna box. below is the exon-intron organization of the b-sarcoglycan gene. the genomic structure was inferred from comparison with the published cdna sequences [ , ] and upon analyses of the genomic sequence with several algorithms. the human b-sarcoglycan gene is organized in exons (none of the programs identified the six) whose size range from to bp, separated by introns (from to bp). fig. . analysis of the ′ untranslated region of the b-sarcoglycan gene. the top lane represents the cos dna region corresponding to the ′-utr of the b- sarcoglycan gene. the scale is in kilobase. the different putative polyadenylation sites aataaa, the stop codon in exon and predicted exon b are indicated with their position. the arrows represent ests and their orientation with the name of the cdna clones from which they were obtained. the position of the . and . kb transcripts detected by northern blot [ ] are shown. f. duclos et al. / neuromuscular disorders ( ) – sence of mrnas of . , . and . kb, respectively [ ]. the . and . kb molecules could result from the use of these alternative polyadenylation sites (fig. ), whereas the . kb transcript still remains unaccounted for. the latter may correspond to the ′ end est of the cdna clones and (see fig. ), although no polyadenyla- tion site was identified in the corresponding genomic sequence. use of the promoter scan algorithm predicted a promo- ter-like structure (from nucleotide to ; score . ) upstream of the gene, containing a gc box (position − with respect to the initiator atg), and spanning a bp long cpg rich island ( %) covering exon and part of intron . these deductions were in agreement with the pre- dictions of the tssw program of bcm gene finder (human genome center, baylor college of medicine, houston, tx, usa). three prediction algorithms, grail a, fexh bcm and hexon bcm were applied to the genomic dna sequences; table shows that none of them detected all known exons with at least a good score, and that several exons were not detected by one or two of these algorithms. in addition, these programs also revealed additional poten- tial exons, two of which had good or excellent scores, respectively. exon a was ranked good by two algorithms and mapped between exon and exon and would lead, if authentic, to another isoform of this gene. however, its location within an alu area and its content in repetitive dna diminished the likelihood that it is translated. in con- trast, exon b was scored excellent by the three algorithms. this bp long exon b lies downstream of the recognized b-sarcoglycan cdna sequence on the same strand (fig. ). it could therefore correspond to an as yet undocumented isoform. alternatively, it could be part of another gene, although no promoter or initiating atg was significantly identified in silicio. to test these hypotheses, northern blot as well as rt-pcr experiments on human skeletal, cardiac and brain mrna were conducted. all tests performed to visualize the corresponding transcripts were negative (data not shown). either the corresponding transcript was present at too low levels to be detected, or its restricted spatio-tem- poral pattern of expression prevented its detection. whereas no homologous amino-acid sequence was recognized upon screening public domain (tigr, genbank, est, c.elegans and yeast) databases, two regions showed amino-acid homology with sequences internal to the b-sarcoglycan exon . the most significant was the rxxnilf motif which is also found in mycoplasma (accession: u ); escherichia coli (accession: ae ), and chloroplast s ribosomal protein (e.g. accession: p ). although this homology does not imply an actual functional role for exon b, it seems likely that the latter is somehow related to the b-sarcoglycan gene. given the difficulty to assess whether exon b is an authentic exon, it may be worthwhile to investigate the corresponding sequence in other mam- mals. additionally, one exon was scored excellent with pro- grams fexh, hexon and fgeneh of bcm gene finder and good with grail a (reverse position: – , data not shown), on the opposite strand of the cosmid insert. this putative ‘orphan’ exon showed no hit with any pub- lished sequence and hence was not integrated onto the map (table ). . . b-sarcoglycan gene mutations in the lgmd e amish community in agreement with the common haplotype displayed by the first families analyzed, a missense mutation (t r) common to all southern indiana amish patients tested has been found and was associated with a mild form of muscular dystrophy [ ]. no evidence of cardiomyopathy has been found, and joint contractures have been infrequent in these patients. since then, two sibs from an amish family exhibit- ing heterozygosity for the common haplotype with chromo- some q markers and a second distinct haplotype were identified among the community of southern indiana. sub- sequently, mutation screening of these patients revealed in addition to the common t r mutation, a c to t transition corresponding to the substitution of arginine by a cysteine. these two patients ( a and b) had a late onset of the disease (table ), characterized by muscle weakness and enlargement of calves. at age and years, respec- tively, both individuals were still able to carry on work requiring physical exertion and were still able to walk. five other children in the family have not been found to be affected. it is unclear whether the unaffected members of this family are carriers of mutations (i.e. a penetrance issue) or just unaffected genetically (i.e. carrying one or two copies of the normal allele). surprisingly, two other familial cases not belonging to the amish community car- ried this mutation in the homozygous state (table , cases a and b). no relationship was ascertained between the father and mother, however amish names were reported in their ancestry. patient a and her affected sister ( b) presented leg cramps and weakness after exercise at age and years, respectively. calf hypertrophy was noted in early childhood in both patients. patient a was still able to ambulate at age years whereas her affected sister has used a walker since age years. they have two unaffected brothers. the status of the r c mutation was analyzed by restriction digest of pcr amplified genomic dna using hinf , which is ablated as a result of the mutation. analysis of chromosomes from the ceph reference families confirmed that the r c was a mutation and not a poly- morphism. . . analysis of the expression of the components of the dgc since the original characterization of the b-sarcoglycan gene defect in lgmd e amish families from southern indiana [ ], two additional sarcoglycan genes have been f. duclos et al. / neuromuscular disorders ( ) – cloned and new antibodies developed against these mole- cules. we examined the expression of the sarcoglycan and dystroglycan complexes, dystrophin, and the a chain of laminin in a biopsy from an amish patient homozygote for the t r mutation. this analysis extended previous examinations [ , , ] by using a monoclonal antibody against g-, and d-sarcoglycan. in addition, a newly affinity purified anti-a-dystroglycan antibody was developed. the antibody against a-dystroglycan allowed us for the first time to specifically look at the expression of one of the key proteins of the dgc on skeletal muscle cryosections of lgmd patients. the expression level of dystrophin, b-dys- troglycan, and the laminin a chain in the amish patient was comparable to the levels found in normal human control muscle. on the other hand, expression of the sarcoglycan complex was strongly reduced in the patient’s biopsy com- pared to control muscle. all sarcoglycan proteins showed a similar level of reduction at the sarcolemma. in addition to the secondary loss of a-, g-, and d-sarcoglycan, we also found a reduced expression of the a-subunit of the dystro- glycan complex. this unexpected and interesting finding may unveil for the first time a direct or indirect link between the sarcoglycan complex and a-dystroglycan. . . patient screening for b-sarcoglycan gene mutations in order to identify additional patients carrying a primary b-sarcoglycan deficiency, we examined a total of new independent cases of autosomal recessive myopathy char- acterized by normal immunostaining of dystrophin and markedly elevated serum creatine kinase level. we primar- ily screened these patients by immunostaining of skeletal muscle cryosections for a-sarcoglycan deficiency and iden- tified new cases. all these sporadic cases originate in the usa. in all patients, the serum creatine kinase levels were elevated but the clinical pattern was of variable severity, ranging from severe childhood to late onset progressive muscular dystrophy (data not shown). there were no intel- lectual impairments, nor cardiomyopathy. skeletal muscle sections of these patients were further analyzed by immuno- fluorescence staining for the other components of the dgc and a concomitant loss or a drastic reduction of b, g and d- sarcoglycan was observed (data not shown). the immuno- fluorescence data did not indicate the primary defect caus- ing the observed phenotype. all sporadic lgmd cases were analyzed for mutations in the b-sarcoglycan gene as well as in the other known sar- coglycan genes (data not shown), due to the absence of linkage data. for one of the sporadic cases (t ), only one mutant allele corresponding to the substitution of an isoleucine by a phenylalanine at position (i f), was found in the b-sarcoglycan gene although all coding sequences were investigated. this patient developed a prox- imal muscle weakness as well as progressive scoliosis with onset in early childhood. at age years, he was confined to a wheelchair and presented more severe clinical symptoms as compared to the amish patients. in this isolated case, the second mutation could be in the unexplored untranslated region or possibly in one of the alternative exons. allele specific hybridization with either a control oligonucleotide or an oligonucleotide including this latter mutation, revealed that this substitution was not found in inde- pendent chromosomes from ceph reference families (data not shown) and that the sporadic lgmd patient was thus in all likelihood a compound heterozygote for the i f and a second, as yet unidentified mutation. we also determined the primary sarcoglycan gene defect in out of remaining isolated cases: four patients have a mutated a-sarcoglycan gene and three have a mutated g-sarcoglycan gene (data not shown). . discussion in order to facilitate further gene expression analysis and the identification of functional regulatory elements, we per- formed sequence analysis of a kb genomic fragment containing the b-sarcoglycan gene. interestingly, several putative additional exons were predicted by the computer programs xgrail and/or bcm in the ′-utr of the b-sar- coglycan gene, which may be part of different tissue-speci- fic transcripts. one gc box was detected at position using promoter scan, and several polyadenylation sites were found (table and fig. ). the relationship between these putative polyadenylation sites and the different transcripts detected by northern hybridizations at . , . , . – . kb requires further probing with specific ′-utr fragments to be ascertained. the tissue-specificity of expression should also be determined. although experiments to confirm the existence of the two predicted exons gave negative results it was not possible to conclude whether these two exons form part of real mrna. this study constitutes a good example of what to expect from the human genome sequen- cing project, since it illustrates how far one can get inferring relevant genomic information just from sequencing data. on the one hand it highlights some of the limits of the current prediction algorithms, and on the other hand it points also to the presence of potential false positives. although a common chromosome q –q haplotype is displayed by most of the southern indiana amish lgmd e families around the disease locus [ ], two patients from one family were compound heterozygotes in this region. mutation screening revealed that these patients carried the common t r mutation [ ] together with another mis- sense mutation, r c. in addition, we identified the same mutation (r c) in an unrelated familial case (table , amish ) who does not belong to the southern indiana amish community, but whose ancestors have amish names on both paternal and maternal sides. the presence of a second mutation in the amish community was unex- pected, although the presence of multiple mutations in a small ethnic community has already been observed before. f. duclos et al. / neuromuscular disorders ( ) – the presence of several mutations in the calpain gene among lgmd a patients from la réunion island was puz- zling [ ]. a hypothesis that could account for this and other related observations has been reported [ – ]. the identi- fication of a second mutation in another family of probable amish origin suggests a relatively ancient occurrence of this mutation, in contrast to the possibility of a high mutation rate coupled with a recent occurrence of these mutations in this isolate as proposed by zlotogora et al. [ ] for hurler syndrome, metachromatic leukodystrophy and lgmd a. in such highly inbred populations, one mutation associated with a founder haplotype may become frequent by result of a genetic drift. this frequent haplotype/mutation can now uncover other rare events leading possibly to these multiple disease alleles we observe now in the amish isolate from southern indiana. these homozygotes (r c; codon cgc mutated to tgc) displayed a mild muscular dystrophy phe- notype (table , amish a and b). surprisingly, bönnemann et al. [ ] reported a r p mutation (codon cgc changed to ccc) in three homozygous sibs from a brazilian family associated with a severe form of lgmd. secondary structure prediction analysis suggested that the mutation r c did not affect the protein structure whereas the proline at position did. these findings suggest that the arginine in b-sarcoglycan may constitute a critical site of interaction in the complex. these homozygote cases may be helpful in unraveling some genotype/phenotype correlations in lgmd e and in providing clues on the sarcoglycan/ dystroglycan interaction. we have also analyzed patients with normal dystro- phin and identified sporadic cases with a sarcoglycan complex deficiency. these cases represent % of the mus- cular dystrophy patients tested and this correlates with the ratio obtained by a recent study [ ]. these patients were investigated for sarcoglycan mutations in the a-, b-, g- and d-sarcoglycan genes and a sarcoglycan molecular defect was revealed in out of (data not shown). these two latter may carry mutations in another yet unidentified sar- coglycan gene. we identified one lgmd patient (table , case t ) carrying on one chromosome a novel missense mutation (i f) in the b-sarcoglycan gene, the second allele remaining unidentified despite the fact that all coding sequences were investigated. interestingly, many of the t r and r c amish cases are associated with a milder course of the disease in contrast to the severe phenotype of lgmd e patients reported [ , ] and one lgmd e case (t ) reported in this study. all mutations mapped to the extracellular domain of the protein. the b-sarcoglycan gene mutation r c (cgc to tgc) abolished a cpg site. the functional importance of the region in which the mutation occurred has not yet been defined. on the other hand, mis- sense mutations can cause abnormal protein processing or stability, as was seen in the majority of cases with lgmd [ – ]. in lgmd d, a broad spectrum of missense muta- tions in the a-sarcoglycan gene have been reported which affected protein targeting to the sarcolemma and resulted in muscular dystrophy of varying degrees of severity [ , ]. this phenomenon has already been observed with other transmembrane proteins, including cftr [ ]. the close association of the components of the sarco- glycan complex [ , ] and their concomitant loss in lgmd c–f have been reported. in the bio . cardio- myopathic hamster, an animal model of d-sarcoglycan defi- cient lgmd [ ], a secondary reduction of a-dystroglycan expression had been reported [ ]. to test whether the sta- bility of a-dystroglycan is also affected in human sarcogly- can deficient skeletal muscle, we used a new anti-a- dystroglycan antibody and demonstrated for the first time that a missense mutation in the b-sarcoglycan gene (t r) can modify a-dystroglycan expression (fig. ). this result suggests a possible link between the sarcoglycan complex and the peripheral membrane protein a-dystroglycan. this link could either be mediated through direct interaction or via an intermediate. the maintenance of b-dystroglycan expression in the sarcolemma of this patient supports the hypothesis that the sarcoglycan complex is required for high table clinical and biochemical data of patients with lgmd case no. age (years) sex onset (years) ck value at (age) calf muscle hypertrophy activity b-sarcoglycan gene defect amish f presymptomatic ( ) + ambulant t r amish a f ( ) + ambulant t r–r c amish b f ? ? ambulant t r–r c amish a f ? + walker r c amish b f . ( ) + ambulant r c t m ? ? ? wheelchair i f–? creatine kinase levels (ck) measured at (age) of the patients are indicated. normal ck values are estimated to be lower than . severity of the condition of the patients at present is indicated by their activity. no cardiomyopathy nor intellectual impairment have been reported in these cases. the second allele mutation in the sporadic case t was not determined despite the analysis of the whole coding sequence of the b-sarcoglycan gene. amish refers to one patient from one of the families originally characterized with the common mutation t r for which we had a biopsy available. amish refers to the two lgmd e patients from an amish family heterozygote for the common t r and the r c mutations. amish refers to two affected sibs from a lgmd e family of probable amish descent which was identified independently, and shown to carry an homozygous r c mutation. f. duclos et al. / neuromuscular disorders ( ) – affinity interaction of a-dystroglycan with the sarcolemma. further investigation of muscle biopsies from sarcoglycan deficient patients are currently being performed. the genetic and clinical heterogeneity of the limb-girdle muscular dystrophies has broad implications for the diag- nosis and management of persons afflicted with this disease. because defects in several distinct proteins can cause simi- lar clinical symptoms and biochemical manifestations, a definitive diagnosis can so far only be achieved through genetic testing of all candidate genes. analysis of additional cases will provide more insights in genotype/phenotype cor- relations and key domains of the proteins. acknowledgements we are grateful to delphine samson, corinne cruaud, colleen campbell, chris snyder, cindy leveille and herve crespeau for their help. we are grateful to all the clinicians who provided the patient samples for our study. v.s. is supported by a grant from the deutsche forschungs- gemeinschaft. c.e.j., g.l.f., j.-c.k., f.p. and k.p.c. are supported by three grants from afm (association francaise contre les myopathies). l.e.l. is supported by a grant from the iowa affiliate of the american heart association. this work was also supported by the muscular dystrophy asso- ciation. k.p.c. is an investigator of the howard hughes medical institute. references [ ] campbell kp, kahl sd. association of dystrophin and an integral membrane glycoprotein. nature ; : – . [ ] ervasti jm, ohlendieck k, kahl sd. et al. deficiency of a glycopro- tein component of the dystrophin complex in dystrophic muscle. nature ; : – . [ ] yoshida m, ozawa e. glycoprotein complex anchoring dystrophin to sarcolemma. j biochem ; : – . [ ] ervasti jm, kahl sd, campbell kp. purification of dystrophin from skeletal muscle. j biol chem ; : – . fig. . immunofluorescence analysis of dgc components in human control and amish lgmd e muscle. the lgmd e patient (patient) carried a homozygous t r mutation. seven micrometer cryosections were stained with dystrophin (dys), a-dystroglycan (a-dg), b-dystroglycan (b-dg), laminin a (lam-a ), a-, b-, g-, and d-sarcoglycan antibodies (a-sg, b-sg, g-sg and d-sg) as described in section . the entire sarcoglycan complex is greatly reduced at the sarcolemma of skeletal muscle from the b-sarcoglycan mutated patient compared to control. additionally, the sarcolemmal staining for a- dystroglycan is reduced. bar, mm. f. duclos et al. / neuromuscular disorders ( ) – [ ] ervasti jm, campbell kp. membrane organization of the dystrophin glycoprotein complex. cell ; : – . 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[ ] jorgensen ao, arnold w, shen ac. et al. identification of novel proteins unique to either transverse tubules (ts ) or the sarco- lemma (sl ) in rabbit skeletal muscle. j cell biol ; : – . [ ] shapiro mb, senapathy p. rna splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. nucleic acids res ; : – . [ ] kislauskis eh, zhu x, singer rh. sequences responsible for intra- cellular localization of beta-actin messenger rna also affect cell phenotype. j cell biol ; : – . [ ] richard i, broux o, allamand v. et al. mutations in the proteolytic enzyme calpain , cause limb girdle muscular dystrophy type a. cell ; : – . [ ] allamand v, broux o, bourg n, et al. genetic heterogeneity of autosomal recessive limb-girdle muscular dystrophy in a genetic isolate (amish) and evidence for a new locus. hum mol genet ; ( ): – . [ ] beckmann js. the réunion paradox and the digenic model. am j hum genet ; : – . 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[ ] roberds sl, ervasti jm, anderson rd. et al. disruption of the dystrophin-glycoprotein complex in the cardiomyopathic hamster. j biol chem ; : – . f. duclos et al. / neuromuscular disorders ( ) – wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ pnas correction .. corrections evolution correction for “the abo blood group is a trans-species poly- morphism in primates,” by laure ségurel, emma e. thompson, timothée flutre, jessica lovstad, aarti venkat, susan w. margulis, jill moyse, steve ross, kathryn gamble, guy sella, carole ober, and molly przeworski, which appeared in issue , november , , of proc natl acad sci usa ( : – ; first published october , ; . /pnas. ). the authors note that fig. appeared incorrectly. some abo polymorphism statuses have been corrected. the corrected fig- ure and its legend appear below. www.pnas.org/cgi/doi/ . /pnas. fig. . the phylogenetic distribution of abo phenotypes and genotypes. shown is a phylogenetic tree of primate species, with a summary of phenotypic/ genotypic information given in the first column, and the genetic basis for the a versus b phenotype provided in the second column (functionally important codons at positions and are in uppercase letters). see dataset s for the source of information about phenotypes/genotypes. only species with available divergence times are represented here ( of ). the phylogenetic tree is drawn to scale, with divergence times (on the x axis) in millions of years taken from ref. . owm, old world monkeys; nwm, new world monkeys. under a model of convergent evolution, these data suggest that a is the ancestral allele, and a turnover (e.g., a neutral substitution) occurred on the branch leading to old world monkeys. if instead, b were ancestral, all old world monkeys would have had to serendipitously converge from atg to ttg to encode a leucine, whereas all new world monkeys and hominoids would have had to converge to the ctg codon. www.pnas.org pnas | april , | vol. | no. | – c o r r ec ti o n s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , www.pnas.org/cgi/doi/ . /pnas. evolution; earth, atmospheric, and planetary sciences correction for “mass extinction of lizards and snakes at the cretaceous–paleogene boundary,” by nicholas r. longrich, bhart-anjan s. bhullar, and jacques a. gauthier, which appeared in issue , december , , of proc natl acad sci usa ( : – ; first published december , ; . / pnas. ). the authors note the following: “the genus name lamiasaurus, which we proposed for a new lizard from the late cretaceous of wyoming, is preoccupied by lamiasaurus watson ( ), a tapinocephalid therapsid from the permian of africa. we therefore propose the name lamiasaura for the wyoming lizard; its type species is lamiasaura ferox, also proposed in our paper. fur- thermore, holotypes were figured for this and other newly pro- posed species but not explicitly identified in the text. we designate types and provide diagnoses as follows. cemeterius monstrosus, holotype: united states national museum . diagnosis: large stem varanoid characterized by a deep, massive jaw, teeth short, unserrated, robust, and labiolingually expanded. cerberophis robustus, holotype: university of california museum of paleon- tology . diagnosis: medium-sized (∼ m) alethinophidian, trunk vertebrae with broad, flat ventral surface, hypertrophied synapophyses, large, massive prezygapophyses with rudimentary prezygapophyseal processes and anterior ridges; neural arch with dorsolateral ridges, moderately tall neural spine. lamiasaura ferox, holotype: university of wyoming a, left dentary with four teeth. diagnosis: dentary straight, tapered in lateral view; teeth widely spaced, crowns weakly recurved, crowns with bottleneck constriction between the base and apex, low mesial and distal cusps, and ridged lingual surface. lonchisaurus trichurus, holotype: american museum of natural history . diagnosis: dentary long, low, and weakly bowed in lateral view; tooth crowns robust, weakly recurved, with weakly pointed crowns; tooth bases wider la- bially than lingually, tooth replacement reduced, coronoid overlaps dentary laterally. obamadon gracilis, holotype: university of cal- ifornia museum of paleontology . diagnosis: small poly- glyphanodontian characterized by the following combination of characters: dentary slender, symphysis weakly developed, tooth im- plantation subpleurodont, teeth lack basal expansion, tooth crowns with a tall central cusp separated from accessory cusps by deep lin- gual grooves. pariguana lancensis, holotype: american museum of natural history . diagnosis: small iguanid; teeth tall, slender, with tapering crowns and weak accessory cusps; coronoid extended onto lateral surface of jaw below last tooth, meckelian groove con- stricted suddenly ahead of anterior inferior alveolar foramen. so- cognathus brachyodon, holotype: yale peabody museum (princeton university collection) . diagnosis: socognathus with posterior teeth having strongly swollen, weakly tricuspid crowns.” “this correction formally validates the taxa proposed in our paper; thus, those taxa should be attributed to this note and accordingly dated as march , .” “we thank christian kammerer and christopher taylor for bringing these two issues to our attention.” . watson dms ( ) the deinocephalia, an order of mammal-like reptiles. proceedings of the zoological society of london – . www.pnas.org/cgi/doi/ . /pnas. neuroscience, psychological and cognitive sciences correction for “sensory adaptation as optimal resource allocation,” by sergei gepshtein, luis a. lesmes, and thomas d. albright, which appeared in issue , march , , of proc natl acad sci usa ( : – ; first published february , ; . /pnas. ). the authors note that, due to a printer’s error, some text ap- peared incorrectly. on page , right column, third full paragraph, line “in fig. a” should instead appear as “in fig. b”. on page , right column, second full paragraph, line “in fig. a” should instead appear as “in fig. a”. on page , left column, fourth full paragraph, lines – “in fig. a, sensitivity changes are plotted for two speeds (fig. a, upper) and for the entire domain of the sensitivity function (fig. a, lower)” should instead appear as “in fig. b, sensitivity changes are plotted for two speeds (fig. b, upper) and for the entire domain of the sensitivity function (fig. b, lower)”. on page , left column, first full paragraph, line “cortical visual area middle temporal (mt)” should instead appear as “middle temporal (mt) cortical visual area”. on page , right column, second full paragraph, lines – “as illustrated in fig. a for experiment and fig. a for ex- periment ” should instead appear as “as illustrated in fig. a for experiment and fig. a for experiment ”. www.pnas.org/cgi/doi/ . /pnas. chemistry correction for “enhanced surface hydrophobicity by coupling of surface polarity and topography,” by nicolas giovambattista, pablo g. debenedetti and peter j. rossky, which appeared in issue , september , , of proc natl acad sci usa ( : – ; first published august , ; . / pnas. ). the authors note the following: “recalculations confirm some of the conclusions reported in our paper but do not confirm others. specifically, we confirm that (i) polar surfaces can be hydrophobic, (ii) capillary evaporation can occur when water is confined between “polar hydrophobic” nanoscale surfaces, and (iii) inversion of surface polarity can alter the surface hydro- phobicity (i.e., water contact angle). however, because of the sensitivity of the results to the value of the ewald wave vector cutoff parameter mmax for the model surface studied, the re- ported observation that adding polarity to an apolar silica-based surface can enhance hydrophobicity beyond that of the original apolar surface is not confirmed. “we thank zhonghan hu for bringing to our attention dis- crepancies between his computer simulation results and some of our calculations reported in the above-cited pnas paper; richard c. remsing and john d. weeks for generously sharing their results with us; and sapna sarupria, amish patel, and sumit sharma for useful discussions. additional details regarding the recalculations are available from the authors upon request.” www.pnas.org/cgi/doi/ . /pnas. | www.pnas.org d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , www.pnas.org/cgi/doi/ . /pnas. www.pnas.org/cgi/doi/ . /pnas. www.pnas.org/cgi/doi/ . /pnas. untitled original research an update to the raymond–roy occlusion classification of intracranial aneurysms treated with coil embolization justin r mascitelli, henry moyle, eric k oermann, maritsa f polykarpou, aanand a patel, amish h doshi, yakov gologorsky, joshua b bederson, aman b patel department of neurosurgery, icahn school of medicine at mount sinai, new york, new york, usa department of radiology, icahn school of medicine at mount sinai, new york, new york, usa correspondence to dr aman b patel, department of neurosurgery, mount sinai medical center, one gustave l levy place, annenberg building , box , new york, ny , usa; aman.patel@mountsinai.org received april revised april accepted may published online first june to cite: mascitelli jr, moyle h, oermann ek, et al. j neurointervent surg ; : – . abstract background the raymond–roy occlusion classification (rroc) is the standard for evaluating coiled aneurysms (class i: complete obliteration; class ii: residual neck; class iii: residual aneurysm), but not all class iii aneurysms behave the same over time. methods this is a retrospective review of patients with intracranial aneurysms treated with coil embolization. a modified raymond–roy classification (mrrc), in which class iiia designates contrast within the coil interstices and class iiib contrast along the aneurysm wall, was applied retrospectively. results class iiia aneurysms were more likely to improve to class i or ii than class iiib aneurysms ( . % vs . %, p< . ) and were also more likely than class ii to improve to class i ( . % vs . %, p< . ). class iiib aneurysms were more likely to remain incompletely occluded than class iiia aneurysms ( . % vs . %, p< . ). class iiib aneurysms were larger with wider necks while class iiia aneurysms had higher packing density. class iiib aneurysms had a higher retreatment rate ( . % vs . %, p< . ) and a trend toward higher subsequent rupture rate ( . % vs . %, p= . ). conclusions we propose the mrrc to further differentiate class iii aneurysms into those likely to progress to complete occlusion and those likely to remain incompletely occluded or to worsen. the mrrc has the potential to expand the definition of adequate coil embolization, possibly decrease procedural risk, and help endovascular neurosurgeons predict which patients need closer angiographic follow-up. these findings need to be validated in a prospective study with independent blinded angiographic grading. introduction endovascular coiling is being used increasingly for the treatment of intracranial aneurysms. the raymond–roy occlusion classification (rroc; also known as the montreal scale, modified montreal scale, or the raymond montreal scale) is a widely accepted system for evaluating aneurysm occlusion class but was not designed to predict recurrence. in this scheme, class i is defined as complete obliteration, class ii as residual neck, and class iii as residual aneurysm. it has been shown that class iii aneurysms have a higher propensity to remain incompletely occluded and potentially rebleed. – since only a proportion of class iii aneurysms remain incompletely occluded, however, it would be useful to understand the factors influ- encing progression to complete occlusion. our experience with coil embolization suggests that class iii aneurysms exhibit heterogeneous behavior, with one subset likely to occlude over time and another likely to remain patent or to grow. we propose the modified raymond–roy classification (mrrc), in which class iiia designates contrast opaci- fication within the coil interstices of a residual aneur- ysm and class iiib designates contrast opacification outside the coil interstices, along the residual aneur- ysm wall (figure ). we hypothesize that class iiia aneurysms progress to occlusion more frequently than class iiib aneurysms. methods study design this is a retrospective review of patients treated with coil embolization at a single institution. a database containing patients with intracranial aneur- ysms treated by endovascular techniques from to was examined. after excluding atypical aneur- ysms (eg, fusiform), those that were previously treated, and those that were not treated with coil embolization (eg, flow diversion), the final study population included patients with aneurysms. patients, aneurysms, and procedural characteristics subject and aneurysm characteristics were deter- mined by review of subjects’ medical charts and angiographic data. the study population was diverse, including both ruptured and unruptured aneurysms and a wide range of aneurysm sizes and locations. aneurysm size was determined both qualitatively and quantitatively. the qualitative system involved using the maximum dome diameter and is as follows: small: < mm; large: – mm; and giant: > mm. posterior communicating artery aneurysms were included in the anterior circulation. all aneurysm embolizations were performed at a single institution by two interventional neurosur- geons and one interventional neuroradiologist. a variety of coil and stent types were used as well as different degrees of procedural assistance including stand-alone, balloon-assisted, and stent-assisted coiling. editor’s choice scan to access more free content neuroimaging mascitelli jr, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n ju n e . d o w n lo a d e d fro m http://crossmark.crossref.org/dialog/?doi= . /neurintsurg- - &domain=pdf&date_stamp= - - http://www.snisonline.org/ http://jnis.bmj.com http://jnis.bmj.com/ figure modified raymond–roy classification (mrrc). class i: complete obliteration; class ii: residual neck; class iiia: residual aneurysm with contrast within coil interstices; class iiib: residual aneurysm with contrast along aneurysm wall. figure examples of three aneurysms treated with stand-alone coiling. all three aneurysms were graded as class iiia because there was contrast within the coil interstices (first column) and because coils opposed the wall of the entire aneurysm (second column). all three aneurysms progressed to occlusion at first follow-up (third column). neuroimaging mascitelli jr, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n ju n e . d o w n lo a d e d fro m http://jnis.bmj.com/ angiographic review all initial and follow-up angiograms were reviewed by either interventional neurosurgeon abp or hm and the mrrc was applied. although the angiographic review was not blinded, the initial angiogram was always evaluated before the follow-up to reduce bias. the following considerations were taken into account when applying the mrrc: ▸ all aneurysms were graded in at least two angiographic views. ▸ class iiia or iiib designation took precedence over the status of the aneurysm neck. ▸ flow stasis was not included in the mrrc. ▸ any contrast seen within the aneurysm at the end of the pro- cedure was defined as class iii, even if believed to be a result of heparinization or use of antiplatelet therapy. ▸ class iiia designation was only given if coils opposed the wall of the entire aneurysm dome on the unsubtracted view (figure ). class iiib designation was only given if there was a gap between the coils and the aneurysm wall on an unsub- tracted view (figure ). ▸ class iiib aneurysms usually had contrast opacification through a portion of the aneurysm neck with continuation along a portion of the aneurysm wall (figure ). occasionally, however, class iiib designation was used when the neck was completely occluded (eg, complete coiling of a proximal aneurysm lobule with persistent filling of a distal lobule). statistical methods the rroc was calculated for each aneurysm at each angiogram as previously discussed. pretreatment, aneurysm, and treatment variables were assessed for differences between classes iiia and iiib using the mann–whitney test or pearson χ test for con- tinuous and categorical variables respectively. kaplan–meier figure examples of three aneurysms treated with coil embolization except for the ophthalmic aneurysm (c) that was treated with stent assistance. all three aneurysms were graded as class iiib because there was contrast along the aneurysm dome wall (first column) and because there was a gap between the coils and the aneurysm wall (second column). all three aneurysms remained incompletely occluded at first follow-up (third column). the anterior communicating artery (a) and ophthalmic artery aneurysms (c) required retreatment and the posterior communicating artery aneurysm (b) reruptured and required surgical clipping. neuroimaging mascitelli jr, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n ju n e . d o w n lo a d e d fro m http://jnis.bmj.com/ curves were constructed to graphically depict time to closure or improvement, and the log rank test was used for subsequent evaluation. all reported p values are two sided with a standard α set at . . all averages are the mean average with a sd. all data management and analyses were conducted using spss v. . (ibm, armonk, new york, usa). multivariate logistic regression analysis was also performed using spss v. . . categorical variables were reclassified as indi- cator variables, and multivariable models incorporating both the rroc and mrrc were constructed and ors and p values were calculated for individual predictors within each model. results patients, aneurysms, and procedural data the majority of patients were women ( . %) and the major- ity of aneurysms were ruptured ( . %), small ( . %), and in the anterior circulation ( . %). stent and balloon assist- ance was used in . % and . % of cases, respectively. a variety of coil and stent types were used. aneurysms were treated with bare platinum coils in . % of cases (table ). compared with class iiia aneurysms, class iiib aneurysms were larger ( . vs . mm , p= . ) with wider necks ( . vs . mm, p= . ). class iiia aneurysms were treated with higher packing density (pd; . % vs . %, p< . ). otherwise, no other patient, aneurysm, or proced- ural characteristics were different between these two groups. angiographic data angiographic follow-up was available in . % of patients at . months, . % of patients at . months, and . % of patients at . months. the mrrc was determined imme- diately after the procedure and at all follow-ups (table ). class iiia aneurysms were more likely than class iiib aneur- ysms to improve to either class i ( . % vs . %, p< . ) or to improve to class ii ( . % vs . %, p< . ). class iiia aneurysms were also more likely than class ii to improve to class i ( . % vs . %, p< . ; figures and ). class iiib aneurysms were more likely than class iiia aneurysms to remain incompletely occluded ( . % vs . %, p< . ; figures and ). class iiia aneurysms were more likely to remain incompletely occluded compared with class ii and i aneurysms ( . % vs . % and . %, p< . ; table ). multivariate analysis demonstrated that class iiia and iiib status was independently predictive of progression to occlusion (p= . ) and recurrence (p< . ), respectively. in addition, multivariate analysis demonstrated that mrrc class iiib status was more predictive of recurrence than rroc class iii (or vs ). clinical data the complication rate in the class iiib group compared with the class iiia group was not statistically different ( . % vs . %, p= . ), nor were the rates of death or permanent disability secondary to the complication ( . % vs . %, p= . ). the vasospasm rate was not statistically different between class iiia and class iiib ( . % vs . %, p= . ). the percentage of patients with discharge modified rankin scale scores of – was similar in both groups ( . % vs . %, p= . ). there were more retreatments in the class iiib group ( . % vs . %, p< . ). there was a trend toward more post-treatment ruptures in the class iiib group but this did not reach statistical significance ( . % vs . %, p= . ; table ). discussion initial angiographic occlusion class is a predictor of aneurysm recurrence and rehemorrhage. – many studies have addressed aneurysm recurrence, but there is less literature examining aneurysms that progress to occlusion. in the current study, a high proportion of coiled aneurysms with residual contrast opa- cification within the coil interstices at the end of the procedure (class iiia) were found to be completely occluded or to exhibit only neck filling at follow-up. this subgroup appeared to have a favorable angiographic outcome in comparison with those aneurysms with contrast opacification outside the coil inter- stices, along the aneurysm wall (class iiib). there are a few possible explanations for the observations in this study. the prothrombotic environment that coils create are likely to have a greater effect on blood within the coil mass than outside the coil mass. coil compaction and aneurysm growth are two methods of aneurysm recurrence. blood table patient, aneurysm, and procedure data general patients: aneurysms: age: . years women: . % (n= ) men: . % (n= ) rupture status ruptured: . % (n= ) unruptured: . % (n= ) aneurysm size small: . % (n= ) large: . % (n= ) giant: . % (n= ) dome volume: . mm neck size: . mmaspect ratio: . aneurysm location anterior circulation: . % (n= ) ▸ cavernous ica: . % (n= ) ▸ ophthalmic: . % (n= ) ▸ superior hypophyseal: . % (n= ) ▸ posterior communicating: . % (n= ) ▸ anterior choroidal: . % (n= ) ▸ ica other: . % (n= ) ▸ ica bifurcation: . % (n= ) ▸ anterior communicating: . % (n= ) ▸ pericallosal: . % (n= ) ▸ aca other: . % (n= ) ▸ mca: . % (n= ) posterior circulation: . % (n= ) ▸ vertebral: . % (n= ) ▸ pica: . % (n= ) ▸ basilar trunk: . % (n= ) ▸ sca: . % (n= ) ▸ basilar tip: . % (n= ) ▸ pca: . % (n= ) procedure assistance stand-alone: . % (n= ) balloon: . % (n= ) stent: . % (n= ) coil type galaxy: . % (n= ) gdc: . % (n= ) hydrocoil: . % (n= ) matrix: . % (n= ) orbit: . % (n= ) penumbra: . % (n= ) target: . % (n= ) trufill: . % (n= ) unknown: . % (n= ) multiple coil types: . % (n= ) stent data enterprise: . % (n= ) liberty: . % (n= ) neuroform: . % (n= ) multiple stents: . % (n= ) aca, anterior cerebral artery; ica, internal carotid artery; mca, middle cerebral artery; pca, posterior cerebral artery; pica, posterior inferior cerebellar artery; sca, superior cerebellar artery. neuroimaging mascitelli jr, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n ju n e . d o w n lo a d e d fro m http://jnis.bmj.com/ along the wall of a class iiib aneurysm may promote coil com- paction and/or lead to increased wall stress causing aneurysm growth. there is evidence that the mechanism of aneurysm occlusion following coil embolization involves remodeling/ healing of the blood vessel wall along the aneurysm neck. class iiib aneurysms usually have an area in the aneurysm neck without coils that can be potentiated by the inflow jet over time and prevent neck healing. an experimental model designed to simulate class iiia and iiib aneurysms is needed to determine the exact mechanism by which these aneurysms either progress to occlusion or recur. it is not surprising that the class iiib group had larger aneur- ysms with wider necks. these two features inherently make cir- cumferential packing of the entire aneurysm with coils more difficult. it is also not surprising that pd was lower in the class iiib group, as low pd has been associated with recurrence in previous studies. – it is important to note, however, that the predictive nature of the mrrc was maintained in a multivariate model that removed the influence of aneurysm size, neck size, and pd. in addition, the multivariate model demonstrated that the mrrc was able to predict recurrence better than the stand- ard rroc model. the two groups had similar clinical courses except that the class iiib group had more retreatments and a trend toward more post-treatment ruptures, which are two sources of morbid- ity for this patient population. this is an important finding because it suggests that the mrrc has an angiographic and also a clinical impact. there have been previous efforts to augment the rroc. in , deshaies et al evaluated patients with aneurysms treated with hydrocoils. the initial rroc was class i in %, class ii in %, and class iii in %. in this paper, the patients were stratified by size and the authors looked directly at aneur- ysm recurrence and improvement in rroc class. they found table modified raymond-roy classification immediately after procedure and at follow-up a. first follow-up occlusion class ( . months) i ii iiia iiib total initial occlusion class i . % ( ) . % ( ) . % ( ) . % ( ) ii . % ( ) . % ( ) . % ( ) . % ( ) iiia . % ( ) . % ( ) . % ( ) . % ( ) iiib . % ( ) . % ( ) . % ( ) . % ( ) total b. second follow-up occlusion class ( . months) i ii iiia iiib total initial occlusion class i . % ( ) . % ( ) . % ( ) . % ( ) ii . % ( ) . % ( ) . % ( ) . % ( ) iiia . % ( ) . % ( ) . % ( ) . % ( ) iiib . % ( ) . % ( ) . % ( ) . % ( ) total figure kaplan–meier curve demonstrating progression to occlusion over time for class iiia and iiib aneurysms. figure kaplan–meier curve demonstrating recurrence over time for class iiia and iiib aneurysms. neuroimaging mascitelli jr, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n ju n e . d o w n lo a d e d fro m http://jnis.bmj.com/ that three large and three very large aneurysms initially desig- nated as class iii ultimately improved to class i. in the discus- sion, the authors claimed that incompletely treated aneurysms with stasis of the contrast material in the neck or dome tended to improve whereas those with contrast flow in the neck or dome recurred. taking these observations into account, the authors went on to develop their own five-point aneurysm embolization grade (aeg) to help predict recurrence. in , singla et al reported an internal validity study to evaluate the aeg described above. in this system, aeg a desig- nates no filling of the aneurysm neck or dome, aeg b contrast stasis in the neck and no filling of the dome, aeg c contrast stasis in the neck and dome, aeg d contrast flow in the neck and no filling of the dome, and aeg e contrast flow in the neck and dome. they found that aeg a had significantly better follow-up than all other groups and that the contrast stasis group also had significantly better follow-up than the contrast flow group. they also found that only the rroc class i desig- nation could be used to predict follow-up durability, and that there was no significant difference between rroc classes ii and iii in their predictive value. stent assistance did not have an effect on initial or long-term durability. the authors favored the aeg system over the rroc system because of its ability to account for hemodynamic flow within the aneurysm neck and dome. while the aeg system accounts for contrast stasis, it does not account for contrast location within the aneurysm dome as is the case with the mrrc. stent assistance has been investigated in its ability to promote progression to occlusion. in , lawson et al investigated the capability of stent assistance to augment the progression from incomplete occlusion (class ii or iii) to complete occlu- sion (class ). of patients available for angiographic follow-up, ( %) in the stented group and ( %) in the unstented group progressed from either class ii or iii to class i. the authors concluded that stent-assisted coiling promotes a progression to occlusion and complete thrombosis of incom- pletely coiled aneurysms via a flow remodeling effect. this phe- nomenon has also been observed in other studies of stent-assisted coiling. interestingly, stent assistance was not identified as an independent factor that promotes progression to occlusion in our study. in , abdihalim et al investigated contrast stasis as a factor that predicts progression to occlusion. forty-four patients were identified as having contrast stasis at the end of emboliza- tion. of the patients who underwent follow-up angiography, patients had spontaneous thrombosis of the aneurysm. size of the contrast stasis (p= . ) was the only statistically significant factor that influenced this trend. dome to neck ratio > (p= . ) and washout on the initial angiogram (p= . ) only approached statistical significance. finally, the knowledge that a class iiia result following the procedure is safe and frequently has a favorable progression over time could potentially increase the safety profile of the pro- cedure. aspiring to always achieve a class i result requires increased fluoroscopic time, increased number of coils placed, and increased attempts at catheterizing the aneurysm (if the catheter were to herniate out of the aneurysm during the pro- cedure). increased angiographic time and prolonged attempts at achieving complete aneurysm occlusion have been shown to be associated with an increased ischemic stroke rate. on the other hand, a class iiia result can be achieved more quickly, with fewer coils, and frequently progresses to a class i or ii result at follow-up. certainly, a class i result is the most desirable and provides the most protection against recanalization (as seen in our results) or rerupture. it is important to know, however, that, in the vast majority of cases, a class iiia result can also provide a safe and stable outcome. fortunately, none of our class iiia aneurysms presented with rerupture. we understand that this study is limited by its retrospective nature, the subjective nature of the rroc/mrrc, and the lack of an independent blinded angiographic review. certainly many occlusion grades are debatable, and it has been shown that there is frequently disagreement between observers when using these grading scales (especially as the number of categories in the scale increases). some authors have advocated replacing the rroc system with computerized grading scales due to the sub- jective nature of the classification and interobserver vari- ation. in addition, the mrrc does not differentiate table angiographic follow-up a. class iiia class iiib p value improved to class i or ii . % ( ) . % ( ) < . remained class iii . % ( ) . % ( ) < . b. class i class ii class iiia p value improved to class i – . % ( ) . % ( ) < . worsened to or remained class iii . % ( ) . % ( ) . % ( ) < . table clinical data class iiia class iiib p value complications . % ( ) . % ( ) . death or permanent deficit from complication . % ( ) . % ( ) . vasospasm . % ( ) . % ( ) . discharge mrs – . % ( ) . % ( ) . retreatment . % ( ) . % ( ) < . post-treatment rupture . % ( ) . % ( ) . mrs, modified rankin scale. neuroimaging mascitelli jr, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n ju n e . d o w n lo a d e d fro m http://jnis.bmj.com/ between contrast flow and stasis, which may also be an import- ant factor. conclusions we propose the mrrc to further differentiate class iii aneur- ysms into those likely to progress to complete occlusion and those likely to remain incompletely occluded or to worsen. the mrrc has the potential to expand the definition of adequate coil embolization, reduce procedural risk, and stratify patients with residual aneurysms into those at higher and lower risk for recurrence. understanding this phenomenon should prove useful to other interventional neurosurgeons. these findings need to be validated in a prospective study with angiographic grading by an independent blinded neuroradiologist or interven- tional neurosurgeon. acknowledgements we would like to acknowledge margaret panzer for her artwork in figure and michael chary for his assistance with statistics. contributors all authors had integral participation in the study. aap, jrm, hm, ad, jbb and yg were involved in conception of the project. jrm, mfp and aap performed the data collection. abp, hm, ad and jrm performed the angiographic review. eko performed the statistical analysis and figure/table presentation. all authors were involved in manuscript preparation. competing interests none. ethics approval ethics approval was obtained from the institutional review board. provenance and peer review not commissioned; externally peer reviewed. data sharing statement we are not participating in data sharing. references molyneux aj, kerr rs, yu lm, et al.; international subarachnoid aneurysm trial (isat) collaborative group. international subarachnoid aneurysm trial (isat) of neurosurgical clipping versus endovascular coiling in patients with ruptured intracranial aneurysms: a randomised comparison of effects on survival, dependency, seizures, rebleeding, subgroups, and aneurysm occlusion. lancet ; : – . lin n, cahill ks, frerichs ku, et al. treatment of ruptured and unruptured cerebral aneurysms in the usa: a paradigm shift. j neurointerv surg ; : – . roy d, milot g, raymond j. endovascular treatment of unruptured aneurysms. stroke ; : – . raymond j, guilbert f, weill a, et al. long-term angiographic recurrences after selective endovascular treatment of aneurysms with detachable coils. stroke ; : – . raymond j, roy d. safety and efficacy of endovascular treatment of acutely ruptured aneurysms. neurosurgery ; : – . johnston s, dowd c, higashida r, et al. predictors of rehemorrhage after treatment of ruptured intracranial aneurysms: the cerebral aneurysm re-rupture after treatment (carat) study. stroke ; : – . ries t, siemonsen s, thomalla g, et al. long-term follow-up of cerebral aneurysms after endovascular therapy 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density, improved biomechanical stability, and midterm angiographic outcome. ajnr am j neuroradiol ; : – . otani t, nakamura m, fujinaka t, et al. computational fluid dynamics of blood flow in coil-embolized aneurysms: effect of packing density on flow stagnation in an idealized geometry. med biol eng comput ; : – . deshaies em, adamo ma, boulos as. a prospective single-center analysis of the safety and efficacy of the hydrocoil embolization system for the treatment of intracranial aneurysms. j neurosurg ; : – . singla a, villwock mr, jacobsen w, et al. aneurysm embolization grade: a predictive tool for aneurysm recurrence after coil embolization. acta neurochir (wien) ; : – . lawson mf, newman wc, chi yy, et al. stent-associated flow remodeling causes further occlusion of incompletely coiled aneurysms. neurosurgery ; : – ; discussion – . santillan a, greenberg e, patsalides a, et al. long-term clinical and angiographic results of neuroform stent-assisted coil embolization in wide-necked intracranial aneurysms. neurosurgery ; : – . hwang g, park h, bang js, et al. comparison of -year angiographic outcomes of stent- and nonstent-assisted coil embolization in unruptured aneurysms with an unfavorable configuration for coiling. ajnr am j neuroradiol ; : – . abdihalim mm, chaudry sa, tariq n, et al. the natural history of “contrast stasis” within aneurysm after embolization. j vasc interv neurol ; : – . willinsky ra, taylor sm, terbrugge k, et al. neurologic complications of cerebral angiography: prospective analysis of , procedures and review of the literature. radiology ; : – . bradac gb, bergui m, stura g, et al. periprocedural morbidity and mortality by endovascular treatment of cerebral aneurysms with gdc: a retrospective -year experience of a single center. neurosurg rev ; ( ): – ; discussion – . cloft hj, kaufmann t, kallmes df. observer agreement in the assessment of endovascular aneurysm therapy and aneurysm recurrence. ajnr am j neuroradiol ; : – . tollard É, darsaut te, bing f, et al. outcomes of endovascular treatments of aneurysms: observer variability and implications for interpreting case series and planning randomized trials. ajnr am j neuroradiol. ; : – . sherif c, gruber a, schuster e, et al. computerized occlusion rating: a superior predictor of aneurysm rebleeding for ruptured embolized aneurysms. ajnr am j neuroradiol ; : – . milavec h, gruber a, schuster e, et al. computerized occlusion rating of embolized ruptured intracranial aneurysms: relation to intra- and postinterventional aneurysm rehemorrhage. acta neurochir suppl ; : – . scan here to listen to a podcast with justin mascitelli on this article neuroimaging mascitelli jr, et al. j neurointervent surg ; : – . doi: . /neurintsurg- - o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn is.b m j.co m / j n e u ro in te rve n t s u rg : first p u b lish e d a s . /n e u rin tsu rg - - o n ju n e . d o w n lo a d e d fro m http://dx.doi.org/ . /s - ( ) - http://dx.doi.org/ . /jnis. . http://dx.doi.org/ . /hs . http://dx.doi.org/ . / .str. . .e http://dx.doi.org/ . / - - http://dx.doi.org/ . /strokeaha. . http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /radiology. . .r oc http://dx.doi.org/ . /jns- / / http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /strokeaha. . http://dx.doi.org/ . / .str. . . http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . / http://dx.doi.org/ . / http://dx.doi.org/ . /s http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /jns. . . . http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /neu. b e c b http://dx.doi.org/ . /neu. b e a http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /radiol. http://dx.doi.org/ . /s - - -z http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /ajnr.a http://dx.doi.org/ . /ajnr.a http://jnis.bmj.com/ 	an update to the raymond–roy occlusion classification of intracranial aneurysms treated with coil embolization 	abstract 	introduction 	methods 	study design 	patients, aneurysms, and procedural characteristics 	angiographic review 	statistical methods 	results 	patients, aneurysms, and procedural data 	angiographic data 	clinical data 	discussion 	conclusions 	references microsoft word - ross_note_ - shall businesses profit if their owners lose their souls? examining whether closely held corporations may seek exemptions from the contraceptive mandate christopher s. ross* may for-profit, secular corporations claim the protection of the religious freedom restoration act (rfra)? this question is central to numerous lawsuits against the federal government in which business owners argue that certain regulations under the patient protection & affordable care act substantially burden the exercise of their religion. this note examines the threshold hurdle that for- profit business owners must clear to successfully state a claim under rfra: the question of whether the businesses are “persons” the statute protects. this is an issue of first impression for the u.s. supreme court, and it has split the circuit courts of appeal. first, this note provides an overview of free exercise jurisprudence, with a focus on the ebbs and flows of the supreme court’s exemption doctrine. this overview includes a discussion of the religious freedom restoration act and the laws, regulations, and religious objections that form the basis of the current disputes. second, this note introduces the conflict among circuit courts and their varying interpretations of whether for-profit corporations are “persons” under rfra. third, this note assesses this conflict by examining rfra’s text and the context in which congress enacted the statute. nothing within this context precludes corporations from stating rfra claims. in addition, this note examines legislative history that supports application of the dictionary act, which explains that the word “person” in federal statutes includes corporations. this note ultimately concludes that rfra does indeed grant corporations the ability to seek exemptions, but that the statute will require courts to undertake the task of ascertaining the proper contours of the law as applied to different corporate forms.   * j.d. candidate, , fordham university school of law; b.a., , the king’s college. i am grateful to my family and my friends for their constant love and support. i would like to thank my adviser, professor abner greene, for his guidance as i wrote this note. fordham law review [vol. table of contents introduction ........................................................................................   i. free exercise jurisprudence, legislative exemptions, and corporate constitutional rights: a background .........   a. the historical understanding of the free exercise clause ............................................................   b. the court giveth and the court taketh away: a history of exemptions in the supreme court .........................................   . the creation of the exemption regime: sherbert v. verner and wisconsin v. yoder .......................................   . the fall of the exemption regime: employment division v. smith .............................................................   c. congress restores the exemption regime: the religious freedom restoration act ......................................................   d. an overstep of authority: the court limits rfra’s reach .......................................................................   e. free exercise claims by profit-seeking individuals and corporations .........................................................................   . claims from individuals in the pursuit of profit ..............   . claims from religious nonprofit corporations ...............   . theories of the corporation and corporate first amendment claims .........................................................   f. a new challenge: the affordable care act and its perceived threat to religious liberty ..................................   ii. dissension in the ranks: the circuits differ on corporate rfra claims ...................................................   a. the tenth and seventh circuits hold that a for-profit corporation may state a claim under rfra ......................   . the tenth circuit finds that rfra protects corporations through a textual analysis ......................   a. statutory analysis indicates that rfra’s “person” includes corporations ..............................................   b. constitutional principles of free exercise do not require excluding for-profit corporations from rfra’s protection ....................................................   . the seventh circuit agreed that a secular corporation could invoke rfra’s protection ...................................   b. the third circuit holds that a for-profit corporation cannot exercise religion .....................................................   . the third circuit rejects for-profit corporations’ ability to exercise religion under the first amendment .....................................................................   a. the direct exercise of religion by a corporation ....   b. the third circuit rejects the ninth circuit’s pass- through method .......................................................   ] close corporations and contraceptives . the third circuit rejects corporate personhood for purposes of rfra ..........................................................   c. the d.c. circuit holds that the owners of a closely held corporation can assert a rfra claim on behalf of the corporation ..........................................................................   iii. finding the proper framework: putting statutory interpretation before constitutional interpretation ............................................   a. the context of rfra and the nature, history, and purpose of the free exercise clause do not preclude corporate rfra claims ........................................................................   . context matters: congress’s intent to restore the pre-smith regime ........................................   . “person” v. “person’s exercise of religion”: the result is the same ...........................................................   a. most narrow interpretation: purely textual analysis and the dictionary act ...............................   b. most broad interpretation: the entire free exercise jurisprudence and bellotti .........................   . a brief note on legislative history ................................   . the corporate form and its relevance to the hhs mandate ...........................................................................   . why the common law model is crucial ........................   a. congress has the final say .......................................   b. courts should develop a jurisprudence to determine the contours of corporate rfra claims ..........................................   b. if the court precludes all corporations from rfra, then the d.c. circuit provides an alternative approach .............   conclusion ...........................................................................................   “in reality, the [religious freedom restoration act] will not guarantee that religious claimants bringing free exercise challenges will win, but only that they have a chance to fight.” introduction religious observers typically act according to their religion’s view of the ultimate good. this pursuit of the good can affect how an individual orders his or her entire life. sometimes, however, obedience to religious   . h.r. rep. no. - , at ( ) (additional views of hon. henry j. hyde, hon. f. james sensenbrenner, hon. bill mccollum, hon. howard coble, hon. charles t. canady, hon. bob inglis, hon. robert w. goodlatte). . see alan brownstein, taking free exercise rights seriously, case w. res. l. rev. , ( ). . see id. fordham law review [vol. dictates might require action that appears questionable to others or is even against the law. some native american traditions, for example, suggest that followers ingest certain hallucinogens as part of sacramental ceremonies, even though the government classifies these hallucinogens as controlled substances. in addition, many faiths require action outside of ritualistic exercise. thus, members of pacifist religions might refuse to produce weapons in their work. some religious acts, such as a roman catholic nurse objecting to performing abortions, even offend the sensibilities of those with different opinions on social or religious matters. for each of these religious actors, faith governs more than merely association with the divine; it governs relationships, interactions with society, personal development, and even careers. a law or regulation might hinder or restrict religious exercise. following a shift in the u.s. supreme court’s jurisprudence that congress believed might result in increased burdens on religion, congress enacted the religious freedom restoration act (rfra). rfra sought to minimize the burdens that the nation could place on religious believers and gave courts the ability to review burdensome laws and exempt claimants burdened by those laws. but congress was silent on exactly who it envisioned could take refuge within rfra. federal courts now face a novel interpretive question: whether for-profit, closely held corporations may claim the protection of this law. indeed, in march of , the u.s. supreme court will hear a consolidated appeal of cases from the third and tenth circuits, which disagreed on the approach to this question. plaintiffs in these cases object to a regulation promulgated by the department of health and human services (hhs) under the patient protection and affordable care act (aca). over plaintiffs claim   . see emp’t div. v. smith, u.s. , ( ). . see thomas v. review bd., u.s. , ( ). . see cenzon-decarlo v. mount sinai hosp., f. d , ( d cir. ). . see the opposition is overwhelming but will the administration listen?, planned parenthood (sept. , ), http://www.plannedparenthood.org/about-us/newsroom/ press-releases/opposition-overwhelming-but-will-administration-listen- .htm. . see, e.g., smith, u.s. at (examining whether a state law classifying peyote possession as a felony burdened the free exercise of the claimants’ religion). . u.s.c. §§ bb to bb- ( ). . see infra part i.c. . this note’s analysis focuses on closely held corporations, see infra note , in discussing corporate rfra claims. it does not analyze whether other business organizations can also state rfra claims. for a discussion of the relevance of corporate form to issues of standing, see generally steven j. willis, corporations, taxes, and religion: the hobby lobby and conestoga contraceptive cases, s.c. l. rev. ( ). . see hobby lobby stores, inc. v. sebelius, f. d ( th cir.) (en banc), cert. granted, s. ct. ( ); conestoga wood specialties corp. v. sec’y of the u.s. dep’t of health & human servs., f. d ( d cir.), cert. granted, s. ct. ( ). . pub. l. no. - , stat. ( ) (codified in scattered sections of and u.s.c.). ] close corporations and contraceptives that the hhs regulation burdens their religious exercise. the regulation, known colloquially as “the hhs mandate,” requires employer-sponsored insurance plans to provide some women’s preventive services, including contraceptives and sterilization procedures, without requiring the employee to share the cost. but it exempts companies below a certain size, as well as religious and nonprofit organizations that meet a stringent test. both for-profit and nonprofit organizations have made religious objections to the hhs mandate —most coming from various christian traditions that have long opposed the use and distribution of contraception, particularly methods that they believe have an abortive tendency. this note examines a threshold hurdle that stands in the way of for-profit corporations’ ability to state rfra claims: the question of whether corporations are “persons” under the statute. this is an issue of first impression for the supreme court, and it has split the circuits. part i of this note provides an overview of free exercise jurisprudence, with a focus on the ebbs and flows of the supreme court’s exemption doctrine and congress’s legislative response. part i also explains the laws and regulations—and the religious objections—that form the basis of the current disputes. part ii introduces this conflict more thoroughly by analyzing four appellate decisions with differing opinions. part iii assesses this conflict, concludes that the purposes anticipated by rfra do not exclude corporations, and provides the outline of a basic framework for courts as they begin the daunting task of ascertaining rfra’s contours for corporate claimants.   . see hhs mandate information central, becket fund for religious liberty, http://www.becketfund.org/hhsinformationcentral/ (last visited feb. , ). . see id. . see group health plans and health insurance issuers relating to coverage of preventive services under the patient protection and affordable care act, fed. reg. (feb. , ) (to be codified at c.f.r. pt. , c.f.r. pt. , c.f.r. pt. ); see also u.s.c. § gg- (a)( ) (supp. v ). . see hobby lobby, f. d at . for a description of the nonprofit exemption, see infra notes – . . see, e.g., briscoe v. sebelius, f. supp. d , (d. colo. ) (confronting the argument that a for-profit corporation run by an evangelical christian was bound by faith not to provide access to abortion); univ. of notre dame v. sebelius, no. : -cv- (rlm), wl , at * (n.d. ind. dec. , ) (confronting the argument that a nonprofit, catholic educational institution was bound by religious doctrine not to provide access to abortion, sterilization, or contraception). . see, e.g., pope paul vi, humanae vitae ¶ ( ), available at http://www.vatican.va/holy_father/paul_vi/encyclicals/documents/hf_p- vi_enc_ _humanae-vitae_en.html (“[t]he direct interruption of the generative process already begun and, above all, all direct abortion, even for therapeutic reasons, are to be absolutely excluded as lawful means of regulating the number of children. equally to be condemned . . . is direct sterilization . . . .” (emphasis added)). . see infra part ii. fordham law review [vol. i. free exercise jurisprudence, legislative exemptions, and corporate constitutional rights: a background the supreme court’s free exercise jurisprudence and congress’s subsequent reactions create a complex ebb and flow of tests and doctrines. this part first examines the history of free exercise and the court’s cases that established and then disestablished a regime of exempting religious adherents from laws that burdened their religious exercise. it then examines the legislative response to this line of cases and introduces how corporations fit into the picture. a. the historical understanding of the free exercise clause the question of exemptions for religious believers has roots in the historical understanding of the free exercise clause, which represents a fusion of various political theories. a number of american colonies protected religious dissenters and afforded members of religious sects the opportunity to escape the religious persecution of both england and the other colonies. the most protective colonies were the first to formulate the free exercise of religion as a legal principle. eventually, certain colonial charters also allowed colonial proprietors to grant “indulgences and dispensations” to religious believers burdened by the colony’s laws. this exemption power was discretionary. following the revolutionary war, the new states and eventually the federal government had to determine how best to protect religious liberty. for example, james madison successfully proposed that virginia’s bill of rights protect “the full and free exercise of [religion].” the american system would no longer be based on mere tolerance—religion was not a gift from government, but rather a duty to god that no government could abridge. by , every state except connecticut provided some protection of religious freedom. this generally extended to religious   . see john witte, jr., the essential rights and liberties of religion in the american constitutional experiment, notre dame l. rev. , – ( ). . see michael w. mcconnell, the origins and historical understanding of free exercise of religion, harv. l. rev. , – ( ). . see id. at . . see id. at (quoting carolina charter of , reprinted in federal and state constitutions, colonial charters, and other organic laws of the united states , (benjamin perley poore ed., washington, gov’t printing office d ed. )). for examples of legislatures and executives granting exemptions, see id. at – . . see id. at . . see id. at , . . see id. at . . see stephen pepper, taking the free exercise clause seriously, byu l. rev. , (alteration in original) (quoting gaillard hunt, james madison and religious liberty, in annual report of the american historical association for the year , at , – ( )). . see mcconnell, supra note , at – . . see id. at . ] close corporations and contraceptives practice, in addition to belief. accordingly, some states offered accommodations to minorities whose beliefs were at odds with societal mores. free exercise rights, however, were subject to certain limitations that served the interests of the states. for example, a state might allow free exercise rights for peaceable actions, but not for licentious ones. when the several states joined to form the federal government, they drafted a constitution banning religious tests for office and allowing for either oaths or affirmations. beyond these, however, the federalists writing the constitution saw little need to enact protection of religious rights, as they believed that the nature of the federal system, checks and balances between government branches, and the numerous religious sects would ensure the protection of religious minorities. this argument did not convince such groups as the quakers, who feared the effects of laws passed without consideration of minority beliefs. later, to protect individual liberty expressly, congress enacted the bill of rights and, within this set of amendments, the religion clause. initially, the delegates discussed religious liberty in terms of the “rights of conscience”   . see id. at (explaining that the dictionaries of the day included “action” in their definitions of “exercise”). . mcconnell acknowledges that his examples of religious accommodation “were initiated by the legislature.” id. at . he suggests, however, that it is reasonable to suppose that framers of state and the federal free exercise provisions understood that courts would create and enforce similar protections. id. . see id. at . for example, an oath requirement might ensure honest testimony in court, but it would violate the religious beliefs of quakers. id. a quaker in a lawsuit, therefore, would not be able to testify in court. see id. to protect conscientious objectors, most state governments allowed alternative procedures. id. . see id. at – . . see id. . see u.s. const. art. vi. . see u.s. const. art. ii, § (providing the text of an oath or affirmation the president must take before entering office); u.s. const. art. vi (requiring that all government officials take an oath or affirmation to support the u.s. constitution). . see mcconnell, supra note , at – ; see also the federalist no. (alexander hamilton). . see mcconnell, supra note , at . the quakers’ concern comports with a view of the free exercise clause described by christopher eisgruber and lawrence sager, who propose that the free exercise clause should protect minority viewpoints rather than privilege religion. see generally christopher l. eisgruber & lawrence g. sager, the vulnerability of conscience: the constitutional basis for protecting religious conduct, u. chi. l. rev. ( ). . see u.s. const. amends. i–x. . see u.s. const. amend. i (“congress shall make no law respecting an establishment of religion, or prohibiting the free exercise thereof . . . .”). scholars divide this section of the first amendment into two bodies of law. see, e.g., abner s. greene, the political balance of the religion clauses, yale l.j. , ( ); ira c. lupu, threading between the religion clauses, law & contemp. probs. , ( ). each clause has its own corresponding jurisprudence. compare lemon v. kurtzman, u.s. , ( ) (explaining a test for whether a law violates the establishment clause), with emp’t div. v. smith, u.s. , – ( ) (holding that neutral laws of general applicability do not violate the free exercise clause). fordham law review [vol. but eventually decided on the “free exercise of religion.” as used in the colonies and states, “free exercise” protected public conduct emanating from religious motivation, including religious speech, worship, assembly, publication, and education. exercise also included the right to join with other members of the faith to worship. this understanding of the free exercise clause fused views of both the importance of protecting religion and the prevailing enlightenment philosophy of the day by allowing individuals to worship according to their conscience and restricting government from interfering with religious practice. b. the court giveth and the court taketh away: a history of exemptions in the supreme court at its core, the free exercise clause, made applicable to the states through the fourteenth amendment, means the right to believe and to profess religious doctrine. the clause therefore prohibits the government from any regulation of religious beliefs, such as compelling beliefs or punishing religious expression. the extent to which the clause protects conduct, however, has been the subject of much debate. the supreme court initially protected matters of belief, but not necessarily conduct. for example, the mormon practice of polygamy was religious, and mormons sought to use religion as a defense while on trial for violating an antipolygamy statute. the court disagreed and held that the constitution did not require the government to allow   . see mcconnell, supra note , at – (internal quotation marks omitted). “free exercise” protects more than the “rights of conscience,” according to mcconnell. see id. at . the former denotes the application of the religious belief, while the latter indicates the knowledge or rationale of the belief. id. at . for the first amendment, this is a key distinction. beliefs are definitively protected, but it would appear that actions undertaken as a matter of sincere, religious belief receive protection too. see id. at – . . see witte, supra note , at – . . see id. at . the liberty to join in religious association included the liberty to govern the religious body without governmental interference. see id. . see mcconnell, supra note , at – (“the religious view emphasizes the importance of the individual; the enlightenment, the incapacity of the government.”). for a description of additional political and theological understandings that form the foundation of the american conception and dedication to religious liberty, see generally witte, supra note (describing theories that yielded principles of liberty of conscience, free exercise of religion, pluralism, equality, separationism, and disestablishment of religion). . see cantwell v. connecticut, u.s. , ( ). . see emp’t div. v. smith, u.s. , ( ). . see sherbert v. verner, u.s. , ( ). . see smith, u.s. at . . see infra part i.b–d. . see davis v. beason, u.s. , ( ) (“whilst legislation for the establishment of a religion is forbidden, and its free exercise permitted, it does not follow that everything which may be so called can be tolerated. crime is not the less odious because sanctioned by what any particular sect may designate as religion.”); reynolds v. united states, u.s. , ( ) (holding that laws banning polygamy were within congress’s legislative power). . see reynolds, u.s. at . ] close corporations and contraceptives mormons to state this belief as a defense before a jury. this distinction between belief and conduct did not stand, however, in the latter half of the twentieth century. beginning in sherbert v. verner, the court construed the first amendment to require exemptions from otherwise valid laws that substantially burdened individuals’ religious beliefs or practice. sherbert and its progeny, including wisconsin v. yoder, presented cases where an individual claimed that a law had the effect of hampering his or her religious practice. . the creation of the exemption regime: sherbert v. verner and wisconsin v. yoder the supreme court first articulated a standard for exemptions in sherbert. adell sherbert was a member of the seventh-day adventist church when her employer adjusted the number of days in its work week. because sherbert refused to report to work on the adventists’s holy day, her employer discharged her. she sought unemployment benefits from south carolina’s employment security commission, but the agency found that her refusal to take saturday work disqualified her. in response to sherbert’s free exercise challenge, the south carolina supreme court upheld the commission’s decision, holding that the unemployment statute did not restrict her ability to observe her religious beliefs. the u.s. supreme court reversed south carolina’s court, noting that the free exercise clause prevents the government from regulating “religious beliefs as such.” it also acknowledged that it had rejected free exercise challenges on the ground that legislation may restrict religious practices if such practices threaten public safety, peace, or order. despite this precedent, the court found that south carolina’s withholding of unemployment benefits was a substantial burden on the free exercise of sherbert’s religion. the state denied benefits because of her religiously motivated action, and the pressure to forego her beliefs to obtain the benefit was a substantial burden.   . see id. at , (explaining that congress’s legislation was “free to reach actions which were in violation of social duties or subversive of good order” and that a person may not “excuse his practices . . . because of his religious belief”). . see, e.g., sherbert v. verner, u.s. , ( ). . id. . see eugene volokh, a common-law model for religious exemptions, ucla l. rev. , ( ). . u.s. ( ). . see u.s. at . . id. at . . id. . id. at – . . see id. at . . id. at . . id. at – . . id. at . . id. fordham law review [vol. because the law substantially burdened sherbert’s religion, the court examined whether south carolina had a compelling interest to justify infringing upon her first amendment rights. in light of the claim’s constitutional basis, the court explained that “[o]nly the gravest abuses, endangering paramount interests, give occasion for permissible limitation.” the state argued that its interests were to prevent fraudulent claims that might cause a dilution of the unemployment fund, but the court stated that this interest did not justify abridging sherbert’s free exercise rights. the state, therefore, had to exempt sherbert from its law and provide unemployment benefits. the supreme court later affirmed this analysis in wisconsin v. yoder. there, amish parents declined to send their children to school after their children completed the eighth grade. wisconsin’s compulsory attendance law required parents to send their children to school through age sixteen. the amish parents were convicted of violating this law and fined $ . each. the court noted that education is a strong state interest, but even such strong interests are still subject to a balancing process. to determine the balance in this case, the court examined the amish religion and found that its commitment to not sending children to school beyond the eighth grade was religiously motivated behavior. indeed, despite increasing regulation that had encroached upon the traditional amish way of life, the religion’s traditions had not changed. amish families continued to believe that additional schooling interfered with their way of life. the court held that the wisconsin compulsory attendance law, which carried the threat of criminal sanction, substantially burdened the petitioners’ religion. to   . see id. at . . id. at (quoting thomas v. collins, u.s. , ( )). . see id. at . . see id. . id. the court did not directly state an intent to create a constitutionally compelled exemption regime. in dissent, however, justice john marshall harlan ii explained that the court’s holding meant that “if the state chooses to condition unemployment compensation on the applicant’s availability for work, it is constitutionally compelled to carve out an exception—and to provide benefits—for those whose unavailability is due to their religious convictions.” id. at (harlan, j., dissenting). . u.s. ( ). . id. at . the amish “believed that by sending their children to high school, they would not only expose themselves to the danger of the censure of the church community, but . . . also endanger their own salvation and that of their children.” id. at . . id. at . . id. at . . id. at – (“[o]nly those interests of the highest order and those not otherwise served can overbalance legitimate claims to the free exercise of religion.”). . see id. at – . . id. at . . id. at . . see id. ] close corporations and contraceptives require compliance, the court explained, would endanger or destroy amish religious exercise. wisconsin argued that its interest in compulsory education was sufficiently compelling to override the amish religious practices. the court did not accept such a sweeping claim, however, and stated that the government must present a specific interest and the harm that would result if the government granted an exemption. the resulting analysis showed that, as applied to the amish petitioners, the additional years of school required by the law would do little to serve the state’s interests, because the amish way of life centered around a separate agrarian community. therefore, even though wisconsin had a strong interest in education, the amish families satisfactorily demonstrated a need for an exemption from the education law. together, sherbert and yoder stood for the proposition that a religious believer may obtain an exemption from a law by showing that the law substantially burdens his or her free exercise of religion, and that the state has no compelling interest that outweighs the burden in question. this protection of religious faith extended to matters of action or conduct, and not just belief. moreover, if the state action could not withstand a court’s scrutiny, the constitution compelled the exemption.   . id. at . . id. at . . see id. in other words, the court established that it would view free exercise exemption claims with strict scrutiny. see volokh, supra note , at . in cases concerning constitutional law, the court views different types of claims with various levels of scrutiny. see erwin chemerinsky, constitutional law ( th ed. ). when important rights are at stake, the government must meet a heavy burden and show that the law is “necessary to achieve a compelling government purpose.” see id. at (emphasis omitted). this is the strict scrutiny explained in sherbert and yoder, as well as the test codified in rfra. see infra part i.c. the court uses a more deferential level of review in other contexts. the rational basis test requires that the government show that it is “rationally related to a legitimate government purpose.” see chemerinsky, supra, at (emphasis omitted). . see yoder, u.s. at . wisconsin also attacked the “ignorance” that amish parents would bequeath to their children. see id. the court rebuked this critique of a minority religion. id. at (“a way of life that is odd or even erratic but interferes with no rights or interests of others is not to be condemned because it is different.”). . see id. at . . see id. at (stewart, j., concurring) (“decisions in cases such as this . . . will inevitably involve the kind of close and perhaps repeated scrutiny of religious practices, as is exemplified in today’s opinion, which the court has heretofore been anxious to avoid.”). but see eisgruber & sager, supra note (arguing that religion should not receive special privilege, but rather that the law should regard religions equally). in a context in which administrative schemes provide discretion to decisionmakers, therefore, courts should have discretion to grant exemptions to protect religious minorities from veiled discrimination. see eisgruber & sager, supra note , at – . . see yoder, u.s. at ; sherbert v. verner, u.s. , – ( ); see also mark l. rienzi, god and the profits: is there religious liberty for moneymakers?, geo. mason l. rev. , n. ( ) (explaining that the key for protected activity is motivation by religious belief). . see volokh, supra note , at (explaining that before sherbert, it was for legislatures to grant exemptions, but that sherbert “launched the constitutional exemption fordham law review [vol. following sherbert and yoder, courts heard a variety of cases in which petitioners sought exemptions, claiming that laws burdened their religious practice. for example, the court granted an exemption to a jehovah’s witness who was denied unemployment insurance after he quit his job in a metal factory when he discovered that he was producing armaments. the court explained that it would not “dissect” his beliefs in order to determine whether his understanding of his religion was “unreasonable.” instead, the court found that indiana’s law would require thomas to modify his beliefs in order to continue working, and that conditioning unemployment benefits on this choice substantially burdened his religious exercise. similarly, a woman who changed religions during the course of her employment stated a valid free exercise claim when the unemployment commission denied her benefits for failing to work on her sabbath day. claimants did not, however, always find success before the supreme court. for example, claimants who were part of the armed forces could not seek the same level of protection as civilians, even if a law substantially burdened their religion, because of the military’s compelling interest in “instinctive obedience” and the “esprit de corps.” a jewish claimant, therefore, could not challenge a regulation prohibiting his religious use of a yarmulke. the court also denied free exercise claims when the exemption would require the government to adjust its internal affairs to account for interference with—and not necessarily a burden on—an individual’s spiritual development. for example, the government’s use of social security numbers for administrative efficiency did not amount to impairing religious beliefs or exercise. finally, the meaning of “burden” was in flux, and different interpretations of the term yielded different results   regime” under which the constitution would compel an exemption if a law failed to pass strict scrutiny). . see, e.g., mozert v. hawkins cnty. bd. of educ., f. d , ( th cir. ) (explaining that requiring students to read certain materials without requiring them to affirm or deny a belief or to participate in an objectionable practice is not an unconstitutional violation of students’ free exercise); bethel baptist church v. united states, f. supp. , – (m.d. pa. ) (holding that requiring churches to pay social security taxes was an indirect burden justified by a compelling governmental interest in a sound tax program). . see thomas v. review bd., u.s. , – , ( ). . see id. at . . see id. at – . . see hobbie v. unemployment appeals comm’n, u.s. , , – ( ). . see, e.g., united states v. lee, u.s. ( ) (holding that a member of the old order amish religion could not receive an exemption from requirements to pay social security taxes). . see goldman v. weinberger, u.s. , ( ). . see id. at (stevens, j., concurring). . see lyng v. nw. indian cemetery protective ass’n, u.s. , ( ) (holding that while construction on government land that was sacred to native americans might hinder spiritual development, it did not burden religious beliefs or exercise); see also bowen v. roy, u.s. , – ( ) (explaining that the first amendment did not “require the government itself to behave in ways” to further individuals’ spiritual development). . see bowen, u.s. at . ] close corporations and contraceptives for claimants. these inconsistences in the application of the doctrine caused some scholars to state that it rarely operated as strict scrutiny. . the fall of the exemption regime: employment division v. smith the sherbert and yoder exemption regime appeared to end in , when the court shifted its free exercise clause analysis to hold that neutral laws of general applicability did not violate claimants’ free exercise rights. in employment division v. smith, two members of the native american church lost their jobs at a drug rehabilitation facility after ingesting peyote, a felony offense in oregon, as part of a religious ceremony. relying on the sherbert line of cases, the respondents sought an exemption from oregon’s law classifying possession of a controlled substance as a felony. the court distinguished their case from sherbert, however, where the religious behavior was not criminal. the court explained that granting respondents’ exemption claim would result in a large expansion of free exercise rights because the law did not target their religious practice and was otherwise constitutional against other peyote users. the court stated that it had never allowed an individual’s beliefs to excuse that person from complying with valid laws within the state’s purview. when it previously held that the first amendment required exemption from a neutral law of general applicability, such as in yoder, it was “in conjunction with other constitutional protections,” such as the right to free speech or parental rights. the court limited sherbert to   . see ira c. lupu, where rights begin: the problem of burdens on the free exercise of religion, harv. l. rev. , ( ). . see richard f. duncan, free exercise is dead, long live free exercise: smith, lukumi, and the general applicability requirement, u. pa. j. const. l. , ( ); laura underkuffler-freund, the separation of the religious and the secular: a foundational challenge to first amendment theory, wm. & mary l. rev. , – ( ). . see emp’t div. v. smith, u.s. , ( ). . peyote is a hallucinogen that comes from the plant lophophora williamsii lemaire. see id. at . . see id. the court accepted that the smith petitioners were members of the native american church. see id. a new york times article, however, indicates that alfred smith and galen black were guests at the ceremony in which they ingested peyote and that they did so to learn about the religious practice, not to participate in a sacrament. see oregon peyote law leaves defendant unvindicated, n.y. times, july , , at a . . see smith, u.s. at . . see id. . see id. at . . see id. at – ; see also id. at (explaining that recent cases upheld neutral laws of general applicability against free exercise challenges in braunfeld v. brown, u.s. ( ), and united states v. lee, u.s. ( )). . id. at . some, however, critique this “hybrid rights” view of the court’s free exercise jurisprudence, stating that it represents a misreading of such cases as yoder. see james g. dwyer, the good, the bad, and the ugly of employment division v. smith for family law, cardozo l. rev. , – ( ) (explaining that yoder rejected substantive due process claims as the basis of its holding and relied wholly upon free exercise). fordham law review [vol. unemployment cases, and explained that where state unemployment benefits programs allowed for individual exemptions, the state must describe a compelling reason for extending the system to cases of religious hardship. outside of this limited scope, the free exercise clause did not require court-created exemptions from otherwise neutral, generally applicable laws. the court thus rejected the idea that the constitution compelled application of the sherbert balancing test to general laws that incidentally burdened individuals’ free exercise of religion. free exercise claims that challenged neutral laws of general applicability would receive only rational basis review going forward. justice antonin scalia bolstered his smith holding in two additional ways. first, he explained the problems associated with judicial determination of questions about religion, even in case-by-case proceedings. second, he explained that subjecting generally applicable laws to the compelling interest test whenever such a law burdened a person’s religious exercise “would be courting anarchy.” he did, however, note that even if the first amendment did not require judicially crafted exemptions for these reasons, the legislature might be more accommodating. indeed, he pointed to state accommodations of sacramental peyote use as a “solicitous” protection of the value of religious freedom. between and , the court created a process of exempting religious behavior from burdensome laws and then later claimed not to have such power. legal scholars fiercely debated whether the first amendment required such exemptions. following smith, however, free exercise   . smith, u.s. at – . . see id.; see also eisgruber & sager, supra note , at . . smith, u.s. at – . . see, e.g., korte v. sebelius, f. d , ( th cir. ); fortress bible church v. feiner, f. d , ( d cir. ). . see smith, u.s. at (“repeatedly and in many different contexts, we have warned that courts must not presume to determine the place of a particular belief in a religion or the plausibility of a religious claim.”). . see id. at . . see id. at . . id. (“it is therefore not surprising that a number of states have made an exception to their drug laws for sacramental peyote use.”). . compare gerard v. bradley, beguiled: free exercise exemptions and the siren song of liberalism, hofstra l. rev. , ( ) (arguing that constitutionally compelled exemptions are “manifestly contrary to the plain meaning of the free exercise clause”), and philip a. hamburger, a constitutional right of religious exemption: an historical perspective, geo. wash. l. rev. , ( ) (asserting that individuals historically sought occasional exemptions from certain laws but that this did not amount to a general or a constitutional right to an exemption), with mcconnell, supra note , at – (arguing that the first amendment requires religious exemptions). ] close corporations and contraceptives claims required courts to analyze whether a law was neutral and generally applicable. the constitution did not allow for exemptions. c. congress restores the exemption regime: the religious freedom restoration act justice scalia’s opinion in employment division v. smith inspired intense debate. in response, a strongly bipartisan congress passed rfra, which reinstated the sherbert regime and instructed the courts to analyze laws burdening religious practice with strict scrutiny. professor douglas laycock, a major academic force behind the law, explained that the drafters intended rfra to ensure that people need not abandon their religious beliefs to comply with the law. in its findings, congress explained that neutral laws “may burden religious exercise as surely as laws intended to interfere with religious exercise,” that “governments should not substantially burden religious exercise without compelling justification,” and that the smith court “virtually eliminated the requirement that the government justify burdens on religious exercise imposed by laws neutral toward religion.” the act’s purpose, then, was to restore sherbert and yoder’s compelling interest test. to achieve its goals, rfra provided in its operative section as follows: (a) in general—government shall not substantially burden a person’s exercise of religion even if the burden results from a rule of general applicability, except as provided in subsection (b) of this section.   . see, e.g., church of the lukumi babalu aye, inc. v. city of hialeah, u.s. ( ) (holding that a law directly targeting religious practices was unconstitutional). . see duncan, supra note , at (“[t]he key to understanding the constitution’s protection of religious liberty in the post-smith world is to locate the boundary line between neutral laws of general applicability and those that fall short of this standard.”). . see, e.g., william p. marshall, in defense of smith and free exercise revisionism, u. chi. l. rev. , ( ) (defending smith against mcconnell and others); michael w. mcconnell, free exercise revisionism and the smith decision, u. chi. l. rev. , ( ) (critiquing the analysis in smith). . see remarks on signing the religious freedom restoration act of , weekly comp. pres. doc. – (nov. , ) (explaining that the bill passed in the senate ninety-seven to three and had overwhelming support in the house of representatives on a voice vote). . see the religious freedom restoration act of , pub. l. no. - , stat. , invalidated by city of boerne v. flores, u.s. ( ), amended by religious land use and institutionalized persons act of , pub. l. no. - , stat. (codified at u.s.c. §§ bb to bb- ( )). . see douglas laycock, rfra, congress, and the ratchet, mont. l. rev. , ( ) (explaining, among other examples, that rfra might protect those who run a day care, because the government might not see day care management as religious exercise). . see u.s.c. § bb(a)( ). . see id. § bb(a)( ). . see id. § bb(a)( ); see also korte v. sebelius, f. d , ( th cir. ) (“rfra represents a congressional judgment that the rule of smith is insufficiently protective of religious liberty.”). . see u.s.c. § bb- (b). fordham law review [vol. (b) exception—government may substantially burden a person’s exercise of religion only if it demonstrates that application of the burden to the person— ( ) is in furtherance of a compelling governmental interest; and ( ) is the least restrictive means of furthering that compelling governmental interest. to apply this test, courts must examine the application of the interest to the individual claimant, rather than to the general population. in addition, the nature of compelling interest as articulated in yoder, sherbert, and other pre-smith cases places a high burden on the government to articulate concrete reasons for the law’s application. this strict scrutiny applies only if a law is a substantial burden of religious exercise. thus, neither an insubstantial burden nor a burden on nonreligious activities qualifies for the protection afforded by rfra. in the committee report that accompanied rfra, congress explained that it expected courts to rely on free exercise cases decided before smith to determine whether religious exercise has been burdened. moreover, the committee explained that it did not intend to codify any specific free exercise decision, but rather to restore the court’s legal standard in those cases. a court must still consider relevant facts and circumstances. federal courts have interpreted rfra to expressly require accommodation instead of insisting on smith neutrality. congress defined certain operative terms in rfra. its broad scope was clear through its definition of “government,” which included the various parts of federal, state, and local governing bodies. in addition, congress defined the “exercise of religion” as “the exercise of religion under the first amendment to the constitution.” scholars noted that defining “religion” any other way would prove difficult to adjudicate, and that courts had already shown the ability to differentiate between religiously   . see id. § bb- . any person may state a claim for judicial relief if a law burdens the exercise of their religion. see id. § bb- (c). . see laycock, supra note , at . . see douglas laycock & oliver s. thomas, interpreting the religious freedom restoration act, tex. l. rev. , – ( ). . see id. at . . see id. . h.r. rep. no. - , at ( ). . see id.; see also laycock & thomas, supra note , at . . see laycock & thomas, supra note , at . . see korte v. sebelius, f. d , – ( th cir. ) (quoting o’bryan v. bureau of prisons, f. d , ( th cir. )). . see u.s.c. § bb- ( ). notably, congress did not define “person.” see infra part ii. . see the religious freedom restoration act of , pub. l. no. - § , stat. , invalidated by city of boerne v. flores, u.s. ( ), amended by religious land use and institutionalized persons act of , pub. l. no. - , stat. (current version at u.s.c. § bb- ). . see id. this language was amended by the religious land use and institutionalized persons act of . the current version can be found at u.s.c. § cc- ( )(a). ] close corporations and contraceptives motivated and nonreligious behavior. principal organizers of rfra indicated, however, that it would protect individuals and that the dictionary act’s default definition of “person” meant it would also cover religious organizations. defining the statute in such broad terms was not only a political necessity to keep congress out of the weeds of specific applications; it was also a matter of principle because the statute performed a constitutional function by enacting the congressional understanding of the free exercise clause. the statute’s sweep was also clear from its broad applicability; as originally enacted, rfra “applie[d] to all federal and state law, and the implementation of that law, whether statutory or otherwise, and whether adopted before or after [november , ],” unless congress excluded applicability through explicit reference to u.s.c. § bb- . because of its applicability to every law passed by any local, state, or federal government, rfra garnered a reputation as a “super-statute.” because the law had such breadth and swept across the entirety of american law, it was not simply a change from smith. rather, it represented a substantially different vision of religious liberty and indicated congress’s intent to allow people of all religious persuasions to enjoy a fundamental right by restoring pre-smith free exercise protection.   . see laycock & thomas, supra note , at – . . see u.s.c. § ( ). for further explanation of the dictionary act, see infra note . . see laycock & thomas, supra note , at . interestingly, early drafts of rfra defined “person,” whereas the enacted version did not. see h.r. , st cong. § ( ) ( ) (defining person as “natural persons and religious organizations, associations, or corporations); h.r. , d cong. § ( ) ( ) (defining person as “natural persons and organizations, association [sic], corporations, or other entities”). the use of legislative history in statutory interpretation is frequently suspect, especially when the history is unexplained, prior versions of a statute. see joseph l. gerken, what good is legislative history? justice scalia in the federal courts of appeals ( ); max radin, statutory interpretation, harv. l. rev. , ( ). the difficulty lies in weighing the relative value of various sources. see christian e. mammen, using legislative history in american statutory interpretation ( ). if a judge can determine a reason for the change, however, it might be useful in interpreting the statute. see john f. manning & matthew c. stephenson, legislation & regulation – ( st ed. ). . see laycock & thomas, supra note , at . . see the religious freedom restoration act of § . . see michael stokes paulsen, a rfra runs through it: religious freedom and the u.s. code, mont. l. rev. , – ( ) (“rfra is thus a powerful current running through the entire landscape of the u.s. code.”). . see laycock & thomas, supra note , at – . . see id. there is some debate about whether congress intended to expand pre-smith protection through rfra, or whether it intended to restore this protection. compare michael c. dorf, incidental burdens on fundamental rights, harv. l. rev. , ( ) (“rfra does not, however, simply restore the pre-smith law.”), with h.r. rep. - , at (additional views of hon. henry j. hyde, hon. f. james sensenbrenner, hon. bill mccollum, hon. howard coble, hon. charles t. canady, hon. bob inglis, hon. robert w. goodlatte) (“[t]he bill does not reinstate the free exercise standard to the high water mark as found in sherbert v. verner and wisconsin v. yoder, but merely returns the law to the state it existed prior to smith.”). because the court frequently rejected pre-smith free exercise claims using the sherbert test, see supra notes – and accompanying text, the difference between expanding and restoring pre-smith protection matters for resolving the merits of fordham law review [vol. professor eugene volokh explained that rfra sought to restore the courts as decisionmakers on claims of religious freedom, contrary to smith’s claim that courts were not the proper body to make such decisions. sherbert launched a constitutional model in which courts decided who would receive exemptions, but smith instructed that exemptions should be purely statutory. rfra, then, adopted a model in which courts would evaluate claims individually and continue to build upon sherbert and its progeny. volokh called this the “common-law exemption model” because it granted courts the “initial discretionary decision-making power” and asked them to design a jurisprudence in accordance with congress’s vision for religious liberty. in addition, rfra claimed that the pre-smith compelling interest test provided a sensible balance between religious liberty and the government’s interests. a judge striking this balance does not necessarily have the final say, however, because the decision would be statutory and not constitutional. in matters of statutory interpretation, judges must interpret the law and might make incremental changes, but legislatures retain ultimate authority to reverse judicial action. if congress determined that a court was “too stingy” in withholding an exemption from a statute that substantially burdens a person’s religious exercise, then it could enact a specific exemption to address that situation. conversely, if congress found that a court overreached in granting an exemption, it could amend the relevant statute to withhold exemptions. although the decisions in individual rfra cases would be final, the underlying statute could be amended for future application, placing claims for religious liberty into the political process, which is how american law generally developed in a common law system. thus, the law acts to overcome legislative inertia by granting individuals the ability to petition courts for redress rather than wait for legislatures to act. rfra may also encourage courts to   rfra claims. this note, however, does not address the substance of the hhs mandate claims and therefore does not attempt to resolve this issue. . see volokh, supra note , at . . see id. . see id. . see id. at . . see u.s.c. § bb(a)( ) ( ). . see douglas laycock, the religious freedom restoration act, byu l. rev. , (“if the court strikes the balance in an unacceptable way, congress can respond with new legislation.”). . see guido calabresi, a common law for the age of statutes – ( ). . see volokh, supra note , at . . see id. . see id. at . like volokh, this note does not refer to common law “in the specific sense of the particular body of contract, tort, and property law” developed over centuries by anglo-american courts, but rather “in the general sense of ‘law made initially by judges but subject to statutory override.’” id. at n. . . see id. at . ] close corporations and contraceptives grant exemptions more generously, sound in the knowledge that the legislature possesses an additional level of review. d. an overstep of authority: the court limits rfra’s reach although congress enacted rfra with strong bipartisan support, the supreme court held that congress exceeded its power by extending the law’s reach to local and state governments. in city of boerne v. flores, a catholic church in texas sought review of a local zoning board decision denying its application for a building permit. in response, the city challenged the constitutionality of the law. the court explained that congress relied on its enforcement power in section five of the fourteenth amendment, which congress can use to address constitutional violations in the states. this enforcement power, however, has limits. any enforcement mechanism passed pursuant to section five must actually enforce the constitution, not substantively change its meaning. the court defined this requirement by saying there must be congruence and proportionality between the injury the law seeks to prevent and the means it adopts to effect that end. without congruence and proportionality, a legislative act effects a substantive change beyond enforcement. for rfra, specifically, the court found a dearth of examples where legislatures passed laws because of religious bigotry. rather, the court found that rfra addressed occasional, incidental effects that general laws can have on religious exercise.   . see id. at . critics of rfra suggested that congress asked the court to do precisely what the court rejected in smith: to engage in a balancing of religious claims with governmental interests. for examples of such critiques, see id. at n. (quoting various scholars critiquing rfra). . see generally city of boerne v. flores, u.s. ( ); see also christopher l. eisgruber & lawrence g. sager, congressional power and religious liberty after city of boerne v. flores, sup. ct. rev. , – . . u.s. . . see id. at . . see id. at . the court’s analysis in boerne appeared to limit the constitutional question to rfra’s legitimacy as applied to state and local governments. the court later applied rfra to the federal government in gonzales v. o centro espirita beneficente uniao do vegetal, u.s. ( ). . u.s. const. amend. xiv, § (“the congress shall have power to enforce, by appropriate legislation, the provisions of this article.”). . see boerne, u.s. at – . . see id. at . . see id. at (“congress does not enforce a constitutional right by changing what the right is.”). . see id. at ; see also eisgruber & sager, supra note , at . . see eisgruber & sager, supra note , at . the court confirmed this analysis by analyzing the history of the fourteenth amendment’s adoption, see boerne, u.s. at – , as well as its early section five jurisprudence. id. at – (explaining that in the civil rights cases, u.s. ( ), the court held that section five allowed for legislation that corrects state constitutional violations, but does not allow general legislation). . see id. at . . id. at – (citing examples from the legislative history, including “anecdotal evidence of autopsies performed . . . in violation of” certain religious beliefs). fordham law review [vol. rfra, it held, was not proportional to what it sought to remedy. instead, its “[s]weeping coverage” included local, state, and federal laws without temporal limitation. for these reasons, the congruence and proportionality required by section five were not present. according to the court and some scholars, this decision was necessary on federalism grounds because it protected the states from overweening action by the federal government. on the other hand, justice anthony kennedy’s opinion elevated the court’s interpretation of the constitution above the interpretation of the other branches and gave little to support his assertions that the other branches cannot engage with the court on the meaning of a constitutional provision. the court did note, however, that in its proper spheres of responsibility, congress “has not just the right but the duty to make its own informed judgment on the meaning and force of the constitution.” thus, the court’s precedent compelled the application of smith in boerne, but the court’s reasoning left the possibility that if rfra were enacted in accordance with congress’s powers, then it could apply to the federal government. boerne’s federalism rationale also inspired a wave of state analogues to rfra that reflected what the court stripped from the federal rfra. the court confirmed rfra’s applicability to federal laws and regulations in gonzales v. o centro espirita beneficente uniao do vegetal . there, the court entertained a claim by a brazilian christian spiritist sect, o centro espirita beneficente uniao do vegtal (udv), seeking an exemption from the federal controlled substances act (csa). part of the sect’s worship was ingesting hoasca, a tea brewed from two plants. one of the plants in the tea contained dimethyltryptamine, which the csa classified as a schedule i drug. the tea was central to udv’s rituals, and banning it indisputably burdened the church’s religious exercise. the court explained that rfra requires the government to justify a compelling interest to burden a person seeking an exemption. the   . see id. at . . id. . id. at (“the stringent test rfra demands of state laws reflects a lack of proportionality or congruence between the means adopted and the legitimate end to be achieved.”). . see, e.g., marci a. hamilton, city of boerne v. flores, a landmark for structural analysis, wm. & mary l. rev. , – ( ). . see abner s. greene, against obligation ( ). . boerne, u.s. at . . id. at . . see volokh, supra note , at n. (compiling state analogues to rfra). . u.s. , ( ). . see id. at (citing u.s.c. §§ – ( )). . see id. . see u.s.c. § (c) sched. (c)( ). . see gonzales, u.s. at . . see id. at . ] close corporations and contraceptives government must state more than “broadly formulated interests,” and the court “must searchingly examine” the government’s interests, as well as any impediments flowing from granting an exemption. this level of strict scrutiny results in a highly fact-intensive analysis for each claim. in gonzales, the court held that the government’s argument for a compelling interest in uniform application of the law failed. the csa contained a provision allowing for nonuniform enforcement, so the government could not claim uniform enforcement as a compelling interest. the court thus found that the law could support exemptions and held that the application of the csa to the members of the udv sect violated rfra. rfra granted the power of exemption to judges, and the court used this power to exempt the udv. e. free exercise claims by profit-seeking individuals and corporations the court has not yet used its rfra exemption power to entertain a claim from a secular corporation. the court has heard, however, free exercise claims from individuals involved in the pursuit of profit and corporations organized around religious ends. in addition, it has articulated a standard for applying first amendment rights to corporations, which have long been considered “persons” in many aspects of statutory law. this section briefly explores each of these aspects.   . id. at (quoting wisconsin v. yoder, u.s. , ( )). . see id. at . . see id. at – . . see id. similarly, a longstanding exemption on the use of peyote for religious purposes supported the argument that uniform application of the controlled substances act was a weak argument. it showed that congress expected certain limitations on the act and that the act did not require uniform application. id. at – . . see id. at – . . see id. the gonzales case did not challenge the constitutionality of rfra, so whether the law violates the establishment clause or represents an overreach of congress’s powers applied to the federal government is still a possible source of future litigation. . see hobby lobby stores, inc. v. sebelius, s. ct. , ( ) (sotomayor, circuit justice) (denying application for an injunction pending appellate review). . see, e.g., braunfeld v. brown, u.s. ( ) (adjudicating a claim by merchants who believed a law burdened their religious exercise). . see, e.g., gonzales, u.s. at (adjudicating free exercise claim by incorporated church group); church of the lukumi babalu aye, inc. v. city of hialeah, u.s. ( ) (same); see also scott w. gaylord, for-profit corporations, free exercise, and the hhs mandate, wash. u. l. rev. (forthcoming) (manuscript at ), available at http://ssrn.com/abstract= . . see, e.g., first nat’l bank of bos. v. bellotti, u.s. ( ); see also julie marie baworowsky, note, from public square to market square: theoretical foundations of first and fourteenth amendment protection of corporate religious speech, notre dame l. rev. , – ( ) (explaining three theories of corporations and the availability of corporate speech under each). fordham law review [vol. . claims from individuals in the pursuit of profit in several cases, the supreme court has heard free exercise challenges to laws that allegedly burdened the religion of individuals engaged in business. the court decided braunfeld v. brown before it created the sherbert regime, but it contained seeds of the compelling interest test. the plaintiffs were orthodox jews who claimed that a law requiring their stores to close on sundays violated their free exercise of religion; the plaintiffs already closed for the sabbath on saturdays, and would therefore lose further economic benefit. although the court accepted that this would be a result, it held that the state had a compelling interest in sunday closing laws, and that the interest in a uniform day of rest outweighed the business owners’ free exercise claims. in united states v. lee, decided after sherbert and yoder, the court examined the claims of an old order amish farmer and carpenter who did not pay social security taxes for several employees on the grounds that the amish faith forbids receipt of and contributions to social security. the court held that the law burdened lee’s religious exercise, but it also found that the government’s interest in a uniform social security system justified this burden. the court distinguished the feasibility of exemptions from social security from yoder, where the exemption was from an education system. in short, the court found that exemptions from a tax system would be difficult to manage if myriad faith groups sought exemptions. in these cases, an individual in his capacity as either an employee or an employer sought judicial protection from a law that burdened the exercise of his religion. without that protection, the petitioners would face the choice of following the instructions of their religion and losing a government benefit, or abandoning their religion to gain a benefit.   . see united states v. lee, u.s. , ( ); thomas v. review bd., u.s. , ( ); braunfeld, u.s. at . . see braunfeld, u.s. at – (“[l]egislative power over mere opinion is forbidden but it may reach people’s actions when they are found to be in violation of important social duties or subversive of good order, even when the actions are demanded by one’s religion.”). . see id. at . . see id. at – (explaining that allowing an exemption from the single day of rest for business owners of other religions could create enforcement problems or unfair economic advantage); see also gonzales, u.s. at (explaining the meaning of braunfeld for the court’s analysis of individual exemptions to state’s interests). . u.s. . . see id. at – . . see id. at – . . id. at – . . see id. . see supra notes – and accompanying text; see also korte v. sebelius, f. d , – ( th cir. ). . see hobbie v. unemployment appeals comm’n, u.s. , ( ). ] close corporations and contraceptives . claims from religious nonprofit corporations in addition to claims from individuals in the pursuit of profit, the supreme court has entertained claims from corporations organized for religious purposes, even though such nonprofit corporations do not pray, worship, or undertake any act independent of the individuals comprising them. gonzales was the most recent rfra claim by a nonprofit organization. there, the petitioner was a new mexico corporation. thirteen years earlier, the court heard a free exercise claim from a florida nonprofit corporation in church of lukumi babalu aye, inc. v. city of hialeah. the church in that case was part of the santeria religion and sued the city over an ordinance forbidding the sacrifice of animals in the town. the court held that such laws intentionally targeting religious exercise were not neutral, and therefore received strict scrutiny rather than smith’s rational basis review. ultimately, the law did not survive that level of scrutiny. in each of the cases described in part i.e. and i.e. , the court did not question whether the religious person or corporation had the ability to assert a claim. both individuals in the search of profit and corporations organized around religious principles could equally seek exemptions under the free exercise clause, and a religious corporation could state a claim under rfra. the court did not always grant such exemptions, but when it did, the availability of an exemption hinged on the balancing test rather than on who stated the claim. . theories of the corporation and corporate first amendment claims american law regularly considers corporations to be “persons” for purposes of statutory construction. corporations are separate legal entities with rights, obligations, and liabilities that are different from those of their owners and operators. the court has explained that a   . see gaylord, supra note , at . rather, nonprofits act according to their directors’ instructions. see id. at . . see supra notes – and accompanying text. . joint appendix at , gonzales v. o centro espirita beneficente uniao do vegetal, u.s. , ( ) (no. - ), wl (reproducing plaintiff’s complaint). . u.s. ( ). . see id. at . . see id. at – (explaining the town’s distaste for the religion’s practices of ritualistic animal slaughter and the process by which it created laws curtailing the practice). . see id. at – . . id. at . but cf. bob jones univ. v. united states, u.s. , ( ) (rejecting a religious freedom claim on the grounds of compelling interest, not standing). . see, e.g., u.s.c. § ( ) (defining the word “person” in the u.s. code as inclusive of corporations, partnerships, trusts, and other entities unless the context of the law dictates otherwise); am. jur. d corporations § ( ); antonin scalia & bryan a. garner, reading law ( ) (explaining the artificial person canon of construction, which mirrors the dictionary act). . william meade fletcher et al., fletcher cyclopedia of the law of corporations § (rev. vol. ). fordham law review [vol. corporation is “an artificial being, invisible, intangible, and existing only in contemplation of law.” black’s law dictionary defines “corporation” as an entity “having authority under law to act as a single person distinct from the shareholders who own it and having rights to issue stock and exist indefinitely.” a corporation, therefore, is a separate legal entity with distinct rights and obligations and is the product of positive law. corporations do business through a board of directors, not through shareholders. many corporations elect to make business decisions based on values and principles outside of the bottom line, and many corporations have adopted a theory of corporate social responsibility as a meaningful purpose of doing business. a corporation might be a “person” for purposes of a statute if such a construction will give effect to the purpose or the spirit of the law. the u.s. code recognizes this in the dictionary act by providing a rebuttable presumption that the word “person” in a statute includes corporations, and courts have held that a corporation does not literally need to be the actor for such statutes, because corporations do nothing without human actors. language imparting personhood to corporations has existed as long as corporations have, although the prevailing theories of corporations have changed. three dominant theories of corporations include the artificial person theory, the contractual theory, and the real entity theory. the artificial person theory was the earliest theory of corporations in american law. it taught that a corporation was an artificial entity created through legislative grant and controlled by the state for public reasons.   . trs. of dartmouth coll. v. woodward, u.s. ( wheat.) , ( ). . black’s law dictionary ( th ed. ) (listing and defining numerous types of corporations). . see thomas e. rutledge, a corporation has no soul—the business entity law response to challenges to the ppaca contraceptive mandate, wm. & mary bus. l. rev. (forthcoming) (manuscript at – ), available at http://ssrn.com/abstract= . but see jonathan t. tan, comment, nonprofit organizations, for-profit corporations, and the hhs mandate: why the mandate does not satisfy rfra’s requirements, u. rich. l. rev. , ( ) (arguing that the separate legal status of a corporation is true for both nonprofit and for-profit corporations). . see rutledge, supra note , at ; see also lucian a. bebchuk & robert j. jackson, jr., corporate political speech: who decides?, harv. l. rev. , ( ). . see rienzi, supra note , at – (providing the examples of whole foods and chipotle that have adopted social purposes as part of their business platforms). . see fletcher et al., supra note , § . . . u.s.c. § ( ). . see jeremy m. christiansen, note, “the word[] ‘person’ . . . includes corporations”: why the religious freedom restoration act protects both for- and nonprofit corporations, utah l. rev , – . . see margaret m. blair, corporate personhood and the corporate persona, u. ill. l. rev. , . . see id. at – (explaining three theories of corporations). . see id. . see katherine lepard, note, standing their ground: corporations’ fight for religious rights in light of the enactment of the patient protection and affordable care act contraceptive coverage mandate, tex. tech l. rev. , ( ). . see baworowsky, supra note , at . ] close corporations and contraceptives indeed, corporate charters frequently went to the politically connected elite. under this theory, the corporation, with purposes dictated by the state, could not assert a claim against the entity that allowed its existence. the contractual theory arose in backlash to the artificial person theory, which granted corporate status as a privilege. corporations desired protection from the regulatory power of the government, so states created statutes through which individuals could join together and incorporate businesses to pursue legal business activities. this theory asserted that the sole goal of a corporation is profit and ostensibly viewed corporate rights as those of the individuals joined together for sake of the business. the contractual theory crumbled, however, as publicly traded companies increased the number of shareholders involved in a business venture. the real entity theory replaced the dying contractual theory and is a middle ground between the contractual and the artificial entity theories. the theory accounts for the natural human inclination to associate and to form purposeful groups, and it holds that individual owners are different from the directors. under this theory, a corporation can develop an identity, pursue certain purposes defined by its board of directors, and serve a mediating function in society between individuals and government. the real entity theory provides a framework for conceiving corporations as more than mere profit-generating machines. especially in closely held corporations, where individual owners also participate in the   . see blair, supra note , at . . see baworowsky, supra note , at . . see id. at – . . see id.; see also blair, supra note , at – . . see baworowsky, supra note , at ; see also conestoga wood specialties corp. v. sec’y of u.s. the dep’t of health & human servs., f. d , ( d cir.), cert. granted, s. ct. ( ). . see blair, supra note , at . . see id. . see baworowsky, supra note , at . . see id. at (calling the corporation “a new group body naturally forming from the cooperation of its individual members”); see also blair, supra note , at (explaining that scholars and courts developed this theory to account for the shareholder becoming more of an investor than a owner). . see baworowsky, supra note , at – (citing alexis de toqueville, democracy in america (harvey c. mansfield & delba winthrop eds. & trans., univ. of chi. press ) ( )). this position is far from gaining universal acceptance, however, as many view corporations as a source of economic inequality and societal injustice. see, e.g., kent greenfield, daniel j.h. greenwood & erik s. jaffe, should corporations have first amendment rights?, seattle u. l. rev. , – ( ); we the people, not we the corporations, move to amend, https://movetoamend.org (last visited feb. , ). . see blair, supra note , at – (explaining that the existence of a corporate person is necessary for companies like facebook and google to achieve their goals of emphasizing “the importance of such factors as ‘culture’ and ‘reputation’ and ‘innovativeness’ in the value creating process” rather than simply acting in accordance with a number of contracts). . see a fletcher et al., supra note , § . (rev. vol. ) (“courts generally identify common law close corporations by three characteristics: ( ) a small number of fordham law review [vol. management of the company, a corporate identity can emerge from the deliberate actions of those directing the corporation. in the context of a company run by those sharing a religion, “these individuals can choose to maintain a religious identity.” the supreme court has relied on each of these theories in its various decisions on corporate rights, so it is unclear which theory it will use to examine corporate exercise of religion under either the constitution or rfra. the best compass for its potential treatment of corporate religion is its treatment of corporate speech. in first national bank of boston v. bellotti, the court heard an argument by a corporation alleging that a massachusetts statute restricting use of corporate funds to influence elections violated its free speech rights. at the outset of its analysis, the court instructed that the proper framing of the question was not whether the corporation has the right, but rather whether the regulated activity was something “that the first amendment was meant to protect.” certain constitutional rights are “purely personal,” meaning that historically, only individuals received protection under that right. to determine whether the right was purely personal, the court examined “the nature, history, and purpose of the particular constitutional provision.” in its analysis of the free speech clause, the court explained that first amendment freedoms comprise “fundamental components of liberty” and that speech has no “separate source” when asserted by a corporation. because of this, the court held that the first amendment protected the bank and struck down the state statute as unconstitutional. the court recently upheld bellotti in citizens united v. federal election commission. the citizens united court held that the free speech clause protects a corporation’s ability to speak in the electoral context. specifically, it overturned a statute that limited corporate expenditures on   shareholders; ( ) no ready market for corporate stock; and ( ) active shareholder participation in the business.”). . see baworowsky, supra note , at . . id. . see lepard, supra note , at . . see u.s. const. amend. i (“congress shall make no law . . . abridging the freedom of speech.”). . u.s. ( ). . see id. at – . . id. at ; see also gaylord, supra note , at . in bellotti, the court explained that the issue was whether “the corporate identity of the speaker deprives this proposed speech of what otherwise would be its clear entitlement to protection.” bellotti, u.s. at . . see bellotti, u.s. at n. (citing united states v. white, u.s. , – ( ), for the example that the right against self-incrimination applied only to individuals). . see id. . see id. at . . see id. at . . u.s. ( ). . see id. at . ] close corporations and contraceptives speech related to elections. justice anthony kennedy explained that the first amendment’s protections extend to corporations and that bellotti’s holding means that corporate speech does not lose constitutional protection merely because it comes from a corporation. in the corporate context, political speech decisions are made by directors through “the same rules as ordinary business decisions.” in citizens united, the corporation was a nonprofit, but the court explicitly stated that the constitution forbids suppression of the freedom of speech, regardless of whether the corporation is for profit or not for profit. the citizens united holding reflected two views of corporate personhood: that corporations possess the aggregate rights of their shareholders and that corporations possess autonomous rights separate from their shareholders. the court used the aggregate rights theory to determine that corporations are essentially associations of individuals and that restrictions on corporate speech are not permissible simply because the source of the speech is a corporation. the court also used the entity theory of corporations by explaining that corporate political speech can have value in the marketplace of ideas. thus, under either theory, the first amendment protects corporate speech. f. a new challenge: the affordable care act and its perceived threat to religious liberty current litigation alleges that certain provisions of the affordable care act substantially burden the free exercise of religion by those who provide insurance plans to employees and students. the affordable care act requires group insurance plans provided by employers to ensure certain levels of health services, including coverage without cost-sharing of preventive care and screenings for women, according to regulations promulgated by the health resource and services administration (hrsa) and hhs. hhs sought the recommendations of the institute of medicine (iom), an arm of the national academy of sciences, to define guidelines   . see id. at – . . see id. at . . see bebchuk & jackson, supra note , at . . see citizens united, u.s. at . . see anne tucker, flawed assumptions: a corporate law analysis of free speech and corporate personhood in citizens united, case w. res. l. rev. , – ( ). . see id. at ; see also citizens united, u.s. at (quoting first nat’l bank of bos. v. bellotti, u.s. , ( )). . see tucker, supra note , at – ; see also citizens united, u.s. at (“on certain topics corporations may possess valuable expertise, leaving them the best equipped to point out errors or fallacies in speech of all sorts, including the speech of candidates and elected officials.”). . see infra part ii. for an overview of litigation in the federal district courts and the early stages of appellate litigation, see generally tan, supra note . . see u.s.c. § gg- (a)( ) (supp. v ); u.s.c. § d. fordham law review [vol. for women’s preventive care and screening. the iom recommended that any fda-approved contraceptive method be part of the coverage, and hhs adopted this recommendation in its rulemaking process. the fda has approved twenty contraceptive methods. these include barrier methods (such as condoms), hormonal methods (such as oral contraceptives), emergency contraception (such as plan b or ella), implanted devices (such as intrauterine devices (iuds)), and permanent methods (such as vasectomies). the aca and the regulatory agencies enforcing it have provided authority for the hrsa to exempt religious employers from complying with this law if they object to providing contraception. the regulation provides that a “religious employer” is an organization that: ( ) has a purpose of inculcating religious values, ( ) primarily employs those who share its religious tenets, ( ) primarily serves those who share its religious tenets, and ( ) meets the internal revenue code’s definition of a nonprofit organization. for these reasons, it does not cover for-profit corporations, including closely held corporations. like religious nonprofit corporations, some for-profit corporations also state religious objections to providing contraception on the grounds that some methods can cause a fertilized egg to fail to implant in the uterus and that this is equivalent to facilitating an elective abortion. the methods to which plaintiffs in the tenth circuit object, for example, include plan b, ella, and two iuds. other methods, which simply prevent fertilization, may not be objectionable, depending on the religious background of the plaintiff. there is medical and scientific debate concerning whether these devices actually prevent implantation, but in hobby lobby stores, inc. v. sebelius, the government conceded that certain contraceptive methods   . see hobby lobby stores, inc. v. sebelius, f. d , ( th cir.) (en banc), cert. granted, s. ct. ( ). . see id. . see group health plans and health insurance issuers relating to coverage of preventive services under the patient protection and affordable care act, fed. reg. , (feb. , ) (to be codified at c.f.r. pt. ). . see birth control: medicines to help you, food & drug admin., http://www.fda.gov/forconsumers/byaudience/forwomen/freepublications/ucm .ht m (last visited feb. , ). . id. . see c.f.r. § . ( )(a)(iv)(a) ( ). . id. § . ( )(a)(iv)(b). . see id. . see, e.g., hobby lobby stores, inc. v. sebelius, f. d , – ( th cir.) (en banc), cert. granted, s. ct. ( ). . see id. . see conestoga wood specialties corp. v. sec’y of the u.s. dep’t of health & hum. servs., f. d , – ( d cir.) (explaining that the mennonite plaintiffs object to providing plan b and ella), cert. granted, s. ct. ( ); univ. of notre dame v. sebelius, no. : -cv- (rlm), wl , at * (n.d. ind. dec. , ) (explaining that the catholic plaintiffs object to all “abortifacients, contraception (when prescribed for contraceptive purposes), and sterilization”). . f. d . ] close corporations and contraceptives can prevent uterine implantation, rendering a full scientific analysis unnecessary. nevertheless, amici to the tenth circuit disagreed about the effect that plan b, ella, and iuds have on ovulation, conception, and implantation. in the brief for the physicians for reproductive health and other groups, amici explained that the scientific and legal definition of pregnancy is implantation in the uterine lining, which can occur up to five to nine days after fertilization of an egg by a sperm. because the emergency contraceptives at issue in hobby lobby almost always act before implantation, labeling the methods “abortifacients” was improper. amici also explained that a copper, fda-approved iud creates a toxic environment for sperm and that any changes to the endometrial lining would only prevent, not disrupt, the implantation necessary to create a scientific pregnancy. in contrast, amici for the plaintiff explained that fda-approved contraceptive methods might prevent either conception or implantation. these amici explained various studies in an effort to persuade the court that certain contraceptive methods might prevent implantation of a fertilized cell. for example, they cited studies showing that ella has the potential to impair or prevent a fertilized egg from implanting in the uterine wall. they also argued that the scientific definition of pregnancy as a fully implanted embryo does not negate the fact that certain contraceptive methods might prevent the implantation of an embryo. the plaintiffs in the hhs mandate litigation have stated that their opposition to the hhs regulation is that it requires them to pay for contraception that they believe can cause the death of a fertilized embryo. the plaintiffs in these cases claim that requiring them to pay even part of the fees for insurance plans that cover contraception constitutes a substantial burden on the exercise of their religion. the hobby lobby plaintiffs, for example, stated that their religion barred causing the death of an embryo, and, by extension, providing certain contraceptives that could cause an embryo to fail to implant. the regulation imposes a burden   . see id. at n. . . compare brief for physicians for reproductive health et al. as amici curiae supporting appellees, hobby lobby, f. d (no. - ), wl [hereinafter brief for physicians for reproductive health et al.], with brief for ass’n of american physicians & surgeons et al. as amici curiae supporting appellants, hobby lobby, f. d (no. - ), wl [hereinafter brief for ass’n of american physicians & surgeons et al.]. . see brief for physicians for reproductive health et al., supra note , at * – . . id. at * – . . see id. at * . . see brief for ass’n of american physicians & surgeons et al., supra note , at * . . see id. at * – . . see id. at * . . see id. * – . . see hobby lobby stores, inc. v. sebelius, f. d , ( th cir.) (en banc), cert. granted, s. ct. ( ). . see brief of appellants at , hobby lobby, f. d (no. - ), wl , at * . . see hobby lobby, f. d at . fordham law review [vol. because it requires the company to participate in an activity prohibited by a sincere religious belief, or, at the very least, substantially pressures the company to act contrary to sincere religious beliefs. such pressure comes as a result of large, annual fines for a corporation that fails to comply with the regulation. the government, in contrast, argues that the regulation’s burden is too attenuated to be a substantial burden because the requirement is to provide many types of insurance and because the decision to use contraceptives is between a woman and her doctor. ii. dissension in the ranks: the circuits differ on corporate rfra claims the hhs mandate cases have led to deeply fractured opinions in the circuits. for-profit corporate plaintiffs have sought injunctions against application of the hhs mandate to varying degrees of success. some courts have found that corporations will not succeed on a rfra claim. other courts have found the possibility of success. the major source of conflict is whether corporations are “persons” exercising religion. the answer to this question is crucial, as rfra contemplates strict scrutiny of laws that substantially burden a person’s religion. the issue in these cases, then, is a matter of statutory interpretation of the term “person” in rfra. this part examines four circuits’ answers to this question. the tenth and seventh circuits each held that secular corporations may state claims under rfra. in contrast, the third circuit held that corporations could not exercise religion, barring them from stating claims under rfra. the d.c. circuit fell between these two approaches, holding that individual plaintiffs may state the rfra claims of a closely held corporation.   . see brief of appellants, supra note , at * – . . see hobby lobby, f. d at (explaining that hobby lobby’s fine for failing to comply would be $ million annually). . see petition for writ of certiorari, hobby lobby, f. d (no. - ), wl , at * . . see, e.g., hobby lobby, f. d at (sitting en banc and resulting in six different opinions). . among other factors, a litigant seeking a preliminary injunction must prove a likelihood of success on the merits of the claim. see, e.g., conestoga wood specialties corp. v. sec’y of u.s. the dep’t of health & human servs., f. d , ( d cir.), cert. granted, s. ct. ( ). . see generally conestoga wood, f. d at ; autocam corp. v. sebelius, f. d ( th cir. ); eden foods, inc. v. sebelius, f. d ( th cir. ). . see generally korte v. sebelius, f. d ( th cir. ); gilardi v. u.s. dep’t of health & human servs., f. d (d.c. cir. ); hobby lobby, f. d at . . see korte, f. d at . an additional question is whether the distinction between for-profit and nonprofit corporations is relevant for rfra. see id. at – . . see infra part ii.a. . see infra part ii.b. . see infra part ii.c. ] close corporations and contraceptives a. the tenth and seventh circuits hold that a for-profit corporation may state a claim under rfra the tenth and the seventh circuit each held that for-profit corporate plaintiffs were “persons” exercising religion and therefore protected by rfra. this section examines each circuit’s opinion. . the tenth circuit finds that rfra protects corporations through a textual analysis in hobby lobby stores, inc. v. sebelius, the court considered a rfra claim brought by hobby lobby stores, a chain of craft stores; mardel, inc., a chain of book stores; and the family members who own both corporations. together, these corporations employed over , people. five members of the green family owned and ran both chains according to their christian beliefs, as described in hobby lobby’s statement of purpose, which recited the family’s commitment to operating their business according to biblical principles. their managerial decisions stemmed from their faith. for example, the stores did not open on sundays, and hobby lobby placed evangelistic advertisements in newspapers. moreover, their corporate structure included a management trust that had religious requirements: “the trust exists ‘to honor god with all that has been entrusted’ to the greens and to ‘use the green family assets to create, support, and leverage the efforts of christian ministries.’” the plaintiffs objected to the hhs mandate because their faith included the belief that the fertilization of an egg by a sperm is the beginning of human life and that facilitating acts that cause a human embryo to die was immoral. in light of this belief, the plaintiffs sought relief from enforcement of the mandate as applied to four of the fda-approved contraceptive methods that they believed prevented uterine implantation. if hobby lobby and mardel failed to comply with the regulation, each would be exposed to tax penalties, regulatory activity, and possible lawsuits. the regulatory taxes, for example, would amount to $ per day for each uncovered individual. because the plan covered more than   . f. d . . see id. . . see id. at . . see id. . see id. . see id. . see id. . see id. at – . . the plaintiffs did not object to contraception generally, as a catholic organization might, see supra note , but rather objected only to four of the twenty fda-approved methods. see hobby lobby, f. d at . . see hobby lobby, f. d at . the government did not dispute the sincerity of this belief. see id. . see id. (citing u.s.c. §§ d, h (supp. v ); u.s.c. §§ , d). . see id. (citing u.s.c. § d(b)( )). fordham law review [vol. , individuals, this would be a fine of almost $ million annually. alternatively, the plaintiffs could have eliminated health insurance coverage and would face penalties of $ million annually. a. statutory analysis indicates that rfra’s “person” includes corporations the tenth circuit held that the corporate plaintiffs were “persons” who may state a claim under rfra, and a plurality found that these plaintiffs were likely to succeed on the merits of their claim. the government advanced two arguments that hobby lobby and mardel did not fall under rfra’s scope. first, it argued that the existence of religious exemptions for nonprofit organizations in civil rights and labor laws indicated that congress intended rfra to carry forward a distinction between for-profit and nonprofit organizations. second, the government argued that the for- profit/nonprofit distinction came from the supreme court’s jurisprudence and that congress did not intend for rfra to expand the free exercise clause’s scope. the court rejected both arguments as a matter of statutory interpretation. because rfra did not specifically define “person,” the court relied on the dictionary act’s default definition, which includes corporations unless the statute’s context dictates otherwise. contrary to the government’s position, rfra’s context did not include statutes that explicitly provided only for religious nonprofit exemptions. the court explained that exemptions for religious organizations in such acts as title vii of the civil rights act of and the americans with disabilities act (ada) show that congress “knows how to craft a corporate religious exemption.” the court claimed that to state that congress intended to carry forward title vii and the ada’s distinction without expressly providing so is to strain the text. thus, the default dictionary act definition applied, and rfra protected the corporate plaintiffs.   . id. . see id. (citing u.s.c. § h). . id. at – . the tenth circuit remanded to the district court to evaluate the other preliminary injunction factors, which it did, granting the injunction. see hobby lobby stores, inc. v. sebelius, no. - -he, wl , at * (w.d. okla. july , ). . see hobby lobby, f. d at – . . see id. . see id. at . . see id.; see also u.s.c. § ( ). . see hobby lobby, f. d at – . . see u.s.c. §§ e to e- ( ). . see u.s.c. §§ – ( & supp. ii ). . see hobby lobby, f. d at ; cf. korte v. sebelius, f. d , ( th cir. ) (“if congress intended a nonprofit limitation in rfra, surely that would be some hint of it in the statutory text.”). . see hobby lobby, f. d at – . the court characterized this reading this way: “in short, the government believes congress used ‘person’ in rfra as extreme shorthand for something like ‘natural person or ‘religious organization’ as that term was ] close corporations and contraceptives b. constitutional principles of free exercise do not require excluding for-profit corporations from rfra’s protection the court next addressed the argument that congress’s understanding of the free exercise clause informed its passage of rfra, and that this understanding precluded protection of for-profit corporations. the court rejected this analysis on the grounds that rfra’s purpose was to restore the pre-smith balancing test for whether a neutral law of general applicability can withstand a strict scrutiny analysis to justifiably burden religious exercise. congress did not intend to alter who could bring a claim under pre-smith free exercise jurisprudence, which included the guarantee to protect associations and not just individuals. the protection of associational freedom is important for protecting other individual liberties. moreover, the first amendment protects such associations as churches, which incorporate. the court rejected the government’s bright-line approach that precludes a rfra claim for a corporation that does not have nonprofit status through the internal revenue service. this, the court explained, cannot be a position rooted in the first amendment, because its drafters rejected the term “conscience” in favor of “exercise.” the chosen term carried a connotation of action and thus protected “religiously motivated conduct, as well as religious belief.” in light of this action-oriented history, the court rejected the argument that incorporation alters first amendment rights while individuals maintain protection. the court also rejected the district court’s argument that a business corporation cannot partake in “religiously- motivated actions separate and apart from the intention and direction of   used in exemptions for religious organizations as set forth in title vii, the ada, and the nlra.’” id. at . . see id. at . . see id. at . . see id. . see id. (“an individual’s freedom to speak, to worship, and to petition the government for the redress of grievances could not be vigorously protected from interference by the state unless a correlative freedom to engage in group effort toward those ends were not also guaranteed.” (quoting roberts v. u.s. jaycees, u.s. , ( ))). . id. at ; see also citizens united v. fec, u.s. , – ( ) (explaining that the court does not accept the argument that corporate first amendment rights do not differ “simply because such associations are not ‘natural persons’”). . hobby lobby, f. d at . (citing church of the lukumi babalu aye, inc. v. city of hialeah, u.s. , ( )). in addition, the supreme court has decided free exercise cases for individuals running for-profit businesses. see supra part i.e. . . see hobby lobby, f. d at . . see id. . see id. (citing mcconnell, supra note , at – ). an additional distinction between conscience and religion is the corporate or institutional aspect of religious belief. see id. at – (citing mcconnell, supra note , at ). the court also noted that free exercise includes proselytizing. see id. (citing emp’t div. v. smith, u.s. , ( )). . see id. at – (“[w]e see no reason why one must orient one’s business toward a religious community to preserve free exercise protections.”). fordham law review [vol. their individual actors.” this could not be right, according to the court, because churches and other entities that incorporate clearly exercise religion. rfra thus includes corporations in its definitions, because the for-profit/nonprofit distinction is irrelevant. . the seventh circuit agreed that a secular corporation could invoke rfra’s protection using a similar analysis to the hobby lobby court, the seventh circuit held that secular corporations could challenge the hhs mandate. in korte v. sebelius, the court heard a consolidated appeal involving two for-profit corporations. the first, korte & luitjohan contractors, inc., was a closely held construction company that employs ninety full-time workers. cyril and jane korte were the majority shareholders of k&l contractors, as well as its only officers and directors. as catholics, they believed that artificial contraception and abortifacient drugs were contrary to their religion and objected to providing any health plan that facilitated the use of such contraceptive methods. to emphasize this objection, the couple, acting in their managerial capacity, enacted ethical guidelines describing limits on employee healthcare benefits. failing to comply with the hhs mandate would result in penalties totaling $ , annually. in the second case, the corporate plaintiffs were grote industries, inc., which acted as the managing member for grote industries, llc, which manufactures vehicle safety systems. six individually named plaintiffs, along with other family members, fully owned grote industries, inc. the individual plaintiffs were the officers and directors of the corporation. grote industries employed employees in the united states and provided a self-insured healthcare plan that did not carry contraception and sterilization procedures before the hhs mandate became law. the plaintiffs managed grote in accordance with their catholic faith, objected to the mandate in light of that faith, and would face an annual penalty of nearly $ million for failing to comply.   . id. at (quoting hobby lobby stores, inc. v. sebelius, f. supp. d , (w.d. okla. )). . see id. (citing gonzales v. o centro espirita beneficente uniao do vegetal, u.s. ( )). . see korte v. sebelius, f. d , ( th cir. ). . f. d . . see id. at . . see id. at . . see id. . id. at – . . see id. at . . see id. . see id. at – . . see id. at n. . . see id. at . . see id. . see id. ] close corporations and contraceptives judge diane s. sykes began her opinion by explaining the breadth of rfra, including its applicability to all federal law unless congress explicitly excludes application of rfra to a statute. rfra, she explained, should alleviate “the inevitable clashes between religious freedom and the realities of the modern welfare state.” against this backdrop, the court examined the meaning of “person” in rfra by invoking the dictionary act. in its analysis, the court sought to determine whether allowing secular corporations to be “persons” for rfra would be a poor fit with the statute’s purposes, and it held that because some corporations qualify for rfra protection, the corporations in this litigation could qualify as well. as evidence of this claim, the court pointed to gonzales, church of the lukumi babalu aye, inc., and the hhs mandate exemption for religious organizations. like the tenth circuit, judge sykes rejected the government’s argument that rfra carried forward the for-profit/nonprofit distinction within title vii and the ada. instead, the court examined the history of rfra and free exercise more broadly to see if any pre-smith cases rejected secular corporations’ ability to exercise religion. recognizing that the question of secular corporate religious exercise was novel, the korte court nonetheless explained that unless something about seeking profit hinders the ability to practice religion, “a faith-based, for-profit corporation can claim free-exercise protection to the extent that an aspect of its conduct is religiously motivated.” an examination of thomas, sherbert, braunfeld, and lee showed that individuals in the pursuit of profit had their claims examined by the supreme court—not for standing, but on the merits of the argument.   . see id. at (“the affordable care act does not explicitly exclude application of rfra.”). . see id. (citing thomas c. berg, what hath congress wrought? an interpretative guide to the religious freedom restoration act, vill. l. rev. , – ( )). . see id. at – . . see id. at (“a corporation is just a special form of organizational association. no one doubts that organizational associations can engage in religious practice. the government accepts that some corporations—religious nonprofits—have religious-exercise rights under both rfra and the free-exercise clause.”). . u.s. ( ) (incorporated religious sect). . u.s. ( ) (incorporated church). . see c.f.r. § . (a) ( ) (granting an exemption with or without incorporated status). . see korte, f. d at – (explaining that title vii and the ada’s nonprofit exemptions rely on the church autonomy doctrine that provides complete immunity to religious organizations from compliance with these laws); cf. hosanna-tabor evangelical lutheran church & sch. v. eeoc, s. ct. , ( ). . see korte, f. d at . . see id. (citing hobby lobby stores, inc. v. sebelius, s. ct. , ( ) (sotomayor, circuit justice) (denying an extraordinary writ on grounds including the novelty of the issue presented)). . see id. . see id. at – . fordham law review [vol. the court further rejected the government’s argument by rebuking its attempt to cast united states v. lee as dispositive of the claim that for-profit corporations cannot pursue religious exercise. the lee court stated: congress and the courts have been sensitive to the needs flowing from the free exercise clause, but every person cannot be shielded from all the burdens incident to exercising every aspect of the right to practice religious beliefs. when followers of a particular sect enter into commercial activity as a matter of choice, the limits they accept on their own conduct as a matter of conscience and faith are not to be superimposed on the statutory schemes which are binding on others in that activity. judge sykes called the government’s interpretation of this passage “a far- too-narrow view of religious freedom,” because it assumes that the religious believer compartmentalizes his or her life. having explained that neither the supreme court’s free exercise jurisprudence nor rfra’s text precluded for-profit corporate religious exercise, the court turned to the supreme court’s “general jurisprudence of corporate constitutional rights” and found that nothing automatically limits such rights to nonprofit corporations. the korte court took note of the supreme court’s decisions granting and withholding certain constitutional rights to all corporations and explained that bellotti was the closest thing to a proper decision framework. namely, if the right in question has a historical function of guaranteeing rights to individuals only, the provision in question will not be available to corporations. because the issue at bar, however, was strictly statutory, the court declined to further analyze the free exercise rights of the corporate plaintiffs. based on all of these findings, the korte court held that k&l contractors and grote industries could state rfra claims. in dicta, the court seemed to limit its holding to closely held corporations, because the individual owners set company policy as its directors, and they are intimately involved in directing the company. both the seventh and the tenth circuits, therefore, held that rfra covered corporate claimants in light of the statute’s text and purpose.   . see id. at . . united states v. lee, u.s. , ( ) (emphasis added). . see korte, f. d at ; see also christiansen, supra note , at (explaining that the business judgment rule allows directors to make decisions in the interests of nonshareholders). . see korte, f. d at . . see id. at – . . see id. at (citing first nat’l bank of bos. v. bellotti, u.s. , ( )). . see id. . see id. . see id. . see id. at n. (dictum). judge sykes implied that in a larger corporation, ownership will be separate from controllers, and will have more members of the board, presumably making it more difficult to define religious beliefs. see id. ] close corporations and contraceptives b. the third circuit holds that a for-profit corporation cannot exercise religion in contrast to the hobby lobby and korte courts, the third circuit disagreed that for-profit corporations receive rfra’s protection. in conestoga wood specialties corp. v. secretary of the u.s. department of health & human services, the individual plaintiffs were members of the hahn family, which was mennonite. the family owned percent of the shares of a corporation that manufactured wood cabinets and employed workers. as mennonites, the hahns believed that it was intrinsically evil and a sin to take a human life, including the termination of a fertilized embryo. in light of this belief, the hahns and conestoga objected to providing plan b and ella, which the plaintiffs believed could cause a fertilized embryo to fail to implant in a woman’s uterus. . the third circuit rejects for-profit corporations’ ability to exercise religion under the first amendment the third circuit began its analysis with the issue of whether corporations can exercise religion under the first amendment. the plaintiffs suggested two theories: ( ) that a corporation can exercise religion directly under the first amendment, and ( ) that a corporation can exercise religion indirectly by asserting the owners’ rights under a theory established by the ninth circuit. a. the direct exercise of religion by a corporation conestoga premised its direct exercise theory on citizens united, which held that the first amendment applied to corporations in the context of a free speech case. corporations may exercise a number of constitutional rights, but the guarantee must not be one that is “purely personal.” the third circuit examined bellotti’s relationship to citizens united and explained that the latter’s holding was specific to the court’s history of protecting corporate free speech. moreover, free speech cases pose a   . see conestoga wood specialties corp. v. sec’y of the u.s. dep’t of health & human servs., f. d , ( d cir.), cert. granted, s. ct. ( ). . f. d . . see id. at . . see id. . id. at – ; see also first amended verified complaint ¶ , conestoga wood, f. d (no. - ), wl (explaining that the board of directors of conestoga adopted “the hahn family statement on the sanctity of human life,” which explains the family’s belief that life begins at conception and their moral opposition to participating in any act involving the termination of human life). . see conestoga wood, f. d at . . see id. at . . see id. . see id. . see id. at (quoting first nat’l bank of bos. v. bellotti, u.s. , ( )). . see id. fordham law review [vol. separate analytical and interpretive framework than free exercise cases. the conestoga court, therefore, found that bellotti had no influence on the hhs mandate cases. for these reasons, it found that bellotti’s “nature, history, and purpose” test was not met for corporate free exercise rights. the court particularly emphasized a corporation’s inability to hold religious beliefs. in addition, it distinguished cases relied on in the petitioners’ and dissent’s arguments that showed recent supreme court cases allowing free exercise claims by religious organizations. these cases were inapposite for the majority because none involved a for-profit corporation. religious organizations are a means through which individuals can practice their religion, and that such organizations receive protection within the free exercise clause is not determinative for for- profit corporations. b. the third circuit rejects the ninth circuit’s pass-through method after dispensing with the plaintiffs’ first theory, the court examined their second theory. the plaintiffs cited two cases from the ninth circuit that allowed closely held corporations to state the free exercise claims of their owners. under this “pass-through theory,” the ninth circuit explained that closely held corporations may convey by extension the beliefs of the family members who own them. the corporate plaintiffs in these cases did not have any free exercise rights separate from or greater than their   . see id. at . . see id. at . . see id. (“we do not see how a for-profit ‘artificial being, invisible, intangible, and existing only in contemplation of law,’ that was created to make money could exercise such an inherently ‘human’ right.” (quoting consol. edison co. of n.y., inc. v. pataki, f. d , ( d cir. ))). . see id. (“it might also be added that corporations have no consciences, no beliefs, no feelings, no thoughts, no desires.” (quoting citizens united v. fec, u.s. , ( ) (stevens, j., concurring in part and dissenting in part))); sch. dist. v. schempp, u.s. , ( ); hobby lobby stores, inc. v. sebelius, f. supp. d , (w.d. okla. ) (“general business corporations do not, separate and apart from the actions or belief systems of their individual owners or employees, exercise religion. they do not pray, worship, observe sacraments or take other religiously-motivated actions separate and apart from the intention and direction of their individual actors.”), rev’d, f. d ( th cir.) (en banc), cert. granted, s. ct. ( ). . see conestoga wood, f. d at – . . see id. . see id. at . . see id. at – (citing eeoc v. townley eng’g & mfg. co., f. d ( th cir. ), which held that for-profit corporations can assert the free exercise claims of their owners, and stormans, inc. v. selecky, f. d ( th cir. ), which affirmed townley). . see id. . see townley, f. d at (“townley is merely the instrument through and by which mr. and mrs. townley express their religious beliefs.”); stormans, f. d at (“stormans, inc. does not present any free exercise rights of its own different from or greater than its owners’ rights. we hold that, as in townley, stormans has standing to assert the free exercise rights of its owners.”). ] close corporations and contraceptives owners’ rights. the third circuit rejected this analysis, claiming it rested on a faulty analysis of the corporate form, which creates a distinct entity. in exchange for limited liability, the corporate owner gives up certain prerogatives. the pass-through theory, in contrast, ignores this separate legal entity. on this basis, the third circuit found that the pass-through theory was legally incorrect, and that the mandate’s burden falls squarely on conestoga and not the hahns. because conestoga itself cannot exercise religion, it has no free exercise recourse to seek an exemption from the mandate. . the third circuit rejects corporate personhood for purposes of rfra the third circuit also rejected conestoga’s ability to state a rfra claim with a brief, plain-language analysis. because the statute is for a “person’s exercise of religion,” and because the court held that a corporation cannot exercise religion under the free exercise clause, it also held that a corporation is unable to state a claim under rfra. c. the d.c. circuit holds that the owners of a closely held corporation can assert a rfra claim on behalf of the corporation in gilardi v. united states department of health & human services, the d.c. circuit held that the individual plaintiffs who owned the corporate plaintiff could state a rfra claim on behalf of their corporation, although it rejected the ninth circuit’s pass-through theory. the individual plaintiffs were two brothers who owned two companies equally. freshway foods and freshway logistics together employed people, who could participate in a self-insured health plan. as catholics, the gilardi brothers opposed the coverage mandated by the hhs regulation, but failure to provide it to their employers would result in fines amounting to over $ million annually. judge janice rogers brown first analyzed whether the corporate plaintiffs could state a claim under rfra. she took note of the hobby lobby court’s opinion but explained that its analysis of the term “person,” relying on the dictionary act, was too narrow. rather, she explained,   . see conestoga wood, f. d at – (citing stormans, f. d at ). . id. . see id. . see id. . see id. at . . see id. . see id. . see id. . f. d (d.c. cir. ). . see id. at – . . see id. at . . see id. . see id. . see id. at . . see id. fordham law review [vol. the proper focus is on a “person’s exercise of religion” rather than “person” alone. the court therefore examined the full free exercise jurisprudence to determine the “nature, history, and purpose” of the free exercise clause and whether it applied to a secular corporation. noting that the clause protected “exercise” rather than merely “conscience,” the court found that the clause’s protection extended to conduct undertaken either individually or institutionally. indeed, the clause regularly protected individuals and religious organizations. no supreme court case had yet held, however, that a secular corporation could exercise religion. the gilardi court thus refused to extend the free exercise right directly to the secular corporations, even if corporations might receive that right in the future. here, judge brown noted that citizens united was the culmination of decades of case law. the free exercise cases, on the other hand, militated against extending the free exercise right directly to corporations. like the third circuit, the d.c. circuit rejected the ninth circuit’s pass- through theory as legally “dubious.” rather, the court held that the gilardis have standing to sue because they are shareholders of the corporation injured separately from the corporation, and because the corporation did not have the capacity to sue for relief separately. although the government argued that incorporation always involves creating a distinct legal entity for which the owners forego certain rights, the court explained that an important part of this rights bargain is that corporations “can generally exercise some analogue of the forgone right.” in the case of free exercise, then, if the corporation cannot exercise that right, the shareholder should not give up the right upon incorporation. to hold otherwise would cause an important right to “disappear[] into the ether” depending on how a person ran his or her   . rfra defines the exercise of religion by referring to the religious land use and institutionalized persons act, which states that “‘religious exercise’ includes any exercise of religion, whether or not compelled by, or central to, a system of religious belief.” u.s.c. § cc- ( )(a) ( ). . see gilardi, f. d at (citing rasul v. myers, f. d , (d.c. cir. )). . see id. at . . id. . see id. at – (explaining that the word “religion” carries a connotation of a community of believers). . see id. at – (collecting free exercise cases). . see id. at . but see id. (“but that the court has never seriously considered such a claim by a secular corporation or other organizational entity is not to say it never will.”). . id. at . . see id. . see id. . see id. at – . . see id. at . . see id. at – . . see id. at . . see id. ] close corporations and contraceptives business. judge brown rejected the government’s argument because she could not believe that congress intended free exercise rights to disappear when it instituted rfra’s compelling interest balancing test. iii. finding the proper framework: putting statutory interpretation before constitutional interpretation this note proposes two approaches for the issue of whether secular corporations are “persons” protected by rfra. first, agreeing with the tenth and seventh circuits, it proposes that the absence of case law examining free exercise claims by for-profit corporations is not, as some suggest, dispositive of the cases currently under review in the federal courts. based on the dictionary act’s “context” requirement and a proper bellotti analysis, certain for-profit corporations should be able to state rfra claims, and the federal courts should continue building a jurisprudence to determine the contours of such claims. second, this note proposes that even if the supreme court completely forecloses the possibility for any corporation to have rights under rfra, the d.c. circuit’s approach to allowing the shareholders to state a claim under rfra is correct, because it comports with the nation’s broad protections of religious freedom in accordance with formal theories of corporate law. a. the context of rfra and the nature, history, and purpose of the free exercise clause do not preclude corporate rfra claims rfra is a “super-statute” implementing congress’s interpretation of the free exercise clause by incorporating a particular jurisprudence of the supreme court. interpreters of the statute, then, must be careful not to intertwine the necessary statutory and constitutional analyses. courts will properly take notice of pre-smith jurisprudence and adjudicate the hhs mandate cases according to principles of substantial burden and compelling interests that are outside the scope of this note. in terms of who can bring claims under the statute, however, rfra’s terms must govern, and rfra grants judicial relief to persons burdened in their religious exercise. unless the government can rebut the dictionary act’s presumption that the word “person” includes corporations, the corporate plaintiffs should be able to directly state rfra claims. this section explains why congressional intent, textual interpretation, and legislative history support corporations’ ability to state rfra claims and concludes that the federal judiciary should begin developing a jurisprudence of corporate rfra rights.   . see id. . see id. at – . . see supra part ii.a. . see supra part ii.c. . see supra note and accompanying text. . see supra note and accompanying text. . see supra note and accompanying text. . see supra note and accompanying text. fordham law review [vol. . context matters: congress’s intent to restore the pre-smith regime determining who are the “persons” exercising religion under rfra requires a proper statutory analysis. this note suggests that the analytic frameworks of the seventh and tenth circuits and the third circuit present opposite forms of analysis, and that the former frames the issue correctly. the hobby lobby and korte courts first approached the hhs mandate issue by determining what rfra protected and then by analyzing its text and purpose. in contrast, the conestoga court proceeded directly to the free exercise clause, determined that the clause failed the bellotti test for protecting corporations, and determined that corporations could receive neither constitutional nor statutory protection. the framework matters because rfra appears to place a wider fence around religious liberty than the smith court did in its explanation of the free exercise clause. in theory, this will help religious actors avoid clashes between their beliefs and the regulatory state. the context of a rfra analysis should include an understanding of what the statute aimed to restore. from sherbert to smith, the courts engaged in an enterprise that looked similar to a common law adjudicative process that entertained individual claims, resulting in a jurisprudence that later courts could apply. unlike a true common law system, however, the sherbert line of cases carried the imprimatur of constitutional prescription. the smith court rejected the sherbert compelling interest test in part because it believed that judges were not suited to make the type of case-by-case balancing determinations required by sherbert. using its own considered judgment of the constitution, congress enacted rfra against the background of smith precisely to instruct courts that they should make such determinations in the first instance, because congress believed that smith would not otherwise provide satisfactory protection of americans’ religious liberty. to rectify the wrong it perceived, congress’s legislation instructed courts to use a balancing test that measured the government’s compelling interest against the alleged burden on believers’ exercise of their religion. this concept of legislative accommodation has existed since colonial days and reinforces the   . see supra part ii.a. . see supra part ii.b. . . see supra notes – and accompanying text. but see discussion supra note (explaining that whether congress intended to expand or restore pre-smith doctrine is a subject of debate that could affect the resolution of the merits of the hhs mandate cases). . see supra note . . see supra note . . see supra note and accompanying text. . see supra part i.b. . . see supra note . . see supra note and accompanying text. . see supra part i.c. . see supra part i.c. . see supra notes – and accompanying text. ] close corporations and contraceptives american dedication to religious liberty beyond mere toleration. in addition, it mirrors the understanding—again dating from colonial times— that compelling government interests justify burdening religious exercise. rfra thus restores not only the strict scrutiny regime articulated in sherbert and yoder, but also restores the common law model in which courts can and should examine particular claims in light of previous claims. this determination involves asking whether the new claim is similar enough to previous claims, and, if so, to extend the law’s protection. if it is not similar enough, a court can distinguish and dismiss the claim. courts making such determinations should take account of cases in which courts granted exemptions, in addition to cases where courts found that a law was not a substantial burden or where the government satisfied the compelling interest test. . “person” v. “person’s exercise of religion”: the result is the same by understanding this background, it becomes clear that the seventh and tenth circuits’ analysis of persons covered by rfra is stronger than the third circuit’s approach. the analysis at this phase of the hhs mandate litigation requires statutory interpretation of rfra rather than a discussion of the free exercise clause’s contours. the key issue in each case is whether rfra’s “person” exercising religion includes secular, for-profit corporations. because the statute does not define this operative term, courts must analyze rfra’s context to determine if the default definition that includes corporations, partnerships, trusts, and other associations is appropriate, in accordance with the dictionary act. regardless of how broadly courts construe rfra’s “context,” corporations arguably fall within rfra’s reach. a. most narrow interpretation: purely textual analysis and the dictionary act at the most narrow level of interpretation, a court will look purely within the statute’s text. “person” standing alone would clearly receive the dictionary act’s default definition, because nothing in the text explicitly disclaims application to corporations. the court’s artificial person canon bolsters this position. as used in rfra, however, the word person includes the modification “person’s exercise of religion,” so the   . see supra note and accompanying text. . see supra notes – and accompanying text. . see supra notes – and accompanying text. . see supra notes – , and accompanying text. . see supra part i.b. . . see supra notes – and accompanying text. . see supra note and accompanying text. . see supra part ii.a. .a. . see supra note and accompanying text. . see supra note . fordham law review [vol. dictionary’s act context requirement might entail a broader approach. the federal courts’ definition of exercise of religion is any conduct that is motivated by a religious belief, and the free exercise clause undoubtedly protects both individual and institutional religious exercise. the corporations involved in this litigation pass this test through their actions, which include proselytization, charitable giving, closing on holy days, and providing health insurance plans that may withhold drugs and devices contrary to certain religious beliefs. these are religiously motivated actions that either for-profit or nonprofit corporations can take. in addition, protecting corporations generally fits within the purposes of rfra: among other things, congress intended to protect those who engaged in activities that a secular government would not deem “religious” but which might be important to the claimant acting in accordance with a belief. the obvious objection here is that the corporation itself does not have beliefs. this objection, however, fails to appreciate that a board of directors can direct—and many in this litigation have directed—the corporation’s bylaws, ethical statements, or statements of purpose to include religious tenets in accordance with which the corporation operates. this is similar to the free speech context, where a corporation’s “speech” comes from the direction of its officers and directors in their decisionmaking capacity. no special rules or laws govern corporate speech; it is the corporation itself that “speaks” at the direction of its board. similarly, here, when corporate directors institute bylaws, statements, or guidelines, they define corporate beliefs upon which a corporation can act. this is true of secular corporations, such as whole foods, chipotle, and facebook, seeking to institute a specific moral culture, and it is true of faith-based corporations, such as hobby lobby, seeking to institute a specific religious culture. an imperfect analogy is that the directors and officers are the “conscience” of the corporation because they direct conduct in accordance with religious belief. b. most broad interpretation: the entire free exercise jurisprudence and bellotti even at the broadest possible level of context—the supreme court’s entire free exercise jurisprudence—the argument that rfra can include corporations succeeds. because application of rfra to for-profit   . see supra notes – and accompanying text. . see supra notes – and accompanying text. . see supra note and accompanying text. . see supra part ii. . see supra note . . see supra part ii.b. . see supra notes – and accompanying text. . see supra note and accompanying text. . see supra notes , – , and accompanying text. . see supra notes , and accompanying text. ] close corporations and contraceptives corporations is a novel question, the best indication of how the court would analyze this approach is the bellotti test. here, again, the tenth and seventh circuits more fully captured the spirit of religious liberty than the third circuit. bellotti instructs that application of a certain constitutional provision depends on whether the provision is “purely personal,” as measured by the nature, history, and purpose of the clause. the third circuit examined the history of the free exercise clause and correctly found that the supreme court has never found that a secular corporation can exercise religion. this analysis, however, is incomplete because it fails to consider the nature and purpose prongs of bellotti and relies almost exclusively on the history prong. this leads to a weak appreciation of instances in which individuals pursuing profit successfully petitioned courts for relief from laws that burdened their religious practice. in addition, the third circuit’s analysis of the nature and purpose of religious liberty is lacking, because it fails to appreciate that the nature of free exercise is to protect conduct more broadly than conscience. moreover, additional purposes of religious liberty are to ensure that government does not encroach on religion and to maintain the vibrant pluralism that has been part of the american fabric since the nation’s founding. instead, the conestoga court simply held that no prior cases of constitutional corporate religious exercise meant no future cases of statutory corporate religious exercise. the conestoga court’s rejection of bellotti was premature because bellotti contained language about corporate first amendment rights generally, and not just about the free speech clause. rfra, in turn, explicitly instructs courts to engage in an assessment of free exercise claims according to pre-smith jurisprudence, which includes the potential for a bellotti analysis. the third circuit, therefore, missed a crucial part of the analysis by engaging in a purely constitutional and historical discussion and not engaging with rfra’s text. the hobby lobby, korte, and gilardi courts provided a more robust analysis of the nature, history, and purpose of what the right to free exercise protects. indeed, they addressed the issue in the manner that the bellotti court instructed: by analyzing what the right protects, rather than whether a corporation can claim that right. the conestoga court did exactly the opposite. a bellotti analysis should take account of what congress would   . see supra notes , and accompanying text. . see supra note and accompanying text. . see supra part ii.b. .a. . see supra part ii.b. . see supra part i.e. . . see supra note and accompanying text. . see supra note and accompanying text. . see supra part i.a. . see supra part ii.b. . . see supra part ii.b. . . see supra note and accompanying text. . see supra note and accompanying text. . see supra part ii.a. . see supra note and accompanying text. fordham law review [vol. know in legislating rfra: america’s commitment to religious liberty from colonial days, the fact that free exercise protects conduct over conscience, the long-standing definition of “persons” as including corporations unless otherwise specified, and past statutes that show congress knows how to create corporate or religious exemptions. the tenth and seventh circuits incorporate these principles and show how rfra can and should cover at least some corporations. the government’s position includes distinguishing between for-profit and nonprofit corporations and calling that distinction determinative. the practice of allowing nonprofit corporations to state claims for violations of their free exercise of religion during the sherbert regime, however, shows that the mere act of incorporation does not destroy religious liberty. moreover, a profit motive cannot be determinative, as the supreme court has allowed individual business owners to state free exercise claims. in addition, as a normative matter, first amendment issues can trump formal issues concerning the corporate form. each of these points form a part of the pre-smith jurisprudence that congress restored with rfra, and nothing in that jurisprudence expressly excludes for-profit corporations. rather, congress wanted courts to engage in a balancing act of rfra’s contours, and it firmly offered its support to religious liberty by enacting a strict scrutiny test. this is the important context for bellotti purposes and should inform any analysis of rfra. . a brief note on legislative history some have noted that rfra’s legislative history, such as its committee reports, does not mention its application to corporations. few, however, have noted that prior versions of rfra introduced in congress did, in fact, define the persons the statute meant to protect. prior legislative history is frequently an unreliable source of legislative intent, especially in the absence of materials explaining changes and amendments, but rfra’s history may present a plausible lesson to a judge willing to engage in this form of analysis. at a minimum, the prior versions of the statute show that   . see supra part i.a. . see supra part i.b. . see supra note and accompanying text. . see supra notes – and accompanying text. . see supra notes – and accompanying text. . see supra part i.e. . . see supra part i.e. . . see supra notes – and accompanying text. . see steven d. smith & caroline mala corbin, debate, the contraception mandate and religious freedom, u. pa. l. rev. online , ( ), available at http://www.pennlawreview.com/online/ -u-pa-l-rev-online- .pdf. . see supra note . for others recognizing the definition of “persons” in proposed rfras, see lloyd hitoshi mayer, politics at the pulpit: tax benefits, substantial burdens, and institutional free exercise, b.u. l. rev. , ( ), and caroline mala corbin, corporate religious liberty (august ) (manuscript at – ), available at http://ssrn.com/abstract= . . see supra note . ] close corporations and contraceptives congress at times contemplated defining “person.” in the initial draft, rfra protected individuals and religious organizations, corporations, and associations. the next version omitted the religious modifier and, without any modification, protected individuals, corporations, organizations, associations, and other entities. this analysis yields the argument that successive versions of the proposed statute protected respectively larger categories of potential claimants. the initial version protected individuals and religious groups; the next version dropped the religious modifier, seemingly offering the proposed statute’s protection to a broader constituency. indeed, the potential claimants in the second version would largely mirror those protected if the word “person” were not defined and the default dictionary act definition applied. congress plausibly dropped the definition to avoid excess language where the dictionary act would apply. . the corporate form and its relevance to the hhs mandate notably, the court’s corporate first amendment jurisprudence has not settled on a theory of corporate personhood. indeed, in , the citizens united court appeared to rely on two different theories to reach its holding. rfra could accommodate either theory. if the court relies on the contractual theory that views a corporation as an association of shareholders, it could hold that a closely held corporation can exercise religion in much the same way it has allowed churches or religious groups to state rfra and free exercise claims. the court has not previously held that the mere act of incorporation restricts such a claim. if the court uses the real entity theory of corporations, then corporate religious exercise, such as proselytizing through advertisements or refusing to fund contraception through insurance plans, is religious conduct performed directly by the corporation. the individuals who direct the corporation would elect to maintain such a religious entity. . why the common law model is crucial having explored whether rfra’s context precludes corporations from stating rfra claims, this note returns again to the common law model congress directed the courts to create. two points will reinforce that this analysis of corporate rfra rights has important limits.   . see supra note . . see supra note . . see supra notes – and accompanying text. . see supra notes – and accompanying text. . see supra notes – and accompanying text. . see supra note and accompanying text. fordham law review [vol. a. congress has the final say first, if courts allow secular corporations to state rfra claims, then congress can react. it did, after all, expressly legislate a balancing test to replace the supreme court’s abandoned, constitutionally compelled sherbert jurisprudence. moreover, it seemingly did so in response to smith’s invitation to be solicitous of religious liberty and in accordance with boerne’s allowance for congressional action based on its own interpretation of the constitution. congress, however, retained final say over courts’ decisionmaking, at least for federal purposes, as it always does when courts interpret its laws. as enacted, rfra appears to grant protection to corporations. if the supreme court holds this to be true and congress disagrees, then congress can amend the law accordingly. conversely, if the court denies standing under rfra, then congress can determine otherwise and adjust the law as needed. the important point is that the court should examine this claim as a statutory matter and not a constitutional one. b. courts should develop a jurisprudence to determine the contours of corporate rfra claims second, rfra allows the court to develop a statutory jurisprudence. this is in contrast to the sherbert regime, which was based in the constitution. as justice sotomayor explained in her in-chambers opinion, the hhs mandate litigation presents a novel claim for religious liberty. the court should not attempt to answer the rfra question for all corporate forms in one fell swoop. corporate free speech provides an analogue to this point. as the d.c. circuit noted, citizens united only emerged after decades of excruciating analysis of the corporate speech right. the court did not allow corporations to claim the protections of the free speech clause overnight. similarly, courts addressing the hhs mandate cases should not define these cases as determining whether all—or even most—corporations have rfra rights. the corporate plaintiffs in the cases percolating in the federal courts are organized as closely held corporations. the owners of such corporations also frequently tend to be the directors and managers, and they might make business decisions that subvert their desire for profit to follow the commands of their religion. for this reason, a closely held corporation might state a claim under rfra if its directors and owners instruct it to follow religious conduct, and a law burdens that conduct. as judge sykes   . see supra note and accompanying text. . see supra note and accompanying text. . see supra note and accompanying text. . see supra note and accompanying text. . see supra note . . see supra note and accompanying text. . see supra part ii. . see supra notes – and accompanying text. ] close corporations and contraceptives indicated, the individual owners do not compartmentalize their lives into business and religion, so rfra’s purposes could plausibly accommodate an expansion of existing law to cover certain business. in fact, the court might find that allowing closely held corporations to state rfra claims will encourage individual flourishing, support religious pluralism, and provide an important mediating layer between persons and government—all of which are purposes of religious liberty. for the same reasons, a multinational, publicly held corporation will likely struggle to have the same dedication to a religious belief and could very easily fall outside of rfra’s protection. its owners and directors likely will not present a unified religious face, and the profit-generating capacity of the business will act by necessity to compartmentalize the individual shareholders and directors’ religious and business lives. simply put, a more diverse ownership will likely not tolerate a religious accommodation in its pursuit of profit. moreover, such corporations are unlikely to be seen as promoting human flourishing or religious pluralism in the way that small, community-oriented businesses might be. only after a developed jurisprudence emerges, however, will the full contours of this issue be visible. to cut such a nascent jurisprudence off now, however, would be an injustice to religious liberty and those religious believers who see their small businesses as an extension of their religious life. corporations should have the ability to state rfra claims, and the court should allow the federal judiciary to engage in an adjudicative process that seeks to determine the contours between religious liberty and corporate form. b. if the court precludes all corporations from rfra, then the d.c. circuit provides an alternative approach if the supreme court rejects the above analysis, it should affirm the d.c. circuit’s shareholder-standing exception. although the details of this standing rule are beyond the scope of this note, the gilardi court provided a persuasive reason for why a close corporation should be able to state the claims of its shareholders. incorporation should not equal the disappearance of a constitutional or statutory right without a corporate analogue. if a sole proprietor or a nonprofit corporation may claim protection, then it is a perverse incentive to religious believers to withhold protection upon incorporation. this shareholder exception would allow owners to state claims that their corporations are otherwise precluded from litigating. doing this would preserve the american tradition of protecting   . see supra note and accompanying text. . see supra note and accompanying text. . see willis, supra note , at – . . cf. supra note (listing scholars and an activist group that reject the notion of rights for corporations). . see supra part ii.c. . see supra notes – and accompanying text. fordham law review [vol. religious liberty while also maintaining a formal respect for the corporate form. conclusion some other aspect of the rfra analysis might conceivably doom hobby lobby and other for-profit corporations in their quest for an exemption from the hhs mandate. courts might find that providing the contraceptives to which they object may not be a substantial burden or that the government has a compelling interest to override the plaintiffs’ objections. this will undoubtedly be the source of much future litigation. the question of these corporations’ ability to exercise religion and their standing under rfra, however, should not be the fatal aspect. the support given to religious liberty by congress, the supreme court, the founders, and, ultimately, the american people is simply too great for such a result to be satisfactory. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . 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publishing corporation case reports in medicine volume , article id , pages doi: . / / case report successful tricuspid valve replacement in a patient with severe pulmonary arterial hypertension and preserved right ventricular systolic function jamil a. aboulhosn, ronald j. oudiz, amish s. dave, abbas ardehali, and david j. ross division of cardiology, david geffen school of medicine at ucla, los angeles, ca , usa los angeles biomedical research institute at harbor, ucla medical center, los angeles, ca , usa division of cardiothoracic surgery, david geffen school of medicine at ucla, los angeles, ca , usa division of pulmonary and critical care medicine, david geffen school of medicine at ucla, los angeles, ca , usa correspondence should be addressed to jamil a. aboulhosn, jaboulhosn@mednet.ucla.edu received february ; accepted may recommended by frank a. pigula a -year-old patient with severe pulmonary hypertension developed severe tricuspid regurgitation, right-sided heart failure, and congestive hepatopathy. she was transferred for possible lung transplant and/or tricuspid valve surgery. clinical and echocardiographic assessment provided confidence that acute tricuspid valve failure was responsible for the decompensation and that tricuspid valve replacement despite pulmonary hypertension could be performed. copyright © jamil a. aboulhosn et al. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. . case presentation a- -year old hispanic female with idiopathic pulmonary arterial hypertension (pah) for years was stable (who class ii) on subcutaneous treprostinil for years. in , diagnostic right heart catheterization had shown elevated right atrial (mean mmhg) and pulmonary artery (pa) pressure ( / mmhg) with normal pulmonary capillary wedge pressure ( mmhg). the patient began complaining of fatigue, lethargy, and dyspnea on minimal exertion in january and was admitted in february for syncope, anorexia, abdominal pain, jaundice, and decreasing urine output. an echocardiogram revealed right ventricular hyper- trophy with severe tricuspid regurgitation (tr) and pre- served right ventricular (rv) systolic function. in may she was admitted to an outside hospital for persistent symp- toms and resistant volume overload. physical examination demonstrated jaundice, ascites, and right heart failure. her jugular venous pressure was cm h o. a systolic rv heave was present. there was a / holosystolic murmur at the left sternal border, a loud pulmonary component of the second heart sound and an rv s gallop. the patient was moderately hypotensive (systolic blood pressure of mmhg), and room air oxygen saturation was %. an echocardiogram demon- strated severe tr with a flail anterior leaflet (figure ), which was not previously noted. estimated pa pressure had fallen to / of systemic (figure (a)). m-mode echocardiography demonstrated diastolic leftward septal motion consistent with rv volume overload (figure ). rv systolic function was unchanged, and there was no tv annular dilation. retrospective review of hepatic function aroused sus- picion for acute decompensated rv failure with conges- tive hepatopathy occurring several months earlier, possibly related to the flail tricuspid leaflet. upon review of an echocardiogram performed three months prior, the flail tricuspid leaflet was indeed found to be present. because of the temporal relationship between the severe tr and worsening symptoms as well as rv failure and hepatic dysfunction despite preserved rv systolic function, it was felt that tricuspid valve (tv) failure alone could explain the patient’s symptoms and that tv repair or replacement might benefit the patient. the patient was continued on subcutaneous treprostinil ( ng/kg/min) and treated with diuretics and digoxin then case reports in medicine rv ra tv flail (a) tr . . (b) figure : (a) transthoracic d echo parasternal long-axis systolic view of a flail anterior tricuspid valve leaflet (tv). right atrium (ra) and right ventricle (rv) are labeled. the tricuspid valve annulus is not dilated, measuring cm in maximum diameter. (b) color flow doppler demonstrating severe tricuspid regurgitation (tr). cw focus = mm cw gain = v = - . m/s pg = . mmhg m/s cw: mhz . db (a) pw: mhz . . m/s db ./ / / pw depth = mm pw gate = . mm pw gain = - db v = . m/s pg = . mmhg (b) figure : (a) continuous wave doppler profile of tricuspid regurgitation in the parasternal long-axis view. the tricuspid valve regurgitant velocity is . m/s. using the modified bernoulli equation (Δp = v ) and assuming a right atrial pressure of mmhg, the estimated pulmonary artery systolic pressure is mmhg. (b) tissue doppler profile of the tricuspid valve annulus demonstrates a systolic velocity of . cm/sec consistent with preserved right ventricular function [ , ]. transferred to our institution for evaluation for possible lung transplant and/or tv surgery. repeat right heart catheterization demonstrated elevated right atrial pressures (v = mmhg, mean = mmhg), moderately elevated pa pressure ( / mmhg), pulmonary capillary wedge pressure at upper limits of normal (mean mmhg), decreased cardiac index by thermodilution ( . l/min/m ), and elevated pulmonary vascular resistance ( . wood units). chest ct demonstrated marked dilation of the main and proximal branch pulmonary arteries with mural calcification and a moderate pericardial effusion. ventila- tion/perfusion scan showed low probability for thromboem- bolism. laboratories showed platelet count of /µl, total bilirubin of . mg/dl, serum creatinine of . mg/dl, and albumin of . g/dl. repeat echocardiogram with agitated saline demonstrated excellent biventricular systolic function, absence of tricuspid annular dilation, and late (> cycles) right-to-left shunting suggesting a pulmonary right-to-left shunt. the patient received empiric steroids for possible idiopathic thrombocytopenic purpura after other case reports in medicine rv cal = mm v c h . mhz ucla echo ucla echo store in pro hr = bpm sweep = mm db ./ / ./ m gain = db s pw pef lv figure : m-mode echo, parasternal short-axis, demonstrating leftward displacement of the septum during diastole (white arrows), consistent with right ventricular volume overload. right ventricle (rv), septum (s), left ventricle (lv), posterior wall (pw), and pericardial effusion (pef). etiologies were excluded, with improvement of her platelet counts. the patient also developed recurrent atrial flutter. because of her hyperbilirubinemia and thrombocytope- nia, she was felt to be a poor candidate for lung trans- plantation. it was concluded that without tv surgery, the patient’s long-term outcome would be poor and that surgery was indicated. the recommendation was buttressed by the presence of normal rv systolic function with normal tv tissue doppler systolic annular velocity (figure (b)) and the abrupt clinical deterioration suggesting sudden disruption of valve integrity leading to poorly tolerated severe tr. the patient was presented with the option of high-risk surgical intervention and consented to tv surgery. two weeks later the patient underwent tv replacement. treprostinil was held immediately prior to surgery. intra- operative inspection of the tv revealed ruptured chor- dae with a flail, prolapsing anterior leaflet, and normal annular size. a patent foramen ovale was not present. valve repair was not performed due to friability of the subvalvular apparatus and concern regarding longevity of repair given her pulmonary hypertension. a stented mm magna (edwards life sciences) bioprosthesis was selected for tv replacement. right atrial maze cryoablation and ligation of the left atrial appendage were also performed. pa systolic pressure increased to mmhg postvalve replace- ment (preintervention baseline mmhg). the patient was successfully weaned from cardiopulmonary bypass on intra- venous epinephrine, dopamine, milrinone, inhaled nitric oxide ( parts per million), and intravenous prostacyclin ( ng/kg/min). total cardiopulmonary bypass time was minutes, and aortic cross-clamp time was minutes. catheterization on postoperative day demonstrated pa pressure of / mmhg and improved cardiac index of . l/min/m and a pulmonary vascular resistance of . wood units. repeat echo demonstrated decreased rv size, normal systolic function, and mild high pressure tr. she was extubated on postoperative day and discharged from the intensive care unit days postoperatively. she was transitioned from intravenous prostacyclin to subcutaneous treprostinil ( ng/kg/min) and oral sildenafil ( mg twice daily). she was discharged on postoperative day on the above medications as well as digoxin, prednisone, spironolactone, bosentan, and furosemide. she was seen in clinic three months postoperatively and had returned to her baseline functional status (who class ii). the jaundice, ascites, hypoalbuminemia, rv failure, and liver abnormalities had resolved. . discussion there is a paucity of data on the incidence, prevalence, and prognosis of severe tr in patients with pah not due to elevated left heart filling pressures. it has long been thought that tv surgery in patients with severe pulmonary hypertension without mitral valve disease carried an unac- ceptably high risk of mortality. this patient’s presentation is not unique; although uncommon, tricuspid chordal rupture can occur in patients with pah. however, the use of multiple clinical and imaging modalities to determine that tv surgery without concomitant lung transplantation could be successfully performed is novel. the relatively sudden deterioration after a long period of clinical stability was suspicious for an abrupt event leading to right heart failure and hepatic dysfunction. chronic tr secondary to rv enlargement and systolic failure is more likely to result in gradual functional decline. absence of rv systolic dysfunction or tricuspid annular dilation and presence of a flail leaflet all pointed to chordal rupture as the index event leading to clinical deterioration. in patients with implantable pacemakers and cardioverter-defibrillators, disruption of the tricuspid valve apparatus by the device lead must also be considered [ ]. echocardiography with doppler is a readily available, cost-effective, and accurate tool for the anatomic, hemody- namic, and functional assessment of cardiopulmonary status in patients with pulmonary hypertension [ , , , ]. at centers that do not employ tissue doppler, tricuspid annular plane systolic excursion (tapse) may be used to evaluate right ventricular function [ ]. computed tomography and magnetic resonance imaging can also provide an excellent assessment of right ventricular size and function [ , ]. pulmonary vasodilator therapy in pah is associated with improved quality of life and functional performance [ ]. this patient was managed with subcutaneous and intravenous prostacyclin prior to, during, and following surgery. both phosphodiesterase inhibitions with sildenafil and endothelin antagonism with bosentan were utilized in this patient. perioperative mortality and morbidity risk is not low, and difficult intra- and postoperative courses are common in patients with advanced pah. in conclusion, this report describes successful tv replacement in a patient with severe pah unrelated to mitral valve disease presenting with severe rv failure and hepatic dysfunction. clinical and echocardiographic assessments suggested that increased tr severity was secondary to abrupt case reports in medicine chordal rupture. rv systolic function and tricuspid annular diameter were preserved, supporting the hypothesis that tv competence would be well tolerated. importantly, in patients with significantly impaired rv function, tricuspid valve surgery carries an extremely high risk of mortality and is generally contraindicated. valve replacement was chosen over valve repair given the absence of annular dilation, the friable nature of the subvalvular apparatus, and the concern regarding long term durability of valve repair given her pulmonary hypertension. a bioprosthetic valve was selected over a more durable mechanical prosthesis because of the patient’s thrombocytopenia and the wish to avoid chronic anticoagulation. the patient has done remarkably well in the twelve months following surgery. however, the long- term durability of a bioprosthetic valve in the tricuspid position in a patient with severe pulmonary hypertension is unclear. finally, the availability of oral, subcutaneous, inhaled, and intravenous pulmonary arterial vasodilators in the current era, in addition to improved multimodality diagnostic accuracy necessitates reconsideration of a long discarded surgical option. references [ ] n. rajagopalan, n. saxena, m. a. simon, k. edelman, m. a. mathier, and a. lopez-candales, “correlation of tricuspid annular velocities with invasive hemodynamics in pulmonary hypertension,” congestive heart failure, vol. , no. , pp. – , . [ ] s.-h. hsiao, s.-k. lin, w.-c. wang, s.-h. yang, p.-l. gin, and c.-p. liu, “severe tricuspid regurgitation shows significant impact in the relationship among peak systolic tricuspid annular velocity, tricuspid annular plane systolic excursion, and right ventricular ejection fraction,” journal of the american society of echocardiography, vol. , no. , pp. – , . [ ] g. lin, r. a. nishimura, h. m. connolly, j. a. dearani, t. m. sundt iii, and d. l. hayes, “severe symptomatic tricuspid valve regurgitation due to permanent pacemaker or implantable cardioverter-defibrillator leads,” journal of the american college of cardiology, vol. , no. , pp. – , . [ ] e. k. louie, s. rich, s. levitsky, and b. h. brundage, “doppler echocardiographic demonstration of the differential effects of right ventricular pressure and volume overload on left ventricular geometry and filling,” journal of the american college of cardiology, vol. , no. , pp. – , . 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[ ] d. b. badesch, s. h. abman, g. simonneau, l. j. rubin, and v. v. mclaughlin, “medical therapy for pulmonary arterial hypertension: updated accp evidence-based clinical practice guidelines,” chest, vol. , no. , pp. – , . zurich open repository and archive university of zurich main library strickhofstrasse ch- zurich www.zora.uzh.ch year: microbiome and asthma sokolowska, milena ; frei, remo ; lunjani, nonhlanhla ; akdis, cezmi a ; o’mahony, liam abstract: the mucosal immune system is in constant communication with the vast diversity of microbes present on body surfaces. the discovery of novel molecular mechanisms, which mediate host-microbe communication, have highlighted the important roles played by microbes in influencing mucosal immune responses. dendritic cells, epithelial cells, ilcs, t regulatory cells, effector lymphocytes, nkt cells and b cells can all be influenced by the microbiome. many of the mechanisms being described are bacterial strain- or metabolite-specific. microbial dysbiosis in the gut and the lung is increasingly being associated with the incidence and severity of asthma. more accurate endotyping of patients with asthma may be assisted by further analysis of the composition and metabolic activity of an individual’s microbiome. in addition, the efficacy of specific therapeutics may be influenced by the microbiome and novel bacterial- based therapeutics should be considered in future clinical studies. doi: https://doi.org/ . /s - - -y posted at the zurich open repository and archive, university of zurich zora url: https://doi.org/ . /uzh- journal article published version the following work is licensed under a creative commons: attribution . international (cc by . ) license. originally published at: sokolowska, milena; frei, remo; lunjani, nonhlanhla; akdis, cezmi a; o’mahony, liam ( ). micro- biome and asthma. asthma research and practice, : . doi: https://doi.org/ . /s - - -y https://doi.org/ . /s - - -y https://doi.org/ . /uzh- http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / https://doi.org/ . /s - - -y review open access microbiome and asthma milena sokolowska , , remo frei , , nonhlanhla lunjani , , , cezmi a. akdis , and liam o’mahony * abstract the mucosal immune system is in constant communication with the vast diversity of microbes present on body surfaces. the discovery of novel molecular mechanisms, which mediate host-microbe communication, have highlighted the important roles played by microbes in influencing mucosal immune responses. dendritic cells, epithelial cells, ilcs, t regulatory cells, effector lymphocytes, nkt cells and b cells can all be influenced by the microbiome. many of the mechanisms being described are bacterial strain- or metabolite-specific. microbial dysbiosis in the gut and the lung is increasingly being associated with the incidence and severity of asthma. more accurate endotyping of patients with asthma may be assisted by further analysis of the composition and metabolic activity of an individual’s microbiome. in addition, the efficacy of specific therapeutics may be influenced by the microbiome and novel bacterial-based therapeutics should be considered in future clinical studies. keywords: asthma, microbiome, bacteria, mucosal immune system, immune tolerance, short-chain fatty acids, histamine background an enormous number of microbes colonize the skin and mucosal body surfaces. these microbes are highly adapted to survive within complex community struc- tures, utilizing nutrients from other microbes and/or host processes. the microbiome is defined as the sum of these microbes, their genomic elements and interactions in a given ecological niche. the composition and diver- sity of the microbiome varies across body sites, resulting in a series of unique habitats within and between indi- viduals that can change substantially over time [ ]. the establishment of stable microbial communities closely tracks host growth and immune development during the first few years of life. factors that influence this evolu- tion include antibiotic use, birth mode, infant nutrition and biodiversity in the home, surrounding environment and in family members [ ]. delayed or altered establish- ment of these microbial communities’ leads to micro- biome immaturity and has been associated with increased risk of allergies and asthma later in life. highly sophisticated mucosal immune cellular and molecular networks need to be constantly coordinated in order to tolerate the presence of a large number and diversity of bacteria, while protective immune responses to potential pathogens must be maintained and induced on demand. the balance between immune tolerance and inflammation within tissues is regulated in part by the crosstalk between immune cells and the microbiome [ ]. disrupted communication between the microbiome and the host due to altered microbiome composition and/or metabolism is thought to negatively influence immune homeostatic networks. this can be clearly seen in mice bred under germ-free (gf) or sterile conditions, whereby mucosal tolerance mechanisms do not fully develop and these mice display increased allergic responses to aller- gen challenge. in this review, we will examine the potential mechanisms by which the microbiome influences immune responses within the lung and assess the evidence for a dysbiotic microbiome in the gut and the respiratory tract of asthma patients. in addition, we will summarize the current thera- peutic approaches and challenges associated with microbial-based therapies in asthma patients and highlight the future research and clinical needs in the field. immune mechanisms influenced by the microbiome multiple mechanisms have now been described, through which bacteria can induce regulatory responses or dampen inflammatory processes. both bacterial cell wall components and metabolites from the microbiome have * correspondence: liam.omahony@siaf.uzh.ch swiss institute of allergy and asthma research, university of zürich, obere strasse , davos, switzerland full list of author information is available at the end of the article © the author(s). open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/ . /) applies to the data made available in this article, unless otherwise stated. sokolowska et al. asthma research and practice ( ) : doi . /s - - -y http://crossmark.crossref.org/dialog/?doi= . /s - - -y&domain=pdf mailto:liam.omahony@siaf.uzh.ch http://creativecommons.org/licenses/by/ . / http://creativecommons.org/publicdomain/zero/ . / been associated with immunoregulatory effects within the mucosa. certain commensal microbes such as spe- cific bifidobacterium, lactobacillus and clostridium strains have been shown to increase the proportion of t regulatory cells in mice [ – ]. in addition, clostridia have been shown to stimulate ilc s to produce il- , which helps to reinforce the epithelial barrier and reduces the permeability of the intestine to dietary pro- teins [ ]. furthermore, bifidobacteria and lactobacilli can stimulate metabolic processes in dendritic cells, such as vitamin a metabolism, tryptophan metabolism and heme oxygenase- , which promote induction of t regu- latory cells [ – ]. the capsular polysaccharide a from bacteroides fragilis has been shown to interact directly with mouse plasmacytoid dendritic cells and thereby promoted il- secretion from cd + t cells [ ]. in addition, an exopolysaccharide from bifidobacterium longum was recently shown to suppress th responses within the gut and within the lung [ , ]. notably, consumption of bifidobacterium longum , by healthy human volunteers increased foxp + t regula- tory cells in peripheral blood, while administration of this bacterial strain to psoriasis patients, chronic fatigue syndrome patients or ulcerative colitis patients consist- ently resulted in reduced levels of serum proinflamma- tory biomarkers such as crp, possibly mediated by increased numbers of t regulatory cells [ , ]. in addition to bacterial-associated components, bacterial-derived metabolites have significant effects on immunoregulatory processes. short-chain fatty acids (scfas), such as acetate, propionate and butyrate, are produced by the gut microbiota and have been shown to influence dendritic cell and t cell responses, via their binding to g protein-coupled receptors and their inhib- ition of histone deacetylases, thereby promoting epigen- etic changes [ ]. bacteria within the human gut can produce a wide range of biogenic amines (due to metab- olism of amino acids), which can also influence immune and inflammatory responses [ ]. interestingly, in murine models, microbiota-derived taurine, histamine, and spermine were shown to influence host-microbiome interactions by co-modulating nlrp inflammasome signaling, epithelial il- secretion, and downstream anti-microbial peptide secretion [ ]. microbiome in animal models of asthma a number of different animal studies support the con- cept for a role of the microbiome in development of airway diseases. in particular, valuable insights for the mechanistic role of the microbiome in the development of allergic airway inflammation comes from gf animals, lacking any exposure to pathogenic or nonpathogenic microorganisms. herbst et al. observed that ova- induced type airway inflammation and airway hypersensitivity is much stronger in gf mice as com- pared to the mice from a specific pathogen-free environ- ment (spf) that were colonized with commensal microbes. moreover, the exaggerated allergic inflamma- tion in gf mice could be reduced to the same level observed in spf mice, when gf mice were co-housed for weeks with spf mice, suggesting that gut and airways recolonization with commensal microbes had protective effects [ ]. in addition, early life colonization of gf mice prevented invariant natural killer t cell accu- mulation in the gut lamina propria and the lungs thereby reducing the severity of allergic airway re- sponses. later life colonization had no effect on disease phenotypes nor on the development of regulatory t cells or on invariant natural killer t cells [ ]. furthermore, antibiotic treatment of neonatal mice resulted in fewer regulatory t cells and a more pronounced t helper cell type response, which was prevented by re-introducing a commensal intestinal microbiota [ – ]. gollwitzer et al. examined the susceptibility to house dust mite (hdm)-induced allergic airway inflammation in mice of different ages ( , and days), simulating the conditions of gradual colonisation of the human infant airways [ ]. neonatal mice were prone to de- velop exaggerated airway eosinophilia, they released more type cytokines and exhibited higher airway hyper-responsiveness following exposure to hdm com- pared to mice that were older. this protective effect in older mice was associated with the colonization of the mouse lungs with increased numbers of bacteria and the shift from a predominance of gammaproteobacteria and firmicutes to bacteroidetes. the maturation of the lung microbiota was associated with the pdl- -dependent emergence of helios-negative t regulatory cells. this study suggests that the absence of specific bacterial species early in life could influence appropriate regulatory mechanisms later in life and subsequently shift the immunological balance towards allergy instead of tolerance [ ]. oral supplementation of mice with specific microbes such as bifidobacterium breve, clostridium clade iv and xiv species, or with the capsular polysaccharide psa of bacteroides fragilis induced an anti-inflammatory re- sponse associated with induction of regulatory t cells and il- secretion that attenuated allergic airway in- flammation [ , ]. in addition to the gut-induced regulatory t cells that could migrate to the lungs to pro- vide anti-inflammatory effects, there are metabolites produced by the microbiome such as scfas that are absorbed and potentially have direct effects on lung im- mune responses [ ]. deliberate administration of scfas, or dietary fibers that are metabolized to scfas, has repeatedly been shown to reduce airway inflamma- tion in murine models. a high-fiber diet increased the sokolowska et al. asthma research and practice ( ) : page of level of colonic bacteroidetes and actinobacteria species and decreased firmicutes and proteobacteria, which was associated with increased scfa serum levels and sup- pression of allergic airway-inflammation in mice [ ]. the beneficial effect was transferred to the offspring after treatment of pregnant mice via epigenetic mecha- nisms [ , ]. the influence of the microbiota on d- tryptophan, vitamin a or biogenic amine metabolism can also modulate t helper cell type mediated allergic airway inflammation within the lung [ , , , ]. several studies have suggested that direct exposure of the murine respiratory tract to microbial products such as endotoxin, cpg-containing oligonucleotides or other toll-like receptor ligands could inhibit the classical fea- tures of asthma [ , ]. for example, intranasal exposure to the bacterium escherichia coli was protective in the ova-induced allergic airway inflammation model [ ]. these studies were recently expanded by novel findings that linked the protective effect of the farm environment with the microbiota and endotoxin levels in the house dust. schuijs et al. demonstrated that prolonged exposure to low-dose endotoxin or farm dust protected mice from hdm-induced asthma via a (tnfaip )-dependent air- way epithelial cells-dendritic cells interactions [ ]. stein et al. further demonstrated that intranasal installation of the dust from amish houses, but not dust from hutterite homes, reduced ova-induced allergic airway inflamma- tion in mice, via myd and trif-dependent mechanisms [ ]. the dust from the amish homes had different bacterial populations (especially higher in bartonellaceae) and higher endotoxin levels as compared to hutterite houses’ dust [ ]. the role of the gut microbiome in asthma the human gut microbiome is the largest collection of bacteria in the body, consisting of – distinct bacterial species with more than million genes poten- tially influencing the host immune system [ , ]. euro- pean adults’ gut microbiota is predominantly colonized by bacteroidetes, firmicutes, actinobacteria, proteobac- teria, and verrucomicrobia. the stomach, duodenum, and proximal small intestine are mainly colonized with aerobic bacteria including streptococci species, lactoba- cilli species, and enterobacteriaceae while anaerobes such as bacteroides, bifidobacterium, prevotellaceae, rikenellaceae, lachnospiraceae, ruminococcaceae, and clostridium species dominate the distal small intestine and the colon [ , ]. the gut microbiota can influence immune responses at distant sites (such as the lung) via multiple mechanisms. for example, it was recently shown that there is an increase in the number of bac- teria capable of secreting histamine from the gut of adult asthma patients, compared to healthy volunteers [ ]. however, it is not clear if increased secretion of histamine by gut microbes can have an overall detrimen- tal or protective effect as histamine can induce protect- ive responses in the lung via histamine receptor and detrimental effects via histamine and receptors [ ]. the composition of the gut microbiome is thought to reach an adult-like diversity by years of age. develop- ment of the early life gut microbiome is influenced by many environmental factors, such as living in microbial rich environments (e.g. on a farm or with frequent contact to livestock and pets), or a diverse diet, which have been inversely associated with childhood asthma [ – ]. it is thought that exposure to and colonization by certain mi- crobes at the correct time during early life is important for gut development, immune cell maturation and resistance to pathogens, all of which may protect against the devel- opment of asthma [ , , ]. the mode of delivery has a significant influence on colonization. babies delivered via caesarean section typically have more staphylococcus spe- cies, bacillales, propionobacterineae, corynebacterineae, firmicutes and acinetobacter species with fewer actino- bacteria and bacteroidetes, while vaginal delivery has been linked to increased colonization with clostridia [ , ]. clostridia metabolize fibers to scfas, which can have systemic anti-inflammatory effects as described above. in addition to delivery mode and diet, maternal antibiotic use during pregnancy or antibiotic treatment in early childhood significantly disrupts the microbiota and was associated with long-lasting effects such as decreased actinobacteria and increased bacteroidetes and proteo- bacteria [ ]. several studies have linked early life dysbiosis of the gut microbiota with an altered risk of asthma later in life. colonization by clostridium difficile at month of age was associated with wheeze throughout the first to years of life and with asthma at age to years [ ]. children that developed asthma at school age, had a lower gut microbiome diversity at week or month of age, but not at year of age, compared to non-asthmatic children [ ]. in another study, the early life relative abundance of the bacterial genera lachnospira, veillo- nella, faecalibacterium, and rothia was significantly decreased in children at risk of asthma. this dysbiosis was accompanied by reduced levels of fecal acetate and dysregulation of enterohepatic metabolites [ ]. in addition, neonates with the lowest relative abundance of bifidobacteria, akkermansia and faecalibacterium and a higher relative abundance of particular fungi (candida and rhodotorula), had the highest risk to develop atopy and asthma [ ]. thus, early life dysbiosis of the gut microbiota has been consistently associated with an in- creased risk of asthma later in life. however, it remains unclear if microbial dysbiosis within the gut can actually drive relevant disease promoting mechanisms or if dys- biosis simply reflects associated phenomena such as sokolowska et al. asthma research and practice ( ) : page of altered patterns of immune response to microbes and environmental stimuli. role of the respiratory microbiome in asthma the human microbiome project, launched in , did not include airway tissue sampling as healthy human lung tissue at that time was assumed to be sterile [ ]. however, shortly afterwards a number of pioneering publications in this field appeared and several research consortia and individual groups subsequently started in- tensive studies to characterize and understand the com- position and function of airway microbiota in health and disease [ – ]. currently, it is known that the healthy respiratory mucosa is inhabited by niche-specific bacter- ial communities [ ]. the highest densities of bacterial communities are found in the upper respiratory tract, reaching up to viable bacteria per nasal swab from the nasal cavity and nasopharynx, with even up to / ml viable cells from oropharynx lavages [ – ]. in the trachea and lungs, the estimated numbers of bacteria are lower with approximately bacterial cells per ml being found in bronchoalveolar lavages (bal) from healthy lungs [ ]. the six dominant phyla routinely found in the lung are firmicutes, proteobacteria, bacteroides, fusobacteria, acidobacteria, and actinobacteria [ ]. the original proof-of-concept study from hilty et al., with microbiome assessments of the nose, oropharynx, bronchial brushings and bal samples from the lower airways revealed that the proteobacteria phylum and es- pecially haemophilus species are more often present in upper and lower airways of asthmatic and copd adults and asthmatic children [ ]. the study performed by huang et al. in patients with suboptimal controlled asthma, defined as persistent symptoms on the asthma control questionnaire after weeks of standardized treatment with inhaled fluticasone, showed a greater air- way microbiota diversity in these patients compared to control subjects that correlated positively with bronchial hyperresponsiveness [ ]. specifically, there was an increase in the phylum proteobacteria in asthma patients, including comamonadaceae, sphingomonada- ceae, nitrosomonadaceae, oxalobacteraceae, and pseu- domonadaceae families [ ]. interestingly, adult patients who benefited most from clarithromycin treatment, as assessed by the reduction in bronchial hyperactivity to methacholine were those who had significantly greater bacterial diversity prior the intervention [ ]. subse- quent studies also confirmed that proteobacteria were present in higher proportions in the asthmatic airways [ , ]. in addition, klebsiella species were enriched in patients with severe asthma as compared to patients with mild-to-moderate asthma and controls [ ]. more- over, within severe asthma patients, proteobacteria was associated with th -related gene signature in airway epithelium, worsening asthma control and total leuco- cytes in the sputum, while bacteroides/firmicutes were more abundant in obese patients with severe asthma. in contrast, the presence of actinobacteria correlated with improvement and/or no change in asthma control [ ]. severe asthma had long been associated with the pres- ence of mycoplasma pneumoniae and chlamydophila pneumoniae, resulting in several clinical trials with macrolide antibiotics in this group of patients [ ]. yet, in the face of controversial study results and the possi- bility that beneficial microbial species are also affected, further clinical trials that include detailed microbiome studies are needed [ , ]. the composition of the airway microbiome develops ex- ponentially very early in life and later in life can be influ- enced by the environment, health status and age. birth mode (vaginal or via caesarean section), the exposures during the first hours of life and the environment of the following – months of life have been shown to be of utmost importance in shaping the development of stable respiratory and gut microbiota to ensure respiratory health later in life [ , – ]. both human and animal studies have shown that inhaled dust particles can carry a complex mixture of microbes and microbial factors, which influence susceptibility to asthma development via their effects on innate and adaptive immune responses [ , ]. the important research questions that are currently being addressed in children include: i) what is the longitudinal process of upper airways colonization in healthy infants? ii) how do environmental factors such as breast feeding, living on a farm, number of siblings, day-care, pets at home, smoking and antibiotic usage impact the respira- tory microbiome? iii) are there correlations between pat- terns of respiratory microbial colonization in early life with the occurrence of acute respiratory infections such as respiratory syncytial virus (rsv), rhinovirus and influenza virus and their further impact on chronic non infectious- associated recurrent wheeze, atopic sensitization and asthma? [ , – ]. teo et al. analyzed the nasopharynx microbiome in a prospective cohort of children at several time-points up to years of age and correlated the presence of specific groups of bacteria with acute respiratory infections [ ]. healthy infants from this cohort were initially colonized with staphylococcus or corynebacterium species up to months of age with subsequent stable colonization by allociococcus or moraxella. in contrast, streptococcus, moraxella or haemophilus colonization were correlated with virus-associated acute respiratory infections in the first weeks of life. early asymptomatic streptococcus colonization, rare in children from dog and cat-owning families, increased the risk of asthma at years of age [ ]. early upper respiratory tract colonization with s. pneumoniae, h. influenzae and/or m. catarrhalis in sokolowska et al. asthma research and practice ( ) : page of children at weeks of age from other prospective birth cohorts was also found to be associated with an increased risk of pneumonia and bronchiolitis or asthma at years of age [ , ]. additional studies have also noted associations between h. influenza, streptococcus species and s. aureus nasopharyngeal colonization with rsv infection and hospitalization in children independ- ently of their age [ – ]. furthermore, early colonization of the upper respiratory tract of healthy in- fants with staphylococcus species, subsequently followed by corynbactrium/dolosigranulum and moraxella, were described for infants who were breastfed and who had lower rates of respiratory infections in the first years of life [ , , ]. indeed, airway microbial diversity ap- pears to be inversely associated with sensitization to house dust mites in early childhood [ , ]. of particu- lar interest is a recent study comparing amish children raised on traditional farms, who have a low prevalence of asthma and atopy, with hutterite children coming from highly industrialized farms who have a higher prevalence of asthma and atopy, even though these two populations are genetically similar. one striking differ- ence was the microbial composition and endotoxin load of dust from those two housing environments, associ- ated with the enhanced induction of innate immune pathways in amish children. the high-endotoxin dust from amish houses was able to inhibit ova-induced al- lergic airway inflammation in mice, as described above [ ]. several other studies have also confirmed that the farm environment is associated with increased bacterial diversity in the house dust samples and nasal micro- biome diversity of the same children who have lower risk of developing asthma [ – ]. it is currently unknown if the protective effect of the dust-associated microbiome is due to inhalation of multiple bacteria species and fur- ther colonization of the airways, or if inhaled bacterial metabolites may also play a role. microbiome strategies for asthma prevention, treatment and management alterations in the lung and gut microbiome of asthma patients have been well described previously in this review. the deliberate restoration of lung and gut micro- biota through the use of prebiotics; probiotics or synbio- tics is one potential strategy currently being assessed. interest in probiotics and prebiotics for their potential benefits in protecting against airway inflammation is relatively recent but increasing significantly, particularly as several lines of evidence suggest that a “healthy” microbial community facilitates the development of im- mune tolerance [ ]. in vitro studies and animal models have repeatedly shown the protective effects of certain probiotic strains on lung inflammatory responses, but have also shown that not all probiotics will induce the same effects [ ]. intervention and prevention studies in humans are inconsistent in their findings, possibly be- cause many factors complicate the analysis of dysbiosis in patients with asthma. comparison between human studies are difficult, because of considerable heterogen- eity in the probiotics and/or prebiotics used, study design, sample size, age of study population, geographic location and lifestyle factors (including diet). one pre- liminary study did suggest that symbiotic (pre and pro- biotic) use improved peak expiratory flow and reduced the systemic production of th cytokines in allergic asthmatics [ ]. another recent study using a combin- ation of nutritional interventions (fish oils and vegetable extracts) with a probiotic led to significant improvement in pulmonary function parameters and significantly reduced requirement for short-acting inhaled bronchodi- lators and inhaled corticosteroids in children with asthma, suggesting that the combination of multiple ap- proaches may lead to the most optimal benefits [ ]. these findings are promising, however more definitive studies are needed to determine whether modification of gut and lung microbiota can be attributed to pre and/or probiotic use. currently, there is no recommendation to use pre- or probiotics for treatment or prevention of asthma. nevertheless, there is accumulating evidence that antenatal interactions between maternal diet, gut bacteria and bacterial metabolites may lead to immuno- logical imprinting on the developing fetal immune system that could influence the development of allergy and asthma later in life [ ]. thus, further studies are required to determine if appropriate prebiotic and pro- biotic use during pregnancy may functionally impact the maternal gut microbiome with subsequent effects on maternal immune function and risk of asthma in the offspring [ ]. in addition to using single probiotic bacterial strains, the manipulation of the entire gut microbiome with fecal microbiota transplants (fmt) is currently being explored. fmt has been successfully used for the treatment of clos- tridium difficile infection and research into its use for other inflammatory diseases such as type diabetes, inflamma- tory bowel diseases and non-alcoholic steatohepatitis is well under way [ ]. the use of fmt beyond intestinal dis- orders requires additional studies and currently there is no data supporting its use in allergic disease or asthma [ ]. the role of the microbiome in influencing precision medicine approaches to patient care has been best explored to date in the oncology field. accumulating evidence sug- gests that the microbiome not only influences the severity of treatment-associated side effects in cancer patients, but also has a dramatic effect on treatment efficacy via pharma- codynamic and immunological mechanisms [ ]. notably, a melanoma mouse model showed commensal microbe- derived antitumor immunity evidenced by higher sokolowska et al. asthma research and practice ( ) : page of intratumoral cd + t cell accumulation. from this micro- biota, bifidobacteria were identified as having the strongest association with antitumor t cell immunity and the ability to maximize the efficacy of the cancer immunotherapeutic anti-pd-l -specific antibody treatment. bifidobacteria aug- mented dendritic cell function leading to enhanced cd + t cell priming and accumulation within the tumor [ ]. while there is a growing amount of data on the compos- itional differences in lung microbiota in health and disease, there is a dearth of research into the functional role of the microbiome on treatment efficacy in patients with chronic respiratory disorders [ ]. one important study did correl- ate corticosteroid use and corticosteroid sensitivity in asthma patients with the presence of specific microbes in the lower airways. at the genus level, neisseria species, haemophilus species, campylobacter species and leptotri- chia species were present in the lower airways of patients with corticosteroid-resistant asthma, but not in patients with corticosteroid-sensitive asthma [ ]. others have dem- onstrated that corticosteroid use, particularly the combin- ation of inhaled and oral corticosteroids, is associated with an increased abundance of proteobacteria and the genus pseudomonas, and decreased abundance of bacteroidetes, fusobacteria, and prevotella species [ ]. one recent study suggests that microbiome-related functions might affect re- sponsiveness to corticosteroid treatment in asthma patients [ ]. pre-steroid treatment haemophilus levels were in- creased in asthma patients with diminished responses to corticosteroids. furthermore, the predicted metagenome metabolic functions in inhaled corticosteroid nonre- sponders suggested increased microbiome-associated xeno- biotic degradation capacity [ ]. further profiling and characterization of the micro- biome associated with different asthma phenotypes is necessary for identifying novel microbiota-related mech- anisms of disease. in addition, identification of these key microbial species and their associated functional effects will contribute to a more precise definition of asthma phenotypes and may help identify more suitable “pheno- type-specific” management strategies [ ]. future perspectives while it is clear that the microbiome significantly influ- ences host immune maturation and immune activity, the molecular basis for these immunomodulatory mecha- nisms are only beginning to be elucidated. it still remains unclear whether and, if so, to what extent pat- terns of airway microbial dysbiosis actually drives rather than merely reflects associated patterns of immune re- activity within the lung. current studies in prospective birth cohorts and cross-sectional studies in children have heightened our awareness of time-sensitive patterns of colonization of seemingly protective or detrimental bacteria in the gut or airways of healthy and diseased children. however, further mechanistic and epidemio- logical studies are needed to uncover the functional, multidirectional associations between the specific bacter- ial strains, host, allergens and viruses. respiratory micro- biome assessments in adults have so far been performed in a cross-sectional manner, comparing the airway microbiota composition between healthy controls and patients with asthma and often with other chronic airway diseases. some studies have provided detailed clinical characteristics of patients, allowing for the cor- relation of microbiota differences across different asthma phenotypes. however, longitudinal and prospective ana- lyses of adult airway microbiome in bigger cohorts of well-characterized patients are still needed to under- stand the relationships between the course of the disease, its phenotype and endotype, susceptibility to exacerbations and disease progression as well as its response to treatment. compositional profiling needs to be complemented with metagenomics, transcriptomics, physiological, bio- chemical and function-oriented analyses of both the host response and microbial communities as interactions between the host and microbiome are almost certainly bidirectional, with species- and strain-specific behaviors shaped by the genetic background and microenviron- ment in which they exist. in addition, current compos- itional analysis at the genus level is not sufficient and future analysis needs to be conducted at greater depth to include information at the species and strain level. the immune response to a bacterium is often strain-specific and results from one strain cannot be extrapolated to other strains even within the same species. thus, the traditional methods for microbiological classification, based on s sequencing and certain biochemical prop- erties, of a bacterium into a given genus or species do not currently help us to predict immunological out- comes. culturing methods need also to be improved in order to isolate and grow lung-derived bacterial strains in vitro, particularly the obligate anaerobes, in order to facilitate strain-specific immunological assessments. conclusions the last few years have resulted in pivotal studies that clearly associate changes in gut or lung microbial popu- lations with asthma. however, mechanistic studies are still necessary to elucidate how members of the micro- biota induce or modulate inflammatory responses in asthmatic patients. we anticipate that the continuing identification of novel bacterial strains, their compo- nents and metabolites, which modulate mucosal immu- noregulatory responses, will open up new possibilities for a bug-to-drug approach in the treatment of asthma patients and the prevention of airway diseases. sokolowska et al. asthma research and practice ( ) : page of abbreviations bal: bronchoalveolar lavages; fmt: fecal microbiota transplant; gf: germ- free; hdm: house dust mite; nlrp : nod-like receptor family pyrin domain containing ; rsv: respiratory syncytial virus; scfas: short-chain fatty acids; spf: specific pathogen-free acknowledgements none funding the authors are supported by swiss national science foundation grants (project numbers crsii _ , , _ , , – , and , _ ) and christine kühne – center for allergy research and education (ck-care). availability of data and materials not applicable authors’ contributions all authors contributed to the writing of the review. all authors read and approved the final manuscript. ethics approval and consent to participate not applicable consent for publication not applicable competing interests lom is a consultant to alimentary health ltd. and has received research funding from glaxosmithkline. ca has received research support from novartis and stallergenes and consulted for actellion, aventis and allergopharma. ms, rf and nl have no competing interests. publisher’s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. author details swiss institute of allergy and asthma research, university of zürich, obere strasse , davos, switzerland. christine kühne – center for allergy research and education (ck-care), davos, switzerland. university of cape town, cape town, 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next manuscript to biomed central and we will help you at every step: sokolowska et al. asthma research and practice ( ) : page of 	abstract 	background 	immune mechanisms influenced by the microbiome 	microbiome in animal models of asthma 	the role of the gut microbiome in asthma 	role of the respiratory microbiome in asthma 	microbiome strategies for asthma prevention, treatment and management 	future perspectives 	conclusions 	abbreviations 	funding 	availability of data and materials 	authors’ contributions 	ethics approval and consent to participate 	consent for publication 	competing interests 	publisher’s note 	author details 	references ucla ucla previously published works title coronary artery disease in patients with hiv infection permalink https://escholarship.org/uc/item/ qr s d journal american journal of cardiovascular drugs, ( ) issn - authors patel, aa budoff, mj publication date - - doi . /s - - - peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ qr s d https://escholarship.org http://www.cdlib.org/ american journal of cardiovascular drugs issn - volume number am j cardiovasc drugs ( ) : - doi . /s - - - coronary artery disease in patients with hiv infection amish a. patel & matthew j. budoff your article is protected by copyright and all rights are held exclusively by springer international publishing switzerland. this e- offprint is for personal use only and shall not be self-archived in electronic repositories. if you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. you may further deposit the accepted manuscript version in any repository, provided it is only made publicly available months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on springer's website. the link must be accompanied by the following text: "the final publication is available at link.springer.com”. l e a d i n g a r t i c l e coronary artery disease in patients with hiv infection amish a. patel • matthew j. budoff published online: february � springer international publishing switzerland abstract hiv-infected patients are known to be at risk for premature coronary artery disease. this emerging paradigm is a rising concern for clinicians. due to advances in the treatment of hiv, this once fatal infection has been trans- formed into a chronic illness. traditional risk factors paired with the long-term use of antiretroviral therapy (art) and chronic inflammation leads to premature atherosclerosis, particularly progression of atherosclerotic plaque. this population of patients requires early recognition of sub- clinical atherosclerosis, as well aggressive primary and sec- ondary prevention strategies among the multi-disciplinary team of physicians caring for them. we sought to present a comprehensive review of the available literature related to hiv and atherosclerosis and cardiovascular risk. key points recognize non-traditional and emerging risk factors for coronary artery disease (cad) in the hiv population. the benefit of continuous antiretroviral therapy remains the standard in cad reduction. statins are suggested in the hiv population to reduce cad but caution must be taken due to drug–drug interactions. due to possible underestimation of cad in the hiv population, consider computed tomography and coronary angioplasty for preventative and therapeutic guidance, respectively. introduction cardiovascular disease (cvd) has emerged as a significant threat to the hiv-infected population, in large part due to the effectiveness of antiretroviral therapy (art) in treating hiv infection and extending life expectancy [ ]. currently, cvd is the second most frequent cause of death among hiv patients, with the first being cancer [ ]. the sub- clinical makers of atherosclerosis, such as carotid, femoral, or iliac intima-media thickness are consistently greater and progress earlier among the hiv-positive population than among the general population [ , ]. the d:a:d (data collection in adverse effects of anti- hiv drugs) study showed that risk for cardiovascular event was statistically increased by such traditional risk factors as age, male sex, greater body mass index (bmi), family history of cvd, and smoking [ ]. this is exacerbated by hiv-specific risk factors such as low cd t-lymphocyte cell (cd ) count, co-infection with hepatitis c virus, and certain arts (table ). d:a:d showed that the dys- metabolic effects of hiv protease inhibitors (pi) increased the risk of diabetes mellitus, hypertension, and dyslipi- demia [ ]. in hiv-infected patients, the risk of myocardial infarction (mi) is approximately twofold [ ], and the risk of sudden cardiac death is approximately fourfold [ ] compared with the general population. the macs (multicenter aids cohort study), initiated in , is an on-going multicenter prospective, observa- tional cohort study. it continues to conduct studies and publish key papers on the natural history of untreated and treated hiv infection [ ]. a number of significant results a found that hiv-infected men had a greater extent of non- calcified coronary artery plaque (ncap) [ ]. in another study also using computed tomography coronary a. a. patel � m. j. budoff (&) los angeles biomedical research institute at harbor-ucla, w carson st, torrance, ca , usa e-mail: mbudoff@labiomed.org am j cardiovasc drugs ( ) : – doi . /s - - - author's personal copy angiography (coronary cta), an increased prevalence of vulnerable plaque features among relatively young hiv- infected men was found [ ]. these findings are not only limited to the male hiv-infected population. a study that focused on the female hiv population found that young, asymptomatic hiv-infected women also demonstrate in- creased ncap [ ]. this has led to a surge in interest in the association between hiv infection and cvd, specifically ncap due to the increase of major adverse cardiac events (mace) associated with it [ ]. it should be pointed out that the coronary plaque analysis from the macs database was concluded using cross-sectional studies. this leaves the possibility that hiv-infected indi- viduals have an overall greater risk profile than non-in- fected individuals. the macs cohort is currently undergoing a second cta at years to evaluate for plaque progression in the hiv group using different retroviral therapies. pathophysiology of coronary artery disease in patients with hiv infection hiv acts by mainly targeting the cd ? t cells and, to a lesser extent, macrophages and dendritic cells, which all play an essential role in immunity. the infection and massive depletion of cd ? t cells represents the funda- mental pathogenesis of hiv infection [ ]. the patho- physiology of coronary artery disease (cad) in hiv- infected patients is multifactorial and complex, combining traditional and non-traditional risk factors in addition to emerging risk factors. to date, factors associated with cad in hiv have been determined indirectly or by asso- ciation, which makes our understanding of the overall process limited. prospective studies evaluating long-term cad outcomes are needed. cad is the result of chronic inflammation of the arterial wall, which is lined by endothelial cells (ecs) and smooth- muscle cells [ ]. hiv infection is associated with an in- crease in systemic inflammation and damage to the vascular endothelium [ ]. this leads to hiv-positive in- dividuals being prone to premature atherosclerosis. in atherosclerosis, the activation of ecs in response to an inflammatory state leads to the expression and release of cytokines and chemokines, specifically interleukin (il)- and monocyte chemoattractant protein (mcp)- . these have been suggested to play the key role in the initial phase of atherosclerosis [ – ]. an increase in il- levels has been associated with an increase in cardiovascular mor- tality in hiv-infected individuals [ ], and mcp- has been shown to play a part in the attraction, migration, and activation of monocytes [ ]. when using mice deficient in mcp- or its receptor, c-c chemokine receptor (ccr)- , to examine atherosclerosis, it was demonstrated that, in the absence of mcp- or its receptor, ccr- , there was a substantial reduction in atherosclerosis [ ]. proinflammatory monocytes cd ? cd ? have also been implicated in the atherosclerosis disease process [ ]. in a large study involving more than patients with cad, increased percentages of the cd ? cd ? monocytes were seen in the patient cohort [ ]. a recent study analyzing coronary artery calcium (cac) and in- flammatory markers found that higher frequencies of cd ? monocytes (lacking cd expression) predicted greater cac progression [ ]. cac progression has been shown to be associated with increased cardiovascular morbidity and all-cause mortality [ , ]. cac has also been proven as an independent predictor of future risk of cvd events [ ]. in hiv-in- fected patients, the disease process is thought to begin with an inflammatory state. inflammation initiates the recruit- ment of monocytes; monocytes then migrate to the en- dothelium and differentiate to macrophages and foam cells. foam cells transform and undergo apoptosis because of calcium-dependent endoplasmic reticulum stress, thus leading to atherosclerosis [ ]. excessive death of foam cells overloaded with cholesterol eventually forms the plaques in the arteries, induces further inflammation, and exacerbates metabolic dysregulation [ ]. it is speculated that the hiv virus, which activates inflammation and lipid- markers, also triggers endoplasmic reticulum (er) stress through its interaction with host genes that result in an imbalance of calcium [ ]. histopathologically, the vas- cular changes associated with hiv infection include ec- centric atheromatous plaques composed of fibrous tissue, lipid, and calcium, with variable degrees of chronic in- flammation and accelerated atherosclerosis [ ]. a post- mortem study observed an unusual pattern of dystrophic vascular calcification in hiv? patients who were treated with art from all major class for a mean ± standard deviation (sd) duration in years of . ± . , speculating that the metabolic derangements in hiv? patients re- ceiving art may predispose them to this type of table cardiovascular risk factors in hiv [ – ] traditional nontraditional age low cd count sex lidodystrophy syndrome greater body mass index hepatitis c co-infection family history of premature heart disease metabolic syndrome smoking end-stage renal disease diabetes antiretroviral therapy dyslipidemia hypertension a. a. patel, m. j. budoff author's personal copy atherosclerosis [ ]. art essentially acts to suppress the hiv viral load and is recommended for all hiv-infected individuals to reduce the risk of disease progression. current data on the effect of cd count and viral load on cardiovascular mortality have not been entirely consis- tent. the d:a:d study found there was no association be- tween either the nadir cd ? lymphocyte count or peak hiv- rna level and the risk of mi [ ]. in the prospectively recorded fhdh (french hospital database on hiv) study, lower nadir cd cell count and higher plasma viral load were associated with a statistically sig- nificant increased rate of mi, independent of exposure to art and presence of traditional risk factors [ ]. there- fore, it appears that age-related changes in combination with hiv infection and long-term art therapy all likely contribute in a cumulative effect on the arterial walls to produce atherosclerosis. diagnosis the first step in diagnosis of cvd in the hiv population begins with a thorough medical history, which would in- clude a complete history of prior antiretroviral exposure. a further detailed assessment of cad risk factors is then to be performed, including smoking history, family history of cad, and fasting determinations of total cholesterol, high- density lipoprotein (hdl) and low-density lipoprotein (ldl) cholesterol, triglycerides, framingham risk score (frs) or the atherosclerotic cardiovascular disease (ascvd) risk assessment. as mentioned, recent analysis has suggested that chronic inflammation can partially at- tribute to the increased cardiovascular risk correlated with hiv infection [ , ]. the underlying mechanism of this disease process is currently being studied and remains unclear. however, the atherosclerotic byproduct of this process can be detected with current cardiac imaging modalities, particularly cardiac ct. cardiac ct estimates cac, which provides a noninva- sive assessment of subclinical atherosclerosis that also correlates with the extent of histologically confirmed nacp [ , ]. additionally, cac progression is inde- pendently associated with future risk of cvd events and all-cause mortality [ , ]. the risk classification for cad or stroke significantly improves by adding cac assessment along with traditional risk factors [ , , ]. however, in the hiv population, the generalized use of only cac scoring could be deceptive. recently demon- strated among an hiv population receiving art therapy for at least years, the extent of calcification was sig- nificantly reduced compared with hiv-seronegative con- trols [ ]. in a study using coronary cta, hiv-infected men were found to have a greater extent of non-calcified plaque after cad risk factor adjustment [ ]. only a small number of studies have characterized the degree of both cac plaque and ncap in hiv-infected adults [ , ]. one study found an increased ncap volume in hiv-in- fected men compared with uninfected controls with a trend toward higher agatston calcium scores among those pa- tients with hiv. within the hiv-infected group, plaque volume was associated with traditional markers of cvd risk and hiv-specific risk factors [ ]. in another study, the data found an increased prevalence of vulnerable plaque features among a relatively young population of hiv-in- fected patients [ ]. though the routine use of coronary cta to assess the degree of coronary atherosclerosis is still debated, it can provide preventive and therapeutic guidance in this high- risk population. furthermore, specific risk factors identified in hiv-related atherosclerosis, such as chronic inflamma- tion, immune activation, and effects of art are not cal- culated with available risk scores. this can lead to underestimation of true cardiovascular risk in the hiv-in- fected population. prevention the most important preventive measure for a high-risk population is lifestyle modification and statin use. a study analyzing data from macs and the wihs (women’s in- teragency hiv study) found hiv-infected individuals to have a higher prevalence of smoking (up to %) and to meet criteria for being overweight or obese (bmi [ ), which increased predicted cardiovascular risk [ ], thereby giving clinicians an opportunity to intervene on modifiable risk factors. the first of these is smoking cessation, as smoking is a well-known cardiovascular risk factor. the american heart association has set forth guidelines for lifestyle management to reduce cardiovascular risk in the general population and these shouldalso be applied to this sub- group. these include improved diet and increased physical activity, both of which contribute to weight loss, lowering blood pressure, reduction in insulin resistance, and blood lipid modification [ ]. due to the increased risk of premature atherosclerosis associated with hiv infection, an aggressive implementa- tion of cardiovascular therapies is required soon after di- agnosis. as mentioned earlier, art remains the first-line treatment for hiv infection. combination art is currently defined as any combination of three antiretroviral drugs, usually two nucleos(t)ide reverse transcriptase inhibitors (nrtis) plus a pi or a non-nucleoside reverse transcriptase inhibitor (nnrti) plus an integrase inhibitor [ ]. effec- tive art reduces viral load, which is thought to decrease cad in hiv author's personal copy the inflammatory effects of hiv and therefore reduce atherosclerosis [ ]. the d:a:d study indicated that cumulative exposure to specific pis (lopinavir–ritonavir and indinavir) and two nrti drugs (abacavir and didanosine) was associated with an increased risk of mi [ , ]. as the duration of exposure to pis increases, the risk for mi also increases, as seen in the fhdh and d:a:d cohorts [ , ]. the cumulative exposure to all studied pis was associated with a higher risk of mi, with an odds ratio (or) of . per years of exposure [ % confidence interval (ci) . – . ] [ ]. however, the pi risk–benefit ratio continues to remain positive, as the increase in life expectancy in pi-based art outweighs the risk of mi [ ]. neither of these cohorts found any significant association between the development of mi and cumulative exposure to an nnrti [ , ]. of the nrtis, the only significant association between mi risk and cumulative exposure was with abacavir [rela- tive risk (rr) . ( % ci . – . )]; recent exposure (less than months) to abacavir [rr . ( % ci . – . )] or didanosine [rr . ( % ci . – . )] were both associated with an increased risk of mi. there were no significant associations between mi risk and recent exposure to any of the other nrtis, particularly tenofovir [ ]. the smart (strategies for management of anti- retroviral therapy) study reported that the current use of abacavir was associated with an excess risk of cvd compared with other nrtis. adjusted hazard ratios for clinical mi or major cvd were . ( % ci . – . ) and . ( % ci . – . ), respectively [ ]. a canadian study also found an increased risk of mi with any exposure to abacavir [or . ( % ci . – . )] [ ]. in contrast, a retrospective study using the us veterans administration’s clinical case registry showed no sig- nificant association between abacavir use and mi risk [ ]. the prospective fhdh study did not find any causal re- lationship, as the observed association with recent exposure to abacavir or didanosine was unstable in sensitivity ana- lysis [ ]. three meta-analyses also showed no significant association between abacavir use and mi [ – ]. in a study analyzing the macs and women’s interagency hiv study cohorts found abacavir use was not independently associated with elevated inflammatory markers (high sen- sitivity c-reactive protein, il- , or d-dimer) at months after initiation [ ]. the differences in these studies can be explained by the presence of confounding factors, such as smoking, kidney disease, cocaine and/or intravenous drug use, and potential for selection biases. therefore, it is not currently possible to draw any conclusions regarding a causal relationship between treatment with abacavir and the risk for develop- ing an mi [ ]. this should be taken into account when considering specific art therapy. the recommendations to initiate medications for pri- mary prevention do not differ from those for the general population. statins are recommended for the increased risk of cvd in the hiv infection [ ], although caution needs to be taken due to possible drug interactions. for instance, certain hmg-coa reductase inhibitors are contraindicated in combination with pis [ ]. lower-dose fluvastatin, ro- suvastatin, pravastatin, and atorvastatin are recommended to avoid the increase of drug concentration induced by cytochrome p (cyp)- a inhibition by pis (table ). evidence is clear that art therapy significantly decreases the overall mortality associated with hiv infection. de- spite concern that certain art agents might be associated with cardiovascular risks, the discontinuation of hiv-sup- pressive therapy may result in an even greater risk of dis- ease. a potential explanation for this finding is that hiv suppression in itself is cardioprotective by reducing proinflammatory cytokines (i.e., il- ), which play a role in arterial inflammation [ ]. the smart study found that patients receiving episodic art were at greater risk for cardiovascular events than were those receiving continuous therapy [ ]. therefore, the prevention of cvd should be focused on continuous art, lifestyle modification, and consideration of lipid-lowering agents. conclusion the life expectancy of the hiv-infected population con- tinues to improve with art therapy and nearly matches the general population when viral load is controlled [ ]. mortality from aids-related illnesses is steadily decreas- ing, while age-related disease such as cvd continues to increase in this population [ ]. more profound is the evidence to suggest that subclinical atherosclerosis is being table antiretroviral drug combinations to avoid [ , ] protease inhibitor hmg-coa reductase inhibitors to avoid ritonavir (rtv) lovastatin, simvastatin atazanavir (atv)/rtv lovastatin, simvastatin darunavir (drv)/rtv lovastatin, simvastatin fosamprenavir (fpv)/rtv lovastatin, simvastatin lopinavir/ritonavir (lpv/ rtv) lovastatin, simvastatin saquinavir (sqv)/rtv lovastatin, simvastatin tipranavir (tpv)/rtv atorvastatin, lovastatin, simvastatin alternative recommendations: use pravastatin or fluvastatin, which have the least potential for drug–drug interactions (except for pravastatin with drv/rtv, which needs careful monitoring). may use atorvastatin or rosuvastatin with caution (start with the lower possible dose and titrate based on tolerance and lipid-lowering effi- cacy). avoid atorvastatin with tpv a. a. patel, m. j. budoff author's personal copy found in relatively young hiv-infected patients [ , ]. hiv suppression remains the standard in cvd reduction, as studies have suggested that higher cd cell counts and lower hiv rna levels are associated with a decrease in mi risk [ , ]. opportunities for risk factor reduction such as lifestyle modification are required, and the imple- mentation of pharmacologic therapy such as statins can be considered. once primary and secondary preventions have been exhausted and further risk stratification is needed, we suggest the novel use of coronary cta to assess atherosclerotic plaque morphology among hiv-infected patients. the ability to compare plaque morphology be- tween hiv-infected patients and non-hiv-infected patients with similar traditional cardiovascular risk factors can be a useful tool not only in epidemiological studies but also in clinical applications. finally, we hope continued interest remains to verify current data and associations with prospective studies involving coronary plaque analysis. conflict of interest m. j. budoff is a consultant for general electric. a. a. patel has no conflict of interest to declare. references . wada n, jacobson lp, cohen m, french a, phair j, munoz a. 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and mortality in hiv-infected patients. plos one. ; ( ):e . . hare cb, vu mp, grunfeld c, lampiris hw. simvastatin-nelfi- navir interaction implicated in rhabdomyolysis and death. clin infect dis. ; ( ):e – (epub oct ). . dubé mp, stein jh, aberg ja, fichtenbaum cj, gerber jg, tashima kt, henry wk, currier js, sprecher d, glesby mj, adult aids clinical trials group cardiovascular subcommittee, hiv medical association of the infectious disease society of america. guidelines for the evaluation and management of dyslipidemia in human immunodeficiency virus (hiv)-infected adults receiving antiretroviral therapy: recommendations of the hiv medical association of the infectious disease society of america and the adult aids clinical trials group. clin infect dis. ; ( ): – . . el-sadr wm, lundgren jd, neaton jd, et al. cd ? count- guided interruption of antiretroviral treatment. n engl j med. ; : – . . palella fj jr, baker rk, moorman ac, chmiel js, wood kc, brooks jt, holmberg sd, hiv outpatient study investigators. mortality in the highly active antiretroviral therapy era: changing causes of death and disease in the hiv outpatient study. j acquir immune defic syndr. ; : e . cad in hiv author's personal copy 	coronary artery disease in patients with hiv infection 	abstract 	introduction 	pathophysiology of coronary artery disease in patients with hiv infection 	diagnosis 	prevention 	conclusion 	conflict of interest 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page 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least one member with an age at diagnosis of < years. these families included a total of affected sibpairs and other affected relative pairs. nonparametric linkage analysis revealed seven regions showing nominal evidence for linkage with type diabetes (logarithm of odds [lod] > . , ppointwise < . ): chromosome at . cm (lod � . ), chromosome at . cm (lod � . ), chromosome at . cm (lod � . ), chromosome at . cm (lod � . ), chromosome at . cm (lod � . ), chromosome at . cm (lod � . ), and chromosome at . cm (lod � . ). simulation studies showed genome-wide signifi- cant evidence for linkage of the chromosome region (pgenome-wide � . ). we have confirmed the results of previous studies for the presence of a susceptibility locus on chromosome q -q ( . cm) and suggest the locations of other loci that may contribute to the development of type diabetes in hong kong chinese. diabetes : – , t ype diabetes is a heterogeneous disease char- acterized by insulin resistance and pancreatic �-cell dysfunction ( ). genetic factors play an important role in the development of type diabetes. despite considerable effort, there has been rela- tively little progress in identifying the genes that affect risk. this may be due, at least in part, to phenotypic heterogeneity; i.e., type diabetes is not one disease but many, each characterized by hyperglycemia. genome scans to map type diabetes susceptibility loci have been conducted in many different populations ( – ). some of the mapped loci have been observed in multiple popula- tions, including those on chromosomes q -q , q, and . other regions, however, may be unique to specific populations, e.g., mexican americans and chromosome q . and the amish and chromosome q . it is unclear if this reflects underlying phenotypic heterogeneity, racial/ ethnic differences in susceptibility allele frequencies, or differences in sample size, study design, and analytical methods. the prevalence of diabetes in hong kong is among the highest in asia, with an age-adjusted rate of . % in ( ). this high prevalence is thought to be due to the affluent and westernized lifestyle of hong kong chinese, resulting from rapid socioeconomic development over the last few decades. this is compatible with the thrifty genotype hypothesis, in which favorable genetic traits that facilitate energy storage and mobilization in times of famine are detrimental during time of plenty, leading to the development of modern diseases such as diabetes ( ). we carried out an autosomal genome scan in families with type diabetes ( affected sibpairs and other affected relative pairs). we also considered glucose intol- erance (git) as a trait (defined as type diabetes, im- paired fasting glucose, or impaired glucose tolerance) because impaired fasting glucose and impaired glucose tolerance may be precursors to type diabetes. we carried out an autosomal scan for git in families ( affected sibpairs and affected relative pairs). the clinical char- acteristics of affected subjects involved in linkage analyses were similar except for the higher bmi and fasting glucose levels in the type diabetes group (table a in the online appendix [available at http://diabetes.diabetesjournals. org]). seven regions showed nominal evidence for linkage with type diabetes (logarithm of odds [lod] � . , ppointwise � . ) (table , fig. , and online appendix table a ). the region showing the strongest evidence was on chromosome q -q at . cm (lod � . , ppointwise � . , -lod ci � – cm). the lod scores in these seven regions were not increased when we used git as the trait. we used simulation to assess the significance of the from the department of medicine and therapeutics, the chinese university of hong kong, prince of wales hospital, shatin, hong kong; the department of biochemistry and molecular biology, university of chicago, chicago, illinois; the department of human genetics, university of chicago, chicago, illinois; and the department of medicine, university of chicago, chicago, illinois. address correspondence and reprint requests to dr. maggie c.y. ng, howard hughes medical institute, university of chicago, s. maryland ave., mc , chicago, il . e-mail: maggieng@uchicago.edu. received for publication march and accepted in revised form march . † j.a.j.h.c. passed away in . additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. git, glucose intolerance; ilr, independent linked region; lod, logarithm of odds. © by the american diabetes association. diabetes, vol. , june results and found the lod score threshold of . for � independent linked regions (ilrs) for type diabetes, yielding an estimated pgenome-wide � . . the chromo- some q region demonstrated genome-wide significant linkage for type diabetes (lod � . , pgenome-wide � . ). chromosomes q (lod � . ), q (lod � . ), and p (lod � . ) showed suggestive evidence for linkage based on observation in our simulations of an average of one ilr per genome scan ( ) at a lod score of . (table ). we also used locus-counting methods to evaluate the linkage results ( ). we observed seven, five, and two loci with lod � . (ppointwise � . ), � . (ppointwise � . ), and � . (ppointwise � . ), respectively. the simulations showed that of the replicates ( %) had seven or more loci at lod � . , whereas ( %) had five or more loci at lod � . , and ( %) had two or more loci at lod � . . overall, the results suggest that there may be one or more susceptibility loci in the other six regions with nominal evidence for linkage. could our results be due to the presence of genotyping error or missing data? in general, these problems are believed to reduce the power to detect linkage ( ). our genotyping error rate as assessed by blind duplicates is comparable with that of previous genome screens for type diabetes ( ). lod scores obtained before and after removal of merlin-derived potential genotyping errors were similar in the seven regions with nominal evidence for linkage (chromosome , . vs. . ; chromosome , . vs. . ; chromosome , . vs. . ; chromosome , . vs. . ; chromosome , . vs. . ; chromosome , . vs. . ; and chromosome , . vs. . ). our missing data rate is comparable with that observed in another type diabetes genome scan ( ), where it was demonstrated that % missing data did not substantially decrease the power to detect linkage. it should be noted that our moderately sized pedigrees allow recovery of some missing information through haplotype reconstruc- tion. finally, our study assessed significance through simulation, and thus the consequences of the observed missing data patterns are appropriately taken into account. in this study, we used both strict (type diabetes) and loose (git) criteria of glucose homeostasis to define affection status. the former should yield a more homoge- neous sample with respect to disease, whereas the latter provides a larger sample size. our results generally indi- cate overlapping linkage signals for type diabetes and git (chromosomes , – , and ) but the linkage signals for type diabetes tended to be stronger than those for git (chromosomes , , and ) (fig. ), despite the fact that the sample size for type diabetes was smaller. the higher signals for type diabetes suggest that the reduced heterogeneity is important for detecting the larger signals. the region on chromosome q ( . cm from p-termi- nus) has also been linked to type diabetes and related traits in chinese from shanghai, pima indians, and euro- peans ( – , – ) (table ). there are � known genes in this region. results from association studies on genes encoding for potassium inwardly rectifying channel j (kcnj ) ( ), c-reactive protein (crp) ( ), and phos- pholipase a a (pla g a) ( ) suggest there are asso- ciations that require confirmation. this region is the focus of an international collaborative project to positionally clone the gene(s) that affect type diabetes risk. at least one or more of the six other regions showing nominal evidence for type diabetes may harbor true susceptibility loci based on the results of locus-counting methods ( ) and linkage to these regions in other studies (table ). the identification of the genes and alleles that affect risk to type diabetes may lead to a better understanding of the genetic basis of type diabetes in hong kong chinese and other populations. research design and methods since , all patients attending the diabetes clinic of the prince of wales hospital have been documented for detailed phenotypes and family history (parents, siblings, and offspring), which formed the prince of wales hospital diabetes registry ( ). through this registry, hong kong family diabetes study ascertained families through a diabetic proband with available first- degree relatives for screening at two stages. at the first stage, families were recruited through a diabetic proband with age at diagnosis � years (early-onset group). at the second stage, an additional families were recruited through a diabetic proband with age at diagnosis � years and with positive family history of diabetes (late-onset group). the second cohort aimed to increase the sample size for gene-mapping study. a total of families were recruited. among the young-onset families, of the families were screened for antibodies to gad. individuals with clinical type diabetes (diabetic ketoacidosis or heavy ketonuria [� ] or required continuous insulin treatment within year of diagnosis) or with antibodies to gad were excluded. subsequently, families without any git or diabetic members remaining were excluded. in addition, of the probands with positive family history had participated in a previous study to screen for mutations in the mody and mody genes and in the mitochondrial dna nucleotide a g mutation ( ). families with these gene mutations were also excluded. a total of families remained. from these families, studies on the dichotomous traits of type diabetes and git included and families, respectively, due to the requirement for at least two affected (nonparent offspring) relatives. the probands, all of their first- and second-degree relatives, and the probands’ spouses were recruited between january and march . the clinical characteristics, family structure, and distribution of the affected relative pairs of these families are summarized in tables a , a , and a , respectively, in the online appendix. of special note, of the families used in the type diabetes studies and of the in the git studies table regions showing nominal evidence of linkage to type diabetes in hong kong chinese* chromosome flanking markers position (cm)† lod ppointwise apoa –d s . . . d s . . . d s –d s . . . d s . . . d s –d s . . . d s . . . d s . . . *evidence of linkage is determined as lod � . , ppointwise � . ; †marker positions are indicated in haldane centimorgans from the p-terminus obtained from the marshfield medical research foundation. scan for type diabetes in hong kong chinese diabetes, vol. , june fig. . multipoint linkage analyses for diabetes and glucose intolerance. the horizontal axis is centimorgans from the p-terminus. m.c.y. ng and associates diabetes, vol. , june had at least one affected member diagnosed before age years. informed consent was obtained for each participating subject. this study was approved by the clinical research ethics committee of the chinese university of hong kong. clinical studies. all available family members were assessed for blood pressure and standard anthropometric parameters and completed a question- naire on demographic data, family and medical histories, and lifestyle. fasting blood samples were collected for measurement of plasma glucose, insulin, c-peptide, lipid profile, liver and renal function, and dna extraction. family members with no known history of type diabetes were tested with a -g oral glucose tolerance test. type diabetes, impaired glucose tolerance, and impaired fasting glucose were diagnosed using the world health orga- nization criteria ( ). plasma glucose was measured by a glucose oxidase method (diagnostic chemicals, charlottetown, pei, canada). plasma insulin and c-peptide were measured by enzyme-linked immunosorbent assay (dako diagnostics, glostrup, denmark). genotyping. a total of autosomal microsatellite markers (research genetics, huntsville, al) were typed. these included markers from the human screening set, version , and an additional markers that replaced discarded markers from the screening set due to lack of amplification, monomorphism, or significant departure from hardy-weinberg equilibrium (p � . ). the average intermarker distance was cm, and average heterozygosity was %. the largest gap was . cm between d s and d s due to removal of two markers not in hardy-weinberg equilibrium. microsatellite markers were amplified with fluorescent-labeled primers using a universal multiplex pcr protocol. genotypes were performed by capillary electrophoresis (abi dna analyzer). alleles were called and scored using the genescan . and genotyper . software (abi) followed by manual checking. sixteen percent of genotype data were missing. blind duplicates were included, with an allele-wise genotyping error rate of . %. genetic relationships among family members were checked by relpair ( ) and prest ( ). after correction of family relationships and removal of unrelated individuals, mendelian incompatibilities for all markers were iden- tified by pedcheck (version . ) ( ). the genotypes that were most likely to have errors within a family were removed, if possible. otherwise, the genotypes of the incompatible marker were removed in all subjects within the family. potential genotyping errors observed in . % of the data using merlin (version . . b) were removed ( ). linkage analyses. marker allele frequencies were estimated from all subjects in families, including those involved in type diabetes and git studies. marker position was based on the sex-average maps from the marshfield medical research foundation (http://research.marshfieldclinic.org/genetics/ map_markers/maps/indexmapframes.html). nonparametric multipoint link- age analyzes were performed with merlin (version . . b) using the score- pairs statistic ( ). lod scores with associated pointwise p values are reported ( ). two linked regions were defined as independent if their maximum lod score positions differed by � cm. simulation studies were conducted to assess the significance of the observed linkage signals. five hundred simulated genotype datasets were generated by gene dropping with merlin under the null hypothesis of no linkage. the maps, marker allele frequencies, pedigree structures, and missing genotype patterns used in these simulations were identical to those in the actual data. the linkage analyzes were then conducted on the simulated datasets. in each simulation, the number of ilrs greater than a particular lod score threshold was tallied. pgenome-wide for n ilrs at a lod score threshold was calculated as the proportion of simulations with �n ilrs at that particular lod score. a significant excess of ilrs in the observed dataset was defined as pgenome-wide � . . acknowledgments this work was supported by grants from the hong kong research grants committee, the chinese university of hong kong strategic grant program, the innovation and technology support fund, hong kong (its/ / ), and u.s. public health service grants dk- , dk- , and dk- . g.i.b. is an investigator of the howard hughes medical institute. we thank emily poon and patty tse for their technical support and dr. june li and cherry chiu for their help with the recruitment of patients and their family members. we also thank william wen for computational support and cheryl roe for simulation data analysis. we are also indebted to the late professor robert turner, oxford university, for his collaboration in our family study. we thank all of the nursing and medical staff at the prince of wales hospital diabetes and endocrine centre for their dedication and professionalism. this work is dedicated to the memory of professor julian critchley. references . zimmet p, alberti kg, shaw j: global and societal 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wijmenga c: a genome-wide scan in type diabetes mellitus provides independent replication of a susceptibility locus on p and suggests the existence of novel loci on q and q . j clin endocrinol metab : – , m.c.y. ng and associates diabetes, vol. , june wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the 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time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ symptom profiles and illness course among anabaptist and non-anabaptist adults with major mood disorders gill et al. int j bipolar disord ( ) : doi . /s - - - r e s e a r c h symptom profiles and illness course among anabaptist and non-anabaptist adults with major mood disorders kelly e. gill , *, stephanie a. cardenas , layla kassem , thomas g. schulze , and francis j. mcmahon abstract background: anabaptists comprise large and growing amish and mennonite populations with a unique genetic heritage and cultural background. little is known about the symptoms and course of major mood disorders in ana- baptists. even less is known about the impact of potential moderators on symptom severity and course. methods: a sample of amish and mennonite participants with bipolar, recurrent unipolar, or schizoaffective bipo- lar disorder (n = ) were systematically evaluated with a well-validated instrument. cases were compared with non-anabaptist participants (n = ) matched for age, sex, and psychiatric diagnosis and evaluated by the same methods. results: despite substantial cultural differences, the profile of manic and depressive symptoms during illness epi- sodes did not significantly differ between the two groups. alcohol use disorder (aud) was significantly less frequent among anabaptists, and was associated with more major depressive episodes and more hospitalizations for major depression in anabaptist, but not non-anabaptist participants. lifetime history of head injury showed a trend toward association with more episodes of major depression in both anabaptist and non-anabaptist groups that did not withstand multiple test correction. conclusions: the presentation of a highly heritable psychiatric illness such as bipolar disorder does not differ in cases drawn from genetically unique anabaptist populations. however, alcohol comorbidity, head injury, and their effects on illness course suggest some differences that deserve further investigation. keywords: depression, mania, bipolar disorder, amish, mennonite, alcohol, head injury, concussion © the author(s). this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. background as psychiatry enters the age of genomic medicine, inter- est in genetically isolated populations is growing once again. each genetically isolated population represents an unique “experiment of nature,” characterized by unusual gene frequencies, increased rates of homozygosity, and a greater prevalence of some otherwise rare inherited dis- eases (mckusick et  al. ). a recent simulation study of genetic isolates (zuk et al. ) illustrated how other- wise rare alleles that influence disease risk may rise to fre- quencies large enough to be detectable by genome-wide association studies. here we present the first clinical results from an ongoing study of bipolar disorder among genetically isolated anabaptist populations, known as the amish mennonite bipolar genetics study, or ambigen (hou et  al. ). the ultimate aim of ambigen is to identify alleles conferring substantial risk for bipolar dis- order and related conditions. anabaptists include such well-known groups as the amish and mennonites, who originated in western europe, as well as several less well-known groups of var- ied ancestries (e.g. hutterites, schwarzenau brethren). some anabaptists who migrated to north america in the early s have opted to remain separate from the gen- eral population, marrying within their traditional com- munities and raising large families. they suffer from high open access *correspondence: kegill @gmail.com department of psychology, the catholic university of america, michigan ave ne, washington, d.c. , usa full list of author information is available at the end of the article http://creativecommons.org/licenses/by/ . / http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf page of gill et al. int j bipolar disord ( ) : rates of some otherwise rare diseases (mckusick ) and may also have an increased risk of mood disorders (egeland and hostetter ). tightly knit communities, a traditional agrarian life- style, and distinct religious beliefs distinguish many ana- baptists from the surrounding population (brewer and bonalumi ). among the amish and other traditional (“plain”) anabaptists, for example, men commonly work as farmers, carpenters, or other craftsmen, while women take care of the household and the children (kreps et al. ). children typically begin working for the family business at age , and their formal education ends after the eighth grade (kreps et al. ). most do not use elec- tricity, communicate by telephone, or drive motor vehi- cles at home (weyer et al. ). they follow plain dress codes and grooming habits, which they believe reflect a rejection of vanity and materialism (brewer and bona- lumi ). daily life is centered on the home. low rates of divorce and contraceptive use leads to large families ( – offspring), and multiple generations may share the same household or live as neighbors. some of the distinctive aspects of anabaptist lifestyles may decrease vulnerability to develop certain mental ill- nesses. potential protective factors include increased religiosity (smith et  al. ), increased social support from family, friends, and community members (john- son et al. ), and lower rates of alcohol and substance use than in surrounding communities (trimble ). however, it remains unclear to what extent lifestyle dif- ferences characteristic of anabaptists affect the risk of mood disorders, illness course, or symptom severity. a series of studies have already provided a detailed description of bipolar disorder and related conditions in the old order amish of eastern pennsylvania (ege- land and hostetter ; egeland et  al. ; hostetter et  al. ). the amish study of major affective dis- order began in and examined amish individuals with mood disorders, along with family members (ege- land et al. ). overall,  % of the individuals studied had a major mood disorder:   % were diagnosed with bipolar i or bipolar ii disorder, and  % were diagnosed with unipolar depression (egeland et  al. ). diagno- ses in these individuals were found to be valid and reli- able, although there were expected cultural factors that influenced assessment of some symptoms (egeland et al. ; hostetter et  al. ). further research on this group included the search for genetic markers (egeland et  al. ; ginns et  al. ; kelsoe et  al. ), and studies of prodromal symptoms of mood disorders (shaw et  al. ). while the amish study of major affective disorders provided the first and most detailed account of mood disorders in this population, the absence of a non-amish comparison group precluded any direct com- parisons of symptoms or course of illness. the present study examined the symptoms and course of major mood disorders in anabaptists ascertained from several communities in the united states. comparisons were performed with a set of age and sex-matched cases assessed with the same instrument, but ascertained from the general (non-anabaptist) population (potash et  al. ). we also examined potential moderating vari- ables affecting the course and severity of mood disorders in both the anabaptist and non-anabaptist groups we studied. methods sample and procedures data pertaining to the anabaptist group (n =  ) were collected over the course of   years ( – ). par- ticipants were mainly recruited through clinical settings in pennsylvania, indiana, and ohio. some participants responded to advertisements placed in publications tar- geting anabaptist households or were referred from oth- ers in the community who had knowledge of the study. the comparison group (here called “non-anabaptists”) was drawn from individuals with a major mood dis- order previously ascertained from to by the national institute of mental health (nimh) genetics initiative and collated in the bipolar disorder phenome database (potash et al. ). clinical symptoms in both groups were assessed by direct interview with the diagnostic interview for genetic studies (digs), a widely used instrument with established reliability (nurnberger et  al. ). the digs is a semi-structured assessment of major depres- sion, mania, psychosis, alcohol/drug abuse and depend- ence, suicidal behaviors, and anxiety disorders. the digs assesses symptoms over the lifetime, as well as for the most severe episodes of major depression and mania. final diagnoses were assigned in a best estimate proce- dure by at least two clinicians who reviewed the digs along with information from family informants and avail- able medical records. each case in the anabaptist sample was matched on age (±   years), gender, and dsm-iv diagnosis with a case drawn randomly from the phenome database using spss (v. ). in both the anabaptist and non-anabaptist groups,   % (n  =  ) were diagnosed with bipolar i,   % (n  =  ) with bipolar ii,   % (n  =  ) with major depression (recurrent),  % (n =  ) with schizoaffective bipolar, and  % (n =  ) with single episode major depres- sion. this matching procedure was designed to minimize differences in clinical course and symptoms that could be attributable to the matched variables. page of gill et al. int j bipolar disord ( ) : statistical analyses all statistical analyses were performed using spss (v. ). selected demographic and lifetime psychiatric his- tory variables were assessed using χ or t tests, as appro- priate, with bonferroni correction for tests (α =  . was declared significant). individual symptoms reported during the most severe episodes of mania and major depression were compared using χ tests, with bonfer- roni correction for the variables tested (α =  . was declared significant). dependent variables chosen to give a picture of the lifetime severity and course of the mood disorder included: age at first major depression, number of symptoms during the most severe episode of major depression, number of hospitalizations for major depres- sion, number of major depressive episodes, and frequency of major mood episodes (calculated as the total number of manic or major depressive episodes per year of illness) (fisfalen et  al. ). the dependent variables were con- verted into standardized z-scores for all analyses. predictor (independent) variables included: group (anabaptist vs. non-anabaptist), reported head injury, and presence of an alcohol use disorder (dsm-iv alco- hol abuse or dependence). there were too few anabaptist cases with other comorbidities to support any powerful comparisons. a history of admission to a psychiatric hos- pital was included as a covariate, since the majority of the anabaptist sample was recruited from hospital settings, but many of the non-anabaptists were not. categorical predictor variables were analyzed using two-way analysis of covariance (ancova) with bonferroni correction for  ×   =  tests (α =  . ). results demographics and comorbidity as expected, there were demographic differences between the anabaptist and non-anabaptist cases (table  ). anabaptists had more children and fewer years of education. anabaptists reported a significantly higher rate of major role impairment due to their illness and had higher rates of psychiatric hospitalization and psycho- tropic medication use. some differences in comorbidity were also observed (table  ). anabaptists reported fewer occurrences of medical illness, less alcohol and drug use, and were less likely to experience delusions outside of a mood episode. interestingly, anabaptists reported a higher lifetime inci- dence of head injury. the symptom profiles for the most severe episodes of major depression (fig.  ) and mania (fig.  ) were very similar between the anabaptists and non-anabaptists. however, anabaptist participants did report higher rates of psychomotor retardation during major depressive epi- sodes (χ  =  . , bonferroni p <  . ). factors influencing mood disorder severity alcohol use disorder (aud) was associated with greater severity of major depression within anabaptists, but not non-anabaptists, in this study. aud within the anabap- tists was associated with more hospitalizations for depres- sion, f( , )  =  . , p  =  . , corrected p  =  . (fig.  ). there was also a significant interaction between group and number of “clean” depressive episodes (not directly associated with alcohol use): aud was associated with more episodes among anabaptists but fewer episodes among non-anabaptists, f( , ) =  . , p =  . , cor- rected p =  . (fig.  ). there were no significant effects of aud on age at first major depression, number of symp- toms during the most severe episode of major depression, or frequency of major mood episodes in either group. lifetime history of head injury showed a trend toward association with more episodes of major depression in both anabaptist and non-anabaptist cases, f( , )  =  . , p  =  . , but this main effect was not significant after bonferroni correction. out of ana- baptists with a head injury, reported their first head injury before onset of major mood disorder, with a mean interval between injury and onset of  years. most of the reported head injuries were reported to be without loss of consciousness or need for medical attention. there were no significant effects of head injury on age at first major depression, number of symptoms during the most severe episode of major depression, number of hospi- talizations for major depression, or frequency of major mood episodes in either group. discussion we sought to fill several important gaps in the literature by comparing the course and symptom profiles of major mood disorders between anabaptists, a unique popula- tion characterized by varying degrees of cultural and genetic isolation, and cases drawn from the general pop- ulation who were matched on age, gender, and diagno- sis. two main findings emerged: ( ) despite substantial cultural and genetic differences, manic and depressive symptom profiles were very similar among anabaptist and non-anabaptist participants. ( ) alcohol use dis- orders were associated with greater severity of major depression in the anabaptist, but not the non-anabap- tist, participants we studied. the results demonstrate that the symptomatology of bipolar disorder and major depression shines through cultural and genetic differ- ences, while alcohol comorbidity may differentially influ- ence the course and severity of major mood disorders in important ways. the findings of this study should be interpreted in the context of several limitations. anabaptist participants were mostly recruited from psychiatric hospitals, which page of gill et al. int j bipolar disord ( ) : may oversample cases with more severe mood disorders within this population. individuals who are ill enough to require hospitalization may not be representative of mood disorders in the community, especially where awareness of mental illness and access to mental health services are limited. non-anabaptist cases were pre- viously enrolled on the basis of a diagnosis of bipolar i table demographic and clinical characteristics of anabaptists and non-anabaptists * bonferroni p <  . anabaptist non-anabaptist analysis n (%) n (%) χ married ( . ) ( . ) . medical illness* ( . ) ( . ) . ever treated ( . ) ( . ) . unable to function* ( . ) ( . ) . ever hospitalized* ( . ) ( . ) . medication ( . ) ( . ) . polarity* . st mania before st depression ( . ) ( . ) – st depression before st mania ( . ) ( . ) – visual hallucinations ever ( . ) ( . ) . auditory hallucinations ever ( . ) ( . ) . delusions ever* ( . ) ( . ) . alcohol use disorder* ( . ) ( . ) . suicide attempt ( . ) ( . ) . head injury ( . ) ( . ) . mean (sd) mean (sd) t value # children* . ( . ) . ( . ) − . # school years* . ( . ) . ( . ) . age illness onset . ( . ) . ( . ) . age st depressive episode . ( . ) . ( . ) . age st manic episode . ( . ) . ( . ) . # psych hospitalizations . ( . ) . ( . ) − . # illicit drugs used* . ( . ) . ( . ) . # suicide attempts . ( . ) . ( . ) . fig. symptoms reported during the most severe episode of major depression in anabaptist and non-anabaptist participants. dfa diffi- culty falling asleep, ema early morning awakening, pma psychomotor agitation, pmr psychomotor retardation, pdw passive death wishes; *bonferroni p < . fig. symptoms reported during the most severe episode of mania in anabaptist and non-anabaptist participants page of gill et al. int j bipolar disord ( ) : or schizoaffective bipolar disorder, and many were not ascertained in hospital settings (potash et  al. ). this ascertainment difference probably explains the higher overall rates of major role impairment, greater number of psychiatric hospitalizations, and increased use of psycho- tropic medications in the anabaptist cases we studied. ascertainment differences are unlikely to account for the observed effects of alcohol use or head injury, how- ever, since those analyses were controlled for group dif- ferences in psychiatric hospitalization rates. assessment procedures relied on the digs, which is a well-validated instrument (nurnberger et  al. ), but does not pro- vide good quantitative measures of psychiatric symptom- atology outside major dsm-based diagnostic categories. our ongoing work in the anabaptist communities incor- porates additional measures of neurocognition (card- enas et al. ) and subsyndromal symptoms, but these measures are not available for the non-anabaptists in the phenome database (potash et al. ). despite these limitations, several conclusions can be drawn. we found that the symptom profiles of severe major depressive and manic episodes were remark- ably similar in the anabaptists and non-anabaptists we studied. these findings are in broad agreement with the previous studies of egeland and colleagues performed in the pennsylvania amish (egeland and hostetter ; egeland et  al. ; hostetter et  al. ). differences in rates of marriage, number of children, and education level were all expected based on the previous literature (brewer and bonalumi ; kreps et al. ) and ana- baptist lifestyles and beliefs. the higher rate of psycho- motor retardation reported by anabaptist participants may reflect a greater awareness of this symptom owing to active lifestyles and widespread reliance on physical labor. we found that alcohol use disorder (aud) worsened the course of mood disorder, but only in the anabaptist participants we studied. prior work has shown that alco- hol use increases incidence of major depression (gilman and abraham ). we ran an analysis of the entire non-anabaptist database (phenome database n =  ), and found that the presence of an aud was related to increased depressive episodes, similar to previous find- ings. therefore, our findings are likely the result of a gen- der distribution that differs from the overall phenome sample. gender distributions were significantly differ- ent in our matched sample compared to those in the full phenome database (matched sample =   % male, overall phenome database =   % male, p <  . ). therefore, the results should be interpreted with the greater proportion of females in mind. since there are more practical and cultural barriers to alcohol use among anabaptists, aud in this group may reflect poorer social adjustment and other psychosocial vulnerabilities that contribute to increased depressive episodes. aud may also lower the threshold for psychiat- ric hospitalization among anabaptists. we also observed that aud was associated with increased number of clean depressive episodes among the anabaptists we studied. fig. main effects of group and alcohol use disorder on number of hospitalizations for major depression in anabaptist (n = ) and non- anabaptist (n = ) participants fig. interactive effects of group and alcohol use disorder on num- ber of “clean” major depressive episodes in anabaptist (n = ) and non-anabaptist (n = ) participants page of gill et al. int j bipolar disord ( ) : this suggests that those anabaptists who do develop aud are vulnerable to increased depressive episodes, that depressive episodes predispose to aud, or that some third factor—such as social isolation or deviance— is involved. further studies that use a longitudinal design would be needed to clarify this association. we found that the course and severity of mood dis- order was associated with head injury at a trend level in both anabaptist and non-anabaptist participants, although head injuries were more common among ana- baptists. previous studies have also found a higher rate of head injuries in anabaptists. one study found that   % of injuries in amish children aged   months to   years involved the head and central nervous system (vitale et al. ). previous work has also shown that head inju- ries are an important contributor to mood disorder mor- bidity (mrazek and haggerty ). between and  % of individuals who experience a traumatic brain injury develop a mood disorder within a year of the injury (deb et  al. ; jorge and robinson ). our results are consistent with these findings, and highlight the poten- tial importance of head injuries that might be considered “minor” since they do not lead to loss of consciousness or medical attention. if confirmed in larger samples, the results might suggest that public health measures aimed at reducing head injuries could have a beneficial impact on mood disorder morbidity. conclusion this is the first study to directly compare major mood disorders in anabaptist and non-anabaptist individuals. despite cultural differences, there was a remarkable simi- larity in symptom profiles for severe episodes of mania or major depression. however, alcohol comorbidity, head injury, and their effects on illness course suggest some differences that deserve further investigation. these results also show that the presentation of a highly herit- able psychiatric illness such as bipolar disorder does not differ much in the genetically isolated anabaptist popu- lation, implying that genetic insights enabled by isolated populations might also inform our understanding of psy- chiatric disorders in the general population. abbreviations aud: alcohol use disorder; ambigen: amish mennonite bipolar genetics study; ancova: analysis of covariance; digs: diagnostic interview for genetic studies; dsm-iv: diagnostic and statistical manual of mental disorders, th edition; nimh: national institute of mental health; spss: statistical package for the social sciences. authors’ contributions all authors contributed to the design of the study. kg wrote the initial draft of the manuscript. all authors reviewed and approved the final text of the manuscript. all authors read and approved the final manucsript. author details human genetics branch, section on genetic basis of mood and anxiety disorders, national institute of mental health intramural research program, national institutes of health, new york city, usa. department of psychol- ogy, the catholic university of america, michigan ave ne, washington, d.c. , usa. institute of psychiatric phenomics and genomics (ippg), medical center of the university of munich, munich, germany. acknowledgements this work was supported by the national institute of mental health (nimh) intramural research program (zia-mh ; nct ). we thank the study participants who gave generously of their time to make this study pos- sible. some data and biomaterials were collected as part of ten projects that participated in the national institute of mental health (nimh) bipolar disorder genetics initiative. from – , the principal investigators and co-investiga- tors were: indiana university, indianapolis, in, r mh , john nurnberger, m.d., ph.d., marvin j. miller, m.d., elizabeth s. bowman, m.d., n. leela rau, m.d., p. ryan moe, m.d., nalini samavedy, m.d., rif el-mallakh, m.d. (at university of louisville), husseini manji, m.d. (at wayne state university), debra a. glitz, m.d. (at wayne state university), eric t. meyer, m.s., carrie smiley, r.n., tatiana foroud, ph.d., leah flury, m.s., danielle m. dick, ph.d., howard edenberg, ph.d.; washington university, st. louis, mo, r mh , john rice, ph.d, theodore reich, m.d., allison goate, ph.d., laura bierut, m.d.; johns hopkins university, baltimore, md, r mh , melvin mcinnis, m.d., j. raymond depaulo, jr., m.d., dean f. mackinnon, m.d., francis m. mondimore, m.d., james b. potash, m.d., peter p. zandi, ph.d, dimitrios avramopoulos, and jennifer payne; university of pennsylvania, pa, r mh , wade berrettini, m.d., ph.d.; university of california at irvine, ca, r mh , william byerley, m.d., and mark vawter, m.d.; university of iowa, ia, r mh , william coryell, m.d., and raymond crowe, m.d.; university of chicago, il, r mh , elliot gershon, m.d., judith badner, ph.d., francis mcmahon, m.d., chunyu liu, ph.d., alan sanders, m.d., maria caserta, steven dinwiddie, m.d., tu nguyen, donna harakal; university of california at san diego, ca, r mh , john kelsoe, m.d., rebecca mckinney, b.a.; rush university, il, r mh , william scheftner, m.d., howard m. kravitz, d.o., m.p.h., diana marta, b.s., annette vaughn-brown, m.s.n., r.n., and laurie bederow, m.a.; nimh intramural research program, bethesda, md, z mh - , francis j. mcmahon, m.d., layla kassem, psyd, sevilla detera-wadleigh, ph.d, lisa austin, ph.d, den- nis l. murphy, m.d. competing interests the authors declare that they have no competing interests. ethical standards the authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the helsinki declaration of , as revised in . all participants signed informed consent before participating in this study, which was approved by the national institute of mental health. received: july accepted: september references brewer ja, bonalumi nm. cultural diversity in the emergency department: health care beliefs and practices among the pennsylvania amish. j emerg nur. ; ( ): – . cardenas sa, brotman ma, kassem l, leibenluft e, mcmahon fj. neurocogni- tive functioning in euthymic patients with bipolar disorder and unaf- fected relatives: a review of the literature. neurosci biobehav rev. . doi: . /j.neubiorev. . . . deb s, lyons i, koutzoukis c, ali i, mccarthy g. rate of psychiatric illness year after traumatic brain injury. am j psychiatry. ; ( ): – . egeland ja, gerhard ds, pauls dl, sussex jn, kidd kk, alien cr, housman de. bipolar affective disorders linked to dna markers on chromosome . nature. ; ( ): – . http://dx.doi.org/ . /j.neubiorev. . . page of gill et al. int j bipolar disord ( ) : egeland ja, hostetter am. amish study, i: affective disorders among the amish, – . am j psychiatry. ; ( ): – . egeland ja, hostetter am, eshleman sk. amish study iii. the impact of cultural factors on diagnosis of bipolar illness. am j psychiatry. ; ( ): – . fisfalen me, schulze tg, depaulo jr jr, degroot lj, badner ja, mcmahon fj. familial variation in episode frequency in bipolar affective disorder. am j psychiatry. ; ( ): – . gilman se, abraham hd. a longitudinal study of the order of onset of alcohol dependence and major depression. drug alcohol depend. ; ( ): – . ginns ei, jean ps, philibert ra, galdzicka m, damschroder-williams p, thiel b, murray ma. a genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affec- tive disorder among the old order amish. proc natl acad sci usa. ; ( ): – . hostetter am, egeland ja, endicott j. amish study, ii: consensus diagnoses and reliability results. am j psychiatry. ; ( ): – . hou l, faraci g, chen dt, kassem l, schulze tg, shugart yy, mcmahon fj. amish revisited: next-generation sequencing studies of psychiatric disor- ders among the plain people. trends genet. ; ( ): – . johnson sl, winett ca, meyer b, greenhouse wj, miller i. social support and the course of bipolar disorder. j abnorm psychol. ; ( ): . jorge r, robinson rg. mood disorders following traumatic brain injury. int rev psychiatry. ; ( ): – . kelsoe jr, ginns ei, egeland ja, gerhard ds, goldstein am, bale sj, conte g. re-evaluation of the linkage relationship between chromosome p loci and the gene for bipolar affective disorder in the old order amish. nature. ; ( ): – . kreps gm, donnermeyer jf, kreps mw. a quiet moment in time a contempo- rary view of amish society. sugarcreek: carlisle printing; . mckusick va. the amish. endeavour. ; ( ): – . mckusick va, hostetler ja, egeland ja. genetic studies of the amish. bull johns hopkins hosp. ; : – . mrazek pj, haggerty rj. reducing risks for mental disorders frontiers for preventive intervention research. washington, d.c.: national academies press; . nurnberger ji, blehar mc, kaufmann ca, york-cooler c, simpson sg, harkavy- friedman j, reich t. diagnostic interview for genetic studies: rationale, unique features, and training. arch gen psychiatry. ; ( ): – . potash jb, toolan j, steele j, eng b, miller eb, pearl j, simpson sg. the bipolar disorder phenome database: a resource for genetic studies. am j psychi- ary. ; ( ): – . shaw ja, egeland ja, endicott j, allen cr, hostetter am. a -year prospective study of prodromal patterns for bipolar disorder among amish youth. j am acad child psychiary. ; ( ): – . smith tb, mccullough me, poll j. religiousness and depression: evidence for a main effect and the moderating influence of stressful life events. psychol bull. ; ( ): . trimble j. cultural variations in the use of alcohol and drugs psychology and culture. boston: allyn & bacon; . p. – . vitale ma, rzucidlo s, shaffer ml, ceneviva gd, thomas nj. the impact of pedi- atric trauma in the amish community. j pediatr. ; ( ): – . weyer sm, hustey vr, rathbun l, armstrong vl, anna sr, ronyak j, savrin c. a look into the amish culture: what should we learn? j transcult nurs. ; ( ): – . zuk o, schaffner sf, samocha k, do r, hechter e, kathiresan s, lander es. searching for missing heritability: designing rare variant association stud- ies. proc natl acad sci. ; ( ):e – . 	symptom profiles and illness course among anabaptist and non-anabaptist adults with major mood disorders 	abstract 	background: 	methods: 	results: 	conclusions: 	background 	methods 	sample and procedures 	statistical analyses 	results 	demographics and comorbidity 	factors influencing mood disorder severity 	discussion 	conclusion 	authors’ contributions 	references innate immunity in asthma e d i t o r i a l t h e n e w e n g l a n d j o u r n a l o f m e d i c i n e n engl j med ; nejm.org august , innate immunity in asthma talal a. chatila, m.d., m.sc. it is appreciated that the marked increase in the prevalence of asthma over the past few decades reflects changes in environmental exposures and living conditions associated with modern lifestyles. of particular interest is the documen- tation of a protective effect of exposures associ- ated with traditional farming, the influence of which has waned with increased urbanization and the advent of mechanized agriculture. on small family-based farms where children are reared in close proximity to farm animals and their sheds, increased exposure to the microbial products found in these environments, including lipopolysaccharides, has been associated with protection against asthma. , it remains unclear how exposure to a traditional farming environ- ment confers protection against asthma and whether such protection also applies in the con- text of large-scale industrialized farming. stein et al. now advance our knowledge on both ac- counts in this issue of the journal. in their study, stein et al. took advantage of a lifestyle attribute that differentiates two other- wise closely related u.s. populations in which the incidence of asthma is dissimilar. the amish and the hutterites are reproductively isolated farming communities that are linked by ances- try, having originated in german-speaking alpine regions of europe. they also share a similar lifestyle that includes environmental exposures that often affect the risk of asthma, with one notable exception — whereas the amish have maintained a traditional farming practice that revolves around single-family dairy farms and eschews mechanization, the hutterites practice large-scale, highly mechanized communal farm- ing. the prevalence of asthma and allergic sen- sitization among the amish is low, but among the hutterites the prevalence of both conditions is strikingly high, similar to that in the u.s. population at large. , as such, these two com- munities are ideally suited for analysis of the influence of environmental exposures on sus- ceptibility to asthma. by studying children from these two commu- nities, stein et al. confirmed the discrepancy that exists in the communities’ incidences of allergy and asthma. the researchers also estab- lished the presence of a distinct microbial com- position and an increased burden of lipopolysac- charides in dust samples collected from the houses of the amish as compared with those of the hutterites. after exposing samples of periph- eral-blood lymphocytes from both populations to lipopolysaccharides, the samples from the amish expressed more innate immunity-related cytokines than those from the hutterites. the peripheral-blood lymphocytes of amish children also exhibited a genetic signature characterized by higher levels of the gene transcripts associ- ated with innate-type immune responses, includ- ing those involved in the innate immune re- sponse to microbial products such as tumor necrosis factor and irf . in addition, dust sam- ples collected from amish households suppressed the induction of airway inflammation in a mouse model of allergic asthma. this protection was abrogated in mice lacking myd and trif, adaptor proteins that mediate signaling by mi- crobial products through toll-like receptors (tlrs). in agreement with these findings are previous studies on protective bacterial species isolated from farm-dust samples, including the gram-negative bacterium acinetobacter lwof f ii, whose application to the airways of mice trig- gers local and systemic inflammatory innate the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. t h e n e w e n g l a n d j o u r n a l o f m e d i c i n e n engl j med ; nejm.org august , immune responses in a tlr-dependent manner. overall, the results of the study by stein et al. are consistent with the idea that the protective effect of amish dust is related to its distinct microbial composition. what mechanisms account for the protective effect of long-term, innate, immune-cell activa- tion by farming-related microbial products (fig. )? studies have delineated a network of innate im- mune cells, including epithelial cells, type in- nate lymphoid cells, mast cells, dendritic cells, and others, that are activated by allergens. this network programs a proallergic adaptive immune response involving allergen-specific type helper t cells and ige-producing b cells that sustains disease activity. the findings of stein et al. sup- port the notion that exposure to microbe-rich farm dust directs an alternative, proinflamma- tory, innate immune response involving tran- scriptional pathways and mediators, including nuclear factor κb and irf , that prevents the emergence of asthma. it should be pointed out that microbial products may also act through tlrs and myd to activate the formation of regulatory t cells that enforce tolerance at mu- cosal surfaces. thus, both long-term, low-level activation of innate immune cells and possibly t-cell–related immune regulatory mechanisms may contribute to the protective effects of the amish farm dust against asthma. stein et al. also leave several questions unan- swered. it is unclear whether continuous expo- sure to farm dust is required to maintain its protective effect against allergic asthma. in mice, pregnant mothers exposed to a. lwoffii transmit protection against allergic asthma to their off- spring through mechanisms that involve mater- nal tlr activation, which suggests that an epi- genetic effect may be acquired in utero. it is also unclear whether the protective effect of mi- crobial exposure requires live microbes that may colonize the airways or can be reproduced with purified microbial products. the answers to these questions will help to harness the insights gleaned from the studies of stein et al. and others for the purpose of treating and preventing asthma. disclosure forms provided by the author are available with the full text of this editorial at nejm.org. from the division of immunology, boston children’s hospital, and the department of pediatrics, harvard medical school, boston. . eder w, ege mj, von mutius e. the asthma epidemic. n engl j med ; : - . . von mutius e, vercelli d. farm living: effects on childhood asthma and allergy. nat rev immunol ; : - . . braun-fahrländer c, riedler j, herz u, et al. environmental exposure to endotoxin and its relation to asthma in school-age children. n engl j med ; : - . . ege mj, mayer m, normand a-c, et al. exposure to environ- mental microorganisms and childhood asthma. n engl j med ; : - . . stein mm, hrusch cl, gozdz j, et al. innate immunity and asthma risk in amish and hutterite farm children. n engl j med ; : - . . holbreich m, genuneit j, weber j, braun-fahrländer c, was- er m, von mutius e. amish children living in northern indiana have a very low prevalence of allergic sensitization. j allergy clin immunol ; : - . . motika ca, papachristou c, abney m, lester la, ober c. rising prevalence of asthma is sex-specific in a us farming population. j allergy clin immunol ; : - . figure . farming lifestyle, the activation of innate immunity, and protection against asthma. differences in the prevalence of asthma and allergic sensitization in amish and hutterite communities are linked to their distinct farming practices. the traditional farming practices in amish communities protect against asthma by inducing a long-term, low-level, proinflammatory innate immune response. this protection involves the activation by microbial signals, act- ing through myd and trif and the production of tumor necrosis factor (tnf) and interleukin- . the hutterites, who practice mechanized farming and are not exposed to the same microbial influences, are not protected. the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. edit or i a l s n engl j med ; nejm.org august , . conrad ml, ferstl r, teich r, et al. maternal tlr signaling is required for prenatal asthma protection by the nonpathogenic microbe acinetobacter lwoffii f . j exp med ; : - . . pulendran b, artis d. new paradigms in type immunity. science ; : - . . wang s, charbonnier lm, noval rivas m, et al. myd adaptor-dependent microbial sensing by regulatory t cells pro- motes mucosal tolerance and enforces commensalism. immu- nity ; : - . doi: . /nejme copyright © massachusetts medical society. the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. international journal of pharmaceutical sciences and research (ijpsr) pages menu 	home 	about us 	contact us 	search articles issn (online): - , issn (print): - international journal of pharmaceutical sciences and research an international journal published monthly an official publication of society of pharmaceutical sciences and research categories menu 	editorial board 	current issues 	archives 	instructions to authors 	manuscript submission 	conference proceedings international journal of pharmaceutical sciences and research international journal of pharmaceutical sciences and research (ijpsr) is an publication of society of pharmaceutical sciences & research. it is an open access online and print international journal published monthly. website: www.ijpsr.com projected impact factor ( ): . , icv : . , : . , : . doi: . /ijpsr. - sj impact factor ( ): . global impact factor ( ): . , ( ): . indexing - embase- elsevier's international journal of pharmaceutical sciences and research panchkula (hr), india panchkula, hr in editorijpsr@gmail.com / ijpsronline@gmail.com phone: + - (ist am – pm) http://ijpsr.com/wp-content/themes/ijpsr/images/logo.png 	 			 					 												 					 				 	 					 												 					 				 	 					 												 					 				 	 			 				 						 							thu 							benzothiazole – a magic molecule 															posted in blog 													 					 	 						 							tue 							chemical synthesis of bile acids and their physico-chemical properties 															posted in blog 													 					 	 						 							tue 							an overview on covid- outbreak: epidemic to pandemic 															posted in blog 													 					 		 	 abstracting and indexing information 			embase -elsevier, pubmed (selected citations), , thomson reuters, web of science - emerging sources citation index (jan ), scopus ( - ), corss ref.,hinari- who, chemical abstract, scirus - elsevier's, gale- expanded academic asap, ebsco, google, google scholar, international consortium for the advancement of academic publication (icaap), scientific common, pharmaceutical sciences open access resources (psoar), index copernicus, ulrich's international periodical directory, proquest, new york university health sciences libraries, research gate, open-j-gate, geneva foundation for medical education & research, ayush research portal and genamics journal seek. 		 ijpsr updates 				volume , issue , april of ijpsr is available online 	an embase - elsevier indexed journal 	our associate monthly journals : 	international journal of pharmacognosy - www.ijpjournal.com 	international journal of life sciences and review - www.ijlsr.com 	join us on www.facebook.com/spsr 		 why publish with us? 				worldwide dissemination through open access 	immediate access of research of global audience 	authoritative & constructive peer review process 	prompt review 	indexed with most international bibliographic databases 	regular e- alert and sms alert 		 volume ( ) - issue , april review articles . overview of favipiravir and remdesivir treatment for covid- the current coronavirus disease (covid- ) outbreak caused by severe acute respiratory syndrome coronavirus (sars-cov- ) emerged in the wholesale market in wuhan, china in the last months of and spread to almost all countries of the world. although several vaccines have already been developed in different countries of the world, there is currently no specific treatment for covid- . so... e. eroglu * and c. toprak department of pharmacology, faculty of pharmacy, lokman hekim university, ankara, turkey. doi: . /ijpsr. - . 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( ). - - 	 abstract 	 html full text 	 pdf 	 read more spsr national webinar – 		 			 			‘impact of covid on pharmaceutical education and research’ on th january ; time pm onwards. for registration, kindly open the following link and fill the required details. https://forms.gle/ygrknna dhyskev 		 	 citescore ( ) : . 			 . citescore   powered by   		 spsr youtube channel 			subscribe to youtube channel of spsr https://www.youtube.com/c/societyofpharmaceuticalsciencesandresearch 		 	home 	about us 	editorial board 	current issues 	instructions to authors 	manuscript submission 	contact us 	gallery 	manuscript tracking all © are reserved by international journal of pharmaceutical sciences and research this journal licensed under a creative commons attribution-noncommercial-sharealike . unported license report resumes ed subcultural differences in children's language development. entwisle, doris r. johns hopkins univ., baltimore, md. report number br- - - pub date may grant oeg .. ... ecrs price mfs . hc -$ . p. descriptors- *verbal development, *rural urban differences, racial differences, *language development, *environmental influences, language.ability, *sociolinguistics, linguistics, interviews, socioeconomic status, culturally disadvantaged, baltimore patterns of the linguistic development of children of different socioeconomic environments were determined by a study of word associations. the relation of residential area, social class, or subcultural group membership to linguistic development was the main concern of the study. each membership group was further categorized according to iq level, sex, and grade. groups were compared by holding constant age and iq. the word associations were obtained in response to a list of stimulus words. each child. was interviewed alone, and was asked to respond with the first word thought of as the interviewer said a word aloud. results of the study support the following conclusions - -( ) there are negligible differences. between surbursan children from upper middle class and blue collar neighborhoocs, ( ) rural maryland children tend to develop more slowly than the suburban children, especially those whose iq is average or below, ( ) amish children develop even more slowly than the rural maryland children, and ( ) white slum children are advanced compared to suburban children at first grade, but retarded at third grade. negro slum children are generally behind white slum children, but at first grade the negro slum children are on a par with white suburban children. related 'reports are aa and aa . (al) the johns hopkins university subcultural differences in children's language development doris r. entwisle the johns hopkins university u. s. department of health, education and welfare office of education this document has been reproduced exactly as received from the person or organization originating it. points of view of opinionsstated do not necessarily, represent official office of education position or policy subcultural differences in children's language development doris r. entwisle the johns hopkins university subcultural differences in children's language development project no. - contract no. oeg- - - - doris r. entwisle may the research reported herein was performed pursuant to a grant with the office of education, u.s. department of health, education and welfare. contractors undertaking such projects under govern- ment sponsorship are encouraged to express freely their professional judgment in the conduct of the project. points of view or opinions stated do not, therefore, necessarily represent official office of education position or policy. the johas hopkins university baltimore, maryland contents acknowledgments i. introduction ii. method iii. results school differences racial differences urbanization iv. discussion v. summary references cited footnotes map (pennsylvania - maryland) "geographical locations of sample clusters" tables table . design of clusters of children living in and around baltimore, maryland table . percentage of paradigmatic responses , , acknowledgments the preparation of this paper was supported by the center for the study of social organization of schools, the johns hopkins university. most of the research upon which it is based was supported by the national institute of child health and human development, grant hd- . i am grateful to james coleman, robert gordon, edward mcdill, and arthur stinchcombe for critical reading of this paper. i. introduction word associations, although long a topic for psychological inquiry, have only within the last decade assumed prominence among the tools of developmental psycholinguistics. two pioneering studies, one by brown and berko ( ) and another by ervin ( ), show cor- relation between the prevalence of paradigmatic associations and the grammatical competence of the child. independent evidence of the validity of word associations as an index of language maturity is that children's free associations are highly related to word comprehension and ability to place words in appropriate contexts (riegel, riegel, smith and quarterman, ). also a high correlation is observedbe- tween semantic meanings and primary associates (di vesta, ). other studies (entwisle, forsyth, and muuss, ; entwisle a, b, a, b) confirm both the sharp rise in paradigmaties over ages to and the replacement of syntagmatic with paradigmatic associates. (the syntagmatic-paradigmatic shift, although discovered years ago (woodrow and lowell, ), has received little attention until lately.) high-speed data-processing equipment has opened the door to cross-cul- tural study of linguistic development using word associations of chil- dren from different social classes and different cultural groups. this paper reports subcultural variations in language development for chil- dren from to years of age, and speculates upon the socialization and cultural factors that spawn the differences. it is not very clear what specific environmental factors are important in verbal development and the major concern here is to elucidate those factors. this research, which began in , was undertaken partly to pro- vide normative word association data for sizeable groups of young chil- dren, and partly to shed light on the course of linguistic development. the present research differs from other studies of associations in that the intelligence level of various samples of children is specified rather closely and the socio-cultural environment, including the school milieu, is described in detail. as expected, intelligence (measured iq) is highly related lo appearance of paradigmatic associates, espe- cially for the younger children. intelligence is of secondary interest here, however. residential locus, social class, or subcultural group membership, and their relation to linguistic development is the main focus of this paper. insofar as possible, social class is conceived in fairly specific terms--median income, father's educational level, quality of school attended, housing conditions, and so forth. ii. method several different groups of children at first-, third-, and fifth- grade have been studied. rural american children, both amish-pennsyl- vanian and a cross-section of typical maryland farm children, and some rural german children, are represented. urban baltimore children of three different socioeconomic levels, upper middle class, blue collar, and slum, are also represented. baltimore city has a population of about one-million persons. entirely surrounding the city is baltimore county, and the city and county have separate school systems. the racial composition of schools is vastly different, with about per cent negro pupils in the county compared to per cent negro in the city, but in neither place are negroes homogeneously distributed. for upper middle and working class, only white children are studied and they live in the suburbs (baltimore county). for the lowest socio- economic group, slum residents of baltimore city, equal numbers of negro and white children were procured. each residential or socioeconomic group is further categorized according to iq level, sex, and grade, and groups can be compared holding constant age and iq. three iq strata are defined: low iq ( or below); medium iq ( - ); and high iq ( or higher). iq data were mostly taken from school records. not all iq strata are repre- sented in all residential groups. for instance, there are no "high iq" slum children. background data on the different groups are given in table which displays the incomplete factorial design of the sampling plan. figure shows geographical locations of sample clusters. the amish, a distinct subcultural group who develop in a con- siderably different cultural stream from rural maryland children, re- side on farms north of the maryland-pennsylvania line (see fig. ). they are descended from anabaptist immigrants of years ago, and are of special interest because religious scruples cause them to iso- late themselves from tv sets, radios, and other mass media. amish child-rearing practices also reduce exposure to spoken language be- cause conversation between adults and children is limited and educa- tion is not valued. these children are not economically deprived, for the farms are well-run and prosperous, but automobiles and other "luxuries" are fnrbidden. the amish children were interviewed in exactly the same way as the maryland children, with an amish woman serving as interviewer. briefly, stimulus words, chosen to represent different form classes and, where possible, different degrees of rarity, are used to elicit associations. the thorndike-lorge j count was used to estimate frequency ( thorndike and lorge, ). for nouns, adjectives, and verbs, stimuli are divided into strata, one with frequency greater than , a second with frequency to , and a third with frequency below . no frequency division was possible for adverbs and pronouns. a young woman interviewed children one at a time, saying aloud each word on the list and then recording the child's response. the child is instructed that he is going to play a "word game", and when the interviewer says a word aloud, he is to respond with the first word he thinks of. details of administration are given in entwisle ( b). figure i i urban slum low ses suburban high ses suburban rural maryland rural amish table design of clusters of children living in and around baltimore, md. suburban white suburban white high-ses (income est. $ p.a.) low-ses (income est. $ p.a.) grade high-iq med.-iq med.-iq low-iq kindergarten n mean age . mean iq mean grade completed by father . a . b grade n mean age . . . . mean iq . . . . mean grade completed by father . . . . grade n mean age . . . . mean iq . . . . mean grade completed by father . . . . grade n mean age . . . . mean iq . . . . mean grade completed by father . . . . a. for ninety-nine subjects b. for eighty-eight subjects .....wwntvlnlmmlnawrwowprmwmpgwpkimp.....p.w...y table (cont.) baltimore slum rural maryland amish white negro high med. low med. low med. low grade iq iq iq iq iq iq iq iq grade n mean age . . . . c . . . - . mean iq . . . . . . . . . mean grade . . . . . d . e . f . g completed by father grade n mean age . . . . . . . . mean iq . . . . . h . . . . . mean grade . . . . . . . . k completed by father grade n mean age . . . . , . . . . mean iq . . . . . . . . mean grade . . . . . .gm . n . ( completed by father c. for two of the twenty children iq scores were not. available . d. for seven subjects e. for fifteen subjects f. for ten subjects g. for six subjects h. for seventeen subjects i. for six subjects i. for five subjects k. for six subjects . for ten subjects m. air eleven subjects n. for three subjects o. for nine subjects r. iii. results relative positions of subcultural groups. word associations were analyzed in a number of ways (see entwisle, a, b, a, b), but the percentage of paradigmatic responses in the various sample strata offers the most convenient way to study relative rates of development (see table ). analyses of variance of paradigmatic races support ale following conclusions about relative rates of devel- opment. (all comparisons assume children of the same iq level.) ( ) there are negligible differences between suburban children from upper middle class and blue collar neighborhoods. ( ) rural maryland children tend to develop more slowly than the suburban children, especially those who fall in the lower two iq strata. ( ) amish children develop even more slowly than the rural maryland children. ( ) white slum children are advanced compared to suburban chil- dren at first-grade, but retarded at third-grade. negro slum children are generally behind white slum children, but at first-grade the negro slum children are on a par with white suburban children. these observations in american children suggest a relation be- tween rate of development and degree of urbanization. at first-grade the children rank as follows: white slum children, negro slum chil- dren and suburban middle class and working class children of both ses levels, rural maryland children, and amish children residing in rural pennsylvania. study of rural german children living in schleswig- holstein (entwisle and muuss, ) and of parisian children (rosen- zweig and menahem, ) suggests a similar rural-urban difference in non-english-speaking children. appearance and prevalence of paradigmatic responses is taken as an index of linguistic development, and rates of development among various groups are compared. differences among groups have been pointedup, but these are related to timing rather than to qualitative factors. it should be emphasized that the sequence of changes and the eventual level of paradigmatic responding are theosame for all groups. the paradigmatic response rate is a rough measure and only appropriate over early childhood. other more refined measures would probably reveal differences at ages - when paradigmatic response rates have attained fairly constant levels. the increase in paradigmatic re- sponses, the increase in commonality or percentage of primaries, and the diminution in syntagmatic responses are common features in all groups. pronouns and adjectives develop sooner than verbs and adverbs, and there is only a small increase for nouns. the overall picture looks much the same for all groups and the process itself seems in- variant. some children just take longer than others to pass through the sequence of changes. ,.....a, ripm.nown,r s u b u r b a n b a l t i m o r e c h i l d r e n - k i n d e r g a r t e n f i r s t - g r a d e t h i r d - g r a d e h i g h - l o w - h i g h - s e s l o w - s e s h i g h - s e s l o w - s e s f i f t h - g r a d e h i g h - s e s l o w - s e s s e s s e s h i g h i q m e d . i q m e d . i q l o w i q h i g h i q m e d . i q m e d . i q l o w i q h i g h i q m e d . i q m e d . i q l o w i q ' n o u n s . . . . . . . . . . . . . . a d j . . . . . . . . . . . . . . . v e r b s . . . . . . . . . . . . . . a d v e r b s . . . . . . . . . . . . . . p r o n o u n s . . . . . . . . . . . . . . f i r s t - g r a d e t h i r d - g r a d e f i f t h - . g r a d e r u r a l m a r y l a n d a m i s h r u r a l m a r y l a n d a m i s h r u r a l m a r y l a n d a m i s h h i g h i q m e d . i q _ _ l o w i q i q h i g h i q m e d . i q l o w i q i q h i g h i g m e d , i q l o w i q i q n o u n s . . . . . . . . . . . . a d j e c t i v e s . . . . . . . . . . . . . v e r b s . . . . . . . . . . . . a d v e r b s . . . . . . . . . . . . . p r o n o u n s . . . . . . . . . . . . b a l t i m o r e s l u m c h i l d r e n - f i r s t - g r a d e t h i r d - g r a d e f i f t h - g r a d e w h i t e n e g r o w h i t e n e g r o w h i t e n e g r o m e d . i q l o w i q m e d . i q l o w i q m e d . i q l o w i q m e d . i q l o w i q m e d . i q l o w i q m e d . i q l o w i q n o u n s . . : . . . . . . . . . . , a d j e c t i v e s . . . . . . . . . . . . v e r b s . . . . . . . . . . . . a d v e r b s . . . . . . . . . . . . p r o n o u n s . . . . . . . . . . . a.z.ares: weasa_a- results are very surprising in two respects: ( ) rural maryland children develop more slowly than suburban maryland children even 'though the children are of the same age and the same tested intelli- gence, and are closely matched in terms of schools attended, father's educational level and general economic status; ( ) first-grade white children living in the slums of baltimore city are accelerated com- pared to first-grade white children living in the suburbs, and first- grade negro children are not behind white suburban children. it is not so surprising, but it is informative, that rural amish children lag behind rural maryland children. slum children being behind suburban children at third- and fifth- grades is not very surprising either, because reports of verbal or intellectual deficit associated with economic deprivation are legion (see especially reports by . deutsch and his co-workers ( to ) and by coleman ( ). in fact, study of slum children was undertaken after study of upper middle class and working class children had shown minimal differences (entwisle, a, b). it was thought that more extreme economic and cultural deprivation might be associated with language deficits even though mild relative deprivation was not. results directly counter to expectation are both exciting and challenging. school differences. a few words are in order first about sclools the children attended. many of the suburban children had attended kindergarten but rural children had not. this could account, at least in part, for rural-urban differences. it could not account for rural maryland vs. amish differences, because neither group attended kindergarten. a very large proportion of inner city children ( - per cent) attend free kindergartens. we estimate that per cent of the middle class and per cent of the working class children had attended kinder- garten. kindergarten attendance cannot explain the superiority of slum children over suburban upper middle class children, then, because a very large proportion of both groups attended kindergarten. another fact negating the importance of kindergarten is the equivalence at first-grade of working class and upper middle class suburban children even though many fewer working class children attend. nevertheless, at first-grade there is some correspondence between the per cent of children in a given residential group attending kindergarten and level of paradigmatic response rates because there are no reversals and some consistencies between paradigmatic level and proportion of kindergarten attendance. a favorable impact of kindergarten is reported by lee ( ) and deutsch and brown ( ). a strong reason for starting research in baltimore county was that one large school system enrolls children residing in a - square -mile area, and this permits "school factors" to be held con-. stant. (such a large school district is rare and not typical of the u.s.a.) rural maryland children, upper middle class suburban children, and working class suburban children all attend schools under the same jurisdiction. there is bound to be variation favoring schools nearer the'city over rural areas,, but this variation is small compared to variations between systems. salary scales, curricula, textbooks, testing programs, and many other conditions are the same throughout one system. it seems unlikely, therefore, that school quality is a major factor in rural-urban differences. the amish, of course, at- tend schools in pennsylvania (some parochial) and so differences in school quality are completely confounded with rural maryland-amish differences. big differences at first-grade, however, suggest that more than just school factors are involved, because at first-grade the school has had little time to exert an effect. it is hard to make an overall comparison between schools in baltimore city and baltimore county, but generally teacher qualifica- tions in the city are equivalent to those in the county. the physical condition of schools in the inner city is mostly inferior to schools in the county (or to schools in other parts of the city) but this relates more to lack of ancillary facilities like cafeteria, audito- rium, sinks in classrooms, and so on, than to lack of educational materials or the basic classroom essentials. the relative advance- ment of suburban third- and fifth-graders compared to inner city chil- dren could be owing, at least in part, to differences in school quality. first-grade slum children being superior is just the opposite from what could be predicted from school quality, but as was mentioned in the case of the rural maryland-amish differences, the school's effect has not had much time to be felt at first-grade. one might questidn the assignment of children to iq groups as a possible cause of differences. the same iq test (pma) is used for first-graders by the two school systems, but rural children and inner city children may both be in "culturally unfair" positions vis-a-vis standard tests. thus these children's "true intelligence level" might be higher than the test score indicates. for rural children such a bias would lead one to predict an elevation in verbal develop- ment rather than the lag actually observed, because to score at an average level on an unfair verbal test implies that their endowment is underestimated. for slum children it is the pattern of results that vitiates the bias argument. first-graders of average iq are responding like high iq ( ) suburban children, and third-graders of average iq are responding like low iq ( ) suburban children. there is no reason to believe that a strong bias at one age would be re- versed in direction two years later. racial differences. it is not fully clear what subcultural factors could lead to the differences in paradigmatic rates between white and negro slum children. they attend similar schools and live close to one another, except that the negro slum dweller is probably more deprived. (number of children per family, mothers working and fathers absent, illegitimacy rates, or almost any other measure of social class turns out to be less favorable for negro slum dwellers (see keller, ).) for census tracts where these particular slum children's schools are located, percentage of unemployment, number of occupants per room, age of dwelling, and number of women in the labor force are all directly related to percentage of negroes in the tract. but there seem to be many more similarities than differences between negro and white slum dwellers, especially when they are being con- sidered with respect to suburbanites. the dialect cleavage between middle class interviewers and lower class negro children may be greater than that between these same interviewers and lower class white children, and this might favor the white children. the speech of negro college girls who served as interviewers resembles that of the white community more than that of the negro slum. the most provocative difference that exists between the white and negro slum groups is in terms of in-migration. for persons who resided outside the baltimore area years previously, most lived to the south, irrespective of race membership. but proportionately many more negroes than whites are recent migrants. in the largely negro ( per cent) tract, about per cent had moved up from the south in the previous years compared to .to per cent in the predominantly white tracts. persons coming from the south probably come mostly from rural areas of virginia and other southern states, baltimore being the first large industrial city going north on the eastern sea- board. the relative advancement of the white over the negro slum children may thus reflect a rural-urban rather than a racial differen- tial. this will be discussed more fully later. racial differences of another kind are related to interviewer effects. there is considerable evidence that word associations are sensitive to administration procedures (see entwisle, a, p. - ) probably because of social pressure that inheres in the role relations of the interviewer and respondent. for this reason the basic factorial design incorporating sex, grade, and iq as variables, was replicated four times with slum children: white interviewers with white children, negro interviewers with' negro children, white interviewers with negro children, and negro interviewers with white children. generally higher rates of paradigmatic responding are noted when the race of the inter -.. viewer differs from that of the respondent, and low iq students' are most sensitive. (labov ( ) illustrates at length the differences in oral speech that people exhibit under varying conditions of for- mality such as speaking to an interviewer, compared to scolding their children.) both white and negro children tend to give more paradig- matic responses (more mature behavior) when the race of the inter- viewer differs from their own. this suggests that racial differences could be at least partly owing to a performance-vs.-knowledge dis- crepance. some preliminary evidence exists that semantic systems of white and negro children do not completely overlap (entwisle, b). for instance the primary associate of negro slum children to "music" is "dance", while less than per cent of white slum children and per cent of suburban white children give this response. also "chicken" is a high frequency response to "wing" for negro children but not for white children. future work is planned to try to point up differences in semantic and hierarchical. systems between negroes and whites, and aiso between' urban and rural dwellers. urbanization. degree of urbanization, perhaps because of its relation to opportunities for verbal interaction, seems to be generally correlated with rate of development. opportunities for verbal inter- change may be limited for the rural pre-schooler because of isolation of dwellings, lack of kindergartens, and lower exposure to television and radio. older rural children may be hampered by long bus rides to and from school, and a large number of home and farm chores. exposure to mass media, especially television, may be a crucial factor in the rural-urban difference because tv reception is less dependable in rural areas and also the time available to watch tv is less for rural children. tv became prevalent later in rural areas than in urban areas, too, so its cumulative impact to date could be smaller. the amish, who develop even more slowly than the rural maryland groups, lack tv entirely and this is a tempting explanation for the amish- rural maryland difference. (genetic differences, also a possibility, will be discussed later.) there is little doubt that in terms of word associations (entwisle, et al., ; palermo and jenkins, ; entwisle, a, b) or other measures (templin, ) american children of fifty years ago were not as advanced linguistically as modern children, modern children may be ahead as much as five years, if we take either primary associates or degree of paradigmatic responding as an index. this is a huge discrepancy. the relative acceleration of modern children was one observation that prompted study of the amish children, for in many ways conditions of life for amish children resemble conditions for minneapolis chil- dren studied by woodrow and lowell.( ). the amishare the slowest to develop of any american group we have observed (even negro third- grade slum children are faster), but they are considerably in advance of minneapolis children of years ago. unfortunately the early data are based mainly on responses to nouns and adjectives, so many kinds of syntagmatic responses that might help in estimating linguis- tic maturity are not available. nevertheless, thete are more immature patterns (more sequence-type responses) in old data collected before the days of mass media, and also there are more syntagmatic responses of the adjective-noun variety for modern groups living under "old fashioned" conditions. it is very unfortunate that there are no data available for american children around (just before television was introduced) so that the effect of tv alone, aside from other changes such as magazines, radios, improved curricula, and so on, could be estimated. life conditions in the american urban slum may favor rapid development of basic language skills. houses are crowded, with many children sharing one bed or bedroom, and time outside the house is usually spent surrounded by other children on the street or playground. rq although there is little evidence to cite, and certainly our data shed no light on the point, the pressures upon young slum children to at- tain some kinds of verbal proficiency may be more powerful than those impinging upon the suburban child. the slum child usually needs to find his own way around and it is not at all uncommon to see pre- school children unattended on sidewalks, near busy streets. anec- dotes are often told of - year old slum children who are very articulate when stopped on the street and asked for directions by strangers. lack of close supervision both inside and outside the home could force the young slum child to develop skills in verbal com- munication at an early age. several lines of evidence attest to the high exposure of modern american children to television,, some estimates being as high as ' hours per day, roughly equal to the hours spent in school. in the united states it is estimated that three-year old children already average about minutes of tv a day, and average daily watching time increases to a peak at ages to . the cumulative statistics are staggering: during the years of school attendance, the total time spent watching television ranges from , and , hours (schramm, ). we guess that the sheer quantity of exposure (mostly via tele- vision) may be the most inportant factor in accounting for the verbal facility of -year-old slum children, with the narrowness of exposure as a secondary but important cause. these two factors will now be discussed in turn. the slum children were selected to represent the most severe degree of economic, and cultural deprivation in the city of baltimore. as many as percent of children in some schools are on public assist- ance and median income in some neighborhoods is as low as $ per year. notwithstanding, it would be very unusual for a house to have no television set. (keller's ( ) survey of a similar group in new york city showed per cent tv ownership.) middle class children also have television, but there is differential exposure (bailyn, ) with lower class children having much higher rates than middle class children. pre-school slum children spend a large portion of their time watching tv. most'of the verbal models presented on tv emanate from adult speakers. even cartoon programs are cast in an adult idiom, and situation comedies, also popular with children, deal almost exclusively with adults. commercials which are both strident and frequent are also good sources of simple models, especially since the are repeated verbatim over and over. (it is no accident that a large number of children respond "living" to "pleasant" because this is a part of a commercial linked to a local product.) the slum child, although deprived in many ways, is thus afforded a very large amount of vicarious exposure to simple discourse. there is little reason to doubt that high tv exposure would enhance language development in young children, because children who come from towns with tv have larger vocabularies than children from non-tv'towns (schramm et al., ). secondly, the direct exposure to adult speech (parents, neighbors, etc.) is in terms af simple verbal models. some experimental work by hess and his co-workers, directly relevant to this point, describes the constricted verbal environment of the slum child. following a hypothesis stated earlier by bernstein ( ), hess and shipman ( ) arranged for negro mothers of four social levels (college-educated to lower-lower class) tointeract verbally with their children. inter- action was recorded as the mothers taught their children sorting and simple classification tasks. later the mothers were also questioned about how they would deal with certain hypothetical situations, like preparing the child to enter school. differences in language by social class were marked, with middle class mothers using more speech, and more elaborate speech expressing alternatives and contingencies. the lower class mothers preferred simple, short sentences and gestures. use of abstract words was directly correlated with social class level. lower status mothers often conceptualized the child's behavior in terms of role prescriptions rather than in.terms of individual needs and situations. they were unlikely to allow sufficient time before a decision to permit the child to assess contingencies or to analyze a situation. a rather different cognitive panorama is seen by upper status and low status mothers, and they relay these views directly to their children. the language deprivation is essentially a depri- vation of meaning. this work suggests that verbal models presented to a slum child in direct verbal interaction are apt to be simple and uncomplicated. we have already remarked that through tv a slum child may also be hearing mostly simple models. thus, unlike the suburban child, the slum child may be exposed almost entirely to straightforward and redundant sets of utterances. such exposure could favor early develop- ment, and lead to an early appreciation of form class properties, particularly for very common words and for verbal concepts at a law level of abstraction. by the same reasoning, a lack of redundance and possibly the negative transfer generated by the more complex verbal environment of the suburban preschooler might be a temporary handicap (seen in his short-lived inferiority to the slum child). hierarchical and abstract meanings, partially learned, would not be very evident in word association measures. as the child continues to develop and requires more elaborate verbal models to stimulate growth, however, the suburban youngster begins to profit' from his rich environment whereas the slum child falls progressively behind and fails to redeem his early promise. an uncensored and unlimited exposure to tv, plus a world popu- lated with persons who speak a simple and uncomplicated idiom, may be optimum for laying down form class concepts of the most basic and elementary kind, but a handicap for ultimate development. the quan- tity and the quality of the slum child's early verbal environment may be superior to that of the suburban child for development of language at its most basic stages. the disadvantages only become apparent subsequently. the syntax that the slum child is not exposed to may hinder communication later in a most insidious wty by depriv- ing the child ,of the tools of thought. the plain talk that aids early development may lead to a conceptual poverty that is more than just a restriction in vocabulary. degree of urbanization, or a rural-urban differential, has been recently reported (greenfield, reich, and olver, ) to influence cognitive growth on a much broader scale than we have been able to observe directly in the development of form class concepts. th difference is one between abstractness and concreteness, and the authors speculate that it is derived from a differential exposure to problem solving and communication in situations not supported by context. in senegal and mexico there are differences in abstracting ability between rural and urban children, and although the difference is'small, it is similar in nature to larger differences separating children who have been to school from those who have not. the rural- urban dimension appears to overshadow any kind of strict ethnic grouping, because city-living eskimos are much like urban children studied elsewhere. the conceptual development of unschooled wolof children resembles that of lower class american children, particularly as differences increase with age. (it is not hard to equate poor schooling to lack of schooling, especially when absence rates are high as in slum children.) because of this widely observed rural-urban difference in broad areas of cognitive development plus our own observation of rural-urban differences in children very closely matched on other variables (entwisle, b), the relative in-migration rates of white and negro slum dwellers, mentioned earlier, gains favor as an explanation for white-negro differentials. negro slum dwellers in baltimore are much more likely than white slum dwellers to have come recently from the south, largely rural. patterns observed in rural german children are also consistent with this rationale. the slum school's impact cannot be isolated, but its influence is apparently not sufficient to maintain the advanced rate of develop- merit found in first-graders. many studies document the increasing distance between disadvantaged and other children, and a huge survey by coleman ( ) suggests that school quality per se has very little impart); on verbal skills. whatever the slum home lacks, the slum school may be unable to compensate for. perhaps class-wise dialect cleavage .prevents effective communication, because middle class teachers have semantic systems that differ from those of their lower class pupils. the curriculum perhaps is not designed either to capitalize upon the advantages of a lower class environment or to compensate for its deficiencies. the school environment could even be highly favorable in terms of cognitive factors but cognitive factors may become irrel- evant as affective factors become increasingly pressing. the slum child's experience of failure and strong feelings of inablility to influence the environment (see coleman, ) could completely over- shadow even optimum cognitive factors. iv. discussion. discussion. practically nothing is known about the transmission of linguistic habits from one generation to the next, although the wide differences in linguistic usage between social classes and sub- . cultural groups must be initiated early in life. these differences are far from being matters of simple dialect geography or pronuncia- tion. they are matters of such far-reaching consequence as different semantic systems and differing perceptual and cognitive styles. evidence is accumulating that non-standard english is a cognitive as well as a social handicap. it hinders the child in problem solving efforts as well as in reading, and the deficits seem to accumulate. the research summarized here is concerned with acquisition of fundamental concepts about words that will permit simple communica- tion. much further work is required to specify exact kinds of language deficits and their precise relation to environmental deprivation. for this purpose it seems most expedient to conceive social class or subcultural membership as a series of specific experiences to which a child is exposed. actually there is little research directed at social class or subcultural differences in american children's language behavior that cannot be explained by the well-documented association between socioeconomic status and intelligence level. the rare samples where iq is controlled find class differences to be small but to increase with age (cherry-peisach, ; deutsch and cherry-peisach, ). a specific cognitive deficit that might be class-related is a low level of auditory discrimination. some data of ours indicate that children who live in very noisy environments may not develop the requisite discrimination abilities to learn to read well. recent work stresses process rather than status variables as components of social class, i.e. the educational aspirations of parents for their children vs. income level (bloom, ). although income level is correlated with school attainment, it is easy to see how the two variables could be only indirectly related. educational aspirations are only realistic if income is sufficient to finance continuing education. there is no direct evidence available from our work relative to genetic influences. that very young children are not retarded, though, whereas older slum children are, suggests that it is chiefly environmental factors which are responsible for social class differences in language ability on our measures. genetic influences as far as rural children are concerned are not so easily disposed of. rural children may be drawn from a reservoir of persons with lower- than-average verbal talents because of selective migration. that is, over the years rural persons with the highest verbal abilities may have been most successful at making a rural-to-urban move, and those that remain have relatively low verbal ability. although our data are certainly incapable of testing this notion, one would expect that inter-class mobility might be selectla in the same manner as rural-urban mobility. persons capable of upward mobility . . ., by reason of superior verbal skills should rise from the slum population or the blue collar population to higher class strata, and thus lead to interclass differences in the urban and suburban groups. such differences are not found, of course, especially at first-grade when such effects should be most apparent. the genetic argument as an explanation for the rural-urban differen-. tials thus loses some cogency although it cannot be completely discounted. finding of strong rural-urban differences on word association measures, and of minimal differences between urban groups, is congruent with results from a whole series of studies on cognitive style by bruner and his co- workers cited earlier.= they find rural- urban differences that resemble differences noted between schooled and unschooled children, and inter-cultural differences appear small when degree-of-urbanization is held constant. one reason advanced for the rural-urban divergencies is the greater abstractness of urban life which forces practice of verbal models not supported by context. our observation of a falling-behind by slum children between first- and third-grades, and the evidence of impoverished verbal models to which the slum child attends, point to a similar cause for the slum child's rising verbal deficiencies. further study in more detail is now being undertaken to see how the responses themselves differ from one group to another. (it is known, for instance, that amish children at third-grade not only give fewer paradigmatic responses but give more syntagmatic responses.) labeling is generally considered a less complex mental activity than categorizing, and an examination of superordinate responses, now under way, may show that groups of the same level in paradigmatic develop- ment nevertheless differ in conceptual structuring. also the total lexicon available for each group may give some clue as to the percep- tual efficiency of the group as well as the extent of overlap between semantic systems. finally, it should be noted that our findings are entirely consistent with chomsky's ( ) notion that exposure in no way decrees the direction which language acquisition takes, whatever its effect on rate. all the groups we have studied, including german- speaking, show growth in paradigmatic responses to about the same asymptotic levels, and the levels,of development from one group to another are fairly stable even though groups differ in rate. if chomsky's notion is correct, slum children may just not receive sufficient exposure to certain kinds of language ever to attain an asymptote in other areas of verbal competence. it could be true as well that there are stages in grammatical development where exposure to instruction could be "most efficient" for children whose extra-school exposure is insufficient, and this hypothesis is amenable to experiment.. v. summary children's word associations, which are closely related to grammatical competence and verbal development, seem to evolve .through similar stages for several different american subcultural groups. the groups do differ in rate of evolution, however, and slum children are advanced at first grade and behind at third grade. the intelligence of the child and the geographical loca- tion of his home (urban vs. rural) are both influential factors. school differences and differential exposure to mass media are considered in relation to social class. references cited bailyn, l., mass media and children: a study of exposure habits and cognitive effects. psychol. monogr., , , whole no. , - . bernstein, b. linguistic codes, hesitation phenomena, and intelligence. language and speech, , , - . bloom, b.s. stability and change in human characteristics. new york: john wiley, . brown, r. and berko, j. word association and the acquisition of - grammar. child developm., , , - . cherry-peisach, e. children's comprehension of teacher and peer speech. child developm., , , - . *chomsky, n. aspects of a theory of syntax. cambridge: m.i.t. press, . coleman, j.s. equality of educational ortunity. washington, d.c.: office of education, . deutsch, m. and brown, b. social influences in negro-white intelligence differences. . soc. issues, , , - . deutsch, m. and cherry-peisach, e. a study of language patterns. the instructor, . deutsch, m., malirer, a., brown, b., and cherry, e. communica- tion of information in the elementara school classroom. 'coopera- tive research project no. . office of education, u.s. dept. of health, education and welfare, . di vesta, f.j. a normative study of concepts rated on the semantic differential by children in grades through . j. genet. psychol., , , - . entwisle, d.r. word associations of young children. baltimore: the johns hopkins press, (a). entwisle, d.r. developmental sociolinguistics: a comparative study in four subcultural settings. sociometry, , , - (b). entwisle, d.r. form class of children's word associations. j. verb. learn. verb. behay., , in press (a). entwisle, d.r. developmental sociolinguistics: inner city children. in preparation, (b). references cited entwisle, d.r., forsyth, d.f., and muuss, r. the syntactic-paradig- matic shift in children's word associations. j. verb. learn. verb. behay., , , - . entwisle, d.r. and muuss, r. word associations of rural german children. j. verb. learn. verb. behay., in press, . ervin, s.m. changes with age in the verbal determinants of word association. amer. j. psychol., , , - . greenfield, p.m., reich, l.c., and olver, r.r. on culture and equivalence: ii. in studies in cognitive growth. bruner, j.s., olver, r.r., and greenfield, p.m. (eds.), new york: wiley, . hess, r.d. and shipman, v. early experience and socialization of cognitive modes in children. child developm., , , - . keller, s. the sacial world of the urban slum child. amer. j. orthopsychiatry, , , - . labov, w. the social stratification of english in new york city. washington, d.c.: center for applied linguistics, . lee, e.s. negro intelligence and selective migration: a philadelphia test of the klineberg hypothesis. amer. soc. rev., , , - . meumann, e. intelligenzprilfungen an kindern der volksschule. z. exp. padagogik, , , - . palermo, d.s. and jenkins, j.j. frequency of superordinate responses to a word association test as a function of age. j. verb. learn. verb. behay., , , - . palermo, d.s. and jenkins, j.j. changes in the word associations of fourth and fifth grade children from to . j. verb. learn. verb. behay., , , - . riegel, k.f., riegel, r.n., smith, h.e., and quarterman, c.j. an analysis of differences in word meaning and semantic structure between four educational levels. mimeo. dept. of psych., university of michigan, . rosenzweig, m.r. and menahem, r. age, sexe et niveau d'instruction comme facteurs determinants dans les associations de mots. l'annee psychol., , , - . schramm, w., lyle, j., and parker, e.b. television in the lives of our children. stanford, calif,: stanford university press, references cited schramm, w. (ed.) the effects of television on children and adolescents. new york: unesco, . templin, m.c. certain language skills in children. minneapolis: university of minnesota press, . thorndike, e.l. and lorge, i. the teacher's word book of , words. new york: bureau of publications, teachers college, columbia university, . woodrow, h. and lowell, f. children's association frequency tests. psychol. monogr., , no. , - . c.. .: see acknowledgments, see acknowledgments, footnotes paragraph one. paragraph two. the exact percentage is unknown because many children attend public kindergarten and then go to catholic parochial first-grade. the total first-grade enrollment exceeds the kindergarten enroll- ment in spite of the departures to parochial school. there is also a net out-migration from the school population which has averaged over per cent in recent years. these data are taken from census tracts where inner city schools are located. the tracts do not coincide exactly with the boundaries of areas that schools draw their pupils from, but neighboring city tracts are closely related. census data are'for , and our school survey was carried out in - . some fragmentary data cited by meumann ( ) suggests that german children around the turn of the century may have similarly given sequence-type responses at considerably later ages than modern german children (see entwisle and muuss, ). we tried, unsuccessfully, to study slum children with hearing deficiencies, with the idea that their exposure to language would be greatly reduced. hearing losses spring from many causes, but only those due to conduction defects would be suitable for study. it is a commentary on the success of modern public health measures that we could not locate enough children with hearing losses to form samples, altholsh twenty years ago this probably would have been an easy task. archives of disease in childhood ; : - byler-like familial cholestasis in an extended kindred b bourke, n goggin, d walsh, s kennedy, k d r setchell, b drumm abstract progressive familial intrahepatic cholestasis (pfic) occurs in many com- munities and races. a form ofpfic in five children from two consanguineous mar- riages in an irish kindred is described. in addition, a review of clinical information from the records of three deceased mem- bers of the kindred strongly implies that they also suffered from pfic. the chil- dren had a history of neonatal diarrhoea, sepsis, and intermittent jaundice that ultimately became permanent. they suf- fered intractable pruritus and growth retardation. despite evidence of severe cholestasis, serum y-glutamyl transferase and cholesterol were normal in these chil- dren. sweat sodium concentration were raised in three children. liver histology showed severe intrahepatic cholestasis and hepatocellular injury. urinary bile acid analysis revealed a non-specific pat- tern consistent with chronic cholestasis. these children suffer from a form ofpfic remarkably similar to that occurring in members ofthe byler kindred. (arch dis child ; : - ) keywords: familial cholestasis, byler disease. department of paediatrics, university college dublin, republic of ireland b bourke n goggin d walsh b drumm department of pathology, st vincent's hospital, dublin, republic ofireland s kennedy clinical mass spectrometry center, children's hospital medical center, cincinnati, ohio, usa k d r setchell correspondence to: dr billy bourke, division of gastroenterology, hospital for sick children, university avenue, toronto, ontario, canada m g x . accepted april in clayton et al described seven members of an amish family descended from jacob byler with progressive familial intrahepatic cholestasis (pfic).' although there have been reports of a similar disease occurring in various individuals and, races - - many of these patients differed clinically from the byler children. therefore, it is recognised that pfic comprises a heterogenous group of condi- tions." we describe eight affected children from a single kindred with a form of familial intrahepatic cholestasis very similar to that of the original byler family. this kindred is part of a group of highly intermarried (fig ) irish travellers (an indigenous irish nomadic com- munity). familial cholestasis affecting eight related children offered a unique opportunity for a detailed clinical and pathological study of the natural history of this rare disease. patients clinical and biochemical profiles for the five members of the kindred reviewed in this hospi- tal were obtained from other hospital records and appear in tables and . before cholestasis became clinically apparent these children suffered from episodes of sepsis and severe, protracted diarrhoea. these features continued after cholestasis was established and caused significant morbidity throughout early child- hood years. in three of the children, episodic jaundice appeared within the first two months of life. however, the onset of jaundice was delayed until and months respectively in the other two children. for each of the children, the established illness was dominated by the complications of chronic cholestasis. severe, intractable pruritus that was unresponsive to trials of phenobarbi- tone,rifampicin,ursodeoxycholic acid,orphoto- therapy was a particularly distressing symp- tom. marked hepatosplenomegaly was present in all children. the liver was palpable between and cm below the right costal margin and spleens were palpable up to cm below the left costal margin. in all cases the liver was of very firm consistency. short stature and failure to thrive were also prominent (table ). recurrent epistaxis was common to all five children. however, haematemesis occurred in only one child. intellectual development was normal in all of these children. a number of additional features were also present. each of the five living children had fine hypopigmented hair. these children also manifested a spectrum of dystrophic nail changes. mild fingernail pitting, which was evident in the younger children, was super- ceded by gross dystrophic changes in the older two. nail scrapings taken from the eldest child grew candida inconspicua. results varying degrees of conjugated hyperbilirubi- naemia and liver enzyme elevation were present (table ). however, in all of the children, serum albumin and prothrombin time were normal, suggesting hepatic synthetic function was relatively preserved. during intercurrent illness liver enzyme values in- creased markedly. y-glutamyl transferase and cholesterol were normal or near normal in all five children studied. all of the children had markedly raised faecal fat content and severe fat soluble vitamin deficiency. abnormal sweat tests were documented in three of these children (table ). results of sweat tests performed during infancy were available for four living children (patients , , , and ) and for two of the children now dead. these sweat tests were all in the normal range during infancy. in view of the abnormal sweat o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ http://adc.bmj.com/ http://adc.bmj.com/ bourke, goggin, walsh, kennedy, setchell, drumm table clinicalfindings in (living) members ofkindred with byler disease onset of jaundice pruritus nail hepatomegaly patient no age (years) (months) height centile weight centile ( - + *) dystrophy (cmt) haematemesis < < - + severe - yes < < < - + moderate - no < < < + mild no < - + mild - no < < < + mild no * pruritus scoring: = none, + = rubbing or scratching when undistracted, + = scratching without abrasions, + = abrasions evident, + = cutaneous mutilation, haemorrhage, and scarring. t measurements indicate liver size below the right costal margin. table biochemicalfindings in (living) members ofkindred with byler disease sweat patient age ast alp bilirubin cholesterol albumin prothrombin ggt 'nt sodium chloride no (years) (iuil) (iuli) (pmol/l) (mmol/l) (gll) time (s) (iul) (iul) (mmol/l) (mmolfi) . . . . . normal - - - < . - - . - < < < < values biochemical parameters refer to values at presentation to this hospital (at age as given). ast = aspartate aminotransferase; alp = alkaline phosphatase; ggt = y-glutamyltransferase; 'nt = '-nucleotidase. tests the four siblings, patients , , , and , and their mother were all screened for cystic fibrosis af mutation and other muta- tions common among irish cystic fibrosis patients including ri h, di , g x, g d, r t/r k, n k, w x, r x, - g-a and + g->t. each of the children and their mother were negative for all mutations. exhaustive investigations were undertaken to rule out other causes of cholestasis. screen- ing studies for hepatitis a, b, and c were nega- tive. serum and urinary amino acid chroma- tography and urinary organic acid profile gave normal reults. the serum al-antitrypsin values and phenotype were also normal. extrahepatic * * affected female i affected male q unaffected female flunaffected male * marriage between second or third cousins t deceased figure pedigree ofirish byler kindred illustrating high degree of intermarriage. manifestations of alagille's syndrome, such as posterior embryotoxin and butterfly vertebrae, were sought but not found. karyotype was normal in all of the children. three other children from this kindred died from sepsis. two children were aged months and one was months. clinical information was available for two of these children. the month old child was diagnosed in another hos- pital with pfic. she had recurrent diarrhoea and failure to thrive before the onset of jaundice and pruritus at age months. her disease was clinically and biochemically identi- cal to that of the five living children described above. the month old child suffered with severe diarrhoea and failure to thrive from age month. at the time of death ( months) he was anicteric but had markedly raised transaminases (alanine aminotransferase = u/ ). no clinical notes were available for the third child. however, he was jaundiced at the time of death. each of the five living children were followed up at this institution for three years since the initial referral; they were maintained on fat soluble vitamin replacements. the eldest child required intermittent hospitalisations during intercurrent infections, which were commonly accompanied by bouts of watery diarrhoea. although copious, stool output diminished appropriately during fasting. invariably, patho- genic organisms were absent from stools during these episodes. furthermore, immuno- logical investigations performed on a subgroup of these children including immunoglobulin concentrations (patients and ) and t cell subsets (patient ) were normal. igg , igg , and igg subclasses were raised in the one child (patient ) in whom they were evaluated. despite the severity of cholestasis during this period there was no clinical or biochemical evi- dence of disease progression among any of the children. t ( i o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ byler-like familial cholestasis in an extended kindred liver histology liver biopsy specimens were obtained from each of these children. the specimens from the youngest children (patients , , and ) showed moderate periportal inflammation and portal expansion. in these specimens there was apparently normal bile duct number and calibre with neocholangiole formation at the periphery of the portal tracts. changes within the lobular parenchyma included pseudoro- sette or pseudoacinar formation with bile thrombi and canalicular bile stasis (fig a). in the specimens from the older children (patients and ), pseudoacinar change and cholangi- olar bile stasis were equally prominent with giant cell transformation in the children aged and years (fig b). steatosis was not seen. thin fibrous bands extended out from the portal tracts in these specimens, although bridging fibrosis and regenerative nodules were not present (fig c). in specimens from these older children there were inflammatory aggre- gates within the hepatic parenchyma and occa- sional acidophil bodies. hepatocellular archi- tectural changes such as irregular liver cell plates were seen at this stage. an acquired pau- city of intralobular bile ducts was also present in these specimens. urinary bile acid analysis urinary bile acids were analysed for four of the children (patients , , , and ). bile acids were extracted, concentrated, and analysed by fast atom bombardment ionisation-mass spec- trometry (fab-ms) and gas chromatography- mass spectrometry (gc-ms).'s' the results of fab-ms and gc-ms analysis revealed similar patterns for each of the four children. all children were capable of synthesising the two primary bile acids. each had markedly increased total urinary bile acid excretion. raised excretion of glycine, taurine, and sulphate conjugates of dihydroxy, trihydroxy, and tetrahydroxy cholanoic acids was detected. the pattern of bile acid excretion was consist- ent with chronic cholestasis rather than an inborn error of bile acid synthesis. discussion since the report of clayton et al in ,' the terms pfic and byler disease have been used synonymously for this condition. however, it is clear that many cases described subsequently demonstrate considerable clinical, biochemi- cal, and pathological heterogeneity. a therefore, it is now speculated that pfic includes a group of conditions of varying aeti- ologies." in this report we have described a form of pfic affecting an extended kindred of irish travellers. members of this kindred suffered with a form ofpfic identical in many respects to that of the byler kindred. specific features common to this irish family and the original byler kindred include early onset of diarrhoea followed by episodic cholestatic jaundice, which ultimately becomes permanent, intrac- table pruritus, growth, but not mental, retarda- tion, fine blond hair, the complications of fat soluble vitamin deficiency, normal serum cholesterol, and recessive inheritance pat- tern.' it is also noteworthy that a high degree of intermarriage is common among both the amish and irish travelling people. however, some features specific to the children in this report (for example nail dystrophy) suggest that this irish family may suffer from a unique form of pfic. death in early life is a feature among some children with this condition. three chil- dren in this kindred are dead, two in infancy and one at months old. of the five living children, all spent the greater part of the first year of life in hospital. true byler disease and byler-like familial cholestasis associated with low serum y-glutamyltransferase is associated with a poor prognosis.' however, it is noteworthy that survival past infancy in this irish kindred has been followed by relatively slow disease progression, albeit in the face of severe cholestasis and a high level of morbidity. figure liver biopsy specimens. (a) biopsy specimen taken from patient (aged years). pseudoacinar transformation (arrows) and intracanalicular bile are prominent (magnification x ). (b) biopsy specimen taken from patient (aged years); there is hepatocellular disarray with multinucleate hepatocytes scattered throughout the liver parenchyma (arrows) (magnification x ). (c) biopsy specimen from patient ; there is stellate periportalfibrosis and inflammation spilling into the adjacent hepatic parenchyma. transformation of hepatocytes to neocholangioles can be seen at the edges of the portal tracts (arrows) (magnification x ). o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ bourke, goggin, walsh, kennedy, setchell, drumm recurrent diarrhoea and sepsis were features of this disease in the original byler kindred. a recent report from switzerland describes a similar case presenting with idiopathic diar- rhoea and recurrent sepsis. although we are not told of any relationship between this case and the byler family, it is of interest that the byler kindred were of swiss descent." sweat electrolytes are reported to be raised in some children with pfic.' in this study, raised or high normal sweat sodium values were observed in all except one child (patient ) over months old. frankly raised sweat sodium was found in three children aged , , and years. normal sweat test results were documented elsewhere for five of these chil- dren during infancy. this suggests that sweat sodium is normal in early life when diagnostic confusion with cystic fibrosis would be most likely to occur. conversely raised sweat sodium or chloride may be a useful marker for this condition in later childhood. despite some phenotypic similarities these children do not appear to have cystic fibrosis as they have no evidence of respiratory disease and do not manifest any of the more common cystic fibro- sis mutations found in the irish population. in addition, the immune reactive trypsinogen value was not depressed in one child (patient ) in whom it was measured, consistent with intact pancreatic ftunction. cell mediated immunity has been docu- mented to be abnormal in patients with pfic," which may account for the recurrent episodes of sepsis in these children. although dystrophic nail changes associated with chronic candidiasis have not previously been reported in pfic, nail channel osteomyelitis occurred in a very similar case. although we failed to observe an abnormality in immuno- globulin concentrations or t cell subsets in members of this family, chronic nail infection offers further evidence for a defect in cell mediated immunity in pfic. the relevence of the observed increase in igg sublasses in one of the kindred is as yet uncertain. in this series the liver disease is characterised at the histological level by a combination of intrahepatic cholestasis and hepatocellular in- jury consistent with the toxic affect of accumu- lated bile acids. giant cell transformation was not remarkable in the biopsy specimens of the original byler kindred.' ' however, giant cell transformation has been commented on in other cases of pfic," including a case typical of the original family in other respects. pseudoacini or rosettes are a prominent feature of the disease in most reports. paucity of bile ducts has been a prominent feature in some reports of pfic.' ductular proliferation rather than paucity was present in the early infancy biopsy specimens in the original report of this condition.' however, ductular paucity (with focal ductular proliferation) was present in a child aged . with the typical disease. there is considerable disagreement about the typical pattern of bile acid excretion in pfic.' however, there is no evidence to implicate an inborn error of bile acid synthesis as a cause of this disease. we found no evidence to suggest a primary defect in bile acid synthesis in this family. we were also unable to detect a raised cholic acid to cheno- deoxycholic acid ratio in urine from this family with pfic as has been described for other patients with pfic. a recent report from france implicates a defect in canalicular secre- tion of bile acids in the aetiology of pfic." this assumption was based on the finding of very low concentration of biliary bile acids in the presence of raised serum bile acids coupled with normal serum y-glutamyltransferase in seven unrelated children with pfic. further studies comparing the bile acid profiles of urine, serum, and bile from patients of the original byler kindred and others displaying a similar disease (such as those described in this report) will help confirm this finding. such studies would also help to more precisely establish the relationship of byler disease to other forms of pfic. although not phenotypically identical, this extended non-amish family suffers from a form of pfic remarkably similar to that of the byler kindred. an understanding of these rare diseases may provide insights into the aetio- pathogenic events underlying more frequently encountered cholestatic conditions such as that associated with cystic fibrosis. therefore, further investigations are warranted to eluci- date the aetiology of these conditions. we thank dr j murphy, dr m taylor, and dr e naughten for allowing us the opportunity to examine these patients. we are grateful to dr s mcquaid and dr d barton for providing molecular genetic analysis and dr bernard portmann for addi- tional histopathological consultations during the management of these children. clayton rj, iber fl, ruebner bh, mckusick va. byler dis- ease. fatal familial intrahepatic cholestasis in an amish kindred. amj dis child ; : - . williams cn, kaye r, baker l, hurwitz r, senior jr. pro- gressive familial cholestatic cirrhosis and bile acid metabo- lism. jpediatr ; : - . ballow m, margolis cz, schachtel b, hsia ye. progressive familial intrahepatic cholestasis. pediatrics ; : - . odievre m, gautier m, hadchouel m, alagille d. severe familial intrahepatic cholestasis. arch dis child ; : - . de vos r, de wolf-peeters c, desmet v, eggermont e, van acker k. progressive intrahepatic cholestasis (byler's disease): case report. gut ; : - . jones ea, rabin l, buckley ch, webster gk, owens d. progressive intrahepatic cholestasis of infancy and child- hood. gastroenterology ; : - . nakagawa m, tazawa y, kobayashi y, et al. familial intrahepatic cholestasis associated with progressive neu- romuscular disease and vitamin e deficiency. j pediatr gas- troenterol nutr ; : - . winklhofer-roob bm, shmerling dh, soler r, briner j. progressive idiopathic cholestasis presenting with profuse watery diarrhoea and recurrent infections (byler's disease). acta paediatr ; : - . whitington pf, freese dk, alonso em, fishbein mh, emond jc. progressive familial intrahepatic cholestasis (byler's disease). in: lentze m, reichen j, eds. paediatric cholestasis-novel approaches to treatment. dordrecht: kluwer academic publishers; : - . gray op, saunders ra. familial intrahepatic cholestatic jaundice in infancy. arch dis child ; : - . dahms bb. hepatoma in familial cholestatic cirrhosis of childhood. arch pathol lab med ; : - . riely ca. familial intrahepatic cholestasis: an overview. in: walker wa, durie pr, hamilton jr, walker-smith ja, watkins jb, eds. pediatric gastrointestinal disease. vol . philadelphia: bc decker, : - . whitington pf, freese dk, alonso em, schwarnenberg sj, sharp hl. clinical and biochemical findings in progressive familial intrahepatic cholestasis. j pediatr gastroenterol nutr ; : - . setchell kdr, worthington j. a rapid method for the quan- titative extraction of bile acids and their conjugates from serum using commercially available reverse phase octade- cylsilane bonded silica cartridges. clin chim acta ; : - . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ byler-like familial cholestasis in an extended kindred sjovall j, lawson am, setchell kdr. mass spectrometry of bile acids. in: law jh, rilling hc, eds. methods and enzy- mology. vol . london: academic press, : - . setchell kdr, matsui a. serum bile acid analysis: the application of liquid-gel chromatographic techniques and capillary column gas chromatography and mass spectrom- etry. clin chim acta ; : - . linarelli lg, williams cn, philips mj. byler's disease: fatal intrahepatic cholestasis. j pediatr ; : - . maggiore g, bernard , riely c, hadchouel m, lemonnier a, alagille d. normal serum y-glutamyl-transpeptidase activity identifies groups of infants with idiopathic cholestasis with poor prognosis. j pediatr ; : - . hillemeier ac, hen j, riely ca, dolan tf, gryboski jd. meconium peritonitis and increasing sweat chloride deter- minations in a case of familial progressive intrahepatic cholestasis. pediatrics ; : - . lloyd-still jd. familial cholestatic syndrome with elevated sweat electrolytes. in: sturgess jm, ed. proceedings ofthe th international congress on cystic fibrosis. toronto: imperial press, : a. maggiore g, de giacomo c, scotta ms, siena s, maccario r, vitiello a. cell-mediated immunity in children with chronic cholestasis. j pediatr gastroenterol nutr ; : - . alonso em, snover dc, montag a, freese dk, whitington pf. histologic pathology of the liver in progressive familial intrahepatic cholestasis. j pediatr gastroenterol nutr ; : - . jacquemin e, dumont m, bernard , erlinger s, had- chouel m. evidence for defective primary bile acid secretion in children with progressive familial intrahepatic cholestasis. eurjpediatr ; : - . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ archives of disease in childhood ; : ing theme: bronchiolitis, cystic fibrosis and bronchopulmonary dysplasia, among others, are listed together as causes of wheezing but would rarely be considered together as possi- ble diagnoses. in the text i would have welcomed more emphasis on differential diagnosis and more information about the relative incidence of the conditions mentioned under each head- ing. there are some excellent concise ac- counts. these are particularly useful where information in textbooks is lacking, for example munchausen disease by proxy, and where an overview directs further reading, for example headaches and convulsions. how- ever, combining an attempt to say something about every condition with a need for brevity often results in simplistic accounts, for exam- ple, 'crohn's disease is a rare though impor- tant cause of abdominal pain in childhood'. this book, despite its limitations, usefully supplements standard texts. paediatricians may be helped when encountering less com- mon symptoms and signs and when there is a need to consider the rarer causes of common symptoms. i have found it helpful when con- sidering the differential diagnosis of such diverse symptoms and signs as livedo reticu- laris, excessive sweating, and the rarer causes of apnoea in infancy. the enthusiastic student may also usefully refer to the book when starting to face diagnostic dilemmas. carolyn j adcock senior registrar paediatric pathology. rd ed. edited by sir colin berry. (pp ; £ hardback.) springer-verlag, . isbn - - - . professor berry's paediatric pathology now enters its third edition, only six years after its second. over the three editions the size has increased significantly with a smaller text size on more pages. as a paediatric book, it largely avoids those diseases and conditions associ- ated more with the process of birth or prema- turity, although of course congenital malfor- mation features large. while some overlap is inevitable, it is a stablemate of, and largely complementary to, keeling's fetal and neo- natal pathology. the book is directed prima- rily towards the general rather than the specialist paediatric pathologist and, as there is no direct competition in paediatric pathol- ogy, should find a receptive audience. as a paediatric pathologist, i have found previous editions a little too thin to be ofvery much practical help when faced with a prob- lem. exception are those chapters that describe a very practical approach to a prob- lem whether of description (cardiac) or of specimen handling and diagnostic require- ments (metabolic). if described at all, conditions have been covered too briefly with little discussion of differential diagnosis. as i doubt a general pathologist will need less information and explanation to understand a problem than a specialist, i suspect my experience is true for the target readership. but that is the past, what of the third edition? there is no significant change to the overall format. as before, chapters cover organ and system pathology in a conventional manner but also with chapters on sudden unexpected infant death, embryonal tu- mours, and theoretical aspects of congenital malformation. there are some changes in authorship and new chapters on the pathol- ogy of aids and bone marrow pathology. the text is well set out and the illustrations generally of good quality. however, the most significant alteration since the first edition is a cumulative one. the modification of chapter titles together with a gradual expansion of some chapter lengths, not necessarily extensive, has led to a text that will be a better resource to general and specialist pathologists needing an introduc- tion to less familiar areas. it may be premature to look forward to the fourth edi- tion, but i hope this trend towards expansion continues. steven gould consultant paediatric pathologist gellis and kagan's current pediatric therapy. th ed. edited by f burg, j ingel- finger, e wald, and r colin. (pp ; £ hardback.) wb saunders, . isbn - - - . over the years successive editions of gellis and kagan's classic work, just like topsy, have 'growed and growed'. thirty years on and now in its th edition, this magnum opus has four editors and contributors. it seemed a little incongruous (and more than a little overwhelming) for a single reader to comment on such a body of scholarship and, in an attempt to redress the numerical imbal- ance, i enlisted a handful ofwilling colleagues to help me undertake the task! these included a couple of general paediatricians, one with an interest in rheumatology, a paediatric oncologist, and a senior registrar. we each chose relevant sections of the book to read. i then collected comments, allowed them to simmer for several weeks and finally tried to prepare a distillate which was representative of our views. firstly, the design characteristics of the book were appreciated; printing was clear, subheadings stood out, tables were easily assimilated, and key references were ap- pended after each author's contribution. inevitably the style of the text was a little uneven with such a huge authorship but it was easy to find one's way around the volume. the book is truly comprehensive with sections on fetal and adolescent medi- cine, behavioural and social medicine, and balanced consideration is given to emergency management of acute disorders as well as long term management of chronic condi- tions. i failed to find guidance on one topic only-pain relief in the dying child. the consensus view is that gellis and kagan is a good reference book for providing the historic perspective on treatment as well as current concepts, although precise practi- cal advice on challenging problems is some- times lacking. it should be noted that despite our reserva- tions, my willing helpers have extracted a promise that the copy of this book that we are allowed to keep as a reward for our labours is generally available ! gaynor f cole consultant paediatric neurologist correction byler-like familial cholestasis in an extended kindred an error unfortunately occurred in this paper by bourke et al ( ; : - ). a vertical line indicating descent of the father of the larger sibship and his sister, the mother of the smaller affected sibship, from the second consanguineous grandparental marriage was inadvertently omitted from figure . the correct depiction of the figure is shown below. t t t * affected female affected male unaffected female flunaffected male * marriage between second or third cousins t deceased figure pedigree ofirish byler kindred illustrating high degree of intermarriage. genetics, medicine, and the plain people anrv -gg - ari july : genetics, medicine, and the plain people kevin a. strauss , , ,∗ and erik g. puffenberger , ,∗ clinic for special children, strasburg, pennsylvania ; email: kstrauss@clinicforspecialchildren.org, epuffenberger@clinicforspecialchildren.org department of biology, franklin and marshall college, lancaster, pennsylvania lancaster general hospital, lancaster, pennsylvania annu. rev. genomics hum. genet. . : – first published online as a review in advance on july , the annual review of genomics and human genetics is online at genom.annualreviews.org this article’s doi: . /annurev-genom- - copyright c© by annual reviews. all rights reserved - / / - $ . ∗equal contributors. key words anabaptist, deme, haplotype, identity-by-descent, mendelian, single-nucleotide polymorphism abstract the old order amish and old order mennonite populations of pennsylvania are descended from swiss anabaptist immigrants who came to the new world in the early eighteenth century. today they live in many small endogamous demes across north america. geneti- cally, these demes have dissimilar allele frequencies and disease spectra owing to unique founders. biological and social aspects of old order communities make them ideal for studies in population genetics and genomic medicine, and over the last years, advances in genomic sci- ence coincided with investigational studies in plain populations. newer molecular genetic technologies are sufficiently informative, rapid, and flexible to use in a clinical setting, and we have successfully integrated these tools into a rural pediatric practice. our studies with the pennsyl- vania plain communities show that population-specific genetic knowl- edge provides a powerful framework in which to prevent disease, reduce medical costs, and create new insights into human biology. a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : knowledge of genetic anatomy is having a com- parably strong and pervasive influence on all of medicine. . . . genomic anatomy permits medicine to become more predictive and preventive. at the same time, diagnosis and treatment are rendered more sensitive, specific, effective, and safe. –victor a. mckusick, ( ) be not conformed to this world, but be ye trans- formed by the renewing of your mind. romans : introduction the old order amish and old order mennonite (plain) people of north america have contributed much to our understanding of human genetics. although wary of technol- ogy, they have cooperated with clinical inves- tigators for more than years, believing the knowledge gained could help alleviate suffer- ing in their own communities and elsewhere. the history of genetic discovery among plain sects has broad relevance: it charts the trans- formation of human genetics from an esoteric discipline into a foundation of clinical medicine ( ). beginning in , inherited disorders among old order communities were the sub- ject of exceptional observational studies, col- lected in the landmark medical genetic studies in the amish ( ). between and , the advent of southern blotting ( ), genetic markers ( ), linkage analysis, dna sequenc- ing ( , ), and the polymerase chain reaction (pcr) ( ) enabled investigators to describe these disorders in more precise molecular terms ( , , , , , , ). recently, single- nucleotide polymorphism (snp) microarray technology has accelerated this process ( , , , ) and provided fascinating details about the genetic structure of inbred populations. over the past two decades, the focus has shifted to treatment. genetic studies within old order populations have revealed how per- sonal genetic information can be integrated into everyday clinical practice ( , , ). here, we present original as well as previously published data that inform the larger field of genomics and fulfill a central promise of the human genome project—to harness genetic knowledge to heal the sick ( ), prevent disability ( ), and reduce medical costs ( , – ). the plain populations contemporary old order groups fall under the collective term plain people, which refers to christian groups that live simply, dress plainly, and live in the modern world but remain sep- arate from it. they include the amish, old order and conservative mennonites, old order brethren, and hutterites (d. kraybill, personal communication). our clinical practice serves old order amish and mennonite peo- ple of pennsylvania and maryland, who are the focus of this review. the anabaptist movement began in amid the protestant reformation. it was based on the idea that baptism should be an inten- tional act of adults who wanted to join an austere christian community ( , , ). for this and other beliefs, early anabaptists were tortured and killed as heretics for more than years as they moved throughout europe in exile. by the s, most had settled in switzerland ( ). jakob amman ( – ), a mennonite bishop living in the canton of berne, believed that many anabaptists had compro- mised their religious discipline to avoid per- secution ( ). he insisted upon the practice of shunning (meidung), the social exclusion of noncompliant church members, and in forced a division among mennonite preachers that marked the formation of the amish church ( ). william penn offered anabaptists asylum in the new world, and several thousand ar- rived in the port of philadelphia between and . the mennonites generally appeared earlier in the records, whereas the amish ar- rived in the latter half of this migration ( – ). several hundred amish and mennonites strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : eventually settled in lancaster county ( , , ). the present-day lancaster county amish population of approximately , individu- als is derived from perhaps to of these original founders ( ). most early anabaptist settlements in pennsylvania failed, and resid- ual members joined later anabaptist immi- grants who moved west to ohio, indiana, and illinois ( ), where they remain concentrated. about % of contemporary amish occupy just four counties: lancaster county, pennylvania; holmes and geauga counties, ohio; and lagrange county, indiana ( ). the old order amish in america have doubled in the last years to approximately , individ- uals (d. kraybill, personal communication). following the migration to north america, amish culture became extinct in europe ( ), while forces of assimilation and change reached anabaptist immigrants. some regional amish and mennonite churches were further divided by ideological conflicts ( ), and by the s groups that held fast to tradition were des- ignated the old order by their more pro- gressive counterparts ( , ). philosophical differences, reinforced by geographical separa- tion resulted in many endogamous subdivisions called demes. medical genetic studies in , dr. victor a. mckusick reviewed john hostetler’s amish society for johns hopkins university press and recognized the potential for using the amish to study medical genetics (table ) ( ). that same year, he read about david krusen, a lancaster county doctor who reported a high prevalence of achondroplasia among the amish. these events sparked a productive collaboration between mckusick and hostetler ( ), and mckusick made his first field trips to lancaster county with dr. krusen ( ). he soon recognized that dwarfism among the amish was not achondroplasia, but rather two distinct recessive conditions: ellis-van creveld syndrome and cartilage-hair hypoplasia (figure ) ( , ). figure this amish boy with cartilage-hair hypoplasia has the characteristic physical features of short-limbed dwarfism, sparse hair, and small nails. he suffered from neuronal intestinal dysplasia and combined immune deficiency, rare complications of the disorder, and had a bone marrow transplant at months of age. he died of intractable autoimmune hemolytic anemia one year later (photo used with parent permission). over the next years, the amish were subjects of diverse genetic studies. many of these were detailed clinical descriptions of re- cessive disorders, but there were also studies about blood groups ( ), hla antigens ( ), immunoglobulin levels ( ), chromosomal vari- ation ( ), and the heritability of complex traits (e.g., blood pressure, glucose tolerance, and cancer risk) ( , , ). seminal works from this era are collected in the publication medical genetic studies in the amish ( ), edited by mckusick, which describes previously rec- ognized and newly diagnosed mendelian dis- orders among the amish of pennsylvania, ohio, and midwestern states. medical genetic studies in the amish established many known principles of population genetics that remain pertinent today (table ). www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : table characteristics of plain populations conducive to genetic studies mckusick et al. ( ) • a self-defined, closed population (little gene inflow) • western european origin • small number of founding members • concentrated population settlements (relative immobility) • extensive geneological records • high standards of living (i.e., nutrition, hygiene) and physical health • strong interest in inherited illness and the healing arts • relatively uniform social, economic, and occupational circumstances • large sibships (e.g., average – per family) • low illegitimacy rate • children with birth defects or genetic disorders cared for at home • multiple discrete subpopulations (demes) allow comparative analysis molecular update, • large haplotype blocks (small population of recent origin) from identity-by-descent around disease loci • mutation homogeneity (founder effect) • population-specific allele frequencies before the advent of molecular genetic tech- nology, mckusick used genealogical records to infer identity-by-descent for pathogenic alleles. he traced the mutation for pyruvate kinase deficiency to “strong jacob” yoder and his wife, who immigrated in , and traced the ellis-van creveld allele through sibships, affected individuals, and parents to samuel king and his wife, who also immigrated in the s ( ). although complex pedigree analysis is seldom necessary today, these studies highlighted the advantages of genetic linkage analysis and disease gene mapping in plain populations. molecular genetic studies population genetics early investigators recognized the distinct na- ture of amish demes based on their immi- gration history and surname distribution ( ). surnames are inherited through the pater- nal line, largely independent of selection or inbreeding. deviations in their frequency within a population over time simulate ran- dom genetic drift. presently, the name stoltzfus accounts for % of amish households in lancaster county. three other surnames (king, fisher, and beiler) each account for %– % of the total. similarly, among weaverland and groffdale mennonites, the surname martin accounts for % of house- holds. mitochondrial dna haplotypes, inher- ited through the maternal line, demonstrate similar drift over the last – generations (table ). within each anabaptist subpopula- tion, pathogenic alleles may also increase (or de- crease) in frequency by random chance. when the founding population is small, random allele frequency shifts from one generation to the next can be profound; some rare mutations become quite common, while others become extinct. this underlying genetic structure made plain populations ideal for early genetic map- ping studies, which relied on large collections of patients and traditional linkage analysis. formal linkage analysis was necessary because dna markers were sparse and genotyping was time- consuming and expensive. typically, – microsatellite markers were genotyped in af- fected individuals, their parents, and siblings in an attempt to link a phenotype to a genomic region. many mapping studies in plain popula- tions have identified disease genes in this way ( , , , , , , , , , , , , ). it has long been known that isolated pop- ulations have a high incidence of rare genetic strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : table a frequency distribution of surnames and mitochondrial hypervariable region (hvr ) haplotypes among four pennsylvania amish and mennonite demes groffdale weaverland lancaster juniata and mifflin mennonites mennonites amish amish surname martin % % % % zimmerman % % % % hoover % % % % nolt % % % % weaver % % % % stoltzfus/stoltzfoos % % % % king % % % % fisher % % % % beiler/byler % % % % peachey % % % % swarey % % % % kanagy % % % % yoder % % % % mtdna haplotype c c % % % % t c t % % % % cambridge reference % % % % c % % % % c c % % % % t c % % % % c t t % % % % a c g % % % % c c % % % % t c % % % % g c c c c c % % % % a t c % % % % c c a % % % % c % % % % these data demonstrate significant genetic drift and distinct paternal and maternal lineages within plain demes. in our local amish population, the t c haplotype accounts for % of hvr haplotypes. by genealogical analysis, it can be shown that all copies of this haplotype were derived from a single ancestral female who migrated to pennsylvania in the eighteenth century. in the groffdale mennonites, hvr c c predominates ( %), whereas t c t is the most common haplotype among the weaverland mennonites ( %). thirty-eight percent of the juniata and mifflin county amish harbor the hvr cambridge reference sequence. disorders. this, along with inbreeding and a restricted founder pool, has been taken as evidence of low genetic diversity. however, microsatellite marker ( ) and snp data challenge this view. using affymetrix k genechip arrays, we found that calculated heterozygosity across autosomal snps was . % and . % for amish (n = ) and mennonite (n = ) individuals, respectively. these values are similar to the . % measured in an european control population (these data refer to the affymetrix european control set for the k arrays; from affymetrix, santa clara, ca). the comparisons may be affected by drift, www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : which can lead to allele fixation. among men- nonites, snps are monoallelic; of these are also monoallelic in the european control population. thus, (i.e., – ) snp al- leles have become fixed in the mennonite pop- ulation, whereas different snp alleles have reached fixation in the amish. further inspection of snp allele frequen- cies shows that amish and mennonite popu- lations are genetically dissimilar. we compared snps for significant allele frequency dif- a b c mennonite vs. european amish vs. european amish vs. mennonite percentagenumber of snps . . . chromosome number sn p x va lu e sn p x va lu e sn p x va lu e ferences (chi-square > . , alpha = . ) among lancaster county amish, lancaster county mennonites, and the affymetrix eu- ropean control set. amish and mennonite sam- ples were more similar to european controls than to each other (figure ). this signif- icant finding underscores the importance of choosing proper control groups for association and linkage mapping studies (figure ) ( ). it is also relevant to clinical work; we have long appreciated that genetic diseases of amish and mennonite populations are quite distinct (table ) ( , ). of the disorders we de- lineated among the pennsylvania amish and menonnite people, only five are found in both populations and only two share the same molec- ular defect. thus, for any particular child who presents with a medical problem, the genetic differential diagnosis depends critically on pop- ulation of origin ( ). random genetic drift is a compelling and parsimonious explanation for allele frequency ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− figure single-nucleotide polymorphism (snp) allele frequencies are highly dissimilar between old order amish and mennonites. we performed a χ analysis of allele frequencies for snps from the affymetrix genechip human mapping k arrays. we used amish and mennonite females and performed the analysis with high cut-off values (χ > . , alpha = . ). we also compared the two plain populations with affymetrix european controls. genome-wide snp χ values are plotted in panels (a), (b), and (c) to highlight overall differences. (a) the mennonite population was moderately dissimilar to the european controls. one hundred ninety snps ( . %) demonstrated statistically significant differences between these two groups. (b) the amish demonstrated greater allele frequency differences; roughly . % of snps exhibited significant differences with european controls. (c) the amish and mennonite comparison was most striking as nearly in snps showed significant allele frequency differences. we surmise that these large differences are due to genetic drift within each population. since the amish founding population was smaller, we expect larger shifts due to drift over time. this is evident by the comparison with european controls, where the amish show a nearly fourfold increase in allele frequency differences as compared to the mennonites. strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : shifts within a small, isolated population. how- ever, allele frequencies might have been shaped by selection as well. for example, anabaptist settlers were exposed to new pathogens that likely exerted significant selective pressures. although difficult to measure, allele frequency shifts point to interesting gene candidates. from our genechip k genotype data, the snp rs demonstrates the largest allele frequency differences between plain popula- tions and the affymetrix european controls and hapmap data sets. the a allele frequency is % in amish and % in mennonites, compared to % in affymetrix european controls. this snp resides within an intron of lpar , a gene associated with immunity and inflammation. when compared to control data (americans of european descent), amish and mennonite samples are skewed toward larger blocks of homozygous snps (figure ). within any isolated population, homozygous markers that occur in “runs” or blocks are likely autozygous (identical-by-descent); i.e., both the maternal and paternal alleles derive from a common ancestor. now, we can use microarray data to quantify this—the genome-wide estimate of au- tozygosity is . % and . % in amish and mennonite individuals, respectively. similar es- timates based on homozygous megabases of ge- nomic dna yield comparable results ( . % and . %). this implies that the average amish individual harbors ∼ mb of autozygous dna. the probability that any two random amish individuals would be autozygous for the same genomic region and the same haplotype is very remote. relatively high genetic diversity and low allele sharing help facilitate successful autozygosity mapping studies, even with very small sample sizes. modern disease gene mapping large-scale snp genotyping methodologies are a major technological breakthrough because they allow simultaneous and rapid genotyping of many markers randomly distributed across the entire genome. this reduces analytical time chromosome number x sn p x va lu e sn p x va lu e gcdh a b figure population-specific snp allele frequencies are critical for association mapping within plain populations. as a test case, we analyzed snp profiles of amish children with glutaric aciduria type (ga ), who were all homozygous for gcdh c. c>t. the gcdh gene maps to a region of chromosome that has poor snp coverage on genechip k arrays, making homozygosity mapping impossible. as an alternative, we attempted to map the disease using x analysis of snp allele frequency differences between affected children and controls. this analysis depends on accurate control allele frequencies as well as extended linkage disequilibrium surrounding the disease gene. when we compared affected ga children to old order amish controls, we easily identified the location of gcdh. however, when we used old order mennonite allele frequency data, background noise increased and the mapping signal on chromosome was obscured. and cost, and allows autozygosity analysis to be done with fewer affected patients. with suf- ficiently dense snp coverage, it is no longer necessary to infer genotype and haplotype shar- ing between distant markers; increased marker density provides enough information to reveal sharing directly. higher marker density also simplifies analysis. large shared blocks of ho- mozygous snps can be detected with simple counting and graphing algorithms using stan- dard spreadsheet software ( , ). we have www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : table thirty-seven amish and mennonite disorders understood at the molecular level among demes of pennsylvania, ohio, and maryland disorder gene dna variant protein variant population deme∗ -methylcrotonylglycinuria mccc g>c glu gln amish a -ß-oh-steroid dehydrogenase deficiency hsd b g>a gly glu amish a aldosterone deficiency cyp b bp deletion amish a amish microcephaly slc a g>c gly ala amish a bardet-biedl syndrome bbs t>g met arg amish a bartter syndrome clcnkb gene deletion amish a, b cardiomyopathy, dilated, with av block lmna c>t arg trp amish a cartilage-hair hypoplasia rmrp a>g amish a, o cortical dysplasia and focal epilepsy cntnap delg amish a, b, o crigler-najjar syndrome ugt a c>a tyr ter amish a ellis-van creveld syndrome evc ivs + g>t amish a familial hypercholanemia baat a>g met val amish a familial hypercholanemia tjp t>c val ala amish a galactosemia galt a>g gln arg amish a, e gittelman syndrome slc a c>a arg gly amish a, b gittelman syndrome slc a bp deletion amish a, b gutaric aciduria, type gcdh c>t ala val amish a gutaric aciduria, type c orf c>t arg trp amish a, b mckusick-kaufman syndrome mkks [ c>t + g>t] his tyr/ala ser amish a nemaline rod myopathy tnnt g>t glu ter amish a nephrotic syndrome nphs g>a arg gln amish a osteogenesis imperfecta col a g>t gly cys amish a pelizaeus-merzbacher-like syndrome gja a>g tyr cys amish a phenylketonuria pah - delatc amish a phenylketonuria pah g>a arg gln amish a propionic acidemia pccb a>g asn asp amish a, b severe combined immune deficiency rag a>g lys glu amish a sitosterolemia abcg g>a gly arg amish a weil-marchesani syndrome adamts exon del amish a chronic granulomatous disease cybb c>a cys ter amish e troyer syndrome spg dela amish o -hydroxylase deficiency cyp b g>a arg his amish b adenosine deaminase deficiency ada g>a gly arg amish b byler disease atp b g>t gly val amish b cockayne syndrome ercc ivs + g>t amish b gm synthase deficiency st gal c>t arg ter amish b, o primary ciliary dyskinesia dnah c>t gln ter amish b pyruvate kinase deficiency pklr g>a arg his amish b sudden infant death with dysgenesis of the testes tspyl insg amish b homocystinuria mthfr c>t arg cys amish j (continued ) strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : table (continued ) disorder gene dna variant protein variant population deme∗ -methylcrotonylglycinuria mccc inst mennonite m, x alpha- antitrypsin deficiency serpina g>a glu lys mennonite m biotinidase deficiency btd t>c trp arg mennonite m congenital nephrotic syndrome nphs delc mennonite m congenital nephrotic syndrome nphs delg mennonite m crigler-najjar syndrome ugt a c>a tyr ter mennonite m cystinuria slc a ivs + t>c mennonite m cystinuria slc a c>t arg trp mennonite m cystinuria slc a c>t ile ile mennonite m cystinuria slc a c>t thr met mennonite m deafness, nonsyndromic gjb delg mennonite m factor deficiency f c>t arg cys mennonite m fragile x syndrome fmr (cgg)n expansion mennonite m glycogen storage disease, type pygl ivs + g>a mennonite m hirschsprung disease ednrb g>t trp cys mennonite m, s lyk deficiency lyk bp deletion mennonite m maple syrup urine disease bckdha t>a tyr asn mennonite m, s medium-chain acyl-coa dehydrogenase deficiency acadm a>g lys glu mennonite m medium-chain acyl-coa dehydrogenase deficiency acadm ivs - a>g mennonite m mevalonate kinase deficiency mvk t>c ile thr mennonite m mevalonate kinase deficiency mvk g>a ala thr mennonite m phenylketonuria pah ivs + ga mennonite m phenylketonuria pah g>a arg gln mennonite m propionic acidemia pccb a>g asn asp mennonite m, s restrictive dermopathy zmpste inst mennonite m severe combined immune deficiency il r t>g mennonite m spinal muscular atrophy smn exon deletion mennonite m tyrosine hydroxylase deficiency th g>a arg his mennonite m tyrosinemia, type hpd g>a ala thr mennonite m vitamin b deficiency amn bp deletion mennonite m properdin deficiency pfc t>g cys gly mennonite x osteoporosis-pseudoglioma syndrome lrp a>g thr ala mennonite s osteoporosis-pseudoglioma syndrome lrp g>a trp ter mennonite s phenylketonuria pah ivs - g>a mennonite s cardiomyopathy, hypertrophic slc a delc mennonite w periodic fever (traps) tnfrsf a g>a arg gln mennonite w salla disease slc a c>t arg cys mennonite w tyrosinemia, type hpd a>g tyr cys mennonite w tyrosinemia, type hpd c>g ile met mennonite w ∗a, lancaster county (pa) amish; b, juniata and mifflin county (pa) amish; o, holmes and geauga county (oh) amish; j, somerset county (pa) amish; m, weaverland and groffdale conference mennonites (pa and elsewhere); s, stauffer mennonites (pa/md); w, western md; x, other unspecified mennonites; e, eastern shore of maryland amish. www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : a b a ve ra g e n u m b er o f b lo ck s d ev ia ti o n fr o m c o n tr o l – – – – – – – – amish mennonite control block size - - - - - - - - > amish mennonite block size figure plain populations demonstrate increases in homozygous block size and number compared to americans of european descent. blocks of homozygosity were quantified by their snp content and physical size. (a) the average number of homozygous snp blocks per person is shown. amish and mennonite individuals have more (and larger) runs of homozygosity than do controls. (b) the figure plots deviations from the average control block data by size (i.e., number of snps contained in each homozygous block). the plain populations are relatively deficient in small homozygous runs, which are replaced by larger blocks. the excess runs of homozygosity range in size from about to over adjacent snps (highlighted area). for each plain population, excess homozygosity relative to european controls is found in a relatively small number of large blocks, comprising roughly . and . mb of homozygous dna in amish and mennonite genomes, respectively. used these techniques to map disorders with as few as two affected children ( ). we have applied various methods to iden- tify different mutations segregating in the plain populations of southeastern pennsylva- nia and elsewhere. the most straightforward method is candidate gene analysis—sequencing a candidate gene known to cause a pheno- type. phenylketonuria mutations in several unrelated old order amish patients from lan- caster county were identified in this way, and the same strategy was applied to disorders such as , -methylenetetrahydrofolate reduc- tase (mthfr) deficiency, factor xi deficiency, and crigler-najjar syndrome. a related approach, candidate gene local- ization, uses snp data to narrow a list of candidate genes that are associated with a particular phenotype and scattered across the genome, provided the child is autozygous sur- rounding the disease gene locus. by correlating homozygous blocks to the location of can- didate genes, we can often identify a single gene for targeted sequencing. this method has been used successfully to map autosomal reces- sive deafness, cockayne syndrome, megaloblas- tic anemia, and hereditary spastic paraplegia (figure ). finally, disease gene mapping uses genetic marker data to map unknown recessive pheno- types by comparing homozygous blocks among multiple affected individuals ( , , , ). this strategy exploits genetic properties com- mon to young populations with a small number of founders, and also assumes mutation homo- geneity. we often delineate locus boundaries rapidly and efficiently, but the shared homozy- gous blocks tend to be quite large. the average shared block is . mb (mean . patients per study) and contains dozens and sometimes hun- dreds of genes. thus, although finding the rele- vant block can be simple, identifying the disease gene within it can be daunting. we have initiated over separate ge- netic mapping studies in plain populations of pennsylvania, maryland, indiana, and ohio. for of these, we unequivocally mapped the disease gene and identified the pathogenic se- quence variant. for others, we delineated a chromosomal region but have yet to identify the causative gene (table ). for the remainder, mapping results are inconclusive due to insuf- ficient sample size or the presence of multiple shared homozygous blocks. strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : locus location number of homozygous snps location score spg q q q q p q q q q q q q q q spg spg spg spg spg a spg spg spg spg spg spg spg spg spg spg b spg chromosome number h o m o zy g o u s b lo ck s iz e figure two brothers from a midwestern amish deme presented to our clinic with developmental delay, short stature, and symmetric spastic diplegia. recessive hereditary spastic paraplegia (spg) was considered in the genetic diagnosis, but a review of online mendelian inheritance in man revealed multiple spg loci scattered throughout the genome. this daunting gene list would likely preclude further genetic testing. however, assuming identity-by-descent for the disease gene, we used k snp data to exclude the large majority of spg loci and show that both boys were homozygous for a large block of dna surrounding the locus for troyer syndrome (spg ); sequencing confirmed homozygosity for the known single-base-pair deletion ( dela) in spg . mendelian disorders in plain populations the evolution of clinical methods amish genetic studies between and predated the era of molecular mapping and did not deal explicitly with the problem of providing care. many research subjects died or were crippled because they lacked appro- priate medical services ( ). in , holmes and caroline morton established the small nonprofit clinic for special children to serve amish and mennonite children who suffered from genetic disorders ( , ). the clinic was sited in rural lancaster county (figure ), which gave families nearby access to affordable care ( ) and provided a framework for study- ing genetic disorders in a natural setting ( , ). it is a paradigm of community genetics ( ), designed to serve particular people living in a specific place and time ( , , , ). the old order communities support the work as a way to care for their own ( , ). today, clinic staff manage active patients representing over different mendelian disorders. from the clinic’s inception, on-site labo- ratory studies were continually shaped by the needs of patients; research and clinical care were inseparable. between and , a gas chromatograph-mass spectrometer donated by hewlett-packard was used to screen amish children for glutaric aciduria type (ga ) ( ). in , two mennonite churches donated a high-performance liquid chromato- graph that has run more than , amino acid samples for the diagnosis and management of maple syrup urine disease (msud) ( ). in the early years of the clinic, operating these in- struments on the front line of clinical practice meant that ga could be diagnosed within a few hours of life (i.e., from amniotic fluid ob- tained at delivery) ( ), and msud could be detected in asymptomatic newborns less than day old ( ). these methods were a crucial www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : table twenty-three genetic disorders mapped using snp microarrays at the clinic for special children since . the causative gene was identified for conditions, whereas other disorders are mapped to a region but the gene remains unknown disease chromosome gene nephrotic syndrome, amish nphs pelizaeus-merzbacher-like syndrome gja bartter syndrome clcnkb hypertrophic cardiomyopathy slc a primary ciliary dyskinesia dnah sudden infant death with dysgenesis of the testes tspyl salla disease slc a cortical dysplasia and focal epilepsy cntnap glutaric aciduria, type c orf severe combined immune deficiency rag deafness, mennonite gjb gitelman syndrome slc a polyhydramnios, megalencephaly, and symptomatic epilepsy strada weil-marchesani syndrome adamts posterior column ataxia with retinitis pigmentosa congenital nystagmus usher-like syndrome hydrocephalus lethal seizure syndrome mental retardation, nonsyndromic junctional ectopic tachycardia spastic paraplegia “yoder” dystonia “schwartz” syndrome microcephaly first step toward disease prevention, allowing affected newborns to start therapy days before state screening results were complete. in we began direct dna sequencing, which led quickly to mutation identification for numerous disorders and carrier testing for ga , msud, and several other conditions ( , , , ). affymetrix donated a genechip scanner in . it proved a powerful tool for mapping ( , , ) and also revealed important details about allele dynamics within plain populations. in , we acquired a roche real-time pcr system (lightcycler) through charitable contributions from the a.j. stamps foundation and csl behring. this instrument uses melting curve analysis to detect mutations in about an hour ( ). it is ideal for community-based genetic testing [see sidebar , , -methylenetetrahydrofolate reductase (mthfr) deficiency] and can be adapted to practical clinical problems such as low-resolution hla matching ( ). spectrum of mendelian disease table lists mennonite and amish disorders understood at the molecular level, restricted for the purpose of this review to populations of pennsylvania and maryland. al- though there is some crossover, demes of ohio, strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : indiana, and elsewhere suffer from different genetic disorders. even within pennsylvania, there are several genetically distinct groups, and these distinctions are clinically important (see sidebar , genetic epidemiology). ninety percent of the conditions listed in table are recessive, and % of these result from a single allele restricted to a particular deme ( , ). however, there are several exceptions. among old order mennonites, who stem from a larger founding popula- tion, we have identified multiple mutations for phenylketonuria, cystinuria, medium- chain acyl-coa dehydrogenase deficiency, mevalonate kinase deficiency, osteoporosis- pseudoglioma syndrome, and tyrosinemia type . among the amish, biotinidase de- ficiency, galactosemia, gitelman syndrome, and phenylketonuria demonstrate mutation heterogeneity, and familial hypercholanemia exhibits locus heterogeneity. although most of the disorders we manage are recessive, we have also identified several x- linked (e.g., fragile x syndrome, chronic granu- lomatous disease, and properdin deficiency) and dominant conditions (e.g., lmna cardiomy- opathy and polycystic kidney disease), and recognize others yet to be mapped (sympha- langism, multiple exostoses, arteriovenous mal- formation). although fewer in number, these disorders nevertheless exact a heavy toll when they occur in large extended families. we have also found de novo mutations in a child with lissencephaly (pafah b deletion), a child with apert syndrome (fgfr ), an adult with idiopathic torsion dystonia (dyt ), and sev- eral children with prader-willi or angelman syndrome (chromosome deletions). finally, using k snp arrays we have identified chro- mosomal abnormalities at the resolution of a standard karyotype in numerous children with syndromic developmental delay (figure ). although certain mutations are concen- trated in amish and mennonite demes, they are seldom unique to these groups. about % of individuals leave the old order each generation, allowing mutations to flow into figure the clinic for special children is sited on an untillable corner of farmland in rural lancaster county, pennsylvania. the land was the gift of an amishman who has two granddaughters with glutaric aciduria type . it was built with the donated labor of amish and mennonite workers. the clinic is readily accessible to most of its patients and has on-site biochemical and molecular testing. sidebar . , -methylenetetrahydrofolate reductase (mthfr) deficiency an amish boy, born in somerset county in , had psychomo- tor delay and slow head growth by months of age, and an mri showed global cerebral atrophy and hypomyelination. he was seen by numerous pediatric subspecialists but remained without a diagnosis after more than $ , of diagnostic testing. we saw him in october , and based on a biochemical workup identified a pathogenic c>t change in mthfr ( ). de- spite treatment with high-dose betaine, he remained severely re- tarded, autistic, and mute. we made several field trips to somer- set county where we identified mthfr c. c>t carriers among healthy members of the deme (estimated population carrier frequency %). in collaboration with dr. naylor and his colleagues, we developed a lightcycler method for detecting mthfr c. c>t in dried filter paper blood spots ( ). mid- wives offered amish parents the option of getting a “fifth blood spot” to test for the mthfr mutation. the first newborn diag- nosed by lightcycler screening in was sister to the proband ( ). she started therapy during her second week of life and has no signs of brain damage after four years of follow-up. www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : sidebar . genetic epidemiology most amish babies are delivered at home, breastfeed, and do not receive parenteral vitamin k. in lancaster county, two recessive alleles in the amish (tjp c. t>c and baat c. a>g) dis- rupt bile salt metabolism and result in vitamin k malabsorption ( ). thus genetic factors interact with cultural practice to deter- mine actual risk: over the past years, three amish infants with bile salt disorders presented with massive intracerebral and retinal hemorrhages from vitamin k deficiency. one child survived with dense spastic hemiparesis and unilateral blindness. the other two died of acute brain injuries. in one case, the parents were wrong- fully accused of child abuse and temporarily lost custody of their other children. screening for bile salt disorders and neonatal vita- min k administration are pressing public health concerns against this genetic background. in juniata and mifflin counties, a mu- tation in tspyl ( insg) among amish children causes brain disease and a dysautonomia syndrome (siddt) that ac- counts for % of overall infant mortality and perhaps half of all sudden infant deaths in the region ( ). to prevent sudden infant death in mifflin county, efforts to understand and treat siddt syndrome are at least as important as the “back to sleep” campaign (http://nichd.nih.gov/sids/). the outbred population ( ). moreover, most pathogenic alleles probably existed in europe before the anabaptist migration. for example, all four plain mutations of the pah gene were first identified in europeans with phenylke- tonuria (table ), and the only gcdh variant known to cause glutaric aciduria type in the amish (c. c>t) is among the more com- mon mutations found throughout europe and the united states. curiously, several founder mutations from finland are also distributed in north american plain sects (i.e., rmrp c. a>g and slc a c. c>t). there are no known genealogical ties between swiss an- abaptists and finns, suggesting that these mu- tations are either ancient or recurrent ( ). for ( %) conditions listed in table , an experienced clinician can make a correct diagnosis based on the clinical presentation and a single confirmatory test. a few oth- ers are distinctive and easily recognized (e.g., amish lethal microcephaly, ellis-van creveld syndrome, weil-marchesani syndrome, etc.). only ( %) are reliably detected by state newborn screening. importantly, about % of the disorders cannot be diagnosed by rou- tine clinical methods; they present to physi- cians as nonspecific problems such as failure to thrive, jaundice, bruising, itching, develop- mental delay, cerebral palsy, mental retardation, autism, seizures, short stature, or weakness. an understanding of genetic risks within a popu- lation makes it clear that these general terms apply to many discrete causes (table ) (see sidebar , glutaryl-coa dehydrogenase defi- ciency). the same is likely true in outbred pop- ulations, but it is much harder to investigate. when we encounter a novel phenotype, in- vestigational studies are needed to determine the molecular cause and then translate this in- formation into useful clinical testing ( , , ). many core genetic laboratories offer dna isolation and storage as well as biochemical ge- netics testing. however, few offer the flexible genotyping and sequencing services needed to map a new disease. fewer still achieve these aims within a clinically relevant time frame or at an acceptable cost to self-pay families. our laboratory functions as a specialized core fa- cility with the sole purpose of solving patient- and community-centered problems quickly and economically ( ). we have moved the tools of genetic research to the clinical laboratory and invested heavily in their continued use and ef- ficacy. consequently, when evaluating a new patient we are as likely to sequence a gene or generate snp genotypes as we are to order an electrolyte profile. translational medicine and the treatment of genetic disease treatment of genetic disease just over years ago, half of known genetic disorders were considered untreatable and only about % could be treated in a highly effective strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : manner ( ). we have seen extraordinary progress in plain populations over the past two decades. for about % of the disorders we manage, simple dietary intervention has a deci- sive effect. these include conditions such as bi- otinidase deficiency, galactosemia, and vitamin b malabsorption. protocols for the treatment of maple syrup urine disease (msud), crigler- najjar syndrome, and severe combined immune deficiency (scid) are more complex but still quite effective ( , , , ). these latter con- ditions can now be cured by tissue transplant, currently the only reliable form of gene therapy ( , ). problems such as pyruvate kinase de- ficiency, cystinuria, and deafness are treatable, but not perfectly so. nevertheless, like diabetes, depression, and cancer, skillful medical care al- lows people to live longer, suffer less, and enjoy more independence. for ( %) genetic conditions listed in table , eventual outcome is not affected by treatment; five of these disorders are lethal dur- ing infancy. they are among the most common genetic disorders we encounter; among the −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ figure affymetrix genechip human mapping k arrays are useful for copy-number assessment. although the k arrays lack fine resolution for this application, our data show that they perform at least as well as a standard karyotype. we readily identify copy-number abnormalities in the – mb range, validated by standard metaphase karyotype (http://www.mayomedicallaboratories.com/). our analyses use the copy-number data generated by the affymetrix genotype software. we calculate mean and standard deviations for the fluorescence at each snp marker and then generate z scores for the patient at each snp marker. this partially corrects for polymorphic copy-number variation within the genome. panels (a) and (b) show two small deletions of ∼ . and ∼ . mb, respectively. the first patient had multiple congenital anomalies, and the second patient was diagnosed with angelman syndrome after snp analysis detected monosomy on chromosome . panels (c) and (d ) depict trisomy of chromosome q markers and a complex rearrangement involving partial deletion and duplication of chromosome . data in panels (b) and (d ) are from males, and hemizygosity for x chromosome markers is apparent. amish, for example, we have a record of chil- dren with lethal microcephaly born between and and children born with ne- maline rod myopathy (tnnt ) since – – – – – – – – c a d b chromosome number x sn p c o p y n u m b er z s co re sn p c o p y n u m b er z s co re sn p c o p y n u m b er z s co re sn p c o p y n u m b er z s co re www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : sidebar . glutaryl-coa dehydrogenase deficiency before , amish children with ga were commonly misdi- agnosed with cerebral palsy or acute viral encephalitis. the first genetic study at the clinic for special children identified ga as the cause of “amish cerebral palsy” in disabled children who traced to a single founding couple ( ). beginning in , the clinic offered amniotic fluid and urine organic acid screening for newborns, comprehensive pediatric follow-up care, and inpa- tient management for illnesses. dr. naylor introduced statewide supplemental screening for ga in . between and the present, the rate of brain injury in ga decreased from % to % ( ). thus the efficacy of treatment in ga , as with many genetic conditions, is relative. although ga is the most com- mon mimic of cerebral palsy in lancaster county, other hered- itary conditions can produce a similar phenotype and, equally importantly, not every child with dystonia has a mendelian dis- order. acquired causes of brain injury are intermingled with, and difficult to distinguish from, genetic disease. ( , ). even for such “untreatable” prob- lems, medical care is important ( , ). before the clinic was established, amish children with nemaline rod myopathy were repeatedly sub- jected to invasive and costly interventions (e.g., muscle biopsies, nerve conduction testing, elec- tromyography, magnetic resonance imaging, echocardiograms, etc.) that collectively cost the community millions of dollars. a thoughtful physician informed with the right genetic di- agnosis can help a family determine appropri- ate limits of medical care while also protecting the child from futile interventions and the com- mon miseries of hunger, thirst, dyspnea, and pain. presymptomatic diagnosis and disease prevention children born today with conditions such as ga , mthfr deficiency, or msud— once thought untreatable ( )—now grow up healthy if diagnosed early in life (see sidebars , , and ) ( , , ). presymptomatic diagnosis creates a window of opportunity ( , , , ). this was the idea behind guthrie & susi’s test for phenylketonuria in newborns, published in ( ). thirty years later, shortly after dr. morton began his work in lancaster county, edwin naylor and colleagues were using tan- dem mass spectrometry to screen newborns for a much broader spectrum of genetic conditions ( ). screening does not, however, assure good outcome. early diagnosis is of no value with- out a pragmatic follow-up plan designed to deal with the dynamic interplay among genes, envi- ronment, and disease as it unfolds over time. genes encode proteins embedded in complex biological networks. these networks are con- tained within cells, grouped into tissues, and able to interact within physiological systems. understanding disturbances at all of these lev- els, as experienced by intact humans in their natural setting, is the basis for designing effec- tive therapies ( , ). this is vividly illustrated by the history of msud in lancaster county ( ). before , one-third of mennonite chil- dren with msud died during childhood. most who survived were severely disabled [see side- bar , changing the natural history of maple syrup urine disease (msud)] ( , , ). since the clinic started offering local services, outcomes for msud have improved ( ). no child died while under our care for metabolic crisis, and all mennonite children with msud born after attained normal developmen- tal milestones. knowledge of pathophysiology, not genetics, fueled this progress. through careful study of many individuals in diverse clinical circumstances, we learned how defi- ciency of branched-chain ketoacid dehydrogen- ase disrupts critical biological processes such as metabolic adaptation to fasting and illness ( ), cerebral amino acid transport ( , ), and cell volume control ( ). these processes change with age and are influenced by many nutri- tional and environmental variables. our treat- ment protocol evolved in lockstep with our un- derstanding of this complex physiology ( ) and allowed us to prevent many serious neurologi- cal consequences of msud. strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : table gene mutations result in specific medical conditions that are often described with nonspecific terms: mental retardation, cerebral palsy, autism, sudden infant death, sepsis, and epilepsy clinical diagnosis disorder gene cerebral palsy glutaric aciduria type gcdh propionic acidemia pccb gap junction deficiency gja crigler-najjar syndrome ugt a hereditary spastic paraplegia spg dopamine-responsive dystonia th idiopathic torsion dystonia dyt mental retardation phenylketonuria pah mthfr deficiency mthfr maple syrup urine disease bckdha salla disease slc a bardet-biedl syndrome bbs fragile x syndrome fmr epilepsy biotinidase deficiency btd cdfe syndrome (caspr ) cntnap gm synthase deficiency st gal pmse syndrome strada stroke/hemorrhage hypercholanemia,tjp type tjp hypercholanemia, baat type baat factor v leiden f sitosterolemia abcg alpha- -antitrypsin deficiency serpina sudden death siddt syndrome tspyl -beta-hsd deficiency hsd b hypertrophic cardiomyopathy slc a lethal infection galactosemia galt properdin deficiency pfc scid, il receptor type il r adenosine deaminase deficiency ada rag deficiency, omenn rag this table lists a selection of genetic syndromes that cause developmental disorders in amish and mennonite children. recognizing genetic predispositions that underlie such disorders is a key step toward planning effective prevention strategies. disease prevention has substantial eco- nomic repercussions ( ). based on our expe- rience with msud and several other treatable genetic conditions that affect the nervous system (table ), we have prevented severe mo- tor disability or mental retardation in an es- timated children over a -year period. this saved the plain communities about $ million in direct and indirect costs ( ). thus annually, our operating budget of $ . million reduces the community economic burden by over $ million. to help uninsured patients, preventative therapies must be affordable. for example, early www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : sidebar . changing the natural history of maple syrup urine disease (msud) between and , mennonite children were born with msud in lancaster county. they all became encephalopathic within the first week of life and required weeks or months of intensive hospital treatment that cost tens of thousands of dollars. follow-up care was expensive, far away, and disorganized ( , ). msud patients were often sick at home for days before arriving comatose in philadelphia with critical cerebral edema. no aspect of therapy dealt specifically with this complication, and ( %) of these children died of brain herniation within h of hospitalization ( , ). since , we have managed mennonite infants with msud. thirty-two ( %) were diagnosed between and h of life by on-site amino acid analysis or, more recently, di- rect mutation detection from umbilical cord blood using real- time pcr. among infants diagnosed before days of age, % were treated safely at home. to manage crises locally, we made msud hyperalimentation solution available h a day at lancaster general hospital and had on-site amino acid analysis with a -min turnaround time. we have successfully managed over metabolic crises at lancaster general with an average hospital stay of just days. by combining general pediatric care and specialized services, a much larger number of metabolic crises were averted out of hospital; the overall hospitalization rate for our msud cohort is currently only . hospital days per patient per year. studies of vitamin b deficiency led to reli- able and inexpensive forms of replacement ther- apy ( ). now, for mennonite children with hereditary b malabsorption (amn deletion), once daily sublingual methyl-b prevents life- threatening anemia and spinal cord injury for about $ per year. however, if sublingual methyl-b were to be developed as a new thera- peutic today, it would cost between $ million and $ billion to bring to the u.s. market, and its cost would be prohibitive for many self-pay families ( ). although we can expect exciting new advances in the treatment of genetic dis- ease, attendant costs will put effective therapies beyond the reach of some families. plain people at the frontier of genomic medicine when mckusick and colleagues began their studies of the amish in ( ), medical ge- netics was just a fledging discipline and molec- ular studies were limited to descriptions of ab- normal chromosome number and structure ( ). polymerase chain reaction was not introduced until ( ), and polymorphic microsatel- lite markers were not widely used until the early s ( ). the human genome project started in october , and the complete draft of the human genome was published in febru- ary ( , ). thus, genetic discovery in the plain communities developed in parallel with larger events taking place in the field of ge- nomics. genetic science has become a cornerstone of medicine, but the practice of medicine has been equally vital to progress in genetic science. bench scientists and clinicians have different interests and responsibilities. basic scientists investigate physiologic systems, tis- sues, membranes, pathways, and molecules. they are primarily concerned with patterns and mechanisms. physicians, in contrast, are prin- cipally concerned with the welfare of individ- uals, “for the patient is no mere collection of symptoms, signs, disordered functions, dam- aged organs, and disturbed emotions. [the pa- tient] is human, fearful, and hopeful, seeking relief, help, and reassurance” ( ). these differing perspectives influence how one views progress in genomic science ( ). consider three decades of progress in cellu- lar immunology. over the past years, im- munogenetics research has expanded at an ex- plosive pace; we now understand many cellular immune responses in exquisite molecular de- tail ( ). yet among amish and mennonite children born with scid during the same time period, only half received a bone marrow trans- plant and % died by years of age ( ). there was little funding for patient-centered research ( ), and hospitals with expertise in scid were far away and cost too much ( ). strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : we came face to face with this problem in january , when an amish newborn pre- sented to our clinic with omenn syndrome, a variant of scid (figure ) ( ). she was sick and uninsured, and needed timely, affordable care. we were able to use k snp genotypes from the child and her siblings to delineate the rag /rag locus, and used these same data to quickly and inexpensively find an hla- matched stem cell donor among her siblings. this entire process—from clinical presentation to genetic diagnosis to donor identification— took less than two weeks and saved the fam- ily between $ , and $ , . applied ge- nomics enabled access to life-saving therapy. in a medical-industrial research environ- ment that views case reports and small cohorts as poor evidence ( ), this one amish child gave us a glimpse of mckusick’s vision for ge- nomic medicine (figure ) when he wrote: “[g]enomics is likely to render medicine more predictive and, therefore, more preventive. . . on the traditional turf of clinical medicine, di- agnosis will become more specific and pre- cise, and treatment also more specific and safer. genomics-based individualization of medical care aims to achieve the right treatment for the right patient” ( ) (italics added). thus, the fu- ture of translational genomics is about patient discovery, not gene discovery ( , , ). the idea has a modern emphasis but is not new; hippocrates believed that diseases as such did not exist in nature. to him, what existed natu- rally were sick people, and no two people were sick in exactly the same way ( ). the ethical imperative to care for individ- uals with genetic disease is apparent, but the scientific value of such work should not be over- looked. the connection of a gene to a human phenotype is the only way to fully appreciate a gene’s function in vivo ( , , , , ). in this respect, mice, fish, and flies will always fall short of modeling human disease (see sidebar , new disease pathways in human brain development). the astute physician, working daily to care for one or a few patients with a rare and complex disorder, can develop key insights that deepen our understanding ( , ), rag /rag c h ro m o so m e hla gene cluster c h ro m o so m e a b c chromosome number figure an amish girl was born in with erythroderma, hepatosplenomegaly, hypereosinophilia, hypogammaglobulinemia, and b cell deficiency. she grew poorly and had recurrent infections. she was provisionally diagnosed with omenn syndrome, a variant of severe combined immune deficiency (scid), but died at months of age before the diagnosis could be confirmed or a transplant attempted. in january , her sister presented as a newborn with alopecia and erythroderma, developed a systemic staphylococcus aureus infection at three weeks of age, and had immunologic studies consistent with scid. (a) a homozygosity plot was generated for the proband ( gray peaks); identity- by-state (ibs) plots for each pairwise comparison between the proband and her unaffected siblings were superimposed on this plot. genotype-identical regions (colored lines) between the proband and her siblings (i.e., ibs = ) denote regions where the disease gene cannot reside. the red line at the top is a summary of the exclusion mapping. this analysis demonstrated that only a single, homozygous genomic region on chromosome was consistent with linkage within the pedigree. (b) the rag and rag loci mapped to the homozygous interval on chromosome . rag sequencing revealed homozygosity for a pathogenic c. a>g variant in the patient. (c) the homozygosity plot of chromosome in the proband and her seven unaffected siblings. compared to the proband, only one sibling is haplo-identical for the major hla loci on chromosome . subsequently, hla typing of the patient and her potential sibling donor was performed using standard serotyping and molecular probes; these data corroborated the snp matching. the patient had a successful unmodified bone marrow transplant at days of age. she is now years old, fully engrafted, and healthy. dna extracted from paraffinized bone marrow showed that her sister, who died years earlier, was also homozygous for rag c. a>g. www.annualreviews.org • genetics, medicine, and the plain people a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : sidebar . new disease pathways in human brain development in , we discovered a homozygous frameshift mutation of the cntnap gene in a group of amish children with cortical dys- plasia, focal epilepsy, and developmental regression ( ). remark- ably, mice with homozygous cntnap mutations have no central nervous system disease. nevertheless, the findings prompted new studies about the role of cntnap in human brain development. within two years this gene was linked to idiopathic autism ( , , ), schizophrenia ( ), and mutism ( ) in outbred populations across the country and throughout the world. thus, the study of rare mendelian disorders links specific gene families or protein networks—i.e., “interactomes”—to relevant processes in human biology. in the words of william harvey (ca. ): “nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path; nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of nature by careful investigation of cases of rarer forms of disease.” expand our diagnostic capabilities ( , ), and create new opportunities for prevention ( , , , ). in the era of genomic medicine, physicians and molecular biologists who work closely together can translate these insights into real clinical benefits. such collaborations will help us tackle big problems at the frontier of genomic medicine, including multigenic dis- ease (e.g., atherosclerosis, hypertension, men- tal illness, etc.), modifier genes, and epigenetics ( , , , , ). disclosure statement the authors are not aware of any biases that might be perceived as affecting the objectivity of this review. acknowledgments we thank donna robinson and christine hendrickson for assistance in data collection and for their exceptional contributions to patient care. drs. nicholas rider and terry sharrer provided important insights and helpful suggestions. don kraybill and his colleagues at the young center for anabaptist studies, elizabethtown college, provided demographic data. holmes and caroline morton envisioned the clinic for special children and had the tenacity to make it real; we are figure victor a. mckusick (left), d. holmes morton (right), and bowie sitting on the front porch at the clinic for special children, spring . finally, it is important to remember that ad- vances in molecular science only reveal what can be done with genetic information. perhaps the plain people can teach us something impor- tant about what should be done with it ( , ). the old order communities measure the value of medical research not in grant awards, publi- cations, or academic promotions, but in human terms: alleviation of pain, prevention of disabil- ity, equitable delivery, and fair cost. they un- derstand that to translate genetic information into better public health, one must first com- mit to caring for individuals. strauss · puffenberger a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . anrv -gg - ari july : deeply indebted to them for their extraordinary example and ongoing contributions to people of the plain communities. finally, we thank the children and families whom we serve; they continue to teach us much about science, medicine, and meaningful work ( ). literature cited . mckusick va, ed. . medical genetic studies of the amish. baltimore: johns hopkins univ. press . martin j, martin vm, eds. . what is wrong with our baby? ephrata, pa: grace press. pp. . honeycutt a, dunlap l, chen h, al homsi g, grosse s, schendel d. . economic costs associated with mental retardation, cerebral palsy, hearing loss, and vision impairment—united states, . morb. mortal. wkly. rep. 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. d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . ar -gg -fm ari august : annual review of genomics and human genetics volume , contents chromosomes in leukemia and beyond: from irrelevant to central players janet d. rowley � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � unraveling a multifactorial late-onset disease: from genetic susceptibility to disease mechanisms for age-related macular degeneration anand swaroop, emily y. chew, catherine bowes rickman, and gonçalo r. abecasis � � � syndromes of telomere shortening mary armanios � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � chronic pancreatitis: genetics and pathogenesis jian-min chen and claude férec � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � the genetics of crohn’s disease johan van limbergen, david c. wilson, and jack satsangi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � genotyping technologies for genetic research jiannis ragoussis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � applications of new sequencing technologies for transcriptome analysis olena morozova, martin hirst, and marco a. marra � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � the posttranslational processing of prelamin a and disease brandon s.j. davies, loren g. fong, shao h. yang, catherine coffinier, and stephen g. young � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � genetic testing in israel: an overview guy rosner, serena rosner, and avi orr-urtreger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � stewardship of human biospecimens, dna, genotype, and clinical data in the gwas era stephen j. o’brien � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � schistosoma genetics: new perspectives on schistosome biology and host-parasite interaction ze-guang han, paul j. brindley, sheng-yue wang, and zhu chen � � � � � � � � � � � � � � � � � � � � evolution of genomic imprinting: insights from marsupials and monotremes marilyn b. renfree, timothy a. hore, geoffrey shaw, jennifer a. marshall graves, and andrew j. pask � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � v a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . ar -gg -fm ari august : methods for genomic partitioning emily h. turner, sarah b. ng, deborah a. nickerson, and jay shendure � � � � � � � � � � � � � biased gene conversion and the evolution of mammalian genomic landscapes laurent duret and nicolas galtier � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � inherited variation in gene expression daniel a. skelly, james ronald, and joshua m. akey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � genomic analyses of sex chromosome evolution melissa a. wilson and kateryna d. makova � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � sequencing primate genomes: what have we learned? tomas marques-bonet, oliver a. ryder, and evan e. eichler � � � � � � � � � � � � � � � � � � � � � � � � � � � genotype imputation yun li, cristen willer, serena sanna, and gonçalo abecasis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � genetics of athletic performance elaine a. ostrander, heather j. huson, and gary k. ostrander � � � � � � � � � � � � � � � � � � � � � � � � genetic screening for low-penetrance variants in protein-coding diseases jill waalen and ernest beutler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � copy number variation in human health, disease, and evolution feng zhang, wenli gu, matthew e. hurles, and james r. lupski � � � � � � � � � � � � � � � � � � � � � the toxicogenomic multiverse: convergent recruitment of proteins into animal venoms bryan g. fry, kim roelants, donald e. champagne, holger scheib, joel d.a. tyndall, glenn f. king, timo j. nevalainen, janette a. norman, richard j. lewis, raymond s. norton, camila renjifo, and ricardo c. rodríguez de la vega � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � genetics, medicine, and the plain people kevin a. strauss and erik g. puffenberger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � indexes cumulative index of contributing authors, volumes – � � � � � � � � � � � � � � � � � � � � � � � � � � � � � cumulative index of chapter titles, volumes – � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � errata an online log of corrections to annual review of genomics and human genetics articles may be found at http://genom.annualreviews.org/ vi contents a nn u. r ev . g en om . h um an g en et . . : - . d ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by d r. k ev in s tr au ss o n / / . f or p er so na l us e on ly . 	search annual reviews 	annual reviews online 	annual review of genomics and human genetics online 	most downloaded genomics and human genetics
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errata 	view current editorial committee 	all articles in the annual review of genomics and human genetics, vol. 	chromosomes in leukemia and beyond: from irrelevantto central players 	unraveling a multifactorial late-onset disease: from genetic susceptibility to disease mechanisms for age-related maculardegeneration 	syndromes of telomere shortening 	chronic pancreatitis: genetics and pathogenesis 	the genetics of crohn’s disease 	genotyping technologies for genetic research 	applications of new sequencing technologies for transcriptome analysis 	the posttranslational processing of prelamin a and disease 	genetic testing in israel: an overview 	stewardship of human biospecimens, dna, genotype, and clinical data in the gwas era 	schistosoma genetics: new perspectives on schistosome biology and host-parasite interaction 	evolution of genomic imprinting: insights from marsupials and monotremes 	methods for genomic partitioning 	biased gene conversion and the evolution of mammalian genomic landscapes 	inherited variation in gene expression 	genomic analyses of sex chromosome evolution 	sequencing primate genomes: what have we learned? 	genotype imputation 	genetics of athletic performance 	genetic screening for low-penetrance variants in protein-coding diseases 	copy number variation in human health, disease, and evolution 	the toxicogenomic multiverse: convergent recruitment of proteinsi nto animal venoms 	genetics, medicine, and the plain people wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ doi: . /j.bpj. . . a monday, march , provide valuable insights into its mode of action. the molecular framework of dfbp resembles that of levosimendan, thus it was chosen to mimic levosimen- dan to establish how the ctnc-ctni binding equilibrium is modulated. we have utilized d { h, n} hsqc and d { h, c} hsqc nmr spectroscopy to examine the binding of dfbp to cntnc�ca þ in the absence and presence of ctni - and of ctni - to cntnc�ca þ in the absence and presence of dfbp. the results show that dfbp and ctni - bind cntnc�ca þ concurrently and the affinity of dfbp for cntnc�ca þ is increased ~ - - fold by ctni - . we are in the process of determining the nmr solution structure of cntnc�ca þ�ctni - �dfbp. this structure will contribute to the understanding of the mechanism of action of levosimendan in the therapy of heart disease. it will also provide a structural basis for the design of ca þ- sensitizing drugs in general. -pos board b decreased fatigue tolerance in diaphragm muscle of slow troponin t knockdown mice hanzhong feng, bin wei, j.-p. jin. northshore university healthsystem and northwestern university feinberg school of medicine, evanston, il, usa. the loss of slow skeletal muscle troponin t (tnt) results in a severe type of nemaline myopathy in the amish (anm). the genes encoding tnt and tropo- nin i (tni) are closely linked in pairs in which the -enhancer region of the slow tnt gene overlaps with the cardiac tni gene. in a mouse line with the en- tire cardiac tni gene deleted, a partial destruction of the slow tnt gene pro- moter produces a knockdown effect. by crossing with transgenic mouse lines that over-express a core structure of cardiac tni (ctni-nd) under the control of cloned alpha-mhc promoter, we rescued the postnatal lethality of the car- diac tni gene-deleted mice with no detrimental cardiac phenotypes or leaking expression in non-cardiac tissues. the double transgenic mice exhibited de- creased expression of slow tnt mrna and protein in adult diaphragm muscle. functional analysis of isolated muscle strips showed that the slow tnt deficient (stnt-kd) diaphragm had significantly decreased fatigue tolerance evident by the faster decrease in force and slower rate of recovery as compared with that in wild type controls. as a consequence of slow tnt deficiency, the stnt-kd di- aphragm muscle contained a higher proportion of fast tnt, decreased slow tni with increased fast tni, and decreased type i myosin with increased type ii my- osin. consistent with the switch toward fast myofilament contents, the stnt- kd diaphragm muscle produced higher specific tension in twitch and tetanic contractions as well as shorter time to develop peak tension in twitch contrac- tions. the decreased fatigue tolerance of stnt-kd diaphragm muscle explains the terminal respiratory failure seen in virtually all anm patients and this dou- ble transgenic mouse model provides a useful experimental system to study the pathogenesis and treatment of anm. -pos board b troponin isoforms and stretch-activation of insect flight muscle uros krzic , gian de nicola , vladimir rybin , annalisa pastore , kevin leonard , wolfgang linke , belinda bullard . embl, heidelberg, germany, nimr, london, united kingdom, ebi, cambridge, united kingdom, university of muenster, muenster, germany, department of biology, university of york, united kingdom. oscillatory contraction of insect indirect flight muscle (ifm) is activated by si- nusoidal length changes. work done by oscillating fibres is measured from the area of loops on a length-tension plot. at [ca þ] above mm, progressively less oscillatory work is produced because fibres contract isometrically and are unable to relax fully after each cycle of oscillation. periodic stretches during oscillations activate fibres through the action of tnc f , which binds one ca þ in the c-lobe. activation of isometric contraction by ca þ acts through f , which binds ca þ in both n- and c-lobes. lethocerus ifm fibres substituted with f gave oscillatory work, which did not decline at high [ca þ], while fibres substituted with f produced more isometric tension as [ca þ] was increased. varying proportions of f and f gave maximal work with an f :f ratio of : , which is higher than the in vivo ratio of : . the structure of f , and the interaction with tni, were determined by nmr. the n-lobe of f is in the closed conformation in apo and ca þ- bound forms and does not bind tni. unexpectedly, the c-lobe is open in both states, and binds the n-terminal domain of tni independently of ca þ. the affinity of f and f for a complex containing tropomyosin, tnt and tnh (lethocerus tni) were measured by isothermal calorimetry in the presence of ca þ. the affinities of f and f for the complex were . mm and nm respectively. this difference is likely to be due to a single tni binding site on f and two sites on f . stretch may be sensed by an extended c-terminal domain of tnh, and transmitted to the c-lobe of f , resulting in a change in the interac- tion of the tni inhibitory domain and actin. -pos board b tracking of qdot conjugated titin antibodies in single myofibril stretch experiments reveals ig-domain unfolding at physiological sarcomere lengths anika grützner , pallav kosuri , julio m. fernandez , wolfgang a. linke . university of münster, münster, germany, columbia university, new york, ny, usa. the mechanical characteristics of titin in muscle sarcomeres were previously studied by us in single myofibril stretch experiments, where the extensibility of i-band titin segments was usually measured under static conditions. here we investigated the behavior of i-band titin during and after stretch of single rabbit psoas myofibrils in real-time. the focus was on titin’s proximal ig-do- main region, whose stretch dynamics were analyzed by labeling the myofibrils specifically in the n a-titin domain using antibody-conjugated quantum dots, which stained the periphery of the myofibril but did not enter the myofilament lattice. qdot labels were tracked to obtain the stretch-dependent change in epi- tope distance (across z-disc) and sarcomere length (sl) over time. in contrast to what was expected from the current titin extensibility model, at sarcomere lengths of . and . mm, titin’s proximal ig-domain region elongated contin- uously, in proportion to the half i-band length. already at ~ . mm sl the prox- imal ig-segment length exceeded the value expected if all ig-domains remain folded. our results suggest that ig-domains unfold in parallel with pevk-titin extension at physiological sarcomere lengths and under relatively low forces. by reducing the antibody-qdot concentration, we succeeded in observing titin ig-domain dynamics in myofibrils at the single-molecule level. -pos board b constitutive phosphorylation of cardiac myosin binding protein-c increases the probability of myosin cross-bridge interaction with actin brett a. colson , tanya bekyarova , matthew r. locher , carl w. tong , daniel p. fitzsimons , patricia a. powers , thomas c. irving , richard l. moss . university of wisconsin medical school, madison, wi, usa, illinois institute of technology, chicago, il, usa. protein kinase a-mediated (pka) phosphorylation of cardiac myosin binding protein-c (cmybp-c) accelerates the kinetics of cross-bridge cycling and ap- pears to relieve the tether-like constraint of myosin heads imposed by cmybp-c (colson et al., , circ res., : - ). we favor a mechanism in which phosphorylation of the pka sites in cmybp-c modulates cross- bridge kinetics by regulating the proximity and interaction of myosin with actin. to test this idea, we used synchrotron low-angle x-ray diffraction and mechanical measurements in skinned myocardium isolated from a mouse model with phosphomimetic substitutions in cmybp-c, i.e., the ctsd mouse. the substitutions were introduced by transgenic expression of cmybp-c with ser-to-asp mutations on a cmybp-c null background. western blots showed that expression of ctsd cmybp-c was % of wild-type (wt), and the heart weight to body weight ratio was similar ( . . mg/g) in ctsd and wt mice. expression of wt cmybp-c on the knockout background served as con- trol (i.e., the ctwt mouse). skinned myocardium from ctsd and ctwt mice exhibited similar maximum active forces (mn/mm : . . vs . . ), ca þ-sensitivities of force (pca : . . vs . . ), and maximum rates of force development (ktr, sec - : . . vs . . ; kdf, sec - : . . vs . . ). i /i intensity ratios and d lattice spacings determined from equatorial reflections from ctsd and ctwt myocardium were used to determine the effect of constitutive cmybp-c phosphorylation on the distribution of cross-bridge mass between the thick and thin filaments and on interfilament lattice spacing. the results suggest that interactions between cmybp-c and the s domain of myosin heavy chain are dynamically regulated by phosphorylations in the cmybp-c motif. (aha-predoctoral fellowship (bac); nih-hl-r - ) -pos board b obscurin interacts with a novel isoform of myosin binding protein c-slow to regulate the assembly of thick filaments maegen a. borzok, rebecca hu, amber l. bowman, john strong, robert j. bloch, aikaterini kontrogianni-konstantopoulos. university of maryland, baltimore, md, usa. obscurin is a multidomain protein composed of adhesion and signaling do- mains that plays key roles in the organization of contractile and membrane structures in striated muscles. we used adenoviral-mediated gene transfer to overexpress its extreme nh -terminus in developing myofibers, followed by immunofluorescence and ultrastructural methods to study its effects in sarco- merogenesis. we found that overexpression of obscurin’s second immunoglob- ulin domain (ig ) inhibits the assembly of a- and m-bands, but not z-disks and 	decreased fatigue tolerance in diaphragm muscle of slow troponin t knockdown mice 	troponin isoforms and stretch-activation of insect flight muscle 	tracking of qdot conjugated titin antibodies in single myofibril stretch experiments reveals ig-domain unfolding at physiological sarcomere lengths 	constitutive phosphorylation of cardiac myosin binding protein-c increases the probability of myosin cross-bridge interaction with actin 	obscurin interacts with a novel isoform of myosin binding protein c-slow to regulate the assembly of thick filaments book reviews horst penner, weltweite bruderschaft. ein mennonitisches geschichtsbuch. ueberarbeitet von horst gerlach u n d horst quir- ing ( weierhof, verlag mennonitischer geschichtsverein, ); hardcover, pp. horst penner's weltweite bruderschaft, written to provide a survey of mennonite history for german readers, is an update of the book which was first published in , and subsequently reprinted in and . although the scope of the book is world-wide mennonitism, the amount of space allocated to the various areas is quite uneven. the sixteenth century anabaptist-mennonite movements are covered in considerable detail, as are also the danzig-west prussian and russian mennonite experiences. the usa and canadian mennonite surveys are quite brief, the latin american mennonite history is very sketchy, and only a few pages are devoted to the one-third of the world's mennonites who do not originate in europe. a third of the book consists of general regional surveys of mennonite history since . this section is written by horst gerlach, and includes only those mennonites who originated in europe. the strength of the book lies in the ability of the writers to place the european anabaptist-mennonite history into its historical and geo- graphical context. the writers' detailed knowledge of the history of the various german states, their familiarity with the terrain, trade routes and boundary changes allow them to portray the mennonite story within its context in a way that many other writers are unable to do. the authors have also included numerous maps which aid the reader to follow the discussion. the liberal use of pictures is also helpful for the reader, although the selection of the pictures is uneven and does not illustrate the various sections equally. woven into the copy are numerous and frequently quite long quota- tions from letters and other source materials. some of them duminate the topics well, others, however, illustrate only one side of an issue and may help to confuse the reader. a further difficulty with the quotations is that no footnotes are attached, and it is thus not possible to go to the entire documents from which the selections are made. the fact that there are no journal of mennonite studies vol. , journal of mennonite studies footnotes in the book is generally a weakness. the authors have included a brief bibliography of articles and books for each section in the book, but this does not substitute for documentation. the selection of articles and books is also quite dated. for the west prussian and russian sections no studies after are listed, and for north america no work after the edition of c. henry smith's die geschichte der mennoniten europas is included. it is hard to understand why recent anabaptist mennonite scholarship has not been included in the content or in the bibliographies in this book. the factual content of the non-european sections also leaves some- thing to be desired. in discussing the immigration of mennonites from russia into manitoba, the book indicates that the immigrants primarily came from the old colony in russia. this ignores the fact that they came from four settlements in russia: bergthal, fuerstenland, borosenko and the old colony. this error creates problems in dealing with the emigra- tion from canada to latin america in the s. the authors say that all the people emigrating were old colonists, some of whom moved to mexico and some to paraguay. in the s three groups actually em- igrated from manitoba to latin america, old colonists to mexico, sorn- merfelder to mexico and paraguay, and chortitza to paraguay. in the emigration to mexico, only the settlements near cuauhtemoc are men- tioned. the settlement in the mexican state of durango is omitted. in manitoba the conflict over control of the schools is identified as a conflict between mennonites and the canadian government. in actual fact educa- tion is the responsibility of the provinces, and the conflict was between mennonites and the provincial government. coming back to the emigration of mennonites from russia to man- itoba in , the authors say mennonites landed in the usa, whereas they actually landed in quebec. they also state that the russian men- nonites stayed over winter among fellow mennonites in pennsylvania and settled in manitoba in the spring in , whereas in fact they proceeded directly from quebec to manitoba in , arriving in august. this is a sample of factual inaccuracies from only one small section and suggests that closer attention should have been given to detail especially in those sections outside the expertise of the authors. the authors overplay' the degree of identification between men- nonites throughout the world and the german culture and german nationalism. at numerous points, e.g. in the manitoba section, the conflict between mennonites and their host society is portrayed as a conflict to maintain the german language and german culture. although it is true that statements to this effect can be found, a closer analysis will reveal that especially for the more conservative branches of the men- nonite community in russia and in the americas, the stronger motivation was separation from nationalism and from what they called the "world." ~ o o k reviews retention of german language was more frequently due to unwillingness or even inability to translate their religious concepts into another lan- guage, rather that the desire to maintain a german national and cultural identity. the area in which the adoption of german culture and german nationalism becomes an important issue is in the history of the danzig- west prussian mennonite churches. here too, questions need to be raised about the emphasis. the identification of the danzig-west prus- sian mennonites with german culture and national ideals are presented as having always been the case. in actual fact the earliest mennonites living in that area were mainly from the netherlands and lived under polish rule. the temptation to become part of the german ethos was resisted for centuries. the point of resistance was the insistence of ex- emption from military service. it was not until the order in council of and the franco prussian war when prussian mennonites accepted military service that mennonite identification with the german cultural and national destiny became pronounced. this process of identification found its high point in the era of national socialism. this long process of ever greater identification is not clearly enough presented as a process in which the original attitudes were transformed. some of the interpretations in the book are difficult to square with the facts. for example, the authors seem to want to distance the sixteenth century anabaptist movement from the chiliasm of hans hut and the chiliasm which resulted in muenster. in the section of south and central german anabaptist leaders hut does not appear, despite the fact that he was one of the most active and successful anabaptist missionaries in the south-central german states. regarding muenster, the argument is made that the teachings of hofmann could not have been responsible for muenster. maybe in detail muenster differed from hofmann, but the chiliastic approach was certainly similar. another interpretation which raises questions is the attempt to show a positive relationship between anabaptists and jews. the exam- ples which are cited of anabaptists relating positively to jews in the sixteenth century, must certainly be balanced by the opposite, namely the anti-jewish sentiment among at least some early anabaptists in strassburg and balthasar hubmaier's participation in anti-jewish cam- paigns. the sections which deal with the era of national socialism in ger- many include numerous references to a history which is clearly painful for german mennonites. the war against the netherlands and the treat- ment of the dutch, as well as the conquest of poland and the fact that mennonites in poland fought against mennonites in germany are diffi- cult aspects of german history. in the sections about danzig-west prussia most of the attention is, however, focussed on the human pain and journal o f mennonite studies suffering that resulted at the end of the war. it is true that the suffering and eventual loss of the homeland is a story of almost unbearable anguish. yet, one would also have hoped for more critical analyses of the relation- ship of mennonites to nationalism, to national socialism, and to its anti- jewish policies and programmes. on the basis of the discussions of the historical mennonite emphasis on peace and non-resistance in the earlier sections of the book, some critical analyses of mennonite life under national socialism could have been made. such analyses would have been helpful for german mennonites in their attempt to understand and come to terms with their painful past. neither stout defense of men- nonite actions under national socialism, nor harsh condemnation are likely to bring about this understanding and eventual healing. while there are aspects of the book which are helpful, it is question- able whether the book will adequately enable german mennonites and returnees from russia, as the writers claim, to discover the roots of their past. john friesen canadian mennonite bible college c. arnold snyder, the life and thought o f michael sattler. studies i n anabaptist a n d mennonite history, n o . (scottdalel kitchener: herald press, ). hardcover, pp. arnold snyder's book on michael sattler is most important in that it either revises many hitherto held views concerning anabaptist begin- nings or confirms the work of such historians as walter klaassen, hans- jiirgen goertz, james stayer and werner packull. snyder's revision pro- ceeds from a careful rereading of the available sources surrounding sattler's life and thought and particularly from his incisive analysis of the social-religious milieu of south-western germany and switzerland. as prior of st. peter's, a benedictine monastery in the black forest, sattler came in touch with the peasants who favoured the reformation, but who interpreted the gospel in terms of "economic justice and christian equal- ity" (p. ). opposing the entrenched catholicism of nearby freiburg and the "sola fide" of martin luther, the peasants confronted st. peter's with their demands for economic and social reforms. snyder shows that anabaptists from the waldshut region were among the rebelling peas- ants who occupied the monastery in the spring of . snyder argues convincingly that when sattler decided to leave the monastery and join the waldshut anabaptists he was in agreement with ~ o o k reviews the anticlericalism of the peasants and sympathized wit them and the anabaptists in their reform drives. in fact, according to snyder, both sattler and the early swiss brethren at first hoped to extend their reforma- tion to society at large, similar to the intentions of the mainline reformers. the idea of a separate community of believers, as expressed in the schleitheim articles of , came to sattler and the swiss brethren after the defeat of the peasants and their failure to convince the magisterial reformers and the civil authorities of their brand of christianity. the important schleitheim articles reflect the new sectarian view of the church and sattler's monastic background. such points and empha- ses as withdrawal from a sinful world, rejection of the sword and the oath, and imitating christ in daily living, derived from the monastic tradition. according to snyder, sattler's other writings emphasize man's cooperation with god's grace in salvation and the centrality of christ, which is also more catholic than protestant. the influence of the peasants on sattler is seen in his application of the gospel to all areas of life. sattler rejected, however, the peasants' violence in their attempts to right their situation, accepting instead the nonresistance of the swiss anabaptists. while the monastic tradition and the demands of the common man had a profound influence on the life and thought of sattler, the genius of the young reformer was to put his stamp on a movement which was "neither protestant, nor catholic, nor monastic: [but] anabaptist" (p. ). snyder is one of the first scholars to show clearly that it was not grebel, mantz and blaurock who in the end determined the direction of early anabaptism, but michael sattler. in modifying the biblicism of the grebel group by emphasizing the role of the holy spirit and by clearly separating the church from the world - as reflected in the schleitheim union - sattler gave direction to south-european and dutch anabaptism for centuries to come. historically sattler thus emerges as a more important early anabaptist leader than any of the swiss brethren, including conrad grebel. there are at least two questions with regard to snyder's interpreta- tion of anabaptist origins that need to be raised. first, if sattler stood somewhere in between the biblicism of the swiss anabaptists and the spiritualism of hans denck, as snyder claims, why is this not more clearly reflected in sattler's writings, particularly in the schleitheim arti- cles? snyder obviously favours deppermann's view that at schleitheim the anabaptists tried to distance themselves from those of their brethren who followed denck and questions yoder's view that the "false brothers," mentioned in the articles, refer to the strasbourg reformers bucer and capito. it seems that snyder has not quite resolved the question of sattler's emphasis on the work of the holy spirit and his harsh language against the spiritualistic "false brothers." secondly, if sattler favoured the demands of the peasants with lournal o f mennonite studies regard to social and economic reform, should this not have been more fully reflected in his writings, especially in the programmatic schleitheim articles? it seems that there is little, if any, of thomas muntzer's spirit in the articles. on the other hand, the articles do express a repudiation of all violent force, advocate a total withdrawal from worldly society, and to a large extent internalize the faith and life of believers. sattler's with- drawal theology thus not only renounces all carnal force but also seems to dispair of affecting much needed change in society. the book includes a foreword by cornelius j. dyck, a map of the lands in which sattler lived and ministered, copious end notes, an up-to- date bibliography of sources and secondary works, and a useful index of names and places. the book is handsomely bound and relatively free of misprints. harry loewen university of winnipeg anne chislett, quiet in the land (toronto: coach h o u s e press, ); pages, paperback. $ . . quiet in the land is a well-constructed and beautifully written play by the canadian writer anne chislett. this drama was initially written for the blyth summer festival (ontario). its first production was so successful that it was revived the following year for a longer run and has since then been performed in many important centres. in it won the coveted chalmers award for the best play presented in toronto. chislett's drama about an amish community addresses a number of important issues. on the one hand it explores the age-old predicaments associated with the generation conflict. at the same time the problems experienced in following the ideals of non-resistance, of aloofness from worldly things and the perennial attempts of religious groups to keep out corrupting influences by the erection of moral and social "fences", figure prominently. while an investigation of these problems is hardly in itself a novel undertaking, chislett does manage to use the vehicle of her play to force us to take a hard new look at them and to become personally involved. the play explores the predicament of a small amish community in southwestern ontario during the final war years - . the author shows us how the conscription issue stirs u p the latent hostilities of the outside world and, at the same time, brings to a head the problems already smoldering within the amish group itself. the older members of the community cling obstinately to the established customs and beliefs, whereas the younger generation wishes to bring about enlightened ~ o o k reviews change, see genuine attempts made to answer their many troubling questions and honest, open discussion of common problems. as the play opens eighteen-year-old menno and katie are being admitted to full membership in the church. yock (jacob) bauman, who is the same age or even a little older, on the other hand, has been steadily refusing to seek baptism despite intense pressure from his orthodox father christy (christian) because of deeply-felt doubts. while his friend menno becomes a member of the church and, with visiting bishop ely's permission, starts a sunday school for the young people where they can openly discuss their problems, yock remains an outsider, torn within himself and in daily conflict with his father. this conflict is hightened when christy is elected bishop and puts further pressure on yock to conform without asking questions. the alienation finally leads to a public confrontation when yock defiantly tells his father in the presence of fellow church members that he for one never asked christ to die for him in the first place and is physically chastized by his outraged father. although yock has come to love katie, who returns his love, and has previously made plans to marry her and settle down in the community, he now despairs and runs away to join the army as a soldier. not having heard from yock for a long time, katie submits to community pressure and marries the upright and progressive menno. yock's father christy has become an alcoholic and submits his flock to a tyrannical fanaticism which alienates even his best friends. when yock returns from the war he has become a "hun-killing" hero to the outside world but to his own people he is nothing but a common murderer. however, yock has learned his lesson over the dead body of a german soldier he killed and is now a pacifist by conviction. when he remorsefully seeks his father's forgive- ness he is rejected selfrighteously and not permitted to return to the fold. as the play ends yock has to leave once more and we see a lonely, worn- out christy who has turned away his only son and his community. his only triumph now can be the fact that his son has finally admitted that his father has been right all along. chislett's staging is very straightforward and proves most effective. the homes of the brubachers and the baumans and their yards are visible side by side. actions often take place simultaneously in both places and throw an interesting light on one another. this technique also serves to underline the community aspect of the plot. the language of the charac- ters is simple and borrows heavily from scripture. yet, this same simple language can turn quite poetic as well, particularly in such emotionally charged scenes as the love encounter between yock and katie and the final confrontation between yock and christy. the characters come across as very real people and keep us intimately involved with their problems. the audience is permitted to become part of the small community por- trayed. journal o f memonite studies while the play ably creates the atmosphere of a small community, its most impressive aspect is that the author manages to go beyond the immediate problems of a particular amish group whose spiritual fences threaten to collapse. the disturbing questions raised by the soul-search- ing of the characters are of great concern to us today as well. the interper- sonal conflicts shown are those of any tightly knit group with a common goal. we too are made to see, along with the characters, that material and technological progress, so often sought with a plea for personal freedom, can indeed become pandora's box. the book is a well-made paperback. it sports a most attractive cover and is printed on high quality paper. excellent black and white photo- graphs showing us the characters as they were portrayed in the original production stimulate the reader's imagination. an introduction and pro- duction notes by the author give the reader and potential director the necessary tools to a full understanding of the play. quietin the land does not provide any new answers but it is sure to generate a lot of discussion. this excellent play is not only to be recom- mended to those who are deeply concerned with the issues raised but also to all who enjoy good theatre. andre oberle university of winnipeg patrick friesen, unearthly horses (winnipeg: t u r n s t o n e press, ); paperback, pp. unearthly horses is the fourth volume of patrick friesen's poems to be published by turnstone press. the previous collections, the lands i am ( ), bluebottle ( ) and the shunning ( ), all include poems in which friesen deals with his mennonite past. in this volume friesen takes a more personal approach to this ethnic theme than he did in the shunningand develops it in a more mature, more thoughtful way than he did in the first two books. in unearthly horses friesen charts his own highly individualistic spiritual pilgrimage. it is also the archetypal jour- ney from eden, or the garden-like world of childhood, to the fallen adult world and, finally, to a state that both combines and transcends the previous two. this final state is achieved by coming to terms with his own human limitations, his mortality. ~ o o k reviews the first state is preserved in a series of word photographs, often focusing on his mother: in the garden leaning on.her hoe pushing back strands of hair her eyes lock on mine where i lie on my stomach between rows of raspberry bushes this is how i was chosen this innocent world occasionally includes his father: in the garden pa making straight lines with string scooping a shallow trench and dropping seeds he waters it from a can stands u p satisfied turning when the back door opens he smiles watching ma walk toward him their arms lock around each other's waists he swings her off her feet she gasps and he sets her down turning to where i stand in twilight she sings 'the sandman look the sandman's here' it is a world of trees and grass and summer foliage: ma's voice floating in july air across lawns past ' s bungalows along a dirt road through knee-deep grass spreading among poplar leaves where i sit at the top above homes seeing beyond the outskirts of town to grandfather's farm the second world, a fallen state of consciousness, is most vividly portrayed in the "pa poems." the stern yet loving father is reminiscent of old testament justice and new testament grace. in "pa poem " the poet recalls an encounter with his father that has profound religious con- notations: and there we were in front of the furnace me pleading across your knee both of us wishing we were someplace else but you not spoiling the child and you swung that leather high journal o f mennonite studies me twisting to look u p your arm flung out seeing you naked and nailed like a child to a tree how could there be so much love? the second state is one of suffering and death. in the suffering and death of his father the poet perceives his own mortality but, at the same time, he also finds his place in the eternal scheme of things. "pa poem " describes the night following his father's death: after i stood the night through in pa's basement writing his obituary as if it was mine handed it to the minister watched him stroke out 'died' and scribble in 'passed away' as if there was no end to it even for me the third state, though hinted at in some of the "pa poems," is anticipated more strongly in the three easter poems. it is a state in which the poet finds himself out of tune with the "solemn surrender" of his ancestors, "that certainty of theirs" ("easter morning "), their pious attitude which excludes and denies all those who do not adhere to a particular creed. the poet celebrates instead the broader, mythical im- plications of the event. "easter morning " concludes as follows: the sky is faultlessly blue on easter morning the dove has flown and father calls me for church i jump the shattered ice and water each death gives life ,to me i want to commemorate this morning this nostalgia for the momentary man in his pain in the name of the father for his bereavement in the name of the son and the ghost this broader, mythical interpretation of the resurrection can also be seen in the poem "easter": i can saj that my birthday each summer is here in this field ~ o o k reviervs that i see how the world turns here dawn to dusk and this is it how the world happens for me this is the place i am here wherever i am i am here for the poet, easter symbolizes the constant cycle of death and renewal within the life-span of the individual, a miracle in the here and now. its celebration leads to a celebration of life in general and ushers in a redeemed state of forgiveness and love. however, this state is not as idyllic as the garden of childhood. it is one in which the fallen and the unfallen, good and evil, must coexist, illustrated by conflicting images such as lions and lambs, shadows and sunlight, winter and summer, grey hydro lines and lilacs. the poem "celebration," addressed to his father, is the poet's acceptance of this truth: carol's reading a book on bulbs pa this spring she planted offshoots of your old glads i'm reading a book of saints last night on television i saw marshal zhukov sending infantry into minefields to save his armour to authenticate his resolve an image of seven peasants dangling from a gallows almost off-camera a german officer with frost on his breath stamps his rag-wound feet at this level of consciousness the poet celebrates the here and now in spite of its uncertainties and ambiguities. it is a disordered garden but not totally without hope. the poet's as yet innocent children in many ways compensate for the fallen world all around him: we're planning marijke's birthday possibly an afternoon on the beach she'll be nine if you could see her pa you'd be so proud just at that age of long legs and grace you should see her dance and learning about the rest of the world too arab bazaars sombreros and the violin and first inklings that the world's coming down around us is the war coming here she asks marijke's "first inklings" suggest that her innocent world is about to be destroyed too. and so the cyclical process continues. journal o f mennonite studies patrick friesen is always striving to reach an audience beyond the narrowly ethnic. he knows he must root his poetry in his own particular ethnic experience. at the same time, he knows he must also transcend the restrictions of a specific faith or creed. in unearthly horses friesen describes his own personal spiritual journey but it is one that is also universal. peter pauls university of winnipeg wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ mini-clusters of potentially prodromal symptoms may identify psychiatrically well amish children at higher risk of developing bipolar i disorder egeland ja, shaw ja, endicott j, et al. prospective study of prodromal features for bipolarity in well amish children. j am acad child adolesc psychiatry ; : – . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q do frequencies of potential early prodromal clinical features for bipolar i (bpi) disorder in psychiatrically well amishchildren correlate with family history, and therefore inferred risk, of bpi? methods design: prospective cohort study; outcome assessors were blinded as to family history, however, it was impossible to blind data collectors. follow up period: seven years. setting: amish families in rural pennsylvania, usa; to . people: children from families. bpi families ( children) had one parent with bpi. the families of same sex, psychiatrically unaffected siblings of each bipolar parent were selected as matched controls where possible. control siblings were selected to be as close in age, marriage date, and number of children to the bipolar sibling. in the absence of a suitable matched sibling control, the family of a non-related amish participant matched for sex, age, and family size, but without a family history of bpi, was selected. there were sibling control families ( children) and family history negative control families ( children). risk factors: the child and adolescent research evaluation (care) interview was administered annually to parents of participating children. the care interview comprised three parts: part a (yes/no questions) pertained to the pregnancy/delivery, medical, and developmental history of the child; part b was narrative and asked parents whether they had any concerns about their child’s health, wellbeing, or behaviour; part c (yes/ no questions) was used in alternate years and comprised questions about the child being ‘‘noticeably different’’ from other children for potentially prodromal or antecedent items, and about the child’s functioning and ‘‘wellness’’. outcomes: presence of prodromal symptoms and predicted risk of developing bpi disorder as determined by independent, blinded assessors (one psychiatrist, two child psychiatrists, and one clinical psychologist). main results children with one bpi parent were significantly more likely to have or more potentially prodromal symptom/behaviour items than children with well parents (ar: % for bpi family child v % for control family child; p = . ). symptoms significantly more frequent in children with one bpi parent than in children with well parents were: anxiety, poor attention span in school, low energy, excitability, hyper-alertness, mood lability, school role impairment, sensitivity, somatic complaints, and stubbornness (p( . ). assessors identified more of the children with one bpi parent as ‘‘at risk’’ of bpi than children with well parents (ar: % for bpi family child v % for control family child; overall p value for liability class distribution , . ). conclusions mini-clusters of early potential prodromal features occur at higher frequencies in psychiatrically well amish children with one parent with bpi than in those with well parents. notes the authors note that the major limitation of this interim report of their study is that although these findings do identify potential early prodromal features for bpi, the true test of the outcome will be whether children designated as ‘‘at risk’’ do eventually develop bpi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . for correspondence: dr egeland, university of miami north research office, briarcrest square, hershey, pennsylvania, usa; ooamish@aol.com sources of funding: the care (child and adolescent research and evaluation) programme was supported in part by the stanley medical research institute and with funding from the university of miami school of medicine. commentary t his paper describes a prospective study of healthy old-order amish children, designed to determine the pattern of psychiatric symptoms that may be prodromal for bipolar disorder. data are the result of years of annual interviews with families begun in as part of the child and adolescent research evaluation (care) programme, designed for the above purpose. it is a novel study design producing useful data for several reasons. while several studies have collected retrospective data from inpatients on selected prodromal bipolar symptoms or prospective data on such symptoms in children from affected families, none has used a community sample including children of varying levels of risk for bipolar disorder. further, as they have studied an amish group, they have been able to collect multigenerational data in large families useful for genetic studies, within a cultural community with much social stability. these group data on early bipolar risk may not be applicable to a general population, as symptoms of disruptive behaviour disorders—common in bipolar children—are possibly mitigated by cultural influences. however, this study has allowed the egeland team to describe a spectrum of prodromal symptoms that may be specific for bipolar disorder and prove to be of relevance to clinical practice. of clinical importance, they found that children with bipolar parents have high risk of developing bipolar disorder— %, similar to rates cited in other risk studies. children of unaffected siblings of a bipolar adult had intermediate risk compared with control children, suggesting reduced penetrance of bipolar symptomatology in families. symptoms of children of a bipolar parent, which are significantly more common than in children of non-bipolar parents, are described. it is suggested that some prodromal symptoms for bipolar disorder are episodic in % of the children, such as mood and energy changes, need for sleep, and anger/ temper outbursts. these findings will be helpful for clinicians assessing bipolar risk in children with subsyndromal symptoms combined with known or suspected family history of the illness. khrista boylan, md department of psychiatry and behavioural neurosciences, mcmaster university, hamilton, ontario, canada aetiology www.ebmentalhealth.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://e b m h .b m j.co m / e vid b a se d m e n ta l h e a lth : first p u b lish e d a s . /e b m h . . . o n f e b ru a ry . d o w n lo a d e d fro m http://ebmh.bmj.com/ introduction and comments introduction and comments jennifer l. hochschild this is my final introduction as editor of perspectives on politics, and i’ll conclude with a few thanks and hopes. but my main task here is to introduce the articles in this issue. they cluster around two themes—leadership and dilemmas of action (those themes are, of course, inti- mately related). it does not take much discernment to find the first cluster; three articles have “leadership,” an american president’s name, or both in the title. nannerl keohane has been a political theory professor, college and univer- sity president, and member of boards of trustees, and she is returning to the professoriate. she is therefore well suited to channel niccolò machiavelli and to update the prince. keohane concurs with some of machiavelli’s precepts: a leader must be willing to accept distress among followers in pursuit of a worthy goal; a leader should use power rather than shirk from it, even at some personal sacrifice. but she is not a sixteenth century courtier: keohane insists on inclusion of those previously left out, and she enter- tains the possibility that one should remain a member of virginia woolf ’s society of outsiders. i discovered woolf ’s three guineas in the company of nan keohane many years ago; it has shaped much of my teaching and been a moral beacon (as well as being one of the funniest books i have ever read). so it has been a pleasure to help shepherd into print keohane’s most recent thoughts about this book. stephen skowronek reflects on leadership from the out- side rather than the inside, in “leadership by definition: george w. bush and the politics of orthodox innova- tion.” but he is no less analytic, and his ability to place president bush’s style of governance in a long historical context gives us a great deal of leverage on understanding such a politically controversial figure. skowronek’s con- cept of leadership by definition explains much of what seem baffling about bush to skeptics, such as his insis- tence on maintaining a stance once chosen rather than learning from new evidence—an insistence especially dif- ficult to accept for those of us who earn a living by teach- ing and persuading. leadership by definition also explains much of bush’s impressive political and policy success, and puts the definition-shattering events of september , in stark relief. i predict that historians will one day point to this article as the most prescient of all those written during the bush presidency. historians’ judgment of a president and his behavior under stress is a central theme of benjamin kleinerman’s “lincoln’s precedent: executive power and the survival of constitutionalism.” did lincoln act outside the consti- tution when he suspended the writ of habeas corpus and jailed people accused of treason during the civil war— or did he act within his constitutional mandate? and if he acted outside the constitution, was he justified in so doing? when are other presidents justified in taking extraordi- nary, perhaps extra-constitutional, measures to address national crises? in tackling these questions, kleinerman adduces several lessons from lincoln’s speeches and action. most importantly, lincoln claimed that political neces- sity—not popular approval or constitutional mandate— legitimated his actions; that claim sets a standard that is correct in kleinerman’s view, but is extraordinarily diffi- cult for both leaders and their followers to live up to. leaders face dilemmas, and our judgment of their lead- ership largely depends on how effectively they resolve (or escape) them. but non-leaders also face quandaries, and the rest of the articles in this issue of perspectives analyze an array of dilemmas in widely dispersed contexts. michael barnett, in “humanitarianism transformed,” dissects the vexed relationship between political action and would-be apolitical assistance to individuals in desperate straits. in theory, humanitarians ignore wars, factions, ideologies, and power plays in their efforts to feed the starving and care for the ill. but increasingly in practice, humanitarian organizations are drawn into political conflicts, whether because they are co-opted by states or nonstate actors or because they seek a way to eliminate human rights viola- tions rather than endlessly alleviating them. humanitar- ian organizations are also becoming more, well, organized as they grow. barnett lays out the many defects and occa- sional virtues of this seemingly inexorable politicization of aid, and calls for new ways to help preserve humanitarian organizations from drowning in standard operating pro- cedures and lobbying. janice stein’s commentary on barnett’s article, entitled “humanitarianism as political fusion” is a little more san- editor’s note december | vol. /no. https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core guine about this dilemma. she interprets institutionaliza- tion and standardization as a process of maturing—and those of us of a certain age certainly hope that maturation includes benefits as well as costs. politicization provides opportunities as well as constraints, largely because it makes explicit what has been implicitly the case all along. there is no neutral, apolitical stance, stein argues, and by com- ing to grips with that fact, humanitarian organizations can do their job more effectively and creatively. stein does not, however, have many encouraging words about the increasing bureaucratization of aid agencies; under some conditions, “the demand for accountability is profoundly corrupting.” this dilemma is not easily resolved. nor is that of individuals deciding whether to remain in an all-encompassing community in which they have been raised. in “consenting adults? amish ‘rumspringa’ and the quandary of exit in liberalism,” steven mazie considers the situation of teen-aged members of the amish society after a rumspringa—a mandated year of loose liv- ing in secular america. the amish insist that people com- mit to join the church after experiencing alternatives; theirs is a genuine social contract. but is it really? asks mazie. he uses this unusual case to raise larger questions about the relationship between illiberal groups and the would-be liberal society in which they are located. roughly speak- ing, liberal political theorists promote either autonomy for such groups, on the condition that people may freely exit from them, or a requirement of some tolerance within illiberal groups. but in mazie’s view, the amish case shows that both sets of liberal theorists have made it too easy on themselves—genuine freedom of exit is much more diffi- cult than the former imagine, and real liberalism within groups would require excessive intervention. like barnett, mazie does more to lay out than to resolve the dilemma discussed in his article, but it is a wonderful demonstration of how a particular case can disrupt a large theory. vivien schmidt paints on a larger canvas than either barnett or mazie; her field is the continent of europe. in “democracy in europe: the impact of european integra- tion,” she considers the tensions facing democratic euro- pean governments as they seek to enforce mandates of the european union (eu), respond to the needs and demands of national constituents, and negotiate with other govern- ments facing the same tensions. she parses this array of difficulties by dividing states into simple and compound democracies; the former have relatively tight governance structures with a single accountable authority, and the latter have loose governance structures with many loci of political engagement such as states or multiple branches of government. the eu is compound, so its institutional fit is more disruptive to simple than to compound mem- ber states. however, the eu’s messages are more difficult to diffuse in compound than in simple provinces. no insti- tutional fit is ideal. but the biggest problem in the eu (as demonstrated by the recent rejection of its constitution by voters in france and the netherlands) is that leaders in all states evade discussion of the genuine disruptions that creation of a supranational government inevitably entails. all europeans (except currently elected officials, perhaps) would benefit from stein’s commitment to make implicit politics explicit, or mazie’s attention to the dynam- ics of group membership— or simply from the sorts of bolder leadership described by keohane, skowronek, or kleinerman. helen marrow returns us to the difficulties facing individuals—in this case new immigrants to the united states who are settling in places where there are few others like them. in “new destinations and immigrant incorpo- ration,” marrow transcends the usual disciplinary divide among sociology, economics, and political science with the simple observation that a given person lives his or her life in all three domains of society, economy, and polity simultaneously. but that is all that is simple about this review of the new literatures on immigrant incorporation. complexities multiply—across nationality or racial groups, among different types of destinations, among forms of political or social engagement, between qualitative and quantitative scholars, and more. marrow is a steady, reli- able guide through this maze; she carefully documents when and how immigrants are able to sort through the dilemmas facing them and move toward successful incor- poration. her main conclusion should be welcome to polit- ical scientists; researchers and activists should focus more on structures and contexts of reception, and perhaps less on the qualities that a given immigrant brings to his or her new home. luckily for us, the immigrants are cooperating with this research agenda by moving to many different locations, which elegantly sets the scene for comparative research. in a review essay about several important new books, helen milner moves back to the global arena, to consider the dilemmas facing international organizations seeking to help developing nations. in “globalization, develop- ment, and the role of international institutions,” milner asks whether the world bank, international monetary fund, and similar operations really benefit their supposed beneficiaries. that is, of course, their mandate and pre- sumably a sincere commitment of the workers within the organizations. but just as humanitarian organizations get caught up in politics and organizational straitjackets, so banking organizations become caught up in the norms and niceties of banks—perhaps to the detriment of the people and nations who they aim to assist. milner works through the arguments in the books that she reviews, and then contributes her own resolution to this dilemma, including importantly an analysis of the normative issues surrounding nongovernmental organizations involved in international development. in his book review essay, george thomas takes a rather different tack, with not much attention to either leader- editor’s note | introduction and comments perspectives on politics https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core ship or dilemmas of governance and action. instead, in “the qualitative foundations of political science: mov- ing beyond kkv,” he reminds us of another mission of perspectives on politics. rather than using political science to address issues in the real world, he uses political sci- ence to address issues within the discipline. typically, the standard for excellent scholarship in our discipline is taken to be the scientific rigor exemplified by the best research analyzing a large number of discrete variables. qualitative research with purportedly less rigor and fewer variables (both terms as understood through quantitative lenses) is thought to be best suited for generating hypotheses, describ- ing how variables operate, or providing striking examples of a relationship that “science” has demonstrated. thomas stands this whole logic on its head. in his review of several recent books on qualitative methodology, he argues that qualitative logic and research provide the foundation on which “scientific”— or at least quantitative—work neces- sarily builds. and more than just being the starting point, qualitative research, according to thomas and most of the authors he reviews, provides more of the genuine infor- mation and insight that political scientists seek. king, keo- hane, and verba (“kkv”) wonderfully provoked an argument, and thomas and the books he discusses have eagerly taken up the gauntlet; readers and the next gener- ation of scholars will judge the tournament. as always, we have a full complement of informative book reviews; perhaps this is the moment to confess that i always turn to them before rereading articles in the front half of perspectives. i want to take this chance to thank greg mcavoy (and earlier in my term as editor, susan bickford), for their wonderful leadership in resolving the dilemmas of being a book review editor. they were a plea- sure to work with—fun, professional, responsible, cre- ative. i, like other authors and aspiring authors, look forward eagerly to the book review editorship of jeffrey isaac of indiana university. he has a high standard to meet, and will undoubtedly do so. i want also to thank the other people without whom my editorship, and the journal itself, could not have hap- pened. we had in succession three excellent managing editors, lisa burrell, kevin mckenna, and thomas koza- chek, who all did a great deal to begin and constantly improve this complex operation. the ph.d. student assis- tants to the editor were also invaluable—and if any read- ers are seeking junior colleagues, they are now expert researchers and evaluators! they were, in alphabetical order, michael fortner, brian glenn, melissa kayongo, daniel kenney, jason lakin, eric lomazoff, anna nelson, and meg rithmire. dan was there from beginning to end (and is still working, as i write). the apsa staff in washington did their usual heroic job with their usual efficiency and good humor. editors at cambridge university press, espe- cially mark zadrozny and ed barnas, provided essential expertise. and i want especially to thank the five associate editors—henry brady, william galston, atul kohli, paula mcclain, and jack snyder—with whom it was an honor and a deep pleasure to work. despite their overloaded schedules and their own agendas, they gave a great deal of time and care to the editorial mission. we went through some difficult moments–more than once, i thought about the painted signs on old barns urging drivers-by to “impeach earl warren”—but never with each other. and they never wavered in their commitment to excellent schol- arship, political science, and perspectives on politics. i may write a more extensive reflection on what i learned during the past very full three and a half years, so i will conclude here with just two thoughts. first, there is a lot of talent, good cheer, graciousness, and hard work in our profession, and i have been privileged to see and learn from it. second, and more grouchily, we risk selling our- selves short by settling too often for poor writing, under- developed arguments, inattention to questions of “so what?”, and unwillingness to take seriously the views of people who disagree with our position. perspectives on pol- itics exists to take advantage of the former and contest the latter. i have loved my time as editor, but am now happily immersed in wrestling with my own writing demons. per- spectives has a fine new editor, james johnson of univer- sity of rochester, and “now sits expectation in the air” (chorus, henry v ). december | vol. /no. https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ health needs assessment of five pennsylvania plain populations international journal of environmental research and public health article health needs assessment of five pennsylvania plain populations kirk miller ,* , berwood yost , christina abbott , scottie thompson buckland , emily dlugi , zachary adams , varun rajagopalan , meryl schulman , kimberly hilfrank and mara a. cohen department of biology, franklin & marshall college, harrisburg ave., lancaster, pa , usa floyd institute center for opinion research, franklin & marshall college, lancaster, pa , usa department of psychology, franklin & marshall college, lancaster, pa , usa johns hopkins hospital, baltimore, md , usa u.s. news & world report, washington, dc , usa ernst and young advisory services, new york, ny , usa center for health care strategies, trenton, nj , usa school of public health, the university of michigan, ann arbor, mi , usa * correspondence: kirk.miller@fandm.edu; tel.: + - - - received: june ; accepted: july ; published: july ���������� ������� abstract: we performed a health needs assessment for five plain communities in pennsylvania from a random sample of households, comparing them to the general population of pennsylvania adults. plain respondents were more likely to drink well water, as likely to eat fruit and vegetables and much more likely to drink raw milk and be exposed to agricultural chemicals. plain respondents were less likely to receive screening exams compared to the general population and there was variation from settlement to settlement in whether respondents had a regular doctor, whether they received preventive screenings or had their children vaccinated, with mifflin county amish generally lowest in these and plain mennonites highest. plain respondents reported good physical and mental health compared to the general population but groffdale mennonite respondents had a high proportion of diagnoses of depression and were more likely to be receiving treatment for a mental health condition. most plain respondents would want a spouse tested for genetic disease with mifflin county amish least in favor of these tests. despite their geographic and genetic isolation, the health of plain communities in pennsylvania is similar to that of other adults in the state. keywords: amish; old order mennonite; health needs; household survey . introduction pennsylvania is home to a number of communities that isolate themselves from the larger society and that are descended from anabaptist sects that migrated to america between about and to avoid religious persecution in europe [ ]. lancaster county, in the southeastern part of the state, is the center of distribution of these amish and mennonite groups that, partly due to shortages of farmland and partly from a desire to isolate themselves, have spread throughout pennsylvania, much of the rest of the united states and canada, mexico, the caribbean and central america [ ]. these groups do not have health care providers of their own because of restrictions on education but they do make use of modern medicine and health care providers from outside their communities. because of their isolation, the health needs of these communities are often overlooked in assessments of large geographic areas in the service region of hospitals. because they do not assimilate, have rigid rules about the use of technology and often do not receive formal education beyond the int. j. environ. res. public health , , ; doi: . /ijerph www.mdpi.com/journal/ijerph http://www.mdpi.com/journal/ijerph http://www.mdpi.com https://orcid.org/ - - - https://orcid.org/ - - - http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /ijerph http://www.mdpi.com/journal/ijerph int. j. environ. res. public health , , of eighth grade, the demographic characteristics of these communities are often very different from those of the larger society. the old order amish and old order mennonite groups that eschew many of the trappings of modern life call themselves “plain.” because these plain groups were all founded by small numbers of families, they carry high numbers of certain disease-causing genes (and small numbers of others). researchers have identified over disease-causing mutations in the plain communities [ ] but life expectancy and infant mortality in these communities are unknown. we have previously reported on a health needs assessment of the amish and plain mennonites of lancaster county [ ]. here we report a health needs assessment of two isolated and relatively conservative pennsylvania amish communities and compare them directly to the amish and mennonites of lancaster county and, when possible, to the general population of pennsylvania. amish families founded a settlement in somerset county, pa, in the western part of the state, perhaps as early as [ ]. while this is the second oldest extant amish community, it is small and, uniquely, sunday services are held in meeting houses rather than homes. members migrated west to found the now very large amish settlements in holmes county, ohio and elkhart and lagrange counties, indiana [ ]. the somerset county settlement has an unusually high carrier frequency of a mutation in mthfr that can lead to severe cerebral atrophy [ ]. amish families settled in mifflin county, pa starting in [ ]. this region, called the big valley, includes at least groups that are amish or descendants of amish groups [ , ]. this settlement has a high prevalence of propionic acidemia, which can lead to seizures and severe heart problems and of a mutation in tspyl that can lead to sudden infant death [ , ]. there is little evidence-based research on plain people’s health, health care and preventive care and behaviors and exposures that affect health. this research provides data on some of these important health indicators. . materials and methods the center for opinion research at franklin & marshall college conducted a random sample household survey by mail between august and may that assessed the health needs of adult old order amish and old order mennonite individuals ( years of age and older) living in five settlements in pennsylvania. the purpose of the survey was to gain an understanding of the current health and health needs of these plain communities, to assess differences between settlements and to measure how their perceptions of modern medicine and technology may be altering the plain way of life. the study was approved by the institutional review board of franklin & marshall college (# approved / / ). . . populations and sample the address book of the lancaster county amish [ ], directory of the groffdale conference mennonite churches [ ], directory of the weaverland conference mennonite churches [ ], old order amish directory of somerset county, pa letard, west virginia [ ], old order amish directory of mifflin and juniata counties [ ] and nebraska amish directory [ ] were used as the sampling frames. these contained the most complete available listing of households in their respective groups. estimated population sizes, sample sizes and response rates are given in table . for the purposes of this analysis the groups of mifflin county amish were combined. . . survey methods and measures survey methods and measures have been described elsewhere [ ] (a copy of the survey is available from the corresponding author). the response rate averaged % and varied from settlement to settlement (table ). we report here the proportion of respondents for each survey question for the five surveyed settlements. where possible, we also report the proportion answering similarly from surveys of int. j. environ. res. public health , , of pennsylvania residents. we compare responses from plain settlements using pearson chi-square or analysis of variance. table . population estimates, sample sizes and response rates for five surveyed plain communities. settlement population sample response lancaster county amish , a % groffdale mennonite b % weaverland mennonite c % mifflin county amish d % somerset county amish e % a young center for anabaptist and pietist studies. available online: http://groups.etown.edu/amishstudies/ (accessed june ). b lancaster county total population kraybill and hurd horse-and-buggy mennonites p. . c adult members landis, ira d. and richard d. thiessen. “weaverland mennonite conference.” global anabaptist mennonite encyclopedia online. october . available online: http://gameo.org/index.php?title=weaverland_ mennonite_conference&oldid= (accessed june ). d kraybill, johnson-weaver and nolt, the amish p. . e yoder, samuel l. “meyersdale-springs old order amish settlement (somerset county, pennsylvania, usa).” global anabaptist mennonite encyclopedia online. . available online: http://gameo.org/index.php?title= meyersdale-springs_old_order_amish_settlement(somerset_county,_pennsylvania,_usa)&oldid= (accessed june ). . results the plain respondents ranged in age from to , mean . years. plain respondents were more likely to be married and to have large families than the general population of adults living in pennsylvania (table ). plain respondents were more likely to live on a farm and to drink water from a private well. table . demographic characteristics of pennsylvania plain communities and the general pennsylvania population. settlement general populationlancaster county amish groffdale mennonite weaverland mennonite mifflin country amish somerset country amish average age of respondent . . . a married % % % % % % b average number of children of respondent . . . . . . c live on a farm % % % % % . % d home built before % % % % % % e drinking water from a private well % % % % % % f a median age of pa residents and older us census . b married pa residents and older pa state data center. c average number of children per pa family with children us census . d pa housing units on farms us census . e pa houses built before american community survey . f private water wells in pa penn state extension private water systems faqs. plain respondents were as likely to eat fruit and vegetables as the general population of adults in pennsylvania (table ) and probably much more likely to drink raw milk. there was variation among settlements in these measures with mifflin county amish less likely to eat fruit and vegetables and weaverland mennonites less likely to drink raw milk. plain respondents were also highly likely to report being exposed to agricultural chemicals. there was considerable variation from settlement to settlement in whether respondents had a regular doctor, whether they availed themselves of preventive screening and whether they had their children vaccinated, with the mifflin county amish generally the lowest in these measures and the plain mennonite groups the highest. plain respondents were generally http://groups.etown.edu/amishstudies/ http://gameo.org/index.php?title=weaverland_mennonite_conference&oldid= http://gameo.org/index.php?title=weaverland_mennonite_conference&oldid= http://gameo.org/index.php?title=meyersdale-springs_old_order_amish_settlement(somerset_county,_pennsylvania,_usa)&oldid= http://gameo.org/index.php?title=meyersdale-springs_old_order_amish_settlement(somerset_county,_pennsylvania,_usa)&oldid= int. j. environ. res. public health , , of less likely to receive screening exams compared to the general population of pennsylvania. plain respondents had a body–mass index (bmi: weight kg/height m ) comparable to the adult population of pennsylvania although there was variation among settlements, somerset county amish having higher and lancaster county amish and groffdale mennonites having lower proportions of overweight and obese bmi. plain respondents almost universally received prenatal care. table . behaviors and exposures of pennsylvania plain communities and the general pennsylvania population. settlement general population pa lancaster county amish groffdale mennonite weaverland mennonite mifflin country amish somerset country amish in a typical week how often do you eat (% responding once a day or more) fruit * % % % % % % a vegetables * % % % % % % a salad * % % % % % raw milk * % % % % % exposed to agricultural chemicals % % % % % has a regular doctor or health professional * % % % % % % b received the following health services in the past months blood cholesterol check * % % % % % % c physical checkup * % % % % % % d blood pressure test * % % % % % test for diabetes * % % % % % % e flu shot * % % % % % % b test for bacterial vaginosis % % % % % dental exam * % % % % % % f pelvic exam * % % % % % pap smear % % % % % % e physical breast exam * % % % % % % g mammogram * % % % % % g prostate exam % % % % % % h if you have children, have they been vaccinated? (% responding yes) % % % % % % i during your or your wife’s last pregnancy, did she visit a midwife or other health professional? (% yes) % % % % % % j bmi (% overweight or obese) * % % % % % % g * significant differences between settlements. a cdc state indicator report on fruits and vegetables . b pa dept. health behavioral health risks of pennsylvania adults. c past years, pa dept. health behavioral health risks of pennsylvania adults. d past years pa dept. health brfss/eddie. e past years pa dept. health brfss/eddie. f pa dept. health healthy people . g pa dept. health brfss/eddie. h psa test pa dept. health brfss/eddie. i fully immunized children - months pa dept. health healthy people . j prenatal care in first trimester pa dept. health healthy people . plain respondents report good physical health and generally fewer diagnosed health conditions compared to the general population of pennsylvania adults (table ). notably, plain groups report fewer diagnoses of asthma and hypertension compared to adults in pennsylvania and many diagnoses of urinary tract infections, vaginal yeast infections and thyroid problems. plain respondents also report fewer diagnoses of obesity and arthritis and fewer pregnancy complications. plain mennonite int. j. environ. res. public health , , of groups generally report more diagnosed conditions compared with amish groups and strikingly higher diagnoses of cancer. about % of plain respondents needed medical care in the past months. of these, the most common answers to questions about why care might be delayed were because of expense ( % of respondents) and because of uncertainty about where to go for care ( % of respondents). table . health and health conditions of pennsylvania plain communities and the general. pennsylvania population. settlement general population pa lancaster county amish groffdale mennonite weaverland mennonite mifflin county amish somerset county amish in general, would you say your health is . . . self-reported health fair or poor % % % % % % a has a doctor ever told you that you have . . . asthma % % % % % % a hypertension * % % % % % % a high cholesterol * % % % % % % a coronary disease % % % % % % a stroke % % % % % % a blood clot % % % % % epilepsy % % % % % copd % % % % % % a obesity % % % % % % a anemia % % % % % anxiety or depression * % % % % % % a arthritis % % % % % % a thyroid problems % % % % % chlamydia % % % % % . % a herpes % % % % % gonorrhea % % % % % . % a syphilis % % % % % . % a diabetes * % % % % % % a cervical cancer % % % % % . % a urinary tract infection * % % % % % endometriosis % % % % % bacterial vaginosis % % % % % vaginal yeast infection % % % % % pelvic inflammatory disease % % % % % pregnancy complications % % % % % . % b cancer * % % % % % . % a * significant differences between settlements. a pa dept. health brfss/eddie. b pa dept. health healthy people . plain respondents generally report better mental health compared with the general population of adults living in pennsylvania. very few scored high on measures of depression and bipolar disorder and many scored very high on measures of life satisfaction and social support (table ). of plain respondents, ( . %) had one or more poor mental health days, compared with % of adults in pennsylvania. groffdale mennonite respondents have a high proportion of diagnoses of depression, have a high number of poor mental health days and are more likely to be receiving treatment for a mental health condition but are also likely to go to a doctor if they felt they had a mental health problem. despite having a high number of poor mental health days, they receive the social support they need and are very satisfied with their life. int. j. environ. res. public health , , of table . mental health and social support in pennsylvania plain communities and the general pennsylvania population. settlement general population pa lancaster county amish groffdale mennonite weaverland mennonite mifflin county amish somerset county amish has a doctor ever told you that you have anxiety, depression or bipolar disorder * % % % % % % a in past weeks have you accomplished less than you wanted because of emotional problems, % often or all the time % % % % one or more poor mental health days % % % % % % a taking medication or receiving treatment for a mental health condition * % % % % % cesd depression score, % scoring – on a scale of – b % % % mood disorder score, % scoring on a scale of – c % % % % % would go to a doctor if felt had a mental problem, % very likely or likely * % % % % % phq- depression scale, % moderate or severe symptoms d % % % % social support how often do you get the social and emotional support you need? % usually or always % % % % % % e how satisfied are you with your life? % satisfied or very satisfied * % % % % % % e % responding often or all the time on measures of social support f % % % % % * significant differences between settlements. a pa dept. health brfss/eddie . b questions from the center for epidemiologic studies depression scale. c. questions from the mood disorder questionnaire screening for bipolar disorder. d personal health questionnaire depression scale. e pa dept. health brfss/eddie . f medical outcomes study social support survey. respondents’ attitudes towards genetic testing and fatalism are summarized in table . although there was variation among settlements, with mifflin county amish least in favor, most respondents would want a spouse tested for genetic disease. fewer respondents would want an unborn child tested for genetic disease and, again, mifflin county amish were least in favor. mifflin county amish babies were also least likely to have received newborn genetic screening. there was broad agreement among respondents on questions about fatalism with only very small proportions agreeing that their health was determined by fate (table ) and only about one-fourth agreeing that following medical advice would not affect the likelihood of a serious disease. int. j. environ. res. public health , , of table . attitudes towards genetic testing and fatalism in pennsylvania plain communities. settlement lancaster county amish groffdale mennonite weaverland mennonite mifflin county amish somerset county amish if you found out you were a carrier for a genetic disease, would you want your spouse tested for it? (% yes) * % % % % % would you want to know if your child was going to be affected by a particular genetic disease before he or she was born if a test could tell you this? (% yes) * % % % % % after your baby was born, did he or she receive a newborn screening test? (% yes) * % % % % % fatalism if someone is meant to have a serious disease, it doesn’t matter what doctors and nurses tell them to do, they will get the disease anyway. (% strongly agree or agree) a % % % % % my health is determined by fate. (% strongly agree or agree) * % % % % % * significant differences between settlements. a questions from a question fatalism scale. the relationships between respondents’ views on fatalism and genetic testing are in table . there does not appear to be a relationship between views on fatalism and the desire to test a spouse or unborn child for genetic disease. table . the relationship between attitudes on genetic testing and fatalism in pennsylvania plain communities. if someone is meant to have a serious disease, it doesn’t matter what doctors and nurses tell them to do, they will get the disease strongly agree or agree disagree or neutral if you found out you were a carrier for a genetic disease, would you want your spouse tested for it? yes ( %) ( %) p = . no ( %) ( %) would you want to know if your child was going to be affected by a particular genetic disease before he or she was born if a test could tell you this? yes ( %) ( %) p = . no ( %) ( %) my health is determined by fate strongly agree or agree disagree or neutral if you found out that you were a carrier for a genetic disease, would you want your spouse to be tested for it? yes ( %) ( %) p = no ( %) ( %) would you want to know if your child was going to be affected with a particular genetic disease before he or she was born if a test could tell you this? yes ( %) ( %) p = . no ( %) ( %) fifty-one respondents had someone in their family who is a patient at the clinic for special children. there were no differences between those who had or did not have a family member a patient at the clinic on answers to questions about fatalism (p = . and p = . ), nor in their desire to test an unborn child for genetic disease (p = . ). those with a family member a patient at the clinic were slightly less likely to want a spouse tested for genetic disease ( % vs. %, p = . ). int. j. environ. res. public health , , of . discussion there was considerable variation from settlement to settlement in health and health behaviors. in general, mennonite groups were most likely to receive preventive screening and most likely to have received diagnoses of disease, and mifflin county amish were least likely to receive screenings and least likely to have received diagnoses of disease. among plain respondents, groffdale mennonite respondents have a high proportion of diagnoses of depression and are more likely to be receiving treatment for a mental health condition. groffdale and weaverland mennonites report higher diagnoses of cancer. differences between groups may reflect differences in diagnosis rather than different underlying disease prevalence. few plain respondents seem to have attitudes that suggest fatalism, and most are in favor of genetic testing. with little variation between settlements, only about one-fourth of respondents agree that “if someone is meant to have a serious disease, it doesn’t matter what doctors and nurses tell them to do, they will get the disease anyway.” and only one-tenth agree that “my health is determined by fate.” even among those who agree with the statements, at least four-fifths would want a spouse tested for genetic disease and about half would want an unborn child tested. mifflin county amish, the most geographically isolated settlement, scored highest on measures of fatalism and lowest on the desire for genetic testing. despite some variation between plain groups, the health of members of old order groups appears remarkably similar to members of the general population of pennsylvania, despite living a very different lifestyle and being genetically isolated from them. there are some notable differences, though, between plain respondents and pennsylvania adults. plain respondents are more likely to live on a farm, drink water from a private well and be exposed to agricultural chemicals than other pennsylvania adults; they have a high prevalence of certain conditions (notably, anemia, thyroid problems and urinary tract and vaginal yeast infections) and lower prevalence of others (notably, depression and asthma). plain respondents were less likely than members of the general population to receive preventive screening, which may reflect attitudes towards healthcare or its difficulty and expense. asthma prevalence may be lower in plain communities [ , ]. the lancaster county amish community also has lower rates of low birth weight compared with women in central pennsylvania generally [ ]. plain communities may also have different numbers of diagnoses of various mental illnesses compared with the general population. fuchs et al. [ ] report higher prevalence of depression among the amish while miller et al. [ ] reports lower. bipolar disorders may be more prevalent among the amish and some plausibly causative pathogenic alleles have been identified [ , ]. differences in the prevalence of mental health conditions between plain people and the general population have been of interest to investigators both because of social influences on mental health and to isolate genetic influences on mental health. this work shows, we think, that surveying plain communities by mail is feasible. thus, knowledge of other, more isolated and poorer, plain communities might be easily and relatively cheaply obtained. furthermore, this work shows that, despite their relative geographic and genetic isolation and with a few notable exceptions, the health of adult members of the plain communities in pennsylvania is similar to that of other adults in the state. the desire of plain people to farm, combined with the expense of farm land, will continue to cause migration to less populated areas as plain populations continue to grow. thus, contact between plain communities and other parts of the general population and particularly with local medical care providers will continue to increase. providers will be particularly interested in those areas where plain people’s health differs from their neighbors. why do plain families apparently have fewer low birth weight babies and less asthma and depression, for example? what are the public health and medical care implications of the high prevalence of maple syrup urine disease, glutaric aciduria type i and other genetic disease in these communities? what are the implications for urgent care and chronic disease management? future work should explore these questions and survey other, smaller, int. j. environ. res. public health , , of poorer, more isolated plain groups to better understand the variation that exists within and between plain populations. . conclusions a health needs assessment of five pennsylvania plain communities was performed on a random sample of households. there was variation from settlement to settlement in whether respondents had a regular doctor, whether they received preventive screenings or had their children vaccinated, with more conservative groups generally lowest in these and least conservative groups highest. plain respondents reported good physical and mental health compared to the general population. despite their geographic and genetic isolation, the health of plain communities in pennsylvania is similar to that of other adults in the state. author contributions: conceptualization, k.m., b.y. and c.a.; methodology, k.m., b.y., c.a., s.t.b., e.d., z.a. and v.r.; validation, s.t.b., e.d., m.s., k.h. and m.a.c.; formal analysis, k.m., b.y., e.d., m.s. and k.h.; investigation, e.d., z.a. and v.r.; resources, s.t.b., m.s. and m.a.c.; data curation, b.y. and s.t.b.; writing—original draft preparation, k.m.; writing—review and editing, b.y., c.a., s.t.b., e.d., z.a., v.r., m.s., k.h. and m.a.c.; visualization, k.m. and s.t.b.; supervision, b.y.; project administration, b.y., c.a. and s.t.b.; funding acquisition, k.m. funding: this research was made possible by the generous support of the howard hughes medical institute (no. ). acknowledgments: donald kraybill and edsel burdge, jr. of the elizabethtown college young center for anabaptist and pietist studies generously helped us to understand the plain way of life and modern pressures on it and plain settlement populations. holmes morton, kevin strauss, erik puffenberger and karlla brigatti of the clinic for special children in strasburg, pa generously supported our work. rebecca bascom and her group at the penn state hershey medical center, anne dimmock, judie howrylak and kristen walters, engaged us in important discussions about asthma in the plain community. conflicts of interest: the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results. references . smith, c.h. the mennonites of america; wipf and stock publishers: goshen, in, usa, . . kraybill, d.b. concise encyclopedia of amish, brethren, hutterites, and mennonites; johns hopkins university press: baltimore, md, usa, . . strauss, k.a.; puffenberger, e.g. genetics, medicine, and the plain people. annu. rev. genomics hum. genet. , , – . 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[crossref] [pubmed] © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . /j.jaci. . . http://www.atsjournals.org/doi/abs/ . /ajrccm-conference. . . _meetingabstracts.a http://www.atsjournals.org/doi/abs/ . /ajrccm-conference. . . _meetingabstracts.a http://dx.doi.org/ . /j.whi. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . / http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /journal.pgen. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /hmg/ddu http://www.ncbi.nlm.nih.gov/pubmed/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	materials and methods 	populations and sample 	survey methods and measures 	results 	discussion 	conclusions 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ springer a++ viewer publisherinfo publishername : biomed central publisherlocation : london publisherimprintname : biomed central the thousand doors to disease articleinfo articleid : articledoi : . /gb- - - -reports articlecitationid : reports articlesequencenumber : articlecategory : web report articlefirstpage : articlelastpage : articlehistory : registrationdate : – – received : – – onlinedate : – – articlecopyright : biomed central ltd articlegrants : articlecontext : colin semple abstract online mendelian inheritance in man is a database of human genes and genetic disorders. content online mendelian inheritance in man is a database of human genes and genetic disorders. in the foreword to his classic work mendelian inheritance in man ( , john hopkins university press), victor a. mckusick cites a th century comment by sir thomas browne on the fragility of the human constitution ''... and considering the thousand doors that lead to death, do thank my god that we can die but once''. mckusick's book was the first to attempt comprehensively to classify and catalog the thousand doors, with particular attention to the genetic component of human disease. it is a measure of the trend towards the digital in genetics that mckusick's book, which now thrives as a website, is the subject of a report in an online journal. the mendelian inheritance in man catalog began in the early s and was originally confined mainly to rare recessive disorders discovered in old order amish populations. as early as the complexity of even this limited catalog necessitated computational methods for assembling and indexing data. the catalog was published online in , and since then has expanded to become the central authority on phenotypic and mapping data for human genes. as of july , it contained , entries and a substantial fraction of these entries represent known disease genes. for instance in february , omim celebrated passing the , disease-gene entries mark. the information held in omim is extracted by human curators from the scientific and medical literature and includes reports of the genetics, physiology and biochemistry of diseases along with mapping, phenotypic and phylogenetic data on human genes. navigation text searches of the complete database are possible and may be restricted to certain fields (for example, references) and/or entries (such as those that have changed recently). bookmarking of individual entries is possible. omim also curates a gene map and a 'morbid map'; the latter lists genetic diseases and their cytogenetic map locations. both maps can also be searched with simple text queries and all data are available to download via ftp. reporter's comments timeliness omim is updated monthly. on average the last year has seen about a hundred new entries and around six hundred changes to existing entries per month. best feature the annotation of gene and disease entries in omim is second to none, as a result of their policy of manual curation. it will be interesting to see how the database deals with the coming deluge of data, both on the identity of genes from the emerging human genome and on their associations with disease from large-scale studies of expression and single-nucleotide polymorphisms. omim contains an impressive array of links to sequence data and the literature via the ncbi entrez search system. it also lists a number of useful links to locus-specific mutation databases. wish list omim is really the only comprehensive record of how human dna sequence variation relates to disease phenotypes so criticisms inevitably sound churlish. having said that, certain improvements could be made. it would be nice to have better integration of the database with the currently available mapping data. for instance, if i want to find all diseases that have susceptibility loci including or close to a given marker, i have no way of easily finding them all from the database. many, though not all, disease loci could be placed on a genetic map given the data already in omim. but why stop there? the more pertinent challenge now is to integrate the information in omim about susceptibility loci with the emerging genomic sequence and mapping resources, such as the entrez homo sapiens genome view genome map viewer also maintained by the ncbi. related websites disease-associated mutations are also catalogued in the human gene mutation database (hgmd), locus-specific mutation databases are listed on the omim home page, and to some extent also by the genome database (gdb). the ncbi genes and disease map provides a friendly introduction to the study of disease genes. http://www.ncbi.nlm.nih.gov/cgi-bin/entrez/hum_srch?chr=hum_chr.inf&query http://www.uwcm.ac.uk/uwcm/mg/hgmd .html http://gdbwww.dkfz-heidelberg.de/ http://gdbwww.dkfz-heidelberg.de/ http://www.ncbi.nlm.nih.gov/disease/ table of links omim: online mendelian inheritance in man homo sapiens genome view human gene mutation database the genome database genes and disease map references . omim: online mendelian inheritance in man. this pdf file was created after publication. http://www.ncbi.nlm.nih.gov/omim/ http://www.ncbi.nlm.nih.gov/cgi-bin/entrez/hum_srch?chr=hum_chr.inf&amp;query http://www.uwcm.ac.uk/uwcm/mg/hgmd .html http://gdbwww.dkfz-heidelberg.de/ http://www.ncbi.nlm.nih.gov/disease/ http://www.ncbi.nlm.nih.gov/omim/ 	the thousand doors to disease 	content 	navigation 	reporter's comments 	timeliness 	best feature 	wish list 	related websites 	table of links 	references untitled revista romÂnÅ de pediatrie – volumul lxi, nr. , an adresa de corespondenţă: asist. univ. dr. anamaria burlea, universitatea de medicină şi farmacie „gr. t. popa“, str. universităţii nr. , iaşi e-mail: anamburlea@yahoo.com particularitĂŢi clinico-evolutive Şi terapeutice ale tulburĂrilor afective bipolare la copii Şi adolescenŢi asist. univ. dr. alexandra boloş, asist. univ. dr. anamaria burlea, prof. dr. roxana chiriţă universitatea de medicină şi farmacie „gr. t. popa“, iaşi rezumat tulburările afective bipolare reprezintă o condiţie medicală încă subdiagnosticată sau nediagnosticată la copii şi adolescenţi. trăsăturile clinice specifi ce, cum ar fi ciclarea rapidă şi episoadele mixte, complică, de obicei, posibilităţile de diagnostic clinic. În plus, comorbidităţile asociate sau supraadăugate pot avea impact asupra diagnosticului diferenţial. astfel, vor fi necesare studii specifi ce diferitelor grupe de vârstă care vor genera criterii de diagnostic particulare fi ecărei vârste. acest lucru este necesar pentru tinerii pacienţi deoarece un diagnostic adecvat cât mai precoce va determina şi un management terapeutic adecvat. cuvinte cheie: tulburare afectivă, diagnostic, adolescenţi, terapie referate generale tulburările psihice ale copiilor sunt mult mai difi cil de caracterizat decât cele ale adulţilor. deşi s-au realizat progrese în ceea ce priveşte diagnosticul tulburărilor psihice la copii, multe tratamente sunt administrate pentru o simptomatologie vagă, cum ar fi agresivitate, depresie sau manifestări discom- por tamentale ( ). o serie de factori contribuie la această situaţie, cum ar fi : • mult timp copiii au fost neglijaţi de serviciile medicale psihiatrice; • conceptul de anormalitate la copii este in fl u- enţat de procesul de dezvoltare a acestora, ceea ce face mult mai difi cilă interpretarea unor indicatori ai disfuncţionalităţi cere brale; • diagnosticul diferenţial este mult mai difi cil de realizat la copii comparativ cu adulţii, din cauza lipsei expresivităţii modifi cărilor com- portamentale din psihopatologia copiilor; • copiii prezintă multe difi cultăţi în ceea ce priveşte descrierea simptomatologiei psihia- trice. debutul simptomatologiei tulburărilor afective bipolare se realizează la vârste foarte diferite, în special între şi de ani, dar % dintre adulţii bipolari au prezentat un prim episod afectiv înaintea vârstei de ani. simptomatologia speci fi că tulburărilor afective bipolare este variabilă la copii şi adolescenţi şi prezintă o serie de simptome supra- adăugate şi comorbidităţi, cum ar fi abuzul de dro- guri sau adhd. astfel, se constată că la un număr important de copii tulburarea afectivă bi polară este greşit diagnosticată; de aceea, se încearcă găsirea unei soluţii în ceea ce priveşte diagnosticarea pre- coce a acestei tulburări psihice pentru a asigura un tratament adecvat. diagnosticarea cât mai pre coce a tulburării afective bipolare reprezintă un obiectiv clinic important, avându-se în vedere ur mă toarele motive ( ): • tulburarea afectivă bipolară reprezintă o sursă importantă de disfuncţionalitate psihosocială pentru copii şi adolescenţi, cu importante con secinţe asupra vieţii acestora; • există evidenţe clinice care arată faptul că un sindrom psihiatric, cu cât este mai prelungit, cu atât acesta este mai refractar la tratament. un diagnostic precoce al tulburării afective bi- polare la copii şi adolescenţi este asigurat de o serie de factori, şi anume ( ): revista romÂnÅ de pediatrie – volumul lxi, nr. , an • identifi carea simptomelor sugestive pentru episodul maniacal; • când nu există simptome psihotice, se va asigura un diagnostic diferenţial adecvat între manie şi adhd; • când există simptome psihotice, diagnosticul diferenţial se va realiza între manie şi schi- zofrenie. estimarea prevalenţei tulburărilor afective la copii şi adolescenţi este foarte difi cilă deoarece există foarte puţine studii pe acest tip de populaţie. În trecut, se considera că tulburările afective bi- polare ar fi rar întâlnite la copii şi adolescenţi, dar în prezent se ştie faptul că această tulburare psihică este frecventă, prevalenţa sa fi ind încă necu noscută ( ). rata prevalenţei depinde de criteriile de diagnostic utilizate. dsm iv prezintă aceleaşi criterii de diagnostic pentru toate tipurile de pacienţi cu tulburare afectivă bipolară, indiferent de vârstă ( ). cu toate acestea, clinicienii trebuie să ia în con- siderare toate aspectele referitoare la dezvoltarea co respunzătoare a copiilor şi adolescenţilor, atunci când aceştia sunt evaluaţi din punct de vedere psi- hiatric. de aceea, este important să înţelegem această tulburare psihică din punct de vedere al dezvoltării şi creşterii adecvate a acestora. de exemplu, unii tineri care prezintă ciclare rapidă a episoadelor sau episoade hipomaniacale nu înde- plinesc criteriile de diagnostic pentru tulburarea afectivă bipolară a adultului, dar un diagnostic cât mai precoce pentru o astfel de categorie de pacienţi şi un tratament adecvat determină o îmbunătăţire mai rapidă a simptomatologiei. pe de altă parte, în situaţia în care debutul simptomatologiei este sub vârsta de ani, apar forme atipice şi subclinice ale tulburării afective bipolare ( ). În cazul prezenţei unei ciclări rapide, simptomatologia include epi- soade mixte, evoluţie cronică, labilitate emoţională şi episoade maniacale sau depresive cu manifestări discrete. copiii pot prezenta manifestări explozive, iar modifi cările comportamentale sunt mai degrabă continue, decât episodice. În timpul unui episod ma niacal, copiii manifestă în general iritabilitate şi mai rar dispoziţie euforică. adolescenţii diagnos- ticaţi cu tulburare afectivă bipolară prezintă o simp- tomatologie similară cu cea a adulţilor şi episoade afective distincte, cu un debut rapid al simpto ma- tologiei. În cazul episoadelor afective, simptoma- tologia este clasică şi diagnosticul este mai uşor de identifi cat. tabelul prezintă diferenţiat tulburarea afectivă bipolară în funcţie de vârstă (după academy of child and adolescent psychiatry) ( ). toate aceste simptome ale tulburării afective bi- polare pot fi infl uenţate de contextul cultural. astfel, un studiu efectuat pe pacienţii din populaţia amish a descoperit că tinerii pacienţi diagnosticaţi cu un episod maniacal prezintă o serie de simptome, cum ar fi ideaţia de grandoare, reduse în intensitate, din cauza limitelor de natură religioasă. de ase menea, studiile au arătat faptul că acei copii pro veniţi din minorităţile etnice cu un nivel socio economic redus au un risc mai mare de diagnosticare greşită a unei schizofrenii comparativ cu alţi pa cienţi cu tulburare afectivă bipolară, deoarece epi soadele maniacale prezintă frecvent şi simptome psihotice. În plus, diferenţele rasiale pot infl uenţa şi managementul terapeutic ( ). un studiu asupra particularităţilor terapeutice a descoperit faptul că adolescenţii afroamericani cu tulburare afectivă bi polară primesc de două ori mai multe antipsihotice decât cei de origine caucaziană, din cauza interpre tării greşite a unor simptome. ( ) scopurile prin cipale ale mana- gementului terapeutic din tulburarea afec tivă bipolară sunt reprezentate de îmbunătăţirea simp to- matologiei şi de prevenirea recăderilor, avându-se în vedere reducerea morbidităţii pe ter men lung şi asigurarea unei dezvoltări cât mai nor male a acestor tineri. trialurile clinice controlate, efectuate pe tineri, sunt limitate, dar s-a constatat că cele mai utilizate medicamente cu rol normo sta bilizator sunt reprezentate de litiu, carbamazepină şi valproat. de obicei, strategiile terapeutice în ca zul copiilor şi adolescenţilor sunt bazate pe expe rienţa clinică cu pacienţii adulţi. dar, simpto ma tologia afectivă prezentă la tineri nu se suprapune întotdeauna cu cea a adulţilor şi ar fi necesare trialuri clinice specifi ce fi ecărei vârste pentru a putea identifi ca un tratament adecvat. Înaintea ini ţierii oricărei terapii psihofarmacologice este ne cesar să se obţină un consimţământ informat adec vat, să se evalueze corect faza de evoluţie a tulburării afective şi să se estimeze, pe cât po sibil, durata tratamentului. ale- gerea terapiei adec vate se bazează pe următoarele principii: ( ) • evidenţierea efi cacităţii medicaţiei alese; • faza de evoluţie a tulburării psihice; tabelul . evoluţia clinică a tulburării afective bipolare în funcţie de vârstă perioada prepubertală și adolescenţa precoce perioada de adolescenţă tardivă și adult episod iniţial depresiv maniacal tipul episoadelor ciclare rapidă, episod mixt discret cu debut brusc și sfârșit disti nct durata cronic, ciclare conti nuă săptămâni funcţionalitate episodică fără episoade funcţionalitate bună revista romÂnÅ de pediatrie – volumul lxi, nr. , an • prezenţa altor tipuri de simptome cum ar fi ciclarea rapidă, simptomele psihotice sau mo difi cările dispoziţionale; • efectele adverse ale medicaţiei; • istoricul pacientului legat de răspunsul la medicaţia anterioară; • preferinţele pacientului şi ale familiei. psihoterapia reprezintă un alt element important, parte integrantă a managementului terapeutic. se pot dezvolta măsuri educaţionale adecvate în urma con- sultării familiei şi a educatorilor şi astfel pa cientul şi rudele sale vor învăţa să managerizeze această tul- burare psihică. măsurile psihoterapeutice trebuie să fi e adaptate la necesităţile pacientului şi implică mo- dalităţi de învăţare de către pacient a simptomelor pro dromale ce vor prezice un viitor episod afectiv, ba- zându-se pe o serie de factori pre dictori, cum ar fi de privarea de somn, modifi cările situaţionale, patternul sezonier, abuzul de droguri sau noncomplianţa la tra- tament ( ). psihiatrul trebuie să realizeze un plan tera peutic folosind un algoritm asemănător celui din fi gura următoare ( ): litiu sau valproat (nu este răspuns) ↓ litiu+valproat (nu este răspuns) ↓ carbamazepină (nu este răspuns) ↓ carbamazepină + litiu (nu este răspuns) ↓ olanzapină sau risperidonă (nu este răspuns) ↓ noile anticonvulsivante (nu este răspuns) ↓ terapia electroconvulsivantă pentru psihiatri este foarte important să înţeleagă când să iniţieze şi când să discontinue terapia cu normostabilizatori. deoarece există doar câteva studii la copii şi adolescenţi cu tulburare afectivă bipolară privind evoluţia acesteia, experienţa clinică este cea care furnizează cele mai utile informaţii le gate de terapie. astfel, experienţa clinicienilor su- gerează faptul că nivelul terapeutic al timosta bili- zatorilor trebuie menţinut pentru minimum ani, după ce s-a obţinut remisia simptomatologiei. uneori, în cazul adolescenţilor este necesară discon- tinuarea trata mentului. această discontinuitate tre- buie efec tuată foarte lent, în timp, cu o reducere a dozelor pe parcursul a minimum luni. ( ) diagnosticarea şi tratarea tulburărilor afective bipolare la copii şi adolescenţi rămâne o problemă difi cilă avându-se în vedere complexitatea feno- meno logiei şi evoluţiei acestei tulburări. terapia farmacologică este necesară în scopul asigurării unei remisiuni adecvate, dar acest tip de pacienţi sunt frecvent subdiagnosticaţi şi trataţi necores- punzător. astfel, vor fi necesare şi în continuare nu- meroase cercetări pentru a evalua necesitatea mo- noterapiei sau a combinaţiilor terapeutice la tinerii pacienţi, deoarece este absolut necesară o intervenţie precoce şi chiar agresivă în cazul acestora. revista romÂnÅ de pediatrie – volumul lxi, nr. , an clinical and therapeutical approaches of children and adolescent bipolar disorders alexandra bolos, anamaria burlea, roxana chirita universitatea de medicină şi farmacie „gr. t. popa“, iasi abstract bipolar disorder remains a condition which it is underdiagnosed and misdiagnosed among children and adolescents. the specifi c traits, like rapid cycling and mixed episodes often complicate the diagnosis. in additional, overlapping and comorbid conditions may infl uence the differential diagnosis. thus, it is necessary that age specifi c studies may produce diagnostic criteria specifi c for younger patients because an earlier and accurate diagnosis will determine an adequate therapeutically management. key words: bipolar disorder, children, diagnosis, therapy child psychiatric disorders are more diffi cult to characterize than those of adults. although there are important advances in diagnosis of psychiatric disorder at children, many treatments are prescribed for vaguely defi ned disorder like aggressiveness, diffi cult behavior or depression. there are some factors which contributed to this ( ): • children have been neglected in psychiatric services; • abnormality in children is infl uenced by maturation and development, that make more diffi cult for doctors to interpret indicators of brain dysfunction; • differential diagnosis is more diffi cult to realize compared with adult caused to lack of richness of behavioral expressions of psycho- pathology at children; • children had a lot of diffi culties to describe psychopathological symptoms. affective bipolar disorders had a variable age of onset and it is especially diagnosed between ages of and , but % of adults experienced their fi rst episode under the age of . the symptoms of bipolar disorders are variable among children and adolescence and they had a lot of overlapping and comorbid conditions like adhd or substance abuse. ( , ) thus, we want to point on fact that a good number of children presented with bipolar disorder are misdiagnosed and this review offers a little solution to the problem of an early diagnosis, which it is critical for a good effi cacy of treatment. an early diagnosis of bipolar disorder is a very important clinical objective for psychiatrists for some reasons ( ): • this psychiatric disorder is a source of serious psychosocial dysfunction for children and adolescents with important consequences for their lives; • there is evidence that a psychiatric syndrome is longer it will become more refractory to treatment. there are three main diagnostic issues which are very important to facilitate an early diagnostic of bipolar disorder in children and adolescents ( ): • to identify symptoms suggestive for mania; • to differentiate between mania and adhd, when there are not psychotic symptoms; • to discriminate between mania and schi- zophrenia, when there are psychotic sym- ptoms. to estimate the prevalence of bipolar disorder among children and adolescent is very diffi cult because there are only a few studies on this type of population. even, in the past, it is believed that bipolar disorder occurred rarely among children and adolescent, nowadays it is recognized that this disorder is frequently, but prevalence is still unknown. the prevalence rates depend on diagnostic criteria used. ( ) the dsm iv presents the same criteria of diagnosis for all types of patients with bipolar disorders regardless of age. however, the clinicians should consider developmental issues when it is necessary to evaluate children or adoles- cents. ( ) thus, it is important for all of us to understand this disorder from a developmental perspective. for example, some young persons who express rapid cycle episodes and hypomania may do not have criteria for an adult bipolar disorder, but an early diagnosis and treatment for such revista romÂnÅ de pediatrie – volumul lxi, nr. , an patients it is important because early intervention means an improved outcome. on the other hand, often, patients with onset of the disorder younger than years old had atypical and subthreshold forms of bipolar disorder. ( ) for those with rapid cycling form, symptoms include mixte state, chronic evolution, emotionally labile behavior and less discrete episode of mania or depression. children can experience explosive outbursts and changes in mood are continuous in course, rather than episodic. during a manic episode, children manifest irritability more than euphoric mood. adolescent patients with bipolar disorder had symptoms like adult patients and experience distinct episode, unlike children with rapid onset of the symptoms. also, they present classical symptoms of mania and can be easily diagnosed with bipolar disorder. table summarizes differences in bipolar disorder based on age of onset (from american academy of child and adolescent psychiatry).( ) table . clinical course of bipolar disorder by age of onset prepubertal and young adolescent older adolescent and adult initi al episode depressive manic type of episodes rapid-cycling, mixed discrete with sudden onsets and clear off sets durati on chronic, conti nuous cycling weeks interepisodic functi oning nonepisodic improved functi oning all these expression of bipolar symptoms and behavior may be infl uenced by cultural context. thus, a study on old order amish patients found that among amish youths manic symptoms, like grandiosity were diminished by religious ties. also, studies had shown that children from ethnic mi- norities with lower socioeconomic backgrounds had a greater risk of misdiagnosis of schizophrenia than other patients with bipolar disorder because manic episodes include frequently psychotic fea- tures. in addition, racial differences can infl uence treatment patterns. ( ) a study on treatment pat- terns found that african american adolescents with bipolar disorder were twice as likely as caucasians ones to receive treatment with antipsychotic cause to misinterpretation of the symptoms. ( ) therapeutic management of bipolar disorder had two principals’ goals: to improve patient symptoms and to prevent relapses in order to reduce long term morbidity and to have a normal growth for these children. controlled medication trials in younger patients are limited, but the most common drugs used are lithium, valproat, carbamazepine. therapeutically strategies for children and adoles- cents with bipolar disorder are based mostly on clinical experience with adult patients. but, mani- festation of bipolar disorder in youths doesn’t mime adult type of bipolar disorder and it is necessary more controlled trial age specifi c to determine which therapy is most useful for youths. before initiating psychopharmacological treatment it is recommended to obtain an appropriate informed consent, to evaluate the phase of disorder and to estimate the length of treatment. the choice of me- dication is based on the following guidelines ( ): • evidence of effi cacy of the drug; • phase of the disorder; • the presence of other symptoms like rapid cycling, mood changes or psychotic features; • side effects of the drug; • the history of the patient regarding the response to drug; • preferences of patient and family. psychosocial treatment represents also a part of an integrated approach of the therapeutically ma- nagement. it can be develop an appropriate learning environment by consultation with families and educators, thus patients and family are taught to cope with this disorder. psychotherapy should be fl exible on the necessities of the patient and it involves teaching the patient to predict future epi sode relapses based on some factors like sleep de privation, situational changes, seasonal patterns, sub stance abuse and non- compliance to treatment. ( ) psychiatrists should realize a plan of treatment using an algorithm like in the next fi gure. ( ) lithium or valproat (if no response) ↓ lithium+valproat (if no response) ↓ carbamazepine (if no response) ↓ carbamazepine+lithium (if no response) ↓ olanzapinum or risperidonum (if no response) ↓ newer antiepileptic drugs (if no response) ↓ electroconvulsive therapy for psychiatrists, it is also important to understand when to initiate and when to discontinue the therapy. because there are only a few studies performed in children and adolescents with bipolar disorders regarding the course of the disorder, clinical experience provide us a lot of information useful for treatment. thus, clinical experience revista romÂnÅ de pediatrie – volumul lxi, nr. , an suggests that therapeutic levels of mood stabilizers should be maintained for at least years after the resolution of the symptoms. also, adolescent pa- tients may request discontinuation of the treatment. discontinuation must occur very slowly and doses should be tapering over month period. ( ) diagnosis of bipolar disorder in children and adolescents is complicated by the complexity of the phenomenology and course of the disorder. the pharmacological treatment is necessary for a good outcome, but often this kind of patients are mis- diagnosed and undertreated. thus, more research is required to evaluate monotherapy or combination of therapies at youths because it is important to have an early and even aggressive intervention at these patients. . gagan joshi, carter petty, janet wozniak, stephen v. faraone, et al. – a prospective open-label trial of quetiapine monotherapy in preschool and school age children with bipolar spectrum disorder journal of affective disorders. oct . o. bonnot, l. holzer – utilisation des antipsychotiques chez l’enfant et l’adolescent neuropsychiatrie de l’enfance et de l’adolescence. sep . howard y. liu, mona p. potter, k. yvonne woodworth, dayna m. yorks, carter r. petty, et al. – pharmacologic treatments for pediatric bipolar disorder: a review and meta-analysis journal of the american academy of child & adolescent psychiatry. aug , vol. , no. : - . . jonathan c. pfeifer, robert a. kowatch, melissa p. delbello – pharmacotherapy of bipolar disorder in children and adolescents cns drugs. jul , vol. , no. : - . eric taylor – managing bipolar disorders in children and adolescents nature reviews neurology. sep , vol. , no. : - . n.c. patel, d.m. patrick, e.a. youngstrom, s.m. strakowski, m.p. – delbello response and remission in adolescent mania journal of the american academy of child & adolescent psychiatry. may , vol. , no. : - . diagnostic and statistical manual of mental disorders, th edition ( ) american psychiatric association, washington dc. . edith m. jolin, elizabeth b. weller, ronald a. weller – the public health aspects of bipolar disorder in children and adolescents current psychiatry reports. apr , vol. , no. : - . michael strober, boris birmaher, neal ryan, david axelson, sylvia valeri, henrietta leonard, et al. – pediatric bipolar disease: current and future perspectives for study of its long-term course and treatment bipolar disorders. aug , vol. , no. : - . nick c. patel, melissa p. delbello, robert a. kowatch, stephen m. strakowski – preliminary study of relationships among measures of depressive symptoms in adolescents with bipolar disorder journal of child and adolescent psychopharmacology. jun , vol. , no. : - . cassano g.b., mcelroy s.l., brady k., nolen w.a., placidi g.f. – current issues in the identifi cation and management of bipolar spectrum disorders in special populations. j affect disord. ; (suppl ): s - . saunders and goodwin – the course of bipolar disorder adv. psychiatr. treat. ; : - . . geller et al. – child bipolar i disorder: prospective continuity with adult bipolar i disorder; characteristics of second and third episodes; predictors of -year outcome arch gen psychiatry ; : - . goldstein – recent progress in understanding pediatric bipolar disorder arch pediatr adolesc med ; : - . . rick t. bowers, christina g. weston, julia jackson – child and adolescent affective disorders and their treatment journal of affective disorders. mar : - references effectiveness of video feedback and self-management on inappropriate social behavior of youth with mild mental retardation effectiveness of video feedback and self- management on inappropriate social behavior of youth with mild mental retardation petri j.c.m. embregtsa,b,* auniversity of nijmegen, he nijmegen, the netherlands bjan pieter heye residential facility, oosterbeek, the netherlands accepted december abstract the effectiveness of a video feedback and self-management package was assessed with various inappropriate behaviors exhibited by six youth with mild mental retardation. the procedure consisted of (a) videotaping participants’ inappropriate behavior, (b) having them self-monitor and record their behavior, (c) prompting them to evaluate their behavior against a criterion, and (d) allowing to reinforce themselves for appropriate behaviors. data were collected within a nonconcurrent multiple baseline design across participants. results showed a statistically significant decrease of the percentage intervals of inappropriate behavior when the procedure was in effect. the total number of interactions remained stable across the different phases of the study. video feedback and self-management contributed to generali- zation across settings. © elsevier science ltd. all rights reserved. keywords: mild mental retardation; inappropriate social behavior; video feedback; self-management . introduction during the last two decades, much attention has been given to the social behaviors of persons with mild mental retardation. these people tend to exhibit more social behavior deficits and inappropriate behaviors as compared to peers without disabilities (e.g., schumaker, pederson, hazel, & meyen, ). defi- * correspondence. tel.: - - - - ; fax: - - - - . e-mail address: p.embregts@ped.kun.nl research in developmental disabilities ( ) – - / /$ – see front matter © elsevier science ltd. all rights reserved. pii: s - ( ) - cient social behaviors hinder their integration into the mainstream of society. as integration of people with a handicap receives high priority it is clear that this area deserves attention from behavior analysts. various strategies have been employed to improve social behaviors, such as instruction, modeling, role play- ing, behavior rehearsal, coaching, feedback, homework, and reinforcement (e.g., amish, gesten, smith, clark, & stark, ; bates, ). although these procedures have demonstrated their effectiveness on the ac- quisition of social behaviors, they are also associated with several shortcomings. due to their reliance on external control, the effects obtained have, by definition, limited generalizability to natural settings. the validity of procedures to establish social behaviors depends therefore, upon the degree to which the behaviors to be taught generalize across settings, time, and responses. stokes and osnes ( ) indicate that generalization of behaviors is more likely to occur if a procedure is at least partly conducted in natural settings. a problem of this approach is, however, the staff time needed to conduct such procedures in natural settings. moreover, external contingencies may then interfere with ongoing interactions. self-management is one of the strategies that may provide a solution to this problem (ferretti, cavalier, murphy, & murphy, ; harchik, sherman, & sheldon, ). self-management has been defined as the application of operant procedures to one’s own behavior (skinner, ). procedures of self-manage- ment, such as self-monitoring, self-recording, self-evaluation, and self-adminis- tering consequences have shown to be effective with various populations and target behaviors (e.g., cavalier, ferretti, & hodges, ; dunlap, dunlap, koegel, & koegel, ). research has shown that self-monitoring can be helpful in reducing external control and by consequence, to facilitate generali- zation across stimulus dimensions (e.g., koegel & koegel, ; lonnecker, brady, mcpherson, & hawkins, ; rhode, morgan, & young, ). despite these advantages, self-monitoring requires immediate self-recording of target behavior(s). it might be argued that such recording may disturb ongoing social interactions. for establishing social behaviors the use of videorecording may be consid- ered, which involves recording participants while they are interacting with others and then having them view a playback (e.g., booth & fairbank, ). research on this means has shown that video feedback improves responding of behavior- ally disordered children (osborne, kiburz, & miller, ), on-task performance of children with emotional and behavioral disorders (walther & beare, ), peer interactions of students with emotional and behavioral disorders (falk, dunlap, & kern, ; kern-dunlap et al., ; kern et al., ), and productivity of an adult worker with moderate mental retardation (cavaiuolo & gradel, ). the purpose of the present study was to assess the effectiveness of a video feedback and self-management package on the frequency of inappropriate social behavior exhibited by youth with mild mental retardation. the procedure en- compassed (a) videotaping participants during lunch and dinner time and during group meetings, (b) while viewing the video, having them monitor and record p.j.c.m. embregts / research in developmental disabilities ( ) – https://isiarticles.com/article/ book reviews culture and equality: an egalitarian critique of multiculturalism b. barry polity press, cambridge, , pp. paperback, isbn: - - - / - . citizenship and national identity d. miller polity press, cambridge, , pp. paperback, isbn: - -x/ - . brian barry’s book is an extended critique of the prevailing multiculturalist orthodoxy in anglophone political theory. david miller’s book is a collection of his pieces, most of them previously published, addressing the themes of deliberative democracy, nationality, community, and justice. barry writes as an egalitarian liberal, miller as a socialist communitarian. each has reasons, albeit very different ones, not to endorse multiculturalism as a normative political programme. both, in their distinct ways, illuminate contemporary debates in political philosophy. barry’s book is the more substantial of the two. it is a trenchant, robust, vigorously and rigorously argued counterblast in the name of liberal egalitarianism against those, especially fellow egalitarian liberals, who defend the politics of multiculturalism. barry’s two-sided claim is simple: the politics of ‘recognition’ or of ‘difference’ are not demanded by liberal principles of equality; furthermore, the pursuit of these politics obscures and obstructs the realisation of these principles through more appropriate social and economic policies. the usual suspects f will kymlicka, bhikhu parekh, iris marion young, and charles taylor amongst others f are rounded up and roundly chastised for their illiberalism, inconsistency, invalid argumentation, or inexactitude. the tone is headmasterly f impatient with the poor presentation of a case, quick to correct, often abrasive, and always magisterial. charles taylor, for instance, is indicted in passing for his ‘characteristic imprecision’ (p. ). strictly speaking, barry is not opposed to multiculturalism. the fact of cultural diversity is, he thinks, inescapable and ineradicable, but he is happy to ‘privatise’ the problem. this means that individuals are free to associate with others of a similar persuasion and indeed they can as a group engage in illiberal practices. a liberal state, however, is not required to give special recognition to groups by, for instance, granting them exemption from the burdens of contemporary political theory, , , ( – ) r palgrave macmillan ltd - / $ . www.palgrave-journals.com/cpt conforming to all laws or by the provision of exceptional benefits. barry can find no liberal warrant for this kind of public multiculturalism. liberal principles rest on universal enlightenment tenets of reason and he deprecates those writers whose appeal to the value of culture is a poor disguise for their moral relativism. no short review can do full justice to the richness of barry’s book. it combines erudition (it is refreshing to be reminded of exactly what the much discussed old amish culture amounts to), unabashed defence of liberalism, a passion for the point of liberal politics, and a finely tuned sensitivity to the idiocies of simple-minded multiculturalist apologetics. there are excellent discussions of, inter alia, education, ‘culture’, the costs of group membership, and the free exercise of religion. more would have been welcome on one matter. whilst prizing autonomy, barry does not see a plurality of ways of life as a necessary precondition for its fullest exercise. the thought is that membership of a particular culture provides a context within which its members’ autonomy is exercised, and thus has value, and, further, that a variety of cultures provides a broader context for the autonomy of all those within a society. this is probably the most influential liberal defence of multiculturalism to be found in kymlicka whom barry castigates for not being a liberal and raz whom barry does not consider. moreover, both writers think of membership of a culture as a primary social good whereas barry restricts himself to rawls’s narrower list of such goods. barry also owes an account of how a liberal polity can sustain the civic solidarity which is necessary if diversity is not to prove destabilising and liberal principles of justice are to be generally supported. he does not supply one in this book though he does offer an illuminating sketch of a civic national identity which is additive and not assimilationist. he does, however, need to say more about how it fortuitously comes to be that the core of a common national identity is ‘a common commitment to the welfare of the larger society...and mutual trust in others to abide by that commitment’ (p. ). david miller, as is well-known, has developed a theory of nationality which would allow a democratic society to pursue the goal of social justice. for miller nationality is the only remotely plausible candidate for supplying the shared and bounded identity needed to underpin citizenship and shared subscription to principles of justice. the essays published here take up this idea and consider threats to it from pluralism, globalisation, and identity politics. miller is sceptical about the last of these not, as barry is, because they are not required by liberal justice, but because he thinks that they erode the common identity which is instrumentally necessary for realising justice. in a number of places he responds to the challenge of the difference theorist, iris marion young, who must be doing something interesting to provoke the very different criticisms of barry and miller. book reviews contemporary political theory miller’s book contains some clear, well-ordered discussions of the claims of global justice, the limits of secession, and the nature of delibe-rative democracy. his discussion of ‘nested nationalities’ f those, such as the scots who simultaneously think of themselves as having a narrow (‘scottish’) and an encompassing broader (‘british’) identity f is a welcome addition to the literature of political philosophy on nationality. again, however, more would have been welcome on the question of how a national culture can be subject to democratic deliberation and scrutiny so that it serves, and does not hinder, the ends of justice. miller recognises that this is a difficult subject (pp. – ) but does not pursue it at any length. but there seem good reasons to doubt that an identity can be collectively moulded by those who inherit it in the same way as institutions can be democratically designed. one would also have welcomed further discussion by miller of the extent to which his moral particularism f the content of principles of justice as rooted in the culture to which they apply f is an ‘uncomfortably relativistic view’ (p. ). miller is evidently and explicitly indebted to the work of michael walzer but he does not make clear how far he shares walzer’s relativism. at the heart of both these books is a worry as to how a polity can realise principles of justice and deal with difference without betraying these principles. the arch multiculturalist will kymlicka has, in recent publications, declared that we are all multiculturalists now. barry and miller offer plausible but very different reasons for doubting that assertion. david archard department of philosophy, university of st. andrews. book reviews contemporary political theory 	book reviews 	culture and equality: an egalitarian critique of multiculturalism 	citizenship and national identity from the question concerning technology to the quest for a democratic technology: heidegger, marcuse, feenberg symposium: andrew feenberg’s questioning technology* from the question concerning technology to the quest for a democratic technology: heidegger, marcuse, feenberg iain thomson university of new mexico, albuquerque andrew feenberg’ s most recent contribution to the critical theory of technology, questioning technology , is best understood as a synthesis and extension of the critiques of technology developed by heidegger and marcuse. by thus situating feenberg’ s endeavor to articulate and preserve a meaningful sense of agency in our increasingly technologized lifeworld, i show that some of the deepest tensions in heidegger and marcuse’ s relation re-emerge within feenberg’ s own critical theory. most signi� cant here is the fact that feenberg, following marcuse, exaggerates heidegger’ s ‘fatalism’ about technology. i contend that this mistake stems from feenberg’ s false ascription of a technological ‘essentialism’ to heidegger. correcting this and several related problems, i reconstruct feenberg’ s ‘radical democratic’ call for a counter-hegemonic democratization of technological design, arguing that although this timely and important project takes its inspiration from marcuse, in the end feenberg remains closer to heidegger than his marcuseanism allows him to acknowledge. i. introduction richard wolin has remarked that ‘[t]he full story of marcuse’s relation to heidegger has yet to be written’. indeed, there are at least two stories to be told about the marcuse–heidegger relationship: the story of its historical past and the story of its philosophical future. let us hope that intellectual historians like wolin will continue to bring the past of this important relation to light; in the meantime, andrew feenberg has already begun writing the philosophical story of its future. the goal of his questioning technology is to # taylor & francis inquiry, , – * andrew feenberg, questioning technology (london and new york: routledge, ), xvii + pp., pb. $ . . unprefixed page references are to this work. earlier versions of this paper were presented at the society for philosophy of technology conference in san jose, ca, on july , and at the eastern division meeting of the american philosophical association in boston, december . i thank bert dreyfus, jerry doppelt, andy feenberg, wayne martin, david stump, and richard wolin for helpful comments and criticisms. articulate a critical theory capable of responding to ‘[t]he fundamental problem of democracy today’, namely, the question of how to ‘ensure the survival of agency in this increasingly technological universe’ (p. ). to meet this challenge, feenberg synthesizes and extends the critiques of technology developed by heidegger and post-heideggerian thinkers like marcuse and foucault. my approach will seek to situate feenberg’s project within this historical perspective. ii. the history behind feenberg’s heidegger–marcuse dialectic marcuse studied with heidegger from to , and feenberg was a marcuse student during the late s. this, of course, makes feenberg one of heidegger’s intellectual grandchildren. but this is a genealogy fraught with political and philosophical tensions, tensions which occasionally make themselves felt in feenberg’s interpretations and which point back to the fact that marcuse himself broke with heidegger bitterly – and permanently – in . to marcuse, heidegger’s strong early support of national socialism represented a fundamental betrayal of heidegger’s own ‘existential’ philosophy, and thus an abandonment of ‘the greatest intellectual heritage of german history’, and he said so at the time. in and , while heidegger was making political speeches on hitler’s behalf, marcuse was � eeing hitler’s rise to power, � rst from frankfurt to geneva in , then emigrating to new york in , where he served as the philosophical specialist for the now exiled frankfurt school. during this period, marcuse wrote reason and revolution, defending hegel’s notion of the state – as ‘a social order built on the rational autonomy of the individual’ – against the ‘pseudo-democratic ideology’ characteristic of fascism, which pays lip service to the direct rule of the ‘people’ [volk], while in fact ‘the ruling groups control the rest of the population directly, without the mediation of . . . the state’. hitler had abolished all such democratic mediation, so marcuse concludes reason and revolution by quoting carl schmitt’s proclamation that on january th, , ‘the day of hitler’s ascent to power, “hegel, so to speak, died”.’ marcuse’s post-heideggerian return to hegel was of course also a return to marx; he was elaborating the major philosophical sources of frankfurt school critical theory. but around this time max horkheimer, who directed the institute for social research and controlled its � nances, began working closely with another philosopher, theodore adorno. adorno, whose hatred for heidegger apparently spilled over onto marcuse, wrote to horkheimer in to remind him of the ‘illusions’ marcuse had so recently had ‘of herr heidegger, whom he thanked all-too-heartily in the foreword to his ( ) hegel book’. adorno went so far as to accuse marcuse of being ‘hindered iain thomson [only] by judaism from being a fascist’! whether or not adorno’s vicious intrigue succeeded, marcuse soon found institute funds in too short a supply to continue supporting him and his family. thus it was that marcuse, the philosopher now best remembered as the intellectual guru of the new left (and thus the mentor of new left philosophers like feenberg, angela davis, and douglas kellner), found himself working for various american intelligence agencies from to . this is less strange than it sounds. marcuse actually spent the � nal years of the second world war doing ‘de-nazi� cation studies’ for the of� ce of strategic services. here, with two other prominent members of the frankfurt school (the legal scholar and economist franz neumann and the political theorist otto kirchheimer), marcuse engaged in an intensive interdisciplinary effort to uncover and ‘eliminate the root causes that had produced fascism’. looking back in , however, marcuse would conclude that: ‘the defeat of fascism and national socialism has not arrested the trend toward totalitarianism.’ the fundamental political threat to democracy had not been rooted out; it had merely changed forms and continued to spread after the war. marcuse called this new, post-fascist form of totalitarianism ‘technocracy’. a technocracy is a political state in which ‘technical considerations of imperialistic ef� ciency and rationality supersede the traditional standards of pro� tability and general welfare’. for the rest of marcuse’s long and fruitful career, his overriding question became: how can the increasingly global technocracy be subverted, that is, democratized? this is precisely the quest behind questioning technology, the project that feenberg takes up – with heidegger’s help. of course, marcuse himself would not have looked to heidegger for help. marcuse was deeply dissatis� ed by heidegger’s private admission of a ‘political error’; he expected heidegger to publicly announce his political change of ‘allegiance’ (as nazi opportunists like schmitt and alfred bäumler had done right after the war, a disingenuous act that heidegger, the thinker of authenticity, found simply ‘loathsome’). marcuse warned heidegger that his refusal to make such an apology would be interpreted as a continuing ‘complicity’ with nazism, but heidegger obstinately refused. thus a controversial stalemate was reached, and marcuse and heidegger would remain personally and professionally estranged for the rest of their lives. unfortunately, as feenberg shows, this mutual estrangement led them to neglect the important insights contained in each other’s work on technology. feenberg brings out remarkable similarities between marcuse’s critique of technocracy , the technologically mediated production and maintenance of a one-dimensional society, and heidegger’s ontological critique of enframing, the technological understanding of being which turns everything it touches into a mere resource. indeed, feenberg stages a forceful post-marcusean heidegger, marcuse, feenberg return to heidegger, and thus presents in absentia much of marcuse and heidegger’s missing interlocution on the essence of modern technology. true to the philosophical spirit of marcuse, feenberg’s critique of heidegger is thoroughly dialectical. its negative or critical moment seeks to isolate heidegger’s deepest insights into technology, preserving these insights from distortions feenberg blames on heidegger’s ‘techno-phobic’ (p. ) and ‘essentialist’ (p. ) understanding of technology. in the positive moment of his critique, feenberg appropriates several of heidegger’s insights, incorporating these in a powerful new way into his own critical theory of technology. in so doing, he demonstrates the continuing importance of the heideggerian critique of technology while going beyond heidegger – and marcuse – in signi� cant respects. iii. feenberg’s marcusean critique of heidegger feenberg argues that the four major types of theories of technology (determinism, instrumentalism, substantivism, and critical theory) can be differentiated by the answers they each give to two basic questions (p. ). for feenberg, heidegger’s � rst answer represents an unsurpassable historical advance beyond determinism and instrumentalism, but heidegger’s second response pinpoints where his ‘substantivist’ view goes wrong and needs to be superseded by feenberg’s own critical theory. the � rst question is: is technology neutral or is it value-laden? as feenberg argues, heidegger undermines once and for all the belief that technology is neutral by showing that the technological doer comes to be historically ‘transformed by its acts’ (p. ). heidegger’s understanding of technology thus overturns both traditional marxist determinism (according to which technological advance will inevitably usher in the golden age of communism), and liberal instrumentalism (which understands technology merely as an instrument of progress, a set of tools which can be used transparently to achieve independently chosen ends). as feenberg puts it, heidegger shows that ‘technology is not merely the servant of some prede� ned social purpose; it is an environment within which a way of life is elaborated’ (p. ). and thus, ‘for good or ill, the human manner of inhabiting the environment can only be [an] ethical’ question. heidegger’s answer to this ethical question concerning technology argues that technology has an ontological impact which is far from neutral. as technology colonizes the lifeworld, everything ‘sucked up’ into its purview, including the modern subject, is reduced to the ontological status of a resource to be optimized. within our current technological ‘constellation’ of intelligibility, ‘[o]nly what is calculable in advance counts as being’. this technological understanding of being produces a ‘calculative thinking’ which iain thomson quanti� es all qualitative relations, reducing all entities to bivalent, programmable ‘information’, digitized data, which increasingly enters into what baudrillard calls ‘a state of pure circulation’. as this historical transformation of beings into resources becomes more pervasive, it increasingly eludes our critical gaze; indeed, we come to treat even ourselves in the terms underlying our technological refashioning of the world: no longer as conscious subjects in an objective world but merely as resources to be optimized, ordered, and enhanced with maximal ef� ciency (whether cosmetically, psychopharmacologically, genetically, or cybernetically). for heidegger, the ‘greatest danger’ of our spreading technological under- standing of being is the possibility that we will lose the capacity to understand ourselves in any other way. feenberg seems to agree with heidegger’s basic diagnosis of technology’ s ontological impact, but thinks that heidegger overstates the danger because he ignores resources internal to technological society capable of combating this ontological devastation. this brings us to the second question feenberg uses to categorize the � eld of technological theories, the question which differentiates feenberg from heidegger: can the historical impact of technology be humanly controlled, or does it operate according to its own autonomous logic? is humanity capable of guiding the historical direction in which technology is taking us? no, heidegger answers; what is most essential about technology – namely, the way in which it alters how reality shows up for us – cannot be controlled. as heidegger writes: ‘no single man, no group of men, no commission of prominent statesmen, scientists, and technicians, no conference of leaders of commerce and industry, can brake or direct the progress of history in the atomic age.’ this answer reveals what feenberg most fundamentally objects to in heidegger’s approach: heidegger attributes an autonomous logic to technology. this fatalistic ‘substantivism’ stems ultimately from heidegger’s essentialism, feenberg contends (p. ), and it leads heidegger to advocate ‘liberation from [the technological order] rather than [its] reform’ (p. ). but feenberg’s reading is never so hermeneutically violent as when he accuses heidegger of being a technological ‘essentialist’. heidegger’s paradoxical-sounding claim that ‘the essence of technology is nothing technological’ does not mean that technology leaves no room for ‘re� exivity’ (p. ). heidegger is really expressing the paradox of the measure; height is not high, treeness is not itself a tree, and the essence of technology is nothing technological. to understand the ‘essence of technology’ , heidegger says, we cannot think of ‘essence’ the way we have been doing since plato (as what ‘permanently endures’), for that makes it seem as if ‘by the [essence of] technology we mean some mythological abstraction’. we need, rather, to think of ‘essence’ as a verb, as the way in which things ‘essence’ [west] or ‘remain in play’ [im spiel bleibt]. ‘the essence of technology’ thus means heidegger, marcuse, feenberg the way in which intelligibility happens for us these days, that is, as ‘enframing’ (the historical ‘mode of revealing’ in which things show up only as resources to be optimized). heidegger’s historical understanding of the ‘essence’ of technology may actually put his position closer to the ‘constructivist’ than the ‘essentialist’ camp, and it becomes clear that feenberg shares a similar view when he advocates ‘a historical concept of essence’ in the book’s concluding chapter (p. ). what feenberg really objects to, it seems, is heidegger’s claim that the appropriate response to technology is best characterized by the comportment toward phenomena heidegger calls gelassenheit , that is, releasement, equanimity, composure, or ‘letting-be’ (p. ) – not ‘resignation and passivity’, as feenberg rather polemically translates the term at one point (p. ). but feenberg gives a more sympathetic treatment of the notion of gelassenheit later, when he writes: ‘heidegger’s undeniable insight is that every making must also include a letting-be, an active connection to the meanings that emerge with the thing and which we cannot “make” but only release through our productive activity’ (p. , my emphasis). if the ‘criteria for constructive reform’ (p. ) feenberg seeks are to be found anywhere in heidegger’s view, it is here. in fact, gelassenheit is one of the main criteria that the amish use when deciding for or against the integration of a new technological device into their community. to some this example may seem its own refutation, but the critical theorist of technology can learn much from the amish, who are not ‘knee-jerk technophobes’ , but rather ‘very adaptive techno-selectives who devise remarkable technologies that � t within their self-imposed limits’. the amish may actually have achieved heidegger’s ideal of a ‘free relation to technology’ , according to which we should ‘af� rm the unavoidable use of technical devices, and yet also deny them the right to dominate us, and so to warp, confuse, and lay waste to our nature’. heidegger is not a luddite, but rather advocates a non-addicted ‘proper use’ of technical devices in which we keep ourselves ‘so free of them that we may let go of them at any time’. he says we should ‘let technical devices enter our daily life, and at the same time leave them outside’; the amish take this advice quite literally when they leave their cellular phones in the outhouse overnight so that phone calls will not interrupt the face-to-face communal relations they cherish. the amish do not reject new devices like the cell phone out of hand, but live re� exively with them, sometimes for years, before deciding ‘what will build solidarity and what will pull them apart’, what can be adapted to � t the needs and values of their community (like high-tech electric barbecues) and what cannot (like cars), and in such adaptation they can be quite creative. but for feenberg, heidegger’s faith in gelassenheit is too ‘nostalgic’ (p. ) and passive; heidegger’s ‘fatalism’ gives over too much human autonomy to the technological order. in fact, feenberg’s fundamental iain thomson objection appropriates marcuse’s most powerful political criticism of heidegger. as marcuse put it, heidegger succumbed to a ‘hopeless heteronomism’, that is, he lost faith in the enlightenment’s understanding of freedom as the capacity for substantive rational self-determination, the ability to direct the ends as well as the means of human life. feenberg expresses this marcusean criticism in a marxist register: heidegger is a ‘technological fetishist’ (p. viii). in the marxist vocabulary, fetishism occurs when a ‘social relation between men’ assumes ‘the fantastic form of a relation between things’. for a marxist (and let us not forget that critical theory is post-marxian marxism), to fetishize something is to detach it from the human labor that produced it but to continue nevertheless to project human meanings upon it, mistaking these projections for an independent reality. the fetishist’s anthropomorphic projection endows a humanly created thing with the magical appearance of possessing a telos independent of human ends. heidegger’s technological fetishism is visible in the fact that, in his view (as feenberg reconstructs it), ‘technology rigidi� es into destiny’ (p. ). but just as feenberg downplays the active element in gelassenheit , so here he overlooks the fact that for heidegger enframing is our ‘destiny’, but it is not necessarily our ‘fate’. as dreyfus puts it, ‘although our understanding of things and ourselves as resources to be ordered, enhanced, and used ef� ciently has been building up since plato and dominates our practices, we are not stuck with it. it is not the way things have to be, but nothing more or less than our current cultural clearing’. in fact, the critical force of heidegger’s ‘history of being’ comes from his hope for a new historical beginning in which we would no longer treat everything as resources to be optimized. feenberg argues, however, that heidegger succumbs to the ‘deterministic illusion’ because he fails to notice the ‘speci� c technical choices’ which are in fact always involved in processes like ‘the deskilling of work, the debasement of mass culture, and the bureaucratization of society’ (p. ). if heidegger ‘allows no room for a different technological future’ (p. ), a future which would avoid ‘the gloomy heideggerian prediction of technocultural disaster’ (p. ), it is because he overlooks the speci� c choices that always go into the process of ‘technological design’, and thus cannot envision the possibility that technologization could come to serve democratization. again, i do not think feenberg is right about heidegger’s supposed fatalism. this objection ignores heidegger’s hope for an ‘other beginning’ to western history (this is not surprising, since for feenberg the political direction in which this hope led heidegger disquali� es the hope itself). second, it rests on feenberg’s polemical characterization of gelassenheit as ‘heidegger’s outright rejection of agency’ (p. ). but, as feenberg recognizes subsequently, heidegger’s more balanced insistence on ontological receptivity is in fact better understood as heidegger’s later heidegger, marcuse, feenberg ‘corrective to his overemphasis on the role of dasein in disclosure’ in his early work (p. ). for heidegger it is crucial that we recognize our ontological receptivity if we are to get beyond our ‘willful’ technological ontology and envision an alternative future. still, feenberg’s conclusion – that heidegger’s own suggestions about this alternative future leave no room for a democratization of technology – is probably right for another reason, namely, heidegger’s excessively dim view of democracy. at any rate, feenberg’s critique of heidegger becomes the springboard for his own alternative, which seeks to expand democratic control over the technological design process. here feenberg again draws his inspiration from marcuse. unlike heidegger, marcuse learned from hitler’s rise to power about the importance of maintaining strong democratic institutions capable of mediating the will of the people and ensuring that the national voice is as inclusive as possible. still, marcuse was deeply concerned that the technological colonization of these democratic institutions discouraged rational autonomy. as marcuse looked around himself in , he saw that ‘[i]ndividualistic rationality has developed into ef� cient compliance with the pre-given continuum of means and ends’. indeed, one revealing difference between heidegger and marcuse can be seen in heidegger’s interpretation of a massive highway interchange on the autobahn as a ‘thing’ capable of putting us in touch with the meanings of the world it embodies. pace feenberg, here heidegger recognizes that: ‘devices are things too’ (p. ), that is, he acknowledges that it is possible to attain a ‘re� exive relation’ to technological devices (p. ). heidegger thus helps raise the question concerning the world of meanings opened and transformed by technological phenomena such as the ‘information superhighway’, the internet. unlike heidegger, however, marcuse thought that: ‘in manipulating the machine, man learns that obedience to the directions is the only way to get the desired results. . . . there is no room for autonomy.’ i think this shows that in fact heidegger thought further in the direction of feenberg’s project than did marcuse, even though this project is inspired by marcuse’s notion of a technological ‘democratization of functions’ (the only development marcuse could point to within western democracies that seemed capable of reversing our slide toward a ‘totally-administered society’). iv. feenberg’s alternative feenberg uses the work of bruno latour to uncover the way in which substantive political choices are embedded into technology during the design process. think for example of the moral content locked into the ‘technical code’ of the ‘speed-bump’: rather than appealing to our rational autonomy through the imposition of speed-limits, the technical device simply decides iain thomson for us and forces us to comply. as feenberg writes: ‘design comes to re� ect a heritage of . . . choices. . . . [i]n a very real sense there is a technical historicity; technology is the bearer of a tradition that favors speci� c interests and speci� c ideas about the good life’ (p. ). in short, technological ‘design mirrors back the social order’ (p. ). thus, against heidegger’s supposed technological essentialism, feenberg argues that we need to recognize the historical ‘malleability of technology’ (p. ), the possibility that technology could come to embody more democratic values. as an example of such technical historicity, feenberg describes the struggle between ibm and macintosh over text versus graphics user interfaces. early on, the text-based interface nicely represented the values of computer users, who were mostly programmers. but as the democratization of computers spread computer use beyond programmers, the graphics interface came to better represent the values of the broader community of users. why is it then that when we look at today’s computers we see no sign of this struggle, which only recently ended? feenberg’s answer to this question explains why he thinks heidegger missed what he missed. when the design process is complete, the value-laden choices that went into it are ‘black- boxed’, sealed into ‘the technical code’ (p. ). this hard-wiring of speci� c cultural values into our technical devices obscures the fact that these values were chosen, and this reinforces a fatalistic attitude toward technology. such an analysis leads feenberg to suggest that heidegger falls victim to the ‘deterministic illusion’ technological ‘closure’ produces (p. ) because he ‘doesn’t view modern technology from within’ (p. ). it is certainly true that heidegger did not have much internal experience with technology (he did not own a television and wrote his more than one hundred book-length manuscripts all by hand; he would not even type, let alone ‘word-process’, and it is not hard to imagine what he would have thought of the voice- recognition software feenberg himself uses). this becomes a decisive point for feenberg, who concludes that heidegger has unknowingly adopted the top-down ‘strategic standpoint of the systems manager’ rather than the bottom-up ‘tactical standpoint of the human beings’ enrolled within the technological network (p. ). thus feenberg responds to heidegger with foucault, supplementing the view from above with the ‘view from below’, adding the perspective of the many ‘subjugated knowledges that arise in opposition to a dominating rationality’ (p. ). every program has its ‘anti-program’ (p. ), feenberg shows, because the dominating rational order only comes into existence in opposition to a subjugated group. the hope for a democratization of technology is thus placed with such subjugated groups who, feenberg convincingly argues, could increasingly come to intervene in the design process. of course, to do so they must overcome the technocratic inertia produced by the vested interests embodied in the technical code (which, like heidegger, marcuse, feenberg bentham’s panopticon, eliminates the need for someone actually to occupy the dominant subject position). can feenberg tell us how we are to do this? he should be able to, since he is so critical of the fact that heidegger ‘offers no criteria for constructive reform’ (p. ). in fact, there is a tension in feenberg’s positive view which re� ects the difference between the marcusean and heideggerian positions he has synthesized. he vacillates between an optimistic, marcusean, may ‘ , ‘progress will be what we want it to be’ view which exalts the human capacity to control our future through strategic interventions in the design process (p. ), and a more pessimistic heideggerian view which suggests that while we cannot directly control the historical direction in which technology is taking us, we can nevertheless impact the future in small ways by learning to recognize, encourage, and support technological democratizations when they occur. but in the end, feenberg’s optimism wins out, and takes him beyond the alternatives envisioned by marcuse and heidegger. for feenberg holds that ‘[w]hile the technocratic tendency of modern societies is no illusion, it is nowhere near as total as its adversaries once feared’ (p. ). the birmingham school has taught him that the ‘power structure of advanced societies’ is ‘a contestable “hegemony” rather than a “total administration” ’ (p. ). in so far as the technocracy is not totalizing (as both marcuse and heidegger thought it would be), resistance to it need not take the utopian form of trying to transform the entire system at once. so feenberg replaces heidegger’s epochal view of revolutionary historical change with a progressivist, evolutionary model. clearly, feenberg does not like heideg- ger’s idea that we must wait for ‘another god’, that is, a radically transformative cultural event which would successfully realign our values in one fell swoop. yet here i can’t help wondering, isn’t ‘may ’ ’ the name for an event in which such a god seemed for a time to arrive? feenberg’s own project is certainly deeply motivated by the experiences of this event and the historical possibilities it revealed. feenberg nevertheless claims to be content to advocate an activism which is ‘far more modest in its ambitions’ (p. ). he does not follow marcuse’s emphasis on possible resistances to technocracy which come from ‘“without” (art, philosophical critique, the instincts, the third world)’ (p. ); rather, he advocates a progressive reform which taps into the ‘radical political resources immanent to technologically advanced societies’ (p. ). feenberg’s goal is what he calls ‘deep democratization’, that is, a short-circuiting of the administrative ‘suppression’ of resistances which would ‘permanently open the strategic interiority to the � ow of subordinates’ initiatives’ (p. ). but feenberg does not rid himself of all revolutionary ambitions; as he calls for the establishment of this permanent democratic voice in the design process, he situates his project within the broader movement known as radical democracy. feenberg’s hope is that the proliferation of situated micro- iain thomson struggles will eventually lead to a ‘convergence’ in which aids patients join together with environmentalists, minitel hackers, progressive medical researchers, and the like, in order to form a ‘counter-hegemony’ capable of permanently democratizing technological design and so gaining some control over the historical impact of technology. but if the goal is not simply democratic control for the sake of control, if, rather, this endeavor is ‘pre� gurative’, that is, if its goal is ‘to open up a possible future’ other than enframing or technocracy (p. ), then in the end feenberg’s powerful and important project may remain closer to heidegger than his marcuseanism allows him to acknowledge. n o t e s richard wolin (ed.), the heidegger controversy (new york: colombia university press, ), p. . see douglas kellner, herbert marcuse and the crisis of marxism (london: macmillan, ), pp. – ; and rolf wiggershaus, the frankfurt school: its history, theories, and political signi� cance, trans. m. robertson (cambridge, ma: mit press, ), pp. – . herbert marcuse, ‘the struggle against liberalism in the totalitarian view of the state’ ( ), negations: essays in critical theory, trans. j. j. shapiro (london: free association books, ), p. . marcuse, reason and revolution: hegel and the rise of social theory, nd ed. (new york: humanities press, ), p. ; ibid., p. ; franz neumann, behemoth: the structure and practice of national socialism, – (new york: oxford university press, and ), p. (marcuse clearly shared neumann’ s view; see kellner, ‘technology, war, and fascism: marcuse in the s’, in herbert marcuse, technology, war, fascism, kellner [ed.], [london: routledge, ], p. ); marcuse, reason and revolution , p. (schmitt, of course, was celebrating rather than bemoaning this fact; see marcuse, ‘the struggle against liberalism in the totalitarian view of the state’, p. , note ). adorno’s may , letter to horkheimer is quoted by kellner in ‘technology, war, and fascism’, p. note . kellner, ‘technology, war, and fascism’, op. cit., pp. – ; see also wiggershaus, the frankfurt school, op. cit., pp. – . kellner, ‘technology, war, and fascism’, ibid., p. ; marcuse, reason and revolution , op. cit., p. . marcuse, ‘some social implications of modern technology’ ( ), in marcuse, tech- nology, war, fascism, op. cit., p. . cf. feenberg (p. ). heidegger, january , letter to marcuse (in wolin [ed. and trans.], the heidegger controversy, p. ); ibid., p. . marcuse indiscriminately associated heidegger with schmitt and bäumler in ’s ‘the struggle against liberalism in the totalitarian view of the state’, op. cit. (see pp. – ). marcuse, may , letter to heidegger (in wolin [ed.], the heidegger controversy , ibid., p. ; i am reading marcuse’ s self-reference as a displaced warning to his old mentor). the irony of their estrangement is compounded by the fact that heidegger understood his critique of technology as his philosophical repudiation of nazism; see hubert l. dreyfus, ‘heidegger on the connection between nihilism, art, technology, and politics’ , in c. guignon (ed.), the cambridge companion to heidegger (cambridge: cambridge university press, ). augustin berque, e^ tre humains sur la terre (paris: gallimard, ), p. ; translated and quoted by feenberg (p. ). heidegger, marcuse, feenberg heidegger, the question concerning technology , trans. w. lovitt (new york: harper & row, ), p. ; martin heidegger, ‘traditional language and technological language’ , trans. w. gregory, journal of philosophical research xxiii ( ), p. . heidegger, discourse on thinking, trans. j. anderson and e. freund (new york: harper & row, ), p. ; heidegger, ‘traditional language and technological language’ , ibid., p. ; jean baudrillard, the transparency of evil, trans. j. benedict (london: verso, ), p. . but cf. feenberg (p. ); and albert borgmann, holding on to reality (chicago: university of chicago press, ), pp. – . heidegger, the question concerning technology , op. cit., p. . ‘humanity does not have control over unconcealment itself’ (heidegger, the question concerning technology , ibid., p. ). indeed, the very attempt to control technology – ‘the will to mastery which becomes all the more urgent the more technology threatens to slip from human control’ (ibid., p. ) – is for heidegger part of the problem; willful ontic attempts to control or manage technology risk reinforcing the nietzschean ontotheology of eternally recurring will-to-power ultimately responsible for our technological epoch of ‘the atomic age’. martin heidegger, discourse on thinking, op. cit., p. . heidegger, the question concerning technology , p. ; ibid., pp. – ; ibid., p. . howard rheingold, ‘look who’s talking’, wired (jan. ), p. ; ibid., p. ; heidegger, discourse on thinking, p. ; ibid. (my emphasis). here we have at least one of the heideggerian ‘criteria’ feenberg seeks: to relate comportmentally to technological things with gelassenheit means, minimally, to be able to let them go, to be able to live without the television, cell phone, pager, fax machine, internet hook-up, etc. of course, counter-examples like the pace-maker and hearing-aid suggest that heidegger’ s criterion needs further re� nement. donald kraybill, the riddle of amish culture (quoted by rheingold, ‘look who’s talking’ , op. cit., p. ). herbert marcuse, ‘the struggle against liberalism in the totalitarian view of the state’, op. cit., p. ; karl marx, capital (volume one), in the marx-engels reader , nd, r. tucker (ed.) (new york: norton, ), p. . hubert l. dreyfus, ‘heidegger on gaining a free relation to technology’ , in andrew feenberg and alastair hannay (eds), technology and the politics of knowledge (bloomington, in: indiana university press, ), p. . for a philosophical defense of heidegger’ s hope for a ‘new beginning’ , see my ‘ontotheology? understanding heidegger’ s destruktion of metaphysics’ , forthcoming in the international journal of philosophical studies . on heidegger’ s understanding of this alternative, see ibid., pp. – . marcuse, ‘some social implication of modern technology’ , op. cit., pp. – ; see heidegger, poetry, language, thought, trans. a. hofstadter (new york: harper & row, ), pp. – . if television has been the best ‘opiate of the people’ since religion, the internet has the potential to function more like a psychedelic, opening minds and increasing rather than diminishing the interaction between self and world. feenberg himself steadfastly defends the democratic potentials emerging within recent forms of cyber-optimized political networking, paying less attention to the political dangers ‘lurking’ here as well. marcuse, ‘some social implication of modern technology’ , p. ; ibid., p. . thanks for this example go to john senion. cf. charles spinosa, fernando flores, and hubert l. dreyfus, disclosing new worlds: entrepreneurship, democratic action, and the cultivation of solidarity (cambridge, ma: mit press, ), pp. – . feenberg pokes fun at heidegger’ s critique of typewriting (see heidegger, parmenides , trans. a. schuwer and r. rojcewicz [bloomington, in: indiana university press, ], pp. – ). i � nd it remarkable, however, that in heidegger already recognizes (in the replacement of handwriting by typewriting) a symptom of our ontological transformation toward enframing, a transformation which only becomes obvious once typewriting itself is replaced by word-processing. for a convincing argument to this effect, see hubert l. dreyfus and charles spinosa, ‘highway l. bridges and feasts: heidegger and borgmann iain thomson on how to af� rm technology’ , man and world ( ), no. . when heidegger looked out at the highway interchange and the powerplant on the ister and found words which now seem to describe those developments we associate with the internet, genetic research, and cloning, his was not what auden called ‘the dazed uncomprehending stare / of the danubian despair’ . here feenberg follows the foucaultian thinker michel de certeau rather than ‘the � nal foucault’, who abandoned his own earlier focus on the power-resistance isomorphism in favor of an ‘aesthetics of the self’ after his concrete genealogies taught him that such resistances are too often re-inscribed into the system so as to expand and reinforce its rule. (for a particularly ironic example, we might think of the way in which the new left student movement inadvertently catapulted reagan to power.) for heidegger, history does not � ow in a smooth, uniform succession; the wheel of history turns in starts and stops, revolutions catalyzed by climactic events which set the tone for the epoch which follows; cf. thomas kuhn, the structure of scienti� c revolutions (chicago: university of chicago press, ). on heidegger’ s understanding of a new ‘god’, see my ‘the silence of the limbs: critiquing culture from a heideggerian understanding of the work of art’, enculturation ( ), no. . see ernesto laclau and chantal mouffe, hegemony and socialist strategy: toward a radical democratic politics (london: verso, ). one problem with staking the future of the new left on the hope that local, situated micro- struggles will converge into a democratizing counter-hegemony is the fact that our recent political history seems to demonstrate that egalitarian groups have great dif� culty building and maintaining large-scale alliances. leftist anti-authoritarianism and distaste for coercion often generate an insistence on communal unanimity which (especially when combined with the tendency toward radical self-critique) tends to splinter and divide egalitarian alliances. see michael thompson, richard ellis, and aaron wildavsky, cultural theory (boulder: westview press, ), pp. – . for heidegger, such democratization for the sake of control would be, at best, an attempt to roll back the wheel of history, reconstituting modern subjects out of post-modern resources . received august iain thomson, department of philosophy, university of new mexico, humanities building, albuquerque, nm , usa heidegger, marcuse, feenberg microsoft word - hhe ibf.rtf special section hum. hered. ; :i-vi contents, vol. , founded as ‘acta genetica et statistica medica’ by gunnar dahlberg editors: m. hauge, odense l. beckman, umeå editorial board: l. gedda, rome r. grubb, lund k. henningsen, københavn d. klein, geneve t. larsson, stockholm h. lehmann, cambridge margareta mikkelsen, københavn j. mohr, københavn a. e. mourant, london h. nachtsheim, berlin j. v. neel, ann arbor, mich. p. o. pedersen, københavn marianne rasmuson, umeå s. refsum, oslo l. d. sanghvi, bombay m. siniscalco, napoli t. sjörgen, göteborg e. t. o. slater, london a. c. stevenson, oxford e. strömgren, risskov r. turpin, paris f. vogel, heidelberg m.whittacker, exeter a. c. allison, harrow a. g. beam, new york, n.y k. berg, oslo j. a. book, uppsala l. l. cavalli-sforza, stanford, calif. t. j. david, manchester e. essen-möller, lysekil f. c. fraser, montreal j. a. fraser roberts, london n. freire-maia, curitiba j. frézal, paris s·karger ·basel ·münchen •paris •london · new york· sydney drug dosage. the author and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. however, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. this is particularly important when the recommended agent is a new and/or infrequently employed drug. all rights reserved. no part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. s.kargerag, p.o.box, basel (switzerland) printed in switzerland by buchdruckerei national-zeitung + basler nachrichten ag, basel contents vol. , no. original paper alpha- -antitrypsin phenotypes in french canadian newborns joly, j.; richer, g.; boisvert, f., and laverdière, m survey on haemoglobin variants, ß-thalassaemia, glucose- -phosphate dehydrogenase deficiency and haptoglobin types in turkish people living in manavgat, serik and boztepe (antalya) aksoy, m.; dinçol, g., and erdem, s interaction of sickle cell trait and glucose- -phosphate dehydrogenase deviciency in cameroon bernstein, s.c.; bowman, j.e., and kaptue noche, l transferrin, haptoglobin and group-specific component types in tribal populations of andhra pradesh goud, j. d. and rao, p.r the’ ’ variant of human placental alkaline phosphatase is identical to the ‘d-variant’ doellgast, g.j.; kennedy, m., and donald, l.j sjögren-larsson syndrome in dizygous twin sisters david, tj human red cell galactose- -phosphate uridylyltransferase (ec . . . ). electrophoretically determined polymorphism in denmark and its use in paternity cases eriksen, b. and dissing, j genetic studies of red cell glutamic-pyruvic transaminase in some japanese populations nishigaki, i.; 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dey, b.; das, s.k.; roy, m.; banerjee, s., and gupta, r maternal age and dermatoglyphic asymmetry in man: another look young, r.s. and reed, t partial lipodystrophy and familial c deficiency mclean, r.h. and hoefnagel, d serum ige levels in twins sistonen, p.; johnsson, v.; koskenvuo, m., and aho, k mitochondrial malic enzyme polymorphism in an indian population ghosh, u.; banerjee, p.k., and saha, n genetic characteristics of hemophilia a in chile cruz-cokem, r. and rivera, l study of red blood cell and serum enzymes in five pyrenean communities and in a basque population sample vergnes, h.¦constans, j.iquilici, j.c.; levèvre-witier, p.; sevin, j., and stevens, m protein and enzyme polymorphisms in affective disorders in northern greece parisi, h.a.; lanara, e.c.; triantaphyllidis, cd effects of parental consanguinity on mortality and reproductive function lindelius, r. comparing the ‘family set’ approach to estimate heritability with other conventional estimators: a monte carlo simulation rodriguez, a.; chakraborty, r., and schull, w.j contents v no. original paper is the hpo phenomenon in tropical populations really genetic? rougemont, a.; quilici, m; delmont, j., and ardissone, j.p some biochemical polymorphisms in members of the gaddi tribe of himachal pradesh kaur, h.; sehajpal, p.k.; khanna, a.k., and shrivastava, p.k isoenzymes of red cell glyoxalase i (ec . . . ) in a finnish population uotila, l. and koivusalo, m polymorphism of the sixth component of complement (c ) in norwegian lapps olving, j.h.; teisberg, p., and olaisen, b search for linkage between dermatoglyphic sole characters and blood groups bener, a plasma protein and enzyme polymorphisms in belgium brocteur, j.; hoste, b., and andre, a mohr-wriedt (a ) brachydactyly. analysis of a large brazilian kindred freire-maia, n.; maia, n.a., and pacheco, c.n.a erythrocyte glyoxalase i and esterase d polymorphisms in four french populations vergnes, h.; meyer, s.; weil, d.; goudemand, j.; brevière, d.; sevin, j., and constans, j frequencies of gm and km allotypes in the population of singapore, sri lanka and punjabis in north india daveau, m.; rivat, l.; lalouel, j.m.; langaney, a.; roberts, d.f., and simons, m.j polymorphism of the second component of complement (c ) in graves’ disease noel, e.p.; sampson, l.; pepper, b.m., and farid, n.r electrophoretic investigation of γ-glutamyl-cyclotransferase from human erythrocytes board, p.g population studies in cameroon. hemoglobin s, glucose- -phosphate dehydrogenase deficiency and falciparum malaria bernstein, s.c.; bowman, j.e., and kaptue noche, l short communications glyoxalase i (glo; ec . . . .). gene frequency variation in iraq al-agidi, s.k.; papiha, s.s., and shukri, s.m high frequency of ina antigen among iranians and arabs badakere, s.s.; vasantha, k.; bhatia, h.m.; ala, f.; clarke, v.a.; moesri, r.; sommai, s., and amin, a.b book review no. original paper dermatoglyphic traits in an amish-mennonite population in ontario tutton, d.a. and zachariah, k glucose- -phosphate dehydrogenase in an afro-american population calvert, a.f. and trimble, g.e a note on the frequency of consanguineous marriages in reading, england in / coleman, d.a properdin factor b (bf) as an exclusion determinate in parentage testing dykes, d. and polesky, h vi contents genetic studies on the chenchu tribe of andhra pradesh, india ramesh, a.; blake, n.m.; vijayakumar, m., and murty, j.s alpha- -antitrypsin types and chronic obstructive lung disease in an industrial community in northern sweden beckman, g.; beckman, l.; mikaelsson, b.; rudolphi, o.; stjernberg, n., and wiman, l.-g variations on dermal ridges in nine population groups of maharashtra, india. i. intra- and interpopulation diversity malhotra, k.c.; chakraborty, r.; bhanu, b.v., and fulmali, p.m transferrin c subtypes and spontaneous abortion beckman, g.; beckman, l., and sikström, c probability calculations in pedigrees under complex modes of inheritance lalouel, j.m ß + -thalassemia intermedia. genetic and biochemical study of a family including cases philip, t.; souillet, g.; philippe, n.; freycon, f.; bektas, s.; morlé, l.; trabuchet, g., and godet, j. short communication distribution of abo and rh (d) blood groups among four endogamous groups of andhra pradesh reddi, a.p.; mukherjee, b.n., and ramachandraiah, t no. original paper a new unstable pi m variant of dp antitrypsin in a finnish isolate frants, r.r. and eriksson, a.w studies on the immunoglobulin allotypes of asiatic populations. viii. immunoglobulin allotypes among the tuvinians of the ussr schanfield, m.s.; alexeyeva, t.e., and crawford, m.h segregation analysis of schizophrenia under a mixed genetic model carter, c.l. and chung, c.s slow-moving serum albumin variant in a south indian tribal population walter, h.; veerraju, p., and hilling, m glucose- -phosphate dehydrogenase deficiency in south vietnamese panich, v.; bumrungtrakul, p.; jitjai, c; kamolmatayakul, s.; khoprasert, b.; klaisuvan, c; kongmuang, u.; maneechai, p.; pornpatkul, m.; ruengrairatanaroje, p.; surapruk, p.; and viriyayudhakorn, s dermatoglyphic peculiarities in hypospadias micle, s. and fried, k absent dtriradius and dotting of the ridges in siblings wertelecki, w.; plato, c, and plato, c.c heritable salivary proteins and dental disease friedman, r.d.; azen, e.a.; yu, p.l.; green, p.a.; karn, r.c., and merritt, a.d transferrin c subtypes in us blacks and whites kueppers, f. and harpel, b.m short communication glo polymorphism in iceland karlsson, s.; arnason, a., and jensson, Ó announcements author index subject index book reviews journal of medical genetics, , , - medical genetic studies of the amish. selected papers by v. a. mckusick. (pp. x + ; figures + tables. £ - .) baltimore and london: johns hopkins university press. . to read this edited collection of papers is both a fascinating experience and, for one already familiar with the amish, a pleasurable reminder of the persistence and success of their remarkable way of life in th century america. a visitor to the moore clinic at johns hopkins hospital in the late s might have been excused if he had thought that the amish formed a majority of the baltimore popula- tion, but this book shows clearly how worthwhile the intensive study of this group has proved in both medical and population genetics. the papers are reproduced unchanged, but their value is greatly increased by the addition of notes on subsequent developments. they are grouped into those dealing with general and population genetics, studies of previously recognised mendelian syn- dromes, and (the largest section) 'new recessively inherited entities, many of which would have been difficult, if not impossible, to recognise outside the setting of a defined, closed, and well documented community as provided by the amish'. among these delineations of new disorders, that of 'cartilage hair hypoplasia' still stands out for its clarity and its masterly combination of clinical and genetic information. the value of the studies in this book extends far beyond the demonstration that the disorders in question are recessively inherited. the range of clinical expression produced by what clearly must be a single gene is something that is difficult to document in the more heterogeneous patients seen in most populations. the studies of autosomal recessive limb-girdle muscular dystrophy and of the 'troyer syndrome' provide excellent examples of this. another particularly impressive feature of the amish studies is the detail and accuracy of the genealogical and demographic work which underlies them, much of it due to the attachment of amish people themselves to this type of documentation. it is of interest that a number of the main contributors to the book are themselves of amish origin, and it is fortunate that the amish antipathy to further education has not prevented the emergence of those individuals to transmit the unique aspects of amish culture to the world in general. anyone working in medical genetics to whom the amish are unfamiliar should read this book without delay; it will be a revelation as to the value of studying genetic isolates. those who do know of the amish will not need encouragement to refresh their memory of the subject and gain new ideas. it should also serve as a stimulus for people to study their own equivalents of the amish. though few of us have access to such an ideal population, there are many minority groups which would repay much closer investigation, where studies of genetic diseases have not been attempted. many of these groups, less robust than the amish, may soon disappear for ever if the opportunity is not taken soon. p. s. harper british medical bulletin: the hla system vol. , no. , september . scientific editor, w. f. bodmer. (figures + tables. £ o .) london: the british council. . medical journals are now replete with articles on hla and, though weary voices have been heard to exclaim 'not hla again', we should take heart. cytogenetics had similarly inauspicious beginnings when, years ago, pictures of 'squashed spiders' began to appear in medical journals. there are many aspects of hla which are relatively simple, though others seem comprehensible only to experienced travellers in the exotic lands of lymphomania. the latest issue of the british medical bulletin provides an excellent overview of the hla system and will do much to enlighten the weary. it is a convenient introductory package for this fascinating genetic system. i recommend reading 'evolution and function of the hla system' at an early stage as, in it, central issues relating to the system are dealt with succinctly and comprehensibly. the other articles can be divided into several categories. firstly, the 'technology' of hla: serology, cellular typing, the chemistry of hla antigens, and the 'new' hla- drw locus are considered in articles. secondly, the role of hla as part of the major histocom- patibility system (mhs) is discussed in articles devoted to complement genetics and to the immune response. thirdly, the somewhat variable importance of hla in human organ transplantation is described objectively. the fourth category, that of the associa- tion between hla and various diseases, takes up a large part of the bulletin in a total of papers o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . -a o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ reviewing arthropathies, multiple sclerosis, diabetes whole, as a few examples will show. single immuno- mellitus, coeliac disease, liver disease, acute leuk- globulin polypeptides are coded for by two or more aemia and hodgkin's disease, and finally tropho- genes; families of multiple closely linked genes are blastic tumours. unfortunately, the genetic linkage characteristic of immunology, but may well be a between hla and -hydroxylase deficiency was basic phenomenon of mammalian germ lines; discovered too late for inclusion in the bulletin. immunoglobulins demonstrate the importance of -hydroxylase deficiency is a paradigm of genetic somatic mutations; the interaction between different disease as the genetics, biochemistry, and treatment lymphocyte types exerts control over protein syn- are so well worked out. never before have we had thesis, presumably by some form of genetic feedback. such razor-sharp dissection of a segment of a human last, but not least, allelic exclusion (involving auto- chromosome and been able to discern, even if we do somes) was discovered in a study of antibody not always fully understand, the interactions of molecules. closely linked genes, the effects of linkage disequili- fudenberg's book covers all these areas with great brium, and the emergence of disease associations. clarity, tackling with skill a plethora of experimental without doubt, hla will be of the greatest practical data from both comparative and human studies. an importance in the prediction of those at risk of abundance of tables and diagrams is a particularly developing many diseases with a clear genetic basis praiseworthy feature of the book. as in the first but which depend on environmental triggers. as our edition, chapter is an introductory essay which is understanding of this system increases, we will be well worth reading. chapter describes immuno- able to detect and potentially remove offending globulin structure and evolution and goes on to a environmental agents thus allowing true prevention. lucid exposition of the admittedly complicated this issue of the british medical bulletin is first genetics of immunoglobulin molecules. chapter rate and is obligatory reading for every clinical tackles the generation of antibody variability and geneticist. it is remarkable value at £ - and is describes what is known of the genetics of antibody perfectly complementary to 'basic immunogenetics' specificity. chapter deals succinctly with lymphoid by fudenberg and his colleagues which is reviewed membrane antigens including their genetic control below. and relation to immune response, t and b cells, and r. harris lymphocyte interactions. the potted version of mouse h- is excellent. chapter is an adequate basic immunogenetics review of the human blood group systems and the nd edition. by h. h. fudenberg, j. r. l. pink, book finishes, as before, with a number of useful an-chuan wang, and s. d. douglas. appendices. (pp. ix + ; figures + tables. £ * .) the index is generally good, though there are new york and oxford: oxford university press. occasional omissions. for example, i encountered . 'fd' in the text but could not find an entry in the index or in the appendices. this edition went to press since the first edition was published in , a great before the seventh international histocompatibility deal has happened in the field of immunogenetics workshop and before most of the new information and it is remarkable that dr fudenberg and his on hla-dw and -drw loci became available. colleagues have managed to limit the expansion of (this gap is filled admirably by 'the hla system' the second edition to only pages. the use of finer in no. of vol. of the british medical bulletin quality paper has also kept the overall dimensions which should be read in parallel with this book and of the book about the same. one has to confess at is reviewed above.) the section on hlawill no doubt the outset that immunogenetics is difficult. i wonder, be extended in a third edition. these criticisms do not for example, howmany immunochemists feel at home detract significantly from 'basic immunogenetics' in the new and rapidly expanding sister-field of cell which remains an essential for the libary of gene- immunology? taken together, the subject matter of ticists of all persuasions, both graduate and under- this book, which covers the chemistry and genetics graduate. of antibody molecules, cell mediated immunology, r. harris and human blood group serology, is a highly concentrated collection of facts and theories. one the genetics of agig should not, however, succumb to the temptation of edited by e. l. schneider. (pp. xvi + ; leaving immunogenetics to the experts in the hope figures + tables. $ - .) new york and that it will become easier as time goes by, for already london: plenum press. . the subject has produced a number of unexpected homer likened the generation of man to the fall of observations with wide relevance to genetics as a leaves. addison introduced a continuous stochastic bo-ok reviews o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . -a o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ © indian journal of plastic surgery | published by wolters kluwer - medknow introduction osteomas are slow growing, innocuous, benign osteogenic tumours composed of compact and/or cancellous bone. the three variants described are peripheral, central and extraskeletal types. [ ] the central osteoma arises from the endosteum, the peripheral osteoma from the periosteum, and the extraskeletal osteoma usually develops within a muscle. generally, osteoma is seen as a solitary lesion though multiple osteomas are found in gardner’s syndrome. they are mostly restricted to the craniofacial skeleton involving the paranasal sinuses and mandible and rarely involve the maxilla. it is usually found over the angle and inferior border of the mandible; an osteoma developing in the sigmoid notch of the mandible is extremely rare. [ ] we report a case of a solitary periosteal osteoma arising from the sigmoid notch of the mandible in a -year- old woman. case report a -year-old female presented with a gradually enlarging painless swelling on the left cheek in the preauricular region for the past years. there was no other significant medical history and no history of any trauma. on examination, there was facial asymmetry but no trismus, deviation of the jaw on mouth opening, difficulty with mastication or neurological deficits. access this article online quick response code: website: www.ijps.org doi: . / - . case report bony tumour in an unusual location on the mandible aashish sasidharan, amish gohil, santosh koshy , ashish kumar gupta departments of plastic and reconstructive surgery and oral and maxillofacial surgery, christian medical college and hospital, vellore, tamil nadu, india address for correspondence: dr. aashish sasidharan, department of plastic surgery, christian medical college and hospital, vellore - , tamil nadu, india. e-mail: draashish @gmail.com abstract osteomas are benign osteogenic tumors that are seen in the facial bones, but uncommonly in the mandible. in the facial bones, both central and peripheral osteomas have been described. peripheral osteomas have been described to occur in the frontal, ethmoid, and maxillary sinuses but are not common in jawbones. when in the mandible, they are usually found over the angle and inferior border of the mandible. we report on a solitary peripheral osteoma located unusually in the sigmoid notch of the left mandible causing facial asymmetry. key words mandible; mandibular notch; osteoma; sigmoid notch; tumor this is an open access article distributed under the terms of the creative commons attribution-noncommercial-sharealike . license, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. for reprints contact: reprints@medknow.com how to cite this article: sasidharan a, gohil a, koshy s, gupta ak. bony tumour in an unusual location on the mandible. indian j plast surg ; : - . published online: - - sasidharan, et al.: bony tumour in an unusal location on the mandible on extra-oral examination, a swelling was observed in the left preauricular region which became more prominent on opening the mouth. palpation revealed a single, hard, non-tender, fixed swelling . cm × . cm × . cm large that appeared to be arising from the mandible [figure ]. the overlying skin was free. a non-contrast computed tomography (ct) scan showed a well-circumscribed, bone-like hyper dense, sclerotic image with a broad base and lobulated surface on the left mandibular notch [figure ]. a provisional diagnosis of osteoma was made. under general anesthesia, the lesion was approached intraorally, vestibular incision taken on left side mandible ramus. the lateral surface of ascending ramus was exposed up to the coronoid process. as the medial placement of the coronoid prevented access to the base of the tumor, a coronoidectomy was performed,. the lesion was then excised using an oscillating saw blade. figure shows the excised specimen. primary closure of the wound was achieved with - vicryl. the postoperative period was uneventful. histopathology findings showed the tumor to be composed of trabeculae of dense, sclerotic, mature lamellar and woven bone with a pauci-cellular fibro collagenous stroma [figure ]. there was no evidence of malignancy. based upon the clinical, radiological and histological findings, a definitive diagnosis of peripheral osteoma was established. the patient has presently been followed up regularly over the past months and there has been no recurrence of the swelling [figures and ]. discussion osteoma is a benign, slow growing bone tumor that presents as a protruding mass, frequently arising from the craniomaxillofacial bones and characterised by proliferation of compact, lamellar cortical bone. it arises from the paranasal sinuses, most commonly the frontal sinus. peripheral osteomas have been reported to occur figure : swelling over left preauricular region, prominent on opening mouth figure : three-dimensional computed tomography face showing a radio- opaque well defined swelling . cm × . cm × . cm at left mandible notch figure : excised specimen measuring . cm × . cm × . cm figure : interwoven trabeculae of mature lamellar bone with intervening lose stroma (h and e × ) indian journal of plastic surgery may-august vol issue sasidharan, et al.: bony tumour in an unusal location on the mandible in the mandible, maxilla, external auditory meatus, temporal bones and pterygoid plates. the mandible is more frequently involved than the maxilla with reports of incidence ranging from % [ ] to . %, respectively. [ ] lesions are usually located in the angle and margin of the mandible. osteoma is most frequently observed in the second and fifth decades of life. osteomas are usually solitary; if multiple, the patient should be evaluated for gardner’s syndrome. the etiology of osteomas is still unknown. while some investigators consider it a true neoplasm; others classify it as a developmental anomaly. osteomas are often asymptomatic and detected incidentally. however, secondary to the location of the tumor, problems with occlusion, asymmetry of the face, headaches, facial pain, and impaired mandibular movements may occur. ct is the best imaging modality for determining the location, attachment, extension and anatomical relationships of the lesion. [ ] peripheral osteomas are easy to recognise by their radiographic findings. they appear as round or oval radiopaque masses with a broad base and distinct borders. [ ] if a peripheral osteoma is pedunculated, a narrow contact area can be seen between the lesion and the compact bone. in this case, the lesion consisted of dense, uniformly opaque compact bone with a broad base. histological classification differentiates among compact (dense, compact bone), cancellous (soft, spongy bone), and mixed-type osteomas. the compact osteoma consists of mature lamellar bone with few marrow spaces. the cancellous/trabecular osteoma is characterised by bony trabeculae and fibro-fatty marrow enclosing osteoblasts, surrounded by a cortical bone margin. although the compact type is the most common type seen in the mandible, in this case, the osteoma was a mixed variant. treatment includes observation with periodic clinical and radiological examination [ ] for small, non-progressing, asymptomatic, solitary lesions. surgery should be considered for large, deforming osteomas. in this case, excision was considered as the lesion was progressive and causing facial asymmetry. we employed an intraoral approach for aesthetic purposes, which also allowed easy access to this unusually placed osteoma. recurrence of peripheral osteoma after surgical excision is extremely rare. malignant transformation of peripheral osteoma has not been reported in the literature. sigmoid notch osteomas are rare and to the best of our knowledge, this is only the sixth such case reported in table : comparison between the reported cases of the osteoma of the sigmoid notch reference age (years) sex side location size (in cm) symptoms treatment bessho et al. male right lateral . × . × . swelling excision schulze female left medial . × . × . pain not available sekerci et al. male right middle not available no conservative iwai et al. female right medial . × . × . no conservative rao et al. female right lateral . × . swelling excision (intraoral) our case female left lateral . × . × . swelling excision (intraoral) figure : post-operative photo, absence of swelling over left pre-auricular region figure : three-dimensional computed tomography face postoperative at months, with absence of residual tumour/recurrence indian journal of plastic surgery may-august vol issue sasidharan, et al.: bony tumour in an unusal location on the mandible english literature. [ , - ] among the previously reported cases [table ], the mean age of presentation was years. in contrast to the lateral location of the osteoma in our case and that reported by bessho et al. [ ] and rao et al.,[ ] the others were in the middle [ ] or the medial aspect of the sigmoid notch. [ , ] conservative management was adopted in cases where the lesion was asymptomatic; in this case progressive enlargement of the lesion and facial asymmetry warranted surgical excision. financial support and sponsorship nil. conflicts of interest there are no conflicts of interest. references . bessho k, murakami ki, iizuka t, ono t. osteoma in mandibular condyle. int j oral maxillofac surg. ; : - . . kaplan i, calderon s, and buchner a. “peripheral osteoma of the mandible: a study of new cases and analysis of the literature,” journal of oral and maxillofacial surgery, vol. , no. , pp. - , . . shulze d.radiographic diagnostic: osteoma of the left mandibular notch. quintessence int, ; : - . . sekerci ae, sahman h, ertas et, sisman y. an unusal peripheral osteoma of the mandibular notch: a case report with computed tomography evaluation. oral radiology. ; : - . . iwai t, izumi t, baba j, maegawa j, mitsuda k, tohnai i. peripheral osteoma of the mandibular notch: report of a case. iran j radiol. ; : - . . rao s, rao s, pramod dsr. transoral removal of peripheral osteoma at sigmoid notch of the mandible. j maxillofac oral surg. mar; : - . indian journal of plastic surgery may-august vol issue document resume ed so author paulston, rolland g. title separate education as an ethnic survival strategy: the finlandssvenska case. institution pittsburgh univ., pa. international and development education program. pub date nov ' note p.; paper presented at the annual meeting of the american anthropological association (washington, d.c., november ) edrs price mf-s . hc- . plus postage. descriptors *case studies; comparative education; controlled environmentz culture conflict; *educational programs;- educational research; elementary secondary education; ethnic grouping; *ethnic groups; ethnic studies; ethnocentrism; foreign countries; group status; *group unity; minority groups; nationalism; social change; *typology identifiers *finland abstract a typology of ethnic education programs is suggested and a case study is presented where separate education for an ethnic group has been used to maintain and defend a separate ethnic identity. the typology rates ethnic education programs both by degree of normative and structural change being sought and by degree of control by ethnic groups. supplemental ethnic studies education programs have low structure change and low ethnic control; alternative prograns, such as black, chicano, and native american studies, have high structure change and low ethnic control; defensive programs, such as the amish who seek to control the content and ideological orientation of their educational programs, have low structure change and high ethnic control; and transformative programs, such as the american indian movement which takes a militant approach, have high structure change and high ethnic control. the case study examines a declining elite group which is striving to maintain ethnic identity and boundaries through defensive programs--the svedish-speaking community in finland. the minority community uses a variety of formal and nonformal educational programs to sustain group solidarity and ethnic identity. history and description of the rationale, ideology, goals, organization, and various data of the social movement show how and why the groupls educational programs are classified as defensive. although the swedish-speaking population has declined in numbers and influence since world war ii, their separatist policy has defended their ethnicity. if decline continues, however, ethnic integration might occur. an extensive bibliography is included. (nd) documents acquired by eric include many informal unpublished materials not available from other sources. eric makes every effort to obtain the best copy available. nevertheless, items of marginal reproducibility are often encountered and this affects the quality of the microfiche and hardcopy reproductions eric makes available via the eric document reproduction service (edrs). edrs is not responsible for the quality of the original document. reproductions supplied by edrs are the best that can be made from the original. permission to reproduce this copy righted material has been granted by fonaxal. ata/si-oa..) tg eric and organizations operating under agreements with the national in st,tote of education further repro duction outside the eric system re. suires permission of the copyright owner u s department of health, education &welfare national institute op education this document has been repro- duced exactly as received from the person or organization origin- ating it points of view or opinions state do not necessarily repre sent official national institute of education position or policy separate education as an ethnic survival strategy: the finlandssvenska case rolland g. paulston international and development education program school of education university of pittsburgh pittsburgh, pennsylvania november paper presented at the american anthropological association annual meeting, washington, d. c. , november i .ase do not use without permission. although the c.thnicity-educational change nexus would seem to be of increasing interest to educational anthropologists, there has been surprisingly little effort made to systematically delineate variables or examine relationships in specific field settings. we have, for example, little knowledge of how and to whai effect various ethnic groups have influenced ethnic-education programs in public schools or have created and used their own non-formal educational programs outside of super- ordinate control. we need to determine under what conditions groups mobilizing around cultural symbols- . e., the "old" ethnicity,.as well as groups seeking to use ethnicity in resource competition--i.e., the "hew" ethnicity--have sought to use education in formal schools, in non-formal programs, and via the media and resocialization, et al, to help achieve ethnic-movement goals for individual and social change (bennett, ; despres, ;, collins, ). a second research question might well ask about the pedagogical aims, processes, and outcomes of ethnic-education programs in various configurations of ideology and autonomy. in addition, we will also need to ask a third question concerning evaluation of ethnic-education programs. here, we need to ascertain both anticipated and. unan,ticipated outcomes, and to produce generalizations concerning key factors influencing relative educational-program effectiveness.. in this paper, i would like to suggest a typology of ethnic-education programs and examine a case where separate education has been used since wprld war to maintain and defend a separate ethnic identity. where most anthropological studies of ethnicity and education examine the educational problems of weak, low-status ethnic groups in,superordinate efforts to assimilate, enfold, or isolate, this case study will examine a rather unusual situation where a declining et. ltural, economic, and social elito has sought to maintain ethnic ideralty and boundaries through control of a separate swedish-speaking school system and widespread non-formal educational efforts. before we examine the swede-finn case, however, it may be helpful to very briefly delineate the entire range of the phenomena under study, and to suggest some useful categories. one approach might be to sort out cases along the two dimensions of change and control as in figure one below. here, the vertical dimension is the degree of normative and structural change sought by program authorities and reflected in explicit learning goals. the horizontal dimension indicates the degree of control or autonomy from central educational authorities held by ethnic groups or movements seeking to influence ethnic-education ideology, outcomes, and the like. in the lower-left quadrant, for example, supplemental "ethnic studies, " ethnic-heritage programs, and/or "bilingual education" programs are offered in educational settings--in north america and northern europe-- dominated by superordinate elements with long traditions of using public schooling for acculturative and assimilationist ends. .where these elite elements come under pressure for greater cultural pluralism yet continue to control, the response will be type i programs,lpitfralistic educational experiences that kjolseth ( ) argues are increasingly viewed by school authorities as the most effective route to assimilation. supplemental programs are also discussed in anderson ( ), seifer ( ), and gambino ( ). where such programs come under greater influence from ethnic-revival activists, however, as in numerous black studies enclaves in higher ededucation, and numerous bilingual programs/inlocat southwestern communities whcre ethnic elements have made power gains, then ethnic-educational programs may reject essentially status-quo orientations and ethnic tokenism to seek greater ethnic consciouiness, solidarity, . and eradication of supposedly oppressive relationi with the creation of alternative programs in formal educational settings. figure one a typology of ethnic education programs alternative programs; i. e., black, chicano, native american, ef.al, studies enclaves in higher education; same formal-school bilingual education programs supplemental programs: . e., bilingual and ethnic heritage programs in formal schools transformative programs; black panther, american indian movement, and other militant ethnic--movement programs. defensive programs: i. e., knish, swede-finn, saxon german, and most reservation indian programs, danish- american folk high school programs, hebrew- schools, and nation of islam low degree of ethnic contial high if, however, the pressure for change exceeds the tolerance of superordinate control elements, alternative programs as in the upper-left quadrant will, as the structural-functionists rightly tell us, one way or another, be brought back to conformance with the consensus, or eliminated (sizemore, ). defensive programs in the lower-right-side quadrant are high in autonomy yet seek low to molleracvange. : ather, they control the content and ideological orientation of their educational programs either within their own formal-school programs--as with the amish or swede-finnsor within such ethnic formal and non-formal education programs as afternoon hebrew schools, german schools, et al, to maintain valued cultural behaviors, preserve ethnic boundaries, and resist total assimilation into mainstream society (wirth, ; dozier, ; medding, ; jonsson, , mortenson, ; hostetler, and ; national indian brotherhood, nria' i; weinman, ; gray, ; and mcarthur, ). transformative programs in'the upper-right quadrant, because of their rejection of the existing cultural hegemony and high priority on individual change and social reconstruction, will always be found, if at all, in the non-formal, .or out-of-school educational sector where greater program autonomy is possible. some examples of the more militant approach of transformative programs are included in mecarelli ( ), lejeune ( ), mason ( ), and paulston ( b). although neither space nor time is available here to place all ethnic- education programs into the typolcjy suggested, we have'noted some illustrative cases. although all ethnic-education programs should fit somewhere in this schema, exact location will, of course, shift with changes in the educational goals and program autonomy of ethnic groups involved. the, swede-finn (sf) case anas we have noted, u.. s. examples of separate education aset/hnic- survival strategy can be found in hostetler's amish studies, mortensen's work on the danish lutheran folk high schools in america, and in a number of related works on ethnic minorities. these groups have sought, with varying success, to avoid complete assimilation into the american "melting pot" by providing alternative educational programs. where the ownership of land and compact settlement reinforce ethhic-boundary maintenance, as with the amish, separate education has powerfully contributed to ethnic survival. where settlement has been scattered and ethnic-boundary maintenance weak, as with the danes, separate education, at best, served to ease acculturation and assimilation' int-) the-national culture. we must go to europe, however, for examples of relatively high- status ethnic minorities that control separate educational programs in efforts to maintain what is left of fading cultural and economic dominance. one group, for example, the "saxon" ethnic germans of southern transylvania in romania, are now facing disintegration after some eleven centuries of educational and political autonomy (mcarthur, p. ). the swedish-speaking community in finland presents, in some ways, a comparable case. yet, while they are clearly in decline, they continue to use a variety of formal and non-formal educational programs to sustain group solidarity and separate ethnic identity. in the remainder of this paper, i will address --using a culture-conflict perspective--the three research questions proposed at the outset. the swede-finn ethnic movement in finland ethnic movemenrs.as a type of.social movement begin with the articulation of a structural bind. this is most often a shared perception of injustice, . e., a painful discrepancy between the "way it is" and the "way it should be. " following a period .of more or less general discontent and unrest, movements pass through a somewhat disorganized popular state and, if successful in securing adherents and resources, they develop leadership, ideology, and movement organizations, as well as slogans, symbols, and doctrine. the final stage is often institutionalization and a tendency towards oligarchy, a lessening of the we/they polarity, and maintenance of a new status quo. the sf ethnic movement ( . e., sfem) in finland has offered--during the past half-century or so, a clear example of this process, and especially of organizational development and goal displacement as the movement pro- gressed. we should note, however, that because the sfem adherents represented a high percentage of finland's economic and social elite as well as all swedish-speaking farmers and fishermen, it is clearly an atypical ethnic movement. with its superior resources, historical dominance, and psychological advantage, and continuing, if diminished, political power, the movement presents a valuable "extreme case" of ethnic-movement efforts to use education for defense and survival under extremely advantageous conditions (myhrman, ): the rationale put forward by sfem leaders for separate education is best viewed in terms of the movement's ideology and goals. we shall, accordingly, first.examine the ideational counterparts of these observable phenomena as presented by movement intellectuals and then cbscribe and assess educational contributions to advancement of movement goals. ideology and goals of the sfem although sweden lost political control of finland to russia following the napoleonic wars, the swedish-speaking elements, some percent of the total population, continued to dominate the universities, the civil service, and economic and urban life (wuorinen, ). since the early middle ages, swedes occupied extensive coastal areas of western and southern finland where the vast majority were free peasants and fishermen. the finns, a non-scandinavian people, came from the east and settled the interior forest regions. contact between the two language-cultural 'groups took place largely through swedish burghers who tended to dominate the few urban centers in the finnish areas. swedish was also the language of government and administration, of the courts and legal system, and of all higher education and intellectual life. although considerably isolated from 'the main currents of th-century romanticism and nationalism, a finnish literary movement developed in the 's and 's. this group of young intellectuals drew on fegelian philosophy and finnish folk culture to advocate the creation of a national unity ano a national culture based on exclusive uee of the finnish language. although they wrote largely in swedish, the young finns rejected swedish as foreign and viewed finnish as the natural means of expressing the spiritual unity of the nation (hama lain, ). the swedish upper-class response to intense finnish nationalism was, to, use a swedish expression, a kulturaristokratisk policy. in the 's, for example, this small elite continued to dominate two of the four ruling estates, . e., the nobility and the burghers. thus, the emerging swedish movement at that time represented only swedish upper-class interests in defending the historical position of the swedish language and the swedish cultural heritage as well as its own privileged status and rule in the country. the swedish-speaking common folk of the town, countryside, and littoral found no place in this movement. rather, they were the only dimly realized reserves thought best left undisturbed by the raging culture conflict of the time (eklund, ). where swedish.elite perceptions of the swedish lower class was paternalistic, their view of finnish nationalists and their call for "one language-one mind" was hostile. professor c. g. est lender, a spokesman for the educated swedish classes defined this group's attitudes in his newspaper article, "my position on the language question, " quoted in myhrman ( , p. ): what i want to emphasize is that my upbringing was thoroughly swedish. "i came into conta'cts with finns all the time. my playmates were sometimes finns; the maids and the hired men were finns. so i learned the finnish language as well as the swedish. but i never regarded it very highly. to me it was the language of the maids and the hired men--and of the peasants. it is hare to describe the curious attitude i had-- and have--toward the finns. i was very fond of some of them, and i was willing to use their language to a certain extent. sometimes i even felt a passionate patriotism that included the finns as well as the swedes of my'country. but all the time there was a feeling of superiority, that i belonged to a better race, and was different from them. as i grew older, this feeling was emphasized because of the finnish attitude toward us swedes. it became absolute contempt. by the time i left finland i had no use at all for either the finns or their language, because of the behavior politically. i felt they had betrayed the country. by , however, the constitutional campaign in ppposition to mounting russification and ensuing jemocratization had ended swedith political control. with swedish control of legislation and the ar%ninistration swept away and with intensifying finnish nationism overcorning all obstacles, the swedish leaders, facing total defeat, sought a new basis on which to rebuild their cause. this effort saw the creation of the swedish people's party in ma y , a largely successful effort to reject class divisions and to mobilize the entire sf population. the need to draw the indifferent and inarticulate swedish masses into active participation in a swedish nationality move- ment to secure their heightened perception of the ethnic-threat-survival bind has subsequently been the task of a variety of movement organizations down to the present day. the movement, thus, has sought, especially before world war ii, to include all swedish-speaking finns within an ethnic solidarity group where "we-they" distinctions will be heightened and maintained. this mobiliza- tion of ethnic identity became necessary to support the movement's ideological argument that there exists in finland a political finlandic nationality. this political nationality, or ethnicity, is claimed to be based on common historical traditions, a common government,* and geographical unity. it includes the two cultural nationalities, swedish and finnish, which are based primarily on language. in this respect, the swede-finns mak tain, finland is a culturally pluralistic society like switzerland, be.,..tium, or great britain. they view finland as a "state of rights" ( ttsstat) where rights and relationships are established by law, rather than a population organized primarily on the primacy of language. they argue that both nationalities in finland have existed side by side since the beginning of finland's history. both have contributed to its development. with inde- pendence and the constitution of , both swedish and finnish are accorded the legal status of national languages. with this crucial legal equality of swedish as a national language, the sfem has aggressively pursued a policy of separatism and cultural autonomy. this has included demands for civil-service appointments of the basis of merit, and mit ethnic affiliation, and state aid for cultural and educational institutions of both nationalities in accordance with identical principles. equally important, the sf movement secured control of separate swedish-language schools, and with generous support from wealthy swede-finn industrialists, the movement has created a number of non-formal educational institutions and cultural foundations. it is to these movement-education programs that we now turn. organization ! swedish ethnicity in the struggle to survive the attempt to counter finnish nationalism with a mobilization of the entire sf population required the creation of a variety of new or adapted ethntc-movement organizations. the first swedish cultural organization in finland, the friends of the swedish public school, foutided in ,. became more widee'pread and overtly concerned with the survival of swedish ethnicity not only in the swedish districts butwtlhe financial support of educationally disadvantaged swedish minorities in finnish communes as well. with the establishment of a special swedish department in the central bureau of schools in april , finnish and swedish schools were put in an equal legal position. since then swede-finns have maintained considerable autonomy in the running of the swedish-language elementary and secondary schools (hamalianen, p. ). the extent of this autonomy stands in notable contrast to the strong and persistent efforts of educational authorities in sweden to acculturate and assimilate the finnish minority in northern sweden (kuusela, ; lundegard, ; guikkila, ). with the legal provision of swedish control of swedish schools, however, the struggle for self-determination and autonomy increasingly took place in the national university and through the sfem-controlled folk high schools. during the 's and 's finnish university students in. league with the agrarian party battled to secure "the finnification'of the university of helsinki. " they complained that the continuing predominance of a swedish faculty and instruction in swedish placed an intolerable burden on finnish students. the disproportionately large size of the swedish-speaking educated class and the numerous swedish secondary schools, moreover, caused, they further claimed, an overproduction of swedish-speaking uni- versity students in comparison to the total swede-finn population. in this bitter conflict the swede-finns were able to continue a bilingual policy at the university of helsinki--there are still some sf professors teaching in swedish--and, in addition, secured a government-supported swedish-language university, the abo academy in the swedish west-coast heartland. with the advent of state-supported compulsory schooling, the friends of the swedish public school society, in their work for everything swedish, shifted their support and initiated a great number of other largely non-formal and informal educational activities. chief among these have been the society's support of sf folk high schools, ole publication of popular literature, and the founding of popular libraries, the provision of popular lectures, and, to great effect, the organization of the swedish singing and music festivals. since this work has been supported by the more comprehensive sf movement organczation--the swedish-finland's folkting. this sf pecnze's congress of elected representatives meets every other rear and maintains permanent and active sections on culture policy, social policy, economic policy and information, sand on constitutional matters. the folkting forcefully represents interests of the sf population. it coordinates provision of social and cultural services, serves as a forum for debate and information within the movement and between the movement and its larger national, rerional, and international contexts. we will now take note of two especially effective movement ethnic- education programs, the folk high schools and music festivals. where the earlier swedish class movement used its monopoly of the national uni- versity to maintain its privilege, the broader swedish ethnic movement has been more directly served by the swedish-language folk high schools. these residential colleges offer courses in practical subjects and domestic con- cerns. they offer opportunities for continuing education in a swedish ethnic context, and they have "served as sources and centers of the ideology and sentiments of the swedish nationality movement." (myhrman, , p. ) the first swedish folk high schools were founded by the swedish youth movement in . this group of university,students, without parallel in scandinavia, sought to advance popular arid civic education ln rural areas and mixed districts so as to promote the swedi,ih language and culture. by fourteen swedish folk high schools with'iover students continued the youth movement's work to create ln young swede-finns'a desire to be swedish ln mind and language and to actively support the aims and organized efforts of the whole swedish ethnic movement. although sf folk high schools continue to serve the rural and small- town population, the extension of compulsory education and the near disappearance of culture conflict in the post-world war ii ora has meant , that they must seek a new role in providing adult education. the ratio of / autonomous folk high schools to the swedish population in different regions ( ) remains impressive, however. it is presented in figure below: figure two region swedish population number of fhs fhs/ swedish population dstra nyland , , mellersta nyland , : , va stra nyland , , nyland , , aboland , , aland , , södra osterbotten , , mellersta osterbotten , , norra osterbotten , , osterbotten , . total , , source: svenska kulturfond ( , p. ). the sf singing and music festivals sought more directly to develop consciousness of ethnic identity and group solidarity. swedish-speaking finlanders had participated in bilingual singing and music festivals on a large scale until they became dominated by the fennomen, or finnish nationalism movement, members who used them as occasions for fennomen propaganda. after , separate swedish music festivals were held yearly and reached theirwaihe/% t clitifot in the 's. on midsummer day in , for example, some , youth from swedish areas gathered in helsingfors to sing traditional swedish and sf songs and unite rural and urban swedes in a sense of ethnic community and movement consensus. since then, and to a much lesser extent, sf music festivals have taken place at the local, or provincial, level. they are supported--along with amateur theater and literary activitiesby a number of young peoples' societies and choruses where winter rehearsals are an important social activity. related sf cultural organizations concerned with folk culture and social welfare use the summer music festivals, moreover, as occasions to meet and discuss their work and concerns. the swede-finns continue to surpass national educational norms, and especially those s-f youth who live in towns and regional urban centers.,, artiong the rural population, the relative educational advaniage of the swede- finns is slight, or nonexistent. in figure , following, the continuing high priority of urban swede-finns on formal schooling is apparent. figure three educational levels of language groups in finland ( ) middle school exam by location total over yrs. higher secondary exam total over yrs. national population . % . % . % . % sf pop. only . % . % . % . % large towns & cities . % . % . % . % sf pop. only . % . % .. % . % rural & villages . % . % . % . % sf pop. only . % . % . % . % source: svenska finlands folkting, p. . we do have doraparable data on . languages of folk-high-school pupils, the number of schools related to the respective languages, and the proportion of finnish and swedish language in the general population as presented in figure below. here we see, for the year - , enrollment in sf folk high schools . times greater than the percent of swede-finns.in the total population. in the number of schools, there were . times as many sf folk high schools in finland than would be a simple representation according to language spoken in the country. this disproportionate participation of the sf minority in all sectors of the national educational complex continues, but at a rapidly declining figure with the grundskola reforms and increasing educational participation among the finnish-speaking population (ulfvens, ; svenska kulturfond, ). figure four folk high school enrollments by language group in finland, - years item finnish number percent swedish number percent total number percent language . . . * - enrollment , . . , . . s - q enrollment , . . , . - enrollment , . - schools . . . *other . percent. sources; leskinen, p. ; larson, p. . evaluation: educational contributions to the swedish ethnic movement this paper has briefly examined a little-known example of ethnic- movement education that might be best described, using the typology presented, as "defensive. " as such, it would fit in the lower right of figure one along with other ethnic groups seeking to maintain ethnic boundaries and solidarity. while a variety of educational programs have clearly helped to mobilize sf ethnic identity and reinforce ethnic consciousness, other favorable factors have been more central in securing the conditions favorable to swedish ethnic survival. here we must mention the great economic and intellectual resources of the swedish population, the effective parliamentary strategy of the swedish people's political party after , the continued sf control of large areas of productive agricultural land, and the land- defense organizations that bought and kept land in sf areas under sf ownership. equally noteworthy have been the swedish cooperatives, agri- cultural societies, banks, rural credit and insurance companies, and, not least, the cultural foundations. the above indicate something of the pene- tration of ethnic or nationality sentiments into the field of econciinic ancl financial activities, efforts that have successfully provided the funds necessary to support the movement's educational work in popular education, folk high schools, cultural activities, and in the media. swedish-ethnic educational activities have, perhaps, contributed most directly to movement goals through their efforts to create a common ethnic identity that included both the upper class and the 'masses (wrede, ). by the 's this key objective had been won. educational efforts were equally successful in securing the obverse of this mutual recognition, le., the sense of being different from finns.: as we have noted, upper-class swedes shared by and large attitudes of superiority over the finns, who were largely laborers and peasants. swedish independent peasants, in contrast, rarely came in contact with finns of their own class. ethnic consciousness among this group or.ilv developed with a threat to cultural survival and a systematic educational effort to articulate this bind and teach how collective efforts might ease it (eklund, ; lille, ). since world war ii, the swedish-speaking population has continued to decline in numbers iind influence. their separtist policy has clearly served to defend ethnicity, but at the same time it has limited opportunities to gain influence at the national level. increased contact between the two cultural groups with resettlement of finnish refugees and post-war reconstruction, along with the increased economic power and legitimacy of finnish nationalism, has meant increased intermarriage and the rejection of swedish culture for a national 'identity by a relatively small but growing number of young swede-finns. with lack of pressure for assimilation from finnish nationalists, the bind experienced by older swede-finns has lost nearly all meaning for the younger generations. and with the disappearance of culture conflict, the sfem organizations .have been gradually transformed into service organizations that find their reason for existence in the maintenance of a new status quo. while the swede-finn population remains at , , or a bit less than it was a century ago, the sf percentage of the total population had declined to some . percent in . recent rese.a h indicates that this decline in the swedish-speaking population--the loss was . 'percent from to and . percent from to --resulted in large part from language shift among sf youth in late adolescence and early adulthood (see figure below). it further suggests that important factors influencing language shift from swedish to finnish were the experiences of secondary education, labor-market entry, and the selection of a marriage partner (devries, ). figure five year percent of swedish speakers . (estimated) . (estimated) . . . as the minority of swedish speakers declines in absolute numbers as well as in proportion to the total pupulation, the finnish nationalists stand to gain by a policy of tolerance for ethnic pluralism and s%pport for increased bilingualism what they have been unable to gain by force in nearly a century of struggle and culture conflict. in concluslon,..the swede- finn_case might_also be viewed as an instructive example of what bronfenbrenner ( ) calls a "transforming experiment, " or "an experiment that radically restructures the environ- ment producing a new configuration that activates previously unrealized behavioral potentialities in the subject" (p. ). where most anthro- pological study of ethnic education has viewed the ecological 'contexts of such efforts as sociological givens, a "transforming experiment" or social-movement approach would view ethnic relations with mobilization as subject to significant and novel transformation. where we have in the past focused our research on the ethnicity-education nexus as.it exists with system elements that make up the status quo, we will also need to examine how educatio:t in conflict situations contributes to ethnic movement attempts to secure and defend "a restructuring of established institutional forms and values. 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impresora sahuaro. smith, john kenneth jewish education in colombia: group survival versus assimilation. doctoral dissertation, university of wis- consin, madison. suikkila, juhani pinar, i sverige: en studie av social identitet. stockholm: sociology department, university of stockholm. suomen kansanopistoyhdistys suomen kansanopistot, - . helsinki: the association. svenska finlands folkting svenskt i finland. helsingfors: the folkting. svenska kulturfond fran grundskola till vuxenutbildning i finlandssvenskt perspektiv. heisingfors: holger schildts förlag. ulfvens, levi de finlandssvenska folkhdgskolornas framtid. in folkhogskolan inför framtiden. helsingfors: svenska kulturfonden, pp. - . weinman, janice j. local control over formal education in two american-indian communities: a preliminary step toward cultural survival. review of educational research, ( ): - . wilson, william j. , et al racial solidarity and separate education. school and review, ( ): - . wir, louis education for survival. american journal of sociology, ( ): - . wrede, r. a. svenskheten finland och medlen fr dess bevarande. helsingfors: eken s. wuorinen, john h. nationalism in modern finland., new york: columbia university press. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in 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help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ science should drive vaccine policy invited commentary | infectious diseases science should drive vaccine policy tara c. smith, phd; maimuna s. majumder, mph, phd using agent-based modeling, sinclair et al have demonstrated the risk that many texas children face because of their classmates’ religious or philosophical exemptions from measles vaccination. in texas, vaccine exemption rates have increased -fold since , and texas allows for exemptions “for reasons of conscience,” which include religious and philosophical objections to vaccinations. because of these exemptions, others have warned that many texas cities are at risk of measles outbreaks. several cities in texas were previously identified as possible hotspots for outbreaks of vaccine-preventable diseases, including houston, fort worth, plano, and austin. the article by sinclair et al examines just how large these outbreaks could be given current vaccination rates, finding that epidemics of up to several hundred children could occur. further, they note that while the group that is unvaccinated would have the most cases, bystanders—including those for whom vaccination failed and students who are medically exempt—would also be susceptible, demonstrating the importance of herd immunity to protect those who do not respond to vaccines or who cannot be vaccinated. nevertheless, these results must be considered with some caution. although agent-based models describe heterogeneity well in populations for which highly granular data (eg, individual-level data) are available, these models are prone to assumptions that may not reflect real-world conditions when such data are not available (as noted by sinclair et al in their discussion). further, the authors’ choice to use the basic reproduction number (r ) to run their model likely yielded higher simulated outbreak sizes than would have been estimated if the more appropriate effective reproduction number (reff) had been used instead. unlike r , which describes the number of infections that a case will cause in a fully susceptible population, reff describes the number of infections that a case will cause in a population that is not fully susceptible (ie, a population in which some fraction has been effectively vaccinated, such as the state of texas). however, as partial empirical validation for the modeling method employed by sinclair et al, texas has already seen cases of measles in counties from january to mid-july . indeed, has been a record year for measles infections in the united states, with cases reported as of july , the largest nationwide annual count since . what can be done to prevent this outcome? although the authors did not include interventions in their model, we can consider aspects of prevention: that which takes place in the midst of an epidemic and that which occurs otherwise. during an epidemic, strict public health interventions may be put in place. during outbreaks of measles, cases are typically isolated, and those who have been exposed are subject to quarantine. in new york, new york, these measures were not enough to stop the outbreak that began in october ; unvaccinated children were banned from schools, and mandatory vaccination orders for measles were put in place. an outbreak situation may also encourage individuals to catch up on vaccinations. during ohio’s measles outbreak in the amish population, more than individuals were vaccinated with the measles, mumps, and rubella vaccine ; similarly, more than measles cases in early led to a -fold spike in measles vaccinations in washington. the more challenging aspect of prevention is maintaining vaccination rates in the absence of ongoing epidemics, which is complicated by the continued spread of misinformation about vaccines. texas is an unfortunate leader in this aspect and the current home of andrew wakefield, the researcher whose fraudulent study first erroneously linked the measles, mumps, and rubella + related article author affiliations and article information are listed at the end of this article. open access. this is an open access article distributed under the terms of the cc-by license. jama network open. ; ( ):e . doi: . /jamanetworkopen. . (reprinted) august , / downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / https://jama.jamanetwork.com/article.aspx?doi= . /jamanetworkopen. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . https://jama.jamanetwork.com/article.aspx?doi= . /jamanetworkopen. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . https://jama.jamanetwork.com/article.aspx?doi= . /jamanetworkopen. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . vaccine to autism. texas is also home to an effective antivaccine lobbying group, texans for vaccine choice, which has worked to curtail any changes in legislation regarding vaccine exemptions in the state. countering such antivaccine rhetoric is not a simple task. research into the factors driving vaccine hesitancy has not led to any easy answers or readily scalable interventions. as such, many in public health are looking to change state laws regarding vaccine exemptions to increase vaccination rates. mississippi and west virginia have long allowed only for medical exemptions; religious and/or philosophical exemptions to vaccines are present in other states. california became the third state to eliminate nonmedical exemptions in with the passage of california senate bill following the disneyland measles outbreak in to . because of the measles epidemic, maine and new york have similarly modified their vaccine laws, removing nonmedical exemptions. we suggest that studies like sinclair et al may aid legislators in making these changes proactively, before such outbreaks are experienced. article information published: august , . doi: . /jamanetworkopen. . open access: this is an open access article distributed under the terms of the cc-by license. © smith tc et al. jama network open. corresponding author: tara c. smith, phd, college of public health, kent state university, hilltop dr, lowry hall, kent, oh (tsmit @kent.edu). author affiliations: college of public health, kent state university, kent, ohio (smith); department of health care policy, harvard medical school, boston, massachusetts (majumder). conflict of interest disclosures: none reported. references . sinclair dr, grefenstette jj, krauland mg, et al. forecasted size of measles outbreaks associated with vaccination exemptions for schoolchildren. jama netw open. ; ( ):e . doi: . / jamanetworkopen. . . texas department of state health services. exemption information: school immunizations. https://www.dshs. texas.gov/immunize/school/exemptions.aspx. accessed july , . . hotez pj. texas and its measles epidemics. plos med. ; ( ):e . doi: . /journal.pmed. . olive jk, hotez pj, damania a, nolan ms. the state of the antivaccine movement in the united states: a focused examination of nonmedical exemptions in states and counties. plos med. ; ( ):e . doi: . / journal.pmed. . us centers for disease control and prevention. measles cases and outbreaks. https://www.cdc.gov/measles/ cases-outbreaks.html. accessed july , . . pager t, mays jc. new york declares measles emergency, requiring vaccinations in parts of brooklyn. new york times. april , . https://www.nytimes.com/ / / /nyregion/measles-vaccination-williamsburg.html. accessed july , . . gastañaduy pa, budd j, fisher n, et al. a measles outbreak in an underimmunized amish community in ohio. n engl j med. ; ( ): - . doi: . /nejmoa . foden-vencil k. in a measles outbreak, demand for vaccine spikes. morning edition. february , . https:// www.npr.org/ / / / /in-a-measles-outbreak-demand-for-vaccine-spikes. accessed july , . . deer b. how the case against the mmr vaccine was fixed. bmj. ; :c . doi: . /bmj.c . smith tc. vaccine rejection and hesitancy: a review and call to action. open forum infect dis. ; ( ): ofx . doi: . /ofid/ofx jama network open | infectious diseases science should drive vaccine policy jama network open. ; ( ):e . doi: . /jamanetworkopen. . (reprinted) august , / downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / https://jama.jamanetwork.com/article.aspx?doi= . /jamanetworkopen. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . https://jamanetwork.com/journals/jamanetworkopen/pages/instructions-for-authors#secopenaccess/?utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . mailto:tsmit @kent.edu https://jama.jamanetwork.com/article.aspx?doi= . /jamanetworkopen. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . https://jama.jamanetwork.com/article.aspx?doi= . /jamanetworkopen. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . https://www.dshs.texas.gov/immunize/school/exemptions.aspx https://www.dshs.texas.gov/immunize/school/exemptions.aspx https://dx.doi.org/ . /journal.pmed. https://dx.doi.org/ . /journal.pmed. https://dx.doi.org/ . /journal.pmed. https://dx.doi.org/ . /journal.pmed. https://www.cdc.gov/measles/cases-outbreaks.html https://www.cdc.gov/measles/cases-outbreaks.html https://www.nytimes.com/ / / /nyregion/measles-vaccination-williamsburg.html https://dx.doi.org/ . /nejmoa https://www.npr.org/ / / / /in-a-measles-outbreak-demand-for-vaccine-spikes https://www.npr.org/ / / / /in-a-measles-outbreak-demand-for-vaccine-spikes https://dx.doi.org/ . /bmj.c https://dx.doi.org/ . /ofid/ofx https://jama.jamanetwork.com/article.aspx?doi= . /jamanetworkopen. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . https://jama.jamanetwork.com/article.aspx?doi= . /jamanetworkopen. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamanetworkopen. . microtissue size and hypoxia in hts with d cultures drudis – fe a tu re s � p e r s p e c t iv e feature microtissue size and hypoxia in hts with d cultures amish asthana and william s. kisaalita*, williamk@engr.uga.edu the three microenvironmental factors that characterize d cultures include: first, chemical and/or biochemical composition, second, spatial and temporal dimensions, and third, force and/or substrate physical properties. although these factors have been studied individually, their interdependence and synergistic interactions have not been well appreciated. we make this case by illustrating how microtissue size (spatial) and hypoxia (chemical) can be used in the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (hts) in drug discovery. we further show how transcriptomic and/or proteomic results from heterogeneously sized microtissues and scaffold architectures that deliberately control hypoxia can misrepresent and represent in vivo conditions, respectively. we offer guidance, depending on hts objectives, for rational d culture platform choice for better emulation of in vivo conditions. drug discovery today �volume , number �april perspective traditionally, the meaning of three-dimension- ality in cell culture has been simply associated with providing a d spatial microenvironment. in our recent work, the meaning has been extended to providing the total microenviron- ment that supports the formation of microtissue that exhibit ‘complex’ physiological relevance (cpr) or better emulation of the in vivo micro- tissue functionality in a manner not possible in d cultures [ ]. the literature has provided guidance that lead to three main categories or microenvironment factors (mefs) or ‘three- dimensions’ of: first, chemical or biochemical composition, second, spatial (geometric d) and temporal dimensions, and third, force and sub- strate physical properties [ – ]. although much attention has been paid to biochemical factors, such as integrin adhesion and presence of rdg peptides (integrin recognition site) in d cultures please cite this article in press as: asthana, a. microtissue s - / /$ - see front matter � published by elsevier ltd. d and biophysical cues, such as loading and unloading of collagen matrices, interactions among the factors have not been well studied. the effect of the aforementioned microen- vironmental factors is not exclusive, but they act synergistically to propel the cells towards a specific outcome. for instance, although regu- lating the size of the microtissue might seem trivial, it might indirectly have major implications on the functional response of the cells. if the tissue formed is too small, it might lack the physiologically relevant complexity and might not emulate the complex functionality present in vivo. conversely, if the tissue is large (spatial factor), the oxygen diffusion (biochemical factor) limitation might lead to a necrotic core, reducing the viability and influencing the phenotypic outcome. it is known that oxygen can diffuse across – mm of tissue thickness and it is ize and hypoxia in hts with d cultures, drug discov today oi: . /j.drudis. . . generally desirable to maintain the optimal aggregate size approximately mm to pre- vent hypoxia [ ]. however, this size should not be treated as a gold standard, as the optimal size might depend on the application. in the field of regenerative medicine, circumventing hypoxia to produce larger tissues with higher viability for implantation in vivo is relentlessly pursued, however, the field of drug discovery might benefit from incorporating hypoxia in platform design. after all, hypoxia is a physiologically relevant phenomenon and is important for many in vivo processes, such as development and tumor progression. indeed hypoxia has been widely implicated as the initiator of angiogenesis in avascular tumors, vasculogenesis during embryonic development and regulator of terminal differentiation [ ]. as such, it is impor- tant to regulate the size of the tissue in d ( ), doi: . /j.drudis. . . www.drugdiscoverytoday.com http://dx.doi.org/ . /j.drudis. . . http://dx.doi.org/ . /j.drudis. . . perspective drug discovery today �volume , number �april drudis – table microtissue size control in the commercially available d platforms company trade name type and material cell aggregate/ scaffold pore size dbiomatrix perfecta d plates hanging drops na perfecta d scaffolds hydrogel na insphero gravityplus plates hanging drops na bd matrigel laminin, collagen na glycosan biosystems extracel hyaluronic acid and collagen na globalcellsollutions/hamilton gem magnetic alginate microcarrier na trevigen cultrex d matrix bme, laminin, collagen na sigma hydromatrix synthetic peptide hydrogel na maxgel human ecm na qgel mt d matrix hydrogel na kollodis biosciences maptrix hygel chemically defined hydrogel na synthecon inc. biofelt pga, plla, plga, custom nr biomerix d scaffold polycarbonate polyurethane-urea – invitrogen geltrex laminin, collagen na algimatrix alginate – zellwerk sponceram ceramic na amsbio alvetex polystyrene nr dm inc. puramatrix peptide – corning ultraweb polyamide – dbiotek d insert pcl polycaprolactone ; d insert ps polystyrene ; d insert plga poly(dl-lactide-co-glycolide) b-tcp disc b-tricalcium phosphate nr microtissues inc. d petri dish agarose multiple abbreviations: na, not applicable; nr, not reported. fe a tu re s � p e r s p e c t iv e culture to include or eliminate the effects of hypoxia depending on the physiological phe- nomenon of interest. to illustrate further, in anticancer drug discovery, test compounds might be scored ‘hits’ with early stage tumor progression models, however, these compounds might be incompetent if tested with a micro- tissue tumor model having a larger size, owing to the drug resistance associated with hypoxic tumors. to make the case for the importance of microtissue size, we present many d and/or d comparative transcriptomic and proteomic stu- dies where the effect of the culture platform studied, might have been confounded by the heterogeneous microtissue sizes and thus the effect of hypoxia on gene expression was affected. taken together, we stress the impor- tance of inclusion of hypoxic conditions in constructs through size regulation for certain applications. such a realization is important in rational design and/or choice of a d platform, where the need for strict control of microtissues is balanced against the need for flexibility to alter microtissue size to better emulate the in vivo conditions. please cite this article in press as: asthana, a. microtissue s www.drugdiscoverytoday.com spatial constraints in d platforms most of the d platforms that are commercially available (table ) can be broadly classified into three categories based on the spatial constraints that they impose. first, hydrogel-forming (algi- nate, agarose, chitosan, fibrin, hyaluronan and collagen to name a few) that cannot exert control on the size of the microtissues giving rise to heterogeneous microtissues (type i; fig. a). in such constructs, the microtissues range from being just a cluster of few cells to larger tissues that are above the crucial size for oxygen dif- fusion and might provide an adulterated response as discussed in the previous section. the sizes of the tissues depend on the seeding density and the proliferation rate of the cells and because the scaffold material is pliable there is no physical constraint on the size of the aggregates formed. recently, a gradient depressurization strategy has been used to control the porosity and pore diameter in chit- osan hydrogels. however whether this technol- ogy is successful with hydrogels made of other substrates remains to be seen [ ]. scaffold-free spheroid cultures also fall into the same category ize and hypoxia in hts with d cultures, drug discov today because their size also depends on the afore- mentioned factors rather than physical restric- tions. the sizes of the aggregates growing in rotary wall vessels (rwv) or continuous stirred- tank reactors (cstr) can be regulated by physical factors, such as revolutions per minute (rpm) and fluid shear stress, however; this control is acti- vated only when the spheroids grow above a crucial size. moreover, the larger tissues are broken down into random smaller sizes giving rise to heterogeneously sized microtissues. also, smaller tissues present in the culture, ranging from single cells to just below the threshold limit are not affected by this mechanism. second, synthetic microporous scaffolds made of stiff materials (e.g. polymers, such as polystyrene) that put a physical restraint on the size of the microtissues but the range of the pore size is large, again resulting in a variable sized popu- lation (type ii; fig. b). however, the extent of variation is lower than type i cases and the variability is defined as the range of pore sizes is known to the user. third, polymeric scaffolds that have a defined geometry and homogeneous pore sizes and provide a strict spatial control on ( ), doi: . /j.drudis. . . http://dx.doi.org/ . /j.drudis. . . drug discovery today �volume , number �april perspective drudis – a b c drug discovery today figure three categories of d platforms. (a) hydrogel (algimatrix) with heterogeneous (c a human hepatocytes) microtissues stained with live/dead dye kit. dead cells (stained red) are visible at the center of large aggregates [ ]. (b) polystyrene scaffold that imposes a size constraint but offers a wide range of pore sizes. the scaffold was fabricated following procedures described in [ ]. (c) su- (photoresist material) microwell scaffold that provides a uniform pore size. the scaffold was fabricated following procedures described in [ ]. scale bars = mm. fe a tu re s � p e r s p e c t iv e the size of the microtissues (type iii; fig. c). if seeded at the optimal cell density, such scaffolds produce a population of equally sized aggre- gates; however they might lack flexibility and can be application specific. for instance, a please cite this article in press as: asthana, a. microtissue s scaffold having a defined pore size of mm might not be suited for studying or developing a drug against a late stage cancer, where the tumor is hypoxic and adapting for angiogenesis. conversely, while developing tissue models for ize and hypoxia in hts with d cultures, drug discov today drug testing by differentiating stem cells, a larger pore size ( mm) might produce microtissues with hypoxic cores and this might influence the differentiation capacity of the cells, giving rise to a heterotypic model. the impact of hypoxia in both these scenarios is discussed below. owing to such variability, the response generated due to a specific treatment by cells growing on different d platforms might be different and too difficult to compare. one must consider that variability in tissue size might manifest itself in the form of hypoxia or other unknown forms and perturb gene expression leading to an adulter- ated response to the administered treatment. relationship between microtissue size and gene expression as discussed above, if the size of the microtissue grows beyond the threshold for oxygen diffu- sion, the cells in the core of the aggregate become hypoxic. hypoxia can manifest itself in the form of gene expression perturbation because it regulates the expression of a wide variety of genes associated with oxygen trans- port and iron metabolism, glycolysis and glucose uptake, angiogenesis, extracellular matrix (ecm) and coagulation systems, drug resistance, ph regulation, transcription and growth factors and cytokines (table ) [ , ]. the relationship between microtissue size and gene expression was established by kelm et al. [ ] where it was shown that larger myocardial spheroids ( � mm in diameter) produced high levels of vascular endothelial growth factor (vegf; a marker of hypoxia) while it was absent in smaller spheroids ( � mm in diameter) although both the sizes showed cpr (synchronized beating frequencies). this relationship is further sub- stantiated by a transcriptomic study [ ] showing that neural progenitor (np) cells growing as neurospheres (larger size) had a higher number of upregulated genes than those growing in d microporous scaffolds (controlled smaller size) when compared with d cultures. it was sug- gested that this might be owing to hypoxia associated with the larger size of neurospheres as was evident by the upregulation of macrophage inflammatory protein- (mip- ) gene, which is induced by hypoxic conditions [ ]. several transcriptomic and proteomic studies that have compared gene expression variations between a variety of cells grown in d and d formats are listed in table . these differential gene expression events have generally been solely attributed to the transition from d to a d platform, but hypoxia associated with larger tissue size might also be responsible for these gene perturbations as several of them might be ( ), doi: . /j.drudis. . . www.drugdiscoverytoday.com http://dx.doi.org/ . /j.drudis. . . perspective drug discovery today �volume , number �april drudis – table hypoxia controlled genes/proteins found upregulated in d/ d comparative studies. cell line d scaffold size (mm) genesa refs fibroblasts collagen matrix nd il- [ ] imr- collagen–gag matrix – hig , il- , cxcl , vegf, fth , ftl [ ] mg- , saos- si-hpmc polymer hydrogel nd il- [ ] na spheroids on phema plates nd il- , cxcl , angiopoetin like , ca- , lox, adm, hig , bnip , igfbp , jun, itga [ ] r cytomatrix rw-spinner culture jun, igf- [ ] l rada-oligopeptide matrix nd ccl , tnf [ ] pdac pecm nd il- , il- [ ] oscc , u mda-mb plg, rgd alginate, matrigel nd il- , vegf [ ] hfsf, crl- hes hal mrc- spheroids on agarose plates nd cox- , ccl ,ccl , cxcl cxcl cxcl cxcl [ ] bmsc spheroids on agarose plates cxcl [ ] abold italic indicates protein results, other results are only transcriptomic. abbreviations: il, interleukin; cxcl , macrophage inflammatory protein ; fth , ferritin heavy subunit; ftl, ferritin light subunit; hig , hypoxia-inducible gene protein; nd, not defined. fe a tu re s � p e r s p e c t iv e missing in d cultures of smaller sizes [ , ]. the genes listed in table were found to be upre- gulated in d cultures when compared to d but they were also induced by hypoxia (table ) suggesting that gene expression due to hypoxic conditions might have been involved in these studies. thus, to assess the genes whose expression is significantly altered specifically owing to d culture conditions, the size of the microtissue should be controlled; else the genes influenced by hypoxia might augment the total number of differentially expressed genes and mask the ones that are really of interest. physiological relevance of hypoxia – need for inclusion in construct design hypoxia can be a physiologically relevant phe- nomenon because it is a major characteristic of d microenvironments both in vivo and in vitro. oxygen concentration in d tissues depends on the balance between oxygen delivery and con- sumption. in vivo, this balance is tightly regu- lated by evenly distributed capillary networks but in vitro homotypic d microtissues lack vasculature and therefore develop a hypoxic core as their size increases. this might lead to cells, producing chemical signals (cytokines) and programming themselves for developmental, adaptive or neoplastic angiogenesis depending upon their type (stem, committed or malignant, respectively). this is similar to the response generated by in vivo hypoxic tissues where balanced signaling cascades lead to vascular remodeling and angioadaptation until the tissue oxygen concentration is back within its normal please cite this article in press as: asthana, a. microtissue s www.drugdiscoverytoday.com range [ ]. the central connection between physiological hypoxia and the cellular response is mediated by hypoxia inducible transcription factors (hifs). under hypoxic conditions, hif- a and hif- b are translocated to the nucleus [ ] where they dimerize and bind to target gene motifs called hypoxia responsive elements (hres) to alter gene expression [ ]. in vivo, hypoxia is generally associated with the tumor microenvironment where it is responsible for angiogenesis, drug resistance and increased metastatic potential of the malignant cells and also with development where it regulates vas- culogenesis, stem cell renewal and terminal differentiation. as such, hypoxic conditions need to be included in the d scaffold design, where applicable, to better mimic these conditions in vitro. this can be done in a physiologically relevant manner by strictly regulating the size of the microtissue. hypoxia and the tumor microenvironment neoplastic angiogenesis is an essential process in tumor progression and the initiation of metastasis [ ]. the phenomenon of angiogen- esis comprises a series of linked and sequential steps that finally leads to the development of a neovascular blood supply to the tumorous tissue [ ]. angiogenic growth factors secreted by infiltrating immune cells, adjacent stroma and tumor cells themselves bind to specific receptors on endothelial cells which leads to endothelial cell proliferation, migration and invasion, even- tually culminating in capillary formation. ize and hypoxia in hts with d cultures, drug discov today angiogenesis regulation by hypoxia is an important homeostatic mechanism that links vascular oxygen supply to metabolic demand. lately, molecular characterization of angiogenic pathways, establishment of hifs as their key transcriptional regulators and the identification of hydoxylases that regulate hif corresponding to the oxygen availability have provided novel insights into this process [ ]. hypoxia in the tumor microenvironment is of specific impor- tance in drug discovery and development because optimal oxygenation is a primary requisite for many chemotherapeutic drugs, such as alkylating agents (melphalan), antibio- tics (bleomycin) and podophyllotoxins (etopo- side) to act at their maximum efficiency [ ]. most alkylating agents act by transferring alkyl groups to dna during cell division, following which the dna strand breaks or cross-linking of the two strands occurs, preventing subsequent dna synthesis [ , ]. hypoxic conditions can lead to resistance against these drugs directly by increased production of nucleophilic sub- stances, such as glutathione, which might compete with the target dna for alkylation, subsequently reducing the drug efficacy [ ]. another class of anticancer agents acts at spe- cific phases of the cell cycle. as hypoxia causes the cell cycle to slow down or lead to pre-s- phase arrest in extreme conditions [ ], it can also indirectly affect the apoptotic potential of these agents. also, hypoxia leads to changes in the genome that can confer growth advantage to cells with p mutations [ ] or deficient in dna mismatch repair [ ], while inhibiting ( ), doi: . /j.drudis. . . http://dx.doi.org/ . /j.drudis. . . drug discovery today �volume , number �april perspective drudis – table genes induced by hypoxia biological function gene (abbreviation) refs o transport and iron metabolism erythropoietin (epo) [ ] ferritin (fth , ftl) [ ] heme oxygenase- [ ] transferrin [ ] transferrin receptor (tfr) [ ] ceruloplasmin [ ] angiogenesis vascular endothelial growth factor (vegf) [ , ] vegf receptor- [ ] cyclooxygenase (cox)- [ ] leptin (lep) [ ] endothelin- , - [ ] fibroblast growth factor (fgf)- [ ] angiopoietin- (ang- ) [ ] nitric oxide synthase (nos) [ ] placental growth factor (plgf) [ ] transforming growth factor (tgf)-a [ ] tgf-b [ ] tgf-b [ ] matrix metabolism and coagulation metalloproteinases [ ] matrix metalloproteinase (mmp)- [ ] plasminogen activator inhibitor- [ ] urokinase receptor [ ] collagen prolyl hydroxylase [ ] a-integrin [ ] glycolysis and glucose metabolism adenylate kinase- [ ] aldolase-a,c (alda,c) [ ] carbonic anhydrase- (ca- ) [ ] enolase- (eno ) [ ] glucose transporter- , (glut , ) [ , ] glyceraldehyde phosphate dehydrogenase (gapdh) [ ] hexokinase , (hk , ) [ ] lactate dehydrogenase-a (ldha) [ ] pyruvate kinase m (pkm) [ ] phosphofructokinase l (pfkl) [ ] phosphoglycerate kinase (pgk ) [ ] -phosphofructo- -kinase/gructose- , -bisphosphate- (pfkfb ) [ ] transcription factors hypoxia-inducible factor (hif)- a [ ] hif- a [ ] activator protein (ap- ) [ ] jun [ ] nuclear factor-kb (nf-kb) [ ] insulin-like growth factor (igf) binding protein- ,- , - [ ] cyclic amp responsive-element-binding protein (creb) [ ] drug resistance multi-drug resistance (mdr ) [ ] apoptosis bcl- /adenovirus eib kd-interacting protein (bnip ) [ ] nip -like protein x (nix) [ ] growth factors and/or cytokines insulin-like growth factor- (igf- ) [ ] platelet-derived growth factor (pdgf) [ ] adrenomedullin (adm) [ ] interleukin- (il- ) [ ] interleukin- (il- ; cxcl ) [ , ] tumor necrosis factor a (tnfa) [ ] macrophage inflammatory protein -a (ccl ; mip- a) [ ] macrophage inflammatory protein (mip- ; cxcl ) [ ] stromal cell-derived factor (sdf- ; cxcl ) [ ] chemokine (c–c motif) ligand (ccl ; rantes) [ ] fe a tu re s � p e r s p e c t iv e apoptosis, which also contributes to their therapeutic resistance. screening of such drug compounds on the aforementioned type i and ii d platforms, that produce microtissues of variable sizes, can lead to please cite this article in press as: asthana, a. microtissue s skewed responses because the compounds might be selectively effective against a certain popula- tion of cells. for such compounds, type iii plat- forms seem to be the more obvious choice, but the size cut-off that is meant for this particular ize and hypoxia in hts with d cultures, drug discov today application must be carefully chosen. also, the difference between hypoxic cancer cells and normal cells provides a novel target upon which cytotoxic drugs can be designed. these drug designing strategies include: targeting the hif- ( ), doi: . /j.drudis. . . www.drugdiscoverytoday.com http://dx.doi.org/ . /j.drudis. . . perspective drug discovery today �volume , number �april drudis – fe a tu re s � p e r s p e c t iv e transcription factor (thioredoxin- inhibitors – pleurotin, px- ; [ ]), hypoxia-selective gene therapy, pro-drugs activated by hypoxia (aq n, nlcq- ) and the use of recombinant obligate anaerobic bacteria (non-pathogenic clostridia) [ ]. as such, these novel strategies require a consistent population of hypoxic microtissues to be tested upon. one might argue that culturing cells under reduced oxygen pressure or adding a hypoxia-mimetic agent, such as desferrioxamine mesylate (dfx; iron chelator) to the media can produce hypoxic growth conditions but this might not recreate the natural oxygen and nutrient gradients that are associated with the tumor microenvironment and a d culture of larger hypoxic microtissues might be a better emulation of the in vivo situation, rendering the platforms that do not provide control on size or produce microtissues below the required size, less desirable for such applications. recently, a report published by rohwer and cramer [ ] listed several studies where hypoxic conditions and hifs have been directly implicated in chemothera- peutic resistance leading to inefficacy of drugs along with the resistance phenotypes and the molecular mechanisms underlying the resistance. had the potency of these drugs been validated on d microtissues of a particular size that incorpo- rated a hypoxic core, it is plausible that they might have been rejected during the initial phase of discovery and this would have saved considerable resources. hypoxia and stem cell niches the availability of reliable cell types is the pri- mary concern in the successful development of therapeutics against cellular targets in the drug discovery process. these cells are typically obtained from primary tissue, immortalized tumor cells or genetically transformed cell lines. compared with primary and immortalized cells, stem cells are more advantageous as they are genetically normal and can be maintained in culture for a longer time, increasing their applicability in the screening process. however, to fully exploit their potential, successful and consistent protocols need to be developed for their renewal and maintenance and also differ- entiation into committed lineages. recently, many studies have shown that d cultures consistently outperform d monolayer cultures in terms of promoting stem cell growth, differ- entiation and development of complex physio- logically relevant structures and functionality. when compared with d, embryonic stem cells (esc) grown in d cultures, differentiated into hepatocytes that had closer resemblance to their in vivo counterparts in terms of morphology, please cite this article in press as: asthana, a. microtissue s www.drugdiscoverytoday.com gene expression and biological behavior [ , ]. similar results have been obtained with d cultures of stem cells differentiated into cells of neural [ – ], epithelial [ ], endothelial [ , ], chondrogenic [ , ], and hematopoie- tic [ ] lineages. moreover, d cultures have also been shown to sustain long-term self-renewal of human escs, maintaining them in undifferen- tiated state, preserving their normal karyotype, and conserving their differentiation capacity as indicated by embryoid body formation [ ]. considering an increasing shift from the usage of traditional cultures towards d platforms for maintaining or differentiating stem cells to generate complex models of particular tissue types for drug testing, the hypoxic conditions associated with the d microenvironment should be taken into account as oxygen tension has been known to influence stem cell quies- cence, proliferation and differentiation both in vivo and in vitro [ ]. it has been shown that hematopoietic stem cells (hscs) are more likely to reside in the low oxygen areas of the marrow, away from blood vessels and hypoxia appears to regulate hematopoiesis in the bone marrow by influencing the survival, metabolism and cell cycle of hsc and also provides protection against oxidative stress [ ]. also, the differen- tiation capability of bone marrow-derived mesenchymal stem cells (mscs) is decreased in hypoxic culture conditions along with an increase in oct- expression and telomerase activity, further substantiating the notion that low oxygen tension promotes an undifferen- tiated state of these stem cells [ ]. similarly, hypoxia also promotes survival, proliferation and maintenance of an undifferentiated state in neural crest stem cells and nscs [ ]. hypoxic (physiologically normoxic) culture conditions also seem to maintain full pluripotency and enhance embryoid body formation of human escs [ ] whereas normal environmental oxygen levels lead to a significant decrease in the expression of stem cell markers, sox , nanog and oct- [ ]. furthermore, silencing of hif- a and hif -a, but not of hif- a, also led to a substantial reduction in the expression of the aforementioned pluripotency markers [ ] further implicating a role of hypoxia in the maintenance of pluripotency and stemness. recently, hif- a has been shown to bind to the oct- promoter and induce its expression and transcriptional activity [ ], thus hypoxia can contribute to generation of induced pluripotent stem cells (ipsc). as hypoxia has such an important role in determining stem cell fate, it is essential to regulate the size of the microtissue in d to either maintain the differentiation capacity ize and hypoxia in hts with d cultures, drug discov today of the cells or induce differentiation towards a particular lineage. therefore a platform that does not maintain a homogenous population of equally sized microtissues might have a mixed heterotypic population of cells, some differen- tiated while others maintaining their differentia- tion capability, giving rise to differences in drug response. a mixed population is a physiologically relevant condition; however each microtissue should have the same distribution of each type of cells to generate a valid drug response. if the size of the aggregates is different, the smaller ones might have a higher proportion of differentiated cells whereas the larger ones might be replete with stem cells as their cores become hypoxic and maintain the stemness of the cells. concluding remarks the tale of the spatial and the biochemical microenvironmental factors in d cell culture is that they are not mutually independent, because an increase in size leads to a depletion of oxygen and creates hypoxic growth conditions. most of the commercially available d growth platforms either do not regulate the size of the microtissue or restrict it to prevent hypoxia. however, hypoxia is a physiologically relevant phenomenon encountered in avascular tumors and stem cell niches and affects the gene expression profiles of the cells. as such, it should be a key factor in rational design or selection of d hts platforms for preclinical drug discovery. for example, to test the cytotoxic potential of chemotherapeutic agents, it might be more desirable to use hypoxic rather than smaller normoxic microtissues that might be more susceptible to their apoptotic actions and produce false positives. similarly, for generation of tissue models from stem cells for drug screening, a size cutoff should be chosen according to the state of cells (renewal or differ- entiation) required for the application. finally, in future transcriptomic and/or proteomic compar- ison studies, a marker for hypoxic conditions, such as lysyl oxidase (lox) should be included or a fluorescent hypoxyprobe, such as oxylite (oxford optonics; http://www.oxford-optronix.com/) should be used to determine whether the cells are hypoxic. also, a subset of genes (table ) dedi- cated to hypoxia can be established and its hierarchy or the rank assigned to it by clustering programs might suggest the degree to which hypoxia might be responsible for results from differential gene expression studies. references kisaalita, w.s. 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( ) expression and secretion of rantes (ccl ) in human adipocytes in response to immunological stimuli and hypoxia. horm. metab. res. , – amish asthana, william s. kisaalita cellular bioengineering laboratory, driftmier engineering center, university of georgia, athens, ga , united states ( ), doi: . /j.drudis. . . http://dx.doi.org/ . /j.drudis. . . 	microtissue size and hypoxia in hts with d cultures 	spatial constraints in d platforms 	relationship between microtissue size and gene expression 	physiological relevance of hypoxia - need for inclusion in construct design 	hypoxia and the tumor microenvironment 	hypoxia and stem cell niches 	concluding remarks 	references attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp a wednesday, february , of cardiac troponin c’s defunct ca þ -binding site (site ). in this study, we combined in vitro and in situ structural and functional techniques to elucidate the role this mutation may play in the modulation of troponin’s function. we used nuclear magnetic resonance to solve the structure and characterize the backbone dynamics of the regulatory lobe of troponin c with this mutation. the overall structure and dynamics of troponin c was not significantly altered by l q; however there was a slight rearrangement of site making it more similar to trout cardiac troponin c, which also has a glutamine at position at residue and displays increased ca þ sensitivity. backbone dynamics mea- surements indicated that q was more flexible than l . the structure and function of l q was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. the structure and/or orientation of the regulatory lobe of troponin c was slightly perturbed by l q in relaxing conditions and was unaffected at activating ca þ concentrations. the ca þ sensitivity of the structural change and contractility were both unaltered by the l q mutation, suggesting that while this may cause a small change in the structure of troponin c, this does not translate to a large functional effect in cardiac muscle. -pos board b troponin i ser- phosphorylation sustains troponin ca d sensitivity in an acidic environment benjamin r. nixon, shane d. walton, jonathan p. davis, brandon j. biesiadecki. physiology and cell biology and davis heart and lung research institute, the ohio state university, columbus, oh, usa. a hallmark of cardiac ischemia is decreased intracellular ph which can affect a number of cellular processes. such an acidic environment alters cardiac troponin (tn) myofilament regulation to decrease ca þ sensitive force produc- tion. tn also undergoes cardiac ischemia-induced ampk troponin i (tni) ser- phosphorylation. we recently characterized the effects of tni ser- phos- phorylation demonstrating that it blunted the functional effects of canonical tni ser- / phosphorylation; however, the role of ser- phosphorylation in ischemia remains unknown. as an initial step, we sought to investigate the effect of acidic ph on myofilament regulation in the presence of tni ser- phos- phorylation alone and in combination with ser- / phosphorylation. we first investigated the effect of in vivo cardiac ischemia on levels of tni ser- and ser- / phosphorylation. exposure to minutes of regional ischemia re- sulted in elevation of both tni ser- and ser- / phosphorylation. next we determined the effects of tni ser- pseudo-phosphorylation (s d) on the myofilament by measuring troponin c (tnc) ca þ binding properties at normal and acidic ph. results demonstrate acidic ph decreases steady- state ca þ binding to tnc in reconstituted thin filaments across all tn (wt, s d, s / d, and s / / d) such that tni s d ca þ sensitivity at ph . is similar to wt at ph . decreasing the ph had no effect on ca þ disso- ciation such that compared to wt, s / / d remained fast while s d was slowed. we conclude that tni ser- phosphorylation imparts resistance to acidic ph-induced myofilament ca þ desensitization while retaining increased tn ca þ dissociation when in combination with ser- / phosphorylation suggesting the potential for an increase in force while maintaining accelerated ca þ dissociation. future investigations are aimed at examining the effect of tni ser- and ser- / phosphorylation on protease cleavage of tni. -pos board b deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue bin wei, yingru lu, j.-p. jin. physiology, wayne state university, detroit, mi, usa. loss of slow skeletal muscle troponin t (sstnt) due to a nonsense mutation at codon glu in exon of the tnnt gene causes a severe form of recessive nemaline myopathy (amish nemaline myopathy, anm). to investigate the pathogenesis and muscle pathophysiology of anm, we studied the phenotypes of partial and total loss of sstnt in tnnt gene targeted mice. an insertion of neomycinr cassette in intron of tnnt caused approximately % decrease in sstnt protein expression whereas deletion of exons - using cre-loxp approach resulted in total loss of sstnt as that seen in the muscle of anm pa- tients. in diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that sstnt deficiency resulted in significantly decreased levels of other slow fiber-specific myofilament proteins while fast fiber-specific myofilament proteins were increased. histology studies revealed that sstnt deficiency caused significant atrophy of type i slow fibers and a hypertrophic growth of type ii fast fibers. along with the slow fiber atrophy and the changes in myofilament protein isoform contents, sstnt deficiency in soleus muscle shifted the force-frequency relationship toward the fast muscle type and significantly reduced the tolerance to fatigue. sstnt deficient soleus muscle also exhibited a significant number of smaller size central nuclei type i fibers, indicating an adaptive regeneration. sstnt deficient mouse soleus muscle contained apparently normal number of spindles, in which intrafusal fibers were positive for type i myosin with a trend of atrophic morphology. the results demonstrate the essential function of sstnt in skeletal muscle and the causal effect of its loss on the pathology of anm. -pos board b attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp thomas j. longyear , matthew a. turner , brandon j. biesiadecki , joseph lopez , jonathan p. davis , edward p. debold . university of massachusetts, amherst, ma, usa, the ohio state university, columbus, oh, usa. repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, which defines fatigue. the decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains unclear. we sought to gain novel insight into the decrease in velocity by determining if the depressive effect of acidosis could be altered by ) introducing ca þþ -sensitizing mutations into troponin (tn) or ) by agents that directly affect myosin function, including inorganic phosphate (pi) and -deoxy-atp (datp) in an in vitro motility assay. acidosis reduced regulated thin filament velocity (vrtf) at both maximal and sub- maximal caþþ levels in a ph-dependent manner. a truncated construct of the inhibitory subunit of tn, r , and a caþþ-sensitizing mutation in the caþþ- binding subunit of tn, v q, increased vrtf at sub-maximal ca þþ under acidic conditions, but had no effect on vrtf at maximal ca þþ levels. in contrast, both mm pi and replacement of atp with datp reversed much of the acidosis- induced depression of vrtf at saturating ca þþ ( . . control, . . withpi, . . withdatp, . . withbothpi anddatp),withthecombined effectfully restoringthevrtf to thevalue under control conditions.interestingly, despite producing similar magnitude increases in vrtf, the combined effects of pi and datp were additive, suggesting different underlying mechanisms of action. these results suggest that the major mechanism by which acidosis slows vrtf is through directly slowing myosin’s rate of detachment from actin. -pos board b the effect of truncated troponin components on activation of lethocerus flight muscle belinda bullard , bogos agianian , gian-felice de nicola , annalisa pastore , kevin leonard . department of biology, university of york, york, united kingdom, molecular biology and genetics, democritus university of thrace, alexandroupolis, greece, national institute of medical research, london, united kingdom, european bioinformatics institute, cambridge, united kingdom. indirect flight muscle (ifm) of lethocerus is activated by periodic stretches at a constant priming concentration of calcium. the muscle is unusually stiff and stress is transmitted to the thick and thin filaments by kettin, which reinforces links between both filaments and the z-disc. the activating effect of stress on thin filaments is likely to affect troponin. the isoforms of troponin in ifm differ from those in other muscles. tnt has a c-terminal extension not present in vertebrate tnt; tnh is an isoform of tni with a c-terminal extension rich in pro and ala; tnc is present in two isoforms: f binds a single calcium in the c-lobe and is needed for stretch-activation; f binds one calcium in both n- and c-lobes and is needed for isometric force. under conditions of low ionic strength, native fibres have a force-pca curve that shows high calcium- sensitivity and low cooperativity (pca = . , nh = . ). fibres with f alone have a pca curve similar to that of cardiac muscle, (pca = . , nh = . ). a fragment of f without the n-lobe (f -ct) inhibits stretch-activation; therefore the n-lobe of f is necessary, although it does not bind calcium or tnh. f -ct is displaced by f and isometric force is restored, but not stretch-activation. we hope to show the effect of replacing endogenous troponin in fibres with a com- plex containing tnt truncated at the c-terminus, tnh with tni sequence but without the pro-ala extension, and either f or f . this will show how impor- tant the ifm isoforms of troponin are to the stretch-activation response. -pos board b changes in the orientation of the myosin light chain domain (lcd) associated with thick filament-based regulation of skeletal muscle luca fusi, zhe huang, malcolm irving. randall division of cell and molecular biophysics, king’s college london, london, united kingdom. the dependence of myosin lcd orientation on temperature, myofilament lattice spacing and sarcomere length was determined using fluorescence 	troponin i ser- phosphorylation sustains troponin ca + sensitivity in an acidic environment 	deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue 	attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp 	the effect of truncated troponin components on activation of lethocerus flight muscle 	changes in the orientation of the myosin light chain domain (lcd) associated with thick filament-based regulation of skelet ... wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ veloso v, et al. colestasis intrahepática familiar progresiva tipo . an facultad med (univ repúb urug). ; ( ): - anfamed - issn: - caso clínico colestasis intrahepática familiar progresiva tipo , una causa rara de cirrosis en el adulto joven resumen se describe el caso de una paciente joven con una enfermedad hepática diagnosticada como colestasis intrahepática familiar progresiva tipo , seis años luego de su debut clínico, basado en los hallazgos histológicos y el estudio de las mutaciones en el gen abcb . palabras clave abcb , colestasis, colestasis intrahepática familiar progresiva. title progressive intrahepatic familial cholestasis type , an atypical cause of cirrhosis in a young patient. abstract this study describes a young female patient diagnosed with progressive familial intrahepatic cholestasis type , six years after her clinical debut based in histological findings and gene analysis which revealed multiple mutations in the abcb gene. key words abcb , cholestasis, progressive familial intrahepatic cholestasis. . clínica de gastroenterología, facultad de medicina, universidad de la república, uruguay. . departamento de gastroenterologia, hepatología y nutricion pediatrica, centro hospitalario pereira rossell, facultad de medicina, universidad de la república, uruguay. * contacto: victoria veloso giribaldi. e-mail: victoriavelosogiribaldi@gmail.com introducción la colestasis intrahepática familiar progresiva (cifp) es un trastorno hepático de herencia auto- sómica recesiva que aparece como consecuencia de mutaciones en genes que codifican para pro- teínas encargadas del transporte hepatocelular [ - ]. se traduce en colestasis crónica, con el prurito como rasgo clínico dominante [ ]. la in- cidencia estimada es de cada . a . nacimientos pero su prevalencia exacta se desco- noce [ ]. afecta a ambos sexos y se ha registrado victoria veloso giribaldi *, paula daniela chiodi , carola lópez , nelia hernández en todas las áreas geográficas, con predominan- cia en ciertos grupos étnicos (amish, esquimales, indios canadienses) [ ]. constituye el - % de las colestasis neonatales y el a % de las indicaciones de trasplante hepático en niños [ ]. su curso natural es hacia el desarrollo de hiper- tensión portal, falla hepática, cirrosis, carcinoma hepatocelular y la necesidad de trasplante hepá- tico a edades tempranas [ ]. sobre la base de la presentación clínica, los hallazgos de laborato- rio, la histología hepática y el defecto genético, anfamed - issn: - caso clínico veloso v, et al. colestasis intrahepática familiar progresiva tipo . an facultad med (univ repúb urug). ; ( ): - se divide en tres tipos (tabla ): tipo , también llamada enfermedad de byler´s (deficiencia de la proteína de colestasis intrahepática familiar ); tipo o síndrome de byler´s (deficiencia de la bomba exportadora de sales biliares) y la tipo en la que se observa un defecto en la proteína de multirresistencia a drogas (pmrd ) [ - ]. el defecto básico en cifp y es la alteración de característica cifp cifp cifp edad de presentación infancia período neonatal, infancia temprana infancia tardía (~ %), edad adulta temprana estadio final de enfermedad hepática primera década tempranamente, primeros años ª – ª década curso de la enfermedad moderadamente severo severo insidioso prurito severo muy grave moderado manifestaciones extrahepáticas diarrea, pancreatitis, retraso de crecimiento ausentes ausentes riesgo de desarrollo de cáncer hepático no alto aumento leve riesgo de cálculos de colesterol ausente aumento aumento alt sérica elevación leve elevación moderada ( vn) elevación leve alfa feto proteína normal elevado normal ggt normal normal elevada Ácidos biliares elevado ++ elevado+++ elevado + composición de la bilis Ácidos biliares primarios bajo ( - mm) muy bajo (< mm) normal fosfolípidos normal normal baja histología hepática colestasis , fibrosis lobular leve colestasis, células gigantes, necrosis hepatocelular, fibrosis porta proliferación ductal biliar, infiltrado inflamatorio y fibrosis biliar tabla . principales características de los tipos de colestasis intrahepática familiar progresiva. cifp: colestasis intrahepática familiar progresiva; alt: alanina aminotransferasa; vn: valores normales ; ggt: gamma-glutamil transpeptidasa; mm: mili molar. veloso v, et al. colestasis intrahepática familiar progresiva tipo . an facultad med (univ repúb urug). ; ( ): - anfamed - issn: - caso clínico la secreción de sales biliares mientras que en la cifp , se debe a una reducción de la secreción de fosfolípidos biliares [ , ]. la cifp y re- presentan / de los casos, mientras que la cifp representa el tercio restante [ - ]. aunque el tratamiento con ácido ursodeoxicólico tiene be- neficio, una vez establecida la cirrosis es frecuen- te la necesidad de trasplante hepático [ ]. caso clínico mujer de años sin antecedentes persona- les ni familiares a destacar, que fue referida a la clínica de gastroenterología del hospital de clínicas, de la facultad de medicina, de la uni- versidad de la república, uruguay, para estudio de una colestasis crónica. su enfermedad co- menzó años atrás, con reiterados episodios de dolor abdominal de características inespecíficas y prurito en palmas y plantas. el examen físico era normal. la bioquímica al ingreso mostraba: bilirrubina . mg/dl, fosfatasa alcalina ( - u/l), gama-glutamil-transpeptidasa (< u/l), aspartato-transferasa (< u/l), alanino-transferasa (< u/l), albúmina . ( . - g/dl), tasa de protrombina %, hemog- lobina . ( - g/dl), glóbulos blancos ( - /mm ), plaquetas mil ( - mil/mm ), colesterol , (< g/l). la ecogra- fía de abdomen y la colangiografía endoscópica retrógrada no mostraban alteraciones y la fibro- gastroscopía evidenció várices esofágicas peque- ñas. la serología infecciosa (virus de hepatitis b y c, virus de inmunodeficiencia humana y ) y los autoanticuerpos (anti-nucleares, anti-mus- culo liso, anti-microsomales hígado-riñón, anti- citoplasma del neutrófilo y anti-mitocondriales) fueron negativos. no presentaba anillo de keyser fleischer, la cupruria de hs y la dosificación de alfa antitripsina fueron normales. la biopsia hepática puso en evidencia una arquitectura dis- torsionada, con nódulos y tractos fibrosos porto- porta, infiltrado mononuclear, proliferación duc- tal e infiltrado polimorfonuclear en la pared de los pequeños ductos (imágenes colangiolíticas). la inmunohistoquímica fue concordante con de- ficiencia de pmrd , y el estudio genético evi- denció heterocigosidad compuesta para el gen abcb (transición g>a en nucleótidos y en el exón ). aun bajo tratamiento con mg/k/día de ácido ursodeoxicólico, la evolu- ción fue al desarrollo de insuficiencia hepática, hipertensión portal y ascitis, ingresando a lista de espera para trasplante hepático. discusión la colestasis es un síndrome clínico y bioquími- co determinado por la detención o disminución del flujo biliar al duodeno. puede determinar ictericia, figura .inmunohistoquímica sobre tejido hepático (cortesía del dr. luis Álvarez) a la izquierda: proteína de resistencia a múltiples drogas ; en el centro: bomba exportadora de sales biliares; a la derecha: proteína de multirresistencia a drogas anfamed - issn: - caso clínico veloso v, et al. colestasis intrahepática familiar progresiva tipo . an facultad med (univ repúb urug). ; ( ): - prurito, aumento de la fosfatasa alcalina y gama- glutamil-transpeptidasa. el origen extrahepático fue descartado mediante los exámenes de imagen. entre las causas intrahepáticas, la colangitis escle- rosante primaria, hipótesis a tener presente, fue alejada por la ausencia de episodios de colangitis, de enfermedad inflamatoria intestinal y el hallazgo de una vía biliar normal en la colangiografía. los hallazgos histológicos permitieron el diagnóstico de la cifp . a diferencia de las otras formas, los pacientes con cifp cursan con niveles altos de gama-glu- tamil-transpeptidasa, ácidos biliares y transamina- sas, con niveles de colesterol normales o poco ele- vados. es rara la ictericia neonatal, presentándose en la infancia o adolescencia con complicaciones de la cirrosis o hallazgo de hepato-esplenomegalia [ ]. el prurito es leve y la colestasis puede acom- pañarse o no de ictericia. la evolución a cirrosis en la primera década de la vida es propia de pa- cientes con mutaciones que causan una ausencia completa de función de pmrd . las mutaciones que conllevan una actividad residual permiten ma- yor supervivencia y podrían ser la causa de cirrosis inexplicada en adultos. el carcinoma hepatocelu- lar puede desarrollarse en la adolescencia [ ]. la cifp es una enfermedad autosómica rece- siva, causada por mutaciones en el gen abcb , lo- calizado en el cromosoma ( q ) [ - ]. el diag- nóstico se realiza mediante inmunohistoquímica y el estudio de las mutaciones permite identificar casos con escasa o ninguna expresión histoquími- ca. las alteraciones del abcb abarcan un gran espectro de enfermedades como la colestasis intra- hepática del embarazo, la colestasis inducida por drogas, la colestasis neonatal transitoria, litiasis biliar intrahepática recurrente en jóvenes [ ]. así, ante una adolescente con colestasis de inicio en la infancia tardía, descartadas otras causas más fre- cuentes, que desarrolla cirrosis con hipertensión portal, en ausencia de manifestaciones extrahepá- ticas, con hallazgos bioquímicos (aumento de ga- ma-glutamil-transpeptidasa, sin marcado aumento de colesterol) e histológicos congruentes, se debe sospechar el diagnóstico de cifp y su confir- mación debe ser mediante la inmunohistoquími- ca (deficiencia de pmrd ) y el análisis genético (abcb ). referencias . hierro l, jara p. colestasis infantil y trans- portadores biliares. j gastroenterol he- patol. ; ( ): - . http://dx.doi. org/ . / . strautnieks ss, kagalwalla af, tanner ms, gar- diner rm, thompson rj. locus heterogeneity in progressive familial intrahepatic cholestasis. j med genet. ; : - . . deng bc, lv s, cui w, zhao r, lu x, wu j, et al. novel atp b mutation in an adult male with progressive familial intrahepatic cholesta- sis. world j gastroenterol. ; ( ): - . http://dx.doi.org/ . /wjg.v .i . . hori t, nguyen jh, uemoto s. progressive fami- lial intrahepatic cholestasis. hepatobiliar pancreat dis int. ; ( ): - . . jacquemin e, hadchouel m. genetic basis of pro- gressive familial intrahepatic cholestasis. j hepa- tol ; ( ): - . http://dx.doi.org/ . / s - ( ) - . stapelbroek jm, van erpecum kj, klomp lw, houwen rh. liver disease associated with cana- licular transport defects: current and future thera- pies. j hepatol. ; ( ): - . http://dx.doi. org/ . /j.jhep. . . . hirschfield gm, heathcote ej, gershwin me. pathogenesis of cholestatic liver disease and therapeutic approaches. gastroenterology. ; ( ): – . . de vree jm, jacquemi e, sturm e, cresteil e, bos- ma pj, aten j, et al. mutations in the mdr gene cause progressive familial intrahepatic cholestasis. proc natl acad sci usa. ; ( ): - . . jansen pl, strautnieks ss, jacqemin e, hadchouel m, sokal em, hooiveld gj, et al. hepatocanali- cular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis. veloso v, et al. colestasis intrahepática familiar progresiva tipo . an facultad med (univ repúb urug). ; ( ): - anfamed - issn: - caso clínico gastroenterology. ; ( ): – . . bezerra ja, balistreri wf. intrahepatic cho- lestasis: order out of chaos. gastroenterology. ; ( ): - . . jansen plm, muller mm. progressive fami- lial intrahepatic cholestasis types , , and . gut. ; : - . http://dx.doi.org/ . / gut. . . . whitington pf, whitington gl. partial external diversion of bile for the treatment of intractable pruritus associated with intrahepatic cholestasis. gastroenterology. ; ( ): - . . srivastava a. progressive familial intrahepatic cholestasis. j clin exp hepatol. ; ( ): - . http://dx.doi.org/ . /j.jceh. . . . hori t, egawa h, takada y, ueda m, oike f, ogu- ra y, et al. progressive familial intrahepatic cho- lestasis: a single-center experience of living-donor liver transplantation during two decades in japan. clin transplant. ; ( ): – . http://dx. doi.org/ . /j. - . . .x . jacquemin e. progressive familial intrahepa- tic cholestasis. clin res hepatol gastroente- rol. ; suppl :s -s . http://dx.doi. org/ . /s - ( ) - news & views nature genetics • volume • march as described on pages – of this issue, judith goodship and a multi- national group of collaborators have identified the gene that is mutated in people with a form of dwarfism, ellis-van creveld (evc) syndrome. they discov- ered five different mutations, including one that underlies the disorder in the old order amish. originally described in (ref. ) by paediatricians richard ellis and simon van creveld, evc syndrome is an autosomal reces- sive disorder, involving postaxial poly- dactyly of the hands (see figure), short stature with shortening especially of the forearms and lower legs and, in at least half of all cases, congenital heart malformation. the mutation in the amish of lan- caster county, pennsylvania, in whom the disorder occurs at unprecedentedly high frequency, is predicted to cause aberrant splicing. it occurs in the fifth nucleotide of intron of a novel gene, evc, that is predicted to encode a pro- tein containing a leucine zipper, three putative nuclear localization signals and a putative transmembrane do- main. the pathogenic ‘status’ of the amish mutation is supported by the fact that mutation of analogous nucleotides effect disease: according to the human gene mutation database , intronic mutations, causing a total of separate disorders, have been reported at the + position of introns. of history and heritage the amish have several characteristics , that recommend them to medical geneti- cists. first, they are descended from a lim- ited number of founders who immigrated, during the eighteenth century, to the united states from the rhineland (in the southwest of germany) where they had settled temporarily following emigration in the s from the canton of berne, switzerland. second, the amish observe strict endogamy (they marry only within the community), with gene flow being exclusively centrifugal (that is, members may leave the community but ‘outsiders’ do not join it and thereby introduce exogenous genes). third, like the ice- landers, they keep excellent genealogic records and have a restricted geography. finally, they tend to have large families, with many children. it was therefore possible to trace the lineage of both parents of all evc cases back to a single couple, samuel king and his wife (regrettably, her name is no longer known), who immigrated to east- ern pennsylvania in —thus demon- strating founder effect and a recessive pattern of inheritance. epidemiological data indicate that the frequency of the mutated gene is approximately . and that heterozygotes make up about . % of the population . at the time of these estimates, . % of lancaster county amish carried samuel king’s surname, and samuel was the only male founder of that name. during studies carried out in the mid- sixties , , it became apparent that the amish are distributed in three consanguineal kin groups (demes) across the united states. at that time, each was made up of about , members. the deme in lan- caster county was founded by those who immigrated before the american revolution. the deme of holmes county (ohio), and the deme com- prised of groups in lagrange and elkhart counties (northern indiana) descended, for the most part, from post- revolutionary immigrants who, upon finding the land taken up in eastern pennsylvania, moved to points west. the genetic distinctness of the three major demes is supported by different patterns of blood-group frequencies , , different family names— % of people in the lancaster deme have the name stoltzfus, which is absent in the other demes—and different frequencies of rare recessive disorders. for example, evc syndrome was found to be limited to the lancaster-county deme. haemophilia b, on the other hand, was (and still is) unusually frequent in the holmes-county deme, and almost completely limited to that group. it is as though the amish immigrants were streaked like bacteria on a culture plate across the waist of america, with the genetic profile of each deme depending on the genetic constitution of the founders, for whom the present popula- tions represent a bioassay. cartilage-hair hypoplasia a second recessive form of dwarfism, dis- tinct from evc, is prevalent in the lan- caster-county deme . cartilage-hair hypoplasia (chh) was previously unrec- ognized until the amish came to the attention of clinical geneticists in the mid- sixties. in contrast with evc syndrome, it occurs in all amish demes. moreover, it is impossible to trace its origin to a single founder couple, indicating that the muta- tion was introduced by several immi- grants. it turns out that chh is also frequent in finland ; the odds favour a mutation of independent origin with ellis-van creveld syndrome and the amish victor a. mckusick institute of genetic medicine, johns hopkins hospital, baltimore, maryland , usa. e-mail: mckusick@peas.welch.jhu.edu genetic studies often involve the cooperation of large numbers of affected persons and their families. the discovery of the gene that, when mutated, causes a form of dwarfism (ellis-van creveld syndrome) has been accelerated through a collaborative effort between geneticists and the old order amish, of lancaster county, pennsylvania. amish mother and child. the child has ellis-van creveld syndrome, which is characterized by polydactyly (six fingers on each hand), short stature, and shortening of the fore- arms and lower legs. (image reproduced with permission from johns hopkins university press). © nature america inc. • http://genetics.nature.com © n a tu re a m e ri c a i n c . • h tt p :/ /g e n e ti c s .n a tu re .c o m news & views nature genetics • volume • march respect to that carried by the amish— albeit one that has achieved a high fre- quency through the same mechanisms: founder effect and perhaps genetic drift. whereas the ‘causative’ gene’s locus is known , its identity yet eludes the assidu- ous efforts of positional cloners in helsinki and bethesda. after discovery of chh in the amish, rare cases of chh were recognized in non-amish. for example, billy barty, an actor and founder of little people of america, a support group for persons of short stature, has chh. so did michael (‘pat’) bilon, who played et in the movie of that name. medical genetics is indebted to the amish for their cooperation in studies that have led to an improved understand- ing of genetic disorders. the physicians who carried out the studies in the s and s approached the amish with a view to helping them. arrangements were made, for example, for surgical repair of the cardiac defect in evc patients and for orthopedic correction of their knee defor- mities. aid was also provided to family members with non-evc related problems of great diversity. how could knowledge of the amish ‘evc’ mutation help? pre- marital and pre-natal counselling should now be possible, based on testing for the splice-site mutation or a nearby marker— ideally one within the gene. goodship and colleagues discovered a polymor- phism that is in linkage disequilibrium with the ‘causative’ mutation, and could therefore serve as such a marker. whether the amish would acquiesce to premarital testing is uncertain, and it is unlikely that they would accept prenatal testing because of the implication of abortion. because a specific evc muta- tion is limited to the lancaster county amish, marriage between an evc carrier with an amish from another community might be recommended but may generate logistical difficulties. alternatively, know- ledge of carrier status could inform choice of partner within the lancaster county amish community. the amish acceptance of the geneti- cists was achieved by their being intro- duced by local physicians and by sociologists whom they trusted. the rela- tionships were maintained through com- munication with the bishops and others in authority and by the assistance of amish who served as guides and intro- ducers during home visits. another notable example of beneficial collabora- tion between geneticists and religous community is that between the ashke- nazi jewish groups who use screening for mutations that cause tay-sachs disease as the basis of marriage advice by rabbis. the evc syndrome in the amish has become a favourite elementary genetics textbook example of several aspects of human genetics. now, to founder effect, consanguinity, recessive inheritance and so on, one can add linkage mapping, positional cloning and the molecular nature of mutation, as well as carrier detection and the social implications thereof. possibly, it will not be long before the student can be informed of the way in which the mutation disturbs development, leading to polydactyly, heart defect and skeletal dysplasia. � . ruiz-perez, r.-l. et al. nature genet. , – ( ). . ellis, r.w.b. & van creveld, s. arch. dis. child. , – ( ). . krawczak, m. & cooper, d.m. trends genet. , – ( ). . hostetler, j.a. amish society (johns hopkins university press, baltimore, ). . mckusick, v.a., hostetler, j.a., egeland, j.a. & eldridge, r. cold spring harbor symp. quant. biol. , – ( ). . mckusick, v.a. in medical genetic studies of the amish: selected papers assembled, with commentary (ed. mckusick, v.a.) – (johns hopkins university press, baltimore, ). . cross, h.e. & mckusick, v.a. social biol. , – ( ). . murdock, g.p. social structure (macmillan, new york, ). . mckusick, v.a., bias, w.b., norum, r.a. & cross, h.e. humangenetik , – ( ). . juberg, r.c., schull, w.j., gershowitz, h. & davis, l.m. human biol. , – ( ). . mckusick, v.a., eldridge, r., hostetler, j.a., ruangwit, u. & egeland, j.a. bull. johns hopkins hosp. , – ( ). . kaitila, i. & perheentupa, j. in population struc- ture and genetic disorders (eds eriksson, a.w., forsius, h.r., nevanlinna, h.r., workman, p.l. & norio, r.k.) – (academic, new york, ). . sulisalo, t. et al. nature genet. , – ( ). making the most of microarray data terry gaasterland & stefan bekiranov laboratory of computational genomics, the rockefeller university, york ave, new york, new york, , usa. e-mail: gaasterland@genomes.rockefeller.edu and bek@genomes.rockefeller.edu the impact of microarray technology on biology will depend on computational methods of data analysis. a supervised computer- learning method using support vector machines predicts gene function from expression data—and shows promise. microarray assays can measure the tran- scriptional effects of changes in gene func- tion under different conditions. they can reveal genes that characterize tissue type, developmental stage, or responses to envi- ronmental conditions or genetic modifica- tions. microarray assays will therefore become a general feature of experimental protocols in genetics and cell physiology. as array data burgeon, new questions arise: if we, as a research community, col- lect all array hybridization data in a central location , can we assign new genes of unknown function to known functional classes? can we correlate gene expression with gene function? can we find new classes of co-regulated genes? can we extract complete gene regulatory networks from microarray gene expression data? computation is our only hope, and an article by michael brown and colleagues in a recent issue of the proceedings of the national academy of sciences describes an approach to microarray data analysis that addresses the first question. the authors use support vector machines (svms; fig. ), a supervised computer-learning method, to train a ‘classification machine’ to recognize new genes that are similar in expression pattern to groups of genes known to be co-regulated. in contrast with classical unsupervised clustering methods and pure self-organizing maps, the approach builds on existing knowledge (fig. ) and has the potential to refine and correct it. © nature america inc. • http://genetics.nature.com © n a tu re a m e ri c a i n c . • h tt p :/ /g e n e ti c s .n a tu re .c o m the university of manchester research focal congenital hyperinsulinism as a cause for sudden infant death doi: . / document version accepted author manuscript link to publication record in manchester research explorer citation for published version (apa): chinoy, a., banerjee, i., flanagan, s. e., ellard, s., han, b., mohamed, z., dunne, m. j., & bitetti, s. ( ). focal congenital hyperinsulinism as a cause for sudden infant death. pediatric and developmental pathology. https://doi.org/ . / published in: pediatric and developmental pathology citing this paper please note that where the full-text provided on manchester research explorer is the author accepted manuscript or proof version this may differ from the final published version. if citing, it is advised that you check and use the publisher's definitive version. general rights copyright and moral rights for the publications made accessible in the research explorer are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. takedown policy if you believe that this document breaches copyright please refer to the university of manchester’s takedown procedures [http://man.ac.uk/ y bo] or contact uml.scholarlycommunications@manchester.ac.uk providing relevant details, so we can investigate your claim. download date: . apr. https://doi.org/ . / https://www.research.manchester.ac.uk/portal/en/publications/focal-congenital-hyperinsulinism-as-a-cause-for-sudden-infant-death( d e d -a c - c-b -dbf a ).html /portal/mark.j.dunne.html https://www.research.manchester.ac.uk/portal/en/publications/focal-congenital-hyperinsulinism-as-a-cause-for-sudden-infant-death( d e d -a c - c-b -dbf a ).html https://www.research.manchester.ac.uk/portal/en/publications/focal-congenital-hyperinsulinism-as-a-cause-for-sudden-infant-death( d e d -a c - c-b -dbf a ).html https://doi.org/ . / for peer review focal congenital hyperinsulinism as a cause for sudden infant death journal: pediatric and developmental pathology manuscript id pdp- - .r manuscript type: case report date submitted by the author: -dec- complete list of authors: chinoy, amish; royal manchester children's hospital, paediatric endocrinology banerjee, indraneel; royal manchester children's hospital, paediatric endocrinology flanagan, sarah; university of exeter medical school, institute of biomedical and clinical science ellard, sian; university of exeter medical school, institute of biomedical and clinical science han, bing; university of manchester, faculty of biology, medicine and health mohamed, zainab; royal manchester children's hospital, paediatric endocrinology; university of manchester, faculty of biology, medicine and health dunne, mark; university of manchester, faculty of biology, medicine and health bitetti, stefania; st mary's hospital, paediatric histopathology keywords: hypoglycaemia, sudden infant death, post-mortem, congenital hyperinsulinism, pancreas, insulin abstract: congenital hyperinsulinism (chi) is the commonest cause of persistent and severe hypoglycaemia in infancy due to unregulated insulin secretion from pancreatic beta cells. prompt early diagnosis is important, as insulin reduces glucose supply to the brain, resulting in significant brain injury and risk of death. histologically, chi has focal and diffuse forms; in focal chi, an inappropriate level of is secreted from localised beta-cell hyperplasia. we report a four-month old boy, who presented with sudden illness and collapse without a recognised cause and died. post-mortem examination revealed pancreatic histopathology compatible with focal chi. immunofluoresence-staining showed limited expression of p kip beta-cells reinforcing the diagnosis. mutation testing for genes associated with chi from dna from the focal lesion was negative. this case highlights the recognition of focal chi as a possible cause for sudden infant death. in children dying suddenly and unexpectedly, post-mortem pancreatic sections should be carefully examined for focal chi. https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review pediatric and developmental pathology case report title: focal congenital hyperinsulinism as a cause for sudden infant death authors: dr amish chinoy (amishchinoy@doctors.org.uk), dr indraneel banerjee (indi.banerjee@cmft.nhs.uk), dr sarah e flanagan (s.flanagan@exeter.ac.uk), professor sian ellard (s.ellard@exeter.ac.uk), bing han (bing.han- @postgrad.manchester.ac.uk), dr zainab mohamed , (zainab.mohamed@nuh.nhs.uk), professor mark j dunne (mark.j.dunne@manchester.ac.uk), dr stefania bitetti (stefania.bitetti@cmft.nhs.uk) institutions: . department of paediatric endocrinology, royal manchester children’s hospital, manchester, uk . institute of biomedical and clinical science, university of exeter medical school, exeter, uk . faculty of biology, medicine & health, university of manchester, manchester, uk . department of paediatric histopathology, st mary’s hospital, manchester, uk corresponding author: dr stefania bitetti; stefania.bitetti@cmft.nhs.uk; department of paediatric histopathology, st mary’s hospital, oxford road, manchester, uk, m wl; running head: focal chi and sudden infant death page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review grant support: the authors received support from the norchi charitable fund (m.j.d, i.b.) , the national institute for health research (m.j.d., i.b.), the million dollar bike fund (pilot award number: mdbr- - -chi; m.j.d.,i.b.) and the sir henry dale fellowship jointly funded by the wellcome trust and the royal society (grant number: /z/ /z; sef) acknowledgements: none declarations of conflicts of interest: the authors declare that there is no conflict of interest focal congenital hyperinsulinism as a cause for sudden infant death abstract congenital hyperinsulinism (chi) is the commonest cause of persistent and severe hypoglycaemia in infancy due to unregulated insulin secretion from pancreatic beta cells. prompt early diagnosis is important, as insulin reduces glucose supply to the brain, resulting in significant brain injury and risk of death. histologically, chi has focal and diffuse forms; in focal chi, an inappropriate level of is secreted from localised beta-cell hyperplasia. we report a four-month old boy, who presented with sudden illness and collapse without a recognised cause and died. post-mortem examination revealed pancreatic histopathology compatible with focal chi. immunofluoresence-staining showed limited expression of p kip beta-cells reinforcing the diagnosis. mutation testing for page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review genes associated with chi from dna from the focal lesion was negative. this case highlights the recognition of focal chi as a possible cause for sudden infant death. in children dying suddenly and unexpectedly, post-mortem pancreatic sections should be carefully examined for focal chi. key words: congenital hyperinsulinism, hypoglycaemia, post-mortem, sudden infant death, pancreas, insulin running head: focal chi and sudden infant death introduction congenital hyperinsulinism (chi) is the commonest cause of persistent and severe hypoglycaemia in infancy. chi typically presents in the neonatal period, although cases in infancy and even childhood have been reported [ ]. these infants typically present with symptoms of hypoglycaemia, with detectable/inappropriate insulin and c-peptide levels for hypoglycaemia, along with suppressed beta-hydroxybutyrate and free fatty acids. prompt diagnosis is important, as the presence of insulin inhibits ketone formation, resulting in significant brain injury if the hypoglycaemia is persistent or profound [ ]. acute management involves provision of high concentration glucose, either in feeds or parenterally, and glucagon as an emergency measure. long-term medical management includes diazoxide and octreotide as first-line and second-line options respectively. failing response to medical therapy, pancreatectomy may be required [ ]. page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review chi refers to a heterogenous group of conditions, with genetic aetiology in around % of patients with mutations in known genes, those affecting the atp sensitive k + -channel (abcc /kcnj ) being most frequent [ , ]. histologically, chi can be classified broadly into focal and diffuse forms. in the diffuse form, hyper-functioning pancreatic beta cells with nucleomegaly are distributed throughout the pancreas, whereas in the focal form there is nodular hyperplasia of islet cells surrounded by normal tissue [ ]. we report a four-month old male infant who died in hospital after presentation with an acute illness and collapse. pancreas histology at post mortem revealed an isolated focal lesion in keeping with focal chi. case report a four-month old boy presented to hospital unresponsive. the background to this episode was birth at + weeks of gestation following normal vaginal delivery, with a weight of . kg ( th centile) and satisfactory apgar scores ( at minute and at minutes). he spent days on the neonatal unit, requiring antibiotics for suspected sepsis. he had received phototherapy for unconjugated hyperbilirubinaemia. hypoglycaemia was identified by point of care testing during this neonatal period on more than one episode and was promptly treated with frequent feeds, although a venous sample for confirmation was not obtained and the specific value of glucose was not recorded in the case notes. the cause for hypoglycaemia at the time was not investigated. hypoglycaemia was corrected for inadequate glucose intake on the page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review background of prematurity, as per the unit policy. sepsis was presumed to be a cause for hypoglycaemia and treated robustly, although blood cultures were negative indicating that infection was an unlikely aetiology. following discharge from the neonatal unit, he had reportedly been well until just prior to representation. prior to representation, the parents had noted blood in his nappy. balanitis was diagnosed, and treated with oral antibiotics in the community. the following day he fed poorly, and suffered from vomiting and diarrhoea. his condition deteriorated rapidly and he became unresponsive. he was rushed to hospital, where he was noted to be hypothermic, pale, with circulatory failure. there were no external signs of bleeding. initial blood tests showed profound hypoglycaemia (serum glucose < . mmol/l), acidosis (ph . ) and pancytopaenia (haemoglobin g/l, white cell count . x /l, platelet count x /l). despite extensive resuscitative measures, including ventilatory support, fluid boluses, inotropes and antibiotics, he died within hours of presentation. a coroner’s post-mortem examination was conducted. autopsy revealed a male infant with normal growth, with crown heel length ( cm) and weight ( . kg) between th and th centiles for his corrected age for gestation ( months and weeks). no congenital abnormalities of the internal organs were identified at autopsy. a male karyotype was noted in keeping with male genitalia. post mortem examination did not identify liver or kidney tumours; there were no clinical features of beckwith wiedemann syndrome. a widespread petechial rash was present. bone marrow histology showed a severely left shifted marrow with immature precursor cellular profile, thus raising the possibility of sepsis/acute infective terminal illness. however, no positive bacterial culture or tissue inflammation was ever identified. page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review the most significant finding was found on pancreas histology. this revealed a localised (focal) expansion of islets interspersed within normal pancreatic tissue suggestive of focal chi. targeted next generation sequencing of dna isolated from pancreatic tissue did not reveal any coding mutations or changes in the copy number of the atp sensitive k + channel genes, abcc and kcnj . coding mutations and partial/whole gene in glud , gck, hadh, hnf a, hnf a, insr and trmt a were also excluded. immunohistochemistry and immunofluorescence studies were performed on µm-thick sequentially sectioned tissue slides (heat-mediated antigen retrieved) as previously described [ , ]. insulin (abcam : rabbit), glucagon (rtu biogenex pa - psg) and somatostatin (dako : a ) staining were performed to examine islet cell expression. all slides were digitised as previously described [ , ]. figure a illustrates the histological characteristics of the pancreas, with the focal domain associated with islet cell hyperplasia, in contrast to the non-focal region. figure b demonstrates the immunological characteristics - how the focal lesion is associated with a marked expansion of insulin-expressing islet cells and the location of glucagon-expressing cells within the lesion. glucagon expression is localized to the peripheral regions of the focal islets. figure shows the expression and localization of islet insulin, glucagon and somatostatin within the lesion and the non-lesion domains of the pancreas. note the expansion of insulin-positive cells within the lesion and how the non-β-cells are located around the periphery of the islet and islet-like structures. to address the mechanism of islet β-cells expansion we assessed the expression of p kip by immunohistochemistry ( : , mouse; thermo scientific, uk), also demonstrated in figure . p kip is normally expressed in β-cell nuclei where it acts as a negative repressor of the cell cycle. focal chi is caused by the loss of heterozygosity on ch. p and loss of p kip expression. this was confirmed in tissue samples from the lesion domain. page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review on the balance of probabilities the cause of death was felt to be related to the focal lesion in the pancreas most probably resulting in hypoglycaemia. discussion we have reported focal chi as a probable cause of death in a child with infantile hypoglycaemia, presenting with sudden illness and collapse. focal chi is well recognised as a cause for early and late presenting hypoglycaemia [ ]. while neonatal hypoglycaemia is detected early when the child is still in hospital, infantile hypoglycaemia due to late presenting chi can be missed and the diagnosis therefore delayed. we have highlighted in our case report an extreme scenario where focal chi was not diagnosed until after post-mortem examination. the incidence of focal chi as a cause of sudden unexpected infant death has not been reported. it is important to raise awareness that focal chi may cause hypoglycaemia severe enough to cause death from delayed recognition. for histopathologists, it is important to consider the possibility of focal chi when examining pancreatic sections in post-mortem examinations. it was not possible to establish the diagnosis of chi in the neonatal period as samples for serum insulin had not been drawn at the time of hypoglycaemia. further, samples for glucose and insulin were not analysed in the post-mortem period as measurements several hours after death would be too unreliable. however, the diagnosis of focal chi in our case was comprehensively confirmed by standard examination of pancreatic sections, followed by immunofluorescence staining for insulin and p kip . although a genetic aetiology was not ascertained, the diagnosis of focal chi was unequivocal. page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review in most cases of focal chi, diagnosis is made following the identification of paternal heterozygous mutations in abcc /kcnj in peripheral blood dna and confirmation by -fluoro-dopa pet-ct scanning [ ]. in the presented case, a diagnosis of chi was not suspected at any stage of life and the above investigations of gene mutation testing and imaging were not undertaken. focal chi was identified only at post-mortem on histology. conclusions this case demonstrates the consequences and severity of focal chi; if not recognised, investigated and treated, focal chi can lead to death. this case also highlights the need for careful assessment of the pancreas in post-mortem examination in children dying suddenly and unexpectedly. references (max ) . arnoux jb, verkarre v, saint-martin c, et al. congenital hyperinsulinsm: current trends in diagnosis and therapy. orphanet j rare dis. ; ( ): - . . avatapalle hb, banerjee i, shah s, et al. abnormal neurodevelopmental outcomes are common in children with transient congenital hyperinsulinsm. front endocrinol. ; ( ): - . . roženková k, gűemes m, shah p, hussain k. the diagnosis and management of hyperinsulinaemic hypoglycaemia. j clin res pediatr endocrinol. ; ( ): - . . stanley ca. perspective on the genetics and diagnosis of congenital hyperinsulinsm disorders. j clin endocrinol metab. ; ( ): - . page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review . flanagan se, kapoor rr, hussain k. genetics of congenital hyperinsulinaemic hypoglycaemia. semin pediatr surg. ; ( ): - . . han b, newbould m, batra g, et al. enhanced islet cell nucleomegaly defines diffuse congenital hyperinsulinism in infancy but not other forms of the disease. am j clin pathol. ; ( ): - . . salisbury rj, han b, jennings re et al. altered phenotype of β-cells and other pancreatic cell lineages in patients with diffuse congenital hyperinsulinism in infancy caused by mutations in the atp-sensitive k-channel. diabetes. ; ( ): – . . banerjee i, skae m, flanagan s et al. the contribution of rapid katp channel gene mutation analysis to the clinical management of children with congenital hyperinsulinism. eur j endocrinol. ; ( ): - . . banerjee i, avatapalle b, padidela r, et al. integrating genetic and imaging investigations into the clinical management of congenital hyperinsulinism. clinical endocrinology ; : - . page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review figure . histological and immunological characteristics of the pancreas. panel a high and low power h&e images of the pancreas. the focal domain ‘lesion’ is associated with islet cell hyperplasia. in the non-focal region – ‘non-lesion’, islets can be readily observed, indicated by dotted region. panel b illustrates the expression of insulin by immunohistochemistry in the tissue block. note the enrichment of insulin-expressing cells in the designated lesion domain compared to the expression of insulin in the remainder of the pancreas, panel a low power image (x ), scale bar mm; high power images (x ), scale bars µm. panel b magnification . x, scale bars mm. x mm ( x dpi) page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology for peer review figure . islet hormone expression in the focal and non-focal domains of the chi pancreas. in the non- lesion domains of the tissue insulin expression is largely found within islet structures with a central location within the islet domain. glucagon and somatostatin expression is localized to the periphery of the islets. the focal lesion is characterized by extensive expression of insulin-positive cells, with glucagon- and somatostatin-expressing cells around the peripheral structures of the extended structures. islets outside of the focal lesion have nuclei localization of p kip , whereas islet cells within the lesion do not express p kip . images - x magnification; scale bars: µm for all images, except p kip ; µm. x mm ( x dpi) page of https://mc.manuscriptcentral.com/pedpath pediatric and developmental pathology wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") 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intersection of vitamin d and coronary artery disease? editorial is coronary artery calcification at the intersection of vitamin d and coronary artery disease? zari dastani, j. brent richards cardiovascular disease is the leading cause of deathamong men and women in developed countries. most of this premature mortality occurs in a subgroup of the popula- tion that is prone to accelerated atherogenesis caused by genetic, lifestyle, and environmental factors, along with their interactions. growing evidence suggests that vitamin d deficiency is associated with coronary artery disease (cad) development. however, mechanistic evidence supporting this association is lacking. coronary artery calcification (cac) is strongly associated with risk of vascular disease, , and several studies report an inverse relationship between levels of the active form of vitamin d and atherosclerotic calcification. therefore, it is important to understand if vitamin d metabolism influences degree of cac; insights into this relationship would provide support for a role of vitamin d in the pathogenesis of cad. see accompanying article on page in this issue of arteriosclerosis, thrombosis, and vascular biology, shen et al present a well-designed candidate gene study to investigate the association of single-nucleotide poly- morphisms (snps) in key genes involved in vitamin d metabolism with cac. the cac phenotype was quantified as the sum of the calcification scores in both left and right coronary arteries. the researchers selected genes, cyto- chrome p , family , subfamily r (cyp r ), cytochrome p , family , subfamily b (cyp b ), cytochrome p , family , subfamily a (cyp a ), vitamin d receptor (vdr), viatmin d-binding protein (gc), known to be in- volved in vitamin d homeostasis. – in the discovery phase, they conducted the association analysis in samples from amish families (n� ), with genotyped snps from available genes using a chip (humancvd beadchip v ). although no snps in the cyp b , vdr, or gc genes were associated with cac score, snps in the cyp a gene were nominally associated with cac score (p� . to p� . ) in the discovery phase. then, these snps were tested for replication in samples from the genetic epidemi- ology network of arteriopathy (n� ) and the penn coronary artery calcification (n� ), independent cohorts of european white ancestry (figure). in the replica- tion phase, of these snps, rs , demonstrated evidence of an association with cac in both the genetic epidemiology network of arteriopathy and the penn coro- nary artery calcification cohorts (p� . and p� . , respectively). the subsequent meta-analysis of the data from these populations yielded a probability value of . � � for rs . however, in further analysis, they could not find any association between circulating levels of - hydroxy-vitamin d ( [oh]d) levels and this snp in rela- tively small populations. the results of the study conducted by shen et al raise the possibility of the role of vitamin d homeostasis in cac development. the cyp a gene product is central to vitamin d regulation because it degrades the active form of vitamin d, , -dihydroxy-vitamin d ( , [oh] d). despite the fact that previous transgenic studies have revealed the role of the cyp a gene on stability plasma level of , (oh) d and (oh)d, the current study did not estab- lish any association between rs and (oh)d levels. in addition, , (oh) d levels were not available for re- searchers to assess this possible association. it remains possible that the identified snp may influence , (oh) d levels and, consequently, cac, without affecting vitamin d stores, as reflected by (oh)d levels. therefore, it is important that the association between rs , (oh)d, , (oh) d, and other coronary disease outcomes be clari- fied in adequately powered consortia designed to identify the genetic determinants of vitamin d levels or cad. further- from the department of epidemiology and biostatistics (z.d. and j.b.r.), faculty of medicine, mcgill university, montréal, quebec, canada; the department of human genetics (j.b.r.), faculty of medi- cine, lady davis institute, jewish general hospital, mcgill university, montréal, quebec, canada; and the department of medicine (j.b.r.), faculty of medicine, lady davis institute, jewish general hospital, mcgill university, montréal, quebec, canada. correspondence to j. brent richards, md, msc, lady davis institute, jewish general hospital, pavillon e- , cote st catherine, montréal, qc h t e , canada. e-mail brent.richards@mcgill.ca (arterioscler thromb vasc biol. ; : - .) © american heart association, inc. arterioscler thromb vasc biol is available at http://atvb.ahajournals.org doi: . /atvbaha. . discovery phase amish samples (n= )y p snps achieved p value < . p ( ) replica�on phase penncac samples (n= , )genoa samples (n= ) one snp (rs ) achieved p value < . meta-analysis in three cohorts (n = , )meta-analysis phase rs , p value = . x - figure. the strategy used by shen and colleagues to investigate whether dna sequence variants in the candidate genes in vitamin d metabolism contribute to coronary artery calcification. d ow nloaded from http://ahajournals.org by on a pril , more, it will be worthwhile to investigate whether this snp affects the transcription and protein product levels of the genes involved in the maintenance of , (oh) d and (oh)d levels. this finding raises interesting questions about the direct role of vitamin d in the progression or initiation of athero- sclerosis. other research has discovered that vitamin d lowers the activity of the inflammatory activator nuclear factor �b, inhibiting foam cell formation and suppressing macrophage cholesterol uptake in patients with type diabetes mellitus. , on the other hand, vitamin d levels are correlated with other cad risk factors, such as hypertension, hyperlipidemia, and diabetes. – therefore, the described association may occur directly through vitamin d metabolism or indirectly through other pathways, regardless of their effect on (oh)d, by influencing known risk factors for cardiovascular disease (ie, hypertension, diabetes, and inflammation). this study suggests a role for vitamin d homeostasis in cac and provides an important signpost on the road toward understanding the role of calcium and vitamin d metabolism in risk of cad. disclosures none. references . castelli wp. epidemiology of coronary heart disease: the framingham study. am j med. ; : – . . levin a, li yc. vitamin d and its analogues: do they protect against cardiovascular disease in patients with kidney disease? kidney int. ; : – . . greenland p, labree l, azen sp, doherty tm, detrano rc. coronary artery calcium score combined with framingham score for risk prediction in asymptomatic individuals. jama. ; : – . . taylor aj, bindeman j, feuerstein i, cao f, brazaitis m, o’malley pg. coronary calcium independently predicts incident premature coronary heart disease over measured cardiovascular risk factors: mean three-year outcomes in the prospective army coronary calcium (pacc) project. journal of the american college of cardiology. ; : – . . watson ke, abrolat ml, malone ll, hoeg jm, doherty t, detrano r, demer ll. active serum vitamin d levels are inversely correlated with coronary calcification. circulation. ; : – . . shen h, bielak lf, ferguson jf, streeten ea, yerges-armstrong lm, liu j, post w, o’connell jr, hixson je, kardia sl, sun yv, jhun ma, wang x, mehta nn, li m, koller dl, hakonarson h, keating bj, rader dj, shuldiner ar, peyser pa, reilly mp, mitchell bd. association of the vitamin d metabolism gene cyp a with coronary artery calcification. arterioscler thromb vasc biol. . . tashiro k, abe t, oue n, yasui w, ryoji m. characterization of vitamin d-mediated induction of the cyp transcription. mol cell endocrinol. ; : – . . ponchon g, kennan al, deluca hf. “activation” of vitamin d by the liver. j clin invest. ; : – . . takeyama k, kitanaka s, sato t, kobori m, yanagisawa j, kato s. -hydroxyvitamin d alpha-hydroxylase and vitamin d synthesis. science. ; : – . . kasuga h, hosogane n, matsuoka k, mori i, sakura y, shimakawa k, shinki t, suda t, taketomi s. characterization of transgenic rats con- stitutively expressing vitamin d- -hydroxylase gene. biochem biophys res commun. ; : – . . psaty bm, o’donnell cj, gudnason v, lunetta kl, folsom ar, rotter ji, uitterlinden ag, harris tb, witteman jc, boerwinkle e. cohorts for heart and aging research in genomic epidemiology (charge) consortium: design of prospective meta-analyses of genome-wide association studies from cohorts. circ cardiovasc genet. ; : – . . oh j, weng s, felton sk, bhandare s, riek a, butler b, proctor bm, petty m, chen z, schechtman kb, bernal-mizrachi l, bernal-mizrachi c. , (oh) vitamin d inhibits foam cell formation and suppresses macrophage cholesterol uptake in patients with type diabetes mellitus. circulation. ; : – . . cohen-lahav m, shany s, tobvin d, chaimovitz c, douvdevani a. vitamin d decreases nfkappab activity by increasing ikappabalpha levels. nephrol dial transplant. ; : – . . sepulveda jl, mehta jl. c-reactive protein and cardiovascular disease: a critical appraisal. curr opin cardiol. ; : – . . mccarty mf. secondary hyperparathyroidism promotes the acute phase response – a rationale for supplemental vitamin d in prevention of vascular events in the elderly. med hypotheses. ; : – . . holick mf. high prevalence of vitamin d inadequacy and implications for health. mayo clin proc. ; : – . . li yc, kong j, wei m, chen zf, liu sq, cao lp. , - dihydroxyvitamin d( ) is a negative endocrine regulator of the renin- angiotensin system. j clin invest. ; : – . . forman jp, curhan gc, taylor en. plasma -hydroxyvitamin d levels and risk of incident hypertension among young women. hypertension. ; : – . key words: atherosclerosis � calcification � calcium � coronary artery disease � vitamin d arterioscler thromb vasc biol december d ow nloaded from http://ahajournals.org by on a pril , doi: . /j.bpj. . . a monday, march , provide valuable insights into its mode of action. the molecular framework of dfbp resembles that of levosimendan, thus it was chosen to mimic levosimen- dan to establish how the ctnc-ctni binding equilibrium is modulated. we have utilized d { h, n} hsqc and d { h, c} hsqc nmr spectroscopy to examine the binding of dfbp to cntnc�ca þ in the absence and presence of ctni - and of ctni - to cntnc�ca þ in the absence and presence of dfbp. the results show that dfbp and ctni - bind cntnc�ca þ concurrently and the affinity of dfbp for cntnc�ca þ is increased ~ - - fold by ctni - . we are in the process of determining the nmr solution structure of cntnc�ca þ�ctni - �dfbp. this structure will contribute to the understanding of the mechanism of action of levosimendan in the therapy of heart disease. it will also provide a structural basis for the design of ca þ- sensitizing drugs in general. -pos board b decreased fatigue tolerance in diaphragm muscle of slow troponin t knockdown mice hanzhong feng, bin wei, j.-p. jin. northshore university healthsystem and northwestern university feinberg school of medicine, evanston, il, usa. the loss of slow skeletal muscle troponin t (tnt) results in a severe type of nemaline myopathy in the amish (anm). the genes encoding tnt and tropo- nin i (tni) are closely linked in pairs in which the -enhancer region of the slow tnt gene overlaps with the cardiac tni gene. in a mouse line with the en- tire cardiac tni gene deleted, a partial destruction of the slow tnt gene pro- moter produces a knockdown effect. by crossing with transgenic mouse lines that over-express a core structure of cardiac tni (ctni-nd) under the control of cloned alpha-mhc promoter, we rescued the postnatal lethality of the car- diac tni gene-deleted mice with no detrimental cardiac phenotypes or leaking expression in non-cardiac tissues. the double transgenic mice exhibited de- creased expression of slow tnt mrna and protein in adult diaphragm muscle. functional analysis of isolated muscle strips showed that the slow tnt deficient (stnt-kd) diaphragm had significantly decreased fatigue tolerance evident by the faster decrease in force and slower rate of recovery as compared with that in wild type controls. as a consequence of slow tnt deficiency, the stnt-kd di- aphragm muscle contained a higher proportion of fast tnt, decreased slow tni with increased fast tni, and decreased type i myosin with increased type ii my- osin. consistent with the switch toward fast myofilament contents, the stnt- kd diaphragm muscle produced higher specific tension in twitch and tetanic contractions as well as shorter time to develop peak tension in twitch contrac- tions. the decreased fatigue tolerance of stnt-kd diaphragm muscle explains the terminal respiratory failure seen in virtually all anm patients and this dou- ble transgenic mouse model provides a useful experimental system to study the pathogenesis and treatment of anm. -pos board b troponin isoforms and stretch-activation of insect flight muscle uros krzic , gian de nicola , vladimir rybin , annalisa pastore , kevin leonard , wolfgang linke , belinda bullard . embl, heidelberg, germany, nimr, london, united kingdom, ebi, cambridge, united kingdom, university of muenster, muenster, germany, department of biology, university of york, united kingdom. oscillatory contraction of insect indirect flight muscle (ifm) is activated by si- nusoidal length changes. work done by oscillating fibres is measured from the area of loops on a length-tension plot. at [ca þ] above mm, progressively less oscillatory work is produced because fibres contract isometrically and are unable to relax fully after each cycle of oscillation. periodic stretches during oscillations activate fibres through the action of tnc f , which binds one ca þ in the c-lobe. activation of isometric contraction by ca þ acts through f , which binds ca þ in both n- and c-lobes. lethocerus ifm fibres substituted with f gave oscillatory work, which did not decline at high [ca þ], while fibres substituted with f produced more isometric tension as [ca þ] was increased. varying proportions of f and f gave maximal work with an f :f ratio of : , which is higher than the in vivo ratio of : . the structure of f , and the interaction with tni, were determined by nmr. the n-lobe of f is in the closed conformation in apo and ca þ- bound forms and does not bind tni. unexpectedly, the c-lobe is open in both states, and binds the n-terminal domain of tni independently of ca þ. the affinity of f and f for a complex containing tropomyosin, tnt and tnh (lethocerus tni) were measured by isothermal calorimetry in the presence of ca þ. the affinities of f and f for the complex were . mm and nm respectively. this difference is likely to be due to a single tni binding site on f and two sites on f . stretch may be sensed by an extended c-terminal domain of tnh, and transmitted to the c-lobe of f , resulting in a change in the interac- tion of the tni inhibitory domain and actin. -pos board b tracking of qdot conjugated titin antibodies in single myofibril stretch experiments reveals ig-domain unfolding at physiological sarcomere lengths anika grützner , pallav kosuri , julio m. fernandez , wolfgang a. linke . university of münster, münster, germany, columbia university, new york, ny, usa. the mechanical characteristics of titin in muscle sarcomeres were previously studied by us in single myofibril stretch experiments, where the extensibility of i-band titin segments was usually measured under static conditions. here we investigated the behavior of i-band titin during and after stretch of single rabbit psoas myofibrils in real-time. the focus was on titin’s proximal ig-do- main region, whose stretch dynamics were analyzed by labeling the myofibrils specifically in the n a-titin domain using antibody-conjugated quantum dots, which stained the periphery of the myofibril but did not enter the myofilament lattice. qdot labels were tracked to obtain the stretch-dependent change in epi- tope distance (across z-disc) and sarcomere length (sl) over time. in contrast to what was expected from the current titin extensibility model, at sarcomere lengths of . and . mm, titin’s proximal ig-domain region elongated contin- uously, in proportion to the half i-band length. already at ~ . mm sl the prox- imal ig-segment length exceeded the value expected if all ig-domains remain folded. our results suggest that ig-domains unfold in parallel with pevk-titin extension at physiological sarcomere lengths and under relatively low forces. by reducing the antibody-qdot concentration, we succeeded in observing titin ig-domain dynamics in myofibrils at the single-molecule level. -pos board b constitutive phosphorylation of cardiac myosin binding protein-c increases the probability of myosin cross-bridge interaction with actin brett a. colson , tanya bekyarova , matthew r. locher , carl w. tong , daniel p. fitzsimons , patricia a. powers , thomas c. irving , richard l. moss . university of wisconsin medical school, madison, wi, usa, illinois institute of technology, chicago, il, usa. protein kinase a-mediated (pka) phosphorylation of cardiac myosin binding protein-c (cmybp-c) accelerates the kinetics of cross-bridge cycling and ap- pears to relieve the tether-like constraint of myosin heads imposed by cmybp-c (colson et al., , circ res., : - ). we favor a mechanism in which phosphorylation of the pka sites in cmybp-c modulates cross- bridge kinetics by regulating the proximity and interaction of myosin with actin. to test this idea, we used synchrotron low-angle x-ray diffraction and mechanical measurements in skinned myocardium isolated from a mouse model with phosphomimetic substitutions in cmybp-c, i.e., the ctsd mouse. the substitutions were introduced by transgenic expression of cmybp-c with ser-to-asp mutations on a cmybp-c null background. western blots showed that expression of ctsd cmybp-c was % of wild-type (wt), and the heart weight to body weight ratio was similar ( . . mg/g) in ctsd and wt mice. expression of wt cmybp-c on the knockout background served as con- trol (i.e., the ctwt mouse). skinned myocardium from ctsd and ctwt mice exhibited similar maximum active forces (mn/mm : . . vs . . ), ca þ-sensitivities of force (pca : . . vs . . ), and maximum rates of force development (ktr, sec - : . . vs . . ; kdf, sec - : . . vs . . ). i /i intensity ratios and d lattice spacings determined from equatorial reflections from ctsd and ctwt myocardium were used to determine the effect of constitutive cmybp-c phosphorylation on the distribution of cross-bridge mass between the thick and thin filaments and on interfilament lattice spacing. the results suggest that interactions between cmybp-c and the s domain of myosin heavy chain are dynamically regulated by phosphorylations in the cmybp-c motif. (aha-predoctoral fellowship (bac); nih-hl-r - ) -pos board b obscurin interacts with a novel isoform of myosin binding protein c-slow to regulate the assembly of thick filaments maegen a. borzok, rebecca hu, amber l. bowman, john strong, robert j. bloch, aikaterini kontrogianni-konstantopoulos. university of maryland, baltimore, md, usa. obscurin is a multidomain protein composed of adhesion and signaling do- mains that plays key roles in the organization of contractile and membrane structures in striated muscles. we used adenoviral-mediated gene transfer to overexpress its extreme nh -terminus in developing myofibers, followed by immunofluorescence and ultrastructural methods to study its effects in sarco- merogenesis. we found that overexpression of obscurin’s second immunoglob- ulin domain (ig ) inhibits the assembly of a- and m-bands, but not z-disks and 	decreased fatigue tolerance in diaphragm muscle of slow troponin t knockdown mice 	troponin isoforms and stretch-activation of insect flight muscle 	tracking of qdot conjugated titin antibodies in single myofibril stretch experiments reveals ig-domain unfolding at physiological sarcomere lengths 	constitutive phosphorylation of cardiac myosin binding protein-c increases the probability of myosin cross-bridge interaction with actin 	obscurin interacts with a novel isoform of myosin binding protein c-slow to regulate the assembly of thick filaments [pdf] automated computer-derived prostate volumes from mr imaging data: comparison with radiologist-derived mr imaging and pathologic specimen volumes. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /radiol. corpus id: automated computer-derived prostate volumes from mr imaging data: comparison with radiologist-derived mr imaging and pathologic specimen volumes. @article{bulman automatedcp, title={automated computer-derived prostate volumes from mr imaging data: comparison with radiologist-derived mr imaging and pathologic specimen volumes.}, author={j. bulman and r. toth and amish d. patel and b. bloch and c. mcmahon and lan v. ngo and a. madabhushi and n. rofsky}, journal={radiology}, year={ }, volume={ }, pages={ - } } 	j. bulman, r. toth, + authors n. rofsky 	published 	medicine 	radiology purpose to compare prostate gland volume (pv) estimation of automated computer-generated multifeature active shape models (mfas) performed with -t magnetic resonance (mr) imaging with that of other methods of pv assessment, with pathologic specimens as the reference standard. materials and methods all subjects provided written informed consent for this hipaa-compliant and institutional review board-approved study. freshly weighed prostatectomy specimens from patients (mean age, years… expand view on pubmed pubs.rsna.org save to library create alert cite launch research feed share this paper citationsbackground citations methods citations view all figures, tables, and topics from this paper 	 figure 	 table 	 figure 	 table 	 figure view all figures & tables 	specimen 	health insurance portability and accountability act 	master of fine arts 	cdisc send biospecimens terminology 	compliance behavior 	polycythemia vera 	slope 	estimated 	reference standards 	fifty nine 	prostatectomy citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency fully automated prostate segmentation on mri: comparison with manual segmentation methods and specimen volumes. 	b. turkbey, s. fotin, + authors p. choyke 	medicine 	ajr. american journal of roentgenology 	 	 	view excerpt, cites methods save alert research feed comparison of a commercially available prostate segmentation application to traditional 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imaging signatures on tesla endorectal, in vivo t ‐weighted mr imagery 	s. viswanath, n. b. bloch, + authors a. madabhushi 	medicine 	journal of magnetic resonance imaging : jmri 	 	 	pdf save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency determinations of prostate volume at -tesla using an external phased array coil: comparison to pathologic specimens. 	j. sosna, n. rofsky, s. gaston, w. dewolf, r. lenkinski 	materials science, medicine 	academic radiology 	 	 save alert research feed modified prostate volume algorithm improves transrectal us volume estimation in men presenting for prostate brachytherapy. 	p. macmahon, a. kennedy, d. murphy, michael maher, m. mcnicholas 	medicine 	radiology 	 	 	 save alert research feed accuracy of in‐vivo assessment of prostatic volume by mri and transrectal ultrasonography 	a. rahmouni, a. yang, + authors e. zerhouni 	medicine 	journal of computer assisted tomography 	 	 save alert research feed accurate prostate volume estimation using multifeature active shape models on t -weighted mri. 	r. toth, b. bloch, + authors a. madabhushi 	mathematics, medicine 	academic radiology 	 	 	pdf save alert research feed prostate cancer: incremental value of endorectal mr imaging findings for prediction of extracapsular extension. 	l. wang, m. mullerad, + authors h. hricak 	medicine 	radiology 	 	 save alert research feed comparison of prostate volume measured by transrectal ultrasonography and mri with the actual prostate volume measured after radical prostatectomy 	c. jeong, h. park, s. hong, seok-soo byun, h. lee, s. lee 	medicine 	urologia internationalis 	 	 save alert research feed transrectal ultrasound versus magnetic resonance imaging in the estimation of prostate volume as compared with radical prostatectomy specimens 	j. lee, b. chung 	medicine 	urologia internationalis 	 	 save alert research feed intraobserver and interobserver variability of mr imaging- and ct-derived prostate volumes after transperineal interstitial permanent prostate brachytherapy. 	d. dubois, b. prestidge, l. a. hotchkiss, j. j. prete, w. bice 	medicine 	radiology 	 	 save alert research feed impact of prostate volume evaluation by different observers on ct-based post-implant dosimetry. 	b. al-qaisieh, d. ash, d. bottomley, b. carey 	medicine 	radiotherapy and oncology : journal of the european society for therapeutic radiology and oncology 	 	 save alert research feed a magnetic resonance spectroscopy driven initialization scheme for active shape model based prostate segmentation 	r. toth, p. tiwari, + authors a. madabhushi 	computer science, medicine 	medical image anal. 	 	 	pdf save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue apolipoprotein c-iii nearly years have transpired since albrink and man first observed an association between high triglyceride levels and coronary artery disease (cad). yet, the mecha- nisms underlying this association gained minimal traction until decades later and even after many years of exhaus- tive investigative work, remains incompletely understood. what has been clearly demonstrated, however, is that triglyc- eride serves as a primary mammalian energy source and is not directly atherogenic. furthermore, triglyceride-rich lipo- proteins (eg, chylomicrons, very low-density lipoprotein) develop atherogenic characteristics on their conversion to cholesterol-enriched remnant particles. in this issue of atvb, van capelleveen et al demonstrate that apoc-iii also plays a central role in promoting vascular risk. see accompanying article on page human apoc-iii is an . -kda polypeptide that contains amino acids and is considerably smaller than other major apolipoproteins regulating lipoprotein metabolism, includ- ing apob ( kda, amino acids), apoe ( kda, amino acids), and apoa ( kda, amino acids). apoc-iii resides on the surface of triglyceride-rich lipoproteins and serves several important metabolic functions. they include triglyceride raising by direct inhibition of lipoprotein lipase and via a recently identified lipoprotein lipase–independent mechanism. apoc-iii also inhibits hepatic lipase, delays clearance of apob containing particles, and accelerates the conversion of light to dense low-density lipoprotein (ldl) particles. these proatherogenic particles gain facilitated entry through the endothelial intima followed by oxidation and uptake by vascular macrophages lining the arterial wall. ironically, the clinical relevance of apoc-iii in humans was initially uncovered with low triglyceride, rather than hypertri- glyceridemia. specifically, sisters with familial apoa -c deficiency exhibited triglyceride levels of and mg/dl (≈ %– % lower than the median triglyceride during that period), nondetectable apoc-iii levels, and increased fractional turnover of very low-density lipoprotein triglyceride consis- tent with increased lipoprotein lipase activity. more recently, very low triglyceride (mean, mg/dl) combined with a % reduction in apoc-iii levels was discovered in an amish cohort who had inherited a single defective allele (apoc . r x) that also correlated with reduced coronary calcification. carriers of several different rare apoc loss-of-function mutations (including r x) were subsequently found to have low triglyc- eride and a significantly reduced risk of vascular disease. , in contrast, studies examining the effect of apoc-iii enrich- ment in apob containing lipoproteins (ie, very low-density lipo- protein and ldl) identified increased coronary arteriographic progression and recurrent cad events. , among the large prospective studies that previously examined plasma apoc-iii levels and cad risk, one found a significant association with apoc-iii enrichment of ldl but not with plasma apoc-iii after adjustment for triglyceride. in the second study, apoc-iii lev- els in the top quartile at baseline were predictive of cardiovas- cular death over the -year follow-up period. however, this effect was attenuated after adjustment for triglyceride and was of borderline statistical significance in the fully adjusted analy- sis. finally, a recent meta-analysis that incorporated the afore- mentioned studies found an ≈ % increased risk of cad for each -mg/dl increment in plasma apoc-iii levels although the vast majority of these studies did not adjust for triglyceride. the study by van capelleveen et al confirms the asso- ciation between plasma apoc-iii levels and incident cad. although these effects were attenuated after adjustment for tri- glyceride, subgroup analysis found apoc-iii to remain indepen- dently associated with cad in subjects with high triglyceride (median, > . mmol/l or mg/dl). interestingly, the combi- nation of high triglyceride and low apoc-iii was not associated with increased cad risk. this supports the concept that high apoc-iii may potentiate vascular risk, especially in the setting of hypertriglyceridemia. as illustrated in the figure, there are at least potential pathways promoting these effects. inhibition of lipoprotein lipase and hepatic lipase results in delayed clearance of triglyceride-rich lipoproteins and atherogenic remnants, the latter of which are incorporated by vascular macrophages lining the vascular wall. apoc-iii also inhibits removal of large ldl and remnants by inhibiting hepatic lipoprotein receptors that interact with apob and apoe, culminating in facilitated conver- sion to smaller proatherogenic particles (eg, small dense ldl). finally, apoc-iii induces proinflammatory cellular signaling by activating vascular cell adhesion molecule- and nuclear factor κb. in effect, the association between apoc-iii and incident cad reported by van capelleveen et al was largely attributable to increased triglyceride-rich lipoprotein, remnants, sdldl (small, dense ldl), and high sensitivity c-reactive pro- tein, a biomarker of systemic inflammation. overall, the current study suggests that high apoc-iii levels provide incremental discriminatory power in the assessment of cad risk, particularly in hypertriglyceridemia states. however, before routine screening for high apoc-iii apolipoprotein c-iii the small protein with sizeable vascular risk michael miller (arterioscler thromb vasc biol. ; : - . doi: . /atvbaha. . .) © american heart association, inc. arterioscler thromb vasc biol is available at http://atvb.ahajournals.org doi: . /atvbaha. . from the department of cardiovascular medicine, university of maryland school of medicine and veterans affairs medical center (vamc), baltimore. correspondence to michael miller, md, university of maryland school of medicine, s. paca st, suite – , baltimore, md . e-mail mmiller@som.umaryland.edu editorial d ow nloaded from http://ahajournals.org by on a pril , mailto:mmiller@som.umaryland.edu arterioscler thromb vasc biol june is recommended, it would seemingly be most reasonable to first establish whether lowering triglyceride (+/- apoc-iii) reduces cad risk. fortunately, ongoing randomized clinical trials are well underway , and together with novel therapies targeting apoc-iii should move us a step closer to satisfac- torily addressing this clinical conundrum in the near future. disclosures dr miller serves on the steering committee for the reduce it study (reduction of cardiovascular events with icosapent ethyl- intervention trial). references . albrink mj, man eb. serum triglycerides in coronary artery disease. ama arch intern med. ; : – . . zilversmit db. atherogenesis: a postprandial phenomenon. circulation. ; : – . . miller m, stone nj, ballantyne c, bittner v, criqui mh, ginsberg hn, goldberg ac, howard wj, jacobson ms, kris-etherton pm, lennie ta, levi m, mazzone t, pennathur s; american heart association clinical lipidology, thrombosis, and prevention committee of the council on nutrition, physical activity, and metabolism; council on arteriosclerosis, thrombosis and vascular biology; council on cardiovascular nursing; council on the kidney in cardiovascular disease. triglycerides and cardio- vascular disease: a scientific statement from the american heart association. circulation. ; : – . doi: . /cir. b e . . van capelleveen jc, bernelot moens sj, yang x, kastelein jjp, wareham nj, zwinderman ah, stroes esg, witztum jl, hovingh gk, khaw k- t, boekholdt sm, tsimikas s. apolipoprotein c-iii levels and incident coronary artery disease risk: the epic-norfolk prospective population study. arterioscler thromb vasc biol. ; : – . doi: . / atvbaha. . . . kwiterovich po. lipid, apolipoprotein and lipoprotein metabolism. in: kwiterovich po, ed. the johns hopkins textbook of dyslipidemia. baltimore, md: lippincott, williams & wilkins; : – . . brown wv, baginsky ml. inhibition of lipoprotein lipase by an apo- protein of human very low density lipoprotein. biochem biophys res commun. ; : – . . gaudet d, brisson d, tremblay k, alexander vj, singleton w, hughes sg, geary rs, baker bf, graham mj, crooke rm, witztum jl. targeting apoc in the familial chylomicronemia syndrome. n engl j med. ; : – . doi: . /nejmoa . . windler e, havel rj. inhibitory effects of c apolipoproteins from rats and humans on the uptake of triglyceride-rich lipoproteins and their remnants by the perfused rat liver. j lipid res. ; : – . . mendivil co, zheng c, furtado j, lel j, sacks fm. metabolism of very- low-density lipoprotein and low-density lipoprotein containing apolipo- protein c-iii and not other small apolipoproteins. arterioscler thromb vasc biol. ; : – . doi: . /atvbaha. . . . mudd jo, borlaug ba, johnston pv, kral bg, rouf r, blumenthal rs, kwiterovich po jr. beyond low-density lipoprotein cholesterol: defining the role of low-density lipoprotein heterogeneity in coronary artery disease. j am coll cardiol. ; : – . doi: . /j.jacc. . . . . norum ra, lakier jb, goldstein s, angel a, goldberg rb, block wd, noffze dk, dolphin pj, edelglass j, bogorad dd, alaupovic p. familial deficiency of apolipoproteins a-i and c-iii and precocious coronary-artery disease. n engl j med. ; : – . doi: . /nejm . . ginsberg hn, le na, goldberg ij, gibson jc, rubinstein a, wang- iverson p, norum r, brown wv. apolipoprotein b metabolism in subjects with deficiency of apolipoproteins ciii and ai. evidence that apolipopro- tein ciii inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo. j clin invest. ; : – . doi: . /jci . . pollin ti, damcott cm, shen h, ott sh, shelton j, horenstein rb, post w, mclenithan jc, bielak lf, peyser pa, mitchell bd, miller m, o’connell jr, shuldiner ar. a null mutation in human apoc confers a favorable plasma lipid profile and apparent cardioprotection. science. ; : – . doi: . /science. . . tg and hdl working group of the exome sequencing project, national heart, lung, and blood institute, crosby j, peloso gm, auer pl, et al. loss-of-function mutations in apoc , triglycerides, and coronary dis- ease. n engl j med. ; : – . . jørgensen ab, frikke-schmidt r, nordestgaard bg, tybjærg-hansen a. loss-of-function mutations in apoc and risk of ischemic vascular dis- ease. n engl j med. ; : – . doi: . /nejmoa . . blankenhorn dh, alaupovic p, wickham e, chin hp, azen sp. prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts. lipid and nonlipid factors. circulation. ; : – . . sacks fm, alaupovic p, moye la, cole tg, sussex b, stampfer mj, pfeffer ma, braunwald e. vldl, apolipoproteins b, ciii, and e, and risk of recurrent coronary events in the cholesterol and recurrent events (care) trial. circulation. ; : – . . mendivil co, rimm eb, furtado j, chiuve se, sacks fm. low-density lipoproteins containing apolipoprotein c-iii and the risk of coronary heart disease. circulation. ; : – . . scheffer pg, teerlink t, dekker jm, bos g, nijpels g, diamant m, kostense pj, stehouwer cd, heine rj. increased plasma apolipopro- tein c-iii concentration independently predicts cardiovascular mortal- ity: the hoorn study. clin chem. ; : – . doi: . / clinchem. . . . wyler von ballmoos mc, haring b, sacks fm. the risk of cardiovascular events with increased apolipoprotein ciii: a systematic review and meta- analysis. j clin lipidol. ; : – . doi: . /j.jacl. . . . . zheng c, azcutia v, aikawa e, figueiredo jl, croce k, sonoki h, sacks fm, luscinskas fw, aikawa m. statins suppress apolipoprotein ciii- induced vascular endothelial cell activation and monocyte adhesion. eur heart j. ; : – . doi: . /eurheartj/ehs . . bhatt dl, steg pg, brinton ea, jacobson ta, miller m, tardif jc, ketchum sb, doyle rt jr, murphy sa, soni pn, braeckman ra, juliano ra, ballantyne cm; reduce-it investigators. rationale and design of reduce-it: reduction of cardiovascular events with icosapent ethyl- intervention trial. clin cardiol. ; : – . doi: . /clc. . . https://clinicaltrials.gov/ct /show/nct . accessed may , . key words: editorials ◼ apolipoprotein c-iii ◼ coronary artery disease ◼ hypertriglyceridemia ◼ lipoprotein lipase ◼ lipoproteins, vldl figure. potentiation of vascular risk induced by apoc-iii. the molecules of apoc-iii on each of the depicted lipopro- teins is for illustration purposes only. it is estimated that there are to apoc-iii molecules for each low-density lipopro- tein (ldl) particle. there is only apob molecule (as depicted) for each lipoprotein particle. cm indicates chylomicron; cmr, chylomicron remnant; hl, hepatic lipase; idl, intermediate density lipoprotein; lpl, lipoprotein lipase; nf-κb, nuclear factor-κb; sdldl, small, dense ldl; trl, triglyceride-rich lipoprotein; vcam, vas- cular cell adhesion molecule; vldl, very low-density lipoprotein; and vldlr, very low-density lipoprotein receptor. d ow nloaded from http://ahajournals.org by on a pril , a novel form of lethal microcephaly with simplified gyral pattern and brain stem hypoplasia � wiley-liss, inc. american journal of medical genetics part a a: – ( ) new syndrome a novel form of lethal microcephaly with simplified gyral pattern and brain stem hypoplasia anna rajab, * m. chiara manzini, ganeshwaran h. mochida, , christopher a. walsh, , and m. elizabeth ross genetic unit, dgha, ministry of health, muscat, sultanate of oman, oman department of neurology and howard hughes medical institute, beth israel deaconess medical center and harvard medical school, boston, massachusetts pediatric neurology unit, department of neurology, massachusetts general hospital, boston, massachusetts division of genetics, children’s hospital, boston, massachusetts laboratory of neurogenetics and development, weill medical college of cornell university, new york, new york received march ; accepted june we report on four patients from the same family affected by a lethal form of autosomal recessive microcephaly of prenatal onset. symptoms include low birth-weight and length with disproportionately small head, fetal distress, apnea, seizures and facial features reminiscent of amish microcephaly and bowen–conradi syndrome. brain imaging revealed a simplified gyral pattern with normal to slightly thinned cortical gray matter, thin corpus callosum, mild brainstem and cerebellar hypoplasia. no abnormalities of the internal organs, eye, or skeleton, and no striking dysmorphic facial features were found to be associated with this syndrome. all patients died within hours to weeks after birth following severe apnea attacks and central hypoventilation. recessive primary microcephaly with lethality in early infancy is rarely reported. the patients described here do not resemble any other published cases of such clinical severity and the locus for the only reported early lethal microcephaly gene found in amish families was excluded. therefore, this appears to be a distinct genetic cause of lethal microcephaly. � wiley-liss, inc. key words: lethal microcephaly; simplified gyral pattern; fetal distress; apnea; autosomal recessive inheritance how to cite this article: rajab a, manzini mc, mochida gh, walsh ca, ross me. . a novel form of lethal microcephaly with simplified gyral pattern and brain stem hypoplasia. am j med genet part a a: – . introduction microcephaly is defined as small head size characterized by occipito-frontal circumference (ofc) at least standard deviations (sd) below the mean. it is a common developmental defect, which can have either prenatal or postnatal onset. it is anatomically, clinically, and etiologically a hetero- geneous disorder [mochida and walsh, ]. micro- cephaly has been reported in numerous syndromes with a large spectrum of clinical presentations, inheritance patterns, lifespan, degree of psycho- motor abnormalities and variety of associated anomalies including various inborn errors of meta- bolism [mochida and walsh, ; dobyns, ; woods, ]. it can be of environmental origin and it has been associated with different teratogenic agents [woods, ]. while microcephaly is often observed in multi- organ genetic syndromes, isolated microcephaly is not as common [mochida and walsh, ; woods, ]. primary microcephaly is detectable prenatally, and several autosomal recessive forms of primary microcephaly have been described with varying severity and clinical presentation. microcephaly vera is characterized by normal cerebral cortical thick- ness, normal or mildly disrupted gyral pattern, mild developmental delay and moderate mental retarda- tion [mochida and walsh, ]. at the other end of the spectrum, amish lethal microcephaly presents extremely profound microcephaly (less than � sd), agyria and hypoplasia of the cerebellum and pons. anna rajab and m. chiara manzini contributed equally to the work. grant sponsor: nih; grant numbers: po ns , r -ns . *correspondence to: dr. anna rajab, consultant clinical geneticist, genetic unit, dgha, ministry of health, p.o. box , muscat , sultanate of oman, oman. e-mail: drarajab@omantel.net.om doi . /ajmg.a. affected children are unresponsive to light and noise, and usually die by months of age because of metabolic acidosis, associated with -ketoglutaric aciduria [kelley et al., ]. smaller head size is usually correlated with reduced brain growth [mochida and walsh, ; cox et al., ]. brain development is a complex process determined by multiple factors regulating cell proliferation, migration and neuronal circuit formation. primary microcephaly is first detectable during pregnancy when the brain fails to grow as neurons are generated, suggesting that defects in neuronal proliferation are the principle cause of this malformation. as expected, the analysis of families affected by autosomal recessive primary micro- cephaly has led to the identification of genes involved in the regulation of neuronal mitosis [cox et al., ]. aspm (abnormal spindle-like, micro- cephaly-associated), cdk rap (cyclin-dependent kinase regulatory subunit-associated protein ) and cenpj (centromere protein j) are all important for the correct assembly of the mitotic spindle [bond et al., , ], while microcephalin regulates chromosome condensation in prophase [jackson et al., ]. the amish lethal microcephaly gene, on the other hand, encodes for a mitochondrial deoxynuleotide carrier (dnc), and it is associated with increased cell death in the developing brain [rosenberg et al., ]. mitochondrial dysfunction explains the accumulation of a-ketoglutarate and the metabolic defects in the patients, but it is not clear how perturbation of dnc function causes such severe reduction in the number of neurons. functional studies of the identified genes and further investigation of the genetic causes of autosomal recessive microcephalies will help define the mechanisms responsible for the control of brain size. the emergence of nuclear magnetic resonance imaging (mri) opened a new avenue to study morphological and structural brain anomalies. microcephaly is evident upon physical examination, but the spectrum of brain malformations associated with different forms of microcephaly can only be diagnosed by imaging the brain. the combination of clinical and radiological findings has proven a powerful approach in grouping patients for mole- cular studies and gene identification [mochida and walsh, ; barkovich et al., ]. here, we present a clinical study of four patients from a consanguineous couple with a lethal form of microcephaly characterized by simplified gyral pattern and small corpus callosum, brainstem, and cerebellum. most patients died in the neonatal period and the longest survival did not exceed days. clinical and radiological features are not consistent with any previously described syndrome, indicating that this is a novel form of lethal micro- cephaly. patients and methods clinical studies the parents and four affected children in one omani family were examined by a.r. and the brain imaging results were reviewed independently by m.e.r, g.h.m., and c.a.w. microsatellite marker analysis genomic dna was purified from patient after obtaining informed consent in accordance with protocols approved by the institutional review board of children’s hospital and beth israel deaconess medical center, and the office of human research protection. peripheral blood was subject to lymphocyte separation using lymphocyte separa- tion medium (organon technica, durham, nc) and dna was extracted from lymphocytes using com- mercial kits (qiagen, valencia, ca). highly poly- morphic microsatellite markers associated with the amish lethal microcephaly (mcpha) locus on chromosome q were chosen from the marsh- field database in the ucsc genome browser (http:// genome.ucsc.edu/). fluorescently labeled pcr primers (abi, foster city, ca) were purchased for each marker and used to amplify the patient dna sample using standard conditions. pcr products were resolved on an abi xl genetic analyzer. alleles were assigned using the genotyper software package (abi). results the parents belong to a large tribal unit from oman and are first cousins. they are both healthy with no family history of brain malformation. occipitofrontal circumference (ofc) was cm ( th centile) for both parents. at the time of the first pregnancy, the mother was years old and the father was . the mother did not receive prenatal vitamin supplementation before the first pregnancy, but was administered prenatal vitamins prior to all successive conceptions. she had no history of any medication, infection or irradiation exposure during any of the pregnancies. the couple had five children: four affected by lethal microcephaly (patients – , below) and one unaffected sibling born after patient . patient ultrasound serial scans during pregnancy showed inappropriately small biparietal diameter (bpd) measurements. the proband was a male, delivered at weeks gestation by cesarean because of fetal distress. birth weight was . kg ( th centile), ofc cm (< rd centile, � . sd below the mean, rajab et al. american journal of medical genetics part a: doi . /ajmg.a [fenton, ]), length cm ( rd centile, � . sd) and chest circumference cm ( th centile, mean for gestational age). apgar scores were at min and at min. the newborn had a disproportionately small head compared to normal size shoulders and normal body proportions. the anterior fontanelle was almost fused. the forehead was narrow, short and sloping, and the anterior hairline was low. there were bitemporal dimples and areas of scalp hair growth over the temples in close proximity to distal ends of the brows. the patient had irregular respirations and required oxygen. no intubation was attempted. he died at the age of hr and min because of poor respiratory drive and central apnea. blood gases were normal after face-mask oxygena- tion for min on occasions. chest radiograph and skeletal survey were unremarkable. patient patient was a younger sister of patient . head size was closely monitored during pregnancy and it was found to be falling behind from the th week of pregnancy. in the third trimester bpd measurements were � . sd below the mean. the patient was born by normal vertex delivery at term. birth weight was . kg ( rd centile), ofc cm (< rd centile, � . sd below the mean), length cm (< rd centile, � sd) and chest circumference cm ( th centile, � sd). apgars were at min and at min after birth. the facial and body appearance was similar to patient . there were no joint contractures to suggest oligohydramnios. there were irregular respirations and apnea attacks, but no abnormali- ties of heart or lungs were detectable on clinical examination. the baby required face-mask oxygen and after bagging, blood gases were within normal range. chest radiograph and electrocardiogram were entirely normal. she died at the age of hr following a prolonged apnea attack. patient the bpd monitored on fetal ultrasound during the third pregnancy was � sd below the mean at weeks of gestation and continued to lag on subsequent sonograms. cesarean was performed at weeks gestation because of the deterioration of the fetal movements and abnormal cardiotoco- gram (ctg). the female baby weighed . kg ( th centile), had an ofc of cm (< rd centile, � . sd below the mean) and length cm ( th centile, � sd). apgars were at min and at min, respiratory efforts were adequate and blood gas parameters were maintained with head box oxygen. the baby had a normal body habitus and normal chest size, which contrasted with disproportionately small head size. the anterior fontanelle was almost closed. the forehead was flat, narrow and short with low anterior and temporal hairline. there were temporal dimples and orbital ridges appeared prominent. the nose appeared large but had average length of . cm, which is proportionate to the body length at this developmental stage [siebert, ]. there was no evidence of pulmonary infection on chest x-ray examination. ultrasound examination of the abdomen and an echocardiogram were normal. jitteriness was noticed in the first day of life. seizures, poor responsiveness to stimuli, inactivity, apnea, truncal hypotonia, marked hyperreflexia and ankle clonus became evident from the first week of life. the patient startled to loud noises and had complete visual inattention. she was not able to breastfeed sufficiently and was given supplementary tube feeds, but she failed to thrive. no biochemical abnormalities were found, and ammonia, lactate, serum amino acids and urinary organic acids were all reported as normal. a computerized tomogram (ct) of the head revealed simplified gyri with shallow sulci and normal to thinned cortex, suggesting microcephaly with simplified gyri (msg) [dobyns, ]. there was thinning of the corpus callosum that was apparent anteriorly (fig. b). the extra-axial spaces were generous. the brainstem was only mildly hypo- plastic compared with the cortex and there was cerebellar hypoplasia including the vermis (fig. c, arrow) that was proportional to the cortical micro- cephaly. apneic attacks increased in frequency after the first month. repeated blood counts, urinalysis, bacterial cultures of urine, blood and surface swabs were all normal. the patient died at the age of days, likely because of central hypoventilation. patient the fourth pregnancy was uneventful, but head size of the foetus measured cm (� sd below the mean) during the first visit at weeks of pregnancy. a female child was born by emergency cesarean at weeks because of fetal distress. apgars were at min and at min. birth weight was . kg ( th centile), ofc cm ( th centile, � . sd below the mean), length cm ( th centile, � sd) and chest circumference cm ( th centile, mean for gestational age). the baby had similar facial features to patients – with a disproportionately small cranium and normal body proportions (fig. a,b). nasal length was . cm. there was also a sacral dimple. no skeletal anomalies noted on radiological survey. echocardiogram and chest x-ray were unremarkable; abdominal ultrasound did not reveal any liver or renal abnormalities. arterial blood gases, blood counts, blood cultures, serum electrolytes, urinalysis, serum amino acids and urinary organic acids were all normal. paucity of movements, jitteriness on stimulation and attacks of apnea were observed from the first a novel form of autosomal recessive lethal microcephaly american journal of medical genetics part a: doi . /ajmg.a day of life. brisk knee jerks and ankle clonus could be elicited at the age of week. there was no response to visual stimuli, but there was response to auditory stimulation. ct scan was similar to patient . head growth was reduced with only cm increase in ofc in the first weeks of postnatal life. the baby was inactive and fed poorly. breastfeed- ing was attempted but was insufficient and supple- mentary tube feeds were required. the patient became ill with gasping respirations and apnea at the age of days. aspiration pneumonia was diagnosed on chest radiogram. parenteral antibiotics and supplementary oxygen were administered. apnea attacks became more frequent and prolonged. after bagging with oxygen blood gases were normal. it was decided not to ventilate the patient and she died at the age of days. upon radiological analysis, pulmonary changes due to pneumonia were not found to be extensive enough to cause demise. central hypoventilation was the likely cause of death. exclusion of the mcpha (dnc) locus the only known gene responsible for lethal microcephaly was mapped in an amish kindred to a locus on chromosome (mcpha, q ; fig. . neuroimaging and pedigrees from a family with lethal microcephaly. a–c: ct images of patient reveal a simplified gyral pattern with normal to thinned cortical gray matter and mildly enlarged extra-axial space (a). anteriorly (b) the corpus callosum is significantly thinned. brainstem hypoplasia is mild compared to the reduced cortical volume while the cerebellar hemispheres and vermis (c, arrow) are small but proportional to the cerebral cortex. d: the simplified pedigree from the omani family shows four affected siblings, all of whom died by weeks of age. age of death for each child is listed below the pedigree. fig. . physical appearance. patient in three-quarter (a) and side profile (b) shows a disproportionately small neocranium compared with normal facial features, making ears and nose appear large. low hairline, sloping forehead, and temporal hollowing are consequences of the disproportion between calvarium and skull base. rajab et al. american journal of medical genetics part a: doi . /ajmg.a [rosenberg et al., ]). the lack of metabolic acidosis in the patients described here suggests that this form of lethal microcephaly may have a different cause than amish microcephaly, which is associated with mitochondrial failure. however, to exclude that these children had a different form of the same syndrome, we analyzed the mcpha locus. since the parents are first cousins, it is expected that the founder mutation, originated in a common ancestor, will be transmitted in homozygosity to the affected children [lander and botstein, ]. genomic dna was obtained from patient # and two highly polymorphic microsatellite markers flanking the dnc gene, d s and d s , were tested. both markers were present in hetero- zygozity in patient # (d s : allele ¼ , allele ¼ ; d s : allele ¼ , allele ¼ ), indicating that dnc is not associated with this disorder. discussion the four patients described in this article had a lethal form of microcephaly of prenatal onset, with ofcs at birth measuring � . to � . sd for gestational age. one baby (patient # ) had a birth ofc of � . sd with birth weight and chest circumference in the th centile, brain ct findings of msg and succumbed by weeks. this relatively larger ofc of the four cases may reflect the fact that this baby was the most premature of the live births and the head size would continue to fall off the curve, as indeed it did in the neonatal period despite supplemental tube feeding. reduced ofc was detectable in our patients as early as the th week of pregnancy on fetal ultrasound examination. reduced fetal movements were noted by the mother in all affected pregnancies. fetal distress with ctg abnormalities were detected in three monitored deliveries and were indication for deliveries by caesarean. it is uncertain if intrauterine death was the possible outcome in patients # , , and , if caesarean had not been performed. the birthweight for all of these patients was small to average for age ( th to th centile). they had a similar appearance with small neocranium, short sloping forehead, low anterior and temporal hairline, small glabellar angle and relatively large nose. since it is possible that next to a sloping forehead and small ofc the nose may appear larger and the glabellar angle smaller, the nasal length was measured in two of our patients (patients # and ) and was found to be proportionate to body length [siebert, ]. similarly, the head appeared particularly small in comparison with an average chest size and normal body proportions (table i). all four patients had failure of adaptation after delivery, irregular respirations, apnea attacks and jitteriness from the first days of life. flexions of the limbs, head jerking and often mouth and limb twitching was observed. they all required face-mask oxygen and died within the first hours, days, or weeks of life following apnea attacks and central hypoventilation. lung function proved to be satis- factory in all patients and early demise in our patients could not be attributed to pulmonary causes as oxygen and co exchange were easily supported with facemask o or bagging. therefore, brain stem hypoplasia and malfunction of respiratory centers could be the cause of recurrent apnea. substantial clinical evaluation did not find evidence of skeletal anomaly, liver or renal abnormality, or evidence of a defect in metabolic homeostasis or amino aciduria. brain imaging is difficult to obtain in these children. they survive for a short period of time, during which they are clinically unstable due to apneic episodes and the risk of further depressing respirations precludes the sedation required for ct or careful mri study. ct images that are available on two of our patients (patients # and # ), and share similar features. these include a simplified pattern of cortical gyri with normal or slightly reduced thick- ness of the cortical gray matter. there is thinning of the corpus callosum, especially anteriorly and cerebellar volume reduction, including the vermis that is proportional to the cortical microcephaly. the brainstem is also reduced in volume but to a lesser table i. clinical features in four patients (three females and one male) patients gestation age birth-weight , g , g , g , g length at birth cm cm cm cm small ofc/chest circumference ratio þ þ þ þ microcephaly cm cm cm cm small neocranium compared to facial structures þ þ þ þ sloping narrow forehead þ þ þ þ low frontotemporal hairline þ þ þ þ prominent nose þ þ þ þ inactivity þ þ þ þ apneic attacks þ þ þ þ seizures ? ? þ ? days survived hr min hr days days a novel form of autosomal recessive lethal microcephaly american journal of medical genetics part a: doi . /ajmg.a degree than the reduced overall brain size. the extra- axial spaces around the brain are generous, but not strikingly enlarged. this indicates that the growth of the brain is at least relatively parallel to the cranial growth and suggests a primary problem with neural proliferation or combination of proliferation and cell death rather than excessive neural apoptosis as the cause of microcephaly here [woods, ]. we cannot absolutely exclude an unknown inborn error of metabolism that was not corrected by maternal-fetal blood exchange or circulation and that produced a defect of neuronal proliferation and patterning. however, most patients with inborn errors of metabolism present with severe metabolic imbalances and microcephaly is typically postnatal rather than congenital [dobyns, ]. our metabolic screens found no evidence of pancreatic, hepatic or renal system failure, aminoaciduria or acidosis beyond that readily corrected with respiratory support for apneic episodes. severe microcephaly (ofc � � sd below the mean), such as observed in these patients is more likely to have a genetic origin, as microcephaly attributable to the intrauterine environment, for example, following maternal diabetes, drug expo- sure or intrauterine infection, is typically less pronounced (� sd below the mean) [dobyns, ]. when compared with the reports of autosomal recessive microcephaly with simplified gyral (msg) pattern available in the literature, these patients appear clearly distinct. some features observed in the patients described here resemble those in bowen–conradi syndrome (bcs), a lethal auto- somal recessive syndrome first described by bowen in the hutterite community [bowen, ] and more recently reported in india [gupta and phadke, ]. however, while the bcs patients encountered by bowen [ ] and lowry et al. [ ] presented with microcephaly with micrognathia and low birth weight, they were also characterized by overlapping fingers, joint contractures and an average survival to months [hunter et al., ; bowen, ; lowry et al., ]. early demise and the absence of joint contractures in our patients, combined with the normal appearance of the brain on ct scans of examined bcs patients [lowry et al., ], suggest that bcs represents a different condition from our patients. peiffer et al. [ ] described microcephaly in six related children with onset of seizures at – months of age and mental deficiency with similarity in phenotypic appearance, similar head size at birth and joint contractures. however, none of our patients had joint contractures and all cases reported by peiffer had better survival in the first year of life and half reached adult age. sztriha et al. [ ] reported a single case of extreme microcephaly with mri abnormalities of cortex, pontocerebellar dysplasia and partial agen- esis of corpus callosum. however, sztriha’s patient had an apparently different condition as he lived at least a year and brain imaging was distinct from our patients. mr brain images in the sztriha report showed a cortex with variable thickness (normal in some areas and significantly thickened in others) consistent with a neuronal migration defect and there was more severe midline cerebellar hypoplasia. like our patients, children with amish lethal microcephaly (mcpha) [kelley et al., ] also died in early infancy. however, brain images of our patients differ from the single published mri associated with mutation in the deoxynucleotide carrier gene (dnc) [kelley et al., ; rosenberg et al., ]. that image was quite distinct from the present cases in that there was only a thin remnant of cerebral cortex and marked cerebellar hypoplasia with a relatively preserved brainstem and diencephalon. moreover, our patients did not have metabolic derangements characteristic of mcpha and the survival in the reported amish cases was better than for our patients. most importantly, our patients did not map to the same locus as amish lethal microcephaly, ruling out mutation in the dnc gene as a possible cause of microcephaly in these omani children. the incidence of congenital lethal microcephaly is difficult to assess and may be more prevalent than currently appreciated. this condition may escape accurate reporting due to third trimester or neonatal lethality and/or the challenges of investigating critically ill newborns. indeed, we have observed another five patients with a similar phenotype, but these could not be included in this study because investigations were incomplete. the selection of patients for molecular studies who survive for a very short time poses challenges for obvious reasons. the diagnosis of congenital lethal microcephaly can be reached by exclusion of other metabolic and syndromic causes, neuroimaging and comparison of neurodevelopment and growth patterns with normative data. in conclusion, we described novel form of lethal microcephaly of prenatal onset with simplified gyri and unique set of brain abnormalities detectable on mri, which does not map to amish locus. acknowledgments the authors would like to thank the family for their participation in this study; bernard chang for assistance in reviewing the radiological findings, and christina austin for technical help. this work was supported by nih grants po ns (mer) and r -ns (caw). caw is an investigator of the howard hughes medical institute. ghm is supported by the narsad young investigator award. rajab et al. american journal of medical genetics part a: doi . /ajmg.a references barkovich aj, kuzniecky ri, jackson gd, guerrini r, dobyns wb. . a developmental and genetic classification for malformations of cortical development. neurology : – . bond j, 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agyria-pachygyria, partial agenesis of corpus callosum, and pontocerebellar dysplasia. j child neurol : – . woods cg. . human microcephaly. curr opin neurobiol : – . a novel form of autosomal recessive lethal microcephaly american journal of medical genetics part a: doi . /ajmg.a pone. .. ucsf uc san francisco previously published works title a common variant in the telomerase rna component is associated with short telomere length. permalink https://escholarship.org/uc/item/ zh h journal plos one, ( ) issn - authors njajou, omer t blackburn, elizabeth h pawlikowska, ludmila et al. publication date - - doi . /journal.pone. peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ zh h https://escholarship.org/uc/item/ zh h #author https://escholarship.org http://www.cdlib.org/ a common variant in the telomerase rna component is associated with short telomere length omer t. njajou , elizabeth h. blackburn , ludmila pawlikowska , massimo mangino , coleen m. damcott , pui-yan kwok , timothy d. spector , anne b. newman , tamara b. harris , steven r. cummings , richard m. cawthon , alan r. shuldiner , ana m. valdes , wen-chi hsueh * department of medicine, university of california san francisco, san francisco, california, united states of america, department of anesthesia and perioperative care, university of california san francisco, san francisco, california, united states of america, department of biochemistry and biophysics, university of california san francisco, san francisco, california, united states of america, cardiovascular research institute, university of california san francisco, san francisco, california, united states of america, twin research and genetic epidemiology unit, king’s college london, st thomas hospital, london, united kingdom, division of endocrinology, diabetes and nutrition, department of medicine, university of maryland, baltimore, maryland, united states of america, department of epidemiology, university of pittsburgh, pittsburgh, pennsylvania, united states of america, laboratory of epidemiology, demography, and biometry, national institute on aging, bethesda, maryland, united states of america, research institute, california pacific medical center, san francisco, california, united states of america, department of human genetics, university of utah, salt lake city, utah, united states of america abstract background: telomeres shorten as cells divide. this shortening is compensated by the enzyme telomerase. we evaluated the effect of common variants in the telomerase rna component (terc) gene on telomere length (tl) in the population- based health aging and body composition (health abc) study and in two replication samples (the twinsuk study and the amish family osteoporosis study, afos). methodology: five variants were identified in the terc region by sequence analysis and only one snp was common (rs , g/a). the frequency of the g allele was . and . in white and black, respectively. testing for association between tl and rs was performed using linear regression models or variance component analysis conditioning on relatedness among subjects. results: the adjusted mean tl was significantly shorter in white carriers of the g allele compared to non-carriers from the health abc study ( . . kbp vs. . . kbp, measured by quantitative pcr, p = . ). this association was replicated in another white sample from the twinsuk study ( . . kbp in carriers compared to . . kbp in non-carriers, measured by southern blots, p = . ). a similar pattern of association was observed in whites from the family-based afos and blacks from the health abc cohort, although not statistically significant, possibly due to the lower allele frequency in these populations. combined analysis using , white subjects from studies showed a significant association between tl and rs (b = . . kbp, p = . ). conclusion: our study shows a significant association between a common variant in terc and tl in humans, suggesting that terc may play a role in telomere homeostasis. citation: njajou ot, blackburn eh, pawlikowska l, mangino m, damcott cm, et al. ( ) a common variant in the telomerase rna component is associated with short telomere length. plos one ( ): e . doi: . /journal.pone. editor: amanda ewart toland, ohio state university medical center, united states of america received april , ; accepted august , ; published september , copyright: � njajou et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. funding: this study was supported in part by the national institutes of health (nih) contracts (n ag- - , n ag- - and n ag- - ) and grants (k ag , r ag and u ag ). this research was also supported in part by the intramural research program of the nih, national institute on aging and by the wellcome trust; nihr (tds), nihr biomedical research centre (grant to guys’ and st. thomas’ hospitals and king’s college london). dr. omer t. njajou is supported by the training in molecular and genetic epidemiology of cancer grant from the national cancer institute (r ca ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. competing interests: the authors have declared that no competing interests exist. * e-mail: wen-chi.hsueh@ucsf.edu introduction telomerase is a ribonucleoprotein polymerase that maintains telomere ends by addition of the telomere dna repeat ttaggg in humans. the core enzyme consists of a protein component with reverse transcriptase activity (tert), and an rna component (terc) located on chromosome q . that provides the template for the telomere repeat. several lines of evidence in experimental and epidemiological research suggest that the human telomerase rna component is involved in telomere homeostasis [ , , ]. the wild-type telomerase rna is assembled with small nucleolar rnas (snorna), small cajalbody rna (scarna) proteins, the putative cab box binding protein and telomerase reverse transcriptase to form a functional telomerase complex that will later be recruited to the chromosome ends and maintain telomere length [ ]. terc is a small gene, basepairs (bp) long, that is highly expressed in the germline and in tumor cell lines, in which there is plos one | www.plosone.org september | volume | issue | e high telomerase activity, and at lower levels in tissues such as kidney, prostate, and liver in which there is little detectable telomerase activity. mutations in terc result in a reduction of telomerase activity leading to premature telomere shortening and have been linked to the autosomal dominant form of dyskeratosis congenita and aplastic anemia [ , ]. goldman et al. have observed that individuals haploinsufficient for terc have very short telomeres [ ]. they found that when terc activity is limiting, this leads to the accelerated shortening of telomeres. the limited amount of active telomerase in individuals with terc rna haploinsufficiency may not be able to maintain the minimal critical telomere length in cells with already short telomeres. a recent study reported that a common haplotype in the other telomerase component, tert, is associated with longer telomere length in centenarians [ ]. following previous studies of rare mutations in terc, this study aimed at investigating whether common polymorphisms in or around the terc gene region may play a role in the regulation of telomere length (tl) in normal populations. materials and methods study populations three independent samples were used to investigate the association between common variants in or around terc and tl. the first population is a biracial cohort ( % black and % white) from the health, aging, and body composition (health abc) study. as we observed a significant association in the white samples only, we sought to replicate our initial findings in another outbred white population from the twinsuk study and a white founder population from the amish family osteoporosis study (afos). the health abc study the health abc study population is a community-based cohort of , healthy, well functioning men and women aged to years. to be eligible for participation in the study, subjects had to report no difficulty in walking one-quarter mile ( . kilometers) or climbing stairs without resting. participants were identified from a random sample of white medicare beneficiaries and all age-eligible black residents in designated zip code areas surrounding pittsburgh and memphis. exclusion criteria included reported difficulty performing basic activities of daily living, obvious cognitive impairment, inability to communicate with the interviewer, intention of moving within three years, or participa- tion in a trial involving a lifestyle intervention. a total of , subjects with tl measures and the terc genotypic data available were included in this study. all participants gave written informed consent. the committee on human research at both study sites approved the protocol and written consent for the study. the twinsuk study the twinsuk study recruited white monozygotic (mz) and dizygotic (dz) twin pairs from the twinsuk adult twin registry, a group designed to study the heritability and genetics of age-related diseases (www.twinsuk.ac.uk). these twins were recruited from the general population through national media campaigns in the uk and shown to be comparable to age-matched population singletons in terms of clinical phenotype and lifestyle character- istics. a subset of female subjects with tl measures and the terc genotypic data were included in this study. the study was approved by st. thomas’ hospital research ethics committee and all twins provided informed written consent that was approved by the ethics committee. the amish family osteoporosis study a total of old order amish subjects for this study were recruited through the amish research clinic in strasburg, pa, as part of the amish family osteoporosis study (afos), whose aim was to identify genetic determinants of osteoporosis. the recruitment methods and study objectives and design have been described in details previously [ ]. briefly, individuals with low bone mineral density or history of fracture were recruited into the study as probands (n = ). their spouses and all first-degree relatives aged years and over were invited to participate in the study. genealogical information was obtained from the fisher family history and the larger anabaptist genealogy database version . [ , ]. the protocol for the afos was approved by the institutional review board of the university of maryland, and verbal informed consent as well as permission to contact relatives was obtained from all participants. (amish people do not use signature, so only verbal consent was given and this was approved by the review board). measurement of leukocyte telomere length in the health abc study and afos samples the mean tl in leukocytes (peripheral blood mononucleocytes) was measured using a validated quantitative pcr (q-pcr) method [ , ] which measures the relative average tl in genomic dna by determining the ratio of telomere repeat copy number to single copy gene copy number (t/s ratio) in experimental samples relative to a reference sample. the t signal for an experimental dna sample is the number of nanograms of the reference dna that matches the experimental sample for copy number of the telomere repeats. the s signal is the number of nanograms of the reference dna that matches the experimental sample for copy number of the single copy gene. experimental samples with t/s ratio . . have longer average telomere lengths than the reference dna. experimental samples with t/s ratio , . have shorter average telomere lengths than the reference dna. the reference dna is a pooled sample of dnas from several normal utah whites, aged years or older. all samples were measured in triplicate and the mean value was used. the coefficient of variation (cv) for this assay was %. results obtained using this method correlate very well with those obtained with the traditional terminal restriction fragment length (trfl) by southern blot technique [ ]. in comparison with the trfl method, the q-pcr method is simple, fast, and less expensive, and requires a significantly lower amount of dna. to convert the t/s value to basepairs, the t/s value was multiplied by a conversion factor of , bp, which was the tl for the reference dna. to obtain the tl for the reference dna, we used the t/s ratios of dna samples with known mean trf lengths. the slope of the linear regression line through a plot of t/s ratio (the x axis) vs. mean trf length (the y axis) is the number of basepairs of telomeric dna corresponding to a single t/s unit. since the reference dna has a t/s of . , by definition, this slope is also the average tl of the reference dna sample, , bp in our case. among the , participants at baseline, , subjects had dna available and tl was successfully measured in , individuals. measurement of leukocyte telomere length in the twinsuk study samples the mean leukocyte tl was assessed using the trfl method, which was measured using the southern blot method in duplicates. briefly, each sample was digested using restriction enzymes hinfi ( u) and rsai ( u) (roche, indianapolis, indiana, usa) and resolved terc snp and telomere length plos one | www.plosone.org september | volume | issue | e on . % agarose gels. dna was then depurinated and denatured. hybridization with digoxenin -end-labeled telomeric probes was conducted overnight and probes detected using a digoxenin luminescent detection procedure (roche). the autoradiographs were scanned and the trfl signal was digitized at molecular weight – kb. conversion of the optical density versus dna migration distance to optical density adjusted for background/molecular weight yielded a histogram from which the mean trfl was calculated. the cv of the trfl assay in this study was . %. the laboratories conducting the telomere length measurements were blinded to all characteristics of the leukocyte donors, who were identifiable only by coded id numbers. discovery and genotyping of the terc variants variants in terc covering the entire gene of bp plus bp upstream and bp downstream of the coding region were identified by conventional sanger sequencing in pools of pcr products ( pools of white and pools of black, with individuals per pool). because no protein is encoded by terc, the "coding" region refers to the sequence encoding the mature rna transcript. sequencing was performed using the forward primer and standard procedures on the abi capillary sequencer. sequencing traces and subsequent genotyping were scored by investigators blinded to phenotype (telomere measurement status). five snps were identified by sequence analysis and the allele frequencies were estimated from the trace heights in pools (see table ). the snps at base # , , and , , have been previously reported and correspond to base # and in those reports [ , ]. the other three snps were newly discovered. we genotyped these five snps in the full health abc cohort by sequencing or by illumina golden gate and subsequently we genotyped the only common snp in the twinsuk and the afos samples by the taqman method. statistical analyses the associations between leukocyte tl and common terc snps were modeled using linear regression models. age, sex, race, and the recruitment site were added to this model to control for potential confounding. to account for the relatedness of twins in the twinsuk cohort, we used mixed linear models where the random effect was the family of origin. analyses for the health abc and uk cohort data were carried out using the software spss for windows (chicago, il) and analyses for the afos cohort were conditioning on their relatedness and carried out using the program solar [ ]. since significant associations were observed only in white, we also conducted combined analysis by pooling data from all white populations. the combined analysis was carried out in solar so that the relatedness among subjects (in the twinsuk and afos cohorts) can be taken into account. in addition to covariates used in population-specific analysis, the type of tl assay was also included as a covariate. a p value , . was considered statistically significant. results of the five variants identified by sequence analysis of the terc gene region, only one snp (i.e. rs , g/a) had a minor allele frequency (maf) . % (see table ). the frequency of the g allele was . – . in white populations, and was . in blacks (see table ). a total of , individuals ( , white and , black) from the health abc cohort, female caucasian participants of the twinsuk cohort and caucasian subjects from the afos cohort were included in our analysis. table shows the baseline characteristics of the study populations. participants in the health abc study were older (mean age = . . years) compared to those in the twinsuk (mean age = . . ) and the afos (mean age = . . ) popula- tions. however, the age range was much narrower in the health abc cohort compared to the other two cohorts comprised of younger participants. nearly half of the health abc cohort and % of afos participants were female, while all participants of the twinsuk cohort were female. overall, the mean tl (kbp) measured by q-pcr was longer among amish subjects ( . . kbp) compared to that of the health abc participants ( . . kbp), but in the same age group ( – years), the mean tl in populations were similar. the mean tl in the twinsuk cohort measured by southern blot appeared to be longer ( . . kbp). the frequency of the g allele of rs was , % in outbred white populations while it was much rarer in the white founder population (the amish, . %) and black samples ( . %). table shows the mean tl by rs genotypes. there was a significant difference in the mean tl by genotypes ( -df test) in the health abc white (p = . ) and the twinsuk cohort (p = . ). a dose-response of tl with respect to the number of g alleles was observed in the health abc whites and the afos. modeling of the association between tl and rs genotypes suggested that a dominant mode of inheritance for the minor g allele was the best fit to the data. as shown in table , among the health abc study white participants, the adjusted mean tl was significantly shorter in carriers of the g allele ( . . kbp vs. . . kbp in non-carriers, p, . ). no significant difference was observed in blacks ( . . kbp in carriers of the g allele vs. . . kbp in non-carriers, p = . ). we successfully replicated the association in whites from the twinsuk study. the adjusted mean tl was significantly (p, . ) shorter in carriers of the g allele compared to non-carriers. in the afos, carriers of the g allele appeared to have shorter tl on average compared to non- carriers, but the difference was not statistically significant. table . sequence variants identified in the terc gene. base # location base pair change minor allele frequency in white minor allele frequency in black sequence location , , upstream g/a . . tagaaaaaag[g/a]ccctctgata , , rna coding g/a . . aaccctaact[g/a]agaagggcgt , , rna coding g/a . cgggtcgcct[g/a]cccagccccc , , (rs ) flank a/g . . ctcgccggca[a/g]tgggggcttg , , flank g/a . ggggttgcct[g/a]gagccgttcc doi: . /journal.pone. .t terc snp and telomere length plos one | www.plosone.org september | volume | issue | e as the effect of rs appeared similar among white study populations, we pooled data from all these populations (n = , ) and found that the mean tl for carriers of the minor g allele was . kbp shorter than non-carriers (b = . . kbp, p, . ). discussion we observed that the g allele of the snp rs near terc ( bps upstream from the start site) was significantly associated with shorter telomere length in a us white population and replicated the findings in another younger white population in the uk. on the other hand, we did not observe such an association in whites from the family-based afos or in blacks from the health abc cohort. the g allele was rarer in the afos sample (frequency = . %) and in the blacks ( . %) compared to whites from health abc study ( . %) or the twinsuk study ( . %). thus, the non- significant associations in these two populations are likely due to the lack of sufficient statistical power (we had only % power) to detect the same effect size observed in the health abc cohort. a recently published genome-wide association study of telomere length by codd et al. reported their top signal to be rs , . kb downstream of terc [ ]. this snp resides in the same ld block of kb and is , bps away (ncbi build . ) from our reported snp rs . we looked into this region more closely for all typed snps in the kb block, including rs . all of these snps were significantly associated with telomere length, but rs still gave the strongest signal and is in high ld (r = . ) with rs . the snp rs is not typed by the hapmap and therefore not included in any high- throughput genotyping platform. as this snp is closer to the mature rna transcript of terc, it might be a better target for further functional studies. both this report and the report by codd et al. detected the rna template of telomerase as major determinant for human telomere length variation. it is interesting that the yeast terc ortholog, tlc , is a major telomere length qtl in the natural population [ ]. it does appear that this gene is prone to be a determinant for telomere length variation. this could be due to its dynamic structure or by differences in gene table . characteristics of study participants from three cohorts. traits (mean ± sd or number, %) health abc (white) health abc (black) twinsuk (white) afos (white) n (% female) , ( %) , ( %) ( %) ( %) age (years) . . . . . . . . range (years) – – – – telomere length (kbp) . . . . . . * . . range (kbp) . – . . – . . – . . – . frequency of the g allele of rs . % . % . % . % rs genotypes aa ( . %) ( . %) ( . %) ( . %) ag ( . %) ( . %) ( . %) ( . %) gg ( . %) ( . %) ( . %) ( . %) *including the subtelomeric segment. doi: . /journal.pone. .t table . association between telomere length and rs . ethnicity caucasian black study health abc afos twinsuk health abc n (%) tl ± se n (%) tl ± se n (%) tl ± se n (%) tl ± se means by genotype aa ( . ) . . ( . ) . . ( . ) . . ( . ) . . ga ( . ) . . ( . ) . . ( . ) . . ( . ) . . gg ( . ) . . ( . ) . . ( . ) . . ( . ) . . p value . ns . ns aa ( . ) . . ( . ) . . ( . ) . . ( . ) . . gg + ga ( . ) . . ( . ) . . ( . ) . . ( . ) . . p value>* . ns . ns association* b se . . . . . . . . p value . ns . ns *based on a dominant genetic model for the g allele, adjusted for age, sex, relatedness, telomere length assay batch. doi: . /journal.pone. .t terc snp and telomere length plos one | www.plosone.org september | volume | issue | e expression. indeed, a number of telomerase rna template molecules appear to be a limiting factor of telomere elongation in budding yeast. similarly, there is a pronounced terc haploinsufficiency effect in human telomeres and this agrees with our findings for the rs snp. one possible issue of our replication sample is that the twinsuk study subjects consisted only of females. however, in the health abc cohort the reported association did not differ by sex. another issue has to do with different methods for measuring tl. tl was measured by the q-pcr method in the health abc and amish cohorts while it was measured by the trfl method in the twinsuk study. the trfl method tends to make the tl about . kb longer as it includes the subtelomeric region in its measurement. nevertheless, the strength of the association and the effect size estimates appeared comparable based on either method. there have been debates regarding tl measurements as to whether different methods for tl measurement could affect the informativeness of tl measures for genetic and epidemiological studies involving tl [ , , ]. therefore, it is reassuring that we were able to replicate our observed association in the health abc study (tl measured by the q-pcr method) and in the twinsuk study (tl measured by the trfl method). in addition, in the combined analysis adjusted for the method of tl measurement, the observed association remained significant and in the same direction. some environmental factors like smoking are found to affect tl [ ]. in our study, tl was not associated with smoking and adjusting for smoking did not alter our observations. we however adjusted our analysis for the tl assay’s plate effects which has been associated to tl measurements in several laboratories. the terc and tert genes encode the core essential elements of the telomerase complex that replicates telomeres and maintains their length. mutations in terc have been previously linked to telomere shortening in mouse models [ ]. studies in human have also shown that rare mutations in terc are associated with telomere shortening in individuals with dyskera- tosis congenita [ , , ]. our study extends these observations to populations free of congenital or other severe diseases. the biological relevance of our observations is further supported by a recent report that variants in another telomerase rna compo- nent, tert, are also associated with tl in humans. both tert and terc are essential for telomerase function. the terc region does not appear to be conserved in evolution. there is an open reading frame (orf) antisense to terc. however, the likelihood that there is a protein encoded on either strand of the terc locus is extremely remote, as indicated by codon usage/substitution patterns in the orfs, the lack of peptides in mass spectrometry databases that map to these orfs, and the lack of homology of any of the orfs to any known protein sequence. there is not yet functional information on rs and it is beyond the scope of our current investigation. one possible functional study is to take advantage of the induced pluripotent stem (ips) cells technique since terc upregulation is a feature of the pluripotent state and several telomerase components are targeted by pluripotency- associated transcription factors [ ]. conventionally, genes that do not encode proteins are thought to be of little relevance with clinically measurable phenotypes. however, a growing number of human studies of rna genes such as terc [ ] and microrna genes [ ] suggest that non-coding regions such as these rna genes may harbor genetic variants of clinical relevance. taken together, our findings suggest that common variants in the terc gene in normal healthy populations may be involved in telomere homeostasis. our observations further re-enforce the idea that variants nearby genes encoding functional rnas may potentially be biologically important. author contributions conceived and designed the experiments: otn tds abn tbh src rc ars amv wch. performed the experiments: lp cmd pyk abn rc. analyzed the data: otn mm wch. contributed reagents/materials/ analysis tools: eb lp pyk tds src rc ars amv wch. wrote the paper: otn wch. references . hoare sf, bryce la, wisman gb, burns s, going jj, et al. 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hal id: hal- https://hal.archives-ouvertes.fr/hal- submitted on dec hal is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. the documents may come from teaching and research institutions in france or abroad, or from public or private research centers. l’archive ouverte pluridisciplinaire hal, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. dyt dystonia: review of the literature and creation of the umd locus-specific database (lsdb) for mutations in the thap gene arnaud blanchard, vuthy ea, agathe roubertie, mélanie martin, coline coquart, mireille claustres, christophe béroud, gwenaelle collod-beroud to cite this version: arnaud blanchard, vuthy ea, agathe roubertie, mélanie martin, coline coquart, et al.. dyt dystonia: review of the literature and creation of the umd locus-specific database (lsdb) for mutations in the thap gene: review. human mutation, wiley, , ( ), pp. - . � . /humu. �. �hal- � https://hal.archives-ouvertes.fr/hal- https://hal.archives-ouvertes.fr databases www.hgvs.org dyt dystonia: review of the literature and creation of the umd locus-specific database (lsdb) for mutations in the thap gene arnaud blanchard, , vuthy ea, , agathe roubertie, , , mélanie martin, , coline coquart, mireille claustres, , , christophe béroud, , , and gwenaëlle collod-béroud , ∗ inserm u , montpellier, f- , france; université montpellier , ufr de médecine, montpellier, f- , france; chu montpellier, hôpital gui de chauliac, service de neuropédiatrie, montpellier, f- , france; chu montpellier, hôpital arnaud de villeneuve, laboratoire de génétique moléculaire, montpellier, f- , france communicated by alastair f. broun received january ; accepted revised manuscript june . published online july in wiley online library (www.wiley.com/humanmutation).doi: . /humu. abstract: by family-based screening, first fuchs and then many other authors showed that mutations in thap (thap [thanatos-associated protein] domain- containing, apoptosis-associated protein ) account for a substantial proportion of familial, early-onset, nonfo- cal, primary dystonia cases (dyt dystonia). thap is the first transcriptional factor involved in primary dysto- nia and the hypothesis of a transcriptional deregulation, which was primarily proposed for the x-linked dystonia- parkinsonism (dyt dystonia), provided thus a new way to investigate the possible mechanism underlying the de- velopment of dystonic movements. currently, fami- lies present with a thap mutation; however, no geno- type/phenotype relationship has been found. therefore, we carried out a systematic review of the literature on the thap gene to colligate all reported patients with a spe- cific thap mutation and the associated clinical signs in order to describe the broad phenotypic continuum of this disorder. to facilitate the comparison of the identified mutations, we created a locus-specific database (umd- thap lsdb) available at http://www.umd.be/thap /. currently, the database lists probands and rela- tives with the associated clinical phenotype when avail- able. the identification of a larger number of thap mutations and collection of high-quality clinical informa- tion for each described mutation through international collaborative effort will help investigating the structure– function and genotype–phenotype correlations in dyt dystonia. hum mutat : – , . c© wiley-liss, inc. ∗correspondence to: gwenaëlle collod-béroud, inserm u , institut universitaire de recherche clinique, av du doyen gaston giraud, montpellier cedex , france. e-mail: gwenaelle.collod-beroud@inserm.fr contract grant sponsors: université montpellier ; inserm; french ministry of health (national phrc -a - ); amadys-lfcd, alliance france dystonie; lions club la grande motte; the french dystonia network; the european community’s sev- enth framework programme (fp / – under grant agreement no. —the gen phen project). key words: dystonia; dyt ; thap , database; umd introduction dystonia defines a heterogeneous group of movement disorders due to central nervous system (cns) dysfunction leading to sus- tained involuntary muscle contractions that cause abnormal pos- tures and twisting movements. since secondary dystonia is often linked to striatal injuries, it has been hypothesized that hereditary dystonia also could be due to a dysfunction of these structures and more broadly of the basal ganglia. accordingly, many stud- ies have reported electrophysiological, metabolic, and structural abnormalities in these brain areas [vidailhet et al., ]. how- ever, basal ganglia are not the only brain region that has been found to be altered in hereditary dystonia. indeed, dysfunction of cerebral cortex, cerebellum, spinal cord, and other structures that are mainly involved in motor function has also been reported [breakefield et al., ; defazio et al., ]. therefore, the con- cept that dystonia is a disorder linked to a specific cns struc- ture appears to be too restrictive and it should rather be consid- ered as the result of a general disturbance of the motor circuit functions. hereditary dystonias are clinically and genetically heterogeneous. to date, different genetically forms are known (dyt to dyt ; with dyt = dyt ) and mutations in nine genes have been in- volved [breakefield et al., ; norgren et al., ]. all the inheri- tance modes are found (autosomic recessive, autosomic dominant, and x-linked) but no epistatic relation between these genes was discovered except for dyt and dyt forms [gavarini et al., ; kaiser et al., ]. since their products have different cell functions, it is very difficult up to now to propose a scheme of cell dysfunction common to all the dystonia forms and particularly to propose a unitary pathophysiological mechanism even if links with dopamine deregulation are often found [blanchard et al., ; park et al., ]. the last identified gene, thap for thap [thanatos-associated protein] domain-containing, apoptosis-associated protein , was first found to be mutated in amish–mennonite families with pri- mary dystonia and its implication was then extended to other c© wiley-liss, inc. populations [almasy et al., ; fuchs et al., ]. thap muta- tions cause dyt dystonia, an autosomal dominant primary form with about % penetrance. the main clinical characteristics of this form are early onset (mean = . years of age [calculated from patients with available data]) and symptoms often localized to one upper limb at the beginning with tendency to extend to other body regions. fifty-three different mutations in the thap gene have been re- ported so far in families (table ). these mutations are mainly private, essentially missense or out-of-frame deletions, usually non- recurrent, and widely distributed throughout the gene. to date, no genotype/phenotype relationship has been observed. from this per- spective, we have compiled a database that gather all the available information on this gene and on the patients with thap mutations and created a software package to facilitate the mutational analysis of thap . this database is available at http://www.umd.be/thap /. thap expression two spliced mrna variants that produce functional proteins have been reported (thap a: ccds and thap b: ccds ) [girard et al., ]. the first . -kb isoform contains three exons (fig. ), whereas the second corresponds to an alternatively spliced isoform that lacks exon ( -kb mrna). this second isoform en- codes a truncated thap protein without the c-terminus of the thap domain. the two isoforms are expressed in many tissues, suggesting that thap has a widespread (although not ubiquitous) distribution in humans. in mouse brain tissue, immunoblot analysis revealed a highest concentration in embryonic whole brain tissue (at e ), which declines after birth in the different tested brain regions (at p ) [gavarini et al., ]. thap a, which contains the thap domain with dna-binding properties, is the most studied. thap is a transcription factor thap was first identified by two-hybrid screen of a high en- dothelial venule cell cdna library using a cytokine bait [roussigne et al., ]. thap is a amino-acid-long protein characterized by an n-terminal thap domain (amino acids to ) [bessiere et al., ] with dna-binding properties, followed by a proline- rich region (amino acids to ), and a nuclear localization signal (amino acids to ) (fig. a). the protein is only found in the nucleus where it can display both a diffuse distribution and a discrete localization in nuclear dots. thap colocalization with daxx, a well-characterized pro- tein that is expressed in promyelocytic leukemia (pml) nuclear bodies (nbs), indicates that these subnuclear regions are pml nbs. pml nbs are spheres of . – . μm, heterogeneous in composition, mobility, and function, that are present in most mammalian cell nu- clei [lallemand-breitenbach and de the, ]. the pml protein is the key organizer that recruits an ever-growing number of proteins, residing constitutively or more often transiently. pml nbs facil- itate partner protein posttranslational modification (acetylation, sumoylation, or phosphorylation) resulting in partner sequestra- tion, activation, or degradation. they are proposed to fine-tune a wide variety of processes: induction of apoptosis and cellular senes- cence, inhibition of proliferation, maintenance of genomic stability, and antiviral responses [bernardi and pandolfi, ]. thap interacts with the prostate apoptosis response- (par- , hgnc-approved gene symbols pawr), a pro-apoptotic protein that acts as a transcriptional regulator [roussigne et al., ]. pawr is present in low amounts in dendrites and synapses throughout the brain under normal conditions and its amount in- creases rapidly in neurons subjected to several triggers of apop- tosis including trophic factor deprivation, oxidative stress, and excitotoxins [duan et al., a; guo et al., ]. pawr induc- tion has been linked to neuronal death in various neurodegenera- tive diseases as parkinson’s [duan et al., b], alzheimer [guo et al., ], or amyotrophic lateral sclerosis [pedersen et al., ]. more interestingly, pawr has been shown to serve other important function in nervous system. it interacts with d dopamine recep- tor (d dr) suggesting its potential role in modulating calcium- mediated dopaminergic signaling [park et al., ]. thap and pawr exhibit pro-apoptotic activities (they increase the apoptosis sensitivity of mouse t fibroblasts to tnf-α and serum withdrawal when overexpressed) and they are both indirectly recruited to pml nbs by pml [roussigne et al., ]. roussigne and coworkers proposed that pml nbs regulate the functions of thap and pawr. they may be the site of assembly and/or post- translational modifications of the thap /pawr complex or they may serve as a site of storage or degradation, thus regulating the free nucleoplasmic pool of pawr and thap . extensive database search using the thap domain identified about thap proteins among seven model animal organisms [clouaire et al., ]. interestingly, some of these proteins are tightly linked to the prb (retinoblastoma protein)/e f pathway, which regulates commitment of mammalian cells to dna replica- tion. the thap domain is present in the zebrafish ortholog of e f and in five caenorhabditis elegans proteins (lin- , lin- b, lin- a, him- , and gon- ), which are known to be genetically linked to lin- , the only rb ortholog found in c. elegans. based on these data, cayrol et al. [ ] investigated whether thap could be linked to the prb/e f pathway. they found that silenc- ing as well as overexpression of thap led to cell cycle arrest at the g /s transition. the similar effects following thap silenc- ing/overexpression suggest that, like many other proteins, thap needs an optimal range of concentration to fulfill its physiologi- cal roles. dna microarray analysis showed in both cases (upreg- ulation or downregulation of thap ) a reduction of the mrna levels of cell cycle regulators and about prb/e f-target genes [cayrol et al., ], including rrm (ribonucleotide reductase m ), birc (survivin), cdc (cell division cycle ), ccnb (cy- clin b ) [lacroix, ]. rrm is activated at the g /s transition and contains two predicted thap -binding sites (thabs) in its promoter. dnase i footprinting, electrophoretic mobility shift assay (emsa) protein/dna binding, and chromatin immunoprecipita- tion (chip) in proliferating endothelial cells demonstrated that en- dogenous thap directly binds to the promoter of rrm [cayrol et al., ]. moreover, mazars et al. [ ] showed that thap recruits hcf- (host cell factor ) to the rrm promoter to allow its expression, thus adding further evidence for a role of thap in cell cycle regulation. although the list of cofactors that participate in thap regulation of target genes is certainly not exhaustive, the available data clearly link thap to apoptosis (through its interac- tion with pawr) and to cell cycle (through hcf- ). thap structure the most extensive structural studies have focused on the thap domain of thap and implicitly serve as a model for the other hundred thap proteins discovered in the animal kingdom. in many aspects, the thap domain, which is about -amino-acid long, human mutation, vol. , no. , – , ta bl e . m ut at io ns r ep or te d in th e th a p g en e in ea ch fa m il y/ p ro ba nd m u ta ti o n n am e n o m en cl at u re (p ro te in ) w il d t yp e co d o n m u ta n t co d o n m u ta ti o n al ev en t a m in o ac id p o si ti o n e xo n n o . st ru ct u re h c d r el at iv es in th e d at ab as e r ef er en ce n o . in sd el m u ta ti o n c. _ d el in sg g g t t t a ,a p .p h e l eu fs x c c c in d el s in d el s st o p at l -t h a p d o m . [f u ch s et al ., ;b re ss m an et al ., ] m is se n se m u ta ti o n s c. a > g p .m et ? a t g g t g a -> g t h a p d o m . [d e c ar va lh o a gu ia r et al ., ] c. t > c p .s er p ro t c c c c c t -> c l -t h a p d o m . [c lo t et al ., ] c. c > t p .s er p h e t c c t t c c -> t l -t h a p d o m . [h o u ld en et al ., ] c. a > g p .t yr c ys a t g t g c a -> g l -t h a p d o m . [h o u ld en et al ., ] c. g > t p .g ly c ys g g c t g c g -> t l -t h a p d o m . [x ia o et al ., ] c. c > a p .a sn l ys a a c a a a c -> a l -t h a p d o m . [b re ss m an et al ., ] c. g > a p .a rg h is c g c c a c g -> a l -t h a p d o m . [z it te l et al ., ] c. a > g p .a sp g ly g a c g g c a -> g l -t h a p d o m . [x ia o et al ., ] c. t > a p .s er t h r t c t a c t t -> a l -t h a p d o m . [b re ss m an et al ., ] c. a > c p .h is p ro c a c c c c a -> c b s -t h a p d o m . [k ai se r et al ., ] c. c > g p .p ro a rg c c t c g t c -> g l -t h a p d o m . d n a b in d in g [h o u ld en et al ., ] c. g > a p .a rg g ln c g a c a a g -> a l -t h a p d o m . d n a b in d in g [p ai sa n -r u iz et al ., ] c. g > a p .a rg g ln c g a c a a g -> a l -t h a p d o m . d n a b in d in g [p ai sa n -r u iz et al ., ] c. g > c p .a rg p ro c g a c c a g -> c l -t h a p d o m . d n a b in d in g [b re ss m an et al ., ] c. c > g p .p ro a rg c c c c g c c -> g l -t h a p d o m . [j ec h et al ., ] c. t > a p .l eu h is c t t c a t t -> a l -t h a p d o m . a a in te ra ct io n s [s ch n ei d er et al ., ] c. g > a p .a la t h r g c t a c t g -> a a h -t h a p d o m . a a in te ra ct io n s [b re ss m an et al ., ] c. g > a p .c ys t yr t g t t a t g -> a b s -t h a p d o m . c c h m o ti f [g av ar in i et al ., ] c. c > a p .h is a sn c a c a a c c -> a a h -t h a p d o m . c c h m o ti f [s ö h n et al ., ] c. c > t p .t h r il e a c t a t t c -> t t h a p d o m . [g ro en et al ., ] c. t > g p .l eu a rg c t g c g g t -> g l -t h a p d o m . [c lo t et al ., ] c. a > t p .a sn il e a a t a t t a -> t l -t h a p d o m . [c h en g et al ., ] c. t > c p .p h e l eu t t t c t t t -> c a h -t h a p d o m . a a in te ra ct io n s [f u ch s et al ., ] c. t > c p .c ys a rg t g t c g t t -> c [s ö h n et al ., ] c. a > g p .l ys a rg a a g a g g a -> g [b re ss m an et al ., ] c. t > c p .p h e se r t t c t c c t -> c [x ia o et al ., ] c. a > g p .a sn se r a a c a g c a -> g h c f - b in d in g [h o u ld en et al ., ] c. c > g p .a sn l ys a a c a a g c -> g h c f - b in d in g [g ro en et al ., ] c. a > g p .t yr c ys t a t t g t a -> g h c f - b in d in g [s ö h n et al ., ] c. a > g p .m et v al a t g g t g a -> g c o il ed -c o il d o m . [s ö h n et al ., ] c. t > c b p .i le t h r a t t a c t t -> c n l s [x ia o et al ., ] c. a > c p .h is p ro c a t c c t a -> c n l s [c h en g et al ., ] c. g > a p .a la t h r g c a a c a g -> a c o il ed -c o il d o m . [x ia o et al ., ] c. g > a p .a rg g ln c g a c a a g -> a c o il ed -c o il d o m . [h o u ld en et al ., ] c. t > c p .c ys a rg a t g c g c t -> c c o il ed -c o il d o m . [b o n et ti et al ., ] c. c > a p .g ln l ys c a g a a g c -> a c o il ed -c o il d o m . [x ia o et al ., ] c. g > a p .a sp a sn g a c a a c g -> a [s ö h n et al ., ] (c on ti n u ed ) human mutation, vol. , no. , – , ta bl e . c on ti nu ed m u ta ti o n n am e n o m en cl at u re (p ro te in ) w il d t yp e co d o n m u ta n t co d o n m u ta ti o n al ev en t a m in o ac id p o si ti o n e xo n n o . st ru ct u re h c d r el at iv es in th e d at ab as e r ef er en ce n o . n o n se n se m u ta ti o n s c. c > t p .g ln x c a g t a g c -> t l -t h a p d o m . [h o u ld en et al ., ] c. c > t p .a rg x c g a t g a c -> t l -t h a p d o m . d n a b in d in g [b re ss m an et al ., ] c. t > g p .t yr x t a t t a g t -> g l -t h a p d o m . d n a b in d in g [h o u ld en et al ., ] c. c > t p .g ln x c a g t a g c -> t [s ö h n et al ., ] s m al lo u t- o f- fr am e d el et io n s c. d el t p .m et ? a t g d el b st o p at t h a p d o m . [b re ss m an et al ., ] c. _ d el p .a la g lu fs x g c c d el b st o p at l -t h a p d o m . [c lo t et al ., ] c. d el t p .p h e l eu fs x t t t d el c st o p at t h a p d o m . d n a b in d in g [h o u ld en et al ., ] c. _ d el a g p .g lu v al fs x g a g d el b st o p at l -t h a p d o m . [s ö h n et al ., ] c. d el c p .t h r l ys fs x a c a d el b st o p at a h -t h a p d o m . a v p t if m o ti f [h o u ld en et al ., ] c. _ d el c t p .p ro a rg fs x c c t d el b st o p at [b la n ch ar d et al ., ] c. _ d el t c p .v al p h ef sx t c a d el a st o p at [s ö h n et al ., ] c. _ d el t c b p .v al p h ef sx t c a d el a st o p at [d ja rm at i et al ., ] c. _ d el p .a rg a sp fs x c g g d el a st o p at n l s [c lo t et al ., ] c. d el c p .g ln se rf sx c a g d el a st o p at n l s [b re ss m an et al ., ] c. d el a b p .l ys a sn fs x a a a d el c st o p at n l s [d ja rm at i et al ., ] s m al li n -f ra m e d el et io n c. _ d el c a a p .a sn _ a sn d el a a c d el c in -f ra m e d el l -t h a p d o m . [g ro en et al ., ] c. _ d el c a a p .a sn _ a sn d el a a c d el c in -f ra m e d el l -t h a p d o m . [c lo t et al ., ] s m al lo u t- o f- fr am e in se rt io n c. d u p p .a rg l ys fs x a g g in s b st o p at c o il ed -c o il d o m . [b la n ch ar d et al ., ] f o r ea ch m u ta ti o n ,t h e n u cl eo ti d e an d p ro te in n am es ar e gi ve n ac co rd in g to th e n o m en cl at u re gu id el in es (h tt p :/ /w w w .h gv s. o rg /) . a n o te th at th e c. _ in sg g g t t ; _ d el a a c a m is h m u ta ti o n h as b ee n re n am ed in c. _ d el in sg g g t t t a to fo ll o w th e gu id el in e fo r “i n sd el ” m u ta ti o n (h tt p :/ /w w w .h gv s. o rg /) . b sa m e p at ie n t d es cr ib ed in th e tw o gi ve n re fe re n ce s; “t h a p d o m .” :t h a p d o m ai n ; “l -t h a p d o m .” :t h a p -d o m ai n lo o p ; “l -t h a p d o m .” :t h a p -d o m ai n lo o p ; “l -t h a p d o m .” :t h a p -d o m ai n lo o p ; “l -t h a p d o m .” :t h a p -d o m ai n lo o p ; “a h -t h a p d o m .” :t h a p -d o m ai n α -h el ix ; “h -t h a p d o m .” :t h a p -d o m ai n h el ix ; “h -t h a p d o m .” :t h a p -d o m ai n h el ix ; “h -t h a p d o m .” :t h a p -d o m ai n h el ix ; “c o il ed -c o il d o m .” :c o il ed -c o il d o m ai n ; “n l s” :n u cl ea r lo ca li za ti o n se q u en ce ; “d n a b in d in g -” :a m in o ac id s p ro ve n to b e in vo lv ed in d n a -b in d in g ac ti vi ty ; “c c h m o ti f” :a m in o ac id s in vo lv ed in th e zi n c- d ep en d en t d n a -b in d in g ac ti vi ty o f t h a p th ro u gh th e c c h m o ti f; “h c f - b in d in g” :a m in o ac id s in vo lv ed in th e in te ra ct io n w it h h c f - ; “a a in te ra ct io n s” :i n te ra ct io n s b et w ee n t h a p am in o ac id s as d es cr ib ed in f ig u re b .e xa m p le :t h a p am in o ac id is in vo lv ed in in te ra ct io n s w it h t h a p am in o ac id s , ,a n d . f o r ea ch p ro b an d ,t h e n u m b er o f re la ti ve s in cl u d ed in th e d at ab as e is gi ve n . human mutation, vol. , no. , – , amino acid: exon nucleotide: exon exon thap domain proline-rich region nuclear localization sequence coiled-coil domain l l l l b b h h h h b : mvqscsaygcknrydkdkpvsfhkfpl rpslckeweaavrrknfkptkyssicsehftpdcfkrecnnkllkenavptift c c k p r w r f t c hf p k l e v y no binding activity: binding activity diminished: known amino acid interactions: a: thap domain amino acid: thap b thap a amino acid: s s y k pslcke r kn kp k ss st no change in activity when mutated to ala: loop beta strand alpha helice thap b : figure . thap and its thap domain. a: schematic representation of the thap gene and its two isoforms. isoform (thap a) and isoform (thap b) result from alternative splicing of exon ; b : schematic representation of the thap domain with amino acid positions for each structure. the upper figure represents the α-helix h , the three helices (h , h , and h ) (all in orange), the two β-sheets (b and b ) (in red), and the four loops (l to l , in gray). “no binding activity”: when these amino acids of the thap domain are mutated to alanine, thap shows loss of zinc-dependent dna-binding activity; amino acids that are part of the c ch motif are in red; “binding activity diminished”: when these amino acids are mutated to alanine, there is partial loss of dna-binding activity; “no binding activity modification when mutated to ala”: mutation of these amino acids to alanine does not change the dna-binding activity of thap [bessiere et al., ; clouaire et al., ]; “known amino acid interactions”: structural representation of the known amino acid interactions [bessiere et al., ]; b : topology diagram of the thap domain of human thap showing the secondary structure elements: the α-helix h , the three helices (h , h , and h ) (all in orange), the two β-sheets (b and b ) (in red), and the location of the four loops (l to l ). the zinc finger and the four ligands are shown in gray. modified from bessiere et al. [ ]. exhibits some specific features (fig. b ). it is characterized by a c ch signature (cys-x – -cys-x – -cys-x -his) associated with four invariant residues (pro , trp , phe , pro ) in the thap sequence. direct mutagenesis of each of these eight amino acids showed their critical identity for the zinc-dependent specific binding of the thap domain to a precise dna sequence (txxxggca: thabs consensus sequence) [clouaire et al., ] (fig. b ). an avptif box (ala -phe , in the thap sequence), also essential for dna binding, is located at the c-terminus of the thap domain. the nmr solution structure of the thap zinc finger of thap [bessiere et al., ] indicates that this domain consists of a first loop (l : gln -ser ), followed by two short antiparallel β-sheets (b : phe -lys and b : ser -cys ) separated by a loop (l : phe -lys )-helix (h : cys -val )-loop (l : arg -ser ) struc- ture (fig. b ). the four residues of the c ch motif (cys -cys - cys -h ) are three-dimensionally brought together to form the zinc-dependent dna-binding site. after the second β-strand, there are three helices: h (ser -his ), h (arg -phe ), and h (thr -phe ). h contains part of the avptif motif and is pre- ceded by a last mobile loop (l : lys -pro ). the alpha-helix h can be considered as an important inter- action hub and it is essential for the maintenance of the three- dimensional ( -d) shape of the thap domain (fig. b ). indeed, it makes contacts with many residues distributed throughout the do- main allowing a spatial connection between h (trp , ala , val ), l (phe , pro , arg ), l (phe ), ∼h (phe ), h (phe ), l (pro ), and h (phe ). residue trp , which is located in the core of h , seems to be a key amino acid for this function. it makes hydrophobic contacts with four amino acids (phe , arg , phe , and phe ), and nmr data strongly suggest the presence of interac- tion also with the two highly conserved prolines (pro and pro ). four of these residues have been functionally tested (pro , arg , phe , pro ) and their replacement by alanine strongly decreases the thap domain ability to bind to dna, suggesting that they have a critical role in the maintenance of thap -d shape [clouaire et al., ]. the -d structure of the dna–thap domain complex has re- cently been solved [campagne et al., ], showing that the thap domain contacts dna by insertion of its two antiparallel β-sheets into the major dna groove. this ensures the connection of the second half of loop l (residues lys to ser ) with the core of the thabs sequence. upon binding to dna, the folding of thap is changed, enabling loop l to contact the dna minor groove. based on their nonconservation in the thap family, specific base-contact with the core of the thabs motif and chemical behavior with non- specific dna, some thap domain residues have been proposed to play a crucial role in the specificity of dna recognition: tyr and ser of loop l , ser of b , gln and ser of the n-terminal tail and, finally arg of l . other residues are involved in the nonspecific interaction with dna, but their role remains absolutely necessary for the targeting of thap to the thabs sequence. for example, thr , which is located in the middle of the dna-interaction area of loop l , shows a chemical shift perturbation when the thap domain is incubated with an unrelated dna sequence, suggesting that thr is involved in nonspecific interactions. however, muta- tion of this residue led to total loss of the dna-binding activity of the thap domain and it has been proposed that, with lys , it may be important for positioning the protein onto dna. human mutation, vol. , no. , – , table . clinical characteristics of carriers of thap mutations amish patients a n = non-amish patients n = all patients b n = sex: f ( %) f c ( %) f c ( %) age at onset (y): median . ( – ) median ( – ) median ( – ) age at last examination (y): median ( – ) median ( – ) median ( – ) family history: % . % . % site at onset: patients patientsd patientsd upper limb ( %)e ( %)e ( %) lower limb ( %)e ( %)e ( %) cervical ( %)e ( %)e ( %) cranial ( %) ( %) ( %) site at examination: patients patients patients upper limb ( %) ( %) ( %) lower limb ( %) ( %) ( %) cervical ( %) ( %) ( %) cranial ( %)e ( %)e ( %) speech ( %)e ( %)e,f ( %)e,f distribution: patients patients patients fd: ( %) fd: ( %) fd: ( %) sd: ( %) sd: ( %) sd: ( %) md: ( %) md: ( %) md: ( %) gd: ( %) gd: ( %) gd: ( %) gender: f, female; m, male. dystonia distribution: “gd”: generalized dystonia; “fd”: focal dystonia; “md”: multifocal dystonia; “sd”: segmental dystonia. a four families with common ancestry, patients. data from fuchs et al. [söhn et al., ]. b thirty-five families, sporadic, total patients. c sex is unknown for one patient from söhn et al. [söhn et al., ]. d for some patients, data were unavailable [bressman et al., ; gavarini et al., ; kaiser et al., ]. e data were estimated from bressman et al. [ ] and fuchs et al. [ ] when possible. f of note, two patients from groen et al. [ ] have oromandibular dystonia without knowing if there was speech disturbance or not. thap is involved in dyt dystonia dyt dystonia has been first described in the genetically iso- lated amish–mennonite population [almasy et al., ] as an autosomal dominant trait with incomplete penetrance. in two amish–mennonite families, the site of onset of the disease was lo- calized with similar frequency to upper limb, cervical, or cranial muscles and symptoms began between and years of age (aver- age . ) with a tendency to progressive involvement of other body regions. hence, dyt dystonia was named “idiopathic torsion dys- tonia of ‘mixed’ type.” these families were large enough to identify by linkage analysis the morbid locus, which maps in both families to chromosome p - . the locus was further narrowed by the discovery of other affected amish–mennonite families [saunders-pullman et al., ] and, in , fuchs et al. [ ] demonstrated the involvement of a thap mutation in dyt dystonia: an insertion/deletion founder muta- tion found in four related amish–mennonite families. a point mu- tation was then identified in an unrelated non-amish–mennonite german family (family s, c. t>c), indicating that dyt dysto- nia was present also in populations of different ancestry. indeed, the complex insertion/deletion mutation was later identified also in a german family with no known amish–mennonites origin but sharing the same haplotype [bressman et al., ]. several teams have reported to date different mutations in the coding sequence of thap in families ( probands and rela- tives) of american [bressman et al., ; xiao et al., ], chinese [cheng et al., ; xiao et al., ], czech [jech et al., ], dutch [groen et al., ], english [houlden et al., ], french [blanchard et al., ; clot et al., ; houlden et al., ], german [bressman et al., ; djarmati et al., ; fuchs et al., ; houlden et al., ; söhn et al., ; zittel et al., ], greek [bonetti et al., ; houlden et al., ], iranian [schneider et al., ], jewish [houlden et al., ], mauritian/indian [houlden et al., ], spanish [paisan-ruiz et al., ], and brazilian [de carvalho aguiar et al., ] ancestry. unfortunately, clinical data are not available for probands implemented in the database [bressman et al., ; gavarini et al., ; kaiser et al., ] and insufficient clinical and molecular data for relatives do not allow to implement them in the database [bressman et al., ]. one of the mutation has been described in an asymptomatic individual control (c. _ delag) [söhn et al., ]. the enlargement of the group of patients with thap muta- tions ( up to now without the asymptomatic carrier) has pro- vided new data for delineating the phenotype of dyt dystonia, which nevertheless has hardly changed since the original description (table ). dyt dystonia has been reported slightly more in females than males. the age distribution at onset is broad, ranging from to years (mean . years [calculated from patients with available data]). onset before years of age occurs in . % of the patients, and onset after years of age in one-sixth ( %) of the patients. in almost half ( %) of the patients, the first symptoms of dystonia concern an upper limb, while cranial or cervical onset is almost equally observed in the other half of the patients. onset in a lower limb is less common ( / patients reported in the litera- ture). dyt dystonia tends to spread slowly to other body regions. although the symptom distribution varies widely, the involvement of the cranial region is frequent, and disability mainly results from cranial dystonia with speech difficulties. at last follow-up, almost half of the reported patients with dyt dystonia suffered from gen- eralized dystonia, but most of them remained ambulatory. to date, patients underwent deep brain stimulation; one good response is reported in one patient [jech et al., ], although deep brain stimulation (dbs) resulted in only mild or moderate improvement human mutation, vol. , no. , – , in the other patients, with poor effect on speech or dysarthria [clot et al., ; djarmati et al., ; groen et al., ; jech et al., ; zittel et al., ]. early onset in a limb and progressive generalization in few years are shared features between dyt and dyt dystonias [valente and albanese, ]. nevertheless, several differences emerge. in dyt dystonia, cervical or cranial muscles are involved most of the time, whereas lower limbs are frequently spared. the dyt clinical spectrum is more variable, and some adult-onset cases with stable focal dystonia are reported as well. onset (mean age) is somewhat later and with a wider age range in dyt than in dyt patients. the penetrance in dyt is difficult to evaluate; it was performed only in the amish–mennonite population (around % [saunders- pullman et al., ]). recently, tor a has been demonstrated as a direct target of thap by emsa, chip, and luciferase reporter gene assays [gavarini et al., ; kaiser et al., ]. specific modulation of torsina expression was not reported in nonneuronal cells after thap knockdown or overexpression, nor in fibroblasts or lym- phoblast cells from dyt patients [gavarini et al., ; kaiser et al., ]. the lack of effect could be explained by tissue and/or developmental stage specificity. however, thap is predicted to po- tentially regulate numerous gene targets [data not published] and tor a deregulation in dyt patients should then be responsible of only one part of the phenotype. nevertheless, human wild-type torsina can lead to a cellular dysfunction of mice neurons when overexpressed at high levels (abundant inclusion-like structures, ab- normalities of the ne ultrastructure, behavioral abnormalities, sig- nificant reduction of dopamine levels, serotonin, and homoranillic acid) as shown in transgenic mice line hwt [grundmann et al., ]. the umd-thap database we have used the universal mutation database (umd) software [beroud et al., , ] to create a computerized locus-specific database that contains information about the published mutations of the thap gene. codons were numbered (e.g., + = a of atg) based on the cdna sequence of the thap variant (isoform with three exons) (accession number nm_ . , “homo sapiens thap domain containing, apoptosis associated protein (thap ), transcript variant , mrna”) obtained from the genbank database. intron–exon boundaries as well as intronic sequences were defined by matching the cdna sequence to the corresponding genome sequence (nt_ . ) and to the module organization from swissprot (accession number q nvv ). the database follows the guidelines on mutation databases of the hugo mutation database initiative including the latest nomenclature (http://www.hgvs.org/). the thap mutation database can list point mutations, large and small deletions, insertions, indels, and mutations affecting splicing (intronic mutations) in the thap gene. it cannot accommodate mutations from the utr and promotor regions. in addition, two mutations that affect the same allele are entered as two different records linked by the same sample id. it allows to link specific poly- morphisms identified in the course of sequencing to one mutation for a specific patient. separate entries are made for each probands and for each relative (with different sample id) in order to de- scribe specific phenotypes. if the same mutation in a single patient has been reported in different papers (example: the same patients , l- , l- , and l- were first reported in [djarmati et al., ] and subsequently in [zittel et al., ]), only one entry was made. if the same mutation has been reported in apparently unrelated patients (example c. _ delcaa identified in french and dutch patients [clot et al., ; groen et al., ]), separate entries were made for each patient as recurrent mutations, in the absence of haplotypes demonstrating common ancestor. for each mutation, different types of information are provided: genetic (exon and codon number, wild type and mutant codon, mutational event, mutation name), protein (wild type and mutant amino acids, af- fected domain, mutation name), clinical data (age of onset, site of onset, dystonia distribution, sites involved, genealogic tree when available), and experimental data. we have annotated the thap sequence with indirect arguments in order to determine whether potential missense mutations are really causative using the umd-predictor r© tool [frederic et al., ]. the umd-predictor r© tool is dedicated to the analysis of nucleotide substitutions in cdna sequences. it provides a com- binatorial approach that includes localization of the mutation in the protein, conservation, biochemical properties of the mutant and wild type residue, and potential impact of the variation on mrna (http://www.umd.be). the indirect arguments, listed in the “highly conserved domain” (hcd) of umd-predictor r© are: ( ) amino acids involved in the zinc-dependent dna-binding activ- ity of thap through the c ch motif [clouaire et al., ]; ( ) amino acids involved in dna binding of the thap domain of hu- man thap [bessiere et al., ; clouaire et al., ]; ( ) amino acids involved in the interaction with hcf- (hbm consensus se- quence) [mazars et al., ]; and ( ) thap amino acids involved in interactions with other thap amino acids [bessiere et al., ] (fig. b ). mutations that potentially affect splicing can be highlighted with dedicated algorithms that have been recently implemented in the human splicing finder (hsf) tool [desmet et al., ]. hsf is also available for any gene as a web resource (http://www.umd.be/hsf/) and is dedicated to the prediction of the impact of a mutation on splicing signals and/or to the identification of these motifs in any human sequence. it contains all available matrices for auxiliary sequences prediction, as well as new position weight matrices to evaluate the strength of ′ and ′ splice sites and of branch point sequences. the umd-thap database can be used with different routines described at http://www.umd.be/thap /. it is possible to analyze the mutation distribution by exon or by mutation type and the mechanism for deletion/insertion. with the “hcd” annotation, it is possible to know whether an amino acid has a known reported function in the protein. the umd software has already been used successfully since for the analysis of multiple genes implicated in genetic diseases, such as ldlr [villeger et al., ], tgfbr [frederic et al., ], dys [krahn et al., ], and in cancer, such as tp [cariello et al., a, b; hamroun et al., ] or men [wautot et al., ]. twenty-nine umds are available through the web. more information concerning the umd software is available at http://www.umd.be. the current database and the ensuing updated versions are avail- able online at http://www.umd.be. notifications of omissions and errors in the current version as well as specific phenotypic data will be gratefully received by the corresponding author. the software will be expanded as the database grows and according to the users’ requirements, and new functions may be implemented. mutation analysis to date, a total of different thap mutations are known (table ) since two of the three patients and their specific mutation reported by zittel et al. [ ] were already described by djarmati human mutation, vol. , no. , – , et al. [ ], and the patient reported by van gerpen et al. [ ] is part of the cohort described by xiao et al. [ ]. these mutations are mainly private, missense, and widely distributed throughout the gene. only three apparently recurrent mutations have been de- scribed. for the c. g>a, described in two patients by paisan-ruiz et al. [ ], no other polymorphism has been described ruling out common ancestor [coro paisan-ruiz, personal communication]. the c. _ deltc mutation [djarmati et al., ; söhn et al., ] and the c. _ delcaa mutation [clot et al., ; groen et al., ] have been characterized by different teams, but until haplotype analyses become available, it is unclear whether these are truly recurrent mutations or whether they result from a founder effect. these mutations have then been considered as recurrent. it is noteworthy that two mutations, c. a>g [houlden et al., ] and c. t>a [schneider et al., ] have been found to be ho- mozygous. insdel mutations the heterozygous amish mutation, described as a -base (gggtt) insertion followed by a -base deletion (aac) (c. _ insgggtt; _ delaac) [fuchs et al., ], has been detected in four related amish–mennonite families (fami- lies m, c, r, and w). this mutation was later identified also in a german family without known amish–mennonite ancestry (fam- ily ) but sharing the amish haplotype [bressman et al., ]. this patient has then been implemented as a relative. to follow the international guideline for “insdel” mutation nomenclature (http://www.hgvs.org/), the name of the mutation has been cor- rected to c. _ delinsgggttta, that is deletion of five bases (taaac) and insertion of seven bases (gggttta), which results in the same predicted mutant protein. nonsense and missense mutation four reported mutations are nonsense mutation: c. c>t [houlden et al., ], c. c>t [bressman et al., ], c. t>g [houlden et al., ], and c. c>t [söhn et al., ]. thirty- seven are missense mutations. five mutations are predicted to im- pair thap dna-binding activity: c. c>g [houlden et al., ]; the two recurrent mutations c. g>a [paisan-ruiz et al., ]; c. g>c [bressman et al., ]; and c. c>a in the c ch motif [söhn et al., ]. three mutations are located in the sequence involved in thap binding to hcf- : c. a>g [houlden et al., ], c. c>g [groen et al., ], and c. a>g [söhn et al., ]. one mutation (c. t>c) is located in the avptif motif [fuchs et al., ]. the homozygous mutation c. a>g [houlden et al., ] creates a potential acceptor splice site in exon , but its strength and the presence of many upstream good candidate accep- tor splice sites make it unlikely that it will be used. the c. a>g mutation [bressman et al., ] is located in the penultimate nucleotide of exon . this mutation does not impair the donor acceptor splice site, neither auxiliary splicing sequences (exonic sequence enhancer [ese] or silencer [ess]). for the remaining mutations, the mutated codon does not seem to be clearly involved in a particular function. among all the missense mutations, only nine were not predicted by the umd- predictor r© tool to be pathogenic based on a combinatorial ap- proach [frederic et al., ] (table ): c. g>a [zittel et al., ], c. t>a [bressman et al., ], c. c>a [söhn et al., ], c. t>c [fuchs et al., ], c. a>g [bressman et al., ], c. a>g [söhn et al., ], c. g>a [houlden et al., ], c. c>a [xiao et al., ], and c. g>a [söhn et al., ]. nevertheless, mutation c. t>c, reported in dbsnp without frequency data, shows a decreased dna-binding activity in vitro [fuchs et al., ]. emsa experiments for c. t>a show that the mutation abolishes binding of thap to tor a [bressman et al., ; gavarini et al., ], while mutation c. g>a is shown to significantly reduce thap -mediated repression of tor a ( % of residual activity)[kaiser et al., ; zittel et al., ]. umd-predictor r© tool has a very high positive predictive value and a high negative predictive value. mutations experimentally demonstrated by gel-shift assays (table ) [bessiere et al., ; clouaire et al., ] to have ( ) no binding activity (p.cys ala, p.cys ala, p.lys ala, p.pro ala, p.arg ala, p.trp ala, p.arg ala, p.phe ala, p.thr ala, p.cys ala, p.his ala, p.phe ala, and p.pro ala) are all predicted as pathogenic; ( ) a diminished binding activity were also all predicted as pathogenic (p.glu ala, p.lys ala, p.leu ala, and p.tyr ala) or proba- bly pathogenic (p.val ala); ( ) no change in activity were ei- ther predicted: as pathogenic (p.ser ala, p.tyr ala, p.lys ala, p.leu ala, p.arg ala, p.asn ala, p.lys ala, p.lys ala); as probably pathogenic (p.thr ala, p.pro ala, p.glu ala, p.lys ala, p.lys ala); as polymorphism (p.ser ala); or as prob- able polymorphism (p.ser ala, p.pro ala, p.ser ala, p.cys ala, p.ser ala, p.ser ala). nevertheless, these gel-shift assays demon- strate no modification of the dna-binding activity “within the lim- its of detection of the present assay” and do not exclude “a role in the binding affinity and selectivity” [bessiere et al., ]. amino acids ser , ser , and ser are described to be involved in the specificity of dna recognition and lys proposed, with trp , to be impor- tant for positioning the protein onto dna [campagne et al., ]. therefore, the predictions are in full agreement with in vitro stud- ies for pathogenic mutations ( / ). for the alanine substitutions classified as “non modifying binding activities,” some discrepancies are observed but their interpretation is difficult as they could result from wrong predictions by umd-predictor or wrong interpretation of experimental data. these experimental results show that in spite of the high efficiency of the umd-predictor tool [frederic et al., ] and other predic- tion tools [sift, polyphen, data not shown] for several genes (fbn , fbn , tgfbr , tgfbr [frederic et al., ], dys [krahn et al., ], cdkn a [kannengiesser et al. ], and alas [ducamp et al. ]), more functional data will have to be collected to im- prove the prediction of missense variations in thap gene. insertion/duplication only one reported mutation is an insertion that corresponds to the duplication of a single base (a ). this insertion is predicted to create a premature termination codon (ptc), codon seven amino acids downstream of the mutation (position ) [blanchard et al., ]. deletions among the small deletions, create a ptc and two are in-frame deletions resulting in loss of the asn residues (c. _ delcaa). two single-base deletions, c. delt [houlden et al., ] and c. dela [djarmati et al., ], might be the result of slipped mispairing. five mutations are deletions of a re- peated sequence: c. _ delag [söhn et al., ], the recurrent mutations c. _ deltc [djarmati et al., ; söhn et al., ] and c. _ delcaa [clot et al., ; groen et al., ]. one mutation, c. _ del, is flanked by direct repeats [clot et al., ]. for the five other mutations, the mechanisms are unknown human mutation, vol. , no. , – , ta bl e . a na ly si s of m is se ns e m ut at io ns w it h th e u m d -p re di ct or r© to ol n o m en cl at u re c. n o m en cl at u re p . st ru ct u re h c d c o n se rv at io n si f t p ro b ab il it y b l o su m b io ch em ic al va lu es e se m o d ifi ca ti o n sp li ce si te p at h o ge n ic it y c o n cl u si o n c. c > t p .s er p h e l -t h a p d o m . . . - . . n o im p ac t p at h o ge n ic c. a > g p .t yr c ys l -t h a p d o m . . . - . . pa ss c [ . ] p at h o ge n ic c. g > t p .g ly c ys l -t h a p d o m . . . - . . n o im p ac t p at h o ge n ic c. a > g p .a sp g ly l -t h a p d o m . . . - . . n o im p ac t p at h o ge n ic c. a > c p .h is p ro b s -t h a p d o m . . . - . . n o im p ac t p at h o ge n ic c. c > g p .p ro a rg l -t h a p d o m . d n a b in d in g - . . - . . -s c [ . ] n o im p ac t p at h o ge n ic c. g > c p .a rg p ro l -t h a p d o m . d n a b in d in g - . . - . . pa ss c [ . ] p at h o ge n ic c. c > g p .p ro a rg l -t h a p d o m . . - . . n o im p ac t p at h o ge n ic c. t > a p .l eu h is l -t h a p d o m l -t h a p d o m . . - . . n o im p ac t p at h o ge n ic c. g > a p .a la t h r a h -t h a p d o m . l -t h a p d o m . . . . -s f /a sf [ . ] n o im p ac t p at h o ge n ic c. g > a a p .c ys t yr b s -t h a p d o m c c h m o ti f. . . - . . n o im p ac t p at h o ge n ic c. t > g p .l eu a rg l -t h a p d o m . . . - . . n o im p ac t p at h o ge n ic c. a > t p .a sn il e l -t h a p d o m . . . - . . n o im p ac t p at h o ge n ic c. t > c p .c ys a rg . . - . . n o im p ac t p at h o ge n ic c. t > c p .p h e se r . - . . n o im p ac t p at h o ge n ic c. c > g p .a sn l ys h c f - b in d in g . . . n o im p ac t p at h o ge n ic c. a > g p .t yr c ys h c f - b in d in g . - . . n o im p ac t p at h o ge n ic c. t > c p .i le t h r n l s . . - . . n o im p ac t p at h o ge n ic c. a > c p .h is p ro n l s . . - . . n o im p ac t p at h o ge n ic c. t > c p .c ys a rg c o il ed -c o il d o m . . . - . . n o im p ac t p at h o ge n ic c. a > g p .m et ? t h a p d o m . . . . . n o im p ac t p ro b ab ly p at h o ge n ic c. t > c p .s er p ro l -t h a p d o m . . . - . . n o im p ac t p ro b ab ly p at h o ge n ic c. c > a p .a sn l ys l -t h a p d o m . . . . . n o im p ac t p ro b ab ly p at h o ge n ic c. g > a p .a rg g ln l -t h a p d o m . d n a b in d in g - . . . . pa ss c [ . ] p ro b ab ly p at h o ge n ic c. c > t p .t h r il e t h a p d o m . . . - . . n o im p ac t p ro b ab ly p at h o ge n ic c. a > g p .a sn se r h c f - b in d in g . . . p a s s c [ . ] p ro b ab ly p at h o ge n ic c. g > a p .a la t h r c o il ed -c o il d o m . . . . . n o im p ac t p ro b ab ly p at h o ge n ic c. g > a a p .a rg h is l -t h a p d o m . . . . . n o im p ac t p ro b ab le p o ly m o rp h is m c. c > a p .h is a sn a h -t h a p d o m . c c h m o ti f . . . . n o im p ac t p ro b ab le p o ly m o rp h is m c. t > c a p .p h e l eu a h -t h a p d o m . a a in te ra ct io n s . . . . n o im p ac t p ro b ab le p o ly m o rp h is m c. t > a a p .s er t h r l -t h a p d o m . . . . . n o im p ac t p o ly m o rp h is m c. a > g p .l ys a rg . . . . p a s s c [ . ] p o ly m o rp h is m c. a > g p .m et v al c o il ed -c o il d o m . . . . . n o im p ac t p o ly m o rp h is m c. g > a p .a rg g ln c o il ed -c o il d o m . . . . . n o im p ac t p o ly m o rp h is m c. c > a p .g ln l ys c o il ed -c o il d o m . . . . . pa ss c [ . ] p o ly m o rp h is m c. g > a p .a sp a sn . . . . n o im p ac t p o ly m o rp h is m t h e u m d -p re d ic to r r© to o l co m p u te s al l el em en ts (s tr u ct u re ,b io ch em is tr y, sp li ci n g, an d co n se rv at io n ) an d p ro vi d es fo r ea ch o f th em a sp ec ifi c st re n gt h b as ed o n it s re la ti ve im p ac t. “c o n se rv at io n ”: t h e sc o re ra n ge s fr o m fo r n o n co n se rv ed to fo r fu ll y co n se rv ed . “s if t ”: (s o rt in g in to le ra n t fr o m to le ra n t) :u se s se q u en ce h o m o lo gy to p re d ic t w h et h er an am in o ac id su b st it u ti o n w il la ff ec t p ro te in fu n ct io n an d ,h en ce ,p o te n ti al ly co n fe r a p h en o ty p e [n g an d h en ik o ff , ]. sc o re s th at ar e ≤ . ar e co n si d er ed to b e d el et er io u s (i n b o ld ). “b l o su m ” is an am in o ac id su b st it u ti o n m at ri x b as ed o n lo ca l m u lt ip le al ig n m en ts o f an u n se le ct ed p ro te in se t o f re la te d se q u en ce s [h en ik o ff an d h en ik o ff , ]. p o si ti ve sc o re s ar e as so ci at ed w it h co n se rv at iv e ch an ge s an d n eg at iv e sc o re s (i n b o ld ) w it h le ss co n se rv at iv e ch an ge s. “b io ch em ic al va lu e” is an am in o ac id su b st it u ti o n m at ri x d ep en d in g o n q u al it at iv e p h ys ic o ch em ic al p ro p er ti es d es cr ib in g si d e ch ai n st ru ct u re an d fu n ct io n al gr o u p s, o p ti ca l p ro p er ti es ,h yd ro p h o b ic it y/ ch ar ge /a ci d – b as e p ro p er ti es ,a n d si ze (v o lu m e an d si d e ch ai n le n gt h ) [y u , ]. a va lu e b el o w . d efi n es an in va li d su b st it u ti o n co n ce rn in g th e p h ys ic o -c h em ic al p ro p er ti es (i n b o ld ). “e se ” (e xo n ic sp li ci n g en h an ce r) an d “s p li ce si te ” m o d ifi ca ti o n s ar e ev al u at ed b y th e u m d to o l. “p a ss c ”: p o te n ti al ac ce p to r sp li ce si te cr ea te d ;p o te n ti al d el et er io u s m o d ifi ca ti o n s ar e sh o w n in b o ld . “p at h o ge n ic it y” :t h e u m d -p re d ic to r r© to o l co m p u te s al l th e el em en ts (s tr u ct u re ,b io ch em is tr y, sp li ci n g, an d co n se rv at io n ) p ro vi d in g fo r ea ch a sp ec ifi c st re n gt h b as ed o n th ei r re la ti ve im p ac t. p re d ic ti o n s ar e n o rm al iz ed o n a sc al e fr o m to . in “c o n cl u si o n ”: a va lu e o f le ss th an is as so ci at ed w it h th e p re d ic ti o n o f a n o n p at h o ge n ic m u ta ti o n an n o ta te d as “p o ly m o rp h is m ”; a va lu e o f – is as so ci at ed w it h th e p re d ic ti o n o f a “p ro b ab le p o ly m o rp h is m ”; a va lu e o f – is as so ci at ed w it h th e p re d ic ti o n o f a “p ro b ab ly p at h o ge n ic ” m u ta ti o n ;w h il e a va lu e ab o ve is as so ci at ed w it h th e p re d ic ti o n o f a “p at h o ge n ic ” m u ta ti o n .f o r “s tr u ct u re ” an d “h c d ” d es cr ip ti o n s se e th e le ge n d s to t ab le .f o r m o re d et ai ls se e f re d er ic et al .[ ]. a t h es e m u ta ti o n s h av e b ee n st u d ie d in vi tr o [f u ch s et al ., ;g av ar in i et al ., ;k ai se r et al ., ]. human mutation, vol. , no. , – , and have to be determined by searching, among others, for the presence of quasi-palindromic sequences, inverted repeats, or sym- metric elements that facilitate the formation of secondary-structure intermediates [krawczak and cooper, ]. splice-site mutations only one splice site variation is described in the literature: c. - t>c (or c.ivs - t>c) in association with the missense mu- tation c. c>g [houlden et al., ]. the splice site variation has been analyzed with the umd algorithm implemented in hsf tool [desmet et al., ]. the software computed the variation of the consensus value (cv) of the mutant “splice site” (cv = . ) rel- ative to the wild-type site (cv = . ). as the variation between these values was not higher than %, it is highly probable that such cv variation does not disrupt the wild-type splice site and thus this variation corresponds to a polymorphism as suggested also by the authors [houlden et al., ]. the pathogenic mutation is thus likely to be c. c>g and consequently the c. - t>c (or c.ivs - t>c) polymorphism was associated to the c. c>g mutation in the database. conclusion the umd-thap database contains different mutations (ta- ble ) in probands and relatives. these mutations are mainly private, essentially missense, usually nonrecurrent, and widely dis- tributed in the thap domain and the rest of the thap gene. the currently reported thap mutations are mainly missense mu- tations ( . %, / ) and small out-of-frame deletions ( . %, / ), with a minority of other mutation types ( % of nonsense [ / ], . % of small in-frame deletions [ / ], . % of small out- of-frame insertions [ / ], and . % of complex mutations). to date, no clear genotype/phenotype relationship has been identified. this is not surprising when considering past experience with other disease genes, such as the fibrillin- gene (fbn ), for which many mutations were accumulated before the genotype/phenotype rela- tionships emerged. indeed, the umd-fbn database was created in [collod et al., ], and the first report about the correlations between genotype and phenotype was published in [faivre et al., ] and more followed [detaint et al., ; faivre et al., , , a, b, c; stheneur et al., ]. from this perspective, the umd-thap locus-specific database will facilitate the muta- tional analysis of thap gene as previously demonstrated for other genes. acknowledgments a.b. and v.e. are supported by phd studentships from mesr (ministère de l’enseignement supérieur et de la recherche). the research leading to these results has also received funding from the université montpellier , inserm, the french ministry of health (national phrc -a - ), amadys-lfcd, alliance france dystonie, lions club la grande motte, the french dystonia network, and from the european 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http://cmgm.stanford.edu/biochem / projects% /yu.pdf. zittel s, moll ck, bruggemann n, tadic v, hamel w, kasten m, lohmann k, lohnau t, winkler s, gerloff c, schonweiler r, hagenah j, klein c, munchau a, schneider sa. . clinical neuroimaging and electrophysiological assessment of three dyt dystonia families. mov disord : – . human mutation, vol. , no. , – , diabetologia ( ) : – doi . /s - - - article b. i. freedman . s. s. rich . m. m. sale . g. heiss . l. djoussé . j. s. pankow . m. a. province . d. c. rao . c. e. lewis . y. d. i. chen . s. r. beck . on behalf of the hypergen investigators genome-wide scans for heritability of fasting serum insulin and glucose concentrations in hypertensive families received: july / accepted: november / published online: march # springer-verlag abstract aims/hypothesis: the heritability of fasting serum insulin and glucose concentrations in non-diabetic members of multiplex hypertensive families is unknown. methods: we calculated the familial aggregation of fast- ing serum glucose and insulin concentrations and performed a genome-wide scan to assess whether quantitative trait loci contribute to these phenotypes in , non-diabetic individuals from , families enrolled in the hyperten- sion genetic epidemiology network (hypergen) in the family blood pressure program. results: the heritability (±se) of fasting serum insulin was . ± . in european americans and . ± . in african americans (p< . for both), after adjusting for age, sex, and bmi. a genome- wide scan for fasting serum insulin yielded a maximum log of the odds (lod) score of . on chromosome at cm (p= . ) in european americans, and an lod score of . on chromosome at cm (p= . ) in african americans. the heritability of fasting serum glu- cose was . ± . in the former and . ± . in the latter (p< . for both) after adjusting for age, sex and bmi. a genome-wide scan for fasting serum glucose re- vealed a maximum lod score of . on chromosome at cm (p= . ) in european americans. conclusions/ interpretation: these analyses demonstrate the marked her- itability of fasting serum insulin and glucose concentrations in families enriched for the presence of members with hy- pertension. they suggest that genes associated with fasting serum insulin concentration are present on chromosomes and , and that genes associated with fasting serum glucose concentration are on chromosome , in families enriched for hypertension. keywords african americans . essential hypertension . fasting blood sugar . heritability . linkage analysis . serum insulin concentration abbreviations aa: african american . ea: european american . fbpp: family blood pressure program . h : heritability . homa: homeostasis model assessment . hypergen: hypertension genetic epidemiology b. i. freedman (*) . m. m. sale department of internal medicine, sections on nephrology and endocrinology the wake forest university school of medicine, medical center boulevard, winston-salem, nc, - , usa e-mail: bfreedma@wfubmc.edu tel.: + - - fax: + - - s. s. rich . s. r. beck public health sciences wake forest university school of medicine, winston-salem, nc, usa m. m. sale center for human genomics wake forest university school of medicine, winston-salem, nc, usa g. heiss department of epidemiology, university of north carolina, chapel hill, nc, usa l. djoussé evans department of medicine, section of preventive medicine and epidemiology, boston university, boston, ma, usa j. s. pankow department of epidemiology, university of minnesota, minneapolis, mn, usa m. a. province . d. c. rao division of biostatistics washington university school of medicine, st. louis, mo, usa c. e. lewis department of preventive medicine, university of alabama, birmingham, al, usa y. d. i. chen department of internal medicine, cedars-sinai medical center, los angeles, ca, usa network . ibd: identity-by-descent . lod: log of the odds . nhlbi: national heart, lung and blood institute . solar: sequential oligogenic linkage analysis routines introduction hyperinsulinaemia is a major risk factor for the subsequent development of type diabetes mellitus [ , ]. fasting serum insulin concentrations are excellent estimates of insulin re- sistance and the resulting hyperinsulinaemia in population studies, and have been shown to aggregate in families [ ]. insulin resistance is associated with increased serum triglyc- eride levels, reduced high-density lipoprotein concentrations and reduced low-density lipoprotein particle size; these meta- bolic parameters predict increased cardiovascular morbidity and mortality [ , ]. the heritability (h ) and roles of inherited and envi- ronmental factors in causing hyperglycaemia and elevated serum insulin concentrations among hypertensive subjects remain unknown. in non-hypertensive populations, an ad- justed h value of . for fasting serum glucose [ ] and . for fasting serum insulin concentrations [ ] were re- ported in the framingham offspring study. similar high familial correlations for these prediabetic measures were reported in amish families [ ], the heritage family study [ ], and caucasian twins [ ]. elevated serum insulin and glucose concentrations represent an increased risk of developing overt type diabetes mellitus, with increased rates of cardiovascular morbidity and mortality [ ]. it is likely that both genetic and environmental factors determine fasting serum insulin and glucose concentrations in hypertensive subjects. the hypertension genetic epide- miology network (hypergen) is a family-based consor- tium seeking to identify the genes responsible for elevated blood pressure [ ]. we performed maximum likelihood var- iance component linkage analysis of fasting serum glucose and insulin concentrations and homeostasis model assess- ment (homa) to identify loci contributing to the variance of these traits in the non-diabetic relatives of families en- riched for the presence of essential hypertension. subjects and methods population participants in the hypergen network of the family blood pressure program (fbpp), sponsored by the national heart, lung and blood institute (nhlbi), were evaluated. hypergen study methods have previously been reported [ ]. in brief, family members were recruited from five clinical centers (framingham, ma; minneapolis, mn; salt lake city, ut; forsyth county, nc; and birmingham, al). participants provided written informed consent and the project was approved by the institutional review boards at all of the institutions. a sibship was ascertained if it had two or more siblings with hypertension (defined as blood pres- sure ≥ / or the use of anti-hypertensive medications), with an age at diagnosis of less than years. participants reporting a personal history of diabetes mellitus, having a fasting blood glucose concentration of . mmol/l or high- er, or being treated with insulin or oral hypoglycaemic agents were excluded from analyses. phenotyping morning fasting serum samples from study participants were collected in a resting state and run in du- plicate for insulin concentration on an automated immuno- assay instrument and its ultra-sensitive insulin kit (beckman coulter, fullerton, ca, usa) [ ]. the sensitivity of this assay is . mu/l ( . pmol/l) and the dynamic range is . – mu/l ( . – pmol/l). there is zero cross- reactivity with pro-insulin and c-peptide, % with bovine insulin, and % with porcine insulin. serum glucose concentrations were measured using elan glucose reagent (hexokinase method) [ ]. assay sensitiv- ity is . – . mmol/l and the detection limit . mmol/l is documented through the repetitive assay of a diluted serum control. the observed detection limit, calculated as two standard deviations of a -replicate within-run preci- sion study, is . mmol/l and is below the claimed limit of . mmol/l. homa was calculated as (fasting serum insulin*fasting serum glucose)/ . . genotyping genotyping was performed by the nhlbi- funded mammalian genotyping service. for additional information regarding the genotyping methods see the website of the center for medical genetics at the marsh- field medical research foundation http://www.research. marshfieldclinic.org/genetics/). the genome screen was performed by means of an automated technique with the scanning fluorescence detector. the cooperative human linkage center screening set , including microsatellite markers spaced approximately every cm throughout the genome, was used, with an average marker heterozygosity of . . statistical analysis the distributions of fasting serum in- sulin and glucose concentrations were positively skewed. the natural logarithm transformed both insulin and glucose to approximate normality and was used for all analyses and model building. any highly influential outliers were exclud- ed from the analyses after adjusting for covariates. pedigree and genotype data were screened for possible errors using aspex software, version . [ ], mapmaker/sibs, version . [ ], pedcheck, version . [ ], and prest, version . [ ]. the heritability of serum measures was estimated sep- arately in each race and jointly, using variance component modelling as implemented in solar software, version . . [ ]. covariates in the model for fasting glucose were age, sex, bmi, age , and age×sex. covariates in the model for fasting insulin were age, sex, bmi, age , age×sex, sex×bmi, and sex×age . these were selected using a backward elimi- nation approach allowing for re-entry of eliminated covari- ates at each step (significance level= . for backward and forward steps). for both analyses, covariates were selected among age, sex, and bmi, age , age×sex, age×bmi, sex× bmi, sex×age , and bmi×age . centred values were used to http://research.marshfieldclinic.org/genetics/ http://research.marshfieldclinic.org/genetics/ model the effects of continuous covariates, and indicator variables ( / ) were used for discrete covariates. the heri- tability estimate adjusted for the effects of covariates is re- ported together with corresponding estimates of standard error, p value, and proportion of variance due to covariates. we considered p values of . or less to be significant. markov chain monte carlo methods were used to esti- mate race-specific multipoint ibd matrices as implemented in the loki software package, version . . [ ]. variance component multipoint and bivariate linkage analysis as implemented in solar software, version . . [ ], was performed to detect and localise quantitative trait loci that influence variation in fasting serum insulin and glucose concentrations, using lodadj to account for slight departures from normality. this approach has been described in detail [ – ]. results genotype data were available from , individuals in recruited families who participated in the fbpp. eight sub- jects were excluded due to fasting serum glucose concentra- tions above . mmol/l (six aa, two ea). after correcting family relationships based upon the genetic data, , dis- tinct pedigrees ( of whom were african american) were used in the analysis. after exclusion of all influential outliers, , of the genotyped individuals who were nondiabetic had measurements of fasting serum insulin and/or glucose concentrations. the mean age (±sd) of these individuals was . ± . years, . % (n= , ) were women, % were aa (n= , ), and a majority were hypertensive ( . %) and had a mean bmi (±sd) of . ± . kg/m (table ). participants had a mean fasting serum glucose concentration table demographic charac- teristics of hypergen study population data listed as means±standard deviation (n) for continuous measures and % (n) for dichot- omous measures adefined as blood pressure > / mmhg or use of antihypertensive medication characteristic african american caucasian race combined sex female . ( ) . ( ) . ( , ) male . ( ) . ( ) . ( ) age, years . ± . ( , ) . ± . ( , ) . ± . ( , ) bmi, kg/m . ± . ( , ) . ± . ( , ) . ± . ( , ) fasting serum glucose, mmol/l . ± . ( , ) . ± . ( , ) . ± . ( , ) fasting serum insulin, mu/l . ± . ( , ) . ± . ( , ) . ± . ( , ) hypertensiona . ( ) . ( ) . ( , ) fig. genome scan plots for fasting serum insulin concentra- tions. dotted line african americans; solid line caucasians t a b le l in k ag e re su lt s: fa st in g in su li n , g lu co se , h o m a an al y si s an d b iv ar ia te an al y si s l o ca ti o n h o m a a f as ti n g in su li n a f as ti n g g lu co se a b iv ar ia te b m ar k er c h r a a e a a a e a a a e a a a e a d s / d s . @ . . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . . @ . d s . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . . @ . . @ . d s . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c . @ . . @ . . @ . . @ . d s / d s . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c d s / d s . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . . @ . . @ . ( . , . ) . c d s . @ . ( . , – ) . c . @ . . @ . . @ . . @ . . @ . . @ . . @ . d s . @ . ( . , . ) . c . @ . . @ . . @ . . @ . . @ . . @ . . @ . d s . @ . . @ . . @ . . @ . . @ . . @ . ( . ,– ) . c . @ . . @ . ( . ,– ) . c d s / d s . @ . . @ . . @ . . @ . . @ . . @ . ( . ,– ) . c . @ . . @ . d s . @ . . @ . . @ . . @ . . @ . . @ . . @ . ( . ,– ) . c . @ . d s / d s . @ . . @ . . @ . . @ . . @ . . @ . . @ . (– , . ) . c . @ . d s / d s . @ . . @ . . @ . . @ . . @ . (– , . ) . c . @ . . @ . (– , . ) . c . @ . d s / d s . @ . . @ . . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c d s . @ . . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . . @ . d s . @ . (– , . ) . c . @ . . @ . (– , . ) . c . @ . . @ . . @ . . @ . (– , . ) . c . @ . a a a fr ic an a m er ic an , e a e u ro p ea n a m er ic an , c h r ch ro m o so m e a s ig n if ic an t re su lt s re p o rt ed as m ax im u m l o d sc o re @ lo ca ti o n in ce n ti m o rg an s (l o d - in te rv al ) em p ir ic al p v al u e b s ig n if ic an t re su lt s re p o rt ed as m ax im u m l o d sc o re @ lo ca ti o n in ce n ti m o rg an s (l o d - in te rv al ) p v al u e c l o d - su p p o rt in te rv al an d p v al u es fo r m ax im u m l o d sc o re s ab o v e . of . ± . mmol/l and a mean fasting serum insulin con- centration of . ± . mu/l. among these , individuals, there were , sibling pairs ( aa), avuncular pairs ( aa), half-sibling pairs ( aa), first cousins ( aa), and ea monozygotic twins. the mean family size with insulin and/or glucose data was . members (aa . , ea . ). the h of serum insulin was . ± . in eas and . ± . in aas (p< . for both), after controlling for the significant main and interactive effects of age, sex and bmi. an additional % and % of the variance in eas and aas, respectively, was due to measured covariates. figure contains the univariate genome-wide scan results for fasting erum insulin concentration in eas and aas. a maximum lod score of . was observed on chromosome at . cm (marker d s /d s , p= . ), with lesser peaks of lod . on chromosome ( cm, marker d s ) and . on chromosome ( cm, marker d s ) in eas, and . on chromosome ( cm, marker d s ) in aas, p< . for all (fig. ). table contains the re- sults of the genome scan for fasting serum insulin (as well as for homa, fasting serum glucose and the bivariate analysis), reporting maximum lod scores of more than . , position, lod- interval and p value in aas and eas (see symbols and footnotes) and maximum lod scores with position in all other scans in proximity to the sig- nificant results. the results of the homa genome scan are presented in fig. and table . similar regions of linkage were observed for fasting serum insulin concentrations on chromosomes and in eas and on chromosome in aas. the h of serum glucose concentration was . ± . in eas and . ± . in aas (p≤ . for both), after controlling for the significant main and interactive effects of age, sex, and bmi. an additional % and % of the variance in eas and aas, respectively, was due to mea- sured covariates. the univariate genome-wide scan results for fasting serum glucose are depicted in fig. . a maxi- mum lod score of . was observed on chromosome at . cm (marker d s /d s , p= . ) in eas and a lesser peak was observed on chromosome : lod . at . cm (marker d s /d s ), p< . in aas (fig. ; table ). in aas, the genetic correlation between fasting serum glucose and insulin was nonsignificant (p= . ), while the environmental correlation was re= . ± . (p< . ). in eas, the genetic correlation between fasting serum glucose and insulin was also nonsignificant (p= . ), while the environmental correlation was re= . ± . (p= . ). these data suggest that the primary genetic determinants of fasting serum glucose concentration are different from those that contribute to the variation in fasting serum insulin concentration. a bivariate genome-wide scan for loci con- tributing to both fasting serum glucose and insulin con- centrations in both races demonstrated eight regions with maximum lod scores above . (four in aas and four fig. genome scan plots for homa. dotted line african americans; solid line caucasians in eas). these regions are depicted, separately by race, in fig. and table . discussion this report is the first to reveal that inherited factors appear to play important roles in the regulation of fasting serum insulin and glucose concentrations in hypertensive, nondia- betic aas and caucasians. the heritability of fasting serum insulin and glucose concentrations remained highly signif- icant even after controlling for the effects of significant covariates. the marked heritability of fasting serum glucose and insulin concentrations appears consistent with other reports in framingham [ , ], amish [ ], and heritage families [ ]. additionally, the genome scans in aas and eas provided suggestive evidence that genes regulating fasting serum insulin concentrations and insulin sensitivity (homa) were present on chromosomes , , , and , and genes regulating fasting glucose concentration were present on chromosomes , , , and . in general, the linkage peaks for fasting insulin, homa and fasting glu- cose differed by ethnic group (see table ). race-combined analyses added little, as results were driven by a single eth- nic group (data not shown). although the bivariate genome scan demonstrated several regions with suggestive evidence for linkage, these results were probably driven by linkage to either fasting serum insulin or fasting serum glucose con- centrations alone, based on the low genetic correlation ob- served between these measures. several genome-wide scans have been performed in non- diabetic families evaluated for prediabetic phenotypes and in multiplex type diabetes families. regions of linkage in several reports overlap with those observed in this hy- pergen analysis. on chromosome , linkage was detected at cm for fasting insulin in pima indians (lod . ) [ ], and for type diabetes and glucose intolerance in young-onset french families at cm (lod . ) [ ]. linkage with fasting glucose, and combined fasting and non-fasting glucose, was detected on chromosome at cm in the framingham offspring study (lod . ) [ ], and framingham heart study (lod . ) [ ]. the hy- pergen fasting serum glucose scan loci on chromosome are near reported loci for related phenotypes in finnish families from the fusion study [ ], i.e., -h insulin (lod . at cm) and insulin secretion (insulin/ glucose) (lod . at cm), and loci for type diabetes in aas with earlier age at diagnosis (p< . at cm) [ ] and japanese families (lod . , cm) [ ]. link- age for these phenotypes resides within, or near, the peaks where we found evidence for linkage to fasting insulin (chromosomes and ) and fasting glucose ( and ) in the univariate analyses. therefore, the regions identified in the hypergen genome scan as influencing fasting serum insulin and glucose concentrations may reflect genes that also play major roles in susceptibility to diabetes mellitus fig. genome scan plots for fasting serum glucose concen- trations. dotted line african americans; solid line caucasians and/or related phenotypes. our data are unique, however, in that they are from a biracial population enriched for the presence of essential hypertension. fasting serum glucose concentrations clearly fluctuate in individuals with insulin resistance and in those with diabe- tes mellitus. in addition, little is known about the natural history of insulin resistance among treated and untreated hypertensive subjects. these difficulties are encountered in all cross-sectional studies. the overlapping regions of link- age observed for the diabetes-related phenotypes in pima [ ] and finnish [ ] families, and the framingham off- spring [ ] and heart studies [ ], support the existence of genes affecting fasting serum insulin concentrations on chro- mosomes and and fasting serum glucose on chromo- some . in conclusion, this report analysed the heritability of fasting serum insulin and glucose concentrations in non- diabetic members of multiplex hypertensive families. ele- vated serum insulin concentrations and fasting blood sugars (insulin resistance) are well recognised risk factors for the development of heart attack and stroke. the heritabilities of fasting serum insulin and glucose concentrations were signif- icant after controlling for the main and interactive effects of age, sex, and bmi. additionally, suggestive evidence for genetic linkage to fasting serum insulin concentrations and insulin sensitivity (homa) were detected on chromosomes , , , and ; and suggestive evidence for linkage to fasting serumglucoseconcentrationswasdetectedonchromosomes , , , , and . these results suggest that the genes regulating susceptibility to insulin resistance, hyperglycaemia and dia- betes-related phenotypes may reside in these regions and play an important role in the observed familial aggregation of cardiovascular disease. it is important that additional large, family-based analyses in hypertensive cohorts attempt to re- produce these results and identify the causative genes. acknowledgements hypergen participating institutions and prin- cipal staff: s.c. hunt, r.r. williams (deceased), h. coon, p.n. hopkins, j. hood, l. wu, j. skuppin (network center/university of utah field center); a. oberman, c.e. lewis, m.t. weaver, p. johnson, s. walker, c. oden (university of alabama at birmingham field center); r.c. ellison, r.h. myers, y. zhang, l. djoussé, j.b. wilk, g.l. splansky (boston university/framingham field center); d. arnett, a.r. folsom, m. miller, j. pankow, g. feitl, b. lux (university of minnesota field center); g. heiss, b.i. freedman, k. north, k. rose, a. haire (university of north carolina field center); d.c. rao, m.a. province, i.b. borecki, a. adelman, d. morgan, k. schwander, d. lehner, a. kraja, s. mandel (data coordinating center, washington university); j.h. eckfeldt, c. leiendecker-foster, r.c. mcglennen, g. rynders, m.y. tsai, j. bucksa (central biochemistry lab, university of minnesota); m. leppert, s.c. hunt, j.m. lalouel, r. weiss (molecular genetics laboratory, university of utah); s.e. old, m. higgins (retired), c. jaquish, m. lundberg, m. gerschenson (na- tional heart, lung, and blood institute). this hypertension network is funded by cooperative agreements (u ) with nhlbi: hl , hl , hl , hl , hl , hl , hl , hl . fig. genome scan plots for bivariate analysis of fasting serum glucose and insulin con- centrations. dotted line african americans; solid line caucasians references . haffner sm, valdez ra, hazuda hp, mitchell bd, morales pa, stern mp ( ) prospective analysis of the insulin-resistance syndrome (syndrome x). diabetes : – . laaksonen de, lakka hm, niskanen lk, kaplan ga, salonen jt, lakka ta ( ) metabolic syndrome and development of dia- betes mellitus: application and validation of recently suggested definitions of the metabolic syndrome in a prospective cohort study. am j epidemiol : – . snieder h, boomsma di, van doornen lj, neale mc ( ) bivariate genetic analysis of fasting insulin and glucose levels. genet epidemiol : – . ferrannini e, haffner sm, mitchell bd, stern mp ( ) hyperinsulinaemia: the key feature of a cardiovascular and met- abolic syndrome. diabetologia : – . haffner sm, d’agostino r jr, mykkanen l et al ( ) insulin sensitivity in subjects with type diabetes. relationship to car- diovascular risk factors: the insulin resistance atherosclerosis study. diabetes care : – . meigs jb, panhuysen ci, myers rh, wilson pw, cupples la ( ) a genome-wide scan for loci linked to plasma levels of glucose and hba( c) in a community-based sample of cau- casian pedigrees: the framingham offspring study. diabetes : – . panhuysen ci, cupples la, wilson pw, herbert ag, myers rh, meigs jb ( ) a genome scan for loci linked to quantitative insulin traits in persons without diabetes: the framingham offspring study. diabetologia : – . hsueh wc, st jean pl, mitchell bd et al ( ) genome-wide and fine-mapping linkage studies of type diabetes and glucose traits in the old order amish: evidence for a new diabetes locus on chromosome q and confirmation of a locus on chromo- some q –q . diabetes : – . hong y, weisnagel sj, rice t et al ( ) familial resemblance for glucose and insulin metabolism indices derived from an intravenous glucose tolerance test in blacks and whites of the heritage family study. clin genet : – . edelstein sl, knowler wc, bain rp et al ( ) predictors of progression from impaired glucose tolerance to niddm: an analysis of six prospective studies. diabetes : – . williams rr, rao dc, ellison rc et al ( ) nhlbi family blood pressure program: methodology and recruitment in the hypergen network. hypertension genetic epidemiology net- work. ann epidemiol : – . allauzen s, mani jc, granier c, pau b, bouanani m ( ) epitope mapping and binding analysis of insulin-specific mono- clonal antibodies using a biosensor approach. j immunol methods : – . pesce aj, kaplan la (eds) ( ) 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j hum genet : – . almasy l, dyer td, blangero j ( ) bivariate quantitative trait linkage analysis: pleiotropy versus co-incident linkages. genet epidemiol : – . pratley re, thompson db, prochazka m et al ( ) an autosomal genomic scan for loci linked to prediabetic pheno- types in pima indians. j clin invest : – . vionnet n, hani el h, dupont s et al ( ) genomewide search for type diabetes-susceptibility genes in french whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome q -qter and independent replication of a type -diabetes locus on chromosome q –q . am j hum genet : – . mcqueen mb, bertram l, rimm eb, blacker d, santangelo sl ( ) a qtl genome scan of the metabolic syndrome and its component traits. bmc genet (suppl ):s . watanabe rm, ghosh s, langefeld cd et al ( ) the finland–united states investigation of non-insulin-dependent diabetes mellitus genetics (fusion) study. ii. an autosomal genome scan for diabetes-related quantitative-trait loci. am j hum genet : – . sale mm, freedman bi, langefeld cd et al ( ) a genome- wide scan for type diabetes in african-american families reveals evidence for a locus on chromosome q. diabetes : – . iwasaki n, cox nj, wang yq et al ( ) mapping genes influencing type diabetes risk and bmi in japanese subjects. diabetes : – http://aspex.sourceforge.net/usage.html http://aspex.sourceforge.net/usage.html 	genome-wide scans for heritability of fasting serum insulin and glucose concentrations in hypertensive families 	abstract 	abstract 	abstract 	abstract 	abstract 	introduction 	subjects and methods 	population 	phenotyping 	genotyping 	statistical analysis 	results 	discussion 	references << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (none) /calrgbprofile (srgb iec - . ) /calcmykprofile (iso coated) /srgbprofile (srgb iec - . ) /cannotembedfontpolicy /warning /compatibilitylevel . /compressobjects /off 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/ccittfaxencode /monoimagedict << /k - >> /allowpsxobjects false /pdfx acheck false /pdfx check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ . . . . ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ . . . . ] /pdfxoutputintentprofile (none) /pdfxoutputcondition () /pdfxregistryname (http://www.color.org?) /pdfxtrapped /false /syntheticboldness . /description << /deu <feff a f f f e f f c c e d f c f c e e e f e c e e d e d c d e d d d d c f e e f e c f a f f f f e e e e f f d f e c f e c e f f c f f f f e c e f f c e f c f a f d f e e> /enu <feff a f f f e f f c c e d f c f c e e e f e c e e d e d c d e d d > >> >> setdistillerparams << /hwresolution [ ] /pagesize [ . . ] >> setpagedevice ollier and kennedy canine genetics and epidemiology , : http://www.cgejournal.org/content/ / / editorial open access every dog has its day: a new journal for canine genetics and epidemiology william er ollier* and lorna j kennedy the launch of a new journal dedicated to canine genetics and epidemiology is both timely, and needed, to support research communities within veterinary medicine, wild- life conservation, evolutionary biology and canine com- parative health genetics. in this context, we are delighted to announce the launch of canine genetics and epi- demiology, an open access journal published by biomed central with the support and backing of the uk kennel club. the last decade has witnessed an unprecedented in- crease in genetic analysis of canine species especially the domestic dog. this has largely been driven through major advances in molecular biology, high throughput dna sequencing and genotyping, comparative genom- ics, statistical methodology and the development of high powered computers. sequencing of the dog genome has been a critical factor for driving this progress. the im- portance of the dog, both to science and veterinary medicine, is underscored by the fact that its genome was one of the first mammalian species to be sequenced after man. having established the dog genome sequence, we are now defining the level of genetic variability both within and between breeds, and investigating and refining the evolutionary relationships between canine species. such knowledge is now having an impact on research, translating into clinical benefit through the introduction of new screening initiatives for disease related gene vari- ants and also informing breeding programmes. eventually genetic knowledge will help tailor and optimise thera- peutic dosage and indicate which drugs should be used or avoided. these advances are now leading to major discoveries, which need to be published as quickly as possible, and communicated to the widest possible audience. it can be difficult to publish canine genetic and epidemiological studies in specialist (mainly human) journals, as they are likely to be assessed by referees who are more experienced * correspondence: bill.ollier@manchester.ac.uk centre for integrated genomic medical research, university of manchester, manchester m pt, uk © ollier and kennedy; licensee biomed c creative commons attribution license (http:/ distribution, and reproduction in any medium domain dedication waiver (http://creativecom article, unless otherwise stated. with human rather than canine research. some aspects of study design for canine genetic analyses differ significantly from the way human population studies are currently in- vestigated. this is because human populations tend to be mostly unrelated, in contrast to the dog, where within breeds, dogs are often closely related. human disease asso- ciation studies can require sample sizes of many thousands of cases and controls to generate the statistical power needed to detect genetic risk factors; in canine studies it has been shown that sample sizes of a hundred or fewer may suffice. furthermore, statistical analyses used for hu- man studies may be inappropriate for use in canine studies of inbred populations. existing journals are often too gen- eral to assess canine genetic and epidemiology papers ap- propriately and may not have sufficient specialist referees since each journal receives a limited number of canine pa- pers. currently, it is rare to have rapid publication of ca- nine genetics research. epidemiological studies exploring canine disease risk are now expanding rapidly, arising from analyses of data from second opinion referral centres and pet insurance compan- ies. more robust approaches, such as the use of longitu- dinal cohort studies, are also being introduced. reporting both canine genetic and epidemiology research reports in a single journal is appropriate, as both genetic and envir- onmental factors contribute and interact to trigger disease processes. it is important that both genes and environment should not be studied in isolation. through publishing both canine genetic and epidemiological research in a sin- gle journal, we hope to bring research communities closer. domestication of the dog from the wolf some – , years ago, is a recent event in terms of evolution- ary time scale. sequencing genomes for different canine species is underway and is revealing their evolutionary relationships. this will lead to important insights into canine physiology, health and disease, and help facilitate conservation and wildlife management policies. comparative genetic approaches using studies in one species to help identify disease related genes in another species are well established. comparative maps of the entral ltd. this is an open access article distributed under the terms of the /creativecommons.org/licenses/by/ . ), which permits unrestricted use, , provided the original work is properly credited. the creative commons public mons.org/publicdomain/zero/ . /) applies to the data made available in this mailto:bill.ollier@manchester.ac.uk http://creativecommons.org/licenses/by/ . http://creativecommons.org/publicdomain/zero/ . / ollier and kennedy canine genetics and epidemiology , : page of http://www.cgejournal.org/content/ / / genomes of a wide range of species exist, and it is pos- sible to cross reference the chromosomal position of a disease causing gene found in one species to its location in another species. the term “animal model” is often used to describe animal based pathology which repre- sents that seen in human diseases; many of these models are experimentally induced rather than occurring natur- ally. furthermore, many experimentally induced animal disease models represent only partial aspects of their counterpart human condition. the standard model spe- cies are usually mice or rats. however, the dog genome is more similar to that of the human than rodents. it is now recognised that domestic dogs spontaneously de- velop many clinical conditions that are also seen in humans, and these may have similar if not identical underlying pathological processes. examples include can- cers, autoimmune diseases, allergies, eye diseases, mus- culoskeletal and neurological conditions. veterinarians involved in research benefit from specialist training to clinically describe their canine patients, unlike research scientists working with rodent animal disease models. the research potential that the dog offers for comparative studies has started to bring dog owners and breed clubs together with veterinary and human clinical scientists to develop collaborations which benefits both human and canine medicine. the existence of a wide range of dog breeds where some are particularly predisposed to certain diseases represents great potential for conducting comparative studies. in many ways they are similar to isolated inbred human populations, such as the amish, which have been critical for identifying disease causing mutations. most domestic dog breeds are of recent origin having been de- veloped over the last years. selective breeding of dogs with particular physical or behavioural attributes, have been fixed by restricting breeding to within defined pedigrees. across all dog breeds there is now a wide range of gene mutations encoding the extremes of many physical and behavioural characteristics; e.g. the size variation between great dane and chihuahua. one con- sequence of breed formation has been that undesirable and/or unrecognised disease causing genes have been in- advertently included along with desirable attributes. the potential of the domestic dog for comparative dis- ease studies formed the basis for a four year eu frame work research award called lupa. this brought to- gether research geneticists, epidemiologists and veterinar- ians across europe with the sole mission identifying the genetics of canine diseases that also represent important human conditions. a wide range of diseases were included. two aspects are seen to be paramount for the scope and focus of this new journal. firstly, that the journal should be open access, so it can disseminate research and know- ledge to a wide, international readership. secondly that it should completely embrace “public engagement with sci- ence”. there are many dog owners and breeders around the world committed to understanding and improving the health and welfare of domestic dogs. further potential readers include those who are committed to wildlife and conservation biology. this journal will provide an oppor- tunity to bring scientists, veterinarians, dog owners and an interested general public closer together. every article published will have a lay summary and we will occasionally include lay interpretations of com- plex scientific terminology. received: march accepted: march published: april doi: . / - - - cite this article as: ollier and kennedy: every dog has its day: a new journal for canine genetics and epidemiology. canine genetics and epidemiology : . submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www.biomedcentral.com/submit rp- - .pmd t he most commonly used drugs in the treatment of nephrotic syndrome (ns) are steroids. children exposed to steroids for a prolonged period may experience adverse effects such as growth failure, infections, hypertension and osteoporosis. in order to reduce steroid toxicity, alternative drugs such as cyclophosphamide, cyclosporine and levamisole are used [ , ]. levamisole is an immunomodulator that has been used for more than two decades, and is often used as the first option in the treatment of steroid-dependent or frequently relapsing ns in children. its major advantages are steroid-sparing property and less toxicity in comparison to the other immunosuppresants [ ]. this study was conducted to evaluate the efficacy of levamisole in steroid-dependent nephrotic syndrome (sdns) and frequently relapsing nephrotic syndrome (frns) in children. the specific objectives were: (a) to determine the efficacy of daily levamisole in sdns and frns children; (b) to compare the efficacy of levamisole between sdns and frns children; and (c) to evaluate the response of levamisole in sdns/frns children post- cyclophosphamide therapy. methods the records of children, who attended the pediatric efficacy of levamisole in children with frequently relapsing and steroid-dependent nephrotic syndrome sudha ekambaram, vijayakumar mahalingam, prahlad nageswaran, amish udani, sangeetha geminiganesan and shweta priyadarshini from the department of pediatric nephrology, mehta children’s hospital, chennai, india. correspondence to: dr m vijayakumar, consultant pediatric nephrologist, mehta children’s hospitals, no. (e) mc nichols road, rd lane, chetpet, chennai , tamilnadu, india. doctormvk@gmail.com received: august , ; initial review: august , ; accepted: march , . objectives: to assess the efficacy of levamisole in frequently relapsing nephrotic syndrome and steroid-dependent nephrotic syndrome. study design: retrospective analysis of hospital case records. setting: pediatric nephrology department of a tertiary referral pediatric hospital. participants: children with frequently relapsing nephrotic syndrome and children with steroid-dependent nephrotic syndrome. methods: case records of children who were diagnosed as steroid-dependant or frequently-relapsing nephrotic syndrome from june to june , were reviewed. levamisole was given daily ( mg/kg/d) along with tapering doses of alternate day steroids after remission on daily steroids. results: levamisole was effective in . % children with a better ( . %) efficacy in frequently relapsing nephrotic syndrome. a total of children completed year follow-up post levamisole therapy. the cumulative mean (sd) steroid dose -year before therapy was ( ) mg/m and -year post therapy was ( ) mg/m (p< . ). the relapses were also less during the period of post-levamisole therapy. conclusion: levamisole is an effective alternative therapy in frequently relapsing and steroid-dependent nephrotic syndrome. keywords: treatment, steroids, outcome, relapse, nephrotic syndrome. rrrrr eeeee sssss eeeee aaaaa rrrrr ccccc h ph ph ph ph p aaaaa ppppp eeeee rrrrr indian pediatrics volume __may , nephrology clinic at a tertiary pediatric referral hospital, in southern india between june and june were analyzed retrospectively. children aged - years receiving levamisole for at least six months for treatment of sdns or frns were included. infantile ns, congenital ns and ns secondary to systemic illnesses were excluded. sdns was defined when there were two consecutive relapses while on alternate day steroids or within days of their discontinuation. frns was defined by two or more relapses in six months or more than three relapses in any twelve months. relapses were treated according to indian pediatric nephrology group guidelines [ ]. levamisole was started in sdns/frns children daily at a dose of mg/kg/day at the end of two weeks of daily steroids on inducing remission. prednisolone was given at a dose of . mg/kg every other day for weeks and then gradually tapered to a maintenance dose of . mg/kg every other day at months and . mg/kg every other day at end of year. all children were monitored every three months for response and side effects; urinalysis, total and differential blood cell counts, and liver enzymes were done. at the end of one year, serum albumin, serum cholesterol and urine albumin were checked, and if under remission, ekambaram, et al. levamisole in nephrotic syndrome indian pediatrics volume __may , prednisolone was discontinued. levamisole was stopped at the end of two years and these children were followed up for at least one year following cessation of therapy. levamisole was considered effective, if the children were able to maintain remission when steroids were tapered and stopped. it was considered ineffective if child developed two or more relapses while on every other day steroids or when steroids could not be withdrawn. levamisole was stopped when it was considered ineffective. children in post-immunosuppressive therapy (cyclophosphamide), presenting with sdns/frns were included and their response to levamisole was analyzed. statistical analyses were done using repeated measures anova, f-test, and chi-squared test. p < . was taken as significant. results a total of children ( boys) completed months of levamisole therapy; ( %) of these were frns. none of the children had renal failure, hypertension or gross hematuria. the baseline characteristics at the start of levamisole therapy are shown in table i. the duration of levamisole therapy ranged from to months with a mean (sd) duration of . ( . ) months. levamisole was effective in . % children, was stopped in ( . %) children as it was ineffective, and ( . %) children were lost to follow-up. frequent relapsers showed a better efficacy to levamisole in comparison to steroid-dependent ns ( . % vs. . %; p= . ). prednisolone was tapered to . mg/kg/day at the end of months with a mean (sd) duration of ( . ) months. sixty-five children completed one year of therapy and prednisolone was stopped with a mean (sd) duration of . ( . ) months. mean (sd) serum albumin at the start of therapy was . ( . ) g/dl and at completion of therapy was . ( . ) g/dl. at the end of months, children completed therapy and these children were kept under surveillance for at least a year. a total of children were followed-up for year post-therapy and the cumulative steroid dose and relapse rates are shown in table ii. the steroid dose and relapse rates were significantly less after levamisole therapy. relapse free period was observed in ( . %) children during therapy and in ( . %) children during the one year period of post-levamisole therapy. before the administration of levamisole, sdns children had received cyclophosphamide. renal biopsy was performed in all these children. four children had minimal change disease and had diffuse mesangial proliferation by histopathology. levamisole therapy was effective in children. discussion in this retrospective study of children with sdns or frns, levamisole was found to be effective in majority ( . %), with a better efficacy in children with frns as compared to those with sdns. in our study, levamisole was administered in daily dosing schedule based on personal experience; most guidelines suggest alternate day therapy in nephrotic syndrome. fu, et al. [ ], in a comparative study between daily and alternate day levamisole usage in children with frns and sdns, reported that daily levamisole usage can be considered when response to alternate day usage is unsatisfactory. we did not have any comparison group as this study was a retrospective analysis. madani, et al. [ ] evaluated the efficacy of levamisole among children and demonstrated that it was effective in children with both sdns and frns. in their study, the relapse rates reduced by about one-half after levamisole therapy. alsaran, et al. [ ] documented response in . % children with frns/sdns. sumegi, et al. [ ] followed children for a duration of – months and documented a reduction in relapse rate after levamisole therapy. our results are in concordance with the above studies. in children with effective therapy, we were able to taper and stop steroids in majority of patients. bagga, et al. [ ] also showed that levamisole was effective in children with sdns. in a meta-analysis table ii cumulative steroids and relapse rates in children with frns/sdns duration of dose of steroids no. of relapses follow up (mg/m )mean (sd) mean (sd) y before levamisole . ( ) . ( . ) during levamisole . ( ) . ( . ) y after levamisole . ( . ) . ( . ) table i baseline characteristics of the study population characteristic sdns frns no. (%) ( . %) ( . %) male gender, n (%) ( . %) ( . %) age at diagnosis, y . ( . ) . ( . ) age at beginning of therapy, y . ( . ) . ( . ) sdns -– steroid-dependent nephrotic syndrome; frns – frequently relapsing nephrotic syndrome; * values in mean (sd). indian pediatrics volume __may , ekambaram, et al. levamisole in nephrotic syndrome of randomized controlled trials [ ], durkan, et al. showed that prolonged course of levamisole reduces the incidence of relapses. various studies have reported side effects while on alternate day levamisole schedule, though these were not life-threatening and were reversible on discontinuing levamisole [ , , , ]. we did not observe any side effects, even in those who completed years of daily levamisole therapy. to conclude, daily levamisole along with initial low dose steroid therapy can be effective in children with frns/sdns with a better efficacy in children with frns. it significantly reduces the cumulative dose of steroid intake and relapse rates. levamisole can be used as an effective steroid-sparing agent in children with frequently-relapsing and steroid dependent nephrotic syndrome. references . davin jc, merkus mp. levamisole in steroid-sensitive nephrotic syndrome of childhood: the lost paradise? pediatr nephrol. ; : - . . hodson em, craig jc, willis ns. evidence-based management of steroid-sensitive nephrotic syndrome. pediatr nephrol. ; : - . . van husen m, kemper mj. new therapies in steroid- sensitive and steroid-resistant idiopathic nephrotic what is already known? • levamisole is commonly used as a steroid sparing agent in children with steroid dependent or frequently relapsing nephrotic syndrome. what this study adds? • daily levamisole along with initial low dose steroid therapy can be effective in frns/sdns children with a better efficacy in children with frns. • cumulative dosage of steroids reduces with levamisole therapy in frns/sdns. syndrome. pediatr nephrol. ; : - . . management of steroid sensitive nephrotic syndrome: revised guidelines. indian pediatric nephrology group, indian academy of pediatrics. indian pediatr. ; : - . . fu ls, shien cy, chi cs. levamisole in steroid-sensitive nephrotic syndrome children with frequent relapses and/or steroid dependency: comparison of daily and every-other- day usage . nephron clin pract. ; :c –c . . madani a, isfahani st, rahimzadeh n, fereshtehnejad sm, hoseini r, moghtaderi m, et al. effect of levamisole in steroid-dependent nephrotic syndrome. iran j kidney dis. ; : - . . al-saran k, mirza k, al-ghanam g, abdelkarim m. experience with levamisole in frequently relapsing, steroid-dependent nephrotic syndrome. pediatr nephrol. ; : - . . sumegi v, haszon i, ivanyi b, bereczki c, papp f, turi s. long-term effects of levamisole treatment in childhood nephrotic syndrome. pediatr nephrol. ; : - . . bagga a, sharma a, srivastava rn. levamisole therapy in corticosteroid-dependent nephrotic syndrome. pediatr nephrol. ; : - . . durkan am, hodson em, willis ns, craig jc. immunosuppressive agents in childhood nephrotic syndrome: a meta-analysis of randomized controlled trials. kidney int. ; : - . . tenbrock k, muller-berghaus j, fuchshuber a, michalk d, querfeld u. levamisole treatment in steroid-sensitive nephrotic syndrome. pediatr nephrol. ; : - . [pdf] negotiating agency: amish and ultra-orthodox women's responses to the internet | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / corpus id: negotiating agency: amish and ultra-orthodox women's responses to the internet @article{shahar negotiatingaa, title={negotiating agency: amish and ultra-orthodox women's responses to the internet}, author={rivka neriya-ben shahar}, journal={new media soc.}, year={ }, volume={ }, pages={ - } } 	rivka neriya-ben shahar 	published 	sociology, computer science 	new media soc. this study explores how women in two devout religious communities cope with the internet and its apparent incompatibility with their communities’ values and practices. questionnaires containing both closed and open-ended questions were completed by participants, approximately half from each community. while their discourses included similar framings of danger and threat, the two groups manifested different patterns of internet use (and nonuse). rigorous adherence to religious dictates is… expand view via publisher brandeis.edu save to library create alert cite launch research feed share this paper citationshighly influential citations background citations view all topics from this paper 	internet 	software incompatibility 	nonlinear gameplay citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency the medium is the danger: discourse about television among amish and ultra-orthodox (haredi) women 	rivka neriya-ben shahar 	sociology 	 	 	 save alert research feed offline: the possible effects of internet-related behavior on work values, expectations, & behavior among ultra-orthodox millennials 	avi kay, l. 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please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ bet bromodomain inhibition triggers apoptosis of nf -associated malignant peripheral nerve sheath tumors through bim induction cell reports article bet bromodomain inhibition triggers apoptosis of nf -associated malignant peripheral nerve sheath tumors through bim induction amish j. patel, , chung-ping liao, zhiguo chen, chiachi liu, yong wang, and lu q. le , , ,* department of dermatology cancer biology graduate program harold c. simmons cancer center utsw neurofibromatosis clinic university of texas southwestern medical center at dallas, dallas, tx, - , usa *correspondence: lu.le@utsouthwestern.edu http://dx.doi.org/ . /j.celrep. . . this is an open-access article distributed under the terms of the creative commons attribution-noncommercial-no derivative works license, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. summary malignant peripheral nerve sheath tumors (mpnsts) are highly aggressive sarcomas that develop sporad- ically or in neurofibromatosis type (nf ) patients. there is no effective treatment for mpnsts and they are typically fatal. to gain insights into mpnst pathogenesis, we utilized an mpnst mouse model that allowed us to study the evolution of these tumors at the transcriptome level. strikingly, in mpnsts we found upregulation of a chromatin regulator, brd , and show that brd inhibition profoundly sup- presses both growth and tumorigenesis. our find- ings reveal roles for bet bromodomains in mpnst development and report a mechanism by which bromodomain inhibition induces apoptosis through induction of proapoptotic bim, which may repre- sent a paradigm shift in therapy for mpnst patients. moreover, these findings indicate epige- netic mechanisms underlying the balance of anti- and proapoptotic molecules and that bromodomain inhibition can shift this balance in favor of cancer cell apoptosis. introduction neurofibromatosis type (nf ) is one of the most common hu- man genetic disorders of the nervous system and affects one in , individuals around the world regardless of ethnicity and gender (wallace et al., ). nf manifests through inheritance or sporadic mutation of the nf tumor suppressor gene, a nega- tive regulator of oncogenic p -ras, which predisposes nf patients to a wide spectrum of symptoms including develop- mental, neurological, dermatological, and cardiovascular defects and tumor development (le and parada, ; martin et al., ). although nf patients are susceptible to devel- oping various neoplasms (juvenile myelomonocytic leukemia, optic glioma, astrocytoma, rhabdomyosarcoma), the most common occurring are benign neurofibromas, which can be stratified into two subgroups: plexiform and dermal (albers and gutmann, ; bajenaru et al., ; le and parada, ; shannon et al., ). plexiform neurofibromas can progress to malignant sarcomas known as malignant peripheral nerve sheath tumors (mpnsts), which account for % of all soft tissue sarcomas (king et al., ). they are highly aggressive, incurable through conventional chemotherapy or surgical resection, and a leading cause of mortality in the nf patient population (duong et al., ). although significant progress in understanding nf tumor development has been made, surgery remains the standard of care for mpnst patients, and prognosis remains bleak (zou et al., ). neurofibroma progression to mpnst in nf patients is asso- ciated with additional genetic changes including amplification/ overexpression of oncogenic receptor tyrosine kinases (i.e., egfr, pdgfr, met) or growth factors (i.e., neuregulin- , hepa- tocyte growth factor) and loss of tumor suppressors ink a, pten, or p (the latter being the most common) (cichowski et al., ; endo et al., ; gregorian et al., ; huijbregts et al., ; joseph et al., ; keng et al., ; ling et al., ; perrone et al., ; perry et al., ; torres et al., ; vogel et al., ). modeling these genetic changes along- side loss of nf in mice has been reported to promote mpnst development, which confirms their contributions to mpnst pathogenesis. this has led to the identification of therapeutic targets regulating the cell cycle, thus allowing for inhibition of proliferation, but eventual resistance or tumor burden are likely to hinder the efficacy of such agents (albritton et al., ; jessen et al., ; johannessen et al., ; mo et al., ; patel et al., ; wu et al., a). selective inhibition of both proliferation and survival may offer mpnst patients a better prognosis. however, limited capability to culture human mpnsts and lack of a model system that permits genome- wide analysis or functional interrogation of mpnsts and their pre-tumorigenic counterparts have hindered the elucidation of cell reports , – , january , ª the authors mailto:lu.le@utsouthwestern.edu http://dx.doi.org/ . /j.celrep. . . http://crossmark.crossref.org/dialog/?doi= . /j.celrep. . . &domain=pdf survival dependencies in mpnsts. we and others have developed mouse models in which genetic loss of tumor sup- pressors nf and p leads to spontaneous initiation of mpnsts (cichowski et al., ; vogel et al., ) (l.q.l., unpublished data). more recently, we identified skin-derived precursors (skps) with nf deficiency to be a cell of origin for dermal neuro- fibromas (le et al., ). serendipitously, we found that nf -deficient skps are also capable of giving rise to plexiform neurofibromas when trans- planted into a nerve, and further loss of p readily allows for malignant transformation into mpnsts with histological and molecular features consistent with human mpnsts (mo et al., ; chau et al., ). this mpnst mouse model affords us the opportunity to monitor the evolution of these tumors from stem cell to benign neurofibroma to mpnst. here, we utilized our skp-derived mpnst model to study the evolution of these tumors by comparative transcriptome analysis of skp-derived mpnsts (smpnsts) and their pretumorigenic ancestors, nf ;p -deficient skps (np-skps), and identify upregulation of brd in mpnsts. we investigated the role of brd in mpnst pathogenesis. brd is a bet (bromodomain and extraterminal) family member that contains two bromodomains in tandem, which permit recognition and binding to acetylated histones, and subsequent recruitment of cofactors (including ptefb) for rna polymerase- ii-dependent transcription elongation (dey et al., ; jang et al., ; wu et al., b; yang et al., ). brd is reported to regulate expression of mitotic genes required for cell-cycle progression, and its fusion to nut (nuclear protein in testis) has been implicated in the pathogenesis of nut-midline carci- nomas (filippakopoulos et al., ; mochizuki et al., ; yang et al., ). the development of selective small molecule inhibitors of brd , called jq , i-bet , and cpi , has allowed for selective inhibition of c-myc expression and self- renewal in hematopoietic malignancies (acute myeloid leukemia, mixed lineage leukemia, multiple myeloma, and t cell acute lymphoblastic leukemia), thus establishing jq or i-bet - mediated inhibition of brd as therapeutic strategy to disable oncogenic c-myc (dawson et al., ; delmore et al., ; king et al., ; zuber et al., ). recent evidence indicates that brd and mediator together occupy mostly active genes in multiple myeloma cells, and at high concentrations at superen- hancers for key oncogenic drivers or cell identity genes (lovén et al., ). these superenhancer-regulated genes have been shown to be highly sensitive to levels of brd or its cofactors, such that selective inhibition with jq leads to substantial sup- pression of their transcription elongation, whereas active genes with typical enhancers are less affected (lovén et al., ). all together, these findings suggest that the concentration/levels of brd and its cofactors are important for the maintaining high levels of cancer cell specific oncogenes, thus indicating a general strategy for identifying dependencies in diverse cancer types (lovén et al., ). whether the levels of brd and its cofactors are important for solid tumor initiation and progres- sion, including mpnsts, remains unknown. here, we show that increased levels of brd accompany the progression of pretumorigenic np-skps to mpnsts in vivo. genetic and pharmacological inhibition of brd substantially cell reports , – , january , ª the authors inhibits both in vitro growth and in vivo tumorigenesis of nf - associated mpnsts. transcriptome and genomic occupancy analysis of brd inhibition in mpnsts reveals downregulation of cyclin d and bcl expression along with derepression of bim transcription, which we demonstrate to suppress tumori- genesis and initiate apoptosis of mpnsts. collectively, our findings reveal a role for bet bromodomains in maintaining the growth and survival of nf -associated mpnsts and identify a therapeutic strategy for selective inhibition of mpnst tumori- genesis and survival, which may serve to improve the prognosis for nf -associated mpnst patients. results transcriptome analysis of nf �/� p �/� skp progression to mpnst identifies upregulation of brd previously, we established a mouse model of dermal neurofi- broma (dnf) where we demonstrate that nf -deficient skps can give rise to dnfs, which is contingent on their local micro- environment (le et al., ). in the course of these skp and neurofibroma studies, we serendipitously found that we could faithfully generate mpnsts from skps doubly deficient in nf and p (np-skps) (mo et al., ; chau et al., ). this finding creates a powerful mpnst model that affords us the opportunity to genetically monitor the evolution of mpnsts from their pretumorigenic precursors (np-skps). these skp- derived mpnsts (smpnsts) were found to have a robust poten- tial for giving rise to tumors when either transplanted as tumor fragments or as few as , cells, whereas their ancestors (np-skps) required at least , cells and longer time in vivo to become smpnsts. these observations led us to hypothesize that dual loss of tumor suppressors nf and p is required but not sufficient for progression of np-skps to smpnsts. we envisioned that further genetic or epigenetic changes, the micro- environment, cells of origin, or multiple factors are required for progression to mpnst. although these complexities pose challenges to addressing our hypothesis, we reasoned that underlying transcriptome changes would allow us to molecularly understand tumor initiation, maintenance, and progression regu- lated in this model system. advantageously, our mpnst mouse model allows us to take our transcriptome insights and system- atically and rapidly determine how mpnst tumorigenesis is regulated. therefore, we compared the gene expression profiles of smpnst tumors to their pretumorigenic ancestors (np-skps) via microarray analysis to understand what transcriptome changes occur after mpnst tumor initiation/progression (fig- ure a). as anticipated, comparative microarray analysis indicates that smpnst tumors had numerous genes up- and downregulated when compared to their ancestors (np-skps). however, we found substantially more genes upregulated, and with greater magnitude of fold change expression (figure b), which was associated with upregulation of rna polymerase ii (rnap ii) regulator brd (figure c). quantitative rt-pcr and western blot analyses confirm these findings (figures d and e). consistent with these data, we also observed abundant expression of brd in human mpnst primary tissue and xeno- graft (figure s ). brd along with mediator and ptefb are all implicated in promoting rna polymerase-ii (rnap ii)-dependent figure . identification of brd upregulation and roles in mpnsts (a) diagram of microarray experiment for transcriptome analysis. (b) bar graph representation of the absolute fold change in expression of all genes from microarray experiment with fold change of r . (c) pictorial representation of positive regulators of transcriptional elongation by rna polymerase ii and identification of brd upregulation in mpnsts (from microarray experiment). (d and e) qrt-pcr and western blot analysis of mpnst cells and precursors for expression of brd . (f and g) qrt-pcr and western blot analysis for brd knockdown in smpnst-ptripz cells with or without doxycycline (dox). (h) effect of brd shrna induction on mpnst cell growth/viability using atp celltiter glo assay. all statistics are represented as the mean ± sem (*p % . , **p % . , ***p % . , ***p % . ). cell reports , – , january , ª the authors transcriptional elongation (donner et al., ; jang et al., ; wu and chiang, ) (figure c). brd has been previously implicated in cancer biology (dawson et al., ; delmore et al., ; filippakopoulos et al., ; firestein et al., ; lovén et al., ; zuber et al., ) and is currently amenable to pharmacological inhibition through small molecule bromodo- main inhibitor jq (filippakopoulos et al., ), which may present a novel therapeutic modality for treating nf -associated mpnsts. thus, we focused on elucidating the role of brd in mpnsts. brd is critical for growth and tumorigenic capacity of mpnsts we sought to dissect the function of brd in mpnsts by employing a doxycycline (dox)-inducible small hairpin rna (shrna) system to acutely knockdown brd mrna levels. smpnst cells were transduced with lentivirus harboring either scrambled shrna (ptripz-shcontrol) or brd shrnas (ptripz-shbrd . or ptripz-shbrd . ), and then treated with puromycin to select for stably infected cells. treatment of these cells with doxycycline to induce shrna expression re- veals > % reduction of brd mrna levels via brd shrnas compared to scrambled shrna (shcontrol) induction, which is consistent with protein levels (figures f and g). to evaluate the effect of acute depletion of brd on smpnst cellular growth, we evaluated atp levels as a surrogate for cell numbers before and after acute knockdown of brd and observed significantly reduced growth upon brd shrna induction with doxycycline (figure h). to study the influence of brd on the tumorigenic capacity of mpnst cells, we subcutaneously injected ptripz-shcontrol and ptripz-shbrd smpnst cells (luciferase tagged) into nude mice. two days later, both scrambled and brd shrnas were turned on in smpnst allografts by administration of doxycycline ( mg/ml) through drinking water of the mice. by days of shrna induction in vivo, we found that brd shrna-smpnst cells had significantly delayed tumor burden/progression compared to control as indicated by periodic measurements of tumor bioluminescence and volume and final weight of excised tumors (figures a– e). remarkably, induction of brd shrna expression in established tumors ( days after subcutaneous implantation) halted smpnst tumor progression/growth when compared to shcontrol smpnst tumors (figures f and g). western blot analysis of these tumors indicates that these findings are consistent with reduced brd protein levels in shbrd +dox tumors (figure h). through molecular analysis of tumor proliferation, we found that shbrd tumors had signifi- cantly fewer bromodeoxyuridine (brdu)-positive cells than shcontrol tumors (figures i and j). all together, these data indicate an important role for brd in maintaining tumori- genic capacity of mpnsts in vivo. pharmacological inhibition of brd suppresses mpnst growth and tumorigenesis the remarkable inhibition of mpnst tumorigenesis through brd knockdown prompted us to evaluate the effect of inhibiting brd with small molecule bet bromodomain inhibitor jq (filippakopoulos et al., ). we tested the effect of jq on cell reports , – , january , ª the authors pretumorigenic np-skps, smpnst cells, and cis mpnst cells (derived from spontaneous mpnst arising in cis nf +/� p +/� mice) (mo et al., ; vogel et al., ). all mpnst cells and np-skps had decreased cellular viability/growth in a jq dose-dependent manner with ic values less than nm, whereas skps (both wild-type and nf null) were relatively unaffected (figure a). these data may suggest a role for brd in maintaining in vitro growth and survival signaling prop- agated by loss of both nf and p in mpnsts and their pretu- morigenic precursors (np-skps). collectively, these promising findings suggest that jq may have important therapeutic value in the treatment of mpnsts. in that regard, to investigate the therapeutic efficacy of jq on mpnst tumor progression, we generated palpable ( mm average) smpnst allografts (luciferase expressing) in nude mice (two tumors per mouse). prior to drug administra- tion, we measured tumor volume and bioluminescence to sepa- rate tumor-bearing mice into two groups ( tumors per group), in which tumor size and mouse gender/weight are equally rep- resented (figure b). mice were treated with either vehicle or jq for days and then sacrificed. during this treatment period, we found that growth of all jq -treated tumors (n = ) had been severely blunted compared to vehicle-treated tumors (n = ) as indicated by delayed progression of tumor bioluminescence and volume (figures c and d). interestingly, during the course of the experiment, we observed smpnst tumor regression in jq -treated mice both visually and through bioluminescence imaging (figure e). remarkably, we observed % to near complete regression of tumor volume in as little as days of jq treatment, which resulted in much smaller tumors compared to vehicle tumors (figures f and g). moreover, analysis of tumor proliferation revealed significantly fewer brdu-positive cells in jq -treated smpnst allografts compared to vehicle (figures h and i). importantly, we observed no significant changes in body weight nor behavior of mice during jq treatment (data not shown), which is consis- tent with jq tolerance observed in published mouse studies (cheng et al., ; delmore et al., ; filippakopoulos et al., ; zuber et al., ). these data strongly suggest great therapeutic potential for jq in the treatment of nf - associated mpnsts. brd regulates mpnst cell-cycle progression and cyclin d expression to gain insight into the mechanism of action for brd in mpnst tumorigenesis, we first evaluated the effect of genetic and pharmacological inhibition of brd on smpnst cell num- ber. on average, we found that induction of brd shrnas led to %– % reduction in cell number after days in culture compared to cells without induction or shcontrol cells (fig- ure a). we observed a similar effect on smpnst cells treated days with jq (figure b). these data suggested inhibition of mpnst proliferation through brd inhibition. indeed, we found that brd inhibition restrains mpnst cell- cycle progression. analysis of proliferation via brdu incorpora- tion and dna content by flow cytometry led us to find that brd depletion leads to significant reduction of brdu incorpo- ration, a predominant increase in percentage of cells in g figure . brd maintains tumorigenic capacity of mpnsts in vivo (a) growth of shcontrol and shbrd . smpnst tumors relative to ‘‘day ’’ value. values represent luminescence counts (tumor bioluminescence imaging, n = tumors per group). (b) representative pictures of smpnst tumor bioluminescence in mice over time, which indicate that acute brd knockdown suppresses mpnst tumorigenesis in vivo. mice were started on doxycycline water on day and kept on this treatment until the end of the experiment. (c) smpnst tumor volume measurements (each data point represents the average measurement from six different tumors per group). (d) top panel: mice at days postsubcutaneous implantation of smpnst tumor cells (shcontrol on left flank and shbrd . on right flank). bottom panel: tumors excised from mice in the top panel. (e) average weight of excised tumors from bottom panel of figure d. (f) tumor volume of shcontrol and shbrd . smpnst-ptripz tumors in mice. at day , when tumors were established ( – mm ), mice were started on doxycycline water. (g) representative picture and average weight of excised smpnst tumors from end of experiment in figure f. (h) western blot analysis of brd protein levels in shcontrol and shbrd . smpnst tumors in mice given doxycycline water. (i) representative images of smpnst tumor sections stained with either hematoxylin and eosin (h&e) or brdu antibody. (j) quantification of the percentage of brdu(+) cells from smpnst tumor sections (scale bars represent mm). all statistics are represented as the mean ± sem (*p % . , **p % . , ***p % . , ***p % . ). cell reports , – , january , ª the authors figure . jq induces mpnst regression in vivo (a) dose-response curves for day jq treatment on primary murine skps (wild-type, nf �/�, nf �/� p �/�) and mpnst cells (skp model and cisnp model). atp celltiter-glo assay was used to measure cell viability and normalized to dmso (vehicle) for each cell type. (b) overview of jq drug trial with nude mice bearing smpnst allografts. (c) average smpnst tumor volume measured during jq drug trial (n = tumors per treatment group). (d) average smpnst tumor bioluminescence counts measured during jq drug trial (n = tumors per treatment group). (e) bioluminescence imaging of smpnst allografts in mice before and after the jq drug trial. (f) waterfall plot showing the percentage change in smpnst tumor volume from before starting (day ) and after days of jq treatment. (g) representative pictures of smpnst allografts excised from mice treated with vehicle or jq for days. (h) staining of sections from smpnst allografts (vehicle or jq treated) with hematoxylin and eosin (h&e) or immunostaining with brdu antibody. (i) quantification of brdu(+) cells from vehicle- and jq -treated smpnst tumor sections (scale bars represent mm). all statistics are represented as the mean ± sem (*p % . , **p % . , ***p % . , ***p % . ). phase, and modest affect on the percentage of cells in g /m phase (figure c). we observed similar results in both smpnst cells and s human mpnst cells treated with jq (figures c and d). collectively, these data suggest that genetic and pharmacological inhibition of brd impedes mpnst cell-cycle progression. cell reports , – , january , ª the authors previously, we and others described a role for the cxcr / b-catenin signaling pathway in stimulating mpnst cell-cycle progression via control of cyclin d mrna expression, which highlights the importance of cyclin d maintenance in mpnst cell-cycle control (mo et al., ). how cyclin d transcription is regulated in mpnsts remains unknown, but previous reports figure . brd maintains cyclin d expres- sion and cell-cycle progression in mpnsts (a) smpnst cells harboring doxycycline (dox)- inducible shrnas were counted after days culture (+ or – dox) and normalized to cell count of ‘‘-dox’’ cells. (b) smpnst cells were counted days after culturing in the presence of vehicle (dmso) or jq . (c) smpnst cells were harvested days after brd shrna induction or days after jq treat- ment for processing, and subsequent analysis of brdu uptake and dna content by flow cytometry was conducted to determine the percentage of cells in the indicated cell-cycle phases. (d) cell-cycle analysis of hr treated s cells through flow cytometry for brdu(+) cells and dna content (pi). (e) smpnst cells treated hr with vehicle or , nm jq were harvested for chromatin immunoprecipitation (chip)-pcr analysis at different regions relative to cyclin d transcription start site (tss). (f) western blot analysis of cyclin d protein levels in smpnst cells with brd knockdown or jq treatment. (g and h) qpcr analysis of cell-cycle regulatory genes in smpnst cells with brd knockdown or jq treatment, respectively. all statistics are represented as the mean ± sem (*p % . , **p % . , ***p % . , ***p % . ). indicate cyclin d as a potential target of brd (mochizuki et al., ; yang et al., ). therefore, we sought to determine if brd directly regulates cyclin d transcription in mpnsts. through chromatin immunoprecipitation quantitative pcr (chip-qpcr) analysis, we found that brd occupies the pro- moter of cyclin d , and that displacement of brd from chromatin by jq treatment led to reduced promoter occupancy in smpnst cells (figure e). consequently, we found that shrna-mediated knockdown or pharmacological inhibition of brd leads to substantial decrease in cyclin d mrna and protein abundance in mpnst cells, whereas other cell-cycle regulators are less affected by brd shrna or jq in smpnst cells (figures f– h). together, these data point to a mechanism of brd -mediated epigenetic control of cyclin d transcription and suggest brd as a therapeutic target for inhibiting onco- genic cyclin d in mpnsts. brd regulates expression of proapoptotic bim cell-cycle arrest can lead to subsequent cellular apoptosis (pie- tenpol and stewart, ). further analysis of acute knockdown of brd in mpnst led us to observe increased floating cells in culture (figure a), which was suggestive of apoptosis induction. indeed, we found an increase in apoptotic cells and activation of apoptotic markers in both mouse and human mpnst cells with cell reports , – brd inhibition (figures b– d). on the other hand, we observed that both wild- type skps and nf �/� skps do not undergo robust apoptosis when treated with jq (figure s ). to elucidate how brd inhibition triggers apoptosis of mpnsts, we performed gene expression microarray analysis to first iden- tify differentially expressed genes in mpnst cells with or without brd inhibition (both shrna and jq ), which led us to identify upregulation of proapoptotic bim (bcl l ) (figure e). we also found that brd inhibition decreased expression of antiapop- totic bcl in our microarray data (data not shown). quantitative rt-pcr and western blot analyses confirm that brd inhibition (shrna or jq ) leads to induction of bim and downregulation of bcl- , and relatively minor effect on the expression of additional apoptosis regulators evaluated (figures f and g). bim is a pro- apoptotic, bh -domain-containing protein that is thought to play a central role in apoptosis through activation of proapoptotic bax and bak, which leads to mitochondrial permeabilization that is followed by activation of caspases and apoptosis (bean et al., ; tait and green, ; wei et al., ). bcl- is an antia- poptotic protein that is thought to prevent bax/bak activation (cheng et al., ). one of the mechanisms is through inhibi- tion/sequestration of proapoptotic bh -only proteins, such as bim and puma (cheng et al., ; letai et al., ; youle and strasser, ). to determine if brd -inhibition-mediated downregulation of bcl expression promotes mpnst apoptosis, we treated , january , ª the authors figure . bet bromodomain inhibition triggers mpnst apoptosis through bim induction (a) microscopy images of smpnst cells after days of shrna induction in vitro. (b) percentage apoptosis in smpnst cells with and without brd shrna induction ( days) by flow cytometry analysis of annexin v (+) cells. (c) apoptosis induction by days of jq treatment in mouse and human mpnsts cells through flow cytometry analysis for annexin v (+) cells. (d) western blot analyses of lysates from smpnst cells with ( days) brd knockdown or ( days) jq treatment for activation of apoptosis (ccasp = cleaved caspase , cparp = cleaved parp). (legend continued on next page) cell reports , – , january , ª the authors mpnst cells with abt- , a selective small molecule that inhibits bcl- /bcl-xl, which prevents sequestration of proap- optotic bim or puma. we found that bcl- /bcl-xl inhibition with abt- is not sufficient to trigger robust apoptosis of mpnst cells (figure s ), which suggests that perhaps inhibition of brd leading to induction of bim initiates mpnst apoptosis. indeed, we found that constitutive knockdown of bim attenuates/rescues jq -induced apoptosis in multiple mpnst cell types as indicated by reduced caspase- cleavage and fewer apoptotic cells (figures h and i). collectively, these find- ings lead us to propose a model in which brd inhibition or jq treatment initiates apoptosis through induction of proapoptotic bim, and suppression of antiapoptotic bcl to trigger apoptosis of nf -associated mpnsts (figure j). furthermore, the dual restraint on proliferation and survival may indicate how brd inhibition is exquisitely effective against mpnsts. discussion here, we employed a mouse model of mpnst that allowed us to study the evolution of these tumors at the transcriptome level, which permitted us to identify and elucidate a mechanistic basis for brd in maintaining the tumorigenic capacity and survival of mpnsts. importantly, this study translates basic insights into a therapeutically tractable target for impeding the progression and survival of incurable nf -associated mpnsts, which may represent a significant paradigm shift for mpnst patient therapy. we also provide a mechanism of action as a framework for developing new therapeutic strategies to disarm remaining cell-survival networks that mpnsts may rely upon. although traditional genetic engineered mouse models (gemms) with loss of tumor suppressors nf and p have pro- vided a genetic basis for mpnst pathogenesis, their utility for discovering therapeutic targets to impede both progression and survival of these tumors has remained limited. in addition, the traditional route to study the function of additional genes in a gemm would require generating new mouse strains (knockout, knockin, or conditional alleles), which incurs a longer time horizon and costs toward studying gene function (heyer et al., ). moreover, if one wishes to study gene expression at different stages of tumor evolution, isolation of relatively homog- enous cells from spontaneously arising tumors in traditional gemms would prove difficult (heyer et al., ). although skps are a cell of origin for dermal neurofibromas, at present it is not clear that skps are a cell of origin for mpnsts in humans. nevertheless, observing that nf �/� p �/� skps can undergo malignant transformation to mpnsts suggested a novel oppor- tunity to delineate what molecular changes underlie mpnst development. thus, in this study, we took advantage of our nongermline gemm of nf -associated mpnst to study the (e) expression microarray analysis comparing the effect of brd shrna or jq o (f) qpcr analysis of the effect of ( days) brd shrna or ( days) jq treatmen (g) western blot validation of bim induction and bcl- downregulation through (h) western blot analysis of bim knockdown leading to attenuation of cleaved ca (i) flow cytometry analysis of annexin v (+) cells reveals attenuated apoptosis th (j) model for how bet bromodomain inhibition modulates the ratio of proapopto all statistics are represented as the mean ± sem (*p % . , **p % . , ***p % transcriptome of relatively homogenous smpnsts and their pre- tumorigenic precursors (np-skps). the elevated expression of brd in our smpnst transcriptome data captured our attention. our ability to genetically and pharmacologically inhibit brd in a temporal manner with great haste and ease in our stem/ progenitor transplantation model allowed us to elucidate the role of brd in mpnst tumorigenesis. these results highlight the strength and speed in which our nongermline gemm can accelerate discovery of tractable therapeutic targets for rare malignancies that represent an unmet medical need. as a proof of principle, using this smpnst model, we and our colleagues previously delineated a signaling pathway in which cell-surface receptor cxcr mediates intracellular activation of b-catenin, which leads to expression of cyclin d and estab- lished a critical role for cyclin d in promoting cell-cycle pro- gression and tumorigenesis of mpnsts (mo et al., ). in agreement, another group identified wnt/b-catenin signaling as a potential driver of mpnsts through a sleeping beauty genetic screen and found that this pathway regulates cyclin d expres- sion in mpnsts as well (rahrmann et al., ; watson et al., ). in the current study presented here, we demonstrate that brd occupies the promoter of cyclin d in mpnsts, and that pharmacological inhibition with jq inhibits brd occu- pancy at that promoter leading to decreased expression of cyclin d , which correlates with decreased proliferation observed. in contrast, brd has been shown to be required for maintaining transcription of oncogenic c-myc in hematopoietic malignancies, but we did not observe loss of c-myc expression in mpnsts with brd inhibition (data not shown). this cancer cell type specificity for regulating tumor oncogenes through brd can now be attrib- uted to the recent discovery of brd -regulated superenhancers in multiple myeloma and other cancers (lovén et al., ). those findings suggest that brd may maintain the cancer cell state through transcriptional regulation of specific oncogenes depend- ing on the cell type of origin (c-myc in the case of leukemia and multiple myeloma, and cyclin d in nf -associated mpnsts) (delmore et al., ; zuber et al., ). most importantly, our findings point to a mechanism of action by which bromodomain inhibition induces apoptosis through induction proapoptotic effector molecule bim. we demonstrate that knockdown of bim can rescue mpnst cells from jq - induced apoptosis, which further highlights the importance of this finding. interestingly, in support for the role of bim in mpnst tumor development, we observed that smpnst cells with bim knockdown had elevated tumor bioluminescence when compared control smpnst cells. thus, further highlighting the importance of bim regulation through brd (figure s ). albeit, additional mechanisms underlying jq -induced death in mpnsts may exist. for example, we observed downregula- tion of bcl expression in brd -inhibited mpnsts, which is n smpnst cells reveals induction of proapoptotic effector bim. t on the expression of apoptosis regulators in smpnst cells. brd shrna ( days) or jq ( days) treatment in smpnst cells. spase in smpnst and cis mpnst cells treated with jq ( days). rough bim shrnas in smpnst cells treated with jq for days. tic and antiapoptotic molecules in favor of apoptosis. . , ***p % . ). cell reports , – , january , ª the authors consistent with a study involving bromodomain inhibition in mll-fusion leukemia (dawson et al., ). however, through direct inhibition of bcl- using abt- we did not observe sig- nificant apoptosis compared to jq , which suggests that bcl- inhibition alone does not induce mpnst apoptosis robustly, but, in combination with bim induction, can efficiently induce apoptosis. our observations suggest that bromodomain inhibi- tion with jq induces bim and downregulates bcl expression, leading to an imbalance of pro- and antiapoptotic effectors that favors induction of apoptosis (figure j), and supports the model for an antiapoptotic/proapoptotic bcl- rheostat (bean et al., ; corcoran et al., ). consistent with this idea, we found that further inhibition of bcl- with abt- syner- gizes with jq or shbrd to more potently induce mpnst apoptosis (figure s ). on the other hand, we observed that a majority of jq -regu- lated genesdo not overlapping with shbrd -regulated genes (fig- ure s ), which is expected given that jq also inhibits brd and brd . it remains unknown whether additional bet bromodomain proteins (brd , brd ) (also targeted by jq ) play a role in mpnst pathogenesis. however, in our nf �/� p �/� skp and mpnst microarray data, differential expression of neither brd nor brd was observed, which is contrary to what we observed for brd (data not shown). nonetheless, the role of additional bet bromodomain family members (including brd and brd ) will be of interest in future studies, but at present, it remains clear from our findings that bim induction plays a central role in shbrd / jq -induced apoptosis in mpnsts. more broadly, these findings suggest an epigenetic mechanism underlying the balance of pro- apoptotic/antiapoptotic proteins that can be exploited by bet bromodomain inhibitors for inducing cancer cell apoptosis. all together, the dual effect of growth inhibition and apoptosis leading to tumor regression via jq may allow physicians to utilize jq along with surgery to better manage these tumors in nf patients. although jq remains to be optimized for clinical development, its equivalent bromodomain inhibitor i-bet is currently being evaluated in phase i clinical trials and we hope will reach patients in the clinic (http://www. clinicaltrials.gov). future studies to molecularly understand how jq induces proapoptotic bim should lead to new targets or additional approved drugs for therapeutically inducing this apoptotic pathway, and we speculate those studies would allow more rapid or synergistic induction of tumor regression. there- fore, this may allow physicians to limit drug exposure to patients while maintaining or boosting therapeutic efficacy. in conclusion, we took advantage of an mpnst mouse model that permits the study of tumor evolution for transcriptome analysis, which allowed us to identify and elucidate mechanisms for brd in mpnst growth and survival. our findings collectively provide a strong preclinical basis for evaluating bet bro- modomain inhibitors as novel therapies for these life-threatening mpnsts in nf patients. experimental procedures cells and reagents primary smpnst and cis mpnst cells were established from skp-mpnst and cisnp model mice as described (mo et al., ; vogel et al., ). cell reports , – , january , ª the authors s human mpnst cell line is a gift from dr. karen cichowski (brigham and women’s hospital, ma). all mpnst cells (mouse and human) are cultured in dmem ( % fetal bovine serum, % penicillin-streptomycin, % l-gluta- mine, % sodium pyruvate). skps were prepared and cultured as described (biernaskie et al., ). animal studies all mice were housed in the animal facility at the university of texas south- western medical center at dallas (utsw). animal care and use were in compli- ance with regulations of the institutional animal care and research advisory committee at utsw. athymic nude mice were used for tumor studies. for shrna induction in smpnst-ptripz tumors in vivo, mice were given water containing mg/ml doxycycline (sigma-aldrich) in % sucrose. for daily drug administration, a single dose of vehicle or mg/kg jq (cayman chemical) were prepared as described (filippakopoulos et al., ; zuber et al., ). tumor volume was calculated as described (mo et al., ). d-luciferin ( mg/kg) was administered by intraperitoneal injection followed by bioluminescent imaging of mice min later with ivis spectrum system (caliper life sciences). lentiviral constructs mouse brd shrnas were generated by synthesizing -mer sequences cor- responding to brd shrnas described previously (zuber et al., ) for pcr cloning into ptripz lentiviral vector. for lentivirus production, pspax and pmd .g (addgene plasmids and ) packaging vectors were used. in vitro growth assays atp celltiter glo assay (promega) was performed as per manufacturer’s instructions. the fluostar optima -well plate reader (bmg labtech) was used for luminescence measurements. brdu cell-cycle analysis and annexin v flow cytometry cell-cycle studies were conducted using brdu flow kit (bd biosciences) as per manufacturer’s instructions. for analysis of cellular apoptosis/death, annexin v-fitc kit (miltenyi biotec) was used as per manufacturer’s instruc- tions. all flow cytometry was performed using facscalibur flow cytometer (bd biosciences) at the utsw flow cytometry core facility. data were analyzed using flowjo software (tree star). rna isolation, cdna synthesis, qrt-pcr rneasy mini kit (qiagen) was used to isolate total rna from cells, followed by cdna synthesis with iscript select cdna synthesis kit (bio-rad), and then qrt-pcr using itaq universal sybr green supermix (bio-rad) on the cfx connect real-time pcr platform (bio-rad). data were quantified by dct method and normalized relative to gapdh. see figure s for primers used. expression microarray analysis for figure , total rna was isolated from smpnst tumors and np-skps from which those tumors were derived from (three biological replicates were prepared for both groups). for microarray analysis of shbrd and jq effect on smpnst cells, technical replicates (n = ) were used for the experiment. rna quality and microarray experiments using mouse genome . microarrays (affymetrix) were conducted by the ut southwestern microarray core facility. data were analyzed with genespring gx software (agilent technologies). western blot protein isolation and subsequent western blot analysis were performed as described previously (mo et al., ). antibodies used were as follows: brd (epitomics, bethyl labs); bim, cleaved caspase- (cell signaling technology); cleaved parp, cyclin d (millipore); and bcl , gapdh (santa cruz biotechnology). immunohistochemistry tumor tissue sample preparation, immunohistochemistry, and data quantifi- cation was performed as described previously (mo et al., ). antibodies used were brd (bethyl laboratories) and brdu (dako). http://www.clinicaltrials.gov http://www.clinicaltrials.gov chromatin-immunoprecipitation qpcr chip experiments were conducted as described in detailed protocol from abcam. briefly, chromatin equivalent to mg dna was -fold diluted in ip dilution buffer, precleared by hr incubation with chip-grade protein a/g plus agarose beads (thermo scientific), and then incubated overnight with brd or control immunoglobulin g (igg) antibody, hr with protein a/g agarose beads, followed by wash, elution, and dna purification (phenol/chloroform extraction followed by ethanol precipitation). for qpcr analysis, each ip signal was normalized to input signal to plot data as percentage of input. antibodies used were as follows: brd (bethyl labs) and control igg (cell signaling technology). statistical analyses all data are displayed as the mean ± sem unless specified otherwise. a two- tailed t test was used to evaluate statistical significance (p < . was deemed statistically significant). accession numbers geo accession number for data in this paper is gse . supplemental information supplemental information includes six figures and can be found with this article online at http://dx.doi.org/ . /j.celrep. . . . acknowledgments we thank all members of the le lab and wei mo for helpful suggestions and discussions. a.j.p. is a recipient of the young investigator award from children tumor foundation. l.q.l. holds a career award for medical scien- tists from the burroughs wellcome fund. this work was partially supported by funding from the dermatology foundation, disease-oriented clinical scholar program, national cancer institute of the national institutes of health grant no. r ca , and u.s. department of defense grant no. w xwh- - - to l.q.l. received: july , revised: september , accepted: december , published: december , references albers, a.c., and gutmann, d.h. 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information 	acknowledgments 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ full terms & conditions of access and use can be found at http://tandfonline.com/action/journalinformation?journalcode=hjmr download by: [t&f internal users], [laura kochman] date: march , at: : journal of media and religion issn: - (print) - (online) journal homepage: http://tandfonline.com/loi/hjmr the medium is the danger: discourse about television among amish and ultra-orthodox (haredi) women rivka neriya-ben shahar to cite this article: rivka neriya-ben shahar ( ) the medium is the danger: discourse about television among amish and ultra-orthodox (haredi) women, journal of media and religion, : , - , doi: . / . . to link to this article: http://dx.doi.org/ . / . . published online: feb . submit your article to this journal article views: view related articles view crossmark data http://tandfonline.com/action/journalinformation?journalcode=hjmr http://tandfonline.com/loi/hjmr http://tandfonline.com/action/showcitformats?doi= . / . . http://dx.doi.org/ . / . . http://tandfonline.com/action/authorsubmission?journalcode=hjmr &show=instructions http://tandfonline.com/action/authorsubmission?journalcode=hjmr &show=instructions http://tandfonline.com/doi/mlt/ . / . . http://tandfonline.com/doi/mlt/ . / . . http://crossmark.crossref.org/dialog/?doi= . / . . &domain=pdf&date_stamp= - - http://crossmark.crossref.org/dialog/?doi= . / . . &domain=pdf&date_stamp= - - the medium is the danger: discourse about television among amish and ultra-orthodox (haredi) women rivka neriya-ben shahar sapir academic college abstract this study shows how old order amish and ultra-orthodox women’s dis- course about television can help develop a better understanding of the creation, construction, and strengthening of limits and boundaries separat- ing enclave cultures from the world. based on questionnaires containing both closed- and open-ended questions completed by participants, approximately half from each community, i argue that both communities can be understood as interpretive communities that negatively interpret not only television content, like other religious communities, but also the medium itself. their various negative interpretive strategies is discussed and the article shows how they are part of an “us-versus-them” attitude created to mark the boundaries and walls that enclave cultures build around themselves. the comparison between the two communities found only a few small differences but one marked similarity: the communities perceive avoidance of a tool for communication, in this case television, as part of the communities’ sharing, participation, and common culture. when my son was hospitalized after a procedure, there was a television in his room. i stayed with him the entire week, and i knew that in this situation i was allowed to watch a little bit. but i decided to call the hospital staff and ask them to turn it off. with this thing in the room, i couldn’t take care of my son, i couldn’t pray or read my bible, and i couldn’t think about god. the amish woman quoted here sheds light on the conflict caused by living a very strict and devoted religious life in a modern, western, and secular society. the main question this article addresses is how amish and ultra-orthodox women’s discourse about television can help develop a better understanding of the creation, construction, and strengthening of limitations and boundaries separating enclave cultures from the world. i argue that both communities can be understood as interpretive communities that negatively interpret not only television content, like other religious communities, but also the medium itself. their various negative interpretive strategies will be discussed, and the article will show how those strategies are part of an “us-versus-them” attitude created to mark the boundaries and walls that enclave cultures build around themselves. the old order amish and the ultra-orthodox the old order amish (hereafter, amish) and the jewish ultra-orthodox comprise the case studies of this research. because both are part of the unique communities that avoid the television medium, they differ from other religious communities that mostly negotiate with its content. the amish are an ethno-cultural religious group affiliated with the anabaptist church, residing in the united states contact rivka neriya-ben shahar rivka.nbs@gmail.com department of communication, sapir academic college, d.n. hof ashkelon, israel . this research does not include the habad and breslav ultra-orthodox jewish sects because their use of technology is different than that of the other sects. © taylor & francis group, llc journal of media and religion , vol. , no. , – http://dx.doi.org/ . / . . and canada. they number close to , , or less than one-tenth of % of the american population (kraybill & bowman, ; kraybill, johnson-weiner, & nolt, ). their religious and social lives are dictated by the ordnung (literally, order), a set of rules that stresses humility, simplicity, obedience (hostetler, ; kraybill, ), and deep commitment to gelassenheit, “the idea of yielding fully to god’s will and forsaking all selfishness” (kraybill et al., , p. ). the israeli ultra-orthodox are a jewish religious group that constitutes . % of the adult population (ages and over) of israel (friedman et al., ; central bureau of statistics–israel, ). their religious and social lives are bound by a stringent interpretation of jewish religious law, a commitment to the study of torah (especially the talmud), and to unquestioning faith in rabbinic authority (el-or, ; friedman, ). the amish and ultra-orthodox women who participated is this study will enable us to view the world from the unique perspective of the real educators and gatekeepers in religious societies – the women (neriya-ben shahar, , , )—“the main socializers […] transmitters and guar- dians of cultural norms and traditions, including religion, in their maternal role” (beit-hallahmi & argyle, , p. ). literature on ultra-orthodox women is abundant (e.g., davidman, ; el- or, ; neriya-ben shahar, ; feder, ), with some dealing with their exposure patterns and conceptions of mass communication (neriya-ben shahar, , ). literature on amish women, on the other hand, is sparse. studies deal with historic aspects (schmidt, zimmerman-umbel, & reschly, ), the status of women in various amish societies (johnson-weiner, ; schmidt & reschly, ; van ness, ), and home births (jolly, ). none have dealt with mass commu- nication in general, or television in particular. the few comparative studies of the amish and the ultra-orthodox conducted to date pointed to differences in levels of schooling, attitudes toward state involvement in health and education, relationships with the state (neuberger, ; neuberger & tamam, ; spinner, ), and perceptions of the internet (neriya-ben shahar, ). the comparisons showed that the amish are an agrarian working society (kraybill, ) while ultra-orthodox are a society of scholars (friedman, ). the ultra-orthodox make pragmatic use of technology while amish reject innovations, do not use electricity, and travel by horse and buggy. the reality of the latter dimension is in fact much more complex in both communities, involving an intricate combination of accep- tance, rejection, and adaptation (caplan, ; cooper, ; hurst & mcconnell, ; kraybill et al., ; lev-on & neriya-ben shahar, ; neriya-ben shahar & lev-on, , ; zimmerman-umble, ). technology among religious communities marvin ( ) argued that technology and media play an important part in the creation of the limits of every community, with old and new groups negotiating issues like power, authority, and knowl- edge regarding new technologies. users share information, ideas, problems, and solutions about technologies, and those interpretations are an important part of the relationships between indivi- duals, groups, communities, and technologies. political and sociocultural situations have an impor- tant influence on the value norms of every group, and all affect the artifact’s meaning among them (leonardi, , ; pinch & bijker, ). these technology–community relationships are more complicated in religious societies (stout, ). campbell ( ) wrote about the values and priorities of religious communities as reflected in their discourse and decision making toward all new technologies: “the success, failure or redesign of a given technology by a specific group of users is based, not simply on the innate qualities of the technology, but also on the ability of users to socially construct the technology in line with the moral economy of the user community or context” (p. ). when the technology has features with potential problematic influences on the community, and if it could open the community to the bender ( ) and stoltzfus ( ) are interesting reads, but they are not based on academic studies. r. neriya-ben shahar secular world, it will be rejected, especially if the technologies “encourage the cultivation of values and practices antithetical to the communities’ prescribed religious life” (p. ). many studies have dealt with religious people and television (bobkowski, ; davies, ; golan & baker, ; hamilton & rubin, ; roberts, ; warren, ). most focused on content limits and content-reading strategies or watching behaviors. some researchers have written about the phenomenon of nonexposure or excluded media, people who have decided to live without cinema or television, but this was mostly found to be a personal decision (ammerman, ; atkin, ; lepter & lindlof, ). both amish and ultra-orthodox communities prohibit television and use limits and sanctions to enforce this prohibition. the amish prohibit the use of electricity, which is a technical limit (kraybill et al., ). ultra-orthodox communities use electricity, so they choose an important social sanction: their education system accepts only children without a television at home. if the school finds out the family owns a television, the child is removed from the school the same day (neriya-ben shahar, ). interpretative communities and enclave cultures one of the key terms in this article is interpretive community (fish, ), “a collectivity of people who share strategies for interpreting, using, and engaging in communication about a media text or technology. the strategies are devised with respect to norms and standards that evolve among the community members through innovation and the influence of argument” (lindlof, , p. ). even though these communities do not need to be geographically based and can be diffused, “any inter- pretation is the property of the community as a historical social body” (lindlof & meyer, , p. ). religious communities are interesting sites from which to examine the strategies of interpretive communities. these strategies are a resource for identity creation, ethos, and spiritual worldviews (lepter & lindlof, ; lindlof, ). for example, stout ( ) examined how an interpretive community of mormons who live in las vegas created various rich strategies to cope with the secular environment around them. the interpretative strategies of religious communities can be understood as part of the segrega- tionist patterns created by enclave cultures. enclave culture is a concept derived from cultural theory that refers to a dissenting minority, which often tends to be sectarian (douglas, ). in anthro- pological studies, it is customary to make a binary distinction between such cloistered religious communities and the open, free outside world. almond, appelby, and sivan ( ) developed and deepened the enclave concept, applying it to fundamentalist muslim, christian, and jewish societies: the enclave … is usually the response to the community’s problem with its boundary. its future seems to be at the mercy of members likely to slip away. for some reason, usually the appeal of the neighboring central community, it cannot stop the members from deserting … the only control to be deployed in order to shore up the boundary is moral persuasion. the interpretation developed by this type of community thus stands in opposition to outside society. (p. ) enclave culture theory highlights the boundaries between the “threatened” community and the surrounding society. it frequently employs such images and concepts as “fences,” “boundaries,” or “walls,” which can be both physical and spiritual, as in the “wall of virtue” they build around themselves. these help to create clear definitions of “us” and “them,” the “pure” versus the “impure” (douglas, ). one way of preserving the community’s boundaries is to monitor mainstream media and their content, which can breach the walls (almond et al., ). the combination terms interpretative communities and enclave cultures will enable us to better understand one of the main collision points between religious communities and the modern world: technology. a review of the literature shows that study about religious communities and television has focused on interpretation of television’s content, more than on the medium itself. this article focuses on two of the unique religious communities that live among modern and secular societies but refuse to use one of the most important symbols of modern technological society: the television. journal of media and religion the study not only describes the various discourses within these communities but will also enable us to understand one of the main interpretative strategies of the creation and preservation of enclave communities’ walls: the rejection of the medium itself. therefore, the main research question is, “what interpretative strategies are used by amish and ultra-orthodox women regarding the televi- sion?” the secondary research question asks what we can learn from a comparison of amish and ultra-orthodox women’s interpretation strategies. method sample eighty-two women participated in this study: belonging to the lancaster pa old order amish community and to various israeli ultra-orthodox communities. because of the ongoing discus- sion revolving around the definition of ultra-orthodox and amish, for this study i relied on the self- definition of the respondents (friedman et al., ; pew research center, ). snowball (or referral) sampling was used because it is well suited to closed communities (lee, ). to overcome the internal homogeneity of each group, research assistants (women from the respective communities) were asked to set a number of “snowballs” in motion among women with different demographic characteristics. the women’s ages and number of children were compared by indepen- dent t-test to ensure that differences in their answers were the product of cultural differences and not demography. no significant differences were found between the ages of the two groups (t( . ) = . , p > . ) or number of children (t( ) = . , p > . ). research instrument and method women who agreed to participate in the study were explained its purpose, that is, to understand how they view and use new media, and signed an informed consent to participate. they were assured there were no correct or incorrect answers and were each paid $ for their time when the questionnaire was completed. as with hurst and mcconnell ( ), who were aided by the cooperation of bishops in their fieldwork with the amish community in ohio, i hired assistants from the community to help recruit participants and to administer the questionnaires, usually in the subjects’ homes. the questionnaire consisted of both yes/no and open-ended qualitative questions. the amish women, who usually only have eight years of schooling, seemed less familiar with responding to questionnaires than the ultra-orthodox women, who had an average of years of formal education. the two groups were given similar questionnaires, the amish in english and the ultra-orthodox in hebrew. the quantitative data were analyzed using descriptive statistics and a chi-square test; the qualitative analysis followed strauss and corbin’s ( ) grounded-theory approach. i encountered a number of difficulties and issues working on this project that shed light on the challenges associated with research on cloistered communities (lee, ; rier, schwartbaum, & heller, ; steinmetz & haj-yahia, ; kraybill, ). the first was gaining access, especially to the amish community. i was fortunate in that i had a personal connection with a family from lancaster who agreed to host me on their farm six separate times. my activities mainly included washing dishes, folding laundry, working in the fields, and doing errands in my car. working side-by- side with the women affording me the opportunity to conduct numerous interviews, and some women even agreed to help distribute the questionnaires. finding ultra-orthodox participants was easier because i have extensive personal and family ties to the community. from personal conversations with the amish women and experience with the ultra-orthodox community, i assumed the respondents would be concerned they could be identified through their responses, would be asked questions offensive to them and their lifestyle, that my research assistants would appear in immodest dress, and that it would take up too much time. the first concern was r. neriya-ben shahar assuaged by complete anonymity; like cooper ( ), the numbered questionnaires contained no identifying information and were formulated with sensitivity to language and values. in addition, the research assistants adhered to the dress codes, language, and conduct of the community (as a religiously observant jew, i dress in a manner suited to both groups). since the research assistants in both groups belonged to the respective communities, their manner of dress and behavior were acceptable. findings and discussion interpretative strategies toward television to examine the interpretative strategies of the amish and ultra-orthodox women toward television, the questionnaire asked simply: “what do you think about television?” the women’s answers fell into to three strategies: television contains bad content, the medium itself is a danger, and the “us- versus-them” attitude. television contains bad content: the first interpretative strategy this strategy, found in both communities’ answers, focuses on television content. it can be further divided into two agendas: bad content and the influence of the content. bad content “things i hear that you can watch on there are shameful”; “the content is very bad” (amish). “an endless stream of false messages” (ultra-orthodox). women from both communities used various negative terms to describe the content: bad—negative, terrible, the worst; junk—trash, garbage, filth, nauseating, and dirt; danger and destruction—a very real threat, distorted, obstruc- tive, risky, burns, a hindrance, a destroyer, distraction, detrimental, spoils, interrupts, disastrous, killer, harmful; as well as various other terms, such as not true, shallow, cheap, low quality, disgraceful, unacceptable, hacks, slander and gossip, levity and laughternon-educational, and not for children. influence of the content “programs on the tv can pollute our minds”; “it crowds the mind with worldly issues”; “too much soul damaging content on it” (amish). “it loads un-filtered information on my soul, which distances me from my self, from god, and from my role in this world”; “nothing has more impact than sitting and watching, filling your head with other people opinions”; “i don’t want to watch things that spur us to leave the path of truth”; “i don’t want to fall from grace and watch things that have a bad effect on me”; “people watch bad things and it gives them ideas, they can’t understand that it’s a show. they really do the horrible things”; “we can’t get out of our minds the things we watched there” (ultra-orthodox). the women also mentioned the influence of violent content: “i think watching violence on tv can be very harmful to children and adults”; “if there were no television, there would be less violence” (amish); “too much violence in the shows, it makes the person immoral and brutal” (ultra-orthodox). the first strategy, marking television content as bad and dangerous, isn’t new or unique to the amish and ultra-orthodox. religious communities have traditionally been afraid of the influence of television content on their community’s values because of their incompatible worldviews (valenti & stout, ). they are afraid of worldly influences (lamberts-bendorth, ) and perceive television news as including immoral content (golan & baker, ). therefore, religious audiences use various kinds of selective exposure (bobkowski, ; hamilton & rubin, ; iorio, ; mcfarland, ), which is frequently a subject of religious leaders’ sermons (cohen, ; stout, ). journal of media and religion the medium is the danger: the second interpretative strategy the second strategy, however, is unique to very few communities and is widespread among the amish and ultra-orthodox: a community-encompassing decision to ban the medium itself. this strategy frames the medium as a dangerous device with multilevel influences. the findings show that both communities perceive it as exceedingly influential on many levels. the respondents’ answers included five perceived deep-seated effects of the medium: it influences morals and the soul, it affects the mind, it involves visuals, influences behavior, and wastes time. morals and the soul the tv is a very real threat to the morals of any individual, home, or nation”; “television […] can do a lot of harm to your normal standards”; “it destroys morals” (amish). the amish women used the word “soul” once (“it could be damaging to your soul”) as compared to times among the ultra-orthodox: “the television is a tool that defiles the human soul”; “television watchers are miserable, their souls are filled with evil and abominations”; “it’s a weapon that destroys the holy jewish soul”; “the television pollutes our soul, while we try hard to keep it pure”; “the soul of television is secular and worldly”; “i don’t want to corrupt my soul”; “watching has a huge impact on the human soul”; “the devastating effects of television can subvert the soul and numb it ”; “when a person watches those disgraceful things, it influences his soul, his outlook, and then his actions.” influence on the mind “i was dismayed at how it stayed in my mind”; “it fills your mind with trash”; “plus all the junk that they can fill their minds with”; “disrupts pure and creative thinking” (amish). “it’s brainwashing”; “there is something in the watching that blocks thinking”; “everything you watch on television is so tangible that it’s impossible to forget and take out of your head (it works a lot on the subconscious, therefore it’s hard to forget)”; “it’s harmful to the ability to think abstractly” (ultra-orthodox). visuality the ultra-orthodox stressed the effect of the visual experience, “the sound and vision have a bad effect”; “it makes a greater impression”; “the eye’s nature is to be attracted to movies. what we watched is engraved. it’s hard to describe and hard to make it pure”; “it’s very effective: the terrible pictures could remain for one’s entire life”; “what we see penetrates”; “the pictures penetrate the soul and confound it; “the visual experience enters deeper into the person’s soul”; “i want to keep my eyes”; “the eyes watch and the heart desires”; “watching something negative leads to a negative personality”; “i don’t want to be under the influence of visions”; “i wish i could delete everything i watched”; “some stuff i watched as a little girl, i can’t forget till today”; “a soap that could clean those visions doesn’t exist.” behavior “tv can lead a person to do wrong.” (amish): laziness – “it makes people lazy”; “it can turn you into a ‘couch potato’ (amish). “it turns people into plants; it turns intelligent people into ‘couch potatoes” (ultra-orthodox). addictiveness – “it’s addictive” (amish); “the screen is addicting people”; “it vacuums the watcher”; “you can’t stop; you can’t hold back” (ultra-orthodox). the impossibility of controlling content, in keeping with the traits of the medium: “it is impossible to censor what they absorb” (amish) “no control”; “no critic”; “no filter”; “it’s open and accessible”; “i don’t have any option of knowing what i will watch the next minute” (ultra-orthodox). waste of time the word time was mentioned times by amish women and times by ultra-orthodox women in the context of wasting time. we can understand that time is an appreciated commodity in the communities. in both communities the women wrote they do not want to spend time that could be r. neriya-ben shahar used for better things. “it wastes a lot of time” (ultra-orthodox); “it takes precious time”; “it would take up time that could be used for more important things” (amish). the amish women especially stressed family time: “it takes away valuable family time” and godly time: “it would take time away from bible reading”; “takes away precious time from god”; “don’t we have enough reasons to spend our time here on earth thinking and working for good (or godly) interests in this short life?” a discursive tool marking the medium itself as a danger is a clever discursive tool: by characterizing the television as the devil or a sin, they make clear that the device is itself the demon. this is an important difference between the evangelistic christian and ultra-orthodox habad communities who accept the medium and adapt the content to their purposes (campbell, ; hendershot, ) and the amish and ultra-orthodox who portray the medium itself as a tool of the devil and toss it outside their communities’ fences. i would like to suggest three possible explanations for their use of this tool. the first derives from the technology of the medium itself: while the internet affords the opportunity to “make it kosher” or otherwise block and censor usage so it can be used for work out of the home in both communities, the technology of television does not lend itself to limitations and controls. of course, religious people can chose not to subscribe to regular television channels, but they have no control over immodest pictures on, say, the news channels. censorship of content in newspapers and magazines is technically much easier than for television—the ultra-orthodox mostly read their communities’ newspapers and the amish try to control the reading of out-of- community magazines (neriya-ben shahar, , ). moreover, without taking content into account, it is difficult to watch television and work at the same time. it may therefore be more problematic than the other media technologies in terms of guilt feelings (davies, ; panek, ). printed newspapers and magazines can be censored and their technology is old. radio enables work while listening, and even the internet with appropriate limits can be a work tool. television, however, is a modern technology used mostly for entertainment that does not facilitate appropriate censorship or simultaneous work. the second explanation is that making the medium instead of its content a demon enables the community to create and maintain strong and stable control mechanisms. the prohibition against electricity among the amish and the social sanctions levied by the ultra-orthodox educational system on children whose families own a television leads to almost complete control over those communities’ members. compared to other religious communities that carry out discussions about selective exposure and whose members can actually watch an unlimited amount of legitimated content, the amish and ultra-orthodox understand that any discussion of the topic could lead to negotiation and compromise. therefore, marking the medium as the devil eliminates the discussion itself. the third explanation derives from the multilevel effects the women attribute to the medium itself. through them we can understand how those communities create, increase, and maintain the discourse and consciousness of danger. these effects are ostensibly so clearly rational and simple that the women believe they must protect themselves from their danger. it could be that they name these effects because there is no actual religious prohibition against watching television, like owning a car to the amish or eating pork to the ultra-orthodox, but they have decided to frame it as such. in addition, for these devoted communities, which are used to living a life of black or white, the gray area is very dangerous. this is apparently the reason why their “focus in the medium” multilevel discourse around the television was developed. it teaches us how the devoted religious enclave community defends itself from the outside modern world even without total prohibition, using only moral terms and conditions. when faced with a gray area, the community develops very strong sanctions and a strict discourse. the medium itself must be depicted as having multi-level effects and being a dangerous demon, without any positive facets. journal of media and religion “us-versus-them”: the third interpretative strategy this interpretative strategy, found in both communities, can also be expressed as the black-and- white perceptions that mark the fences and limits between the communities and the television- watching world: “we,” are the amish or ultra-orthodox and “them” are the english or non-ultra- orthodox, respectively. amish women wrote: “it is something we do not want among our people”; “that’s something we don’t believe in”; “it makes me thankful for our simple society—that our forefathers and our parents chose to live without them and not be entangled in the world as much” and one woman cited: “love not the world, nor the things that are in the world. if any man loves the world, the love of the father is not in him (i john : ).” ultra-orthodox women wrote: “we don’t want this in our camp”; “it’s not our sector’s custom to watch television”; “the ultra-orthodox community should keep its holy character, and rotten journalism [she didn’t even mention the word television] hits our holy walls” [my emphases]. part of the “our” feeling was connected to “our leaders.” in both groups, women cited their leaders’ opposition to television: “our church doesn’t allow it”; “because our leaders have decided it, it is something we do not want among our people and why not respect leaders who are appointed above you!” (amish). “the television is opposite of the rabbis’ attitude”; “if the rabbis prohibited it, it means that this is not a good thing”; “our rabbis prohibited television, even though we can’t understand the reasons” (ultra-orthodox). another perspective of “we” is “our values”: “it is the opposite of the tora’s values, opposite our pure view, our education, our walls”; “inappropriate to our values, the content is bad from a moral view and from the perspective of jewish law”; “subjects we will never speak about”; “you can see every serious sin that our community keeps away from”; “it’s contrary to our religious principles” (ultra-orthodox). “it distracts our views and values”; “i would lose my spiritual values” “detracts from our ethic, not for amish christians” (amish). the “them” discourse focuses on the “others,” that is, the “english” or “israelis” who watch television and are part of “the world.” amish women wrote: “to hear or see all this stuff that’s going on in the world is not good”; “i don’t have to know everything that’s going on in the world”; “too much of a distraction from the real world” “i’m not an updated person on the latest world issues.” the word “worldly” was used in “worldly issues,” “worldly music,” and “worldly things.” the ultra- orthodox referred to the israeli population: “the television is a weapon that helps the israeli population become so violent and shallow […] their youngsters are interested only in alcohol and violence.” lindlof ( ) wrote that formation of an interpretative community assures group solidarity and creation of the community’s limits. the women’s answers help us understand that one of the most important and strong tools for separation of religious communities are the walls that the community creates between “we” and the “other” (douglas, ; lepter & lindlof, ; mcguire, ). the attitudes and beliefs toward technologies are an important part of the communities’ self-definition and the creation of these walls (campbell, ). if the primary requirement of technology is seen as the ability to accomplish a social task (jackson, ), the communities’ attitudes toward technology are rooted in “group-specific beliefs about how the world could be known, and how other groups than one’s own imagined it to be” (marvin, , p. ). these beliefs lead to specific media consumption, create collective identities, and mark taste and belonging (rauch, ). however, these results lead to the argument that technology avoidance is the other side of the coin and enable a pleasure derived from the distinction itself. technology avoidance creates a sense of identity, “a binary opposition belying an ‘us-versus-them’ worldview, to reinforce symbolic boundaries” (rauch, , p. ). social capital, which usually focuses on what people have (bourdieu, ), can turn into social capital that focuses on what people do not have or do not use (author, in press). this distinction can create homogeneity; protect self-esteem; and engage in perceptual, affective, and behavioral continual identity (hildebrand, demotta, sen, & kongsompong, ). r. neriya-ben shahar amish versus ultra-orthodox interpretation strategies the second research question asked what we can learn from comparing amish and ultra-orthodox women’s interpretation strategies. the similarity between the communities is reflected in the negative terms they both used toward the television—the multilevels effects of the medium, televi- sion content that is antithetical to the communities’ values, and the bad influences of content. moreover, in both communities we can find the “us-versus-them” discourse that enables them to protect community walls and limits. but perhaps the most interesting similarity is reflected in their answers to the next question: “do you think that watching television is in keeping with amish/ultra-orthodox values?” all of the women from both communities ( %— ultra-orthodox and amish) answered “no.” such a result is rare in social science research. in this case it could be read as a great success for the communities’ education. the main differences between the two communities were found in a careful reading of the women’s answers about television content. only two amish women wrote about the danger of worldly issues and music compared to ultra-orthodox references, which usually included the term secular. the amish did not mention modesty, while the ultra-orthodox mentioned it times in a negative context, like outrageous immodesty, immodest vision. while women from both communities wrote about the medium’s multilevel deep-seated effects—to the mind, morals, the soul, and behavior—only the ultra-orthodox wrote about the visual. the amish women used the word soul once compared to times among the ultra-orthodox. whereas the ultra-orthodox wrote much about the low quality of television content, it was not mentioned by the amish at all. i would suggest some possible explanations for these differences. the first could derive from differences in education between the communities. while amish women receive only eight years of schooling, the ultra-orthodox women had an average of years of formal education. this difference could have had an effect on their writing skills, knowledge, and the critical thinking they presented. if this were true, how is it possible that the amish wrote detailed and rich answers about the medium compared to what they wrote about the content? perhaps the reason is simply that the amish do not have electricity, and therefore the temptation is less powerful than among the ultra-orthodox, who technically could have a television at home and had to develop a more powerful, scary, and rich discourse to maintain their values. but still, if in both communities there are prohibitions and sanctions toward consumption of the medium, why did the ultra-orthodox women develop such a discourse? most of the ultra-orthodox women work outside the home compared to the amish women, who usually are stay-at-home mothers. this fact obviously provides the ultra-orthodox women with more opportunities for television consumption. therefore, they developed a detailed negative discourse not only toward the medium, which in any case cannot enter the homes in both communities, but also toward the content, which the ultra-orthodox women might consume outside the home. conclusion the amish and ultra-orthodox women who participated in this study shared with us their enclave cultures’ interpretative strategies toward one of the most important modern technologies: the television. similar to other religious communities (campbell, ; hendershot, ), they deal with television content and see it as a bad influence. in contrast to other religious communities that negotiate the content but use the medium, however, the amish and ultra-orthodox have decided to reject the medium itself. since this is not an actual religious prohibition, their rejection includes a sophisticated discourse that frames the multiple effects of television on morals and the soul, the mind, vision, behavior, and time. moreover, they use this discourse as a sociological strategy, maintaining their “us-versus-them” point of view and marking the boundaries and walls they build between their safe enclave cultures and the outside world. comparison between the groups found they are more similar than different. journal of media and religion the result that showed a % agreement among both groups to the question if they think television is in keeping with the community’s values is much more meaningful than the differences, which mostly derive from differences in the women’s educational and occupational lives. stout ( ) argued that cultural war is a dual term that is not really appropriate for describing the complicated relationship between religious communities and institutions and popular culture. this argument is correct when referring to most of the religious groups that have deeply and bravely negotiated television content while adapting the medium. on the other hand, we can see two religious communities that have decided to mark the medium itself as a danger, using discursive strategies that have not only created interpretative communities, but also enclave cultures that repeatedly mark the walls, fences, and boundaries between their sacred communities and the outside secular world. this point of view expands our understanding by showing the communities that deal with the daily drudgery of raising the walls, compared to others that build and maintain windows and portholes. carey ( ) defined the media as a ritual. “in a ritual definition, communication is linked to terms such as ‘sharing,’ ‘participation,’ ‘association,’ ‘fellowship,’ and ‘the possession of a common faith’” (p. ). he asked us to pay attention to the common root of the terms “commonness,” “communion,” “community,” and “communication.” taking these common roots as a point of departure, i argue that a community can perceive avoidance of a tool for communication, in this case television, as part of sharing, participation, and commonness. if carey saw a person sitting down and watching television for entertainment ending up establishing solidarity with the larger community, the insight from this research is that the person who almost never sits down to watch television ends up establishing solidarity within the community. the issues addressed here need to be investigated further with larger samples that include amish and ultra-orthodox men, and that compare men and women from diverse devout orthodox communities with more liberal communities—jewish, christian, and muslim. the st century and the new media technologies continue to create unlimited challenges for religious groups, and as campbell ( ) wrote, the values and priorities of the religious communities are reflected in their discourse and decision making toward every new technology. acknowledgments i would like to thank sapir academic college and the israeli second authority for television and radio for their support of this research. i am also grateful to helene landau for her skillful editing. references almond, g. a., appelby, r. s., & sivan, e. 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( ). the amish and the telephone: resistance and reconstruction. in r. silverstone & e. hirsh (eds.), consuming technologies: media and information in domestic spaces (pp. – ). london, england: routledge. r. neriya-ben shahar http://nms.sagepub.com/content/early/ / / / .abstract http://ijoc.org/index.php/ijoc/article/view/ / http://www.pewforum.org/ / / /jewish-american-beliefs-attitudes-culture-survey/ http://www.pewforum.org/ / / /jewish-american-beliefs-attitudes-culture-survey/ 	abstract 	the old order amish and the ultra-orthodox 	technology among religious communities 	interpretative communities and enclave cultures 	method 	sample 	research instrument and method 	findings and discussion 	interpretative strategies toward television 	television contains bad content: the first interpretative strategy 	bad content 	influence of the content 	the medium is the danger: thesecond interpretative strategy 	morals and the soul 	influence on the mind 	visuality 	behavior 	waste of time 	a discursive tool 	“us-versus-them”: the third interpretative strategy 	amish versus ultra-orthodox interpretation strategies 	conclusion 	acknowledgments 	references hydrometrocolpos and polydactyly: a common neonatal presentation of bardet-biedl and mckusick-kaufman syndromes albert david, pierre bitoun, didier lacombe, jean-claude lambert, annie nivelon, jacqueline vigneron, alain verloes abstract mckusick-kaufman syndrome (mkks) is a rare, recessively inherited syndrome reported mainly in young children and is characterised by vaginal atresia with hydrometrocolpos, postaxial polydactyly, and congenital heart defect. bardet-biedl syndrome (bbs) is the generic name for a genetically heterogeneous group of auto- somal recessive disorders characterised by retinal dystrophy or retinitis pigmen- tosa (appearing usually between and years of age), postaxial polydactyly, obes- ity, nephropathy, and mental distur- bances, or, occasionally, mental retardation. typically, mkks is diagnosed (and reported) in very young children, whereas the diagnosis of bbs often is delayed to the teenage years. we report here a series of nine patients diagnosed in infancy with mkks because of the presence of vaginal atresia and postaxial polydactyly, who later developed obesity and retinal dystrophy, thus turn- ing out to be instances of bbs. the overlap of bbs and mkks is a real diagnostic pitfall and its importance has to be stressed, for genetic counselling, for clinical management and follow up, and for molecular approaches. the diagnosis of mkks should be considered with caution in all published cases described exclusively in the neonatal period and in those with mental retardation. we strongly recommend all children seen in infancy with a diagnosis of mkks to be re-evaluated for rp and other signs of bbs. (j med genet ; : – ) keywords: bardet-biedl syndrome; mckusick- kaufman syndrome; hydrometrocolpos mkks was first delineated by mckusick et al in in two amish sibships and rapidly confirmed. – over cases have now been reported – and autosomal recessive inheritance is clearly established. cardinal features of mkks are hydrometrocolpos and polydactyly and it is often reported as the “hydrometrocolpos-polydactyly syndrome”. hydrometrocolpos is present in - % of females and results from either vaginal atresia or imperforate hymen, which leads to the development of an abdominopelvic mass with regional compression and secondary hydrone- phrosis. hydrometrocolpos is sometimes asso- ciated with urogenital sinus. in males, hypospa- dias is the only uncommon anomaly. postaxial polydactyly or, rarely, mesoaxial polydactyly or syndactyly is present in % of cases. congeni- tal heart defects (atrioventricular canal, vsd, hypoplastic left heart) are seen in - % of cases. mental prognosis is favourable. in two pedigrees derived from the original work of mckusick et al in the old order amish, a locus for mkks syndrome has recently been mapped to p , close to the jagged gene. bbs is a well known combination of hypogenitalism, obesity, postaxial polydactyly, renal dysplasia, retinal degeneration, and men- tal impairment, reported in more than patients. – except for visual loss, expression of bbs is highly variable. the diagnosis can only be made if four of the five major manifes- tations are present in a person or in sibs, and remains a diycult diagnosis in infancy, as the appearance of several key features is delayed. polydactyly, which is present in % of cases, may only avect one limb and is sometimes associated with syndactyly and commonly with brachydactyly. renal problems are present in % of cases, but often remain undiagnosed for years in children with normal renal function if ivp is not performed. abnormalities include calyceal clubbing, cysts or diverticulae, fetal lobulations, renal cortical loss, and a reduced ability to concentrate urine (sometimes mim- icking juvenile nephronophthisis) that may lead to renal failure. genital anomalies are uncom- mon, but include vaginal atresia and hypoplasia of the uterus and fallopian tubes. hirschsprung disease – and anal atresia are rarely observed. mental retardation is present in fewer than half of cases. endocrine anomalies include obesity ( %), diabetes mellitus ( %), hypogonad- ism in males ( %), and menstrual problems in females ( %). visual abnormalities charac- teristically consist of atypical retinitis pigmen- tosa (rp) with early macular involvement. electrophysiological studies show a cone-rod dystrophy. visual impairment is constant but onset is often delayed to the second or the third decade. most patients are registered blind by the age of years. from a genetic point of view, bbs is a heterogeneous, recessively inherited disease, with at least four loci in q -q , q , p -p , and q . there seem to be only weak genotype-phenotype correlations: chromosome associated cases have polydac- tyly of all four limbs, while in chromosome j med genet ; : – department of paediatrics and human genetics, nantes university hospital, france a david department of paediatrics and genetics, hôpital jean verdier, bondy, france p bitoun department of paediatrics and human genetics, bordeaux university hospital, france d lacombe department of genetics, centre hospitalier universitaire de l’archet, nice, france j c lambert genetic centre, children’s hospital, dijon university hospital, france a nivelon department of neonatology and genetics, regional maternity antoine pinard, nancy, france j vigneron wallonia centre of human genetics, liège university, chu sart tilman, b- liège, belgium a verloes correspondence to: professor verloes. received july revised version accepted for publication march o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ associated cases polydactyly is mostly confined to the hands. chromosome associated cases are least inclined to obesity, whereas chromo- some associated cases have early onset, morbid obesity. – we report here a collaborative, retrospective study of nine unrelated bbs girls misdiagnosed as mkks in the neonatal period, in the presence of genital and digital anomalies. case reports case case was born in to non- consanguineous french parents. at birth, postaxial polydactyly was noted in both feet. at the age of months, the development of an abdominal mass led to the discovery of vaginal atresia complicated by bilateral ureterohy- dronephrosis. a diagnosis of mkks was suggested at that time. the patient was lost to follow up until the age of , when she was referred for genetic counselling. she presented as a short, obese girl ( cm, kg). excessive weight started in infancy. she had rp, a flat erg, and mild bilateral cataract. she was totally blind on one side and had residual visual acuity of less than / on the other side, with a ° tubular visual field. further investigations showed bilateral kidney atrophy with enlarged calyces. there was no mental retardation. family history was unremarkable. bbs was finally diagnosed. case this girl was born in to non- consanguineous french parents. at term, birth weight was g, length was . cm, and ofc cm. she was noted to have bilateral postaxial polydactyly of both feet and syndac- tyly iv-v of the left foot. at the age of months, patent urinary tract infection and oedema of the legs led to the discovery of a hydrometrocolpos with vaginal atresia and severe ureterohydronephrosis. a diagnosis of mkks was made. at the age of years, obes- ity, developing progressively since the age of , and severe myopia were recorded. at the age of years, she was re-evaluated in a genetic cen- tre. she was cm tall and weighed . kg. she had myopia (− dioptres) and rp with low visual acuity ( to / ), tunnel vision of °, a flat erg, and a squint. she was of low normal intelligence. the family history was unremark- able. a diagnosis of bbs was then suggested. case an abdominal mass was found in utero in this girl born in to non-consanguineous french parents. at weeks of gestation, birth weight was g, length was . cm, and ofc cm. mkks was diagnosed in the neo- natal period, based on hydrometrocolpos, bilateral ureterohydronephrosis, intercalate polydactyly of the upper limbs, and postaxial polydactyly of the lower limbs. relative micro- melia, hip dislocation, and iugr were noted. bbs was suggested in infancy because of early obesity, and the erg, at the age of year, con- firmed retinal dysfunction. mild conductive deafness and scoliosis were noted at ages and years respectively. she menstruated sponta- neously at years. at the age of ⁄ years, she was cm tall and weighed kg. she had rp with visual acuity < / . the family history was unremarkable. case this girl was born in after an uneventful pregnancy to non-consanguineous french par- ents. she was operated on for hymenal atresia on the second day of life. postaxial polydactyly was present in the left foot, leading to a diagnosis of mkks. impaired renal function was noted at the end of the first week. at the age of months, intestinal occlusion occurred and hirschsprung disease was found, which required transient ileostomy. hemeralopia, loss of visual acuity, and progressive reduction of the visual field were noted at the age of years. visual evoked potentials showed increased latencies and the erg was flat. a diagnosis of bbs was proposed at that time. renal insuy- ciency rapidly worsened and made a renal transplant necessary at the age of years. a rapidly evolving scoliosis was noted in infancy ( ° at age , ° at age ), for which surgi- cal treatment is foreseen. at the age of , she was cm tall and weighed kg. she was mildly dysmorphic with a long face, microg- nathia, high arched palate, small teeth, short neck, and brachydactyly. she had normal puberty with irregular menses. basal gonado- trophins and steroid hormones were normal. she was of normal intelligence. the family his- tory was unremarkable. case this girl was born in to first cousin moroccan parents. birth weight was g, length was cm, and ofc cm. prenatal ultrasonographic investigations showed an ab- dominal mass, enlarged kidneys, and postaxial polydactyly of all four limbs. these anomalies led the gynaecologist to suggest a diagnosis of mkks. at birth, vaginal atresia complicated by upper vaginorectal fistula and ureterohy- dronephrosis seemingly confirmed the diagno- sis. a correct diagnosis of bbs was made by the geneticist in the neonatal period, after evalua- tion of the patient’s brother. in infancy, progressive renal insuyciency appeared, with abnormal corticomedullary diverentiation on us scan, but no malformations. obesity was obvious by the age of , and hemeralopia was noted before the age of . at the age of years, she had developed atypical rp with pale papil- lae and gracile retinal vessels. the erg was flat. visual acuity was / . at the age of , she was cm tall (< rd centile) and weighed kg. ofc was . cm. iq was cm and she required special schooling. examination of the older brother of case was prompted by her birth. this boy, aged years at that time, had postaxial polydactyly of all four limbs, obesity (onset at age ), micropenis, and poor vision. investigations showed atypical rp with abnormal erg, allowing a firm diagnosis of bbs, and switching the diagnosis of his newborn sister to bbs. at the age of , he was cm tall and weighed david, bitoun, lacombe, et al o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ kg. ofc was cm. iq was . visual acu- ity was / bilaterally, the appearance of his kidneys on us was similar to case , but with- out functional consequences. after the birth of case , the parents had two further avected girls (born in and ), both with upper limb polydactyly, but no genital anomalies. at the age of , the older one was cm tall and weighed kg. ofc was cm. iq was . she showed an asymptomatic renal anomaly similar to her brother and visual impairment, whereas the youngest was already obese ( cm, kg). visual impairment has not yet been assessed in the latter. case at birth, this girl, born in at weeks of gestation to non-consanguineous malian par- ents, was noted to have hydrometrocolpos and an imperforate anus. birth weight was g. she developed renal failure in the first few months of life, causing severe failure to thrive. she was started on dialysis by the age of months, and required left sided nephrectomy at the age of and right sided renal transplant at the age of . rp was diagnosed at the age of months, with a flat erg. profound right sided conductive hearing loss was diagnosed at months. at examination at the age of years months, her height was . cm (− sd), her weight was . kg (+ . sd), and her ofc was cm (+ . sd). she was of normal intel- ligence and had normal speech. visual acuity was < / bilaterally. her fundi showed a grey- ish retina without pigment clumps bilaterally. a diagnosis of bbs was suggested, although the manifestations in this patient had obviously occurred earlier than usually reported, whereas obesity and polydactyly were not present. although obesity often starts in infancy in bbs, the present case illustrates the fact that early onset of renal insuyciency may delay its development as a consequence of the nephro- genic failure to thrive. case this girl was born in to non- consanguineous french parents. postaxial polydactyly of the feet, hydrometrocolpos, bilateral ureterohydronephrosis, urogenital sinus, imperforate hymen, and vaginourethral fistula were noted. birth weight was g and length cm. she was diagnosed as having mkks. at years, obesity and severe myopia (− /− dioptres) were noted, with poor vision and an abnormal erg. when evaluated at the age of years, she was cm tall and weighed . kg. erg showed photopic and scotopic anomalies compatible with a mixed retinal degeneration and the fundi showed rp with a salt and pepper type of pigmentation. subval- vular aortic stenosis was surgically corrected at the age of years. she required special school- ing. at that time, a diagnosis of bbs seemed appropriate. the family history was unremark- able. case this girl was born in to non- consanguineous algerian parents. in the prena- tal period, an abdominal mass was noted. at birth, the presence of postaxial polydactyly in the right upper limb and in both feet, as well as urogenital sinus and hydrometrocolpos, led to a diagnosis of bbs. birth weight was g and ofc cm. progressive hydronephrosis developed, which was surgically corrected at months. at the age of , she was cm tall (− sd) and weighed kg (+ sd). ofc was normal ( cm). the fundi, at age , showed retinal dystrophy with atrophic pigmentary epithelium but without pigment clumps, pale papillae, and gracile retinal vessels, compatible with atypical retinitis pigmentosa. she had mild mental retardation. bbs was highly likely. the family history was unremarkable. case an abdominal mass was noted antenatally in case . at birth, in , this girl weighed g, with a length of cm. ofc was cm. postaxial polydactyly of the left hand and of both feet was noted, as well as vaginal atresia and a huge hydrometrocolpos ( ml). a diagnosis of mkks was made. at the age of years, she was cm tall (+ sd) and weighed kg (+ sd). she had psychomotor retarda- tion, recurrent uti despite normal urinary tree imaging, intermittent nystagmus, and abnormal erg (microvolted potentials), com- patible with bbs. a diagnosis of bbs was likely. the family history was unremarkable and the parents were non-consanguineous, but came from the same french village. discussion hydrometrocolpos is a rare malformation, which may occur in association with other malformations (table ). eight of the nine patients reported here showed a convincing bbs phenotype associated with vaginal atresia, and patient was felt to belong to this spectrum because of rp, renal impairment, and vaginal atresia. although rare, genital anomalies have been reported in bbs for a long time, but their importance in early infancy has never been stressed. the most common problems are vaginal atresia or transverse septum noted in patients reported before our own series (table ). rarely, anomalies involving the müllerian derivatives have been described: müllerian duct hypoplasia, dupli- cation of the uterus, and hypoplastic uterus with unilateral ovary. lower urinary tract anomalies include ectopia of the urinary meatus, urethral atresia, and urogenital sinus. rp is a hallmark of bbs. in reports of mkks, rp has been reported twice. in the report of goecke et al, a sibship with consan- guineous parents was described, consisting of one girl with postaxial polydactyly and table syndromes with hydrometrocolpos with polydactyly mckusick-kaufman syndrome bardet-biedl syndrome pallister-hall syndrome ellis-van creveld syndrome - orofaciodigital syndrome, type iv without polydactyly langer-giedion syndrome hydrometrocolpos and polydactyly o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ hydrometrocolpos who had a brother with atypical rp. the report of chitayat et al (family b) describes a year old girl with postaxial polydactyly, hydrometrocolpos, hydronephro- sis, retinal dystrophy, short stature, and normal menarche. obesity was not mentioned. re- cently, kumar et al described a highly inbred pakistani family in which at least four sibs and two related persons had mkks. this family showed unusually severe polysyndactyly for mkks. males presented with small penis/ hypospadias. one of the sibs had hirschsprung disease (total colonic aganglionosis), bilateral renal cystic dysplasia, and mental delay. from the table in that report, retinal problems are present, although this does not appear in the text of the article. in the light of the present report, we suggest that these three reports deal with bbs instead of the proposed diagnosis, and we have some doubt about the patient reported by davenport et al, who was decribed as mkks with hirschsprung disease. heart defect has often been considered as a useful clue to the diagnosis of mkks in the polydactylous male, but is of uncertain use in the diverential diagnosis of bbs and mkks. chd is rarely mentioned in older publications on bbs, / in the reviews of mcloughlin et al and uncommon ( %) in the review by chitayat et al, but it has been noted with high frequency ( / ) in a series of bedouin bbs patients, where the most frequent diagnoses were bicuspid aortic valve, asd, pulmonary stenosis, and cardiomyopathy. despite the fact that genital anomalies had already been reported in bbs in , the overlap of bbs with mkks has not received attention until very recently, and the possible confusion between both disorders is still poorly appreciated. the only clear mention of this overlap was in a letter by schaap et al report- ing the erroneous diagnosis of mkks in their previous case report. a reason for this oversight could be that bbs and mkks have been published in diverent areas of paediatric and genetic publications, as they are seen (and diagnosed) at diverent ages by diverent doctors (neonatologists and paediatric sur- geons in infancy, neurologists, endocrinolo- gists, and ophthalmologists later). lurie and wulfsberg in their recent review of mkks did not discuss bbs at all. they noted that “the frequency of some findings is underestimated [in mkks]. it is evident, for instance, that not all patients had ophthalmo- logic examination, and cases of rp may be only ‘the tip of the iceberg’”. considering our own experience with mkks, we cannot accept these statements, but we rather think that mkks is an overdiagnosed condition, perhaps because of the misleading evect of descriptive names in syndromology: our message is that not all children with hydrometrocolpos and polydactyly have the hydrometrocolpos- polydactyly syndrome. the consequences of this are straightfor- ward. as long as genetic tests for mkks or bbs are unavailable routinely, genetic counsel- ling for parents of newborns with hydrometro- colpos and polydactyly should be much more cautious, even when congenital heart defect is present, considering the poor visual prognosis of bbs and the risk of mental impairment. a firm diagnosis of mkks should be deferred to to years and the possibility of delayed complications should be discussed accordingly. similarly, this clinical overlap in infancy makes it necessary to establish systematic ophthalmo- logical and neurodevelopmental follow up of all newborns presenting as mkks. it should be stressed that a part of the overestimation of the mental handicap in bbs probably results from their “slow” behaviour and delayed diag- nosis of their poor vision. renal ultrasonogra- phy combined with urography should be performed in all cases of mkks and detection of impaired concentration ability with the ddavp test should probably be performed in every case. for the same reasons, retrospective studies of mkks should be considered with caution if cases are presented in the neonatal period or are mentally retarded, and all mkks cases should be re-evaluated for rp and other signs of bbs, as some of these children could be avected by bbs, as illustrated by our experi- ence. the significant overlap of bbs and mkks could be of importance for teams dealing with mkks gene mapping in non-amish popula- tions. the similarities of mkks and bbs indi- cate that the mkks gene products are likely to act in the same developmental pathway and to interact with the proteins involved in the pathogenesis of bbs. mckusick v, bauer bl, koop ce, scott rb. hydrometro- colpos as a simply inherited malformation. jama ; : - . dungy ci, aptekar rg, cann hm. hereditary hydromet- rocolpos with polydactyly in infancy. pediatrics ; : - . kaufman rl, hartmann hf, mcalister wh. family stud- ies of congenital heart disease ii: a syndrome of hydromet- rocolpos, postaxial polydactyly and congenital heart disease. birth defects ; : - . robinow m, shaw a. the mckusick-kaufman syndrome: recessively inherited vaginal atresia, hydrometrocolpos, uterovaginal duplications, anorectal anomalies, postaxial polydactyly, and congenital heart disease. j pediatr ; : - . cantani a, tacconi ml, benincori n, et al. rare syndromes. the kaufman-mckusick syndrome. a review of the cases reported in the literature. ann genet ; : - . chitayat d, hahm sy, marion rw, et al. further delineation of the mckusick-kaufman hydrometrocolpos- polydactyly syndrome. am j dis child ; : - . lurie iw, wulfsberg ea. the mckusick-kaufman syndrome: phenotypic variation observed in familial cases as a clue for the evaluation of sporadic cases. genet couns ; : - . stone dl, agarwala r, schäver aa, et al. genetic and physical mapping of the mckusick-kaufman syndrome. hum mol genet ; : - . table reported cases of vaginal atresia in bbs reference vaginal septum and uterus duplex in a year old woman , case vaginal atresia detected at age vaginal atresia + haematocolpos vaginal atresia, hydrocolpos, and urogenital sinus diagnosed at age one girl with hydrometrocolpos and polydactyly, her brother with rp absence of vagina vaginal atresia ( cases) vaginal atresia neonatal hydrometrocolpos + meatal ectopia hydrometrocolpos detected at age + urogenital sinus persisting urogenital sinus vaginal atresia + vaginorectal fistula, sister with bbs david, bitoun, lacombe, et al o n a p ril , b y g u e st. p ro te 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of the bardet-biedl syndrome. am j med genet ; : . unsinn km, neu n, krejci a, et al. pallister-hall syndrome and mckusick-kaufmann syndrome: one entity. j med genet ; : - . akoun r, bagard m. la maladie d’ellis-van creveld. algerie med ; : - . yang ss, langer loj, cacciarelli a, et al. three conditions in neonatal asphyxiating thoracic dysplasia (jeune) and short rib-polydactyly syndrome spectrum: a clinicopatho- logic study. am j med genet ;suppl : - . yapar eg, ekici e, aydogdu t, senses e, gökmen o. diag- nostic problems in a case with mucometrocolpos, polydac- tyly, congenital heart disease, and skeletal dysplasia. am j med genet ; : - . meinecke p, hayek h. orofaciodigital syndrome type iv (mohr-majewski syndrome) with severe expression ex- panding the known spectrum of anomalies. j med genet ; : - . fryns jp. trichorhinophalangeal syndrome type : another syndromic form of hydrometrocolpos. am j med genet ; : . hydrometrocolpos and polydactyly o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ familial hypercholesterolemia and type diabetes in the old order amish familial hypercholesterolemia and type diabetes in the old order amish huichun xu, kathleen a. ryan, thomas j. jaworek, lorraine southam, , jeffrey g. reid, john d. overton, aris baras, marja k. puurunen, eleftheria zeggini, simeon i. taylor, alan r. shuldiner, and braxton d. mitchell , diabetes ; : – | https://doi.org/ . /db - alleles associated with lower levels of ldl cholesterol (ldl-c) have recently been associated with an increased risk of type diabetes (t d), highlighting the complex relationship between ldl-c and diabetes. this observa- tion begs the question of whether ldl-c–raising alleles are associated with a decreased risk of t d. this issue was recently addressed in a large familial hypercholester- olemia (fh) screening study, which reported a lower prev- alence of self-reported diabetes in fh subjects than in age-matched relatives without fh. to extend this obser- vation, we tested the association of fh with diabetes sta- tus and glycemia in a large amish population enriched for the fh-associated apob r q variant that in- cluded apob r q carriers and , noncarriers. each copy of the r q t allele was associated with a . mg/dl increase in ldl-c. there was little difference in t d prevalence between subjects with ( . %) and with- out ( . %) the r q allele (p = . ), and there was no association between r q variant and impaired fasting glucose, fasting glucose or insulin, or oral glucose tolerance test–derived measures. our data provide no evidence supporting an association between the apob r q var- iant and t d or glycemia and highlight the asymmetry of the ldl-c–t d relationship and/or the gene/variant- dependent specificity of the ldl-c–t d association. the observation that treatment of hypercholesterolemia with statins to reduce ldl cholesterol (ldl-c) levels leads to an ; % increased risk of type diabetes (t d) ( ) has generated a large dialogue on the relation of ldl-c low- ering and t d. studies showing that ldl-c–lowering variants in hmgcr, the gene encoding hmg-coa reductase and the molecular target of statins, are also associated with increased risk of t d ( ) implicated the gene itself as the driver for the increased t d risk rather than an off-target effect of statins. subsequent studies have since shown ldl- c–lowering alleles at other genes also to be associated with increased risk of t d, further suggesting that the ldl low- ering itself is related somehow to increased t d risk. an intriguing recent finding is the appearance of heterogeneity among ldl-c–lowering variants in different genes of the degree to which they increase t d risk. for example, recent meta-analyses have revealed that ldl-c–lowering alleles at pcsk and ldlr are associated with a small ( %, p = . , and %, p = . , respectively) increased risk of t d per ldl-c reduction of mmol/l ( . mg/dl), while ldl-c– lowering alleles at npc l appear to impose a greater t d risk ( %, p , . ) per ldl-c reduction of mmol/l ( ). these observations and others ( , ) suggest that mech- anisms both dependent and independent of ldl-c may be associated with increased t d risk. uncovering these mech- anisms may reveal potentially targetable pathways for di- abetes prevention or treatment. the fact that ldl-c–lowering alleles are associated with an increase in t d risk begs the question as to whether ldl-c–raising alleles are associated with a decrease in t d risk. this hypothesis was recently tested by besseling et al. ( ) in familial hypercholesterolemia (fh) cases identified from the national dutch fh screening program. fh is as- sociated with extremely high levels of ldl-c due to muta- tions in the ldl receptor (ldlr) pathway, with mutations occurring mainly in apob, ldlr, and pcsk . in the study program in personalized and genomic medicine, and division of endocrinology, diabetes & nutrition, department of medicine, university of maryland school of medicine, baltimore, md wellcome trust sanger institute, hinxton, u.k. wellcome trust centre for human genetics, university of oxford, oxford, u.k. regeneron genetics center, regeneron pharmaceuticals, inc., tarrytown, ny geriatrics research and education clinical center, baltimore va medical center, baltimore, md corresponding author: braxton d. mitchell, bmitchel@som.umaryland.edu. received february and accepted april . © by the american diabetes association. readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. more information is available at http://www.diabetesjournals .org/content/license. diabetes volume , july g e n e t ic s /g e n o m e s /p r o t e o m ic s /m e t a b o l o m ic s https://doi.org/ . /db - http://crossmark.crossref.org/dialog/?doi= . /db - &domain=pdf&date_stamp= - - mailto:bmitchel@som.umaryland.edu http://www.diabetesjournals.org/content/license http://www.diabetesjournals.org/content/license by besseling et al. ( ), there was a % lower rate of (self- reported) t d in fh cases (adjusted prevalence . %) compared with unaffected relatives (adjusted prevalence . %). in subanalyses, the relative protection against t d was observed in patients with either the ldlr or apob mutations, with the strongest effect in those with ldlr null mutations, which are more severe in disturbing ldl-c metabolism. because of inherent biases in using self-reported diabetes history and the potential for differential preventive care given to subjects already known to have a lipid disorder, we sought to evaluate the association of fh with t d and glycemia in a well-phenotyped cohort. our analyses were carried out in , old order amish (ooa) individuals from lancaster county, pennsylvania, a population enriched for one of the known fh mutations, the r q variant in the apob gene (rs ). we have previously reported that ldl-c levels are increased by mg/dl per copy of the apob r q allele in the ooa ( ). this mutation is more common in the ooa population than in other caucasian populations because of a founder effect. our sample in- cluded apob r q carriers, a number far larger than the apob mutation carriers reported in the dutch study. we compared across apob r q genotypes t d prevalence and mean levels of glycemia-related traits, in- cluding glucose and insulin values measured during oral glucose tolerance tests (ogtts) in a subset of subjects. we found no evidence supporting an association between the apob r q variant and t d or glycemia. research design and methods all , subjects included in this study were phenotyped for one or more of the study outcomes (diabetes, fasting glucose, and/or hba c) and genotyped for apob r q by either the illumina humanexome beadchip or by whole- exome sequencing. paired-end bp exome sequencing was performed by the regeneron genetics center as pre- viously described ( ) but using xgen (integrated dna tech- nologies) capture followed by sequencing on the illumina hiseq system with v chemistry. for samples with both exome sequence and chip genotype data (n = , ), the concordance rate for the r q variant was . %. all study subjects had participated in at least one of the community-based studies our group had carried out in this population over the past years as part of our amish complex disease research program ( – ). among these were subjects heterozygous (n = ) or homozygous (n = ) for apob r q, corresponding to an allele frequency of % and carrier frequency of %, consistent with what we have previously reported ( ). for the analyses described in this report, t d was diag- nosed according to american diabetes association criteria of fasting glucose . mmol/l ( mg/dl) or hba c $ . % (if available). subjects reporting current use of antidiabetes medications (n = ) were also considered to have diabetes regardless of glucose testing. a -h ogtt was administered to individuals who had previously participated in the amish family diabetes study ( ), and subjects having a -h glucose level . . mmol/l ( mg/dl) were also consid- ered to have t d. impaired fasting glucose was defined in individuals without diabetes as a glucose level between . and . mmol/l ( and mg/dl). fasting glucose was measured by a beckman glucose analyzer using the glu- cose oxidase method or the spectrophotometry method as implemented by quest diagnostics, fasting insulin by radio- immunoassay (linco research, inc., st. charles, mo), and hba c by immunoturbidimetry (quest diagnostics, hor- sham, pa). homa of insulin resistance (homa-ir) was defined as homa-ir = [fasting insulin (mu/l) fasting glucose (mmol/l)]/ . . area under the curve (auc) in ogtt was computed using the trapezoid rule. we tested the association of glucose and hba c levels with apob r q genotype using a mixed model that accounts for relatedness among individuals as a random effect. age and sex were included as covariates. analyses were carried out under an additive genetic model using the mmap software program ( ). logistic regression was used to test the as- sociation of t d with genotype. results in the sample described in this report, each copy of the apob r q t allele was associated with a . mg/dl increase in ldl-c levels ( % ci . – . ), a slightly higher estimate than the mg/dl per allele effect size we have reported previously in a smaller sample size ( ). this effect size was present across all ages studied (i.e., ages years and older). the overall prevalence of t d in the sample was . %, and there was virtually no difference by r q genotype (wild type . %, heterozygotes . %, and homozygotes . % across genotypes, p = . ) (table ). in fact, the r q t allele was associated with an in- creased, not decreased, risk of t d (per allele odds ratio [or] . [ % ci . – . ] for additive model; per allele or . [ % ci . – . ] for dominant model), although in both models the cis were wide and included . further analyses revealed no evidence for genotype ∗ age or genotype ∗ bmi interactions. results of the -h ogtts are shown in fig. for the subset of subjects who underwent this procedure, and as indicated, the response profiles, measured as auc for glucose and insulin changes following glucose challenge, were virtually indistinguishable across genotypes (p = . and . , respectively). table compares prevalence of im- paired fasting glucose and mean values of fasting glucose, fasting insulin, and hba c as well as homa-ir in individ- uals without diabetes according to genotype. these results also reveal little differences between genotypes (p = . – . ). discussion our study is unique in its large sample size of fh subjects (n = ) carrying a single apob mutation who were diabetes.diabetesjournals.org xu and associates recruited as part of a community-based population survey and tested for diabetes. our data provide no evidence supporting an association (protective or otherwise) of the apob r q variant with either t d or any other glycemia-related trait. notably, the % ci surrounding our estimate of the t d–apob r q association ( . – . ) is not consistent with a % reduced odds of having t d as reported by besseling et al. ( ) but could be associated with as much as a % decreased odds of t d or as much as a % increased odds. our analysis is also con- sistent with the lack of association reported for this var- iant with t d from the got d consortium (or . , p = . ) based on allele carriers and . , noncar- riers (http://www.type diabetesgenetics.org). the absence of association observed in our study (and got d) contrasts with the results obtained by besseling at al. ( ) that were based on self-reported diabetes history in the netherlands fh registry. the protective association of fh with t d reported in the dutch study was present for both ldlr and apob mutations; notably, the r q variant present in the amish also appears to be the predominate apob variant observed in the netherlands study ( ). a possible explanation for this discrepancy is that heightened surveil- lance of fh cases and carriers in the netherlands fh reg- istry may have altered behaviors related to t d risk, while this was unlikely to be the case for amish apob r q carriers as subjects were unaware of their genotypes at the time of subject enrollment and few amish seek primary care; in fact, only . % of our amish fh subjects reported taking lipid-lowering medications. the absence of an association observed in our study between the r q variant and t d contrasts with the increased risk of t d that has been reported in mendelian randomization studies of hmgcr, pcsk , npc l , and ldlr. one speculation is that the critical determinant of lipid-associated t d risk is more closely tied to an ldl receptor–regulated mechanism than to an upstream defect affecting the quality of the apob ligand. as elucidated in the pioneering research of brown and goldstein ( ), a dimeric complex between srebpf chaperone (scap) and insulin-induced genes (insig and insig ) functions as an intracellular receptor for cholesterol. together, these proteins mediate effects of cholesterol on srebp pathway– mediated regulation of gene expression. it is plausible that this srebp pathway might mediate some of the observed associations between ldl-c and the risk of diabetes. be- cause the insig/scap/srebp pathway is regulated by in- tracellular levels of cholesterol, this raises the question of whether the r q alters the rate at which ldl-c enters cells. boren et al. ( ) reported that the r q substitu- tion decreases the affinity of apob for the ldl receptor by ; . -fold, suggesting that a ; . -fold higher concentra- tion of q -apob would compensate for the lower bind- ing affinity as compared with r -apob. in this study, we report an ; . -fold increase in ldl-c level, which is less than the theoretical prediction of a . -fold increase in the concentration of apob. at least two biological factors may t a b le — l e ve ls o f m e ta b o lic va ri a b le s a c c o rd in g to a p o b r q g e n o ty p e t ra it a ll su b je ct s (n = , ) c c (n = , ) t c (n = ) t t (n = ) b p a g e (y ea rs ) . . . . . . . . . . m al e se x (% ) ( , / , ) ( , / , ) ( / ) ( / ) . b m i (k g /m ) . . (n = , ) . . (n = , ) . . (n = ) . . (n = ) . . l d l (m g /d l ) . . (n = , ) . . (n = , ) . . (n = ) . . (n = ) . , . w ai st ci rc u m fe re n ce (c m ) . . (n = , ) . . (n = , ) . . (n = ) . . (n = ) . . d ia b et es p re va le n ce (% ) . ( / , ) . ( / , ) . ( / ) . ( / ) . † . t ak in g d ia b et es m ed ic at io n (% ) . ( / , ) . ( / , ) . ( / ) ( / ) . . d at a ar e m ea n s d u n le ss o th er w is e in d ic at ed . † o r . ( % c i . – . ). fh and t d in the old order amish diabetes volume , july http://www.type diabetesgenetics.org contribute to the apparent discrepancy. first, it is possible that the ratio of apob:ldl-c may be higher in individuals who are homozygous for q -apob. second, a decrease in ldl receptor occupancy would be predicted to induce upregulation of the number of ldl receptors on the cell surface by decreasing the rate of ligand-induced receptor degradation ( ). taken together, an increase in the num- ber of ldl receptors and an increase in the concentration of apob would be predicted to preserve relatively normal levels of receptor-mediated endocytosis. several limitations of our study should be acknowledged. first, the amish subjects included in our study were relatively young (mean age years), and some subjects destined for future t d may not have developed it yet. second, our sample included only subjects with t d, and the minimal detectable or at % power in our sample is only . . however, this estimate is approximately iden- tical to the or of . reported by besseling et al. ( ). moreover, our sample provides . % power to detect a significant association of the r q variant with fasting glucose if this variant accounted for as little as . % of the variation in fasting glucose levels, corresponding to a change of , mg/dl in glucose levels. thus, our sample size is well powered for detecting associations between the r q variant and glycemic traits. the lack of association of this fh mutation with t d adds to the complexity of the ldl-c–t d relationship. while ldl-c–lowering alleles appear to be associated with a modest increase in t d risk, recent studies also indicate that the increased t d risk is not necessarily correlated with the magnitude of the ldl-c–lowering effect. our study adds to this story by considering a single ldl-c– raising variant, albeit one leading to a very large increase in ldl-c. notably, we find no association with either t d or any other glucose parameter. in contrast, other fh-associated variants, including some in apob, have been associated with self-reported diabetes. these discrep- ancies could reflect an asymmetry in the ldl-c–t d re- lationship, with t d risk increasing with lower ldl-c levels but unchanged with markedly higher ldl-c levels, or these discrepancies could highlight a gene (or variant)-dependent specificity of the ldl-c–t d association similar to the het- erogeneous effect shown by lotta et al. ( ). given these possibilities, mendelian randomization studies that have shown genetic risk scores for high ldl-c to be inversely correlated with t d risk ( , , ) should be interpreted cautiously, as the risk score–driving risk alleles included in these analyses typically fall in a range of different genes and their effects on t d risk may represent widely varying mechanisms rather than a single unified one. sorting out table —fasting and ogtt-derived measurements in individuals without diabetes trait all subjects cc tc tt b p impaired fasting glucose (%) . ( / , ) . ( / , ) . ( / ) . ( / ) . † . glucose (mg/dl) . . (n = , ) . . (n = , ) . . (n = ) . . (n = ) . . insulin (mu/l, ln) . [ . , . ] (n = , ) . [ . , . ] (n = , ) . [ . , . ] (n = ) . [ . , . ] (n = ) . . homa-ir . [ . , . ] (n = , ) . [ . , . ] (n = , ) . [ . , . ] (n = ) . [ . , . ] (n = ) . . hba c (%, ln) . [ . , . ] (n = , ) . [ . , . ] (n = , ) . [ . , . ] (n = ) . [ . , . ] (n = ) . . ogtt-derived measures glucose auc . . (n = ) . . (n = ) . . (n = ) . (n = ) . . insulin auc . . (n = ) . . (n = ) . . (n = ) . (n = ) . . data are mean sd except for insulin, homa-ir, and hba c (median [q , q ]) unless otherwise indicated. sd not presented for glucose and insulin auc in the tt genotype group because of the small number of subjects. ln, natural logarithm. †or . ( % ci . – . ). figure —glucose and insulin response to a -h ogtt by apob r q (rs ) genotype. diabetes.diabetesjournals.org xu and associates the mechanisms and pathways involved may reveal impor- tant insights for diabetes prevention and treatment. funding. this study was supported by national institutes of health grants r dk , r ag , r hl , u hl , u gm , r hl , and p dk and by the wellcome trust (wt ). duality of interest. no potential conflicts of interest relevant to this article were reported. author contributions. h.x. and b.d.m. contributed to study design and management. l.s., j.d.o., and e.z. contributed to genotyping. a.r.s. and b.d.m. contributed to subject recruitment. h.x., k.a.r., and t.j.j. analyzed data. h.x., j.g.r., a.b., e.z., s.i.t., a.r.s., and b.d.m. interpreted the results. h.x., k.a.r., s.i.t., and b.d.m. drafted the manuscript. h.x., k.a.r., t.j.j., l.s., j.g.r., j.d.o., a.b., m.k.p., e.z., s.i.t., a.r.s., and b.d.m. critically reviewed the manuscript. b.d.m. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. references . sattar n, preiss d, murray hm, et al. statins and risk of incident diabetes: a collaborative meta-analysis of randomised statin trials. lancet ; : – . swerdlow di, preiss d, kuchenbaecker kb, et al.; diagram consortium; magic consortium; interact consortium. hmg-coenzyme a reductase inhibition, type diabetes, and bodyweight: evidence from genetic analysis and randomised trials. lancet ; : – . lotta la, sharp sj, burgess s, et al. association between low-density lipo- protein cholesterol-lowering genetic variants and risk of type diabetes: a meta- analysis. jama ; : – . schmidt af, swerdlow di, holmes mv, et al. pcsk genetic variants and risk of type diabetes: a mendelian randomisation study. lancet diabetes endocrinol ; : – . ference ba, robinson jg, brook rd, et al. variation in pcsk and hmgcr and risk of cardiovascular disease and diabetes. n engl j med ; : – . besseling j, kastelein jj, defesche jc, hutten ba, hovingh gk. association between familial hypercholesterolemia and prevalence of type diabetes mellitus. jama ; : – . shen h, damcott cm, rampersaud e, et al. familial defective apolipoprotein b- and increased low-density lipoprotein cholesterol and coronary artery calci- fication in the old order amish. arch intern med ; : – . dewey fe, murray mf, overton jd, et al. distribution and clinical impact of functional variants in , whole-exome sequences from the discovehr study. science ; : aaf . hsueh wc, mitchell bd, aburomia r, et al. diabetes in the old order amish: characterization and heritability analysis of the amish family diabetes study. di- abetes care ; : – . streeten ea, mcbride dj, lodge al, et al. reduced incidence of hip fracture in the old order amish. j bone miner res ; : – . post w, bielak lf, ryan ka, et al. determinants of coronary artery and aortic calcification in the old order amish. circulation ; : – . mitchell bd, mcardle pf, shen h, et al. the genetic response to short-term interventions affecting cardiovascular function: rationale and design of the heredity and phenotype intervention (hapi) heart study. am heart j ; : – . bozzi lm, mitchell bd, lewis jp, et al. the pharmacogenomics of anti-platelet intervention (papi) study: variation in platelet response to clopidogrel and aspirin. curr vasc pharmacol ; : – . mmap user guide [article online], . available from http://edn.som. umaryland.edu/mmap/index.php. accessed april . kusters dm, huijgen r, defesche jc, et al. founder mutations in the neth- erlands: geographical distribution of the most prevalent mutations in the low-density lipoprotein receptor and apolipoprotein b genes. neth heart j ; : – . brown ms, goldstein jl. cholesterol feedback: from schoenheimer’s bottle to scap’s meladl. j lipid res ; (suppl.):s –s . boren j, lee i, zhu w, arnold k, taylor s, innerarity tl. identification of the low density lipoprotein receptor-binding site in apolipoprotein b and the modulation of its binding activity by the carboxyl terminus in familial defective apo-b . j clin invest ; : – . goldstein jl, brown ms. binding and degradation of low density lipoproteins by cultured human fibroblasts. comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. j biol chem ; : – . fall t, xie w, poon w, et al.; genesis consortium. using genetic variants to assess the relationship between circulating lipids and type diabetes. diabetes ; : – . white j, swerdlow di, preiss d, et al. association of lipid fractions with risks for coronary artery disease and diabetes. jama cardiol ; : – fh and t d in the old order amish diabetes volume , july http://edn.som.umaryland.edu/mmap/index.php http://edn.som.umaryland.edu/mmap/index.php doi: . / letters to the editor am. j. hum. genet. : – , inter- and intrachromosomal rearrangements are priate informed consent, from the patient, both parents, and paternal grandparents. dna was isolated by use ofboth involved in the origin of q -q deletions in prader-willi syndrome a qiaamp blood kit (qiagen). we employed markers d s and d s , both mapping to yac to the editor: a a , proximal to the deletion region, and markers prader-willi syndrome (pws) is due to an interstitial de d s and d s , both mapping distal to the novo deletion at q -q in Ç % of cases. the common deletion region (hudson et al. ). in addi- deletion spans a region of Ç mb and invariably in- tion, marker atc c , mapping õ mb from the dis- volves the paternally derived homologue (robinson et tal deletion breakpoint (s. l. christian and d. h. led- al. ). for most patients the distal breakpoint ap- better, unpublished data), was used. pcr assays were pears to be located within a single yac (kuwano et al. performed as described elsewhere (christian et al. ; ), whereas two consistent breakpoint hot spots hudson et al. ). the results of the microsatellite have been identified on the proximal side; one (class i) analysis are shown in table , and examples of the analy- lies in the region between the centromere and d s / sis performed on two independent families are shown d s , and the other (class ii) lies between in figure . d s /d s and d s , with each account- three patients were deleted for the d s /d s ing for approximately half of the deletions (christian markers, thus being classified as class i – deletion patients. et al. ). this finding was consistent with the known frequency the relatively high frequency of deletions, significant of the class i breakpoints among pws patients. the lack clustering of the breakpoints in pws deletion patients, of a proximal marker on the deleted allele in these pa- and the finding of a similar location for the breakpoints tients precluded assessment of the haplotype for the re- in small inv dup( ) has led to the hypothesis that small gion comprising the deletion. of the seven class ii pa- regions in proximal q may contain sequences leading tients, five demonstrated a paternal recombination event to instability (knoll et al. ; huang et al. ). between the markers flanking the common deletion re- recently, preliminary data for a low-copy repeat associ- gion. the genetic distance between marker d s and ated with a novel evolutionarily conserved gene family marker d s has been estimated as . cm in males spanning the proximal and distal breakpoint regions has (robinson and lalande ). marker d s maps been reported (ji et al. ). cm proximal to d s (hudson et al. ). there- in order to analyze the mechanism underlying dele- fore, when the genetic distance between d s and tions in pws, we genotyped three-generation families d s in male meiosis is taken into account, the iden- of pws-deletion patients, using microsatellite markers tification, in five of seven cases, of a different grandparen- flanking the common deletion region. each patient was tal origin for the alleles flanking the deletion is signifi- known to be deleted for the interval from d s to cantly different from the expected frequency (x gabrb , by fish and/or other molecular techniques. Å . , p Å . ). this finding is highly suggestive of an unequal crossover occurring in the paternal meio-peripheral blood samples were obtained, with appro- / a d$$jy - - : : ajhga uc-ajhg letters to the editor table microsatellite analysis of chromosome markers in pws families proximal markersa distal markersa grandparental- grandparental- allele allele mechanism of family d s d s inheritance d s d s inheritance rearrangement n.t. gf (ab), gm (cd), gm gf (be), gm (ad), n.t. gf interchromosomal f (ad), p(bd), f (bd), p (bc), m (bc) m (bc) n.t. gm (cd), f (bd), gf gm (ab), f (bd), gm (bb), f (ab), gm interchromosomal p (ab), m (aa) p (bc), m (bc) p (bb), m (ab) n.t. gf (ab), gm (ac), gm gf (ac), gm (bc), gf (ac), gm (bc), gm intrachromosomal f (bc), p (cd), f (bc), p (bb), f (bc), p (bb), m (bd) m (bb) m (bb) n.t. gf (cd), gm (ab), gf gf (bd), gm (ad), gf (de), gm (ac), gm interchromosomal f (ad), p (bd), f (ad), p (ac), f (ad), p (ab), m (bb) m (bc) m (bc) n.t. gm (ab), f (bc), gf gm (aa), f (ac), gm (bb), f (bc), gf intrachromosomal p (cd), m (de) p (cc), m (bc) p (ac), m (ac) gf (ad), gm (ce), gf (ab), gm (bb), gm gf (bd), gm (ce), gf (cd), gm (be), gf interchromosomal f (cd), p (bc), f (bb), p (bb), f (de), p (ad), f (bc), p (ac), m (bb) m (bc) m (ab) m (ae) n.t. gm (cd), f (bd), gm gm (bb), f (bc), gm (cd), f (cd), gf interchromosomal p (ad), m (ab) p (cc), m (ac) p (bc), m (ab) a n.t. Å not tested; gf Å grandfather; gm Å grandmother; f Å father; p Å patient; and m Å mother. sis, at the breakpoint, as being the mechanism leading junction fragment suggests the occurrence of strand-ex- change events mediated through the transposase activityto the deletion. asymmetrical exchanges between nonsister chroma- (reiter et al. ). duplications of q -q have been reported in only a few instances (reviewed in clay-tids in meiosis i have previously been demonstrated in humans and are the basis of a number of genetic dis- ton-smith et al. ), and it is unclear whether any of these represent the reciprocal event of deletion by un-eases. when the related sequences are part of tandemly arrayed homologous genes, nonhomologous recombi- equal crossing-over. the paucity of duplication cases compared with deletions of this region is interesting andnation may lead to the formation of chimeric genes, such as the globin-chain variants in some hemoglobin- indicates either that duplications occur much less fre- quently or that a milder phenotype causes them to beopathies (weatherall et al. ) and the red-green pigment genes involved in color-vision abnormalities ascertained much less often. conversely, our study also has shown that in two(nathans et al. ). in other instances, the deletion/ duplication event may arise from the unequal recombi- pws families the data were consistent with an intrachro- mosomal mechanism being responsible for the deletion.nation between repetitive elements interspersed throughout a genomic region. a misalignment between it cannot be ruled out that this observation might be due to a classical crossover occurring, at meiosis i, betweenalu-repetitive sequences has been demonstrated in du- plications of the ldl-receptor gene (lehrman et al. d s /d s and d s /d s , followed or preceded by an unequal homologous recombination. ) and the hypoxanthine phosphoribosyltransfer- ase gene (marcus et al. ). however, a recombination event occurring twice in such a small interval would be unlikely. intrachromosomalrecent studies have demonstrated the presence of two copies of a large repetitive element (cmt a-rep) rearrangements have been infrequently demonstrated as a mechanism leading to human diseases. one exampleflanking the region duplicated in charcot-marie-tooth disease type ia (cmt a) patients and deleted in pa- is the intrachromosomal recombination occurring at xq , between gene a, a small intronless gene withintients with hereditary neuropathy with liability to pres- sure palsies (hnpp), at p (pentao et al ; intron of the factor viii gene, and one of the two copies of gene a located on the same chromosome, chance et al. ). a model of unequal crossing-over between misaligned cmt a-rep homologues has been kb telomeric to the factor viii gene, a recombination causing severe hemophilia (lakich et al. ). molecu-proposed for the generation of both the cmt a dupli- cation and the hnpp deletion. interestingly, the pres- lar studies have demonstrated that this rearrangement arises almost exclusively in male meioses (rossiter et al.ence of a mariner transposon – like element at the / a d$$jy - - : : ajhga uc-ajhg letters to the editor romeo carrozzo, , , elena rossi, susan l. christian, kirk kittikamron, chiara livieri, andrea corrias, lucia pucci, alberto fois, paolo simi, laura bosio, luciano beccaria, orsetta zuffardi, , and david h. ledbetter servizio di genetica medica, laboratorio di citogenetica, clinica pediatrica iii, universita degli studi milano, ospedale san raffaele, and telethon institute of genetics and medicine (tigem), san raffaele biomedical science park, milan; clinica pediatrica, biologia generale e genetica medica, universita degli studi, pavia; divisione di endocrinologia pediatrica, ospedale infantile regina margherita, turin; clinica pediatrica universita degli studi, siena; u.o. azienda ospedaliera pisana, pisa; figure representative microsatellite analysis of two families, and center for medical genetics, the university of illustrating an interchromosomal deletion event and an intrachromo- chicago, chicago somal deletion event. data for one proximal and one distal marker are presented. in each example an arrow indicates the inheritance of the patient’s paternal chromosome that bears the pws deletion. a, acknowledgmentsinterchromosomal mechanism is inferred where the proximal marker, d s , shows grandpaternal inheritance whereas the distal marker, we wish to thank drs. g. casari and a. ballabio for helpful atc c , shows grandmaternal inheritance of the deleted chromo- discussion, and we wish to thank nehal bhatt for assistance some. b, intrachromosomal mechanism, demonstrated by the fact that in preparation of the figures.both the proximal marker, d s , and the distal marker, d s , show grandmaternal inheritance of the deleted chromosome. gf Å grandfather; gm Å grandmother; f Å father; p Å patient; and m referencesÅ mother. chance pf, abbas n, lensch mw, pentao l, roa bb, patel pi, lupski jr ( ) two autosomal dominant neuropa- thies result from reciprocal dna duplication/deletion of a ). the in-cis mechanism leading to the deletions in region on chromosome . hum mol genet : – pws patients can be related either to an exchange of christian sl, robinson wp, huang b, mutirangura a, linechromosomal material between sister chromatids or to mr, nakao m, surti u, et al ( ) molecular characteriza-the formation of an intrachromosomal loop, either dur- tion of two proximal deletion breakpoint regions in bothing meiosis or as a somatic event, followed by an exci- prader-willi and angelman syndrome patients. am j humsion of the chromosomal material lying between the re- genet : – combining regions. clayton-smith j, webb t, cheng xj, pembrey me, malcolm the overall findings of this study are similar to s ( ) duplication of chromosome in the region those of dutly and schinzel ( ) for williams syn- q - in a patient with developmental delay and ataxia drome; this latter syndrome is due to a -kb inter- with similarities to angelman syndrome. j med genet : stitial deletion at q . . segregation analysis of – grandparental markers flanking the deleted region in dutly f, schinzel a ( ) unequal interchromosomal rear- rangements may result in elastin gene deletions causing the patients and their parents demonstrated a recom- williams-beuren syndrome. hum mol genet : – bination between grandmaternal and grandpaternal huang b, crolla ja, christian sl, wolf-ledbetter me, machamarkers on chromosome , at the site of the deletion me, papenhausen pn, ledbetter dh ( ) refined molec-in two of the three cases, whereas an intrachromoso- ular characterization of the breakpoints in small inv dup( )mal recombination appeared to have occurred in the chromosomes. hum genet : – remaining cases. hudson tj, stein ld, gerety ss, ma j, castle ab, silva j, the deletion occurring at the q -q band and slonim dk, et al ( ) an sts-based map of the human leading to pws syndrome appears, therefore, to be due genome. science : – to both inter- and intrachromosomal rearrangements. ji y, buiting k, walkowicz mj, johnson dk, amos-langraf given the similar frequency and extent of the maternal jm, stubbs l, horsthemke b, et al ( ) an evolutionarily deletion at q -q , which is responsible for % conserved gene associated with the common deletion of angelman syndrome cases, it will be interesting to breakpoint regions in the prader-willi/angelman syn- determine whether these two deletion mechanisms are dromes. am j hum genet suppl :a knoll jhm, wagstaff j, lalande m ( ) cytogenetic andsimilarly responsible for that disorder. / a d$$jy - - : : ajhga uc-ajhg letters to the editor molecular studies in the prader-willi and angelman syn- usually first evident in the pelvic girdle and then spread- drome: an overview. am j med genet : – ing to the upper limbs while sparing facial muscles. on- kuwano a, mutirangura a, dittrich b, buiting k, horsthemke set of symptoms is variable (mean age years), and b, saitoh s, niikawa n, et al ( ) molecular dissection creatine kinase (ck) levels are elevated from early in- of the prader-willi/angelman syndrome region ( q - ) fancy and remain elevated until the individual is well by yac cloning and fish analysis. hum mol genet : – past this age (jackson and strehler ). affected indi- viduals are often wheelchair-bound – years afterlakich d, kazazian hh, antonarakis s, gitschier j ( ) the onset of symptoms. there is variability in the age ofinversions disrupting the factor viii gene are a common death, and most individuals die in middle age.cause of severe haemophilia a. nat genet : – the gene for lgmd a was first linked to chromo-lehrman ma, goldstein jl, russel dw, brown ms ( ) duplication of seven exons in ldl receptor gene caused by some by beckmann et al. ( ). allamand et al. alu-alu recombinatiion in a subject with familial hypercho- ( ) narrowed the region to q . -q . , using lesterolemia. cell : – large kindreds from the isle of la réunion and the marcus s, hellegren d, lambert b, fallstrom sp, wahlstrom northern indiana amish. the muscle-specific calcium- j ( ) duplication in the hypoxanthine phosphoribosyl- activated neutral protease or calpain (canp ) gene, transferase gene caused by alu-alu recombination in a pa- a possible candidate gene in the q . -q . region, tient with lesch nyhan syndrome. hum genet : – was examined by richard et al. ( ). fifteen different nathans j, piantanida tp, eddy rl, shows tb, hogness ds mutations, including missense, splice-site, frameshift,( ) molecular genetics of inherited variation in human and nonsense mutations, were identified in lgmd acolor vision. science : – patients, and many others have subsequently been iden-pentao l, wise ca, chinault ac, patel pi, lupski jr ( ) tified. since the affected patients in la réunion belongcharcot-marie-tooth type a duplication appears to arise from recombination at repeat sequences flanking the . mb to a genetic isolate presumed to derive from a single monomer unit. nat genet : – ancestor who immigrated to the island during the late reiter lt, murakami t, koeuth t, pentao l, muzny d, gibbs s, it was expected that all affected patients from ra, lupski jr ( ) a recombination hotspot responsible la réunion would have the same lgmd a mutation. for two inherited peripheral neuropathies is located near a paradoxically, six different mutations were identified. mariner transposon-like element. nat genet : – this paradox led the investigators to propose digenic robinson wp, bottani a, yagang x, balakrishnan j, binkert inheritance, in which the founder effect is due to an f, mächler m, prader a, et al ( ) molecular, cytogenetic, as-yet-unidentified modulating gene (either nuclear orand clinical investigation of prader-willi syndrome patients. mitochondrial) that permits mutations in canp to ex-am j hum genet : – press lgmd a. this hypothesis does not require therobinson wp, lalande m ( ) sex-specific meiotic recom- presence of multiple mutations, since the genetic princi-bination in the prader-willi/angelman syndrome imprinted region. hum mol genet : – ples of digenic inheritance should apply to all popula- rossiter jp, young m, kimberland ml, hutter p, ketterling tions with lgmd caused by calpain- mutations. rp, gitschier j, horst j, et al ( ) factor viii gene inver- in the amish of northern indiana, richard et al. sions causing severe hemophilia a originate almost exclu- ( ) identified a single mutation in canp sively in male germ cells. hum mol genet : – (cggrcag, r q) in a homozygous state in affected weatherall dj, clegg jb, higgs dr, wood wg ( ) the patients. the authors speculated that the complete pene- hemoglobinopathies. in: scriver cr, beaudet al, sly ws, trance of this disease in the amish and in the other valle d (eds) the metabolic and molecular bases of inherited lgmd a pedigrees might also be under the control of adisease, th ed. mcgraw-hill, new york, pp – second locus. one expectation of the digenic hypothesis address for correspondence and reprints: dr. david h. ledbetter, center for would be that some individuals homozygous for the mu- medical genetics, the university of chicago, east th street, room r , tation would be clinically unaffected (i.e., ck is normalchicago, il - . e-mail: dhl@genetics.uchicago.edu � by the american society of human genetics. all rights reserved. and there are no physical findings suggestive of lgmd). - / / - $ . because of the possible implications in genetic testing and counseling, we analyzed dna samples from amish individuals in one northern indiana county foram. j. hum. genet. : – , the presence of the r q mutation, looking for evi- dna studies of limb-girdle muscular dystrophy dence of phenotypically normal r q homozygotes. type a in the amish exclude a modifying we initiated the countywide screen by first identifying mitochondrial gene and show no evidence for a carrier couples. appropriate informed consent was ob- modifying nuclear gene tained from all individuals. in order to identify r q to the editor: carriers in this population, we specifically approached members of previously studied nuclear lgmd alimb-girdle muscular dystrophy type a (lgmd a) is characterized by slowly progressive muscle weakness, families from this county. we obtained blood samples / a d$$jy - - : : ajhga uc-ajhg wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ r e v i e w p a p e r genetics of healthy aging and longevity angela r. brooks-wilson received: december / accepted: july � the author(s) . this article is published with open access at springerlink.com abstract longevity and healthy aging are among the most complex phenotypes studied to date. the heritability of age at death in adulthood is approximately %. studies of exceptionally long-lived individuals show that herita- bility is greatest at the oldest ages. linkage studies of exceptionally long-lived families now support a longevity locus on chromosome ; other putative longevity loci differ between studies. candidate gene studies have identified variants at apoe and foxo a associated with longevity; other genes show inconsistent results. genome-wide asso- ciation scans (gwas) of centenarians vs. younger controls reveal only apoe as achieving genome-wide significance (gws); however, analyses of combinations of snps or genes represented among associations that do not reach gws have identified pathways and signatures that con- verge upon genes and biological processes related to aging. the impact of these snps, which may exert joint effects, may be obscured by gene-environment interactions or inter-ethnic differences. gwas and whole genome sequencing data both show that the risk alleles defined by gwas of common complex diseases are, perhaps sur- prisingly, found in long-lived individuals, who may toler- ate them by means of protective genetic factors. such protective factors may ‘buffer’ the effects of specific risk alleles. rare alleles are also likely to contribute to healthy aging and longevity. epigenetics is quickly emerging as a critical aspect of aging and longevity. centenarians delay age-related methylation changes, and they can pass this methylation preservation ability on to their offspring. non- genetic factors, particularly lifestyle, clearly affect the development of age-related diseases and affect health and lifespan in the general population. to fully understand the desirable phenotypes of healthy aging and longevity, it will be necessary to examine whole genome data from large numbers of healthy long-lived individuals to look simul- taneously at both common and rare alleles, with impecca- ble control for population stratification and consideration of non-genetic factors such as environment. background the basis of human longevity and healthy aging, and how to achieve these desirable phenotypes, remain among the principal challenges of biology and medicine. while an understanding of lifestyle and environmental factors will maximize our ability to prevent disease and maximize health in the general population, studying the genetic basis of longevity and healthy aging in exceptional individuals is providing important biological insights. in model organ- isms it has been possible to demonstrate effects of muta- tions in genes that can extend lifespan nearly tenfold (ayyadevara et al. ). studies of inbred lab strains and of natural genetic variants in model organisms including yeast and worms (tissenbaum and guarente ), flies (paaby and schmidt ) and mice (yuan et al. ) have clearly implicated many specific genes in the lifespan of these organisms. our understanding of human lifespan stands in contrast to this, with only one consistently rep- licable genetic association, apoe, observed to date in a. r. brooks-wilson (&) canada’s michael smith genome sciences centre, bc cancer agency, west th avenue, vancouver, bc v z l , canada e-mail: abrooks-wilson@bcgsc.ca a. r. brooks-wilson department of biomedical physiology and kinesiology, simon fraser university, university drive, burnaby, bc v a s , canada hum genet doi . /s - - -z several genome-wide association scans (gwas) of lon- gevity-related traits. this may be because healthy aging and longevity are particularly complex traits, involving not only maintenance of long-term function but also absence or reduction of disease and other morbidities. it has been proposed that human lifespan is influenced not only by longevity assurance mechanisms and disease susceptibility loci but also by the environment, gene–environment interactions, and chance (cournil and kirkwood ). it will be important to understand the effects of environment (lifestyle) and of genetics, as well as how they interact to affect health and lifespan. the importance of age and aging is underscored by the recognition that all common complex diseases increase with age. questions remain about whether aging is the cause or effect of such diseases (hekimi ). the study of desirable phenotypes like longevity and healthy aging has been referred to as ‘positive biology’ (farrelly ). its premise is that understanding the basis for such desir- able traits may allow us to design interventions to improve human health. this review was intended to summarize our current understanding of genetic factors affecting the phenotypes of longevity and healthy aging in humans, including the defi- nition and heritability of these traits, and linkage, associa- tion, and sequencing studies. the surprising and novel findings that centenarians do not appear to have a relative lack of common complex disease risk alleles, and that some genetic variants appear to ‘buffer’ or protect against specific risk alleles, are discussed in detail. shorter summaries of the findings related to somatic mosaicism and the promising study of epigenetics of aging are included for completeness. aging, healthy aging, and longevity the phenotypes used in studies of the genetics of human aging are usually lifespan (age at death), longevity (long life, usually defined as being a specific advanced age or older at the time of study), exceptional longevity (defined as attaining or exceeding a specific exceptional age), or healthy aging (a combination of old age and health, often defined as freedom from specific disorders or desirable performance levels on functional tests). longevity studies focus on long-lived individuals (lli), often centenarians aged or more years. one advantage of such studies is the simplicity of phenotype definition. healthy aging can be defined in various ways, usually with regard to reaching an at least moderately old age in the absence of certain diseases or disabilities, and/or in the presence of desirable traits such as intact cognition or mobility. both types of studies should be differentiated from the study of the fundamental biological processes of aging (for example, cellular senescence). a major difference between longevity and healthy aging studies is that the former focuses on lifespan, whereas the latter is focused on healthspan. lifespan and healthspan are intimately related, however, and individuals who live exceptionally long also tend to be healthy for much of their lives. a landmark study of the health of supercentenarians (aged – ), semisupercentenarians (aged – ), centenarians (in this context aged – ), nonagenari- ans, and younger controls found that the older the age group, the greater the delay in onset of major disease (andersen et al. ). remarkably, for every category of increasing age, the hazard ratio for each of six disorders (cancer, cardiovascular disease (cvd), dementia, hyper- tension, osteoporosis, and stroke) was \ . relative to the next oldest group. this delay in disease development and postponement of cognitive and physical decline in the oldest group amounted to a compression of morbidity (fries ). based on these findings, andersen et al. ( ) suggest that a realistic and practical limit of human lifespan is – years, close to that of the oldest doc- umented person in the world to date, who lived to (robine and allard ). women have a lower mortality rate than men at every age, and women live longer than men in most human populations. any given exceptional age, therefore, is more exceptional for men than for women. as noted by sebas- tiani and perls ( ) % of us women (but only . % of men) born circa the turn of the last century lived to be . potential explanations for this difference include hormonal and immune differences, hemizygosity of the x-chromo- some in men (which may allow manifestation of unfavor- able sex-linked variants), and unrecognized confounders [reviewed in newman and murabito ( )]. the heritability of lifespan and related traits age at death in adulthood has a heritability of approxi- mately % (summarized in murabito et al. ). a population-based study of , danish twin pairs born between and found that the heritability of adult lifespan was . in men and . in women (herskind et al. ). this cohort was not only population-based but nearly non-censored and, with follow-up for years, encompassed essentially the entire human lifespan. importantly, the heritability of longevity increases with greater age. the heritability of living to at least has been estimated at . in women and . in men (sebastiani and perls ). male and female siblings of us centenarians were -fold and eightfold more likely (compared with us social security data) to reach the age of , respectively (perls et al. ). the increase in heritability of longevity at greater age is consistent between several studies. in over , scandanavian twins, hum genet heritability of longevity was negligible from age – , but increased with age thereafter (hjelmborg et al. ). long life was heritable in icelanders aged over years (gudmundsson et al. ). the siblings of okinawan centenarians show increased adult survival probability that starts at age and increases with age (willcox et al. ); the authors speculate based in part on absence of many age-related diseases from okinawan (bernstein et al. ) and other centenarians (evert et al. ), that these individuals have genetic factors that confer resistance to such diseases and increase the likelihood of reaching exceptional old age. the estimation of heritability also depends on how it is studied; murabito et al. ( ) note that the framingham heart study cohorts give much greater estimates of heritability when longevity is studied as a dichotomous trait ( % heritability for survival to and % for survival to ), compared with % heritability when age at death is treated as a continuous trait. clustering of longevity and healthy aging is observed in families. parents of centenarians born in approximately were sevenfold more likely than their contemporaries to have lived to age – ; offspring of centenarian parents showed lower prevalence of age-related disease than age- matched control groups (atzmon et al. ). exceptional familial clusters of extreme longevity have also been reported (perls et al. ). healthy aging is also heritable. reed and dick ( ) defined ‘wellness’ in male twins as achieving the age of free of heart attack, coronary surgery, stroke, diabetes or prostate cancer, and showed that this trait had a heritability exceeding %. environment and lifestyle likely constitute much of the remaining influence on human lifespan and healthspan. these factors have varied greatly over time and may not reflect the extrinsic factors that will affect the lifespan of babies born today. many members of the elderly and centenarian cohorts under study today lived through times of caloric restriction (e.g., the great depression) and grew up before the use of antibiotics and vaccines became commonplace. the selective pressures that influenced their mortality are not identical to those experienced by later generations, and this is an important consideration for study design. the importance of study design phenotype definition is particularly important in genetic studies; it affects the interpretation and meaning of results, and the ability to compare to the results of other studies. studies of longevity can include extreme longevity (defined as living beyond a specific extreme age) or age at death. studies of healthy aging may use age to disease onset, successful aging or wellness (which can also have a variety of definitions), or other phenotypes (manolio ). linkage or family-based association study designs, longi- tudinal cohort studies, or case/control designs have been used. family-based designs have the advantage of being robust to population stratification. longitudinal cohorts have the advantage of limiting sampling bias, but take time and due to practical limits of size may not contain many individuals of extreme age. sample size is a consideration for all these study designs. to date, the largest studies of lli are in the low thousands of subjects; this is much smaller than the largest studies of common complex dis- eases (which now include over , subjects), despite the likely similar modest size of many of the genetic fac- tors being sought. choice of a comparison group to contrast with excep- tionally long-lived or exceptionally healthy elderly indi- viduals is also critical. health data for lli can be compared with archived data for deceased individuals of the same birth cohort, but dna samples from an ideal comparison group (such as their birth cohort) are not available. case/control molecular genetic studies of long- lived or healthy aged individuals often compare elderly cases to younger controls. potential pitfalls of such studies include inadequate detection and control for population stratification, particularly for populations that have expe- rienced immigration of different ethnicities over time (nebel and schreiber ). the use of principal compo- nents analysis (price et al. ) or genomic controls (devlin and roeder ) can mitigate this problem, as can the conduct of studies within specific ethnic groups (bar- zilai et al. ). in the case/control design, the control group is also expected to contain individuals who will go on to become equivalent to cases; their presence in the control group reduces power. environmental factors must be acknowledged in such studies as potential confounders; inevitably, the cases and controls have lived in different times and experienced different lifestyles. a way to miti- gate some of these problems; however, is to choose con- trols that are no older than (halaschek-wiener et al. ) because in modern day developed countries, mor- tality before age is minimal. choosing a comparison group\ years of age makes the control group essentially an ‘unselected’ group with regard to mortality from age- related diseases. choosing a control group in their or , however, would exacerbate this issue, as the control group would fail to include individuals who died in their ’s or ’s. several studies, such as the longevity gene study (barzilai et al. ), the leiden longevity study (lls) (mooijaart et al. ), the new england centenarian study (necs) (terry et al. ), and the long life family study (llfs) (newman et al. ) include com- parisons of the offspring of lli (who are assumed to have inherited some longevity factors) to contemporary age- hum genet matched controls. they have observed that the offspring of lli have more favorable blood lipid profiles (barzilai et al. ; newman et al. ) and lower prevalence of hypertension and metabolic and cardiovascular disease (atzmon et al. ; westendorp et al. ; newman et al. ) and all-cause mortality (terry et al. ) than age-matched controls. comparison of the offspring of lli with their contemporaries controls for cohort effects such as variation in bmi in human populations over time; it has the limitation, however, of under-estimating the difference in phenotypes and genotypes that would presumably be observed if the lli could be compared with their largely long-deceased birth cohort. linkage studies of longevity and healthy aging linkage studies of long-lived sibships or extended pedi- grees with exceptionally long-lived individuals have iden- tified several putative and one replicated longevity linkage. in , the necs (puca et al. ) reported a -cm sib- pair based linkage scan of individuals in sibships with exceptional longevity (defined as having a proband of at least and a -year-old male or -year-old female sib). they found significant evidence for linkage of lon- gevity to a region around d s . suggestive support for this region was obtained through analysis of concordant pairs of fraternal male twins with a wellness phenotype (age at least with no overt cvd or prostate cancer) (reed et al. ). initial convergence of two linkage studies with very different phenotypes led to excitement about this region and its suspected role in longevity and health. subsequent study of the region focused in part on a regional biological candidate gene, microsomal triglyceride transfer protein (mtp), identified through haplotype anal- ysis (geesaman et al. ). in a larger and higher density linkage study (boyden and kunkel ) expanded on the initial necs resource, with a genome-wide linkage study of fami- lies with multiple long-lived sibs years and older, including / of those previously described (puca et al. ). a limitation of this study was the use of expected life span (estimated from age- and gender-specific life expectancies) for the % of subjects who were still living. this analysis of , snps found just-significant lod scores at p - and q - , as well as modest evidence for linkage at the original site, q - and possibly at q. a larger study (kerber et al. ) repli- cated the linkage of p - to extreme longevity and identified possible additional loci. working with sub- jects from the utah population database and database and population controls, including caucasian individuals aged – who showed a phenotype including both excess individual longevity (the difference between observed and expected lifespan) and excess familial lon- gevity (a weighted average of excess longevity for all family members), they used a linkage screen with , microsatellite markers to identify a strongly suggestive peak at p at the same position as boyden and kunkel. meta-analysis of linkage in the utah and new england data sets supported linkage at the chromosome locus. other linkage peaks were observed in the utah data at q - , q , and q ; meta-analysis provided additional sup- port but not outright replication for q, q, and q. the new data; however, did not support linkage to chromosome or chromosome . larger sample sets and denser and more informative linkage analyses were pointing away from the original chromosome linkage observation, and converging instead most strongly at q - . two linkage studies of successful aging in amish individuals over years of age within a single -gen- eration pedigree showed linkage to chromosomes , , and , different regions than those found in the longevity linkage studies. successful aging was defined as cogni- tively intact and without depression, high functioning, and satisfied with life. these studies (edwards et al. ; edwards ) analyzed cognitively intact amish over years old ( successfully aged and normally aged) within sub-pedigrees using , autosomal snps. linkage was found at q - , as well as association of a snp, rs , in the interval linked to the ‘successfully aged’ phenotype. the chromosome linkage identified in the amish is different from those identified in the utah and new england studies; this may reflect the different phe- notype, or may be due to genetic factors specific to the amish founder population. the largest linkage study to date was done in the multi- site european genetics of healthy aging (geha) study, which looked at european full sib-pairs over years old (beekman et al. ). geha found linkage at regions: q . , q -q , p . -p . , and q . -q . . the chromosome linkage is at a dif- ferent site from that observed in the amish study; the large chromosome region overlaps the q locus observed by kerber et al. fine mapping of these linkage regions using gwas data in a subset of unrelated nonage- narians and controls identified a snp near apoe at the q locus as significantly associated with longevity. apolipoprotein e (apoe) isoforms are known risk factors for cardiovascular disease (cvd) and alzheimer disease (ad), likely due to their involvement in inflammation, elevated lipid levels, and oxidative stress (huebbe et al. ). apoe has three main isoforms: apoe , apoe and apoe . combined modeling in the geha study showed that apoe (p = . ) and apoe (p = . - ) account for the linkage at q. the apoe linkage was characterized by absence of apoe , but enrichment for hum genet apoe among the nonagenarians. in this study the apoe allele is the stronger association, and the authors refer to apoe as a longevity gene. the multiple linkage signals observed in these studies likely indicate genetic heterogeneity of longevity and healthy aging in human populations. interestingly, the geha study observed heterogeneity among its multiple geographic regions; northern european subjects contribute most to some of the linkage peaks they observe, including the apoe locus. gender-specific effects were also observed, with a male-specific linkage peak at p and female-specific ones at q and the q apoe locus (beekman et al. ). while the lack of association at the other linkage regions in the geha study may be due to power limitations, it could also imply that multiple rare or ‘private’ variants contribute to linkage but not association at these loci. candidate gene association studies candidate genes examined for association with longevity or healthy aging or related phenotypes fall into several categories. they include genes nominated based on observations of lifespan extension in model organisms; and genes involved in lipid metabolism, immune response and inflammation, stress response, and others. candidate genes tested for association with longevity and related pheno- types have been the subject of several excellent reviews (christensen et al. ; wheeler and kim ; ferrario et al. ; newman and murabito ); an exhaustive listing is beyond the scope of this review. of the candidate genes assessed for association with longevity, variants in apoe and foxo a have been most consistently replicated, though some candidate genes have been associated with longevity phenotypes in more than one population but not in all populations tested; many more have been associated in a single study but failed to repli- cate in others (reviewed by (christensen et al. )). in a study of , healthy italians aged – , apoe was found at lower frequency and apoe at higher frequency in elderly and centenarians than in younger individuals (e.g., seripa et al. ); apoe is a putative protective factor in this context and apoe can be considered a ‘frailty’ allele (gerdes et al. ). foxo a is a homo- logue of the c. elegans daf- gene that is important in control of lifespan in the worm (hsin and kenyon ); it is part of the insulin/igf signaling pathway. foxo a variants have been associated with longevity in many populations (reviewed in wheeler and kim ). additional genes show promise of great relevance to healthy aging. a variant at cetp, for example, though inconsistently associated with longevity in different popu- lations (reviewed by (christensen et al. )), in ashkenazi jewish individuals of average age is asso- ciated not only with longevity but also with additional aging-related phenotypes including a desirable lipid profile (barzilai et al. ) and preservation of cognitive function (barzilai et al. ). other recent studies with extensive replication data are also encouraging. association of a snp in a heat shock factor gene, hsf with all-cause mortality was seen in the longitudinal rotterdam study ( , par- ticipants and , deaths), with replication in eight pop- ulation-based cohorts (broer ). other candidate genes have been associated with lon- gevity or healthy aging phenotypes in some but not all studies. mtp, identified as a regional candidate at the q locus, failed to show replication of association with lon- gevity in larger studies of approximately lli each (beekman et al. ; bathum et al. ; nebel et al. ). progeria genes have shown association with lon- gevity in some studies. a haplotype of snps at lmna, the gene that is mutated in hutchinson-gilford progeria, was associated with long life (age [ years) in lli and controls, and remained significant upon meta-analysis of , subjects from four independent samples (conne- ely et al. ). polymorphisms at wrn have shown inconsistent associations with age (castro et al. ; kuningas et al. ). sirtuins mediate the effects of caloric restriction, a non-genetic factor known to increase life span in many organisms. the effect of polymorphisms in sirtuin genes (sirt - ) on longevity and age-related diseases was reviewed by polito et al. ( ). there is evidence that variants in sirt (rose et al. ) are associated with longevity. a functional promoter variant at dna repair gene exo was associated with longevity in female centenarians (nebel et al. ), but tagsnps in the gene showed no association with longevity in men (morris ). given the multifactorial nature and likely genetic het- erogeneity of healthy aging and longevity, as well as environmental influences on these complex traits, it may not be reasonable to expect that replication of candidate gene studies would be uniform between populations. rea- sons for lack of replication include limitations of sample size, rarity (low minor allele frequency) of actual variants, and small true effect sizes. poorly designed or under- powered studies will result in false positives that legiti- mately fail to replicate. for studies of longevity and heal- thy aging, in particular, differences in phenotype or type of study will also result in findings that are non-uniform between studies. while larger case/control studies are fre- quently suggested as a solution to the limitations of pres- ent-day association studies, combining data from populations with different lifestyles and genetic back- grounds, even if well-matched for ethnicity, may obscure true association signals. hum genet genome-wide association studies to date, snps in or near apoe are the only ones to achieve genome-wide significance (gws, generally p b - ) in genome-wide association studies (gwas) of lifespan-related traits. in three gwas of long- lived individuals vs. younger controls, apoe was signifi- cantly associated with longevity at the genome-wide level. the first of these included long lived ( – years) and , control ( – years) from german biobanks and replication in an independent set of german samples ( cases aged – , controls aged – ) (nebel et al. ). only rs near apoc and in linkage disequilibrium (ld) with apoe achieved gws. gwas of unrelated nonagenarians (average age ) from lon- gevity families in the lls vs. , controls (average age ) showed similar results (deelen et al. a). only one of snps carried forward to meta-analysis with , nonagenarian cases and , younger controls from the rotterdam study, the leiden ? study and the danish cohort reached gws, rs at tomm near apoe. meta-analysis of the apoe and apoe snps showed significant associations of both snps with lon- gevity, with e being protective of long life (or . , ci . – . , p = . - ), and e being deleterious (or . , ci . – . , p = . - ). a third lon- gevity gwas (sebastiani et al. ) included three phases: a discovery phase with new england cente- narians (aged – , many with a family history of extreme longevity) vs. controls genetically matched by means of principal components analysis; a first replication in centenarians ( – ) vs. genetically matched controls; and a second replication with additional cen- tenarians ( – ) and unmatched controls. of , snps analyzed only one, tomm snp rs near apoe, reached gws. inverse association of apoe with longevity (p = . - ) was also detectable in the southern italian centenarians study (sics) of llis aged – and young controls aged – (malovini et al. ), despite the known lower frequency of the e allele in southern, as compared with northern, europe (haddy ). other gwas of lifespan-related phenotypes revealed no associations that were significant at the genome-wide level. a gwas of the framingham health study (lun- etta et al. ) ( original cohort and , off- spring individuals, members of the largest families in the study) revealed no gws snps for any of five aging-related phenotypes. newman et al. (newman et al. ) meta-analyzed four cohort studies in the cohorts for heart and aging research in genomic epidemiology (charge) consortium for survival to at least years of age. cases were , people who achieved survival to at least ; controls were , participants who died aged – . snps were genotyped and imputed in sub- jects of european ancestry, with systematic elimination of outliers and correction for population stratification. replication was carried out in the lls ( long-lived probands and partners of their offspring and blood bank donors) and the danish cohort survey ( , long-lived participants and danish twin study controls aged – ). no snps reached genome- wide significance. walter et al. ( ) conducted a meta-analysis of gwas of nine longitudinal cohort studies in the charge consortium, including , unselected people of euro- pean ancestry. they analyzed two continuous traits, all- cause mortality, and event-free survival (where ‘event’ was defined as myocardial infarction, heart failure, stroke, dementia, hip fracture, or cancer). no snps reached gws for either phenotype. snps near apoe reached only nominal significance in the charge study (walter et al. ), in contrast to the results of gwas of centenarians, in which apoe has been a significant and replicable finding. the charge meta-analysis contained few extremely old individuals, and so in comparison with centenarian studies or those targeting long-lived healthy individuals, has examined earlier mortality and events, a different phenotype. the framingham study gwas (lunetta et al. ), which also showed no gws snps also represents a much younger group, on average, than studies of oldest old or centenarians. this may mean that different genes and variants may come into play in dif- ferent phases of aging, with apoe being most relevant at older ages. earlier mortality is often related to lifestyle as well, and the heritability of aging is lower at younger ages, as described above. a genome-wide association study of copy number variants (cnvs) in the rotterdam study rs cohort, with replication in the rs cohort and the fhs, found that large common deletions are associated with mortality (vs. sur- vival) at old age (kuningas et al. a). they tested common cnv regions and measures of cnv burden for association with mortality during follow-up. a higher burden of cnvs of kb or more in size was associated with mortality. two specific regions were also associated with mortality, p . and q . . the p . associ- ation, which would survive bonferroni correction for tests, includes insertions and deletions which were ana- lyzed together relative to non-carriers; it contains genes including some related to longevity or complex diseases. the q . region contains no genes and is characterized only by deletions. runs of homozygosity, which can indicate presence of recessive loci, were not associated with survival to old age in this cohort (kuningas et al. b). hum genet analyses of phenotypes that may influence long-term good health have also been undertaken. personality traits are associated with healthy aging and longevity (terrac- ciano et al. ). in the llfs, a gwas of five personality factors in families with , individuals and repli- cation in , other subjects identified a locus associated with agreeableness, and identified several significant age snp interactions that may affect longevity through effects on personality (bae et al. ). in contrast to the results of longevity gwas, gwas of common complex diseases have revealed hundreds of snps associated with cancers, cvd, diabetes and other age-related diseases, albeit with increasing numbers of associations found with increasing gwas size. one explanation for this may be that the phenotypes of healthy aging and longevity may be much more complex than those of these complex diseases, in part because they often (depending on phenotype definition) involve absence of specific complex diseases. if gwas studies of survival to elderly ages are even more confounded by environmental (e) factors than gwas of diseases, combining studies from different populations in pooled or meta-analyses may complicate the e effects even more. in studies of older individuals, it is particularly hard to control for e factors experienced over many decades of life. effects of variants that do not achieve p b - snps at only one locus, apoe, have achieved bonferroni- corrected levels of gws in gwas of longevity. by current standards these gwas, which involved fewer than , centenarians, or a few , nonagenarians, are modest in size. larger gwas may in theory allow additional snps to achieve this threshold. there are other indications, how- ever, that support the idea that snps that do not reach this threshold of gws may be biologically important, either individually or through their joint effects. several studies used a variety of techniques to analyze collections of nominally longevity-associated snps to determine if they act in concert to affect lifespan. in the framingham study gwas of aging-related phenotypes (lunetta et al. ) observed that snps in some candidate genes, including snps near the werner syndrome gene wrn and foxo a, as well as gapdh, kl, lepr, pon , psen , and sod were associated with age at death. kulminski and culminskaya ( ) used framingham affymetrix k snp data to perform gwas of four endophenotypes (cvd, cancer, systolic blood pressure, and total cholesterol) to identify snps that were associated at p \ - with at least one endopheno- type. genes at or near these snps were enriched in terms of gene ontology annotations related to aging-relevant processes. yashin et al. ( ) hypothesized that lifespan depends on the number of small-effect longevity alleles present in individual genomes. they re-analyzed fra- mingham k snp data and identified snps asso- ciated at p \ - . the number of these snps carried by an individual correlated with lifespan and explained % of its variance; in contrast, randomly chosen snps did not correlate with lifespan. gene set analysis of gwas data from the lls and rotterdam studies was used to show that genes in the insulin/igf- signaling (iis) and telomere maintenance tm pathways are associated with longevity (deelen b). and gwas snps were identified within kb of iis and tm genes, respectively. both pathways were associated with longevity. nine iis genes (akt , akt , foxo , igf , ins, pik ca, sgk, sgk , and ywhag) and one tm gene (pot ) were the main determinants of the association. sebastiani et al. ( ) constructed a model in which snps showed % sensitivity and % specificity to predict longevity in their gwas discovery set, and – % specificity and – % sensitivity in indepen- dent sets. they call this a ‘genetic signature of exceptional longevity’. these snps explain nearly % of the heri- tability of extreme longevity. they find that the tomm snp near apoe alone has poor predictive value; removing it from the model reduces specificity and sensitivity by only %. the snps include in genes, including lmna, wrn, sod , cdkn a, sorcs and sorcs , and gip. this set of genes is highly and significantly enriched for those related to alzheimer dis- ease ( genes), related to dementia, to tauopathies, to cad, and several to neoplasms. gwas of the sics study of lli and younger controls identified snps that reached a permutation- defined level of genome-wide significance of p \ - (malovini et al. ). among them was rs at the calcium/calmodulin-dependent protein kinase iv (cam- kiv) that replicated in additional lli and con- trols. malovini et al. demonstrate that camk phosphorylates and activates survival proteins foxo a, akt, and sirt . homozygous carriers of the minor allele had lower camkiv protein expression and were under- represented among lli’s, consistent with a deleterious effect of this allele on longevity. the biological relevance of other snps besides those at apoe is also strongly supported by similarities between the results of human gwas and mouse lifespan studies. eight of the ten top charge snps detected by gwas, but which did not achieve gws, correspond to mouse lifespan quantitative trait loci (qtl) (murabito et al. ). these studies connect gwas findings that do not reach gws with many genes that are relevant to aging or age-related diseases. in several cases, this convergence hum genet with genes of biological interest is statistically unlikely to be due to chance and is likely to reflect the presence of true association signals that are not consistent enough to be replicated predictably as candidate genes or achieve gws, or have effects that are too subtle to be detected individ- ually. such potential true signals may be more affected by ‘e’ factors than those that have been replicated, i.e., apoe and foxo a. as pointed out by yashin et al., the same sets of variants would not be expected to work in all populations because of differences in environment (yashin et al. ). the extremely long-lived do not lack risk alleles for common complex diseases several recent studies have shown that centenarians do not carry smaller numbers of risk alleles for common complex diseases than average people. in an important paper in , beekman et al. ( ) studied two case/control collections: ( ) nonagenarian siblings (mean age ) from the lls vs. unrelated younger controls (mean age ), and ( ) long-lived individuals over (mean age ) from the pop-based leiden ? study vs. , younger controls (mean age ) from the netherlands twin register. they looked at snps known to be associated with cvd, cancer or type diabetes (t d). the cases and controls each carried an average of disease risk alleles. the distribution of risk alleles was the same in elderly and young subjects. beekman et al. note that ‘‘gwas-identi- fied disease risk alleles do not compromise human lon- gevity’’ and suggest that a lack of rare disease factors, or the presence of protective factors, is at work in the long- lived individuals. it is important to note, however, that cvd, cancer, and t d are diseases that have very clear lifestyle components and that part of the effect could be due to lifestyle differences. mooijaart et al. ( ) extended this observation the following year, showing that ‘‘snps associated with t d and identified by gwas are not major determinants of the beneficial glucose tolerance that characterizes familial longevity.’’ they compared the offspring of the lls long- lived individuals with the offspring’s spouses and other controls. the lls offspring had a better metabolic profile and better glucose tolerance than same-age controls, although the frequency of known t d snps did not differ between the two groups. when individuals were compared within each group, however, glucose levels did correlate with the number of t d snps. they speculate that the lls offspring may have protective factors that improve their metabolic profile and glucose tolerance in spite of the presence of t d gwas snps. this compar- ison, using same-age groups of individuals, clearly points to protective genetic factors contributing to preservation of a healthy phenotype, rather than lifestyle and environ- mental factors that should be very similar (at least in adulthood) between the offspring and their spouses. sebastiani et al. ( ) also noted that there was not a substantial difference in the numbers of , known dis- ease-associated snps in centenarians and controls. a similar observation was made in their whole genome sequence data from one male and one female supercente- narian (sebastiani et al. ). these important and perhaps surprising results show that extreme longevity, and the long-term good health that often accompanies it, is not incompatible with the presence of many disease risk alleles. at least for the common snps associated with common complex diseases, it is not the absence of ‘bad’ alleles, but more likely the presence of ‘good’ alleles that influences longevity, though effects of ‘good’ environmental factors may also contribute. protec- tive factors of some kind may allow these risk variants to not be manifest. these results also have implications beyond the study of longevity—in an age when substantial effort is being invested in personalized disease risk pre- diction, the presence of many disease alleles that are non- penetrant in some individuals potentially complicates pre- dictions of disease. do ‘good’ variants protect against ‘bad’ ones? one mechanism for a lack of effect by an undesirable allele is the ‘buffering’ mechanism explored by barzilai et al. they propose that some individuals who show exceptional longevity may do so despite the presence of unfavorable alleles because those alleles are buffered by favorable alleles in other genes (bergman et al. ). they suggest that buffering gene variants (longevity variants) will show a monotonic increase in frequency from early old age ( ) to later ages; examples of buffering genotypes are cetp vv, apoc cc, and a ? deletion in adipoq. buffered alleles, in contrast, should show a u-shaped fre- quency curve, higher at younger ages, dipping low in early old age, and then increasing in the exceptionally old (who have the ‘buffering’ protective genotype that allows dis- ease-related variants to accumulate); examples of buffered genotypes are heterozygotes for deleterious alleles of klotho and lpa. importantly, bergman et al. use a cross-sectional study design, with , subjects in their – th decades of life to show experimental support for the buffering hypothesis; their data support the idea that cetp vv genotype buffers the deleterious effects of an lpa genotype. they show a genetic interaction between cetp genotype and lpa; lpa heterozygotes with the cetp iv/ ii genotypes monotonically decrease in frequency with age, but those in cetp vv individuals increase from age onward. they argue that case/control analyses are hum genet insufficient to reveal this effect because it does not reveal the shape of the allele frequency age curve. earlier observations are also explained by a buffering mechanism. de benedictis et al. ( ) described an age- related convex trajectory of a apob-vntr genotype that they interpret as consistent with crossing mortality curves relevant to subgroups of individuals with different genotypes. a x-sectional study of healthy aging sub- jects from to years free of clinically apparent dis- ease genotyped variants in apoa , apoc , and apoa (garasto et al. ). they noted that an allele of apoa that correlated with higher serum ldl-c was paradoxi- cally increased in frequency in the oldest old. the authors called it ‘‘another genetic paradox of centenarians.’’ while this observation could reflect population stratification in the different age groups, it may also be due to the u-shaped curve of a buffered gene. the buffering mechanism may also explain some of the inconsistency in the findings for mtp. huffman et al. ( ) find that mtp cc is a deleterious genotype that is buffered by any of three longevity genotypes of cetp, apoc , or adipoq. mtp cc shows a u-shaped curve, declining ages – , and then dramatically increasing in those who live or more years. if this mtp genotype is observed at high frequency in centenarians, but only in the presence of specific protective variants, this may in part explain why the linkage at chromosome was not observed consistently between studies. buffering has been described in model organisms. the heat-shock protein hsp is known to buffer genetic var- iation in drosophila, allowing it to accumulate under neutral conditions (rutherford and lindquist ). such a gene is known as a phenotypic capacitor, and it masks the presence of phenotypic variation. it is interesting to spec- ulate that protective genetic variants carried by centenari- ans may be capacitors for the disease risk variants we now know they carry at, on average, the same frequency as other people. identification of buffering/capacitor genes and study of their function will be necessary to understand the longevity phenotype. it will also be important to determine if such capacitors operate in healthy aging as well as extreme longevity. because such variants are likely rare, intensive study of rare individuals at the upper ends of the human lifespan and healthspan, perhaps by whole genome sequencing and examination of unusual variants they carry, is paramount. the interaction between buffering and buffered genes and genotypes also has implications for study design. the exquisite studies carried out by barzilai’s group are done in a single well-defined ethnicity, ashkenazi jewish individ- uals. since a buffered gene will only show a distinctive u-shaped curve in the presence of its buffer, and a buffer may only be advantageous in the presence of a deleterious gene that it buffers, this underscores the importance of avoiding population stratification in such studies. if some of the associations detected to date in case/control studies of healthy aging and longevity are actually underlain by genotypes with u-shaped curves, the choice of ages for the cases and controls will greatly affect whether an associa- tion is detected, and may explain some failures of associ- ations to replicate. finally, the concept of buffering genes has implications for the use of centenarians, or excep- tionally healthy elderly individuals as super-controls for disease studies; if the exceptional elderly are healthy because of a protective factor rather than lack of a disease allele, their use as an extreme comparison group may not necessarily be helpful. do differences in lifestyle affect these studies? given that lifestyle is expected to have a greater impact than genetics on healthy aging, it seems unlikely that dif- ferences in lifestyle are not confounding association studies of longevity and healthy aging. it is challenging to quantify lifestyle in an optimal comparison group for, for example, centenarians. younger control groups inevitably have dif- ferent lifestyles than the elderly had at their age. for example, the charge consortium (newman et al. ), which compared individuals who survived to at least to those who died aged – , found that the younger con- trols had higher rates of smoking. the longevity gene study overcame the birth cohort limitation using pre-existing lifestyle data from , nhanes controls of the same birth cohort as ash- kenazi jewish individuals aged – (rajpathak et al. ). they found no obvious differences in lifestyle and suggested that the long-lived individuals may interact with lifestyle factors differently than others. this study, how- ever, did note subtle differences between the long-lived and comparison groups. they saw significantly fewer obese men, more overweight women, and fewer obese women in the long-lived group; in addition, more control men smoked. these differences, combined with recall limita- tions of the long-lived group, imply that this analysis may have missed many small lifestyle differences that could add up to substantial health differences. it will likely be diffi- cult to take into account all but the largest lifestyle factors when planning gxe studies of longevity and healthy aging. biomarkers of exposures may vary not only with exposure but also over time, complicating the use of such methods for these phenotypes. association studies of mitochondrial variants mitochondria are thought to be important to aging due to their key roles in oxidative phosphorylation, cell hum genet metabolism, and apoptosis. a relationship of variation in the mitochondrial genome with health and/or longevity is implied by the observation that age at death correlates more closely with the age at death of a person’s mother more so than that of the father (brand et al. ). asso- ciations of mitochondrial genome sequence variants or haplogroups (combinations of specific variants that corre- late with specific populations) with healthy aging or lon- gevity have been noted in many populations including, for example, italian (de benedictis et al. ), japanese (tanaka et al. ), amish (courtenay et al. ), chi- nese uygur (ren et al. ), costa rican (castri et al. ), ashkenazi jewish (iwata et al. ), irish (ross et al. ), and finnish individuals (niemi et al. ). the associations observed are inconsistent between popu- lations and do not involve the same variant or haplogroup. this lack of consistency may be due in part to the relatively small size of many of these studies. three common prob- lems have been noted about such studies: inadequate matching of cases and controls, inadequate correction for multiple tests, and undetected population stratification (shlush et al. ). interestingly, when the frequencies of different mito- chondrial haplogroups are plotted for italian individuals aged to over , the curve shapes observed include monotonic increase for haplogroup j, and a u-shaped curve for haplotype h (de benedictis et al. ), reminiscent of the ‘longevity’ and ‘buffered’ variants described earlier. a variant at the origin of replication of the mitochondrial heavy strand, c t, has been observed at higher fre- quencies in centenarians, both through inheritance and through somatic increase in frequency, with some indi- viduals achieving homoplasmy for this variant in their lymphocytes and monocytes, but not in granulocytes; a selective advantage of achieving high frequency of this variant in at least some cell types has been suggested (zhang et al. ). interactions between nuclear genome variants and both inherited and somatic mitochondrial variants have also been suggested to play a role in aging and longevity (santoro et al. ; tranah ). genome sequencing sebastiani et al. ( ) recently reported the whole genome sequencing of one male and one female supercentenarian of european ancestry from the necs. the genomes of these exceptionally long-lived individuals were similar, in terms of the rate of nonsynonymous snps and number of indels, to other genomes sequenced to date. they have a similar number of known disease-associated variants to other genomes showing that their exceptional lifespan does not seem to be due to lack of known disease-associated variants. it is possible, though, that they failed to inherit a combination of variants that would have acted together to cause disease. both supercentenarians lacked apoe alleles. they do not carry most of the longevity variants reported previously in the literature, implying that these known variants are not necessary for longevity. it is pos- sible that they carry as yet undiscovered protective vari- ants. one per cent of the variants observed were novel. interestingly, an excess of coding region variants was seen in genes closest to gwas-identified longevity variants, an observation that supports the idea that rare variants of these genes may contribute to the longevity phenotype. telomeres in healthy aging and longevity telomeres are indisputably important to aging. telomeres shorten with age and are considered to be a biomarker of age. the role of telomere biology in healthy aging and disease was recently reviewed (zhu et al. ). leukocyte telomere length (ltl) has been correlated with measures of health and ability in elderly individuals. in a commu- nity-based cohort of - to -year-olds, ltl was associ- ated with more years of healthy life; ltl was suggested to be a biomarker of healthy aging (njajou et al. ). louisiana healthy aging study results concurred with this observation; ltl was correlated with measures of healthy aging in an age-dependent way (kim et al. ). ltl was also found to correlate positively with physical ability (but not cognitive function) in danish twins aged at least years (bendix et al. ) and inversely with disability in american seniors (risques et al. ). ashkenazi centenarians and their offspring also showed longer telo- meres, for their age, than controls; longer telomeres cor- related with less disease (atzmon et al. ). in contrast, in a study of canadian ‘super-seniors’ (individuals aged at least and never diagnosed with cancer, cardiovascular disease, alzheimer disease, major pulmonary disease or diabetes) the healthy oldest-old did not have exceptional telomere length for their age, but showed less variability in telomere length than mid-life controls, implying that they may be selected for optimal rather than extreme telomere length (halaschek-wiener et al. ). variation in genes involved in telomere maintenance has also been associated with longevity. one snp at sirt (kim et al. ) and one in terc (soerensen et al. ) are associated with both ltl and longevity. detailed analysis of tert and terc in ashkenazi centenarians showed an excess of genetic variation in both genes in the centenarians and identified a tert haplotype associated with extreme longevity (atzmon et al. ). gene set analysis of gwas data also supported the relevance of telomere maintenance (deelen et al. ). overall, the relationship between telomeres, aging, healthy aging, and longevity is multi-layered. telomere maintenance is an hum genet important process in aging, and also a biomarker of it. ltl is a biomarker of aging and of healthy aging. variation in telomere maintenance genes appears to affect both telo- mere length, and life span and health span in humans. somatic genetics of aging two recent large-scale analyses of data from gwas studies have established that mosaicism for large genomic alterations increases with age (laurie et al. ; jacobs et al. ). in one study, data for , subjects found that \ . % of people aged \ , and – % of elderly ( . % in subjects [ years), have detectable mosaicism in peripheral blood. age was a significant predictor of mosaic status, but sex, ancestry, and smoking status were not. the second study used data from , cancer cases and , controls from gwas studies and found detectable clonal mosaicism in . % of individuals. in the cancer-free controls, they found mosaicism in . % of those \ years old and in . % of those aged – , a significant difference (p = . - ). somatic mosa- icism (heteroplasmy) of the mitochondrial genome also increases over the lifespan (sondheimer et al. ). of course, telomere shortening is another somatic genomic change that occurs over the human lifespan. such somatic changes are both a genetic aspect of aging and an aging- related phenotype. epigenetics and longevity/aging epigenetics, at the interface between the genome and the environment, is emerging as an important factor in lon- gevity, and has been the subject of recent excellent reviews (gravina and vijg ; ben-avraham et al. ). methylation patterns change with age, and discordance in methylation between mz twins also increases with age (talens et al. ), an observation consistent with the effect of environment and lifestyle on the epigenome. studies of dna methylation support the idea that aging is associated with a relaxation of epigenetic control and that this epigenetic drift may affect the development of aging- related diseases (gravina and vijg ). an epigenome- wide association scan (ewas) identified age-related dif- ferentially methylated regions as well as differentially methylated regions associated with age-related phenotypes (bell et al. ). whole genome bisulfite sequencing of dna from cd ? t cells of a centenarian and a newborn identified differentially methylated regions that were usu- ally hypomethylated and less correlated with methylation of adjacent cpg dinucleotides in the centenarian (heyn et al. ). these results support the idea that small cumulative dna methylation changes accumulate over a lifetime. age-related temporal changes in dna methylation also show significant familial clustering, indicating that methylation maintenance is a familial trait (bjornsson et al. ). a study of dna methylation in centenarians and their offspring compared with the off- spring of non-long-lived individuals and young individuals showed that the offspring of the centenarians delay age- related methylation changes (gentilini ). a landmark paper by hannum et al. ( ) offers an explanation for this familiality. they used methylome analysis to compare human aging rates in individuals of age – and iden- tify methylation qtls (meqtls) (including one at methyl- cpg binding domain protein ) that affect it. indeed, trans- generational epigenetic inheritance of extended lifespan has been demonstrated in c. elegans (greer et al. ). it is likely that the effects of epigenetic changes mani- fest in part by effects on gene expression. longevity- selected lines of drosophila show gene expression profiles that are similar to younger control flies (sarup et al. ). this type of observation is more difficult to make in humans, however. several human studies have compared gene expression between lli and younger individuals. blood mirna expression differences between lli and younger controls identified genes known to be differen- tially expressed in age-related diseases (elsharawy et al. ). this study design, however, does not allow dis- crimination between genes that are differentially expressed because they are involved in longevity, related to chrono- logical age, or affected by environmental differences between the old and young groups. a cross-sectional analysis of individuals aged – , and centenarians, was used to identify a mirna, mir- *, whose expression declined with age but was preserved at youthful levels in the centenarians (gombar et al. ). the leiden lon- gevity study, however, used lli and their offspring to show that rptor in the mtor pathway is differentially expressed between the offspring of the lli and their spouses (passtoors et al. ). the study design issues that are important to avoid confounding by lifestyle factors in studies of inherited factors will be even more important in gene expression studies. it seems likely that as yet unidentified genetic factors and lifestyle practices that help us maintain a favorable epigenetic profile and optimal gene expression will be important in longevity and healthy aging. conclusions to date, studies of longevity and healthy aging have shown few consistent and many inconsistent results. this is probably due in part to the nature of the variants we seek to find, which may be rare or even private, may act in concert or as a ‘signature’, and which may buffer against the hum genet presence of other variants. it is also due to inter-ethnic differences, population stratification, differences in phe- notype definition and study design, effects of and con- founding by known and unrecognized non-genetic factors in part due to cohort effects, and exacerbation by insuffi- cient sample size. nevertheless, the results of longevity studies to date establish their importance for our under- standing of health and disease. only one gene has emerged as consistently found in studies of lli, apoe, though another, foxo a, was replicated in multiple candidate gene studies. additional candidate genes are associated in more than one but not all studies. many of these correspond to signals that hover below gws in gwas studies, and when analyzed together for commonalities of pathways or other processes by means such as gene set analysis, as a group are significantly associated with these phenotypes. epigenetics is quickly emerging as a critical aspect of aging and longevity. centenarians delay age-related methylation changes, and they can pass this methylation preservation ability on to their offspring, probably via genetic variants that affect methylation qtls. importantly and perhaps surprisingly, centenarians appear to have the same numbers of gwas-identified common disease variants as ordinary people, and yet they have lived long and, to a great extent, free of disease. specific examples support the idea that centenarians may have advantageous alleles of ‘buffering’ genes that allow them to remain healthy despite the presence of ‘buffered’ deleterious alleles in other genes. it will be important to determine how generalizeable this is—does it apply only to a few genes, or is it a general mechanism for suppression of disadvantageous alleles of many genes? as others have pointed out (bergman et al. ), a longevity gene that buffered frailty alleles in several other genes would be a desirable drug development tool. understanding this will affect not only personalized medicine but also our overall interpretation of genomes, and could even give us the information needed for rational design of agents to re- create this desirable scenario in those not lucky enough to inherit it. open access this article is distributed under the terms of the creative commons attribution license which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. references andersen sl, sebastiani p, dworkis da, feldman l, perls tt ( ) health span approximates life span among many supercentenar- ians: compression of morbidity at the approximate limit of life span. j gerontol a biol sci 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variants on human longevity. aging (albany ny) : – yuan r, peters ll, paigen b ( ) mice as a mammalian model for research on the genetics of aging. ilar j : – zhang j, asin-cayuela j, fish j, michikawa y, bonafe m, olivieri f, passarino g, de benedictis g, franceschi c, attardi g ( ) strikingly higher frequency in centenarians and twins of mtdna mutation causing remodeling of replication origin in leukocytes. proc natl acad sci usa : – zhu h, belcher m, van der harst p ( ) healthy aging and disease: role for telomere biology? clin sci (lond) : – hum genet 	genetics of healthy aging and longevity 	abstract 	background 	aging, healthy aging, and longevity 	the heritability of lifespan and related traits 	the importance of study design 	linkage studies of longevity and healthy aging 	candidate gene association studies 	genome-wide association studies 	effects of variants that do not achieve p le x minus 	the extremely long-lived do not lack risk alleles for common complex diseases 	do ‘good’ variants protect against ‘bad’ ones? 	do differences in lifestyle affect these studies? 	association studies of mitochondrial variants 	genome sequencing 	telomeres in healthy aging and longevity 	somatic genetics of aging 	epigenetics and longevity/aging 	conclusions 	open access 	references virtual mentor virtual mentor american medical association journal of ethics june , volume , number : - . journal discussion the ethics of genetic testing in psychiatry aaron d. besterman, md hoop jg. ethical considerations in psychiatric genetics. harv rev psychiatry. ; ( ): - . before the nazi regime focused its efforts on the extermination of european jewry, one of its early eugenics directives aimed at cleansing the gene pool of psychiatric disease. thousands of mentally ill patients were either sterilized against their will or murdered as part of the german racial hygiene movement. shamefully, this movement was largely fueled by the research and ideologies of early psychiatric geneticists, such as ernst rudin [ ]. similar efforts were undertaken in the united states to prevent the reproduction of those deemed mentally insufficient. this practice was famously upheld by the supreme court in buck v. bell in , with justice oliver holmes jr. concluding his argument by declaring that “three generations of imbeciles are enough” in reference to the plaintiff carrie buck, her mother, and her daughter [ ]. given a past marred by such ethically deplorable behavior, it is vital for current medical professionals to have a thorough understanding of the ethical issues involved in psychiatric genetics and a structured framework with which to evaluate new psychiatric genetic tests. in “ethical considerations in psychiatric genetics,” jinger hoop provides a succinct introduction to this topic with practical suggestions on how to assess the ethical acceptability of psychiatric genetic tests. summary of “ethical considerations in psychiatric genetics” in her review article, dr. hoop touches on four broad ethical issues in psychiatric genetics: ( ) the use of genetic testing to predict future health outcomes, ( ) the psychosocial consequences of genetic testing, ( ) the effect of genetic testing on family members and communities, and ( ) the ethics of the use of emerging genetic technologies [ ]. she begins by describing how the landscape of clinical psychiatric genetics has changed from one focused on rare, monogenic disorders such as huntington’s disease (hd) to one that will be dedicated increasingly to common, polygenic diseases, such as schizophrenia. in hd, the ethical debate concerns the risks and benefits of providing a patient with precise information about his or her medical future—if you have the hd gene, you will get the disease if you live until its onset, typically middle age. if little can be done to prevent the onset or mitigate the effects of the disease being tested for, is it ethical to test for the disease at all? virtual mentor, june —vol www.virtualmentor.org in contrast, hundreds or thousands of genes each confer a small increase in risk for developing schizophrenia. testing a patient for such genes provides only an estimate of his or her increased risk for developing schizophrenia (e.g. percent, when risk is percent in the general population) with much uncertainty remaining. hoop highlights the difficulty patients are likely to have in interpreting such results. some patients may think they are completely free of risk if they test negative for a risk gene, while others may believe that they are destined to develop a disease if they test positive, when really they may have only a slightly greater-than-average risk. hoop identifies psychological consequences, insurance and employment discrimination, and social stigmatization as the three main psychosocial risks of psychiatric genetic testing. while genetic testing in general certainly has psychological consequences, the consequences of genetic testing for psychiatric disorders may be more profound. many view the psyche as an inextricable component of one’s being. thus, being told of a genetic “defect” in one’s psyche may be particularly distressing. the findings of several studies hoop cites are consistent with this theory; they suggest that “learning one has a ‘good’ or ‘bad’ [psychiatric] genotype may have a more profound psychological impact than learning one’s absolute risk of illness”[ ]. another potential hazard of genetic testing in psychiatry is that employers, insurers, and the general public may discriminate against individuals based on their psychiatric genotypes. hoop discusses a case of three young men who lost their jobs because their employer discovered that they had first-degree relatives with schizophrenia [ ]. there is a risk that similar discrimination could occur based on one’s genotype, a risk that may be more likely to happen with mental than with physical disease. in hopes of limiting such discrimination, the united states congress enacted the genetic information and nondiscrimination act of [ ]. genetic information can also have serious implications for relatives of a person being tested and for those who come from a similar ethnic background. for example, if a grandparent developed alzheimer’s disease, his or her grandchild (of adult age) may wish to get tested for disease-related genes to determine the risk for developing alzheimer’s. if the grandchild’s parent does not want to know his or her risk, an ethical dilemma arises in which the parent’s right to not to know must be weighed against the child’s right to know. similarly, ethical conflicts may arise during population-based genetic testing in reproductively isolated groups, such as the amish or ashkenazi jews. testing can have great public health benefits by identifying severe genetic conditions that are present in these groups. but this benefit must be carefully weighed against the risk that finding a genetic predisposition for illness, especially mental illness, could reinforce preexisting stigmas and provide a false basis for discrimination and bigotry. www.virtualmentor.org virtual mentor, june —vol at the time of hoop’s writing, many candidate genes had been identified, but virtually no genes had been conclusively linked to psychiatric disease, with the exception of apoe for alzheimer’s disease. without a definitive link between gene and disease, genetic testing may be highly unethical. with significant advancements in the field, however, direct-to-consumer or physician-requested genetic testing will certainly play a future role in the management of psychiatric illness. it will fall to the clinician to interpret the results and advise patients on how to proceed. unfortunately, data from the most recent studies published (up to ) [ ] indicate that the vast majority of psychiatrists are insufficiently trained in genetics to provide this service and that there are too few genetic counselors to fill in the gaps. hoop then applies burke et al.’s framework for categorizing ethical considerations of genetic tests to psychiatric testing specifically. according to burke’s frameworks, the primary ethical consideration in psychiatric genetic testing is nonmaleficence, doing no harm, as few effective treatments can be provided based on test results and tests have low predictive power. hoop proposes an expanded framework that includes additional factors, such as psychosocial risk, the level of stigma of the condition, and the newness of the test to provide a more robust evaluation of whether genetic tests for psychiatric disorders are ethical. lastly, hoop emphasizes the importance of designing “prospective evaluations of the outcomes of psychiatric genetic counseling and testing…[to] complement empirical ethics research methods”[ ]. she argues that the knowledge gained from such studies will help prevent genetic discrimination and improve public trust in psychiatric genetic research and testing. discussion hoop’s piece stands out in the field of bioethics not only as a review of important ethical issues in psychiatric genetics but also as a source of a novel, structured framework for evaluating the ethical nuances of new psychiatric genetic tests. this is a particularly timely contribution to the field, as our knowledge about the genetic underpinnings of mental illness has been advancing at a furious pace. for example, many genomic deletions and duplications have been detected in patients with autism spectrum disorders [ ]. this has led to recent guideline changes recommending chromosomal microarray analysis as part of an initial work-up for children who display autistic-type behaviors [ ]. similar advances have been made in schizophrenia, with strong evidence for associations with vasoactive intestinal peptide receptor (vipr ), neurexin (nrxn ), and transcription factor (tcf ) [ ]. many more genes involved in psychiatric disease are likely to emerge in the near future, with the thousand-dollar genome, once a distant dream, now at our fingertips [ ]. furthermore, some genetically informed pharmacotherapies are now in development, from mglur antagonists for fragile-x syndrome to pi k inhibitors for schizophrenia [ , ] with such advances, nonmaleficence will no longer be the prevailing ethical principle dictating psychiatric genetic testing. instead, as hoop virtual mentor, june —vol www.virtualmentor.org proposes, justice, respect for autonomy, psychosocial risk and stigma of disease will all have to be strongly considered before pursuing genetic testing for psychiatric illness. references . schulze tg, fangerau h, propping p. from degeneration to genetic susceptibility, from eugenics to genethics, from bezugsziffer to lod score: the history of psychiatric genetics. int rev psychiatry. ; ( ): - . . kevles dj. from eugenics to patents: genetics, law, and human rights. ann hum genet. ; ( ): - . . hoop jg. ethical considerations in psychiatric genetics. harv rev psychiatry. ; ( ): - . . hoop, . . hoop, - . . hoop, . . hoop, . . morrow em. genomic copy number variation in disorders of cognitive development. j am acad child adolesc psychiatry. ; ( ): - . . shen y, dies ka, holm ia, et al. clinical genetic testing for patients with autism spectrum disorders. pediatrics. ; ( ):e -e . . gejman pv, sanders ar, kendler ks. genetics of schizophrenia: new findings and challenges. annu rev genomics hum genet. ; : - . . eisenstein m. oxford nanopore announcement sets sequencing sector abuzz. nat biotechnol. ; ( ): - . . krueger dd, bear mf. toward fulfilling the promise of molecular medicine in fragile x syndrome. annu rev med. ; : - . . marder sr, roth b, sullivan pf, et al. advancing drug discovery for schizophrenia. ann n y acad sci. ; : - . aaron d. besterman, md, graduated from new york medical college in and entered the residency program in psychiatry at the university of california, san francisco. his interests lie at the intersection of child psychiatry and medical genetics. related in vm a physician’s duty to warn third parties of hereditary risks, september emerging dilemmas in newborn testing, september acknowledgments the author would like to thank fan lee, jeffrey besterman, md, and paul nestadt, md, for their helpful comments and feedback. the viewpoints expressed on this site are those of the authors and do not necessarily reflect the views and policies of the ama. copyright american medical association. all rights reserved. www.virtualmentor.org virtual mentor, june —vol http://virtualmentor.ama-assn.org/ / /hlaw - .html http://virtualmentor.ama-assn.org/ / /pfor - .html wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // 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phd; john a. rumberger, md, phd; patrick f. sheedy ii, md; alan r. shuldiner, md; patricia a. peyser, phd; braxton d. mitchell, phd background—coronary artery calcification (cac) is associated with an increased risk of cardiovascular disease; little is known, however, about thoracic aortic calcification (ac). our goal was to characterize risk factors for cac and ac and to estimate the genetic contribution to their variation. methods and results—the presence and quantity of cac and ac were measured with electron beam computed tomography and fasting blood tests and cardiovascular risk factors were obtained in asymptomatic amish subjects. cac prevalence was higher in men than women ( % versus %; p� . ), although there was no sex difference in ac prevalence ( % and % in men and women, respectively; p� . ). age was more strongly associated with ac presence (odds ratio [or], . for years) than cac presence (or, . for years) (homogeneity p� . ). subjects with ac had a . -fold higher odds of having cac. heritabilities of cac and ac presence were . � . (p� . ) and . � . (p� . ), respectively, whereas the heritabilities of quantity of cac and ac were . � . (p� . ) and . � . (p� . ), respectively. the genetic correlation between cac and ac quantity was . � . , whereas the environmental correlation between these traits was . � . . conclusions—cac and ac have similar risk factors, except male gender is associated only with cac and age is more strongly associated with ac. the patterns of correlations suggest that cac and ac share some common sets of genes and environmental factors, although it is likely that separate genes and environmental factors also influence calcification at each site. (circulation. ; : - .) key words: aging � aorta � atherosclerosis � coronary disease � epidemiology � genetics � imaging atherosclerosis is a systemic disease that can affectmultiple vascular beds. noninvasive imaging of coro- nary artery calcification (cac) can be used to assess cardio- vascular disease (cvd) risk, especially in intermediate-risk patients. the quantity of cac by electron beam computed tomography (ct) correlates directly with the quantity of coronary atherosclerotic plaque in necropsy studies. quan- tification of cac in asymptomatic and symptomatic adults by electron beam ct predicts risk for future cvd events. – clinical perspective p aortic calcification (ac) frequently is seen on ct scans, although its importance is not well understood. there is moderate concordance between the presence of cac and ac ; however, calcification may be detectable years ear- lier in the aorta than in the coronary arteries. , it has been established that the presence of ac by plain radiograph or ct scanning is associated with cvd, – although it is not clear whether this association is secondary to the presence of cac. the tendency of ac and cac to occur together, especially in older individuals, also has made it difficult to sort out the degree to which there may be site-specific differences in the risk factors for development of calcifi- cation. there are different types of vascular calcification based on the location of the calcification within the arterial wall: intimal calcification, which occurs within atheroscle- rotic plaques, and medial calcification, which is less associated with atherosclerosis and more related to meta- bolic processes such as diabetes and renal disease. although cac is almost always found in the intima, ac may found in either the intimal or medial layers. to contrast the determinants of calcification in these vascular beds, we measured calcification in the coronary arteries and thoracic and upper abdominal aortas in an amish received may , ; accepted november , . from the division of cardiology, department of medicine, johns hopkins university, baltimore, md (w.p.); department of epidemiology, university of michigan school of public health, ann arbor (l.f.b., p.a.p.); division of endocrinology, diabetes, and nutrition, department of medicine, university of maryland, baltimore (k.a.r., h.s., a.r.s., b.d.m.); department of epidemiology, johns hopkins university bloomberg school of public health, baltimore, md (w.p., y.-c.c.); department of cardiovascular diseases, ohio state university, columbus (j.a.r.); department of diagnostic radiology, mayo clinic and foundation, rochester, minn (p.f.s.); and geriatrics research and education clinical center, veterans administration medical center, baltimore, md (a.r.s.). reprint requests to wendy post, md, ms, blalock h, n wolfe st, baltimore, md . e-mail wpost@jhmi.edu © american heart association, inc. circulation is available at http://www.circulationaha.org doi: . /circulationaha. . epidemiology d ow nloaded from http://ahajournals.org by on a pril , population from lancaster county (pennsylvania). the old order amish are a socially and culturally homogeneous population characterized by large families. they eschew modern technology, including many modern cvd prevention therapies. the goals of the present study were to characterize and contrast risk factors for the presence of cac and ac and to test explicitly for homogeneity of risk factor effects in this unique population of subjects not selected for clinical symp- toms. in addition, we assessed the genetic contribution to variation in calcification at the sites and then further estimated the extent to which the same genes jointly contrib- ute to this variation. methods the amish family calcification study (afcs) was initiated in to identify the determinants of vascular calcification and to evaluate the relationship between calcification of bone and vascular tissue among members of the old order amish community in lancaster county. subjects were initially recruited into the afcs on the basis of their participation in an earlier family study of bone mineral density, although recruitment guidelines were later modified to allow other interested individuals in the community to participate. all first- and second-degree relatives of these new participants also were invited to participate in the afcs. recruitment efforts were made without regard to cvd health status. the protocol was approved by the institutional review boards of the university of maryland and other participating institutions. informed consent, including permis- sion to contact relatives, was obtained before participation. the analyses presented in this report are based on afcs participants � years of age examined from the start of recruitment in march through july (n� ). excluded from the analysis were individuals with a self-reported prior cvd event. the final sample included individuals. all afcs participants underwent a detailed clinical examina- tion at the amish research clinic in strasburg (pa), including assessment of potential risk factors for cvd and a medical history interview. examinations were conducted after an over- night fast. height and weight were measured with a stadiometer and calibrated scale with shoes removed and in light clothing. body mass index (kg/m ) was calculated. systolic (first phase) blood pressure (bp) and diastolic (fifth phase) bp were obtained in triplicate with a standard sphygmomanometer with the subject sitting for at least minutes. for these analyses, bp was defined as the mean of the second and third measurements. pulse pressure was defined as the difference between the systolic and diastolic bps. medication lists were obtained at the participant’s home by a study nurse. smoking habits were recorded by questionnaire; subjects were classified as current smokers or not. blood samples were obtained for determination of fasting glucose and lipid levels. glucose concentrations were assayed with a beckman glucose analyzer using the glucose oxidase method. lipid concentrations were assayed by quest diagnostics (baltimore, md). low-density lipoprotein cholesterol levels were calculated using the friedewald equation. diabetes mellitus was defined as a fasting glucose � mg/dl or use of diabetes medications; impaired fasting glucose was defined as glucose � mg/dl. the electron beam ct scans were performed on an imatron c- scanner (ge, south san francisco, calif) in timonium (md). cac scanning was performed using a standard protocol that included to three-mm contiguous transverse slices between the aortic root and the apex of the heart, gated to % of the rr interval and obtained during a single breathhold. the extent of calcium in the thoracic aorta was assessed by scanning between the superior aspect of the aortic arch and the superior pole of the kidney at -mm intervals. we elected to scan only the thoracic and upper abdominal aorta to limit radiation exposure. cac was quantified using the agatston method, which incorpo- rates both density and area. the presence of calcification was defined as a density � hounsfield units in � contiguous pixels (� mm ). the sum of the scores in the left main, left anterior descending, circumflex, and right coronary arteries was considered the cac score. ac also was measured using the agatston method, and the sum of all the ac lesions was considered the ac score. all scans were scored by a single experienced cardiologist (j.r.) using accuimage (accuimage diagnostic corp, san francisco, calif) software. interscan repro- ducibility for quantification of cac with this software was previously reported to range from % to %. the inter- reader and intrareader reproducibilities were each � %. re- producibility of accuimage measures of ac has not been reported; however, the median reproducibility of ac agatston score using a similar scoring software system was % with interreader and intrareader reproducibilities of % and %, respectively. we defined presence of calcification as a cac (or ac) score � . statistical analyses age- and sex-adjusted associations of each risk factor with cac and ac presence were assessed with logistic regression. initial analyses assessed quadratic effects of age and interaction effects of risk factors with sex and age on calcification. none of the quadratic effects of age achieved statistical significance and thus were omitted in the final models. to test whether the magnitude of association of each risk factor with calcification differed between the sites, we tested for equivalence of the odds ratios (ors) by computing a mantel-haenszel � statistic based on the weighted sum of the squared deviations of the stratum-specific log ors from their weighted mean. sibship membership was included in these models as a random effect to account for residual correlations in calcification liability existing among siblings. we performed multivariate analy- ses using a forward stepwise procedure, including variables that were significant in the age- and sex-adjusted analyses as eligible for inclusion. we assessed the correlation between cac and ac using the quantitatively distributed calcification scores. to minimize skew- ness, we transformed the calcification score before analysis by adding and obtaining the natural logarithm of the value—ie, ln (score� ). age- and sex-adjusted pearson correlations were estimated in the entire group, and age-adjusted pearson correla- tions were estimated stratified by sex. similarly, age- and sex-adjusted pearson correlations were estimated for subjects � and � years of age, and age-adjusted pearson correlations were estimated stratified by sex in the age groups. to evaluate possible genetic effects on cac and ac, we made more full use of the family structures using the variance compo- nent framework to partition the total variance in calcification into effects attributable to the measured covariates (eg, age, sex, and risk factors), the additive genetic variance (estimated from the covariance among relatives), and a residual environmental effect corresponding to the amount of unexplained variation in the phenotype. the additive genetic variance, or heritability, corre- sponds to the proportion of trait variance attributable to the additive effects of genes after accounting for the effects of measured covariates. the heritability of calcification presence (and score) was assessed by comparing the likelihood of a model in which the polygenic (heritability) component was included as an independent variable with a nested model in which the effect of this component was constrained to . the likelihood ratio statistic is distributed asymptotically as a � statistic with degrees of freedom equal to the difference in number of parameters in the models being compared. significance testing was carried out by likelihood ratio test using the solar software program. we computed the relative proportions of the total variance in quantity of cac and ac, ie, ln (score� ), explained by the measured covariates and unmeasured genes. these components of variance were computed by evaluating the proportionate reduc- tion in the total variance in calcification scores associated with adding in each component. the residual variance that was not circulation february , d ow nloaded from http://ahajournals.org by on a pril , accounted for by the components corresponds to the residual environmental variance or the proportion of the variance attrib- utable to unmeasured environmental factors, including measure- ment error. we extended the univariate variance component analysis of a single calcification trait to a bivariate analysis to estimate potential shared genetic and environmental effects on the joint distribution of quantitative cac and ac scores. the transformed cac and ac scores were treated as a joint dependent variable, and the joint trait variance was deconstructed into components attributable to measured covariates, additive genetic effects, and residual environmental effects (as before) and to a shared genetic and environmental component. , these latter components, corresponding to the genetic and environmental correlations between these traits, reflect the degree to which shared genes and environmental factors influence their distribution. the genetic correlations may be interpreted as a measure of the degree of pleiotropy between the traits. the hypothesis of polygenic pleiotropy was evaluated by a likelihood ratio test, calculated as the difference in � �ln likelihoods between a restricted model (the value of the genetic correlation fixed at , indicating no shared genetic variance) and an unrestricted model (all parame- ters are estimated). the authors had full access to take full responsibility for the integrity of the data. all authors have read and agree to the manuscript as written. results the final sample size of asymptomatic subjects without a history of cvd includes individuals from sibships, with sibships ranging in size from to . additional relationship types were identified by linking study subjects into larger pedigrees through their unexamined (or examined) parents. these individuals could be combined into multiplex pedigrees, ranging in size from to examined individuals and representing sibpairs, parent-offspring pairs, avuncular pairs, and first-cousin pairs. characteristics of subjects are presented in table . the prevalence of diabetes mellitus in this sample was low ( . %); therefore, we combined subjects with impaired fasting glucose (n� ) and diabetes mellitus (n� ) into a single category for analysis. twenty percent of men reported that they currently smoked (primarily pipes and cigars). few subjects reported current use of bp- or cholesterol-lowering medications ( . % and . %, respectively). the prevalence of cac was markedly higher in men than women ( . % versus . %; age-adjusted p� . ), although there was no sex difference in the prevalence of ac ( . % and . % in men and women, respectively; age- adjusted p� . ). figure shows the prevalences of calci- fication in men and women by age group. similarly, median calcification scores were higher for men than women in the coronary arteries but not in the aorta. ors showing the degree of association between each risk factor and presence of detectable calcification at each site are shown in table . presence of cac and presence of ac were each significantly associated with increasing age and total and low-density lipoprotein cholesterol. additionally, pres- ence of cac was significantly associated with male gender, higher systolic bp and pulse pressure, higher triglycerides, lower high-density lipoprotein cholesterol, and history of smoking. we tested for sex and age by risk factor interactions on cac or ac, except we could not test for sex interactions with smoking because none of the women smoked. the association between diabetes/impaired fasting glucose and cac was significantly stronger in women (age-adjusted or, . ; % ci, . to . ) than men (age- adjusted or, . ; % ci, . to . ; interaction p� . ). comparisons of ors, carried out to test whether the magnitude of the associations differed between cac and ac, revealed differences in risk factor association patterns. first, male gender was strongly associated with presence of cac (or, . ; % ci, . to . ) but not with presence of ac (or for male gender, . ; % ci, . to . ; homogeneity p� . ). second, age was more strongly associated with ac presence (or, . for -year age difference; % ci, . to . ) than with cac presence (or, . for -year age difference; % ci, . to . ; homogeneity p� . ). virtually iden- tical results were obtained when these analyses were repeated including an additional adjustment for the pres- ence of calcification at the other site. to identify the subset of risk factors independently associated with the presence of cac or ac, we performed a multivariate analysis in which all risk factors signifi- cantly associated with cac or ac in the age- and sex-adjusted analyses were eligible for inclusion. after forward stepwise elimination, age, sex, pulse pressure, total and high-density lipoprotein cholesterol, and smoking status remained independently associated with the pres- table . characteristics of the study population men (n� ) women (n� ) age, y . � . . � . systolic bp, mm hg . � . . � . diastolic bp, mm hg . � . . � . pulse pressure, mm hg . � . . � . bmi, kg/m . � . . � . diabetes/ifg . . cholesterol, mg/dl . � . . � . triglyceride, mg/dl . � . . � . hdl cholesterol, mg/dl . � . . � . ldl cholesterol, mg/dl . � . . � . current smoking, % . . cholesterol medications, % . . bp medications, % . . diabetes medications, % . . presence of cac,* % . . presence of ac, % . . median cac score ( %, %)* . ( , ) ( , ) median ac score ( %, %)† . ( , ) . ( , ) bmi indicates body mass index; ifg, impaired fasting glucose; hdl, high-density lipoprotein; and ldl, low-density lipoprotein. values are mean�sd or frequency. *p� . , adjusted for age. †ac scores were missing in participants. post et al determinants of coronary and aortic calcification d ow nloaded from http://ahajournals.org by on a pril , ence of cac. the variables independently associated with the presence of ac in multivariate analysis included age, diabetes/impaired fasting glucose, low-density lipoprotein cholesterol, and smoking status. the presence of calcification in site was associated with the presence of calcification at the other site (age-, sex-, and sibship membership–adjusted or, . ; % ci, . to . ; p� . for subjects with ac having cac). additional analyses revealed this association to be stronger among those � years of age (adjusted or, . ; % ci, . to . ; p� . ) than among those � years of age (adjusted or, . ; % ci, . to . ; p� . ), possibly because the prevalence of calcification is low before years of age, having not yet appeared in many susceptible individuals. these same trends were apparent when the correlations in calcification scores at the sites were compared. as shown in table , the magni- tudes of the correlations in calcification scores are approx- imately similar between men and women and are stronger in subjects � years of age than in subjects � years of age. we next evaluated the genetic contributions to calcifi- cation at the sites. after the variation in age and sex was accounted for, the residual heritability of cac presence was estimated at . � . (p� . ), and the residual heritability of ac presence was estimated at . � . (p� . ). these residual heritability estimates in- creased slightly with additional adjustment for other co- variates (to . � . and . � . for cac and ac presence, respectively). additional analyses were con- ducted on the quantitative calcification scores. the resid- ual heritability of the cac score was . � . (p� . ), whereas heritability of the ac score was . � . (p� . ). the heritability of cac and ac scores was considerably higher in those � years of age (cac; h � . , p� . ; ac: h � . , p� . ) than in those � years of age (cac: h � ; ac: h � . , p� . ). figure shows the components of variance for cac and ac scores. the sectors in the pie chart correspond to the proportion of the total phenotypic variance attributable to variation in age and sex, other measured covariates (ie, those achieving statistical significance in table ), addi- tive genetic effects, and residual effects (ie, unmeasured environmental factors, including measurement error). the residual environmental component was computed as the remainder of the phenotypic variance that was not ex- plained by the measured covariates and genetic effects. the proportion of the variance attributable to additive genetic effects estimated from this analysis was less than the residual heritability estimated in the previous analyses because the residual heritability corresponds to the propor- tion of the unexplained variation accounted for by genes (ie, after accounting for all measured covariate effects), whereas the proportion of the variance attributable to genetic effects shown in figure reflects the proportion of the total phenotypic variation accounted for by genes. age accounted for a larger proportion of the total phenotypic variation for ac than cac ( % versus %, respec- tively), with a larger proportion attributable to sex for cac than ac ( % versus . %, respectively). the other measured covariates explained % of the variability for both cac and ac. genetic factors accounted for % and % of the total phenotypic variation for cac and ac score, respectively. further analyses were carried out to assess whether common genetic and environmental determinants influ- ence variation in calcification scores at both sites. the genetic and environmental correlations between cac and ac score were . � . and . � . , respectively. these correlations both differed significantly from (ge- netic correlation, p� . ; environmental correlation, p� . ), indicating that genes and environmental fac- tors unique to each site contribute to variation in both traits. the environmental correlation between cac and ac scores also differed significantly from (p� . ), suggesting that some common environmental factors jointly influence variation in these traits. in contrast, the genetic correlation between cac and ac scores did not differ significantly from (p� . ), although the standard error associated with this estimate was large. discussion our analyses revealed not only many similar epidemiological patterns between calcification in the coronary arteries and figure . unadjusted prevalences of cac (a) and ac (b) by age decades stratified by gender. circulation february , d ow nloaded from http://ahajournals.org by on a pril , thoracic aorta but also some significant differences. the most striking difference in risk factor associations between the presence of detectable cac and the presence of detectable ac was the lack of a gender difference in the prevalence of ac. in contrast, there is a well-known male excess in the prevalence of cac seen in the present study and others, reflecting the known gender differences in cvd events in the united states. in line with our findings, dixon et al previously reported little overall difference in the prevalence of abdominal ac between men and women. little is known regarding the relationship between ac and peripheral artery disease; it is interesting to note, however, that a recent analysis of the national health and nutrition examination survey population– based survey demonstrated a similar table . age- and sex-adjusted ors for the association between selected risk factors and presence of cac and thoracic ac age- and sex-adjusted cac (n� ) age- and sex-adjusted ac (n� ) or ( % ci) p or ( % ci) p homogeneity p* (orcac�orac) male gender . ( . to . ) � . . ( . to . ) . . age ( y) . ( . to . ) � . . ( . to . ) � . . systolic bp ( mm hg) . ( . to . ) . . ( . to . ) . . diastolic bp ( mm hg) . ( . to . ) . . ( . to . ) . . pulse pressure ( mm hg) . ( . to . ) . . ( . to . ) . . bmi ( kg/m ) . ( . to . ) . . ( . to . ) . . diabetes/ifg vs normal† women . ( . to . ) . . ( . to . ) . . men . ( . to . ) . . total cholesterol ( mg/dl) . ( . to . ) � . . ( . to . ) . . natural log of triglycerides ( . )‡ . ( . to . ) . . ( . to . ) . . hdl cholesterol ( mg/dl) . ( . to . ) . . ( . to . ) . . ldl cholesterol ( mg/dl) . ( . to . ) � . . ( . to . ) . . smoking status (current vs not) . ( . to . ) . . ( . to . ) . . or indicates odds ratio; bmi, body mass index; ifg, impaired fasting glucose; hdl, high-density lipoprotein; and ldl, low-density lipoprotein. all p values (except age and sex) were adjusted for age, sex, and family structure. *see text for a description of the computation of the homogeneity probability value. †association between diabetes/ifg and cac was stronger in women than men (gender interaction p� . ); therefore, results are presented stratified by gender. these results are adjusted only for age. there was no sex interaction of diabetes/ifg with ac (interaction p� . ). for ac, the age-adjusted ors for diabetes/ifg are . ( % ci, . to . ) for men and . ( % ci, . to . ) for women. homogeneity p values are stratified by gender. ‡triglycerides were log-transformed for analysis. table . correlation* between quantities of cac and thoracic ac in the entire study group and stratified by age and gender total age � y age � y men . (� . ) . ( . ) . (� . ) number women . (� . ) . ( . ) . (� . ) number total . (� . ) . ( . ) . (� . ) number values in parentheses are p values. *estimated pearson correlation coefficients for natural log-transformed calcification score: ln(calcification score� ). correlation coefficients in the total sample are adjusted for age and gender; gender-specific correlation coeffi- cients are adjusted for age. figure . components of variance for cac (a) and ac (b) scores (ln [calcification score� ]). other measured covariates include all those achieving statistical significance in table other than age and sex. genetic variance corresponds to the proportion of the total phenotypic variation accounted for by genes. post et al determinants of coronary and aortic calcification d ow nloaded from http://ahajournals.org by on a pril , prevalence of peripheral arterial disease in both men and women, consistent with our ac findings. in addition, we found that age was more strongly associated with ac than cac. these results are similar to those of allison et al, who found that the strongest association between age and calcifi- cation was seen in the proximal aorta compared with other vascular beds. the pathology of ac may sometimes reflect a different process than cac. calcification in the coronary arteries generally occurs in the intimal layer, probably reflecting a healing response to inflammation of an atherosclerotic plaque; however, calcification in noncoronary arteries such as the aorta can reflect calcification in both the intimal and medial tunica layers of the artery. medial calcification is not associated with atherosclerotic plaque but is strongly associ- ated with aging, diabetes, and end-stage renal disease. , in the present study, it is unclear how much of the calcification detected in the aorta was in calcified atherosclerotic plaque and how much was nonatherosclerotic because intimal calci- fication cannot be differentiated from medial calcification on electron beam ct scanning. the presence of ac as detected by plain radiographs predicts risk for future clinical cvd, , especially in diabetic populations , ; however, the predictive ability of ac measured by ct compared with cac is unknown. it also is unclear whether ac is an independent predictor of cvd risk after accounting for traditional risk factors and cac. we found that calcification in both the coronary arteries and thoracic aorta is moderately heritable in the amish. our heritability estimate for cac score is similar to those previously published in whites from rochester (minn) and those obtained from families with type diabetes in north carolina. our estimate of heritability of thoracic ac score also is similar to that obtained for abdominal ac measured by lateral radiograph in the framingham heart study. the residual heritabilities for ac presence ( . � . ) and ac score ( . � . ) were higher than the corre- sponding estimates for cac presence ( . � . ) and score ( . � . ). the higher residual genetic heritability for ac (presence and score) compared with cac (pres- ence and score) may be related to the fact that measured environmental risk factors, particularly age, account for a higher proportion of the total variance in ac than cac (see figure ). consequently, the proportion of unex- plained or residual variance is smaller for ac, making the proportion of the unexplained variance attributable to genetic effects relatively larger for ac compared with cac. in contrast, there was little difference between ac and cac in the proportion of the total variation in the calcification score that was accounted for by genes ( % of the total variation in ac score, % of the total variation in cac score). the heritability of calcification scores was significantly higher in older compared with younger subjects. a likely explanation is that calcification prevalence is relatively low in younger people (see figure ), and if many susceptible subjects have not yet developed detectable calcification, then the correlations in calcification scores among younger related individuals may not have differed substantially from the correlations between younger unre- lated individuals. a novel, although perhaps not surprising, result from the present study is that there appears to be a moderate degree of joint genetic and environmental contribution, supporting the idea that common genes and environmental risk factors likely account for a moderate degree of variation in both cac and ac scores. one could speculate that if we were able to separate out calcification in the intimal layer of the aorta from calcification in the media, the correlations might be higher. even though there was a moderate degree of correlation between cac and ac, our results also suggest that there are site-specific differences in the contribution of genes and environmental factors for calcification. we found a few important differences in predictors of cac versus ac, namely gender and age, but there might be other factors that we did not measure. identifying factors associated with calcification at both sites versus factors that are unique to a single vascular bed might provide important new insights into the cause of cardiovascular diseases. the present study has several notable strengths and limitations. the relative social, cultural, and lifestyle homogeneity of the amish reduces variability resulting from unmeasured factors. additionally, the frequency of conventional bp- and cholesterol-lowering medication use is considerably less among the amish than in the general us population, allowing more informative estimates of the associations between bp, lipids, and calcification. amish families also tend to be very large, providing informative estimates for heritability. potential limitations of the pres- ent study include measurement of thoracic but not abdom- inal ac, which may be more strongly associated with symptomatic peripheral arterial disease. thoracic ac is detected routinely in patients receiving heart and lung ct scans, however. the cohort is largely a convenience sample rather than population based. because this is a cross-sectional analysis, associations cannot necessarily be interpreted as causally related. the power to detect differ- ences in the associations of risk factors with the presence cac and ac was relatively modest, particularly for the dichotomous variables, diabetes/impaired fasting glucose and smoking. additionally, we had very low power to assess the effects of diabetes on calcification presence because of the very low prevalence of diabetes in this population. finally, measurement error exists for both cac and ac quantity as shown by others. – this measurement error would be included in the residual environmental contribution to variation and, assuming it were uncorrelated among family members, would deflate the heritability estimate. conclusions in the present study, we have demonstrated that unlike cac, there are no gender differences in the presence of thoracic ac, that age is more strongly associated with ac than cac, and that both cac and ac are moderately circulation february , d ow nloaded from http://ahajournals.org by on a pril , heritable and share some genetic and environmental ori- gins. studies are needed to assess the independent predic- tive power of cac and thoracic ac for determining risk for future cvd events and to identify their common genetic and environmental origins as well as those that differ between the sites. acknowledgments we gratefully acknowledge our amish liaisons and field workers and the extraordinary cooperation and support of the amish community, without which the present study would not have been possible. sources of funding this work was supported by research grants r hl and u hl and the university of maryland general clinical research center (grant m rr ), general clinical research centers program, national center for research resources, national insti- tutes of health, and the baltimore veterans administration geriatric research and education clinical center. dr post is supported by the paul beeson physician faculty scholars in aging program and the johns hopkins donald w. reynolds cardiovascular research center. disclosures none. references . taylor aj, merz cn, udelson je. th bethesda conference: executive summary: can atherosclerosis imaging techniques improve the detection of patients at risk for ischemic heart disease? j am coll cardiol. ; : – . . rumberger ja, simons db, fitzpatrick la, sheedy pf, schwartz rs. coronary artery calcium area by electron-beam computed tomography and coronary atherosclerotic plaque area: a histopathologic correlative study. circulation. ; : – . . greenland p, labree l, azen sp, doherty tm, detrano rc. coronary artery calcium score combined with framingham score for risk pre- diction in asymptomatic individuals. jama. ; : – . . raggi p, callister tq, cooil b, he zx, lippolis nj, russo dj, zelinger a, mahmarian jj. identification of patients at increased risk of first unheralded acute myocardial 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. iribarren c, sidney s, sternfeld b, browner ws. calcification of the aortic arch: risk factors and association with coronary heart disease, stroke, and peripheral vascular disease. jama. ; : – . . wilson pw, kauppila li, o’donnell cj, kiel dp, hannan m, polak jm, cupples la. abdominal aortic calcific deposits are an important predictor of vascular morbidity and mortality. circulation. ; : – . . lehto s, niskanen l, suhonen m, ronnemaa t, laakso m. medial artery calcification: a neglected harbinger of cardiovascular compli- post et al determinants of coronary and aortic calcification d ow nloaded from http://ahajournals.org by on a pril , cations in non-insulin-dependent diabetes mellitus. arterioscler thromb vasc biol. ; : – . . douglas ja, erdos mr, watanabe rm, braun a, johnston cl, oeth p, mohlke kl, valle tt, ehnholm c, buchanan ta, bergman rn, collins fs, boehnke m, tuomilehto j. the peroxisome proliferator- activated receptor-gamma pro a a variant: association with type diabetes and trait differences. diabetes. ; : – . . peyser pa, bielak lf, chu js, turner st, ellsworth dl, boerwinkle e, sheedy pf nd. heritability of coronary artery calcium quantity measured by electron beam computed tomography in asymptomatic adults. circulation. ; : – . . wagenknecht le, bowden dw, carr jj, langefeld cd, freedman bi, rich ss. familial aggregation of coronary artery calcium in families with type diabetes. diabetes. ; : – . . o’donnell cj, chazaro i, wilson pw, fox c, hannan mt, kiel dp, cupples la. evidence for heritability of abdominal aortic calcific deposits in the framingham heart study. circulation. ; : – . clinical perspective prospective observational studies have demonstrated that the presence and extent of coronary artery calcification measured by rapid computed tomography scanning predict the risk for future cardiovascular disease events, even after traditional risk factors are accounted for. less is known about aortic calcification, which is a common finding with computed tomography imaging. in the present study, we show that aortic calcification and coronary artery calcification share some common risk factors such as cholesterol levels. aortic calcification, however, is influenced more strongly by advancing age than is coronary artery calcification, and there are no gender differences in the prevalence and quantity of aortic calcification as there are with coronary artery calcification. furthermore, we show that genes contribute to the variation in both coronary and aortic calcification. the sites of calcification share some genes in common, but there are also genes that contribute to only site. studies are under way to determine the independent predictive power of aortic compared with coronary artery calcium measurements and traditional risk factor assessment (especially age) for future cardiovascular events and to identify site-specific calcification susceptibility genes. event data will be available in the near future from prospective studies such as the national heart, lung, and blood institute–funded multi-ethnic study of atherosclerosis. until then, it is difficult to determine the relative clinical utility of extent of aortic calcification versus extent of coronary artery calcification to identify high-risk individuals. circulation february , d ow nloaded from http://ahajournals.org by on a pril , mutations in the na-cl cotransporter reduce blood pressure in humans mutations in the na-cl cotransporter reduce blood pressure in humans dinna n. cruz, david b. simon, carol nelson-williams, anita farhi, karin finberg, laura burleson, john r. gill, richard p. lifton abstract—the relationship between salt homeostasis and blood pressure has remained difficult to establish from epidemiological studies of the general population. recently, mendelian forms of hypertension have demonstrated that mutations that increase renal salt balance lead to higher blood pressure, suggesting that mutations that decrease the net salt balance might have the converse effect. gitelman’s syndrome, caused by loss of function mutations in the na-cl cotransporter of the distal convoluted tubule (ncct), features inherited hypokalemic alkalosis with so-called “normal” blood pressure. we hypothesized that the mild salt wasting of gitelman’s syndrome results in reduced blood pressure and protection from hypertension. we have formally addressed this question through the study of members of a large amish kindred with gitelman’s syndrome. through genetic testing, family members were identified as inheriting (n ), (n ), or (n ) mutations in ncct, permitting an unbiased assessment of the clinical consequences of inheriting these mutations by comparison of the phenotypes of relatives with contrasting genotypes. the results demonstrate high penetrance of hypokalemic alkalosis, hypomagnesemia, and hypocalciuria in patients inheriting mutant ncct alleles. in addition, the ncct genotype was a significant predictor of blood pressure, with homozygous mutant family members having significantly lower age- and gender-adjusted systolic and diastolic blood pressures than those of their wild-type relatives. moreover, both homozygote and heterozygote subjects had significantly higher -hour urinary na than did wild-type subjects, reflecting a self-selected higher salt intake. finally, heterozygous children, but not adults, had significantly lower blood pressures than those of the wild-type relatives. these findings provide formal demonstration that inherited mutations that impair renal salt handling lower blood pressure in humans. (hypertension. ; : - .) key words: blood pressure n sodium, dietary n hypokalemia n human n diuretics n genetics despite decades of investigation, determination of the roleof salt in blood pressure variation in humans has proved difficult. observational studies in large populations have often relied on recollections or a small number of measure- ments of salt intake as a surrogate of long-term dietary habits; interventional studies have typically studied small cohorts for short duration, making their interpretation difficult. as a result, advocating reduction in dietary salt in the general population has been controversial. one fundamental ques- tion in this debate has remained paramount: does alteration in net salt balance alter blood pressure in humans? studies of rare inherited forms of hypertension have begun to provide insight into this issue. in recent years, the molec- ular basis of glucocorticoid-remediable aldosteronism, lid- dle’s syndrome, and the syndrome of apparent mineralocor- ticoid excess have been defined. all result from mutations that lead to increased renal reabsorption of salt by the epithelial na channel of the distal nephron. this initiates a rise in blood pressure by the expansion of plasma volume and the consequent increased cardiac output. the cosegregation of hypertension with these mutations has demonstrated a causal link between them. these observations raise the question of whether mutations that diminish renal salt reabsorption have the opposite effect, ie, lowering blood pressure. patients with bartter’s and gitelman’s syndromes have salt wasting with hypokalemic alkalosis but, in contrast to the above disorders, remain free of hypertension, with so-called normal blood pressure. – these observations raise the question of whether the normal blood pressure in these syndromes actually reflects diminished blood pressure resulting from reduced salt balance. studies to test this possibility have been difficult to perform for several reasons: these disorders are rare autosomal recessive traits, making the investigation of many individuals under a uniform protocol problematic; second, there has been marked clinical variation among patients with hypokalemic alkalosis, raising the question of how to define distinct disease entities within this group. third, for rare disorders with affected subjects of received september , ; first decision october , ; revision accepted november , . from the departments of medicine (d.n.c., d.b.s., r.p.l.) and genetics (c.n.-w., a.f., k.f., l.b., r.p.l.), howard hughes medical institute, yale university school of medicine, new haven, conn; and the national institutes of health (j.r.g.), bethesda, md. correspondence to dr richard p. lifton, howard hughes medical institute, boyer center for molecular medicine, congress ave, new haven, ct . e-mail richard.lifton@yale.edu © american heart association, inc. hypertension is available at http://www.hypertensionaha.org d ow nloaded from http://ahajournals.org by on a pril , diverse ethnic and geographic backgrounds, identifying ap- propriate control populations for comparison has proved difficult. this situation has changed with the recent demonstration of the molecular basis of gitelman’s and bartter’s syndromes. bartter’s syndrome is caused by mutation in any of genes involved in salt reabsorption in the thick ascending limb of henle. these patients are typically diagnosed in the neonatal period with severe intravascular volume depletion. in con- trast, gitelman’s syndrome is caused by loss of function mutations in the na-cl cotransporter of the distal convoluted tubule (ncct); a wide range of mutations causing disease have been identified. , patients with gitelman’s syndrome typically have a more benign clinical course, presenting with neuromuscular signs and symptoms in adolescence or adult- hood; clinical signs of volume depletion are typically not apparent. , in both disorders, the renal salt wasting activates the renin-angiotensin system, increasing na reabsorption via the epithelial na channel in the distal nephron. this provides the electrical gradient for increased secretion of k and h , accounting for the hypokalemic alkalosis seen in affected patients. in addition, patients with gitelman’s syndrome typically have hypomagnesemia and hypocalciuria, in con- trast to the normal serum mg levels and hypercalciuria typically seen in bartter’s syndrome. , , , it is important to point out that these effects of ncct mutations have been identified only in patients presenting with these biochemical abnormalities, leaving open the possibility of ascertainment bias. thus, although all patients with these clinical features of gitelman’s syndrome appear to have ncct mutations, it has not been possible until now to determine the penetrance of the various clinical features resulting from ncct mutations. we have identified an amish kindred with gitelman’s syndrome spanning generations with many consanguine- ous loops; the ancestry of this kindred can be traced to a common pair of founders in the th century. identification of the mutations underlying the disease in this kindred affords the opportunity to unambiguously follow the inheritance of mutant ncct alleles and to then determine the impact of these alleles on biochemical features and blood pressure. this within-family study design provides an ideal control popula- tion, substantially eliminating the effects of differences in genetic backgrounds of cases and controls and permitting the assessment of clinical features free of ascertainment bias. methods family studies and clinical investigation the study population consisted of members of a single amish kindred ascertained through an index case, a -year-old white male who was diagnosed with gitelman’s syndrome at age , when he presented with complaints of muscular weakness. his laboratory values at presentation revealed the following: serum k . mmol/l, mg . mg/dl ( . mmol/l), bicarbonate mmol/l, and urine ca /creatinine ratio . mmol/mmol. in the absence of thiazide diuretics, these clinical features are now recognized to be diagnostic of gitelman’s syndrome. the extended kindred of the index case was investigated. most members lived in pennsylvania dutch country and had similar lifestyles. the research protocol was approved by the yale human investigation committee, and all subjects provided written informed consent. blood pressure was measured in a standardized fashion. all blood pressures were measured by a single physician (d.n.c.). three oscillometric blood pressure readings were measured at -minute intervals, by using the left arm of the patient, who was seated. the first reading was discarded, and the average of the second and third readings was used in data analysis. hypertension was defined as a blood pressure . / or the use of antihypertensive medications. valid measurements were obtained in subjects. three young children could not cooperate for valid measurement. two additional patients were excluded from analysis: patient for chronic steroid use for multiple sclerosis and patient for diltiazem use for cardiac dysrhythmia. twenty-nine months after the initial blood pressure recordings, measurements were repeated in subjects by the same methods. the repeated blood pressures, measured in a blinded fashion, showed a highly significant correlation with the initial readings (r . and p, . for systolic blood pressure, r . and p, . for diastolic blood pressure). biochemical measurements were all performed in the same laboratory by using standard procedures with subjects consuming their typical ad libitum diets. serum k and mg levels were determined in all subjects. measurements of serum bicarbonate (hco ) levels were performed in a subset of the subjects (n ). in a subset of patients, -hour urinary na , urinary k , and urinary ca levels were also measured (n , , and , respectively). these values are expressed as the millimolar ratios of urine electro- lytes/creatinine (ie, urine na /creatinine, k /creatinine, and ca /creatinine). mutation identification and genetic testing genomic dna was prepared from venous blood samples by standard procedures. mutations were sought by single-strand conformational polymorphism of the ncct gene as previously described. identified variants were subjected to dna sequencing according to standard procedures. the missense mutation identified in this kindred was genotyped in family members by polymerase chain reaction (pcr), followed by single-strand conformational polymorphism in kindred members. the deletion identified in this kindred was genotyped in family members by pcr. deletion homozygotes were identified by the absence of ncct pcr products, whereas exons from other genes in the same pcr reaction were successfully amplified, providing a positive control. these results were confirmed by southern blotting, hybridizing probes from the deleted ncct exons to genomic dna of kindred members. heterozygous carriers of the ncct exon- to - deletion were identified by quantitative comparison of pcr amplification of exons to in family members with obligate heterozygotes and in normal control subjects. inheritance of specific mutations was further confirmed by genotyping polymorphic mark- ers tightly linked to ncct. importantly, all genotypic assignments were performed by individuals blinded to clinical data. statistical analysis the data are presented as mean sem. the primary analysis compared the clinical characteristics of the genotype groups by use of anova for continuous variables (age, serum k , mg , hco , and urinary ca /creatinine) and x for categorical variables (gender and the presence of hypertension), with a -tailed probability value. comparison of continuous variables between any groups was performed by use of the student t test. univariate linear regression analysis (pearson correlation) was used to examine the relationship between serum k , mg , hco , and urinary ca /creatinine among affected patients. this was also used to examine the corre- lation between repeated blood pressure measurements (see above). systolic and diastolic blood pressure was analyzed for the effect of genotype by using forward stepwise regression, with age and gender considered a priori as covariates. calculations were performed by using spss . for macintosh (spss inc). a value of p, . was considered statistically significant. cruz et al blood pressure in gitelman’s syndrome d ow nloaded from http://ahajournals.org by on a pril , results mutations causing gitelman’s syndrome in k two different ncct mutations were identified in k (figure ). one mutation introduced a single base substitution (cgc ggc) in codon ; this mutation substitutes gly- cine for the normal arginine in the cytoplasmic c-terminus of the encoded protein. this arginine residue lies in a –amino acid segment that is completely conserved among flounder, rat, and human nccts, , , and the observed substitution is nonconservative, eliminating a positive charge. this mutation is not a simple polymorphism in the population, inasmuch as it was absent among control chromosomes studied. from these observations, as well as the cosegregation of this mutation with the biochemical features of gitelman’s syn- drome (see below), we infer that this mutation leads to loss of ncct function. the other allele in the index case contains a deletion that eliminates exons to of the gene. seventeen individuals in the kindred were found to be homozygous for this deletion by pcr, and the absence of this segment of the gene in these individuals was confirmed by southern blotting (figure ). because this deletion removes the start codon as well as the first transmembrane domains of the encoded protein, this mutation is also inferred to result in a loss of function of ncct. nine individuals were compound het- erozygotes with copy of the deletion and copy of the missense mutation. there were no significant phenotypic differences between the classes of individuals homozygous for defective ncct alleles. no other mutations in ncct were identified in this kindred. genotypes in the extended kindred the identification of these mutations and marker haplotypes segregating with them permitted unambiguous assessment of the inheritance of these mutations through the extended kindred as described in methods. in sum, members of this kindred were studied. all members were classified as inheriting , , or copies of ncct mutations (figure ). this analysis revealed that members had inherited defective copies of the gene (referred to as genotypically affected subjects), had inherited defective copy (het- erozygotes), and had inherited neither mutation (homozy- gous wild types). among the genotypically affected patients, there were males and females; among the heterozygous subjects, males and females; and among the wild-type subjects, males and females. the gender ratio of affected subjects was not significantly different from the expected / ratio, and the gender ratios among the groups were not significantly different. individuals inheriting mutant alleles were older (mean age, . years) than their heterozygous and wild-type relatives ( . and . years, respectively; p . ), attributable to pedigree structure (figure ). biochemical features of members of k heretofore, identification of patients with gitelman’s syn- drome has relied on the identification of abnormal serum chemistries, potentially introducing ascertainment bias in assessment of the severity of disease; ie, previously, one would not have been able to identify patients inheriting these figure . mutations causing gitelman’s syndrome in kindred k . a, heterozy- gous variant in k . a segment of the ncct gene was amplified by pcr, and the products were fractionated by elec- trophoresis under nondenaturing condi- tions as described in methods. the results in subjects from kindred k with gitelman’s syndrome are shown, flanked by normal (unaffected) relatives. the arrow indicates the location of a het- erozygous variant found in these patients but not in normal control subjects. b, r g mutation. a -base segment of the antisense strand of the dna sequence of the normal and variant frag- ments in panel a is shown. the first bases of the sequence are intronic, shown in lower case, and the last are from the adjacent exon. the mutated base is indicated by an asterisk. the sequence of the sense strand is shown in black above, and the to orienta- tion of the sense strand is indicated. the encoded amino acid sequence is indi- cated in single-letter format above the dna sequence. codon is split by an intron, with the first bases in exon (shown) and the last base in the exon . the single base substitution mutates the first base of codon from g to c, altering the wild-type (wt) arginine (r) to glycine (g). c, deletion of exons to of ncct. an au- toradiogram of a southern blot of genomic dna of selected members of kindred k is shown. genomic dna was digested with restriction enzyme bglii, fractionated by electrophoresis on . % agarose gel, denatured, and transferred to nylon membrane. p- labeled ncct probes corresponding to exons to of the ncct cdna were hybridized to the filter as described in methods. in con- trast to normal controls, who show hybridization to fragments of . and . kb, kindred members shown have complete deletion of these sequences. hypertension june d ow nloaded from http://ahajournals.org by on a pril , mutations who had no biochemical abnormalities. conse- quently, identification of genotypically affected family members based solely on their relationship to the index case permits an unbiased assessment of the effect of inheritance of these mutations on clinical and biochemical parameters. moreover, tracing the inheritance of mutations through the family for the first time permits unambiguous genotypic distinction of heterozygous from wild-type homozygous in- dividuals. the laboratory values in patients of different genotypic classes are shown in figure . individuals inherit- ing defective copies of ncct all had significant hypoka- lemia, (mean k . mmol/l, range . to . mmol/l), whereas the means of both the homozygous wild-type and heterozygous individuals were . mmol/l, with no individ- ual , . mmol/l (p, . , figure a). similarly, the mean serum bicarbonate levels were also significantly higher than those of the homozygous wild-type and heterozygous individuals (figure c). in addition to these effects on k and bicarbonate, signif- icant effects were also seen on mg and ca handling. serum mg was markedly diminished among genotypically affected subjects, with a mean of . mg/dl ( . mmol/l, range . to . mg/dl or . to . mmol/l) compared with . mg/dl ( . mmol/l) in unaffected relatives (p, . , figure b). patients with gitelman’s syndrome have been noted to have diminished urinary ca excretion, regardless of diet. the ratio of urinary ca /creatinine was markedly diminished in gitelman’s patients compared with their unaffected relatives (p, . , figure d). in the chronic state, patients’ -hour urinary na and k levels reflect their self-selected dietary consumption. the results demonstrated a highly significant difference in urinary na and k among the genotype classes, with genotypically affected subjects having the highest urinary na (p . ) and k (p, . ) values (figure e and f). among the genotypically affected gitelman’s patients, there was no significant difference between males and fe- males in mean serum k ( . versus . mmol/l, respective- ly), bicarbonate ( versus mmol/l), mg ( . versus . mg/dl or . versus . mmol/l), or urinary ca /creati- nine ratio ( . versus . mmol/mmol). among the af- fected patients, there were no significant correlations among these laboratory values. effects of mutations on blood pressure none of the genotypically affected subjects had a diagno- sis of hypertension, none was being treated with antihyper- tensive medication, and none had a blood pressure . / mm hg. the mean blood pressure in this group was / mm hg for adult males and / mm hg for adult females. the quantitative impact of gitelman’s mutations on blood pressure was analyzed by multiple linear regression, compar- ing blood pressures of relatives of contrasting ncct geno- figure . pedigree of gitelman’s syn- drome kindred k . the family relation- ships of members of the extended kin- dred of k are shown, and the number of mutant ncct alleles each has inher- ited, as determined by molecular geno- typing, is indicated. males are indicated by squares, and females are indicated by circles. individuals who have inherited mutant ncct alleles (homozygous mutant) are indicated by completely filled symbols; those with mutant allele and normal allele (heterozygotes) are indi- cated by half-filled symbols; and those with normal ncct alleles (homozygous wild types) are indicated by unfilled sym- bols. dotted symbols indicate kindred members who were not studied. because the deletion and missense mutation both result in loss of ncct function, they have not been distin- guished from one another on the figure. the index case is indicated by an arrow. cruz et al blood pressure in gitelman’s syndrome d ow nloaded from http://ahajournals.org by on a pril , types. ncct genotype, age, and gender were all significant predictors of diastolic and systolic blood pressure (table). the effect of genotype on diastolic blood pressure was highly significant (p . ), with genotypically affected individu- als having age- and gender-adjusted diastolic blood pressures . and . mm hg lower than those of their heterozygous and wild-type relatives, respectively (figure ). the effect of genotype on systolic blood pressure was also significant and quantitatively similar (table, p . ). effects in heterozygous individuals we compared the phenotypes of heterozygous individuals with those of their wild-type relatives. there were no statis- tically significant differences in the heterozygous and the homozygous wild-type subjects in terms of serum k , mg , bicarbonate, and urinary ca and k excretion (figure ). however, -hour urinary na excretion was significantly higher in the heterozygous individuals (urinary na /creati- nine . versus . mmol/mmol, heterozygous versus wild-type individuals, respectively; p . ). this observa- tion is consistent with these patients having mild salt wasting. although analysis of blood pressure for the entire study population did not detect a significant difference in age- and gender-adjusted diastolic blood pressure between heterozy- figure . effect of ncct mutations on laboratory parameters. the mean sem values of indicated laboratory parameters are shown for individuals inheriting ( / ), ( / ), or ( / ) mutations in ncct. values for serum mg can be converted to mmol/l by multiply- ing by . . cr indicates creatinine. p values for anova among different genotype classes are indicated. figure . effect of ncct mutations on blood pressure. the mean sem values of age- and gender-adjusted diastolic blood pressures for members of kindred k with different ncct ge- notypes are shown. p values for anova among different geno- type classes are indicated. individuals who have inherited defective copies of ncct have blood pressure that is signifi- cantly lower than that of their wild-type relatives. predictors of bp in kindred git variable coefficient (b) standard error % ci p diastolic bp intercept . . . to . . genotype . . . to . . age . . . to . . gender . . . to . . systolic bp intercept . . . to . . genotype . . . to . . age . . . to . . gender . . . to . . bp indicates blood pressure. hypertension june d ow nloaded from http://ahajournals.org by on a pril , gous and homozygous wild-type individuals, the higher urinary na excretion in the heterozygous group suggests that the heterozygous individuals have self-selected a higher na intake to compensate for a mild salt-wasting defect. because young individuals may have less opportunity to self-select their salt intake and may therefore have less ability to compensate for a mild defect, we tested the genotypic effect on blood pressure in children (aged , years). young heterozygous individuals (n ) had mean age and gender- adjusted diastolic blood pressure . mm hg lower than that of homozygous wild-type relatives (n , p . ). al- though there were only genotypically affected children in the kindred, their mean diastolic blood pressure was . mm hg lower than that of homozygous wild-type individuals, consistent with a stepwise effect of mutant gene dose on blood pressure in children. together, these results provide evidence of a significant effect of the heterozygous genotype on salt homeostasis and blood pressure. discussion gitelman’s syndrome is caused by a wide variety of loss of function mutations in ncct, the na-cl cotransporter of the distal convoluted tubule (dct). the results of the present study of a very large extended kindred indicate effects of homozygous and heterozygous mutations in the thiazide- sensitive na-cl cotransporter on electrolyte homeostasis and blood pressure. prior studies of this syndrome have largely relied on the identification of cases via abnormalities in electrolytes, po- tentially introducing ascertainment bias. – , identification of patients with inherited ncct mutations based solely on their relationship to an index case and the finding that all of these subjects have hypokalemia, metabolic alkalosis, hypo- magnesemia, and hypocalciuria demonstrate the complete penetrance of these features among such individuals. more- over, the cosegregation of this constellation of biochemical abnormalities with mutant ncct alleles across this extended kindred constitutes proof that the observed hypomagnesemia and hypocalciuria, findings of uncertain etiology from known physiology, are in fact attributable to inheritance at the ncct locus, underscoring the relationship between salt reabsorption in the dct and renal mg and ca homeostasis. the effects on mg homeostasis are particularly striking. renal mg reabsorption has been believed to be mediated largely through paracellular conductance in the thick ascend- ing limb of henle, with only a small fraction of reabsorption occurring in the dct. it is consequently surprising that a defect in salt reabsorption in the dct should result in hypomagnesemia. this observation raises the possibility that the dct normally mediates the final fine tuning of mg homeostasis, analogous to the role played by the epithelial na channel for salt homeostasis in the distal nephron. the mechanism underlying this defect is uncertain. similarly, hypocalciuria is a consistent finding among patients with these mutations, indicating a consistent influence of salt handling in the dct on renal ca homeostasis. the results of investigation of this kindred provide formal demonstration that the homozygous state for gitelman’s syndrome lowers blood pressure in humans. the salt wasting of gitelman’s syndrome is relatively mild. nonetheless, these individuals have blood pressure lower than that of their unaffected relatives. this reduction in blood pressure is highly significant and is seen in both genders. moreover, because these subjects have self-selected a markedly higher salt diet, it is likely that their blood pressures would be even lower without this adaptation. although thiazide diuretics have long been recognized to have blood pressure–lowering effects, the present study indicates that loss of a single target, the gene product of ncct, is sufficient to account for the blood pressure–lowering effects of these agents. moreover, these findings indicate some of the inherent limits in the use of antagonists of the ncct gene product. for example, complete inhibition of this target can be predicted to almost invariably lead to hypomagnesemia. therefore, these obser- vations predict both the utility and the limits in the use of antagonists of this target. these findings are unlikely to be unique to the kindred studied. we have investigated a cohort of unrelated adult patients with gitelman’s syndrome in whom mutant ncct alleles have been identified. in this group, % of subjects had blood pressures that fell below the median diastolic blood pressure of the national health and nutrition examination survey (nhanes) participants of comparable age and gen- der (d.n. cruz and r.p. lifton, unpublished data, ), supporting the general effect of ncct mutations in lowering blood pressure. previous studies have demonstrated that mutations that increase renal salt reabsorption increase blood pressure. the present observations formally demonstrate the opposite side of this equation, namely, that mutations that reduce renal salt reabsorption reduce blood pressure. these findings together demonstrate a strong and consistent effect of salt balance on blood pressure in humans that operates in both directions, demonstrating that alteration in salt balance represents a final common pathway for altering blood pressure in humans. in addition, the present studies demonstrate a significant effect of the heterozygous state, with significantly increased dietary salt intake and, in younger individuals, lower blood pressure. this is of relevance because ' % of the population is likely to be heterozygous for mutations in this gene. these observations raise the question of whether the heterozygous state might underlie additional phenotypes. for example, diuretic-induced hypokalemia is a relatively common com- plication of loop diuretics; it is possible that ncct heterozy- gous individuals may be more susceptible to this complication. with regard to observational studies of the relationship between salt and blood pressure, it is worth noting that although primary salt wasting leads to lower blood pressure in gitelman’s syndrome, in these patients there is actually an inverse relationship between dietary salt and blood pressure that is due to compensatory self selection of a high salt diet. complexities such as these underscore the difficulties in interpreting observational studies of the salt– blood pressure relationship that do not investigate the underlying physiology of individual subjects. these observations raise the question of whether identification of individuals with very high salt cruz et al blood pressure in gitelman’s syndrome d ow nloaded from http://ahajournals.org by on a pril , intake but very low blood pressure might identify other subsets of patients with impaired renal salt reabsorption. the demonstration of a consistent relationship between altered renal salt handling and blood pressure variation in humans identifies one of the final common pathways for altered blood pressure. these findings are beginning to put a molecular face on the physiological studies of guyton, who demonstrated the requirement for an active role of the kidney in the development of hypertension. these observations in rare mendelian disorders raise the possibility that common variants in genes that mediate or regulate salt homeostasis in humans might underlie blood pressure variation in the general population. moreover, they identify targets for the develop- ment of improved antihypertensive agents that may more closely address the abnormal physiology contributing to disease pathogenesis. acknowledgments this study was supported by a grant from the national institute of diabetes and digestive and kidney diseases, an nih specialized center of research in hypertension, and the yale general clinical research center. dr lifton is an investigator of the howard hughes medical institute. we gratefully acknowledge the members of the family studied for their invaluable contribution to this project, helen brickel for assistance with the kindred evaluation, joan buenconsejo and david silver for assistance in statistical analysis, and the staff of the general clinical research center at yale university. references . chrysant gs, bakir s, oparil s. dietary salt reduction in hypertension: what is the evidence and why is it still controversial? prog cardiovasc dis. ; : – . . lifton rp. molecular genetics of human blood pressure variation. science. ; : – . . bettinelli a, vezzoli g, colussi g, bianchetti mg, sereni f, casari g. genotype-phenotype correlations in normotensive patients with primary renal tubular hypokalemic metabolic alkalosis. j nephrol. ; : – . . de heide l, birkenhager j. bartter’s syndrome, hypomagnesemia and chondrocalcinosis. neth j med. ; : – . . puschett j, greenberg a, mitro r, piraino b, wallia r. variant of bartter’s syndrome with a distal tubular rather than loop of henle defect. nephron. ; : – . . simon db, lifton rp. ion transporter mutations in gitelman’s and bartter’s syndromes. curr opin nephrol hypertens. ; : – . . simon db, nelson-williams c, bia mj, ellison d, karet fe, molina am, vaara i, iwata f, cushner hm, koolen m, et al. gitelman’s variant of bartter’s syndrome, inherited hypokalemic alkalosis, is caused by mutations in the thiazide-sensitive na-cl cotransporter. nat genet. ; : – . . bell g, karam j, rutter w. polymorphic dna region adjacent to the end of the human insulin gene. proc natl acad sci u s a. ; : – . . gamba g, saltzberg sn, lombardi m, miyanoshita a, lytton j, hediger ma, brenner bm, hebert sc. primary structure and functional expression of a cdna encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter. proc natl acad sci u s a. ; : – . . gamba g, miyanoshita a, lombardi m, lytton j, lee ws, hediger ma, hebert sc. molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)- chloride cotransporter family expressed in the kidney. j biol chem. ; : – . . simon db, lu y, choate ka, velazquez h, al-sabban e, praga m, casari g, bettinelli a, colussi g, rodriguez-soriano j, et al. paracellin- , a renal tight junction protein required for paracellular mg resorption. science. ; : – . . guyton ac. blood pressure control: special role of the kidneys and body fluids. science. ; : – . hypertension june d ow nloaded from http://ahajournals.org by on a pril , pnas .. letter cerebral vasculopathy is a common feature in aicardi–goutières syndrome associated with samhd mutations in their recent interesting paper, xin et al. ( ) described an autosomal recessive condition in individuals of old order amish ancestry characterized by cerebral vasculopathy and early onset stroke. the affected patients presented a heterogeneous phenotype, including variable developmental disability, irrita- bility in infancy, chilblain lesions, glaucoma, and arthritis. through genome-wide homozygosity mapping and candidate gene sequencing, the authors identified the homozygous muta- tion c. - a > g in samhd being associated with this entity. additionally, they detected this mutation in of patients with developmental delay (phenotypes not further described). although mutations in samhd have been found to be disease- causing in aicardi–goutières syndrome (ags) ( ), xin et al. ( ) stated that “the phenotype reported here is apparently in- compatible with aicardi–goutières syndrome” and that “none of our patients has been diagnosed with aicardi– goutières syndrome.” although, as xin et al. ( ) explained, ags is most commonly recognized as “a type of encephalopathy whose clinical features mimic those of acquired in utero viral infection,” there now exists an extensive literature highlighting the diverse spectrum of phenotypes that can occur in the context of ags (an overview is provided in ref. ). thus, for example, neurological dysfunc- tion in ags is not always progressive or, indeed, necessarily present at all; microcephaly is not always seen; onset is not al- ways in the first year of life; intracranial calcification and white matter changes are not inevitable; and a cerebrospinal fluid (csf) lymphocytosis is absent in a considerable number of af- fected individuals. we accept that such diversity makes clinical diagnosis challenging. however, we consider that the clinical findings presented by xin et al. ( ), most particularly the chil- blain lesions, early irritability, and glaucoma, should prompt consideration of the diagnosis. we suggest that it might be sensible for xin et al. ( ) to confirm that intracerebral calcifi- cations were not evident on computed tomography and that reliable csf indices (elevated white cells and increased titers of ifn-α and pterins) of ags were not present in any of their cases (with recognition of the age-dependent nature of the csf findings). irrespective of diagnostic classification, there are two earlier independent descriptions of cerebral arterial stenoses, stroke, and cerebral vasculopathy in patients with ags carrying muta- tions in samhd ( , ). these two papers fully encompass the phenotypes described in the article by xin et al. ( ). in our view, recognizing that the old order amish disorder is part of the ags-related phenotype means that future understand- ing of ags disease pathogenesis will have relevance to the amish community also. in summary, we would argue that xin et al. ( ) did not present a new clinical condition but described a heterogeneous group of old order amish individuals with ags and in- tracerebral arteriopathy. based on our previous work and their findings, we conclude that intracerebral large artery disease is a common phenomenon in patients with samhd mutations. interestingly, we have never observed large artery disease in association with mutations in trex , rnaseh a, rnaseh b, or rnaseh c, perhaps indicating a particular role for samhd in blood vessel integrity and homeostasis. acknowledgments. this work was supported by a grant from the inter- disciplinary center for clinical research münster (to m.d.m and f.r.). y.j.c. acknowledges the manchester national institute for health research bio- medical research centre, the european leukodystrophy association, and the european union’s seventh framework programme (fp / - ) under grant agreement number . marcel du moulina, peter nürnbergb, yanick j. crowc, and frank rutscha, adepartment of general pediatrics, münster university children’s hospital, d- münster, germany; bcologne center for ge- nomics, university of cologne, d- cologne, germany; and cgenetic medicine, university of manchester, manchester aca- demic heath science centre, central manchester foundation trust university hospitals, manchester m wl, united kingdom . xin b, et al. ( ) homozygous mutation in samhd gene causes cerebral vascu- lopathy and early onset stroke. proc natl acad sci usa : – . . rice gi, et al. ( ) mutations involved in aicardi-goutières syndrome implicate samhd as regulator of the innate immune response. nat genet : – . . aicardi j, crow yj, stephenson jbp ( ) aicardi–goutières syndrome. genereviews. available at http://www.genetests.org/ [updated nov , ]. . ramesh v, et al. ( ) intracerebral large artery disease in aicardi-goutières syndrome implicates samhd in vascular homeostasis. dev med child neurol : – . . thiele h, et al. ( ) cerebral arterial stenoses and stroke: novel features of aicardi- goutières syndrome caused by the arg x mutation in samhd are associated with altered cytokine expression. hum mutat :e –e . author contributions: m.d.m., p.n., y.j.c., and f.r. wrote the paper. the authors declare no conflict of interest. to whom correspondence should be addressed. e-mail: rutschf@ukmuenster.de. e | pnas | june , | vol. | no. www.pnas.org/cgi/doi/ . /pnas. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.genetests.org/ mailto:rutschf@ukmuenster.de genetic mapping of glutaric aciduria, type , to chromosome and identification of mutations in c orf report genetic mapping of glutaric aciduria, type , to chromosome and identification of mutations in c orf eric a. sherman, kevin a. strauss, , , silvia tortorelli, michael j. bennett, ina knerr, d. holmes morton, , , and erik g. puffenberger , ,* while screening old order amish children for glutaric aciduria type (ga ) between and , we found three healthy children who excreted abnormal quantities of glutaric acid but low -hydroxyglutaric acid, a pattern consistent with glutaric aciduria type (ga ). none of these children had the gcdh c. c/t mutation that causes ga among the amish. using single-nucleotide polymorphism (snp) genotypes, we identified a shared homozygous . mb region on chromosome . this region contained genes including c orf , an open reading frame with a putative mitochondrial targeting sequence and coenzyme-a transferase domain. direct sequencing of c orf revealed that the three amish individuals were homozygous for a nonsynonymous sequence variant (c. c/t, arg trp). we then sequenced three non-amish children with ga and discovered two nonsense mutations (c. c/t, arg ter, and c. c/t, arg ter) in addition to the amish mutation. two pathogenic alleles were identified in each of the six patients. there was no consistent clinical phenotype associated with ga . in affected individuals, urine molar ratios of glutarate to its derivatives ( -hydroxyglutarate, glutarylcarnitine, and glutarylglycine) were elevated, suggesting impaired formation of glutaryl-coa. these observations refine our under- standing of the lysine-tryptophan degradation pathway and have important implications for the pathophysiology of ga . glutaric aciduria type (ga ) is one of the most common genetic disorders of the old order amish of lancaster county, pennsylvania. infants with ga have elevations of glutarate, -hydroxyglutarate, and glutarylcarnitine in blood and urine. without timely diagnosis and therapy, ga results in striatal degeneration and severe dystonia. , between and , we screened amish infants for ga by urine organic-acid analysis with gas chromatog- raphy-mass spectrometry. in the process, we identified three healthy children who excreted large quantities of glutarate but low -hydroxyglutarate, consistent with the phenotype of glutaric aciduria type (ga [omim ]), first described in . these children received no therapy and remained healthy over more than years of follow-up. in their original description of ga , bennett et al. pos- tulated that glutaryl-coa degradation in vivo occurred in two compartments, mitochondria and peroxisomes, corre- sponding to the ga and ga phenotype, respectively. they showed that fibroblast homogenates from a ga patient did not produce hydrogen peroxide in the presence of labeled glutaryl-coenzyme a (coa) and took this as evi- dence of a defective peroxisomal glutaryl-coa oxidase. this was a compelling idea; it suggested that neurodegen- erative consequences of ga (in contrast to the benign phenotype of ga ) were rooted in the mitochondrial locus of the disturbance. however, no gene encoding a glutaryl-coa oxidase has been identified, and subsequent work showed that, the american journal of human genetics , – , novemb in vitro, a small amount of glutaryl-coa is oxidized in per- oxisomes by an inducible acyl-coa oxidase (acox , a.k.a. palmitoyl-coa oxidase), whereas this enzyme probably mediates little or no glutaryl-coa degradation in vivo. , as a further complication, case reports in described disabled children with alpha-ketoadipic and alpha-amino- adipic aciduria who were thought to have a defect in the oxidative decarboxylation of alpha-ketoadipate to form glutaryl-coa. – however, no enzyme mediating this reac- tion was ever found. in an effort to clarify the metabolic pathology of the lysine-tryptophan degradation pathway, we took advan- tage of the natural occurrence of both ga and ga among the pennsylvania old order amish. this study was approved by the institutional review board of lancas- ter general hospital and parents consented in writing to molecular genetic testing. in addition to the three old order amish patients, three other ga patients were stud- ied, including an american child of mixed european ances- try and two previously described patients, one german and one pakistani. single-nucleotide polymorphism (snp) genotyping was performed with the genechip mapping k assay kit (af- fymetrix, santa clara, ca, usa) as previously described. data were analyzed in microsoft excel spreadsheets (micro- soft corporation, redmond, wa, usa) that were custom formatted at the clinic for special children. snp positions came from affymetrix genome annotation files, and geno- type data came from the affymetrix genechip human ephrata high school, ephrata, pa , usa; clinic for special children, strasburg, pa , usa; biology department, franklin and marshall col- lege, lancaster, pa , usa; lancaster general hospital, lancaster, pa , usa; biochemical genetics laboratory, department of laboratory med- icine and pathology, mayo, clinic school of medicine, rochester, mn , usa; department of pathology and laboratory medicine, university of penn- sylvania and children’s hospital of philadelphia, philadelphia, pa , usa; children’s and adolescent’s hospital, university of erlangen-nuremberg, erlangen , germany *correspondence: epuffenberger@clinicforspecialchildren.org doi . /j.ajhg. . . . ª by the american society of human genetics. all rights reserved. er , mailto:epuffenberger@clinicforspecialchildren.org mapping k xba arrays. data analyses were designed for identification of genomic regions that were identically homozygous between all three affected old order amish individuals. such analyses assume mutation and locus ho- mogeneity. two-point lod scores were calculated for each genotyped snp with an approach similar to broman and weber. cumulative two-point lod scores for blocks of homozygous snps were considered the location score for that region, providing a relative measure that the region harbors the disease gene. genotype data from healthy old order amish samples as well as from previous stud- ies , – were used for estimation of population-specific snp allele frequencies. genome-wide autozygosity mapping using three dis- tantly related old order amish children with ga identi- fied a homozygous . mb region on chromosome p bounded by snps rs and rs (figure ). the region was queried for candidate genes with both the uni- versity of california santa cruz (ucsc) and national cen- ter for biotechnology information (ncbi) genome browsers. the region contained known or hypothetical genes. for each gene, we assessed function and expression to generate a priority list for sequencing. we chose c orf (omim ) as a candidate based on its puta- tive coa transferase function (ncbi gene) and mitochon- drial targeting sequence (mitoprot). c orf was subjected to polymerase chain reaction (pcr) amplification and sequencing. pcr primers were de- signed (primer ) for amplification of the coding regions and adjacent intron-exon boundaries. dna sequence from ga patients was compared to the human reference sequence and dbsnp so that potential pathogenic sequence variants could be identified. all three amish ga subjects were ho- mozygous for a nonsynonymous c. c/t (arg trp) change in exon . this sequence variant is not a known polymorphism (dbsnp) and is highly conserved in all species tested. among amish control individuals, we identified who were heterozygous and one who was figure . two mapping parameters, the number of consecutive homozygous snps and the location score, were max- imized on chromosome p . -p . in three old order amish ga patients the region comprised snps and con- tained known genes including c orf . homozygous for the c orf c. c/t variant. retrospectively, we collected a urine sample from this c. c/t homozygote, a healthy -year-old man, and confirmed high glutaric acid excretion ( mmol/mol creatinine; control . . mmol/mol cr). we subsequently analyzed dna from three non-amish ga subjects and identified c orf mutations in all of them (figure ). one patient was heterozygous for the amish c. c/t allele and a second variant, c. c/t (arg ter). the lat- ter, a nonsense change, is predicted to produce a truncated, nonfunctional protein. a second child, from germany, was homozygous for the same c. c/t mutation found in the amish patients. surprisingly, the intragenic snps indi- cated that the mutation in this patient resided on a different haplotype (figure ). this circumstance could be explained by an old intragenic recombination or a recurrent muta- tion; however, the observation that the snp genotypes were identical in patients and at the end of c orf (and beyond) lends support to the hypothesis of intragenic recombination. finally, sequence analysis of the pakistani child originally described by bennett et al. revealed homo- zygosity for c. c/t (arg ter). this exon nonsense mutation is predicted to result in a nonfunctional protein. urine organic acids were measured by gas chromatogra- phy-mass spectrometry. glutarylglycine and glutarylcar- nitine were measured with electrospray-ionization tandem mass spectrometry. , to quantitate -hydroxyglutarate and glutarylglycine levels, we added labeled -hydroxyglu- taric acid and c -glutarylglycine, respectively, as internal standards. for biochemical comparisons, we used urine samples from six amish ga patients and amish ga patients (gcdh c. c/t homozygotes), ages week to years, as well as healthy amish siblings and parents of ga subjects (designated controls). urine metabolite values for three groups (control, ga , and ga ) were studied with one-way analysis of variance (anova). for anova p values < . , we used tukey’s posttest to make pairwise comparisons among groups. we log transformed urine-metabolite-concentration ratios to produce normal distributions for anova testing. amish ga patients had high glutarate excretion ( . mmol/mol cr; control . . mmol/mol cr) relative to controls (table ). urine glutarate levels were not distinguishable between ga and ga subjects; the american journal of human genetics , – , november , figure . single-nucleotide polymorphism and c orf mutation table for six ga patients snp genotypes were derived from affymetrix k genechips and direct sequencing of c orf ; snp physical locations were from human genome build . the red box delimits the c orf locus, and the gray highlighting indicates homozygous genotype blocks in each patient. glutarate excretion was highly variable within these groups (i.e., across three orders of magnitude) and overlapped broadly between them. in contrast, individuals with ga had normal urinary -hydroxyglutarate, glutarylcarnitine, and glutarylglycine values, whereas ga patients had ele- vations of all three derivatives, particularly -hydroxyglu- tarate. urine acetylcarnitine levels were also high in ga patients ( . . mmol/mol cr; control . . mmol/mol cr), possibly reflecting l-carnitine supplemen- table . urine metabolites as mean and standard deviation for ga , ga , and healthy control subjects metabolites (mmol/mol cr) controls (n ¼ ) ga (n ¼ ) ga (n ¼ ) anova p value glutarate . ( . ) . ( . ) . ( . ) a . -hydroxyglutarate . ( . ) . ( . ) . ( . )a,b < . glutarylcarnitine . ( . ) . ( . ) . ( . ) a,b < . acetylcarnitine . ( . ) . ( . ) . ( . )a . glutarylglycine . ( . ) . ( . ) . ( . ) a,b . metabolite ratios (mol:mol)c glutarylcarnitine/ total acylcarnitines . ( . ) . ( . ) . ( . ) a,b . glutarate/ -hydroxyglutarate . ( . ) . ( . )a . ( . )b < . glutarate/ glutarylcarnitine . ( . ) . ( . ) a . ( . ) b < . a different from control (tukey, p < . ). b different from ga (tukey, p < . ). c log transformed for anova test. the american journal of human genetics , – , novemb tation. compared to both control and ga subjects, molar ratios of glutarate to -hydroxyglutarate and gluta- rylcarnitine were markedly elevated in ga (figure ), sug- gesting that the loss of c orf function interferes with the formation of glutarate derivatives through a glutaryl-coa intermediate (figure ). in striking contrast to ga , individuals with ga have no consistent disease phenotype. this fact, together with urine metabolite data, provides some insight into the path- ophysiology of ga . relative to individuals with ga , those with ga produce comparatively little -hydroxy- glutarate, glutarylcarnitine, or glutarylglycine; in ga the ratios of glutarate to these metabolites are - to -fold higher than they are in ga (table and figure ). this suggests that individuals with ga produce little or no glutaryl-coa because this compound is the predicted pre- cursor of glutarylcarnitine, glutarylglycine, and -hydrox- yglutarate in tissues (figure ). such a finding shows that glutaryl-coa or one of its downstream derivatives is likely to be the primary neurotoxin in ga , an idea consistent with recent in vitro work. we identified four different mutations in c orf that cause ga . one of these, c. c/t, underlies the amish form of the condition. there is no existing functional data on the c orf protein, but analysis of its amino acid se- quence suggests that it functions in mitochondria rather than peroxisomes (mitoprot) and that one of its actions might be to transfer coa to glutarate (ncbi gene). on the basis of these findings, we postulate that alpha-ketoa- dipic aciduria, ga , and ga arise from sequential defects along a common mitochondrial lysine-tryptophan degra- dation pathway. within this pathway, c orf may act independently or as part of a multiunit complex (figure ). future studies to ascertain the exact enzymatic function er , and cellular localization of the c orf gene product are critical to advance our knowledge of this metabolic pathway. the incidence of ga in the general population is unknown. because these individuals do not produce abnormal quantities of glutarylcarnitine, they are not de- tected by newborn screening methods based on tandem mass spectrometry analysis of blood spots on dried filter paper. however, clinical laboratories commonly encounter isolated elevations of urine glutaric acid during routine or- ganic-acid analysis and, because ga does not appear to figure . urine metabolite quantitation in patients with ga and ga and normal controls individuals with ga (black triangles) and ga (white circles) have elevated urine glutarate relative to controls (gray squares). how- ever, compared to subjects with ga , those with ga have much lower levels of glutarate derivatives: -hydroxyglutarate (upper panel), glutarylcarnitine (middle panel), and glutarylglycine (lower panel). note that all axes are in log scale. the american cause disease, the incidence is certainly underestimated. our findings provide a rationale for sequencing c orf in individuals with isolated, persistent, and unexplained glutaric aciduria. perhaps more importantly, however, the present study brings the pathophysiology of ga into sharper focus. future in vitro and in vivo studies of ga should concentrate on elucidating precisely how the intra- mitochondrial formation of glutaryl-coa interferes with the metabolism and survival of striatal neurons. in conclusion, we used , marker snp microarrays to genetically map ga to chromosome p and demon- strated that the disorder is caused by mutations in c orf . on the basis of our findings, we postulate that alpha-ketoadipic aciduria, ga , and ga arise from sequen- tial molecular lesions along a common mitochondrial lysine-tryptophan degradation pathway. consistent with recent in vitro studies, our results suggest that the forma- tion of glutaryl-coa in mitochondria is an integral part of the histotoxic process in ga . received: september , revised: september , accepted: september , published online: october , web resources the urls for data presented herein are as follows: dbsnp, http://www.ncbi.nlm.nih.gov/snp/ mitoprot, http://ihg .helmholtz-muenchen.de/ihg/mitoprot. html ncbi gene, http://www.ncbi.nlm.nih.gov/sites/entrez?db¼gene ncbi genome browser, http://www.ncbi.nlm.nih.gov/projects/ mapview/map_search.cgi?taxid¼ online mendelian inheritance in man (omim), http://www.ncbi. nlm.nih.gov/sites/entrez?db¼omim primer , http://frodo.wi.mit.edu/ ucsc genome browser, http://genome.ucsc.edu/cgi-bin/ hggateway references . morton, d.h., bennett, m.j., seargeant, l.e., nichter, c.a., and kelley, r.i. ( ). glutaric aciduria type i: a common cause of episodic encephalopathy and spastic paralysis in the amish of lancaster county, pennsylvania. am. j. med. genet. , – . . strauss, k.a., lazovic, j., wintermark, m., and morton, d.h. ( ). multimodal imaging of striatal degeneration in amish patients with glutaryl-coa dehydrogenase deficiency. brain , – . . bennett, m.j., pollitt, r.j., goodman, s.i., hale, d.e., and va- mecq, j. ( ). atypical riboflavin-responsive glutaric acidu- ria, and deficient peroxisomal glutaryl-coa oxidase activity: a new peroxisomal disorder. j. inherit. metab. dis. , – . . van veldhoven, p.p., vanhove, g., assselberghs, s., eyssen, h.j., and mannaerts, g.p. ( ). substrate specificities of rat liver peroxisomal acyl-coa oxidases: palmitoyl-coa oxidase (inducible acyl-coa oxidase), pristanoyl-coa oxidase journal of human genetics , – , november , http://www.ncbi.nlm.nih.gov/snp/ http://ihg .helmholtz-muenchen.de/ihg/mitoprot.html http://ihg .helmholtz-muenchen.de/ihg/mitoprot.html http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene http://www.ncbi.nlm.nih.gov/projects/mapview/map_search.cgi?taxid= http://www.ncbi.nlm.nih.gov/projects/mapview/map_search.cgi?taxid= http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim http://frodo.wi.mit.edu/ http://genome.ucsc.edu/cgi-bin/hggateway http://genome.ucsc.edu/cgi-bin/hggateway figure . alpha-ketoadipic aciduria, ga , and ga possibly arise from sequential enzymatic defects along a common mitochon- drial lysine-tryptophan degradation pathway the c orf protein has a putative mitochondrial targeting sequence and a coa transferase domain. it may function as part of a multiunit enzyme complex (a), similar to the alpha-ketoglutarate dehydrogenase system, or as the second of two independent enzymes, the first of which decarboxylates alpha-ketoadipate to glutarate (b). in either case, c orf defects would block the production of glutaryl-coa, which we believe to be the source of -hydroxyglutarate, glutarylcarnitine, and glutarylglycine in patients with ga . ka, ketoadipic aciduria; ga , glutaric aciduria type ; ga , glutaric aciduria type ; and gcdh, glutaryl-coa dehydrogenase. (non-inducible acyl-coa oxidase), and trihydroxycoprosta- noyl-coa oxidase. j. biol. chem. , – . . wanders, b.j., denis, s.w., and dacremont, g. ( ). studies on the substrate specificity of the inducible and non-inducible acyl-coa oxidases from rat kidney peroxisomes. j. biochem. , – . . przyrembel, h., bachmann, d., lombeck, i., becker, k., wendel, u., wadman, s.k., and bremer, h.j. ( ). alpha- ketoadipic aciduria, a new inborn error of lysine metabolism; biochemical studies. clin. chim. acta , – . . wendel, u., rudiger, h.w., przyrembel, h., and bremer, h.j. ( ). alpha-ketoadipic aciduria: degradation studies with fibroblasts. clin. chim. acta , – . . wilson, r.w., wilson, c.m., gates, s.c., and higgins, j.v. ( ). alpha-ketoadipic aciduria: a description of a new metabolic error in lysine-tryptophan degradation. pediatr. res. , – . . puffenberger, e.g. ( ). genetic heritage of the old order mennonites of southeastern pennsylvania. am. j. med. genet. c. semin. med. genet. c, – . . knerr, i., zschocke, j., trautmann, u., dorland, l., de koning, t.j., muller, p., christensen, e., trefz, f.k., wundisch, g.f., rascher, w., et al. ( ). glutaric aciduria type iii: a distinc- tive non-disease? j. inherit. metab. dis. , – . . puffenberger, e.g., hu-lince, d., parod, j.m., craig, d.w., dobrin, s.e., conway, a.r., donarum, e.a., strauss, k.a., dunckley, t., cardenas, j.f., et al. ( ). mapping of sudden infant death with dysgenesis of the testes syndrome (siddt) by a snp genome scan and identification of tspyl loss of function. proc. natl. acad. sci. usa , – . the american journal of human genetics , – , novemb . broman, k.w., and weber, j.l. ( ). long homozygous chromosomal segments in reference families from the centre d’etude du polymorphisme humain. am. j. hum. genet. , – . . puffenberger, e.g., strauss, k.a., ramsey, k.e., craig, d.w., stephan, d.a., robinson, d.l., hendrickson, c.l., gottlieb, s., ramsay, d.a., siu, v.m., et al. ( ). polyhydramnios, megalencephaly and symptomatic epilepsy caused by a homo- zygous -kilobase deletion in lyk . brain , – . . strauss, k.a., puffenberger, e.g., craig, d.w., panganiban, c.b., lee, a.m., hu-lince, d., stephan, d.a., and morton, d.h. ( ). genome-wide snp arrays as a diagnostic tool: clinical description, genetic mapping, and molecular charac- terization of salla disease in an old order mennonite popula- tion. am. j. med. genet. a. a, – . . strauss, k.a., puffenberger, e.g., huentelman, m.j., gottlieb, s., dobrin, s.e., parod, j.m., stephan, d.a., and morton, d.h. ( ). recessive symptomatic focal epilepsy and mu- tant contactin-associated protein-like . n. engl. j. med. , – . . rinaldo, p., hahn, s.h., and matern, d. ( ). inborn errors of amino acid, organic acid, and fatty acid metabolism. in tietz textbook of clinical chemistry and molecular diagnos- tics, e.r. ashwood, d.e. bruns, and c.a. burtis, eds. (st. louis: elsevier saunders). . tortorelli, s., hahn, s.h., cowan, t.m., brewster, t.g., rinaldo, p., and matern, d. ( ). the urinary excretion of glutarylcarnitine is an informative tool in the biochemical diagnosis of glutaric acidemia type i. mol. genet. metab. , – . er , . itoh, t., ito, t., ohba, s., sugiyama, n., mizuguchi, k., yamagu- chi, s., and kidouchi, k. ( ). effect of carnitine administra- tion on glycine metabolism in patients with isovaleric acide- mia: significance of acetylcarnitine determination to estimate the proper carnitine dose. tohoku j. exp. med. , – . the america . sauer, s.w., okun, j.g., schwab, m.a., crnic, l.r., hoffmann, g.f., goodman, s.i., koeller, d.m., and kolker, s. ( ). bio- energetics in glutaryl-coenzyme a dehydrogenase deficiency: a role for glutaryl-coenzyme a. j. biol. chem. , – . n journal of human genetics , – , november , 	genetic mapping of glutaric aciduria, type , to chromosome and identification of mutations in c orf 	web resources 	references diagnostic accuracy of administrative data algorithms in the diagnosis of osteoarthritis: a systematic review research article open access diagnostic accuracy of administrative data algorithms in the diagnosis of osteoarthritis: a systematic review swastina shrestha , amish j. dave , , elena losina , , , and jeffrey n. katz , , , * abstract background: administrative health care data are frequently used to study disease burden and treatment outcomes in many conditions including osteoarthritis (oa). oa is a chronic condition with significant disease burden affecting over million adults in the us. there are few studies examining the performance of administrative data algorithms to diagnose oa. the purpose of this study is to perform a systematic review of administrative data algorithms for oa diagnosis; and, to evaluate the diagnostic characteristics of algorithms based on restrictiveness and reference standards. methods: two reviewers independently screened english-language articles published in medline, embase, pubmed, and cochrane databases that used administrative data to identify oa cases. each algorithm was classified as restrictive or less restrictive based on number and type of administrative codes required to satisfy the case definition. we recorded sensitivity and specificity of algorithms and calculated positive likelihood ratio (lr+) and positive predictive value (ppv) based on assumed oa prevalence of . , . , and . . results: the search identified studies that used algorithms. of these algorithms, were classified as restrictive and as less restrictive. restrictive algorithms had lower median sensitivity and higher median specificity compared to less restrictive algorithms when reference standards were self-report and american college of rheumatology (acr) criteria. the algorithms compared to reference standard of physician diagnosis had higher sensitivity and specificity than those compared to self-reported diagnosis or acr criteria. conclusions: restrictive algorithms are more specific for oa diagnosis and can be used to identify cases when false positives have higher costs e.g. interventional studies. less restrictive algorithms are more sensitive and suited for studies that attempt to identify all cases e.g. screening programs. keywords: osteoarthritis, diagnostic accuracy, administrative data, systematic review background administrative health care data are collected by health care providers, insurers, and governments for enrollment, reimbursement, and payment purposes [ , ]. sources of administrative data include physician billing databases, hospitalization discharge records, prescription drug records, private insurers, managed care plan data systems, medicare, and medicaid [ ]. administrative data are used increasingly in health services research as they tend to be less expensive than manual medical rec- ord review, available for large populations, and unaffected by recall or selection biases [ , , ]. researchers also use administrative health care data to identify patients for inclusion in study cohorts as these data provide a less costly approach to identifying subjects than screening in person or by phone [ ]. along with these advantages, however, administrative data have limitations, such as misclassification, which may jeopardize study results [ ]. an international consortium of researchers and administrative health care data users has identified validation of administrative data coding as a research priority [ ]. to strike a balance between the spe- cificity and sensitivity of administrative data, investigators * correspondence: jnkatz@partners.org department of orthopedic surgery, orthopaedic and arthritis center for outcomes research, brigham and women’s hospital, francis st, bc - , boston, ma , usa harvard medical school, boston, ma, usa full list of author information is available at the end of the article © the author(s). open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/ . /) applies to the data made available in this article, unless otherwise stated. shrestha et al. bmc medical informatics and decision making ( ) : doi . /s - - -y http://crossmark.crossref.org/dialog/?doi= . /s - - -y&domain=pdf mailto:jnkatz@partners.org http://creativecommons.org/licenses/by/ . / http://creativecommons.org/publicdomain/zero/ . / create algorithms, which typically involve ‘and’ and ‘or’ statements to focus on diagnosis or procedures of interest. the us food and drug administration’s (fda) mini- sentinel initiative has highlighted the importance of understanding the validity of administrative data algo- rithms for identifying health outcomes of interest [ , ]. the accuracy of algorithms for identifying cases with specific diagnoses depends on features of the database, condition, study population, and reference standard for confirming the diagnosis. many of the studies that establish the accuracy of administrative data algorithms lack consistent methodology and reporting standards, making it difficult to compare the data accuracy across studies [ ]. these issues are of concern to investigators and policy makers worldwide as many health systems across the globe are making increasing use of administrative data. this study examines the accuracy of administrative health care data algorithms for identifying patients with osteoarthritis (oa). oa is associated with significant bur- den, affecting million adults in the us and more than million adults worldwide [ , ]. administrative data play an important role in research on disease burden, treatment outcomes, and quality improvement across a range of conditions including oa [ – ]. however the accuracy of administrative data for the diagnosis of oa has received sparse study. one systematic review reported the accuracy of administrative data-based diagnosis in a wide range of rheumatologic conditions but provided limited detailed information on oa [ ]. the goal of the present study is to perform a systematic review of studies of administrative data algorithms to diagnose oa and to evaluate the diagnostic characteristics of these algorithms based on restrictiveness and reference standards. methods study identification this systematic review was performed based on the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines [ ]. a search of all titles available in medline, embase, cochrane, and pubmed was conducted using the following major keywords: administrative data, validation studies, and osteoarthritis (additional file : table s for search strings) [ ]. we carried out the search on january and two reviewers (ajd and ss) screened every reference to determine whether the study met the inclusion criteria. we also reviewed the bibliographies of relevant articles to identify articles that might have been missed by our initial search. the search was repeated to include references published from january through february . inclusion and exclusion criteria we included english-language studies that reported both sensitivity and specificity of administrative data algorithms to identify cases of symptomatic oa by comparing the algorithm with a reference standard. if the studies pre- sented by tables of positive and negative cases (based on a reference standard) crossed with positive and negative putative cases (based on an administrative data algorithm), we used the table to calculate sensitivity and specificity of the algorithm using the formulas below [ ]. true positives were cases that were identified by both algorithm and gold standard and true negatives were cases that were not identified by both. false positives were cases that were identified by the algorithm but not the gold standard and false negatives were cases that were identified by the gold standard but not by the algorithm. sensitivity ¼ true positives true positives þ false negatives specificity ¼ true negatives true negatives þ false positives studies only reporting positive predictive value (ppv) without reporting by tables (or including sensitivity and specificity values) were excluded. if the algorithm classified oa positive cases as definite and possible, we calculated the sensitivity and specificity based on only the definite cases. in studies that evaluated diagnostic algorithms for oa in multiple anatomic locations (e.g. hip, knee, hand and combinations of these joints), algorithms that combined all anatomic locations of oa were preferentially selected. algorithms that used only imaging as the reference standard were excluded due to the variability in oa imaging classification criteria and frequent occurrence of positive imaging findings in asymptomatic persons. we contacted the authors of studies that reported other diagnostic measures such as kappa value to obtain the crude by table data for computing the sensitivity and specificity of the algorithms. discrepancies between reviewers regarding the reasons for abstract and study exclusions were resolved by consult- ation with senior coauthors (jnk and el). data abstraction and quality assessment from the articles that met our inclusion criteria, we extracted information on: author, year of publication, country of study, administrative data source and setting, location of oa, cohort characteristics (age, gender, size), description of the algorithm (minimum number of out- patient, prescription, and hospitalization codes, use of diagnosis information entered in electronic medical rec- ord, and years of administrative data), reference standard, disease prevalence in the sample, algorithm and reference standard positive and negative cases, and performance characteristics of the algorithms (positive predictive value, sensitivity and specificity with % confidence intervals). when % confidence intervals were not provided, we shrestha et al. bmc medical informatics and decision making ( ) : page of calculated them using the binomial distribution when pos- sible. we considered oa diagnosis in the medical record as a proxy for physician diagnosis. for quality assessment of all included studies, we used the point modified standards for reporting of studies of diagnostic accuracy (stard) criteria [ ]. if the study results were in abstract form prior to manuscript submission, we contacted the author for quality assessment of the study. the two reviewers (ajd and ss) independently completed all screening, data extraction, and quality reporting activities. analysis we classified the algorithms as restrictive or less restrictive based on the number and use of stringent codes such as procedural, hospitalization, or prescription codes to ascer- tain the diagnosis of oa. the algorithm was classified as restrictive if it required more than one code of any kind or if it required one or more stringent code such as proced- ural, prescription, or hospitalization codes. for example, each of the following algorithms would be classified as restrictive ) an algorithm that required oa codes from two separate outpatient visits; ) an algorithm that required one code from an outpatient visit and one prescription code; and ) an algorithm that required a single hospitalization visit. algorithms that only required a single oa code from one outpatient visit were classified as less restrictive. additionally, an algorithm that required a single oa code from one outpatient visit or one prescription record would be deemed less restrictive because the more stringent prescription code was not required to identify oa diagnosis. we recorded the sensitivity and specificity of all the oa ascertainment algorithms. for studies that did not report sensitivity and/or specificity, we calculated these values from by tables that stratified the sample based on algorithm positivity and reference standard positivity. we calculated the positive likelihood ratio (lr+) of the algorithms using the formula [ ]: positive likelihood ratio lrþð Þ ¼ sensitivity −specificity in order to calculate the positive likelihood ratios of algorithms with perfect specificity, we used the lower end of the confidence interval of specificity. additionally we calculated positive predictive values (ppv) for different oa prevalence rates in order to highlight the prevalence dependence of the algorithm ppvs [ ]. the ppv of an algorithm determines the probability that an individual identified by the algorithm truly has oa. we used the hypothetical proportion of . to approximate oa preva- lence in general population, . to approximate oa prevalence in adults over , and . to approximate oa prevalence in specialty clinic settings [ , , ]. results search results our search strategy identified unique articles. upon screening the titles, we identified articles for abstract review. % ( / ) of abstracts were excluded be- cause they addressed other administrative data; % ( / ) were studies of quality of care, therapy, and cost-effectiveness; and % ( / ) used no adminis- trative data. we identified references for full article review. of these fully reviewed articles, studied other administrative data, did not include quantita- tive validation of the algorithm, only reported the ppv of the algorithms but not sensitivity or specifi- city, was a review, combined codes for oa and rheumatoid arthritis, and study compared self- reported oa diagnosis with medical records. we in- cluded articles from this search in our final analysis. in addition, we identified peer-reviewed article, abstract, and research report from searching the bibliographies of relevant articles. the updated search on february identified eligible article, which was included in the review. figure outlines the study selec- tion process. features of abstracted studies table describes the characteristics of the included studies. study sample size ranged from to and sources of administrative data included medicare claims, health maintenance organizations (hmo), primary care surveillance network, and health data repositories. five studies were published in peer-reviewed journals [ – ], one was published as a research report [ ], and one as an abstract [ ]. the reference standards for posi- tive oa diagnosis were self-report, american college of rheumatology (acr) classification criteria for oa, and physician diagnosis. one study compared the diag- nostic accuracy of algorithms using multiple reference standards, including plain radiograph, mri, self-report, and acr classification criteria. algorithms from these studies were included in the final analysis, of which were classified as restrictive and were classi- fied as less restrictive. performance characteristics stratified by reference standard type the sensitivity, specificity, lr+, and ppv at assumed preva- lence values of . , . , and . of individual algorithms are shown in table . table reports the same diagnostic performance characteristics aggregated across restrictive versus less restrictive algorithms and across types of refer- ence standard. the sensitivity and specificity of the algo- rithms with % ci is shown as forest plots in figs. and respectively. shrestha et al. bmc medical informatics and decision making ( ) : page of performance characteristics stratified by reference standard type self-report the four assessments of restrictive algorithms with reference standard of self-report had lower sensitivity (median . ) and higher specificity (median . ) compared to two assessments of less restrictive algorithms (median sensitivity . ) and (median specificity . ). the restrictive algorithms had higher lr+ and ppvs compared to less restrictive algorithms (table ). acr criteria the one assessment of restrictive algorithms with refer- ence standard of acr criteria had lower sensitivity ( . ) and higher specificity ( . ) compared to two assessments of less restrictive algorithms (median sensitivity . ) and (median specificity = . ). physician diagnosis all the algorithms that had reference standard of phys- ician diagnosis were less restrictive. among these, studies used emr based algorithms and study used a non-emr based algorithm. the emr based algorithms were highly specific ( . ) and modestly sensitive ( . ) and lr+ of emr based algorithms ranged from . to . . the non-emr based algorithm was highly specific ( . ) but less sensitive ( . ) and lr+ of non-emr based algorithm was . . quality assessment table shows the number of studies that met each of the data quality and reporting criteria (modified stard cri- teria). all studies reported the type of study and location, described patient sampling, details of data collection, dis- ease classification, methods of calculating accuracy, and discussed the applicability of findings. most studies provided the age of the cohort, identified the diagnosis of the validation cohort, and described the inclusion and exclusion criteria. only one study reported the severity of disease, studies provided flow charts and no study revali- dated the algorithm in a different population. the most commonly reported study statistics were positive predict- ive value (n = ), sensitivity (n = ), specificity (n = ), and negative predictive value (n = ). of these, studies provided the % confidence interval for all reported diagnostic measures. only study reported the likelihood titles met search criteria medline: embase: pubmed: cochrane abstracts screened reason for exclusion: –duplicate: –not english: –animal study: –title includes other condition: –study of instrument/ index/scale: –genetics/ biochemistry study: –study population younger than years: reason for exclusion: –study of other administrative data: –no quantitative validation of oa algorithm: –no sensitivity, specificity or by table: –combines oa & rheumatoid arthritis: –review study: –no administrative data: reason for exclusion: –duplicate: –study of other conditions: –study of instrument/ index/scale: –genetics/ biochemistry study: –no mention of administrative data: –study of other administrative data: –study of therapy/ cost & quality of care: –unable to locate reference: peer-reviewed article, abstract, and research report identified through references of relevant articles, and article identified in update articles reviewed articles included in systematic review fig. study search and selection process shrestha et al. bmc medical informatics and decision making ( ) : page of ratio and studies calculated the prevalence of oa in the study population. discussion we conducted a systematic literature review of diagnos- tic accuracy studies of administrative data algorithms for osteoarthritis diagnosis and compared their accuracy based on restrictiveness and reference standards employed in the studies. more restrictive algorithms had lower sensitivities and higher specificities compared to less restrictive algorithms when the reference standards were acr criteria and self-report. all the algorithms that were validated against physician diagnosis were less restrictive and had very high specificities. the high positive likeli- hood ratios in this group was driven by studies that validated oa diagnosis in the electronic medical record (emr) based primary care database, canadian primary care sentinel surveillance network (cpcssn), designed for chronic disease surveillance [ ]. the database com- bined billing icd- codes with information from the emr that allowed for more rigorous case definitions. widdifield et al. conducted a systematic review of studies that validated administrative data algorithms to identify rheumatic diseases [ ]. they included osteoarth- ritis among the conditions studied but did not provide any analyses of the performance characteristics of oa algorithms. the authors reported high variability in pa- tient sampling, reference standards, and measures of diag- nostic accuracy among studies [ ]. they found that use of pharmaceutical codes across the range of rheumatic conditions increased algorithm specificity slightly but compromised sensitivity; we observed similar patterns in studies of oa [ ]. our study included five additional co- horts not included in widdifield et al., and excluded oa studies that did not provide adequate data to calculate likelihood ratios [ ]. these differences notwithstanding, the two studies concurred in finding that greater restrict- iveness increased specificity of the administrative data algorithm. widdifield and colleagues also suggested that study algorithms using self-report as the reference stand- ard had lower sensitivity compared to studies that used medical record review as the reference standard [ ]. our study found that the algorithms had similar sensitivity when the reference standard was self-reported diagnosis ( . ) compared to physician diagnosis in the medical record ( . ). we calculated the positive likelihood ratio (lr+) and positive predictive values (ppv) of each algorithm at assumed prevalence rates of . , . , and . . many validation studies of administrative data algorithms only report ppv. however, the sensitivity and specificity of the algorithm are generally not influenced by disease fig. forest plot of sensitivity of oa diagnosis algorithms. table provides description of each algorithm error bars show % confidence intervals (ci). error bars are missing for rahman as they did not report ci. lix r : hospitalization or physician visits or physician visit and rx icd- -cm diagnostic codes in years lix r : hospitalization or physician visits icd- -cm diagnostic codes in years lix r : physician visits icd- -cm diagnostic codes in years rahman r : physician visits in years or hospitalization icd- -cm diagnostic code compared with self report rahman r : physician visits in years or hospitalization icd- -cm diagnostic code compared with acr criteria lix l : physician visit icd- -cm diagnostic code in years rahman l : physician visit or hospitalization icd- -cm diagnostic code com- pared with self report rahman l : physician visit or hospitalization icd- -cm diagnostic code compared with acr criteria shrestha et al. bmc medical informatics and decision making ( ) : page of prevalence [ ], the ppvs depend on the underlying prevalence of the condition in the study population [ ]. our results show that for the same algorithm, the ppv improves when the underlying oa prevalence increases from . to . and . . this suggests that when studies report high ppv, we cannot ascertain whether the high ppv stems from a good algorithm or the underlying high oa prevalence in the study sample. therefore, qualification of the algorithm solely based on ppv may be misleading. thus, the underlying oa preva- lence of the study sample needs to be clearly specified to evaluate the ppv of administrative data algorithms. oa is a common comorbidity in the older population and has been frequently cited as an underreported diag- nosis in studies that use administrative data to identify medical conditions [ ]. the performance characteristics of administrative data algorithms diagnosing oa were influ- enced by reference standard and algorithm restrictiveness. table characteristics of included studies study country sample size diagnosis age description % female admin data source study population fowles et al. [ ] us unspecified oa and above not reported medicine parts a and b claims primary care patients in maryland harrold et al. [ ] us unspecified oa and above % health maintenance organization (hmo) multispecialty group practice patients lix et al. [ ] canada unspecified oa and above not reported population health research data respiratory general manitoba population rahman et al. [ ] canada knee oa range – not reported bc linked health database subjects with knee pain from a population based study of oa kadhim-saleh et al. [ ] canada unspecified oa mean age % canadian primary care sentinel surveillance network ontario primary care research network williamson et al. [ ] canada unspecified oa % above . % canadian primary care sentinel surveillance network primary care research network in canada coleman et al. [ ] canada unspecified oa % above % canadian primary care sentinel surveillance network mantibo primary care research network fig. forest plot of sensitivity of oa diagnosis algorithms. table provides description of each algorithm. error bars show % confidence intervals (ci). error bars are missing for rahman as they did not report ci. lix r : hospitalization or physician visits or physician visit and rx icd- cm diagnostic codes in years lix r : hospitalization or physician visits icd- -cm diagnostic codes in years lix r : physician visits icd- -cm diagnostic codes in years rahman r : physician visits in years or hospitalization icd- -cm diagnostic code compared with self report rahman r : physician visits in years or hospitalization icd- -cm diagnostic code compared with acr cri- teria lix l : physician visit icd- -cm diagnostic code in years rahman l : physician visit or hospitalization icd- -cm diagnostic code compared with self report rahman l : physician visit or hospitalization icd- -cm diagnostic code compared with acr criteria shrestha et al. bmc medical informatics and decision making ( ) : page of table descriptive and diagnostic characteristics of administrative data algorithms algorithm restrictiveness refence standard study algorithm definition years spanned by admin data sensitivity specificity positive likelihood ratio calculated ppv at % prevalence calculated ppv at % prevalence calculated ppv at % prevalence % ci % ci restrictive lix [ ] hospitalization or physician visits or physician visit and rx icd- -cm diagnostic codes in years . . – . . . – . . . . . lix [ ] hospitalization or physician visits icd– –cm diagnostic codes in years . . – . . . – . . . . . lix [ ] physician visits icd– –cm diagnostic codes in years . . – . . . – . . . . self-report rahman [ ] physician visits in years or hospitalization icd- -cm diagnostic code b . . . . . . acr criteria rahman [ ] physician visits in years or hospitalization icd- -cm diagnostic code b . . . . . . less restrictive medical record review fowles [ ] physician visit icd- -cm diagnostic code . . – . . . – . . . . . kadhim–saleh a [ ] icd- -cm diagnostic code or problems list in emr unspecified . . – . ( . δ) . – . . . . . coleman a [ ] icd- -cm diagnostic code or problems list in emr unspecified . . – . . . – . . . . . williamson a [ ] icd- -cm diagnostic code or problems list in emr unspecified . . – . . . – . . . . . self-report lix [ ] physician visit icd- -cm diagnostic code in years . . – . . . – . . . . . acr criteria rahman [ ] physician visit or hospitalization icd- -cm diagnostic code b . . . . . . harrold [ ] inpatient or outpatient icd- -cm diagnostic code . . – . . . – . . . . . rahman [ ] physician visit or hospitalization icd- -cm diagnostic code b . . . . . . acase definitions are developed in emr based database bvisit codes were restricted to years and timespan of hospitalization code was unspecified. rahman did not report % ci δlower confidence interval of specificity (instead of ) was used to calculate lr+s and ppvs sh resth a et a l. b m c m ed ica l in fo rm a tics a n d d ecisio n m a kin g ( ) : p ag e o f table medians and ranges of diagnostic characteristics of administrative data algorithms algorithm restrictiveness reference standard no of algorithms median sensitivity sensitivity range median specificity specificity range median positive likelihood ratio positive likelihood ratio range median ppv at % prevalence median ppv at % prevalence median ppv at % prevalence restrictive self-report . . – . . . – . . . – . . . . acr criteria . na . na . na . . . less restrictive physician diagnosis . . – . . . – . . . – . . . . self-report . . – . . . – . . . – . . . . acr criteria . . – . . . – . . . – . . . . sh resth a et a l. b m c m ed ica l in fo rm a tics a n d d ecisio n m a kin g ( ) : p ag e o f table number of studies meeting individual stard modified criteria for validating health administrative data reported/total title, keywords, abstract identify article as study of assessing diagnostic accuracy / identify article as study of administrative data / introduction: state disease identification & validation one of goals of study / methods: participants in validation cohort: describe validation cohort (cohort of patients to which reference standard was applied) / age / disease / severity / location/jurisdiction / describe recruitment procedure of validation cohort / inclusion criteria / exclusion criteria / describe patient sampling (random, consecutive, all, etc.) / describe data collection / who identified patients and did selection adhere to patient recruitment criteria / who collected data / a priori data collection form / disease classification / split sample (i.e. re-validation using a separate cohort) / test methods: describe number, training and expertise of persons reading reference standard / if > person reading reference standard, quote measure of consistency (e.g. kappa) / blinding of interpreters of reference standard to results of classification by administrative data e.g. chart abstractor blinded to how that chart was coded / statistical methods: describe methods of calculating/comparing diagnostic accuracy / results: participants: report when study done, start/end dates of enrollment / describe number of people who satisfied inclusion/exclusion criteria / study flow diagram / test results: report distribution of disease severity / report cross-tabulation of index tests by results of reference standard / estimates: report at least estimates of diagnostic accuracy / diagnostic accuracy measures resported: sensitivity / spec / ppv / npv / shrestha et al. bmc medical informatics and decision making ( ) : page of we found that most of the algorithms that identify oa are relatively insensitive, potentially missing about % of the cases [ – ]. several reasons could account for the low sensitivity. for example, the physician might record oa as a secondary diagnosis but not enter the billing code, choosing instead to focus on the primary diagnosis. this situation might arise when the primary diagnosis is semi urgent such as active coronary heart disease with congestive heart failure, physicians may not be inclined to code for oa in such a circumstance. it has been shown that when physicians see patients for more pressing prob- lems they often do not code less pressing problems [ ]. the specificity of the algorithms was relatively high and algorithms that were validated against physician diagnosis had the highest specificity. as a result, the likelihood ra- tios of the algorithms with physician diagnosis as the ref- erence standard were very high. the specificity of algorithms that validated the diagnosis against acr cri- teria might have been lower because acr classification criteria for oa are stringent and not widely used in clin- ical settings to diagnose oa. the restrictive algorithms had lower sensitivity and higher specificity compared to the less restrictive algo- rithms. therefore, when the purpose of the algorithm is to identify and recruit a patient cohort for a research study such as a treatment trial, it is crucial that each subject has the disease in question. thus, restrictive al- gorithms with high specificity are most useful. however, if the aim is to identify all positive cases of oa, such as a screening program, less restrictive algorithms with high sensitivity may be more useful – especially if a sec- ond, more specific can be applied to those that screen positive on the algorithm in order to reduce the number of false positive cases. limitations of this review include the exclusion of stud- ies written in languages other than english. we did not report youden index of the algorithms as only one study reported this statistic. we did not include studies that reported only kappa values, as we lacked the information to compute sensitivity and specificity for these algorithms. we did not include algorithms with radiographs as a reference standard as radiographs can be both insensitive and non-specific in persons with oa [ – ]. as a conse- quence diagnoses made on the basis of radiographic find- ings may be inaccurate. such misclassification would bias findings of this review to the null. also, we did not con- duct a meta-analysis of the diagnostic accuracies due to substantial heterogeneity in the methodologies of the in- cluded studies. we did not select algorithms based on site of oa, as majority of the studies did not specify the site of oa. the studies were heterogeneous with respect to population characteristics (e.g. age range), settings (e.g. primary care, specialty clinics), and administrative data sources (e.g. medicare, health maintenance organization, primary care surveillance database, and state database). these differences enhance generalizability of findings but the heterogeneity precludes formal quantitative synthesis of the study findings. finally, we recognize that each of the reference standards used in these studies (self-report, physician diagnosis, acr criteria) has advantages and drawbacks. the observation that restrictive algorithms were less sensitive and more specific across multiple refer- ence standards supports the robustness of this finding. conclusions administrative data algorithms with restrictive case defini- tions are more specific for the diagnosis of oa whereas algorithms with less restrictive case definition are more sensitive. in general, published algorithms designed to identify positive oa cases have low sensitivity, missing more than half the cases. algorithms assessed with refer- ence standard of physician diagnosis have higher sensitivity and specificity than algorithms assessed with reference of self-reported diagnosis or acr criteria. our assessment of article quality revealed variable and sparse reporting of sev- eral key methodological features such as oa severity and oa prevalence in the underlying population. our work has implications for research and policy. from a research standpoint, the most appropriate algo- rithm for a particular study will depend on whether the study would best be served by optimizing sensitivity (missing as few cases as possible) or optimizing positive table number of studies meeting individual stard modified criteria for validating health administrative data (continued) likelihood ratios / kappa / area under the roc curve/c-statistic / accuracy/agreement / report accuracy for subgroups (e.g. age, geography, differen sex, etc.) / if ppv/npv reported, ratio of cases/controls of validation cohort approximate prevalence of condition in the population / report % confidence intervals for each diagnostic measure / discussion: discuss the applicability of the validation findings / shrestha et al. bmc medical informatics and decision making ( ) : page of predictive value (increasing the likelihood that a person characterized by the algorithm as having oa indeed has oa). our data suggest that requiring more than one oa outpatient code or a specialized code (e.g. a pharmacy or a hospitalization claim) will increase specificity and ppv, whereas requiring simply a single outpatient oa code will enhance sensitivity at the expense of specificity. from a policy standpoint, in circumstances that employ administrative data to portray burden of disease without actually intervening in individuals, the overall level of misclassification may be the most relevant parameter as the goal would be to have as accurate a count as pos- sible. if an algorithm is used to target a subgroup of pa- tients for a specific intervention (such as a prevention or education program), an algorithm with high ppv may be the best approach to ensure that program resources are spent on persons who indeed have oa. additional file addition file : table s . medline, embase, cochrane and pubmed search strategies; list of search strings and keywords used to identify studies in different databases. (docx kb) abbreviations acr, american college of rheumatology; fda, us food and drug administration’s; lr+, positive likelihood ratio; oa, osteoarthritis; ppv, positive predictive value; prisma, preferred reporting items for systematic reviews and meta-analyses; stard, standards for reporting of studies of diagnostic accuracy funding funded in part by brigham and women’s hospital. availability of data and materials the datasets supporting the conclusions of this article are included within the article and its additional file. authors’ contributions jnk conceived of the study. ss and ajd collected and summarized the data. el made substantial contributions to the conception and design, analysis and interpretation of data. ss drafted the manuscript. ajd, el, and jnk revised the manuscript for critically important intellectual content. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. consent for publication not applicable. ethics approval and consent to participate not applicable. author details department of orthopedic surgery, orthopaedic and arthritis center for outcomes research, brigham and women’s hospital, francis st, bc - , boston, ma , usa. harvard medical school, boston, ma, usa. division of rheumatology, immunology and allergy, brigham and women’s hospital, boston, ma, usa. department of epidemiology, harvard school of public health, boston, ma, usa. department of biostatistics, boston university school of public health, boston, ma, usa. received: october accepted: june references . iezzoni li. assessing quality using administrative data. ann intern med. ; 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( ): – . doi: . /art. . epub / / . . harrold lr, yood ra, straus w, andrade se, reed ji, cernieux j, et al. challenges of estimating health service utilization for osteoarthritis patients on a population level. j rheumatol. ; ( ): – . epub / / . . kopec ja, rahman mm, sayre ec, cibere j, flanagan wm, aghajanian j, et al. trends in physician-diagnosed osteoarthritis incidence in an administrative database in british columbia, canada, – through – . arthritis rheum. ; ( ): – . doi: . /art. . epub / / . . katz jn, barrett j, mahomed nn, baron ja, wright rj, losina e. association between hospital and surgeon procedure volume and the outcomes of total knee replacement. j bone joint surg am. ; -a( ): – . epub / / . . katz jn, losina e, barrett j, phillips cb, mahomed nn, lew ra, et al. association between hospital and surgeon procedure volume and outcomes of total hip replacement in the united states medicare population. j bone joint surg am. ; -a( ): – . epub / / . . widdifield j, labrecque j, lix l, paterson jm, bernatsky s, tu k, et al. systematic review and critical appraisal of validation studies to identify rheumatic diseases in health administrative databases. arthritis care res (hoboken). ; ( ): – . doi: . /acr. . epub / / . . moher d, liberati a, tetzlaff j, altman dg, group p. preferred reporting items for systematic reviews and meta-analyses: the prisma statement. j clin epidemiol. ; ( ): – . doi: . /j.jclinepi. . . . epub / / . . leong a, dasgupta k, bernatsky s, lacaille d, avina-zubieta a, rahme e. systematic review and meta-analysis of validation studies on a diabetes case definition from health administrative records. plos one. ; ( ): e . doi: . /journal.pone. . pubmed pmid: , pubmed central pmcid: pmc , epub / / . shrestha et al. bmc medical informatics and decision making ( ) : page of dx.doi.org/ . /s - - -y http://dx.doi.org/ . /mlr. b e c aa http://dx.doi.org/ . /mlr. b e c aa http://dx.doi.org/ . /j.jclinepi. . . http://dx.doi.org/ . /jrheum. http://dx.doi.org/ . /pds. http://dx.doi.org/ . / - - - http://dx.doi.org/ . /pds. http://dx.doi.org/ . /pds. http://dx.doi.org/ . /art. http://dx.doi.org/ . /art. http://dx.doi.org/ . /art. http://dx.doi.org/ . /art. http://dx.doi.org/ . /acr. http://dx.doi.org/ . /j.jclinepi. . . http://dx.doi.org/ . /journal.pone. . altman dg, bland jm, diagnostic tests. : sensitivity and specificity. bmj. ; ( ): . epub / / . pubmed pmid: ; pubmed central pmcid: pmc . . simel dl, samsa gp, matchar db. likelihood ratios with confidence: sample size estimation for diagnostic test studies. j clin epidemiol. ; ( ): – . epub / / . . brenner h, gefeller o. variation of sensitivity, specificity, likelihood ratios and predictive values with disease prevalence. stat med. ; ( ): – . epub / / . . mikkelsen wm, dodge hj, duff if, kato h. estimates of the prevalence of rheumatic diseases in the population of tecumseh, michigan, – . j chronic dis. ; ( ): – . epub / / . . fowles jb, lawthers ag, weiner jp, garnick dw, petrie ds, palmer rh. agreement between physicians’ office records and medicare part b claims data. health care financ rev. ; ( ): – . . kadhim-saleh a, green m, williamson t, hunter d, birtwhistle r. validation of the diagnostic algorithms for chronic conditions in the canadian primary care sentinel surveillance network (cpcssn): a kingston practice- based research network (pbrn) report. j am board fam med. ; ( ): – . doi: . /jabfm. . . . epub / / . . harrold lr, yood ra, andrade se, reed ji, cernieux j, straus w, et al. evaluating the predictive value of osteoarthritis diagnoses in an administrative database. arthritis rheum. ; ( ): – . . williamson t, green me, birtwhistle r, khan s, garies s, wong st, et al. validating the cpcssn case definitions for chronic disease surveillance in a primary care database of electronic health records. ann fam med. ; ( ): – . doi: . /afm. . pubmed pmid: , pubmed central pmcid: pmc , epub / / . . coleman n, halas g, peeler w, casaclang n, williamson t, katz a. from patient care to research: a validation study examining the factors contributing to data quality in a primary care electronic medical record database. bmc fam pract. ; : . doi: . /s - - -z. pubmed pmid: , pubmed central pmcid: pmc , epub / / . . lix l, yogendran m, burchill c, metge c, mckeen n, moore d, et al. defining and validating chronic diseases: an administrative data approach. winnipeg: manitoba centre for health policy; . . rahman ja m, kopec ja, cibere j. abstract no. the validation of administrative osteoarthritis diagnosis using a clinical and radiological population-based cohort from british columbia, canada. osteoarthritis cartilage. ; (supplement ):s . . birtwhistle r, keshavjee k, lambert-lanning a, godwin m, greiver m, manca d, et al. building a pan-canadian primary care sentinel surveillance network: initial development and moving forward. j am board fam med. ; ( ): – . doi: . /jabfm. . . . epub / / . . van walraven c, austin p. administrative database research has unique characteristics that can risk biased results. j clin epidemiol. ; ( ): – . doi: . /j.jclinepi. . . . epub / / . . jencks sf, williams dk, kay tl. assessing hospital-associated deaths from discharge data. the role of length of stay and comorbidities. jama. ; ( ): – . epub / / . . kim c, nevitt mc, niu j, clancy mm, lane ne, link tm, et al. association of hip pain with radiographic evidence of hip osteoarthritis: diagnostic test study. bmj. ; :h . doi: . /bmj.h . epub / / . pubmed pmid: ; pubmed central pmcid: pmc . . hannan mt, felson dt, pincus t. analysis of the discordance between radiographic changes and knee pain in osteoarthritis of the knee. j rheumatol. ; ( ): – . epub / / . . bedson j, croft pr. the discordance between clinical and radiographic knee osteoarthritis: a systematic search and summary of the literature. bmc musculoskelet disord. ; : . doi: . / - - - . pubmed pmid: , pubmed central pmcid: pmc , epub / / . • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal • we provide round the clock customer support • convenient online submission • thorough peer review • inclusion in pubmed and all major indexing services • maximum visibility for your research submit your manuscript at www.biomedcentral.com/submit submit your next manuscript to biomed central and we will help you at every step: shrestha et al. bmc medical informatics and decision making ( ) : page of http://dx.doi.org/ . /jabfm. . . http://dx.doi.org/ . /afm. http://dx.doi.org/ . /s - - -z http://dx.doi.org/ . /jabfm. . . http://dx.doi.org/ . /j.jclinepi. . . http://dx.doi.org/ . /bmj.h http://dx.doi.org/ . / - - - 	abstract 	background 	methods 	results 	conclusions 	background 	methods 	study identification 	inclusion and exclusion criteria 	data abstraction and quality assessment 	analysis 	results 	search results 	features of abstracted studies 	performance characteristics stratified by reference standard type 	performance characteristics stratified by reference standard type 	self-report 	acr criteria 	physician diagnosis 	quality assessment 	discussion 	conclusions 	additional file 	show [abbreviations] 	funding 	availability of data and materials 	authors’ contributions 	competing interests 	consent for publication 	ethics approval and consent to participate 	author details 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ microsoft word - shah_toidj the open infectious diseases journal, , , - - / bentham open open access parvovirus b infection in pregnancy: implications for childhood outcomes? amish jain , edmond kelly , and vibhuti shah *, , department of paediatrics, mount sinai hospital, university of toronto, canada faculty of medicine, university of toronto, mount sinai hospital, canada abstract: background: maternal parvovirus b infection can lead to serious consequences in the fetus including fetal death, non-immune hydrops fetalis and possibly long-term adverse neurodevelopmental outcome. to date, there has been no systematic review of the literature regarding long-term outcomes of these infants and children. objective: to systematically review the literature on the long-term neurodevelopmental outcomes of infants and children born to mothers with acute parvovirus b infection in pregnancy. methods: electronic databases medline, embase, cinahl, psychinfo, cochrane database of systematic reviews (cdsr) and cochrane central register of controlled trials (ccrt) were searched to identify studies that reported on long-term neurodevelopmental outcome of infants and children born following maternal parvovirus b infection in pregnancy. no language restrictions were applied. data synthesis was performed using qualitative description and was categorized based on ) outcome of infants and children with clinical presentation of non-immune hydrops fetalis and ) outcomes of infants and children born to mothers with serologic evidence of acute infection. results: eleven studies were included. five studies reported on outcomes in infants and children with non-immune hydrops fetalis while six studies reported on outcomes in infants and children born to mothers with serologic evidence of acute infection. there is a suggestion that the incidence of adverse neurodevelopmental outcome is higher in those who present with non-immune hydrops fetalis [ % ( / ), range from individual studies - . %] compared to asymptomatic infants and children born to mothers with acute parvovirus b infection in pregnancy [ . % ( / ) range from individual studies - . %]. conclusion: even though the outcome of parvovirus b in pregnancy in most cases is favorable, there is some supportive data that fetal parvovirus b infection can cause central nervous system damage. future prospective studies needs to be conducted to address this issue. background parvovirus b , named from latin “parvum” meaning small, is a single-stranded dna virus responsible for erythema infectiosum, also known as “slapped cheek disease” or “fifth disease” common in young children [ ]. the virus was discovered in by cossart et al. [ ] during routine screening of serum for hepatitis b and identified as the cause of erythema infectiosum in [ ]. since then the link between maternal parvovirus b infection and non- immune hydrops and fetal death has been well established [ - ]. the virus attaches to the “p” antigen that is linked to intracellular lytic and/or apoptotic pathways leading to cellular destruction and permitting viral replication [ ]. the “p” antigen has been identified on erythroid progenitor cells, megarkaryocytes, endothelial cells, placental cells and fetal liver and myocardial cells which are involved in the complex of symptoms that characterize the infection. parvovirus tend *address correspondence to this author at the department of paediatrics, room a, mount sinai hospital, university avenue, toronto, ontario m g x , canada; tel: - - ; fax: - - ; e-mail: vshah@mtsinai.on.ca to be species specific and animal strains are not associated with disease in the humans [ ]. seroprevalent studies have documented that approximately - % of woman of reproductive age are susceptible to parvovirus b infection and at risk of vertically transmitting the infection to the fetus [ , ]. approximately - % of susceptible pregnant women without a known history of exposure to parvovirus b will seroconvert during pregnancy [ - ]. up to % of susceptible pregnant women exposed to parvovirus b through household contacts will develop seropositivity while the rate of seroconversion is - % when the exposure occurs in day care centres or school [ - ]. the risk of transmission to the fetus has been reported in the range of - % [ - ] but infection remains asympto- matic in most cases [ ]. however, a wide range of adverse outcomes have been reported in association with intrauterine parvovirus b infection including: spontaneous abortions (rate of . % if infected prior to weeks gestation and . % if the infection occurs after weeks gestation) [ - ]; occurrence of non-immune hydrops fetalis (nihf) [ , - ]; congenital anomalies including central nervous system, craniofacial and eye anomalies [ , ]. the presen- ting features on ultrasound include generalized edema/ana- the open infectious diseases journal, , volume jain et al. sarca, ascites, pleural and pericardial effusion as well as placental edema. other ultrasonographic manifestations of b infection include placentomegaly, polyhydramnios and doppler findings consistent with anemia. various mechanisms have been implicated in the occurrence of nihf including severe fetal anemia leading to hypoxia and high output cardiac failure, development of fetal viral myocarditis leading to cardiac failure and impaired hepatic function [ , ]. the diagnosis of maternal parvovirus infection is based on clinical symptoms and signs and evidence of infection on serological testing for parvovirus specific immunoglobulin (ig) g and ig m. table provides a guide to interpretation of parvovirus ig g and ig m serology results in the diagnosis of infection [ ]. however, serology may not be useful if infection is suspected after onset of hydrops fetalis due to the lag period between immune response and occurrence of hydrops. therefore, the method of choice for detection of intrauterine infection is polymerase chain reaction (pcr) amplification of viral dna via amniocentesis or cordocentesis [ ]. prevention of exposure is ideal, but not feasible, in susceptible pregnant individuals as the period of contagion is prior to the onset of symptoms. women with confirmed exposure should be evaluated in a tertiary care unit by a maternal-fetal medicine specialist. the fetus should be assessed weekly for to weeks for evidence of anemia using middle cerebral artery dopplers and for hydrops. if hydrops develops at or near term, consideration should be made for delivery or intrauterine transfusion. if hydrops develops in late first or early second trimester, when directed fetal therapy is impractical, management should be expectant or termination of pregnancy could be considered. management of the hydropic fetus is by cordocentesis to assess fetal hemoglobin and reticulocyte count and facilitate intrauterine transfusion. currently there are no randomized controlled trials to evaluate the best management for fetal hydrops secondary to parvovirus b infection but intrauterine transfusion appears to be a reasonable option [ , ]. to date, there have been practice guidelines published in canada [ ] and uk [ ] regarding management of parvovirus b infection in pregnancy but none of these statements systematically review the literature regarding the long-term neurodevelopmental outcomes of infants and children. further, the impact of clinical presentation on long-term neurodevelopment has not been addressed. the availability of this information is vital to physicians in order to provide appropriate counseling regarding management and therapeutic options to the pregnant couple. we undertook this systematic review to provide a summary on the long-term neurodevelopmental outcomes of infants and children born to mothers with parvovirus b infection in pregnancy. material and methods search strategies a comprehensive literature search using the ovid search platform was conducted in the following databases: medline ( – march ), embase ( – march ), cinahl ( – march ), psychinfo ( – march ), cochrane database of systematic reviews (cdsr) and cochrane central register of controlled trials (ccrt) ( st quarter ). no language restrictions were applied. the search terms used to identify studies for inclusion in this review are described in appendix. the articles were scanned initially based on titles and abstracts by two reviewers (aj, vs). articles that reported on the long-term neurodevelopmental outcomes following maternal parvovirus b infection in pregnancy were retrieved in full and assessed for eligibility by two reviewers (aj, vs). the reference lists of eligible studies were checked to identify additional studies for inclusion in this review. a log of search strategy, articles scanned, articles retrieved and included and excluded are reported in the results section. inclusion criteria studies that reported on long-term neurodevelopmental outcome of infants and children born following maternal parvovirus b infection in pregnancy were included irrespective of the type of study design. we included cohort studies (prospective or retrospective), case-control studies, case series and case reports if they described the neuro-developmental outcomes. information from studies published as full/short report was included. exclusion criteria information presented as abstracts, letters, commentaries and editorials were excluded. primary outcome the primary outcome was neurodevelopmental outcome of infants and children born following maternal parvovirus b infection in pregnancy assessed using a validated tool or based on information provided by their physicians or caregivers using a questionnaire. examples of validated tools include: snijders-oomen non-verbal intelligence test [ ], kaufmann assessment battery for children [ ], griffiths test for infant development [ ], denver developmental screening test [ ] and bayley scales of infant development [ ]. table . guide to interpretation of parvovirus serology values parvovirus b ig g result parvovirus b ig m result interpretation clinical implication negative negative no antibody detected susceptible to parvovirus b infection positive negative indicative of past exposure not susceptible to parvovirus b infection positive positive indicative of infection infection most likely within the last to months negative positive indicative of infection evidence of acute/recent infection with b . consider repeat testing in to weeks parvovirus b infection in pregnancy the open infectious diseases journal, , volume data abstraction and presentation data from each eligible study were extracted individually by two reviewers (aj, vs) and compared. the reviewers resolved any disagreements through discussion or, if required, consulted a third person (ek). data synthesis was performed using qualitative description from the included studies and was categorized based on the clinical presentation: ) infants and children with clinical presentation of nihf and ) infants and children born to mothers with serologic evidence of acute infection during pregnancy. results the electronic searches yielded citations from all six databases. all references were stored in an endnote™ library that identified duplicates. the remaining unique references were screened by two authors (aj, vs) against the inclusion criteria and articles [ , , , - ] were retrieved for additional review (fig. ). eleven studies [ , , , - , , ] met the eligibility criteria and were included in the systematic review. one study was excluded as it was a letter to the editor [ ]. information on the baseline characteristics of included studies is presented in table . the results from studies are described below based on ) outcomes of infants and children with nihf and ) confirmed maternal infection during pregnancy (based on maternal serology). outcomes based on clinical presentation of nihf five studies reported on the neurodevelopmental outcome in infants and children who presented with nihf secondary to congenital parvovirus b infection (table ) [ - , , ]. the type of study designs include retrospective case series (n= ) [ , , ] and case reports (n= ) [ , ]. results from individual studies are presented below. cameron et al. [ ] reported outcome of pregnancies complicated by nihf secondary to parvovirus infection. three fetuses received intrauterine transfusion (iut) of packed red blood cells of which only one survived. the only live born infant in this study was followed up regularly and was reported to have normal development at years of age. dembinski et al. [ ] reported neurodevelopmental outcomes of infants and children with nihf who received iut from a single tertiary care center in germany. of the confirmed cases of congenital infection (based on pcr or specific ig m positive), cases developed nihf and received iut. in this group, intrauterine deaths occurred in five while the remaining were born alive with one infant dying in the neonatal period. amongst survivors, the authors were able to obtain neurodevelopmental outcome in ( % loss to follow up). the age of infant at the time of assessment ranged from . - . years (median, . years). a variety of validated assessment tools were used to calculate the developmental quotient (dq) and intelligence quotient (iq) depending on the age of the child. for children years (n= ), griffiths test for infant development was used to calculate the dq scores [mean , standard deviation (sd) ]. intelligence quotient was calculated using either the kaufmann assessment battery for children > years of age (n= ) or snijders-oomen non-verbal intelligence test for children between - years of age (n= ) with a mean iq of and sd of . the mean dq/iq score of all children was (range - ). two children had iq/dq score of (below one sd) while one child had an iq/dq score that exceeded one sd. neurological examination was reported to be normal in all children. using a multivariate analysis, the following variables were found not to influence the iq/dq scores: minimum fetal haemoglobin (p= . ) or fetal haematocrit (p= . ), gestational age at first iut (p= . ) or at delivery (p= . ) and number of iut (p= . ). donders et al. [ ] reported neurodevelopmental outcome on a hydropic fetus secondary to parvovirus infection delivered at weeks gestation for fetal distress. following aggressive neonatal management and repeated red blood cell transfusion the infant recovered. the infant continued to have persistent anemia necessitating red blood cell transfusion until the age of months. the infant had a positive test for pcr at months which became negative on follow-up. the child was reported to have a normal neurodevelopmental outcome when assessed at months [method of assessment not reported (nr)]. nagel et al. [ ] reported adverse neurodevelopmental outcome in fetuses with nihf treated with iut. of the cases in which iut was performed, information on neurodevelop- mental outcome was available in all survivors (n= ). thirteen children were assessed by the investigators in the out- patient clinic; one was assessed at home while for the remaining two children who were evaluated elsewhere information was retrieved. age of children at the time of follow up was between . - years. for infants and children between . - . years, the bayley scale of infant development (n= ) was used to calculate the mental developmental index (mdi) and psycho- motor developmental index (pdi). the snijders-oomen non verbal intelligence test was used to calculate iq for children between . - years of age (n= ). the mean (sd) for these scores (mdi, pdi, iq) is ( ). eleven children ( %) had scores within normal limits with normal neurological examination. three children had mild delay (defined as scores of - ) while two children had severe delay (defined as scores < ). all these children were also noted to have neurological deficits on examination. these deficits include: marked hypotonia of lower limbs (n= ), delayed fine motor coordination (n= ), marked hypertonia and hyperreflexia of upper limbs (n= ) and generalized hypotonia, ataxia and strabismus (n= ). the child with ataxia was further evaluated by mri scan which showed cerebellar vermis atrophy. metabolic and laboratory evaluation in this child did not detect any other etiology for the cerebellar vermis atrophy. no correlation was demonstrated between neurodevelopmental outcome and pre transfusion fetal haemoglobin (p= . ), platelet count (p= . ) and intrauterine ph (p= . ) at the time of cordocentesis. sheikh et al. [ ] reported two cases of nihf secondary to parvovirus infection which resolved spontaneously and were reported to be normal at one year of age. in summary, only two included studies reported adverse neurodevelopmental outcomes in infants and children who presented with nihf. [ , ] in the study by dembinski et al. [ ], even though / ( %) children had mild delay (iq/dq score < ), the authors concluded that these the open infectious diseases journal, , volume jain et al. table . long term outcomes following maternal parvovirus b infection in pregnancy study, year, country and design number of cases and study period fetal intervention and outcome age at follow-up and method of assessment results conclusion and limitations studies reporting outcomes based on clinical presentation of nihf cameron et al. , uk retrospective case series n= cases nihf (n= ) - iud (n= ) iut (n= ) survival (n= / ) years nr normal development (n= ) nihf associated with poor outcome in terms of mortality interpretation regarding long- term outcome limited due to single survivor dembinski et al. , germany retrospective case series n= cases nihf treated with iut (n= ) of those treated with iut, n= survived and follow-up data available for n= - spontaneous resolution (n= ) iud following iut (n= ) live births (n= ) neonatal death (n= / ) to months (median months) assessment tools: snijders-oomen non- verbal intelligence test (n= ) kaufmann assessment battery for children (n= ) griffiths test for infant development (n= ) mild delay (n= ) neurodevelopmental scores ranged within two standard deviation of a normal population and exceeded one standard deviation in three children children with successful iut have good neurodevelopmental outcome limitations: ) conclusion based on small number of participants ) different values of scores reported in the abstract and the results section. in addition, unclear whether scores are reported as mean or media value ) missing neurodevelopmental outcome on / ( %) children donders et al. , belgium nihf (n= ) follow-up data (n= ) study period nr nihf at weeks follow-up until months method of neuro- developmental assessment nr developmentally normal no definitive conclusions based on a single case report nagel et al. , netherlands retrospective case series nihf with iut for anemia or thrombocytopenia (n= ) follow-up data (n= ) - iud during the procedure (n= ) iud after the procedure (n= ) death at birth (n= ) . to years assessment tools: bayley scales of infant development or snijders-oomen test mild developmental delay (n= ) severe developmental delay (n= ) fetal parvovirus b infection may result in central nervous system damage limitations: ) small sample size with a wide range of follow-up age sheikh et al. , usa case report nihf (n= ) follow-up data (n= ) study period: nr spontaneous resolution preterm birth at weeks (n= ) follow-up at and months for case and respectively method of assessment nr developmentally normal no definite conclusions can be made based on cases studies reporting outcomes based on serologic evidence of acute maternal infection bruu and flugsrud , norway retrospective case series acute maternal infection in pregnancy based on serology (n= ) follow up data (n= ) - miscarriages (n= ) years information from local health authority normal development (n= ) hyperactivity and delayed language (n= ) no evidence of increased adverse neurodevelopmental outcome limitations: ) none had evidence of congenital infection ) small sample size ) method of assessment not reported gratacós et al. , spain prospective cohort study acute maternal infection in pregnancy based on serology (n= ) live born infants (n= ) follow-up data (n= ) study period: dates nr (duration years) miscarriages (n= ) and confirmed secondary to b infection (n= ) serological evidence of congenital neonatal infection at year of age (n= / tested) year information on neuro- developmental outcome obtained from paediatrician no severe neuro- developmental outcome reported in any infant adverse fetal outcome rare in infants born to mothers with acute parvovirus b infection in pregnancy limitations: ) no standardized method of neurodevelopmental assessment with short duration of follow-up ) no report of whether there was mild to moderate adverse neuro-developmental outcome parvovirus b infection in pregnancy the open infectious diseases journal, , volume (table ) contd….. study, year, country and design number of cases and study period fetal intervention and outcome age at follow-up and method of assessment results conclusion and limitations studies reporting outcomes based on serologic evidence of acute maternal infection koch et al. , usa prospective cohort study acute maternal infection in pregnancy based on serology (n= ) live born infants (n= ) follow-up data (n= ) - no evidence of fetal hydrops or death - months denver developmental screening normal examination acute parvovirus b infection in pregnancy can be associated with normal neurodevelopmental outcome limitations: ) information limited to small sample size ) no information on scores obtained form the screening tool phl service working party on fifth disease , uk prospective cohort study acute maternal infection in pregnancy based on serology (n= ) live births (n= ) follow-up data (n= ) - therapeutic abortion (n= ) spontaneous abortion or iud (n= ) stillbirth (n= ) preterm (n= ) year information on neuro- developmental outcome obtained from family physicians using questionnaire further follow-up of / children - . years information on neuro- developmental outcome obtained from family physicians using questionnaire no developmental delay in any infant developmental delay (n= ) majority of the children will have normal neurodevelopment limitations: ) information based on questionnaire only rather than formal assessment miller et al. , uk prospective cohort study acute maternal infection in pregnancy based on serology (n= ) live born infants (n= ) follow-up data (n= ) - therapeutic abortion (n= ) spontaneous abortion or fetal death (n= ) at least year of age information on neuro- developmental outcome obtained from family physicians using questionnaire mild developmental delay (n= ) majority of the children will have normal neurodevelopment limitations: ) information based on questionnaire only rather than formal assessment and short follow-up duration rodis et al. , usa retrospective case- control study cases: women with serologic evidence of recent parvovirus infection (n= ) follow-up data (n= ) control: pregnant women who were non-immune or with serologic evidence of past infection (n= ) follow-up data (n= ) - cases: spontaneous abortion (n= ) stillbirth (n= ) ectopic pregnancy (n= ) twins (n= ) nihf (n= ) control: spontaneous abortion (n= ) stillbirth (n= ) twins (n= ) months to years information based on questionnaire to caregivers (mothers) cases: delays in motor, speech or language development or significant attention deficits require special education (n= ) cerebral palsy (n= ) motor delays (n= ) attention, speech, learning delay (n= ) both language and motor delay (n= ) controls: significant developmental delays (n= ) cerebral palsy (n= ) motor delays (n= ) attention, speech, learning delay (n= ) both language and motor delay (n= ) no statistically significant difference in the proportion of developmental delay between cases and control (p> . ) no apparent increase in the occurrence of adverse neurodevelopmental outcomes in children exposed to in utero parvovirus b infection limitations: ) inadequate power to demonstrate a two fold difference in the risk of adverse neurodevelopmental outcome between groups iud: intrauterine death; iut: intrauterine transfusion; nihf: non-immune hydrops fetalis; nr: not reported. [ - , , ]. [ , , , , , ]. further follow-up data at - . years obtained using same methodology and reported in a separate publication by miller et al. [ ]. the open infectious diseases journal, , volume jain et al. findings were insignificant with all children having normal neurological examination. in contrast, nagel et al. [ ] reported that / ( %) infants and children had evidence of delayed psychomotor development [ / ( %) with mild delay and / ( %) with severe delay]. all five of these infants and children had tone abnormalities with ataxia andstrabismus reported in one. none of these studies had a healthy control group for comparison. in total, / ( %) infants and children who presented with nihf had adverse neurodevelopmental outcome with a range of - . %. outcomes based on serologic evidence of acute maternal infection six studies reported on the neurodevelopmental outcome in infants and children born to mothers with serologic evidence of acute parvovirus infection during pregnancy (table ) [ , , , , , ]. the type of study designs include prospective cohort studies (n= ) [ , , , ], retrospective case series (n= ) [ ], and case control study (n= ) [ ]. results from individual studies are presented below. bruu and flugsrud [ ] reported on pregnant women with serologic evidence of acute parvovirus infection. there were two cases of miscarriage. follow up data was available for children at two years of age. except for one child with hyperactivity disorder and delayed language, all were reported to have normal growth and development. there was no evidence of congenital infection in these children based on serological testing. gratacós et al. [ ] identified pregnant women with serologic evidence of acute parvovirus infection (n= ). the incidence of fetal loss secondary to parvovirus infection was reported to be . %. of the five cases with spontaneous abortion, only one fetus was positive for pcr dna. six of the live born infants (one was a twin pregnancy) were positive for ig m antibodies suggesting congenital infection. information was obtained for of these infants at year of age from paediatricians. none of the infants were reported to fig. ( ). flow diagram for selection of studies. parvovirus b infection in pregnancy the open infectious diseases journal, , volume have severe neurodevelopmental impairment or congenital anomalies. koch et al. [ ] prospectively evaluated pregnant women with confirmed acute parvovirus infection (ig m positive). all women delivered healthy term infants. neonatal information was obtained retrospectively from medical records. of the four infants who had serology testing at birth, three had evidence of congenital infection (ig m positive). follow-up evaluation included: physical and neurological examination, developmental assessment using denver developmental screening and hemato- logical testing. the age range of follow-up was . - . years. all infants were reported to be normal with no evidence of anemia. the first report on the long-term neurodevelopmental out- comes of infants born to pregnant women with acute parvovirus infection in pregnancy was published from uk. the study was a prospective cohort study conducted during - by public health laboratory (phl) service working party on fifth disease [ ]. of the pregnant women enrolled in the study, four underwent elective termination of pregnancy, had either spontaneous abortion or intrauterine fetal death, and one pregnancy ended in stillbirth. out of the , fetuses were tested for b dna and six were confirmed positive. rate of fetal loss was more common if infection occurred before compared to after weeks of gestation though the difference was not statistically significant [ % vs %, p= . , relative risk (rr) . , % confidence interval (ci) . to . ]. out of infants eligible for follow up (age one year), information was obtained from family physicians in cases ( % loss to follow up). details on infants’ physical and neurodevelopment was obtained by requesting family physicians to complete a simple form. no details were provided regarding the method of assessment. none of the infants had any congenital malfor- mation or serious neurodevelopmental problem reported. of the infants, ( %) had some evidence of congenital parvo- virus infection (ig g positive at year). in a subsequent study using the same methodology and questionnaire, miller et al. [ ] conducted a prospective cohort study following an outbreak of erythema infectiosum in uk between and . information was obtained regarding neurodevelopmental outcome from infants at one year of age. the rate of mild delay was . % (n= ). evidence of congenital infection was found in % of infants tested (salivary sample positive of ig g at year of age). miller et al. [ ] also obtained neurodevelopmental outcome from family physicians of children who were enrolled in the phl study published in [ ]. the rate of some developmental delay was % (n= ) at the age of - . years. in a retrospective case-control study, rodis et al. [ ] compared neurodevelopmental outcomes of children born to pregnant women with evidence of recent parvovirus infection during pregnancy (ig m positive) to pregnant women with evidence of past infection (ig g positive) or who were susceptible to infection (both ig g and ig m negative) during the same period. there were two cases of nihf in study group (one was a still birth while the second survived following iut) compared to none in control group. the median age (range) of follow-up was ( . - ) years. neurodevelopmental outcomes were available in children (cases) compared to (controls) from mothers using a questionnaire. there was no statistically significant difference in the rate of children with developmental delays between the study and control group ( . % vs . %, p > . ). two infants in the study group developed cerebral palsy (one with mild spasticity and other with severe ataxic cerebral palsy) compared to none in the control group. difference in occurrence of cerebral palsy was not statistically significant. in summary, of the included studies reported adverse neurodevelopmental outcomes in infants and children born to mothers with serologic evidence of acute infection. bruu and flugsrud [ ] reported hyperactivity and delayed language in / ( . %) children. miller et al. [ ] reported / ( . %) children with mild delay. rodis et al. [ ] reported adverse outcome in / ( . %) children, however when compared to a control group the incidence was not statistically different. two of the study infants had cerebral palsy compared to none in the control group which the authors found concerning. miller et al. [ ] also obtained information at - years of age for the original cohort of phl study [ ] and reported the incidence of some delay as / ( %) children (this cohort was reported by phl to be normal at year of age). the only study [ ] that evaluated neurodevelopmental outcome using validated tools did not report any abnormalities in the study population. the other included studies [ , ] only reported on absence or presence of severe adverse neurodevelopmental outcomes. in total, / ( . %) infants and children who were born to mothers with serologic evidence of acute infection had adverse neuro- develpmental outcome with a range of - . %. discussion in this review of combined data from studies selected on the basis of well defined inclusion criteria, there is a suggestion that the incidence of adverse neurodevelopmental outcome is higher in infants and children who present with nihf compared to infants born to mothers with serologic evidence of acute parvovirus infection. of those with adverse neurodevelopmental outcome, delayed psychomotor development and tone abnormalities seemed to predominate. methodological challenges of this review include data derived from few studies with small number of participants, not all infants with nihf received iut, the diagnostic criteria of congenital parvovirus infection in the neonate was heterogeneous, there was a marked variation in the methods used to assess neurodevelopmental outcome (ranging from validated assessment tools to infor- mation derived from parents and physicians using questionnaire or simply reporting that these infants and children were followed at regular intervals), variation in reporting adverse outcomes (i.e. some studies reported on the presence or absence of severe outcome), wide range of age at which neurodevelop- mental outcome was assessed making comparison between studies difficult and in most studies a healthy control group was not included as a comparator. in addition, neurodevelopmental outcome was not available for all infants and children with nihf or those born to mothers with acute parvovirus b infection from the initial cohort. therefore, caution should be exercised in interpreting this finding as the conclusion is based on included studies with methodological differences. the open infectious diseases journal, , volume jain et al. of the five studies [ - , , ] that report on outcomes in infants with nihf, adverse neurodevelopmental outcome was reported in two studies [ - ]. five of the ( . %) survivors had adverse outcome which included mild and severe delay in three and two children respectively in the study by nagel et al. [ ] in addition, all these children had neurological deficits. in the study by dembinski et al. [ ] children were reported to have mild delay / ( %) however in this case series % of subjects were lost to follow-up. parents of infants and children with adverse outcome are less likely to be compliant with their follow-up visits and this could have influenced the results of this study [ ]. further, in both these studies there was a wide range in age of follow up and different assessment tools were used. in the remaining three studies [ , , ], the conclusion of normal development was based on case reports/series. based on individual study information, the range of adverse neurodevelopmental outcome was % to . %. six studies report on neurodevelopmental outcome in infants and children born to mothers with serologic evidence of acute parvovirus b infection in pregnancy. the type of outcomes reported varied from no severe adverse outcome [ , ] to delays in motor, speech or language development or significant attention deficits requiring special education [ , , ]. except for the study by koch et al. [ ] in which denver developmental testing was used to assess outcomes, in all the remaining studies information was obtained using questionnaire either from physicians, local health authority or caregivers. none of the studies described the type of questionnaire used. this could put the validity of the study in question. the range of adverse neurodevelopmental outcome varies from % to . % based on individual studies. the mechanisms by which parvovirus b can cause central nervous system damage is unclear. postulated mechanisms include teratogenic effect [ , ], vasculitis [ ], hypoxic- ischemic effects secondary to anemia and intracranial bleeding secondary to thrombocytopenia [ ]. in animals, parvovirus b has a teratogenic effect causing cerebellar hypoplasia and ataxia in cats and anencephaly, microcephaly, facial defects and ectopic hearts in hamsters [ , ]. several studies have reported on the neuropathological findings in fetuses, neonates and adults with parvovirus b infection. the findings include development of fetal ventriculomegaly [ ] and presence of multinucleated giant cells predominantly in the cerebral white matter and calcifications with a perivascular predilection in the white matter, cerebral cortex, basal ganglia, thalamus and germinal layer. parvovirus b genome dna was detected in the nucleus of the multinucleated giant cells by pcr amplification and in situ pcr methods [ ]. barah et al. [ ] tested cerebrospinal fluid samples from patients ( day to years of age) with undiagnosed meningoencephalitis who presented during an outbreak of parvovirus b . seven patients were found to be positive for parvovirus ( . %) [ ]. in a subsequent publication from the same population, kerr et al. documented elevated cytokine levels in serum and cerebrospinal fluid in subjects with proven b meningoencephalitis [ ]. two of these subjects were newborns with possible perinatally acquired infection. in four cases cerebellar involvement was suggested either clinically (ataxia) or pathologically (atrophy of the molecular and granular layers of the cerebellum with focal loss of purkinjie cells). neuroimaging (mri or ct) was performed in six cases and showed evidence of demyelination. the consequences of anemia secondary to parvovirus b can be comparable to neonates with blood group incompatibility where adverse neurodevelopmental outcome have been reported to be in the range of . % to % [ - ]. therefore, one may speculate that the a combination of each of the above pathophysiological mechanisms may be implicated in the occurrence of adverse neurodevelopmental outcome. despite a comprehensive review of the literature, we conclude that there is paucity of systematically collected data regarding long-term outcome of children following parvovirus b infection on which to base evidence based counseling. however, in spite of these concerns it can be concluded that the outcome of parvovirus b in pregnancy is favorable in most cases. most women exposed to parvovirus b do not become infected and most infected mothers will not transmit the infection to the fetus. further, most infected fetuses remain asymptomatic and do not become hydropic. with the availability of iut, survival of hydropic fetus has improved. there is some data suggesting that fetal parvovirus b infection can cause central nervous system damage. in order to obtain definitive evidence regarding long-term neurodevelopmental outcome with parvovirus b infection, future studies with adequate sample size using standardized neurodevelopmental assessment tools at - months and school age need to be conducted. in addition, the role of neuroimaging such as mri (both fetal and neonatal) in identifying the effects of parvovirus b on cranial structures should be elucidated. these findings should be correlated with neurodevelopmental outcomes to provide appropriate counseling to women with acute parvovirus infection in pregnancy. it would be unethical to conduct a randomized trial evaluating the efficacy of iut in fetuses with anemia as its role in improving mortality is already proven. due to the rarity of number of cases born to mothers with acute parvovirus b infection seen at each center/year, a multicenter prospective cohort trial will be needed to address this issue. definitions susceptible individual: an individual without evidence of previous immunity against parvovirus b (i.e. absence of ig g) and therefore at risk of developing infection if exposed to the virus. history of exposure: a significant contact with a person suffering from acute parvovirus b infection. significant contact is defined as contact in the same room (e.g. in a house or classroom or a - bed hospital stay) for a significant period of time ( minutes or more), or face-to- face contact with a laboratory confirmed case of parvovirus b infection during the period of maximum infectivity (from days prior to appearance of a rash to the date of appearance of the rash) in the absence of respiratory isolation precautions [ ]. acknowledgement we would like to acknowledge ms. elizabeth uleryk, chief librarian at the hospital for sick children who performed the literature search for this review. parvovirus b infection in pregnancy the open infectious diseases journal, , volume appendix: literature search strategy we ran searches using the ovid search platform in the following databases: medline, embase, cinahl, psycinfo, cdsr and cctr. we retrieved a total of references from all databases. all references were saved in an endnote library used to identify the duplicates. author aj and vs reviewed the remaining unique references against our inclusion criteria. the following tables and text record the search strategies and terms used. medline the search strategy for medline ( to march week ) retrieved references of which were unique and not duplicated in our other searches. we used a combination of mesh and free text terms for set # history results comments parvoviridae/ or parvovirus b , human/ or erythema infectiosum/ parvovirus mesh terms exp pregnancy complications/ or exp pregnancy/ pregnancy mesh terms exp "congenital, hereditary, and neonatal diseases and abnormalities"/ congenital abnormality mesh terms and and congenital abnormalities base set exp mental disorders/ or exp "behavior and behavior mechanisms"/ or exp "psychological phenomena and processes"/ or exp neurologic manifestations/ or exp human development/ or exp fetal alcohol syndrome/ or exp nervous system/ or exp nervous system diseases/ or child development/ or exp diagnostic techniques, neurological/ or exp neuropsychological tests/ or cerebral arteries/ or cerebrovascular circulation/ or brain chemistry/ or exp nerve growth factors/ or exp nervous system physiology/ or exp neurotransmitter agents/ or exp receptors, neurotransmitter/ or circadian rhythm/ or mental health/ or exp psychological tests/ or exp social behavior disorders/ or child psychiatry/ or exp psychophysiology/ or exp neuropsychological tests/ or hallucinations/ or illusions/ or fetal alcohol syndrome/ or exp psychology/ or communication/ or language/ or exp verbal behavior/ or exp nonverbal communication/ or exp child development/ or fetal alcohol syndrome/ or exp movement/ or exp nervous system physiology/ or exp vision tests/ neurocognitive mesh terms and neurocognitive outcomes base set or total unique citations embase the search strategy for embase ( to week ) retrieved references of which were unique and not duplicated in our other searches. there were duplicates from medline. we used a combination of embase and free text terms for the following embase search strategy retrieved citations of which were duplicates from medline set # history results comments parvovirus/ or erythema infectiosum/ or "parvovirus b ".mp. ( ) parvovirus embase terms exp pregnancy/ or exp fetus disease/ or exp pregnancy complication/ pregnancy embase terms exp congenital disorder/ or exp prenatal disorder/ or prenatal exposure/ or exp postnatal development/ or congenital abnormality embase terms and and congenital abnormalities base set exp "disorders of higher cerebral function"/ or exp motor dysfunction/ or exp sensory dysfunction/ or exp speech disorder/ or exp mental function/ or cerebral palsy/ or exp communication disorder/ or exp psychomotor disorder/ or sensation/ or neurocogniti*.ti,ab. neurocognitive embase terms and and neurocognitive outcomes base set or total unique citations the open infectious diseases journal, , volume jain et al. references [ ] levy r, weissman a, blomberg g, hagay zj. infection by parvovirus b during pregnancy: a review. obstet gynecol surv ; : - . [ ] cossart ye, field am, cant b, widdows d. parvovirus-like particles in human sera. lancet ; : - . 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[ ] leads from the mmwr. risks associated with human parvovirus b infection. jama ; : - . cinahl (ebscohost) the search strategy for cinahl ( to march week ) retrieved references of which were unique and not duplicated in our other searches. we used a combination of cinahl and free text terms for set # history results comments (mh "parvovirus infections+") parvovirus cinahl terms (mh "pregnancy+") or (mh "pregnancy complications+") pregnancy cinahl terms (mh "neonatal, fetal diseases and abnormalities (non-cinahl)+") congenital abnormality cinahl terms and and congenital abnormalities base set (mh "delirium, dementia, amnestic, cognitive disorders+") or (mh "behavior and behavior mechanisms+") or (mh "behavioral and mental disorders (non-cinahl)+") or (mh "neurodegenerative diseases+") or (mh "nervous system diseases+") neurocognitive cinahl terms and and neurocognitive outcomes base set or total unique citations psycinfo the search strategy for psycinfo ( to march week ) retrieved citations of which was a duplicate from medline, embase and cinahl set # history results comments parvovir*.mp. parvovirus textword terms ebm reviews - cochrane database of systematic reviews the following cdsr ( st quarter ) search strategy retrieved citations none of which were duplicated in medline, embase, cinahl and psycinfo set # history results comments parvovir*.mp. parvovirus textword terms ebm reviews - cochrane central register of controlled trials the following cctr ( st quarter ) search strategy retrieved citations all of which were duplicated in medline, embase, cinahl, and psycinfo, but not cdsr set # history results comments parvovir*.mp. parvovirus textword terms parvovirus b infection in pregnancy the open infectious diseases journal, , volume [ ] gillespie sm, cartter ml, asch s, et al. occupational risk of human parvovirus b infection for school and day-care personnel during an outbreak of erythema infectiosum. jama ; : - . 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[ ] crowcroft ns, roth ce, cohen bj, miller e. guidance for control of parvovirus b infection in healthcare settings and the community. j public health med ; : - . received: july , revised: august , accepted: august , © jain et al.; licensee bentham open. this is an open access article licensed under the terms of the creative commons attribution non-commercial license (http://creativecommons.org/licenses/ by-nc/ . /) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. biomed centralbmc genetics ss open accemethodology article robust physical methods that enrich genomic regions identical by descent for linkage studies: confirmation of a locus for osteogenesis imperfecta peter brooks* , charles marcaillou , maud vanpeene , jean-paul saraiva , daniel stockholm , stephan francke , reyna favis , nadine cohen , francis rousseau , frédéric tores , pierre lindenbaum , jörg hager and anne philippi address: integragen sa, rue pierre fontaine, evry, france, généthon, cnrs umr , rue de l'internationale, evry , france and department of pharmacogenomics, johnson & johnson prd, po box , raritan, new jersey , usa email: peter brooks* - peter.brooks@parisbc.com; charles marcaillou - charles.marcaillou@integragen.com; maud vanpeene - maud.vanpeene@integragen.com; jean-paul saraiva - jean-paul.saraiva@integragen.com; daniel stockholm - stockho@genethon.fr; stephan francke - sfranck @prdus.jnj.com; reyna favis - rfavis@prdus.jnj.com; nadine cohen - ncohen @prdus.jnj.com; francis rousseau - francis.rousseau@integragen.com; frédéric tores - frederic.tores@integragen.com; pierre lindenbaum - pierre.lindenbaum@integragen.com; jörg hager - jorg.hager@integragen.com; anne philippi - anne.philippi@integragen.com * corresponding author abstract background: the monogenic disease osteogenesis imperfecta (oi) is due to single mutations in either of the collagen genes cola or cola , but within the same family a given mutation is accompanied by a wide range of disease severity. although this phenotypic variability implies the existence of modifier gene variants, genome wide scanning of dna from oi patients has not been reported. promising genome wide marker-independent physical methods for identifying disease- related loci have lacked robustness for widespread applicability. therefore we sought to improve these methods and demonstrate their performance to identify known and novel loci relevant to oi. results: we have improved methods for enriching regions of identity-by-descent (ibd) shared between related, afflicted individuals. the extent of enrichment exceeds - to -fold for some loci. the efficiency of the new process is shown by confirmation of the identification of the col a locus in osteogenesis imperfecta patients from amish families. moreover the analysis revealed additional candidate linkage loci that may harbour modifier genes for oi; a locus on chromosome q includes cox- , a gene implicated in osteogenesis. conclusion: technology for physical enrichment of ibd loci is now robust and applicable for finding genes for monogenic diseases and genes for complex diseases. the data support the further investigation of genetic loci other than collagen gene loci to identify genes affecting the clinical expression of osteogenesis imperfecta. the discrimination of ibd mapping will be enhanced when the ibd enrichment procedure is coupled with deep resequencing. published: march bmc genetics , : doi: . / - - - received: september accepted: march this article is available from: http://www.biomedcentral.com/ - / / © brooks et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. page of (page number not for citation purposes) http://www.biomedcentral.com/ - / / http://creativecommons.org/licenses/by/ . http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ http://www.biomedcentral.com/info/about/charter/ bmc genetics , : http://www.biomedcentral.com/ - / / background mapping of regions identical-by-descent (ibd) is a power- ful method for the identification of genetic loci shared within families and implicated in disease. classically, typ- ing of individual genetic markers throughout the genomes of the afflicted individuals mapped shared haplotypes and has been successful in finding loci linked with numerous monogenic traits [ ]. an alternative physical method, genomic mismatch scanning (gms) [ ], physically com- pares genomes of two affected individuals, related by a not too distant common ancestor, and enriches for the ibd regions they share. despite its promise, the technique has not been exploited due to technical complexities. as gms offers the potential to avoid certain ambiguities associated with genotyping and may be applicable to pools of dna samples from afflicted, related individuals, we aimed to improve the technique to reduce its inherent noise and to render it robust. as compared with linkage discovery by genotyping, phys- ical methods based on direct comparison of genomic sequences would enable more complete access to all ibd regions of the genome. such methods rely on the fact that non-ibd regions are densely polymorphic between two individuals. a conceptually attractive approach for such a direct comparison involves the formation of duplex heter- ohybrid dna fragments from the dnas of related indi- viduals sharing a trait of interest, and then challenging these fragments with reagents actuated by mispairings in the heterohybrids. such reagents may bind to the mis- matched fragment or may introduce strand breaks to per- mit separation of hybrid fragments that are not perfectly complementary from those that are perfectly paired from the ibd regions. physical comparison has been success- fully used with a variety of technologies that exploit the chemical or structural differences between perfectly matched and mismatched hybrids. these include chemi- cal mismatch cleavage [ ] and various attempts to harness proteins that respond to mismatches such as resolvase [ , ], single-stranded dna-specific nucleases [ , ], muts mismatch binding [ - ] or cleavage by the mismatch- specific enzymatic activity of e. coli muts, mutl and muth [ ]. in addition, transfection of hybrid molecules into bacteria enables enrichment for perfectly paired frag- ments via an in vivo process dependent on mismatch repair activities [ , ]. the use of these mismatch recognition methods has gen- erally been limited to the analysis of a few targeted frag- ments, but adapting them to genome-wide analysis is conceivable. global treatment of fragments from the entire genome, however, also requires elimination of reannealed homohybrid fragments (fragments between strands of dna from the same individual formed during hybridization, including both mismatch-bearing hybrids formed with one paternal and one maternal strand and isohybrids comprising strands from the same parent) whose presence would confound the identification of ibd dna. ford and colleagues[ , ], in presenting the con- cept of genome-wide enrichment of ibd fragments, intro- duced the strategy of tagging one genome with methyl groups, and then using restriction enzymes specific for either methylated or non-methylated dna, but inactive with hemimethylated dna, to remove homohybrid dna. in addition, they suggested using various mismatch-spe- cific agents, either by immunoprecipitation or by the e. coli mismatch repair system to eliminate mispaired frag- ments [ , ]. subsequently, nelson et al. [ ] described gms, a combination of the methylation-dependent homohybrid elimination method with in vitro cleavage by the mismatch repair proteins muts, mutl and muth, and digestion by exonuclease iii. application of gms to pairs of related yeast strains, including mapping of the ibd-enriched regions by hybridization to arrayed clones, correctly identified meiotic recombination crossover points [ ]. use of gms with mammalian genomes was demonstrated by enrichment of microsatellite alleles shared between related individuals [ , ] and confirmation of loci con- taining previously documented disease-related genes with mapping of ibd regions by hybridization to dna arrays targeted to the chromosome of the known locus [ ] or by microsatellite allele recovery [ ]. despite the recognition of the potential of gms [ , ], the approach has not been widely exploited, due to a lack of availability of the mismatch repair proteins, various technical challenges inherent in a multi-step procedure and the lack of appro- priate means to map the ibd-enriched dna. a fundamen- tal problem has been an apparent lack of appreciation of the need for a highly efficient process to ensure elimina- tion of non-identical hybrid fragments. such residual frag- ments will hybridize to the dna features on microarrays and so increase non-specific noise. modifications to gms and ibd mapping have been reported but the results from genome wide studies showed a substantial lack of con- cordance to confirm ceph family meiotic crossovers [ ]. as linkage studies require processing of many samples, an additional limitation has been the use of reagent volumes and methods unsuitable to high throughput microtiter plate-based procedures. we present here an improved protocol for physical enrich- ment of ibd regions. the monogenic disease osteogenesis imperfecta (oi; brittle bone disease) provides a context for demonstrating that the protocol could correctly iden- tify a well-established monogenic locus and an opportu- nity to discover novel loci relevant to oi etiology. oi includes a heterogeneous group of autosomal dominant page of (page number not for citation purposes) bmc genetics , : http://www.biomedcentral.com/ - / / inherited disorders characterized by bone fragility and other generalized connective tissue abnormalities [ ]. as analysis of locus-specific restriction fragment length poly- morphisms had shown that nearly all cases of oi segre- gated with the two collagen genes [ - ], genome-wide linkage analysis was not perceived as necessary. however, given the wide variety of clinical expression of oi, it is possible that variants of modifier genes may also cosegre- gate with oi [ ]. therefore a genome wide analysis might detect loci harbouring such genes. we have implemented an ibd enrichment protocol that overcomes many of the difficulties of the original gms protocol. we report that application of the improved pro- tocol to dna from oi patients has correctly identified the shared ibd locus that includes col a bearing the dis- ease-causing mutation and additional loci that may be rel- evant to oi etiology. the protocol is now suitable for finding linkage loci in applications ranging from mono- genic disease to complex multigenic disease. results overview of ibd enrichment process the procedure for ibd enrichment can be applied to any related pair of individuals and is outlined in figure . affected relatives may have the same phenotype because they share a particular set of predisposing alleles inherited from common ancestral chromosomal regions. the pro- cedure enriches for loci that, due to patterns of parental meiotic recombination events, are identical between the siblings, and thus include the predisposing alleles. the final ibd-enriched product is generically amplified and the ibd regions are then mapped by various methods, such as by hybridization to dna microarrays or high throughput sequencing. genomic dna (gdna) from each individual is cleaved with restriction enzymes that yield fragments of sufficient length to ensure a high probability that they will include common polymorphisms. the two sets of fragments are tagged in different ways such that homohybrid molecules with identical tags are targeted for subsequent elimina- tion, but heterohybrids, with differentially tagged strands, are conserved. after tagging, the dnas are mixed, dena- tured and reannealed to form hybrid fragments of differ- ent types as shown in figure . strands from one individual may reanneal with strands from the same indi- vidual to generate self-self hybrids (as isohybrids or as mixed parental hybrids), and with strands from the other individual to produce heterohybrids, either with strands of different parental origin (m/p heterohybrids) or with both strands derived from the same parent (m/m or p/p heterohybrids). any hybrid with strands from different ancestral chromosomes, hence bearing mispairs at poly- morphic sites, is a target for subsequent enzymatic attack by muts, mutl and muth (lshase) and exo iii. the dna that survives the procedures that eliminate self-self hybrids and mismatch specific enzymatic digestion is enriched for the perfectly paired ibd dna. necessity for high efficiency of removal of non-specific dna as shown in table , for any relative pair whose ancestral chromosomes are not related, at least % of the dna mass is targeted for removal. with sibling pairs, for a given locus, depending on the sharing status, the process aims to eliminate / , / or all of the dna fragments from the locus. this represents a major challenge because system- atic inefficient removal of unwanted dna reduces the physical ibd enrichment processfigure physical ibd enrichment process. genomic dnas (gdna) are isolated from two related, afflicted individuals and digested with a restriction enzyme that leaves exonucle- ase iii-resistant ends and generates fragments of about kb. dna fragments are modified (diamonds or ovals) to permit discrimination between the individuals, for example by pres- ence or absence of methylation at gatc sequences. the fragments are mixed, denatured and renatured under condi- tions to favour unique copy reannealing. non-annealed strands and hybrids of strands from the same individual are eliminated. reannealed dna fragments with one strand from each individual are called heterohybrids, which may be per- fectly paired due to inheritance of both strands from the same ancestral sequence or mismatched due to variation between different ancestral sequences. mismatched hetero- hybrid fragments are removed by lshase, a nucleolytic cock- tail of mutl, muts and muth, and subsequent digestion by exonuclease iii. the resulting ibd-enriched dna is generi- cally amplified, labelled and mapped by two-colour hybridiza- tion to genomic topographic arrays, using the reannealed dna as reference. the process is repeated for other afflicted pairs in the same family and in additional families. variations include use of oligonucleotides as discrimination tags, reducing the number of steps by combining similar intermediate purification procedures, and eventually mapping ibd regions by high throughput redundant sequencing, with or without amplification. genomic dna digest with -cutter rest. enz. family a individual gdna add individual discrimination tags denature & anneal unique copy sequences remove ssdna reference dna . μg . μg amplify label map ibd dna by microarray hybridization remove self-self hybrids remove mismatched dna eliminated fragments family a individual gdna x x non-annealed dna cy- cy- x x x ibd-enriched dna page of (page number not for citation purposes) bmc genetics , : http://www.biomedcentral.com/ - / / sensitivity of detection of ibd regions. this is because for any of the procedures used for ibd mapping that measure the mass of dna at specific sets of sequence positions, such as those represented on bac clone microarrays, a positive signal at a given position indicating ibd enrich- ment for some pairs must be compared to the signal for pairs where there is no sharing at the position. previous reports have not addressed a quantitative definition of this problem. therefore we determined the level of efficiency required to adequately detect ibd enrichment. we express the sensi- tivity of detecting ibd sharing as a discrimination factor (df), dependent on the overall efficiency of the removal of homohybrid and mismatched fragments. for an expected ibd mass fraction, (i), and a residual surviving fraction of the dna that was targeted for removal, r, with r = - overall efficiency, the behaviour of this function (figure ) for the different classes of dna fragments (table ) shows that the overall process efficiency for loci with monoparental ibd sharing must be at least % to achieve a minimum of about two- fold discrimination, a reasonable goal for microarray analysis or for real time quantitative pcr. this level of overall efficiency mandates much higher efficiencies for the individual sequential steps of the process. therefore, we designed various assays, each specific for a different step, to test and select optimum reaction parameters. some of the same assays are also routinely performed both as periodic quality controls to monitor reagents and enzyme specific activities and as internal and parallel con- trols during batch processing of multiple dna sample pairs. robust enrichment of ibd dna to produce adequate yields of reannealed fragments, we ensured that all restriction digested gdnas had a consist- ent range of fragment sizes, indicating intact gdna. we optimized several components of the process, notably the choice and concentration of reagents for reannealing in a formamide emulsion [ , , ] and conditioning of the resin that eliminates fragments resulting from nucleolytic digestion. quality control during the process also includes df i r ( i) r = + ⋅ − ( ) table : partition of hybrid fragments after random reannealing of sibling pair dnas. heterohybrid homohybrid maternal or paternal maternal/paternal ibd non-identical all loci / / / / biparental ibd locus / / / monoparental ibd locus / / / / non-identical locus / / / variation in the distribution of reannealed dna mass depending on the class of locus sharing. the mass distribution for a monoparental ibd locus is the same as that expected for annealing of grandparent and grandchild dnas. discrimination between ibd loci and loci with no sharing as a function of overall process efficiencyfigure discrimination between ibd loci and loci with no sharing as a function of overall process efficiency. the discrimination factor (df) is the ratio of a value measured when the signal is derived from pairs when the fragments are enriched for ibd sharing to the value for pairs with no shar- ing. the overall process efficiency indicates the extent that the process has eliminated homohybrids and non-identical heterohybrids. plots of df as a function of the efficiency (see equation ) are shown for biparental sharing (ibd- , gray) and monoparental sharing (ibd- , black). the ratio of the dfs for ibd- versus ibd- is also shown (dashed). overall pr oces s efficienc y (%) d is c ri m in a ti o n f a c to r ibd- / ibd- ibd- / n o n -id e n tical ibd- / n o n -id e n tical page of (page number not for citation purposes) bmc genetics , : http://www.biomedcentral.com/ - / / monitoring of the dna concentrations after digestion by the fragmenting restriction endonuclease, after reanneal- ing, after the final enrichment process and after generic amplification to detect any atypical losses or excess yields. several steps require dna purification and/or concentra- tion and with a view to automation and robust process- ing, we perform the process entirely in microtiter plates using ultrafiltration instead of alcohol precipitation. the number of steps has been significantly reduced. using mismatched dna substrates and perfectly matched controls, we found protein concentrations and reaction conditions that would eliminate all measurable mis- matched dna. we designed model mismatched dna substrates with a gatc density typical for the human genome (about per . kb), but with only one mismatch per kb, to ensure that the enzymatic activities that remove non-identical dna were efficient even for genomic dna fragments containing a lower than typical density of snps. as shown in figure , elimination of mis- matched substrates, linear or circular, is dependent on both the nuclease activity of lshase and subsequent treat- ment with exonuclease and a resin that removes the digested dna. we titrated the lshase activity and the exo- nuclease/resin procedure such that a significant fraction of the control perfectly paired dna was also degraded. we also designed two model substrates as internal quality controls in each sample. one substrate is a kb duplex with a single mismatch (mm), and the second is perfectly matched (pm), but with an additional kb of inserted sequence. to each hybrid dna sample, we added amounts of mm and pm that mimicked single copy gdna equivalents. subsequent evaluation by quantitative pcr using primers specific for each substrate ensured that in each sib pair sample all enzymatic functions were suffi- ciently active to provide adequate discrimination (data not shown). we also included the mm and pm substrates as parallel controls at concentrations sufficient to observe the elimination of the mismatched dna and conserva- tion of the control dna (data not shown). discrimination efficiency evaluated by quantitative pcr to determine the extent of discrimination achieved by the ibd enrichment process, we performed quantitative pcr (qpcr) on generically amplified ibd-enriched dna from ceph family grandfather-grandchild and grandmother- grandchild pairs. meiotic recombination points in ceph family pedigrees have been extensively documented [ , ] and so provide a high resolution map of expected ibd regions between related pairs of individuals. process- ing ceph grandparent-grandchild pairs mimics sib pair analysis in that reannealing produces the same expected mass distribution of hybrid fragments as for monoparen- tal ibd loci sharing between sib pairs (table ). as a result of parental meiotic crossing over, the sharing status of each region for a grandfather-grandchild pair is always the opposite to that of the grandmother-grandchild pair. fig- ure shows that ibd loci can be distinguished from non- identical loci over a range of copy number concentrations. differences in ct values of up to to cycles between ibd dna and non-identical depleted dna from the two com- plementary relative pairs indicate discrimination factors ranging from about to . therefore for some frag- ments, there remains less than % of the dna that is evaluation and quality control of lshase activityfigure evaluation and quality control of lshase activity. substrates of . kb that mimic reannealed hybrid restriction fragments, either perfectly complementary (ibd) or with a single base pair mismatch (n-id), and either as covalently closed circular (ccc) or linear (lin) forms, were incubated with lshase and/or exo iii (exo) and treated with the dap procedure (resin), as indicated. agarose gels of reaction products were stained with ethidium bromide. lane m – high mass ladder, invitrogen. quantitative pcr detects an extensive range of enrichment of ibd dnafigure quantitative pcr detects an extensive range of enrichment of ibd dna. eight ca microsatellite sites were assayed by qpcr using a universal (ca)n-specific probe and ibd-enriched dna from complementary grandparent- grandchild pairs. loci – represent shared loci for the grandfather-grandchild pair and loci – represent shared loci for the grandmother-grandchild pair. ct is the cycle number at which a common threshold level of amplification was achieved. page of (page number not for citation purposes) bmc genetics , : http://www.biomedcentral.com/ - / / expected to be depleted by the process (equation ( ), fig- ure ). based on results from such qpcr assays, we con- clude that the ibd enrichment procedure is highly efficient and that generic amplification of the ibd dna adequately conserves copy number differences. identification of ibd regions for grandparent-grandchild pairs by mapping on bac microarrays to map all ibd regions, amplified reannealed hybrid dna, labelled with cy , and amplified ibd-enriched dna, labelled with cy , were hybridized to genome-wide bac microarrays [ , ]. as described in methods, after initial filtering, we calculated ratios of the cy -labeled ibd-enriched dna signals to the cy -labeled reannealed dna signals, and normalized the ratios. we standardized the variance between arrays using the variance of all the ratios on each array. figure shows the profiles of the ratios for complementary grandparent-grandchild pairs. the performance of an immobilized dna clone is vali- dated when its ratio in an ibd-enriched region is signifi- cantly greater than its ratio when the region represents non-identical dna. regions of sequential clones with ratios that consistently discriminate the sharing status between the two pairs correspond to regions where the grandparent and grandchild share common microsatellite alleles due to the child's inheritance of the region from the grandparent. therefore, parental meiotic crossover loci identified by microarray mapping of ibd-enriched dna are the same as those identified by microsatellite genotyp- ing. such experiments with pairs of known ibd status vali- dated the enrichment process and the behaviour of immo- bilized dna clones. however, due to uneven distribution of snps and different amounts of unique and efficiently annealable sequence, each clone displays its particular characteristics, and hence there is a wide variability in the ratio values between clones. indeed, as shown in figure , the ratios for some clones detecting enrichment are simi- lar to those of other clones detecting depletion. therefore, as described in methods, in subsequent experiments to discover unknown ibd regions, we standardized the ratios by mean centering using the variance of the ratios of each clone obtained from experiments with ceph sib pairs. application of ibd enrichment and mapping to identify monogenic disease loci osteogenesis imperfecta patients from four old order amish families, descended from a single founding couple, have a single gly cys mutation in col a , and yet have variable clinical expressivity of the disease. a sample family pedigree is shown in figure . dna was available for patients and we analyzed affected pairs as shown in table . in parallel with the affected pairs, independent control sib-pairs from the ceph family collection were processed. no linkage is expected at any locus in this collection and as shown in figure , none of the clones showed signifi- cant evidence for linkage at the nominal p-value < × - . thus the protocol is not inherently prone to generate false positive results. analysis of the oi family pairs revealed more than peaks of increased ibd sharing with nominal p-values < × - (figure ). however, the analysis of non-independ- ent pairs from a small number of families descended from a common founder will increase the observed ibd sharing between individuals. therefore, in the context of a mono- genic disease the genome-wide threshold for significant confirmation of known meiotic crossoversfigure confirmation of known meiotic crossovers. the ibd enrichment process was applied to ceph family grandpar- ent-grandchild pairs. amplification, labelling, hybridization and data analysis were performed as described in methods, except standardization was by division of the normalized ratios by the standard deviation of the ratios of all clones on each array. for chromosome , the ratio values for the ibd- enriched dna (solid lines) are compared with the regions where microsatellite genotypes are shared between a grand- parent and grandchild (dotted lines); grandfather (gf)/child, blue and grandmother (gm)/child, red. the arrow indicates a meiotic recombination crossover region. crossover gf / child gm / child microsat. ibd-enriched table : osteogenesis imperfecta relative pairs type of pair number of pairs expected shared ibd fraction sibling . avuncular . cousins . page of (page number not for citation purposes) bmc genetics , : http://www.biomedcentral.com/ - / / linkage is not useful. some peaks may be linked to specific traits that segregated only within a single family. we there- fore considered that the loci more likely to include any cosegregating genes would be represented by peaks with clones showing p-values < - , for which all pair-wise tests were informative and furthermore where all clones showed ibd sharing. by these criteria, there were two prominent loci on chromosomes and (figure ). the chromosome locus spanned about mb ( . to . mb) and although this may represent a chance shar- ing of ibd within these families associated with some trait, it is possible that such a locus harbours a gene that is relevant to the expression of oi. the chromosome locus, ( . to . mb) includes col a at . mb. as this is the gene bearing the oi causative mutation, we conclude that the ibd enrichment and mapping procedures have successfully identified the disease locus in these families. discussion here we have described several critical modifications to a physical positional cloning process to ensure its reliability and to reduce noise in the final mapping analysis. we have recognized a major challenge in applying this meth- odology, namely that the procedure must eliminate a large fraction of the reannealed dna, mainly comprised of strands derived from different chromosomes and from potentially confounding self-self isohybrid fragments. we have formally defined the dependence of the discrimina- tion of ibd dna from non-identical dna as a function of the efficiency of the process of ibd selection. therefore, we designed the procedure to ensure highly efficient and specific intermediate yields, and included step-specific quality control assays. improvements included a reduc- tion in the number of steps, optimization of reannealing, and conditioning of a dna binding resin to render this key reagent suitably reliable. osteogenesis imperfecta, an autosomal dominant diseasefigure osteogenesis imperfecta, an autosomal dominant disease. pedigree of amish family a (coriell) showing individuals with osteogenesis imperfecta as filled symbols. ? linkage test results for ceph control populationfigure linkage test results for ceph control population. genome-wide ibd sharing with standardized ratios was determined as described in methods. based on sib pairs from independent families, no clones in chromosomes through surpass the threshold of significance at × - (red line). – lo g p v a lu e - - - - - - page of (page number not for citation purposes) bmc genetics , : http://www.biomedcentral.com/ - / / page of (page number not for citation purposes) sharing of ibd regions among individuals from amish osteogenesis imperfecta familiesfigure sharing of ibd regions among individuals from amish osteogenesis imperfecta families. genome-wide ibd shar- ing with standardized ratios was determined as described in methods. – lo g p v a lu e - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - bmc genetics , : http://www.biomedcentral.com/ - / / the essential component for ibd selection is the enzy- matic activity that recognizes and hydrolyzes mismatched dna fragments. we cloned the three proteins into high yield overexpression vectors, and with various methods, including surface plasmon resonance studies, character- ized their activities and optimized storage and reaction conditions [ - ]. we over-titrated the enzymatic activi- ties and other reagents that eliminate unwanted dna to provide sufficient discrimination for mapping of ibd regions. because the yield of the ibd enrichment process is low, we validated a generic amplification method that produced sufficient dna for mapping. deviations in copy number representation introduced by the amplification method are not detrimental to the final array ratio determinations because increases or decreases in copy number of different fragments are roughly balanced throughout the long bac clone sequences and hence in contiguous clones repre- senting an ibd region. any clone-specific or sequence- dependent deviations are expected to be of similar direc- tion and magnitude in both sample and reference dnas. the range of discrimination factors between . and that we observed by bac clone hybridization might be inter- preted as representing the efficiency of the enrichment process. however, microarray analysis is subject to numerous perturbing factors [ ] resulting in dynamic range dampening. to exclude any microarray-related fac- tors, we assessed discrimination by qpcr and showed that the process can enrich some ibd fragments at least - to -fold. even though numerous hybrid fragments might escape the steps designed to eliminate non-identical dna and so contribute to noise, the improvements we have introduced to the overall process ensure that the prepon- derant hybridization signal from the bac clones is due to strongly enriched ibd fragments. we have also developed a related protocol, genome hybrid identity profiling, which incorporates these improvements and the use of self-self hybrid discrimination via ligation of oligonucle- otide tags; this method enables initial fragmentation of the gdna with any restriction enzyme that generates effi- ciently ligatable termini. the distinction between mendelian monogenic disease and complex polygenic disease has blurred in recent years [ ]; modifier genes may influence the clinical pheno- types of monogenic conditions [ ]. to identify candidate loci harbouring disease or modifier genes, extended pedi- grees are particularly useful, as any ibd regions conserved in most or all of the afflicted individuals are large, whereas the probability of finding any ibd loci shared among many distant relatives is small [ , ]. to demonstrate adequate performance for genome-wide disease gene mapping, we applied the ibd-enrichment process to old order amish oi family pairs from an extended pedigree and successfully identified a chromosome locus con- taining col a , the gene bearing the mutation responsi- ble for oi in these families. another ibd-enriched locus, mapping to chromosome q, includes ptgs (cox- ), located at . mb. cox- is expressed in a regulated manner in osteoblasts and is a key regulator in bone for- mation, interacting with various key proteins of bone metabolism. variants of cox- might affect the clinical outcome of collagen mutations and so may be involved in some of the pleiotropic phenotypes of oi [ , - ]. the phenotype of mice homozygous for the col a gly cys mutation mimics the milder clinical expres- sion of the mutation in the amish families, and the phe- notype of heterozygous mice is inconsistent with autosomal dominance [ ]. as phenotypic severity of the mutated mice varies substantially depending on the genetic background [ ], these mice would be appropriate for constructing additional mutations in candidate modi- fier genes, such as cox- . the potential of family-base studies to identify complex disease genes has yet to be realized; numerous reports have identified linkage peaks of only suggestive p-values and loci are often not replicated [ ]. due to modest gene effects, loci for complex diseases will be difficult to iden- tify unless at least families are genotyped [ ]. therefore, much effort has been invested in the genome wide association approach using high density snp geno- typing arrays, and many credible associations have been described in case-control studies [ ]. nevertheless, in contrast to linkage analysis, failure to find significant association in a region, even with high density arrays, can- not exclude a disease-related gene in the region [ ]. gen- otyping several hundred thousand markers generates thousands of significant associations [ ], such that the most significant p-values most likely correspond to gene regions unrelated to the disease; whereas the rare true associations are likely to be among the lower ranking p- values of the significant associations [ ]. hence linkage studies that whittle the genome to a few regions, followed by association studies limited to these regions, decrease the multiple testing burden and the likelihood of false associations. genotypes from high density snp arrays are also useful for ibd detection by comparing data from two or more related individuals to find long runs that contain no gen- otypes inconsistent with common inheritance from the same ancestral chromosome, and that exceed some calcu- lated or simulated cut-off length, thus confidently exclud- ing runs of random ibs [ , , ]. this approach has been applied with small pedigrees to confirm a previously identified locus for prostate cancer [ ], and to identify page of (page number not for citation purposes) bmc genetics , : http://www.biomedcentral.com/ - / / candidate loci for kidney cancer [ ]. this novel means to exploit snp data, together with the physical method we present here, provide alternative or complementary strat- egies for gene hunts in family collections and pedigrees. by addressing both the set of variants represented on snp arrays and additional variants, such as those typically revealed only by fine mapping of regions of associated haplotypes, the physical method may detect shorter ibd regions than the genotyping approach, including regions that might be excluded as likely ibs, or regions of genuine ibd runs broken by erroneous genotypes. to identify small ibd regions, the resolution of the map- ping method must approach the expected mapping reso- lution of the physical ibd enrichment procedure. in addition, genome-wide mapping of ibd-enriched dna should preserve the high level of relative enrichment revealed by qpcr. we expect that next-generation rese- quencing methods [ ] will permit mapping ibd regions by scoring sequence read depth. analysis, including geno- typing of sequenced snps, would be restricted to unique sequences and restriction fragments with some minimum density of snps of high minor allele frequencies, thus ignoring the sequences that contribute to dampening of discrimination in microarray analysis. in addition, sequence selection based on resequencing performance with pools of ibd-enriched dna from ceph family pairs would generate a genome-wide set of sequences that accu- rately report ibd sharing. considering that reannealing to generate unique sequence hybrids reduces the genomic representation at least five-fold and that the level of ibd enrichment for well-behaved sequences is - to -fold, very few sequencing runs would be sufficient to obtain a depth of coverage comparable to the depth that permitted calling heterozygous snps after resequencing an entire human genome, requiring about runs [ ]. sufficiently deep coverage may permit distinction between monopa- rental and biparental sharing in ibd-enriched regions. conclusion we have established a robust process that physically selects and maps genomic regions that are shared between family members. the methodological approach, origi- nally proposed by ford and colleagues to isolate "inherit- ance units" [ , ], is now practicable for the simultaneous processing of several hundred relative pairs such as sibling pairs under rigorous quality control condi- tions. the improved process enabled mapping of loci for a monogenic trait, osteogenesis imperfecta. using the physical enrichment process, we have previously reported the identification of loci for autism, including a locus on chromosome p. with subsequent high density genotyp- ing in this locus, we found that prkcb , protein kinase beta, is associated with autism [ ]. thus the procedure is now suitable for enriching for ibd dna in applications ranging from monogenic to complex diseases. coupled with mature deep resequencing methods to map the ibd- enriched dna, the technology will enable increased dis- crimination that will be required to analyze more dis- tantly related individuals and cost-efficient pools of ibd- enriched dna samples. methods reagents and dna reagents and suppliers included: restriction endonucle- ases, dam methylase and exonuclease iii, ne biolabs; templiphi kit, amersham; repli-g wga kit, molecular staging/qiagen; sybr green, sybr gold and picogreen, molecular probes/invitrogen; genomehip reagent com- ponents including hyfast gdna reannealing reagent (hyf), dna affinity polymer (dap, a reconditioned derivative of benzoyl-naphthoyl-deae cellulose, sigma), dap buffer, lshase (a formulation of e. coli muts, mutl and muth), enzyme buffer (eb), a formamide-based array hybridization buffer (ahb), coverslip removal buffer (crb) and stringent wash buffer (swb), integragen; mul- tiscreen filtration plates, millipore; qiaquick glass-fibre purification plate, qiagen; ultragaps slides, corning. ceph family genomic dnas were either prepared from immortalized tissue culture cells with the recoverease kit (stratagene), a procedure that yields highly intact dialyzed dna [ ], or obtained from the coriell repository. bac clones were obtained from invitrogen or from the central national de séquencage, genoscope (evry, france). cori- ell provides dna from osteogenesis imperfecta families a, b, c and d, (for pedigrees and phenotypes, see http:// ccr.coriell.org/nigms/phenotype/oi.html). dna quantification we measured dna concentrations with picogreen in a -well fluorescent plate reader using calf thymus dna as standard. hybrid reannealed dna we digested genomic dna (gdna) from pairs of relatives with pst i and purified and concentrated the digests with multiscreen manu- ultrafiltration. we quantified the purified, digested dna and verified the expected range of fragment sizes by agarose electrophoresis. we used dam methylase to tag one of the pst i-digested gdnas. we combined . μg of each of the samples, denatured the dna by incubation in . m naoh at rt for min, and neutralized the solution by addition of hyf buffer and phenol. we reannealed complementary strands by shaking the emulsion for hours and recovered the aqueous phase after mixing with chloroform. we immo- bilized duplex fragments on glass fibre (qiaquick) in the presence of a chaotropic salt to eliminate non-renatured single stranded dna [ ]. we eluted the hybrid renatured page of (page number not for citation purposes) http://ccr.coriell.org/nigms/phenotype/oi.html http://ccr.coriell.org/nigms/phenotype/oi.html bmc genetics , : http://www.biomedcentral.com/ - / / fragments with te and removed an aliquot for labelling and hybridization to microarrays. enrichment for ibd dna we incubated the reannealed hybrid dna with lshase in eb at °c for min and heated for min at °c. we incubated the product of the lshase reaction with mbo i and dpn i for min at °c and min at °c. then, we added exo iii, incubated for min at °c, added dap buffer and treated with dap to isolate ibd-enriched duplex dna free of single-stranded digestion products. amplification of ibd-enriched dna to amplify the ibd-enriched dna, we used either tem- pliphi or repli-g, as recommended by the manufacturers with minor modifications. we denatured the reannealed hybrid dna or the ibd-enriched dna with naoh and amplified for hrs at °c. we purified the amplified products by ultrafiltration and quantified the dna. the extent of amplification ranged from about - to - fold, or the equivalent of about to doublings. array design with the aim of choosing clones with mb spacing, we used the program clonetrek (integragen). clone trek's input includes essential features of each clone and two parameters, the distance between two clones on the tiling path and the minimal distance accepted between two clones. initially, all clones in the ncbi clone registry are placed on a tiling path. clone trek's iterative algo- rithm: for each chromosome, while there are clones whose removal would create acceptable gaps, identify and remove the least favoured clone. the least favoured clone has the lowest score with respect to various defined crite- ria, including fish data, sts content, sequencing status, size and mapping status. the arrays had replicates of clones, including mapped to a unique genomic position with the build assembly (may, ). average spacing was . mb and median spacing . mb. duplicate sets of blocks were printed in two zones in order to maximally separate the two sets of dupli- cates printed in each block. various controls including rice bacs were also printed in quadruplicate. subsequent versions of the arrays have clones. array printing we purified bac dna using alkaline lysis, filtration and isopropanol precipitation. we amplified bac dna as described above for amplification of ibd-enriched dna. clone identity was verified by end sequencing of all clones and restriction digest fingerprinting of some clones. we checked the fidelity of amplification by verify- ing that sample bac fingerprint patterns matched before and after amplification. we digested to μg of each amplified bac with alu i and purified the digests by ultra- filtration. the dna was dried by vacuum centrifugation and resuspended in μl of % dmso. we printed the dna on gaps slides (corning) with a biorobotics microgridii arrayer using biorobotics quill pins; spot diameters were about microns. we irradiated arrays with mj of nm uv light and then baked them at °c for two hours. we scanned all slides and examined the images of auto-fluorescence to identify any pin-spe- cific problems. we validated the printing batches of about slides by sybr green staining and hybridization tests on selected slides. mapping of ibd-enriched dna on microarrays amplified reannealed hybrid dna, labelled with cy , and amplified ibd-enriched dna, labelled with cy , were hybridized to the bac microarrays to enable a ratio- metric analysis [ , ]. labelling reactions of μl were for to hours at °c with klenow (exo-) dna polymerase (ne biolabs) and contained μg of dna, μm random octamers, μm datp, dgtp, ttp, μm dctp and either μm cy -dctp or μm cy -dctp. we purified the labelled dna by spun gel filtration through sephadex g (apb biotech) in hv micro- plates (millipore). the specific fluorescence (fluoro- chromes/kbp, fl/kb), of each probe, as determined by dna quantification and cy-specific fluorescence readings in a fluorescence plate reader using cy-dctps as stand- ards, ranged from to fl/kb. we prepared hybridiza- tion mixes by mixing cy - and cy -labeled dna, concentrating by vacuum centrifugation and resuspend- ing in μl of ahb containing cot dna. array slides were blocked by incubation in % bsa, . % sds at °c for min. we pre-hybridized slides with μl ahb containing mg/ml salmon sperm dna at rt for min. we removed about μl of the prehybridization mix, deposited the hybridization mixes on the arrays, cov- ered with hybrislips (grace) and placed the arrays in indi- vidual hybridization chambers (corning) in a water bath at °c for to days. we removed coverslips by gentle agitation in crb, rinsed in × ssc, soaked in swb at °c for min, and then briefly rinsed slides in a series of baths: . × ssc, filtered . × ssc and % isopropanol. we have also tested and validated various commercial labelling kits, hybridization buffers and alternative proto- cols, including washing at higher temperatures in the absence of formamide. image and microarray data analysis arrays were scanned using an agilent scanner and fluores- cent intensities were corrected by subtraction of local background using genepix® pro . . spots with fluores- cent signals indicating partial saturation (> , ) or sig- nals less than times the mean of the backgrounds from all autosomal clones were excluded. a ratio value of ibd- enriched dna versus reannealed dna was determined page of (page number not for citation purposes) bmc genetics , : http://www.biomedcentral.com/ - / / based on the four spot replicates for each clone. clones with less than three morphologically acceptable replicates or with excessive variance of replicate ratios (var(repli- cate) - mean(var(replicates)) > * var(var(replicates)) were eliminated. median ratios of the replicates for each clone were computed and data were normalized between arrays by dividing the ratio of each clone by the mean of the ratios of all autosomal clones. unless otherwise noted, the ratios of each clone were standardized using full sib pair controls by subtracting the mean of the control ratios of the clone and dividing by the variance of the con- trol clone ratios. only the clones mapped to a unique genomic position were used in the analysis. ibd determination and linkage analysis we determined a moving average (ma) ratio for each clone. with rk as the standardized ratio of clone k, di the physical distance weight for neighbouring clone i (d is inversely proportional to the distance in mb from the clone, using a weighting function based on a normal dis- tribution), and m the number of flanking clones on each side of clone k, then , the ma ratio of clone k is obtained: we set m as three, corresponding to a moving window of seven adjacent clones. as we expect that the status of an average of % of the clones is ibd, a threshold ratio t was determined such that % of the ma-ratios from all clones and all sib pair controls were greater than t. we set the ibd status to one for clones with ma-ratios greater than t and to zero for clones with ma ratios less than t. after these binary ibd scores were determined for each clone for each affected relative pair, we then counted the number of pairs that were ibd at each clone. a region rep- resented by a clone or a series of consecutive clones is linked to the trait only if the number of affected pairs that are ibd for the region exceeds the number of pairs that by chance could have received copies of the same ancestral region. the null distribution of chance sharing appropri- ate for studies with different types of relative pairs was determined as described [ ]; alternative methods to determine the null distribution may be appropriate for various experimental designs, including those with inbred pedigrees [ - ]. to limit the genome-wide probability of false linkage to %, we used the p-value of × - to set the pointwise significance threshold for declaration of significantly increased sharing [ ]. quantitative pcr pcr amplifications were quantified by real time qpcr, using a common probe specific for ca microsatellite repeat sequences [ , ]. reactions of μl contained ng of amplified ibd-enriched dna, amplitaqgold buffer, . mm each datp, dctp, dgtp, ttp, . mm dutp, mm mgcl , . u/μl uracil-n-dna glycosy- lase (sigma), . u/μl amplitaqgold dna polymerase (roche), . μm each primer and the probe oligonucle- otide ' fam-(ca) -tamra. incubations were at °c for min, °c for min, followed by cycles at °c for sec and °c for min. availability the microarray gpr files and three annotation files can be obtained from http://bmc_genetics .integragen.org. the file "osteogenesis_samples.txt" lists the coriell osteo- genesis imperfecta samples. the file "osteogenesis_relative_pairs.txt" lists the experi- mental pairs of relatives and the identity codes for the microarray gpr files. the file "bac_clones.txt" lists the clone names and their chromosomal positions. if it was not possible to confi- dently position a clone, it is annotated as "null". authors' contributions pb wrote the manuscript and, with expert participation by cm, directed the development of the enzymology and technology for ibd enrichment, dna amplification and microarray printing and hybridization. mv prepared dna and performed experiments and hybridizations. jps man- ufactured the arrays, prepared dna and performed exper- iments and hybridizations. fr directed the implementation of the protocol in a production setting. ds guided the qpcr experiments. sr, rf and nc partici- pated in the design of the oi study. pl designed and wrote the clonetrek program and managed data bases. jh con- ceived of the study and participated in drafting the manu- script. ap participated in drafting the manuscript and designed and performed the statistical analysis with assist- ance by ft. all authors read and approved the final man- uscript. acknowledgements we thank jan mous and elke roschmann for critical reading and comments; lon aggerbeck and david rickman, cnrs, gif-sur-yvette for initial dna array printing; stephanie maillard and virginie decaulne for expert technical assistance, rachel rousseau for performing qpcr experiments, jolanta luberda for assistance in array printing, safa saker, isabelle lambert, isa- belle bezier and thierry larmonier, genethon, association française con- tre les myopathies, for cell culture; abdel benajou for help with array data analysis; bruno copin for help with figure preparation; gabor gyapay, jean weissenbach and colleagues, genoscope, for contributions to dna prep- r k r d r i m m d i m mk i k i i = ⋅ + =− ∑ =− ∑ ( ) page of (page number not for citation purposes) http://bmc_genetics .integragen.org bmc genetics , : http://www.biomedcentral.com/ - / / arations and sequencing; mark lathrop and colleagues, centre national de genotypage for access to their facilities; and the reviewers for constructive criticism of the manuscript. references . botstein d, risch n: discovering genotypes underlying human phenotypes: past successes for mendelian disease, future approaches for complex disease. nat genet , (suppl): - . . nelson sf, mccusker jh, sander ma, kee y, modrich p, brown po: genomic mismatch scanning: a new approach to 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- . page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= 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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ http://www.biomedcentral.com/info/publishing_adv.asp http://www.biomedcentral.com/ 	abstract 	background 	results 	conclusion 	background 	results 	overview of ibd enrichment process 	necessity for high efficiency of removal of non-specific dna 	robust enrichment of ibd dna 	discrimination efficiency evaluated by quantitative pcr 	identification of ibd regions for grandparent-grandchild pairs by mapping on bac microarrays 	application of ibd enrichment and mapping to identify monogenic disease loci 	discussion 	conclusion 	methods 	reagents and dna 	dna quantification 	hybrid reannealed dna 	enrichment for ibd dna 	amplification of ibd-enriched dna 	array design 	array printing 	mapping of ibd-enriched dna on microarrays 	image and microarray data analysis 	ibd determination and linkage analysis 	quantitative pcr 	availability 	authors' contributions 	acknowledgements 	references [pdf] preliminary results of a novel hay-hole fall prevention initiative | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /ta. corpus id: preliminary results of a novel hay-hole fall prevention initiative @article{batra preliminaryro, title={preliminary results of a novel hay-hole fall prevention initiative}, author={erich k. batra and b. gross and shreya jammula and e. bradburn and ronald d. baier and michael j reihart and d. murphy and kay moyer and joseph hess and s. lackmann and j. miller and f. rogers}, journal={journal of trauma and acute care surgery}, year={ }, volume={ }, pages={ – } } 	erich k. batra, b. gross, + authors f. rogers 	published 	medicine 	journal of trauma and acute care surgery background hay-hole falls are a prevalent source of trauma among anabaptists—particularly anabaptist youth. we sought to decrease hay-hole falls in south central pennsylvania through the development and distribution of all-weather hay-hole covers to members of the at-risk anabaptist community. methods following the creation of a rural trauma prevention syndicate, hay-hole cover prototypes co-designed and endorsed by the pennsylvania amish safety committee were developed and distributed… expand view on wolters kluwer lancastergeneralhealth.org save to library create alert cite launch research feed share this paper citationsbackground citations view all topics from this paper 	accidental falls 	hay 	wounds and injuries 	community 	traumatic injury one citation citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency injuries among amish children: opportunities for prevention 	stephen strotmeyer, abigail koff, j. honeyman, b. gaines 	medicine 	injury epidemiology 	 	 	 	view excerpt, cites background save alert research feed references showing - of references sort byrelevance most influenced papers recency childhood injuries due to hay-hole falls: a -year experience at a rural pediatric trauma center 	b. engbrecht, a. kulaylat, m. dias, james w. kendig, r. cilley 	medicine 	pediatric emergency care 	 	 	highly influential 	view excerpts, references background save alert research feed injury analyses in rural children: comparison of old-order anabaptists and non-anabaptists. 	k. forward, m. chan, t. c. stewart, j. gilliland, c. campbell, d. fraser 	medicine 	the journal of trauma 	 	 	pdf save alert research feed farm-related injuries among old order anabaptist children: developing a baseline from which to formulate and assess future prevention strategies 	jerene m gilliam, p. jones, w. field, donald b. kraybill, stephen scott 	engineering, medicine 	journal of agromedicine 	 	 save alert research feed falls from heights among children: a retrospective review. 	m. lallier, s. bouchard, d. st-vil, j. dupont, m. tucci 	medicine 	journal of pediatric surgery 	 	 save alert research feed pediatric fall injuries in agricultural settings: a new look at a common injury control problem 	w. pickett, s. dostaler, r. berg, j. linneman, r. brison, b. marlenga 	medicine 	journal of occupational and environmental medicine 	 	 save alert research feed injuries from falls in the pediatric population: an analysis of cases. 	m. wang, k. a. kim, + authors g. mahour 	medicine 	journal of pediatric surgery 	 	 save alert research feed the mortality of childhood falls. 	j. r. hall, h. reyes, m. horvat, j. l. meller, r. b. stein 	medicine 	the journal of trauma 	 	 save alert research feed pediatric injuries attributable to falls from windows in the united states in – 	vaughn harris, l. m. rochette, g. smith 	medicine 	pediatrics 	 	 	pdf save alert research feed farm safety issues in old order anabaptist communities: unique aspects and innovative intervention strategies. 	p. j. jones, w. e. field 	engineering, medicine 	journal of agricultural safety and health 	 	 	pdf save alert research feed american academy of pediatrics: falls from heights: windows, roofs, and balconies. 	medicine 	pediatrics 	 	 save alert research feed ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue antimicrobial resistance in the environment. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / x corpus id: antimicrobial resistance in the environment. @article{waseem antimicrobialri, title={antimicrobial resistance in the environment.}, author={h. waseem and m. r. williams and r. stedtfeld and s. hashsham}, journal={water environment research : a research publication of the water environment federation}, year={ }, volume={ }, pages={ - } } 	h. waseem, m. r. williams, + author s. hashsham 	published 	biology, medicine 	water environment research : a research publication of the water environment federation this review summarizes selected publications of with emphasis on occurrence and treatment of antibiotic resistance genes and bacteria in the aquatic environment and wastewater and drinking water treatment plants. the review is conducted with emphasis on fate, modeling, risk assessment and data analysis methodologies for characterizing abundance. after providing a brief introduction, the review is divided into the following four sections: i) occurrence of amr in the environment, ii… expand view on pubmed onlinelibrary.wiley.com save to library create alert cite launch research feed share this paper citationsbackground citations view all citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency recent advances in treatment technologies for antibiotics and antimicrobial resistance genes 	h. waseem, s. jameel, jafar ali, a. jamal, m. ali 	business 	 	 save alert research feed antibiotic resistance, its health impacts and advancements in their removal techniques with a focus on biological treatment 	r. kumar, payal mazumder, m. jawed 	business 	 save alert research feed what are the effective solutions to control the dissemination of antibiotic resistance in the environment? a systematic review protocol 	anaïs goulas, b. livoreil, + authors a. andremont 	business 	environmental evidence 	 	 	 save alert research feed fate of antibiotics and amr/args in the environment 	z. markiewicz, m. popowska 	biology 	 save alert research feed antimicrobial resistance in the gulf cooperation council region: a proposed framework to assess threats, impacts and mitigation measures associated with amr in the marine and aquatic environment. 	w. j. f. le quesne, c. baker-austin, d. verner-jeffreys, h. al-sarawi, h. balkhy, b. lyons 	business, medicine 	environment international 	 	 	 save alert research feed antimicrobial resistance in wildlife 	m. vittecoq, sylvain godreuil, + authors f. renaud 	biology 	 	 	 save alert research feed otter fecal enterococci as general indicators of antimicrobial resistance dissemination in aquatic environments 	t. semedo-lemsaddek, nuno m. pedroso, + authors m. oliveira 	biology 	 	 save alert research feed comparison of antibiotic-resistant bacteria and antibiotic resistance genes abundance in hospital and community wastewater: a systematic review. 	nasreen hassoun-kheir, yoav stabholz, + authors m. paul 	biology, medicine 	the science of the total environment 	 	 save alert research feed environmental dissemination of carbapenemase-producing enterobacteriaceae in rivers in switzerland. 	stephanie bleichenbacher, marc j a stevens, + authors m. nüesch-inderbinen 	biology, medicine 	environmental pollution 	 	 	 save alert research feed baseline screening for the presence of antimicrobial resistance in e. coli isolated from kuwait's marine environment. 	h. al-sarawi, a. jha, c. baker-austin, m. al-sarawi, b. lyons 	biology, medicine 	marine pollution bulletin 	 	 	pdf save alert research feed ... ... related papers 	abstract 	 citations 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue [pdf] individual-based modeling of potential poliovirus transmission in connected religious communities in north america with low uptake of vaccination. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /infdis/jit corpus id: individual-based modeling of potential poliovirus transmission in connected religious communities in north america with low uptake of vaccination. @article{kisjes individualbasedmo, title={individual-based modeling of potential poliovirus transmission in connected religious communities in north america with low uptake of vaccination.}, author={k. kisjes and r. d. duintjer tebbens and g. wallace and m. pallansch and s. cochi and s. wassilak and k. thompson}, journal={the journal of infectious diseases}, year={ }, volume={ suppl }, pages={ s - } } 	k. kisjes, r. d. duintjer tebbens, + authors k. thompson 	published 	geography, medicine 	the journal of infectious diseases background pockets of undervaccinated individuals continue to raise concerns about their potential to sustain epidemic transmission of vaccine-preventable diseases. prior importations of live polioviruses (lpvs) into amish communities in north america led to their recognition as a potential and identifiable linked network of undervaccinated individuals. methods we developed an individual-based model to explore the potential transmission of a lpv throughout the north american amish population… expand view on pubmed ncbi.nlm.nih.gov save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all figures, tables, and topics from this paper 	 figure 	 table 	 table 	 figure 	 figure 	 figure 	 figure view all figures & tables 	poliomyelitis 	community 	genetic heterogeneity 	varicella virus vaccine live (oka-merck) strain 	lopinavir 	human poliovirus 	hiv infections citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency managing population immunity to reduce or eliminate the risks of circulation following the importation of polioviruses. 	k. thompson, d. a. kalkowska, r. d. duintjer tebbens 	medicine 	vaccine 	 	 	 save alert research feed characterization of the impacts of heterogeneity in high-risk populations for infectious disease transmission: modeling polio and measles in the north american amish 	k. thompson, k. kisjes 	 	pdf 	view excerpts, cites methods and background save alert research feed lessons from the polio endgame: overcoming the failure to vaccinate and the role of subpopulations in maintaining transmission 	k. thompson, r. d. duintjer tebbens 	medicine 	the journal of infectious diseases 	 	 	pdf save alert research feed community structure mediates sabin polio vaccine virus transmission 	m. famulare, w. wong, + authors m. taniuchi 	geography, medicine 	medrxiv 	 	 	pdf 	view excerpt, cites background save alert research feed modeling measles transmission in the north american amish and options for outbreak response. 	k. thompson, k. kisjes 	medicine 	risk analysis : an official publication of the society for risk analysis 	 	 	view excerpts, cites methods, background and results save alert research feed review of poliovirus modeling performed from to to support global polio eradication 	k. thompson, d. a. kalkowska 	political science, medicine 	expert review of vaccines 	 	 	 	view excerpts, cites methods and background save alert research feed an economic analysis of poliovirus risk management policy options for – 	r. d. duintjer tebbens, m. pallansch, s. cochi, s. wassilak, k. thompson 	medicine 	bmc infectious diseases 	 	 	 	view excerpt, cites background save alert research feed risk analysis for the reintroduction and transmission of measles in the post-elimination period in the americas 	d. m. lemos, a. franco, + authors luciano pamplona de góes cavalcanti 	medicine 	revista panamericana de salud publica = pan american journal of public health 	 	 	pdf save alert research feed lessons from a decade of individual-based models for infectious disease transmission: a systematic review ( - ) 	l. willem, f. verelst, j. bilcke, n. hens, p. beutels 	medicine, biology 	bmc infectious diseases 	 	 	highly influenced 	pdf 	view excerpts, cites background and methods save alert research feed modeling and managing the risks of measles and rubella: a global perspective, part i. 	k. thompson, s. cochi 	medicine 	risk analysis : an official publication of the society for risk analysis 	 	 	view excerpts, cites methods save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency the potential impact of expanding target age groups for polio immunization campaigns 	r. d. duintjer tebbens, d. a. kalkowska, s. wassilak, m. pallansch, s. cochi, k. thompson 	medicine 	bmc infectious diseases 	 	 	pdf save alert research feed trends in the risk of u.s. polio outbreaks and poliovirus vaccine availability for response 	k. thompson, g. wallace, + authors s. wassilak 	biology, medicine 	public health reports 	 	 	pdf 	view excerpts, references methods, background and results save alert research feed transmission of imported vaccine-derived poliovirus in an undervaccinated community in minnesota. 	j. alexander, k. ehresmann, + authors h. hull 	biology, medicine 	the journal of infectious diseases 	 	 	pdf 	view excerpt, references background save alert research feed characterizing poliovirus transmission and evolution: insights from modeling experiences with wild and vaccine-related polioviruses. 	r. d. duintjer tebbens, m. pallansch, d. a. kalkowska, s. wassilak, s. cochi, k. thompson 	geography, medicine 	risk analysis : an official publication of the society for risk analysis 	 	 	pdf 	view excerpts, references methods, background and results save alert research feed risks of paralytic disease due to wild or vaccine-derived poliovirus after eradication. 	r. d. tebbens, m. pallansch, + authors k. thompson 	medicine 	risk analysis : an official publication of the society for risk analysis 	 	 	pdf 	view excerpts, references background save alert research feed modeling population immunity to support efforts to end the transmission of live polioviruses. 	k. thompson, m. pallansch, r. d. tebbens, steve g f wassilak, s. cochi 	biology, medicine 	risk analysis : an official publication of the society for risk analysis 	 	 	pdf save alert research feed expert review on poliovirus immunity and transmission. 	r. d. duintjer tebbens, m. pallansch, + authors k. thompson 	medicine 	risk analysis : an official publication of the society for risk analysis 	 	 	pdf 	view excerpts, references methods and background save alert research feed review and assessment of poliovirus immunity and transmission: synthesis of knowledge gaps and identification of research needs. 	r. d. duintjer tebbens, m. pallansch, + authors k. thompson 	medicine 	risk analysis : an official publication of the society for risk analysis 	 	 	pdf 	view excerpts, references methods and background save alert research feed preeradication vaccine policy options for poliovirus infection and disease control. 	k. thompson, m. pallansch, r. d. duintjer tebbens, steve g f wassilak, j. kim, s. cochi 	medicine 	risk analysis : an official publication of the society for risk analysis 	 	 	pdf save alert research feed from emergence to eradication: the epidemiology of poliomyelitis deconstructed 	n. nathanson, o. kew 	medicine 	american journal of epidemiology 	 	 	pdf 	view excerpt, references background save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue inquiry and virtue: a pragmatistliberal argument for civic education inquiry and virtue: a pragmatist-liberal argument for civic education phillip deen liberal democracy and virtue have an uncomfortable relationship. the repub- lican tradition ranging from the ancient world through the atlantic tradition of the founding fathers, to present-day communitarians, argues that democracy relies uniquely on the virtue of its citizens. they hold that republics are not defeated from without, but corrupted from within. there is a dire need for a shared conception of the good life. only then are citizens willing to act together and to make the sacrifices necessary for the common weal. liberals, however, argue that a shared conception of the good life can be bought only at the price of freedom. within a nation-state of our size and possessing our level of freedom, differing images of the good life are inevitable and worth respecting. therefore, the state should not craft souls, but provide the conditions for all to flourish according to their own desired ends. liberals are not tolerant of all forms of life. moral doctrines, communities, and traditions that do not accept liberal values of tolerance, respect, liberty, equality, and free inquiry do not have a place in the liberal public sphere (although anti-liberal beliefs may be retained in one’s private life). however, liberals are in a uniquely difficult position because, while they may possess deeply held com- mitments regarding the need for openness, courage, or respect in a good life, part of that life includes not imposing their image of a good life on others. liberals must find reasons for coercing fellow citizens that do not require a prior “conver- sion” to liberalism. they must find impartial reasons for their partiality. more sardonically, robert frost defined a liberal as someone “too broadminded to take his own side in a quarrel.” the contest between liberalism and virtue is particularly heated in the area of public education. because public education is coerced, both by requiring attendance by the young and by taxation of adults, it is a test case for liberalism. seemingly, public schools must refrain from imposing an image of human excel- lence on children of disparate faiths and traditions since the state sanctions those who do not comply. take the example of california’s recent mandate that the historical contribution of homosexuals be included in the curriculum. as a liberal, it is possible to believe not only that toleration and respect of homosexual citizens are inherently good and necessary for the health of our democracy, but also that imposing these virtues through a coercive system of education is intolerant and disrespectful since it violates the right of illiberal parents to raise their children bs_bs_banner journal of social philosophy, vol. no. , winter , – . © wiley periodicals, inc. as they see fit. the difficulty is that civic education—that is, education for good citizenship, including the cultivation of certain virtues—is both necessary if liberal democracy is to continue and hard to justify on liberal terms. i argue that recent political philosophy rooted in the tradition of american philosophical pragmatism offers a way to reconcile the liberal image of a good life with the pluralism of modern, liberal democracies. pragmatism has clear histori- cal links to the rise of progressive education and political liberalism. in particular, john dewey is a pivotal figure in both. widely known as the father of progres- sive education—although he chafed at the label—dewey was also a key figure in interwar american politics. one biographer goes so far as to name his work john dewey and the high tide of american liberalism (ryan ). a great deal of excellent work has been written on dewey’s philosophy of education (e.g., gar- rison ). with apologies to those authors, i will intentionally avoid a discus- sion of pragmatist philosophy of education. rather, in this article, i discuss the political problem of how we might justify education for civic virtue in public schools while avoiding the problem of liberal pluralism. i present two types of argument for liberal-democratic virtues, both of which are grounded in the pragmatist thought of charles peirce and john dewey. the first type relies on their substantive teleological accounts of truth and moral flourishing. the second type, however, is based on their account of the general conditions of inquiry and not on any substantive ontology or moral ideal. i argue that, under conditions of liberal pluralism, the latter is more practically viable and morally justifiable. i conclude by applying the general argument for liberal- democratic virtue to the case of civic education and by addressing some objec- tions. the argument from a pragmatist account of inquiry shows the legitimacy of coerced civic education while giving due respect to the plurality of moral tradi- tions among those being coerced. . teleological pragmatist accounts of virtue this section sets out one nonexclusive account of the relationship between pragmatism and virtue. here, the emphasis is on how liberal-democratic virtues may be rooted in substantive pragmatist metaphysical and moral commitments. for now, i will present only thick metaphysical versions of dewey’s and peirce’s pragmatism, and how liberal-democratic virtues arise from them. this section is intentionally partial and does not claim to describe the sole or primary means for a pragmatist to justify liberal virtues. as will be discussed in the next section, the pluralism of the modern nation-state makes the following substantive approach problematic. before proceeding, however, i must clarify what is meant by “liberal- democratic virtues.” virtue generally means those qualities of character deserving of praise, exhibiting excellence, or conducive to a good life. virtues include piety, magnanimity, and prudence, among many more. by “liberal-democratic” inquiry and virtue i mean the subset of those virtues central to the maintenance and justice of a liberal-democratic society. democracy requires, for example, that citizens exhibit concern for the whole and moderate their selfish desires, while also courageously defending their beliefs in the public arena. liberal-democratic societies particu- larly emphasize toleration and mutual respect. if we are to protect basic rights and liberties, including rights to privacy, practice of religion, and free expression, then toleration of competing images of a good life is essential. ironically, if we are to have a liberal society that refrains from imposing a singular image of the good on its citizens, those citizens must have a set of liberal-democratic virtues conducive to that end. in what follows, i attempt a justification of civic education in a cluster of those virtues—openness, a willingness to experiment, courage, perseverance, sociality, inclusiveness, moderation, self-reflection, and toleration. . dewey: virtue as attentiveness to experience “experience,” as william james noted, is a double-barreled word. it includes the passive and active, the experienced and the experiencer. based in an openly darwinian model, dewey presents experience as the nexus of meaning where an organism makes contact with, manipulates, and is acted upon by its natural and social environment. this transaction is disrupted when previously fit habits no longer result in a fruitful relation with the organism’s environment. dewey describes this as a “problematic situation” and calls for its “reconstruction.” if the organism is to survive, it must develop new habits, and, in more developed species, habits of reflection. the latter are second-order habits that allow the organism to take the formation of primary habits as an active concern. we may then draw certain broad conclusions about experience in general. experience has an irreducibly temporal dimension. recalling peirce’s notion of tychism, dewey argues that time matters, and that novelty and chance are real. the next moment is not strictly determined. we find that it has phases that are, to borrow from a chapter heading in experience and nature, precarious and stable. at times, our transactions sustain us and provide nutrition. at others, we starve. our relationships with others sometimes provide the conditions for flourishing, or they may stunt further growth. perhaps the most sustained account of deweyan democratic virtue is provided by gregory pappas in his john dewey’s ethics: democracy as experience (pappas , especially chapter ; see also teehan ; pappas ; rice ; olson ). pappas reveals how a compelling theory of virtue develops out of dewey’s concept of experience. virtues of open-mindedness, sensitivity, con- scientiousness, and courage are all qualities of character that suit a world that is both precarious and stable. a character that is not open to future experience is unprepared to deal with, or learn from, novel situations. open-mindedness and sensitivity are virtues because they show “hospitality to the new,” the ability to learn from experience, and a “willingness to let experiences accumulate and sink in and ripen” (dewey phillip deen – /mw : ). and to hear what experience has to teach, we must have a refined sensitivity. a blunt or dogmatic approach to the world closes one off from the situation at hand in all of its complexity and richness. the conscientious person then attends to the implications of his/her acts and seeks the greater significance of what he/she experiences. but to counter the instability of life, a person must develop virtues of persistence and courage. sheer vulnerability to the new is not enough to sustain a fulfilling relation to an environment. there is also the need to press on when experience does not satisfy. courage without openness produces dogmatism, while openness without courage dissolves the self. both results are failures of transaction (pappas , chapter ; dewey – /lw : chapter – ). an implication of the novelty and precariousness of experience is that morality is not a matter of identifying fixed moral ends, principles, or virtues. each of these is derived from the needs, relations, and tensions of the situation at hand. moral reflection arises when the fund of valued goods or social duties no longer harmonize under novel conditions. this morally problematic situation must then be reconstructed. through inquiry, we determine which goods and duties should have priority at this time, which need to be set aside and which must be invented. moral is not a matter of identifying and applying the tran- scendent good, but of constructing the good so that human practice may proceed in a fruitful manner. virtues are those qualities of character that allow experience to grow in “ordered richness.” scattered through his many works, dewey catalogues a substantial table of such virtues, including honesty, courage, kindness (mw : – and mw : ), open-mindedness (lw : ; mw : , , ), curiosity and respectfulness (mw : ), sympathy, sensitivity, persistence (mw : – ), imagination (mw : – , lw : – ), and finally the cardinal virtues of justice, courage, temperance, and wisdom (mw : – ). as pappas writes of openness and courage, but is true of the virtues in general, [f]or pragmatism they are virtues because ( ) they are part of the concrete set of habits which make possible the amelioration of experience through its own means (what i will call the “pragmatist faith”), ( ) they are dispositions of the kind of character and community that is faithful to experience, and ( ) a checking-complementary relation of these disposi- tions in a character is the only way to avoid extremes that pragmatism considers undesir- able. (pappas , ) or, as stated by another author, “[t]hese are virtues required by any moral agent (understood transactionally) attempting to maintain the integrity of their moral image in an uncertain and changing world” (teehan , ). ultimately, dewey defined education as “that reconstruction or reorganization of experience which adds to the meaning of the experience, and which increases ability to direct the course of subsequent experience” (mw : ). education is the growth of experience. democratic virtues are those that are required for personal growth in a constantly changing environment. inquiry and virtue . peirce: virtue as concrete reasonableness charles peirce provided a similarly thick account. despite some noble attempts to correct the disparity, peirce’s normative theory still receives little attention compared with the effort spent to plumb the depths of his semiotics and logic. although this brief discussion will not eliminate that gap, i hope to sketch out peirce’s own account of what we might call a “virtue ethics,” although peirce did not believe himself to be doing anything of the sort. i argue here that peirce’s model of reality as increasingly self-controlled and self-reflective, and as oriented toward an ultimate good, undergirds his account of virtue. ironically, peirce did not believe that scientific inquiry had much to offer ethics. he believed that ethical behavior was largely the result of instinct and tradition. it is a matter of habit or convention, and virtue does not depend on rational reflection (peirce – / . ). further, in matters of vital impor- tance such as morality and religion, we have neither the time nor inclination to apply science ( . – ). belief is a commitment, an urge to act in the name of some ideal, but scientists hold their beliefs at abeyance as hypotheses that are open to experimentation. the scientist whose first commitment is to a moral or political belief then undercuts the clinical detachment required for inquiry. science then requires a certain laxity of moral commitment. as a consequence, peirce amus- ingly informs his audience at the beginning of his lecture on ‘philosophy and the conduct of life’ that philosophy must be kept away from practice, and “i do not hold forth the slightest promise that i have the philosophical wares to offer you which will make you either better men or more successful men” ( . ). of course, this is not the whole story. although he did not believe that scientific philosophy would help us directly, it was shot through with commit- ments of its own, and these may indirectly describe the admirable character. where the moralist is governed by some particular conventional or instinctual ideal, the scientist cares only for truth and reason, and this love shapes the moral character of the scientist. the eternal forms, that mathematics and philosophy and the other sciences make us acquainted with, will by slow percolation gradually reach the very core of one’s being; and will come to influence our lives; and this they will do, not because they involve truths of merely vital importance but because they are ideal and eternal verities. ( . ) let us then examine peirce’s account of why scientific inquiry has a normative dimension and what bearing this has for a pragmatist virtue ethics. all conduct desires to achieve some end, to satisfy some desire. in order to act according to these ideals, agents develop rules of conduct. such rules are resolutions to act in a certain manner in future situations. when desire inevitably arises and we act in pursuit of it, we are then in a position to reflect on whether our conduct accorded with our prior resolution, and in so doing provided a feeling of pleasure. delving deeper, we may examine the intention behind the resolution or, phillip deen of particular importance to our present discussion, the character of the man behind the intention. right thought and right behavior are structurally similar. as peirce stated, “[t]he phenomena of reasoning are, in their general features, parallel to those of moral conduct. for reasoning is essentially thought that is under self-control, just as moral conduct is conduct under self-control” ( . ; for more on self-control in peirce’s thought, see petry ). that conduct is governed by some ideal is not sufficient to make it right conduct. ends or ideals may be desired without being desirable. right conduct pursues what is desirable in itself without any ulterior motive. we must necessar- ily postulate an ultimate end if ethical conduct is possible ( . – ). ethics is, therefore, dependent upon the deeper level of esthetics. as peirce understands it, esthetics is not the evaluation of artworks but the deliberate formation of taste, the creation of habits of feeling that will govern future conduct. esthetics concerns the truly desirable, that ultimate aim that ought to serve as the ideal of our conduct ( . , . ). truly right conduct, either logical or ethical, is that which seeks the right end, the ultimate aim. this aim must be general in nature, unified, pure, and precise. it is real, not a postulate. it is self-satisfied, but it is also not static. it is inherently good, not instrumentally so ( . – ). for peirce, only one thing satisfies these conditions, and it is reason. reason is perfect in itself, but it is inexhaustible as an object of desire and devotion. the scientific endeavor is the perpetual attempt to think and act according to the real while never fully succeeding. peirce clearly understands this as a sacred task, an act of devotion: the creation of the universe, which did not take place during a certain very busy week, in the year bc, but is going on today and will never be done, is the very development of reason. the one thing whose admirableness is not due to an ulterior reason is reason itself comprehended in all its fullness, so far as we can comprehend it. ( . ) elsewhere, he writes that nature is “something great, and beautiful, and sacred, and eternal, and real,” and that the scientist’s aim is “simply and solely knowledge of god’s truth” ( . , . ). a logical and ethical life is then a life spent in service of reason and the real. we may now see why peirce believed that science required its own ideal and its own way of life. “the ideal of conduct will be to execute our little function in the operation of the creation by giving a hand toward rendering the world more reasonable whenever, as the slang is, it is ‘up to us’ to do so” ( . ). ethical conduct contributes to the growth of concrete reasonableness, of the self-control and self-reflection that peirce, as an objective idealist, saw as the development of god. this process requires the development of habits of self-control in general, and greater control of one’s reasoning in particular. dedication to the ultimate aim of reason entails moral conduct and scientific inquiry. therefore, despite peirce’s claim that the scientific way of life must refrain from thick commitments, such a life does place requirements on the character of inquiry and virtue the scientist. peirce has sketched an image of human flourishing. for a person to be committed to reason, he/she must show love and devotion to the truth, or what peirce calls as “the true scientific eros” ( . ; see also reilly, , – ). inquiry requires dedication to one’s highest ideals in the face of the temptation to accept easy and comforting beliefs. he/she must be industrious and dedicated to the work before him/her, despite the fact that the project will not be completed in his/her lifetime ( . , . ). this way of life requires selflessness, courage, and humility. the scientist “must prefer the truth to his own interest and well- being and not merely to his bread and butter, and to his vanity too” ( . ). lastly, there must be a social sentiment, an overcoming of our natural feeling of self-love through the unification of sentiment into communities of feeling and interpretation ( . , . – ). this all culminates in peirce’s panegyric to the methods of scientific inquiry: the genius of a man’s logical method should be loved and reverenced as his bride, whom he has chosen from all the world. . . . and having made it, he will work and fight for her, and will not complain that there are blows to take, hoping that there may be as many and as hard to give, and will strive to be the worthy knight and champion of her in the blaze of whose splendors he draws his inspiration and his courage. ( . ) of course, to anyone familiar with his many indiscretions—ranging from physical abuse of his wife and employees, to personal abuse of drugs and alcohol, and abuse of federal funds as a member of the u.s. oceanic and geodetic survey— peirce’s praise of self-control and devotion to one’s beloved might seem hollow, hypocritical, or just laughable (brent ). but setting the character of the man aside, it is clear that his placement of scientific inquiry within a broader meta- physic entailed a further account of the virtuous life. . democracy and the virtues of inquiry these substantive arguments for particular virtues are rooted in an image of reality and humanity’s place in it. for dewey, it is a novel, natural world that requires openness, respectfulness, and courage. for peirce, we must develop self-control and a love of truth so that we might be in touch with reason and, in part, bring it about. these are the sort of virtues one might want to see inculcated in children if we are to have a sustainable liberal-democratic culture. they must be open to the experience of others, yet willing to defend their beliefs in a process of political debate. they must be tolerant of those with whom they disagree, yet demand that respect be returned. from a substantive position, the argument would run as follows: because this thick description of the true and the good is correct, liberal-democratic virtues instrumental to that end are true and good as well. therefore, parents should be required to financially support democratic education, and children should be required to receive it, under threat of sanction. in this section, i argue that dewey’s phillip deen and peirce’s grounding of virtue in thick metaphysical accounts of experience, novelty, the real and/or concrete reasonableness are unhelpful within the context of liberal public reason and within deliberation about what should be done with the force of law. i then justify democratic virtue education by turning to another, equally important aspect of their thought—their pragmatist account of inquiry. . critiquing the teleological pragmatist approach now that we have a sketch of two pragmatist, substantive attempts to ground democratic virtues in a thick account of reality and humanity’s place in it, we may turn to the problem of liberal pluralism. stated simply, pluralism raises a problem because, in liberal democracies, (i) the use of coercion must be justified to the citizens affected, but (ii) the freedom afforded to democratic citizens ensures that they will not agree on one comprehensive metaphysical or moral doctrine. there- fore, (iii) justifying coercion on the basis of such a thick account is certainly impractical and likely unjust, as it will exclude many reasonable ways of life. appeal to a substantive metaphysical and moral doctrine is problematic not because people are too stubborn or unreasonable to accept its truth. this is not a failure of understanding, although such failures do explain many political dis- agreements. if that were true, then we would have to assert that those who disagree are irrational, ignorant, or insincere. but this does not seem to represent many cases where reasonable people disagree. rather, disagreement over the good is a result of freedom. in any sufficiently free and large society, there will be many incompatible yet reasonable doctrines (and an endless number of unreasonable ones). given that no one doctrine will be shared by the citizenry, yet all will be affected by the law, the law’s authority cannot rest on any particular image of the universe and the good (rawls ). as it bears on the discussion at hand, the result is that we may not coerce individuals into being liberal-democratic citizens on the basis of dewey’s and peirce’s arguments (at least, not on the substantive accounts presented so far). whether i, as a partisan deweyan, find his account true or argument persuasive is beside the point. as a matter of prudence, it will neither persuade those of other moral and metaphysical traditions nor secure their participation in our project of social cooperation. as a matter of justice, i must accept that others may be wrong, yet still entitled to their beliefs. i must accept that others reasonably disagree, even with the truth—assuming, of course, that i am in possession of the truth. it is not that we are forbidden from bringing moral commitments into the public sphere. rather, acceptance of a substantive doctrine must not be a precon- dition for acceptance of an argument. if an argument is brought into the public sphere, it must be adjudicable by means of public reason. a deweyan may bring his/her darwinian ontology into the public arena as part of his/her argument that we must educate for democratic citizenship, but that ontology must be open to debate. he/she may try to persuade fellow citizens to see the truth of the darwin- ian ontology but, ultimately, there are limits. not everyone will be persuaded. out inquiry and virtue of respect for the experience of others and the requirements of political prudence, the pragmatist must abide by certain argumentative constraints, just as they would ask the fundamentalist to do, despite the gulf between pragmatist fallibilism and fundamentalism. . virtues as conditions of inquiry within recent pragmatist political philosophy, there have been attempts to find a way to justify democracy without relying on a shared image of the good life. an alternative to grounding justification of democratic virtues in a thick account of experience and human nature is to ground it in inquiry. robert talisse and cheryl misak have made such an attempt. they argue that there are general norms of inquiry that provide a firm foundation for basic principles that bind even the harshest critic of democracy, yet do not assume a prior commitment to some ideal. their argument is that the mere assertion of belief commits the person to a process of inclusive, public justification (misak ; talisse ; see also westbrook , anderson , and putnam , – ). misak argues that “the requirements of genuine belief show that we must, broadly speaking, be democratic inquirers” ( , ). although my argument bears a resemblance to talisse and misak’s, there are significant differences. first, they appeal to the conditions of belief assertion, while i emphasize the conditions of successful experimental inquiry. their argu- ment and mine share a quasi-transcendental character. as the “quasi” indicates, neither argument is a traditional deduction of the necessary conditions for the possibility of some given phenomena. misak explicitly rejects the transcendental- pragmatic argument by habermas and apel (misak , – ). rather, demo- cratic inquiry is a necessary condition for genuine belief in the way that oxygen is a necessary condition for combustion. oxygen is not derived conceptually from combustion; however, it is practically necessary. misak and talisse argue that democratic inquiry is necessary for genuine belief, as refusal to participate in a democratic community of inquiry undercuts one’s claim to be reasonable. i argue that liberal-democratic character is a necessary condition for good inquiry; further, i do not appeal to the conditions of genuine belief, but of successful problem-solving. these conditions have been revealed during the long history of attempts to solve problems. second, as noted, i extend the quasi-transcendental argument to considerations of character. if we are to inquire well, then we must have a certain character. in particular, liberal-democratic virtues are the necessary conditions for the possibility of good inquiry. and the need for good inquiry is inescapable. therefore, we are justified in coercing civic education upon the basis of these reasons. let us turn to the argument itself. for both peirce and dewey, inquiry is the attempt to reconstruct a formerly habitual and fruitful transaction with the world. our ruts are usually satisfying and lead where we expect. however, particular habits sometimes break down as we encounter new phenomena, or our standard phillip deen actions bring about new consequences. when our relationship to our environment is sufficiently out of sorts, we feel uneasy and compelled to investigate. inquiry is that process of instituting new habits through changing ourselves and our environments. once the concrete situation is corrected, which allows us to achieve a fruitful transaction with our world, inquiry ends. peirce describes this as the doubt–belief arc, while dewey describes it as the movement from a problematic situation to a reconstructed one. moral and political inquiry, specifically, addresses the conflict of competing goods. the goal is not to attain the good, but to harmonize the various goods that are already in play. for example, were it not for the preexistence of goods, such as the welfare of children or the preservation of families and moral traditions, then there would be no need to deliberate about what school policy should be. such inquiry attempts to resolve a situation already shot through with competing goods into one where goods are articulated, ranked, and harmonized. to get from the problematic to the reconstructed, we must develop certain techniques to “fix” belief. peirce dismisses many possible methods. one may tenaciously hold to the standard way of behaving, rely on the authority of others to tell them what is best to do, or submit them to the test of abstract reason by asking themselves what seems most reasonable. however, in trying each, we find ourselves inevitably running into brute facts. the world resists our desires, we find our beliefs are not shared by others, and so on. only the scientific method of inquiry allows problems to be consistently resolved, including problems of value. it is the only method that tests beliefs against the world. beliefs, both old and new, become the hypotheses that are proposed to alleviate the original uneasy breakdown of habit. they are put into practice and their consequences evaluated. if they work, then the unease goes away and we have a new equilibrium. if not, then we learn from the failure and propose a new hypothesis. scientific inquiry then has certain stages: there is felt unease, an attempt to articulate the problem, proposal of hypotheses, testing them, and finally evaluation for future action (lw : passim, especially chapter ). scientific inquiry not only tests against the natural environment, but also against the social one. hypotheses are tested in public, and others may repeat the experiments, evaluate the results of previous tests, or add their wisdom by proposing their own solutions to the problems at hand. the best inquiry is con- ducted by a community of inquirers, and the larger that community, the more likely a resolution of the problem—assuming they are committed both to resolv- ing the problem and conduct themselves according to the best methods currently known. we need not say with peirce that the true is that which the community of inquiry is “fated” to believe, given infinite time and an infinite number of inquirers (cp : ). however, we may say that when certain conditions are met, good inquiry follows and problems are more likely to be resolved. the long history of humanity’s attempts to overcome problems both big and small has shown that some conditions are more conducive to trustworthy habits and beliefs than others. inquiry and virtue improvement of inquiry then requires determination of those conditions under which inquiry is more likely to succeed. regarding method, science has shown itself more valuable than imploring the gods or unquestioning faith in community authorities. these conditions also include objective, institutional conditions, such as freedom of expression and the press so that information might be more easily exchanged. free institutions of learning, such as universities or publicly funded research centers, lessen political and economic distortion of experimentation. the lessening of violence in political decision making produces outcomes that are more likely to harmonize competing goods. but they also include certain subjective conditions. that is, improved inquiry requires that those involved possess certain virtues, such as openness, respect, and courage (see bohman , passim but especially p. ). consider the basic arc of inquiry just outlined. to engage in this process, the inquirer must exhibit certain qualities of character. first, he/she must be open to new information. unwillingness to acknowledge novel situations or facts results in the inability either to identify sources of unease or to discover sources of new hypotheses or evidence. as peirce and other philosophers of science have noted, scientific inquiry is a creative enterprise, requiring flights of imagination. to use more technical language, in addition to induction and deduction, inquiry requires abduction, or inference to the best explanation, or “educated guessing.” following openness is the willingness to experiment, to test beliefs and proposals against the natural world and the experience of others. willingness to experiment entails other virtues, including, on the one hand, perseverance and courage, and on the other, moderation and willingness to reflect. perseverance and courage are required by those who must engage in the arduous task of forming tests and gathering evidence, and face the recriminations of those who question received authorities or tenaciously held beliefs. moderation and reflection are similarly required because the inquirers must limit their regard for their own beliefs if they are to question or test them. these are virtues of obedience, submission, or scientific objectivity. as inquiry is a social process of public experimentation, inquirers must be open to the broadest acceptable range of input. the community of inquiry must be maximally inclusive if we are to maximize the chances of success. to exclude participation on the basis of arbitrary considerations like gender, class, race, or nationality would be to artificially limit sources for hypotheses and experimental feedback. in other words, inquiry requires toleration and mutual respect, whenever reasonable. this is not to say that it is infinitely tolerant. the pragmatist model finds those who are unwilling to engage in the process of proposing, critiquing, and defending hypotheses unreasonable. but limits on participation, toleration, and respect are themselves determined only in the course of inquiry, so there must always be a way to question them in the course of future inquiries. inquiry then entails a certain character, and what qualifies as virtue depends on its value to inquiry. put simply, “[a] character trait (habit, disposition or human potency) is a virtue if it facilitates intelligent problem solving. a character trait is phillip deen a vice if it hinders intelligent problem solving” (olson , ). or, to cite james gouinlock at length, [i]n dewey’s ideal, experimental inquiry and democratic behavior become fused. the nature of their combination can perhaps be best suggested by thinking of them as a union of certain moral and intellectual virtues . . . the virtues include the willingness to question, investigate, and learn; a determination to search for clarity in discourse and evidence in argument. there is also a readiness to hear and respect the views of others, to consider alternatives thoroughly and impartially, and to communicate in a like manner in turn. one is not irrevocably committed to antecedent conditions but is ready to qualify or change his views as a consequence of inquiry and communication. there is an urgency to persist in shared discourse in the direction of agreement. these virtues embrace novelty, innovation, growth, regard for the concerns of others, and scientific discipline. (gouinlock , ; see also dewey lw : , , and macgilvray , ) hence, the virtues that make for good inquiry are also many of those that typify liberal-democratic citizens. dewey long held that scientific and democratic prac- tice was similar in structure: “i would not claim that any existing democracy has ever made complete or adequate use of scientific method in deciding upon its policies. but freedom of inquiry, toleration of diverse views, freedom of commu- nication, the distribution of what is found out to every individual as the ultimate intellectual consumer, are involved in the democratic as in the scientific method” (lw : ). to simplify the argument, inquiry is the process by which doubt or the problematic is converted into belief or the reconstructed. because problems are unavoidable, inquiry is too. although doubts may be resolved in various ways, pragmatists believe that experience has shown that scientific inquiry is by far the most successful. such inquiry requires the virtues of openness, willingness to experiment, perseverance, courage, moderation, reflection, sociality, inclusive- ness, toleration, and mutual respect. these are also many of the virtues required by a liberal-democratic society. because the need for successful inquiry is inevitable, virtues required for such inquiry are necessary as well, and a society may justi- fiably cultivate these virtues in its citizens—not because they are the substantive values of a liberal-democratic community, but because they are the virtues of inquiry. of course, history has shown that individual scientists, even brilliant ones, may be vicious people. nevertheless, the community of inquiry must exhibit these virtues as a whole. as regards educational policy, the result is that if we are to produce good inquirers, they must be educated in certain (liberal-democratic) virtues. this is particularly true, given the problems that the community is trying to solve—those that are felt widely, and require cooperation and mutual concern. . method, not end this approach from the conditions of inquiry differs importantly from the first pragmatist way to ground virtue. admittedly, by linking scientific inquiry with inquiry and virtue character, this argument also recalls peirce’s substantive one. peirce went so far as to challenge the reader to identify any great man of truth who was a criminal, claiming that they are mutually exclusive ( . ). critically, however, the con- nection to the objective idealist metaphysic has been dropped. these virtues are not valuable because they are instrumental to the end of concrete reasonableness or the self-organization of a loving god. second, this differs from the substantive deweyan account above in that they are the virtues of a method, not of any particular end, nor is this argument based on a substantive and prior commitment to liberal ideals. it does not argue that citizens must be educated for tolerance because it maximizes the greatest utility (mill), we are endowed with natural rights (locke and jefferson), or we are rationally autonomous beings (kant). rather, the justification is based on the necessity of inquiry in light of the permanent need to address problems. further, in distinction from epistemic justifications of democracy as the best form of government because it is the only form that provides the epistemic conditions for truth, the argument from inquiry does not assert the value of truth, and then claim that inquiry and democracy are valuable because they serve that end (estlund ). democracy is not justified because it produces truth; that is, democracy is not valuable simply because it is a means to something inherently valuable outside of it. inquiry is not motivated by anything other than the attempt to reconstruct a situation, to solve a felt problem. truth is internal to this process. for peirce, truth is what arrives at the end of an endless, well-conducted inquiry, while for dewey, it is (notoriously) “warranted assertibility.” the argument from the conditions of inquiry is then non-teleological, except in the general sense that problem-solving has a generic telos: to resolve felt problems. democratic virtues are conditions for the possibility of inquiry, not the true or good. they govern the attempt to understand one’s present ideals, to resolve tensions between them, and to formulate new ideals in response to the problems that arise in the course of living according to one’s ideals. as inquiry is the result of problematic situations, the only way to avoid it is to never encounter a tension between one’s ideals and the world, or between the ideals themselves. such a life is simply unavoidable, given limited resources, the existence of others, and a world that frustrates our desires. most importantly, for the problem at hand—the tension between the liberal image of the good life and its commitment to toleration—there is no imposition of an ideal life on illiberal citizens. the telos is provided from within the illiberal community as it attempts to resolve internal tensions and conflicts with the larger world. . educating for democratic virtue we now have an alternative pragmatist-liberal argument for the superiority of certain virtues central to the democratic character: good inquiry requires certain liberal-democratic virtues, and the need for good inquiry is inescapable. further- phillip deen more, this argument is less vulnerable to the liberal objection that this argument appeals to prior belief in a liberal moral and metaphysical framework, and therefore is disrespectful to illiberal citizens. one does not have to be a liberal to accept this argument. one must only value inquiry. in principle, we may reasonably expect others to be persuaded by it, even if there is no prior conversion to liberalism. let us return to the case of civic education. if the argument above is cogent, then liberals have a compelling argument on behalf of certain virtues. this argu- ment can be used in service of civic education in public schools, despite the fact that attendance by children and taxation of parents is backed by force of state sanction. schools must cultivate liberal-democratic virtues of toleration, openness to novelty, respect, courage, and honesty because these virtues are required for good inquiry and good inquiry is necessary. . the case of civic education of course, not every parent wants their child to be educated in these virtues. what strikes the liberal as obviously virtuous may strike another as equally obviously vicious. the tolerance and mutual respect valued by the liberal may be seen as the celebration of depravation or sin. given that, from within the liberal framework, the state has a prima facie duty to respect the values of such families, churches, and other moral communities, this poses a problem for those liberals who would require liberal civic education in public schools. consistent with the liberal value of toleration and the practical necessity of maintaining social stability, the state has affirmed a right to be exempt from compulsory attendance in public schools, either entirely or from certain parts of the curriculum. there are many bases for exemption, but i focus here on the first amendment right to the free exercise of religion. the pivotal court decision in this matter is the well-known wisconsin v. yoder (although michigan v. dejonge and duro v. district attorney also address the matter). in the yoder case, members of the old order amish religion and the conservative amish mennonite church objected to a wisconsin law mandating attendance in public schools until the age of sixteen, and petitioned to remove their children at the end of eighth grade on the grounds that compulsory attendance violated their religious beliefs. their amish faith required a separation from the “evil world” and a commitment to a tightly knit community threatened by supposed virtues, such as openness, toleration, and a willingness to experiment. rather, amish youth must develop the virtues of purity, piety, and discipline. the supreme court unanimously ruled that the right to the free exercise of religion outweighed any state interest in compelling chil- dren to attend another two years of public education (wisconsin v. yoder, us [ ]). interestingly, in his dissent, justice william douglas made a classic liberal argument, contending that the child’s right to determine their own way of life was being overlooked: “he may want to be a pianist or an astronaut or an oceanog- rapher. to do so he will have to break from the amish tradition. it is the future of inquiry and virtue the students, not the future of the parents, that is imperiled by today’s decision. if a parent keeps his child out of school beyond the grade school, then the child will be forever barred from entry into the new and amazing world of diversity that we have today” (ibid.). however, as i have tried to argue, such a substantive appeal to the value of autonomy is problematic if pluralism is to be taken seriously. according to the argument i have put forth, the amish children from the yoder case would be compelled to receive education in those liberal-democratic virtues necessary for inquiry, that is, to be provided a character that will allow them to test their beliefs against the “evil world.” even from a liberal position, such coercion is unnerving. however, it is, in principle, justifiable. the amish community has historically had to come to terms with the modern, secular world. from within, there have always been tensions between those who would be more open and those who would not, between those who interpret their tradition more strictly and those who have not. particularly in the case of technological appropriation, the amish have a long history of slowly testing the effect of new technologies against communal and traditional values (kelly , chapter ). the inescapability of the need for inquiry entails children’s inescapable need for certain virtues. this need is not imposed from without, but arises from within. rather than present a harmless supplement to traditional beliefs, this argument concludes that public reasoning, and the democratic virtues it requires, must be taught to children even when it does pose a threat to traditional pieties. for simply trying to live according to these pieties in a conflicted and frustrating natural and social environment entails a commitment to liberal-democratic virtues. . objecting to inquiry given this conclusion, one might reasonably object that the shift from sub- stantive arguments to arguments grounded in the structure of inquiry does not avoid the problem of pluralism. rather, it obscures it by a false appeal to universal norms that are, in fact, surreptitiously partisan. the argument from inquiry circumvents problematic moral virtues with inquiry-based ones, but the latter are also hotly contested. the argument from inquiry assumes that illiberal citizens still accept public inquiry. i believe this describes the majority of conservative american citizens. for example, parents may believe that tolerance of homosexuality should not be taught to children because promotion of tolerance of homosexuality has a corro- sive effect on the health of the child and on basic institutions like the family and their church. the sheer fact that their position is illiberal does not make them unreasonable (as some have charged of rawls’s account of “reasonableness”). their position is reasonable because they believe that this is a publicly defensible position and that the facts will bear it out. they are willing to submit their beliefs to a process of public inquiry and experimentation. in cases such as this, the justification of coerced civic education has force. phillip deen however, some are put off when dewey claims that there is “but one method for ascertaining fact and truth—that conveyed by the word ‘scientific’ in its most general sense,” or that “democracy thus appears as the means by which the revelation of truth is carried on” (lw : and ew : ). commitment to a public and discursive model of truth is to reject peirce’s other means of fixing belief—the methods of tenacity, authority, and a priori reasoning. the pragmatist assumes an image of truth and how it is attained, a deep metaphysical commitment that other citizens may not share and that results in a civic totalism with undesirable con- sequences for religious communities (macedo , – ). rather than accept the idea that truth arises through the public testing of hypotheses of reasons and evidence—that is, that there is an essential link between truth and public experimentation—fundamentalist communities argue that truth is given by means of divine revelation, private intuition, or sacred texts and traditions. they may further argue that the use of public reason exposes one’s sacred beliefs to corruption by those who have not received the revelation. recall the case mozert v. hawkins, in which parents claimed the right to reject any public education that exposed their children to any beliefs other than their own, as this might lead children to respect other faiths and to question their own (mozert v. hawkins ). if others are corrupt, then we might be better off not exposing truths to criticism. in other words, they may appeal to the methods of “fixation” rejected by peirce: tenacity, authority, and appeal to subjective reason. the criticism is ultimately that, while children and parents are inevitably compelled to inquire, they are not compelled to engage in pragmatist-scientific inquiry. therefore, they are not compelled to cultivate liberal-democratic virtues. robert talisse has argued that, while reasonable disagreement may exist more than substantive moral and metaphysical beliefs, it may not in matters of seeking truth. recall his quasi-transcendental argument that mere assertion of beliefs as true entails a process of presenting and evaluating reasons. there are implicit norms in this process. anyone who asserts “i believe that p, but i have never subjected p to the type of scrutiny that would allow counterevidence to challenges to p to emerge, and have never attended to the reasons that others have for holding beliefs opposed to p” has undercut their own claim to be reasonable (talisse , ). one could tweak this to hold that those who assert “i believe p, but i am unwilling to test p experimentally” would have similarly given up on the norms implicit to asserting their beliefs. upon self-assessment, it signals an epistemic failure. however, as noted in section . , the argument i present here is not the same as talisse’s. i do not appeal to the conditions for the possibility of belief assertion but the conditions of good inquiry. therefore, this response is inconsistent with the argument at hand. in addition, although his argument may conceivably appeal to our imagined fundamentalist, it is unlikely to do so. tal- isse’s argument may be sufficient to convince a liberal-democrat that the funda- mentalist has sacrificed his/her epistemic authority, but the fundamentalist rejects the assumption connecting truth and public argumentation. if the goal is to inquiry and virtue persuade the fundamentalist to send his/her children to public schools, then it is unlikely to succeed. a more modest alternative is to concede that if not everyone assents to a necessary connection between truth and public argumentation, then we must appeal to an empirical connection. in that case, the pragmatist would have to concede that the fundamentalist may reject his/her account of truth and its internal connection to public inquiry, but in doing so, the fundamentalist is also rejecting the scientific and democratic revolutions. in that vein, amy gutmann argues that those who reject the teaching of evolution in public schools reject not only a particular scientific theory, but also the form of scientific reasoning itself. “the religions that reject evolution as a valid scientific theory also reject the secular standards of reasoning that make evolution clearly superior as a theory to creationism” (gutmann , ). they reject the standards of cogent reasoning that form the core of scientific method. in this case, one might point out the great successes of public experimen- tation and the corresponding decline of the methods of revelation and authority in science and public life, and argue that the open exchange of reasons has been shown a far superior method of solving problems. the appeal is to the history of problem- solving and to the benefits of scientific inquiry they currently enjoy. given the historical successes of experimental inquiry, we may argue that those who enjoy its benefits while denying it a place in education are being unreasonable. in the extreme case, believers may reject the modern world remade by public reason as corrupt. they may see scientific-technological and democratic inquiry, and the “progress” that follows, as a fall from a simpler, more divine time. reasonable agreement may not be possible. at that point, there would be the need for bargaining, voting, or other nondiscursive means of decision making, even as we hold that those who are unwilling to engage in experimental inquiry are being unreasonable. but, even if the argument from inquiry is not universally persuasive, it is at least less controversial than more substantive ones. while deep moral disagreement is easy to find, there is certainly less dispute over the assertions that coercive acts must be open to public scrutiny, that hypotheses must be tested, or that problems are best resolved by those who exhibit certain qualities of character (even if we must admit the existence of extreme epistemic positions that reject these conclusions). consistent with pragmatism’s commitment to meliorism, the argument from inquiry offers hope for greater agreement, even if universal agree- ment always evades us. the claim here is modest—that arguments based in an account of inquiry are on less controversial ground, both prudentially and norma- tively. given the need to secure broad consensus if virtue education is to be instituted, and given the further ethical demand (within the liberal model) to respect, when possible, all comprehensive doctrines affected by the policy, our reasons should strive to be as “public” as possible. nevertheless, the inquiry-based, pragmatist account allows the liberal democrat to argue that virtues necessary for inquiry are not open to reasonable disagreement. that is, the underlying necessity of inquiry implies that even anti-democrats admit that liberal-democratic virtues are required of all. parents phillip deen are not previously obliged to accept the ideal of consciously reproducing a liberal society. rather, they need only admit that next generation must be able to inquire. therefore, the state may legitimately use coercion to cultivate a genera- tion of children who are able to test their beliefs. from a comprehensive deweyan position, this is so because the world is both precarious and stable, while for the comprehensive peircean, it is a requirement of the pursuit of truth. but from the inquiry-based pragmatist position, it is entailed by the need for any moral tradition to engage objections both from within and without, or to deal with new problems. even the fundamentalist parent must acknowledge external tensions with the modern, secularizing world, and internal tensions between those who interpret their tradition. both call for inquiry. despite the difficulties that arise from liber- alism’s central commitment to toleration, there is at least one impartial reason for compulsory civic education. notes in this, i am engaged in a project similar to that of eric macgilvray and robert talisse (macgilvray ; talisse ). for a nice summary of the attempt by pragmatists to develop post-rawlsian justifications of political liberalism, see festenstein ( ). i have ignored the contributions of other pragmatists, specifically james and mead. i have done this only for reasons of economy. stephen macedo has offered his own defense of liberal virtues, including sympathy, self-reflection, willingness to experiment, openness, self-control, altruism, and appreciation of social inheritance (macedo , – ). see also galston ( ) and kloppenberg ( ). following standard practice, all further references to dewey’s works will indicate the volume of the early works (ew), middle works (mw), or late works (lw) of the collected works of john dewey, followed by the page or chapter number. sadly, conscientiousness and openness are not easily joined. a study of the geographic distribution of the “big five” personality traits in the united states found that where agreeableness and consci- entiousness are more prominent—the southeast—openness is lacking. the reverse is found where openness is more prominent—the northeast and west coast metropolitan areas (rentfrow, gosling, and potter ), graphically represented at http://mapscroll.blogspot.com/ / /new-and- improved-geography-of.html. dewey elsewhere defines democracy as the “belief in the ability of human experience to generate the aims and methods by which further experience will grow in ordered richness” (lw : ). a complementary deweyan argument is made by juan carlos mougán rivero that social cooperation, as a necessary element of human flourishing, grounds other democratic virtues (mougán rivero ). this section is indebted to hookway , – ; sheriff , – ; reilly , – ; and particularly mullin , – . following standard practice, all further references to peirce’s works will indicate the volume and paragraph number of the collected papers. for more on peirce’s substantive moral and metaphysical commitments, see potter ( ) and mayorga ( ). despite presenting an excellent account of dewey’s moral theory, pappas fails to address the real force of the pluralist objection ( , – ). he sees disagreement as the result of dogged partiality or stubbornness rather than as an outcome of free reason. disagreement is presented as the result of irrationality, despite pappas’ insight that, for dewey, tension and conflict are a fundamental quality of moral life. inquiry and virtue admittedly, most deweyans do not believe that the rawlsian problem of reasonable pluralism poses a problem for a pragmatist justification of democracy. in particular, they have objected to robert talisse’s claim that pluralism requires that we abandon dewey entirely for a peircean epistemic approach (talisse ). because of limited space, i cannot attempt to defend the rawlsian objection here. however, despite sharing his belief that rawls’s pluralist problem must be taken seriously, i should note that i disagree with talisse’s conclusion: both dewey and peirce have much to offer a pragmatist philosophy of democracy, and it need not be strictly epistemic in form (deen ). the contention that scientific inquiry is the best method to resolve not only matters of fact but also of value is a controversial one. central to pragmatist moral philosophy are a rejection of the fact-value split and a call to extend experimental methods to morals. defense of these central beliefs would require far more space than this article allows, as it spans much of dewey’s vast corpus, not to mention the work of other pragmatists. however, those interested should consult dewey’s ethics (lw ), theory of valuation (lw : – ), and “logic of judgments of practice” (mw : – ). the inquiry-based virtues discussed here do not encompass all democratic virtues. as michael walzer and others have noted, democratic politics is not the same as a democratic deliberation. it requires virtues appropriate to political mobilization and realpolitik (walzer , chap. ). however, they are among the central liberal-democratic virtues. this argument from inquiry differs from stephen macedo’s position in liberal virtues that liberal virtues are entailed by the process of public justification. although i find that argument compel- ling, i differ in an emphasis on inquiry, which does not run the risk of emphasizing discourse to the detriment of experiment (macedo ). the federal appeals court ultimately ruled that the parents did not have legitimate claim. a variant of this argument is that, while the method of experiment and inquiry is appropriate for natural scientific facts, it is not proper for moral values. however, as already pointed out in footnote , the pragmatist tradition has a long history of rejecting the fact–value dichotomy and of calling for an experimental approach to values. references anderson, elizabeth. . “the epistemology of democracy.” episteme : – . bohman, james. . “ethics as moral inquiry: dewey and the moral psychology of social reform.” in cambridge companion to dewey, ed. molly cochran, – . cambridge: cambridge university press. brent, joseph. . charles sanders peirce: a life. bloomington: indiana university press. deen, phillip. . “a call for inclusion in the pragmatic justification of democracy.” contemporary pragmatism : – . dewey, john. – . the collected works of john dewey. volumes. carbondale: southern illinois university press. estlund, david. . democratic authority: a philosophical framework. princeton, nj: princeton university press. festenstein, matthew. . “pragmatism, inquiry and political liberalism.” contemporary political theory : – . galston, william. . liberal purposes: goods, virtues and diversity in the liberal state. new york: cambridge university press. garrison, jim. . dewey and eros: wisdom and desire in the art of teaching. charlotte, nc: information age. gouinlock, james. . “what is the legacy of instrumentalism? rorty’s interpretation of dewey.” in rorty and pragmatism: the philosopher responds to his critics, ed. herman saatkamp, jr., – . nashville, tn: vanderbilt university press. gutmann, amy. . democratic education. princeton, nj: princeton university press. phillip deen hookway, christopher. . peirce. london: routledge and kegan paul. kelly, kevin. . what technology wants. new york: viking press. kloppenberg, james. . the virtues of liberalism. new york: oxford university press. macgilvray, eric. . “experience as experiment: some consequences of pragmatism for demo- cratic theory.” american journal of political science : – . macgilvray, eric. . reconstructing public reason. cambridge, ma: harvard university press. macedo, stephen. . liberal virtues: citizenship, virtue and community in liberal constitution- alism. oxford: clarendon press. ——. . diversity and distrust: civic education in a multicultural democracy. cambridge, ma: harvard university press. mayorga, rosa maria. . “on talisse’s ‘peirceanist’ theory.” transactions of the charles s. peirce society : – . misak, cheryl. . truth, politics, morality: pragmatism and deliberation. new york: routledge. mougán rivero, juan carlos. . “social cooperation as civic virtue.” in self and society: central european pragmatist forum, ed. alexander kremer and john ryder, vol. , – . amsterdam: rodopi. mozert v. hawkins county public schools. . f.supp. ( ) united states district court, eastern district of tennessee, northeastern division (march , ). mullin, richard. . the soul of classical american philosophy: the ethical and spiritual insights of william james, josiah royce, and charles sanders peirce. albany: state university of new york press. olson, philip. . dewey’s virtues. paper presented at the meeting of the society for the advancement of american philosophy, march . pappas, gregory fernando. . “open-mindedness and courage: complementary virtues of prag- matism.” transactions of the charles s. peirce society : – . ——. . john dewey’s ethics: democracy as experience. bloomington: indiana university press. peirce, charles s. – . collected papers. cambridge, ma: harvard university press. petry, edward s., jr. . “the origin and development of peirce’s concept of self-control.” transactions of the charles s. peirce society : – . potter, vincent. . charles s. peirce on norms and ideals. amherst: university of massachusetts press. putnam, hilary. . renewing philosophy. cambridge, ma: harvard university press. rawls, john. . political liberalism. new york: columbia university press. reilly, francis e. . charles peirce’s theory of scientific method. new york: fordham university press. rentfrow, peter j., gosling, samuel d., and potter, jeff. . “a theory of the emergence, persis- tence, and expression of geographic variation in psychological characteristics.” perspectives on psychological science : – . rice, suzanne. . “dewey’s conception of ‘virtue’ and its educational implications.” in philoso- phy of education, ed. frank margonis, – . urbana, il: philosophy of education society. ryan, alan. . john dewey and the high tide of american liberalism. new york: w. w. norton. sheriff, john k. . charles peirce’s guess at the riddle: grounds for human significance. bloomington: indiana university press. talisse, robert. . a pragmatist philosophy of democracy. new york: routledge. ——. . “reply to festenstein.” contemporary political theory ( ): – . teehan, john. . “character, integrity and dewey’s virtue ethics.” transactions of the charles s. peirce society : – . walzer, michael. . politics and passion: toward a more egalitarian liberalism. new haven, ct: yale university press. westbrook, robert. . democratic hope: pragmatism and the politics of truth. ithaca, ny: cornell university press. inquiry and virtue wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ uc irvine western journal of emergency medicine: integrating emergency care with population health title characterization of regional poison center utilization through geospatial mapping permalink https://escholarship.org/uc/item/ d n hf journal western journal of emergency medicine: integrating emergency care with population health, ( ) issn - x authors olives, travis d. westgard, bjorn c. steinberg, lila w. et al. publication date doi . /westjem. . . license https://creativecommons.org/licenses/by/ . / . peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ d n hf https://escholarship.org/uc/item/ d n hf#author https://creativecommons.org/licenses/https://creativecommons.org/licenses/by/ . // . https://escholarship.org http://www.cdlib.org/ volume , no. : november western journal of emergency medicine original research characterization of regional poison center utilization through geospatial mapping travis d. olives, md, mph, med*†‡ bjorn westgard, md†‡§ lila w. steinberg, ba*†‡ jon b. cole, md*†‡ section editor: jeffrey suchard, md submission history: submitted april , ; revision received july , ; accepted july , electronically published october , full text available through open access at http://escholarship.org/uc/uciem_westjem doi: . /westjem. . . introduction poison centers (pc) accredited by the american association of poison control centers (aapcc) offer high- minnesota poison control system, minneapolis, minnesota hennepin healthcare, department of emergency medicine, minneapolis, minnesota university of minnesota medical school, department of emergency medicine, minneapolis, minnesota regions hospital, department of emergency medicine, st. paul, minnesota * † ‡ § introduction: penetrance is the annual rate of human exposure calls per persons, a measure that historically describes poison center (pc) utilization. penetrance varies by sociodemographic characteristics and by geography. our goal in this study was to characterize the geospatial distribution of pc calls and describe the contribution of geospatial mapping to the understanding of pc utilization. methods: this was a single-center, retrospective study of closed, human, non-healthcare facility exposure calls to a regional pc over a five-year period. exposure substance, gender, age, and zone improvement plan (zip) code were geocoded to us census data (household income, educational attainment, age, primary language) and spatially apportioned to us census tracts, and then analyzed with linear regression. penetrance was geospatially mapped and qualitatively analyzed. results: from a total of , exposure calls during the study period, we identified , non-healthcare exposure calls. of those records, , included zip codes. after exclusions, we analyzed , records. penetrance ranged from . - . calls/ population/year (median . calls/ persons/year). regression revealed positive associations between >eighth- grade educational attainment (β = . , p = . ), non-hispanic black (β = . , p = . ) and american indian (β = . , p = . ) populations, suggesting that regions with higher proportions of these groups would display greater pc penetrance. variability explained by regression modelling was low (r = . ), as anticipated. geospatial mapping identified previously undocumented penetrance variability that was not evident in regression modeling. conclusion: pc calls vary substantially across sociodemographic strata. higher proportions of non-hispanic black or american indian residents and >eighth-grade educational attainment were associated with higher pc call penetrance. geospatial mapping identified novel variations in penetrance that were not identified by regression modelling. coupled with sociodemographic correlates, geospatial mapping may reveal disparities in pc access, identifying communities at which pc resources may be appropriately directed. although the use of penetrance to describe pc utilization has fallen away, it may yet provide an important measure of disparity in healthcare access when coupled with geospatial mapping. [west j emerg med. ; ( ) - .] quality information to callers seeking information and medical consultations for poisoned patients. they serve critical roles in real-time epidemiological surveillance of poison exposures western journal of emergency medicine volume , no. : november poison center utilization through geospatial mapping olives et al. population health research capsule what do we already know about this issue? poison centers (pc) serve large populations, but call penetrance may vary. what was the research question? we sought to characterize the geospatial distribution of calls to a single regional pc. what was the major finding of the study? calls to a pc vary substantially by sociodemographic strata, with significant geospatial variation in call origin, while regression modelling suggested greater penetrance in regions with higher proportions of non-hispanic black and american indian populations, and > th grade educational attainment, however variability explained by the model was low. how does this improve population health? statistical analyses describe patterns to regional pc callers, but spatially mapping call density may identify areas of low call penetrance to guide outreach efforts. and disease epidemics, and are a key component of our national health surveillance system. increased pc utilization has been associated with decreased emergency medical services utilization and unnecessary hospitalizations and with shortened hospital stays following exposure. , pc utilization has historically been assessed in terms of penetrance, defined as the annual number of calls per persons in a defined call area. , penetrance rises with changes in united states census bureau (uscb) population estimates and live birth rates, suggesting a correlation between population growth and childhood poison exposures. low pc penetrance is associated with increased healthcare utilization, particularly in children. , variations in penetrance have been attributed to seasonality, changing pediatric population proportions, limited awareness of pc services, and suspicion regarding pc cost and safety of personal information. social determinants of pc penetrance are less well-defined, although several racial (black and native american) and linguistic (low english proficiency and native spanish-speaking) characteristics are associated with lower pc utilization when compared to white and english- speaking populations. , , , the use of penetrance has been disputed over time, largely due to a perceived limited efficacy in assessing both pc efforts and outcomes. although the aapcc discontinued its use of penetrance as one of multiple methods to ascribe efficacy to individual pc outreach and promotion efforts in , it was done prior to the advent of easily accessible, geospatial mapping tools to provide a more refined data than at a county level, suggesting that penetrance may once again serve a purpose in identifying areas in which pcs are underused. the same variation in penetrance attributed to sociodemographic variables that led to its discontinuation as a metric for pc accreditation is suggestive of its value in further exploring predictors of pc utilization. few studies describe geographic penetrance at a level more granular than county-wide, despite intra-county variability in race, income, ethnicity, and other socioeconomic determinants of health. – focal exposure clusters may be localized within close proximity and overlooked within county-wide analyses. therefore, exposure patterns may be better understood when mapped geospatially. we hypothesized that highly granular geospatial mapping would reveal previously unidentified sociodemographic predictors of penetrance. the goal of the study was to characterize pc call penetrance by uscb tracts to better characterize variation across the pc catchment area. methods setting this was a retrospective study characterizing the group- level demographic characteristics and geospatial distribution of human exposure calls to a regional pc from locations other than healthcare facilities over a five-year period, from january , –december , . the minnesota poison control system covers a catchment area of nearly , square miles (greater than , square kilometers) and serves approximately . million people. it receives more than , calls annually; a majority of these originate in sites other than healthcare facilities. minnesota is a diverse state. smaller proportions of the population than the national average live in poverty ( . % vs . %) and fewer report non-english language use ( . % vs . %), but racial disparities are profound: higher proportions of blacks and native americans in our state live in poverty than nationally ( . % vs . % and . % vs . % in , respectively). , attainment of a bachelor’s degree or higher varies substantially across racial groups, from % among ojibwe to % among asian-indian residents. additionally, the state is home to the country’s largest somali population, second largest hmong population, third largest lao population, and fifth largest burmese population. one in seven liberians in the us resides in minnesota, while one in of ethiopian descent resides here. overall, . % of our service population possesses limited english proficiency. data characterizing statewide health literacy is limited, but suggest that up to one in five patients seeking emergency services possesses limited health literacy. , recent multi-patient toxicological exposures in minority communities , have highlighted the importance of pc penetrance in historically underserved populations. these outbreaks have been concentrated in small geographic areas volume , no. : november western journal of emergency medicine olives et al. poison center utilization through geospatial mapping incompletely captured by county-level geospatial mapping, suggesting a potential benefit to improved understanding of the spatial distribution of pc calls. despite known multi-patient exposures in language- and ethnic-minority communities, telephonic interpretive services are engaged on average fewer than five times per month in the pc, or less than . % of all calls. while it is plausible that linguistically under-represented and economically disadvantaged segments of the population experience fewer poisonings than others, such disparities raise suspicion for a lack of access to pc services. thus, following approval from the governing institutional review board, we queried the national poison data system (npds) for all closed human-exposure calls originating within minnesota (caller site/exposure site: own residence, other residence, workplace, school, restaurant / food service, public area, unknown, null). the npds maintains all call data generated by the nation’s pcs, with nearly continuous real- time database updates. because the goal of this study was to characterize non-healthcare penetrance across an entire pc call area, we excluded calls coded as originating from healthcare facilities or referencing exposures occurring in healthcare facilities, as their inclusion would have over-represented a small number of census tracts rather than accurately describe exposure distribution. calls originating outside of minnesota, calls without zone improvement plan (zip) codes, and calls for which zip code geocoding was not possible were excluded from analysis. no further exclusions were made. data analysis patient-level data including zip code, gender, age, exposure reason (intentional or unintentional), caller and exposure sites were electronically abstracted from npds. we then imported call records to microsoft excel (microsoft corporation, redmond, wa). pc data, including postal zip code, were geocoded to uscb tract data ( ) for household income, educational attainment, age, ethnicity and primary language. the resulting dataset was spatially apportioned to uscb tracts based on quantifiable spatial and population overlaps. arcgis . (esri, redlands ca) was then used to generate heat maps defining the penetrance of callers to the pc over uscb tracts overlapping minnesota counties. we developed a multiple regression model of penetrance using clinically important variables within the uscb dataset, including the continuous ( to . ) proportions of households reporting greater than eighth-grade educational attainment, population < years of age, households below the federal poverty line, and households that reported speaking a language other than english. the proportion of the population identifying as hispanic, non-hispanic black, non-hispanic american indian, and non-hispanic asian were also included. we evaluated the distributions of predictor variables for normality using standardized normal probability and kernel density plots (pnorm, qnorm, and kdensity commands). those with non- normal distributions were considered for transformation prior to multivariate analysis using linear regression modeling in order to meet the assumptions of the model. all data were analyzed using stata (statacorp, college station, tx). we compared the resulting penetrance heat map to known geographic, political, and sociodemographic maps of the state. a qualitative comparison of penetrance “hot spots” (areas of increased penetrance) and “cold spots” (areas of decreased penetrance) to areas of known sociodemographic or geographic importance was then made. the assessment of importance was made by pc staff, medical toxicologists and medical toxicology fellows, based on pc-identified areas of interest. as discussion of heat mapping of each of more than uscb tracts was infeasible for the purpose of a single study, we highlighted previously unidentified geospatial findings of potential clinical importance to the pc as exemplars of the utility of geospatial analysis for pcs. results annual call volume to the pc ranged from approximately , to , calls during the study period; of these, approximately - % were exposure calls annually, and - % were unintentional. over the five-year study period, , exposure calls to the pc were identified (figure ). of these we excluded , , largely accounted for by those originating figure . study flow diagram of phone calls made to minnesota poison control system, using geospatial analysis to pinpoint origin. western journal of emergency medicine volume , no. : november poison center utilization through geospatial mapping olives et al. from healthcare facilities ( . %). smaller exclusions were due to missing zip codes ( . %) or zip codes that were not mappable to the state ( . %). the remaining , exposure calls not originating from healthcare facilities were included for regression analysis and geospatial mapping. non-normal distributions of observations were noted for all variables but the proportion of population less than five years of age. numerical and graphical evaluation suggested square root variable transformations as most appropriate to meet the regression assumption of normally distributed data for all but the population proportion reporting educational attainment of eighth-grade or better to the uscb. in that case, transformation did not meaningfully impact observation distribution, and was not applied. post-hoc model assessment revealed heteroskedastic distribution of regression residuals (estat hettest, breusch-pagan χ . , p = . ), and thus robust standard errors were applied to the model. linear regression revealed significant associations between pc penetrance and uscb tracts with higher proportions of eighth-grade educational attainment or higher (β = . , p = . ), non-hispanic blacks (β = . , p = . ), and american indians (β = . , p = . ), indicating that census tracts with higher proportions of these demographic groups would be expected to display greater pc penetrance. no significant association was noted between pc penetrance and population proportions below the federal poverty line, proportions identifying as asian, hispanic, non-english speaking, or proportions of population less than five years of age. variance in penetrance explained by regression modelling was low (r = . ). previous county-based geospatial penetrance mapping (figure b) revealed a caller distribution profoundly more complex than previously available county-wide penetrance maps (figure a), with substantial intra-county variability in pc penetrance. “cold spots,” or regions of low penetrance, were identified in southern, southeastern, and west central regions of the state, while “hot spots,” or regions of increased penetrance, were identified in small north central and northern areas of the state, and within the state’s two largest urban centers. these consequential variations in the geospatial distribution of pc calls were not captured by statistical modelling. case examples elucidate nuances to call distribution not captured by regression analysis. case examples leech lake reservation an isolated penetrance “hot spot” in north central minnesota correlated with the intersection of cass, beltrami, calls per , residents county boundary for reference calls per , residents figure . a) distribution of poison center penetrance (calls per population) prior to geospatial mapping of all calls. legend reports penetrance as calls per residents per year. b) – census tract geospatial mapping of poison control call penetrance. legend reports penetrance as calls per residents per year over the study period. a. b. volume , no. : november western journal of emergency medicine olives et al. poison center utilization through geospatial mapping and itasca counties (figure a). no regional suggestion of increased calls was apparent by county-based spatial mapping of call penetrance (figure a). census-tract spatial distribution of penetrance revealed a hot spot substantially and uniquely overlapping the legally designated leech lake indian reservation. this finding suggests a previously undetected variation in penetrance within the reservation with no clearly apparent etiology. southeast minnesota a “cold spot” was identified in far southeastern minnesota correlating with fillmore, houston, and winona counties (figure b). all three counties were low penetrance by county- based mapping; however, census tract mapping revealed that the extreme southeastern component of the area had considerably lower penetrance than the northern and western portions of the counties. this subregion represents the most sparsely populated area of the three low-penetrance counties, and correlates with one of the largest amish settlements in the us as a percentage of county population ( . % of fillmore county in ). amish communities commonly de-emphasize ownership or use of private telephones, , and pc penetrance within this community may thus be constrained by technology, suggesting a need for further exploration of this finding, and for consideration of alternate communication methods in areas of low telephone availability. cedar-riverside a “cold spot” was identified in central minneapolis overlapping cedar-riverside (figure c), a triangular neighborhood contained on two sides by freeways, and on the other by the mississippi river. forty-eight percent of the cedar- riverside population is black, while . % of the population there speaks a language other than english. this diverse neighborhood is the epicenter of minnesota’s somali diaspora, estimated between , born in somalia and , reporting somali ancestry. the western and southern regions of cedar- riverside are more heavily populated by the somali population, while the northern and eastern regions are occupied by the university of minnesota campus. low pc penetrance appears limited to areas of cedar-riverside with the highest somali population density, while the remaining neighborhood heat map displays no observable low penetrance. discussion in a regional pc in a state with significant racial and cultural disparities, uscb-defined characteristics of greater than eighth- grade educational attainment, non-hispanic black identity, and non-hispanic american indian identity were associated with increased call penetrance to a pc. this suggests increased pc utilization among those with higher educational attainment and those who identify as black or american indian. our findings share some of the findings reported in by litovitz et al, who noted an increase in penetrance in populations with high figure . case examples. a) high penetrace region at the confluence of three rural counties and overlying leech lake reservation. b) low penetrance region in far southeastern minnesota. c) low penetrance region correlating with the cedar- riverside neighborhood of minneapolis. percentages of residents with asian background, residents younger than five years of age, and residents holding bachelor’s degrees, among others. our studies stand in distinction to the findings reported by vassilev et al, who identified high population density and high proportions of non-white races as predictors of low, rather than high, pc utilization. still other studies have identified hispanic background as a negative predictor of pc utilization ; our results describing this association did not achieve statistical significance, despite suggesting a similar relationship. western journal of emergency medicine volume , no. : november poison center utilization through geospatial mapping olives et al. nonetheless, in the context of finite pc resources, the results of regression modeling are useful but insufficient to plan strategic and cost-effective pc outreach. regression modeling alone cannot identify specific geographic regions of low penetrance, and ultimately this is inadequate to implement fully informed, ground-level decisions regarding resource utilization and geographic targeting of pc outreach. routine statistical modeling, therefore, provides a conceptual framework for understanding pc penetrance, while geospatial mapping offers a direct assessment of low and high penetrance areas of interest on which pcs may focus outreach resources. the three cases of leech lake, southeast minnesota, and the cedar-riverside neighborhood of minneapolis provide unique examples of regions inadequately described by statistical modeling and prior county-level geospatial descriptions of pc penetrance. the etiology of increased pc penetrance in the leech lake region is obscure but consistent with regression modeling, and this “hot spot” was not identified prior to granular pc penetrance mapping. while a culture of increased utilization may exist across residents of this geographic region, a single “super user” in a sparsely populated region may also be responsible for this finding. alternately, a higher than expected volume of exposures reported from non-healthcare locations may be related, warranting further public health outreach and pc investigation. finally, in a resource-poor area of the state, access to expert medical opinion regarding poisonings may be more feasible by phone than by physical presentation to a medical provider. in southeast minnesota, multiple plausible explanations for decreased penetrance exist. a relatively large proportion of the regional population is of the amish faith, and many are likely without telephone service in their homes. while other regions of minnesota are home to significant amish populations, few are as large or established, and most are much more recently founded. this raises the possibility of important cultural differences, including telephone ownership, between older and more conservative amish communities in southeast minnesota and more recently founded, more progressive communities in other regions. despite prior studies identifying mass-mailing campaigns as ineffective in reaching rural populations and increased rural call volumes following the implementation of toll- free access to pcs, this region may stand in contradistinction given the higher than normal proportion of residents with minimal access to technology including telephones and electricity. lastly, our findings may simply identify an area where pc outreach efforts have heretofore been inadequate, where lower than expected rates of poisonings occur, or where poisoned patients and those around them more commonly present to healthcare facilities than contact the pc. finally, cedar-riverside represents an area of particular concern for the pc, and likely reflects challenges experienced by other pcs. while the volume of pc calls using a telephonic language-interpreting line remained very low as a percentage of all calls over the study period, no prior efforts had been made to objectively study our poor penetrance into language minority groups. the present study strongly suggests that the pc is not attending to one of the largest regional minority groups. during the study period, somali language interpreters were used for only four calls, and as recently as – , somali interpreters were used for - calls annually despite a known population of more than , . whether this poor penetrance represents sociocultural or linguistic barriers, low awareness of pc services, or a low rate of poisonings in this subgroup is unclear, and suggests an avenue to which outreach resources may be directed. limitations several limitations govern the interpretation of these findings. this cross-sectional study in a single state identifies associations between pc penetrance and uscb-defined variables, but causal relationships between demographic variables and penetrance variation cannot be inferred. generalizability to other pc catchment areas is not described. similarly, a high risk of type i statistical error is inherent to large datasets such as this: many ucsb component variables are available for statistical modeling, raising the risk of inappropriately focusing on unexpected associations or findings. to mitigate this, we identified variables of interest a priori, and did not add to our model thereafter. while our resulting regression model explained little of the variability seen in our study, this was likely a result of confounding by multiple factors, one of which is the geographic distribution of callers that we sought to study through geospatial mapping. indeed, the limited utility of statistical modeling, absent geospatial mapping, is an important and central finding of this study. additionally, the assessment of penetrance in this study is rooted in its historical utilization both as a marker of pc efficacy and for accreditation through the aapcc. the use of penetrance as an accreditation metric was discontinued in absent data to support its use. however, data from this era are characterized largely by evaluations of penetrance as it relates to differences in populations’ ages, specifically the proportion of the population younger than two years old, at a time when counties were largely considered the unit of measurement, and when further geographic subanalyses would have been less accessible. penetrance, described at a much more granular level of analysis, better defines areas of low pc utilization, inviting further evaluation prior to the redistribution of pc resources and suggesting that penetrance may yet hold value for pcs. an additional limitation of our dataset is the predefined nature of uscb data. within uscb-defined variables such as “non-hispanic black,” more nuanced associations, unique to our state, may exist between pc penetrance and subgroups otherwise subsumed under uscb variables (for example, both karen and hmong cultural groups coding to “non-hispanic asian”). this limitation is at the root of the present study, which seeks to better identify underserved groups through geospatial mapping. we excluded calls coded as originating from healthcare facilities, but miscoded or misreported calls may have been inadvertently included in the study. nonetheless, a small number volume , no. : november western journal of emergency medicine olives et al. poison center utilization through geospatial mapping of miscoded cases is likely mitigated by the overall large number of observations. similarly, callers from mobile phones with area codes mapping to minnesota may have called the pc from outside the state, causing inclusion of calls from an unintended region. callers from mobile phones with area codes mapping outside of minnesota, but residing within the state, may have been inadvertently excluded. this is likely addressed, however, by exclusion of such calls when documenting caller-reported zip codes not mapping to minnesota at call initiation. finally, spatial apportionment of us zip codes to uscb tracts is a good measure of population parameters, but it imparts a small degree of imprecision when combining these datasets, both of which are characterized by similar but unique geographic boundaries. in describing penetrance, this imprecision, likely to occur on the edges of identified boundaries, is unlikely to meaningfully affect the interpretation of results intended to geographically guide outreach efforts. while some case records report addresses, far more contained zip codes, making this a more adequate data point to map calls. further, the extraction of addresses was not feasible due to limitations in data extraction from local call management software. additionally, zip codes may change periodically, but it was beyond the scope of this investigation to identify small changes to zip code areas, potentially imparting further imprecision to our findings. conclusion in this investigation, historically employed statistical and county-based methods to define poison center penetrance fail to recognize systematic failures to reach specific demographic and geospatially defined groups. higher american indian and non- hispanic black population proportions, and greater than eighth- grade educational attainment, are characteristics associated with increased pc penetrance in this study. evaluating the geospatial distribution of calls to other pcs may enhance understanding of penetrance patterns, improve resource allocation and elucidate previously unknown predictors of pc penetrance. this novel and detailed visual account of pc penetrance, uniquely interpretable when contextualized in a knowledge of the state served by the poison center, offers a new tool to optimize pc outreach. address for correspondence: travis d olives, md, mph, med, park ave, rll - poison center, minneapolis, mn . email: travis.olives@hcmed.org. conflicts of interest: by the westjem article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. no author has professional or financial relationships with any companies that are relevant to this study. there are no conflicts of interest or sources of funding to declare. copyright: © olives et al. this is an open access article distributed in accordance with the terms of the creative commons attribution (cc by . ) license. see: http://creativecommons.org/ licenses/by/ . / references . gummin dd, mowry jb, spyker da, et al. annual report of the american association of poison control centers’ national poison data system (npds): th annual report. clin toxicol (phila). ; ( ): - . . miller tr and lestina dc. costs of poisoning in the united states and savings from poison control centers: a benefit- cost analysis. ann emerg med. ; ( ): - . . friedman ls, krajewski a, vannoy e, et al. the association between u.s. poison center assistance and length of stay and hospital charges. clin toxicol. ; ( ): - . . offerman sr. the clinical management of acetaminophen poisoning in a community hospital system: factors associated with hospital length of stay. j med toxicol. ; : - . . litovitz t, benson be, youniss j, et al. determinants of u.s. poison center utilization. clin toxicol. ; ( ): - . . albertson te, tharratt 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communication: sociodemographic differences between counties with high and low utilization of a regional poison control system. j toxicol environ health. ; (a): - . . everson g, es r, kendrick m, et al. ineffectiveness of a mass mailing campaign to improve poison center awareness in a rural population. vet hum toxicol. ; ( ): - . . krenzelok ep and mrvos r. the initial impact of toll-free access on poison center volume. vet hum toxicol. ; ( ): - . https://mn.gov/bms-stat/assets/the-economic-status-of-minnesotans-chartbook-msdc-jan -post.pdf https://mn.gov/bms-stat/assets/the-economic-status-of-minnesotans-chartbook-msdc-jan -post.pdf https://www.census.gov/data/tables/ /demo/ - -lang-tables.html https://www.census.gov/data/tables/ /demo/ - -lang-tables.html measurement without theory: a response to bailey and collins greenwood, jeremy, ananth seshadri, and guillaume vandenbroucke working paper no. february university of rochester measurement without theory: a response to bailey and collins jeremy greenwood university of pennsylvania ananth seshadri university of wisconsin guillaume vandenbroucke university of iowa february department of economics, university of iowa, w pbb, iowa city, ia - , usa. email: guillaume-vandenbroucke@uiowa.edu. abstract bailey and collins (forth.) argue that greenwood, seshadri and vandenbroucke ( )�s hypothesis that the baby boom was partly due to a burst of produc- tivity in the household sector is not supported by evidence. this conclusion is based upon regression results showing that appliance ownership is negatively correlated with fertility. they also argue that the amish, who limit the use of modern technology, had a baby boom. first, it is demonstrated that a nega- tive correlation between appliance ownership and fertility can arise naturally in greenwood et al.�s model. second, evidence is presented casting doubt upon the presumed technological phobia of the amish. keywords: amish, appliances, baby boom, bailey and collins, fertility, model laboratory, monte carlo simulations, regressions introduction greenwood, seshadri and vandenbroucke ( ) hypothesize that the baby boom was partly due to a burst of productivity in the household sector. the idea is that the introduction of appliances (for example dryers, refrigerators and washing machines) and new products (such as frozen and packaged foods and infant formula) reduced the cost of having children. equally important was the home economics movement, that introduced the principles of scienti�c management into the home. albanesi and olivetti ( ) argue that advances in obstetrics and pediatric medicine had much the same e¤ect. by lowering the cost of having children, such forces promoted fertility. bailey and collins (forth.) argue that this hypothesis is not supported by evidence. they make two points. first, they report results of regressions show- ing that appliance ownership is negatively correlated with measures of fertility. according to them, gsv�s theory warrants a positive correlation. second, they argue that the amish, who limit the use of modern technology, had a baby boom. they conclude that it could not have been caused by technological progress, another point against gsv�s theory. these points are addressed in or- der. section demonstrates that bc�s empirical strategy is not well designed. therefore, it does not deliver a suitable test of gsv�s hypothesis. section presents evidence casting doubts on the well-accepted notion that the amish are �anti-technology.� quantitative strategy bc�s strategy is to regress fertility on adoption rates, controlling for income and other variables. this is done for a set of u.s. counties. they assert that the gsv theory of the baby boom implies a positive coe¢ cient of adoption on fertility. a negative coe¢ cient is found. this is interpreted as evidence against the theory. bc are, in fact, misinterpreting their results. the negative correlation they �nd between the adoption rate for appliances and fertility is perfectly consistent with gsv�s model. this is shown below by constructing a simple example that illustrates how fertility and the adoption of appliances are determined. the idea is that technological adoption di¤ers by income. richer people will tend to adopt electricity and appliances earlier than poorer ones. they will also have lower levels of fertility than poorer people. so, a regression may associate low levels of fertility with the adoption of appliances, if the rich adopt �rst. two monte carlo experiments are conducted running the bc�s regressions on simulated data generated from a simpli�ed version of gsv�s model. they yield the same negative correlations that bc �nd. if such regressions don�t perform the baby boom is now an active area of research in macroeconomics. for example, doepke, hazan and maoz ( ) and jones and schoonbroodt ( ) analyze the impact of world war ii and the great depression on the baby boom. their hypotheses are not mutually exclusive with the one discussed here. well in a dust-free model laboratory it is hard to understand why they should be expected to work well in the real world. the idea that regression equations can be �tested�on simulated data drawn from economic models is not new in macroeconomics. an early example is baxter and jermann ( ), who examine tests of the permanent income hypothesis. a more recent one is chari, kehoe and mcgrattan ( ) who study the use of vars. the constructed example is realistic in that it displays a secular decline in fertility that is brie�y interrupted by a baby boom. it is important to stress that as logical matter all that is needed is a counter example to the hypothesis that the gsv model implies a positive relationship between fertility and adoption in a regression of the form run by bc; i.e., there is no need for the example to be realistic to disprove the hypothesis, as any logician knows. . a simple model suppose that individuals live for one period of time. individuals earn the wage wi, which di¤ers by income class i. they have preferences over consumption, ci, and the number of kids, ni, represented by u (ci;ni) = � c �� i �� +( ��)ni, with � ( ; ) and � > . there are two available household technologies. the �rst is free and implies a time cost q for raising children. the second costs e units of the consumption good and implies a time cost z < q. the budget constraints for users and nonusers are ci +qwini = wi; for nonusers, ci +zwini = wi �e; for users. the optimal consumption and fertility decisions for an individual of income class i are given by: cni = wi �qwinni ; for nonusers, nni = =q � [�=( ��)] =� (qwi) ( ��)=� ; cui = wi �e�zwinui ; for users, nui = (wi �e)=(zwi)� [�=( ��)] =� (zwi) ( ��)=� ; where the superscript u (n) denotes the decision for a (non) user. the adoption decision is summarized by ai = � ; if u (cui ;n u i ) > u (c n i ;n n i ) ; user; ; if u (cui ;n u i ) � u (cni ;nni ) ; nonuser. the following example illustrates how the above model can generate a secular decline in fertility that is punctuated by a baby boom, the latter due to the adoption of a labor-saving household technology. in the example, there are three types of individuals, viz, �poor,� �middle-income,� and �rich�. their wages grow at percent a year. the price of appliances starts o¤very high and remains so for years. it then proceeds to decline at percent a year. example: parameterization tastes � = : ;� = : technology q = : ;z = : initial wages w = : ;w = : ;w = : growth in wages wi(t) = w : (t� ) i ; for t > initial price e = growth in price e(t) = � e; for t � ; e : (t� ); for t > figure shows the time path of fertility for these individuals. fertility displays a secular decline for all income classes due to rising wages. observe that richer individuals tend to have lower fertility, both in the cross section and the time series, a fact �rmly established in jones and tertilt ( ). this is because the opportunity cost of having children is increasing in the wage rate. notice also that richer individuals adopt the new technology earlier. at the time of adoption there is a jump in fertility because the new technology reduces the time cost of raising children. . bailey and collins�first regression to test the above theory, bc run a cross-sectional regression of the form: nj = constant + �aj +�yj +"; with " � n( ;�); where nj is fertility in county j, aj is the county�s rate of adoption for appliances, and yj is per-capita income. they postulate that the theory implies that � should be positive. a monte carlo experiment can be conducted to test this regression on simulated data generated from the model. to do this, assume that there are j = ; counties indexed by j and that each county is populated by n = ; individuals indexed by i. in each county j draw individual i�s wage, wij, for i = ; : : : ;n, from a lognormal distribution. speci�cally, let lnwij � n(�wj; : ). note that each county j has its own mean level of wages, �wj. let this mean also be lognormally distributed. in particular, ln �wj � n( ; : ). these numbers are chosen so that the coe¢ cient of variation of wages is in line with the u.s. data. in particular, in the monte carlo experiment considered here the coe¢ cient of variation of wij is : . kopecky (table , forth.) reports that the coe¢ cient of variation in earnings, from census data, is between : and : . take the price for the new technology to be given by e = . for each individual generate the following data points for fertility, adoption and income: nij = ( (wij �e)=(zwij)� [�=( ��)] =� (zwij) ( ��)=� ; if user, =q � [�=( ��)] =� (qwij) ( ��)=� ; if nonuser, aij = � ; if u � cuij;n u ij � > u � cnij;n n ij � ; user; ; if u � cuij;n u ij � � u � cnij;n n ij � ; nonuser. yij = � wij ( �znij) ; if user, wij ( �qnij) ; if nonuser, where cij +qwijnij = wij; for nonusers, cij +zwijnij = wij �e; for users. note that adoption, aij, fertility, nij, and income, yij, are all simultaneously determined endogenous variables. from this individual-level data, county-wide averages can be constructed. specially, let nj = n � nx i= nij;aj = n � nx i= aij, and yj = n � nx i= yij. nowtheabove regressioncanbeestimatedusingmodel-generatedcross-sectional county-level data. the estimation yields �̂ = � : . to gather intuition about this result, contemplate figure again. is it true that individuals who adopted the time-saving technology have higher fertility than those who did not? not necessarily. focus on the year . the rich (circles) have adopted the technology and their fertility increased as a result, but yet it remains below that of the poor (diamonds) who has not yet adopted the technology. such observation is consistent with a negative correlation be- tween adoption and fertility. observe also that the �rst intersection between the poor and middle-income classes fertility (squares) occurs in the s. at this moment the poor have not adopted while the middle-income class has. yet, their fertility is the same: a zero correlation. . bailey and collins�second regression bc argue that the issue raised above can be dealt with by regressing di¤erenced variables. in particular, they now run a regression of the form �nj = constant + ��aj +��yj +"; with " � n( ;�): consider, then, redoing the above experiment. add another time period to the earlier analysis and let wij increase by percent (i.e., % per year for years) for each individual i in each county j, and let e decrease by percent. compute n j, a j and y j, or the new values for fertility, adoption and income in county j, and build �nj = n j � nj, �aj = a j � aj and �yj = y j � yj. estimating this equation on model-generated data yields �̂ = � : . so, this does not cure the problem. . upshot these examples show that gsv�s model is not appropriately �tested�by regres- sions such as those used by bc. regressions of these types are not implied by the model proposed in gsv on many grounds: they are linear while the gsv model is not; they are based on static and incomplete theorizing about fertility alone, whereas in gsv�s model forward-looking people solve complicated dynamic op- timization problems involving both adoption and fertility, where current and future wages and prices will matter; �nally, they overlook the endogeneity of both adoption and income. a long time ago, koopmans ( , p. ) railed against �measurement without theory�: �the various choices as to what to �look for,�what economic phenomena to observe, and what measure to de�ne and compute, are made with a minimum of assistance from theoretical conceptions or hypothesis regarding the nature of the economic processes by which the variables studied are generated.� by dispensing with theoretical guidance it is easy to misinterpret the results from empirical measurement, in this case the observed correlation between fer- tility and adoption rates. the amish bc present data on amish fertility. according to them, the amish also experi- enced a baby boom. bc assert that the amish do not use modern labor-saving technologies in their households. certainly, this is conventional wisdom. it is suspect, though. an expert on amish culture, d. b. kraybill ( , p. ), relates the following on this matter: �consider some of the household changes in the last �fty years. amish women no longer wash clothes in hand-operated machines. they use washing machines powered by hydraulic pressure or gaso- line engines. gas refrigerators have replaced iceboxes, indoor �ush toilets have replaced outdoor privies, hydraulic water pumps have re- placed windmills, and gas water heaters have replaced the �re under wrought-iron kettles. modern bathtubs have superseded old metal tubs. kerosene lanterns have given way to gas lights. wood-�red cookstoves have yielded to modern gas ranges. hardwood �oors and no-wax vinyl have replaced linoleum and rag carpets. spray starch, detergents, paper towels, instant pudding, and instant co¤ee have eased household chores. permanent-press fabrics have lifted the burden of incessant ironing. although canning still predominates, some foods are preserved by freezing. air-powered sewing machines are replacing treadle machines. battery-powered mixers do the job of hand-operated egg beaters,and air-powered food processors have replaced hand grinders.� figure shows an amish kitchen, sometime prior to . the presence of lights (perhaps from a gas generator), a refrigerator, and range can be seen immediately. less apparent is the fact the kitchen is arranged in a modern, rationalized, time-saving manner, as evidenced by the built-in wood cabinets, continuous counter tops, and modern sink. such a picture perfectly illustrates the following quote from gsv (p. ): �take the kitchen, for example. the kitchen of the s was characterized by a large table and isolated dresser. an organized kitchen with continuous working surfaces and built-in cabinets began to appear in the s, after a period of slow evolution. in the s, the kitchen became connected with the dining room and other living areas, ending the housewife�s isolation.� such innovations, while hard to quantify, did save time. often they can be seemingly small. for example, gsv discuss how christine frederick, an early advocate of applying the principals of scienti�c management to the home, �discovered that dishwashing could be accomplished more e¢ - ciently by placing drainboards on the left, using deeper sinks, and connecting a rinsing hose to the hot-water outlet; she estimated that this saved minutes per dinner.� in fact, today, in some areas the amish are on the forefront of technology adoption. holmes county, oh, hosts the largest amish community in the world. about % of the amish living there use solar power. apparently, the amish�s main concern about the use of electricity is its connection with a publicly shared grid, which reduces independence from the outside world. this can be avoided through the use of diesel generators, windmills, and, in modern times, solar panels. to conclude, while there is some truth in the conventional wisdom about the amish being technophobic, the true story is much more nuanced. to the extent that they adopted labor-saving practices in the home one would expect that they should also experience a baby boom. it is interesting to note that conventional wisdom also believes that the amish do not use any form of birth control. the presence of an amish baby boom casts doubt on this too (unless one believes that amish women became more fecund during this period). references [ ] albanesi, stefania and olivetti, claudia. �maternal health and the baby boom.�nber wp , . [ ] bailey, martha j. and collins, william j. �did improvements in household technology cause the baby boom? evidence from electri�cation, appli- ance di¤usion, and the amish.�american economic journal: macroeco- nomics, forthcoming. [ ] baxter, marianne and urban j. jermann. �household production and the excess sensitivity of consumption to current income.� american eco- nomic review ( ), : pp. - . [ ] chari, v.v.; kehoe, patrick j. and mcgrattan, ellen r. �are structural vars with long-run restrictions useful in developing business cycle the- ory?�journal of monetary economics, ( ), : pp. - . [ ] doepke, matthias; hazan, moshe and maoz, yishay, �the baby boom and world war ii: a macroeconomic analysis.�nber working paper no , . [ ] greenwood, jeremy; seshadri, ananth and vandenbroucke, guillaume. �the baby boom and baby bust.� american economic review ( ), : pp. - . [ ] jones, larry e. and tertilt, michele. �an economic history of fertility in the u.s.: - ,�in peter j. rupert, ed, frontiers of family eco- nomics. bingley ,uk: emerald group publishing, : pp. - . [ ] jones, larry and schoonbroodt, alice, �baby busts and baby booms: the fertility response to shocks in dynastic models.�mimeo, university of minnesota, . [ ] koopmans, tjalling c. �measurement without theory.� the review of economics and statistics, ( ), : pp. - [ ] kopecky, karen a. �the trend in retirement.� international economic review, forthcoming. [ ] kraybill, donald b. the riddle of amish culture. baltimore, md: the johns hopkins university press, and . . . . . fe rt ilit y rich middle income poor figure : baby boom and baby bust. the simulation starts in . wages grow at percent annually. appliance prices are constant until , and then decline at percent a year. figure : an amish kitchen sometime prior to . source: kraybill ( , p. ) brief genetics report polymorphisms in both promoters of hepatocyte nuclear factor -� are associated with type diabetes in the amish coleen m. damcott, nicole hoppman, sandra h. ott, laurie j. reinhart, jian wang, toni i. pollin, jeffrey r. o’connell, braxton d. mitchell, and alan r. shuldiner , hepatocyte nuclear factor -� (hnf a) is a transcrip- tion factor located on chromosome q that regulates expression of genes involved in glucose metabolism and homeostasis. recently, two groups independently iden- tified single nucleotide polymorphism (snps) in an alternate upstream promoter (p ) of hnf a that were associated with type diabetes in ashkenazi jews and finns. we genotyped haplotype-tagging snps (htsnps) across the two promoter regions and the coding region of hnf a in individuals with type diabetes (n � ), impaired glucose tolerance (igt) (n � ), and normal glucose tolerance (n � ) from the amish family diabetes study (afds) to test for association with type diabetes. in the p promoter region, we observed a significant association between the a allele of rs and type diabetes (odds ratio [or] . , p � . ). furthermore, the mean age of type diabetes onset was, on average, . years earlier in those with the aa or ga genotype at snp rs than in those with the gg genotype ( . vs. . years, p � . ). in the p promoter, the htsnp rs showed borderline association with both type diabetes (or . , p � . ) and the combined type diabetes/igt trait ( . , p � . ). in an expanded set of nondiabetic afds subjects, we found association between rs and glucose area under the curve during an oral glucose tolerance test (additive model, p � . ; dominant model, p � . ). the results of this study provide evidence that variants in both the p and p promoters of hnf a increase risk for typical type diabetes. diabetes : – , h epatocyte nuclear factor -� (hnf a) is a transcription factor that is expressed in several tissues, including liver and pancreas, where it regulates expression of genes involved in glu- coneogenesis and glucose-stimulated insulin secretion, respectively ( – ). relatively rare mutations in hnf a have been identified that cause maturity-onset diabetes of the young type (rev. in ), a dominantly inherited, early-onset form of type diabetes characterized by im- paired glucose-induced insulin secretion due to pancreatic �-cell dysfunction ( – ). hnf a expression patterns are complex as a result of alternative splicing and transcrip- tion from two different promoters, the proximal p pro- moter and the p promoter, which lies � kb upstream of the p promoter ( – ). the coding exons of hnf a span � kb on chro- mosome q , a region of overlapping linkage to type diabetes in several caucasian ( – ) and asian ( , ) populations. recently, through fine-mapping efforts in this region of chromosome q, two groups concurrently iden- tified single nucleotide polymorphisms (snps) in the p and p promoter regions and coding exons of hnf a that are associated with type diabetes in the ashkenazi jews ( ) and finns (fusion [finland-united states investi- gation of niddm genetics ]) ( ). silander et al. ( ) identified snps across a -kb region spanning the p and p promoter regions and exons – of hnf a that were associated with type diabetes in the fusion population. in the ashkenazi jewish cohort, the snps closer to the p promoter and coding exons were not associated with type diabetes ( ); however, four snps spanning a � -kb region encompassing the p promoter were associated with type diabetes (rs , rs , rs , and rs ). these four snps are located in a -kb region of strong linkage disequilibrium (ld), including � kb upstream of the p promoter ( ). a second haplotype block was observed within the hnf a coding region, while ld tended to decay across the � -kb gap separating hnf a from p ( , ). in addition to the observed association with type diabetes, these p snps appeared to explain a significant portion of the linkage to chromosome q -q observed in both the ashkenazi jews and finns. the replicating evidence pre- from the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland; and the geriatric re- search and education clinical center, veterans administration medical cen- ter, baltimore, maryland. address correspondence and reprint requests to alan r. shuldiner, md, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood st., room , baltimore, md . e-mail: ashuldin@medicine.umaryland.edu. received for publication june and accepted in revised form august . afds, amish family diabetes study; hnf, hepatocyte nuclear factor; htsnp, haplotype-tagging single nucleotide polymorphism; igt, impaired glucose tolerance; ld, linkage disequilibrium; ngt, normal glucose tolerance; ogtt, oral glucose tolerance test; snp, single nucleotide polymorphism. © by the american diabetes association. diabetes, vol. , december sented by these studies suggests that snps near the p promoter of hnf a increase susceptibility to type diabetes. although no evidence for linkage to type diabetes was detected on chromosome q -q in our genome-wide scan (average marker density � . cm) in the old order amish (logarithm of odds � . between markers d s and d s , which are � cm apart) ( ), we tested whether snps in hnf a and its promoters were associated with type diabetes in the amish. we selected six haplotype-tagging snps (htsnps) spanning the p and p promoters and the hnf a coding region from the ld blocks defined in the ashkenazi jews ( ) and finns ( ). given that the amish are a young founder population, we hypothesized that haplotype blocks would be as large as or larger than those in the other populations, thus allowing us to capture most or all of the variation across the gene with these snps. these snps were genotyped in individuals enrolled in the amish family diabetes study (afds), which included subjects with type diabetes, individuals with impaired glucose tolerance (igt), and control subjects with normal glucose tolerance (ngt). the ngt control subjects selected were � years of age in order to increase the probability of their capacity for diabetes resistance. table summarizes the allele frequen- cies in individuals with type diabetes, igt, and ngt and the results of genotypic association analysis for each snp. all snps conformed to hardy-weinberg expectations. for rs , one of the snps located in the p promoter region, the frequency of the a allele was significantly higher in the type diabetic group than in the control group in the amish (genotypic odds ratio [or] . , p � . ). furthermore, the mean age of diabetes onset was . years in subjects with the aa genotype for snp rs , . years in those with the ga genotype, and . years in those with the gg genotype. the mean age of type diabetes onset was, on average, . years earlier in those with the aa or ga genotype at snp rs than in those with the gg genotype ( . vs. . years, p � . ). we genotyped one (rs ) of the four p promoter snps reported to be associated with type diabetes and in near-perfect ld with each other in both the ashkenazi jewish and finnish populations. in the amish, the frequency of the a allele at the rs snp was lower in control subjects with ngt than in both the the type diabetic group (genotypic or . , p � . ) and the combined type diabetic/igt group (genotypic or . , p � . ), although these differences did not achieve statistical significance, as observed in the ashkenazi jews and finns. none of the other snps in the p promoter region or the coding region were associated with type diabetes in the amish, including the other snps observed to be associated with type diabetes in the finns (rs and rs ) and ashkenazi jews (rs ). haplotype analysis revealed that only those haplotypes containing the rs a allele and rs a allele were associated with increased type diabetes prevalence (results not shown). table shows the pairwise ld (�d�� and r ) among the genotyped snps in the amish. the haplotype block struc- ture in the amish appears very similar to that reported in finns and ashkenazi jews, suggesting that the snp den- sity we chose is adequate for the detection of most of the common variation in hnf a. as shown in the ashkenazi jews and the finns, the snp representing the p haplotype block that was genotyped in the amish (rs ) was table allele frequencies and results of association analysis in subjects with type diabetes, igt, and ngt for snps in the hnf a region minor allele frequency diabetes vs. ngt* diabetes � igt vs. ngt* snp location (kb)† snp name major/minor allele type diabetes (n � ) igt (n � ) ngt (n � ) or p or p 	 . rs g/a . . . . . . . . rs g/t . . . . . . . . rs g/a . . . . . . . . rs t/c . . . . . . . . rs g/t . . . . . . . . rs g/t . . . . . . . *p values are based on genotype frequencies, and ors reflect the odds of disease associated with having two copies of the minor allele versus the odds of disease associated with having two copies of the major allele and were adjusted for age, sex, and pedigree structure. reported p values are not adjusted for multiple comparisons. p values . are shown in bold. table pairwise ld among hnf a snps in the amish* rs rs rs rs rs rs rs — . . . . . rs . — . . . . rs . — . . . rs . . . — . . rs . . . — . rs . . . . . — *values in the upper right represent �d�� , while values in the bottom left represent r . shown in bold are the two snps, rs and rs , in the p and p promoters, respectively, that were associated with type diabetes and glucose traits. hnf a and type diabetes in the amish diabetes, vol. , december clearly not in ld with rs in the p promoter or with the other hnf a snps. in addition to the case/control analysis, we genotyped rs and rs in an additional nondiabetic amish subjects to create an expanded set of nondia- betic individuals (ngt [n � ] and igt [n � ]) and performed an association analysis with diabetes-related quantitative traits. figure shows the mean plasma glu- cose levels at -min intervals during a -h oral glucose tolerance test (ogtt) according to genotype at the rs snp. carriers of the a “risk” allele for the rs snp exhibited significantly higher total glucose area under the curve during the ogtt (additive model, p � . ; dominant model, p � . ). higher glucose levels in nondiabetic a carriers provides additional evi- dence that the a allele of rs (or a haplotype marked by this allele) influences glucose homeostasis and type diabetes risk. however, presence of the a allele was not associated with either fasting insulin or total insulin area under the ogtt curve (fig. ). although we do not have direct measures of insulin secretion, the finding of increased ogtt glucose levels without differences in ogtt insulin levels in carriers of the a “risk” allele suggests a relative deficiency in insulin secretion in these individuals. this interpretation is consistent with the hy- pothesis that the a allele, present in the islet-specific p promoter, may decrease expression of hnf a in insulin- secreting �-cells, thus affecting �-cell function. the quan- titative trait analysis for rs in the p promoter showed no association with glucose- or insulin-related traits. in conclusion, we found that htsnps in the p and p regions of hnf a are associated with type diabetes and diabetes-related traits in the amish. rs in the p region was also associated with type diabetes in the finns; however, contrary to our findings in the amish, in which the a allele was the at-risk allele for both type diabetes risk and an earlier onset of diabetes, the fre- quency of the g allele was significantly higher in finnish subjects with type diabetes. this discrepancy between the two populations may indicate that this snp is not the functional snp but is marking an at-risk haplotype that differs between the amish and finns. of note, this snp was not associated with type diabetes in the ashkenazi jews, but others in the region were associated with type diabetes, suggesting again that the snps thus far examined may be marking at-risk haplotypes in the different popu- lations. alternatively these discrepancies between popula- fig. . mean plasma glucose (a) and insulin (b) levels at -min intervals during a -h -g ogtt according to snp rs genotype groups. carriers of the a “risk” allele exhibited higher total glucose area under the curve during the ogtt (additive model, p � . ; dominant model, p � . ). there was no association with total insulin area under the curve during the ogtt (additive model, p � . ; dominant model, p � . ). c.m. damcott and associates diabetes, vol. , december tions could represent false-positive or false-negative results. rs , an htsnp in the p region of hnf a was associated with glucose levels during an ogtt in the amish and was also associated with type diabetes in both the ashkenazi jews and the finns. in all populations studied to date, the p and p snps reside in different haplotype blocks, suggesting the presence of two indepen- dent variants influencing type diabetes risk. this repli- cation across several studies lends further support to the possibility that variation in the p and p regions of hnf a, or snps in strong ld with these regions, contrib- utes to the pathogenesis of type diabetes. of note, our genome scan did not provide any evidence for linkage to type diabetes or related traits to this region of chromo- some in the amish ( ). this observation is likely due to the relative insensitivity of linkage analysis compared with association analysis and suggests that this allele may also influence type diabetes risk more broadly in other populations. although hnf a is the strongest candidate gene for type diabetes in this region, the p snps reside in a large haplotype block that contains several other known and predicted genes and expressed sequence tags; therefore, the possibility must be considered that the pathogenic snp(s) may reside in another gene. further studies in other populations, as well as functional analysis, will be required to further define the role of variation in hnf a in type diabetes pathogenesis. research design and methods the afds was initiated in with the goal of identifying susceptibility genes for type diabetes and related traits in a cohort of individuals from the old order amish population in lancaster county, pennsylvania. details of the afds design, recruitment, phenotyping, and pedigree structure have been described previously ( ). briefly, probands with previously diagnosed type diabetes (onset between and years of age) and all first- and second- degree relatives of probands and spouses over the age of were recruited. phenotypic characterization of study participants included medical and family history, anthropometry, and a -h -g ogtt with insulin levels. the diagnosis of type diabetes was defined on the basis of the ogtt using criteria of the american diabetes association ( -h glucose � . mmol/l or fasting glucose � mmol/l), by current treatment with diabetes medications, or by a previous physician-documented diagnosis of diabetes. igt was defined by a -h ogtt glucose between . and . mmol/l. ngt was defined by a fasting glucose . mmol/l and a -h ogtt glucose . mmol/l. the total glucose and insulin areas under the curve during the -h ogtt were calculated using the trapezoid method. bmi was calculated as weight (in kilograms) divided by height (in meters) squared. the mean age of diagnosis of diabetes in the afds cohort was . � . years, and the mean bmi was . � . kg/m (range . – . ). informed consent was obtained from all study subjects, and the institutional review board at the university of maryland school of medicine approved the study protocol. genotyping. genotyping was completed using the orchid/beckman snpstream ultra high throughput genotyping platform. this genotyping method is described in detail elsewhere ( ). briefly, the protocol involved pcr amplification of target sequences surrounding the snps to be assayed in panels of -plex reactions. following enzymatic purification, the pcr prod- ucts were subjected to single-base primer extension with fluorescent-labeled dye terminators. each extension primer contained a unique -nucleotide tail at its � end whose sequence was designed to hybridize to its complementary probe immobilized in a mini-array within each well of a -well snp-it plate (beckman coulter, fullerton, ca). the microarray plate was imaged by the snpscope reader (beckman coulter). the two-color system allowed the detection of the snp by comparing signals from the two fluorescent dyes. the image signals were then transferred to genotyping software that translated the images of the arrays into genotype calls. the error rate based upon blind replicates for the snps examined in the present study was – . %. statistical analysis. before analysis, genotypes were checked for mendelian consistency using the pedigree information and inconsistencies ( . % of genotypes) were resolved or removed before analysis. allele frequencies were calculated for each snp by gene counting, and observed genotypes were tested for fit to the expectations of hardy-weinberg using the � test. pairwise ld was computed between the snps using the two most commonly used statistics �d�� and r , and haplotypes were inferred for each individual using an expectation maximization algorithm implemented in the zaplo software program ( ). we evaluated the association between snp genotype and disease status (type diabetes versus ngt and type diabetes/igt versus ngt) using a variance component approach, in which we modeled the probability that the subject was a case or control subject, as a function of the individual’s age, sex, and genotype, conditional on the correlations in phenotype among relative pairs. for the primary analysis, we considered an additive genetic model in which the genotype was coded as , , or , depending on whether the subject was homozygous for the minor allele (genotype � ), heterozygous (geno- type � ), or homozygous for the major allele (genotype � ). statistical testing was accomplished using the likelihood ratio test, in which we compared the likelihood of the data under a model in which the genotype effect was estimated against the likelihood of a nested model in which the genotype effect was constrained to be zero. secondary analyses were carried out under the dominant and recessive genetic models by imposing appropriate constraints on the genotypic effects. parameter estimates (i.e., � coefficients) were obtained by maximum likelihood and ors by taking the inverse log of the � coefficient. the or for the additive model was scaled to reflect the odds that a case was homozygous for the minor allele versus the odds that the case was homozygous for the major allele. the variance components analysis was carried out using the solar software program ( ). finally, mean levels of glucose (fasting and glucose area under the curve during a -h ogtt) and insulin (fasting and insulin area under the curve during a -h ogtt) were estimated according to hnf a genotypes in an expanded set of nondiabetic afds subjects (n � ). to account for the relatedness among family members, the measured genotype approach was used ( ), in which we estimated the likelihood of specific genetic models given the pedigree structure. parameter estimates were obtained by maximum likelihood methods, and the significance of association was tested by likeli- hood ratio tests. within each model, we simultaneously estimated the effects of age and sex. insulin values were transformed by their natural logarithms (ln) to reduce skewness. quantitative trait analyses were conducted using the solar program ( ). acknowledgments this work was supported by research grants r - dk , k -dk , u -dk , and k -ca ; the university of maryland general clinical research center grant m rr ; the general clinical research centers program; the national center for research re- sources (ncrr); the national institutes of health; and the baltimore veterans administration geriatric research and education clinical center. we gratefully acknowledge our amish liaisons and field workers and the extraordinary cooperation and support of the amish community, without whom these studies would not be possible. references . stoffel m, duncan sa: the maturity-onset diabetes of the young (mody ) transcription factor hnf alpha regulates expression of genes required for glucose transport and metabolism. proc natl acad sci u s a : – , . wang h, maechler p, antinozzi pa, hagenfeldt ka, wollheim cb: hepato- cyte nuclear factor alpha regulates the expression of pancreatic beta-cell genes implicated in glucose metabolism and nutrient-induced insulin secretion. j biol chem : – , . bartoov-shifman r, hertz r, wang h, wollheim cb, bar-tana j, walker md: activation of the insulin gene promoter through a direct effect of hepatocyte nuclear factor alpha. j biol chem : – , . rhee j, inoue y, yoon jc, puigserver p, fan m, gonzalez fj, spiegelman bm: regulation of hepatic fasting 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general pedigrees. am j hum genet : – , . boerwinkle e, chakraborty r, sing cf: the use of measured genotype information in the analysis of quantitative phenotypes in man. i. models and analytical methods. ann intern med : – , c.m. damcott and associates diabetes, vol. , december development of carrier testing for common inborn errors of metabolism in the wisconsin plain population education report © american college of medical genetics and genomics introduction the plain population represents “christian groups that live simply, dress plainly, and live in the modern world but remain separate from it.” these communities originated from the anabaptist movement in europe in the s. a small number of each group immigrated to the united states in the s and s and eventually settled in wisconsin. in wisconsin, the plain population includes individuals from both the old order amish and mennonite communities. in , the state’s plain population was approximately , , including approximately , amish and , mennonite members (d kraybill, per- sonal communication). wisconsin’s amish population is the fourth largest in the united states. as a result of religious separation and a restricted founder gene pool from subsequent consanguinity within these communities, there is a high prevalence of certain autosomal- recessive conditions, including several disorders currently detected by newborn screening programs (see table ). , newborn screening is aimed at early identification of various genetic disorders, including several inborn errors of metabo- lism, and allows early treatment and thus improved long-term clinical outcomes. however, the plain population’s culture and way of life lead to several challenges in collecting newborn screens from neonates and treating those affected with identi- fied disorders. lack of medical follow-up due to financial (e.g., lack of medical insurance), logistical (e.g., the need to hire driv- ers to get to medical centers or home births that require a sepa- rate midwife visit for newborn collection), or cultural beliefs (e.g., the viewpoint that a physician need not be seen unless a child is seriously ill) is a common barrier. this project is an initiative through the university of wisconsin biochemical genetics clinic and the wisconsin newborn screening program to help families in the plain pop- ulation identify family members who may be at risk for having a child with maple syrup urine disease (msud; omim ) or propionic acidemia (pa; omim ) or who may be at risk for having pa themselves. these conditions were chosen for this project because of their high prevalence in the plain population as well as the importance of detection and the poten- tial for early intervention for these life-threatening conditions. pa and msud are inborn errors of branched-chain amino acid metabolism caused by the deficiency of propionyl- coa-carboxylase (pcc) and branched-chain dehydroge- nase (bckdha), respectively, leading to the accumulation of amino acids and by-product organic acids. each of these enzymes has multiple subunits, with variability in presentation associated with the subunit. in pa, reduced enzyme activity leads to an accumulation of propionic acid and its associated metabolites that are particularly toxic to the central nervous system and cardiac tissue. in msud, accumulation of all three submitted january ; accepted june ; advance online publication august . doi: . /gim. . purpose: this community project is an initiative through the uni- versity of wisconsin biochemical genetics clinic and the wisconsin newborn screening program to identify members of the plain popu- lation who are at risk for having children with maple syrup urine dis- ease (msud) or propionic acidemia (pa) or who have pa. methods: because of the high prevalence of metabolic condi- tions in the plain population and the importance of early inter- vention, a statewide outreach project was developed to provide targeted variant analysis of the common msud and pa patho- genic variants in this population through health-care provider distribution of blood spot testing kits. awareness was achieved through outreach efforts with the state midwives guild and plain population meetings. results: eighty individuals were tested; diagnosis was confirmed for three adults with pa and one couple was identified as being at risk for having a child with pa. genetic counseling was provided to those identified. follow-up diagnostic testing was completed for the at-risk couple’s children; none were found to be affected. conclusion: this initiative successfully provided accessible clinical testing for msud and pa for a high-risk population. early identifi- cation of at-risk couples sets the foundation for early care of at-risk neonates, thereby improving future clinical outcomes. genet med advance online publication august key words: carrier testing; maple syrup urine disease; propionic acidemia; plain population waisman center, university of wisconsin–madison, madison, wisconsin, usa; department of pediatrics, university of wisconsin–madison, madison, wisconsin, usa; department of molecular and medical genetics, oregon health and science university, portland, oregon, usa; newborn screen lab at wisconsin state lab of hygiene, newborn screen lab madison, madison, wisconsin, usa. correspondence: jessica scott schwoerer (jscottschwoerer@pediatrics.wisc.edu) development of carrier testing for common inborn errors of metabolism in the wisconsin plain population ashley kuhl, cgc , , sandra van calcar, phd, rd , mei baker, md , , christine m. seroogy, md , gregory rice, md , and jessica scott schwoerer, md , volume | number | march | genetics in medicine http://www.nature.com/doifinder/ . /gim. . mailto:jscottschwoerer@pediatrics.wisc.edu carrier testing for common inborn errors of metabolism in wisconsin’s plain population | kuhl et al education report branched-chain amino acids occurs, but elevations in leucine and -ketoisocaproic acid have particularly neurotoxic effects. in the amish population, pa is caused by a com- mon pathogenic variant, c. a>g, in the β-subunit of propionyl-coa-carboxylase, pccb, and the clinical disease can present at any time during the life span. during the neo- natal period, pa presents with poor feeding, lethargy, profound acidosis, and hyperammonemia. it can also present with meta- bolic decompensation in the setting of a stressor, often an inter- current illness. during decompensation, all individuals with pa are prone to brain injury resulting in ataxia, lethargy, coma, and seizures. long-term complications can include developmental delays, seizure disorder, and movement disorders due to basal ganglia injury. more recently, a cardiac phenotype with dilated cardiomyopathy, arrhythmia, and sudden death has been asso- ciated with pa. – the clinical phenotype most commonly seen in the old order amish includes cardiac involvement, although metabolic decompensation and neurologic sequelae have also been described. in the amish population, there are biochemically affected but seemingly clinically healthy indi- viduals who have not had the symptoms associated with the neonatal presentations of pa. they are later recognized to have pa after the sudden cardiac death of a family member prompts testing (j.s.s, g.r., personal experience). treatment includes diet; medications, including carnitine, biotin (pcc cofactor), and citrate; close cardiac monitoring; and standard care for car- diomyopathy and arrhythmia. unlike pa, msud in the old order mennonite population presents most often during the neonatal period. with accu- mulation of leucine, the neonate can quickly become enceph- alopathic—initially presenting with poor feeding, lethargy, and eventually coma. symptoms can develop at as early as – days of life, often before routine newborn screening results are available. immediate initiation of dietary therapy to reduce the leucine concentration is lifesaving; with long-term dietary and illness management, growth and developmental outcomes can be normal. unlike the general population, in which the incidence of msud is / , (carrier frequency of / ), approximately / individuals in the mennonite popula- tion carries a specific pathogenic variant (c. t>a) in the bckdha that causes classic, severe disease. the carrier testing provided through this project enables identification of couples who are at risk for having an infant with one of these conditions as well as preparation for the birth of a potentially affected baby, including diagnosis by variant analy- sis before  h of life and immediate initiation of dietary treat- ment for an affected neonate. this identification of increased risk, through either carrier testing or a known affected family member, has been shown to preempt nicu stays (j.s.s, g.r., personal experience). carrier testing also allows the identifica- tion of previously undiagnosed individuals with pa. this type of carrier testing has previously been performed for msud in the old order mennonite population in pennsylvania but has not previously been performed for pa. materials and methods patients all amish and mennonite families residing in wisconsin are eligible for msud carrier testing and pa carrier and diagnostic testing for the common pathogenic variants found in the plain population. the general population and plain families living outside wisconsin are not eligible for this study. the testing is provided by the wisconsin newborn screening program and is offered free of charge as part of the services the program pro- vides. this genetic testing is clinical testing and therefore did not require institutional review board approval for this study. carrier testing kits the genetic testing was performed using kits that included specimen collection supplies as well as consumer-friendly, con- dition-specific information, provider collection and condition information, and patient consent forms (see supplementary information online). for those who pursued testing, a blood sample obtained by finger prick was collected on a filter paper card, dried, and mailed to the wisconsin state laboratory of hygiene for analy- sis. targeted analysis for the common pathogenic variants found in the plain population for msud (bckdha c. t>a) and pa (pccb c. a>g) was completed using a laboratory- developed test. testing was ordered through a health-care provider but could be collected by a health-care provider or the family. families were made aware that this testing did not table metabolic newborn screening disorders more common in the amish and mennonite populations amish mennonite both galactosemia tyrosinemia type ( pathogenic variants) phenylketonuria glutaric acidemia type tyrosinemia type -methyl crotonyl coa carboxylase deficiency homocystinuria (due to methylenetetrahydrofolate reductase) phenylketonuria ( pathogenic variants) propionic acidemia medium-chain acyl coa dehydrogenase deficiency maple syrup urine disease biotinidase deficiency data from refs. , . genetics in medicine | volume | number | march kuhl et al | carrier testing for common inborn errors of metabolism in wisconsin’s plain populationeducation report replace the standard newborn screen. a diagram of this process can be seen in figure . the plain population does not provide formal education beyond the eighth grade and maintains different cultural norms than those outside of the plain population regarding the discus- sion of certain topics, such as pregnancy. thus, it was important that the material used to inform families about this testing and the testing results be clear, accurate, and culturally sensitive. all forms and information sheets were reviewed by metabolic geneticists, dietitians, genetic counselors, and a licensed mid- wife who follows patients in the plain population. outreach activities to increase accessibility, mail-order kits for both msud and pa testing were provided to midwives and other health-care providers following at-risk families. kits were also distrib- uted at plain population meetings where clinical information about these conditions and the available testing was provided. newborn screening education was also provided at the com- munity meetings through physician-led discussions and mid- wife workshops to increase awareness of these disorders in the plain population. in a deliberate effort to build trust through community rela- tionships, the family information for msud was also reviewed by a respected elder of a wisconsin plain community who has a family history of msud. the larger wisconsin partnership program (wpp) project also includes community elders on its board, and their input was sought for the carrier testing project as well as the other projects associated with this grant (newborn screening awareness and provision of research-based testing for a wider range of clinical conditions in the plain population). to better understand and meet community needs, we uti- lized existing local relationships and the current infrastructure of the wisconsin newborn screening program and wisconsin midwife guild. the majority of prenatal/perinatal care for the plain population is provided by midwives (licensed and unli- censed birth attendants from the community referred to as lay midwives) and sometimes local physicians. working closely with this group of care providers helped to establish trust with community members and facilitated effective and efficient access to and distribution of education materials, testing, and results. patient communication testing results were sent by mail to both the couple and the ordering provider. no letters were returned as undeliverable. those who tested positive or were at increased risk for hav- ing a child with msud or pa were offered further counsel- ing and medical care in an outreach clinic located in la farge, wisconsin, or at the university of wisconsin biochemical genetics clinic in madison, wisconsin. the results and kit dis- tribution were tracked in an excel spreadsheet. results distribution of kits from april to may , testing kits were distributed to four midwives ( kits), one local physician ( kit), and one public health nurse ( kits). these wisconsin health-care pro- viders were from the cities of withee, blue river, unity, athens, and eau claire. thirty-seven of these kits were utilized for test- ing. in addition, approximately kits were used directly by university of wisconsin biochemical genetics clinic and la farge medical clinic providers at outreach clinics and a com- munity meeting. nineteen of these kits were utilized for testing. carrier testing findings results were provided to individuals as well as their mid- wives or local physicians (table ). of these, three siblings in their early s were diagnosed with pa. for these individuals, the diagnosis of pa had been made with biochemical testing in infancy or early childhood but the family was lost to follow-up. they were returning for care and wanted to confirm the patho- genic variant for other family members. similarly, an at-risk couple pursued testing because they had a son who died from suspected pa and wanted to know if their adult children were at risk. they were both determined to be carriers of pa. testing figure flow diagram of the carrier testing process. patient information and consent forms were reviewed by plain community elders as well as healthcare providers and midwives working with the plain community for readability and cultural competency. each kit contained: patient information sheet patient consent form provider information sheet blood spot test card addressed envelope to return card and consent kits were mailed to requesting healthcare providers, including: local physicians and nurses midwives public health nurses healthcare providers were informed about kit availability through: plain community meetings wisconsin guild of midwives patients needed to sign a consent form and send this in with their sample before testing would proceed. consent included: recurrence risk testing only for common mutation does not replace newborn screen testing is voluntary results letter was sent to both the patient (s) and the ordering provider. the provider was also sent the lab report. testing was done at no charge as a service through the wisconsin newborn screen lab. if positive, an in-person appointment was set up at the uw genetics clinic or the la farge medical clinic. testing kit patient consent patient information about condition and test provider information about condition and test patient consent results letter labreport nbs lab pat ien t providertest card test card tonbs lab to nbs lab volume | number | march | genetics in medicine carrier testing for common inborn errors of metabolism in wisconsin’s plain population | kuhl et al education report was subsequently provided to all nine of their adult children. none were found to have pa caused by the common patho- genic variant, but four were found to be carriers. carrier test- ing was offered to their (future) spouses, and none of the tested spouses was found to be a carrier. because this testing looked only for these common patho- genic variants, negative results did not completely eliminate risk for the family or individual, but they did give a family and its health-care providers the ability to prepare for the possibility of an affected newborn. given the possibility of adult-onset pa in the amish population, variant analysis could also provide a diagnosis and allow initiation of treatment prior to the onset of symptoms. discussion targeted carrier screening for genetic disorders in specific pop- ulations is not new and has been successfully completed in other populations. as an example, screening for tay–sachs disease in the ashkenazi jewish population has been shown to be success- ful. using this example, various aspects need to be considered in developing a carrier screening project, including severity of the disease and availability of treatment, testing accuracy, cost- effectiveness, funding for testing and genetic counseling, defini- tion of the target population to be screened, development of a public/professional education program, informed consent for screening, and level of awareness in the community. the measure of success may vary depending on whether the disease is treatable. for the nontreatable tay–sachs disease, suc- cessful screening showed a dramatic decline in the incidence of the disease in the ashkenazi jewish population. for treatable metabolic diseases, however, preventing clinical presentation and need for hospitalization is a successful end point. this cur- rent approach of carrier screening and aggressive management of “at-risk” infants was initiated for infants at risk for msud at the clinic for special children in strasburg, pennsylvania. over years, at-risk neonates (identified by carrier testing or family history) were evaluated within  h and none required hospitalization. without this identification, other infants were identified; had clinical presentations of critical illness requiring hospitalization. because the common msud and pa pathogenic variants are site-specific in the plain population, the testing and its results are straightforward. molecular dna testing for both msud and pa was already offered by the wisconsin newborn screening program, and utilizing this existing service enabled economical testing. the needed outreach efforts in the plains population indicate the importance of education and counseling for any population- based testing program. – indeed, the educational component of this program was the key to its success. as part of a larger project funded by the wisconsin partnership program, sev- eral community meetings hosted by the plain population and attended by community elders, families, local physicians, and midwives were held and provided the opportunity to increase awareness of the disorders, the testing that was available, the benefits of newborn screening, and the treatability of many of these conditions. to encourage midwives to attend, training for the newborn screening process was provided. to establish trust and exemplify the benefit of this testing, panel discussions were held during the meeting and individuals with a family member who had either pa or msud spoke of their experience. communicating the testing information and results was a challenge because many plain communities do not use or have limited access to certain forms of communication (e.g., phones, e-mail). because of this limitation, information outside of meet- ings was communicated through health-care providers and word of mouth. it was unclear what background information the providers and community members had regarding genetic disorders. to overcome this challenge, we created provider and patient information sheets and sent the results letter to both the patients and the providers. we also provided a toll-free number for our clinic on the information sheets to help promote timely communication for those with phone access. although we did not diagnose any unsuspecting individu- als or unsuspecting at-risk couples, plans were in place to fol- low up in person with any patients with abnormal results, at the outreach clinic in la farge or the university of wisconsin clinics in madison. the testing greatly lowered the estimation of occurrence risk for the couples who underwent carrier test- ing (from as high as % to less than %) and promoted the importance of newborn screening to all who underwent this testing. there are limited survey data about the view of genetic test- ing among the amish and mennonites, and much of it is based on clinicians’ experience with the communities. a survey of amish, mennonites, and hutterites about genetic testing for cystic fibrosis showed that it is important to understand cul- tural differences as well as variability in the attitudes for test- ing. this fits with our current experience with the amish and mennonites in that this population is heterogeneous in its view of western medicine and the use of its services (unpublished data). for example, a survey performed through this wisconsin table results of testing over the course of year propionic acidemia msud molecular dna testing completed age in years (median age) – ( ) – ( ) known at-risk couplea b type of testing   carrier   diagnostic wild-type (no pathogenic variant) heterozygous (one pathogenic variant) homozygous (two pathogenic variants) c aat-risk couple is a couple with both partners who are at least heterozygous for the common pathogenic variant; therefore, risk for an affected offspring is at least %. bfamily identified as being at risk due to family history. cprior biochemical diagnosis as infant/young child and molecular dna testing to confirm diagnosis. msud, maple syrup urine disease. genetics in medicine | volume | number | march kuhl et al | carrier testing for common inborn errors of metabolism in wisconsin’s plain populationeducation report partnership program found that more than % of the plain families surveyed felt that newborn screening was encour- aged or common within their community, whereas just under % were unsure about their community view about newborn screening or felt it was uncommon or (rarely) discouraged. this project illustrates the importance of developing relation- ships with health-care providers who work closely with plain population members, community elders, and the community members themselves. future plans include improving the general communication of results and testing information through continued educational and training efforts with local health-care providers and mid- wives, continuing to work with the plain population to improve our cultural competency in order to optimize the offerings of the current carrier and diagnostic testing, and expanding our test menu to include similar conditions that are common in this pop- ulation, specifically, glutaric acidemia type and galactosemia. supplementary material supplementary material is linked to the online version of the paper at http://www.nature.com/gim acknowledgments we thank catherine reiser, laura birkeland, and gretchen spicer, for reviewing the manuscript. this study was funded, in part, by a wisconsin partnership program opportunity grant. disclosure the authors declare no conflict of interest. the authors have full control of the primary data, which are available for review by the journal if requested. references . strauss ka, puffenberger eg. genetics, medicine, and the plain people. annu rev genomics hum genet ; : – . . hostetler, ja. the amish, rd edn. herald press: harrisburg, va, . . young center for anabaptist and pietist studies, elizabethtown college. amish population by state, . http://www .etown.edu/amishstudies/population _by_state.asp. accessed september . . puffenberger eg. genetic heritage of the old order mennonites of southeastern pennsylvania. am j med genet c semin med genet ; c: – . . saudubray j, van den berghe g, wlater jh (eds). inborn metabolic diseases diagnosis and treatment, th edn. springer: new york, . . omim (online mendelian inheritance in man). johns hopkins university center for medical genetics: baltimore, md, . http://www .ncbi.nlm.nih. gov/ omim/. . pena l, franks j, chapman ka, et al. natural history of propionic acidemia. mol genet metab ; : – . . lee tm, addonizio lj, barshop ba, chung wk. unusual presentation of propionic acidaemia as isolated cardiomyopathy. j inherit metab dis ; suppl :s – . . laemmle a, balmer c, doell c, sass jo, häberle j, baumgartner mr. propionic acidemia in a previously healthy adolescent with acute onset of dilated cardiomyopathy. eur j pediatr ; : – . . jameson e, walter j. cardiac arrest secondary to long qt(c) in a child with propionic acidemia. pediatr cardiol ; : – . . kakavand b, schroeder va, di sessa tg. coincidence of long qt syndrome and propionic acidemia. pediatr cardiol ; : – . . bhan ak, brody c. propionic acidemia: a rare cause of cardiomyopathy. congest heart fail ; : – . . lücke t, pérez-cerdá c, baumgartner m, et al. propionic acidemia: unusual course with late onset and fatal outcome. metabolism ; : – . . mardach r, verity ma, cederbaum sd. clinical, pathological, and biochemical studies in a patient with propionic acidemia and fatal cardiomyopathy. mol genet metab ; : – . . strauss ka, puffenberger eg, morton dh. maple syrup urine disease. january (updated may ). in: pagon ra, adam mp, ardinger hh, et al. (eds). genereviews. university of washington: seattle, wa. . love-gregory ld, dyer ja, grasela j, hillman re, phillips cl. carrier detection and rapid newborn diagnostic test for the common y n maple syrup urine disease allele by pcr-rflp: culturally permissible testing in the mennonite community. j inherit metab dis ; : – . . vallance h, ford j. carrier testing for autosomal-recessive disorders. crit rev clin lab sci ; : – . . kaplan f. tay-sachs disease carrier screening: a model for prevention of genetic disease. genet test ; : – . . morton dh, morton cs, strauss ka, et al. pediatric medicine and the genetic disorders of the amish and mennonite people of pennsylvania. am j med genet c semin med genet ; c: – . . falik-zaccai tc, kfir n, frenkel p, et al. population screening in a druze community: the challenge and the reward. genet med ; : – . . zlotogora j, carmi r, lev b, shalev sa. a targeted population carrier screening program for severe and frequent genetic diseases in israel. eur j hum genet ; : – . . kaback m, lopatequi j, portuges ar, et al. genetic screening in the persian jewish community: a pilot study. genet med ; : – . . miller sr, schwartz rh. attitudes toward genetic testing of amish, mennonite, and hutterite families with cystic fibrosis. am j public health ; : – . . sieren s, grow m, goodsmith m, et al. cross-sectional survey on newborn screening in wisconsin amish and mennonite communities. j community health ; : – . volume | number | march | genetics in medicine http://www.nature.com/gim http://www .etown.edu/amishstudies/population _by_state.asp http://www .etown.edu/amishstudies/population _by_state.asp http://www .ncbi.nlm.nih. gov/omim/ http://www .ncbi.nlm.nih. gov/omim/ 	development of carrier testing for common inborn errors of metabolism in the wisconsin plain population 	introduction 	materials and methods 	patients 	carrier testing kits 	outreach activities 	patient communication 	results 	distribution of kits 	carrier testing findings 	discussion 	disclosure 	acknowledgements 	references words on fire: the unfinished story of yiddish (review) words on fire: the unfinished story of yiddish (review) daniel j. gilman journal of american folklore, volume , number , winter , pp. - (review) published by american folklore society doi: for additional information about this article [ this content has been declared free to read by the pubisher during the covid- pandemic. ] https://doi.org/ . /jaf. . https://muse.jhu.edu/article/ https://doi.org/ . /jaf. . https://muse.jhu.edu/article/ detailed overview of north african folklore begins with the “ancient egyptian legacy” and continues up to recent decades, highlighting important books and current trends. research- ers looking for a variety of sources and accurate references will also appreciate the entries on education (“folklore in schools”) and libraries, as well as the appendices, especially the com- prehensive treatment of films related to folklore listed in the last pages. since we are dealing with african folklore, there are countless entries for oral traditions and heritage: “jokes and humor,” “oral nar- rative,” “oral performance and literature,” “performance in africa,” “proverbs,” and “ver- bal arts,” plus an original text, “gossip and rumor.” all of these, as well as the accurate entry on popular culture, insist that africa be viewed as a diverse continent, not a cultural monolith. the contributors to the encyclopedia are clearly aware of the developments and crises that have occurred during recent decades in africa, and some authors discuss the presence of folklore in new media and technologies (“electronic media and oral traditions,” “ra- dio and television dramas”). on a tragic note, one article refers to tales of genocide in rwanda. an exemplary effort, african folklore: an encyclopedia is the kind of book that one can read for hours, since each article invites the reader toward another, and so on. one of the work’s strongest points is its accurate portrayal of the current state of research on every topic discussed. readers and critics in the field would be unfair to ask for more entries or to try to indicate the missing elements in such a hefty reference book. perhaps its high price may keep younger scholars from buying it, but most uni- versity libraries should acquire a copy. my only complaint is that there are too many biblio- graphical references in english for an interna- tional encyclopedia; i would have liked to find even more french authors mentioned. this marvelous encyclopedia confirms once again routledge’s expertise as a publisher of fine, up- to-date, readable reference books. i recommend that any scholar in ethnology or african studies be aware of and use this fine work. words on fire: the unfinished story of yid- dish. by dovid katz. (new york: basic books, . pp. xvi + , acknowledgments, notes on transcription, introduction, photographs and illustrations, index.) daniel j. gilman university of texas–austin dovid katz’s words on fire thoroughly lays out the history (and prehistory) of the yiddish lan- guage. starting with earliest antecedents of yid- dish in pre-biblical aramaic and canaanite, the book examines its birth in medieval germany, its development throughout the european con- tinent, the literary flowering of the language during its nineteenth-century “secular outburst,” and its most recent innovations in grammar and internet content. as much a history of the ash- kenazi diaspora as (in his phrase) a “linguogra- phy” (p. ), words on fire is a richly informed, well written, and critically engaged work. in his historiography, katz pays a good deal of attention to the cultural baggage that the language eventually accumulated, in particular the rise of various gendered understandings of yiddish’s place in the world of jewish europe. in one form or another, yiddish speakers have for centuries figured their native language as feminine or feminized. rabbinical judaism dis- couraged women from studying sacred texts, leaving educated women only yiddish writings to enjoy. as ashkenazi culture established itself, the social divide between educated men who mastered the hebrew of the bible and the ara- maic of the talmud (the “real men” of tradi- tional jewish culture) and uneducated men who generally could read only yiddish fo- mented an additional derogatory association with yiddish as the province of the unpresti- gious (and to a degree, unmanly) majority of the population. as katz demonstrates, the dis- missal and derogation of yiddish as an effemi- nate language persists up to the present day in some quarters—this, despite yiddish modern- ists who revalued their literary medium as a constant lover and hasidic sects that declared its sanctity as the proper language for the diasporic faithful. yiddish presents a fascinat- ing case study of the intersections of language book reviews journal of american folklore ( ) ideology and gender politics, especially in its relationship to the classical jewish languages and the majority tongues that surrounded and overlapped its territory. although katz painstakingly charts the his- tory of speakers of yiddish and provides an excel- lent framework for understanding how the lan- guage emerged and grew, he is rather parsimonious with the details of how the lan- guage itself changed over time and distance. he delves into this subject most deeply in the chap- ter “genesis,” in which he shows not only how historical analysis of word variants and grammar allows for a rough estimate of the place and time of the language’s origin but also how newly ar- rived jewish immigrants from southern europe and the near east to germany acclimated to the environment, playfully assigning biblical names to the european territories in which they found themselves. (ashkenaz, the name given to ger- many and later to all of yiddishland, comes from the book of jeremiah.) then again, katz is clearly concerned primarily with elucidating the social contexts of yiddish rather than linguistic nuts and bolts. as a professional linguist, katz has covered the technical details of yiddish in depth in many previous publications. the final chapter of the book addresses the most controversial aspect of yiddish: its future. katz treats this topic with admirable clarity and honesty, and he states unapologetically what yid- dish enthusiasts consider, to put it gently, to be a bitter pill to swallow: the linguistic and demo- graphic evidence suggests that, outside of aca- demia, the world of secular yiddish is doomed to die a natural death, albeit one tragically has- tened by the holocaust and stalin’s purges. the future of yiddish lies with the hasidic sects for whom the language has always been their native tongue and an important literary vehicle. katz makes the claim that yiddish as a living language cannot exist without its speakers maintaining intimate contact with the world of traditional jewish scholarship and its associated classical languages—what he summarizes as the “trilin- gualism of old ashkenaz” (p. ) —as well as retaining a privileged place in the home and in daily life. as he observes, even the most radical leftist yiddish writers were steeped in traditional learning before breaking with religion. without the classical teaching, katz argues, too many of the nuanced expressions of hebrew or aramaic derivation lose their psychosocial significance and disappear from the lexicon. likewise, with- out pride of place in ordinary communication, yiddish will gradually cede ground to the host languages that surround it in every community. in short, yiddish cannot long survive outside of a jewish community that largely keeps to itself and uses yiddish in at least some aspects of daily life. katz therefore ends with a call for linguists to focus seriously upon the living language of the hasidim, even as masters of secular yiddish literature offer a few last pearls of their craft for us to appreciate. mennofolk: mennonite and amish folk tra- ditions. by ervin beck. (scottsdale, ny: herald press, . pp. , foreword, preface, pho- tographs and illustrations, notes, suggested readings, credits.) alan l. chan lutheran theological seminary, hong kong the forty-sixth addition to the studies in ana- baptist and mennonite history book series published by herald press, ervin beck’s men- nofolk: mennonite and amish folk traditions demonstrates that mennonites and amish con- stitute a religious faith with folk traditions that can be traced to the anabaptists in europe in the sixteenth century. the nine chapters cover diverse traditional genres such as ethnic slurs, origin tales and beliefs, trickster tales, urban legends, protest songs, material culture, and festival. the author was an english professor at goshen college from to and is con- sidered to be an insider of the mennonite and amish culture. beck’s purpose for writing this book is to make both mennonites and interested non- mennonites more aware of the group’s cultural traditions. these traditions have been learned by word of mouth or customary example and have been transmitted to succeeding generations of mennonites. they involve both long-estab- lished materials and creative variants, and they express feelings, ideas, and values that are im- cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core modern judaism modem judaism explores modern jewish history, thought, and culture. thejournal examines a wide range of topics relative to t h e j e w i s h e x p e r i e n c e s i n c e t h e haskalah—from the zionist movement and the establishment of the state of is- rael to the rise of modern anti-semitism and its devastating climax in the holo- caust. book reviews and essays are also featured. published three times a year in february, may, and october. volume , number includes: representa- tions of jewish women in the works and life of elizabeth stem, ellen m. umansky • the unlearned lessons of the holocaust, alan milchman and alan rosenberg • the jewish councils of the main ghettos of lithuania, dina porat • franz rosenzweig's critique of buber's i and thou. maurice friedman • conservative halakha in israel, david ellenson s t e v e n t . k s t z , edstof prepayment is required. annual subscriptions: $ . , individuals, $ . , institutions. foreign postage: $ . , canada & mexico, $ . , outside north america. single issue prices: $ . , individuals; $ . , institutions. pay- ment must be drawn on a u.s. bank or made by international money order. md residents add % sales tax. for orders shipped to canada add % gst (# ). send orders to: the johns hopkins university press, po box . baltimore, md . *b call toll free - - - , mon.-fri., : - : et for visa or mastercard orders. or fax your order anytime: ( ) - . w t h e j o h n s h o p k i n s u n i v e r s i t y p r e s s has cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core b k i n n i o l u i v i e b u i r a a n d «rohi¥ i>e^* jê t a.hl a m e r i c a n odyssey daniel i". rice reinhold niebuhr and john dewey frequently have been identified as among the most influential american theologians and philosophers of their respec- tive times. although their direct contact in print and in political action was marginal, their substantive conflict over such issues as religion, naturalism, the liberal tradi- tion, and democracy both reflected and shaped much of america's inner dialogue from to mid-century and beyond. in this intriguing book, daniel rice makes a strong case that, although the clash between niebuhr and dewey was real and important, in a wider context the two shared more insights than either realized. 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" w h o t r v to m a i n t a i n a d e l i b e r a t e . r e l i g i o u s l y focused, c o m m u n i t y - o r i e n t e d life, b e c o m e s i n c r e a s i n g l y f a s c i n a t i n g . \nu\li style p r e s e n t s t h e a r t i f a c t s of a r e p r e s e n t a t i v e g r o u p of a m i s h from n o r t h e r n i n d i a n a , l . x a m p l c s of t h e s e c u l t u r a l a r t i f a c t s a n d art a r e p r e s e n t e d w i t h i n t h e c o n t e x t of a m i s h lives, i n c l u d i n g p h o t o g r a p h s of a m i s h c o m m u n i t y a c t i v i t i e s . the i n d i a n a s t a t e m u s e u m ' s p o t t i n g c r a m i s h c o l l e c t i o n w a s t h e s p r i n g b o a r d for t h i s b o o k . t h i s c o l l e c t i o n is as c o m p l e t e a g a t h e r i n g of a m i s h a r t i f a c t s as e x i s t s a n y w h e r e in t h e w o r l d . it i n c l u d e s q u i l l s , s e w i n g t o o l s , c l o t h i n g , h o m e f u r n i s h i n g s , t o y s , d o l l s , a n d o t h e r i t e m s found in a n d a r o u n d a m i s h c o m m u n i t i e s in n o r t h e r n i n d i a n a . p a g e s . color p h o t o s . b ^ w p h o t o s c l o t h s . .s p a p e r s . i i , i • „ ?-*j at bookstores or from j j v d x a n a . u siversity bloomington, in p r e s s orders: - - - cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the file:///nu/li https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core disruption of sonic hedgehog signaling in ellis-van creveld dwarfism confers protection against bipolar affective disorder original article disruption of sonic hedgehog signaling in ellis-van creveld dwarfism confers protection against bipolar affective disorder ei ginns , m galdzicka , rc elston , ye song , sm paul and ja egeland ellis-van creveld syndrome, an autosomal recessively inherited chondrodysplastic dwarfism, is frequent among old order amish of pennsylvania. decades of longitudinal research on bipolar affective disorder (bpad) revealed cosegregation of high numbers of evc and bipolar i (bpi) cases in several large amish families descending from the same pioneer. despite the high prevalence of both disorders in these families, no evc individual has ever been reported with bpi. the proximity of the evc gene to our previously reported chromosome p bpad locus with protective alleles, coupled with detailed clinical observations that evc and bpi do not occur in the same individuals, led us to hypothesize that the genetic defect causing evc in the amish confers protection from bpi. this hypothesis is supported by a significant negative association of these two disorders when contrasted with absence of disease (p = . , fisher’s exact test, two-sided, verified by permutation to estimate the null distribution of the test statistic). as homozygous amish evc mutations causing evc dwarfism do so by disrupting sonic hedgehog (shh) signaling, our data implicate shh signaling in the underlying pathophysiology of bpad. understanding how disrupted shh signaling protects against bpi could uncover variants in the shh pathway that cause or increase risk for this and related mood disorders. molecular psychiatry ( ) , – ; doi: . /mp. . ; published online october introduction bipolar affective disorder (bpad; manic-depressive illness) is a common psychiatric disorder with primary features of recurrence (cyclicity) and swings (polarity) from high to low of both mood and energy. individuals with bpad can shift from ‘mania to melancholia’ or from high affectivity and excitement to the profound low energy and sadness of depression. – bpad affects – % of the global population and is associated with a high risk of suicide. morbid risk analyses of bpad demonstrate a high prevalence of affective disorder (especially bpi) among first-degree relatives of bipolar probands in the amish study families. because of the long-term, longitudinal nature of the amish study, the unaffected, mentally healthy (well individuals) in these families were also followed, most for a period of years past the age of risk for bpad. although sporadic cases of ellis-van creveld syndrome occur in many populations, evc is frequent among the old order amish (ooa) of pennsylvania. unexpectedly, no evc individual has ever been reported with bpi, despite more than years of research documenting the cosegregation of evc and bpi in the same extended pedigree and descending from the same pioneer. twin, family and adoption studies have all provided strong evidence for an important genetic component in the susceptibility to develop bpad. , however, unlike most common medical illnesses, objective biological markers have not been identified for bpad, and genetic studies have had to rely on only clinical diagnoses. despite compelling clinical-epidemiologic evidence supporting a significant genetic susceptibility to develop bpad, identification of the genetic variants and an associated underlying molecular mechanism or pathophysiology have remained elu- sive. – genetic heterogeneity, phenocopies, genotyping errors and the complexities of performing and interpreting statistical analyses may have contributed to some of the inconsistences observed in genetic studies. , in , we reported the results of a genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bpad among the ooa. we found strong evidence for a locus in the proximity of the evc gene on chromosome p at d s (maximum genehunter-plus nonparametric linkage score = . , p = . × − ; sibpal empirical po × − ) and suggestive evidence for a locus on chromosome q at d s (maximum genehunter-plus nonparametric linkage score = . , p = . × − ; sibpal empirical po × − ; see supple- mentary figure s ). , the genes for evc and hedgehog- interacting protein (hhip), a shh antagonist, were subsequently cloned and found to be located within million bases of our chromosome p (d s ) and q (d s ) putative protective or susceptibility loci for bpad, respectively. taken together, these observations led us to postulate that the molecular mechanism underlying evc is protective against bpi. we now report clinical and statistical evidence that disruption of sonic hedgehog signaling in evc confers protection from bpi and perhaps more generally against affective disorders. departments of clinical labs, neurology, pediatrics, pathology and psychiatry, university of massachusetts medical school/umass memorial medical center, worcester, ma, usa; departments of clinical labs and pathology, university of massachusetts medical school/umass memorial medical center, worcester, ma, usa; department of epidemiology and biostatistics, case western reserve university school of medicine, cleveland, oh, usa; departments of neuroscience, psychiatry and pharmacology, weill cornell medical college of cornell university, new york, ny, usa and department of psychiatry and behavioral sciences, university of miami miller school of medicine, miami, fl, usa. correspondence: dr ei ginns, departments of clinical labs, neurology, pediatrics, pathology and psychiatry, reed rose and gordon building, university of massachusetts medical school/umass memorial medical center, maple avenue, shrewsbury, ma , usa. e-mail: edward.ginns@umassmed.edu received june ; revised august ; accepted august ; published online october molecular psychiatry ( ) , – © macmillan publishers limited all rights reserved - / www.nature.com/mp http://dx.doi.org/ . /mp. . mailto:edward.ginns@umassmed.edu http://www.nature.com/mp materials and methods patient samples/genotyping blood samples from ooa individuals were obtained with written informed consent approved by the institutional review boards for human subject studies at the university of miami miller medical school, the university of massachusetts medical school and the intramural research program at the national institute of mental health (nimh). lymphoblast and/or fibroblast cell lines were established at the coriell institute for medical research, camden, nj, usa, the clinical neuroscience branch, intramural research program, national institute of mental health, bethesda, md, usa and/or at the university of massachusetts medical school. the coriell nigms human catalogue of cell lines contains updated pedigree and bpad diagnostic information for selected large families from amish pedigrees. dna analysis genomic dna was obtained from peripheral blood samples, immortalized lymphoblastoid cell lines and/or skin fibroblasts. analysis for the amish evc gene intron (ivs + g t) mutation , on dna samples obtained from the amish pedigree subjects under study was performed using sanger sequencing and the massarray maldi-tof (sequenom/agena biosciences, san diego, ca, usa) platforms. evc ascertainment and diagnosis the current research on evc syndrome began years ago. evc families were ascertained systematically by amish church districts. evc subjects were examined and diagnosed at home or at the johns hopkins moore clinic in baltimore, md, usa. deceased were certified by death records. a pedigree trace was conducted for every evc sibship. the resulting progenitor charts tested whether all amish evc cases descended from a common progenitor. at that time, the single existing ooa genealogy covered only two founders (fisher and stoltzfus). it was coupled with archival materials, and subsequent research resulted in a large evc pedigree that has been previously published. , drafting progenitor charts for evc cases ascertained since the original research has benefited from the publication of a major genealogy that incorporates all present amish families tracked along different pioneer family lines. bpi ascertainment and diagnoses the amish study on major affective disorders ( – ) has been conducted with annual irb approval and multiple informed consents. details regarding ascertainment procedures, documentation of clinical materials and reliability of diagnoses have been published widely, including by the coriell nigms human catalogue of cell lines. , two types of clinical data (psychiatric interviews and medical records) were processed for independent and ‘blinded’ assessment by a five-member amish study psychiatric board using research diagnostic criteria (rdc) and diagnostic criteria from dsm-iii-iv (dsm) to make categorical diagnoses (see table ). after consensus diagnosis of bpi cases, their families became the basis of a large, multi-generational pedigree known as amish study bpad ped. / / / . results evc samples past and present the original evc study reported on evc sibships yielding dwarfs ( infant deaths and living to maturity). currently, of the original evc sibships have been updated and an additional families recently ascertained. this doubled the original sample to evc families with dwarfs, surviving to adulthood. among these individuals with evc were a number in the – years of age categories ( % over age years; % over age years) and past the window of risk for onset of bpi disorder. our sample includes nuclear families with one evc dwarf, with two, with three, with four and families with five evc dwarfs (total of dwarfs). data were also assembled regarding evc features among the ‘normal’ relatives in the extended evc families. relatives were identified with a missing digit, toe, arm, foot or leg, as further evidence of the broad spectrum of skeletal dysplasia in evc features for amish families. cosegregation of evc and bpi progenitor traces on confirmed bpi cases yielded the same pioneer, koenig, identified previously as the progenitor for evc and giving early evidence for significant cosegregation of these two illnesses. the individual progenitor traces for each evc family determined their precise location on bpad master pedigree / / / and resulted in a new amish study pedigree designated as evc/bpad pedigree . although the bipolar pedigree comprises bpi sub-pedigrees, the evc families were mainly included in four of them. (see figure ). apart from the documented cosegregation of evc and bpi, case ascertain- ment spanning four decades did not reveal subjects with comorbidity. association analysis two types of association analysis were performed on all individuals in the bpad master pedigree for whom both evc genotype and bpad diagnosis were available: a fisher’s exact test, which ignores all relationships, and large sample tests based on a logistic regression that allowed for the familial dependencies. only one primary hypothesis was tested, that of association between bpi/no disease status and evc/no disease status; all other tests were exploratory and for these p-values are given with no correction for multiple tests. it is important to note that, although individuals were genotyped because of being related to bpad- diagnosed pedigree members, all individuals who were geno- typed were included in the data, regardless of whether they had bpad or not (bpad disease/no disease status) when the sample table . diagnostic hierarchies for bipolar affective disorder (bpad) phenotypic subcategories for bpad (the standard dsm subcategory for the affective disorders) as developed by the amish study psychiatric board, according to research diagnostic criteria (rdc) and the dsm-iv guidelines subcategory (definitely affected): bipolar i (bpi), manic episodes, schizoaffective disorder: bp sub-type. subcategory (affected): plus bipolar ii, atypical bipolar (atyp:bp/bp:nos) major depressive disorder, recurrent subtype, tagged for bp (mddr.tag bp) note: the amish study found that the diagnosis of mddr.tag bp was likely to convert to a bpi over the course of the illness, especially if the patient was a first degree relative of a bpi subcategory (probably affected): + plus add diagnoses assumed to be in a bipolar spectrum: hypomanic, recurrent; major depressive disorder, recurrent (mddr) atypical psychosis, tagged bp, plus undiagnosed psychiatric disorder: tagged bp (undxbp) subcategory (possibly affected): + + plus the common diagnosis of a major depressive disorder, single episode (mdds) (note: given the nature of situational depression, this could be a false positive.) subcategory (unknown psychiatric): the other subcategory: includes hypomanic episodes, minor depression, intermittent minor depression, dysthymia, labile or cyclothymic personality, obsessive compulsive disorder, somatization disorder, generalized anxiety disorder, and other affective subcategory disorders. it also includes psychotic disorder, unspecified (psych.undx) – useful for historic cases. subcategory (definitely unaffected or mentally well): a critical subcategory for analyses of first degree well siblings with a brother/sister diagnosed with bpi. disrupted shh signaling protects against bpad ei ginns et al © macmillan publishers limited molecular psychiatry ( ), – for analysis was selected. thus, no ascertainment bias could result from the way the sample was selected. the primary hypothesis attained significance (p = . , fisher’s exact test, two-sided), verified by permutation to estimate the null distribution of the test statistic (see supplementary materials, especially supplementary table s ). most of the pairs of pedigree members included in this test, as for all the pedigree members, were unrelated at least up to third degree (see supplementary table s in supple- mentary materials). mean age of onset, when known, was – for subcategories – , for subcategory (see table and supplementary table s in supplementary materials). tables ( × ) were then formed from individual and groups of bpad disease/no disease subcategories (as defined in supplementary materials) and the two evc classes of interest, namely carriers of two mutant alleles versus all others, and tested under various mixed effect logistic models as well as by fisher’s exact test. we performed these exploratory logistic regression analyses as imple- mented in the s.a.g.e program assoc (https://code.google.com/ p/opensage/), which differentiates the dependence of siblings from that of the parent-offspring relationship, allows for a spousal correlation and performs two different but asymptotically equi- valent tests—wald tests and likelihood ratio (lr) tests. although the same p-value for testing the same null hypothesis does not guarantee that we can rely on the asymptotic test, different p- values automatically imply that the sample is not large enough for the asymptotic test to be reliable. the latter assumes no numerical inaccuracies, which might occur owing to computer limitations, but would not be relevant for the small number of significant digits we show in our tables. the regression analyses could thus detect a polygenic component of variance, as well as variance components attributable to effects common to spouses, common to siblings over and above that attributable to a polygenic effect, and/or individual-specific effects. the wald and lr tests, unlike fisher’s exact test, are strictly valid only for large samples, but can be compared for samples of approximately the same size. figure summarizes noteworthy features of the numerous analyses performed, showing the percent of the sample variance that can be attributed to evc status under a full model, and p-values that figure . pedigree sample evc families. disrupted shh signaling protects against bpad ei ginns et al molecular psychiatry ( ), – © macmillan publishers limited https://code.google.com/p/opensage/ https://code.google.com/p/opensage/ make large sample assumptions, for the corresponding associa- tion of disease/no disease bpad subcategories with disease/no disease evc status (see supplementary materials, especially supplementary tables s and s ). taken together, these results suggest evc has a protective effect against all subcategories – of bpad. small p-values for association analysis indicate no more than that the null hypothesis of no association is highly unlikely, with no indication of what that association may be due to, as it could be a spurious association due to any number of causes, including an incorrect statistical model. furthermore, p-values are dependent on the sizes of the samples compared. that is why we show in figure the estimated percent of the total sample variance that can be attributed to evc segregation for the subcategory contrasts tested. these estimates, but not their s.e., would be expected to reflect the variance components indepen- dent of sample size. for our primary hypothesis, when evc status was included in the model the polygenic component decreased from . to . %, whereas the individual variance component increased much less, from . to . %. this strongly suggests that the association found by fisher’s exact test is mostly due to segregation at the evc locus. no other variance components were detected for this test, but other tests that were performed detected (non-significant) variance components. it is unlikely that evc is protective against subcategory , as the percentage of total variance attributable to evc segregation is the largest for the comparison of subcategory versus subcategories and combined; here the polygenic variance component decreased from . to . %, whereas the individual variance component increased from . to . %, on including evc in the model (see supplementary table s in the supplementary materials). discussion the cosegregation of evc and bpad in our large, multigenera- tional amish pedigree provided a rare, informative ‘experiment of nature’. decades of careful longitudinal tracking of evc and bpad cases in amish families (same extended pedigree, descending from the same pioneer) led to our observation that through figure . subcategory contrasts that result in the largest estimates of variance attributable to evc, ranked by magnitude from left to right. the model allows up to four variance components, in addition the effect of evc, to be estimated. subcategories are defined in table : diagnostic subcategories. (a) top panel: variance attributable to evc, as a percent of the total sample variance, and approximate s.e. bars. bottom panel: tests of the evc effect when included as a covariate in the model; values of − log p for the wald and likelihood ratio (lr) tests. (b) fourfold tables showing the observed numbers and expected numbers under independence for each of the subcategory contrasts tested. disrupted shh signaling protects against bpad ei ginns et al © macmillan publishers limited molecular psychiatry ( ), – multiple generations no individual with evc had ever been reported with bpi. analyses performed to test our primary hypothesis, that is, that of association between absence of bpi and evc (p = . , fisher’s exact test, two-sided; see supple- mentary materials, especially supplementary table s ), supported our hypothesis that evc confers protection (that is, mental health wellness) from bpi, as well as suggesting a more general protection against the spectrum of affective disorders in these families (see supplementary materials, especially supplementary tables s and s ). attempts to confirm this association can be performed in large gwas data sets, by restricting analyses to genes comprising or interacting with the shh pathway. the significant association between absence of bpi and evc, along with evc causing disruption of shh signaling and linkage evidence for protection/susceptibility genes for bpad at the p evc and q hhip loci suggested the involvement of shh signaling in mood disorders. to date, most genetic studies of affective disorders have been limited to identifying genetic variants that increase the risk of disease. by contrast, we have previously provided evidence that, in addition to rare susceptibility alleles, there may be rare alleles that reduce the risk of developing bpad in a manner similar to that reported for other complex inherited disorders. false- negative genomic study findings could result when individuals inherit disease susceptibility alleles, but are misclassified because they do not manifest the phenotype due to the presence of protective alleles. , although the concept that rare protective alleles could modify (or even prevent) a behavioral phenotype like bpad is relatively novel, several examples for non-psychiatric diseases have been reported. for instance, individuals in limone sul garda in northern italy were discovered to have apo a-imilano, a rare mutant apolipoprotein that is associated with a reduced risk of atherosclerosis. among ecuadorian villagers, the rare, auto- somal recessively inherited laron syndrome (ghrd) dwarfism appears protective of diabetes and cancer due to reduced levels of insulin-like growth factor . – more recently, a rare mutation in the amyloid precursor protein gene has been shown to protect individuals from developing alzheimer's disease. cosegregation of diseases that interact in this way is more likely to occur in a genetic isolate like the amish, where there are limited numbers of founders. our earlier genome-wide linkage data suggested the presence of two loci with rare alleles protective of bpad located on chromosome , one on p at d s and the other on q at d s , respectively (see supplementary figure s ). , the proximity of the evc gene to our previously reported chromosome p bpad protective/susceptability locus, coupled with detailed clinical observations and statistical confirmation that evc and bpi do not occur in the same amish individuals, led us to consider that the genetic defect causing evc in the amish confers protection from bpi. the discovery that evc is the result of disrupted shh signaling focused our attention on the sonic hedgehog (shh) signaling pathway (figure ). – the presence of a shh antagonist gene, hedgehog-interacting protein (hhip), at our figure . the sonic hedgehog signaling pathway. (a) in the off-state, shh is inhibited by hedgehog-interacting protein (hhip). in the absence of shh, smo is inhibited by patched (ptch- ) receptor. smo bound to patched is unable to make a complex with evc, evc , sufu (supressor of fused), fu (fused) and other proteins. the gli proteins are phosphorylated by protein kinase (pka) and form repressors that move to the nucleus and repress the gli-dependent transcription of targeted genes. (b) in the active state, shh covalently linked to cholesterol moiety (n- shh) binds to the patched /smo complex and releases smo. evc and evc are required for smo activation and for releasing gli proteins from their associated cytoplasmic factors. gli activators translocate into the nucleus where they activate transcription of a variety of genes, including (a) gli itself that is responsible for a positive feedback loop, (b) genes such as ptch- and hhip that set up a negative feedback loop and (c) other genes coding for proteins involved in the wnt pathway. glycogen synthase kinase- (gsk- ) negatively regulates the shh signaling pathway by promoting degradation of gli . lithium blocks the dephosphorylation of gsk -β causing activation of target proteins including gli . disrupted shh signaling protects against bpad ei ginns et al molecular psychiatry ( ), – © macmillan publishers limited bpad chromosome q protection/susceptibility locus is addition- ally suggestive of shh signaling involvement in bpad. the association of evc and shh pathway mutations has been mainly with a heterogeneous group of inherited skeletal disorders, holoprosencephaly and a wide range of tumor growth, progression and metastasis. – evc syndrome in the amish of pennsylvania is the result of homozygous intron (ivs + g t) evc gene mutations. the clinical manifestations of evc are diverse: some patients dying a few days after birth, while others live a long and active life. , more recently, attention has focused on the importance of evc and evc in primary cilia signal transduction, structures enriched in key components of the shh- transduction pathway, controlling the kind, numbers and patterning of cells during development in many tissues, including the nervous system. the correct localization and stoichiometry of evc and evc proteins as a complex in primary cilia are required for their normal function as positive modulators of the shh pathway signaling. lack of this normal evc/evc protein complex disrupts shh signaling (figure ). , , it is likely that the mechanism by which this homozygous mutation acts is by overriding abnormal shh signaling to protect against appearance of bpad disorder in these high-risk multigenerational pedigrees. although hedgehog ligands encode signaling molecules in a wide range of tissues, sonic hedgehog (shh) is the only hedgehog family member reported to be expressed in the mammalian central nervous system. – a growing body of evidence suggests that the state-like and oscillatory interactions of gene products within and extending from the shh signaling pathway (figure ), , , , and modu- lated by environmental factors, could constitute the basis for the wide range of phenotypic manifestations of bpad. – antide- pressant drugs, including lithium, and electroconvulsive therapy have been shown to alter shh signaling. glycogen synthase kinase (gsk ; gsk α/gsk β) is a target of lithium and has a central role in shh signaling. in addition to lithium, valproate, selective -ht reuptake inhibitors, monoamine oxidase inhibitors and tricyclic antidepressants have been shown to alter gsk activity. studies on interactions between shh and corticotropin-releasing hormone (crh) signaling networks have related shh signaling to neurotransmitter systems underlying anxiety, stress and depressive disorders. , cholesterol and palmitoic acid, required for appropriate shh processing and long range signaling, – have been associated with suicide, depres- sion and bpad. significantly more suicide attempters and completers have been reported among the biological relatives of smith-lemli-opitz syndrome carriers, a population of indivi- duals with reduced activity of -dehydrocholesterol reductase (dhcr ), an enzyme required for shh processing. altered brain sterol composition, involving cholesterol, -dehydrocholesterol and/or -dehydrodesmosterol, has also been associated with a greater risk of suicidal behaviors. variants in evc have recently been reported to be associated with male completed suicide. interestingly, the amish study ascertained ooa suicides (n = ) for the period – and found that they were heavily clustered in ped , almost all were males with bpad, equally divided between bipolar and major depression. it is possible that bpad, evc and male suicide in these amish study families could be explained by genetic variants that influence shh signaling. , our study of the cosegregation of evc and bpad in the old order amish further implicates shh signaling in the pathophysiol- ogy of bpad and suggests that other genes/proteins in the shh signaling pathway may be involved in protection or susceptibility to developing mood disorders. the increased understanding of the molecular basis of shh signaling and reactivation occurring in a wide range of cancers has led to the identification of shh signaling antagonists that are already in human clinical studies. repurposing of drugs targeting shh signaling that are already in clinical development for other medical conditions could lead to better treatments for affective disorders in the near future. conflict of interest the authors declare no conflict of interest. acknowledgments we thank the essential function of the amish study psychiatric board since : drs abram m hostetter, john j schwab, jon a shaw, jean endicott and the late drs james n sussex and david r offord. we thank cleona r allen for pedigree drafting and editing, bernadette warman as graphic artist, mary f sweger for progenitor charts, martha p hansell for computer consultation and alma becker, project phlebotomist, for collection of all evc blood samples. our deepest respect and appreciation belongs to old order amish patients and families who participated in this research commencing in . we thank betty and irving brudnick, patient families and organizations, and our own families for their encouragement and support. we are grateful for support from the case western reserve university amasa b. ford md chair of geriatric medicine (rae), the brudnick neuropsychiatric research institute (eig; 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: – . egeland ja, sussex jn. suicide and family loading for affective disorders. jama ; : – . hur em, zhou fq. gsk signalling in neural development. nat rev neurosci ; : – . kim wy, wang x, wu y, doble bw, patel s, woodgett jr et al. gsk- is a master regulator of neural progenitor homeostasis. nat neurosci ; : – . sheikh a, alvi aa, aslam hm, haseeb a. hedgehog pathway inhibitors—current status and future prospects. infect agent cancer ; : . supplementary information accompanies the paper on the molecular psychiatry website (http://www.nature.com/mp) disrupted shh signaling protects against bpad ei ginns et al molecular psychiatry ( ), – © macmillan publishers limited 	disruption of sonic hedgehog signaling in ellis-van creveld dwarfism confers protection against bipolar affective disorder 	introduction 	materials and methods 	patient samples/genotyping 	dna analysis 	evc ascertainment and diagnosis 	bpi ascertainment and diagnoses 	results 	evc samples past and present 	cosegregation of evc and bpi 	association analysis 	discussion 	acknowledgements 	note 	references original article analysis of quantitative lipid traits in the genetics of niddm (gennid) study alka malhotra, johanna k. wolford, and the american diabetes association gennid study group* coronary heart disease (chd) is the leading cause of death among individuals with type diabetes. dyslipidemia con- tributes significantly to chd in diabetic patients, in whom lipid abnormalities include hypertriglyceridemia, low hdl cholesterol, and increased levels of small, dense ldl par- ticles. to identify genes for lipid-related traits, we per- formed genome-wide linkage analyses for levels of triglycerides and hdl, ldl, and total cholesterol in cau- casian, hispanic, and african-american families from the genetics of niddm (gennid) study. most lipid traits showed significant estimates of heritability (p < . ) with the exception of triglycerides and the triglyceride/ hdl ratio in african americans. variance components analysis identified linkage on chromosome p . - q . for the triglyceride/hdl ratio (logarithm of odds [lod] � . ) and triglyceride (lod � . ) in caucasian families. statistically significant evidence for linkage was identified for the triglyceride/hdl ratio (lod � . ) on p in hispanic families in a region that showed suggestive evi- dence for linkage (lod � . ) for triglycerides in this population. in african americans, the strongest evidence for linkage (lod � . ) was found on p . - q . for total cholesterol. our findings provide strong support for previous reports of linkage for lipid-related traits, suggesting the presence of genes on p . - q . , p . - p . , and p . - q . that may influence traits underlying lipid abnormalities associated with type diabetes. diabetes : – , c ardiovascular disease is the leading cause of death among individuals with type diabetes ( ). dyslipidemia plays a major role in the development of cardiovascular disease in type diabetic patients, in whom lipid abnormalities are charac- terized by hypertriglyceridemia and reduced levels of hdl cholesterol present mainly in the form of small, dense hdl particles ( , ). levels of ldl cholesterol are typically normal or only mildly elevated; however, an increased level of small, dense ldl particles that are highly athero- genic is frequently a component of diabetic dyslipidemia ( , ). disregulated lipoprotein metabolism and risk for cardiovascular disease most likely precede the develop- ment of glucose intolerance and frank diabetes ( ). haffner et al. ( ) showed that among individuals who were normoglycemic at baseline examination, those who later progressed to diabetes had lower hdl levels and higher levels of ldl and total cholesterol compared with individ- uals who remained diabetes free. in this study, differences in lipid levels persisted following adjustments for bmi and level of glycemia but not fasting insulin concentration. similarly, in analyses using nuclear magnetic resonance spectroscopy, garvey et al. ( ) found that increasing severity of insulin resistance, in both diabetic and nondi- abetic individuals, was associated with ) increased vldl size and levels of large vldl particle concentrations (where circulating triglycerides are predominantly car- ried); ) decreased hdl size due to depletion of large hdl particles and increased levels of small hdl particles; and ) increased levels of small, dense ldl particles and an overall increase in the total number of ldl particles. combined, these findings suggest that insulin resistance, and possibly hyperinsulinemia, likely underlie the lipid- related changes associated with type diabetes. diet and exercise are among the most common environ- mental factors affecting lipid levels ( , ); however, genetic determinants of several monogenic lipid-related disorders have been clearly established ( ). for example, muta- tions in the ldl receptor (ldlr) gene underlie familial hypercholesterolemia, which is marked by excessively high ldl levels ( ), and defects in the atp binding cassette a (abca ) gene lead to tangier disease, char- acterized by reduced hdl levels ( – ). despite the considerable success in identifying mutations underlying monogenic lipid-related disorders, genetic determinants of lipid traits in the general population remain largely un- known. based on twin and pedigree analyses, genetic heritability (the proportion of variance due to genetic factors) of lipid levels has been estimated to range from . to . ( , ), suggesting roles for both environmen- tal and genetic components. over genome scans have been performed to identify loci affecting lipid levels (for a comprehensive review, see bossé et al. [ ]). of these, four were performed in families who were originally ascertained for type diabetes ( – ). duggirala et al. ( ) identified a major susceptibility locus for plasma triglycerides on q in mexican-american families ascertained for type diabetes. linkage to chro- mosome was identified for total cholesterol, triglycer- ides, and ldl in pima indians ( ), non-hispanic caucasians ( ), and old order amish ( ), respectively, in a region linked with lipid traits in nondiabetic study samples. additional regions of linkage for lipid traits in from the translational genomics research institute, phoenix, arizona. address correspondence and reprint requests to johanna k. wolford, diabetes and obesity research unit, translational genomics research insti- tute, n. th st., phoenix, az . e-mail: jwolford@tgen.org. received for publication april and accepted in revised form july . *a list of the american diabetes association gennid study group mem- bers can be found in the appendix. cad, coronary artery disease; chd, coronary heart disease; gennid, genetics of niddm; lod, logarithm of odds. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , october type diabetic families include q -q ( ), q ( ), and q ( ). the goal of this study was to identify quantitative trait loci that affect the diabetes-related plasma lipid profile in ethnically diverse populations. for this purpose, we ana- lyzed genotype data for � microsatellite markers in non-hispanic caucasian, hispanic, and african- american families who participated in the genetics of niddm (gennid) study, which comprises a repository containing extensive phenotypic and genotypic informa- tion on individuals from different ethnic groups ascer- tained by the presence of at least two type diabetic siblings ( ). heritability estimates for lipid traits were obtained in these families, and variance components link- age analyses were performed for log-transformed plasma levels of triglycerides, hdl, ldl, and total cholesterol, as well as triglyceride/hdl and ldl/hdl ratios. research design and methods the gennid study was initiated in to establish a repository of phenotypically well-characterized families ascertained by the presence of at least two siblings with type diabetes. ascertainment was completed in two phases: phase i families contained at least three other first-degree relatives and a nondiabetic ethnically matched control (i.e., an unaffected spouse) in addition to two diabetic siblings; phase ii families had at least two diabetic siblings and both parents or, in cases with no available parent(s), up to two additional siblings ( ). the present study used phenotypic data from both phase i and ii families. clinical and anthropometric measures, as well as family/medical histories for all gennid participants, were collected from different clinical centers throughout the u.s. diabetes was documented according to the national diabetes data group criteria (i.e., either a fasting plasma glucose concentration of � mg/dl on more than one occasion or a plasma glucose concentration of � mg/dl in a -h sample and in at least one other sample taken during an oral glucose tolerance test) ( ). among the clinical data collected, measures for fasting plasma levels of triglycerides and hdl, ldl, and total cholesterol were also obtained ( ). plasma levels of triglycerides, hdl, and total cholesterol were directly measured. triglycerides were measured using a free-glycerol blanking method, hdl was measured following heparin-manganese sulfate precipitation, and total cholesterol was measured enzymatically ( ). plasma ldl concentration was estimated using the friedewald equation [ldl � total cholesterol � hdl � (triglyceride/ )] ( ). this study used phenotypic data from the gennid database for non-hispanic caucasians ( families), hispanics ( families), and african americans ( families). non-hispanic caucasian family size ranged from to members with parent-offspring pairs, sibling pairs, cousin pairs, uncle/aunt-niece/nephew pairs, and grandparent-grand- child pairs. hispanic family size ranged from to members with parent-offspring pairs, sib-pairs, cousin pairs, uncle/aunt-niece/ nephew pairs, and grandparent-grandchild pairs. african-american families consisted of – individuals with parent-offspring pairs, sibpairs, cousin pairs, uncle/aunt-niece/nephew pairs, and grandparent-grand- child pairs. genotypes. whole-genome microsatellite marker (marshfield screening sets and ) genotyping was performed at the marshfield medical research foundation (marshfield, wi), and additional markers were genotyped at glaxo wellcome (research triangle park, nc) to fill gaps in one or more of the sample populations ( ). in total, , , and microsatellite markers were genotyped in the non-hispanic caucasian, hispanic, and african-amer- ican study groups, respectively. average marker spacing ranged from � to cm, and average marker heterozygosity for each sample population was � . ( ). statistical analyses. the simwalk program ( ) was used to identify mendelian genotyping errors, which were then coded with missing values. in analyses of the phenotypic data, outliers with trait values � sds were removed to avoid any bias that might be introduced into the results (n � in caucasians, in hispanics, and in african americans). some of the lipid traits (hdl, triglycerides, and ldl/hdl and triglyceride/hdl ratios) were not normally distributed. we therefore log- or square root–transformed these lipid concentrations, and the statistical analysis system package (sas institute, cary, nc) was used for regression analysis of the transformed lipid traits. the covariates tested were sex, duration of diabetes, bmi, waist circumference, hip circumference, waist-to-hip ratio, age, weight, and their square and cubic terms, smoking status, and alcohol intake status. adjustment of covariates significantly related to plasma lipid levels at the . level was performed separately in individuals affected and unaffected with diabetes to remove any effects of diabetes status. the adjusted and standardized [n ( , )] residuals for all traits were approximately normally distributed with skewness ranging from � . to . and kurtosis ranging from � . to . . in this study population, there were caucasians, hispanics, and african americans taking lipid-lowering drugs; data analysis was performed both including and excluding these individuals. since the most commonly used diabetes drugs were sulfonylureas, which do not exert direct effects on lipid levels, no adjustments were made for individuals on medication for type diabetes. genetic heritability was estimated for the adjusted and standardized residuals using the pedigree analysis package ( ). in addition, the six lipid traits (we will refer to the transformed, adjusted, and standardized lipid levels as total cholesterol, triglycerides, ldl, hdl, and ldl/hdl and triglyceride/ hdl ratios) were subjected to variance components linkage analysis as implemented in the program genehunter ( ). to assess the genome-wide significance of our linkage signals observed in caucasians, african americans, table characteristics of study participants caucasians hispanics african americans men women men women men women n age (years) . � . . � . . � . . � . . � . . � . bmi (kg/m ) . � . . � . . � . . � . . � . . � . triglycerides (mg/dl) . � . . � . . � . . � . . � . . � . ldl (mg/dl) . � . . � . . � . . � . . � . . � . hdl (mg/dl) . � . . � . . � . . � . . � . . � . total cholesterol (mg/dl) . � . . � . . � . . � . . � . . � . data are means � sd. descriptive statistics of lipid levels and covariates in caucasians, hispanics, and african americans are given. lipid concentrations represent untransformed, unadjusted values. table heritability estimates for lipid levels in caucasians, hispanics, and african americans heritability estimate � se lipid level caucasians hispanics african americans total cholesterol . � . . � . . � . hdl . � . . � . . � . ldl . � . . � . . � . triglycerides . � . . � . . � . triglyceride/hdl ratio . � . . � . . � . ldl/hdl ratio . � . . � . . � . heritability (h ) was estimated for caucasian, hispanic, and african-american families. all h estimates were significant (p � . ) except for triglycerides and triglyceride/hdl ratio in african americans (p � . ). linkage analysis of quantitative lipid traits diabetes, vol. , october and hispanics, , replicates of the genome were simulated using the program simulate ( ). the family structure and marker allele frequency information of the original dataset were used to generate the replicates. variance components linkage analysis was then performed on all the repli- cates, and a logarithm of odds (lod) score threshold was estimated for a false-positive rate anywhere in the genome of . . we estimated the lod score threshold by first subjecting each replicate to variance components linkage analysis, then obtaining the maximum lod score for each replicate and ranking these lod scores in ascending order. the lod score threshold was identified as the lod score that was exceeded in � % of replicates. in addition, empirical p values were estimated as the proportion of replicates equal to or exceeding the observed lod score. due to time and computational constraints, we performed simulations only for those traits showing the highest lod scores (i.e., triglycerides, triglyceride/hdl ratio, and total cholesterol). results the characteristics of each study sample used in our analyses are shown in table . diabetes prevalence was % in both caucasians and hispanics and % in african americans. in the caucasian sample, covariates explained – % and – % of the phenotypic variance in diabetic and nondiabetic individuals, respectively. in the hispanic sample, covariates explained less of the phenotypic vari- ance than in caucasians: – % and – % in diabetic and nondiabetic individuals, respectively. covariates in afri- can americans explained levels of phenotypic variance comparable to those seen in the hispanic sample (i.e., – % and – % for diabetic and nondiabetic individuals, respectively). statistically significant estimates of heritability were observed for all plasma lipid traits in caucasians (p � . ) and hispanics (p � . ) as shown in table . in african americans, total cholesterol, ldl, hdl, and ldl/ hdl ratio showed substantial heritability (p � . ), but triglycerides and triglyceride/hdl ratio did not (table ; p � . ). variance components linkage analyses were performed for all lipid traits in each study population. the highest lod scores were found on chromosome for both triglyc- eride/hdl ratio (lod � . ; empirical p value � . ) and triglycerides (lod � . ; empirical p value � . ) in caucasian families. an lod score threshold of . was obtained following simulation of , replicates, making the lod score for both triglycerides and triglyceride/hdl ratio slightly less than the level of statistical significance. suggestive evidence for linkage was observed on chromo- somes , , and for ldl, chromosome for triglycer- ides and triglyceride/hdl ratio, chromosomes and for total cholesterol, and chromosome for the ldl/hdl ratio (table ). statistically significant evidence for linkage was ob- served for triglyceride/hdl ratio (lod � . ; empirical p value � . ) on chromosome p in hispanic families, where suggestive evidence for triglycerides (lod � . ; empirical p value � . ) was also found. simulation results for observing a false-positive rate of . anywhere in the genome identified an lod score threshold of . (p � . ). suggestive evidence for linkage was observed on chromosomes and for total cholesterol, and for ldl/hdl ratio, chromosome for hdl, and chromo- some for triglycerides and triglyceride/hdl ratio (table ). in african americans, three chromosomal regions were identified with multipoint lod scores � . (table ). the strongest evidence for linkage was found on chromosome for total cholesterol (lod � . ; empirical p value � . ). an lod score threshold of . (p � . ) was table regions showing evidence for linkage (multipoint lod � . ) in caucasians chromosome lipid level multipoint lod -point lod closest marker -lod interval (cm) triglyceride . . gata c – triglyceride/hdl ratio . . gata c – triglyceride . . d s – triglyceride/hdl ratio . . d s – total cholesterol . . mfd – ldl . . mfd – total cholesterol . . d s – ldl . . ggaa c – ldl . . d s – ldl/hdl ratio . . d s – variance components linkage analysis was performed using data from families. table regions showing evidence for linkage (multipoint lod � . ) in hispanics chromosome lipid level multipoint lod -point lod closest marker -lod interval (cm) total cholesterol . . d s – total cholesterol . . d s – total cholesterol . . d s – ldl/hdl ratio . . d s – ldl/hdl ratio . . d s – triglyceride/hdl ratio . * . d s – triglyceride . . d s – triglyceride . . gata a – triglyceride/hdl ratio . . gata a – ldl/hdl ratio . . d s – variance components linkage analysis was performed using data from families. *significant evidence for linkage based on a simulation study. a. malhotra and associates diabetes, vol. , october obtained following simulation of , replicates, making the lod score for total cholesterol slightly less than the level of statistical significance. suggestive evidence for linkage for ldl was also detected in the same region on chromosome (lod � . ). additional regions of linkage included chromosome for ldl/hdl ratio (lod � . ) and hdl (lod � . ), chromosome for ldl (lod � . ), chromosome for total cholesterol (lod � . ), and chromosome for triglycerides (lod � . ) and triglyceride/hdl ratio (lod � . ). discussion genetic factors most likely underlie control of lipid traits in the general population. we found substantial genetic heritability for most lipid traits in the three populations comprising this study, and the estimates shown here correspond well with previously reported values ( , ). high estimates of genetic heritability imply familial aggre- gation of lipids; therefore, a gene underlying the inheri- tance of lipid levels may be detectable through the use of linkage and association analyses. the highest lod scores obtained in these analyses were found for triglyceride/hdl ratio (lod � . ) and triglyc- erides (lod � . ) on p . - q . in caucasian fam- ilies. this chromosomal region overlaps with intervals that have been identified by linkage analysis in at least five previously published studies (fig. ). the majority of linkage findings in this region were obtained in caucasian populations (four of five) using different ascertainment schemes as well as different lipid-related traits (i.e., quan- titative and qualitative). analysis of the triglyceride/hdl ratio showed evidence for linkage in a coronary heart disease (chd)-ascertained mauritian population ( ) and a randomly ascertained population from the framingham heart study ( ). in a bivariate analysis of total cholesterol and triglycerides, evidence for pleiotropic effects of a gene residing in this region was found ( ). the interval iden- tified in the present study has also been implicated in discrete trait analyses of familial hypercholesterolemia ( ) and coronary artery disease (cad) ( ). four of the above studies (including the present study), using either randomly or disease-ascertained families, showed evi- dence for linkage on chromosome for triglycerides and/or triglyceride/hdl ratio, and a fifth study identified linkage for cad in this region. because triglycerides levels are generally increased in individuals with cad and dia- betic dyslipidemia, these results strongly support the presence of a common gene on chromosome , which affects triglycerides levels in both randomly and disease- ascertained families. the strongest evidence for linkage (lod � . ) in the hispanic study sample was found for triglyceride/hdl ratio on chromosome p . - p . . based on a simula- tion study for observing a false-positive rate of . anywhere in the genome, this lod score was statistically significant. the linked region on chromosome has also been reported in several other studies, one of which included families ascertained for type diabetes (fig. ). fig. . a summary of studies showing evidence for linkage on chromosome . the ethnic background of the study population is given. horizontal lines with arrows represent the approximate -lod support interval on a cytogenetic map (bottom) for the respective studies. the closest marker and position (cm, vertical line) of the maximum lod score or lowest p value (right) are indicated above the horizontal line. table regions showing evidence for linkage (multipoint lod � . ) in african americans chromosome lipid level multipoint lod -point lod closest marker -lod interval (cm) total cholesterol . . mfd – ldl . . d s – hdl . . d s – ldl/hdl ratio . . d s – variance components linkage analysis was performed using data from families. linkage analysis of quantitative lipid traits diabetes, vol. , october evidence of linkage to this region was observed in families ascertained by the presence of familial combined hyper- lipidemia (lod � . for triglycerides and lod � . for total cholesterol in the same region) ( ) in the quebec family study (lod � . for triglycerides) using both randomly and obesity-ascertained families ( ) and in the old order amish (lod � . for triglycerides) ascertained for type diabetes ( ). linkage on chromosome p was also observed for other lipid traits. in a study combining data from chd and randomly ascertained families, a linkage peak was identified for ldl ( ). this region has also been identified in randomly ascertained individuals for total cholesterol ( ) and familial combined hyperlip- idemia (as a discrete trait) ( ). replication of previously published results supports the presence of a gene affecting lipid levels in this region on chromosome . in addition, the use of different ascertainment schemes, all of which are associated with abnormal lipid levels, may suggest pleiotropic effects of a common underlying gene in these various conditions. several potential candidates for control of lipid metab- olism map to the linked regions on p . - q . and p . - p . . the pregnane x receptor gene (also known as nuclear receptor subfamily , group i, member [nr i ]), which induces expression of genes involved in clearance of cholesterol metabolites, is located on q and has recently been shown to exert direct effects on the detoxification of cholesterol metabolites ( ). the phos- photidylserine-specific phospholipase a -� (pla a) gene, located on q , is involved in the hydrolysis of fatty acids. genes on chromosome include the liver x receptor � (lxra), which plays a key role in the regulation of cholesterol and lipid metabolism; oxysterol-binding pro- tein-like protein (osbpl ), which belongs to a family of intracellular lipid receptors and may play a role in the regulation of cholesterol metabolism ( ); and -dehydro- cholesterol reductase (dhcr ), which catalyzes the con- version of -dehydrocholesterol to cholesterol during the final step of endogenous cholesterol biosynthesis and is linked to control of total cholesterol levels ( ). we observed a statistically significant correlation of . (p � . ) between triglycerides and triglyceride/hdl ratio in both caucasians and hispanics. given that we observed similar lod scores for these traits in the same region but a lower lod score for hdl ( . in hispanics and � in caucasians), we predict that the triglycerides level is responsible for most of the linkage signal on chromosomes and in caucasians and hispanics, respectively; however, it is possible that pleiotropic effects of an underlying gene are present. because hypertriglyc- eridemia is typically observed in type diabetic patients, these regions may harbor genes underlying susceptibility to developing cad in families ascertained for type diabetes. in the african-american study sample, the strongest evidence for linkage was found on chromosome for total cholesterol in a region that also overlapped a linkage peak for ldl in our analyses. interestingly, this region has been reported for lipid traits in at least five independent studies, including three investigations of families origi- nally ascertained for type diabetes (fig. ). in these diabetic families, evidence of linkage was seen for total cholesterol in pima indians ( ), triglycerides in non- hispanic caucasians ( ), and ldl in the old order amish ( ). in addition, linkage for ldl concentrations was found in two non-hispanic caucasian populations, one randomly ascertained ( ) and the other containing a combination of randomly and obesity-ascertained families ( ). in a separate study, rainwater et al. ( ) fractionated ldl components and performed quantitative linkage anal- ysis on the different ldl size fractions. evidence for linkage of two size fractions, ldl- ( . – nm) and fig. . a summary of studies showing evidence for linkage on chromosome . the ethnic background of the study population is given. horizontal lines with arrows represent the approximate -lod support interval on a cytogenetic map (bottom) for the respective studies. the closest marker and position (cm, vertical line) of the maximum lod score (right) are indicated above the horizontal line. one study did not have closest marker information available (see ref. ). a. malhotra and associates diabetes, vol. , october ldl- ( . – . nm), were identified on p . -q and q . -q . , respectively, in mexican americans ( ). although the linkage signal obtained for chromosome in the present study was not statistically significant, the strong support for linkage reported in other studies, particularly those using families with type diabetic individuals, suggests the possibility that this region har- bors genes that may impact the regulation of lipid traits in diabetic individuals. a number of candidate genes with possible effects on lipid metabolism are located in the region of interest on chromosome . these include the genes encoding the ldl receptor (ldlr; mim ), which mediates ldl uptake at the cell membrane; hormone-sensitive lipase (lipe; mim ), which converts cholesteryl esters to free cholesterol and hydrolyzes stored triglycerides to free fatty acids; apolipoprotein e (apoe; mim ), which is essential for lipoprotein metabolism; and the upstream stimulatory factor (usf ; mim ), which belongs to the same nuclear hormone receptor family as usf (mim ), a key factor in the development of familial combined hyperlipidemia ( ). in addition, several mem- bers of the cytochrome p family involved in cholesterol and steroid hormone biosynthesis are located in this region. although the present study provides strong support for the presence of loci on chromosomes , , and that contribute to the control of lipid levels, a number of limitations remain. even though many studies have shown evidence for linkage in regions overlapping the ones reported here, results from other studies do not support these findings ( , ). disparities in linkage findings may result from different ascertainment schemes, dissimilar methods of statistical analysis, and variability in the power of a particular study sample to detect genetic linkage. undoubtedly, failure to replicate findings among indepen- dent studies is also indicative of the complexity involved in the control of lipid levels. furthermore, evidence for linkage of different lipid traits within the same region is suggestive of pleiotropic effects for an underlying gene(s). although beyond the scope of the present study, multivar- iate analysis would be an appropriate method to address this possibility. a second limitation of this study involves the use of lipid-lowering drugs. in the present study, we performed variance components analyses including individuals who self-reported use of lipid-lowering prescription drugs, al- though we recognize that the plasma lipid levels in these individuals may influence our findings. loss of information was the primary reason for not removing these individuals from the analyses. for example, in our analyses, removal of individuals who self-reported lipid-lowering drug use ( individuals from african-american families, individu- als from hispanic families, and individuals from caucasian families) did not eliminate the evidence for linkage but did reduce the lod scores in the correspond- ing regions ( . to . in african americans, . to . in hispanics, and . to . in caucasians; data not shown). our results suggest that these individuals contrib- uted to the evidence for linkage in these regions, and removing them from the variance components analyses would result in significant loss of information. further- more, in this study sample, removal of individuals on lipid-lowering medications may not be the most appropri- fig. . a summary of studies showing evidence for linkage on chromosome . the ethnic background of the study population is given. horizontal lines with arrows represent the approximate -lod support interval on a cytogenetic map (bottom) for the respective studies. the closest marker and position (cm, vertical line) of the maximum lod score or lowest p value (right) are indicated above the horizontal line. two regions of linkage did not have closest marker information available ( ). in addition, in the old order amish study ( ), the markers at the ends of the linkage interval are given. linkage analysis of quantitative lipid traits diabetes, vol. , october ate way to account for drug use. first, all medication information was self-reported by the participants of the gennid study and not confirmed by a qualified physician. second, use of different types of drugs (i.e., statins, niacin, gemfibrozil, and resins), variable duration times of medi- cation usage, and a lack of information on actual drug dosage make adjustments for medication use extremely complicated in the gennid study populations. however, the fact that the results obtained in the present study identify chromosomal regions linked to quantitative lipid traits in other studies of families ascertained for type diabetes suggests that our approach is tenable. finally, we cannot determine whether the loci identified in this study represent genes specifically linked to lipid traits under the control of hyperglycemia, hyperinsulin- emia, or both. because linkage for each trait has been found in both families ascertained for type diabetes and families ascertained for other dichotomous traits such as cad, as well as randomly ascertained samples, it may be possible that the genes underlying linkage peaks for diabetic dyslipidemia also contribute to lipid traits in the general population. that said, chromosomes and may harbor genes playing a role in the inheritance of lipids in both the general population and in individuals with lipid- related disorders, whereas the replication of the chromo- some peak in three other studies of families ascertained for type diabetes suggests the presence of a gene affecting diabetic dyslipidemia in this region. however, until the specific genes within each region are identified and characterized, we cannot assign common loci to linkage signals obtained for lipid traits in differently ascer- tained study samples. acknowledgments this study was supported by the american diabetes association. we thank the investigators and staff who contributed to the gennid repository, as well as the individuals who participated in the gennid study. we also appreciate the expert guidance and assistance of tory asfahani and dr. zhiying wang of the american diabetes association. appendix american diabetes association gennid study group. genetic material was collected 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cholesterol-lowering gene maps to chromosome q. am j hum genet : – , linkage analysis of quantitative lipid traits diabetes, vol. , october course report: intravenous conscious sedation training for the whole team urgent notice for foundation dentists faculty supports faster cancer referrals in april the intercollegiate advisory committee for sedation in dentistry (iacsd) of the dental faculties of the royal colleges of surgeons and the royal college of anaesthetists published new standards for conscious sedation in the provision of dental care which includes updated recommendations on conscious sedation training for the whole dental team. the standards state that ‘patients have the right to expect a high quality service to meet their dental needs. this can only be achieved through robust, validated education and training of the entire dental team.’ as a result, an innovative course was established by health education england, london and the department of sedation & special care dentistry at guy’s and st thomas’ nhs foundation trust. the new course was based on the learning outcomes outlined in the standards and was run over eight days. the first two days were non-clinical and dedicated to increasing and updating theoretical knowledge and improving practical sedation skills. topics covered included relevant physiology and pharmacology, patient assessment and treatment planning, cannulation, administra- tion of midazolam, sedation monitoring and managing sedation related emergencies. the next six days were dedicated to treating patients requiring a wide range of dental treatment under intravenous sedation with midazolam. all the participants were encour- aged to bring a dental nurse to observe the sedation technique, patient management and intravenous conscious sedation training for the whole team course report procedural skills. the course provided the opportunity to treat patients with intra- venous sedation under closely supervised conditions. in addition there were oppor- tunities to experience the use of inhalation sedation and intranasal midazolam sedation and to observe the use of conscious sedation using a propofol infusion. attendees gained confidence in treatment planning patients for sedation and also took part in a seminar on cognitive behavioural therapy. the course was organised and supervised by emily sherwin, specialist in special care dentistry, and david craig, consultant in special care dentistry. having four attendees allowed for close supervision on the clinics with readily available support to assist with any challenging cases. the course involved continuous clinical assessment with both informal and formal feedback including workplace-based assessments and an end- of-course review. there was also an mcq examination to assess theoretical knowledge. the course is designed for ‘new starters’ in dental sedation wishing to progress to inde- pendent (unsupervised) practice but it would also be helpful for experienced dentists who wish to expand their skills. its clinical focus makes this course ideal for primary dental care dentists who are interested in adding conscious sedation to their skill set. by amish patel and emily sherwin . standards for conscious sedation in the provision of dental care. report of the intercollegiate advisory committee for sedation in dentistry. the dental faculties of the royal colleges of surgeons and the royal college of anaesthetists, . (l-r) sheena quille (dental nurse), amish patel (course member), emily sherwin (supervisor), chris vondee (course member), david craig (supervisor), parimal patel (course member), and lena vakil (course member) the faculty of general dental practice (uk) has expressed its support for a new standards document on cancer referrals from the national institute for health and care excellence (nice). in the uk, over people a day are diagnosed with oral cancer, and fgdp(uk) is asking dentists to take note in particular of the recommendation in the new document that ‘people with suspected cancer who are referred to a cancer service are given written information encouraging them to attend’. the faculty is recommending that dental practices make suitable resources, such as the nhs’s ‘patient information for urgent referrals’ leaflet, available for patients. to download the leaflet visit: https://www. myhealth.london.nhs.uk/healthy-london/ cancer/pan-london-suspected-cancer-referrals/ patient-leaflets. the british dental association (bda) is reminding all new graduates who have recently been allocated to a dental founda- tion training post in england or wales starting on september this year that they must apply to be included in the dental performers list before that date. the same applies to inclusion in the hscb dental list in northern ireland. the pcs and area teams/ health boards then have three months to finalise the inclusion of names in the list. for those in northern ireland applying to be on the dental list, full completion of the hs form and attendance at the hscb dental information session is a pre-requisite to a ds number being issued. young dentists and their trainers/educa- tional supervisors should also check with the deaneries and pcs/ats/hbs whether the process for inclusion is moving forward smoothly, so that any problems can be clarified in good time and the inclusion is confirmed by the end of november at the very latest. if trainers/supervisors or foundation dentists are becoming aware of any problems with this process, please get in touch with the bda as soon as possible. british dental journal | volume no. | august upfront © macmillan publishers limited, part of springer nature. all rights reserved. https://www.rcseng.ac.uk/fds/publications-clinical-guidelines/docs/standards-for-conscious-sedation-in-the-provision-of-dental-care- https://www.rcseng.ac.uk/fds/publications-clinical-guidelines/docs/standards-for-conscious-sedation-in-the-provision-of-dental-care- https://www.myhealth.london.nhs.uk/healthy-london/cancer/pan-london-suspected-cancer-referrals/patient-leaflets https://www.myhealth.london.nhs.uk/healthy-london/cancer/pan-london-suspected-cancer-referrals/patient-leaflets https://www.myhealth.london.nhs.uk/healthy-london/cancer/pan-london-suspected-cancer-referrals/patient-leaflets https://www.myhealth.london.nhs.uk/healthy-london/cancer/pan-london-suspected-cancer-referrals/patient-leaflets 	course report: intravenous conscious sedation training for the whole team 	references genetic and functional evidence relates a missense variant in b galt to lower ldl-c and fibrinogen genetic and functional evidence relates a missense variant in b galt to lower ldl-c and fibrinogen may e. montasser , cristopher v.van hout , rebecca mcfarland , avraham rosenberg , myrasol callaway , biao shen , ning li , thomas j. daly , alicia d. howard , wei lin , yuan mao , bin ye , giusy della gatta , gannie tzoneva , james perry , kathleen a. ryan , lawrence miloscio , aris n. economides , regeneron genetics center , nhlbi topmed program , carole sztalryd-woodle , braxton d. mitchell , , matthew healy , elizabeth streeten , norann a. zaghloul , simeon i. taylor , jeffrey r. o'connell , alan r. shuldiner program for personalized and genomic medicine, department of medicine, university of maryland school of medicine, baltimore, maryland, usa regeneron genetics center, llc., tarrytown, ny , usa regeneron pharmaceuticals, inc., tarrytown, ny , usa full list of names and affiliations in the online supplementary materials geriatrics research and education clinical center, baltimore va medical center, baltimore, md , usa .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / abstract increased ldl-cholesterol (ldl-c) and fibrinogen are independent risk factors for cardiovascular disease (cvd). we identified novel associations between an amish- enriched missense variant (p.asn ser) in a functional domain of beta- , - galactosyltransferase (b galt ) and . mg/dl lower ldl-c (p= . e- ), and mg/dl lower plasma fibrinogen (p= . e- ). n-linked glycan profiling found p.asn ser to be associated (p-values from . e- to . e- ) with decreased glycosylation of glycoproteins including: fibrinogen, apob , immunoglobulin g (igg), and transferrin. in vitro assays found that the mutant ( ser) protein had % lower galactosyltransferase activity compared to wild type ( asn) protein. knockdown of b galt in zebrafish embryos resulted in significantly lower ldl-c compared to control, which was fully rescued by co-expression of asn human b galt mrna but only partially rescued by co-expression of ser human b galt mrna. our findings establish b galt as a novel gene associated with lower ldl-c and fibrinogen and suggest that targeted modulation of protein glycosylation may represent a therapeutic approach to decrease cvd risk. cardiovascular disease (cvd) accounts for of every deaths in the usa and is the leading cause of morbidity and mortality worldwide . elevated low-density lipoprotein cholesterol (ldl-c) increases arterial plaque formation and atherosclerosis, and fibrinogen increases risk for blood clotting and thrombosis; both are independent risk factors for coronary artery disease (cad) , . ldl-c and fibrinogen are governed by both genetic and environmental factors, as well as the interplay between them , . rare variants in ldlr, pcsk , apob cause severe forms of familial hypercholesterolemia, and many common genetic variants, each with small effect size, have been identified in large genome-wide association studies of both ldl-c and fibrinogen - . however, few variants have been found with pleiotropic effects on more than one cad risk factor. similarly therapeutic approaches to mitigate cad risk have focused on treating individual risk factors. deeper understanding of the genetic determinants of ldl-c and fibrinogen may unveil novel targets for therapy that may be more efficacious and safe to treat or prevent cad. founder populations can facilitate the identification of novel disease associations with variants that are enriched to a higher frequency through genetic drift. multiple examples have been recently reported of highly enriched variants with large effect sizes associated with complex diseases and traits in homogenous populations in iceland , sardinia , greenland , samoa and the old order amish (ooa) - . while such drifted variants are often rare or absent in the general population, their novel associations can inform biological mechanisms and therapeutic targets relevant to all humans. the ability to perform whole-exome and whole-genome sequencing in founder populations provides the opportunity to identify drifted causative genetic variants hard to identify in general populations. in this study, we performed genetic association analyses for ldl-c in ooa participants with whole-genome and whole-exome sequencing data. we identified a novel genome-wide significant association between an amish-enriched missense variant in the beta- , -galactosyltransferase gene (b galt p.asn ser) and decreased ldl-c, which was also associated with decreased fibrinogen. homozygotes for ser have increased levels of incompletely synthesized glycans on glycoproteins as a result of decreased b galt enzymatic activity suggesting a potential mechanism to modulate lipid metabolism and coagulation. .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / association analyses identify b galt p.asn ser as a novel ldl-c variant to identify genetic variants associated with ldl-c, we performed an exome-wide association scan using , ooa subjects with whole-exome sequencing (wes) performed at the regeneron genetics center. demographic and clinical characteristics of the wes subjects are shown in supplementary table . linear mixed model association analysis identified several previously known loci for ldl-c as well as a novel locus on the short arm of chromosome (supplementary fig. and supplementary table ). as shown in figure a, a missense variant (rs , p.asn ser) in b galt was strongly associated with . mg/dl lower ldl-c in an additive genetic model (p = . e- ). this variant has a minor allele frequency (maf) of % in the ooa population while extremely rare in the general population (only copies were identified in , wgs of non-amish participants in the nhlbi trans-omics for precision medicine (topmed) program (www.nhlbiwgs.org). since wes captures only the coding variants, we performed association analysis using , ooa subjects with whole-genome sequence (wgs) as part of topmed (dbgap accession number: phs ) to exhaustively interrogate all the coding and noncoding genetic variants in this region. despite the much smaller sample size, the wgs analysis identified the same b galt variant rs as the top signal in this region with p-value of . e- and effect size of . mg/dl lower ldl-c (figure b). in addition, wgs analysis revealed variants in the region in high linkage disequilibrium (ld) with rs (r > . , range from . to . ), with ldl-c association p-values ranging from . e- to . e- (supplementary table ). of the variants, rs is the only protein coding variant (b galt p.asn ser), and is classified as damaging or deleterious by in silico protein function prediction algorithms (sift = deleterious, polyphen = possibly damaging, lrt = deleterious, mutation taster = disease-causing, provean = deleterious). these variants and rs comprise a mb amish-specific haplotype extending from approximately . mb to . mb on chromosome (supplementary fig. ). these variants have a maf of approximately % in the ooa while extremely rare or nonexistent in the general population (supplementary table ) the limited sample size of the wgs (n= , ) was not able to differentiate statistically the top missense variant (p = . e- ) from the other highly correlated variants (p from . e- to . e- ). to further differentiate between these highly linked variants, we imputed genotypes in the full ooa dataset of , subjects with genotype chip data to the topmed wgs reference panel. as shown in figure c and supplementary table , the missense variant was the top associated variant with a p value of . e- , two or more orders of magnitude smaller than any of the other variants (p from . e- to . e- ). independent direct genotyping for of these variants gave similar results (data not shown). to determine if rs is the sole signal in the region, conditional analysis was performed. conditional analysis adjusting for rs completely abolished the association of the other variants while conditional analyses adjusting for any of the other variants reduced the association of rs due to the strong correlation (r = . - . ), but did not abolish it (p= . e- - . e- ). our analysis supports rs as the most likely causal variant in this region. association with other traits and b galt human knockout support a functional role of b galt p.asn ser .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / b galt is a member of the beta- , -galactosyltransferase gene family which encode type ii membrane-bound glycoprotein that plays a critical role in the processing of n-linked oligosaccharide moieties in glycoproteins. impairment of b galt activity has the potential to alter the structure of n-linked oligosaccharides and introduce aberrations in glycan structure that have the potential to alter glycoprotein function. a homozygous frame shift insertion in b galt that results in a truncated dysfunctional protein was previously reported to cause congenital disorder of glycosylation type (cdgii) in patients , . these patients exhibited, among other traits, abnormal coagulation and very high levels of aspartate transaminase (ast). interestingly we found b galt p.asn ser to be strongly associated with lower levels of fibrinogen (n= , beta = - mg/dl, p= . e- ). we also found that the mean level of ast in ser homozygotes was two-fold higher than that of asn homozygotes ( . vs . u/l, respectively) (n= , , additive model p= . e- , recessive model p= . e- ), further supporting the functional role of this variant. while the high level of ast might suggest liver injury, we found no association with alanine aminotransferase (alt), alkaline phosphatase (alp), liver to spleen density ratio, or inflammatory markers (supplementary table ). moreover, all ser homozygotes (n= ) had normal levels of gamma-glutamyl transpeptidase (ggt), activated partial thromboplastin time (aptt), prothrombin time (pt), and internationalized normalized ratio (inr) (supplementary table ). b galt p.asn ser is associated with increased levels of incompletely synthesized glycans on glycoproteins to assess the impact of b galt p.asn ser on glycosylation, the carbohydrate deficient transferrin (cdt) test, which assesses glycosylation levels of transferrin and apolipoprotein ciii (apociii), was performed using serum samples from participants from the genotype groups ( ser homozygotes, asn ser heterozygotes and asn homozygotes). as expected, based on the fact that b galt plays a role in processing of n- linked carbohydrate, all samples had normal profiles for apociii o-linked carbohydrate (supplementary table ). all samples also had normal levels of mono-oligosaccharide/di- oligosaccharide transferrin ratio, and a-oligosaccharide/di-oligosaccharide transferrin ratio. however, while all eight asn homozygotes had normal levels of the tri-sialo/di- oligosaccharide transferrin ratio, the ratio in all ser homozygotes was abnormal; heterozygotes were intermediate between the two homozygote groups (p= . e- ) (figure and supplementary table ). since transferrin is mostly tetrasialylated , , this increase in trisialylated transferrin reflects a paucity of tetrasialylated molecules and indicates that the ser allele is associated with increased levels of carbohydrate deficient transferrin, likely due to decreased enzymatic activity of b galt . to determine if the lower levels of sialylation are affecting only transferrin or extending to other glycoproteins, n-linked glycan profiling was performed using plasma samples from participants ( ser homozygotes and asn homozygotes) for global plasma glycoproteins, as well as selected specific plasma glycoproteins - apolipoprotein b- (apob ), fibrinogen and immunoglobulin g (igg) - by hydrophilic interaction liquid chromatography with fluorescent detection and mass spectroscopy (hilic-flr-ms). glycans from ser homozygotes had increased levels of incompletely synthesized oligosaccharides as evidenced by increased percentages of truncated biantennary glycans devoid of galactoses and sialic acids (g f, p= . e- ; bg , p= . e- ), and biantennary glycans with only one galactose and one sialic acid (g s , p= . e- ). reciprocally, ser homozygotes had significantly lower levels of biantennary glycans with two galactose and two .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / sialic acid residues (g s , p= . e- ) (supplementary table ). the results for plasma apob , fibrinogen and igg were similar, where serum from ser homozygotes had significantly increased level of glycans lacking galactose and sialic acid moieties and decreased levels of more mature glycans (supplementary table , , ). overall, there was significantly lower galactosylation (p from . e- to . e- ) and sialylation (p from . e- to . e- ) for global glycoproteins, and enriched apob , fibrinogen and igg among ser homozygotes compared to asn homozygotes, however there was no difference in fucosylation (figure ), which is consistent with the role of b galt in adding galactose moieties that then are capped by sialic acid while having no role in the addition of fucose . in summary, the ser allele is associated with significantly increased levels of incompletely synthesized glycans on glycoproteins indicating defective protein glycosylation. b galt p.asn ser causes reduced enzymatic activity to compare the in vitro enzymatic activities of wild type ( asn) and mutant ( ser) b galt protein, we transiently transfected cos- cells, which express very low endogenous levels of b galt , with cdnas encoding myc-flag epitope-tagged versions of the proteins. we immunoprecipitated with anti-flag antibody, and measured b galt activities in the immune complexes (supplementary fig. ). compared to asn b galt , ser b galt showed on average % decrease in galactosyltransferase specific activity (figure a). as a complementary approach, we used synthesized recombinant human asn and ser b galt protein to test the reduction rate of the substrate uridine diphosphate galactose (udp-gal) incubated with each protein and found that the udp-gal level decreased faster with asn than ser b galt , indicating decreased enzymatic activity of ser b galt compared to asn b galt (figure b). functional validation of the effect of b galt p.asn ser on ldl-c in zebrafish we used a zebrafish model to investigate the effect of b galt p.asn ser on ldl-c using our established assays , . we first generated a genomic knockout of the zebrafish ortholog (b galt ) using crispr/cas -mediated targeting of exon . consistent with mouse reports of embryonic lethality in knockout animals injected f animals were not viable to adulthood and consistently died at juvenile stages. to circumvent the lack of viability, we employed a knockdown approach using a previously reported splice-blocking antisense morpholino oligonucleotide (mo) injected into embryos . after validation of mo efficacy (supplementary fig. ) and ruling out the possibility of off-target toxicity by demonstrating no increases in d p expression and no overt morphological defects (supplementary fig. , a), we quantified changes in ldl-c levels in unfed larvae at days post fertilization (dpf) as per previously published protocols . we observed a significant decrease in ldl-c in mo- injected larvae compared to control larvae, consistent with a role for b galt in ldl-c homeostasis (figure ). we further confirmed this result using a second splice-blocking mo targeting exon of b galt which similarly produced a reduction in ldl-c concentration (supplementary fig. b-d). to validate the specificity of these observations and to test the functionality of human b galt in zebrafish, we co-injected mrna encoding human asn b galt along with .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / b galt mo into embryos and assessed ldl-c in unfed larvae. this resulted in ldl-c levels that were statistically indistinguishable from those in larvae injected only with a control mo (p= . ), suggesting that the human mrna could rescue the effects of knockdown of the zebrafish gene (figure ). these data suggest that human wild type b galt mrna is functional in zebrafish and support the use of this model system for functional interpretation of p.asn ser. using site- directed mutagenesis , we introduced a t to c change in the coding sequence of human b galt and generated full length mrna. co-injection of the ser b galt mrna with b galt mo resulted in a reduced capacity for rescue of the ldl-c phenotype. the resulting ldl-c concentration was % lower than that resulting from co-injection of asn b galt mrna with b galt mo, a statistically significant effect (p= . ). this level of ldl-c was also statistically greater, however, than b galt mo alone (p= . ) (figure ), suggesting only a partial defect in function introduced by the missense variant. to examine the relevance of b galt to other hypercholesterolemic models, we examined the impact of b galt knock down on two previously reported zebrafish models of elevated ldl-c: high cholesterol diet fed larvae and ldlr knockdown animals . for the former, we treated dpf larvae with either a control diet or the same diet supplemented with % w/w cholesterol as previously reported . while b galt knockdown reduced ldl-c in animals fed a control diet, this effect was abolished with introduction of a high cholesterol diet (supplementary fig. ). in contrast, when we introduced b galt suppression on a background of genetically- induced hypercholesterolemia via ldlr mo injection, we observed amelioration of the ldl-c phenotype in those animals (supplementary fig. ). these data suggest that suppression of b galt can abrogate elevated ldl-c resulting from genetic defects in ldl-c metabolism but not that introduced by diet. discussion large genome-wide analyses of approximately , individuals have identified loci associated with lipid traits, none of which identified the b galt gene . using next generation sequencing in , ooa, we found a strong novel association of b galt p.asn ser with lower plasma ldl-c and fibrinogen levels. glycosylation profiling and experimental assays confirmed the functional role of this variant. this variant is very rare in the general population, but has % frequency in the amish, likely due to genetic drift over approximately generations after founding. this report highlights the value of founder populations in identifying novel gene variants that can provide new insights into human biology of common traits. homozygosity for a protein truncating mutation in b galt ( insc) reported in two related patients is known to cause cdg type (cdgii) , . both patients have marked abnormal carbohydrate structures on glycosylated proteins and severe clinical phenotypes manifesting in early childhood with developmental delay, hypotonia, coagulopathy, and elevated transaminases , , . knock-out of b galt in mice results in semi-lethality after birth and several other sever developmental abnormalities , . interestingly, mass spectrometry studies of n- glycans from plasma and hepatic membrane glycoproteins, revealed an unexpected high level of sialylation and galactosylation in b galt -/- (~ % compared to that of b galt +/+ mice). kotani et. al., explained the altered pattern of sialylation and galactosylation with a shift in synthesis from type to type glycan chains in b galt -/- versus b galt +/+ mice, most probably caused by b galt proteins in response to loss of b galt . conversely, the overall decrease in galactosylation and sialylation that we observed in the carriers of ser b galt missense .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / variant, suggests that this missense variant could act as hypomorph. indeed, our preliminary investigation showed that the level of type chain (corresponding to the b , linkage) was very low and comparable between asn and ser homozygotes (data not shown), suggesting that our missense mutation did not cause any glycan chain type switch as shown in kotani et. al., and that compensatory action of b galt may differ between mouse and human. the p.asn ser b galt missense variant that we identified in the amish does not appear to be associated with any severe phenotype. in fact, the self-reported medical and family history of fifteen ser homozygotes did not note previous heart attack, stroke, coronary angiogram, blocked arteries, or heart/carotid artery surgery. none had evidence of coronary ischemia or mi on ekg except for one year old person who showed evidence of a potential previous mi. abdominal ultrasound from five ser homozygotes over years old showed normal abdominal aorta with no atherosclerotic plaque, fatty liver or aneurysm. the variant showed no association with reduced coronary artery calcification (cac) or aortic calcification (ac), however, the sample size was small with relatively young average age of , that included only one ser homozygote (supplementary table ). we also found that ser homozygotes had double the serum ast levels compared to asn homozygotes, however, they all had normal levels of alt, ggt and alp, suggesting that the source of high ast is from tissue other than liver, i.e., muscle. moreover, they also had normal inr, ptt and inflammation markers (supplementary tables and ). the absence of a severe phenotype in the minor homozygotes may be due to the subtle effect of the missense mutation compared to the protein truncating frameshift mutation identified in cdgii patients. the missense variant p.asn ser changes the asparagine to serine at position in the amino acid sequence of the long isoform of human b galt (corresponding to position of the short isoform). the asparagine residue is completely conserved across vertebrate species (gerp score = . ). p.asn ser is located in the highly flexible long c terminal region of the protein that undergoes conformational changes to allow for the exchange of the sugar molecule during glycosylation . hence, a mutation in this region may impede the necessary conformational change and impact glycosylation efficiency as shown by our glycosylation profiling and functional assays. however, it is important to note that the ser mutation had less severe effects on glycosylation than that previously reported in cdgii patients carrying a b galt truncating mutation . the mechanism by which this variant leads to lower ldl-c and fibrinogen remains to be elucidated. b galt is a ubiquitously expressed protein that transfers the galactose from uridine diphosphate galactose (udp-gal) to specific glycoprotein substrates . a defect in b galt that affects glycosylation and sialylation may affect the folding, secretion, stability, activity, and half-life of lipoprotein and coagulation related glycoproteins - ultimately resulting in lower levels of circulating ldl-c and fibrinogen. genome-wide association studies (gwas) have identified common non-coding variants at the b galt locus that are significantly associated with igg glycosylation and ast levels , , but not with any lipid traits. however, several lines of evidence point to protein glycosylation as a major player in lipid metabolism. reduced apob glycosylation leads to shorter ldl-c half-life and rapid clearance from the circulation, which in turn reduces its atherogenic effect , while increased ldl-c glycosylation was associated with increased ldl-c oxidation leading to greater atherosclerosis . glucose mediated n-glycosylation plays a major role in regulating sterol regulatory element-binding proteins (srebps) which are major players in cholesterol metabolism . also, reduced ldlr glycosylation was found to result in reduced lipid binding and endocytosis . recently, changes in igg glycosylation have been associated .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / with blood lipids and dyslipidemia in han chinese . a similar role for glycosylation was also reported in relation to coagulation proteins, where mutations affecting glycosylation were associated with different enzymatic activity . finally, gwas has identified loci containing other enzymes involved in glycoprotein biology associated with lipid traits including galnt , st gal , and asgr . similarly, studies support a role of glycosylation in cvd. for example, a loss of function mutation in asgr was reported to be associated with lower non-hdl cholesterol and reduced coronary artery disease (cad) risk . asgr encodes a subunit of the asialoglycoprotein receptor (the “ashwell receptor”) that mediates binding and endocytosis of asialylated n- glycoproteins. recently menni et.al. reported that the glycosylation profile of igg was associated with cvd risk score and subclinical atherosclerosis in two independent cohorts. while these studies are not specific to b galt , they support an important role of glycosylation in cvd with potential prognostic and/or therapeutic implications. in summary, we discovered a novel missense variant in b galt that is associated with lower ldl-c and fibrinogen, both cardioprotective phenotypes, through enrichment in a founder population. evidence from human data as well as in vitro and animal-based experiments indicate that the variant leads to decreased protein glycosylation. further understanding of the underlying biological mechanism of the variant may provide new insights into the role of glycosylation in cvd risk that may lead to novel therapeutic targets. online content methods, additional references, nature research reporting summaries, statements of data availability and associated accession codes are available in the online paper acknowledgements this work was supported in part from nih grants u hl , u hl , r ag , r hl , p dk , aha grnt and regeneron pharmaceuticals, inc. whole-genome sequencing (wgs) for the trans-omics in precision medicine (topmed) program was supported by the national heart, lung and blood institute (nhlbi). wgs for “nhlbi topmed: genetics of cardiometabolic health in the amish” (phs ) was performed at the broad institute of mit and harvard (hhsn c). centralized read mapping and genotype calling, along with variant quality metrics and filtering were provided by the topmed informatics research center ( r hl- - s ). phenotype harmonization, data management, sample-identity qc, and general study coordination, were provided by the topmed data coordinating center ( r hl- - s ). we gratefully thank the amish community and research volunteers for their long- standing partnership in research, and acknowledge the dedication of the amish liaisons, field workers and the amish research clinic staff, without which these studies would not have been possible. author contributions conceived, designed and supervised the work: mem, ars, ane performed data collection: mem, ars, jo, bs, nl, gdg, gt, lm, ar, rmc, naz, wl, ym .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / contributed to data analysis mem, cvh, bs, nl, by, gdg, gt, lm, mh, jro, rmc, naz, kar, jp interpreted the results: mem, ars, cvh, bs, nl, tjd, gdg, ane, mh, jro, ah, cs, bdm, es, naz, sit preparation of the manuscript: mem, ars competing interests cvh, gdg, ar, mc, tjd, bs, nl, by, gt, lm, ae, mh, and ars are current or former employees of regeneron pharmaceuticals inc. mem, cvh, ars, gdg, and mh are inventors on a patent that was published by the united states patent and trademark office on december , under publication number us - , and international patent application that was published on december , under publication number wo- / regarding b galt variants and uses thereof. corresponding author correspondence and requests for materials should be addressed to mmontass@som.umaryland.edu .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / references . mozaffarian d, benjamin ej, go as, et al. heart disease and stroke statistics- update: a report from the american heart association. circulation 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d, khoder-agha f, peltoniemi m, et al. the dimeric structure of wild-type human glycosyltransferase b galt . plos one ; ( ):e . . global lipids genetics consortium, willer cj, schmidt em, et al. discovery and refinement of loci associated with lipid levels. nat genet ; ( ): - . .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / figures a b c fig. : evidence for genetic association between rs , encoding b galt p.asn ser and ldl-c. a, whole-exome sequencing results for , ooa subjects. b, whole-genome sequencing results for , ooa subjects. c, imputed data from genome- wide genotyping chip results for , ooa subjects. blue line marks a genome-wide suggestive threshold ( . e- ) and red line a genome-wide significant threshold ( . e- ). .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / fig. : b galt p.asn ser is associated with increased carbohydrate-deficient transferrin. the transferrin tri-sialo/di-oligo ratio for the genotype groups ( asn homozygotes, asn ser heterozygotes and ser homozygotes) as measured by the carbohydrate deficient transferrin test. .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / fig. : b galt p.asn ser is associated with decreased glycosylation. the levels of total galactosylation, sialylation and fucosylation in global plasma glycoproteins, fibrinogen, apob , and igg for asn homozygotes and ser homozygotes. data are represented as mean and standard error. .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / a b fig. : b galt p.asn ser decreases galactosyltransferase activity. a, the activity level of asn b galt and ser b galt immunoprecipitated proteins from transfected cos- cells. the data are expressed as the percent of asn b galt activity and represent the mean of four experiments ± the standard error. b, the decrease in udp-gal incubated with recombinant b galt ser (open circles) and asn proteins (solid circles). .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / fig. : quantification of ldl-c in zebrafish. average ldl-c in homogenates of unfed wild type dpf zebrafish larvae (n= per well, duplicated within each experiment, n= for control, and asn rescue, n= for b galt mo, n= for ser rescue). larvae were first injected with either a non-targeting control mo (control) or ng of morpholino (b galt mo) against b galt at - cell stage. rescue conditions were ng mo co-injected with pg asn human b galt mrna ( asn rescue), or ng mo co-injected with pg b galt mrna encoding the ser mutation ( ser rescue). data are represented as mean and standard error, normalized to control mo within each experiment and averaged across experiments. .cc-by-nc-nd . international licensea certified by peer review) is the author/funder, who has granted biorxiv a license to display the preprint in perpetuity. it is made available under the copyright holder for this preprint (which was notthis version posted august , . ; https://doi.org/ . / doi: biorxiv preprint https://doi.org/ . / http://creativecommons.org/licenses/by-nc-nd/ . / wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ shieldsquare captcha we apologize for the inconvenience... ...but your activity and behavior on this site made us think that you are a bot. note: a number of things could be going on here. 	if you are attempting to access this site using an anonymous 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timing of light used to entrain biological rhythms. in humans and other mammals, light is detected by the retina and intrinsically photosensitive retinal ganglion cells project this information both to the circadian system and limbic brain regions. therefore, it is possible that exposure to light at night, which has become pervasive, may disrupt both circadian timing and mood. notably, the rate of major depression has increased in recent decades, in parallel with increasing exposure to light at night. strong evidence already links circadian disruption to major depression and other mood disorders. emerging evidence from the past few years suggests that exposure to light at night also negatively influences mood. in this review, we discuss evidence from recent human and rodent studies supporting the novel hypothesis that nighttime exposure to light disrupts circadian organization and contributes to depressed mood. molecular psychiatry ( ) , – ; doi: . /mp. . ; published online may keywords: circadian; depression; light at night; light pollution introduction life on earth has adapted to a consistent and predictable -h solar cycle. to anticipate patterns in the environment, individuals synchronize internal biological rhythms to the external world, primarily using light information. during the past b years, however, the invention and widespread adoption of electric light has led to ‘round-the-clock’ societies. instead of aligning with the environment, individuals respond to artificial light cycles created by social and work schedules. exposure to artificial light at night (lan) has become pervasive (figure ). virtually every individual living in the united states and europe experiences this unnatural light exposure, and moreover about % of the population performs shift work. , exposure to lan may obscure entrainment of biological processes to external conditions, potentially leading to misalignment among physiology, behavior and the environment. furthermore, chronic disruption of circadian timing may have implications beyond dysregulated biological rhythms. in mam- mals, circadian photoentrainment is mediated by intrinsically photosensitive retinal ganglion cells (iprgcs) that project light information to the suprachiasmatic nucleus (scn) in the hypotha- lamus, regulating circadian rhythms. however, iprgcs also project to regions involved in mood regulation, such as the prefrontal cortex, hippocampus and amygdala, suggesting that unnatural light exposure has the potential to influence mood as well. as modern life has allowed humans to manipulate lighting easily and has led to unnatural exposure to lan, the prevalence of major depressive disorder (mdd) has increased in parallel. accumulating evidence from the past few years suggests that nighttime light exposure may have serious consequences for circadian timing and mood. in this review, we discuss evidence from recent human and rodent studies supporting the novel hypothesis that nighttime exposure to light disrupts circadian organization and contributes to depressed mood. photoentrainment circadian rhythms are generated by the scn, a molecular clock located in the hypothalamus, and entrained to the external environment primarily using light information sent directly from the retina to the clock. a transcription–translation feedback loop in the scn produces endogenous rhythms of approximately h, but the gene and protein components of this cycle can be modulated by light to maintain synchronization with the environment. in the absence of light and dark input, the endogenous clock becomes out of phase with the external environment, so correctly timed light information is essential to biological timekeeping. in mammals, the retina is the sole mechanism of light detection, consisting of image-forming photoreceptors, called rods and cones, and non-image-forming photoreceptors called iprgcs. in contrast to rods and cones, iprgcs are depolarized in response to light and are largely responsible for circadian photoentrainment. light detected by iprgcs activates a unique photopigment called melanopsin, which is maximally sensitive to blue wave- lengths (b nm) and minimally sensitive to longer, red wavelengths ( nm). this means that blue wavelengths exert a more potent influence on the circadian system. notably, in the united states nighttime use of incandescent bulbs, which produce light of longer yellow range wavelengths, is being replaced by compact fluorescent light bulbs, which contain the blue wavelengths that maximally activate iprgcs. activated iprgcs project to the scn directly through the retinohypothalamic tract and indirectly through the intergeniculate leaflet. information sent via the retinohypothalamic tract reaches the scn through a single glutamatergic synapse. this process is similar in both nocturnal and diurnal species; lan triggers fos induction in the scn of both. , the scn projects axons to the paraventricular nucleus of the hypothalamus (pvn) and to the thalamus (figure ). thalamic department of neuroscience, the ohio state university wexner medical center, columbus, oh, usa. correspondence: ta bedrosian, department of neuroscience, the ohio state university wexner medical center, biomedical research tower, west th avenue, columbus, oh , usa. e-mail: bedrosian. @osu.edu received february ; revised march ; accepted april ; published online may molecular psychiatry ( ) , – & macmillan publishers limited all rights reserved - / www.nature.com/mp http://dx.doi.org/ . /mp. . mailto:bedrosian. @osu.edu http://www.nature.com/mp relays of the scn project to regions directly involved in mood regulation, such as the prefrontal cortex, hippocampus and amygdala. projections through the pvn to the pineal gland regulate melatonin secretion, which is responsible for entraining peripheral clocks in cells and tissues throughout the body. and projections through the pvn to the adrenal gland regulate glucocorticoid output. from these examples, it seems reasonable to suggest that temporally aberrant cues in the environment arising from artificial lan could cause desynchronized biological rhythms at multiple levels. melatonin pineal melatonin warrants brief discussion on its own because its production is under direct control by light. typically melatonin is rhythmically secreted by the pineal gland during the night, where it is released directly into systemic circulation, having many different physiological roles throughout the body. its secretion is potently inhibited by light in an intensity-dependent manner. evidence suggests that only h of exposure to b photopic lux can decrease plasma melatonin concentrations by b % in healthy human volunteers. in hamsters, light levels as low as . lx are sufficient to significantly suppress pineal melatonin content. during the night, suppressed melatonin levels can disrupt physiological timekeeping. for example, melatonin regulates clock gene oscillations in the pituitary and pars tuberalis in mice and hamsters. , circadian disruption and mood a role for the circadian system in mood is already well-established. a host of rhythm-related disturbances have been noted in association with major depression and other mood disorders; and on the other hand, environmental perturbations of the circadian system provoke mood disturbances in some indivi- duals. – any unnatural timing of light exposure, or lack of appropriate light/dark cues in the environment, can cause misalignment between internal biological processes and the external environment, putatively leading to impaired mood. to illustrate this point, there are several instances in which obscured environmental lighting cues lead to depressed mood. seasonal affective disorder (sad) is one prominent example. at temperate latitudes, seasonal affective disorder affects nearly % of the population. it is characterized by recurrent winter depression, with symptoms manifesting during the short day lengths of winter when daylight exposure is low, and symptoms remitting during the spring and summer. lack of sufficient daytime light is thought to cause a phase shift in the rhythm of pineal melatonin secretion. without bright morning light to inhibit melatonin production, secretion persists into the daytime, leading to desynchronization between internal timekeeping processes and the external environment. although melatonin concentrations are not suppressed, as they may be with lan exposure, there is misalignment between the rhythm of secretion and the environmental light cycle. the presence of an appropriate and correctly timed melatonin signal may be more important to circadian regulation and mood than absolute melatonin concentrations. morning bright light therapy, particularly blue wavelengths, may be used to synchronize the circadian system to the appropriate time of day. evidence from animal models supports a role for day length in mood. nocturnal siberian hamsters exposed to short, winter-like, day lengths develop depressive-like responses in the forced swim test and anxiety-like responses in the elevated plus maze. two figure . worldwide artificial light at night. inset shows detailed view of north america. images are composites acquired by the nasa suomi npp satellite in . figure . potential pathways through which light at night (lan) may influence mood. hpc, hippocampus; pfc, prefrontal cortex; pvn, paraventricular nucleus of the hypothalamus; scg, superior cervical ganglion; scn, suprachiasmatic nuclei. influence of the modern light environment on mood ta bedrosian and rj nelson molecular psychiatry ( ), – & macmillan publishers limited diurnal rodent species, fat sand rats (psammomys obesus) and nile grass rats (arvicanthis niloticus), also exhibit depression-like behavior in the forced swim test after exposure to very short photoperiods ( h light/ h dark). , bright light therapy, one current treatment for seasonal affective disorder in human patients, reverses some of the depressive responses in fat sand rats. a complete lack of light/dark cues in the environment may also elicit cognitive and behavioral impairments. rodents exposed to constant light become arrhythmic in terms of locomotor activity and other biological rhythms, a major circadian disruption. after weeks of constant light exposure, rats exhibit impaired spatial learning and memory, and mice show impaired performance in the morris water maze and reduced hippocampal neurogenesis. in contrast, light deprivation in the form of constant darkness may provoke depression-like changes in rodents too. mice and rats exposed to constant darkness for several weeks develop immobility in the forced swim test, as well as other depression-like behaviors. there is some evidence that constant darkness causes neuronal damage to monoamine systems, including increases in apoptosis in several brain regions. other evidence suggests that constant darkness increases proinflammatory cytokine levels in the brain and periphery, and that the depression-like responses are interleukin- -dependent and mediated through the nuclear factor-kb signaling pathway. these examples serve to highlight the importance of light in regulating behavior and mood. proper alignment among biologi- cal rhythms, behavior and the environment requires a balance in the amount of light and dark to which an organism is exposed. tipping that balance in either direction can have profound consequences for physiology and mood. populations exposed to lan over % of individuals living in the united states and europe experience nightly light pollution. streetlights illuminate the bedroom, television and computer screens glow in the home at all hours, and work and social demands keep the lights on. lan can be invasive for these individuals, but for certain populations, exposure to lan is even more pronounced. for example, in industrial economies, b % of the population are shift workers. these are often factory workers, medical staff, flight attendants and others working in environments that require bright lights during night shifts. such bright and chronic lan exposure provokes several physiological changes. exposure to lan of p lx, levels easily encountered during night shift work, can suppress melatonin secretion and other circadian responses in humans. shift work is associated with health consequences, including increased negative affect and feelings of helplessness. also, the world health organization (who) recently cited shift work as a probable carcinogen. following this announcement, denmark became the first nation to compensate women who developed breast cancer after working night shifts. by the same token that night shift nurses are affected by lan, their patients may be similarly affected. light readings obtained at a midwest hospital sicu demonstrate that lights were illuminated at least min out of every hour throughout the night, often when no nursing or care activity was being performed. lan in this context may negatively affect patient outcome by disrupting sleep and biological rhythms. although artificial lan is most common, natural lan may be experienced by individuals living at certain latitudes. during the summer at northern latitudes near the arctic circle, a phenom- enon referred to as ‘midnight sun’ occurs, in which the sun remains visible throughout the night. scandinavian countries, for example, experience sunlight almost h each day during certain times of the year. in parts of finland’s territory lying north of the arctic circle, the sun does not set for days during the summer. the circadian system is not adapted to such extremes in the light environment. at least one study has reported that the number of violent suicides in these regions increases dramatically during times of ‘midnight sun’, suggesting that even naturally occurring lan can negatively influence mood and behavior. in contrast, one population in the united states excluded from many types of lan exposure is the old order amish. the amish do not use televisions or computers, which are a major source of lan exposure among the general population, and lan exposure is minimal. interestingly, the amish display greatly reduced cancer rates compared with the general population. incidence of depression and other psychiatric disorders is also reduced. of course, other lifestyle factors may contribute to better health in this population, but the amish represent an interesting opportunity to understand the effects of modern lifestyle choices on mood. physiological effects of lan clearly, exposure to lan is quite pervasive in western societies and several mechanisms exist by which this unnaturally timed exposure may influence mood. for one, at least in diurnal species, lan is likely a sleep disruptor. there is a large literature implicating sleep disruption or deprivation in negative mood regulation (reviewed in tsuno et al.). as many as – % of depressed patients complain of poor sleep quality and b % of insomniacs are clinically depressed. a relationship exists between disrupted sleep and depression and chronic exposure to lan may be one contributor. recent rodent studies suggest, however, that lan can influence mood independent of sleep disruption, and this evidence will be reviewed in detail later. pineal melatonin secretion is also disrupted by lan and accumulating evidence suggests that melatonin is related to mood. agomelatine, a melatonin-receptor agonist and serotonin ( -ht c) receptor antagonist, belongs to a new class of melatonergic antidepressants. , in rodents, melatonin administration prevents stress-induced depressive-like behaviors and reductions in hippocampal dendritic complexity. melatonin has positive effects on hippocampal cell proliferation and can stimulate neurotrophin production in the brain—both of which are responses associated with antidepressants. direct dysregulation of circadian clock genes could also underlie depression associated with lan exposure. some clock genes are directly regulated by light. for example, per expression in the scn is directly stimulated by acute lan exposure. disruption of clock gene oscillations, particularly within limbic regions that receive projections from the scn, may contribute to altered mood. over time, upsetting the daily oscillation of clock genes and their protein products may alter the function of important mood- regulating systems—for example, it may alter expression and activity of neurotransmitter receptors implicated in mood, and clock gene variants have been associated with risk of mood disorders in humans (reviewed by mcclung ). the precise role of clock genes in mood regulation remains undetermined, as does the question of whether acute disruption of clock genes is sufficient to provoke altered mood regulation or whether disruption over the long term is required. similarly, diurnal variations in hormone secretion may be disrupted by lan, particularly within the hypothalamic-pituitary- adrenal (hpa) axis. recall that the pvn receives direct input from the scn, which projects to the pituitary and then the adrenal. cortisol, a major stress hormone released by the adrenal gland, has been implicated in depression and atrophy of the hippocam- pus when levels are chronically elevated. in depressed patients, the diurnal rhythm of cortisol concentrations tends to be lost, and levels are consistently high throughout the day. it is possible that lan may act at any or all of these levels to provoke altered mood regulation. indeed, it is likely that many influence of the modern light environment on mood ta bedrosian and rj nelson & macmillan publishers limited molecular psychiatry ( ), – layers are implicated at once, causing a complex interplay of misaligned systems. disruption at one level could also provoke further dysregulation at another; for example, suppressed melatonin secretion could in turn further disrupt diurnal clock gene oscillations throughout the body, compounding the effects of lan exposure. evidence linking lan to mood humans several studies in shift-working populations have linked night work to negative affect. among us workers, regular shift work has detrimental effects for sleep and social factors, and is associated with high prevalence of mdd, with a higher rate among women than men. the prevalence of mdd during or after night shift work is greater than the general population prevalence, but reflects the same trend of disproportionately affecting women ( . % of female shift workers vs . % of male shift workers). long-term shift work is not necessary to see these effects, however. among young student nurses performing night shift work for the first time, feelings of helplessness, loss of control, apathy and low social support were perceived after only months of night work. with longer term exposure (up to years of shift work), there is an increased lifetime risk of mdd. one of the proposed treatments for problems associated with night shift work is bright light exposure during the night to phase shift the biological clock to align with the work schedule, thus improving alertness at work and facilitating sleep during the day. a limitation of this approach is the difficulty maintaining such a schedule. on weekends and ‘off’ days, workers quickly revert back to synchronize with environmental and social cues. although increased depressive symptoms have been well- documented among shift workers, these studies are limited in establishing an exclusive link to lan due to the other variables involved, such as sleep disruption and episodic (for example, weekend) phase shifts. jet lag is another modern change that is thought to be related to changes in affect. patients admitted for psychiatric emergencies are more likely to exhibit depression or mania after travel across time zones compared with those admitted with no recent travel history, and those traveling westbound show the most symptoms of depression. even from a biological perspective, in flight attendants transmeridian travel with short recovery time between trips is associated with reduced temporal lobe volume that is related to cognitive deficits. unfortunately, these studies are limited by the number of variables, as both shift work and jet lag tend to disrupt sleep and social schedules in addition to lighting exposure. it is difficult to obtain direct data linking lan to mood in human subjects because such studies would entail careful control of several environmental variables over many weeks. animal models animal models allow mechanistic studies into the role of lan in mood regulation, without the confounding variables associated with human studies, and allow investigation into the molecular basis of lan effects. a summary of results from these studies is presented in table . our laboratory recently developed a model of dim ( lx) lan exposure using siberian hamsters. this light intensity is similar to levels that may be easily encountered by the average individual through the use of televisions, computers and e-readers at night. one general limitation of rodent models, however, is that species differ in their responsiveness to a particular intensity. for example, melatonin is suppressed by about . lx in syrian hamsters, whereas lx decreases melatonin levels in humans. , it is not clear how other circadian responses to a given intensity may compare between different species. an alternate model is to study the effects of housing rodents in constant light (ll) without varying light intensity; however, rodents become arrhythmic under these conditions, making it difficult to obtain interpretable results. hamsters exposed to lx dim lan, however, remain entrained to the light–dim light cycle, at least in terms of locomotor activity. some changes in activity occur, however, as homecage locomotor activity is slightly reduced during the dim light phase compared with typical dark phase activity levels and fast fourier transforma- tion analysis reveals slight decrements in the strength of the -h rhythm. eliminating the lan rapidly reverses this effect. entrainment of other diurnal rhythms may be influenced, however. the daily expression patterns of per and per proteins in the scn are altered with lan exposure; in both cases, the rhythm is weakened, but bmal expression remains intact. the daily fluctuation in plasma cortisol concentrations is abolished as well. there are two advantages to using siberian hamsters for lan studies. first, they express melatonin receptors in the brain and, unlike most inbred strains of laboratory mice, they produce detectable levels of pineal melatonin. the extent to which melatonin is implicated in lan effects on mood is still unclear, as depressive responses after exposure to lan have been reported in at least one melatonin-deficient mouse strain. however, because melatonin suppression may likely occur in humans exposed to lan, using a melatonin-proficient animal model may increase the translational relevancy to humans. second, unlike humans, hamsters are nocturnal, meaning that lan exposure occurs during their active period, allowing us to separate the effects of lan and disrupted sleep. lan does indeed influence mood and physiology in at least one diurnal species, nile grass rats; however, sleep quality was not investigated in these studies, making it difficult to attribute solely the findings to lan. our studies have consistently demonstrated that dim lan provokes depressive-like responses in hamsters. , because of the difference in activity levels during the dark or dim phase, behavioral testing for activity-dependent measures must be performed during the light phase, when activity levels are equivalent between groups. after weeks of exposure to nightly lx lan, hamsters tested during the light phase display more immobility in the forced swim test, typically interpreted as behavioral despair, and reduced preference for sucrose solution, an anhedonic-like symptom. , within weeks of eliminating lan, however, behaviors in both of these tests resemble those of hamsters exposed only to dark nights. one method of validation for behavioral assays of depressive-like response is to measure behavior after treatment with an antidepressant drug that is known to be effective in humans. although siberian hamsters housed in dim lan for weeks develop depressive-like symptoms in the forced swim test, weeks of treatment with the selective serotonin reuptake inhibitor, citalopram, ameliorates the symptoms, supporting the validity of this model. the complete mechanism underlying these behavioral responses remains unknown, but we have observed stark changes to the hippocampus, one brain structure implicated in the pathophysiology of mdd. depressed patients often have hippo- campal atrophy – and dysregulation of many hippocampal- related systems, such as stress coping and memory (reviewed in nestler et al.). similarly, loss of hippocampal dendritic spines and reduced dendritic complexity are observed in animal models of chronic stress and depression. – in these models, expression of brain-derived neurotrophic factor is typically reduced in the hippocampus, but antidepressant drugs enhance its expression. hamsters exposed to weeks of dim lan have reduced dendritic spine density on hippocampal ca pyramidal neurons, although no other changes to overall dendritic complexity or to neurons within other hippocampal subregions are apparent, and reduced mrna expression of brain-derived neurotrophic factor in influence of the modern light environment on mood ta bedrosian and rj nelson molecular psychiatry ( ), – & macmillan publishers limited the hippocampus. both of these effects are reversed after eliminating lan. in addition, the hippocampus is disproportionately vulnerable to inflammation compared with other brain structures because of its high expression of receptors for proinflammatory cytokines such as il- b and tumor necrosis factor-a. neuroinflammation may have a role in depressive-like behavior provoked by a variety of types of circadian disruption. as mentioned, exposure to constant darkness induces depression-like behavior and increased interleukin- levels, which are ameliorated by blocking nuclear factor-kb signaling. furthermore, mice with a deletion of interleukin- are resistant to the behavioral effects of exposure to constant darkness. in our experiments, hamsters exposed to dim lan show increased tumor necrosis factor-a expression in the hippocampus; treatment with a pharmacological inhibitor of tumor necrosis factor-a prevents depressive responses in the forced swim test after exposure to lan. this link between depression-like behavior provoked by circadian disruption and neuroinflammation is not surprising. a role for proinflammatory cytokines in the pathogenesis of depression has been proposed and recent evidence demonstrates a direct molecular pathway whereby disruption of circadian clock proteins enhances expression of cytokines within the brain. our results suggest that exposure to lan alters mood by disrupting circadian rhythms, but to answer the question of whether unnatural light exposure on its own can directly impair mood, a model of aberrant light exposure was used that does not strongly influence circadian timing or sleep. mice were exposed to an ultradian cycle of . h light and . h dark (termed t ), which lengthens the period of body temperature and locomotor activity rhythms, but maintains diurnal fluctuations in per expression in the scn and total sleep. after weeks of exposure to the ultradian light cycle, mice exhibited increased depressive-like responses in the forced swim and sucrose preference tests. further, the mice had impaired learning and memory performance accompanied by reductions in hippocampal long-term potentiation. much like the results we have obtained using dim lan, treatment with an selective serotonin reuptake inhibitor reversed the depressive responses. these behaviors seem to occur through iprgc projections because mice lacking the gene for melanopsin do not develop depressive responses after exposure to the ultradian light cycle. the results of this study led the authors to conclude that lan influences mood regulation directly, without disrupting circadian rhythmicity. this is an intriguing hypothesis, particularly in light of evidence that iprgcs project to mood-regulating regions of the brain, although whether these pathways are direct connections or projections through the scn remains unclear. however, several technical considerations may limit the conclusions drawn from this study. for one, time of day for behavioral testing procedures was not described, making it unclear whether differences in locomotor activity levels evident under the t cycle may have contributed to the results. in addition, per expression was measured relative to the activity of the animal. in other words, time points were chosen based on zeitgeiber time for control mice in a -h light cycle, but based on circadian time for t mice. it is not clear whether behavioral testing was similarly coupled. presumably, behavioral measurements were performed in both groups at one time of day, thus uncoupling depressive-like behaviors from the activity rhythm. time of day effects on behavioral performance in these tasks is well-established and particularly the forced swim test is highly dependent on the activity level of the animal. it is possible that the effects ascribed to the t cycle may actually reflect testing occurring during different circadian phases for each group. furthermore, the mechanism through which these changes may occur remains undetermined. although it is tempt- ing to speculate that unnatural timing of light exposure may contribute to impaired mood, more investigation will be necessary to parse out circadian effects from direct light effects. nevertheless, converging evidence seems to point towards negative effects of lan on mood regulation. an important question arising from this collective work is how to prevent the deleterious effects of lan when exposure cannot be avoided. one possibility may be to manipulate the wavelength of light exposure. as mentioned previously, the melanopsin-expressing iprgcs responsible for projecting light information to the circadian system are minimally responsive to red wavelength light. replacing standard bulbs with red ones where possible, or using glasses that only transmit red wavelengths, may be an effective preventative against the disruptive effects of lan. we have observed reduced effects of dim red lan on hamster depressive responses, compared to white or blue lan, along with reduced fos activation in the scn following red lan exposure. clearly, lan has a variety of effects on the brain and circadian rhythms, each potentially contributing individually or in concert with others to regulate mood. implications and future directions the incidence of depressive disorders has increased significantly in recent decades, , , in parallel with the expansion in the use of electric lan. particularly, urban-dwelling individuals and night shift workers are exposed to artificial lan on a chronic basis. for these people, lan may be considered as a modern circadian table . summary of evidence from rodent studies demonstrating effects of lan on mood and cognition species active period light manipulation findings—behavior findings—brain fonken et al. mouse (swiss webster) nocturnal ll m fst immobility none measured k sucrose preference bedrosian et al. siberian hamster nocturnal lx dim lan m fst immobility k hpc spine density k sucrose preference fujioka et al. mouse (c bl/ ) nocturnal ll k spatial learning k hpc neurogenesis fonken et al. nile grass rat diurnal lx dim lan m fst immobility k hpc dendritic length k sucrose preference k spatial learning legates et al. mouse (b / f hybrid) nocturnal ultradian t cycle m fst immobility iprgcs necessary k sucrose preference reversed by fluoxetine k spatial learning bedrosian et al. siberian hamster nocturnal lx dim lan m fst immobility reversed by citalopram bedrosian et al. siberian hamster nocturnal lx dim lan m fst immobility k hpc bdnf mrna k sucrose preference m hpc tnf mrna abbreviations: fst, forced swim test; hpc, hippocampus; iprgcs, intrinsically photosensitive retinal ganglion cells; lan, light at night. influence of the modern light environment on mood ta bedrosian and rj nelson & macmillan publishers limited molecular psychiatry ( ), – disruptor. given the evidence for disrupted circadian processes in depression, excessive lan exposure could be one factor contributing to depressed mood among vulnerable individuals. artificial and unnaturally timed light from the environment could disrupt physiological timekeeping, leading to misalignment of various biological rhythms, or could act directly to influence mood. studies using animal models have been useful thus far to identify a link between lan and mood, but such a link in humans remains to be demonstrated. a first step would be to perform thorough correlational analyses through epidemiological analysis. within this past decade, epidemiological work paved the way toward identifying the relationship between lan and breast cancer, which is now officially recognized by the who and american medical association. such studies should not only focus on shift workers but also include individuals experiencing low levels of lan at home. example studies are already beginning to identify relationships between lan and obesity. from an historical perspective, the widespread adoption of electric light occurred before an understanding of circadian biology. the effects of what we now know to be a major change for circadian biology were simply not considered. it is essential that we learn about the effects of modern technology on the brain and health, so that we might appropriately manage 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circadian clock protein cryptochrome regulates the expression of proinflammatory cytokines. proc natl acad sci usa ; : – . legates ta, altimus cm, wang h, lee hk, yang s, zhao h et al. aberrant light directly impairs mood and learning through melanopsin-expressing neurons. nature ; : – . hattar s, kumar m, park a, tong p, tung j, yau kw et al. central projections of melanopsin-expressing retinal ganglion cells in the mouse. j comp neurol ; : – . bedrosian ta, vaughn ca, galan a, weil zm, nelson rj. role of melatonin in depressive-like behaviors provoked by chronic light at night. society for neu- roscience annual meeting, , new orleansp. . . compton wm, conway kp, stinson fs, grant bf. changes in the prevalence of major depression and comorbid substance use disorders in the united states between – and – . am j psychiatry : – . simon ge, vonkorff m. reevaluation of secular trends in depression rates. am j epidemiol ; : – . obayashi k, saeki k, iwamoto j, okamoto n, tomioka k, nezu s et al. exposure to light at night, nocturnal urinary melatonin excretion, and obesity/dyslipidemia in the elderly: a cross-sectional analysis of the heijo-kyo study. j clin endocrinol metab ; : – . influence of the modern light environment on mood ta bedrosian and rj nelson & macmillan publishers limited molecular psychiatry ( ), – 	title_link 	introduction 	photoentrainment 	melatonin 	circadian disruption and mood 	figure™ worldwide artificial light at night. inset shows detailed view of north america. images are composites acquired by the nasa suomi npp satellite in 	figure™ potential pathways through which light at night (lan) may influence mood. hpc, hippocampus; pfc, prefrontal cortex; pvn, paraventricular nucleus of the hypothalamus; scg, superior cervical ganglion; scn, suprachiasmatic nuclei 	populations exposed to lan 	physiological effects of lan 	evidence linking lan to mood 	humans 	animal models 	implications and future directions 	table 	a 	a _ olson ©chaplaincy today • e-journal of the association of professional chaplains • volume number • spring/summer forgiveness as a core ingredient of spiritual care: an exploration of four resources roy f. olson bcc forgiveness is a journey that many who have been wounded find most difficult to undertake. the author has over thirty years of experience as a behavioral health chaplain during which he developed and conducted spirituality groups focused on everett worthington’s models of moving from unforgiveness to forgiveness and seeking to reconcile broken relationships. in addition to worthington’s five steps to forgiveness, this article presents three other resources that chaplains may use to assist patients in realizing these goals. forgiveness often is a primary spiritual issue for patients, at least for those hospitalized for psychiatric and substance dependence reasons. the challenge to forgive the divine, others and oneself is daunting. yet, if this journey is not undertaken, unforgiveness may grow like dangerous bacteria without the presence of the antibiotic of forgiveness that arrests and sometimes cures it. this article introduces four excellent resources that the author commends to any caregiver who seeks to become a midwife of forgiveness. five steps to forgiveness a middle-aged woman i’ll call miriam was a patient in a hospital-based day treatment program for adults with mood disorders. typical attendance for a spirituality group in this program was twenty or more persons. miriam sat on my far left. i could barely see her out of my peripheral vision. the topic was “forgiving those who have wronged us.” as it unfolded, i noticed miriam was becoming physically agitated. then she began to mutter under her breath. finally she stood up, shouted, “this is a bunch of crap,” and stormed out of the room. in the days that followed, her self-destructive thinking increased to the point that she was hospitalized. i made two attempts to visit her but each time she angrily told me to leave. she did not want to talk with me. eventually, two spirituality groups were developed utilizing the forgiveness models of everett worthington, a psychologist and professor at virginia commonwealth university. the first focused on his “five steps to forgiveness.” the second group described a process for those who seek to reconcile a broken relationship. from my experience with miriam, i learned that a calm, methodical introduction to the first group was essential. i began by asking for a show of hands as to how many in the group had at least one person in their lives that they had never been able to forgive and secondly, how many thought that it had something to do with their need to be in this program. typically half or more raised their hands for the first question and almost all of those hands went up again for the second. i learned to warn the patients that the content of the forgiveness group would be controversial, challenging, radical and possibly triggering and that if they needed to leave the group they had my permission in advance. it was not unusual to have at least one person leave. roy f. olson dmin bcc (retired) currently serves as one of the on-call chaplains at sherman hospital, elgin, il. he is endorsed by the evangelical lutheran church in america (elca). this article stems from his many years of service as a behavioral health chaplain, most recently at alexian brothers behaviorial health hospital from which he retired last june. olson @sbcglobal.net mailto:olson @sbcglobal.net ©chaplaincy today • e-journal of the association of professional chaplains • volume number • spring/summer i learned to inform the program staff whenever the forgiveness group was scheduled so that they could be prepared for possible fallout. i am grateful to that team for their collaboration and the trust and respect they placed in this work. empathy is the basis for worthington’s model and he states very clearly that god must play a role in the healing of unforgiveness. at the core of his model lies a radical idea based upon research he has done with those who are trying to forgive. if one forgives in order to feel better, it does not last. instead, one must make the far more difficult journey toward developing empathy for the perpetrator and eventually give what usually is an undeserved and unearned gift of forgiveness, often because the perpetrators have never taken responsibility for their actions. in the forgiveness sessions i conducted, i briefly outlined worthington’s description of the journey into unforgiveness and more carefully explored the journey to forgiveness. each has five stages. unforgiveness results from • a transgression, • perception of offense and hurt, • resulting hot emotions like anger and fear, • rumination, which lasts until • unforgiveness has made a permanent home in the psyche. worthington devotes a chapter to each of the five stages of forgiveness: • recall the hurt but with enough detachment to explore it from a fresh perspective. • develop empathy for the perpetrator in three ever more challenging levels—shallow, middle and deep. • give the altruistic gift of forgiveness. he explicitly names the importance of his christian background and the necessity of a role for the divine. granting the gift of forgiveness may or may not be done in person, due to issues of safety or to the inaccessibility of the perpetrator. • commit publicly to forgiveness, recognizing that if this is only an internal process, it will not prove to be lasting. • hold on to the forgiveness. he describes strategies to accomplish this. the second spirituality group explored the stages of reconciliation in which two persons may engage in order to restore a broken relationship. briefly, using the metaphor of approaching a bridge from opposite sides and meeting in the middle, worthington devotes a chapter to each of the four stages in this model: • decide whether or not to try to reconcile. • initiate discussion, probably with a therapist guiding the process. • detoxify, i.e., remove the negative elements from the relationship. • restore or reach a new level of devotion. worthington provides many practical suggestions for utilizing both processes. they may be more suitable for a pastoral counseling or therapy setting, as this is primarily a group rather than an individual model. in a psychiatric treatment setting, the goal of these two spirituality groups was to ©chaplaincy today • e-journal of the association of professional chaplains • volume number • spring/summer introduce the difficult path of forgiveness to the patients and to claim a central role for spirituality. often, requests for individual chaplain visits followed the group experience. my third attempt to visit miriam on the inpatient unit met with a more positive response. she had mellowed considerably and spoke openly about the horrific abuse she had received from the parent who had died in the previous year. she thanked me for getting her started on the forgiveness journey, even if had been very difficult for her at the beginning. in time, she returned to the same day treatment program where it all began. when she attended the spirituality group on forgiveness, she smiled and asked if she could address the group. she told her story about how she had reacted initially to the idea of forgiveness and how she was on that forgiveness journey now. beyond revenge michael mccullough begins beyond revenge with the following story. chante mallard became front-page news. after a long night of partying, drunk and high, she drove her car straight into a man walking along the highway. catapulted over the hood, he came to rest wedged into her windshield, his head and upper torso inside her car. after stopping to try to figure out what happened, in her drugged confusion, she panicked. she decided to drive her car home and into her garage. despite his pleas for help, mallard, a nurse’s aide by profession, let him bleed to death in her garage. the medical examiner stated the victim would have lived if she had called for help. instead, she entertained her boyfriend in her home that night. with the help of friends she eventually dumped the body in a nearby park and joked about it all. chante mallard was eventually arrested. she was convicted of murder and sentenced to fifty years in prison. at her sentencing hearing, brandon, the son of the man that she had allowed to die in her garage, made a victim impact statement. instead of insisting on the severest of penalties, the son spoke to the court and mallard’s family, “‘there’s no winners in a case like this. just as we all lost greg, you all will be losing your daughter.’ later, brandon would go on to say, ‘i still want to extend my forgiveness to chante mallard and let her know that the mallard family is in my prayers.’” mccullough, a professor of psychology at the university of miami, asserts three core truths: • revenge is a built-in feature of human nature. • the capacity for forgiveness is also a built-in feature of human nature. • to make the world a more forgiving, less vengeful place, don’t try to change human nature, change the world! firmly planted in evolutionary theory, he amasses a significant body of research from the social and biological sciences, including studies of the behavior patterns of primates, dolphins, hyenas, goats and fish; the experiments social psychologists love to do with university students; game theory; and computer simulations. he challenges the formulations of the monotheistic religions and much of western literature, rejecting their conceptualization of revenge and forgiveness as a disease/cure model. in that model revenge is a virus that invades a vulnerable host; forgiveness is an external force that must enter the host to treat and cure revenge. instead, from an evolutionary perspective, he views revenge as functional. he maintains that revenge was adaptive in that it • deterred aggressors from aggressing a second time by actual or threatened revenge. • warned would-be harm doers to back off, leading to a deterrence that prevented aggression in the first place, i.e., if you don’t take or threaten revenge, you are labeled an easy mark and become susceptible to being taken advantage of. ©chaplaincy today • e-journal of the association of professional chaplains • volume number • spring/summer • coerced “free riders” to cooperate. a free rider is defined as the person in a group that is loafing and not carrying a fair share of the burden. the threat of ostracism (revenge) brings the person back into line. (think of resistant members of cpe groups or congregations!) likewise, from an evolutionary perspective, forgiveness has adaptive functions. animals and humans hesitate to inflict revenge on any blood relative for it threatens to reduce the success of their gene pool. even with nonrelatives, survival in a hostile environment depends upon forming cooperative alliances. we are forgiving towards neighbors and friends because at some level we need to cooperate with them. stress and anxiety increases in humans and primates when conflict erupts and remains unresolved. in what ways is forgiveness instinctual or hard-wired into human existence? while clinical psychology makes the distinction between forgiving without continuing a relationship and reconciliation that seeks to restore a relationship, mccullough believes that both are rooted in the same internal process and exist along a continuum of experience. survival in a hostile environment requires communal living and cooperation, which provides the impetus for seeking forgiveness. anxiety is an unpleasant outcome of unresolved conflict, and both animals and humans are wired to overcome this. biological measurements show that stress and anxiety decrease when one moves in the direction of forgiveness and reconciliation. three conditions awaken the forgiveness instinct: • empathy for those who have harmed them, which is easier if it’s a blood relative. • seeing potential value for maintaining or attempting to restore the relationship. • safety, i.e., having assurance that the other party can no longer inflict harm or is sincere in promising not to do so. three gestures signal the desire to pursue forgiveness: • apologies, including five key ingredients—carefully chosen words, an admission of responsibility, an explanation/clarification of why the hurt occurred, an offer of reparations and a promise not to repeat the action. • self-abasing displays and gestures (body language). • offer of compensation. (the amount is less important than a genuine offer.) mccullough maintains that the major world religions exhibit the desire for revenge and the desire for forgiveness both in their historical behaviors and in their sacred texts. his conclusion about how to move forward deserves a hearing and may resonate with the core convictions of chaplains, pastoral counselors and clinical educators. if you want religious groups around the world … to be forces for forgiveness, you need to create the conditions that will cause them to perceive that forgiveness is in their best interests. when you do, they’ll emphasize the doctrine and traditions that favor forgiveness. if those religious groups perceive instead that revenge is the behavioral option that will work best for them, then that’s what you’ll get from them … . the challenge for harnessing religion’s power to motivate forgiveness is to create the kind of socio-political world in which religious groups can’t help but perceive that forgiveness is in their best interests. in figuring out how to make these kinds of sociopolitical realities happen, we’d be fools not to try to work with reformers within those traditions who can offer help and guidance. ©chaplaincy today • e-journal of the association of professional chaplains • volume number • spring/summer transcending tragedy the bucolic serenity of amish life in lancaster county, pennsylvania was irreparably shattered on october , , when a monstrous deed was done. some would later say it was their / . a lone gunman, charles carl roberts iv, burst into a one-room schoolhouse of twenty-six children, aged six to thirteen, in the community of nickel mines. he brought with him guns and supplies intending to barricade himself for a standoff. the teacher snuck out a side door and ran for help. he dismissed other adults who happened to be present and all the boys. he announced his intention to molest the ten remaining girls. the police arrived more quickly than he expected, disrupting his plans. about twenty minutes later shots were fired inside the school and out the window at them. they rushed into the building as roberts shot himself and discovered roberts had murdered five of the girls, execution style. five survived. as the news spread, large numbers of law enforcement and community service personnel, including grief counselors, responded to the disaster. it also rapidly became a media event. huge vans with satellite dishes and reporters with cell phones further shattered amish life. the media came to report a story of evil, hatred and violence; thus, they were totally unprepared for what gradually became a story of grace and forgiveness. the amish community quickly realized that the surviving roberts family members, which included his wife, three children and the children’s grandparents, were also victims of this tragedy. they searched for them to offer their compassion and forgiveness. when the roberts family gathered to bury charles at the local methodist church, more than half of the attendees were amish. the question lingered as to whether charles roberts was a monster. how could one develop empathy for him, something both worthington and mccullough have suggested is necessary in order to move from revenge toward forgiveness? one of the surviving children reported that roberts said to the girls, “i’m angry at god and i need to punish some christian girls to get even.” during the standoff, roberts called his wife on his cell phone to say he was not coming home and that he had left notes for everyone. he was angry at god, he said, for the death of their firstborn daughter, elise, who had lived for only twenty minutes after her birth nine years earlier. in the note to his wife roberts had written, “i’m not worthy of you, you are the perfect wife, you deserve so much better …. i’m filled with so much hate towards myself, hate towards god, and an unimaginable emptiness. it seems like every time we do something fun i think about how elise wasn’t here to share it with us and i go right back to anger.” there was speculation that roberts might have been abused as a child but no solid evidence of it was uncovered. what would the chaplain’s assessment be of roberts’ spiritual condition at the time of the shooting? in their book, amish grace: how forgiveness transcended tragedy, donald kraybill, et al., have written a meticulously detailed and emotionally moving account of the events surrounding the school massacre and the response of the amish community to the murder of their children. their knowledge of amish life is extensive. after telling the story at some length, they explore the history of the anabaptist movement, including its many early martyrs and how literal adherence to the teachings of jesus, especially forgiveness, is a way of life. the presentation of this tragedy and its aftermath, especially the insistence of the amish that they must forgive, gives spiritual caregivers much upon which to reflect. amish grief is not stoic denial, yet there is no room for anger at god. (the reluctance to support anger in general or anger at god is also part of the spirituality of addiction recovery.) submission or surrender to the will of god is a core element of their spirituality. because this tragedy fell upon more than one family simultaneously, an entire community took up the task of forgiveness together. yet a deeper understanding of amish life reveals that grief and forgiveness are communal tasks whether or not ©chaplaincy today • e-journal of the association of professional chaplains • volume number • spring/summer there were multiple victims. their way of life rejects much of the individualism in mainstream american culture that suggests forgiveness is an individual decision and task. it is not left to one person or one family to manage hate, bitterness and resentment; these are absorbed and overcome by the entire community. chaplains know the difference between ministering to a nuclear family coping with tragedy and joining in the ministry of a large extended family who gathers to grieve together. the amish in this story testify not only to their belief in forgiveness but also to the difficulties inherent in practicing forgiveness. there is no denial of how long or difficult maintaining their stance of forgiveness will be. they fully acknowledge it will be an ongoing struggle. these events and their response to them raise challenging questions. do representatives of the church sometimes counsel or insist upon offering forgiveness too quickly, especially in cases of abuse? is god’s forgiveness conditional or unconditional—does god’s forgiveness depend upon our willingness to forgive? kraybill wrestles with these and other complicated questions. those of us who work alongside counseling colleagues are aware that they perceive their task as moving persons from an identity as a victim of a tragedy to that of a survivor. kraybill suggests this amish story raises the possibility of moving to a third stage. renouncing the right to remain bitter about the outcome of the shooting means one becomes “a hero instead of a victim in the story.” not that the victims claim to be heroes or heroines, but in our telling of their stories, they are perceived as such. i would suggest that our role as caregivers is not only to help victims, when ready, to move toward empathy and walk the perilous path toward forgiveness. it is also to tell to one another—and to all who will listen—the many remarkable stories of people who were able to forgive. the stories of miriam, brandon and the amish are only a few of the stories we could tell. the power of forgiveness these three excellent resources may deepen the chaplain’s understanding of forgiveness both from a psychological/clinical and from a spiritual/theological perspective. the final resource is the power of forgiveness, an excellent dvd production that includes eleven chapters. two of the three authors mentioned earlier in this article are featured. everett worthington is shown teaching in his psychology classroom and conducting a group therapy session. he tells his own tragic story of the brutal murder of his mother and his challenge to forgive the perpetrator. donald kraybill retells the story of the nickel mines tragedy. in addition, a number of spiritual leaders from various faith traditions, including james forbes, elie wiesel, thich nhat hanh, marianne williamson and thomas moore, share their perspectives on the subject of forgiveness. lengthier stories include the portrayal of the legacy of hatred in northern ireland’s long-standing conflict between catholics and protestants. community leaders have come together to address their concern that unresolved conflict and hatred is being passed on from one generation to another without interruption. in response, they have designed forgiveness curricula, which are being implemented in elementary school classrooms. these do not directly address the historic political conflicts. rather, through story telling and conversation they introduce forgiveness concepts and practices into the daily classroom experiences of elementary school children. elie wiesel reflects on his experiences in auschwitz and asks a poignant question. are some human deeds too horrible to be forgiven? included are excerpts from his address to the german bundestag in berlin in which he asks the leader of the bundestag if the time has not come for him to ask for the forgiveness of the jewish people for what the third reich had done in the name of germany. later, in an address to the knesset of israel, the german leader does exactly what wiesel requested. another story focuses on the aftermath of / through the eyes of a widow whose husband died in the twin towers and two mothers whose sons also died that day—one a firefighter and the other an office worker. they question the wisdom of the all-out effort to remove the remaining debris as ©chaplaincy today • e-journal of the association of professional chaplains • volume number • spring/summer quickly as possible. they visit the dump where it was deposited, searching in vain for a gravesite and a sense of closure. after an interview with the priest of st. paul’s parish near ground zero, who hopes to create a garden of forgiveness nearby, the three women visit such a garden, which is under construction in beirut, lebanon. alexandra asseily, who provided the vision for the lebanese garden, sees it as a place where historic religious conflicts existing over many centuries may begin to heal. the final story is that of two remarkable men, who united in a mission to speak to schoolchildren about their experiences. the son of azim khamisa was murdered in a senseless act of violence. ples felix is the grandfather and guardian of the teenage boy who committed that act of murder. azim is a sufi muslim, whose spiritual advisor suggested that if he was ever going to overcome his grief he would have to commit himself to an act of charity. he decided to develop a foundation to educate young people about the evils of violence. he asked ples, a baptist from the american south, to help him. on their journey together, they became best of friends. azim eventually became able to forgive ples’ grandson, which led him to forgive his parents’ abandonment of him in his childhood. ples, in turn, was able to forgive himself for his failures in raising his grandson. this is the story they tell to the schoolchildren. as chaplains we willingly—and sometimes unwillingly—absorb pain, grief, hate, bitterness and resentment. we are committed to steering those who appear ready and able toward the perilous pathway to forgiveness. these four resources provide excellent assistance in our own journeys as agents of forgiveness. everett worthington, five steps to forgiveness: the art and science of forgiving (new york: crown publishers, ). michael e. mccullough, beyond revenge: the evolution of the forgiveness instinct (san francisco: jossey-bass, ). ibid., xiv. for a humorous account of how not to apologize, mccullough cites bill clinton’s many failed attempts to apologize to the american people for his white house dalliance and trent lott’s failed attempts to apologize for his misplaced praise of strom thurmond’s run for president as a segregationist. ibid., - . donald b. kraybill, steven m. nolt, david l. weaver-zercher, amish grace: how forgiveness transcended tragedy (san francisco: john wiley & sons/jossey-bass, ), . ibid., . martin doblmeier, the power of forgiveness (dvd: www.journeyfilms.com, ). http://www.journeyfilms.com mapping of sudden infant death with dysgenesis of the testes syndrome (siddt) by a snp genome scan and identification of tspyl loss of function erik g. puffenberger*†, diane hu-lince†‡, jennifer m. parod†‡, david w. craig‡, seth e. dobrin‡, andrew r. conway§, elizabeth a. donarum¶, kevin a. strauss*, travis dunckley‡, javier f. cardenas‡, kara r. melmed‡, courtney a. wright‡, winnie liang‡, phillip stafford‡, c. robert flynn�, d. holmes morton†, and dietrich a. stephan‡** *clinic for special children, strasburg, pa ; ‡neurogenomics division, translational genomics research institute, phoenix, az ; §silicon genetics, redwood city, ca ; ¶department of neurodevelopmental genetics, barrow neurological institute, phoenix, az ; and �arizona biodesign institute and harrington department of bioengineering, arizona state university, tempe, az edited by albert de la chapelle, ohio state university, columbus, oh, and approved june , (received for review february , ) we have identified a lethal phenotype characterized by sudden infant death (from cardiac and respiratory arrest) with dysgenesis of the testes in males [online mendelian inheritance in man (omim) accession no. ]. twenty-one affected individuals with this autosomal recessive syndrome were ascertained in nine separate sibships among the old order amish. high-density single- nucleotide polymorphism (snp) genotyping arrays containing , single-nucleotide polymorphisms evenly distributed across the human genome were used to map the disease locus. a genome- wide autozygosity scan localized the disease gene to a . -mb interval on chromosome q . -q . . this interval contained genes, including two testis-specific y-like genes (tspyl and tspyl ) of unknown function. sequence analysis of the tspyl gene in affected individuals identified a homozygous frameshift mutation ( � insg) at codon , resulting in truncation of translation at codon . truncation leads to loss of a peptide domain with strong homology to the nucleosome assembly protein family. gfp-fusion expression constructs were constructed and illustrated loss of nuclear localization of truncated tspyl, suggest- ing loss of a nuclear localization patch in addition to loss of the nucleosome assembly domain. these results shed light on the pathogenesis of a disorder of sexual differentiation and brainstem- mediated sudden death, as well as give insight into a mechanism of transcriptional regulation. over two generations, nine families from the bellevilleamish community have lost infants to a recently discovered disorder we have entitled sudden infant death with dysgenesis of the testes syndrome (siddt; fig. ) [online mendelian inheritance in man (omim) accession no. ]. the condition is not seen in the lancaster county old order amish population. three of these infants have been cared for at the clinic for special children. most previous cases were studied extensively at regional medical centers; however, no diagnostic tests were available, and clinical recognition of the syndrome was difficult, particularly in affected females. caretakers say that they can often recognize affected infants at birth by the unusual sound of their cry, which is a staccato sound, similar to the cry of a goat (see materials and methods for a complete description of the syndrome). homozygosity mapping to identify disease genes in autosomal recessive disorders common in founder populations by using traditional methods has often been hampered by microsatellite marker density ( , ). typically, � microsatellite markers are used in such linkage studies spaced at an average -centimorgan density throughout the genome. single-nucleotide polymor- phisms (snp) are present in the genome at an average density of � per , bp and hold enormous potential as a high-density high-throughput genotyping strategy for disease gene mapping ( ). information content is a function of marker heterozygosity, distance between markers, and pedigree structure. although each individual biallelic snp is less informative than a single microsatellite marker in most cases (e.g., average heterozygosity of . on affymetrix k array vs. . on the center for inherited disease research database), the greater number of snps in aggregate leads to higher information content at any particular point in the genome ( ). the affymetrix k array assay requires only ng of dna and generates , snp genotypes with an average resolution of one snp every kb ( ). this new genotyping platform has a throughput -fold greater than microsatellite genotyping and an accuracy of � . % with automated allele calling. the high density and information content of this genotyping platform make it ideal for localizing small regions of homozygosity. blinded validation studies were done to verify that gene mapping could be accomplished using the snp arrays in small disease pedigrees with known map location. the gene for each disease was previously mapped and the causative mutation identified. in each case, we were able to reproduce the mapping results. a software analysis package entitled varia (silicon genetics) was developed to handle the large amount of data inherent to these assays and to correctly localize disease-carrying loci from markers that are in linkage disequilibrium with one another. the genome-wide linkage scan conducted on the multiplex siddt pedigree rapidly and unambiguously mapped this disorder to q with a location score of . [maximum -point logarithm of odds (lod) of . ] in a . -mb interval. sequencing of two candidate genes in the region identified a nonsense mutation in the testis-specific y-like gene (tspyl). functional validation was performed through construction of gfp-fusion proteins of both the full-length and truncated tspyl proteins to investigate the effect of truncation on cellular localization. materials and methods subjects and samples. dna samples used in mapping and se- quencing studies of siddt were acquired by the clinic for special children, with informed consent. peripheral blood was collected from four affected individuals, their parents, siblings, and extended family members. samples from maple syrup urine this paper was submitted directly (track ii) to the pnas office. freely available online through the pnas open access option. abbreviations: siddt, sudden infant death with dysgenesis of the testes syndrome; tspyl, testis-specific y-like; snp, single-nucleotide polymorphism; nap, nucleosome assembly protein; lod, logarithm of odds. data deposition: the disorder reported in this paper has been deposited in the online mendelian inheritance in man (omim) database (accession no. ). †e.g.p., d.h.-l, and j.m.p. contributed equally to this work. **to whom correspondence should be addressed. e-mail: dstephan@tgen.org. © by the national academy of sciences of the usa www.pnas.org�cgi�doi� . �pnas. pnas � august , � vol. � no. � – g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , disease, crigler–najjar syndrome, and congenital nephrotic syndrome families were previously collected with informed consent and described elsewhere ( , ). genealogies for all families in the four diseases described herein were prepared through interviews and several published family histories. ad- ditional research was performed at the lancaster mennonite historical society (lancaster, pa). clinical description of the condition. infants with siddt appear normal at birth, develop signs of visceroautonomic dysfunction early in life, and die before months of age of abrupt cardiorespiratory arrest. one infant died in the hospital while awake and attached to a cardiac-respiratory monitor. the mon- itor showed simultaneous asystole and cessation of respiratory efforts. another infant died suddenly and unexpectedly at home, with no premonitory signs. in both cases, neuropathological examinations were done by experts in sudden infant death syndrome. brain and peripheral nerves were normal. specifi- cally, there was no dysplasia or inf lammation of the brainstem and no pathology of cervical anterior horn cells or lower motor neurons of the hypoglossal nerve. the absence of anterior horn cell pathology contrasts with signs of progressive craniocervical and upper thoracic motor unit dysfunction in affected patients. these signs can include tongue fasciculations, ocular palsies, symmetric weakness of the facial nerve, and diminished upper extremity ref lexes. signs of abnormal autonomic and visceral nerve regulation manifest within the first months of life and include neonatal bradycardia, hypothermia, severe gastroesophageal ref lux, la- ryngospasm, bronchospasm, and abnormal cardiorespiratory patterns during sleep. all affected infants have a hyperactive startle ref lex that can be provoked by loud noise, bright light, movement, or tactile stimulation (e.g., bathing). the ref lex is unresponsive to clonazepam. affected newborns are difficult to feed and may require nasogastric tube alimentation. infant vii- was evaluated twice, at and weeks of age, for feeding and respiratory problems. at age weeks, she presented with poor growth, persistent stridor, and episodes of laryngospasm and cyanosis. a barium swallow excluded tracheoesophageal fistula. a five-channel pneumogram and ph probe showed esophageal ph � for % of study (normal � %), with central, obstruc- tive, and mixed apneas. direct laryngoscopy revealed aretynoid and postcricoid supraglottic erythema. she had persistent bilat- eral perihilar consolidations on serial chest radiographs. this infant underwent gastrostomy tube placement and fun- doplication, which prevented acid ref lux and provided airway protection, but she continued to have life-threatening apnea and obstructive breathing. when a five-channel pneumogram and ph probe was repeated at age weeks, attempted passage of the probe through both left and right nares provoked severe brady- cardia and cyanosis. this -h study showed normal lower esophageal ph, frequent isolated bradycardias, extreme back- ground heart rate variability (range – beats per minute), and multiple bradycardic events associated with disorganized breathing or prolonged apnea. a salivagram showed normal transit of sublingual tracer from mouth to stomach, with no aspiration. a cranial mri was normal and electroencephalo- gram showed normal waking, drowsiness, and slow-wave sleep electrical patterns. despite exclusive gastrostomy tube feeding, growth remained poor, and she died suddenly before her first birthday. genotypic males with siddt syndrome have fetal testicular dysgenesis and ambiguous genitalia and can be mistaken for females. in two genetically male but externally phenotypic female infants with the syndrome, intraabdominal testes were examined at autopsy. they were dysplastic, with tortuous vas- cularity and an arrest of cell development at an early stage. both leydig and sertoli cells were present but diminished in number and nested in rudimentary fibrotic cords. normal regression of mullerian structures in males indicates that sertoli cells secrete mullerian inhibiting hormone. however, leydig cells fail to produce the testosterone and dihydrotestosterone throughout fetal life necessary to sustain growth and development of the external male organ. this can be detected postnatally as an abnormal response to human chorionic gonadotropin challenge. development of male genitalia arrests at variable embryologic stages. at birth, some males were thought to be females on the basis of external genitalia, but other male infants had fusion and rugation of the gonadal sac and partial development of the penile shaft. variable maturation of male genitalia indicates that leydig cell failure can occur at different times throughout fetal development. female sexual development is normal, as is neonatal hypo- thalamic and ovarian function. patient vii- , a genotypic and phenotypic female, had normal internal and external genitalia and normal postpartum elevation of serum estradiol, luteinizing hormone (lh), and follicle-stimulating hormone (fsh). adre- nal and gonadal pathways of steroid biosynthesis were studied extensively in patient vi- and found to be normal. in this child, pituitary and hypothalamic functions were also normal with regard to serum thyroid, corticotropin (acth), cortisol, lh� fsh, and prolactin levels. additional endocrinological and biochemical studies were done on siddt syndrome patients at various stages of investi- fig. . siddt in a consanguineous old order amish pedigree. (a) tspyl � insg mutation status is indicated for available pedigree members (m denotes � insg, whereas � represents wild-type sequence). (b) sequencing of tspyl reveals a homozygous single base-pair insertion ( � insg) in siddt syndrome patients. (i) tspyl sequence from a normal control. (ii) � insg heterozygote. (iii) � insg homozygote. � www.pnas.org�cgi�doi� . �pnas. puffenberger et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , gation. plasma sterol and organic analyses by gas chromatogra- phy mass spectrometry, serum acylcarnitines by tandem mass spectrometry, steroid immunoassays for congenital adrenal hy- perplasias, and plasma amino acid analyses yielded normal results. cerebrospinal f luid amino acids and neurotransmitter metabolites were normal in patient vii- . chromosomes were done in many cases, both to establish genotypic sex and to detect structural aberrations; no abnormalities were found. pcr and gene sequencing excluded spinal muscular atrophy (smn exon deletion). an assay was done for the triplet repeat expansion of the testosterone receptor gene, which in adult males causes testicular failure and progressive bulbar weakness (kennedy syndrome), and results were normal. genotyping. dna from whole blood was isolated by using the puregene dna isolation kit (gentra systems). snp geno- typing was done by using the genechip mapping k array and assay kit (affymetrix). the genotyping protocol was modified from kennedy et al. ( ). all incubations were done by using a tetrad thermal cycler (mj research, cambridge, ma). internal positive and negative controls were performed in parallel by using the supplied genomic dna (affymetrix). two hundred fifty nanograms of double-stranded genomic dna was digested with xbai (new england biolabs) for h at °c followed by heat inactivation for min at °c. digested dna was then incubated with a . m xba adapter (affymetrix) and dna ligase (new england biolabs) in standard ligation buffer (new england biolabs) for h at °c followed by heat inactivation for min at °c. ligated products were amplified in quadruplicate by using . m of the supplied generic primer xbai (affymetrix) in pcr buffer ii (applied biosystems) with . mm mgcl � . mm dntps� units of amplitaq gold polymerase (applied biosystems) under the following pcr conditions: °c for min, followed by cycles ( °c for sec, °c for sec, and °c for sec) and a final extension at °c for min. fragments in the - to , -bp size range are preferentially amplified under these conditions. pcr products were purified with the qiaquick pcr purification kit (qiagen, valencia, ca) according to the manufacturer’s recommendations, with the exception of the elution procedure. dna from each of four pcr replicate samples was bound to separate columns and washed. the eluant collected from column was used to elute the remaining three columns in series. the final purified product is the combination of four purified pcr product samples. eighteen to twenty micrograms of purified pcr products were fragmented with . units of the supplied genechip fragmentation reagent (af- fymetrix) for min at °c followed by a heat inactivation for min at °c. samples were then labeled with units of terminal deoxytransferase (affymetrix) and . mm of sup- plied genechip dna labeling reagent in tdt buffer (af- fymetrix) for h at °c followed by min at °c. after end-labeling, the fragments were hybridized to a genechip human mapping k array for – h at °c while rotating at rpm. microarrays were washed by using the fluidics station (affymetrix) in . � sspe (sodium chloride, sodium phosphate, and edta), followed by a three-step staining pro- tocol. we incubated the arrays first with �g�ml streptavidin (pierce), washed with � sspe and incubated with �g�ml biotinylated antistreptavidin (vector laboratories) and �g�ml streptav idin–phycoer ythrin conjugate (molecular probes), and finally washed with � sspe per the manufactur- er’s recommended times. microarrays were scanned by using the genechip scanner according to the manufacturer’s pro- tocol (affymetrix). data acquisition was performed by using the genechip gcos software. initial data analysis was done by using the genechip dna analysis software and then exported to varia (silicon genetics) for analysis. all raw genotype data and call statistics for the four disorders are in tables – , which are published as supporting information on the pnas web site. the overall performance statistics of the genotyping arrays over the , genotypes in individuals (including the siddt pedigree), yielded average call rates of . % and average signal detection rates of . %. linkage mapping. data analysis was done by using the varia software package developed by silicon genetics. snp positions came from dbsnp build and national center for biotech- nology information build v of the human genome. varia searches for genomic regions that are identical by descent between all affected individuals and assumes no mutation het- erogeneity within affected individuals. several assumptions were made for the analysis, including a genotype error rate of % and hardy–weinberg equilibrium. details on the generation of ‘‘location scores’’ can be found at http:��www.silicongenetics. com�support�autozygosity.pdf (referred to as ‘‘regional lod scores’’ in the pdf; ref. ). brief ly, the two-point lod scores across the autozygous region are summed to produce the loca- tion score, accounting for size of the shared interval as well as the magnitude of allele frequency differences within the interval. one hundred seventy old order amish control chromosomes (including six untransmitted chromosomes from the siddt parents and from the three mapping validation sibships) were used to estimate snp allele frequencies. the three regions of the genome having the highest location scores are illustrated in figs. and . sequencing. mutation analysis was performed for the tspyl and tspyl genes in the linked region on chromosome q . the exon of each target gene was amplified by using specific primers and ng of genomic dna from affected and unaffected family members. the tspyl gene lacks introns and contains a coding region of fig. . snp genotyping reveals a . -centimorgan region ( . mb) on chromosome q . -q . that contains the siddt gene. asterisks denote individuals who were genotyped. the three regions across the genome with the highest location scores are ranked by descending score. the four affected individuals are homozygous for adjacent snps at the q locus. horizontal red bars indicate the autozygous region (with physical distance illustrated), and two-point lod scores for each snp are plotted to illustrate that informa- tion content of biallelic markers in small pedigrees alone make mapping difficult. the region is bounded by snps rs and rs . the prox- imal and distal transmitted haplotypes flanking the homozygous region were not diverse, whereas there was little haplotype sharing in intervals b and c outside the autozygous segment, indicating these were false-positive regions. the two regions with the next best location scores had small numbers of contiguous homozygous snps, smaller homozygous intervals, and little shar- ing outside the homozygous interval. puffenberger et al. pnas � august , � vol. � no. � g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , , bases (genbank accession no. al ). the mrna is � , bases in length (genbank xm� ), and the mature tspyl protein is aa. primer sequences for tspyl amplifica- tion were �-agatctccagtcctgacgacac- � (forward) and �-agga a acagggtgcaga a a agt- � (reverse). tspyl sequencing primers (in addition to the forward and reverse primers, above) were tspyl -f: �-ggccgagtgg- tgtctctttcta- �; tspyl -f: �-ggaggatagattg- gaggaggag- �; tspyl -f: �-tactccccagatc- cgagttgtt- �. in addition to the � insg mutation, two polymorphic variants were incidentally detected while sequencing control samples: a known nonsynonymous snp g�a (a t) and a unique in-frame short tandem repeat [ (gtg) - ] that codes for either two or three adjacent valine residues at positions – in the peptide. the a allele had a frequency of . %, whereas the (gtg) allele had a frequency of . % on control chromosomes. primer sequences for pcr amplification of tspyl were �-aaaactccccttccagactgac- � (forward) and �-cacaatgcagaaaagcatgaag- � (reverse). tspyl sequencing primers (in addition to the forward and reverse primers, above) were tspyl c -f: �-acacaggtgatggcgaac- acag- �; tspyl c -f: �-ccatcgatcaagagttgtc- aaa- �; tspyl c -f: �-caggctcatatccacaga- aacc- �; and tspyl c -r: �-taatgaaacttctgc- gctgcat- �. pcr products were purified by using qiaquick columns (qiagen) and then sequenced using the bigdye terminator cycle sequencing protocol (applied biosystems). extension products were size-fractionated on an applied biosystems genetic analyzer. sequences were compared to normal sequence for each gene using genbank al , which contains both genes. population-based control samples were sequenced in an identi- cal fashion. functional validation. full-length and truncated tspyl cdnas were amplified by using gene-specific primers for the full-length: fig. . accurate disease gene localization using the affymetrix genechip mapping k assay kit and the silicon genetics varia software package. mapping of the congenital nephrotic syndrome locus, maple syrup urine disease locus, and the crigler–najjar syndrome locus was accomplished by using minimal numbers of individuals in each pedigree. the three highest location scores across the genome are indicated for each disorder in descending order. the location score combined with physical distance was found to be the best predictor of the correct region in each case. no affected individuals were genotyped in the nphs pedigree, yet it was still possible to detect the correct locus, and the inferred region of autozygosity is indicated by a green horizontal bar. � www.pnas.org�cgi�doi� . �pnas. puffenberger et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , forward, �-caccatgagcggcctggatggggtca a- gagg- �; reverse, �-tagctcgagaccagactggaac- ccaaagggcctggggatc- �; and the truncated: identical forward to above; reverse, �-tagctcgagtggcggct- gctcctctacctcc- � versions from genomic dna using the expand high fidelity pcr system (roche, indianapolis). cy- cling conditions were °c for min and then cycles ( °c for sec, °c for min, and °c for sec) followed by °c for sec and then cycles ( °c for min, °c for min) and then °c for min. pcr products were cloned directionally into the pentr-d-topo gateway vector (invitrogen) and then into the gateway destination vector pcdna-dest (invitro- gen) containing a c-terminal gfp tag. all clones were sequence verified. helaf cells ( ) were grown in dmem supplemented with % fbs (gibco�brl). the helaf cells were trans- fected with �g of the tspyl full-length or truncated gfp constructs, respectively, using lipofectin reagent (invitrogen) according to the manufacturer’s recommendations. twenty-four hours post-transfection, subconf luent helaf cells were washed three times with pbs. the transiently transformed cells were fixed in % formaldehyde and permeabilized in . % triton x- . nuclear localization was confirmed by using a �, - diamidino- -phenylindole dihydrochloride stain from molecular probes. images were captured with fitc and uv filter sets and a � oil immersion objective in conjunction with a leica (deerfield, il) tcs-nt confocal microscope. results and discussion we first wanted to verify that statistically significant and unam- biguous disease gene mapping could be accomplished with a very small number of affected sibships and a dense genotyping platform. these blinded validation studies used three small disease-mapping sets from the old order amish and old order mennonite populations of southeastern pennsylvania, namely, maple syrup urine disease ( , ), crigler–najjar syndrome ( , ), and congenital nephrotic syndrome ( , ). the gene for each disease had been previously identified. genome-wide linkage scans were performed and regions of autozygosity identified through a location score statistic. heterozygous markers f lank- ing the identical by descent homozygous segment in affecteds define the minimal genetic interval. in each of the three test cases, the interval with the largest location score correctly identified the true gene location (fig. ). the location score is not a formal statistic, but a summation of two-point lod data across a region of homozygosity. equal marker density across physical distances is assumed, which is a f law of the statistic, but this issue should resolve as marker spacing stabilizes in future array iterations. the siddt mapping panel was comprised of four affected individuals (fig. , vi- , vi- , vi- , and vii- ) and their parents from three sibships. the region that received the highest two-point lod score ( . ; fig. ) and the highest location score ( . ; fig. ) was on chromosome q . -q . [genome build v , national center for biotechnology information (ncbi)]. the homozygous segment spanned . mb, corresponding to . centimorgans. examination of the minimal shared region in affecteds, which was f lanked by snps rs and rs , revealed known and hypothetical genes based on both the ncbi and celera annotations, of which are characterized (fig. ). the siddt phenotype includes testicular dysgenesis; thus genes with known or inferred function related to sexual differentiation and testicular development were sought. two genes were possible candidates based on sequence homology to the testis-specific gene tspy at chromosome yp : the paralogs testis-specific y-encoded like genes tspyl and tspyl . unlike tspy, which has testis-restricted expression, tspyl is expressed in the brain and testes (as well as other tissues). complete sequencing of the tspyl gene in an affected individual (vi- ) revealed a homozygous single base insertion, � insg (fig. ). this variant causes premature truncation of the protein at codon . this change was not seen as a polymorphic variant in genbank. sequencing of all individ- uals in the siddt pedigree revealed that all affected individuals were homozygous for the change, all parents of affected indi- viduals were heterozygous, and no unaffected siblings were homozygous for the change. fifty-eight old order amish con- trols were genotyped for the insertion (n � chromosomes). most of these samples were from the lancaster county old order amish population; however, eight controls were available for study from the juniata and miff lin county old order amish. none of the lancaster county old order amish carried the variant, but four heterozygotes were detected from the miff lin and juniata county old order amish. although the sample size is small (n � chromosomes), this suggests that the tspyl � insg variant has an especially high carrier frequency in this genetic isolate. a subset of sudden infant death syndrome and�or ambiguous genitalia in these populations could be caused fig. . mutation of the tspyl gene causes siddt. (a) the gene content of the interval contained characterized and hypothetical genes (not shown). the genomic interval surrounding tspyl and tspyl ( -kb proximal) contains only two documented (ca)n repeats, neither of which is in the standard applied biosystems linkage panel; thus, this locus might have been missed by using the small dna sample set and a standard microsatellite marker screen. (b) individuals with siddt syndrome were found to have a tspyl frameshift mutation at codon causing cessation of translation at codon . the predicted secondary structure shows a series of �-helixes and �-strands in the nap homologous region. (c) the primary amino acid sequence. we speculate that the � domain may be a dna-binding domain or may interact with regulatory complexes within the chromatin. (d) subcellular localization of tspyl is altered through loss of the � portion of the peptide (upper, trun- cated tspyl; lower, full-length tspyl). loss of the nap functional domain probably directly affects the ability of tspyl to shuttle histones from the cytoplasm to the nucleus but also disrupts the nuclear localization signal on the tertiary surface of the peptide. tspyl localizes to the nucleus with a punctate staining pattern, whereas the truncated form illustrates diffuse cytoplasmic staining. the nucleus is counterstained with �, -diamidino- - phenylindole dihydrochloride. puffenberger et al. pnas � august , � vol. � no. � g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , by tspyl loss of function. a second gene family member, tspyl , was found kb proximal to tspyl, but direct sequencing of an affected individual identified no coding vari- ants. the truncated tspyl exhibited inappropriate subcellular targeting in an in vitro assay (fig. ), providing additional evidence that tspyl is causative of siddt. aspects of the molecular function of tspyl can be gleaned from its primary sequence (fig. ). the tspyl nucleosome assembly protein (nap) domain is preceded by a region of low complexity with no known functional motifs. naps are a family of proteins that function as chaperones, shuttling histones from the cytosol to the nucleosome. studies in yeast show that naps are critical to nucleosome assembly, mitotic progression, and chromatin formation ( – ). nap domains may also function as transcription factors during embryogenesis. during the devel- opment of xenopus embryos, the nap-containing protein nap l is broadly expressed at its highest level during develop- ment of hematopoietic tissue. when nap l is overexpressed, genes involved in tissue development are up-regulated, specifi- cally gata- , a gene essential for hematopoiesis ( ). although we focused on tspyl based on its similarity to tspy, the knowledge that genotypic females are affected suggests that tspyl, unlike tspy, is expressed in other tissues ( , ). tspyl may play a role in development by altering regulation of specific developmental genes and contributing to region-specific chromatin remodeling. several data sets within the gene ex- pression omnibus (geo) illustrate that tspyl is highly and almost exclusively (with respect to adult structures) expressed in the fetal brain (geo no. gsm ). in addition, tspyl has been shown to be negatively regulated in the hippocampus in a linear dose-dependent fashion by corticosteroids (geo no. gsm ), sensitively negatively regulated by jnk (geo no. gsm ), and positively regulated by testosterone (geo no. gsm ). elucidation of tspyl function may shed light on fundamental aspects of embryogenesis of the human nervous and reproductive systems through a previously not described signaling mechanism. more important, studies of tspyl ex- pression and function in the developing brain may provide new insight into the genetic basis of apnea, dysphagia, cardiac arrests, and sudden unexplained deaths in infancy. present clinical evidence suggests that in siddt, sudden death may result from dysregulation of the autonomic brainstem systems that control cardiac and pulmonary protective ref lexes ( – ). the lethal event may be profound vagally mediated laryngobronchospasm or asystole. we thank the old order amish families who participated in the research and the old order amish community for their willingness to participate in research studies, robert wells at affymetrix, inc., and g. terry sharrer at the smithsonian institution for facilitating this work, drs. allen ettinger and bruce lidston for bringing this disorder to our attention, phanindra tangirala for posting data on the web, and john pearson for bioinformatics support. this work was supported in part by national institute of neurological disorders and stroke grant u ns - (to d.a.s.) and by national research service award fellowship f ns - (to d.h.-l.). . puffenberger, e. g. 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( ) j. neuropathol. exp. neurol. , – . � www.pnas.org�cgi�doi� . �pnas. puffenberger et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , e d i t o r i a l propelling st-segment elevation myocardial infarction systems of care into the air a greater prevalence of hemodynamic compro‑ mise presumably due to delays in stemi recog‑ nition. although the authors do not character‑ ize in ‑hospital outcomes, the use of thrombo‑ lytic therapy, and how often hems could not be prompt recognition and early coronar y ar‑ tery reperfusion are the cornerstones of any st ‑segment elevation myocardial infarction (stemi) systems of care. american and euro‑ pean guidelines recommend rapid transport of patients to a stemi receiving center capable of high ‑quality primary percutaneous coronary in‑ tervention (pci), if first medical contact to pci reperfusion can be achieved within min‑ utes. , the boundary between rural and urban areas has typically been assigned by civil bound‑ aries. it has been long understood that geograph‑ ic disparities exist for many rural communities that are too distant to reliably provide time‑ ly stemi transport. as urban cities expand in population density and geographic size, time‑ ly stemi transport is jeopardized due to dis‑ tances from fringes of those areas and congest‑ ed traffic patterns. the alternative to primary pci defaults to primary thrombolytic therapy; however, the wealth of clinical trial evidence in‑ dicates that thrombolytic therapy is associated with worse ischemic and bleeding outcomes in comparison with timely pci. the integration of helicopter emergency medical services (hems) in modern stemi systems of care has improved the timeliness of transport of these patients and improved outcomes. , in this issue of kariologia polska (kardiol pol, polish heart journal) Świeżewski et al investigat‑ ed in their retrospective analysis the patient char‑ acteristics and early outcomes of more than patients with stemi that were transferred from rural and urban communities to stemi receiv‑ ing centers in poland. the authors observed that hems facilitated efficient interhospital trans‑ port of patients with stemi for pci, and patients who were transported from rural locations had correspondence to: amish n. raval, md, facc, faha,  division of cardiovascular  medicine, department  of medicine, university  of wisconsin school of medicine  and public health, h /   clinical sciences center,   highland avenue, madison,  wisconsin,    , united states,  phone: +       ,  email: anr@medicine.wisc.edu received: march  ,  . accepted: march  ,  . published online: april  ,  . kardiol pol.  ;   ( ):  - doi: . /kp. copyright by the author(s),  e d i t o r i a l propelling st‑segment elevation myocardial infarction systems of care into the air amish n. raval , amy shepard   division of cardiovascular medicine, department of medicine, university of wisconsin school of medicine and public health, madison, wisconsin, united states   university of wisconsin -health, madison, wisconsin, united states related article by Świeżewski et al, see p. figure box and whisker plot showing the time from the first medical contact to percutaneous coronary  intervention reperfusion in patients transferred from a rural  location to the university of wisconsin -madison stemi receiving center by ground emergency medical services  (gems; n = ), helicopter emergency medical services (hems; n = ), or intercepted by hems from gems (n = ) from january  ,   to september  ,  . despite similar  overall distances travelled, hems intercepting with gems demonstrated the lowest median transfer times (p < . ,  paired t test). the bottom whisker, bottom edge of the box,  top edge of the box, and top whisker indicate the  th,  th,  th, and  th percentiles, respectively. the x symbol  represents the median value ( th percentile). the horizontal  line represents the mean. gems hems intercept m in ut es k ardiologia polsk a ; ( ) Świeżewski sp, wejnarski a, leszczyński pk, et al. characteristics of urban ver- sus rural utilization of the polish helicopter emergency medical service in patients  with st-segment elevation myocardial infarction. kardiol pol.  ;  :  - . delgado mk, staudenmayer kl, wang ne, et al. cost -effectiveness of heli- copter versus ground emergency medical services for trauma scene transport in  the united states. ann emerg med.  ;  :  - .e . dispatched due to weather conditions, the overall hems stemi transport effort seems favorable for patients with stemi in and around poland. notably, the authors describe close coordina‑ tion between ground emergency medical servic‑ es (gems) and hems and this was particular‑ ly evident for rural transport. in our own insti‑ tution’s experience involving stemi transport from rural wisconsin between and , hems intercept with gems at designated sites was associated with the lowest first medical con‑ tact to coronary reperfusion times, in compar‑ ison with gems and hems transfer alone, de‑ spite similar median distances travelled (figure  ). gunnarsson et al observed that having physi‑ cians on board hems for stemi transport im‑ proved in ‑hospital outcomes. modern hems systems are equipped with enhanced aviation safety technology and are now capable of transporting patients in shock with percutaneous circulatory and oxygen‑ ation support devices. unfortunately, little is understood regarding the financial cost implica‑ tions of hems for stemi transport in the unit‑ ed states, europe, and elsewhere. in another example, the cost ‑effectiveness of hems for the transfer of trauma patients is dependent upon the mortality and morbidity reduction that the hems transfer affords and over ‑triage of patients with minor injuries. until such data are available, judicious utilization of hems for stemi transfer to enable timely primary pci seems to be a reasonable use of this resource within the framework of a comprehensive ste‑ mi system of care. article information disclaimer the opinions expressed by the author are not necessarily those  of the journal editors, polish cardiac society, or publisher. conflict of interest none declared. open access this is an open access article distributed under the terms  of  the  creative  commons  attribution -noncommercial -noderivatives  .   in- ternational license (cc by -nc -nd  . ), allowing third parties to download ar- ticles and share them with others, provided the original work is properly cited,  not changed in any way, distributed under the same license, and used for non- commercial purposes only. for commercial use, please contact the journal office  at kardiologiapolska@ptkardio.pl. how to cite raval an, shepard a. propelling st-segment elevation myo- cardial  infarction systems of  care  into  the  air. kardiol pol.  ;  :  - .  doi: . /kp. references o’gara pt, kushner fg, ascheim dd, et al; american college of cardiology  foundation/american heart association task force on practice guidelines.    accf/aha guideline for the management of st -elevation myocardial infarction:  a report of the american college of cardiology foundation/american heart associ- ation task force on practice guidelines. circulation.  ;  : e - . ibanez b, james s, agewall s, et al; group escsd.   esc guidelines for  the management of acute myocardial infarction in patients presenting with st- -segment elevation: the task force for the management of acute myocardial in- farction in patients presenting with st -segment elevation of the european society  of cardiology (esc). eur heart j.  ;  :  - . gunnarsson si, mitchell j, busch ms, et al. outcomes of physician -staffed ver- sus non -physician -staffed helicopter transport for st-elevation myocardial infarc- tion. j am heart assoc.  ;  : e . schneider ma, mcmullan jt, lindsell cj, et al. reducing door -in door -out inter- vals in helicopter st -segment elevation myocardial infarction interhospital trans- fers. air med j.  ;  :  - . https://doi.org/ https://doi.org/ https://doi.org/ https://doi.org/ . /j.annemergmed. . . https://doi.org/ . /j.annemergmed. . . https://doi.org/ . /j.annemergmed. . . https://doi.org/connection: close https://doi.org/connection: close https://doi.org/connection: close https://doi.org/connection: close https://doi.org/connection: close https://doi.org/ . /eurheartj/ehx https://doi.org/ . /eurheartj/ehx https://doi.org/ . /eurheartj/ehx https://doi.org/ . /eurheartj/ehx https://doi.org/ . /eurheartj/ehx https://doi.org/ . /jaha. . https://doi.org/ . /jaha. . https://doi.org/ . /jaha. . https://doi.org/ . /j.amj. . . https://doi.org/ . /j.amj. . . https://doi.org/ . /j.amj. . . biophysical microenvironment and d culture physiological relevance r e v ie w s � g e n e t o s c r e e n drug discovery today �volume , numbers / �june reviews biophysical microenvironment and d culture physiological relevance amish asthana and william s. kisaalita cellular bioengineering laboratory, college of engineering, driftmier engineering center, university of georgia, athens, ga , usa force and substrate physical property (pliability) is one of three well established microenvironmental factors (mefs) that may contribute to the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (hts) in preclinical drug discovery. in d cultures, studies of the physiological relevance dependence on material pliability are inconclusive, raising questions regarding the need to design platforms with materials whose pliability lies within the physiological range. to provide more insight into this question, we examine the factors that may underlie the studies inconclusiveness and suggest the elimination of redundant physical cues, where applicable, to better control other mefs, make it easier to incorporate d cultures into state of the art hts instrumentation, and reduce screening costs per compound. conventionally, d cell culture simply refers to providing a d spatial microenvironment for the cells to grow in. however, in our recent work, the meaning of three-dimensionality has been extended to providing the total microenvironment that supports the formation of microtissue that exhibit ‘complex’ physiological relevance (cpr) or better emulation of the in vivo microtissue functionality in a manner not possible in d cultures [ ]. a good example of cpr outcome is the formation of bile canaliculi-like structures by hepg hepatocyte cells (fig. ) is d but not in d culture formats. the literature has provided guidance that leads to three main categories or microenvironment factors (mefs) or ‘three-dimensions’ of: (i) chemical or biochemical composition, (ii) spatial (geometric d) and temporal dimensions, and (iii) force and substrate physical properties [ – ]. however, as pointed out by lai et al. [ ], because of the lack of a quantifiable entity or biomarkers of three dimensionality, the optimum composition of the microenvironment that is required for the cells to provide a physiologically relevant response has remained elusive. it might be that one microenvironmental factor is more important than the other to emulate in vivo-like functionality or if the cells are provided with some initial cues they might be able to create their own endogenous microenvironment rendering the other exogen- ous factors less important [ ]. corresponding author: kisaalita, w.s. (williamk@engr.uga.edu) - / /$ - see front matter � elsevier ltd. all rights reserved. http://dx.doi.org/ . /j.drudis. evidence in support of varying degrees of importance of mefs comes from the success of the various commercially available d cell culture platforms that provide different mefs that elicit similar functional or structural cpr from the cells. for instance, mam- mary epithelial cells (mcf- a) grown in matrigel (bd) have formed acini-like or hollow lumen, a structural element associated with glandular cells in vivo [ ]. similarly, a spheroid culture of mcf- cell line has also shown the presence differentiation fea- tures like lumen and budding formation [ ]. interestingly, both the growth platforms, although considered d, are very different in nature, with matrigel providing all the three aforementioned microenvironmental cues to the cells while the spheroid culture just provides a d space for them to grow in. this begs the question as to the minimum level of exogenous mefs that give rise to the in vivo conditions that emulate or produce a structurally and func- tionally analogous in vitro tissue model. it is reasonable to argue that the composition of the microenvironment is not standard, rather the optimum mefs’ combination that depends on the application (e.g. cell type, relevant cpr, among others). while in the field of regenerative medicine, a precise emulation might be necessary as the construct is meant for implantation in vivo, on the contrary, in the field of drug discovery, exogenous emulation of the microenvironment to the level found in vivo (e.g. for cpr expression) may not be necessary. if this is true, the redundancy in the mefs of interest may be eliminated which may result in . . www.drugdiscoverytoday.com mailto:williamk@engr.uga.edu http://dx.doi.org/ . /j.drudis. . . reviews drug discovery today �volume , numbers / �june blood inflow basement membrane tight junction bile canaliculus m mvg (b) (a) (c) bc bc sc tj tj hepatocyte sinusoidal endothelial cell kupffer cell stellate cell drug discovery today figure formation of in vivo-like bile canaliculi structures by hepg hepatocytes that is only observed in d but not d culture formats. (a) illustrative schematic of liver tissue in vivo. (b) transmission electron micrographs showing pericanalicular region of two adjoining periportal hepatocytes of adult rat liver tissue – labeled structures are the bile canaliculus (bc), mitochondria (m), golgi apparatus (g) and tight junctional regions (arrowheads). (c) almost identical transmission electron micrograph of hepg cells cultured on d porous polystyrene scaffolds (sc) for days [ ], exhibiting tight junction (tj) complexes between adjacent cell; the void formed in-between cells in (c) closely resembles the in vivo bile canaliculus (bc) in (b) and is similarly lined with microvilli (mv). bar = nm. r e v ie w s � g e n e t o s c r e e n reduction in cost and making it easier to configure for state of the art high-throughput screening (hts) instrumentation. herein we illustrate this ‘conjecture’ with a focus on substrate/scaffold plia- bility mef, one of the three ‘dimensions.’ cell–substrate interaction – biophysical constraints in d platforms cells exert stress on their matrix during morphogenesis, tissue remodeling, differentiation, and normal physiological activities. the rigidity of the matrix along with the number of receptor- mediated adhesions formed by the cells with the microenviron- ment influence the extent to which the matrix can be contracted by them [ ]. this in turn generates intracellular tension which leads to the formation of stress fibers in the cells. if the matrix is rigid, it is more difficult for the cells to contract it, resulting in differential cell functions [ ], wherein lies the importance of providing an optimal biophysical microenvironment to the cells in vitro. most of the d platforms that are commercially available (table ) can be broadly classified into three categories based on their rigidity: (i) hydrogel-forming (alginate, agarose, chitosan, fibrin, hyaluronan and collagen to name a few) that are pliable and provide a ‘soft’ environment for the cells to grow in (type i); (ii) synthetic microporous (‘spongy’) scaffolds or constructs fabri- cated by freeform technology are generally made of stiffer materi- als having high modulus of elasticity such as polystyrene, plla www.drugdiscoverytoday.com (poly-l-lactide acid), plga (poly(lactic-co-glycolic acid)) and pcl (polycaprolactone) (type ii); (iii) scaffold-free d formats such as spheroids or cellular aggregates, produced with hanging drop or the rotating wall vessel (rwv) configurations that lack cell– exogenous material interaction (type iii). advantages and disad- vantages of the three platform types are discussed below. type i the pliability of hydrogels can be altered within the physiological range ( – , pa) by either changing the concentration of the polymer ((poly(ethylene glycol))peg, agarose), the extent of cross- linking or the proportion of the (extracellular matrix) ecm pro- teins (hyaluronan, collagen, fibronectin, laminin) incorporated in the substrate. modifying the concentration of the ecm proteins (also biochemical cues) might also lead to a change in the number of adhesion ligand sites present for the cells to bind to. however, this can be managed by mixing a more compliant ecm compo- nent like matrigel with a stiffer constituent (collagen i) in defined proportions that allow a relatively constant chemical–ligand con- centration within a variable stiffness range. furthermore, the rigidity of the hydrogels can also be increased by using the hydrogel in an ‘attached’ configuration, where the hydrogels are bound to the bottom of the culture dish and resist the forces that are exerted by the cells, rather than a floating raft or sus- pended mode. an increase in gel rigidity generally results in the drug discovery today �volume , numbers / �june reviews table elastic moduli for commercially available d platforms company trade name type and material elastic moduli (kpa) ref. dbiomatrix perfecta d plates hanging drops na perfecta d scaffolds hydrogel nr insphero gravityplus plates hanging drops na bd matrigel laminin, collagen . [ ] glycosan biosystems extracel hyaluronic acid and collagen . – . [ ] globalcellsollutions/hamilton gem magnetic alginate microcarrier . % – . � . . % – . � . . % – . � . [ ] trevigen cultrex d matrix bme, laminin, collagen . [ ] sigma hydromatrix synthetic peptide hydrogel . – . [ ] maxgel human ecm – [ ] qgel mt d matrix peg hydrogel . – . ( – % pegda) [ ] kollodis biosciences maptrix hygel chemically defined hydrogel nr synthecon inc. biofelt pga, plla, plga, custom nr biomerix d scaffold polycarbonate polyurethane-urea nr invitrogen geltrex laminin, collagen . [ ] algimatrix alginate . % – . � . . % – . � . . % – . � . [ ] zellwerk sponceram ceramic nr amsbio alvetex polystyrene [ ] dm inc. puramatrix peptide . – . [ ] corning ultraweb polyamide . [ ] dbiotek d insert pcl polycaprolactone [ ] d insert ps polystyrene , (fibrous) [ ] d insert plga poly(dl-lactide-co-glycolide) , (porous) [ ] b-tcp disc b-tricalcium phosphate . � . to . � . (� ) [ ] microtissues inc. d petri dish agarose . % – . % – . % – [ ] abbreviations: bme: basement membrane extract; na: not applicable; nr: not reported. r e v ie w s � g e n e t o s c r e e n enhancement of proliferation and inhibition of differentiation because of elevation in the phosphorylation of focal adhesion kinase and the formation of focal adhesions, as shown by paszek et al. [ ]. even though such platforms provide an in vivo-like pliable environment for the cells to grow in, they lack a defined geometry and fail to impose any physical constraints on the size of the aggregates. as such, the microtissues formed range from being just a cluster of few cells to larger tissues that are above the crucial size for oxygen diffusion and this might generate an adulterated out- come in response to drug exposure [ ]. type ii because the pliability of these scaffolds is above the physiological range, it is usually assumed that they fail to provide the optimum biophysical cues for the cells, yet they have been successful as shown by their commercial adoption (table ). however, it should be noted that it is not just the material that affects the pliability but also the form in which it is presented. for example, polystyrene in its bulk state as used in tissue culture plates has a very high elastic modulus ( – gpa; [ ]) but when used to fabricate salt leached microporous scaffolds, it exhibits a considerably lower modulus ( kpa; [ ]). also, ecm proteins can be coated on such scaffolds to provide adhesion sites for the cells to attach. the protein coating might provide a more compliant surrounding for the cells, however it has been argued that the coating is very thin and it is a known fact that cells can sense and adapt in response to the topography independent of the adsorbed proteins [ ]. the major advantages of type ii scaffolds are that they have a defined geo- metry and controllable pore sizes that provide a strict spatial control on the dimension of the microtissues [ ] and are better suited for incorporation in hts state of the art instrumentation. type iii scaffold-free d culture production is either achieved in static conditions such as gravity-enforced hanging drops or dynamic conditions, such as rwv (rotating wall vessel), stirred tank reac- tors (cstr), spinner vessels, and microfluidic chambers. in the dynamic configuration, shear stress generated because of the fluid flow constitutes the primary driving biophysical factor in absence of stress created because of the cells pulling onto to the material as in the previous cases. microtissues generated by the above mentioned techniques have been used as in vivo surrogates far and wide [ , ], however, these systems are generally labor intensive and are difficult to adapt in state-of-the-art hts instrumentation. www.drugdiscoverytoday.com reviews drug discovery today �volume , numbers / �june r e v ie w s � g e n e t o s c r e e n cellular complex physiological relevance and drug discovery outcomes moving to second generation d cell culture platforms is being driven by the brief that the responses generated by the cells growing in a d format are not just ‘different,’ but are physiolo- gically more relevant, when compared to cells cultured on tradi- tional d surfaces. it is necessary to conclusively show that these responses produced in d formats are emulations of those that are seen in vivo. to be meaningful, these physiologically relevant outcomes (structural or functional) that are also known in vivo should be absent in d formats and such outcomes (cprs) should be established for cells derived from the four major tissue types (epithelial, muscle, connective, and nerve). these outcomes can serve as a standard for determining how close a d culture is to its native tissue or which out of a given number of d platforms is better suited for a given application. below, we explore some of the well established or provisional cprs for cells derived from the three tissue types of most interest in preclinical drug discovery (epithelial, neuronal and cardiac). cpr in liver tissue-derived cells because liver is the primary organ involved in the metabolism of xenobiotics, d constructs of hepatocytes are often used as a tool to screen for drug toxicity and test compounds that may affect liver cell function. as such, because of the vast amount of literature available, both structural and functional cpr responses for liver cells can be established with ease. one of the basic structural phenomena that distinguish liver cells in the native tissue from those cultured in d formats is polarity. while those in their natural environment possess structural and functional polarity [ , ], the ones that are isolated and cultured on most flat non- porous surfaces do not [ , ]. in their native conditions, hepa- tocytes maintain a cuboidal shape, with two to three basal surfaces facing the sinusoid. the lateral domain between adjacent cells is divided by a polygonal network of microvilli-lined bile canaliculi (fig. ) which is formed by membranes contributed from contig- uous cells and comprises the apical domain of cells. furthermore, position specific processes are carried out in each domain. for instance, proteins involved in the shuttling metabolites from the blood capillaries are centralized to the basal surface, while those involved in bile acid transport are confined to the apical domain. in monolayer, the development of such bile canalicular networks is sparse, heterogeneous and transient [ , ] further differentiat- ing it from native tissue and substantiating this structural element as a valid cpr outcome. in terms of function, hepatocytes in their native environment display high levels of liver specific activities like high cytochrome p activity [ ], transferrin secretion [ ], albumin production [ , ], tyrosine aminotransferase induction [ ] and ureagenesis [ ] than their monolayer counterparts. cells growing in a d format entirely lack or have very low levels of many cytochrome p enzymes (cyps) and transporters found in hepatocytes in vivo [ , ]. the cyps are a family of phase metabolizing enzymes that consist of isoforms, six of which metabolize % of drugs [ ]. the primary isoforms in human liver include cyp a , cyp a , cyp c , cyp c , cyp d , cyp e and cyp a , making them a very important family of enzymes not only for screening purposes but also in validating the cpr of d liver cell cultures. www.drugdiscoverytoday.com cpr in nerve tissue-derived cells the literature on cells of neuronal origin has not provided a consensus toward establishing a neuronal microtissue cpr, however, several phenomena with high cpr outcome potential are more worthy of further exploration. for example, intracel- lular calcium oscillations are an innate characteristic of neural cells in vivo and have a pivotal role in synaptic signal transmis- sion. although calcium oscillations have been observed in both d and d nerve cell cultures, we submit that there should be differences in the nature of oscillations between the two cul- tures. the frequencies of these oscillations found in d cultures are considerably lower (e.g. . / s, brain slices [ ]; / s, np cells derived from neurospheres [ ]) than those found in d cultures (e.g. / s [ ], / s [ ]) and is closer to many in vivo experiences (e.g. . / s [ ]). interestingly, the differ- ences in voltage-gated calcium channel (vgcc) function in d and d cultures might partly be the underlying cause, because these channels are central to the movement of calcium into and out of cells [ ]. it has been shown that intracellular calcium transients are significantly lower in d as compared with d cultures and this is more representative of the in vivo situation [ , ]. also, l-type vgcc agonists and antagonists have pre- viously been shown to enhance and abolish calcium oscilla- tions, respectively, in nerve cells [ – ], substantiating involvement of vgcc in the differential frequency of calcium oscillations found in different culture formats. these findings suggest potential for calcium transients and/or oscillations to serve as nerve cell cpr outcome. cpr in muscle tissue-derived cells myocardial microtissues are of particular importance as they find application in hts q-t elongation assays, required for screening every drug in development [ ]. some recent exam- ples of drugs that have been withdrawn from various markets because of q-t interval prolongation effects include droperidol (inapsine; akorn; ), dofetilide (tikosyn; pfizer; ) and thiordazine (mellari; novartis; ). as such, it is essential to establish a consensus for the cpr of cardiac microtissue to provide an in vivo-like tissue model for drug development and screening. a couple of phenomena that are characteristic of the native cardiac tissue in vivo, found in d cultures but are lacking in traditional monolayers include beat frequency and contraction force. in a study by kelm et al. [ ], neonatal rat cardiomyocytes (nrc) showed rhythmic contractions at a beat frequency of beats per minute (bpm) when they were grown as spheroids in a hanging drop culture. similar beat frequencies ( � bpm) have been seen when nrc were transplanted in vivo in adult rat and formed a microtissue [ ] which is considerably lower as compared with d culture ( . � . bpm [ ], . � . [ ]). this is consistent with the fact that cells in monolayers exhibit exaggerated responses, specifically vgcc function which is responsible for cardiac cell contraction and has been discussed earlier. however, there are a few studies that have suggested otherwise, such as zimmer- man et al. [ ] who reported the beat frequency of nrc growing as engineered heart tissue (eht) of bpm. it should be noted that hearts from -day-old rats have a beat rate of – bpm [ ]. drug discovery today �volume , numbers / �june reviews r e v ie w s � g e n e t o s c r e e n cpr in hts d platform validation the two examples of liver and muscle cell culture used above, to illustrate the cpr concept, have traditionally been associated with low throughput later phases of discovery applications like toxicol- ogy. a relevant question is how relevant the examples are in early hts phases of discovery. in response to this question, we submit that as long as the cells can express a target of interest, their d assay can be used for both hts and low throughput discovery applications. for example, there is no reason why hepg (human hepatocellular carcinoma) cannot be used in both toxicology (low throughput) and chemotherapeutics hts. therefore, cpr should not be thought about only in the context of low throughput applications. as a matter of fact, d culture platforms are slowly finding their way into hts laboratories. for example, studies of hts assay robustness (in terms of z factor) with d cultures in high well-density plates (e.g. – wells) are beginning to appear [ , ]. a drug exposure might generate a different, and in some cases a ‘better,’ response, such as differentiation toward a particular line- age, from cells growing on a d format as compared with those in a monolayer. however, in the absence of a validated cpr outcome, there would be no way of telling if the outcome is predictive of the in vivo response. for example, tung et al. [ ] showed that two cytotoxic drugs had different effects on cells growing as spheroids in hanging drops and those in d. had it been shown that the d spheroids produced in this particular platform emulated the native tissue and were validated using a particular cpr outcome, then the effect of the drugs on the spheroids would have been considered more predictive of its in vivo effect. therefore, it is more desirable table liver cell studies with different platforms, but similar cpr outcome cell line and type scaffold type and material structural cpr rat small hepatocytes (shs) stacked layers on microporous membranes bile canaliculi w tight junctions, hhy alginate beads canaliculi with desmosomes a hepg and hhy alginate beads desmosomes, j and canaliculi l lig- cell line (adult rat liver progenitor) peptide hydrogel (puramatrix) primary rat hepatocytes, hepg chitosan-collagen coated pet mesh scaffold microvilli primary rat hepatocytes nanofibrous/porous plla scaffolds tight junctions junctions hepg porous ps scaffold tight junctions primary rat hepatocytes pmma or pc polymer scaffold junctional com fetal porcine hepatocytes plla scaffolds primary rat astrocytes spheroids bile canaliculi, m abbreviations: pet: poly(ethylene terephthalate); pc: propylene carbonate; pmma: poly(methy that the existing and the upcoming novel d culture platforms are validated with respect to a cpr outcome known in the native tissue and then the results of a drug exposure would be a more physiologically relevant outcome that should lead to a better success rate in the drug development process. relationship between cpr and biophysical factors – are they independent? cases where the biophysical mefs do not seem to influence the cpr outcome have raised the question as to how much importance they command or to what extent they need to be exogenously included in construct design for hts. summarized in table are studies where liver cells were grown in type i, ii and iii scaffolds that provided vastly different biophysical cues with no difference in cellular function outcomes. encapsulation of primary human hepatocytes (hhy ) in alginate beads (pliable; type i) has been shown to promote the growth of these cells leading to the forma- tion of d aggregates and upregulation of liver specific functions [ ]. cells grown in this particular d format have prominent structural cpr features, such as junctional complexes and micro- villi-lined network of canaliculi along with improved secretion of liver specific proteins, cytochrome p function, and urea synth- esis. as the pliability of alginate beads is comparable to that of native hepatic tissue (bovine liver: . � . kpa and . � . kpa by ultrasound and instron young’s modulus, respectively [ ]) and their resultant biophysical mef can be considered optimal. considering this as a standard, a porous polystyrene surface (rigid; kpa) that has an elastic modulus considerably above the physiological range can be considered s functional cpr ref. ith luminal microvilli, desmosomes albumin secretion, tyrosine aminotransferase expression [ ] network of microvilli; nd junctional complexes albumin secretion, fibrinogen, a- -antitrypsin production, cytochrome p a activity, urea synthesis [ ] unctional complexes ined with microvilli albumin secretion, cyp a , cyp a cytochrome p activity [ ] albumin secretion, cyp a , cyp a , and cyp e cytochrome p activity [ ] albumin secretion [ ] , bile canaliculi, gap glycogen storage, hnf- positive, albumin secretion [ ] , channels with microvilli albumin secretion [ , ] plexes, luminal microvilli albumin secretion, cytochrome p activity, tyrosine aminotransferase induction [ ] albumin secretion, cytochrome p a / capacity, ammonia removal, urea synthesis [ ] icrovilli [ ] l methacrylate); ps: polystyrene. www.drugdiscoverytoday.com reviews drug discovery today �volume , numbers / �june r e v ie w s � g e n e t o s c r e e n sub-optimal and the liver cells grown on it are expected to produce an aberrant or at least a significantly different outcome. however, studies by bokhari et al. [ , ] have shown that hepg cells grown on porous polystyrene scaffolds (type ii) exhibit cprs similar to those shown by microtissues grown on softer materials like higher viability, structural integrity and formation of bile canaliculi, enhanced liver function and drug response comparable to in vivo activity. one might argue that the type of cells (e.g. primary hepatocytes versus hepatocellular carcinoma) and substrate rigidity might be a factor in screening results as cancerous cells are known to have a higher elastic modulus than the non-malignant phenotype and can easily adapt to a more rigid environment or substrate (e.g. standard polystyrene plates). however, the modulus of hepg cells ( . , . and . kpa cultured on collagen i-, laminin-, and matrigel-coated substrates, respectively [ ], kpa by micropipette aspiration [ ]) is not considerably higher than that of native tissue (bovine liver – . � . kpa and . � . kpa by ultra- sound and instron ym, respectively [ ]), while the modulus of polystyrene is well beyond the adaptability of the cell, thus ruling out the difference in cell phenotype and respective optimal growth environment elasticity requirement as major factors. this is further substantiated by the fact that primary hepatocytes grown in nanofibrous plla scaffolds have also exhibited comparable morphological and functional cpr outcomes [ ]. interestingly, the level of albumin produced by cells cultured on this scaffold for a day was found to be mg/ cells ( mg/day/ cells in d) which is considerably higher than that found in alignate beads ( mg/day/ cells in d, mg/day/ cells in d [ ]) and closer to that in vivo ( mg/day/ cells in adult normal rat liver [ ]). furthermore, to totally eliminate the effects of substrate pliability and cell–material interactions, spheroid systems that lack any physical scaffolding for cells (type iii) can be considered. such systems, whether employed in static (hanging drops) or dynamic state (rwv [ ] or spinner flasks [ ]) have yielded cpr outcomes similar to the previous cases. hence, it can be inferred from these studies that neither the pliability (soft, rigid or scaffold-free) nor the structure (microporous or nanofibrous) of the substrate has a major effect on the crp phenomena exhibited by the liver cells grown therein. a similar analysis is needed if this is also the case for cells derived from other tissue types. a possible explanation for the above lack of pliability effect comes from the ‘cell-on-cell’ hypothesis [ ], where cell-to-cell contacts appear to have a more pivotal role as neighboring cells provide a soft ‘stroma’ for surrounding cells and produce responses similar to those seen when cells are grown in softer gels. for example, prominent actomyosin striation can be seen in myotubes www.drugdiscoverytoday.com when they are cultured on top of a layer of muscle cells. the lower layer of myotubes that adheres more strongly to the rigid substrate shows formation of ample stress fibers, however, myotubes in the upper layers differentiate to a more physiological, striated state with an elastic modulus in a range similar to that of gels suited for differentiation and also the native muscle tissue [ ]. similarly, a basal layer of astrocytes grown on glass provides a pliable envir- onment optimal for branching of neurons, which is similar to gels having brain-like pliability [ ]. also, when endothelial cells seeded at a high density become confluent, they have similar morphologies on both pliable and rigid substrates [ ], which differ from cells in a monolayer that are attached only to an underlying rigid surface that cause actin cytoskeletal remodeling and spreading. a similar phenomenon might be responsible for the above discussed liver cell cpr outcomes from cells grown on rigid porous scaffolds (type ii/high muduli) where only the out- ermost layer of the microtissue comes in contact and adapts to the pliability of the substrate while the cells in the core grow in the softer environment provided by peripheral cells. however, whether it is only the peripheral layer that is affected by substrate rigidity or a radial gradient in elastic modulus exists within the microtissue is not yet known and requires further investigation. concluding remarks with the field of d cell culture moving into its second generation products, the significance of translational research is increasing. up until now, much thought and effort has been put towards engineering an optimal d microenvironment that can emulate the characteristics of the native milieu. however, adapting state- of-the-art cell-based hts platforms to accommodate d cultures requires a balance between simplistic architecture, control over microenvironmental parameters, structural and functional integ- rity of microtissues and cost effectiveness. based on the current state of knowledge, it is fair to suggest that in some cases, biophy- sical factors might not be necessary for obtaining cpr outcomes in vitro, which are predictive of treatment/drug efficacy in vivo as the microtissues might be creating their own physical domain (endo- genous ecm) rendering the exogenously incorporated factors less important. elimination of such redundant physical cues might lead to a better control over important parameters like aggregate size and hypoxia, easier adaptability to automated handling and a reduction in high costs that are currently associated with d cell culture platforms. however, more translational research spanning different platforms, constituting a vast array of biophysical factors such as pliability (soft, rigid or scaffold free) and structure (micro- porous or nanofibrous) is required to establish a design philosophy consensus. references kisaalita, w.s. 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( ) enhanced functional maturation of fetal porcine hepatocytes in three-dimensional poly-l-lactic acid scaffolds: a culture condition suitable for engineered liver tissues in large-scale animal studies. cell transplant. , – 	outline placeholder 	cell-substrate interaction - biophysical constraints in d platforms 	type i 	type ii 	type iii 	cellular complex physiological relevance and drug discovery outcomes 	cpr in liver tissue-derived cells 	cpr in nerve tissue-derived cells 	cpr in muscle tissue-derived cells 	cpr in hts d platform validation 	relationship between cpr and biophysical factors - are they independent? 	concluding remarks 	references magnetic instabilities along the superconducting phase boundary of nb ∕ ni multilayers amish g. joshi, sergiy a. kryukov, lance e. de long, elvira m. gonzalez, elena navarro, javier e. villegas, and jose l. vicent citation: journal of applied physics , g ( ); doi: . / . view online: http://dx.doi.org/ . / . view table of contents: http://scitation.aip.org/content/aip/journal/jap/ / ?ver=pdfcov published by the aip publishing articles you may be interested in probing the magnetization inside a superconducting nb film by nuclear resonant scattering appl. phys. lett. , ( ); . / . full spin switch effect for the superconducting current in a superconductor/ferromagnet thin film heterostructure appl. phys. lett. , ( ); . / . magnetism and superconductivity in the superconductor/quasimagnet/ferromagnet nb ∕ pd ∕ fe system j. appl. phys. , c ( ); . / . fabrication and physical properties of pb ∕ cu multilayered superconducting nanowires appl. phys. lett. , ( ); . / . laser switch for stroboscopic read-out of magnetic flux rev. sci. instrum. , ( ); . / . [this article is copyrighted as indicated in the article. reuse of aip content is subject to the terms at: http://scitation.aip.org/termsconditions. downloaded to ] ip: . . . on: fri, sep : : http://scitation.aip.org/content/aip/journal/jap?ver=pdfcov http://oasc . realmedia.com/realmedia/ads/click_lx.ads/www.aip.org/pt/adcenter/pdfcover_test/l- / /x /aip-pt/jap_articledl_ /aip-apl_photonics_launch_ x _general_pdf_ad.jpg/ c a a c b f?x http://scitation.aip.org/search?value =amish+g.+joshi&option =author http://scitation.aip.org/search?value =sergiy+a.+kryukov&option =author http://scitation.aip.org/search?value =lance+e.+de+long&option =author http://scitation.aip.org/search?value =elvira+m.+gonzalez&option =author http://scitation.aip.org/search?value =elena+navarro&option =author http://scitation.aip.org/search?value =javier+e.+villegas&option =author http://scitation.aip.org/search?value =jose+l.+vicent&option =author http://scitation.aip.org/content/aip/journal/jap?ver=pdfcov http://dx.doi.org/ . / . http://scitation.aip.org/content/aip/journal/jap/ / ?ver=pdfcov http://scitation.aip.org/content/aip?ver=pdfcov http://scitation.aip.org/content/aip/journal/apl/ / / . / . ?ver=pdfcov http://scitation.aip.org/content/aip/journal/apl/ / / . / . ?ver=pdfcov http://scitation.aip.org/content/aip/journal/jap/ / / . / . ?ver=pdfcov http://scitation.aip.org/content/aip/journal/apl/ / / . / . ?ver=pdfcov http://scitation.aip.org/content/aip/journal/rsi/ / / . / . ?ver=pdfcov magnetic instabilities along the superconducting phase boundary of nb / ni multilayers amish g. joshi,a� sergiy a. kryukov, and lance e. de longb� department of physics and astronomy, university of kentucky, lexington, kentucky - elvira m. gonzalez, elena navarro, javier e. villegas, and jose l. vicent departamento de fisica de materiales, c. c. fisicas, universidad complutense, madrid, spain �presented on january ; received october ; accepted january ; published online may � we report vibrating reed and superconducting quantum interference device magnetometer data that exhibit prominent dips or oscillations of the superconducting �sc� onset temperature, �tc�h� � . – . k, for a �nb� nm� / ni� nm�� multilayer �ml� in dc magnetic fields applied nearly parallel to the ml plane. the vibrating reed data exhibit reproducible structures below tc that may reflect multiple sc transitions, but they are sensitive to ac field amplitude and dc field orientation. this striking behavior poses challenges for theoretical and experimental investigations of interfaces between sc and ferromagnetic layers that involve magnetic pair breaking effects, “pi phase shifts” of the sc order parameter, and exotic �“loff”� pairing states. alternatively, the anomalies may mark dynamical instabilities within a confined, strongly anisotropic abrikosov vortex lattice. © american institute of physics. �doi: . / . � multilayers �mls� composed of alternating supercon- ducting �sc� and ferromagnetic �fm� thin films exhibit re- markable properties related to the destabilizing effect of magnetic interactions on sc pairing. , this work was moti- vated by evidence of oscillations of tc�y� of order of . k for �nb� nm� / ni�y�� ml �nb layer thickness x = nm and ni layer thickness . � y � . nm�. , similar non- monotonic decreases of tc with fm layer thickness have been observed in other sc/fm bilayer, trilayer, and ml sys- tems, and attributed to a magnetic exchange coupling be- tween fm layers that oscillates in magnitude and sign as a function of the fm layer thickness. , – the sc proximity effect and magnetic pair breaking interactions within the ni layers alter the magnitude and phase of the complex sc or- der parameter that determines the stability of the sc state of the ml, and may even induce “pi phase shifts” that can completely suppress superconductivity. a recent letter has reported the resistively measured tc of ni� nm� / nb�x� / ni� nm� trilayers � nm � x � nm� as a function of applied magnetic field that controls the relative orientation of the magnetizations of the two ni lay- ers. although very small, the observed shifts � � �tc � mk� between parallel and antiparallel ni layer orienta- tions were ten times larger than predicted by existing theo- ries for fm/sc/fm trilayers, using measured normal state parameters. we wish to point out that interpreting such shifts �tc � mk is problematic because the experimental definition of tc�h� is not precise in applied magnetic fields that couple the equilibrium of the sc state to the weak stability of the abrikosov vortex lattice. indeed, modest probe currents can initiate dissipative motion or nonlinear dynamics of vortices , in materials with few defects to “pin” them against lorentz forces. consequently, resistive determina- tions of tc�h� require an arbitrary “voltage criterion” that defines when dissipation due to vortex motion under the ap- plied current drive has dropped to negligible levels. the temperature interval between the initial decrease of resis- tance and apparent tc defined by the voltage criterion can be substantial and very sensitive to the probe current amplitude, and inferred tc’s do not necessarily reflect the equilibrium phase boundary between normal and sc states. on the other hand, shifts of order mk are known to result from confinement of supercurrents and quantized mag- netic flux by mesoscopic boundaries such as thin-film cross sections , or submicron holes lithographically patterned in sc films. , in these cases, the tc shifts reflect an equilib- rium phase boundary when carefully measured at “vanish- ing” drive current. , magnetometry techniques offer alternative definitions of the sc onset, such as an initial diamagnetic change of the real part �m�� or an abrupt increase in the dissipative imagi- nary part �m�� of the ac magnetic moment in field-cooling �fc� experiments. vibrating reed �vr� magnetometry is particularly well suited for measurements of thin-film or an- isotropic samples and is essentially a transverse ac suscep- tibility technique that employs very low ac fields generated perpendicular to the dc applied field. , the high sensitivity of the vr to the entire sample bulk �which is not necessarily the case with resistivity measurements� at relatively low ac drives has been exploited to detect subtle transitions between equilibrium vortex lattice phases in bulk fm super- conductors and the onsets of plastic deformation and flow of the vortex lattice. a�present address: national physical laboratory, dr. k. s. krishnan road, new delhi , india b�author to whom correspondence should be addressed; electronic mail: delong@pa.uky.edu journal of applied physics , g � � - / / � �/ g / /$ . © american institute of physics , g - [this article is copyrighted as indicated in the article. reuse of aip content is subject to the terms at: http://scitation.aip.org/termsconditions. downloaded to ] ip: . . . on: fri, sep : : http://dx.doi.org/ . / . http://dx.doi.org/ . / . http://dx.doi.org/ . / . the �nb� nm� / ni� nm�� ml was fabricated by dc magnetron sputtering on a si � � substrate and exhibited textured growth of nb � � and ni � � layers , with only small nb–ni interdiffusion and interface roughness. in order to carry out vibrating reed experiments, a sample ml was glued onto one end of a rectangular reed � . � � . mm , cut from a si� � wafer�, the opposite end of which was mounted onto one side of a piezoelectric transducer to form a cantilever beam arrangement. the transducer was driven by a synthesizer tuned to the funda- mental cantilever mode �frequency f = – hz�, and the reed amplitude was detected capacitively using a resonant rf � mhz� cavity technique. , the si reed was oriented with its long dimension and the ml plane parallel to the dc magnetic field, which results in a transverse ac field �viewed in the moving vr frame� oriented perpendicular to the ap- plied dc field. , shifts of the vr frequency f �comparable to transverse m�� measure a magnetic restoring torque ex- erted on supercurrents that screen the ac field. the inverse vr amplitude a− measures ac losses �comparable to trans- verse m�� generated primarily by vortex motion. in order to complement the transverse vr response per- pendicular to the applied dc field, we also carried out con- ventional longitudinal measurements �with both dc and ac fields applied along the ml plane� using a quantum design mpms supreconducting quantum interference device �squid� magnetometer operated at frequencies . � f � hz and drive amplitudes . � �oho � . mt. the real part �m�� of the ac moment measures the supercurrent �circulating perpendicular to the dc field� that screens the ac field, and the imaginary part �m�� mainly measures ac losses generated by sc vortex motion. we determined the sc onset temperatures using either the vr frequency f or amplitude a for a �nb� nm� / ni� nm�� ml sample in parallel dc fields �see fig. �. the data define two reversible phase boundaries that coincide remarkably well, except where they exhibit large downshifts at . , . , and . t �and smaller shifts at . and . t�, as shown in fig. . additional peaks or abrupt slope changes in f �t� and a�t� were also identified well below the upper sc onset �see fig. � and were gener- ally found �almost independent of dc field� near characteris- tic temperatures of . , . , and . k. these anomalies could be grouped into two other trajectories that closely par- alleled the behavior of the sc onsets, as shown in fig. . the complementary m��h , t� boundary determined from longitudinal squid data for field parallel to the ml plane exhibits strong oscillations � – mk�, and these anoma- lies persist to fields of at least . t with an average period �o�h � mt, as shown in fig. . additional squid mea- surements using ac field amplitudes �oho = . , . , . , and . mt yield different trajectories—probably a result of fig. . �color online� vr frequency f and amplitude a vs temperature t for a �nb� nm� / ni� nm�� ml in parallel magnetic field �oh = . t with vr drive v = mv. open symbols denote initial field-cooled �fc� data, and solid symbols denote subsequent fc-warming cycle �fcw� data. red lines indicate the assumed normal-state base lines in f �t� and a�t�, and vertical arrows denote the temperatures of the sc onset near . k, and slope changes apparent near . and . k. fig. . magnetic field �h�-temperature �t� “phase boundary” between the sc and normal states for a �nb� nm� / ni� nm�� ml, defined by an abrupt decrease in the vr amplitude a �open circles�. data were taken in field-cooled-warming �fcw� experiments with vr drive v = mv and dc field h nearly parallel to the ml plane. the left �open triangles� and middle �solid squares� trajectories mark temperatures of two anomalies in the slope of a�t� �see fig. �. solid lines are guides to the eye, and error bars denote uncertainties in extracting the points from data. dashed lines denote possible phase transition lines between sc states of the entire ml or sc transitions within individual nb layers. fig. . �color online� magnetic field �h�-temperature �t� “phase bound- ary” between the sc and normal states for a �nb� nm� / ni� nm�� ml, determined from squid magnetometer experiments at ac frequency f = hz and amplitude �oho = . mt, with applied dc field h parallel to the ml plane. solid symbols denote the sc onset temperature indicated by an abrupt increase in the imaginary part m�, and open symbols by an abrupt diamagnetic shift in the real part m�, of the ac magnetic moment during fc. the divergence of the m� and m� data near �oh = . t reflects the onset of vortex depinning with increasing dc field. g - joshi et al. j. appl. phys. , g � � [this article is copyrighted as indicated in the article. reuse of aip content is subject to the terms at: http://scitation.aip.org/termsconditions. downloaded to ] ip: . . . on: fri, sep : : vortex depinning and nonequilibrium disorder of the vortex lattice. nevertheless, these data consistently exhibit oscilla- tions with the same average spacing and magnitudes shown in fig. . small oscillations �of order of – mk and rough pe- riod of mt� were previously observed in lower-precision squid measurements on nb / ni ml. these variations were interpreted as “matching anomalies” at applied fields where the vortex density is equal to an integral multiple of the average density of strong vortex pinning centers. , match- ing anomalies are most prominent if pinning centers have a characteristic size ��t� � d � ��t� and are located on a peri- odic lattice. the sc/fm ml is essentially a one- dimensional periodic array of thin sc slabs spaced by ni layers that strongly confine vortices when their planar cross sections are oriented perpendicular to an applied dc magnetic field h. a related matching effect has been predicted and observed in ml �ref. � when nl vortices enter in succes- sive chains aligned parallel to the ml and h �an average oscillation period of mt implies nl � � �. more- over, calculations , , predict that a succession of vortex lattices will form within a thin sc slab in an increasing par- allel field, and these phases will have different packing to- pologies whose ranges of stability may not be strictly peri- odic in applied field. however, matching analyses of parallel dc field data �fig. � do not clearly discriminate between cases for which there is either a nonzero sc order parameter within the ni layers or isolated sc nb layers �having distinct tc’s� sepa- rated by ni layers having zero sc order parameter. additional vr data were acquired at piezoelectric drives of , , , and mv at selected dc fields of . , . , . , and . t, at which thermal hysteresis was observed. the sc onsets exhibited extraordinary downshifts of . – . k with increasing vr drive, indicating weak vortex pinning and nonequilibrium vortex lattice disorder. the line of onsets near . k was found to extend to at least . t for drives of – mv, but disappeared at �oh � . t for drives v 	 mv, whereas a line of anomalies near . k was found to terminate at . t for v 	 mv �see fig. �. the downshift of the sc onset near . t � . t� therefore marks a hysteretic jump from the . to . k � . – . k� “transition line,” possibly due to an increase in sc currents that drive the vortex lattice and have the potential �for criti- cal current densities jc � a / cm � to switch the magneti- zation direction of ni domains. both vr and ac squid magnetometry sensitively probe the pinning and dynamics of abrikosov vortices, and the anomalies of figs. – are clearly affected by vortex dynam- ics, but since the local vortex pinning force is proportional to the spatial gradient of the squared sc order parameter, the anomalies nevertheless reflect unusually strong variations in the stability of the sc state. since both the real and imaginary parts of the vr signal behave similarly, the anomalies of fig. may well reflect sc phase transition lines for the entire ml or for individual nb layers. the possibility remains open that they are caused by changes in the ni-layer magnetizations that alter the phase and coupling between the sc order pa- rameter of adjacent nb layers with applied dc field. research at university of kentucky supported by u.s. department of energy and kentucky science and engineer- ing foundation. research at universidad complutense supported by spanish ministerio de educacion y ciencia, comunidad de madrid y santander-ucm. one of the authors �a.g.j.� was supported by an indian department of science and technology boyscast fellowship no. sr/by/p- / . b. y. jin and j. b. ketterson, adv. phys. , � �. m. b. maple, physica b , � �. j. e. villegas, e. navarro, d. jaque, e. m. gonzalez, j. i. martin, and j. l. vicent, physica c , � �. l. e. de long et al., philos. mag. , � �. yu. a. izyumov, yu. n. proshin, and m. g. khusainov, phys. usp. , � �. see review of early work in c. l. chien and d. h. reich, j. magn. 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rev. sci. instrum. , � �. v. v. moshchalkov, m. baert, v. v. metlushko, e. rosseel, m. j. van bael, k. temst, y. bruynseraede, and r. jockheere, phys. rev. b , � �. a. i. buzdin and a. yu. simonov, physica c , � �. s.-w. han, j. farmer, h. kaiser, p. f. miceli, i. v. roshchin, and l. h. greene, physica b , � �. l. s. levitov, phys. rev. lett. , � �. c. c. de souza silva, l. r. e. cabral, and j. albino aguiar, physica c , � �. g - joshi et al. j. appl. phys. , g � � [this article is copyrighted as indicated in the article. reuse of aip content is subject to the terms at: http://scitation.aip.org/termsconditions. downloaded to ] ip: . . . on: fri, sep : : wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ zeggini genome medicine , : http://genomemedicine.com/content/ / / research highlight using genetically isolated populations to understand the genomic basis of disease eleftheria zeggini abstract rare variation has a key role in the genetic etiology of complex traits. genetically isolated populations have been established as a powerful resource for novel locus discovery and they combine advantageous characteristics that can be leveraged to expedite discovery. genome-wide genotyping approaches coupled with sequencing efforts have transformed the landscape of disease genomics and highlight the potentially significant contribution of studies in founder populations. ways underpinning t d were achieved through the complex trait locus discovery in isolated populations genetically isolated or founder populations have recently returned to the fore of genetic association studies as valuable resources for complex trait gene identification [ ]. population isolates have well-documented character- istics, including reduced phenotypic, environmental and genetic heterogeneity, that can aid in the detection of rare variants associated with complex traits. in isolated populations, where a relatively small number of individ- uals found a new population, rare variants that were present in the founders can drift up in frequency as the population expands, thus increasing power for genetic association studies. the small effective population size, which remains small over time, leads to increased levels of homozygosity and linkage disequilibrium. in addition, isolated population cohorts often provide the opportun- ity to recall subjects by genotype, access detailed genea- logical records, obtain linkage to health records and follow the cohort longitudinally. recent successes in the literature have highlighted how these advantageous characteristics can help with disease gene mapping. researchers studying the ice- landic population have, in recent years, pioneered the correspondence: eleftheria@sanger.ac.uk wellcome trust sanger institute, hinxton, cambridge cb sa, uk © zeggini; licensee biomed central ltd. months following its publication. after this time, t (http://creativecommons.org/licenses/by/ . ), whi the original work is properly credited. the creativ publicdomain/zero/ . /) applies to the data mad use of next-generation association studies, a hybrid of genome-wide genotyping and whole genome sequencing (wgs) approaches, for complex disease gene mapping [ , ]. in iceland, numerous novel loci for complex dis- eases, such as type diabetes (t d) and prostate cancer [ , ], have been identified through a combination of wgs and long-range phasing-assisted imputation on a genome-wide genotype scaffold, together with calcula- tion of genotype probabilities in approximately , untyped individuals by making use of the extended ge- nealogical information available. more recently, novel insights into the biological path- study of a greenlandic founder population [ ]. a non- sense variant in the tbc d gene was found to be strongly associated with postprandial hyperglycemia, im- paired glucose tolerance and t d. these unique insights into the mechanism conferring muscle insulin resistance for this subset of t d was afforded by studying the small greenlandic population, which has experienced a dramatic increase in t d prevalence, and recalling indi- viduals based on their tbc d variant status. this poly- morphism is common in greenland ( % minor allele frequency), but vanishingly rare in other global popula- tions (only encountered in one japanese individual in the genomes project data). this work elegantly demonstrates the value of combining the genetic charac- teristics of founder populations with the potential to re- contact participants for further follow-up of promising results. studies in extensively phenotyped founder popu- lation cohorts, such as the amish, have also demon- strated the value of combining unique population characteristics with recall of subjects to increase our un- derstanding of disease etiopathology. the old order amish are a cultural isolate and geographically localized, genetically homogeneous population with extensive ge- nealogical records available. this deeply phenotyped co- hort has been the subject of long-term genetic studies. for example, in , pollin et al. [ ] reported a mis- sense variant (r x) that abolishes expression of the the licensee has exclusive rights to distribute this article, in any medium, for he article is available under the terms of the creative commons attribution license ch permits unrestricted use, distribution, and reproduction in any medium, provided e commons public domain dedication waiver (http://creativecommons.org/ e available in this article, unless otherwise stated. mailto:eleftheria@sanger.ac.uk http://creativecommons.org/licenses/by/ . http://creativecommons.org/publicdomain/zero/ . / http://creativecommons.org/publicdomain/zero/ . / zeggini genome medicine , : page of http://genomemedicine.com/content/ / / apoc gene and is strongly associated with a cardiopro- tective phenotype (higher high-density lipoprotein and lower blood triglyceride levels). notably, the same missense cardioprotective variant was also found in an independent isolated population from greece in the helic-manolis study [ ]. resi- dents of the mountainous mylopotamos villages on crete have a high fat content diet but anecdotally display lower levels of, for example, t d complications com- pared with the general population. the r x apoc variant was carried by approximately % of the individ- uals studied and reached genome-wide statistical signifi- cance with a sample size of fewer than , . discovery of the same effect in the general population would have required over times the number of subjects. large- scale studies of over , individuals of european descent have recently also established an association of rare variants in the apoc locus with protection against high triglyceride levels and coronary artery disease [ ]. apoc is now becoming a poster child for the power afforded by founder populations and clearly demon- strates the generalizability of findings in isolates into more cosmopolitan populations. a prime example of how founder population charac- teristics coupled with linkage to medical records can ac- celerate discovery was recently produced by studying the finnish population [ ]. in a whole exome sequencing study of about , finns, lim et al. first established that the finns have fewer variable sites overall but more loss-of-function variants compared with non-finnish european individuals, and subsequently identified robust associations with key traits of medical relevance. linkage to national medical records resulted in the demonstra- tion that splice variants in the lpa gene that are associ- ated with low levels of plasma lipoprotein(a) confer protection against cardiovascular disease. future directions going forward, it is clear that founder populations can provide a unique and powerful resource for the identifi- cation of low frequency and rare variants of direct medical consequence. power to detect association is demonstrably boosted for individual sequence variants that have drifted up in frequency. in addition, power to detect a significant accumulation of rare variants at par- ticular loci is further increased in founder populations as neutral rare variation may be lost from the haplotype pool. in this context, meta-analysis at the locus level across different isolates is posited to be important for establishing burden of proof, although this principle requires empirical substantiation. historically, the trans- ferability of findings in isolates across to more cosmo- politan populations has been a topic of debate. however, there is an accrual of emerging examples of loci discovered in founder populations that are more widely generalizable, with replication of signals achieved in di- verse sample sets [ , , - ]. furthermore, invaluable and unprecedented insights into disease pathogenesis can be afforded by findings restricted to genetically isolated populations, as exemplified by the elegant metabolic trait study in greenland [ ]. decreasing costs for deep whole genome sequencing and the increasing availability of deeply phenotyped genetically isolated cohorts sets the scene for further success stories in the near future. abbreviations t d: type diabetes; wgs: whole genome sequencing. competing interests the author has no competing interests to declare. references . zuk o, schaffner sf, samocha k, do r, hechter e, kathiresan s, daly mj, neale bm, sunyaev sr, lander es: searching for missing heritability: designing rare variant association studies. proc natl acad sci u s a , :e –e . . holm h, gudbjartsson df, sulem p, masson g, helgadottir ht, zanon c, magnusson ot, helgason a, saemundsdottir j, gylfason a, stefansdottir h, gretarsdottir s, matthiasson se, thorgeirsson gm, jonasdottir a, sigurdsson a, stefansson h, werge t, rafnar t, kiemeney la, parvez b, muhammad r, roden dm, darbar d, thorleifsson g, walters gb, kong a, thorsteinsdottir u, arnar do, stefansson k: a rare variant in myh is associated with high risk of sick sinus syndrome. nat genet , : – . . zeggini e: next-generation association studies for complex traits. nat genet , : – . . steinthorsdottir v, thorleifsson g, sulem p, helgason h, grarup n, sigurdsson a, helgadottir ht, johannsdottir h, magnusson ot, gudjonsson sa, justesen jm, harder mn, jørgensen me, christensen c, brandslund i, sandbæk a, lauritzen t, vestergaard h, linneberg a, jørgensen t, hansen t, daneshpour ms, fallah ms, hreidarsson ab, sigurdsson g, azizi f, benediktsson r, masson g, helgason a, kong a, et al: identification of low-frequency and rare sequence variants associated with elevated or reduced risk of type diabetes. nat genet , : – . . gudmundsson j, sulem p, gudbjartsson df, masson g, agnarsson ba, benediktsdottir kr, sigurdsson a, magnusson ot, gudjonsson sa, magnusdottir dn, ohannsdottir h, helgadottir ht, stacey sn, jonasdottir a, olafsdottir sb, thorleifsson g, jonasson jg, tryggvadottir l, navarrete s, fuertes f, helfand bt, hu q, csiki ie, mates in, jinga v, aben kk, van oort im, vermeulen sh, donovan jl, hamdy fc, et al: a study based on whole-genome sequencing yields a rare variant at q associated with prostate cancer. nat genet , : – . . moltke i, grarup n, jorgensen me, bjerregaard p, treebak jt, fumagalli m, korneliussen ts, andersen ma, nielsen ts, krarup nt, gjesing ap, zierath jr, linneberg a, wu x, sun g, jin x, al-aama j, wang j, borch-johnsen k, pedersen o, nielsen r, albrechtsen a, hansen t: a common greenlandic tbc d variant confers muscle insulin resistance and type diabetes. nature , : – . . pollin ti, damcott cm, shen h, ott sh, shelton j, horenstein rb, post w, mclenithan jc, bielak lf, peyser pa, mitchell bd, miller m, o'connell jr, shuldiner ar: a null mutation in human apoc confers a favorable plasma lipid profile and apparent cardioprotection. science , : – . . tachmazidou i, dedoussis g, southam l, farmaki ae, ritchie gr, xifara dk, matchan a, hatzikotoulas k, rayner nw, chen y, pollin ti, o’connell jr, yerges-armstrong lm, kiagiadaki c, panoutsopoulou k, schwartzentruber j, moutsianas l, tsafantakis e, tyler-smith c, mcvean g, xue y, zeggini e, uk k consortium: a rare functional cardioprotective apoc variant has risen in frequency in distinct population isolates. nat commun , : . zeggini genome medicine , : page of http://genomemedicine.com/content/ / / . crosby j, peloso gm, auer pl, crosslin dr, stitziel no, lange la, lu y, tang zz, zhang h, hindy g, masca n, stirrups k, kanoni s, do r, jun g, hu y, kang hm, xue c, goel a, farrall m, duga s, merlini pa, asselta r, girelli d, olivieri o, martinelli n, yin w, reilly d, speliotes e, et al: loss-of-function mutations in apoc , triglycerides, and coronary disease. n engl j med , : – . . lim et, wurtz p, havulinna as, palta p, tukiainen t, rehnstrom k, esko t, magi r, inouye m, lappalainen t, chan y, salem rm, lek m, flannick j, sim x, manning a, ladenvall c, bumpstead s, hämäläinen e, aalto k, maksimow m, salmi m, blankenberg s, ardissino d, shah s, horne b, mcpherson r, hovingh gk, reilly mp, watkins h, et al: distribution and medical impact of loss-of-function variants in the finnish founder population. plos genet , :e . doi: . /s - - - cite this article as: zeggini: using genetically isolated populations to understand the genomic basis of disease. genome medicine : . 	abstract 	complex trait locus discovery in isolated populations 	future directions 	abbreviations 	competing interests 	references diabetes care, volume , number , january o b s e rvat i o n s diabetes scre e n i n g practices among i n d i v i d u a l s a g e d ye a r s a nd o l d e r i n , the american diabetes associa- tion (ada) adopted new re c o m m e n d a- tions for screening the general popula- tion aged years for diabetes every years with an emphasis on those at high risk for undiagnosed diabetes ( ). few studies have examined the extent to which this screening has been adopted. this re p o rt describes the results of a telephone s u rvey of montana residents aged years to assess the diabetes screening prac- tices in this population. f rom october to december , the montana department of public health and human services conducted a random household telephone survey of montana residents aged years living in counties. respondents indicated whether they ever had been told by a physician that they had diabetes, the number of visits they made to a health care provider during the past year, their family history of dia- betes, whether they had ever been told they had high cholesterol and/or high blood pre s s u re, and their height and weight. respondents were asked the fol- lowing question to identify whether they had ever been screened for diabetes: “glu- cose or sugar is a substance found in your blood. have you ever had your blood glu- cose or sugar checked to see if you have diabetes?” when respondents re s p o n d e d “yes” to this question, they were asked to identify when screening was completed (“when was the last time your blood glu- cose or sugar level was measured by a health care professional?”). the re s p o n s e categories for this question included within the past year, within the past years, years ago, do not know/not sure, and refused to answer. pearson tests were used to assess associations between dia- betes screening and risk factors for dia- betes. logistical re g ression analyses were conducted to identify independent vari- ables associated with screening for diabetes during the past year. odds ratios ( % cis) were calculated. of the , respondents, ( . %) re p o rted that they had diagnosed diabetes. the remaining , respondents re p o rt e d that they did not have diagnosed diabetes and are included in the following analyses. of the respondents, % re p o rted a family h i s t o ry of diabetes, % re p o rted a bmi kg/m , % re p o rted having hyper- tension, and % re p o rted having high c h o l e s t e rol. excluding age, % of re s p o n- dents had one risk factor for diabetes, and % had two or more risk factors. of the , respondents without diagnosed dia- betes, % re p o rted that they had been s c reened for diabetes during the past year, % re p o rted screening from to years ago, and % re p o rted screening years ago or having never been scre e n e d . respondents who re p o rted being s c reened for diabetes during the past year w e re more likely to be age years and to have a family history of diabetes, two or m o re visits to a health care provider dur- ing the past year, hypertension, and high c h o l e s t e rol levels (table ). we found no association between recent screening and sex ( % men vs. % women), ameri- can indian ancestry ( % yes vs. % no), or bmi ( % kg/m vs. % kg/m ). respondents with three or m o re risk factors (e.g., aged years, american indian ancestry, family history of diabetes, hypertension, high choles- t e rol, or bmi kg/m ) were more likely to be screened for diabetes compared with respondents with only one risk factor ( vs. %, respectively). however, % of individuals with two risk factors for dia- betes and % of individuals with more than three risk factors had not been s c reened during the past years. based on logistical re g ression analysis, t h ree factors were associated with scre e n i n g for diabetes during the past year: two or m o re visits to a health care provider during the past year ( . [ % ci . – . ]), high cholesterol level ( . [ . – . ]), and family history of diabetes ( . [ . – . ]). respondents aged – years were less likely to re p o rt re c e n t s c reening than those aged years ( . [ . – . ] ) . a limitation of this assessment is that these data are self-re p o rted. pre v i o u s studies, however, have found that self- re p o rts of conditions such as diabetes and h y p e rtension are reliable ( , ). in addi- tion, the survey was conducted by tele- phone and does not re flect the experience of individuals in montana homes without t e l e p h o n e s . the findings suggest that diabetes s c reening is being adopted by physicians for individuals aged years at risk for diabetes and that the ada re c o m m e n d a- tions are being implemented in the general c o m m u n i t y. however, these data also indi- cate a need to develop strategies to encour- l e t t e r s table —characteristics of respondents aged years reporting screening for diabetes in montana in s c reening for diabetes past year – years years or never n age (years) – ( ) ( ) ( ) – ( ) ( ) ( ) ( )* ( ) ( ) family history of diabetes ye s ( )* ( ) ( ) n o ( ) ( ) ( ) visits to a health care provider during the past year ( )* ( ) ( ) ( ) ( ) ( ) h y p e rt e n s i o n ye s ( )* ( ) ( ) n o ( ) ( ) ( ) high cholestero l ye s ( )* ( ) ( ) n o ( ) ( ) ( ) data are n (%). *p . . diabetes care, volume , number , january letters age screening among all individuals at high risk for diabetes. todd s. harwell, mph jane g. smilie, ba janet m. mcdowall, rn, bsn steven d. helgerson, md, mph dorothy gohdes, md f rom the montana diabetes project, montana d e p a rtment of public health and human serv i c e s , helena, montana. a d d ress correspondence to todd s. harw e l l , mph, montana department of public health and human services, cogswell building, c- , p. o . box , helena, mt - . e-mail: t h a rw e l l @ s t a t e . m t . u s . a c k n o w l e d g m e n t s — this project was sup- p o rted through a cooperative agreement (u- / ccu- - ) with the centers for disease c o n t rol and prevention, division of diabetes translation, atlanta, georg i a . we thank linda priest and the staff mem- bers at northwest resource consultants for their work on the telephone surv e y. the contents of this letter are solely the responsibility of the authors and do not neces- sarily re p resent the official views of the centers for disease control and pre v e n t i o n . r e f e re n c e s . american diabetes association: screening for type diabetes. diabetes care (suppl. ): s –s , . kehoe r, wu sy, leske mc, chylack lt jr: comparing self-re p o rted and physician- re p o rted medical history. am j epidemiol : – , . jackson c, jatulis de, fortmann sp: the behavioral risk factor survey and the stan- f o rd five-city project survey: a comparison of cardiovascular risk behavior estimates. am j public health : – , h b a c is not recommended as a s c reening test for diabetes in cystic fibro s i s i n the june issue of diabetes care, h u n k e rt et al. ( ) recommend the use of h b a c for early detection of cystic fib ro- s i s – related diabetes (cfrd). their re c o m- mendation is based on the finding that mean hba c is slightly higher in cystic fib rosis (cf) patients requiring insulin t h e r a p y, compared with cf patients with i m p a i red or normal glucose tolerance. h o w e v e r, no data on the validity of this a p p roach for the diagnosis of asympto- matic diabetes in patients with cystic fib ro- sis are pre s e n t e d . our group has a long-standing intere s t in the early diagnosis of cfrd, comparing fasting blood glucose levels with oral glu- cose tolerance test results ( ). in our series, we have now cf patients with newly diagnosed diabetes based on a -h venous plasma glucose value mg/dl ( . mmol/l), and simultaneous determ i n a t i o n of hba c ( h i g h - p e rf o rmance liquid chro- matography method [pharmacia, erlan- gen, germany], normal range . – . %). only out of cf patients ( %) diag- nosed as diabetic according to the ameri- can diabetes association and world health o rganization criteria ( ) had an hba c value above the normal range (individual values . , . , . , and . %). in nine diabetic cf patients with normal hba c, values between . and . % were e n c o u n t e red (mean ± sd, . ± . %). the mean -h blood glucose value after inges- tion of oral glucose was not signific a n t l y d i ff e rent between diabetic cf patients with n o rmal hba c ( ± mg/dl, mean ± sd) and diabetic cf patients with elevated h b a c ( ± mg/dl, student’s t t e s t ) . these data clearly demonstrate that the d e t e rmination of hba c is not able to sub- stitute for the oral glucose tolerance test in the early diagnosis of cfrd. our fin d i n g s a re in agreement with several re p o rts in the l i t e r a t u re ( – ) as well as the consen- sus conference on cfrd ( ). in addition to its low sensitivity when used as a diagnos- tic tool for the detection of cfrd, the mea- s u rement of hba c has the disadvantage of considerable interassay variability and lack of standardization. there f o re, we stro n g l y advise against the use of glycosylated hemoglobin as a screening test for the early diagnosis of diabetes in patients with cystic fib ro s i s . reinhard w. holl, md christian buck, md christine babka, md anna wolf, md angelika thon, md f rom the department of pediatrics (r.w.h.), uni- versity of giessen, giessen; the department of pedi- atrics (c.bu., c.ba., a.w.), the university of ulm, ulm; and the medical school hannover (a.t. ) , h a n n o v e r, germ a n y. a d d ress correspondence to pd dr. reinhard w. holl, universitätskinderklinik giessen, feulgenstr. , d- giessen, germ a n y. r e f e re n c e s . h u n k e rt f, lietz t, stach b, kiess w: potential impact of hba c d e t e rm i n a t i o n on clinical decision making in patients with cystic fib ro s i s – related diabetes (let- ter). diabetes care : – , . holl rw, buck c, cario h, wolf a, thon a, heinze e, kohne e, debatin k-m: diag- nosis of diabetes in cystic fib rosis and tha- lassemia major. diabetes care : – , . the expert committee on the diagnosis and classification of diabetes mellitus: r e p o rt of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care : – , . lanng s, hansen a, thorsteinsson b, n e rup j, koch c: glucose tolerance in patients with cystic fib rosis: five year p rospective study. b m j : – , . deluca f, arrigo t, nibali sc, sferlazzas c, gigante a, dicesare e, cucinotta d: insulin secretion, glycosylated haemoglo- bin and islet cell antibodies in cystic fib ro- sis children and adolescents with diff e re n t d e g rees of glucose tolerance. h o rm metab r e s : – , . moran a, doherty l, wang x, thomas w: a b n o rmal glucose metabolism in cystic fib rosis. j pediatr : – , . moran a: highlights of the febru a ry consensus conference on cfrd. bonn, g e rm a n y, cystic fibrosis foundation, p ro gln p e roxisome p ro l i f e r a t o r- a c t i v a t e d r e c e p t o r- and o b e s i t y r istow et al. ( ) re p o rted an activating mutation in the peroxisome pro l i f- e r a t o r-activated re c e p t o r- g e n e ( p ro gln ppa r - ), which was pre s e n t in % ( of ) of obese and % ( of ) of nonobese german caucasians. these findings may have profound implica- tions, particularly if the presence of this variant and its association with obesity are c o n firmed in other populations. we perf o rmed polymerase chain re a c- t i o n – restriction fragment length polymor- phism analysis for the pro gln ppa r - variant as described ( ) on dna samples f rom several independent populations, including lean and obese caucasians fro m the baltimore, maryland, region; african- diabetes care, volume , number , january letters americans from jackson, mississippi, and forsyth county, north carolina; pima indians from arizona; and old ord e r amish from lancaster county, pennsylva- nia (table ). a pcr fragment corre s p o n d- ing to gastric insulinotropic peptide, which has two known restriction sites for h i n dii, was mixed with each sample as a positive control. among a total of sub- jects ( , alleles), the pro gln variant was not detected in a single subject. these findings were unexpected because there is substantial overlap of gene pools of caucasians from central e u rope and the baltimore and amish caucasians studied ( ). the germ a n caucasians studied by ristow et al. were said to be unrelated and re c ruited fro m the nord rh e i n - westfalen region, but they may be a genetic isolate whose gene pool does not re flect that of other caucasian populations. altern a t i v e l y, if the individ- uals carrying the mutation were re l a t e d , the true frequency of the pro g l n p pa r - variant may have been overe s t i- mated. the absence or very low fre- quency of this variant has also been doc- umented in danish ( ) and german ( ) populations. this study is the first, to our knowledge, to examine american popu- lations for this variant. in summary, the study by ristow et al. demonstrating that the pro g l n p pa r - variant is activating and can influence body weight in people is important. however, this variant appears to be absent or very r a re in the american populations studied. additional studies are re q u i red in other regions of europe and the u.s. to furt h e r d e fine the relevance of this intere s t i n g genetic variant to susceptibility to obesity. alan r. shuldiner, md william nguyen, bs w.h. linda kao, phd brock a. beamer, md ross e. andersen, phd richard pratley, md frederick l. brancati, md, phd f rom the department of medicine (a.r.s., w. n . ) , university of maryland school of medicine; the d e p a rtments of medicine (b.a.b., f.l.b.) and epi- demiology (w.h.l.k.), johns hopkins university school of medicine, baltimore, maryland; and the national institute of diabetes and digestive and kidney diseases (r.p.), national institutes of health, phoenix, arizona. a d d ress correspondence to alan r. shuldiner, md, professor and head, division of endocrinol- o g y, diabetes, and nutrition, department of medi- cine, university of maryland school of medicine, w. lombard st., room s- , baltimore, md . e-mail:ashuldin@medicine.umary l a n d . e d u . a c k n o w l e d g m e n t s — this study was sup- p o rted by nih r dk- , the american diabetes association, glaxowellcome, the b a l t i m o re geriatrics research and education clinical center of the baltimore ve t e r a n s administration medical center. r e f e re n c e s . ristow m, muller- wieland d, pfeiffer a, k rone w, kahn cr: obesity associated with a mutation in a genetic regulator of adipocyte diff e rentiation. n engl j med : – , . c a v a l l i - s f o rza ll, menozzi p, piazza a: t h e h i s t o ry of human genes. princeton univer- sity press, princeton, nj, . elk j, urhammer sa, sorensen ti, ander- sen t, auwerx j, pedersen o: homozygosity of the pro ala variant of the pero x i s o m e p roliferation-activated re c e p t o r- g a m m a ( p par-gamma ): divergent modulating e ffects on body mass index in obese and lean caucasian men. diabetologia : – , . mamann a, munzberg h, buttron p, busing b, hinney a, mayer h, siegfried w, hebe- brand j, greten h: missense variants in the human peroxisome pro l i f e r a t o r- a c t i v a t e d re c e p t o r-gamma gene in lean and obese subjects. eur j endocrinol : – , insulin secre t i o n , insulin sensitivity, and glucose e ffectiveness in nonobese individuals with va ry i n g d e g rees of glucose to l e r a n c e a lthough it is well known that insulin s e c retion, insulin sensitivity, and glu- cose effectiveness are impaired in type diabetic patients ( – ), little is known about the role of each of these fac- tors individually on the evolution of type diabetes. in this context, a major issue is that hyperglycemia per se impairs insulin s e c retion and insulin sensitivity and that obesity observed in type diabetic patients per se causes insulin resistance ( , ). to o v e rcome this problem, we studied u n t reated nonobese subjects classified as having normal glucose tolerance (ngt) (n = ; bmi . ± . kg/m [ r a n g e . – . ], mean ± sem), impaired glu- cose tolerance (igt) (n = ; bmi . ± . kg/m [ . – . ]), and type dia- betes (n = ; fetal bovine serum . ± . mmol/l [range . – . ], bmi . ± . k g / m [ . – . ]), based on the criteria of the world health organization ( ). they were normotensive and had norm a l renal, hepatic, and thyroid function. insulin sensitivity and glucose eff e c t i v e n e s s w e re estimated by the minimal model a p p roach ( – ). insulin secretion was e x p ressed as the area under the insulin c u rve between and min after an intra- venous glucose injection ( ). after the data w e re analyzed by one-way analysis of vari- ance, bonferroni correction was used to evaluate the diff e rences between any two of the three groups we studied ( ). no signifi- cant difference was observed in bmi among the three groups. compared with the subjects with ngt, the subjects with table —characteristics of subjects screened for pro gln ppa r - n ( a l l e l e s age ± sd f e m a l e bmi ± sd p o p u l a t i o n t y p e d ) ( y e a r s ) ( % ) ( k g / m ) c a u c a s i a n s b a l t i m o re longitudinal ( ) . ± . . . ± . study on aging johns hopkins we i g h t ( ) . ± . . . ± . management center amish, lancaster, pa ( ) . ± . . . ± . a f r i c a n - a m e r i c a n s a t h e ro s c l e rosis risk in ( ) . ± . . . ± . communities study pima indians a r i z o n a ( ) . ± . — . ± . all non-amish subjects were unrelated; amish subjects were not fir s t - d e g ree relatives of each other. diabetes care, volume , number , january letters igt had significantly lower insulin secre- tion ( , ± vs. , ± pmol l m i n , p = . ) and glucose eff e c- tiveness ( . ± . vs. . ± . m i n , p . ). insulin sensitivity index was lower in subjects with igt ( . ± . m i n pmol l) than in those with ngt ( . ± . min pmol l ) , but was not statistically significant (p = . ). in con- trast, disposition index calculated by the p roduct of insulin secretion and insulin sensitivity was significantly lower in sub- jects with igt ( , ± ) than in those with ngt ( , ± , p = . ). on the other hand, patients with type dia- betes had significantly lower insulin secre- tion ( ± pmol l m i n , p = . ) compared with subjects with igt. although no significant diff e rence was observed in insulin sensitivity index between subjects with type diabetes and igt ( . ± . vs. . ± . min pmol l, p = . ), disposition index was s i g n i ficantly diminished in type diabetic patients as compared with subjects with igt ( ± vs. , ± , p = . ). glucose effectiveness in type diabetic patients ( . ± . min ) was similar to that in subjects with igt ( . ± . m i n , p = . ) but was signific a n t l y lower than that in the subjects with ngt (p . ). from these results, the fol- lowing may be hypothesized: ) impair- ments in insulin secretion and disposition index and decreased glucose eff e c t i v e n e s s , but not insulin resistance, seem to consti- tute the basic characteristics of patients with igt or type diabetes in nonobese japanese populations. ) risk factors wors- ening to type diabetes in subjects with igt are associated with further impair- ments in insulin secretion and disposition index, but not associated with furt h e r derangement in glucose effectiveness in japanese populations. ataru taniguchi, md mitsuo fukushima, md masahiko sakai, md itaru nagata, md kentaro doi, md shoichiro nagasaka, md kumpei tokuyama, md yoshikatsu nakai, md f rom the first department of internal medicine ( a . t., m.s., i.n.), kansai-denryoku hospital, and the department of internal medicine, hoshida- minami hospital (m.f.), osaka; the second depart- ment of internal medicine (k.d.) and the college of medical technology (y.n.), kyoto university, kyoto; the department of internal medicine (s.n.), jichi medical college, tochigi; and the laboratory of biochemistry of exercise and nutrition (k.t. ) , tsukuba university, tsukuba, japan. a d d ress correspondence to ataru ta n i g u c h i , md, first department of internal medicine, kansai- d e n ryoku hospital, - - , fukushima-ku, fuku- shima, osaka city, osaka - , japan. e-mail: k @ k e p c o . c o . j p . r e f e re n c e s . b e rgman rn: to w a rd physiological under- standing of glucose tolerance: minimal- model approach. d i a b e t e s : – , . welch s, gebhart ssp, bergman rn, phillips ls: minimal model analysis of intravenous glucose tolerance test-derived insulin sensitivity in diabetic subjects. j c l i n endocrinol metab : – , . taniguchi a, nakai y, fukushima m, kawamura h, imura h, nagata i, tokuyama k: pathogenic factors re s p o n s i- ble for glucose tolerance in patients with niddm. d i a b e t e s : – , . nagasaka s, tokuyama k, kusaka i, hayashi h, rokkaku k, nakamura t, kawakami a, higashiyama m, ishikawa s, saito t: endogenous glucose pro d u c t i o n and glucose effectiveness in type diabetic subjects derived from stable-labeled mini- mal model approach. d i a b e t e s : – , . best jd, kahn se, ader m, watanabe rm, ni t-c, bergman rn: role of glucose eff e c- tiveness in the determination of glucose tol- erance. diabetes care : – , . rossetti l, giaccari a, defronzo ra: glu- cose toxicity. diabetes care : – , . taniguchi a, nakai y, doi k, fukuzawa h, fukushima m, kawamura h, to k u y a m a k, suzuki m, fujitani j, tanaka h, nagata i: insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis. m e t a b o l i s m : – , . world health organization: diabetes melli - tus: report of a who study gro u p . g e n e v a , world health org., (tech. rep. ser. , no. ) . winer bj: statistical principles in experimen - tal design. nd ed. new york, mcgraw-hill, , p. – late-onset tro g l i t a z o n e - i n d u c e d hepatic dysfunction r e c e n t l y, iwase et al. ( ) re p o rted a case of liver dysfunction occurring after months of troglitazone therapy. because it was thought before this re p o rt that the risk of liver dysfunction with tro g l i- tazone after months was negligible, we wish to re p o rt another patient who took t roglitazone intermittently and developed hepatic dysfunction after months. a -year-old white man with type diabetes, ischemic heart disease (post angioplasty and stent placement), hyper- tension, degenerative joint disease, benign p rostatic hypert ro p h y, dyslipidemia, and g a s t roesophageal re flux disease had tro g l i- tazone mg daily added to his re g i m e n of glimeperide mg daily and metform i n mg b.i.d. because of poor glycemic c o n t rol (hba c . % [normal – %]). the other medicines he used were aspirin and pravastatin. after months of triple oral therapy, his hba c level dropped to . %, and, after months, to . %. after months, the patient’s hba c began to rise: . % at months, . % at year, and . % at months. liver function tests were normal until months, when his aspartate amino- transferase (ast) was found to be (nor- mal – u/l) and alanine aminotrans- ferase (alt) (normal – u/l). the t roglitazone regimen was discontinued, and testing for hepatitis b and c, h e m a c h romatosis, autoimmune liver dis- ease, and gallbladder disease were nega- tive. two months after discontinuing t roglitazone, the patient’s ast and alt had decreased to and u/l, and, after months, had re t u rned to normal at and u/l, re s p e c t i v e l y. his ast and alt have remained normal since then and he has continued to take pravastatin, aspirin, m e t f o rmin, and glimeperide. when the patient was told to discon- tinue troglitazone, he admitted that he had been taking it only interm i t t e n t l y. he esti- mated that he took the drug regularly at first, but after the first months, he took the drug only once or twice weekly on aver- age. he gave the following three reasons for his lack of compliance: a lack of funds, a fear of liver disease, and a tendency to avoid taking drugs whenever possible. this case, like the case described by iwase et al., illustrates that the hepatic dysfunction caused by troglitazone can occur after months and further sup- p o rts the u.s. food and drug administra- t i o n ’s current recommendation that quar- terly liver function tests should be obtained when troglitazone utilization extends beyond year ( ). in this case, could the onset of hepatic dysfunction have been delayed because the diabetes care, volume , number , january letters d rug was being taken only interm i t t e n t l y after the first months, and the estimated total load presented to the liver would be equivalent to the exposure at months in a compliant patient? we doubt this, since t ro g l i t a z o n e ’s hepatic effects are thought to be idiosyncratic and there f o re the total e x p o s u re should be irre l e v a n t . david s.h. bell, mb fernando ovalle, md f rom the division of endocrinology and metabo- lism, department of medicine, school of medicine, university of alabama, birmingham, alabama. a d d ress correspondence to david s.h. bell, mb, th ave. s., birmingham, al . d.s.h.b. and f.o. have served on an advisory panel for sankyo parke-davis and have received con- sulting fees, re s e a rch grant support, and honoraria for speaking engagements from sankyo parke-davis. r e f e re n c e s . iwase m, yamaguchi m, yoshinari m, oka- mura c, hirahashi t, tsuji h, fujishima m: a japanese case of liver dysfunction after months of troglitazone tre a t m e n t . diabetes care : – , . parke-davis, division of wa rn e r- l a m b e rt : rezulin package insert. morris plains, nj, g l y b u r i d e - i n d u c e d hemolysis in m y e l o d y s p l a s t i c s y n d ro m e g lyburide, also known as gliben- clamide, is a widely used sulfonylure a to treat patients with type diabetes. hemolytic anemia is an extremely rare side e ffect of which there have been only a few re p o rts ( – ). we describe a patient with myelodysplastic syndrome who pre s e n t e d with glyburide-induced hemolysis. a -year-old man with a long history of type diabetes presented with left foot cellulitis of week’s duration. this patient was known to have slowly pro g re s s i v e pancytopenia for years, for which no work-ups had been perf o rmed. his med- ications included the following: glyburide, mg per day, which he had taken for m o re than year; buformine, mg per day; and boglibose, . mg per day. he was afebrile, and the physical examination was normal, except for localized cellulitis on his left foot, for which he was start e d on intravenous antibiotics. the laboratory studies revealed a white blood cell count of . / l , hemoglobin . g/dl, platelet count /l, reticulocyte count . %, mean cor- puscle volume fl, moderate anisocyto- sis, fasting plasma glucose mg/dl, h b a c . %, lactate dehydrogenase iu/l, total bilirubin . mg/dl, and hapto- globin mg/dl. red cell glucose- - phosphate dehydrogenase level was ade- quate. cold agglutinin test, ham’s test, and sugar water test were normal. both dire c t and indirect coombs’ tests were negative. red cell resistance to osmolarity was mildly low (parpart ’s method). urinalysis demonstrated no urobilinogen. endo- scopic studies did not reveal gastro i n t e s t i- nal bleeding. ultrasonography of the abdomen showed no splenomegaly. the result of bone marrow aspiration was equivocal. on the basis of pre s u m p t i v e glyburide-induced hemolysis, glyburide was discontinued and the patient was switched to subcutaneous insulin on the seventh day. there a f t e r, his hemoglobin level increased to . g/dl, re t i c u l o c y t e count decreased to . %, and anisocytosis d i s a p p e a red pro m p t l y. he was discharg e d with insulin therapy after month in the hospital, which is when the cellulitis re s o l v e d . t h ree months later, his hemoglobin level was . g/dl and his haptoglobin remained low. repeated bone marro w aspiration confirmed the diagnosis of myelodysplastic syndro m e . we conclude that this patient devel- oped glyburide-induced hemolysis super- imposed on red cell fragility secondary to an underlying bone marrow disord e r. t h e re have been several re p o rts of hemoly- sis caused by sulfonylureas, most of which have been considered immune-mediated ( , , ). our case points to the possibility that glyburide could cause hemolysis by a non–immune-medicated mechanism. it is i m p o rtant to be aware of this potential side e ffect of glyburide in light of this medica- t i o n ’s widespread prescription, even though such a side effect is rare . hiroshi noto, md kazuhisa tsukamoto, md satoshi kimura, md f rom the department of diabetes and metabolism, tokyo university hospital, tokyo, japan. a d d ress correspondence to hiroshi noto, md, d e p a rtment of diabetes and metabolism, tokyo uni- versity hospital, - - hongo, bunkyo-ku, to k y o - , japan. e-mail: noto-tky@umin.ac.jp. r e f e re n c e s . nataas ob, nesthus i: immune haemolytic anaemia induced by glibenclamide in selective iga defic i e n c y. b m j : – , . abbate sl, hoogwerf bj: hemolytic ane- mia associated with sulfonylurea use. d i a - betes care : – , . meloni g, meloni t: glyburide-induced acute haemolysis in a g pd-defic i e n t patient with niddm. br j haematol : – , . kopicky ja, packman ch: the mechanism of sulfonylurea-induced immune hemoly- sis: case re p o rt and review of the literature . am j hematol : – , e ffects of exposure at an altitude of , m on p e rf o rmance of glucose meters s elf-monitoring of blood glucose is m a n d a t o ry for type diabetic p a t i e n t s who participate in sports to adjust insulin dose and carbohydrate ingestion ( ). sports also include activities p e rf o rm e d at moderately high altitudes, such as hiking or skiing. capillary blood glucose monitors (bgms) have been shown to underestimate blood glucose values at an altitude of , m ( ) and at a simulated altitude of , m with t e m p e r a t u re and humidity kept constant ( ). the aim of the present study was to assess the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and p o can result in errors in blood glucose d e t e rmination ( ). two bgms, the lifescan one touch ii (ot) (ortho diagnostics, milpitas, ca) and the glucometer elite ii (ge) (bayer diagnostics, brussels, belgium), were tested during a study on the effects of acute exposure at an altitude of , m and exercise on blood pre s s u re and albu- min excretion rate in six type diabetic p a t i e n t s . all subjects (four men and two women) were free of disease-related com- plications and in good and stable glycemic control (ghb . ± . %). all subjects gave their informed and written consent to participate in the study pro t o- col. all subjects were investigated both at diabetes care, volume , number , january letters sea level and after ascent by car and cable car to the angelo mosso institute at col d’olen ( , m altitude), gressoney la trinité, italy. at sea level and at a moderately high altitude, bgm reliability at diff e rent blood glucose levels was tested, and blood glu- cose was assessed in fasting and re s t i n g conditions at : a.m.; at : a.m. b e f o re an in-field exercise test; and imme- d i a t e l y, min, and min after the exer- cise stopped. capillary glucose was simul- taneously assessed with the ot and the ge. both of these bgms measure capillary blood glucose through the glucose oxi- d a s e - p e roxidase reaction. bgms were cali- brated at the beginning of each test ses- sion. a venous blood sample was simulta- neously drawn from the contralateral antecubital vein in a sodium fluoride tube, centrifuged, and stored at °c. plasma glucose was assayed with the glucose oxi- dase method (go) within days. this last assessment was taken as a re f e re n c e method. statistical analysis compare d bgm capillary glucose values and go plasma glucose values for each blood col- lection time. measurement linearity was tested with pearson’s correlation coeff i- cient. the mean of the diff e rences between the bgm and go results re p resents the mean bias between the methods with accuracy expressed as percent error (pe): pe (%) = bmg – go % g o the level of statistical significance was c o n s i d e red to be p . . the ge and ot measurements had a good correlation with plasma glucose both at moderately high altitude and at sea level. p e a r s o n ’s correlation coefficients were . and . for the ge and . and . for the ot at sea level and at moderately high altitude, re s p e c t i v e l y. biases between plasma glucose and bgm measure m e n t s w e re as follows: for the ge, . ± . at sea level and . ± . at moderately high altitude; for the ot, . ± . at sea level and . ± . at moderately high alti- tude. at sea level, both the ge and the ot tended to underestimate glucose values (ns); at moderately high altitude, the ge tended to overestimate and the ot tended to underestimate glucose values (ns). mean pes between plasma glucose and bgm m e a s u rements were . (ot) and . (ge) at sea level and . (ot) and . (ge) at moderately high altitude. pe tended to be higher for both bgms at moderately high altitude (ns). figures and show the bias between the single measurements with both devices at sea level and at moderately high altitude, re s p e c t i v e l y. at moderately high altitude (fig. ), the tendency of the ge to o v e restimate was more evident for low ( mg/dl) and intermediate ( – mg/dl) blood glucose values, whereas the ot tended to underestimate mainly high blood glucose values (ns). in our study, bgm perf o rmance was similar and good at sea level. at a moder- ately high altitude, a tendency to overe s t i- mate blood glucose for the ge and to u n d e restimate for the ot was observ e d . the overestimation for the ge involved mainly low ( mg/dl) and interm e d i- ate ( – mg/dl) blood glucose val- ues. this could present a problem in the p resence of symptoms suggesting hypo- figure —relationship between plasma and capillary glucose at sea level. figure —relationship between plasma and capillary glucose at moderately high altitude. lifescan one touch ii glucometer elite ii lifescan one touch ii glucometer elite ii diabetes care, volume , number , january letters glycemia and normal blood glucose val- ues. the ot tended to undere s t i m a t e mainly high blood glucose values, although its perf o rmance with low to i n t e rmediate values was good. the pre s- ent study assessed the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and po can result in errors in blood glu- cose determination ( ). our results are consistent with previous studies ( , ). the decrease in po could alter the sec- ond phase of the chromogen reaction and u n d e restimate blood glucose values ( ); on the other hand, an increase in atmos- pheric pre s s u re could overestimate blood glucose values ( ). in our study, minimal o v e restimation by the ge at low interm e- diate blood glucose values at moderately high altitude cannot be explained by the a l t e red po . an increase in hematocrit, which is known to alter blood glucose m e a s u rements with bgms ( ), may also occur after prolonged exposure to high altitude or as a consequence of dehydra- tion. although our study did not deter- mine hematocrit, the exercise test was s h o rt, and the patients were instructed to drink according to their thirst during the , -m exposure; there f o re, dehydration was not likely to have occurred. in con- clusion, bgm perf o rmance is similar and good at sea level. at a moderately high altitude similar to that experienced during winter skiing or summer hiking, a ten- dency to overestimate low to norm a l blood glucose values for the ge and to u n d e restimate high blood glucose values for the ot was observed. the bias is not clinically meaningful for either bgm, both of which can be safely used by dia- betic patients during exposure to moder- ately high altitudes. some care in the eval- uation of low and intermediate blood glu- cose values measured with the ge is n e v e rtheless re c o m m e n d e d . oriana pecchio, md simona maule, md marco migliardi, md marina trento, bsc massimo veglio, md f rom the italian alpine club medical commission ( o . p.); the department of internal medicine (m.t. ) , university of turin; the s. giovanni battista hospital (s.m.); and the department of endocrinology (m.m., m . v.), mauriziano umberto i hospital, turin, italy. a d d ress correspondence to dr. m. veglio, via mancini , torino, italy. e-mail: veglio@ o n w. n e t . r e f e re n c e s . h o rton es: role and management of exer- cise in diabetes mellitus. diabetes care : – , . g i o rdano bp, trash w, hollenbaugh l, dube wp: perf o rmance of seven blood glucose testing systems at high altitude. diabetes educ : – , . gautier jf, bigard ax, douce p, duvallet a, cathelinau g: influence of simulated alti- tude on the perf o rmance of five blood glu- cose meters. diabetes care : – , . b a rnett c, ryan f, ballonoff l: effect of altitude on the self monitoring of blood glucose (smbg) (abstract). diabetes (suppl.): a, . piepmeier eh, hammett-stabler c, price me, kemper gb, davis mg: atmospheric p re s s u re effects on glucose monitoring devices (letter). diabetes care : – , . b a rreau pb, buttery je: effect of hematocrit concentration on blood glucose value d e t e rmined on glucometer ii. diabetes care : – , c o m m e n t s a n d r e s p o n s e s deterioration of glycemic contro l after long-te rm treatment wi t h troglitazone in nonobese type diabetic patients t roglitazone is an oral antidiabetic d rug used to treat type diabetic patients with insulin re s i s t a n c e . troglitazone improves overall insulin sen- sitivity in the liver and skeletal muscles, which are the largest consumers and metabolizers of glucose in the body ( – ). recent re p o rts showed that troglitazone is also effective in nonobese type diabetic patients whose hyperglycemia could not be controlled with sulfonylurea therapy ( , ). however, we aware that in some patients in whom adequate glycemic con- t rol is obtained during the first several months of troglitazone treatment, their glycemic control deteriorates several months later. we assume that two distinct g roups of type diabetic patients exist who respond diff e rently to long-term administration of troglitazone, one gro u p that maintains a steady response and another group that has a decre a s i n g response after certain periods. in this s t u d y, we re t rospectively examined patients with type diabetes who were t reated with troglitazone for months and whose hba c levels had improved by % with troglitazone by month . in of the patients ( %), hba c levels increased by . % after – months despite continuous tro g l i t a z o n e t reatment (group p). in contrast, the rest of the patients experienced steady glycemic c o n t rol with . % of hba c flu c t u a t i o n ( g roup g). during the first months, h b a c levels decreased from means ± sem . ± . to . ± . % in group p and fro m . ± . to . ± . % in group g, re s p e c- t i v e l y. no significant diff e rences were evi- dent between the two groups re g a rding the d e c rease in hba c during the first months (fig. ). from month onward, hba c l e v- els in group p climbed gradually by . % a month up to the baseline level at month , but hba c levels were stable in group g t h roughout the treatment period. a signifi- cant diff e rence in hba c levels was evident during months – (p . ) . among clinical characteristics, gro u p p had a significantly lower bmi ( . ± . vs. . ± . kg/m , p . ) and s i g n i ficantly lower fasting insulin levels ( . ± . vs. . ± . µu/ml, p . ). of the patients with a bmi of kg/m ( %), exhibited deteriora- tion of glycemic contro l . in this study, we re p o rt a group of patients who showed a renewed decline in glycemic control after long-term tre a t m e n t with troglitazone. these results seemed to suggest a secondary failure of tro g l i t a z o n e . our study demonstrates that this drug is indeed useful for a long-standing obese i n s u l i n - resistant diabetes but not for a nonobese type diabetes. yuko murase, md takanobu wakasugi, md kunimasa yagi, md hiroshi mabuchi, md f rom the department of internal medicine (y. m . , t. w.), fukui perfectural hospital; and the second d e p a rtment of internal medicine (k.y., h.m.), kanazawa university, ishikawa, japan. a d d ress correspondence to yuko murase, md, the second department of internal medicine, kanazawa university, - takara-machi, kanazawa, ishikawa - , japan. e-mail: diabe@med. k a n a z a w a - u . a c . j p . diabetes care, volume , number , january letters r e f e re n c e s . suter sl, nolan jj, wallace p, gumbiner b, olefsky jm: metabolic effects of new oral hypoglycemic agent cs- in niddm subjects. diabetes care : – , . o’rourke cm, davis ja, saltiel ar, corn i- celli ja: metabolic effects of troglitazone in the goto-kakizaki rat, a non-obese and n o rmolipidemic rodent model of nonin- sulin-dependent diabetes mellitus. m e t a b - o l i s m : – , . troglitazone study group: the metabolic e ffects of troglitazone in non-insulin dependent diabetes (abstract). d i a b e t e s (suppl. ): a, . mori k: the effect of troglitazone in combi- nation with sulfonylurea in non-insulin dependent diabetes mellitus (abstract). j japan diabetes soc (suppl. ): , . h o rton es, venable tc, whitehouse f, the troglitazone study group, ghazzi mn, whitcomb rw: troglitazone in combina- tion with sulfonylurea re s t o res glycemic c o n t rol in patients with type diabetes. diabetes care : – , figure —change from baseline in hba c. values are means ± sem. correction for kessler et al., de novo mutations across , diverse genomes reveal mutational insights and reductions in the amish founder population correction genetics correction for “de novo mutations across , diverse genomes reveal mutational insights and reductions in the amish founder population,” by michael d. kessler, douglas p. loesch, james a. perry, nancy l. heard-costa, daniel taliun, brian e. cade, heming wang, michelle daya, john ziniti, soma datta, juan c. celedón, manuel e. soto-quiros, lydiana avila, scott t. weiss, kathleen barnes, susan s. redline, ramachandran s. vasan, andrew d. johnson, rasika a. mathias, ryan hernandez, james g. wilson, deborah a. nickerson, goncalo abecasis, sharon r. browning, sebastian zöllner, jeffrey r. o’connell, braxton d. mitchell, national heart, lung, and blood institute trans-omics for precision medicine (topmed) consortium, topmed pop- ulation genetics working group, and timothy d. o’connor, which first published january , ; . /pnas. (proc. natl. acad. sci. u.s.a. , – ). the authors note that a data error resulted in incorrect values for the mer count totals shown in fig. b and in fig. s of the si appendix. this error does not affect the conclusions of the article. the corrected fig. and its legend appear below. the si appendix has been corrected online to show the corrected fig. s . c>tt>a t>cc>gc>at>g mutation type mers * * * * * * t>g t>a c>a c>g t>c c>t c o u n t g t c .g g c g c g .g g g g ta .g g a t c g .t a g t c g .t g g c ta .c g a g ta .g a a a t c .a g c c ta .c a a a c g .a a g c c g .c a g a c g .a g g c c g .c g g t t g .t a g g t t. g g t c t c .c g c g t c .g a c g t t. g a t c t c .c a c g t g .g a g c t t. c a t g c g .g a g t t c .t a c c t g .c a g g t g .g g g t t c .t g c a ta .a g a g c a .g g a g c c .g g c t ta .t g a c t t. c g t c t g .c g g g c t. g a t a c c .a g c a t t. a g t a t g .a g g c c t. c a t t t g .t g g a c t. a a t c c a .c g a g c t. g g t c c c .c a c t c c .t a c t c a .t a a a ta .a a a a t t. a a t c c a .c a a g t c .g c c g c a .g a a t c c .t g c a t g .a a g g c c .g a c c c c .c g c a t c .a a c t c a .t g a t t t. ta t c t c .c c c t ta .t a a c c t. c g t a c a .a g a a c t. a g t a c a .a a a t c t. ta t t t c .t c c c ta .c c a t t t. t g t a c c .a a c t t g .t c g a t c .a c c g ta .g c a g t g .g c g g t t. g c t c t t. c c t t c t. t g t t ta .t c a c t g .c c g g c c .g t c g c a .g ta c c c .c t c g c t. g t t t c c .t t c t c a .t ta a c c .a t c t t t. t c t t c t. t t t a c t. a t t c c a .c ta c c t. c t t a c a .a ta a t t. a c t a t g .a c g t c g .t t g a ta .a c a g c g .g t g c c g .c t g a c g .a t g * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * a b fig. . distribution of single-base and -mer mutation types across snv dnm call set. (a) the distribution of single-base mutation type counts across our snv dnm call set is shown. colors represent mutation type, and stars represent associations with paternal age (red, p < . after bonferroni correction). (b) the counts across our dnm call set for each of -mer mutation types is shown. colors represent the center base mutation, and are the same as those in a. stars represent associations with paternal age (red, p < . after bonferroni correction). published under the pnas license. published march , . www.pnas.org/cgi/doi/ . /pnas. pnas vol. no. e https://doi.org/ . /pnas. | of c o r r ec ti o n d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , https://www.pnas.org/site/aboutpnas/licenses.xhtml https://www.pnas.org/cgi/doi/ . /pnas. https://doi.org/ . /pnas. https://doi.org/ . /pnas. de novo mutations across , diverse genomes reveal mutational insights and reductions in the amish founder population michael d. kesslera,b,c,d, douglas p. loescha,b,c, james a. perryb,c, nancy l. heard-costae,f, daniel taliung, brian e. cadeh,i, heming wangh,i, michelle dayaj, john zinitik, soma dattak, juan c. celedónl, manuel e. soto-quirosm, lydiana avilam, scott t. weissk,n, kathleen barnesj, susan s. redlineh,o,p, ramachandran s. vasanf, andrew d. johnsonf,q, rasika a. mathiasr,s, ryan hernandezt, james g. wilsonu, deborah a. nickersonv, goncalo abecasisw, sharon r. browningx, sebastian zöllnery,z, jeffrey r. o’connellb,c, braxton d. mitchellb,c,aa, national heart, lung, and blood institute trans-omics for precision medicine (topmed) consortium , topmed population genetics working group , and timothy d. o’connora,b,c,d, ainstitute for genome sciences, university of maryland school of medicine, baltimore, md ; bdepartment of medicine, university of maryland school of medicine, baltimore, md ; cprogram for personalized and genomic medicine, university of maryland school of medicine, baltimore, md ; duniversity of maryland marlene and stewart greenebaum comprehensive cancer center, university of maryland school of medicine, baltimore, md ; edepartment of neurology, boston university school of medicine, boston, ma ; fframingham heart study, framingham, ma ; gdepartment of biostatistics and center for statistical genetics, university of michigan school of public health, ann arbor, mi ; hdivision of sleep and circadian disorders, brigham and women’s hospital, boston, ma ; iprogram in medical and population genetics, broad institute, cambridge, ma ; jdepartment of medicine, university of colorado denver, aurora, co ; kchanning division of network medicine, department of medicine, brigham and women’s hospital, boston, ma ; ldivision of pediatric pulmonary medicine, university of pittsburgh school of medicine, pittsburgh, pa ; mdepartment of pediatrics, hospital nacional de niños, san josé, costa rica; ndepartment of medicine, harvard medical school, boston, ma ; odivision of sleep medicine, harvard medical school, boston, ma ; pdivision of pulmonary, critical care, and sleep medicine, beth israel deaconess medical center, boston, ma ; qpopulation sciences branch, division of intramural research, national heart, lung and blood institute, the framingham heart study, framingham, ma ; rdivision of allergy and clinical immunology, the johns hopkins school of medicine, baltimore, md ; sbloomberg school of public health, the johns hopkins university, baltimore, md ; tquantitative life sciences, mcgill university, montreal, qc h a og , canada; udepartment of physiology and biophysics, university of mississippi medical center, jackson, ms ; vdepartment of genome sciences, university of washington, seattle, wa ; wschool of public health, university of michigan, ann arbor, mi ; xdepartment of biostatistics, university of washington, seattle, wa ; ydepartment of biostatistics, university of michigan, ann arbor, mi ; zdepartment of psychiatry, university of michigan, ann arbor, mi ; and aageriatrics research and education clinical center, baltimore veterans administration medical center, baltimore, md edited by stephen t. warren, emory university school of medicine, atlanta, ga, and approved december , (received for review february , ) de novo mutations (dnms), or mutations that appear in an individual despite not being seen in their parents, are an important source of genetic variation whose impact is relevant to studies of human evo- lution, genetics, and disease. utilizing high-coverage whole-genome sequencing data as part of the trans-omics for precision medicine (topmed) program, we called , single-nucleotide dnms across , trios from an array of diverse human populations, and used them to directly estimate and analyze dnm counts, rates, and spectra. we find a significant positive correlation between local recombina- tion rate and local dnm rate, and that dnm rate explains a substan- tial portion ( . to . %, depending on the model) of the genome-wide variation in population-level genetic variation from k unrelated topmed samples. genome-wide heterozygosity does correlate with dnm rate, but only explains < % of variation. while we are underpowered to see small differences, we do not find significant differences in dnm rate between individuals of european, african, and latino ancestry, nor across ancestrally dis- tinct segments within admixed individuals. however, we did find significantly fewer dnms in amish individuals, even when com- pared with other europeans, and even after accounting for paren- tal age and sequencing center. specifically, we found significant reductions in the number of c→a and t→c mutations in the amish, which seem to underpin their overall reduction in dnms. finally, we calculated near-zero estimates of narrow sense herita- bility (h ), which suggest that variation in dnm rate is significantly shaped by nonadditive genetic effects and the environment. de novo mutations | amish | mutation rate | recombination | diversity de novo mutations (dnms) appear constitutively in an indi-vidual despite not being seen in their parents, and their identification and study are critically important to our un- derstanding of human genomic evolution ( – ). for example, it is necessary to understand the rate at which dnms accumulate in order to calibrate evolutionary models of species divergence. dnms are also implicated in many diseases, including rare genetic disorders ( , , ) and common complex diseases, such as au- tism and schizophrenia ( – ). early studies indirectly inferred mutation rate estimates from patterns of rare mendelian diseases author contributions: m.d.k., n.l.h.-c., b.e.c., m.d., j.c.c., m.e.s.-q., l.a., s.t.w., k.b., s.s.r., r.s.v., a.d.j., r.a.m., r.h., j.g.w., d.a.n., g.a., s.r.b., s.z., j.r.o., b.d.m., n.h.l.a.b.i.t.-o.f.p.m.t.c., t.p.g.w.g., and t.d.o. designed research; m.d.k., d.p.l., and j.r.o. performed research; m.d.k. and j.r.o. contributed new reagents/analytic tools; m.d.k., d.p.l., j.a.p., n.l.h.-c., d.t., b.e.c., h.w., m.d., j.z., s.d., j.c.c., m.e.s.-q., s.t.w., k.b., a.d.j., r.a.m., r.h., g.a., s.r.b., s.z., j.r.o., b.d.m., and t.d.o. analyzed data; and m.d.k. and t.d.o. wrote the paper. the authors declare no competing interest. this article is a pnas direct submission. this open access article is distributed under creative commons attribution-noncommercial- noderivatives license . (cc by-nc-nd). data deposition: raw sequence data are available through dbgap as part of the topmed program, and is available via study-specific accessions. these are: whole genome se- quencing and related phenotypes in the “framingham heart study” (phs ) and “nhlbi topmed, genetics of cardiometabolic health in the amish” (phs ), “nhlbi topmed: the genetics and epidemiology of asthma in barbados” (phs ), “nhlbi topmed: the cleveland family study (wgs)” (phs ), and “nhlbi topmed: the genetic epidemiology of asthma in costa rica” (phs ). centralized alignments and variant calls are also available via these dbgap accessions. after working with the topmed consortium, we have created dnm call-set files with chromosome, position, reference allele, and alternative allele, as well as meta data files with paternal age, maternal age, sequencing center, european ancestry proportion, african ancestry pro- portion, native american ancestry proportion, and self-reported ancestry. the cohort- specific files are available upon request. a complete list of the national heart, lung, and blood institute trans-omics for precision medicine (topmed) consortium can be found in si appendix. a complete list of the topmed population genetics working group can be found in si appendix. to whom correspondence may be addressed. email: timothydoconnor@gmail.com. this article contains supporting information online at https://www.pnas.org/lookup/suppl/ doi: . /pnas. /-/dcsupplemental. first published january , . – | pnas | february , | vol. | no. www.pnas.org/cgi/doi/ . /pnas. http://orcid.org/ - - - http://crossmark.crossref.org/dialog/?doi= . /pnas. &domain=pdf https://creativecommons.org/licenses/by-nc-nd/ . / https://creativecommons.org/licenses/by-nc-nd/ . / http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental mailto:timothydoconnor@gmail.com https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/cgi/doi/ . /pnas. or from dna substitution rates between species ( , , ). more recently, modern sequencing technologies have enabled the use of pedigree data to directly estimate the number of new mutations found across the genome ( – ). these pedigree-based studies have identified both paternal and maternal age effects ( , ), and most recently estimate contributions of . and . dnms per year of paternal and maternal age, respectively ( ). while these effects are often explained on the basis of dna replication errors, recent studies have found that dna repair processes are likely to be major contributors to the mutations that accrue in both paternal and maternal gametes ( , – ). some of this repair-associated mutation accumulation has been found to be due to maternal age- dependent dna damage in oocytes and to maternal age-dependent postzygotic mutation increases ( ). these studies have also iden- tified specific mutation patterns, such as c→g transversions, that strongly associate with dna double-strand breaks and repair ( , ). in addition, these repair-associated mutations have been found to cluster together in distinct patterns, to pre- dominate in certain genomic regions, and to be associated with recombination ( , , ), which has itself been shown to in- fluence mutation rates ( – ). other features have also been reported to influence variation in mutation rates across the genome. gc content was recently shown to directly increase single-nucleotide and structural mu- tation rates, and was also shown to increase recombination rates ( ). chromatin structure has also been shown to associate with dna mutations, with dna replication times associating signif- icantly with point mutations ( ), and nucleotide positioning found to significantly modulate mutation rate ( ). recent work has tied a number of these features together by providing reso- lute and individualized recombination maps, and using them to demonstrate a positive relationship between maternal age and the rates and locations of meiotic crossovers ( ). specifically, older mothers have increased recombination that also shifts to- ward late replicating regions and low gc regions, and numerous loci seem to genetically influence meiotic recombination. other recent work evaluates nucleotide content, histone and chromatin features, replication timing, and recombination to provide one of the clearest pictures to date of the factors that shape genome- wide variability in the human mutation rate ( ). with regard to base content, amos ( ) has proposed the “heterozygote instability” hypothesis, which challenges the assumption that population size and mutation rate are independent, and suggests their interdependence on the basis of heterozygosity. according to this hypothesis, the occurrence of gene conversion events at heterozygous sites during meiosis could locally increase mutation rates, and amos ( ) uses substitution rates to provide support for this. yang et al. ( ) test this hypothesis using parent–offspring sequencing of arabidopsis, rice, and honey bee, and show support for the relationship between heterozygosity and mutation rate by demonstrating an ∼ . -fold higher mutation rate in heterozygotes compared with homozygotes, and mutation occurring closer to heterozygous sites and crossover events. while identifying these mutational correlates have helped us better understand the biological processes that drive mutation, genetic estimates of mutation rate are one-half the magnitude of those originally inferred phylogenetically ( , – , – ). this has raised questions about the accuracy of these genetics esti- mates, as well as about the accuracy of human evolutionary time points calculated using phylogenetic estimates. while it has been proposed that the failure of genetic methods to account for postzygotic mutations in the parent might bias estimates down and partly explain this discrepancy ( ), recent work using sibling recurrence suggests only minor mutation rate estimate increases when accounting for a substantial portion of these mutations ( ). this discrepancy has also raised the possibility that muta- tion rates have evolved more rapidly than previously assumed, and that molecular clock-type analyses are therefore flawed ( , ). for example, analyses of base pair substitution patterns have identified mutational differences between human populations, and showed most notably that the rate of tcc→ttc transitions appears to have increased in europeans thousands of years ago for some finite period of time ( , ). these findings provide a rationale for how mutation rates might differ between human populations. however, since most studies of dnms have used data from small cohorts of individ- uals with predominantly european ancestry ( , , ), little is known about the role of dnms in the evolution and health of populations of predominantly non-european ancestry, and it is unclear whether dnm rates vary across different human pop- ulations. to address this and other questions about mutation, we used a high-coverage whole-genome sequencing (wgs) dataset generated by the national heart, lung, and blood institute (nhlbi) trans-omics for precision medicine (topmed) pro- gram ( ) to directly estimate and analyze dnm accumulation across multiple human ancestries and populations. after ana- lyzing genome-wide patterns of mutation using a call set of , single-nucleotide variant (snv) dnms, we compared dnm counts and rates across five topmed cohorts that rep- resent european, african, and native american (latino) an- cestry individuals, and that include amish individuals from a founder population with european ancestry. we also estimate the correlation between heterozygosity and snv dnm count, and then test whether mutation rate is a heritable trait in an- ticipation of using genome-wide association studies (gwas) to look for mutation rate-modifying loci. results topmed dataset and positive correlation between dnms and parental age. using wgs data for , individuals and their parents from the topmed initiative ( ), we identified a dnm call set and compared dnm accumulation rates across ancestral background (table ). the analyzed individuals belong to five topmed co- horts with varied ancestral backgrounds: ) the amish, which are an isolated european founder population; ) the barbados asthma genetics study (bags), which consists of individuals with pre- dominantly african ancestry; ) the cleveland family study (cfs), which consists of both european and african american individuals; ) the genetic epidemiology of asthma in costa rica and the childhood asthma management program, which are collectively referred to as the cra cohort and consist of admixed latino in- dividuals; and ) the framingham heart study (fhs), which con- sists of individuals with european american ancestry. for our analyses, we treated the cfs study as two separate cohorts (signi- fied as cfs_afr and cfs_eur, respectively). these make up our six analysis cohorts (amish, bags, cfs_afr, cfs_eur, cra, fhs) (table ). after removing samples with dnm counts that were extreme outliers (often due to pedigree errors) (si appendix), we were left with a dnm call set of , snvs across , significance here we provide the most diverse human de novo mutation call set to date, and use it to quantify the genome-wide re- lationship between local mutation rate and population-level rare genetic variation. while we demonstrate that the human single-nucleotide mutation rate is similar across numerous human ancestries and populations, we also discovered a re- duced mutation rate in the amish founder population, which shows that mutation rates can shift rapidly. finally, we find that variation in mutation rates is not heritable, which sug- gests that the environment may influence mutation rates more significantly than previously realized. kessler et al. pnas | february , | vol. | no. | g en et ic s https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental individuals. this equates to an average of about . ( % ci: . to . ) mutations per individual, which is consistent with previous findings ( , , ). these , individuals come from , independent nuclear families, and for our analysis we treated each child and their two parents as a trio. to control for any potential confounding effects from this sibling data, we repeated all of our analyses that focused on per individual dnm measures after randomly choosing one child per family. the results from these repeated analyses are qualitatively the same as those from our full analysis, and for simplicity, we only report results from analyses run with our full dataset (except when noted within our heritability models). using this call set, we calculated per sample mutation rate estimates as snv dnm count divided by the number of auto- somal bases with depth ≥ and quality ≥ , which serves as a good representation of the number of bases evaluated for dnms by our filter heuristic (see si appendix for additional details). this results in a mean snv dnm rate estimate of . × − ( % ci: . × − to . × − ) mutations per base pair per generation. while our dnm rate estimate is slightly lower than previous genetic estimates (see si appendix for additional details), our estimate is generally concordant with recent genetic estimates ( , , ), and lends support to the accuracy of our filtering approach. as expected based on previous studies ( , ), we found a highly significant association between dnm rate per individual and paternal age (linear regression, r = . , p < . × − ) (si appendix, fig. s a), which provides an addi- tional degree of validation for our approach and our call set. while the high correlation in our dataset between paternal and maternal age makes it difficult to evaluate the separate effect of each on dnm count, linear modeling does succeed in identifying significant paternal and maternal age effects that are consistent in magnitude with those of recent studies ( . mutations per year of father’s age, and . mutations per year of mother’s age) ( , , ). dnm totals per individual did not differ significantly on the basis of the sex of the individual for whom the dnms were called (si appendix, fig. s b), their year of birth (si appendix, fig. s c), or the age of their dna (i.e., the individual’s age) at the time of collection (si appendix, fig. s d). dnm mutation types and patterns. using variant effect predictor annotations ( ) from the topmed consortium ( ) for loss-of- function (lof) variants found within the genomes of the , single offspring trios, we found , lof variants (si appendix, fig. s ). these , offspring also have , dnms. looking at the intersection of these dnm and lof call sets, we found lof dnm mutations. therefore, . % of dnms are lof mutations, while . % of lof variants are dnms, which suggests that dnms contribute significantly each generation to the total number of segregating lof variants. using the set of , dnms that remain after counting only once those dnms that are recurrent in siblings, and mapping our dnm set over to hg so as to be compatible with recent genetic maps, we found that , ( . %) dnms are in coding bases, which is consistent with the proportion of the genome comprised of coding sequence. we then used this set of , dnms to evaluate mutational patterns across the genome. consistent with previous findings ( , ), the most frequent mutation types in our dnm call set are c→t ( . %) and t→c ( . %) transitions (fig. a and table s ). all mutations showed robust associations with pa- ternal age, which we used as a proxy for the total parental age effect due to the previously described confounding between pa- ternal and maternal age (fig. a, red stars). while dnm-type composition is similar across the genome (si appendix, fig. s ), regions with significant deviations from the mean may serve as good candidates for the identification and investigation of atypical mu- tational processes (chromosomes [chrs] , , , and ) (si ap- pendix, fig. s b). the influence of base context on mutational frequency can be better appreciated when viewing each dnm as a -mer by considering the bases in the human reference genome immediately preceding and following each mutation ( , , , , ) (fig. b). for example, t→c mutations preceded by an a appear to be more common than might have been predicted based on their central base pair mutation-type alone. while cpg to tpg transitions already comprise four of the five most common -mer dnms, their mutational potential is particularly highlighted by normalizing each -mer dnm count for background -mer frequency, which demonstrates an excess of cpg to tpg transitions com- pared to the expectation based on genome frequency (si appendix, table . cohort characteristics and mutation estimates study name topmed project no. of children (after outlier removal) no. of nuclear families (after outlier removal) average paternal age at conception (years ± sd) average maternal age at conception (years ± sd) populations mutation rate mutation rate % ci parental age effect parental age effect % ci variance explained amish genetics of cardiometabolic health in the amish ( ) ( ) . ± . . ± . old order amish large extended pedigrees . e- . e- , . e- . . , . . bags barbados asthma genetics study ( ) ( ) . ± . . ± . african ancestry (from barbados) . e- . e- , . e- . . , . . cfs_afr the cleveland family study ( ) ( ) . ± . . ± . african american . e- . e- , . e- . . , . . cfs_eur the cleveland family study ( ) ( ) . ± . . ± . european american . e- . e- , . e- . . , . . cra genetic epidemiology of asthma in costa rica (gacrs), childhood asthma management program (camp) ( ) ( ) . ± . . ± . costa rican (latino/ hispanic) . e- . e- , . e- . . , . . fhs whole genome sequencing and related phenotypes in the framingham heart study ( ) ( ) . ± . . ± . european american . e- . e- , . e- . . , . . study cohorts and metadata are described. the six cohorts used in this study derive their names (“study name”) from five topmed projects (“topmed project”), and represent a diversity of populations and ancestries (“populations”). sample sizes are shown (“no. of children”) along with mean paternal and maternal age values per cohort (after the removal of dnm outliers). bags individuals have the highest average paternal age, which seems to explain their elevated dnm rate, and cfs_afr individuals have the lowest maternal ages. the estimated average mutation rate and % ci per cohort is also shown (calculated after removal of outliers), as are parental age effects (estimated using paternal age alone, due to confounding between paternal and maternal ages), and the proportion of dnm variance explained by this parental age effect after accounting for poisson variation. | www.pnas.org/cgi/doi/ . /pnas. kessler et al. https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/cgi/doi/ . /pnas. fig. s ). this postnormalization-inflated mutational pattern can also be seen for other cpg mutations (si appendix, fig. s , cpg to gpg in gold, and cpg to apg in salmon). to evaluate the relationship between mutation and recombina- tion, we used our dnm call set to estimate local mutation rate across -mb windows, and then tested the correlation between these rates and local recombination rate estimates derived from the recently published decode genetic map ( ). we found a significant positive correlation in which regions of higher recombination have more dnms (si appendix, fig. s a) (r = . , p = . × − ) (see si appendix for additional details). this relationship varies across the autosomes, with chrs , , , , and , , showing strong positive correlations in which recombination explains as much as % of the variation in dnm rates (si appendix, fig. s b). conversely, this correlation is either absent or limited across a number of the other autosomes. evolutionary theory predicts that mutation, natural selection, and genetic drift act in concert to shape genomic variation, and that recombination can shape variation by breaking up linked variants and enabling selection to act on them more efficiently ( , ). therefore, we tested the degree to which local recombination and mutation rate estimates explain the distribution of genomic vari- ation seen at the human population level. to do this, we used genomic variation data from the topmed consortium that was ascertained by performing wgs on ∼ k diverse unrelated in- dividuals ( ). we first calculated the total number of rare variants (af < . ) within -mb windows across the genome, and used standardized z-scores derived from these counts as a measure of localized levels of recent human genomic variation. local recombi- nation and mutation rates explain up to . % ( % ci: . to . ) of the local genomic variation in segregating rare variants (p < . × − ) (fig. ). as expected, dnm rate explains the majority of this signal, accounting for . % ( % ci: . to . ) of the variation in rare variants after regressing out the effects of local recombination rate (p < . × − ). regions with the highest levels of rare variation, such as megabases to on chr and to and to on chr , have high dnm rates, and are largely com- prised of regions in the top % of recombination values (fig. ). when including both gc content and replication timing in the model as covariates, we can explain up to . % ( % ci: . to . ) of the variation in rare variant totals. even after adjusting for recombination rate, gc content, and replication timing, which is conservative given the interconnected relation- ships between these variables, dnms still explain . % ( % ci: . to . ) of the variation in rare variants. similar models per chromosome can explain between % and % of the vari- ation in rare variant totals across chromosomes that have seg- ments with high local dnm rates (chrs , , , and ) (table s ), with dnm rate as the dominant explanatory variable. in- terestingly, replication timing seems to best explain variation in rare variant levels across chrs and . in contrast to these rare variant models, when we ran similar models with standardized z-scores derived from the counts of common variants as our dependent variable, the most dominant explanatory variable is recombination rate. that is, when using recombination rate, gc content, replication timing, and dnm rate as covariates to predict the distribution of common variation across the genome, we explain a similar proportion of the vari- ation in common variants as we did in rare variants (r = . , % ci: . to . ), but the relative contributions of dnm rate and recombination rate seem to invert. in the former set of models of the genomic distribution of rare variation, re- combination plus replication timing explain . % ( % ci: . to . ) of variation, whereas dnm rate explains . % ( % ci: . to . ) of the remaining variation. conversely, in the models of the genomic distribution of common variation, recombination rate plus replication timing explain . % ( % ci: . to . ) of variation, whereas dnm rate explains only the remaining . % ( % ci: . to . ). this is consistent with evolutionary predictions, as common variants typically repre- sent older variants, and their distribution has been shaped more by recombination, selection, or drift than by mutation. these patterns hold when using linear regression to implement an adjustment for coding proportion and mappability concerns, with each model (dnm rate, recombination rate, replication timing, and gc as covariates) explaining about % of the variation in common and rare variants, respectively. dnm rates still explain the majority of the variation in rare variant levels ( . %, % ci: . to . , after adjusting for recombination rate, replication timing, and gc content), and recombination rates still explain the largest portion of variation in common variant levels ( . %, % ci: . to . , after adjusting for dnm rate, replication timing, and gc content). the relationship between dnm rates and heterozygosity. in testing the relationship between heterozygosity and dnm rate across all individuals, we found a significant positive correlation (p < . ) (si appendix, fig. s a). however, while this persists after adjusting for parental age (p < . ), heterozygosity only ex- plains ∼ . % of the variation in dnm count. this relationship is also entirely driven by the amish population, and no significant relationship persists when removing amish individuals from the analysis that adjusts for parental age (p > . ). furthermore, we found no relationship between heterozygosity and dnm count when looking intracohort (see si appendix, fig. s b for a repre- sentation of this across individuals from the fhs, our largest cohort). the number of kilobases in an individual’s genome found within runs of homozygosity (roh) also correlates significantly with dnm rate (p < . × − ) (si appendix, fig. s a) and persists after adjusting for parental age (p < . × − ). however, as was the case with heterozygosity, this is entirely driven by the amish, who happen to be outliers for roh. similar to hetero- zygosity, additional analysis suggests that this association is confounded and not reflective of a causal relationship. roh only explains ∼ % of the variation in dnms, and its association with dnms is no longer significant after filtering out the amish (p > . , r = . ) or when evaluating intracohort (p ≥ . ). dnm rate comparisons across ancestrally diverse cohorts. when comparing dnm rate across all six ancestrally diverse cohorts (table ), we found significant differences (p < . × − , anova) (fig. fig. . distribution of single-base and -mer mutation types across snv dnm call set. (a) the distribution of single-base mutation type counts across our snv dnm call set is shown. colors represent mutation type, and stars represent associations with paternal age (red, p < . after bonferroni correction). (b) the counts across our dnm call set for each of -mer mutation types is shown. colors represent the center base mutation, and are the same as those in a. stars represent associations with paternal age (red, p < . after bonferroni correction). kessler et al. pnas | february , | vol. | no. | g en et ic s https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental and table ), even after accounting for parental age (p < . , anova). however, this appears to be driven by the amish, who have significantly fewer dnms per individual than the other cohorts (p < . between the amish and the four largest populations [bags, cfs_eur, cra, fhs] after correcting for multiple test- ing). a reduced dnm rate persists in the amish even when com- paring dnm rates across population within regions of the genome estimated not to be in roh in either parent’s genome (p < . , anova) (si appendix, fig. s b). therefore, while recent work suggests heterozygosity as a mutational driver, and increased roh in the amish is an appealing explanation for their reduced mutation rate, on the whole, differences in heterozygosity or roh do not seem to be sufficient to explain the reduced mutation rate in the amish. to control for sequencing center differences that could potentially influence the number of dnms identified, we performed a sub- analysis comparing dnms between cohorts that were sequenced at the same center. this subanalysis did not include individuals from the bags cohort, since they alone were sequenced directly by illumina. this within-sequence center analysis also revealed significantly lower dnm rates in the amish compared with indi- viduals from fhs (european ancestry; p < . , anova), even after adjusting for parental age effects (p < . , anova), whereas we found no significant differences in dnm rates before and after adjusting for parental age effects between individuals from cfs_eur (european ancestry), individuals from cfs_afr (african ancestry), and individuals from cra (latino ancestry; p > . , anova). while the bags cohort appears to have an elevated mutation rate at first glance, this is a result of them having higher average paternal ages (table ). while we are underpowered to see smaller differences in mutation rate, we have reasonable power to see moderate to large differences between all populations other than cfs_afr (si appendix, fig. s ), so these results do suggest that differences between ancestry thought to influence the dnm rate (such as heterozygosity, demographic history, and so forth) are not driving large differences in the accumulation of dnms. the amount of variation in dnm rate attributable to parental age effects after adjustment for poisson variation is lower in our dataset ( . %) than previously reported (> %) ( ). this seems to be due to the fact that there is heterogeneity in the parental age effects across our sample set (table ), which itself becomes a contributor to the variation in dnm rate. in accor- dance with this, and consistent with the reduced dnm rate we found in the amish, we estimate a lower parental age effect in the amish than in the other cohorts (table ). however, we have limited power to detect this difference, which only reaches sig- nificance when comparing effect sizes determined by poisson regression between the amish and fhs (p < . ). evaluation of batch effects and technical artifacts. to evaluate the robustness of the observation of a dnm rate reduction in the amish to technical artifacts, we took a multipronged approach. first, we validated the consistency of our dnm call set by using two offspring samples from the fhs cohort that had undergone repeat sequencing at another sequence center (university of washington [uw]), as well as one offspring sample from the amish cohort that has a monozygotic twin in our dataset. for these three samples, we evaluated the proportion of dnms that are called as heterozygous sites in the validation sample, as well as the concordance between dnms called in the initial and validation samples. in two of the three samples, % of dnms (one amish, one fhs) were called as heterozygous sites in the validation sample. in the third sample (fhs), of dnms were called as homozygous refer- ence in the validation sample. however, upon further inspection, all five of these dnms had read counts and genotype likelihoods suggesting that they were heterozygous sites. with regard to dnm concordance, . % of dnms were called in both the initial and dnm rate d e n si ty . e . e . e . e+ . e+ . e+ . e+ . e+ mean rate = . e mean rate = . e mean rate = . e mean rate = . e mean rate = . e mean rate = . e anova: p = . amish bags cfs_afr cfs_eur cra fhs fig. . dnm rates across diverse cohorts. dnm rates per individual show significant differences across cohort, which are driven by a reduction in the amish. fig. . city plot of rare variation, recombination rate, and dnm rate across the genome. the relationship between dnm rate (blue to red color range), rare variation (y axis, ranging from − . to . z-scores), and recombina- tion rate (z axis, ranging from . × − to . cm/mb) across the ge- nome (x axis, dotted vertical lines divide autosomes to ) is shown. in moving from low to high rare variation levels across the y axis, a blue to red gradient can be seen, which reflects the significant correlation between dnm rate and population-level rare variation. furthermore, regions with high dnm rates and high variation levels generally have taller bars, which reflects the positive relationship between dnm rate, variation level, and recombination (a few exceptions to this can be seen as taller blueish bars). regions with the highest variation levels in the genome, such as those on chromosomes and , have the highest dnm rates. | www.pnas.org/cgi/doi/ . /pnas. kessler et al. https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/cgi/doi/ . /pnas. validation samples. notably, this number is significantly higher ( . %) when excluding the five dnms noted above as being im- properly called as homozygous reference by the variant caller de- spite strong evidence of heterozygosity, which is unlikely to be a common issue. we also assessed how a number of quality control measures vary across population. specifically, we compared measures of average genome-wide sequence depth (si appendix, fig. s a), average depth of variants called autosome-wide and passing filters (si appendix, fig. s b), number of bases genome-wide with a minimum depth ≥ (si appendix, fig. s c), genome-wide ts/tv ratio (si appendix, fig. s d), number of million reads genome- wide per sample (si appendix, fig. s e), percent of the genome covered (si appendix, fig. s f), percent of the genome with a depth of (si appendix, fig. s g), average depth of genome-wide bases with q ≥ (si appendix, fig. s h), percent of the genome with q ≥ (si appendix, fig. s i), and the number of autosomal bases with depth ≥ and q ≥ (si appendix, fig. s j). on the whole, our study cohorts feature similar quality metrics, and the amish are not a notable outlier in any metric (si appendix, fig. s ). while the bags cohort seems to be a moderate outlier in ts/tv and number of reads (si appendix, fig. s d and e), and a more pronounced outlier with regard to percent genome covered and number of bases with q ≥ (si appendix, fig. s f–h), adjusting for these (as well as all of the other) quality control measures did not significantly influence our signal. similarly, when we used the number of bases per sample with depth ≥ + q ≥ to normalize our dnm counts into dnm rate estimates, as described earlier, we ac- tually saw an increased signal of a reduced dnm rate in the amish. dnm rates by ancestral proportions of individuals from admixed populations. to test more directly how interancestral differences might influence dnm accumulation, we used linear regression to assess the relationships between dnm rate and african ancestry proportion, and dnm rate and native american ancestry pro- portion. within the bags, cfs_afr, and cra individuals, which represent the three cohorts with significant african ances- try, we found no significant correlation between african ancestry proportion and dnm rate after adjusting for parental age (p > . , anova) (si appendix, fig. s a). this absence of a re- lationship persists when looking only within bags individuals (p > . ) or only within cra individuals (p > . ), and there was also no relationship when comparing native american an- cestry proportion and dnm rate before (p > . ) or after (p > . ) adjusting for parental age within latino ancestry individ- uals from the cra cohort (si appendix, fig. s b). since we are also underpowered to see small-to-moderate differences in single-base or -mer mutation frequencies between populations, we instead tested the correlation between -mer mutation type and european ancestry proportion within all indi- viduals of admixed african ancestry (bags, cra, cfs_afr cohorts). this should increase our power to detect mutational associations with ancestral background, and specifically allow us to test for a signal of ancestral association for the tcc→ttc -mer mutation that was previously shown to positively correlate with european ancestry ( , ). notably, we did not find a significant association between tcc→ttc -mer mutation and european ancestry (p ≥ . ). interestingly, when testing this association in individuals from bags, which is our cohort with the highest average proportion of african ancestry, we found a negative correlation (β = − . , % ci: − . to − . , p < . , uncorrected, poisson regression) (si appendix, fig. s ). overall, this result is interesting when contrasted with recent evidence that an ancestry-specific pulse increased the occurrence of these -mer mutations in europeans ( ). shifts in the mutational spectrum of the amish founder population. to further assess the reduced dnm rates seen in the amish, we compared dnm counts for each single-base mutation type across cohort. anova revealed differences in mutation counts across cohort, with differences in c→a (p < . ), t→c (p < × − ), and t→g (p < . × − ) mutations being most significant (si appendix, fig. s ). these differences are largely driven by de- creases in these mutations in the amish and increases in these mutations in individuals from the bags cohort. to further eval- uate these differences while controlling for sequencing center, we compared mutations between the amish and fhs cohorts, and found the reduced number of c→a (p < × − ) and t→c (p < × − ) mutations in the amish to persist (si appendix, fig. s ). interestingly, the reduction in t→c mutations explains about % of the overall dnm reduction seen in the amish. local ancestry analysis does not identify ancestrally distinct mutation rates. to look more closely at whether mutation rates differ between ancestries, we compared the rates of dnm ac- cumulation across ancestrally distinct genomic segments within admixed samples. using local ancestry assignments, we counted dnms across all possible diploid ancestral segment combina- tions (e.g., homozygous african, heterozygous african and eu- ropean, and so forth) (si appendix, fig. s ), and limited each comparison to individuals with at least mb of each diploid category. the resulting intraindividual comparisons allow for the control of unmeasured variables that may otherwise confound interindividual analyses, such as environmental exposures that might influence mutation rate. therefore, the only variable that should differ between diploid segments is their ancestral origin, which should allow us to isolate and test for any effects of this ancestral background on local sequence context (i.e., this is a test for cis effects). after estimating dnm rates per individual by normalizing dnm counts by diploid category base total (i.e., rates per base pair), we did not find evidence of ancestry specific differences in dnm rate (si appendix, fig. s ). while we did find a significant reduction in the mutation rate in african/european segments compared with african/african segments (p = . × − ), other comparisons between african and european seg- ments showed no differences. given the absence of consistent differences in dnm rate across local ancestral segments, these results do not provide compelling support for the existence of ancestry-based differences in dnm rate. the heritability of dnm accumulation. to assess whether we could use gwas to detect any genetic loci that might underpin the interindividual variation we see in dnm accumulation, we first wanted to test for what proportion of the variation in dnm accumulation was explained by genetics. one measure of this is narrow-sense heritability (h ), which represents the proportion of phenotypic variation explained by additive genetic effects ( ), and which can be used as a null model for running associations at every locus (i.e., each locus is tested for the proportion of h that it explains). treating the number of dnms per individual as a phenotype reflecting dnm accumulation, we used the mmap software ( ) to estimate the h of dnm accumulation via a restricted maximum likelihood-based (reml) method (si appendix). when using all samples across all cohorts, we used paternal age at offspring’s conception, maternal age at offspring’s conception, and cohort label as fixed effects, and a genetic relatedness matrix esti- mated from the sequence data. we estimate an h = . (se = . ), and we reaffirmed a paternal age effect (p < . × − ), a maternal age effect (p < . × − ), and an effect of amish cohort status (p < . × − ) (table and si appendix, table s ). when running this base model on each cohort separately, h estimates are zero for each cohort other than cra (h = . , p = . ) (table s ). however, the h result for cra was . when further restricting to only unrelated subjects (table s ). to evaluate whether the portion of mutational variation explained by parental age might be shaped by additive genetic factors, we repeated these heritability models without including kessler et al. pnas | february , | vol. | no. | g en et ic s https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental parental ages as fixed effects. interestingly, these models estimated nonzero heritability (h = . , p < . × − ), with the bags and cfs_eur cohorts contributing most significantly to this nonzero heritability estimate (tables s and s ). however, when repeating analyses with only one offspring randomly chosen per nuclear family (i.e., removing sibling data) (table s ), or with only unrelated samples (table s ), h is zero in nearly all models. exceptions include the amish and cfs_eur per cohort analy- ses, but these have notably small sample sizes as well as non- significant (p > . ) h estimates with wide ses. overall, this suggests that the significant nonzero heritability we see when not adjusting for parental age effects is driven by the shared parental contribution of siblings and or confounding due to relatedness, and not by genotypic similarity. while statistical power is a concern as a result of our sample sizes, we do have reasonable power (≥ . ) to detect moderate h across all samples as well as across larger co- horts and cohort combinations (si appendix, fig. s ). simulations across the known pedigree of nearly , amish individuals also suggest that we would only expect to estimate zero heritability across our samples if the true heritability was ≤ . (empirical p < . ) (si appendix, fig. s ). discussion here we call dnms across , individuals from diverse cohorts sequenced through the topmed program. our call set is de- termined by a filtering heuristic that is similar to previous ap- proaches ( ), its specificity is supported by a bayesian approach implemented in the triodenovo software ( ) that called . % of our dnms, and its overall accuracy is supported by validation sequencing across two repeated samples and one pair of monozygotic twins. in addition to this, quality assessments done as part of the topmed consortium sequence analysis efforts used repeatedly sequenced samples to demonstrate similar geno- typic concordance rates within and between sequence centers ( ). while this was done for both variants passing calling filters and variants failing calling filters, variants passing calling filters have even larger levels of concordance than those failing calling filters, which reflects the efficacy of these calling filters in identifying errors. this suggests that even the differences in some quality measures mentioned above, such as the average depth of q bases in bags, are already effectively handled by our variant calling quality control. this is directly in accordance with the facts that controlling for these covariates doesn’t qualitatively change our signal, and that using dp q metrics to estimate normal- ized per sample mutation rates in fact increases our signal of a reduced mutation rate in the amish. other topmed consortium results using principal components analysis to evaluate the influ- ence of sequence center on genotypic variance further support a very limited influence of sequencing center ( ). despite the predominance of c→t mutations among dnms, we did not find any differences between populations or ancestral backgrounds in c→t mutations. this is notable given the high frequency of this mutational class, and the concomitant in- creased power to detect differences. this suggests that processes driving cytosine deamination are fairly conserved, and is con- sistent with the assertion by others that cpg mutation rates may serve as a better “molecular clock” than the base substitution rates that have previously been used ( , , , ). similarly, we did not find differences in other single-base and -mer mutation types across ancestral background. while we did have somewhat limited statistical power to see smaller effect sizes (si appendix, fig. s ), we did have reasonable power to see moderate-to-large differ- ences when comparing most populations, and potentially even had good power to see small-to-moderate differences when comparing our larger populations. for example, when comparing dnm rates between the bags and fhs cohorts (our largest african ancestry and european ancestry cohorts, respectively), we had % power to see an effect size difference reflected by a cohen’s d of . . this represents an effect size small enough so that only . % of the dnm rates in the population with the larger dnm rate would be larger than the mean in the other population (cohen’s u ), there would only be a . % chance that a random individual from the population with the larger dnm rate has a higher dnm rate than a random person from the other population (probability of su- periority), and there would be an . % overlap between the dnm rate distributions of the two populations ( ). this is gen- erally considered a small-to-moderate effect size, and helps to appreciate what kind of dnm rate differences we have power to see. however, we likely only have this level of power to detect differences in the overall snv dnm rate (and possibly certain table . heritability model across all cohorts variable value variable value mean dnms . βpaternal_age p value . e- sd . βmaternal_age . minimum . βmaternal_age se . maximum . βmaternal_age p value . e- kurtosis . βamish − . dnms > sd . βamish se . dnms < sd . βamish p value . e- sample size , βbags . h . βbags se . h p value . βbags p value . h p value se . βcfs_afr . proportion variance explained by covariates . βcfs_afr se . adjusted proportion variance explained by vovariates . βcfs_afr p value . ln likelihood − , . βcra − . intercept . βcra se . intercept se . βcra p value . intercept p value . e- βfhs − . βpaternal_age . βfhs se . βpaternal_age se . βfhs p value . results are shown from heritability models run with mmap across all samples with paternal and maternal ages available (n = , ). heritability is estimated as zero (h = . ), with an se of . ). these models confirm that paternal age at offspring’s conception (p = . × − ), maternal age at offspring’s conception (p = . × − ), and amish cohort status (p = . × − ) are significantly correlated with dnm total per individual. | www.pnas.org/cgi/doi/ . /pnas. kessler et al. https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/cgi/doi/ . /pnas. single-base mutation rates), as we are notably more underpowered to detect differences in -mer rates due to their reduced count sizes. nonetheless, we did not see significant mutation rate dif- ferences between populations with distinct ancestral backgrounds, which suggests that overall and single-base mutation rate differ- ences are not likely to be large. when leveraging quantitative estimates of genetic ancestry within a framework with increased power to see potential differences, we also did not find significant associations between european ancestry pro- portion and -mer mutation type across individuals from admixed cohorts (i.e., bags, cfs_afr, cra). this result is interesting when contrasted with recent findings of europeans having had a significantly higher rate of tcc→ttc mutations in the past ( , ). first, the recent identification of a significant batch effect in the genomes data used to identify these mutational differences raises some questions about their legitimacy ( ). nevertheless, assuming tcc→ttc mutations did in fact increase in europeans at some point in the past, our results suggest that there is currently no difference, which is consistent with findings that the increase in europeans happened as a pulse that ended around , y ago ( ). it is also possible that differences in the rate of this mutation across populations has been influenced by differential shifts in mean parental age at conception, which is consistent with recent findings by jónsson et al. ( ) and agarwal and przeworski ( ) that this muta- tion is enriched for sex differences. ultimately, additional research is needed to better understand the evolutionary history of tcc→ttc and other mutations across divergent human populations. the correlations we found between local recombination and dnm rates are concordant with research showing that processes underpin- ning recombination are mutagenic ( , , ), and our models comparing population-level variation with recombination rates, dnm rates, replication timing, and gc content add further resolution to the relationship between these intertwined covariates and genomic evo- lution. areas of the genome with the highest local dnm rate are in regions with high recombination that have recently been identified as featuring specific recombination-based processes that drive mutation ( ). alternatively, regions with high recombination rate estimates that have low dnm rates (fig. , tall blue bars) are good candidates for the identification of genomic features that may explain a reduced mutation rate, including contexts in which recombination itself po- tentially drives less mutagenesis. while recent research into the re- lationship between dnms and fine-scale local recombination rates identified up to -fold increases in dnm rates around meiotic crossover events ( ), the fact that these events are rare is likely why we only explain about % of the variation in dnms on the basis of recombination. this recombination study also found loci influ- encing recombination rate, which may lead one to ask why we did not find any signal of genetic influence over dnm accumulation (i.e., we estimate h = ), given that recombination is mutagenic and associates with dnms in our dataset. first, this is likely due to the fact that recombination only explains a small portion of the overall variation in dnms, and small effect-size modulators of recombination are unlikely to be detected as correlates of dnm rate without very large sample sizes. second, we adjusted for parental age effects, which might eliminate from many of our heritability models the portion of varia- tion in dnm rate influenced by meiotic recombination. furthermore, multivariate models have been able to explain > % of the variation in regional mutation rates on the basis of genomic features, such as gc content, exon density, sex chromosome status, recombination, and distance to telomeres ( ). given that we excluded most centromeric/ telomeric segments from our analyses, didn’t consider exon density or sex chromosome status, and used metadata from multiple studies across multiple populations, we expected our model to explain a lower proportion of the variation in mutation rate than these models. nonetheless, these and other covariates potentially explain the exis- tence of high recombination regions with low dnm rates. via these models, we also estimated the quantitative contri- butions of mutation and recombination to population-level variation, and were able to explain up to % of the variation across the ge- nome in population-level rare variation using local recombination and dnm rates, and % when including replication timing as an additional covariate in the model. the remaining proportion of variation is likely the result of a combination of genetic drift, selection, or measurement error. of this explained variation, ∼ . % is attrib- utable to variation in local dnm rates. regions with high mutation rates have the most variation in the genome (e.g., chr , chr ), and often feature high recombination rates that likely increase mutation accumulation. numerous recent studies have found complementary results that describe the types of mutations predominating in high- variation regions, and identify the likely sources of these mutations ( , , ). some of this recent work demonstrated that clustered dnms contribute very significantly to the clustering of genomic snps (specifically c→g snps), although clustered dnms are es- timated to make up less than . % of dnms ( ) and may only have limited impact on the findings presented here. here, we show that single-nucleotide dnms contribute profoundly to the entire genomic distribution of common and rare single-nucleotide vari- ation, but that recombination is a larger driver of the variation in common variant levels than is mutation. this is consistent with evolutionary theory about the ability of recombination to discon- nect linked variants and enable selection to act more efficiently, as well as the expectation that common variants are older and have been subjected to nonmutational evolutionary forces for longer. while the heterozygosity instability hypothesis ( , , , ) and recent related findings ( ) predict that increasingly het- erozygous genomes will have higher mutation rates, we only found evidence of a modest relationship between heterozygosity and dnm rate. furthermore, we did not find the differences in dnm rate between ancestral background that differences in heterozygosity across ancestry would have predicted, nor a significant correlation between heterozygosity and dnm rate between samples from the same population. while runs of homozygosity do seem to shape dnm rates more so than levels of heterozygosity, this relationship is entirely driven by the amish, and additional analysis of mutation rate across non-roh–bearing regions of the genome suggests that this is likely due to confounding between the high roh in the amish and whatever else is reducing their mutation rate. therefore, while this finding of a potential relationship between roh and dnm rate does raise the possibility that the absence of hetero- zygosity may drive significant reductions in dnm rate, additional research is needed to more directly address this question. in our heritability analyses, we estimate the heritability of dnm rates to be zero across nearly all models. due to sample size con- cerns, we conducted power analyses and used simulations to esti- mate the likelihood of estimating zero heritability across increasing values of true heritability. for the full dataset, most power calcu- lation approaches (si appendix, fig. s a–d) suggest we have good power to detect low to moderate levels of heritability [i.e., (h ≥ . )], although we likely have very little power to detect her- itability across the smaller cohorts. similarly, given sample sizes similar to that of our full dataset, a trait with an h ≥ . will be estimated as zero ≤ % of the time. even in circumstances with limited power, we would still expect h estimates centered off of zero when working with traits that have true heritability greater than zero. therefore, given our simulation results, and that our h estimates are consistently centered around , we find it more likely that the true heritability of dnm rates across our dataset are low or approaching zero. while future initiatives might still consider large- scale gwas efforts in order to search for local mutation events under genetic control, and larger studies with increased power are needed to confirm the heritability of dnm rates, we did not find evidence that genetic similarity explains the variation in mutation rates we see within or between populations. despite being a founder population that diverged from other europeans only very recently ( ), the amish show a mutation rate reduction of about %. this reduction persists when controlling kessler et al. pnas | february , | vol. | no. | g en et ic s https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental https://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental for parental age effects and sequence quality metrics, and seems to be driven by reductions in c→a and t→c mutations. together with our estimation that dnm rate has zero narrow-sense heri- tability, this suggests that the environment may play a bigger role in modulating the mutation rate than previously appreciated. the amish lifestyle features preindustrial era aspects, and while modern amish communities are diverse and have adapted to the usage of some modern items, they continue to limit the influence of technology in their daily lives ( , ). given this, it is possible that the amish are exposed to fewer mutagens, and that this “clean living” may be partially responsible for the reduced mutation rate we report here. for example, studies have shown that rural areas, such as those similar to the areas occupied by the amish, have fewer carcinogens and mutagens than industrialized areas ( – ). recent analysis of mutation patterns has also called into question the canonical view that dnms rise predominantly from replicative errors, and suggests that exogenous mutagens may play a larger role in mutation accumulation than previously appreciated ( ). if the amish do in fact experience less environmentally driven mu- tagenesis, then one would predict a significant reduction in the rate of cancer in the amish. this is exactly what has been found in multiple old order amish populations, with a particularly large reduction in cancer rates found in men ( , ). a similar re- duction in overall mortality has been found in amish men com- pared with fhs men, which is hypothesized to be due to lifestyle factors, such as reduced tobacco use and increased physical activity ( ). given that dnm mutation in sperm is the single largest driver of dnm accumulation, an amish environment that potentially limits dna damage in amish men is consistent with a lower dnm rate. in accordance with this, the amish have the lowest estimated parental age effect (table ). this is also consistent with the recent finding of significant variability in parental age effects across an- cestrally similar families, which also suggests the possibility that environmental factors influence dnm rates ( ). it is important to note that considerations of the environment as a potential explanation for the reduced dnm rate we detect in the amish are entirely speculative, and that batch effects or other technical artifacts remain possible despite our significant efforts to control for them. nonetheless, our findings as well as the aforementioned context do suggest environmental influence as a possible explanation, and one that is worthy of additional consid- eration and follow-up. the fact that the only signal of dnm her- itability we detect is driven by siblings when not accounting for parental age effects further suggests that the environmental simi- larity shared by siblings (including parental age effects) is signifi- cantly influencing dnm rates. in sum, the mutational differences we found in the amish stand in contrast to the relative homoge- neity seen across the other diverse human populations we analyzed, and suggest that additional work is needed to better appreciate the forces shaping human mutational processes at fine scales. methods wgs was performed using samples previously collected and consented across nhlbi-funded research projects as part of the topmed program ( ). using variant data from a jointly called variant call set for samples from five of these topmed research projects, we implemented a filtering heuristic to call , dnms across , samples after the removal of outliers and pedigree errors. we then used two resequenced samples and one monozygotic twin pair to validate our call set by evaluating the proportion of dnms in one sample that were called as heterozy- gous sites in the second sample, and the percent of dnms that were called by our filtering approach in both the initial and validation samples. after demonstrating high concordance between our call set and dnms called with the triodenovo bayesian mutation caller, we compared single-nucleotide dnm counts and rates using anova, t tests, and linear and poisson regression. dnm rates were estimated as the number of single-nucleotide dnms autosome-wide divided by the number of autosomal bases with depth ≥ and quality ≥ . to test whether more hetero- zygous genomes have a higher dnm rate, we calculated genome-wide heterozy- gosity scores for each of the , samples included in our analysis, and compared these with estimated dnm rates per individual using linear regression. for com- parison within only a particular ancestry, samples were subset down accordingly. for genome-wide analyses, we used the university of california, santa cruz’s liftover tool ( ) to lift our dnm call set over to hg coordinates, and calculated local dnm rates, local recombination rates ( ), local replication timing rates ( ), gc content, and rare variation levels ( ) for -mb windows across the autosomes. to call local ancestry across our samples, we first phased the data using the eagle algorithm as implemented in the eagle software ( ), combined wgs data from the genomes project ( ) with high coverage wgs data from the peruvian genome project ( ), and used this genotype data as reference input to the rfmix software ( ). heritability was estimated using mmap ( ) and gcta ( ) with dnm count per individual as the quantitative mutation phenotype. additional methodological details are described in si appendix. data can be accessed via dbgap (https://www.ncbi.nlm.nih.gov/gap/) using the “phs” accession numbers listed for each study cohort in the acknowledgments. acknowledgments. whole genome sequencing (wgs) for the trans-omics in precision medicine (topmed) program was supported by the national heart, lung, and blood institute (nhlbi). wgs for “nhlbi topmed: whole genome sequencing and related phenotypes in the framingham heart study” (phs ) and “nhlbi topmed: genetics of cardiometabolic health in the amish” (phs ) were performed at the broad institute of mit and harvard ( r hl - s , r hl - s ). wgs for “nhlbi topmed: the genetics and epidemiology of asthma in barbados” (phs ) was per- formed by illumina, inc. ( r hl - s ). wgs for “nhlbi topmed: the cleveland family study (wgs)” (phs ) and “nhlbi topmed: the ge- netic epidemiology of asthma in costa rica” (phs ) were performed at the university of washington northwest genomics center ( r hl - s , r hl - s ). centralized read mapping and genotype calling, along with variant quality metrics and filtering, were provided by the topmed informatics research center ( r hl- - s ). phenotype har- monization, data management, sample-identity quality control, and general study coordination, were provided by the topmed data coordinating cen- ter ( r hl- - s ). we gratefully acknowledge the studies and par- ticipants who provided biological samples and data for topmed. a full list of topmed collaborators can be found at https://www.nhlbiwgs.org/topmed- banner-authorship. m.d.k. was supported by nih grant t ca . m.d.k. and t.d.o. were supported by funding from the center for health related informatics and bioimaging at the university of maryland school of medicine, institutional support for the institute for genome sciences and program in personalized genomic medicine at the university of maryland school of med- icine, nih genomic commons award ot od - , nhlbi trans-omics for precision medicine program high-performance grant u hl - , and national human genome research institute genomic innovator grant r hg - (to t.d.o.). d.p.l. was supported by nih t hl . this work was further supported by grants to the amish research program (r hl , r ag , and u hl ); a grant for the study of asthma in costa rica ( p hl - to s.t.w.); grants to study sleep apnea (r - hl to s.s.r., r -hl from sleep research society foundation to s.s.r. and with support for h.w., and k -hl from american thoracic society foundation to b.e.c.); and framingham heart study grant hhsn (to r.s.v.). sequencing was funded by grants to the genetics of cardiometabolic health in the amish study ( r hl - s ), the genetics and epidemiology of asthma in barbados study ( r hl - s ), 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nature , – ( ). . d. n. harris et al., evolutionary genomic dynamics of peruvians before, during, and after the inca empire. proc. natl. acad. sci. u.s.a. , e –e ( ). . b. k. maples, s. gravel, e. e. kenny, c. d. bustamante, rfmix: a discriminative mod- eling approach for rapid and robust local-ancestry inference. am. j. hum. genet. , – ( ). . j. yang, s. h. lee, m. e. goddard, p. m. visscher, gcta: a tool for genome-wide complex trait analysis. am. j. hum. genet. , – ( ). kessler et al. pnas | february , | vol. | no. | g en et ic s https://rpsychologist.com/d /cohend/ https://rpsychologist.com/d /cohend/ [pdf] characterizing surface wetting and interfacial properties using enhanced sampling (swipes). | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /c sm d corpus id: characterizing surface wetting and interfacial properties using enhanced sampling (swipes). @article{jiang characterizingsw, title={characterizing surface wetting and interfacial properties using enhanced sampling (swipes).}, author={h. jiang and suruchi fialoke and z. vicars and a. patel}, journal={soft matter}, year={ }, volume={ }, pages={ - } } 	h. jiang, suruchi fialoke, + author a. patel 	published 	materials science, physics, medicine 	soft matter we introduce an accurate and efficient method for characterizing surface wetting and interfacial properties, such as the contact angle made by a liquid droplet on a solid surface, and the vapor-liquid surface tension of a fluid. the method makes use of molecular simulations in conjunction with the indirect umbrella sampling technique to systematically wet the surface and estimate the corresponding free energy. to illustrate the method, we study the wetting of a family of lennard-jones surfaces… expand view on pubmed arxiv.org save to library create alert cite launch research feed share this paper 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on solid surfaces. 	b. shi, v. dhir 	chemistry, medicine 	the journal of chemical physics 	 	 	pdf save alert research feed on the equilibrium contact angle of sessile liquid drops from molecular dynamics simulations. 	srikanth ravipati, b. aymard, s. kalliadasis, a. galindo 	medicine 	the journal of chemical physics 	 	 	pdf save alert research feed ... ... related papers 	abstract 	figures and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and 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archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ mutation screening of the dyt /thap gene in italy mutation screening of the dyt / thap gene in italy monica bonetti, bsc, chiara barzaghi, phd, francesco brancati, md, phd, , alessandro ferraris, md, phd, emanuele bellacchio, phd, alessandro giovanetti, bsc, tamara ialongo, md, phd, giovanna zorzi, md, carla piano, md, martina petracca, md, alberto albanese, md, nardo nardocci, md, bruno dallapiccola, md, anna rita bentivoglio, md, phd, barbara garavaglia, phd, and enza maria valente, md, phd , * irccs casa sollievo della sofferenza, css-mendel institute, rome, italy; unit of molecular neurogenetics, department of child neuropsychiatry and department of neurology, irccs neurologic institute carlo besta, milan, italy; department of biomedical sciences, ce.s.i. aging research center, gabriele d’annunzio university foundation, chieti, italy; institute of neurology, catholic university, rome, italy; department of child neuropsychiatry, irccs neurologic institute carlo besta, milan, italy; department. of neurology, irccs neurologic institute carlo besta, milan, italy; department of medical and surgical pediatric sciences, university of messina, messina, italy video abstract: mutations in the thap gene on chromosome p -p (dyt locus) have been recently reported as causative of autosomal dominant primary torsion dystonia (ptd) in four amish–mennonite families and in addi- tional probands of different ancestry. we sequenced the thap gene in patients with dyt -negative ptd who had onset of symptoms below years and/or posi- tive family history. one sporadic greek male patient, aged years, was found to carry a novel heterozygous missense variant in thap exon (p.cys arg), of likely pathogenic significance. this subject first presented with right writer’s cramp at age of years and, subse- quently, developed left arm dystonia and an extremely severe left laterocollis, without further spreading to other body districts. our findings expand the genotypic spectrum of thap and strengthen the association with upper body involvement, including the cranial and cervi- cal districts that are usually spared in dyt -ptd. � movement disorder society key words: primary torsion dystonia; dyt ; thap ; tor- ticollis primary torsion dystonia (ptd) is characterized by sustained muscle contractions, causing twisting and re- petitive movements and abnormal postures, and can be inherited as an autosomal dominant or recessive trait. only two genes (tor a/dyt and thap /dyt ) have been identified so far that cause autosomal domi- nant ptd with reduced penetrance. a -base pair (gag) deletion in the tor a gene represents a com- mon cause of early-onset dystonia, especially among ashkenazi jews. the dyt phenotype usually presents with onset in a limb and rapid generalization, with sparing of the cranial-cervical district. – recently, mutations in the thap (thanatos-associated protein domain-containing apoptosis-associated protein ) gene have been identified in dyt -linked families, including a founder amish-mennonite mutation. two large screenings of ptd patients have subsequently identified thap mutations in of ( %) dyt -negative families with early-onset nonfocal ptd and in of ( %) patients with ptd that was early-onset and/or generalized and/or familial and/or involving the face and laryngeal districts. , in the patients with thap mutations so far reported, the dyt phenotype appears to be characterized by onset mostly in the first two decades, with generalization in about half cases and frequent involvement of the upper body, including arms, cranial, and cervical districts. in particular, up to % patients presented cranial dysto- nia, of whom a large proportion experienced speech problems related to dysarthria and/or spasmodic dys- phonia. , aim of this study was to investigate the frequency and phenotypic spectrum of thap mutations in a large cohort of ptd patients ascertained in italy. patients and methods the thap gene was first tested in patients with familial or sporadic early-onset ptd, recruited from the movement disorders centers of the catholic university in rome and the c. besta institute in milan. an arbitrary cut-off has been set at years, as > % of known patients with thap mutations had an onset below this age. – we subsequently tested a second group of ptd probands with onset above years the first two authors contributed equally to this work. additional supporting information may be found in the online version of this article. *correspondence to: dr. enza maria valente, neurogenetics unit, css-mendel institute, viale regina margherita , rome , italy. e-mail: e.valente@css-mendel.it potential conflict of interest: none reported. received july ; revised september ; accepted october published online november in wiley interscience (www.interscience.wiley.com). doi: . /mds. m. bonetti et al. movement disorders, vol. , no. , and positive family history. all patients tested negative for the dyt deletion, fulfilled a diagnostic flow-chart for dystonia and had no signs of secondary dystonia. inclusion criteria did not consider either the site of onset or the distribution of dystonia at latest ex- amination (table ). the project was approved by the local ethics committees and written informed consent was obtained by all patients or legal representatives. the three thap exons and exon-intron junctions were amplified from genomic dna by polymerase chain reaction (primers and conditions available upon request), purified and sequenced bidirectionally using the big dye terminator chemistry and an abi prism xl automated sequencer (applied biosystems). multiple sequence alignments of the human thap protein and its orthologues were generated by clus- talw software (http://www.ebi.ac.uk/clustalw/). predic- tion of the possible impact of the identified variant on the protein function was obtained using polyphen (http://genetics.bwh.harvard.edu/pph/) and sift (http:// sift.jcvi.org/) software. the structure of thap in the amino acid range – has been modeled with the program modeller� v , using as a template the crystallographic structure of general control protein gcn (pdb entry: efr). similarly, the putative dime- rization between two thap molecules has been simulated by reproducing the same topology of interac- tions observed for the dimer of gcn . results only patient with early-onset ptd was found to carry a novel heterozygous missense variant in thap exon (c. t>c, p.cys arg). this -year-old sporadic male patient was born in xanthi (in the northern greek region of thraki) from greek ances- tors; pregnancy, delivery, psychomotor development were uneventful. at age , he presented with right writer’s cramp. over the following years, he devel- oped abnormal posturing of the left arm and neck. the latest progressed in a few years to a severe left latero- collis associated with levoscoliosis, much more inva- lidating than the writer’s cramp (see video). dystonia failed to improve with anticholinergics, tetrabenazine, and myorelaxants. botulinum toxin injections improved pain but failed to affect the dystonic posture. no other muscular districts have become affected over time, and dystonia remained segmental. in particular, no cranial or speech involvement could be observed. brain mag- netic resonance imaging and routine testing were nor- mal. his parents died at and years without mani- festing any dystonic feature; as far as the patient recalls, none of his family members (including his - year-old brother and -year-old daughter) is affected by any movement disorder. the p.cys arg variant affects an highly con- served residue in the coiled coil domain (ccd) (aa – ), was predicted as damaging both by poly- phen (psic score: . ) and sift (score: . ), and it was not detected in control chromosomes (fig. ). to gain insights into the effects of this mutation on the protein, we generated a structural model of thap ccd (aa – ). we observed that all hydrophobic amino acids faced the same side of the ccd, while charged residues were regularly placed outwards. this pattern of alternating charged and hydrophobic residues in this specific region of thap is consistent with potential intermolecular interactions between ccds of two proteins. this is in line with the results of a previously reported large scale yeast-two-hybrid assay, that identified several putative thap interactors among which the thap protein itself, suggesting homodimerization. we have therefore generated a possible model of self- interaction between two thap molecules, although it is expected that thap also heterodimerizes with different proteins through the same ccd. in our model, the cys residue fell at the interface between the two molecules and replacement of this residue with an arginine produced a disruptive effect on the predicted intermolecular interaction (fig. ). in addition, the cys arg change introduces a posi- tively charged residue that likely interacts with the negatively charged glu forming an intramolecular salt-bridge that perturbs thap structure. table . demographic and clinical data of tested patients early-onset ptd late-onset ptd number (sex m/f) ( m/ f) ( m/ f) origin italian a all italian age at onset . . ( – ) . . ( – ) age at examination . . ( – ) . . ( – ) family history positive negative distribution of dystonia focal segmental multifocal generalized unknown b ages at onset and at examination are presented as mean stand- ard deviation (range). a one greek, one albanian. b latest examination < year from onset. dyt /thap dystonia in italy movement disorders, vol. , no. , discussion we detected a thap missense variant of potential pathogenic significance in of ptd patients ( . %), which is broadly in line with the % frequency reported in a recent study on european patients. these figures are markedly lower than the % frequency reported by bressman et al who included in the screen- ing only familial cases in which at least patient had nonfocal dystonia with onset below years. yet, it must be noted that both our and the patients carrying thap mutations reported by djarmati et al. were sporadic, although one of these had two asymptomatic relatives with subtle signs of dystonia who also carried the mutation. this lack of family history can be explained by the reduced penetrance of thap muta- tions, although we were not able to confirm this in our patient since both parents were dead and healthy rela- tives were not available for genetic testing. indeed, we found no positive cases among our probands who matched the inclusion criteria of the bressman study, and the mutation frequency in our cohort reached only . % among patients with nonfocal dystonia and onset below years (n ). moreover, the only subject carrying a thap missense change in our cohort was from northern greece, whereas none of the italian patients carried pathogenic mutations. thus, despite two families of italian ancestry have been previously reported bearing thap mutations, it appears that dyt -dystonia represents a rare occurrence in italy. our patient presented with segmental upper limb and neck dystonia in the absence of any facial, lingual, jaw or laryngeal involvement, supporting the conclu- sion that laryngeal dystonia or speech involvement, albeit frequently observed, are not mandatory features of the dyt phenotype. , however, the presence of cranial-cervical involvement may help differentiate this form from dyt -ptd, that could present similar fea- tures but usually spares the cranial-cervical regions. , the thap gene encodes a protein characterized by a conserved putative dna-binding motif at the n-ter- minus, a proline-rich region, and a large coiled coil region at the c-terminus, which includes a nuclear- localization domain (fig. ). overall, eleven muta- tions including six missense, one nonsense, and four frameshift mutations have been described to date, with no obvious genotype-phenotype correlations. the novel fig. . characterization of the p.cys arg mutation. a: schematic of the protein encoded by thap . dbd, thap dna-binding domain; pr, low-complexity proline-rich region; ccd, coiled-coil domain, including the nuclear localisation signal (dashed). b: multiple alignment of thap orthologues (aa – ). hydrophobic residues are highlighted in grey, cys is indicated by an arrow. c model of homodimerization of thap . each monomer (aa – ) is represented as a ribbon. hydrophobic residues and cys are shown respectively as sticks and spheres on each monomer. movement disorders, vol. , no. , m. bonetti et al. missense change identified in our study (p.cys arg) is of likely pathogenic significance because it was not detected in over caucasian control chromosomes (although we did not have access to control samples from northern greece), and it was predicted to be del- eterious by two distinct bioinformatic prediction soft- ware (polyphen and sift) and affected a highly con- served amino acid across species. interestingly, the mutant residue represents the first variant to fall within the ccd of the protein, since all other missense changes clustered within the dna binding domain. although the function of the ccd is still largely unknown, it has been suggested that thap may homo- or heterodimerize with another thap protein or another member of the thap family through this domain, and our molecular modeling results would be in favor of this hypothesis. these protein–protein inter- actions may be critical to enhance the dna binding activity of the thap zinc finger domain, which appears to be relatively weak, and in this light a muta- tion disrupting such intermolecular interactions may severely hamper the key function of dna binding. in conclusion, our results expand the mutational spectrum of dyt -ptd and implicate that this genetic form of dystonia is rare in italy. legend to the video the video illustrates the features of dystonia in the -year-old greek patient carrier of the p.cys arg mutation in the thap gene. segment . (year ), note the marked left latero- collis with subsequent limitation in neck movements, and the typical ‘‘geste antagoniste’’ (moving the right arm to the head) that completely abolishes the dystonic posturing of the neck. when the patient is sitting with his arms out- stretched, there is dystonic posturing of both arms. segment . (year ), the left laterocollis and the arm dystonic posturing have not changed over time. note the writer’s cramp when the patient is writ- ing and the levoscoliosis associated with neck dystonia while the patient is walking. acknowledgments: this work was supported by the ital- ian ministry of health (ricerca corrente ; ricerca final- izzata). the support of fondazione livio patrizi and transge- nomics is also gratefully acknowledged. part of dna sam- ples were obtained from the ‘‘bank for the diagnosis and research of movement disorders’’ (b.g.) of the eurobiobank network (www.eurobiobank.org). financial disclosure: alberto albanese: occasional con- sultancy advisory boards and honoraria from drug companies: teva, allergan, merz, ipsen; anna rita bentivoglio: occa- sional consultancy and honoraria from allergan, ipsen, novartis, boheringer ingheleim, ucb-pharma. monica bonetti, chiara barzaghi, francesco brancati, alessandro ferraris, emanuele bellacchio, alessandro giovanetti, tam- ara ialongo, giovanna zorzi, carla piano, martina petracca, nardo nardocci, bruno dallapiccola, barbara garavaglia, enza maria valente have nothing to disclose concerning any source of financial support and funding (including: stock ownership in medically-related fields, consultancies, advisory boards, partnerships, honoraria, grants, intellectual property rights, expert testimony, employment, contracts, royalties, etc) for the preceding months, regardless of relationship to this manuscript. author roles: m. bonetti and a. ferraris were involved in execution of the project, data analysis, and manuscript writing; c. barzaghi, f. brancati, e. bellacchio, and a. gio- vanetti, were involved in execution of the project and data analysis; t. ialongo, g. zorzi, c. piano, and m. petracca were involved in ascertainment of patients; a. albanese, n. nardocci, b. dallapiccola, and a.r. bentivoglio were involved in ascertainment of patients, review, and critique of the manuscript; b. garavaglia and e.m. valente were involved in conception and organization of the project, review and critique of the manuscript references . müller u. the monogenic primary dystonias. brain ; : – . . ozelius lj, hewett jw, page ce, et al. the early-onset torsion dystonia gene (dyt ) encodes an atp-binding protein. nat genet ; : – . . valente em, warner tt, jarman pr, et al. the role of dyt in primary torsion dystonia in europe. brain ; : – . . bressman sb, sabatti c, raymond d, et al. the dyt pheno- type and guidelines for diagnostic testing. neurology ; : – . . fuchs t, gavarini s, saunders-pullman r, et al. mutations in the thap gene are responsible for dyt primary torsion dystonia. nat genet ; : – . . bressman sb, raymond d, fuchs t, heiman ga, ozelius lj, saunders-pullman r. mutations in thap (dyt ) in early-onset dystonia: a genetic screening study. lancet neurol ; : – . . djarmati a, schneider sa, lohmann k, et al. mutations in thap (dyt ) and generalised dystonia with prominent spas- modic dysphonia: a genetic screening study. lancet neurol ; : – . . albanese a, lalli s. is this dystonia? mov disord ; : – . . sali a, blundell tl. comparative protein modelling by satisfac- tion of spatial restraints. j mol biol ; : – . . rual jf, venkatesan k, hao t, et al. towards a proteome-scale map of the human protein-protein interaction network. nature ; : – . . fasano a, nardocci n, elia ae, zorzi g, bentivoglio ar, albanese a. non-dyt early-onset primary torsion dystonia: comparison with dyt phenotype and review of the literature. mov disord ; : – . . bessière d, lacroix c, campagne s, et al. structure-function analysis of the thap zinc finger of thap , a large c ch dna-binding module linked to rb/e f pathways. j biol chem ; : – . dyt /thap dystonia in italy movement disorders, vol. , no. , wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ 복식문화연구 the research journal of the costume culture 제 권제 호, ( . ), pp. ～ － － † 교신저자 e-mail : hawleyj@missouri.edu textiles, clothing & the human element jana m. hawley † dept. of textile and apparel management, university of missouri, usa ( . . . 접수일 : . . . 수정완료일 : . . . 게재확정일) abstract years ago, anthropologists gave no attention to clothing and dress; but in the s, a new research agenda was created placing the body surface at center stage (tranberg-hansen, ). now culture is seen as a process created through agency, practice, and performance－no doubt, clothing very much has a human element. just as important, we now understand that the globalization process has both local and global impact. highest on this local/global interaction is the process of consumption. certainly we are not surprised by this when we know that clothes may be inspired from france, tech-packed from new york, sourced from all over the world, produced in china, and sold in shops on the other side of the globe. keywords: human element, metatheory Ⅰ. the human element the human element is what makes textiles and costume symbolically, politically, socially, and eco- nomically compelling. “human element” has been defined as a complex multi-dimensional issue that affects the entire spectrum of human activities. recently, dow chemical ran an award-winning, impressive ad campaign using “the human element” to market the chemical industry. the campaign used compelling images and touching voice-overs to illustrate how life depends on countless contributions from humans. the theme purports that the practice of chemistry is ultimately about serving people. in , the clothing and textiles research journal published dr. jean hamilton’s article, dress as a cultural sub-system: a unifying metatheory for clothing and textiles. as a tool for stretching our thoughts, the metatheory seeks a common disciplinary language regardless of our specializations. if we use the metatheory to look at the human element of textiles and clothing we must consider the social framework of technology, social structure, and world view. hamilton’s meta-theory has three cultural com- ponents illustrated like a layer cake, and includes technology, social structure, and world view. these three components represent the ways in which we satisfy our culture and are illustrated in figure . technology satisfies our material needs and provides us with the tools, material culture, and infrastructure to maintain existence. social structure satisfies our social needs and includes the ways in which we organize ourselves. they are the established patterns of behavior. world view or ideology satisfies our psychic needs and has to do with the way we 제 권 제 호 jana m . h awley － － think about things. the three components of culture are interactive. when a change in one category occurs, it is likely to result in change of another category. alongside the cultural components are the organi- zing mechanisms, or the ways in which we adapt to our environment. whatever the unit of analysis, whether it is a local weaver’s guild, or traditional costume, or contracted sweatshops, or gift exchange-- the organizing mechanisms help us to understand how humans, textiles, and costume are intertwined. no doubt, the complexity of humans is embodied in the organizing mechanisms of hamilton’s meta- theory. this includes economic, socialization, communi- cation, arts & aesthetics, world view, technology, and politics. but to focus today’s talk, i will focus on three domains: ( ) social relations, ( ) political significance, ( ) economic significance. i will also address sustainability and the future of textiles and costume. many of the examples i will provide come from my years of research and teaching. throughout history, textiles have impacted human life. whether in the form of personal expression, exploited human labor, or expressive artistry-- textiles and clothing are important to humans. textiles can take many shapes and decorative variations. by changing colors of warp and weft, or layering colors of dye, or sandblasting surfaces the potential for differentiation is endless. textiles can be worn, ex- changed, displayed or used to communicate ideo- logical values and human conditions such as poverty, wealth, status, gender and sexuality, political affilia- tions, ethnicity, age, occupation, or consumer be- havior. and they can create significance and meaning to our lives. in each of these cases…and many others too numerous to mention…textiles and costume are closely linked to human life. two points are important to consider: ( ) textiles and costume are often divided along gender lines, and ( ) the modern capitalist fashion system occurs at break-neck speed in order to meet high consumption <fig. > hamilton's meta-theory. patterns throughout the modern world－often nega- tively impacting human life. the result has been that much of the traditional symbolism as been removed and factory workers have been exploited. for many societies, though, clothing remains imbued with highly significant meaning. Ⅱ. social, political, and economic significance . social significance for the most part, textiles and clothing are couched in a social milieu. across the globe, textiles layer social meaning not only in birth and death, but also in rituals of marriage, religion, social move- ments, or sporting events. dress can identify groups, delineate social classes, and create social distance. as the world becomes increasingly globalized, tex- tiles and clothing traditions have been hybridized. for example, what does it mean to be “french haute couture” or “italian leather” or “indian bandhini”? how has modernity impacted guatemalan backstrap weaving or old order amish dress? in june , i packed my personal belongings and my ten-and eleven-year old boys and moved to jamesport, missouri to do a year of participant observation research living among the old order amish. it was perhaps the most peaceful, reflective, and revealing year of our lives and resulted in lifelong friendships with several amish friends with whom i remain in contact to this day. when we live with a group of people outside our own culture for an extended period of time, we learn all kinds of things about that group. the textiles, clothing & the h uman element 복식문화연구 － － amish are located in rural communities throughout the americas. as a large christian-based group whose core values focus on separateness from the world and commitment to tradition, family, and community, their values have set them apart from the rest of the world making them a cultural curio- sity. the amish often refer to themselves as “plain”. yet each amish community is unique with its own set of rules called the ordnung. outsiders have difficulty understanding why some amish allow a tractor engine mounted on a flatbed wagon to power farm equipment while another amish community strictly enforces the use of only horse-drawn power. these variances reflect specific rules of the ordnung and result in a continuum with orthodox old order on one end and the progressive beechy amish on the other. it is these varying rules that also explain variations in amish dress. for most amish, clothing represents several di- mensions of their cultural values including noncon- formity, humility, modesty, utility, thrift, and group conformity. amish dress is both a separator and an identifier. they also believe that the church, rather than fashion trends should control identity. outsiders might view variances in group wardrobe as confusing, but for the amish their choices make perfect sense. as outsiders interact, some changes in dress have occurred. over the past fifty years, athletic shoes, eyeglasses, and head scarves have been introduced for work days. even though tradi- tional or ethnic dress seems to honor tradition, careful observations reveal that it is dynamic and changing. all the more so, the amish consider notions of style even if the “latest style” is culturally contained. for example, in amish stores, stacks of colored head scarves are sold to the amish teenage girls who buy the scarves to match their dress, with colors changing from year to year. when members from other amish communities visit change occurs. when i was doing my research, a young girl from indiana had moved into the community. in her indiana community, women wore snaps as closures on their dresses. but in jamesport, dresses are held closed with straight pins. concern from the amish elders was raised because young jamesport girls were seen trying to “get by” sewing snaps onto their dresses. a special meeting was held by the church hierarchy and the girl from indiana was told that if she wanted to stay in jamesport, she had to remove all her snaps and replace them with pins like the other jamesport girls. if she refused, she would need to return to indiana. this is one example of how social control is exerted. indeed, while she was in jamesport she did remove her snaps, but she stayed only a few months because she decided jamesport was too strict. in i was among a fulbright-hayes study group to india on a project called “fabric of india life”. our fulbright experience focused most on the rajasthan area with special attention to the rabari textile artisans. the rabari are formerly a nomadic people now scattered in villages throughout northwest india. the textiles of each of these groups evolved through necessity as portable vessels, furnishings, and items of clothing. there is a commonality of dress and artisan textiles among the rabari communities. while, each community and tribal group has its own lexicon of motifs and embroidery stitches, specific differences in colors, fabric, and decoration will signify each community, and also within that age, and marital status. the people of this region share a common dowry tradition. in addition to the usual gifts of jewelry and household utensils, a bride must bring to her husband’s home a large number of richly embroidered textiles which she and the women of her family have created. this dowry will consist of costume for the bride and groom, hangings for her new home and trappings for their domestic animals. a toran is created to hang over the 제 권 제 호 jana m . h awley － － doorway for good luck. traditional dowry require- ments were so onerous that it was causing serious social and marital problems within the community. recently the council elders of the dhebaria commu- nity issued a ruling that banned embroidery on dowry (edwards, ). their primary aim was to reduce the costs of dowry and the increased time it took to create dowry. elders declared that dowry was destroying the community as a whole. by banning embroidery, they could redirect wealth into community development but to do so would require a major revision to the process of rabari marriage. implementation of the ban freed women of social obligation and subsequently they are free to produce embroidery for the market or purely as a pastime. this also means that many young dhebaria women have lost the cultural knowledge of their own traditional embroidery techniques. . political significance governments influence clothing -and clothing influences governments. clothing is used as a tool to demonstrate the power and authority of a nation. many examples exist for illustrating textiles and political significance. easy to store and transport, prestige textiles awe patrons, smooth the rough edges of diplomacy and earn foreign exchange. especially noteworthy were empires that compiled warehouses of textiles as a means for creating revenue and enhancing authority. imperial china was particularly impressive when its divisible units of value in silk became precursors to paper money. throughout history, textiles have been used to bestow, exchange, or sacrifice. but seldom have we thought about the political significance of second hand clothing. in my research in textile recycling, many cases exist where trade laws forbid countries to ship used apparel to certain other countries. the second had clothing business is worth more than $ billion each year in global trade. supporters of second hand clothing point out that the trade increases employment and provides low-cost clothing to people living in poverty. for example in senegal, it is estimated that , people are involved in the trading, distribution, repair, restyling, and washing of used clothing. it has been estimated that $ m worth of second had clothing generates about jobs in a developing country. yet policy makers report that second hand clothing is often contaminated, and under- mines fledgling garment industries. this politically motivated charge impacts our landfills and reduces the possibility of extended life for clothing. . economic significance regardless of a country’s economic situation, the clothing industry－from fiber to costume－is a major economic factor. during the post chinese reform period, the development of china’s textile and apparel industries was a process of liberali- zation in which the government cultivated and encouraged market competition as a way to mo- dernize china and enhance the economy. the deve- lopment of china’s textile and clothing industries is a part of the state agenda (zhao, ). chinese made garments exported to the united states is predicated on the global political economy. as it turns out, clothing is not just a business, but one that involves cultural meaning -both economic and political. when india became interested in an export market, the indian craftsman modified the colors they used to conform to export market tastes. this resulted in such a hugely successful penetration of european markets that european cloth makers responded by industrializing textile printing. india is one of the world’s most environmen- tally hazardous countries ranking th from the bottom. on the fabric of india life fulbright in , we visited the rabari tribes in the rural areas of kutch－a region that seemed an untouched bucolic area. when we returned just years later, textiles, clothing & the h uman element 복식문화연구 － － we were shocked to see two new coal-plants on either side of the small kalaraksha school. the untouched environment had been replaced with an ultra mega power project, one being the largest in asia. while, no doubt, india is in need of modern energy, the impact on the human element must also be considered. pollution from the plants comes in the form of air, water, biological, and noise (goffman, ). during the construction phase, rabari women were hired to help install insulation on the new buildings. one woman came home and complained that the cotton was cutting her arms. in actually, she was not installing “cotton”, but fiberglass insulation without the protection of gloves on their arms. completion of the project means that hours a day, days of the week power plants are now a constant presence in the lives of rabari tribes. Ⅲ. manufacturing processes three essential variables exist in textile weaving: the interlacing of warp and weft, surface decoration, and the colors used. variations result in weaving traditions that reflect local customs and preferences. industrial age innovations in spinning and weaving grew out of an english effort to compete with india cottons－particularly the manufacturer of calico as a less-costly substitute for chintz. in the eighteenth century, it would take indian hand spinners , hours to process lbs. of cotton, the industrial age brought machines that could do pounds in hours. but, by the late th century, textile and garment manufacturing fled from industrial countries to developing countries. much like their western counterparts, third world textile factories exploit labor, predominantly female labor and compete for yarn with hand-loom weavers. the nineteenth and twentieth centuries resulted in an explosion of design possibilities with the availability of industrial yarns and dyestuffs. humans <fig. > rabari tattoos inspire batik designs. ascribe a wide range of social and cultural meaning to cloth with motifs that are often abstracted from life. the rabari attribute color and motif ideas to cultural symbols. figure shows a rabari woman whose tattooed arm serves as inspiration for the artist’s batik design on silk. misinterpretations also happen. for example, ikat traditions of guatemala, indonesia, and asia are culture specific but no doubt labor intensive everywhere. many are stunning pieces of art－ collected, displayed, and worn. in the fashion season, couture designer oscar de la renta used ikat as his central inspiration (http://www.youtube.com/ watch?v=xeiwplgenke). . tourist markets in the case of cloth made for the tourist market, artisans are concerned about sellability resulting in products that no longer are true to the culture. products produced for the tourist market have been commoditized. moreno and littrell ( ) reported that marketing craft objects to local tourists has resulted in signi- ficant change. not only do products change to meet consumer demand, but so do the processes. often it is intermediaries and non-indigenous pro- ducers who envision what the final product should be－ frequently making changes to indigenous craft in order to meet the demands of a commercialized tourist market. the result can lead to tense re- lations between the negotiating parties. where merchants and artisans display their handi- 제 권 제 호 jana m . h awley － － crafts, changes are inevitable. not only do artists need to accommodate tourist’s taste, but also they must change their production processes to meet the higher demands. both negligence and care might co-exist. even though rural artisans might produce low-quality for export markets, they would apply high standards to cloth for indigenous social and ritual purposes. . guatemala in may , i traveled with a group of textile enthusiasts to guatemala to study textile artisans and their work. undoubtedly, some of the most colorfully costumed people in the americas are the highland maya of guatemala. while traditional native dress has disappeared in many parts of the world, guatemala remains a place where a high percentage of the indigenous people still proudly wear their traditional dress called the traje. traje is village-specific or language-group related, thus with different ethno-linguistic groups, the variety of indigenous dress is astonishing. traje consists of the huipil (traditional square-cut blouse), the corte (or skirt), and faja (or belt). each group also adorns with some type of headpiece or hair ribbon. figure shows a mayan woman in traditional traje. if you wander through the highland villages of guatemala, you will see women gathered to weave on their backstrap looms the end products of which are coin purses, placemats, or other domestic textiles for the tourist market. many of these textiles end up at the chi chi market or in tourist cities like antigua. but even when fair trade is considered, the economics of their production is far from ideal. in my visit to guatemala, a handwoven napkin sold in antigua for $ . each. but the woman who produced the napkin made less than $. per hour for her labor. while it is true that textile weaving provides one of only a few jobs that a village woman in guatemala can get, her receipts still keep her at poverty level. <fig. > traditional m ayan traje. for years, mayan women have produced textile products from back-strap looms using colorful cotton yarns acquired through local providers. but when cotton hit an all time high in early , mayan women could not afford the yarns for their weaving. two things resulted: they began using recycled plastics as weft in their backstrap weaving. and they learned how to hook rugs using strips of cotton from the used clothing pacas. pacas are literally a bale of used clothes shipped into guatemala from western countries, primarily the united states. clothes sell for q -q per item (or $. to $ . usd). see figure . the biggest challenge on learning to hook rugs was to honor their traditional designs but transfer designs from their traditional arts to a visually expanded mode and to learn the concept of “back- ground”. using the fist as a unit of measurement, the guatemalan women learned how to hook rugs by identifying sources for designs from their sur- roundings: huipils, the stone pattern on the rock wall, the pattern on the iron gate, the quatrefoil shape of the window－rug inspiration is everywhere. most women found that extracting elements textiles, clothing & the h uman element 복식문화연구 － － from their clothing could honor their own textile heritage while at the same time they learn new skills that would be economically beneficial. these rugs sell for ～$ usd. figure shows a rug that was inspired from traditional dress. soon women were telling stories about how the sale of rugs has impacted their lives. one woman said, “i used to have to go to the mountain and chop firewood that i sold door-to-door. i worked like a man and i still feel the work in my body. now that i am selling my rugs i don’t have to go to the mountain anymore. i am a lucky woman.” another woman said, “i used to be a low person. that is how people see you if you can’t read or have never been to school. now that i am selling my rugs i wee the world differently. i am not a low person anymore.” these strategies were made <fig. > h ooked rugs inspired from traje. possible through workshops taught by outside inter- mediaries from the united states. the mayan artisans who learned the new techniques not only had eco- nomic benefits that could help the women feed their families, but they also learned new skills and design techniques which broadened their product mix. Ⅳ. the future wearing clothing is uniquely a human characteristic. for years it was unknown when humans first began wearing clothing, but new technologies has changed that! a new nuclear dna technology has led to a technological breakthrough that has determined that humans began wearing clothing around , years ago. the discovery was based on a certain type of lice that lives only in threads and hair (mauch, / / from http://www.tulsaworld.com/ news/article.aspx?subjectid= &articleid= _ _a _losang ). until this study, direct dating of clothing has been nearly impossible because clothing does not survive well over time. not only has the dna dating given us a clearer understanding of when humans began to wear clothing, it also has significance for a broader understanding of the human element. in today’s world, with facebook, high-speed internet, and hyper-communication, a global impact has been felt and resulted in breaking down tradi- tional concepts of fashion. understanding fashion as a global phenomenon reveals shifts in global production and vast economic differences between those who produce and those who consume. no doubt, the meaning of clothing varies across regions of the world. there is a world of fashion out there, dominated by the economic power of the west, even if the west no longer fully controls inspiration and production. yet the human signifi- cance of global garment production is enormous. yes, textiles and costume rank high among our 제 권 제 호 jana m . h awley － － most personal possessions. they provide humans with identity, creative expression, protection from the elements, gender distinctions, and economic security. textiles and costume are inextricably linked to the human element. acknowledgements i was trained as a cultural anthropologist. through- out my career i have used culture theory and research methods in my scholarship. i have conducted sys- tematic research on the old order amish, fair trade organizations, and textile recycling. anthropologists often build bridges between cultural worlds. they blend respect for cultural difference and the aware- ness of common humanity. this, combined with interdisciplinary research methods, helps anthropologists to understand, make policies, develop programs, and improve the human condition. for me, under- standing both the global nature of textiles as well as the local cultural nuances has been an important part of my academic career. references beach, j. l., kincade, d. h., & schofield-tomschin, s.( ). human complexity: development of a theoretical framework for the clothing and textile field. clothing and textiles research journal, ( ), - . dow chemical( ). the human element. retrieved august , . from http://www.youtube.com/ watch?v=i byt xmsca edwards, e.( ). marriage and dowry customs of the rabari of kutch: evolving traditions. in foster, h. b. & johnson, d. c.(eds.), weddings dress across cultures. oxford: berg, - . goffman, e. ( ). india and the path to environ- mental sustainability. retrieved august , from http://www.csa.com/discoveryguides/ india/review .php hamilton, j. a.( ). dress as a cultural sub- system: a unifying metatheory for clothing and textiles. clothing and textiles research journal, ( ), - . mauch, t. h.( ). retrieved august , from http://www.tulsaworld.com/news/article.aspx?s ubjectid= &articleid= _ _a _lo sang moreno, a., & littrell, m.( ). negotiating tradi- tion: tourism retails in guatemala. annals of tourism research, ( ), - . oscar de la renta speaks about ikat. you tube. retrieved january , from http://www. youtube.com/watch?v=xeiwplgenke roach-higgins, m. e., & eicher, j. b.( ). dress and identity. clothing and textiles research journal, ( ), - . schneider, j.( ). the anthropology of cloth. annual review anthropology, , - . tranberg-hansen, k.( ) the world in dress: anthropological perspectives on clothing, fashion and culture. annual review of anthropology, , - . zhao, j.( ). fashioning change: the cultural economy of clothing in contemporary china. retrieved august , from http://www. anthropology.pitt.edu/grad/research/zhaophd.ht ml wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ fine-mapping of candidate region in amish and ashkenazi families confirms linkage of refractive error to a qtl on p -p robert wojciechowski, joan e. bailey-wilson, dwight stambolian statistical genetics section, inherited disease research branch, national human genome research institute (nih), baltimore, md; departments of ophthalmology and genetics, university of pennsylvania, philadelphia, pa purpose: a previous genome-wide study in orthodox ashkenazi jewish pedigrees showed significant linkage of ocular refraction to a quantitative trait locus (qtl) on p - . . we carried out a fine-mapping study of this region in orthodox ashkenazi jewish (ashk) and old order amish (ooa) families to confirm linkage and narrow the candidate region. methods: families were recruited from ashk and ooa american communities. the samples included: individuals in ooa families; and members in ashk families. families were ascertained to contain multiple myopic individuals. genotyping of , snps was carried out within a cm (~ . mb) candidate qtl region on p - . multipoint variance components (vc) and regression-based (reg) linkage analyses were carried out separately in ooa and ashk groups, and in a combined analysis that included all families. results: evidence of linkage of refractive error was found in both ooa (vc lod= . , reg lod= . at ~ cm) and ashk families (vc lod= . , reg lod= . at ~ cm). combined analyses showed three highly significant linkage peaks, separated by ~ cm (or mb), within the candidate region. conclusion: in a fine-mapping linkage study of ooa and ashk families, we have confirmed linkage of refractive error to a qtl on p. the area of linkage has been narrowed down to a gene-rich region at p . - . containing ~ genes. ocular refraction is a complex phenotype that is affected by a host of environmental and biological influences. a number of studies in a variety of populations have shown refractive error to be highly heritable [ - ]. almost twenty genetic loci for myopia or refractive error have been identified in linkage studies, but few have been reliably reproduced in independent populations (see e.g., the online mendelian inheritance in man [omim] database for a complete list, and tang et al. [ ] for a good review). one reason for this difficulty in confirming loci for refraction is the inherently complex nature of refractive error, wherein multiple interacting genes and environmental factors likely contribute to differential eye growth and refractive regulation. moreover, many myopia loci were mapped in highly-selected families that aggregated severe forms of myopia, which may have different genetic etiologies than more common types of refractive errors [ - ]. finally, genetic linkage studies generally lack the statistical power to detect loci of small effect, and coding of refraction phenotypes may be inconsistent across studies, making comparisons and generalizations troublesome. in an attempt to address some of these issues, the myopia family study (mfs) was developed to systematically search for the genetic causes of refractive errors in extended families from distinct ethnic groups. correspondence to: robert wojciechowski, inherited disease research branch, national human genome research institute, cassell dr., suite , baltimore, md, ; phone: ( ) - ; fax: ( ) - ; email: robwoj@mail.nih.gov the first genetic linkage studies of refractive phenotypes involved an x-linked syndromic form of myopia termed bornholm disease [ ]. later, young et al. successfully mapped severe myopia loci in a small number of extended families to p . [ ] and q - [ ] using parametric linkage methods. parametric statistics were well-suited to detect linkage in these highly-selected families in which an underlying genetic model–in this case autosomal-dominant– could be reasonably assumed. more recently, linkage studies have also focused on more prevalent refractive errors, such as low-to-moderate myopia [ - ] and ocular refraction, as a quantitative phenotype [ - ]. the quantitative trait locus (qtl) linkage analyses of refractive error were conducted in both population-based cohorts [ , ] and selected samples of families [ , , ]. these various sampling and analytical strategies have yielded numerous loci linked to refraction traits. nevertheless, although some of these mapped linkage regions were successfully reproduced in independent samples [ , , - ], many more loci have not been replicated [ ]. the myopia family study is comprised of families from four american ethnic groups: orthodox ashkenazi jewish (ashk); old order amish (ooa); african american (aa); and caucasian (cau). thus far, analyses of the aa families have shown genome-wide significant linkage of refraction to a qtl on chromosome p [ ]. this region has also been seen in french families with high myopia [ ] and suggested independently in a population-based cohort of sibships from the beaver dam eye study [ ]. analyses of ashk families have identified a locus for myopia on chromosome q molecular vision ; : - <http://www.molvis.org/molvis/v /a > received may | accepted july | published july © molecular vision http://www.ncbi.nlm.nih.gov/omim/ http://www.molvis.org/molvis/v /a [ ]. this finding has since been confirmed by investigators from the beaver dam study [ ], and replicated in an independent cohort of ashk families from the mfs [ ]. these results suggest that, while refractive errors undoubtedly have multifactorial genetic and environmental etiologies, at least some genetic polymorphisms involved in refractive variation may be shared across populations. in an analysis of the ashkenazi jewish sample of the mfs, we previously reported genome-wide significant linkage of ocular refraction to a novel qtl at p - [ ]. a subsequent meta-analysis of all mfs families showed no evidence of linkage to this region in ooa, aa, or cau participants [ ]. this lack of corroboration could be due to several possibilities: allele frequency differences between ethnic groups; locus heterogeneity; false positive results in the original analysis; and/or due to a lack of statistical power for replication. we subsequently genotyped a dense map of snp markers within the area of linkage in families from the ashk and ooa cohorts (see methods) in an attempt to replicate the findings in a fine-mapping linkage study in ooa families, and narrow down the region of interest in both the ooa and ashk for subsequent positional cloning experiments. this manuscript presents results from fine-mapping linkage analyses designed to address these aims. we report replication of chromosome linkage results. in addition, using a lod cutoff as boundaries for the area of linkage in both ashk and ooa families, we narrow down the location of the qtl to a mb area at p . to p . . methods family recruitment and selection criteria have been reported elsewhere and are summarized here [ , ]. briefly, ashk and ooa participants were recruited into the myopia family study primarily from the lakewood, nj (ashk) and lancaster county, pa (ooa) areas. all participating individuals were of either old order amish or orthodox ashkenazi jewish cultural/religious heritage (individuals of sephardic jewish origin and their offspring were not included in the study). for the ooa, myopic individuals were identified through community liaisons and their families were invited to participate in the study; ashk participants were asked to participate through mass mailings sent to all known orthodox jewish families residing in lakewood township, nj. in order to be eligible for inclusion in the study, a nuclear family had to contain only one myopic parent and at least one myopic offspring. these criteria were established to enhance selection of autosomal-dominantly transmitted myopia within families. larger pedigrees were then formed by extending nuclear families through first- and second-degree relatives. extended families were then selected for the linkage study if ) there was at least one affected pair of relatives besides a single parent-offspring pair and ) biological specimens were available for at least these affected individuals. the study protocol adhered to the tenets of the declaration of helsinki and was approved by the university of pennsylvania and the national human genome research institute, national institutes of health institutional review boards. all participants underwent a comprehensive eye examination including: medical and ophthalmic histories; visual acuities; slit lamp examination; goldmann applanation tonometry; fundoscopy; and objective and manifest refraction. individuals under years of age also received cycloplegic refraction using . % cyclopentolate or % tropicamide. when participants could not be examined at our study clinics, we attempted to obtain their most recent ocular examination records from their eye care providers. the quantitative phenotype, ocular refraction, was defined as the manifest spherical equivalent refractive error, averaged between the eyes. cycloplegic refractive error was used to define ocular refraction for participants under age , or when available from ocular examination records. because the initial recruitment strategy focused on myopia as a binary trait, individuals under age with a refractive error between and - d in any principal meridian were classified as "unknown" and were excluded from the study. also ineligible were individuals who had ocular or systemic conditions that could affect ocular refraction or significantly compromise the accuracy of refractive measurements. these conditions included: a history of prematurity; connective tissue disorders; poorly controlled diabetes mellitus; keratoconus; cataract; corneal opacities; and ocular syndromes in which myopia is a defining or common feature. individuals selected for fine-map snp genotyping were drawn from participating ashk and ooa families in the mfs. families were selected based on informativeness for linkage, dna quality and availability, and sample size constraints. most of these families were also included in previously-reported genome-wide linkage studies [ , , , , ]: ashk (out of ) and (out of ) ooa families in the current analysis overlapped with pedigrees from previous genome-wide linkage scans of ocular refraction [ , ]. four ashk and ooa families from the original genome-wide linkages were not included in the fine-mapping for the reasons given above, generally because these families could contribute virtually no information about linkage. marker selection: all snps were chosen a priori by the authors to provide sufficient coverage of a linkage area identified in a previous quantitative-trait analysis of a larger ashk sample [ ]. the linkage peak was located at ~ cm on chromosome p between microsatellite markers d s and d s , and the broadly-defined linkage area spanned roughly cm or . mb (from . to . mb) at p . - p . our final candidate region for fine-mapping extended from mb to . mb. coverage within the candidate linkage area was obtained by identifying haplotype tagging snps from the european population (ceu) in the human international hapmap molecular vision ; : - <http://www.molvis.org/molvis/v /a > © molecular vision http://hapmap.org/ http://www.molvis.org/molvis/v /a consortium [ , ]. tagging snps were picked using the two- snp aggressive tagging algorithm of the online tagger software server [ ] so as to cover common snps (maf >= . ) at a minimum r of . . in addition, coverage gaps larger than kb were filled-in with non-tagging polymorphic snps. we also included for genotyping all known functional snps (i.e., missense and nonsense mutations) with maf of at least . . in total, unique functional snps, with average heterozygosity= . , were genotyped. dna extraction and genotyping: venipuncture was used to collect peripheral blood from participating family members. high molecular weight genomic dna was extracted from the blood samples with a puregene dna purifying kit (gentra systems, inc; minneapolis, mn). the purified dna was then stored in a refrigerated dna repository under a unique sample code. custom snp genotyping was carried out at the center for inherited disease research (johns hopkins medical institutions, baltimore, md) on an illumina beadlab system (illumina, inc., san diego. ca) using goldengate chemistry. quality control and data cleaning: a total of , snps were genotyped in the chromosome candidate region. of these, ( . %) were excluded from analysis because of potential genotyping errors: because of unreliable raw intensity scores or call rates below %; due to atypical intensity clustering patterns; and were found to be monomorphic. also removed from analysis were snps that departed significantly from expected hardy-weinberg proportions (p< . ) in either ashk ( snps) or ooa ( snps) founders. these quality control measures resulted in , and , high quality snps being available for analysis in ashk and ooa families, respectively. the programs pedcheck [ ], pedstats [ ], and relcheck [ , ] were used to check for mendelian transmission inconsistencies and confirm putative pedigree relationships. all mendelian and relationship errors were corrected prior to analysis. when multiple mendelian transmission errors could not be reconciled with pedigree structures, incompatible individuals were removed from the dataset. large pedigrees were split to accommodate the default memory limits of multipoint analysis in the program merlin [ ] (i.e., bits for lander-green algorithm), making sure that no individuals were duplicated across pedigrees. statistical analysis: the statistical package merlin [ ] (version . . ) and its subroutine merlin-regress [ ] were used to perform multipoint quantitative trait locus (qtl) linkage analyses. linkage statistics were estimated using all snps that passed our quality-control filtering. because these tagging snps were chosen to minimize between-marker linkage disequilibrium, an increase in type- error rates was deemed unlikely. however, to guard against this possibility we repeated our analyses using a randomly- chosen subset of snps spaced at least kb apart (average spacing kb). both variance components (vc) and regression-based (reg) qtl linkage analyses were performed on logarithmic transformations of the spherical equivalent refractive error. allele frequencies were estimated using the maximum- likelihood method in merlin. all calculations were performed separately for ashk and ooa families, and after combining families from both populations. the modified haseman-elston approach implemented in merlin- regress [ ] requires that the trait mean, variance, and heritability of the underlying populations be pre-specified. although these parameters are unknown in orthodox ashkenazi jews and the old order amish, we estimated them using previous epidemiological studies in related populations [ , ]. we have also shown, in previous reports, that estimates of linkage statistics in merlin-regress are stable under reasonable population parameter specifications [ ]. however, because population means and variances of refractive error are thought to be significantly different between orthodox ashkenazi jews and the old order amish, combined reg and vc analyses were performed after standardizing refraction errors to the respective empirical means and variances of both groups. hence, values for mean and variance parameters were set to and , respectively, for combined reg linkage analyses, while the heritability for the combined groups was set to . . results population characteristics: sample characteristics of ooa and ashk families are presented in table . there were individuals in ooa pedigrees including: male; female; founders; and non-founders. a total of ( . %) ooa participants were genotyped. ooa families averaged . persons per family and . generations. the mean spherical equivalent refractive error among all ooa participants was - . d (sd= . ) and ( . %) of individuals with known phenotypes had myopia of at least d in both eyes. the ashk sample contained families and individuals ( males and females). the average pedigree for the ashk sample was comprised of . individuals in . generations. genotypes were obtained for ( . %) ashk participants. the mean refractive error among all ashk was - . d (sd= . ) and ( . %) of subjects with known phenotypes were categorized as myopic (≤- d in both eyes). ooa and ashk participants showed significant differences in both the mean refractive error and the proportion of myopic participants. these disparities are likely due to differences in the distribution of refractive error in the underlying populations: whereas the old order amish are molecular vision ; : - <http://www.molvis.org/molvis/v /a > © molecular vision http://www.broadinstitute.org/mpg/tagger http://watson.hgen.pitt.edu/register/docs/pedcheck.html http://www.sph.umich.edu/csg/abecasis/pedstats/index.html http://www.biostat.wisc.edu/~kbroman/software/ http://www.sph.umich.edu/csg/abecasis/merlin/ http://www.sph.umich.edu/csg/abecasis/merlin/ http://www.molvis.org/molvis/v /a thought to be at low risk for myopia [ ], the prevalence of myopia among orthodox jewish communities is considerably higher [ , ]. however, it is important to note that these families were selected for linkage studies of myopia and thus are not representative of refractive error distributions in their populations. marker statistics: the average call rate of all snps was . . after excluding snps with a call rate of , the call rate was . %. the mean minor allele frequency of non- monomorphic snps was . (sd= . ), the average heterozygosity was . % (sd= . ) and the mean between- snp spacing was kb (range . - . ). linkage analysis: results from multipoint vc and reg linkage analyses for ashk and ooa families are presented in figure . the maximum multipoint vc lod scores for mean spherical equivalent refractive error were: . (p= . ) at ~ cm (or . mb) for ooa; and . (p= . ) at ~ . cm (or . mb) for ashk. for reg analyses, the maximum lod scores were: . (p= . ) at ~ . cm (or . mb) for ooa; and . (p< - ) at ~ . cm (or . mb) for ashk families. the -lod support intervals for vc and reg analyses of the ooa sample extend from rs and rs at p . (~ cm), to rs and rs at p . (~ cm); and the -lod support regions in ashk spanned from rs - rs ( p . , ~ cm) to rs ( p . , cm). the -lod support intervals for vc analyses extended from . to . cm in the ooa, and between . and . cm for ashk. results from qtl multipoint linkage analyses after combining families from both populations are shown in figure . three linkage peaks were identified in the vc analysis: lod= . at ~ cm ( . mb); lod= . at ~ cm ( mb); and lod= . at ~ . cm ( . mb). three local linkage peaks were also found in the reg analyses: lod= . at ~ cm ( . mb); lod= . at ~ . cm ( . mb); and lod= . at ~ cm ( . mb). all local linkage signals in the combined analysis were highly statistically significant (all p< - ). analyses using a subset of snp markers yielded similar linkage profiles, although the maximum lod scores were attenuated relative to analyses with the full marker set: the maximum vc lod score was . (p= . ) for ooa; and . (p= . ) for ashk (see figure for vc results). the maximum reg lod score using the restricted marker set was . (p= . ) for ooa and . (p= . ) for ashk. the following discussion will mainly focus on linkage results using all markers. discussion using an independent sample of old order amish families, we have confirmed linkage of a candidate region for ocular refraction, previously mapped to a locus on p -p in ashkenazi jewish families. in the current study, the maximum multipoint lod scores of . (p= . ) for vc, and . (p= . ) for reg are highly significant in the ooa. even when using the most conservative test, namely the analyses of ooa using only the snp markers with no intermarker ld, the p-values were . for vc and . for reg which satisfy the well-known criteria for linkage replication (p ≤ . ) proposed by lander and kruglyak [ ]. in a genomewide linkage study of ashk jewish families, wojciechowski et al. [ ] first reported genome-wide significant linkage of ocular refraction to a broad region spanning from p to p ; and their peak multipoint linkage signal was seen between microsatellite markers d s and d s at p . - . . in the present study, the area of maximum evidence for linkage in ooa families was centered at markers rs -rs on p . (in both vc and reg analyses). this signal coincides with results from the initial genomewide screen in ashk. not surprisingly, the present study also shows evidence of linkage of refractive error to this region in ashk families. this result was expected since most of the same families from the initial genomewide study [ ] were also used in the current analysis. nevertheless, our fine-mapping analyses (with higher information content in the region and additional families) provide further evidence that the strong linkage signal identified using microsatellite markers was not simply due to chance allele sharing in a region with low linkage information. in the current analysis of ashk families, the maximum lod scores were found at table . sample characteristics. family size (range) generations (range) genotyped (%) male (%) female (%) founders (%) mean age (range) number myopic (%) mse (sd) old order amish (ooa) . ( - ) . ( - ) ( ) ( ) ( ) ( ) . ( - ) ( . ) - . ( . ) ashkenazi jewish (ashk) . ( - ) . ( - ) ( ) ( ) ( ) ( ) . ( - ) ( . ) - . ( . ) combined: . ( - ) . ( - ) ( ) ( ) ( ) ( ) . ( - ) ( . ) - . ( . ) myopia is defined as a manifest (or cycloplegic) refraction ≤- d in all four principal meridians. the proportion of myopic individuals (%) estimate excluded unknown phenotypes from the denominator. mse=mean spherical equivalent refractive error. molecular vision ; : - <http://www.molvis.org/molvis/v /a > © molecular vision population families n http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.molvis.org/molvis/v /a rs ( p . ) for both vc (lod= . p= . ) and reg (lod= . , p= . ) analyses, with the -lod confidence regions spanning p . to p . . even though most of the ashk participants from the original genomewide scan were used in the current study, fine- mapping linkage signals of ashk families were further from the original ashk linkage peak than results from the ooa sample. this is not surprising since localization of linkage peaks can be affected by a variety of factors including statistical variation, and a number of sample-specific factors [ - ] (i.e., new families with a different mixture of linked and unlinked families, differing penetrance and heritability due to allelic heterogeneity at the same locus, meiotic recombination events, map precision, marker informativeness, etc.) . moreover, a previous genomewide scan of ooa families showed little evidence of linkage of ocular refraction to chromosome (reg lod= . at cm). this discrepancy can be attributed to the sparseness of microsatellite markers used in the whole-genome study, and the resulting comparatively low marker information content. the low information content in the initial study was one of the motivating factors for the fine mapping performed in the ooa here. in the present analysis, linkage peaks in the ooa and ashk families were separated by approximately cm. although the lod support intervals are separated by - cm, the lod support intervals overlap considerably (figure ). given the inherent imprecision in localizing genetic loci for complex traits in genetic linkage studies [ , ], this result is consistent with the presence of a single qtl for ocular refraction in this region. nonetheless, the possibility of two distinct qtls contributing to refraction variation among ooa and ashk families cannot be ruled out. compared to single population analyses, our combined analyses of ooa and ashk families yielded greater evidence for linkage across the entire candidate region (maximum lod= . for vc; . for reg). however, in the combined analysis, three distinct linkage peaks can be seen from ~ to ~ cm (figure ). this may be simply a statistical artifact that resulted from the merging of genetically heterogeneous subgroups. it is also possible that the three peaks are due to locus heterogeneity, wherein more than one locus in the region is linked to the phenotype. however, statistical artifact is more likely than the presence of three refractive error genes in this region. absent a causative gene(s) or polymorphism(s) for variations in refractive error, it may not be possible to distinguish between these possibilities. further study would be required to make that determination. our results suggest that a single locus may account for variations of refractive error in both ooa and ashk families. given the ubiquity of refractive errors in human populations, a genetic locus that is involved in variation of refractive error in different ethnic subgroups can be expected, especially since linkage analysis can detect such a locus even if different ancestral alleles are involved in the different populations or if the same variant alleles are present but at differing frequencies figure . multipoint variance-components (left) and merlin-regress (right) lod scores for old order amish (ooa) and ashkenazi jewish (ashk) families. locations of linkage peaks are indicated by vertical lines. physical locations of snp markers (bottom axis, in mb) were determined using the ncbi dbsnp reference map, build . genetic map positions (top axis, in cm) were obtained through the rutgers combined linkage-physical map of the human genome. because the relationship between physical position and genetic maps are non-linear, genetic positions in the figure are approximate. molecular vision ; : - <http://www.molvis.org/molvis/v /a > © molecular vision http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?rs= http://www.sph.umich.edu/csg/abecasis/merlin/ http://www.molvis.org/molvis/v /a in the different groups. moreover, both ooa and ashk american populations are descendents of european migrants to the north american continent within the last few centuries [ , ], suggesting a (relatively) recent phylogenetic separation between the groups. nevertheless, the ooa and orthodox ashk are somewhat culturally isolated, largely endogamous, societies; and significant demographic and socio-cultural differences exist between these groups. in fact, genetic clustering analyses of our study participants showed that individuals could be classified into ooa or amish groups with high confidence using a limited number of markers (data not shown). the distribution of refractive errors differs significantly between ooa and orthodox ashk ethnic groups. while orthodox jewish groups suffer from high rates of myopia (especially among men) [ , ], the prevalence of myopia is thought to be low among the ooa [ ]. this is likely due to widely different exposures to environmental and behavioral risk factors for myopia between these groups. jewish orthodoxy emphasizes an intense education in religion and ethics from an early age and, for men, religious scholarship is required throughout life. the ooa, on the other hand, live agrarian lifestyles, eschew technology and oppose any forms of higher education. because of these disparities in environmental exposures (in addition to inherent population genetic differences), we took care to account for these differences in the design, analysis and interpretation of this linkage study. first, all initial analyses were conducted separately for the ooa and ashk, limiting the likelihood of bias in our results. second, phenotypes in combined analyses were normalized within distinct subgroups a priori. otherwise, the distributional and independence assumptions on which linkage statistics are based could have been violated. even after such normalization, however, departures from hardy-weinberg proportions and imprecise allele frequency estimates could invalidate allele sharing calculations required in estimating lod scores in combined analyses. finally, rather than using a binary trait, such as myopia, in our analyses, we analyzed refractive error as a continuous phenotype. in addition to some statistical advantages inherent in quantitative trait linkage statistics, they may be more appropriate for between-group comparisons when distributional differences exist between groups. for instance, quantitative linkage methods assess variations within populations whereas arbitrary thresholding of refractive error to define myopia may not apply equally to ooa and ashk individuals. hence, our results suggest that a quantitative trait locus at p . - . may account for variations in refractive errors in both the ooa and amish despite widely divergent underlying trait distributions. this can occur if both genes and environment contribute separately to refractive error regulation and/or if the frequency of alleles differs between the two populations at this quantitative trait locus. in the former model, environmental and behavioral risk factors would cause a shift in the overall distribution of refractive errors towards myopia, while various alleles at a gene (or figure . multipoint variance-components (left) and merlin-regress (right) lod scores for a combined analysis of ooa and ashk families. locations of local linkage peaks are indicated by vertical lines. physical locations of snp markers (bottom axis, in mb) were determined using the ncbi dbsnp reference map, build . genetic map positions (top axis, in cm) were obtained through the rutgers combined linkage-physical map of the human genome. because the relationship between physical position and genetic maps are non-linear, genetic positions in the figure are approximate. molecular vision ; : - <http://www.molvis.org/molvis/v /a > © molecular vision http://www.sph.umich.edu/csg/abecasis/merlin/ http://www.molvis.org/molvis/v /a genes) would account for variations of refractions within this range. this hypothesis is supported by evidence from a number of heritability studies, which consistently show high heritability estimates across ethnic groups with varying prevalences of myopia [ , - , ] (though mendelian forms of myopia or hyperopia may be less subject to environmental influence). using a -lod drop as a cutoff, the area of linkage in the present study spans from rs (the telomeric boundary in ooa) to rs (the centromeric boundary in ashk). to our knowledge, no other studies have reported linkage of either myopia or refractive error to this region. the linkage region in the current analysis spans approximately mb and contains human genes in the ncbi rna reference sequence collection (refseq). to date, of these genes (fatty acid binding protein [fabp ], gap junction protein, alpha [gja ], gap junction protein, alpha [gja ], glutamate receptor, ionotropic, kainate [grik ], and solute carrier family (copper transporters), member [slc a ]) have been reported in human association studies [ ], three of which have shown positive associations to disease phenotypes. however, no genes in the candidate region have, as of yet, been reported as being associated with refraction or other ocular traits in humans. two mouse orthologs of human genes within the linkage area (collagen, type viii, alpha ; and neurochondrin) have been shown to influence ocular phenotypes in experimental studies in mouse [ ] –though neither of these seem to be particularly strong candidates for refractive error control. in order to follow-up our findings and identify causal polymorphisms for refractive error variation, we are conducting family-based association analyses of snp markers. these analyses will help identify either causal polymorphisms or markers strongly correlated with causal genetic variations (i.e., via linkage disequilibrium). in the future, association analyses of additional snp and copy- number polymorphisms in the region may help to identify the dna variants responsible for this linkage signal. summary: in a fine mapping study in orthodox ashkenazi and old order amish families, we have confirmed linkage of refractive error to a quantitative trait locus on p and have narrowed the region of interested to a ~ mb area spanning p . - . . given that linkage was found in two independent, culturally and genetically isolated, groups, it is probable that the underlying genetic cause is a single genetic locus with variant alleles of large effect that can be detected in both these ethnic groups. it is reasonable to expect that such a locus would also have variant alleles that influence refractive error in other european-derived populations. acknowledgments this study was supported in part by u.s. public health national eye institute grant ey (d.s.) and in part by funds from the intramural program of the national human genome research institute, nih (j.e.b-w., r.w.). r.w. also received a william c. ezell-ciba vision fellowship from the american optometric foundation. genotyping services were provided by the center for inherited disease research (cidr). cidr is fully funded through a federal contract from figure . multipoint variance-components linkage lod scores for ooa (left) and ashk (right) for two different snp densities. all snps analyses used all markers that passed quality control measures ( , for ooa and , for ashk); 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[pmid: ] molecular vision ; : - <http://www.molvis.org/molvis/v /a > © molecular vision the print version of this article was created on july . this reflects all typographical corrections and errata to the article through that date. details of any changes may be found in the online version of the article. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.molvis.org/molvis/v /a jpma august- .qxd presenting as haemoperitoneum. careful evaluation avoids emergency surgery. references . uzair ts, oonwala zu, vellani m. a rare case of cystic hygroma presenting as inguinal hernia. j coll physicians surg pak ; : - . . konen o, rathaus v, dlugy e, freud e, kessler a, shapiro m, et al. childhood abdominal cystic lymphangioma. pediatr radiol ; : - . . steyaert h, guitard i, moscovici i, iuricic m, vaysse p, iuskiwenski s. abdominal cystic lymphangioma in children: benign lesions that can have a proliferative course. j pediatr surg ; : - . . de lagausie p, bonnard a, berrebi d, lepretre o, statopoulos l, delarue a, et al. abdominal lymphangiomas in children: interest of the laparoscopic approach. surg endosc ; : - . . porras-ramirez g, hernandez-herrera mh. hemorrhage into mesenteric cyst following trauma as a cause of acute abdomen. j pediatr surg ; : - . . roganovic j, smokvina m, ahel v, saina g, mavrinac b, jonjic n. intra- abdominelle lymphangiome. klin padiatr ; : - . . gyves-ray k, stein sm, hernanz-schulman m. hemoperitoneum in a newborn secondary to antenatal hemorrhage into a retroperitoneal lymphangioma. pediatr radiol ; : - . case report anaesthetic management of patient with ellis van creveld syndrome faisal shamim, fauzia nasim minai department of anaesthesia, aga khan university, karachi. abstract a known case of ellis-van creveld syndrome was scheduled for emergency repair of obstructed paraumblical hernia. we describe the anaesthetic management of the case with special reference to the classic physical and physiological manifestations of this syndrome present in our patient. introduction ellis-van creveld syndrome or chondroectodermal dysplasia is a form of short-limbed dwarfism. the name chondroectodermal is used because it affects the skeleton (chondro) and the skin (ectoderm). the syndrome was first described by ellis and van creveld in and this disease was found mainly among the amish group of population chiefly in pennsylvania, ohio and indiana. the disorder is characterized by anomalies of the hands and ectodermal dysplasia involving the nails and teeth. reported incidence is one in , , live births. incidence in pakistan and india is very rare. literature search from publication of first case in to date revealed only five case reports from this region. case report a year old female, known case of ellis-van creveld syndrome, presented in the hospital emergency room with vomiting, abdominal swelling and pain. a diagnosis of obstructed paraumblical hernia was made and the patient was scheduled for emergency repair of the obstructed hernia. on preoperative evaluation, patient had short stature, short limbs in relation to the trunk, polydactyly of the hands (figure ), protuberant abdomen, narrow chest and moderate lumbar lordosis. she was morbidly obese with a body mass index (bmi) of . . there was no abnormality of cervical spine and airway examination was unremarkable. blood pressure was / mmhg and heart rate - /min. the white blood cell count was , /cmm and baseline arterial blood gases showed a pao of mm hg and sao of % on room air. an echocardiogram done one year back showed a moderately dilated right atrium, mild right ventricular hypertrophy, large atrial septal defect (secundum type) with left to right shunt and severe pulmonary stenosis. the left ventricular systolic function was normal. the patient was evaluated by cardiologists and placed in high cardiac risk category for any kind of surgical intervention under general anaesthesia. supplemental oxygen was started via face mask preoperatively. aspiration prophylaxis (syrup sodium citrate . m ml, inj. ranitidine mg and inj. figure . polydactyly in hand. ulnar artery cannulated for invasive arterial pressure monitoring. j pak med assoc metoclopramide mg intravenously) was given an hour prior to induction of anaesthesia considering the high risk of regurgitation and aspiration due to obstructed hernia and morbid obesity. general anaesthesia with endotracheal intubation and invasive cardiovascular monitoring was planned. any neuraxial block for pain management was not considered due to thoracic and lumbar skeletal deformity. she was labeled as american society of anesthesiologist (asa) class ive. pre-induction, invasive arterial monitoring was initiated by cannulation of left ulnar artery (figure ) because radial pulse was not easily palpable. after preoxygenation, a modified rapid sequence induction was done with fentanyl µg, etomidate mg and succinylcholine mg. the trachea was intubated with endotracheal tube (ett) size . mm. the right internal jugular vein was cannulated for monitoring of central venous pressure and fluids. a foleys catheter was placed for monitoring of urine output. general anaesthesia with pressure control ventilation was maintained with isoflurane . - % and % oxygen in air. neuromuscular blockade with boluses of atracurium and systemic analgesia with fentanyl was continued during the maintenance period. glyceryl trinitrate (gtn) infusion was used to control blood pressure. the patient remained haemodynamically stable throughout the surgical procedure which lasted for three hours and intraoperative blood loss was insignificant. postoperatively, she was admitted to the intensive care unit (icu) for mechanical ventilation and invasive cardiovascular monitoring due to presence of several risk factors for postoperative respiratory insufficiency as well as early detection and rapid control of cardiovascular instability. she was extubated uneventfully the next day and kept under observation for hours. she was subsequently transferred to the special care unit and discharged from hospital three days later. discussion richard w.b. ellis of edinburgh and simon van creveld of amsterdam first described ellis-van creveld (evc) syndrome. they met in a train compartment while traveling to a paediatrics conference in england in the late s, and discovered that each had a patient with the syndrome. disproportionate dwarfism, postaxial polydactyly, ectodermal dysplasia, a small chest, and a high frequency of congenital heart defects characterize this autosomal recessive syndrome , which has an increased incidence among persons of old order amish descent. pathophysiology is unknown; however, recent identification of the evc gene should lead to a better understanding. histopathologic examination of foetuses with ellis-van creveld syndrome revealed that the cartilage of long bones showed chondrocyte disorganization in the physeal growth zone. in the general population, the frequency is case per , live births. among persons from the old order amish, the incidence is estimated at cases per live births. the frequency of carriers in this population may be as high as %. morbidity and mortality is related to thoracic dysplasia, respiratory insufficiency and cardiac anomalies. this leads to % deaths in infancy. there is no sex preponderance. in our patient significant, cardiac anomalies, thoracic dysplasia and morbid obesity were present. family history may include parental consanguinity or previously affected siblings or family members. neonatal history may reveal small size at birth, slow growth, and skeletal anomalies. natal teeth may be present. heart disease may be manifested as failure to thrive, cyanosis, shortness of breath, cardiac murmur, or other signs suggestive of heart failure. developmentally, most patients have had intelligence in the normal range. diagnosis rests solely upon skeletal anomalies. expected findings include relative shortening of the distal and middle segment of the limbs which is most prominent in the hands, where the distal and middle phalanges are shorter than the proximal phalanx. polydactyly is common on ulnar side, as it was present in our patient while valgus deformity of the knee and fibula disproportionately smaller than the tibia. chest radiograph, ecg and echocardiogram are required to evaluate cardiac anomalies. foetoscopy and ultrasonograpphy may be performed for prenatal diagnosis. occasionally e.v.c. has to be differentiated from asphyxiating thoracic dysplasia or asphyxiating thoracic dystrophy (jeune syndrome) and short limb polydactyly syndrome type because of overlapping features. the primary goal of medical care is supportive and care should be taken for respiratory distress, recurrent respiratory infections, cardiac failure and dental care in childhood. prevention of caries with dietary counseling, plaque control, and oral hygiene instruction should be given to parents. the surgical procedures are usually orthopaedic to correct polydactyly and valgus deformity. final adult height is - inches. usually, some limitation of hand function exists, such as inability to form a clenched fist. dental problems are frequent. the primary goal of anaesthetic management is thorough evaluation of skeletal deformities, airway abnormalities, restrictive ventilatory defect and cardiac vol. , no. , august anomalies. the magnitude of decompensation should be assessed and optimized if possible. the aim of intraoperative management is to gain rapid control of airway, prevention of aspiration, barotrauma, swings of blood pressure, myocardial depression, worsening of pulmonary hypertension and left to right shunt, reversal of shunt and cardiac failure. ventilatory management is directed towards maintaining stable airway pressures, normoxia and normocarbia. postoperative management should constitute adequate analgesia and prevention of adverse cardio-respiratory events. conclusion our patient showed many of the characteristic changes described in literature. no case reports regarding anaesthetic management of these patients have been found. the principal goal of anaesthetic management in these patients is to maintain cardiorespiratory stability, with use of invasive haemodynamic monitoring and ventilatory support, continued into the postoperative period. references . sharma op, saraf r, gupta b. ellis-van creveld's syndrome (a case report). ind j radiol imag ; : - . . ellis rw, van creveld s. a syndrome characterized by ectodermal dysplasia, polydactyly, chondrodysplasia and congenital morbus. arch dis childhood ; : . . varela m, ramos c. chondroectodermal dysplasia (ellis-van creveld syndrome): a case report. eur j orthod ; : - . . howard td, guttmacher ae, mckinnon w, sharma m, mckusick va, jobs en. autosomal dominant postaxial polydactyly, nail dystrophy, and dental abnormalities map to chromosome p , in the region containing the ellis- van creveld syndrome locus. am j hum genet ; : - . . mahoney mj, hobbins jc. prenatal diagnosis of chondroectodermal dysplasia (ellis-van creveld syndrome) with fetoscopy and ultrasound. n engl j med ; : - . . brueton la, dillon mj, winter rm. ellis-van creveld syndrome, jeune syndrome, and renal-hepatic pancreatic dysplasia: separate entities or disease spectrum? j med genet ; : - . case report autoerotic asphyxia by hanging recep fedakar , okan akan , bülent eren forensic medicine department, , görükle , bursa, turkey, council of forensic medicine of turkey bursa morgue department, bursa, turkey, council of forensic medicine of turkey bursa morgue department , bursa, turkey. abstract a case of sexual asphyxial death by hanging is presented. a -year-old man was found dead hanging by a towrope. a mirror was placed in front of a table, reflecting him in full height. a photograph of a celebrity mannequin was found against him. the towrope was arranged for the compression of the neck, and controlled voluntarily by a shower apparatus. autopsy findings revealed an ascending ligature mark, cm in width on the neck. although this is a very ancient behaviour, we are presenting a very rare case from turkey. we aim to discuss sexual asphyxiation phenomenon together with the features of the previous turkish cases on the aspect of the forensic viewpoint, as well as the similarities and differences between different nations and religious. introduction autoerotic asphyxiation is well-known phenomenon described in medical literature, particularly in forensic medicine reports, known as accidental autoerotic deaths. this attitude is defined as a non-psychotic mental disorder where unusual or bizarre imagery acts are necessary for sexual excitement, such kind of imagery or acts tend to be insistently and involuntarily repetitive. although this is a very ancient behaviour, we are presenting the fourth case from turkey. we aim to discuss sexual asphyxiation phenomenon together with the features of the three previous turkish cases on the aspect of the forensic viewpoint, as well as the similarities and differences between different nations and religious. case report a -year-old man was found dead hanging by a towrope at the kitchen of a bank's guesthouse (figure ). a mirror was placed in front of the table, reflecting him in full height and also there was a broken mirror in the kitchen. the victim was found by a bank's official driver. when the driver opened the balcony door of the kitchen, the mirror which was located leaning against the door was broken. the curtains of the kitchen were drawn and the door was locked. his undershirt was bounded as a brassiere over his nipples and underpants were slipped on his head. the towrope was arranged for the compression of the neck, and controlled voluntarily by a shower apparatus (figure ). a photograph of a celebrity mannequin was found against him. his toenails were polished (figure ). the victim's body was partially supported by the ground. there were two bottles of j pak med assoc [pdf] a randomized controlled trial of prochlorperazine versus metoclopramide for treatment of acute migraine. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /j.annemergmed. . . corpus id: a randomized controlled trial of prochlorperazine versus metoclopramide for treatment of acute migraine. @article{friedman arc, title={a randomized controlled trial of prochlorperazine versus metoclopramide for treatment of acute migraine.}, author={b. friedman and d. esses and c. sol{\'o}rzano and n. dua and p. greenwald and radu rădulescu and e. chang and michael l. hochberg and caron campbell and a. aghera and tyson valentin and j. paternoster and p. bijur and r. lipton and e. gallagher}, journal={annals of emergency medicine}, year={ }, volume={ }, pages={ - } } 	b. friedman, d. esses, + authors e. gallagher 	published 	medicine 	annals of emergency medicine study objective we compare prochlorperazine mg intravenously versus metoclopramide mg intravenously for the emergency department (ed) treatment of acute migraine. methods this was a randomized, double-blind, clinical trial comparing parenteral dopamine antagonists. both drugs were administered during minutes with mg intravenous diphenhydramine. pain scores on a numeric rating scale were assessed at baseline, every minutes for hours, and by telephone hours after discharge… expand view on pubmed evidencebasedpractice.osumc.edu save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all figures, tables, and topics from this paper 	 figure 	 table 	 table 	 figure 	 figure 	 table 	 table view all figures & tables 	prochlorperazine 	metoclopramide 	migraine disorders 	diphenhydramine 	dopamine 	headache 	erectile dysfunction 	pain 	accident and emergency department 	confidence intervals 	numbers 	addison disease 	alveolar rhabdomyosarcoma paper mentions interventional clinical trial intravenous fluids in benign headaches trail migraine headache has a -year period prevalence in the us of . % and accounts for approximately . million migraine visits to us emergency departments per year . there are… expand 	conditions	headache 	intervention	drug university medical center of southern nevada may - may interventional clinical trial a comparative efficacy trial of iv acetaminophen versus iv ketorolac for emergency department treatment of generalized headache this randomized, double-blind trial will compare the clinical efficacy of iv acetaminophen to that of iv ketorolac as adjuncts to standard therapy (prochlorperazine and diphenhydramine… expand 	conditions	headache 	intervention	drug lakeland health september - september blog post prochlorperazine, metoclopramide, and diphenhydramine for acute migraine headache aliem january interventional clinical trial the check trial: a comparison of headache treatment in the ed: compazine versus ketamine this study compares the efficacy of low dose ketamine versus compazine for the control of headache in patients presenting to the emergency department.   	conditions	headache 	intervention	drug university medical center of southern nevada march - march citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency randomized trial of iv valproate vs metoclopramide vs ketorolac for acute migraine 	b. friedman, l. garber, + authors e. gallagher 	medicine 	neurology 	 	 save alert research feed metoclopramide for acute migraine: a dose-finding randomized clinical trial. 	b. friedman, l. mulvey, + authors e. gallagher 	medicine 	annals of emergency medicine 	 	 	 save alert research feed a randomized controlled trial of intravenous haloperidol vs. intravenous metoclopramide for acute migraine therapy in the emergency department. 	matthew e gaffigan, david i. bruner, courtney wason, amy pritchard, k. frumkin 	medicine 	the journal of emergency medicine 	 	 	pdf save alert research feed randomized study of iv prochlorperazine plus diphenhydramine vs iv hydromorphone for migraine 	b. friedman, eddie irizarry, + authors e. gallagher 	medicine 	neurology 	 	 	pdf 	view excerpt, cites background save alert research feed comparison of intravenous metoclopramide and acetaminophen in primary headaches: a randomized controlled trial 	g. faridaalaee, s. rahmani, + authors f. rahmani 	medicine 	emergency 	 	 	pdf 	view excerpt, cites background save alert research feed olanzapine versus droperidol for the treatment of primary headache in the emergency department. 	c. hill, j. miner, m. martel 	medicine 	academic emergency medicine : official journal of the society for academic emergency medicine 	 	 	 save alert research feed metoclopramide and diphenhydramine: a randomized controlled trial of a treatment for headache in pregnancy when acetaminophen alone is ineffective (mad headache study) 	k. childress, christina dothager, j. gavard, sara lebovitz, catherine laska, d. mostello 	medicine 	american journal of perinatology 	 	 save alert research feed a comparison of headache treatment in the emergency department: prochlorperazine versus ketamine 	t. zitek, m. gates, + authors j. m. clark 	medicine 	annals of emergency medicine 	 	 	pdf save alert research feed diphenhydramine as adjuvant therapy for acute migraine: an emergency department-based randomized clinical trial. 	b. friedman, lisa cabral, + authors e. gallagher 	medicine 	annals of emergency medicine 	 	 	pdf save alert research feed randomized trial of adding parenteral acetaminophen to prochlorperazine and diphenhydramine to treat headache in the emergency department 	stefan meyering, r. stringer, matthew hysell 	medicine 	the western journal of emergency medicine 	 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency a trial of metoclopramide vs sumatriptan for the emergency department treatment of migraines 	b. friedman, j. corbo, + authors e. gallagher 	medicine 	neurology 	 	 	view excerpts, references methods and background save alert research feed intravenous chlorpromazine vs intravenous metoclopramide in acute migraine headache. 	j. cameron, p. lane, m. speechley 	medicine 	academic emergency medicine : official journal of the society for academic emergency medicine 	 	 	 	view excerpt, references methods save alert research feed intramuscular prochlorperazine versus metoclopramide as single-agent therapy for the treatment of acute migraine headache. 	j. jones, s. pack, e. chun 	medicine 	the american journal of emergency medicine 	 	 	view excerpts, references background save alert research feed intravenous sodium valproate versus prochlorperazine for the emergency department treatment of acute migraine headaches: a prospective, randomized, double-blind trial. 	d. tanen, s. miller, t. french, r. riffenburgh 	medicine 	annals of emergency medicine 	 	 	view excerpt, references methods save alert research feed randomized, placebo-controlled evaluation of prochlorperazine versus metoclopramide for emergency department treatment of migraine headache. 	m. coppola, d. yealy, r. leibold 	medicine 	annals of emergency medicine 	 	 save alert research feed randomized double-blind trial of intravenous prochlorperazine for the treatment of acute headache. 	j. jones, d. sklar, j. dougherty, w. white 	medicine 	jama 	 	 	view excerpt, references background save alert research feed intravenous chlorpromazine in the emergency department treatment of migraines: a randomized controlled trial. 	m. bigal, c. bordini, j. speciali 	medicine 	the journal of emergency medicine 	 	 save alert research feed acute migraine treatment with droperidol 	s. silberstein, w. young, j. e. mendizabal, j. rothrock, a. alam 	medicine 	neurology 	 	 	view excerpt, references background save alert research feed droperidol vs. prochlorperazine for benign headaches in the emergency department. 	j. miner, s. j. fish, s. w. smith, m. biros 	medicine 	academic emergency medicine : official journal of the society for academic emergency medicine 	 	 	view excerpts, references methods and background save alert research feed intravenous chlorpromazine versus intramuscular sumatriptan for acute migraine. 	a. kelly, m. ardagh, c. curry, j. d’antonio, s. zebic 	medicine 	journal of accident & emergency medicine 	 	 	pdf 	view excerpt, references methods save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	paper mentions 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue assessing performance of pathogenicity predictors using clinically relevant variant datasets gunning ac, et al. j med genet ; : – . doi: . /jmedgenet- - original research assessing performance of pathogenicity predictors using clinically relevant variant datasets adam c gunning , , verity fryer, james fasham , andrew h crosby, sian ellard, emma l baple, caroline f wright diagnostics to cite: gunning ac, fryer v, fasham j, et al. j med genet epub ahead of print: [please include day month year]. doi: . / jmedgenet- - ► additional material is published online only. to view please visit the journal online (http:// dx. doi. org/ . / jmedgenet- - ). college of medicine and health, university of exeter medical school institute of biomedical and clinical science, exeter, uk exeter genomics laboratory, royal devon & exeter nhs foundation trust, exeter, uk correspondence to professor caroline f wright, college of medicine and health, university of exeter medical school institute of biomedical and clinical science, exeter ex dw, uk; caroline. wright@ exeter. ac. uk acg and vf contributed equally. received march revised may accepted june © author(s) (or their employer(s)) . re- use permitted under cc by. published by bmj. abstract background pathogenicity predictors are integral to genomic variant interpretation but, despite their widespread usage, an independent validation of performance using a clinically relevant dataset has not been undertaken. methods we derive two validation datasets: an ’open’ dataset containing variants extracted from publicly available databases, similar to those commonly applied in previous benchmarking exercises, and a ’clinically representative’ dataset containing variants identified through research/diagnostic exome and panel sequencing. using these datasets, we evaluate the performance of three recent meta- predictors, revel, gavin and clinpred, and compare their performance against two commonly used in silico tools, sift and polyphen- . results although the newer meta- predictors outperform the older tools, the performance of all pathogenicity predictors is substantially lower in the clinically representative dataset. using our clinically relevant dataset, revel performed best with an area under the receiver operating characteristic curve of . . using a concordance- based approach based on a consensus of multiple tools reduces the performance due to both discordance between tools and false concordance where tools make common misclassification. analysis of tool feature usage may give an insight into the tool performance and misclassification. conclusion our results support the adoption of meta- predictors over traditional in silico tools, but do not support a consensus- based approach as in current practice. introduction as the scale of genomic sequencing continues to increase, the classification of rare genomic variants is the primary bottleneck in the diagnosis of rare monogenic disorder. guidelines published by the american college of medical genetics (acmg) in have helped to bring greater consistency to variant classification. these have been followed by gene/disorder- specific rule- sets, and country/ healthcare system–specific guidance such as the uk association for clinical genomic science (acgs) best practice guidelines for variant inter- pretation. common to all guidelines is the recom- mendation of the use of in silico prediction tools to aid in the classification of missense variants. in silico prediction tools are algorithms designed to predict the functional impact of variation, usually missense changes caused by single- nucleotide vari- ants (snvs). though originally designed for the prioritisation of research variants, the tools are used routinely in clinical diagnostics during variant classification. the tools integrate a number of features in order to assess the impact of a variant on protein function. initially, inter- species conser- vation formed the bulk of the predictions, with some additional functional information, such as substitution matrices of physicochemical distances of amino acids (such as grantham or pam ), and data derived from a limited number of available x- ray crystallographic structures. since the devel- opment of the first in silico prediction tools over a decade ago, large- scale experiments such as the encode project have generated huge amounts of functional data, and we now also have access to large- scale databases of clinical and neutral varia- tion. – these additional sources of data have led to an explosion of new in silico prediction algo- rithms – that purport to increase accuracy. however, the large increase in the number of predictors integrated into classification algorithms has raised concerns about overfitting. overfit- ting occurs when the prediction algorithm is trained on superfluous data or features that are irrelevant to the prediction outcome. while it may appear that an increasingly large feature list leads to improvements in prediction, random variability within the training dataset may result in decreased accuracy when applied to a novel dataset. overfit- ting can be mitigated through the use of increas- ingly large training datasets, and the usage of online variant databases, such as the genome aggregation database (gnomad) and clinvar, allows for sufficiently large training datasets. in addition, reli- ance on additional information—such as protein functional data and allele frequency data such as from gnomad —may be contrary to the standard assumptions of variant classification methodology, namely that each dataset is independent and applied only once during classification. the acmg guidelines recommend the use of a concordance- based approach, where several prediction algorithms are used, and evidence is applied only when there is agreement between tools. there is no guidance on which in silico tools should be used, how many or on what constitutes a consensus, and this ambiguity allows for inconsis- tencies in the application of this piece of evidence across clinical laboratories. studies have previously o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://dx.doi.org/ . /jmedgenet- - http://dx.doi.org/ . /jmedgenet- - http://crossmark.crossref.org/dialog/?doi= . /jmedgenet- - &domain=pdf&date_stamp= - - http://jmg.bmj.com/ gunning ac, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics identified the limitations of applying a strict binary consensus- based approach. in response, multiple groups – have created meta- predictors; tools which integrate information from a large number of sources into a machine- learning algorithm. these tools thereby adhere to the principle of the consensus- based model suggested by acmg without the onerous task of determining tool concordance and reduce discordance when increasingly large numbers of tools are used. unlike a manual consensus- based model, where tools are weighted equally, meta- predictors can apply weighting to features in order to maximise accuracy. the uk acgs guidelines suggest it is likely that a single meta- predictor will replace this concordance- based approach, but a comprehensive analysis using a clinically representative dataset has not yet been done. in order to evaluate the accuracy of in silico prediction tools, precompiled variant datasets such as varibench have been designed to aid in training and benchmarking of pathogenicity predictors. however, the use of standardised datasets may intro- duce inherent biases into prediction algorithms, resulting in overfitting and false concordance. typically, prediction software is trained using machine- learning algorithms, and assessed using variants available from large online public databases – such as exac/gnomad, clinvar and swissprot. it has been previously shown that prediction algorithms have variable perfor- mance when applied to different datasets, and therefore the use of variant datasets derived from online public databases may not be representative of the performance of tools when applied in a clinical setting. while studies emphasise the use of ‘neutral’ variation, the output from a modern next- generation sequencing pipeline is generally far from neutral and includes a large number of variant filtering steps in order to reduce the burden of manual variant assessment. here, we evaluate and compare the performance of two tradi- tional in silico pathogenicity prediction tools commonly used for clinical variant interpretation (sift and polyphen- ), and three meta- predictors (revel, gavin and clinpred ) using a publicly available (‘open’) variant dataset and a clinically rele- vant (‘clinical’) variant dataset (see figure ). while a number of other tools are available, these metapredictors were selected as they were designed in the anticipation of being used in a clinical setting. we show that the tools’ performance is heavily affected by the test dataset, and that all tools may perform worse than expected when classifying novel missense variants. by assessing figure flow diagram of selection and filtering steps used for the generation of the open (a) and clinical (b) datasets. oval—variant source; box— selection criteria; rounded box—dataset. red text (right) shows the number of pathogenic variants, green text (left) shows the number of benign variants. maf, minor allele frequency. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ gunning ac, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics the effect of a consensus- based approach, our results support the use of a single classifier when performing variant classification. materials and methods open dataset open dataset (n= , see figure a) represents the typical training and validation dataset used during in silico predictor design and benchmarking. positive (‘pathogenic’) variants were downloaded from clinvar on november and subscription- based hgmd professional release . ; neutral (‘benign’) variants in omim morbid genes were down- loaded from the gnomad database (exomes only data v . . ). clinvar criteria: stringent criteria were used to increase the likelihood of selected variants being truly pathogenic. missense snvs with either ‘pathogenic’ and/or ‘likely pathogenic’ clas- sification, multiple submitters and no conflicting submissions were included; variants with any assertions of ‘uncertain’, ‘likely benign’ or ‘benign’ were excluded. hgmd pro criteria: single- nucleotide missense variants marked as disease- causing (‘dm’) were taken from hgmd professional release . . gnomad criteria: missense snvs with an overall minor allele frequency (maf) between % and % were selected. these variants were deemed too common to be disease causing but are not necessarily filtered out by next- generation sequencing pipelines depending on the maf thresholds used. chromosomal locations with more than one variant (multiallelic sites) were excluded. any variants found to be present in the ‘pathogenic’ and ‘neutral’ datasets were removed from both. variants present figure in silico pathogenicity predictor feature usage and source. shading indicates that a category of evidence is used by the tool. codes within each box indicate that the feature is inherited from another tool. feature lists were taken from the tools' original publications, supplementary materials and available online material. c, cadd; d, dann; f, fathmm; fc, fitcons; mp, mutpred; mt, mutationtaster; p, polyphen- ; s, sift; v, vest. an extended version is shown in online supplementary figure s . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n a u g u st . d o w n lo a d e d fro m https://dx.doi.org/ . /jmedgenet- - http://jmg.bmj.com/ gunning ac, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics in the sift, polyphen- , revel, gavin and clinpred training datasets were removed to reduce bias and circularity. variants with missing or intermediate scores were also removed. clinical dataset clinical dataset (n= , see figure b and online supplemen- tary table s ) more accurately reflects variants that might require classification in a clinical diagnostics laboratory following iden- tification in an exome or genome sequencing pipeline. variants were selected from three sources. group (‘ddd’) consists of pathogenic (n= ) and benign (n= ) missense variants iden- tified from families in the deciphering developmental disorders (ddd) study that have been through multiple rounds of variant filtering and clinical evaluation. variants were identified through exome sequencing and were reported to the patients’ referring clinicians for interpretation and confirmation in accredited uk diagnostic laboratories. all benign variants from this list were assessed as having no contribution towards the patient’s phenotype, and were present in either as hetero- zygotes in monoallelic genes or homozygotes in biallelic genes classified according to the developmental disorder genotype- - phenotype database (ddg p) (data accessed oct ). group (‘diagnostic’) consisted of pathogenic (n= ) and benign (n= ) missense variants identified through sanger sequencing, next- generation sequencing panel analysis or single gene testing in an accredited clinical diagnostic laboratory. vari- ants were manually classified according to the acmg guidelines on variant interpretation on a - point scale (data accessed apr ). group (‘amish’) consisted of benign missense vari- ants (n= ) identified through a community genomics research study of amish individuals. variants were identified through singleton exome sequencing and were classified as benign based on population frequencies and zygosity within this study. two subgroups were manually selected and annotated based on inher- itance pattern and disease penetrance; subgroup (i) consisted of variants in genes that cause a dominantly inherited disorder with complete penetrance in childhood, for which the individual was clinically unaffected; this list was curated by a consultant in clinical genetics; subgroup (ii) consisted of variants in all other omim morbid genes (including those with incompletely penetrant dominant disorders and recessive and x- linked inher- itance), with maf > % in the amish cohort and maf ≤ . % in gnomad (data accessed oct ). variants with missing or intermediate scores were removed. transcript selection and variant annotation for the open dataset, the canonical transcript was selected for each variant using the variant effect predictor (vep). for the clinical dataset, the hgmd professional refseq transcript was used, unless absent from the database, in which case the mane primary transcript was selected. variants were annotated with variant cdna and protein nomenclature in reference to the selected transcript. polyphen- and sift scores were annotated using vep. revel and clinpred scores were annotated using flat files containing precomputed scores for all possible single- nucleotide substitutions, and in both cases, the combination of nucleotide position, nucleotide change and amino acid change was sufficiently unique to identify a single record, that is, tran- script selection did not affect the scores. gavin scores were generated through a batch submission to the gavin server. tool benchmarking the performance of each of the tools was determined for both datasets. for sift, polyphen- , revel and clinpred, the output of the analysis was a numerical score between and . initially, all tools were analysed according to the criteria defined in their original publications, with the thresholds for pathogenicity being ≤ . for sift, ≥ . for polyphen- and ≥ . for clinpred. for revel, where no threshold is recommended, a threshold of ≥ . was used. the categorical classification of gavin was used directly (‘benign’, ‘pathogenic’). a supplementary analysis was done for table results of variant classification for individual tool, and two consensus- based combinations, for the (a) open (n= ) and (b) clinical (n= ) datasets true positive true negative false positive false negative sensitivity specificity mcc lr+ lr− (a) open dataset individual sift . . . . : : . polyphen- . . . . : : . revel . . . . : : . gavin . . . . : : . clinpred . . . . : : . consensus sift+polyphen- . . . : : . revel+clinpred . . . . : : . (b) clinical dataset individual sift . . . . : : . polyphen- . . . . : : . revel . . . . : : . gavin . . . . : : . clinpred . . . . : : . consensus sift+polyphen- . . . . : : . revel+clinpred . . . . : : . for consensus- based results, non- concordant, where tools disagree on the classification, were considered incorrect. matthews correlation coefficient (mcc) was calculated as follows: mcc = [(tp×tn)−(fp×fn)]√ (tp+fp)×(tp+fn)×(tn+fp)×(tn+fn) lr+ is the positive likelihood ratio; lr− is the negative likelihood ratio. fn, false negatives (ie, pathogenic variants predicted to be benign); fp, false positives (ie, benign variants predicted to be pathogenic); tn, true negatives (ie, benign variants predicted to be benign); tp, true positives (ie, pathogenic variants predicted to be pathogenic). o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n a u g u st . d o w n lo a d e d fro m https://dx.doi.org/ . /jmedgenet- - https://dx.doi.org/ . /jmedgenet- - http://jmg.bmj.com/ gunning ac, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics those tools with a numerical output (sift, polyphen- , revel and clinpred) to more accurately compare their performance. a unique threshold was selected for each tool to calculate the speci- ficity when sensitivity was set to . . in order to include gavin in this analysis, a third analysis was performed, whereby each tool's specificity was measured when the threshold was adjusted to set the sensitivity identical to that of gavin. results classification of variant sources we compared the feature list of all tools benchmarked in this study (polyphen- , sift, revel, gavin and clinpred) and, in the case of the meta- predictors, the tools that they use as part of their algorithm (mpc, mutpred, vest, cadd, dann, snpeff, fathmm, fitcons and mutationtaster ). features were split into five broad categories: conservation, genetic variation, functional evidence (nucleotide), functional evidence (protein) and amino acid properties (see figure and online supplementary figure s ). in general, the meta- predictors employ a wider variety of sources and are less heavily reliant on conservation alone. cadd/dann and fitcons, and by exten- sion gavin and clinpred, are the only predictors with features within the functional (nucleotide) category and are therefore able to predict the pathogenicity of a variant in the context of its nucleotide change, regardless of whether there is a resultant amino acid change. benchmarking predictor performance in the open and clinical datasets initially, each of the tools was benchmarked according to the threshold provided by the tools’ authors. this analysis involved a dichotomisation of scores with no intermediate range (see table ). the distribution of scores from sift, polyphen- , revel and clinpred is shown in figure and receiver operating char- acteristic (roc) curves are shown in figure . of the tools with numerical outputs, clinpred has the highest discriminatory power for the open dataset with an area under the roc curve (auc) of . , while revel has the highest auc for the clin- ical dataset of . . the two meta- predictors outperformed sift and polyphen- in both datasets. in agreement with tool author benchmarking, – the meta- predictors revel, clin- pred and gavin were highly proficient at classifying the vari- ants in the open dataset, achieving sensitivities of . , . and . , and specificities of . , . and . , respectively. for variants in the clinical dataset, although the sensitivity of each tool remained largely constant, the specificity of all tools dropped considerably. for revel, clinpred and gavin, spec- ificity is reduced to . , . and . , respectively (table ). it was apparent that the threshold suggested by the tools’ authors was not well suited to both datasets, given the tools’ high sensitivity but low specificity in the clinical dataset. in order to correct for this, we performed a supplementary anal- ysis for those predictors which gave a numerical output (sift, figure violin plot showing variant scores for sift, polyphen- , revel and clinpred using two datasets. open dataset—blue; clinical dataset—red; pathogenic variants—filled; benign variants—unfilled. plot was generated in r using the 'vioplot' function in the 'vioplot' library. for ease of comparison, sift scores have been inverted. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n a u g u st . d o w n lo a d e d fro m https://dx.doi.org/ . /jmedgenet- - http://jmg.bmj.com/ gunning ac, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics polyphen- , revel and clinpred). here, a variable threshold was allowed for each tool to give a common sensitivity of . (ie, pathogenic variation is called correctly % of the time). the threshold required to give a sensitivity of . in each tool is shown in online supplementary table s . the specificity of each tool at the determined threshold is shown in online supple- mentary figure s . when allowed a variable threshold, the tools’ specificity increased significantly, with polyphen- , sift, revel and clinpred achieving a specificity of . , . , . and . for the open dataset, and . , . , . and . for the clin- ical dataset, respectively. in order to include gavin in this anal- ysis, a third analysis was performed in which each tool was given a threshold to match the sensitivity achieved by gavin in each of the datasets. the specificity of all five tools is shown in online supplementary figure s , and the sensitivity and threshold for each tool is shown in online supplementary table s . use of individual tools versus a consensus-based approach between multiple tools in accordance with current variant classification guidelines, we investigated the effect of performing a consensus- based analysis, using two commonly used tools, sift and polyphen- , and two meta- predictors, revel and clinpred, to determine whether this combined approach has improved sensitivity/specificity over the individual tools. figure shows the true concordance rate (correct classification by all tools), false concordance rate (incor- rect classification by all tools) and discordance rate (disagreement between tools) for each of these tool pairings for the pathogenic and benign variants in both datasets. within the clinically rele- vant dataset, the tools are either falsely concordant or discor- dant for ~ % of pathogenic variants but ~ % of benign variants. the sensitivity and specificity of this approach is shown in table . use of a consensus- based approach may introduce a third ‘discordance’ category to the classification where tools disagree and no in silico evidence can be used, which applied to % and % of variants when considering the concordance of polyphen- and sift, and % and % when considering the concordance between revel and clinpred, for the open and clinical datasets, respectively. an alternative ‘majority rule’ method can instead be applied. here, more than three tools are used, and the result agreed by > % of tools selected. this method eliminates the ‘discordance’ figure receiver operating characteristic (roc) curves for sift, polyphen- , revel and clinpred using two datasets. open dataset—blue; clinical dataset—red. generated in r using the ‘roc’ and ‘plot.roc’ functions in the ‘proc’ library. area under the roc curve (auc) was calculated in r using the ‘roc’ function. for ease of comparison, sift scores have been inverted. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n a u g u st . d o w n lo a d e d fro m https://dx.doi.org/ . /jmedgenet- - https://dx.doi.org/ . /jmedgenet- - https://dx.doi.org/ . /jmedgenet- - https://dx.doi.org/ . /jmedgenet- - https://dx.doi.org/ . /jmedgenet- - https://dx.doi.org/ . /jmedgenet- - http://jmg.bmj.com/ gunning ac, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics category, as dissenting tools are ignored. two majority- based analyses were performed using ( ) all five tools (sift, poly- phen- , revel, gavin and clinpred) and ( ) using only the meta- predictors (revel, gavin and clinpred). the sensi- tivity and specificity of this majority- based approach is shown in online supplementary table s . while this approach did improve on the strict concordance approach outlined previously, and is commonly applied in clinical genomics, the false concordance was still high and the highest specificity in the clinical dataset, achieved using a majority voting based approach with all five tools, was . (in contrast to the specificity of . achieved by revel in the same dataset). discussion we have compared the performance of five in silico pathoge- nicity predictors—two tools used routinely in variant classi- fication (sift and polyphen- ) and three recently developed clinical meta- predictors (revel, clinpred and gavin)—using two variant datasets: an open dataset collated using the selec- tion strategy commonly employed when benchmarking tool performance, and a clinically representative dataset composed of rare and novel variants identified through high- throughput research and clinical sequencing with manual classification. overall, the data herein show that meta- predictors have a greater sensitivity and specificity than the classic tools in both variant datasets. however, despite the increased accuracy of the meta- predictors, all tools performed substantially worse in the clinical dataset compared with the open dataset. this difference in tool performance illustrates the importance of considering the provenance of variants when benchmarking tools and how overfitting of a classifier to the training dataset can occur when increasingly large sets of variant features are used. the two data- sets herein were constructed using very different methodologies, which determine the variants present within each. the open dataset, composed of variants derived from online repositories, is modelled on the methods commonly used when constructing test datasets. the tools performed universally well when char- acterising this dataset, indicating that these variants inherently possess features easily identifiable to the in silico predictors. in contrast, the clinical dataset is composed of variants identified through research and clinical next- generation sequencing pipe- lines, which had undergone multiple rounds of variant filtering figure concordance between tools separated by dataset and classification (pathogenic and benign). open dataset—blue; clinical dataset—red; pathogenic variants—top graph; benign variants—bottom graph. true concordance indicates that the tools agree and were correct. false concordance indicates that the tools agree but were incorrect. discordance indicates that the tools disagreed on the classification. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n a u g u st . d o w n lo a d e d fro m https://dx.doi.org/ . /jmedgenet- - http://jmg.bmj.com/ gunning ac, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics and selection. many variants within the open dataset would be automatically filtered out of the clinical dataset, based on maf for example, and this dataset therefore gives a more representa- tive assessment of the performance of such tools in genomic diag- nostics laboratories—an assessment not previously performed. our analysis suggests that revel performs best when clas- sifying rare variants routinely identified in clinical sequencing pipelines, with an auc for our clinical dataset of . , followed closely by clinpred with an auc of . (figure ) and with a higher specificity than gavin in a direct (although suboptimal) comparison (online supplementary figure s ). while the revel team does not suggest a strict threshold for categorisation, in our analysis for the clinical dataset, a threshold of . gave a sensitivity of . , and a specificity of . , which is comparable with previous studies’ threshold of . . current guidelines on the classification of variants indicate that evidence should only apply when multiple tools are concor- dant. however, the use of concordance may introduce a third category to variants classification (discordance), where there is disagreement between tools and therefore the tools cannot be used as evidence to categorise the variant as either benign or pathogenic. the use of a majority- voting system appears to improve performance over a strict concordance approach, but our data show that both concordance methodologies give a lower sensitivity and specificity than the use of either of these tools in isolation, and furthermore that their performance is below that of the meta- predictors. as with all similar studies, we were limited by the availability of novel variants absent from online databases such as gnomad. the use of under- represented and genetically isolated popula- tions, such as the amish, allowed for the identification of several novel benign variants and suggests that such populations may be a rich source for future studies. we also identified several both pathogenic and benign variants in a clinical population through a translational research study (ddd). while steps were taken to ensure that the benign variants attained from this group were indeed benign (all variants were present within either monoal- lelic genes or in biallelic genes in a homozygous state, and were annotated by the referring clinician as having no contribution towards the patient’s clinical phenotype), nonetheless it cannot be guaranteed that the variants had no impact of protein func- tion. the study underlines the need for improved data- sharing between clinical laboratories, including both pathogenic and benign rare variants. this study supports the adoption of in silico meta- predictors for use in variant classification but recommends the use of a single meta- predictor over a consensus- based approach, as recommended by current acmg guidelines. each of the tools uses different though heavily overlapping data sources and the feature list used by a tool should be carefully considered before the tool is used. our results also suggest that tools that use gnomad data directly may have low specificity when classi- fying rare or novel variants and that care should be taken when using these tools in conjunction with the acgs guidelines, as presence in or absence from the gnomad database is already accounted for in other evidence criteria. although use of a meta- predictor tool offers advantages over the use of previously available and widely adopted in silico tools, there remain issues to be addressed before they can be used at a level greater than supporting evidence for clinical variant interpretation. twitter adam c gunning @gunningac, james fasham @jamesfasham, andrew h crosby @rdexeter, sian ellard @ellardsian, emma l baple @rdexeter and caroline f wright @carolinefwright acknowledgements we wish to thank all the patients and family members who participated in the study. we also thank dr michael cornell and dr angela davies, of the university of manchester. this work was supported by the wellcome trust [wt /z/ /z] and [wt /a/ /z]. se is a wellcome senior investigator. the ddd study presents independent research commissioned by the health innovation challenge fund (grant number hicf- - ), a parallel funding partnership between the wellcome trust and the department of health, and the wellcome trust sanger institute (grant no. wt ). see nature ; : – or www. ddduk. org/ access. html for full acknowledgement. contributors se and cfw conceived of and designed the study. ahc, se, elb and cfw provided the data. acg, vf and jf performed the data analysis. the manuscript was written with input from all authors. funding this work was supported by the wellcome trust [wt /z/ /z] and [wt /a/ /z]. competing interests none declared. patient consent for publication not required. provenance and peer review not commissioned; externally peer reviewed. data availability statement data are available in a public, open access repository. all data relevant to the study are included in the article or uploaded as online supplementary information. the clinical dataset (online supplemental table s ) is released under the cc- by license. open access this is an open access article distributed in accordance with the creative commons attribution . unported (cc by . ) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. see: https:// creativecommons. org/ licenses/ by/ . /. orcid ids adam c gunning http:// orcid. org/ - - - james fasham http:// orcid. org/ - - - caroline f wright http:// orcid. org/ - - - references richards s, aziz n, bale s, bick d, das s, gastier- foster j, grody ww, hegde m, lyon e, spector e, voelkerding k, rehm hl, acmg laboratory quality assurance committee. standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology. genet med ; 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iso- ; iso- . fly ; : – . shihab ha, gough j, cooper dn, stenson pd, barker gla, edwards kj, day inm, gaunt tr. predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden markov models. hum mutat ; : – . gulko b, hubisz mj, gronau i, siepel a. a method for calculating probabilities of fitness consequences for point mutations across the human genome. nat genet ; : – . schwarz jm, rödelsperger c, schuelke m, seelow d. mutationtaster evaluates disease- causing potential of sequence alterations. nat methods ; : – . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n a u g u st . d o w n lo a d e d fro m http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /j.ajhg. . . http://dx.doi.org/ . /j.ajhg. . . http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /j.ajhg. . . http://dx.doi.org/ . /j.ajhg. . . http://dx.doi.org/ . /j.cct. . . http://dx.doi.org/ . /ci http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /humu. http://dx.doi.org/ . /humu. http://dx.doi.org/ . /humu. http://dx.doi.org/ . /nar/gky http://dx.doi.org/ . /nar/gky http://dx.doi.org/ . /hmg/ddu http://dx.doi.org/ . /journal.pcbi. http://dx.doi.org/ . /s - ( ) - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /nar/gki http://dx.doi.org/ . /gim. . http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /bioinformatics/btp http://dx.doi.org/ . / - - -s -s http://dx.doi.org/ . /ng. http://dx.doi.org/ . /ng. http://dx.doi.org/ . /bioinformatics/btu http://dx.doi.org/ . /fly. http://dx.doi.org/ . /humu. http://dx.doi.org/ . /ng. http://dx.doi.org/ . /nmeth - http://jmg.bmj.com/ 	assessing performance of pathogenicity predictors using clinically relevant variant datasets 	abstract 	introduction 	materials and methods 	open dataset 	clinical dataset 	transcript selection and variant annotation 	tool benchmarking 	results 	classification of variant sources 	benchmarking predictor performance in the open and clinical datasets 	use of individual tools versus a consensus-based approach between multiple tools 	discussion 	references personality processes and individual differences moral identity and the experience of moral elevation in response to acts of uncommon goodness karl aquino university of british columbia brent mcferran university of michigan marjorie laven laven communication services, nanaimo, british columbia, canada four studies using survey and experimental designs examined whether people whose moral identity is highly self-defining are more susceptible to experiencing a state of moral elevation after being exposed to acts of uncommon moral goodness. moral elevation consists of a suite of responses that motivate prosocial action tendencies. study showed that people higher (vs. lower) in moral identity centrality reported experiencing more intense elevating emotions, had more positive views of humanity, and were more desirous of becoming a better person after reading about an act of uncommon goodness than about a merely positive situation or an act of common benevolence. study showed that those high in moral identity centrality were more likely to recall acts of moral goodness and experience moral elevation in response to such events more strongly. these experiences were positively related to self-reported prosocial behavior. study showed a direct effect on behavior using manipulated, rather than measured, moral identity centrality. study replicated the effect of moral identity on the states of elevation as well as on self-reported physical sensations and showed that the elevation mediates the relationship between moral identity, witnessing uncommon goodness, and prosocial behavior. keywords: morality, identity, moral identity, moral elevation, prosocial behavior “the evil that men do lives after them; the good is oft interred with their bones.”—william shakespeare, julius caesar conventional wisdom and daily experience hold that people remember the painful and traumatic moments in their lives more vividly and with greater accuracy than those that brought pleasure or joy. most people can readily bring to mind the memory of having been hurt, betrayed, or unfairly treated by another person. when they do so, they are often able to faithfully summon the feelings, the thoughts, and even the physical sensations that ac- companied the event. but ask them to remember a time when someone showed kindness or generosity and they struggle to reclaim the full intensity of these happy moments. the features of human memory and cognition that lead people to recall the bad more easily than the good have been well-documented (baumeis- ter, bratslavsky, finkenauer, & vohs, ). moreover, the pro- pensity of people to attend to the bad more than the good extends into the realm of social perception. when people judge others, they are more sensitive to signs of immorality than morality, both in what information they attend to and how they weigh that informa- tion (e.g., de bruin & van lange, ; reeder & coovert, ; wojciszke, bazinska, & jaworski, ). the causal attribution literature shows that negative behaviors exhibited by others have a greater impact than positive behaviors on how people perceive them (e.g., birnbaum, ; reeder & brewer, ; reeder & coovert, ; ybarra, ), and people often discount positive behaviors by attributing them to normative pressures or social desirability concerns rather than to internal states of goodness or virtue (ybarra, ; ybarra & stephan, ). as shakespeare made plain, the evil that men and women do frequently leaves a more lasting stain on people’s consciousness than the good. but it is also true that people can sometimes be karl aquino, organizational behavior and human resources division, sauder school of business, university of british columbia, vancouver, british columbia, canada; brent mcferran, marketing area, stephen m. ross school of business, university of michigan; marjorie laven, laven communication services, nanaimo, british columbia, canada. this research was supported by grants from the social sciences and humanities research council of canada awarded separately to karl aquino and brent mcferran. we thank julie christine marchand and shelly singh of ipsos interactive custom panels for their assistance, as well as laurie barclay, miranda abild, kiran nagra, david fisette, and susan kuo for their help with data collection. correspondence concerning this article should be addressed to karl aquino, organizational behavior and human resources division, sauder school of business, university of british columbia, main mall, vancouver, british columbia v t z , canada, or brent mcferran, marketing area, stephen m. ross school of business, university of michigan, tappan street, ann arbor, mi . e-mail: karl.aquino@sauder.ubc.ca or mcferran@umich.edu journal of personality and social psychology © american psychological association , vol. , no. , – - / /$ . doi: . /a moved and even transformed when they witness acts of extraor- dinary moral goodness. psychologists have described the experi- ence of being affected by such acts as a state of moral elevation (haidt, , ; keltner & haidt, ). moral elevation consists of a distinctive feeling of warmth and expansion that is accompanied by admiration, affection, and even love for the per- son (or people) whose exemplary behavior is being observed (haidt, , ). moral elevation has been likened to the aesthetic experience felt when one beholds a beautiful object or scene. but unlike purely aesthetic experiences, moral elevation can sometimes lead to behavioral changes, eliciting action tendencies associated with the desire to draw closer to other people and to show greater social responsiveness to the needs and interests of others (haidt, , ). notably, this orientation characterizes what ethicists (e.g., kant, ; singer, ) and psychologists (e.g., eisenberg, ; gilligan, ; kohlberg, ) have de- scribed as the defining feature of morality. our goal with the present research was to investigate whether some persons may be more susceptible than others to experiencing a state of moral elevation following exposure to examples of uncommon moral goodness. the variable we examine as a possible source of this increased susceptibility is moral identity. moral identity has been defined as the degree to which a person’s moral character is experienced as a central part of his or her overall self-concept (aquino & reed, ; blasi, ). moral identity has emerged as a useful construct in developmental, social, and organizational psychology for explaining various aspects of moral functioning (e.g., aquino, freeman, reed, lim, & felps, ; aquino, reed, stewart, & shapiro, ; blasi, , ; det- ert, treviño, & sweitzer, ; hardy, bhattacharjee, reed, & aquino, ; lapsley & lasky, ; reynolds & ceranic, ; skarlicki, van jaarsveld, & walker, ; weaver, ). several writers (e.g., aquino & freeman, ; aquino & reed, ; blasi, ; lapsley & lasky, ) have argued that moral identity is a reliable determinant of moral behavior, and a growing body of empirical evidence supports this claim (see shao, aquino, & freeman, , for a review). there is also evidence that people for whom moral identity is an important basis for self-definition (vs. those for whom moral identity is of low importance) evaluate helping behaviors exhibited by others as being more caring, moral, and socially responsible (reed, aquino, & levy, ). on the basis of empirical evidence and recent theoretical elaborations of moral identity’s role in moral functioning (e.g., aquino & free- man, ), there are reasons to believe that it can partly explain people’s responses to witnessing acts of uncommon moral good- ness. we tested this possibility in four studies using different operationalizations of an act of uncommon goodness and measur- ing several internal states that theorists have associated with the experience of moral elevation. moral elevation and moral identity haidt ( ) suggested that moral elevation, along with grati- tude, is one of the moral emotions that he calls “other praising” (p. ). moral elevation shares properties with other emotions. for example, it results from an eliciting event, produces physical changes in the person, is a phenomenological experience, and motivates a certain type of action tendency (ekman, ; scherer, ; shweder, ). emotions have been described as consist- ing of a suite of related components that include thoughts, feelings, motivations, and physical changes (ekman, ; izard, ). among the thoughts and motivations that have been associated with moral elevation are the desire to become a better person and the wish to open one’s heart to others (keltner & haidt, ; silvers & haidt, ). algoe and haidt ( ) also showed that moral elevation involves physical sensations like a warmth or pleasant feeling in the chest or a lump in the throat. silvers and haidt ( ) speculated that some of these sensations might be caused by the release of the hormone oxytocin, which rises in levels when people receive signals of trust (zak, kurzban, & matzner, ). arguably, seeing someone perform a virtuous act can be interpreted as a signal of trustworthiness, which could explain the link between witnessing such acts and the physiolog- ical sensations associated with moral elevation. according to haidt ( ), the components of the moral eleva- tion experience initiate action tendencies like the wish to emulate the moral exemplar and to act prosocially. algoe and haidt ( ) provided empirical support for this hypothesis by showing that people who experience elevation are more likely to want to help others, give money to charity, and list prosocial actions when asked to write about their life goals. there is also evidence that moral elevation can suppress the effect of ideological beliefs that might otherwise discourage social responsiveness toward certain peo- ple. freeman and his colleagues (freeman, aquino, & mcferran, ) showed that exposing people to acts of moral goodness led them to contribute more money to a charitable cause. notably, they also showed that moral elevation led some white study partici- pants (those high in social dominance orientation, or sdo) to override beliefs that might otherwise lead them to withhold dona- tions to an outgroup cause (the united negro college fund, or uncf). they found that whites who scored high on sdo donated less money to the uncf than did those who scored low; however, they also found that this relationship was attenuated when partic- ipants were exposed to a morally elevating act. other prior studies have also established a causal link between moral elevation and prosocial behavior (e.g., algoe & haidt, ; schnall, roper, & fessler, ). although we expected to replicate this finding in this research, we also sought to learn whether moral identity can influence the intensity of the moral elevation experience and whether this experience mediates the relationship between moral identity and prosocial behavior. moral identity is one of many possible identities that a person can use as a basis for self-definition (aquino & reed, ). there is growing evidence that this particular identity plays a key role in moral functioning by influencing how people interpret and respond to situations involving moral judgment and choice (shao et al., ). blasi ( ) described moral identity as an individual difference reflecting the degree to which being moral is a central or defining characteristic of a person’s sense of self. according to blasi ( ), the felt obligation to engage in a moral action is directly related to moral identity through the desire to maintain self-consistency. aquino and his colleagues (aquino et al., ; aquino & reed, ; aquino & freeman, ) proposed a social cognitive model of moral identity that defines this construct as a self-schema organized around a set of moral trait associations (e.g., honest, caring, compassionate). following other theorists (e.g., blasi, , ; lapsley & lasky, ), aquino and reed ( ) argued that people differ in the degree to which moral aquino, mcferran, and laven identity is experienced as being central to their overall self- definition. from a social cognitive perspective, this difference implies that the moral self-schema is more cognitively accessible for some persons than others (lapsley & narvaez, ). evidence supports the utility of a schema-based conceptualization of moral identity (aquino & reed, ; aquino, reed, thau, & freeman, ; olsen, eid, & johnsen, ; reed & aquino, ; reed et al., ; reynolds & ceranic, ). an assumption of aquino et al.’s ( ) social cognitive model is that certain situations can increase the cognitive accessibility of a person’s moral identity. for example, they showed that priming moral identity by asking people to recall the ten commandments made moral identity relatively more salient than other identities, which, in turn, influenced peoples’ behavioral intentions to act morally. however, other research (e.g., aquino & reed, ) also suggests that moral identity is more chronically active for some people than others, meaning that it tends to be readily available for processing and acting on social information. studies by aquino and his colleagues (aquino et al., ; aquino & reed, ) provide empirical evidence supporting both of these claims. what is missing from their social cognitive model is consideration of whether its accessibility can influence reactions to seeing others’ behavior. the social cognitive principle that peo- ples’ identity schemas can affect their interpretation of social information (e.g., fiske, ; james, / ; smith & semin, ) supports such a possibility, but no previous research has made this argument or tested it empirically in the context of witnessing acts of uncommon goodness. we conduct a first test of this conjecture by hypothesizing that a person’s moral identity can influence his or her responses to being exposed to such acts. reed and his colleagues (reed et al., ) provided an empir- ical basis for making this prediction. in one of the studies reported in their article (study ), they asked participants to evaluate the actions of a fictional company that organized a program for em- ployees to volunteer , hr to a community outreach organiza- tion that provides free job training to unemployed workers. they showed that people whose moral identity was more rather than less central to their self-concept evaluated the company’s actions as being more caring, moral, and socially responsible. one interpre- tation of their finding is that moral identity influenced the degree to which people saw the company as being concerned about the needs of others. it is interesting that reed et al. ( ) found that moral identity did not predict evaluations of a fictional company that donated money to the same outreach organization. they interpreted this result as showing that people assign greater moral weight to giving one’s time or effort to a moral cause than to simply giving money. we extend reed et al.’s ( ) findings by proposing that when people are exposed to acts of uncommon moral goodness, those whose moral identity is more central to their self-definition assign greater psychological weight, relevance, and value to these actions compared with people whose moral identity is less self-defining. as a result, the former will experience a heightened state of moral elevation relative to the latter. by testing whether moral identity can influence peoples’ responses to acts of uncommon moral goodness, we make an important theo- retical refinement to aquino et al.’s ( ) social cognitive model by showing how exposure to a particular type of social behavior— extraordinary moral goodness— can elicit a suite of responses from people whose moral identity occupies a central role in their self- concept. in turn, these responses act as a motivational impetus for prosocial behavior. by testing these relationships, we specify a psy- chological mechanism (moral elevation) that connects the interaction of moral identity and observations of others behavior to prosocial motivation, a connection aquino and his colleagues (aquino et al., ) did not make. what our article shares with their social cognitive model is that they both adopt an identity-situation framework as a basis for explaining some aspects of moral functioning. aquino and reed’s ( ) conceptualization of moral identity as a chronically accessible schema posits that it consists of two dimen- sions, one of which reflects a private experience of moral identity centrality, which they call internalization, and the other its public expression, which they call symbolization. these dimensions corre- spond to theories of the self that posit that self-awareness can be characterized by an internal introspective awareness of one’s inner thoughts and feelings and an externally and active self as a social object that impacts others (fenigstein, scheier, & buss, ). prior research shows that both dimensions of moral identity exhibit positive relationships with various morally relevant constructs. for example, symbolization has been found to be positively related to religiosity, volunteerism, charitable giving, and the willingness to aid outgroups (aquino & reed, ; reed & aquino, ; reynolds & ceranic, ). internalization has been positively related to moral reasoning, volunteering, satisfaction from volunteering, and donating cans of food to the needy (aquino & reed, ; reynolds & ceranic, ). we expected the two dimensions of moral identity to show the same directional relationship to many kinds of moral outcomes; however, in the specific case of peoples’ responses to witnessing an act of un- common moral goodness, we hypothesized that the internalized facet (moral identity centrality) would be a more robust predictor of these responses than the symbolic facet. we speculated that this would occur because the internalized facet should, by logical inference, be more closely connected to subjective, private states or experiences like moral elevation than would symbolization, its publicly oriented counterpart. accordingly, we hypothesized that the interaction of exposure to acts of uncommon goodness and the internalization dimension of moral identity would explain more variance in moral elevation than would the interaction of exposure to the same act and the symbolization dimension of moral identity. overview of studies we tested our hypotheses in four studies. in study , we tested whether moral identity interacts with exposure to an act of uncom- mon moral goodness to predict emotions and thoughts associated with moral elevation. in study , we tested whether moral identity predicted the likelihood that people would recall having witnessed an act of uncommon moral goodness as well as the intensity of moral elevation responses to such acts. study also tested whether the state of moral elevation mediated the relationship between moral identity and self-reported prosocial motivation. study provided causal evidence for the role of moral identity centrality by manipulating rather than measuring it. study also tested following aquino et al. ( ), we treat the internalization dimension of moral identity in aquino and reed’s ( ) model as being synonymous with the concept of moral identity centrality. therefore, we use them interchangeably throughout the article. moral identity and moral elevation whether the interaction of moral identity and exposure to an act of moral goodness could motivate actual behavior. finally, study provided evidence that moral elevation mediates the relationship between the interaction of moral identity and exposure to acts of uncommon goodness and prosocial behavior. study we hypothesized that people high in moral identity would experience a stronger moral elevation experience when exposed to an act of uncommon goodness than would people who were low in moral identity. however, we did not expect moral identity to influence elevation-related responses after exposure to stimuli that elicit positive but nonmoral emotions or to behavior that represents a more common example of moral goodness. finally, we expected the interaction between the internalization dimension of moral identity and exposure to uncommon goodness to be a stronger predictor of moral elevation than the interaction between symbol- ization and such exposure. method sample and procedure. four hundred thirty-six undergrad- uate students ( men, women, not reported) from a canadian university participated in the study for partial course credit. they averaged . (sd � . ) years of age. data were collected using an online survey. participants were recruited for a study titled “understanding reactions to the media” and were told the study was investigating “how individuals react to newspaper articles.” the study consisted of two parts. in the first part, participants were asked to complete measures of moral identity and demographic information. the second part contained the ma- nipulation, with participants being randomly assigned (between subjects) to one of four groups. experimental manipulation. the design was a between- subjects experiment in which participants in each of four condi- tions read a one-page news story. two of the stories were taken from a study by freeman et al. ( ) and were designed to elicit either moral elevation or nonmoral but positive emotions. one of the stories has been shown to more effectively elicit moral eleva- tion than the other (freeman et al., ). in the uncommon goodness (ug) story condition, participants read about how an amish community responded with extraordinary grace and for- giveness after charles roberts opened fire in an amish school- house, killing five girls and injuring another five before killing himself. the story described how hours after their children were killed, several amish went to see roberts’s widow to offer for- giveness and express sympathy and later offered financial assis- tance to her and her children. in the positive emotion (pe) condi- tion, the news story described the reactions of a couple to viewing a sunset over the ocean that was “absolutely incredible.” to ensure that the story seemed newsworthy, the couple was quoted as saying, “i’ve just never seen anything like that . . . witnessing that kind of thing has the effect of changing people’s lives forever.” the amish story of forgiveness is an act of uncommon good- ness that is rare by definition. however, it may be that exposure to more common forms of benevolence might be sufficient to elicit elevation, particularly among people high in moral identity. we tested this possibility by including two additional story conditions in our design. the first was a common goodness non-amish (cgna) condition where participants read a real news story about goodness grows, a group that created and maintains a community garden for their neighborhood and has a plot set aside to grow food for another group that cooks meals for the homeless. the second was a common goodness amish (cga) condition where partici- pants read a real article about how the kentucky amish helped their neighbors during an extended power outage, providing ker- osene lamps and making their neighbors coffee each morning. featuring the amish engaging in an act of common goodness made this story more directly comparable to the ug story. after they read each story, participants completed several measures assessing the extent to which they experienced moral elevation. measures. moral identity. we measured moral identity using a -item instrument developed by aquino and reed ( ) that was de- signed to assess the importance of this identity to the self. their instrument is based on a conceptualization of moral identity as a schema organized around a set of moral trait associations (e.g., compassionate, kind, honest) and loads consistently on two dimen- sions, which they labeled internalization and symbolization. re- spondents answered each item on a -point likert-type scale ( � strongly disagree, � strongly agree). sample items for the internalization subscale include “being someone who has these characteristics is an important part of who i am” and “i strongly desire to have these characteristics.” the symbolization subscale consists of items that include “i am actively involved in activities that communicate to others that i have these characteristics” and “the types of things i do in my spare time (e.g., hobbies) clearly identify me as having these characteristics.” reliabilities were � � . and � � . for internalization and symbolization, respectively. moral elevation. the state of elevation consists of several related components (haidt, , ). we assessed the emo- tional component by asking participants to rate on a -point scale ( � not at all, � very much) how much they felt the following emotions after reading the news story: compassion, inspired, awe, and admiration. the scores were then averaged (� � . ). the items have been used previously (freeman et al., ) and are consistent with the emotions that haidt ( , ) described as resulting from witnessing acts of uncommon moral goodness. we also assessed whether reading the story influenced participants’ views of humanity in general and whether it increased their desire to be a better person (freeman et al., ). it has been suggested that exposure to morally virtuous acts can lead people to adopt a more positive view of humanity (haidt, , ). views of humanity were assessed with the following items: “there is still some good in the world,” “people are really good,” “the world is full of kindness and generosity,” “the actions of most people are admirable,” and “what a nice person (or what nice people).” respondents answered on a -point scale ( � never, � always), and their responses were averaged (� � . ). according to haidt ( , ), the state of moral elevation is likely to involve at least a temporary desire to be a better person, and higher levels should be indicative of having experienced a more intense state of moral elevation. desire to be a better person was assessed with six items: “i want to be more like the person/people in the story,” “the person/people in the story have shown me how to be a better person,” “i am going to try to follow the story’s example,” “i need to do more to help other people,” “i can learn a lot from the aquino, mcferran, and laven person/people in the story,” and “the person/people in the story are my new role models.” participants responded to these items by indicating on a -point scale, ranging from � never to � always, how often they had (or were still having) these thoughts while reading the story. items were averaged to form a scale (� � . ). positive emotions. we assessed whether participants experi- enced the following positive but nonmoral emotions: joy, pleasure, and enthusiasm ( � not at all, � very much). we assessed these emotions to demonstrate that the effects of moral identity on the emotional component of elevation would occur only for moral emotions and not positive emotions in general. the three nonmoral emotions were averaged to form a scale (� � . ). control variables. in this and all subsequent studies, we con- trolled for gender ( � male, � female) to account for possible differences in the value men and women place on acts of caring or benevolence (gilligan, ). we included the measure of positive emotions as a control variable so that we could examine whether the moral elevation components explained additional variance in our dependent variables above and beyond these emotions. results descriptive statistics for the study variables are shown in table . we tested the effectiveness of our manipulations by creating an aggregated moral elevation variable consisting of the three measured components. the three elevation measures were on different scales, so we first standardized them before summing them for analysis. an analysis of variance involving the four story conditions performed on the aggregate moral elevation variable revealed only a difference between the merely positive story (mpe � �. , sd � . ) and all three treatment conditions (mcgna � . , sd � . ; mcga � . , sd � . ; muga � . , sd � . ; all ps � . ; ds ranged from . to . ), none of which differed significantly from each other. hierarchical regression analysis was used to test the hypothe- sized interaction of moral identity and exposure to uncommon goodness. gender and positive emotions measures were entered in step . the internalization and symbolization subscales and the dummy-coded main effects for the cgna, cga, and ug stories were entered in step . the pe condition was treated as the reference category. in step , we entered the two-way interactions involving the two dimensions of moral identity and the cgna story. step added the two-way interactions involving the moral identity dimensions and the cga story. finally, we entered the two interactions involving the moral identity dimensions and the ug story in step . we entered and tested the interaction terms in this order to conduct a conservative test of our hypothesis. by entering the focal interaction terms last in our model, we can conclude that any variance explained by the focal interactions is above and beyond that which is accounted for by general positive emotion and the other interactions involving common goodness (measured by �r and significant coefficients). all interaction terms were mean centered to reduce multicollinearity (aiken & west, ). results are shown in table . we first performed separate regression analyses for each of the elevation components. in all of these regressions, the incremental variance in r in the final step of the regression was significant (all ps � . ). inspection of the individual interaction terms in the final step showed that the hypothesized internalization � ug story inter- action was significant in all four models (all ps � . ). analysis of simple slopes (aiken & west, ) of the significant interactions revealed that the general pattern among the components of elevation was identical, namely, a stronger relationship among those high versus low in moral identity centrality. for this reason, and to simplify the presentation of our results, we performed regression analysis on the aggregated moral elevation measure. aggregation is further jus- tified by the relatively high intercorrelations among the components, which ranged from . to . (all ps � . ). a five-step regression analysis on the aggregated measure found that the interactions involving internalization or symbolization and the two conditions featuring stories of common goodness did not explain significant incremental variance. however, in the final step, significant additional variance was explained (�r � . , p � . ). inspection of the individual interaction terms showed that the interaction between internalization and the elevating story manipulation was significant, b � . , se � . , t � . , p � . . the results of this analysis are shown in table . analysis of simple slopes for the interaction predicting the aggregated measure of moral elevation replicated the patterns found when we analyzed each elevation component separately. participants who were high in internalization experienced greater moral elevation after reading the ug story than the merely positive one (b � . , se � . , t � . , p � . ), whereas those low in internalization experienced significantly less elevation, but the slope was still positive (b � . , se � . , t � . , p � . ). we conducted a hierarchical regression on the positive but nonmoral emotions to see whether our findings for elevation would replicate. they did not. results showed that moral identity and the story about uncommon goodness did not interact to predict positive emotions ( p � . ), indicating that participants high in internalization were not simply experiencing more general positive emotion in response to the uncommon goodness story. discussion study supported our hypothesis that when exposed to an act of uncommon moral goodness, people whose moral identity was more central to their self-definition, as indicated by their score on the internalization dimension of aquino and reed’s ( ) mea- sure, would report stronger moral elevation responses than would those whose moral identity was less central. it is important to note that the interaction of moral identity and experimental condition was found only when people were presented with a story of uncommon moral goodness and not when they read a story about more mundane moral behaviors or a beautiful sunset. of the two dimensions of moral identity measured by aquino and reed’s removing positive emotions as a covariate does not alter the interpre- tation of the results across the studies. for brevity, we present only the aggregated variable. full results at each step of the regression are available from the authors. only the internalization � ug story interaction replicates and explains incremental variance for each subcomponent. full results are available on request. because the ug story did not elicit higher baseline levels of elevation as a main effect, it remains possible that those low in moral identity view the ug story through the lens of tragedy (the massacre) rather than the uplifting act, which is why the this condition’s main effect mean is not higher than that in the cgna or cga treatment conditions. future research should examine this possibility. moral identity and moral elevation ( ) instrument, we found that only the internalization dimen- sion amplified the elevation states of participants who we exposed to an act of uncommon moral goodness. this finding is consistent with our theoretical expectations and makes logical sense given that the internalization dimension has been conceptualized by aquino and reed ( ) as representing the private aspect of moral identity. it should therefore be more closely associated with constructs that are experienced internally rather than projected externally. study supported our hypothesized interaction of moral identity and exposure to an act of uncommon moral goodness, but a limitation of the study is that it did not provide any evidence for the possible consequences of moral elevation, namely, that it should motivate people to be more prosocial. we conducted study to investigate this possibility using a representative sample of adults who recalled a time when they witnessed an act of uncom- mon moral goodness. we also explored whether the two dimen- sions of moral identity predict whether people whose moral iden- tity was more central to their self-definition would more easily recall such an act. if so, it would suggest that moral identity can explain not only immediate responses to uncommon goodness but perhaps the longer term encoding and cognitive accessibility of exemplary moral behaviors in memory. study we tested three hypotheses in study . first, we hypothesized that people who are high rather than low in moral identity inter- nalization would be more likely to recall witnessing an act of uncommon moral goodness. second, we expected moral identity internalization to be positively and more strongly related to the intensity of the moral elevation experience for people who recalled witnessing uncommon goodness compared with symbolization. third, we hypothesized that moral elevation would be positively related to the motivation to act prosocially as a result of witnessing the elevating act. method sample and procedure. the sample for study was ob- tained from the ipsos opinion panel, which includes , cana- dian adult panelists. the sample is representative of the canadian population on the basis of gender, age, region, income, and lan- guage. by using a representative sample, we increase the potential generalizability and external validity of our findings. respondents were invited to join the panel by completing a short online profil- ing questionnaire. an invitation to complete the survey online was table study descriptive statistics and correlations among key variables variable m (scale max) sd . moral elevation (aggregate measure) . (z score) . — . �� . �� . �� . �� . �� . . �� . elevation emotions . ( ) . — . �� . �� . �� . �� . �� . �� . views . ( ) . — . �� . �� . � . �� . �� . desire . ( ) . — . �� . � . �� . �� . positive emotions . ( ) . — . � . �� . �� . internalization . ( ) . — . �� . �� . symbolization . ( ) . — . �� . gender . ( ) . — note. n � . for gender, � male, � female. � p � . . �� p � . . table study hierarchical multiple regression results (aggregate elevation measure only) predictor step step step step step gender . �� . �� . �� . �� . �� positive emotions . �� . �� . �� . �� . �� common goodness non-amish (cgna) . �� . �� . �� . �� common goodness amish (cga) . . . . uncommon goodness (ug) . �� . �� . �� . �� internalization (int) . �. . �. � symbolization (sym) . �� . �� . � . int � cgna . . . �� sym � cgna �. �. �. int � cga �. �� �. sym � cga . . int � ug . �� sym � ug . �r . �� . . . �� model r . . . . . note. unstandardized regression weights are presented. � p � . . �� p � . . aquino, mcferran, and laven sent to all panelists with the opportunity to win a $ cash prize, which was awarded through a random draw of the individuals who participated in the survey. ipsos fielded the questionnaire and made the results available to us for analysis. four hundred forty- three panelists fully completed the survey, which is a response rate of approximately %. the response rate may have been slightly lower than average given the longer length of the survey and because it was launched around the mid-december holiday season. respondents ranged in age from to years (m � . years, sd � . ). of the sample, . % were female and . % were currently employed ( . % full time, . % part time). of the two official languages, respondents primarily identified themselves as english speaking ( . %), with the remainder ( . %) indicating that they primarily spoke french. most respondents had at least some postsecondary education ( . %). participants were first asked the following question: at one time or another many of us have witnessed an unexpected and extraordinary act of human goodness, kindness, or compassion. we are often exposed to such events in movies, television, news magazines, or books. in this study, we are interested in finding out whether people also witness such events in their own lives. if you have witnessed such an act of goodness, it may have left you feeling more or less positive about yourself and your environment, or it may have had no effect at all. take a moment to think of the most recent time you observed such an act being performed. try to imagine the place, the circumstances, and the person or persons involved in the event. when you have brought these images to mind, answer the following questions about your experience. please be as specific as possible when providing your answers. have you ever experienced such an event? participants who recalled a morally elevating event were asked to provide further details about the elevating event to increase its sa- lience; namely, when it occurred, a brief description of the event, and information—such as gender, age, and relationship to the respon- dent—about the primary person(s) who performed the act. this was followed by measures of moral elevation and various prosocial be- haviors they might have engaged in as a result of witnessing the event they described. finally, participants completed the moral identity scale and questions regarding basic demographic information (includ- ing age, gender, occupation, and education). measures. event experience. participants were asked to indicate whether they had witnessed an event that fit the description of a morally elevating experience ( � no, � yes). they were also given space to type a short description of the event. moral identity. we used the same measure as we did in study . cronbach’s alpha reliabilities were . and . for the internal- ization and symbolization subscales, respectively. moral elevation. we assessed whether the event participants described elicited the emotional component of elevation with the same four emotions items used in study (� � . ). to reflect the more general events participants described, the items for views of humanity and desire to be a better person were modified slightly. four items regarding views of humanity were assessed on a -point likert-type scale ( � never, � always): “what a nice person,” “there are still a few good people out there,” “that was an extremely charitable act,” and “there is still some good in the world.” items were averaged to form a scale (� � . ). desire to be a better person was assessed with five items: “i want to follow that example,” “would i have done the same?” “how good a person am i?” “are my priorities in order?” and “i want to be a better person.” items were answered on a -point likert-type scale ( � never, � always) and averaged (� � . ). positive emotions. the items used in study were also used in this study to measure positive emotions (� � . ). motivation to act prosocially. we assessed whether partici- pants felt motivated to engage in prosocial behavior as a direct result of witnessing the morally elevating event with three items: “as a result of this experience, i felt more (or less) like doing good for others/helping someone in need/caring for people less fortunate than me” ( � much less, � much more). items were averaged to form a scale (� � . ). we performed a confirmatory factor analysis on all of the items described above to assess the dimensionality of our measures of elevating emotions, views of humanity, desire to be a better person, prosocial behavior, and positive emotions. the results of the cfa showed that all of the items loaded on their expected factors, and the five-factor model exhibited adequate fit, � ( ) � . , p � . , comparative fit index (cfi) � . , root-mean-square error of approximation (rmsea) � . . control variables. our sample consisted of a diverse group of adults, so we controlled for a number of variables that might influence the likelihood of an elevating experience in the ipsos panel. as in study , we controlled for gender and positive emotions. we controlled for age because developmental differ- ences in moral maturity and life experience might make older people more sensitive to moral acts (kohlberg, ; puka, ). age could also be associated with the probability of witnessing such acts, because it is logical to assume that the longer one is alive, the greater one’s chances of having observed a wide range of social behaviors. finally, we controlled for the length of time that had lapsed since the event occurred, given that it may be that events that occurred in the more distant past might be recalled as being less impactful. this variable was measured with the single item “if you can recall such an act, how long ago did it occur?” ( � less than one week ago, � two weeks to three months ago, � four to eight months ago, � nine to twelve months ago, � more than twelve months ago). results table shows the descriptive statistics and correlations for the study variables. event experiences. of the participants, ( . %) witnessed an event of moral goodness, with the majority of them occurring within the past months ( . %). after indicating whether they recalled such an event, participants briefly described it. examples of event descriptions are as follows: my aunt met a man at work (she barely knew him) who had just found out he had aids— he had no family at all. when he started getting ill and was in the hospital, she went to see him regularly until he passed away. the inclusion (or exclusion) of the control variables has no impact on any of our findings and does not significantly improve model fit. of the control variables, only gender had any effect, b � . , se � . , p � . (women had stronger reactions), and so, for clarity and parsimony, we present the model without the controls. moral identity and moral elevation one of our compatriots has had to take a leave without pay to look after her dying daughter. she has no savings and is a single mother who works hard to look after her family. one of us, who is very shy and rarely takes part in anything of a social nature, took it upon herself to make sure this woman has enough for rent and necessities. a good friend [ . . . ] who had plans to travel the world and live life big has taken custody of [a] -month-old baby whose natural parents was on heroin. i thought she was crazy, but this baby has brought a new meaning to her life. hypothesis tests. we performed a logistic regression with the control variables of gender and age included and the two moral identity subscales to test the likelihood of recalling or experiencing the event. results indicated that high scores on internalization increased the likelihood of reporting an elevating event (b � . , se � . , p � . ), but neither scores on symbolization nor any of the control variables were significant predictors of recall. this result supported our hypothesis that examples of uncommon moral goodness were more readily accessible for people higher versus lower in moral identity centrality. we conducted a path analysis using simultaneous regression to test our hypotheses that moral identity internalization is positively related to the experience of moral elevation and that moral eleva- tion is positive related to the motivation to act prosocially. we chose path analysis as our method for testing these hypotheses because we wanted to account for the interrelationship between the two dimensions of moral identity when testing for mediation. we used manifest indicators rather than taking a latent structural model approach because our relatively small sample size (only the people who reported witnessing a morally elevating event could be included) would result in a ratio of respondents to indicators of less than : , which can reduce the power of our tests in an structural equation modeling framework (chin, ). we estimated a path model that included the three elevation components as mediators of the two dimensions of moral identity and prosocial behavioral intentions (see figure ). the two dimen- sions of moral identity were permitted to covary, as were the disturbance terms for the three mediating variables. this model fit the data well, � ( ) � . , p � . , cfi � . , rmsea � . . the paths from internalization to elevating emotions (b � . , se � . , p � . ), views of humanity (b � . , se � . , p � . ), and desire to be a better person (b � . , se � . , p � . ) were all significant, whereas none of the paths from symbol- ization reached significance (all ps � . ). it is important to note that the paths from these variables to prosocial behavior were all significant (b � . , se � . , p � . , for elevating emotions; b � . , se � . , p � . , for views of humanity; b � . , se � . , p � . , for desire to become a better person), supporting our hypotheses. we tested for mediation in a path-analytic framework by compar- ing the proposed path model with alternative models that specified a direct path from internalization to prosocial behavior and from sym- bolization to prosocial behavior. the results of these analyses showed that neither of the additional paths significantly improved the fit of the model over the original structural model. including a direct path from internalization did not statistically improve the fit, � ( ) � . , � difference test p � . , nor did a second model that included a direct path from symbolization to behavior, � ( ) � . , � difference test p � . . these results indicate that the relationship between the internalization dimension of moral identity and prosocial behavior was fully mediated through the states associated with moral elevation. results from sobel tests verified this conclusion and were significant for each of the three paths: emotional component (sobel z � . , p � . ), views of humanity (sobel z � . , p � . ), and desire to be a better person (sobel z � . , p � . ). discussion study supported our hypothesis that people who witnessed an example of uncommon moral goodness would report stronger elevation-related reactions to the event if they were high rather the results are identical if conducted when the parameters are esti- mated separately and mediation conducted sequentially, using the methods described by baron and kenny ( ). standardizing and combining the three elevation components into the aggregated measure reported in study and running the mediation test results in even stronger effects (sobel z � . , p � . ) that are consistent with treating the components separately. we chose to present the path analytic model involving individual components so that interested readers would be able to see the specific relationship between moral identity and each of the components. table study descriptive statistics and correlations among key variables variable m sd . moral elevation . . — . �� . �� . �� . �� . �� . �� . �� . �� . �� �. . elevation emotions . . — . �� . �� . �� . �� . . . . �� . �� . views . . — . �� . �� . �� . �� . �� �. . �� . � . desire . . — . �� . �� . � . � �. . �� . � . prosocial behavior . . — . �� . �� . �. . �� . � . positive emotions . . — . . �. . � . � . gender . . — �. �. �� . �� . �� . age . . — . . �� . . when occurred . . — �. �. � . internalization . . — . �� . symbolization . . — note. n � . for gender, � male, � female. � p � . . �� p � . . aquino, mcferran, and laven than low in moral identity internalization. study also showed that these elevation states were associated with the motivation to en- gage in prosocial behavior as a result of the elevating event. finally, study showed that people who were higher in moral identity internalization were more likely to recall an event that fit the description of a morally elevating act. together, these results provide evidence that experiencing moral elevation is positively re- lated to behaviors that demonstrate social responsiveness to the needs and interests of others but that the susceptibility to experience the states associated with elevation are influenced by moral identity. despite evidence supporting our hypotheses, study has limi- tations. first, it relied on peoples’ retrospective accounts to assess the relationship between moral identity and the states associated with elevation. this approach does not provide strong causal evidence that witnessing an act of uncommon goodness elicits these states of elevation or, if they do, that they are likely to be stronger among people whose moral identity is more rather than less self-defining. second, although the use of a highly represen- tative field sample of citizens increases the generalizability of our findings, it introduces the possibility that unmeasured variables associated with the contexts in which these respondents frequently interact with others could influence our results. third, the kind of act of uncommon goodness that people experienced was not iden- tical across respondents because we asked them to select their own example of the event. this feature of study raises the question of whether our findings might be explained by the kind of experience people recalled rather than by the theoretical mechanisms we hypothesized. finally, we did not measure actual behavior but only the self-reported motivation to act prosocially. study addresses the first three limitations. we conducted study to address all four concerns by using an experimental design and measuring actual behavior. we also wanted to demonstrate that our findings would be replicated when we experimentally manipulated the accessibil- ity of moral identity via a priming manipulation (see aquino et al., ) rather than measuring it. study the purpose of study was to test whether priming moral identity would increase prosocial behavior when people were exposed to an act of uncommon goodness but not when they were exposed to a positive but nonmoral event. we hypothesized that people would be most likely to act prosocially when (a) their moral identity is highly accessible and (b) they are exposed to an act of uncommon goodness. that is, we expected that exposure to the act of uncommon goodness (as compared with a merely positive story) would motivate prosocial behavior to a greater degree when peo- ple’s moral identity was primed than when it was not. method sample and procedure. sixty-three undergraduate students ( men, women) from a canadian university participated in the study for partial course credit. they averaged . (sd � . ) years of age. participants were recruited for a study titled “stories and people” and were told they would be participating in two unrelated studies. the first study contained the experimental ma- nipulations, both for moral identity centrality and for the story. the experiment was a (prime: moral identity vs. control) � (story: uncommon goodness vs. control) between-subjects design. in the ostensibly unrelated second study, participants were told that they would be playing an economic game with an unknown partner; in actuality, the game contained the measure of prosocial behavior. experimental manipulations. participants were told they would be completing two separate studies, which we refer to as part and part . part contained the experimental manipulations. we first manipulated the accessibility of moral identity using a modified priming technique adapted from srull and wyer ( ). in this task, participants completed a word search puzzle for up to min. each crossword contained words to search for, either the items from aquino and reed’s ( ) moral identity scale (e.g., kind, caring, compassionate) or control words (e.g., desk, car, . (. )** . (. )** . (. )** . (. ) . (. ) . (. ) emotional component views of humanity desire to be a better person internalization symbolization prosocial behavioral intentions . ** . (. )** . (. )** . ** . ** . ** . (. )** figure . study model. � p � . . �� p � . . moral identity and moral elevation chair). note that the word moral does not appear as a word or in any of the words in the moral prime word search, which minimizes the likelihood that responses to the accessibility question were influenced by exposure to this particular word. we validated the effectiveness of the word search manipulation in a separate pretest of participants ( women, men). the accessibility of moral identity was assessed with the item “being a moral person is an important part of who i am” ( � completely disagree, � completely agree), which was asked after participants completed the word search. results showed that those who were assigned to complete the moral word search scored higher (m � . , sd � . ) on this measure than did those who completed the control word search (m � . , sd � . ), f( , ) � . , p � . , d � . , indicating that the manipulation was effective. after the word search manipulation, participants read either the amish ug story used in study or the control story featuring the beautiful sunset. after the two manipulations, participants were asked to recall the words in the word search and to briefly sum- marize the story to reinforce the manipulations. in part , partic- ipants were given a separate booklet for a silent negotiation game containing the dependent measure of interest. measures. prosocial behavior. we measured prosocial behavior by having participants play a modified dictator game. participants were told that they would be randomly assigned to role of either allocator or re- ceiver. the allocator was to decide how to divide $ between him- or herself and the receiver. the receiver would have to accept what- ever division the allocator proposed. participants were run in groups of up to people and told that they would be playing the dictator game with another participant in the session, either from among those in their room or from among participants in a different room. partic- ipants were told that one in dyads would get to keep the money, chosen at random after the experiment was completed. in reality and unknown to participants, all were assigned to the role of allocator. we expected the dictator game to present participants with a powerful incentive to act selfishly because the allocator has total control over the distribution of benefits. in the context of the game, a relatively more prosocial response would therefore be one where the allocator chooses to give more money to the receiver; hence, we treated the amount allocated to the receiver as our measure of prosocial behavior. after the experiment was completed, participants were debriefed, and those who were randomly selected were given the full $ to keep. control variable. we again controlled for gender in the analysis. results we performed an analysis of covariance on the dependent measure to test our hypotheses. results revealed a marginally significant interaction between story and prime, f( , ) � . , p � . , 	p � . . planned contrasts revealed that the uncommon goodness condition lead those primed with moral identity to allo- cate more money to their partner (m � $ . , sd � . ) than the merely positive story did (m � $ . , sd � . ), f( , ) � . , p � . , d � . . however, among those in the control prime condition, the story manipulation produced no differ- ences in allocation behavior (m � $ . , sd � . , in the uncommon goodness condition and m � $ . , sd � . , in the positive emotion condition, f � ). the pattern of this effect is shown graphically in figure and supports our hypothesis that people would be influenced by the presentation of an act of uncom- mon goodness only when they were primed to make their moral identity accessible. discussion study extends our previous studies by showing that priming moral identity using a word search procedure resulted in a pattern of results similar to the results found when measuring moral identity. the findings of study allow us to make stronger causal inferences about the relationship between moral identity and exposure to uncommon goodness than was possible in our previous studies. we also show that the interaction between these variables predicts actual behavior. a limitation of study is that we did not measure the suite of responses associated with moral elevation to show that they were directly related to behavior in the dictator game. we conducted our final study to test this relationship. in addition, we wanted to see if we could replicate the results of study using a different, sensory-rich example of moral goodness that we expected would arouse stronger reactions than reading a story. study we hypothesized that moral elevation responses to a music video depicting an act of uncommon moral goodness would be stronger than such responses to a video that did not depict an act of moral goodness and that this effect would be stronger for those who were high (vs. low) in moral identity. further, we hypothe- sized that moral elevation would mediate the relationship between the interaction of moral identity and exposure to uncommon good- ness and subsequent prosocial behavior. method sample and procedure. one hundred twenty-nine under- graduate students ( men, women) from a canadian university figure . study : amount given to partner by condition. aquino, mcferran, and laven participated in the study for $ remuneration. they averaged . (sd � . ) years of age. participants were recruited for a study titled “brands and the world” and told the study was investigating “brands, products, and the media.” the study consisted of three parts. in the first part, participants were asked to complete the measure of moral identity and demographic information. the second part contained measures unrelated to the present study. in the third part of the study was the manipulation, with participants being randomly assigned (between subjects) to either an experi- mental group or a control group, after which they completed a postmanipulation questionnaire. experimental manipulation. participants in both groups watched a music video chosen to elicit either moral elevation or general positive but nonmoral emotion. both videos were from the same canadian artist, sarah mclachlan; were approxi- mately the same length ( min); and featured music of the same tempo. in the experimental group (uncommon goodness condi- tion), participants watched world on fire (mclachlan & marchand, ). the video describes how all but $ of the $ , budget for the video was donated to various charitable causes around the world. the video then chronicles how the money benefited the impoverished communities that received the money versus how it could have been spent creating a regular music video. in the control condition, participants watched adia (mclachlan & marchand, ). this video depicts mclachlan singing to the camera with various city scenes in the background. after watching the video, participants completed measures assess- ing the extent to which they experienced moral elevation responses as a result of watching the video, including a measure of self- reported physical sensations. a pretest was conducted to validate the video manipulation. one hundred two students ( men, women) were randomly as- signed to one of the two video conditions and rated their emotional responses on the same -point scale used in study . following silvers and haidt ( ), we find that participants who viewed world on fire felt more inspired than did those who viewed adia (mworld � . , sd � . ; madia � . , sd � . ), f( , ) � . , p � . , d � . . they also reported feeling more admiration (mworld � . , sd � . ; madia � . , sd � . ), f( , ) � . , p � . , d � . ; awe (mworld � . , sd � . ; madia � . , sd � . ), f( , ) � . , p � . , d � . ; and gratitude (mworld � . , sd � . ; madia � . , sd � . ), f( , ) � . , p � . , d � . . however, the two were deemed equally happy (mworld � . , sd � . ; madia � . , sd � . ), pleasuring (mworld � . , sd � . ; madia � . , sd � . ), and joyful (mworld � . , sd � . ; madia � . , sd � . ), all ps � . . measures. moral identity. the same items used in our previous studies measured internalization (� � . ) and symbolization (� � . ) in study . moral elevation. the emotional component (� � . ), views of humanity (� � . ), and desire to be a better person (� � . ) were measured using the same items as were used in study . in addition to these components, we included a fourth measure of moral elevation—physical sensations—to more fully capture the range of experiences as described by haidt ( , ). the physical sensations measure consisted of five yes or no items from freeman et al. ( ) and algoe and haidt ( ) pertaining to sensations that participants “may have experienced while watching the video (or may still be experiencing).” the sensations included “lightness or feeling ‘bouncy’,” “warmth in your chest,” “tears in your eyes,” “a lump in your throat,” and “chills or tingles.” yes responses were summed for analysis. as in study , we created a higher order (aggregated) measure of moral elevation by standard- izing and then summing the scores for the four measures of the elevation components. controls. we controlled for gender ( � male; � female) and positive emotions (� � . ). the positive emotions items were identical to those used in our previous studies. prosocial behavior. we measured behavior by giving partic- ipants an opportunity to donate to a local charity any part of the $ remuneration they received for participating in the experiment. the charity we selected was walk bravely forward, a nonprofit organization dedicated to assisting canadian first nations (ab- original) peoples who have been incarcerated to reintegrate into society and become productive citizens. we chose this organiza- tion for two reasons. first, it was unlikely that any of the study participants would socially identify with the persons that the organization attempts to help, thereby increasing the psychological distance between themselves and the intended beneficiaries of their donation. second, the beneficiaries were unlikely to elicit high levels of sympathy because they were former criminal of- fenders. we believe that these two features of the beneficiary group served by walk bravely forward allowed us to conduct a fairly strong test of the possible influence of moral elevation and moral identity on prosocial behavior. the opportunity to donate money was presented to participants immediately after they com- pleted the measures of moral elevation. participants were told that they could donate any amount from $ to $ to the charity and that the researchers would send this amount on their behalf. par- ticipants indicated their preference by checking one of options on a sheet of paper (e.g., $ for myself $ to walk bravely forward, $ for myself $ to walk bravely forward, etc.). donations were made in $ increments. after the experiment, all donations made by participants were sent to walk bravely for- ward by the researchers. results descriptive statistics are shown in table . hypothesis tests. bootstrapped estimation of conditional in- direct effects (see preacher, rucker, & hayes, ) was used to test whether the interaction of moral identity and video predicted prosocial behavior through the mediating construct of moral ele- vation. this approach offers several advantages over the conven- tional procedures for assessing moderated mediation detailed by baron and kenny ( ). among these are that it directly esti- mates the size of the indirect effects; provides confidence intervals (cis) for the estimated effects; demonstrates higher power and greater control over type i error rates; and relies on fewer as- sumptions about the sampling distribution, especially for smaller a regression run on these five items results in the same significant interaction and slope pattern (stronger relationship for high vs. low on internalization), and omitting these items from the aggregate elevation measure does not change our results. moral identity and moral elevation samples like ours (see bollen & stine, ; mackinnon, lock- wood, hoffman, west, & sheets, ; mackinnon, lockwood, & williams, ; preacher & hayes, ; shrout & bolger, ). the approach maintains the required observation of significant relationships between the independent variable and the mediator, as well as the mediator and the dependent variable. however, the observation of a significant, direct effect of the independent vari- able on the dependent variable is not required (see collins, gra- ham, & flaherty, ; mackinnon, krull, & lockwood, ; shrout & bolger, ). following the recommendations of preacher et al. ( ), our analysis specified a moderated media- tion model where we tested (a) whether moral identity interacted with video to predict an increase in moral elevation and (b) whether moral elevation was positively related to donations to the charitable organization. it also involved a formal assessment of the indirect effect of video on prosocial behavior among participants who were high versus low in moral identity. we controlled for gender and positive emotions in all analyses. the experimental conditions were dummy coded ( � world on fire, � adia). results of the regressions are shown in table . the first hierarchical regression in table tests the joint effect of the interactions of the two moral identity dimensions and videos on the aggregated measure of moral elevation. step results showed a positive effect of the uncommon goodness video on moral elevation (b � . , se � . , p � . ). internalization (b � . , se � . , p � . ) and symbolization (b � . , se � . , p � . ) were both positively related to moral eleva- tion. step results show that the internalization � video interac- tion significantly predicted moral elevation (b � . , se � . , p � . ). this result satisfies the first condition for establishing mediation according to preacher et al. ( ). the symboliza- tion � video interaction was not significant. the second hierarchical regression model in table predicts prosocial behavior. step results showed a significant effect of the video (b � . , se � . , p � . ) such that participants who watched world on fire donated more money to the charitable organization than did those who watched adia. step results also showed that women donated more money than men did (b � . , se � . , p � . ), as did people who scored higher in internal- ization (b � . , se � . , p � . ). step results showed that the interactions involving each dimension of moral identity and video did not significantly predict donation behavior. however, as noted above, finding a significant effect of the interaction on the dependent variable is not required to establish mediation (preacher et al., ; shrout & bolger, ). when moral elevation was added in step , it was positively related to donations to the charitable organization (b � . , se � . , p � . ). this finding satisfies the second condition for moderated mediation because the mediator (moral elevation) was associated with the dependent variable (prosocial behavior) in the hypothesized man- ner (positive). formal assessment of conditional indirect effects. we con- ducted a moderated mediation analysis (preacher et al., ) using an spss macro written by hayes ( ) to assess the indirect effect of experimental condition on prosocial behavior at different levels of internalization (i.e., standard deviation from the mean), which was the dimension of moral identity that signif- icantly interacted with video to predict moral elevation. the esti- mation of each ci for the indirect effect involved generating , resamples of the data (with replacement) to derive empirical dis- tributions for assessing statistical significance, which is indicated by an interval that does not include zero (see shrout & bolger, ). gender and positive emotions were treated as covariates, moral elevation was treated as the mediator, and internalization was treated as a moderator of the effect of the video on prosocial behavior. the resulting % ci for the indirect effect of video among participants who were high in internalization ranged from . to . . because the lower bound of the ci is above zero and the direction of the effect is positive, we concluded that people who watched the world on fire video donated more money to the charitable organization than did people who watched adia if they scored high in internalization. furthermore, this effect is mediated through moral elevation. for participants who were low in inter- nalization, the % ci ranged from � . to . . because this interval includes zero, we concluded that there was no indirect effect of video on charitable donations among those low in inter- nalization. discussion study provided evidence that experiencing a state of moral elevation is associated with prosocial behavior. these results are consistent with what we found in studies and . they extend study by showing that the state of elevation is a joint function of regressions run on the individual elevation components produce the same significant interaction pattern for all components. table study descriptive statistics and correlations among study variables variable m sd . gender . . — �. . �� . � �. . . �� . positive emotions . . — �. � . �. . �� . �� . internalization . . — . �� . . . �� . symbolization . . — �. . �� . . video . . — . �� . � . moral elevation � . . — . �� . charitable donation . . — note. n � . for gender, � male, � female. for video, � adia, � world on fire. �p � . . ��p � . . aquino, mcferran, and laven witnessing an act of moral goodness and moral identity (internal- ization). it also extends study by showing an effect of these variables on actual behavior. our final study extends study by showing that the effects of moral identity and exposure to uncom- mon goodness on behavior were mediated through moral eleva- tion. general discussion current theory and empirical research on moral elevation has distinguished it from other types of moral emotions (haidt, ). the studies reported in our article contribute to research on the moral elevation construct by showing that some persons might be more susceptible than others to having this experience. across four studies, we showed that moral identity, as both a measured and a manipulated variable, partly explains why some persons are more likely than others to be affected by exposure to acts of uncommon moral goodness. we also showed that people whose moral identity is experienced as more central to their self-concept recall witnessing such acts more frequently. finally, we replicate the results of previous studies (e.g., algoe & haidt, ; freeman et al., ; schnall et al., ) showing that the states associated with moral elevation can motivate prosocial behavior, thereby testifying to its efficacy as a mechanism for promoting such behavior. much has been written about how people pay more attention to the bad events in their lives than the good. what our studies suggest is that when people are confronted with behavior that is extraordinarily good, it can lead some to experience a set of related responses that leave a lasting imprint in their memory. other researchers (e.g., algoe & haidt, ; freeman et al., ) have shown that the response to witnessing uncommon goodness is perhaps best conceptualized as a related suite of thoughts, emotions, and physical sensations. the complexity of the total elevation experiences makes it difficult to identify a specific process through which moral elevation motivates prosocial behavior. we speculate that several processes can be implicated, including heighted positive affect; a change in goal orientation; a desire to emulate a moral exemplar; and, for people who are high in moral identity centrality, a desire to act in a manner consistent with their sense of self. our data do not permit us to test which of these processes best explains the relationship between moral elevation and prosocial behavior, but they do show that both the emotional and the cognitive components of moral elevation matter. a key theoretical implication of our studies is that acts of uncommon goodness are not universally valued or appreciated. we showed that one boundary condition for producing a state of elevation is when moral identity is neither chronically nor temporarily accessible. previous research using aquino and reed’s ( ) measure of the chronic accessibility of moral identity has shown that it predicts a variety of morally relevant cognitions, emotions, and behaviors (see shao et al., ). our studies add to this body of work by showing that moral identity as measured by aquino and reed’s ( ) instrument predicts how people evaluate the behavior of others. but we also showed that when moral identity is made temporarily salient (study ), it can produce similar effects. it is important to note that the temporal salience of moral identity is not conceptually identical to its being chronically accessible (aquino et al., ), which implies that its effects on moral functioning can occur through different mechanisms, one dispositional, the other situationally induced. our findings support our argument that when moral identity is experienced as an important part of the self-concept, it can lead people to assign greater weight to morally virtuous acts. however, the precise mechanism is not directly tested in our studies. perhaps people high in moral identity are motivated to scan their environ- ment for evidence of morality. our finding that that internalization was positively related to the ability to recall a morally elevating act (study ) supports this conjecture. however, it may also be that people who are high in moral identity centrality experience height- ened moral elevation states because doing so is identity reinforc- ing; that is, they feel that it is how a person whose moral character is a defining aspect of the self should respond. it was beyond the scope of the present study to empirically test the different possible explanations for why people whose moral identity is more self- table study : model estimation results for assessing mediated moderation wherein exposure to uncommon goodness and moral identity interact to influence donation behavior through moral elevation predictor model model step step step step step gender . . . � . � . � positive emotions . �� . �� . . � . internalization (int) . � . �� . �� . . � symbolization (sym) . �� . �� . . � . video . �� . �� . �� . �� . video � int . �� . �. video � sym . . . moral elevation . � �r . �� . . � model r . . . . . note. the dependent variable for model is aggregate elevation and for model is charitable donation. unstandardized regression weights are presented. � p � . . �� p � . . moral identity and moral elevation definitional are more likely to experience heightened states of elevation when exposed to acts of uncommon goodness. having established that the relationship exists, we leave it for future research to isolate the specific process that account for it. a second limitation of our study would seem to be the practical value of our results. acts that elicit the state of elevation are, by definition, rare and fall into the category of what philosophers (e.g., flescher, ) describe as “supererogatory,” meaning they are praiseworthy to do but not worthy of blame if avoided. for this reason, many of the prosocial acts typically studied by psycholo- gists—such as donations to charity, volunteerism, or a willingness to help others—are unlikely to produce the intense emotional responses in observers that are associated with moral elevation. further, a fair percentage of people in study reported never having observed an act that fit our description of an act that was morally elevating. however, even if many people might never witness extraordinary acts firsthand, studies and showed that it is possible to elicit the emotions, thoughts, and physical sensa- tions associated with moral elevation by presenting people with examples of morally exceptional behavior in either written or visual–auditory form and that doing so has implications for sub- sequent behavior (study ). indeed, thomas jefferson ( / ) noted that stories in literature or fiction may be more potent elicitors of elevation than witnessed action, meaning that people need not be present to observe the acts firsthand (nor do the events need to have actually occurred) to experience elevation. thus, we have reason to believe that even a seemingly weak stimulus, like a story of moral goodness, can evoke moral elevation responses in nonexperimental settings. whether doing so produces sustained or lasting change in a person’s thinking or behavior is a question for future research. conclusion people have often looked to the lives of moral exemplars like desmond tutu, mahatma gandhi, or the dalai lama to provide lessons on how they should treat others. fortunately, one does not have to be in the presence of the saintly to be uplifted by moral acts. the world’s literature and cinema are replete with examples of uncommon goodness. think of sydney carton sacrificing his life to save the husband of the woman he loves in a tale of two cities or of liam neeson portraying oskar schindler in the cine- matic depiction of the german businessman’s effort to save jews during the holocaust. but we also found that people from diverse backgrounds were able to recall at least one real-life example of uncommon goodness and to report that it had some influence on their emotions, thoughts, and behavior. this finding tells us that it is not only the evil that men (and women) do that survives them, but sometimes also the good. references aiken, l. s., & west, s. g. 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( ) in the article “perceived past, present, and future control and adjustment to stressful life events” by patricia frazier, nora keenan, samantha anders, sulani perera, sandra shallcross, and samuel hintz (journal of personality and social psychology, , vol. , no. , pp. – ), there is an error on page . in the sentence “present control predicted later event-specific distress in sample (� � . , p � . ) but did not predict later general distress (� � . ) in sample , controlling for earlier distress” the value . should have been �. . doi: . /a aquino, mcferran, and laven wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ the use of tranexamic acid to reduce blood loss in uncemented total hip arthroplasty for avascular necrosis of femoral head: a prospective blinded randomized controlled study research open access the use of tranexamic acid to reduce blood loss in uncemented total hip arthroplasty for avascular necrosis of femoral head: a prospective blinded randomized controlled study javahir a. pachore , vikram indrajit shah , sachin upadhyay , * , kalpesh shah , ashish sheth and amish kshatriya abstract background: the purpose of this prospective, double-blinded, randomized controlled study is to assess the efficacy of administration of intravenous tranexamic acid (txa) for reducing blood loss in uncemented total hip arthroplasty (tha) for the treatment of osteonecrosis of femoral head. methods: between april and march , patients with avascular necrosis of femoral head were treated in our center. the patients were randomized and allocated to study group (n = ; treated with txa) and control group (n = ). intra- and postoperative blood loss, blood transfusion, and incidence of deep vein thrombosis were assessed. a p value less than . was considered statistically significant. results: the intraoperative, postoperative, and total (clinical method and gross’ formula) blood loss were significantly greater in the control group (p < . ). on the first, second, and third postoperative days, the levels of hemoglobin and hematocrit were significantly better in the study group (p < . ). there was a significantly greater number of patients who required blood transfusion in the control group (p = . ). deep vein thrombosis was not found in either group. conclusions: a single dose of txa used preoperatively may minimize intraoperative, postoperative, and total blood loss in uncemented tha for the treatment of osteonecrosis of femoral head, and may not increase the risk of prothrombotic complications. keywords: tranexamic acid, osteonecrosis, total hip arthroplasty, deep vein thrombosis background total hip arthroplasty (tha) may cause postoperative blood loss that may necessitate blood transfusion [ , ]. however, allogenic reactions due to blood transfusion are possible, which may put the patient at the risk of se- vere complications, and add more cost to the treatments [ – ]. several strategies have been devised to reduce perioperative blood loss during tha [ ]. the use of tranexamic acid (txa) can reportedly reduce blood loss and thereby decrease blood transfusion after tha [ , ]. txa is a synthetic derivative of lysine that exerts its antifibrinolytic effect by blocking the lysine binding sites of plasminogen, which ultimately blocks the degradation of fibrin [ ]. this process may also potentially enhance the risk of venous thromboembolic events (vtes) by promoting thrombosis [ ]. most randomized con- trolled trials evaluating the efficacy of tranexamic acid during tha included cohorts with a diagnosis of osteoarthritis or those with either osteoarthritis or osteonecrosis of femoral head [ , – ]. in contrast to caucasian population, in india total hip arthroplasty is © the author(s). open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/ . /) applies to the data made available in this article, unless otherwise stated. * correspondence: drsachinupadhyay@gmail.com department of orthopaedics, nscb medical college, jabalpur, mp, india department of trauma, joint replacement and minimal invasive surgery, shalby hospitals jabalpur, jabalpur, madhya pradesh, india full list of author information is available at the end of the article arthroplastypachore et al. arthroplasty ( ) : https://doi.org/ . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf http://orcid.org/ - - - http://creativecommons.org/licenses/by/ . / http://creativecommons.org/publicdomain/zero/ . / mailto:drsachinupadhyay@gmail.com more commonly performed for avascular necrosis of hip than for primary osteoarthritis [ ]. compromised sub- chondral microcirculation and ischemia through distinct underlying pathophysical mechanism leading to osteo- necrosis [ ]. we found no randomized controlled trial evaluating the efficacy of intravenous tranexamic acid in reducing perioperative blood loss and need for blood transfusions after uncemented tha for a specific diagno- sis of osteonecrosis of the femoral head only and this was the aim of our study. we also wanted to find out if there is any high incidence of dvt in this group. we hypothe- sized that a single pre-incisional dose of intravenous tran- examic acid reduces blood loss associated with uncemented tha for osteonecrosis of femoral head. materials and methods the institutional review boards of the participating hos- pitals reviewed the study and approved the protocol. informed consent was obtained from each patient. between april and march , patients undergo- ing uncemented tha for osteonecrosis of femoral head were considered for the study. participants were recruited into the study, and randomized into two groups: treatment and control group. study was designed as a : case control study. our eligibility criteria were unilateral involvement; avascular necrosis of femoral head with moderate to severe arthritis; hip pain interfering with daily living; and primary tha. patients were excluded if they had one of the follow- ing: ) mild arthritis, rheumatoid arthritis, post-traumatic arthritis; ) thrombocytopenia (platelet count < , / mm ; ) anaemia (hemoglobin (hb) < g%; packed cell volume < %); ) under thrombolytic or anticoagulant therapy; ) haemorrhage or haemorrhagic diathesis; ) hypersensitivity to tranexamic acid; ) a history of adminis- tration of nsaids within week; ) a history of deep vein dvt/pulmonary embolism (pe); ) renal dysfunction; ) major illness and health condition such as severe cardiac disorders; ) previous ipsilateral hip surgery such as syno- vectomy, decompression, grafting, osteotomy, and fracture fixation; ) body mass index (bmi) > ; ) decline to participate; ) unwilling to receive any surgical interven- tion; ) bilateral involvement; and ) osteonecrosis sec- ondary to sickle cell anaemia or renal transplant. sample size was estimated using formula of simple ran- dom sampling for infinite population. assumptions were considered based on the difference in mean volume of blood loss in treatment [ ]. the power analysis deter- mined that the study comprising a minimum of patients required % confidence intervals ( % ɑ), % power, and . absolute precision (marginal error). out of patients, were selected, randomized, and allocated to a study group (n = ) and a control group (n = ). the consort flow chart for the study is shown in fig. . txa administration patients in study group and control group received txa or nothing, respectively, which were packaged in enve- lopes marked with numbers. the envelopes were ran- domly drawn by a third person who did not participate in the treatments and were blinded to the sample enve- lope numbers. the envelopes were handled to the anaes- thetists. the surgeons and assessors were blinded. the sample code was not broken until the study was com- pleted. patients in the study group received a single dose of intravenous txa ( mg/ kg body weight, maximum dose: g) min prior to skin incision. patients in the control group did not receive any drug. all patients were operated on by a single experienced surgeon in lateral position using posterior approach thus ensuring a con- sistent surgical technique. intraoperative haemostasis was completely achieved using electrocoagulation. all patients received spinal anaesthesia for surgery by the same anaesthetist. a closed suction drainage system was used in all cases and was removed in h. blood loss calculation intraoperative blood loss was measured using an electric weighing scale with an accuracy of . mg (alexandra scale pvt. ltd., gujarat, india). all blood-stained mops and the blood in the suction cylinders were included in the blood loss. the postoperative blood loss was calcu- lated using two methods. first, the blood loss was deter- mined based on the blood collected from the suction drain , , and h after surgery. second, the blood loss was estimated based on hematocrit balance using gross’ formula [ ], which included the blood loss of ex- travasation in the tissues. gross’ formula are as follows: estimated blood loss ¼ estimated blood volume � initial hematocrit−final hematocritð Þ =mean hematocrit estimated blood volume ¼ body weight kgð Þ � ml=kg the total blood loss (clinical) was determined by add- ing intraoperative and postoperative blood loss. laboratory data collection the levels of hemoglobin and hematocrit concentration, as well as bleeding time, clotting time, prothrombin time, and activated partial thromboplastin time, were tested at admission as the baseline. the parameters on the first and second postoperative days were also ob- tained. a uniform blood transfusion protocol was used in both groups. postoperative blood transfusion was given if the level of haematocrit was less than % of normal range. intraoperative, postoperative, and total pachore et al. arthroplasty ( ) : page of blood loss (both clinical method and gross’ formula method were used) were the primary analytic focus. patient managements doppler ultrasound was performed prior to surgery and on the th postoperative day to rule out dvt. all pa- tients were given anti- thromboembolic stockings for weeks. chemoprophylaxis for dvt and pe was not used. all patients were ambulated partial weight bearing with support postoperatively as soon as they were comfort- able for the first weeks, which was followed by full weight bearing thereafter. statistical analysis demographic data were presented as mean ± standard deviation (sd) and range. during the analysis, numerically-coded categorical variables were cross tabu- lated, and chi square or fisher’s exact test was applied as required. if the frequency was less than five, a fisher’s exact p value was used; and pearson’s chi square tests were used for other analyses. student t test was used to test the differences between two independent means. the shapiro-wilk test was used to test normality. when the variable significantly deviated from a normal distri- bution, a log transformation was performed for statistical comparison. analyses were performed using a s tatistical software package (spss for windows software, version . , spss, inc., chicago, il, usa). results there were no significant differences between the two groups with regard to demographic or preoperative characteristics (p > . ) (table ). intraoperative and postoperative blood loss, changes in the levels of hemoglobin and hematocrit, the amount of blood trans- fusion and complications are shown in table . in the study group, one patient who refused to receive allocated intervention after randomization was excluded from the final analysis. a total of patients were available for final analysis. the mean between-group discrepancies in intraopera- tive blood loss, postoperative blood loss, total blood loss fig. consort flow chart pachore et al. arthroplasty ( ) : page of (clinical), and total blood loss (gross’ formula) were ml, ml, ml, and ml, respectively. we found that there were significant differences be- tween the two groups with regard to the amount of in- traoperative blood loss (p < . ) and postoperative drainage (p < . ), and total (clinical method and gross’ formula) blood loss (p < . ). we also found significant differences with regard to the levels of hemoglobin concentration immediately after surgery (p = . ), and on the first postoperative day (p = . ), and on the second postoperative (p < . ) day. the levels of hematocrit concentration were also significantly different (p < . ). the decreased amount of hemoglobin and hematocrit concentration on the first (p < . ; p < . ) and second (p < . ; p = . ) postoperative days were significantly different. there were significant dif- ference with regard to the number of patients who received blood transfusion (p = . ). neither deep venous clots nor pes happened, and nor other complications took place in both groups. table patient demographics and preoperative characteristics characteristics study group (mean ± sd) (n = ) control group (mean ± sd) (n = ) p value age (years) . ± . . ± . . sex (male: female) : : . bmi . ± . . ± . . pre-op hb (g/ ml) . ± . . ± . . pre-op hct (%) . ± . . ± . . cause of osteonecrosis (no. of patients) steroid intake . alcoholism idiopathic sd standard deviation, bmi body mass index, hb haemoglobin, hct hematocrit table comparison of intra- and postoperative characteristics between the two groups parameters study group (n = ) control group (n = ) p value operative time (minutes)a . ± . . ± . . intraoperative blood loss (ml) . ± . ± < . drain (ml)- h ± . ± . < . h . ± . ± . < . h ± . . ± . < . total blood loss (clinical method) ± . . ± . < . total blood loss- gross’ formula . ± . ± . < . post-op hb day . ± . . ± . = . day . ± . ± = . drop in hb day . ± . ± . < . day . ± . . ± . =. post-op hct day . ± . . ± . = . day . ± . . ± . < . drop in hct day . ± . . ± . < . day . ± . . ± . . blood transfusion (no. of patients)b . complications none none . hb haemoglobin, hct haematocrit, atime from induction tol wound closure, beach patient was given one unit of whole blood transfusion pachore et al. arthroplasty ( ) : page of discussion we found txa can effectively decrease perioperative bleeding and the need for blood transfusions after tha. in a meta-analysis, zhou et al. [ ] found the total blood loss was ml, intraoperative blood was ml, and postoperative blood loss was ml in tha. another meta-analysis conducted by huang et al. [ ] found that txa decreased the total blood loss of ml. however, our study included patients undergoing tha for osteo- necrosis of femoral head only. the blood loss during tha for osteonecrosis of femoral head is likely to be dif- ferent from tha for osteoarthritis due to the differences in the degree of marrow edema and synovitis [ ]. clave et al. [ ] and yamasaki et al. [ ] reported similar intra- operative blood loss but reduced postoperative blood loss in the tranexamic acid treatment group as compared to the control group. this is in contrast to our findings of reduction in both intraoperative and postoperative blood loss in the tranexamic acid group as compared to the control. furthermore, they included patients with osteo- arthritis only in their study while our study included osteonecrosis of femoral head. johansson et al. [ ], who included patients with osteoarthritis only but performed cemented thas, also showed no significant difference in intraoperative blood loss in the tranexamic acid group. rajesparan et al. [ ] and husted et al. [ ], with predom- inantly osteoarthritic patients in their studies, also re- ported no significant difference in the intraoperative blood loss between tranexamic acid and control groups. it is likely that the indication for tha (osteoarthritis vs. osteonecrosis) might be responsible for the reportedly different pattern of blood loss because the factors, like marrow edema and synovitis, that are likely to influence blood loss, are different in the two pathologies. we ad- ministered a single dose of tranexamic acid minutes prior to skin incision. however, the reduction in blood loss was seen intraoperatively and postoperatively up-to h. surgical trauma and venous stasis causes release of tissue plasminogen activator which initiates fibrinolysis that lasts for an hour [ , ]. however, the pre-incisional administration of tranexamic acid inhibits fibrinolysis by binding itself to plasminogen. the reduction in intraop- erative blood loss as seen in our study might be explained by this effect of tranexamic acid on the coagulation path- way. in our own clinical practice, we have observed re- duced intraoperative bleeding and better surgical field in patients receiving tranexamic acid prior to incision. the fibrinolytic inhibition that occurs at about h is due to an increased release of plasminogen activator inhibitor which inactivates tissue plasminogen activator. the sus- tained effect on reduction in blood loss postoperatively may be in part due to fibrinolytic inhibition and in part due to clot stabilization. this reduces postoperative blood loss after tha in patients receiving tranexamic acid and the result has been widely reported [ , , , , , ]. the two groups in our study were similar (table ). factors that are likely to influence blood loss like age, bmi, gender, and pathology were similar between the two groups. the preoperative hb and hct were also similar be- tween the two groups. all patients underwent uncemented tha using the same approach by the same surgeon and anaesthetist to ensure consistency. both the surgeon and the assessor were blinded to the randomization. thus, all the factors that are likely to cause bias were controlled. an intravenous dose of mg/kg reportedly maintained thera- peutic plasma concentration of tranexamic acid for up to hours [ ] and hence this dose was chosen in our study. a higher dose was avoided due to theoretical concern of pro- thrombotic complications. we used a single dose instead of multiple ones since several studies have confirmed the effi- cacy of a single dose in reducing blood loss during tha [ , ]. benoni et al. have shown that tranexamic acid adminis- tered at the end of surgery has no effect on reduction of postoperative blood loss and hence we administered it min prior to incision [ ]. our study focused on a homogenous group of pa- tients undergoing uncemented tha unlike previous studies [ , , ] that have included cemented, unce- mented, and hybrid thas which may behave differ- ently as far as postoperative bleeding is concerned. uncemented tha behaves differently from cemented or hybrid tha as the femoral canal and possibly the acetabular bony beds are closed off by cement, and the pressurization of cement has a ceasing effect on blood loss from intramedullary circulation. therefore, postoperative bleeding tends to be higher in the unce- mented tha than in the cemented tha due to spontaneous bleeding from intramedullary circulation [ ]. with the use of tranexamic acid, there is a con- cern that it may induce a hypercoagulable state [ ]. an interesting feature of our study was the lack of use of chemoprophylaxis against dvt/ pe. anti-thromboembolic stockings and early ambulation were encouraged to prevent dvt/ pe. most studies evaluating the role of tranexamic acid in tha have used chemoprophylaxis against dvt/ pe and have reported that the use of tranexamic acid was not associated with a significant increase in the risk of dvt/pe [ , , ]. since the chemoprophylaxis may counter the hy- percoagulability effect of tranexamic acid, it is a confounding factor. however, by not using chemoprophylaxis against dvt/pe, our study conclusively shows that the use of tran- examic acid is not associated with an increased risk of dvt/ pe. the preoperative and postoperative day screening for dvt using venous doppler ensured that asymptomatic dvts were not missed. the limitation of our study was the small sample size that may affect the results of the present trial. a multicenter, randomized study with large sample size may yield more statistically significant results. pachore et al. arthroplasty ( ) : page of conclusion the present analysis needs to be put in the perspective of the rapidly increasing number of hip arthroplasties being performed each year. our findings suggest that a single pre-incisional dose of tranexamic acid results in a statistically significant and clinically meaningful reduc- tion in intraoperative, postoperative, and total blood loss from uncemented tha for osteonecrosis of femoral head without increasing the risk of prothrombotic complications. abbreviations bmi: body mass index; dvt: deep vein thrombosis; hb: haemoglobin; nsaids: nonsteroidal anti-inflammatory drugs; pe: pulmonary embolism; tha: total hip arthroplasty; txa: tranexamic acid; vte: venous thrombo- embolism acknowledgements we acknowledge all the patients who participated in the study, nursing, paramedical staff. we also acknowledge the contribution of entire research team. authors’ contributions jap (conceptualization; data curation; investigation; methodology; supervision; writing –review & editing). vis(resources). su (conceptualization; data curation; formal analysis; investigation; methodology; resources; supervision; validation; visualization; writing – review & editing). ks (resources). as (resources). ak (resources). all authors read and approved the final manuscript. funding not applicable availability of data and materials the data that support the findings of this study are available from [shalby hospitals india] but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. data are however available from the authors upon reasonable request and with permission of [shalby hospitals india]. ethics approval and consent to participate the study was approved by the scientific review committee and the institutional review board of the participating health service. written informed consent (about the surgical technique, risks and potential complications) was provided according to the declaration of helsinki and obtained from all participating patients. consent for publication informed consent was obtained from the patients for publication of their case records for providing evidence-based scientific literature for further research. competing interests the authors declare that they have no competing interests. author details department of hip arthroplasty, shalby hospitals, ahmedabad, gujarat, india. department of knee and hip arthroplasty, shalby hospitals, ahmedabad, gujarat, india. department of orthopaedics, nscb medical college, jabalpur, mp, india. department of trauma, joint replacement and minimal invasive surgery, shalby hospitals jabalpur, jabalpur, madhya pradesh, india. received: may accepted: october references . melvin js, stryker ls, sierra rj. tranexamic acid in hip and knee arthroplasty. j am acad orthop surg. ; : – . . kim c, park ss, davey jr. tranexamic acid for the prevention and management of orthopedic surgical hemorrhage: current evidence. j blood med. ; : – . . kleinman s, chan p, robillard p. risks associated with transfusion of cellular blood components in canada. transfus med rev. ; : – . . johansson t, pettersson lg, lisander b. tranexamic acid in total hip arthroplasty saves blood and money: a randomized, double-blind study in patients. acta orthop. ; : – . . blumberg n, kirkley sa, heal jm. a cost analysis of autologous and allergenic transfusions in hip-replacement surgery. am j surg. ; : – . . rajesparan k, biant lc, ahmad m, field re. the effect of an intravenous bolus of tranexamic acid on blood loss in total hip replacement. j bone joint surg br. ; : – . . huang f, wu y, yin z, ma g, chang j. a systematic review and meta- analysis of the use of antifibrinolytic agents in total hip arthroplasty. hip int. ; : – . . dunn cj, goa kl. tranexamic acid: a review of its use in surgery and other indications. drugs. ; : – . . ng w, jerath a, wasowicz m. tranexamic acid: a clinical review. anaesthesiol intensive ther. ; : – . . imai n, dohmae y, suda k, miyasaka d, ito t, endo n. tranexamic acid for reduction of blood loss during total hip arthroplasty. j arthroplast. ; : – . . clave a, fazilleau f, dumser d, lacroix j. efficacy of tranexamic acid on blood loss after primary cementless total hip replacement with rivaroxaban thromboprophylaxis: a case-control study in patients. orthop traumatol surg res. ; : – . . singh j, ballal ms, mitchell p, denn pg. effects of tranexamic acid on blood loss during total hip arthroplasty. j orthop surg. ; : – . . claeys ma, vermeersch n, haentjens p. reduction of blood loss with tranexamic acid in primary total hip replacement surgery. acta chir belg. ; : – . . niskanen ro, korkala ol. tranexamic acid reduces blood loss in cemented hip arthroplasty: a randomized, double-blind study of patients with osteoarthritis. acta orthop. ; : – . . yamasaki s, masuhara k, fuji t. tranexamic acid reduces blood loss after cementless total hip arthroplasty-prospective randomized study in cases. int orthop. ; : – . . benoni g, fredin h, knebel r, nilsson p. blood conservation with tranexamic acid in total hip arthroplasty: a randomized, double-blind study in primary operations. acta orthop scand. ; : – . . husted h, blond l, sonne-holm s, holm g, jacobsen tw, gebuhr p. tranexamic acid reduces blood loss and blood transfusions in primary total hip arthroplasty: a prospective randomized double-blind study in patients. acta orthop scand. ; : – . . pachore ja, vaidya sv, thakkar cj, bhalodia hp, wakankar hm. ishks joint registry: a preliminary report. indian j orthop. ; : – . . shah kn, racine j, jones lc, aaron rk. pathophysiology and risk factors for osteonecrosis. curr rev musculoskelet med. ; : – . . gross jb. estimating allowable blood loss: corrected for dilution. anesthesiology. ; : – . . zhou xd, tao lj, li j, wu ld. do we really need tranexamic acid in total hip arthroplasty? a meta-analysis of nineteen randomized controlled trials. arch orthop trauma surg. ; : – . . song jh, han sk, lee kh, lee jm, lee kh. comparison of perioperative blood loss in primary non-cemented total hip arthroplasty for rapidly destructive coxarthrosis and osteonecrosis of the femoral head. hip pelvis. ; : – . . jansen aj, andreica s, claeys m, d’haese j, camu f, jochmans k. use of tranexamic acid for an effective blood conservation strategy after total knee arthroplasty. br j anaesth. ; : – . . benoni g, lethagen s, nilsson p, fredin h. tranexamic acid, given at the end of the operation, does not reduce postoperative blood loss in hip arthroplasty. acta orthop scand. ; : – . pachore et al. arthroplasty ( ) : page of . reid rw, zimmerman aa, laussen pc, mayer je, gorlin jb, burrows fa. the efficacy of tranexamic acid versus placebo in decreasing blood loss in pediatric patients undergoing repeat cardiac surgery. anesth analg. ; : – . . ekbäck g, axelsson k, ryttberg l, edlund b, kjellberg j, weckström j, carlsson o, schött u. tranexamic acid reduces blood loss in total hip replacement surgery. anesth analg. ; : – . publisher’s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. pachore et al. arthroplasty ( ) : page of 	abstract 	background 	methods 	results 	conclusions 	background 	materials and methods 	txa administration 	blood loss calculation 	laboratory data collection 	patient managements 	statistical analysis 	results 	discussion 	conclusion 	abbreviations 	acknowledgements 	authors’ contributions 	funding 	availability of data and materials 	ethics approval and consent to participate 	consent for publication 	competing interests 	author details 	references 	publisher’s note primary hip arthroplasty for the treatment of alkaptonuric hip arthritis: - to -year follow-ups research open access primary hip arthroplasty for the treatment of alkaptonuric hip arthritis: - to -year follow-ups javahir a. pachore , vikram indrajit shah , , sachin upadhyay , * , kalpesh shah , ashish sheth and amish kshatriya abstract background: the objective of this study is to share our experience in total hip replacement for the treatment of ochronotic hip arthritis, in particular to report how to establish the diagnosis and some tips to limit complications. method: a cohort comprised of patients ( hips) with alkaptonuric hip arthritis. there were six men and four women with the mean age of . ± . years. all patients had a stiff spine, grossly restricted movements of hip joints, and severely limited daily routine activities. total hip replacement was performed in all patients. the patients were evaluated at , , and months after surgery, as well as every years thereafter. harris hip score was used to assess the functional outcome. the level of significance was set at p < . . results: the mean follow-up lasted . ± . years ( to years). at the final available follow-up, nine patients returned to work, ambulate without an orthosis, and achieve complete pain relief. harris hip score was improved from poor to excellent. one patient died years after surgery due to breast cancer. no complication relating to prosthetic failures was detected. conclusion: total hip replacement gives long-term satisfactory results in patients with alkaptonuric hip arthritis, resulting in comparable function of the hips in patients who undergo primary osteoarthrosis. keywords: total hip replacement, alkaptonuric arthritis, ochronosis, harris hip score background first described by virchow in , ochronosis is the con- nective tissue manifestation of alkaptonuria, an autosomal recessive mutation of the hgo gene on chromosome q, caused by deficiency of homogentisate , dioxygenase activity [ ]. homogentisic acid oxidase is responsible for turnover of homogentisic acid (hga) during the course of phenylalanine and tyrosine catabolism [ ]. hga accumu- lates and is polymerized into a blue-black pigment that is ultimately deposited in skin, bones, tendons, articular carti- lages, synovial membranes, lungs, valves, and kidneys [ ]. the accumulation eventually causes severe degeneration of the spine and peripheral joints, which may clinically lead to common arthritic disorders [ ]. alkaptonuria is a rare metabolic disorder characterized by a triad of degenerative arthritis, ochronotic pigmentation, and homogentisic acid- uria, affecting one in , to million people [ , ]. chromatographic, enzymatic or spectrophotometric deter- minations of hga are confirmatory tests. currently, there is no definitive cure for alkaptonuric ochronosis. symptom- atic treatment of the complications of alkaptonuria is the only option, including pain management, physiotherapy, chiropractic care, and instruction regarding a home exer- cise program. a successful treatment for tendon ruptures caused by ochronosis is primary repair [ ]. high dose of vitamin c decreases urinary benzoquinone acetic acid, but has no effect on hga excretion and, moreover, no cred- ible studies have shown that treatment with vitamin c is clinically effective. nitisinone, a potent inhibitor of - hydroxyphenylpyruvate dioxygenase, dramatically reduces © the author(s). open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/ . /) applies to the data made available in this article, unless otherwise stated. * correspondence: drsachinupadhyay@gmail.com institution(s) at which the work was performed: shalby hospitals in ahmedabad, gujarat department of orthopaedics, nscb medical college, jabalpur, mp, india department of trauma, joint replacement and minimal invasive surgery, shalby hospitals jabalpur, jabalpur, madhya pradesh, india full list of author information is available at the end of the article arthroplastypachore et al. arthroplasty ( ) : https://doi.org/ . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf http://orcid.org/ - - - http://creativecommons.org/licenses/by/ . / http://creativecommons.org/publicdomain/zero/ . / mailto:drsachinupadhyay@gmail.com production and urinary excretion of homogentisic acid [ ], however, the effectiveness of nitisinone in treating ochronosis remains unknown. patients with alkaptonuria are usually asymptomatic, and the ochronotic arthropathy appears after the fourth decade [ ]. total replacement of hip, knee, elbow, and shoulder can alleviate pain and in- crease patient’s daily activities [ ]. there is a paucity of studies concerning total hip replacement in the present patient population with adequate follow-up statistics. the primary objective of the present study is to share our experience in total hip replacement (thr) for ochro- notic hip arthritis, in particular to report on establishment of diagnosis and tips to decrease the complications. patients and methods the study was approved by the institutional ethics committee of the hospital. informed consent was ob- tained from each patient. a case series of patients ( hips) with alkaptonuric hip arthritis who presented to our institute were reported. there were six men and four women with the mean age of . ± . years (range, – years). demographic vari- ables, presentation, comorbidities, and preoperative diagno- ses were recorded and analyzed (table ). all patients had pain in groin region for the past months to several years. none of the patients was under specific treatments. clin- ical, imaging, and laboratory assessments were performed in all patients. of the patients, seven patients had a fam- ily history of alkaptonuria, and eight patients had cutaneous signs of ochronosis. nine patients had a history of blackish discoloration of urine on exposure to air. spine examin- ation revealed stiff spine with limited movements in all pa- tients. on local examination, hip movements were painful and severely restricted. all patients reported gross limita- tion of daily routine activities and needed assisted ambula- tion. a dried urine spot (dus) [ ] detects hga in all patients. other laboratory parameters were within the nor- mal ranges. the standard radiographs of hip showed severe joint degeneration and narrowing of the joint space with ir- regularity and flattening of the femoral head (fig. a, b). a lateral spine radiograph revealed osteoporosis, flattened and intra-discal calcification, and variable degrees of fusion of the vertebral bodies (fig. a, b). pre-operatively, the mean harris hip score was . ± . . all patients were diag- nosed with ochronotic hip arthritis, and underwent primary hip arthroplasty. eight patients underwent unilateral sur- gery and two had bilateral procedures. in bilateral cases, the priority was given to the side with more severely affected joint. surgical technique all surgeries were performed by the same senior surgeon and the same arthroplasty team through the posterior approach with the patients in the lateral decubitus pos- ition. the incision was made over the center of the tro- chanteric region and then was adequately lengthened both cranially and caudally for a good exposure to avoid force- ful retraction. the subcutaneous fat was incised deep to the fascia lata. we found black discoloration over the ten- sor fascia lata, and calcification and hard tissue at the in- sertion of gluteus maximus tendon with blackish deposits (fig. ). we found deposition of black tissues with some degrees of fragility in the gluteus medius tendon. the fascia lata was incised and the gluteus maximus tendon was bluntly splited along the direction of fibers. the tro- chanteric bursa overlying the external rotators was incised. the sciatic nerve was carefully protected. the short exter- nal rotators were tagged with a suture for identification and subsequent repair. these rotators were detached at their insertions onto the greater trochanter and reflected posteriorly to expose the posterior joint capsule. the cap- sule was found to be more fibrotic and contracted with blackish discoloration. if the capsule was hard and contracted, we usually incised the capsule from o’clock to o’clock to ease dislocation of the femoral head. when the hip was dislocated, we found the femoral head was covered with typical black painted articular cartilage, but the subchondral bone was not affected (fig. a). the tip of the trochanter was impregnated with thick black pigments. during the preparation of the femur, great care should be taken to avoid fracturing the tip of trochanter. preparation of the acetabular bone was difficult due to the black deposition that looked like a layer of tissue (fig. b). the bone was sclerotic, hard, and difficult to ream, so as to open subchondral bone (fig. b). we conducted either pre-drilling or curettage of the cup and finally reamed it to get punctate bleeding. furthermore, owing to the scler- otic rim cup, expansion of the acetabulum was probably difficult. therefore, additional screws were used to secure the fixation. after trailing, the final cup position was achieved within the safe zone for both anteversion and in- clination. the femoral preparation was done in the rou- tine fashion, because the femoral canal was normal. after placement of the optimal stem and femoral head, thr was completed and hip joint was reduced. closure was done in layers under negative suction drain. postoperative managements the pigmented cartilaginous surface, bone, and soft tis- sues were sent for histomorphological evaluation. our protocol was to remove the drain on the second postoper- ative day. the patient was discharged on the sixth postop- erative day, and then given a standard rehabilitation. postoperative evaluation all patients returned for follow-ups at , , and months, as well as every years thereafter. preoperatively pachore et al. arthroplasty ( ) : page of t a b le c h ar ac te ris ti cs o f p at ie n ts c as e a g e/ se x d u ra ti o n o f p ai n si d e o f h ip c u ta n eo u s m an ife st at io n s u rin e d is co lo ra ti o n fa m ily h is to ry sp in e c o m o rb id it ie s pr es en ta ti o n la st fo llo w u p /m ye ar s le ft pr es en t ye s ye s st iff w it h re st ric ti o n o f m o ve m en ts n il bi la te ra l g ro in p ai n ;d is tu rb ed sl ee p ; n ee d ed as si st an ce in th e fo rm o f el b o w cr u tc h es to w al k; u n d er g o n e b ila te ra l to ta l kn ee re p la ce m en t ye ar s b ac k pa tie n t w as ad vi se d ri g h t si d e u n ce m en te d to ta l h ip re p la ce m en t. . ye ar s /m ye ar ri g h t pr es en t ye s ye s st iff w it h re st ric ti o n o f m o ve m en ts n il ri g h t g ro in p ai n ;u n ab le to w al k fo r m o re th an m in s, sl ee p w as d is tu rb ed . ye ar s /f m o n th s bo th a b se n t ye s n o st iff w it h re st ric ti o n o f m o ve m en ts h yp er te n si o n ,d ia b et es , is ch em ic h ea rt d is ea se , va lv u la r ca lc ifi ca ti o n , h yp o th yr o id is m pa in in le ft si d e o f g ro in fo r p as t m o n th s an d p ai n in b o th th e kn ee s; u n ab le to w al k d u e to p ai n . ri g h t to ta l h ip re p la ce m en t w as d o n e ye ar s b ac k. . ye ar s (r ig h t h ip ) . ye ar s (le ft ) /f ye ar s bo th pr es en t ye s n o st iff w it h re st ric ti o n o f m o ve m en ts h yp er te n si o n ,d ia b et es pa in in b o th g ro in ,a lo n g w it h b ila te ra l kn ee p ai n an d b ac k p ai n fo r p as t ye ar s; pa ti en t w as b ed rid d en an d d is ab le d w it h p ai n . (l ef t) . (r ig h t) ye ar s (b ila te ra l h ip ) p at ie n t d ie d ~ ye ar s af te r th e se co n d su rg er y /f m o n th s le ft pr es en t ye s n o st iff w it h re st ric ti o n o f m o ve m en ts h yp er te n si o n ,d ia b et es , h ia tu s h er n ia ,u m b ili ca l h er n ia ,p ep ti c u lc er pa in in th e le ft g ro in fo r th e p as t m o n th s. sh e al so h ad p ai n in b o th th e kn ee s an d sh o u ld er w it h lim it at io n o f m o ve m en ts . . ye ar s /f m o n th s le ft pr es en t ye s ye s st iff w it h re st ric ti o n o f m o ve m en ts h yp er te n si o n ,d ia b et es ; o b es e pa in in b o th th e g ro in fo r p as t m o n th s. le ft h ip w as m o re p ai n fu l th an th e rig h t si d e ca u si n g d iff ic u lt y in w al ki n g an d n ee d o f as si st an ce in th e fo rm o f w al ke r to w al k. lo w ba ck ac h e . ye ar s /m ye ar s ri g h t pr es en t ye s ye s st iff w it h re st ric ti o n o f m o ve m en ts h yp er te n si o n ,d ia b et es sp o n ta n eo u s an d in cr ea si n g p ai n in b o th h ip s, m o re se ve re o n th e rig h t. lo w b ac ka ch e w it h d iff ic u lt y in w al ki n g . ye ar s /m . ye ar s ri g h t pr es en t ye s ye s st iff w it h re st ric ti o n o f m o ve m en ts h yp er te n si o n ,d ia b et es pr o g re ss iv e p ai n in rig h t h ip jo in t fo r th e la st . ye ar s an d lo w b ac k ac h e fr o m th e la st ye ar s. o rt h o si s as si st ed w al ki n g . ye ar s /m ye ar s ri g h t a b se n t n o ye s st iff w it h re st ric ti o n o f m o ve m en ts n il pa in in rig h t g ro in si n ce ye ar s w ith m ar ke d re st ric ti o n o f m o ve m en ts . . ye ar s /m m o n th s le ft pr es en t ye s ye s st iff w it h re st ric ti o n o f m o ve m en ts h yp er te n si o n pa in w as d u ll ac h in g ,i n si d io u s in o n se t, co n ti n u o u s in n at u re m o re o n th e le ft si d e th an rig h t, in te rf er in g w it h h is d ay to d ay ac ti vi ti es . ye ar s pachore et al. arthroplasty ( ) : page of and at the final follow-up, functional outcome evaluation was performed using hhs. standard anteroposterior and lateral femoral x-rays were obtained to study heterotopic ossification, radiolucent lines, position of stem and subsid- ence, the change in the cup inclination or cup migration, and loosening of implants. all parameters were expressed as mean ± standard deviation. the differences were ana- lyzed with student’s t test. a p < . was considered sta- tistically significant. results in this series, the postoperative course was uneventful. no intraoperative complications other than increased blood loss were observed. histopathological examination of periarticular tissues revealed that tissue pigmenta- tion was most markedly in the deeper layers, with areas of calcification and degeneration. the synovium was thickened, inflamed, and pigmented. hga was detected using dried urine spots [ ] in all patients. in all cases, fig. a & b anteroposterior and lateral x-ray films of the pelvis, with both hip joints showing reduced join space with degenerative changes, irregularity, and flattening of femoral head fig. a & b anteroposterior and lateral spine x-ray films revealed osteoporosis, flattened, and intra-discal calcification (doubling signs), and variable degrees of fusion of the vertebral bodies pachore et al. arthroplasty ( ) : page of there was a macroscopic layer of black pigmented articu- lar cartilage, which was more severe in elderly patients. histopathological, physical, biochemical, and radio- graphic evaluations confirmed the diagnosis of ochrono- tic arthropathy. one patient died years after the second hip surgery due to breast cancer. therefore, a total of hips were followed-up finally. the mean fol- low-up time was . ± . years (range, to years). at -month follow-up, the mean hhs improved signifi- cantly, from . ± . preoperatively to . ± . (p < . ). radiographs showed stable prostheses in situ without any evidence of subsidence (fig. a, b, c, d, e, f, g and fig. a, b, c, d). the latest follow-up showed that nine patients ( hips) went back to work and ambulated without walking aids. all patients had complete pain re- lief. hhs was improved from poor to excellent. al- though patients had complaints pertaining to low backache, none of the patient had major complications requiring a revision surgery. we did not found compli- cations related to prosthetic failures (fig. a, b, c, d, e, f, g and fig. a, b, c, d). discussion alkaptonuria is a rare autosomal recessive inborn meta- bolic disorder of tyrosine metabolism due to deficiency of homogentisic oxidase enzyme. alkaptonuria is charac- terized by excretion of homogentisic acid in urine, and deposition of oxidized homogensitate pigments in the fig. black discoloration over the tensor fascia lata, and calcification of the hard tissues with blackish deposit over the gluteus muscles fig. a a classical black painted articular cartilage of the femoral head; b a black painted articular cartilage of the acetabulum with sclerosis pachore et al. arthroplasty ( ) : page of connective tissues and articular cartilages (ochronosis). the gene for this pathological condition is present at locus q – [ ]. in ochronosis, there is deposition of pigments from homogentisic acid in all types of connect- ive tissues, including cartilage, cardiovascular system, genitourinary system, sclera, skin, and ear cartilage [ , ]. urine discoloration is the first clinical manifestation of alkaptonuria, followed by color changes of the sclera and ears. based on the detailed general examination, cu- taneous signs of ochronosis include color changes of the sclera and ears which can easily be observed (fig. ). % of cases in our series had positive association. there is a paucity of available literature concerning the occur- rence of ochronosis arthropathy without ocular and cu- taneous signs. kusakabe et al. [ ] showed cervical arthropathy without ocular and dermatological findings. retrospective inquiry for the family history of alkapto- nuria usually makes sense. in the present ten patients, seven had a positive family history. patients will respond to the leading question about discoloration of urine. ninety percent of the patients in our series had a history of blackish discoloration of urine on exposure to air. the tendons and ligaments may also be affected be- cause of their high collagen content. it causes inflamma- tory alterations resulting in rupture of the tissues [ ]. early diagnosis of ochronosis is valuable to avoid tendon ruptures. the preoperative cardiac clearance is impera- tive to rule out the risk of valvular calcification [ ]. in our series, one patient who had a history of valvular cal- cification underwent aortic valve replacement and cor- onary artery bypass graft surgery. alkaptonuric arthropathy has previously been shown to be a relatively benign disease. our evidence showed that the patients with this disease were crippled and dis- abled with pain. recent overall increase in life expect- ancy may account for the scenario. although alkaptonuria affects both men and women with ochro- notic arthropathy, the trend is more severe and more frequent for men than for women. the result is also male preponderance (six men), which was compatible with our series [ , ]. patients with alkaptonuria are usually asymptomatic, and arthropathy appears after the third or fourth decade with a sudden onset of pain limiting daily routine activities [ , ]. a late onsets is attributed to the gradual age-re- lated decline in the renal ability to excrete homogentisic acid, resulting in a diseased association with signs and symptoms of accumulation of homogentisic acid. in about % of patients, alkaptonuric cases develops arthropathy [ ]. low backache precedes joint diseases. in our cases, low backache with restricted mobility was on the fore- ground. in most of the cases, spinal examination showed the restricted movements of the spine. back pain and stiff- ness in the thoraco-lumber junction and cervical region may be the initial symptom of ochronosis. the spinal stiff- ness is almost like ankylosing spondylitis but the age group is different. pain is more severe in patients with an- kylosing spondylitis than in patients with ochronosis, which have an increased degeneration that may not pro- portionally increase severity of pain. there is a loss of cer- vical or lumbar lordosis. there may be kyphosis or localized scoliosis. in a few of such cases, root pain or sci- atica could be the presenting or only symptom in ochro- notic arthropathy. spine radiograph shows intra-discal calcifications giving a “doubling” of the outline, which confirmed the diagnosis of ochronosis [ ]. in patients with ankylosing spondylitis, there is extensive ossification of spinal ligaments with little calcification of the interver- tebral discs. ochronotic arthropathy usually involves the large weight-bearing joints (knee and hip joints) rather than the small joints of the hand and foot [ – ]. in our series, the hip joints were severely affected. movements of the hip joints were painful and were restricted due to the ar- thropathies. similar to patients with primary osteoarthro- sis, concentric reduction of the joint space is frequently seen on x-rays. however, the radiological alterations may be much less apparent than the clinical manifestations. intraoperatively, we noticed an increased blood loss, which may be the result of en bloc synovectomy of the hypertrophied synovium. the blood loss was more than what is usually observed in arthritis of other common causes. although the hemoglobin and haematocrit levels were not decreased after surgery, we still advise to avoid total synovectomy without intraoperative bleeding control. fig. cutaneous signs of ochronosis that include changes of the color of the sclera and ears pachore et al. arthroplasty ( ) : page of in ochronotic cases, cebesoy et al. [ ] has recommended complete removal of the joint capsule. we speculated that complete capsular resection could increase the rate of dis- location postoperatively. therefore, the capsule should be preserved and utilized for capsular closure. we did not find any complication with the use of the technique. the bone quality around the hip joint affects the stability of prosthesis. during reamerization, cebesoy et al. [ ] found poor bone quality at both the acetabulum and prox- imal femur irrespective of the patient’s age. in our cases, we noticed the bone quality of the proximal femur was in ac- cordance with the age of patients, but acetabulum wall was sclerotic, which was attributed to deposition of pigments in the deeper layers of articular cartilage (fig. b). as a result, the cartilage loses its elasticity and become sclerotic [ ]. in view of the matter, we advise to secure the cup with screws, because the sclerotic rim may impede the cup expansion. before , we did cemented thr because we did not had other choices, and in the rest of the hips we used cementless thr. because the bone tissue is uncommonly in- volved, we were not suspecting any bone-ingrowth deficits that might affect stability of the cementless implants [ , ]. in the present study, we didn’t observe instability, early loos- ening, subsidence, or protrusion problems on radiographs. radiolucent lines, migration, or change in alignment were not observed on the acetabular socket. these factors are sug- gestive of stable implant with bone growth (spot welding). on femoral side, we did not find subsidence, radiolucent lines, or instability of femoral components (fig. a, b, c, d, e, f, g and fig. a, b, c, d). long-term follows showed fig. pelvis and both hips on x-rays. a preoperative anteroposterior view; b lateral view c anteroposterior view immediately after surgery; d anteroposterior view years after surgery; e lateral view years after surgery; f anteroposterior view . years after surgery; g lateral view . years after surgery fig. pelvis and hips on x-rays. a an anteroposterior view preoperatively (left); follow-up x-rays after years (uncemented asr with s-rom; right); b preoperative lateral view of left hip; c an anteroposterior view immediately after surgery (left) and follow-up after years (right); d anteroposterior view at the final follow-up of . years (uncemented asr with s-rom; right) and left hip ( . years; pinnacle with summit) pachore et al. arthroplasty ( ) : page of that superior pain relief and successful restoration of hip function were achieved (mean follow-up time: . ± . years; range, to years) in patients with ochronotic ar- thropathy. we therefore conclude that both the uncemented and cemented total hip had long term survivorship. our preference is to use a cemented thr in patients with poor bone stock or osteoporotic bone. furthermore, in view of pa- tient’s stiff spine in present series, we strongly believe that in the early era of total hip arthroplasty in india, very little or no significance was attributed to the stiffness of spine and /or deformity of spine. we have operated on the patients in present series for a long time before the inception of concept of spinopelvic mobility and total hip arthroplasty. the con- cept of spinopelvic parameters/movements in relation to thr is new. so, we did not have preoperative lateral spino- pelvic-hip x-rays, nor sitting or standing lateral spine x-rays. in the early years, we had intraoperatively used manual jigs and eyeballing to achieve optimal component alignment with respect to patient’s anatomy and limb length. versions/off- sets were attended independently to ensure optimal compo- nent positioning and a stable hip. in cemented hip, the principals of charnley hip were followed to measure various angles. recently, we used the principles of scott ranawat co- plannar test in uncemented surgery. fortunately, there was no dislocation in these groups of patients, in spite of a - mm femoral head used. there is no specific medical treatment for alkapto- nuria, and hence all therapeutic approaches are symp- tomatic. in severe osteoarthrosis, total hip arthroplasty is the preferred treatment. results from our series are con- sistent with the available literature [ , , – ]. the findings support reliability of thr in patients with ochronotic arthropathy. we hope the rationale behind the present series can guide the surgeons to establish the pre-operative diagnosis and to facilitate the intra-opera- tive procedures. no complications related to implant failure were detected after total arthroplasty, and our re- sults are compatible with patients who underwent pri- mary osteoarthrosis. conclusion primary hip arthroplasty is an effective and preferred treatment for alkaptonuric hip arthritis. the critical factor that directly influences the outcomes of total arthroplasty is surgeons’ acquaintance with the ochronosis. through these cases we have attempted to provide tips pertinent to the establishment of the diagnosis of ochronosis and per- formance of thr for alkaptonuric hip arthritis. those tips can help to lower surgical complications. abbreviations dus: dried urine spot; hga: homogentisic acid; hhs: harris hip score; thr: total hip replacement acknowledgements we acknowledge all the patients who participated in the study, nursing, paramedical staff. we also acknowledge the contribution of entire research team. authors’ contributions jap (conceptualization; data curation; investigation; methodology; supervision; writing–review & editing). vis (resources). su (conceptualization; data curation; formal analysis; investigation; methodology; resources; supervision; validation; visualization; writing – review & editing). ks (resources). as (resources). ak (resources). all authors read and approved the final manuscript. funding not applicable availability of data and materials the data that support the findings of this study are available from [shalby hospitals india] but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. data are however available from the authors upon reasonable request and with permission of [shalby hospitals india]. ethics approval and consent to participate the study was approved by the scientific review committee and the institutional review board of the participating health service. written informed consent (about the surgical technique, risks and potential complications) was provided according to the declaration of helsinki and obtained from all participating patients. consent for publication informed consent was obtained from the patient’s for publication of their case records for providing evidence-based scientific literature for further research. competing interests the authors declare that they have no competing interests. author details department of hip arthroplasty, shalby hospitals, ahmedabad, gujarat, india. department of knee, shalby hospitals, ahmedabad, india. department of orthopaedics, nscb medical college, jabalpur, mp, india. department of trauma, joint replacement and minimal invasive surgery, shalby hospitals jabalpur, jabalpur, madhya pradesh, india. department of knee and hip arthroplasty, shalby hospitals, ahmedabad, gujarat, india. received: march accepted: august references . keller jm, macaulay w, nercessian oa, jaffe ia. new developments in ochronosis: review of the literature. rheumatol lnt. ; : – . . spencer jm, gibbons cl, sharp rj, carr aj, athanasou na. arthroplasty for ochronotic arthritis: no failure of replacements in patients followed - years. acta orthop scand. ; : – . . collins ej, hand r. alkaptonuric ochronosis: a case report. aana j. ; : – . . borman p, bodur h, ciliz d. ochronotic arthropathy. rheumatol int. ; : – . . sahin g, milcan a, bagis s, kokturk a, pata c, erdogan c. a case of ochronosis: upper extremity involvement. rheumatol int. ; : – . . kumar rv, rajasekaran s. spontaneous tendon ruptures in alkaptonuria. j bone joint surg (br). ; : – . . fisher aa, davis mw. alkaptonuric ochronosis with aortic valve and joint replacements and femoral fracture: a case report and literature review. clin med res. ; : – . . demir s. alkaptonuric ochronosis: a case with multiple joint replacement arthroplasties. clin rheumatol. ; : . . jacomelli g, micheli v, bernardini g, millucci l, santucci a. quick diagnosis of alkaptonuria by homogentisic acid determination in urine paper spots. jimd rep. ; : – . pachore et al. arthroplasty ( ) : page of . janocha s, wolz w, srsen s, srsnova k, montagutelli x, guenet jl, grimm t, kress w, mueller cr. the human gene for alkaptonuria (aku) maps to chromosome q. genomics. ; : – . . nas k, gür a, akdeniz s, cevik r, harman m, saraç aj. ochronosis: a case of severe ochronoticarthropathy. clin rheumatol. ; : – . . wauthy p, seghers v, mathonet p, deuvaert fe. cardiac ochronosis: not so benign. eur j cardiothorac surg. ; : – . . kusakabe n, tsuzuki n, sonada m. compression of the cervical cord due to alcaptonuric arthropathy of the atlanto-axial joint. a case report. j bone joint surg am. ; : – . . phornphutkul c, introne wj, perry mb, bernardini i, murphey md, fitzpatrick dl, anderson pd, huizing m, anikster y, gerber lh, gahl wa. natural history of alkaptonuria. n engl j med. ; : – . . laskar fh, sargison kd. ochronotic arthropathy. a review with four case reports. j bone joint surg. ; : – . . ranganath lr, cox tf. natural history of alkaptonuria revisited: analyses based on scoring systems. j inherit metab dis. ; : – . . eisenberg h, et al. arch intern med. ; : . . cervenansky j, sitaj s, urbanek t. alkaptonuria and ochronosis. j bone joint surg. ; : . . resnick d. alkaptonuria. in: resnick d, niwayama g, editors. diagnosis of bone and joint disorders. nd ed. philadelphia: w. b. saunders; . p. – . . o’brien w, la db, bunim jj. biochemical, pathologic and clinical aspects of alcaptonuria, ochronosis, and chronotic arthropathy. am j med. ; : – . . rose gk. ochronosis. br j surg. ; : . . cebesoy o, isik m, subasi m, kaya a, bilgin f, kaya o. total hip replacement for an ochronotic patient: a technical trick. am j case rep. ; : – . . aydogdu s, cullu e, ozsoy mh, sur h. cementless total knee arthroplasty in ochronotic arthropathy: a case report with a -year follow-up. j arthroplast. ; : – . . carrier da, harris cm. bilateral hip and bilateral knee arthroplasties in a patient with ochronotic arthropathy. orthop rev. ; : – . . araki k, sudo a, hasegawa m, uchida a. devastating ochronotic arthropathy with successful bilateral hip and knee arthroplasties. j clin rheumatol. ; : – . . moslavac a, moslavac s, cop r. case report of a patient with ochronosis and arthroplasty of the hip and both knees. reumatizam. ; : – . publisher’s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. pachore et al. arthroplasty ( ) : page of 	abstract 	background 	method 	results 	conclusion 	background 	patients and methods 	surgical technique 	postoperative managements 	postoperative evaluation 	results 	discussion 	conclusion 	abbreviations 	acknowledgements 	authors’ contributions 	funding 	availability of data and materials 	ethics approval and consent to participate 	consent for publication 	competing interests 	author details 	references 	publisher’s note association of cytochrome p c genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy original contribution association of cytochrome p c genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy alan r. shuldiner, md jeffrey r. o’connell, dphil kevin p. bliden, bs amish gandhi, md kathleen ryan, mph richard b. horenstein, md coleen m. damcott, phd ruth pakyz, bs udaya s. tantry, phd quince gibson, mba toni i. pollin, phd wendy post, md, ms afshin parsa, md braxton d. mitchell, phd nauder faraday, md william herzog, md paul a. gurbel, md d ual antiplatelet therapy, including clopidogrel and aspirin, inhibits platelet function, preventing ische- mic events and improving outcomes fol- lowing acute coronary syndromes and percutaneous coronary intervention (pci). , to exert an antiplatelet effect, clopidogrel requires conversion to an active thiol metabolite (sr ) by hepatic cytochrome p (cyp) iso- enzymes, which inhibit adenosine di- phosphate (adp)–stimulated platelet for editorial comment see p . author affiliations: division of endocrinology, dia- betes and nutrition, department of medicine (drs shuldiner, o’connell, gandhi, horenstein, damcott, pollin, and mitchell, mss ryan and pakyz, and mr gib- son) and division of nephrology, department of medi- cine (dr parsa), university of maryland school of medi- cine, baltimore; geriatric research and education clinical center, veterans administration medical cen- ter, baltimore (dr shuldiner); sinai center for throm- bosis research, sinai hospital of baltimore, baltimore (mr bliden and drs tantry, herzog, and gurbel); di- vision of cardiology, department of medicine (drs post and herzog); and department of anesthesiology and critical care medicine (dr faraday), johns hopkins uni- versity school of medicine, baltimore. corresponding author: alan r. shuldiner, md, divi- sion of endocrinology, diabetes and nutrition, uni- versity of maryland school of medicine, w red- wood st, room , baltimore, md (ashuldin @medicine.umaryland.edu). context clopidogrel therapy improves cardiovascular outcomes in patients with acute coronary syndromes and following percutaneous coronary intervention by inhibiting adenosine diphosphate (adp)–dependent platelet activation. however, nonrespon- siveness is widely recognized and is related to recurrent ischemic events. objective to identify gene variants that influence clopidogrel response. design, setting, and participants in the pharmacogenomics of antiplatelet in- tervention (papi) study ( - ), we administered clopidogrel for days to healthy amish persons and measured response by ex vivo platelet aggregometry. a genome-wide association study was performed followed by genotyping the loss-of- function cytochrome p (cyp) c * variant (rs ). findings in the papi study were extended by examining the relation of cyp c * genotype to platelet function and cardiovascular outcomes in an independent sample of patients un- dergoing percutaneous coronary intervention. main outcome measure adp-stimulated platelet aggregation in response to clo- pidogrel treatment and cardiovascular events. results platelet response to clopidogrel was highly heritable (h = . ; p � . ). thirteen single-nucleotide polymorphisms on chromosome q within the cyp c –cyp c –cyp c –cyp c cluster were associated with diminished clopidogrel response, with a high degree of statistical significance (p = . � − for rs , additive model). the rs polymorphism was in strong linkage disequilibrium with the cyp c * variant, and was associated with diminished clopidogrel response, accounting for % of the variation in platelet aggregation to adp (p = . � − ). the relation between cyp c * genotype and platelet aggregation was replicated in clopidogrel-treated patients undergoing coronary intervention (p = . ). furthermore, patients with the cyp c * variant were more likely ( . % vs . %) to have a cardiovascular ischemic event or death during year of follow-up (hazard ratio, . ; % confidence interval, . - . ; p = . ). conclusion cyp c * genotype was associated with diminished platelet re- sponse to clopidogrel treatment and poorer cardiovascular outcomes. jama. ; ( ): - www.jama.com © american medical association. all rights reserved. (reprinted) jama, august , —vol , no. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / activation by irreversibly binding to platelet p y receptors. - variability in clopidogrel response is well established. - patients treated with clopidogrel who demonstrate higher ex vivo platelet reactivity are at increased risk of ischemic events. - variation in platelet function in response to clopi- dogrel has been associated with lipo- philic statins, calcium channel block- ers, proton pump inhibitors, st john’s wort, and smoking. - however, these factors account for only a small frac- tion of the variation in response. re- cently, the loss-of-function cyp c (genbank ) * allele has been shown to be associated with a de- creased activation of clopidogrel , and antiplatelet effect - and with in- creased cardiovascular events in pa- tients receiving clopidogrel. , - to identify genes associated with variation in clopidogrel response, we performed a genome-wide association study of adp-stimulated platelet aggregation in response to clopidogrel in the old order amish, a relatively homogeneous founder population in which confounding factors, including medication usage and lifestyle variabil- ity, are minimized. replication and extension of genetic findings and time-to-event analyses were performed in a population with high risk for car- diovascular disease recruited from a cardiac catheterization laboratory in baltimore, maryland. methods study populations amish pharmacogenomics of anti- platelet intervention study. the amish pharmacogenomics of anti- platelet intervention (papi) study (nct ) recruited gener- ally healthy white participants years or older between august and october (for additional details, see emethods at http://www.jama .org). these individuals comprised a number of relative pairs informative for estimating heritabilities, including parent-offspring pairs, sibling pairs, grandparent-grandchild pair, avun- cular pairs, and first-cousin pairs. medical and family histories, anthro- pometry, physical examinations, and blood samples after an overnight fast were obtained. complete blood count with platelet number and levels of se- rum lipids (total cholesterol, high- density lipoprotein cholesterol, and tri- glycerides) were assayed by quest diagnostics (horsham, pennsylvania); levels of low-density lipoprotein cho- lesterol were calculated using the friede- wald equation. after baseline platelet aggregation measurements were obtained, partici- pants were given a -mg oral load- ing dose of clopidogrel followed by mg per day for days. follow-up platelet aggregation studies were repeated hour following the last dose of clopidogrel. a second follow-up platelet aggregation measurement was made later the same day, hour after oral ingestion of mg of chewable aspirin. platelet function was assessed by optical aggregometry with a pap e aggregometer (bio/data corporation, horsham, pennsylvania) in platelet- rich plasma, stimulated with adp ( µmol/l) or arachidonic acid ( . mmol/l) (emethods). sinai hospital of baltimore study patients. the sinai hospital of balti- more study (nct ) enrolled patients older than years and undergoing nonemergent pci between january and may (emeth- ods). of these, ( . %) were w h i t e , ( . % ) w e re a f r i c a n american, and ( . %) were other race/ethnicity. information on race/ ethnicity was obtained by self-report. on the day of pci, patients received a -mg (n = ) or -mg (n = ) clopidogrel loading dose; were already receiving maintenance therapy with a -mg daily dose at the time of pci and received no loading dose. there were no differences in baseline characteristics or in the long-term out- comes investigated in stratified analy- ses of acute clopidogrel dosing; thus, these groups were combined for fur- ther analyses. patients received bivalirudin or hep- arin therapy, either with (n = ) or without (n = ) eptifibatide. , an- ticoagulant therapy was discontinued at the completion of the procedure in all patients. all patients received to mg of aspirin daily for at least week prior to pci and mg on the day of the procedure. to minimize the effects of acute anticoagulant therapy during pci, platelet function was mea- sured on the day of hospital discharge in patients not treated with eptifi- batide or days or more postdis- c h a rg e i n p a t i e n t s t re a t e d w i t h eptifibatide. in total, results of baseline platelet function studies were available for patients not taking clopidogrel at the time of enrollment, and results of postclopidogrel platelet aggregation s t u d i e s w e r e a v a i l a b l e f o r patients. platelet aggregation was assessed in platelet-rich plasma after stimulation with µmol/l of adp or mmol/l of arachidonic acid using a c h ro n o l o g l u m i - a g g re g o m e t e r (model - d; chronolog, haver- town, pennsylvania), as described previously (emethods). aspirin ( mg/d) and clopidogrel ( mg/d) were prescribed for all patients at the time of hospital dis- charge, according to american col- lege of cardiology/american heart association guidelines. we assessed medication adherence by self-report and by review of source documents from hospitalizations for ischemic events. the enrolled patients were con- tacted at the end of and months post-pci to determine the occurrence of postdischarge cardiovascular ische- mic events (emethods). of these pa- tients, were still taking clopidogrel after year; were not. a physi- cian, blinded to the study results of the patient, adjudicated all end points through review of source documents obtained from medical records. post- discharge ischemic events were de- fined as myocardial infarction (the oc- currence of ischemic symptoms and a troponin i value greater than the up- per limits of normal), ischemic stroke, stent thrombosis (definite stent throm- cytochrome p c genotype and clopidogrel therapy jama, august , —vol , no. (reprinted) © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / bosis according to academic research consortium criteria ), unplanned tar- get vessel revascularization (revascu- larization of vessel treated at time of study enrollment), unplanned nontar- get vessel revascularization (revascu- larization of a vessel different from that treated at time of study enrollment), hospitalization for coronary ischemia without revascularization (hospitaliza- tion for chest pain with evidence of is- chemia on electrocardiogram and no evidence of myocardial infarction as measured by troponin i value), and death secondary to any cardiovascular cause. the study protocols were approved by the respective institutional review boards at the university of maryland and sinai hospital of baltimore. writ- ten informed consent was obtained from each participant; participants were compensated for their participation. genotype analysis genotyping in the papi study was per- formed with the affymetrix genechip human mapping k or m (ver- sion . ) arrays according to the manu- facturer’s instructions (affymetrix inc, santa clara, california). genotype calls were performed using brlmm ( k array) or birdseed version ( m ar- ray). single-nucleotide polymor- phisms (snps) present on both arrays with a minor allele frequency greater than % were included in our analy- ses. the mean genotype call rate of these snps was . %. all snps within the region of interest on chromosome q were in hardy- weinberg equilibrium (p � . ), ex- cept rs (p = . � − ) and rs (p = . � − ). fol- low-up genotyping of the known com- mon loss-of-function cyp c * vari- ant (rs ), as well as other functional variants of cyp c (* [rs ], * [rs ], and * [rs ]) was performed using taqman snp genotyping assays (ap- plied biosystems, foster city, califor- nia). the genotype concordance rate was greater than % in a subset of du- plicate samples. statistical analysis summary statistics (eg, means [sds]) and frequencies for the amish papi and sinai hospital of baltimore popula- tions were generated using sas ver- sion . (sas institute inc, cary, north carolina). for both studies, platelet ag- gregation was expressed as the maxi- mum percentage change in light trans- mittance, using platelet-poor plasma as a referent. amish papi study we assessed the correlates (eg, age, sex, body mass index [bmi], lipid levels, and blood pressure) of clopidogrel re- sponse using a regression-based ap- proach as implemented in the solar version . (southwest foundation for biomedical research, san antonio, texas), in which we accounted for re- latedness among study participants by including a polygenic component as a random effect. triglyceride levels were logarithm-transformed for analysis and back-transformed for presentation. dis- tribution analyses were generated in sas. all statistical tests were -sided. association analyses between snps and adp-stimulated platelet aggrega- tion following clopidogrel administra- tion were performed under a variance component model that assesses the effect of genotype as an additive effect on the quantitative trait, while simultaneously estimating the effects of age, age , sex, preclopidogrel platelet aggregation, and the aforementioned polygenic component. the polygenic component was mod- eled using the relationship matrix de- rived from the complete amish pedi- gree structure available through published genealogical records main- tained by the church. the heritabil- ity of baseline platelet aggregation and clopidogrel response corresponds to the proportion of the trait variance ac- counted for by the polygenic compo- nent. the genomic control � coeffi- cient was . ; thus, the p values reported are unadjusted. a power calculation indicated % power to detect snps with allele fre- quencies of . to . in the initial sample (n = ), accounting for % to % of phenotypic variation at � = − . to determine whether the loss-of- function cyp c * variant could account for the chromosome q association signal, we estimated the in- dependent effects of both rs — the most highly associated snp from the genome-wide association analysis— and the cyp c * variant on plate- let aggregation by including both in the model simultaneously. pairwise link- age disequilibrium correlation statis- tics (|d’| and r ) were computed using haploview (http://www.broad.mit .edu). sinai hospital of baltimore study we estimated the effect of cyp c * genotype on preclopidogrel and post- clopidogrel adp-stimulated platelet ag- gregation under an additive genetic model by classifying participants ac- cording to whether they had , , or risk alleles. the genotype effect was es- timated using analysis of variance with adjustment for age, sex, race, study (peri-procedural myocardial infarc- tion, platelet reactivity, thrombin gen- eration, and clot strength: differen- tial effects of eptifibatide � bivalirudin vs bivalirudin study; clopidogrel loading with eptifibatide to arrest the reactivity of platelets [clear platelets– ] study), and treat- ment group (clopidogrel dose and use of eptifibatide). we similarly compared platelet ag- gregation values between participants who did and did not experience an event while taking clopidogrel. lastly, we constructed survival curves to com- pare -year cardiovascular event-free survival between participants with ( n = ) a n d w i t h o u t ( n = ) a cyp c * risk allele. we analyzed the effect of the allele on outcomes in re- lation to ongoing clopidogrel therapy at the time of the event. we used the proportional hazards model to esti- mate the relative hazard associated with having a risk allele on subsequent is- chemic event rates, adjusting for age, sex, and race. this analysis was car- ried out both with and without adp- cytochrome p c genotype and clopidogrel therapy © american medical association. all rights reserved. (reprinted) jama, august , —vol , no. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / stimulated aggregation included as a mediator variable. s u r v i v a l a n a l y s i s s t r a t i f i e d b y cyp c * genotype was also per- formed in the subset of patients still taking clopidogrel at the time of event or at year of follow-up and the patients who were not receiving clopi- dogrel at the time of event or at year of follow-up. results amish papi study by design, amish papi participants were generally healthy (table ). there was wide interindividual variability in adp-stimulated platelet aggregation at baseline and after clopidogrel admin- istration, with no clear cutoff to de- fine resistance (figure ). poorer clopidogrel response, as defined by adp-stimulated aggregation after days of clopidogrel administration, was as- sociated with increasing age ( . % of variance; % confidence interval [ci], . %- . %; p � . ), greater bmi ( . % of variance; % ci, . %- . %; p = . ), higher triglyceride lev- els ( . % of variance; % ci, . %- . %; p = . ), and nominally lower levels of high-density lipoprotein cho- lesterol ( . % of variance; % ci, . %- . %; p = . ) (table ). the variation explained by these variables combined was less than %. the heri- tability of adp-stimulated platelet ag- gregation at baseline and in response to clopidogrel was . (se, . ; % ci, . - . ; p = . ) and . (se, . ; % ci, . - . ; p � . ), re- spectively, suggesting a substantial ge- netic component. a genome-wide association analysis revealed a cluster of snps spanning . megabases on chromosome q , showing strong evidence for associa- tion with clopidogrel response, with p values � − (figure and etable ; q-q plot shown in efigure). these snps were in strong linkage disequi- librium with each other, eg, pairwise r = . to . with rs , the most significantly associated snp ( p = . � − f o r t h e a d d i t i v e model). the participants homo- z y g o u s f o r t h e m i n o r a l l e l e o f rs had the poorest response to clopidogrel, while the homo- zygous for the major allele had the best response; the heterozygous p a r t i c i p a n t s w e r e i n t e r m e d i a t e between the homozygous groups. none of the chromosome q snps associated with platelet aggre- gation after clopidogrel administra- tion was associated with baseline platelet aggregation measures, con- sistent with this locus as a true de- terminant of clopidogrel response. no other genomic region revealed a s s o c i a t i o n s i g n a l s t h a t m e t o r exceeded genome-wide significance ( p � . � − ) ( f i g u r e a n d etable ). the full results of the genome-wide association study are a v a i l a b l e a t h t t p : / / m e d s c h o o l . u m a r y l a n d . e d u / e n d o c r i n o l o g y /supplementalinfo.asp. the cluster of snps on chromo- some q most significantly associ- ated with clopidogrel response is lo- cated within and immediately flanking the cyp c –cyp c –cyp c – cyp c gene cluster, which encodes a group of cytochrome p enzymes t h a t p l a y a n i m p o r t a n t r o l e i n drug metabolism, including conver- table . characteristics of amish papi study and sinai hospital of baltimore study participants characteristic, units amish papi a sinai hospital of baltimore men women men women no. (%) ( . ) ( . ) ( . ) ( . ) age, mean (sd), y . ( . ) . ( . ) . ( . ) . ( . ) white, no. (%) ( ) ( ) ( . ) ( . ) body mass index, mean (sd) b . ( . ) . ( . ) . ( . ) . ( . ) blood pressure, mean (sd), mm hg systolic . ( . ) . ( . ) . ( . ) . ( . ) diastolic . ( . ) . ( . ) . ( . ) . ( . ) hypertension, no. (%) c ( . ) ( . ) ( . ) ( . ) lipids, mean (sd), mg/dl total cholesterol . ( . ) . ( . ) na na ldl-c . ( . ) . ( . ) na na hdl-c . ( . ) . ( . ) na na triglycerides d . ( . ) . ( . ) na na hypercholesterolemia, no. (%) e ( . ) ( . ) ( . ) ( . ) taking aspirin, no. (%) ( . ) ( . ) ( ) ( ) self-reported diabetes, no. (%) ( . ) ( . ) ( . ) ( . ) hematocrit, mean (sd), % . ( . ) . ( . ) . ( . ) . ( . ) white blood cell count, median (iqr), � /µl . ( . - . ) . ( . - . ) . ( . - . ) . ( . - . ) platelet count, mean (sd), � /µl . ( . ) . ( . ) . ( . ) . ( . ) current smoker, no. (%) f ( . ) ( . ) ( . ) abbreviations: hdl-c, high-density lipoprotein cholesterol; iqr, interquartile range; ldl-c, low-density lipoprotein cho- lesterol; na, not available; papi, pharmacogenomics of anti-platelet intervention. si conversion factors: to convert hdl-c, ldl-c, and total cholesterol values to mmol/l, multiply by . ; triglyc- eride values to mmol/l, multiply by . . a for papi study, all participants were withdrawn from prescription and nonprescription medications, vitamins, and supplements days prior to and for the duration of the study. participants taking prescription antihypertensive, lipid- lowering, and diabetes medications accounted for %, . %, and % of participants, respectively. b calculated as weight in kilograms divided by height in meters squared. c defined as systolic blood pressure greater than mm hg or diastolic blood pressure greater than mm hg or taking prescription medication for previously diagnosed hypertension. d logarithm-transformed for analysis and back-transformed for presentation. e defined as ldl-c level greater than mg/dl or taking prescription medication for previously diagnosed hyper- cholesterolemia. f self-reported history of smoking cigarette, pipe, or cigar. cytochrome p c genotype and clopidogrel therapy jama, august , —vol , no. (reprinted) © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / sion of clopidogrel to its active metabo- lite. , , , , - a guanine�adenine mutation in exon of cyp c ( r s , a l s o k n o w n a s cyp c * ) creates an aberrant splice site that leads to an altered reading frame at amino acid and a prema- ture stop codon amino acids down- stream, producing a nonfunctional truncated protein, lack of translation re- sulting from nonsense-mediated mes- senger rna decay, or both. the frequency of cyp c * was . in the amish papi population, similar to that found in other white populations. cyp c * was in high linkage disequilibrium with the clus- ter of snps on chromosome q identified in the genome-wide asso- c i a t i o n s t u d y ( e g , r = . w i t h rs ). cyp c * was associ- ated with adp-stimulated platelet aggregation after clopidogrel adminis- tration, with a high degree of statisti- cal significance (p = . � − for the additive model) (figure ). adp- stimulated platelet aggregation was reduced to . %, . %, and . % of baseline in response to clopidogrel i n p a r t i c i p a n t s w i t h , , a n d cyp c * alleles, respectively. the cyp c * genotype, present in its heterozygous and homozygous state in . % and . % of the papi study population, accounted for % of the variation in clopidogrel response. when we included cyp c * as a covariate, the association between chromosome q snp rs and clopidogrel response was mark- edly attenuated (p = . � − with- out adjustment for cyp c * geno- type to p = . � − with adjustment for cyp c * genotype). these f i n d i n g s s h o w t h a t t h e l o s s - o f - f u n c t i o n c y p c * m u t a t i o n accounts for most or all of the origi- nal q association signal. other previously described cyp c loss-of-function variants, cyp c * and * , were not polymorphic in the amish population, but there may be ad- ditional variation in cyp c or a nearby gene, further contributing to relatively small effects on response and accounting for the remainder of the as- sociation signal. the gain-of-function variant cyp c * had an allele fre- quency of . and was not associated with adp-stimulated platelet aggrega- tion, either at baseline or in response to clopidogrel (etable ). predictors (age, bmi, and levels of high-density lipoprotein cholesterol and triglycerides) and heritability (h = . [se, . ], p � . ) of adp- stimulated platelet aggregation dur- ing clopidogrel administration were very similar after a single -mg dose of aspirin was added. addition of aspirin to clopidogrel resulted in potent inhibition of platelet aggrega- tion in response to arachidonic acid, indicating effective inhibition of the cyclooxygenase pathway. cyp c * genotype had no effect on aspirin- induced inhibition of platelet aggrega- tion of this pathway (etable ). how- ever, the association observed between c y p c * g e n o t y p e a n d a d p - stimulated platelet aggregation and clopidogrel persisted after administra- tion of aspirin (p = . � − ), sug- gesting that this drug combination, which is standard of care in patients with acute coronary syndromes and following pci, does not overcome the figure . distribution of adenosine diphosphate (adp)–stimulated ( µmol/l) platelet aggregation before and after days of clopidogrel administration in members of the amish pharmacogenomics of anti-platelet intervention (papi) study adp-stimulated platelet aggregation, % % o f s tu d y g ro u p postclopidogrel adp-stimulated platelet aggregation, % % o f s tu d y g ro u p preclopidogrel class intervals include data greater than the lower limit and equal to the upper limit of each interval. table . multivariate analysis of clopidogrel response as measured by adenosine diphosphate–stimulated platelet aggregation in papi study participants (n = ) characteristic � (se) p value a variance of significant predictors, % age . ( . ) �. . sex . ( . ) b . body mass index . ( . ) . . lipids total cholesterol . ( . ) . hdl-c − . ( . ) . . ldl-c . ( . ) . log triglycerides . ( . ) . . blood pressure systolic . ( . ) . diastolic . ( . ) . abbreviations: hdl-c, high-density lipoprotein cholesterol; ldl-c, low-density lipoprotein cholesterol; papi, pharma- cogenomics of anti-platelet intervention. a adjusted for age, sex, and preclopidogrel adenosine diphosphate ( µmol/l)–stimulated platelet aggregation; age adjusted for sex and preclopidogrel platelet aggregation; sex adjusted for age and preclopidogrel platelet aggregation. b indicates that women tend to respond less well than men. cytochrome p c genotype and clopidogrel therapy © american medical association. all rights reserved. (reprinted) jama, august , —vol , no. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / effect of the cyp c * genotype on platelet function. in addition to the cyp c * vari- ant, simon et al identified a variant in abcb (geneid ), which en- codes a transporter that modulates clo- pidogrel absorption, to be associated with poorer clinical outcomes in pa- tients receiving clopidogrel. in our study, this variant (rs ) was not associated with adp-stimulated plate- let aggregation at baseline or after clo- pidogrel treatment (p = . ). sinai hospital of baltimore study we next sought to replicate and ex- tend our findings in a group of indi- viduals from the general us popula- tion with a clinical indication for clopidogrel who underwent pci and were recruited from a cardiac catheter- ization laboratory in baltimore. char- acteristics of these participants are shown in table . similar to the amish papi study group, those with the cyp c * genotype had no differ- ence in baseline platelet aggregation (p = . ) but demonstrated greater re- sidual platelet aggregation after clopi- dogrel therapy (p = . ) compared with those without the loss-of-function al- lele (figure ). after year of follow- up, carriers of the cyp c * geno- type had higher cardiovascular event rates compared with noncarriers ( . % vs . %; hazard ratio [hr], . ; % ci, . - . ; p = . ) (figure ). the increased event rate conferred by the cyp c * genotype was limited to the participants still taking clopidogrel when the event occurred (hr, . ; % ci, . - . ; p = . ), with no in- crease in event rate in those not taking clopidogrel when the event occurred or at year of follow-up (hr, . ; % ci, . - . ; p = . ). inclusion of adp-stimulated platelet aggregation as a covariate in the regression model markedly reduced the relation be- tween the cyp c * genotype and cardiovascular outcome (hr, . ; % ci, . - . ; p = . ), suggesting that the genotype effect on clinical out- comes is mediated through decreased inhibition of platelet function. figure . genome-wide association study of adenosine diphosphate–stimulated platelet aggregation in response to clopidogrel −l o g (p v al u e) −l o g (p v al u e) a chromosome cyp c -cyp c -cyp c -cyp c cluster chromosome location, bp pde c lgi pipsl noc l hells cyp c cyp c cyp c cyp c tbc d plce tmem c orf c b linkage disequilibrium (r ) . rs rs rs rs rs rs rs rs rs rs rs rs rs a, association (plotted as −log p value) of individual single-nucleotide polymorphisms distributed across the autosomes. horizontal dotted line indicates p = . � − . b, enlargement of . -megabase region on chro- mosome q . genes encoded in the region are shown below the plot. c, linkage disequilibrium (r ) among the single-nucleotide polymorphisms showing genome-wide significance with clopidogrel response. in- creasing shades of gray represent increasing linkage disequilibrium, from white (r = ) to black (r = ). cytochrome p c genotype and clopidogrel therapy jama, august , —vol , no. (reprinted) © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / comment our study in a relatively large healthy drug-naive population indicates that clopidogrel response as defined by adp- stimulated platelet aggregation is a nor- mally distributed trait, with no clear cutoff to define resistance. others have reported similar findings in patient populations with wide variation in ath- erosclerotic burden, medication us- age, concurrent illnesses, and compli- ance—all potential confounding influences affecting clopidogrel re- sponse. thus, clopidogrel response is likely caused by multiple factors, in- cluding potentially genetic factors. we found that increased age, bmi, and tri- glyceride levels and decreased levels of high-density lipoprotein cholesterol are predictors of poorer clopidogrel re- sponse; however, these factors com- bined account for less than % of the variation, suggesting other, undiscov- ered factors that contribute to varia- tion in clopidogrel response. since all amish persons are related, we were uniquely able to estimate heri- tability of clopidogrel response by de- termining the extent of variation in adp-stimulated platelet aggregation that could be attributed to familial re- latedness. clopidogrel response was highly heritable. indeed, in an agnos- tic genome-wide association analysis, we found compelling evidence for a ma- jor locus on chromosome q that influences clopidogrel response. this locus extends across the cyp c – cyp c –cyp c –cyp c gene clus- ter. cyp c was a strong biological candidate because this enzyme is a key activator of the antiplatelet function of clopidogrel. indeed, follow-up geno- typing indicated that the common loss- of-function cyp c * variant was as- sociated with clopidogrel response and could account for most of the associa- tion signal detected in the initial ge- nome-wide association study. the cyp c * genotype accounts for ap- proximately % of the variation in clo- pidogrel response. with age, bmi, and lipid levels, approximately % of the variation in clopidogrel response can be explained. although substantial and highly sig- nificant, the majority of the variation in platelet response to clopidogrel re- mains unexplained. presumably, other unmeasured factors that influence clo- pidogrel response (including poten- tially additional genetic variants, given the high heritability estimate) remain to be identified. cyp c * genotype was not associated with preclopido- grel platelet aggregation measures, and the postclopidogrel measures were as- sociated with genotype even after ad- justing for preclopidogrel measures, indicating a true association with clo- pidogrel response. due to linkage dis- equilibrium across this region, we can- not rule out that a variant other than cyp c * may be causative, but this is unlikely in light of the unequivocal effect of this mutation on enzyme pro- duction. furthermore, it is possible that other variants in cyp c or other cyp c genes at this locus may also con- tribute to clopidogrel response. our study is unique in that we used an agnostic genome-wide approach. in our genome-wide association study, we detected no other region associated with clopidogrel response at or exceeding a genome-wide level of statistical signifi- cance, suggesting that common vari- ants in other parts of the genome with similar or greater effect size are un- figure . association of cyp c * (rs ) loss-of-function variant with adenosine diphosphate–stimulated platelet aggregation before and after clopidogrel administration in participants in the amish pharmacogenomics of antiplatelet intervention (papi) study and sinai hospital of baltimore study preclopidogrel no. of participants p la te le t a g g re g at io n , % no. of cyp c ∗ alleles no. of participants p la te le t a g g re g at io n , % postclopidogrel no. of cyp c ∗ alleles preclopidogrel p = . p = . no. of participants p la te le t a g g re g at io n , % no. of cyp c ∗ alleles no. of participants p la te le t a g g re g at io n , % postclopidogrel no. of cyp c ∗ alleles amish papi study sinai hospital of baltimore study p = . p = . × – the horizontal line in the middle of each box indicates the median; the top and bottom borders of each box indicate the interquartile range (iqr). the whiskers above and below the box indicate plus/minus . iqrs, respectively; the points beyond the whiskers indicate outliers beyond . iqrs, except for those carrying alleles in which all data points are plotted. cytochrome p c genotype and clopidogrel therapy © american medical association. all rights reserved. (reprinted) jama, august , —vol , no. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / likely. however, we cannot rule out the possibility that common snps or other types of variants (eg, copy number vari- ants, insertions/deletions), or rare vari- ants with large effect size not tagged by those snps genotyped, may have been missed. genome-wide association studies are prone to false-positive results, owing to the large number of statistical tests per- formed. it is unlikely that our finding represents a false-positive result, be- cause we replicated the association be- tween cyp c * genotype and adp- stimulated platelet aggregation in an independent sample. in addition to the replication of the initial association be- tween cyp c * genotype and clo- pidogrel response, the baltimore sinai hospital cohort demonstrates general- izability to an outbred population with significant atherosclerotic disease. im- portantly, the association between cyp c * genotype and cardiovas- cular event–free survival in this high- risk population provides evidence for clinical relevance. a limitation is that the sinai hospital cohort was a mixed population that received differing regi- mens of antiplatelet agents in the acute setting ( - days). however, our strati- fied analyses did not detect any signifi- cant effect of acute management on the longer-term outcomes investigated. furthermore, we cannot rule out the possibility that a subset of patients were nonadherent to clopidogrel. our data show that there is no relationship be- tween cyp c genotype and platelet aggregation in the absence of clopido- grel. thus, the differences in platelet ag- gregation we observed between geno- types once clopidogrel treatment was initiated strongly suggest that a large proportion of the population did in- deed adhere to the clopidogrel regi- men. lastly, owing to limitations in sample size, we could not determine if cyp c genotype was related to ad- verse bleeding events. using a candidate gene approach, hu- lot et al were the first to report an as- sociation between cyp c * geno- type and adp-stimulated platelet aggregation in response to clopidogrel. more recently, cyp c * genotype was associated with poorer clinical out- comes in patients with coronary artery disease treated with clopidogrel and as- pirin. , - in patients taking clopido- grel and aspirin following myocardial in- farction, those carrying the cyp c * genotype were more likely to experi- ence a second cardiovascular event, with an hr ( . ) similar to that found in our study. in the therapeutic outcomes by optimizing platelet inhibition with pra- sugrel–thrombolysis in myocardial in- farction (triton-timi) trial, pa- tients carrying the cyp c * genotype had lower plasma levels of the active me- tabolite of clopidogrel, reduced maxi- mal platelet aggregation in response to clopidogrel, and a % higher compos- ite primary efficacy outcome of the risk of cardiovascular events and death. in a nationwide french registry of pa- tients with acute myocardial infarction treated with clopidogrel, poorer out- comes were observed in patients who carried cyp c loss-of-function alleles (hr, . ). unlike our study and the other studies, heterozygous per- sons from this french registry did not appear to have a significantly increased event rate, suggesting a more modest effect of the genotype on clopidogrel re- sponse in these patients. the loss-of-function cyp c * genotype is common in diverse popu- lations. in white populations, approxi- mately % have at least cyp c * allele. the frequency of this allele is somewhat lower in mexican ameri- cans (� % with at least cyp c * allele), higher in african americans (� % with at least copy), and mark- edly higher in asian populations (� % with at least copy). - thus, clopidogrel resistance due to this vari- ant may be particularly important in asian and african american popula- tions. however, the strength of effect of cyp c * genotype on clopido- grel response may depend on other fac- tors such as genetic background or en- vironmental exposures, which may differ among ethnic groups. unfortu- nately, our sample size was not suffi- cient to examine ethnicity-specific dif- figure . event-free survival over year of follow-up in sinai hospital of baltimore patients treated with clopidogrel following percutaneous coronary intervention, stratified by cyp c * genotype no. at risk no. of cyp c ∗ alleles days all patients % e xp er ie n ci n g e ve n t no. of cyp c ∗ alleles days patients not taking clopidogrel at time of event days patients taking clopidogrel at time of event postdischarge ischemic events were defined as myocardial infarction (the occurrence of ischemic symptoms and a troponin i value greater than upper limits of normal), ischemic stroke, stent thrombosis (definite stent thrombosis according to the academic research consortium criteria ), unplanned target vessel revasculariza- tion (revascularization of vessel treated at time of study enrollment), unplanned nontarget vessel revascular- ization (revascularization of a vessel different from that treated at time of study enrollment), hospitalization for coronary ischemia without revascularization (hospitalization for chest pain with evidence of ischemia on electrocardiogram and no evidence of myocardial infarction as measured by troponin i value), and death sec- ondary to any cardiovascular cause. patients were further stratified into those who were taking clopidogrel when the event occurred or at year of follow-up and those who were not. all analyses adjusted for age, sex, and race. for all patients, hazard ratio (hr) = . ( % confidence interval [ci], . - . ; p = . ); for pa- tients taking clopidogrel at time of event, hr = . ( % ci, . - . ; p = . ); for patients not taking clo- pidogrel at time of event, hr = . ( % ci, . - . ; p = . ). cytochrome p c genotype and clopidogrel therapy jama, august , —vol , no. (reprinted) © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / ferences in cyp c genotype effects on clopidogrel response. additional studies in diverse populations will be necessary. the commonly prescribed proton pump inhibitors omeprazole and esomeprazole, and some other medica- tions commonly coprescribed with clo- pidogrel, such as cimetidine and fluoxi- tine, are potent inhibitors of cyp c , introducing the possibility that they may attenuate the antiplatelet activity of clopidogrel, especially in individuals heterozygous or homozygous for the cyp c * genotype. , - we could not test this hypothesis directly, be- cause all participants in the amish papi study were drug-naive, and the num- ber of participants taking proton pump inhibitors in the sinai hospital of bal- timore sample was too small. cyp c genotype may prove use- ful in helping clinicians choose the most effective antiplatelet therapy and dose for a given individual. those with the cyp c * genotype may benefit more from an antiplatelet regi- men that does not include clopido- grel, such as the third-generation thienopyridine prasugrel, or ticagrelor and cangrelor. like clopidogrel, these agents inhibit adp-stimulated platelet aggregation but are not as dependent on cyp c for activation. genotype- directed decisions regarding which antiplatelet agent to use in a specific patient may also have an important economic impact if costs of equally efficacious medications differ greatly. whether cyp c * carriers may benefit from increased dosing of clo- pidogrel is not yet known. conclusions we report the first genome-wide asso- ciation study of clopidogrel response and show that the common loss-of- function cyp c * variant is a ma- jor determinant of adp-stimulated platelet aggregation. individuals with this genotype have reduced protec- tion from clopidogrel in preventing car- diovascular disease–related events fol- lowing pci. prospective randomized clinical trials will be necessary to de- termine the efficacy of cyp c geno- type–directed therapy in evidence- based clinical decision making. author contributions: drs shuldiner and gurbel had full access to all of the data in the study and take re- sponsibility for the integrity of the data and the ac- curacy of the data analysis. study concept and design: shuldiner, post, faraday, herzog, gurbel. acquisition of data: shuldiner, bliden, gandhi, horenstein, damcott, pakyz, tantry, herzog, gurbel. analysis and interpretation of data: shuldiner, o’connell, bliden, ryan, damcott, tantry, gibson, pollin, post, parsa, mitchell, faraday, herzog, gurbel. drafting of the manuscript: shuldiner, bliden, tantry, herzog, gurbel. critical revision of the manuscript for important in- tellectual content: shuldiner, o’connell, gandhi, ryan, horenstein, damcott, pakyz, tantry, gibson, pollin, post, parsa, mitchell, faraday, herzog, gurbel. statistical analysis: o’connell, bliden, mitchell, gurbel. obtained funding: shuldiner, gurbel. administrative, technical, or material support: shuldiner, bliden, ryan, damcott, pakyz, tantry, gibson, herzog, gurbel. study supervision: shuldiner, bliden, faraday, gurbel. financial disclosures: dr faraday reported that he is a coinventor of a patent application on novel anti- thrombotic agents and their methods of use. dr gurbel reported receiving grant support from schering- plough, astrazeneca, bayer healthcare, sanofi- aventis, portola pharmaceuticals, daiichi-sankyo, and lilly; and receiving honoraria/consulting income from schering-plough, astrazeneca, bayer healthcare, sanofi-aventis, portola pharmaceuticals, daiichi- sankyo, lilly, and pozen. no other authors reported disclosures. funding/support: this study was supported by na- tional institutes of health grants nih u gm and u hl , the clinical nutrition research unit of maryland (p dk ), the university of maryland general clinical research center (m rr ), the baltimore veterans administration geri- atric research and education clinical center, and si- nai hospital of baltimore. role of the sponsors: the funding organizations had no role in the design and conduct of the study; the collection, analysis, and interpretation of the data; or the preparation, review, or approval of the manu- script. additional information: emethods, etables through , and efigures and are available at http://www .jama.com. additional contributions: we gratefully acknowl- edge our amish liaisons and field workers and the ex- traordinary cooperation and support of the amish com- munity, without which these studies would not have been possible. references . antman em, hand m, armstrong pw, et al; 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(reprinted) © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ genome-wide association study identifies genetic variants in got determining serum aspartate aminotransferase levels original article genome-wide association study identifies genetic variants in got determining serum aspartate aminotransferase levels haiqing shen , coleen damcott , scott r shuldiner , sumbul chai , rongze yang , hong hu , quince gibson , kathleen a ryan , braxton d mitchell and da-wei gong , we carried out a genome-wide association study of serum aspartate aminotransferase (ast) activity in amish participants of the heredity and phenotype intervention heart study and identified significant association of ast activity with a cluster of single nucleotide polymorphisms located on chromosome q . (peak association was rs ; p¼ . e- ), in the vicinity of got , the gene encoding cytosolic ast (cast). sequencing of got revealed an in-frame deletion of three nucleic acids encoding asparagine at position c. _ delaac (p.asn del) in the gene. deletion carriers had significantly lower ast activity levels compared with homozygotes for the common allele (mean±s.d.: . ± . versus . ± . u l� ; p¼ . e- ). further genotyping of the deletion in other amish samples (n¼ ) identified an additional carriers (minor allele frequency (maf)¼ . ). the deletion was not detected in outbred caucasians. asn at codon is conserved among known mammalian casts. in vitro transient transfection of wild-type and mutant cast indicated that mutant cast protein was barely detectable in the cells. furthermore, even after correction for cast expression, mutant cast had markedly diminished enzymatic activity. remarkably, we did not find any association between the deletion and metabolic traits including serum fasting glucose or insulin, fasting and post-meal lipids, inflammatory markers, or sub-clinical markers of cardiovascular disease. in conclusion, we discovered a rare in-frame deletion in got gene, which inactivates cast enzyme in the old order amish. this finding will help us to understand structure and function of the enzyme and would be useful for predicting serum ast levels. journal of human genetics ( ) , – ; doi: . /jhg. . ; published online september keywords: amish; ast; gene; got ; mutation introduction aspartate aminotransferase (ast; ec . . . ) catalyzes the reversible conversion between aspartate/a-ketoglutarate and oxaloacetate/gluta- mate, and has an important role in amino acid metabolism and in the urea and tricarboxylic acid cycles. ast is conserved from all prokaryotic to eukaryotic organisms and is expressed in many types of cells/tissues including liver, red blood cells and cardiac/skeletal muscles. two highly similar isoenzymes are present in mammals, one located in cytoplasm (cast), and the other in mitochondria (mast). in serum, the enzyme activity is largely of cytosolic origin. , serum ast activity elevation is considered a surrogate marker of liver, heart and muscle injury. the locus for soluble ast (got ) is on chromosome q , whereas the mitochondrial form of ast (got ) is on q . twin studies , suggest that serum astactivity is influenced by both genetic and environmental factors. however, to date, specific gene variants that influence serum ast activity and/or metabolic or other pheno- types that may be associated with ast activity have yet to be identified. we sought to determine the genetic determinants of serum ast levels by carrying out a genome-wide association study (gwas) of serum ast in amish subjects from the heredity and phenotype intervention (hapi) heart study. materials and methods population the hapi heart study was initiated in to identify genes that interact with short-term environmental exposures to modify risk factors for cardiovascular disease. details of recruitment and phenotyping have been reported pre- viously. briefly, subjects were members of the old order amish (ooa) community aged years or older and considered to be relatively healthy, based on exclusion criteria of severe hypertension (blood pressure greater than / mmhg), malignancy, and kidney, liver or untreated thyroid disease. hapi heart study participants underwent measurement of cardiovascular disease risk factors and questioning about their prior history of cardiovascular disease. physical examinations, including anthropometry were conducted at the amish research clinic in strasburg, pa, usa. blood samples were obtained received may ; revised july ; accepted august ; published online september division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, md, usa and geriatric research and education clinical center, veterans administration medical center, baltimore, md, usa correspondence: dr h shen, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood street, baltimore, md , usa. e-mail: hshen@medicine.umaryland.edu journal of human genetics ( ) , – & the japan society of human genetics all rights reserved - / $ . www.nature.com/jhg http://dx.doi.org/ . /jhg. . mailto:hshen@medicine.umaryland.edu http://www.nature.com/jhg following an overnight fast. those taking lipid-lowering medications at enroll- ment discontinued usage days before examination. serum lipid levels and high-density lipoprotein cholesterol were assayed by quest diagnostics (hor- sham, pa, usa). all subjects had triglyceride levels less than mgdl� , and low-density lipoprotein cholesterol levels were calculated according to the friedewald formula. hypertension was defined as a systolic blood pressure of mmhg or greater, a diastolic blood pressure of mm hg or greater, or use of prescription blood pressure-lowering medications. diabetes was defined as a fasting glucose of mgdl� or greater, or current use of prescription diabetes medications. smoking habits were recorded; current smoking status included use of cigarettes, pipes or cigars. serum ast and alanine amino- transferase were assayed using spectrophotometry by quest diagnostics. additional amish subjects who had dna samples stored in our biobank (n¼ ) were screened for the mutation identified. six hundred and forty seven non-amish caucasians were screened, who were participants in the type diabetes case–control study conducted by joslin diabetes center (boston, ma, usa) and university of maryland (baltimore, md, usa). the study protocol was approved by the institutional review board at the university of maryland, and other participating institutions. informed consent was obtained from each of the study participants. genotyping genotyping was performed in hapi heart study participants with the affymetrix genechip human mapping k array set (affymetrix, santa clara, ca, usa), including a total of single nucleotide polymorphisms (snps). the affymetrix genechip genotyping analysis software and brlmm genotype-calling algorithm (affymetrix) were used to generate snp data files. mean sample call rate in samples was . % after filtered by relationship check and call rate ( %). a total of snps that passed quality control (snp call rate %, minor allele frequency (maf) ) and hardy–weinberg equilibrium checks (at p . ) were retained for analysis. we confirmed the deletion c. _ delaac (p.asn del) by dideoxy sequence analysis (abi ; applied biosystems, foster city, ca, usa) in the exons of got gene, followed by genotyping using custom taqman snp genotyping assays (applied biosystems) in hapi heart study participants, additional amish dna samples from our biobank, and non-amish caucasians. functional studies of recombinant cast construction of expression vector of wild-type and mutant cast. full-length human cast cdna plasmid (image clone id: ) was purchased from open biosystems (huntsville, al, usa). the plasmid served as template for pcr amplification of the full cast protein-coding region with restriction enzyme hindiii-anchored upstream primer p ¢-cgatgacgacaagcttcatatgg- cacctccgtcagtctttgcc- ¢ and downstream xbai-anchored downstream primer p ¢-cccgggatcctctagtcactggattttggtgactgc- ¢ with high-fidelity phusion polymerase (new england biolabs, ipswich, ma, usa). the resultant pcr product was subcloned into pflag-cmv precut with hindiii and xbai by in-fusion cloning (clontech, mountain view, ca, usa) to make wild- type expression vector pflag-cmv -castwt. for construction of castmut (c. _ delaac), three fragment in-fusion cloning was carried out with mutant ¢-end fragment (pcr amplified with upstream primer p and downstream primer p ¢-ccactcacgattcgaccacttggcagcag- ¢ on wild-type cast cdna template), mutant ¢-end fragment (pcr amplified with upstream primer p ¢-ggtcgaatcgtgagtggcttaaccaccaaa- ¢ and downstream primer p on wtast template), and fragment of hindiii and xba-precut pflag- cmv , to make the mutant cast expression vector pflag-cmv -castmut. both castwt and castmut dna inserts were verified by sequence analysis. expression and enzymatic assay of recombinant castwt and castmut proteins. hek cells were grown in dulbecco’s modified eagle medium containing % fetal bovine serum and antibiotics ampicillin and strepto- mycin. the cells were plated into six-well plates. at b % confluency, the cells were transfected with empty vector pflag-cmv , pflag-cmv -castwt and/or pflag-cmv -castmut with lipod (signagene, gaithersburg, md, usa) according to the manufacturer’s instruction. at h post transfection, the cells were collected in mm tris-hcl buffer (ph . ) and lysed by sonication on ice. the lysates were centrifuged and supernatants were collected for protein quantification using the bradford method, ast activity assay and western blot analysis. ast activity was determined by using the ast kit (catachem, oxford, ct, usa) according to the manufacturer’s instruction. briefly, ml of cell lysate was incubated with a . ml mixture of reagent a and b containing l-aspartate, nadh, malate dehydrogenase and -oxoglutarate at c. absorbance at nm was recorded for min at every s interval after addition of the protein fraction. the slope of absorbance decrease is proportional to ast activity. final ast activities were corrected by protein concentration of cell lysates. one unit of ast activity was defined as the amount of enzyme, which catalyzes the formation of mmol l� of nad per minute under conditions of the assay at c. for western analysis, mg of total proteins from reduced cell lysate were separated by electrophoresis on . % polyacrylamide gels. following electro- phoresis, proteins were transferred onto polyvinylidene fluoride membranes and bound proteins were probed with primary anti-flag antibody (sigma, st louis, mo, usa) or gapdh (millipore, billerica, ma, usa), and developed with an alkaline phosphatase-conjugated second antibody. statistical analysis association analyses of quantitative traits were performed under a variance component model that assesses the effect of genotype, as an additive effect, on the quantitative trait, whereas simultaneously estimating the effects of age, age, sex, and a polygenic component to account for phenotypic correlation due to relatedness. the polygenic component was modeled using the relationship matrix derived from the complete pedigree structure as all the subjects are related. association analysis using the complete pedigree structure was carried out using mixed model analysis for pedigree (mmap) software developed by one of our colleague (jr o’connell). ast, alanine aminotransferase, fasting glucose and fasting insulin were naturally log-transformed. student’s t-test was used to examine differences between castwt and castmut activities. results we carried out a gwas of serum ast activity levels in amish subjects from the hapi heart study. characteristics of study partici- pants are summarized in table . in the gwas, the strongest snp associations with serum ast levels were observed for a group of snps located on chromosome q . . the peak association was with rs (p-value¼ . e- ), in the vicinity of got (b mb downstream), the gene encoding cast (figure ). the frequency of the associated snp is rare (maf¼ . ), with only heterozygotes for rs found in the hapi heart study. the heterozygotes had significantly lower ast levels compared with the homozy- gotes of the wide-type allele (mean±s.d. of ast: . ± . versus . ± . u l� , p¼ . e- ). given the low frequency of the associated snp, its proximity to got , and the very large effect size of the association, we hypothe- sized that rs might tag a functional snp, most likely a mutation changing the protein sequence of cast, in got . we subsequently sequenced the gene and identified an in-frame deletion of three nucleic acids encoding asparagine at position in got gene (figure a). all of heterozygotes for rs were hetero- zygotes for c. _ delaac. the deletion c. _ delaac (p.asn del) was in complete linkage with rs . by construct- ing the pedigree, all the deletion carriers can be traced back to one most likely founder who was born in mid- th century, suggesting a founder effect. genotyping of non-amish caucasians from baltimore area did not identify carriers of this deletion. glutamine at amino acid at is conserved among known cast of vertebrate species, including the human, chimpanzee, mouse and zebrafish (figure b). further genotyping of deletion c. _ delaac (p.asn del) in an additional , amish a mutation in got determines serum ast levels h shen et al journal of human genetics samples identified more carriers (maf¼ . ), among which had phenotype measurements. no homozygotes for the deletion were found. phenotype comparison between the ( from the hapi heart study plus from the additional screening) c. _ delaac (p.asn del) carriers and non-carriers (from the hapi heart study) is shown in table . the deletion carriers had serum ast activity levels that were approximately one- half that of normal homozygotes, suggesting that the deletion resulted in a complete loss of enzymatic function. despite this large difference in ast activity, we did not find any association between the mutation and metabolic traits, including serum fasting glucose or insulin, fasting lipids (table ). neither did we find any association with post-meal lipids, inflammatory markers or sub-clinical markers of cardiovascular disease (data not shown). to further examine the functional consequences of p.asn del, we conducted in vitro functional studies by expressing the recombinant wild-type and mutant cast (p.asn del) in the mammalian kidney cell line hek and measuring ast activity. as shown in figure a, the basal activity of the cells transfected with empty vector (lane ) was barely detectable. ast activity levels were increased by fold in a dose-dependent manner when the cells were expressing wild-type cast (lanes and ). by contrast, lysates from cells transfected with mutant cast (lanes and ) had almost no ast activity, suggesting that the mutant cast is enzymatically inactive. western analysis was conducted to determine the levels of recombinant protein expression in the cells. as shown in figure b, mutant cast only was barely detectable, compared with the wild type (lanes and versus and ). interestingly, the activities from the cells transfected with wild-type and mutant cast at a : ratio were less than half of that of wild-type ast (lane versus ; po . ), suggesting a dominant negative effect. western analysis revealed that less mutant cast protein was expressed after correction for gapdh, suggesting a decreased expres- sion, probably due to decreased rna stability and/or increased protein degradation (figure b). however, even after correcting for ast protein, mutant cast had markedly decreased activity relative to wild-type cast (lane versus , po . ). discussion serum ast activity levels are determined by both genetic and environmental factors. , variation in serum ast activity has a moderate heritable basis ( . – . ) estimated from twin studies. , a gwas of ast protein levels, as one of the group of proteins measured in in chianti study participants, didn’t find any cis- effect associations with snps near or in got or trans-effect associa- tions with snps elsewhere in the genome. in another gwas, no snp was associated with serum levels of ast at or exceeding genome-wide significance. through a gwas of serum astactivity in the ooa, we identified a rare in-frame bp deletion in got that was strongly associated with low serum ast activity in the ooa. subjects hetero- zygous for the got c. _ delaac had serum ast activity levels that were about one-half that of wild-type homozygotes. consistent with this observation, functional studies found that ast activity of the mutant ast recombinant protein was totally abolished. this deletion was not present in outbred caucasians, consistent with negative gwas findings of others. also consistent with negative gwas findings of others, our gwas did not detect any other regions with genome-wide significant association to ast activity levels. elevated serum ast activity is a clinical marker for damage to hepatocytes, myocardial cells or skeletal muscle. by contrast, decreased serum ast and its clinical significance are less known. this loss- of-function mutation in ast provides a unique opportunity to determine if decreased ast is associated with a clinically significant rs -l o g ( p ) chr figure genome-wide association analysis plot for serum ast levels in old order amish subjects. arrow indicates that snp rs on chromosome q . is highly associated with serum ast activity (p¼ . e- ). a full color version of this figure is available at the journal of human genetics journal online. table clinical characteristics of the hapi heart study participants men women n age, mean (s.d.), year . ( . ) . ( . ) bmi, mean (s.d.), kg m� . ( . ) . ( . ) systolic blood pressure, mean (s.d.), mm hg . ( . ) . ( . ) diastolic blood pressure, mean (s.d.), mm hg . ( . ) . ( . ) total cholesterol, mean (s.d.), mmol l� . ( . ) . ( . ) ldl-cholesterol, mean (s.d.), mmol l� . ( . ) . ( . ) hdl-cholesterol, mean (s.d.), mmol l� . ( . ) . ( . ) triglyceride, median (s.d.), mmol l� . ( . ) . ( . ) ast, mean (s.d.), u l� . ( . ) . ( . ) alt, mean (s.d.), u l� . ( . ) . ( . ) hypertension, n (%) ( . ) ( . ) diabetes, n (%) ( . ) ( . ) current smoker, n (%) ( . ) ( ) lipid medication, n (%) ( . ) ( . ) abbreviations: bmi, body mass index; hapi, heredity and phenotype intervention; hdl, high- density lipoprotein; ldl, low-density lipoprotein. a mutation in got determines serum ast levels h shen et al journal of human genetics t t c g t c c g t g a g t got wide-type t t c g t c c g t g a g t ttt t c g t c g t g a a a g t c c got mutation carrier gg gg gg gggg ggaa aa aa aa aa ast _human aqgarivastlsnpelfeewtgnvktmadriltmrselrarlealktpgtwnhitdqigm ast _chimpanzee aqgarivastlsnpelfeewtgnvktmadrilsmrselrarlealktpgtwnhitdqigm ast _mouse aqgarivaatlsdpelfkewkgnvktmadriltmrselrarlealktpgtwshiteqigm ast _xenopus sqgarivattlntpelfdewrdnvktmaervllmraelksrlealktpgtwnhivnqigm ast zebra fish sqgarlvaitlntpelfaewkanvktmadrvllmraqlkeklkalgtpgtwehiteqigm qqq__ ast _c_elegans ahgarivhkvlttparreqwnqsiqamssrikqmraallrhlmdlgtpgtwdhiiqqigm *** * * * * * * ** * * * ***** ** **** ast _human fsftglnpkqveylvnekhiyllpsgrinvsglttknldyvatsiheavtkiq--- ast _chimpanzee fsftglnpkqveylvnekhiyllpsgrinvsglttknldyvatsiheavtkiq--- ast _mouse fsftglnpkqveylvnekhiyllpsgrinmcglttknldyvatsiheavtkiq--- ast _xenopus fsytglnpkqveylikekhiylmasgrinmcglttknidyvaqsiyeastkiq--- ast _zebra_fish fsftglnpkqveymikekhiylmasgrinmcgltsknidyvaesiheavtkvq--- ast _c_elegans fsytgltsaqvdhlianhkvfllrdgrinicglntknveyvakaidetvravksni ** *** ** * **** ** ** *** * * figure detection of got (c. _ delaac) mutation. (a) sequencing identified an in-frame deletion of three nucleic acids encoding asparagine at position in got gene. (b) asparagine (n, indicated by arrow) is conserved in cast protein sequences among human, chimpanzee, mouse, xenopus, zebrafish and c. elegans. peptide sequences are aligned using clustalw with fully conserved amino acid marked with asterisk. partial alignment of cast is shown and amino acid positions are numbered to the left. table clinical characteristics of the got c. _ delaac carriers and non-carriers carrier non-carrier p-value n age, year . ( . ) . ( . ) . bmi, kg m� . ( . ) . ( . ) . waist circumference, cm . ( . ) . ( . ) . ast, u l� . ( . ) . ( . ) . e- alt, u l� . ( . ) . ( . ) . systolic blood pressure, mm hg . ( . ) . ( . ) . diastolic blood pressure, mm hg . ( . ) . ( . ) . total cholesterol, mmol l� . ( . ) . ( . ) . ldl-cholesterol, mmol l� . ( . ) . ( . ) . hdl-cholesterol, mmol l� . ( . ) . ( . ) . triglyceride, mmol l� . ( . ) . ( . ) . fasting glucose, mmol l� . ( . ) . ( . ) . fasting insulin, mmol l� . ( . ) . ( . ) . abbreviations: alt, alanine aminotransferase; ast, aspartate aminotransferase; bmi, body mass index; hapi, heredity and phenotype intervention; hdl, high-density lipoprotein; ldl, low-density lipoprotein. mean (s.d.), adjusted for age, age-square, sex, and family structure for phenotypes other than age. ast and alt measurements were based on got deletion carriers from hapi heart study. the rest of the phenotype measurements were based on got deletion carriers from hapi heart study and additional identified got deletion carriers in amish; non-carriers were from hapi heart study. p-values for fasting glucose, fasting insulin and waist circumference were based on non-diabetics. a mutation in got determines serum ast levels h shen et al journal of human genetics phenotype. although ast has a pivotal role in intermediary metabo- lism, deficiency might be expected to lead to abnormalities in glucose and/or lipid metabolism. interestingly, we did not find any significant differences in a wide variety of metabolic traits in got c. _ delaac carriers compared with wild-type homozygotes. these findings suggest that in humans, ast is not a rate-limiting step in intermediary metabolism, and that a single functional allele is sufficient for normal metabolic function. nevertheless, a total loss of this enzyme could be lethal, as we did not find any of the mutant homozygotes. of possible relevance, fasting glucose levels tended to be higher in deletion carriers than that in non-carriers (p¼ . , after adjusting for age, sex and family structure). recruitment and pheno- typing of additional c. _ delaac carriers will improve the statistical power and help us understand this potential relationship more clearly. rare mutations or genetic diseases have been reported with higher frequency in founder populations, including the ooa. through genetic drift, rare mutations may increase in frequency in founder populations. advances in genetic technology have enabled the identi- fication of many of these rare mutations and has provided the opportunity to glean insights into mechanisms underlying both rare and common phenotypes, which would otherwise be difficult in the general population. , in conclusion, we discovered a rare inactiva- tion mutation in got encoding cytosolic ast in the ooa. as ast is a widely used liver function marker, these mutant carriers will have disguisedly lower or near-normal ast levels in pathogenic liver conditions. the discovery of the got mutation will help to explain and alert physician about lower ast levels under actual liver injury in those carriers. it will also be interesting and clinically and diagnosti- cally significant to examine if such or similar mutations exist in other populations. conflict of interest the authors declare no conflict of interest. acknowledgements the study was supported by grants of maryland clinical nutrition research unit (p dk ), the baltimore diabetes research and training center (p dk ) from the national institutes of health, and aha scientist development grant n. we gratefully acknowledge our amish liaisons and field workers and the extraordinary cooperation and support of the amish community without which these studies would not have been possible. rosenthal, m. d. & glew, r. h. medical biochemistry: human metabolism in health and disease st ed (wiley, nj, usa, ). rej, r. a spartate aminotransferase activity and isoenzyme proportions in human liver tissues. clin. chem. , – ( ). rej, r. measurement of aspartate aminotransferase activity: effects of oxamate. clin. chem. , – ( ). rahmioglu, n., andrew, t., cherkas, l., surdulescu, g., swaminathan, r., spector, t. et al. epidemiology and genetic epidemiology of the liver function test proteins. plos one , e ( ). whitfield, j. b., zhu, g., nestler, j. e., heath, a. c. & martin, n. g. genetic covariation between serum gamma-glutamyltransferase activity and cardiovascular risk factors. clin. chem. , – ( ). mitchell, b. d., mcardle, p. f., shen, h., rampersaud, e., pollin, t. i., bielak, l. f. et al. the genetic response to short-term interventions affecting cardiovascular function: rationale and design of the heredity and phenotype intervention (hapi) heart study. am. heart j. , – ( ). friedewald, w. t., levy, r. i. & fredrickson, d. s. estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracen- trifuge. clin. chem. , – ( ). melzer, d., perry, j. r., hernandez, d., corsi, a. m., stevens, k., rafferty, i. et al. genome-wide association study identifies protein quantitative trait loci (pqtls). plos genet. , e ( ). yuan, x., waterworth, d., perry, j. r., lim, n., song, k., chambers, j. c. et al. population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes. am. j. hum. genet. , – ( ). pollin, t. i., damcott, c. m., shen, h., ott, s. h., shelton, j., horenstein, r. b. et al. a null mutation in human apoc confers a favorable plasma lipid profile and apparent cardioprotection. science , – ( ). shen, h., damcott, c. m., rampersaud, e., pollin, t. i., o’connell, j. r., horenstein, r. b. et al. familial defective apolipoprotein b- and increased low-density lipoprotein cholesterol and coronary artery calcification in the old order amish. arch. intern. med. , – ( ). corpet, f. multiple sequence alignment with hierarchical clustering. nucleic acids res , – ( ). a s t a c ti v it ty (u /g ra m p ro te in ) column vector ast wt . ast mut flag-cast vector castwt gapdh castmut . . . . . . . . . . . figure functional study of recombinant mutant and wild-type cast proteins. (a) ast activities were measured in lysates of hek cells transfected with empty vector (vector), wild-type (castwt), or p.asn del mutant (castmut) at the indicated amount in a six-well plate dish. data are expressed as mean±s.e. (n¼ ). (b) representative western blot of flag- tagged wild-type and mutant cast protein levels in hek cells (same lane order as (a)) detected with anti-flag antibody. anti-gapdh was used as protein loading control. a mutation in got determines serum ast levels h shen et al journal of human genetics 	genome-wide association study identifies genetic variants in got determining serum aspartate aminotransferase levels 	introduction 	materials and methods 	population 	genotyping 	functional studies of recombinant cast 	construction of expression vector of wild-type and mutant cast 	expression and enzymatic assay of recombinant castwt and castmut proteins 	statistical analysis 	results 	discussion 	acknowledgements 	references heritability of choroidal thickness in the amish heritability of choroidal thickness in the amish rebecca j. sardell, phd, muneeswar g. nittala, mphil, larry d. adams, ba, reneé a. laux, ms, jessica n. cooke bailey, phd, denise fuzzell, bs, sarada fuzzell, bs, lori reinhart-mercer, bsn, laura j. caywood, bsn, violet horst, tine mackay, debbie dana, ba, srinivas r. sadda, md, william k. scott, phd, dwight stambolian, md, phd, jonathan l. haines, phd, margaret a. pericak-vance, phd purpose: to evaluate the heritability of choroidal thickness and its relationship to age-related macular degeneration (amd). design: cohort study. participants: six hundred eighty-nine individuals from amish families with early or intermediate amd. methods: ocular coherence tomography was used to quantify choroidal thickness, and fundus photography was used to classify eyes into categories using a modified clinical age-related maculopathy staging (carms) system. repeatability and heritability of choroidal thickness and its phenotypic and genetic correlations with the amd phenotype (carms category) were estimated using a generalized linear mixed model (glmm) approach that accounted for relatedness, repeated measures (left and right eyes), and the effects of age, gender, and refraction. main outcome measures: heritability of choroidal thickness and its phenotypic and genetic correlation with the amd phenotype (carms category). results: phenotypic correlation between choroidal thickness and carms category was moderate (spear- man’s rank correlation, rs ¼ � . ; n ¼ eyes) and significant (glmm posterior mean, � . ; % credible interval [ci], � . to � . ; p ¼ . ) after controlling for relatedness, age, gender, and refraction. eyes with advanced amd had thinner choroids than eyes without amd (posterior mean, � . ; % ci, � . to � . ; p < . ; n ¼ eyes). choroidal thickness was highly repeatable within individuals (repeatability, . ; % ci, . to . ) and moderately heritable (heritability, . ; % ci, . to . ), but did not show significant genetic correlation with carms category, although the effect size was moderate (genetic correlation, � . ; % ci, � . to . ). choroidal thickness also varied with age, gender, and refraction. the carms category showed moderate heritability (heritability, . ; % ci, . to . ). conclusions: we quantify the heritability of choroidal thickness for the first time, highlighting a heritable, quantitative trait that is measurable in all individuals regardless of amd affection status, and moderately phenotypically correlated with amd severity. choroidal thickness therefore may capture variation not captured by the carms system. however, because the genetic correlation between choroidal thickness and amd severity was not significant in our data set, genes associated with the traits may not overlap substantially. future studies should therefore test for genetic variation associated with choroidal thickness to determine the overlap in genetic basis with amd. ophthalmology ; : - ª by the american academy of ophthalmology. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). age-related macular degeneration (amd) is a major cause of blindness in older adults. both demographic and environmental factors, including advanced age, gender, smoking history, and diet, contribute to the risk of amd developing. e intermediate and advanced amd are also heritable (heritability [the proportion of phenotypic variation that is explained by genetic differences], . e . , ), with several common and rare genetic risk factors. , although identified genetic variants explain a relatively large pro- portion ( %e %) of the heritability of advanced disease, a substantial portion remains unexplained. , progression of amd also is poorly understood and highly variable. in addition to unidentified rare variants or interaction effects, ª by the american academy of ophthalmology this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). published by elsevier inc. residual variation in disease risk, heritability, and progression may be partly a reflection of the currently used classification for amd. despite the complexity of the amd phenotype, eyes usually are classified into discrete categories using the age- related eye disease study (areds) , or simplified clinical age-related maculopathy staging (carms) clas- sification systems, which are based largely on the presence and size of key hallmarks of amd, such as drusen or retinal pigmentation. furthermore, most studies of genetic association compare individuals with no or few signs of amd (controls or carms categories and ) with those with late-stage disease (carms categories and ), http://dx.doi.org/ . /j.ophtha. . . issn - / http://creativecommons.org/licenses/by-nc-nd/ . / http://crossmark.crossref.org/dialog/?doi= . /j.ophtha. . . &domain=pdf http://creativecommons.org/licenses/by-nc-nd/ . / http://dx.doi.org/ . /j.ophtha. . . table . modified clinical age-related maculopathy staging classification system category description no drusen < hard drusen > hard drusen or some medium drusen > medium drusen or a single large drusen foveal geographic atrophy choroidal neovascularization ophthalmology volume , number , december whereas only a few studies have considered the genetics of early or intermediate amd or specific amd subtypes. , , such broad-scale classification of disease stages may not adequately represent the biological basis of the disease and may mask important subphenotypes that are linked more directly to the underlying disease process. features found in amd cases also may overlap with other retinal diseases that have a distinct genetic basis, confounding our ability to predict disease risk. we hypothesized that parsing the com- plex amd phenotype into heritable finer-scale retinal traits that are easily measurable in all individuals and that each have a relatively simple genetic basis (endophenotypes ) will increase our understanding of the biological basis of amd, enabling better prediction of disease risk and progression, and aiding the discovery of novel drug targets. , for example, an endophenotype approach was used recently to identify ocular traits and genes associated with glaucoma , and myopia. because of recent technological advances, spectral- domain (sd) ocular coherence tomography (oct) now allows detailed cross-sectional imaging of the retina’s ultrastructure, offering enhanced detection, measurement, and analysis of retinal traits beyond those offered by tradi- tional fundus photography. therefore, sd oct may aid the identification of amd endophenotypes or biomarkers that can be used to predict risk or progression to advanced stages. , traits such as choroidal thickness, , drusen volume, and the presence of reticular pseudodrusen , have been linked previously to amd disease status and progression and may define amd endophenotypes. for example, choroidal thickness was found to decrease with increasing amd severity (areds categories e ). however, most studies have measured only the overall phenotypic correlation between retinal traits and amd, but phenotypic correlation may result from genetic correction (overlapping genes), environmental correlation, or both. if environmental factors drive the correlation between retinal traits and amd, rather than the same genes, then performing genetic association analyses on these fine-scale retinal traits may not be informative for amd. therefore, the relationship between retinal features, amd risk and progression, and genetics is unclear and requires further investigation. specifically, for a trait to be useful as an amd endophenotype requires that the trait is shown to be heritable and genetically correlated, to some extent, with the amd phenotype, that is, that there is some shared genetic basis between the quantitative trait and the disease. , , such analyses can be performed by measuring the phenotypic similarity and relatedness between family members in a pedigree or twin study because this allows phenotypic vari- ation to be separated into genetic variation, environmental variation, and individual-level variation (repeatability). to assess the use of choroidal thickness as an amd endophenotype for future genetic studies, we examined whether the trait is heritable (i.e., whether a significant pro- portion of the phenotypic variation is explained by genetic variation) and phenotypically and genetically correlated with the amd phenotype (carms category) using families from the amish eye study. the amish are genetically and culturally isolated, and experience a relatively uniform environment, reducing genetic diversity and variance in dis- ease risk. additionally, their large extended families provide a powerful tool for heritability analyses. the frequency of smoking (a key environmental risk factor for amd ) also is low. the amish therefore provide an excellent opportunity to examine the genetic architecture of complex disease. methods study population and data collection participants were recruited from amish populations in lancaster county, pennsylvania; holmes county, ohio; and elkhart and lagrange counties, indiana. informed consent was obtained from all individuals. institutional review board approval was obtained, and research complied with the health insurance portability and accountability act and adhered to the tenets of the declaration of helsinki. individuals and their siblings were recruited from fam- ilies with at least affected individuals with early or intermediate amd. recruited families varied in size from nuclear families of up to siblings to extended families of up to individuals. at each clinical center (indiana, ohio, pennsylvania) participants underwent a health history and ophthalmologic examination that included color fundus photography and sd oct volume scans for both eyes where possible. for choroidal thickness assessments, sd oct imaging was performed with the spectralis oct device (hei- delberg engineering, inc., heidelberg, germany) using a �� � field of view centered on the fovea with b-scans each comprising a-scans. images were exported to the doheny image reading center and the choroidal thickness was measured at the foveal center, from the lower border of the retinal pigment epitheliumebruch’s membrane band to the choroidalescleral junction, using the caliper tool in the heyex (heidelberg, germany) software, in accordance with previous reports from the reading center. eyes were classified by a modified carms classification (categories e ) at the doheny center from color fundus photographs (table ). the carms system grades eyes from to and considers eyes with no drusen and few small drusen as category . to achieve a more granular phenotype, for this analysis, eyes with no drusen were assigned to a new category of , whereas only those with a few small drusen were included in category . category included eyes with many small drusen or a few medium drusen, and thus included eyes both without amd and with early amd (using the convention that medium drusen constitute the minimum criteria for amd ). category included eyes with intermediate amd, and categories and included eyes with advanced amd, as in the carms system (table ). statistical analysis to assess the use of choroidal thickness as an amd endopheno- type we quantified ( ) its overall phenotypic correlation with the table . demographic parameters for participants parameter pennsylvania indiana ohio all gender women, men women, men women, men women, men age, yrs (mean � sd) . � . . � . . � . . � . sd ¼ standard deviation. sardell et al � heritability of choroidal thickness amd phenotype, ( ) its heritability, and ( ) its genetic correlation with the amd phenotype to assess the extent to which genetic variation underlying the traits overlapped. our primary analyses treated the amd phenotype as an ordinal trait, carms category ( e ), because this approach was more powerful than dichoto- mizing the phenotype as a binary trait (presence or absence of amd). however, in a secondary analysis we reanalyzed data treating the amd phenotype as a binary trait where possible. broad-sense heritability (the proportion of phenotypic variance that is explained by genetic variance) of both choroidal thickness and carms category and their correlation were quantified in a generalized linear mixed model (glmm) framework. a glmm approach enabled the use of repeated measures (both left and right eyes per subject) and hence an estimate of the proportion of phenotypic variance in each trait that was explained by individual identity (repeatability), the inclusion of covariates such as age, and it maximized the use of relatedness information from a pedigree. analyses were run using the r-package mcmcglmm (available at https://cran.r-project.org/web/packages/mcmcglmm/index.html) that fits models in a bayesian framework using markov chain monte carlomethods. first,aunivariateglmmofchoroidalthicknesswas used to test whether choroidal thickness varied with amd severity (carms category) while controlling for age, gender, spherical equivalent refraction (sphere plus half the cylinder), relatedness, and repeated measures (left and right eyes). a bivariate glmm with a -trait response variable then was fit with carms category ( e ) specified as an ordinal (threshold) trait with probit link and choroidal thickness specified as a gaussian trait to quantify the heritability of each trait and their genetic correlation while controlling for age, gender, and spherical equivalent refraction (as a covariate for choroidal thickness only). a pedigree (participant, mother, father) was used to estimate a genetic varianceecovariance matrix, and a random effect of individual identification was fit to account for repeated measures per person (left and right eyes), allowing phenotypic variance to be partitioned into genetic, individual-level, and residual variance. shared environmental effects between family members were not accounted for and may conflate our estimate of genetic variance, but because amd is a late-onset disease, it was unclear whether accounting for a shared sibship environment was relevant. eyes that were missing of the traits (carms category or choroidal thickness; n ¼ ) were included in analyses because bivariate glmms can handle missing data in the response variable. in the bivariate model, random effect and residual table . number of participants by modified clinical age-related ma eyes) population clinical age-related maculopathy staging c pennsylvania indiana ohio total variances were specified using the us(trait) structure, thereby allowing the variance and covariance to vary between the traits. default priors were used for fixed effects. priors on variance components for residual terms were inverse wishart distributed with variance of and a low degree of belief (nu ¼ . ), with variance fixed at for the ordinal trait. for random effects, we used parameter-expanded priors to facilitate mixing with a mean (mu) of and variance (v) of for choroidal thickness and for carms category. variance estimates were similar when models were run with alternative priors. models were run for iterations with a burn-in interval of and thin of to give an effective sample size of approximately and autocorrelation less than . between consecutive samples. model convergence and mixing were assessed by visual inspection of plots and by using the heidel.diag function in the coda package. all analyses were conducted in r version . . (available at: http://www.cran.r- project.org). the heritability of choroidal thickness was quantitatively similar when run in a (restricted) maximum likelihood framework using the r package pedigreemm. results a total of participants ( women and men) from indiana (n ¼ ), pennsylvania (n ¼ ), and ohio (n ¼ ) were recruited and examined between august and november (n ¼ eyes; table ). mean baseline age of participants was . � . years (range, e years). considering the most severely affected eye per individual, approximately . % of participants were carms category , . % were carms category , . % were carms category , . % were carms category , . % had advanced amd (carms categories or ), and . % were not graded because of a fundus camera malfunction at site (table ). mean spherical equivalent refraction was . � . diopters for right eyes and . � . diopters for left eyes. mean choroidal thickness of right eyes was . � . mm and that of left eyes was . � . mm (fig ). choroidal thickness was strongly correlated between left and right eyes (pearson’s correlation coefficient, r ¼ . ; n ¼ ; fig ). as expected, carms category also was strongly correlated between eyes (spearman’s correlation coefficient, rs ¼ . ; n ¼ ; fig ); therefore, eyes tended to be at the culopathy staging classification system category (most advanced lassification system category not graded all https://cran.r-project.org/web/packages/mcmcglmm/index.html http://www.cran.r-project.org http://www.cran.r-project.org figure . bar graphs showing the distribution of subfoveal choroidal thickness (in micrometers) for (a) right eyes and (b) left eyes. figure . scatterplots showing the phenotypic correlation of (a) clinical age-related maculopathy staging (carms) category and (b) subfoveal choroidal thickness between left and right eyes and the correlation between subfoveal choroidal thickness and carms category for (c) right eyes and (d) left eyes. points have been jittered for visualization. ophthalmology volume , number , december t ab le . p os te ri or m ea n s (a n d % cr ed ib le in te rv al s) fo r v ar ia n ce an d c ov ar ia n ce c om po n en ts e st im at ed fr om a b iv ar ia te g en er al iz ed l in ea r m ix ed m od el of c h or oi da lt h ic kn es s an d m od ifi ed c li n ic al a ge -r el at ed m ac ul op at h y s ta gi n g c la ss ifi ca ti on s ys te m c at eg or y t ra it g en et ic v ar ia n ce (v a ) p er m an en t in di vi du al v ar ia n ce (v p e) r es id u al v ar ia n ce (v e) h er it ab il it y (h ) g en et ic c ov ar ia n ce (c ov , ) g en et ic c or re la ti on (r a ) c h or oi da l th ic kn es s . ( . e . ) . ( . e . ) . ( . e . ) . ( . e . ) � . (� . to . ) � . (� . to . ) c a r m s cl as si fi ca ti on sy st em ca te go ry . ( . e . ) . ( . e . ) (fi xe d) . ( . e . ) c a r m s ¼ c li n ic al a ge -r el at ed m ac ul op at h y s ta gi n g. r ep ea ta bi li ty (r ¼ v a þ v pe /( v a þ v pe þ v e) ), h er it ab il it y (h ¼ v a /( v a þ v pe þ v e) ), an d ge n et ic co rr el at io n be tw ee n ch or oi da lt h ic kn es s an d c a r m s ca te go ry (r a ¼ c ov , /o (v a � v a )) ar e sh ow n , w h er e v a is ge n et ic va ri an ce , v pe is pe rm an en t en vi ro n m en ta l va ri an ce , v e is th e re si du al va ri an ce , an d c ov is th e ge n et ic co va ri an ce be tw ee n tr ai ts . sardell et al � heritability of choroidal thickness same disease stage. however, correlation coefficients suggested that there was sufficient variation to justify the inclusion of both eyes in future analyses. choroidal thickness showed moderate negative phenotypic correlation with carms category (rs ¼ � . ; n ¼ eyes; fig ); eyes more severely affected with amd had thinner choroids. this correlation was significant in a univariate glmm of choroidal thickness controlling for relatedness, repeated measures (left and right eyes), age, gender, and refraction (posterior mean, � . ; % credible interval [ci], � . to � . ; mcmc p value ¼ . ). secondary analyses defining amd severity as a binary trait showed that choroidal thickness was marginally thinner in eyes with amd (categories , , and ; n ¼ eyes; posterior mean, � . ; % ci, � . to . ; mcmc p value ¼ . ) compared with eyes with no amd (carms categories , , and ; n ¼ eyes). interestingly, this difference in choroidal thickness between affected and unaffected individuals was substantially stronger and significant if category (intermediate amd) eyes were excluded (posterior mean, � . ; % ci, � . to � . ; mcmc p value < . ; n ¼ eyes); therefore, eyes with advanced amd had significantly thinner choroids than those without amd. however, note that the sample size for this secondary analysis was small (n ¼ individuals with at least advanced amd eye). a bivariate glmm estimated that the repeatability (proportion of phenotypic variation that was explained by an individual’s identity) of choroidal thickness was high ( . ; % ci, . to . ; n ¼ eyes) and that the heritability of choroidal thickness was moderate ( . ; % ci, . to . ; table ). the carms category also was highly repeatable ( . ; % ci, . to . ; table ) and moderately heritable (heritability, . on the liability scale; % ci, . to . ), similar to that estimated by a twin study (heritability, . ). choroidal thickness (r ¼ � . ; markov chain monte carlo [mcmc] p value < . ; fig ) and carms category (r ¼ � . ; mcmc p value < . ; fig ) were significantly negatively correlated with age, but only choroidal thickness varied with gender; men had slightly thinner choroidal thickness (mcmc p value ¼ . ; table ). choroidal thickness also was correlated positively with refraction (table ). genetic correlation between choroidal thickness and carms category was � . , but % cis overlapped (� . to . ); therefore, the correlation was not statistically significant (table ). the bivariate model also estimated that the overall phenotypic correlation between choroidal thickness and carms category was moderate, negative, and significant (� . ; % ci, � . to � . ). we did not test for the genetic correlation between choroidal thickness and the amd phenotype as a binary trait because the complexity of a bivariate model and the relatively small number of advanced amd cases ( vs. eyes with no amd) prevented model convergence despite long run time. discussion decomposing disease phenotypes into heritable sub- components may be especially useful for unravelling the complex genetic basis of multifactorial diseases such as amd. endophenotypes also may be useful as biomarkers of disease risk or progression, thereby influencing clinical decision making and allowing for intervention to alter disease progression. genetic studies of heritable, novel amd phenotypes also may provide additional biological pathways and therapeutic targets for early or intermediate figure . scatterplots showing the correlation between (a) clinical age-related maculopathy staging (carms) category and age and (b) subfoveal choroidal thickness and age. points are jittered for visualization. ophthalmology volume , number , december amd. one retinal trait that is quantifiable in all individuals using oct imaging, regardless of amd disease status, is choroidal thickness. the choroid performs many of the retina’s essential metabolic functions, and thinning of the choroid previously was associated with age and amd; older individuals e and those with amd , e had thinner choroids, although some studies did not find a difference with amd status. , e here we show that, after controlling for age, gender, and refraction, choroidal thickness is phenotypically negatively correlated with carms category and hence the severity of amd. eyes with amd also had marginally thinner choroids than those without amd. phenotypic but not genetic cor- relation between choroidal thickness and amd has been tested previously. several studies found that advanced amd cases had thinner choroids than controls. , , some studies also found a difference in choroidal thickness table . main effect parameters (posterior mean, % credible interval, and mcmc p values) from a bivariate generalized linear mixed model investigating variation in choroidal thickness and modified clinical age-related maculopathy staging category with respect to age, gender, and refraction trait posterior mean % credible interval mcmc p value choroidal thickness intercept (women) . . to . < . carms category intercept (women) � . � . to � . < . choroidal thickness: age � . � . to � . < . carms category: age . . to . < . choroidal thickness: men � . � . to � . . carms category: men � . to . . choroidal thickness: refraction . . to . < . carms ¼ clinical age-related maculopathy staging. between early amd cases and controls, e whereas others did not, , , and some found that the strength of the correlation depends on the amd subtype. , we found a stronger difference between amd cases and controls when eyes with intermediate amd were excluded, albeit with a small sample of advanced cases, which suggested that the correlation between choroidal thickness and amd severity is driven primarily by the considerable decrease in choroidal thickness in advanced amd cases (fig ). moreover, we showed for the first time that choroidal thickness is significantly heritable and therefore has a substantial genetic component. choroidal thickness there- fore may define an amd endophenotype useful for genetic studies. however, for a trait to define an endophenotype, it also should show some (but not perfect) genetic correlation with the disease phenotype. although the effect size of the genetic correlation between choroidal thickness and carms category was moderate (� . ), suggesting that the phenotypic correlation observed between the traits may reflect some overlap in genetic basis, the genetic correlation was not significantly different from . genetic correlation is the extent to which traits share the same genes, whereas phenotypic correlation also encompasses environmental correlation. it is likely that the absence of significant genetic correlation results from relatively low power to detect correlation with the ordinal trait, carms category, because our study was focused primarily on families whose members demonstrated early or interme- diate amd, and therefore our sample consisted of many controls (approximately % of individuals were cate- gories or ) and relatively few individuals with advanced amd ( %). any genetic correlation between choroidal thickness and amd may be more likely to be detected across a sample with a larger proportion of advanced amd cases or a more variable sample of unrelated individuals using genome-wide trait analysis. finally, carms cate- gory is only measure of amd severity or presence. indeed, many studies dichotomize amd scales into sardell et al � heritability of choroidal thickness case-control status to study the genetics of advanced amd risk. our preliminary analyses of this relatively small sample size suggested that the genetic correlation between these traits was stronger than that for carms category. therefore, the absence of a significant genetic correlation in this data set of patients with early or intermediate dis- ease and their unaffected relatives does not preclude the use of choroidal thickness as an amd endophenotype. because the correlation between choroidal thickness and amd severity was, at most, moderate, choroidal thickness may capture novel genetic and phenotypic variation and therefore may be especially informative for future studies of amd. numerous common and rare variants are associated with advanced amd, although cumulatively they explain no more than % of the heritability of advanced disease, and less for early or intermediate amd. , age-related macular degeneration 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financial disclosures originally received: april , . final revision: august , . accepted: september , . available online: october , . manuscript no. - . john p. hussman institute for human genomics, university of miami miller school of medicine, miami, florida. department of ophthalmology, doheny eye institute, university of california, los angeles, los angeles, california. department of epidemiology and biostatistics, case western reserve university, cleveland, ohio. departments of ophthalmology and genetics, university of pennsylvania, philadelphia, pennsylvania. presented as a poster at: association for research in vision and ophthal- mology annual meeting, may , denver, colorado; and american society for human genetics annual meeting, october , san diego, california; and, october , baltimore, maryland. financial disclosure(s): the author(s) have made the following disclosure(s): s.r.s.: consultant e optosplc (dunfermline, scotland); carl zeiss meditec hq (jena, ger- many); alcon (fort worth, tx); allergan hq (parsippany-troy hills, nj); genentech (san francisco ca); regeneron (tarrytown, ny); novartis (basel, switzerland); financial support e optos; carl zeiss meditec; board member e allergan; genentech supported by the national eye institute, national institutes of health, bethesda, maryland (grant no.: r ey - ; postdoctoral training fellowship no.: t :ey [r.j.s.]), and by a fellowship from phrma informatics, washington, dc (j.n.c.b.). the funding organizations had no role in the design or conduct of this research. author contributions: conception and design: sadda, scott, stambolian, haines, pericak-vance analysis and interpretation: sardell, nittala, sadda, scott, stambolian, haines, pericak-vance data collection: sardell, nittala, adams, laux, cooke bailey, d.fuzzell, s.fuzzell, reinhart-mercer, caywood, horst, mackay, dana, sadda, scott, stambolian, haines, pericak-vance obtained funding: none overall responsibility: sardell, nittala, adams, laux, cooke bailey, d.fuzzell, s.fuzzell, reinhart-mercer, caywood, horst, mackay, dana, sadda, scott, stambolian, haines, pericak-vance abbreviations and acronyms: amd ¼ age-related macular degeneration; carms ¼ clinical age- related maculopathy staging; ci ¼ credible interval; glmm ¼ generalized linear mixed model; oct ¼ ocular coherence to- mography; sd ¼ spectral-domain. correspondence: margaret a. pericak-vance, phd, john p. hussman institute for human genomics, university of miami miller school of medicine, nw th avenue, brb , miami, fl . e-mail: mpericak@med.miami.edu. http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref mailto:mpericak@med.miami.edu 	heritability of choroidal thickness in the amish 	methods 	study population and data collection 	statistical analysis 	results 	discussion 	references apical hypertrophic cardiomyopathy among non-asians: a case series and review of the literature articles © the authors | journal compilation © cardiol res and elmer press inc™ | www.cardiologyres.org this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited case report cardiol res. ; ( ): - resselmer apical hypertrophic cardiomyopathy among non-asians: a case series and review of the literature karan kapoora, c, amal chaudhrya, matthew c. evansa, amish surab abstract apical hypertrophic cardiomyopathy (ahcm) has been rarely de- scribed in the western world. more recently, improved sensitivity of diagnostic modalities and increased diagnostic awareness have in- creased detection rates, suggesting that the prevalence outside of asia may have been previously understated. hallmark features of ahcm include deeply negative, “giant” t-wave inversions on electrocar- diography and a “spade-like” configuration of the left ventricle on ventriculography. we present two cases of ahcm, one in an african- american female and another in a caucasian male. keywords: hypertrophic cardiomyopathy; apical variant; echocar- diography introduction apical hypertrophic cardiomyopathy (ahcm) is found in up to % of japanese patients with hypertrophic cardiomyopathy (hcm), but outside of this population, it represents a markedly uncommon morphologic variant of hcm, with most reports suggesting a prevalence between % and % [ - ]. the basis for differences in the phenotypic expression of apical hypertro- phy between asians and non-asians has not been elucidated. although most patients with ahcm experience minimal to no symptoms, presentations with a variety of signs and symptoms including atrial fibrillation, ventricular tachycardia and angina have been described [ ]. typical features of ahcm include t-wave inversion, particularly in the left precordial leads of the electrocardiogram, and a “spade-like” configuration of the left ventricular cavity at end-diastole on left ventriculography [ , ]. cardiac catheterization (and subsequent ventriculogra- phy) or cardiac magnetic resonance imaging (cmri) is often needed to establish the diagnosis, which can be missed on two-dimensional transthoracic echocardiography. advances in non-invasive imaging techniques, and in particular, cmri, have led to its establishment as the gold standard diagnostic modality given the invasive nature of cardiac catheterization [ , ]. although generally associated with a better prognosis than other forms of hcm, serious cardiac complications have been described, including progressive heart failure, myocar- dial infarction and sudden-cardiac death [ , ]. we describe two cases of ahcm in non-asian patients. case reports case a -year-old african-american woman presented with a sev- eral hour history of typical angina. she provided a history of hypertension, hyperlipidemia, metabolic syndrome, and a fam- ily history of sudden cardiac death in her -year-old brother. associated symptoms included palpitations, progressive or- thopnea, and pedal edema. vital signs were all within normal limits. cardiac examination disclosed a prominent s . electro- cardiography (ekg) revealed left ventricular hypertrophy and deep t-wave inversions in leads v -v , but no st-segment abnormalities (fig. ). cardiac biomarkers and basic labora- tory investigations were normal. transthoracic echocardiography revealed normal left ven- tricular size and function (ejection fraction %), and moder- ate-to-severe left ventricular hypertrophy. although the apex was not well visualized, obstructive physiology was noted de- spite lack of a well-defined gradient. coronary angiography revealed no significant epicardial coronary disease. obliteration of the apex in a “spade-like” fashion and elevation of the left ventricular end-diastolic pres- sure were noted on ventriculography (fig. ). the diagnosis of ahcm was confirmed by cmri (fig. a, b; supplementary video , www.cardiologyres.org). given the family history of sudden cardiac death, she underwent implantation of a single- chamber internal cardioverter defibrillator. case a -year-old caucasian man with a history of paroxysmal atrial fibrillation, obstructive sleep apnea and a family history of sudden cardiac death in two first-degree relatives presented manuscript accepted for publication february , auniversity of maryland medical center, baltimore, md, usa bmercy medical center, baltimore, md, usa ccorresponding author: karan kapoor, department of internal medicine, university of maryland medical center, south greene street, baltimore, md , usa. email: kkapoor@umm.edu doi: http://dx.doi.org/ . /cr w articles © the authors | journal compilation © cardiol res and elmer press inc™ | www.cardiologyres.org kapoor et al cardiol res. ; ( ): - to the outpatient clinic with palpitations. his initial diagnosis of atrial fibrillation was made years prior and was attributed to excessive caffeine consumption, given that he regained a normal sinus rhythm following cessation of caffeine. at the time of atrial fibrillation diagnosis, transthoracic echocardio- gram demonstrated evidence of mild left ventricular hyper- trophy. ten years later, his resting electrocardiogram dem- onstrated recurrence of rate-controlled atrial fibrillation, left ventricular hypertrophy and . mm deep t-wave inversion in leads v -v . he was placed on an event monitor and was not- ed to experience beats of non-sustained ventricular tachy- cardia in addition to periods of atrial fibrillation. he underwent exercise nuclear stress testing according to a standard bruce protocol, during which he completed s of stage iv prior to experiencing shortness of breath at % of his maximal pre- dicted heart rate and concomitant worsening in the degree of t-wave inversions in the inferior and lateral leads (fig. ). no evidence of reversible ischemia was noted. he subsequently underwent cardiac catheterization, which showed no evidence of obstructive coronary disease, but a “spade-like” configura- tion of the left ventricle similar to that seen in case (fig. ). a diagnosis of ahcm was made. given his family history and the occurrence of ventricular tachycardia, implantable cardiac defibrillator therapy was offered. both of our patients and their first-degree family members declined any genetic testing. at subsequent -month follow- up visits, both patients were asymptomatic and reported ad- herence to beta-blocker therapy. no inadvertent defibrillator shocks were reported. discussion the apical variant of hcm in which left ventricular wall thick- ening is confined to the most distal region of the apex has been regarded as a phenotypic expression of non-obstructive hcm largely limited to japanese patients. nevertheless, since its original description in the s [ , ], several studies have been published outside of asia regarding this entity, albeit in- sufficiently powered for robust conclusions regarding natural history, prognosis or long-term management strategies [ , , ]. however, the implication of these studies has been that ahcm among non-asian patients may represent an entirely figure . resting ekg of the patient from case . ekg illustrates marked t-wave inversions in leads ii, iii, avf, and v -v , a typical feature of ahcm. figure . ventriculogram from case demonstrating complete oblitera- tion of the apex in a “spade-like” or “bird’s beak” fashion. elevation of the left ventricular end-diastolic pressure was also noted. articles © the authors | journal compilation © cardiol res and elmer press inc™ | www.cardiologyres.org ahcm cardiol res. ; ( ): - different entity that carries a different prognosis compared to the japanese variant. our first case highlights the potential for ahcm to mani- fest early in adulthood, similar to other more commonly seen variants of hcm [ ]. most other case series involving at least or more patients with the apical variant report a mean age of at least years [ ]. furthermore, this case highlights the po- tential for ahcm to present with angina, which is followed by atypical chest pain ( %), palpitations ( %), dyspnea ( %) and pre-syncope/syncope ( %) in the minority of symptomatic individuals according to one of the larger published series [ ]. notably, smith et al described an initial presentation of ahcm figure . similar ventricular morphology demonstrated on cmri in the sagittal (a) and axial (b) planes. video clip of systole captured during cmri from case , further demonstrating the hallmark morphology of ahcm. figure . ekg from stage iv of an exercise stress test from case . ekg illustrates more pronounced t-wave inversion when compared to the patient’s resting ekg (not shown). articles © the authors | journal compilation © cardiol res and elmer press inc™ | www.cardiologyres.org kapoor et al cardiol res. ; ( ): - similar to case (i.e. severe angina) in a -year-old african- american man, suggesting a potential phenotypic proclivity among young african-american patients [ ]. the second case in our series is representative of the more common, older-onset ahcm [ ]. our patient had antecedent echocardiographic evidence of left ventricular hypertrophy in addition to atrial fibrillation several years prior to his declara- tive presentation. delayed penetrance and late appearing left ventricular hypertrophy have been described among adults diagnosed with ahcm. more frequent surveillance imaging in the -year interval between his initial diagnosis of atrial fibrillation and development of palpitations may have yielded an earlier diagnosis. interestingly, our patient fits the profile of caucasian ahcm patients described in an australian series of patients, regarding his late and relatively abrupt onset of symptomatic ahcm [ ]. both atrial fibrillation and coronary fistulae were noted with increased frequency among these pa- tients, although the latter was absent in our patient. the devel- opment of ahcm in older individuals may represent an exam- ple of gene-environment interaction required for phenotypic manifestations, although the exact environmental triggers are unclear [ ]. additionally, the absence of marked t-wave in- versions on his resting ekg reflects prior studies suggesting that the incidence of this electrocardiographic finding dimin- ishes considerably with advancing age [ ]. finally, pertinent to both of our cases is the fact that female gender and atrial fibrillation have been identified as predictors of mortality in retrospective studies [ ]. ethnically, ahcm accounts for - % of all variants of hcm among asian individuals, whereas the prevalence among non-asians is < % [ ]. however, much of these epidemiologic data are derived from studies conducted in the early s, and the exact prevalence of ahcm in non-asians may be underestimated due to diagnostic unawareness as well as the heterogeneous appearance on transthoracic echocardio- gram, the most frequently utilized diagnostic modality [ ]. contrary to our two cases, previous studies have demonstrated less hypertrophy confined predominantly to the apex and t- wave abnormalities on ekg among north-american ahcm subgroups [ ]. despite the higher prevalence of ahcm among japanese patients, clinical presentation and long-term cardio- vascular outcomes appear to be similar [ ]. specifically, long-term follow up studies have shown co-morbid atrial fi- brillation, apical myocardial infarction, ventricular arrhythmia and apical thrombosis with subsequent embolization to occur in up % of all patients irrespective of ethnicity [ ]. the presence of symptoms in our two cases, severe angina in the first and symptomatic palpitations in the second, bolsters the observations of other investigators that the clinical expression of ahcm is highly variable, and likely subject to a multitude of genetic and environmental influences. no definitive guidelines delineate the role for defibrillator implantation in ahcm patients with family histories of sud- den cardiac death. not surprisingly, expert consensus supports the use of implantable defibrillator for primary prevention in select high risk patients, notably those with one of the five fol- lowing risk factors: a family history of sudden cardiac death, syncope, asymptomatic non-sustained ventricular tachycardia, an abnormal blood pressure response to exercise and a left ventricular wall thickness > mm [ ]. genetic testing has also been offered to both of our patients and their first-degree family members; although contrary to other subtypes of hcm, the apical variant has only occasionally been described as a familial disease manifesting an autosomal dominant inherit- ance. a very limited number of sarcomere gene defects, and in particular, cardiac actin glu lys, have been shown to con- sistently produce the ahcm phenotype [ ]. the widespread availability of genetic testing has led to increased recognition of genotype-positive/phenotype-negative patients. although at present, it is not possible to predict clinical outcomes based on the presence of individual mutations, guidelines do suggest extension of surveillance with cardiac imaging at least through mid-life ( years of age, and beyond, in select circumstances) to detect development of overt disease [ ]. grant support none. references . maron ms, finley jj, bos jm, hauser th, manning wj, haas ts, lesser jr, et al. prevalence, clinical sig- nificance, and natural history of left ventricular apical aneurysms in hypertrophic cardiomyopathy. circulation. ; ( ): - . . kitaoka h, doi y, casey sa, hitomi n, furuno t, maron bj. comparison of prevalence of apical hypertrophic car- diomyopathy in japan and the united states. am j car- diol. ; ( ): - . . klues hg, schiffers a, maron bj. phenotypic spectrum and patterns of left ventricular hypertrophy in hypertroph- ic cardiomyopathy: morphologic observations and signif- icance as assessed by two-dimensional echocardiography in patients. j am coll cardiol. ; ( ): - . figure . cardiac catheterization from case . ventriculogram per- formed during cardiac catheterization reveals “spade-like” configura- tion of the left ventricle, characteristic of ahcm. articles © the authors | journal compilation © cardiol res and elmer press inc™ | www.cardiologyres.org ahcm cardiol res. ; ( ): - . sakamoto t, tei c, murayama m, ichiyasu h, hada y. giant t wave inversion as a manifestation of asymmetri- cal apical hypertrophy (aah) of the left ventricle. echo- cardiographic and ultrasono-cardiotomographic study. jpn heart j. ; ( ): - . . yamaguchi h, ishimura t, nishiyama s, nagasaki f, nakanishi s, takatsu f, nishijo t, et al. hypertrophic nonobstructive cardiomyopathy with giant negative t waves (apical hypertrophy): ventriculographic and echocardiographic features in patients. am j cardiol. ; ( ): - . . patel j, michaels j, mieres j, kort s, mangion jr. echocardiographic diagnosis of apical hypertrophic car- diomyopathy with optison contrast. echocardiography. ; ( ): - . . moon jc, fisher ng, mckenna wj, pennell dj. detec- tion of apical hypertrophic cardiomyopathy by cardiovas- cular magnetic resonance in patients with non-diagnostic echocardiography. heart. ; ( ): - . . eriksson mj, sonnenberg b, woo a, rakowski p, park- er tg, wigle ed, rakowski h. long-term outcome in patients with apical hypertrophic cardiomyopathy. j am coll cardiol. ; ( ): - . . maron bj. apical hypertrophic cardiomyopathy: the con- tinuing saga. j am coll cardiol. ; ( ): - . . maron bj, spirito p, wesley y, arce j. development and progression of left ventricular hypertrophy in chil- dren with hypertrophic cardiomyopathy. n engl j med. ; ( ): - . . klarich kw, attenhofer jost ch, binder j, connolly hm, scott cg, freeman wk, ackerman mj, et al. risk of death in long-term follow-up of patients with apical hypertrophic cardiomyopathy. am j cardiol. ; ( ): - . . smith m, golwala h, hanna eb. apical hypertrophic cardiomyopathy with apical aneurysm in an african- american male. j cardiovasc med (hagerstown). ; ( ): - . . chung t, yiannikas j, freedman sb, kritharides l. unu- sual features of apical hypertrophic cardiomyopathy. am j cardiol. ; ( ): - . . chikamori t, doi yl, akizawa m, yonezawa y, ozawa t, mckenna wj. comparison of clinical, morphological, and prognostic features in hypertrophic cardiomyopathy between japanese and western patients. clin cardiol. ; ( ): - . . stainback rf. apical hypertrophic cardiomyopathy. tex heart inst j. ; ( ): - . . yusuf sw, bathina jd, banchs j, mouhayar en, daher in. apical hypertrophic cardiomyopathy. world j car- diol. ; ( ): - . . prasad k, atherton j, smith gc, mckenna wj, fren- neaux mp, nihoyannopoulos p. echocardiographic pit- falls in the diagnosis of hypertrophic cardiomyopathy. heart. ; (suppl ):iii -iii . . epstein ae, dimarco jp, ellenbogen ka, estes na, rd, freedman ra, gettes ls, gillinov am, et al. accf/ aha/hrs focused update incorporated into the accf/ aha/hrs guidelines for device-based therapy of cardiac rhythm abnormalities: a report of the american college of cardiology foundation/american heart asso- ciation task force on practice guidelines and the heart rhythm society. j am coll cardiol. ; ( ):e - . . arad m, penas-lado m, monserrat l, maron bj, sherrid m, ho cy, barr s, et al. gene mutations in apical hyper- trophic cardiomyopathy. circulation. ; ( ): - . . gersh bj, maron bj, bonow ro, dearani ja, fifer ma, link ms, naidu ss, et al. accf/aha guideline for the diagnosis and treatment of hypertrophic cardio- myopathy: executive summary: a report of the american college of cardiology foundation/american heart as- sociation task force on practice guidelines. circulation. ; ( ): - . ccr- - .. personalized medicine and imaging identification of a variant in kdr associated with serum vegfr and pharmacodynamics of pazopanib michael l. maitland , , , chun-fang xu , yu-ching cheng , emily kistner-griffin , kathleen a. ryan , theodore g. karrison , , soma das , , dara torgerson , eric r. gamazon , vasiliki thomeas , matthew r. levine , paul a. wilson , nan bing , yuan liu , lon r. cardon , lini n. pandite , jeffrey r. o'connell , nancy j. cox , , , , braxton d. mitchell , mark j. ratain , , , and alan r. shuldiner , abstract purpose: vegf receptor (vegfr) kinases are important drug targets in oncology that affect function of systemic endo- thelial cells. to discover genetic markers that affect vegfr inhibitor pharmacodynamics, we performed a genome-wide association study of serum soluble vascular vegfr concen- trations [svegfr ], a pharmacodynamic biomarker for vegfr inhibitors. experimental design: we conducted a genome-wide associa- tion study (gwas) of [svegfr ] in healthy old order amish volunteers. gene variants identified from the gwas were geno- typed serially in a cohort of patients with advanced solid tumor with baseline [svegfr ] measurements, and in patients with renal carcinoma with [svegfr ] measured before and during pazopanib therapy. results: rs (c r) in kdr, the gene encoding svegfr was found to be highly associated with [svegfr ], explaining % of the variance (p ¼ . � � ). association of rs with [svegfr ] was replicated in patients with cancer with comparable effect size (p ¼ . ). furthermore, rs was a significant predictor of changes in [svegfr ] in response to pazopanib (p ¼ . ). conclusion: our findings suggest that genome-wide analysis of phenotypes in healthy populations can expedite identi- fication of candidate pharmacogenetic markers. genotyping for germline variants in kdr may have clinical utility in identifying patients with cancer with unusual sensitivity to effects of vegfr kinase inhibitors. clin cancer res; ( ); – . � aacr. introduction advances in our understanding of the molecular basis of cancer have led to the identification of a number of novel targets and classes of anticancer agents. however, interindividual variability in efficacy and toxicity creates difficulties in optimizing therapy for individual patients. the discovery of germline and/or somatic genetic markers that predict interindividual differences in thera- peutic response promises to improve the efficacy and safety of cancer therapeutics ( – ). these markers also might accelerate the pace and increase the success rate of drug development by iden- tifying subsets of patients who are more likely to respond to a given drug, whereas other markers may define which patients are more likely to experience life-threatening adverse reactions, and in whom that drug should be avoided (or dose attenuated). unfor- tunately, the identification of pharmacogenetic markers for a new compound in early clinical trials is a logistical challenge, and thus the discovery of these markers has typically been deferred to late in the development of new drugs or after the drugs have become commercially available when the numbers of persons having received the drugs is sufficiently large ( ). to date, most pharma- cogenetic studies of anticancer agents have focused on common variants in drug-metabolizing enzymes ( – ). angiogenesis inhibitors are an important new class of anti- cancer agents, but their optimal use requires more detailed understanding of their pharmacology and the biologic basis for section of hematology/oncology, department of medicine, univer- sity of chicago, chicago, illinois. committee on clinical pharmacol- ogy and pharmacogenomics, university of chicago, chicago, illinois. comprehensive cancer center, university of chicago, chicago, illi- nois. glaxo smithkline genetics, stevenage, united kingdom. pro- gram in personalized and genomic medicine, and division of endo- crinology, diabetes and nutrition, school of medicine, university of maryland, baltimore, maryland. department of health studies, uni- versity of chicago, chicago, illinois. department of human genetics, university of chicago, chicago, illinois. section of genetic medicine, department of medicine, university of chicago, chicago, illinois. glaxo smithkline computation biology, stevenage, united kingdom. glaxo smithkline genetics, research triangle park, north carolina. glaxo smithkline oncology, philadelphia, pennsylvania. glaxo smithkline genetics, philadelphia, pennsylvania. glaxo smithkline oncology, research triangle park, north carolina. geriatric research and education clinical center, baltimore veterans admin- istration medical center, baltimore, maryland. note: supplementary data for this article are available at clinical cancer research online (http://clincancerres.aacrjournals.org/). current address for e. kistner-griffin: medical university of south carolina, charleston, sc; and current address for d. torgerson, university of california san francisco, san francisco, ca. corresponding author: michael l. maitland, section of hematology/oncology, department of medicine, university of chicago, mc- , s. maryland avenue, chicago, il . phone: - - ; fax: - - ; e-mail: mmaitlan@medicine.bsd.uchicago.edu doi: . / - .ccr- - � american association for cancer research. clinical cancer research www.aacrjournals.org on april , . © american association for cancer research. clincancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ interindividual differences in resistance and sensitivity to treat- ment ( , ). pazopanib is an oral angiogenesis inhibitor that blocks signaling by vegfr and other kinases, and has been approved for commercial use to treat renal cell carcinoma and soft-tissue sarcoma ( , ). vegfr is a transmembrane receptor tyrosine kinase expressed by endothelial cells, subpo- pulations of bone marrow–derived cells, and some tumor cells ( ). it is the primary transducer of extracellular vegf medi- ating endothelial cell proliferation, migration, and resistance to apoptosis ( ). alternate splicing of kdr, the gene that encodes vegfr , results in a amino acid truncated extracellular domain product which is soluble and circulates in blood (svegfr ; ref. ). vegfr kinase inhibitors decrease serum concentrations svegfr [svegfr ], and these concentrations return to baseline after cessation of drug administration ( – ). in addition, the baseline concentrations among patients with cancer have the same magnitude and distribution as in healthy human subjects. in rodents, the magnitude of these drug-related changes in [svegfr ] is dose-dependent and independent of the presence of tumors ( ). in different human cancer cohorts the change in [svegfr ] was associated with tumor response to vegfr kinase inhibitors ( , ), suggesting that [svegfr ] might serve as a quantitative endo- phenotype with which to better understand differences among humans in response to vegfr inhibitors. furthermore, the discovery of genetic and/or other determinants of [svegfr ] may provide further insights into mechanisms of tumor pro- gression and new targets for therapy. to expedite discovery of gene variants that mark interindi- vidual differences in response to vegfr inhibitors, we per- formed a genome-wide association study (gwas) of [svegfr ] in a noncancer patient population, which identified a locus of linked variants on chromosome associated with the pheno- type. one specific missense variant, rs in kdr, was then identified as a major determinant of [svegfr ], which was also associated with the pharmacodynamics of pazopanib in patients with cancer. materials and methods human subjects this study was approved by institutional review boards (irb) at the university of chicago (chicago, il) and the university of maryland (baltimore, md). all participants from the six dif- ferent studies in the three study cohorts provided informed consent for use of their specimens in pharmacogenetic studies (see supplementary materials and methods for additional detail). study cohort . the amish heredity and phenotype intervention (hapi) heart study. the hapi heart study began in to identify genes that interact with environmental exposures to influence risk for cardiovascular disease. study design and phenotyping procedures have been previously described in detail ( , ). briefly, healthy amish subjects, ages to years, underwent a detailed medical examination including blood pressure, anthropometry, total fat mass by dual energy x-ray absorptiometry, fasting blood draw for lipids and other cardiovascular markers and for extraction of dna for genetic studies, and four short-term interventions designed to chal- lenge cardiovascular function. for this study, fresh-frozen sera, collected at independent time points either hours or week apart, were available for measurement of [svegfr ] in participants. study cohort . biomarker validation and cancer patient replication cohorts. a pharmacokinetic, pharmacodynamic, and pharmacogenetic study of sorafenib and blood pressure elevation in patients with advanced malignancies. study design and patient population have been previously described ( ). sera and dna samples before admin- istration of sorafenib were available for participants with advanced cancer. a randomized phase ii trial comparing cetuximab with concurrent pemetrexed/cetuximab therapy for non–small cell lung cancer refractory to primary treatment. this study enrolled patients with advanced non–small cell lung cancer whose disease progressed after initial platinum-based therapy ( ). sera and dna samples before administration of the first dose of cetuximab were available for participants. a dose-escalation study of sorafenib in normotensive patients with advanced malignancies (nct ). this study enrolled patients with advanced solid tumors and normal blood pressure to receive sorafenib at standard and higher doses. sera and dna samples before administration of sorafenib were available for participants ( ). study cohort . a phase ii study of gw (pazopanib) using a randomized discontinuation design in subjects with locally recurrent or metastatic clear-cell renal cell carcinoma veg (nct ). this study was used to assess the effect of genotype on pharmacodynamics of pazopanib. details of the study design were previously reported ( ). of the patients enrolled, had dna and svegfr serum measure- ments available from baseline and after weeks of pazopanib treatment. serum sample processing for the hapi heart study, venous blood was drawn from an arm vein into serum separator tubes, maintained at �c for to translational relevance germline genetic variation can play an important role in the course of cancer and treatment responses. conventional strat- egies for discovering germline factors in disease and therapeu- tic responses in other health conditions can be challenging to apply to cancer pharmacogenomics research. we employed a step-wise approach of studying a reproducibly measurable serum protein, svegfr , in a population of volunteers who did not have cancer, to identify a genetic variant (rs / c r) as a marker for response to vegfr kinase inhibitors. the association of rs with serum concentrations of svegfr [svegfr ] was replicated first in unrelated cancer patients. in a separate cohort of patients with renal cancer, the uncommon gene variant was associated with lower baseline [svegfr ] measures and carriers experienced greater decline in [svegfr ] over weeks of pazopanib exposure compared with noncarriers. we expect this genetic marker might have a clinically significant effect on treatment outcomes with vegfr kinase inhibitors. maitland et al. clin cancer res; ( ) january , clinical cancer research on april , . © american association for cancer research. clincancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ minutes before centrifugation, and serum separated and stored at � �c storage until assay. for study cohorts – , samples were collected from the upper extremity or a central venous port, incubated at room temperature for minutes and then serum was separated by centrifugation. samples were aliquotted into cryovials and stored at � �c until assay. measurement of serum [svegfr ] serum samples were thawed on ice and processed in triplicate according to manufacturer's specifications (r&d systems). sam- ples in which the results deviated from the manufacturer's per- formance specifications (cv > % among wells) were reana- lyzed. no sample required reanalysis more than once. to control for interplate and interbatch variation, aliquots from a single time point blood draw from two volunteer subjects were run on each plate in triplicate (internal controls). for details of the normal- ization procedure based on internal control sample measure- ments, see supplementary materials and methods. genotyping genome-wide typing and quality control procedures for the amish hapi heart study have been described previously (also see supplementary materials and methods; ref. ). of the individuals with both genome-wide snp data (affymetrix k snp platform) and serum [svegfr ], also had geno- type data on the human cardiovascular disease risk focused beadchip [also known as the itmat-broad-care (ibc) array (illumina); ref. ). this array contains , markers across approximately , cardiovascular disease and metabolic dis- ease candidate genes. twenty-seven additional snps at the kdr locus were present on this platform and analyzed for association with serum [vegfr ]. single snp genotyping of rs and rs in sam- ples from study cohorts and was performed by a combination of taqman allelic discrimination assay and snapshot single base extension assay (applied biosystems). statistical analysis anova was performed to estimate the inter- and intraindivi- dual components of variability in svegfr levels among six healthy volunteers. the basic model for the jth serial measure- ment in the ith subject is yij ¼ m þ ai þ "ij, where ai � n ; s a � � and "ij � n ; s � � ; s a reflects the between individual component and s the within individual component. estimates of s a and s were obtained from the anova breakdown and the ratio of between individual to total variability (or icc) determined as ŝ a = ŝ a þ ŝ � � : group means for [svegfr ] and other measures were com- pared by the t test. mixed-model variance components analysis was performed to identify covariates, estimate heritability, and dominance and household effects of serum [svegfr ] and per- form gwas in the hapi heart study ( ). as described in detail previously, a bonferroni correction–based threshold for genome- wide significance was set at � � ( ). in the university of chicago cancer patients (study cohort ), multivariate regression testing for association between rs and serum [svegfr ] was performed with plink v . (http://pngu.mgh.harvard.edu/ purcell/plink/; ref. ). the q assoc command was run, including age, sex, bmi, and ethnicity (encoded as european american, african american, latino american, and asian/pacific islander american) as covariates. the p value reflects a wald test statistic. for this replication testing, p values < . were considered significant. for the renal cell cancer subjects (study cohort ), linear regression was conducted with an additive genetic model using sas v . , including age as a covariate for baseline and week [svegfr ], and baseline [svegfr ] as a covariate for changes in [svegfr ] after pazopanib exposure. results intra- and interindividual variation in svegfr levels in normal human serum in serial serum samples from healthy volunteers, % of the total measurement variance of [svegfr ] was interindividual, with the remaining small fraction due to within individual measurement differences over time ( ). the high ratio of inter- individual to intraindividual variance suggested that the basis for the interindividual variance could be studied further in larger populations without the need for serial measures. to further characterize the sources of interindividual variance, serum [svegfr ] was measured in old order amish subjects who had participated in the hapi heart study [refs. , , ; men, women; mean age � sd ¼ . � . years;mean body mass index (bmi) � sd ¼ . � . kg/m ]. median serum [svegfr ] was . ng/ml (interquartile range: . – . ) with an approximate normal distribution (fig. ). men had higher concentrations (mean � sd ¼ . � . ng/ml) than women (mean � sd ¼ . � . ng/ml; p < . ). variance component analysis revealed [svegfr ] to be associated with age, bmi, and diastolic blood pressure (table ). sex and age accounted for . % of the variation, with age having the greater impact on [svegfr ]. systolic blood pressure, fat mass, and pulse wave velocity (pwv), a measure of large vessel arterial stiffness, were also associated with variation in [svegfr ] but explained a trivial proportion of the variance. heritability of [svegfr ] the amish subjects of the hapi heart study constituted sibling pairs, parent-offspring pairs, avuncular pairs, . . . . . . . . . . . . f re q ue nc y [svegfr ] (ng/ml) figure . distribution of serum [svegfr ] in amish subjects. histogram plot of serum concentrations. horizontal axis reflects . ng/ml quantiles among the -fold range of measurements from less than . to . ng/ml. the vertical axis represents the number of subjects with measurements in the interval. kdr snp associated with serum vegfr and pazopanib pharmacodynamics www.aacrjournals.org clin cancer res; ( ) january , on april , . © american association for cancer research. clincancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ and first cousin pairs. a polygenic model of [svegfr ], adjusting for sex and age, demonstrated [svegfr ] to be a strongly heritable trait (h ¼ . ). this estimate was virtually unchanged with inclusion of a random household and domi- nance effect into the model. genome-wide association analysis of [svegfr ] the initial agnostic genome-wide association analysis of hapi heart study subjects with the affymetrix k snp genotype data identified a cluster of snps on chromosome significantly associated with [svegfr ]. the top serum [svegfr ]-associated snps were all on chromosome and spanned a . mb region that included several candidate genes, including fip l , pdgfra, kit, and kdr (fig. and table ). for the most significant snp, rs , the effect size of each copy of the minor t allele (allele frequency ¼ . ) was a decrease of . ng/ml [svegfr ] (p ¼ � � ). the strong association of [svegfr ] with multiple snps on chromosome q -q and inferences from the known founder population structure of the lancaster county amish guided further analysis. the causative snp was expected to be in strong linkage disequilibrium with rs and might have been introduced into the lancaster county amish population through a single or small number of founders. the rs mutation therefore marks a broader founder haplotype such that all snps on this haplotype show association with [svegfr ]. because this haplotype would have been passed down from the founder(s) to the present day lancaster county amish for only to generations, opportunity for recombination would be limited and the causative variants could be up to several mb away. consistent with this assumption, we performed multivariate regression association analysis with single timepoint serum [svegfr ] in unrelated advanced solid tumor patients enrolled in clinical trials at the university of chicago (chicago, il). we detected no association between rs (allele fre- quency ¼ . ) and serum [svegfr ] (b ¼ . ng/ml; p ¼ . ) in this heterogeneous cancer patient population. on the basis of the assumption that rs was in linkage disequilibrium (ld) with a functional variant within a broad linkage peak in the amish, but not in ld in the heterogeneous cancer patient population, we pursued two approaches to identify variants with detectable effects on [svegfr ]. first, we identified candidate variants in the larger population within the . mb linkage peak and tested their association with [svegfr ] directly in the patients with cancer. subsequent- ly, we evaluated candidate loci in the kdr gene in the amish using the illumina cardiovascular disease (ibc) chip, which had more extensive coverage of kdr, including rare coding region alleles ( ). evaluation of candidate gene variants for association with [svegfr ] we used the bioinformatics tool scan ( ) to identify all genes within the locus and then accessed the genomes project ( ) to identify all missense polymorphisms within those genes. this approach identified four coding variants (supplementary table s ) within fip l, pdgfra, and kdr with minor allele frequencies > . in populations of european ancestry. in addition, the common polymorphism in the -untranslated region of kdr, rs , had been demonstrated to affect vegfr expression in vitro ( ). none of these candidate variants demonstrated association with [svegfr ] in the heterogeneous cancer patient population (study cohort, see materials and methods; supplementary table s ). table . correlation of serum [svegfr ] with selected variables covariate b � se p age- and sex-adjusted b � se p age (y) � � < . — — bmi (kg/m ) � . � < . fat mass (kg) . � . . . � . . sbp (mmhg) � . � . dbp (mmhg) � . � < . carotid/femoral pwv (m/s) � � . � . note: total fat mass was measured by dual energy x-ray absorptiometry. abbreviations: bmi, body mass index; sbp, systolic blood pressure; dbp, diastolic blood pressure; pwv, pulse-wave velocity; b, effect size (pg/ml); se, standard error. . . . . . . . . . . . chr chr chr chr chr chr chr chr chr chr chr chr chrxchr chr chr chr chr chr chr chr chr chr −l o g p figure . genome-wide association of snps with serum [svegfr ]. manhattan plot of each snp on the genotyping platform by chromosome (horizontal axis) versus the �log p value for association with serum [svegfr ] (vertical axis). chr, chromosome. maitland et al. clin cancer res; ( ) january , clinical cancer research on april , . © american association for cancer research. clincancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ as kdr was within . mb of the top signal, we considered this gene the prime candidate for more in depth analysis. during the course of our investigation, data from the ibc chip became available from of the original amish subjects. in the amish there were nonmonomorphic snps in or near kdr. single snp association analysis with [svegfr ] revealed a missense mutation in exon , c r caused by an a to g change (rs ) to have the lowest p value, . � � (supplementary table s ). among the amish, in an additive genetic model, the minor g allele lowered [svegfr ] approx- imately . ng/ml per allele (fig. a). this finding was consistent with our original hypothesis, that (i) rs would be, in the amish, a proxy on our initial genotyping array in ld with a snp not on the array that has a stronger association with the [svegfr ] phenotype, and (ii) the snp would not be in ld with rs among a more heterogeneous population of european ancestry (such as the university of chicago cancer patient cohort; see supplementary results for a description of the ld assessment). rs is a determinant of [svegfr ] in cancer patients the candidate snp rs met our expectations for a snp that would be associated with serum [svegfr ] in the heteroge- neous population of patients with cancer of european ancestry in the university of chicago cohort. mechanistically, we considered that rs encodes a nonsynonymous variant, c r, located in the fifth of immunoglobulin-like (ig-like) domains of the extracellular region, formed by amino acids – . this cysteine is one of four in the fifth ig-like domain conserved throughout homologues of kdr in the chordates [from t. nigro- viridis (pufferfish), to g. gallus (domestic chicken), to m. musculus (mouse), through h. sapiens; see supplementary fig. s ]. we genotyped rs in advanced cancer patients enrolled in university of chicago trials (study cohort, see materials and methods). using a regression model for additive effects of the allele and incorporating sex, age, body mass index, and self- reported race categories, the rs minor allele was asso- ciated with serum [svegfr ] with the same direction and a similar effect size as in the amish ( . ng/ml lower in g allele carriers; p ¼ . ; fig. b). rs is associated with svegfr response to pazopanib vegfr kinase inhibitors routinely cause circulating [svegfr ] measures to decline within the first weeks of expo- sure ( – ) and the magnitude of change in [svegfr ] has been associated with increased response to therapy in thyroid and lung cancer ( , ). with the biologic relevance of rs established as a significant predictor of [svegfr ] in the amish and unrelated cancer patients, we hypothesized it might also play a role in differential responses to inhibitors of the vegf signaling pathway. in an independent group of patients with renal cell cancer treated on clinical trials with pazopanib (study cohort, see materials and methods), serial [svegfr ] measures were available before, and weeks after the initiation of continuous oral pazopanib therapy mg daily. patients with the g allele not only had lower baseline [svegfr ] measures (fig. a), but also experienced greater decline over weeks in [svegfr ] with table . top snps on genome-wide association scan for [svegfr ] snp chr pos freq b snp p rs . . . e� rs . . . e� rs . . . e� rs . � . . e� rs . . . e� rs . � . . e� rs . . . e� rs . . . e� rs . � . . e� rs . . . e� rs . . . e� rs . . . e� rs . . . e� rs . . . e� rs . � . . e� rs . . . e� rs . � . . e� rs . � . . e� rs . . . e� rs . � . . e� abbreviations: chr, chromosome; pos, position; freq, frequency of the coded allele; b snp, effect size (ng/ml) for the coded allele on serum svegfr concentrations. aa aa ag ag gg n = n = n = svegfr by kdr genotype sv e g f r (n g /m l) sv e g f r (n g /m l) a b figure . association of rs with serum [svegfr ]. a, in amish subjects. cohorts defined by genotype status (aa, homozygous major allele; ag, heterozygous; gg, homozygous minor allele) with boxplots of distribution of serum [svegfr ]. b, in unrelated cancer patients with dotplots of distribution of serum [svegfr ]. given the rarity of gg homozygotes in the population, none were detected among patients with cancer. kdr snp associated with serum vegfr and pazopanib pharmacodynamics www.aacrjournals.org clin cancer res; ( ) january , on april , . © american association for cancer research. clincancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ pazopanib exposure compared with noncarriers (mean decrease � . ng/ml vs. � . ng/ml, respectively; p ¼ . ; fig. b and c). other variants associated with [svegfr ] in the amish, conditional analyses of both the gwas and ibc chips were run, including rs asa covariatein the model to determine the impact on other variants in the chromosome region. several snps on chromosome in the – mb region remained highly significant from the imputed data, but had lower effect size than rs . the most significant of these other snps was rs located kb from rs . the minor allele c hasfrequency . and was associated withdecreasedlevels of [svegfr ] ( . ng/ml per allele; p ¼ . � � ), less than one-third the magnitude of effect of rs . in the primary gwas, there was marginal association between rs and [svegfr ] (p ¼ � ). the ld between rs and rs is r ¼ . and j d j ¼ . suggesting that the two [svegfr ]-lowering alleles act independently in the amish. how- ever, rs was not associated with serum [svegfr ] in either of the cancer cohorts (� . ng/ml, p ¼ . in the study cohort, and . ng/ml, p ¼ . in the study cohort). discussion we performed a gwas of a serum peptide in the amish to discover a snp with reproducible effects on that peptide in patients with cancer. this phenotype [svegfr ] is now recog- nized as a pharmacodynamic marker of vegfr inhibition and this snp affects the pharmacodynamic response to pazopanib. as in genetic studies, for example of grasshopper body size ( ) and human schizophreniform behavior ( ), we employed the approach of endophenotyping to the discovery of a novel cancer pharmacogenetic biomarker. in a step-wise approach, we first characterized the repeatability, interindividual variance, and her- itability of [svegfr ] as an endophenotype for response to vegfr kinase inhibitors. second, we performed an agnostic gwas in a healthy population and identified rs /c r in kdr, the gene encoding the vegfr protein, to be associated with [svegfr ]. third, we replicated these findings in two cancer populations. fourth, we extended these findings by demonstrat- ing that this variant is a marker for pharmacodynamic response to the kinase inhibitor, pazopanib. the svegfr protein is an alternative spliced product of kdr and functions as a physiologic inhibitor of developmental and reparative lymphangiogenesis ( ). in situ hybridization demon- strated mammalian expression of the alternative splice product in epithelium such as in the cornea and skin. the product of this transcript appears to be monomeric and to have higher relative avidity for vegf-c than vegf-a. in the mouse cornea, svegfr preferentially regulates lymphanigogenesis. the extent to which this alternative splice product contributes to the svegfr protein detected in human serum and whether the circulating protein plays additional roles in regulating human angiogenesis and lymphangiogenesis is not known. the function of the rs /c r variant has been pre- viously characterized in studies of hemangiomata ( ). the amino acid is located in the extracellular ig-like domain v, distant from the vegf ligand–binding site ( ). recombinant cell trans- fection studies revealed no differences in vegf-induced phos- phorylation or expression of vegfr . however, the substitution of arginine for cysteine diminished formation of complexes by vegfr with b integrin and the integrin receptor-like protein tem . this complex was associated with vegfr -mediated acti- vation of vegfr transcription and protein expression. with less vegfr expression, endothelial cells demonstrate greater sensi- tivity to activation of vegfr signaling by vegf binding. this amplified sensitivity to vegf/vegfr ligation might explain the potential for greater sensitivity of g allele carriers to pazopanib treatment and potentially greater anticancer activity of vegfr inhibitors in patients with this variant. the rs polymorphism is a common variant in the amish (minor allele frequency ¼ . ) but an uncommon variant in the larger outbred caucasian population (minor allele frequen- cy ¼ . ). we expect this snp might have a clinically significant effect on outcomes of therapy, as the magnitude of change in svegfr has been associated with increased response to therapy in thyroid and lung cancer ( , ). as allele carriers comprise only a small subset (i.e., approximately %) of all patients, almost all individual trials will be underpowered to demonstrate clini- cally significant effects. we have not excluded additional genetic a b c − − − aa ag aa ag n = n = n = n = aa ag n = n = b as el in e sv e g f r (n g /m l) sv e g f r w ee k ( ng /m l) sv e g f r ch an g e fr o m b as el in e to w ee k ( ng /m l) figure . association of rs with changes in serum [svegfr ] after pazopanib therapy. dot and boxplots demonstrate: a, replication of the association between the snp and baseline serum [svegfr ] among an independent cohort of renal cancer patients, (b) the serum [svegfr ] measurements by genotype in the same subjects after weeks pazopanib therapy, and (c) the absolute change in serum [svegfr ] among these subjects by genotype. maitland et al. clin cancer res; ( ) january , clinical cancer research on april , . © american association for cancer research. clincancerres.aacrjournals.org downloaded from published onlinefirst november , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ variants contributing to interindividual differences in baseline [svegfr ] and important interactive covariates for explaining differences in response to vegfr kinase inhibitors is the subject of ongoing investigation. despite the commercial availability of drugs in the class since , a clinically useful biomarker to guide therapy with vegfr inhibitorsremainselusive.ourfindingofagermlinevariantwithin kdr with reproducible effects on the response of the pharmaco- dynamic marker [svegfr ] warrants further investigation. this might provide important insights into mechanisms underlying interindividual variation in response to kinase inhibitors, and approaches to deliver anticancer therapy more effectively. disclosure of potential conflicts of interest c.-f. xu and l. pandite are employees of and hold ownership interest (including patents) in glaxosmithkline. m.j. ratain reports receiving a com- mercial research grant from oncotherapy science and is a consultant/advisory board member for genentech. a. shuldiner is an employee of regeneron genetics center and is a consultant/advisory board member for usds, inc. no potential conflicts of interest were disclosed by the other authors. authors' contributions conception and design: m.l. maitland, t.g. karrison, l.r. cardon, l. pandite, n. cox, m.j. ratain, a. shuldiner development of methodology: m.l. maitland, s. das, n. bing acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): m.l. maitland, c.-f. xu, s. das, n. bing, y. liu, l. pandite, b. mitchell analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): m.l. maitland, c.-f. xu, y.-c. cheng, e. kistner- griffin, k.a. ryan, t.g. karrison, d. torgerson, e.r. gamazon, p.a. wilson, n. bing, y. liu, j.r. o'connell, n. cox, b. mitchell, a. shuldiner writing, review, and/or revision of the manuscript: m.l. maitland, c.-f. xu, e.r. gamazon, v. thomeas, m.r. levine, n. bing, y. liu, l. pandite, j.r. o'connell, b. mitchell, m.j. ratain, a. shuldiner administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): m.l. maitland, k.a. ryan, m.r. levine study supervision: m.l. maitland, l. pandite, a. shuldiner acknowledgments the authors thank carole ober for her initial contributions to the study concept. grant support this project was supported by u.s. nih grants: k ca nci career development award (to m.l. maitland), u ca including study supported by the cancer therapy evaluation program of the nci (to m.j. ratain), nigms u gm pharmacogenetics of anticancer agents research group (to m.j. ratain, n. cox, e. gamazon, s. das, and m.l. mait- land), u hl the amish pharmacogenomics of anti-platelet interven- tion study and u hl the amish heredity and phenotype interven- tion study (to a. shuldiner, b. mitchell, j.r. o'connell, k.a. ryan, y.-c. cheng), a preclinical pilot translational science award from the university of chicago ctsa ul rr , and the university of chicago comprehensive cancer center. additional support was provided by a calgb foundation faculty fellowship (to m.l. maitland), the conquer cancer foundation of the amer- ican society of clinical oncology (to m.l. maitland and m.j. ratain), research funding from the o'connor foundation, and contributions from the friends and families of joseph s. berger jr., and robert wesselhoff. glaxo smithkline supported the contributions of its employees and provided data on pazopanib. the costs of publication of this article were defrayed in part by the payment of 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disease dementia in african and european americans | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /jc. - corpus id: a common dio polymorphism and alzheimer disease dementia in african and european americans @article{mcaninch acd, title={a common dio polymorphism and alzheimer disease dementia in african and european americans}, author={elizabeth a mcaninch and k. rajan and d. evans and sungro jo and l. chaker and r. peeters and d. bennett and d. mash and a. bianco}, journal={the journal of clinical endocrinology & metabolism}, year={ }, volume={ }, pages={ – } } 	elizabeth a mcaninch, k. rajan, + authors a. bianco 	published 	medicine 	the journal of clinical endocrinology & metabolism context a common single nucleotide polymorphism in dio , thr alad , has been associated with a transcriptome typically found in neurodegenerative diseases in postmortem human brain tissue. objective to determine whether thr alad is associated with incident alzheimer disease (ad). design population-based study; human brain tissue microarray. setting community-based cohorts from chicago and northeastern illinois and religious clergymen from across the united states constituted the… expand view on wolters kluwer academic.oup.com save to library create alert cite launch research feed share this paper citationsbackground citations view all figures, tables, and topics from this paper 	 figure 	 table 	 table 	 table 	 table view all figures & tables 	alzheimer's disease 	neurodegenerative disorders 	confidence intervals 	national institute on aging (u.s.) 	manuscripts 	amino acids 	cognition disorders 	lithium 	diabetes mellitus 	inheritance patterns 	dio gene 	autopsy 	conflict (psychology) 	macrophage-activating factors 	hemoglobin north chicago 	doctor of chiropractic 	gastrointestinal system 	biological markers 	stratification 	data table paper mentions news article thyroid gene variation may increase risk for alzheimer’s disease in african americans health medicinet march citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency brain mri findings related to alzheimer's disease in older african american adults. 	o. carmichael, r. newton 	medicine 	progress in molecular biology and translational science 	 save alert research feed low serum concentration of free triiodothyronine (ft ) is associated with increased risk of alzheimer’s disease 	patrick s. quinlan, alexandra horvath, a. wallin, j. svensson 	medicine 	psychoneuroendocrinology 	 	 save alert research feed deiodinases, organic anion transporter polypeptide polymorphisms and ischemic stroke outcomes 	s. taroza, d. rastenytė, a. podlipskytė, vaiva patamsytė, n. 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scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue benchmarking of five commercial deformable image registration algorithms for head and neck patients a corresponding author: jason pukala, department of radiation oncology, uf health cancer center at orlando health, s. orange ave., mp - , orlando, fl , usa; phone: ( ) ; fax: ( ) ; email: jason.pukala@orlandohealth.com benchmarking of five commercial deformable image registration algorithms for head and neck patients jason pukala, a perry b. johnson, amish p. shah, katja m. langen, frank j. bova, robert j. staton, rafael r. mañon, patrick kelly, and sanford l. meeks department of radiation oncology, uf health cancer center at orlando health, orlando, fl, usa; department of radiation oncology, university of miami, miami, fl, usa; department of radiation oncology, university of maryland, baltimore, md, usa; department of neurosurgery, university of florida, gainesville, fl, usa jason.pukala@orlandohealth.com received march, ; accepted january, benchmarking is a process in which standardized tests are used to assess system performance. the data produced in the process are important for comparative purposes, particularly when considering the implementation and quality assurance of dir algorithms. in this work, five commercial dir algorithms (mim, velocity, raystation, pinnacle, and eclipse) were benchmarked using a set of virtual phantoms. the phantoms were previously developed based on ct data collected from real head and neck patients. each phantom includes a start of treatment ct dataset, an end of treatment ct dataset, and the ground-truth deformation vector field (dvf) which links them together. these virtual phantoms were imported into the commercial systems and registered through a deformable process. the resulting dvfs were compared to the ground-truth dvf to determine the target registration error (tre) at every voxel within the image set. real treatment plans were also recalculated on each end of treatment ct dataset and the dose transferred according to both the ground-truth and test dvfs. dosimetric changes were assessed, and tre was correlated with changes in the dvh of individual structures. in the first part of the study, results show mean tre on the order of . mm to mm for all phan- toms and rois. in certain instances, however, misregistrations were encountered which produced mean and max errors up to . mm and mm, respectively. in the second part of the study, dosimetric error was found to be strongly correlated with tre in the brainstem, but weakly correlated with tre in the spinal cord. several interesting cases were assessed which highlight the interplay between the direction and magnitude of tre and the dose distribution, including the slope of dosimetric gradients and the distance to critical structures. this information can be used to help clinicians better implement and test their algorithms, and also understand the strengths and weaknesses of a dose adaptive approach. pacs number(s): . .nj, . .dk, . .qr key words: deformable image registration, virtual phantoms, quality assurance, adaptive radiotherapy, head and neck cancer i. introduction deformable image registration (dir) is the nonaffine process of mapping voxels from one image to another where the individual vectors that describe the mapping may vary in both magnitude and direction from their neighbors. the entire process is encompassed by the deformation vector journal of applied clinical medical physics, volume , number , pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , field (dvf), which aggregates the individual vectors into a single map and specifies the coor- dinate transformation between the two datasets. the dvf facilitates the transfer of information and allows the user to perform a number of useful functions such as contour propagation( ) or dose accumulation.( ) initially, these functions were primarily limited to academic centers where many of the deformation algorithms were developed. over the past several years, however, the use of dir has expanded widely to the point where dir is now included in several commercial treatment planning and contouring platforms. commercial dir algorithms are powerful and complex. given two datasets with inherent differences, dir algorithms are capable of quan- tifying these differences and minimizing them by creating a new deformed image, the result of a process which morphs one image into another. this ability to deform almost anything is also the fundamental limitation of these systems. because dir algorithms are based on com- plex mathematical models, there is no guarantee that the deformation defined by the dvf will represent biological change accurately. thus, as with any new tool intended for use within the radiation oncology clinic, implementation must go hand-in-hand with validation. much research has been done in this area, primarily through the creation of ground-truth models and qa metrics. in relation to the former, ground-truth models consist of two image sets linked via a known dvf or transferred content such as known landmarks. in an effort to provide consistent datasets for algorithm validation, several researchers have made their ground-truth models publicly available. examples include the extended cardiac–torso (xcat) phantom,( ) the point-validated pixel-based breathing thorax model (popi model),( ) the dir-lab thoracic d ct model,( , ) and those provided as part of the empire challenge.( ) in an effort to improve the correlation of computer-based phantoms with the actual anatomical changes seen over a course of radiation therapy, the current authors previously developed synthetic datasets derived from clinical images of real head and neck patients.( ) these phantoms are publicly available for users to download as part of the deformable image registration evaluation project (direp) (http://sites.google.com/site/dirphantoms). with each of the models described in the previous section, it is left to the clinical physicist to test their system, analyze the data, and compare their results with known benchmarks. several metrics have been proposed for this analysis, ranging from the simple (dice similarity) to the abstract (the jacobian of the dvf). one commonly used metric is the target registration error (tre), which describes the difference between co-located voxels once they have been transferred through the ground-truth dvf and a test dvf. in the upcoming report on dir validation by aapm’s task group , the proposed goal (as opposed to “tolerance”) for tre is % of voxels within mm.( ) one limitation shared by all dir metrics, however, is the disconnect between the quantification of dir error and the effect that error has on a given dose distribution. this is a complicated issue akin to relying on the gamma criterion for imrt qa analysis. currently, there are few publications which translate target registration error to dvh error, and thus one of the aims of this research was to investigate this process. in addition to tre/dvh error, the traditional commissioning framework of test, analyze, and compare is hindered by the current lack of benchmarks specifically purposed for the clinical validation of commercial algorithms. due to the fact that the vast majority of usage will be through one of these commercial platforms, it is important to provide clinical physicists comparison data which are directly linked to open-source, ground-truth models. in this way, end users can assess their own implementation using the same tools and metrics as those used during the benchmarking process. in this work, the five most prevalent commercial dir algorithms are assessed using the phantom series of the direp ground-truth model. benchmark data are presented for each algorithm using tre as the dir metric. questions relevant to the commissioning process are discussed, including what constitutes a dir failure, what differences may be expected between different commercial algorithms, and where those differences occur. a further analysis is per- formed to translate tre into dvh error, and the results are discussed in relation to the strengths and weakness of such an approach. http://sites.google.com/site/dirphantoms pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , ii. materials and methods a. ground-truth model the deformable image registration evaluation project (direp) includes a library of virtual phantoms. these phantoms were previously created based on volumetric imaging of head and neck cancer patients.( ) briefly, each patient received a start-of-treatment ct (sot) and a near end-of-treatment ct (eot). the sot dataset for each patient was deformed in a forward fashion to represent the anatomy of the eot dataset (i.e., the eot was the target dataset and the sot dataset was deformed to match it). this was done using a combination of a biomechanical algorithm( , ) and human-guided thin plate splines( ) available as a deformation tool within the imsimqa software package (oncology systems limited, shrewsbury, shropshire, uk). these tools allowed for the modeling of head rotation and translation, mandible rotation, spine flexion, shoulder movement, hyoid movement, tumor/node shrinkage, weight loss, and parotid shrinkage.( ) the combinative approach of utilizing multiple algorithms applied iteratively with human intervention minimizes the bias towards any particular algorithm during subsequent dir using these phantoms. the result of the deformation process was a simulated eot dataset for each patient where the ground-truth deformation was known. together, the sot and simulated eot images form a virtual phantom that may be imported into a third-party dir software package. the dvf from any third-party algorithm may then be compared to the known ground truth to obtain the deforma- tion error for each image voxel. expert drawn contours are also included for several structures in order to provide error statistics for individual organs. table shows the patient characteristics for each of the virtual phantoms, and fig. shows an example of one of the phantoms. table . attributes of patients selected for the development of the virtual phantoms. no. of initial days mean mean weight/ between right left end of planning parotid parotid treatment and patient disease fractions dose dose weight eot no. site stage gender delivered (gy) (gy) (kgs) images base of tongue t n bm m . . . / . base of tongue t n cm f . . . / . tonsil t n bm m . . . / . nasopharynx t n m f . . . / . unknown t n am m . . . / . supraglottic larynx t n m m . . . / . tonsil t n am m . . . / . tonsil t n am f . . . / . nasopharynx t n m m . . . / . base of tongue t n am f . . . / . eot = end of treatment. pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , b. dir algorithm evaluation at the time of evaluation, each software package had only one primary algorithm available for dir represented by the algorithms described below. in order to protect intellectual property, companies that market dir systems typically do not disclose detailed information about their algorithms. therefore, each algorithm will be treated as a “black box” for the purposes of this investigation. the virtual phantom image pairs were imported into each system for registration. to avoid an additional confounding variable, an initial rigid registration was not performed before dir for four out of the five algorithms examined. it was not feasible to disable the initial rigid registration for the pinnacle algorithm, but this should not substantially affect the results because the phantom image pairs were already well-aligned in all of the systems before initiating the deformation. for each registration, the sot dataset was designated as the primary dataset (the target) and the simulated eot dataset was designated as the secondary dataset (to be deformed to match the target). this arrangement was chosen to mimic an adaptive workflow, in which a treatment plan is recalculated on daily igrt imaging and the dose is then transferred back to a planning ct for evaluation. the registration procedure for each algorithm is described in the material & methods sections b. to b. below. after registration, dvfs were exported from each system and compared to the ground-truth dvfs using matlab (mathworks inc., natick, ma). dir error statistics were calculated for all of the voxels contained within the brainstem, spinal cord, mandible, left parotid, right parotid, and external contours. b. algorithm : mim deformable registration was performed using the voxalign algorithm incorporated with mim version . . (mim software inc., cleveland, oh). the voxalign algorithm is a constrained, intensity-based, free-form dir algorithm. because the phantom image pairs were in the same dicom frame of reference, no initial rigid registration was performed. following deform- able registration, the dir information was saved and exported from the system as a dicom deformable spatial registration object. reg reveal and reg refine (mim software inc.) were implemented recently into mim as tools that allow the user to view and refine deformable registrations. these tools were not used in this study. fig. . virtual phantom example (phantom ). the top row shows the planning image set and the bottom row shows the simulated eot image set. comparison of the images reveals the simulated parotid shrinkage (axial view), head rotation and spine flexion (sagittal view), and weight loss (coronal view). pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , b. algorithm : velocityai the deformable multipass algorithm of velocityai version . . (varian medical systems, palo alto, ca) was used to register the virtual phantoms. the deformable multipass algorithm is a multiresolution b-spline algorithm. no initial rigid registration was performed between the image pairs. the dvf data were exported from velocityai as a binary deformation field (bdf) file for analysis. b. algorithm : raystation the hybrid deformable registration algorithm of raystation version . (raysearch laboratories, stockholm, sweden) was used to register the phantoms. the hybrid deformable registration algorithm is an intensity-based algorithm that can also incorporate roi constraints. raystation requires that a rigid registration be performed before initiating a deformable registration. therefore, the “set identity” registration tool was used to mark the initial coordinates of each image pair as aligned. in other words, all initial rigid registration parameters were set to zero. the phantoms’ external contours were defined as required, but no other rois were specified. a deformation grid of . × . × . mm was selected for each case. raysearch provided a script that enabled the export of the calculated dvfs. b. algorithm : pinnacle dir was accomplished using the dynamic planning module of pinnacle . (philips healthcare, fitchburg, wi). the pinnacle system used an implementation of the demons algorithm for deformable registration. before dir, rigid registration and image preprocessing were performed on the virtual phantom image pairs. dvfs were exported from the software as binary files. b. algorithm : eclipse the dir tools in eclipse version (varian medical systems) were used to register the virtual phantom images. the eclipse treatment planning system also uses an implementation of the accelerated demons algorithm. rigid registration was not performed prior to deformable reg- istration. dvf information was exported from the software as a dicom deformable spatial registration object. c. propagation of dose as noted previously, the direp phantoms were created from real patient data. in addition to imaging, this data included treatment plans which were subsequently used in this study to calculate dose on each eot dataset. all of the treatment plans were calculated and delivered using the tomotherapy treatment platform (accuray inc., sunnyvale, ca). this dose was then transferred to the individual sot datasets via the ground-truth dvf. dose was also transferred through the dvfs provided by each dir algorithm. the difference between the two dose propa- gation methods (ground truth vs. test) was evaluated in terms of the difference in mean and maximum (max) dose to each organ at risk. the effect of tre on dvh error was also quantified by calculating the dosimetric difference in the dvh for each organ on a point-by-point basis along the dvh. this metric, termed the mean dvh difference (Δdvhmean), was used along with mean and max dose difference in pearson correlations with both mean (treμ) and max tre. due to the fact that all treatment plans were not prescribed for the same total dose, only the dose as calculated for a single gy fraction was used for this study. all treatment plans were originally prescribed at this fractionation pattern, as indicated in table . at the time of the writing of this manuscript, the pinnacle and eclipse platforms did not perform deformable dose accumulation. they are included here for comparison purposes. pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , iii. results target registration error for each oar is presented in tables – . as shown, the differences between the algorithms are small, although registrations performed using the mim algorithm did consistently produce lower mean errors. this difference was found to be significant (t-test, df = , p < . ) for each oar, except the right parotid. for this organ, no significance was found between any of the five dir algorithms. interestingly, both mim and raystation also generated a large misregistration specific to the right parotid of phantom (treμ > mm and tremax > mm for both algorithms). in order to better visualize this result, histograms of the table . registration error statistics for all of the voxels contained within the brainstems of the virtual phantoms. statistics are listed as the mean ± sd. the maximum errors are shown in parentheses. all errors are reported in mm. phantom no. mim velocity raystation pinnacle eclipse . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) mean . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) table . registration error statistics for all of the voxels contained within the spinal cords of the virtual phantoms. statistics are listed as the mean± sd. the maximum errors are shown in parentheses. all errors are reported in mm. phantom no. mim velocity raystation pinnacle eclipse . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) mean . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) table . registration error statistics for all of the voxels contained within the mandibles of the virtual phantoms. statistics are listed as the mean± sd. the maximum errors are shown in parentheses. all errors are reported in mm. phantom no. mim velocity raystation pinnacle eclipse . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) mean . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , tre of the left and right parotid of phantom are shown in fig. . the long tail of the histogram evident in the right parotid but absent from left parotid indicates the large maximum errors generated by the mim and raystation algorithms for this case. registration error histograms for all of the rois and phantoms are available in the supplementary materials file available on the jacmp website at www.jacmp.org the dvh results from the dose propagation study are shown in fig. – . starting with the spinal cord, fig. illustrates the best (phantom ) and worst (phantom ) case scenarios as scored by Δdvhmean. it is evident from the graph that tre had little effect on the dvh for this structure. this is further supported in the pearson correlations (table and fig. ) which show table . registration error statistics for all of the voxels contained within the left parotids of the virtual phantoms. statistics are listed as the mean± sd. the maximum errors are shown in parentheses. all errors are reported in mm. phantom no. mim velocity raystation pinnacle eclipse . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) mean . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) table . registration error statistics for all of the voxels contained within the right parotids of the virtual phantoms. statistics are listed as the mean± sd. the maximum errors are shown in parentheses. all errors are reported in mm. phantom no. mim velocity raystation pinnacle eclipse . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) mean . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) table . registration error statistics for all of the voxels contained within the external contours of the virtual phantoms. statistics are listed as the mean± sd. the maximum errors are shown in parentheses. all errors are reported in mm. phantom no. mim velocity raystation pinnacle eclipse . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . ( . ) . ± . 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( . ) http://www.jacmp.org pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , several dosimetric parameters having only a weak to very weak correlation with both treμ and tremax. in direct comparison, the brainstem shows a strong to very strong correlation with treμ and a moderate correlation with tremax. four dvhs of the brainstem are highlighted in fig. , showing both similarity and divergence amongst the algorithms for different cases. the remaining oars show a moderate to strong correlation with treμ and a very weak correlation with tremax. the previously mentioned misregistration in the right parotid of phantom is shown in fig. (bottom right). interestingly, while both mim and raystation produced similar mean errors (~ mm), clearly these errors did not affect the dvh in the same way. another interesting case is that of the left parotid of phantom . in this instance, there is a large devia- tion in both directions away from the ground-truth dvh. the net effect on the mean dose to the left parotid is a . % increase when using velocity compared to an % decrease when using pinnacle. this was the largest discrepancy encountered in this study amongst the five commercial algorithms. fig. . tre histograms for the left (top) and right (bottom) parotids of phantom . the histograms show the number of voxels in the designated roi that were deformed with the error specified by the x-axis for each algorithm. note the scale differences of each histogram. fig. . dvh curves of the spinal cord for phantom and phantom after dose has been propagated from the eot to sot dataset through the ground-truth and test dvfs. phantom showed the least disagreement overall with the ground- truth dvh, while phantom showed the most disagreement. pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , fig. . dvh curves of the brainstem for phantoms , , , and after dose has been propagated from the eot to sot dataset through the ground-truth and test dvfs. note the strong convergence with the ground truth for phantom . in this case, the dose distribution did not include the nasopharynx, increasing the distance between the brainstem and the high dose regions of the treatment plan. fig. . dvh curves of the right parotid for phantoms , , , and after dose has been propagated from the eot to sot dataset through the ground-truth and test dvfs. note disagreement for phantom when dose is transferred using the raystation algorithm. in this case, the mean target registration error was equal to . mm and a high dose gradient directly traversed the right parotid. pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , fig. . dvh curves of the left parotid for phantoms , , , and after dose has been propagated from the eot to sot dataset through the ground-truth and test dvfs. note disagreement for difference between the velocity and pinnacle algorithms for phantom . this occurs because the dvf for each algorithm mapped voxels in opposite directions away from the ground truth. fig. . dvh curves of the mandible for phantoms and after dose has been propagated from the eot to sot dataset through the ground-truth and test dvfs. pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , fig. . correlation between mean target registration error and dosimetric error for the spinal cord, right parotid, and brainstem. pearson correlation coefficients can be found for the distributions in table . table . pearson correlation coefficients for several different combinations of target registration error (p ) and dosi- metric error (p ). p treμ	 treμ	 treμ	 treμ	 tremax (mm) (mm) (mm) (mm) (mm) p dμ	 dμ	 dvh dmax dmax (%) (gy) (gy) (gy) (gy) brainstem . . . . . mandible . . . . . parotid rt . . . . . parotid lt . . . . . cord . . . . . external . . . . . pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , iv. discussion benchmarking is a process in which standardized tests are used to assess system performance. in many applications the results are strictly used for intercomparison (i.e., which processor computes the fastest, which business model is more profitable, etc.). in this application, how- ever, the primary purpose is less about the intercomparison of algorithms and more about the establishment of clinically relevant baseline data. this data, as presented in tables – , may be used in a number of ways. first, users of one of these commercial algorithms may download the head and neck datasets and run them as part of their commissioning process, using the benchmarks for comparison. additionally, during upgrades to newer software versions, the cases may be rerun as a way of maintaining self-consistency, as discussed by aapm’s task group .( ) as an example, the large registration errors in the brainstem seen when utilizing the pinnacle algorithm (table ) were reported to the vendor and have reportedly been fixed in current updates to their software. this can be verified using the proposed methodology and will be done in future work. the benchmark data also provide users with a general idea of the magnitude and variation in tre for the specific use case adopted in this study — head and neck dose adaptation. as an example, the mean registration error for the spinal cord using the mim algorithm was . ± . mm with an associated standard error of . mm (n = ). these statistics provide high confidence that the tre for an individual deformable registration using the mim algorithm under similar conditions will be less than mm for this structure. conversely, in looking at the right parotid when applying the raystation algorithm, the mean error was . ± . mm, with an associated standard error of . mm. users in this situation can thus expect higher variability when considering an individual case where the mean error may range between – mm. in comparison to other studies, varadhan et al.( ) and nie et al.( ) used imsimqa to create virtual h&n phantoms and evaluate dir. the varadhan study reported contour comparison metrics between contours deformed using the ground-truth dvf and contours deformed using two different dir algorithms. while these metrics are useful for contour comparison studies, they are of limited use for deformable dose accumulation or other dir applications, and would be difficult to compare to the data presented here. this fact highlights the need for consistent metrics and datasets to compare dir algorithms. the nie study used a single virtual h&n phantom, along with phantoms for other treatment sites, to compare the dvfs generated by mim . . and velocityai . . to the ground-truth provided by the imsimqa dvf. the mim algorithm resulted in . % of voxels having errors greater than mm, . % greater than mm, and only % greater than mm. the velocityai algorithm resulted in . % of voxels having errors greater than mm, . % greater than mm, and only . % greater than mm. these results are consistent with some of the phantom data reported in this study. however, the differences that we observed between phantoms emphasize the need to evaluate multiple cases for comprehensive dir qa. to this point, it is evident that, on an individual basis, h&n dir can result in large spatial errors which may be difficult to detect. all of the commercial systems generated registrations that could be considered “failures” in the sense that they produced a treμ more than two standard deviations (sds) away from the overall system-independent average (treavg) for selected rois. the effects of these failures on a given dvh are not easy to predict. as noted in the results, the strongest correlations between tre and dosimetric error were in the brainstem. in looking at the three instances where the treμ qualified as a failure (pinnacle – phantoms , , and ), two out of the three cases also generated large dosimetric errors (phantoms and ), while the third case did not (phantom ). in viewing the dose distributions for these phantoms it is clear why this occurred. as seen in fig. , the treatment plan for phantom did not encompass the nasopharynx, which resulted in a larger distance between the brainstem and the high-dose regions of the treatment plan. also shown in the figure are dose distributions for phantoms and , which can be compared with the dvh data from fig. and the tre data from table . pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , only the single large error from the pinnacle algorithm ( . mm) appears to affect the dvh for phantom , while smaller errors from all the algorithms ( . – . mm) produced divergence in the dvh for phantom . the common trend amongst the three phantoms is that the closer the brainstem is to the dose gradient, the more sensitive the brainstem becomes to dosimetric error caused by tre. these cases illustrate the difficulty in trying to generalize the correlation between tre and dvh error. clearly, the correlation depends upon not only the magnitude of the registration error, but also the dose distribution itself, including the slope of nearby dose gradients and the distance to critical structures. this is highlighted further in fig. where sharp dose gradients traverse both parotids, but avoid the cord of phantom . in this case, large differences were seen amongst the five algorithms when viewing the dvh for each parotid, but little difference was seen when considering the spinal cord. while treavg for the cord was lower compared to both parotids for this phantom, the trend also held true for other cases such as phantom where treavg for the cord was nearly double that of the parotids. this is further reflected in the pearson correlations for the cord which show only weak to very weak correlation with treμ. fig. . dose distribution shown for phantom (bottom), phantom (middle), and phantom (top). in comparing the dvh and tre data for these patients, it is evident that the distance between the brainstem and dose gradient plays a large role in the sensitivity to dosimetric error caused by tre itself. pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , this is likely attributed to the fact that h&n plans uniformly avoid dosing the cord by applying margins to this structure. the margin increases the distance between high dose gradients and the true cord, thus mitigating the impact of target registration error. for other structures, the direction of the error also played a significant role. the previously mentioned left parotid of phantom is a prime example, where the dvf produced by the velocity algorithm erred towards the low-dose region while the dvf produced by the pinnacle algorithm erred towards the high-dose region (fig. ). this led to dvh curves which were shifted in opposite directions (fig. ). without information on both the direction of the error and the distance to the dose gradient, this result would be difficult to contextualize. it is thus important to note that the benchmark data published in this study should be interpreted for individual cases in combination with a priori knowledge of the dose distribution, keeping in mind all the factors that attribute to dosimetric uncertainty. it should also be noted that the data obtained in this work from the virtual phantoms is likely only applicable to cases involving the same treatment site, imaging modality, and mag- nitude of anatomical changes. for these phantoms, that would include kvct images acquired from h&n patients with appropriate immobilization over a single course of treatment. our experience has shown that deformation algorithms may behave differently depending on these factors. the virtual phantoms presented in this study also have inherent limitations. all of the complexities of the deformation of the human body during a radiotherapy course would be difficult to model. for example, these phantoms do not model sliding interfaces as might be found in the expansion or contraction of the lungs. they also do not model cavities that appear in one image but not the other. fig. . dose distribution shown for phantom . note the steep dose gradients which traverse the parotids but avoid the cord. large differences were seen in the dvh for each parotid, but little difference was seen when considering the spinal cord. pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , v. conclusions in this work, five commercial deformation algorithms have been benchmarked using a set of computation h&n phantoms. the benchmarks have been presented using the error metric tre, which provides a general assessment of dir performance. the benchmarks can be used during the commissioning and qa process to help users validate their systems. a dose adap- tive strategy was also assessed, whereby tre was correlated with dvh error. several factors influenced the results including the magnitude and direction of the registration error, the slope of the dose distribution, and the distance to critical structures. in assessing clinical cases, the latter two pieces of information will be known whereas the former must be estimated based on case studies such as this one. when well-defined trends exist, such as shown for the spinal cord, the dir for a given structure can be utilized with high confidence. in other instances caution is warranted. for these scenarios, strategies which include dir uncertainty into the dose adaptive process should be considered. acknowledgments the authors would like to thank each of the vendors for their support in completing this study. this work was partially funded by a grant from accuray, inc. fig. . coronal view of phantom showing the dvf within the left parotid as determined by the pinnacle (below) and velocity (above) algorithms. note the opposite direction of the vector fields towards and away from the high-dose regions of the plan, which is located medially. pukala et al.: benchmarking of dir algorithms journal of applied clinical medical physics, vol. , no. , copyright this work is licensed under a creative commons attribution . international license. references . zhang t, chi y, meldolesi e, yan d. automatic delineation of on-line head-and-neck computed tomography images: toward on-line adaptive radiotherapy. int j radiat oncol biol phys. ; ( ): – . . yan d, jaffray d, wong j. a model to accumulate fractionated dose in a deforming organ. int j radiat oncol biol phys. ; ( ): – . . segars w, bond j, frush j, et al. population of anatomically variable d xcat adult phantoms for imaging research and optimization. med phys. ; ( ): . . vandemeulebroucke j, sarrut d, clarysse p. the popi-model, a point-validated pixel-based breathing tho- rax model. xvth international conference on the use of computers in radiation therapy (iccr), toronto, canada, . . castillo r, castillo e, fuentes d, et al. a reference dataset for deformable image registration spatial accuracy evaluationusing the copdgene study archive. phys med biol. ; ( ): – . . castillo r, castillo e, guerra r, et al. a framework for evaluation of deformable image registration spatial accuracy using large landmark point sets. phys med biol. ; ( ): – . . murphy k, van ginneken b, reinhardt jm, et al. evaluation of registration methods on thoracic ct: the empire challenge. ieee trans med imaging. ; ( ): – . . pukala j, meeks sl, staton rj, bova fj, manon rr, langen km. a virtual phantom library for the quantifica- tion of deformable image registration uncertainties in patients with cancers of the head and neck. med phys. ; ( ): . . brock k and mutic s. image registration ii: tg —quality assurance for image registration [abstract]. med phys. ; ( ): . . gering d, lu w, ruchala k, olivera g. anatomy driven deformation. proceedings of the ivith iccr, amersterdam, the netherlands, . . gering d, lu w, ruchala k, olivera g. image deformation based on a marionette model [abstract]. med phys. ; ( ): . . bookstein f. principal warps: thin-plate splines and the decomposition of deformations. ieee trans pattern anal mach intell. ; ( ): – . . varadhan r, karangelis g, krishnan k, hui s. a framework for deformable image registration validation in radiotherapy clinical applications. j appl clin med phys. ; ( ): – . . fraass b, doppke k, hunt m, et al. american association of physicists in medicine radiation therapy committee task group : quality assurance for clinical radiotherapy treatment planning. med phys. ; ( ): – . . nie k, chuang c, kirby n, braunstein s, pouliot j. site-specific deformable imaging registration algorithm selection using patient-based simulated deformations. med phys. ; ( ): . https://urldefense.proofpoint.com/v /url?u=http- a__creativecommons.org_licenses_by_ . _&d=awmfag&c=sgmrq dbjbgx e zsshgezx a iaf so aj bnrhlk&r=w os arm hc l cstrk javcs awytd buny_o - c&m=hdrusoqc cbkl t p ahz q hql-wqyeegftlmx c&s=do pkupekbnmnxotw q bu mdb oh o sdjkjmajfc&e= wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, 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/mathematics-for-physicists.pdf diabetologia ( ) : – doi . /s - - - article b. i. freedman . s. s. rich . m. m. sale . g. heiss . l. djoussé . j. s. pankow . m. a. province . d. c. rao . c. e. lewis . y. d. i. chen . s. r. beck . on behalf of the hypergen investigators genome-wide scans for heritability of fasting serum insulin and glucose concentrations in hypertensive families received: july / accepted: november / published online: march # springer-verlag abstract aims/hypothesis: the heritability of fasting serum insulin and glucose concentrations in non-diabetic members of multiplex hypertensive families is unknown. methods: we calculated the familial aggregation of fast- ing serum glucose and insulin concentrations and performed a genome-wide scan to assess whether quantitative trait loci contribute to these phenotypes in , non-diabetic individuals from , families enrolled in the hyperten- sion genetic epidemiology network (hypergen) in the family blood pressure program. results: the heritability (±se) of fasting serum insulin was . ± . in european americans and . ± . in african americans (p< . for both), after adjusting for age, sex, and bmi. a genome- wide scan for fasting serum insulin yielded a maximum log of the odds (lod) score of . on chromosome at cm (p= . ) in european americans, and an lod score of . on chromosome at cm (p= . ) in african americans. the heritability of fasting serum glu- cose was . ± . in the former and . ± . in the latter (p< . for both) after adjusting for age, sex and bmi. a genome-wide scan for fasting serum glucose re- vealed a maximum lod score of . on chromosome at cm (p= . ) in european americans. conclusions/ interpretation: these analyses demonstrate the marked her- itability of fasting serum insulin and glucose concentrations in families enriched for the presence of members with hy- pertension. they suggest that genes associated with fasting serum insulin concentration are present on chromosomes and , and that genes associated with fasting serum glucose concentration are on chromosome , in families enriched for hypertension. keywords african americans . essential hypertension . fasting blood sugar . heritability . linkage analysis . serum insulin concentration abbreviations aa: african american . ea: european american . fbpp: family blood pressure program . h : heritability . homa: homeostasis model assessment . hypergen: hypertension genetic epidemiology b. i. freedman (*) . m. m. sale department of internal medicine, sections on nephrology and endocrinology the wake forest university school of medicine, medical center boulevard, winston-salem, nc, - , usa e-mail: bfreedma@wfubmc.edu tel.: + - - fax: + - - s. s. rich . s. r. beck public health sciences wake forest university school of medicine, winston-salem, nc, usa m. m. sale center for human genomics wake forest university school of medicine, winston-salem, nc, usa g. heiss department of epidemiology, university of north carolina, chapel hill, nc, usa l. djoussé evans department of medicine, section of preventive medicine and epidemiology, boston university, boston, ma, usa j. s. pankow department of epidemiology, university of minnesota, minneapolis, mn, usa m. a. province . d. c. rao division of biostatistics washington university school of medicine, st. louis, mo, usa c. e. lewis department of preventive medicine, university of alabama, birmingham, al, usa y. d. i. chen department of internal medicine, cedars-sinai medical center, los angeles, ca, usa network . ibd: identity-by-descent . lod: log of the odds . nhlbi: national heart, lung and blood institute . solar: sequential oligogenic linkage analysis routines introduction hyperinsulinaemia is a major risk factor for the subsequent development of type diabetes mellitus [ , ]. fasting serum insulin concentrations are excellent estimates of insulin re- sistance and the resulting hyperinsulinaemia in population studies, and have been shown to aggregate in families [ ]. insulin resistance is associated with increased serum triglyc- eride levels, reduced high-density lipoprotein concentrations and reduced low-density lipoprotein particle size; these meta- bolic parameters predict increased cardiovascular morbidity and mortality [ , ]. the heritability (h ) and roles of inherited and envi- ronmental factors in causing hyperglycaemia and elevated serum insulin concentrations among hypertensive subjects remain unknown. in non-hypertensive populations, an ad- justed h value of . for fasting serum glucose [ ] and . for fasting serum insulin concentrations [ ] were re- ported in the framingham offspring study. similar high familial correlations for these prediabetic measures were reported in amish families [ ], the heritage family study [ ], and caucasian twins [ ]. elevated serum insulin and glucose concentrations represent an increased risk of developing overt type diabetes mellitus, with increased rates of cardiovascular morbidity and mortality [ ]. it is likely that both genetic and environmental factors determine fasting serum insulin and glucose concentrations in hypertensive subjects. the hypertension genetic epide- miology network (hypergen) is a family-based consor- tium seeking to identify the genes responsible for elevated blood pressure [ ]. we performed maximum likelihood var- iance component linkage analysis of fasting serum glucose and insulin concentrations and homeostasis model assess- ment (homa) to identify loci contributing to the variance of these traits in the non-diabetic relatives of families en- riched for the presence of essential hypertension. subjects and methods population participants in the hypergen network of the family blood pressure program (fbpp), sponsored by the national heart, lung and blood institute (nhlbi), were evaluated. hypergen study methods have previously been reported [ ]. in brief, family members were recruited from five clinical centers (framingham, ma; minneapolis, mn; salt lake city, ut; forsyth county, nc; and birmingham, al). participants provided written informed consent and the project was approved by the institutional review boards at all of the institutions. a sibship was ascertained if it had two or more siblings with hypertension (defined as blood pres- sure ≥ / or the use of anti-hypertensive medications), with an age at diagnosis of less than years. participants reporting a personal history of diabetes mellitus, having a fasting blood glucose concentration of . mmol/l or high- er, or being treated with insulin or oral hypoglycaemic agents were excluded from analyses. phenotyping morning fasting serum samples from study participants were collected in a resting state and run in du- plicate for insulin concentration on an automated immuno- assay instrument and its ultra-sensitive insulin kit (beckman coulter, fullerton, ca, usa) [ ]. the sensitivity of this assay is . mu/l ( . pmol/l) and the dynamic range is . – mu/l ( . – pmol/l). there is zero cross- reactivity with pro-insulin and c-peptide, % with bovine insulin, and % with porcine insulin. serum glucose concentrations were measured using elan glucose reagent (hexokinase method) [ ]. assay sensitiv- ity is . – . mmol/l and the detection limit . mmol/l is documented through the repetitive assay of a diluted serum control. the observed detection limit, calculated as two standard deviations of a -replicate within-run preci- sion study, is . mmol/l and is below the claimed limit of . mmol/l. homa was calculated as (fasting serum insulin*fasting serum glucose)/ . . genotyping genotyping was performed by the nhlbi- funded mammalian genotyping service. for additional information regarding the genotyping methods see the website of the center for medical genetics at the marsh- field medical research foundation http://www.research. marshfieldclinic.org/genetics/). the genome screen was performed by means of an automated technique with the scanning fluorescence detector. the cooperative human linkage center screening set , including microsatellite markers spaced approximately every cm throughout the genome, was used, with an average marker heterozygosity of . . statistical analysis the distributions of fasting serum in- sulin and glucose concentrations were positively skewed. the natural logarithm transformed both insulin and glucose to approximate normality and was used for all analyses and model building. any highly influential outliers were exclud- ed from the analyses after adjusting for covariates. pedigree and genotype data were screened for possible errors using aspex software, version . [ ], mapmaker/sibs, version . [ ], pedcheck, version . [ ], and prest, version . [ ]. the heritability of serum measures was estimated sep- arately in each race and jointly, using variance component modelling as implemented in solar software, version . . [ ]. covariates in the model for fasting glucose were age, sex, bmi, age , and age×sex. covariates in the model for fasting insulin were age, sex, bmi, age , age×sex, sex×bmi, and sex×age . these were selected using a backward elimi- nation approach allowing for re-entry of eliminated covari- ates at each step (significance level= . for backward and forward steps). for both analyses, covariates were selected among age, sex, and bmi, age , age×sex, age×bmi, sex× bmi, sex×age , and bmi×age . centred values were used to http://research.marshfieldclinic.org/genetics/ http://research.marshfieldclinic.org/genetics/ model the effects of continuous covariates, and indicator variables ( / ) were used for discrete covariates. the heri- tability estimate adjusted for the effects of covariates is re- ported together with corresponding estimates of standard error, p value, and proportion of variance due to covariates. we considered p values of . or less to be significant. markov chain monte carlo methods were used to esti- mate race-specific multipoint ibd matrices as implemented in the loki software package, version . . [ ]. variance component multipoint and bivariate linkage analysis as implemented in solar software, version . . [ ], was performed to detect and localise quantitative trait loci that influence variation in fasting serum insulin and glucose concentrations, using lodadj to account for slight departures from normality. this approach has been described in detail [ – ]. results genotype data were available from , individuals in recruited families who participated in the fbpp. eight sub- jects were excluded due to fasting serum glucose concentra- tions above . mmol/l (six aa, two ea). after correcting family relationships based upon the genetic data, , dis- tinct pedigrees ( of whom were african american) were used in the analysis. after exclusion of all influential outliers, , of the genotyped individuals who were nondiabetic had measurements of fasting serum insulin and/or glucose concentrations. the mean age (±sd) of these individuals was . ± . years, . % (n= , ) were women, % were aa (n= , ), and a majority were hypertensive ( . %) and had a mean bmi (±sd) of . ± . kg/m (table ). participants had a mean fasting serum glucose concentration table demographic charac- teristics of hypergen study population data listed as means±standard deviation (n) for continuous measures and % (n) for dichot- omous measures adefined as blood pressure > / mmhg or use of antihypertensive medication characteristic african american caucasian race combined sex female . ( ) . ( ) . ( , ) male . ( ) . ( ) . ( ) age, years . ± . ( , ) . ± . ( , ) . ± . ( , ) bmi, kg/m . ± . ( , ) . ± . ( , ) . ± . ( , ) fasting serum glucose, mmol/l . ± . ( , ) . ± . ( , ) . ± . ( , ) fasting serum insulin, mu/l . ± . ( , ) . ± . ( , ) . ± . ( , ) hypertensiona . ( ) . ( ) . ( , ) fig. genome scan plots for fasting serum insulin concentra- tions. dotted line african americans; solid line caucasians t a b le l in k ag e re su lt s: fa st in g in su li n , g lu co se , h o m a an al y si s an d b iv ar ia te an al y si s l o ca ti o n h o m a a f as ti n g in su li n a f as ti n g g lu co se a b iv ar ia te b m ar k er c h r a a e a a a e a a a e a a a e a d s / d s . @ . . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . . @ . d s . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . . @ . . @ . d s . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c . @ . . @ . . @ . . @ . d s / d s . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c d s / d s . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . . @ . . @ . ( . , . ) . c d s . @ . ( . , – ) . c . @ . . @ . . @ . . @ . . @ . . @ . . @ . d s . @ . ( . , . ) . c . @ . . @ . . @ . . @ . . @ . . @ . . @ . d s . @ . . @ . . @ . . @ . . @ . . @ . ( . ,– ) . c . @ . . @ . ( . ,– ) . c d s / d s . @ . . @ . . @ . . @ . . @ . . @ . ( . ,– ) . c . @ . . @ . d s . @ . . @ . . @ . . @ . . @ . . @ . . @ . ( . ,– ) . c . @ . d s / d s . @ . . @ . . @ . . @ . . @ . . @ . . @ . (– , . ) . c . @ . d s / d s . @ . . @ . . @ . . @ . . @ . (– , . ) . c . @ . . @ . (– , . ) . c . @ . d s / d s . @ . . @ . . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . ( . , . ) . c d s . @ . . @ . . @ . . @ . . @ . ( . , . ) . c . @ . . @ . . @ . d s . @ . (– , . ) . c . @ . . @ . (– , . ) . c . @ . . @ . . @ . . @ . (– , . ) . c . @ . a a a fr ic an a m er ic an , e a e u ro p ea n a m er ic an , c h r ch ro m o so m e a s ig n if ic an t re su lt s re p o rt ed as m ax im u m l o d sc o re @ lo ca ti o n in ce n ti m o rg an s (l o d - in te rv al ) em p ir ic al p v al u e b s ig n if ic an t re su lt s re p o rt ed as m ax im u m l o d sc o re @ lo ca ti o n in ce n ti m o rg an s (l o d - in te rv al ) p v al u e c l o d - su p p o rt in te rv al an d p v al u es fo r m ax im u m l o d sc o re s ab o v e . of . ± . mmol/l and a mean fasting serum insulin con- centration of . ± . mu/l. among these , individuals, there were , sibling pairs ( aa), avuncular pairs ( aa), half-sibling pairs ( aa), first cousins ( aa), and ea monozygotic twins. the mean family size with insulin and/or glucose data was . members (aa . , ea . ). the h of serum insulin was . ± . in eas and . ± . in aas (p< . for both), after controlling for the significant main and interactive effects of age, sex and bmi. an additional % and % of the variance in eas and aas, respectively, was due to measured covariates. figure contains the univariate genome-wide scan results for fasting erum insulin concentration in eas and aas. a maximum lod score of . was observed on chromosome at . cm (marker d s /d s , p= . ), with lesser peaks of lod . on chromosome ( cm, marker d s ) and . on chromosome ( cm, marker d s ) in eas, and . on chromosome ( cm, marker d s ) in aas, p< . for all (fig. ). table contains the re- sults of the genome scan for fasting serum insulin (as well as for homa, fasting serum glucose and the bivariate analysis), reporting maximum lod scores of more than . , position, lod- interval and p value in aas and eas (see symbols and footnotes) and maximum lod scores with position in all other scans in proximity to the sig- nificant results. the results of the homa genome scan are presented in fig. and table . similar regions of linkage were observed for fasting serum insulin concentrations on chromosomes and in eas and on chromosome in aas. the h of serum glucose concentration was . ± . in eas and . ± . in aas (p≤ . for both), after controlling for the significant main and interactive effects of age, sex, and bmi. an additional % and % of the variance in eas and aas, respectively, was due to mea- sured covariates. the univariate genome-wide scan results for fasting serum glucose are depicted in fig. . a maxi- mum lod score of . was observed on chromosome at . cm (marker d s /d s , p= . ) in eas and a lesser peak was observed on chromosome : lod . at . cm (marker d s /d s ), p< . in aas (fig. ; table ). in aas, the genetic correlation between fasting serum glucose and insulin was nonsignificant (p= . ), while the environmental correlation was re= . ± . (p< . ). in eas, the genetic correlation between fasting serum glucose and insulin was also nonsignificant (p= . ), while the environmental correlation was re= . ± . (p= . ). these data suggest that the primary genetic determinants of fasting serum glucose concentration are different from those that contribute to the variation in fasting serum insulin concentration. a bivariate genome-wide scan for loci con- tributing to both fasting serum glucose and insulin con- centrations in both races demonstrated eight regions with maximum lod scores above . (four in aas and four fig. genome scan plots for homa. dotted line african americans; solid line caucasians in eas). these regions are depicted, separately by race, in fig. and table . discussion this report is the first to reveal that inherited factors appear to play important roles in the regulation of fasting serum insulin and glucose concentrations in hypertensive, nondia- betic aas and caucasians. the heritability of fasting serum insulin and glucose concentrations remained highly signif- icant even after controlling for the effects of significant covariates. the marked heritability of fasting serum glucose and insulin concentrations appears consistent with other reports in framingham [ , ], amish [ ], and heritage families [ ]. additionally, the genome scans in aas and eas provided suggestive evidence that genes regulating fasting serum insulin concentrations and insulin sensitivity (homa) were present on chromosomes , , , and , and genes regulating fasting glucose concentration were present on chromosomes , , , and . in general, the linkage peaks for fasting insulin, homa and fasting glu- cose differed by ethnic group (see table ). race-combined analyses added little, as results were driven by a single eth- nic group (data not shown). although the bivariate genome scan demonstrated several regions with suggestive evidence for linkage, these results were probably driven by linkage to either fasting serum insulin or fasting serum glucose con- centrations alone, based on the low genetic correlation ob- served between these measures. several genome-wide scans have been performed in non- diabetic families evaluated for prediabetic phenotypes and in multiplex type diabetes families. regions of linkage in several reports overlap with those observed in this hy- pergen analysis. on chromosome , linkage was detected at cm for fasting insulin in pima indians (lod . ) [ ], and for type diabetes and glucose intolerance in young-onset french families at cm (lod . ) [ ]. linkage with fasting glucose, and combined fasting and non-fasting glucose, was detected on chromosome at cm in the framingham offspring study (lod . ) [ ], and framingham heart study (lod . ) [ ]. the hy- pergen fasting serum glucose scan loci on chromosome are near reported loci for related phenotypes in finnish families from the fusion study [ ], i.e., -h insulin (lod . at cm) and insulin secretion (insulin/ glucose) (lod . at cm), and loci for type diabetes in aas with earlier age at diagnosis (p< . at cm) [ ] and japanese families (lod . , cm) [ ]. link- age for these phenotypes resides within, or near, the peaks where we found evidence for linkage to fasting insulin (chromosomes and ) and fasting glucose ( and ) in the univariate analyses. therefore, the regions identified in the hypergen genome scan as influencing fasting serum insulin and glucose concentrations may reflect genes that also play major roles in susceptibility to diabetes mellitus fig. genome scan plots for fasting serum glucose concen- trations. dotted line african americans; solid line caucasians and/or related phenotypes. our data are unique, however, in that they are from a biracial population enriched for the presence of essential hypertension. fasting serum glucose concentrations clearly fluctuate in individuals with insulin resistance and in those with diabe- tes mellitus. in addition, little is known about the natural history of insulin resistance among treated and untreated hypertensive subjects. these difficulties are encountered in all cross-sectional studies. the overlapping regions of link- age observed for the diabetes-related phenotypes in pima [ ] and finnish [ ] families, and the framingham off- spring [ ] and heart studies [ ], support the existence of genes affecting fasting serum insulin concentrations on chro- mosomes and and fasting serum glucose on chromo- some . in conclusion, this report analysed the heritability of fasting serum insulin and glucose concentrations in non- diabetic members of multiplex hypertensive families. ele- vated serum insulin concentrations and fasting blood sugars (insulin resistance) are well recognised risk factors for the development of heart attack and stroke. the heritabilities of fasting serum insulin and glucose concentrations were signif- icant after controlling for the main and interactive effects of age, sex, and bmi. additionally, suggestive evidence for genetic linkage to fasting serum insulin concentrations and insulin sensitivity (homa) were detected on chromosomes , , , and ; and suggestive evidence for linkage to fasting serumglucoseconcentrationswasdetectedonchromosomes , , , , and . these results suggest that the genes regulating susceptibility to insulin resistance, hyperglycaemia and dia- betes-related phenotypes may reside in these regions and play an important role in the observed familial aggregation of cardiovascular disease. it is important that additional large, family-based analyses in hypertensive cohorts attempt to re- produce these results and identify the causative genes. acknowledgements hypergen participating institutions and prin- cipal staff: s.c. hunt, r.r. williams (deceased), h. coon, p.n. hopkins, j. hood, l. wu, j. skuppin (network center/university of utah field center); a. oberman, c.e. lewis, m.t. weaver, p. johnson, s. walker, c. oden (university of alabama at birmingham field center); r.c. ellison, r.h. myers, y. zhang, l. djoussé, j.b. wilk, g.l. splansky (boston university/framingham field center); d. arnett, a.r. folsom, m. miller, j. pankow, g. feitl, b. lux (university of minnesota field center); g. heiss, b.i. freedman, k. north, k. rose, a. haire (university of north carolina field center); d.c. rao, m.a. province, i.b. borecki, a. adelman, d. morgan, k. schwander, d. lehner, a. kraja, s. mandel (data coordinating center, washington university); j.h. eckfeldt, c. leiendecker-foster, r.c. mcglennen, g. rynders, m.y. tsai, j. bucksa (central biochemistry lab, university of minnesota); m. leppert, s.c. hunt, j.m. lalouel, r. weiss (molecular genetics laboratory, university of utah); s.e. old, m. higgins (retired), c. jaquish, m. lundberg, m. gerschenson (na- tional heart, lung, and blood institute). this hypertension network is funded by cooperative agreements (u ) with nhlbi: hl , hl , hl , hl , hl , hl , hl , hl . fig. genome scan plots for bivariate analysis of fasting serum glucose and insulin con- centrations. dotted line african americans; solid line caucasians references . haffner sm, valdez ra, hazuda hp, mitchell bd, morales pa, stern mp ( ) prospective analysis of the insulin-resistance syndrome (syndrome x). diabetes : – . laaksonen de, lakka hm, niskanen lk, kaplan ga, salonen jt, lakka ta ( ) metabolic syndrome and development of dia- betes mellitus: application and validation of recently suggested definitions of the metabolic syndrome in a prospective cohort study. am j epidemiol : – . snieder h, boomsma di, van doornen 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linkage studies of type diabetes and glucose traits in the old order amish: evidence for a new diabetes locus on chromosome q and confirmation of a locus on chromo- some q –q . diabetes : – . hong y, weisnagel sj, rice t et al ( ) familial resemblance for glucose and insulin metabolism indices derived from an intravenous glucose tolerance test in blacks and whites of the heritage family study. clin genet : – . edelstein sl, knowler wc, bain rp et al ( ) predictors of progression from impaired glucose tolerance to niddm: an analysis of six prospective studies. diabetes : – . williams rr, rao dc, ellison rc et al ( ) nhlbi family blood pressure program: methodology and recruitment in the hypergen network. hypertension genetic epidemiology net- work. ann epidemiol : – . allauzen s, mani jc, granier c, pau b, bouanani m ( ) epitope mapping and binding analysis of insulin-specific mono- clonal antibodies using a biosensor approach. j immunol methods : – . pesce aj, kaplan la (eds) ( ) methods in clinical chemistry. c.v mosby, st. louis . hinds da, risch n ( ) the aspex package: affected sib- pair exclusion mapping. version . . http://aspex.sourceforge. net/usage.html (accessed / / ) . kruglyak l, lander es ( ) high-resolution genetic mapping of complex traits. am j hum genet : – . o’connell jr, weeks de ( ) pedcheck: a program for identification of genotype incompatibilities in linkage analysis. am j hum genet : – . mcpeek ms, sun l ( ) statistical tests for detection of mis- specified relationships by use of genome-screen data. am j hum genet : – . almasy l, blangero j ( ) multipoint quantitative-trait link- age analysis in general pedigrees. am j hum genet : – . heath sc ( ) markov chain monte carlo segregation and linkage analysis for oligogenic models. am j hum genet : – . goldgar de ( ) multipoint analysis of human quantitative genetic variation. am j hum genet : – . amos ci ( ) robust variance-components approach for assessing genetic linkage in pedigrees. am j hum genet : – . almasy l, dyer td, blangero j ( ) bivariate quantitative trait linkage analysis: pleiotropy versus co-incident linkages. genet epidemiol : – . pratley re, thompson db, prochazka m et al ( ) an autosomal genomic scan for loci linked to prediabetic pheno- types in pima indians. j clin invest : – . vionnet n, hani el h, dupont s et al ( ) genomewide search for type diabetes-susceptibility genes in french whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome q -qter and independent replication of a type -diabetes locus on chromosome q –q . am j hum genet : – . mcqueen mb, bertram l, rimm eb, blacker d, santangelo sl ( ) a qtl genome scan of the metabolic syndrome and its component traits. bmc genet (suppl ):s . watanabe rm, ghosh s, langefeld cd et al ( ) the finland–united states investigation of non-insulin-dependent diabetes mellitus genetics (fusion) study. ii. an autosomal genome scan for diabetes-related quantitative-trait loci. am j hum genet : – . sale mm, freedman bi, langefeld cd et al ( ) a genome- wide scan for type diabetes in african-american families reveals evidence for a locus on chromosome q. diabetes : – . iwasaki n, cox nj, wang yq et al ( ) mapping genes influencing type diabetes risk and bmi in japanese subjects. diabetes : – http://aspex.sourceforge.net/usage.html http://aspex.sourceforge.net/usage.html 	genome-wide scans for heritability of fasting serum insulin and glucose concentrations in hypertensive families 	abstract 	abstract 	abstract 	abstract 	abstract 	introduction 	subjects and methods 	population 	phenotyping 	genotyping 	statistical analysis 	results 	discussion 	references << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (none) /calrgbprofile (srgb iec - . ) /calcmykprofile (iso coated) /srgbprofile (srgb iec - . ) /cannotembedfontpolicy /warning /compatibilitylevel . /compressobjects /off 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/ccittfaxencode /monoimagedict << /k - >> /allowpsxobjects false /pdfx acheck false /pdfx check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ . . . . ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ . . . . ] /pdfxoutputintentprofile (none) /pdfxoutputcondition () /pdfxregistryname (http://www.color.org?) /pdfxtrapped /false /syntheticboldness . /description << /deu <feff a f f f e f f c c e d f c f c e e e f e c e e d e d c d e d d d d c f e e f e c f a f f f f e e e e f f d f e c f e c e f f c f f f f e c e f f c e f c f a f d f e e> /enu <feff a f f f e f f c c e d f c f c e e e f e c e e d e d c d e d d > >> >> setdistillerparams << /hwresolution [ ] /pagesize [ . . ] >> setpagedevice copy number variants encompassing mendelian disease genes in a large multigenerational family segregating bipolar disorder copy number variants encompassing mendelian disease genes in a large multigenerational family segregating bipolar disorder kember et al. kember et al. bmc genetics ( ) : doi . /s - - - kember et al. bmc genetics ( ) : doi . /s - - - research article open access copy number variants encompassing mendelian disease genes in a large multigenerational family segregating bipolar disorder rachel l kember , benjamin georgi , joan e bailey-wilson , dwight stambolian , steven m paul and maja bućan , * abstract background: bipolar affective disorder (bp) is a common, highly heritable psychiatric disorder characterized by periods of depression and mania. using dense snp genotype data, we characterized cnvs in members of an old order amish pedigree with bipolar disorder. we identified cnv regions arising from common ancestral mutations by utilizing the pedigree information. by combining this analysis with whole genome sequence data in the same individuals, we also explored the role of compound heterozygosity. results: here we describe inherited cnv regions, of which are rare in a control population of european origin but present in a large number of amish individuals. in addition, we highlight a set of cnvs found at higher frequencies in bp individuals, and within genes known to play a role in human development and disease. as in prior reports, we find no evidence for an increased burden of cnvs in bp individuals, but we report a trend towards a higher burden of cnvs in known mendelian disease loci in bipolar individuals (bpi and bpii, p = . ). conclusions: we conclude that cnvs may be contributing factors in the phenotypic presentation of mood disorders and co-morbid medical conditions in this family. these results reinforce the hypothesis of a complex genetic architecture underlying bp disorder, and suggest that the role of cnvs should continue to be investigated in bp data sets. keywords: cnv, bipolar disorder, family based studies, mendelian disease genes, genetics loci background bipolar affective disorder (bp) is a serious mental dis- order characterized by periodic changes in mood, energy and activity levels alternating between episodes of de- pression and mania [ ]. the lifetime prevalence of bp type i (bpi) and type ii (bpii) is . % in the united states [ ] and the age of onset is early, at – . years old [ ], making bp responsible for the loss of more disability-adjusted life-years than all forms of cancer [ ] and consequently it is a major public health concern [ ]. as with many complex disorders, the underlying eti- ology of bp is unknown, but is hypothesized to be the result of multiple gene-gene and gene-environment in- teractions [ ]. epidemiological studies using twin data * correspondence: bucan@upenn.edu department of genetics, university of pennsylvania, philadelphia, pa, usa department of psychiatry, perelman school of medicine, university of pennsylvania, philadelphia, pa, usa full list of author information is available at the end of the article © kember et al.; licensee biomed centra commons attribution license (http://creativec reproduction in any medium, provided the or dedication waiver (http://creativecommons.or unless otherwise stated. show that bp has heritability estimates ranging from - % [ , ], although the mode of inheritance is complex. common genetic factors have been shown to contribute substantially to susceptibility for bipolar disorder, with a strong polygenic contribution [ ]. several potential bp candidate genes have been described [ ], but findings are inconsistent and the role of specific genes in bp is currently undetermined. copy number polymorphisms (cnvs) are a common class of genetic variation in the human genome [ - ], and can be readily detected using intensity data from genome-wide snp arrays. like single-nucleotide polymor- phisms (snps), cnvs can affect gene expression, either by encompassing genes or regulatory elements. large, cyto- genetically detectable chromosomal rearrangements, such as aneuploidy, have been historically linked to human dis- ease [ ]. studies of several genomic disorders, associated with inherited or sporadic genomic anomalies which are l. this is an open access article distributed under the terms of the creative ommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and iginal work is properly credited. the creative commons public domain g/publicdomain/zero/ . /) applies to the data made available in this article, mailto:bucan@upenn.edu http://creativecommons.org/licenses/by/ . http://creativecommons.org/publicdomain/zero/ . / kember et al. bmc genetics ( ) : page of smaller (< mb) and therefore can not be detected using conventional cytogenetic methods, revealed that deletions and duplications encompassing several genes may lead to complex and highly pleiotropic clinical syndromes [ ]. several systematic surveys of copy number variation using comparative genomic hybridization (cgh) and high- density snp arrays revealed a large number of benign dele- tions and duplications across the genome, but also revealed the role of a large number of rare potentially pathogenic cnvs in neurodevelopmental and psychiatric diseases, par- ticularly in autism spectrum disorder and schizophrenia [ ]. in autism spectrum disorder, cnvs were found in a number of chromosomal regions [ ], and the burden of rare and de novo cnvs was enriched in affected individuals compared to controls and their unaffected siblings [ ]. similarly, several large, rare cnvs have been associated with schizophrenia [ , ]. among these cnvs several have been observed at elevated rates in multiple neurodevelopmental and psychiatric disorders [ , ]. both linkage and candidate gene analyses, as well as genome-wide association studies, indicate a shared genetic architecture and an overlap of susceptibility between bp and schizophrenia [ ]. however, compared to studies conducted on asd and schizophrenia, there are far fewer examples of cnvs associated with bp [ ]. an analysis of cases and controls reported an increased bur- den of singleton cnvs in early onset bipolar cases [ ]. also, in an independent study of trios, frequencies of de novo cnvs were significantly higher in bipolar disorder as compared to controls, but not as high as in schizo- phrenia [ ]. however, a study using welcome trust case control consortium (wtccc) data found no evi- dence for an elevated burden of cnvs in bipolar individuals (n = ) compared to controls (n = ), although the burden was found to be elevated in schizophrenia [ ]. the same authors recently published the most comprehensive analysis of cnvs in the wtccc revealing a significantly lower rate of rare very large cnvs (> mb) in patients with bipolar disorder (n = , ) compared to reference individ- uals without psychiatric disorder (n = , ) [ ]. al- though the authors state that this result needs to be verified in larger datasets, they propose that a lower cnv burden may underlie differences in the presentation of clinical phenotype between bipolar disorder and schizophrenia. in addition, recent research suggests that de novo cnvs may play a smaller role in bp compared to schizophrenia [ ], but the role of inherited cnvs remains uncertain. the old order amish are a founder population origin- ating in middle europe. since , when victor mckusic and colleagues described the benefits from medical genet- ics studies in the amish [ ], a large number of mendelian disorders have been described in this population [ ]. more recently, next generation sequencing studies of neu- rodevelopmental and psychiatric disorders in the amish provide a unique opportunity to address the role of rarer forms of genetic variation [ , ]. however, these recent studies focus on the role of single nucleotide variants (snvs). apart from a handful of gene deletions associated with mendelian disease [ ], and cnv regions identi- fied in a subset of individuals from the old order amish pedigree with bipolar disorder [ ], global analysis of copy number variation has not been systematically carried out in this genetic isolate. the aim of the present study was to investigate cnvs in the extended old order amish pedigree with bipolar dis- order, and compare these cnvs with cnvs detected in a large collection of unrelated control subjects to identify deletions and duplications private to this family. also, we compared burden and frequency of cnvs in family mem- bers with affective disorders (bpi, bpii and mdd-r) with their unaffected relatives to identify cnvs potentially con- tributing to the locus and allele heterogeneity of bipolar disorder. our systematic analysis revealed rare and moderately rare cnvs encompassing mendelian disease genes that may contribute to the complex and pleiotropic manifestation of mental illness in this founder population. results overall strategy to characterize structural variants in members of a large multigenerational old order amish pedigree with bipolar disorder, we used dense snp genotype data gen- erated using the illumina omni . m platform [ ]. we also performed cnv analysis on , age-related eye disease study (areds) control subjects ( , with european ethnicity) genotyped using the same snp plat- form. a flowchart (figure ) outlines the quality control and analysis pipeline employed to address: a) differences in the allele frequency of cnvs in this genetic isolate compared to a large sample of subjects of european ori- gin; b) the role of cnvs in susceptibility to bipolar ill- ness in this large pedigree; c) an estimate of the total per genome (or person) burden of cnvs, including cnvs that encompass known disease loci. a catalog of inherited cnvs in an amish pedigree segregating bipolar disorder as part of our genetic study of bipolar disorder in the old order amish, we analyzed genome-wide snp genotype data using the penn cnv algorithm [ ] to identify cnvs. we examined the breakpoints of all cnvs (n = , ) and clustered groups of cnvs that have arisen from com- mon ancestral mutations (see methods) into cnv regions (n = ). using the pedigree relationships, we classified all regions as either ‘inherited’ (shown to pass from parent to child), or of ‘unknown origin’ (not seen in either par- ent). to avoid possible technical artifacts in the analysis, we focused on the high quality, inherited cnvs observed figure flowchart outlining the quality control and analysis pipeline of this study. snp data from the control and old order amish populations was used to call cnvs using the penncnv program. the pedigree structure in the amish allowed family based calls to be made, and the cnv frequency in the control data allowed us to determine whether the amish cnv calls were common, rare, or exclusive to the amish population. we sought to determine cnvs in bipolar individuals, burden in individuals, and cnvs in disease loci. in addition, we utilized the whole genome sequence data to find cnvs and snps within the same gene in the same individual. kember et al. bmc genetics ( ) : page of in multiple (more than three) amish family members. these variants are less likely to represent false positives in computational prediction or rearrangements that arose during culturing of lymphoblastoid cell lines [ ]. further- more, in a large pedigree with an excess of bipolar dis- order, we expect that the causal genetic variants will be inherited, rather than de novo. of inherited cnv re- gions identified by the analysis of family members (a subset that contains both parent and child information), overlap with regions previously identified in a small scale study of the core family, i.e. family members [ ]. eight cnv regions (four which are exonic, one which is intronic, and three which are intergenic) were detected on chromosome x (additional file : table s ). among detected inherited cnv regions, the largest category consisted of common cnvs (present in more than % of controls) found throughout the large multi- generational amish pedigree. in addition we detected moderately rare (present in less than % of controls) and rare (present in less than % of controls) cnv regions in subjects, as well as ‘exclusive’ regions that were not present in any controls. of these exclusive re- gions, are deletions and are duplications, and regions include genes (figure , created using circos [ ]). these ‘exclusive’ variants form a key part of the genomic architecture of this pedigree, and could play a role in phenotypic presentation. to illustrate the fre- quency of a cnv in the pedigree, cnv counts are pre- sented for nuclear families only in which the cnv is present, and only for those individuals with bipolar or well phenotypes; individuals with unknown or other phenotypes are excluded. they include a kb duplica- tion on q , present in amish individuals (af- fected / , . %; unaffected / , . %), which encompasses the entire sry (sex determining region y)- box (sox ) gene; a kb deletion on q . , found in individuals (affected / , . %; unaffected / , . %), in an intergenic region upstream of both coiled- coil domain containing (ccdc ) and gm ganglio- side activator (gm a); and a kb deletion on q . , found in individuals (affected / , . %; unaffected / , . %), located downstream of solute carrier family , member (slc a ). we compared the total number, average size, and burden per individual of cnvs in a) all family members (n = ), b) subjects with bipolar disorder (n = ), and unaffected amish and control individuals (amish n = , controls n = ) (table ). analysis was performed on copy number losses (deletions) and gains (duplications) sep- arately. overall number of detected deletions and figure location of cnv regions, burden of rare cnvs, and disease genes. cnv regions are shown as red and green lines (green: heterozygous duplication, dark green: homozygous duplication, red: heterozygous deletion, dark red: homozygous deletion). stacked histogram bars represent the location of specific rare cnvs, and the number, split by phenotype (green background: duplications, red background: deletions; dark grey: bipolar, mid-grey: unknown, light grey: unaffected). inner line plot (blue) shows location and number of disease genes. genes of interest are labeled around the outside of the plot. kember et al. bmc genetics ( ) : page of duplications in the amish pedigree were comparable to those detected in the control subjects. cnvs and disease association no individual cnv segregated fully with bipolar disorder. analysis of cnv data in the linkage regions previously re- ported [ ] identified a single duplication event in the q region. the kb duplication localizes upstream of the maximum lod score marker d s . we confirmed in- heritance of the cnv on the - - - haplotype (d s - d s -d s -d s ). the duplication spans the first exon of the collagen, type xxvi, alpha (col a ) gene. col a has yet to be functionally characterized, with a possible role in aspirin-intolerant asthma [ ]. burden analysis of cnv regions in genes in the amish shows a trend towards an increased number of these table summary of cnv calls for the old order amish (n = ) and controls (n = ) total deletions duplications amish cnvs amish inherited cnvs controls amish cnvs amish inherited cnvs controls amish cnvs amish inherited cnvs controls total number all samples affected - - - unaffected - - - average size (bp) all samples affected - - - unaffected - - - burden (per individual) all samples . . . . . . . . . affected . . - . . - . . - unaffected . . - . . - . . - total number, average size, and burden of cnvs was calculated for all amish cnvs, inherited amish cnvs, and control cnvs. cnvs were analyzed together, and as deletion and duplication events separately. k em b er et a l. b m c g en etics ( ) : p ag e o f kember et al. bmc genetics ( ) : page of cnvs in bipolar individuals (narrow phenotype: bpi and bpii), although this does not reach significance (narrow burden: . , unaffected burden: . , p = . ) (table ). we identified three rare deletions in kcnj , unc c, otol and rare duplications in cntnap /mir f , coro /vasn, dtnb, emid , kcnf , pdpr and sgta/thop that are present in children with bipolar disorder (and their parents). in addition, we find other rare cnvs in genes that are present frequently in individ- uals with bipolar disorder (table ). association analysis for all cnv regions was performed using two different methods: a) fbat [ ] and b) emmax [ ], although no cnv was found to be significantly associated with bp fol- lowing correction for multiple testing. we found no over- all enrichment of large inherited cnvs in affected individuals, although large, rare, cnvs in genes oc- curred more frequently in subjects with bipolar disorder than unaffected family members and control subjects. one of the largest rare genic cnvs is the previously re- ported kb deletion in the q region, which encom- passes the entire prader-willi region non-protein coding rna (pwrn ) gene [ ]. the deletion is present in families, is found on two haplotypes (d s -d s - d s : - - and - - ) and is widely spread through- out the pedigree; / ( . %) of those with bipolar dis- order in these carrier families have the cnv, compared to / ( . %) of well individuals. next, we focused on the analysis of cnvs encompass- ing known disease genes. it has been suggested that het- erozygosity for several mutations in mendelian disease genes may lead to complex disease risk, such as behav- ioral anomalies in neurodevelopmental and psychiatric disorders [ ]. to ask if cnvs in disease genes may con- tribute to the allelic architecture in the amish family segregating bipolar disorder, we mapped known disease loci with respect to cnv breakpoints. specifically, we table burden analysis of cnv regions reveals a higher number of cnvs in genes in individuals with narrow bipolar phenotype (bpi and bpii) unaffected broad narrow no. all cnvs . . . p vs unaffected - . . no. all cnvs in genes . . . p vs unaffected - . . no. rare cnvs in genes . . . p vs unaffected - . . no. cnvs in disease genes . . . p vs unaffected - . . no. rare cnvs in disease genes . . . p vs unaffected - . . a trend towards an increased number of cnvs in disease genes in individuals with narrow bipolar phenotype is also reported. utilized the known disease causing variants (classed ‘dm’ in hgmd) from the human gene mutation database to define genes associated with disease. we identified cnv regions that overlap with genes with known dis- ease causing mutations (additional file : table s ). of these, cnv regions are specific to the amish pedigree, and are rare (< %) in the control population. interest- ingly, the number of cnv regions that encompass disease genes shows a trend towards an increased burden in bipo- lar narrow phenotype individuals (narrow phenotype bur- den: . , unaffected burden . , p = . ), and this is also true for rare (including amish specific) cnvs encompass- ing disease genes (narrow phenotype burden: . , un- affected burden . , p = . ) (table ). in particular, we explored the transmission (from par- ent to child) for rare cnv deletions in disease genes. additional file : table s details the cnv deletions found in these genes, many of which have a behavioral disease phenotype (parkinson disease, autism spectrum disorder, intellectual disability). additional file : figure s displays the extensive genetic heterogeneity within this founder pedigree, focusing on the cnvs in these genes. different branches of the pedigree carry different cnvs, with some nuclear families carrying up to rare deletions in a known disease associated gene. in addition, we find a previously identified schizophrenia associated cnv ( q del) [ ] in an individual with a bpii phenotype. it is striking that in this individual this cnv maps in the vicinity of a recombination site on the paternal chromosome. these results together provide evidence for a complex role of cnvs in the phenotypic presentation within this family. within this set of disease genes, we found an amish exclusive duplication in the hoxd cluster, present in families. of those with bipolar disorder in these carrier families / ( %) have the cnv, compared to around / ( %) of well individuals. our analysis also de- tected three cnvs in genes previously linked to reces- sive disease in the plain populations (cntnap : [ ]; adamts , clcnkb: [ ]). these include a rare in- tronic heterozygous duplication (present in two individ- uals, including one affected) in contactin associated protein-like (cntnap ), a gene associated with aut- ism spectrum disorder. in addition, four individuals (one affected) have an exonic heterozygous deletion not found in controls in adam metallopeptidase with thrombospondin type motif (adamts ), a candidate gene for weill-marchesani syndrome. lastly, individ- uals (four affected) were found to have an exonic hetero- zygous duplication of the chloride channel, voltage- sensitive kb (clcnkb), variants in which are associated with essential hypertension. the availability of a combined dense genotype and whole genome sequence for parent child trios [ ], table cnvs within genes cytoband start stop no. snp length (bp) cn contained genes frequency no. subjects no. affected no. families predicted effect fbat emmax previous disease associations for gene p . & loc rare gene del . q muc , trim amish specific partial gene dup . . kidney disease [ ] q . aldh a very rare exonic del . q . evx , hoxd , hoxd , hoxd , hoxd , hoxd , hoxd amish specific gene dup . . limb and genital abnormalities [ , ] q . dusp , rnpepl very rare partial gene dup . . q . ankmy , dusp , rnpepl very rare partial gene dup for ankmy and rnpepl , gene dup for dusp . . p . gpr , pcbp amish specific partial gene dup . . p . & atg rare intronic del . . frontotemporal dementia, parkinsons disease [ ] q & atp a rare intronic del . . autism [ ] q . fam a amish specific intronic del . . q . ltc s, mgat b, mir , sqstm , maml amish specific partial gene dup for ltc s and sqstm , gene dup for mgat b and mir . . venous thromboembolism and ischaemic stroke [ ], paget disease of bone [ ] q . ltc s, mgat b, mir amish specific gene dup . . venous thromboembolism and ischaemic stroke [ ] p . hla-dqa rare exonic del . p . foxc very rare exonic dup . axenfeld-rieger anomaly [ ] q park very rare exonic del . . parkinsons disease [ ], autism [ ] q . emid rare exonic dup . . q . sspo, znf amish specific partial gene dup . . p . fam b , hr, nudt amish specific partial gene dup for fam b and hr, gene dup for nudt . . alopecia universalis [ ], congenital atrichia [ ] p msr rare partial gene del . . prostate cancer [ ] k em b er et a l. b m c g en etics ( ) : p ag e o f table cnvs within genes (continued) p tusc very rare intronic del . . intellectual disability [ ] q . sh d c, tor a amish specific partial gene dup for tor a, gene dup for sh d c . . q . tmem -as rare partial gene dup . . q . ctnna very rare partial gene del . . arrhythmogenic right ventricular cardiomyopathy [ ] p . c orf , mapk ip , pex amish specific partial gene dup for mapk ip and pex , gene dup for c orf . . diabetes type [ ], zellweger syndrome [ ] p . ascl very rare gene del . . q sox amish specific gene dup . . neuronal development [ ] q gas very rare partial gene dup . . q . ppp r e very rare intronic del . . q . pwrn rare gene del . . prader-willi region [ ] q . idh , znf amish specific partial gene dup . . d- -hydroxyglutaric aciduria, type ii [ ] p . & il r very rare exonic del . . p . abr, bhlha , mir , nxn, timm , tusc very rare partial gene dup for nxn and tusc , gene dup for abr, bhlha , mir and timm . . q nfatc amish specific exonic dup . . tricuspid atresia [ ] q sall very rare gene dup . . q nfatc amish specific exonic dup . . q . sik rare gene dup . . cnvs shown are rare in controls (present in fewer than % of controls. rare: < %, very rare: < %, amish specific: not found in controls), and common in the amish (present in more than % of individuals). contained genes shows all genes in cnv, disease genes are highlighted in bold. fbat and emmax p-values for association analysis for bipolar disorder are included, p< . are in bold. k em b er et a l. b m c g en etics ( ) : p ag e o f kember et al. bmc genetics ( ) : page of permitted the investigation of a combined effect of cnvs with likely deleterious snps on the same and in trans haplotype. table lists disease genes with both a cnv and at least one non-synonymous snp present in the same gene in the same individual. among cnvs in known disease genes are two rare cnvs at the park locus: a kb deletion and a kb deletion spanning the second exon. although these cnvs do not segregate with a bipolar disease status, it is notable that kb distal to the cnv breakpoint, in the neighboring park co-regulated gene (pacrg), we previously de- tected a cluster of snps with a family-based association signal of p-value . x - for the top snp (rs ) (additional file : figure s ) [ ]. in addition, there are six individuals who have both an exonic missense table compound heterozygosity - disease genes with cnvs and variants in the same individual gene number of affected (total = ) number of unaffected (total = ) cnv frequency atg rare sntg common ptprd rare il r rare hla- dqa rare kcnj amish specific ccdc common dicer amish specific galntl rare atp a rare erbb common msr rare park rare rhd common cdh rare prkg rare wwox common smarca common cyp d rare tusc rare ugt a rare ugt a rare ugt a rare ugt a rare ctnna rare cacna c common counts in each column represent the number of individuals with both a cnv and another variant in the same gene. variant (rs ) and a cnv in park . the variant is located upstream of the cnv and in each individual is present on a different haplotype from the cnv. haplotype analysis of the region shows multiple haplo- types containing the snps from the family-based asso- ciation analysis, two of which contain the cnvs, further supporting a proposed clustering of several po- tential risk alleles (snps and cnvs) in a defined chromosomal region [ ]. also among these variants was a rare kb intronic de- letion in autophagy related (atg ), which is present in individuals, out of which have a bipolar phenotype ( / , . %; unaffected / , . %). furthermore, individuals with this cnv also have a possibly damaging exonic missense snp (rs ) present on the same haplotype as the cnv. we also identified a number of individuals (n = , affected / , %; unaffected / , . %) with a rare cnv in the intron of tumor suppres- sor candidate (tusc ). these individuals carry a snp (rs ) within the same gene on the same haplotype as the cnv. although many of these individuals are ‘un- affected’ ( unaffected, affected) within our pedigree, mutations such as these may contribute to the overall burden of disease within the amish population. discussion we recently reported a combined analysis of dense ge- notypes and whole genome sequence for a large old order amish pedigree with bipolar disorder. this study focused on the analysis of missense mutations within linkage peaks and detected a high degree of genetic het- erogeneity of mental illness in this family [ ]. here we re- port results of the analysis of cnvs in the same extended pedigree. the mean burden per individual and size of cnvs were similar between our amish sample and the european controls. while previous studies of the role of cnvs as risk alleles for bipolar disorder have been limited, an increased burden of cnvs in bipolar disorder has been reported by some [ , ], although these findings are not consistent [ , ]. we find a trend towards an increased burden of cnvs in genes in individuals with bp, specific- ally for cnvs that are rare or moderately frequent in the general population. while this finding does not reach sig- nificance, it adds to a body of evidence that suggests that cnvs may have some as-yet undefined role in bp and should be investigated accordingly. we identified cnvs in genes previously associated with psychiatric and developmental disorders, that are present at a higher rate in the amish extended pedigree than in the control sample. the comparison of the fre- quency of these structural anomalies in the extended pedi- gree and in a large control dataset identified disease associated cnvs that are enriched in this population and may serve as a starting point for a future “genotype-first kember et al. bmc genetics ( ) : page of approach” [ ] to defining subtypes of bipolar and other complex diseases in this founder population. for example, rare cnvs were found in sox , a transcriptional activator thought to play a role in neuronal development [ ]; and near gm a, which is highly expressed in the brain and can harbor mutations which result in a variant of tay- sachs disease [ ]; and slc a , an amino acid trans- porter expressed highly in the brain and associated with major depressive disorder [ ]. the largest rare, genic de- letion in our pedigree, encompassing pwrn and the sur- rounding region, was found more frequently in bipolar individuals within carrier families. pwrn lies within a . mb section on the long arm of chromosome found to be deleted in prader-willi syndrome, a neurogenetic disorder with cognitive, behavioral and endocrine pheno- types [ ]. a duplication in the hoxd region on chromo- some was also present more frequently in bipolar individuals. the hoxd genes play important roles in morphogenesis, and deletions in this cluster have been as- sociated with limb and genital abnormalities [ ]. our study design allowed us to interrogate a combination of cnvs and other inherited mutations found within the same gene in a single individual on the same or opposite chromosome. using this method, we identify known disease genes that contain both a cnv and exonic mis- sense snp in one or more individuals. of these, particular genes of interest include atg , which has been associated with frontotemporal dementia [ ]; tusc , a gene associ- ated with intellectual disability [ ]; and park , a gene associated with parkinson’s disease [ , ]. although we do not provide evidence that these cnvs and snps alone are disease causing in this pedigree, they may contribute together with other variants within the same chromo- somal region to the disease risk [ ]. in addition, molecu- lar studies of these reported cnvs would be needed to determine if they have any effect on the gene. larger stud- ies in a non-amish population are also required to deter- mine if cnvs at these loci could be of relevance to bipolar or psychiatric disorder in a general population. although clinical information for the large extended old order amish pedigree is limited to mood disorders, our genetic data permits the analysis of cnvs in genes associated with mendelian diseases. in our initial report on the analysis of cnvs in the core amish pedigree (in family members), we provided proof of principle for a family-based investigation of a combination of structural variants in the same subject as that could confer risk for a disease [ ]. our reported trend for an increased bur- den of disease cnvs in bipolar family members (when compared to their unaffected relatives) needs to be fur- ther investigated with a larger sample size, both in the founder and general population. in many mendelian dis- eases, psychiatric and behavioral symptoms are prevalent [ ] and a wide range of medical co-morbidities are common in psychiatric disorders [ ]. the variants underlying mendelian disease are generally highly pene- trant and less influenced by the environment, while hap- loinsufficiency or heterozygosity at several mendelian disease loci may lead to complex behavioral anomalies in psychiatric disorders. moreover, such a burden of risk alleles could explain the high degree of heritability but rather complex genetic architecture observed in these disorders. in other words, we propose that some of the hidden heritability may reside in gene-by-gene interac- tions and that the analysis of interactions at bona fide disease genes may be well powered by focusing on the impact of genetic variation within these critical classes of genes. we have identified over cnvs with a significant difference in allele frequency between the amish family and the control sample. these structural variants add to an extended list of non-synonymous, likely deleterious variants that are rare in the genomes project data- set (< %), but present in - % of bp subjects and their family members [ ]. owing to the anonymized fashion in which our study was conducted, it will not be pos- sible, at this time, to evaluate possible phenotypic conse- quences of private amish structural variants, those that are present in > % of family members in this pedigree, but rare or absent in the general population. however, several ongoing genetics research initiatives involving the plain populations [ , ] combined with the clinical genetics profiling applied in several clinics that serve these communities, are generating valuable insights that could potentially allow prevention of disability and dis- ease. as reported by the clinic for special children and by colleagues involved in genetic studies in hutterites, providing education and offering clinical carrier status for devastating mendelian diseases would likely be wel- comed by members of the founder communities [ , ]. our study has multiple limitations. first of all, the analysis is focused on a large extended family and power is limited for the statistical assessment. also, it is diffi- cult to determine if cnvs found to be enriched in the pedigree are unique to this founder population or to a cluster of these families, originating primarily from the lancaster area (in pennsylvania). however, the use of a genetic isolate and a large family structure provided us with a higher level of genetic and phenotypic homogen- eity, and permitted the tracing of cnv events within nu- clear families and across generations. also, although the availability of biomaterials through a public cell reposi- tory represents a major advantage of this collection, dna isolated from lymphoblastoid cell lines rather than blood represents a limitation of our study. therefore, to avoid possible cell line artifacts, we excluded all single- ton cnvs from our analysis and we were not able to ad- dress the role of de novo cnvs. other studies have kember et al. bmc genetics ( ) : page of reported a role for de novo cnvs in bp [ ], and we were unable to address this area of research. we note that we utilized cnvs from an eye disease study (areds) as a comparison for our dataset. although we were not directly comparing the levels of cnvs between datasets, a disease free control dataset would have been more desirable. however, as their primary use was to identify cnvs that are present in the amish more fre- quently than a european population, we believe the areds dataset was adequate for this purpose. finally, the cnvs reported here were not experimentally vali- dated, but due to our ability to show inheritance of the cnv from parent to child through the pedigree, we con- sider them to be validated cnvs [ ]. conclusions in summary, we identify a number of cnvs in an old order amish pedigree segregating bp. many of the cnvs found were rare in the general population and present in a large number of amish individuals. some of the cnvs were found in a higher frequency in individ- uals with a bp phenotype, and within genes known to play a role in human development and disease. we con- clude that these cnvs may be contributing factors in the phenotypic presentation and heterogeneity of mental illness in this family. methods sample the genetic-epidemiologic study of bipolar disorder among the old order amish in pennsylvania (the amish study of major affective disorder) has been well documented [ , ]. diagnostic methods included struc- tured interviews (sads-l) that were conducted with the patients and close others. in addition, medical records were obtained following signed, informed consent, these were abstracted and collated for five members of the psychiatric board who were blind to patient names, pedigree, address, admission/discharge diagnosis and treatment. the psychiatric board members used strict research diagnostic criteria (rdc) and the diagnostic and statistical manual of mental disorders, th edition (dsm-iv) for uniform clinical criteria, and reviewed all material every few years as a reliability check on diagno- ses. the majority of affected individuals in the current pedigree are diagnosed as either bpi, bpii, or major de- pressive disorder (mdd, recurrent) with a few schizoaf- fective disorder, bp subtype, although there is a wider spectrum of major affective disorders in the extended amish pedigrees. in this study we place individuals into a number of phenotype categories for analysis: narrow (bpi and bpii only), broad (bpi, bpii and mddr), and well (unaffected only). collection of blood samples followed diagnostic con- sensus. lymphoblastoid cell lines were established by the coriell institute of medical research (cimr). signed informed consents were obtained to access medical re- cords for the amish study clinicians exclusively to do diagnostic evaluations and clinical studies. two forms were used: a) one with yearly institutional review board (irb, university of miami) approval adhering to special guidelines because the amish are defined as a “vulnerable” population; and b) a second using state approved, medical record consent forms for specific mental health clinics and psychiatric hospitals throughout central pennsylvania. collection of blood/tissue samples followed diagnostic consensus, using two informed consent forms: a) one with annual univ. miami irb approval defining (with language appropriate for old order amish) how their cells would be preserved for medical research on major affective dis- orders; and, b) the informed consent form required by the institute for medical research (cimr), later coriell - national institute for general medical sciences (nigms) human genetic cell repository (hgcr). in addition, analysis of whole-genome sequence data from consented individuals in this pedigree was also approved by the irb of the weill cornell medical college and the perelman school of medicine at the university of pennsylvania. control subjects control subjects were selected from the age-related eye disease study (areds) sponsored by the national insti- tutes of health (national eye institute). this prospective study of about participants follows the clinical course of age-related macular degeneration (amd) and age- related cataract. participants in this study were required to be ‘free of any illness or condition that would make long- term follow-up or compliance with study medications un- likely or difficult’ and as such are considered ‘well’ for the purposes of our study of mental illness. in addition, age of participants recruited to this study was between to years old, beyond the age at which presentation of a bi- polar phenotype is to be expected. the individuals studied here were not affected with macular degeneration or cata- ract at the areds baseline examination. collection of blood samples for genetic research was performed following recruitment. lymphoblastoid cell lines were established by the coriell institute of medical research (cimr). genotyping was performed on areds samples using illumina omni . m snp arrays at the center for inherited disease research (cidr). we performed rigorous quality control of the raw genotype calls by applying a series of filters on both markers and samples using plink [ ]. the initial dataset contained , , snps and samples. the following filters were applied in sequence; the numbers of markers or sam- ples excluded is given in parentheses: a) exclude snps with kember et al. bmc genetics ( ) : page of missing rate > . ( , ), b) exclude samples with missing rate > . ( ), c) exclude snps with missing rate > . ( , ), d) exclude snps with maf < . ( , ), and e) exclude snps deviating from hardy-weinberg equi- librium at p < -e ( , ). after quality controls we retained , , snps and samples. we subse- quently performed multi dimensional scaling on a set of ~ k overlapping snps for all available areds genotypes and genomes data. this analysis permitted the selec- tion of subjects with european ancestry (additional file : figure s ) for further cnv calling and analysis. genotyping genotyping was performed on samples from the ex- tended amish pedigree using illumina omni . m snp arrays at the center for applied genomics (children’s hospital of pennsylvania, philadelphia, pa). as with the areds data, we performed rigorous quality control of the raw genotype calls by applying a series of filters on both markers and samples using plink [ ], with the addition of excluding markers based on informative missingness and individuals/markers based on the men- del error rate. the initial dataset contained , , snps and samples. the following filters were ap- plied in sequence; the numbers of markers or samples excluded is given in parentheses: a) exclude snps with missing rate > . ( , ), b) exclude samples with missing rate > . ( ), c) exclude snps with missing rate > . ( , ), d) exclude snps with maf < . ( , , ), e) exclude snps with informative missing- ness p < e- ( ), f) exclude snps deviating from hardy- weinberg equilibrium at p < -e ( ), g) exclude individ- uals with > % mendelian errors ( ) and h) exclude snps with > % mendelian errors ( ). after quality controls we retained , , snps and samples. association analysis for all cnv regions was performed using two different methods: a) fbat [ ] (version . . ), a version of the transmission distortion test adapted for lar- ger families and b) emmax [ ] (version from february ), a statistical test for association analysis using mixed models that accounts for the population structure within the sample. identification of copy number variants (cnvs) cnvs were called by penncnv, a previously described cnv detection algorithm [ ], using the gc model wave adjustment [ ]. cnvs were removed if they had a value > . standard deviation of lrr (lrrsd), a wavi- ness factor (wf) value > . , or < snps. regions that are known to be highly unreliable for cnv calls, such as immunoglobulin regions and the centromeres/telomeres of chromosomes were excluded from the analysis (see additional file : table s ). samples that had a total cnv number greater than sd from the mean, or samples that showed evidence of aneuploidy, were also excluded. after quality control we retained a set of , cnvs in individuals from the amish sample, and , in , individuals from the areds sample. inherited cnv regions from all available amish samples with genotype data, we selected individuals that belong to a nuclear family (parent plus children, parents and children) to as- certain regions containing inherited cnvs. this method consists of two stages. first, we establish the cnv region boundaries from the cnv that has the greatest overlap with other cnvs in the same genomic region. all cnvs that overlap % with this cnv are considered part of that cnv region. second, we trace the inheritance of a cnv region using the pedigree information. for a cnv region to be inherited, it must be present in both the child and at least one parent. human disease catalog the human genome mutation database (hgmd) cata- logs known disease associated variants (http://www.hgmd. org/). most of the clinical phenotypes in the database are monogenic diseases. in its most recent release (june ) it contains , different variants in ~ , genes (“hgmd disease genes”) [ ]. we cataloged all cnv re- gions (detected by the analysis of dense genotypes for the amish family members) that partially or fully overlap hgmd disease genes (‘dm’ tag in hgmd). whole genome sequencing whole genome sequencing (wgs) for old order amish family members (including parent child trios) was performed by complete genomics inc. (cgi; mountain view, ca) using a sequence-by-ligation method [ ]. paired-end reads of length bp ( bp at each end) were mapped to the national center for bio- technology information (ncbi) human reference gen- ome (build . ) using a bayesian mapping pipeline [ ]. variant calls were performed by cgi using version . . . of their pipeline. false discovery rate estimates for snp calls of the cgi platform are . – . % [ ]. gene annotations were based on the ncbi build . seq_gene file contained in a ncbi annotation build. the variant calls within the wgs were processed using the cgatools software (version . . , build ) made available by cgi. the listvar tool was used to generate a master list of the . m variants present in the amish samples. the testvar tool was used to determine presence and absence of each variant within the amish wgs. only variants with high variant call scores (“vqhigh” tag in the data files) were included. for further qc measures see [ ]. http://www.hgmd.org/ http://www.hgmd.org/ kember et al. bmc genetics ( ) : page of as described in georgi et al. [ ], we performed phas- ing and imputation of variants identified by wgs into the omni . m snp genotypes using the genotype im- putation given inheritance (gigi) software version . . gigi performs imputation of dense genotypes in large pedigrees based on a sparse panel of framework markers using a markov chain monte carlo approach. overall performance of our imputation is comparable to the published report [ ]. for a threshold on the genotype imputation posterior probability of . , we observed overall concordance of ~ . with a call rate of ~ . as expected, imputation performance increases for sub-pedigrees with a higher number of samples with wgs, i.e. when considering only nuclear families with wgs samples the performance improves to concord- ance ~ . and call rate ~ . [ ]. additional file additional file : figures and tables illustrating quality control measures, and supplementary results including additional cnv regions found in disease genes and on chromosome x. competing interests the authors declare that they have no competing interests. authors’ contributions rk carried out the cnv analysis and drafted the manuscript. bg participated in the amish project and carried out the whole genome sequence analysis. jebw contributed the areds data and gave advice for the analysis. ds contributed the areds data and gave advice for the analysis. smp and mb participated in the design of the study. mb also conceived the study and helped to draft the manuscript. all authors read and approved the final manuscript. acknowledgements this study was supported by the nih grant r mh . genotyping of areds data was provided through cidr, which is fully funded through a federal contract from the national institutes of health to the johns hopkins university, contract number hhsn c. ds is supported by ro ey . jebw is supported by the intramural research program of the national human genome research institute, national institutes of health. the authors would like to acknowledge xiao ji, philip ginsbach, dusanka lalic and emma greger for help with quality control of the amish data. in addition, they would like to thank erik puffenberger and laura conlin for their discussion, and ingrid lindquist for her contribution to the amish project. the authors are especially indebted to the members of the old order amish settlements who participated in the amish study of major affective disorder and dr. egeland who designed and directed this study since . author details department of genetics, university of pennsylvania, philadelphia, pa, usa. department of ophthalmology, university of pennsylvania, philadelphia, pa, usa. department of psychiatry, perelman school of medicine, university of pennsylvania, philadelphia, pa, usa. appel alzheimer’s disease research 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figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www.biomedcentral.com/submit 	abstract 	background 	results 	conclusions 	background 	results 	overall strategy 	a catalog of inherited cnvs in an amish pedigree segregating bipolar disorder 	cnvs and disease association 	discussion 	conclusions 	methods 	sample 	control subjects 	genotyping 	identification of copy number variants (cnvs) 	inherited cnv regions 	human disease catalog 	whole genome sequencing 	additional file 	competing interests 	authors’ contributions 	acknowledgements 	author details 	references prevalence, control, and treatment of diabetes, hypertension, and high cholesterol in the amish bmj open diab res care ; :e . doi: . /bmjdrc- - open access open access prevalence, control, and treatment of diabetes, hypertension, and high cholesterol in the amish shisi he, kathleen a ryan, elizabeth a streeten, patrick f mcardle, melanie daue, donna trubiano, yvonne rohrer, patrick donnelly, maryann drolet, sylvia newcomer, susan shaub, nancy weitzel, alan r shuldiner, toni i pollin, braxton d mitchell department of medicine, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland, usa correspondence to dr braxton d mitchell; bmitchel@ som. umaryland. edu to cite: he s, ryan ka, streeten ea, et al. prevalence, control, and treatment of diabetes, hypertension, and high cholesterol in the amish. bmj open diab res care ; :e . doi: . / bmjdrc- - received october revised july accepted july original research epidemiology/health services research © author(s) (or their employer(s)) . re- use permitted under cc by- nc. no commercial re- use. see rights and permissions. published by bmj. abstract background the burden of diabetes and cardiovascular risk is not uniform across the usa, with much of this disparity tracking differences in socioeconomic status, cultural practices and lifestyle. to further evaluate disparities in these disorders, we assessed the prevalence of diabetes, hypertension, and hypercholesterolemia in an old order amish community that is characterized by distinctive sociocultural practices that include a very cohesive social structure and limited use of modern technologies and medications. we compared prevalence of these conditions with that of the overall us population. method we performed a community- wide survey in amish individuals aged years and older from the lancaster county, pennsylvania, amish settlement that included a basic physical examination and fasting blood draw during the period – . we then compared the prevalence of diabetes, hypertension, and high cholesterol, defined using standard criteria, between the amish and the european caucasian subsample of the – us national health and nutrition examination survey (nhanes). results prevalence rates for diabetes, hypertension and hypercholesterolemia were . %, . %, and . % in the amish compared with . %, . % and . % in nhanes (p< . for all). among individuals with these disorders, amish were less likely to be aware that they were affected, and among those aware, were less likely to be treated with a medication for their disorder. conclusion there is substantially lower prevalence of diabetes, hypertension and hypercholesterolemia in the amish compared with non- amish caucasians in the usa. possible factors contributing to this disparity include higher physical activity levels in the amish or other protective sociocultural factors, a greater understanding of which could inform risk reduction interventions for these chronic diseases. introduction heart disease is the leading cause of death in the usa, and by the year , over million adults in the us population are projected to have some form of cardiovascular disease (cvd) with total costs projected to reach $ . trillion. among the major risk factors for cvd are hypertension, hypercholesterol- emia, and diabetes. in , it was estimated that hypertension alone was responsible for % of all cardiovascular deaths. deaths due to cvd in the usa increased steadily in the th century until about , after which point cvd- attributable deaths have slowly decreased. alarmingly, however, this rate has leveled off beginning in about . of particular concern is that the prevalence of the major cvd risk factors continues to be high. in fact, the prevalence of hypertension among us adults increased between and to a rate of . %, although this rate has not changed through . diabetes significance of this study what is already known about this subject? ► differences in diabetes and cardiovascular risk exist across many populations, and these differences are often used to speculate about the relative impor- tance of lifestyle and other factors in diabetes and heart disease risk. what are the new findings? ► compared with the overall non- hispanic white population in the usa, the amish community from lancaster, pennsylvania, experiences significantly lower prevalence rates of diabetes, hypertension, and high cholesterol. ► these associations persist even after adjustment for body mass index. ► amish are less lilkely to be aware of having these conditions and, among those aware, are less likely to be treated with a medication. how might these results change the focus of research or clinical practice? ► possible factors contributing to this disparity include higher physical activity levels in the amish or other protective sociocultural factors. a greater under- standing of these may inform risk reduction inter- ventions for these chronic diseases. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m http://drc.bmj.com/ http://orcid.org/ - - - http://crossmark.crossref.org/dialog/?doi= . /bmjdrc- - &domain=pdf&date_stamp= - - http://drc.bmj.com/ bmj open diab res care ; :e . doi: . /bmjdrc- - epidemiology/health services research prevalence among us adults has increased steadily from to , at which point the population prevalence was estimated at . %. in contrast, the proportion of individuals with high total and low- density lipoprotein (ldl) cholesterol levels have decreased over the past years, although this decrease has been accompanied by an increase in use of cholesterol- lowering medications. – the burden of cvd and its risk factors is not uniform across the country, and comparisons across subpopula- tions can be informative in highlighting health dispar- ities and providing insights into disease risk. one such subpopulation is the old order amish (ooa), a rural population isolate with distinctive sociocultural practices that includes a very cohesive social structure and limited use of modern technologies. the oldest amish settlement in the usa is in lancaster county, pennsylvania, which is currently home to ~ amish. through community surveys of cvd and its risk factors, we have estimated the prevalence of the major cvd risk factors. in this paper, we report the prevalence and control of diabetes, hyper- tension, and high cholesterol in the amish, contrasting these estimates with those obtained from the us national health and nutrition examination survey (nhanes). research design and methods the lancaster county amish the ooa of lancaster county, pennsylvania, are a popu- lation isolate. the ooa first immigrated to america from central europe, primarily present- day switzerland, in the early s. the lancaster area community remains the largest of the amish settlements in the usa, with an estimated size of ~ , virtually all descendants of the original founders. the present report is based on subjects participating in the amish wellness study, a community survey carried out to assess cardiovascular and metabolic health, disseminate this information to study participants, and evaluate associations of genetic and other risk factors with common diseases and related traits. these analyses are limited to adult partici- pants of the amish wellness study enrolled between its initiation in january and february . recruitment the amish community in lancaster county is divided into church districts, each consisting of up to house- holds. enrollment into the wellness study was structured around church districts, and data for this report are compiled from subjects in the initial (of a total of ) church districts. we targeted these initial church districts because of their proximity to our amish research clinic in lancaster. for each selected church district, our recruitment teams (consisting of a research nurse and amish liaison) first visited each household and invited the heads of the household to participate. if the head of household agreed, then he or she in turn invited all age- eligible (≥ years) persons in their household to partic- ipate. at this visit, the team obtained informed consent for a clinical exam, administered medical history ques- tionnaires, and scheduled subjects for an appointment. the study examinations were conducted in the amish research clinic or the amish wellness mobile, a refur- bished recreation vehicle that includes walk- in space for participant interviews, anthropometry and blood pressure measurements, a blood drawing station, and a centrifuge for blood processing. blood pressure was measured in triplicate on a sphygmomanometer, and blood pressure was defined as the average of all three readings. fasting bloods were drawn and sent to quest diagnostics (horsham, pennsylvania) for measurement of lipids and glucose. glycosylated hemoglobin (hba c) was not added to the amish protocol until november and so diabetes was assessed in only ( . %) of the amish subjects with hba c, fasting glucose, and medication data. disease definitions disease definitions (diabetes, hypertension, and hyper- cholesterolemia) were harmonized for these analyses to match those used in the nhanes – cycle. subjects had diabetes if they were currently taking blood glucose- lowering medications (and had previously been told by a doctor that they had diabetes) or had a fasting blood glucose ≥ mg/dl or an hba c ≥ . %. all other subjects not meeting these criteria were considered not to have diabetes. subjects were considered to have hypertension if they were currently taking blood pressure- lowering medi- cations (and had previously been told by a doctor that they had high blood pressure) or had a systolic blood pressure (sbp) ≥ mm hg or a diastolic blood pres- sure (dbp) ≥ mm hg. blood pressures were measured in . % ( , / , ) of amish subjects and in . % ( / ) of nhanes subjects. blood pressures were calculated by taking the mean of up to three blood pressure readings. of the subjects with blood pressure data, all amish and . % of nhanes had more than two blood pressure readings. subjects reporting that they were currently taking cholesterol- lowering medica- tions or had total cholesterol levels ≥ mg/dl or ldl cholesterol levels ≥ mg/dl were considered to have high total cholesterol or high ldl cholesterol, respec- tively. forty- nine amish participants reported a negative history of hypertension, but a review of their current medications indicated that they were currently taking a blood pressure- lowering medication. these subjects were therefore considered to have hypertension. similarly, amish subjects reported a negative history of high choles- terol, but a review of their current medications indicated that they were currently taking a cholesterol- lowering medication. these subjects were considered to have high cholesterol. we were unable to review the current medi- cations of nhanes subjects, and therefore all nhanes subjects reporting a negative history of hypertension or high cholesterol were considered to be unaffected o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m http://drc.bmj.com/ bmj open diab res care ; :e . doi: . /bmjdrc- - epidemiology/health services research providing that they met the blood pressure or cholesterol criteria. we considered persons with diagnosed diabetes, hyper- tension, or high cholesterol to be ‘aware’ of their disease if they reported that they had previously been told by a doctor that they had diabetes/hypertension or that they had high levels of fat or cholesterol or triglyceride in their blood. we considered persons with diagnosed disease to be treated if they were ‘aware’ of their disease and also reported that they were currently taking a medication for their disease. european caucasians we compared diabetes, hypertension, and hypercholes- terolemia prevalence in the amish with that in non- amish participants from the – cycle of nhanes. nhanes data are downloaded from https://www. cdc. gov/ nchs/ nhanes. we used only european caucasians from nhanes (n= ) to match the european ancestry of the amish. statistical analysis we compared the frequencies of diabetes, hypertension, and hypercholesterolemia between amish and nhanes stratified by age group using poisson regression to calcu- late the prevalence rate ratio (prr) using the sas v. . software. for total cholesterol, we compared amish and nhanes both in all subjects and after excluding . % of amish who are carriers of the known apob r q mutation that is common in the amish and is a known cause of familial hypercholesterolemia. among subjects meeting diagnostic criteria for diabetes, hypertension, or hypercholesterolemia, we compared by χ test the propor- tion of subjects responding that they had previously been told by a doctor of their disease (ie, were ‘aware’ of their disease), and we compared the proportions of amish and nhanes subjects currently taking medications for their disease (ie, ‘treated’) among those ‘aware’ of their disease. we did not use nhanes sampling weights in these analyses because we used caucasians from nhanes only and stratified all analyses by age and sex. results this report is based on subjects recruited into the amish wellness study between and . the mean household response rate across church districts was . %; that is, ~ / of households in each church district had at least one participating family member (range: %– %). the mean age of study participants was . years (range – ), and mean body mass index was . kg/m (range: . – . ). the study participants were . % male (n= ). the overall prevalence rates for diabetes, hypertension, and hypercholesterolemia were . %, . %, and . %, respectively (table ). for comparison, mean age in the nhanes population was . years ( – ), . % of subjects were male (n= ), and mean bmi was . ( . – . ) kg/m . the overall prevalence rates in nhanes for diabetes, hypertension, and hypercholesterolemia were . %, . %, and . %, respectively (table ). in general, the sex differ- ences in the prevalence of diabetes, hypertension, and high cholesterol observed in the amish mirrored those seen in nhanes. after adjustment for age and sex, the prevalence rates for diabetes, hypertension, and hyper- cholesterolemia were significantly higher in nhanes compared with amish, and these differences persisted with further adjustment for body mass index (table ). the prevalence of diabetes in the amish increased from . % ( % ci . % to . %) to . % ( % ci . % to . %) to . % ( % ci . % to . %) across the three age groups ( – , – , and + years) (figure and table ). the corresponding prevalence rates in nhanes were . %, . %, and . %. the age- adjusted and sex- adjusted prevalence of diabetes was . % and . % in the amish and nhanes, respectively (prr: . ( % ci . to . ), p< . ). among subjects with diabetes, amish (representing . % of all amish subjects with diabetes) and nhanes (representing . % of all nhanes subjects with diabetes) were currently taking insulin and reported an age of diabetes onset before the age of years. thus, a larger proportion of amish subjects with diabetes may potentially have type dabetes mellitus (t dm) compared with nhanes subjects with diabetes. we have previously published that amish individuals with age of diabetes diagnosis ≤ years, but not those with age of diabetes diagnosis > years, are significantly more likely to be glutamic acid decarbox- ylase (gad) antibody positive than amish individuals without diabetes. however, the overall prevalence of diabetes is much lower in amish than nhanes, and in terms of population prevalence, amish were less likely to have potential t dm ( / , or . %) compared with nhanes ( / , or . %). because hba c was not measured in the first years of the wellness study, we assessed the comparability of the wellness participants with hba c values (from november to january ) with the well- ness participants without hba c values (january – october ). the proportion of subjects who were currently using glucose- lowering medications was virtu- ally identical between the two groups ( . % vs . %, p= . ). similarly, the frequency of diabetes based on medication use and fasting glucose criteria only was also similar between the two groups ( . % vs . %, p= . ). the prevalence of hypertension in the amish increased from . % ( % ci . % to . %) to . % ( % ci . % to . %) to . % ( % ci . % to . %) in individuals across the three age groups (figure and table ). the corresponding prevalence rates in nhanes were . %, . %, and . %. the age- sex- adjusted prevalence of hypertension was . % and . % in the amish and nhanes, respectively (prr: . ( % ci . to . ), p< . ). the prevalence of high total and ldl cholesterol was higher in the amish than in nhanes in the – years age group ( . % vs . % for total cholesterol and o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m https://www.cdc.gov/nchs/nhanes. https://www.cdc.gov/nchs/nhanes. http://drc.bmj.com/ bmj open diab res care ; :e . doi: . /bmjdrc- - epidemiology/health services research ta b le c lin ic a l c h a ra c te ri st ic s o f a m is h a n d n h a n e s s u b je c ts ( m e a n ± s d o r % a ff e c te d ) a m is h – n h a n e s – a m is h v e rs u s n h a n e s * n = m e n , n = w o m e n , n = a g e - a d ju s te d p v a lu e † n = m e n , n = w o m e n , n = a g e - a d ju s te d p va lu e † a g e - a d ju s te d a n d s e x - a d ju s te d p va lu e *‡ a g e ( ye a rs ) . ( . ) . ( . ) . ( . ) < . . ( . ) . ( . ) . ( . ) . < . b o d y m a ss in d e x (k g /m ) . ( . ) . ( . ) . ( . ) < . . ( . ) . ( . ) . ( . ) . < . f a st in g g lu c o se ( m g /d l ) . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . < . h b a c ( % ) . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . < . s ys to lic b lo o d p re ss u re (m m h g ) . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) < . < . d ia st o lic b lo o d p re ss u re (m m h g ) . ( . ) . ( . ) . ( . ) < . . ( . ) . ( . ) . ( . ) . < . to ta l c h o le st e ro l ( m g /d l ) . ( . ) . ( . ) . ( . ) < . . ( . ) . ( . ) . ( . ) < . < . to ta l c h o le st e ro l ( m g /d l ) w it h o u t a p o b . ( . ) . ( . ) . ( . ) < . a s a b o ve a s a b o ve a s a b o ve a s a b o ve < . l d l c h o le st e ro l ( m g /d l ) . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . < . l d l c h o le st e ro l ( m g /d l ) w it h o u t a p o b . ( . ) . ( . ) . ( . ) < . a s a b o ve a s a b o ve a s a b o ve a s a b o ve < . % d ia b e te s (a d j % § , a d j % ¶ ) . ( . , . ) . ( . , . ) . ( . , . ) . . ( . , . ) . ( . , . ) . ( . , . ) . < . % h yp e rt e n si o n ( a d j % § , a d j % ¶ ) . ( . , . ) . ( . , . ) . ( . , . ) < . . ( . , . ) . ( . , . ) . ( . , . ) < . < . % h ig h t o ta l c h o le st e ro l ( a d j % § , a d j % ¶ ) . ( . , . ) . ( . , . ) . ( . , . ) < . . ( . , . ) . ( . , . ) . ( . , . ) . . % h ig h l d l c h o le st e ro l ( a d j % § , a d j % ¶ ) . ( . , . ) . ( . , . ) . ( . , . ) . . ( . , . ) . ( . , . ) . ( . , . ) . . in t h e a m is h , d ia b e te s b a se d o n s u b je c ts . h yp e rt e n si o n , h ig h l d l c h o le st e ro l a n d h ig h c h o le st e ro l b a se d o n , , a n d s u b je c ts , re sp e c ti ve ly ( se e t e xt ). in n h a n e s , d ia b e te s b a se d o n s u b je c ts . h yp e rt e n si o n , h ig h l d l c h o le st e ro l, a n d h ig h t o ta l c h o le st e ro l b a se d o n , , a n d s u b je c ts , re sp e c ti ve ly ( se e t e xt ). *p v a lu e c a lc u la te d b y lo g is ti c r e g re ss io n f o r c a te g o ri c a l o u tc o m e o r lin e a r re g re ss io n f o r c o n ti n u o u s o u tc o m e , a d ju st in g f o r a g e a n d s e x. p v a lu e f o r a g e , a d ju st e d f o r se x o n ly . b o ld v a lu e s d e n o te s ta ti st ic a l s ig n ifi c a n c e a t le ve l . . † p v a lu e f o r se x d iff e re n c e c a lc u la te d b y lo g is ti c r e g re ss io n f o r b in a ry o u tc o m e o r lin e a r re g re ss io n f o r c o n ti n u o u s o u tc o m e , a d ju st in g f o r a g e . p v a lu e f o r a g e , a d ju st e d f o r se x o n ly . b o ld v a lu e s d e n o te s ta ti st ic a l s ig n ifi c a n c e a t le ve l . . ‡ p v a lu e s fo r d iff e re n c e s b e tw e e n a m is h a n d n h a n e s w e re u n c h a n g e d f ro m t h e v a lu e s p ro vi d e d w h e n a d d it io n a lly a d ju st e d f o r b o d y m a ss in d e x, w it h t h e e xc e p ti o n o f % h ig h t o ta l c h o le st e ro l ( a g e - a n d s e x- a d ju st e d p = . c h a n g e d t o . w it h a d d it io n a l a d ju st m e n t fo r b m i) a n d % h ig h l d l c h o le st e ro l ( a g e - a d ju st e d a n d s e x- a d ju st e d p = . c h a n g e d t o . w it h a d d it io n a l a d ju st m e n t fo r b m i). § p re va le n c e a d ju st e d f o r a g e a n d s e x u si n g p o is so n r e g re ss io n . ¶ p re va le n c e a d ju st e d f o r a g e , se x, a n d b m i u si n g p o is so n r e g re ss io n . a p o b , a p o lip o p ro te in b g e n e ; b m i, b o d y m a ss in d e x; h b a c , g ly c o sy la te d h e m o g lo b in ; l d l , lo w - d e n si ty li p o p ro te in ; n h a n e s , n a ti o n a l h e a lt h a n d n u tr it io n e xa m in a ti o n s u rv e y. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m http://drc.bmj.com/ bmj open diab res care ; :e . doi: . /bmjdrc- - epidemiology/health services research . % vs . % for ldl cholesterol), although lower in the amish in the two older age categories ( . % vs . % and . % vs . % for total and ldl cholesterol, respectively, at ages – years, and . % vs . % and . % vs . % for total and ldl cholesterol, respec- tively, at ages + years) (figure and table ). overall, the age- adjusted and sex- adjusted prevalence of high total cholesterol was . % and . % in the amish and nhanes, respectively (prr: . ( % ci . to . ), p< . ) and the age- adjusted and sex- adjusted preva- lence of high ldl was . % and . % in the amish and nhanes, respectively (prr: . ( % ci . to . ), p= . ). after excluding amish subjects with the apob r q mutation, the prevalence of high total and ldl cholesterol was lower in the amish at all ages and the prevalence of high total cholesterol was % lower in the amish than in nhanes (prr: . ( % ci . to . ), p< . ) and the prevalence of high ldl choles- terol was % lower (prr: . ( % ci . to . ), p< . ). since the amish are significantly less likely to take physician- prescribed medications than nhanes and thus less likely to be diagnosed on the basis of medication use alone, we considered the possibility that the lower frequency of hypertension and hypercholesterolemia observed in the amish might be an artifact of the diag- nostic criteria for these conditions being less sensitive in the amish. if this were true, then one might expect to see that among those without a diagnosis of hypertension (or high cholesterol), mean levels of blood pressure (or cholesterol) would be higher in amish than in nhanes, although below the diagnosis criteria for disease diag- nosis. to evaluate this possibility, we therefore compared mean blood pressures and cholesterol levels between amish and nhanes among individuals not diagnosed with hypertension or high cholesterol. among individuals without hypertension, mean sbp was . mm hg lower in amish than in nhanes ( . ± . vs . ± . mm hg, age- adjusted and sex- adjusted p< . ), while mean dbp was slightly higher in amish ( . ± . vs . ± . , p= . ). similarly, among individuals without diabetes, mean fasting glucose was . mg/dl lower in amish than in nhanes ( . ± . vs . ± . mg/dl, age- adjusted and sex- adjusted p< . ), although mean hba c was slightly higher in amish ( . ± . vs . %± . %, p< . ). in contrast, among individuals without a diagnosis of high total cholesterol, mean total choles- terol was . mg/dl higher in amish than in nhanes ( . ± . vs . ± . mg/dl, p< . ). similarly, amish had mean ldl cholesterols that were . mg/dl higher ( . ± . vs . ± . mg/dl, p< . ). control and treatment among individuals meeting diagnostic criteria for diabetes, hypertension, or hypercholesterolemia, amish are less likely to be aware of their disease compared with nhanes (figure ). for example, . % of amish with hypertension are aware of their disease compared with . % of nhanes (p< . ) and . % of amish with high total blood cholesterol are aware of their disease compared with . % of nhanes (p< . ). differen- tial awareness of diabetes was much smaller, with . % of amish with diabetes aware of their disease compared with . % of nhanes (p= . ). among those ‘aware’ of their disease, amish were also less likely to report that they were currently taking medi- cations to treat their disease compared with nhanes. for example, . % of amish aware of their diabetes reported taking glucose- lowering medications compared with . % of nhanes (p= . ). similarly, . % of figure prevalence of diabetes and hypertension in amish versus nhanes by age group. see text for diagnostic criteria. nhanes, national health and nutrition examination survey. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m http://drc.bmj.com/ bmj open diab res care ; :e . doi: . /bmjdrc- - epidemiology/health services research amish aware of their hypertension reported taking blood pressure- lowering medications compared with . % of nhanes (p< . ), and . % of amish aware of their hypercholesterolemia reported taking cholesterol- lowering medications compared with . % of nhanes (p< . ). among subjects diagnosed with diabetes the propor- tion of amish and nhanes achieving treatment targets for hba c (< %) were comparable ( . % vs . %; age- adjusted and sex- adjusted prr= . ; % ci . to . ; p= . ). in contrast, only . % of amish with hypertension achieved treatment targets for blood pressure control (target of sbp < mm hg or dbp < mm hg) compared with . % for nhanes (age- adjusted and sex- adjusted prr= . ; % ci . to . ; p< . ) and only . % of amish with high total cholesterol achieved the < mg/dl treatment target for cholesterol compared with . % for nhanes (age- adjusted and sex- adjusted prr= . ; % ci . to . ; p< . ). the proportion of subjects with high ldl achieving the ldl treatment target of < mg/dl was . % in amish compared with . % in nhanes (age- adjusted and sex- adjusted prr= . ; % ci . to . ; p< . ). discussion we have shown that amish have a lower prevalence of diabetes, hypertension, and hypercholesterolemia compared with non- amish caucasians. the magnitudes of the differences in disease rates between the two popu- lations are striking, that is, a % lower prevalence of diabetes, % lower prevalence of hypertension, and a % lower prevalence of high total cholesterol. there is no evidence that the decreased prevalence of hyper- tension in the amish is an artifact of nhanes with subthreshold levels of blood pressure being more likely than amish to be treated with blood pressure lowering medications since sbp among non- hypertensive indi- viduals is on average higher, not lower, in nhanes. however, among individuals not meeting diagnostic criteria for high cholesterol, cholesterol levels are on average slightly higher (by ~ mg/dl) in amish than in nhanes. we cannot therefore rule out the possibility that some of the difference in hypercholesterolemia prevalence rates might be due to non- amish being more likely than amish to have cholesterol levels in the high normal range and therefore more likely to be prescribed cholesterol- lowering medications. despite the lower frequency of diabetes, hypertension, and hypercholesterolemia in the amish, these disorders still constitute significant public health problems in this community because of their associations with adverse events. this is especially true since amish individuals with these disorders are less likely than nhanes to be aware that they are affected and less likely to be treated. amish diagnosed with hypertension and high cholesterol are also less likely than nhanes to meet target treatment t a b le p re va le n c e o f d ia b e te s, h yp e rt e n si o n , a n d h ig h c h o le st e ro l i n a m is h a n d n h a n e s b y a g e g ro u p a m is h n h a n e s p re va le n c e r a te r a ti o ( % c i) a g e a n d s e x a d ju s te d a g e , s e x , a n d b m i a d ju s te d – – ≥ – – ≥ to ta l n o . (d ia b e te s/ h yp e rt e n si o n / to ta l c h o le st e ro l/ l d l c h o le st e ro l) / / / / / / / / / / / / / / / / / / d ia b e te s p re va le n c e ( % c i) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . )* ** . ( . t o . )* ** h yp e rt e n si o n p re va le n c e ( % c i) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . )* ** . ( . t o . )* ** h ig h t o ta l c h o le st e ro l p re va le n c e ( % c i) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . )* ** . ( . t o . )* * h ig h t o ta l c h o le st e ro l p re va le n c e w it h o u t a p o b ( % c i) . ( . t o . ) . ( . t o . ) . ( . t o . ) a s a b o ve a s a b o ve a s a b o ve . ( . t o . )* ** . ( . t o . )* ** h ig h l d l p re va le n c e ( % c i) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . ) . ( . t o . )* * . ( . t o . ) * h ig h l d l p re va le n c e w it h o u t a p o b ( % c i) . ( . t o . ) . ( . t o . ) . ( . t o . ) a s a b o ve a s a b o ve a s a b o ve . ( . t o . )* ** . ( . t o . )* ** ** *p < . ; ** p < . ; *p < . . a p o b , a p o lip o p ro te in b g e n e ; b m i, b o d y m a ss in d e x; l d l , lo w - d e n si ty li p o p ro te in ; n h a n e s , n a ti o n a l h e a lt h a n d n u tr it io n e xa m in a ti o n s u rv e y. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m http://drc.bmj.com/ bmj open diab res care ; :e . doi: . /bmjdrc- - epidemiology/health services research guidelines. these results might be expected given that amish do not participate in government- sponsored medical insurance programs and are less likely to receive medical care compared with non- amish caucasians in the usa. the lower prevalence of hypercholesterolemia in the amish occurs despite the fact that % of amish carry at least one copy of a familial hypercholesterolemia- associated allele at the apob locus that is associated with a ~ mg/dl increase in ldl cholesterol levels. more- over, if individuals with the apob r q mutation, a highly drifted variant associated with familial hyper- cholesterolemia carried by % of the amish popula- tion, are excluded, amish have a % lower prevalence of high total cholesterol. the amish are also enriched for at least one other known genetic variant (near the pseudogene, apoop ) that is also associated with high ldl cholesterol levels, with % of amish carrying at least one copy of this variant and each copy associated with ~ mg/dl increase in ldl cholesterol. because of these mutations, the prevalence of high cholesterol is actually higher in younger amish than in younger non- amish, but as other (non- genetic) causes of high choles- terol become more prominent with age, the prevalence of high cholesterol becomes higher in the non- amish beginning in middle age. it seems likely that there are distinctive features about the amish community or amish lifestyle that contribute to their relative protection against diabetes, hypertension, and high cholesterol. two features in particular stand figure prevalence of high total cholesterol and high ldl cholesterol in amish and nhanes by age group. see text for diagnostic criteria. apob, apolipoprotein b gene; ldl, low- density lipoprotein; nhanes, national health and nutrition examination survey. figure proportion of individuals with diabetes, hypertension, and high total cholesterol disease who are aware of their diagnosis, and among those aware, the proportion who are treated. nhanes, national health and nutrition examination survey. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m http://drc.bmj.com/ bmj open diab res care ; :e . doi: . /bmjdrc- - epidemiology/health services research out. first, the amish tend to be far more physically active than their non- amish counterparts, as we and others have previously documented. the protective effects of physical activity on cardiometabolic health are well docu- mented, including in the amish, and the more active amish lifestyle may contribute to less diabetes, hyperten- sion, and high cholesterol in this population. second, the structure of amish culture is very different from the non- amish, and some aspects of this culture may confer addi- tional benefits to cardiometabolic health. for example, amish culture is very socially cohesive and at its core lies a strong ethos of community and family support. one can imagine that such a culture could be beneficial for dealing with stress. it is also possible that components of the amish diet confer protection against these common cardiovascular risk factors. in sum, the relation of amish culture to health is undoubtedly complex, and this is a topic in need of future study. an intriguing observation from our study is that amish have substantially lower fasting glucose levels compared with nhanes despite slightly higher levels of hba c. we have no clear explanation for this, and it is unlikely to be due to assay issues because glucose and hba c were measured in a central standardized lab (quest). possibly, glycemic excursions are not well captured in the amish by a single fasting glucose. amish do have slightly longer fasting times compared with nhanes, although this alone accounts for very little of the discrepancy. amish, who tend to wake up early, may also be more physically active during the fasting period compared with non- amish potentially contributing further to lower fasting glucose levels. it is also possible that non- glycemic factors (eg, red cell turnover and overall physical activity levels) may be at play that contribute to low fasting glucose levels in the amish or to a mismatch of glucose and hba c levels. one limitation of our study is that it is based on cross- sectional data. the possibility must therefore be consid- ered that the incidence of one or more of these disorders could be similar between amish and nhanes but amish with these conditions might experience reduced survival, thus resulting in a lower prevalence. in fact, we have previously documented that amish in general have less access to medical care. although we are aware of no data specifically addressing case fatality from these condi- tions in the amish, we regard this possibility as unlikely since overall mortality among amish adults is very similar to that observed in the framingham heart study cohort, although this prior analysis was restricted to individuals born between and . our survey is based on a sample of the amish community, and the frequencies of these disorders in this sample may not represent those in the overall community. in summary, we have demonstrated that amish have a substantially lower prevalence of diabetes and hyperten- sion compared with nhanes and a moderately lower prevalence of high cholesterol. the reasons for these prevalence differences are not known although likely relate to distinctive features of the amish lifestyle and community that may be protective for cardiometabolic health, such as the relative high levels of physical activity and the cohesive and supportive structure of the amish community. acknowledgements the authors would like to acknowledge the substantial contributions of other staff members at the amish research clinic, including elizabeth zehr, tracy broderick, grace redcay, fred young, regina guaraldi, mary morrissey, theresa roomet, and mary mclane, as well as the many amish liaisons who have contributed to these studies (sarah fisher, sadie fisher, katie stoltzfus, mary esh, anna esh, naomi esh, susie fisher, hanna king, sylvia king, verna petersheim, barbie a stoltzfus, barbie b stoltzfus, fannie stoltzfus, mary stoltzfus, susie stoltzfus, lydia zook, and esther smucker). contributors conceived and drafted the paper: sh and bdm; analyzed and interpreted data and drafted the manuscript: sh and kar; data collection: med, dt, yr, pd, mad, sn, ss and nw; commented on the drafts of the paper: kar, eas, pfm, ars and tp; all authors have approved the final version for submission. funding this work was supported in part by national institutes of health grant p dk , the regeneron genetics center, and the university of maryland school of medicine program for personalized and genomic medicine. competing interests ars is an employee of regeneron pharmaceuticals, inc and receives compensation. patient consent for publication not required. ethics approval this study was approved by the institutional review board at the university of maryland, baltimore (university of maryland, baltimore irb hp- ). provenance and peer review not commissioned; externally peer reviewed. data availability statement data are available on reasonable request. data may be obtained from a third party and are not publicly available. some deidentified amish data are available on dbgap (dbgap study accession: phs .v .p ). further inquiries may be directed to the communicating author. national health and nutrition examination survey (nhanes) data are available through nhanes. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by- nc . ) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non- commercial. see: http:// creativecommons. org/ licenses/ by- nc/ . /. orcid id braxton d mitchell http:// orcid. org/ - - - references benjamin ej, virani ss, callaway cw, et al. heart disease and stroke statistics- update: a report from the american heart association. circulation ; :e – . danaei g, ding el, mozaffarian d, et al. the preventable causes of death in the united states: comparative risk assessment of dietary, lifestyle, and metabolic risk factors. plos med ; :e . egan bm, zhao y, axon rn. us trends in prevalence, awareness, treatment, and control of hypertension, - . jama ; : – . egan bm, li j, hutchison fn, et al. hypertension in the united states, to : progress toward healthy people goals. circulation ; : – . menke a, casagrande s, geiss l, et al. prevalence of and trends in diabetes among adults in the united states, - . jama ; : – . carroll md, kit bk, lacher da, et al. trends in lipids and lipoproteins in us adults, - . jama ; : – . carroll md, fryar cd, kit bk. total and high- density lipoprotein cholesterol in adults: united states, - . nchs data brief ; : – . gu q, paulose- ram r, burt vl, et al. prescription cholesterol- lowering medication use in adults aged and over: united states, - . nchs data brief : – . adabag as, luepker rv, roger vl, et al. sudden cardiac death: epidemiology and risk factors. nat rev cardiol ; : – . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m http://creativecommons.org/licenses/by-nc/ . / http://orcid.org/ - - - http://dx.doi.org/ . /cir. http://dx.doi.org/ . /journal.pmed. http://dx.doi.org/ . /jama. . http://dx.doi.org/ . /circulationaha. . http://dx.doi.org/ . /jama. . http://dx.doi.org/ . /jama. . http://www.ncbi.nlm.nih.gov/pubmed/http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /nrcardio. . http://drc.bmj.com/ bmj open diab res care ; :e . doi: . /bmjdrc- - epidemiology/health services research pollin ti, mcbride dj, agarwala r, et al. investigations of the y chromosome, male founder structure and ystr mutation rates in the old order amish. hum hered ; : – . shen h, damcott cm, rampersaud e, et al. familial defective apolipoprotein b- and increased low- density lipoprotein cholesterol and coronary artery calcification in the old order amish. arch intern med ; : – . hsueh wc, mitchell bd, aburomia r, et al. diabetes in the old order amish: characterization and heritability analysis of the amish family diabetes study. diabetes care ; : – . montasser me, o'hare ea, wang x, et al. an apoo pseudogene on chromosome q is associated with ldl- c levels. circulation ; : – . hairston kg, ducharme jl, treuth ms, et al. comparison of bmi and physical activity between old order amish children and non- amish children. diabetes care ; : – . bassett dr, schneider pl, huntington ge. physical activity in an old order amish community. med sci sports exerc ; : – . bassett dr, tremblay ms, esliger dw, et al. physical activity and body mass index of children in an old order amish community. med sci sports exerc ; : – . mitchell bd, kalra g, ryan ka, et al. increased usual physical activity is associated with a blunting of the triglyceride response to a high- fat meal. j clin lipidol ; : – . mitchell bd, lee w- j, tolea mi, et al. living the good life? mortality and hospital utilization patterns in the old order amish. plos one ; :e . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://d rc.b m j.co m / b m j o p e n d ia b r e s c a re : first p u b lish e d a s . /b m jd rc- - o n a u g u st . d o w n lo a d e d fro m http://dx.doi.org/ . / http://dx.doi.org/ . /archinternmed. . http://dx.doi.org/ . /diacare. . . http://dx.doi.org/ . /dc - http://dx.doi.org/ . / .mss. . . http://dx.doi.org/ . /mss. b e d aa http://dx.doi.org/ . /mss. b e d aa http://dx.doi.org/ . /j.jacl. . . http://dx.doi.org/ . /journal.pone. http://drc.bmj.com/ 	prevalence, control, and treatment of diabetes, hypertension, and high cholesterol in the amish 	abstract 	introduction 	research design and methods 	the lancaster county amish 	recruitment 	disease definitions 	european caucasians 	statistical analysis 	results 	control and treatment 	discussion 	references 	
   	
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   http://jrmdc.com “if you need a virtual community, something is wrong with your congregation”: institutionalized laestadianism and the use of digital media by stefan gelfgren umeå university, sweden contact: stefan.gelfgren@umu.se keywords: digital religion, digital humanities, laestadianism, church, mediatization. abstract: this article studies how the laestadian movement (a christian confessional revivalist movement that is sceptical of technology) uses digital media in general, and the internet in particular, in its work. in a time when churches on a large scale are concerned with how to communicate with people through digital media, the laestadian movement choses another path, based upon other assumptions and choices. the focus here is on how congregations and representatives use digital media, and not on individual and private use, and this article will focus primarily on sweden and finland. based on interviews with representatives and by mapping the congregations’ online presence, this article provides an interpretation of the use of the internet within laestadianism. through this group, we see how ideology, faith, and practices regulate a restricted, negotiated, and conscious use of the internet, which challenges any preconceptions regarding use and effect of the internet on religion. this case study therefore gives additional perspective for understanding the role of digital media within and in relation to institutionalized christianity. about the author: stefan gelfgren is associate professor of sociology of religion and director of humlab, umeå university, sweden. he has a phd in intellectual history from umeå university, and an mphil in “the history of christianity” from university of birmingham, uk. his work is centered around the relation between social and religious transformations throughout history. he is the co-editor of digital religion, social media, and culture: perspectives, practices downloaded from brill.com / / : : am via free access 	
   	
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   and futures (peter lang: new york, ), together with pauline cheong, charles ess & peter fisher-nielsen. to cite this article: gelfgren, s., . “if you need a virtual community, something is wrong with your congregation”: institutionalized laestadianism and the use of digital media. journal of religion, media and digital culture ( ), pp. - . “if you need a virtual community, something is wrong with your congregation”: institutionalized laestadianism and the use of digital media .   introduction this article will study how the laestadian movement, a christian confessional revivalist movement, uses digital media in general, and the internet in particular, in their work. the movement can, in broad terms, be described as conservative in terms of theology, family values, and morals, but also when it comes to use of digital media as means for communication. this article deals with how the official movement, its institutions, and its representatives use and think around the internet, focusing primarily on laestadianism in sweden (with some detours into finland). this article is not concerned with how individuals with a laestadian affiliation use the internet in their personal and private lives. the internet has permeated, influenced, and thus transformed large parts of our society and individual lives (cf. castells ; jenkins ; rainie & wellman ) – from being one component in the process of overthrowing established authorities in for example iran and tunis (cf. howard & hussain ; lindgren ), to transforming journalism (cf. hayes, singer, & ceppos ), changing education (cf. metzger & flanagin ), empowering marginalized groups (cf. lievrouw ), changing how we interact with people (cf. turkle ), and so on. we live our lives online to a higher degree than just a few years ago, in what is claimed to be a hybrid reality, where the virtual and the physical are mixed (cf. lindgren ). we make friends and sustain relationships online; people gather and spend recreational time, seek pleasure, and perform political actions online, and so on. people also live and practice parts of their religious life and faith online, and religious institutions are increasingly active online. one might think this is a transformation that can only be complied with, just to give in and follow a predestined path, without reflecting upon alternatives or other ways to deal with the inevitable process of the “internetalization” of society. the use of digital media within specific groups is, however, always accompanied by a negotiating process, and in the case of the laestadian movement, we will see how they tend to deliberately restrict the use of digital downloaded from brill.com / / : : am via free access 	
   	
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   media instead of adapting to the situation, and to emphasize how it does not comply with beliefs and practises within the movement. as howard rheingold, an early internet pundit and the founder of the concept of “virtual communities” ( ), points out, perhaps we might even “find better ways to wield technological power, other than simply unleashing it and seeing what happens? what can we learn from a culture that habitually negotiates the rules for new tools?” (rheingold ). a study on the use of digital media within the laestadian movement can give us other perspectives and attitudes toward digital media in a media-saturated world. .   laestadianism: an overview and introduction the confessional laestadian revivalist movement originates from the founder lars levi laestadius ( – ), born and active in the northern part of sweden (literature in english on the background and history of the laestadian movement is scarce, but see for example andreassen ; foltz and bergman ; hepokoski , ; lindmark ). today the movement has its stronghold in finland and in the northern part of sweden, but it is also found in central sweden and parts of norway. it has also been spread and established in the usa, first and foremost through finnish and swedish immigrants. it is found all around the world but in rather small groups. laestadius came from a family of clergymen and after studies in uppsala, in botany and theology, he was ordained in and came to work in the upper north of sweden for the rest of his life. as a clergyman and vicar in the sápmi area, close to the finnish border, laestadius grew up speaking finnish and sami. he gave sermons primarily in finnish, but also in swedish and samii. he is known for his powerful and colorful language in encouraging people to convert, and for converting sami people to christianity. laestadianism thereby occasionally empowered samis to protest against the church of sweden (but they never broke away), the state, and mainstream society, and also to improve their living conditions. as with other revivalist movements influenced by the pietistic and movarian movements, there is an emphasis on conversion to “true” christianity, the atoning death of christ, and reading the bible (bebbington, ). there is also a specific emphasis on the need to confess your sins to a fellow christian, have them forgiven, and try to live a modest and moral life apart from the mundane and sinful world (an aspect also relevant for the use of modern media and technology within the movement). like other revivalist movements of that time, laestadius circumvented the regulations of the conventicle act ( – ) through having lay-led gatherings where his distributed handwritten and, later, printed sermons were read out. readings of laestadius’ preaching, alongside lay preaching, has been strong within the movement since that time. the use of media for spreading the word goes, in other words, back for a long time within the movement. in the years after laestadius’ death, the movement split into three major “branches” (each with different subgroups) in sweden and finland, regardless of national borders: the firstborn laestadianism (västlaestadianism, with no institutionalized umbrella organization – with approx.. , members in the world); the little firstborn group downloaded from brill.com / / : : am via free access 	
   	
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   (gammallaestadianism, organized through laestadianernas fridsföreningars förbund – lff – with a few thousands members); and conservative laestadianism (Östlaestadianism, organized through sveriges fridsföreningars centralorganisation – sfc – about , adherents in sweden and , in finland, and all in all approximately , adherents in the world). the firstborn laestadians broke away from the conservative laestadian branch at the beginning of the th century, while the conservatives formed their own branch at approximately the same time. in sweden they are all still part of the church of sweden (the former state church). among these three groups, the firstborns are the most reluctant to engage with the life and habits associated with the profane world and thus try to live according to the original teachings of lars levi laestadius. when, for example, gerd snellman ( ) characterizes the laestadian movement of today, she emphasizes the community of believers and a shared identity. this is upheld through shared traditions and practices within the family and through meetings in the prayer chapel and within the movement as a whole at larger meetings. their accentuated distinction between true believers and non-believers is strong (similar to but probably stronger than within other revivalist movements). the central role of individual and shared confession is also important. the practice of repenting and forgiving builds trust, identity, and community among the members. reading, hearing, and talking about the word of god and living a humble life accordingly, together with praying, are considered key elements of a christian life for laestadians. the laestadian movement can be considered conservative when it comes to family values and theology and are still today formulated in opposition to secular society, and also to the (former state) church, which the movement still belongs to (see for example kejonen ; nordvik, ). context and perspectives within the field of “digital religion” (cf. campbell ; cheong et al. ), there has been a tendency to study the use of the internet in relation to different religious individuals, collectives, and institutions. less attention has been paid to groups who choose not to use, or are reluctant to use, the internet and other modern means of communication. relatively few studies have been made on religious environments in which the use of technology is contested, restricted, and under negotiation. in studies on, for example, the amish, the mennoites, and the ultra-orthodox jewish communities (see below), it is evident how these seemingly technologically hesitant groups have a moderate attitude towards modern technology rather than a strict anti-modern and forbidding attitude. they are “filtering out its negative aspects, embracing its positive features, and using it to impart religious knowledge”, as rashi puts it ( , p. ). livio and tenenboim weinblatt ( ) study how ultra-orthodox jewish women negotiate the use of the internet in their personal and professional lives in their households. in a study of the so-called “kosher cell phone”, campbell ( ) studies how the use of the cell phone is negotiated in conservative jewish communities. in that particular case, a negotiating process took place to make the use of cell phone technology possible by adding some crucial restrictions and filters (see also rashi ). a similar approach can, for example, be seen in kraybill’s ( ) study of amish and mennonites’ views on computers or umble’s ( ) downloaded from brill.com / / : : am via free access 	
   	
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   study of the negotiated use of the telephone in the same communities (also umble and weaver-zercher ). amish communities, for example, place a communal phone in a shed on the outskirts of the village, or hand their cell phones to their english neighbours for recharging overnight. we see similar thoughts in history. in european th century evangelical revivalism, comparable thoughts of concern flourished regarding the use of fiction and music. novels and what was called “true fiction” were transformed into a means for making the gospel easily accessible for common people instead of spreading the worldly values of contemporary fiction. a separate distributing system, distinct from the worldly literature, with their own colporteurs and bookstores, was established as well (cf. brown ; gelfgren ). these cases show how different (religious) groups test and evaluate each technology according to the effects they have on their life and values. only one study so far has focused on the laestadian movement and the use of the internet, although on a rather general level, with the aim to discuss and present an overview of laestadian official websites with focus on online content (andreassen ). olsen ( ) uses laestadian internet material in his study on norwegian laestadianism and gender, but similar online forums for discussions to those olsen mentions are not to be found in relation to this study on swedish and finnish material. andreassen notes the relevance and long tradition (dating back to lars levi laestadius’ days) of the written text and the monthly bulletins within the movement. even if the use of digital media differs between the different branches, the content of the websites reflects the use of the bulletins – invoking existing knowledge from a sender to a receiver with little interaction, according to andreassen. another study does touch upon the use of television in finland in relation to the laestadian movement, which totally banned the television until the s (alasuutari ). according to alasuutari, people within the laestadian movement are hesitant to watch television since it brings the worldly things into their homes and “distort[s] the picture we get of the world” ( , - ), and that is something to protect oneself from. interestingly, alasuutari compares the laestadian view on television with an overall critical view on television and mass culture among the upper middle class. interpretation in cases mentioned above, we can see how religious bodies and institutions negotiate, in a conscious way, to restrict the use of communication technologies. but how does one interpret the negotiation process when it comes to a rather restricted use of digital media and the internet within a christian movement? based on the work done by ferré ( ), campbell ( ) has developed a four-step religious-social shaping of technology approach for this, emphasizing that there are different ways to interpret the relationship between media and the change in religious faith and practices. according to campbell’s model, attitudes towards digital media within various religious branches and institutions can be interpreted and analysed in four consecutive and distinctive steps: ) “history and tradition”; ) “core beliefs and patterns”; ) “the negotiating process”; and finally ) “communal framing and discourse” ( , pp. - ). what is central, and an important contribution by campbell, is how background, beliefs, and internal context set the framework for use of the internet within a religious group, institution, or movement. campbell’s model thereby questions more downloaded from brill.com / / : : am via free access 	
   	
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   deterministic and mono-causal models and adds consciousness and awareness into the interpretation – thus giving the actors agency, which is important. this article will not use campbell’s model step by step, only as a starting point for emphasizing the necessity of taking internal factors within a movement into account when interpreting why and how digital media is used. in previous work i have also argued for including an external contextual frame for understanding the negotiating process since no religious institution lives in a societal vacuum (gelfgren ). hence, the negotiating process takes place within a structure of how one understands and relates to society. here i would like to refer to the mediatization theory promoted by hjarvard ( ). to start with, modern democratic society has, according to hjarvard, an autonomous media sphere with its own set of communicative rules. anyone with a message or opinion has to relate to this condition, and also go through it, in order to reach out to society and the people who are the foundation of society. this affects how media itself, politics, science, and religion communicate and are perceived and interpreted, thereby affecting the role of religion in society. hjarvard claims that the “mediatization of religion … is changing the representation of religion in late modernity at the same time that secularization … is evoking both a decline and a transformation of religious organizations, practices and beliefs” ( , p. ). in the case of the laestadian movement, we will see how important internal factors, such as history, tradition, and core beliefs, as well as external factors, such as how the surrounding world is understood, are for the use of the internet. when it comes to the use of media, there is a reflective awareness of what is considered advantageous or disadvantageous in relation to tradition and beliefs, which is a part of the negotiation process, not only internally but also in relation to the societal context. .   method and material this article is based upon available online resources published within the laestadian movement, which differ between the different branches (see below). interviews were done with a selection of representatives of the movement. the article focuses first and foremost on sweden and swedish conditions, but includes the fenno-swedish relations when there is added value in this (further explained below). regardless of branch, most congregations have a webpage on www.kyrktorget.se (”kyrktorget” can be translated approximately as “the church square”), which is a portal/noticeboard where all swedish churches and congregations can post announcements, short information, links to additional/external webpages, and contact information. in addition, some of the laestadian congregations have their own webpages, made in different ways, with different functionality. at an initial stage of this research, all webpages were traced and mapped, starting with the lists and contact information available on the wikipedia (swedish and english) entry “laestadianism”, where the different branches are listed and linked. from there i moved on to www.kyrktorget.se for a more complete list of the different congregations. downloaded from brill.com / / : : am via free access 	
   	
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   six interviews were also conducted, with the aim to speak to at least one (preferably two) representative from each branch. i promised them all anonymity and confidentiality, and they are treated accordingly. i assumed six informants would be enough to complement the online material, as well as feasible. through a colleague i found one informant, who then introduced me to another informant. another colleague introduced me to the third informant. the three others were found among the congregations that are most active online, in two different branches, and i contacted them via email. the six informants are: one preacher from the firstborn laestadian movement; one web master from the swedish little firstborn group; two members from the finnish equivalent (one web master and one preacher); and two members from the conservative laestadians (one secretary from the umbrella organization and one with another administrative role in a local congregation). all six labelled themselves it-skilled persons, through their work and personal interests, so their hesitant attitude cannot be said to be a result of ignorance or fear (which one might be tempted to assume).ii they were all happy to honestly discuss their use of the internet in their congregations and context. the interviews were semi-structured and centred around questions of how and why the internet is, or is not, used within the different movements. the interviewees are all male, and their ages range between approximately and . they have different professional backgrounds, varying between theology, business, and it administration, and they live in capital cities, mid-sized towns, and small villages. variables such as gender, class, or ethnicity are not studied and problematized in this article. the interviews were between and minutes long and were conducted and recorded via telephone or skype. each informant gave their consent and was informed about how the content of their interviews would be used. an application for ethical vetting was approved by the etikprövningsnämnden (approx. the swedish ethical vetting board). .   laestadianism online resources and presence: “get acquainted […] and find information about us” the three different branches have a similar approach to the internet, what to put online, and with which purpose, but there are some dissimilarities in extent and also content. the firstborn laestadian movement the firstborns is the most restrictive branch in this respect, and they only have a web presence on www.kyrktorget.se with (often, but not always) a calendar and contact information (telephone, email, postal address, and map). one representative from the movement, a clergyman, was interviewed. there is no umbrella organization for the different congregations. there are eleven congregations listed at ”kyrktorget”. none of them has its own webpage or any content outside ”kyrktorget”. two of the congregations have a picture of their church. in most cases there is also a link to a map showing where to find the prayer house or church and a calendar with the program for coming weeks. in some cases, it is said that some meetings take place in someone’s home. the webpage welcomes people to get in touch in order to be told when and where these meetings take place. downloaded from brill.com / / : : am via free access 	
   	
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   the ”kyrktorget” web usually says something like: “we hope you have the possibility to visit one of our services. if you have questions, please contact us” [my translation, from: “vi hoppas att du har möjlighet att besöka någon av våra gudstjänster. om du har några frågor så får du gärna kontakta oss”], indicating that meetings in the physical world are preferred over virtual conversations and encounters. little firstborn group the branch of the little firstborns group has more detailed information than the firstborns. it is the smallest branch in sweden in terms of members; however, it has congregations and is organized in close relation to the laestadianernas fridsföreningars förbund (lff, www.lff.fi) in finland. it stretches over the finnish-swedish border and unites the fenno- swedish and finnish-speaking communities. the little firstborns group has basic contact information and links to a map to the fourteen different congregations; it also has a calendar with meeting information and a bit more information about some of the congregations, such as background information and some bible verses. all webpages have a picture – either of the church/prayer house, nature, or the bible. four of them link to the local congregation of the church of sweden’s webpage. this indicates the confessional nature of this branch and a closer relationship to the church, compared to the firstborns, who have a more conflicting relationship to the church. six pages have a link to streamed radio from different meetings in the local congregations. three of them have links to other laestadian sites, primarily the finnish lff. only one swedish congregation within the little firstborn group, luleå fridsförsamling, have their own website (http://luleafridsforbund.com). there you can find basic information – contact address, map reference, and calendar. you also find streamed and archived services and links to the congregation’s counterparts in finland (lff) and the usa (the apostolic lutheran church). you can find out about the aim of the congregation. lff in finland, on the other hand, has an even more detailed web presence. one important difference is that lff offers a national umbrella organization under which the different congregations can be gathered. no similar organization exists in sweden. there is information about national and international work, contact details, a calendar, and links to their radio station and newspapers. some of the finnish congregations also show up at ”kyrktorget”. lff in finland has its own local radio station in the area around the city of jakobstad (a fenno-swedish area in finland), where lff is particularly active. the radio station transmits from the local congregations and produces weekly programs for worship. there is also a link to their own paper, “sions missionstidning”, which is published and distributed throughout the movement. the paper itself is not available through the web. there are also links to different youth camps and to a missionary blog run by lff’s youth, relating stories and experiences from missionary activities in finland and abroad. conservative laestadianism the conservative branch in sweden is organized under an overarching organization – sveriges fridsföreningars centralorganisation (sfc, http://sfcorg.se), with six congregations in northern and central sweden (and one in norway). this is also the most web-active of the downloaded from brill.com / / : : am via free access 	
   	
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   swedish branches. there are approximately adult members in sweden. the webpage says: “here you can get acquainted with us and find information about us and our associated congregations”. “everybody is welcome to our meetings. some meetings are streamed online”. the individual congregations are to be found on www.kyrktorget.se, and three (one in norway) of them also have individual webpages, organized under sfc’s web. the webpages have information about the organization, contact details, a calendar, links to ongoing and archived streamed services, and so on. apart from basic information about contacts, calendar, and places to meet, there is a short background history of sfc and the laestadian revivalist movement, as well as a short account of how the organization works with publications (such as cassettes, cds, and books). “[t]he need has grown over the years”, it says, and it features statements regarding the faith of the congregations and information about meetings and services. there are links to their counterparts in finland (srk – suomen rauhanyhdistysten keskusyhdistys) and the usa (llc – the laestadian lutheran church). there is an archive of pictures from different events and the ordinary life of the congregations. there is also a link to google play and the app store where one can download the hymnbook (sions sånger & psalmer). the different congregations are presented, and two of the swedish ones have their own webpages (dalarna fridsförening – www.dalarnasfridsforening.se and stockholm fridsförening – www.stockholmsfridsforening.se). dalarna’s webpage has information about their history, aim and activities, contact details, a map, a calendar, and links to sister organisations, google play and app store for the hymnbook, and streamed and archived services and meetings. stockholm fridsförening’s webpage is similar in content to dalarna’s. summary: the laestadian branches and digital media representation to sum up the content of the different online laestadian resources, even though they differ slightly between the branches, one can conclude that there is primarily information and glimpses of ongoing activities, and that communication is one-way. at the same time, web visitors are openly invited to come to the physical meetings. after a few case studies on different topics related to the use of digital media within christian organisations (gelfgren , , ), and with a good overall sense of what is going on in the religious online sphere (first and foremost christian), it is notable that there are no interactive social media, such as blogs, or facebook groups or twitter, youtube, or instagram accounts. apart from the link to the youth blog on lff’s page, there are no social media and consequently no interaction at all on the web resources hosted by the various laestadian branches. based on web presence, and what is known about the hesitant attitude towards modern technology within laestadianism, one might assume there is a simple rejection within the movement of “anything modern”. but as pointed out in research concerning other technology-hesitant religious groups such as amish, mennonites, or ultra-orthodox jews, the answer is not that simple. instead, it is a deliberate standpoint based on faith, tradition, and prerequisites. this more nuanced approach comes through in the interviews that were conducted. downloaded from brill.com / / : : am via free access 	
   	
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   .   “we use the internet when the advantages supersede other technical solutions” for a complementary view of the laestadian online presence, six interviews were conducted. their answers and reasons for how the net is used on an official level can be divided into seven different themes, which are here called: “consensus communicated through the board”, “use the internet when it supersedes other solutions”, “physical versus virtual meetings”, “information versus communication”, “the problem of being public”, “private use of the internet”, and “thoughts about the future”. physical versus virtual meetings / communication versus information a reoccurring theme is not the non-use of digital technology, but rather the best use of specific media. all six informants mention that they use different kinds of digital media – where it is apt for the intended purposes. there is a difference between sharing information and engaging in relations over the net – similar to the division between web . and . , where the former is roughly associated with publishing and sharing information and the latter is related to social media, building relations, user-created content, participatory culture, converging media, etc. (cf. bruns ; jenkins ). nevertheless, in the internal work of the congregations, emailing is used for communication between associated members, and newsletters are distributed through email (and also “snail mail” if sent to someone without internet access). google drive and dropbox are used for sharing and collaborating with documents, and skype is used for board meetings and similar – locally, nationally and internationally. special events and larger meetings often have their own web page for information sharing and live streaming. in these cases, digital media add an advantage compared to their analogue predecessors. the main reason the internet is used in a limited, but specific, way is how the separation between the virtual and physical is perceived among the informants. recent research claims that the internet dissolves the boundary between the two modes of reality, and consequently, they are intertwined and overlapped (for example, hoover and echchaibi ; lindgren ). in the case of the laestadian representatives, there is a sharp division between the physical and the virtual – and the blurring of this division is not to be encouraged. the physical and the personal meeting and interaction is prioritized, and a possible contact is encouraged to “take contact there and then, so and so, with this or that person”, says one informant. the internet is seen only as a means to send a message or information to someone else. information online is about where to find the actual meeting place, some notes regarding the basics of the movement, and the schedule of activities so someone interested can get an initial overview and find out where to go, according to one informant. one representative of the conservative branch mentions that “the web presence can lower the threshold for someone who is seeking a spiritual home within the laestadian movement”. streamed radio is frequently used online as a means to reach people who cannot attend the actual service – but it is considered by all informants to be the second choice, a choice only if you cannot come to the service in person. they try, for instance, to reach old members downloaded from brill.com / / : : am via free access 	
   	
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   who cannot leave their homes, sick members in hospital, and people who are on journeys far from their spiritual home, but also to reach out to a limited extent. commercial or search engine optimization is not used. the radio transmissions make it possible for anyone to listen to a service, just out of curiosity and to seek god and christian community. live-streamed radio is quite common on a regular basis, but mainly from larger meetings. it is more unusual for streams to be archived, even though it happens. “a service or sermon needs its context and without that, the full meaning and actuality is lost”, according to one informant. that is also why a virtual community is disregarded. as one informant put it: “why do you not seek your fellow brothers and sisters, why do you go online instead? if you are not comfortable within the congregation, something is probably wrong”. the important communication, the meeting and interaction, and, first and foremost, the community of fellow believers cannot be transmitted through a media channel. “we are preaching the gospel, and it is going out, and people who find it have the possibility to listen”, one claims. consequently, the webpage is primarily used to publish and communicate activities in the congregation to a wider audience, and radio transmissions from meetings are streamed online (and only occasionally archived). digital media can, however, never supersede heart-to-heart and face-to-face meetings, according to all the informants. building and maintaining relations, talking about things that matter, sharing faith and community can never be done over the net. you are supposed to find community where you live and not to go online to find it. consensus communicated through the board the informants express that there is consensus within the movement on how to use the net; there are, according to the informants, rarely conflicting interests. resolutions or deliberate discussions rarely occur. structure differs between the groups, but there is a hierarchy, usually with a board or a group of reliable and trustworthy people, a preacher at the local level or a group of preachers on a higher level, who have the final say. people who run and maintain the sites have their defined tasks, stated in guidelines or established practice, to publish and to keep the webpages up to date. if there are questions or initiatives to extend the use of the web further, or to develop new features, it is possible to raise the question and reach a decision in due course. the informants all say that it is no problem to have new ideas and to develop the use of the internet, but simultaneously they all seem unaware of, or do not wish to have, any new features at the present time. the interviewee from the technology conservative firstborn laestadians is also the one who said there is no impetus to develop the use of the internet any further. the others discussed possible ways to go, but referred to a common understanding of how to use the internet and social media. the youngest informant is the one who speaks most about his role as a sole performer of his duties. according to him, it has never been an issue at all to, for example, include social media channels on the webpage, while others mention discussions concerning social media. the problem of being public downloaded from brill.com / / : : am via free access 	
   	
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   even if it is toned down in the interviews and no one wants to make a big thing of it, the representatives and the movement as a whole have had bad experiences from being in the public eye. several of them mention a media discussion in which the laestadian movement has been exposed to negative opinions and writing in media, especially in finland; finnish media, for example, reported a sex abuse scandal in where cases of abuse within families were reviled (most material is in finnish, but see, for example, dave for an overview). in relation to a norwegian case, olsen ( ) mentions how experiences from initially having an open online forum led the movement to close it down and lock it behind registrations and passwords due to a polarized discussion (and also a lack of resources). one of the interviewees assumes members of the laestadian movement can be viewed as old-fashioned and backward, and he mentions in particular the fact that they have large families with many children – since kids are viewed as gifts from god (see also snellman ). in media coverage, it has been assumed that laestadian women are forced into an involuntary role as child-bearers. this is frowned upon by the majority/secular community, and the interviewees claim that laestadian webpages have been exposed to malicious and negative comments and discussion online. they have indeed experienced negative attitudes in blogs and other more open channels in the past. according to the informants, it basically takes too much time and effort to monitor and moderate comments on blogs, facebook, and other places for open discussions. there is also a shared opinion that there is little use in trying to have to a balanced discussion online. “the problem is that discussions cannot be mutual and respectful. it is possible [online] for people to express hateful comments without any consequences. [---] it makes you only sad.” soon discussions become polarized and nuances are lost, which makes it difficult to have a meaningful conversation – an experience shared with other groups such as journalists, feminists, the catholic church, the royal family of sweden, and others who are exposed to malicious attitudes and internet trolls (for example hardaker ). one informant mentions that they want to meet people with openness and respect, but the internet forums make this difficult. private use of internet since the focus of the article is on official use of digital media and the internet, i.e., how laestadian organisations and congregations use the net, questions were not asked about how the net is used privately. however, all interviewees, apart from the person representing the firstborns, mentioned that individuals use facebook, twitter, instagram, etc. the firstborns explicitly encourage members to abstain from private use of the internet. there is a risk that the internet will function as a mediator to the secular, and tempting, world, and as such, it should be avoided, according to the oldest and most conservative informant. in other cases, the use of the internet is discussed and taught about within the congregations, especially for the young ones. there are, however, closed facebook groups, often for internal communication, especially among the younger generation, and some use twitter and so on. however, the recreational use of the net and social media is discouraged – since time spent in front of the screen is time away from your friends, family, and the bible. downloaded from brill.com / / : : am via free access 	
   	
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   all interviewees also mention how easy it is to get access to disturbing and morally dubious material. “even if you go to an innocent site like blocket (swedish site for a private buy and sell market), you find flickering adverts that can be about just anything”, one said. the same person, the youngest of the interviewees, mentioned that he wanted to spend his time with family – meetings that really meant something to him (an attitude comparable with a discussion on the use of television, which was banned) (see, for example, alasuutari ). all mention that they, and others in their context, use the net in their professional life as it administrators, for marketing, or for finding special hymns and melodies as clergymen, but also for seeking specific information about just anything. the informant within the most technology-conservative branch, the firstborns, pointed out that people in his congregation use online bibles and hymnbooks during services and at bible study meetings. technology, even for personal use, is in general not prohibited but still viewed with some concern. tentative thoughts about the future most informants said that there is consensus at the present time about how to use the internet in a rather restricted and conscious way, but they could see changes coming in due course. some changes will come with the young generation who are more tech savvy, while the older generation, less skilled and more traditional, will decline in the near future. change will thereby come gradually and naturally. at the same time, the informants could anticipate and wish for some other changes already today, given the possibility to develop their web presence – if only time and resources were allocated. they could foresee an extended use of social media to some degree in some cases, but it takes time and resources, something they could only wish for at the moment. one informant could see how the live-streamed radio broadcasts could also involve streamed video from services and meetings. he also anticipated it being possible to make short films with preachers, missionaries, and other persons of interest, perhaps on their own youtube channel. .   summary: “if you need a virtual community: something is wrong with your congregation” there is, in other words, an informed and cautious attitude towards digital media within the laestadian movement in general even though it differs between different branches. andreassen ( ) claims in his study that there is a general ambivalence towards new technology, for example the internet. at the same time as the possibilities of digital media are seen, there is always a risk to engaging with the internet. on one hand, the movement can reach out to a wider audience through the internet; on the other hand, the internet is seen as a risk for deception and sin. while andreassen writes about an emerging elaestadianism based upon sources from , it seems like there is less, rather than more, signs of such progress today, in . in olsen’s ( ) study, on the other hand, he notes that the internet is used in work with youth. based on the empirical evidence provided by this article, the attitude is more thought through than just being ambivalent. in interviews, rather than the online material, the voices downloaded from brill.com / / : : am via free access 	
   	
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   from the representatives are quite unanimous. the official use of digital media is restricted to transmitting information (‘push’ media as andreassen calls it, based upon stewart hoover ( )), rather than ‘pull’ media (media more in the hands of the end user to do whatever s- /he wants). there are only a few traces of elaestadianism in the material. at first glance, one might see an overall technology-resistant religious group, but that is too generalized an assumption. we find the movement as a whole on the resistant and reluctant side of the scale; however, while there are also differences within the movement, it does not refuse technology per se. in the case of the laestadian movement, we can see an attempt to resist the almost inevitable movement toward mediatization and the related process of secularization. simultaneously, the laestadian movement also relates to the fact that society is mediatized, but instead of embracing media and an alleged mediatized society in order to reach out, measures are taken to contest and negotiate such assumptions. in order to understand this laestadian position on digital media, we must look at the specificity of the movement, as pointed out by campbell. she writes: “[t]he success, failure, or redesign of a given technology by a specific group of users is based not simply on the innate qualities of the technology, but on the ability of users to socially construct the technology in line with the moral economy of the user community or context.” ( , p. ) campbell calls for attention to the way history, tradition, beliefs, and practices shape religious negotiations with, and discourses about, technology, and not something attributed to technology itself. sharing faith and life among fellow believers is at the centre of laestadian life, and that cannot be achieved online, according to the interviewees. confession and forgiveness take place face-to-face among your brothers and sisters in faith, and consequently cannot be mediated. the distribution of the word of god is important throughout the laestadian tradition and is prevalent in their online activities, in particular through papers and streamed services. the internet is thus not used to build a shared and virtual community among believers, but rather to support the individual believers to take part in the community of believers – in the physical space. the congregation is the people present in the prayer houses or at other physical meetings. as one informant mentioned – “if you need a virtual community, something is wrong with your congregation”. the use of digital media is, as in other cases, negotiated based on traditions, beliefs, and established practices. the laestadian movement come to the conclusion that digital technology is to be used when it is regarded as better than other solutions. the spiritual meeting at a physical place cannot be mediated, but sharing documents and administrative work can be done online, and services can be streamed (although it is still a second choice) to reach people who cannot attend the actual meeting. when it comes to the communal framing and discourse within the movement, it seems quite unproblematic to have a rather hierarchical structure where everybody appears to know what is expected and possible. there are ideas for how to move in other directions, and it is deemed possible, but not always necessary or even desirable. campbell’s model primarily focuses on internal factors, but (as she mentions) there are external factors as well. contemporary society is mediatized, if we follow hjarvard’s ( ) line of thinking, and in many cases, actors and representatives of various institutions might be downloaded from brill.com / / : : am via free access 	
   	
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   obliged to be public and communicate through available media channels in formats established in the media sphere. this does apply to the laestadian movement too, but they choose to deliberately abstain from communicating according to the rules of the mediatized society. as long as the use of digital media does not conflict with ideas and practices within the movement, it is possible to use digital media for communication. publicity and openness are obviously dilemmas for the laestadian movement, reflecting the dual nature of the internet (turner ), due to experiences from being in the public eye and how digital technology is perceived. the internet and its consequences are not, by any means, determined in any direction, but a reflection of how the internet is used in certain contexts. social media are used in private, but for the institutionalized movement, recreational use of the internet and the participatory culture of the internet is problematic, so that aspect is toned down or acted against. this approach bears resemblance to examples from amish and jewish ultra-orthodox communities. a nuanced discussion is considered difficult due to the affordance of the media. living a christian conservative life, with large families in a modern secular context, has spurred unwilling controversies and exposed their members to outspoken prejudices and public condemnations from anonymous people. in other words, even though there is a cautious approach towards technology, with differences regarding the use of digital media between the various branches within the laestadian movement, it is difficult to claim there is a hostile attitude toward technology in general. virtual community is always a secondary option and not an alternative to the “real life”, and based upon that assumption, digital technology is used only when it supersedes the alternatives. interviews (recordings and excerpts in the possession of the author) informant : interview conducted / / informant : interview conducted / / informant : interview conducted / / informant : interview conducted / / informant : interview conducted / / informant : interview conducted / / references alasuutari, p., . television as a moral issue. in crawford, p. i., & sigurjón, b. h., eds. the construction of the viewer: media ethnography and the anthropology of audiences. højbjerg: intervention press. alasuutari, p., . cultural images of the media. in alasuutari, p., ed. rethinking the media audience. london: sage, – . andreassen, b.-o., . from monthly bulletins to elaestadianism? exploring attitudes and use of internet within the laestadian movement. temenos, ( ). bebbington, d., . evangelicalism in modern britain: a history from the s to the s. london: unwin hyman. downloaded from brill.com / / : : am via free access 	
   	
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   hepokoski, w. h., . the laestadian movement: disputes and divisions – . availabe at: <http://users.erols.com/ewheaton/disputes/disputes.pdf>. [accessed january ] hjarvard, s., . the mediatisation of religion: theorising religion, media and social change. culture and religion, , pp. – . hjarvard, s., . the mediatization of culture and society. new york: routledge. hoover, s. m., & echchaibi, n., . the “third spaces” of digital religion. presented at the finding religion in the media: work in progress on the third spaces of digital religion, boulder, co: center for media, religion and culture, university of colorado. jenkins, h., . convergence culture  : where old and new media collide. new york: new york university press. kejonen, e., . sexualitet i en brytningstid: en etisk analys av två finländska väckelserörelsers syn på samhälle, kyrka och homosexualitet åren – . Åbo: Åbo akademis förlag kraybill, d. b., . plain reservations: amish and mennonite views of media and computers. journal of mass media ethics, ( ), pp. – . lievrouw, l. a., . alternative and activist new media. cambridge: polity. lindgren, s., . hybrid media culture: sensing place in a world of flows. abingdon, oxon: routledge. lindmark, d., . väckelse i gränsland  : ur laestadianismens tidigaste historia. umeå: luleå stiftshistoriska sällskap. livio, o., & weinblatt, k. t., . discursive legitimation of a controversial technology: ultra-orthodox jewish women in israel and the internet. the communication review, ( ), pp. – . lotan, g., graeff, e., ananny, m., gaffney, d., pearce, i., & boyd, d., . the arab spring| the revolutions were tweeted: information flows during the tunisian and egyptian revolutions. international journal of communication, ( ), . mcluhan, m., . understanding media: the extensions of man. london: routledge. metzger, m. j., & flanagin, a. j., . digital media, youth, and credibility. mit press. nordvik, t., . between criticism and loyalty. the laestadian lyngen group’s relation to the church of norway. acta borealia, ( ), pp – . olsen, t. a., . kall, skaperordning og makt: en analyse av kjønn i lyngenlæstadianismen. universitetet i tromsø. ong, w. j., . orality and literacy: the technologizing of the word. london: methuen  : pfeffer, j., & carley, k. m., . social networks, social media, social change: in nicholson, d. m., & schmorrow, d. d., eds. advances in design for cross-cultural activities, pp. – . boca raton, fla: crc press. rainie, l., & wellman, b., . networked: the new social operating system. cambridge, mass: mit press. rashi, t., . divergent attitudes within orthodox jewry toward mass communication. review of communication, ( ), pp. – . downloaded from brill.com / / : : am via free access 	
   	
   journal	
  of	
  religion,	
  media	
  and	
  digital	
  culture	
   	
   volume	
   ,	
  issue	
   	
  ( )	
   http://jrmdc.com	
  	
   	
   	
   rashi, t., . the kosher cell phone in ultra-orthodox society: a technological ghetto within the global village? in h. campbell, h., ed. digital religion: understanding religious practice in new media worlds. london: routledge. pp. – . rheingold, h., . the virtual community: homesteading on the electronic frontier. reading, mass.: addison-wesley. rheingold, h., . look who’s talking. wired. available at: <http://www.wired.com/ / /amish/>. [accessed january ]. sjödin, u., . mer mellan himmel och jord?: en studie av den beprövade erfarenhetens ställning bland svenska ungdomar. stockholm: verbum. snellman, g., . sions döttrar: de laestadianska kvinnorna som traditionsförmedlare i norra svenska Österbotten åren - . Åbo: Åbo akademis förlag. turkle, s., . alone together: why we expect more from technology and less from each other. new york: basic books. turner, f., . from counterculture to cyberculture: stewart brand, the whole earth network, and the rise of digital utopianism. chicago: university of chicago press. umble, d. z., . holding the line: the telephone in old order mennonite and amish life. baltimore  ; johns hopkins university press. umble, d. z., & weaver-zercher, d. l. eds. . the amish and the media. baltimore: jhu press. zuckerman, p., . atheism: contemporary numbers and patterns: in michael, m., ed. the cambridge companion to atheism. new york: cambridge university press. 	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
   i	
  there	
  are	
  five	
  different	
  sami	
  languages	
  in	
  sweden	
  (north,	
  south,	
  lule,	
  pite,	
  and	
  ume	
  sami).	
  laestadius	
  wrote	
   down	
  and	
  hence	
  “constructed”	
  the	
  written	
  language	
  for	
  the	
  lule	
  sami	
  language,	
  based	
  upon	
  vernacular	
   language.	
  this	
  is	
  comparable	
  with	
  martin	
  luther’s	
  construction	
  of	
  written	
  german	
  and	
  olaus	
  petri	
  in	
  sweden,	
   when	
  translating	
  the	
  bible	
  into	
  the	
  vernacular.	
  	
   ii	
  anecdotally	
  i	
  can	
  say	
  that	
  i’ve	
  never	
  talked	
  to	
  someone	
  in	
  their	
   +	
  who	
  was	
  so	
  knowledgeable	
  about	
  the	
   internet	
  and	
  its	
  various	
  aspects,	
  while	
  still	
  being	
  a	
  non-­‐user	
  on	
  a	
  recreational	
  basis.	
  	
   downloaded from brill.com / / : : am via free access 	journal of religion, media & digital culture (jrmdc) 	“if you need a virtual community, something is wrong with your congregation”: institutionalized laestadianism and the use of digital media 	abstract: 	about the author: 	to cite this article: 	“if you need a virtual community, something is wrong with your congregation”: institutionalized laestadianism and the use of digital media 	 . introduction 	 . laestadianism: an overview and introduction 	context and perspectives 	interpretation 	 . method and material 	 . laestadianism online resources and presence: “get acquainted […] and find information about us” 	the firstborn laestadian movement 	little firstborn group 	conservative laestadianism 	summary: the laestadian branches and digital media representation 	 . “we use the internet when the advantages supersede other technical solutions” 	physical versus virtual meetings / communication versus information 	consensus communicated through the board 	the problem of being public 	private use of internet 	tentative thoughts about the future 	 . summary: “if you need a virtual community: something is wrong with your congregation” 	interviews (recordings and excerpts in the possession of the author) 	references j med genet ; : - evidence of genetic heterogeneity in the autosomal recessive adult forms of limb-girdle muscular dystrophy following linkage analysis with q probes in brazilian families maria rita passos-bueno, i richard, m vainzof, f fougerousse, j weissenbach, o broux, d cohen, j akiyama, s k n marie, a a carvalho, luiza guilherme, j kalil, a m tsanaclis, mayana zatz, j s beckmann centro de miopatias, departamento de biologia, universidade de sao paulo, caixa postal , cep - , sao paulo, brazil. m r passos-bueno m vainzof j akiyama m zatz genethon, rue de l'internationale, bp , paris cedex, france. i richard f fougerousse broux centre d'etudes du polymorphisme humain (ceph), paris, france. d cohen j s beckmann grupo de doenqas neuromusculares, faculdade de medicina, universidade de sao paulo, sp, brazil. s k n marie a a carvalho a m tsanaclis faculdade de medicina, universidade de sao paulo, sp, brazil. j weissenbach l guilherme j kalil correspondence to dr passos-bueno. received august . revised version accepted november . abstract the autosomal recessive limb-girdle muscular dystrophies (lgmd) represent a heterogeneous group of diseases which may be characterised by one or more autosomal loci. a gene at q has recently been found to be responsible for a mild form of lgmd in a group of families from the isolated island of reunion, now classified as lgmd . based on results of eight out of large brazilian lgmd families of different racial background (which were informative for the closest available probe to the lgmd gene), we confirmed linkage to the lgmd gene at q in two of these families and exclusion in six others. these data provide the first evidence of genetic heterogeneity for the autosomal recessive limb-girdle muscu- lar dystrophies. (j med genet ; : - ) progressive muscular dystrophies (pmd) in- clude a group of at least distinct genetic disorders which display different patterns of inheritance and are characterised by a progres- sive muscle wasting and weakness.' among the different forms of pmd, the limb-girdle muscular dystrophies (lgmd) represent a heterogeneous group of diseases. inheritance is autosomal recessive (ar) in the majority of cases with an increased rate of consanguinity among parents of affected patients. they include affected patients of both sexes and the weakness begins in the shoulder or limb-girdle with no facial muscle involvement. the clinical course is variable from very severe forms with onset in child- hood and very rapid progression - to mild types with onset in the second or third decade and a much slower course. the distinction between the different ar forms is extremely difficult based on clinical or laboratory findings, so the only way to assess the problem of genetic heterogeneity is through linkage analysis in an attempt to verify if there is more than one gene responsible for the different subtypes. the identification of a sequence on chromo- some , which has high homology with the dystrophin locus, led to the suggestion that this gene might be responsible for one of the ar forms of lgmd. however, a linkage analysis study using probes from the q re- gion in brazilian families with lgmd excluded this sequence as the lgmd candi- date gene. a gene at q (using the marker locus d ss ) was found to be responsible for a mild form of lgmd, based on a study of subjects belonging to families from the island of reunion (an isolated island in the indian ocean). this condition has been clas- sified as lgmd . positive lod scores using q probes have also been reported among lgmd families from the amish population indicating that the same gene is apparently involved."i in order to verify if there is genetic hetero- geneity among ar lgmd families, we have genotyped members of brazilian families with six markers near the locus d ss . subjects and methods families a total of subjects ( affected and normal) from brazilian lgmd families was included in the present analysis. except for one family (no ), the pedigrees, clinical data, and laboratory examinations (serum en- zyme determinations and muscle biopsies) from all the patients belonging to these fami- lies have been previously described and corres- pond to pedigrees , , , , , , , , , and from a previous report.' we have used the same numbers in the present paper as shown in the figure. methods dna was extracted from whole blood accord- ing to the method of kunkel et al.' dna analysis was done at ceph. the methodology for southern blotting was the same as de- scribed previously. the following probes were used: d s ( q ), d s ( q . ),' d s ( ql -q ), and diss ( q -q ). in addition, the dna samples were analysed by polymerase chain reaction (pcr) for two loci, actc ( qll-qter)' ' and d s (j s beckmann, unpublished data). the method used for actc has previously been reported,' and was also used for d s except for a lower annealing temperature ( °c). the pcr products were visualised in a % sequencing gel. the last published genetic map of chromo- o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n m a y . d o w n lo a d e d fro m http://jmg.bmj.com/ passos-bueno et al ~~~~~~~~~ t l l[z ll gll g ad oo iii iii iii iv condensed pedigrees of lgmd famzilies. iii iv v q(i t ~~~~ ii iii iii ~~~~~~~~~~~~~~~~i ; r tt 'i ta iv v <>< some suggested the following order for these loci: d s -actc-d s -lgmd - d ss , d s .' linkage analysis a two point analysis between each of these markers and the disease locus was performed using the computer program linkage.' homogeneity was tested through the homog program (including all the french and brazi- lian families) using the marker d s , since it is the most informative among the closest available markers to the disease gene. this marker, which is located at most cm, from the lgmd locus, is apparently between d s and diss (j s beckmann, unpub- lished data). the lgmd gene has been assumed to be autosomal recessive with com- plete penetrance and a gene frequency of . results the total lod score results for each of the six loci in the families and the lod scores per family for locus d s are summarised in tables and respectively. the families are not informative for the probes d s , table two point linkage analysis between each marker locus and the disease gene. recombination fraction ( ) locus - - - - d s -x - - atct -x - - - - - d s - '- - - - - - - - diss - x - - - d s -x - - - - - - d ssi -x - - - - - - - djss , and d s . the loci d s , actc, and diss gave positive lod scores, maxi- mum - at = , at = , and - at = respectively, suggesting evidence of linkage. pedigree alone (figure) gave lod scores of - at = with the locus d s and of - at = with the locus diss (table ), strongly supporting linkage between this locus and the disease gene in this family. these results were confirmed through the homog tests which also showed that another family (no ) has a % probability of being linked to the d s locus (table ). however, in six families ( , , , , , and ) linkage with this locus was excluded, sup- porting genetic heterogeneity (x = - , p= ). the remaining three families ( , , and ) were not informative for d s , but showed negative lod scores with the loci d s , actc, and d s . in addition, diss in family and d ss in family also gave negative lod scores (data not shown). discussion the confirmation of linkage to the putative lgmd gene at q in two of the brazilian lgmd families and exclusion in six others (as indicated by the homog test) provides the first evidence of genetic heterogeneity for the ar limb-girdle muscular dystrophies. all the affected patients from the brazilian families included in the present study have the milder form of lgmd with clinical and labor- atory findings very similar to those reported for the french families (zatz et al, in prepara- tion). the lgmd families from reunion, shown to be linked to the q locus d s , are o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n m a y . d o w n lo a d e d fro m http://jmg.bmj.com/ evidence of genetic heterogeneity in the autosomal recessive adult forms of limb-girdle muscular dystrophy table two point linkage analysis between d s and the disease gene per family. recombination fraction ( ) family - - - - - x - - - - - - - -oc - - - - - - - -oc - - - - - ( -xc - - - - - -xc - - - - - - - -x - - - - - - - inbred and all patients are thought to descend from a common ancestor suggesting a founder effect. it is therefore very likely that in this population all affected subjects carry the same mutated gene, similarly in the lgmd amish families."i it is interesting to note that, in the present study, only two out of the eight informative families for d s apparently carry the same mutated locus as described for the french families. although these two families that showed linkage to the q locus have different racial backgrounds (one is negroid and the other caucasoid), they are both highly inbred, living in small villages about km apart, which represented until recently small geographical isolates. such findings suggest that the q gene may be responsible for only a small proportion of lgmd in our population. once a candidate gene is identified it is of great importance to study a large number of affected families from different populations in order to test for genetic heterogeneity and estimate the relative frequency of different subtypes of lgmd. in this respect, the inbreeding in the brazilian population and the existence of large families with many affected subjects represent a valuable advantage. how- table homog test using locus d s in lgmd brazilian families. confidence limits conditional probability family of linked type lower upper - - - - - - - - x = ,p= ever, it is important to point out that, if genetic heterogeneity exists as suggested by the pre- sent report, lod scores should be analysed independently in large informative families and not pooled from different small pedigrees. the collaboration of the following persons is gratefully acknowledged: ms n bourg, l brenguier, c devaud, p pasturaud for help with the genotyping and sabine eggers, simone campiotto, antonia cerqueira, rei- naldo i takata, marta canovas, martha a b lima, thais zago, and all the staff at abdim for invaluable support. this work was sup- ported by fapesp, cnpq, abdim, and association frangaise contre les myopathies (afm). emery aeh. duchenne muscular dystrophy. oxford: oxford university press, . shields rw jr. limb girdle syndromes. in: engel ag, banker bq, eds. myology. vol ii. new york: mcgraw- hill, . walton jn, gardner-medwin d. the muscular dystro- phies. in: disorders of voluntary muscle. th ed. edin- burgh: churchill livingstone, . ben hamida m, fardeau m, attia n. severe childhood muscular dystrophy affecting both sexes is frequent in tunisia. muscle nerve ; : - . zatz m, passos-bueno mr, rapaport d. estimate of the proportion of duchenne muscular dystrophy with auto- somal recessive inheritance. am jf med genet ; : - . vainzof m, pavanello rcm, pavanello-filho i, et al. dys- trophin immunostaining in muscle from patients with different types of muscular dystrophy: a brazilian study. j neurol sci ; : - . love d, hill df, dickson g, et al. an autosomal high molecular weight transcript in skeletal muscle with homo- logy to dystrophin. nature ; : - . passos-bueno mr, terwilliger j, ott j, et al. linkage analysis in families with autosomal recessive limb-girdle muscular dystrophy (lgmd) and q probes flanking the dystrophin-related sequence. am j med genet ; : - . beckmann js, richard i, hillaire d, et al. a gene for limb- girdle muscular dystrophy maps to chromosome by linkage. c r acad sci iii ; : - . frezal j, schinzel a. report of the committee on clinical disorders, chromosome aberrations and uniparental dis- omy (hgm ). cytogenet cell genet ; : - . young k, williams p, foroud ce, et al. confirmation of linkage of limb-girdle muscu-lar dystrophy to chromo- some . hgm ;a . passos bueno mr, vainzof m, pavanello rcm, pavanello- filho i, lima mabo, zatz m. limb-girdle syndrome: a genetic study of large brazilian families, comparison with x-linked duchenne and becker dystrophies. am j med genet ; : - . kunkel lm, smith ko, boyer sh, et al. analysis of human y chromosome specific reiterated dna in chromosome variants. proc natl acad sci usa ; : - . fougerousse f, richard i, broux , cohen d, beckmann js. mapping of two chromosome microsatellites. genomics ; : - . litt m, luty ja. a hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. am j hum genet ; : - . donlon ta, malcolm s. report of the committee on the genetic constitution of chromosome . cytogenetic cell genet ; : - . lathrop cm, lalouel jm, julier c, ott j. strategies for multilocus linkage analysis in humans. proc natl acad sci usa ; : - . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n m a y . d o w n lo a d e d fro m http://jmg.bmj.com/ spontaneous and engineered compensatory hsv mutants that counteract the host antiviral pkr response viruses , , - ; doi: . /v viruses issn - www.mdpi.com/journal/viruses review spontaneous and engineered compensatory hsv mutants that counteract the host antiviral pkr response amish c. shah , jacqueline n. parker , masako shimamura and kevin a. cassady ,* university of pennsylvania, department of pediatrics, children's hospital of philadelphia, th street and civic center boulevard, philadelphia, pa - , usa; e-mail: amishamish@gmail.com (a.c.s.) university of alabama at birmingham, department of pediatrics, division of infectious disease, th avenue south, chb c, birmingham, al , usa; e-mails: jnparker@uab.edu (j.n.p.); mshimamura@peds.uab.edu (m.s.) * author to whom correspondence should be addressed; e-mail: kcassady@peds.uab.edu; tel.: + - - - ; fax: + - - - . received: august ; in revised form: october / accepted: october / published: october abstract: a virulent recombinant hsv lacking the diploid  . gene ( . ) have been investigated over the last two decades both for anti-tumor therapy and as vaccine vectors. the first generation vectors, while safe, are incapable of sustained replication in the majority of treated patients. an interferon inducible host antiviral kinase, protein kinase r (pkr), limits late viral protein synthesis and replication of  . viruses. this review describes the development of new  . vectors, through serial passage selection and direct viral genome engineering, which demonstrate selective pkr evasion in targeted cells and improved viral replication without restoring neurovirulence. keywords: pkr; oncolytic hsv;  . . introduction serial passage is a time-honored viral laboratory method that has been used to select progeny with attenuating mutations (e.g. oka vaccine strain from varicella zoster virus). in recent years, serial passaging has been used as a method to identify secondary mutations in attenuated hsv oncolytic open access viruses , vectors that mediate improved viral replication and anti-tumor activity. additional uses for the serial passaging techniqueinclude: i) the testing of genetic stability of biologic therapeutics and potential pathogenic mutations; ii) identification of cryptic gene functions that may have served evolutionarily distant functions for the virus; and iii) to identify antiviral escape mutations, that can then provide leads to improved drug design. this review focuses on the interferon-induced, innate antiviral response triggered during viral transcription that limits protein synthesis initiation in the infected cell. in addition to discussing the protein product encoded by the hsv  . gene that counters this host antiviral response, the review also describes how mutant viruses lacking the  . gene ( . ) have been studied as vaccine and anti-tumor therapies because of their safety in humans. while these vectors are safe, their limited late viral protein synthesis and diminished replication limit their efficacy. serial passage of  . recombinants has been used to identify whether compensatory mutations improve viral replication or the anti-tumor properties of the  . mutants. current efforts to improve these vectors’ replication are now focused upon engineering a new generation of hsv recombinants based on the earlier serial passage experiments. . pkr innate antiviral response eukaryotic cells contain innate defenses that target viral infection [ ]. a principal component of this defense is protein kinase r (pkr), which limits viral gene expression and replication in human cells (summarized in figure ) [ ]. low levels of this evolutionarily conserved, interferon-inducible kinase are present in unstressed cells. however, its production is induced by type i interferons or double-stranded rna (dsrna) produced during viral infection [ ]. upon binding dsrna, the kinase activates and phosphorylates itself as well as cellular proteins involved in the antiviral response. the best characterized of the substrates is the α subunit of eukaryotic translation initiation factor (eif- ) [ , ]. phosphorylation of eif-  prevents recycling of a critical translation initiation factor, thus limiting viral and cellular protein synthesis in the infected cell [ ]. in addition to its antiviral functions, the pkr-mediated host protein shutoff response is also involved in cellular homeostasis. consequently, second messenger signaling pathways and pkr modulate one another’s activity in the cell. these pathways can block pkr activation during periods of cellular stress or when the cell is actively replicating. for example, upregulation of mitogen- activated protein kinase (mapk) activity in the cell (mediated by a component of this pathway, mek) blocks pkr activation during growth factor stimulation [ , ]. likewise, pkr has also been shown to act as a signal integration point between signaling pathways [ ]. while regulation of protein synthesis initiation is the best characterized of the pkr antiviral functions, the kinase also modulates other cellular functions, including: bulk protein degradation in the cell (also called autophagy), rna transcription, and signal transduction in the cell [ , - ]. viruses , figure . summary of pkr mediated protein shutoff and hsv icp . defense. ( ) hsv entry, gene expression, ) complementary transcripts anneal and form stable dsrna ) pkr binds dsrna resulting in activation and autophosphorylation, ) activated pkr selectively phosphorylates eif- inhibiting protein translation, ) hsv produces icp . which ) forms a multiprotein complex with the host phosphatase pp  ) and dephosphorylates eif . ) an example of pulse labeling study showing abundant radioactive methionine incorporation in uninfected (m) and wild-type hsv- -infected (f) proteins at hpi but decreased incorporation in icp . (-) infected cells (). . the hsv-  . gene the hsv-  . gene encodes a multifunctional protein called icp . [ , ]. one function, encoded within the ’ gene domain, blocks host pkr-mediated protein shutoff during infection thus allowing continued late viral protein synthesis in infected cells [ ]. during infection, wild-type hsv- produces complementary mrna transcripts that anneal, forming stable dsrna, which triggers the dimerization and activation of dsrna-activated host pkr. the icp . overcomes this pkr- mediated host protein shutoff by binding and recruiting a host phosphatase that specifically dephosphorylates eif- , allowing continued viral protein synthesis (hereafter referred to as the hsv wild-type protein synthesis phenotype) in the infected cell (summarized in figure ) [ , ]. recombinant viruses that lack the  . gene, ( . hsvs) are incapable of maintaining eif-  in an unphosphorylated form and therefore are unable to maintain protein synthesis in the infected cell [ ]. cessation of protein synthesis occurs at the onset of viral dna synthesis late in infection, essentially eliminating bulk synthesis of viral structural proteins necessary for viral capsid formation [ ]. in addition to structural proteins, viral encapsidation, envelopment and maturation are necessary for efficient viral replication. there are examples of  . mutants that are capable of late viruses , viral protein synthesis but that cannot negotiate the other steps necessary for viral replication [ ]. evidence suggests that in addition to protein translation, other cellular processes are disrupted in  . infected cells that are critical for viral egress. consequently,  . hsv replicate inefficiently and produce fewer progeny virions in cells with intact pkr pathways [ ]. while the  . gene product does not interrupt ifn signaling in the infected cell as has been described with the hsv ul and ul gene products, icp . is critical for hsv evasion of the ifn inducible gene product, pkr. consequently,  . viruses are highly sensitive to type i ifn and replicate poorly in vivo or in ifn treated cells [ , ]. restoration of the protein synthesis function allows  . hsv to replicate efficiently in the presence of the type l ifn ( or  interferon) response [ ]. the  . gene also encodes a second function, neurovirulence, enabling efficient viral replication in post-mitotic neuronal cells [ - , ]. the neurovirulence and protein synthesis functions encoded by the  . gene are discrete and separable. late viral protein synthesis can be selectively restored without restoring wild-type neurovirulence [ , , ]. the  . hsv are incapable of efficient replication after direct inoculation in the cns and do not cause encephalitis [ ]. as such,  . hsv vectors have been developed as anti-tumor agents for cns-based malignancies. whereas - pfu of wildtype hsv will lead to encephalitis and death in half of the mice inoculated intracerebrally, more than x pfu are required to produce encephalitis and death with a  . hsv recombinant [ ]. finally, the  . gene also allows hsv- to block autophagy in infected cells [ ]. this function contributes to hsv neurovirulence, maps within the ’ domain of the gene and allows binding and sequestering of beclin- , a host protein necessary for autophagosome formation [ ]. .  . hsv as oncolytic vectors genetically modified hsv are attractive as replication-competent, oncolytic vectors as well as vaccine vectors for a number of reasons: ) procedures for constructing novel hsv are well established; ) genetic modifications (insertions or deletions) do not significantly affect virus replication; ) considerable experience with the biology of hsv and its behavior in humans and nonhuman primates exists; and ) modified herpesviruses retain sensitivity to standard antiviral drug therapy as a “built-in” safety feature [ - ]. deletion of the hsv- neurovirulence gene,  . , allows the safe administration of recombinant vectors intracranially (for brain tumor therapy) or systemically (for peripheral tumors or as a vaccine vector). in addition to inhibiting protein synthesis initiation, other viral functions are inhibited as a consequence of this deletional mutation. the  . mutants also demonstrate changes in glycoprotein processing figure ). the hsv . gene product differs in amino acid sequence between viral strains. adoptive transfer studies have shown that these icp . amino acid differences affect glycoprotein processing and plaque phenotype in cell culture [ , ]. in viruses lacking the  . gene, the differences in glycoprotein processing are even more dramatic. immunostaining studies show that gd in the  . infected cells exist in a single form after separation using denaturing conditions. in contrast, viruses capable of pkr evasion and late viral protein synthesis exhibit multiple slower migratory forms of the glycoprotein indicative of further glycoprotein processing. in figure the chimeric hsv, c , which contains the hcmv pkr-evasion gene trs , viruses , contains multiple forms of glycoprotein d, similar to that observed for wild-type hsv. confocal immunofluorescence microscopy further demonstrates differences in glycoprotein trafficking in the infected cell. in tumor cells infected with recombinants capable of late viral protein synthesis, gd accumulates within the trans golgi network (tgn) and late endosomal compartment based upon gd and ap- staining and colocalization. in contrast, gd expressed in the  . infected cells does not localize with adaptor protein complex (ap- ) in the tgn. there are also fundamental differences in the appearance of the tgn. in the  . infected cells, the tgn appears disrupted throughout the cytoplasm and does not exhibit the more structured perinuclear aggregates seen in cells capable of continued protein synthesis. the  . recombinants also exhibit differences in protein degradation or autophagy within the infected cell [ ]. improved protein production and processing may may enhance the antitumor capabilities of  . mutants either directly (improved antigen expression and replication) or indirectly by improving the expression and processing of foreign gene inserts in recombinants for gene therapy applications. figure . glycoprotein processing differences between  . and hsvs capable of pkr evasion and late viral protein synthesis. a) autoradiograph showing radiolabeled viral proteins produced in infected malignant glioma cells. viruses capable of pkr evasion (hsv- and the chimeric hsv, c [discussed in detail in section . ]) produce abundant s-methionine-radiolabeled proteins at hpi. in contrast, the  . recombinants (c ) and the chimeric hsv repair virus (c ) have reduced protein synthesis. b) immunostaining using ig anti-glycoprotein d (clone h : sc- santa cruz biotechnology) shows abundant late gene accumulation in the hsv- and chimeric hsv (c ) samples as well as multiple migratory gd forms (arrows) indicative of glycoprotein processing in the infected cells. c) confocal immunofluorescence imaging shows gd (green) distribution in  . (left upper panel) and c (right upper panel) -infected u -mg cells and colocalization with the transgolgi network marker ap- (sigma). co- localization detected (yellow) in the chimeric hsv infected samples (lower right panel) but not in the  . infected cells. viruses , in addition to protein processing recent studies also show that the  . gene product also inhibits ifn response in the cell. icp . targets the ability of the tank-binding kinase (tbk ) to activate an antiviral response through interferon regulatory factor (irf ) and cytokine expression. [ ] phosphorylation and transnuclear translocation of irf as well as expression of downstream genes functioning in antiviral responses are thereby inhibited by the  . protein product. suppression of the interferon signaling pathway has consistently been shown to be a major mode of the interaction of hsv with the innate immune system, and its interaction with tbk appears to be a novel but critical mechanism to ensure successful and productive hsv infection. . serial passage and escape mutations . . in vitro passage attenuated  . recombinants have been engineered for use as antitumor agents. while the vectors are safe and replicate in mitotically active cells, they do not exhibit robust replication. this limited replication and spread is one factor thought to limit their efficacy in vivo. studies have described acquisition of mutations that overcome certain limitations of the icp . -negative status of attenuated hsv- [ ]. mohr and gluzman reported selection of the sup- mutant after passage of the  . spbg e (derived from the patton hsv- strain) in the nonpermissive sk-n-sh human neuroblastoma cell line in vitro [ ]. sup- acquired a deletion in the us region, resulting in two fundamental changes in the viral phenotype: ) deletion of us results results in greater mhc i expression (as discussed in detail in section ), and ) earlier expression of the us gene [ , ]. later studies using engineered recombinants and recombinant expressed us protein in biochemical studies demonstrated that this kinetic shift in us expression enabled viral evasion of pkr and late viral protein synthesis [ ]. the us product, although capable of precluding activated pkr from phosphorylating its target substrate eif- , was much more effective and at lower concentrations at blocking the activation of pkr by dsrna [ ]. ultimately these escape mutations produced progeny virus capable of continued viral protein translation in infected sk-n-sh cells [ ]. mohr’s mutant sup- , while it had a protein synthesis phenotype similar to that of wild-type hsv- , retained the neurovirulence profile of its  . parent (ld > x ); however, it was engineered in a more neurovirulent strain of hsv- which limited maximal ld testing [ , ]. r , derived from the parent hsv- (f) strain, was also sequentially passaged in two different cell lines that restricted  . late viral protein synthesis. serial passage in hela cells produced progeny virus with no compensatory mutations [ ]. the parent passage virus and the virus recovered after serial infections in the hela cell line both had a  . phenotype. it was concluded that because  . recombinants are capable of modest viral replication in hela cells that the selection was not sufficiently rigorous to provide compensatory mutant viruses with a selective advantage. serial passage in sk-n-sh cells produced progeny that, like sup- , were capable of blocking the protein kinase r (pkr)-induced shutoff of protein synthesis [ ]. unlike sup- , however, these mutations mapped outside of the us - domain, and at least one isolate had acquired a partially restored neurovirulence profile [ ]. viruses , . . in vivo passage in vivo passage has also been used to develop improved  . anti-tumor activity. m , a  . recombinant that expresses murine il- , was sequentially passaged in flank tumors of the hsv- resistant d -mg cell line. this study was based on the assumption that the virus would, through selective pressure incurred by the tumor microenvironment, acquire mutations allowing for more efficient replication in the resistant tumor cell line [ ]. m and its parent virus, r were each sequentially passaged in d -mg tumor cells in vivo in flank tumors, and in vitro in cell culture [ ]. in vitro passage in tissue culture selected for mutants similar to the passaged virus described originally by mohr. the progeny virus expressed us earlier in infection, exhibited a wild- type protein synthesis phenotype, and was found in in vivo studies to partially restore hsv neurotoxicity (ld - . x – . x ). interestingly, these secondary mutations did not improve the anti-tumor activity of the  . recombinants. tumor-bearing animals had similar survival rates irrespective of whether they were administered the parent virus or a serially passaged virus capable of late viral protein synthesis [ ]. in contrast, in vivo passage in d flank tumors selected for second site  . mutants with different compensatory mutations and a different phenotype. in vivo passaged viruses demonstrated decreased neurovirulence as compared to the non-passaged parent viruses and resulted in significantly improved survival of tumor-bearing mice in two separate murine experimental brain tumor models: human d -mg intracranial xenografts in scid mice and murine neuro- a neuroblastoma syngeneic tumors in a/j mice. the in vivo passaged virus retained strict  kinetic expression of the us gene and  . protein shutoff phenotype. however, the recombinants were more effective in their anti- tumor activity [ ]. in contrast to specifically engineered constructs, it can be more difficult to identify the particular mutation(s) responsible for the phenotypic changes seen in passaged viruses, and therefore, their interaction with the host environment more elusive to explain. currently, the compensatory mutation responsible for this change has not been mapped, re-emphasizing the primary limitation of the serial passage process. in cases where a deletional or insertional mutation is responsible, mapping the mutation can easily be achieved through restriction enzyme digestion and differences in dna fragment migration in agarose gels. in cases where a point mutation or a small deletion mutation occurs, sequencing of the viral genome or using marker transfer of genetic domains must be used to map the mutation. thus, these studies emphasize the importance of the in vivo tumor environment for selecting novel oncolytic hsv strains that mediate improved survival in vivo. . engineered mutations the serial passaging of many viruses has resulted in selection of novel mutations providing unique insights into the complexity of evasion of pkr by hsv vectors. the findings of these studies have helped investigators specifically engineer viruses with the goal of improving their anti-tumor activity and producing more effective vaccine vectors. additionally, other strategies have been investigated which involve the engineering of novel viruses to enhance the oncolytic activity of  . hsv oncolytic vectors. these are summarized in the following subsections. viruses , . . r one strategy employed replacing the carboxyl terminus of the  . gene, responsible for blocking host pkr-mediated protein shutoff, with the murine gene encoding myeloid differentiation gene (myd ), which is homologous to this region [ ]. both myd and the hamster homologue gadd , are primary response genes in hematopoiesis and expressed during growth arrest and dna damage. the virus constructed with this substitution in the  . gene, r , does not induce premature host protein shutoff in infected human cells [ ]. treatment of human gliomas established intracranially in scid mice with r prolonged survival as compared to controls [ ]. . . g – engineering the us us mutation tumor destruction is expected to be determined in part by the properties of the oncolytic virus used, but is also likely strongly influenced by viral-host interaction and induction of an immune response against the tumor. us gene expression from hsv- functions to inhibit transporter associated with antigen presentation (tap) of mhc i. when deleted, mhc class i expression from infected cells is increased. the g virus contains an engineered  gene deletion in a g vector [ ]. like the second site mutants described above, it places us gene expression (normally a  or late gene) under the control of the immediate-early  promoter [ ]. the authors suggest the combination of early us expression (allowing for improved viral replication) along with increased mhc i presentation (allowing enhanced cytopathic effect) result in greater anti-tumor activity of g in various animal tumor models, including metastatic breast cancer to the brain [ ], prostate adenocarcinoma [ ], and schwannomas [ ]. these benefits were achieved without increasing the neurovirulence of the vector [ ]. . . controlled expression of the  . gene or tumor targeting of  . containing hsv deletion of the  . neurovirulence gene allows the safe administration of oncolytic hsv to brain tumors without producing hemorrhagic encephalitis characteristic of wild-type hsv- infection. however, since this deletion also diminishes the replicative and cytotoxic abilities of the virus, groups have investigated the targeted expression of the  . gene to replicating tumors. one method involves placing the  . gene expression under the control of a tumor specific promoter or enhancer thereby limiting  . gene expression in complementing tumor cells. one copy of the  . gene was reintroduced under the control of the cellular b-myb promoter, which responds to e f regulation [ ]. as expected, late viral protein synthesis was maintained after infection with this myb . virus, which also has a deletion of the icp ribonucleotide reductase gene, and there was greater viral replication. anti-tumor effect was increased relative to  . hsv- in a murine model of liver metastasis [ ]. more importantly, the study suggests the potential of regulating  . gene expression by promoters regulated by the cell-cycle to permit hsv- to demonstrate its efficacy within cycling tumor cells yet retaining an advantageous neurovirulence profile. further development of the targeted expression of the  . has led to the development of two other conditional expressing hsv recombinants that regulate  . gene expression using nestin enhancers or musashi promoter (both viruses , active in gbm tumors and cns stem cells) [ , ]. another method is to limit hsv ( . +) viral entry to tumor cells. to achieve this, recombinant r was created and replaces the hsv entry molecule gd with a tumor specific fusion entry molecule (gd-il- r  ) [ ]. cell culture studies show that r replicate specifically in tumor [ ]. . . chimeric hsv a different method hypothesized that transfer of the human cytomegalovirus (hcmv) pkr evasion genes trs or irs gene to a  . virus (producing viruses c and c , respectively) would improve viral replication and anti-glioma activity. the trs and irs protein products are hcmv’s tools for blocking the dsrna-dependent pkr response pathway [ , ]. these “chimeric” hsv vectors expressing hcmv genes improved late viral protein synthesis by preventing the shutdown of protein synthesis after viral infection. this attribute proffers the ability to replicate to wild-type levels in human malignant glioma cells in vitro. in vivo, c and c were found to be aneurovirulent with ld measurements from four to over six logs higher than that of wild-type hsv- (ld = . x for c , > x for c ) [ ]. therefore, despite restoration of late viral protein synthesis and greater replication of progeny virus, expression of trs and irs did not restore wild-type neurovirulence. indeed, incorporation of trs and irs into the attenuated  . virus produced a more robust vector, permitting more efficient destruction of tumor cells and resulting in enhanced anti- tumor activity. improved survival was noted in two brain tumor models: a human malignant glioma in severe combined immune deficient (scid) mice and a syngeneic immunocompetent murine neuroblastoma model [ ]. both chimeric viruses demonstrated enhanced anti-glioma activity compared to their parent  . virus, and performed superiorly at lower doses. a similar greater anti-tumor benefit was seen comparing the viruses against a murine neuroblastoma model [ ]. these studies suggest that replication of oncolytic hsv- vectors in partially restrictive tumor cells due to anti-viral pkr responses can be significantly improved by encoding pkr-evasion genes from a related herpesvirus. . conclusions viral protein synthesis is critical for efficient viral replication. as a consequence, hsv has evolved numerous genes involved in maintaining cellular functions necessary for viral replication. despite the extensive viral control of the cellular environment, intrinsic intracellular defense systems are nevertheless active and can disrupt viral gene expression. efforts over the last decade have translated some of the principal work involving hsv innate immune evasion toward development of hsv- viral vectors for use as both vaccine and anti-tumor therapy. fundamental scientific research elucidating these viral mechanisms has made these clinical applications possible. acknowledgements these studies were aided by grants from the ruth l. kirschstein nrsa fellowship f ns - and medical scientist training program (acs), the nih nci p ca viruses , (jnp), nih 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promoter increases survival of animals even when symptomatic from a brain tumor. cancer res. , , - . . zhou, g.; ye, g.j.; debinski, w.; roizman, b. engineered herpes simplex virus is dependent on il ralpha receptor for cell entry and independent of glycoprotein d receptor interaction. proc. natl. acad. sci. u s a , , - . . zhou, g.; roizman, b. characterization of a recombinant herpes simplex virus designed to enter cells via the il ralpha receptor of malignant glioma cells.j. virol. , , - . . child, s.j.; hakki, m.; de niro, k.l.; geballe, a.p. evasion of cellular antiviral responses by human cytomegalovirus trs and irs . j. virol. , , - . . cassady, k.a. human cytomegalovirus trs and irs gene products block the double-stranded- rna-activated host protein shutoff response induced by herpes simplex virus type infection. j. virol. , , - . © by the authors; licensee molecular diversity preservation international, basel, switzerland. this article is an open-access article distributed under the terms and conditions of the creative commons attribution license (http://creativecommons.org/licenses/by/ . /). case reports.pmd indian pediatrics volume __august , case reports administration of oral calcium and calcitriol remains the mainstay of treatment. the goals of therapy are to maintain serum calcium levels within the reference range so as to avoid hypercalciuria. thyroid function tests should be evaluated periodically even in absence of features of aho, as hypothyroidism develops rarely, as seen in our patient. acknowledgement: we are grateful to dr y k amdekar , medical director b j wadia hospital for children for allowing us to publish this case report. contributors: rj diagnosed and treated the patient. mk was involved in treatment of the patient. both of them wrote the article. funding: none; competing interests: none stated. references . mantovani g, spada a. mutations in the gs alpha gene causing hormone resistance. best prac res clin endocr metab. ; : - . . “online mendelian inheritance in man (omim), a knowledgebase of human genes and genetic disorders”. nucleic acids research (database issue): d –d . available from: http://en.wikipedia.org/wiki/omim. accessed july , . mariot v, maupetit-mehouas s, sinding c, kottler ml, linglart a. a maternal epimutation of gnas leads to albright osteodystrophy and parathyroid hormone resistance. j clin endocr metab. ; : - . . levine ma, downs rw jr , moses am, breslau na, marx sj, lasker rd, et al. resistance to multiple hormones in patients with pseudohypoparathyroidism. association with deficient activity of guanine nucleotide regulatory protein. am j med. ; : - . . heinsimer ja, davies ao, downs rw, levine ma, spiegel am, drezner mk, et al. impaired formation of beta-adrenergic receptor-nucleotide regulatory protein complexes in pseudohypoparathyroidism. j clin invest. ; : - . chronic myeloid leukemia in a child with iga nephropathy amish udani, vijayakumar mahalingam, prahlad nageswaran and *sudha ekambaram from the department of pediatric nephrology and *pediatrics, mehta children’s hospitals, chennai, india. we report an year old boy with iga nephropathy developing chronic myeloid leukemia on follow-up. this association suggests that a b cell defect might be involved in the pathogenesis of these two conditions. key words: chronic myeloid leukemia, iga nephropathy. correspondence to: dr m vijayakumar, consultant pediatric nephrologist, mehta children’s hospitals, no. (e) mc nichols road, rd lane, chetput, chennai , tamilnadu, india. doctormvk@gmail.com, received: september , ; initial review: september , ; accepted: february , . t here is increasing evidence of abnormal glycosylation of immunoglobulin a (iga ) subclass due to b-cell defect in the pathogenesis of immune-complex mediated iga nephropathy [ ]. the occurrence of iga nephropathy and leukemia has been reported rarely in children [ ]. here we report a child with iga nephropathy developing chronic myeloid leukemia (cml) on follow-up. case report an -yr-old boy was diagnosed acute nephritic syndrome at year back in view of hypertension, hematuria, proteinuria (spot urine protein to creatinine ratio . ), mild renal insufficiency (serum creatinine . mg/dl), and normal serum albumin and cholesterol. he had no anemia, leukocytosis or electrolyte disturbances. he was treated with salt and fluid restriction and oral nifedepine for hypertension. serum complement c level was normal, anti nuclear antibody and anti double standard dna was negative. in view of persistent hypertension, renal insufficiency, microscopic hematuria and proteinuria he was referred for evaluation. on examination, the patient was well nourished (weight kg, and height cm) with periorbital edema and blood pressure / mm hg. systemic examination was normal. urinalysis showed + albumin, red blood cells and up/uc ratio of . . blood investigations showed a creatinine of . mg/dl, albumin . g/dl and potassium . meq/l. ultrasonogram of the kidneys showed normal size kidneys. renal biopsy showed seven glomeruli of which two were completely sclerosed and one showed segmental sclerosis and proliferation. remaining indian pediatrics volume __august , case reports glomeruli were normal-sized with increase in mesangial cellularity and thin capillary walls; tubules, interstitium and blood vessels were unremarkable. immuno- fluorescence examination showed mesangial granular deposits of iga, and c c and igm. a diagnosis of class iii iga nephropathy was made [ ]. the patient received treatment with angiotensin converting enzyme inhibitors and fish oil supplements without steroids. later, he received therapy with an angiotensin receptor blocker. ten months later, he was admitted with fever, generalized edema, anemia, hepatosplenomegaly and a soft systolic murmur. the blood pressure was / mm hg. investigations showed a serum creatinine level of . mg/ dl, urea mg/dl, uric acid . mg/dl, sodium meq/l, potassium . meq/l and albumin . g/dl. the hemoglobin level was . g/dl, with leukocytes / cu mm, and differential count of % polymorphs, % lymphocytes, % eosinophils, % basophils, % stab neutrophils, % myelocytes, % metamyelocytes, % promyelocytes, % myeloblasts and % nucleated red cells; platelets were normal. a diagnosis of cml with blast crisis was made (fig ), and the patient received intravenous and oral fluids of l /day along with alkalization of urine and allopurinol. bcr-abl translocation assay showed hybrid transcript in leukocytes suggesting chronic phase of cml. genomic breakpoint observed at e a corresponds to p . the patient was treated with imatinib mg once a day [ , ]. after month follow-up, there was no hepatosplenomegaly, and leukocyte counts and renal functions were normal. discussion iga nephropathy is a common chronic primary glomerular disease, which rarely presents as acute nephritic syndrome. systemic diseases with iga deposits include systemic lupus erythematosus, henoch- schonlein purpura, cystic fibrosis, ankylosing spondylitis, dermatitis herpetiformis, inflammatory bowel disease, celiac disease, chronic liver disease, infections like mycoplasma, leprosy and toxoplasmosis, as well as neoplasms like non-hodgkin lymphoma, monoclonal iga gammopathy and carcinoma of the lung and colon [ ]. various hypotheses suggested in the pathogenesis of iga nephropathy are predisposing genetic factors, iga immune complex disease due to abnormal iga glycosylation and adhesion molecules on mononuclear cells and lymphocyte subpopulation [ , , ]. chromosome aberrations identified by genome- wide linkage analysis in families with iga nephropathy cases is suggested to be predisposing genetic factor for development of disease [ ]. abnormal galactosylation in the hinge region of iga subclass results in formation of circulating immune complex and its deposition in mesangium. these deposits release cytokines, growth factors and adhesion molecules, which lead to proliferation of mesangial cells, inflammation and sclerosis [ , ]. the recognition of b-cell defect and the role of adhesion molecules/growth factors enables targeting treatment with bone marrow transplant or neutralizing antibodies [ , ]. findings that predict progression to end stage renal disease include heavy proteinuria, diffuse mesangial proliferation, a high proportion of glomeruli showing sclerosis, crescents or capsular adhesions, and the presence of moderate or severe tubulointerstitial changes [ ]. there are increasing reports of association between chronic glomerulo- nephritis and leukemia in adults [ , ]. renal infiltration by leukemic cells is rare and presents with abdominal pain or hematuria with renal biopsy showing presence of abnormal cells as seen in peripheral blood. the present patient had acute nephritic syndrome, which was followed three years later by the occurrence of cml. this association suggests that a b-cell defect might be involved in the pathogenesis of iga nephropathy and cml. acknowledgement: histopathology department of apollo hospitals, chennai, india for preparation and reporting of renal biopsy, and pediatric hematology and histopathology departments of mehta hospitals, chennai, india for preparation and reporting of peripheral smear. contributors: all the authors were involved in the case management. mvk, np and au were involved in review of literature and preparation of the manuscript. mvk will act as guarantor. funding: none;competing interest: none stated. references . nakanishi k and yoshikawa n. immunoglobulin a nephropathy. in: avner ed, harmon we, niaudet p, yoshikawa n eds. pediatric nephrology th ed. springer- verlag berlin heidelberg; . p. - . fig. peripheral smear showing features of chronic myeloid leukemia. indian pediatrics volume __august , case reports . motoyama o, kojima y, ohara a, tsukimoto i, ishikawa y, iitaka k. iga nephropathy associated with leukemia and lymphoma: report of two cases. clin exp nephrol. ; : - . . churg j, bernstein j, glassock r. in: renal disease: classification and atlas of glomerular diseases nd ed. tokyo: igaku-shion medical publishers . p. - . . floege j and ostendorf t. cytokines and growth factors. lai kn ed. recent advances in iga nephropathy. world scientific publishing co. pte. ltd: singapore; : p. - . . nelson pj and shankl sj. therapeutics in renal disease: the road ahead for antiproliferative targets. nephron exp nephrol. ; : - . . noris m and remuzzi g, editor. iga nephropathy: a stem cell disease? kidney int. ; : - . . wiercinski r, zoch-zwierz w, stasiak-barmuta a, wasilewska a, tomaszewska b, winiecka w. assessment of selected adhesion molecules and lymphocyte subpopulations in children with iga nephropathy. annales academiae medicae bialostocensis. ; : - . . yoshikawa n, ito h, nakamura h. prognostic indicators in childhood iga nephropathy. nephron. ; : - . . hu sl, colvin ga, rifai a, suzuki h, novak j, esparza a, et al. glomerulonephritis after hematopoietic stem transplant: iga nephropathy with increased excretion of dn iga . nephrol dial transplant. ; : - . . iwata y, wada t, uchiyama a, miwa a, nakaya i, tohyama t, et al. remission of iga nephropathy after allogeneic peripheral blood stem cell transplantation followed by immunosuppression for acute lymphocytic leukemia. intern med. ; : - . facio- auriculo- vertebral sequence in association with congenital hypoparathyroidism mandar bhausaheb patil *and sunita mandar patil from department of pediatrics, dr dy patil medical college and *sangeeta hospital for children, kolhapur, maharashtra, india. although, facio-auriculo-vertebral sequence (favs) is a well recognized condition with cranio-facial, ocular and vertebral anomalies, extreme variability of expression is characteristic. association of cardiac, cns, lungs, kidneys and limb defects are described. we report a neonatal case with favs in association with congenital hypoparathyroidism. key words: branchial arch anomaly, congenital hypoparathyroidism, embryology, facio- auriculo-vertebral sequence. correspondence to: dr mandar bhausaheb patil, anita vandan sahanivas colony, , opposite rajhans printing press, near hari puja puram, nagala park, kasaba karveer, kolhapur , maharashtra, india. drmandarpatil@hotmail.com received: january , ; initial review: february , ; accepted: march , . f acio-auriculo- vertebral sequence (favs) is a spectrum of developmental disorders involving oculo- auriculo- vertebral disorder, hemifacial microsomia, fav syndrome and goldenhar syndrome [ , ]. favs consists of facial asymmetry, maxillary and mandibular hypoplasia, cleft palate, macrostomia, microtia or anotia and pre- auricular ear tags or pits, in addition to vertebral anomalies. goldenhar syndrome consists of above defects plus epibulbar dermoids and/ or lipodermoids. association of anomalies of heart, kidneys, cns, lungs, limbs have been described [ , ]. there is only one fetal autopsy case report of fav sequence with associated digeorge sequence (with hypoplasia of parathyroid glands) [ ]. case report a -day-old neonate, second child of a non- consanguineous marriage, presented to us with two days history of multiple brief episodes of seizures. on clinical examination, baby had facial asymmetry with hypoplasia of the right mandible and right macrostomia, cleft palate, small deformed and very low set right pinna with a pre- auricular tag and atresia of the right external auditory canal (fig. ). apart from anti-mongoloid slant and hypertelorism, both the eyes were normal. the neonatal reflexes (including moro’s, sucking, rooting, etc) and other systemic examination were normal. on evaluation, his sepsis screen (including total white cell count, band count, random blood sugar, c- reactive proteins, blood culture and csf study) was negative. biochemical evaluation revealed a serum calcium concentration of mg /dl, the serum phosphorus concentration of mg /dl and serum alkaline phosphatase concentration of u/l. the serum concentration of magnesium was . mg /dl, the serum concentration of -hydroxyvitamin d was ng /ml (normal range- - ) and the serum concentration of spatially discordant alternans in cardiomyocyte monolayers spatially discordant alternans in cardiomyocyte monolayers carlos de diego, rakesh k. pai, amish s. dave, adam lynch, mya thu, fuhua chen, lai-hua xie, james n. weiss, and miguel valderrábano cardiovascular research laboratory, departments of medicine (cardiology), pediatrics (cardiology), and physiology, david geffen school of medicine at university of california los angeles, los angeles, california submitted october ; accepted in final form january de diego c, pai rk, dave as, lynch a, thu m, chen f, xie l-h, weiss jn, valderrábano m. spatially discordant alternans in cardiomyocyte monolayers. am j physiol heart circ phys- iol : h –h , . first published january , ; doi: . /ajpheart. . .—repolarization alternans is a har- binger of sudden cardiac death, particularly when it becomes spatially discordant. alternans, a beat-to-beat alternation in the action potential duration (apd) and intracellular ca (cai), can arise from either tissue heterogeneities or dynamic factors. distinguishing between these mechanisms in normal cardiac tissue is difficult because of inherent complex three-dimensional tissue heterogeneities. to evaluate repo- larization alternans in a simpler two-dimensional cardiac substrate, we optically recorded voltage and/or cai in monolayers of cultured neonatal rat ventricular myocytes during rapid pacing, before and after exposure to bay k to enhance dynamic factors promoting alternans. under control conditions (n � ), rapid pacing caused detectable apd alternans in % of monolayers, and cai transient alternans in all monolayers, becoming spatially discordant in %. after bay k (n � ), conduction velocity restitution became more prominent, and apd and cai alternans developed and became spatially discordant in all monolayers, with an increased number of nodal lines separating out-of-phase alternating regions. nodal lines moved closer to the pacing site with faster pacing rates and changed orientation when the pacing site was moved, as predicted for the dynamically generated, but not heterogeneity-based, alternans. spatial apd gradients during spatially discordant alternans were sufficiently steep to induce conduction block and reentry. these findings indicate that spatially discordant alternans severe enough to initiate reentry can be readily induced by pacing in two-dimensional cardiac tissue and behaves according to predictions for a predominantly dynamically generated mechanism. calcium cycling; arrhythmias t-wave alternans, an important marker of arrhythmia risk ( ), arises from beat-to-beat alternation in the electromechan- ical response at the cellular level ( ). alternans can be spatially concordant, when the entire tissue alternates in phase, or spatially discordant, when adjacent regions alternate out of phase, separated by a nodal line at which no alternation occurs. spatially discordant alternans amplifies dispersion of repolar- ization and can precede conduction block and reentry in whole heart mapping studies ( , , ). two mechanisms have been proposed to explain how alternans becomes spatially discordant in cardiac tissue: ) inherent heterogeneous tissue properties, including structural ( ), electrophysiological ( , ), and/or intracellular calcium (cai) cycling ( ) heteroge- neities; and ) dynamic factors, such as steep action potential (ap) duration (apd) restitution slope or a cai cycling insta- bility, in combination with conduction velocity (cv) restitu- tion ( , , – , ). since both factors are naturally present in real cardiac tissue, it has been difficult to evaluate their relative importance in producing alternans. recent modeling studies, however, have suggested criteria by which the behav- ior of nodal lines in response to pacing interventions can be used to distinguish between these mechanisms ( , ). these criteria, applied to intact rabbit ventricles ( ), supported a key role of dynamic factors in the genesis of spatially discordant alternans. however, in three-dimensional ( d) studies in which only the surface can be mapped, it cannot be excluded that unmapped subsurface events may influence the properties of spatially discordant alternans. neonatal rat ventricular myocyte monolayers provide a sim- ple two-dimensional ( d) tissue model to explore the relevance of dynamic factors to spatially discordant alternans. here we used optical mapping of membrane voltage and cai in mono- layers to determine whether spatially discordant alternans could be induced, and, if so, whether its behavior supported a role of dynamic factors. in addition to control conditions, we studied the effects of the l-type ca current agonist bay k , which enhances dynamic factors by steepening apd restitution slope and promoting cai cycling instability by loading the sarcoplasmic reticulum (sr) with high levels of ca. our findings provide strong additional experimental sup- port for the role of dynamic factors in the generation of spatially discordant alternans and directly demonstrate that spatially discordant alternans can lead to conduction block and initiation of reentry in a preparation without any unmapped subsurface tissue. methods monolayer preparation neonatal rat ventricular myocytes from - to -day-old sprague- dawley rats were obtained by standard methods ( ), plated on � -mm plastic coverslips ( myocytes per coverslip), and cultured for – days. monolayers with insufficient cellular confluence, as assessed by phase-contrast microscopy, or uneven propagation pat- terns during pacing at hz were excluded from analysis. monolayers were studied under two different conditions: control (n � ) and during exposure to bay k (racemic, . �m, sigma, n � ). the use and care of the animals in these experiments were approved by the chancellor’s animal research committee at the university of california los angeles. stimulation protocol unipolar point stimuli were delivered at the edge of the coverslip using a grass stimulator triggered by computer-controlled pacing sequences. specimens were paced at hz. then a rapid pacing address for reprint requests and other correspondence: j. n. weiss, division of cardiology, mrl bldg., david geffen school of medicine at ucla, los angeles, ca (e-mail: jweiss@mednet.ucla.edu). the costs of publication of this article were defrayed in part by the payment of page charges. the article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. am j physiol heart circ physiol : h –h , . first published january , ; doi: . /ajpheart. . . - / $ . copyright © the american physiological societyhttp://www.ajpheart.org h downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . protocol was performed, initially at -ms cycle length, decremented by ms every eight beats until reaching -ms cycle length. optical mapping system monolayers were stained by immersion into oxygenated tyrode’s solution (in mm, nacl, . kcl, . cacl , . nah po , mgcl , hepes, and glucose, ph . ) at °c, containing the fluorescent voltage dye rh- ( �m for min) and/or the ca dye rhod -am or rhod-ff ( �m for min) plus . % (wt/wt) pluronic (molecular probes, eugene, or), and then placed in a perfusion bath. experiments were conducted at °c. fluorescence was excited by two light sources (each with light-emitting diodes; luxeon, ontario, canada) filtered at � nm. the emitted fluorescence was separated using a dichroic mirror (at nm), directed to two separate cameras with their corresponding emission filters ( nm for rh- and nm for rhod /rhod-ff). charge-coupled device camera recordings. we used electron- multiplying, back-illuminated, cooled charge-coupled device (ccd) cameras (photometrics cascade �), with � spatial resolution at . - to -ms per frame. signals were digitized with bits of precision. single-dye staining with rhod -am (n � ) or rh- (n � ) confirmed the absence of cross talk between voltage and ca signals, and simultaneous voltage and ca mapping were performed in specimens. photodiode array. in nine additional experiments, voltage (rh- staining) was also recorded optically using a photodiode array (pda) (wutech), consisting of hexagonally arranged sites with -�m spacing. data analysis raw data were processed with custom-written software. when the goal was to optimize spatial resolution (at the expense of temporal resolution), ccd recordings were subjected to ) spatial filter ( � binning); ) -point median temporal filter; ) polynomial curve fitting to eliminate baseline drift caused by photobleaching; and ) range normalization. after processing, this yielded a final spatial resolution of � ( � �m/pixel) and a temporal resolution of – ms ( – ms per frame � ). when the goal was to optimize temporal resolution (at the expense of spatial resolution), ccd re- cordings were subjected to spatial filter ( � binning), and the other same algorithms were applied. this yielded a postprocessing spatial resolution of � ( � �m/pixel) and a final temporal resolution of ms ( . ms per frame � ). as an alternative method to enhance temporal resolution, we also used pda recordings in place of ccd recordings in some experiments, with no spatial or temporal filtering applied to the pda traces. apd was measured at % repolarization (apd ) using custom- written software to detect the time to repolarize to % from the peak amplitude of the ap back to the take-off point of the same ap. this may have modestly underestimated the amplitude of apd alternans when the take-off point was also alternating, but did not affect the detection of the onset or offset of apd alternans. we chose apd rather than apd at % repolarization (apd ) to minimize measure- ment ambiguity when full repolarization was not achieved at rapid pacing rates. diastolic interval (di) was measured from % repolar- ization of the previous ap to the next ap upstroke. cai transient amplitude was measured as the absolute increase in fluorescence (minimum to peak in arbitrary units). apd and cai alternans were considered to be present when apd and cai transient amplitude alternated by � ms and � %, respectively, on a beat-to-beat basis, and persisted until the end of the pacing protocol. difference maps between apd and cai transient amplitude on successive beats were generated and shaded red if the differences were positive, and green if negative. thus, during spatially discordant alternans, red and green regions alternated, separated by a nonalternating nodal line (white). cv was estimated using voltage signal with custom-written software, which detected the activation time of each pixel and mea- sured the difference in activation time between two points separated by a known distance located orthogonally to propagation direction. to avoid artifacts from direct activation of myocytes near the pacing site (virtual electrode), cv was calculated from the average difference in activation time between a site mm from the pacing site and three equidistant sites as remote as possible from the pacing site. apd and cv restitution curves were generated by plotting apd or cv vs. di and fitting to a single exponential. apd restitution slopes were calculated by differentiating the exponential fits using origin (microcal) software. maximum apd restitution slope was ob- tained by calculating the value of this slope at the shortest di that elicited an ap. the conduction block at the nodal line was defined as block within mm of the nodal line. statistics data are shown as means � sd. statistical tests included � test and student’s t-test. p values � . were considered significant. results electrophysiological properties before and after bay k we examined monolayers under control conditions, with optical mapping of cai alone (n � ), voltage alone (n � ), or both simultaneously (n � ). a bright-field image of a representative confluent monolayer meeting our inclusion cri- teria is shown in fig. a. figure b shows representative optical traces of membrane voltage [voltage fluorescence (fv)] and cai [cai fluorescence (fca)]. apd averaged � ms, and cai transient duration (full width at half-maximal ampli- tude) averaged � ms. in all monolayers meeting our fig. . cardiomyocyte monolayers. a: phase-contrast microphotograph of a typical cardiomyocyte monolayer meeting the inclusion criteria of homoge- neous confluence. b: representative optical voltage [voltage fluorescence (fv)] traces [photodiode array (pda) or charge-coupled device (ccd) camera as labeled, using rh- ] and intracellular ca (cai) [cai fluorescence (fca)] trace (ccd camera, using rhod -am) from representative sites in a monolayer. c and d: isochronal maps obtained from the voltage data show uniform propagation during pacing at -ms pacing cycle length (pcl) from orthog- onal pacing sites at the top (c) or right (d) side of the same monolayer. h discordant alternans in monolayers ajp-heart circ physiol • vol • march • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . inclusion criteria, propagation was uniform without conduction block when paced at hz from either of two orthogonally positioned sites (fig. , c and d). cv averaged . � . m/s in both orthogonal directions. figure , a and b, shows the effects of bay k on apd and cai transient amplitude and duration. during pacing at hz, bay k increased apd by % from � to � ms (p � . ). the net amplitude of the cai transient fluorescence signal increased by � % (p � . ), which is likely to be an underestimate of the change in absolute ca concentration during the cai transient due to the nonlinearity of the dye. the cai transient duration increased by % from � to � ms (p � . ). after bay k , cv during pacing at hz was similar to control ( . � . vs. . � . m/s, p � not significant). figure b compares apd restitution curves before and after bay k . maximum apd restitution slope averaged . � . under control conditions and . � after bay k (fig. b). the range of dis over which apd restitution slope was � was – ms (correspond- ing to pacing cycle lengths � ms) in control conditions, and – ms (corresponding to pacing cycle lengths � ms) after bay k (fig. d). figure c shows that significant cv restitution was present (solid black line), and cv was further reduced by bay k at short dis (red line). fig. . spatially concordant and discordant cai transient alternans during rapid pacing. top: spatial patterns of cai transient alternans over the surface of a monolayer for two successive beats during pacing at ms (two left panels) and ms (two right panels). red and green indicate positive and negative beat-to-beat changes in cai transient amplitude, respectively. at -ms pcl, alternans is spa- tially concordant (i.e., alternans map shows all green on the first beat, and all red on the next beat). as pcl was shortened to ms, alternans became spatially discor- dant. two areas (green and red) alternated out of phase, separated by a white nodal line (nl; dotted line). fca traces recorded at sites a and b in the monolayer (see top) show spatially concordant alternans at ms, transition- ing to sda at ms. large (l) and small (s) cai transients are labeled. 	, change; a.u., arbitrary units. fig. . action potential duration (apd) and conduction velocity (cv) restitution curves before and after bay k . a: optical fv and fca traces before (black) and after (red) bay k . apd at % repolarization (apd ) and cai transient amplitude increased after bay k . (changes in diastolic fca after bay k could not be measured accurately due to photobleaching.) b: dynamic apd restitution (apdr) curve [apd vs. diastolic interval (di)] before (black) and after (red) bay k . the onset of apd alternans after bay k is indicated by the red arrow (alt). the di at which apdr slope exceeds (slope � ) before (black arrow) and after (red arrow) bay k is also shown. note that the apdr slope was � at the onset of apd alternans. c: cv restitution before (solid black line and symbols) and after (solid red line and symbols) bay k . also shown are the cv restitution curves in monolayers that did (dashed line and open squares) or did not (dotted line and open circles) develop spatially discordant alternans (sda) before bay k . all monolayers developed sda after bay k . for all cases, cv is shown as a percentage of its value at -ms pcl. d: slope of apdr vs. di calculated from monoexponential fits, before (black) and after (red) bay k . dotted line indicates slope � . h discordant alternans in monolayers ajp-heart circ physiol • vol • march • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . thus, by increasing the l-type ca current, bay k increased apd and steepened apd restitution slope, increased cai transient amplitude and duration, and caused cv to slow to a greater extent at short dis. all of these effects are predicted to promote dynamically induced spatially discordant apd and cai alternans ( , ). pacing-induced cai transient and apd alternans under control conditions spatially concordant and discordant alternans. monolayers could be paced to a cycle length of � ms before : capture was lost. spatially concordant cai transient alternans was induced in all monolayers, at an average pacing cycle length of � ms, and became spatially discordant in of specimens ( %) at an average pacing cycle length of � ms. cv restitution was more prominent in mono- layers that transitioned to spatial discordant alternans (fig. c, open squares) than in those that did not (open circles). figure shows an example of the cai transient progressing from spatially concordant to discordant alternans. since fast et al. ( , ) reported that the high-ca affinity of rhod -am can lead to spuriously prolonged cai transients, we also stained three monolayers with rhod-ff-am (lower ca affinity) in place of rhod -am. the incidence or onset of ca alternans did not differ significantly (data not shown). under control conditions, we were limited in our ability to detect apd alternans using the standard high-spatial resolution ccd configuration because of the postprocessing temporal resolution of only – ms per frame. however, when we sacrificed spatial resolution (see methods) to improve the postprocessing temporal resolutions to ms (ccd camera) or . ms (pda system), apd alternans was detected in of monolayers ( %). the maximum apd alternans magnitude averaged � ms and progressed from spatially concordant fig. . spatially concordant and discordant apd alternans during rapid pacing. a: spatially concordant apd alternans. apd difference maps between consecutive beats show that the entire tissue alternated in phase at -ms pcl. tracings show optical fv recordings at sites a and b, with l and s labels indicating long and short apd, respectively. b: spatially discordant apd alternans. apd difference maps between consecutive beats demonstrate two regions al- ternating out of phase, separated by a nl (black line) oriented perpendicular to propagation di- rection. tracings show optical recordings at sites a and b. fig. . effects of pcl and site on nl behav- ior during spatially discordant apd alternans. a: pcl effect on nl position. left panel shows the isochronal activation map during pacing at -ms cycle length (note slow cv relative to fig. b). right panels show apd difference maps between successive beats at pcls of , , and ms. the nl (dashed line) moved progressively closer to the pacing site as the pcl decreased. b: pac- ing site dependence of nl position. first and third panels show ischronal activation maps when the monolayer was paced at -ms cycle length from either the top left corner (first panel) or bottom right corner (third panel). second and fourth panels show cai transient amplitude difference maps between successive beats during sda at a pcl of ms for the two different pacing sites. note that the nl (dashed line) reoriented its position to remain roughly circumferential with respect to the pacing site. h discordant alternans in monolayers ajp-heart circ physiol • vol • march • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . to spatially discordant apd alternans in of monolayers ( %). figure shows examples. nodal line behavior. theoretical studies ( , ) predict that during spatially discordant alternans, nodal lines generated dynamically by cv restitution should move closer to the pacing site at faster pacing rates and change their orientation to remain perpendicular to the pacing site when the pacing site is altered, unlike nodal lines due to tissue heterogeneity. we found that apd and cai nodal lines exhibited both of these features. figure a shows an example of an apd nodal line moving closer to the pacing site as the pacing cycle length was shortened. figure b shows an example of a cai transient nodal line reorienting when the pacing site was moved from the top left to bottom left corner of the monolayer. these findings indicate that nodal lines formed during spatially discordant alternans arise predominantly from a dynamic mechanism, rather than tissue heterogeneity ( ). effects of bay k on apd and cai transient alternans spatially concordant and discordant alternans. we exam- ined monolayers after exposure to bay k , mapping either ca alone (n � ), voltage alone (n � ), or both simultaneously (n � ). after bay k , : conduction failed at a longer pacing cycle length of � ms, compared with � ms before bay k (p � . ). spartially concordant cai transient and apd alternans were induced in all ( of ) monolayers after bay k , with the onset at an average pacing cycle of � ms, compared with � ms in control conditions (p � . ). bay k also increased the incidence of spatially discordant cai transient and apd alternans from to % (p � . ), which occurred at an average pacing cycle length of � ms, compared with � ms before bay k (p � . ). the maximum amplitude of apd alternans was signif- icantly greater ( � vs. � ms in control, p � . ), as was the maximum amplitude of cai transient alternans ( � vs. � % in control, p � . ). nodal line behavior after bay k . pacing rate and site had similar effects on nodal line position and orientation after bay k (not shown) as under control conditions. bay k significantly increased the maximum number of nodal lines. figure shows an example of multiple apd nodal lines after bay k . the average number of cai nodal lines increased from . � . to . � . (p � . ; fig. c).), and the average number of apd nodal lines from . � . to . � . (p � . ; fig. c). thus bay k significantly shortened the length scale over which nodal lines formed during spatially discordant cai transient and apd alternans. spatially discordant alternans, conduction block, and reentry we next investigated whether spatially discordant alter- nans could create steep enough apd gradients to cause conduction block and initiation of reentry. under control conditions, as pacing rate increased, : conduction with normal propagation was followed by : conduction with lines of block, and finally : conduction block, in of specimens ( %). spatially discordant apd or cai transient alternans preceded the development of lines of conduction block in % ( of specimens), and the site of block occurred near the nodal line in % of the cases ( of specimens). localized conduction block resulted in initia- tion of reentry in of specimens ( %). bay k increased the incidence of conduction block during rapid pacing to % ( of , p � . ), and lines of block were preceded by spatially discordant alternans in all cases. bay k also increased the incidence of reentry during rapid pacing from to % ( of specimens, p � . ). the mechanism of conduction block during spatially discor- dant alternans is illustrated in fig. . after bay k exposure, two regions (sites a and b) alternated out of phase in both cai and apd, separated by a nodal line. note that the red and green regions of the change (	) in cai and 	apd maps (fig. a) match each other, indicating that larger cai transients are associated with long apd and vice versa. this positive coupling relationship between cai transient amplitude and apd was observed in all monolayers in which voltage and cai were simultaneously recorded, both in control and after bay fig. . effects of bay k on nls. after bay k , the maximum number of nls during spatially discordant apd and cai transient alternans increased significantly. a: an example from a monolayer during pacing at -ms cycle length after bay k . apd difference maps between two successive pairs of beats ( – and – ) show three nls separating regions alternating out of phase. b: fv traces at sites a, b, and c are shown. bumps in the traces are artifacts, but the alternation between l and s apd is clearly visible. c: left side compares the number n of cai nls before and after bay k in individual preparations. right side shows the average number of apd nls under control (con) conditions and after bay k obtained from different preparations. h discordant alternans in monolayers ajp-heart circ physiol • vol • march • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . k . figure b shows that, for beat (black line), apd become shorter as the paced impulse propagated from site a with long apd to site b with short apd. for beat (red line), the apd gradient was reversed. as beat propagated from site a to site b, the gradient in refractoriness left over from beat was too steep for successful propagation, causing a line of conduction block to develop close to the nodal line. the graph of apd along the line ab in fig. b shows that block occurred where the spatial gradient in apd was steepest, reaching ms/mm. thus ) spatially discordant alternans can promote a large enough apd gradient over a -mm span in monolayers to induce conduction block ( ); ) the nodal line marks the location of steepest spatial apd gradient; ) conduction block occurs at the nodal line when the new impulse propagates from short-to-long apd direction of the prior beat, which corre- sponds to the short-to-long cai transient direction (i.e., positive voltage-cai coupling) ( ); and ) cai transient alternans maps correlate highly with apd alternans maps in their ability to predict the site of nodal lines and conduction block. figure illustrates the initiation of reentry in a monolayer exposed to bay k , using ca mapping alone. propagation was initially uniform (fig. a). as pacing cycle length de- creased to ms, spatially discordant alternans appeared, with three discordantly alternating regions separated by two nodal lines, nl and nl (fig. b). for beat , unidirectional conduction block occurred immediately past nl as the im- pulse propagated from site a toward site b, in the small-to-large cai transient direction (corresponding to short-to-long apd direction for positive voltage-ca � coupling), as shown in the spatiotemporal plot in fig. c. figure d shows the isochrome map for beat , illustrating that conduction block was localized to the central region of the monolayer, allowing propagation around the edges to reenter the region of block from the opposite direction, initiating reentry. discussion spatially discordant alternans in cardiac monolayers the novel findings of this study are that spatially discordant apd and cai alternans ) can be induced by rapid pacing in a relatively isotropic d cardiac tissue in which the entire sur- face can be optically mapped, so as to exclude unmapped subsurface electrical activity from influencing the outcome, and can create sufficient repolarization gradients to cause conduction block and reentry; ) share features with intact d ventricular muscle, indicating that it originates primarily from dynamic factors such as cv restitution ( , ) rather than tissue heterogeneity; and ) are exacerbated by the ca channel agonist bay k , as predicted theoretically for an agent fig. . conduction block during sda after bay k . a: simultaneous apd (left) and cai transient (right) alternans amplitude difference maps for beats – during pacing at ms, demonstrating sda with one nl (dashed line). note that the red and green regions in the 	apd and 	cai maps are matched, indicating that the larger cai transients are associated with longer apd, and vice versa (i.e., positive cai-apd coupling). b: the apd gradient along the line between sites a and b from panel a for beats (black) and (red). the apd gradient was steepest near the nl, reaching ms/mm. c: a space-time plot of fca for beats – . fca along the line between sites a and b is shown on the vertical axis (with red-yellow indicating highest ca), and time is shown on the horizontal axis. during beats – , spatially discordant cai alternans is present, as seen on beat by the red-yellow fca, indicating high ca amplitude near site a, and blue-red fca indicating lower ca amplitude near site b, which then reverses on beat . the nl is indicated by the dashed white line. during beat , the cai transient is small (corresponding to short apd) near site a and large (corresponding to long apd) at site b. when the beat propagates from site a toward b, in the direction of the increasing cai transient and apd gradient during beat , conduction block occurs near the nl (dashed white line). d: simultaneous optical fv and fca traces at sites a and b for beats – , showing conduction block : from beat onwards. note again the positive coupling relationship between cai transient amplitude and apd. e: isochrone activation map illustrating the crowding of isochrone lines before block near site b in the top right corner. h discordant alternans in monolayers ajp-heart circ physiol • vol • march • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . that steepens apd restitution, promotes cai overload, and increases cv restitution. the following observations strongly support a dynamic, rather than tissue heterogeneity-based, mechanism in this prep- aration. ) under control conditions, cv restitution was more prominent in the % of monolayers that transitioned from spatially concordant to spatially discordant alternans, com- pared with the % of monolayers that did not (fig. d). by increasing cv restitution, bay k increased the inci- dence of spatially discordant alternans to %. ) bay k also increased the number of nodal lines (i.e., decreased the spacing between nodal lines), consistent with theoretical predictions that the length scale of nodal line spacing is decreased by increasing cv restitution and the amplitude of apd alternans ( , ). ) computer simulations ( , ) predict that nodal lines generated dynamically by cv restitution should move toward the pacing site as pacing cycle length decreases and should reorient to remain circumferential with respect to the pacing site when the pacing site is changed. in contrast, nodal lines formed as a result of tissue heterogeneity do not share these features ( ). in our preparation, we found that nodal lines behaved according to the predictions for the dynamic cv restitution mechanism (fig. ), consistent with recent observations on nodal line behavior in intact d rabbit ventricular tissue ( ). in the case of intact heart tissue, signif- icant macroscopic tissue heterogeneities are unavoidably present. monolayers, when carefully prepared to be fully con- fluent, lack gross macroscopic tissue heterogeneity. however, microscopic heterogeneities still exist and may exert important effects. this is evident from our finding that spatially discor- dant alternans was able to induce localized conduction block (fig. ), as required for reentry initiation (rather than circum- ferential conduction block along the whole wavefront, which cannot induce reentry). the magnitude of the heterogeneity required for this symmetry-breaking effect is unclear. possibly, it could be very minor at normal heart rates, but amplified by dynamic factors at rapid pacing rates ( ), which remains an interesting area to explore in future studies. cellular mechanisms of repolarization alternans although apd alternans is always dually influenced by both apd restitution steepness ( , ) and cai cycling dynamics ( , ), the onset of apd alternans is driven by whichever factor becomes unstable first ( ). in neonatal rat ventricular myocyte monolayers, the following observations favor the cai-driven instability as responsible for the onset of alternans. first, the onset of alternans occurred at a pacing cycle length at which apd restitution slope was considerably � (fig. ), although short-term memory effects ( ) or transient alternans (see below) might conceivably account for this. second, under control conditions, the onset of ca alternans preceded the detection of apd alternans. this could be due to subtle apd alternans below our detection threshold ( . – ms), but also could be explained if primary cai transient alternans had balanced effects on ca-sensitive currents tending to prolong and shorten apd (e.g., na-ca exchange and l-type ca current inactivation, respectively). although cai-driven alternans might seem less likely in neonatal rat ventricular myocytes because of their immature sr, recent studies have shown that, after several days in culture (equivalent to the time monolayers were studied here), neonatal ventricular myocytes resemble adult ventricular myocytes more closely than freshly dissoci- ated neonatal myocytes, both histologically and functionally with respect to t tubule and sr ca cycling properties ( , ). the mechanism underlying prominent cv restitution ob- served in the monolayers is unclear. cv restitution is usually attributed to the kinetics of the na current’s recovery from inactivation. in intact d ventricular tissue, cv restitution is typically not engaged until very short dis, consistent with normally rapid na current recovery kinetics. in monolayers, however, cv varied almost continuously over a wide range of dis (fig. d). although it is possible that cultured neonatal rat ventricular myocytes have much slower na channel recovery kinetics than adult ventricular cells, another potential explana- tion may involve heart rate sensitivity of gap junctional con- ductance, the other key determinant of cv. during rapid pacing, either intracellular acidosis or elevated cai levels could reduce gap junction conductance and progressively slow cv. the observation that bay k exacerbated cv restitution (fig. d) is consistent with this possibility, since bay k increases diastolic cai in chick embryonic myocytes ( ), and cabo et al. ( ) reported that bay k caused cv slowing due to decreased gap junctional conductance in infarcted hearts. a limitation of our study, however, is that we could not reliably measure changes in diastolic cai in this preparation, as previously noted by fast et al. ( ) in this preparation, although it is possible in other preparations ( , ). fig. . conduction block initiating reentry during sda. a: isochrone activation map at a -ms pcl, showing uniform propagation after bay k . fca only was recorded in this monolayer. b: at -ms pcl, cai transient amplitude difference maps between beats – (second panel) and beats – (third panel) showed spatially discordant cai alternans, with nls (nl and nl ) separating out-of-phase regions. c: space-time plot along the line between sites a and b shows that conduction block occurred at beat near nl (dashed white line), as the impulse propagated into the region with the larger cai transient on beat (corresponding to longer apd). the next activation occurs from the opposite direction. d: isochrone activation maps for beats , , and show that conduction block during beat was localized to the center of the monolayer, allowing beat to propagate around the edges and reenter the area of conduction block from the other direction, initiating reentry (beats and ). h discordant alternans in monolayers ajp-heart circ physiol • vol • march • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . spatially discordant alternans and initiation of reentry pastore et al. ( ) demonstrated that spatially discordant alternans can produce spatial repolarization gradients steep enough to cause unidirectional conduction block and reentry. we have extended their findings by demonstrating the rele- vance of the nodal line to the site of conduction block leading to initiation of reentry. under control conditions, conduction block occurred near the nodal line in % of cases. after bay k , which shortened the spacing between nodal lines, the incidence of conduction block increased and was preceded by spatially discordant alternans in % of cases. as predicted theoretically ( , ) and demonstrated experimentally (fig. a), the nodal line marks the location of steepest apd and ca transient gradients. the apd gradient at the nodal line in fig. a reached ms/mm, which agrees well with the experimental estimate of � . ms/mm necessary for conduction block mea- sured in intact ventricular tissue ( ), as well as with theoret- ical predictions ( ). in monolayers, nodal lines consistently predicted the location of conduction block, such that block occurred when propagation was in the direction of short-to- long apd (i.e., increasing refractoriness) and small-to-large ca transient of the previous beat. bay k increased the incidence of conduction block by shortening the length scale of spatially discordant alternans, which is equivalent to steepen- ing the spatial apd gradient. limitations at any given pacing rate, apd and cai transient alternans can be either transient or persistent. any sudden change in heart rate produces transient alternans, which then decays exponentially back to the steady-state apd or cai transient amplitude for the new heart rate, typically with a beat constant of three to four beats ( ). for practical reasons, we used a pacing protocol in which pacing cycle length was decreased every eight beats by ms, which may not unequivocally delineate between transient and persistent alternans. thus it is possible that the alternans induced by our pacing protocol was really transient alternans maintained by the continually increas- ing pacing rate. to eliminate very transient alternans, we required that alternans, once started, had to persist throughout the remainder of the pacing protocol (or until conduction block occurred), but we cannot state conclusively that the monolay- ers exhibited truly persistent alternans. however, from the perspective of arrhythmogenesis, the key factor is whether the gradient in refractoriness caused by spatially discordant apd alternans is steep enough, even transiently, to cause conduction block and initiating reentry. our findings show unequivocally that the gradients in refractoriness achieved during spatially discordant alternans in d monolayers, whether transient or persistent, were sufficient to cause conduction block and reen- try, especially after bay k . nevertheless, neonatal myocytes have different electrophysiology and cai cycling features than adult human ventricular myocytes, so that the relevance to the arrhythmogenic consequences of spatially discordant alternans in humans remains speculative. however, if the fundamental dynamics conferred by apd restitution, cai cycling, and cv restitution play comparable roles in the human heart, they must be considered in the future development of antiarrhythmic therapies. acknowledgments we thank yohannes shiferaw, zhilin qu, and alan garfinkel for helpful discussions. grants this study was supported by the spanish society of cardiology (electrophysiology fellowship grant to c. de diego), the american heart association (grant y to c. de diego; grants y and y to m. valderrábano), the national heart, lung, and blood institute (grants p hl and p hl to j. n. weiss), and the laubisch and kawata endowments (to j. n. weiss). present address of m. valderrábano: cardiology division, methodist debakey heart center, fannin st., suite , houston, tx . references . cabo c, schmitt h, wit al. new mechanism of antiarrhythmic drug 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wh, schneiderbanger k, schubert p, didie m, munzel f, heubach jf, kostin s, neuhuber wl, eschenhagen t. tissue engineering of a differentiated cardiac muscle construct. circ res : – , . h discordant alternans in monolayers ajp-heart circ physiol • vol • march • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . validating culture-and gender-specific constructs: a mixed-method approach to advance assessment procedures in cross-cultural settings validating culture- and gender-specific constructs: a mixed-method approach to advance assessment procedures in cross-cultural settings john h. hitchcock caliber, an icf international company sreeroopa sarkar bonnie k. nastasi gary burkholder walden university kris varjas georgia state university asoka jayasena university of peradeniya, sri lanka an earlier version of this work was presented in a symposium conducted at the annual meeting of the american education research association. funding for the early phases of this work was provided through grants to the third author from the soci- ety for the study of school psychology and the university at albany. the authors ac- knowledge bill disch for his support with statistical analysis. address correspondence to: john hitchcock, caliber, an icf international com- pany, rosehaven street, suite , fairfax, va (e-mail: jhitchcock@ icfcaliber.com). [haworth co-indexing entry note]: “validating culture- and gender-specific constructs: a mixed-method approach to advance assessment procedures in cross-cultural settings.” hitchcock, john h. et al. co-pub- lished simultaneously in journal of applied school psychology (the haworth press, inc.) vol. , no. , , pp. - ; and: multicultural issues in school psychology (ed: bonnie k. nastasi) the haworth press, inc., , pp. - . single or multiple copies of this article are available for a fee from the haworth doc- ument delivery service [ - -haworth, : a.m. - : p.m. (est). e-mail address: docdelivery@ haworthpress.com]. available online at http://japps.haworthpress.com  by the haworth press, inc. all rights reserved. doi: . /j v n _ http://japps.haworthpress.com summary. despite on-going calls for developing cultural compe- tency among mental health practitioners, few assessment instruments consider cultural variation in psychological constructs. to meet the challenge of developing measures for minority and international stu- dents, it is necessary to account for the influence culture may have on the latent constructs that form a given instrument. what complicates matters further is that individual factors (e.g., gender) within a culture necessi- tate additional refinement of factor structures on which such instruments are based. the current work endeavors to address these concerns by demonstrating a mixed-methods approach utilized to assess construct val- idation within a specific culture; and in turn develop culturally-specific in- struments. qualitative methods were used to inform the development of a structured self-report by gaining detailed knowledge of the target culture and creating items grounded in interview and observational data. factor analysis techniques and triangulation with qualitative analyses validated these findings. previous work (sarkar, ) suggested a number of gen- der-specific perceptions of mental health constructs within the target culture and these were investigated using additional mixed-method anal- yses. this article demonstrates an emerging mixed-method technique for developing culturally sound assessment tools, offers guidance on how to incorporate the overall approach in assessment, and provides a basis for thinking critically about the use of existing instruments when working with diverse populations. doi: . /j v n _ [article copies available for a fee from the haworth document delivery service: - -haworth. e-mail address: <docdelivery@haworthpress.com> web- site: <http://www.haworthpress.com> © by the haworth press, inc. all rights reserved.] keywords. assessment, environment, gender, culture, validation researchers, policy makers, professional organizations, and mental health practitioners have repeatedly called attention to the lack of cul- turally appropriate instruments for ethnic minorities, and the need to consider cultural factors in mental health programming (american psy- chological association [apa], , ; hall & okazaki, ; tanaka, ebreo, linn, & morera, ; united states department of health and human services [usdhhs], , ). an example eas- ily demonstrates why such calls are made. egeland, hostetter and eshleman ( ) describe how culture can influence assessment multicultural issues in school psychology http://www.haworthpress.com when describing manic behaviors of old order amish suffering from bipolar disorder. suppose a client presents some of the height- ened psychomotor activities associated with the disorder, but otherwise engages in such behaviors as using pay phones, dresses as a typical american might, is sexually active with more than one partner and uses machinery. absent knowledge of amish cultural norms, such behavior might easily be viewed as typical and a critical diagnosis could be missed. in this example, even cursory knowledge of the culture reveals just how suspect these behaviors might be, and such recognition would facilitate proper diagnosis. unfortunately, however, dealing with cul- tural factors is almost never this straightforward because cultural varia- tion extends into a myriad number of groups and related effects can be subtle (see castillo, ). recognizing the complexities of culture represents a critical compo- nent of school psychology practice (rogers et al., ); indeed, an em- pirical base supports the idea that cultural factors play an important role in influencing mental health (apa ; nastasi, moore, & varjas, ). one explanation is that mental health constructs can be viewed differently by individuals from varied cultures, and perception is influ- enced by different agents of socialization (e.g., parents, school, and peers) that shape people’s beliefs, motives, attitudes, and behavior via socialization practices and expectations. other variables such as gender can influence how mental health constructs are formed (brannon, ; sarkar, ). clear and well-replicated gender differences in mental illnesses like depression, anxiety or schizophrenia have been reviewed by mental health researchers (romans, ). in addition, researchers have shown that physiological, social, psychological, and environmental factors each predict gender differences in terms of mental health constructs (anshell, porter, & quek, ; bird & harris, ; block & robins, ; knox, funk, elliott, & bush, ; ptacek, smith, & dodge, ). broader socio-cultural factors also play an important role in in- fluencing mental health of individuals through the process of socializa- tion. for instance, socialization practices within a particular culture influence the process of gender role socialization, and researchers have shown how this socialization process has made women more vulnerable to mental health problems (das & kemp, ; das dasgupta, ). through its various agents of socialization, culture fosters development of gender role attitudes, beliefs/stereotypes, and gender-specific behav- iors which contribute to the gender differences in mental health con- structs (sarkar, ). hitchcock et al. in view of the findings of gender-role socialization, investigating the gender differences in mental health constructs in the context of culture is important in providing appropriate services (baxter, ; usdhhs, ; ). however, there is a dearth of research exploring the role of culture and particularly gender as a cultural variable influencing men- tal health of individuals; this is especially true in school psychology (henning-stout & brown-cheatem, ). in culturally diverse set- tings, it is therefore important to explore if there is variation in the defi- nition of mental health constructs. because many u.s. public school systems represent multicultural contexts (rogers et al., ), the meth- ods described below have clear applications for practice. what complicates matters is that most psychological instruments do not adequately address the influence of culture on functioning, especially for ethnic minority groups (apa, ; padilla, ; usdhhs, , ). some literature suggests that members of a cultural majority tend to be unaware of cultural influences (sue, bingham, porche-burke & vasquez, ); not surprisingly, researchers often generate instru- ments with little regard for these factors (rogler, ). failure to ad- dress cultural differences in assessment may therefore lead to problems with construct validity and subsequent efforts to develop interventions based on such assessment. the objective of this article is, therefore, to address the shortcom- ings in typical assessment in multicultural settings by demonstrating a mixed-methods approach (see tashakkori & teddlie, ) for devel- oping and validating culturally specific instruments. the approach combines the use of ethnographic and factor analytic methods to de- velop and test psychological instruments for students located in sri lanka. some findings presented here will not have immediate relevance outside of sri lanka, although the methods can be readily applied to fu- ture studies concerned with mental health assessment. the overall approach entailed several stages: (a) a two-year ethnog- raphy conducted in the target culture for the purpose of understanding self-concept issues of adolescents, (b) developing and administering a series of culture-specific instruments that further assess these issues, and (c) demonstrating a mixed-method approach for identifying and vali- dating psychological constructs that are specific to the target culture. an overview of this process can be found in hitchcock and colleagues ( ). the primary purpose of the earlier publication was to illustrate the methodology with a specific focus on the substantive area of self-con- cept (as defined by harter, ) and concerns for adolescents in sri lanka. this paper extends the previous work through reanalysis of quali- tative data (that have not been previously published) and quantitative multicultural issues in school psychology analysis of a different dataset. the earlier work focused on students’ per- ceptions of how their parents valued various competencies and behav- iors; this article focuses on student perceptions of their competencies, and identifying gender-specific differences of these perceptions. foundations the work described herein employed the ecological-developmental model of mental health (edmmh; nastasi et al., ) as the concep- tual framework for investigating individual and cultural factors related to mental health. the edmmh has its foundations in psychology and an- thropology (nastasi & dezolt, ; nastasi, varjas, sarkar, jayasena, ) and has been used to guide the development of culture-specific definitions of key constructs through the integration of cultural factors related to the experiences of participants. an ecological approach to hu- man development involves scientific study of a ‘progressive, mutual ac- commodation’ between an individual and the environment, in view of the social, cultural and historical contexts (bronfenbrenner, ; ). reflected in the edmmh is the conceptualization of individual mental health as an interaction among the personal factors–self-perceptions of culturally valued competencies, personal resources, and personal vul- nerability, with the cultural factors–cultural norms, competencies val- ued within the culture, social-cultural stressors and resources, and socialization agents and practices. the model promotes the idea of de- veloping a sense of normative cultural values and any conflicts in values among groups within a context. this allows for an assessment of devi- ant behavior given cultural norms and stressors derived from lack of re- sources and competing group needs, both of which are representative of mental health concerns. edmhh can help assess what might be cultur- ally deviant, but more importantly, it is a framework that is consistent with well-established conceptions of risk and protective factors related to mental health/illness of individuals and prevalence within communi- ties (e.g., elias & branden, ), models of stress and coping (e.g., lazarus & folkman, ) and personal-social competence (nastasi & dezolt, ). it also is consistent with recent developments in the field of positive psychology (frydenberg, ; snyder & lopez, ). qualitative methodology and context the research involved the combined use of qualitative (e.g., inter- views, focus groups) and quantitative (rating scales) data collection hitchcock et al. methods to assess the contextual environment of adolescents in sri lanka. the study was conducted within the municipality of kandy, the second largest city in the country with a population of approximately , . its schools draw students from the urban community and sur- rounding villages and the socioeconomic status of community members ranges from lower to upper class (nastasi et al., ).the qualitative study was formative in nature and involved the assessment of the mental health needs of adolescents (see nastasi et al., ). this study was conducted with the long-term goal of developing school-based mental health programs in that country. the findings from that work were used to develop and evaluate a culture-specific mental health assess- ment tool and intervention program for promoting mental health among school-age children in sri lanka. sri lanka has experienced advances in education, health and general standard of living (united nations development programme, ; the world bank in sri lanka, ). however, the last twenty years of sri lankan history has been marked by internal tension resulting from high unemployment rates, civil war, ethnic tension and the youth insur- rection ( - ; gunaratna, ). these factors have contributed to an unstable economy with dwindling per capita income as well as po- litical unrest and mental health problems among the youth. in an effort to gather data on the mental health of students, researchers conducted group interviews with adolescents ( groups) and teachers ( groups), and individual interviews with principals from schools to explore the following constructs: (a) cultural definition of mental health/personal- social competence and adjustment difficulties, (b) cultural mechanisms for socialization and development of mental health/personal-social com- petence, (c) social stressors as viewed by the adolescents, (d) mecha- nisms/personal resources for coping with those and other everyday life stressors, and (e) existing resources within the schools and community for securing mental health services. the interviews focused on gather- ing a wide range of data with regard to the definition of key constructs. interviews were therefore conducted in an open-ended, semi-structured format (nastasi, varjas, sarkar & jayasena, .) interview questions were generated in collaboration with an educa- tional sociologist/teacher educator and a child psychiatrist in sri lanka who had experience in schools (nastasi et al., ). additionally, questions were asked regarding more specific mental health related is- sues (e.g., stressors the youth face with regard to academics, types of ac- ademic adjustment difficulties that are prevalent among adolescents, family problems and related adjustment difficulties in the children, and multicultural issues in school psychology the ways in which children and adolescents cope with family problems). data were coded to identify and define the culture-specific individual and cultural constructs. efforts were made to understand the educa- tional and psychological concerns of students through their own per- spective, as well as those of school professionals. specific issues noted were high rates of mental health concerns such as substance abuse among peers and parents, stress associated with dating and teacher/par- ent interest in limiting interaction between the genders, and suicide. a variety of educational concerns associated with a high stakes examina- tion system and limited school resources also were noted. finally, the data reflected gender differences in several mental health constructs. for example, girls discussed sexual harassment, lack of freedom com- pared to their male peers and differential expectations for women; whereas boys expressed concern about violence, corruption, and unem- ployment. [see nastasi et al., for full report of methods and initial results.] in view of these findings, a re-analysis of qualitative data was performed to explore the gender differences in the definition of mental health constructs. these analyses formed the basis for a key approach that can combine quantitative and qualitative findings–ethnographic surveys. quantitative methodology ethnographically informed instruments are designed to reflect the experiences and constructs relevant to the target population (schensul & lecompte, ), thus yielding culture-specific measures. from the qualitative data, we developed self-report measures designed to as- sess adolescents’ perceived competencies and perceived value of the competencies from their own perspective and that of parents, teachers, and peers. the instruments used the likert-scale ( - point; = not at all, = some, = a lot) response format. on the perceived competencies measure (the focus of this paper), adolescents were asked to rate them- selves on a set of culturally defined competencies. scale and item con- struction were designed to reflect the full variation of data across age, gender, ethnicity, and ecological context. after piloting, back translat- ing (e.g., english Æ sinhalese Æ english, to insure accuracy of mean- ing) and refining via consultation with local experts with knowledge of the target culture, the instrument was administered in written form to students (n = ; males, females), grades - , ages - , hitchcock et al. across six schools which represented the range of the student population in terms of ethnicity, religion, and socioeconomic status. for further discussion of instrument development, see hitchcock and colleagues ( ). the strategy used for the quantitative analysis included a principal components analysis (pca) of the items in the survey instrument. promax (oblique) rotation was used to account for an expected correla- tion among components. items loading less than . on any scale, as well as complex items (those whose factor loadings across scales dif- fered by less than . ) were removed. cronbach’s alpha, a measure of internal consistency, was computed for all scales for the total sample and by gender. multivariate analysis of variance (manova) was used to compare first the scale scores resulting from the factor analysis by gender, then the individual items on any scale that demonstrated signifi- cant differences between boys and girls. findings from qualitative analyses formative qualitative data were collected by asking students and teachers to describe characteristics of socially-acceptable and unaccept- able behavior patterns for boys and girls. analysis of these data provided evidence of ( ) socially-acceptable “suitable” behavior, ( ) socially-un- acceptable “unsuitable” behavior and ( ) behavior that reflected atten- tion to the personal and interpersonal needs of adolescents (see table ). overall, gender differences in how unsuitable behavior is defined were noted. that is, both males and females recognized a broader unsuitable behavior construct but differed when defining it. in terms of suitable be- havior, both genders defined it the same way but differences were noted in the degree of expected compliance, with girls generally reporting that they were expected to engage in socially-acceptable behaviors more so than boys. in the area of personal/interpersonal needs, minor gender dif- ferences were reflected in the restricted movement and lack of inde- pendence and freedom of girls. following is an overview of each construct. suitable behavior in group interviews, both male and female adolescents described suit- able behavior to include “good” behavior and obedience (see table ). for example, suitable behavior entails following school rules and lis- multicultural issues in school psychology tening to adults. both male and female students in sri lanka argued that respect for elders is a major attribute of culturally valued suitable behaviors. suitable behavior also was characterized as conducting one- self well, such as by being well disciplined, humble, loyal, and trustwor- thy. respondents from both genders revealed that culturally acceptable suitable behavior also included being performance-oriented (e.g., per- forms well and assumes leadership in extracurricular and competitive activities). as previously noted, there were differences between boys and girls in terms of suitable behavior that focused on degree of compliance ex- pectations. that is, females were expected to exhibit more of these be- haviors than boys. female students described a girl showing suitable behavior as one who listens to what parents and teachers say, and ob- serves the rules and regulations. examples of such behaviors might be trying to avoid troubling others, giving up a seat to a teacher when they are on the bus, and observing customs and tradition of the country. girls indicated that such an individual talks nicely with others, sets a good ex- ample for others, performs community services, and loves the country. male students also indicated that such a person obeys the law and does not harm others, but focused more on avoiding disruptive behavior, as opposed to following a broad set of expectations. hitchcock et al. table . definition of suitable and unsuitable behavior with gender differ- ences highlighted cross-gender definitions of suitable behavior well behaved, obedient, disciplined, humble, respect elders, helpful, loyal, trustworthy, supportive, guides others (e.g., to correct path), makes effort for best performance in academics male-specific reponses female-specific responses obey laws of the country, leader, possesses the ability to address and solve problems, avoids trouble follows rules, talks and behaves nicely with people, loves the country–protects it, performs community services, sets example for others, treats and loves everyone equally–irrespective of ethnic origin cross-gender definitions of unsuitable behavior ill behaved (e.g., rude, tease girls), abusive (e.g., use abusive/foul languages), disruptive (e.g., interrupt others' work), noncompliance, school truancy, neglects responsibilities and/or obligations, not helpful (e.g., do not help friends in trouble) male-specific reponses female-specific responses throws tantrum, pouts, runs away from home, betrays friends, aggressive (e.g., fights, argues; abusive), abuses alcohol, drugs, steals or robs, joins the gang, carries weapons does not follow etiquette, lies, stubborn, interfering, disruptive, mistreats others, slanders, does not love the country, having romantic relationship with boys, acts like a boy unsuitable behavior both males and females referred to unsuitable behavior as being “ill behaved” (e.g., gets drunk and misbehaves with girls), aggressive (e.g., fights with other people), abusive (e.g., uses foul languages), and/or dis- ruptive (e.g., disrupts the classroom). noncompliance (e.g., refuses to comply with rules; does not obey elders) and school truancy were other attributes of unsuitable behavior among adolescents. overall, descrip- tions of unsuitable behavior appeared to be readily identifiable in inter- view responses. despite this finding, the qualitative data indicated important gender differences in how the construct is defined. unsuit- able behavior among sri lankan girls was described in terms of dishon- esty (e.g., lying to parents), stubbornness, not following etiquette, and engaging in romantic relationships or love affairs. furthermore, girls did not approve of cross-gender behavior. they indicated that “girls who act or speak like boys or dress like boys” have behavioral prob- lems. males described the construct in different terms. they focused on behaviors such as delinquency (e.g., substance abuse, stealing, running away from home, joining gangs) and aggression (e.g., fighting, quarrel- ing with adults or peers, assaulting others). personal/interpersonal needs students provided information about personal and interpersonal needs during their interviews. although few gender differences were found in terms of this construct, it is does provide an interesting perspective of some of the pressures adolescents report. respondents indicated that poverty limits their access to basic necessities of life such as proper food, clothing, housing and education. they also indicated that family support, both material and emotional, was very important for them to thrive. meanwhile, several respondents, particularly girls, indicated that many mothers leave to work in the middle east (e.g., saudi arabia, jordan, kuwait, qatar, united arab emirates) for financial reasons. they find jobs as household workers or maids and send money to their families in sri lanka, leaving children to assume several domestic re- sponsibilities without maternal support. respondents were vocal about their academic needs in terms of in- structional support, guidance, and emotional support that they received from parents, teachers, peers, relatives, and private tutors. they particu- larly emphasized the importance of effective teaching in the classroom and the support from teachers to meet their academic needs (recall sri multicultural issues in school psychology lanka uses a high stakes examination system). they also described the lack of mental health services in school. they indicated that such ser- vices were only available through psychiatric centers at hospitals, which are often not readily accessible. furthermore, mental illness is stigmatized in the culture and this is thought to reduce the likelihood that services will be accessed. respondents spoke about their need for extracurricular activities. in particular, they expressed the need for recreation and complained about lack of recreational opportunities due to academic pressure from par- ents. sample quotes include: [our] worst stress is tuition [private tutoring; additional instruc- tion outside of the school context] . . . here all girls get tuition. we finish school at p.m. and go home and grab a snack and go to tui- tion [meaning class] . . . we need other things like sports. but we don’t have the time. [female respondent] [there is] so much competition. we don’t have lots of time to do extracurricular activities. there is not time to do things kids are supposed to do. no hobbies. we do not have time to do such things . . . we have little time, we get tired from going to classes. [male respondent] students expressed a desire to go out with their friends, party, go on va- cations, and watch sports and television. interview data from adoles- cents and adults indicated that indulging in recreational activities is typically not permitted by parents because of the strong emphasis on ac- ademic preparation. several students mentioned interest in interacting with opposite-sex friends; meanwhile, sri lankan society does not encourage free interac- tion of males and females. for example, boys and girls mentioned that they were not allowed by their parents to interact with the opposite gen- der or have any relationship with them. many students thought that this practice needs to be changed. they also indicated the importance of ro- mantic relationships during adolescence and how their parents, relatives and the society did not approve of such relationships. female respon- dents also indicated the lack of freedom and independence for girls in sri lankan society. they suggested that girls enjoyed much less free- dom and independence in contrast with the boys. their activities were restricted and supervised by parents and other elders. the girls ex- pressed the need for more freedom and removal of restrictions that hitchcock et al. would allow them to function independently and would promote self-efficacy and self-confidence in girls. although adults emphasized the importance of suitable and unsuitable behaviors as critical to definitions of culturally-valued competencies, ad- olescents also emphasized behaviors that related to their personal and in- terpersonal needs. for this reason, when developing the perceived competence instrument, we included items related to effective engage- ment in recreational activities and interpersonal relationships (i.e., re- flecting a more “well-rounded” adolescent or, as described in the culture, the “all-rounder”). findings from quantitative analyses principal components analysis (pca). recall that qualitative data were used to develop surveys. analysis of survey responses yielded an initial scale structure that was congruent with the qualitative findings. three factors were identified. unsuitable behavior (socially unaccept- able behavior) [a = . ; a = . (female); a = . (male)] consisted of fifteen items (see table ). these items described behaviors viewed to be inappropriate within this culture (e.g., drug use, stealing, and fight- ing). suitable behavior (socially acceptable behavior) [a = . ; a = . (female); a = . (male)] consisted of thirteen items. this scale de- scribed desirable adolescent behaviors within this culture, including studying, following school rules, and remaining clean in appearance. the final factor, personal/interpersonal needs (i.e., behaviors/compe- tencies related to fulfilling personal and social needs of sri lankan ado- lescents) [a = . ; a = . (female); a = . (male)], consisted of nine items that described behaviors/competencies for meeting these needs. these needs included spending the day with friends and going to par- ties. these factor structures were expected both because of the qualita- tive analyses but also because they have been previously established with a similar survey (student perceptions of the degree to which par- ents value the behaviors) using the same sample (hitchcock et al., ). note, however, that the previous work did not examine the data for gender differences. table contains factor loadings for males and females as well as for the total sample. manova analyses were performed to test for gender differences. in the first manova, gender was used as the independent variable, and the three scale scores were used as dependent variables. the overall manova was significant, wilks lamdba = . , f( , ) = . , p < multicultural issues in school psychology table . factor loadings resulting from principal components analysis (pca) and differences of scale scores and individual items by gender scale/item factor loadings overall male female overall female male mean (se) mean(se) mean(se) suitable behaviors cronbach alpha(a) . . . . (. ) . (. ) . ( )*** i am honest. . . . . (. ) . (. ) . (. )*** i obey school and classroom rules. . . . . (. ) . (. ) . (. )*** when somebody shows me a mistake, i like to accept it and correct myself. . . . . (. ) . (. ) . (. )*** i like to look after others who are sick or hurt. . . . . (. ) . (. ) . (. )*** i study regularly. . . . . (. ) . (. ) . (. )** i pay attention to the studies in the school. . . . . (. ) . (. ) . (. ) i persevere even when faced with a difficult task. . . . . (. ) . (. ) . (. )** i help the poor through good works. . . . . (. ) . (. ) . (. )*** i interact well with my teachers. . . . . (. ) . (. ) . (. ) i move with respectable peers. . . . . (. ) . (. ) . (. ) i follow rules and expectations according to the situation. . . . . (. ) . (. ) . (. ) i observe customs and traditions of the country. . . . . (. ) . (. ) . (. )* i am clever (intelligent). . . . . (. ) . (. ) . (. ) unsuitable behaviors cronbach alpha (a) . . . . (. ) . (. ) . (. ) i use drugs. . . . . (. ) . (. ) . (. ) i drink alcohol. . . . . (. ) . (. ) . (. ) i steal. . . . . (. ) . (. ) . (. ) i carry weapons. . . . . (. ) . (. ) . (. ) i smoke cigarettes. . . . . (. ) . (. ) . (. ) i persuade others to join a gang. . . . . (. ) . (. ) . (. ) table (continued) scale/item factor loadings overall male female overall female male mean (se) mean(se) mean(se) unsuitable behaviors cronbach alpha(a) . . . . (. ) . (. ) . (. ) i persuade others to engage in bad habits. . . . . (. ) . (. ) . (. ) i insult others. . . . . (. ) . (. ) . (. ) i use profane language . . . . (. ) . (. ) . (. ) i scold or criticize teachers. . . . . (. ) . (. ) . (. ) i interrupt others’ work. . . . . (. ) . (. ) . (. ) i associate with bad peers. . . . . (. ) . (. ) . (. ) i am a member of a gang. . . . . (. ) . (. ) . (. ) i waste time. . . . . (. ) . (. ) . (. ) i don’t carry out my responsibilities. . . . . (. ) . (. ) . (. ) personal/interpersonal needs cronbach alpha (a) . . . . (. ) . (. ) . (. ) i like to spend the day with friends. . . . . (. ) . (. ) . (. ) i like to have fun with others. . . . . (. ) . (. ) . (. ) i listen to others’ problems. . . . . (. ) . (. ) . (. ) i like to listen to music or sing. . . . . (. ) . (. ) . (. ) i like to go on trips. . . . . (. ) . (. ) . (. ) i help others to solve their problems. . . . . (. ) . (. ) . (. ) i am sensitive to others’ feelings and needs. . . . . (. ) . (. ) . (. ) i like to go to parties. . . . . (. ) . (. ) . (. ) i safeguard others’ secrets. . . . . (. ) . (. ) . (. ) note: * p < . ; **p < . ; ***p < . . . the follow-up analysis indicated that one of the scales, suitable behaviors, was different by gender [f( , ) = . , p < . ], with girls scoring higher than boys. the other two factors were not statisti- cally significant at the . level (unsuitable behaviors–f( , ) = . , p = . ; personal/interpersonal needs–f( , ) = . , p = . ). subsequent analyses were performed to examine the individual items in the suitable behaviors factor by gender. the overall manova, as ex- pected, was statistically significant (wilks lamdba = . , f( , ) = . , p < . ). table contains more detailed information of the analy- sis, including the items in each factor, the factor loading on the individ- ual factor, the means and standard errors for each factor and item by gender, and significant differences (determined by the manova) by gender. to summarize, results of the factor analyses provided construct validation of perceived competencies consistent with cultural concepts reflected in the qualitative data. a key finding is that the suitable behav- ior construct differs by gender; in general, girls reported more suitable behaviors. examination of the factor loadings for boys and for girls indicated that there might be differences in factor structure. such differences can illuminate ways in which boys and girls interpret items within a con- struct differently. tests for factor (construct) invariance using structural equation modeling can be used to help locate such statistical differences that can then be compared to qualitative findings (see byrne, ). hy- potheses are developed that test the equivalence of factor loadings across groups (levels of gender, in this case). while the details of the analysis are beyond the scope of this paper, we conducted preliminary analyses using this technique. table contains a summary of the anal- yses. the strategy is to initially test the equivalence of the factor struc- ture across groups; then, successively restrictive equality constraints are imposed on the models. for example, the constraints of interest here are the factors loadings. if item factor loadings are constrained to be equal across models, and the change in the degrees of freedom and chi-square are examined, a statistically significant chi-square statistic suggests dif- ferences in structure. the results of table show that, while suitable behaviors and personal & interpersonal needs factor items load equiv- alently for boys and girls, there are differences in the factor structure of unsuitable behaviors. this finding is supported by the qualitative data which indicated that several behaviors such as joining gangs, robbing, carrying weapons, as- saulting people, alcohol and drug abuse were described only by male re- spondents and viewed as only relevant to males. this occurrence may hitchcock et al. be explained by the cultural emphasis on gender-appropriate behavior which is comparatively more rigid and less permissive for women. some caution is needed to avoid over-interpretation. the assessment tool used for quantitative data collection was a self-report instrument and the female respondents may have included only the socially desir- able responses to conform to the cultural expectations. these dif- ferences in definition and interpretation of unsuitable behavior do, how- ever, require further examination. discussion when dealing with populations with distinct cultural variables, re- searchers typically employ preexisting clinical or research instruments developed for mainstream american children and adolescents, in some instances with minimal modifications (e.g., language translation) and without re-validation. this is problematic because of the likelihood that such instruments will miss nuanced issues important to a target popula- tion. the work described here provides an alternative and represents theoretical and methodological approaches for investigating the role of individual and cultural factors in mental health of a distinct culture, par- multicultural issues in school psychology table . constrained models cfa tests summary of analyses model c df dc ddf cfi rmsea full, factor model, no constraints . . . full, factor model, factor loadings, variance, covariances constrained to be equal . . * . . full, factor model, factor loadings only for all three factors constrained . . * . . unsuitable behaviors only constrained . . * . . suitable behaviors only constrained . . . . p & ip needs only constrained . . . . * p < . note. models were compared to the full models with no constraints. the results suggest (and are consistent with exploratory analysis) that structures for unsuitable behaviors are different for boys and girls. ticularly the combined use of ethnographic and factor analytic tech- niques. as a result of this effort, a culturally specific instrument that measures self-concept issues in sri lanka was developed and yielded insights into the culture. there is compelling data that show girls report that they endeavor to follow societal expectations for culturally-defined suitable behavior when compared to boys. this could be attributed to the cultural emphasis on a traditional gender role behavior that encour- ages submissiveness, tolerance and a caring attitude in women (das dasgupta, ). the findings dealing with girls’ perceptions of suitable and unsuit- able behavior may prove useful when engaged in future work in the cul- ture. should a girl appear to not be following expectations, we can now recognize that follow-up may be important. the girl may simply be un- usually independent, or she might be reacting to some difficult life cir- cumstances. whatever the case, these analyses provide a basis for recognizing the behavior as unusual and follow-up may be warranted. this work also suggests a need to revisit the unsuitable behavior con- struct in light of gender-specificity. as noted above, the items that form the factor structure for the construct load differently by gender. this is a subtle difference from the manova results indicating differences in suitable behavior. in this latter construct, the data do not suggest the genders define the construct differently; girls simply scored higher than boys. for unsuitable behavior, however, table shows the factor load- ings for some items were higher for girls than boys (e.g., i carry weap- ons, i insult others, i scold or criticize others, i associate with bad peers, and i don’t carry out my responsibilities). this suggests greater variability in how males responded to these items and the issue can be further explored in future studies. the personal/interpersonal needs construct appeared in the factor analysis, but this was not gender-spe- cific. these findings were congruent with qualitative data; there was no reason to suspect there would be gender differences on this factor. the identification of this third factor can also inform future intervention work. the data suggest that sri lankan students recognize a need for recreational time with friends, yet this is generally not allowed by par- ents and educators. communicating this finding to stakeholders may alone be beneficial as allowing for more recreation may reduce stress among adolescents (a long-term goal of this research program). more generally, the psychological constructs presented here have been vali- dated by qualitative and factor analytic data analyses, so researchers can proceed with confidence using this instrument for wider research proj- ects within the country. hitchcock et al. one limitation of the present study is that our data did not completely capture indicators of socioeconomic status (ses); qualitative work did not highlight this as an important factor. ancillary analyses (not shown) including mother and father education as ses indicators as covariates did not change any of the conclusions made here, and gender did not in- teract with ses variables. however, the results of those analyses sug- gested that ses indicators may offer important ways to understand elements of students’ self-concept in sri lanka; this issue should be ex- plored in future research. we opened with a discussion as to why having knowledge of a target culture facilitates assessment. a specific example was provided using old order amish and the data here further demonstrate the point. now suppose that, in sri lanka, an interventionist meets a girl who reports she is rejecting behavior expectations imposed upon her by parents by actively seeking out recreational activity in lieu of studying and engag- ing in cross-gender behavior (e.g., acting or dressing like a boy). in the united states this behavior would not be considered unusual and possi- bly even be encouraged; in sri lanka, such behavior would be non-nor- mative and a caregiver would do well to pay closer attention to the girl’s circumstances. this raises the issue of whether it is better to change the local culture to facilitate such independence or to get the girls to con- form. this represents a complex debate, but the importance of interven- ing on the girl’s behalf at some level would be clear and the caregiver should not dismiss the behavior. the point is that, as with the case of the old order amish, knowledge of cultural variation would solicit very different behaviors among practitioners. in other words, detailed knowledge of a culture facilitates assessment and the development of sensitive interventions, which is very much in-line with calls for devel- oping cultural competency. as a side note, we have confidence in these findings because there is cross-method consistency (i.e., triangulation) that supports their validity. in-depth qualitative findings, obtained from smaller groups, were consistent with the results of the various quantita- tive analyses described above, using a large sample. finally, these findings should not be of interest only to those with in- terests in mixed-methods and cross-cultural work; there are implica- tions here for school psychologists. psychologists are being pushed to develop cultural competencies and the methods described above sup- port this endeavor. when working in a multicultural setting, the qualita- tive procedures can serve as models for service planning and identifying relevant cultural issues, and the development of a survey from this in- formation can be used to quantify such information in the event a large multicultural issues in school psychology enough sample warrants the additional effort. these skills can help school psychologists understand the idiosyncratic needs of a local cul- ture, develop nuanced assessment skills and in turn develop highly tar- geted interventions. note . more detailed findings can be obtained from the first author. references american psychological association ( ). guidelines for providers of psychologi- cal services to ethnic, linguistic, and culturally diverse populations. washington, dc: author. american 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( ). handbook of mixed methods in social and behav- ioral research. sage: thousand oaks, ca: sage. the world bank in sri lanka (april ). the world development indicator data- base. retrieved april , , from the world bank in sri lanka website; http://lnweb .worldbank.org/sar/sa.nsf/srilanka united nations development programme ( ). human development indicators, . retrieved on april , . http://hdr.undp.org/reports/global/ /en/indicator/ indicator.cfm?file=index.html. u.s. department of health and human services ( ). mental health: a report of the surgeon general. rockville, md: u.s. department of health and human services, substance abuse and mental health administration, center for mental health ser- vices, national institutes of health, national institute of mental health. u.s. department of health and human services ( ). mental health: culture, race, and ethnicity–a supplement to mental health: a report of the surgeon general. rockville, md: u.s. department of health and human services, substance abuse and mental health administration, center for mental health services. doi: . /j v n _ hitchcock et al. http://lnweb .worldbank.org/sar/sa.nsf/srilanka http://hdr.undp.org/reports/global/ /en/indicator/ untitled proc. r. soc. b ( ) , – d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il *rer @e electron . doi: . /rspb. . published online january received accepted religion, fertility and genes: a dual inheritance model robert rowthorn* faculty of economics, cambridge university, sidgwick avenue, cambridge cb dd, uk religious people nowadays have more children on average than their secular counterparts. this paper uses a simple model to explore the evolutionary implications of this difference. it assumes that fertility is determined entirely by culture, whereas subjective predisposition towards religion is influenced by gen- etic endowment. people who carry a certain ‘religiosity’ gene are more likely than average to become or remain religious. the paper considers the effect of religious defections and exogamy on the religious and genetic composition of society. defections reduce the ultimate share of the population with religious allegiance and slow down the spread of the religiosity gene. however, provided the fertility differential persists, and people with a religious allegiance mate mainly with people like themselves, the religiosity gene will eventually predominate despite a high rate of defection. this is an example of ‘cultural hitch- hiking’, whereby a gene spreads because it is able to hitch a ride with a high-fitness cultural practice. the theoretical arguments are supported by numerical simulations. keywords: religion; fertility; evolution; genetic predisposition; cultural hitch-hiking; evolution . introduction it is widely agreed that religion has biological foundations—that belief in the supernatural, obedience to authority or susceptibility to ceremony and ritual depend on genetically based features of the human brain [ – ]. however, there is a disagreement about the extent to which causality also flows in the opposite direc- tion, from religion to biology. one view is that religion is a ‘spandrel’—a cultural phenomenon based on features of the brain that were already in existence in their present form when religion first appeared (e.g. [ – ]). another view is that the existence of religion promotes the evol- ution of genes that predispose people towards religious belief or behaviour. the latter view rests upon two plaus- ible assumptions: (i) individuals have diverse endowments of the genes that predispose humans towards religion; and (ii) religion-induced selection is strong enough to have an appreciable impact within the relevant time-frame on the frequency of such genes. the fact that religion has a genetic basis of great antiquity does not imply that genetic variation has disappeared. koenig & bouchard [ ] survey twin studies that quantify the genetic and environmental determinants of what they call the ‘traditional moral triad’ of auth- oritarianism, conservatism and religiousness. in most cases, to per cent of the observed variation in such personality traits is explained by genotypic variation. the authors argue that these are large genetic effects in comparison with typical findings in the social sciences. there is no direct evidence regarding the speed with which religion affects genetic evolution. however, there is evidence that the rise of complex human culture in gen- eral has greatly accelerated the pace of genetic evolution, by altering the frequency of long-standing genes in the con.cam.ac.uk ic supplementary material is available at http://dx.doi.org/ /rspb. . or via http://rspb.royalsocietypublishing.org. november december global population and by spreading or preserving new mutations [ – ]. for religion to influence genetic evolution it must convey some kind of selective advantage. such an effect might come about through social bonding via ritual, formation of group identity through myth, honest signalling through participation in costly ceremonies and adherence to social norms through love or fear of god [ – ]. in most cases, religious individuals gain personal advantage from their activities or beliefs. however, religion may also induce behaviour that has a fitness cost to the individual but is beneficial to the group. if group selection is strong, this should favour the emergence of a type of religion that induces adherence to pro-social norms, which in turn should favour the evolution of genes that predispose individuals towards this type of religion [ – ]. another channel through which religion might influ- ence genetic evolution is via its impact on fertility. the link between religion and fertility has been extensively dis- cussed by demographers, but, as bouchard [ ] notes, the potential genetic implications of this link have been largely ignored by evolutionary theorists. (a) religion and fertility in the modern world, religious people, even controlling for income and education, have more children on average than people without religion [ , ]. the reasons are cul- tural in the broad sense, and include social norms and the influence exerted by religious organizations [ – ]. the more devout people are, the more children they are likely to have. the world values survey for nations over the period – reveals that adults attending divine service more than once a week averaged . children, those attending once per month averaged . and those never attending averaged . (cited in [ ]). similar findings for a variety of european countries are reported by philipov & berghammer [ ]. sects such as the amish, the hutterites and haredi (‘ultra-orthodox’) jews have this journal is q the royal society mailto:rer @econ.cam.ac.uk http://dx.doi.org/ . /rspb. . http://dx.doi.org/ . /rspb. . http://dx.doi.org/ . /rspb. . http://rspb.royalsocietypublishing.org http://rspb.royalsocietypublishing.org r. rowthorn religion, fertility and genes d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il total fertility rates three to four times greater than the secular average [ , ]. modern fertility differentials partly reflect diverse responses to the ‘demographic transition’. global birth rates have fallen dramatically in recent times and in a number of countries, including japan and most of europe, they are now well below replacement [ – ]. however, the transition to lower fertility has been slower and less complete among religious people than among those without religion, since it is driven partly by individualistic values of self-fulfilment that are inimical to traditional religious teaching [ , ]. the pace and extent of the transition also vary across religions and denominations [ – ]. some religious groups have been largely unaffected by the demographic transition and still have extremely high birth rates, which explains why there is now such an enormous fertility gap between them and the rest of the population. the impact of differential fertility on the religious composition of society depends on the scale of switching between religious groups, and between them and the secular population. with no defections at all, ultra-high fertility groups would rapidly outgrow the rest of the population and soon become a majority. in practice, there may be limits to their expansion. as they get larger, their members may come into closer contact with other members of society and acquire secular values, causing them to have fewer children [ ]. some members may leave to join a less strict group, whereas others may give up religion altogether. for example, the old order amish have a total fertility rate of . , whereas the more modern new order amish have a somewhat lower total fertility rate of . (p. in [ ]). what will happen in the future is a matter of speculation. kaufmann [ ] argues that the stricter religious groups will continue to exhibit high fertility and will also be effective at trans- mitting their beliefs to their children, so that relatively few of them will defect in later life. this would represent a continuation of recent history, which has seen the number of amish in the usa rise from in to in —due almost entirely to internal growth [ ]. sustained growth at this rate would take the amish population to almost million by the end of the century and million by . rapid fertility-driven growth has also been observed among other groups such as hutterites and haredi jews [ , ]. if growth at such rates were to continue, it would speedily turn what is currently a tiny fraction of the population into a majority. indeed, this will inevitably happen unless the forces of secularism constrain the growth of these groups by reducing their birth rates or increasing their defection rates. (b) genetic implications there are two channels through which a high fertility group can influence the composition of the overall gene pool: internal growth and defection. if most of the chil- dren born within a high fertility group remain in the group when they grow up, the size of this group will increase rapidly and its share of the overall gene pool will rise accordingly. conversely, if most of the children eventually leave, this will limit the size of the group. how- ever, such defections will also spread the group’s genes proc. r. soc. b ( ) into the surrounding population, thereby increasing their share in the overall gene pool. the role of defection in this context is similar to that of migration in sewall- wright’s [ ] shifting balance theory of evolution, whereby a mutation may spread outwards from a high- fitness group to the rest of the population. these two channels—internal growth and diffusion through defec- tion—may have radically different implications for the eventual size of the high fertility group, but their long- run genetic implications may be similar. in each case, the genes associated with the high-fertility group may eventually predominate in the overall gene pool. the pic- ture is further complicated by the existence of conversion, whereby individuals join the high-fertility group, and by exogamy, whereby individuals remain in the group but marry an outsider who does not convert. to explore these issues we shall consider some simple mathematical models. these models are in the gene-culture tradition [ ] and draw heavily on the work of boyd & richerson [ ] regarding direct bias, vertical transmission and the natural selection of cultural variants. the models are of the dual inheritance type, in which children inherit both their genes and their initial religious (or non-reli- gious) allegiance from their parents. they retain their genes throughout their lives, but they may eventually change their allegiance. children who are brought up as religious may abandon religion when they grow up, whereas those brought up without religion may later con- vert and become religious. the probability of switching allegiance in our models depends on the genetic endow- ment of the individual concerned. a child who is genetically predisposed towards religion is more likely than other children to remain or become religious as an adult. throughout the analysis we shall assume that ferti- lity is an entirely cultural phenomenon: genes affect the likelihood that particular individuals will be religious, but do not directly influence their reproductive behaviour. all religious adults (‘believers’) have the same fertility irrespective of their genes; likewise non-believers have the same fertility irrespective of their genes. there is some evidence from a danish twin study that genes have an independent influence on the desire of people to have children [ ], but we shall ignore this compli- cation. we initially assume complete assortative mating, whereby religious people mate only with other religious people. one or both of the partners may be converts, but there are no truly mixed couples in which the partners retain distinct allegiances. this assumption is realistic for ultra-high-fertility jewish and christian groups [ ], and also for muslims in general [ ], but is less realistic for mainstream christian churches whose members nowa- days frequently marry members of other churches or people of no religion at all. we later consider how the existence of mixed marriages between religious and non-religious people affects the results. we begin with a haploid model in which the rules of genetic transmission are simple and rates of religious defection and conversion are fixed. this model is later modified to allow for more complex genetic transmission and for variable rates of defection and conversion. such modifications do not affect the qualitative results, although they may have important quantitative impli- cations. in all of the models we consider, religious predisposition (‘religiosity’ for short) is determined by a religion, fertility and genes r. rowthorn d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il single gene. this is unlikely to be true in practice, but without this simplification the analysis would be intractable. . a haploid model of dual inheritance society is divided into two distinct allegiance groups indexed by i ¼ r, n. members of group r are religious ‘believers’ whereas members of group n are ‘non-believ- ers’. all individuals have two cultural parents who are also their genetic parents. individuals live for two periods. they acquire their genes from their parents and during the first period as children they acquire their initial alle- giance from their parents. they then become adults and choose whether to retain or modify their allegiance. next they have children of their own, bring them up and then die. the probability that a child will switch alle- giance on becoming an adult depends on its genetic endowment, which is specified by a single gene at a specific locus. each individual carries exactly one copy of this gene. the gene comes in two forms (‘alleles’) that are indexed by j ¼ r, n. the ‘religiosity’ allele r codes for religious predisposition and allele n codes for non-religious predisposition. thus, there are four distinct types in the population: ðr; rÞ;ðr; nÞ;ðn; rÞ;ðn; nÞ: dynamics depend on fertility and mating. we assume that adults mate only with adults from the same allegiance group. each couple in group i has ci (. ) children. these children initially have the same allegiance as their parents, and the genetic endowment of each child is inherited with equal probability from either parent. let x ij ðtÞ be the number of adults at time t with allegiance i and allele j. every adult of type (i, j) gives rise to ci chil- dren of type (i, j). for example, if two adults of type (r, r) mate they will have cr children of type (r, r), which is equivalent to cr children of this type for each parent. if an adult of type (r, r) mates with an adult of type (r, n) they will on average have cr children of type (r, r). the total number of children of type (r, r) is therefore cr x r rðtÞ. the genetic composition of an individual is fixed for life, but there may be a change of allegiance when a child reaches adulthood. this will alter the distribution of adult types in the next period. let srjð j ¼ r; nÞ be the probability that a child of type (r, j) will switch allegiance to type (n, j) as an adult. likewise, snj is the probability that a child of type (n, j) will switch to type (r, j) as an adult. in principle, these switching parameters could vary through time as a result of wider social trends or they might be density-dependent. for the moment we shall assume they are constant. taking switches into account, the number of adults of each type in the next period is given by the system of difference equations x rrðt þ Þ¼ ð � s r rÞcr x r rðtÞþ s n rcnx n rðtÞ; x nrðt þ Þ¼ s r rcr x r rðtÞþð � s n rÞcnx n rðtÞ; x rnðt þ Þ¼ ð � s r nÞcr x r nðtÞþ s n n cnx n nðtÞ and x nnðt þ Þ¼ s r n cr x r nðtÞþð � s n nÞcnx n nðtÞ >>>= >>>; : ð : Þ proc. r. soc. b ( ) the right-hand side of each equation contains two com- ponents. one component denotes individuals who have retained their childhood allegiance and the other denotes newcomers who have switched allegiance from the other group. for any type (i, j) define the fraction of this type in the adult population as follows: pijðtÞ¼ x ij ðtÞp l;m x l mðtÞ : thus, pijðtÞ is the fraction of the adult population who belong to group i(¼ r, n) and carry allele j(¼r, n). also, define piðtÞ¼ pirðtÞþ p i nðtÞ for i ¼ r; n and pjðtÞ¼ prjðtÞþ p n j ðtÞ for j ¼ r; n: thus, p i (t) is the fraction of the adult population who belong to group i and pj(t) is the fraction who carry allele j. note that p r (t) þ pn(t) ¼ pr(t) þ pn(t) ¼ . (a) modelling predisposition the religiosity allele r codes for a genetic predisposition towards religion. in the present context, this means that at least one of the following conditions must hold: — among children who are brought up without religion, those who carry allele r are more likely to become religious in adult life than those with allele n. — among children who are brought up as religious, those who carry allele r are less likely to abandon their religion in adult life than those with allele n. to express the above conditions mathematically we impose the following conditions on the switching parameters: snr � s n n and s r r � s r n; ð : Þ with at least one strict inequality. these conditions are symmetric. if r codes for a predisposition towards mem- bership of group r then allele n codes for a predisposition towards membership of group n. the above definition of predisposition is similar to that of lumsden & wilson [ ]. (b) long-run behaviour we can now state a simple but powerful result. we assume that , sij , for all i; j and also prð Þ¼ prrð Þþ pnrð Þ . ; pnð Þ¼ prnð Þþ pnnð Þ . : thus, for each type there is a non-zero switching between groups and both alleles are initially present in the population. proposition . . suppose that the predisposition conditions ( . ) are satisfied. suppose further that members of group r have more children than those of group n, such that cr . cn: ð : Þ table . effect of fertility on the evolution of religion and genes. this table illustrates how differential fertility affects social and genetic evolution. the table assumes that srr ¼ : ; srn ¼ : ; snr ¼ : ; snn ¼ : . all trajectories begin at the point prrð Þ¼ : ; prnð Þ¼ ; pnrð Þ¼ ; pnnð Þ¼ : . differential fertility generations elapsed cr/cn share of population who are religious ¼ p r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cr/cn frequency of religiosity allele r ¼ pr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r. rowthorn religion, fertility and genes d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il then, in the long-run, the distribution of types in the population will converge to a limit of the following kind: lim t! prrðtÞ¼ p̂ r r . ; lim t! pnrðtÞ¼ p̂ n r . ; lim t! prnðtÞ¼ and lim t! pnnðtÞ¼ : hence lim t! prðtÞ¼ and lim t! pnðtÞ¼ : ð : Þ proof. see the electronic supplementary material. b thus, if religious allegiance is associated with a higher- than-average birth rate, and there is an allele that predisposes individuals towards religion, this allele will eventually predominate. the fraction of individuals who are ‘believers’ will stabilize at less than per cent and there will remain a finite percentage of adults who are without religion. in the limit, all of the latter will carry the religiosity allele r. it must be stressed that this result is contingent on the assumption that cr . cn. in the opposite case (cr , cn), allele n would eventually predominate. (c) role of the predisposition conditions to understand the role of the predisposition conditions ( . ), consider what happens when these conditions do not hold. suppose the probability of switching between groups is positive and independent of a person’s geno- type, so that snr ¼ snn and srr ¼ srn: suppose also that both alleles are initially present in the population. in this case, the system will eventually settle down to a stable genetic polymorphism in which the genetic compo- sition of groups r and n is identical, and alleles r and n coexist in finite proportions. the ultimate frequency of these alleles in the population as a whole will depend on the starting point. the outcome is different if the predisposition con- ditions ( . ) hold. these conditions establish a bias in proc. r. soc. b ( ) the pattern of inter-group flows and ensure that, irrespec- tive of starting point, allele r will eventually become over-represented in the high fertility group r. from then onwards r will enjoy a growth advantage over allele n, whose share in the overall gene pool will converge to zero. the correlation between cultural phenotype (alle- giance r) and genotype (allele r) is endogenous and is subject to forces that ensure that, in the long run, this correlation is positive. this is an example of ‘cultural hitch-hiking’, whereby a gene spreads because it is able to hitch a ride with a high-fitness cultural practice [ ]. (d) fertility and the pace of evolution the pace of evolution and the eventual share of religion in the population both depend on the fertility differential between religious and non-religious people. this is illus- trated in table . when the relative fertility of the religious group is ultra-high (cr/cn ¼ ), the pace of change is extremely fast. within generations, the share of adults with a religion rises from . to per cent and the share of the religiosity allele r in the overall gene pool rises to more than per cent. with cr/cn ¼ , the pace of change is still fast. within generations, the share of adults with religion rises from . initially to . per cent and the share of allele r in the gene pool rises to . per cent. as relative fertility is further reduced, the pace of change slows down dramatically and the eventual share of religion is much lower. with cr/cn ¼ . , the share of adults with a religion is only . per cent after generations and the share of allele r is only . per cent. it takes generations for the share of r to surpass per cent. (e) ultra-high fertility sects the rate of expansion of ultra-high fertility sects depends on their ability to retain their membership. if all of their children were to remain members for life and had the same ultra-high fertility as their parents, then within just a few generations these sects would dominate the social landscape. if a large number of children leave these sects on reaching adulthood, this will limit their expan- sion. however, it will not prevent the spread of the . . (a) . . generations elapsed (b) figure . how religious defections influence social and gen- etic evolution. the diagrams refer to an ultra-high-fertility religious group. (a) share of believers; (b) share of allele r. a high defection rate reduces the eventual share of religious believers (group r) in the population. whatever the defection rate, the religiosity allele r eventually tends to fixation. the diagram assumes: both variants: cr/cn ¼ . ; pr r ( )¼ . , p r n ( )¼ , pr n ( )¼ , pn n ( )¼ . ; low defection: sr r ¼ . , sn r ¼ . , sr n ¼ , sn n ¼ ; high defection: sr r ¼ . , sn r ¼ . , srn¼ , sn n ¼ . line without diamonds, low defection; line with diamonds, high defection. religion, fertility and genes r. rowthorn d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il genes that predispose individuals towards religion (assuming such genes exist). figure provides a numerical illustration of a closed ultra-high fertility sect that recruits no one from the out- side. the birth rate of its members is three times as high as that of outsiders. the membership of the sect is initially . per cent of the adult population. all members of the sect carry the religiosity allele r, whereas initially no one outside the sect carries this allele. what happens in the course of time depends on the ability of the sect to retain its members. two scenarios are shown in figure . in the low defection scenario, per cent of adult members leave the sect each generation. this is the observed defection rate for old order amish children born in the s [ ]. in the high defection scenario, the figure is per cent. the most striking feature of the low defection scenario is the pace of change. within four gen- erations, one fifth of the adult population belong to the sect and after generations its share stabilizes at per cent. by then almost every adult in the population carries allele r, and the other allele is well on the way to oblivion. under the high defection scenario, the pace of change is much slower and the eventual share of the population who are religious is much lower. even so, after generations, the vast majority of the population carry allele r. such a situation comes about because in each generation a large number of adults who are carrying r abandon their religion and feed this allele into the non- religious population. this example illustrates the paradoxical role of secularization in spreading the religios- ity allele. secularization reduces the growth rate of the ultra-high fertility sect, but it does so by importing into the non-religious population a large number of people who carry the religiosity allele. religion may be kept in check by defections from the sect, but this does not prevent the ultimate triumph of the religiosity allele. . a diploid model the above analysis can be extended to the diploid case in which individuals carry two alleles rather than one. in this version, there are three genetic types and two cultural types, which can be combined as follows: ðr; rrÞ;ðr; rnÞ;ðr; nnÞ;ðn; rrÞ;ðn; rnÞ;ðn; nnÞ: we assume that adults mate at random with adults from the same group and that one gene is inherited with equal probability from each parent. each type (i, jk) has a given probability sijk of switching to the other allegiance group. note that sijk ¼ sikj . the difference equations are more complicated than in the haploid case (see the electronic supplementary material). in the diploid case, allele r codes for a genetic predis- position towards r provided the following inequalities are satisfied: snrr; s n rn � s r nn and s r rr; s r rn � s r nn; ð : Þ with at least one strict inequality. the level of dominance is specified by the equations srrn ¼ h r srrr þð � h rÞsrnn and snrn ¼ h nsnrr þð � h nÞsnnn ) : ð : Þ proc. r. soc. b ( ) if � hr, hn � , then snrr � s n rn � s r nn and s r rr � s r rn � s r nn; ð : Þ with at least one strict inequality. allele r is completely dominant if h r ¼ h n ¼ and completely recessive if h r ¼ h n ¼ . the properties of the diploid version were explored by simulation, using distinct parameter combinations for r dominant and the same number for r recessive, together with a further for the inter- mediate case h r ¼ h n ¼ . . all of these parameter combinations satisfy the predisposition conditions ( . ) and in all of them cr . cn. initial gene frequencies are prrð Þ¼ ; prnð Þ¼ : ; pnrð Þ¼ : and pnnð Þ¼ : : thus only a very small proportion of the adult population are initially religious and all of these individuals carry allele n. even so, in every case, the gene r eventually takes over and the other allele goes to oblivion. this is identical to the result obtained in the haploid case. however, the pace of convergence is slower in the diploid case, especially when r is recessive. figure shows what happens to the overall frequency of r in one particular example. (a) density dependence the decision to switch may depend on the relative size of the two groups. we model this by assuming that, for i ¼ r, n and j, k ¼ r, n sijk ¼ max½slow; minða i jk½ þ b i piðtÞ�; shighÞ�; ð : Þ where aijk and b i are constant and , aijk; slow; shigh , . these assumptions ensure that , sijk , . if b i . , the probability of defecting from group i increases as this group gets larger. in the case of religion, this could occur because the religious group becomes ‘softer’ and less able to police its boundaries as its relative size increases. the group may also become less isolated as . . . . . . . . . . generations elapsed figure . what happens to the share of allele r in the hap- loid and diploid versions of the model. r achieves fixation fastest in the haploid version of the model and slowest in the recessive diploid version. the diagram assumes: all var- iants: cr/cn ¼ . ; pr r ( )¼ . , pn r ( )¼ , pr n ( )¼ , pn n ( )¼ . ; haploid: sr r ¼ . , sn r ¼ . , sr n ¼ . , sn n ¼ . ; diploid: srr r ¼ . , snn r ¼ . , srr n ¼ . , snn n ¼ . ; r domi- nant: srn r ¼srr r , srn n ¼srr n ; r recessive: srn r ¼snn r , srn n ¼snn n . line with circles, diploid þ r dominant; line with dia- monds, diploid þ r recessive; line without circles or diamonds, haploid. . . . . . . (a) (b) . . generations elapsed figure . the evolutionary effects of a constraint on the growth of a high-fertility religious group. defections increase as the group gets bigger, which limits the eventual size of this group. however, large-scale defections spread the religiosity allele r into the rest of the population. the diagram assumes: both variants: cr/cn ¼ . ; pr r ( )¼ . , pn r ( )¼ , pr n ( )¼ , pn n ( )¼ . ; arr r ¼arn r ¼ . , ann r ¼ . , arr n ¼arn n ¼ann n ¼ ; b n ¼ ; no constraint: b r ¼ , constraint: b r ¼ . note: sjk i (t)¼ajk i [ b i p i (t)]. line without diamonds, no constraint; line with diamonds, constraint. r. rowthorn religion, fertility and genes d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il it gets larger and a greater fraction of its members come into contact with the secular world [ ]. if b i , , the probability of defecting from group i decreases as this group gets larger. this could be because group i becomes a world unto itself as it gets larger, so that individuals from this group have fewer contacts with outsiders and, in consequence, are less frequently exposed to ideas or pressures that subvert their allegiance. for r to code for a predisposition towards religion, the following conditions must be satisfied: anrr; a n rn � a r nn and a r rr; a r rn � a r nn; ð : Þ with at least one strict inequality. (b) simulations the simulations reported in the online material indicate that proposition ( . ) holds even in the diploid model with density-dependent behaviour. the allele r will even- tually take over, provided it satisfies the relevant predisposition conditions and cr . cn. moreover, the share of the religious group r will eventually stabilize at less than per cent. figure provides an illustration in which the growth of the religious group is severely con- strained by defection as it gets larger, so that it never attains more than . per cent of the total population. even so, because of this group’s high birth rate, there is a stream of defectors who take the religiosity allele r with them into the non-religious population. (c) mixed marriages the discussion has so far assumed strict endogamy within the two allegiance groups. the partners in a couple may come from different backgrounds, but they have a common allegiance by the time they mate. in this section, we consider briefly what happens if there are mixed mar- riages in which the two partners retain distinct proc. r. soc. b ( ) allegiances. our approach draws on the biology literature on imprinting (especially [ ], but also [ , ]). we make the following assumptions. a constant fraction ai of adults from allegiance group i will only mate with someone from their own group. this preference is unre- lated to their genetic type. the remaining adults are indifferent towards the allegiance of their partner and they mate randomly with each other. some of them find a partner from their own group and some a partner from the other group. mixed couples have cm children, of which a fraction fr receives a religious upbringing. the relevant difference equations are derived in the electronic supplementary material. the evolution of the system was explored numerically for a number of parameter combinations. the con- clusions are as follows. provided the religious group has higher fertility than the non-religious group, there is always a cultural polymorphism: the population share of each group will eventually stabilize at a non-zero level. if mixed marriages are ruled out, as in the preceding sec- tions of this paper, allele r always tends to fixation. however, if mixed marriages are permitted, there are some parameter combinations for which the frequency of allele r tends to . these are characterized by a low preference for endogamy, combined with a low-to- medium fertility advantage for the religious group. this can be seen in the examples shown in rows ( ), ( ), ( ), ( ), ( ) and ( ) of table . in most cases, even where mixed marriages occur, r tends to fixation. more- over, the greater is the preference for endogamy, as indicated by the a parameters, the faster is the approach to fixation. this can be seen from the penultimate column of the table. the effect of mixed marriages in these examples is to slow down the fixation of r and sometimes prevent fixation of this allele altogether. simi- lar effects were observed in other simulations not reported here. none of these simulations gave rise to a stable genetic polymorphism. table . the effect of endogamy on the evolution of genes. this table illustrates how preferences for endogamy influence the overall frequency pr of allele r in the adult population. the fraction of religious (non-religious) adults with a preference for endogamy is ar (an). the number of children per mixed marriage is cm of which a fraction fr receive a religious upbringing; couples in which both partners are religious (non-religious) have cr (cn) children. the table assumes that srrn ¼ srrr ¼ : ; srnn ¼ : ; snrn ¼ snrr ¼ snnn ¼ : : trajectories all start from the point prrð Þ¼ : ; prnð Þ¼ pnrð Þ¼ ; pnnð Þ¼ : . row ar an fr cr/cn cm/cn pr( ) pr( ) pr( ) ( ) . . . ( ) . . . . . ( ) . . . . . ( ) . . . ( ) . . . ( ) . . . . . ( ) . . . . . ( ) . . . ( ) . . . . ( ) . . . . . . ( ) . . . . . . ( ) . . . . ( ) . . , . ( ) . . . . . . ( ) . . . . . . ( ) . . . . ( ) . . . . . ( ) . . . . . . . ( ) . . . . . . . ( ) . . . . . ( ) . . . . . ( ) . . . . . . . ( ) . . . . . . . ( ) . . . . . religion, fertility and genes r. rowthorn d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il . discussion this paper assumes that there exist genetic differences between individuals that affect their predisposition towards religion. in the same cultural environment, some individuals are for genetic reasons more likely to become or remain religious than others. it also assumes that religious people have more children on average than secular people. this gap may currently be quite small in the case of mainstream churches, but there are some reli- gious groups that have fertility rates several times larger than their secular counterparts. they also have high rates of endogamy. if individuals from these groups marry outsiders they must normally either leave the group or else their partner must convert. some of these groups have been growing rapidly for the past century but their future is uncertain. there are several possible scenarios. such groups may continue on their present tra- jectory for the rest of this century and beyond, leading to a spectacular and ultimately unsustainable increase in their numbers. or maybe their growth rates will slow down quite soon because they experience more defections as members leave to join more liberal religious groups or give up religion altogether. or perhaps their fertility will decline as they absorb secular values or face practical obstacles to expansion. these scenarios have different implications for the genetic evolution of society. if reli- gious people continue to have a higher birth rate on average than secular people and they are sufficiently endo- gamous, then any genes which predispose people towards religion will spread. this may be true even if mainstream religions shrink or become increasingly secular in their proc. r. soc. b ( ) breeding habits. provided a core of high-fertility sects continues to exist, and those sects remain highly endoga- mous, they will transform the genetic composition of society through either internal growth or defection. this has been demonstrated in the present paper using a single gene for religiosity. in reality, a phenomenon as complex as religious predisposition is likely to be influ- enced by many different genes, but this does not alter the main argument. one issue of interest in this context is genetic poly- morphism. such a phenomenon is widespread in nature. it may arise through genetic drift if some trait is selectively neutral or because different variants enjoy selective advantage in different environments. it may also arise because heterozygotes have superior fitness when compared with homozygotes. alternatively, an observed polymorphism may be transitory and scheduled to disappear as some particular variant eventually tri- umphs. in the case of religion, heritability studies suggest there is currently significant variation in genetic predisposition towards religion [ ]. there are several possible explanations for this. religious predisposition may be associated with genes that were, or still are, differ- entially advantageous in specific natural or social environments. the current variation in predisposition may simply reflect environmental diversity. to the extent that religious predisposition is the result of group selection, variation is to be expected, since group selec- tion requires variation to operate. or perhaps we are living in a transitional era in which selective forces will eventually marginalize certain variants. this is one r. rowthorn religion, fertility and genes d ow nl oa de d fr om h tt ps :/ /r oy al so ci et yp ub li sh in g. or g/ o n a pr il possible interpretation of the analysis presented in this paper. however, even within our framework, there are several avenues through which a stable polymorphism could arise. if there is a convergence of religious and non-religious birth rates, the share of the religiosity allele r will eventually stabilize at less than unity. or there might be some form of heterozygote advantage, whereby heterozygotes have a stronger religious predis- position than homozygotes. this will be the case within our framework if h r , h n . . simulations not reported here suggest that even a modest effect of this kind will generate a stable polymorphism and allow n to survive. however, heterozygote advantage is merely a theoretical possibility and there is currently no evidence to support it. the most plausible mechanism that could generate a stable polymorphism is a future convergence of religious and non-religious birth rates. some of the present fertility differentials are so large that, if they persist, they may have a significant genetic effect within the space of a few generations. this may not be because of an increase in the share of individuals who are actively religious. if high fertility rates are com- bined with high defection rates, the share of the population who belong to high-fertility religious groups may stabilize at quite a low level. however, defections from such groups will spread religiosity genes to the rest of society. there will be an increasing number of people with a genetic predisposition towards religion but who lead secular lives. it is interesting to speculate how such a predisposition might manifest itself in a secular context. the findings of koenig & bouchard [ ] suggest that a genetic predisposition towards religion is associated with obedience to authority and conservatism. if this is correct, then the diffusion of religiosity genes into the rest of society should see an increase in the number of secular people who are genetically inclined towards such values. the implications of such a development are beyond the scope of this paper to consider. i should like to thank samuel bowles, herbert gintis, peter richerson, paul seabright and two anonymous referees for their comments on a draft of this paper; also seminar participants at the santa fe institute and the 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(doi: . / ) http://dx.doi.org/ . /s - - -y http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /j. - . . .x http://www.biomedcentral.com/ - / / http://dx.doi.org/ . / http://dx.doi.org/ . / http://dx.doi.org/ . /nature http://dx.doi.org/ . / http://www .etown.edu/amishstudies/pdf/statistics/population_change_ _ .df http://www .etown.edu/amishstudies/pdf/statistics/population_change_ _ .df http://www .etown.edu/amishstudies/pdf/statistics/population_change_ _ .df http://www .etown.edu/amishstudies/pdf/statistics/population_change_ _ .df http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . / http://dx.doi.org/ . / http://dx.doi.org/ . / http://dx.doi.org/ . / http://dx.doi.org/ . /hdy. . http://dx.doi.org/ . / 	religion, fertility and genes: a dual inheritance model 	introduction 	religion and fertility 	genetic implications 	a haploid model of dual inheritance 	modelling predisposition 	long-run behaviour 	role of the predisposition conditions 	fertility and the pace of evolution 	ultra-high fertility sects 	a diploid model 	density dependence 	simulations 	mixed marriages 	discussion 	i should like to thank samuel bowles, herbert gintis, peter richerson, paul seabright and two anonymous referees for their comments on a draft of this paper; also seminar participants at the santa fe institute and the university of siena. 	references spontaneous recovery of superhydrophobicity on nanotextured surfaces spontaneous recovery of superhydrophobicity on nanotextured surfaces suruchi prakasha, erte xia, and amish j. patela, adepartment of chemical & biomolecular engineering, university of pennsylvania, philadelphia, pa edited by xiao cheng zeng, university of nebraska–lincoln, lincoln, ne, and accepted by the editorial board march , (received for review november , ) rough or textured hydrophobic surfaces are dubbed “superhydro- phobic” due to their numerous desirable properties, such as water repellency and interfacial slip. superhydrophobicity stems from an aversion of water for the hydrophobic surface texture, so that a water droplet in the superhydrophobic “cassie state” contacts only the tips of the rough surface. however, superhydrophobicity is re- markably fragile and can break down due to the wetting of the surface texture to yield the “wenzel state” under various conditions, such as elevated pressures or droplet impact. moreover, due to large energetic barriers that impede the reverse transition (dewetting), this breakdown in superhydrophobicity is widely believed to be irrevers- ible. using molecular simulations in conjunction with enhanced sam- pling techniques, here we show that on surfaces with nanoscale texture, water density fluctuations can lead to a reduction in the free energetic barriers to dewetting by circumventing the classical dewetting pathways. in particular, the fluctuation-mediated dewetting pathway involves a number of transitions between distinct dewetted morphologies, with each transition lowering the resistance to dewetting. importantly, an understanding of the mechanistic path- ways to dewetting and their dependence on pressure allows us to augment the surface texture design, so that the barriers to dewetting are eliminated altogether and the wenzel state becomes unstable at ambient conditions. such robust surfaces, which defy classical expec- tations and can spontaneously recover their superhydrophobicity, could have widespread importance, from underwater operation to phase-change heat transfer applications. cassie | wenzel | dewetting | fluctuations | barriers surface roughness or texture can transform hydrophobic surfacesinto “superhydrophobic” surfaces and endow them with prop- erties such as water repellency, self-cleaning, interfacial slip, and fouling resistance ( , ). each of these remarkable properties stems from the reluctance of water to penetrate the hydrophobic surface texture, so that a drop of water sits atop an air cushion in the so- called cassie state, contacting only the top of the surface asperities. however, water can readily penetrate the surface texture, yielding the wenzel state ( , ) at elevated pressures ( , ) or temperatures ( ), upon droplet impact ( , ), as well as due to surface vibration ( ), localized defects ( ), or proximity to an electric field ( ); superhydrophobicity is thus remarkably fragile and can break down due to the wetting of the surface texture under a wide variety of conditions. to facilitate the recovery of superhydrophobicity and to afford reversible control over surface properties, significant efforts have focused on inducing the reverse wenzel-to-cassie dewetting transition. however, a true wenzel-to-cassie transition has been elusive ( ), with most reported instances making use of trapped air ( , – ) or generating a gas film using an external energy source ( , ) to jump-start the dewetting process. insights into why achieving a wenzel-to-cassie transition remains challenging are provided by macroscopic interfacial thermodynamics ( ), which suggests that the dewetting transition is impeded by a large free energetic barrier. this “classical” barrier is attributed to the work of adhesion for nucleating a vapor–liquid interface at the base of the textured surface. consequently, the breakdown of superhydrophobicity upon wetting of the surface texture is widely believed to be irreversible ( , , ), so that once the texture wets it remains in the wet state, even when the pressure is subsequently lowered or the electric field is switched off. by using atomistic simulations in conjunction with specialized sampling techniques, here we challenge this conventional wisdom and uncover principles for the design of nanotextured surfaces that can spontaneously recover their superhydrophobicity by dewetting their surface texture at ambient conditions. our work builds upon recent theoretical and simulation studies that have shown that water density fluctuations, which are not captured in macroscopic mean- field models, are enhanced at hydrophobic surfaces ( – ) and situate the interfacial waters at the edge of a dewetting transition ( ). such enhanced fluctuations have also been shown to modulate the pathways to dewetting and lead to reduced dewetting barriers in several confinement contexts ( – ). to investigate how fluctua- tions influence cassie–wenzel transitions on nanotextured surfaces, here we perform atomistic simulations of water adjacent to pillared surfaces, and use the indirect umbrella sampling (indus) method ( ) to characterize the free energetics of the transitions and the corresponding pathways, as well as their dependence on pressure. by comparing our results to macroscopic theory, we find that although water density fluctuations do not influence the pressure at which the cassie-to-wenzel wetting transition occurs, they are nevertheless crucial in the wenzel-to-cassie dewetting transition, that is, in the process of recovering superhydrophobicity when it breaks down. in particular, fluctuations stabilize a nonclassical dewetting pathway, which features cross-overs between a number of distinct dewetted morphologies that precede the formation of the classical vapor–liq- uid interface at the basal surface; the nonclassical pathway offers a lower resistance to dewetting, leading to reduced dewetting barriers. significance due to its aversion to the hydrophobic surface texture, a water droplet makes minimal contact with, and readily rolls off of, a superhydrophobic surface, conferring it with beneficial properties such as water repellency and self-cleaning. however, the surface texture can readily wet in response to conditions such as elevated pressures, leading to a breakdown of superhydrophobicity that is widely believed to be irreversible. by using specialized molecular simulations to study surfaces with nanoscale texture, here we find that the dewetting of the surface texture is strongly influenced by water density fluctuations. furthermore, an understanding of the dewetting pathways allows us to design novel surface textures on which fluctuations can facilitate a spontaneous recovery of superhydrophobicity. author contributions: s.p. and a.j.p. designed research; s.p. performed research; e.x. contributed new reagents/analytic tools; s.p. and e.x. analyzed data; and s.p. and a.j.p. wrote the paper. the authors declare no conflict of interest. this article is a pnas direct submission. x.c.z. is a guest editor invited by the editorial board. to whom correspondence should be addressed. email: amish.patel@seas.upenn.edu. this article contains supporting information online at www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental. – | pnas | may , | vol. | no. www.pnas.org/cgi/doi/ . /pnas. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://crossmark.crossref.org/dialog/?doi= . /pnas. &domain=pdf mailto:amish.patel@seas.upenn.edu http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental www.pnas.org/cgi/doi/ . /pnas. importantly, by uncovering the nanoscale dewetting pathways, and in particular by finding regions of the surface texture that are hardest to dewet, our results provide strategies for augmenting the surface texture to further destabilize the wenzel state and reduce the bar- riers to dewetting. on such rationally designed surfaces the barriers to dewetting the texture can be eliminated altogether, so that the wenzel state is no longer metastable but has been rendered unstable at ambient conditions, and the superhydrophobic cassie state can be spontaneously recovered from the wet wenzel state. free energetics of cassie–wenzel transitions fig. contrasts the behavior of water on textured surfaces in the cassie and wenzel states; water does not penetrate the surface texture in the cassie state but does so in the wenzel state. in fig. a, right, the particular textured surface morphology that we study here is shown and consists of square pillars of height h and width w, arranged on a square lattice, and separated by a distance s. also highlighted is the textured volume, v, which is devoid of water molecules in the cassie state but is filled with water in the wenzel state. the normalized water density, ρn in v, thus serves as a reliable order parameter to distinguish the cassie and the wenzel states. in fig. a, we show the free energy, ΔfðρnÞ, of a system in the par- tially wet state relative to that in the wenzel state, obtained using molecular dynamics simulations in conjunction with indus ( ). here, ρn ≡ n=nliq, with n and nliq being the number of water molecules in v in the partially and fully wet states, respectively. details pertaining to our simulation setups, the force-field parame- ters, and the algorithms used are included in materials and methods. the simulated free energy profile, ΔfðρnÞ, clearly shows two basins, cassie at ρn ≈ and wenzel at ρn ≈ , separated by a large barrier. to uncover the importance of water density fluctuations on the free energetics of cassie–wenzel transitions, we first compare the sim- ulated ΔfðρnÞ with classical expectations based on macroscopic in- terfacial thermodynamics, which does not account for fluctuations. macroscopic theory envisions dewetting being initiated with the nucleation of a vapor–liquid interface at the base of, and perpen- dicular to, the pillars; dewetting then proceeds through the ascent of this interface along the pillars ( ). the height of the interface above the base of the pillars is thus given by hðρnÞ = hð − ρnÞ, and the theoretical free energy profile, ΔfthðρnÞ, is given by ΔfthðρnÞ = Δfadh + ½γ cos θaside + Δpv�ð − ρnÞ, [ ] where γ is the vapor–liquid surface tension, θ is the water droplet contact angle on a flat surface, and Δp is the difference between the system pressure and the coexistence pressure at the system temperature, t. in addition, Δfadh ≡ γabaseð + cos θÞ is always un- favorable (positive) and corresponds to the work of adhesion for creating the vapor–liquid interface, with abase = sðs + wÞ being the basal area and aside = wh being the area of the vertical faces of the pillars. because cos θ < for hydrophobic surfaces, the sec- ond term could be favorable (negative) if Δp is sufficiently small, that is, if Δp ≤ Δpint ≡ − γ cos θ aside=v. thus, the two key features of ΔfthðρnÞ are (i) a large adhesion barrier at ρn ≈ , which must be overcome to nucleate the vapor–liquid interface, and (ii) a linear a b fig. . water on textured hydrophobic surfaces can exist in either the cassie or the wenzel state. (a) in the cassie state, water is unable to penetrate the surface texture (blue) so that a water droplet (red) sits on a cushion of air, contacting only the top of the pillars. as a result, there is minimal contact between water and the solid surface, leading to a small contact angle hysteresis and a large contact angle, which are critical in conferring superhydrophobicity to the sur- face. also shown is a simulation snapshot of the pillared surface that we study here (right), which consists of square pillars arranged on a square lattice and is made of atoms (blue spheres) arranged on a cubic lattice. the textured volume, v, as well as the dimensions that characterize the pillared nanotextured surface are highlighted; the width of the pillars is w = nm, their height is h = . nm, and the interpillar spacing is s = nm. (b) in the wenzel state, water wets the texture, so that there is extensive contact between water and the solid surface, leading to a large contact angle hysteresis and a smaller contact angle; in this state, the surface is no longer superhydrophobic. we define the normalized density, ρn, in the textured volume to be the number of waters in v, normalized by the corresponding number of waters in the wenzel state. a c b fig. . free energetics and pathways of wetting–dewetting transitions on a pillared surface. (a) the simulated free energy, ΔfðρnÞ (in units of the thermal energy, kbt ≡ β− , with kb being the boltzmann constant and t the tempera- ture), features two basins that are separated by a large barrier. for . < ρn < . , Δf varies linearly with ρn, in agreement with macroscopic theory. however, the simulated barrier for dewetting ( kbt) is found to be smaller than the clas- sical barrier ( kbt). (b) between the wenzel state and the barrier ( . < ρn < ), ΔfðρnÞ is marked by several kinks, which demarcate five regions with distinct dewetted morphologies (dashed lines are a guide to the eye). (c) representative configurations corresponding to these regions are shown as interfaces encompassing the dewetted volumes (shown in orange, waters omitted for clarity). region v (ρn ≈ ) displays gaussian fluctuations resulting in a parabolic basin. region iv ( . < ρn < . ) is characterized by vapor pockets at the base of the pillars. as ρn is reduced, vapor pockets grow, break symmetry, and merge to form a striped vapor layer between the pillars. the stripe expands laterally in region iii ( . < ρn < . ) until a nearly intact vapor–liquid interface is formed. region ii ( . < ρn < . ) is characterized by water molecules sticking to the center of the cell and also contains the nonclassical barrier, which even- tually gives way to the classical region i at ρn ≈ . . prakash et al. pnas | may , | vol. | no. | a p p li ed p h y si c a l sc ie n c es d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , portion of ΔfðρnÞ corresponding to the vapor–liquid interface ris- ing along the pillars as ρn decreases. a derivation of eq. and details of the comparison between simulation and theory are in- cluded in si appendix. simulated configurations with . < ρn < . (fig. c, ia and ib) are consistent with the macroscopic expectation and display dewetted regions with the vapor–liquid interface being perpendicular to the pillars, and at heights that are consistent with hðρnÞ = hð − ρnÞ; see si appendix. theory also predicts that Δf ought to vary linearly with ρn. at Δp = , the pressure at which our simulations are performed, the corresponding slope is expected to be ð−γ cos θÞ aside. as seen in fig. a, the simulated ΔfðρnÞ is indeed linear for . < ρn < . (dashed line). the fitted slope yields a surface tension, γfit = mj/m , which agrees well with that of our water model, γspc=e = . mj/m ( ). the behavior of the system for . < ρn < . is thus classical. macroscopic theory also associates the barrier to dewetting with the work of adhesion to nucleate the vapor–liquid interface, which can be estimated as γfitð + cos θÞabase ≈ kbt. in contrast, the cor- responding simulated barrier is only kbt (fig. a). thus, even though the ascent of the vapor–liquid interface along the pillars is classical, the nucleation of that vapor–liquid interface and the as- sociated dewetting barriers, which are central to the recovery of superhydrophobicity, appear to be nonclassical. fluctuations facilitate nonclassical dewetting pathways with reduced barriers to understand why the dewetting barrier is smaller than the classical expectation, we take a closer look at Δfðρn > . Þ (fig. b) as well as the corresponding representative configurations (fig. c, ii–iv), which are shown as instantaneous interfaces encompassing the dewetted regions (orange). a description of the algorithm used to compute the instantaneous interfaces and the corresponding aver- aged interfaces are included in si appendix. interestingly, we ob- serve a host of nonclassical partially wet configurations preceding the formation of the classical vapor–liquid interface. as ρn is de- creased from , vapor pockets first form at the base of the pillars, then grow to round the corners around the pillars (fig. c, iv). on further reducing ρn, symmetry is broken as vapor pockets from opposite pillars merge to form stripes of vapor spanning the inter- pillar region (fig. c, iii). this change in the dewetted morphology coincides with a kink in ΔfðρnÞ, suggesting that the system adopts a lower free energy path by transitioning from the vapor pocket to the stripe morphology. subsequent decrease in ρn results in another transition to a donut-shaped vapor layer (fig. c, ii). ΔfðρnÞ displays a maximum in the donut morphology; the barrier thus corresponds to a configuration with water molecules sticking to the central region of the basal surface that is farthest from the pillars, rather than an intact vapor layer. expelling the remaining water molecules to form an intact vapor layer is energetically favorable, as is the subsequent (classical) rise of the vapor–liquid interface along the pillars. this novel and clearly nonclassical pathway preceding the formation of a vapor–liquid interface, which is facilitated by nano- scopic water density fluctuations and involves transitions between various dewetted configurations ( , ), results in a smaller barrier for the wenzel-to-cassie transition than anticipated by macroscopic theory (eq. ). we note that the kinks in ΔfðρnÞ are a consequence of the high- dimensional free energy landscape being projected onto the scalar order parameter, ρn. consequently, while we expect ρn to suitably describe the transition pathway within the different regions (i–v), capturing transitions between regions with different dewetted morphologies would require consideration of additional order pa- rameters ( ). we further note that the use of periodic boundary conditions in our simulations leads to the correlated dewetting of adjacent cells. we have ensured that the nonclassical dewetting pathway discussed above is also observed in the absence of such correlations by additionally studying the dewetting of the first unit cell in a simulation setup containing four unit cells; see si appendix, fig. s . how pressure influences barriers to wetting and dewetting the implications of this nonclassical pathway on the pressure de- pendence of the dewetting transition are even more interesting. because pressure favors configurations with higher densities in a well-defined manner, its effect on ΔfðρnÞ can be readily estimated, as shown in si appendix. as shown in fig. a, the most striking effect of changing pressure is seen in the slope of region i; as pressure is increased, the slope decreases and the cassie state is destabilized. the increase in pressure also leads to a decrease in the barrier to transition from the cassie to the wenzel state, as shown in fig. c. the pressure at which this barrier for wetting disappears, Δpint, corresponds to the limit of stability (or spinodal) of the cassie state; at Δp = Δpint, a system in the cassie state will spontaneously descend into the wenzel state, as shown in the cassie-to-wenzel hysteresis curve (blue) in fig. d. the exact agreement between the theoretical and simulated Δpint values seen in fig. c and d is not coincidental, but a consequence of the fact that the value of vapor–liquid surface tension, γfit, used in the macroscopic theory, was obtained by fitting the simulated free energy profile in region i; see si appendix. the agreement between γfit and γspc=e nevertheless suggests that the macroscopic theory prediction of intrusion pressure, Δpint = −γ cos θ aside=v ( , ), should be reasonably accurate even for surfaces with nanoscale texture, a finding that is in harmony with recent experiments ( ). this success of macroscopic theory in describing Δpint, the pressure at which superhydrophobicity fails, is a direct consequence of its ability to capture region i of ΔfðρnÞ accurately. although regions ii to iv play no role in determining Δpint, their role is profoundly important in the reverse process, that is, the wenzel-to-cassie (dewetting) transition. as pressure is decreased, not only does the slope of region i increase (fig. a), but the slopes of regions ii–iv that are negative at Δp = also increase, approaching zero at sufficiently negative pressures. fig. b zooms in on the liquid basin of ΔfðρnÞ and highlights that as pressure is de- creased the location of the peak in the free energy shifts to higher ρn and is accompanied by a gradual decrease in the height of the dewetting barrier (fig. c). this decrease in the dewetting barrier with decreasing pressure is in stark contrast with the classical ex- pectation that a constant adhesion barrier must be overcome to go from the wenzel to the cassie state ( ). under sufficient tension (negative pressure), the barrier goes to zero as the wenzel basin reaches the limit of its stability. our simulations thus suggest that superhydrophobicity can be recovered, that is, a system in the wenzel state can spontaneously and remarkably transition back into the cassie state below a so-called extrusion pressure, Δpext (fig. d). informing the design of robust superhydrophobic surfaces the term “robust” is used here to describe the superhydrophobic cassie state (not the mechanical robustness of the surface texture), and in particular the ability of a surface to recover its super- hydrophobicity once the surface texture is wet. for the surface studied here, the extrusion pressure, Δpext = − bar, is quite small. although water can sustain significant tension, and experi- ments have been able to access Δp k − , bar before cavitation occurs ( ), from a practical standpoint robust superhydrophobic surfaces with significantly larger Δpext values that exceed atmo- spheric pressure are desirable. clues for designing such surfaces are contained within fig. , which suggests that it is hardest to remove water molecules from the center of the simulation cell. to de- stabilize those waters and facilitate the formation of the vapor– liquid interface, we modify the pillared surface by adding a spherical nanoparticle at the center of the cell (fig. a, inset). a comparison of the free energy profile, ΔfðρnÞ, of this novel surface with that of the pillared surface (fig. a) highlights that the introduction of the nanoparticle has a dramatic influence on ΔfðρnÞ; the wenzel state | www.pnas.org/cgi/doi/ . /pnas. prakash et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf www.pnas.org/cgi/doi/ . /pnas. is destabilized to such an extent that it is no longer metastable but has been rendered unstable at Δp = . in fact, as shown by the hysteresis curves in fig. b, the wenzel state remains unstable up to an extrusion pressure, Δpext = + bar. the pressure dependence of ΔfðρnÞ for this surface as well as the corresponding barriers to wetting/dewetting are included in si appendix. thus, a system prepared in the wet (wenzel) state, by condensation or by starting at a high pressure, for example, should spontaneously dewet on de- creasing the pressure below bar, resulting in a recovery of the superhydrophobic cassie state. to investigate this possibility and rule out the existence of any additional barriers to dewetting, we per- formed equilibrium (unbiased) simulations starting with ρn ≈ . in each case, the system spontaneously descends into the cassie state within ps, indicative of a barrierless transition from the wenzel to the cassie state. details of these simulations are provided in si ap- pendix and one of the dewetting trajectories is included as movie s . the dewetting trajectory as well as characteristic configurations along the dewetting pathway (fig. c) highlight that the strongly confined region between the nanoparticle and the basal surface nu- cleates a vapor bubble (ρn ≈ ), which then grows to facilitate the spontaneous formation of an intact vapor–liquid interface (ρn = . ). once formed, the vapor–liquid interface begins to rise, and al- though it adheres to the top of the nanoparticle, the interface continues to rise along the pillars; the unfavorable density gradient between the center and the edges of the cell then facilitates the barrierless depinning of the interface. this dewetting pathway suggests that the spontaneous recovery of superhydrophobicity re- quires (i) a confined (negative curvature) region of nanoscopic di- mensions to nucleate a vapor bubble, which (ii) must grow to a large enough size to facilitate the formation of an intact vapor–liquid interface, which in turn (iii) must not be pinned by surface features as it rises along the pillars. to test the validity of these criteria, we study three additional surfaces that each destabilize the central waters but violate exactly one of the above criteria (fig. d). by running unbiased simulations initialized in the wenzel state, we show that none of the three surfaces is able to spontaneously transition to the cassie state (fig. e). in si appendix, fig. s we also show ΔfðρnÞ for these modified surfaces. in each case, the destabilization of the central waters by the nanoparticle leads to a substantial reduction in the dewetting barrier; however, this de- stabilization alone is insufficient to lead to the spontaneous recovery of the superhydrophobic cassie state. because augmenting the surface texture alters the stability of the barrier and the wenzel states in different ways, and can also change the partially dewetted morphologies observed along the dewetting pathway, the relation- ship between the surface texture geometry and the height of the dewetting barrier is nontrivial. in particular, the extent to which a change in surface texture influences the dewetting barrier does not depend on its ability to (de)stabilize the barrier state or the wenzel state alone; rather, how the surface texture modification influences the entire dewetting pathway is important. indeed, by following the three design criteria outlined above, which pay attention to the dewetting pathway (fig. c), we were able to design a second surface texture that displays an unstable wenzel state at ambient conditions (see si appendix, fig. s ). the fact that each of these complex criteria have to be satisfied for spontaneous dewetting could help explain why nanotextured surfaces that spontaneously recover superhydrophobicity have not been discovered serendipitously, despite the use of both nanoparticles and nanoscale pillars to texture surfaces ( , ). outlook the last decade has seen an explosion of studies pertaining to superhydrophobic surfaces, ranging from the development of novel techniques for their synthesis to those aimed at understanding the properties of these surfaces and their putative applications. our work informs both fundamental and applied aspects of super- hydrophobicity, not only uncovering nonclassical cassie–wenzel transition pathways, but also suggesting strategies for the rational design of robust superhydrophobic surfaces that can spontaneously recover their superhydrophobicity. in particular, we show that the free energetics of wetting–dewetting transitions on nanotextured surfaces, the corresponding mechanistic pathways, and their de- pendence on pressure are all strongly influenced by collective water density fluctuations. although the importance of fluctuations at the nanoscale is clear, the extent to which fluctuations influence dewetting pathways for larger texture sizes remains an interesting open question. we note, however, that nonclassical effects stem from the stabilization of dewetted morphologies that precede the formation of an intact vapor–liquid interface at the basal surface; once a vapor–liquid interface is nucleated, the remainder of the dewetting process is classical. because the width of this nascent vapor–liquid interface is not expected to depend on the texture size, and should always have nanoscale dimensions, fluctuations may continue to play an important role in the dewetting of surface textures with significantly larger feature sizes. our results also clarify that even though certain aspects of dewetting on nanotextured surfaces are nonclassical, other aspects can be classical. in particular, because the intrusion pressure depends on the free energetics of the (clas- sical) ascent of the vapor–liquid interface along the pillars, it is well described by macroscopic theory. similarly, macroscopic models ( , ) have also been shown to the capture apparent contact angle of a liquid droplet localized in the cassie or the wenzel basin ( – ). a c d b fig. . effect of pressure on cassie–wenzel transitions. (a) ΔfðρnÞ is shown for pressures ranging from − to bar, with the arrow pointing in the di- rection of increasing pressure (purple, − bar; blue, − bar; green, − bar; red, bar; brown, bar). as pressure is increased, the slope of the classical region i decreases, destabilizing the cassie state; conversely, as pressure is de- creased, the wenzel state is destabilized. (b) this destabilization of the wenzel state is manifested not only in an increase in the slope of region i but also in a corresponding increase in the slopes of the nonclassical regions ii–iv, from negative toward zero to eventually being positive. as a result, a decrease in pressure shifts the location of the barrier (○) to higher ρn and leads to a con- comitant decrease in the height of the barrier. (c) the barriers for the wetting and dewetting transitions are shown here as a function of Δp (simulation, solid lines; theory, dashed lines). both the simulated and the classical cassie-to-wenzel barriers (blue) decrease on increasing pressure, eventually disappearing at the in- trusion pressure, Δpint. on the other hand, although the classical wenzel-to-cassie barrier (magenta) is predicted to be independent of pressure, simulations suggest that the barrier to dewetting disappears at a sufficiently small extrusion pressure, Δpext. (d) pressure-dependent hysteresis curves for ρn, assuming the system remains in its metastable basin and is unable to surmount barriers larger than kbt. prakash et al. pnas | may , | vol. | no. | a p p li ed p h y si c a l sc ie n c es d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://movie-usa.glencoesoftware.com/video/ . /pnas. /video- http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf an understanding of the cassie–wenzel transition pathway also facilitates the rational design of surfaces with superior super- hydrophobicity. although numerous methods have been suggested for texturing hydrophobic surfaces, from top-down fabrication to bottom-up self-assembly techniques ( – ), a key bottleneck in their widespread adoption has been the fragility of the super- hydrophobic cassie state and the associated irreversible breakdown of superhydrophobicity upon wetting ( ). our findings represent a major step in addressing this challenge, highlighting the importance of water density fluctuations in stabilizing nonclassical pathways on nanotextured surfaces, which reduce dewetting barriers and enable the spontaneous recovery of superhydrophobicity. our results also suggest a general strategy for augmenting the design of existing nanotextured surfaces. by identifying the pathways to dewetting, and in particular the regions that are hardest to dewet, we were able to inform the location of sites where the introduction of additional texture would be optimal. the rational design of nanotextured sur- faces with superior superhydrophobicity could be further bolstered by investigating the extent to which the dewetting of a unit cell is influenced by whether an adjacent cell is wet or dry. in particular, if a dry cell could facilitate the dewetting of adjacent cells, then not all cells would have to possess a nanoparticle for the spontaneous re- covery of superhydrophobicity; once a nanoparticle-containing cell dewetted spontaneously, it could facilitate the dewetting of cells adjacent to it. such cooperative effects would also have interesting implications on whether dewetting is nucleated at the edges or the center of a water droplet, and on the pinning of the three phase contact line and the associated contact angle hysteresis ( , ). in conjunction with recent advances in introducing texture at the nanoscale ( , ), our results thus promise to pave the way for robust superhydrophobic surfaces with widespread applicability un- der the most challenging conditions. one example of such an ap- plication involves sustained underwater operation ( ), which would require the surface texture to not only remain dry under hy- drostatic pressure but also be able to return to the dry state if the texture wets in response to a perturbation. another example pertains to condensation heat transfer ( ), wherein an unstable wenzel state would facilitate the immediate roll-off of condensing water droplets and enable dropwise condensation to be sustained at higher fluxes. materials and methods simulation setup. each of the surfaces that we study is composed of atoms arranged on a cubic lattice with a lattice spacing of . nm; the surface atoms are constrained to remain in their initial positions throughout the simulations. the basal surface, which is situated at the bottom of the simulation box, is made of eight layers of atoms and is nm thick. on the pillared surface, square pillars of height h = . nm and width w = nm are used to introduce surface texture. the pillars are placed on a square lattice with interpillar spacing s = nm. we also study surfaces that additionally contain spherical or hemispherical nano- particles that are also made of atoms on a cubic lattice and are placed at the center of the simulation cell, touching the top of the basal surface. each system is periodic in all three spatial directions and is initialized in the wenzel state. the surfaces are hydrated with roughly , water molecules, so that even in the wenzel state a -nm-thick water slab rests above the pillars. we also pro- vide a buffering vapor layer at the top of the simulation box, which is roughly nm thick in the wenzel state. as waters leave the textured region the vapor layer shrinks to roughly nm in the cassie state. the vapor layer, which is thus present in our system at all times, ensures that water is in coexistence with its vapor, and Δp = ( ). to ensure that the vapor layer remains at the top of the × × nm simulation box, we include a repulsive wall at z = . nm. simulation details. we have chosen the spc/e model of water ( ) because it adequately captures the experimentally known features of water, such as surface tension, isothermal compressibility, and the vapor–liquid equation of state near ambient conditions, all of which are important in the study of dewetting on hydrophobic surfaces ( , , , ). the surface atoms interact with the water oxygens through the lennard-jones (lj) potential (σ = . nm, « = . kj/mol). as shown in si appendix, this choice leads to a flat surface water droplet contact a c e b d fig. . designing surface textures for the spontaneous recovery of superhydrophobicity. (a) the free energetics of a surface with a . -nm diameter spherical nanoparticle at the center of the cell (inset) are compared with the corresponding pillared surface; the surface modification stabilizes the superhydrophobic cassie state and renders the wenzel state unstable. (b) the modified surface features a positive extrusion pressure, Δpext = bar, suggesting spontaneous recovery of superhydrophobicity at ambient conditions. (c) representative instantaneous interface configurations highlight that dewetting commences at the center of the cell and spreads outward, facilitating the spontaneous formation of the vapor–liquid interface, which then rises along the pillars. although waters stick to the top surface of the nanoparticle, the unfavorable gradient in water density between the edges and the center of the cell facilitates the depinning of the vapor–liquid in- terface. (d) we simulate three additional variants of the pillared surface, which displace waters from the center of the cell using (i) a . -nm hemispherical particle, (ii) a -nm spherical nanoparticle, and (iii) an inverted . -nm hemispherical particle. the three surfaces (top row) all violate one of the three criteria for spontaneous dewetting outlined in the text. when initialized in the wenzel state, these surfaces are unable to undergo complete dewetting, as evidenced by the final configurations of -ns-long unbiased simulations (bottom row). (e) the time dependence of ρn for such unbiased simulations highlights that the wenzel state is unstable only for the pillared system with a . -nm spherical nanoparticle. | www.pnas.org/cgi/doi/ . /pnas. prakash et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf www.pnas.org/cgi/doi/ . /pnas. angle, θ = . °, in accord with contact angles observed on typical hydrophobic surfaces, such as alkyl-terminated self-assembled monolayer surfaces ( ). we use the gromacs molecular dynamics simulation package ( ), suitably modified to perform indus simulations in the canonical ensemble. a detailed description of the indus calculations ( , ), which we use to characterize the free energetics of wetting–dewetting transitions on nanotextured surfaces, is included in si appendix. to maintain a constant temperature of t = k ( ), the canonical velocity-rescaling thermostat with a time constant of . ps is used. lj interactions and the short-ranged part of the electrostatic interactions are truncated at nm, and the particle mesh ewald algorithm is used to treat the long-ranged part of electrostatic interactions ( ). the shake algorithm is used to con- strain the bond lengths of the water molecules ( ). acknowledgments. a.j.p. thanks john crocker, shekhar garde, and pablo debenedetti for helpful discussions. this work was supported by national science foundation grants dmr - and cbet . . quéré d ( ) wetting and roughness. annu rev mater res : – . . nosonovsky m, bhushan b ( ) superhydrophobic surfaces and emerging applica- tions: non-adhesion, energy, green engineering. curr opin colloid interface sci : – . . sbragaglia m, et al. 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( ) a smooth particle mesh ewald method. j chem phys : – . . ryckaert jp, ciccotti g, berendsen hjc ( ) numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. j comput phys : – . prakash et al. pnas | may , | vol. | no. | a p p li ed p h y si c a l sc ie n c es d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf upcoming events acknowledgments. the authors acknowledge with thanks dr. j. l. steiner, usda-ars, soil and water conservation laboratory, bushland, texas, and dr. c. w. richardson, usda-ars, grassland soil and water research laboratory, temple, texas, for providing pre- cipitation and evaporation data. references . anderson, j. r., j. l. dillon, and j. b. har- daker. . agricultural decision analysis. the iowa state university press, ames, iowa. . bloodgood, d. w., r. e. patterson, and r. l. smith. . water evaporation in texas. texas agricultural experiment station bulle- tin , college station, texas. . cooley, k. r. . evaporation suppression for conserving farm water supplies. in g. w. frasier (ed.). proc. water harvesting sympo- sium, phoenix, arizona, march - , . ars-w- . agricultural research service, u.s. department of agriculture, washington, dc. . frasier, g. w. . water harvesting for live- stock, wildlife and domestic use. in g. w. fra- sier (ed.). proc. water harvesting symposium, phoenix, arizona, march - , . ars- w- . agricultural research service, u.s. de- partment of agriculture, washington, dc. . krishna, j. h., g. f. arkin, and j. r. martin. . runoff impoundment for supplemental irrigation in texas. water resources bulletin : - . . laing, i. a. f. . sealing excavated tanks on farms in western australia. in g. w. fra- sier (ed.). proc. water harvesting symposium, phoenix, arizona, march - , . ars- w- . agricultural research service, u.s. de- partment of agriculture, washington, dc. . lansford, v. d., w. l. harmon, and j. t. musick. . the texas high plains: adjust- upcoming events may - . an amish study tour will be held in berlin, ohio. contact ed martsolf/arlene musselman, a whole new approach; ( ) - . may - . fourth north american symposium on society and resources management will be held in madison, wisconsin. contact donald r. field, school of natural resources, lin- den drive, madison, wi . june - . diversity in food, agricul- ture, nutrition and environment will be held at the kellogg center, michi- gan state university, east lansing, michigan. sponsors are the associa- tion for the study of food and society and the agriculture, food, and human values society. contact lawrence ments to changing economic and resource con- ditions - . texas agricultural experi- ment station, college station, texas. . ritchie, j. t. . model for predicting evap- oration from a row crop with incomplete cover. water resources research : - . . scrimgeour, f. g. . a model for the eco- nomic evaluation of water harvesting. in p. w. unger, w. r. jordan, and t. v. sneed (eds.). proc. international conference on dryland farming, amarillo, texas, august, . ag- ricultural research service, u.s. department of agriculture, washington, dc. . steiner, j. l. . simulation of evaporation and water use efficiency of fallow based crop- ping systems. in p. w. unger, w. r. jordan, and t. v. sneed (eds.). proc. international conference on dryland farming, amarillo, texas, august, . agricultural research service, u.s. department of agriculture, washington, dc. . stewart, b. a., j. t. musick, and d. a. dusek. . yield and water use efficiency of grain sorghum in a limited irrigation-dryland farm- ing system. agronomy journal : - . . texas agricultural extension service. . texas crop enterprise budgets. texas panhan- dle district, texas a&m university, college station, texas. . thornton, p. k., and j. b. dent. . ibs- nat crop models in a socio-economic whole farm framework. agrotechnology transfer : - . . usda-ars. (undated). the usda conser- vation and production research laboratory. u.s. department of agriculture, bushland, texas. . williams, j. r., c. a. jones, and p. t. dyke. . the epic model. texas agricultural experiment station, college station, texas. busch, program chair, department of sociology, msu, east lansing, mi - . june - . a theology for coming home, sponsored by american baptist churches, usa board of national ministries, american baptist churches of the central region, and the land institute, will be held in salina, kansas. contact dr. a. david stewart, anderson, manhattan, ks ; ( ) - . july - . international crop sci- ence congress will be held at iowa state university in ames, iowa. con- tact brian meyer, isu ag information, ( ) - ; or steve jones, isu news service, ( ) - . july -august . participatory on-farm research and education for agricultural sustainability, sponsored by agricultural research institute, university of illinois college of agri- culture, american society of agron- omy, usda-epa, and lisa-ace program, will be held in champaign, illinois. contact dr. john m. gerber, ui agricultural experiment station, mumford hall, west greg- ory drive, urbana, il ; ( ) - . august - . resource management in a dynamic world, the soil and wa- ter conservation society's annual meeting, will be held in baltimore, maryland. contact tony vrana, swcs, northeast ankeny road, ankeny, ia - ; ( ) - . august - . rural society in the changing world order, the th world congress for rural sociology, will be held at pennsylvania state university, followed on august - by rurality and the global environment, the th annual meeting of the rural sociolog- ical society. contact world congress/ rss, penn state, agricultural ad- ministration building, university park, pa ; ( ) - . august - . th annual confer- ence, natural organic farmers associ- ation, will be held at hampshire col- lege, amherst, massachusetts. contact julie rawson, sheldon road, barre, ma ; ( ) - . august - . industrial and third world environmental assessment: the urgent transition to sustainability, the th annual meeting of the inter- national association for impact as- sessment, will be held at the world bank, washington, dc. contact mau- rice e. voland, iaia, p.o. box , belhaven, nc; ( ) - . november - . organic agricul- ture, a key to a sound development and a sustainable environment, a sci- entific conference of the international federation of organic agriculture movements, will be held in sao paulo, brazil. contact soma rel. e com. ltda, rua atlantica, , , sao paulo, sp, brazil. - a volume , number , https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms update on the genetics of congenital myopathies update on the genetics of congenital myopathies katarina pelin, phd,*,† and carina wallgren-pettersson, md, phd† the congenital myopathies form a large clinically and genetically heterogeneous group of disor- from the *molecular ulty of biological helsinki, finland. ythe folkh€alsan inst department of m helsinki, finland. supported in part b française contre l€akares€allskapet, , and the med address reprint reque environmental sci ), fi- helsi https://doi.org/ - / ders. currently mutations in at least different genes have been reported to cause a congenital myopathy, but the number is expected to increase due to the accelerated use of next-generation sequencing methods. there is substantial overlap between the causative genes and the clinical and histopathologic features of the congenital myopathies. the mode of inheritance can be auto- somal recessive, autosomal dominant or x-linked. both dominant and recessive mutations in the same gene can cause a similar disease phenotype, and the same clinical phenotype can also be caused by mutations in different genes. clear genotype-phenotype correlations are few and far between. semin pediatr neurol : - © elsevier inc. all rights reserved. introduction the application of next-generation sequencing methods,such as whole-exome sequencing, targeted gene panels, and whole-genome sequencing has resulted in an accelerated discovery of novel disease genes and disease-causing variants underlying the various types of congenital myopathies. further- more, the use of custom high-density oligonucleotide arrays for comparative genomic hybridization has enabled the discovery of large copy number variations (cnvs) causing, for example, nemaline myopathy and centronuclear myopathy. � the inheritance of congenital myopathies can be autosomal dominant, autosomal recessive or x-linked. de novo domi- nant disease-causing variants are common in some genes, for example, acta and tpm . , both dominant and recessive variants have been described in several genes, for example, acta , tpm , tpm , ryr , myh , and ttn. � interest- ingly, epigenetic silencing of a wild-type allele can result in and integrative biosciences research programme, fac- and environmental sciences, university of helsinki, itute of genetics, folkh€alsan research center, and edical and clinical genetics, university of helsinki, y the sigrid jus�elius foundation, the association les myopathies grant no. , the finska muscular dystrophy uk grant no. nem-pg - icinska underst€odsf€oreningen liv och h€alsa. sts to katarina pelin, phd, faculty of biological and ences, university of helsinki, p.o.box (viikinkaari nki, finland. e-mail: katarina.pelin@helsinki.fi . /j.spen. . . /© elsevier inc. all rights reserved. monoallelic expression of a mutant allele causing a congenital myopathy. this has been described for ryr and core myopa- thies. furthermore, it has been suggested that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital myopathies. mutations in the same gene can result in more than clini- cal phenotype, and the same clinical phenotype can result from mutations in several different genes. there is also sub- stantial variation in the severity of the clinical phenotype, even within genetic entity, seldom with any discernible genotype-phenotype correlations. nemaline myopathies including cap myopathy and fiber-type disproportion the clinical spectrum of nemaline myopathies (nm) is wide, ranging from severe congenital forms, sometimes already detect- able in utero, through the typical form to milder childhood- onset and even adult-onset forms. nemaline rods, derived from sarcomeric z discs, and often type fiber predominance, are characteristic pathological features of nm. cap myopathy is pathologically characterised by cap-like structures of disorgan- ised myofibrils and thickened z discs, but usually no large rods. following the description of families and patients with variable presence of nemaline rods and/or caps, , nm and cap myopathy are considered to be overlapping entities. fiber- type disproportion (ftd), that is, type hypotrophy in the http://crossmark.crossref.org/dialog/?doi= . /j.spen. . . &domain=pdf mailto:katarina.pelin@helsinki.fi https://dx.doi.org/ . /j.spen. . . congenital myopathy genetics presence of larger type fibers, but in the absence of specific pathological features, may be caused by the same genes as nm and cap myopathy. ftd and type fiber predominance are common features in the other congenital myopathies also, caused by mutations in other genes. eleven nm-causing genes have been described to date � (fig., table). seven of these genes, that is, acta , neb, tpm , tpm , tnnt , lmod , and mypn, encode structural proteins of the skeletal muscle sarcomere, cfl regulates actin filament dynamics and is essential for muscle maintenance, whereas three of the genes, that is, kbtbd , klhl , and klhl , encode proteins involved in the maintenance of sarcomeric integrity by regulating turnover of sarcomeric proteins. the nebulin gene (neb) disease-causing variants in the nebulin gene (neb) are the most common cause of autosomal recessive nm, accounting for approximately % of all nm cases, and the most com- mon cause of the typical form. the majority of the patients are compound heterozygous for different neb mutations. point mutations causing aberrant splicing, small indels caus- ing frameshifts, and nonsense mutations are the most com- mon mutation types in neb. a custom high-density oligonucleotide array, the nm-cgh array, has revealed sev- eral large, - kb, cnvs in neb, including recurrent cnvs in the triplicate region spanning exons - . , eight exons are repeated times in the -kb triplicate (tri) region of neb, and the normal copy number is ( copies in each allele). deletion or duplication of one tri copy is non- pathogenic, but gains of or more tri copies segregate with nm in % of the families studied, and are, thus, interpreted to be pathogenic. the cnvs in the tri region of neb can currently be detected only using the nm-cgh-array. we have estimated that a large pathogenic cnv in neb is present in %- % of nm patients. figure congenital myopathy-causing genes. the diagram show myopathies, and the overlap between different entities. cor between nemaline myopathy and core myopathy. missense variants are very common in neb. in the current release of the exac browser (http://exac.broadinstitute.org), % of the variants in the coding region (including splice sites and utrs) of neb are missense, % are synonymous changes, and % are apparent pathogenic variants (nonsense, splice site, frameshift, indels). most of the missense variants are rare, % of the variants being present in only - heterozygous carriers (allele frequencies well below . ). this makes the interpreta- tion of the pathogenicity of missense variants extremely difficult. our current recommendation is that only variants affecting con- served actin- and tropomyosin-binding sites in neb can readily be considered as pathogenic, but all the others require functional studies for assessment of their pathogenicity. actin- and tropo- myosin-binding experiments may be used for this purpose. in addition to the “classical” forms of nm, recessive disease- causing variants in neb may cause distal nebulin myopathy without nemaline rods, core-rod myopathy, distal forms of nm, and lethal multiple pterygium syndrome. to date, only clearly dominant neb variant has been found. it is a » kb in-frame deletion spanning neb exons - resulting in the expression of substantially smaller nebu- lin proteins, expected to have a dominant-negative effect. this variant segregates with a distal form of nm in a -generation finnish family. the skeletal muscle alpha-actin gene (acta ) according to our estimate, % of nm cases are caused by mutations in acta . most of the pathogenic variants in acta are dominant ( %) missense variants, most often causing severe nm. of the sporadic cases with acta variants, approxi- mately % have been shown to be caused by de novo mis- sense variants. autosomal recessive variants are rarer ( %), and result in null alleles (splice site, nonsense, frameshift, and some missense variants). dominant variants inherited across s genes implicated in various forms of the congenital e-rod myopathy was included to illustrate the overlap http://exac.broadinstitute.org table genes causing congenital myopathies disorder gene inheritance other entities caused by mutations in the gene nemaline myopathy neb ar, ad* distal nebulin myopathy, distal nemaline myopathy, core-rod myopathy, lethal multiple pterygium syndrome acta de novo, ad, ar actin-accumulation myopathy, core-rod myopathy, intranuclear rod myopathy, zebra body myopathy, cftd, progressive scapuloperoneal myopathy, distal nemaline myopathy tpm ad, de novo, ar cftd tpm ad, de novo, ar† cftd, core-rod myopathy, distal arthrogryposis, escobar syndrome (ar) tnnt ar cfl ar lmod ar mypn ar cardiomyopathy kbtbd ad klhl ar klhl ar core myopathy ryr ad, ar core-rod myopathy, cftd, malignant hyperthermia, multi-minicore disease with ophthalmoplegia, arthrogryposis multiplex congenita sepn ar rigid spine muscular dystrophy, cftd, desmin-related myopathy with mallory body-like inclusions, myofibrillar myopathy ttn ar tmd, lgmd j, hmerf, adult-onset recessive proximal mus- cular dystrophy, emery-dreifuss-like phenotype without cardiomyopathy, cardiomyopathy myh ad laing distal myopathy, cftd, myosin storage myopathy (hyaline body myopathy), cardiomyopathy megf ar centronuclear myopathy mtm x-linked dnm ad cmtdib, cmt m ryr ar see above bin ar, ad ttn ar see above speg ar ccdc ad myh-related myopathy myh ad, ar myh ad see above myh ad distal arthrogryposis myh ad distal arthrogryposis other congenital myopathies cacna s ad, ar hypokalemic periodic paralysis type , malignant hyperthermia scn a ar hypokalemic periodic paralysis type , congenital myasthenic syndrome , myotonia congenita, paramyotonia congenita zak ar split-foot malformation with mesoaxial polydactyly ad, autosomal dominant; ar, autosomal recessive; cftd, congenital fiber type disproportion; cmt, charcot-marie-tooth neuropathy; hmerf, hereditary myopathy with early respiratory failure; lgmd, limb-girdle muscular dystrophy; tmd, tibial muscular dystrophy. *only dominant neb mutation has been identified to date. †only recessive tpm mutation has been identified to date. k. pelin and c. wallgren-pettersson or more generations have been identified in less than % of acta families, while mosaicism has been observed in a few families. - we have recently described a dominant acta missense variant segregating in a -generation family with clini- cally variable nm, illustrating the clinical and histological vari- ability of nm between patients sharing the same mutation. in addition to nm, dominant, mostly de novo, disease-caus- ing variants in acta can cause actin-accumulation myopa- thy, cap myopathy, congenital fiber type disproportion, core-rod myopathy, intranuclear rod myopathy, , zebra body myopathy, progressive scapuloperoneal myopathy, and distal myopathy with nemaline rods. the alpha- and beta-tropomyosin genes (tpm and tpm ) mutations in tpm and tpm are relatively rare causes of nm, accounting for less than % of the cases. in addition to nm, mutations in tpm and tpm can cause cap myopathy, core- rod myopathy, congenital fiber type disproportion, distal congenital myopathy genetics arthrogryposes, and escobar syndrome. the majority of the mutations in both tpm and tpm are dominant missense variants or in-frame deletions removing one amino acid. a few recurrent mutations have been described in both genes; p.lys del and p.glu del in tpm , and p.arg his, p.arg cys, and p.arg gly in tpm . the mutations alter the conserved coiled-coil structure of the tropomyosins, resulting in aberrant tropomyosin-actin-binding. , recessive mutations are more common in tpm than in tpm . in tpm only recessive homozygous nonsense muta- tion has been described, causing escobar syndrome associated with nm. in tpm , a few recessive mutations, including non- sense, frameshift, and stop-lost mutations have been described. � nms caused by mutations in tpm usually have milder presentations than nms caused by mutations in tpm . recessive disease caused by mutations in these genes appears mostly to be severe. no clear correlation was found between the type of mutation and the clinical phenotype. cap formation in the muscle biopsy may be seen in disorders caused by mutations in either gene, and type fiber hypotrophy and predominance is common in both groups. furthermore, we have identified a large, - kb homozygous deletion that removes the promoter and the first exons of tpm , causing a severe form of nm. the troponin t gene (tnnt ) the first mutation in tnnt , a recessive nonsense mutation causing a severe form of nm with tremor in the first months of life and contractures in the old order amish, was described almost years ago. not until recently have a few other nm-causing mutations in tnnt been identified, all showing recessive inheritance. compound heterozygosity for a splice site mutation resulting in skipping of tnnt exon , and an exon deletion was identified in a dutch patient with a similar clinical phenotype as in amish nm. a clinical phenotype similar to amish nm was also observed in a hispanic patient homozygous for a nonsense mutation (different from the amish one) in tnnt . homozygosity for a complex indel mutation in tnnt causing premature truncation of the protein has been described in unrelated palestinian patients with a severe form of nm. the cofilin- gene (cfl ) recessive mutations in the cfl gene are rare causes of nm. the first cfl mutation was described in . the homozygous missense mutation, p.ala thr, was found to cause nm with some minicores in a large consanguineous family of middle east- ern origin showing congenital onset, delayed milestones and no facial weakness or foot drop. the second cfl mutation was published in . again a homozygous missense mutation, in this case p.val met, was found in sisters of iraqi kurdish origin with axial and limb girdle weakness who were born to consan- guineous parents. the sisters had nm with features of myofibril- lar myopathy. a third mutation described in cfl is a homozygous base pair deletion causing a frameshift, p.lys glnfs* . the mutation had caused a severe form of nm in a saudi arabian consanguineous family. the leiomodin- gene (lmod ) recessive mutations in lmod have hitherto been described in families with severe, often lethal forms of nm, which in some cases were associated with perinatal fractures. , most of the mutations are nonsense or frameshift variants causing loss of leiomodin- protein expression. the patients were homozygous or compound heterozygous for the mutations. the myopalladin gene (mypn) recessive mutations in mypn have been described in four fami- lies with childhood or adult-onset mild nm, and in families with congenital slowly progressive cap myopathy. , all mypn mutations described to date are loss-of-function variants, either nonsense, frameshift or splice site variants, leading to no or very low expression of myopalladin in skeletal muscle. the patients are either homozygous or compound heterozygous for the muta- tions. intranuclear rods, previously only associated with acta mutations, were observed in the muscle biopsies of of the patients with mild nm. interestingly, dominant mypn muta- tions have been reported to cause dilated, familial hypertrophic or familial restrictive cardiomyopathy. � contrary to the nm- and cap myopathy-causing mutations, the cardiomyopathy- causing mypn mutations lead to the expression of mutant proteins with dominant-negative effects in cardiomyocytes. the kelch repeat- and btb/poz domain- containing protein , the kelch-like and the kelch-like genes (kbtbd , klhl , and klhl ) kbtbd , klhl , and klhl encode proteins of the kelch superfamily including altogether genes and protein members. kbtbd interacts with cullin ubiquitin ligase, and this interaction is required for the formation of a func- tional cul ring ubiquitin ligase complex, which is involved in the ubiquitination of proteins destined for degradation. three different missense variants, p.arg ser, p.lys asn, and p.arg cys in kbtbd have been found to cause auto- somal dominant nm with cores, and unusual clinical presen- tations including a characteristic slowness of movement. klhl has been shown to bind and stabilize nebulin and lmod in the sarcomere, as well as prevent ubiquitination of lmod . recessive mutations in klhl are a fairly com- mon cause of severe nm, often with fetal akinesia or hypoki- nesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. mutations in klhl account for up to % of severe cases of nm in the japanese popula- tion due to a founder mutation, p.glu lys. one patient with a mild form of nm has been reported to be homozygous for a missense mutation, p.arg cys, in klhl . this mutation has not been found in the severe cases published to date. , , furthermore, one patient with severe nm due to compound heterozygous mutations in klhl showed pro- longed beneficial response to treatment with high-dose acetyl- cholinesterase inhibitors (pyridostigmine). such a response k. pelin and c. wallgren-pettersson has also been observed in other congenital myopathies, for example, myotubular/centronuclear myopathy. klhl shows high homology to klhl , but klhl preferentially stabilizes nebulin rather than lmod . five families with clinically different forms of nm have been found to have recessive mutations in klhl . frameshift mutations correlated with severe phenotypes with neonatal death, whereas missense variants resulted in impaired motor function with survival into late childhood and/or early adult- hood compatible with mild, typical or intermediate nm. core myopathies central core disease, minicore myopathy, and multiminicore disease are historical definitions of congenital myopathies with cores, that is, areas devoid of mitochondria and, thus, lack of oxidative enzyme activity in muscle biopsies. there is patho- logic, clinical and genetic overlap in congenital myopathies with cores, and thus the term “core myopathy” is nowadays preferentially used. five genes have been reported to cause core myopathies. the ryanodine receptor encoding gene, ryr , was the first one to be discovered, , and is now known as the major core myopathy-causing gene. the sec- ond most common core myopathy-causing gene, sepn enco- des selenoprotein n. occasional mutations causing core myopathies have also been described in the satellite cell gene megf , the titin gene ttn, and the myosin heavy chain encoding gene myh (fig., table). the ryanodine receptor gene (ryr ) ryr encodes the skeletal muscle specific ryanodine receptor ryr , which is a calcium release channel involved in excitation- contraction coupling activating muscle contraction. both domi- nant and recessive mutations in ryr have been found to cause core myopathies, but also related disorders such as core-rod myopathy, congenital ftd and centronuclear myopathy, as well as malignant hyperthermia susceptibility. , ryr is a large gene with exons encoding a polypeptide of amino acids, which forms the subunits of the tetramer calcium release channel. more than ryr mutations have been reported. , most mutations causing core myopathies and malignant hyperthermia are dominant missense variants changing conserved amino acids, many of them clustered in specific hotspot regions in the n-terminus, central region and in the c-terminal transmembrane region of ryr . , de novo dominant ryr variants have been reported to cause core-rod myopathy. - recessive ryr mutations are widespread throughout the gene and patients with such mutations are generally more severely affected than those with a dominant mutation. , the recessive mutations include null mutations, but also com- binations of missense variants. among the recessive variants, one recurrent allele carrying different missense variants (p. ile val, p.arg his, and p.tyr cys) has been reported in the dutch population, but it is unclear whether one of the variants is causative, or if a combination of or all variants cause disease. furthermore, this missense variant- carrying allele, as well as some other missense variants are associated with the malignant hyperthermia trait in heterozy- gous individuals, but cause recessive ryr -related myopathies in homozygous or compound heterozygous individuals. in addition to variants affecting a single or a few base pairs, recessive large-scale ryr deletions associated with myopa- thies have been published. , the first one, an in-frame deletion of out of ryr exons, was identified in a child with a congenital myopathy with lethal neonatal weak- ness and atypical histopathologic features. the child was compound heterozygous for the deletion and a single amino acid duplication. the second large deletion starts in ryr exon and ends within exon , causing a frameshift. the deletion was detected in a family with recessive late-onset core myopathy, the patients being compound heterozygous for the deletion and a missense variant. in addition, a reces- sive deletion of ryr exons - has been described in a family with severe arthrogryposis multiplex congenita. tissue-specific epigenetic silencing of the maternal ryr allele has been documented in a cohort of patients with recessive core myopathies. silencing of the maternal allele in skeletal muscle tissue unmasked the recessive paternal allele causing the disease. the selenoprotein n gene (sepn ) sepn encodes selenoprotein n, which is an integral mem- brane glycoprotein of the endoplasmic reticulum. sepn is expressed at high levels in several human fetal tissues, and is thought to have a role in early muscle development. sepn is physically associated with ryanodine receptors and modifies ryr channel activity. furthermore, it has recently been shown that sepn is a key component of redox- regulated calcium metabolism in the endoplasmic reticulum, through its interaction with the serca calcium pump. recessive loss-of-function mutations in sepn have caused entities termed rigid spine muscular dystrophy, core myopathy, congenital fiber type disproportion, and desmin- related myopathy with mallory body-like inclusions. , , due to the overlap of clinical and histopathologic features these disorders are now collectively referred to as sepn - related myopathies. all types of mutations have been identi- fied in sepn , many being truncating nonsense or frameshift variants, but missense variants affecting conserved amino acids are also common. homozygous mutations seem to be surprisingly prevalent, also in affected children born to non- consanguineous parents. , the multiple egf-like domain gene (megf ) a recessive congenital myopathy with minicores has been described, caused by missense variants in megf . three sib- lings were compound heterozygous for different missense var- iants, p.cys arg and p.cys arg in megf . megf regulates myoblast function via the notch signalling pathway, congenital myopathy genetics and the interaction between megf and notch is impaired by the p.cys arg variant. the titin gene (ttn) the huge ttn gene with exons encodes titin, the largest polypeptide in nature. one titin molecule reaches from the z disc to the m line in the skeletal and cardiac muscle sarco- meres. , given the size of titin, it is not surprising that sev- eral clinically distinct disorders affecting skeletal and/or cardiac muscle are caused by dominant or recessive muta- tions in ttn. most of these disorders have adult onset. however, five patients from families with congenital mus- cle weakness, minicore-like lesions and abundant centrally located nuclei, and severe childhood-onset dilated cardiomy- opathy were found to be homozygous for truncating muta- tions in the c-terminus of ttn. the parents were consanguineous in both families. furthermore, in a cohort of patients with congenital core myopathy combined with primary heart disease, pathogenic ttn variants were iden- tified in patients from families. the variants included missense and truncating mutations. the patients were homo- zygous or compound heterozygous for the mutations. the myosin heavy chain gene (myh ) the majority of the more than missense mutations iden- tified in the slow skeletal muscle fiber myosin heavy chain encoding gene myh cause cardiomyopathy. a subset of the mutations cause skeletal muscle disease, including laing distal myopathy and myosin storage myopathy. more recently mutations in myh have been reported in dominant core myopathies. , cullup al. described patients from families affected by multiminicore disease caused by novel dominant missense mutations in myh . romero et al described four patients in a -generation family with autoso- mal dominant central core disease. they identified a novel missense mutation in myh that segregated with the disease in the family. the mutations identified in these families are located in the myh tail region, close to previously described mutations causing laing distal myopathy. centronuclear myopathies centrally located nuclei in the muscle fibers are hallmarks of centronuclear (myotubular) myopathies (cnm), but some muscle biopsies may also show additional pathological fea- tures such as type fiber predominance, type fiber hypo- trophy, and cores. the most common genes causing centronuclear myopathies are mtm , dnm , ryr , and ttn. minor causative genes are bin , ccdc , and speg (fig., table). ryr and ttn variants identified in cnm will be discussed briefly below. the other cnm genes will be the focus of separate paragraphs. mutations in ryr have turned out to be a fairly common cause of autosomal recessive cnm (arcnm). the patients are usually compound heterozygous for mutations, often one truncating mutation on one allele and a missense one on the other allele. the mutations are spread all across the ryr gene. some of the ryr mutations found in arcnm patients have previously been reported in core myopathy or malig- nant hyperthermia susceptibility. � compound heterozygous truncating mutations causing arcnm have also been identified in the ttn gene. to date, unrelated patients with arcnm due to mutations in ttn have been described. , , the cnm-causing mutations are spread all along the ttn gene. one of the mutations has previously been reported to cause tibial muscular dystrophy, and another caused adult-onset cardiomyopathy in the het- erozygous state. the myotubularin gene (mtm ) mutations in mtm , encoding myotubularin, a ubiquitously expressed lipid phosphatase, cause x-linked myotubular myopathy (xlmtm). myotubularin colocalizes with ryr at the junctional sarcoplasmic reticulum in skeletal muscle, and it is a key regulator of sarcoplasmic reticulum remodelling together with its lipid substrate phosphatidyli- nositol -monophosphate (ptdins p). lack of mtm activity leads to disorganisation of the sarcoplasmic reticulum, which is considered to be the primary cause of most of the organelle positioning defects observed in muscles biopsies from xlmtm patients. the xlmtm-causing mutations in mtm are loss-of-function mutations spread across the exons of the gene. the majority of the patients are neonatally severely affected boys. most muta- tions are truncating, but missense variants affecting conserved amino acids essential for mtm activity are common also. , a few large deletions removing one or more mtm exons, as well as whole-gene deletions of mtm including neighbouring genes have been reported. the latter causes contiguous gene syndromes. , several different types of mtm pre-mrna splicing affecting mutations have also been described. , , germ line mosaicism for de novo mtm mutations has been documented in a few families, in some cases manifesting as paternal transmission of the x-linked pathogenic variant. , evidence is accumulating that there is a higher number of females manifesting xlmtm than previously antici- pated. , females with xlmtm are usually less severely affected than males, but the clinical phenotype is highly vari- able in age of onset and severity. the most severely affected females can show a similar clinical course as a severely affected xlmtm male. in general, those mtm mutations that cause a severe phenotype in males, cause a milder phe- notype in females, probably due to the normal pattern of approximately - x-chromosome inactivation in females. however, there is an increased prevalence of highly skewed x-chromosome inactivation in females affected by xlmtm, although it has not been possible to determine which of the x chromosomes is preferentially inactivated. not all mani- festing females show any skew, even in muscle tissue. interestingly, dynamin (dnm ) expression levels are increased in the muscles of xlmtm patients, as well as in mtm knock-out mice, indicating that mtm may be a k. pelin and c. wallgren-pettersson negative regulator of dnm expression. this finding has led to the development of a potential therapeutic approach aiming towards reducing dnm levels in the muscles of xlmtm patients. proof-of-principle has been achieved with antisense oligonucleotide-mediated dnm knockdown in a mouse model for xlmtm. the dynamin gene (dnm ) dnm encodes dynamin , a large gtpase involved in diverse cellular processes, among others endocytosis, cytoki- nesis, phagocytosis, and cell migration. mutations in dnm cause autosomal dominant adcnm with onset usually in adolescence or early childhood, with ptosis, distal weakness and contractures, and often radial strands in muscle fibers on biopsy, and charcot-marie-tooth (cmt) peripheral neu- ropathy (cmtdib and cmt m). � however, cases with earlier onset have been reported due to de novo muta- tions in the pleckstrin homology domain of dnm . the adcnm-causing mutations in dnm are gain-of -function mutations, predominantly missense variants. one in-frame deletion of one amino acid, as well as one splice site mutation causing an in-frame deletion of three amino acids in addition to an in-frame insertion of new amino acids have been identified. many of the missense variants are recurrent and present in several unrelated families. the mutations causing adcnm are distinct from the ones causing cmt. functional studies of common adcnm dnm mutations show abnormal self-assembly of mutant dnm resulting in abnormally high gtpase activity of the protein, which in turn leads to t-tubule fragmentation. the hyperactive mutant dnm protein is a potential therapeutic target in adcnm, that is, downregulation of dnm activity should have a similar beneficial effect in adcnm muscle as in xlmtm muscle. , the bridging integrator gene (bin ) bin encodes for amphiphysin , a protein involved in mem- brane tubulation. the membrane tubulation activity of bin is enhanced by its interaction with mtm . nicot et al described the first disease-causing variants in bin years ago. two missense variants and nonsense variant were shown to cause arcnm with congenital or childhood onset in consanguineous families. the patients were homozygous for the mutations. subsequently, novel homozygous missense mutation and novel homozygous nonsense muta- tion have been published as causative for arcnm. , furthermore, a homozygous acceptor splice site mutation in intron causing abnormal splicing of the skeletal muscle- specific bin exon was identified in patients with rapidly progressive arcnm in consanguineous family. the corre- sponding splice site was found to be mutated in canine inherited myopathy of great danes, which, thus, represents a mammalian model for bin -related cnm. dominant mutations in bin have been reported to cause mild and adult-onset forms of cnm. , three of the mutations are single base pair deletions in the stop codon of bin , causing read-through and extension of the protein with novel amino acids. two other dominant mutations were in-frame deletion of amino acid, and missense mutation, located in the n-terminus of bin . a second dominant missense mutation, also in the n-terminus, was recently published. the dominant bin mutations are dis- tinct from the recessive ones, with different impacts on pro- tein function, suggesting different pathomechanisms for dominant and recessive bin -related cnm. the speg complex locus gene (speg) speg interacts with myotubularin at the junctional sarcoplas- mic reticulum in skeletal muscle. speg is also expressed in car- diac muscle. the first speg mutations were described in cnm patients from families. in addition to cnm, unre- lated patients had dilated cardiomyopathy. the mutations were recessive loss-of-function mutations (nonsense or frameshifts), and the patients were compound heterozygous or homozygous for the mutations. two novel speg mutations were recently reported in unrelated cnm patients. one of the patients had dilated cardiomyopathy also. the patient with cnm and car- diomyopathy was homozygous for a nonsense mutation, and the patient with cnm without cardiac involvement was homo- zygous for a frameshift mutation in speg. the coiled-coil domain-containing protein gene (ccdc ) only dominant mutation in ccdc causing cnm with atypical cores has been described in family with patients in generations. the mutation changes the acceptor splice site of intron in ccdc , causing retention of the intron, which is in-frame with the coding sequence. this is predicted to result in the addition of amino acids to the protein. myosin-related myopathies myosin heavy-chain genes, especially myh , myh , myh , and myh are implicated in various myopathies affecting skel- etal and/or cardiac muscle (fig., table). some of these myopa- thies are congenital. myh was already discussed in the context of the core myopathies, but dominant mutations in myh can also cause, for example, congenital fiber type dis- proportion without any other specific histological features. one such case was recently reported to be due to a de novo heterozygous splice site mutation causing skipping of myh exon . a few cases of myh -related congenital myopathy were also found in a cohort of italian patients. these patients had dominant missense mutations in myh . both dominant and recessive mutations in myh can cause a usually mild congenital myopathy with external oph- thalmoplegia. the dominant cases are caused by missense mutations, and the recessive ones usually by truncating mutations in myh . a homozygous splice site mutation causing skipping of myh exon , leading to a frameshift, congenital myopathy genetics was recently reported in a consanguineous family where patients had a congenital myopathy with ophthalmople- gia. a novel homozygous frameshift mutation in myh has also recently been described to cause a congenital myop- athy with chronic aspiration pneumonia in infancy. myh and myh encode embryonic and fetal myosin heavy-chain isoforms. dominant missense mutations in myh and myh cause distal arthrogryposis syndromes, probably as the result of a severe muscle weakness already during fetal development. , , other genes causing congenital myopathies two genes, cacna s and scn a, previously known channelopathy-causing genes, have now been implicate in congenital myopathies as well. � a third gene, zak, has also recently been identified as a novel congenital myopathy-causing gene (fig., table). a dihydropyridine receptor (dhpr) congenital myopathy caused by dominant or recessive mutations in the cacna s gene was recently described in patients from families. the muscle biopsies showed features of centralised nuclei, focal zones of sarcomeric disorganisation, and cores. dhpr directly regulates the ryr calcium release channel. both the dominant and recessive mutations identified in cacna s are hypothesised to cause a decrease in overall dhpr func- tion in skeletal muscle. recessive loss-of-function mutations in the scn a gene encoding the alpha-subunit of the skeletal muscle voltage-gated sodium channel (nav . ) have been identified in patients from families with a congenital myopathy of variable severity, severe or “classical.” histological features were unspecific; abnormal fiber size variability, in some with type predominance, and no pathognomic findings. � partial loss-of-function mutations were associated with a milder disease phenotype. a congenital myopathy with fiber type disproportion caused by recessive loss-of-function mutations in the mito- gen-activated protein triple kinase encoding gene, zak, was recently reported in six patients from three families. the patients were homozygous for frameshift or nonsense muta- tions in zak. the parents were consanguineous in all fami- lies. all mutations are located in the kinase domain of zak. 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) - /sbref http://refhub.elsevier.com/s - ( ) - /sbref http://refhub.elsevier.com/s - ( ) - /sbref http://refhub.elsevier.com/s - ( ) - /sbref http://refhub.elsevier.com/s - ( ) - /sbref 	update on the genetics of congenital myopathies 	introduction 	nemaline myopathies including cap myopathy and fiber-type disproportion 	the nebulin gene (neb) 	the skeletal muscle alpha-actin gene (acta ) 	the alpha- and beta-tropomyosin genes (tpm and tpm ) 	the troponin t gene (tnnt ) 	the cofilin- gene (cfl ) 	the leiomodin- gene (lmod ) 	the myopalladin gene (mypn) 	the kelch repeat- and btb/poz domain-containing protein , the kelch-like and the kelch-like genes (kbtbd , klhl , and klhl ) 	core myopathies 	the ryanodine receptor gene (ryr ) 	the selenoprotein n gene (sepn ) 	the multiple egf-like domain gene (megf ) 	the titin gene (ttn) 	the myosin heavy chain gene (myh ) 	centronuclear myopathies 	the myotubularin gene (mtm ) 	the dynamin gene (dnm ) 	the bridging integrator gene (bin ) 	the speg complex locus gene (speg) 	the coiled-coil domain-containing protein gene (ccdc ) 	myosin-related myopathies 	other genes causing congenital myopathies 	conclusions 	references novel autosomal dominant tnnt mutation causing nemaline myopathy novel autosomal dominant tnnt mutation causing nemaline myopathy the harvard community has made this article openly available. please share how this access benefits you. your story matters citation konersman, chamindra g., fernande freyermuth, thomas l. winder, michael w. lawlor, clotilde lagier#tourenne, and shailendra b. patel. . “novel autosomal dominant tnnt mutation causing nemaline myopathy.” molecular genetics & genomic medicine ( ): - . doi: . /mgg . . http:// dx.doi.org/ . /mgg . . published version doi: . /mgg . citable link http://nrs.harvard.edu/urn- :hul.instrepos: terms of use this article was downloaded from harvard university’s dash repository, and is made available under the terms and conditions applicable to other posted material, as set forth at http:// nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of- use#laa http://osc.hul.harvard.edu/dash/open-access-feedback?handle=&title=novel% autosomal% dominant% tnnt % mutation% causing% nemaline% myopathy&community= / &collection= / &owningcollection / &harvardauthors= cebe cf fe d c &department http://nrs.harvard.edu/urn- :hul.instrepos: http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa original article novel autosomal dominant tnnt mutation causing nemaline myopathy chamindra g. konersman , fernande freyermuth , , thomas l. winder ,a, michael w. lawlor , clotilde lagier-tourenne , & shailendra b. patel ,b department of neurosciences, university of california san diego, san diego, california massgeneral institute for neurodegenerative disease, department of neurology, massachusetts general hospital, harvard medical school, charlestown, massachusetts broad institute of harvard university and mit, cambridge, massachusetts prevention genetics, marshfield, wisconsin division of pediatric pathology, department of pathology and laboratory medicine and neuroscience research center, medical college of wisconsin, milwaukee, wisconsin division of endocrinology, metabolism and clinical nutrition, medical college of wisconsin, and clement j. zablocki vamc, milwaukee, wisconsin keywords congenital myopathy, nemaline myopathy, tnnt , troponin t correspondence chamindra g. konersman, va san diego healthcare system, la jolla village drive, neurology service , room b - , san diego, ca . tel: + ; fax: + ; e-mail: ckonersman@ucsd.edu current addresses ainvitae corporation, san francisco, california bdivision of endocrinology, diabetes and metabolism, university of cincinnati, cincinnati, ohio funding information c. l.-t. received salary support from the ludwig institute for cancer research, the massachusetts general hospital, and ninds/ nih (r ns ). received: may ; revised: july ; accepted: july molecular genetics & genomic medicine ; ( ): – doi: . /mgg . abstract background nemaline myopathy (nem) is one of the three major forms of congenital myopa- thy and is characterized by diffuse muscle weakness, hypotonia, respiratory insuf- ficiency, and the presence of nemaline rod structures on muscle biopsy. mutations in troponin t (tnnt ) is of genes known to cause nem. to date, only homozygous nonsense mutations or compound heterozygous truncat- ing or internal deletion mutations in tnnt gene have been identified in nem. this extended family is of historical importance as some members were reported in the s as initial evidence that nem is a hereditary disorder. methods proband and extended family underwent sanger sequencing for tnnt . we performed rt-pcr and immunoblot on muscle to assess tnnt rna expres- sion and protein levels in proband and father. results we report a novel heterozygous missense mutation of tnnt c. a>t (p.e v) that segregated in an autosomal dominant fashion in a large family residing in the united states. extensive sequencing of the other known genes for nem failed to identify any other mutant alleles. muscle biopsies revealed a characteristic pattern of nemaline rods and severe myofiber hypotrophy that was almost entirely restricted to the type fiber population. conclusion this novel mutation alters a residue that is highly conserved among vertebrates. this report highlights not only a family with autosomal dominant inheritance of nem, but that this novel mutation likely acts via a dominant negative mechanism. introduction nemaline myopathy (nem; mim# ) is a clinically and genetically heterogeneous form of congenital myopa- thy characterized by muscle weakness and the presence of nemaline rods on histologic examination (greenfield et al. ; shy et al. ; romero et al. ). nem is one of the three major types of congenital myopathy that include nemaline myopathy, centronuclear myopathy, and core-related myopathies. the incidence of nem is ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. this is an open access article under the terms of the creative commons attribution license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://creativecommons.org/licenses/by/ . / estimated at : , (romero et al. ). the proto- typic clinical features include congenital hypotonia, weak- ness of proximal, bulbar, facial, and neck flexor muscles that may result in death secondary to respiratory insuffi- ciency (romero et al. ). mutations in genes are currently known to cause nem: actin alpha (acta ) (nowak et al. ), nebulin (neb) (pelin et al. ), al- pha-tropomyosin (tpm ) (laing et al. a,b), beta-tro- pomyosin (tpm ) (donner et al. ), troponin t (tnnt ) (johnston et al. ), cofilin- (cfl ) (agra- wal et al. ), kelch repeat and btb domain-containing (kbtbd ) (sambuughin et al. ), kelch-like fam- ily member (klhl ) (ravenscroft et al. ), kelch- like family member (klhl ) (gupta et al. ), and leiomodin- (lmod ) (yuen et al. ). the proteins encoded by these genes are all key to the function of sar- comeric thin filaments. tnnt -related nem (nem , mim ) is a reces- sive disorder that, until recently, was known to occur only in the old order amish of pennsylvania. in this popula- tion all patients carry a homozygous nonsense founder mutation, c. g>t (p.e x), resulting in a premature stop codon in exon (johnston et al. ). amish nemaline myopathy presents with tremors of the jaw and lower limbs in the neonatal period that gradually subside with age, development of progressive proximal muscle contractures, diffuse atrophy, delays in gross motor devel- opment, pectus carinatum, and respiratory insufficiency by the second year of life (johnston et al. ). the p.e x mutation results in complete loss of troponin t in muscle (jin et al. ). in , van der pol et al. described the first case of nem known outside of the old order amish in a dutch family with a compound heterozygous splice site c. + g>a mutation and an exon deletion in tnnt . the c. + g>a mutation in splice donor site of exon results in abnormal skip- ping of this exon and the production of a shortened mrna transcript (van der pol et al. ). deletion of exon was shown to alter the function of tropomyosin- binding site and the deletion of exon is predicted to destabilize the tnnt protein, thereby reducing binding affinity and incorporation into the thin filament (amaras- inghe et al. ). the dutch clinical phenotype was sim- ilar to amish nem with hypotonia, delayed gross motor milestones, progressive contractures, diffuse weakness with atrophy, absent reflexes, pectus carinatum, severe kyphosis, spinal rigidity, and absence of tremors, eventu- ally causing respiratory insufficiency requiring home ven- tilation at age (van der pol et al. ). marra et al. ( ) reported a second case of nem in a hispanic boy born from consanguineous parents and carrying a homozygous nonsense c. c>g (p.s x) mutation. this variant occurs in exon and results in a truncated troponin t protein with missing amino acids at its c-terminus, thereby damaging tropomyosin-binding site (amarasinghe et al. ). the clinical phenotype of this hispanic male is reminiscent of the amish nem with hypotonia, delayed motor development without achieving crawling or independent walking and dysarthria with mild expressive language delay without cognition abnormality by . years of age (marra et al. ). other salient fea- tures include a high-arched palate, pectus carinatum, tho- racic kyphoscoliosis, hip and knee contractures, facial weakness with tented mouth, prominent head lag, promi- nent proximal muscle weakness, and need for noninvasive ventilation and gastrostomy tube placement by . years (marra et al. ). finally, abdulhaq et al. described nine palestinian patients from seven unrelated families all carrying a similar homozygous c. _ indel tagtgctgt (l *) in tnnt leading to a c-terminal truncation of the protein which reduces affinity of tro- ponin t protein to tropomyosin (abdulhaq et al. ; amarasinghe et al. ). the palestinian patients’ phe- notype also resembled the amish population with the exception of rigid spine with kyphosis, stiff neck, and a transient tremor (abdulhaq et al. ). all five tnnt mutations described so far have involved the functionally important and evolutionarily highly conserved c-terminal region of the troponin t protein (jin et al. ; johnston et al. ; van der pol et al. ; marra et al. ; mondal and jin ). we report a novel autosomal dominantly inherited missense mutation in tnnt gene (c. a>t, p.e v) with considerable intrafamilial clinical heterogeneity in a large family with ashkenazi jewish ancestry residing in the united states. this family is of historical importance since various members of the family were clinically and pathologically described by spiro and kennedy ( ) and gonatas et al. ( ) as the first cases of hereditary nemaline myopathy. patients and methods patient cohort members of an extended family with ashkenazi jewish ancestry, living in distant parts of the united states, had their blood samples drawn after obtaining informed con- sent. information regarding the history, clinical manifesta- tions and rate of progression, age of onset, and genetic relationships were collected. some of the participants were evaluated personally by one of the authors (cgk, indicated by asterisk on pedigree, fig. ). clinical data were obtained either personally, by chart review, or via telephone for subjects iii. , iii. , iii. , iii. , iii. , iii. , iii. , iii. , iii. , iv. , iv. , iv. , iv. , iv. ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. c. g. konersman et al. tnnt mutation causing nem (table ). the institutional review board of the medical college of wisconsin approved this study and all partici- pants provided written informed consent. mutation screening genomic dna was extracted as per standard protocols from blood in all individuals who agreed to participate. the proband underwent sanger sequencing of full coding regions and ~ bases of flanking noncoding sequences of nine of the nem-related genes (acta [nm_ . ], cfl [nm_ . ], klhl [nm_ . ], kbtbd [nm_ . ], klhl [nm_ . ], neb [nm_ . ], tnnt [nm_ . ], tpm [nm_ . ], tpm [nm_ . ]). lmod was not evaluated as it was identified as a cause of nem after this genetic analysis had been completed (yuen et al. ). the proband also underwent analysis of neb exon dele- tion using appropriate primers that flank or lie within the deleted exon (anderson et al. ). to determine which variants found in the proband segregated with disease, the remaining subjects were subsequently analyzed by pcr amplification and sequencing for tnnt exon and neb exon . primer sequences used are available on request. muscle pathology vastus lateralis open muscle biopsies of three affected family members (fig. ; subjects iv. [proband], iv. , iii. ) were analyzed at time of presentation or if genetic analysis indicated presence of the suspected segregating mutation. one specimen was frozen in isopentane pre- cooled by liquid nitrogen and a small piece of muscle was fixed in % glutaraldehyde. standard techniques were applied for hematoxylin and eosin (h&e) and enzyme histochemical staining. biopsies from subjects iii. and iv. underwent further structural analysis via electron microscopy using standard techniques at the medical col- lege of wisconsin (mcw) electron microscopy core facility. rna isolation and expression analysis via reverse transcription and pcr total rna was isolated from human muscle tissue using trizol reagent (thermo fisher scientific, carlsbad, ca, usa). genomic dna was removed from rna by incubation with turbo dna-free kit (thermo fischer scientific, vilnius, lithuania) at °c for min. using superscript iii reverse transcriptase (thermo fisher scientific, carlsbad, ca, usa), ng of total rna was reverse transcribed to generate cdna. pcr was per- formed with taq polymerase, one denaturation step at °c for min, cycles of amplification at °c for sec, °c for sec, °c for min, and a final step at °c for min using the specific human primers for tnnt exon (forward: ggctcagcctcaagattcac, reverse: tccagcaggtctttctccat), for tnnt exon (forward: tggagctgcagacactcatc; reverse: ttaccacgcttctgttctgc), and for tnnt exon figure . family pedigree with current age or age at time of death. solid black circles and squares designate affected female and male subjects, respectively, as proven by skeletal muscle biopsy or muscle weakness. arrow indicates proband. subject i. is the half-uncle of i. , and iii. and iii. are third cousins. *examined personally by author cgk. †genetically tested. subjects published previously are cited. ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. tnnt mutation causing nem c. g. konersman et al. t a b le . c lin ic a l, g e n e ti c, p a th o lo g ic a l, a n d la b o ra to ry fi n d in g s o f a ff e ct e d a n d u n a ff e ct e d in d iv id u a ls in th e e xt e n d e d fa m ily . p e d ig re e d e si g n a ti o n a g e h is to ri ca l p u b lic a ti o n m u ta ti o n (s ) p e rt in e n t cl in ic a l fi n d in g s a n d o th e r st u d ie s m u sc le b io p sy fi n d in g s ii. d ie d a t a g e s p ir o a n d k e n n e d y ( ) c lin ic a l h is to ry o b ta in e d th ro u g h d a u g h te r ii. d e ce a se d � in ab il it y to h ee l w al k an d w ad d le at ag e � m il d sh o u ld er /p el v ic g ir d le w ea k n es s at ag e � in te rm it te n t u se o f w al k er an d d o e at ag e � n o rm al d t r th ro u g h o u t at y ea rs o f ag e � s le n d er b u il d � a b le to as ce n d st ai rs w it h m ar k ed d if fi cu lt y p ri o r to d ea th � d ie d o f a st ro k e an d co m p li ca ti o n s o f h ea rt fa il u re at ag e n e m a lin e ro d s p re se n t in ty p e a n d fi b e rs a n d d is tr ib u te d u n e ve n ly th ro u g h o u t sa rc o p la sm q u a lit a ti ve ly fe w e r ro d s th a n e a rl ie r m a n if e st in g d a u g h te r iii : iii . ye a rs n o m u ta ti o n in t n n t n e b c. t > c , p .s p n o sy m p to m s iii . ye a rs s p ir o a n d k e n n e d y ( ) t n n t c. a > t , p .e v n e b c. t > c , p .s p � l o rd o ti c p o st u re in in fa n cy w it h g o w er s’ si g n , d if fi cu lt y cl im b in g st ai rs an d ru n n in g , w ad d li n g g ai t � p es ca v u s an d in ab il it y to w al k o n h ee ls at y ea rs � m il d d o e si n ce te en s � m o d er at e d if fi cu lt y w al k in g p ro lo n g ed d is ta n ce s � d ec re as ed d t r in u p p er ex tr em it ie s � m il d o cc as io n al d y sp h ag ia si n ce ~ ag e � s le n d er b u il d � a t ag e , st il l am b u la to ry an d ca n w al k fo r m o re th an h p er d ay � n o ti ce d im p ro v em en t in d o e w it h d ai ly su st ai n ed ex er ci se fo r se v er al h o u rs n e m a lin e ro d s p re se n t in ty p e a n d fi b e rs d is tr ib u te d u n e ve n ly th ro u g h o u t sa rc o p la sm iii . ye a rs n o m u ta ti o n in t n n t n e b c. t > c , p .s p n o sy m p to m s iii . ye a rs t n n t c. a > t , p .e v n e b c. t > c , p .s p � g o w er s’ si g n an d d if fi cu lt y ru n n in g , cl im b in g , ju m p in g si n ce ag e � c h il d h o o d to e w al k in g w it h m in o r d if fi cu lt y h ee l w al k in g at ag e � g ra d u al w ea k n es s in le g b u t in d ep en d en tl y am b u la to ry at ag e � s le n d er b u il d � n o p ec tu s d ef o rm it y � h ig h ar ch ed p al at e � u n ab le to ru n at ag e ; m in o r d if fi cu lt y sw al lo w in g n e m a lin e ro d s m o st ly p re se n t in ty p e a n d ra re ly fo u n d in ty p e fi b e rs . fi b e r ty p e d is p ro p o rt io n w it h a tr o p h ic ty p e a n d h yp e rt ro p h ic ty p e fi b e rs . iii . ye a rs g o n a ta s e t a l. ( ) t n n t c. a > t , p .e v n e b c. t > c , p .s p � g o w er s’ si g n in la te te en s � d if fi cu lt y w al k in g lo n g d is ta n ce , ri si n g fr o m ch ai r, cl im b in g st ai rs � s le n d er b u il d � p ec tu s ca ri n at u m � d t r w er e n o rm al at ag e � s n o ri n g an d o b st ru ct iv e sl ee p ap n ea � a t ag e , ca n w al k in d ep en d en tl y � u n ab le to h ee l w al k n e m a lin e ro d s m o st ly p re se n t in ty p e a n d ra re ly fo u n d in ty p e fi b e rs . fi b e r ty p e d is p ro p o rt io n w it h a tr o p h ic ty p e a n d h yp e rt ro p h ic ty p e fi b e rs . (c o n ti n u e d ) ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. c. g. konersman et al. tnnt mutation causing nem t a b le . c o n ti n u e d . p e d ig re e d e si g n a ti o n a g e h is to ri ca l p u b lic a ti o n m u ta ti o n (s ) p e rt in e n t cl in ic a l fi n d in g s a n d o th e r st u d ie s m u sc le b io p sy fi n d in g s iii . * ye a rs g o n a ta s e t a l. ( ) t n n t c. a > t , p .e v n e b c. t > c , p .s p � s lo w ru n n er , d if fi cu lt y ri d in g b ic y cl e co m p ar ed to p ee rs as a te en ag er � m il d g ra d u al p ro x im al m u sc le w ea k n es s w it h ag e � h ig h ar ch ed p al at e � p ec tu s ca ri n at u m � m y o p at h ic , el o n g at ed fa ci es � d t r ab se n t o n ly at a ch il le s an d b ra ch io ra d ia li s b il at er al ly � a t ag e , n o lo n g er ab le to ru n o r ju m p , h as m in o r d if fi cu lt y as ce n d in g st ai rs b u t ca n w al k ~ m il es p er d ay � n o g o w er ’s m an eu v er at ag e � k y p h o si s; m il d sc o li o si s si n ce ag e � s ca p u la r w in g in g � s le n d er b u il d � a b le to h ee l w al k , b u t m il d ly u n st ea d y � c k (r ef – iu /l ) n e m a lin e ro d s m o st ly p re se n t in ty p e a n d ra re ly fo u n d in ty p e fi b e rs . fi b e r ty p e d is p ro p o rt io n w it h a tr o p h ic ty p e a n d h yp e rt ro p h ic ty p e fi b e rs . t yp e fi b e r p re d o m in a n ce . iii . * ye a rs t n n t c. a > t , p .e v n e b c. g > a , p .r h n e b c. t > c , p .s p � in re tr o sp ec t, n o te d in ab il it y to “b u lk u p ” in h is s (n o fo ca l m u sc le w ea k n es s) d u ri n g w ei g h t tr ai n in g � v er y ac ti v e in h is y o u th b u t le an m u sc u la tu re � w it h ag e, fe lt m in o r fa ti g u in g in le g s w it h p ro lo n g ed ex er ci se � e lo n g at ed fa ci es , h ig h ar ch ed p al at e � n o rm al re fl ex es � n o p ec tu s d ef o rm it ie s � n o d if fi cu lt y w al k in g o n h ee ls � m in o r d y sp h ag ia n o te d at ag e � r ep o rt ed sy m p to m at ic im p ro v em en t in en d u ra n ce w it h l -t y ro si n e g /d ay fo r y ea r � c k at ag e (r ef – u /l ) n e m a lin e ro d s a lm o st e xc lu si ve ly p re se n t in ty p e fi b e rs a n d ve ry ra re ly in ty p e fi b e rs . e vi d e n ce o f fi b e r ty p e d is p ro p o rt io n w it h a tr o p h ic ( – l m ) ty p e a n d h yp e rt ro p h ic ( – l m ) ty p e fi b e rs . t yp e fi b e r p re d o m in a n ce . iii . * ye a rs n o n e in t n n t a n d n e b g e n e s n o sy m p to m s o r si g n s iii . ye a rs n o m u ta ti o n in t n n t n e b c. t > c , p .s p n o sy m p to m s iv . ye a rs n o m u ta ti o n in t n n t n e b c. t > c , p .s p n o sy m p to m s (c o n ti n u e d ) ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. tnnt mutation causing nem c. g. konersman et al. t a b le . c o n ti n u e d . p e d ig re e d e si g n a ti o n a g e h is to ri ca l p u b lic a ti o n m u ta ti o n (s ) p e rt in e n t cl in ic a l fi n d in g s a n d o th e r st u d ie s m u sc le b io p sy fi n d in g s iv . * ye a rs t n n t c. a > t , p .e v n o m u ta ti o n in n e b � m in o r o cc as io n al tr ip p in g � m in o r d if fi cu lt y h ee l w al k in g w it h m il d a ch il le s co n tr ac tu re s b il at er al ly � h ig h ar ch ed p al at e w it h el o n g at ed fa ci es � n o p ro x im al m u sc le w ea k n es s, al l d t r s p re se n t � c k (r ef – ) b io p sy sl id e s p o o r q u a lit y to co n fi rm p re se n ce o f n e m a lin e ro d s, b u t n e m a lin e ro d s re p o rt e d b y p a ti e n t. iv . * ye a rs n o m u ta ti o n in t n n t n e b c. g > a , p .r h n e b c. t > c , p .s p n o sy m p to m s iv . * ye a rs t n n t c. a > t , p .e v n o m u ta ti o n in n e b � m il d d if fi cu lt y w it h lo w er ex tr em it y w ei g h t li ft in g at ~ ag e � h ig h ar ch ed p al at e � m il d m y o p at h ic fa ci es � r ed u ce d re fl ex es in u p p er ex tr em it ie s co m p ar ed to lo w er , h o w ev er ab se n t at an k le s � n o rm al st re n g th w it h ab il it y to sq u at , ju m p , ri se fr o m k n ee li n g p o si ti o n � u n ab le to h ee l w al k � n o p ec tu s d ef o rm it y � n o sc o li o si s n e m a lin e ro d s a lm o st e xc lu si ve ly in ty p e fi b e rs a n d ve ry ra re ly in ty p e fi b e rs . e vi d e n ce o f co n g e n it a l fi b e r ty p e d is p ro p o rt io n w it h re la ti ve ly a tr o p h ic ( – l m ) ty p e a n d h yp e rt ro p h ic ( – l m ). e m d e m o n st ra te d m it o ch o n d ri a l in cl u si o n s b u t n o n e m a lin e ro d s (l ik e ly se co n d a ry to sa m p lin g a rt if a ct ). iv . * p ro b a n d ye a rs t n n t c. a > t , p .e v n e b c. g > a , p .r h � f at ig u in g o f lo w er ex tr em it y m u sc le s w it h p ro lo n g ed ex er ci se an d m il d fa ci al w ea k n es s � h ig h ar ch ed p al at e � e lo n g at ed m y o p at h ic fa ci es � p ec tu s ca ri n at u m � m il d sc o li o si s � m il d w ad d li n g g ai t an d in ab il it y to w al k o n h ee ls at ag e � s le n d er b u il d � m il d p ro x im al m u sc le w ea k n es s w it h p o o r ju m p an d q u ad ri ce p s w ea k n es s � r ed u ce d d t r in u p p er ex tr em it ie s � e m g d o n e at ag e w as m y o p at h ic � r ep o rt ed sy m p to m at ic im p ro v em en t in en d u ra n ce w it h l -t y ro si n e g /d ay fo r y ea rs � c k (r ef – iu /l ) n e m a lin e ro d s a lm o st e xc lu si ve ly p re se n t in ty p e fi b e rs . s tr ik in g fi b e r ty p e d is p ro p o rt io n w it h a tr o p h ic ty p e fi b e rs a n d ty p e p re d o m in a n ce * e xa m in e d b y c g k ; c k , cr e a ti n e k in a se ; n /a , n o t a va ila b le ; d o e , d ys p n e a o n e xe rt io n ; t n n t , tr o p o n in t g e n e ; n e b , n e b u lin g e n e ; d t r , d e e p te n d o n re fl e xe s; e m g , e le ct ro m yo g ra p h y; e m , e le ct ro n m ic ro sc o p y. ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. c. g. konersman et al. tnnt mutation causing nem (forward: gcagaacagaagcgtggtaa, reverse: gcc atcaggtcgaacttctc). the pcr products were ana- lyzed by electrophoresis with % polyacrylamide native gel, stained with sybr gold nucleic acid gel stain (thermo fisher scientific, eugene, or, usa, : , ) and visual- ized with gel imaging system (bio-rad, hercules, ca, usa). the imaging quantification of each pcr product was performed using imagej quantification. western blotting of tnnt protein total protein extracts isolated by trizol reagent (thermo fisher) from biopsied vastus lateralis muscle from subjects iii. (biopsied at age ), iv. (biopsied at age ), an age-matched -year-old healthy male control, and a - year-old healthy female control were obtained. muscle biopsies from subjects iii. and iv. were obtained under an irb-approved protocol through the congenital muscle disease tissue repository (http://www.mcw.edu/ congenital-muscle-disease-cmd-tissue-repository.htm). control biopsies were obtained from dr. denise malicki and dr. karra jones under an irb-approved research pro- tocol at ucsd. the protein samples were mixed with x laemmli sample buffer and denatured at °c for min. the muscle protein extracts were resolved on % laemmli gel with an acrylamide to bis-acrylamide ratio : and transferred onto pvdf membrane. after block- ing with % nonfat dry milk (nfm) in tris-buffered sal- ine (tbs, mmol/l nacl, mmol/l tris-hcl, ph . ), the membrane was either incubated with mouse monoclonal antibody anti-slow troponin t (clone ct , santa cruz, sc- , : ) or with rabbit monoclonal anti-gapdh (clone c , cell signaling, , : ). then, the membrane was washed three times and incu- bated with anti-mouse or anti-rabbit peroxidase antibody (ge healthcare life sciences, : , ) for h in tbs- % nfm, followed by revelation using ecl chemilumi- nescence system (thermo fisher scientific, rockford, il, usa). results clinical features the proband, an -year-old ashkenazi jewish male (fig. , table , iv. ) born to nonconsanguineous par- ents, presented at years of age with mild facial and palatal weakness and minor fatigue of his lower extremity muscles when swimming. he exhibited very mild progres- sion of proximal weakness with age, manifesting as fati- gue in the legs during prolonged exercise. examination showed a slender build, high-arched palate, elongated facies, pectus carinatum, mild scoliosis, mild bilateral isolated achilles contractures, myopathic facies, mild quadriceps and ankle dorsiflexion weakness, poor jump, and reduced reflexes only in the upper extremities. of note, his intellect, extraocular muscle function, cardiac function, and breathing and swallowing functions were normal. emg performed at age years demonstrated normal nerve conductions with evidence of a nonirritable myopathy in proximal and distal lower extremity muscles (data not shown). creatine kinase (ck) was mildly ele- vated at (reference – iu/l). the family history of the proband revealed multiple cases of myopathy in three different generations and a history of consanguinity with individual i. being the half-uncle of i. and individuals iii. and iii. being third cousins (fig. ). table summarizes the age of onset, presenting symptoms, associated clinical features, and rel- evant studies of studied members of this large family. symptoms in the proband’s brother (iv. ) and father (iii. ) started at and years of age, respectively, both of whom represented milder phenotypes than the pro- band with self-reported complaints of poorer endurance in the legs during weight training compared to their peers (fig. and table ). both exhibited no focal weakness and were fairly athletic, although mild myopathic facies with high-arched palates were noted. the proband’s brother could not heel walk and had reduced reflexes in the upper extremities. the proband’s father could heel walk and had intact reflexes, but developed intermittent mild dysphagia at years of age with normal levels of creatine kinase (ck) at iu/l (reference – iu/l). a broad spectrum of clinical heterogeneity was observed in this family. the earliest onset of weakness was in childhood ranging from ages to years for sub- jects iv. (proband; onset at age years), ii. (onset at age years), iii. (onset at age years), iii. (onset at age years) ranging from a gower’s maneuver for subject iii. to mild waddling gait and inability to walk on heels for subject iii. (table ). the oldest affected member of the family, subject ii. died at age years from presum- ably unrelated stroke and complications of heart failure, despite having onset of muscle weakness at years of age (table ). the overall pattern of weakness was proximal, manifesting as a trendelenburg gait, gower’s maneuver, or difficulty in performing repeated exercises that require proximal muscle strength. however, isolated ankle dorsi- flexion weakness was a common early distal manifesta- tion. several subjects exhibited pectus carinatum (iii. , iii. , iv. ; table ). mild scoliosis or kyphosis was seen in iv. and iii. with onset in the teens (table ). high-arched palate and/or myopathic elongated facies was seen in subjects iv. , iii. , iii. , iii. , iv. , and iv. (table ). the weakness progressed slowly over time and the most severely affected members were still ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. tnnt mutation causing nem c. g. konersman et al. http://www.mcw.edu/congenital-muscle-disease-cmd-tissue-repository.htm http://www.mcw.edu/congenital-muscle-disease-cmd-tissue-repository.htm independently ambulatory in their – s (iii. , ii. , iii. ) and subject ii. was ambulatory over short distances with a walker until her time of death at age years (table ). subjects iv. and iii. reported subjective symptomatic improvement in exercise endurance after taking l-tyrosine g/day for at least year (table ). subject iv. was unavailable for clinical or molecular testing, however, his mother reported weakness (table ). subject ii. died at the age of years of encephalitis and ii. died at the age of years from glomerulonephritis prior to developing any significant symptoms (table ). muscle pathology skeletal muscle biopsies of the vastus lateralis in subjects iii. , iv. , and iv. identified severe type fiber hypotrophy (diameter – microns), with numerous red/purple-staining rod-like structures on gomori tri- chrome exclusively in type fibers (fig. a–c, muscle histopathology). these inclusions were distributed unevenly throughout the sarcoplasm. type fibers in the specimens were hypertrophic (diameter – microns), and there was type fibers predominance (ranging from % to % type fibers) across all three biopsies. mild increased internal nucleation was present. there were no cores, inflammation, or increased endomysial connective tissue. electron microscopy for iii. and iv. confirmed the presence of electron dense bodies consistent with nemaline rods in the former, however, not in the latter (likely due to sampling; fig. d). unlike the patients studied here (iii. , iv. , and iv. ), histopathology of subjects ii. and iii. published in demonstrated presence of nemaline rods in both type and fibers (fig. , table ) (spiro and kennedy ). in contrast, muscle pathology for subjects iii. and iii. revealed nemaline rods mostly in type and rarely in type fibers with type fiber hypertrophy, recapitulating our findings (fig. , table ) (spiro and kennedy ; gonatas et al. ). mutation detection genetic testing in the proband demonstrated the presence of a novel heterozygous missense variants of unknown clinical significance in exon of the gene tnnt c. a>t (p.e v) (nm_ . ) (fig. a and b, electropherogram). the tnnt c. a>t change is located at the splicing site of the evolutionarily con- served exon among vertebrates (jin et al. ). this variant was absent from the nhlbi evs and the genomes database (genomes project c, abecasis, et al., ) and the exome aggregation consortium (exac, figure . pathological analysis of quadriceps muscle (a–d) reveals considerable fiber size disproportion and presence of nemaline rods. h&e (a) demonstrates clusters of atrophic type fibers and hypertrophic type fibers with nemaline rods as dark red inclusions (arrowheads). these same inclusions are better visualized on gomori trichrome (b) as purple/blue inclusions (arrowhead) almost exclusively localized to the atrophic type fibers. the atpase stain (c) at ph . demonstrates absence of staining in the location of rods (yellow circles) in the hypotrophic type fibers (dark staining). electron microscopy (d) demonstrates the electron dense nemaline rods (arrowhead). scale bar = lm for panels a–c and nm for panel d. ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. c. g. konersman et al. tnnt mutation causing nem cambridge, ma; url: http://exac.broadinstitute.org). furthermore, molecular analysis in the proband revealed no significant sequence variants or unknown variants in acta , neb, cfl , tpm , tpm , kbtd , klhl , and klhl genes. pcr detection using primers that flank the documented deletion of neb exon , an ashkenazi jewish founder mutation, was also negative (anderson et al. ). subjects iii. , iii. , iii. , iii. , and iii. , all of whom have known nemaline rods on pathology, also have the tnnt c. a>t (p.e v) change (table ). subject ii. was deceased at the time of this study, thus mutation analysis was not possible. none of the six unaffected family members tested had the tnnt c. a>t mutation. two variants already reported in individuals not affected with congenital myopathy, neb c. t>c (p.s p) and c. g>a (p.r h), were found in several individuals of the fam- ily but did not segregated with disease. therefore, only the tnnt mutation segregated with clinical and patho- logical phenotype. the glutamic acid residue at position is highly con- served in human slow skeletal muscle troponin t , car- diac troponin t , and fast skeletal muscle troponin t (jin and chong ; wei and jin ). the e is located in the highly conserved tropomyosin-binding site region among vertebrates of the troponin t protein, suggesting its importance in the structure and/or the function of troponin t (amarasinghe et al. ; mon- dal and jin ; wei and jin ). splicing and protein level analysis tnnt consists of exons and encodes for a - to - kda protein with variable n-terminal regions and con- served middle and c-terminal regions (jin et al. ). the mutation c. a>t is located in exon , two nucleo- tides from the intron –exon junction at the splice acceptor site (figs. b, a). we first determined whether the mutation was associated with abnormal splicing of exon in rna extracted from vastus lateralis muscle biopsies of the proband (iv. ), affected father (iii. ), and two age-matched controls (fig. b). by reverse tran- scriptase pcr using primers in exons and , we found that tnnt exon was normally included in tnnt transcripts from both patients (fig. b; upper panel). we then examined the inclusion of exon and exon (a longer version of exon ) which were reported to be alternatively spliced exons (gahlmann et al. ; samson et al. ; jin et al. ; zhang et al. ). indeed, previous work reported that resistance training increased expression of tnnt mrna isoforms without exons and in vastus lateralis, whereas a sedentary lifestyle figure . electropherogram of tnnt mutation in unaffected (a) and affected (b) family member showing a c. a>t (p.e v) in the latter. (c) alignment of troponin t protein showing complete conservation of residue across species. ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. tnnt mutation causing nem c. g. konersman et al. http://exac.broadinstitute.org increased the inclusion of these exons (zhang et al. ). in addition, larsson et al. determined that expres- sion of the tnnt isoform missing exon was increased in the demyelinating forms of charcot–marie–tooth (cmt type ) but not in axonal form of the disease (cmt type ) (larsson et al. ). we did not observe any inclusion of exon in vastus lateralis muscle biop- sies of the proband (iv. ) and his affected father iii. (fig. b; lower panel). in contrast, transcripts without exon were increased in both patients compared to age- matched controls (fig. b, middle panel and fig. c). consistent with this result, immunoblot of proteins extracted from vastus lateralis muscle biopsies identified increased levels of a low-molecular-weight band in both patients (fig. d). this short isoform of troponin t protein was previously shown to result from skipping of exon (larsson et al. ). notably, the c. a>t (p.e v) was not associated with reduced level of the protein in muscles from patients compared to controls (fig. d). discussion this study describes the first tnnt mutation that trans- mits in an autosomal dominant fashion to cause nemaline myopathy. tnnt encodes troponin t type , which is exclusively found in slow skeletal muscle (type fibers) and serves to anchor the troponin complex (along with troponin c and i) onto the tropomyosin–actin thin fila- ments (nadal-ginard and mahdavi ; jin et al. ; wei et al. ). three homologous genes encode differ- ent isoforms of troponin t in specific tissues: slow skele- tal muscle troponin t (tnnt ), fast skeletal muscle troponin t (tnnt ), and cardiac troponin t (tnnt ) with alternative splicing of each gene conferring protein variants with slightly different functional abilities (samson et al. ; jin et al. ; wei et al. ). troponin t regulates the conformational changes in the thin filaments during excitation–contraction–coupling of slow skeletal muscle (wei et al. ; wei and jin ). all of the previously described mutations were autoso- mal recessive cases of tnnt -related nem (nem ) (johnston et al. ; van der pol et al. ; abdulhaq et al. ; marra et al. ). the tnnt c. a>t nucleotide substitution predicts a p.e v missense mutation which alters the nature of the amino acid from a polar to a nonpolar residue and is predicted to be pathogenic by polyphen (adzhubei et al. ). the amino terminal segment undergoes alternative splicing, with studies indicating that although this region does not bind any other thin filament protein, it plays a regulatory role in the conformational changes in troponin t , thus modulating muscle contraction and interactions with the myofilament (jin et al. ; amarasinghe et al. ). in contrast to the previously described recessive tnnt mutations resulting in truncation or reduction of tro- ponin t protein levels, we describe the first heterozygous missense mutation with evidence of intact (albeit appar- ently functionally altered) tnnt protein. levels of tro- ponin t were not affected by the mutation, and the tnnt c. a>t mutation located at the second nucleo- tide position of exon did not alter the constitutive splic- ing of tnnt exon containing the mutation. troponin t binds tropomyosin to link the troponin complex to actin via two tropomyosin binding sites present in the highly conserved regions corresponding to residues to and residues to , respectively (jin and chong ; amarasinghe et al. ; mondal and jin ). the p.e residue is located within tropomyosin-binding site (jin and chong ; mondal and jin ). we hypothesize that the mutation of a glutamic acid residue into valine at this particular position can affect the affin- ity of troponin t for tropomyosin. a low-molecular-weight (lmw) troponin t protein isoform that results from alternative splicing of exon of tnnt (jin et al. ; zhang et al. ) was observed in both the affected proband and his father. the shorter troponin t is developmentally regulated: troponin t lmw is absent in the fetus, appears in term the newborn, and decreases with age in human quadriceps muscle (jin et al. ; zhang et al. ). larsson et al. ( ) determined that expression of the lmw troponin t was increased in the demyelinating forms of charcot–marie– tooth (cmt) type , presumably due to compensatory overuse of the muscle, in the setting of a reinnervating and intact axon, in comparison to cmt type , which has axonal destruction. we hypothesize that the shorter band identified on the immunoblot from the affected individuals could represent an upregulation of lmw tnnt as a physiological adaptation in patients carrying the tnnt c. a>t mutation. if this alternative splicing is a physiological response to nemaline myopathy in patients with the c. a>t mutation, further investiga- tion is necessary to determine whether this mechanism extends to other tnnt mutations or other nem-related gene mutations. clinically, the tnnt c. a>t heterozygous missense autosomal dominant mutation results in a mild pheno- type with considerable clinical heterogeneity, whereas the recessive truncating and internal deletion mutations with loss of function are associated with severe, early-onset phenotypes (mondal and jin ). variable expression is a common feature of autosomal dominant diseases (stra- chan and read ). in our family, severity ranged from mild difficulty with weight-training exercises to a gower’s maneuver in early childhood (age ). while the severe ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. c. g. konersman et al. tnnt mutation causing nem recessive mutations resulted in respiratory insufficiency and ventilatory assistance by years of age, none of the members of this family have required ventilatory assis- tance even into their late s. pectus carinatum, a com- mon finding in individuals with the autosomal recessive form of the disease, is found in several members of this family and seem to correlate with increased severity. although nonspecific, blunted upper extremity reflexes, high-arched palate, ankle dorsiflexion weakness resulting in poor heel walk, and mild myopathic facies were the figure . analysis of tnnt mrna splicing and troponin t protein expression in nemaline myopathy patients harboring a>t mutation. (a) schematic representation of the tnnt pre-mrna. the a>t mutation is located in the splicing site of the constitutive exon of tnnt gene, with the boxes representing the exons and the black lines representing the introns. the alternative exon , the constitutive exon , and the alternative exon (partial retention of intron ) are shown in blue, orange, and green boxes, respectively. forward (f , f , f ) and reverse primers (r , r , r ) used for the detection of the tnnt splicing isoforms by reverse transcriptase rt-pcr are represented with arrows. primers f and r , f and r , f and r were used to analyze the splicing of the exons , , and , respectively. (b) rt-pcr analysis of the splicing of tnnt exon (upper panel), exon (middle panel), and exon (lower panel) in vastus lateralis muscle from control (black) and affected subjects iv. and iii. (red) carrying the a>t mutation. exons , , and inclusions (+ , + , + ), exclusions (� , � , � ), and expected sizes of the pcr products are indicated on the right of each panel. (c) quantification of tnnt transcripts with exclusion of exon in vastus lateralis muscle from control and affected subjects iv. and iii. carrying the a>t mutation. the percentage of the exon exclusion was calculated by the ratio of the intensity of the upper pcr product relative to the sum of two products on acrylamide gel. (d) total protein extracted from vastus lateralis muscle biopsies was analyzed by immunoblotting for troponin t protein with the monoclonal ct antibody. full- length protein is observed at the expected molecular weight. a low-molecular-weight troponin t isoform reported to result from mrna with exon skipping (larsson et al. ) is detected (asterisk). gapdh (glyceraldehyde- -phosphate dehydrogenase) was used as a loading control. ª the authors. molecular genetics & genomic medicine published by wiley periodicals, inc. tnnt mutation causing nem c. g. konersman et al. most common clinical hallmarks of our affected subjects. the impact on life span appears fairly negligible in this family, since multiple affected family members are inde- pendently ambulatory into in their s and subject ii. survived to years of age despite having mild symptoms since childhood. although contractures were mild and limited to the achilles tendon in this cohort, they were progressive and present proximally in the recessive forms of the disease. tremors were characteristically absent in our cohort. the fiber type disproportion found on most of the muscle biopsies suggests that the abnormality more pro- foundly impacts type fibers. this is not surprising, as troponin t localizes to the type muscle fibers and wei et al. ( ) demonstrated that tnnt deficiency results in approximately % reduction in cross-sectional area of type fibers. while the previous reports in the amish and dutch nem families recapitulate the fiber type disproportion with type hypotrophy, it is unclear whether the nemaline rods are primarily restricted to type fibers in all individuals with tnnt mutations (johnston et al. ; van der pol et al. ). marra et al. described the presence of nemaline rods in roughly % of type and fibers in their case study (marra et al. ). the same pathologic finding of fiber type disproportion with atrophic type fibers has been described in patients with mutations in tpm , another protein known to cause congenital myopathy and pri- marily expressed in slow skeletal muscle (laing et al. ; lawlor et al. ; ottenheijm et al. ; marttila et al. ). this report illustrates that the newly identi- fied tnnt c. a>t mutation can produce nem with hypotrophy and nemaline rods restricted to the type fiber population, and such cases should prompt genetic screening for both the tnnt and tpm genes in patients. the type fibers in the three cases reported here display marked hypertrophy, which we presume is a compensatory adaptation to hypotrophic type fibers. this compensatory hypertrophy may mitigate symp- tomatic weakness and suggest a role for exercise or hypertrophic agents in the management of these patients. three members of the family (iii. , iv. , iv. ) reported subjective improvement in endurance after tak- ing l-tyrosine – g/day for several months. ryan et al. reported improved sialorrhea and muscle strength in infants with acta nemaline myopathy, presumably due to increased peripheral catecholamine synthesis improving sympathetically mediated salivary gland function (ryan et al. ). of note, other sympathetic systemic features, such as tachycardia and hypertension, did not occur in the australian cohort (ryan et al. ) or in our patients. tnnt appears to be emerging as a causative gene for both recessive and dominant nemaline myopathy outside the old order amish population. a pattern of myofiber hypotrophy and nemaline rods restricted to type fibers was specifically seen in muscle biopsies of these tnnt missense mutant patients. in addition to classic infantile nem, we propose that tnnt should be considered in mild cases with nemaline rods and an autosomal domi- nant pattern of inheritance. acknowledgments we thank the family members for participating and the medical college of wisconsin neurology department for financial assistance. c. l.-t. received salary support from the ludwig institute for cancer research, the mas- sachusetts general hospital, and ninds/nih (r ns ). additional project support provided via internal fund- ing by dr. shailendra patel’s laboratory. conflict of interest dr. lawlor is a member of advisory boards for audentes therapeutics and receives research support from audentes therapeutics, solid gt, and demeter therapeutics. drs. lawlor and konersman 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article candidate high myopia loci on chromosomes p and q do not play a major role in susceptibility to common myopia grace ibay , betty doan , lauren reider , debra dana , melissa schlifka , heping hu , taura holmes , jennifer o'neill , robert owens , elise ciner , joan e bailey–wilson and dwight stambolian* address: inherited disease research branch, national human genome research institute, national institutes of health, cassell dr., suite , baltimore, md , usa, dept. of ophthalmology, university of pennsylvania, market st., suite , philadelphia, pa , usa, owens optometrics, e. main st., new holland, pa , usa and pennsylvania college of optometry, old york rd., elkins park, pa , usa email: grace ibay - ibayg@mail.nih.gov; betty doan - bdoan@jhsph.edu; lauren reider - lreider@mail.med.upenn.edu; debra dana - ddana@mail.med.upenn.edu; melissa schlifka - schlifka@mail.med.upenn.edu; heping hu - hhu@cceb.upenn.edu; taura holmes - tnholmes@mail.nih.gov; jennifer o'neill - joneill@jhsph.edu; robert owens - stamboli@mail.med.upenn.edu; elise ciner - eciner@pco.edu; joan e bailey–wilson - jebw@mail.nih.gov; dwight stambolian* - stamboli@mail.med.upenn.edu * corresponding author abstract background: to determine whether previously reported loci predisposing to nonsyndromic high myopia show linkage to common myopia in pedigrees from two ethnic groups: ashkenazi jewish and amish. we hypothesized that these high myopia loci might exhibit allelic heterogeneity and be responsible for moderate /mild or common myopia. methods: cycloplegic and manifest refraction were performed on jewish and amish families. individuals with at least - . d in each meridian of both eyes were classified as myopic. genomic dna was genotyped with markers on chromosomes q - and p . . parametric and nonparametric linkage analyses were conducted to determine whether susceptibility alleles at these loci are important in families with less severe, clinical forms of myopia. results: there was no strong evidence of linkage of common myopia to these candidate regions: all two-point and multipoint heterogeneity lod scores were < . and non-parametric linkage p- values were > . . however, one amish family showed slight evidence of linkage (lod> . ) on q; another amish families each gave lod > . on p; and jewish families each gave lod > . on q. conclusions: significant evidence of linkage (lod> ) of myopia was not found on chromosome p or q loci in these families. these results suggest that these loci do not play a major role in the causation of common myopia in our families studied. background myopia is one of the leading causes of vision loss around the world[ ]. in the united states, myopia affects approx- imately % of adult americans[ ]. ethnic diversity published: august bmc medical genetics , : doi: . / - - - received: march accepted: august this article is available from: http://www.biomedcentral.com/ - / / © ibay et al; licensee biomed central ltd. this is an open-access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= . / - - - http://www.biomedcentral.com/ - / / http://creativecommons.org/licenses/by/ . http://www.biomedcentral.com/ http://www.biomedcentral.com/info/about/charter/ bmc medical genetics , : http://www.biomedcentral.com/ - / / appears to distinguish different groups with regard to prevalence. caucasians have a higher prevalence than african americans[ ]. asian populations have the highest prevalence rates with reports ranging from – %[ , , ]. jewish caucasians, one of the target popula- tions of the present study, have consistently demonstrated a higher myopia prevalence than the general caucasian population in both u.s. and european population sur- veys; orthodox jewish males in particular show increased susceptibility[ , ]. despite many decades of research, little is known about the precise molecular defects and abnormal biochemical pathways that result in myopia. compelling data from familial aggregation and twin studies indicate that suscep- tibility to myopia is inherited. several familial aggregation studies have reported a greater prevalence of myopia in children of myopic parents compared to children of non- myopic parents [ - ]. several twin studies have demon- strated a very high heritability (estimates ranging from to %) for myopia [ - ]. other recent genetic studies of families with - . d or more of myopia (termed high or pathological myopia) have reported significant linkage to regions on chromosome p . , q - , q - and q [ - ]. the p candidate region has been confirmed in an independent study of high myopia [ ]. mutti et al.[ ] examined the hypothesis that families with milder, juvenile onset myopia might show linkage to these same candidate regions. they found no evidence to support such a role in this more common form of myopia but their study was not highly powered in the presence of heterogeneity. evidence also exists that myopia may be under environmental influences. the rapid increase in the prevalence of myopia over the last several decades sug- gests that environmental factors are important [ , ]. furthermore, studies have shown a positive correlation of specific environmental factors, such as nearwork, with myopia [ , ]. it has been postulated that myopia devel- ops in a person who engages in significant periods of sus- tained nearwork as an adaptive response to achieve better focus for near images[ ]. interestingly, cordain et al.[ ] suggest a positive correlation for myopia with increased consumption of carbohydrates, hyperinsulinemia and type ii diabetes. finally, experimental findings from ani- mal studies show that the refractive state of young chicks will adapt to compensate for refractive errors induced by spectacle lenses[ ]. the combination of genetic and environmental influences on the development of myopia suggests that myopia is a complex disorder and should not be classified as a simple mendelian trait. further evidence is shown by studies that have reported correlation coefficients for myopia between offspring and parents and between pairs of siblings to lie between . – . [ - ]. due to the possible complex- ity of myopia, population isolates offer many advantages for genome-wide mapping studies[ ]. first, they have reduced genetic complexity. second, the people in most isolates share a common environment and culture. differ- ences in diet, exercise, sanitary conditions, and exposure to infectious diseases are minimized. a common language and religion usually promote social cohesion. therefore, some of the environmental noise surrounding complex diseases that are determined by a combination of nature and nurture may be avoided. to avoid some of the complexity in mapping genes for myopia we have collected refractive measurements and dna samples from amish and orthodox jewish families with myopia. the old order amish are mostly rural farm- ers and craftsmen. they lead a culturally and technologi- cally distinct lifestyle. they are a genetically well-defined founder population with large families and well-docu- mented genealogies [ , ]. family history records of the amish in lancaster county, pennsylvania, beginning from are highly preserved[ ]. other features of this population include a relatively high standard of living, low migratory tendencies, and no practice of birth con- trol, which facilitate the recruitment of large and extended families. the orthodox jewish families in this study are all of ashkenazi descent, a population with known founder effects in other common diseases[ ]. this population also has somewhat larger family sizes than average in the us. in this initial report, we describe the design of our study and show that two regions ( p and q) previ- ously reported to be linked to high myopia cannot explain the familial aggregation in these families with mostly moderate to milder forms of myopia. we had hypothe- sized that allelic heterogeneity might exist at these candi- date loci such that in addition to highly penetrant alleles for extreme high myopia, there might also exist other sus- ceptibility alleles of (possibly) lower penetrance that pro- duce milder phenotypic forms of myopia. however, we found no strong evidence in support of this hypothesis. methods family screening the study protocol adhered to the tenet of the declaration of helsinki and was approved by the university of penn- sylvania and the national human genome research institute, national institutes of health institutional review boards. informed consent was obtained from the subjects after explanation of the nature and possible con- sequences of the study. the collection of orthodox jewish individuals was begun by a mass mailing of letters to all the known orthodox jewish families living in lake- wood, new jersey. questionnaires were sent with letters explaining the study. if willing to participate, individuals page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / completed and returned questionnaires that included their contact and physician information. second and third mailings went out to individuals who did not respond – either positively or negatively – to the first mailing. the total number of questionnaires returned was , . all jewish individuals included in the study were of ashkenazi heritage. collection of amish families was done by an advertisement in an amish newspaper, refer- rals from local eye doctors in the lancaster county com- munity and word of mouth. criteria for entry into the study included the following: ) negative history of sys- temic or ocular disease which may predispose to myopia, ) negative history of a premature birth, ) proband must be affected and must have a family history of myopia in either their parents or children, ) only one parent (as opposed to both parents) of the proband can be myopic. for the orthodox jewish population, an individual's myopic status was obtained from the most recent (within years) measurement of refractive error. if not recent, an individual was given a repeat exam by their local eye doc- tor or one of the study investigators (d.s.). for the amish subjects, all participants were examined by a study inves- tigator (d.s.) at the amish eye clinic in strasburg, pa. amish participants were brought to the study site because they do not have phone access making it difficult to obtain a past history and records. cycloplegic refractions were done on all individuals less than years of age with one drop each of % cyclogyl, % mydriacyl and . % phenylephrine. a manifest refraction was performed if an individual was older than years of age. classification as myopic required at least - . d in each meridian of both eyes. individuals were classified as nonmyopes if they were over years of age and did not meet the above cri- teria for myopia. other individuals were classified as non- myopic if they were – years old and had ≥ + . d in each meridian, – years old with ≥ + . d in each meridian or – years old with ≥ + . d in each merid- ian. all other individuals were classified as unknown for the trait. this ascertainment protocol resulted in the collection of amish families and orthodox jewish families. of the amish families, phenotype data were available on persons ( individuals were affected and were unaffected) but only dna samples were available to be genotyped. in the jewish families, phenotype data were available for persons ( affected, unaf- fected and of indeterminate phenotype) and dna samples were available and genotyped for of these family members. dna extraction and genotyping peripheral blood was collected from family members. high molecular weight genomic dna extraction from the blood samples was performed with a kit (puregene; gen- tra systems, inc.; minneapolis, mn, usa). polymerase chain reactions were performed in a . ul volume con- taining – ng/ul of dna; um each of datp, dctp, dgtp, and dttp; mm mgcl ; mm tris/hcl (ph . ); mm kcl; . um of each primer; and . units/ul of taq polymerase. standard thermocycling was as follows: °c for sec., °c for sec. and °c extension time for sec. markers used included d s , d s , d s , d s , d s , d s , d s , d s , d s , d s , d s , and d s located in the p and q regions implicated in high myopia [ , ]. power studies a simulation study was conducted on the first ashkenazi jewish families collected in this study, using the computer program simlink [ , ], to compare the projected power from alternative parametric trait models (five of these families contributed no information about linkage and so were not genotyped and the sixth family was dropped after genotyping because of sample prob- lems that resulted in inadequate linkage information). it was assumed that the myopia trait is controlled by an autosomal dominant bi-allelic locus and the frequency of the high risk allele was varied in different simulations, using both . and . . the actual observed pedigree structures, trait phenotypes and dna sample availability were used to simulate the trait locus genotypes and linked and unlinked marker loci were also simulated. a highly polymorphic marker locus ( equally frequent alleles) was assumed. the power available from these families to detect linkage was evaluated using different models for penetrance and sporadic rates. for each of the models tested, simulations were performed assuming that the underlying proportion of families linked to the same marker locus (α) was %, %, % and %. further- more, for each model at each specified level of α, simula- tions were performed for six recombination distances (θ) between the disease and the marker loci (i.e., θ = . , . , . , . , and . ); for three maximum penetrances ( . , . and . ) for gene carriers; and for two pheno- copy rates ( . and . ). lod scores assuming homo- geneity were calculated for each of replicates. the average lod score over all replicates (elod) and its stand- ard deviation were calculated for each model simulated. the power of these families to detect a linkage (i.e., to obtain a lod score ≥ . ) was tabulated for the linked marker and the probability of obtaining a lod score greater than . when no linkage exists (type i error) was tabulated for the unlinked marker in all simulations. linkage analysis the data on amish and ashkenazi jewish families were checked for misspecification of family structures, data entry errors and genotyping errors using the program page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / sibpair[ ]. this program was also used to estimate allele frequencies at marker loci from the unrelated founder individuals in the families. parametric two-point linkage analysis was performed with the mlink program of the fastlink package [ , ] and the utility programs makeped, linkage control program, and linkage report program from linkage . [ - ]. intermarker dis- tances (kosambi cm) of the microsatellite markers were obtained from the marshfield database http:// research.marshfieldclinic.org/genetics/map_markers/ mapmaker/mapformframes.html: d s - . - d s - . -d s - . -d s - . -d s - . -d s ; d s - . -d s - . -d s - . -d s - . -d s - . -d s . to care- fully explore the possibility of linkage of common myopia to these high myopia candidate regions, we utilized different parametric models (table ). analyses were per- formed assuming all combinations of three different fre- quencies for the myopia susceptibility allele ( . , . and . ) and four different sets of genotypic penetrances for the gene carriers and non-gene carriers, respectively: . and . ; . and . ; . and . ; and . and . . models – (table ) assume an allele frequency for the putative myopia susceptibility allele of . , which is the same value used by young et al. in their linkage studies of high myopia [ , ] and close to the value of . that showed good power in our power simulation (note that a more frequent allele frequency of . resulted in similar but always lower predicted power in our simulations than the power obtained when an allele frequency value of . was used; note also that this allele frequency applies only to the linked trait locus, so that if there are multiple loci and environmental factors involved in causing myopia under a heterogeneity model, any single locus might only account for a small propor- tion of all myopia cases). no sex difference was assumed in any of these models. all persons younger than age were coded as unknown for the trait. this analysis assumed autosomal dominant inheritance of a myopia susceptibility allele. recombination fractions were assumed to be equal in men and women. the program homog[ ] was used to test for evidence of heterogene- ity in the presence of linkage in the two-point parametric linkage analyses. the heterogeneity testing was performed separately in the jewish and amish families and also in a joint analysis of lod scores from the two datasets com- bined. multipoint parametric and nonparametric linkage analyses were performed with the genehunter pro- gram[ ]. because of program memory constraints, one large amish pedigree was split into three small ones for the genehunter analysis. the parametric analyses in genehunter used the same models described above, while allowing for locus heterogeneity. the nonparamet- ric statistic nplall, which estimates the statistical signifi- cance of alleles shared ibd between all affected family members, was calculated also, together with an estimated p value for the amish and jewish datasets separately. a nonparametric analysis combining the amish and jewish families was then performed by calculating the sum of npl scores for each family (obtained in the separate amish and jewish analyses just described) divided by the square root of the total number of families (n = )[ ] to obtain an overall combined npl score. results power simulation as expected, the estimated average maximum lod score decreases with the distance between the linked marker locus and the trait locus, and with increasing heterogene- ity. however, only minimal changes in projected power for our ashkenazi families were observed as penetrance, phenocopy rate and disease allele were varied. projected power was always higher when an allele frequency of . was used for the susceptibility allele at the trait locus than table : different parametric models utilized for the linkage analysis model allele frequency penetrance in dd:dd susceptibility allele carriers penetrance in dd normal homozygotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page of (page number not for citation purposes) http://research.marshfieldclinic.org/genetics/map_markers/mapmaker/mapformframes.html http://research.marshfieldclinic.org/genetics/map_markers/mapmaker/mapformframes.html http://research.marshfieldclinic.org/genetics/map_markers/mapmaker/mapformframes.html bmc medical genetics , : http://www.biomedcentral.com/ - / / when an allele frequency of . was used; however, these differences in power were very small. table shows a rep- resentative sample of the predicted power results for detecting linkage to a marker cm from the trait locus (the average maximum distance that a trait locus would be from our genotyped markers if it fell within the confines of either of these two candidate regions on p and q) assuming an autosomal dominant susceptibility allele fre- quency of . . if all families were linked to one locus, these families were predicted to have % power to detect linkage to a marker cm away from the trait locus with a lod of or more (the elods were all ≥ ). as less families were linked to the marker locus (i.e., as genetic heterogeneity increased) the power decreased but was still good (≥ %) if % or more of the families were linked. even when only % of families were linked to the same locus, the expected lod score was over . for all models. of course, these lod scores were calculated assuming homogeneity, and it is well known that power can be sub- stantially increased when heterogeneity exists if lod scores are calculated assuming heterogeneity (hlods) as we have done in this study. so we would expect our actual power to detect linkage to be even higher than our simu- lations of heterogeneity predict. observed type i error rates were compatible with the nominal type i error levels for all models. between and % of replicates produced a lod score > at any test map distance for unlinked markers. linkage parametric and nonparametric lod scores were calcu- lated for amish pedigrees and jewish pedigrees. the six markers on chromosome q -q spanned cm, and the six markers on chromosome p . -p . spanned cm. markers d s , d s , d s , d s and d s were previously reported by young et al. [ , ] as showing evidence of linkage to autosomal dominant high myopia. under all parametric models, the evidence in favor of linkage to these candidate regions was minimal and this evidence varied only slightly as the assumptions of the trait model were changed across the models. therefore, only the results from model are pre- sented here. two-point linkage analyses in the amish and jewish populations results of two-point parametric linkage analysis of myo- pia assuming linkage heterogeneity to the chromosome and markers in amish families are presented in table . statistically significant or suggestive linkage under locus homogeneity was not observed for either chromosome or chromosome . only one marker, d s showed a two-point lod ≥ . (lod = . at θ = . ). testing for linkage heterogeneity using hlods in homog did not significantly improve the evidence for linkage to any of these markers. the same markers on chromosome q -q and chro- mosome p . -p . were analyzed using ashkenazi jewish families (table ). statistically signifi- cant or suggestive linkage was not observed on either chromosome, no homogeneity lod scores ≥ . were observed, and testing for linkage in the presence of heter- ogeneity (hlods in homog) did not alter this result. heterogeneity testing using homog in the combined jewish and amish families also did not yield any signifi- cant evidence of linkage in these two regions, with the maximum hlod's being . and . on chromosomes and respectively. furthermore, nonparametric two-point npl scores did not show any significant evidence for linkage in either the amish or jewish populations. the observed combined npl score of . for d s approached nominal sig- nificance at p = . but was not close to the significance level of at least p = . needed to provide confirmation of a prior linkage[ ]. table : power and elods from replicates of simulated data, dominant susceptibility allele frequency of . penetrance in dd:dd susc. allele carriers penetrance in dd normal homozygote % families linked % % % % power elod ± s.d. power elod ± s.d. power elod ± s.d. power elod ± s.d. . . . ± . . ± . . ± . . ± . . . . ± . . ± . . ± . . ± . . . . ± . . ± . . ± . . ± . . . . ± . . ± . . ± . . ± . . . . ± . . ± . . ± . . ± . . . . ± . . ± . . ± . . ± . power = x proportion of replicate samples that yielded a homogeneity lod score ≥ . elod = average homogeneity lod score over all replicates ± its standard deviation page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / multipoint linkage analyses in the amish and jewish populations multipoint parametric linkage analyses assuming homo- geneity were consistently negative in both the amish and jewish datasets. a maximum multipoint parametric hlod of . was observed at d s in the amish population. however, multipoint parametric hlod scores were essentially zero for the chromosome region in the amish and for both the chromosome and regions in the jewish families. the multipoint nonparametric analyses did not show sta- tistically significant evidence for linkage of myopia to either candidate region in the amish (figures and ) or the jewish (figures and ) families. only very mild evi- dence for linkage of myopia in the amish was observed between markers d s and d s (npl= . , p = . ) (figure ). individual families showing linkage only one amish family ( ) showed marginal evidence for linkage (lod > . ) to the region previously reported on chromosome (d s and d s ) for both two-point and multipoint parametric analyses (table ). three amish families gave lod > . at markers on chromosome p. in both two-point and multipoint par- ametric analyses, family showed mild evidence of linkage (lod = . ) to marker d s , and families and showed slight evidence of linkage to marker d s (two-point lod= . and . , respec- tively). simulations using simlink (model ) showed that for these individual families, the maximum two- table : two-point parametric lod scores for myopia (model ) in amish families recombination fraction, θ marker . . . . . . . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . . d s - . - . - . - . - . . . d s - . - . - . - . - . - . . d s - . - . - . - . - . - . - . d s - . - . - . - . . . . table : two-point parametric lod scores for myopia (model ) in ashkenazi jewish families recombination fraction, θ marker . . . . . . d s - . - . - . - . - . . . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . . d s - . - . - . - . - . . . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . d s - . - . - . - . - . - . - . page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / point lod score obtained when a linked marker was sim- ulated at a recombination fraction of . ranged from . to . , and the probability of obtaining a lod over . for an unlinked marker ranged from < . to . . the nominal significance level corresponding to a lod score of . is approximately . . a total of three jewish families showed marginal evidence for linkage to chromo- some q markers for both two-point and multipoint analyses, with two-point and multipoint parametric lod > . . simulations using simlink (model ) showed that for these individual families the maximum two-point lod score obtained when a linked marker was simulated at a recombination fraction of . ranged from . to . , and the probability of obtaining a lod over . for an unlinked marker ranged from < . to . . discussion the overall results of these preliminary studies do not indicate any strong evidence for linkage of myopia in these families to the candidate regions on chromosomes or . although some families show marginal evidence multipoint nonparametric linkage analysis of myopia to chro-mosome q in amish familiesfigure multipoint nonparametric linkage analysis of myopia to chro- mosome q in amish families multipoint nonparametric linkage analysis of myopia to chro-mosome p in amish familiesfigure multipoint nonparametric linkage analysis of myopia to chro- mosome p in amish families . . . . . . d s d s d s d s d s d s cm n p l s c o r e . . . . . . . . d s d s d s d s d s d s cm n p l s c o r e p = . multipoint nonparametric linkage analysis of myopia to chro-mosome q in ashkenazi jewish familiesfigure multipoint nonparametric linkage analysis of myopia to chro- mosome q in ashkenazi jewish families multipoint nonparametric linkage analysis of myopia to chro-mosome p in ashkenazi jewish familiesfigure multipoint nonparametric linkage analysis of myopia to chro- mosome p in ashkenazi jewish families - . - . - . . . . . d s d s d s d s d s d s cm n p l s c o r e - . - . - - . - . - . - . d s d s d s d s d s d s cm n p l s c o r e page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / of linkage to one of these regions, the results could be due to chance. our negative results for these candidate regions have several possible explanations. first, the diagnostic criteria used in the previous studies [ , ] that implicated these candidate regions were based on limiting the affec- tion status to the sphere component of a plus cylinder refraction. an individual was considered affected with high myopia if the sphere was equal to or greater than - d regardless of the astigmatic error. our study required an individual to have - d in each meridian to be considered affected. therefore, the criterion for being affected was quite different between the two studies. second, the study population in our study included moderate and low myopes in addition to a small number of high myopes. none of our families showed strong aggregation of high myopia. therefore, there were no families in our study recruited exclusively for high myopia and no families that would have been highly powerful for the detection of linkage to a high myopia trait. we utilized this study design to search for allelic heterogeneity with regard to the p and q loci thinking that one or both loci may pre- dispose to moderate/mild forms of myopia. thus, the cur- rent linkage analysis was done to test the hypothesis that other alleles at the candidate high myopia loci on chro- mosomes and might contribute to the etiology of moderate/mild myopia. the mild evidence of linkage in a few families indicates that this hypothesis cannot be fully ruled out for a very small proportion of families with mild forms of myopia. however, there is no strong evidence in favor of this hypothesis and strong negative evidence against linkage in most of the families in this study. previous studies attempting to confirm the high myopia loci on chromosomes and have yielded inconsistent results. naiglin et al.[ ]collected french families with high myopia (spherical equivalent ≥ - d) and performed a genome scan with markers. significant linkage was not found on p and q. lam et al.[ ] mapped families with high myopia, ≥ - . d, using only p mark- ers. statistically significant (lod > ) linkage was not demonstrated although a multipoint lod over . was observed, thus giving evidence of confirmation of the p candidate region. mutti et al.[ ] collected families with varying degrees of myopia (affected ≥ - . d in each meridian) and genotyped the family members with p and q loci markers implicated in high myopia. no evi- dence of linkage to milder forms of myopia was found to the chromosome p and q loci previously associated with high myopia. our study, although consistent with the results of mutti et al.[ ] was significantly different in design. first, mutti et al. used a heterogeneous population that could decrease the chances of obtaining significant linkage for a minor gene effect from p or q if substan- tial ethnic heterogeneity exists. both the amish and ashkenazi populations used in our study are more homo- geneous and each sample was analyzed using marker table : families showing slight evidence for linkage of myopia (model ) to chromosome q or p amish families family id two-point lod multipoint marker zmax lod npl p value d s . . * n/a d s . . * n/a d s . . * n/a d s . . . . d s . . -* n/a d s . . -* n/a jewish families family id two-point lod multipoint marker zmax lod npl p value d s . . . . d s . . . . d s . . . . d s . . . . d s . . . . * only single affected parent-offspring pairs were genotyped in these families so the npl analysis was uninformative in these families. however, the parametric lod score analysis that uses both affected and unaffected family members was informative for linkage. page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / allele frequencies estimated from the sample. second, their study utilized samples while we genotyped individuals. our power simulations predicted higher power in the presence of heterogeneity in our ashkenazi families than was predicted for the mutti et al. study. our amish families were of similar size and structure and so should have similar predicted power as the ashkenazi families, and our combined analyses of the two data sets should provide much more power than that predicted by the simulations of the ashkenazi families alone. the com- bination of the mutti et al. study with the results presented here strongly suggest that these two candidate regions do not play a large role in the causation of moderate/mild myopia in several populations examined. these studies suggest that myopia is complex and proba- bly caused by the interaction of multiple genes with the environment. therefore, to understand myopia it is neces- sary to apply the equation: genes + environment=out- come. the difficulty here is the uncertainty surrounding both terms in the equation; ideally, one set of genetic fac- tors will interact with one set of environmental influences to produce identical outcomes, but it is unknown whether this is always going to be the case. therefore, to lessen the problem of multiple gene interaction as well as gene-envi- ronment interaction confounding the results, strategies to limit this problem should be utilized in the genetic map- ping of myopia. the use of isolated populations is one approach to limiting the heterogeneity across populations and is the approach we are using for a genome wide scan in these families. furthermore, the definition of myopia needs to be standardized so comparisons across studies can be made accurately. previous studies have utilized dif- ferent requirements with regard to affection status making cross comparisons difficult. in conclusion, we find little evidence implicating previously described susceptibility loci for high myopia on chromosomes and as being important in the etiology of common, moderate/mild myopia in our two population samples. competing interests none declared. authors' contributions dwight stambolian, lauren reider, debra dana, robert owens, and elise ciner recruited patients for the study. melissa schlifka carried out the genotyping in chromo- somes and . dwight stambolian and joan e. bailey- wilson performed the study design and wrote part of the manuscript. joan bailey-wilson oversaw all statistical analyses; grace ibay performed statistical analyses and wrote part of the manuscript; taura holmes, betty doan and jennifer o'neill assisted with analyses of the data. all authors read and approved of the final manuscript. acknowledgment this work was supported by the national eye institute grant ey . references . fredrick dr: myopia. bmj , : - 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/ / /prepub http://www.biomedcentral.com/ http://www.biomedcentral.com/info/publishing_adv.asp http://www.biomedcentral.com/ 	abstract 	background 	methods 	results 	conclusions 	background 	methods 	family screening 	dna extraction and genotyping 	power studies 	table 	linkage analysis 	table 	results 	power simulation 	linkage 	two-point linkage analyses in the amish and jewish populations 	table 	table 	multipoint linkage analyses in the amish and jewish populations 	table 	individual families showing linkage 	discussion 	competing interests 	authors' contributions 	acknowledgment 	references 	pre-publication history cover godly republic a centrist blueprint for america's faith-based future john j. diiulio, jr. a former white house official explodes ten polarizing myths about religion and government in america today "dilulio rejects the kind of evangelical triumphalism that declares america to be a christian nation, even as he turns from that secularity which leares no room for religion in the pyfcfic sector. writing in a lucid style, h i shows how religion and government can maintain legal relationship needs r campolo the search for meaning a short history dennis ford "around its centerpiece—meaning—this book weaves a tapestry so encompassing, so intriguingly beautiful, i am stunned by its accomplishment. ford approaches this many angles, touching istorical, spiritual ernes." on smith, author of world's religions of the greate: wildavsky ft s s a l f p thfwewfa social engage donald e. tetsunao includes a c "wri with vid an n co-|uthorof h&m* sacred at bookstores or order ( ) - • www.ucpres .edu p r e s s terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core http://www.ucpres .edu https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core '••-- ^ $ s i k *^w ,# ^ "baker has long specialized in sports. in this worthy addition to his works, he again considers sports in america, this time studying the mutual influence of sports and religion...well written and highly informative." —james f. deroche, library journal "that old quarterback bill baker, once the passing preacher, plays with the old question, is sports america's true religion?, then scores with this readable and fascinating study of their interlocking history." —robert lipsyte, former new york times sports columnist harvard university press terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core spending to religion ^ : ^ ^ - -- --v. mm **w*-l."-̂ j r • ••;;• ; "cfcfgi&ijr tetyt&f&wd ike sways i tw& p&yskal affliction.. i k§a!ifc.,cttrtts encour- iwjmrt^'ts^ theological, p&iees that come into i; aw purpose of • •?*£?•:iu- •-.vr-v.r *.-••• . ' •-. <u»d^vetwss,lb*d the • - tî jettgcics'of gtgtary influence." —john^on%^>,'!llfflada state university mennonites, amish, and the american civil war james o. lehman and steven m. nolt "a well researched and carefully argued treatment that reminds us that not all churches fell into lockstep support for either the union or the confederacy." —george c. rable, university of alabama, author of fredericksburg! fredericksburg * '•.•"..; ( enter books in anabaptist and pietist studies: i >•••:.,:'.•' li. kraybill, series editor i l j s hardcover american catholics in the protestant imagination rethinking the academic study of religion michael p. carroll "carroll's challenge to the 'protestant degradation narrative' is creative, credible, and one that is long overdue. carroll is insight- ful in illuminating discrepancies between actual historical fact and (protestant) theological assumptions in regard to both the definition and understanding of god and that of religion." —william d. dinges, catholic university of america $ . hardcover v the johns hopkins university press - - - • www.press.jhu.edu terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core http://www.press.jhu.edu https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core aw * « > . * • • ; .«*. * . : ^«.." *&'.- *^:. - i , , - i ; - k ^ « terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core cultural transmission, segregation and the distribution of minority groups integration policy: cultural transmission with endogenous fertility.* sagit bar-gill** and chaim fershtman*** the eitan berglas school of economics tel aviv university january , "but the more they were oppressed, the more they multiplied and spread" (exodus , ). abstract: we live in heterogeneous societies with many cultural and ethnic minorities. the cultural composition of our societies changes over time as a result of immigration fertility choices and cultural assimilation. studying such population dynamics, we examine the effect of integration policies, which increase the cost of direct cultural transmission, on the size of the cultural minority. we show that integration policies, while often aimed at reducing the minority's size, may have the opposite effect of increasing minority fertility and its growth rate. key words: minorities, fertility, cultural transmission, integration policies. *we would like to thank the editor of this journal and two anonymous referees for many valuable comments. **sagit bar-gill: the eitan berglas school of economics, tel aviv university, tel aviv israel, email: sagitb@post.tau.ac.il. ***chaim fershtman: the eitan berglas school of economics, tel aviv university, tel aviv, israel and cepr, email: fersht@post.tau.ac.il. . introduction the biblical excerpt "but the more they were oppressed, the more they multiplied and spread" (exodus , ) has become central in jewish culture. it is studied in jewish schools, referred to in jewish history classes and quoted in contemporary political debates. the excerpt describes the israelites in ancient egypt ( th century b.c.) who were oppressed as slave laborers on the one hand, but on the other hand grew in numbers. the phenomenon described in exodus , is clearly counterintuitive. is it possible that oppressing a minority leads to an increase in its size? this question is not merely an historical one. we live in heterogeneous societies with different types of minority groups. the cultural composition of our societies keeps changing over time, and many governments implement different forms of integration and assimilation policies aiming to change the cultural composition of their societies. the interpretation of the biblical excerpt in a modern day context, is whether it is possible that such integration policies may result in even larger minorities? integration policies are commonly used to affect the fabric of society. these policies are usually designed to speed up or even force the assimilation process of minority groups. f in the ancient world (and in some countries even these days) physical oppression was commonly used in order to force social integration. in present times the emphasis is on legislation that affects minorities' cultural identity. such policies target minorities' cultural institutions, and make the process of identity preservation and transmission more costly. other policies may target minority segregation forcing minorities to interact with the majority group. examples for integration policies include restricting the teaching or usage of minority languages (e.g., the russian language in estonia and the basque language in the basque province in spain) f . in other cases governments may impose restrictions or eliminate subsidies for minority schools, theaters or cultural clubs, and may even prohibit although exceptions exist in the form of "multicultural policies" through which the government encourages a pluralistic society, rather than attempting to assimilate its minorities. for example a governmental integration program in estonia mandated that all russian-speaking schools teach at least % of their curriculum in the estonian language, starting the / school year. see krimpe ( ). certain cultural rituals or dress codes (the burqa ban in france or turkey is a prominent example). f evidences that integration policies are not always effective are described in bisin, patacchini, verdier and zenou ( ) who study the ethnic identity of muslim immigrants in the uk and show that muslims integrate less and more slowly than non-muslims. the paper studies the possible effects of integration policies in a society characterized by a minority and a majority group f . we do so in a framework of population dynamics, where individuals choose both their fertility level and their investment in their children's cultural identity. our main results demonstrate the need to exercise caution when introducing integration policies, as these may "backfire". namely, when minority fertility is endogenous, a policy aimed at minority assimilation may result in a larger fertility rate and a larger minority group. the population dynamics of minority groups are governed by three (possibly related) processes: (i) migration which is beyond the scope of this paper, (ii) the relative fertility of each cultural group and (iii) the preservation of group identity of future generations through cultural transmission. f the role of cultural identity and ethnicity in determining minorities’ fertility rates and the difference between minority and majority fertility rates have been extensively discussed in the sociological and demographic literature. three major explanations for minorities' relative fertility patterns dominate the literature. f (i) the assimilation (or characteristics) hypothesis suggests that differences in fertility rates between majority and minority groups are due to differences in socioeconomic and demographic factors. therefore, once these differences in characteristics vanish, the fertility rates of the two groups would converge. (ii) the cultural hypothesis yet, even policies that aim to support multiculturalism and strengthen the minority's identity may lead to social unrest. this is usually the case when the minority group's lifestyle is in odds with that of the majority group. see buruma ( ) for a discussion on such policies in the netherlands and vaisse ( ) for a discussion on such policies in france. by cultural integration we refer to a decline in the size of minority groups. under this definition there is no change in the set of cultural identities but only in the relative size of each identity group. an alternative view is presented in kuran and sandholm ( ). intermarriage between cultural groups can be viewed as part of this process (see bisin, topa and verdier ( ). for an extensive discussion see goldscheider and uhlenberg ( ), ritchey ( ), mosher and goldschider ( ) kollehlon ( ) and forste and tienda ( ). postulates that difference in fertility rates between cultural groups are due to different norms and attitudes towards fertility which is typically referred to as “taste for children”. f (iii) the “minority group status hypothesis” asserts that minority group status exerts an independent influence on fertility behavior (see goldscheider and uhlenberg ( )). f the economic approach to fertility emphasizes individuals’ rational fertility choice (see becker ( ), becker and lewis ( ), becker and tomes ( ) and for surveys see easterlin ( ) and nerlove, razin and sadka ( )). f fertility, in this approach, depends on the economic variables that determine the alternative cost of bearing and rearing children (e.g. women’s wages). the second process that determines the dynamics of minority groups is cultural transmission or assimilation process by which individuals may choose different cultural identity and this cultural identity is transmitted from one generation to the next. the paper adopts the dynamic cultural transmission framework introduced by bisin and verdier ( ). f in this setting parents would like their children to inherit their own cultural type. cultural identity is transmitted from one generation to the next via a two-stage socialization process. the first stage is direct socialization, where parents directly invest in each child, determining the probability that this child would adopt their own cultural identity. whenever direct socialization fails, children are subject to "oblique socialization", where they adopt the cultural identity of a role for a discussion on the effect of social norms and social interaction on fertility choice see manski and mayshar ( ). an interesting example was provided by johnson and nishida ( ) who considered the following natural experiment: in in the state of hawaii the population distribution was such that the japanese were . %, the chinese were . % and the whites were . %. at the same time in california the japanese were only . %, the chinese were . % while whites were . %.comparing the fertility rates of all these groups they showed that minority status matters and it may have depressed the fertility of married japanese and chinese women in california relative to these groups in hawaii. another interesting example was described by day ( ) who noted that a strong catholic- protestant fertility differential was substantially reduced in west germany after world war ii when the partition of germany gave catholic and protestants equal shares of the west german society. for a discussion on the different sociological and economic approach to fertility see andorka ( ) and hammel ( ). while most of the literature asserts that minority fertility rate will be lower this may not be the general case and minority group status may lead in some cases to higher fertility rate. this population dynamics is based on earlier work in anthropology by cavalli-sforza and feldman ( , ) and boyd and richardson ( ). an alternative setup for population and preferences dynamics would be based on evolutionary sociobiology see becker ( ), dawkins ( ) and frank ( ). according to this approach individuals may change their cultural traits to adopt those of “successful individuals”. model randomly selected from the entire population. f as an example consider the problem of religious intermarriage as discussed by bisin, topa and verdier ( ). in this paper parents are paternalistically altruists such they prefer their children to have the same religion as they do. marriage is determined by social interaction and parents may exercise some segregation effort in order to reduce the probability of religious intermarriage. this segregation effort is equivalent to the direct socialization in the general model and if it fails children are choosing a partner randomly from the entire population (this is the oblique socialization part). the reason individuals are engaged in direct socialization is that parents would like their children to inherit their own cultural trait. this has been the standard assumption in the sociological literature (e.g. cavalli-sforza, ), and has been further established in the context of ethnic-racial socialization (e.g. speight and thomas, , verkuyten, , baley and schecter, and the survey by hughes et al. ). following bisin and verdier ( ), we refer to this assumption as paternalistic altruism. this assumption implies that parents would like their children to carry their main cultural traits (e.g. religion and cultural heritage). f yet, when individuals have several children, paternalistic altruism may take on different forms. individuals from different cultures may have different intolerance to having only some of their children adopting different cultural identities. for example, what is the relative disutility of a catholic, protestant, jewish or muslim individual when only one of their two children converts to another religion relative to the case where both her children convert? in some cultural groups the emphasis is on having at least one child that remains loyal to the parents’ cultural group – we denote this type as a "survivalist type". in other cultural groups the emphasis is on having all the children maintaining the group's cultural identity and there may even be a collective penalty on families, where even a single child deviates and adopts a different identity – we denote this type as a "zealot type". most groups are in between these two bisin and verdier ( , ) identify the conditions for having a stable population with a non- degenerate cultural structure. religion, for example, represents a cultural trait that most people are keen to transmit to their children (see for example glazer ). extreme characterizations. f note that the meaning of a zealot type in our setting is only with respect to the attitude towards conversion of one child and not with respect to fertility norms. the main result of the paper is that individuals’ fertility decision and their direct socialization effort crucially depend on their degree of zealousness. while there is not much empirical literature on the degree of zealousness of different cultural groups we believe that this is an important and interesting cultural variable that explain fertility, socialization and segregation choices of individuals from different cultures. as an example one can compare the following two cultural groups: the amish and the orthodox jewish groups. both have high fertility rates and both are considered to be very conservative with strict behavior and dressing codes. however these two groups have different types of paternalistic altruism. in the orthodox jewish tradition there are very strong social norms against any conversion. these norms are best illustrated by the following biblical quotations: “do not intermarry with them. do not give your daughters to their sons or take their daughters for your sons, for they will turn your children away from following me to serve other gods, and the lord’s anger will burn against you and will quickly destroy you.” deuteronomy : - . "if thy brother, the son of thy mother, or thy son…. saying let us go and serve other gods,…, thou shalt not consent unto him; …, but thou shalt surely kill him; thine hand shall be first upon him to put him to death.” deuteronomy : - . consequently, in the orthodox jewish community there is a collective harsh penalty for any conversion. if one of the children converts, marry with a non-jewish person or becomes a secular the marriage prospects for all of his/her siblings are affected, the family may be boycotted by people from the community, their children may be expelled from their schools etc. this reality is illustrated in a bbc documentary on the high cost of leaving ultra-orthodox judaism: f “the fear of secular society is so strong that if a son or daughter chooses to leave, for parents it can be "the end of the when there are several cultural groups an individual may be zealot with respect to conversion of her child to group a and relatively survivalist with respect to group b. we believe that measuring the different degrees of zealousness between different cultural groups is an important part of studying the dynamics and the coexistence of these groups. see http://news.bbc.co.uk/ /hi/ .stm http://news.bbc.co.uk/ /hi/ .stm world", she says. if one child leaves, it can harm the marriage prospects of their brothers and sisters, or influence siblings to make the break too, she explains. in one case she knows of, a father told his daughter he would rather kill her than see her become secular. she eventually committed suicide.” f given these preferences against any conversion the orthodox jewish group would be considered as zealous in terms of our model. in contrast to the orthodox jewish community in the amish community there is no such penalty and young amish are encouraged to sample other ways of life before voluntarily committing to the amish church. this aspect of amish culture is best described in the book on amish society by hostetler ( ): “during this period the young person must come to terms with two great decisions: whether to join the amish church, and whom to marry. to make these decisions, the individual must establish a certain degree of interdependence from family and community. the family relaxes some of its control. the church has no direct control over the young person who has no voluntarily become a member… the young people are thereby allowed some freedom to taste the outside world …” (p. ). as a result some young people leave the amish community but there is no collective punishment imposed on their family. clearly this freedom is not given in the orthodox jewish community that tries to segregate young individuals from the world outside their community. this different attitude does not mean that the amish and the orthodox jewish communities have different levels of paternalistic altruistic preferences, it just emphasis that these preferences have a different structure (i.e. different zealousness). combining the fertility and the cultural transmission processes, parents in our model have two important decisions that may affect the evolution of their cultural group; fertility rate and direct socialization effort. we focus on integration policy that affects the direct socialization cost and makes it more costly or less effective. an alternative approach would be to focus on a policy that targets the oblique socialization process by reducing segregation and facilitating more social interaction between members of different cultural groups. the focus of this paper is on the the first story in this documentary was about an orthodox jewish man that became secular and as a result was unable to see his two children for five years. conditions under which integration policies are ineffective and result in even larger minority groups. f for example, whenever individuals are relatively survivalists an integration policy may induce higher fertility rates as individuals would switch from having one child with a high (and costly) direct socialization level to having two children with much lower direct socialization level. this change may result in larger minority size whenever the effect of higher fertility is stronger than the effect of lower socialization level. our results depend on having endogenous fertility and assuming that whenever there are several children the paternalistic preferences are not separable. bar-el et al. ( ) study secularization dynamics in a cultural transmission framework with endogenous fertility assuming that the minority and the majority groups have different preferences for fertility. extending their analysis would indicate that integration policies would have the expected of effect of resulting in smaller minority size. bisin and verdier ( ) also consider a model with endogenous fertility but they assumed that the utility from children is additive and independent. that is, the conversion of one child does not affect the utility function from the second child. in their model endogenous fertility is relevant because there is a cost of raising children which is a function of the number children. considering integration model in such a setup would always result in having smaller minorities. . a model of cultural transmission with endogenous fertility choice consider a society which consists of two cultural groups; the minority and the majority groups denoted by r and m respectively. the fraction of the minority in the population is ( , . )rq ∈ (and respectively m rq q= − ). our cultural transmission model is based on bisin and verdier ( , ) into which we introduce endogenous fertility and integration policy. individuals are asexual and live for two an interesting example is given by clark ( ) that documented that the basque nationalist party was growing only slightly in areas where basque is widely spoken, but is making significant gains in area of low usage. there are clearly other forms of integration policies that are aimed to affect the relationship between cultural groups, the acceptance of cultural minorities or the attitude of minorities towards the majority group. as it was documented by joppke ( ) there are also limits to such integration policies. periods. in the first period, the childhood period, their cultural identity is determined. in the second period, the adulthood period, individuals choose the number of their children (fertility choice) and engage in socialization activities that may affect their children’s cultural type. we assume for simplicity that individuals may have either one or two children. we assume that individuals have paternalistic altruistic preferences such that each individual would like her children to be of her own type (or belong to her own cultural group). f when an individual of type { },i r m∈ has only one child we let ( )iv j be her utility from having a child of type { },j r m∈ . this utility includes all the costs of child bearing, as well as the joy of having a child, and can therefore be either positive or negative. we let ( ) ( )r rv r v mβ = − be the gain of a minority group member from having one child who maintains his parent's type. paternalistic altruism implies that β > . when people have two children we let ( )iv j j be the value for type i individual of having children of types and j j , where { } , , ,i j j r m∈ . we focus on the minority group choices and frame our presentation in terms of group r – the minority group. clearly the choices of the majority group are analogous. paternalistic altruistic preferences imply that ( ) ( ) ( )r r rv rr v rm v mm≥ ≥ . we let ( ) ( )r rb v rr v mm≡ − captures the gains of a minority type individual from having two children of the minority type relative to a situation in which both her children convert to the majority type. we define ( , ]δ ∈ such that b δβ= . we assume that ( ) ( )r rv rr v r> which captures a taste for children, i.e., individuals prefer two children of their own type to one child of their own type. we further use the normalization ( ) ( )r rv m v mm= . f similar inequalities hold for the majority group. while this is a standard assumption in the sociological and economic literature there are clearly counter examples, such as new immigrants that would like their children to be assimilated into the larger society. but even in such cases parents may prefer that their children will carry on with their main cultural traits, like religion. we need to normalize the utility from having one child and two children as the fertility decision will be based on such a comparison. we therefore assume that ( ) ( )r rv m v mm= . we can change this normalization letting ( ) ( )r rv m v mmξ= without any qualitative change in our analysis. an important aspect of paternalistic altruism is the parents’ attitude towards having children of mixed types. that is, how would parents react, or what would be their disutility, when only one of their children converts to the other group. we distinguish between two extreme cases. in the first, the emphasis is on having at least one child who maintains the parent's cultural type. in such societies the emphasis is on continuation and survival of the cultural trait. on the other hand, there are societies in which there is a strong emphasis on having all children maintaining their parent's type. in such societies there might be a collective penalty on families whenever one of their children converts to the majority type. we denote these two extreme types as “survivor” and “zealot”. specifically, (i) survivor type: the most important consideration for this type is to have at least one child of her own type. we capture such preferences by assuming that for this type ( ) ( )r rv rm v rr= . (ii) zealot type: for an individual in this type of societies, the main consideration is that all her children will be of her own type i.e., ( ) ( )r rv rm v mm= . f the general paternalistic preferences are in between the above two extreme types. in order to capture this aspect of paternalistic preferences we introduce the measurement [ , ]µ ∈ that captures the cultural group zealousness. formally we let µ be the proportion of b which is "lost" by individual of type 𝑟 when one of her two children converts to the opposite cultural type. f that is ( ) ( )r rv rr v rm bµ− = (and respectively ( ) ( ) ( )r rv rm v mm bµ− = − ). therefore a survivor type is characterized by µ = while a zealot type is characterized by µ = . we refer to µ as the level of zealousness of members of the minority group 𝑟. we assume that µ is given and not a subject to evolutionary forces. note that under our assumptions for the zealot type having two children one of his own type and one of the other type is worse than having one child of his own type. while this may be viewed for some readers as an extreme assumption it would be interesting to note that in some zealot communities it is customary to treat the converted child as dead to mourn him/her and never to meet with him/her. clearly the minority and the majority type may have different zealousness levels but for convenience we use hereinafter the term µ for the minority type. remark: since there are only two cultural groups in our setting for individuals of group r there is only one type of conversion; from group r to group m. but when there are several cultural groups, paternalistic preferences may depend on the new identity of the converted child. for example, a jewish parent might have a different degree of zealousness if her child would convert to islam or catholicism (see for example bisin, topa and verdier ( ) for a discussion on the attitude of different cultural groups to intermarriage of their children). therefore whenever there are multiple cultural groups, paternalistic altruistic preferences need to define the parents’ attitude towards different combinations of mixed type children. f clearly different fertility rates may be the outcome of different tastes for children, that vary across societies (see andorka ( ) and hammel ( ) for a summary of the debate on the effect of social norms on fertility decisions and manski and mayshar ( ) for the effect of social interaction and norms for bearing children on the fertility rate). it is possible that such fertility norms will be the major factor that determines the fertility of individuals our focus is on the decision to deviate from these norms. the fertility norm is typically vague and does not necessarily distinguish between six or seven children and therefore there is still a possibility for individuals to change their fertility choice as a respond to changes in the environment in which they live. cultural transmission: there are two types of cultural transmission. the first is direct socialization which occurs either inside the family or at special schools. this socialization is an outcome of parental costly effort. we denote the degree of direct socialization of group r by rτ , which represents the probability that a child of a type r parent becomes type r through the process of direct socialization. we assume that the cost of such direct socialization is / rατ and assume that α > β. f when a household has two children we assume that it cannot discriminate between the children and thus this would clearly complicate our analysis. we thus chose to simplify by assuming that individuals have either one or two children, and there are only two cultural groups. assuming α > β guarantee an internal solution for the direct socialization problem. it must provide for both of them the same level of direct socialization. f this is clearly a simplifying assumption but it can be modified in different ways and still capture the main intuition of our results. f we also assume that there are no economies of scale in direct socialization and the cost of direct socialization of two children is rατ . f children whose cultural type has not been determined by the direct socialization process are subject to oblique socialization - they randomly choose a role model from the entire population, and adopt the cultural identity of this role model. letting ( )rp j be the probability that a child of type r parent becomes type j individual ( where { , },j m r∈ ) we have: ( a) ( ) ( )r r r rp r qτ τ= + − ( b) ( ) ( )( )r r rp m qτ= − − when there are two children we assume that there is no correlation between the children's conversion probabilities beyond the one generated from having the same rτ . that is, our assumption that households cannot discriminate between children with respect to direct socialization clearly creates a conversion correlation between siblings. our assumption is that for a given rτ there is conversion correlation. f consequently, ( c) ( ) ( ) ( ), where , { , }r r rp j j p j p j j j m r≡ ⋅ ∈ . we begin by examining the direct socialization effort for each fertility choice, and then proceed to find the optimal fertility choice. while in principal individuals are free to discriminate between their children the question is if empirically this is a common behavior and if parents are comfortable with such discrimination. the critical part of this assumption is that parents are not able to condition the direct socialization effort of their second child on the cultural trait choice of their first child. this assumption can be easily justified as in most cases the relative small age difference between children will make such a conditioning impossible. the intuition of our results will hold even when there are economies of scale as long as direct socialization of two children is more costly than direct socialization of one child. the intuition of our main results would hold even when there is such a correlation as long as it is not a perfect correlation. . the direct socialization choice the optimal direct socialization choice depends on the number of children, the size of the minority group and its zealousness level µ. one child: the utility from having one child and choosing the direct socialization level rτ is denoted by ( | )r r ru qτ and is given by: ( ) ( | ) [ ( ) ] ( ) ( )( ) ( ) r r r r r r r r r r r u q q v r q v mτ τ τ τ ατ≡ + − + − − − . maximizing ( ) with respect to rτ yields the optimal direct socialization effort when an individual has one child, denoted ( )r rqτ : ( ) ( )( ( ) ( )) ( ) ; ; r r r rr r q v r v m q q min minτ β α α − − −   = =        our assumption that α β> guarantees an interior solution of ( ). two children: the utility of an individual of type µ who has two children is denoted by ( | , )r r ru qτ µ and is given by f : ( ) ( | , ) [ ( ) ] ( ) ( ) ( ) ( ) ( )( )[ ( ) ] ( ) . r r r r r r r r r r r r r r r r r u q q v rr q v mm q q v rm τ µ τ τ τ τ τ τ ατ = + − + − − + − − + − − maximizing ( ) yields the optimal direct socialization level with two children, ( ) ,r rqτ µ : ( ) [ ] ( ) ( )( ) ( , ) ( ) ( ) r r r r r r b q q q q q b µ µ τ µ α µ − + − − = − − − . our assumption that bα > implies that ( , ) r rqτ µ≤ < . since cultural substitution plays an important role in describing the cultural transmission process our first proposition identifies the conditions under which this property holds (or does not hold) in our population dynamics setup. note that µ enters ( ) via ( )iv ij (as ( ) ( ) ( )i iv ij v jj bµ= + − ). proposition (cultural substitution): (i) when individuals have only one child the direct socialization effort ( )r rqτ is decreasing with rq (cultural substitution). (ii) when individuals have two children the cultural substitution property does not necessarily hold. specifically, when . µ > there is a critical ( )eq µ such that whenever ( )erq q µ< the optimal direct socialization effort ( , )r rqτ µ increases with rq . proof: see the appendix. the intuition of part (i) is simple. when rq increases there are lower incentives to invest in direct socialization since a higher qr implies that if the direct socialization fails there is a higher probability that the child will still maintain the minority culture trait through the process of oblique socialization. this is the cultural substitution effect which holds whenever individuals have one child (see bisin and verdier, ). but when there are two children the probability of having one child of type r and one child of type m, denoted by ( ) ( ) ( )r r rp rm p r p m≡ ⋅ , is non-monotonic in rq . while ( )rp r increases in rq and ( )rp m decreases in rq , ( )rp rm increases for low values of rq and decreases for high values of rq . this implies that for low levels of rq , an increase in rq results in a relatively higher level of ( )rp rm , which increases the incentives for a relatively zealous type to invest in direct socialization, as the biggest gain for such a type is to transform his children from (rm) types to (rr) types. thus for a small and zealous minority the cultural substitution effect does not hold whenever there are two children. do parents invest more (per child) in direct socialization when they have one child than when they have two children? this question relates to the discussion on quantity/quality tradeoffs in child education (see for example becker and lewis ( ) and becker and tomes ( )). the typical tradeoff suggests that when there are more children the education "investment" per child is lower. however, when accounting for the effect of the individuals’ level of zealousness, this tradeoff may not hold. proposition : zealot type individuals (high µ ) will have a higher direct socialization effort when they have two children than when they have only one child. specifically, there is a ( )rqµ , such that for every ( )rqµ µ> the optimal direct socialization effort with one child is lower than the optimal direct socialization effort with two children. on the other hand, when ( )rqµ µ< , the optimal direct socialization effort is higher whenever there is only one child. proof: see appendix. the intuition of the above result is based on the complementarity between the two children’s types. when a parent is relatively zealous (high µ ) and one child maintains the parent’s type, the marginal utility from the type of the second child is high and increases with the parent’s zealousness. since the optimal effort is a function of the minority’s relative size, then the threshold zealousness, from which the direct socialization effort will be higher for two children, is a function of the minority’s size as well. throughout the remainder of the paper we will continue to focus on the minority group, omitting the subscript r whenever it does not cause ambiguity. . the fertility choice. we now turn to individuals' fertility decision. let ( ) u q be the expected utility from having one child and choosing the optimal direct socialization level ( ) ( ) /q qτ β α= − . ( ) ( ) ( ) ( ) r u q q q v m β β α = − + + . similarly, let ( , )u q µ be the expected utility of a minority group individual of type µ that has two children and chooses the optimal direct socialization level ( , )qτ µ . ( ) ( ) [ ( )( )] ( , ) [ ( )( )] ( ) ( ) ( ) r b q q q u q qb q q v mm q b µ µ µ µ µ α µ − + − − = + + − − + − − − individuals' fertility decision is based on the comparing the utilities ( ) and ( ). in figure we describe the utility from having one child and two children as a function of q . since we focus on the minority group, we restrict our attention to . q < . we graph the utility for three levels of zealousness: for the zealot type ( )µ = , for the survivor type ( µ = ) and for a moderate type µ . f figure : the utility from one vs. two children comparing ( ) and ( ) yields that when individuals are of the survivor type (low µ ) their utility from having two children is always higher than their utility when they have one child. the reason is that the cost of direct socialization is convex and they suffer little or no utility loss when one of two children switches to the other type. thus the survivor type always prefers having two children on having one child. on the other hand, an individual with a high µ (zealot type individuals) suffers a large utility loss whenever one of their two children converts to the other type and thus wishes to minimize the risk of such a conversion. thus, as long as he is part of the minority group, the zealot type will always prefer having one child (eq. ( ) is above eq. ( )). however when a zealot individual is part of the majority type, and mq is sufficiently large, she would be better off having two children. f note that the utility from having two children or one child are always increasing with q as a larger minority leads to a higher probability of a successful oblique socialization. also note that the utility from having two children (eq. ( )) is by an intermediate type we mean ( ),l hµ µ µ∈ , where and l hµ µ are defined in proposition . note that when q= all players would be better off having two children as in this case it is guarantee that these children would be of their own type and there is no need for direct socialization. in this case the utility does not depend on the level of zealousness. µ = µ = ( ),l hµ µ µ∈ q . ( , )u q µ ( )u q u * ( )q µ decreasing in the level of zealousness µ . in terms of figure , the line that represents the utility from having two children as a function of q shifts downward when µ increases. as seen in figure , for a moderate level of µ there is an intersection of the two utility lines. when q is relatively large, the moderate types will choose to have two children, and when q is small, they will choose to have only one child. furthermore, an increase in the level of zealousness µ would increases the range of q for which individuals would choose to have only one child, and decreases the range of q for which individuals choose two children. this intuition is summarized in the proposition . proposition : there are , l hµ µ ; l hµ µ< such that: (i) for lµ µ< , ( , ) ( )r ru q u qµ > for all q . that is, survivors (low µ ) always choose to have two children regardless of their size q . (ii) for hµ µ> , ( , ) ( ) r ru q u qµ < for all q . that is, zealots (high µ ) always choose to have one child. (iii) whenever l hµ µ µ≤ ≤ there exists * ( , ) . q µ α < , such that ( , ) ( )r ru q u qµ > whenever * ( , )q q µ α> and ( , ) ( ) r ru q u qµ < for * ( , )q q µ α< . that is, for such a µ, a low q induces individuals to have only one child, while for a large q they will have two children. furthermore, * ( , )q µ α is increasing in µ . proof: see appendix. ■ the interpretation of proposition is not necessarily that zealot types would have less children but that given the group’s fertility norm and other potential factors that affect fertility (like economic success), zealousness may provide incentives to reduce the number of children. therefore having zealot types with many children (like the orthodox jewish community) does not contradict proposition . . integration policy and endogenous fertility choice integration policy is a policy that aims to reduce the size of the minority group. there are several types of integration policies. a government may attempt to make direct socialization more difficult or more costly. in terms of our model this integration policy would increasesα . such a policy may be implemented by reducing or eliminating government subsidies for special minority clubs, books, newspapers, theater and other cultural activities that are instrumental in maintaining the minority's cultural identity. an alternative integration policy would be to try to affect the oblique socialization process. in our model we assume that oblique socialization process is random without any segregation biases. in reality many minority groups try to affect this socialization process by adopting different types of segregation practices. minorities sometimes prefer to live in segregated neighborhoods or send their children to special schools. these segregation practices affect the oblique socialization process as they increase the probability that it will be biased in favor of one’s own cultural group. integration policies such as promoting neighborhood integration and enforcing uniform public schooling may interfere with these segregation practices. a third type of integration policy may target the minority incentives to maintain its own cultural trait and therefore it may reduce their incentives to invest in direct socialization. we focus in this paper only on the first type of integration policies targeting direct socialization. when fertility is exogenous any increase in the cost of direct socialization will lower the optimal socialization effort, thereby decreasing the size of the minority group in the following period. when fertility is endogenous the effect of integration policy on fertility depends on the assumed paternalistic preferences. when the utility from children is additive and independent such that the utility from one child does not depend on the type of the other child, as in bisin and verdier ( ), then increasing the cost of direct socialization leads to lower levels of direct socialization and possibly to a reduction in fertility rates. the question however, is whether raising the cost of direct socialization could result in a higher fertility rate. proposition (integration policy and fertility rate): there exists ( )qαµ , such that for minority type µ ; ( )qαµ µ< increasing α decreases * ( , )q µ α . that is, increasing the cost of direct socialization,α , may encourage members of the minority group to increase their fertility rate. f proof: see appendix. ■ the intuition of the above result is as follows: consider a relative survivalist individual (i.e., ( )qαµ µ< ). assume that for a givenα , this individual would prefer to have one child, and in order to ensure a high probability that this child would retain the minority identity this individual chooses a high level of direct socialization. but when direct socialization becomes more expensive this individual might prefer to change her fertility choice and to have two children with a low level of direct socialization, hoping that at least one of the these two children would maintain the minority identity. figure : the utility from one vs. two children, before and after an increase in the cost of direct socialization ( 'α α> ), for an α -policy that encourages minority fertility. note that * ( , )q µ α is defined by proposition as the critical fraction of population such that whenever q is below * ( , )q µ α minority members choose to have only one child and if q is above this level they choose to have two children. a lower * ( , )q µ α implies that there is a larger range of population fractions for which individuals choose to have two children. note that this result will hold only for µ such that ( ),l hµ µ µ∈ , that is a µ for which optimal fertility depends on the size of the minority population q. q . u * ( , )q µ α* ( , ')q µ α ( , , )u q µ α ( , )u q α ( , ')u q α ( , , ')u q µ α figure depicts the utility for each fertility choice for two levels of α . an increase of α to 'α (where 'α α> ) implies a decline of these utility functions as the cost of direct socialization goes up. when two children are chosen, the level of zealousness mitigates the effect of an increase in cost. thus, for a low level of zealousness both the decline in the optimal effort and the resultant utility when having two children is relatively lower compared to the decline in effort and utility with one child. consequently, when the minority is not too zealous, an increase in the cost of direct socialization may cause individuals to switch from having one to two children whenever ( ) ( )( )* *, ' , ,q q qµ α µ α∈ . numerical example: consider the fertility choice problem when the parameter values are , and = . α β δ= = . for this case ( ) . qαµ ≈ , and thus whenever . lµ µ≤ < there exists a range of minority sizes for which integration policy will induce an increase in the fertility rate. for example, whenever . µ = and the minority group is . % of the population, members of the minority group choose to have one child when α = . increasing the cost of direct socialization by % to ' . α = will induce them to switch to two children. . integration policy and the minority growth rate the growth rate of the minority group is determined by the fertility rates of the majority and the minority groups as well as the cultural transmission process of both groups which determine the rate of conversion from one group to the other. our analysis will be carried out in two steps. we first examine the effect of integration policy on the minority's one period growth rate. we will then consider the stable steady state structure. . the minority’s one period growth rate our focus is on the effect of integration policy on the one period growth rate defined by /t tr rq q + . one period dynamics is of particular interest as in our setting a period is actually a generation. one period dynamics are thus the main interest of a policy maker, who wishes to assess the effect of different policies on the minority's expected growth rate from one generation to the next. maintaining our focus on the minority's decisions we assume that members of the majority group have only one child and apply no direct socialization effort, relying solely on oblique socialization. in the previous section we consider the effect of integration policy on fertility. but a high fertility minority does not necessarily grow faster as high fertility may be accompanied by a low level of direct socialization which may result in a high conversion rate that may, in turn, reduce the size of the minority group. we therefore start by stating the conditions under which a low fertility minority would enjoy a faster growth rate than a high fertility minority. lemma : there exist ( ), , and ( , , , )g gq qα β δ µ α β δ such that a minority in which individuals have only one child would grow faster than a minority in which individuals have two children whenever: (i) ( ), , and ( , , , )g gq q qα β δ µ µ α β δ< > , or – (ii) ( ), , and ( , , , )g gq q qα β δ µ µ α β δ> < proof: see appendix. ■ a one-child minority will grow faster than a two-child minority only when individuals choose a significantly higher level of direct socialization effort when having one child. in these cases the effect of a high direct socialization effort overrides the effect of lower fertility. as lemma states, this is characteristic of a small and zealous minority who will invest heavily in the direct socialization of their only child. it is also characteristic of a large and survivalist minority, which will put in only a little effort whenever choosing to bear two children, relying on oblique socialization. alternatively, note that a small and survivalist minority will choose a relatively low level of direct socialization when having either one or two children, and will grow at a faster rate when choosing to have two children. finally, note that for a large and zealous minority, the two effects are aligned. this minority will choose a high fertility rate along with high levels of direct socialization effort, which will result in a higher growth rate when two children are chosen. when integration policy does not change the minority fertility choice it will always result is a lower growth rate as it induces a lower direct socialization effort. but, as discussed above, integration policy may change minority’s fertility rate. we identify two situations in which integration policy results in a higher growth rate. as in lemma , the first case occurs when an integration policy induces higher fertility rates, which result in higher growth rates. in the second case, the integration policy induces lower fertility rates, but the conditions of lemma hold and such lower fertility rates are accompanied by high levels of the direct socialization effort, leading to higher growth rates. case (higher growth rate with higher fertility rate): assume a minority characterized by ( )qαµ µ< (where ( )qαµ is defined in proposition ). figure a graphs the minority's growth rate as a function of its relative size, and the optimal fertility choice, for two levels of the direct socialization cost α and 'α , such that 'α α> . figure a: minority growth rate as a function of its size and fertility choice, pre and post integration policy- the case of increased growth rate due to higher fertility. when ( )qαµ µ< proposition implies that ( )* , 'q µ α < ( )* ,q µ α . thus there are three regions of q (see figure a): (i) when ( )* , 'q q µ α≤ integration policy does not affect fertility and members of the minority group choose to have one child. as a result of higher cost of direct socialization these individuals reduce their direct socialization effort, resulting in a lower growth rate. tq . t t q q + * ( , ')q µ α * ( , )q µ α children growth rate α growth rate 'α child α 'α child childrenα 'α . . fert gr ↑ ↑ . . fert gr → ↓ . . fert gr → ↓ (ii) when ( ) ( )(* *, ' , ,q q qµ α µ α ∈  the higher cost of direct socialization induces minority group members to increase their fertility and have two children (see proposition ). thus the higher cost of direct socialization results in a higher minority growth rate. (iii) when ( )* ,q q µ α> individuals choose to have two children for both and 'α α . a higher cost of socialization 'α leads to lower socialization effort and to a lower growth rate. case (higher growth rate with lower fertility): assume a minority characterized by ( )qαµ µ> . figure b graphs the minority's growth rate as a function of its relative size, and the optimal fertility choice, for two levels of the direct socialization cost α and 'α , such that 'α α> . when ( )qαµ µ> proposition implies that ( )* , 'q µ α > ( )* ,q µ α . figure b: minority growth rate as a function of its size and fertility choice, for two levels of socialization cost 'α α< - increased growth rate with lower fertility. whenever ( )* , 'q q µ α> or ( )* ,q q µ α≤ the integration policy does not affect the fertility choice and therefore it results in a lower growth rate due to a lower direct socialization effort. however whenever ( ) ( )( * *, , , 'q q qµ α µ α ∈  integration policy induces a reduction in the minority's fertility rate from two children to one child (see tq . t t q q + * ( , )q µ α * ( , ')q µ α children growth rate α growth rate 'α child α 'α child childrenα 'α . . fert gr ↓ ↑ . . fert gr → ↓ . . fert gr → ↓ proposition ). whenever the conditions of lemma hold, this lower fertility leads to a higher growth rate as depicted in figure b. we summarize the effect of integration policy on the minority growth rate in the following proposition: proposition (integration policy and minority growth rate): increasing the cost of direct socialization,α , will increase the minority’s growth rate in the following cases: (i) when it induces a switch from one to two children and the fertility effect dominates the lower direct socialization effect, such that a two-child minority grows faster (figure a). (ii) when it induces a switch from two children to one but with a higher direct socialization effort, and the direct socialization effect dominates the fertility effect, such that a one-child minority grows faster (figure b). numerical example: to illustrate the possibility that an integration policy may increase the minority's growth rate, we consider the baseline case (in which ,α = , . and . β δ µ= = = ) and examine the effect of a % increase in the cost of socialization to ' . α = (see table ). when the minority is only % of the population the integration policy does not affect fertility and minority members choose to have one child. the higher costs of socialization induce a reduction in the direct socialization effort which results in a lower growth rate. a similar effect occurs when the minority is % of the population, and members of the minority choose to have two children. the interesting effect occurs for a mid-range minority. for example when the minority is . % of the population the integration policy induces individuals to switch from one child to two children but to lower their direct socialization effort. the overall effect however is a higher growth rate. q , 'α α fertility choice τ /t tq q+ %q = α = . . ' . α = . . . %q = α = . . ' . α = . . %q = α = . . ' . α = . . table : integration policy and growth rate ( , . and . β δ µ= = = ) . steady state population structures we turn to discuss the effect of integration policy on the long-run steady state population structure. we assume that the majority group is characterized by . mµ = and that both the minority and majority group members choose their direct socialization effort and fertility level optimally. the steady state minority size, denoted by sq , should therefore satisfy the following steady state condition: ( ) ( ) ( ) ( ) ( ) s s r r m ms s s r m q n p r q n p r q q n q n + − = + − where ( )rp r and ( )mp r are respectively the probability that a minority child would maintains his type and the probability that a majority child would switch to the minority's type. the fertility choice of the two groups are given by and r mn n . members of the majority and minority groups may differ in their child-bearing costs and benefits. throughout this section group subscripts will be added to the cost and benefit parameters, now denoted by { } and , for ,i i i r mα δ ∈ . f the only symmetry between the groups is assumed with respect to β , the utility loss when having one child that switches types, i.e., ( ) ( ) ( ) ( )r r m mv r v m v m v rβ = − = − . we now turn to consider the fertility decision of the majority group. since . mq > and . mµ = the majority's fertility choice depends on mδ . lemma : there exists *mδ such that for * m mδ δ≥ members of the majority group choose mn = regardless of its size. when * m mδ δ< , there exists a threshold ( )* , ,m m m mq µ α δ such that when * m mδ δ< and ( )* , ,m m m m mq q µ α δ≥ , members of the majority group choose mn = , and only when * m mδ δ< and ( )* , ,m m m m mq q µ α δ< they choose mn = . proof: see appendix. ■ recall that .r rb δ β= note that while, in principle, the majority group may choose to have one child, this would occur only for very low values of mδ . for most parameter values that we examined *mδ is close to . a choice of high fertility seems intuitive for the majority group, as its type of . mµ = implies a relatively low penalty for a deviation of one child, which, combined with the group's high probability of successful oblique socialization, results in an overall low child-bearing risk. we will therefore restrict our analysis hereinafter to the case of mn = . we now proceed to study the steady state population structures in two steps. at the first step we take the fertility rate as given and show that for each fertility choice pair ( ), rn there exists a unique stable steady state population structure denoted by ( ), , r s nq ∈ (see lemma a in the appendix). we then check for each , r s nq whether for such a minority size the individuals' optimal fertility choice would indeed be rn . in order to guarantee that at the steady state . rq < such that the minority maintains its minority group status, we need to further assume that m m rα δ α< and ( ) . r r m mµ α δ β α δ β< + − . proposition : the population will converge to one of the stable structures , , or s sq q depending on the initial population structure q : (i) when lrµ µ≤ the population will converge to , sq . (ii) when hrµ µ≥ the population will converge to , sq . (iii) when l hrµ µ µ< < : a. if ( )* , , , ,s s r r r rq q q µ α δ< ≤ the population will converge to , sq . b. if ( )* , , , ,s sr r r rq q qµ α δ≤ < the steady state will depend on the initial population structure q . if ( ) * , ,r r r rq q µ α δ≤ the population will converge to , sq and if ( ) * , ,r r r rq q µ α δ> the population will converge to , sq . c. if ( )* , , , , s sr r r rq q qµ α δ < < the population will converge to , sq . part (i) and (ii) of proposition are immediately derived from proposition according to which whenever lrµ µ≤ individuals always choose to have two children and whenever hrµ µ≥ they always prefer to have one child. part (iii) is illustrated in figure . figures a and c correspond to cases (iii)a and (iii)c (respectively), and figure b corresponds to case (iii)b where the steady state depends on the initial population structure. for this latter case, when the initial minority size is ( ) * , ,r r r rq q µ α δ≤ the population will converge to the * , q steady state and when the initial minority size is ( ) * , ,r r r rq q µ α δ> the population will converge to the steady state of * , q , which is characterized by a larger minority. figure : convergence to the steady state, from different initial minority sizes. an integration policy which imposes a higher rα , may change all the threshold levels in proposition . we therefore do not describe all the possible cases, but provide one example where the onset of an integration policy results in a new steady state, with a larger long-run minority. proposition : integration policy may result in a larger minority in the long run steady state. specifically, whenever ( )* , , , ,s sr r r rq q qµ α δ≤ < , and ( ) * , ,r r r rq q µ α δ≤ the stable steady state is , sq . in this case, introducing an integration policy could lower ( )* , ,r r r rq µ α δ , and may thus increase the minority's fertility rate, thereby leading to convergence to the new steady state , sq , with a larger minority. numerical example: assume that , , , . r m r mα α β δ δ= = = = = . this case is illustrated in figure b. for these parameter values, the threshold minority size above which the minority chooses high fertility is . % of the population. therefore a minority which is just below the threshold and consists of . % of the population , tq + , tq + (c) low rµ(b) intermediate rµ(a) high rµ tq ° tq + . , sq , sq * rq tq ° tq + . , sq , sq * rq . tq ° tq + , sq , sq * rq will converge to the steady state of , sq , with a long run population size of %. following the onset of an integration policy which increases the minority's cost of direct socialization by %, the threshold ( )* , ,r r r rq µ α δ decreases to . %. for the above minority, this implies a change from low to high fertility, and will thus result in a convergence to the higher steady state of , sq with a minority size of % of the population. . the effect of minority's zealousness level on its growth rate combining the result on fertility choice with the result on growth rate we can now discuss the effect of minority zealousness on its growth rate. figures a and b graph the minority's growth rate as a function of its relative size for two minority types, and ; µ µ µ µ< . figures a and b differ in the maximal benefit from two children, δ . when δ is sufficiently large, individuals apply a higher effort, and the growth rate with two children is higher than the growth rate with one child, for all minority sizes. however, when δ is small, individuals apply a smaller effort with two children, and there exists a range of minority sizes for which a two-child minority will exhibit a lower growth rate than a one-child minority. figure a: minority growth rate depending on fertility choice, µ µ< . figure a presents the case in which the fertility effect dominates the direct socialization effect and consequently a two-child minority grows faster than a one- (more zealous)µ tq . t t q q + µ * ( )q µ * ( )q µ µ childrenµ − µ − children child (less zealous)µ child minority (lemma does not hold). for this case, we compare the growth rate of the two minority types, for three ranges of q : (i) ( )* q q µ≤ - both types choose to have one child and consequently have the same growth rate. (ii) ( ) ( )( * * ,q q qµ µ ∈  - only the less zealous minority (i.e., µ ) chooses to have two children while the more zealous minority ( µ ) chooses to have only one child. in this case the less zealous minority grows faster. (iii) ( )* q q µ> - both minority types will choose to have two children, and the more zealous minority grows faster due to a higher level of direct socialization effort. figure b: minority growth rate depending on fertility choice, µ µ< . figure b demonstrates the case in which a two-child minority will not necessarily enjoy a faster growth rate than a one-child minority, due to the lower direct socialization effort applied with two children as described in lemma . again, we compare the growth rate of the two minority types, for three ranges of q : (i) ( )* q q µ≤ - both types choose to have one child and have the same growth rate. tq . t t q q + µ * ( )q µ * ( )q µ µ µ − µ − children child (more zealous)µ (less zealous)µ children q̂ (ii) ( ) ( )( * * ,q q qµ µ ∈  - only the less zealous minority (characterized by µ ) chooses to have two children. there is a q̂ , such that: a) for ( )( )* ˆ,q q qµ∈ - the less zealous minority has a higher growth rate. b) for ( )( * ˆ,q q q µ ∈  - the more zealous minority enjoys a higher growth rate despite its lower fertility, as the direct socialization effect dominates the fertility effect. (iii) ( )* q q µ≥ - both minority types choose to have two children. the minority type which applies a higher effort will grow at a faster pace. following lemma , the more zealous minority applies a higher effort only when the minority's size is large enough. concluding remarks the focus of our paper has been on the effects of an "integration policy" on the fertility, assimilation process and growth of minority groups. the integration policy is modeled as an exogenous increase in the cost of the direct socialization effort, which could be the outcome of a decrease in government subsidy to the minority's schools, newspapers, theater or other cultural establishments. a naive policy maker would expect such policies to decrease the socialization effort chosen by the minority, and a subsequent decrease in the minority's size. however, taking into account both fertility and effort choices, we show that the integration policy could have the opposite effect. our results therefore suggest that integration policies may have counter intuitive results and may lead to a larger minority. the cultural transmission model assumes only two forms of socialization; direct socialization and oblique socialization, with individuals choosing only the direct socialization effort. the standard cultural transmission framework may be generalized by allowing individuals to affect the oblique socialization level as well. this may be done by introducing different levels of voluntary segregation. such a segregation implies that when the direct socialization process fails, the probability that a child adopts the majority's identity would also be a decreasing function of the segregation level imposed by his parents. such a framework may give rise to yet another detrimental effect of integration policies, as these policies could result in a more segregated minority. references andorka, r. 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( ), pp. – . http://onlinelibrary.wiley.com/doi/ . /sode. . .issue- /issuetoc appendix: proof of proposition : (note that subscripts are omitted from this and following proofs whenever it causes no ambiguity.) the optimal effort with one child is given by ( ) ( )q q β τ α = − , and the derivative w.r.t. q is ( ) d q dq τ β α = − < . the optimal effort with two children is given by [ ] ( ) ( )( ) ( , ) ( ) ( ) b q q q q q b µ µ τ µ α µ − + − − = − − − , and the derivative w.r.t. q is given by: [ ] ( )( ) ( ) ( )( , ) ( ) ( ) q q b qq b q b q α µ µ µ µτ µ α µ − + − − − − −∂ = ∂  − − −  this derivative is negative if and only if the following inequality holds: [ ] ( )( ) ( ) ( ) q q b qα µ µ µ µ− + − − − − − < when . µ ≤ , the above inequality holds for all values of q. when . µ > , the above inequality holds for ( ), where ( ) . e eq q q b b b α µ µ µ α αµ µ µ µ > = − + − < − summarizing, ( , ) for . : iff ( ) ( , ) iff ( ) ( , ) for . : for all [ , ]. e e q q q q q q q q q q q τ µ µ µ τ µ µ τ µ µ ∂ > ≤ ≥ ∂ ∂ > < ∂ ∂ ≤ < ∈ ∂ ■ proof of proposition : we compare the optimal direct socialization effort with one child to the optimal effort with two children. the optimal effort with one child is larger whenever: [ ] ( ) ( )( ) ( ) q q q δβ µ µβ α α µ δβ + − − > − − − solving yields the threshold eµ , given by: ( ) ( ) ( ) ( ) e q q q q q α δβ αδ µ αδ δβ αδ + − − − ≡ + − − − such that for eµ µ> the optimal effort with two children is higher, and for eµ µ< it is lower than the effort with one child. note that ( )( ) ( ) ( ) ( ) e q q q q q αδ δ β αµ αδ δβ αδ − − −∂ = ∂  + − − −  therefore, eµ (weakly) increases in q whenever ( )qα β≤ − , and decreases in q otherwise. ■ proof of proposition : we write the utility for minority group members for extreme values of q , for each fertility choice: ( ) ( ) ( . ) ( ) ( ) ( , ) ( ) ( ( ) ) ( . , ) . . ( ) ( ) r r r r v m u v m b u v mm b b u b v mm b u β µ µ α µ µ µ α µ α β β α = + = − = + − − = + − + + − + − since ( )u q and ( , )u q µ are monotonically increasing in q, we show that there is a single crossing of ( )u q and ( , )u q µ for ( ) , . q ∈ , by showing that ( ) ( , )u u µ> and ( . ) ( . , )u u µ< . the first condition, ( ) ( , )u u µ> , yields the following inequality: ( ) ( ) b b β µ α α µ − − > − − . this inequality is quadratic in µ and yields ,µ µ such that for µ µ µ≤ ≤ the inequality holds. since and µ µ> < < , we denote lµ µ≡ : l b b b α β β β α µ α α − − + − = . and ( ) ( , )u u µ> for all lµ µ> . substituting , ( , ]b δβ δ= ∈ , we can show that for , . lδ µ> > , and for , . lδ µ< < . the second condition, ( . ) ( . , )u u µ< , yields the following inequality: . ( . ) . ( ) b b b β µ β α µ α + − > + − − . this inequality is quadratic in µ as well, and yields ,µ µ such that for and µ µ µ µ< > the inequality holds. since and µ µ> < < , we denote hµ µ≡ : ( ) ( ) h b b b α α αβ β α α αβ β α αβ β µ α    + − − − − + + + +   = . and ( . ) ( . , )u u µ< for all hµ µ< . we have therefore identified the lower and upper bounds for µ : for l hµ µ µ< < there is single crossing of ( )u q and ( , )u q µ for ( ) , . q ∈ . further note that l hµ µ< is non-empty – a sufficient condition for l hµ µ< is ( ) b α β β α + + < or α β δ β α < + + . denote the single crossing of ( )u q and ( , )u q µ by * ( , )q µ α . it remains to show that * ( , )q µ α is increasing in µ . clearly, * ( , )q µ α increases in µ if and only if ( , )u q µ decreases in µ . we write the derivative of ( , )u q µ w.r.t µ : ( ) ( ) ( ) ( ) ( , ) ( ) ( ) b q q b q b qu q b q α µ α µµ µ α µ  − − + − − − − ∂   = < ∂  − − −  which completes our proof. ■ proof of proposition : increasingα decreases both ( ) and ( , )u q u q µ . when the effect of α on ( )u q is stronger * ( , )q µ α decreases, and there exist minority sizes for which this implies a change in the fertility choice from one to two children. we proceed by identifying the condition for ( , ) ( ) u q u qµ α α ∂ ∂ − > ∂ ∂ : ( , ) ( ) [ ( )( )] ( ) [ ( ) ( )] [ ( )( )] [ ( )( )] ( ) ( ) ( ) ( ) ( ) a u q u q b q q q b q b q q b q q q b q b q µ β µ µ α α α α µ β µ µ β µ µ α µ α µα α > ≡  ∂ ∂ + − − − = − − = ∂ ∂ − − −    + − − + − − = − + −  − − − − − −    we denote [ ( )( )] ( ) ( ) b q q a b q β µ µ α µα + − − ≡ − − − − . ( , ) ( ) u q u qµ α α ∂ ∂ − > ∂ ∂ if and only if a > . further arranging yields that a > if and only if ( )qαµ µ< , where: ( ) ( ) ( ) ( ) ( ) q q q q q α α δβ αδµ δβ αδ + − − − = − − − this completes our proof. ■ proof of lemma : we solve r r t t n n q q+ + = = > : ( ) ( ) ( ) ( ) ( ) ( ) q q q q q q q q q q q q τ τ τ τ   − + + − − + + −   > − + − + this reduces to q τ τ> + : ( ) ( )[ ] ( ) ( )( ) ( ) q q q q q q δβ µ µβ α α δβ µ − + − − − > + − − − (*) ( ) ( ) ( ) ( ) ( ) ( ) ( )q q q q q q qα αδ δβ µ αδ δβ + − − + − + > − + − −   the expression marked (*) is increasing in q , and there exists ( , , )gq α β δ such that (*) is negative for all ( , , )gq q α β δ< and positive for all ( , , )gq q α β δ> . the threshold value for µ is derived by dividing the lhs of the inequality by (*). denote this threshold by gµ : ( ) ( ) ( ) ( ) g q q q q α δ µ αδ δβ − + ≡ + − + − + . we conclude that r r t t n n q q+ + = = > whenever: (i) ( , , )gq q α β δ< and ( ), , ,g qµ µ α β δ> , or – (ii) ( , )gq q α β> and ( ), , ,g qµ µ α β δ< . ■ proof of lemma : we begin by writing a majority group member's utility from having one child and from having two children (under the assumption that . mµ = ): ( ) ( ) ( ) m m m mm u q q q v r β β α = − + + ( ) ( ) ( ) m m m m m m m u q q q v rr δ β δ β α = − + + two children are chosen whenever: ( ) ( ) ( ) ( ) m m m m m m m m m m q q v rr q q v r δ β β δ β β α α − + + > − + + arranging yields: ( ) ( ) mm m m m q q βδ δ β α −  − + − >    note that when mδ ≥ the above inequality holds for all mq , and more generally, there exists a threshold mδ , denoted ( )* , mδ ∈ such that when *m mδ δ≥ the inequality holds for all mq and the majority will always choose mn = . solving the inequality for mq , we find that one root is positive and smaller than and the other is larger than . denote these roots by q q< . when or q q q q< > the inequality holds. we denote the smaller root by ( )* ,m m mq α δ and conclude that for * m mδ δ< and ( )* ,m m m mq q α δ< the majority will choose mn = , and otherwise mn = . ■ lemma a : for each fertility choice pair ( ), rn there exists a unique stable steady state population structure, with ( ), , r s nq ∈ : (i) for ( ) , : ( ) , s m m m rq α α δ α= + , thus , . iff s m m rq α δ α< < . (ii) for ( ) , : ( ) , , sq ∈ . , . sq < iff r m r m α α µ δ β δ β   < + −    . proof of lemma a : the condition for a stable population structure, given a fertility choice pair ( ),r mn n is mr nnr r m mn nτ τ= . we solve this condition for fertility choice pairs ( ) , and ( ) , : (i) for ( ) , : r mτ τ= implies – m r m q qδ β β α α − = arranging yields , s m m m r q α α δ α = + . note that , iff s m m rq α δ α< < . furthermore, this steady state is stable, since , sq q< implies r mτ τ> , and thus minorities that are smaller than , sq will grow towards this size, while larger minorities will decrease towards , sq . (ii) for ( ) , : r mτ τ= implies – ( ) ( )( ) ( ) ( ) r m mr r q q q q q δ β µ µ δ β αα δ β µ  − + − −  = − − − solving for q is equivalent to finding the zeros of the following function: ( ) ( ) ( )( ) ( ) ( ) , , r m r m m m r m r r m r m m r f q q q qµ δ δ β µ δ α δ β µ α δ µ α δ α δ δ δ β µ α δ µ  = − + + − + − − + − +  + − we find values of ( ) , ,f q µ for { } , . , q ∈ : ( ) ( ) ( ) ( ) ( ) , , , , , . , . . iff m r r m r m r m m r r m r m f f f µ α δ µ µ α δ α α µ δ δ β µ α δ α δ µ δ β δ β = − ≥ = − <   = − + − < < + −    by the intermediate value theorem, we can state that there exists a zero in ( ) , . denote this zero by , sq . furthermore, ( ) , , . sq ∈ when ( ) , . , f µ < , i.e. if and only if r m r m α α µ δ β δ β   < + −    . also note that , sq is the unique zero of ( ) , ,f q µ in ( ) , (this is straight forward for . µ ≥ and can be shown numerically for . µ < ). stability of , sq follows from similar arguments as in (i). ■ 	bayley, robert and sandra r. schecter ( ) "language socialization practices and cultural identity: case studies of mexican-descent families in california and texas," tesol quarterly, vol. ( ), pp. - . 	speight, suzette l. and anita j. thomas ( ), "racial identity and racial socialization attitudes of african american parents," journal of black psychology, vol. 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ whole-exome capture and sequencing identifies heatr mutation as a cause of primary ciliary dyskinesia report whole-exome capture and sequencing identifies heatr mutation as a cause of primary ciliary dyskinesia amjad horani, , todd e. druley, , , maimoona a. zariwala, anand c. patel, , benjamin t. levinson, laura g. van arendonk, katherine c. thornton, joe c. giacalone, alison j. albee, kate s. wilson, emily h. turner, deborah a. nickerson, jay shendure, philip v. bayly, margaret w. leigh, michael r. knowles, steven l. brody, susan k. dutcher, , and thomas w. ferkol , ,* motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (pcd). despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of pcd remain elusive. we identified nine related subjects with pcd from geographically dispersed amish communities and performed exome sequencing of two affected individuals and their unaffected parents. a single autosomal-recessive nonsynonymous missense mutation was identified in heatr , an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. airway epithelial cells isolated from pcd-affected individuals had markedly reduced heatr levels, absent dynein arms, and loss of ciliary beating. microrna-mediated silencing of the orthologous gene in chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. these findings were recapitulated by small hairpin rna-mediated knockdown of heatr in airway epithelial cells from unaffected donors. moreover, immunohistochemistry studies in human airway epithelial cells showed that heatr was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. the identification of heatr contributes to the growing number of genes associated with pcd identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease- causing gene mutations. dysfunction of cilia contributes to the pathophysiology of a growing number of syndromes that are collectively known as ciliopathies. , rapid progress in genetics has uncovered the basis of many rare syndromes involving the primary (sensory and nonmotile) cilium, but the genetic etiology of the prototypical disease of motor cilia, primary ciliary dyskinesia (pcd) (cild : mim ), remains elusive for many individuals with pcd. , , pcd is a rare, genetically heterogeneous disorder that is usually inherited as an autosomal-recessive trait and typically presents with neonatal respiratory distress, sinopulmonary disease, otitis media, male infertility, and left-right laterality defects. – mutations in genes are associated with pcd, but these account for fewer than half of all cases. , traditionally, genetic causes of pcd have been identified through sequencing of human homo- logs of genes discovered by screening for ciliary dysfunc- tion in model organisms, including chlamydomonas rein- hardtii. candidate genes found via this approach include proteins with roles in structural elements of the ciliary axoneme or in the preassembly of dynein arms. , , – department of pediatrics, washington university school of medicine, st. lo washington university school of medicine, st. louis, mo , usa; depar at chapel hill school of medicine, chapel hill, nc , usa; departmen , usa; department of genetics, washington university school of med and material science, washington university, st. louis, mo , usa; depa seattle, wa , usa; department of pediatrics, university of north ca department of internal medicine, university of north carolina at chapel h biology and physiology, washington university school of medicine, st. louis these authors contributed equally to this work *correspondence: ferkol_t@kids.wustl.edu http://dx.doi.org/ . /j.ajhg. . . . � by the american societ the americ once identified, a putative pcd-causing gene mutation is typically authenticated if it is a member of the ciliome, a collection of genes that code for proteins in the ciliary axoneme. , however, recent identification of a cilia defect in the ortholog dnaaf (mim ) in c. reinhardtii and zebrafish (danio rerio) clearly showed that mutations in genes encoding nonciliary proteins also cause pcd. amish communities present a unique population for genetic research owing to intermarriage, relative isolation, and detailed family recordkeeping. , we identified nine related subjects (six males) with pcd in six subfamilies of geographically dispersed amish communities (fig- ure a). permission for research was obtained from the washington university in st. louis human research protection office (institutional review board). all subjects provided informed consent for diagnostic evaluation and genetic characterization. the diagnosis of pcd was consid- ered on the basis of classic clinical features, which included chronic sinopulmonary disease, persistent neonatal hyp- oxemia, infertility, and laterality defects. each subject uis, mo , usa; center for genome sciences and systems biology, tment of pathology and laboratory medicine, university of north carolina t of medicine, washington university school of medicine, st. louis, mo icine, st. louis, mo , usa; department of mechanical engineering rtment of genome sciences, university of washington school of medicine, rolina at chapel hill school of medicine, chapel hill, nc , usa; ill school of medicine, chapel hill, nc , usa; department of cell , mo , usa y of human genetics. all rights reserved. an journal of human genetics , – , october , mailto:ferkol_t@kids.wustl.edu http://dx.doi.org/ . /j.ajhg. . . figure . family pedigree and genetic analysis (a) pedigree of consanguineous kindred with related nuclear families in amish communities from the midwestern united states. solid symbols represent affected individuals; central dots represent heterozygous individuals. the following abbreviations are used: si, situs inversus totalis; sa, situs ambiguus. viii- specifies the proband. asterisks indicate subjects who underwent exome sequencing. (b) nucleotide sequences in affected heatr region confirming exome sequencing results. heatr mutation, nm_ . (heatr ): c. t>c, resulting in t-to-c transition at base position chr : (hg /grch ), is shown on the chromatograms for normal and pcd-affected subjects. arrow indicates base change. this region does not have any reported nonsynonymous snp or repeat elements. the heatr variant allele segregated perfectly in the cohort and was homozygous mutant in all nine affected individuals. (c) heatr mutation leads to an amino acid change from leucine to proline, leu pro, an amino acid conserved from humans to microalgae. arrow indicates amino acid change. had reduced nasal nitric oxide measurements and electron microscopy of the ciliary axonemes (table ) that showed absent outer dynein arms. in addition, most outer doublets lacked inner dynein arms, and a very small number ( . %, range %– %) assembled normal or truncated inner dynein arms. these data suggest a combined inner and outer dynein arm defect. sequencing of known candidate genes failed to identify a known pcd-causing mutation. with the use of genechip genome-wide human snp array . , dna snp analysis of nine affected and unaffected family members identified homozygous regions on chromosome : , , – , , and chromosome : , , – , , , which included candidate genes associated with ciliary or flagellar defects in humans and preclinical models, such as cep (mim , centrosomal protein of kda), wdr b (wd repeat-containing protein b), and rb (mim , retinoblastoma tumor suppressor protein). we sequenced the exons for all genes within the regions of interest—c orf , c orf , cep , tmtc , kitlg (mim ), sucla (mim ), nudt , med (mim ), itm b (mim ), the american journal of human genetics , – , october rb , and lpar (mim ) —and no mutations were detected. exome capture and sequencing was performed with the agilent sureselect mb all exon hybridization array on two affected children (vii- and vii- ), who were double first cousins, and their parents (vi- , vi- , vi- , and vi- ) (table ). the sureselect protocol was used to prepare libraries for paired-end sequencing ( bp) on an illumina hiseq instrument. exome sequencing identified an average of , total coding variants with greater than x coverage per exome, which were filtered against the genomes project and dbsnp build for removal of known variants. our filtering strategy only retained a variant if ( ) it was not identified as a systematic artifact and ( ) the variant was heterozygous in all four unaffected parents and homozygous in both affected offspring. we identified sequence variants that segre- gated in an autosomal fashion, and from these, variants were known and relatively common, with minor allele frequencies ranging from % to %. however, a rare, non- synonymous variant in the heatr also fit a model of autosomal-recessive inheritance (table ). this variant , table . clinical characteristics of amish pcd-affected subjects with heatr mutant alleles subject age gender clinical manifestations laterality ultrastructural defect nasal no (nl/min) vii- m nrd, rs, cb, in ss oda-ida . vii- m om, rs, cb si oda-ida . vii- m nrd, rs, cb, in si oda-ida . vii- m nrd, rs ss oda-ida . viii- . f nrd, rs ss oda-ida . viii- f nrd, rs si oda-ida . viii- . m nrd, rs si oda-ida . viii- . f nrd, rs ss oda-ida . viii- . m chd, nrd, om sa oda-ida . normal nno level > nl/ml. the following abbreviations are used: no, nitric oxide; nl, nanoliter; m, male; f, female; nrd, neonatal respiratory distress or hypoxemia; rs, rhinosinusitus; cb, chronic bronchitis; in, infertility; om, chronic or recurrent otitis media; chd, congenital heart disease; ss, situs solitus; si, situs inversus totalis; sa, situs ambiguus; oda, outer dynein arm; and ida, inner dynein arm. created a t-to-c transition on chromosome , base posi- tion (hg /grch ), and changed a highly conserved amino acid in heatr from leucine to proline (nm_ . [heatr _i ]; p.leu pro) (figures b and c). exome sequencing results were confirmed in the affected individuals, their parents, and a group of related individuals that included seven additional pcd cases (table s available online). dideoxy sequencing of a bp pcr fragment spanning chr : – used primers tcccgtgaggtgtgggtttcttagg and ggtcacacggctcagctacagatgc. the heatr variant allele (nm_ . [heatr ]: c. t>c) was homozygous in all nine affected individuals and heterozy- gous in the parents. these results were highly significant (fisher’s exact test, p ¼ . � ) for segregation of the variant allele in the individuals genotyped. a search of the exome variant server, which contains variants from , unrelated individuals including , euro- pean americans, did not identify mutations in the heatr sequence (chr : ). moreover, the genomes browser did not reveal variation at this position. notably, despite genotype confirmation through additional rese- quencing and appropriate segregation of the heatr variant, this region was not identified with homozygosity mapping, an approach that can have limitations when applied to inbred amish kindred. table . data filtering analysis stepwise filtering vi variants in coding regions with > x coverage (n) variants after filtering against genomes or dnsnp build (n) variants after filtering against exomes (n) variants that fit model (n) the total number of identified variants remaining after each step in the data filte nonsynonymous, and heterozygous in all four parents and homozygous in both the americ heatr is a member of a family of ten other uncharac- terized heat-repeat-containing proteins in humans. the heat repeat is related to the armadillo/beta-catenin-like repeats and found in many other eukaryotic and pro- karyotic proteins across a broad range of functional classes, – including the four that give rise to the acronym heat (huntingtin, elongation factor , pp a, mtor). none of the heatr proteins have been previ- ously linked to axonemal ultrastructure or have been related to other proteins associated with ciliary function. however, recent studies have implicated the heat- containing protein transportin (tnpo , mim ) in kinesin import into the cilium. preliminary analyses showed that heatr is a highly conserved gene and protein that is enriched in organisms with motile cilia and flagella (figure a and table s ). furthermore, the amino acid modified by the observed variant is highly conserved in organisms with motile cilia and flagella (figure c). we next sought to examine the function of its c. reinhardtii ortholog (chlre gene model ; phytozome v . gene id cre .g .t ). artificial microrna (amirna) directed to bp sequences in the coding region were designed with the use of the web microrna designer website, cloned into pchlamirna vector, and verified via sanger sequencing with estab- lished protocols. the inserts for hybridization were i- vi- vi- vii- vi- vi- , , , , , , , , , , , , , , , , ring analysis is listed for each of the six exomes. only one variant was novel, affected offspring. an journal of human genetics , – , october , figure . ultrastructural and functional defects in silenced c. reinhardtii (a) phylogenetic tree showing the relation- ship of heatr among diverse organisms generated from itol v . a blast search identified two to four proteins with simi- larity to heatr in organisms that have motile cilia or flagella. in contrast, none of the species that lack an orthologous heatr have motile cilia or flagella, which suggests a conserved role for heatr -like proteins in the formation of motile cilia and flagella. the numeral on the right side of each species indicates the number of heatr -like proteins found in the relevant genome database. (b) ultrastructural appearance of outer microtubule doublets and dynein arms from c. reinhardtii flagella isolated from wild-type and amirna transformants with reduced heatr mrna. outer dynein arms were absent in the transform- ant. blue and white arrows indicate inner and outer dynein arms, respectively. (c) isolated axonemes from wild-type (wt) and heatr -silenced (htr ) strains were resolved by sds-page and stained with coomassie blue. protein molecular weights are shown on the right, and asterisks indicate bands with obvious differences between the preparations. the polypeptide at kda is probably a membrane protein that is often variable in axonemal preparations, whereas identi- ties of polypeptides at kda are un- known. immunoblots for structural pro- teins were normalized to a-tubulin, and values are indicated as fractions. dic is a component of the outer dynein arms and is nearly absent in htr axonemes; however, dic is a component of the inner dynein arms and is equivalent. cnt is a component of the inner dynein arms and is overrepresented in the axonemes from silenced strains, which may reflect their shorter length. (d) flagellar beat frequencies in wild-type uniflagellate (uni - ) cells (wt) and unifla- gellate double mutant (uni - :htr ) cells (htr ) (p < . ). (e) swimming speeds of wt, htr -silenced, and oda (oda, outer dynein arm mutant) cells (p < . ). ctagtaagtcagtggatgagaaatgatctcgctgatc ggcaccatgggggtggtggtgatcagcgctatcatatc tcatccactgacttg and ctagcaagtcagtgg atg agatatgatagcgctgatcaccaccacccccatggtgc cgatcagcgagatcatttctcatccactgactta, which recognize bases – on chromosome and are located in the terminal exon of the gene the american journal of human genetics , – , october , cre .g .t . two transformed strains with . - and . -fold reduc- tions in mrna levels in biological replicates were obtained. flagella were isolated from cells grown on low-sulfur medium for days in light and days in the dark, then differentiated into gametes over hr with the use of medium that lacks nitrogen, before deflagellation was induced through ph shock. electron microscopy of htr -silenced axonemes from these strains revealed missing outer dynein arms (figure b) compared to wild- type c. reinhardtii axonemes. this ultrastructural defect was confirmed by immunoblot analyses of isolated silenced and wild-type flagella. the blots were incubated with primary antibodies oda (dic ) ( : , , a gift from d. mitchell ), centrin (cnt ) ( : , , h a, a gift from j. salsibury ), ic (dic ) ( : , , a gift from w. sale ), and a-tubulin ( : , , dm a, sigma-aldrich, st. louis) for characterization of outer and inner dynein arm assembly. antibodies were detected with enhanced chemiluminescnece, and band intensities from immuno- blots were determined with the use of photoshop soft- ware (adobe systems, san jose, ca, usa), normalized, and reported as the fraction of a-tubulin. a markedly reduced level of the outer dynein arm intermediate chain, dic , was present compared to dic , a component of the inner dynein arm (figure c). however, centrin, a protein found in a subset of inner dynein arms, was present and elevated in the amirna-silenced strain (figure c). these findings are in line with the ultrastructural analysis by electron microscopy and suggest that htr plays a role in outer dynein arm assembly. to test this postulate, we assessed htr levels following deflagellation in wild-type cells. expression of htr was increased . -fold min following deflagellation compared to pretreatment values, consistent with the well-known transcriptional upregula- tion of flagellar genes during cillogenesis. to characterize the functional phenotype of the htr -silenced strains, we performed motion analysis of swimming wild-type (cc- ) and amirna transformant cells (amirna:htr and uni - ;amirna:htr cells), using established methods. , swimming velocity was mea- sured in biflagellate wild-type and htr algal cells, whereas rotation rate and beat frequency were measured in unifla- gellate mutants (uni - ) or double mutants (uni - ;htr ). rotation speed and beat frequency of uniflagellate cells were obtained from the mean slope and oscillation fre- quency of time series of angular displacement (figure s , movies s and s ). uniflagellate double mutant cells (uni - ;htr ; n ¼ ) had a decreased beat frequency, . . hz, and a slower rotation rate, . . revo- lutions per second (rps), compared to the htr þ uniflagel- late cells, at . . hz (uni - ; n ¼ ) (unpaired two- tailed student’s t test, p < . , figure d) and . . rps, respectively (figure s ). moreover, biflagellate cells from silenced strains exhibited decreased cell veloci- ties ( . . mm/s; n ¼ cells) compared to wild- type ( . . mm/s; n ¼ cells) (single-factor anova, p < . , figure e). these motion parameters in htr -silenced cells corresponded closely to measured values in an oda mutant, which also has absent outer dynein arms. to elucidate the importance of heatr in cilia assembly and its association with pcd, we evaluated the expression of heatr in human multiciliated airway epithelial cells. normal human lung, obtained from excess tissue of healthy lung donated for transplantation, was fixed and immunostained as previously described. , primary anti- bodies used included rabbit anti-heatr ( : , sigma- the americ aldrich), mouse anti-acetylated a-tubulin ( : , , clone - -b , sigma-aldrich), and mouse anti-a-tubulin ( : , , clone b- - - , sigma-aldrich) and were imaged with secondary antibodies conjugated to alexa fluor dyes (life technologies, grand island, ny, usa). nuclei were stained with . mg/ml of dapi (vector laboratories, bur- lingame, ca, usa). images were acquired with the use of epifluorescent microscopy with band-pass filter cubes opti- mized for the secondary dyes, interfaced with qcapture pro software (qimaging, surrey, bc, canada), and adjusted globally with photoshop software. heatr was observed only in the cytoplasm of ciliated airway epithelial cells and was absent in cilia (figures a and b). these findings are consistent with published proteomic analyses that did not include heatr in cilia. , furthermore, within the cytoplasm, heatr was observed in a diffuse punctate pattern (figure b), which did not colocalize with endo- somes, lysosomes, or basal bodies with the use of mouse anti-lamp ( : , abcam, cambridge, ma, usa), mouse anti-eea ( : , bd biosciences, san jose, ca, usa), and mouse anti-g-tubulin ( : , clone gtu- , sigma- aldrich), respectively (figure s ). the punctate pattern was also present in nasal epithelial cells recovered from healthy non-pcd-affected subjects (figure c). to exclude secondary effects of chronic rhinosinusitis on heatr levels in freshly collected samples from affected individ- uals, nasal epithelial cells were scraped from the inferior turbinate, expanded in culture, and then differentiated on semiporous supported membranes (transwell, corning, ny, usa) at an air-liquid interface. preparations were maintained in culture for to weeks. cultured nasal epithelial cells were harvested from membranes and immunostained as previously described. , to assess heatr levels in cultured cells, proteins were isolated and analyzed by immunoblot. the blots were incubated with the heatr antibody ( : dilution) and detected with enhanced chemiluminescence. cultured nasal epithelial cells from an individual with pcd (viii- in figure a) had markedly lower levels of heatr compared to cells from non-pcd-affected normal subjects, as determined by both immunofluoresence and immunoblot analysis (figures d and e). the actual fate of the mutant heatr is unknown, though the leu pro substitution is pre- dicted to render the protein unstable. prolonged immu- noblot exposures showed that the size of the mutated heatr was similar to that of the normal control (figure s ). we evaluated the effect of heatr mutation on dynein arm function by examining cilia beat frequency. with a nikon ti-s inverted phase-contrast microscope enclosed in a customized environmental chamber maintained at �c, images were captured by a high-speed video camera and processed with the sisson-ammons video analysis system (ammons engineering, mt. morris, mi, usa). five fields were captured for each sample, and whole fields were analyzed for generating a mean and sd. simul- taneous fluorescent images were obtained with a ccd an journal of human genetics , – , october , figure . heatr localization in cili- ated airway epithelial cells (a and b) photomicrographs of normal human lung section (scale bar ¼ mm) (a) and bronchial epithelium (b) following immunofluorescent staining for heatr , which reveals the cytoplasmic localization of the protein (heatr , red) only in ciliated cells (cilia marker, acetylated a-tubulin (a-tub), green; nuclei stained with dapi, blue) (scale bar ¼ mm). (c and d) immunofluorescent staining of nasal epithelial cells cultured at an air- liquid interface from a healthy subject (nl) showing the presence of heatr (c) and an individual with pcd showing a reduction of heatr levels (d) (scale bar ¼ mm). (e) immunoblot analysis of tracheo- bronchial (tr) epithelial cells and nasal epithelia (np) cells from a healthy subject and an individual with pcd (pcd) con- firms the very low level of the mutant form of heatr . (f and g) immunoflourescent staining in representative human airway epithelial cells transfected with nontargeted, scram- bled shrna sequences (nt-shrna) (f) and heatr -specific shrna sequences (g) for heatr (red) and acetylated a-tubulin (green) (scale bar ¼ mm). (h) immunoblot analyses of airway epi- thelial cells transfected with each of three different heatr -specific shrna ( – ) or nontargeted shrna (nt) sequences and nontransfected control cells (m). (i–k) ultrastructural appearance of cilium from the proband (i), and cilia from airway epithelial cells following transfection with (j) nontargeted and (k) heatr -targeted shrna sequence . blue and red arrows indicate inner and outer dynein arms, respectively. retiga- rv camera (qimaging) for determining cell viability through staining with calcein and ethidium homodimer- activity (life technologies). live imaging showed that the nasal epithelial cells from a subject with a heatr mutation had virtually immotile cilia, in con- trast to those from healthy subjects (unpaired two-tailed student’s t test, p < . , n ¼ fields examined) (figure s , movies s and s ). to define the role of heatr in differentiation, cilia assembly, and function of ciliated airway epithelial cells, we silenced heatr expression with an rnai approach in primary airway epithelial cells obtained from excess tracheal and bronchial tissue from lung donors. human airway epithelial cells were transfected with nontargeted (scrambled) or heatr -specific small hairpin rna (shrna) sequences with the use of recombinant lentivirus that contained a cassette for conferring puromycin resistance. the shrna-targeted sequences used were those generated by the rnai consortium (trc) in the plko. hairpin rna sequence transfer plasmid. shrna sequences used were trcn (shrna # ), trcn (shrna # ), and trcn (shrna # ) (obtained through sigma-aldrich). a nontar- geted sequence with a yellow fluorescent protein reporter gene (gift from y. feng and g.d. longmore, washington university, st. louis ) was used as a control sequence and for measuring transfection efficiency. for generating the american journal of human genetics , – , october lentivirus for shrna delivery, each vector was cotrans- fected with the phr’ . dr packaging and vsv-g envelope plasmids in hek t cells with the use of fugene (promega, madison, wi, usa) according to the standard trc protocol. cultured, undifferentiated human airway epithelial cells (early passage) seeded onto supported transwell membranes were incubated with medium con- taining freshly produced lentivirus with mg/ml prot- amine sulfate for hr. cells were expanded for to days, then selected in puromycin ( . mg/ml) for an additional days. once confluent, an air-liquid interface condition was created to allow cell differentiation. heatr expression was reproducibly inhibited by each of three different heatr -specific shrna sequences in three independent preparations compared to cells transfected with the nontargeted shrna sequence, as determined by immunoblot analysis and immunostaining (figures f– h). each shrna provided different degrees of heatr inhibition; shrna # and shrna # were the most effec- tive. similar to the subject’s nasal epithelial cells, cilia were present on the apical surface of preparations treated with each of the different shrna sequences, which indi- cates that heatr was not required for cilia formation (figure s ), a finding that was analogous to our observa- tions in the algal silenced strains. moreover, the human airway epithelial cells had cilia with markedly reduced motility when assessed with high-speed video microscopy , figure . expression and localization of specific inner and outer dynein arm proteins in ciliated airway epithelial cells (a) immunofluorescent staining of nasal epithelial cells cultured at an air-liquid interface from a healthy subject (nl) showing the presence of outer dynein arm protein dnai (red) and a subject with pcd (pcd) showing absence of heatr expression. (b) expression of dnah (red), an inner dynein arm protein, as seen in cultured nasal epithelial cells from an individual with pcd (pcd) and a healthy subject (nl). dnah was expressed and localized to cilia in both normal and pcd airway epithelial cells. cilia were identified by im- munofluorescent staining with a-tubulin (green), and nuclei were stained with dapi (blue). scale bar ¼ mm. and compared to the preparations transfected with the control nontargeted sequences (unpaired two-tailed student’s t test, p < . , n ¼ fields) (figure s , movies s and s ). ultrastructural analysis of heatr shrna-silenced cells performed with the use of previously described protocols revealed absent outer dynein arms and truncated or possibly absent inner dynein arms (figure k). these findings were similar to the axonemal defect observed in the proband (figure i). to better define the changes observed in the dynein arms, we immunostained ciliated cells with antibodies for dnai , an outer dynein arm polypeptide, using mouse anti-dnai ( : , , gift from lawrence ostrowski, university of north carolina, chapel hill, nc ), as well as dnah , an inner dynein arm polypeptide, using rabbit dnah ( : , novus biologicals, littleton, co, usa). the american journal of human ge dnai was absent in ciliated airway epithelial cells from both the indi- vidual with pcd (figure a) and heatr shrna-silenced cells (fig- ure s ). this lack of dnai con- firms the findings in the silenced c. reinhardtii and the ultrastructural studies in the human cells that the outer dynein arm does not assemble. however, the localization of the inner dynein arm protein dnah to cilia was comparable in the normal and pcd airway epithelial cells (figure b). this finding suggested that part or subsets of the inner dynein arms were still present, or that the protein was mislocalized elsewhere in the cilium. these data are consistent with our observa- tion that silenced c. reinhardtii may have an abnormal distribution of inner dynein arms, reflected by al- tered centrin expression found in amirna-treated cells (figure c). by applying a whole-exome sequencing strategy to analyze the genome of several individuals with pcd from a large endogamous amish family, we identified a previ- ously uncharacterized gene, heatr , which encodes a highly conserved protein that is essential for the preas- sembly or stability of the axonemal dynein arms. specifi- cally, we found that the leu pro substitution in heatr reduced expression in ciliated airway epithelial cells in affected individuals to produce classic findings of pcd that include ultrastructural defects of the dynein arms and markedly dysmotile cilia. we confirmed that the func- tion of heatr is highly conserved by reproducing similar defects in dynein arm ultrastructure and markedly altered function in amirna-silenced c. reinhardtii flagella and netics , – , october , human airway epithelial cilia. these studies and earlier proteomic analyses clearly establish that heatr is not a structural protein in cilia and flagella, but its presence in the cytoplasm indicates a role in the preassembly of the dynein arms, similar to dnaaf (mim ), dnaaf (mim ), and dnaaf , or in their transport to the basal bodies, such as oda . – significantly, the ciliary proteome did not predict heatr involvement in ciliary biogenesis or assembly, and thus this tool has limitations for gene discovery in pcd. the precise mechanism by which heatr interacts with other ciliary proteins and its involvement in motor dynein arm assembly remain unknown. thus, heatr joins the growing number of genes linked to pcd. supplemental data supplemental data include six figures, two tables, and six movies and can be found with this article online at http://www.cell.com/ ajhg/. acknowledgments the authors thank huawen lin and anne sun for preliminary screens for heatr amirna and zhengyan zhang for technical assistance with the c. reinhardtii silencing experiments. david mitchell, jeff salsibury, win sale, felix y feng, greg longmore, and lawrence ostrowski generously provided reagents for these studies. the authors also acknowledge kimberly burns, wandy beatty, and marilyn levy for assistance with electron micros- copy; milan hazucha and johnny carson for their diagnostic expertise; susan minnix, jane quante, kathryn akers, lu huang, michelle farberman, hauw lie, marianna schmajuk, adriana lori, michael armstrong, and hilda metjian for their expert clin- ical and scientific support; and cassie mikols and jian xu from the children’s discovery institute airway epithelial cell core at washington university school of medicine for assistance with cell culture. the authors were funded by the children’s discovery institute (t.e.d., p.v.b., s.l.b., s.k.d., and t.w.f.), the national institutes of health (nih) awards hl (t.w.f.), hl (s.l.b.), gm (s.k.d.), hl (m.r.k., m.a.z.), hl (d.a.n., j.s.), hg (d.a.n., j.s.), and hl (a.c.p.), and the ruth l. kirschstein national research service awards fellowship f gm (a.j.a.). in addition, this work was advanced by the genetic disorders of mucociliary clearance consortium, hl (m.a.z., m.w.l., m.r.k., s.l.b., and t.w.f.), part of the nih rare diseases clinical research network, with programmatic support from the national heart, lung, and blood institute and the nih office of rare diseases research. the views expressed do not neces- sarily reflect the official policies of the department of health and human services, nor does mention of trade names, commercial practices, or organizations imply endorsement by the u.s. government. received: june , revised: august , accepted: august , published online: october , the american journal of human genetics , – , october web resources the urls for the data presented herein are as follows: genomes browser, http://browser. genomes.org ciliary proteome database, http://v .ciliaproteome.org/cgi-bin/ index.php exome variant server, http://evs.gs.washington.edu/evs interactive tree of life (itol) v , http://itol.embl.de mustab for predicting mutant protein stability change, http:// bioinfo.ggc.org/mustab online mendelian inheritance in man (omim), http://www. omim.org references . fliegauf, m., benzing, t., and omran, h. 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( ). conditional dele- tion of dnaic in a murine model of primary ciliary dyskinesia causes chronic rhinosinusitis. am. j. respir. cell mol. biol. , – . . gherman, a., davis, e.e., and katsanis, n. ( ). the ciliary proteome database: an integrated community resource for the genetic and functional dissection of cilia. nat. genet. , – . an journal of human genetics , – , october , 	whole-exome capture and sequencing identifies heatr mutation as a cause of primary ciliary dyskinesia 	supplemental data 	acknowledgments 	web resources 	references books: did he save lives? a surgeon’s story chasing the sun: the new science of sunlight and how it shapes our bodies and minds linda geddes wellcome collection, , pb, pp, £ . , - let there be light the sun has had bad press. as gps we correctly declaim grim warnings to our patients about skin cancer. we rightly warn of the sharp rise in malignant melanomas, the risks of sunbathing on holidays, and frequenting those tanning salons that benight our high streets. on the other hand, we are aware of the sun’s benefits in the production of vitamin d. my results inbox daily has at least one patient with low vitamin d, and we seem to be testing it for a range of conditions, physical and psychological, with a corresponding increase in prescribed vitamin d supplementation. the health benefits of sunlight have been known for centuries. hippocrates built a solarium at his treatment centre on the greek island of kos, and a fellow physician, aretaeus of cappadocia, recommended sunlight for ‘lethargics.’ more recently, florence nightingale would position patients close to windows, arguing that ‘second only to their need of fresh air is their need for light … not only light but direct sun-light’. in the early th century, niels finsen developed light therapy for the treatment of skin tuberculosis, and robert koch showed that the bacterium responsible for tb could be killed by sunlight. there followed a fashion for heliotherapy, until the rise of critical voices such as those of the british surgeon john lockhart-mummery, who, in his book nothing new under the sun, dismissed sunlight therapy as ‘pseudo-magic’. linda geddes argues that we are missing the point in this age-old binary discussion, and instead we should focus on the integral role of the sun in the development of our circadian rhythms. this starts in utero, but, as all new parents are well aware, circadian rhythms do not fully develop until months after birth, and they continue to evolve throughout life. in adolescence the biological rhythms are shifted later, making it harder for teenagers to fall asleep at night and get up in time for school. adults generally have well-developed rhythms, with defined changes in body temperature throughout the day and night. in order to illustrate the importance of circadian rhythms for our health, geddes travelled far and wide. she starts with the amish, an american people whose rhythms are completely dictated by the sun. she describes a yard-sale at . am: ‘already a man with a chin-curtain beard, and the distinctive amish uniform of straw hat, plain shirt and braces, is firing up a barbecue, and the smell of smoke and grilled chicken intermingle with the sweet smell of desserts’. living their lives in the outdoors, the amish are exposed to far greater levels of natural light than the rest of us, and geddes suggests that this may help account for their lower levels of depression. in complete contrast, geddes describes her trip to a conference in las vegas in when, suitably, the international agency for research on cancer added night-shift work to the list of ‘probable’ human carcinogens. she describes her experience of vegas as lurching ‘like a confused moth, through underground malls and vast casino floors, my sense of time becoming ever more distorted’. she explains that exposure to bright light at night ‘forces the body to feel alert when it should be sleeping, setting off a cascade of damaging effects’. as an example of how we can all re-engage with our circadian rhythms, geddes travels to the german spa town of bad kissingen, bavaria, historically a place of health and healing, which has now refashioned itself along the lines of ‘chronobiology’. guided by the ‘chronobiologist’ thomas kantermann, the town starts schools later in the day, holds classes outdoors, and encourages businesses to offer flexitime for the larks and owls in their workforce. most controversially, bad kissingen has proposed unilaterally abandoning daylight saving time, which curtails teenagers’ sleep even further, and may well have an adverse effect on their exam results. geddes ends her travels where she began, at stonehenge. there she describes the -year-old understanding we have had of the sun and its importance for the ‘circularity of our biology’. justifiably, after writing a book full of complex biomedical theories intertwined with delightful history and travel writing, we learn that she has graduated from a mere visitor to a guest of the cotswold order of druids. rhodri evans, gp, newport, south wales. email: evans.rhodri@gmail.com doi: https://doi.org/ . /bjgp x * * * * * did he save lives? a surgeon’s story david sellu sweetcroft publishing, , pb, pp, £ . , - a miscarriage of justice one ordinary evening in february an experienced consultant colorectal surgeon saw his last patient of the day, a -year old retired builder who had developed abdominal pain shortly following an elective knee replacement. three years later on a cold november day david sellu was sentenced to prison for and a half years for unlawfully killing john hughes. did he save lives? charts the events between these two dates that would lead to the conviction of a surgeon with a previously unblemished record of gross negligence manslaughter. sellu’s methodical, sparse yet descriptive prose depicting the day-to-day of prison life belies the quiet horror of a life stripped of freedom, autonomy, and dignity. born in a rural village in sierra leone to illiterate farmers it was not in sellu’s destiny to become an eminent surgeon in england. life & times books british journal of general practice, february two things changed that. the first was moving to the capital to be raised by his aunt, and the second was winning a scholarship to study medicine in manchester. after years of surgical training and marrying his wife catherine, a staff nurse at hammersmith hospital, sellu had it all: four successful children, one of whom was reading medicine at his alma mater, a happy marriage, and a thriving nhs and private practice. john hughes, a private inpatient, was seen by sellu late one thursday evening. hughes was days post-total knee replacement but had developed abdominal pain. sellu’s plan included antibiotics, bloods, and an urgent ct scan. at home later that evening sellu called the hospital several times to enquire about available anaesthetists. shockingly, unlike nhs hospitals, many private hospitals do not have -hour anaesthetic cover. sellu also called the residential medical officer to enquire about the results of the tests and to advise on antibiotics. the rmo reassured sellu that the bloods were normal and the antibiotics prescribed. the bloods were never done and the antibiotics were never issued — sellu was later blamed for both these failures. the following morning, sellu called radiology to chase the ct scan but it was not done until later that afternoon and showed a perforation of the large bowel. sellu would later be held responsible for the delay in the scan. sellu tried to book the patient for theatre but the earliest he could secure both a theatre and an anaesthetist was . pm that evening. unfortunately, the anaesthetist got delayed on another case. on a friday evening with no -hour anaesthetic cover sellu tried in vain to find another anaesthetist. hughes was eventually operated on hours later than planned and passed away days later in the intensive care unit. the coroner later referred sellu to the police, believing he had committed a crime, and sellu was subsequently convicted of that crime — gross negligence manslaughter. how could this happen? how could a surgeon who acted in accordance with what a body of his peers would have done at that time, with no access to an emergency anaesthetist, with no power to arrange a ct scan any earlier — be held culpable for systemic failures? how can a jury who openly expressed their confusion about exactly what issue they were deliberating on be allowed to determine the fate of a man? how can a judge be allowed to use a report commissioned by the hospital whose agenda was to exonerate itself at whatever cost instead of using the original case notes? the conviction and incarceration of david sellu is one of the biggest miscarriages of justice in british history; it is also a stark reminder of our own vulnerability — as clinicians whose decisions can be scrutinised in a vacuum devoid of the systemic context, for some as persons of colour who do not fit the establishment image, as law-abiding citizens who find ourselves on the wrong side of the law. sellu’s story is also a testament to the power of perseverance, determination, and faith — faith in family, justice, and the future. sellu wryly observes, ‘prison taught me that whatever obstacles man can invent, man can circumvent.’ sellu’s determination to maintain his sanity and sense of hope has taught me that, whatever circumstances befall a person, they can overcome them. maryam naeem, gp, tulasi medical centre, essex. email: maryam.naeem@icloud.com doi: https://doi.org/ . /bjgp x * * * * * the intelligence trap: why smart people do stupid things and how to make wiser decisions david robson hodder & stoughton, , hb, pp, £ . , - brain power ‘a great many people think they are thinking, when they are merely rearranging their prejudices.’ (william james, th-century psychologist) the intelligence trap is written for anyone who wants to escape the above mistake — a user’s guide to both the science and art of wisdom. the author asks three questions: why do smart people act stupidly? what skills and dispositions are they missing that can explain these mistakes? and, how can we cultivate those qualities that protect us from these errors? robson is an award-winning science journalist working with bbc future, where he specialises in psychology, neuroscience, and medicine. his skill as a journalist makes him readable and entertaining while his scientific approach makes him credible. robson engages us with stories; he reviews the scientific literature (notes and references of over pages) and also describes his interviews with researchers exploring intelligence and wisdom. part defines the problem. we explore the flaws in our understanding of intelligence and the ways that even the brightest of minds can backfire — from arthur conan doyle’s dogged beliefs in fairies to the fbi’s flawed investigation into the madrid bombings of — and the reasons why knowledge and expertise can exaggerate these errors. part presents solutions to these problems by introducing the new discipline of ‘evidence-based wisdom’ (ebw), which outlines those other thinking dispositions and cognitive abilities crucial for good reasoning. it offers some practical techniques to cultivate ebw. we discover why our intuitions often fail and the ways we can correct those errors to fine-tune our instincts. we explore strategies to avoid falling for misinformation and fake news so that we can be sure that our choices are based on solid evidence rather than wishful thinking. part turns to the science of learning and memory. despite their brain power, intelligent people sometimes struggle to learn well, reaching a plateau in their abilities that fails to reflect their potential. ebw can help to break that vicious cycle by offering three rules for deep learning — rules that explain why east asian education systems are so successful. part explores the reasons why talented groups can act stupidly — from the failings of the england football team to the crises of huge organisations like bp, nokia, and nasa. there is a section on intuitive-based diagnostic errors by doctors, and how simple rational measures can reduce the rate of error. however, diagnostic errors take up only a few pages — this is a book that explores individuals and organisations from all areas of society. the intelligence trap may help us not only make better decisions about patients, but also in our personal lives. it could be useful in our teaching by inspiring students to improve their thinking and learning skills. however, beyond being useful, i just found this book fascinating. ‘life is short, and the art long; the occasion fleeting; experience fallacious; and judgement difficult’. (hippocrates). hilary lavender, retired gp, london. email: hilary.lavender@gmail.com doi: https://doi.org/ . /bjgp x british journal of general practice, february thorax ; : - candidate gene loci in asthmatic and allergic inflammation medical genetics was revolutionised during the s by the application of genetic mapping to locate the genes responsible for simple mendelian diseases. most diseases, however, do not follow simple inheritance patterns, and geneticists have now taken up the even greater challenge of the genetic dissection of complex diseases such as hypertension, ischaemic heart disease, and asthma. this editorial sets out to review recent work which has attempted to determine the genetic basis of atopy and asthma with a focus on immunopharmacological mechanisms. the review excludes recent reports of b adrenoceptor polymorphisms, asthma severity, and bronchodilator re- sponsiveness, nor do we discuss the genetics of chronic obstructive pulmonary disease (copd) which have re- cently been reviewed elsewhere.' one of the first problems encountered in this endeavour is definition of the phenotype. asthma is a clinical diagnosis with no foolproof diagnostic test, so surrogate markers for the disease are used including atopy, bronchial hyper- responsiveness, and clinical history. inevitably this leads to disagreement between various research groups and an inability to compare results achieved using different defin- itions. genetic heterogeneity, incomplete penetrance, and environmental factors may also confound statistical analysis and make it difficult to reproduce positive findings. strategies for determining the genetic basis for asthma and allergy include using random markers to screen the entire genome looking for evidence of linkage to the disease or disease associated traits; this approach is now feasible given the increasing number ofdna polymorphisms avail- able for such a purpose and has been used successfully by todd's group in oxford in their search of the human genome for genes that predispose to type insulin de- pendent diabetes mellitus. another approach would be to examine markers in and around candidate loci whose products are thought to be important in the pathogenesis of the disease; examples of this include the ige receptor on chromosome , the cytokine cluster on chromosome , and the t cell receptors on chromosomes and . both these approaches have been used in recent studies on the genetics of asthma and atopy. ige, atopy and asthma problems with definition of the asthma phenotype have led researchers to study atopy, a major risk factor for the development of asthma, as characterised by a persistent ige-mediated response to common environmental al- lergens. atopy, which contributes to diseases such as asthma, eczema, and allergic rhinitis, is defined as a dis- order of the ige response to common allergens such as pollen, animal dander, house dust mites, and fungi. these diseases are frequently detected by a raised total serum ige level, a raised specific ige level, and positive skin tests to common aeroallergens. burrows et al investigated the association of self-re- ported asthma or allergic rhinitis with serum ige levels and skin test reactivity to allergens in subjects in a general population study. regardless of the atopic status of the subjects or their age group, the prevalence of asthma was closely related to the serum total ige level standardised for age and sex. no asthma was present in the subjects with the lowest ige levels for their age and sex. the conclusion reached was that asthma is almost always as- sociated with some type of ige-related reaction and there- fore has an allergic basis. further evidence for the relationship between ige levels and asthma has been pro- vided by sears et al who studied the relationship between serum total ige levels and airway responsiveness to me- thacholine challenge in the presence or absence of asthma in a birth cohort ofnew zealand children. the prevalence of diagnosed asthma was significantly related to the serum ige level, and airway hyperresponsiveness was still related to an allergic diathesis as reflected by the serum total ige level even in children who had been asymptomatic throughout their lives and had no history of atopic disease. bronchial hyperresponsiveness, atopy and asthma further work by sears et al on the same cohort of children has looked at the relationship between airway hyper- responsiveness, asthma and atopy. airway hyper- responsiveness (methacholine pc fev, < mg/ml) was found to be strongly correlated with reported asthma and wheezing and with atopy as defined by positive skin prick test, particularly to house dust mite and cat. furthermore, all the children with diagnosed asthma and airway hyper- responsiveness were atopic. they concluded that atopy was a major determinant of airway hyperresponsiveness in children, not only in those with a reported history of asthma and wheezing, but also in those without any history suggestive of asthma and rhinitis. there is clearly a link between atopy, airway hyper- responsiveness, and asthma, although the precise re- lationship remains a source of considerable debate. there is a tendency to dichotomise subjects as hyperresponsive or non-responsive on the basis of whether or not their forced expiratory volume in one second (fevi) falls by % at a given dose of inhaled histamine or methacholine, and different cut off doses have been used by different workers. on the basis of this, one would hope to be able to discriminate clearly between asthmatics and non- asthmatics. however, some atopic subjects with no evid- ence of symptomatic asthma will also demonstrate bron- chial hyperresponsiveness according to the same criteria, as will a small percentage of normal subjects. enhanced bronchial responsiveness has a strong association with clinically defined asthma, and the association appears to be stronger in those with more immediate and severe symptoms and with greater treatment requirements, al- though the overlap between groups is large. moreover, there is documentation from longitudinal studies that bron- chial hyperresponsiveness may not be present in some people at a time when they have unmistakable asthma symptoms and airway obstruction and, conversely, that greatly enhanced bronchial responsiveness may be present in the absence ofsymptoms, or may develop after symptoms have become manifest as occurs in seasonal asthma.' " thus, although bronchial hyperresponsiveness and asthma are related, the two are not synonymous. a number of variables have been shown to affect both serum ige levels and bronchial hyperresponsiveness. smoking, for example, has been shown to lead to an increase in total serum ige levels.' the effects of age on o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th x. . . o n ja n u a ry . d o w n lo a d e d fro m http://thorax.bmj.com/ wlkinson,holgate serum ige levels are not clear, with studies showing variably a decline, no change, or an increase. environmental factors undoubtedly influence basal levels of ige and will vary depending on the age of the subject and on the time of year the sample was taken. ideally, therefore, multiple samples should be taken and looked at for seasonal vari- ation. the prevalence of atopy has been shown to be higher in boys than girls,' although mean ige levels do not differ significantly between the sexes. bronchial hyperreactivity may also be affected by smoking and previous history of respiratory illness.' these variables should be taken into account in any analysis which uses ige or bronchial hyperresponsiveness as surrogate markers of atopy or asthma. genetics of ige the familial incidence of atopic disease is high; most family studies report a positive family history in approximately % ofcases. however, the mode ofinheritance is disputed, and a single dominant gene with partial penetrance, a single recessive gene with partial penetrance, and multigene inheritance have all been suggested. studies looking at ige levels in twins have found that monozygotic twins are substantially more similar to each other than are otherwise comparable dizygotic twins of a pair.' - there are several theories as to the mode of inheritance of ige. gerrard et al studied ige levels in parents and children. ' the results from an analysis of families were consistent with low levels of ige being determined by two dominant genes, the absence of one or the other permitting high levels to occur. further data acquired from nuclear families supported the hypothesis of a regulatory locus for ige occupied by two alleles with the dominant allele suppressing persistently high levels of ige. their data were subsequently reanalysed and the findings not confirmed. a further study of ige distribution in three large pedigrees suggested a strong hereditary involvement with high levels being determined by a dominant allele. a study of ige levels in five pedigrees selected through breast cancer probands provided evidence for the presence of a polygenic component in the determination of ige with no evidence of a major gene effect. meyers et al attempted to resolve the confusion over the genetic basis of ige production by studying families ( individuals) de- liberately not selected for the presence of atopy. segregation analysis showed that the mixed model of recessive in- heritance of high levels was most appropriate for their data, with approximately % of the total phenotypic variation in log (ige) attributable to genetic factors equally divided between mendelian and more general polygenic com- ponents. it is apparent from the plethora of studies addressing this issue and reaching different conclusions that the study ofthe genetics ofige production is fraught with difficulties. ascertainment of the sample population to be analysed may introduce a substantial source of potential bias into any results. a study published by cookson et a! found evidence for vertical transmission of atopy as defined by a positive skin prick test (> mm than the negative control), a serum total ige level > - standard deviations above the mean for the normal population, and one or more positive rast tests to a panel of allergens. only one of the above criteria had to be met for the patient to be designated atopic. it was concluded from an analysis of the data that atopy is inherited as an autosomal dominant trait. the study was based on a sample of nuclear families recruited via asthmatic probands from outpatients at a chest clinic with a control group recruited from patients admitted to hospital for other reasons. in addition, three large families with asthmatic members were recruited - two by means of letters to general practitioners and one in response to an article in the local newspaper. it was felt that the inheritance pattern of atopy with particular regard to the extended families clearly indicated autosomal dominant transmission. with such a broad definition of atopy and the inclusion of extended pedigrees with such a strong history of atopy, it is difficult to reject an autosomal dom- inant model, but this does not necessarily imply that it is correct. furthermore, when the same group undertook segregation analysis on an australian cohort of random families "adjusting" total ige for atopy, they reported a major recessive gene which does not seem consistent with the previously held view of atopy as an autosomal dominant trait. chromosome llq in the oxford group presented evidence for a single major autosomal dominant "atopy gene" linked to the d i i s marker on chromosome qi with strong evid- ence for linkage (lod score= ) at a recombination fraction of - . most ofthe lod score ( ) was contributed by a single family having meioses. a second study to confirm this finding was undertaken on a sample of nuclear families recruited from asthma and allergy clinics and by appeal in the media. linkage analysis to dl s this time provided a lod score of - at a recombination fraction of . . pooling of these two samples gave a lod score of at a recombination fraction of - . in a summary paper, a lod score of was reported on a sample of "over individuals from over nuclear and extended british families". this agrees substantially with the sum of their two published samples. shortly afterwards a lod score of - was reported with a recombination fraction of . . no explanation has been advanced for the loss of more than % of the information between the studies. support for linkage data to chromosome q comes from a study published by shirakawa et al on four families with meioses selected from families using an ex- treme definition of atopy, and a report by collee et al in a small sample of affected sibling pairs. following on from initial linkage studies, the oxford group showed that the unit of the high affinity receptor for ige (fc,r ) was also located on chromosome qi and was in close linkage with the gene for atopy. ige- mediated mast cell or basophil activation occurs through the interaction of multivalent antigen with antigen-specific ige bound to high affinity ige receptors (fc,ri). the fclr receptor consists of one alpha chain, one beta chain, and two gamma chain subunits (fig ). the ct chain contains one membrane-spanning region and the single ige binding site. the chain has four membrane-spanning domains with both the amino and carboxy terminals pro- truding into the cell cytoplasm. the two y chains have a single membrane-spanning region. the f subunit is in- volved in the critical process of tyrosine phosphorylation of cross-linked fcgrl subunits leading to the assembly of a "signalling complex" of receptor associated proteins. this, in turn, leads to the production of inositol phos- pholipids which cause a rise in intracellular calcium es- sential for the release of vasoactive mediators from in- flammatory cells. stimulation of the receptor on mast cells and basophils also provokes the release of interleukin which, through a cognate interaction with cd and its ligand on b cells, may lead to isotype b cell switching to ige synthesis. shirakawa et al performed dna sequence analysis on six atopic and six non-atopic individuals and revealed one o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th x. . . o n ja n u a ry . d o w n lo a d e d fro m http://thorax.bmj.com/ candidate gene loci in asthmatic and allergic inflammation figure schematic representation of fc,r showing the leu substitution in the fourth transmembrane domain of the ,b subunit. patient with a variant sequence in which a leucine was substituted for an isoleucine at position in the fourth transmembrane domain of the subunit (fig ). ° the presence of this variant and its association with atopy was examined in a random sample of patients unselected for atopy and families ascertained through a young asth- matic proband with at least one other sibling and both parents available for study. associations were reported between the presence of leu and high total serum ige levels. from the nuclear families with allergic asthmatic probands, leu was identified in ( %), was ma- ternally inherited in each, and was reported to show a strong association with atopy. while reporting linkage to q, collee et al failed to reveal evidence of significant linkage to the fc,r locus implying that other genes on this chromosome may be involved. these potentially exciting findings were consistent with a further observation that the transmission of atopy at the q locus was only detectable through the maternal line. ' it was argued that this was consistent with paternal im- printing or with maternal modification of the developing immune response. their data showed that ( %) of sibling pairs affected by atopy shared the maternal qi allele and ( %) did not. this distribution differs significantly from the expected / distribution (p= - ), suggesting an excess sharing of maternal alleles in affected siblings. intrauterine environmental factors may in part account for the maternal influence over the immune response of the progeny in favour of atopy. work from the mrc environmental epidemiology at southampton has pro- vided interesting data concerning possible maternal effects on the developing immune response of the fetus. it has been shown that a raised serum ige concentration in adult men and women was strongly correlated with a large head circumference at birth. this association was independent of adult size, social class, smoking, or gestational age at birth. one hypothesis that might explain this phenomenon is that disproportionate fetal growth resulting in a larger head circumference at birth may be associated with im- paired thymic development with a diminished production of thl cells which are more sensitive to adverse en- vironmental stimuli. thl cells produce interferon y, low levels of which at birth are thought to be a risk factor for the development of atopy. although several groups have failed to replicate the findings of the oxford group, all the studies cited have used different definitions and protocols, and on the whole sample sizes were small. we have also attempted to replicate the oxford data on a large sample of families recruited at random from general practitioner registers in wessex. the families were selected solely for having three or more children, and all were typed for three markers on chro- mosome q. using combined segregation and linkage analysis, no evidence was found for linkage to atopy, and mother-child and father-child correlations were virtually identical, making it difficult to substantiate imprinting of either parent. ' neither is the theory of paternal imprinting supported by expression studies of imprinting from the homologous region ofthe mouse genome. to substantiate the findings reported by the oxford group on chromosome iq, further studies are needed in populations enriched for asthma. chromosome atopic individuals differ in the allergens to which they react. the difference is clinically important, since asthma and bronchial hyperresponsiveness may be associated with allergy to house dust mite antigen but not necessarily to grass pollen. genetic regulation of specific ige responses is probably different from that of the general atopic re- sponse. specific ige reactions might be constrained by variation in the hla or t cell receptor (tcr) proteins, since these molecules are central to the handling and recognition of foreign antigens. the role of the tcr in allergic reactions is unclear. the receptors consist of ot and chains; the former arises from chromosome and the latter from chromosome . genetic linkage has recently been shown between specific ige reactions to highly puri- fied major allergens and the tcr-oc complex on chro- mosome . antigens tested included highly purified proteins from the house dust mite, dermatophagoides ptery- nissinus, the domestic cat and dog, grass pollen, and the mould alternaria alternata. two independent sets of famil- ies, one british and one australian, were studied. no linkage of ige serotypes to tcr-,b microsatellite alleles was found, but significant linkage to tcr-oc microsatellite alleles was seen in british sibling pairs with ige responses to the house dust mite (p = - ). in australian subjects there was excess sharing of alleles in siblings responsive to grass pollen (p< ). it has been concluded that a gene in the tcr-ot region modifies the specific ige response. chromosome there have been many studies looking at the relationship of hla d encoded mhc class genes and specific ige responses suggesting linkage of certain haplotypes with individual responses to purified allergens. this is re- ferred to as the cognate, antigen specific arm of the ige response. from mossman's initial studies in mice, t helper lymphocytes in humans designated thl-like and th -like play a crucial role in facilitating the immune response. atopic subjects preferentially expand t cell clones with a "th " phenotype. the antigen-cognate interaction of th cells with b cells involves the cd - o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th x. . . o n ja n u a ry . d o w n lo a d e d fro m http://thorax.bmj.com/ wilkinson, holgate a p q b u o <,m zacmb ll -~~~~~~~ucl c j * lc)j)cc j (d j /) < j centromere telomere co) a) (o ) ) cv) cy) cy) . u) (n u) n l c cn lo o lo lo r ( ic ln lo lo lo lo lo i j i v , q q q irfl: interferon releasing factor gm-csf: granulocyte macrophage colony stimulating factor fgf-a: fibroblast growth factor acidic csf- r: colony stimulating factor adrb : beta adrenergic receptor adra b: beta adrenergic receptor linkage figure (a) schematic representation of the banding pattern on chromosome . (b) simplified genetic map of q -q showing il- cytokine gene cluster. (c) map of genetic markers on chronmosome . cd l binding, b cell activation, and the release of il- and il- from the th cells. this process leads to ig heavy chain class switching to the c isotype, resulting in specific ige antibody responses. ' interleukin is crucial for the development and functioning ofth cells, including their ability to express il- , the cytokine largely responsible for eosinophilia in allergic disorders. it has also been shown that basophils and mast cells both produce il- . the interaction of basophils and mast cells with b cells via the cd -cd l also leads to the production of ige. this interaction is not antigen driven and is therefore said to be non-cognate. the il- gene has emerged as a major candidate for ige responsiveness and atopy, with other candidate genes for atopic disease including il- and il- mapping within the "il- gene cluster" in chromosome q . , or within q . -q . marsh et al looked for linkage between total serum ige and multi-allergen ige antibody and several polymorphic genetic markers in and around chromosome q . -q , with a primary focus on markers mapping within the il- gene itself and in or close to the il- cluster (fig ). the analysis centred on subjects from large caucasian amish families who were selected on the basis of detectable serum ige to common inhalant allergens in at least one child. ' using the sibling pair method of analysing the data, marsh found significant evidence for linkage for il -r , interferon releasing factor (irf- ), il- , d s , and d s located in q . with the total serum level of ige. the p value for il -r was - , and with adjustment for multiallergen ige antibody this improved slightly to - . however, some- what strangely, none of the markers showed evidence for linkage with specific or cognate ige antibody responses. subsequent exclusion of all siblings who had detectable ige antibody by the multiallergen test and analysis of only "non-atopic" individuals strengthened the evidence for linkage with total ige, the p value for il -r improving further to . marsh employed complex segregation analysis to analyse the distribution of total ige among the amish family members and found significant evidence for the genetic determination of total ige with a dominant high ige model being slightly favoured over the dominant low model. when both ofthese models were used in lod score analyses, the data were consistent with linkage to the q . markers in each case, with the maximum lod scores being in the range of - - . these workers interpret their findings as demonstrating a likely role for il- or neighbouring gene in determining non-cognate ige production. further evidence for the potential importance of the cytokine cluster on chromosome q has been provided by the study of meyers et al. segregation analysis was performed on data from families from northern holland ascertained through a parent with a diagnosis of asthma who were first studied approximately years ago. families were selected through a proband with symptomatic asthma who met the following criteria at the time of the first study: < years of age, bronchial hyperresponsiveness to histamine, and non-smoking. the original evaluation included skin tests to a variety of allergens, blood and sputum eosinophil counts, pulmonary function testing, and bronchial responsiveness to histamine. total serum ige levels were not measured during the initial evaluation. current evaluation included a standardised respiratory questionnaire, pulmonary function testing, bronchial re- sponsiveness to inhaled histamine, skin tests, specific ige to house dust mite and grass mix, and total ige. genotyping was performed on families and the following markers were tested for linkage: il- , d s , fibroblast growth factor acidic (fgf-a), d s , colony stimulating factor- r (csf- r), d s , d s , and d s . the first two of these were also used by marsh and both were found to be significant. linkage was looked for using the sibling pair method and the lod score method using the genetic model obtained from the segregation analysis of recessive inheritance of high ige levels. positive evidence for linkage of a gene for ige production was found to il- (p =ns), d s (p= - ), d s (p< ), and csf- r (p< . ). lod scores for these markers ranged from - to - . the work by marsh et al was carried out in the amish population, a genetically isolated group, and the study by meyers et al looked at a population selected for asthma but examined for atopy. with a trait as common as atopy affecting up to % of the population, it is important to perform linkage studies in a random population selected without reference to atopy. this was undertaken in a random sample of families with three or more children from general practice registers in and around southampton. the families comprised individuals ( parents and offspring). the mean age of the parents was years (range - years) and of the children - years (range - years). seventeen ( - %) of the parents had self- reported asthma, while ( %) of children had self or parent-reported asthma, consistent with the prevalence of asthma in southern england. one hundred and seven ( %) of the parents and ( - %) of the children had a skin prick test of mean weal diameter of mm or more. each family member completed a structured written questionnaire on atopic symptoms and disease. participants underwent skin prick testing for common allergens, and bronchial responsiveness to histamine was measured. total ige levels were also assessed. a number of markers around candidate loci, namely il- receptor (d s ), cd o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th x. . . o n ja n u a ry . d o w n lo a d e d fro m http://thorax.bmj.com/ candidate gene loci in asthmatic and allergic inflammation (d s ), interferon a (ifncx), il- (d s ), tumour necrosis factor (tnf- ), il- receptor, ifny, fcrloo (di s ), and il- , were examined. the alleles were sized using an abi a automated dna sequencer. in considering the phenotype to be analysed the approach we adopted to define atopy in the analysis of the data was the use of stepwise principal component regression of six traits (log ige, skin prick test, bronchial hyper- responsiveness, history of wheezing, history of eczema, and history of seasonal rhinitis)." atopy was defined as the derived first principal component of the age and sex- adjusted traits. this definition of atopy was dominated by serum ige, and so linkage and association analysis are based on age and sex-corrected log ige. linkage and association analysis on the sample of random families demonstrated allelic association between the serum ige level and il- on chromosome q (p< ). given the numbers of markers tested, significant association is to be expected in some cases by chance, and these findings may be due to a type error. however, this is unlikely for two reasons. firstly, the interleukin cluster around il- contains many attractive candidate loci including il- . the markers used in this study were not randomly selected, and candidate loci in this region have biological plausibility. secondly, linkage for serum total ige levels has previously been shown in this region of chromosome in the two studies mentioned above. " further work is now under- way on a sample of multiplex families in which two or more members have a diagnosis of asthma. markers in and around the cytokine cluster on chromosome will be examined including d s , d s , d s , il- , and il- . the fcgrl marker on chromosome q will also be tested in addition to another random marker on chro- mosome llq, d . conclusion new techniques for scanning the human genome promise great advances in tracking the origins of disorders caused by multiple genes. however, it is clear from the studies presented in this overview that we are far from under- standing the genetic basis of asthma and atopy and their interaction with the environment. it is also clear that agreement must be reached on definition of the phenotype and methods of ascertainment in order to carry out large multicentre collaborative studies. positive findings need to be validated in different populations selected for the presence of the disease and then confirmed in a random population where the prevalence of asthma and atopy will also be expected to be significant. university medicine, centre block, southampton general hospital, southampton s xy, uk j wilkinson s t holgate ohe m, munakata m, hizawa n, itoh a, doi i, yamaguchi e, et al. beta, adrenergic receptor gene restriction fragment length polymorphism and bronchial asthma. thorax ; : - . luisetti m, pignatti pf. the search for susceptibility genes of copd. monaldi arch chest dis ; : - . davies jl, kawaguchi y, bennett st, copeman jb, cordell hj, pritchard le, et al. a genome-wide search for human type diabetes susceptibility genes. nature ; : - . pepys j. 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; : - . meyers da, postma ds, panhuysen cim, xu j, amelung pj, levitt rc, et al. evidence for a locus regulating total serum ige levels mapping to chromosome . genonnics ; : - . doull ijm, lawrence s, watson m, begishvili b, beasley r, lampe f, et al. allelic association of markers on chromosomes q and q with atopy and bronchial hyperresponsiveness. anti rezv respir cit care med (in press). o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th x. . . o n ja n u a ry . d o w n lo a d e d fro m http://thorax.bmj.com/ poster presentation open access left atrial scar burden determined by delayed enhancement cardiac magnetic resonance at post radiofrequency ablation: association with atrial fibrillation recurrence gerd brunner , *, lucien abboud , kamran a shaikh , amish s dave , joel morrisett , william a zoghbi , miguel valderrábano , dipan j shah from th annual scmr scientific sessions orlando, fl, usa. - february background left atrial (la) radiofrequency (rf) ablation has become routine treatment for atrial fibrillation (af) but still suf- fers from af recurrence requiring a repeat procedure. la-rf ablation success rates vary between % and %. delayed-enhancement cardiac magnetic reso- nance (de-cmr) can be used to noninvasively visualize la hyperenhancement (scar). we have utilized de- cmr to quantify la scar extent post la-rf-ablation and related this measure to af recurrence. methods twenty-seven patients ( . ± . years, males) with paroxysmal and chronic af underwent la-rf-ablation and subsequent de-cmr, an average of . ± . days post procedure. the de-cmr procedure was per- formed utilizing a navigated d inversion recovery gra- dient echo sequence (siemens . t avanto or . t verio) approximately minutes after administration of . mmol/kg diethylenetriaminepentaacetic acid−gado- linium (dtpa-gd, magnevist, berlex laboratories, wayne, nj). all scans were electrocardiographically (ecg)-gated and acquired during a ms window in mid-diastole with navigator-gating and fat suppression. we have developed an image analysis method and gra- phical user interface to semi-automatically quantify hyperenhanced regions in the la wall (scar). la scar was quantified by a single experienced observer blinded to patient data. la-scar measurements were normalized by la size. the intra-class correlation coefficient (icc) was used to assess intra-observer variability of ran- domly selected scans which were re-read one week later. variables were tested for normality with the shapiro- wilk test and a p-value< . was considered statistically section of atherosclerosis and vascular medicine, department of medicine, baylor college of medicine, houston, tx, usa full list of author information is available at the end of the article table la-scar quantification in af patients. variable af-recurrence [n= , mean, std] af-free [n= , mean, std] p-value la-volume [ml] . ± . . ± . . lvef [%] . ± . . ± . . . la-scar [cm ] . ± . . ± . . age [years] . ± . . ± . . gender [no. males] - la= left atrium; la scar (hyperenhanced area) was normalized by la volume. n= number of patients; std=standard deviation; rf= radio frequency; lvef: left ventricle ejection fraction; af: atrial fibrillation. brunner et al. journal of cardiovascular magnetic resonance , (suppl ):p http://www.jcmr-online.com/content/ /s /p © brunner et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by/ . significant (all tests were -sided). all patients provided informed consent. results the de-cmr scans were performed . ± . days after the initial la-rf- ablation procedure (figure). af recurrence was noted to occur in ( %) patients whereas ( %) patients demonstrated no af recur- rence. there was a trend toward a larger la-volume in the af-recurrence group ( . ± . ml vs. . ± . ml; p= . , see table ). left ventricle ejection fractions (lvef) were smaller in the af-recurrence group but the difference was not statistically significant ( . ± . % vs. . ± . . %, p= . ). average analysis time per scan was . ± min and intra-observer variability was excellent (icc= . ). la-scar was normally distributed (p= . ). average la scar extent, quantified in post la-rf-ablation de-cmr scans, was significantly larger in recurrence-free af patients ( . ± . cm ) when compared with individuals with af-recurrence ( . ± . cm ; p= . ). the results indicate that there is a significant inverse relationship between la-scar burden and af-recurrence. conclusions la scar extent can be reproducibly quantified with de- cmr; and a lower scar burden post la-rf-ablation is associated with af recurrence. funding this work was supported in part by nih grant t hl . author details section of atherosclerosis and vascular medicine, department of medicine, baylor college of medicine, houston, tx, usa. the methodist debakey heart & vascular center, houston, tx, usa. published: february doi: . / - x- -s -p cite this article as: brunner et al.: left atrial scar burden determined by delayed enhancement cardiac magnetic resonance at post radiofrequency ablation: association with atrial fibrillation recurrence. journal of cardiovascular magnetic resonance (suppl ):p . submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www.biomedcentral.com/submit figure de-cmr images of the left atrium (la) obtained with a siemens . t avanto (left and middle panels). the right panel shows the result of the semi-automated la scar segmentation for the center panel. the left atrium is indicated by the red contour and the blue area highlights hyperenhanced regions (scar). brunner et al. journal of cardiovascular magnetic resonance , (suppl ):p http://www.jcmr-online.com/content/ /s /p page of 	background 	methods 	results 	conclusions 	funding 	author details leukotriene b mediates macrophage influx and pulmonary hypertension in bleomycin-induced chronic neonatal lung injury leukotriene b mediates macrophage influx and pulmonary hypertension in bleomycin-induced chronic neonatal lung injury mong tieng ee, , crystal kantores, julijana ivanovska, mathew j. wong, amish jain, , , and robert p. jankov , , , program in physiology and experimental medicine, research institute, hospital for sick children, toronto, ontario, canada; division of neonatology, department of pediatrics, university of toronto, toronto, ontario, canada; department of physiology, university of toronto, toronto, ontario, canada; heart and stroke richard lewar centre of excellence in cardiovascular research, university of toronto, toronto, ontario, canada; and lunenfeld-tanenbaum research institute, mount sinai hospital, toronto, ontario, canada submitted march ; accepted in final form june ee mt, kantores c, ivanovska j, wong mj, jain a, jankov rp. leukotriene b mediates macrophage influx and pulmonary hypertension in bleomycin-induced chronic neonatal lung injury. am j physiol lung cell mol physiol : l –l , . first pub- lished june , ; doi: . /ajplung. . .—systemical- ly-administered bleomycin causes inflammation, arrested lung growth, and pulmonary hypertension (pht) in the neonatal rat, similar to human infants with severe bronchopulmonary dysplasia (bpd). leukotrienes (lts) are inflammatory lipid mediators produced by multiple cell types in the lung. the major lts, ltb and cysteinyl lts, are suggested to contribute to bpd, but their specific roles remain largely unexplored in experimental models. we hypothesized that lts are increased in bleomycin-induced bpd-like injury, and that inhibition of lt production would prevent inflammatory cell influx and thereby ameliorate lung injury. rat pups were exposed to bleo- mycin ( mg·kg� ·day� ip) or vehicle (control) from postnatal days – and were treated with either zileuton ( -lipoxygenase inhibitor), montelukast (cysteinyl lt receptor antagonist), or sc a (lta hydrolase inhibitor) mg·kg� ·day� ip. bleomycin led to increased lung content of ltb , but not cysteinyl lts. bleomycin- induced increases in tissue neutrophils and macrophages and lung contents of ltb and tumor necrosis factor-� were all prevented by treatment with zileuton. treatment with zileuton or sc a also prevented the hemodynamic and structural markers of chronic pht, including raised pulmonary vascular resistance, increased fulton in- dex, and arterial wall remodeling. however, neither treatment pre- vented impaired alveolarization or vascular hypoplasia secondary to bleomycin. treatment with montelukast had no effect on macrophage influx, pht, or on abnormal lung structure. we conclude that ltb plays a crucial role in lung inflammation and pht in experimental bpd. agents targeting ltb or ltb -mediated signaling may have utility in infants at risk of developing bpd-associated pht. rat; newborn; inflammation; lung injury the survival of extremely low-birth-weight infants has im- proved over recent decades, but at the cost of a high risk of developing chronic lung injury, known as bronchopulmonary dysplasia (bpd) ( ). chronic pulmonary hypertension (pht) is common in infants with severe bpd, heralding a greatly increased morbidity and mortality ( , , , ). the patho- genesis of bpd is multifactorial, with upregulation of inflam- matory mediators leading to, or caused by, infiltration of inflammatory cells playing a major role ( , , ). however, the specific mediators contributing to inflammatory neonatal lung injury remain unclear, and there are presently no effective treatments. leukotrienes (lts) are potent lipid mediators, first described in by borgeat and samuelsson ( ) as a new lineage of arachidonic acid-derived metabolites. lts are produced by, recruit, and activate immune cells, thus initiating, augmenting, and sustaining tissue inflammation. the concerted action of -lipoxygenase ( -lpo) and -lpo-activating protein (flap) on arachidonic acid produces lta , which is converted either by lta hydrolase (lta h) to ltb or is conjugated with reduced glutathione by ltc synthase to produce cysteinyl (cys) lts (ltc , ltd , and lte ) ( ) (illustrated in fig. ). -lpo activity requires perinuclear translocation from the cytosol ( ) and association with flap homodimer ( , , ) and is increased by phosphorylation at serine ( ). ltb and cyslts exert their actions by binding to distinct g protein- coupled receptors: blt and blt for ltb , and cyslt and receptors (cyslt r and cyslt r) for cyslts (fig. ). ltb is a particularly potent chemoattractant for multiple inflamma- tory cell types, especially neutrophils and macrophages. due to the wide-ranging pathological effects of lts, including in- creased production of matrix proteins, increased smooth mus- cle contractility and proliferation, and enhanced cell survival, critical roles have been proposed for a number of lung disor- ders, including asthma, pulmonary fibrosis, and chronic pht ( , ). a critical role for lts has been established in adult rat models of chronic pht ( – , ) and is suggested by observational studies in adult humans with primary pht ( , , ) and in newborn infants with evolving or established bpd ( , , , ). in neonatal animals, ltb receptor blockade prevented acute hyperoxic lung injury in preterm guinea pigs ( ), and a -lpo inhibitor decreased lung inflam- mation and edema induced by saline lavage in newborn piglets ( ). inhibitors of -lpo or flap prevented inhibited alveolar development secondary to severe hyperoxia ( ), whereas mon- telukast (a cyslt r antagonist) had no effect on chronic neonatal lung injury secondary to moderate hyperoxia ( ) in neonatal rats. in premature human infants with evolving bpd, ltb and lte were increased in tracheal aspirate fluid ( ) and urine ( , ), respectively. ex-premature infants with severe bpd also had increased urinary lte ( ). montelukast given as preventive therapy in at-risk preterm infants did not reduce the incidence of moderate-severe bpd ( ); however, address for reprint requests and other correspondence: r. p. jankov, hospital for sick children, . peter gilgan centre for research and learning, bay st., toronto, on, canada m g a (e-mail: robert. jankov@sickkids.ca). am j physiol lung cell mol physiol : l –l , . first published june , ; doi: . /ajplung. . . - / copyright © the american physiological society http://www.ajplung.orgl downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . mailto:robert.jankov@sickkids.ca mailto:robert.jankov@sickkids.ca when employed as rescue therapy for severe late-stage bpd, it conferred a survival advantage ( ). increased cyslts have also been reported in tracheal aspirates of infants with persis- tent pht of the newborn, an acute form of pht ( ). however, the specific role of various lts in the pathogenesis of chronic neonatal pht, particularly in the context of bpd or experi- mental bpd-like lung injury, has not been previously studied. bleomycin sulfate is a chemotherapeutic agent that produces a dose-dependent pulmonary inflammatory and fibrotic re- sponse when administered systemically or intratracheally ( , ). in neonatal rats, our laboratory and others have reported that repeated systemic administration of bleomycin leads to a selective decrease in lung growth, along with arrested alveo- larization, vascular hypoplasia, and chronic pht ( , , , ), similar to the lung pathology observed in human infants with severe bpd. we have employed this model to demon- strate a critical role for macrophage influx and macrophage- derived tumor necrosis factor (tnf)-� in the pathogenesis of chronic pht ( , ). in the present study, we hypothesized that lts are upregulated in bleomycin-induced lung injury, and that inhibition of lt biosynthesis or signaling would attenuate inflammation and consequent chronic pht. our observations reported herein suggest a critical role for ltb , but not for cyslts, in experimental chronic neonatal pht. materials and methods materials. bleomycin sulfate was purchased from calbiochem (san diego, ca). zileuton, montelukast, and c solid-phase extrac- tion cartridges (catalog no. ) were purchased from cayman chemical (ann arbor, mi). sc a was purchased from tocris biosciences (bristol, uk). acids, alcohols, organic solvents, parafor- maldehyde, permount, and superfrost/plus microscope slides were from fisher scientific (whitby, on, canada). weigert’s resorcin- fuchsin stain was from rowley biochemical (danvers, ma). anti- chemokine (c-c motif) ligand (ccl ) (catalog no. sc- ), anti-glyceraldehyde- -phosphate dehydrogenase (gapdh; catalog no. sc- ), and anti-flap (catalog no. sc- ) were from santa cruz biotechnology (santa cruz, ca). anti-cluster of differentiation (cd) (catalog no. mca r) was from serotec (raleigh, nc). anti-tnf-� (catalog no. hp ) was from hycult biotech (uden, the netherlands). anti- -lpo (catalog no. ), anti-phospho-ser- ine -lpo (catalog. no. ), and goat anti-rabbit igg-peroxi- dase antibody were from cell signaling technology (beverly, ma). anti-lta h (catalog no. nbp - ) was purchased from novus biologicals (littleton, co). anti-myeloperoxidase (mpo; catalog. no. a ) was from dako agilent (mississauga, ontario, canada). elisa kits for ltb (catalog no. adi- - ) and cyslts (catalog no. adi- - ) were from enzo life sciences (farmingdale, ny). tris-glycine precast gels and polyvinylidene difluoride (pvdf) mem- branes were from thermo scientific (rockford, il). unless otherwise specified, all other chemicals and reagents were from bioshop canada (burlington, ontario, canada). animal exposures and interventions. all procedures involving animals were performed in accordance with the standards of the canadian council on animal care and were approved by the animal care committee of the hospital for sick children research institute. timed-pregnant sprague-dawley dams were purchased from taconic farms (germantown, ny). commencing on the day after birth, pups received mg/kg bleomycin sulfate ( . mg/ml suspended in . % saline � % dmso; �l/g body wt by g needle in the right iliac fossa) or . % saline � % dmso (control) daily intraperitoneally for days, as previously described ( ), with or without zileuton ( -lpo inhibitor), montelukast (cyslt r antagonist), or sc a (lta h inhibitor; all at mg/ml � mg/kg) (see fig. ). for zileuton, dose-response studies ( , , or mg/kg ip daily) were conducted, demonstrating maximal inhibitory effect on lung macro- phage influx at mg/kg (data not shown). for both montelukast and sc a, which are orally active, the intraperitoneal route of delivery was necessitated by an inability to safely perform daily gavage in newborn rats. for montelukast, dose-response studies ( , , or mg/kg ip daily) were conducted, with the lowest dose based on a previous negative study in neonatal rats ( mg/kg ip daily) ( ). these studies demonstrated maximal inhibitory effect on lung ccl content at mg/kg (data not shown). for sc a, dose-response studies were conducted using a range of doses surrounding mg/kg ( , , and mg/kg ip daily) based on equivalence of ic with zileuton and similar oral bioavailability ( ). these studies demon- strated maximal inhibitory effect of sc a on lung ltb content at mg/kg (data not shown), the same (orally administered) dose and dose interval reported to have inhibitory effects on inflammatory injury in adult rodents ( ). each litter was maintained at n � – pups to control for nutritional effects. at the end of each - or -day exposure period, pups either were killed by pentobarbital overdose, or were exsanguinated after anesthesia. cardiac ventricular weights. right ventricular (rv) hypertrophy was quantified by measuring the rv-to-left ventricle and septum dry weight ratio (fulton index), as previously described ( ). two-dimensional echocardiography-derived measurements of pul- monary hemodynamics. pulmonary vascular resistance (pvr) was evaluated noninvasively using two-dimensional echocardiography and doppler ultrasound (vivid cardiac ultrasound system and i l linear probe; ge medical systems, milwaukee, wi), as previously described ( , ). briefly, following induction of anesthesia with % (vol/vol) isoflurane, the animal was laid supine while spontaneously breathing – % (vol/vol) isoflurane through a modified face mask. a short-axis view of the heart was acquired at the level of the aortic valve, and the pulmonary artery was identified by color flow doppler. the pulsed doppler gate was placed proximal to the pulmonary valve leaflets and aligned with an insonation angle of � ° to obtain a doppler profile. the pulmonary artery acceleration time (paat) was measured as the time from the onset of systolic flow to peak velocity and the rv ejection time (rvet) as the time from onset to comple- tion of systolic flow. pvr index was derived using the formula, arachidonic acid lta ltb ltc ltd lte cyslt r -lpo lta hydrolase ltc synthase zileuton sc a montelukast flap cyslt r/ blt / blt fig. . illustration of the arachidonic acid-leukotriene (lt) pathway and drug targets. the concerted action of -lipoxygenase ( -lpo) and -lpo-activating protein (flap) on arachidonic acid produces lta , which either is converted by lta hydrolase to ltb , or is conjugated by ltc synthase to produce cysteinyl (cys) lts (ltc , ltd , and lte ). attenuated production of ltb by inhibition of -lpo with zileuton or by inhibition of lta hydrolase with sc a prevented bleomycin-induced pulmonary hypertension. antago- nism of the cyslt receptor (cyslt r), for which lts c , d , and e act as ligands, had no effect on bleomycin-induced pulmonary hypertension. l leukotriene b mediates neonatal pulmonary hypertension ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . rvet/paat. all values were averaged from three doppler traces per animal using an offline analysis system (echopac, ge medical systems). histological studies. lungs from six animals from each group ( from each of separate litters) were air-inflated and perfusion-fixed at constant pressure, embedded in paraffin, sectioned, and immuno- stained for cd (to identify macrophages), or were stained with hematoxylin-eosin, or for elastin, as previously described ( , , , ). for all analyses, measurements were carried out on four noncon- tiguous left lung sections per animal by an observer blinded to group identity. for assessment of percentage of arterial medial wall area, pulmonary arteries were identified by the presence of both inner and outer elastic lamina using hart’s elastin stain, as previously described in detail ( , ). analyses of tissue macrophage (cd -positive) cell numbers, mean linear intercept (using hematoxylin and eosin-stained sections), and counts of peripheral arteries (identified as vessels of external diameter between and �m with both internal and external elastic laminae visible on elastin staining) were conducted as previously described in detail ( , , ) from random, non- overlapping, low-power fields captured from each section. paraform- aldehyde-fixed cardiac ventricular tissues were also embedded in paraffin oriented in the short axis and stained with hematoxylin-eosin. for measurement of tissue neutrophils, immunostaining for mpo was performed as previously described ( ), and low-power images ( per section, sections per slide, and slides per animal) were digitally captured using identical magnification, white balance settings, and exposure times. a tissue neutrophil index was derived representing the percentage of tissue area reaching a preset staining intensity threshold (for mpo-positive cells, normalized to total tissue area), using immunoratio online software (http:// . . . : /immu- noratio). quantitative pcr. rna was extracted and reverse transcribed, and quantitative pcr was performed as previously described ( ). primer sequences of genes of interest are listed in table . a standard curve for each primer set was run to ensure that reaction efficiency was equivalent to primers for the housekeeping genes, �-actin and gapdh, the expressions of which were determined in preliminary experiments to be unaffected by exposure to bleomycin. samples were run in duplicate, and fold or fraction change in expression relative to control samples was calculated by the �	ct method. elisa. lung lysates from six animals per group ( from each of separate litters) were purified and analyzed according to the manu- facturer’s instructions. western blot analyses. lung tissues from six animals per group ( from each of separate litters) were lysed in ripa buffer containing protease and phosphatase inhibitors. samples were fractionated by sds-page, transferred to pvdf membranes, and blotted, and band densities were measured as previously described ( ). differences in protein loading were compensated for by reblotting for gapdh, the expression of which was found, in preliminary studies, to be unaf- fected by chronic exposure to bleomycin. dilutions of primary anti- sera were : for flap, : for ccl and tnf-�, : for mpo, : , for -lpo and phospho-serine -lpo, : , for gapdh, and : , for lta h and secondary antisera. protein bands were identified using enhanced chemiluminescent substrate (supersignal west dura, thermo fisher scientific), and images were digitally captured using a microchemi chemiluminescent system (dnr bio-imaging systems, jerusalem, israel). bands were quanti- fied by digital densitometry of nonsaturated images with background density removed (imagej, nih, bethesda, md). data presentation and analysis. all values are expressed as means se. statistical analyses were performed using sigma plot . (systat software, san jose, ca). where three or more groups were compared, statistical significance (p � . ) was determined by one-way anova, followed by pairwise multiple comparisons using the tukey test. where two groups were compared, the student t-test was used. results temporal changes in effects of bleomycin on lung mrna expression and protein content of key enzymes and receptors mediating lt signaling and on inflammatory cell influx. as shown in table , relative changes in mrna expression were examined after or days of bleomycin (or vehicle) expo- sure. relative to controls, exposure to bleomycin for (but not ) days led to significantly increased alox , alox ap, ltc s, ltb r, and ltb r mrna expression. as shown in fig. , lung content of -lpo, flap, and lta h were also examined after (fig. a) or days (fig. b) of bleomycin (or vehicle) exposure. relative to vehicle, exposure to bleomycin for days led to significantly increased lung content of -lpo and flap and no change in lta h (fig. a). in contrast, -lpo and flap content in bleomycin-exposed lungs was no longer significantly increased after days, whereas there was a small, but significant, increase in lung lta h (fig. b). lung content of phospho-serine -lpo did not differ between vehicle- and bleomycin-exposed animals at either time point (data not shown). numbers of lung tissue (cd -positive) macrophages (normalized to tissue fraction) secondary to bleo- mycin were increased by day of exposure (fig. c), to a similar extent as at day . as shown in fig. d, tissue table . rat primer sequences for quantitative pcr gene refseq accession no. common name forward =- = reverse =- = alox nm_ -lipoxygenase cttcctggcatgactttgct ctaggctgcactccaccatt alox ap nm_ -lipoxygenase activating protein (flap) gggtctacactgccaaccag ctcccagatagccgacaaag ltc s nm_ leukotriene c synthase agctcttctggctaccgtca atttacctgggctcggaaga lta h nm_ leukotriene a hydrolase agcatcgaagacctgaagga gccgtaaccatctgaatcgt ltb r nm_ leukotriene b receptor ggcatgtccctgtctctgtt ccgagccagaaagtgtagga ltb r nm_ leukotriene b receptor cgtctttactgcgggtgatt tgccctgacccactactttc cyslt r nm_ cysteinyl leukotriene receptor tggagctgaaaatatgacagca ggaaggctgatttctcatgg table . relative changes in mrna expression secondary to bleomycin exposure gene day day alox . . * . . alox ap . . * . . ltc s . . * . . lta h . . . . ltb r . . * . . ltb r . . * . . * cysltr . . . . values are means se of samples per group relative to control (vehicle-treated) group, which was assigned a value of . *p � . , by t-test, compared with control. l leukotriene b mediates neonatal pulmonary hypertension ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . http:// . . . : /immunoratio http:// . . . : /immunoratio neutrophils were also increased in the bleomycin-exposed lung by days, with a further significant increase at days. effects of bleomycin exposure and zileuton ( -lpo inhibitor) treatment on lung ltb and cyslt content. exposure to bleomy- cin for days had no effect on ltb content (data not shown); however, ltb content was significantly increased after days of bleomycin exposure (fig. a). the bleomycin-induced increase in ltb at days was completely prevented by treatment with zileuton (fig. a). cyslt (ltc , ltd , and lte ) contents were unaltered by bleomycin exposure or by zileuton treatment, both at (data not shown) and at days (fig. b). effects of zileuton on bleomycin-induced pht. as previously reported ( , ), exposure to bleomycin for days led to significantly increased pvr index (fig. a), fulton index (fig. b), and percentage of medial wall area in pulmonary resistance arteries (fig. c), relative to vehicle-treated controls. treatment of bleomycin-exposed animals with zileuton restored these parame- ters to values comparable to controls (fig. ). effects of montelukast (cyslt r antagonist) or sc a (lta h inhibitor) on bleomycin-induced pht. treatment with montelukast did not prevent bleomycin-induced pht, as evi- denced by a lack of effect on pvr (fig. a) and fulton indexes (fig. b) or on percentage of medial wall area (fig. c). in contrast, treatment with sc a completely normalized pvr index (fig. a), fulton index (fig. b), and percentage of medial wall area (fig. c). as shown in fig. e, treatment with sc a completely prevented the bleomycin-induced in- crease in lung ltb , which was unaffected by montelukast. to determine whether a lack of effect of montelukast on cyslt r signaling was responsible for a failure to prevent bleomycin- induced pht, we examined lung content of ccl (also known as macrophage inflammatory protein- �), a chemokine known fig. . exposure to bleomycin increases lung contents of -lipoxygenase ( -lpo), -lpo activating protein (flap), and leukotriene a hydrolase (lta h), and numbers of tis- sue macrophages and neutrophils. pups were exposed to daily intraperitoneal bleomycin ( mg/kg) or % dmso in . % saline vehicle from postnatal day . western blot analyses are shown of -lpo ( kda), flap ( kda), or lta h ( kda) on day (a) or day (b) of bleomycin exposure. representative immunoblots are shown be- low each graph, with noncontiguous gel lanes demarcated by black lines. n � – samples/group. c: tissue macrophage counts per field normalized to tissue fraction on days or of vehicle or bleomycin expo- sure. n � animals/group. d: tissue neu- trophil index on days or of vehicle or bleomycin exposure. n � animals/group. values are means se. *p � . , by t-test, compared with vehicle-treated group. #p � . , by t-test, compared with vehicle- treated group. †p � . , by anova, compared with all other groups. l leukotriene b mediates neonatal pulmonary hypertension ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . to be stimulated by cyslt r activation ( ). despite there being no increase in lung cyslt content secondary to bleo- mycin exposure (fig. b), ccl was found to be significantly increased in the bleomycin-exposed lung (fig. f). increased lung ccl secondary to bleomycin was completely normal- ized by treatment with montelukast (fig. f), in keeping with effective blockade of the cyslt r. effects of zileuton, montelukast, or sc a on bleomycin- induced lung inflammation. the lungs of animals exposed to bleomycin had greatly increased cd -positive cell numbers fig. . zileuton prevented bleomycin-induced increase in leukotriene (lt) b content. pups were exposed to daily intraperitoneal bleomycin ( mg/kg) or . % saline in % dmso vehicle and treated with daily intra- peritoneal zileuton ( mg·kg� ·day� ) or vehicle (control) from postnatal days – . lung content of ltb (a) or cysteinyl lts (b) was quantified by elisa. values are means se for n � – samples per group. *p � . , by anova, compared with all other groups. fig. . zileuton prevented bleomycin-induced pulmonary hypertension. pups were exposed to daily intraperitoneal bleomycin ( mg/kg) or . % saline in % dmso vehicle and treated with daily intraperitoneal zileuton ( mg·kg� ·day� ) or vehicle (control) from postnatal days – . a: pulmonary vas- cular resistance (pvr) index, as estimated by the right ventricular (rv) ejection time (rvet)-to-pulmonary arterial acceleration time (paat) ratio. n � – animals/ group. b: rv-to-left ventricle � septum (lv�s) dry weight ratios (fulton index) as a marker of rv hypertrophy. n � – animals/group. tiled low-power photomi- crographs of hematoxylin and eosin-stained cardiac sections, oriented in the short axis below the atrioventricular valves (rv cavity � rv), are shown to demonstrate differences in rv wall thickness between groups. c: percentage of arterial medial wall area. val- ues (for n � animals/group) are a marker of pulmonary arterial remodeling. represen- tative photomicrographs of elastin staining (dark brown inner and outer elastic laminae delineating the medial vascular wall; scale bar � �m) are shown demonstrating medial wall thickening in bleomycin-ex- posed animals (bleomycin control), which was largely prevented by concurrent treat- ment with zileuton (bleomycin zileuton). values are means se. *p � . , by anova, compared with all other groups. #p � . , by anova, compared with vehicle groups. l leukotriene b mediates neonatal pulmonary hypertension ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . (fig. a) and increased lung tnf-� content (fig. b). treat- ment of bleomycin-exposed animals with zileuton or sc a significantly decreased tissue macrophage numbers (fig. a), while montelukast had no effect (fig. a). treatment of bleomycin-exposed animals with zileuton also normalized lung tnf-� content (fig. b), in keeping with previous find- ings indicating that tnf-� is predominantly macrophage de- rived ( ). treatment with zileuton also completely prevented neutrophil influx in the bleomycin-exposed lung (fig. c). effects of zileuton, montelukast, or sc a on bleomycin- induced changes in lung morphology. as demonstrated by representative low-power elastin-stained sections (fig. a), the lung structure of bleomycin-exposed animals was character- ized by septal thinning, arrested alveolarization [manifesting as “emphysematous” distal air spaces, quantified as increased mean chord length (fig. b)], and vascular hypoplasia (fig. c). treatment with zileuton, montelukat, or sc a had no effects on these parameters (fig. ). discussion in human preterm infants with respiratory distress, early in- creases in lung macrophages are known to persist in infants who develop severe bpd, while declining in those who do not ( ). the fig. . bleomycin-induced pulmonary hyper- tension was prevented by sc a, a leuko- triene (lt) a hydrolase inhibitor, but not by montelukast, a cysteinyl lt receptor antago- nist. pups were exposed to daily intraperitoneal bleomycin ( mg/kg) or . % saline in % dmso vehicle and treated with daily intraperi- toneal montelukast ( mg·kg� ·day� ) or sc a ( mg·kg� ·day� ) from postnatal days – . a: pulmonary vascular resistance (pvr) index, as estimated by the right ventric- ular (rv) ejection time (rvet)-to-pulmonary arterial acceleration time (paat) ratio. n � – animals/group. b: rv-to-left ventricle � septum (lv�s) dry weight ratios (fulton in- dex) as a marker of rv hypertrophy. n � – animals/group. c: percentage of arterial me- dial wall area (n � animals/group) as a marker of pulmonary arterial remodeling. d: representative photomicrographs of elastin staining (dark brown inner and outer elastic laminae delineating the medial vascular wall). scale bar � �m. e: lung content of ltb , quantified by elisa. n � samples/group. f: western blot analyses of ccl ( kda). rep- resentative immunoblots are shown below the graph with noncontiguous gel lanes demar- cated by black lines. n � samples/group. values are means se. *p � . , by t-test, compared with vehicle group. #p � . , by anova, compared with all other groups. l leukotriene b mediates neonatal pulmonary hypertension ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . fig. . effects on inflammation in the bleomycin-exposed lung. pups were exposed to daily intraperitoneal bleomycin ( mg/kg) or . % saline in % dmso vehicle and treated with daily intraperitoneal zileuton ( mg·kg� ·day� ), montelukast ( mg·kg� ·day� ), sc a ( mg·kg� ·day� ), or vehicle (control) from postnatal days – . a: tissue macrophage counts per field normalized to tissue fraction. representative medium-power photomicrographs are shown of cd immunostaining demonstrating increased numbers of tissue macrophages (large dark brown cells, some highlighted by arrows) in bleomycin-exposed animals (bleomycin control), which was prevented by treatment with zileuton (bleomycin zileuton) or sc a (bleomycin sc a), but not by montelukast (bleomycin montelukast). scale bar � �m. b: western blot analyses of lung tnf-� ( kda) content. representative immunoblots are shown adjacent to the graph with noncontiguous gel lanes demarcated by black lines. c: tissue neutrophil index. all values are means se for animals or samples per group. *p � . , by t-test, compared with vehicle group. #p � . , by t-test, compared with vehicle group. †p � . , by anova, compared with all other groups. ‡p � . , by anova, compared with all other groups. l leukotriene b mediates neonatal pulmonary hypertension ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . potential role of lts in this inflammatory process has received limited attention. we report in bleomycin-exposed neonatal rat pups, a model with similarities to human bpd, that treatment with zileuton, a -lpo inhibitor, or sc a, a lta h inhibitor, prevented upregulation of ltb , inflammatory cell influx, and the hemodynamic and structural changes of chronic pht. our laboratory has previously shown that macrophage- derived tnf-� is critical to bleomycin-induced chronic pht ( ). in the present study, increased tnf-� was also attenuated by -lpo inhibition. treatment with montelu- kast, a cyslt r inhibitor, had no effect on macrophage influx or on markers of chronic pht. these findings are in agreement with studies in adult experimental animals of inflammatory injury, which favor a dominant role for ltb , rather than for cyslts ( – ). we did not observe any effects of lt pathway inhibition on arrested alveolarization or vascular hypoplasia. this diver- gence is consistent with our laboratory’s own previous obser- vations employing alternative interventions [e.g., therapeutic hypercapnia, arginase inhibition ( , )] that were also effi- cacious in preventing inflammation and vascular remodeling, but not in preventing vascular hypoplasia or emphysematous lung structure. it appears, therefore, at least in bleomycin- exposed neonatal rats, that inflammation and vascular remod- eling are regulated by pathways distinct from those that regu- late angiogenesis and alveologenesis. whether this also holds true in humans remains unclear. we observed increased mrna expression of -lpo, flap, blt , and blt , which was confirmed at the protein level for -lpo and flap in the lungs of bleomycin-exposed animals. we did not observe increased lung content of phospho-serine -lpo, which increases -lpo activity and favors ltb synthesis ( , ). nonetheless, these changes were accompa- nied by increased lung ltb in the bleomycin-exposed lung. the reasons for our observations regarding temporal changes in -lpo and flap (increased at day , but not at day ) are unclear, but could represent a negative feedback effect of increased ltb , which was only elevated at day . the degree of increase in ltb secondary to bleomycin exposure was small, which we speculate may have reflected a dilutional effect inherent to measurement in whole tissue. ltb has been previously described to be predominantly produced by inflammatory cells, particularly activated macro- phages and neutrophils ( , ), which are both present in fig. . targeting leukotriene pathways did not prevent abnormal lung morphology induced by bleomycin. pups were exposed to daily intraperitoneal bleomycin ( mg/kg) or . % saline vehicle and treated with daily intraperi- toneal zileuton ( mg·kg� ·day� ), montelu- kast ( mg·kg� ·day� ), sc a ( mg·kg� ·day� ), or % dmso in . % sa- line (control) from postnatal days – . a: representative low-power photomicrographs of elastin-stained sections demonstrating marked distal airway simplification, septal thinning, and decreased numbers of small peripheral arteries (outlined by dark brown stain for elas- tin) in bleomycin-exposed (bleomycin control) animals, which were not improved by treatment with zileuton (bleomycin zileuton), montelukast (bleomycin montelukast), or sc a (bleo- mycin sc a). scale bar � �m. mor- phometric analyses of mean chord length (lm; b) and peripheral arteries per field (c), cor- rected for tissue fraction, are shown. values are means se for n � animals/group. *p � . , by t-test, compared with vehicle group. l leukotriene b mediates neonatal pulmonary hypertension ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . abundance in the bleomycin-exposed lung ( , , , ). in the present study, we were unable to determine the cellular sources of ltb , due to a lack of commercially available antibodies suitable for in situ immunostaining. however, it appears unlikely that macrophages are a major source of ltb in the lungs of bleomycin-exposed neonatal rats, given our present findings that tissue macrophage numbers plateaued by days of bleomycin exposure when ltb was not increased. increased ltb production by pulmonary artery endothelial cells has also been described ( , ). as nonmyeloid cells are poor in -lpo, lt production in such cells is believed to be facilitated by intercellular transfer of lta ( , , ). the pattern of temporal changes in inflammatory cell number and the observation that treatment with zileuton decreased both macrophage and neutrophil influx opens the possibility that one inflammatory cell type may recruit the other, particularly that macrophages may recruit neutrophils; how- ever, our laboratory’s previous work in the present model suggests that this is not the case. for example, an interven- tion (therapeutic hypercapnia) that prevented macrophage influx had no effect on tissue neutrophils ( ). conversely, a cxcr antagonist intervention that prevented neutrophil influx had no effect on increased tissue macrophages or on the development of pht ( ). our present results are consistent with previous work by tian and colleagues ( ), in which blockade of lta h- expressing macrophages prevented endothelial injury and re- versed pht in sugen-exposed, athymic rats. based on in vitro observations, the suggested mechanism of ltb -mediated in- jury was endothelial cell apoptosis via inhibited expression of sphingosine- -phosphate and endothelial nitric oxide synthase ( ). whether changes in sphingosine- -phosphate and endo- thelial nitric oxide synthase represented a direct effect of ltb or the result of limiting macrophage influx and thereby other macrophage-derived products was unclear. ltb has also been reported to stimulate pulmonary artery smooth muscle cell proliferation and migration ( ) and to activate adventitial fibroblasts ( ), thus potentially playing a direct role in vas- cular remodeling. there are several limitations to this study. first, a major feature of bleomycin-induced lung injury is collagen deposi- tion, which is not a consistent feature of modern bpd. our use of this model was predicated on a striking degree of arrested lung growth and development, which is disproportionate to the effects on other organs, despite systemic administration ( ). second, our intervention studies demonstrating an effect in preventing chronic pht were limited to inhibition of -lpo and lta h. confirmation of the marked upregulation of blt mrna in the bleomycin-exposed lung at the protein level, localization of blt -expressing cells in the bleomycin- exposed lung, and further intervention studies employing spe- cific blt antagonists ( , , ) are a focus of ongoing and future work. the cellular localization of lt pathway mediators upregulated in the bleomycin-exposed lung and whether ltb plays a direct role in the pathogenesis of vascular remodeling, in addition to an indirect role via macrophages, are also issues worthy of future consideration. finally, our early preventive strategy in the present study was to treat throughout the -day period of injury. as macrophage influx in this model plateaued by days of life, it is possible that a shorter duration of therapy may be sufficient to confer a protective effect. in conclusion, ltb , but not cyslts, appears to play a crucial role in lung inflammation and pht in experimental bpd-like injury secondary to systemic bleomycin. agents targeting ltb or ltb -mediated signaling may have utility in human infants at risk of developing bpd-associated pht. grants this work was supported by operating funding from the canadian institutes of health research (mop- to r. p. jankov) and by infrastructure funding from the canada foundation for innovation (to r. p. jankov). a. jain was supported by a clinician-scientist training program award from the hospital for sick children research training centre and by a queen elizabeth ii/heart and stroke foundation of ontario graduate scholarship in science and technology. m. j. wong was supported by a lorne-phenix award from the cardiovascular sciences collaborative pro- gram and a graduate scholarship from the department of physiology, university of toronto. disclosures no conflicts of interest, financial or otherwise, are declared by the author(s). author contributions m.t.e. and r.p.j. conception and design of research; m.t.e., c.k., j.i., m.j.w., a.j., and r.p.j. performed experiments; m.t.e., c.k., j.i., m.j.w., a.j., and r.p.j. analyzed data; m.t.e. and r.p.j. interpreted results of experiments; m.t.e. and r.p.j. prepared figures; m.t.e. and r.p.j. 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omentin plasma levels and gene expression are decreased in obesity celia m. de souza batista, , rong-ze yang, mi-jeong lee, nicole m. glynn, dao-zhan yu, jessica pray, kelechi ndubuizu, susheel patil, alan schwartz, mark kligman, susan k. fried, , da-wei gong, , alan r. shuldiner, , , toni i. pollin, and john c. mclenithan , , central obesity and the accumulation of visceral fat are risk factors for the development of type diabetes and cardiovascular disease. omentin is a protein expressed and secreted from visceral but not subcutaneous adipose tissue that increases insulin sensitivity in human adipocytes. to determine the impact of obesity-dependent insulin resis- tance on the regulation of two omentin isoforms, gene expression and plasma levels were measured in lean, over- weight, and obese subjects. omentin was shown to be the major circulating isoform in human plasma. lean subjects had significantly higher plasma omentin levels than obese and overweight subjects. in addition, higher plasma omen- tin levels were detected in women compared with men. plasma omentin levels were inversely correlated with bmi, waist circumference, leptin levels, and insulin resis- tance as measured by homeostasis model assessment and positively correlated with adiponectin and hdl levels. both omentin and omentin gene expression were decreased with obesity and were highly correlated with each other in visceral adipose tissue. in summary, de- creased omentin levels are associated with increasing obe- sity and insulin resistance. therefore, omentin levels may be predictive of the metabolic consequences or co-morbid- ities associated with obesity. diabetes : – , o besity is a chronic pathological condition and a risk factor for type diabetes and cardiovascu- lar disease ( – ). several studies have shown that visceral obesity in particular is strongly associated with insulin resistance, hyperglycemia, dyslip- idemia, and hypertension. subcutaneous fat deposition has also been associated with decreased risk of cardiovas- cular disease in some studies ( – ). in light of the diver- gent pathological consequences of differences in adipose tissue distribution, it is of great interest to resolve the molecular differences between visceral and subcutaneous adipose tissue depots. although anatomical location and vascularization are clearly different ( ), the molecular basis of differences in metabolism and secretory profile between visceral (omental) and subcutaneous adipose tissues and their impact on whole-body physiology are still not totally understood. omentin is a newly identified secretory protein that is highly and selectively expressed in visceral adipose tissue relative to subcutaneous adipose tissue ( – ). omentin has been identified in other tissues at lower expression levels and named intelectin ( ), intestinal lactoferrin receptor ( ), or endothelial lectin ( ). it is expressed in intestinal paneth cells ( ), endothelial cells ( ), and visceral adipose stromal-vascular cells ( ). in vitro stud- ies have shown that omentin increases insulin signal transduction by activating the protein kinase akt/protein kinase b and enhancing insulin-stimulated glucose trans- port in isolated human adipocytes. thus, omentin may play a paracrine or endocrine role in modulating insulin sensitivity. a homolog of omentin has been identified that shares % amino acid identity with omentin/intelectin ( ) and will be referred to as omentin . the two omentin genes, omentin and omentin , are localized adjacent to each other in the q -q chromosomal region ( ), which has been previously linked to type diabetes in several populations ( – ). based on the preferential expression of omentin in visceral fat, the presence of omentin in the circulation, and its potential role as an insulin sensitizer, we evaluated the association of omentin levels with measures of obesity, insulin resistance, and related features. we developed and validated a quantitative western blotting assay that allowed us to measure omentin levels in the plasma. we found that omentin is the major circulating form of omentin and that its plasma levels and adipose tissue gene expression are decreased with obesity. in addition, plasma omentin is positively correlated with plasma adiponectin and hdl and negatively correlated with homeostasis model assessment (homa), a measure of insulin resistance. research design and methods these studies involved healthy volunteers. subjects who had malignant disease, diabetes, or major renal, hepatic, and/or thyroid dysfunction were excluded. none were on hormonal replacement therapy. the study protocols were approved by the institutional review boards for human subjects from the division of endocrinology, diabetes and nutrition, department of medicine, university of maryland school of medicine, baltimore, maryland; the department of physiology, university of maryland school of medicine, balti- more, maryland; the department of biology, university of maryland baltimore county, baltimore, maryland; the department of medicine, johns hopkins university school of medicine, baltimore, maryland; the division of general surgery, department of surgery, university of maryland school of medicine, baltimore, maryland; and geriatric research, education and clinical center, baltimore veterans affairs medical center, baltimore, maryland. address correspondence and reprint requests to john c. mclenithan, west redwood st., room , baltimore, md . e-mail: jmcle @ umaryland.edu. received for publication october and accepted in revised form february . published ahead of print at http://diabetes.diabetesjournals.org on feb- ruary . doi: . /db - . afds, amish family diabetes study; homa, homeostasis model assess- ment; pi, isoelectric point. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , june research at university of maryland and johns hopkins university. informed consent was obtained from all subjects. effect of obesity on omentin gene expression. human visceral (omental) adipose tissues were obtained from subjects ( men and women) over a range of age ( – years) and bmi ( . – . kg/m ) undergoing intra- abdominal surgery at the university of maryland medical center or the johns hopkins bayview medical center. after excision, tissue was immediately frozen in liquid nitrogen and stored at � °c for subsequent rna extraction and quantitative rt-pcr analysis. in a subgroup of these subjects (n � , men and women) over a range of age ( – years) and bmi ( . – . kg/m ), blood samples were collected after overnight fasting on a visit before the day of the surgery. plasma was separated and stored at � °c for subsequent quantification of omentin levels. effect of obesity on human plasma omentin levels. a subset of healthy subjects ( women and men) from the previously described amish family diabetes study (afds) ( ) were selected as sibpairs that best fit the criterion of age-matched (within years) and sex-matched bmi discordance (� . kg/m difference). the bmi-discordant sibpair design was chosen to maximize the power to detect association with bmi. one subject did not fall within the detection limits of the assay, and two subjects were considered outliers (� sd from the mean). consequently, these three subjects were eliminated from all analyses. therefore, pairs were used in the sibpairs analysis. for additional analyses, the participants (n � , women and men) were divided into three groups: ) lean (n � ; bmi � kg/m ), ) overweight (n � ; kg/m � bmi � kg/m ), and ) obese (n � ; bmi � . kg/m ). identification of omentin and omentin in human plasma by two- dimensional gel electrophoresis. human plasma samples, purified recom- binant omentin ( ), and omentin were separated by two-dimensional gel electrophoresis followed by western blotting with anti-omentin monoclonal antibodies. omentin cdna was subcloned into pcdna (invitrogen, carls- bad, ca) and transfected into hek- a cells using lipofectamine plus (invitrogen). stably transfected cells were selected with �g/ml g (invitrogen). omentin – expressing cells were cultured in % fetal bovine serum/dulbecco’s modified eagle’s medium until % confluent and then switched to serum-free medium for days. conditioned medium containing omentin was then harvested and concentrated using centricon centrifugal concentrators (millipore, billerica, ma). plasma samples, omentin , and/or omentin standards were diluted in rehydration buffer (bio-rad, hercules, ca). first-dimension isoelectric focusing was conducted using immobilized ph gradient strips (ph – , cm; bio-rad) in a bio-rad ief cell. after focusing, strips were equilibrated, and they were subjected to % sds-page. proteins were then transferred to immobilon-polyvinylidine fluoride mem- brane (millipore), blocked with starting block solution (pierce, rockford, il) plus . % tween (pierce), and incubated with g b , a human omentin- specific monoclonal primary antibody ( ), followed by horseradish peroxi- dase-conjugated anti-mouse secondary antibody (kpl, gaithersburg, md). immunoreactive spots were visualized by chemiluminescent detection with the femto-west kit (pierce) on a fluorchem chemiluminescent/fluores- cent imager (alpha innotech, san leandro, ca) (fig. ). quantification of human plasma omentin levels. the quantification of human plasma omentin levels was performed in three steps. initially, purified omentin was quantitated against a bsa standard curve using % sds-page. after electrophoresis, protein bands were visualized with sypro ruby fluorescent protein stain (bio-rad). a standard curve was constructed using the log- transformed bsa concentrations versus the band intensities, and purified omen- tin was quantitated by extrapolation from this curve. in the next step, a subset of plasma samples was analyzed by quantitative western blotting (described above) using the previously quantified purified omentin as a standard. a standard curve was constructed using the purified omentin concentrations versus the optical densities, and “standard plasma samples” were quantitated by extrap- olation from this curve (fig. ). in the last step, “standard plasma samples” were then used to quantify the rest of the samples by quantitative western blotting. a standard curve was constructed using the “standard plasma” concentrations versus the optical densities, and plasma samples were quantitated by extrapola- tion from this curve. fluorescent or immunoreactive band intensities were visualized and quantified on a fluorchem chemiluminescent/fluorescent imager (alpha innotech). duplicate measurements differing by � % were rejected, and the samples were retested. quantification of human plasma adiponectin levels. total adiponectin levels were determined in fasting plasma samples by a commercial radioim- munoassay kit (linco research, st. louis, mo). the samples were analyzed in duplicate and measured on an automated packard cobra-ii auto gamma counter (perkin-elmer, wellesley, ma). the duplicate measurements with counts differing by � % were rejected, and the samples were retested. quantitative real-time pcr analysis. omentin and omentin mrna levels were measured by real-time quantitative rt-pcr in a lightcycler (roche applied science, indianapolis, in). total rna was extracted from omental adipose tissue samples with trizol (invitrogen). total rna was reverse tran- scribed using the roche transcriptor cdna kit. cdna ( ng) was quantified using lightcycler probes master kit (roche applied science) and taqman probe/primer sets (omentin , hs _m ; omentin , hs _m ; applied biosystems, foster city, ca). s mrna ( s rna, hs _sl) was used as an internal control for normalization. each sample was run in duplicate. duplicates exhibiting sds of � . cycle threshold were repeated. copy numbers of the specific mrnas were obtained using standard curves generated with omentin – containing ( ) and omentin – containing (pcdna ) plasmids. nor- malized gene expression values were obtained using lightcycler relative quan- tification software (lightcycler ; roche applied science). fig. . identification of omentin as the major omentin isoform in human plasma. purified omentin (a), human plasma (b), and human plasma plus exogenous omentin (c) were subjected to two-dimen- sional gel electrophoresis and western blotting with anti-human omen- tin monoclonal antibody. spots indicate immunoreactive omentin and with pis of � . and . , respectively. fig. . quantification of omentin levels in human plasma samples. a: plasma samples (“standard plasma”) from human subjects (marked p , p , and p ) and purified omentin at six different concentrations (marked o –o ) were electrophoresed in duplicate on % sds-page gel followed by western blotting with anti-omentin antibodies. b: the standard curve was constructed by plotting known omentin concentra- tions (f) versus their optical densities in arbitrary units. omentin concentrations in plasma (‚) were extrapolated from this standard curve. decreased omentin levels and obesity diabetes, vol. , june statistical analysis. to adjust for family structure, variance component analysis as implemented in solar (sequential oligogenic linkage analysis routines), which allows the inclusion of a kinship matrix to allow for residual intra-pair correlations between relatives, was used for all analyses involving the association of circulating omentin levels with other obesity- and metabolic syndrome–related traits in amish subjects ( ). before analysis, data were tested for normality of distribution by the shapiro-wilk test. consequently, homa, leptin, insulin, and triglyceride levels were natural log-transformed to obtain a normal distribution. spearman rank correlations were used to estimate relationships between omentin gene expression and other quantita- tive variables in the non-amish (unrelated) subjects using sas . software package (sas, cary, nc). results human plasma samples contain detectable omentin levels but not omentin by two-dimensional western blotting. because two highly homologous isoforms of omentin/intelectin exist ( , ) and may contribute to total circulating immunologically detectable omentin lev- els, two-dimensional western blots were used to deter- mine specificity of the g b monoclonal antibody ( ) and the relative abundance of omentin and in plasma. two-dimensional gel electrophoresis of omentin purified from conditioned medium ( ) followed by western blot- ting resolved three spots with isoelectric point (pi) � . and an apparent molecular weight of kda (fig. a). all immunoreactive species, most likely resulting from post- translational modification, were confirmed to be omentin by matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis (data not shown). a similar pattern was observed when human plasma samples were loaded (fig. b). when omentin – conditioned me- dium was analyzed alone, only one spot with a slightly lower molecular weight and a pi of . was observed (data not shown). when omentin – conditioned medium was added to human plasma, three spots with pi � . plus one spot with pi � . were observed in a similar molecular weight range (fig. c). the experimentally determined pis were identical to the predicted pi values for omentin and based on amino acid sequence ( ). these data suggest that the g b antibody recognizes both omentin and and that omentin is present in human plasma but that omentin is either absent or below the limit of detection of the quantitative western blot. human plasma omentin levels negatively correlate with obesity and insulin resistance markers. meta- bolic and demographic characteristics of the afds sub- jects adjusted for family structure are presented in table . plasma omentin levels, determined by quantitative west- ern blotting, were significantly higher in the leaner mem- bers of the bmi-discordant sibpairs than in their heavier siblings (paired t test, n � sibpairs, p � . ). after regrouping subjects into established bmi categories, a variance component analysis, adjusted for age, sex, and family structure, was performed to study the differences between lean and overweight subjects and between lean and obese subjects. plasma omentin levels (se) were shown to be significantly higher in the lean group ( . � . �g/ml, n � ) than in overweight ( . � . �g/ml, n � , p � . ) and obese ( . � . �g/ml, n � , p � . ) groups. in a sex-stratified analysis in which adiposity level was coded on a linear scale ( , lean; , overweight; and , obese), in both women and men, increasing levels of adiposity were associated with de- creased plasma omentin levels (p � . and . , respectively; fig. ). a sex difference in circulating omen- tin levels was observed when comparing all women to all men, adjusted for bmi category ( . � . vs. . � . �g/ml, p � . ), and when comparing lean women with lean men ( . � . vs. . � . �g/ml, p � . ; fig. ). significant correlations based on variance components analysis adjusted for sex, age, and family structure were found between plasma omentin levels and bmi (r � � . , p � . ), waist circumference (r � � . , p � . ), ln homa index (r � � . , p � . ), ln leptin (r � � . , p � � . ), ln fasting insulin (r � � . , p � . ), adiponectin (r � . , p � . ), and hdl levels (r � . , p � . ) (fig. ; table ). the correlation coefficients were calculated by taking the square root of the percent reduction in variance in omentin when adding in the specified predictor variable in a model already containing age and sex. when the variance components analysis was ad- justed for bmi or waist circumference as well as sex, age, and family structure, only leptin retained a significant al- though reduced correlation with omentin levels (r � � . , p � . and r � � . , p � . , respectively) (table ). table demographic and clinical characteristics of the afds subjects divided into lean, overweight, and obese groups variable lean overweight obese n sex (men/women) / / / age (years) � � � bmi (kg/m ) . � . . � . * . � . * waist circumference (cm) . � . . � . * . � . * insulin (pmol/l) . � . . � . * . � . * glucose (mmol/l) . � . . � . * . � . homa index . � . . � . * . � . * triglycerides (mmol/l) . � . . � . * . � . * total cholesterol (mmol/l) . � . . � . . � . hdl cholesterol (mmol/l) . � . . � . * . � . * ldl cholesterol (mmol/l) . � . . � . . � . systolic blood pressure (mmhg) . � . . � . * . � . * diastolic blood pressure (mmhg) . � . . � . * . � . * adiponectin (�g/ml) . � . . � . * . � . * leptin (ng/ml) . � . . � . * . � . * omentin (�g/ml) . � . . � . * . � . * data are means � sd. *p � . ( adjusted for family structure). c.m. de souza batista and associates diabetes, vol. , june omentin gene expression correlates negatively with bmi. omentin and omentin mrna levels (copy numbers) were measured in visceral fat from surgical subjects by real-time quantitative rt-pcr using standard curves gener- ated with omentin – containing ( ) and omentin – contain- ing (pcdna ) plasmids. using spearman regression analysis adjusted for sex and age, significant negative correlations between bmi and omentin (n � , rs � � . , p � . ) and omentin mrna levels (n � , rs � � . , p � . ) were found (fig. a and b). additionally, when comparing omentin and omentin gene expression, a highly significant positive correlation was observed (n � , rs � . , p � . ; fig. c). although omentin and gene expressions are highly correlated in visceral adipose tissue, the mrna level of omentin is � times greater than omentin . in light of the small number of men ( of ), the same analysis adjusted for age was performed only with women and revealed essentially the same results. both omentin and gene expressions correlated negatively with bmi (n � , rs � � . , p � . and n � , rs � � . , p � . , respectively). in addition, omentin and correlate posi- tively with each other (n � , rs � . , p � . ). omentin gene expression correlates positively with omentin plasma levels. to determine whether a linear relationship exists between omentin gene expression and plasma omentin levels, visceral adipose gene expression of omentin was compared with plasma omentin levels from a subgroup of the surgical subjects. omentin mrna levels were positively correlated with omentin plasma levels (n � , rs � . , p � . ; fig. ), thus illustrating a positive relationship between omentin gene regulation and omentin levels in the circulation. discussion visceral obesity is highly associated with risks for type diabetes, cardiovascular disease, hypertension, and hyper- lipidemia ( – ). expression of adipose-derived factors such as leptin ( ), plasminogen activator inhibitor ( ), and adiponectin ( , ) are modulated by obesity. there- fore, our study of circulating omentin levels and omentin gene expression was undertaken to better understand the regulation and role of omentin, a new visceral fat depot- specific adipokine, in obesity. two-dimensional gel electrophoresis followed by western blotting was conducted to determine which omentin iso- forms or other cross-reactive species contributed to the immunoreactivity in the one-dimensional western assay that was subsequently used to quantitate plasma omentin levels. based on the cross-reactivity of the g b antibody and the differing pis between omentin and , we demonstrated that omentin is the major circulating form in plasma. other apparent cross-reactive species were not observed in the same molecular weight range of the omentins, thus validating the specificity of the monoclonal antibody g b for omen- tin in human plasma by two-dimensional gel electrophoresis and its utility in the quantitative western blotting assay. as previously reported ( ), visceral fat is the tissue exhibiting the highest level of omentin expression. however, compared with omentin , omentin is ex- pressed in considerably lower levels in visceral fat and in fig. . circulating omentin levels are decreased with obesity. data are means � se. lean (n � ), overweight (ovw, n � ), and obese (ob, n � ) values for women were . � . , . � . , and . � . , respectively. lean (n � ), overweight (ovw, n � ), and obese (ob, n � ) values for men were . � . , . � . and . � . , respectively. p values for the sex-stratified analysis between the three groups in men and women (*p � . and **p � . , respec- tively) were determined by variance components analysis adjusted for age, sex, and family structure. plasma omentin levels were higher in lean women than in lean men (***p � . ). table correlations of metabolic syndrome–related traits with plasma omentin levels in amish subjects covariate age and sex age, sex, and bmi age, sex, and waist r p r p r p bmi (kg/m ) � . . — — � . . adiponectin (�g/ml) . . . . . . ln leptin (ng/ml) � . � . � . . � . . ln homa index � . . � . . � . . hdl cholesterol (mmol/l) . . . . . . waist circumference (cm) � . . � . . — — ldl cholesterol (mmol/l) � . . � . . � . . ln triglycerides (mmol/l) � . . � . . � . . total cholesterol (mmol/l) � . . � . . . . ln insulin (�u/ml) � . . � . . � . . glucose (mg/dl) � . . . . . . systolic blood pressure (mmhg) � . . � . . � . . diastolic blood pressure (mmhg) � . . . . . . all analyses incorporate residual correlations between family members using variance components analysis as implemented in solar. ln, natural log transformed. decreased omentin levels and obesity diabetes, vol. , june higher levels in the intestine (data not shown). release of omentin into the intestinal lumen and the difference in visceral fat expression of the two isoforms may explain why omentin was not detected in human plasma. al- though omentin and omentin exhibit a different pattern of tissue expression, they may share some similar regula- tion by obesity as evidenced by the negative correlations between the gene expression of both isoforms and bmi and the positive correlation with each other in visceral adipose tissue. plasma omentin levels were measured in a well- characterized genetically homogeneous population of old order amish ( ). as expected for a visceral depot- specific adipocytokine that positively affects insulin sensi- tivity, higher plasma omentin levels were observed in lean versus obese and overweight subjects independent of age, sex, and family structure. further evidence to suggest that omentin may be involved in some aspect of insulin sensitivity is the negative correlation between omentin plasma levels and measurements of insulin resistance (homa). moreover, negative correlations between plasma omentin levels and bmi, waist circumference, and leptin values were observed. these data suggest that some aspect of obesity negatively regulates omentin expression and release into the circulation. it is interesting to note that this pattern of results for omentin is similar to adiponectin, an insulin sensitizer and cardio-protective adipokine ( – ). in fact, circulat- ing total plasma adiponectin levels were positively corre- lated with plasma omentin values in this study. the inverse relationship between obesity and both omentin and adiponectin may suggest similar regulation. it is also possible that regulation of omentin may be dependent on adiponectin or vice versa. visfatin, the proposed insulin- mimetic adipokine, also shows negative correlations be- tween plasma levels and bmi ( ). however, despite similar relationships with bmi for omentin , adiponectin, and visfatin, omentin solely exhibits visceral fat depot– specific expression. a difference in plasma omentin levels between lean women and men was observed. this difference was not highly significant probably because of the small sample size. therefore, future studies will be required to address fig. . plasma omentin levels correlations with obesity and insulin resistance markers. significant correlation coefficients based on variance components analysis adjusted for sex, age, and family structure were found between plasma omentin levels with bmi (n � , r � � . , p � . ) (a); waist circumference (n � , r � � . , p � . ) (b); ln homa index (n � , r � � . , p � . ) (c); and plasma adiponectin levels (n � , r � . , p � . ) (d). c.m. de souza batista and associates diabetes, vol. , june this apparently divergent regulation of omentin between men and women. when plasma omentin is adjusted for bmi or waist circumference in the covariate analysis, there is a loss of significance with most omentin-associated traits. plasma leptin was still significant after adjustment although re- duced in magnitude. these results reinforce the concept that plasma omentin levels are highly regulated by obesity and will lose significant associations with obesity- dependent variables when adjusted by bmi or waist cir- cumference. the modestly significant residual correlation between leptin and omentin independent of bmi or waist circumference may be indicative of a regulatory relation- ship between these two adipokines outside of the effects of adiposity. to extend the relationship between plasma omentin and obesity to omentin gene expression, omentin and mrna levels were measured in visceral adipose tissue from surgical subjects. similar to plasma omentin levels, visceral adipose omentin gene expression for both iso- forms, omentin and omentin , is lower in the obese state than in the lean state, as evidenced by negative correla- tions between gene expression and bmi. in fact, mrna levels for omentin and are highly correlated with each other in omental adipose tissue. in contrast with omentin and adiponectin ( ), which show obesity-dependent de- creases in gene expression, visfatin was previously shown to have obesity-dependent increases in visceral fat gene expression ( , ). correlation between omentin mrna and plasma omentin levels was also observed, suggesting that the regulation of omentin gene expression in vis- ceral fat can predict the circulating levels of omentin . although the data clearly support regulation of omentin by obesity, omentin may also be regulated by inflamma- tion. other studies have shown that omentin (also known as intelectin ) expression is altered in inflamma- tory states ( , ). obesity itself is associated with low levels of chronic inflammation, which may contribute to the regulation in the role of omentin in human physiology ( – ). consequently, weight loss and different inflam- matory states could be modulators of omentin expression and function. in summary, we find that plasma omentin and both omentin and mrnas are inversely related to obesity. women have higher levels of circulating omentin and a larger range of variation with bmi. plasma omentin correlates negatively with bmi, leptin, waist circumfer- ence, fasting insulin, and homa and positively with adiponectin and hdl. association with these metabolic indexes suggests that higher omentin levels may be seen as a marker for leanness or as a positive factor that opposes the obese state and its pathophysiological consequences. acknowledgments this study has received national institutes of health grants dk- , ag- , gm- , and p -dk- and support from the veterans administration, geriatric research, education, and clinical center. we thank peter gutierrez (university of maryland greenebaum cancer center) and julie ray (university of fig. . visceral adipose omentin and gene expression levels are decreased with bmi. visceral adipose omentin (a) and omentin (b) mrna copies–to– s rna ratio, measured by quantitative rt-pcr, were negatively correlated with bmi (n � , r s � � . , p � . ; n � , r s � � . , p � . , respectively; spearman correlations ad- justed for sex and age). c: there was a significant positive correlation between omentin and gene expression levels (n � , r s � . , p � . ; spearman correlation adjusted for sex and age). fig. . omentin gene expression was positively correlated with human plasma omentin concentrations. visceral omentin mrna copies–to– s rna ratio, measured by quantitative rt-pcr, was pos- itively correlated with human plasma omentin levels (n � , r s � . , p � . ; spearman correlation). decreased omentin levels and obesity diabetes, vol. , june maryland school of pharmacy) for the mass spectrometry analysis. references . sharma am: adipose tissue: a mediator of cardiovascular disease. int j obes (suppl. ):s –s , . felber jp, golay a: pathways from obesity to diabetes. int j obes (suppl. ):s –s , . kershaw ee, flier js: adipose tissue as an endocrine organ. j clin endocrinol metab : – , . galuska da, khan lk: obesity: a public health perspective. in present knowledge in nutrition. th ed. 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perspectives. the third section criticizes three normative (what is named ‘‘self- actional’’) theories of altruism, viz., the kantian, the socialization argument, and ‘‘warm glow’’ story. the fourth section elaborates on three implications of altruism qua charity. first, while altruism differs from self-interest, it is still within the domain of rational theory. second, altruism should not be confused with parental care or, what is the same thing, philanthropy. third, altruism should be distinguished from honesty. � elsevier b.v. all rights reserved. psycinfo classification: ; ; jel classification: d keywords: three rationalistic/interactional theories; three normative/self-actional theories; three implications (sentimental foolishness, parental care, honesty) q a longer version has benefited from the comments of gary becker, ulrich witt, robert goldfarb, mark wilhelm, timothy crippen, john davis, thomas nitsch, roger masters, (especially) robert frank, three anonymous referees, and participants of seminars at the university of freiburg, the university of chicago, and the american economic association meeting. this work was made possible by a research fellowship from the alexander von humboldt foundation (germany). the usual disclaimer applies. e-mail address: elk@aier.org (e.l. khalil). url: http://www.brc-aier.org - /$ - see front matter � elsevier b.v. all rights reserved. doi: . /s - ( ) - journal of economic psychology ( ) – www.elsevier.com/locate/joep mail to: elk@aier.org http://www.brc-aier.org . introduction on august , , an amish family of members was traveling on a horse- drawn buggy on a north-central ohio road. a pickup truck, driven by an intoxicated driver, slammed into the back of the buggy, killing six of the instantly. the rest – all children aged, , , , and – were left parentless. the amish family did not carry insurance, and they do not believe in lawsuits. a local bank in mansfield, ohio, set up an account on their behalf that was announced in the mansfield news journal, the local newspaper. the newspaper reported that as of october , , the account received many donations, totaling $ , . the act of charity by anonymous donors can, but with some difficulty, find an ac- commodation in the homo economicus house of neoclassical economists. the diffi- culty originates from the fact that the tools of neoclassical economics have not been originally developed to account for anonymous donation. this failing can be overlooked if it is not for the fact that anonymous donation is not a rare event. the donation of blood, for instance, is a common practice in many parts of the world – despite the fact that the donation receives little public fanfare and little pe- cuniary reward. interestingly, some authors have shown theoretically (e.g., stewart, ) and empirically (titmuss, ) that the supply of blood would decline if it were sold via the market. the phenomenon of altruism is generally a considerable component of any society. the phenomenon started to attract the attention of neo- classical economists only recently (e.g., becker, ; bergstrom, ). altruism can be witnessed at small-scale ‘‘gift’’ sharing in small villages in the developing world to institutionalized charity organizations, associated with community groups, in mod- ern societies. the paper does not provide a theory of altruism. it only exposes the failings of existing theories, heightened by the view of altruism qua charity – as illustrated in the amish case. the paper reviews the limitations of existing theories given some of the ramifications, discussed in the paper, of the view of altruism qua charity: . insofar as charity is motivated by the concern over the welfare of the recipient, the benefactor should end up with a lower pecuniary benefit. . on the other hand, if the donor is solely motivated by the concern for the welfare of the other, how to distinguish charity from sentimental foolishness? a relatively poor person who gives most of his income to charity would be judged as a senti- mental fool. that is, one needs a rational choice model to analyze altruism. so, contrary to some positions (e.g., helms & keilany, ), altruism does not pose a serious anomaly to the neoclassical approach. . if altruism is about charity, altruism cannot be the defining element of the parent– child transfer of wealth. otherwise, it would be more efficient – as measured by the amount of welfare per dollar – to support the homeless instead of raising chil- dren. . one should not model altruism as about honesty (the origin of justice). when one pays his debts or discloses the defects of his products, one is not necessarily acting out of altruism qua charity. any behavior stemming from the concern over fair- e.l. khalil / journal of economic psychology ( ) – https://isiarticles.com/article/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ diabetes care, volume , number , january o b s e rvat i o n s diabetes scre e n i n g practices among i n d i v i d u a l s a g e d ye a r s a nd o l d e r i n , the american diabetes associa- tion (ada) adopted new re c o m m e n d a- tions for screening the general popula- tion aged years for diabetes every years with an emphasis on those at high risk for undiagnosed diabetes ( ). few studies have examined the extent to which this screening has been adopted. this re p o rt describes the results of a telephone s u rvey of montana residents aged years to assess the diabetes screening prac- tices in this population. f rom october to december , the montana department of public health and human services conducted a random household telephone survey of montana residents aged years living in counties. respondents indicated whether they ever had been told by a physician that they had diabetes, the number of visits they made to a health care provider during the past year, their family history of dia- betes, whether they had ever been told they had high cholesterol and/or high blood pre s s u re, and their height and weight. respondents were asked the fol- lowing question to identify whether they had ever been screened for diabetes: “glu- cose or sugar is a substance found in your blood. have you ever had your blood glu- cose or sugar checked to see if you have diabetes?” when respondents re s p o n d e d “yes” to this question, they were asked to identify when screening was completed (“when was the last time your blood glu- cose or sugar level was measured by a health care professional?”). the re s p o n s e categories for this question included within the past year, within the past years, years ago, do not know/not sure, and refused to answer. pearson tests were used to assess associations between dia- betes screening and risk factors for dia- betes. logistical re g ression analyses were conducted to identify independent vari- ables associated with screening for diabetes during the past year. odds ratios ( % cis) were calculated. of the , respondents, ( . %) re p o rted that they had diagnosed diabetes. the remaining , respondents re p o rt e d that they did not have diagnosed diabetes and are included in the following analyses. of the respondents, % re p o rted a family h i s t o ry of diabetes, % re p o rted a bmi kg/m , % re p o rted having hyper- tension, and % re p o rted having high c h o l e s t e rol. excluding age, % of re s p o n- dents had one risk factor for diabetes, and % had two or more risk factors. of the , respondents without diagnosed dia- betes, % re p o rted that they had been s c reened for diabetes during the past year, % re p o rted screening from to years ago, and % re p o rted screening years ago or having never been scre e n e d . respondents who re p o rted being s c reened for diabetes during the past year w e re more likely to be age years and to have a family history of diabetes, two or m o re visits to a health care provider dur- ing the past year, hypertension, and high c h o l e s t e rol levels (table ). we found no association between recent screening and sex ( % men vs. % women), ameri- can indian ancestry ( % yes vs. % no), or bmi ( % kg/m vs. % kg/m ). respondents with three or m o re risk factors (e.g., aged years, american indian ancestry, family history of diabetes, hypertension, high choles- t e rol, or bmi kg/m ) were more likely to be screened for diabetes compared with respondents with only one risk factor ( vs. %, respectively). however, % of individuals with two risk factors for dia- betes and % of individuals with more than three risk factors had not been s c reened during the past years. based on logistical re g ression analysis, t h ree factors were associated with scre e n i n g for diabetes during the past year: two or m o re visits to a health care provider during the past year ( . [ % ci . – . ]), high cholesterol level ( . [ . – . ]), and family history of diabetes ( . [ . – . ]). respondents aged – years were less likely to re p o rt re c e n t s c reening than those aged years ( . [ . – . ] ) . a limitation of this assessment is that these data are self-re p o rted. pre v i o u s studies, however, have found that self- re p o rts of conditions such as diabetes and h y p e rtension are reliable ( , ). in addi- tion, the survey was conducted by tele- phone and does not re flect the experience of individuals in montana homes without t e l e p h o n e s . the findings suggest that diabetes s c reening is being adopted by physicians for individuals aged years at risk for diabetes and that the ada re c o m m e n d a- tions are being implemented in the general c o m m u n i t y. however, these data also indi- cate a need to develop strategies to encour- l e t t e r s table —characteristics of respondents aged years reporting screening for diabetes in montana in s c reening for diabetes past year – years years or never n age (years) – ( ) ( ) ( ) – ( ) ( ) ( ) ( )* ( ) ( ) family history of diabetes ye s ( )* ( ) ( ) n o ( ) ( ) ( ) visits to a health care provider during the past year ( )* ( ) ( ) ( ) ( ) ( ) h y p e rt e n s i o n ye s ( )* ( ) ( ) n o ( ) ( ) ( ) high cholestero l ye s ( )* ( ) ( ) n o ( ) ( ) ( ) data are n (%). *p . . diabetes care, volume , number , january letters age screening among all individuals at high risk for diabetes. todd s. harwell, mph jane g. smilie, ba janet m. mcdowall, rn, bsn steven d. helgerson, md, mph dorothy gohdes, md f rom the montana diabetes project, montana d e p a rtment of public health and human serv i c e s , helena, montana. a d d ress correspondence to todd s. harw e l l , mph, montana department of public health and human services, cogswell building, c- , p. o . box , helena, mt - . e-mail: t h a rw e l l @ s t a t e . m t . u s . a c k n o w l e d g m e n t s — this project was sup- p o rted through a cooperative agreement (u- / ccu- - ) with the centers for disease c o n t rol and prevention, division of diabetes translation, atlanta, georg i a . we thank linda priest and the staff mem- bers at northwest resource consultants for their work on the telephone surv e y. the contents of this letter are solely the responsibility of the authors and do not neces- sarily re p resent the official views of the centers for disease control and pre v e n t i o n . r e f e re n c e s . american diabetes association: screening for type diabetes. diabetes care (suppl. ): s –s , . kehoe r, wu sy, leske mc, chylack lt jr: comparing self-re p o rted and physician- re p o rted medical history. am j epidemiol : – , . jackson c, jatulis de, fortmann sp: the behavioral risk factor survey and the stan- f o rd five-city project survey: a comparison of cardiovascular risk behavior estimates. am j public health : – , h b a c is not recommended as a s c reening test for diabetes in cystic fibro s i s i n the june issue of diabetes care, h u n k e rt et al. ( ) recommend the use of h b a c for early detection of cystic fib ro- s i s – related diabetes (cfrd). their re c o m- mendation is based on the finding that mean hba c is slightly higher in cystic fib rosis (cf) patients requiring insulin t h e r a p y, compared with cf patients with i m p a i red or normal glucose tolerance. h o w e v e r, no data on the validity of this a p p roach for the diagnosis of asympto- matic diabetes in patients with cystic fib ro- sis are pre s e n t e d . our group has a long-standing intere s t in the early diagnosis of cfrd, comparing fasting blood glucose levels with oral glu- cose tolerance test results ( ). in our series, we have now cf patients with newly diagnosed diabetes based on a -h venous plasma glucose value mg/dl ( . mmol/l), and simultaneous determ i n a t i o n of hba c ( h i g h - p e rf o rmance liquid chro- matography method [pharmacia, erlan- gen, germany], normal range . – . %). only out of cf patients ( %) diag- nosed as diabetic according to the ameri- can diabetes association and world health o rganization criteria ( ) had an hba c value above the normal range (individual values . , . , . , and . %). in nine diabetic cf patients with normal hba c, values between . and . % were e n c o u n t e red (mean ± sd, . ± . %). the mean -h blood glucose value after inges- tion of oral glucose was not signific a n t l y d i ff e rent between diabetic cf patients with n o rmal hba c ( ± mg/dl, mean ± sd) and diabetic cf patients with elevated h b a c ( ± mg/dl, student’s t t e s t ) . these data clearly demonstrate that the d e t e rmination of hba c is not able to sub- stitute for the oral glucose tolerance test in the early diagnosis of cfrd. our fin d i n g s a re in agreement with several re p o rts in the l i t e r a t u re ( – ) as well as the consen- sus conference on cfrd ( ). in addition to its low sensitivity when used as a diagnos- tic tool for the detection of cfrd, the mea- s u rement of hba c has the disadvantage of considerable interassay variability and lack of standardization. there f o re, we stro n g l y advise against the use of glycosylated hemoglobin as a screening test for the early diagnosis of diabetes in patients with cystic fib ro s i s . reinhard w. holl, md christian buck, md christine babka, md anna wolf, md angelika thon, md f rom the department of pediatrics (r.w.h.), uni- versity of giessen, giessen; the department of pedi- atrics (c.bu., c.ba., a.w.), the university of ulm, ulm; and the medical school hannover (a.t. ) , h a n n o v e r, germ a n y. a d d ress correspondence to pd dr. reinhard w. holl, universitätskinderklinik giessen, feulgenstr. , d- giessen, germ a n y. r e f e re n c e s . h u n k e rt f, lietz t, stach b, kiess w: potential impact of hba c d e t e rm i n a t i o n on clinical decision making in patients with cystic fib ro s i s – related diabetes (let- ter). diabetes care : – , . holl rw, buck c, cario h, wolf a, thon a, heinze e, kohne e, debatin k-m: diag- nosis of diabetes in cystic fib rosis and tha- lassemia major. diabetes care : – , . the expert committee on the diagnosis and classification of diabetes mellitus: r e p o rt of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care : – , . lanng s, hansen a, thorsteinsson b, n e rup j, koch c: glucose tolerance in patients with cystic fib rosis: five year p rospective study. b m j : – , . deluca f, arrigo t, nibali sc, sferlazzas c, gigante a, dicesare e, cucinotta d: insulin secretion, glycosylated haemoglo- bin and islet cell antibodies in cystic fib ro- sis children and adolescents with diff e re n t d e g rees of glucose tolerance. h o rm metab r e s : – , . moran a, doherty l, wang x, thomas w: a b n o rmal glucose metabolism in cystic fib rosis. j pediatr : – , . moran a: highlights of the febru a ry consensus conference on cfrd. bonn, g e rm a n y, cystic fibrosis foundation, p ro gln p e roxisome p ro l i f e r a t o r- a c t i v a t e d r e c e p t o r- and o b e s i t y r istow et al. ( ) re p o rted an activating mutation in the peroxisome pro l i f- e r a t o r-activated re c e p t o r- g e n e ( p ro gln ppa r - ), which was pre s e n t in % ( of ) of obese and % ( of ) of nonobese german caucasians. these findings may have profound implica- tions, particularly if the presence of this variant and its association with obesity are c o n firmed in other populations. we perf o rmed polymerase chain re a c- t i o n – restriction fragment length polymor- phism analysis for the pro gln ppa r - variant as described ( ) on dna samples f rom several independent populations, including lean and obese caucasians fro m the baltimore, maryland, region; african- diabetes care, volume , number , january letters americans from jackson, mississippi, and forsyth county, north carolina; pima indians from arizona; and old ord e r amish from lancaster county, pennsylva- nia (table ). a pcr fragment corre s p o n d- ing to gastric insulinotropic peptide, which has two known restriction sites for h i n dii, was mixed with each sample as a positive control. among a total of sub- jects ( , alleles), the pro gln variant was not detected in a single subject. these findings were unexpected because there is substantial overlap of gene pools of caucasians from central e u rope and the baltimore and amish caucasians studied ( ). the germ a n caucasians studied by ristow et al. were said to be unrelated and re c ruited fro m the nord rh e i n - westfalen region, but they may be a genetic isolate whose gene pool does not re flect that of other caucasian populations. altern a t i v e l y, if the individ- uals carrying the mutation were re l a t e d , the true frequency of the pro g l n p pa r - variant may have been overe s t i- mated. the absence or very low fre- quency of this variant has also been doc- umented in danish ( ) and german ( ) populations. this study is the first, to our knowledge, to examine american popu- lations for this variant. in summary, the study by ristow et al. demonstrating that the pro g l n p pa r - variant is activating and can influence body weight in people is important. however, this variant appears to be absent or very r a re in the american populations studied. additional studies are re q u i red in other regions of europe and the u.s. to furt h e r d e fine the relevance of this intere s t i n g genetic variant to susceptibility to obesity. alan r. shuldiner, md william nguyen, bs w.h. linda kao, phd brock a. beamer, md ross e. andersen, phd richard pratley, md frederick l. brancati, md, phd f rom the department of medicine (a.r.s., w. n . ) , university of maryland school of medicine; the d e p a rtments of medicine (b.a.b., f.l.b.) and epi- demiology (w.h.l.k.), johns hopkins university school of medicine, baltimore, maryland; and the national institute of diabetes and digestive and kidney diseases (r.p.), national institutes of health, phoenix, arizona. a d d ress correspondence to alan r. shuldiner, md, professor and head, division of endocrinol- o g y, diabetes, and nutrition, department of medi- cine, university of maryland school of medicine, w. lombard st., room s- , baltimore, md . e-mail:ashuldin@medicine.umary l a n d . e d u . a c k n o w l e d g m e n t s — this study was sup- p o rted by nih r dk- , the american diabetes association, glaxowellcome, the b a l t i m o re geriatrics research and education clinical center of the baltimore ve t e r a n s administration medical center. r e f e re n c e s . ristow m, muller- wieland d, pfeiffer a, k rone w, kahn cr: obesity associated with a mutation in a genetic regulator of adipocyte diff e rentiation. n engl j med : – , . c a v a l l i - s f o rza ll, menozzi p, piazza a: t h e h i s t o ry of human genes. princeton univer- sity press, princeton, nj, . elk j, urhammer sa, sorensen ti, ander- sen t, auwerx j, pedersen o: homozygosity of the pro ala variant of the pero x i s o m e p roliferation-activated re c e p t o r- g a m m a ( p par-gamma ): divergent modulating e ffects on body mass index in obese and lean caucasian men. diabetologia : – , . mamann a, munzberg h, buttron p, busing b, hinney a, mayer h, siegfried w, hebe- brand j, greten h: missense variants in the human peroxisome pro l i f e r a t o r- a c t i v a t e d re c e p t o r-gamma gene in lean and obese subjects. eur j endocrinol : – , insulin secre t i o n , insulin sensitivity, and glucose e ffectiveness in nonobese individuals with va ry i n g d e g rees of glucose to l e r a n c e a lthough it is well known that insulin s e c retion, insulin sensitivity, and glu- cose effectiveness are impaired in type diabetic patients ( – ), little is known about the role of each of these fac- tors individually on the evolution of type diabetes. in this context, a major issue is that hyperglycemia per se impairs insulin s e c retion and insulin sensitivity and that obesity observed in type diabetic patients per se causes insulin resistance ( , ). to o v e rcome this problem, we studied u n t reated nonobese subjects classified as having normal glucose tolerance (ngt) (n = ; bmi . ± . kg/m [ r a n g e . – . ], mean ± sem), impaired glu- cose tolerance (igt) (n = ; bmi . ± . kg/m [ . – . ]), and type dia- betes (n = ; fetal bovine serum . ± . mmol/l [range . – . ], bmi . ± . k g / m [ . – . ]), based on the criteria of the world health organization ( ). they were normotensive and had norm a l renal, hepatic, and thyroid function. insulin sensitivity and glucose eff e c t i v e n e s s w e re estimated by the minimal model a p p roach ( – ). insulin secretion was e x p ressed as the area under the insulin c u rve between and min after an intra- venous glucose injection ( ). after the data w e re analyzed by one-way analysis of vari- ance, bonferroni correction was used to evaluate the diff e rences between any two of the three groups we studied ( ). no signifi- cant difference was observed in bmi among the three groups. compared with the subjects with ngt, the subjects with table —characteristics of subjects screened for pro gln ppa r - n ( a l l e l e s age ± sd f e m a l e bmi ± sd p o p u l a t i o n t y p e d ) ( y e a r s ) ( % ) ( k g / m ) c a u c a s i a n s b a l t i m o re longitudinal ( ) . ± . . . ± . study on aging johns hopkins we i g h t ( ) . ± . . . ± . management center amish, lancaster, pa ( ) . ± . . . ± . a f r i c a n - a m e r i c a n s a t h e ro s c l e rosis risk in ( ) . ± . . . ± . communities study pima indians a r i z o n a ( ) . ± . — . ± . all non-amish subjects were unrelated; amish subjects were not fir s t - d e g ree relatives of each other. diabetes care, volume , number , january letters igt had significantly lower insulin secre- tion ( , ± vs. , ± pmol l m i n , p = . ) and glucose eff e c- tiveness ( . ± . vs. . ± . m i n , p . ). insulin sensitivity index was lower in subjects with igt ( . ± . m i n pmol l) than in those with ngt ( . ± . min pmol l ) , but was not statistically significant (p = . ). in con- trast, disposition index calculated by the p roduct of insulin secretion and insulin sensitivity was significantly lower in sub- jects with igt ( , ± ) than in those with ngt ( , ± , p = . ). on the other hand, patients with type dia- betes had significantly lower insulin secre- tion ( ± pmol l m i n , p = . ) compared with subjects with igt. although no significant diff e rence was observed in insulin sensitivity index between subjects with type diabetes and igt ( . ± . vs. . ± . min pmol l, p = . ), disposition index was s i g n i ficantly diminished in type diabetic patients as compared with subjects with igt ( ± vs. , ± , p = . ). glucose effectiveness in type diabetic patients ( . ± . min ) was similar to that in subjects with igt ( . ± . m i n , p = . ) but was signific a n t l y lower than that in the subjects with ngt (p . ). from these results, the fol- lowing may be hypothesized: ) impair- ments in insulin secretion and disposition index and decreased glucose eff e c t i v e n e s s , but not insulin resistance, seem to consti- tute the basic characteristics of patients with igt or type diabetes in nonobese japanese populations. ) risk factors wors- ening to type diabetes in subjects with igt are associated with further impair- ments in insulin secretion and disposition index, but not associated with furt h e r derangement in glucose effectiveness in japanese populations. ataru taniguchi, md mitsuo fukushima, md masahiko sakai, md itaru nagata, md kentaro doi, md shoichiro nagasaka, md kumpei tokuyama, md yoshikatsu nakai, md f rom the first department of internal medicine ( a . t., m.s., i.n.), kansai-denryoku hospital, and the department of internal medicine, hoshida- minami hospital (m.f.), osaka; the second depart- ment of internal medicine (k.d.) and the college of medical technology (y.n.), kyoto university, kyoto; the department of internal medicine (s.n.), jichi medical college, tochigi; and the laboratory of biochemistry of exercise and nutrition (k.t. ) , tsukuba university, tsukuba, japan. a d d ress correspondence to ataru ta n i g u c h i , md, first department of internal medicine, kansai- d e n ryoku hospital, - - , fukushima-ku, fuku- shima, osaka city, osaka - , japan. e-mail: k @ k e p c o . c o . j p . r e f e re n c e s . b e rgman rn: to w a rd physiological under- standing of glucose tolerance: minimal- model approach. d i a b e t e s : – , . welch s, gebhart ssp, bergman rn, phillips ls: minimal model analysis of intravenous glucose tolerance test-derived insulin sensitivity in diabetic subjects. j c l i n endocrinol metab : – , . taniguchi a, nakai y, fukushima m, kawamura h, imura h, nagata i, tokuyama k: pathogenic factors re s p o n s i- ble for glucose tolerance in patients with niddm. d i a b e t e s : – , . nagasaka s, tokuyama k, kusaka i, hayashi h, rokkaku k, nakamura t, kawakami a, higashiyama m, ishikawa s, saito t: endogenous glucose pro d u c t i o n and glucose effectiveness in type diabetic subjects derived from stable-labeled mini- mal model approach. d i a b e t e s : – , . best jd, kahn se, ader m, watanabe rm, ni t-c, bergman rn: role of glucose eff e c- tiveness in the determination of glucose tol- erance. diabetes care : – , . rossetti l, giaccari a, defronzo ra: glu- cose toxicity. diabetes care : – , . taniguchi a, nakai y, doi k, fukuzawa h, fukushima m, kawamura h, to k u y a m a k, suzuki m, fujitani j, tanaka h, nagata i: insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis. m e t a b o l i s m : – , . world health organization: diabetes melli - tus: report of a who study gro u p . g e n e v a , world health org., (tech. rep. ser. , no. ) . winer bj: statistical principles in experimen - tal design. nd ed. new york, mcgraw-hill, , p. – late-onset tro g l i t a z o n e - i n d u c e d hepatic dysfunction r e c e n t l y, iwase et al. ( ) re p o rted a case of liver dysfunction occurring after months of troglitazone therapy. because it was thought before this re p o rt that the risk of liver dysfunction with tro g l i- tazone after months was negligible, we wish to re p o rt another patient who took t roglitazone intermittently and developed hepatic dysfunction after months. a -year-old white man with type diabetes, ischemic heart disease (post angioplasty and stent placement), hyper- tension, degenerative joint disease, benign p rostatic hypert ro p h y, dyslipidemia, and g a s t roesophageal re flux disease had tro g l i- tazone mg daily added to his re g i m e n of glimeperide mg daily and metform i n mg b.i.d. because of poor glycemic c o n t rol (hba c . % [normal – %]). the other medicines he used were aspirin and pravastatin. after months of triple oral therapy, his hba c level dropped to . %, and, after months, to . %. after months, the patient’s hba c began to rise: . % at months, . % at year, and . % at months. liver function tests were normal until months, when his aspartate amino- transferase (ast) was found to be (nor- mal – u/l) and alanine aminotrans- ferase (alt) (normal – u/l). the t roglitazone regimen was discontinued, and testing for hepatitis b and c, h e m a c h romatosis, autoimmune liver dis- ease, and gallbladder disease were nega- tive. two months after discontinuing t roglitazone, the patient’s ast and alt had decreased to and u/l, and, after months, had re t u rned to normal at and u/l, re s p e c t i v e l y. his ast and alt have remained normal since then and he has continued to take pravastatin, aspirin, m e t f o rmin, and glimeperide. when the patient was told to discon- tinue troglitazone, he admitted that he had been taking it only interm i t t e n t l y. he esti- mated that he took the drug regularly at first, but after the first months, he took the drug only once or twice weekly on aver- age. he gave the following three reasons for his lack of compliance: a lack of funds, a fear of liver disease, and a tendency to avoid taking drugs whenever possible. this case, like the case described by iwase et al., illustrates that the hepatic dysfunction caused by troglitazone can occur after months and further sup- p o rts the u.s. food and drug administra- t i o n ’s current recommendation that quar- terly liver function tests should be obtained when troglitazone utilization extends beyond year ( ). in this case, could the onset of hepatic dysfunction have been delayed because the diabetes care, volume , number , january letters d rug was being taken only interm i t t e n t l y after the first months, and the estimated total load presented to the liver would be equivalent to the exposure at months in a compliant patient? we doubt this, since t ro g l i t a z o n e ’s hepatic effects are thought to be idiosyncratic and there f o re the total e x p o s u re should be irre l e v a n t . david s.h. bell, mb fernando ovalle, md f rom the division of endocrinology and metabo- lism, department of medicine, school of medicine, university of alabama, birmingham, alabama. a d d ress correspondence to david s.h. bell, mb, th ave. s., birmingham, al . d.s.h.b. and f.o. have served on an advisory panel for sankyo parke-davis and have received con- sulting fees, re s e a rch grant support, and honoraria for speaking engagements from sankyo parke-davis. r e f e re n c e s . iwase m, yamaguchi m, yoshinari m, oka- mura c, hirahashi t, tsuji h, fujishima m: a japanese case of liver dysfunction after months of troglitazone tre a t m e n t . diabetes care : – , . parke-davis, division of wa rn e r- l a m b e rt : rezulin package insert. morris plains, nj, g l y b u r i d e - i n d u c e d hemolysis in m y e l o d y s p l a s t i c s y n d ro m e g lyburide, also known as gliben- clamide, is a widely used sulfonylure a to treat patients with type diabetes. hemolytic anemia is an extremely rare side e ffect of which there have been only a few re p o rts ( – ). we describe a patient with myelodysplastic syndrome who pre s e n t e d with glyburide-induced hemolysis. a -year-old man with a long history of type diabetes presented with left foot cellulitis of week’s duration. this patient was known to have slowly pro g re s s i v e pancytopenia for years, for which no work-ups had been perf o rmed. his med- ications included the following: glyburide, mg per day, which he had taken for m o re than year; buformine, mg per day; and boglibose, . mg per day. he was afebrile, and the physical examination was normal, except for localized cellulitis on his left foot, for which he was start e d on intravenous antibiotics. the laboratory studies revealed a white blood cell count of . / l , hemoglobin . g/dl, platelet count /l, reticulocyte count . %, mean cor- puscle volume fl, moderate anisocyto- sis, fasting plasma glucose mg/dl, h b a c . %, lactate dehydrogenase iu/l, total bilirubin . mg/dl, and hapto- globin mg/dl. red cell glucose- - phosphate dehydrogenase level was ade- quate. cold agglutinin test, ham’s test, and sugar water test were normal. both dire c t and indirect coombs’ tests were negative. red cell resistance to osmolarity was mildly low (parpart ’s method). urinalysis demonstrated no urobilinogen. endo- scopic studies did not reveal gastro i n t e s t i- nal bleeding. ultrasonography of the abdomen showed no splenomegaly. the result of bone marrow aspiration was equivocal. on the basis of pre s u m p t i v e glyburide-induced hemolysis, glyburide was discontinued and the patient was switched to subcutaneous insulin on the seventh day. there a f t e r, his hemoglobin level increased to . g/dl, re t i c u l o c y t e count decreased to . %, and anisocytosis d i s a p p e a red pro m p t l y. he was discharg e d with insulin therapy after month in the hospital, which is when the cellulitis re s o l v e d . t h ree months later, his hemoglobin level was . g/dl and his haptoglobin remained low. repeated bone marro w aspiration confirmed the diagnosis of myelodysplastic syndro m e . we conclude that this patient devel- oped glyburide-induced hemolysis super- imposed on red cell fragility secondary to an underlying bone marrow disord e r. t h e re have been several re p o rts of hemoly- sis caused by sulfonylureas, most of which have been considered immune-mediated ( , , ). our case points to the possibility that glyburide could cause hemolysis by a non–immune-medicated mechanism. it is i m p o rtant to be aware of this potential side e ffect of glyburide in light of this medica- t i o n ’s widespread prescription, even though such a side effect is rare . hiroshi noto, md kazuhisa tsukamoto, md satoshi kimura, md f rom the department of diabetes and metabolism, tokyo university hospital, tokyo, japan. a d d ress correspondence to hiroshi noto, md, d e p a rtment of diabetes and metabolism, tokyo uni- versity hospital, - - hongo, bunkyo-ku, to k y o - , japan. e-mail: noto-tky@umin.ac.jp. r e f e re n c e s . nataas ob, nesthus i: immune haemolytic anaemia induced by glibenclamide in selective iga defic i e n c y. b m j : – , . abbate sl, hoogwerf bj: hemolytic ane- mia associated with sulfonylurea use. d i a - betes care : – , . meloni g, meloni t: glyburide-induced acute haemolysis in a g pd-defic i e n t patient with niddm. br j haematol : – , . kopicky ja, packman ch: the mechanism of sulfonylurea-induced immune hemoly- sis: case re p o rt and review of the literature . am j hematol : – , e ffects of exposure at an altitude of , m on p e rf o rmance of glucose meters s elf-monitoring of blood glucose is m a n d a t o ry for type diabetic p a t i e n t s who participate in sports to adjust insulin dose and carbohydrate ingestion ( ). sports also include activities p e rf o rm e d at moderately high altitudes, such as hiking or skiing. capillary blood glucose monitors (bgms) have been shown to underestimate blood glucose values at an altitude of , m ( ) and at a simulated altitude of , m with t e m p e r a t u re and humidity kept constant ( ). the aim of the present study was to assess the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and p o can result in errors in blood glucose d e t e rmination ( ). two bgms, the lifescan one touch ii (ot) (ortho diagnostics, milpitas, ca) and the glucometer elite ii (ge) (bayer diagnostics, brussels, belgium), were tested during a study on the effects of acute exposure at an altitude of , m and exercise on blood pre s s u re and albu- min excretion rate in six type diabetic p a t i e n t s . all subjects (four men and two women) were free of disease-related com- plications and in good and stable glycemic control (ghb . ± . %). all subjects gave their informed and written consent to participate in the study pro t o- col. all subjects were investigated both at diabetes care, volume , number , january letters sea level and after ascent by car and cable car to the angelo mosso institute at col d’olen ( , m altitude), gressoney la trinité, italy. at sea level and at a moderately high altitude, bgm reliability at diff e rent blood glucose levels was tested, and blood glu- cose was assessed in fasting and re s t i n g conditions at : a.m.; at : a.m. b e f o re an in-field exercise test; and imme- d i a t e l y, min, and min after the exer- cise stopped. capillary glucose was simul- taneously assessed with the ot and the ge. both of these bgms measure capillary blood glucose through the glucose oxi- d a s e - p e roxidase reaction. bgms were cali- brated at the beginning of each test ses- sion. a venous blood sample was simulta- neously drawn from the contralateral antecubital vein in a sodium fluoride tube, centrifuged, and stored at °c. plasma glucose was assayed with the glucose oxi- dase method (go) within days. this last assessment was taken as a re f e re n c e method. statistical analysis compare d bgm capillary glucose values and go plasma glucose values for each blood col- lection time. measurement linearity was tested with pearson’s correlation coeff i- cient. the mean of the diff e rences between the bgm and go results re p resents the mean bias between the methods with accuracy expressed as percent error (pe): pe (%) = bmg – go % g o the level of statistical significance was c o n s i d e red to be p . . the ge and ot measurements had a good correlation with plasma glucose both at moderately high altitude and at sea level. p e a r s o n ’s correlation coefficients were . and . for the ge and . and . for the ot at sea level and at moderately high altitude, re s p e c t i v e l y. biases between plasma glucose and bgm measure m e n t s w e re as follows: for the ge, . ± . at sea level and . ± . at moderately high altitude; for the ot, . ± . at sea level and . ± . at moderately high alti- tude. at sea level, both the ge and the ot tended to underestimate glucose values (ns); at moderately high altitude, the ge tended to overestimate and the ot tended to underestimate glucose values (ns). mean pes between plasma glucose and bgm m e a s u rements were . (ot) and . (ge) at sea level and . (ot) and . (ge) at moderately high altitude. pe tended to be higher for both bgms at moderately high altitude (ns). figures and show the bias between the single measurements with both devices at sea level and at moderately high altitude, re s p e c t i v e l y. at moderately high altitude (fig. ), the tendency of the ge to o v e restimate was more evident for low ( mg/dl) and intermediate ( – mg/dl) blood glucose values, whereas the ot tended to underestimate mainly high blood glucose values (ns). in our study, bgm perf o rmance was similar and good at sea level. at a moder- ately high altitude, a tendency to overe s t i- mate blood glucose for the ge and to u n d e restimate for the ot was observ e d . the overestimation for the ge involved mainly low ( mg/dl) and interm e d i- ate ( – mg/dl) blood glucose val- ues. this could present a problem in the p resence of symptoms suggesting hypo- figure —relationship between plasma and capillary glucose at sea level. figure —relationship between plasma and capillary glucose at moderately high altitude. lifescan one touch ii glucometer elite ii lifescan one touch ii glucometer elite ii diabetes care, volume , number , january letters glycemia and normal blood glucose val- ues. the ot tended to undere s t i m a t e mainly high blood glucose values, although its perf o rmance with low to i n t e rmediate values was good. the pre s- ent study assessed the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and po can result in errors in blood glu- cose determination ( ). our results are consistent with previous studies ( , ). the decrease in po could alter the sec- ond phase of the chromogen reaction and u n d e restimate blood glucose values ( ); on the other hand, an increase in atmos- pheric pre s s u re could overestimate blood glucose values ( ). in our study, minimal o v e restimation by the ge at low interm e- diate blood glucose values at moderately high altitude cannot be explained by the a l t e red po . an increase in hematocrit, which is known to alter blood glucose m e a s u rements with bgms ( ), may also occur after prolonged exposure to high altitude or as a consequence of dehydra- tion. although our study did not deter- mine hematocrit, the exercise test was s h o rt, and the patients were instructed to drink according to their thirst during the , -m exposure; there f o re, dehydration was not likely to have occurred. in con- clusion, bgm perf o rmance is similar and good at sea level. at a moderately high altitude similar to that experienced during winter skiing or summer hiking, a ten- dency to overestimate low to norm a l blood glucose values for the ge and to u n d e restimate high blood glucose values for the ot was observed. the bias is not clinically meaningful for either bgm, both of which can be safely used by dia- betic patients during exposure to moder- ately high altitudes. some care in the eval- uation of low and intermediate blood glu- cose values measured with the ge is n e v e rtheless re c o m m e n d e d . oriana pecchio, md simona maule, md marco migliardi, md marina trento, bsc massimo veglio, md f rom the italian alpine club medical commission ( o . p.); the department of internal medicine (m.t. ) , university of turin; the s. giovanni battista hospital (s.m.); and the department of endocrinology (m.m., m . v.), mauriziano umberto i hospital, turin, italy. a d d ress correspondence to dr. m. veglio, via mancini , torino, italy. e-mail: veglio@ o n w. n e t . r e f e re n c e s . h o rton es: role and management of exer- cise in diabetes mellitus. diabetes care : – , . g i o rdano bp, trash w, hollenbaugh l, dube wp: perf o rmance of seven blood glucose testing systems at high altitude. diabetes educ : – , . gautier jf, bigard ax, douce p, duvallet a, cathelinau g: influence of simulated alti- tude on the perf o rmance of five blood glu- cose meters. diabetes care : – , . b a rnett c, ryan f, ballonoff l: effect of altitude on the self monitoring of blood glucose (smbg) (abstract). diabetes (suppl.): a, . piepmeier eh, hammett-stabler c, price me, kemper gb, davis mg: atmospheric p re s s u re effects on glucose monitoring devices (letter). diabetes care : – , . b a rreau pb, buttery je: effect of hematocrit concentration on blood glucose value d e t e rmined on glucometer ii. diabetes care : – , c o m m e n t s a n d r e s p o n s e s deterioration of glycemic contro l after long-te rm treatment wi t h troglitazone in nonobese type diabetic patients t roglitazone is an oral antidiabetic d rug used to treat type diabetic patients with insulin re s i s t a n c e . troglitazone improves overall insulin sen- sitivity in the liver and skeletal muscles, which are the largest consumers and metabolizers of glucose in the body ( – ). recent re p o rts showed that troglitazone is also effective in nonobese type diabetic patients whose hyperglycemia could not be controlled with sulfonylurea therapy ( , ). however, we aware that in some patients in whom adequate glycemic con- t rol is obtained during the first several months of troglitazone treatment, their glycemic control deteriorates several months later. we assume that two distinct g roups of type diabetic patients exist who respond diff e rently to long-term administration of troglitazone, one gro u p that maintains a steady response and another group that has a decre a s i n g response after certain periods. in this s t u d y, we re t rospectively examined patients with type diabetes who were t reated with troglitazone for months and whose hba c levels had improved by % with troglitazone by month . in of the patients ( %), hba c levels increased by . % after – months despite continuous tro g l i t a z o n e t reatment (group p). in contrast, the rest of the patients experienced steady glycemic c o n t rol with . % of hba c flu c t u a t i o n ( g roup g). during the first months, h b a c levels decreased from means ± sem . ± . to . ± . % in group p and fro m . ± . to . ± . % in group g, re s p e c- t i v e l y. no significant diff e rences were evi- dent between the two groups re g a rding the d e c rease in hba c during the first months (fig. ). from month onward, hba c l e v- els in group p climbed gradually by . % a month up to the baseline level at month , but hba c levels were stable in group g t h roughout the treatment period. a signifi- cant diff e rence in hba c levels was evident during months – (p . ) . among clinical characteristics, gro u p p had a significantly lower bmi ( . ± . vs. . ± . kg/m , p . ) and s i g n i ficantly lower fasting insulin levels ( . ± . vs. . ± . µu/ml, p . ). of the patients with a bmi of kg/m ( %), exhibited deteriora- tion of glycemic contro l . in this study, we re p o rt a group of patients who showed a renewed decline in glycemic control after long-term tre a t m e n t with troglitazone. these results seemed to suggest a secondary failure of tro g l i t a z o n e . our study demonstrates that this drug is indeed useful for a long-standing obese i n s u l i n - resistant diabetes but not for a nonobese type diabetes. yuko murase, md takanobu wakasugi, md kunimasa yagi, md hiroshi mabuchi, md f rom the department of internal medicine (y. m . , t. w.), fukui perfectural hospital; and the second d e p a rtment of internal medicine (k.y., h.m.), kanazawa university, ishikawa, japan. a d d ress correspondence to yuko murase, md, the second department of internal medicine, kanazawa university, - takara-machi, kanazawa, ishikawa - , japan. e-mail: diabe@med. k a n a z a w a - u . a c . j p . diabetes care, volume , number , january letters r e f e re n c e s . suter sl, nolan jj, wallace p, gumbiner b, olefsky jm: metabolic effects of new oral hypoglycemic agent cs- in niddm subjects. diabetes care : – , . o’rourke cm, davis ja, saltiel ar, corn i- celli ja: metabolic effects of troglitazone in the goto-kakizaki rat, a non-obese and n o rmolipidemic rodent model of nonin- sulin-dependent diabetes mellitus. m e t a b - o l i s m : – , . troglitazone study group: the metabolic e ffects of troglitazone in non-insulin dependent diabetes (abstract). d i a b e t e s (suppl. ): a, . mori k: the effect of troglitazone in combi- nation with sulfonylurea in non-insulin dependent diabetes mellitus (abstract). j japan diabetes soc (suppl. ): , . h o rton es, venable tc, whitehouse f, the troglitazone study group, ghazzi mn, whitcomb rw: troglitazone in combina- tion with sulfonylurea re s t o res glycemic c o n t rol in patients with type diabetes. diabetes care : – , figure —change from baseline in hba c. values are means ± sem. jrmed genet ; : - short reports meiotic instability associated with the cagr trinucleotide repeat at q nicholas t potter abstract cagr is a recently characterised poly- morphic trinucleotide repeat localised to qi , which has been suggested as a pos- sible candidate gene for neurological dis- orders that manifest genetic anticipation. to provide evidence in support of this hypothesis, a large number of chromo- somes (n= ) from patients with a wide variety of neurological diseases were screened for evidence of repeat expansion and meiotic instability. one person with a cagr repeat number of was identi- fied (normal range - ). subsequent molecular analyses of cagr repeat number in additional family members showed meiotic instability of a (cag) allele through three generations. while cagri repeat number did not correlate with a readily discernible phenotype in this family, the finding of meiotic stability and mendelian inheritance of normal cag alleles and meiotic instability of larger repeats fulfil several criteria thought essential for pathologically rel- evant mutations of this type. thus, these data strengthen the hypothesis for a role of cagr in the development of an as yet molecularly uncharacterised human neurological disease. (i med genet ; : - ) keywords: cagri; trinucleotide repeat; meiotic insta- bility developmental and genetic center, the university of tennessee medical center, alcoa highway, knoxville, tn , usa n t potter received october revised version accepted for publication december the expansion of reiterated cag sequences is associated with phenotypic expression in at least six inherited neurological disorders' and mutations of this type are suspected candidates for several other neuropsychiatric and neuro- degenerative disorders which manifest genetic anticipation. ' efforts to identify and charac- terise candidate genes containing trinucleotide repeats have generally relied upon the screen- ing ofhuman brain cdna libraries and human expressed sequence tags (ests) and the use of repeat expansion detection (red) or fish for the identification of abnormally long or highly polymorphic sequences, -' and the causative mutation in at least one neurodegenerative dis- ease, dentatorubral-pallidoluysian atrophy, was subsequently identified using one of these approaches. recently margolis et al,' screening a human retinal cdna library, iden- tified a highly polymorphic cag repeat (called cagri) contained within the ' untranslated region of a gene that shares sequence homology with the caenorhabditis elegans cell fate deter- mining protein mab- . the highly polymor- phic nature of this repeat (normal repeat range from - copies), its high observed heterozy- gote frequency, its predominant expression in brain (cerebellum), and their subsequent iden- tification of a single allele with repeats in a person with an idiopathic movement disorder and an affective disorder provide compelling arguments for consideration of this locus as a candidate gene for molecularly uncharacter- ised inherited neurodegenerative, neuropsychi- atric, or neurodevelopmental disorders. whereas previous studies have elucidated the polymorphic nature of this repeat,' ' there have not been any reports on the identification of meiotically unstable cagr alleles, an essential feature for any trinucleotide repeat locus presumed to be involved in the aetiology of disorders of this type. r oo cn a) in a, a) co cagr repeat number figure distribution of cagri repeat length from the alleles. (cagri), represented the most common allele ( % of all alleles tested) followed by (cagri), ( % of alleles). excluding the one patient with the (cagri),s there were no differences in allele distribution between the nine different patient groups. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n m a y . d o w n lo a d e d fro m http://jmg.bmj.com/ potter ;:. ..f i..... wif figure six percent sequencing gel analysis showing meiotic stability an inheritance ofcagr repeats in the normal range through meioses in t pedigree . pcr primers for cagr and conditions for analysis were es described in margolis et all. with the exception that p-dctp was incorpo) reaction and the annealing temperature for cagjm- '/cag m- ' ( pi reaction) was lowered to °c. similar analysis of the segregation of a stab through five meioses was also performed on dna samples obtainedfrom ft (data not shown). allele sizes were as follows: ( / ); l ( );il. ( );iii. ( );iii. ( );iii. ( );iii. ( );iii. ( );iii. ( );iii. ( );iii. ( );iii. ( );iii ( ); l ( );l. ( ). dna samples from the ceph/amish, family de were obtainedfrom the nigms human genetic mutant coriell institute for medical research, camden, nj . a figure meiotic instability of a cag than through three meioses. dna w. peripheral blood lymphocytes. allele size ( ); ii. ( ); iii. i ( ). a were as described in fig . in an effort to identify such alleles, a total of chromosomes were studied for the deter- mination of cagri repeat number. this included people with inherited neurodegenera- tive disorders of unknown aetiology who tested negative for cag expansions at the hd, drpla, sca- , sca- , and sca- loci (n= ), patients with parkinson's disease (n= ), progressive supranuclear palsy (n= ), multiple system atrophy (n= ), nigrostriatal degeneration (n= ), multiple sclerosis (n= ), alzheimer's disease (n= ), molecularly nega- * tive fragile xa patients with developmental delay/mental retardation (n= ), and normal controls (n= ). the distribution of normal cag repeat size in this cohort was - (fig ), with a modal repeat length of and heterozy- gosity of %. one person from the fragile xa negative group was found to have an allele with cagr repeats (fig , iii. ). as this allele was almost twice the size of the largest normal allele found in this cohort, additional dna samples were obtained from other family members to examine for evidence of meiotic instability. in contrast to the stable inheritance of cag repeat lengths in the normal range (fig !d mendelian ), this family clearly showed meiotic instability the ceph/amish for cagr alleles of or larger with a acag ssentially as of at least five repeats observed through three)rated into the mol of each/ ild generations (fig ). ble (gcag) repeat although the index case in this family had a tinily de : ( ); iii. phenotype of idiopathic mental retardation ( ); iii. with an emergent attention deficit/ f. ( ); h. hyperactivity disorder, there are several reasons pedigree andpcell repository why it may be premature to presume his phenotype is related to the presence of the (cagr ) allele. first, and most significant, is the lack of any readily discernible phenotype in his grandmother, mother, or aunt despite the presence of similarly sized cagri alleles. sec- ond, the localisation of the repeat in an untranslated region of the gene, the small increase in cag length over three generations in a manner consistent with the "slippage mediated model" for repeat sequence instability,' and the finding of meiotically unstable alleles in the - range collectively suggest that the cagr expansion has mo- lecular characteristics reminiscent of the pre- mutations found in the type ii repeat expan- sion mutation diseases fragile xa and e, myotonic dystrophy, and jacobsen syndrome.' whether cagr alleles are prone to further expansion into a pathological size range is cur- rently unknown, although our finding of cagr locus instability and the recent identi- fication of a large ( . kb) expansion in a novel cag repeat found in the non-coding region of hc q - segregating in a family with schizophrenia' suggests that larger expansions are possible. in summary, our data provide evidence of meiotic stability for normal sized cagr alle- les and meiotic instability for larger alleles. collectively these data strengthen the ri repeat greater possibility that cagri expansion may be eas: olate(d )ii i involved in the development of an as yet ssay conditions molecularly uncharacterised human neurological disease. i! i, o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n m a y . d o w n lo a d e d fro m http://jmg.bmj.com/ meiotic instability associated with the cagri trinucleotide repeat at q i would like to thank richard greene, md and virginia frye, edd for their assistance with the evaluation of the index case and enid bowlin for her technical assistance with the fragile xa studies. i would also like to thank the other family members for their participation in this study. postmortem tissue samples were obtained from the harvard brain tissue resource center, bel- mont, ma which is supported by nih award ro -mh and the national neurological research specimen bank, vamc, los angeles, ca , which is sponsored by ninds/nimh, national multiple sclerosis society, hereditary disease foundation, comprehensive epi- lepsy program, tourette syndrome association, dystonia medical research foundation, and veterans health services and research administration, department of veterans affairs. this study was supported in part by grants from the physicians' medical and education research foundation (pmerf), knoxville, tn and the state of tennessee. zoghbi hy the expanding world of ataxins. nat genet ; : - . ross ca, mcinnis mg, margolis rl, li sh. genes with triplet repeats: candidate mediators of neuropsychiatric disorders. trends neurosci ; : - . o'donovan mc, guy c, craddock n, et al. expanded cag repeats in schizophrenia and bipolar disorder. nat genet ; : - . junck l, fink jk. machado-joseph disease and sca : the genotype meets the phenotypes. neurology ; : - . riggins gj, lokey lk, chastain jl, et al. human genes containing polymorphic trinucleotide repeats. nat genet ; : - . li sh, mcinnis mg, margolis rl, antonarakis se, ross ca. novel triplet repeat containing genes in human brain: cloning, expression, and length polymorphisms. genomics ; : - . jiang jx, deprez rhl, zwarthoff ec, riegman phj. char- acterization of four novel cag repeat-containing clones. genomics ; : - . adams md, kervalage ar, fleishmann rd, et al. initial assessment of human gene diversity and expression patterns based upon million nucleotides of cdna sequence. nature ; : - . schalling m, hudson tj, buetow kh, houseman de. direct detection of novel expanded trinucleotide repeats in the human genome. nat genet ; : - . haaf t, sirugo g, kidd kk, ward dc. chromosomal localization of long trinucleotide repeats in the human genome by fluorescence in situ hybridization. nat genet ; : - . koide r, ikeuchi t, onodera , et al. unstable expansion of cag repeat in hereditary dentatorubral-pallidoluysian atrophy (drpla). nat genet ; : - . nagafuchi s, yanagisawa h, sato k, et al. dentatorubral and pallidoluysian atrophy expansion of an unstable cag trinucleotide on chromosome p. nat genet ; : - . margolis rl, stine oc, mcinnis mg, et al. cdna cloning of a human homologue of the caenorhabditis elegans cell fate-determining gene mab- : expression, chromosomal location and analysis of a highly polymorphic (cag)n tri- nucleotide repeat. hum mol genet ; : - . neri c, albanese v, lebre as, et al. survey of cag/ctg repeats in human cdnas representing new genes: candidates for inherited neurological disorders. hum mol genet ; : - . richards ri, sutherland ga. simple repeat dna is not replicated simply. nat genet ; : - . warren st the expanding world of trinucleotide repeats. science ; : - . breschel ts, sirugo g, pleasant n, et al. a novel, heritable, expanding cag repeat in a non-coding region of hc q - in ceph and bipolar pedigrees. am j hum genet ; :a . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n m a y . d o w n lo a d e d fro m http://jmg.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ generation of d surface models from scanning electron microscope images timothy amish , bryan t. hansen , and elizabeth r. fischer electron microscopy unit, research technologies branch, rocky mountain laboratories, national institute of allergy and infectious diseases, nih hamilton, mt the study of host-pathogen interactions begins with the attachment of a bacteria or virus to a host cell. studying this initial interaction which often involves significant cytoskeletal rearrangements and membrane changes can provide clues for entry and egress mechanisms. scanning electron microscopy (sem) provides a widely used tool to qualitatively determine surface changes that occur but is limited quantitatively as the resulting image is still a two- dimensional output. optimizing an image capture strategy and utilizing autodesk’s memento software, allowed -dimensional topographic reconstructions from sem images. [ref. ] new software is becoming increasingly sophisticated and capable of accurate qualitative and quantitative analyses. autodesk has created programs that utilize images from multiple orientations to create a d model for a variety of applications: d printing, medical, industrial, and media. a user inputs a grouping of images at different tilts and rotations around an object. the autodesk programs are able to pick out similar points in the images and compare them which results in correlating the location of photos relative to each other in d space. however, these d to d programs have inherent limitations. the programs cannot reliably generate volumes from objects with little surface texture or symmetry as it may misplace points during correlation. the more unique and complex features are the more likely they will stitch correctly. these programs were primarily designed to collect images by physically moving a camera around a fixed point. by contrast we wanted to develop a data collection scheme in an sem where the camera position is fixed and the specimen is either physically or raster rotated, tilted or combinations thereof relative to the fixed secondary electron detector. many trials were performed with variables that included specimen variability, magnification, and number of images, capture settings, tilts and rotations to optimize the resulting models. multiple tilts with little rotation or fixed tilts with only physical rotation showed little promise with many large gaps and distortions within the model using autodesk’s d catch program. (fig. , ref. ) in contrast, the best results were achieved by rotation around a fixed angle by physically tilting the sample to plus degrees relative to the detector. (fig a & b) moving the stage in a positive direction in degree increments and countering with a negative degree raster rotation through degrees around the sample gave the most complete model generation using autodesk’s memento software. (fig ) once the positions of the images were determined by cross-correlation, points were arranged and projected into d space. the created points were then used to produce thousands of interconnected triangles which provides the model with depth and contours. the volumes can be visualized with a variety of surface textures. (fig a-c) the resulting d models can be useful for quantitative analysis of surface structures. complex measurements can be made with ease. a qualitative understanding of specimens can be observed from a computed environment or by d printing the resulting d model. typically it takes a day to generate images and create a model without expensive software or hardware, making the model generation easily accessible with a standard sem. doi: . /s microsc. microanal. (suppl ), © microscopy society of america https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms microsc. microanal. (suppl ), https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s https://www.cambridge.org/core https://www.cambridge.org/core/terms 	abstract-ta 	amish-fischer-fig erratum to: what is new in cdg? erratum erratum to: what is new in cdg? jaak jaeken & romain péanne published online: june # ssiem erratum to: j inherit metab dis doi . /s - - - due to a typesetting error, part of table is missing in the pdf file. the online version of table is correct. the original article was corrected. the online version of the original article can be found under doi: . / s - - - * jaak jaeken jaak.jaeken@kuleuven.be center for metabolic diseases, university hospital gasthuisberg, ku leuven, herestraat , be leuven, belgium department of human genetics, university hospital gasthuisberg, ku leuven, leuven, belgium j inherit metab dis ( ) : – doi . /s - - - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf table list of all cdg reported up to date according to their cellular location and the different pathways. the asterisk (*) refers to recently reported cdg which are highlighted in this review. the cdg for which there is a causative treatment are highlighted in yellow table . defects localized in the cytosol name clinically affected organs and tissues defective protein tf-ief moi protein n-glycosylation disorders synthesis of monosaccharides gmppa-cdg autonomic nerve fibers of distal oesophagus (achalasia) and lacrimal glands (alacrimia), neurons (brain, hearing system, visual system) guanosine diphosphate mannose pyrophosphorylase a gmppb-cdg brain, skeletal muscles, eyes, heart guanosine diphosphate mannose pyrophosphorylase b mpi-cdg intestine, liver mannose -phosphate isomerase type pmm -cdg nervous system, fat tissue, and nearly all other organs phosphomannomutase type protein o-glycosylation disorders defects in o-n-acetylglucosaminylglycan synthesis eogt-cdg skin (aplasia cutis congenita), skeleton (terminal transverse limb defect) egf domain-specific o-glcnac transferase defects in multiple and other glycosylation pathways defects in dolichol synthesis dhdds-cdg* (retinitis pigmentosa ) retina dehydrodolichyl diphosphate type defects in monosaccharide synthesis cad-cdg* brain, blood cells carbamoyl-phosphate synthetase (cps ) and aspartate transcarbamylase (atcase) activities of the trifunctional enzyme cad gfpt -cdg (limb girdle congenital myasthenic syndrome) neuromuscular junction, skeletal muscles glutamine:fructose -phosphate amidotransferase gne-cdg (hereditary inclusion body myopathy) skeletal muscles (with sparing of quadriceps muscles), rarely cardiac muscles udp-glcnac -epimerase/man-nac kinase nans-cdg* brain, skeleton n-acetylneuraminic acid synthase pgm -cdg uvula (palate, lips), heart, liver, muscles, endocrine organs phosphoglucomutase type pgm -cdg* brain, immune system, skeleton phosphoglucomutase defects in nucleotide-sugar synthesis cps -cdg brain, intestine, kidneys, erythrocytes carbamylphosphate synthetase deficiency table . defects localized in the er name clinically affected organs and tissues defective protein tf-ief moi protein n-glycosylation disorders alg -cdg brain, and variable involvement of eyes, heart, liver, beta cells, kidneys, gonads mannosyltransferase type alg -cdg brain, eyes, skeletal muscles, neuromuscular junction (congenital myasthenic syndrome) mannosyltransferase type alg -cdg brain, skeleton mannosyltransferase type alg -cdg brain, and variable involvement of eyes, gastrointestinal system, liver, heart and skeleton glucosyltransferase type alg -cdg brain, and variable involvement of eyes, skin, liver and intestine glucosyltransferase type alg -cdg* brain, liver, kidneys, and variable involvement of adipose tissue, heart, skeleton, intestine mannosyltransferase / type alg -cdg brain, hearing system mannosyltransferase / type alg -cdg brain, skeleton, heart, genitalia and immune system mannosyltransferase type alg -cdg brain, eyes, liver udp-glcnac:dol-p-glcnac-p transferase type x-linked alg -cdg neuromuscular junction (congenital myasthenic syndrome) udp-glcnac:dol-pp-glcnac transferase type ddost-cdg brain, eyes, liver oligosaccharyltransferase subunit ddost type dpagt -cdg brain, neuromuscular junction (congenital myasthenic syndrome) udp-glcnac: dol-p-glcnac-p type mogs-cdg brain, skeleton, immune system mannosyl-oligosaccharide glycosidase (glucosidase ) ganab-cdg* liver, kidneys (polycystic) glucosidase ii subunit α ad prkcsh-cdg liver, kidneys (polycystic) glucosidase ii subunit β ad j inherit metab dis ( ) : – rft -cdg brain, hearing system flippase of man glcnac -pp-dol type stt a-cdg brain, gastrointestinal tract oligosaccharyltransferase subunit stt a type stt b-cdg brain, optic nerve, gastrointestinal tract oligosaccharyltransferase subunit stt b type (mild) ssr -cdg brain, lungs, gastrointestinal system signal sequence receptor of trap complex type (disialo increase only) ssr -cdg* brain, respiratory system, skeleton signal sequence receptor of trap complex type (mild) x-linked tusc -cdg brain (non-syndromic autosomal recessive mental disability) oligosaccharyltransferase subunit tusc type protein o-glycosylation disorders defect in o-xylosyl/n-acetylgalactosaminylglycan synthesis slc d -cdg (schneckenbecken dysplasia) skeleton (generalized; radiographic snail-like configuration of iliac bones)( stillborn or lethal in the neonatal period) solute carrier family (udp-glucuronic acid/udp-n- acetylgalactosamine dual transporter) member d defect in o-glucosylglycan synthesis poglut -cdg* skin (progressive reticular hyper- and hypopigmentation) protein o-glucosyltransferase ad defects of lipid glycosylation and of glycosylphosphatidylinositol synthesis defects in glycosylphosphatidylinositol synthesis piga-cdg* brain, heart, liver, kidneys, skin udp-glcnac:phosphatidylinositol n-acetylglucosaminyltransferase subunit x- linked pigc-cdg* brain udp-glcnac:phosphatidylinositol n-acetylglucosaminyltransferase subunit pigg-cdg* brain glycosylphosphatidylinositol ethanolamine phosphate transferase pigl-cdg (chime syndrome) brain, eyes, hearing system, heart, skin glcnac-phosphatidylinositol de-acetylase pigm-cdg brain, hepatic veins dol-p-man:phosphatidylinositol mannosyltransferase pign-cdg brain, skeleton (including palate, fingers), cardiovascular system, kidneys glycosylphosphatidylinositol ethanolamine phosphate transferase pigo-cdg brain, lips, fingers, toes, anus/rectum, hearing system, cardiovascular system glycosylphosphatidylinositol ethanolaminephosphate transferase pigq-cdg brain udp-glcnac:phosphatidylinositol n-acetylglucosaminyltransferase subunit pigt-cdg brain, eyes, heart, kidneys, skeleton pigt transamidase subunit pigv-cdg brain, fingers, toes, and less frequent involvement of lips, palate, anus/rectum, hearing system dol-p-man:phosphatidylinositol mannosyltransferase pigw-cdg* brain, skeleton phosphatidylinositol acylase pigy-cdg* brain, fingers, toes udp-glcnac:phosphatidylinositol n-acetylglucosaminyltransferase subunit pgap -cdg brain phosphatidylinositol deacylase defects in multiple and other glycosylation pathways defects in dolichol synthesis dolk-cdg brain, heart, skin dolichol kinase type nus -cdg* brain, eyes (bilateral macular lesions), skeleton nogo-b receptor (subunit of cis-prenyltransferase) type srd a -cdg brain, eyes, heart, skin, joints steroid α-reductase type defects in dolichol utilization/recycling dpm -cdg brain, eyes, skeletal muscles gdp-man:dol-p-mannosyltransferase (dol-p-man synthase ) type dpm -cdg brain, skeletal muscles gdp-man:dol-p-mannosyltransferase (dol-p-man synthase ) type dpm -cdg skeletal and cardiac muscles gdp-man:dol-p-mannosyltransferase (dol-p-man synthase ) type mpdu -cdg brain, eyes, skin man-p-dol utilization type defects in the v-atpase complex atp ap -cdg* brain, b cells, liver (muscles, hearing system) accessory protein ac of the v-atpase type x-linked other defects trappc -cdg* muscles (limb girdle muscular dystrophy, type s) trafficking protein particle complex (trappiii), subunit na. j inherit metab dis ( ) : – table . defects localized in the golgi name clinically affected organs and tissues defective protein tf-ief moi protein n-glycosylation disorders man b -cdg brain, cranial skeleton, fat tissue golgi α - mannosidase type mgat -cdg brain, skeleton, intestine, immune system n-acetylglucosaminyltransferase type protein o-glycosylation disorders defect in o-xylosylglycan synthesis b galt -cdg brain, skeleton (short stature, bowing of extremities), articulations (hyperlaxity, dislocations), skin (premature aging phenotype) b- , -galactosyltransferase b galt -cdg skeleton (spondyloepimetaphyseal dysplasia with bone fragility, severe kyphoscoliose), joints, skin (fragility, delayed wound healing) Β- , -galactosyltransferase b gat -cdg brain, aorta, heart, skeleton, joints, skin, teeth b- , -glucuronyltransferase chsy -cdg (tentamy preaxial brachydactyly syndrome) brain, teeth, skeleton (particularly brachydactyly), hearing system chondroitin β- , -n-acetylgalactosaminyltransferase (chondroitin synthase ) ext -cdg (multiple cartilaginous exostoses) cartilage (osteochondromas of the ends of long bones) exostosin ad ext -cdg* (multiple cartilaginous exostoses) cartilage (osteochondromas of the ends of long bones) exostosin ad xylt -cdg* brain, skeleton (short stature, advanced bone age), articulations (joint laxity), fat xylosyltransferase xylt -cdg* brain, eyes, heart, hearing system, bones xylosyltransferase defect in o-n-acetylgalactosaminalglycan synthesis galnt -cdg (familial hyperphosphatemic tumoral calcinosis) subcutaneous tissue (painful calcified masses) udp-n-acetyl-α-d-galactosamine:polypeptide n- acetylgalactosaminyltransferase defects in o-fucosylglycan synthesis lfng-cdg (spondylocostal dysostosis type ) axial skeleton, associated muscles o-fucose-specific β- , -n-acetylglucosaminyltransferase pofut -cdg (dowling-degos disease ) skin (progressive reticular hyper- and hypopigmentation) protein o-fucosyltransferase ad defects of lipid glycosylation and of glycosylphosphatidylinositol synthesis defects in lipid glycosylation b galnt -cdg (spastic paraplegia , autosomal recessive) brain, peripheral nerves (spastic paraplegia), gonads b- , -n-acetylgalactosaminyltransferase (gm synthase) st gal -cdg (amish infantile epilepsy; salt and pepper syndrome) brain, hearing system, skin lactosylceramide α- , -sialyltransferase (gm synthase) defects in glycosylphosphatidylinositol synthesis pgap -cdg brain phosphatidylinositol glycerol acylase pgap -cdg* brain, skeleton phosphatidylinositol glycerol deacylase defects in multiple and other glycosylation pathways defects in glycosyltransferases b galt -cdg face (dysmorphism), eyes (myopia) b- , -galactosyltransferase type st gal -cdg brain b-galactoside α- , -sialyltransferase defects in nucleotide-sugar transporters slc a -cdg brain, heart, kidneys, platelets cmp-sialic acid transporter type slc a -cdg brain, eyes, gastrointestinal system, skeleton udp-galactose transporter type x-linked slc a -cdg brain, skeleton udp-glcnac transporter slc c -cdg brain, cranial skeleton, neutrophils gdp-fucose transporter defects in the cog complex cog -cdg brain, skeleton cog component type cog -cdg brain, liver cog component type cog -cdg brain, face cog component type j inherit metab dis ( ) : – the pdf file has been corrected. we apologize for this error. cog -cdg brain, hearing system, vision, liver, bladder cog component type cog -cdg brain, gastrointestinal system including liver, immune system cog component type cog -cdg brain, skeleton, skin, gastrointestinal system including liver, heart cog component type cog -cdg brain, eyes, peripheral nervous system cog component type defects in the v-atpase complex atp v a -cdg (autosomal recessive cutis laxa type ii; wrinkly skin syndrome) skin (cutis laxa becoming less obvious with age), brain (mental development mostly normal), eyes, neuromuscular system, skeleton v subunit a of v-atpase type other defects tmem -cdg brain, skeleton (particularly cartilage), joints, heart, liver, kidneys transmembrane protein type vps b-cdg* (cohen syndrome) brain, eyes (chorioretinal dystrophy with myopia), joints, immune system (neutropenia), fat tissue vacuolar protein sorting-associated protein b table . defects localized in the ergic name clinically affected organs and tissues defective protein tf-ief moi defects in multiple and other glycosylation pathways defects in copii sec b-cdg (congenital dyserythropoietic anemia type ii) red cell lineage (secondary involvement of heart, liver, beta cells) copii component sec b other defects ccdc -cdg* liver, spleen, brain coiled-coil domain containing type tmem -cdg* liver transmembrane protein type table . defects localized at the plasma membrane name clinically affected organs and tissues defective protein tf-ief moi defects in multiple and other glycosylation pathways slc a -cdg* brain, skeleton, immune system manganese and zinc transporter type table . defects localized at the sarcolemma membrane name clinically affected organs and tissues defective protein tf-ief moi defects in o-mannosylglycan synthesis b galnt -cdg brain, eyes, skeletal muscles b- , -n-acetylgalactosaminyltransferase fktn-cdg* brain, eyes, skeletal muscles ribitol- -phosphate transferase fkrp-cdg* brain, eyes, skeletal muscles ribitol- -phosphate transferase ispd-cdg* brain, eyes, skeletal muscles isoprenoid synthase domain-containing protein (cdp-ribitol synthase) large-cdg brain, eyes, skeletal muscles acetylglucosaminyltransferase-like protein pomgnt -cdg (muscle-eye-brain disease, isolated rp) brain, eyes, skeletal muscles protein o-mannose β- , -n- acetylglucosaminyltransferase pomt -cdg (cerebro-ocular dysplasia- muscular dystrophy syndrome) brain, eyes, skeletal muscles, heart protein o-mannosyltransferase pomt -cdg (cerebro-ocular dysplasia- muscular dystrophy syndrome) brain, eyes, skeletal muscles protein o-mannosyltransferase tmem -cdg* brain, eyes, skeletal muscles, gonades o-mannosylation β- , -xylosyltransferase j inherit metab dis ( ) : – 	erratum to: what is new in cdg? wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ diabetes care, volume , number , january o b s e rvat i o n s diabetes scre e n i n g practices among i n d i v i d u a l s a g e d ye a r s a nd o l d e r i n , the american diabetes associa- tion (ada) adopted new re c o m m e n d a- tions for screening the general popula- tion aged years for diabetes every years with an emphasis on those at high risk for undiagnosed diabetes ( ). few studies have examined the extent to which this screening has been adopted. this re p o rt describes the results of a telephone s u rvey of montana residents aged years to assess the diabetes screening prac- tices in this population. f rom october to december , the montana department of public health and human services conducted a random household telephone survey of montana residents aged years living in counties. respondents indicated whether they ever had been told by a physician that they had diabetes, the number of visits they made to a health care provider during the past year, their family history of dia- betes, whether they had ever been told they had high cholesterol and/or high blood pre s s u re, and their height and weight. respondents were asked the fol- lowing question to identify whether they had ever been screened for diabetes: “glu- cose or sugar is a substance found in your blood. have you ever had your blood glu- cose or sugar checked to see if you have diabetes?” when respondents re s p o n d e d “yes” to this question, they were asked to identify when screening was completed (“when was the last time your blood glu- cose or sugar level was measured by a health care professional?”). the re s p o n s e categories for this question included within the past year, within the past years, years ago, do not know/not sure, and refused to answer. pearson tests were used to assess associations between dia- betes screening and risk factors for dia- betes. logistical re g ression analyses were conducted to identify independent vari- ables associated with screening for diabetes during the past year. odds ratios ( % cis) were calculated. of the , respondents, ( . %) re p o rted that they had diagnosed diabetes. the remaining , respondents re p o rt e d that they did not have diagnosed diabetes and are included in the following analyses. of the respondents, % re p o rted a family h i s t o ry of diabetes, % re p o rted a bmi kg/m , % re p o rted having hyper- tension, and % re p o rted having high c h o l e s t e rol. excluding age, % of re s p o n- dents had one risk factor for diabetes, and % had two or more risk factors. of the , respondents without diagnosed dia- betes, % re p o rted that they had been s c reened for diabetes during the past year, % re p o rted screening from to years ago, and % re p o rted screening years ago or having never been scre e n e d . respondents who re p o rted being s c reened for diabetes during the past year w e re more likely to be age years and to have a family history of diabetes, two or m o re visits to a health care provider dur- ing the past year, hypertension, and high c h o l e s t e rol levels (table ). we found no association between recent screening and sex ( % men vs. % women), ameri- can indian ancestry ( % yes vs. % no), or bmi ( % kg/m vs. % kg/m ). respondents with three or m o re risk factors (e.g., aged years, american indian ancestry, family history of diabetes, hypertension, high choles- t e rol, or bmi kg/m ) were more likely to be screened for diabetes compared with respondents with only one risk factor ( vs. %, respectively). however, % of individuals with two risk factors for dia- betes and % of individuals with more than three risk factors had not been s c reened during the past years. based on logistical re g ression analysis, t h ree factors were associated with scre e n i n g for diabetes during the past year: two or m o re visits to a health care provider during the past year ( . [ % ci . – . ]), high cholesterol level ( . [ . – . ]), and family history of diabetes ( . [ . – . ]). respondents aged – years were less likely to re p o rt re c e n t s c reening than those aged years ( . [ . – . ] ) . a limitation of this assessment is that these data are self-re p o rted. pre v i o u s studies, however, have found that self- re p o rts of conditions such as diabetes and h y p e rtension are reliable ( , ). in addi- tion, the survey was conducted by tele- phone and does not re flect the experience of individuals in montana homes without t e l e p h o n e s . the findings suggest that diabetes s c reening is being adopted by physicians for individuals aged years at risk for diabetes and that the ada re c o m m e n d a- tions are being implemented in the general c o m m u n i t y. however, these data also indi- cate a need to develop strategies to encour- l e t t e r s table —characteristics of respondents aged years reporting screening for diabetes in montana in s c reening for diabetes past year – years years or never n age (years) – ( ) ( ) ( ) – ( ) ( ) ( ) ( )* ( ) ( ) family history of diabetes ye s ( )* ( ) ( ) n o ( ) ( ) ( ) visits to a health care provider during the past year ( )* ( ) ( ) ( ) ( ) ( ) h y p e rt e n s i o n ye s ( )* ( ) ( ) n o ( ) ( ) ( ) high cholestero l ye s ( )* ( ) ( ) n o ( ) ( ) ( ) data are n (%). *p . . diabetes care, volume , number , january letters age screening among all individuals at high risk for diabetes. todd s. harwell, mph jane g. smilie, ba janet m. mcdowall, rn, bsn steven d. helgerson, md, mph dorothy gohdes, md f rom the montana diabetes project, montana d e p a rtment of public health and human serv i c e s , helena, montana. a d d ress correspondence to todd s. harw e l l , mph, montana department of public health and human services, cogswell building, c- , p. o . box , helena, mt - . e-mail: t h a rw e l l @ s t a t e . m t . u s . a c k n o w l e d g m e n t s — this project was sup- p o rted through a cooperative agreement (u- / ccu- - ) with the centers for disease c o n t rol and prevention, division of diabetes translation, atlanta, georg i a . we thank linda priest and the staff mem- bers at northwest resource consultants for their work on the telephone surv e y. the contents of this letter are solely the responsibility of the authors and do not neces- sarily re p resent the official views of the centers for disease control and pre v e n t i o n . r e f e re n c e s . american diabetes association: screening for type diabetes. diabetes care (suppl. ): s –s , . kehoe r, wu sy, leske mc, chylack lt jr: comparing self-re p o rted and physician- re p o rted medical history. am j epidemiol : – , . jackson c, jatulis de, fortmann sp: the behavioral risk factor survey and the stan- f o rd five-city project survey: a comparison of cardiovascular risk behavior estimates. am j public health : – , h b a c is not recommended as a s c reening test for diabetes in cystic fibro s i s i n the june issue of diabetes care, h u n k e rt et al. ( ) recommend the use of h b a c for early detection of cystic fib ro- s i s – related diabetes (cfrd). their re c o m- mendation is based on the finding that mean hba c is slightly higher in cystic fib rosis (cf) patients requiring insulin t h e r a p y, compared with cf patients with i m p a i red or normal glucose tolerance. h o w e v e r, no data on the validity of this a p p roach for the diagnosis of asympto- matic diabetes in patients with cystic fib ro- sis are pre s e n t e d . our group has a long-standing intere s t in the early diagnosis of cfrd, comparing fasting blood glucose levels with oral glu- cose tolerance test results ( ). in our series, we have now cf patients with newly diagnosed diabetes based on a -h venous plasma glucose value mg/dl ( . mmol/l), and simultaneous determ i n a t i o n of hba c ( h i g h - p e rf o rmance liquid chro- matography method [pharmacia, erlan- gen, germany], normal range . – . %). only out of cf patients ( %) diag- nosed as diabetic according to the ameri- can diabetes association and world health o rganization criteria ( ) had an hba c value above the normal range (individual values . , . , . , and . %). in nine diabetic cf patients with normal hba c, values between . and . % were e n c o u n t e red (mean ± sd, . ± . %). the mean -h blood glucose value after inges- tion of oral glucose was not signific a n t l y d i ff e rent between diabetic cf patients with n o rmal hba c ( ± mg/dl, mean ± sd) and diabetic cf patients with elevated h b a c ( ± mg/dl, student’s t t e s t ) . these data clearly demonstrate that the d e t e rmination of hba c is not able to sub- stitute for the oral glucose tolerance test in the early diagnosis of cfrd. our fin d i n g s a re in agreement with several re p o rts in the l i t e r a t u re ( – ) as well as the consen- sus conference on cfrd ( ). in addition to its low sensitivity when used as a diagnos- tic tool for the detection of cfrd, the mea- s u rement of hba c has the disadvantage of considerable interassay variability and lack of standardization. there f o re, we stro n g l y advise against the use of glycosylated hemoglobin as a screening test for the early diagnosis of diabetes in patients with cystic fib ro s i s . reinhard w. holl, md christian buck, md christine babka, md anna wolf, md angelika thon, md f rom the department of pediatrics (r.w.h.), uni- versity of giessen, giessen; the department of pedi- atrics (c.bu., c.ba., a.w.), the university of ulm, ulm; and the medical school hannover (a.t. ) , h a n n o v e r, germ a n y. a d d ress correspondence to pd dr. reinhard w. holl, universitätskinderklinik giessen, feulgenstr. , d- giessen, germ a n y. r e f e re n c e s . h u n k e rt f, lietz t, stach b, kiess w: potential impact of hba c d e t e rm i n a t i o n on clinical decision making in patients with cystic fib ro s i s – related diabetes (let- ter). diabetes care : – , . holl rw, buck c, cario h, wolf a, thon a, heinze e, kohne e, debatin k-m: diag- nosis of diabetes in cystic fib rosis and tha- lassemia major. diabetes care : – , . the expert committee on the diagnosis and classification of diabetes mellitus: r e p o rt of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care : – , . lanng s, hansen a, thorsteinsson b, n e rup j, koch c: glucose tolerance in patients with cystic fib rosis: five year p rospective study. b m j : – , . deluca f, arrigo t, nibali sc, sferlazzas c, gigante a, dicesare e, cucinotta d: insulin secretion, glycosylated haemoglo- bin and islet cell antibodies in cystic fib ro- sis children and adolescents with diff e re n t d e g rees of glucose tolerance. h o rm metab r e s : – , . moran a, doherty l, wang x, thomas w: a b n o rmal glucose metabolism in cystic fib rosis. j pediatr : – , . moran a: highlights of the febru a ry consensus conference on cfrd. bonn, g e rm a n y, cystic fibrosis foundation, p ro gln p e roxisome p ro l i f e r a t o r- a c t i v a t e d r e c e p t o r- and o b e s i t y r istow et al. ( ) re p o rted an activating mutation in the peroxisome pro l i f- e r a t o r-activated re c e p t o r- g e n e ( p ro gln ppa r - ), which was pre s e n t in % ( of ) of obese and % ( of ) of nonobese german caucasians. these findings may have profound implica- tions, particularly if the presence of this variant and its association with obesity are c o n firmed in other populations. we perf o rmed polymerase chain re a c- t i o n – restriction fragment length polymor- phism analysis for the pro gln ppa r - variant as described ( ) on dna samples f rom several independent populations, including lean and obese caucasians fro m the baltimore, maryland, region; african- diabetes care, volume , number , january letters americans from jackson, mississippi, and forsyth county, north carolina; pima indians from arizona; and old ord e r amish from lancaster county, pennsylva- nia (table ). a pcr fragment corre s p o n d- ing to gastric insulinotropic peptide, which has two known restriction sites for h i n dii, was mixed with each sample as a positive control. among a total of sub- jects ( , alleles), the pro gln variant was not detected in a single subject. these findings were unexpected because there is substantial overlap of gene pools of caucasians from central e u rope and the baltimore and amish caucasians studied ( ). the germ a n caucasians studied by ristow et al. were said to be unrelated and re c ruited fro m the nord rh e i n - westfalen region, but they may be a genetic isolate whose gene pool does not re flect that of other caucasian populations. altern a t i v e l y, if the individ- uals carrying the mutation were re l a t e d , the true frequency of the pro g l n p pa r - variant may have been overe s t i- mated. the absence or very low fre- quency of this variant has also been doc- umented in danish ( ) and german ( ) populations. this study is the first, to our knowledge, to examine american popu- lations for this variant. in summary, the study by ristow et al. demonstrating that the pro g l n p pa r - variant is activating and can influence body weight in people is important. however, this variant appears to be absent or very r a re in the american populations studied. additional studies are re q u i red in other regions of europe and the u.s. to furt h e r d e fine the relevance of this intere s t i n g genetic variant to susceptibility to obesity. alan r. shuldiner, md william nguyen, bs w.h. linda kao, phd brock a. beamer, md ross e. andersen, phd richard pratley, md frederick l. brancati, md, phd f rom the department of medicine (a.r.s., w. n . ) , university of maryland school of medicine; the d e p a rtments of medicine (b.a.b., f.l.b.) and epi- demiology (w.h.l.k.), johns hopkins university school of medicine, baltimore, maryland; and the national institute of diabetes and digestive and kidney diseases (r.p.), national institutes of health, phoenix, arizona. a d d ress correspondence to alan r. shuldiner, md, professor and head, division of endocrinol- o g y, diabetes, and nutrition, department of medi- cine, university of maryland school of medicine, w. lombard st., room s- , baltimore, md . e-mail:ashuldin@medicine.umary l a n d . e d u . a c k n o w l e d g m e n t s — this study was sup- p o rted by nih r dk- , the american diabetes association, glaxowellcome, the b a l t i m o re geriatrics research and education clinical center of the baltimore ve t e r a n s administration medical center. r e f e re n c e s . ristow m, muller- wieland d, pfeiffer a, k rone w, kahn cr: obesity associated with a mutation in a genetic regulator of adipocyte diff e rentiation. n engl j med : – , . c a v a l l i - s f o rza ll, menozzi p, piazza a: t h e h i s t o ry of human genes. princeton univer- sity press, princeton, nj, . elk j, urhammer sa, sorensen ti, ander- sen t, auwerx j, pedersen o: homozygosity of the pro ala variant of the pero x i s o m e p roliferation-activated re c e p t o r- g a m m a ( p par-gamma ): divergent modulating e ffects on body mass index in obese and lean caucasian men. diabetologia : – , . mamann a, munzberg h, buttron p, busing b, hinney a, mayer h, siegfried w, hebe- brand j, greten h: missense variants in the human peroxisome pro l i f e r a t o r- a c t i v a t e d re c e p t o r-gamma gene in lean and obese subjects. eur j endocrinol : – , insulin secre t i o n , insulin sensitivity, and glucose e ffectiveness in nonobese individuals with va ry i n g d e g rees of glucose to l e r a n c e a lthough it is well known that insulin s e c retion, insulin sensitivity, and glu- cose effectiveness are impaired in type diabetic patients ( – ), little is known about the role of each of these fac- tors individually on the evolution of type diabetes. in this context, a major issue is that hyperglycemia per se impairs insulin s e c retion and insulin sensitivity and that obesity observed in type diabetic patients per se causes insulin resistance ( , ). to o v e rcome this problem, we studied u n t reated nonobese subjects classified as having normal glucose tolerance (ngt) (n = ; bmi . ± . kg/m [ r a n g e . – . ], mean ± sem), impaired glu- cose tolerance (igt) (n = ; bmi . ± . kg/m [ . – . ]), and type dia- betes (n = ; fetal bovine serum . ± . mmol/l [range . – . ], bmi . ± . k g / m [ . – . ]), based on the criteria of the world health organization ( ). they were normotensive and had norm a l renal, hepatic, and thyroid function. insulin sensitivity and glucose eff e c t i v e n e s s w e re estimated by the minimal model a p p roach ( – ). insulin secretion was e x p ressed as the area under the insulin c u rve between and min after an intra- venous glucose injection ( ). after the data w e re analyzed by one-way analysis of vari- ance, bonferroni correction was used to evaluate the diff e rences between any two of the three groups we studied ( ). no signifi- cant difference was observed in bmi among the three groups. compared with the subjects with ngt, the subjects with table —characteristics of subjects screened for pro gln ppa r - n ( a l l e l e s age ± sd f e m a l e bmi ± sd p o p u l a t i o n t y p e d ) ( y e a r s ) ( % ) ( k g / m ) c a u c a s i a n s b a l t i m o re longitudinal ( ) . ± . . . ± . study on aging johns hopkins we i g h t ( ) . ± . . . ± . management center amish, lancaster, pa ( ) . ± . . . ± . a f r i c a n - a m e r i c a n s a t h e ro s c l e rosis risk in ( ) . ± . . . ± . communities study pima indians a r i z o n a ( ) . ± . — . ± . all non-amish subjects were unrelated; amish subjects were not fir s t - d e g ree relatives of each other. diabetes care, volume , number , january letters igt had significantly lower insulin secre- tion ( , ± vs. , ± pmol l m i n , p = . ) and glucose eff e c- tiveness ( . ± . vs. . ± . m i n , p . ). insulin sensitivity index was lower in subjects with igt ( . ± . m i n pmol l) than in those with ngt ( . ± . min pmol l ) , but was not statistically significant (p = . ). in con- trast, disposition index calculated by the p roduct of insulin secretion and insulin sensitivity was significantly lower in sub- jects with igt ( , ± ) than in those with ngt ( , ± , p = . ). on the other hand, patients with type dia- betes had significantly lower insulin secre- tion ( ± pmol l m i n , p = . ) compared with subjects with igt. although no significant diff e rence was observed in insulin sensitivity index between subjects with type diabetes and igt ( . ± . vs. . ± . min pmol l, p = . ), disposition index was s i g n i ficantly diminished in type diabetic patients as compared with subjects with igt ( ± vs. , ± , p = . ). glucose effectiveness in type diabetic patients ( . ± . min ) was similar to that in subjects with igt ( . ± . m i n , p = . ) but was signific a n t l y lower than that in the subjects with ngt (p . ). from these results, the fol- lowing may be hypothesized: ) impair- ments in insulin secretion and disposition index and decreased glucose eff e c t i v e n e s s , but not insulin resistance, seem to consti- tute the basic characteristics of patients with igt or type diabetes in nonobese japanese populations. ) risk factors wors- ening to type diabetes in subjects with igt are associated with further impair- ments in insulin secretion and disposition index, but not associated with furt h e r derangement in glucose effectiveness in japanese populations. ataru taniguchi, md mitsuo fukushima, md masahiko sakai, md itaru nagata, md kentaro doi, md shoichiro nagasaka, md kumpei tokuyama, md yoshikatsu nakai, md f rom the first department of internal medicine ( a . t., m.s., i.n.), kansai-denryoku hospital, and the department of internal medicine, hoshida- minami hospital (m.f.), osaka; the second depart- ment of internal medicine (k.d.) and the college of medical technology (y.n.), kyoto university, kyoto; the department of internal medicine (s.n.), jichi medical college, tochigi; and the laboratory of biochemistry of exercise and nutrition (k.t. ) , tsukuba university, tsukuba, japan. a d d ress correspondence to ataru ta n i g u c h i , md, first department of internal medicine, kansai- d e n ryoku hospital, - - , fukushima-ku, fuku- shima, osaka city, osaka - , japan. e-mail: k @ k e p c o . c o . j p . r e f e re n c e s . b e rgman rn: to w a rd physiological under- standing of glucose tolerance: minimal- model approach. d i a b e t e s : – , . welch s, gebhart ssp, bergman rn, phillips ls: minimal model analysis of intravenous glucose tolerance test-derived insulin sensitivity in diabetic subjects. j c l i n endocrinol metab : – , . taniguchi a, nakai y, fukushima m, kawamura h, imura h, nagata i, tokuyama k: pathogenic factors re s p o n s i- ble for glucose tolerance in patients with niddm. d i a b e t e s : – , . nagasaka s, tokuyama k, kusaka i, hayashi h, rokkaku k, nakamura t, kawakami a, higashiyama m, ishikawa s, saito t: endogenous glucose pro d u c t i o n and glucose effectiveness in type diabetic subjects derived from stable-labeled mini- mal model approach. d i a b e t e s : – , . best jd, kahn se, ader m, watanabe rm, ni t-c, bergman rn: role of glucose eff e c- tiveness in the determination of glucose tol- erance. diabetes care : – , . rossetti l, giaccari a, defronzo ra: glu- cose toxicity. diabetes care : – , . taniguchi a, nakai y, doi k, fukuzawa h, fukushima m, kawamura h, to k u y a m a k, suzuki m, fujitani j, tanaka h, nagata i: insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis. m e t a b o l i s m : – , . world health organization: diabetes melli - tus: report of a who study gro u p . g e n e v a , world health org., (tech. rep. ser. , no. ) . winer bj: statistical principles in experimen - tal design. nd ed. new york, mcgraw-hill, , p. – late-onset tro g l i t a z o n e - i n d u c e d hepatic dysfunction r e c e n t l y, iwase et al. ( ) re p o rted a case of liver dysfunction occurring after months of troglitazone therapy. because it was thought before this re p o rt that the risk of liver dysfunction with tro g l i- tazone after months was negligible, we wish to re p o rt another patient who took t roglitazone intermittently and developed hepatic dysfunction after months. a -year-old white man with type diabetes, ischemic heart disease (post angioplasty and stent placement), hyper- tension, degenerative joint disease, benign p rostatic hypert ro p h y, dyslipidemia, and g a s t roesophageal re flux disease had tro g l i- tazone mg daily added to his re g i m e n of glimeperide mg daily and metform i n mg b.i.d. because of poor glycemic c o n t rol (hba c . % [normal – %]). the other medicines he used were aspirin and pravastatin. after months of triple oral therapy, his hba c level dropped to . %, and, after months, to . %. after months, the patient’s hba c began to rise: . % at months, . % at year, and . % at months. liver function tests were normal until months, when his aspartate amino- transferase (ast) was found to be (nor- mal – u/l) and alanine aminotrans- ferase (alt) (normal – u/l). the t roglitazone regimen was discontinued, and testing for hepatitis b and c, h e m a c h romatosis, autoimmune liver dis- ease, and gallbladder disease were nega- tive. two months after discontinuing t roglitazone, the patient’s ast and alt had decreased to and u/l, and, after months, had re t u rned to normal at and u/l, re s p e c t i v e l y. his ast and alt have remained normal since then and he has continued to take pravastatin, aspirin, m e t f o rmin, and glimeperide. when the patient was told to discon- tinue troglitazone, he admitted that he had been taking it only interm i t t e n t l y. he esti- mated that he took the drug regularly at first, but after the first months, he took the drug only once or twice weekly on aver- age. he gave the following three reasons for his lack of compliance: a lack of funds, a fear of liver disease, and a tendency to avoid taking drugs whenever possible. this case, like the case described by iwase et al., illustrates that the hepatic dysfunction caused by troglitazone can occur after months and further sup- p o rts the u.s. food and drug administra- t i o n ’s current recommendation that quar- terly liver function tests should be obtained when troglitazone utilization extends beyond year ( ). in this case, could the onset of hepatic dysfunction have been delayed because the diabetes care, volume , number , january letters d rug was being taken only interm i t t e n t l y after the first months, and the estimated total load presented to the liver would be equivalent to the exposure at months in a compliant patient? we doubt this, since t ro g l i t a z o n e ’s hepatic effects are thought to be idiosyncratic and there f o re the total e x p o s u re should be irre l e v a n t . david s.h. bell, mb fernando ovalle, md f rom the division of endocrinology and metabo- lism, department of medicine, school of medicine, university of alabama, birmingham, alabama. a d d ress correspondence to david s.h. bell, mb, th ave. s., birmingham, al . d.s.h.b. and f.o. have served on an advisory panel for sankyo parke-davis and have received con- sulting fees, re s e a rch grant support, and honoraria for speaking engagements from sankyo parke-davis. r e f e re n c e s . iwase m, yamaguchi m, yoshinari m, oka- mura c, hirahashi t, tsuji h, fujishima m: a japanese case of liver dysfunction after months of troglitazone tre a t m e n t . diabetes care : – , . parke-davis, division of wa rn e r- l a m b e rt : rezulin package insert. morris plains, nj, g l y b u r i d e - i n d u c e d hemolysis in m y e l o d y s p l a s t i c s y n d ro m e g lyburide, also known as gliben- clamide, is a widely used sulfonylure a to treat patients with type diabetes. hemolytic anemia is an extremely rare side e ffect of which there have been only a few re p o rts ( – ). we describe a patient with myelodysplastic syndrome who pre s e n t e d with glyburide-induced hemolysis. a -year-old man with a long history of type diabetes presented with left foot cellulitis of week’s duration. this patient was known to have slowly pro g re s s i v e pancytopenia for years, for which no work-ups had been perf o rmed. his med- ications included the following: glyburide, mg per day, which he had taken for m o re than year; buformine, mg per day; and boglibose, . mg per day. he was afebrile, and the physical examination was normal, except for localized cellulitis on his left foot, for which he was start e d on intravenous antibiotics. the laboratory studies revealed a white blood cell count of . / l , hemoglobin . g/dl, platelet count /l, reticulocyte count . %, mean cor- puscle volume fl, moderate anisocyto- sis, fasting plasma glucose mg/dl, h b a c . %, lactate dehydrogenase iu/l, total bilirubin . mg/dl, and hapto- globin mg/dl. red cell glucose- - phosphate dehydrogenase level was ade- quate. cold agglutinin test, ham’s test, and sugar water test were normal. both dire c t and indirect coombs’ tests were negative. red cell resistance to osmolarity was mildly low (parpart ’s method). urinalysis demonstrated no urobilinogen. endo- scopic studies did not reveal gastro i n t e s t i- nal bleeding. ultrasonography of the abdomen showed no splenomegaly. the result of bone marrow aspiration was equivocal. on the basis of pre s u m p t i v e glyburide-induced hemolysis, glyburide was discontinued and the patient was switched to subcutaneous insulin on the seventh day. there a f t e r, his hemoglobin level increased to . g/dl, re t i c u l o c y t e count decreased to . %, and anisocytosis d i s a p p e a red pro m p t l y. he was discharg e d with insulin therapy after month in the hospital, which is when the cellulitis re s o l v e d . t h ree months later, his hemoglobin level was . g/dl and his haptoglobin remained low. repeated bone marro w aspiration confirmed the diagnosis of myelodysplastic syndro m e . we conclude that this patient devel- oped glyburide-induced hemolysis super- imposed on red cell fragility secondary to an underlying bone marrow disord e r. t h e re have been several re p o rts of hemoly- sis caused by sulfonylureas, most of which have been considered immune-mediated ( , , ). our case points to the possibility that glyburide could cause hemolysis by a non–immune-medicated mechanism. it is i m p o rtant to be aware of this potential side e ffect of glyburide in light of this medica- t i o n ’s widespread prescription, even though such a side effect is rare . hiroshi noto, md kazuhisa tsukamoto, md satoshi kimura, md f rom the department of diabetes and metabolism, tokyo university hospital, tokyo, japan. a d d ress correspondence to hiroshi noto, md, d e p a rtment of diabetes and metabolism, tokyo uni- versity hospital, - - hongo, bunkyo-ku, to k y o - , japan. e-mail: noto-tky@umin.ac.jp. r e f e re n c e s . nataas ob, nesthus i: immune haemolytic anaemia induced by glibenclamide in selective iga defic i e n c y. b m j : – , . abbate sl, hoogwerf bj: hemolytic ane- mia associated with sulfonylurea use. d i a - betes care : – , . meloni g, meloni t: glyburide-induced acute haemolysis in a g pd-defic i e n t patient with niddm. br j haematol : – , . kopicky ja, packman ch: the mechanism of sulfonylurea-induced immune hemoly- sis: case re p o rt and review of the literature . am j hematol : – , e ffects of exposure at an altitude of , m on p e rf o rmance of glucose meters s elf-monitoring of blood glucose is m a n d a t o ry for type diabetic p a t i e n t s who participate in sports to adjust insulin dose and carbohydrate ingestion ( ). sports also include activities p e rf o rm e d at moderately high altitudes, such as hiking or skiing. capillary blood glucose monitors (bgms) have been shown to underestimate blood glucose values at an altitude of , m ( ) and at a simulated altitude of , m with t e m p e r a t u re and humidity kept constant ( ). the aim of the present study was to assess the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and p o can result in errors in blood glucose d e t e rmination ( ). two bgms, the lifescan one touch ii (ot) (ortho diagnostics, milpitas, ca) and the glucometer elite ii (ge) (bayer diagnostics, brussels, belgium), were tested during a study on the effects of acute exposure at an altitude of , m and exercise on blood pre s s u re and albu- min excretion rate in six type diabetic p a t i e n t s . all subjects (four men and two women) were free of disease-related com- plications and in good and stable glycemic control (ghb . ± . %). all subjects gave their informed and written consent to participate in the study pro t o- col. all subjects were investigated both at diabetes care, volume , number , january letters sea level and after ascent by car and cable car to the angelo mosso institute at col d’olen ( , m altitude), gressoney la trinité, italy. at sea level and at a moderately high altitude, bgm reliability at diff e rent blood glucose levels was tested, and blood glu- cose was assessed in fasting and re s t i n g conditions at : a.m.; at : a.m. b e f o re an in-field exercise test; and imme- d i a t e l y, min, and min after the exer- cise stopped. capillary glucose was simul- taneously assessed with the ot and the ge. both of these bgms measure capillary blood glucose through the glucose oxi- d a s e - p e roxidase reaction. bgms were cali- brated at the beginning of each test ses- sion. a venous blood sample was simulta- neously drawn from the contralateral antecubital vein in a sodium fluoride tube, centrifuged, and stored at °c. plasma glucose was assayed with the glucose oxi- dase method (go) within days. this last assessment was taken as a re f e re n c e method. statistical analysis compare d bgm capillary glucose values and go plasma glucose values for each blood col- lection time. measurement linearity was tested with pearson’s correlation coeff i- cient. the mean of the diff e rences between the bgm and go results re p resents the mean bias between the methods with accuracy expressed as percent error (pe): pe (%) = bmg – go % g o the level of statistical significance was c o n s i d e red to be p . . the ge and ot measurements had a good correlation with plasma glucose both at moderately high altitude and at sea level. p e a r s o n ’s correlation coefficients were . and . for the ge and . and . for the ot at sea level and at moderately high altitude, re s p e c t i v e l y. biases between plasma glucose and bgm measure m e n t s w e re as follows: for the ge, . ± . at sea level and . ± . at moderately high altitude; for the ot, . ± . at sea level and . ± . at moderately high alti- tude. at sea level, both the ge and the ot tended to underestimate glucose values (ns); at moderately high altitude, the ge tended to overestimate and the ot tended to underestimate glucose values (ns). mean pes between plasma glucose and bgm m e a s u rements were . (ot) and . (ge) at sea level and . (ot) and . (ge) at moderately high altitude. pe tended to be higher for both bgms at moderately high altitude (ns). figures and show the bias between the single measurements with both devices at sea level and at moderately high altitude, re s p e c t i v e l y. at moderately high altitude (fig. ), the tendency of the ge to o v e restimate was more evident for low ( mg/dl) and intermediate ( – mg/dl) blood glucose values, whereas the ot tended to underestimate mainly high blood glucose values (ns). in our study, bgm perf o rmance was similar and good at sea level. at a moder- ately high altitude, a tendency to overe s t i- mate blood glucose for the ge and to u n d e restimate for the ot was observ e d . the overestimation for the ge involved mainly low ( mg/dl) and interm e d i- ate ( – mg/dl) blood glucose val- ues. this could present a problem in the p resence of symptoms suggesting hypo- figure —relationship between plasma and capillary glucose at sea level. figure —relationship between plasma and capillary glucose at moderately high altitude. lifescan one touch ii glucometer elite ii lifescan one touch ii glucometer elite ii diabetes care, volume , number , january letters glycemia and normal blood glucose val- ues. the ot tended to undere s t i m a t e mainly high blood glucose values, although its perf o rmance with low to i n t e rmediate values was good. the pre s- ent study assessed the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and po can result in errors in blood glu- cose determination ( ). our results are consistent with previous studies ( , ). the decrease in po could alter the sec- ond phase of the chromogen reaction and u n d e restimate blood glucose values ( ); on the other hand, an increase in atmos- pheric pre s s u re could overestimate blood glucose values ( ). in our study, minimal o v e restimation by the ge at low interm e- diate blood glucose values at moderately high altitude cannot be explained by the a l t e red po . an increase in hematocrit, which is known to alter blood glucose m e a s u rements with bgms ( ), may also occur after prolonged exposure to high altitude or as a consequence of dehydra- tion. although our study did not deter- mine hematocrit, the exercise test was s h o rt, and the patients were instructed to drink according to their thirst during the , -m exposure; there f o re, dehydration was not likely to have occurred. in con- clusion, bgm perf o rmance is similar and good at sea level. at a moderately high altitude similar to that experienced during winter skiing or summer hiking, a ten- dency to overestimate low to norm a l blood glucose values for the ge and to u n d e restimate high blood glucose values for the ot was observed. the bias is not clinically meaningful for either bgm, both of which can be safely used by dia- betic patients during exposure to moder- ately high altitudes. some care in the eval- uation of low and intermediate blood glu- cose values measured with the ge is n e v e rtheless re c o m m e n d e d . oriana pecchio, md simona maule, md marco migliardi, md marina trento, bsc massimo veglio, md f rom the italian alpine club medical commission ( o . p.); the department of internal medicine (m.t. ) , university of turin; the s. giovanni battista hospital (s.m.); and the department of endocrinology (m.m., m . v.), mauriziano umberto i hospital, turin, italy. a d d ress correspondence to dr. m. veglio, via mancini , torino, italy. e-mail: veglio@ o n w. n e t . r e f e re n c e s . h o rton es: role and management of exer- cise in diabetes mellitus. diabetes care : – , . g i o rdano bp, trash w, hollenbaugh l, dube wp: perf o rmance of seven blood glucose testing systems at high altitude. diabetes educ : – , . gautier jf, bigard ax, douce p, duvallet a, cathelinau g: influence of simulated alti- tude on the perf o rmance of five blood glu- cose meters. diabetes care : – , . b a rnett c, ryan f, ballonoff l: effect of altitude on the self monitoring of blood glucose (smbg) (abstract). diabetes (suppl.): a, . piepmeier eh, hammett-stabler c, price me, kemper gb, davis mg: atmospheric p re s s u re effects on glucose monitoring devices (letter). diabetes care : – , . b a rreau pb, buttery je: effect of hematocrit concentration on blood glucose value d e t e rmined on glucometer ii. diabetes care : – , c o m m e n t s a n d r e s p o n s e s deterioration of glycemic contro l after long-te rm treatment wi t h troglitazone in nonobese type diabetic patients t roglitazone is an oral antidiabetic d rug used to treat type diabetic patients with insulin re s i s t a n c e . troglitazone improves overall insulin sen- sitivity in the liver and skeletal muscles, which are the largest consumers and metabolizers of glucose in the body ( – ). recent re p o rts showed that troglitazone is also effective in nonobese type diabetic patients whose hyperglycemia could not be controlled with sulfonylurea therapy ( , ). however, we aware that in some patients in whom adequate glycemic con- t rol is obtained during the first several months of troglitazone treatment, their glycemic control deteriorates several months later. we assume that two distinct g roups of type diabetic patients exist who respond diff e rently to long-term administration of troglitazone, one gro u p that maintains a steady response and another group that has a decre a s i n g response after certain periods. in this s t u d y, we re t rospectively examined patients with type diabetes who were t reated with troglitazone for months and whose hba c levels had improved by % with troglitazone by month . in of the patients ( %), hba c levels increased by . % after – months despite continuous tro g l i t a z o n e t reatment (group p). in contrast, the rest of the patients experienced steady glycemic c o n t rol with . % of hba c flu c t u a t i o n ( g roup g). during the first months, h b a c levels decreased from means ± sem . ± . to . ± . % in group p and fro m . ± . to . ± . % in group g, re s p e c- t i v e l y. no significant diff e rences were evi- dent between the two groups re g a rding the d e c rease in hba c during the first months (fig. ). from month onward, hba c l e v- els in group p climbed gradually by . % a month up to the baseline level at month , but hba c levels were stable in group g t h roughout the treatment period. a signifi- cant diff e rence in hba c levels was evident during months – (p . ) . among clinical characteristics, gro u p p had a significantly lower bmi ( . ± . vs. . ± . kg/m , p . ) and s i g n i ficantly lower fasting insulin levels ( . ± . vs. . ± . µu/ml, p . ). of the patients with a bmi of kg/m ( %), exhibited deteriora- tion of glycemic contro l . in this study, we re p o rt a group of patients who showed a renewed decline in glycemic control after long-term tre a t m e n t with troglitazone. these results seemed to suggest a secondary failure of tro g l i t a z o n e . our study demonstrates that this drug is indeed useful for a long-standing obese i n s u l i n - resistant diabetes but not for a nonobese type diabetes. yuko murase, md takanobu wakasugi, md kunimasa yagi, md hiroshi mabuchi, md f rom the department of internal medicine (y. m . , t. w.), fukui perfectural hospital; and the second d e p a rtment of internal medicine (k.y., h.m.), kanazawa university, ishikawa, japan. a d d ress correspondence to yuko murase, md, the second department of internal medicine, kanazawa university, - takara-machi, kanazawa, ishikawa - , japan. e-mail: diabe@med. k a n a z a w a - u . a c . j p . diabetes care, volume , number , january letters r e f e re n c e s . suter sl, nolan jj, wallace p, gumbiner b, olefsky jm: metabolic effects of new oral hypoglycemic agent cs- in niddm subjects. diabetes care : – , . o’rourke cm, davis ja, saltiel ar, corn i- celli ja: metabolic effects of troglitazone in the goto-kakizaki rat, a non-obese and n o rmolipidemic rodent model of nonin- sulin-dependent diabetes mellitus. m e t a b - o l i s m : – , . troglitazone study group: the metabolic e ffects of troglitazone in non-insulin dependent diabetes (abstract). d i a b e t e s (suppl. ): a, . mori k: the effect of troglitazone in combi- nation with sulfonylurea in non-insulin dependent diabetes mellitus (abstract). j japan diabetes soc (suppl. ): , . h o rton es, venable tc, whitehouse f, the troglitazone study group, ghazzi mn, whitcomb rw: troglitazone in combina- tion with sulfonylurea re s t o res glycemic c o n t rol in patients with type diabetes. diabetes care : – , figure —change from baseline in hba c. values are means ± sem. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ j med genet ; : - annotations low segregation ratios in autosomal recessive disorders the recent simultaneous publication of an account of a reduced segregation ratio in carti- lage-hair hypoplasia (chh) in finland' and of one explanation for some examples of isolated cases of autosomal recessive (ar) diseases has led to a reconsideration of this interesting and important subject. it is notable that a low segregration ratio has often been found in genetic studies of suppo- sedly recessive diseases, such as those on chro- nic spinal muscular atrophy, osteogenesis imperfecta, and ataxia telangiectasia. such unexpectedly low proportions of affected sibs of patients with presumed recessive diseases may have many causes which can be con- sidered under the headings of parental be- haviour, observer error, and biological factors. parental behaviour parents may be responsible, albeit often inad- vertently, in three ways. firstly, the affected child may have a different father from the other children in the sibship. secondly, par- ents may not report earlier affected sibs, per- haps because of fear of stigmatisation, or alternatively earlier affected sibs may not have been diagnosed. this may be more likely to happen if many earlier sibs have been born in another less well developed country, and may account for the small proportion of earlier affected sibs (five out of ) seen in pakistani families whose index child with an ar disease was born in the uk. thirdly, the parents may have been deterred from having further children after the first affected, even in the absence of genetic coun- selling. such a deterrent effect was observed in families in which a child with phenylketonuria was diagnosed in a neonatal screening pro- gramme. during the period of ascertainment families with an affected child contained an excess of first affected among second or later born children and a deficit of affected first borns. it thus appeared that some families were deterred from having further children after their first affected child. a deterrent effect of this nature is likely to exist only for those conditions in which the symptoms mani- fest themselves sufficiently early and suffi- ciently seriously to influence the parents' sub- sequent plans for childbearing. observer error can geneticists erroneously produce a segrega- tion ratio greater or lower than - ? segrega- tion analysis is a notoriously difficult subject in which mode of ascertainment is critical. for example, analysis of published cases, in which there is likely to be a bias towards reporting familial cases, can easily lead to a ratio greater than . this also applies if no account is taken of the fact that even the most complete ascertainment is 'truncate' in that sibships in which both parents are carriers but which contain no affected cases are not included. the 'a priori' and 'maximum likelihood' methods of segregation analysis account for truncate ascertainment and should therefore give a more reliable estimate of the true ratio, but these methods rely on full ascertainment which is rarely achievable. however, in the presence of complete ascertainment geneticists can easily produce a lower than true segrega- tion ratio by using an inappropriate method of analysis such as the 'single' or 'sib' method. biological factors most interesting of all are the possible biologi- cal explanations for a low segregation ratio in a presumed ar disease. several mechanisms can be considered. non-penetrance this has been suggested for chh by makitie in finland' and by mckusick et al who studied this condition in old order amish. in the finnish series there was complete ascer- tainment of chh among children, but incom- plete ascertainment in adults. using truncate analysis, the number of affected sibs was signi- ficantly less than expected, and the best fit for the segregation ratio was . the 'single method', not unexpectedly, gave a much lower proportion of - . the author agreed with mckusick et a who suggested that reduced penetrance in some homozygotes could explain the low proportion of affected sibs. the best evidence for such a phenomenon was mild chh discovered radiologically in three amish children who were presumed homozygotes as both their parents were affected. however, no possibly mild manifestations were found in the finnish families.' the disease is relatively common in finland, in live births, and so the finding of two instances of parental consanguinity and out of families who had interfamilial relationships is consistent with all cases having an autosomal recessive condition arising from a few heterozygous an- cestors. the concept of reduced penetrance in o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ bundey, younig a congenital autosomal recessive disease is un- expected, but it gains credence from the obser- vation that a skeletal dysplasia in ancon sheep also displays reduced penetrance. early lethality in these studies of chh,' as in the study mentioned earlier on ataxia telangiectasia, there was no observable increased incidence of abortions or stillbirths, either of which could in theory account for a low proportion of affected sibs. however, pregnancy loss in utero need not always be recognised and early death because of homozygosity for a lethal gene closely linked to the chh locus or to an ataxia telangiectasia locus is another possibility. gamete selection (meiotic drive) selection for or against gametes bearing a recessive gene could obviously result in a dis- torted segregation ratio. to our knowledge no examples of such selection have been docu- mented in man. uniparental disomy molecular techniques have made it possible to identify several examples of uniparental dis- omy in man. woods and edwards have pre- dicted that a small proportion of rare ar disease could be the result of uniparental isodisomy. this would be significant for indi- vidual families for whom there would be no recurrence and emphasises the importance of considering this explanation when counselling parents who have had an affected child with unusual additional features or who have had only one affected child in a very large sibship. however, in segregation analysis, uniparental isodisomy is likely to have only a small effect if many families are being studied, or if the condition is relatively common as applies to chh in finland. monosomy owing to a deletion the occurrence of a recessive allele on one chromosome, paired by a de novo deletion of the homologous region of the other parent's chromosome, is a rare cause of non-recurrent ar disease. such a phenomenon is unlikely to have much effect on the segregation ratio of large family studies but is very important on an individual basis, since it provides a clue to the localisation of the disease gene. the occur- rence of oculocutaneous albinism in some patients with qll- deletions was one of several clues leading to the isolation of the p gene.'° " genetic heterogeneity probably the most common explanation for a low segregation ratio is that the disease is not always recessively inherited, with some cases being the result of new dominant mutations and some examples of recurrence in a sibship result- ing from gonadal mosaicism. it is therefore wise to check if the parental consanguinity rate equals that expected on the basis of ar inherit- ance and the known disease frequency.f such genetic heterogeneity is unlikely to be the explanation for chh where in the amish, and probably also in finland, present day cases originate from a few heterozygous ancestors. however, in more heterogeneous populations an admixture with new dominant mutations could well occur. for example, it is now recog- nised that most cases of type ii osteogenesis imperfecta, a disorder once believed to show ar inheritance, result from new heterozygous (dominant) mutations in one of the type i collagen genes.' four other disorders are worth considering in this respect: achondrogenesis, retinitis pigmentosa, chronic spinal muscular atrophy, and ataxia telangiectasia. there are several types of achondrogenesis, some of which show ar inheritance, whereas the mildest forms result from new dominant mutations in type ii collagen.' similarly retinitis pigmentosa is genetically heterogeneous, with mutations in the rhodopsin gene causing a severe ar condition as well as being responsible for some dominant forms.' it may well be that some dominant types of chronic spinal muscular atrophy result from heterozygous mutations of the gene at ql . - . , which causes the acute and chronic forms. (however, some dominant types do not map to this locus.' ) the explanation of the low segregation ratio in ataxia telangiectasia is not clear but could well be because of admixture with dominant muta- tions. note that the very low fertility of patients with chronic spinal muscular atrophy and ataxia telangiectasia means that transmis- sion of disease from affected parent to affected child is rarely observed. nevertheless, the hypothesis that some cases are the result of new dominant mutations should not be ignored. conclusion a low segregation ratio in a supposedly ar disorder is an important observation. for the geneticist this should trigger off a search for alternative explanations leading ultimately to better understanding of the underlying disease mechanism(s). for the individual family the implications could be enormous. there is a cruel irony in parents being deterred from further child bearing by a quoted recurrence risk of in , only to be presented in years to come by an affected grandchild born to their own affected child whose disorder has arisen as a consequence of a new dominant mutation. it is therefore important not to ignore a sib segregation ratio that is less than - . the investigator's question should not be "is this proportion consistent with ?" but rather "what is the range of this observed proportion, and what are its likely mechanisms?" sarah bundey department of clinical genetics, birmingham maternity hospital, edgbaston, birmingham b tg, uk. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ low segregation ratios in autosomal recessive disorders i d young department of clinical genetics, city hospital, hucknall road, nottingham ng ipb, uk. makitie . cartilage-hair hypoplasia in finland: epidemio- logical and genetic aspects of patients. med genet ; : - . woods cg, edwards jh. the need for care in the use of linkage analysis for genetic diagnosis in small families, with particular reference to uniparental disomy (letter). am hum genet ; : - . peam j, bundey s, carter co, wilson j, gardner-medwin d, walton jn. a genetic study of subacute and chronic spinal muscular atrophy in childhood. neurol sci ; : - . thompson em, young id, hall cm, pembrey me. recurrence risks and prognosis in severe sporadic osteo- genesis imperfecta. med genet ; : - . woods cg, bundey se, taylor amr. unusual features in the inheritance of ataxia telangiectasia. hum genet ; : - . bundey s, alam h. a five-year prospective study of the health of children in different ethnic groups with particu- lar reference to the effect of inbreeding. eurj hum genet (in press). brookfield jfy, pollitt rj, young id. family size limi- tation: a method for demonstrating recessive inheritance. med genet ; : - . emery aeh. methodology in medical genetics. nd ed. edin- burgh: churchill livingstone, : - . mckusick va, eldridge r, hostetler ja, ruangwit u, egeland ja. dwarfism in the amish. ii. cartilage-hair hypoplasia. in: mckusick va, ed. medical genetic studies of the amish. baltimore: johns hopkins university press, : - . gardner jm, nakatsu y, gondo y, et al. the mouse pink- eyed dilution gene: association with human prader-willi and angelman syndromes. science ; : - . rinchick em, bultman sj, horsthemke b, et al. a gene for the mouse pink-eyed dilution locus and for human type ii oculocutaneous albinism. nature ; : - . prockop dj, constantinou cd, dombrowski ke, et al. type i procollagen: the gene-protein system that harbors most of the mutations causing osteogenesis imperfecta and probably more common heritable disorders of con- nective tissue. am med genet ; : - . godfrey m, keene dr, blank e, et al. type ii achondroge- nesis-hypochondrogenesis: morphologic and immunohis- topathologic studies. am hum genet ; : - . rosenfield pj, cowley gs, mcgee tl, sandberg ma, berson el, dryja tp. a null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa. nature genet ; : - . kausch k, muller cr, grimm t, et al. no evidence for linkage of autosomal dominant proximal spinal muscular atrophies to chromosome q markers. hum genet ; : - . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ virtual mentor virtual mentor american medical association journal of ethics september , volume , number : - . history of medicine a brief history of environmental bioethics cristina richie as a response to anthropogenic—that is, human-caused—climate change, nearly every sector of public and private life has been scrutinized by ecologists. automobile pollution, greenhouse gas emissions from livestock, aerosol cans, and individual reproduction have all been topics of environmental policy and practice in the united states since the s [ ]. nearly years ago medicine’s attention was first brought to the effects of ecological matters such as pollution and carbon emissions on human health, concerns that have come to be referred to as “environmental bioethics.” efforts to quantify “the environmental impact of health care…to determine the potential value of mitigation efforts and to reduce harm associated with health care delivery” [ ] have come primarily from several scholars and a few notable journals. this article will provide background on the integration of environmental ethics into health care [ ]. i will move chronologically through a brief history of the field. van rensselaer potter van rensselaer potter’s first book bioethics: bridge to the future expanded the concerns of medical ethics—such as responsibility and rational action—to other branches of life like ecology [ ]. potter saw the interconnectedness of human life and nature as self-evident, given that we humans are situated in a natural environment, and sought to connect us not just to health within the hospital, but to holistic life in the world as well. in he published his second and last book global bioethics: building on the leopold legacy [ ]. global bioethics attempted to link the medical industry back to our earthy origins. although medicine was made by and for humans, we had come to dominate “nature” instead of live harmoniously with it. in the opening pages potter laments that, “with the focus on medical options, the fact that bioethics had been proposed to combine human values with ecological facts was forgotten by many” [ ]. potter considered continuation of the species to be of the utmost importance, but he recognized that there was an “ecological need to limit the exponential increase in the human population…[and] no program [of conservation or advancement] can hope to succeed without the acceptance of controlled human fertility as a basic ethical imperative for the human species” [ ]. potter’s work located bioethics in the bios—the life in the world—and drew a connection between medicine and conservation. his foundational writings opened the door for multidirectional progress in environmental bioethics in the years to follow. www.virtualmentor.org virtual mentor, september —vol http://www.virtualmentor.org/ jessica pierce in the late s, about a decade after potter’s second book, dr. jessica pierce appeared as a major advocate for environmentally sustainable advances in medical and hospital practices, taking up potter’s work for a “second generation” of environmental bioethicists. in pierce examined the idea of “greening” health care products [ , ]. connecting what happens within the walls of hospitals to the natural world suggested avenues for change. she discussed reducing the use of hazardous chemicals in facilities and using environmentally friendly cleaning products, now common practices in many hospitals and other businesses. noting that “about percent of health problems are already environmental in origin” [ ], in pierce demanded that the health care industry examine the way in which human health was inextricably linked to our ecosystem and dependent on a healthy planet. she called for bioethics to examine “health care’s shared responsibility for the environmental problems created by the acquisition, processing and transportation of natural resources required to make the supplies and energy used by consumers” since “health care services represent a significant sector of intensive north american economies” [ ]. her vision for sustainable health care combined the conservationist sensibilities of ecology, the call for slowing the rapid development of the global marketplace, and the more specific conclusion that health care, too, must become “smaller.” the canadian medical association journal and the journal of medical humanities the years between and saw growing academic interest in environmental bioethics and issues related to human health and planetary sustainability. the canadian medical association journal and the journal of medical humanities both ran series exploring ecology and medical ethics. the editorial piece introducing the canadian medical association journal’s - series on environmental bioethics credited michael mccally, md, for suggesting the articles on human health, the economy, social justice, and national environmental security [ ]. the nine articles in the series covered population and consumption, climate change, ozone depletion, cancer, war, endocrine disruption, species loss, sustainable health care, and risk assessment [ ]. the diversity of publications attested to the varied concerns of environmental bioethics. dovetailing with this effort, in the journal of medical humanities dedicated an entire issue to the declining environment and health care [ ]. authors wrote on such varied topics as “environmental thinking,” the role of natural light in human health, the amish ethos of placing communal needs above individual rights to prolonged life, the need for simple living and a restructuring of the global economy to aid public health, connections between ecofeminism and feminist bioethics, the allure of biotechnologies and implications of resource diversion on a planet with limited resources, and the role of nature and childlike wonder in our declining years. this virtual mentor, september —vol www.virtualmentor.org expanded the notion of environmental bioethics from a field solely relevant to medicine to one that was also of interest to literature, religion, feminism and global justice, thus solidifying the interdisciplinary character of environmental bioethics. in , jessica pierce co-authored (with andrew jameton) the ethics of environmentally responsible health care [ ]. this full-length treatment of environmental bioethics was one of the first books since potter’s to address the health needs of human beings—both current and future—and the limits of the shared ecosystem that sustains us. focusing specifically on the united states health care system, the book highlights the tensions between health care needs in developed and the developing world, the individual and the community, and the limits of the planet and the demands of a growing human population. recent trends nearly ten years passed before another “generation” of environmental bioethics emerged. in two promising developments brought environmental bioethics back to the forefront of medicine. david resnik’s environmental health ethics revisited the trails that potter and pierce had blazed and expanded on issues of nutrition, natural disasters, and public health [ ]. in addition, the american society for bioethics and humanities (asbh), which has an affinity group for environmental bioethics, sponsored an undergraduate conference entitled “bioethics: intersections of global health and environmental policy” [ ]. the impact of climate change on the human population has continued to receive interest in the medical industry, urging conservation to better the lives of those who currently suffer under the effects of global warming, including conditions of food scarcity, respiratory disease and drought. it seems that at every turn there is a new organization [ ], ethicist [ , ], or initiative [ ], like the healthy hospitals initiatives [ ] and practice greenhealth [ ], ready to take on the challenge of environmental degradation and human health, health care, and personal responsibility. health care professionals and those who teach them must be prepared to examine the implications of carbon dioxide emissions on human well-being and make decisive steps towards sustainability. references . many trace the inception of the environmental movement in the us back to the seminal text by carson r. silent spring. boston: houghton mifflin; . . chung jw, meltzer do. estimate of the carbon footprint of the us health care sector. jama. ; ( ): . . i have integrated ecological ethics in two different settings: in the course interdisciplinary approaches to bioethics at the experimental college of tufts university in the spring of and om the course health care ethics at massachusetts college of pharmacy and health sciences. . potter v. bioethics: bridge to the future. upper saddle river, nj: prentice hall; . . potter v. global bioethics: building on the leopold legacy. east lansing, mi: michigan state university press; . www.virtualmentor.org virtual mentor, september —vol http://www.virtualmentor.org/ . potter, global bioethics, - . . potter, global bioethics, . . pierce j. can you use a “greener” cleaner? hosp mater manage. : - . . product review yields cleaner, greener use of chemicals. healthc facil manag. : - . . pierce j, jameton a. sustainable health care and emerging ethical responsibilities. cmaj. ; ( ): . . pierce, jameton, . . ecosystem evasion and health (editorial). cmaj. ; ( ): , . . a list of the article in the series can be found at bailer j, bailer a. environment and health: . the science of risk assessment. cmaj. ; ( ) - : . . see the environment-themed articles in j med humanit. ; ( ): - . . pierce j, jameton a. the ethics of environmentally responsible health care. new york: oxford university press; . . resnik d. environmental health ethics. new york: cambridge university press; . . american society for bioethics and humanities. national undergraduate bioethics conference at georgetown university. april - , . http://nubc .org/. accessed july , . . catholic health association and practice greenhealth. environmental sustainability: getting started guide. st. louis: the catholic health association of the united states; . http://www.chausa.org/docs/default-source/general-files/gettingstartedguide- pdf.pdf?sfvrsn= . accessed july , . . most recently discussions have turned from environmental bioethics to “green bioethics.” see richie c. building a framework for green bioethics: integrating ecology into the medical industry. health care ethics usa. ; ( ): - . . richie c. what would an environmentally sustainable reproductive technology industry look like? j med ethics. epub ahead of print july , . . roberts i. the nhs carbon reduction strategy. bmj. ; :b . . healthier hospitals initiative. lead your community to a healthier future: - . http://healthierhospitals.org/sites/default/files/imce/public_files/pdfs/hhi-brochure.pdf accessed may , . . practice greenhealth website. https://practicegreenhealth.org/. accessed may , . cristina richie is writing her phd dissertation on green bioethics in the theology department at boston college. she has taught health care ethics at massachusetts college of pharmacy and health sciences (mcphs) in boston and published in more than a dozen journals. related in vm medicine’s role in mitigating the effects of climate change, june greener clinics, better care, september caring for the health of the community means caring for the health of the environment, june the viewpoints 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ tf-lebm .. http://informahealthcare.com/ebm issn: - (print), - (electronic) electromagn biol med, early online: – ! informa healthcare usa, inc. doi: . / . . o r i g i n a l a r t i c l e evidence that dirty electricity is causing the worldwide epidemics of obesity and diabetes samuel milham retired washington state department of health, olympia, wa, usa abstract the epidemics of obesity and diabetes most apparent in recent years had their origins with thomas edison’s development of distributed electricity in new york city in . his original direct current (dc) generators suffered serious commutator brush arcing which is a major source of high-frequency voltage transients (dirty electricity). from the onset of the electrical grid, electrified populations have been exposed to dirty electricity. diesel generator sets are a major source of dirty electricity today and are used almost universally to electrify small islands and places unreachable by the conventional electric grid. this accounts for the fact that diabetes prevalence, fasting plasma glucose and obesity are highest on small islands and other places electrified by generator sets and lowest in places with low levels of electrification like sub-saharan africa and east and southeast asia. keywords body mass index, diabetes, diesel generator sets, dirty electricity, fasting plasma glucose, islands, obesity, oceania, transients history received august revised january accepted january published online june introduction of the children who used inhalers for asthma in a mid- western us school, only needed the inhalers after the dirty electricity levels were reduced, in an attempt to cure a ‘‘sick building’’ syndrome (braggia, ). in researching asthma incidence and its possible connection to electricity, i became interested in disease on islands after learning that the highest asthma rate in the world is in the population of the island of tristan da cunha (zamel et al., ). the island’s electricity is provided by six diesel generator sets, which are a major source of dirty electricity. i hypothesize that the electricity supplied to residences on the island has high levels of dirty electricity which is causing symptomatic asthma. in , the global burden of metabolic risk factors of chronic diseases collaborating group published two papers in the lancet funded by the bill and melinda gates foundation, one on the national, regional and global trends in fasting plasma glucose (fpg) and diabetes prevalence (danaei et al., ) and the other on the global distribution of obesity, measured by body mass index (bmi) (finucane et al., ). remarkably, the worldwide distributions and trends of diabetes and obesity were nearly identical, favoring the small islands of pacific oceania. i hypothesized that the island excess was due to dirty electricity from diesel generator sets used almost universally to electrify small islands and places unreachable by the conventional electric grid. method the imperial college, london, interactive website (http:// www .imperial.ac.uk/publichealth/departments/ebs/projects/ eresh/majidezzati/healthmetrics/metabolicriskfactors/) has extensive worldwide data on cholesterol, high blood pressure, diabetes prevalence, fasting plasma glucose (fpg) and body mass index. the cholesterol and blood pressure data were unremarkable, but a cursory examination of the diabetes and obesity data showed a strong link with islands. small islands were identified among the countries ranked for bmi, fpg and diabetes prevalence. a search identified all the countries in the highest and lowest disease rankings for males age plus in . a web search identified the extent of their electrical grids and the sources of their electricity. wave forms of the electrical output of commercial diesel generator sets were obtained using a fluke b oscilloscope, and dirty electricity levels in the generators’ outputs were measured with a graham/stetzer microsurge meter. results table shows that of countries in the world with the highest bmis in are small islands, in pacific oceania (nauru, cook island, tonga, samoa, palau, marshall islands, kiribati) and in the caribbean (st. kitts and nevis). all of these places are electrified by diesel generator sets. seven of the places with the highest fpg are also small islands in oceania. seven small islands in oceania are also among the places with the highest diabetes prevalence. the cook islands, tonga, marshall islands, palau and kiribati and samoa are among the top places for bmi, fpg and address correspondence to samuel milham md, gravelly beach loop nw, olympia, wa , usa. e-mail: smilham@dc.rr.com e le ct ro m ag n b io l m ed d ow nl oa de d fr om i nf or m ah ea lt hc ar e. co m b y . . . on / / f or p er so na l us e on ly . http://informahealthcare.com/ebm mailto:smilham@dc.rr.com diabetes prevalence in men in . because about % of the countries listed are small islands, . islands would be expected in the highest countries when or were observed (p . ). the non-islands with high bmi, fpg or diabetes prevalence are saudi arabia, kuwait and jordan and the united states, all three mid-eastern countries have sizable off-grid populations served by generator sets, and generator sets are also used to electrify portions of the grids in these countries. the high obesity levels in the united states is not surprising, because the united states uses the earth as the major conduit for neutral return currents, allowing dirty electricity to enter homes through conductive water and sewer pipes and through the grid. diesel generators are also used extensively for back-up power in peak load periods. many utilities have dozens of standby generators. with the single exception of timor leste (east timor), there are no islands in the places in the world with the lowest bmis, fpgs or diabetes prevalence. their electric grid was damaged by wars and only % of rural residents of timor leste currently have electricity. most of the other low prevalence places are in sub-saharan africa and in east and southeast asia, places with a low levels of electrification. the fact that the united kingdom and the netherlands are among the low prevalence countries may be due to their grids being of the closed delta type with no use of the ground for current return. figure is an oscilloscope tracing of the electrical output of a large commercial diesel generator set. it shows a ‘‘dirty’’ wave form and high dirty electricity levels. two other gasoline-powered generator sets also had ‘‘dirty’’ wave forms and high dirty electricity levels (tracings available on request). table . country trends in metabolic risk factors, males age þ, . ten highest and lowest countries of body mass index (bmi) fasting plasma glucose (fpg) diabetes prevalence (a.s.) country kg/m country mmol/l country percent highest countries of : nauru* . marshall islands* . marshall islands* . cook islands* . kiribati* . kiribati* . tonga* . samoa* . saudi arabia . samoa* . saudi arabia . samoa* . palau* . cook islands* . cook islands* . marshall islands* . palau* . palau* . kiribati* . jordan . jordan . kuwait . tonga* . solomon islands* . usa . kuwait . kuwait . st. kitts and nevis* . solomon islands* . tonga* . lowest countries of : india . central african r. . indonesia . viet nam . philippines . dem. rep. congo . eritrea . united kingdom . philippines . nepal . dem. rep. congo . timor leste* . zambia . timor leste* . malawi . timor leste* . myanmar . burundi . afghanistan . rwanda . myanmar . bangladesh . burundi . netherlands . ethiopia . peru . peru . democratic r. congo . cambodia . cambodia . * ¼ islands; (a.s.)¼age standardized figure . oscilloscope tracing of electrical output of a katolight kv diesel generator set. s. milham electromagn biol med, early online: – e le ct ro m ag n b io l m ed d ow nl oa de d fr om i nf or m ah ea lt hc ar e. co m b y . . . on / / f or p er so na l us e on ly . discussion dirty electricity is a term coined by the electrical utilities to describe electrical pollution contaminating the hz and hz electricity on the electrical grid. it is generated by arcing, sparking and any device which interrupts current flow. on september , thomas edison began generating electricity at the pearl street plant in manhattan, new york city. from the beginning, his ‘‘jumbo’’ generators were plagued with brush arcing and commutator wear (edisonia): there was considerable sparking at the copper commutator brushes of the ‘‘jumbo,’’ due to the odd number of commutator bars, necessitated by the form of armature winding employed. . . this means that dirty electricity was being sent to edison’s customers through the wires that lit their lamps as early as . since the wertheimer–leeper study (wertheimer and leeper, ), there has been concern that exposure to power frequency ( / hz) electromagnetic fields (emfs), espe- cially magnetic fields, may contribute to adverse health effects including cancer. until recently, the most commonly used exposure metric in emf studies has been the time- weighted average of the power frequency ( and hz) magnetic field. however, the low risk ratios in most studies ( . ) suggested that magnetic fields might be a surrogate for a more important metric. in , dirty electricity was shown to be a potent universal carcinogen in a cohort study of teachers (milham and morgan, ) at a california middle school. cancer incidence analysis of the teacher population showed a positive trend of increasing cancer risk with increasing cumulative exposure to high-frequency voltage transients measured on the classroom’s electrical wiring, with high risk ratios ( . ) for a number of cancers. the attributable risk of cancer associated with this exposure was %. a single year of employment at this school increased a teacher’s cancer risk by %. dimmer switches, compact fluorescent lights, computers, copy machines, all transmitters, including cell towers, and all devices containing switching power supplies generate dirty electricity. dirty electricity generated by electrical equipment in a building is distributed throughout the building on the electric wiring. dirty electricity generated outside the build- ing enters the building on electric wiring and through ground rods and conductive plumbing. in recent years in the united states, nearly % of neutral return currents travel to the substations via the earth, carrying an increasing load of dirty electricity. dirty electricity in building wiring is measured with a simple plug-in graham/stetzer microsurge meter (graham, ), which measures the average magnitude of the rate of change of voltage as a function of time (dv/dt). this preferentially measures the higher frequency transients. dirty electricity has been associated with diabetes (havas, ), asthma (sbraggia, ), attention deficit hyperactiv- ity disorder (adhd) (milham, ) and cancer (milham and morgan, ). although much attention has been paid to the obesity and diabetes epidemics recently, there is good evidence that both epidemics started at the beginning of electrification in the united states. most large cities in the united states and the world had electricity by . in the united states, the great distances and the expense slowed rural electrification. it took until for us farms to reach urban and rural non-farm electrification levels. both populations were covered by the us vital registration system. the us census of population, , and contained information on residential electrification. in , urban (electrified) death rates were much higher than rural (unelectrified) rates for cardiovascular diseases, malignant diseases, diabetes and suicide. rural death rates were significantly correlated with level of residential electric service by state for most causes examined. between and , the death rates from all of the so-called diseases of civilization, including diabetes increased steadily in the united states. i hypothesized that the th-century epidemic of the diseases of civilization, includ- ing cardiovascular disease, cancer and diabetes and suicide, was caused by some facet of electrification (milham, ). there is no historical vital record source for bmi, but komlos and brabek (komlos and brabek, ), using historical data from the west point military academy and the citidel military academy in charleston sc, showed that there was very little change in cadet weight in the th century, but that -year-old men had a body weight increase of kg ( . pounds) during the th century, with half of the increase occurring in those born before world war ii. the – citidel birth cohort was kg heavier than the – birth cohort. before world war ii, the major source of dirty electricity was generator and motor brush arcing and arcing and sparking from bad electrical connec- tions. microwaves were not invented until shortly before world war ii. there is some recent evidence that emf exposure from cell towers, which generate both microwaves and dirty electricity, affects both neurotransmitters and adrenal hormones (buchner and eger, ; eskander et al., ), which can impact blood sugar, appetite and obesity. testing the hypothesis the three commercial generator sets i have access to have dirty wave forms and high dirty electricity levels in their electrical output. the islands in oceania with high levels of fpg, diabetes and bmi should all have high levels of dirty electricity in their generator outputs. the more generators operating in a power plant, the higher the output dirty electricity levels should be, because the parallel electrical connection of multiple generators should make their dirty electricity additive. the populations of one or more of these islands should be examined before and after a clean-up of residential dirty electricity with capacitive filters and followed for a generation. long-term follow-up is required because childhood leukemia (greaves, ), asthma (li et al., ) and obesity (li et al., ) have all been associated with in utero emf exposure. symptoms associated with emf and dirty electricity exposures should improve rapidly, and type diabetics should use less insulin. the old order amish in the united states and canada, a mennonite sect who live without electricity and therefore with low levels of dirty electricity exposure, gives a strong doi: . / . . dirty electricity obesity and diabetes e le ct ro m ag n b io l m ed d ow nl oa de d fr om i nf or m ah ea lt hc ar e. co m b y . . . on / / f or p er so na l us e on ly . indication of the sort of mortality and morbidity improvement that is possible. their type diabetes prevalence rates (hsueh, ) and cancer rates (westman, ) are about half those of the non-amish. there is no adhd and little obesity in amish children (ruff, ). i conclude that the epidemics of diabetes and obesity are caused by exposure to dirty electricity. declaration of interest i declare no conflicts of interest. references buchner, k., eger, h. ( ). changes of clinically important neuro- transmitters under the influence of modulated rf fields – a long-term study under real-life conditions. umwelt-medizin-gesellschaft. : – . (original in german). danaei, g., finucane, m. m., lu, y., et al. ( ). national, regional, and global trends in fasting plasma glucose and diabetes prevalence since : systematic analysis of health examination surveys and epidemiological studies with countries and . million participants. lancet. : – . ‘‘edisonia’’, a brief history of the early edison electric lighting system (google ebook). p. . available from: http://www.archive.org/ stream/edisoniaabriefh expogoog/edisoniaabriefh expogoog_dj- vu.txt (accessed may ). eskander, e. f., estefan, s. f., abd-rabou, a. a. ( ). how does long term exposure to base stations and mobile phones affect human hormone profiles? clin. biochem. : – . finucane, m. m., stevens, g. a., cowan, m. j., et al. ( ). national, regional, and global trends in body-mass-index since : systematic analysis of health examination surveys and epidemiological studies with country-years countries and . million participants. lancet. : – . graham, m. h. ( ). circuit for measurement of electrical pollution on power line. united states patent , , b . greaves, m. ( ). pre-natal origins of childhood leukemia. rev. clin. exp. hematol. : – . havas, m. ( ). dirty electricity elevates blood sugar among electrically sensitive diabetics and may explain brittle diabetes. electromagn. biol. med. : – . hsueh, w. c., mitchell, b. d., aburomia, r. ( ). diabetes in the old order amish: characterization and heritability analysis of the amish family diabetes study. diabetes care. : – . komlos, j., brabec, m. ( ). the evolution of bmi values in us adults: – . available from: http://www.voxeu.org/article/ -years- us-obesity. li, d. k., chen, h., odouli, r. ( ). maternal exposure to magnetic fields during pregnancy in relation to the risk of asthma in offspring. arch. pediatr. adolesc. med. : – . li, d. k., ferber, j., odouli, r. et al. ( ). prospective study of in-utero exposure to magnetic fields and the risk of childhood obesity. sci. rep. doi: . /srep . milham, s., morgan, l. l. ( ). a new electromagnetic exposure metric: high frequency voltage transients associated with increased cancer incidence in teachers in a california school. am. j. ind. med. : – . milham, s. ( ). historical evidence that electrification caused the th century epidemic of ‘‘diseases of civilization’’. med. hypotheses. : – . milham, s. ( ). attention deficit hyperactivity disorder and dirty electricity. j. dev. behav. pediatr. : . ruff, m.e. ( ). available from: http://www.additudemag.com/adhd/ article/ .html. sbraggia, c. ( ). letter regarding health effects of graham-stetzer filter installation. available from: www.electicalpollution.com (accessed may ). wertheimer, n., leeper, e. ( ). electrical wiring configurations and childhood cancer. am. j. epidemiol. : – . westman, j. a., ferketich, k. a., kauffman, r. m., et al. ( ). low cancer incidence rates in ohio amish. cancer causes control. : – . zamel, n., mcclean, p., sandell, p. r., et al. ( ). asthma on tristan da cunha: looking for the genetic link. the university of toronto genetics of asthma research group. am. j. respir. crit. care med. : – . s. milham electromagn biol med, early online: – e le ct ro m ag n b io l m ed d ow nl oa de d fr om i nf or m ah ea lt hc ar e. co m b y . . . on / / f or p er so na l us e on ly . waghorn et al. radiation oncology , : http://www.ro-journal.com/content/ / / research open access a margin-based analysis of the dosimetric impact of motion on step-and-shoot imrt lung plans benjamin j waghorn, amish p shah*, justin m rineer, katja m langen and sanford l meeks abstract purpose: intrafraction motion during step-and-shoot (sns) imrt is known to affect the target dosimetry by a combination of dose blurring and interplay effects. these effects are typically managed by adding a margin around the target. a quantitative analysis was performed, assessing the relationship between target motion, margin size, and target dosimetry with the goal of introducing new margin recipes. methods: a computational algorithm was used to calculate , motion-encoded dose distributions and dvhs within the patient’s ct dataset. sinusoidal motion tracks were used simulating intrafraction motion for nine lung tumor patients, each with multiple margin sizes. results: d % decreased by less than % when the maximum target displacement beyond the margin experienced motion less than mm in the superior-inferior direction and mm in the anterior-posterior direction. for target displacements greater than this, d % decreased rapidly. conclusions: targets moving in excess of mm outside the margin can cause significant changes to the target. d % decreased by up to % with target motion mm outside the margin, with underdosing primarily limited to the target periphery. multi-fractionated treatments were found to exacerbate target under-coverage. margins several millimeters smaller than the maximum target displacement provided acceptable motion protection, while also allowing for reduced normal tissue morbidity. keywords: imrt, motion, step-and-shoot, lung, dosimetry pacs: . .dk introduction during radiation therapy, intrafraction motion has the po- tential to affect target dosimetry [ ], sometimes with signifi- cant consequences to target dose coverage [ , ]. this is especially true during intensity modulated radiation therapy (imrt) [ ], where increases in target conformality can pro- duce more desirable dose distributions for the static patient, but at the same time can make the target more susceptible to underdose due to intrafraction target motion [ ]. a num- ber of techniques exist to minimize the effects of intrafrac- tion motion, with the addition of a planning target volume (ptv) being the most commonly used technique to main- tain uniform dosimetry to the target even during motion [ ]. several methods have been developed to estimate the dosimetric impact of intrafraction motion including monte * correspondence: amish.shah@orlandohealth.com department of radiation oncology, uf health cancer center at orlando health, south orange avenue mp , orlando, florida , usa © waghorn et al.; licensee biomed centr commons attribution license (http://creativec reproduction in any medium, provided the or carlo simulations [ ], experimental phantom measurements [ , ] and computational methods [ - ]. one recent com- putational technique applied a given motion track to a static treatment plan to estimate the three-dimensional ( d) dose distribution within the patient anatomy, allow- ing for performance of dvh-based analyses [ ]. this current study utilizes the same technique to investigate the impact of intrafraction motion on sns imrt, spe- cifically to study the relationship between motion and dosimetric effect. motion and planning parameters were varied to test a range of combinations, some of which were similar to those used clinically while others were, by design, beyond those used clinically. the ct dataset and target contours from nine lung cancer patients were inves- tigated, encompassing a representative range of tumor shapes and sizes. in total , motion-encoded dose dis- tributions were calculated for different sinusoidal motion track, margin size, and treatment plan combinations for al ltd. this is an open access article distributed under the terms of the creative ommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and iginal work is properly credited. mailto:amish.shah@orlandohealth.com http://creativecommons.org/licenses/by/ . waghorn et al. radiation oncology , : page of http://www.ro-journal.com/content/ / / the nine patients. the cumulative effects of motion during multi-fractionated deliveries were also considered. methods and materials under an institutional review board (irb)-approved pro- tocol, nine lung cancer cases were retrospectively reviewed for this motion study, all of which had been selected for mv sns imrt treatments. treatment planning the initial target volume determination for the nine patients were contoured by a radiation oncologist using information from the individual dct phases, the average ct, and the maximum intensity projection (mip) reconstruction from the dct, resulting in volumes ranging from . to . cm (average volume ± standard deviation = . ± . cm ). target and ptv combinations with target to ptv margin expansions of , , , and mm were uniformly applied. clinically relevant optimization objectives were used to create sns imrt treatment plans for each ptv/target combination within a research version of a com- mercial treatment planning system (pinnacle, version . x, philips healthcare, andover, ma). the treatment plans were calculated on the patient’s free-breathing ct. the number of beams (all coplanar), treatment prescription, average number of segments per beam, and the average patient plan mu/dose (assumed to be proportional to the plan complexity [ ]) are summarized in table . calculating the motion-encoded dose distribution a computational algorithm was developed in matlab to estimate the dosimetric effect of intrafraction motion [ ]. based upon a chosen motion track, individual segment flu- ence maps were shifted in the opposite direction of the physical target’s beams-eye-view displacement in order to account for this motion during each delivered monitor unit (mu). mu timing was calculated using a mu/minute dose rate, assuming a second interval between each seg- ment (a typical inter-segment treatment time acquired from table patient prescription and treatment plan information patient number prescription dose (cgy) number of fractions number of beams primar volum , , , , , , , , , dynalog files) and a second interval between each gantry angle. a final dose calcula-tion was performed within the ct dataset using the modified fluence maps, creating the d motion-encoded dose distribution. previous work has validated the accuracy of the motion-encoded dose distributions [ ]. motion tracks ideally, this analysis would be performed using clinically acquired tumor motion tracks. it has been demonstrated, however, that respiratory motion can be approximated as sinusoidal motion of the form shown in eq. [ ]: displacement tð Þ mm½ � ¼ a mm½ �⋅ sin � π t sec½ � t sec½ � þ φ ���� þ d mm= sec½ �⋅ t sec½ �ð Þ þ o mm½ � ð Þ each parameter from eq. (namely amplitude (a, half peak-to-peak motion), drift (d), offset (o), period (t) and phase (φ)) was investigated separately to determine its effect on the dosimetry. the specific motion variables for each patient are shown in table , with default values of a = . mm, t = . sec [ ], φ = °, d = mm/min and o = mm being used when not being explicitly stated. for ex- ample, when the effect of target drift was being investigated, values of a = . mm, t = . sec, φ = ° and o = mm were held constant while the drift rate was varied as shown in table , column . for each motion track, data was calculated for every margin size listed in table , creating a total of , motion-encoded dose distributions. intrafraction motion dosimetric impact analysis motion-encoded target dvhs were calculated from the motion-encoded dose distribution, and were compared to the static dvhs by calculating target Δd % and Δd % values; these represent the difference between static and motion-encoded d % and d %, respectively. by definition, Δd % and Δd % equal unity if the motion had no dosimetric impact. y target e (cm ) margin sizes (mm) average segments per beam average mu/dose (mu/cgy) . , , , & . . . , , , & . . . , , & . . . , , & . . . , , & . . . , , & . . . , , & . . . , , & . . . , , & . . table motion track parameters per patient patient number motion direction amplitude (half peak-to-peak, mm) drift (mm/min) offset (mm) period (sec) phase (radians) si/ap/ d , , , . , , − , − . , , . , − , − , − , , , , . , . , . , , π/ , π/ , π/ , π/ si/ap/ d , , , . , , − , − . , , . , − , − , − , , , , . , . , . , , π/ , π/ , π/ , π/ si/ap , , . , , − , , , , . si/ap , , . , , − , , , , . si/ap , , . , , − , , , , . si/ap , , . , , − , , , , . si/ap , , . , , − , , , , . si/ap , , . , , − , , , , . si/ap , , . , , − , , , , . waghorn et al. radiation oncology , : page of http://www.ro-journal.com/content/ / / the quantity ‘max displacement – margin’ (mm) was calculated for each motion calculation and represents the maximum displacement of the target outside the ptv (figure ). this parameter was used to assess the effective- ness of the margin concept for motion management, with an acceptable target deviation selected as Δd % > . (i.e. less than a % reduction in d %). the study was designed to provide a wide range of sinusoidal tumor displacements, encompassing a majority of the maximum tumor displacement expected clinically. therefore, ana- lysis of the ‘max displacement – margin’ parameter was used to create a new approach to the margin size decision making process. multi-fractionation analysis to determine the cumulative impact of motion on mul- tiple fractions, accumulated dvhs were calculated for the following scenarios: figure a schematic representation of intrafraction target motion. ) patient ( mm margin), five fractions each with the same target motion track ( mm amplitude si), but with different starting phases. ) patient ( mm margin), with seven randomly selected si motion tracks. ) patient ( mm margin), with thirty randomly selected si motion tracks. individual fraction motion-encoded dose distributions and dvhs were calculated, as well as the accumulated dose distribution and dvh. results the static plan complexity (characterized by the num- ber of mus required to deliver a cgy of dose) [ ] in- creased approximately linearly with increasing ptv volume (table ), with the linear least square best fit function shown in eq. (r = . ). larger ptvs required more complex static treatment plans (table ). waghorn et al. radiation oncology , : page of http://www.ro-journal.com/content/ / / mu=dose mu=cgyð Þ ¼ � ‐ � ptv volume cm � � þ : ð Þ two example treatment plans are shown in figures a and b, along with mm amplitude si motion-encoded dose distributions in figures c and d, respectively. the corresponding target and ptv dvhs for these two cases are shown in figures e and f. motion amplitudes (half peak-to-peak displacement) used in this study ranged from to mm (table ). these motion tracks corresponded to ‘max displace- ment – margin’ values ranging from − mm ( mm margin, mm amplitude) to + mm ( mm margin, mm amplitude). similarly, drift velocities from − to + mm/min were investigated with the absolute target displacement dependent on the treatment time. the aver- age target displacement due to drift was . ± . mm (‘max displacement – margin’ values ranged from − . to + . mm for the default . mm amplitude drifting tracks). offsets of − to + mm were also studied (‘max displacement – margin’ ranged from − . to + . mm with . mm amplitude). figure dosimetric comparison between patient and patient . sam ( mm margin) are shown in a and b, respectively. the dosimetric effect o for patient and , respectively. the corresponding target (thick lines) and without motion (dashed lines) and with motion (solid lines). figure shows the effect of ‘max displacement – margin’ on target d %, with the results from the ampli- tude, drift and offset studies shown separately in figures a, b and c respectively, and combined in d. least-square fits of a quadratic function were applied to the data for each test and motion direction (for ‘max displacement – margin’ values > ), with the resultant curves shown. all of the data are included in figure d, ranging from the motion track with mm margin and mm ampli- tude motion to the worst-case scenario of zero margin and mm/min drift. by converting these data points to ‘max displacement – margin’ the full spectrum of motion tracks studied here can be compared together without skewing the results; these outliers simply form the ex- tremes within the plot, and help to define the relationship between displacement, margin size and target dosimetry. it can be seen from figure that the dosimetric effect of motion is dependent on the direction of motion. for example, when ‘max displacement – margin’ was between and mm ( . ± . mm), average Δd % values for motion in the ap, si and d directions were . ± . , . ± . and . ± . respectively. using the least- square best-fit quadratic curves, d % was reduced by ple static treatment plans for patients ( mm margin) and f a mm amplitude si sinusoidal motion track is shown in c and d ptv (thin lines) dvhs for these two cases are shown in e and f, both figure the effect of varying the motion amplitude, drift and offset on Δd % are shown separately in a, b and c respectively, and combined in d. each of the plots shows the effect of increasing the target displacement outside the ptv (‘max displacement – margin’) on target d %. the shaded regions represent < ± % change in d %. least-square quadratic fits are shown for each motion direction. waghorn et al. radiation oncology , : page of http://www.ro-journal.com/content/ / / more than % (Δd % < . ) when ‘max displacement – margin’ exceeded . , . and . mm for motion in the ap, si and d directions respectively. the shaded regions in figure represent a < ± % change in d % due to motion. this data can be directly used as a new margin recipe, dependent on the direction and magnitude of the maximum expected tumor displacement. changes in target Δd % were observed for patients and with varying periods. Δd % values for each indi- vidual period deviated from the average by only − . % to + . %. similarly, all of the different starting-phase Δd % values for each margin size and motion direction were within ± . % of the average Δd % value. the effect of motion on d % was also studied, with only . % of the tracks experiencing more than a % increase in Δd %, and less than % increasing by more than %. a majority of the high Δd % values were caused by d motion with ap motion causing the smallest changes. unlike the results for Δd %, Δd % was rela- tively independent of the amount of motion present when the motion size was larger than the margin size. figure displays the effect of varying the drift rate (absolute) on target Δd % for various margin sizes in the si, ap and d directions (figure a, b and c, respectively), as well as the effect of increasing the total si drift during treatment (figure d). in contrast to figure , the data shown in figure distinguishes directly between margin size and the drift, as opposed to combining the variables to form the ‘max displacement – margin’ quantity. with an amplitude of . mm present in each motion track, almost identical motion direction and margin size dependencies on the target dosimetry are ob- served, as were seen in figure and as described above. figure d also takes into account the total treatment time by converting the dose rate into the maximum drift dis- placement (drift rate × treatment time) for each data point. the maximum total displacement would be the sum- mation of the total drift and . mm for the sinusoidal amplitude. similar conclusions can be drawn regarding the effect of margin size on target dosimetry, as described above. finally, the results of the multi-fraction study are shown in figure . results from the three different scenarios , , and listed above are shown in figures a, b, and c, respectively. while the cumulative d % for multiple fractions was approximately equal to the average d % of the individual fractions, the cumulative d % was less than the average d % of the individual fractions. discussion this investigation was devised to provide a more thorough understanding of the potentially detrimental dosimetric effects of intrafraction motion and to investigate the effectiveness of target margins to minimize these dosimetric figure the effect of varying the drift rate on Δd % for si, ap and d motions are shown in a, b and c respectively, as well as the effect of increasing the total si drift during treatment (d). each of the plots shows the effect of changing the drift rate of a . mm amplitude sinusoidal motion track on target dosimetry for various margin sizes. the shaded regions represent < ± % change in d %. note that the total tumor displacement is equal to the drift displacement plus the sinusoidal amplitude. figure the dosimetric effects of multi-fractionated treatments are shown with dvhs for the static plan (thick dashed line), the individual fractions (thin lines) and the cumulative dose (thick solid line). a shows five fractions with the same motion track ( mm amplitude si motion), but different starting phases per fraction. b and c show and fractions respectively, with each fraction experiencing a different, randomly selected, si motion track. waghorn et al. radiation oncology , : page of http://www.ro-journal.com/content/ / / waghorn et al. radiation oncology , : page of http://www.ro-journal.com/content/ / / effects. adequate ctv to ptv margins for treatment of moving lung targets has been extensively discussed in the literature. there are two main thought processes for margin calculations; ( ) include all possible target positions during the breathing cycle [ ] or ( ) make a probabilistic margin calculation based on the target motion [ ]. this study pre- sents a third approach as a compromise between the two common techniques, as well as provides a simple method for margin calculation. a range of motion amplitudes and target margins was investigated, incorporating situations where the target remained within the ptv during treat- ment to where the target deviated up to . mm outside the ptv. this range was chosen to incorporate the broad spectrum of tumor motions; for example, although an upper lobe tumor may move considerably less than a lower lobe tumor, the smaller displacement data points from this study can be used for analysis, and vice versa. a relatively large upper limit of . mm displacement is useful to provide a full range of clinically possible motion tracks, although a tumor with this magnitude of displacement would most likely use alternative motion management techniques. in general, changes in d % were less than % if the margin sizes followed eq. . margin size mmð Þ ≥ max displacement mmð Þ ‐ mm ð Þ based on this investigation, the target can be displaced by up to mm outside the original ptv with less than % change in the target d %. additionally, this suggests that there is little benefit in adding a margin equal to or greater than the anticipated maximum displacement, versus a margin that is mm smaller than the extent of motion. further, this could potentially improve margin- size optimization, allowing for better sparing of normal tissue. clinically, margins are less likely to be created isotropically as presented in this study, but typically might be larger in the craniocaudal plane than the axial plane, for example. the data presented within this paper is still valid for anisotropic expansions: the formula pre- sented in equation will need to be separated into si and ap motion directions to create optimal margin sizes dependent on the three-dimensional nature of the motion. equation is not universally true for all motion tracks and for all patients; therefore, care has been taken to study a wide range of different lung tumors in order to make these conclusions as robust as possible. additional studies considering actual patient motion tracks are warranted to test the integrity of eq. , but are beyond the scope of this current investigation. equation provides a useful guideline for the treatment planning phase of sns imrt under the assumptions that the motion at time of simulation is the same as the motion at time of treatment. in figure it can be seen that the detrimental effects of motion are strongly dependent on motion direction. ap motion provides a small reduction in d %, even when the maximum target displacement is considerably larger than the margin size (Δd % ≈ . when the max- imum target displacement is mm outside the ptv). si and d motion caused much larger reductions in d %. the most likely explanation for this effect is due to directional differences in dose gradients for co-planar treatment plans, with a relatively steep dose fall-off out- side the target volume in the si direction compared to ap. movement of the target through these steep dose gradients with si motion would likely cause a greater dose blurring and therefore a larger reduction in d %. another explanation for the motion-direction depend- ence occurrence can be realized when considering the cumulative effect of multiple gantry angles on the inter- play and dose blurring effects. with zero couch rotation, si motion acts perpendicularly to the beam and in the plane of the mlcs; this increases the contribution of the interplay effect. depending on the gantry angle, ap mo- tion could potentially be moving parallel to the beam and perpendicular to the mlc plane, eliminating the interplay effect and reducing intra-fraction motion effects. in this scenario, the only effect of motion would be an inverse square correction (which is accounted for in the algorithm), a small effect compared to a physical displacement of the target perpendicular to the beam. with non-coplanar beam configurations, these results will clearly be different. figure demonstrates that the effect of motion on tar- get dosimetry is dependent on the maximum sinusoidal target displacement, independent of the type of sinusoidal motion leading to this maximum (e.g. offset, amplitude or drift). in other words, equation , and the corresponding data shown in figure , hold true regardless of whether the motion tracks creating the displacement were gener- ated with a drift, offset or variable amplitude. data from the independent variable studies are indistinguishable from each other when plotted in the format shown in figure d. the cumulative effect of motion over several fractions for several different starting phases (figure a) or motion tracks (figures b and c) demonstrated that, while the cumulative d % was approximately equal to the average d % of the individual fractions, the cumulative d % was typically less than that of the individual fractions. the sys- tematic peripheral cooling effect per fraction is present for most motion tracks so the cumulative effect of multiple fractions results in an average under-dosing in these regions. conversely however, the more random, smaller hot spots near the center of the target become less prominent with increasing fractionation. other combina- tions of motion tracks and fractionation schemes were waghorn et al. radiation oncology , : page of http://www.ro-journal.com/content/ / / calculated (data not shown), displaying similar effects to those shown in figure . the data shown in figure is representative of the observed effects of multiple treatment fractions, but a more thorough analysis look- ing into the complex relationships between margins, drifts and fractionation schemes is beyond the scope of this current study. conclusions motion-encoded dose distributions were calculated for multiple sinusoidal motion tracks applied to sns imrt plans for nine lung tumor patients. further study needs to be done using actual tumor motion tracks as they become available. however, the results using these simulated data provide valuable insight regarding the relationship between treatment dynamics and tumor motion for sns treatments, and also about the protective value of internal margins. as expected, the addition of an internal margin around the target forming the ptv reduced the poten- tially detrimental dosimetric impact of motion. for si motion the margin can be reduced by an additional mm while maintaining an acceptable dosimetry in the target (a change in d % of less than %), allowing for increased normal tissue sparing. this reduction can be increased even further for ap motion where sns imrt motion sensitivity appears to be less significant, with a co-planar beam arrangement. even in the presence of moving mlc leafs, data from this investigation suggest that with careful selection of an internal margin, the dosi- metric effects of motion can be successfully managed and the desired dose can be delivered to the target. additionally, it was found that clinical target volume under-coverage due to motion is neither reduced nor truly propagates as treatment fractionation is increased. consent this study was reviewed and approved by orlando health’s institutional review board. it was determined by the irb that no consent was necessary, as this study was a retrospective review of nine lung cases. competing interests the authors declare no conflicts of interests related to this investigation. authors’ contributions each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content. slm, kml, bjw designed the study. slm, bjw, aps performed the study and analysis. jmr, aps provided clinical assistance with patient planning. the manuscript was written by bjw; all other authors helped and approved the final manuscript. acknowledgements the study was supported in part by industry funding from .decimal, inc. received: july accepted: february published: february references . bortfeld t, jiang sb, rietzel e: effects of motion on the total dose distribution. semin radiat oncol , : – . . chen h, wu a, brandner ed, et al: dosimetric evaluations of the interplay effect in respiratory-gated intensity-modulated radiation therapy. med phys , : – . . duan j, shen s, fiveash jb, popple ra, brezovich ia: dosimetric and radiobiological impact of dose fractionation on respiratory motion induced imrt delivery errors: a volumetric dose measurement study. med phys , : – . . yu cx, jaffray da, wong jw: the effects of intra-fraction organ motion on the delivery of dynamic intensity modulation. phys med biol , : – . . court le, wagar m, ionascu d, berbeco r, chin l: management of the interplay effect when using dynamic mlc sequences to treat moving targets. med phys , : – . . keall pj, mageras gs, balter jm, et al: the management of respiratory motion in radiation oncology report of aapm task group . med phys , : – . . oliver m, staruch r, gladwish a, craig j, chen j, wong e: monte carlo dose calculation of segmental imrt delivery to a moving phantom using dynamic mlc and gating log files. phys med biol , :n –n . . ehler ed, nelms be, tome wa: on the dose to a moving target while employing different imrt delivery mechanisms. radiother oncol , : – . . kanagaki b, read pw, molloy ja, larner jm, sheng k: a motion phantom study on helical tomotherapy: the dosimetric impacts of delivery technique and motion. phys med biol , : – . . waghorn bj, shah ap, ngwa w, et al: a computational method for estimating the dosimetric effect of intra-fraction motion on step-and- shoot imrt and compensator plans. phys med biol , : – . . litzenberg dw, hadley sw, tyagi n, balter jm, ten haken rk, chetty ij: synchronized dynamic dose reconstruction. med phys , : – . . chui cs, yorke e, hong l: the effects of intra-fraction organ motion on the delivery of intensity-modulated field with a multileaf collimator. med phys , : – . . craft d, suss p, bortfeld t: the tradeoff between treatment plan quality and required number of monitor units in intensity-modulated radiotherapy. int j radiat oncol biol phys , : – . . suh y, dieterich s, cho b, keall pj: an analysis of thoracic and abdominal tumour motion for stereotactic body radiotherapy patients. phys med biol , : – . . underberg rw, lagerwaard fj, slotman bj, cuijers jp, senan sj: use of maximum intensity projections (mip) for target volume generation in dct scans for lung cancer. int j radiat oncol biol phys , : – . . van herk m: errors and margins in radiotherapy. sem rad onc , : – . doi: . / - x- - cite this article as: waghorn et al.: a margin-based analysis of the dosi- metric impact of motion on step-and-shoot imrt lung plans. radiation oncology : . submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www.biomedcentral.com/submit 	abstract 	purpose 	methods 	results 	conclusions 	introduction 	methods and materials 	treatment planning 	calculating the motion-encoded dose distribution 	motion tracks 	intrafraction motion dosimetric impact analysis 	multi-fractionation analysis 	results 	discussion 	conclusions 	consent 	competing interests 	authors’ contributions 	acknowledgements 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ i med genet ; : - syndrome of the month cartilage-hair hypoplasia makitie, t sulisalo, a de la chapelle, i kaitila cartilage-hair hypoplasia (chh) or mckusick type metaphyseal chondrodysplasia (mim no )' is a rare autosomal recessive chon- drodysplasia with short limbed short stature, hypoplastic hair, and defective immunity and erythrogenesis. ' the major radiological ab- normalities are confined to the metaphyseal regions of the tubular bones. the disease is prevalent among the old order amish in the united states and in the finnish population. while genealogical data confirm autosomal re- cessive inheritance, segregation analysis shows a lack of affected persons among both the amish and finnish chh families; this has been interpreted as a possible example of reduced penetrance. the chh gene was recently as- signed to chromosome by linkage analysis, and its localisation refined.' this has allowed prenatal diagnosis in four cases. ioa department of medical genetics, helsinki university hospital, haartmaninkatu b, sf- helsinki, finland o makitie i kaitila children's hospital, helsinki university hospital, helsinki, finland o makitie department of medical genetics, po box , university of helsinki, finland t sulisalo a de la chapelle i kaitila folkhiilsan institute of genetics, helsinki, finland t sulisalo a de la chapelle correspondence to: dr kaitila. clinical description growth failure the growth failure has its onset prenatally; the shortness of limbs or stature or both was noticed neonatally in % and by the age of year in % of finnish chh patients." all segments of the limbs are affected. the growth failure is progressive, owing in part to a weak or absent pubertal growth spurt; pubertal maturation is normal. adult heights range from - cm to - cm with medians of cm for males and cm for females. the growth failure is disproportionate with a long trunk in relation to the short limbs. the more severe the growth failure, the greater the disproportion." proportionate short stature has been described in some chh patients." median relative weight is above the normal mean in childhood and is further augmented around puberty; most adult patients are clin- ically obese. the head circumference is close to normal at all ages." other clinical features the majority of affected persons have sparse, fine, and silky hair; the eyebrows, eyelashes, and body hair are similarly affected. in some patients, however, the hair appears normal. this was observed in % of the finnish chh patients.' other clinical features include laxity of lig- aments, limited extension of the elbows, in- creased lumbar lordosis, bowing of the lower limbs, chest deformity (narrow thorax, har- rison's grooves, prominent sternum, or asym- metry), and mild scoliosis (figs and ).' immune deficiency the defective cellular immunity is char- acterised by mild to moderate lymphopenia, decreased delayed hypersensitivity, and im- paired in vitro responsiveness of lymphocytes to pha; humoral immunity is usually intact.""" a few patients with normal immunity" or combined immuno- deficiency" - have been reported. defective cellular immunity results in suscepti- bility to and mortality from infections. the infection problems are most pronounced in early childhood but occasionally persist to adult age. varicella infection has been fatal in a few cases. in the finnish series impaired in vitro cellular immunity was observed in % of the patients.' fifty-six percent had been unusually prone to infections. six patients ( %) had died of primary infections. on the other hand, % of the patients had shown no unusual sus- ceptibility to infections even though deficient cellular immunity had been confirmed in half of them.' the incidence of malignancies is increased as observed in the finnish series' and cases described earlier. - francomano et al ' re- ported an incidence of % of malignancies in amish chh patients: lymphoma in three patients, leukaemia in two, skin neoplasms in five, ocular cancer in one, and bile duct car- cinoma in one patient. ' among the finnish patients the incidence was six out of patients ( %): skin neoplasms in three patients, lymphoma in one, lymphosarcoma in one, and testicular tumour in one patient.' anaemia deficient erythrocyte production presents usu- ally as mild macrocytic anaemia in early child- hood with spontaneous recovery before adulthood. occasionally, however, the patients have severe, even fatal, congenital hy- poplastic anaemia. ' - sixty-seven out of finnish patients ( %) had been anaemic during childhood; in patients ( %) the anaemia had been severe (lowest haemoglobin value - g/l) with a fatal course in six patients. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ makitie, sulisalo, de la chapelle, kaitila .x..x...r x~~.. ..... i i! @ :':..a'......e...tww; nfijff ; gfiff;fffffffg;; j gffff ; gaffgfff ff;~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.......... figure i six chh patients, front view. age and absolute and relative values of height (from left to right): ( ) years, cm (- sd); ( ) years, - cm (- sd); ( ) years, cm (- sd) ( ) years, cm (- - sd); ( ) - years, - cm (- sd); ( ) - years, - cm (- - sd). intestinal manifestations there have been several descriptions of chh patients with congenital megacolon (hirsch- sprung's disease) - the finnish series included eight patients ( %) with con- genital megacolon. these cases clearly indicate increased prevalence of hirschsprung's disease among chh patients. intestinal malabsorption was suspected in six amish patients who had diarrhoea and failure to thrive in the first two years of life. however, primary malabsorption was not observed in any of the finnish chh patients; instead, gastrointestinal infection was confirmed in two patients with symptoms suggesting mal- absorption. these and other reported cases suggest that primary malabsorption is only ex- ceptionally associated with chh; mimicking symptoms may reflect an underlying gastro- intestinal infection. orthopaedic problems orthopaedic problems in chh are rare com- pared with many other chondrodysplasias. lig- amentous laxity and increased lumbar lordosis may cause arthralgic pains in the knees, ankles, or lumbar region of the spine. bow legs may necessitate corrective osteotomy (in % of the finnish patients).' radiographic features the tubular bones are short for age and their metaphyseal ends are flared, scalloped, and irregularly sclerotic, often with cystic areas (fig ); the epiphyses are less affected. the meta- physeal changes are most pronounced in the knees and ankles; the hips are only mildly affected (fig ). the metaphyseal irregularities disappear after the closure of the epiphyseal plates but the ends remain somewhat flared and angulated. the spine shows only minor abnormalities: the vertebral bodies are usually normal and caudal widening of the inter- pediculate distances, though less obvious than normal, is present in most patients (fig ). lumbar lordosis is increased and further ac- 'u.k~~~~~~~ me e, |w ,| af_se.tlm |--. f i _ _ _f l:nnew liss|c~~~~~~~-.mmwg ~aal_;:lm figure six chh patients, side view. same patients as in fig . note the variation in the degree of disproportion, hair hypoplasia, lumbar lordosis, and chest deformity. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ cartilage-hair hypoplasia figure radiograph of the legs of a year old boy. the metaphyseal ends are flared and irregularly sclerotic. note the excessive distal length of the fibula. figure radiograph of the hips of a year old boy (same patient as in fig ) showing only mild metaphyseal involvement in the proximal femora. figure constant interpediculate distances of mm at the levels offirst, third, and fifth lumbar vertebrae. the moderate lumbar lordosis ( ) is accentuated by a horizontally tilted sacrum (male, years). centuated by a horizontally tilted sacrum (fig ). differential diagnosis most of the different osteochondro- dysplasias result in disproportionate short stat- ure and progressive problems in the joints and spine. the clinical features of chh resemble those of hypochondroplasia (mim ) which, however, is a dominantly inherited con- dition and does not present with abnormal hair. it is easily differentiated from chh by normal metaphyses in childhood skeletal radiographs. murk jansen (mim ) and schmid (mim ) metaphyseal dysplasias are dominantly inherited conditions and result in severe growth failure but patients have normal immunity and erythrogenesis; these are easily distinguished from chh. recently, a type x collagen mutation was reported as the cause of schmid chondrodysplasia. shwachman-bod- ian syndrome (mim ), an autosomal recessive condition with metaphyseal in- volvement, is associated with pancreatic in- sufficiency, malabsorption, and leucopenia; the mild skeletal changes are most evident in the proximal femora. epidemiology chh was originally described among the old order amish, a religious isolate in the united states. at least amish chh patients have been recognised ; the incidence is estimated at - : corresponding to a carrier fre- quency of : . another accumulation of the disease has been observed among finns with patients in a population of million; the incidence is estimated at : live births and the carrier frequency at : . the number of diagnosed patients among other populations is low: patients have been reported among the french, eight among the dutch, seven among the british, and sporadic cases among the germans, danes, algerians, italians, pol- ish, spanish, and mexicans. whether this is because of underdiagnosis, under-reporting, or low incidence cannot be determined with cer- tainty. mapping of the gene for chh by linkage and linkage disequilibrium analysis assignment of the chh gene to the proximal part of p was accomplished by a random search for linkage in finnish families. sub- sequently, linkage to the same locus was shown in a large series of amish families.' no signs of heterogeneity within or between the two populations were detected. as there were no recombinations within the highly polymorphic marker d s (lod score - at = ) chh could be placed in the vicinity ofd s in the cm interval between markers d s and d s . as both the finnish and amish populations had few found- ers and have remained highly isolated, it could be assumed that the number of ancestral mut- ations would be small, possibly with a single o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ mdkitie, sulisalo, de la chapelle, kaitila mutation accounting for the majority of the patients in each population. haplotype analyses and linkage disequilibrium studies confirmed this assumption.' moreover, the strength of linkage disequilibrium was used to assess the distance between d s and chh by a re- cently developed method. - ' the current best estimate of this distance is - cm. genetics and penetrance genetic studies among amish and finnish fam- ilies have confirmed the recessive mode of in- heritance in chh. however, segregation analyses showed a slightly lower number of affected members than expected according to the recessive hypothesis. this has been ex- plained by reduced penetrance or by the loss of affected children in utero or in infancy. the mapping of chh and availability of closely linked polymorphic markers provide a tool to examine the question of penetrance. if reduced penetrance were responsible for the unexpectedly low proportion of affected chil- dren in chh sibships, healthy persons with haplotypes identical to their affected sibs should be found. such a phenomenon was looked for in a total of unaffected sibs but was not found. this finding is indeed credible as reduced penetrance appears to be very rare in recessively inherited disorders. several other mechanisms might account for the observed under-representation of affected children, which is more pronounced in the amish than in the finnish series. the haplotype analyses done in unaffected sibs appear to exclude not only reduced penetrance, but also genetic heterogeneity. remaining explanations are uniparental disomy, monosomy owing to de- letion, biased ascertainment of families, early lethality in a fraction of homozygotes, and gam- etic selection. prenatal diagnosis as chh may in some cases be severe or even fatal, some families with an affected child have requested prenatal testing for chh. the pre- cise mapping of the chh gene with respect to several highly polymorphic dna markers provides an excellent opportunity for accurate predictive testing based on the segregation of those markers in families with a previous history of chh. four prenatal determinations have so far been done; in three cases the fetus was predicted to be unaffected, and in one case an affected fetus was predicted.l"a in all cases the outcome was as predicted. hypotheses regarding the pathogenesis the pathogenesis of the disorder remains un- known. however, since previous studies have established defective cellular proliferation of t and b lymphocytes and fibroblasts, and defective erythrogenesis, it is suggested that the clinical manifestations (growth failure, sparse hair, recurrent infections, anaemia, hirschsprung's disease) in chh may result from a generalised defect of cellular pro- liferation. it is intriguing to note that a gene causing isolated hirschsprung's disease located on chromosome has recently been cloned. it follows that one of the pleiotropic effects of the chh gene, hirschsprung's dis- ease, arises either by a different mechanism from that in the isolated form of the disease, or by the interaction of the two genes. additional studies are needed to elucidate further the mo- lecular mechanisms in the pathogenesis of chh. the solution will probably have to await the cloning and characterisation of the gene itself. these studies have been supported by the academy of finland, the sigrid juselius foundation, the march of dimes birth defects foundation, the human growth foundation, the pai- vikki and sakari sohlberg foundation, the finnish medical foundation, the university of helsinki, and the foundation for paediatric research. mckusick va. mendelian inheritance in nman. baltimore: johns hopkins university press, . mckusick va, eldrige r, hostetler ja, ruangwit u, ege- land ja. dwarfism in the amish. ii. cartilage-hair hy- poplasia. bull johns hopkins hosp ; 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: - . ioa sulisalo t, sillence d, wilson m, ryynanen m, kaitila i. early prenatal diagnosis of cartilage-hair hypoplasia (chh) with polymorphic dna markers. prenatdiagn (in press). makitie , perheentupa j, kaitila i. growth in cartilage- hair hypoplasia. pediatr res ; : - . savage mo. metaphyseal dysplasia in siblings: a variant of cartilage-hair hypoplasia (case report). proc r soc med ; : . van der burgt i, haraldsson a, oosterwijk jc, van essen aj, weemaes c, hamel b. cartilage hair hypoplasia, metaphyseal chondrodysplasia type mckusick: de- scription of seven patients and review of the literature. amjmed genet ; : - . trojak je, polmar sh, winkelstein ja, et al. immunologic studies of cartilage-hair hypoplasia in the amish. johns hopkins medj ; : - . ranki a, perheentupa j, andersson lc, hayry p. in vitro t- and b-cell reactivity in cartilage hair hypoplasia. clin exp immunol ; : - . lischka a, frisch h, weissenbacher g. radiologische ver- anderungen bei metaphysarer chondrodystrophie typ mckusick (knorpel-haar-hypoplasie). monatsschr kin- derheilkd ; : - . ammann aj, sutliff w, millinchick e. antibody-mediated immunodeficiency in short-limbed dwarfism. _ pediatr ; : - . rubie h, graber d, fischer a, et al. hypoplasie du cartilage et des cheveux avec deficit immunitaire combine. ann pediatr (paris) ; : - . saulsbury ft, winkelstein ja, davis le, et al. combined immunodeficiency and vaccine-related poliomyelitis in a child with cartilage-hair hypoplasia. _ pediatr ; : - . steele rw, britton ha, anderson ct, kniker wt. severe combined immunodeficiency with cartilage-hair hy- poplasia: in vitro response to thymosin and attempted reconstitution. pediatr res ; o: - . wilson wg, aylsworth as, folds jd, whisnant jk. car- tilage-hair hypoplasia (metaphyseal chondrodysplasia, type mckusick) with combined immune deficiency: vari- able expression and development of immunologic func- tions in sibs. birth defects ; : - . harris re, baehner rl, gleiser s, weaver dd, hodes me. cartilage-hair hypoplasia, defective t-cell function, and diamond-blackfan anemia in an amish child. am _ med genet ; : - . hensley mf. mckusick's dwarfism and resistant hodgkin's disease (letter). jam osteopath assoc ; : . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ cartilage-hair hypoplasia roberts ma, arnold rm. hodgkin's lymphoma in a child with cartilage-hair hypoplasia (case report). milit med ; : - . francomano ca, trojak je, mckusick va. cartilage-hair hypoplasia in the amish: increased susceptibility to malig- nancy (abstract). am j hum genet ; : a. makitie , rajantie j, kaitila i. anaemia and macrocytosis - unrecognized features in cartilage-hair hypoplasia. acta paediatr ; : - . sacrez r, levy jm, godar g, castanier j. anemie de blackfan-diamond associe a des malformations multiples. med infantile ; : - . roberts pal, mann tp, rubin j. hirschsprung's disease associated with a variant form of achondroplasia, in sister and brother (case report). proc r soc med ; : . fauchier c, regy jm, combe p. nanisme diastrophique familial avec maladie de hirschsprung. ann pediatr (paris) ; : - . boothby cb, bower bd. cartilage hair hypoplasia. arch dis child ; : - . le merrer m, briard ml, chauvet ml, maroteaux p. autosomal recessive metaphyseal chondrodysplasia and hirschsprung's disease. ann pediatr (paris) ; : - . wood dj, david tj, chrystie ll, totterdell b. chronic enteric virus infection in two t-cell immunodeficient chil- dren. jmed virol ; : - . lux se, johnston rb, august cs, et al. chronic neutropenia and abnormal cellular immunity in cartilage-hair hypo- plasia. n engl _j med ; : - . warman ml, abbot m, apte ss, et al. a type x collagen mutation causes schmid metaphyseal chondrodysplasia. nature genet ; : - . makitie . cartilage-hair hypoplasia. clinical, radiological and genetic study of an inherited skeletal dysplasia. thesis, university of helsinki, . luria se, delbruck m. mutations of bacteria from virus sensitivity to virus resistance. genetics ; : - . hastbacka j, de la chapelle a, kaitila i, sistonen p, weaver a, lander e. linkage disequilibrium mapping in isolated populations: diastrophic dysplasia in finland. nature genet ; : - . lehesjoki ae, koskiniemi m, norio r, et al. localization of the epm gene for progressive myoclonus epilepsy on chromosome : linkage disequilibrium allows high resolution mapping. hum molec genet ; : - . sulisalo t, klockars j, makitie, , francomano ca, de la chapelle a, kaitila i. high-resolution linkage-dis- equilibrium mapping of the cartilage-hair hypoplasia gene. am jhum genet ; : - . bundey s, young id. low segregation ratios in autosomal recessive disorders. med genet ; : - . romeo g, ronchotto p, luo y, et al. point mutations affecting the tyrosine kinase domain of the ret proto- oncogene in hirschsprung's disease. nature ; : - . edery p, lyonnet s, mulligan lm, et al. mutations of the ret proto-oncogene in hirschsprung's disease. nature ; : - . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ pii: s - ( ) - pergamon pii: s - ( ) - .hmrnal of rural studies, vol. , no. , pp. - , © elsevier science ltd. all rights reserved printed in g r e a t britain - / $ .[~ + o.(x) thirty years of farmland preservation in north america: discourses and ideologies of a movement michael bunce department of geography, university of toronto at scarborough, military trail, scarborough, ontario, canada, m c i a abstract - - thirty years after it first captured public attention, farmland preserva- tion in north america remains a contentious issue which has failed to mature into an integrated element of rural land use planning. this paper argues that the explanation for this lies in the examination of the public discourses of the farmland preservation movement and the ideologies that underpin them. the evolution of popular and academic discourses and the influence of environmental and agrarian ideology are explored. this reveals an expanding discourse with ideological foun- dations riven with internal contradictions yet intersecting in different ways. the result has been a policy agenda influenced by a shift to increasingly broader moti- vations for farmland preservation and controlled by largely non-farm interests. farmers, however, remain at the centre of the issue, cast in roles ranging from guarantors of food supply to guardians of nature, open space and rural community. yet farm voices are barely detectable in the discourse of the farmland preservation movement. this illustrates the representative power of discourse and suggests why farmland preservation remains a contentious policy issue. © elsevier science ltd. all rights reserved introduction it is almost three decades since farmland preserva- tion first b e c a m e a serious public issue in c a n a d a and the united states. in that time a variety o f poli- cies have b e e n e n a c t e d to p r o t e c t farmland fro m development. y e i it has r e m a i n e d an issue; a contentious policy idea which has had limited success, continues to provoke d e b a t e about its p u r p o s e and effectiveness and which has never quite m a t u r e d into an integrated e l e m e n t o f rural land use planning. this is in distinct contrast to the situa- tion in much of western e u r o p e where farmland has not only b e e n subsumed by general countryside planning, but has also b e e n o v e r t a k e n by the belief that it is m o d e r n agricultural land use itself which represents the main t h r e a t to the countryside (e.g., shoard, ). this transatlantic contrast can be explained in part, o f course, by basic geographical, historical and political differences in the nature and use o f rural land which have fashioned divergent meanings o f countryside. it is also a function o f the relative recency o f the e m e r g e n c e o f agricultural land conversion as a planning issue in n o rth america. t h r o u g h o u t the s and s the d o m i n a n t public p e r c e p t i o n was o f a continent, with a limitless supply o f farmland and u n b o u n d e d technological capabilities, which was the b read b ask et o f the world. with large surpluses and the potential to p r o d u c e even g r e a t e r ones, the possibilities o f a declining farmland base were furthest fro m the thoughts of policy makers, and certainly w ere not a considera- tion in local land use planning. yet, by the early s, evidence o f a growing p r o b l e m o f soil degra- dation and urban sprawl p r o m p t e d the first academic rumblings o f c o n c e r n ab o u t the true productive capacity o f the n o r t h a m eri can agricul- tural land base (bogue, ; crerar, ). a few jurisdictions in the u n i t ed states - - maryland was the first - - a d o p t e d limited measures to r e d u c e urban pressure on state farmlands ( l e h m a n , ). but it t o o k a n o t h e r years for the issue o f the urban conversion o f farm l an d to cap t u re serious public attention. a flurry o f studies in the early michael bunce s revealed rapidly increasing rates of conversion (e.g., clibbon, ; real estate research corpora- tion, ; reilly, ; simpson-lewis et al., ). these revelations were quickly turned into media headlines and political action inevitably followed. canadian and us governments sponsored studies to measure rates of change in farmland which provided further ammunition for those who had begun to argue for more land use regulation (diderikson et al., ; manning and mccuaig, ; national agricultural lands study, ). and even before these reports were made public, state, provincial and local governments across the continent had begun to respond with various farmland protection policies. by the end of the decade most state and provincial and many local jurisdictions had some form of legis- lation in place (furuseth and pierce, ). much of this legislation, especially at the local level, was driven by concerns over wasteful patterns of urban development (lehman, ) in which farm- land preservation was used as a growth management tool to regulate urban sprawl. this urban-centred perspective on farmland preservation is a thread which has continued to run through local land use planning (daniels and nelson, ; bunce, ). yet, while urban sprawl was a significant factor in early policy initiatives, the emergence of farmland preservation as a public issue was fuelled more by concerns over the impact of sprawl on agricultural land itself and in particular on the productive capa- city of the resource base. productionist arguments dominated the emerging discourse of the farmland preservation movement. if so much farmland was being converted to non-agricultural uses, it seemed obvious that food production would be threatened. yet little evidence was produced to show that the overall level of agricultural output or the integrity of the agricultural economy was being seriously affected. the few predictive models of the impact of a declining land base provided no conclusive evidence of a serious threat to productive capacity (e.g., cocklin et al., ). to be sure, in regions with especially scarce and specialized agricultural lands, such as british columbia, ontario's niagara peninsula and parts of new england, it was not difficult to establish the necessity of strong farmland preservation strategies. and there was general agreement about the negative impacts of urbaniza- tion on agriculture in most metropolitan fringes. however, by the s it was apparent that the main threats to regional and national agricultural econo- mies came from overproduction and global competi- tion rather than land shortages. and in the decade since, liberalization of continental and global agri- cultural trade has further weakened the produc- tionist argument. moreover, challenges to the validity of at least the us farmland conversion data have prompted several prominent scholars to ques- tion whether there ever was a farmland crisis (crosson, ; fischel, ; platt, ; simon and sudman, ). given the weakness of the productionist argument, what then has sustained farmland preservation as a separate and contentious rural planning issue? that there are other motivations for restricting the urba- nization of agricultural land has been widely recog- nized (bryant and russwurm, ; bunce, ; jackson, ). these range across the control of urban sprawl, preservation of countryside amenity, protection of natural environment, maintenance of rural communities and the farming way of life, even, as in quebec, the guarding of national identity (mccallum, ). indeed bryant and russwurm ( ) went as far as to suggest that the apparent lack of resolution to the debate over the need for farmland preservation policy could be attributed, in part, to the multiplicity of values surrounding agri- cultural land. three years later, furuseth observed that as the number of farmland protection programs has proliferated, most recently diffusing downward to the local level, the controversy surrounding the rationale and the need for these activities has become increas- ingly intense. (furuseth, , p. ) however, while the varied agendas of farmland preservation have been acknowledged, there has been little attempt to explore in any depth their role in the farmland preservation debate. one of the exceptions is lehman's recent analysis of the history of federal government farmland policy in the united states (lehman, , ). he maintains that the farmland preservation issue has really been an ideo- logical struggle, the nature of which has been obscured by the dominance of the debate over the statistics of farmland loss. in this paper i argue that it is in the examination of this ideological dimension and of its influence on the broader valuation of farmland that a large part of the explanation for the peculiar status of farm- land preservation policy in north america lies. i argue further, however, that these values have been articulated through the public discourse of a loose- knit farmland preservation movement. it is this which defines the broad social and political context which, as cloke and little ( ) have rightly stressed, is where we should look for an under- standing of policy development. the discourse consists of a veritable babble of different voices, speaking at the national, regional, local and even personal level, representing academic, government, pressure-group and community viewpoints and arguing for the protection of agricultural land from thirty years of farmland preservation in north america a diversity of perspectives. yet running through all of these is the common thread of the socially constructed primacy of farmland. whatever its specific motivation, the language of farmland preser- vation articulates ideals for which farmland itself acts as a physical symbol and thus elevates the meaning and significance of agricultural life and landscape above that of a basic productive resource. this representative power of the discourse, that is its power to construct 'systems of meanings' (foucault, ) around the urbanization of agri- cultural land, has had a significant influence over farmland preservation policy. it is a discourse which taps the broader idealization of the countryside about which i have written elsewhere (bunce, ). but it rests more specifically on two paradoxically intersecting ideological foundations: environmen- talism and agrarianism. from resourcism to amenity: the environmentalist perspective ), which first resurrected the malthusian spectre of rapid population growth outstripping global food production capacity. but it was in the s and early s that this became, with nuclear war and the energy crisis, one of the central anxieties of an era replete with doomsday scenarios. books with sensationalist titles such as the population bomb (ehrlich, ), exploding humanity (regier and falls, ), the hungry planet (borgstrom, ) and famine- ! (paddock and paddock, ) and even the more measured analyses of lester brown ( ) and the fao (united nations, food and agricultural organization, ) engendered a general sense of imminent global food crisis. for the most part this was seen as a developing world rather than north american crisis. yet it had a profound impact on north american environmental attitudes and especially on attitudes to agricultural land. the prospect of declining per capita land supply, linked to the rhetoric of global famine, established a general unease about food production capacity which environmental groups readily exploited in spreading the gospel of resource stewardship. it is hardly surprising that farmland preservation, as lehman suggests in his recent study of its political history in the united states, should have emerged as a land use planning issue in north america with the rising environmentalism of the s (lehman, ). after all, environmentalism is an intellectual and social movement (evernden, ) which at its heart is about the reform of people-land relation- ships. and what could embody all of the aspects of this relationship more than farmland? at once a resource for satisfying basic human needs, a land- scape which retains natural elements and amenities, and an environment which still embodies our funda- mental connections with the earth, agricultural land inevitably became part of the broader environ- mentalist discourse; of the language, that is, of resource management, ecological conservation and amenity protection. resource management as i have already indicated, it was the threat to the capacity of the land base to sustain agricultural output which initiated farmland preservation as a land use policy issue in north america. this per- spective is firmly embedded in the resourcist ideology which dominated the burgeoning environ- mental movement of the s and s, and in particular in the neo-malthusianism which centred on the population-land equation. it was fairfield osborn's book, limits to the earth (osborn, ), followed by karl sax's, standing room only (sax, this resourcist environmentalist atmosphere, highly charged as it was with neo-malthusian rhetoric, provided ideal conditions for the germination of concern over north american agricultural land resources. but more importantly, it also furnished much of the language of the early farmland preser- vation movement. as academic concern over urban sprawl and evidence of urban conversion of agri- cultural land mounted during the s, the anxiety over global farmland capacity was transferred to the north american setting. the growing problem of soil erosion and other forms of land degradation had already been publicized by conservational organizations and, to some extent, recognized by government agencies. however, while the soil- degrading practices of an increasingly intensive north american agriculture presented a long-term threat to productive capacity, the promotion of improved management practices was generally seen to be a guarantee of restoring the renewability of agricultural land. with urbanization, however, the land was lost forever. even the relatively limited evidence of this was enough to spark the attention of a public which was already sensitized to the language of global famine and general resource scarcity. much of the initial public perception of the farm- land preservation issue therefore was shaped by the pessimistic scenario of running out of agricultural land in much the same way as we were running out of other resources. the language of farmland preservation presented images of an agricultural land base which was literally 'shrinking', 'dis- michael bunce appearing', even 'vanishing' before our eyes. much of this emanated from the popular media. headlines such as 'shrinking farmlands: sprawl of cities stirs fears that agriculture will run out of space' (presto, ), 'vanishing farmlands: selling out the soil (oguibene, ), 'the vanishing land' (macgregor, ), and 'ontario's farming land dwindling' (globe and mail, ) helped to keep these images in the public mind in both the united states and canada. nor were academics averse to using this language. as early as , krueger was writing of the 'disappearance' of the niagara fruit belt in ontario, a theme which he continued to pursue until the s (krueger, , ). lester brown, arguably the pre-eminent analyst of global cropland changes, wrote of the prospect of vanishing north american croplands (e.g., brown, ). and, not surprisingly, the farmland preservation move- ment adopted this language in its public campaigns. publications such as disappearing farmlands." a citi- zen's guide to agricultural land preservation from the national association of counties research foundation (thompson, ), claims about the 'rapid depletion of the nation's farmland resource' and of 'disappearing farmland' from the american farmland trust ( ), and dire warnings from the ontario coalition to preserve foodland that if the rate of urbanization were to continue 'by the year all our foodland would be lost' (ontario coali- tion to preserve foodland, ), typify the willing- ness of citizens' groups to employ the rhetoric of imminent farmland shortages. in the words of one skeptic, 'the united states was said to be running out of itself' (easterbrook, ). strongly influenced by what paarlberg ( ) has referred to as the 'scarcity syndrome', which domi- nated resourcist environmental thinking throughout the s and early s, the predictions of vanishing farmland were presented largely as a production capacity problem. a pamphlet, where have all the farmlands gone, which publicized the establishment of the us national agricultural lands study, re-echoed the disappearing farmland theme and raised the question 'how long will it be before the farm land loss severely cripples farm land production?' (national agricultural lands study, ). charles little, one of the leading figures in the nals programme, argued: it may now be asserted for the first time in this nation's history, that each new subdivision, highway, dam, factory, power plant or shopping centre threatens permanently to reduce the productive capacity of american agriculture. (national agricultural lands study, ) the popular media was quick to pick up this theme. 'america's capacity to feed itself and a burgeoning global population', wrote oguibene ( ) in the saturday review, 'now depends entirely on the amount of land we are willing to devote to agri- culture' (p. ). others were prepared to go as far as predicting domestic food shortages. as scientific an organiza- tion as the ontario institute of agrologists ( ) issued a report entitled foodland." preservation or starvation? its warning of the threat of urbanization to canadian food production capacity echoed the persistent theme of the country's seriously limited agricultural land resource base in the farmland preservation discourse in canada. nowhere is this more apparent than in the publications of the lands directorate of environment canada. set up in to 'keep an eye on the nation's land resources', for the next years it had a powerful influence on the development both of public awareness of and policy towards agricultural land. as i have already pointed out, no evidence could be produced of an immediate decline in agricultural output. much of the resourcist argument therefore was presented as a long-term food security issue. the introduction of the ontario foodland guide- lines, for example, was justified in terms of the need for policies which 'ensure that, as much as possible, land with the capability for agriculture is kept avail- able for farming when it is needed' (ontario, ministry of agriculture and food, , p. ), while another rationale for a national farmland policy presented by the nals study was public uncertainty 'about the capacity of the u.s. agricultural land base to supply food and fiber at the high levels that are likely to be demanded in the coming years' (national agricultural lands study, , p. ). in its illustration of the problem (fig. ), the american farmland trust presents the issue in terms of a 'narrowing margin of safety' highlighted with a large question mark. an argument that draws directly on the wise stewardship philosophy of resourcism, it exploits the uncertainties rather than the certainties of the impact of farmland conversion on production capacity. in particular it stresses the folly of paving over land that might be needed for future food needs: it seems to be only common sense to keep our options open...our ability to cope with the resource and food needs of the future will depend upon our present actions. (simpson-lewis and manning, ) at its height between the mid- s and mid- s, then, the farmland preservation discourse was domi- nated by the language of resource scarcity. that the arguments were at once simplistic and alarming made them all the more persuasive, especially in the thirty years of farmland preservation in north america e.. .o o ( > " o" uj o o m o < disappearing farmlands our narrowing margin of safety improved ;~ today technology i i available cropland current demand ( ) tomorrow remaining i future demand cropland ( ) assumes . million acre annual loss of cropland due to conversion and soil depletion. percent aggregate growth in domestic and export consumption, and i percent average annual growth in per acre crop yields through year . current data from usda national resources inventory. s o u r c e : a m e r i c a n f a r m l a n d t r u s t , . figure . molding of public opinion to support preservationist land use policies. yet this also generated the counter-argument that the resource scarcity predic- tions were smokescreens for other farmland preser- vation agendas (fischel, ; hart, ; simon, ). that the farmland preservation movement, both inside and outside government, could make no direct statistical link between conversion and declining food production not only aided the skep- tics but also led the movement towards broader rationales for its policy position. in the first instance this took the form of the food security issue that have just described. it was also driven by concerns about the profitability of farm enterprises and the sustainability of local agricultural economies. but the arguments for preserving agricultural land to ensure future food needs, as i have suggested, were influenced also by the wise-use philosophy of resourcism. the nals report emphasized the costs of 'farming the wrong acres'. prime farm lands, the report declared 'are our most energy-efficient acres, producing the most food, feed, fiber, forage and oilseed crops with the least amount of fuel, fertiliser and labour' (national agricultural lands study, ). the american farmland trust pointed out that bringing marginal lands into production would be 'prohibitively expensive' (american farmland trust, ). these arguments were strongly influ- enced by the growing evidence by the mid- s of diminishing returns to intensification. in the words of charles little: w h a t is becoming increasingly clear is that if high levels of productivity are needed, and if yields are not increasing, then it becomes important to look at the acres themselves. ( q u o t e d in lehman, , p. ) these kinds of statements were linked to the repeated assertion that the main threat came from the conversion of prime land. this played particu- larly well in canada where the lands directorate studies showed that half the conversion between and occurred on the best cropland (warren and rump, ). that this might push canadian agriculture onto more marginal land was declared to be self-evidently economic and environ- mental folly (mcquaig and manning, ). ecological conservation with this shift in attention from the quantity to the quality of land the farmland preservation issue was drawn under a broader, more ecologically oriented environmentalist banner. as i have already pointed out, early concern over the urbanization of farmland emerged in part from the soil conservation move- ment. the involvement of the soil conservation service in the united states in the first surveys of agricultural land conversion reveals the connection that was made from the outset between the paving over of farmland and the degradation of its soils. and this has been a persistent theme in the subse- quent farmland preservation discourse. lester brown has written of soil erosion and conversion to non-farm uses in the same context (brown, ). r. neil sampson, the vice-president of the national association of conservation districts in the united states, has echoed this, most notably in his paper- back, f a r m l a n d or wasteland: a t i m e to c h o o s e , in which the urbanization of agricultural land is portrayed as yet another degradation of a resource base already ravaged by intensive cultivation michael bunce (sampson, ). a tune which has been replayed again and again in the popular press, it also defines the purpose of the american farmland trust, which was established to 'address the twin threats of urban sprawl and soil erosion' (american farmland trust, ). in drawing parallels between these two threats, the argument being made was that agricultural land was under attack from within and without; from inten- sive agriculture and urban development. while much of the discussion of this was influenced by a resourcist perspective, it also attracted the attention of those who saw it as a more general warning about our relations with the natural environment. in arguing that the farmland preservation movement in the united states emerged with the environmental movement of the s, lehman ( ) stresses the influence of aldo leopold's land ethic. but in fact it is not until a decade later that this environmental philosophy finds its way into the farmland preserva- tion discourse. it was with the weakening of the production capacity arguments for farmland protec- tion that attention began to turn to more funda- mental agricultural land management questions. the call for farmland preservation as part of a new agri- cultural land ethic begins to appear in the late s. one of the earliest allusions to it came from mary rawson who in , in the context of farm- land protection policy in british columbia, wrote that in taking seriously the critical task of preserving food-producing lands, we do so in the belief that there are broader and less animal concerns...that nurture the growth of a new understanding of man's relationship to land, a new land ethic. (rawson, , p. ) similar rhetoric was employed by some of the leading protagonists of us farmland preservation policies. writing in a volume published by the soil conservation society of america, sampson proclaimed: what is needed is a new land ethic - - an ethic forged of our twin concerns for the land's proper use and proper care. farmlands should be given a 'place of honor' and looked at 'in the same way we would a van gogh painting'. (sampson, , p. ) sampson is extensively quoted in the publicity brochure which announced the national agricul- tural lands study, along with the opening lines of woody guthrie's popular song this land is your land and an extract from aldo leopold's writings. with this reinvocation of leopold's philosophy of an ecological relationship with agricultural land (leopold, ), the protection of good agricultural land from developers' shovels was now promoted as part of the more general argument for ecologically sustainable agriculture which had begun to emerge in the late s. although the leading protagonists of this philosophy rarely made direct reference to the urbanization issue, their writings helped to elevate agricultural land to the status proposed by sampson. arguably the most influential figure in this was wendell berry, whose prolific writings promoted the re-establishment of reverential rela- tionships with farmland (berry, , ). these ideas established an ecological stewardship philo- sophy which became a strong undercurrent in the farmland preservation movements of the s. take for example the ontario coalition to preserve foodland, an organization made up of farmland preservation and environmental groups, which began its campaign by arguing the usual food security line, but by the mid s was promoting a broader 'conservation ethic for those who manage foodland' and for a 'stewardship which means hugging the soil as if you were a child' (ontario coalition to preserve foodland, ). support for this ecocentric position came from the mainstream environmental organizations. in the second edition of its handbook for a conserver society in , friends of the earth devoted almost pages to the issue of agricultural land resources, adding its considerable influence to the argument that the protection of prime farmland from urban development was a necessary condition for an ecologically sustainable agriculture (friends of the earth, ). in the late s, the sierra club's national land use committee formed the 'farm- lands task force' to 'organize and assist local and regional efforts to protect america's valuable farm- land' (asbaugh, ). and it was the sierra club which, in coalition with the national association of soil conservation societies of america, national resources defense council and the national wild- life federation, formed the american farmland trust in (lehman, ). by the early s too, farmland preservation was being increasingly presented not just as a prerequi- site for sustainable agriculture, but also as an integral element of natural environmental protec- tion. in billing itself as the 'only national organisa- tion dedicated to conserving land for agricultural purposes - - with scenery and habitat protection as incidental public benefits', the american farmland trust implied that farmland preservation and the protection of the natural environment are synony- mous (american farmland trust, ). the preservation of agricultural lands society in ontario has gone further in broadening farmland preservation into a general conservational agenda with a high-minded strategy for the niagara fruit- lands which includes the 'preservation and enhance- thirty years of farmland preservation in north america ment of genetic diversity, through the protection, enlargement and linkages of natural areas, good conservation practices and the preservation of our genetic base for agriculture' (preservation of agri- cultural lands society, , p. ). and, displaying extraordinary ignorance of modern agriculture, the save the rouge valley organization on the outskirts of toronto argues for 'colourful rows of farmers' fields, majestic cliffs, meandering watercourses, dense forests, lush wetlands, and a flurry of wildlife activity. this is a well-balanced ecosystem...' (save the rouge valley system, , p. ). local amenity protection of a rapidly expanding community conservation movement, which in large measure has hijacked the farmland preservation agenda for its own amenity ends. a widely quoted example is the farmlands conservation project ('save the farmbelt') of the people for open space organization in california's bay area. in answer to its own question, 'how do people in cities benefit by having farms and ranches near them?' the project proposed a broad amenity role for farmland as the basis of a permanent green- belt, 'to produce a portion of our food; to supply high-quality water; to reduce the threat of floods and other hazards; to maintain the richness of plant and animal life; to give us room to wander and to breathe' (people for open space, , p. ). while this perspective may well reflect an altruistic environmental ethic it is one which is dominated by urban and intellectual values (lehman, ), which are readily translated into language which plays well with a public increasingly concerned about the environmental quality of its surroundings. for long the subtext of productionist and resourcist argu- ments, the protection of the general amenity quality of rural land and community has become an increas- ingly important theme. while this has influenced the policy debate at the national, state and provincial level, it is at the local scale that the amenity pers- pective has become dominant. indeed in the united states especially, it is local rather than state govern- ments which have generally been most successful in implementing measures to protect farmland (alterman, ). the amenity rationale has been widely recognized in the literature on farmland preservation. indeed pierce and s guin have argued that the weakening of the resource adequacy issue in the public mind led directly to the shift in support for protecting the amenity values of farmland (pierce and s guin, ). although this is a largely a development which emerged in the mid- , the advantages of combining the two causes of open- space and farmland protection have always rumbled beneath the surface of the farmland preservation movement. it was at the heart of the refer- endum campaign in new jersey (esseks, ) and in the early s, the save our farms committee which was instrumental in the implementation of the pioneering purchase of development rights programme in suffolk county, new york, set out to 'secure through the farmland preservation program- ...clean water, clean air, agriculture, a balanced economy, lower taxes, sound planning, open space and recreation' (quoted in esseks, ). as local governments have played an increasingly central role in the initiation of preservation policies and programmes, especially at the county level in the united states, they have done so in the context however, it is in the upsurge of community protec- tionism in gentrified exurbia, rather than in regional greenbelt planning and certainly the agricultural heartland, that the amenity perspective is most evident. much of the contemporary farmland preser- vation campaign now occurs in the context of local amenity activism (furuseth, ; peters, ), which wright argues is the consequence of the 'failure of governmental land-use planning programs to protect cherished places from urbanization' (wright, , p. ). the past decade has seen the emergence of grass-roots initiatives in which farm- land preservation is embedded in a broader move- ment to protect rural environment and character. in outlining a set of strategies for local rural initiatives, the national trust for historic preservation argues in its 'save america's countryside campaign' that 'rural conservation should integrate natural resource conservation, farmland retention, historic preserva- tion and scenic protection' (stokes, , p. ). in placing its emphasis on the 'historic countryside' the trust reflects sentiments which blend natural environment, farmland and old buildings into a single notion of rural heritage. this perspective has found its most influential expression in the land trust movement which has spread across rural america at a phenomenal rate in the past decade. local non-profit organizations which 'are directly involved in protecting land for its natural, recrea- tional, scenic, historical or productive value, through direct acquisition, conservation easements and other forms of community control over land development, land trusts now number almost a thousand and are the principal vehicle for organising local conserva- tion (elfring, ). many trusts, such as the marin agricultural land trust just north of california which was instru- mental in negotiating conservation easements on property owned by the movie producer george lucas (peters, ), do indeed focus their attention michael bunce on farmland preservation (elfring, ) usually in cooperation with local government protection programmes. yet agricultural land is generally presented as an essential part of the overall conservation package, fulfilling the role of guardian of open space, nature, scenery and rural character, and, by extension, of bulwark against urban develop- ment. the flathead land trust uses conservation easements, to keep the land 'in its agricultural use and still protect its scenic value and its natural habitat' (land trust alliance, ). the open space programme in boulder, colorado has preserved acres of open space to protect scenery, create recreational trails and to safeguard farmland (elfring, ). the american farmland trust has taken an increasingly active role in promoting the link between natural and cultural amenity and the preservation of farmland. in mary- land, for example, it recently funded a conservation easement which prevented residential development of sugarloaf farm and thereby ensured an essential link in the greenway connecting washington, dc with sugarloaf mountain, a popular scenic attrac- tion. nor are these activities restricted to eastern conurbations. in northeastern nevada, the trust has been working with the nature conservancy in the ruby valley to protect an ecosystem which can sustain both wildlife and food production (thompson, ). it is this undercurrent of broader rural amenity conservation which has prompted much of the criti- cism of the farmland movement. one critic of the us national agricultural lands study has gone so far as to suggest that the productionist argument has merely been a ploy used by local amenity groups to 'elevate farmland preservation to the national agenda' and that 'the real beneficiaries are local anti-development interests' (fischel, ), while the most outspoken critic of farmland preservation poli- cies, julian simon, has declared that 'the famine- protection claims are simply a smoke screen for property owners who want a bucolic v i e w . . . ' (simon, ). these arguments ignore the role that concern over the survival of the local agricultural economy has played in sustaining the farmland preservation movement. yet they are hard to refute in the face of local rural conservation agendas which are so obviously dominated by amenity perspectives, especially when these are largely controlled by non-farm people. local rural conservation is at its most active in exurban communities, in which the protection of open space and of rural character is inextricably bound up in lifestyle and property values. when this is expressed in the language of groups like the pickering rural association just east of toronto, which opposes the conversion of farmland and open space to 'keep pickering rural' and thus to ensure the rights of a largely exurbanite population to the 'peaceful enjoyment' of its properties, then the subtext of farmland preservation is quite trans- parent (pickering rural association, ). the pickering organization, like most of its counterparts in canada, is far less sophisticated as well as worse funded than most of the land trusts and other rural conservation groups in the united states. yet its fledgling attempts to influence the local planning agenda mirror the self-interest of much of the rural preservation movement across the continent. the desire to preserve rural atmosphere is entirely consistent with the values of those who have invested directly in their private version of the coun- tryside ideal (bunce and walker, ). however, as these groups have come to dominate local preserva- tion movements, their influence has spilled over the boundaries of private amenity into the public land- scape. at its most sophisticated this is articulated through a preservation ethic which aims to impose its own socially constructed notions of rural authen- ticity on the community as a whole. in his eloquent study of an exurban pennsylvania community, dorst describes how environmental conservation, farmland protection and historic preservation are combined under a broad heritage umbrella. this is intended to do more than merely shelter the community from further development, but rather to develop it to conform to an 'agrarian historical ideal' (dorst, , p. ). agrarian ideals the important point in dorst's interpretation is that agriculture is retained because it is an essential ingredient of rural authenticity. and the retention, even the re-establishment of farms and of a local agricultural economy and culture, as well as the farmscapes they support, is dependent on the preservation of farmland. at its most superficial, this is simply another way of justifying the private amenity agenda. but when we examine it more closely we can detect underlying values which reach back into the persistent agrarian ideologies which, as numerous studies have shown, are entrenched in american, and, less explicitly, in canadian political and social culture (e.g. flinn and johnson, ; dalecki and coughenour, ; rohrer and douglas, ). from its early nineteenth century origins, agrar- ianism has evolved today into a diverse set of values. while these have in common the belief that farmers and farming are valuable elements of society and thirty years of farmland preservation in north america economy (montmarquet, ), this is a vague notion which has been readily manipulated to serve a variety of agendas from the promotion of agri- business to the protection of the traditional family farm and rural community (beus and dunlap, ), from its outset, the farmland preservation move- ment has invoked the progressive agrarianism that argues for the maintenance of a strong and produc- tive agricultural economy. it is with this perspective that the movement has been able to draw some support from farmers who have otherwise tended to oppose its more general regulatory objectives (furu- seth, ). the resourcist environmental rationale which dominated the general farmland preservation discourse at its height was, in large measure, matched by an economic discourse which portrayed agriculture as a strategic sector of national and regional economies. as a president of the american farmland trust put it: at stake is nothing less than the world's food supply and the stability of our nation's most basic industry. (wheeler, , p. ) in this language we can recognize the physiocratic agrarianism - - the belief that the true wealth of the nation is drawn from the land - - w h i c h underlies the progressive side of jeffersonian ideology. but jefferson saw agriculture as also serving political and social goals. in declaring, in his much-quoted phrase, that 'those who labour the earth are the chosen people of god', jefferson saw the indepen- dent yeoman farmer as the source of moral and civic responsibility, the foundation of an harmonious yet bountiful agrarian republic. the farmland preserva- tion discourse is replete with references to the protection of the farm enterprise, and especially of the family farm. in the popular media the urbaniza- tion of farmland and the demise of the family farm have invariably been presented as a coterminous issue. this was especially true of the height of the first upsurge of public concern over farmland in the mid- s when articles with headlines such as "saving the farms' (time, ) and 'suddenly an alarm over vanishing farms' (us news and world report, ) appeared in the popular media with as much frequency and with much the same language as commentaries on the loss of farmland. the nostalgic tone of this language represents a corrupted version of the jeffersonian ideal, a senti- mental strand of agrarian ideology which looks to the family farm and its land as the lingering symbols of an era of rural virtue, simplicity and self-reliance, the loss of which threatens the very heart and soul of the nation and leads to the severing of rural roots, it is this agrarian perspective which local rural preservationists invoke in drawing farmland preser- vation into the broader agricultural heritage preser- vation agenda. farmland preservation is assimilated into the process of what dorst ( ) calls 'tradi- tionalisation', through the extension of heritage preservation to a living agrarian society. historic farmhouses and barns are more interesting if they are still used by farm families and surrounded by active farmland. (stokes, , p. ) this taps into what has been termed the romantic stream of agrarianism (danbom, ; mont- marquet, ), which promotes the culture rather the economics of farming - - agriculture as a way of life - - at the heart of which are notions of connect- edness to land, nature and community. inspired, as danbom ( ) has suggested, more by thoreau than jefferson, this is an ideology which has its roots in the anti-urbanism and anti-industrialism of the back-to-the-land movements of the early part of this century. this has been a recurrent theme in the growth of a gemeinschaft-inspired communitar- ianism (naples, ), in which the ills of urban- industrial society are to be solved by returning to thc land, which in turn w i l l lead to the re-establishment of the ideal rural community. the modern exponents of this ideal extend it into a broad thesis which argues that it is both urbaniza- tion and agricultural industrialization which repre- sent the real threat to true community. only by preserving and restoring traditional family farm culture - - 'the good work of good farmers' - - can we re-establish the traditional rural community (berry, ). the academic contribution to this particular discourse is epitomized by the gold- schmidt thesis which links the demise of the family farm with the decline of rural community (gold- schmidt, ). while this thesis remains controver- sial amongst rural sociologists it has long influenced a stream of research which focuses on the problems of the decline of family farming and which reveals a deep-rooted popular agrarian sentiment which is most frequently associated with the family farm- based community ideal (e.g., flinn and johnson, ; buttel and flinn, ; willits et al., ). indeed this research has revealed such strong agrarian sentiments in american society at large that it has prompted molner and wu to characterize the family farm as a 'national icon' and a 'sacred object' (molnar and wu, ). perhaps the best illustration of the strength of this ideal is the popularity of amish and mennonite culture areas and images. amish society in particular, with its explicit adherence to traditional ways of agrarian life, satisfies "a very strong american nostalgia for historical roots, traditional values and 'good old days'" (cong, ). but above michael bunce all it is the amish perpetuation of the family farm and the ideals with which it is associated which has captured the public imagination. nowhere is this more evident than in lancaster county, pennsyl- vania where the artifacts of amish culture have been commodified into a highly developed tourist region, complete with an internet web site, which promotes an 'amish country' offering a wide range of nostalgic tourist experiences (www. padutch. corn/). at the same time the amish cultural land- scape has become the symbol of rural authenticity of the local historic preservation movement to which i referred earlier. and it is this landscape which the lancaster farmland trust sets out to preserve in its farmland protection activities, campaigning for the protection of farms and farmland to ensure the 'productivity, character and quality of life that exists here' (lancaster farmland trust, ). the main- tenance of working amish farms through the acqui- sition of conservation easements on surrounding farmland is integral to this objective (lancaster farmland trust, ; niemeyer and kraybill, ). the link between the protection of farms and the preservation of farmland has been made explicit in the recent policies of the american farmland trust. in a document entitled saving the farm: a handbook for conserving agricultural land, the trust argues for a comprehensive agenda which places the sustainability of the family farm at the centre of its farmland preservation activities (american farmland trust, ). to a large extent this represents an agrarian ideal which reveals the growing support for an agriculture which operates in the local interest, not just in terms of protecting landscape and heritage, but also in satisfying demand for local produce through farmers' markets, farm-gate sales and pick-your-own operations. these are integral to fabrication of rural authenticity, providing both the flavour of the traditional market place as well as direct connections to the land. both experientially and symbolically they tie into an agrarianism which supports a food system based upon family farms which serve local markets. but it also supports an alternative agriculture which is defined by its ecological sustainability. the growth in demand for local food markets is generally accompanied by demands for produce, and there- fore farming, which is environmentally and livestock friendly. free-range eggs, organically grown veget- ables, hand-churned butter and stone-ground flour have become an essential part of exurban lifestyle. much of the support for this version of agrarian values comes from those who have sought back- to-the-land experiences in small-scale operations which intentionally function in an alternative mode of production and marketing. the restoration of the organic links of farming is thus an essential plank in the broader rural conservation agenda. but what is important here is that farmers are cast in the role of stewards of both land and community. when they are regarded as guarantors of community we can recognize the influ- ence of the values of agrarian fundamentalism, of the myth of farming as the foundation of a sound and sustainable civilization, and of the 'intrinsic value of farming and the economic, political and social value of the family farm to american society' (singer and de sousa, , p. ). this harks back to the southern agrarianism of jefferson with his emphasis on the moral and social values of the inde- pendent family farm (montmarquet, ). this is the image of the steadfast and responsible yeoman - - 'agrarianism's soft-side...which seeks a renais- sance of the self-sufficient, craftsman-naturalist who manages sustainable ecosystems' (dalecki and coughenour, , p. ). viewed from this perspective, farmers are assigned the moral responsibility for caring for the land over which they have been given control, in other words the role of land stewardship. this is a strand of agrarian fundamentalism which is strongly influ- enced by christian theology (bruegemann, ), in which farmers are reminded of 'their responsibility as god's stewards' of the land' (ontario conference of catholic bishops, , p. ). this is a philosophy which has found a comfortable home in the farm- land preservation movement itself. in ontario, for example, the christian farmers federation has played an active role in the province's principal farmland preservation citizen's group, arguing frequently for farmland protection as part of a broader christian duty to ensure the perpetuation of family farmers as 'earthkeepers' (ontario coalition to preserve foodland, ). this notion of land as a sacred trust provides links to the environmentalist arguments for farmland preservation, for it invokes elements of leopold's land ethic, of a relationship with land which involves 'an ethical obligation on the part of the private owner' to act for the good of what leopold calls the 'land community' (leopold, ). although leopold has had an enormous influence on the environmental movement in general, his philosophy is articulated in terms of personal rather than societal relations with nature; with the rela- tions, that is, that come with land and especially farm ownership. but what is more important is that it is a philosophy which regards stewardship not as a productionist goal, but rather as an ethical and spiritual end. for wendell berry, arguably the most eloquent proponent of an ethical, ecological alterna- tive to industrial agriculture, this translates into the thirty years of farmland preservation in north america old-fashioned practice of good husbandry, of a way of farming which is based on harmony with nature and therefore with family and community. for berry agriculture is a cultural not an economic activity, best pursued on small farms producing for them- selves and their community (berry, ). this organic connection between land and community is a theme which can be detected in much of the local rural conservation movement. 'the farmbelt as culture' was one of the six reasons listed by the people for open space organization for protecting farmland in the san francisco bay area: with farmland goes a farming culture, an agricultural way of life... in rural towns where the family farm tradi- tion is strong, people do seem to get involved easily in civic groups and community affairs: there are plenty of joiners. (people for open space, , p. ) d i s c u s s i o n in this paper i have argued for understanding the development of the farmland preservation issue in north america in terms of an expanding discourse, the language of which reveals the influence of two ideological streams: environmentalism and agrar- ianism. these are powerful yet complex ideologies, riven both with their own internal contradictions and their conflicts with each other. yet they also reveal points of convergence which help to explain t h e interrelationships between what has been previously treated as a disparate list of rationales for farmland preservation. within each of these ideologies we can recognize two apparently competing sets of values which have shaped the farmland preservation discourse. the influence of environmentalism divides into two fairly distinct streams. the first of these situates farmland preservation in resourcist environmental philosophy, emphasizing (especially in the early campaigns to generate public and official support) the importance of protecting the resource base as a guarantee of maintaining food production. alongside this per- spective, however, has emerged an ecological environmentalism which promotes farmland preser- vation as an issue of general environmental protec- tion. agrarianism follows similarly divergent paths. on the one hand it is the progressive agrarianism which promotes the centrality of a productive agri- cultural economy to the national interest. on the other it is the romantic and fundamentalist agrar- ianism with its emphasis on the culture of farming and its centrality to notions of rural authenticity and the restoration of connections between nature, land use and community. while at first glance these may appear as four largely separate and competing philosophies, under the broad banner of farmland preservation we can discern a common theme between resourcist envir- onmentalism and progressive agrarianism on the one hand and between romantic agrarianism and ecological environmentalism on the other. certainly the mainstream argument for protecting farmland from conversion to other uses has long been made in terms both of maximizing the supply of agri- cultural land resources and of maintaining a strong agricultural economy. this essentially brings the two ideologies together under a productionist and utili- tarian rationale. by the same token, there is an obvious synchronicity between promoting farmland preservation as a prerequisite for general environ- mental conservation and regarding it as an essential element in protecting rural heritage and local amenity. here the rationale is cultural and ecological. of course there are tensions within these two cate- gories as well as overlaps between them. the productionist perspective embraces both the intensi- fication of land use and the wise management approaches to land resources. the cultural/ ecological framework supports an even broader range of values, from self-interested amenity motives to ecocentric and moralistic ideals of sustainable community. it is with the notions of sustainability of land use and community that we can recognize some of the overlap between the two sets of philosophies. indeed, as the farmland preser- vation movement has matured in north america it has increasingly brought together issues of resource management, environmental protection, farm and community survival under the sustainability umbrella. this is especially true where farmland preservation has been subsumed by more general rural conservation activity, but, as we have seen, it has also been a developing undercurrent in the farmland preservation organizations themselves as they have broadened their message beyond the productionist rationale. within this confusion of conflicting, overlapping and merging ideologies, however, there is one constant: the centrality of farmers. whatever the argument for farmland preservation, farmers and their land have been placed at the centre of the issue, cast in the role of guarantors of food supply, of national, regional and local economic stability and of our connections with the earth, as well as in the role of guardians of nature, landscape, open space, rural heritage and community values. they have been both coerced and co-opted into fulfilling these roles. coercion has occurred through land use regulations - - restrictive zoning, severance and subdivision michael bunce control, compulsory agricultural districting - - which are intended to restrict farmers' rights of land disposal and therefore keep them on the land, so that it can continue to serve the various farmland preservation objectives. faced with the loss of development rights, farmers have tended to be hostile or at the very least ambivalent towards the circumscription of their property rights (bryant and johnston, ). a more subtle approach, therefore, has been to co-opt farmers to the farmland preser- vation agenda through incentives and voluntary agreements. this is far more common in the united states than it is in canada, largely because of the constitutional enshrinement of american property rights, but also because of the strength of american agrarian ideology which continues to support the independence of the property-owning family farm. and so, first with tax incentives, the transfer and purchase of development rights, and more recently with voluntary agricultural districting, conservation easements and management agreements, farmers have been drawn into the farmland preservation process as more or less willing participants. this is particularly apparent in local rural conservation activity, in which conservation and land trusts operate through negotiated agreements with land- owners. in some instances, farmers have become fully co-opted into the conservation process. a recent example is the chesapeake farms for the future board which includes farmers in a programme to protect farmland in maryland and delaware as part of the sustainable agriculture project of the future harvest project (future harvest, ). the determination of the degree of circumscription of property rights in farmland preservation policy presents the problem of resolving the main internal contradiction of agrarianism; between the independ- ence of the family farm and the interests of society. jefferson resolved this by arguing that family farmers are fixed on their land by the benefits of generational succession and the economic constraints on expansion and mobility (browne et al., ). they therefore have a vested interest in maintaining the land and supporting the community. although a serious distortion of the nature and objectives of family farming, even in jefferson's time, it is a myth which has had a powerful influence on the development of north american tarmland preservation policy. if farmers can be fixed on their land, then the protection of farmland in all of its dimensions will naturally follow. paradoxically this has had the effect of entrenching, rather than diminishing, farmers' property rights. as braden has put it, 'the major theme of farmland preservation programs is to enhance private values attached to agricultural land rights' (braden, , p. ). conclusion if, as i have suggested, the inherent conflicts between the various objectives and ideologies which constitute the farmland preservation agenda are resolved through the agency of farmers themselves, then the question that follows is, where do farmers fit in the discourses of the farmland preservation movement? to answer this question empirically would require an analysis which goes beyond the scope of this paper. however, what i have shown is that the movement to protect farmland is dominated and controlled by the intersection of popular and professional discourses (jones, ), and therefore of largely non-agricultural voices. this is not to say that farmers are excluded in the way, for example, the environmental movement takes an appositional stance towards corporate industry and agribusiness. on the contrary, as i have argued, the infusion of agrarian ideologies into the farmland preservation discourse portrays farmers, at least family farmers, as natural allies, who simply require the protection of their main resource, land, in order to fulfill their prescribed and chosen roles. whether farmers accept this role is doubtful. a few studies have shown principled farmer support for farmland preservation (furuseth, t ) while there is occa- sional farmer involvement in preservation organiza- tions (e.g., friends of foodland in ontario which is led by a farmer from the christian farmers associa- tion). farmers too have been supportive of farmland preservation when neighbouring non-farm uses impinge on their activities (pfeffer and lapping, ). but most evidence suggests general resistance from farmers' groups to significant restrictions on their development rights. what is certain is that mainstream farm voices are barely detectable in the farmland preservation movement. this leaves us with the question of who really defines and controls the farmland preservation agenda and whose interests it really serves. if, as this paper implies, it is an agenda which has come to be defined in terms of the socially constructed primacy of farmland as a physical symbol of a mix of ideolo- gies and values held largely by non-farm people, especially those occupying the urban fringe, then it is hardly surprising that it remains a distinct and contentious planning issue. as troughton has so perceptively put it: ...the true nature and the real needs have not been identified. at a very basic level, no farmland use policy is likely to have a satisfactory long-term effect in the absence of parallel attention to farm income and its maintenance. (troughton, , p. ) it is this discontinuity between what is needed and what has actually happened that explains the thirty years o f f a r m l a n d p r e s e r v a t i o n in n o r t h a m e r i c a p a t e n t l y limited success in n o r t h a m e r i c a o f m o s t f a r m l a n d p r e s e r v a t i o n policy a n d the failure to inte- g r a t e it with b r o a d e r rural land use policy. but b e y o n d this it illustrates the p o w e r o f public discourse to r e p r e s e n t the value o f f a r m l a n d in t e r m s o f its own interests a n d ideologies and thus to have significant influence o v e r the policy agenda. r e f e r e n c e s alterman, r. 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[pdf] a multi-institutional study of brainstem gliomas in children with neurofibromatosis type | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /wnl. corpus id: a multi-institutional study of brainstem gliomas in children with neurofibromatosis type @article{mahdi ams, title={a multi-institutional study of brainstem gliomas in children with neurofibromatosis type }, author={j. mahdi and a. shah and aimee sato and s. morris and r. mckinstry and r. listernick and r. packer and m. fisher and d. gutmann}, journal={neurology}, year={ }, volume={ }, pages={ - } } 	j. mahdi, a. shah, + authors d. gutmann 	published 	medicine 	neurology objective: to define the clinical and radiologic features of brainstem gliomas (bsgs) in children with neurofibromatosis type (nf ). methods: we performed a retrospective cross-sectional study of children with nf and concurrent bsgs cared for at nf referral centers. bsg was determined using radiographic criteria. age at diagnosis, tumor location and appearance, clinical symptoms, treatment, and presence of a concurrent optic pathway glioma were assessed. results: the average age at… expand view on wolters kluwer europepmc.org save to library create alert cite launch research feed share this paper citationsbackground citations results citations view all figures, tables, and topics from this paper 	 table 	 figure 	 figure 	 table 	 figure view all figures & tables 	neoplasms 	neurofibromatosis 	neurofibromatoses 	cessation of life 	bsg gene 	midbrain structure 	visual pathway glioma 	numerous 	brainstem glioma 	eye 	hypertrophy citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency nonoptic pathway tumors in children with neurofibromatosis type 	j. mahdi, m. goyal, j. griffith, s. morris, d. gutmann 	medicine 	neurology 	 	 save alert research feed increased prevalence of brain tumors classified as t hyperintensities in neurofibromatosis 	j. griffith, s. morris, j. mahdi, m. goyal, t. hershey, d. gutmann 	medicine 	neurology. clinical practice 	 	 	 	view excerpt, cites background save alert research feed cns tumors in neurofibromatosis. 	j. campian, d. gutmann 	medicine 	journal of clinical oncology : official journal of the american society of clinical oncology 	 	 	 save alert research feed pediatric malignancies in neurofibromatosis type : a population‐based cohort study 	s. peltonen, roope a. kallionpää, + authors j. peltonen 	medicine 	international journal of cancer 	 	 	 save alert research feed brain tumors in neurofibromatosis type 	amanda de andrade costa, d. gutmann 	medicine 	neuro-oncology advances 	 	 	pdf save alert research feed neurofibromatosis french national guidelines based on an extensive literature review since 	christina bergqvist, a. servy, l. valeyrie-allanore, s. ferkal, p. combemale, p. wolkenstein 	medicine 	orphanet journal of rare diseases 	 	 	 save alert research feed an update on neurofibromatosis type -associated gliomas 	m. lobbous, j. bernstock, + authors l. nabors 	medicine 	cancers 	 	 	pdf save alert research feed available therapies for patients with neurofibromatosis-related nervous system tumors 	r. strowd 	medicine 	current treatment options in oncology 	 	 save alert research feed favorable prognosis in pediatric brainstem low‐grade glioma: report from the german siop‐lgg cohort 	johannes holzapfel, daniela kandels, + authors a. gnekow 	medicine 	international journal of cancer 	 	 	 save alert research feed brainstem glioblastoma multiforme in a patient with nf 	john t fortunato, brian d reys, p. singh, edward pan 	medicine 	anticancer research 	 	 	pdf save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency optic pathway gliomas in neurofibromatosis type : the effect of presenting symptoms on outcome 	a. king, r. listernick, j. charrow, l. piersall, d. gutmann 	medicine 	american journal of medical genetics. part a 	 	 	view excerpts, references results save alert research feed brainstem lesions in neurofibromatosis type 	n. ullrich, a. i. raja, m. irons, m. kieran, l. goumnerova 	medicine 	neurosurgery 	 	 save alert research feed the management of brainstem gliomas in patients with neurofibromatosis 	i. pollack, b. shultz, j. mulvihill 	medicine 	neurology 	 	 save alert research feed prognostic factors of cns tumours in neurofibromatosis (nf ): a retrospective study of patients. 	j. guillamo, a. créange, + authors p. wolkenstein 	medicine 	brain : a journal of neurology 	 	 	pdf 	view excerpt, references background save alert research feed natural history of optic pathway tumors in children with neurofibromatosis type : a longitudinal study. 	r. listernick, j. charrow, m. greenwald, m. mets 	medicine 	the journal of pediatrics 	 	 	view excerpts, references background save alert research feed optic gliomas in children with neurofibromatosis type 	a. lund, f. skovby 	medicine 	european journal of pediatrics 	 	 	view excerpts, references background save alert research feed optic pathway gliomas in neurofibromatosis‐ : controversies and recommendations 	r. listernick, r. ferner, g. liu, d. gutmann 	medicine 	annals of neurology 	 	 	pdf 	view excerpt, references results save alert research feed pediatric midbrain tumors: a benign subgroup of brainstem gliomas. 	p. robertson, k. muraszko, j. brunberg, r. axtell, r. dauser, a. turrisi 	medicine 	pediatric neurosurgery 	 	 save alert research feed clinico-radiologic characteristics of long-term survivors of diffuse intrinsic pontine glioma 	s. jackson, z. patay, + authors a. broniscer 	medicine 	journal of neuro-oncology 	 	 	 save alert research feed neurofibromatosis type : brain stem tumours 	l. bilaniuk, p. molloy, + authors m. needle 	medicine 	neuroradiology 	 	 save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue networked religion: metaphysical redemption or eternal regret? commentaries networked religion: metaphysical redemption or eternal regret? peter mclaren , # springer nature switzerland ag , corrected publication keywords postdigital . technology. religion . christianity. liberation theology. cyberspirituality can we doubt that only a divine providence placed this land, this island of freedom, here as a refuge for all those people in the world who yearn to breathe free? president ronald reagan ( ) from reaganomics to reaganligion i’d like to set the tone of this commentary commenting upon the above quotation by reagan: can we doubt that war criminals in your administration, under the tutelage of your ‘reagan doctrine,’ transformed the country into a harrowing neoliberal horror show starring the nomenklatura of corporate america? they achieved this world- historical assault on the working-class through foreign and domestic policies and acts of clandestine military outlawry that can only provoke a rational mind to contemptu- ously scoff at your use of the word ‘refuge’ and ‘freedom’ to describe the usa. reagan was a religious man who believed in the prophecy of the apocalypse and armageddon, who used technology to destroy the lives of thousands of innocents and who normalized the stigmatization of the poor, mostly people of color, whom he gleefully condemned as welfare cheaters, creating an atmosphere linked to what lifton ( ) calls ‘malignant normality’—an atmosphere intensified by those who feel entitled and superior enough to claim ownership of another’s reality, a toxic milieu that trump has expanded exponentially through his primal scream presidency. postdigital science and education https://doi.org/ . /s - - - * peter mclaren peter.mclaren @gmail.com chapman university, orange, ca, usa northeast normal university, changchun, china http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf mailto:peter.mclaren @gmail.com technology and religion are not incompatible, they are not inevitable disputants in a social universe divided between the word of god (now accessible through our transactive memory banks we call databases) and the world (as it is processed into being by computer programmers, search engines and stored in the wikiverse). in fact, religious impulses and technology have a fierce synergistic potential. but in a social universe where protagonists are fighting for socialism, this synergism can have dan- gerous consequences. socialism is an antiquated and blighted term in contemporary parlance throughout the usa and one drenched in political controversy; it’s a term carefully stage-managed by the corporate media who have made it the pale object of unsparingly wicked manipulation by right wing pundits. the term socialism has become a widely knowable trigger term by those who would all too gleefully compare it to soviet union style communism or the national socialist party in germany whose nefarious architects were the nazis. we can only speculate as to how we can implement socialism, whether through meticulously monitored increments or full-blown commu- nal councils, sectoral councils, or grand experiments in participatory democracy, direct democracy, and worker cooperatives. what is clear, however, is that we must keep trying to develop the idea as we become more securely locked into a world of uberized industrial complexes using data analytics, mobile apps and new advances in artificial intelligence to help ensure you remain uploaded as a hard working, church going, productive consumer. while mainstream democrats are decrying the possibility of an intrepid social- ist candidate for the presidency of the usa, there barely is any observable deterioration of the term among youth activists and the coordinates for a socialist future are shifting more toward the realm of possibility than at any other time in recent history. despite the unvarnished countervailing powers in the media pitching a ‘better dead than red’ attack on socialism, the subterranean yearnings for a socialist future remain inexorably palpable among our young. they appear to be in this struggle for the longue durée. trump embodies the führerprinzip (leadership principle), that stipulates that he, as president, should rule with abso- lute white- knuckled power and authority, emphasizing a raw, jaw-jutting mascu- linity that commands unflinching deference and that frequently goes to absurd lengths of personal presidential depravity. even from those members of the senate whom he has ridiculed and humiliated in the mainstream media, trump demands that they repeatedly ‘kiss the whip’ to the point of exhibiting a frictionless compliance and bubble-headed obsequiousness which often ends up in spectacles of fawning adoration and unvarnished glorification. his sup- porters must demonstrate their ardent loyalty by flattering trump in the most cringeworthy ways, such as promoting the spiritually audacious idea that trump was called by god to rescue america from assured destruction by liberals, socialists, feminists, pro-immigration advocates, and those journalists whom he has described as ‘enemies of the people’ (which boils down to any journalist who dares to criticize trump). is there anything that will satisfy his toxic narcissism? mike pence does not need any reminders from trump since this fundamentalist christian seems naturally transfixed in the presence of trump, ready at any moment to drop to his knees and pepper trump’s fleshy and dimpled buttocks with impassioned kisses, secure in the knowledge that his farts will not smell (or perhaps might carry a celestial perfume, even an ‘odor of sanctity’ reserved only for saints), and all the while weeping profusely and postdigital science and education crying out, ‘let me praise you my gloriously sun-tanned lord, how shall i worship thee? how might i serve thee forever my ruddy american king?’ the master of chaos trump is currently engaged in an orgy of revenge against democrats who tried to have him thrown out of office. it is impossible to ignore his unfettered glee in pardoning white collar criminals (one of whom is michael milken, whose company once threat- ened to sue me for calling him a junk bond king) and in punishing any and all political opponents even if it means blatant lying, fabricating evidence, and manufacturing conspiracy theories. a dissimulating deification of the military is part of trump’s fascist imaginary—a virtual space created after reality-based television ‘it’ factors presented in cinéma verité mode, emphasizing events in such a way that they have a magnetic pull on viewers, usually through the makeshift milieu of the twitterverse but also in televised mass rallies that appeal to the scurrilous far-right of das man. these are usually mudslinging conservatives, and persons of pedestrian discernment who feel nothing but pride when they are compared to troglodytes or paleo-nazis. trump’s rallies also include frequent shout-outs to farmers, coal miners, truckers, and police officers where he can brag about rewarding white collar criminals with pardons and ennoble the racist, misogynist, and homophobe, rush limbaugh, whom he presented with the nation’s highest civilian honor to the disgust of anyone with a thread of decency. trump does not love america; he is tearing apart its moral fabric; he is forming the american public on the basis of social media’s fake news and bringing vile and excremental ideas into the mainstream. trump is as fake as the dreamy models in a charlotte tilbury ‘pillow talk’ advertisement although he is definitely not as easy to look at. trump looks and acts as if he is on drugs, frequently slurring his words as if his thoughts, like clumps of sludge from a backed-up toilet, are making their way through the runnels of some ancient roman aquifer and then dumped into the storage basin of america’s social brain. yet he remains energetic enough to pitch an ‘us against them’ ideology that has helped to shore up his electoral base. trump’s anti-globalist, antiimmigrant, and nationalist perspectives, his shamefaced retreat from the constitu- tion and the rule of law, his damning of the media outlets that give him unfavorable coverage as ‘enemies of the people’, and his pusillanimous, two-faced commitment to the betterment of the working-class have given heft to the possibility of american fascism being realized in a single generation, more so than at any other time since the decades prior to world war ii. trump has been proclaimed as a messenger of god by his evangelical christian base that has distinguished itself by its all-embracing and petrifying lunacy. devega ( ) remarks: ‘trump also manifests a condition where he thinks of himself a type of god. he leads a political cult united by cruelty and collective narcissism’. even prior to this, it was clear that trump has a god complex. consider the comment by justin frank: ‘trump does have a god complex. trump reveals this through his constant use of the phrase “we’ll see what happens.” the only person who can say something like that is a person who believes they are a god, because only god can be all-knowing and see the future’ (in devega ) we can imagine without undue strain that trump is a charlatan and master of chaos. he lives in his own self-contained world with its own laws, logic, and symbolic order, twisted into fanciful distortion when set against a postdigital science and education system of intelligibility that requires a logic external to his vainglorious inner world that purports to bear any resemblance to sanity. here the invariant coordinates of trump’s narcissism lead him directly into the vicinity of demagoguery and the playground of madmen. frank points out, i believe correctly, that in addition to being an anger and rage addict, ‘[o]n a basic level donald trump is the jim jones of american presidents’ (in devega ). and his base is drinking the poison carefully calibrated by mar-a- lago chefs into a kool aid unsweetened sharkleberry fin powdered drink mix. social life is heterogeneous and trump and his critics certainly inhabit separate orders of reality resulting from a lifetime of different preunderstandings of the world. both groups have been emplotted in the narrative of citizenship in different, often violently contrasting, ways. for trump’s base, the world is driven by the ‘deep state’ which is set up to keep elites in power at any cost, while trump is putatively dedicated to destroying the deep state (notwithstanding the idea that trump may, in fact, be the very apotheosis of the deep state he is attempting to demolish). for trump’s critics, the usa is hewed to democratic principles and organizations after centuries of hard-won struggle, and trump is quickly unraveling these accomplishments with his temper tantrum presidency. here opposing ideological predilections from these two distinct political tribes are sedimented over time into our cultural memory, spawning vastly different worldviews. trump is sometimes described as an abusive father in relation to his republican administration family, who, after being themselves tormented and publicly harassed by trump, eventu- ally begin to cross the dividing line to identify with their abusive father and then begin to abuse others in a similar fashion. likewise, the democrats began to perceive adam schiff as their father figure who was going to save them from trump through the mueller report and then the impeachment process. predictably, democrats sat at home nervously waiting for schiff’s heroic endeavors to come to fruition instead of taking to the streets to protest. was this the plan all along? all this was made possible by the narratives that framed the daily news both in the mainstream press and in a great deal of the social media. so now the two families have ramped up their antagonism toward each other and we are seeing what amounts to a postdigital rendition of the hatfields versus the mccoys. the task then faced by the critical cultural worker and educator becomes manifold: how do we prevent the coming-into-being of an american volksgemeinschaft, a racially unified and hierarchically organized nation state in which the interests of the rank-and-file population would be stringently subordinate to the nationalist imperatives of a militarized fatherland swimming in patriotic fervor and on a permanent war footing? richard wolf writes: the left needs to respond in three key ways. first, it should stress how world war and holocaust resulted the last time post-crash capitalism used nationalism for scapegoating. second, it should expose scapegoat politics as aimed to deflect working class anger from a crash-prone capitalism. immigration, trade, tariff policies, or european integration define capitalism’s preferred terrain of debate, not a critical left’s. the left’s core response to capitalist nationalism should be this: capitalism is the problem and transition to a new, different, and fundamen- tally democratic system is the answer. (wolff ) i fully agree with wolff. there will be pitched battles ahead. battles over ideology, and battles over what is real and what is not. social justice warriors are feared by those postdigital science and education whose intellectual trajectory leads them to feed off the ideological ordure common among paleoconservatives—that belief in egalitarianism and embracing progress under liberal auspices leads straight to work camps or the executioner. social justice and totalitarianism appear inextricably braided together, eliciting a warning from the right that the left is coming to take away their freedoms and to eat their babies. this makes the right much more forthrightly hostile toward the left and as their souls begin to rot, they become plagued by their own imposing paranoia, turning themselves into vendors of downfall, desolation and degeneration, intellectual charlatans, and deskbound ped- ants unable to grasp the nettle of crisis-prone capitalist relations of production in triggering the alienation of the masses and culminating in the political cataclysms of the twentieth century. their threadbare, aerosol theology has turned christianity into a pagan ritual, and they would prefer to wear a red maga hat to a torrid trump rally, enraptured by trump’s adderall-infused embroidering of narcissistic narratives onto the political events of the day, than to read a line or two from paul’s epistles, or letters, to his various congregations (the romans, corinthians, galatians, ephesians, philippi- ans, colossians, and thessalonians). trump is worried that white people in the usa will soon be overrun by people of color carrying clubs made of oak spiked with nails and in this regard he is no different that the pathetic and cowardly members of the patriot front who visit my campus under the cover of darkness and distribute their odious white supremacist literature. trump views today’s ‘american carnage’ as directly related to the ‘demographic winter’ in which the birthrate of the white race in america is in decline. and as the white folks decline in numbers, so goes the neighborhood. for trump and for many of his followers who proclaim the doctrine of american exceptionalism, america is god’s instrument for saving the world. maybe they assume that god keeps a tiny globe on his office desk that brandishes a big pin with ‘number one’ taped to its head, designating the location of the usa. now that would not be such a bad idea if the pin could actually perform double duty as an acupuncture needle and bring some political ballast to the country. trump has reconstituted the notion of american exceptionalism, remaking it into a trumpist exceptionalism, which is little more than a con game where established institutional policies and practices are all delegitimated and then rejiggered for its main purpose: to keep trump in power. this requires that institutions be structurally repurposed—a process the nazis called gleichschaltung which can be translated to mean ‘reconstructing, reordering, re-gearing’ (lifton in moyers ). the nazis ‘got rid of those who were unreliable, not reliable nazis, [and] replaced them with reliable nazis. and the institutions became sources of nazi concepts and nazi behavior’ (lifton in moyers ). trump has reorganized many us government institutional structures by replacing top administrators and directors with his most fanatical supporters who often lack any relevant skills to justify their appointments. it is unequivocally true that american evangelical christianity (which is notoriously anti-catholic) is a syncretic religion merging or assimi- lating american-style free market capitalism into its belief system, and thus asserting wide divergence on interpreting jesus of the new testament with regard to differentiating wealth, the idea that some people are poor while others are rich. but this syncresis is, nevertheless, not authorized in relation to other forms of capitalism, such as keynesianism, since fox news could never condone such a heresy. political rhetoric feverishly delivered from the pulpit about god’s master plan for american greatness creates a liminal space in which both the state and church can be postdigital science and education brought into a divine harmony. this is especially true during wartime, when the nation itself and the bible join together to become a collective ‘object of worship’ revealing the destiny of america as an unerring march toward victory in its undiminished conquest of evil. in contrast, open exceptionalism views america as just one of many nations, none of which can lay claim to being god’s sole or privileged instrument of salvation. the founding process of america was, for these early settler colonialists, an event that was caused by god’s direct intervention into history, hence, predisposing ‘authentic’ christians against liberals and progressives that has continued over the centuries right up to this day. christian principles, therefore, had to be integrated into governmental bureaucratic oper- ations in order to keep the nation from falling apart and the struggle is still ongoing, evoking the nation’s putative providential past. a recent report by max boot, cited by mathew chapman ( ), reveals that american jingoists are nothing short of delusory when it comes to boasting about the current world-historical greatness of the united states. blaming decades of republican anti-government ideology for its failure to handle the current covid- pandemic, boot offers a sober assessment of the nation’s status as a whole: ‘….we lag in almost every measure of societal well-being among the wealthy nations (now ) of the organization for economic cooperation and develop- ment (oecd),’ wrote boot. ‘as of , we had the second-highest poverty rate, the highest level of income inequality and the highest level of obesity. we spent the most on education but produced less-than-average results. we were also below average on renewable energy, infrastructure investment and voter turnout. we are the only oecd nation that doesn’t mandate paid family leave. one area where we do lead is gun violence. our homicide rate is nearly percent above the oecd average.’ (boot in chapman ) americans by and large do not want to recognize the dark side of their history. do they remember how reverend jerry falwell in the s opposed sanctioning the apartheid regime of south africa, calling bishop desmond tutu a phony? are many evangelical christians who proudly sport their red maga hats aware of richard nixon and henry kissinger’s christmas bombing of cambodia in that saw b- bombers unloading , tons of bombs over hanoi and haiphong that lasted for days, hitting a hospital and numerous urban centers? villages and half a million civilians were decimated. the us bombings of cambodia created the conditions of possibility for the khmer rouge to take over the country and begin years of mass killings. helmed by henry kissinger, the us government supported indonesia’s campaign of genocide of roughly , east timorese (who were seeking their independence from portugal at that time). the usa provided weapons and logistical support. in , henry kissinger and richard nixon gave the cia orders to initiate a covert operation to overthrow president-elect salvador allende in chile and he was subsequently killed and replaced by general augusto pinochet who initiated a seventeen-year reign of terror. the con-sensualisation of cyberspirituality i have been reminding american educators about this for years. with my pen as a lance, i can tilt at the evangelical christian windmill all that i want, but we are also postdigital science and education talking about religio-corporate enterprises that have grown exponentially under the trump administration and have helped to influence government policy, including a restrictive immigration program, appointment of anti-abortion judges, rollback of environmental regulations, and a middle east peace plan that reveals nothing but contempt and distain for the palestinians. technology has certainly played a part in this process that ravi kumar refers to as ‘con-sensualisation’: the con-sensualisation is achieved through the political formation that dominates it – the ruling class that devises interesting and innovative methods during these times of digital revolution and whatsapp universities, as well as through the large bureaucracy that is put to task to ensure that the state’s ideas reach the people. it is a grand exercise in pedagogy. (kumar ) these ‘con-sensualized’ issues are indeed pedagogical, and can spit fire, fueling further evangelical support for trump. as far as their splendid inattention to any hermeneutics of suspicion goes, or their bad faith theology is concerned, i am not preoccupied with lingering aporias or irresolvable paradoxes braided into various scriptural garments and insistently thrust into our national brainpan. nor am i occupying a position that pushes paradoxicality to the point that denies all claims to truth. and i’m not working alongside a skunk works project with a bunch of technologically determinist nerdballs and end- timers who are desperately trying to create an app that will enable the public to connect directly with god. the closest that this sky marshall commando cody cyber breed of industry oddballs has come to creating such an app is when they created a twitter account for donald trump’s self-worship. as devotees of donald trump struggle to survive the covid- , from the dirt paths of tumbledown hillside ghettos to the luxury vinyl tiled floors of high-end shopping plazas in major metropolises, the pastor of church in baton rouge, louisiana, by the name of rev. tony spell, is defying an order from louisiana governor john bel edwards against large gatherings in the midst of the coronavirus outbreak, because the good reverend spell believes the handling of the coronavirus pandemic is ‘politically motivated’ (palma ). in fact, he noted that on sundays he brings in buses packed with parishioners. he recently boasted: ‘i had , in attendance sunday’ (palma ). he further justified his actions by claiming that his church heals a wide array of ailments: ‘our church is a hospital where the sick can come and get healing… he said. cancers are healed here, people are healed of hiv in these services. … and tonight, we’re also going to pass out anointed handkerchiefs to people who may have fear, who may have a sickness — and we believe that when those anointed handkerchiefs go, a healing virtue is going to go on them as well’ (palma ). the self-promoting pastor has since been charged with a misdemeanor violation of the governor’s orders. as political power tilts towards the malignant forces of authoritarian capitalism, it is no secret that big-box revivalist preachers want in on the action, even if that puts the lives of their flock and other constituencies at potential risk during a pandemic. as a catholic who has visited a wide array of religious sites all over the world and occasionally witnessed events that appeared to defy scientific explanation, i am not against the idea of miracles, but i am against pitting triumphalist faith against scientific common sense. there is some work being done on a responsible use of technology by different religious groups. drawing on the work of vincent miller, campbell and garner note that ‘across postdigital science and education amish and other mennonite communities there is a common practice of discernment and understanding around technology and media.’ and that this discernment is ‘grounded in an ecclesiology structured to produce full accountability between individual members of the church and the will and discernment of the larger group’ (campbell and garner : ). miller’s work is instructive on the importance of communitarian decision-making. as indicated by campbell and garner, miller implicates technology here in the perception of time in that, we tend to focus on a more instrumental view of time (chronos), which allows us to order, manage, and control the world, rather than a more relational view of time (kairos) that comprises moments of meaning within a narrative of life. it is this relational view of time, seen in the new testament (e.g., gal. : ; titus ; – ) to represent defining moments in history, that is captured in the approach to appropriate technology. rather than concentrating on managing time and rela- tionships, we should work on using and applying technology to create meaning and true relationships in our individual and communal lives. in this respect, the amish community’s approach to technology is connected to an experience or notion of time that serves the community rather than the other way around. (campbell and garner : ) campbell and garner expand on the values that the amish community wishes to defend by selectively using only the most appropriate technology: ‘thus the use of technology such as a mobile phone is weighed against how it would impact home life. would it contribute to authentic and sustainable human relationships? would it lead to an incessant need to have the next best thing? would it privilege the individual over against the community?’ for the amish community, therefore, ‘the community rather than individuals makes technological decisions; technology is not necessarily evil, so it can be used with caution; and the use of technology can potentially undermine the community and its core values’ (campbell and garner : – ). further, campbell and garner see almost all commodified objects as having some potential for becoming props in a christian catechism designed to deepen our worship god: the activity of worshipping god—father, son, and holy spirit—has been and continues to be intricately linked to technology and media. examples include the construction of sites of worship such as cathedrals; the development of written texts such as the bible or prayer books; (…) the technological environment we inhabit shapes the way we worship and the way we behave in the context of worship and the christian life. (campbell and garner : ) i admit that there is something meaningful about these statements. i regularly show videos in my doctoral seminars, the most significant being the feature film, romero (duigan ) produced by the paulist fathers, whose major narrative culminates in the assassination of archbishop romero in el salvador in . it is indispensable to my portraying the debates over liberation theology since it sets an emotional tone, provides an invocatory power and creates a dramatic resonance among the class that enables them to appreciate the project of liberation theology even, and sometimes especially, among atheists. but i also consider the fact that magicians possess theurgic powers, and create their own props for their stage shows postdigital science and education that help the audience willingly suspend disbelief in the existence of magic. but is this the same thing? the argument that technology is neutral is clearly false. the argument that guns do not kill people, people kill people, that is sometimes hijacked to describe media technology reminds me of the logic embedded in the argument that technology is neutral. but technology cannot be separated from the myriad ways in which it is situated in its deployment. technology does not stand above history as some insurmountable universal tool, like the lightning bolt of zeus; it is not forged solely through the inner determination of capital, nor should it be capable playing the final role of the arbiter of history. each use we make of technology has to be understood within its contextual specificity. in the case of using the film romero in the classroom, one has to consider the ideological presuppositions of the viewers, the interplay between long-past and present- day structural determinations that make manifest our ability to interrogate the themes, and what aspect of the political informs the pedagogical praxis of the educator—just to name a few considerations. to argue that technology is neutral could easily promote the idea of placing religious symbols in public spaces and to make the argument that religion must have a privileged place in the public sphere if civilization is to be kept in place. and we must continually ask: how can a christian claim to be against christian nationalism, while failing to protest and vociferously denounce the banning of immigrants from muslim countries from entering the usa? clearly, this is just another step in trump’s strategy of creating a majoritarian society of white ethno-nationalists. how do we situate christian nationalism in the sociohistorical and cultural context of today? in what ways is it linked to growing fears of socialism, social justice agendas, and the gender and sexuality movements that took place in the s and s? how have various technologies re-presented socialism, social justice and the civil rights struggles? have they created a mythical ‘ s society that makes the protest movements of the ‘ s and ‘ s to seem like gross and destructive interlopers into a benign and noble history? isn’t this what trump’s maga campaign is all about? campbell and garner don’t render such questions problematic when they conclude that the ‘potential for christians to become critical and discerning consumers and producers of digital technologies and media opens the door for a variety of religious expressions in online environments’ ( : ). here they are not simply referring to facebook, twitter, blogging, google, or youtube, but to an online site using labyrinths as meditative exercises that contribute to new forms of cyberspirituality. speaking of cyberspirituality, i would place the work of antonio spanaro in this category. his work, cybertheology ( ), is heavily grounded in the work of teilhard de chardin and marshall mcluhan. spandaro argues that it is precisely teilhard de chardin’s theological framework that provides the necessary theo- logical mindset and aggregate of conceptual religious categories to enable us to understand both the history of technology and technological advances up to the present, in particular the web. for spanaro, the internet marks an important connective stage in humanity’s journey of spiraling upwards toward god, guided by a cyberspatial eschatology provided by avatars of episteme who glide down the corridors of google fame like the ghosts of hogwarts, cloaked in robes festooned with algorithms. thanks to the sacred and sanctifying web, people now can ‘interface’ with their religious beliefs while secretly listening to mutter by rammstein and attend churches postdigital science and education that have been transformed into a virtual or simulated reality. saving one’s soul is favorably compared by spanaro to saving a personal computer file. the web, which has become part of a divine milieu, operates out of a particular system of intelligibility (participative logic and user-generated content) that only theological intuition can most fully explain. the virtual world now represents the divine intellect, of a type of self- thinking thought (thought that thinks itself) such that what was once theological has now become technological. technology has helped to bring civilization closer to the omega point, an open vision of transcendence, a point of divine convergence with the noo- sphere and the cosmic christ. this journey, strangely enough, began with a microphone placed on a church altar which allowed the congregation to enter into an immediate relation with the speaker. the author frequently turns to mcluhan for an explanation: ‘we observe in the liturgy that the acoustic amplification overloads our auditory sensorial channels, lowering the threshold of attention to the visual and individual experience of the liturgy so that it isolates the individual in a sound bubble within the architectural space’ (sparado : ). of course, spadaro also addresses the problems of ‘forking’ with the use of open source software and discusses issues inherent to what he calls open source theology. while i find some of spanaro’s observations provocative, the entire work, which is admittedly christocentric, does give off the odor of christian triumphalism on one too many occasions. i immediately wanted to know whether the theological probes (a term i have borrowed from mcluhan) used by spanaro can be applied, say, to buddhism, or to islam, or possibly to umbanda (santería, candomblé) or wicca. and i am highly skeptical that the creation of the internet has, according to spandaro, proven marx’s critique of political economy to be wrong. are we truly on an evolutionary path to cosmic intelligence via self-organization, auto-catalytic networks and biotechnological advances? can we find christ in technology and evolution, as teilhard de chardin proclaims? do we need to wait for theologians to set the terms for merging with the pleroma or can we robustly engage the transhumanists without the vatican’s approval? or is there a way for christian communities and transhumanists to work together cheek by jowl? the emperor’s religious garments while i have been wildly critical of trump’s evangelical base since trump ascended the throne in (mclaren and jandrić a), i am in agreement with jon meacham ( ) that the sphere of religion offers the most effective portal through which the naked emperor can be exposed for all of his perverse and self-indulgent inhumanity. while clearly trump should be adjudicated into a penal colony, that is very unlikely to happen while trump is still president and considered by inveterate end-timers to be chosen by god to rule the american throne with an iron fist. a critique of trump from a christian perspective, using christ’s teachings as a measure would, for instance, be the most effective way to reveal the danger he poses to democracy and civilization itself. for example, the crumbs that trump threw to his base with his tax cuts can be revealed to be a charade when one considers the emphasis that the bible places on economic conditions and poverty. meacham writes: [martin luther] king had been deeply influenced by the theologian walter rauschenbusch and his book ‘christianity and the social crisis,’ which postdigital science and education argued that jesus called the world not simply to contemplate but to act. ‘the gospel at its best deals with the whole man, not only his soul but his body, not only his spiritual well-being, but his material well-being,’ king wrote in a rauschenbusch-inspired passage. ‘any religion that professes to be concerned about the souls of men and is not concerned about the slums that damn them, the economic conditions that strangle them and the social conditions that cripple them is a spiritually moribund religion awaiting burial’ (meacham ) meacham further emphasizes the importance of seeing the new testament as a social gospel in action. he writes: representative john lewis, democrat of georgia, was perhaps king’s most devoted disciple. growing up in pike county, ala., he overcame a childhood stutter by preaching to the chickens on his parents’ tenant farm. hearing king on the radio, mr. lewis was moved to action, and came to share the older minister’s philosophy of christian nonviolence. their inspiration came from the new testament: ‘blessed are the meek, for they shall inherit the earth’; ‘blessed are they who are persecuted for righteousness’ sake, for theirs is the kingdom of heaven.’ as mr. lewis recalled, the struggle within time and space was about ‘heaven and earth. this was the social gospel in action. this is was love in action, what we came to call in our workshops soul force.’ the goal? ‘the beloved community,’ which was, he said, ‘nothing less than the christian concept of the kingdom of god on earth.’ (meacham ) while it is true that technology may have initially been created by millenarianist over- achievers to ‘recover eden’, whose adamic myths compelled them to reverse the fall of adam and eve from god’s most sacred real estate property (noble ), today technology can be used both to transgress the moral maxims of the day, or impel us as good technoscientists to transcend our hatred and embrace a loving faith that includes technolog- ical innovations braided to the teachings of christ and marx. nevertheless the same questions remain: who owns the media conglomerates that control the technolo- gies? and who benefits from such arrangements? what is the relationship between the owners of the means of technological production and those strug- gling for a socialist alternative to capitalism (mclaren and jandrić b)? i have no problem with technology being invented or used to pursue worldly dreams that might help ease the needless suffering of humanity, or to participate in finding hope in a hopeless world, but it is clear to me that more needs to be done to liberate technology from the white-knuckled vice-grip of corporate christian fundamentalists and to liberate christianity from the perils and pitfalls of technological ‘progress’ and to encourage a more nuanced and granular understanding of how technology might enable a project of faith and hope— including how the gospel messages themselves might be better embodied (enfleshed) through technological advances, without having to retrofit humanity on a massive scale with biomechatronic body parts. it is incorrect to say that the collectivization or nationalization of individually owned property will usher in a socialist revolution because when this occurs the basic nature of capitalist society is still present—value augmentation, when labor assumes a value form. postdigital science and education marx of course supports collective ownership of the means of production. but by this he does not mean simply transferring ownership deeds from private to collective entities, but rather ensuring that the working class owns and controls the means of production. we need to transform the very nature of human relations. we need to push past distributive economics in our fight for a socialist society. the struggle is such a daunting task that many of us are left to live our lives fluctuating between hope and despair. justin frank makes some very insightful remarks about hope that we would do well to take to heart: there are several levels of hope. on one level, hope is the denial of anxiety and fear and the denial of helplessness. the irony about hope is that it combines the denial of helplessness with an expression of hopelessness. that’s what’s para- doxical. there are people who hope for things but do not do anything to achieve that outcome. when a person is denying helplessness by hoping that things will work out, they are also acting helpless by hoping that somebody else is going to save them and somehow everything will work out. hope can be an abdication of responsibility as a way of protecting oneself against anxiety. the hope-peddlers are behaving as though they are addicted on an unconscious level to death, because they are denying the work that is necessary to stay alive by protecting the usa. (frank in devega ) many of the experiments to build a socialist society have throughout history ended in failure. whether or not advances in technology will help in this endeavor—or whether they will overall tighten the death grip of capitalist social relations—is a debate that is far from being resolved. this has driven many politically progressive christians to embrace a fatalistic despair and to cling to an abstract hope divorced from the arena of concrete struggle. for americans facing an epic choice during the presidential election in november , the stakes are brutally high. hope is not something that can be invoked as a magical talisman that calls upon supernatural forces to intervene on our behalf. to activate revolutionary change, hope must always be bonded with struggle. and if this includes communing with god in cyberspace, then i can only wish that the ultrahumanist vision of our dear jesuit paleontologist, teilhard de chardin, will lead to the eventual enhancement and deepening of biological life rather than its transcendence. it is time for a de-transcendence, a time for us to meet the poor on their own terms, a time for us to repristinate the commons and expand its democratic sphere, a time to deepen its connection to the people rather than be blown through the history of pain and suffering like benjamin’s angel of history, with a storm caught in its wings, its neck wrenched backwards, forced to look upon the dung heap of humanity's glorious achievements. references campbell, h.a., & garner, s. ( ). networked theology: negotiating faith in digital culture. grand rapids, mi: baker academic. chapman, m. ( ). the gop has demolished the ability of america to lead in crisis: conservative columnist. rawstory, march. https://www.rawstory.com/ / /the-gop-has-demolished-the- ability-of-america-to-lead-in-crisis-conservative-columnist/?utm_source=push_notifications. accessed march . postdigital science and education https://www.rawstory.com/ / /the-gop-has-demolished-the-ability-of-america-to-lead-in-crisis-conservative-columnist/?utm_source=push_notifications https://www.rawstory.com/ / /the-gop-has-demolished-the-ability-of-america-to-lead-in-crisis-conservative-columnist/?utm_source=push_notifications devega, c. ( ). on a fundamental level, donald trump does not believe in america: ‘god complex’ author justin frank. salon, february. https://www.rawstory.com/ / /on-a-fundamental-level- donald-trump-does-not-believe-in-america-god-complex-author-justin-frank/. accessed march . duigan, j. ( ). romero [motion picture]. los angeles: warner bros. kumar, r. ( ). barbarity in the name of religion: delhi violence and a pedagogy of hate. newsclick, march. https://www.newsclick.in/barbarity-name-religion-delhi-violence-and-pedagogy-hate. accessed march . lifton, r. j. ( ). losing reality: on cults, cultism, and the mindset of political and religious zealotry. new york and london: the new press. mclaren, p., & jandrić, p. ( a). the fellowship of the crooked cross: trump’s evangelical hounds of hell. postdigital science and education. https://doi.org/ . /s - - - . mclaren, p., & jandrić, p. ( b). postdigital dialogues on critical pedagogy, liberation theology and information technology. london: bloomsbury. meacham, j. ( ). why religion is the best hope against trump. the salt lake tribune, february. https://www.sltrib.com/opinion/commentary/ / / /jon-meacham-why-religion/. accessed march . moyers, b. ( ). losing reality: can we get the truth back? bill moyers in conversation with robert jay lifton. https://billmoyers.com/story/losing-reality-can-we-get-the-truth-back/. accessed march . noble, d. ( ). the religion of technology: the divinity of man and the spirit of invention. new york: penguin books. palma, s. ( ). baton rouge pastor defies order and holds service amidst coronavirus outbreak: ‘the virus is politically motivated.’ rawstory, march. https://www.rawstory.com/ / /baton-rouge-pastor- defies-order-and-holds-service-amidst-coronavirus-outbreak-the-virus-is-politically-motivated/. accessed march . reagan, r. ( ). acceptance of the republican nomination for president. https://usa.usembassy. de/etexts/speeches/rhetoric/rraccept.htm. accessed march . sparado, a. ( ). cybertheology: thinking christianity in the era of the internet. trans. maria way. new york: fordham university press. wolff, r. ( ). capitalism’s political servants: trump and johnson. counterpunch, february. https://www.counterpunch.org/ / / /capitalisms-political-servants-trump-and-johnson/. accessed march . postdigital science and education https://www.rawstory.com/ / /on-a-fundamental-level-donald-trump-does-not-believe-in-america-god-complex-author-justin-frank/ https://www.rawstory.com/ / /on-a-fundamental-level-donald-trump-does-not-believe-in-america-god-complex-author-justin-frank/ https://www.rawstory.com/ / /the-gop-has-demolished-the-ability-of-america-to-lead-in-crisis-conservative-columnist/?utm_source=push_notifications https://doi.org/ . /s - - - https://www.sltrib.com/opinion/commentary/ / / /jon-meacham-why-religion/ https://billmoyers.com/story/losing-reality-can-we-get-the-truth-back/ https://www.rawstory.com/ / /the-gop-has-demolished-the-ability-of-america-to-lead-in-crisis-conservative-columnist/?utm_source=push_notifications https://www.rawstory.com/ / /the-gop-has-demolished-the-ability-of-america-to-lead-in-crisis-conservative-columnist/?utm_source=push_notifications https://usa.usembassy.de/etexts/speeches/rhetoric/rraccept.htm https://usa.usembassy.de/etexts/speeches/rhetoric/rraccept.htm https://www.counterpunch.org/ / / /capitalisms-political-servants-trump-and-johnson/ 	networked religion: metaphysical redemption or eternal regret? 	from reaganomics to reaganligion 	the master of chaos 	the con-sensualisation of cyberspirituality 	the emperor’s religious garments 	references psm .. a pocket of very high suicide rates in a non-violent, egalitarian and cooperative population of south-east asia f. jollant *, a. malafosse , r. docto and c. macdonald mcgill university, department of psychiatry; and douglas mental health university institute, mcgill group for suicide studies, montréal (québec), canada department of psychiatry, genetics laboratory, academic hospital of geneva, hôpital belle-idée, chêne-bourg, switzerland palawan state university, puerto princesa city, philippines unité d’anthropologie bioculturelle, national center for scientific research (cnrs), marseille, france background. extremely high rates of suicide localized within subgroups of populations where suicide is rare have been reported. we investigated this intriguing observation in a population of south-east asia, where local culture should theoretically be preventative of suicide. method. a team including an anthropologist and a psychiatrist surveyed all cases of suicide that had occurred over years in four isolated regions. a psychological autopsy was carried out comparing each suicide case with two matched control cases. results. in a region of inhabitants, suicides occurred, leading to an annual suicide rate of / which is times the rate in the usa or canada. by contrast, three ethnically similar distant communities showed low to null rates. the gender ratio was three males to one female and two-thirds of cases were aged below years. methods of suicide were poisoning and hanging and motives mainly included interpersonal discord. the pattern of developmental and clinical risk factors was somewhat different from western countries, showing no childhood maltreatment, only one case of alcohol/substance abuse and impulsive–aggressive personality but elevated rates of social anxiety. suicide cases had very high frequencies of second-degree biological relatives who committed suicide. conclusions. our study confirms a persistent phenomenon of high suicide rates restricted to a subgroup of a pre- industrialized population. we hypothesized this might be explained by isolation and endogamy, which may have promoted the selection/amplification of genetic vulnerability factors, or a contagion effect. these findings shed light on suicide from both a singular and a universal perspective, suggesting that particular local conditions may significantly modulate the rate of this complex behavior. received august ; revised december ; accepted december ; first published online january key words: culture, genetics, human behavior, indigenous people, suicide. introduction suicide is a universal human phenomenon. however, it occurs at various frequencies according to location. suicide rates range from below / inhabitants in southern europe and in most developing countries, including the philippines, up to – / in hungary or lithuania (hawton & van heeringen, ). although cultural, genetic and methodological hypotheses have emerged, the origins of these varia- tions are largely unknown. to address some of these issues, we report here an investigation of suicide among a non-violent, cooperat- ive and egalitarian indigenous population living on the island of palawan in the philippines. the palawan peo- ple form an ethnolinguistic entity of approximately to inhabitants (macdonald, ). forty years of anthropological study of this population by c.m. has led to the unexpected observation of a very high frequency of suicide cases, between and , among a small subgroup of palawans (to protect this population, we have limited references to loca- tions) (macdonald, ). this phenomenon seems to have existed within this subgroup for several decades. by contrast, neighboring palawan subgroups of similar culture report suicide very rarely. the first aim of this * address for correspondence: dr f. jollant, douglas mental health university institute, boulevard lasalle, montréal, québec h h r , canada. (email: fabrice.jollant@mcgill.ca) these findings of this paper were presented at the international academy for suicide research (iasr) meeting in montreal, canada, on june . psychological medicine ( ), , – . © cambridge university press doi: . /s original article https:/www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https:/www.cambridge.org/core. university of basel library, on may at : : , subject to the cambridge core terms of use, available at https:/www.cambridge.org/core/terms https://doi.org/ . /s https:/www.cambridge.org/core study was therefore to assess the persistence of this intriguing phenomenon over the past decade, and compare suicide rates in the aforementioned region to rates in three distant control palawan regions. the second objective of our study was to investigate risk factors for suicide in this population. suicide risk factors have mainly been studied in modern western societies, limiting the generalizability of findings. risk factors usually include both proximal (e.g. nega- tive life events, mental disorders) and distal factors (e.g. childhood abuse, genetic factors) (hawton & van heeringen, ). a generally accepted model is that proximal factors act as triggers on pre-existing vulnerabilities (mann, ). a previous anthropological investigation of suicide among the high-risk subgroup of palawans indicated the absence of many classical risk factors, including childhood maltreatment and alcohol/substance abuse. however, no systematic structured assessment of these risk factors (including psychiatric disorders) has been performed. therefore, we conducted standar- dized psychological autopsies of all suicide cases reported within this subgroup over the past years. apart from the logistic complexity and physical challenge of such studies, studying relatively isolated populations presents interesting advantages (shifman & darvasi, ), notably ( ) a genetically and cul- turally homogeneous population living in a uniform environment, ( ) little migration and thus more intact families, allowing good genealogical records, and ( ) the possibility of investigating a pre-industrialized non-western population having limited contact with modern lifestyles. on the basis of previous investigations, we hypo- thesized that ( ) suicide would remain at a very high rate in the aforementioned region in comparison to the three control regions and ( ) risk factors for suicide in this population would differ, in part, from those reported in western countries. method a unique international team including a psychiatrist, an anthropologist, a geneticist and local scholars orga- nized this project. the field missions were conducted in and . ethics this study was approved by the provincial health office, puerto princesa city and the national commission on indigenous peoples, quezon city, philippines. in addition, the douglas institute research ethics board conducted a scientific assess- ment of the project. an information sheet and consent forms were translated into both tagalog (the official language of the philippines) and palawan. in all three villages investigated, the protocol was first explained to, and then approved by, the tribal councils, who gave their signed consent (signature or thumb mark). then, each main participant gave their signed informed consent prior to assessment. census of the total population and suicide cases our investigation focused on three villages of the region previously shown to have a very high rate of suicide (macdonald, ). these villages have a close relationship with each other, with an almost complete absence of non-palawan immigrants. travel to these villages takes day from puerto princesa, the main city of palawan island, and includes car, motorcycle, walking and buffalo. the climate is tropical and the region is in a malaria-resistant zone. we conducted our own census of the investigated villages to determine the number of inhabitants per household and their gender. because we had limited time, we did not determine age in the census. we counted inhabitants ( males, females) in village a, ( / ) in b and ( / ) in c, making a total population of inhabitants. a pre- vious raw census in suggests a small increase in population over the past years. however, consider- ing the lack of precise statistics, we opted to keep our current population number as the base rate for the calculation of the -year suicide rate, with the risk of underestimating this rate. as no official registry exists, data on all suicide cases that occurred from june to may in the three villages were collected from multiple local informants. data on suicide cases before june were collected and published previously (macdonald, ) and were therefore excluded with confidence. control regions we also conducted an investigation of three distant palawan regions as controls. reports of suicide cases during the same period were collected by the same methods described earlier. however, for the control regions, census of the related population was based on administrative sources and not on direct counting because of time restrictions. psychological autopsy all suicide cases from the target region were assessed by one or more informants who were well acquainted with the victim. importantly, as the palawan people have very close relationships with one another, highly concordant information from different sources was f. jollant et al. https:/www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https:/www.cambridge.org/core. university of basel library, on may at : : , subject to the cambridge core terms of use, available at https:/www.cambridge.org/core/terms https://doi.org/ . /s https:/www.cambridge.org/core almost always provided, enabling cross-validation of the information obtained. no medical records or notes left by the victims were available to complete the assessment because of lack of medical services and illiteracy. each suicide case was matched with two controls of the same sex and in the same age group. all controls came from the same three villages. great care was taken to exclude controls who were close relatives (i.e. less than three genetic degrees, which also re- presents the incest threshold) of the investigated sui- cide cases or other controls to avoid overestimation of family genetic risk. each control was assessed by one or more informants using the same proxy-based procedure as for the assessment of suicide completers. this method has been used with success in numerous previous psychological autopsies (dumais et al. ). we had no direct contact with the controls during the assessment. all of the informants contacted agreed to participate. for each case, a genealogical tree was constructed to detect cases of suicide, suicide attempt, ‘madness’ or ‘mental deficiency’ in the family, and to assess the degree of closeness with other cases assessed. as there were no palawan psychiatrists or psycho- logists, the main psychiatric diagnoses were carried out by f.j., a psychiatrist, with the help of c.m., an anthropologist who speaks palawan fluently, has written the only existing palawan–tagalog–english dictionary (www- .sil.org/asia/philippines/works-pal. html) and has a deep knowledge of local concepts including the expression of emotions and feelings. a palawan translation of the mini-international neuro- psychiatric interview (mini) . . (sheehan et al. ) was provided by c.m., with the help of two native palawan people. a back-translation was then provided by c.m. and verified by f.j. to ensure ad- equacy with the original version. all diagnoses were current for the controls and at the time of death of the suicide victims. impulsive–aggressive personality disorders were clinically assessed by systematically asking about a personal history of self or externally directed ag- gression, theft, incarceration, interpersonal arguments or anger propensity. a checklist was used for all other variables, as shown in table . sociodemo- graphic variables were obtained by interviewing in- formants. a suicide attempt was defined as ‘any act carried out with some intent to die’, as defined by mann ( ). early parental loss was defined as the loss of one parent before the age of years. the popu- lation usually discriminates between ‘madness’ and ‘mental deficiency’, the latter term referring to intellec- tual disability. although they lack precise definition, we chose to keep these terms given by informants because, in many cases, the individual to whom they were referred was not sufficiently known by the informant to clarify the diagnosis. statistical analyses the suicide rate was calculated as the number of suicides over years, divided by for an average annual number, divided by the total population ( ) and multiplied by . statistical analyses were carried out using spss version (spss inc., usa). univariate analyses were conducted first. comparisons of quantitative data between the two groups were made by means of a mann–whitney test because of the small size of the groups. associations between qualitative variables and groups were calculated with the χ test. variables significantly associated with group at the univariate stage were then separately entered into a logistic re- gression model for multivariate analysis, using a step- wise procedure. the p level was set a priori at . . results suicide rates and description of cases sixteen suicides were reported between june and may , giving a global suicide rate of / inhabitants per year. suicide victims were males and four females (giving a gender ratio of / ) and sui- cide rates were / in males and / in females. all suicides occurred between and . we could not find any close informant for one suicide case (male, ∼ years old) who seemed to have lived surprisingly isolated. therefore, the psycho- logical autopsy was conducted on suicide completers and controls. the main method of suicide was poisoning by tuba ( cases), a vegetal poison produced from a readily available plant (genus derris) traditionally used to hunt and fish. the three remaining cases hanged them- selves. one man first killed his pregnant wife before committing suicide. reported motives were marital conflicts/separation (six cases), painful disease (severe migraines; painful and intractable tongue lesion, possibly cancer; and painful motor handicap; three cases), poverty or debt (three), death of husband (one), sentimental rebuttal (one) and unknown (one). nine suicide victims were aged between and years ( . %), two between and years ( . %), three between and ( . %) and two between and ( . %). among the suicide completers assessed, four were close relatives (three siblings and one first cousin) and suicide in an isolated population https:/www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https:/www.cambridge.org/core. university of basel library, on may at : : , subject to the cambridge core terms of use, available at https:/www.cambridge.org/core/terms https://doi.org/ . /s https:/www.cambridge.org/core two others were first cousins. other cases were not directly related to each other. in the first control region, two suicides occurred over the past years among a population of inhabi- tants, leading to a suicide rate of . / inhabi- tants. in the most extreme underestimation of the number of suicide cases by % and overestimation of the population size by %, this would lead to a suicide rate of / well below the rate in the target subgroup. in the two other control com- munities, no suicide could be detected among a popu- lation of – inhabitants each over the past years. psychological autopsy higher rates of panic disorder, agoraphobia and social phobia, and first- and second-degree biological relatives who committed suicide were found in suicide completers compared to controls (table ). there was also a trend toward increased depressive disorders within this group. social phobia, but not panic disorder and agoraphobia, remained significant when depression was taken into account in the logistic regression (social phobia: wald= . , p= . ; depression: wald= . , p= . ). the two groups did not differ in terms of other vari- ables. importantly, no case of childhood maltreatment and only one case of alcohol substance abuse and impul- sive–aggressive personality disorder was detected in either group. a burden of risk in terms of number of psychiatric disorders was calculated for each individual as the sum of all available diagnoses (among possible). between-group comparison showed a significantly higher number of diagnoses in suicide victims versus controls [median= (range – ) vs. (range – ), u= . , p= . ]. table . comparison of sociodemographic and clinical variables between suicide completers and controls matched for gender and age suicide (n= ) controls (n= ) mann–whitney u or χ p early parental loss ( . ) ( . ) . . childhood maltreatment ( ) ( ) – – attended elementary school ( . ) ( . ) . . marital status (married) ( . ) ( . ) . . number of children ( – ) ( – ) . . financial debts ( . ) ( . ) . . current physical disease ( . ) ( . ) . . depressive disorders ( . ) ( . ) . . bipolar disorders ( ) ( . ) . . panic disorder ( . ) ( ) . . agoraphobia ( . ) ( . ) . . social phobia ( . ) ( . ) . . ptsd ( ) ( ) – – generalized anxiety ( ) ( ) – – alcohol abuse ( . ) ( ) . . substance abuse ( ) ( ) – – tobacco use ( . ) ( . ) . . psychotic disorder ( ) ( ) – – impulsive–aggressive pd ( . ) ( ) . . expressed suicidal ideas (lifetime) ( ) ( . ) . . previous suicide attempt ( . ) ( . ) . . family history of suicidal act first genetic degree ( . ) ( . ) . . at least second degree ( . ) ( . ) . . at least third degree ( . ) ( . ) . . family history of ‘madness’ or ‘intellectual deficiency’ a ( . ) ( . ) . . ptsd, post-traumatic stress disorder; pd, personality disorder. a no clear data for two cases. values given as number (percentage) or median (range). diagnoses are at time of death for suicide victims and current for controls. f. jollant et al. https:/www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https:/www.cambridge.org/core. university of basel library, on may at : : , subject to the cambridge core terms of use, available at https:/www.cambridge.org/core/terms https://doi.org/ . /s https:/www.cambridge.org/core discussion suicide is usually considered to be extremely low in pre-industrialized populations, leading to the notion that this is a problem of modern societies. although this may be true in many instances, we report here a suicide rate of / inhabitants, that is times the rate of suicide in canada, the usa and many european countries, among a small subgroup of indigenous people in the philippines. this extremely high suicide rate seems to have persisted for the past decade (and probably over the past years) and, importantly, is very localized. indeed, the rest of the palawan population, of similar culture, does not seem to share this feature. hence, in this subgroup suicide does not seem to be related mainly to cultural factors. indeed, the social organization of this population should theoretically represent a protective factor against suicide. for instance, palawans show a high degree of family ties, frequent interactions and a sense of community. cooperation is a core principle of societal functioning. violence is avoided and palawans are usually de- scribed as a shy people, with no rigid code of honor, who value escape when facing aggression. further- more, the group will identify a leader based on that person’s ability to settle disputes. in this egalitarian population, the status of leader entails no power or privilege. moreover, palawan culture does not promote suicide as a way to solve problems and palawans usually express perplexity and fatality regarding suicide. finally, this population has been living more or less in similar conditions for many years and, so far, has not suffered brutal displacement, forced assimilation or ‘westernization’. other reasons have, therefore, to be sought. of note, other similarly striking differences in rates of suicide among identical ethnolinguistic groups have previously been reported worldwide in the ethnological literature (macdonald, ), includ- ing the baruya (suicide rate= / ) versus the ankave (almost none) in new guinea (bonnemère, ) and the aguarunas (suicide rate= / ), a subgroup of the jivaro people (very low suicide rates) (brown, ). these populations differ between them in several respects from the use of violence to gender relationships (symmetry versus asymmetry) or the structure of society (egalitarian versus hierarchi- cal) (macdonald, ). however, all of these popula- tions share the common feature of a high degree of isolation and endogamy (macdonald, ), a factor that may play a significant role in this high rate of suicide. this could explain the significantly higher frequency of suicide cases among first and second biological relatives of suicide completers (reaching % of cases). of note, the family rate of suicide is also very high among controls, up to almost % at the third biological degree. unfortunately, we do not have a precise index of endogamy. therefore, at this stage, its role remains theoretical. future investigation of this population will attempt to measure this factor. two non-mutually exclusive hypotheses may be drawn from this. first, this high level of isolation may increase the risk of contagion effect, a recognized phenomenon in suicidal behavior (brent & melhem, ). however, this phenomenon is usually limited in time. a second explanation may be that an original founding effect or a genetic drift (stone et al. ) has selected particular alleles conferring a higher risk of suicide, the frequency of which is amplified by iso- lation. genetic factors have been robustly implicated in the vulnerability to suicide in family, twin and adoption studies (brent & mann, ). these genetic factors may render carriers more sensitive to stressful events and more likely to experience pain, hopeless- ness and suicidal ideas, and to act upon these negative thoughts. more investigations are necessary to identify these factors. many suicide risk factors described in western populations are absent here, suggesting a particular developmental and clinical profile, in comparison to the ‘suicidal trajectories’ previously described in the literature (seguin et al. ). one important risk factor is childhood maltreatment, previously found in up to % of suicide cases in western countries (turecki et al. ) but in none of the suicide and control cases in palawan. external informants and prolonged observation tend to confirm the absence of childhood maltreatment in this population. moreover, palawan people are known to be non-violent and children are usually raised by an extended family. this may reduce the risk of childhood abuse and consequently the risk of transmission of these behaviors across generations. mental disorders are major risk factors for suicide (cavanagh et al. ), which is also the case in the palawan subgroup, with a higher burden of risk in sui- cide victims. however, the profile of mental disorders found here differs from those previously described in western studies, with the exception of increased de- pression. this is notably the case for social phobia, which was associated with suicide independently of depression. although this association has been de- scribed previously (kanwar et al. ), it seems to be a particular signature of palawan suicide, accounting for half of all suicide cases. it is important to mention that palawan people tend to be shy in general. nevertheless, people clearly identified individuals with social phobia as different from being simply shy (e.g. ‘he never ate with the group during feasts’ or ‘she would never gather with us to discuss in group’). suicide in an isolated population https:/www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https:/www.cambridge.org/core. university of basel library, on may at : : , subject to the cambridge core terms of use, available at https:/www.cambridge.org/core/terms https://doi.org/ . /s https:/www.cambridge.org/core it is noteworthy that motives for suicide are not unique to this population (foster, ), with inter- personal conflicts accounting for almost half of cases, followed by painful diseases. poverty and debt may also be in play through a feeling of failure to take care of family. the ‘social feature’ of suicide is there- fore also found among the palawans, and social phobia may play a significant role in suicidal tendencies by making some individuals more sensitive to their social environment and less likely to seek help from others. alcohol and drug abuse were not significant risk factors, mainly because these substances are not readily available. in addition, we could not identify any impulsive or aggressive personality disorders among the studied groups, with the exception of one individual who had alcohol problems, a long history of arguments with neighbors and who killed his wife before committing suicide. the lack of these major risk factors again supports the idea of a different developmental trajectory to suicide. psychotic and bipolar disorders were rarely reported in our sample, possibly because of the small sample sizes. suicide attempts, although reported, are rare in this population. as a consequence and contrary to what is found in western countries, a history of suicide attempt is not a risk factor of future suicide in this population, which is significant in the perspective of prevention. many explanations may be put forward, including the high lethality of the means used being associated with the lack of closed medical settings, the absence of available medications, which is the pri- mary method used in non-fatal acts, and the lack of available medical services, which has been incrimi- nated as facilitating acts carried out as a ‘cry for help’. to our knowledge, only two other studies have examined risk factors for suicide in endogamous popu- lations. the first one (egeland & sussex, ) studied the old order amish in pennsylvania. like the palawans, the amish are non-violent, show high social cohesion, live in isolation, and show little use of al- cohol and drugs. contrary to the palawans, however, suicide among the amish is taboo and rates of suicide have remained low over the past years. suicide was associated with mood disorders, although it was found to cluster in some families, while other families showed high rates of mood disorders without suicide. we could not assess this latter aspect from our data, and this constitutes a suitable topic for future research. the second study involved an endogamous arab kin- dred in israel that showed high rates of suicide (around / ) but also suicide attempts (hamdan et al. ). like the palawan suicide completers, suicide attempters in this population showed high levels of de- pression and anxiety, high frequencies of first-degree relatives who attempted suicide, and low rates of alcohol and substance abuse. however, they also showed high levels of impulsive aggression and child- hood abuse. in these two groups and in the palawans, endogamy, family transmission and higher burden of mental disorders seem to be major risk factors, although the precise picture varies from one group to another. our study has several limitations. first, language barriers may limit the reliability of some psychiatric diagnoses. however, we believe that many concepts, such as low mood or social phobia, were perfectly translated and understood in palawan. we also took great care to ensure that cultural factors were taken into consideration when determining disorder thres- hold. second, as in many studies, recall issues may bias some data collection. this issue is probably exacer- bated by the fact that palawans do not make written records of any event. nonetheless, the habit of relying on their memory and the fact that many informants were usually present during interviews may have lim- ited this effect. third, suicide was not confirmed by any medical authorities, nor was there any evidence of suicide notes. therefore, false positives may have altered the accuracy of certain findings. fourth, although the control villages were similar in size and culture to the high-risk villages, we cannot exclude the possibility that they differ from each other by fac- tors we did not measure. however, our long-time observations suggest that the rate of suicide is usually low among the palawans. finally, as many informants were usually present during interviews, it cannot be excluded that a desirability effect may have biased some reports. future psychological autopsies in this population may be conducted in different conditions to examine the importance of this effect in this other- wise very social population. we hope that the unique investigation reported here will shed light on the existence of isolated groups at very high risk of suicide across the world, and in- crease our understanding of suicide from both a singu- lar (e.g. low level of impulsivity, violence, childhood abuse and alcohol/drug use; high level of anxiety; the potential effect of isolation among palawans) and a universal (e.g. family history of suicide and personal burden of mental disorders) perspective. acknowledgments f.j. received a fond de recherche du québec santé (frqs) salary grant and c.m. a cnrs travel grant. the study sponsors had no role in designing the study, collecting data, writing the report or in the decision to submit the report. we express our sincere gratitude to edie and botoy for their help in translating the consent forms, f. jollant et al. https:/www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https:/www.cambridge.org/core. university of basel library, on may at : : , subject to the cambridge core terms of use, available at https:/www.cambridge.org/core/terms https://doi.org/ . /s https:/www.cambridge.org/core questionnaires and instructions into the palawan language, to edie and his wife for hosting us during several weeks, and to the palawan people for welcom- ing us and for their genuine interest in participating in the study. f.j. thanks his colleagues for taking care of his patients during his long leave in the field. we thank c. nagy for her valuable help in editing the manuscript. declaration of interest none. references bonnemère p ( ). suicide et homicide: deux modalités vindicatoires en nouvelle-guinée. stanford french review , – . brent da, mann jj ( ). family genetic studies, suicide, and suicidal behavior. american journal of medical genetics. part c, seminars in medical genetics , – . brent da, melhem n ( ). familial transmission of suicidal behavior. psychiatric clinics of north america , – . brown mf ( ). power, gender, and the social meaning of aguaruna suicide. man , – . cavanagh jt, carson aj, sharpe m, lawrie sm ( ). psychological autopsy studies of suicide: a systematic review. psychological medicine , – . dumais a, lesage ad, alda m, rouleau g, dumont m, chawky n, roy m, mann jj, benkelfat c, turecki g ( ). risk factors for suicide completion in major depression: a case-control study of impulsive and aggressive behaviors in men. american journal of psychiatry , – . egeland 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and young adult suicide: a study of life trajectory. journal of psychiatric research , – . sheehan dv, lecrubier y, sheehan kh, amorim p, janavs j, weiller e, hergueta t, baker r, dunbar gc ( ). the mini-international neuropsychiatric interview (m.i.n.i.): the development and validation of a structured diagnostic psychiatric interview for dsm-iv and icd- . journal of clinical psychiatry (suppl. ), – ; quiz . shifman s, darvasi a ( ). the value of isolated populations. nature genetics , – . stone l, lurquin pf, cavalli-sforza ll ( ). genes, culture, and human evolution: a synthesis. wiley-blackwell: chichester. turecki g, ernst c, jollant f, labonte b, mechawar n ( ). the neurodevelopmental origins of suicidal behavior. trends in neurosciences , – . suicide in an isolated population https:/www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https:/www.cambridge.org/core. university of basel library, on may at : : , subject to the cambridge core terms of use, available at https:/www.cambridge.org/core/terms https://doi.org/ . /s https:/www.cambridge.org/core back to the family: a renewed approach to rare variant studies back to the family: a renewed approach to rare variant studies the harvard community has made this article openly available. please share how this access benefits you. your story matters citation zielinski, dina, melissa gymrek, and yaniv erlich. . “back to the family: a renewed approach to rare variant studies.” genome medicine ( ): . doi: . /gm . http://dx.doi.org/ . / gm . published version doi: . /gm citable link http://nrs.harvard.edu/urn- :hul.instrepos: terms of use this article was downloaded from harvard university’s dash repository, and is made available under the terms and conditions applicable to other posted material, as set forth at http:// nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of- use#laa http://osc.hul.harvard.edu/dash/open-access-feedback?handle=&title=back% to% the% family:% a% renewed% approach% to% rare% variant% studies&community= / &collection= / &owningcollection / &harvardauthors=d c b c a d b cde a&department http://nrs.harvard.edu/urn- :hul.instrepos: http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa introduction th e annual meeting of the american society of human genetics is a major  ­ some would say overwhelming  ­ conference that aims to present the state of the fi eld as a whole, with presentations on nearly all aspects of human genetics. th is year, the meeting had a record number of nearly , participants, with approximately scien­ tifi c presentations and over , posters. in addition, more than vendors presented their products, includ­ ing all major dna sequencing companies, bioinformatic services for data analysis, and clinics specializing in genetic medicine. in order to navigate this scientifi c maze, the organizers developed a smartphone application to allow users to browse abstracts based on schedule, presenter, and topic, and to generate a snapshot of the current events at the conference. intriguing buzzwords had participants running back and forth between rooms to hear speakers from parallel sessions. some of the most frequent terms used throughout the meeting, according to quantitative text analysis of #ashg tweets, were ‘rare’ and ‘common’ with both almost equally represented. indeed, in the spirit of the us presidential election, which coincided with the fi rst day of the meeting, there seemed to be a common and rare variant party division. entire sessions were devoted exclusively to either class of variant, such as ‘gwas from head to toe’ and ‘cancer genetics i: rare variants’. in an attempt to fi nd a bipartisan resolution, one session was dedicated to ‘common variants, rare variants, and everything in between’, and presented the advantages of an integrated approach that examines association signals of both variant classes. interestingly, several talks showed that such integrative studies based on whole exome sequencing can simultaneously replicate known genome­ wide association study (gwas) signals and uncover a distinct set of genes that harbor rare etiological variations. the interpretation gap a recurrent issue throughout the meeting was the gap between data generation and data interpretation, espe­ cially for rare variants. th is challenge is exacerbated by the application of sequencing in the clinic, where evaluation of pathogenic variants and incidental fi ndings may determine the course of treatment. a signifi cant number of talks described potential techniques to over­ come this interpretation gap. some speakers, such as heidi rehm (harvard medical school, usa) suggested the development of specifi c databases for clinical inter­ pre tation. others, including marc greenblatt (university of vermont, usa), focused on the development of standards for variant interpretation. another set of presen tations described the recent successes of commu­ nity­based interpretation contests, such as boston children’s hospital’s clarity challenge and berkeley’s critical assessment of genome interpretation. th e main challenge when studying rare variants is that robust statistical inference of their eff ects in case­control studies requires a large amount of sequencing data. daniel macarthur (massachusetts general hospital, usa) suggested a brute force approach that includes sequencing a large number of individuals, postulating that ‘in order to understand one genome, we need to sequence tens of thousands of genomes.’ family studies: a shortcut to analyze rare variants other speakers suggested alternative, more effi cient approaches to rare variant interpretation. in fact, one of the prevailing themes at this year’s meeting was the renewed interest in large pedigrees and isolated popu la­ tions to assess the eff ect of rare variants on common traits. michael province (washington university, usa) abstract a report on the nd annual meeting of the american society of human genetics, san francisco, california, usa, - november . © biomed central ltd back to the family: a renewed approach to rare variant studies dina zielinski , melissa gymrek , , , and yaniv erlich ,* m e e t i n g r e p o r t *correspondence: yaniv@wi.mit.edu whitehead institute for biomedical research, cambridge center, cambridge, ma , usa full list of author information is available at the end of the article zielinski et al. genome medicine , : http://genomemedicine.com/content/ / / © biomed central ltd presented one potential problem of the brute force approach in a session centered on family studies as a means to investigate complex traits. citing recent studies about rapid population growth in humans, he noted that the number of extremely rare alleles in the population is much higher than thought. even doubling the sample size does not help very much, as the new sample will simply present new rare alleles, rather than add statistical power. as an alternative, he suggested focusing on large pedi­ grees, where the allelic diversity is smaller: ‘pedigrees make the needle [rare variants] in the haystack bigger’ and thus easier to find. further, the extensive identity­by­ descent (ibd) between individuals helps to distinguish true rare variant calls from sequencing errors and pro­ vides a means to verify novel alleles in multiple related individuals. robert elston (case western reserve university, usa) was the most insistent about the current appeal of family studies in a special session recognizing his th birthday. he went so far as to say that ‘somehow, for the last decade or so, we were misguided into thinking that families were not necessary, and we have seen epidemiologists having a ball with case­control studies and honestly believing that they are doing genetic research!’ elston did say that family studies may one day in the distant future be dispen sable, but maintained that, for now, it is crucial to study variants in the context of inheritance, rather than simply as dna. from interpretation bottlenecks to genetic bottlenecks in addition to the renewed interest in family studies, several speakers highlighted the value of studying isolated populations. jeffrey o’connell (university of maryland, usa) described a study of complex traits in the amish population, in which he found a steady increase in the inbreeding coefficient in the last years. he showed that, on average, a pair of individuals is as genetically similar as first cousins once removed. with such a strong genetic bottleneck, rare variants that segregate in the european population may increase by orders of magnitude in the amish population, enabling robust statistical inference about their roles. to stress this point, he concluded his talk with a reminder that ‘we study the amish not because they are different but because they are us.’ william scott (university of miami, usa) and cornelia van duijn (erasmus university rotterdam, the nether­ lands) presented an integrative approach for identifying pathogenic variants of complex disorders in isolated populations. their method starts with linkage analysis to find large segments that segregate with a given pheno­ type, followed by whole exome sequencing to pinpoint the pathogenic variant in the linkage interval. this tech nique showed mixed results. they were able to uncover a rare pathogenic variant in a study of depression but found no coherent signal in a study of parkinson’s disease, suggesting a potential role for non­coding variants. other presenters tried to reconcile the advantages of both traditional case­control and family studies. hua zhou (ucla, usa) discussed combining genome­wide association mapping with pedigrees for quantitative trait locus analysis. similarly, richard spritz (university of colorado denver, usa) presented an approach that integrates gwas with the sequencing of siblings under a linkage peak. elizabeth thompson (university of wash­ ing ton, usa) discussed using ibd within and between pedigrees, echoing robert elston’s emphasis on the need for information on relatedness. she concluded that, eventually, pedigree and population studies will be equivalent, in the sense that we can use techniques for analyzing ibd to obtain the same information. conclusion in the past few years, we have witnessed the emergence of large­scale sequencing projects to study common diseases, such as the nhgri’s clinseq study, nhlbi’s exome sequencing project, and the personal genome project. the renewed interest in large pedigrees and isolated populations for complex trait studies at this year’s meeting was refreshing. several speakers high­ lighted the advantages of such designs in interpreting the role of rare genetic variations. in addition to facilitating the ascertainment of multiple individuals with the same rare variant, the substantial ibd in the samples promotes imputation and increases confidence in the sequencing results. further, these designs afford a set of comple men­ tary tools, including linkage analysis and heritability measurements, that can accelerate genetic investigation. by definition, personalized medicine entails drawing conclusions based on the study of a single genome from the general population. despite the tremendous advan­ tages of family and isolated population study designs, we should also remember that they do not entirely reflect the general population. for instance, the substantial ibd between participants in these studies increases the likeli­ hood of overestimating the effect of a variant due to epistasis, a point that was emphasized in the opening talk at ashg by eric lander (broad institute, usa). another potential complication is the sampling of indi­ viduals from narrow environmental conditions, which is more prone to confounding gene­environment inter­ actions. evidently, nothing comes free in human genetics. each study design has its own limitations and advantages, necessitating an integrative approach to bridge the inter­ pretation gap and effectively handle today’s population­ scale datasets. meeting tweets are available online at #ashg . zielinski et al. genome medicine , : http://genomemedicine.com/content/ / / page of abbreviations gwas, genome wide association study; ibd, identity-by-descent. competing interests the authors declare that they have no competing interests. acknowledgments this publication was supported by the national defense science & engineering graduate fellowship and by a national human genome research institute grant r hg . ye is an andria and paul heafy family fellow. author details whitehead institute for biomedical research, cambridge center, cambridge, ma , usa. harvard-mit division of health sciences and technology, mit, cambridge, ma , usa. program in medical and population genetics, broad institute of mit and harvard, cambridge, ma , usa. department of molecular biology and diabetes unit, massachusetts general hospital, boston, ma , usa. published: december doi: . /gm cite this article as: zielinski d, et al.: back to the family: a renewed approach to rare variant studies. genome medicine , : . zielinski et al. genome medicine , : http://genomemedicine.com/content/ / / page of fea-gray.indd april notices of the ams mathematics and home schooling kathleen ambruso acker, mary w. gray, behzad jalali, and matthew pascal e mpowered by the belief that an educated citizenry made for a strong nation, co- lonial governments as early as mandated compulsory education for school-age children (hiatt ). numer- ate citizens were needed for strong commerce and successful farming, and the governments saw their education as an important objective. today parents can choose from public schools, magnet schools, charter schools, private schools, and parochial schools. despite the varied pub- lic and private opportunities available, over . million students are engaged in home school programs, some of whom will choose to pursue a postsecondary education. given the increasing attention to national standards for the prepara- tion of students, it is important to understand the climate for, and results of, home schooling insofar as mathematics is concerned. in our analysis, we address the legal framework surrounding modern home schooling, noting varia- tions in state regulations and curriculum options, with particular attention to mathematics. we then examine how well the structure can be said to prepare students for postsecondary education and whether there are legal remedies if it does not. state mandated education motivated generally by religiosity, ensuring liter- ate children was a high priority among founding colonists (gaither ). toward that goal children were taught in the home or in small groups in a religious setting, where in addition to learning how to run a household, farm, or small business, they learned to read, primarily from the bible. a shift away from home schooling first came in with the establishment of the first public school in boston. in massachusetts passed a law re- quiring parents to teach their children how to read and write; the statute gave the state the authority to provide education if the need arose. a second measure, known as the old deluder satan law or the general school law of (martin ), re- quired towns with more than fifty families to hire a teacher and with more than one hundred families to support a grammar school. massachusetts thus fostered literacy, which in turn meant the popu- lation could read and understand the bible and undertake civic and economic responsibilities or, more colorfully, stay out of the hands of satan. the costs of running the school and paying the teacher were placed upon the adults responsible for the children to be educated. the passage of this law marks the beginning of compulsory education in the united states. through the country’s history, a basic principle of federalism, namely that the federal government would have limited constitu- tionally defined powers with other governmental functions reserved to the states, has guided the development of education. in composing the bill of rights, the founding fathers notably did not address education; the tenth amendment states: the powers not delegated to the united states by the constitution, nor prohib- ited by it to the states, are reserved to the states respectively, or to the people. kathleen ambruso acker is an independent researcher in mathematics education, who teaches mathematics for american university part time. her email address is acker@american.edu. mary w. gray is professor of mathematics at american university. her email address is mgray@american.edu. behzad jalali is director of educational services at ameri- can university. his email address is bjalali@american. edu. matthew pascal is assistant professor of mathematics at park point university. his email address is mpascal@ pointpark.edu. doi: http://dx.doi.org/ . /noti notices of the ams volume , number as colonies and territories became states and drew up their own constitutions, each came to include a clause assuming for the state the responsibilities for education. some of these clauses were limited to providing a basic level of education to assure effective civil and economic participation, while others were broader and more prescriptive as to how this was to be achieved. for example, vermont in was the first state to require that educa- tors hold a teaching certificate (cubberly ); other states specified subjects to be taught, often including mathematics. although the responsibility for education is clearly assigned to the states, in practice educa- tion in the united states has always been a local operation, generally with schools organized into districts by city or county. as the country ex- panded, so also did enthusiasm for inspiring a sense of civic responsibility through education. over time home schooling a child was no longer widely prevalent; complementary to the constitu- tionally mandated state responsibility for educa- tion, compulsory attendance laws were instituted. the laws generally authorized private (including religious) school enrollment as meeting these requirements, sometimes prescribing that the education they provide should be equivalent to that in the public schools. nineteenth- and early twentieth-century litigation upheld the right of the state to carry out its mandate through these laws, including prohibiting home schooling to the extent that the parents who chose to home school were sometimes pursued by the courts on the charge of truancy (gaither ). in state v. bailey ( ) the indiana supreme court declared that the natural rights of the par- ent with regard to the custody and control of his children are subordinate to the power of the state, so that no parent can be said to have the right to deprive his child of the advantages of public education. on the other hand, the lower court in indiana, in state v. peterman ( ), found it sufficient to employ a qualified teacher to teach children in the home under the private school provision of the compulsory attendance law, the court declaring that a school is any place where instruction is imparted to the young, a finding echoed by the illinois supreme court half a century later in people v. levisen ( ). but the attitude of courts was not always so tol- erant. even conceding the defendant’s claim that he was qualified to teach all grades and all subjects taught in the public schools, in washington v. cou- nort ( ) the washington supreme court held that teaching his children at home did not qualify as attending a private school and thus home school- ing failed to conform to the compulsory attendance law. directly addressing home schooling again, the supreme court of new hampshire in hoyt v. dan- iels ( ) declared that being schooled at home was not schooling in a private school, the only alternative to public school permitted in the state’s school attendance law. nearly ninety years later, a lower court in california came to the same conclu- sion (in re rachel ), but in the climate of the political power of the home schooling movement, that decision was overturned in jonathan v. supe- rior court of los angeles county ( ), with the court declaring that home schooling was consid- ered to be a private school and could be forbidden only on safety grounds, for example to prevent child abuse (in the matter of william aa, ). an oregon case is often cited as supporting home schooling, although actually the freedom to run a private school was the issue. the state’s compulsory attendance statute required enroll- ment in the public school system. in fact, although its stated intent included ensuring full opportunity for immigrant children to assimilate as well as mandating integration of children from all eco- nomic strata in an effort to achieve equality of edu- cational opportunities, many viewed the law as an anti-catholic, anti-immigrant measure, no matter how compelling the argument for universal expo- sure to common values and mingling with diverse populations might be. the u.s. supreme court in pierce v. society of sisters ( ) was careful to make clear the state’s right and responsibility to regulate education, although not to the extent that the oregon law contemplated. although often cited as upholding the freedom of religion clause of the first amendment, the decision concerned itself more with the business interests of the so- ciety of sisters school. a few years earlier, inspired by an excess of nationalistic fervor, nebraska had passed a law forbidding teaching in a language other than eng- lish to students who had not completed the eighth grade. once again, the supreme court (meyer v. nebraska ) strongly endorsed the right of the state to regulate education as a part of its mandate to provide for it, but found on fourteenth amend- ment equal protection grounds that the nebraska law was arbitrary and served no rational purpose. upholding the right of parents to direct the educa- tion of their children, at the same time the court clearly did not contemplate home schooling, as- serting, “education of the young is only possible in schools conducted by especially qualified persons who devote themselves thereto.” another case often cited for limiting state regu- lation of school attendance actually also strongly emphasized the state’s responsibility to assure the education of children. in balancing the right of parental control with the rights of the state, the u.s. supreme court in wisconsin v. yoder ( ) allowed an exemption from wisconsin’s compul- sory attendance law for the amish community only because it believed that the very nature of the religion would be undermined with exposure april notices of the ams of its young people to the worldly culture of education beyond the eighth-grade level. if the state’s purpose in education is to prepare children for life, asserted the court, the limiting nature of education espoused by the amish was adequate for their separate agrarian way of life. attempts by other religious groups to claim an “amish exemp- tion” for their educational practices or lack thereof have not been well received by the courts (fellow- ship baptist church v. benton ( ), johnson v. charles city community schools ( )). the rise of home schooling we have seen something of the ups and downs of litigation, but it is ironic that the state where public education originated helped set modern legal precedence for home schooling as an educa- tion option with the decision in perchemlides v. frizzle ( ). the perchemlides family had home schooled their older children while living in boston. after moving to amherst, they sent their youngest son to the public school system for second grade, where he appeared to regress intellectually and socially. the family opted to continue their son’s education at home and filed the required notice with the school district that the family intended to home school. the school district denied the request for four reasons: (a) the parents were not teachers; (b) the curriculum outlined by the family was not linked to the child’s current developmental level; (c) the curriculum did not provide for interaction with other groups of children; and d) the child’s difficulties were the result of his earlier education, not of the current public school setting. the district superintendent brought a charge of truancy against the parents when they chose to keep their son at home. the court ruled that parents were competent to teach their children and that as long as the curriculum covered those subjects mandated by state law on the mandated time schedule and there was a level of accountability in place, home schooling was a legal option for education in massachusetts. the case established that home schooling was protected as a state constitutional right subject only to state regulation that must be essential to providing an adequate education. this turnaround in litigation concerning home schooling marked the rise not only of home schooling as an edu- cational philosophy but as a political movement. although not a decision with precedential value, the cause of the perchemlides family, particularly their resistance to home inspection visits, was widely embraced by the home schooling commu- nity nationally. taking a somewhat different approach, the federal court in jeffery v. o’donnell ( ) ruled that the pennsylvania compulsory attendance laws in place were constitutionally vague in the require- ment that the curriculum must be “satisfactory” and that a parent must be “properly qualified” and thus constituted a threat to first amendment freedoms. the aftermath, however, has not been less vague requirements but rather essentially no requirements at all. teacher certification as home schooling became more popular, par- ticularly among religious conservatives, courts in different states adopted varying approaches to the practice. some states focused on teacher certification. in florida v. buckner ( ), the florida court of appeal declared that the school attendance statute clearly prohibited an unquali- fied parent from teaching a child at home under the guise of being a private school. on the other hand, in delconte v. north carolina ( ), the north carolina supreme court declared that ab- sent a clear legislative intent, the relevant statute could not be interpreted to prohibit home school- ing, but it did not rule whether in fact the state could constitutionally prohibit it were the statute more carefully drafted. the court in blackwelder v. safnauer ( ) upheld a statute requiring instruction in home schools to be “substantially equivalent” to that in public schools and to be given by “competent” instructors. additional rul- ings upheld a teaching certification requirement for home schooling (clonlara, inc. v. runkel ; hanson v. cushman ; jernigan v. state ; people v. dejonge ). however, it is not clear how similar cases would fare today. in particular, between and more than twenty states repealed their teacher certification requirements for home schooling (dwyer ). all states require public school teachers to hold a teaching certificate, granted after completing a regime of college courses accredited by the state and often including an exam or series of exams covering basic subject matter. in general the curric- ulum for certification requires that teachers learn about childhood development and psychology and that secondary school teachers be immersed in courses that allow for subject specialization. the no child left behind act (nclb) required states to ensure that education should come from a “highly qualified” person, described thusly: to be deemed highly qualified, teachers must have: ) a bachelor’s degree, ) full state certification or licensure, and ) prove that they know each subject they teach (nclb ). all indications are that the nclb successor federal legislation will have similar provisions. private schools are generally exempted from teacher certification requirements, and home schooling is specifically exempted from all provisions of the nclb. in the states where legal criteria for home school providers are listed, generally the notices of the ams volume , number qualifications are broadly described and include some or all of the following: parents must be mini- mally competent, to have taken an education class, to have a ged, to have completed a bachelor’s degree. some states require that homeschoolers successfully complete an interview or periodic meetings with a public school representative, requirements not easily nor uniformly enforced. in any case, federal legislation is likely to take a narrower view of education than do many home- schoolers. the nclb does not mention virtue, morality, or religious conviction as a purpose of education but speaks mainly of quantifiable aca- demic assessment and achievement, exempting home schooling from any accountability for such standards. should less controversial standards be adopted, home schooling is still likely to be ex- empt. establishing the basic right to home school seems beyond effective challenge in the current climate. uniform enforcement of compulsory education laws is difficult in a home school situa- tion, particularly with public education strapped for funding for the public schools themselves. state regulations or standards to be met are often vague, and overseers generally represent the very school system the homeschoolers seek to escape. moreover, there are generally no health and safety safeguards, such as compulsory vaccinations, in place. not only is there a fierce lobby in support of home schooling, but given the inadequacy of many public schools with overcrowding, high failure rates, and confusing standards enforced by tests of questionable value, forcing those cur- rently home schooled into public education might be a cure worse than the disease. however, “dis- ease” does not describe the nature of much home schooling that is an option that is clearly valuable for many children, particularly those with special needs. nonetheless, there are concerns centered on the acknowledged government obligation to assure education of children for full civic and economic participation. to further our discussion, we consider the ques- tion, who is a home school student in the united states?, and then we examine two aspects of these concerns: the adequacy of home schooling from a subject matter perspective and the potential for gender discrimination. who chooses to home school? to provide a definition of a home-schooled stu- dent, we choose to follow the definition used by the national center for education statistics (nces), which states: students were considered to be home schooled if their parents reported them being schooled at home instead of a public or private school, if their en- rollment in public or private schools did not exceed hours a week, and if they were not being home schooled solely because of a temporary illness. (nces ) the results of the nces study found a small percentage ( . %) of all school-age children, less than one million, were home schooled, and approximately percent of those students took advantage of services offered at the public school. services available included curriculum materials, texts, meetings for parents of students, and extra- curricular activities (nces ). follow-up studies conducted by nces show that by that number was increasing and by there were in excess of . million students home schooled (nces ). recent reporting from national home education research institute (nheri) estimates the number of students in home schooling to be more than two million (www.hslada.org). data show that families who choose home schooling tend to be white, non-hispanic, two- parent households with only one parent working. religious beliefs are only one component of why parents report choosing to home school; data in the study also suggest that parents feel they can give their children a better education at home, that the curriculum offered in schools is not challeng- ing their child, and that the school environment is poor (nces ). although there are no reliable national sta- tistics, it is the case that many homeschoolers supplement what is taught individually in the home with group study and opportunities for field trips as well as participation in public and private school activities. for the most part the involve- ment has been in such areas as sports (there are also home school leagues in some sports in some regions) and music. entitlement to such participa- tion is not clear, although there has been legisla- tion introduced in several states to ensure it under certain conditions. curriculum concerns the largest group of homeschoolers consists of christian evangelicals whose opposition to enroll- ing their children in public schools is based on two beliefs. one is their desire to avoid the schools’ secular nature, in particular their children’s po- tential exposure to and mingling with those whose religious and cultural background differs from their own. they may also object to particular sub- ject matter, such as evolution or the teaching of the validity of and need for respect for different cultures. it even has been said that the teaching of probability should be avoided, since it evolved see, e.g., friendship baptist church, f. supp at : “tests primarily determine knowledge of content of the subject matter. they do not test other aspects of education necessary to prepare a student for life in today’s society.” http://www.hslda.org april notices of the ams states do not even require home schooling parents to notify the state of their intent to home school, currently all states have legislated some rules for providing home schooling, with the stringency of the requirements varying widely, as illustrated by the following examples (www.hslda.org). pennsylvania apparently demands much from home school families. in addition to submitting an affidavit of intent to home school, applicants must assure that topics are taught in english, provide immunization records, develop an instructional plan, and maintain a portfolio of accomplishments covering state requirements, including english and mathematics. at the end of each academic year, portfolios are evaluated by experts from the state education authority. however, home school teachers simply must hold the equivalent of a high school diploma. in addition, students in grades , , and are required to participate in the pennsylvania system of school assessment, a statewide requirement for public school students. enforcement of these requirements appears to be at best sporadic and superficial. north carolina requires home-schooled stu- dents to maintain immunization records and to keep attendance records. students are not re- quired to follow a state prescribed curriculum, but they must submit to annual standardized testing in several topics, including english and mathemat- ics, and instructors must hold the equivalent of a high school diploma. for follow-up, the state’s rights are limited to simply inspecting test scores. nebraska has very little regulation with regard to the policies and procedures home schools must follow. teachers do not need to meet any quali- fications, unless the family hires outside tutors. the state does not require visitation or testing, because it claims it cannot apply the standards for such uniformly across the state. parents, under oath, swear to provide sequential instruction in several subjects, including language arts and mathematics. california requires instruction to be in english and attendance records must be kept. subjects to be studied include english; additional topics may be chosen from the topics covered in the public schools. mathematics is required only during grades through . no standardized testing is required. looking at the states as a whole, we find that currently twenty-eight states and the district of columbia use “mathematics” for a topic to be learned by those who are home schooled, although content is not specified. north dakota and penn- sylvania are two states that detail the number of mathematics credits and mention algebra as required study. seven states note that subjects covered should be comparable to those in the cur- riculum of the public school system. three other states only require that students learn arithmetic, from and is associated with gambling. the highly effective opposition to regulation of home school- ing is led by this group through the home school legal defense association and its lobbying arm, the congressional action program (stevens ). other homeschoolers are primarily concerned with the inadequacy of the public schools in meet- ing the needs of their children, especially those with special needs at either end of the learning spectrum. it is the first group that engenders the concerns cited earlier. in the s there was a spate of litigation concerning the material covered in public schools. general lack of success was, no doubt, bound to lead to opting out of the system. in mozert v. hawkins county board of education ( ) plain- tiffs objected to requiring the reading of certain textbooks, claiming that it was a burden on the free exercise of religion. since the first amend- ment guarantees this fundamental right, the court chose to adopt an intermediate standard (between rational and strict scrutiny): do defendants have a compelling interest in requiring all students in grades to to read the basic series of books of a specific publisher? the objections were, not surprisingly, based on the treatment of evolution, but also on what was perceived as endorsement of magic. although plaintiffs’ claim was a lack of balance, testimony at trial also revealed objections to teaching tolerance of religious views other than their own. finding that public schools serve the purpose of teaching fundamental values “essen- tial to a democratic society,” the court concluded that reading the texts was not a burden on free- dom of religion and hence that it need not decide whether the compelling interest standard was met, although a concurring opinion asserted that such a state interest would prevail even if there were to be a such a burden. lacking the ability to tailor the public school curriculum to their views has been a motivating factor in the rise of home schooling. few states mandate that home school- ing cover topics such as tolerance for the religion and culture of others, but many homeschoolers fear any attempt to proscribe curriculum as a threat to their values. there has been very little research on how well home-schooled students who choose to attend col- lege do or what areas of study they choose. scores on sats are generally above the average of those of students as a whole, but the home-schooled students who take such exams are a relatively small, self-selected group. home schooling requirements by state as noted, although until the s most states actually prohibited home schooling, it is now the case that every state permits home schooling subject to varying degrees of regulation as an op- tion under compulsory attendance laws. while ten http://www.hslda.org notices of the ams volume , number and no mention is made of higher mathematics. in vermont the statutory language for required topics notes that students should study the use of numbers. although the state of oklahoma strongly recommends the study of mathematics, it does not require it by law. only three states impose on home-schooled students, by law, high school grad- uation requirements that include mathematics. the lack of consistent mathematics require- ments and assessment for home-schooled students by some states may seem surprising given the current climate of assessment of educa- tional achievement required by the public school system. nclb mandated that all students who attend institutions that receive federal subsidies must measure and demonstrate improvement in mathematics and reading ability in the students they educate. in detailing requirements for assess- ment, nclb states: nothing in this section shall be con- strued to affect home schools, whether or not a home school is treated as a home school or a private school under state law, nor shall any home schooled student be required to participate in any assessment referenced or autho- rized under this section (nclb ). in essence, the home-schooled student does not participate in assessments required by public schools to ensure federal funding. however, twenty-four states require students to be assessed either by standardized exams for math and english or in the form of a personal evaluation or student portfolio review by the school district. how this is enforced is not generally prescribed nor systemi- cally recorded. contrasting with the “hands-off” approach to home schooling, at the national level there is a push for the development of a common core standard for language arts and mathematics in the k– curriculum. the goal of the core standards is to better prepare students for a college educa- tion as well as to compete successfully in a global economy. this state-led effort is not a push for national standards but rather an effort designed to give the fifty states a clear common guideline to discuss what is expected in terms of curricu- lum and student success in two subjects that are often the focus of assessment. the goal of the standards is to make instruction consistent as well as to provide comparable assessment regardless of geography (www.corestandards.org). states reserve the right to adopt or reject the standards developed. undoubtedly, state adoption of the core cur- riculum standards will result in changes and chal- lenges to educational law and curriculum materi- als. it is unclear how adopting the standards will affect the home school community; however, the home school legal defense association (hslda) suggests that adopting a common core is simply a step toward nationalizing education, a move toward which they strongly oppose (www.hslda. org). gender equity the major issue we address here is, of course, the adequacy of home schooling with respect to mathematics, especially in light of the limited re- quirements coupled with apparently nonexistent supervision in most states. there seems little if any prospect of the new impetus for national standards through the core standards or the suc- cessor to the nclb legislation to alter the current situation. moreover, a concern that has received little attention is the possibility that a substantial portion of the home schooling community may not be providing equal education to girls, in particular in mathematics and science. one commonly used series of texts advises girls who may be good in mathematics not to dream of becoming engineers or scientists but rather to consider how their tal- ent might be used to assist their future husbands (dwyer ). the traditional american inclination to avoid any federal involvement in education has eroded over the years, tied to federal funding of specific programs but also through enforcement of anti- discrimination laws. in public schools and in private schools that receive federal funds and in some cases even when they do not, constitutional protections (see, e.g., runyon v. mccrary ), federal laws, and some state laws mandate non- discrimination; however, the major statute, title ix of the education act of , permits exemptions on the basis of religious tenets. in any state action, including education, the fourteenth amendment and the due process clause of the u.s. constitution also provide a basis for assuring equal treatment. once private actors take on what is a fundamental state function such as education, then they too are bound by constitutional provisions, in particular when the state affirmatively invests parents with the state responsibility for education. hence one can argue that home schooling parents must provide the same education for their daughters as for their sons. in norwood v. harrison ( ), the state’s provision of textbooks to schools with racially discriminatory admission policies was found impermissible as inducing, encouraging, or promoting private persons to accomplish what is constitutionally forbidden to the state. how- ever, since the parents are the “state actors”, the doctrine affords only intrafamily equity and not interfamily equity (yuracko ). the concept of homeschoolers as state actors might also be useful if one argues that state con- stitutional and statutory provisions for education mandate the responsibility of the state to assure at http://www.corestandards.org http://www.hslda.org http://www.hslda.org april notices of the ams possibly inferior, form of schooling. the tradition of parental control and, in some cases, free exer- cise of religion infringes on their children’s equal protection rights (dwyer ). in a case involv- ing the right of undocumented children to public education, the supreme court found a texas law excluding them to be a denial of equal protection (plyler v. doe ). the court found that the exclusion served no rational state interest, much less a compelling interest. the situation of the children was obvious, and they were represented in the litigation by a guardian, but who might learn of and then bring a legal challenge in the case of inadequate home schooling? courts are reluctant to confer representation on outside advocates in parent-child conflicts. were litigation to occur, religious beliefs of the parents, while sufficient for the amish when it comes to exemption from high school education, would be unlikely to prevail in the case of basic minimal education. although an inferior educa- tion may not be as life-threatening as is denial of certain medical procedures such as blood transfusions (jehovah’s witnesses v. king county hospital ), religious objections are unlikely to withstand the scrutiny given to bypassing the state's responsibility, assumed by parents, for basic education as preparation for life. in the contexts of ensuring the right to vote or to obtain an abortion, courts have held that not only may states not block access but must at a minimum take steps to prevent private interfer- ence with these rights (ex parte yarbrough, u.s. ( ), planned parenthood of central missouri v. danforth ). of course, in the case of home schooling, the interference with the right to a fundamental education comes from parents, and how the state can protect the rights of the home-schooled children is problematic. permit- ting third party complaints on behalf of the child or establishing judicial procedures (as in the abortion context) to allow children themselves to challenge the adequacy of their parents’ choices is more likely to engender fierce opposition than public support in light of the political power of the home schooling movement. however, it is likely that the majority of responsible and committed homeschoolers would also argue for minimum standards somehow to be ensured, albeit in a nonintrusive manner. in the case of inferior education for girls, such enunciated fundamentalist beliefs as “sexual equality denies god’s word” and failure of a wife to accept a subordinate, obedient role in the home means “the doors are wide open to satan” may well exclude girls from the level of mathematics known to be a critical filter for many careers and confine them to low-paying, servile occupations if em- ployed outside the home (yuracko , p. ). in so much more: the remarkable influence of least a basic minimal education. does not the equal protection clause impose a lower limit on state regulation of education (yuracko , p. )? for instance, one could argue that to be a full actor in today’s technological world, home-schooled students need computer training and courses in statistics and even calculus (not to mention evolu- tion and other science). the “amish exemption” from providing an education adequate for the modern world has not in the past been available to those who argue that they do not want their children exposed to concepts that are in conflict with their beliefs, but in the present context of the political strength of the home school movement it is not surprising no one is really challenging the adequacy of home schooling for the state respon- sibility of full civic and economic participation. the “state actor” doctrine is not available for every function of government undertaken by pri- vate actors—for example, parochial schools may teach religion—but in a responsibility as funda- mental as basic education it might be held to apply (as it has to privately operated prisons). and if states can be sanctioned for failing to provide the basic minimum (abbott v. burke ), why not sanction homeschoolers? probably insisting on the equivalent of public school education, however defined, is not possible given the finding of san antonio v. rodriguez ( ) that while a minimum standard must be met, equity is not required. but can homeschoolers be held to some minimum standard of equitable mathematical training for boys and girls? the focus on obligations for education of citi- zens is generally on states. federal education laws like the nclb act and its likely successor have specific statements that they do not authorize any federal control over home schools. however, it has been argued that the “guarantee clause of the fourteenth amendment authorizes and obli- gates congress to ensure a meaningful floor of educational opportunity throughout the nation. the argument focuses on the amendment’s open- ing words, the guarantee of national citizenship. this guarantee does more than designate a legal status. together with section [which assigns to congress the power to enforce the amendment], it obligates the national government to secure the full membership, effective participation, and equal dignity of all citizens in the national community” (liu ). but whether the issue is that home schooling provides an adequate mathematical education to meet the responsibility that the state has ceded to homeschoolers or that girls are receiving an inferior education to that of their brothers, an underlying concern is that the children them- selves have no legal or practical control over the decision whether they receive the state proffered benefit of a public school education or some other, notices of the ams volume , number visionary daughters on the kingdom of god, popu- lar in the christian home schooling community, authors anne sofia botkin and elizabeth botkin claim that college is dangerous for young women because it diverts them from their god-ordained role as helpmeets for their fathers and husbands (quoted in yuracko , p. ). stacy mcdon- ald ( ) supplies even more explicit guidance for potentially discriminatory education: a girl’s education “should be focused on assisting her future husband as his valuable helpmate, not on becoming her ‘own person’.” girls are counseled to “[r]emember that a strong desire to be a doctor or a seeming by-god-given talent in mathematics is not an indication of god’s will for you to have a career in medicine or engineering. sometimes god gives us talents and strengths for the specific purpose of helping our future husbands in their calling.” (quoted in yuracko , p. , note .) when the united states sued virginia for violat- ing the equal protection clause of the fourteenth amendment by denying women admission to the virginia military institute, the supreme court, re- versing the decision of the federal appeals court, declared “[s]uch sex classifications may not be used as they once were, to create or perpetuate the legal, social, and economic inferiority of women” (united states v. virginia at ). the court went on to say that the state’s important interests in education, in order to be constitutional, must undermine sex hierarchy and never reinforce hierarchy or promote sex stereotypes that foster hierarchy. nonetheless, private single-sex higher education continues without constitutional chal- lenge. however, in the case of basic education for children, the state has taken on the responsibility for its provision but has delegated its authority to parents who home school their children, thus making the parents state actors and subject to the equal protection requirement of the fourteenth amendment. also relevant to the fundamental na- ture of education and the state’s responsibility for it is the contrast between griffin v. county school board of prince edward county ( ), where the supreme court refused to countenance the clos- ing of the public schools to avoid integration, and palmer v. thompson ( ), where closing the public swimming pools was permitted. conclusion mcmullen ( ) offers a remedy for the deficien- cies such as those identified above: fair-minded, fairly minimal regulation aimed at the minority of bad actors in the home schooling community. she proposes that those who wish to home school be required to file an application with the local school district, to be approved automatically if the name, address, and proof of vaccination are in order (and the parents have not been convicted of child abuse). age-appropriate competence testing in reading and mathematics would be mandatory in order to maintain home school status, prefer- ably exams like the iowa basic skills test, which is hard to teach to. there should be independent monitoring of home schooling, since local school personnel could be said to have a conflict of interest. teacher certification or more detailed regulation would be difficult to institute and not necessarily a solution, leaving only the ultimate remedy of litigation for the very few children who may be being deprived of basic rights. what can be done to assure adequate train- ing in mathematics and sciences specifically for home-schooled girls? in particular, who has the responsibility and the ability to secure legal pro- tection for them? it can be argued that the state constitutional assumption of education as a state function implies that there is in fact recourse when parents take on the functions of the state. will concern for children’s rights eventually swing the pendulum of home schooling back to universal substantial regulation if not to outright prohibition? this seems unlikely in the current political climate despite the attention being given to education as a national resource and to the necessity to provide a larger, more diverse, and better-trained stem workforce if america’s global position is not to decline. whether home schooling contributes to the goal of better-educated students on the whole or proportionally to the number of students in nationally important stem disciplines remains to be determined. references anne sofia botkin and elizabeth botkin, so much more: the remarkable influence of visionary daugh- ters in the kingdom of god, the vision forum, inc., san antonio, texas, . ellwood patterson cubberly, public education in the united states: a study and interpretations of ameri- can educational history, houghton mifflin company, cambridge, massachusetts, . james g. dwyer, parents’ religion and children’s welfare: debunking the doctrine of parents’ rights, california law review ( ), – . milton gaither, homeschool: an american history, palgrave macmillan, new york, . goodwin liu, education, equality, and national citizen- ship, yale law journal ( ), – . diana buell hiatt, parent involvement in american public schools: an historical perspective – , the school community journal ( ) (fall/winter ), – . http://www.corestandards.org http://www.hslda.org george henry martin, the evolution of the massa- chusetts public school system: a historical sketch, d. appleton & co., new york, . stacy mcdonald, raising maidens of virtue: a study of feminine loveliness for mothers and daughters, books on the path, barker, texas, . no child left behind, u.s.c. §§ et seq. ( ). http://www.corestandards.org http://www.hslda.org april notices of the ams mitchell l. stevens, kingdom of children: culture and controversy in the homeschooling movement, prince- ton university press, princeton, new jersey, . u.s. department of education, nces, home schooling in the united states: , statistical analysis report, july . u.s. department of education, nces, home schooling in the united states: , statistical analysis report, july . u.s. department of education, nces, home schooling in the united states. issue brief, december . kimberly yuracko, education off the grid: constitu- tional constraints on homeschooling, california law review ( ), – . cases cited abbott v. burke, a. d (nj ). blackwelder v. safnauer, f. supp. (ndny ). clonlara, inc. v. runkel, f. supp. (e.d. mich. ). delconte v. north carolina, s.e. d (n.c. ). ex parte yarbrough, u.s. 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setpagedevice genetics and genetic testing for age-related macular degeneration eye ( ) : – https://doi.org/ . /eye. . review article genetics and genetic testing for age-related macular degeneration a. warwick ● a. lotery received: september / accepted: october / published online: november © the royal college of ophthalmologists abstract considerable advances have been made in our understanding of age-related macular degeneration (amd) genetics over the past decade. the genetic associations discovered to date are estimated to account for approximately half of amd heritability, and functional studies of these variants have revealed new insights into disease pathogenesis, leading to the development of potential novel therapies. there is furthermore growing interest in genetic testing for predicting an individual’s risk of amd and offering personalised preventive or therapeutic treatments. we review the progress made so far in amd genetics and discuss the possible applications for genetic testing. introduction age-related macular degeneration (amd) is the commonest cause of blindness in the developed world, affecting % of those aged > years old and an estimated -million people worldwide [ , ]. disease onset is in later life, typically after the age of years, and is characterised clinically by retinal pigmentary change and the appearance of drusen at the macula. central vision may subsequently decline either gradually with progressive geographic retinal atrophy (ga), or acutely due to retinal haemorrhage and fluid exudation from choroidal neovascularisation (cnv) if neovascular amd (nvamd) develops. while the latter may now be effectively treated with intravitreal anti-vascular endothelial growth factor (vegf) injections, there is cur- rently no therapy for ga available in clinical practice [ ]. over the past decade, considerable progress has been made in elucidating the genetic architecture of amd. the discovery of multiple genetic associations and studies of their downstream functional consequences has helped reveal new underlying pathophysiological mechanisms in amd, affording the potential to identify molecular targets for novel therapies. the complement pathway in particular, an important component of the innate immune system, has been consistently implicated [ ]. furthermore, there is growing interest in genetic testing to predict either an individual’s risk of developing amd or how well they will respond to treatment. ultimately, this could mean that in the future, patients are offered personalised preventive or ther- apeutic treatments, tailored to their individual genetic makeup. in this review, we summarise the current literature and possible future directions for amd genetics and genetic testing. genetics of amd amd is a complex disease with multiple environmental and genetic risk factors. the most consistently associated environmental risk factors are age and smoking, although gender, race, cardiovascular disease, and diet have also been implicated [ ]. evidence for a genetic component was supported by family aggregation studies and twin studies. in family aggregation studies, the prevalence of amd was higher in first-degree relatives of amd patients ( . %) compared with relatives of controls ( . %), with an odds ratio (or) of . [ ]. by comparing disease concordance rates between monozygotic and dizy- gotic twins, the heritability of early and advanced amd has been estimated at % and %, respectively, implying that – % of amd variation may be explained by genetic factors [ ]. in early efforts to explore amd genetics, association studies were performed for candidate genes known to cause mendelian macular diseases such as best’s disease, * a. lotery a.j.lotery@soton.ac.uk moorfields eye hospital, london, uk clinical neurosciences research group, clinical and experimental sciences, faculty of medicine, university of southampton, southampton, uk () ;, : () ;,: http://crossmark.crossref.org/dialog/?doi= . /eye. . &domain=pdf http://crossmark.crossref.org/dialog/?doi= . /eye. . &domain=pdf http://crossmark.crossref.org/dialog/?doi= . /eye. . &domain=pdf mailto:a.j.lotery@soton.ac.uk stargardt’s disease, and sorsby’s fundus dystrophy. how- ever, no consistent significant associations were made [ – ]. other groups undertook genetic linkage studies of patient families to identify genomic regions containing susceptibility loci for amd. a meta-analysis of these results showed that the most replicated findings were on chromosomes q - and q [ ]. their impor- tance was then validated by subsequent findings of specific amd-associated common genetic variants at these two loci—the complement factor h (cfh) gene on chromosome and the age-related maculopathy suscept- ibility /htra serine peptidase (arms /htra ) genes on chromosome . common variants technological advancements enabling the analysis of whole genomes, rather than individual genes, have greatly accel- erated the discovery of new genetic associations with common complex genetic diseases such as amd. genome- wide association studies (gwas) examine a genome-wide set of genetic variants, typically single-nucleotide poly- morphisms (snps), for associations with the disease of interest. while genetic linkage studies are effective for investigating high-penetrance single-gene defects under- lying rare monogenic disorders, gwass are better able to detect low-penetrance common genetic variants (with a minor allele frequency (maf) > %) associated with com- plex genetic diseases. in , landmark studies associated a common poly- morphism (tyr his or y h) in the cfh gene on chromosome with amd [ – ]. one of these was the first gwas performed for amd, showing that the y h risk allele had a large effect size ( . and . increased the likelihood of amd in heterozygous and homozygous individuals, respectively) [ ]. of note, this was the first successful gwas of a ‘complex disease’, that is, one with both genetic and environmental factors contributing significantly to the disease. as such, it represented a major success for genetic approaches to studying common diseases. subsequent gwass and candidate gene studies have since associated several other common variants in complement-related genes with amd, including c /cfb [ ], c [ ], c [ ], cfi [ ], and serping [ ]. the complement cascade encompasses a family of more than circulating proteins and their regulators which form an important part of the innate immune system. activation of the complement cascade ultimately results in formation of the membrane attack complex (mac), which induces cell lysis. while the liver is the major source of systemic complement, retinal cells synthesise their own complement [ ], and complement proteins have been detected in drusen from human eyes [ – ]. raised systemic levels of com- plement have also been reported in amd patients vs con- trols [ ], and interestingly, patients with dense deposit disease, a renal condition associated with systemic com- plement dysregulation and glomerular c deposition, often develop drusen [ , ]. however, a locally produced, rather than systemic, complement likely appears to be more important in amd [ , ]. chronic intraocular complement-mediated inflammation in genetically susceptible individuals exposed to environ- mental triggers, such as cigarette smoke and oxidative stress, may contribute to the progressive retinal changes observed in amd. while the functional relevance of most common amd-associated genetic variants remains unknown, a common missense polymorphism in c has been shown to result in reduced binding to cfh, a plasma regulator of complement, and increased activity of the alternative complement pathway [ ]. furthermore, a number of studies have looked at the functional effects of the non-synonymous cfh y h polymorphism. the minor allele, encoding a histidine amino acid at residue of the cfh protein, alters its affinity for crp [ ], glyco- saminoglycans in bruch’s membrane [ , ], mal- ondialdehyde [ ], and zinc [ ]. these changes are thought to decrease its ability to regulate complement. reduced complement regulation may lead to increased mac deposition and choroidal endothelial cell death, impairing the ability of the choriocapillaris to remove debris and allowing the accumulation of waste products in drusen [ ]. in support of this theory, eyes homozygous for the cfh y h risk variant have thinner choroids [ ] and increased mac deposition [ ] at the choriocapillaris compared with eyes with low-risk cfh genotypes. genes not involved in the complement pathway have also been associated with amd. the arms /htra locus on chromosome q has been strongly associated to risk alleles conferring an or of . and a population-attributable risk up to % [ , ]. as both genes at this region are in strong linkage disequilibrium with each other and both harbour functional variants which could plausibly be rele- vant to amd, dissecting out which is responsible for the observed association with amd has proved to be challen- ging [ ]. recently, however, analysis of the largest data set for amd genetics to date suggested that genetic variants at arms , but not htra , are responsible for amd sus- ceptibility at the q locus [ ]. further functional ana- lysis of arms is required to understand its role in amd pathogenesis. other non-complement associations with amd include genes implicated in angiogenesis (tgfbr , vegfa), the extracellular collagen matrix (col a , col a ), the high-density lipoprotein cholesterol pathway (apoe, cetp, and lipc), and immune regulation (pilrb) [ – ]. a. warwick, a. lotery rare variants in , the amd gene consortium published the largest gwas for amd conducted up until that time, evaluating > . million snps in > , cases and > , controls. however, they estimated that only – % of amd her- itability was explained by the loci discovered [ ]. several theories seeking to explain the missing heritability exist, including gene–environment interactions, gene–gene interactions, epigenetics, copy number variation, and rare variants [ ]. in particular, the focus of genetic research for complex diseases in general has shifted towards identifying low-frequency (maf – %) and rare variants (maf≤ %) with relatively large effect sizes [ ]. furthermore, a recent simulation study demonstrated that the clustering of amd in densely affected families was insufficiently explained by the genotypic load of common genetic risk variants and that rare variants may be more important [ ]. various approaches have been successful in discovering new rare genetic variants associated with amd. next- generation sequencing technology can be used to compre- hensively analyse variation within candidate genes between cases and controls. the first rare variant to be associated with amd, cfh r c was identified in this way [ ]. it demonstrated high penetrance (present in cases vs control, p= . × − , and or . ) and was associated with an earlier onset of the disease. similar studies have also identified rare variants in cfi, c , and c [ – ]. whole-exome or whole-genome sequencing is expensive to carry out in large numbers but has been performed in an icelandic case–control cohort [ ], as well as in large amd families, thought to be enriched for rare variants [ – ]. in , the international amd genomics consortium (iamdgc) identified independently associated common and rare variants distributed across loci [ ]. the group performed a gwas using an exome chip, customised to analyse > million variants (including > , directly genotyped, mostly rare, protein-altering variants) in > , patients and > , controls. this is the largest study of amd genetics performed to date, more than doubling the number of known associated variants and identifying the first variant specific to one advanced amd phenotype (mmp and nvamd). rare variants were dis- covered in cfh and cfi, as well as the non-complement genes timp and slc a [ ]. compared with common variants, highly penetrant rare variants often have clearer functional effects [ ]. for example, in a large advanced amd cohort, rare cfh var- iants tended to be located in functional domains and resulted in low cfh serum levels [ ]. rare variants in cfi, another regulator of the complement system, have also been associated with decreased serum cfi levels [ ], and carriers of rare variants in both cfh and cfi have impaired ability to regulate complement activation [ , ]. furthermore, the rare c lys gln variant has been shown to impair c b regulation by cfi with bound cfh [ ]. overall, these findings support the hypothesis that increased complement activity may contribute to amd pathogenesis. carriers of rare variants appear to have differing phe- notypes compared with non-carriers. a number of studies have shown rare variants to be associated with earlier onset of advanced amd [ , , , , ]. carriers of rare cfh variants have increased drusen load, are more likely to have extramacular drusen, drusen nasal to the optic disc, and crystalline or calcified drusen [ , , ]. in addition, rare variants in cfh, cfi, c , and c have been more frequently observed in patients with ga than those with nvamd [ , ]. interestingly, a rare missense mutation in timp (c g), identified by the iamdgc, is associated with earlier age of disease onset (average age years) and bilateral cnv [ ]. other mutations in timp cause sorsby’s fundus dystrophy, an autosomal dominant fundus dystrophy characterised by a similar clinical phenotype to amd, although typically, age of onset is in the fourth decade of life. further, genotype–phenotype correlations are needed to determine whether the phenotype associated with this mutation more closely resembles amd or sorsby’s fundus dystrophy. the iamdgc estimates that the currently known variants account for approximately half the genomic herit- ability of amd [ ]. despite this significant progress, there is still therefore a large portion of missing heritability. the iamdgc highlights the need for very large sample sizes and extensive genome coverage in population studies looking for novel rare variants in complex diseases. future studies looking at other potential sources of missing herit- ability and functional studies of known associated variants are also needed. genetic testing for amd as the list of known amd-associated genetic variants continues to grow, so too does interest in developing predictive risk models incorporating these alleles. the ability to accurately predict a person’s risk of developing amd would be an important step towards personalised medicine, allowing appropriate preventive measures to be taken in high-risk individuals. pharmacogenetic testing could furthermore help identify which amd patients are most likely to benefit from certain treat- ments. for example, it is conceivable that novel therapies modulating the complement system may be most effective in patients harbouring complement-related risk alleles. genetics and genetic testing for age-related macular degeneration genetic risk models for amd in contrast to rare monogenic disorders, complex genetic diseases such as amd are associated with multiple environmental and genetic risk factors. possession of an allele known to be associated with the disease increases an individual’s risk of developing the condition and possessing multiple risk alleles, or exposure to certain environmental triggers may further increase this risk. such individuals could however either remain healthy or only ever progress to a mild asymptomatic stage. risk models for complex diseases therefore aim to accurately predict risk by incorporating multiple alleles or environ- mental risk factors. a common method for describing the accuracy of a risk model is to calculate the area under the receiver-operating characteristic (roc) curve (auc), the roc being a plot of all possible (sensitivity, specificity) pairs for the model (fig. ). the auc can theoretically take values between and , but in practice varies from . to . a score of means perfect accuracy, whereas a score of . indicates that the predictive ability of a model is equivalent to random chance or zero accuracy. a model with a score between . and . therefore has greater ability than chance to dis- criminate between cases and controls. it has been suggested that an auc> . is acceptable and > . is excellent [ ]. existing risk models for amd predict either the risk of developing amd or of progressing from the early and intermediate stages to advanced disease [ ]. models incorporating only genetic information have achieved aucs> . [ , ], and models combining both genetic and environmental risk factors have reported aucs> . [ , , ]. interestingly, a model which only included environmental risk factors predicted the risk of developing advanced amd with a similar auc of . [ ], leading some to question the usefulness of genetic data in predictive models [ ]. baseline macular phenotype in particular is a strong predictor of amd progression. while some groups have shown improved risk model accuracy for disease progression when the genotype is included with fundus phenotype [ , ], a recent study found only a small addition to predictive power [ ]. future studies looking at which genetic variants associate specifically with progres- sion of amd may be useful. while the auc broadly measures the accuracy of a model to distinguish between cases and controls, additional factors need to be considered when evaluating a model’s ability to estimate the risk for individuals. one method is to use the reclassification approach, which classifies individuals into risk strata. in a worked example, jakobsdottir et al. [ ] applied a genetic risk model for amd with an auc of . to a theoretical population with an amd prevalence of . % (corresponding approximately to amd prevalence in patients older than years). for a sensitivity of and specificity of %, the model classified individuals with > % risk of amd as cases (‘high risk’) and those with < % risk as controls (‘low risk’). the cor- responding positive predictive value was only %, mean- ing that % of those patients classified as ‘high risk’ should actually have been in the ‘low risk’ group. the authors also demonstrated how the model’s predictive ability is heavily influenced by disease prevalence, which is highly age dependent for amd. applying their model with the same sensitivity and specificity to a population with a % amd prevalence (the approximate prevalence for patients older than years) classified individuals as > % risk (cases) or < % risk (controls) of amd, with an improved positive predictive value of % [ ]. as illustrated by the example above, a risk model with a seemingly acceptable auc may therefore not be useful clinically as a predictor of individual risk. in addition, some genetic risk variants discovered in one population may not be as important in others. to date, the majority of amd genetic associations have been studied in populations of european ancestry. the risk loci discovered by the amd gene consortium in a non-amish caucasian population appear to account for a lower proportion of amd in amish individuals, and a rare cfh variant (p a) associated with amd in the amish population was absent in non- amish cases and controls [ , ]. the common cfh y h risk variant is present in ~ % of utah residents of northern and western european ancestry, but only ~ % of fig. an example of a receiver-operating characteristic (roc) curve. the solid black line represents an area under the roc (auc) of . , which indicates random chance. the dashed black line indicates an auc of . , a perfect model. the dotted grey line represents an roc curve with an auc between . and . , as may be generated by a predictive risk model. specificity decreases with increasing sensitivity. a. warwick, a. lotery japanese and chinese individuals [ ]. a recent study showed both the cfh y h and arms a s variants to be associated with amd in european americans but not in african americans, mexican americans, or singaporeans [ ]. furthermore, in another study, the common arms a s variant was associated with increased risk of amd in non-hispanic whites and mexican americans but was found to be protective in non-hispanic black individuals [ ]. this implies that differing genetic loci underlie amd in different populations, and predictive risk models should account for these ethnic variations accordingly. despite the limitations of predictive testing in amd, a number of commercial companies now provide genetic amd tests. improved accuracy of these tests could in the future aid the detection of high-risk individuals who may benefit from early preventive measures. however, there is currently no evidence that changes to the management of such patients beyond current practice are beneficial. for these reasons, the current recommendations from the american academy of ophthalmology (aao) include avoidance of routine genetic testing for complex disorders like amd, until prospective clinical trials have shown specific surveillance or treatment strategies to be of benefit [ ]. pharmacogenetics and amd another potential use for genetic testing is to identify which amd patients may respond best to specific treatments. the age-related eye disease study (areds) antioxidant and zinc formulation is the only therapeutic intervention that has been shown prospectively to significantly reduce the risk of progression to advanced amd [ ]. genetic testing to guide preventive treatment with the areds formulation has proved to be controversial. awh et al. [ , ] recently published retrospective analyses on patients from the areds trial, reporting that certain cfh and arms genotypes were associated with potentially harmful responses to dietary antioxidant and zinc supplementation. the authors therefore concluded that favourable outcomes may be achieved by assigning nutritional supplementation based on cfh and arms genotypes. subsequent analyses by the original areds investigators, however, found the areds formulation to be beneficial for all genotypes, as well as pointing out statistical errors made by awh et al. [ , ]. other studies have shown the effectiveness of nutritional supplementation to differ by genotype, but no harmful effects were observed [ , ]. prospective clinical trials are needed to more definitively address this issue, but at present, there is insufficient evidence to support genetic testing prior to recommending areds nutritional supple- mentation [ , ]. a number of studies have also examined whether amd-associated risk variants and vegf-related gene polymorphisms affect response to anti-vegf therapy in nvamd. although the majority of patients do well with anti-vegf treatment, – % respond poorly and lose vision [ ]. variation in treatment outcomes is related to clinical characteristics, such as baseline visual acuity and central retinal thickness, and may also be influenced by genetic factors. a single gwas identified an association between a variant in the or b gene and response to anti-vegf; however, this is yet to be replicated [ ]. small retrospective studies have found statistically significant associations between variants in candidate genes, either related to angiogenesis or known to confer amd risk, and response to anti-vegf [ ]. in contrast, no such genetic associations were observed in the major comparison of amd treatments trial (catt) and inhibit vegf in patients with age-related cnv study (ivan) randomised control trials [ – ]. recent meta-analyses pooling data from these smaller studies with the major trials have reported positive associations between anti-vegf treatment response and the cfh y h and amrs a s variants, as well as polymorph- isms in vegf-a and vegfr- , but all acknowledge the need for large prospective trials to validate their findings [ – ]. genetic testing in amd research routine genetic testing for amd is therefore currently not advised in clinical practice, and further supportive evidence from prospective clinical trials is needed before recom- mending genetic testing to guide treatment with areds nutritional supplementation or anti-vegf [ , , ]. genetic testing may however be useful as a research tool, for example, to select suitable patients for future clinical trials of novel therapeutics designed to prevent amd. restricting patient recruitment to those individuals at the highest risk of developing advanced amd is likely to decrease the sample size requirements for adequate pow- ering of such studies. reconsidering the predictive model with an auc of . by jakobsdottir et al. [ ] discussed earlier, while a positive predictive value of – % may be poor in the clinical setting, it might prove cost-effective to help improve the proportion of participants at high risk of developing amd in a clinical trial. genetic testing could also be useful in therapeutic clin- ical trials. to date, the complement system has been most frequently implicated in amd, and consequently, several trials of complement inhibitors have been initiated [ ]. amd is genetically heterogeneous and it is possible that patients with certain risk variants in complement-related genes may respond differentially to complement inhibition. genetics and genetic testing for age-related macular degeneration recently reported findings from the phase ii mahalo trial of lampalizumab, a monoclonal antibody inhibitor of complement factor d, in ga support this concept. pro- gression of ga lesion size over months follow-up was significantly less in patients treated with monthly intravi- treal injections of lampalizumab; however, this response was only observed in those patients carrying a common cfi risk allele (rs ) [ ]. results from phase iii trials are currently awaited, although preliminary results from one of these phase iii trials, spectri, showed no benefit of lampalizumab therapy overall. it will be interesting to see full results including results in genetic subgroups [ ]. summary and conclusions the proportion of amd heritability now explained by known genetic risk variants is estimated at ~ %. identification of disease-associated common and rare variants has implicated various biological pathways in amd pathogenesis. the complement system in particular appears to play an important role, and functional studies are beginning to elucidate how complement dysregulation may contribute to amd. these discoveries have furthermore led to the development of potential novel therapies modulating complement activity. promising results have now been reported in a phase ii trial of the anti-complement factor d antibody lampalizumab in a genetic subgroup [ ], (although early results from the phase iii spectri trial showed no benefit overall) ], and also in a phase ii trial inhibiting c in amd patients [ ]. further work is needed to uncover the remaining heritable component of amd and better understand how genetic and environ- mental factors interact to cause the disease. an improved understanding of amd genetics will also aid the development of risk models that can more accurately predict disease occurrence and progression. it is possible that such predictive tests may guide amd patient man- agement in the future if effective preventive therapies and pharmacogenetic associations are discovered, although, for now, there is insufficient evidence to recommend genetic testing in clinical practice [ , , ]. genotype-restricted sampling for clinical trials, however, may help accelerate progress in translational amd research. compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest. references . smith w, assink j, klein r, mitchell p, klaver cc, klein be et al. risk factors for age-related macular degeneration: pooled findings from three continents. ophthalmology ; 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https://www.roche.com/ media/store/releases/med-cor- - - b.htm. accessed september . . apellis. apellis pharmaceuticals announces that apl- met its primary endpoint in a phase study in patients with geographic atrophy, an advanced form of age-related macular degeneration. available at: http://apellis.com/pdfs/press% release% filly% % month% results% final% final % .pdf. accessed september . genetics and genetic testing for age-related macular degeneration 	genetics and genetic testing for age-related macular degeneration 	abstract 	introduction 	genetics of amd 	common variants 	rare variants 	genetic testing for amd 	genetic risk models for amd 	pharmacogenetics and amd 	genetic testing in amd research 	summary and conclusions 	compliance with ethical standards 	acknowledgments 	references high treadmill workload in patients with exercise-induced st depression predicts a negative result on exercise echocardiography jacc march , abstracts - cardiac function and heart failure a pacmg at each pacing site did not improve lv diastolic function. conclusion: lvp and bvp improved lv systolic function in patients with heart failure regardless of the baseline rhythm. in patients with af, only bvp improved lv diastolic function. these results indicate that bvp may be more beneficial than single-site lvp in patients with heart failure and af. groups #of patients exercise duratlon(min) met’s peak hr + spect, . * . . f . f % cl +spect, . * . . a . ’ * < % cl - spect, . f . ll.li . * so% cl -spect, . * . ’ . * . ” * < % cl poster session exercise testing: cardiac rehabilitation monday, march , , noonq:oo p.m. mccormick place, hall a presentation hour: :oo p.m.- :oo p.m. - predictors of negative exercise echocardiography in women with positive exercise electrocardiograms amish j. desai, amogh bhat, dallit bagha, mrudula guthikonda, ezra a. amsterdam university of california, davis medical center, sacramento, ca background: exercise (ex) electrocardiography (ecg) is the most widely used non- invawe test for evaluating symptoms suggestive of coronary artery disease (cad). in women. however, ex ecg has limited reliability due to an increased rate of false positive results. therefore, a stress-imaging study is frequently the initial test for evaluation of chest pal” in women. we have previously reported that certain exercise test variables predict a negative ex echocardlogram (echo). to further enhance this predictive value, we report additional exercise test variables predictive of negative ex echo. methods we analyzed the results of simultaneous ex echo and ex ecg in women with no known cardiac disease es part of the initial evaluation for chest pain suggestive of cad. all patients (pts) had a normal resting ecg and adequate exercise capacity by his- tory. all tests were symptom-limited utilizing a bruce protocol. a positive ex echo was defined es an ex-induced regional wall motion abnormality and a positive ex ecg was defined as ex-induced .o mm st segment depression - msec after the j point. results: the study group comprised consecutive women (mean age years [ - ) evaluated by simultaneous ex echo and ex ecg. ex echo was positive i” % ( / ) and negative in % ( / ). ex ecg was positive in % ( / ) and negative in % ( ). in % ( / ) pts with a positive ex ecg, ex echo was negative, suggesting a false positive ex ecg. in this group of pts, non-echo ex test data associated with a negative ex echo included: mets, double product z. . , st depression cl. mm, no ex-induced chest pain, st segment resolution <i minute into recovery, < leads with st depression. four or more of these factors were present in % ( / ) of the pts with a positive ex ecg and a negative ex echaconclusions: these results indicate that ( ) ex ecg was negative and concordant with ex echo in a majority of women and ( ) ~ % of positive ex ecg associated with negative ex echo were characterized by multiple indicators consistent with a false positive or low risk result. the evaluation of chest pain in women can be simple and cost effective utilizing ex ecg in those with a normal baseline egg and adequate exercise capacity. - abnormal peripheral thermoregulatory response to exercise substantially impairs exercise tolerance among patients with and without lschemia on myocardial perfusion single-photon emission computed tomography ehtasham a. qureshi, veeranna merla, pierre charouqi, umber burhan, amy . armenia. george reed, george diamond, alan rozanski, st. luke’s_roosevelt hospital center, new york, ny background: whereas spect myccardial ischemia predicts patients (pts) outcome, exercise (ex) duration generally adds substantial incremental information to event pre- diction. the pathophysiological factors mediating this association are unclear. paradoxi- cal vasoconstriction in finger blood flow during ex has recently been demonstrated, using peripheral arterial tonometry (pat), among cad patients. the relation between such vasoconstriction and ex duration is not known. methods: we employed pat to assess finger pulsewave amplitude (pwa) at rest and continuously during ex spect testing in cad pts (mean age * years. % males) divided into pts with ischemic and pts with non-ischemic spect studies. pwa patterns during ex were compared to previous normal limit values for ex pwa response; with values < % confi- dence intervals (cl) constituting abnormakty. resufts: abnormal finger blood flow response to ex was associated with a substantial reduction in ex duration and achieved ex mets (table). notably, this association was equally significant among patients with and without spect evidence of ischemia. conclusions: paradoxical reduction in finger blood flow during exercise is associated with significantly reduced exercise duration among cad patients. accordingly, this abnormal peripheral vascular response may rep- resent a potential, spect-independent, pathophysiological mechanism linking exercise duration to cardiac events. ‘pco. forz % cl vs. < % cl subgroups - using estimated functional capacity to optimize stress testing for diagnosis and prognosis of cardiovascular disease in women: the nhlbi-sponsored wise study leslee j. shaw, marian olson, sheryl kelsey, bernard r. chaitman. george sopko, c. noel bairey merz, wise study group, atlanta cardiovascular research institute. atlanta, ga, university of pittsburgh, pittsburgh, pa background: functional capacity, measured in mets, is an important component that affects the diagnostic and prognostic value of exercise stress testing. the aim of this study was to compare the prognostic value of predicted functional capacity based on the duke activity status index (dasi) compared with peak exercise mets methods: from the nhlbi-sponsored wise study, a total of wmen with chest pain (average age= years) underwent exercise testing and completed the -item dasi questionnaire. results: average exercw time was . i . minutes on the (modified) bruce or acip protocols and average dasi functional capacity was mets. . .year cardiovascular events (unstable angina, death, myocardial infarction, heart failure, or stroke) were reported in women. exertional lschemia occurred in % and % had a coronary stenosis+o%. in women with dasi-predicted mets < and > . , no inducible ischemia occurred more ( % vs. %), with % predicted maximum heart rate more ( % vs. %) often in functionally impaired women (p=o.o ); despite similar disease prevalence. in conclusion, among women referred for coronary angiography for suspected myocar- dial ischemia, marked functional impairment estimated by a simple estimate of functional capacity is associated with an adverse prognosis. use of the dasi prior to exercise stress testing may stratify candidates for exercise testing or pharmacologic stress. ll w . .year event rates % < . . - . . - . > . p value dascestimated mets . exercise mets . l-l - high treadmill workload in patients with exercise- induced st depression predicts a negative result on exercise echocardiography amish j. desai. amogh bhat, daljit bagha. mrudula guthikinda, ezra a. amsterdam, university of california, davis medical center, sacramento, ca background although exercise (ex) electrocardiography (ecg) is the most com- monly employed initial test to assess patients with symptoms suggestive of coronary artery disease (cad), it has limited diagnostic accuracy. therefore, patients (pts) with positive tests for myocardial ischemia are frequently referred for further evaluation by noninvasive stress imaging such as ex echocardiography (echo). a negative ex echo is considered evidence of absence of high risk cad and of low clinical risk. it has recently been shown that functional capacity is a strong predictor of prognosis. thus, in a group of pts with positive ex ecg but high treadmill workload, we investigated the results of sec- ondary evaluation by ex echo.methods we analyzed the results of consecutwe pts ( males, females; mean age yrs [ - ) referred for ex echo after a posi- tive ex ecg and a treadmill workload of mets. all pts had a normal resting ecg. exercise tests utilized a bruce protocol and were symptom-limited. a positwe ex ecg was defined as ex-induced .o mm st segment depression - msec after the j point and a positive ex echo was defined by an ex-induced regional wall motion abnor- mality. results: ex echo was negative in % ( / ) of pts and positive in % ( / ). conclusions: ex ecg performed to a high workload is highly predictive of a negative ex echo and thus low prognostic risk in pts referred because of positive ex ecg. pts with st depression on ex ecg, who achieve at least mets during treadmill ex. may not require additional noninvasive or invasive evaluation. - impact of obesity on inflammation and metabolic syndrome in coronary patients and effects of cardiac rehabilitation carl j. lavie, richard v. milani,ali morshedi, ochsner clinic foundation, new orleans, la background: obesity is epidemic in the us and represents a major risk factor for cad and type ii diabetes. limited data, however, exist on the eflects of obesity on such risk factors es inflammation and components of the metabolic syndrome (ms) es defined by atp ill in cad patients, and the effects of cardiac rehabilitation and exercise training programs &ret) in these patients. _ _ _article .. review insights into metabolic disease from studying genetics in isolated populations: stories from greece to greenland eleftheria zeggini & anna l. gloyn , , & torben hansen , received: january /accepted: january /published online: march # the author(s) . this article is published with open access at springerlink.com abstract over the last years substantial progress has been made in our understanding of the genetic basis for type diabetes and related traits. these developments have been facilitated by technological advancements that have allowed comprehensive genome-wide assessments of the impact of common genetic variation on disease risk. current efforts are now focused on extending this to genetic variants in the rare and low-frequency spectrum by capitalising on next- generation sequencing technologies. this review discusses the important contributions that studies in isolated populations are making to this effort for diabetes and metabolic disease, drawing on specific examples from populations in greece and greenland. this review summarises a presentation given at the ‘exciting news in genetics of diabetes’ symposium at the annual meeting of the easd, with topics presented by eleftheria zeggini and torben hansen, and an overview by the session chair, anna gloyn. keywords genetic variants . genome-wide association studies . isolated populations . low-frequency variants . next-generation sequencing technology . review . type diabetes abbreviation lof loss-of-function the search for low-frequency and rare variants associated with complex traits requires very large sample sizes to attain the necessary power to detect these effects. population isolates offer the opportunity to discover such signals with much lower sample sizes. isolated, or founder, populations have typically gone through a bottleneck event, followed by population ex- pansion with limited rates of migration. these characteristics can offer power advantages in the study of complex trait ge- netics, especially with respect to the identification of associa- tion signals at low frequency, and rare variants that would otherwise be difficult or impossible to detect in cosmopolitan populations [ , ]. some rare variation present in the source population may have been lost in the isolated population, and this can reduce the number of neutral rare variants, which in turn can increase the power of burden tests (i.e. association tests looking for an aggregation of multiple rare variants in a functional unit, e.g. gene or regulatory region). other rare variants that have made it through the bottleneck can increase in frequency due to drift, or in some cases, selection. this in turn increases the power of association studies as smaller sam- ple sizes are required to detect individual risk loci for complex diseases and traits. in addition, the genomes of isolates have been shaped by population history to typically have longer stretches of linkage disequilibrium. founder populations also tend to have more homogeneous environmental exposures * eleftheria zeggini eleftheria@sanger.ac.uk wellcome trust sanger institute, wellcome trust genome campus, hinxton, cambridge cb hh, uk oxford centre for diabetes endocrinology & metabolism, university of oxford, oxford, uk wellcome trust centre for human genetics, university of oxford, oxford, uk oxford nihr biomedical research centre, churchill hospital, oxford, uk the novo nordisk foundation center for basic metabolic research, faculty of health and medical sciences, university of copenhagen, copenhagen, denmark faculty of health sciences, university of southern denmark, odense, denmark diabetologia ( ) : – doi . /s - - - http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf and, in some cases, deep information on genealogy. these attributes can be further enhanced through the ability to recall participants for further characterisation based on genotype and through access to their medical records. over the last few years, the field of complex trait genetics has witnessed a flourishing of genetic association studies focusing on isolated populations, with the majority of findings replicating across cosmopolitan populations [ – ]. here, we outline examples from two founder populations stemming from geographical coordinates in europe ranging from the extreme north and south: greek individuals from the island of crete in the south mediterranean and inuit individuals from the island of greenland in the arctic (fig. ). the inhabitants of the island of crete in greece have in the past been investigated through epidemiological studies focus- ing on dietary and nutrition patterns with respect to metabolic disease [ , ]. the hellenic isolated cohorts (helic) minoan isolates (manolis) study was launched in with the explicit aim of studying the genetic basis of metabol- ically relevant complex traits in the isolated population resid- ing in the villages of the mountainous mylopotamos region of crete (www.helic.org). anecdotally, this population is known to enjoy good health in old age despite a diet high in animal fat. a total of recontactable individuals (making up > % of the total population) were recruited into the study and subjected to extensive phenotyping, including anthropometry, detailed diet and lifestyle information, medical history, and blood sample collection for dna extraction as well as biochemical, haematological and other biomarker analyses. following genome-wide genotyping and low-depth whole genome sequencing of the entire cohort, as- sociation analysis was carried out for quantitative traits of metabolic relevance measured in the population (such as fasting glucose and insulin levels, lipid traits and anthropometry). initial population genetic analyses revealed statistically sig- nificant enrichment of missense (and therefore potentially functional coding) variants that have substantially risen in frequency in the isolate compared with the general greek population [ ]. a genome-wide scan for association with lipid traits identified a cardioprotective effect at a nonsense variant, p.arg ter, in the apoc gene. the variant abro- gates expression of all three isoforms of the gene and has a -fold higher frequency in manolis compared with the . % % . % . % % . % % % % apoc (p.ar ter) tbc d (p.ar ter) fads - (rs ) apoc p.arg ter arg/arg n= , arg/ter n= t ri a cy lg ly ce ro l ( m m o l/l ) arg/arg arg/ter ter/ter tbc d p.arg ter h p la sm a g lu co se ( m m o l/l ) n= , n= n= fig. allele frequencies (displayed on log scale) of isolate-enriched variants in greenland, crete and the uk general population (uk k frequencies are taken from www.uk k.org [ ]). frequency bars are pink for apoc p.arg ter (rs ) [ ], blue for tbc d p.arg ter (rs ) [ ] and green for fads rs [ ]. the larger graphs show box plots of blood triglyceride levels by apoc p.arg ter genotype in crete, and plasma glucose levels by tbc d p.arg ter in greenland diabetologia ( ) : – http://www.helic.org/ http://www.uk k.org/ source population (fig. ) [ ]. the same variant had previ- ously been found to have risen to similar frequency and to have a similar protective association with lipid levels in the old order amish, an independent founder population [ ]. haplotype analysis demonstrated that the variant predates the divergence of both isolates from the general european population. interestingly, the amish also have a diet high in animal fat, indicating that the variant may have risen in fre- quency because of its cardioprotective effect; however, further analyses were not powered to detect selection signatures due to the low allele frequency (~ %) [ ]. no homozygous indi- viduals were observed in either founder population. recent work has shown that oligonucleotide-mediated inhibition of apoc has therapeutic potential as it decreases blood triacyl- glycerol (triglyceride) levels [ ]. further recent studies have also identified associations between a burden of multiple loss-of-function (lof) variants in the apoc gene and lipid levels in the general european population [ , ]. this story represents a proof-of-principle for the power of isolated pop- ulations in medically relevant trait locus discovery, exempli- fied by an early finding with significant translational potential that is transferable to cosmopolitan populations. the greenlandic population is a historically small and iso- lated founder population. it stands out in at least one important way in comparison with other well-studied founder popula- tions, such as the finnish and icelandic populations: these other founder populations are all genetically similar to at least one large population, whereas the greenlandic inuit are not closely related to any large population [ ]. the greenlandic inuit and indigenous populations throughout the arctic have different fat distribution compared with europeans [ ]. furthermore, the isolated greenlandic inuit population has lower levels of plasma glucose and serum insulin and higher levels of hdl cholesterol than danes at any given level of obesity [ ]. little is known about how genetics and lifestyle behaviour, particularly physical activity, interact to influence metabolic health in inuit and whether a recent risk increase of developing type diabetes and related cardiometabolic con- ditions is driven by a transition from a traditional subsistence lifestyle to a modern, industrialised way of life. a common greenlandic inuit-specific nonsense p.arg ter variant (in which arginine is replaced by a ter- mination codon) in the gene tbc d , with an allele frequen- cy of %, was recently discovered (fig. ). it was shown that homozygous carriers of this variant have markedly elevated h serum insulin levels, postprandial hyperglycaemia, im- paired glucose tolerance and a tenfold increased risk of type diabetes [ ]. the variant explains about % of type dia- betes occurrence in greenland. analyses of skeletal muscle biopsies in groups of three individuals carrying zero, one or two copies of p.arg ter revealed that hetero- and homozy- gous carriers of this stop-gained variant in the gene coding for tbc d are characterised by a marked decrease in the rna and protein expression of the skeletal muscle-specific long isoform of tbc d . other tissues such as pancreatic beta cells, adipose tissue and liver tissue expressing the short iso- form of tbc d were not affected by the stop-gained muta- tion [ ]. thus, homozygous carriers of p.arg ter could be considered as human muscle-specific knockouts of tbc d . since the variant is common in greenland (allele frequency of %) and rare/absent in all other populations, it is possible that selection has historically favoured it in inuit, possibly because of the low carbohydrate diet associated with traditional inuit lifestyle. indeed, weak evidence for positive selection was found [ ]. the finding of a common tissue-specific and inuit-specific lof variant constitutes proof-of-concept that conducting genetic association studies in small and isolated populations can reveal population-specific type diabetes risk variants and novel biological insights. while other lof variants in inuit may have increased in frequency by chance (genetic drift), it is conceivable that some variants are common specifically in inuit because of adapta- tion to the special climate and environment in the arctic. in fact, a recent study of signs of selection identified genetic variants in fat metabolism in the region of the fads - genes that alter metabolism of the large amounts of polyunsaturated fatty acids found in the traditional inuit seafood diet. gene variants under selection have a strong effect on height and weight, of up to cm and kg, respectively, as well as a protective effect on cholesterol and triacylglycerol levels [ ]. this study illustrates the utility of evolutionary studies of locally adapted populations for understanding the genetic basis of phenotypic variation among humans. what is becoming increasingly clear is that rare variants of large effect are most likely to be specific to or enriched in particular ancestries. these illustrations from greece and greenland provide elegant examples of the opportunities for translational biology offered through the discovery of rare coding alleles that alter disease risk. detailed physiological studies in carriers of these alleles offer the potential to under- stand the impact of perturbing particular biological pathways in humans, offering the chance to understand the interplay of proteins in multiple regulatory pathways shedding light on likely adverse on-target effects of therapeutic manipulation. acknowledgements the authors thank a. gilly and i. tachmazidou (wellcome trust sanger institute) for generating and contributing fig. , which makes use of data generated by the uk k consortium, derived from samples from uk k_cohorts_twinsuk (the twinsuk cohort) and uk k_cohort_alspac (the avon longitudinal study of parents and children). a full list of the investigators who contributed to the generation of the data is available from www.uk k.org. funding ez is funded by the wellcome trust ( ) and the european research council (erc- -stg -sepi). alg is a wellcome trust senior fellow in basic biomedical research ( / z/ /z). funding for uk k was provided by the wellcome trust diabetologia ( ) : – http://www.uk k.org/ under award wt . th was supported by research grants from the novo nordisk foundation center for basic metabolic research, an independent research center at the university of copenhagen partially funded by an unrestricted donation from the novo nordisk foundation. duality of interest the authors declare that there is no duality of interest associated with this manuscript. contribution statement all authors were responsible for drafting the article and revising it critically for important intellectual content. all authors approved the version to be published. open access this article is distributed under the terms of the creative commons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. references . peltonen l, palotie a, lange k ( ) use of population isolates for mapping complex traits. nat rev genet : – . zuk o, schaffner sf, samocha k et al ( ) searching for missing heritability: designing rare variant association studies. proc natl acad sci u s a :e –e . 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health and disease. nature : – diabetologia ( ) : – 	insights into metabolic disease from studying genetics in isolated populations: stories from greece to greenland 	abstract 	references untitled international journal of spine surgery, vol. , no. , , pp. – https://doi.org/ . / �international society for the advancement of spine surgery predictive factors and rates of fusion in minimally invasive transforaminal lumbar interbody fusion utilizing rhbmp- or mesenchymal stem cells samuel c. overley, md, steven j. mcanany, md, muhammad a. anwar, mbbs, robert k. merrill, bs, andrew lovy, md, javier z. guzman, md, sergey zhadanov, md, amish doshi, md, edward rothenberg, bs, avani vaishnav, mbbs, catherine gang, mph, sheeraz a. qureshi, md, mba department of orthopedic surgery, icahn school of medicine at mount sinai, new york, new york, department of orthopedic surgery, hospital for special surgery, new york, new york abstract background: several fusion adjuncts exist to enhance fusion rates during minimally invasive transforaminal lumbar interbody fusion (mi-tlif). the objective of this study was to compare fusion rates in patients undergoing mi- tlif with either rhbmp- or cellularized bone matrix (cbm). methods: we conducted a single surgeon retrospective cohort study of patients who underwent mi-tlif with either rhbmp- or cbm placed in an interbody cage. single and multilevel procedures were included. fusion was assessed on computed tomography scans at -month follow-up by an independent, blinded, board-certified neuroradiologist. fusion rates and rate of revision surgery were compared with a fisher exact test between the groups. a multivariate regression analysis was performed to identify patient factors that were predictive of radiographic nonunion after mi-tlif. results: a total of fusion levels in patients were reviewed. thirty-nine patients received cbm, and patients received rhbmp- . the patients receiving rhbmp- were older on average ( . vs . , p¼ . ). the overall fusion rate was % in the cbm group ( / levels) and % in the rhbmp- group ( / ) (p ¼ . ). only preoperative hypertension was predictive of radiographic nonunion (odds ratio¼ . , p¼ . ). there were smokers in the cbm group and smokers in the bmp group, and in each group experienced radiographic pseudarthrosis. a total of patients, in the cbm group and in the bmp group (p¼ . ), required revision for symptomatic pseudarthrosis. all of these patients had a single-level index procedure. conclusions: there were no differences in radiographic fusion and rate of revision surgery in patients who underwent mi-tlif with either rhbmp- or cbm as fusion adjuncts. level of evidence: clinical relevance: both rhbmp- and cbms can be used as effective fusion adjuncts without any clear advantage of one over the other. minimally invasive surgery keywords: minimally invasive transforaminal lumbar interbody fusion, bone morphogenetic protein, cellularized bone matrix, stem cells, lumbar spine fusion, rhbmp- , tlif introduction minimally invasive transforaminal lumbar inter- body fusion (mi-tlif) is a well-accepted surgical treatment option for patients with degenerative conditions of the lumbar spine. – while several studies have reported on the short-term advantages of minimally invasive techniques, proponents of open surgery cite poor fusion success as a major deterrent to widespread adoption. – in an effort to improve fusion in the interbody space in mi-tlif, surgeons have employed a variety of fusion sub- strates. the rates of successful fusion in mi-tlif have varied, with most reports relying on surgeon interpretation of plain radiographs to determine fusion success. in an effort to improve fusion rates in spine surgery, on- and off-label use of recombinant human bone morphogenetic protein- (rhbmp- ) has become widespread. although rhbmp- has improved fusion rates across the realm of spinal fusion procedures, concerns of side effects of rhbmp have led to bioengineering efforts to produce efficacious alternatives, especially in trans- foraminal interbody fusion. – specifically, in mi- tlif, the literature lacks direct comparison of rhbmp- over other fusion substrates. mesenchymal stem cell containing cellular bone matrices (cbms) were recently introduced as an alternative to rhbmp- . cbms offer some theoret- ical promise by combining osteogenic, osteoconduc- tive, and osteoinductive characteristics that mimic the biologic profile of iliac crest bone graft. in vitro studies have demonstrated that cbms contribute to repair and regeneration of bones, though clinical trials validating this capability in the human intervertebral disc space are sparse. – trinity evolution is a cbm comprised of a cancellous bone matrix containing viable osteoprogenitor stem cells derived from cadaveric donor tissues. it has proven to be a safe and efficacious substrate for bone grafting procedures in some orthopedic procedures outside of the spine, though only industry-sponsored studies currently exist detailing its use and efficacy in spinal fusion procedures. – the purpose of this study was to directly compare fusion success and complications in patients under- going mi-tlif surgery with use of rhbmp- (infuse) or cbm (trinity evolution) in a single surgeon series. independent, blinded neuroradiolo- gist review of ct scans to assess fusion was employed, and predictive factors for nonunion were identified. to our knowledge, this study is the first of its kind to directly compare rhbmp- versus cbm as fusion adjuncts in mi-tlif. methods the institutional review board approved this retrospective review of a single surgeon’s patients at institution between july and december . patients included in this study underwent an mi-tlif with either rhbmp- (infuse) or cbm (trinity elite) used as the primary fusion substrate in the interbody space. initial cases were all treated with cbm until a specific point in time after which the remaining cases received rhbmp- . the elec- tronic patient medical records and the paper office charts were queried for data collection. all patients included in this study underwent an appropriate course of nonoperative treatment, including but not limited to activity modification, physical therapy, nonsteroidal anti-inflammatory drugs, limited opioid analgesics, or epidural steroid injections, for at least months. operative indica- tions included degenerative spondylosis or spon- dyolisthesis resulting in central, lateral recess or foraminal stenosis, radiculopathy, or neurogenic claudication as well as failure of nonoperative care. postoperative -mm-cut computed tomography (ct) scans were obtained year after surgery for all patients in the study to assess fusion status. two independent, blinded, board-certified neuroradiolo- gists assessed fusion at the operative level(s). fusion was defined as evidence of bony bridging from end plate to end plate within the cage as well as bony bridging lateral to the cage. a representative ct cut of a fused level is shown in figure , while a case of pseudarthrosis is depicted in figure . ibm spss statistics (armonk, new york) was utilized for statistical analysis. continuous variables were compared between cohorts with independent- sample t tests, and categorical variables were compared between groups with fisher exact test. separate analyses were performed for single-level fusions. a multivariate regression analysis was performed to identify patient factors that were predictive of radiographic nonunion after mi-tlif. all levels were individually entered into the regression model. operative procedure mi-tlif was performed in all cases in equivalent fashion. patients were placed prone on a jackson table with wilson frame. neuromonitoring was used in all cases. guide wires were placed into the pedicles bilaterally under ap and lateral fluorosco- figure . representative -mm sagittal computed tomography cuts showing bridging bony trabeculae both within the cage and lateral to the cage. this is indicative of a solid arthrodesis. overley et al. international journal of spine surgery, vol. , no. py. a tubular retractor was then placed over the facet joint on the side of most significant symptoms. a complete facetectomy was performed. thorough discectomy and end plate preparation was per- formed using a combination of distractors, shavers, and curettes; cm of morecellized allograft bone was placed anteriorly in the disc space. this was followed by placement of an appropriately sized polyetheretherketone (peek) interbody cage. the cage was packed either with an x-small rhbmp- sponge or with cm of trinity evolution cbm. in the rhbmp- group, another cm of morcellized allograft bone was packed behind the cage in the interbody space in attempts to mitigate direct exposure of the neural elements to the rhbmp- . in the trinity evolution cbm group, the remainder of the cm of cbm not permitted in the peek cage was combined with cm of morcellized allograft bone and placed posterior to the interbody cage. cannulated pedicle screw instrumentation was performed over the previously placed guide wires. rods and endcaps were inserted and a standard closure was performed. no patients had an addi- tional posterolateral fusion performed. results patient characteristics a total of fusion levels in patients were reviewed. thirty-nine patients received trinity elite cbm in the interbody cage; patients received infuse rhbmp- in the interbody cage. the epide- miological variables for all cases as well as single- level-only procedures are summarized in tables and . the average age in the rhbmp- cohort was . years as compared with . in the trinity elite cohort (p ¼ . ). there were no statistical differ- ences between the groups with respect to bmi, insurance status, number of operative levels, smok- ing status, or associated medical comorbidities. fusion rates with either rhbmp- or cbm the fusion rate assessed at year with ct scan was found to be % in the cbm group ( / levels) and % in the rhbmp- group ( / levels). the rate of fusion was not found to be statistically significant between the groups (p ¼ . ) (table ). when examining single-level cases only, the fusion rates for the cbm and rhbmp- groups were % ( / ) and % ( / ), respec- tively. although trending toward significance in favor of bmp, these results failed to demonstrate statistical difference (p ¼ . ). a total of patients ( %) required a revision surgery for symptomatic pseudarthrosis, in the cbm group and in the rhbmp- group (p¼ . ). all of these patients had a single-level index procedure. risk factors for nonunion a multivariate analysis was performed to identify the factors that were predictive of progressing to radiographic nonunion. basic descriptive statistics and regression coefficients for all cases are shown in table . the results of the regression analysis figure . representative -mm sagittal computed tomography cuts showing lack of bridging bony trabeculae. there is no bridging bone through or outside of the interbody cage indicative of pseudarthrosis. table . patient demographic and operative information for all cases. variables trinity (cbm) (n ¼ ) rhbmp- (n ¼ ) p value age (average) . . . . . * bmi (average) . . . . . total levels multilevel procedure . l - . l - . l - . l - . l -s . male . female private insurance . average follow-up (mo) . . . . . * smoker . hypertension . diabetes . levels fused / / . reoperation for pseudo / / . abbreviations: cbm, cellular bone matrix; rhbmp- , recombinant human bone morphogenetic protein- ; bmi, body mass index. *statistically significant value at a threshold of p , . . mi-tlif utilizing rhbmp- or mesenchymal stem cells international journal of spine surgery, vol. , no. indicated that only preoperative hypertension was predictive of developing a radiographic pseudar- throsis (odds ration ¼ . , p ¼ . ). complications of the cases performed, a total of complications ( %) occurred. the complications included adjacent-level herniation, pseudarthro- ses that required a return to the operating room, cases of adjacent segment disease, cases of recurrent stenosis, and failures of hardware. there was no statistically significant difference in the proportion of complications between the cbm group ( / ) and the bmp group ( / ) (p ¼ . ). discussion mi-tlif has been validated in the literature as a safe and effective alternative to open tlif as well as traditional instrumented posterolateral lumbar fusion with equal fusion rates and patient-reported outcome measures. – however, to date, no study has directly compared rhbmp- and cbm as fusion adjuncts and their effect on mi-tlif fusion rates. furthermore, the vast majority of relevant studies do not employ the use of stringent ct-based radiologic criteria performed by independent, board-certified, blinded neuroradiologists. this in- troduces the possibility of considerable observer bias. the results of our study found utilization of rhbmp- to trend toward improved fusion rates in single- and multilevel mi-tlif as compared to cbm, though results failed to reach statistical significance. independent studies evaluating fusion rates after mi-tlif with rhbmp- as a fusion enhancer demonstrate fusion rates ranging from % to %. however, there exists significant interstudy heterogeneity with different applications of type of interbody device, addition of posterolat- eral fusion techniques, and unilateral versus bilat- eral instrumentation. villavecencio et al were one of the first groups to publish on fusion rates of mi- tlif with rhbmp- without posterolateral fusion, achieving successful arthrodesis in % of patients. they were the only group to objectively utilize radiologist review of dynamic radiographs, though they did not employ ct. anand et al published on a cohort of patients who underwent mi-tlif with rhbmp- as a fusion adjunct and achieved a solid fusion on % of their subjects. however, their fusion designation was based primarily on dynamic plain radiographs, reserving ct only for equivocal interpretations, though the literature favors ct analysis of fusion against plain radio- graphs. additionally, fusion assessment was per- formed by the operating surgeon. while rhbmp- is a validated fusion enhancer, the use of cbms clinically for fusion application, though extremely prevalent in clinical practice, is sparse in the literature. paradoxically, grabowski et al estimated that cbms were used in almost / of all spinal fusion procedures performed in . currently, only published studies exist evaluating the use of cbms for spinal arthrodesis proce- dures. – of these studies, did not disclose conflicts of interest, , and the remaining study was industry sponsored. fusion rates in these studies ranged from % to %, paralleling previously published mi-tlif fusion data trends. however, these studies employed only plain radiog- table . patient demographic and operative information for single-level procedures only. variables trinity (cbm) (n ¼ ) rhbmp- (n ¼ ) p value age (average) . . . . . bmi (average) . . . . . l - . l - . l - . l - . l -s . male . female private insurance . average follow-up (mo) . . . . . a smoker . hypertension . diabetes . levels fused / / . reoperation for pseudo / / . abbreviations: cbm, cellular bone matrix; rhbmp- , recombinant human bone morphogenetic protein- ; bmi, body mass index. *statistically significant value at a threshold of p , . . table . regression analysis for predictors of developing radiographic nonunion. variable odds ratio p value lower % ci upper % ci age . . . . bmi . . . . multilevel procedure . . . . private insurance . . . . cbm . . . . smoking . . . . hypertension . . a . . diabetes . . . . abbreviations: bmi, body mass index; cbm, cellular bone matrix. *statistically significant value at a threshold of p , . . overley et al. international journal of spine surgery, vol. , no. raphy and relied on surgeon interpretation of fusion status, which introduces considerable observer bias. in our study, blinded, fellowship-trained neuroradiologists assessed fusion by ct scan. fusion, if present, was given a grade ranging from i to iv, in accordance with the spinal fusion classification system proposed by shah et al. for purposes of statisitical analysis, only shah grades iii and iv (bony bridging from end plate to end plate both within and around the cage) were considered fused. of the levels evaluated using cbm as a fusion adjunct, ( %) were designated as fused, while / ( %) levels that employed rhbmp- achieved solid arthrodesis. one may hypothesize that these comparatively low rates of arthrodesis can, at least in part, be attributed to the assimilation of surgeon-assessed fusion assignments in the surgical community and existing literature. when comparing our rates of fusion to those in other studies, the definition of fusion must be considered. for example, the only other study that employed the use of blinded radiologists using flexion/exten- sion radiographs reported a % fusion rate for single-level mi-tlif. despite a nonunion rate of % for all levels as measured on ct scan, only patients ( %) required revision surgery for symptomatic pseudar- throsis, which is similar to reported rates in the literature. these findings suggest that radiographic nonunion may not prognosticate poor outcomes or the development of painful nonunion. it may be more important for the surgeon to consider revision surgery based on patient symptomatology rather than radiographic pseudarthrosis alone, as a radio- graphic pseudarthrosis, when independently identi- fied by a neuroradiologist, may not correlate to a clinical, symptomatic pseudoarthrosis. our regression analysis revealed only preopera- tive hypertension to be predictive of pseudarthrosis. while an increased number of comorbidities, including hypertension, has been previously shown to predict lumbar pseudarthosis, to our knowledge, hypertension alone has never been shown to be a single predictor for nonunion. , other patient- specific variables previously implicated in poor radiological outcomes, such as smoking, diabetes, and obesity, were not found to predict poor fusion status. limitations of our study include the retrospective study design as well as lack of patient-reported outcome measures. while patient-reported out- comes would have been helpful to differentiate radiographic pseudarthrosis from clinical, symp- tomatic pseudarthrosis, the primary purpose of this article was to compare radiographic fusion between fusion substrates. thus, the lack of patient- reported outcomes does not take away from the objective of this study. additionally, the end-point time of the study, months, for fusion designation may have contributed to underreporting of fusion rates in those patients who require . months for fusion maturation. finally, our patient sample was not randomized, which may subject certain out- comes, such as fusion rates and complications, to the effect of learning the procedure, which would bias the results. however, this study was conducted when the operating surgeon was mature in the learning curve. hence, any bias due to the learning curve would have been minimal. conclusions mi-tlif is a proven safe and efficacious means of indirectly decompressing lumbar nerve roots, restoring lordosis, and achieving a solid fusion. we found no significant difference in fusion rates between cellularized bone matrix and recombinant human bone morphogenetic protein in mi-tlif procedures. we found hypertension to be a statistically significant risk factor for nonunion. our stringent definition of fusion that is entirely ct scan based and requires multiple sites of bridging boney trabeculae, both within and outside of the interbody cage, as well as the employment of blinded neuroradiologists to make this designation, may suggest the possibility that previous studies have overestimated fusion rates. further longitudi- nal blinded studies utilizing a standard metric for fusion as designated by third-party neuroradiolo- gists directly comparing fusion enhancers in mi- tlif procedures are needed to validate these claims. references . adogwa o, parker sl, davis bj, et al. cost-effectiveness of transforaminal lumbar interbody fusion for grade i degenerative spondylolisthesis. j neurosurg spine. ; ( ): – . . crandall dg, revella j, patterson j, huish e, chang m, mclemore r. transforaminal lumbar interbody fusion with rhbmp- in spinal deformity, spondylolisthesis, and degener- ative disease—part : bmp dosage-related complications and long-term outcomes in patients. spine (phila pa ). ; ( ): – . mi-tlif utilizing rhbmp- or mesenchymal stem cells international journal of spine surgery, vol. , no. . crandall dg, revella j, patterson j, huish e, chang m, mclemore r. transforaminal lumbar interbody fusion with rhbmp- in spinal deformity, spondylolisthesis, and degener- ative 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scan to evaluate interbody fusion following the use of titanium interbody cages and transpedicular instrumentation. eur spine j. ; ( ): – . . gordon l, patil cg, lad sp, ho c, tian w, boakye m. effects of age and comorbidities on complication rates and adverse outcomes after lumbar laminectomy in elderly patients. spine (phila pa ). ; ( ): – . . kim yj, bridwell kh, lenke lg, et al. pseudarthrosis in long adult spinal deformity instrumentation and fusion to the sacrum: prevalence and risk factor analysis of cases. spine (phila pa ). ; ( ): – . disclosures and coi: an institutional review board approved this study. no funding was received to conduct this study. sheeraz qureshi is currently overley et al. international journal of spine surgery, vol. , no. receiving grant/research support from the cervical spine research society; consulting fees from zimmer-biomet, stryker spine, globus medical, inc.; and shareholder interest in avaz surgical and royalties from rti, zimmer-biomet, stryker spine. corresponding author: sheeraz a. qureshi, md, mba, hospital for special surgery, east th street, new york, ny . phone: ( ) - ; email: sheerazqureshimd@gmail.com. published february this manuscript is generously published free of charge by isass, the international society for the advancement of spine surgery. copyright � isass. to see more or order reprints or permis- sions, see http://ijssurgery.com. mi-tlif utilizing rhbmp- or mesenchymal stem cells international journal of spine surgery, vol. , no. horse manure management by commercial and old-order amish equine operators: economic and conservation implications sustainability article horse manure management by commercial and old-order amish equine operators: economic and conservation implications justin dijak, laura mccann * and caroline brock division of applied social sciences, university of missouri, mumford hall, columbia, mo - , usa; dijakj@missouri.edu (j.d.); brockcc@missouri.edu (c.b.) * correspondence: mccannl@missouri.edu received: july ; accepted: october ; published: october ���������� ������� abstract: horse operations may produce high amounts of manure per acre/ha and be less aware of recommended manure management practices than livestock farmers, leading to negative environmental impacts. this study compared the manure management practices of two populations of horse owners in the usa state of missouri, commercial horse operations and an old-order amish community, using data from a mail survey with a % response rate. in commercial operations, manure was more likely to be piled rather than spread directly on fields, which was the amish practice. the amish were more likely to use manure for crop production, to indicate that was why they had not explored markets for manure, and to test soil for nutrients. regression results for factors affecting previous sales/transfers of manure or compost showed that selling was more likely for commercial operations, female operators, and those who had composted manure. compared to respondents who agreed that manure management had an impact on water quality, those who did not know or were neutral about that statement were more likely to have sold manure. while both groups can improve manure management and are underserved by traditional agricultural information channels, educational efforts should be tailored to their different circumstances. keywords: amish; equine; horse; manure management; value-added; water quality . introduction manure is a source of nutrients for crops, but also has a variety of negative environmental consequences, including impacts on surface water and groundwater quality, local air quality, and climate change. sustainable utilization of this resource is therefore crucial [ ]. the most recent scientific findings on these environmental problems and options for the sustainable utilization of manure as a natural resource are discussed in a recent book published by the american society of agronomy and the soil science society of america [ ]. this study examines horse manure management, which exhibits many of the same environmental problems as manure from other species [ ], but from a social science perspective. the overall equine population in the united states (usa) decreased between and according to the census of agriculture [ ]. generally, equines (primarily horses) are kept for pleasure, farming, or equine businesses. in-depth surveys of operations with more than five equids by usda-aphis found that the percentage of equids used for pleasure was similar in and ( . % and . %, respectively), while the percentage used for farm/ranch work increased ( . % versus . %). small operations ( – equids) accounted for the majority of operations during this period, but accounted for a smaller percentage of total equids; in , they represented . % of operations and . % of equids. large operations (over equids) were more likely to have a primary sustainability , , ; doi: . /su www.mdpi.com/journal/sustainability http://www.mdpi.com/journal/sustainability http://www.mdpi.com https://orcid.org/ - - - http://dx.doi.org/ . /su http://www.mdpi.com/journal/sustainability https://www.mdpi.com/ - / / / ?type=check_update&version= sustainability , , of function of boarding/training, breeding, or being a riding stable than small operations or those with – equids [ ]. equine operations thus tend to be diverse in both their activities and their size. however, it is not clear if amish equine owners were delineated or even included in the usa equine survey (as explained in more depth later, the amish are a christian group that has value-based prohibitions against personal ownership and use of certain technologies, such as cars, as well as tractors for field work). for example, when discussing the primary use of equines, transportation was not even listed as an option [ ], and the amish are the main population who would use horses for transportation in the us. however, other data sources indicate that amish equine operator numbers are growing, since all amish own horses for local transportation, and their population is doubling every years [ ]. these distinct types of operations may have different goals as well as different levels of knowledge regarding the environmental impacts of their operations. in general, equine operations are neglected regarding education and outreach efforts about conservation practices compared to other types of livestock farming [ ]. one of the environmental concerns around equines is manure management [ ]. the types of environmental impacts of horse manure on air and water quality, their sources, and recommended management practices to reduce these impacts were reviewed by westendorf et al. [ ]. as with other types of manure, excess nutrients and pathogens can degrade water quality [ ]. for example, kleinman et al. [ ] indicate that animal manure is a major contributor to phosphorous pollution in the chesapeake bay watershed, and horses represent % of animal units in the watershed (one animal unit represents . kg liveweight). however, appropriate manure management also presents an opportunity to recycle nutrients back to the soil and to improve organic matter and other soil characteristics valued by farmers [ ]. historically, this occurred in integrated crop–livestock operations. the benefits of manure as a soil amendment also provide the opportunity to create a value-added product for agricultural and residential uses. horse manure can be composted, which reduces the volume of waste by – %, and horse manure and bedding can potentially have ideal nitrogen/carbon ratios for composting [ ] (while beyond the scope of this research, feedstuffs used, the type and quantity of bedding products, and the intensity of horse manure management can affect the quality of the manure products, including the nitrogen/carbon ratio [ , ]). the existing research on horse manure management practices in the usa usually focuses on small-scale operations or recreational horses kept in confined and pastured settings [ , ]. while this focus is important given the large number of operations where horses are kept for personal recreational use and the environmental impacts associated with them, their motivations may differ from those of equine businesses. commercial equine operations derive the majority of their income from equine training, boarding, breeding, and sales [ ], and may have less knowledge of the use of manure for agricultural production. the amish are another group that is distinct in that they use horses in their agricultural enterprises and value manure as a soil amendment. there are also geographic limitations to previous studies on horse manure management, which have focused on the mid-atlantic [ ], indiana, and kentucky [ , ]; only one of these studies discussed amish horse operators. however, missouri has the seventh largest number of horses in the usa [ ], and no surveys relating to horse manure have been conducted. missouri also has the seventh highest population of amish people in the usa [ ]. this study was a pioneering effort to compare horse manure management practices among commercial equine operations and amish communities, as well as their current activities and attitudes regarding value-added horse manure products. this research examined the characteristics of these operations, information sources, and manure management practices, as well as the importance each group places on manure as a soil amendment. previous research indicated that there is often a disconnection between extension educators and horse farm operators with respect to environmental management issues, as most education efforts seem to be focused on horse management and well-being rather than manure management [ ]. westendorf et al. [ ] indicate that equine facility managers in the usa are less aware of soil fertility and nutrient management issues, have less contact with conservation professionals, and face unique challenges compared to farmers. understanding the knowledge levels sustainability , , of and decision-making processes of these underserved groups with different motivations and structural considerations is important in order to design targeted educational programs to improve horse manure management and sustainability. this study also examined the current situation regarding sales of horse manure by these two groups, as well as the potential for new uses and new markets for their horses’ manure. more specifically, this research explored some of the barriers to the creation of a value-added manure product, such as access to equipment and current knowledge about composting practices. manure transfers may also improve water quality if the source operation has excess nutrients relative to agronomic requirements [ ]. the research questions are as follows: . how do commercial equine operations and old-order amish differ in the characteristics of the operators, their access to equipment for manure management and composting, and their production systems? . how is horse manure used and what kinds of manure management practices are implemented in these operations? . to what extent are these operators currently selling their manure and what are the barriers to value-added products, such as composted manure? . what factors affect the sale of manure products? it was hypothesized that while manure may be an under-utilized resource in both types of operations, the amish are more likely to understand and appreciate manure’s value as a nutrient source and soil amendment and to use it on their own farms or provide it to neighbors for crop production. in addition, there may be technological barriers for these conservative, old-order amish to pursue value-added manure products for sale. it was hypothesized that commercial horse operations would be more interested in sales and transfers of manure due to their lesser integration with crop production, and they would thus have lower awareness of the nutrient and soil amendment benefits of the manure. it was also hypothesized that higher-value manure products, such as compost, would be more likely to be marketed. . background the trend of specialization in usa agriculture has meant that there are fewer integrated crop–livestock farming systems [ ]. this implies that livestock manure is less likely to be seen as a nutrient source and soil amendment and more likely to be seen as a waste product, which has implications for efficient use of manure and water quality [ , , , ]. this view of manure as a waste product rather than a resource is especially likely among commercial equine operations for several reasons. this section examines the characteristics of this group, followed by those of amish farmers. . . commercial equine operations for a variety of reasons, commercial equine operations may have issues with manure management. horse operations use many of the same agricultural inputs (hay, grains, straw, and various other bedding materials) that livestock and dairy producers use; however, they purchase the majority of them rather than relying on crop production. the managers of these operations may, therefore, be less aware of agricultural information sources, less knowledgeable about crop production, and, thus, less knowledgeable about manure as a resource than typical livestock producers in rural areas. previous research indicates that equine managers are not well connected to organizations that may address larger-scale farm management issues like manure management and its environmental impacts [ ]. many of these enterprises are managed by women who do not have agricultural backgrounds, which means that they are more likely to be overlooked by extension professionals [ ]. owners of many equine operations also have substantial off-farm income, and previous research on sustainability , , of livestock farmers in the usa’s midwest has found that those with more off-farm income are less likely to adopt manure management practices, such as manure testing [ ]. commercial equine training and boarding operations may house a substantial number of horses that produce manure, which can have ecological and economic implications. due to the reliance on revenue generated by instruction and training fees, commercial equine operations are usually located near urban and suburban areas, which means higher land values [ ]. these operations thus tend to have more intensive equine management and smaller acreages [ ]. extension professionals surveyed by perry-hill and prokopy [ ] indicated that a major problem in kentucky and indiana was that there were too many horses on too few hectares. in new jersey, only about one fourth of equine farms that spread manure had more than . hectares of land [ ]. ali et al. [ ] found that livestock density (animal units per unit of surface area, multiple species) was negatively associated with the adoption of manure testing in iowa and missouri, perhaps since nutrients were not limiting for crop production. commercial equine operations are typically too small to be subject to usa water quality regulations, which are relevant for farms with more than animal units, which implies that they may be less motivated to manage the manure effectively for conservation and soil quality purposes. in addition to environmental implications, there may be an unrealized economic opportunity for horse managers to sell composted manure as a soil amendment. for example, compost from horse manure has long been used in mushroom production [ ]. one problem with livestock manure in general that limits marketability is the higher water content and, thus, higher transportation costs for a given amount of nutrients [ ]. buyers of manure also have preferences by animal species; norwood et al. [ ] found a preference for dairy versus swine manure. there is evidence of equine manure being marketed as a value-added soil amendment. one equine facility in washington stopped paying for waste disposal and instead began selling compost at usd/cubic meter, and they recovered their investment in composting technology in three years [ ]. however, there may be economies of scale and access to markets that make composting more feasible for some equine operations than others. . . amish operations the old-order amish are a christian group formed out of the anabaptist movement in the protestant reformation. they maintain distinct guidelines around technology (e.g., restricting individual car ownership, hence the reliance on horses) to maintain key values such as community, family, and a strong work ethic. all old-order amish own and utilize horses for local transportation, and some also use horses for plowing and harvesting operations. while not all amish in the usa are farmers, those who move to more rural areas for affordable land and property tax rates, such as in missouri, are more likely to be involved in farming as part of their economic portfolio. amish farms tend to be small, which is due in part to their reliance on horses for traction as well their desire to live in close proximity to each other [ , ]. for amish farmers, manure represents a valuable input for crop production as part of a diversified livelihood strategy, so they may be more motivated to manage manure as a nutrient source than commercial equine operations. the amish have a long history of utilizing manure for innovative soil fertility practices [ , ], although they may also use commercial fertilizers [ ]. while using both commercial fertilizers and manure is not unique to the amish, people often assume that they produce organically [ ]. they tend to have more diversified farms; earlier research found that amish dairy farmers are also growing vegetables, which was very uncommon for similar non-amish dairy farms [ ]. in these farming systems, outputs from one enterprise can be used as inputs in another, rather than purchasing inputs such as fertilizer or feedstuffs for horses. given their smaller farms, the amish tend to intensively farm the land they do have, and to focus more on crops like vegetables, which are labor-intensive. missouri, in particular, has a sizable group of amish farmers producing vegetables, as indicated by the nine amish produce auctions in the state. vegetables have high nutrient requirements, which may provide an incentive for utilizing manure effectively. given the integrated nature of their communities sustainability , , of and their operations, the amish may be more able to realize the potential of horse manure or composted manure as a nutrient source and soil amendment. however, horse manure can be a source of controversy for local communities [ ] as well as a contributor to water quality issues, especially if these amish farmers also own larger livestock operations. relying on horse-drawn equipment may limit how far the manure is spread [ ]. previous research indicates that conservation awareness around manure management may be lower for amish farmers than non-amish ones, and they are not as familiar with organizations that could assist with manure management decisions [ , ], which is similar to commercial equine operations. there may also be less time for amish to invest in conservation efforts given that many also have non-farming occupations [ ], again similar to commercial operations. the old-order amish also typically complete their formal education after the eighth grade (i.e., after eight years of schooling) and can be harder to reach for extension and conservation agents than other farmers [ ], especially as the internet is not a useful outlet [ ]. social networks for the amish are highly integrated and overlapping, considering that the amish may attend church, go to school, and attend other meetings with many of the same families, which may limit the exchange of information with non-amish farmers. given the descriptions of these two populations, they are expected to differ in their manure management practices. in particular, the old-order amish of clark, missouri are expected to be more likely to have integrated crop–livestock systems than commercial horse operations, and thus have more on-farm uses for the manure. well over half ( %) of the households in clark are involved in an agricultural enterprise as part of their economic portfolio [ ]. in addition, given the greater density of horses in commercial operations, they may have manure in excess of crop or pasture requirements, and may thus be more interested in sales or transfers of manure products. the amish may have less access to manure management machinery, which will negatively affect their likelihood of selling manure products compared to commercial operations. . data and methods in january and february of , a mail survey of commercial equine operators in missouri and of farmers from an old-order amish settlement in near clark, missouri was implemented. a mail version of the survey was chosen rather than an internet-based survey, which would have been inappropriate for the amish. the amish are much more likely to read something that comes in the mail [ ]. a mailing list of commercial equine operators was compiled using the commercial facility locator features of equestrian organizations in the state of missouri. these organizations were the missouri horse shows association, the united states equestrian federation, the american quarter horse association, and equine now.com. the clark settlement is one of the largest and oldest communities in missouri, started in and consisting of church districts (which consist of – people). a mailing list was compiled using all entries in the amish of missouri directory for the settlement of clark [ ]. a combined list was created that included the population of commercial equine operations in missouri and members of the clark settlement for a total of operations. a paper mail survey was developed and implemented using modifications of the dillman method [ ]. the seven-page, -question survey obtained information about the overall operation and detailed information about horse manure management. the question format was primarily closed-ended, with some fill-in-the-blank questions, e.g., numbers of horses in each size category. the questions were related to operation attributes, current manure management practices, agricultural production, equipment and labor used for manure, experience with manure as a value-added product, marketing manure, barriers to composting and sales, and attitudes about water quality and soil conservation, and the survey ended with demographic questions. the reminder postcard, cover letters, and surveys were approved by the university of missouri institutional review board. the survey packet consisted of a cover letter, the survey, and a self-addressed return envelope. the cover letters were signed by hand and the packets were mailed using stamps. the surveys were color-coded to distinguish amish from commercial operations. reminder postcards were sent after three weeks, and a sustainability , , of second survey mailing was sent three weeks after the postcard. one hundred seventy surveys were returned, indicating a . % response rate after correcting for undeliverable surveys or people who did not have any horses. the survey data were modified to create variables for the analyses. detailed explanations are provided for variables used in the regressions (in surveys, it is recommended to have closed-ended rather than open-ended response options and to have categories for sensitive continuous variables, such as age and education [ ], which affect the statistical analysis; categorical or dummy variables are used with a designated base category to which the other categories are compared). over % of the amish respondents left the open-ended gender question blank. given that the names to whom the amish surveys were sent were exclusively male, as well as the patriarchal nature of the amish community, the blank gender responses were changed to male. the total number of animal unit equivalents (aue) on the farm/operation was calculated based on the data on equine numbers in various weight ranges, following manske [ ]. the aue was determined using . kg for small horses and ponies, kg for light horses, and . kg for heavy or draft horses [ ]. the aue was calculated for each weight category and then summed for each respondent across all three categories, and the natural log of this number was used in the regression. four response options for current manure management practices were: “spread fresh over fields”, “composted”, “piled”, or “piled then later spread over fields.” the base category, which included the most responses, was “spread fresh over fields”. a dummy variable for manure management time was constructed by using – h/week as the base, since there were substantial numbers across both types of operations, and the other time categories were combined into > h as the categorical variable. their level of agreement with the statement “manure management practices affect water quality” was also included as a dummy variable with “agree”/“strongly agree” combined as the base category, with dummy variables for “disagree”/“strongly disagree”, “neither agree nor disagree”, and “i do not know”. another dummy variable was created by combining questions about testing soil, manure, or compost for nutrient content into one question, with the base being no nutrient testing. agricultural producers can use manure to improve crop production, which would provide an alternative use for selling manure or compost, so participants were asked whether they “produced and marketed crops in the previous year”. participants were asked about equipment used for manure management. farm machinery can significantly reduce the labor required to turn and move large amounts of manure and compost. for the regression of only commercial operators, the machinery was also grouped by functions related to manure management to form categorical variables. the base category was managers that use manure spreaders, which was the most common type of equipment found in the survey. the categorical variables were “no use of machinery” and “tractors with buckets, bobcats, and skid loaders”, i.e., equipment that will only pile, turn, and load manure. this subset has access to the complete complement of equipment needed for handling piles of manure or compost, but no access to equipment to apply manure or compost to fields. larger commercial equine operations often only use barn staff that are “hired by the primary operator” to perform manure management duties. these operations may have more intensive manure management practices than smaller operations, where horse owners or boarders are responsible for manure management duties. manure management in a paid or professional setting may thus be predictive of manure or compost sales. operations with only hired staff for this task formed the categorical variable, while operations that included boarders, owners, or family members were used as the base. descriptive statistics were examined for a wide variety of questions in order to compare characteristics of operators and operations, manure management practices, and conservation attitudes of commercial equine operations and amish farms. statistical comparison of the means of the variables was conducted using two-tailed t-statistics. before further analysis, a correlation coefficient matrix was examined to identify multicollinearity among variables (data available upon request). sustainability , , of to rigorously examine determinants of manure transfers, bivariate logistic regression was conducted using the r programming language. logistic regression is a method used for categorical dependent variables. previous experience with marketing manure or value-added manure products was used as the dichotomous dependent variable. pseudo-r squared and the akaike information criterion (aic) were used to diagnose model fit and explanatory power of the model. for the combined group regression, observations were used due to missing data for some respondents. thirty-one of these respondents had sold manure or compost; the base of the bivariate logistic regression was those who had not sold a manure product. percentage of income from horses could not be used, nor could crop hectares or variables incorporating it (e.g., animal units/crop ha) because of extensive missing data. there were also cases where the model failed to converge due to a lack of variation. for example, it was not possible to have a commercial–gender interaction term due to the negligible number of female amish farmers. given the differences between the groups and the inability to compare machinery variables across groups, a separate regression with only commercial equine operators was developed, which included responses. . results and discussion given the dearth of information available on these two populations of horse owners and the fact that this is the first study to compare them, an extensive examination of the descriptive statistics is presented, followed by the presentation of the regression results from combining both groups using a more restricted set of variables. an additional regression focusing on commercial operators was analyzed to focus on the effects of machinery. . . summary statistics data relating to the demographic and general farm/operation characteristics are presented in table (values for categorical variables are either —the respondent’s answer corresponds to that category—or —it does not—so this is the range for these variables; one can usually interpret the mean of a categorical variable as the percentage of respondents in that column who are in that category). all differences discussed are significant unless otherwise indicated. operators of commercial equine operations in missouri are older than amish farmers in clark, missouri. this finding of amish farmers being younger on average was also found by brock and barham [ ]. commercial operators also have a higher level of education, with none having only an eighth grade education, as opposed to the amish, of whom % had only an eighth grade education, in line with expectations. the majority of the commercial operators are female and the majority of the amish operators are male. table . summary statistics: operator demographic characteristics (n = ). variables mean comparison range commercial amish difference a age in years – – . . − . ** – – . . − . *** – – . . . ** + – . . . *** education (highest level) th grade – . − . *** high school – . . − . some college – . . . *** college graduate – . . *** post-graduate degree – . . *** gender female – . . . *** male – . . − . *** a difference of means; **, %; ***, . %. sustainability , , of table presents the operation characteristics. the majority ( %) of the commercial equine operations are indeed professional, defined as breeding, training, boarding, selling, or recreation. the majority of the amish operations ( %) are utility operations (horses used for transportation or farm work). the percentage of total income from horse-related activities was % for the commercial operators and % for the amish farmers, indicating that the amish have more diversified operations. commercial operations had more cropland, which was not in line with expectations. the average number of horses in the . to kg range was higher in the commercial operations. table . summary statistics: operation characteristics (n = ). variables mean comparison range commercial amish difference a farm operation type of horse operation hobby – . . . * utility (transportation or farming) – . . − . *** professional (training, boarding, etc.) – . . . *** horse-related income (% of total income) – . . . *** farm size hectares of pasture – . . . hectares of crop land – . . . *** number of equines on farm < . kg – . . . . − kg – . . . *** > kg – . . . # bedding type used wood shavings – . . . *** straw – . . − . *** sawdust – . . hay sources grow – . . − . ** purchase – . . . *** grow and purchase – . . − . *** crop production produced and marketed crops in yes – . . − . *** no – . . . *** crops produced corn – . . − . *** soybeans – . . − . *** wheat – . − . ** barley or oats – . − . *** alfalfa or other hay – . . − . *** vegetables – . . − . *** a difference of means; superscript # indicates % level of significance; *, %; **, %; ***, . %. the commercial equine operations were more likely than the amish to use wood shavings as bedding and less likely to use straw. this may be because commercial operators would have to purchase bedding, and wood products are easier to clean from stalls. according to the review by westendorf et al. [ ], use of wood shavings and sawdust may result in a lower-quality manure product in commercial operations. the hay source for feeding the horses also varied between the types of operation. commercial operators were more likely to have only purchased hay, while the amish farmers were more likely to have grown their own or supplemented with purchased hay. the commercial operators are thus less likely to know how their hay was produced, especially since sustainability , , of the amish who do purchase hay are likely to purchase the hay locally from other amish farmers in the clark community. one problem with compost is that broadleaf herbicides from bedding, feed, or manure may kill plants on which it is applied [ , ] so testing for residual herbicide may be required if forage was purchased [ ]. crop production for the market was much less common among commercial operators compared to the amish. the amish were more likely to have produced all the crops examined. this finding, as well as the lower percentage of income from horses, again indicates the diversified nature of amish operations. commercial equine operations seldom apply manure or manure products to their crops; a maximum of % indicated that they sometimes applied composted manure to crops, which was significantly less than the amish (table ). the survey did not ask about pasture application specifically. the most common application method for both groups was top-dressed application, which indicates that runoff of nutrients may be a problem [ ]. table . summary statistics: manure application on cropland (n = ). variables mean comparison range commercial amish difference a manure application frequency on cropland manure always – . . − . *** sometimes – . . − . *** never – . . * mature manure always – . . . sometimes – . . − . *** never – . . − . compost always – . . . sometimes – . . − . ** never – . . − . application method top-dressed – . . − . *** tilled into soil – . . − . *** other – a difference of means; *, %; **, %; ***, . %. there were distinct differences in manure management practices between the groups (table ). commercial operations were significantly more likely to pile manure and compost than the amish, while the amish were more likely to spread the manure fresh over their fields, which implies that it may not be applied in the location and at the time when it is most needed. westendorf et al. [ ] indicate that horse manure storage has substantial environmental impacts. respondents were asked about their use of equipment and machinery for manure management. on the commercial side, the most frequently used equipment for manure management included tractors with buckets and power take-off (pto)-drive manure spreaders, which can be used to pile and/or turn manure. as expected, none of the commercial operations used horse-drawn equipment, while % of the amish did. commercial operations were also less likely to use ground-drive manure spreaders. a reasonable inference from the data is that many amish producers use horse-drawn ground-drive manure spreaders for their manure management, but do not have equipment that would facilitate piling and turning manure piles. given that the amish do not use tractors themselves, the comparison of means for that equipment is not indicated. sustainability , , of table . summary statistics: manure management practices (n = ). variables mean comparison range commercial amish difference a manure management practices on-farm manure management practices piled – . . . *** spread – . . − . *** piled then spread later – . . . # composted – . . . *** machinery and labor for manure management machinery used for manure management no machinery or equipment – . . . horse-drawn equipment – . − . *** tractor with bucket – . b track loader or bobcat – . b manure spreader with ground drive – . . − . *** manure spreader with pto drive – . b other – . . . ** number of people responsible for manure management primary operators – . . . horse boarders – . . * hired laborers – . . . *** unpaid volunteers or family members – . . − . hours per week spent on manure management (total) – – . . − . *** – – . . − . * – – . . . ** + – . . . *** a difference of means; superscript # indicates % level of significance; *, %; **, %; ***, . %. b comparison of means is inappropriate since the amish do not use tractors. manure management can be a time-consuming and unpleasant task. respondents were asked how many people were responsible for manure management each week, as well as about the roles of those people. the number of people involved did not take account of how much time they spent on manure management, i.e., it is not a full-time equivalent measure. commercial operations were more likely than the amish to indicate that manure management was done by horse boarders and hired laborers. respondents were also asked about how much time each week was spent on manure management by all those involved. the most common category for commercial operators was over h, while the most common category for the amish was – h, and these differences were highly significant. sources of information on manure management also differed between groups (table ). the most common source indicated by commercial equine operations was the internet, while no amish indicated that that was a source for them. commercial operations were also more likely than the amish to use other horse owners, government agencies, university extension services, and magazines, while the amish were more likely to use other farmers as well as family and community members. these results imply that educational efforts may need to be designed differently for the two groups; programming could be online for commercial operators and via mailed documents or local in-person meetings for the amish. an encouraging finding is that the majorities of both groups would like to receive more information about manure management practices, and this is significantly higher for commercial operation. sustainability , , of table . summary statistics: manure management knowledge and attitudes. variables mean comparison range commercial amish difference a information sources about manure management previous employment – . . . other horse owners – . . . # other farmers – . . − . # government agencies – . . ** university extension services – . . . * internet – . . *** magazines or newspapers – . . . # family, neighbors, community members – . . − . *** other – . . . want more information on improved manure practices yes – . . . ** no – . . − . motivation for composting manure protecting soil health – . . . * protecting water quality – . . . *** income opportunities – . . . laboratory tested for chemical composition never tested – . . . *** tested manure – . . tested compost – . . tested soil – . . − . *** sold, bartered, or traded manure or manure products yes – . . . *** no – . . − . *** if you haven’t sold manure or compost, why not? use it for agriculture – . . − . *** didn’t know it is an option – . . − . too much time to compost – . . . too much time to find buyer – . . . ** don’t know how to compost – . . . too busy with other activities – . . . no place to compost – . . − . no equipment to compost – . . − . other – . . . ** manure management practices impact water quality strongly agree – . . . *** agree – . . neither agree nor disagree – . . disagree – . − . strongly disagree – . − . # don’t know (need more information) – . . − . *** a difference of means; superscript # indicates % level of significance; *, %; **, %; ***, . %. for those that do compost manure, protecting soil health was the most important reason indicated by both commercial operations and the amish, but was significantly higher for the commercial operations. this group was also more likely to indicate that protecting water quality was a reason for composting. commercial operations were more likely to have never tested manure, compost, or soil for nutrient content. the amish were more likely to have tested their soil for nutrients in the sustainability , , of past, indicating that they are aware of crop nutrient needs (in missouri, standard soil tests include ph, organic matter, phosphorous, potassium, calcium, and magnesium, but do not include nitrogen). testing rates for manure and compost were very low for both groups, similarly to other research findings that indicate that soil is more likely to be tested than manure, even though testing of manure enables application in line with crop requirements (e.g., [ ]). a major research question was whether these groups sold or bartered manure products, as well as to identify the barriers for those who did not in order to determine the potential for value-added manure products. commercial equine operators were more likely to market manure products than the amish. among those who had not sold manure or compost, the most common reason for the commercial group was that they use it for agriculture or that they are too busy with other activities, followed by a lack of equipment for composting. these were also the most important reasons for the amish; however, the amish were significantly more likely to indicate that they use it for agriculture. one reason that was more important for commercial operators was that it took too much time to find buyers. about % of both groups had not thought of selling manure or compost as an option; a similar percentage indicated that they did not know how to compost, and there were no significant differences between the means. given that commercial operators have a higher likelihood of piling manure and of possessing the necessary equipment, targeting educational efforts regarding value-added manure products towards this group may be appropriate. one question asked the level of agreement with the statement “manure management practices impact water quality.” the majority of the commercial respondents indicated that they agreed or strongly agreed, significantly more than the amish respondents. this is in line with some previous research on the amish [ , ]. commercial respondents were less likely to indicate that they did not know whether there were impacts. . . regression results for determinants of manure sales/transfers: commercial and amish as expected, commercial horse operations were significantly more likely to have sold manure or compost compared to amish farmers, partly due to the latter ’s use of horse manure in crop production, as discussed earlier (table ). men were less likely to have sold manure or compost than women. if manure was composted, rather than being spread immediately over the fields, it was more likely that the operator sold manure or compost. as indicated above, this is potentially a more valuable product that is easier to transport. for the variables relating to the impact of manure management on water quality, those who were neutral or said they did not know were more likely to have sold manure than those who agreed or strongly agreed. the implication is not that we should reduce understanding of the impact of manure on water quality to increase manure sales, but rather that those who sell manure may know less about manure in general. the results were robust to alternative specifications of the model. robustness checks with age, animal units rather than the log of that measure, the presence of a tractor with bucket, the number of people responsible for manure management, and soil testing alone did not change the variables that were significant in the model. . . regression results for determinants of manure sales/transfers: commercial group in order to examine the effect of equipment in greater depth, a separate logistic regression was analyzed for only commercial operations (table ). the same bivariate regression response variable (sales/transfers of manure products) and base (no sales of manure products) were used as in the combined amish and commercial group regression (a regression attempting to use the same or similar variables for the amish group was attempted, but was unable to converge due to the lack of variation in the survey responses). no correlation coefficients exceeded | . |. the significant variables in the combined regression were also significant for only commercial operators. the model fit statistics were slightly better. among the commercial respondents, male managers were more likely to sell manure or composted manure products than female managers. the number of horses at the facility was not a predictor, as the natural log on animal units was not a significant variable, nor were having marketed sustainability , , of crops or only having draft horses. composting manure had a positive and significant relationship with selling manure products relative to those who spread manure immediately on fields. table . results of logistic regression for the sale/transfer of manure or compost by all respondents (n = ). independent variables coefficients a se b farm and farmer characteristics commercial (base = amish) . * . male (base = female) − . # . ln animal units . . marketed crops in (base = no) − . . manure management manure is piled (base = spread immediately) . . piled then spread over fields . . manure is composted . * . more than h per week spent on manure management (base < ) − . . tested soil, manure, or compost for nutrient content (base = no) − . . manure management practices impact water quality disagree/strongly disagree (base = agree/strongly agree) − . . neither agree/disagree . * . do not know . * . intercept − . *** . goodness of fit aic . nagelkerke r . lr chi . a note: superscript # indicates % level of significance; *, %; **, %; ***, . %. b standard error. table . results of logistic regression for sale of manure or compost by commercial facilities (n = ). independent variables coefficients a se b farm and farmer characteristics male (base = female) − . * . ln animal units . . marketed crops in (base = no) − . . only draft horse operation − . . manure management manure is piled (base, spread immediately) . . piled then spread over fields . . manure is composted . * . only machinery to pile, turn and load (base, other machinery) − . ** . no machinery is used . . more than h per week spent on manure management (base < ) − . . only hired laborers are responsible for manure management . . tested soil, manure, or compost for nutrient content (base = no) − . . manure management practices impact water quality disagree/strongly disagree (base = agree/strongly agree) − . . neither agree/disagree . ** . do not know . ** . intercept − . . goodness of fit aic . nagelkerke r . lr chi . a note: superscript # indicates % level of significance; *, %; **, %; ***, . %. b standard error. sustainability , , of for this regression, categorical variables related to machinery were created as indicated in the methods section. those with only access to equipment that would pile, turn, and load manure (versus having other equipment related to spreading) had a significant negative relationship with selling compost. this seems counter-intuitive to the expectation that the presence of equipment needed for composting would have a positive relationship with selling, as composting (versus spreading immediately) has a positive relationship. one possible explanation that could be explored in future research is that facilities sell manure or compost (letting the buyers load/transport) because they are ill-equipped to handle it in any other way. all those who sold manure products indicated that the buyers hauled it, while % also indicated separate haulers, and only % also indicated horse owners (data not shown). if this is because the sellers lack the necessary equipment, a third-party manure recycler would be of even greater value to commercial equine managers. variables testing the intensity of manure management, i.e., time spent on manure management and whether employees were the only people responsible for manure management, did not have a significant relationship to manure marketing activities, nor did nutrient testing. the same question regarding whether manure management has impacts on water quality was included in the commercial-only regression. the base was “agree”/“strongly agree”. there were no commercial responses for “disagree”/“strongly disagree”, so that variable was not included in the regression. those who responded “neither agree nor disagree” and “i do not know” were significantly more likely to have sold manure or compost compared to those who indicated that they agreed that manure impacts water quality. . conclusions and implications sustainable utilization of manure, including horse manure, depends on recycling valuable components while reducing negative impacts on the environment [ ]. moving towards this goal requires an understanding of the motivations and behavior of those managing the manure in order to design tailored educational efforts. this study contributed to a better understanding of manure management by two distinct groups of horse owners, which can enable educators to tailor their efforts. from the examination of the summary statistics, commercial equine operations in missouri differ fundamentally from amish farmers who use horses for traction and transportation. they differ in the number of horses, uses of manure, manure storage, equipment for manure management, information sources, environmental attitudes, and the likelihood of selling manure or compost. there are also some similarities, such as low levels of nutrient testing of manure and compost and a desire for more information on best management practices for manure. both of these groups are underserved by the typical channels used to reach other livestock managers. these results imply that effective educational efforts to reduce negative environmental impacts of horse manure and capture soil amendment value will take into account these differences. there is evidence that commercial managers are less likely than the amish to view manure as a valuable nutrient resource, but through targeted education efforts, they could be encouraged to view it as a value-added product. providing education about proper composting techniques could improve their current manure management practices and increase the likelihood that they engage in marketing activities. another implication is that reducing transaction costs in finding buyers for their manure, such as the creation of local internet-based information exchanges on manure product availability, could facilitate manure markets. markets are likely to be localized due to the volume and weight of manure relative to the nutrient content. there may be an opportunity for intermediaries to collect or purchase manure, compost it, and then retail the value-added product. because the major source of manure management information for this group is the internet, educational programs need to focus on that medium. since they realize that manure management affects water quality, these programs should also explain the environmental benefits of improved management practices. implications from the research for educational programs oriented towards the amish differ from those for commercial equine operations. extension personnel could work with amish producers sustainability , , of within their cultural context to develop methods of utilizing manure nutrients most efficiently on farms. for example, due to the de-emphasis on formal education and because english is typically a second language for the amish (it is the language of instruction in their schools), extension materials should target resources to the eighth grade reading level. it is also helpful to work with local leaders within the community to help distribute written material and/or to organize workshops. complementary research to this study could evaluate the demand for horse manure in order to provide information to producers on the desired attributes of manure products and realistic estimates of prices that consumers would be willing to pay. detailed examination of composting technologies for smaller farms in the usa’s midwest would be useful in determining the feasibility under alternative circumstances. in addition, equine operators in missouri may differ from those in other regions, which means that studies in other areas are needed. old-order amish are also diverse and locally organized, so the results may not transfer to other groups or other regions [ , ]. author contributions: conceptualization, j.d. and l.m.; data curation, j.d.; formal analysis, j.d.; funding acquisition, j.d. and l.m.; methodology, c.m.; project administration, l.m.; resources, c.b.; supervision, l.m.; writing—original draft, j.d. and l.m.; writing—review & editing, j.d., l.m. and c.b. all authors have read and agreed to the published version of the manuscript. funding: we acknowledge funding from the missouri agricultural experiment station and the dudley and virgie alexander scholarship fund, college of agriculture, food and natural resources, university of missouri. conflicts of interest: the authors declare no conflict of interest. references . he, z.; pagliari, p.; waldrip, h.m. advances and outlook of manure production and management. in animal manure: production, characteristics, environmental concerns and management; waldrip, h.m., pagliari, 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ postgraduate medical journal (august ) , - . session iii chairman: professor j. h. middlemiss cartilage hair hypoplasia d. c. siggers m.b., b.s., m.r.c.p. b. morris f.r.c.p. j. m. tanner m.d., ph.d., d.sc., f.r.c.p. j. b. burke m.d., f.r.c.p., d.c.h. i. c. s. normand d.m., f.r.c.p. d. a. j. williamson m.d., f.r.c.p., d.c.h. department of child health, university of southampton, east wing, southampton general hospital summary six cases of cartilage hair hypoplasia from five kindreds are described. they demonstrate variation in the expression of clinical features such as sparsity of hair, hair calibre, radiological changes, short stature and the extent of the disproportion between sitting height and stature. cartilage hair hypoplasia is one of a group of metaphyseal chondrodysplasias. it is distinguished from the others by the eponymous qualification- 'mckusick type'. in mckusick published a paper describing seventy-seven cases amongst the amish communities of north america. it is an autosomal recessive condition. mckusick et al. ( ) described the syndrome in the following terms: 'the dwarfism is of the short limbed variety. the hands are short and pudgy, the fingers loose jointed and the finger nails are short, but of nearly normal width. the elbow joints do not fully extend, harrison's grooves can often be seen in the thorax and the head hair is sparse, fine and silky in most persons and its diameter is reduced. histology of the costochondral junction shows a paucity of cartilage cells which fail to be aligned in orderly cell columns at the growth line'. he concluded that failure of cartilage growth was the main fault. amongst the amish community the incidence of correspondence: dr d. c. siggers, department of child health, university of southampton, centre block, south- ampton general hospital, southampton. cartilage hair hypoplasia is about - per thousand persons. outside the amish community this disorder is rare (mckusick, ). since mckusick's de- scription in only one definite case, so far as the authors know, has been diagnosed in britain, that being published by boothby and bower in , and this was a typical case. before that time others may have been reported under different names and since then, of course, further cases may have been diagnosed but not published. patients studied the authors have observed six cases in southern england and these are the basis of their experience, brief details being shown in table . see figs - . radiological features the main abnormalities are the metaphyseal changes, particularly at the knees and ankles (fig. ) and mildly at the hips, which distinguish the dis- order from other members of this group. these changes diminish with advancing age and in a child of - years of age (fig. ) were seen to be relatively minor. there is some discrepancy in bone age between the bones of the hand and the wrist, e.g. in the hands of a child aged years months, the digital bones had an age of years and months, whilst the carpal bones were only years and months (fig. ). one could also note shortened and wide phalangeal metaphyses with other metaphyseal changes. co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://p m j.b m j.co m / p o stg ra d m e d j: first p u b lish e d a s . /p g m j. . . o n a u g u st . d o w n lo a d e d fro m http://pmj.bmj.com/ d. c. siggers et al. table . no. of standard age deviations below the case no. habitat sex (years) mean height i reading f (fig. ) luton m (fig. ) southampton f (fig. ) southampton f - (fig. ) cambridge m - - (fig. ) cambridge f (fig. ) cases and were siblings. the hips show relatively little abnormality. the vertebrae are described as small, more so in the a-p diameter and are said to be columnar. changes are also seen in the upper and lower end plates of the vertebrae, tending to appear in adolescence. the lesions may resemble schmorl's nodes and may also appear in the adult. the vertebrae are an exception to the general rule that the radiological changes diminish with advancing age. the hair mckusick et al. ( ) figured both normal hairs and those of patients with cartilage hair hypoplasia, giving a graph depicting the differences in hair calibre and its variation in his patients. he also, in his amish patients, noted that the hair, although sparse in many, may not show marked deficiency in others. this point was confirmed in the present patients (e.g. figs and ). nevertheless, the hair is fine and microscopically typical. however, on comparing the calibre ranges of the th to rd centiles for normal hair and that from patients with this disorder, some overlap was noted. the absence of any pigmented core in the hair is also stated to typify this condition. but in one of the patients, a boy with the disorder, several hairs were examined and found to have both a normal diameter and a pigmented core (fig. ). his mother, how- ever, stated that she had observed the presence of several unusually dark hairs and provided these as a sample when requested to do so, thinking that they were rare and maybe interesting. thus there may be, in fact, a certain degree of variation within, as also between, individual patients. the demonstration of a relatively wide hair diameter with a pigment core present therefore does not by any means exclude the diagnosis. an amish girl, with darker abundant hair was in fact amongst mckusick's later patients. he stated: 'in most cases, the dwarfed person's head hair is sparse, fine and silky, but the light colour is not such a consistent feature as the others and as the individual ages, the hair becomes darker. since the pigment core is correlated with hair colour, the lack of a pigmented core may be a function of the small hair diameter'. stature mckusick's original patients were stated to vary in height between and cm. one hundred and fifty-six centimetres is not particularly short and height, like hair calibre, is another feature which displays considerable phenotypical variation. this was the height range within the amish community and so there is not likely to be genetic hetero- geneity. it is interesting that in the original seventy-seven amish patients, mckusick found amongst the sibships only % of the expected number of affected sibs. he ruled out several possible explanations, concluding that this discrepancy was one of reduced gene penetrance. he also noted one family with both parents homozygous for cartilage hair hypoplasia whose three children were at first thought to be normal, but in whom characteristic radiological changes were eventually demonstrated despite their normal stature. it is moreover striking to compare this recessive disorder with considerable pheno- typical variation, not supposed to occur in recessives, with achondroplasia which, for a dominant con- dition, has surprisingly little. the heights of the author's six patients have ranged from - to - s.d. below the mean. the body proportions mckusick emphasizes that the dwarfism is short limbed. two girls of the present six patients were charted for sitting height against total height and were at the th centile, as were also the various recorded heights of one boy. the brother and sister from cambridge were at the th centile for a younger age group. so if these two sibs were not of disproportionate stature, as they were at the th centile, did they have cartilage hair hypoplasia? they were reported by savage and tizard ( ) as having a variant of cartilage hair hypoplasia. one might perhaps deduce that each child has equally co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://p m j.b m j.co m / p o stg ra d m e d j: first p u b lish e d a s . /p g m j. . . o n a u g u st . d o w n lo a d e d fro m http://pmj.bmj.com/ cartilage hair hypoplasia ... .... ... ru.n.-ii. mint n m. . ii. -ne, ........ ... ...... n" mll'."!!.,'illilll jim. wxw fig. . case . age - years; height, cm. fig. . case . age - years; height, cm. fig. . case . age years; height, cm. fig. . case . age - years;height, - cm. fig. . case . age - years; height, cm. fig. . case . age - years; height, cm. co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://p m j.b m j.co m / p o stg ra d m e d j: first p u b lish e d a s . /p g m j. . . o n a u g u st . d o w n lo a d e d fro m http://pmj.bmj.com/ d. c. siggers et al. i. j.f~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ fig. . case . radiographs of knees and ankles at age - years showing irregularity, sclerosis and flaring of metaphyses, and mild varus bowing of the tibiae. $ fig. . case . radiographs of hips, knees and ankles at age showing mild metaphyseal changes of lower femur, upper and lower tibia and fibula. ::. fig. . case . radiograph of left hand showing a bone age of years months in the metacarpals and phalanges, and of years months in the carpals. co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://p m j.b m j.co m / p o stg ra d m e d j: first p u b lish e d a s . /p g m j. . . o n a u g u st . d o w n lo a d e d fro m http://pmj.bmj.com/ cartilage hair hypoplasia of r# d {-e $ ;n r ~~~-* i; ,p r ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. . fig. . case . photomicrograph of two hair shafts (diameters- left- [um: right- ,um), to show the range of calibres in a single patient. - c.h. a - . j.l.o~~~~~~~~~~~~~~~ d^ -__ * - l l l i adult age (years) fig. . centile chart showing the sitting height to stature ratio of case (c.h.), case (p.h.) and case (j.l.) (girls) in relation to their ages. - q e aa a amm.j - - adult age (years) fig. . centile chart showing the sitting height to stature ratio of case (m.j.) and case (j.d.) (boys) in relation to their ages. co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://p m j.b m j.co m / p o stg ra d m e d j: first p u b lish e d a s . /p g m j. . . o n a u g u st . d o w n lo a d e d fro m http://pmj.bmj.com/ d. c. siggers et al. th o l) th a | a r ~~~~ah ' - u) age (yeors) fig. . centile chart showing the sitting height to stature ratio of case (a), compared with five boys of similar age with growth hormone-deficiency (a). proportioned dwarfism, both sitting and standing height being below the rd centile. using other charts the sitting height/total height ratios were calculated for these two sibs according to their age (figs and ). it must be remembered that if an older person is proportionately short in both total stature and limbs, the data may be superimposed upon those of a younger child, i.e. a -year-old upon a -year-old. when plotted accord- to age, the data for the girl p.d. (case no. ) were in fact on the th centile for the age-related ratio (fig. ), indicating that she did have disproportion, although not so clearly defined as the other two girls (cases nos and ). similarly, her brother (case ) was found to be at the th centile (fig. ). clearly an age-related body-proportions chart is mandatory. comparing with similar data for growth hormone-deficient children of the same age the boy of the sib pair (case no. ) with cartilage hair hypoplasia was noted to be at the th centile and the growth hormone-deficient children above and below the th, some being close to case no. (fig. ). it appears likely, therefore, that there may be some overlap between some patients with cartilage hair hypoplasia and others with so-called propor- tionate dwarfism. differential diagnosis this is not discussed here in full detail since it is covered by professor spranger ( ) in his paper on metaphyseal chondrodysplasias in this symposium. there is, however, need to stress the variation in the phenotypic expression of cartilage hair hypoplasia. it is also necessary to mention its differentiation from another disorder with several names-one being 'pancreatic deficiency neutropenia syndrome' (burke et al., ). this has some features in common with cartilage hair hypoplasia but never- theless they are clearly different conditions. both are autosomal recessive. no alteration of hair is re- ported in pancreatic deficiency neutropenia syn- drome. the stature is disproportionate in cartilage hair hypoplasia and proportionate in pancreatic deficiency neutropenia. coeliac syndrome, with no deficiency of the pancreatic enzymes, and hir- schprung's disease have been reported in cartilage hair hypoplasia but not in pancreatic deficiency neutropenia. metaphyseal chondrodysplasia and neutropenia occur in either disorder but, in contrast to the pancreatic deficiency neutropenia syndrome, significant radiological changes in the upper femur and humerus are absent in cartilage hair hypoplasia. to conclude, cartilage hair hypoplasia is a recessively inherited condition with considerable phenotypical variation in the hair, height and bodily proportions. should one see a short child in one's clinic, one should not immediately call for an insulin, a bovril or an arginine tolerance test, an intestinal biopsy and so on-one need but ask for radiological examination of a knee, which will suggest the correct diagnosis. references boothby, c.b. & bower, b.d. ( ) cartilage-hair hypo- plasia. archives of disease in childhood, , . burke, v., colebatch, j.h., anderson, c.m. & simons, m.j. ( ) association of pancreatic insufficiency and chronic neutropenia in childhood. archives of disease in childhood, , . mckusick, v.a. ( ) heritable disorders of connective tissue, th edn. c. v. mosby co., st louis. mckusick, v.a., eldridge, r., hostetler, j.a., ruang- wit, u. & egeland, j.a. ( ) dwarfism in the amish. ii. cartilage hair hypoplasia. bulletin of the johns hopkins hospital, , . savage, m.o. & tizard, j.p.m. ( ) metaphyseal dys- plasia in siblings: a variant of cartilage-hair hypoplasia. proceedings of the royal society of medicine, , . spranger, j.w. ( ) metaphyseal chondrodysplasia. postgraduate medical journal, , . co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://p m j.b m j.co m / p o stg ra d m e d j: first p u b lish e d a s . /p g m j. . . o n a u g u st . d o w n lo a d e d fro m http://pmj.bmj.com/ untitled do farm-grown lungs breathe better? jon genuneit, erika von mutius in thorax, campbell et al report an asso- ciation of growing up on a farm with better adult lung function. on closer investigation, this effect was confined to the fev in female participants in the study. a novel finding and a paper worth reading—but what may be harder to glean is its relevance and implication for future research. the ‘farm-effect’ on allergic disease has been well established in numerous studies. recent systematic reviews with meta-analyses show a strong protective effect of growing up on a farm on child- hood atopy and a lesser effect on child- hood asthma. in these meta-analyses, the effect estimates for the ‘farm-effect’ on asthma were much more heterogeneous than those on any atopic sensitisation. this may be driven by the mix of differ- ent farm exposures shown to be import- ant. it has also been suggested that the increased heterogeneity in estimates of the ‘farm-effect’ on asthma is partly driven by the mix of asthma phenotypes in the respective studies. indeed, the present study by campbell et al is one of the few studies presenting results completely stratified by atopy to investigate this further. here, the authors conclude that protective effects on atopic asthma, atopic bronchial hyper- responsiveness (bhr) and atopic nasal symptoms in comparison to non-atopic subjects without asthma, bhr and nasal symptoms, respectively, exist. on closer investigation of the presented data, these associations are fully driven by the ‘farm effect’ on atopy. no separate ‘farm-effect’ on asthma, bhr or nasal symptoms shows up when comparing diseased with non-diseased separately within the two strata of atopic and non-atopic subjects. it has been previously shown that effect esti- mates for atopic asthma depend on the choice of the reference group in case of strong overall effects on atopy. it is remarkable that early life exposure to farming environments has sustained effects on adult atopic sensitisation. however, added effects on asthma, bhr and nasal symptoms as suggested by previ- ous studies in childhood and adulthood are not supported by the present study. the result of a ‘farm-effect’ on lung function has been investigated to a lesser extent, and direct comparison with previ- ous studies is hindered by differing scaling of the lung function parameters. higher fev /fvc values among those growing up on a farm have been found in one pre- vious study among atopic children only but not in two other studies among all children. the association in the present study was attenuated after further adjust- ment, including adjustment for parental smoking, but no stratification for atopy was presented. personal smoking did not seem to confound the association but residual confounding due to crude mea- sures of smoking history cannot be excluded. the stronger association with higher fev in the present study was more apparent in women and varied across par- ticipating centres. such an isolated lung function parameter is hard to interpret in the absence of findings for fvc and the fev /fvc ratio, as fev can represent lung volume and airway obstruction. whether the on-average ml higher fev in women who grew up on a farm can be replicated in other studies, whether this effect persists after accounting for atopy, and whether it is clinically meaning- ful remains, thus, to be elucidated. what may be more interesting in this context is the sex-specificity of the ‘farm- effect’. a stronger effect on women has been previously shown among adults – as well as children and adolescents, although it has been reversed, absent or only marginally statistically significant in the data of other studies. the sex- specificity may point towards ( ) unaccounted-for disease heterogeneity between women and men, ( ) differing exposure to farming environments, including occupational exposure in adult life but also other exposure in early life, ( ) differing confounding lifestyle factors, such as smoking and ( ) differing physio- logical, hormonal or growth character- istics affecting lung physiology. whereas personal smoking, body weight and body height have been adjusted for in the present study, other proposed factors remain as possible explanations. age at onset of asthma may also play a role since one report on childhood and adolescence has shown that sex-specific associations of exposure to farming with asthma are age-dependent. of note, the study population in the present study covers a wide age range. although age is adjusted for, previous publications, including another one also using european community respiratory health survey (ecrhs) data, have indi- cated cohort effects. cohort effects may exist if the mode of farming changed over time, for example, farms were recently only run part-time which attenu- ates the effect. moreover, no infor- mation on age at onset of atopy or atopic asthma is shown. the ‘farm-effect’ docu- mented in the present study may be a sus- tained effect from childhood onwards. alternatively, it may be an effect on later- onset atopy or atopic asthma, as indicated by data from the rhine study which includes part of the ecrhs study popula- tion. arguably, a substantial portion of adult-onset or late-onset disease may be misclassified due to lack of recall of child- hood disease in mid-adult ages. while critics may argue for alternative explanations of the ‘farm-effect’ (eg, gen- etics, access to or utilisation of healthcare), its environmental component seems estab- lished beyond reasonable doubt. one good example is the dramatic increase of atopy in a rural region of poland accompanied by a decrease of exposure to farming during the same time. another prominent example is the exploitation of two popula- tions, the amish and the hutterites. in this comparison, the model of exposure to farming environments is distilled to its environmental core. not all protection from atopy relates to farming in this study. campbell et al also define a ‘biodiversity score’ based on child- hood exposure to cats, dogs, day care, bedroom-sharing and older siblings. one might argue that the aforementioned factors are proxies for other than microbial exposures and that the simple dichotomi- sation and assumed additive effects are not the optimal operationalisation. moreover, upbringing in a village also shows some protection, but it remains unclear whether this effect is to some extend attributable to the ‘biodiversity score’. possibly, the effect of such a score is only discernible in inner- city environments where other outdoor exposures (eg, greenness) are lacking. nonetheless, the analyses show that the protective ‘farm-effect’ is stronger than what individuals brought up in inner cities can experience by exposure to pets, day care and siblings. also, given that these institute of epidemiology and medical biometry, ulm university, ulm, germany; dr. von hauner children’s hospital, ludwig maximilians university munich, munich, germany correspondence to dr jon genuneit, institute of epidemiology and medical biometry, ulm university, helmholtzstr. , ulm d , germany; jon.genuneit@uni-ulm.de genuneit j, von mutius e. thorax march vol no editorial o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th o ra xjn l- - o n d e ce m b e r . d o w n lo a d e d fro m o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th o ra xjn l- - o n d e ce m b e r . d o w n lo a d e d fro m o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th o ra xjn l- - o n d e ce m b e r . d o w n lo a d e d fro m https://www.brit-thoracic.org.uk/ http://thorax.bmj.com/ http://thorax.bmj.com/ http://thorax.bmj.com/ http://thorax.bmj.com/ exposures are probably as difficult to inter- vene on as is the place of upbringing, we should consider keeping on searching for the most important environmental expo- sures underlying the farm-effect for future preventive efforts. so, do farm-grown lungs breathe better? the present study does not add much to answer this question. but in add- ition to all the previous evidence, it is another reminder from a large-scale study that effects of childhood exposure to farming environments on atopy and thereby atopic disease may well extend into adulthood. this makes the mechan- isms underlying the ‘farm-effect’ particu- larly important for future preventive efforts. how shall we proceed? exploring disease heterogeneity and sub-phenotypes including age at onset as well as investigat- ing sex-specificity should be added to the investigative toolbox. in addition, making use of model populations may be promis- ing in future efforts to unravel elements in the mix of exposures or pathophysio- logical mechanisms that are associated with growing up on a farm. contributors jg and evm drafted this manuscript together and both approved the final version. competing interests none declared. provenance and peer review commissioned; externally peer reviewed. to cite genuneit j, von mutius e. thorax ; : – . received september revised november accepted november published online first december ▸ http://dx.doi.org/ . /thoraxjnl- - thorax ; : – . doi: . /thoraxjnl- - references campbell b, raherison c, lodge cj, et al. the effects of growing up on a farm on adult lung function and allergic phenotypes: an international population-based study. thorax ; : – . campbell be, lodge cj, lowe aj, et al. exposure to “farming” and objective markers of atopy: a systematic review and meta-analysis. clin exp allergy ; : – . genuneit j. exposure to farming environments in childhood and asthma and wheeze in rural populations: a systematic review with meta-analysis. pediatr allergy immunol ; : – . ege mj, frei r, bieli c, et al. not all farming environments protect against the development of asthma and wheeze in children. j allergy clin immunol ; : – . pekkanen j, lampi j, genuneit j, et al. analyzing atopic and non-atopic asthma. eur j epidemiol ; : – . fuchs o, genuneit j, latzin p, et al. farming environments and childhood atopy, wheeze, lung function, and exhaled nitric oxide. j allergy clin immunol ; : – .e . schulze a, van strien rt, praml g, et al. characterisation of asthma among adults with and without childhood farm contact. eur respir j ; : – . ernst p, cormier y. relative scarcity of asthma and atopy among rural adolescents raised on a farm. am j respir crit care med ; : – . merchant ja, naleway al, svendsen er, et al. asthma and farm exposures in a cohort of rural iowa children. environ health perspect ; : – . masley ml, semchuk km, senthilselvan a, et al. health and environment of rural families: results of a community canvass survey in the prairie ecosystem study (pecos). j agric saf health ; : – . douwes j, travier n, huang k, et al. lifelong farm exposure may strongly reduce the risk of asthma in adults. allergy ; : – . timm s, frydenberg m, janson c, et al. the urban-rural gradient in asthma: a population-based study in northern europe. int j environ res public health ; : . lawson ja, rennie dc, senthilselvan a, et al. occurrence of new-onset wheeze in a -year follow-up study of schoolchildren. ann allergy asthma immunol ; : – . genuneit j. sex-specific development of asthma differs between farm and nonfarm children: a cohort study. am j respir crit care med ; : – . adler a, tager i, quintero dr. decreased prevalence of asthma among farm-reared children compared with those who are rural but not farm-reared. j allergy clin immunol ; : – . pekkanen j, xu b, järvelin mr. gestational age and occurrence of atopy at age —a prospective birth cohort study in finland. clin exp allergy ; : – . smit lam, zuurbier m, doekes g, et al. hay fever and asthma symptoms in conventional and organic farmers in the netherlands. occup environ med ; : – . leynaert b, neukirch c, jarvis d, et al. does living on a farm during childhood protect against asthma, allergic rhinitis, and atopy in adulthood? am j respir crit care med ; : – . bråbäck l, hjern a, rasmussen f. trends in asthma, allergic rhinitis and eczema among swedish conscripts from farming and non-farming environments. a nationwide study over three decades. clin exp allergy ; : – . braun-fahrländer c, gassner m, grize l, et al. prevalence of hay fever and allergic sensitization in farmer’s children and their peers living in the same rural community. scarpol team. swiss study on childhood allergy and respiratory symptoms with respect to air pollution. clin exp allergy ; : – . von ehrenstein os, von mutius e, illi s, et al. reduced risk of hay fever and asthma among children of farmers. clin exp allergy ; : – . sozańska b, błaszczyk m, pearce n, et al. atopy and allergic respiratory disease in rural poland before and after accession to the european union. j allergy clin immunol ; : – . stein mm, hrusch cl, gozdz j, et al. innate immunity and asthma risk in amish and hutterite farm children. n engl j med ; : – . genuneit j, von mutius e. thorax march vol no editorial o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://th o ra x.b m j.co m / t h o ra x: first p u b lish e d a s . /th o ra xjn l- - o n d e ce m b e r . d o w n lo a d e d fro m http://crossmark.crossref.org/dialog/?doi= . /thoraxjnl- - &domain=pdf&date_stamp= - - http://dx.doi.org/ . /thoraxjnl- - http://dx.doi.org/ . /thoraxjnl- - http://dx.doi.org/ . /cea. http://dx.doi.org/ . /j. - . . .x http://dx.doi.org/ . /j.jaci. . . 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additional conflicts of interest for their article: erika von mutius is listed as inventor on the following patents: ► publication number ep : composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases. ► publication number ep : stable dust extract for allergy protection ► publication number ep : pharmaceutical compound to protect against allergies and inflammatory diseases erika von mutius is listed as inventor and has received royalties on the following patent: ► publication number ep : specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders. © author(s) (or their employer(s)) . no commercial re-use. see rights and permissions. published by bmj. thorax ; : . doi: . /thoraxjnl- - corr prayle ap. thorax september vol no http://crossmark.crossref.org/dialog/?doi= . /thoraxjnl- - &domain=pdf&date_stamp= - - 	do farm-grown lungs breathe better? 	references long term mortality rates in united states veterans with coronary risk factors, with or without significant coronary artery disease stable ischemic heart disease a jacc march , volume , issue s long term mortality rates in united states veterans with coronary risk factors, with or without significant coronary artery disease poster contributions poster hall b saturday, march , , : a.m.- : a.m. session title: risk markers, cad, prognosis abstract category: . stable ischemic heart disease: clinical presentation number: - authors: swarnalatha kanneganti, thein aung, amish patel, ronald markert, ajay agarwal, wright state university, dayton, oh, usa, dayton veterans affairs medical center, dayton, oh, usa background: coronary artery disease (cad) is a major cause of death in united states. mortality rate in us veterans with coronary risk factors, with or without cad are not well known. we did a retrospective study to compare the all-cause mortality rate in patients with obstructive cad, nonobstructive cad and normal coronaries based on angiographic findings. methods: cardiac catheterization data in a veterans affairs hospital was retrospectively collected on patients (oct -jul ). based on angiographic data, patients were divided into groups, obstructive cad, nonobstructive cad and normal coronaries, and all- cause mortality rate was assessed after a mean follow up of . ± . years. results: the clinical characteristics are listed in table . all-cause mortality rates were . % (obstructive cad), . % (non-obstructive cad), and . % in normal coronaries. these rates are higher compared to non-veteran population as shown in reach registry, where the all-cause mortality rate in patients with risk factors only was . % after a mean follow up of years. relatively high mortality rate in this cohort, without significant cad may be due to excess burden of major cardiovascular risk factors as shown in table . conclusion: in this study, high mortality rate was noted in all groups with or without significant cad. the association of risk factor burden with mortality needs further evaluation in this population. table variable obstructive cadn= nonobstructive cad n= normal coronaries n= age (years) . ± . . ± . . ± . bmi . ± . . ± . . ± . hypertension (%) ( . ) ( . ) ( . ) diabetes (%) ( . ) ( . ) ( . ) hyperlipidemia (%) ( . ) ( . ) ( . ) smoking (%) ( . ) ( . ) ( ) ldl (mg/dl) . ± . . ± . . ± . creatinine (mg/dl) . ± . . ± . . ± . ckd (%) ( . ) ( . ) ( . ) lvef (%) ± ± ± lbbb (%) ( . ) ( . ) ( . ) [pdf] a mutation in the serine protease tmprss in a novel pediatric neurodegenerative disorder | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / - - - corpus id: a mutation in the serine protease tmprss in a novel pediatric neurodegenerative disorder @article{lahiry ami, title={a mutation in the serine protease tmprss in a novel pediatric neurodegenerative disorder}, author={p. lahiry and l. racacho and j. wang and john f. robinson and g. gloor and c. a. rupar and v. siu and d. bulman and r. hegele}, journal={orphanet journal of rare diseases}, year={ }, volume={ }, pages={ - } } 	p. lahiry, l. racacho, + authors r. hegele 	published 	biology, medicine 	orphanet journal of rare diseases backgroundto elucidate the genetic basis of a novel neurodegenerative disorder in an old order amish pedigree by combining homozygosity mapping with exome sequencing.methods and resultswe identified four individuals with an autosomal recessive condition affecting the central nervous system (cns). neuroimaging studies identified progressive global cns tissue loss presenting early in life, associated with microcephaly, seizures, and psychomotor retardation; based on this, we named the condition… expand view on springer ojrd.biomedcentral.com save to library create alert cite launch research feed share this paper citationshighly influential citations background citations view all figures, tables, and topics from this paper 	 figure 	 table 	 table 	 figure 	 figure 	 figure 	 figure view all figures & tables 	neurodegenerative disorders 	microcephaly 	global developmental delay 	cns disorder 	seizures 	chromosome q 	whole exome sequencing 	rare diseases 	neuroimaging 	name 	homozygote 	autosomal recessive inheritance 	biopolymer sequencing 	serine proteases 	parkinson disease 	nervous system structure 	tmprss gene citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency george lyman duff memorial lecture 	r. hegele, jacqueline s. dron 	medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 save alert research feed transcriptome sequencing in a -hydroxydopamine rat model of parkinson's disease. 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	e. puffenberger, diane hu-lince, + authors d. stephan 	biology, medicine 	proceedings of the national academy of sciences of the united states of america 	 	 	pdf 	view excerpt, references background save alert research feed an integrated genetic and functional analysis of the role of type ii transmembrane serine proteases (tmprsss) in hearing loss 	m. guipponi, min-yen toh, + authors h. scott 	biology, medicine 	human mutation 	 	 	pdf 	view excerpt, references background save alert research feed truncating neurotrypsin mutation in autosomal recessive nonsyndromic mental retardation 	f. molinari, m. rio, + authors l. colleaux 	biology, medicine 	science 	 	 save alert research feed early onset west syndrome with cerebral hypomyelination and reduced cerebral white matter 	j. tohyama, noriyuki akasaka, h. osaka, y. maegaki, k. ohno 	psychology, medicine 	brain and development 	 	 	view excerpt, references background save alert research feed progressive atrophy of the cerebrum in japanese sisters with microcephaly with simplified gyri and enlarged extraaxial space. 	m. hirose, k. haginoya, + authors s. tsuchiya 	medicine 	neuropediatrics 	 	 	view excerpt, references background save alert research feed genomic overview of serine proteases. 	g. yousef, ari d kopolovic, m. elliott, e. diamandis 	biology, medicine 	biochemical and biophysical research communications 	 	 	pdf save alert research feed prognostic value of tmprss expression in patients with breast cancer 	b. liang, mingzhe wu, yuehua bu, a. zhao, fang xie 	biology, medicine 	medical oncology 	 	 	view excerpt, references background save alert research feed trypsin and trypsin-like proteases in the brain: proteolysis and cellular functions 	y. wang, w. luo, g. reiser 	biology, medicine 	cellular and molecular life sciences 	 	 	view excerpt save alert research feed cdd: a conserved domain database for the functional annotation of proteins 	a. marchler-bauer, s. lu, + authors s. bryant 	biology, medicine 	nucleic acids res. 	 	 , 	pdf 	view excerpt, references background save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue association of patent ductus arteriosus ligation with death or neurodevelopmental impairment among extremely preterm infants copyright american medical association. all rights reserved. association of patent ductus arteriosus ligation with death or neurodevelopmental impairment among extremely preterm infants dany e. weisz, md, msc; lucia mirea, phd; erin rosenberg, bsc; maximus jang, bsc, md; linh ly, md; paige t. church, md; edmond kelly, md; s. joseph kim, md, phd; amish jain, md; patrick j. mcnamara, md, msc; prakesh s. shah, md, msc importance observational studies have associated patent ductus arteriosus (pda) ligation among preterm infants with adverse neonatal outcomes and neurodevelopmental impairment in early childhood, with a resultant secular trend away from surgical treatment. however, to our knowledge, studies have inadequately addressed sources of residual bias, including survival bias and major neonatal morbidities arising before exposure to ligation. objective evaluate the association between pda ligation vs medical management and neonatal and neurodevelopmental outcomes. design, setting, and participants this retrospective cohort study of preterm infants younger than weeks gestational age born between january , , and december , , with clinical and echocardiography diagnoses of hemodynamically significant pda was conducted at tertiary neonatal intensive care units and affiliated follow-up programs. exposure surgical ligation vs medical management. main outcomes and measures the primary outcome was a composite of death or neurodevelopmental impairment (ndi) at to months corrected age. secondary outcomes included death before discharge, ndi, moderate-severe chronic lung disease, and severe retinopathy of prematurity. multivariable logistic regression analysis was used to adjust for perinatal and postnatal confounders. results of infants with hemodynamically significant pda (mean [standard deviation] gestational age . [ . ] weeks and birth weight [ ] grams), ( %) underwent ligation. infants who underwent ligation had a higher frequency of morbidities before pda closure, including sepsis, necrotizing enterocolitis, and a dependence on mechanical ventilation. after adjusting for perinatal characteristics and preligation morbidities, there was no difference in the odds of death or ndi (adjusted odds ratio (aor), . ; % ci, . - . ), ndi (aor, . ; % ci, . - . ), chronic lung disease (aor, . ; % ci, . - . ) or severe retinopathy of prematurity (aor, . ; % ci, . - . ). ligation was associated with lower odds of mortality (aor, . ; % ci, . - . ). conclusions and relevance patent ductus arteriosus ligation among preterm neonates younger than weeks gestational age was not associated with the composite outcome of death or ndi, and there were no differences in chronic lung disease, retinopathy of prematurity, or ndi among survivors. mortality was lower among infants who underwent ligation, though residual survival bias could not be excluded. previously reported associations of ligation with increased morbidity may be because of bias from confounding by indication. jama pediatr. ; ( ): - . doi: . /jamapediatrics. . published online march , . editorial page supplemental content author affiliations: author affiliations are listed at the end of this article. corresponding author: dany e. weisz, md, msc, department of newborn and developmental paediatrics, sunnybrook health sciences centre, bayview ave, toronto, on m n m , canada (dany.weisz@sunnybrook.ca). research jama pediatrics | original investigation (reprinted) copyright american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . mailto:dany.weisz@sunnybrook.ca copyright american medical association. all rights reserved. o ver the past decade, retrospective studies have asso-ciated patent ductus arteriosus (pda) ligation with in-creased neonatal and neurodevelopmental morbid- ity, including chronic lung disease (cld), retinopathy of prematurity (rop), cerebral palsy, and cognitive, hearing, and visual impairments. - these results have been associated with a secular trend toward a reduction in surgical ligation for per- sistent symptomatic pda. , however, ligation has also been associated with lower mortality compared with medical management. while the collective effect of previous studies has raised concern about surgical ligation, significant methodological shortcomings exist. to our knowledge, no contemporary ran- domized clinical trial has examined the effectiveness of pda ligation. importantly, previous observational studies investi- gating the association of pda ligation and outcomes have only adjusted for antenatal and perinatal covariates in multivari- able analyses. while this approach may be sufficient to bal- ance confounders for interventions occurring shortly after birth, ligation often occurs weeks after birth. during the post- natal preligation period, infants may acquire multiple mor- bidities of prematurity (eg, sepsis and a dependence on inva- sive mechanical ventilation) that influence the decision to treat infants with ligation and outcomes. failing to adjust for such confounders results in a high risk of residual bias against in- fants who underwent ligation because of confounding by in- dication and increased preligation illness severity. on the other hand, using ligation as a rescue treatment after the failure or contraindication of medical therapy confers a risk of survival bias in favor of infants who underwent ligation which has not, to our knowledge, been addressed in previous studies. the objective of this study was to evaluate the associa- tion of pda ligation with neonatal and neurodevelopmental outcomes after accounting for antenatal, perinatal, and post- natal confounders. methods we conducted a retrospective cohort study of extremely pre- term infants born at weeks plus days gestational age or younger and treated at tertiary neonatal units in toronto, canada (mount sinai hospital, sunnybrook health sciences centre, and the hospital for sick children) from january , , to december , . infants were included if they had a clinically significant pda and had undergone at least echo- cardiogram demonstrating a hemodynamically significant pda, defined as a ductal diameter of . mm or larger. infants with major congenital anomalies were excluded. this study was ap- proved by the respective research ethics boards from all neo- natal intensive care unit institutions and the university of to- ronto, who also approved a waiver of informed consent. management of pda treatment for pda was at the discretion of the attending neo- natologist and typically occurred for a clinically and echocar- diographically significant pda. medical treatment aimed to- ward fac ilitating ductal closure (with indomethacin or ibuprofen) was used as first-line therapy. pharmacotherapeu- tic treatment could be repeated at the discretion of the attend- ing team. infants with hemodynamically significant pda (hspda) were considered for surgical ligation after the fail- ure of, or contraindication to, medical therapy. the decision to refer an infant for pda ligation was made by the attending neonatologist at each site in collaboration with a neonatologist with expertise in echocardiography. before li- gation, infants at the perinatal centers were transported to the hospital for sick children for surgical ligation. the sur- gery was performed via a left lateral thoracotomy, an intra- or extrapleural approach, and the pda was closed using either a clip or ligature at the discretion of the attending surgeon. post- operative intensive care was supported by targeted neonatal echocardiography including the targeted administration of in- travenous milrinone for infants with critically low cardiac out- put in the immediate postoperative period. outcomes and assessment surviving infants underwent neurodevelopmental assess- ments at to months corrected age, which consisted of a clinical examination, visual and hearing assessment, and cog- nitive evaluation using the bayley scales of infant develop- ment, third edition (bsid iii), and/or the ages and stages ques- tionnaire (asq). clinical examinations and standardized motor assessments identified the presence of cerebral palsy, which was classified according to the gross motor functional clas- sification system. cognition and language abilities were as- sessed predominantly using the bsid iii, with a small num- ber of infants assessed exclusively using the asq. the primary outcome was a composite of death or mod- erate-severe neurodevelopmental impairment (ndi), evalu- ated at to months corrected age. moderate-severe ndi was defined as a composite of neuromotor, neurocognitive, and/or neurosensory impairment (etable in the supple- ment). secondary outcomes included death before dis- charge, moderate-severe ndi, cld (defined as treatment with supplemental oxygen or positive pressure support at weeks key points question is patent ductus arteriosus ligation associated with adverse neonatal outcomes and neurodevelopmental impairment among extremely preterm infants? findings in this cohort study of extremely preterm infants, when postnatal preligation morbidities were properly accounted for, there was no difference in the composite outcome of death or neurodevelopmental impairment among infants who underwent ligation compared with those who were medically treated. while mortality was lower among infants who underwent ligation, there was no difference in neurodevelopmental impairment, chronic lung disease, or severe retinopathy or prematurity. meaning patent ductus arteriosus ligation may reduce mortality and is not associated with neurodevelopmental impairment. previously reported associations of ligation with increased morbidity may be because of bias from confounding by indication rather than a detrimental causal effect of ligation. research original investigation outcomes of patent ductus arteriosus ligation in extremely preterm infants jama pediatrics may volume , number (reprinted) jamapediatrics.com copyright american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://www.jamapediatrics.com/?utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . copyright american medical association. all rights reserved. corrected gestational age) and severe rop (defined as treat- ment with laser surgery or intravitreal vascular endothelial growth factor inhibitor). data sources and collection the hospital records of all eligible infants were reviewed to ab- stract antenatal, neonatal, and outcome data. the echocardi- ography reports of each infant were reviewed to identify the infants with an echocardiographically significant pda, de- fined as at least echocardiogram with pda diameter . mm or larger. the date and hemodynamic significance of the pda in all echocardiograms were recorded from study reports to define the onset and longitudinal course (duration and severity) of each infant’s exposure to a ductal shunt (etable in the supple- ment). the dates of echocardiography and/or clinical pda clo- sure were recorded. for infants who underwent ligation, the date of pda closure was recorded as the date of surgical liga- tion. for medically-treated infants, echocardiography clo- sure was recorded as the earliest date of the echocardiogram demonstrating ductal closure without a subsequent reopen- ing. clinical closure was determined as the earliest date of the disappearance of established clinical signs of pda (murmur, active precordium, and bounding pulses) in an infant in whom these signs were definitively present. the pda of an infant was considered no longer hemodynamically significant if it under- went clinic al or echo closure or was “small” or “small- moderate” in size on echocardiography. data on postnatal morbidities was collected for each day of life for all infants from birth until death or discharge from the neonatal intensive care unit. specifically, the date of on- set of all morbidities was abstracted to include them as time- dependent covariates and characterize the timing of these morbidities in relation to ductal closure. these included in- traventricular hemorrhage and periventricular echogenicity, necrotizing enterocolitis stage or higher, systemic dexameth- asone for the prevention or treatment of cld (defined as a mini- mum -day course), seizures, systemic hypotension treated with inotropes, culture positive sepsis (defined as clinical sep- sis accompanied by the growth of a pathogenic organism from a sterile site), culture negative sepsis (defined as clinical sep- sis without a positive culture that was treated with at least days of systemic antimicrobials), administration of inhaled ni- tric oxide, pneumothorax, and spontaneous intestinal perfo- ration. daily information on respiratory support (invasive posi- tive pressure, noninvasive positive pressure, low flow oxygen, or none) and the average mean airway pressure was ab- stracted. data were abstracted by study investigators at each site who collected the complete data from each infant's neo- natal intensive care unit course before reviewing neurodevel- opmental evaluations. for analyses, postnatal factors were considered time- dependent and were aggregated to represent cumulative ill- ness severity that was estimated for each day of life when in- fants were considered “at risk” for ligation. for infants who underwent ligation, the “at risk” period consisted of the days before surgical ligation. medically-treated infants were con- sidered “at risk” for ligation during the period that the pda he- modynamic significance was at least ”moderate.” this period for medically-treated infants with a persistent pda was trun- cated at days of age, as this was the latest postnatal day of surgery among the group who underwent ligation. statistical analysis preliminary estimates indicated approximately ex- tremely preterm infants were treated with ligation, and more than infants received medical treatment for a clinically and echocardiographically significant pda. assuming % of infants would be lost to follow-up, we estimated that in- fants who underwent ligation would have had the primary out- come assessed. presuming a % event rate among the group who underwent ligation, a -sample, -sided test of propor- tions with % power and % type i error, using infants who underwent ligation and medically treated infants, was estimated to detect an % absolute difference in the primary outcome. study infants were categorized according to whether they underwent surgical ligation (with or without prior pharmaco- therapy) or were not treated with ligation (“medically treated,” composed of cyclooxygenase inhibitor and/or conservative therapy). the distribution of perinatal and postnatal charac- teristics between groups who underwent ligation vs medical treatment was compared using the χ test for categorical vari- ables and the t test or wilcoxon rank-sum test for continu- ous variables. the time to pda closure for infants who under- went ligation vs medically treated infants was compared using the kaplan-meier analysis. the associations between pda ligation and adverse out- comes were estimated using logistic regression analyses. ini- tially, unadjusted analyses estimated crude odds ratios and % confidence intervals (ci) (model ). multivariable logistic re- gression analysis was used to adjust for possible confound- ing. a multivariable model was constructed using only ante- natal and perinatal covariates (model ) to prov ide a comparison with the results of previous studies. the final model (model ) included postnatal covariates representing morbidities that occurred during the period an infant was at risk of ligation. variable selection for the final model was de- termined by backward elimination. subcohort analyses to explore the effect of survival bias (in which medically- treated infants had to survive to be eligible for ligation), subcohort analyses were conducted including only infants who survived with a hspda beyond specific time periods (day of life , , , , and ). the longest periods of exclu- sion ( and days) were selected as they represented the earliest date of death of an infant in the group who under- went ligation (day of life ) and the time period of major early morbidities of prematurity such as sepsis (day of life ). an additional analysis was performed to explore the effect of possible information bias because of using the asq rather than the bsid among a small minority of infants. sta- tistic al analyses were performed using sas version . (sas). all statistical tests were -sided with significance evaluated at the % level. outcomes of patent ductus arteriosus ligation in extremely preterm infants original investigation research jamapediatrics.com (reprinted) jama pediatrics may volume , number copyright american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://www.jamapediatrics.com/?utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . copyright american medical association. all rights reserved. results during the study period, preterm infants born at + ges- tational weeks or younger had a clinical diagnosis of pda. of these, infants were excluded from the study (figure ). a total of preterm infants had a clinical and echocardiography di- agnosis (≥ . mm) of pda, of whom received medical treat- ment only and were treated with surgical ligation. the pri- mary outcome of death or ndi at to months corrected age was known for infants ( . %). compared with the infants who survived to discharge with known neurodevelopmental out- comes (n= ), the surviving infants who were lost to follow- up (n = ) were more likely to have been singleton and less likely to have been treated at sunnybrook health sciences centre. how- ever, there were no differences in other perinatal characteristics, postnatal morbidities, or neonatal outcomes of cld and severe rop (etable in the supplement). patient characteristics the antenatal and perinatal characteristics of medically and surgically treated infants are presented in table . infants treated with ligation had lower gestational age and birth weight and were more likely to be female, require intensive delivery room resuscitation, and receive indomethacin prophylaxis. in- fants who underwent ligation had a longer exposure to an hspda, with most medically-treated infants experiencing duc- tal closure prior to the timing of ligation in surgically treated infants (efigure in the supplement). infants who underwent ligation had higher rates of mor- bidity during the postnatal period of ductal patency com- pared with medically treated infants. (table ) the burden of invasive mechanical ventilation was higher among infants who underwent ligation, who required a significantly higher aver- age daily mean airway pressure ( . cm h o [± . ] vs . cm h o [± . ]; % ci, . - . ), mean difference ( . cm h o; % ci, . - . ), and more days of invasive mechanical ventilation table . antenatal and perinatal characteristics of the cohort of extremely preterm infants with clinically and echocardiographically significant patent ductus arteriosus characteristic no. (%) difference ( % ci) p valuea surgical ligation (n = ) medical treatment only (n = ) ga, mean (sd), wk . ( . ) . ( . ) − . (− . to − . ) <. bw, mean (sd), g ( ) ( ) − (− to − ) <. sga, < th percentile ( . ) ( . ) . % (− . to . ) . initial level hospital na . sb ( . ) ( . ) msh ( . ) ( . ) hsc ( . ) ( . ) multiple gestation ( . ) ( . ) − . % (− . to . ) . male ( . ) ( . ) − . % (− . to − . ) . antenatal corticosteroids, full ( . ) ( . ) − . % (− . to . ) . vaginal delivery ( . ) ( . ) . % (− . to . ) . not born at institution ( . ) ( . ) . % (− . to . ) . intensive delivery room resuscitationb ( . ) ( . ) . % ( . - . ) . -min apgar < ( . ) ( . ) . % (− . to . ) . indomethacin prophylaxis ( . ) ( . ) . % ( . - . ) . snapii score ≥ ( . ) ( . ) . % (− . to . ) . abbreviations: bw, birth weight; ga, gestational age; hsc, hospital for sick children; msh, mount sinai hospital; na, not applicable; sb, sunnybrook health sciences centre; sga, small for gestational age; snapii, score for neonatal acute physiology ii. a t-test or wilcoxon test for continuous data, or χ test for categorical data. b defined as intubation, chest compressions, or epinephrine administration. figure . flow diagram of infants included in the study infants (ga ≤ + ) with diagnosis of pda clinical and echo diagnosis of significant pda medical treatment only infants excluded no echo performed pda < . mm on all echo congenital anomaly incomplete data surgical ligation conservative management nsaid treatment follow-up complete follow-up complete nsaid treatment and ligation primary ligation follow-up complete follow-up complete ga indicates gestational age; nsa, nonsteroidal anti-inflammatory drug; and pda, patent ductus arteriosus. research original investigation outcomes of patent ductus arteriosus ligation in extremely preterm infants jama pediatrics may volume , number (reprinted) jamapediatrics.com copyright american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://www.jamapediatrics.com/?utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . copyright american medical association. all rights reserved. ( . [ ± . ] vs . [± . ]; % ci, . - . ), mean differ- ence ( . days; % ci, . - . ) in the postnatal period be- fore ductal closure (figure ). main outcomes on univariable analysis (model ), ligation was associated with lower mortality but increased cld, rop and ndi, and there was no difference in the composite outcome of death or ndi (table ). after adjusting for antenatal and perinatal confounders only (model ), the associations between ligation and ndi, cld and rop were attenuated but remained significant (table ). liga- tion remained associated with lower mortality and there was no difference in the composite outcome of death or ndi. however, after further adjustment for postnatal, preductal closure confounders (model ), the associations between surgi- cal ligation and ndi (adjusted odds ratio [aor] . ; % ci, . - . ), cld (aor . ; % ci, . - . ), and rop (aor . ; % ci, . - . ) were no longer significant (table ). ligation re- mained associated with lower mortality (aor . ; % ci, . - . ). and there was no significant association with the compos- ite outcome of death or ndi (aor . ; % ci, . - . ). the causes and timing of death (n = in the ligation group; n = in the medically-treated group) are reported in etable in the supplement. among those who died, medically-treated infants had higher rates of sepsis and early respiratory failure, and mortality occurred earlier in the postnatal period. subcohort analyses in the subcohort analyses (to address survival bias) that in- cluded infants who survived beyond a specific age, ligation was associated with lower mortality among all subcohorts and re- duced odds of the composite outcome of death/ndi in the figure . average daily mean airway pressure over the first days of life . . m ea n a ir w ay p re ss ur e, c m h o postnatal age, d . . . . . . medianq date of ligation q ligation no ligation average daily mean airway pressure (cm h o) with % and % cis over the first days of life for medically-treated infants (black line) vs infants who underwent ligation (orange line) before ductal closure. infants no longer contributed data after the date of ductal closure, leading to the widening of cis over time as the number of infants with persistent hemodynamically significant patent ductus arteriosus diminished with time. the median date of ligation was day of life , with the interquartile range (day of life to day of life ) (solid gray box). the earliest date of ligation was on day of life . table . morbidity arising during the nicu course, period of ductal patency (before surgical ligation or medical closure), and at-risk period for surgical ligation among the infant cohort (n = ) morbidity no. (%) p valueasurgical ligation (n = ) medical treatment (n = ) incidence of morbidity before discharge ( ) incidence of morbidity before ligation, ( ) incidence of morbidity before discharge ( ) incidence of morbidity before medical pda closure ( ) incidence of morbidity before medical closure or dol ( ) ( ) vs ( ) ( ) vs ( ) ( ) vs ( ) culture positive sepsis ( . ) ( . ) ( . ) ( . ) ( . ) <. <. <. culture negative sepsis ( . ) ( . ) ( . ) ( . ) ( . ) . . . grade / ivh ( . ) ( . ) ( . ) ( . ) ( . ) . . . hypotension inotropes ( . ) ( . ) ( . ) ( . ) ( . ) . . . nec, stage ≥ ( . ) ( . ) ( . ) ( . ) ( . ) . . . pneumothoraxb ( . ) ( . ) ( . ) ( . ) ( . ) . . . nitric oxide ( . ) ( . ) ( . ) ( . ) ( . ) . . . seizure ( . ) ( . ) ( . ) ( . ) ( . ) . . . sip ( . ) ( . ) ( . ) ( . ) ( . ) . . . dexamethasonec ( . ) ( . ) ( . ) ( . ) ( . ) <. . . abbreviations: dol, day of life; ivh, intraventricular hemorrhage; nec, necrotizing enterocolitis; nicu, neonatal intensive care unit; pda, patent ductus arteriosus; sip, spontaneous intestinal perforation. a χ test or fisher exact test. b pneumothorax requiring thoracostomy tube insertion. c systemic dexamethasone administered for the prevention or treatment of chronic lung disease, minimum d course. outcomes of patent ductus arteriosus ligation in extremely preterm infants original investigation research jamapediatrics.com (reprinted) jama pediatrics may volume , number copyright american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://www.jamapediatrics.com/?utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . copyright american medical association. all rights reserved. subcohorts of infants with persistent hspda beyond the th day of life (etable in the supplement). the association of pda ligation and death/ndi (aor . ; % ci, . - . ) or ndi alone (aor . ; % ci, . - . ) in the subcohort of infants evaluated at neurodevelopmental follow-up using the bsid only (n = , underwent liga- tion) was similar to results from the primary analysis (which included infants evaluated using the bsid or asq). discussion in this retrospective cohort study of extremely preterm infants with hspda, surgical ligation was not associated with higher odds of the composite outcome of death or ndi, compared with medi- cal management alone. ligation was also not associated with ndi, cld, or severe rop; however, pda surgery was associated with reduced odds of mortality. these findings contrast with multiple large cohort studies over the past decade that have strongly as- sociated surgical ligation with ndi, cld, and rop. - , , methodological differences likely account for the diver- gence in results. previous studies did not adjust for postna- tal, preligation confounders, which represent increased ill- ness severity. we observed similar findings as previous studies when we only included antenatal and perinatal covariates (model ) which changed with the inclusion of postnatal co- variates measured before ductal closure (model ). the at- tenuation of the associations with the inclusion of the post- natal covariates suggests that bias because of confounding by indication (as sicker infants are more likely to be referred for ligation), rather than a detrimental causal effect of the sur- gery itself, may explain the previously reported associations of ligation and adverse outcomes. local clinical practices may also have contributed to the absence of an association between ligation and adverse out- comes. targeted neonatal echocardiography was used to guide management aimed at ductal closure, including the use of a delayed, selective approach in referring infants for surgical li- gation, a clinical practice which has been associated with im- proved neonatal and neurodevelopmental outcomes. , in this study, mortality was significantly lower for infants who underwent ligation compared with medically-treated in- fants, which corroborates previous studies. , although this finding is compelling, the association between ligation and lower mortality is, like prior studies, possibly influenced by survival bias or confounding by contraindication (in which in- fants with the highest illness severity are considered unsuit- able for surgery and do not survive). for example, medically- treated infants were more likely to have a cerebral injury (etable in the supplement), from which several died early in life af- ter the withdrawal of life-sustaining medical therapy. in ad- dition, medically-treated infants were more likely to die from sepsis, suggesting that increased illness severity may have ren- dered them too unstable for pda surgery. to reduce survival bias, we conducted subcohort analyses excluding infants who died or experienced pda closure within the first weeks of life, though infants who underwent ligation continued to have significantly lower mortality compared with medically-treated infants. however, these findings should be interpreted with caution because of the possibility of residual survival bias. for example, it is unknown for an infant with a large pda who died of sepsis on the th day of life whether prior pda ligation may have reduced the mortality risk. the poten- tial for residual survival bias means that the association of li- gation with outcomes that include mortality requires further evaluation before clinicians can be confident that ligation im- proves survival. ultimately, the effect of survival bias and an es- timate of any mortality benefit of ligation may only be reliably evaluated in a randomized clinical trial. the strengths of this study include the in-depth abstrac- tion of postnatal morbidities and indices of illness severity, such as daily respiratory support, in a large cohort of extremely pre- term infants. this data collection included key covariates in the multivariable analyses to minimize confounding by indi- table . neonatal and neurodevelopmental outcomes of infants who underwent ligation vs medically treated infantsa outcome no. (%) model : crude or ( % ci) model : aor ( % ci)b antenatal/perinatal covariates only model : aor ( % ci)c model and postnatal, preductal closure covariates ligation (n = ) medical treatment (n = ) death or moderate-severe neurodevelopmental impairmentd ( . ) ( . ) . ( . - . ) . ( . - . ) . ( . - . ) death before dischargee ( . ) ( . ) . ( . - . ) . ( . - . ) . ( . - . ) moderate-severe neurodevelopmental impairment ( . ) ( . ) . ( . - . ) . ( . - . ) . ( . - . ) chronic lung disease ( . ) ( . ) . ( . - . ) . ( . - . ) . ( . - . ) severe retinopathy of prematurity ( . ) ( . ) . ( . - . ) . ( . - . ) . ( . - . ) abbreviations: aor, adjusted odds ratio; ga, gestational age; nec, necrotizing enterocolitis; nicu, neonatal intensive care unit; or, odds ratio; sga, small for gestational age; snap, score for neonatal acute physiology. a reference is medically treated infants. b adjusted for ga, sga, antenatal corticosteroids, sex, multiple gestation, a snap score of or higher, and treatment center. c adjusted for covariates in model plus the following morbidities if they occurred before pda closure: culture positive sepsis, severe intraventricular hemorrhage, inotrope use, nec stage or greater, average daily mean airway pressure, proportion of days of invasive mechanical ventilation, total dose of indomethacin, and systemic dexamethasone. d primary composite outcome includes death before to mo neurodevelopmental follow-up. e death before discharge from the nicu. research original investigation outcomes of patent ductus arteriosus ligation in extremely preterm infants jama pediatrics may volume , number (reprinted) jamapediatrics.com copyright american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://www.jamapediatrics.com/?utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . copyright american medical association. all rights reserved. cation. in addition, the comprehensive collection of all echo- cardiography data permitted identification of the “at-risk” pe- riod for ligation for each infant based on an objective estimate rather than clinician preference or perception. finally, the pro- portion of infants lost to neurodevelopmental follow-up was low ( . %) and these infants had similar characteristics as those for whom follow-up was complete, increasing the con- fidence in our estimates (etable in the supplement). limitations this study is limited by possible ascertainment bias, as medi- cally-treated infants were considered eligible for ligation until the date of confirmed pda closure, which may have been de- layed relative to the true date of ductal closure. this may have led to overestimation of the “at-risk of ligation” period for some medically-treated infants and introduced bias in favor of the medically-treated group, as all morbidities that occurred be- fore this date were adjusted for in the multivariable analyses. in addition, a small minority of infants were evaluated using the asq rather than the bsid for the cognition and language as- sessments. however, results from a subcohort analysis includ- ing only infants evaluated using the bsid were similar to those from the full cohort, suggesting minimal ascertainment bias ow- ing to variable neurodevelopmental testing methods. finally, there remains the potential for confounding by contraindica- tion, residual survival bias, and residual confounding because of unmeasured covariates in this analysis. conclusions compared with medical management, surgical ligation of hspda is associated with lower mortality without increased cld, rop, ndi, or the composite of death or ndi. the previously reported associations of ligation with adverse neonatal outcomes and neu- rodevelopmental impairment may be because of confounding by indication rather than a detrimental causal effect of pda sur- gery. however, well-designed randomized clinical trials are needed to evaluate the relative effects of medical and surgical treatment of hspda on neonatal and early childhood outcomes. article information accepted for publication: december , . published online: march , . doi: . /jamapediatrics. . author affiliations: department of newborn and developmental pediatrics, sunnybrook health sciences centre, toronto, canada (weisz, church); department of pediatrics, university of toronto, toronto, canada (weisz, ly, church, kelly, jain, mcnamara, shah); phoenix children's hospital, phoenix, arizona (mirea); department of pediatrics, hospital for sick children, toronto, canada (rosenberg, ly, mcnamara); department of pediatrics, mt. sinai hospital, toronto, canada (jang, kelly, jain, shah); department of medicine, university of toronto, toronto, canada (kim); department of physiology, university of toronto and physiology and experimental medicine, sickkids research institute, toronto, canada (mcnamara); institute of health policy, management and evaluation, university of toronto, toronto, canada (shah). author contributions: dr weisz had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. concept and design: weisz, mirea, ly, kim, mcnamara, jain, shah, church. acquisition, analysis, or interpretation of data: all authors. drafting of the manuscript: weisz, mirea, shah. critical revision of the manuscript for important intellectual content: all authors. statistical analysis: weisz, mirea. administrative, technical, or material support: weisz, shah. supervision: mirea, ly, kim, jain, mcnamara, shah. conflict of interest disclosures: none reported. additional contributions: the authors thank dr william benitz, md, stanford university, for his review of the study results and insightful comments. dr benitz was not compensated for his contributions. references . kabra ns, schmidt b, roberts rs, doyle lw, papile l, fanaroff a; trial of indomethacin prophylaxis in preterms investigators. neurosensory impairment after surgical closure of patent ductus arteriosus in extremely low birth weight infants: results from the trial of indomethacin prophylaxis in preterms. j pediatr. ; ( ): - , .e . . madan jc, kendrick d, hagadorn ji, frantz id iii; national institute of child health and human development neonatal research network. patent ductus arteriosus therapy: impact on neonatal and -month outcome. pediatrics. ; ( ): - . . mirea l, sankaran k, seshia m, et al; canadian neonatal network. treatment of patent ductus arteriosus and neonatal mortality/morbidities: adjustment for treatment selection bias. j pediatr. ; ( ): - .e . . chorne n, leonard c, piecuch r, clyman ri. patent ductus arteriosus and its treatment as risk factors for neonatal and neurodevelopmental morbidity. pediatrics. ; ( ): - . . bourgoin l, cipierre c, hauet q, et al. neurodevelopmental outcome at years of age according to patent ductus arteriosus management in very preterm infants. neonatology. ; 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( ): - .e . . palisano r, rosenbaum p, walter s, russell d, wood e, galuppi b. development and reliability of a system to classify gross motor function in children with cerebral palsy. dev med child neurol. ; ( ): - . . clyman ri. surgical ligation of the patent ductus arteriosus: treatment or morbidity? j pediatr. ; ( ): - . . wickremasinghe ac, rogers ee, piecuch re, et al. neurodevelopmental outcomes following two different treatment approaches (early ligation and selective ligation) for patent ductus arteriosus. j pediatr. ; ( ): - . . jhaveri n, moon-grady a, clyman ri. early surgical ligation versus a conservative approach for management of patent ductus arteriosus that fails to close after indomethacin treatment. j pediatr. ; ( ): - , .e . . kristman v, manno m, côté p. loss to follow-up in cohort studies: how much is too much? eur j epidemiol. ; ( ): - . outcomes of patent ductus arteriosus ligation in extremely preterm infants original investigation research jamapediatrics.com (reprinted) jama pediatrics may volume , number copyright american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . http://jama.jamanetwork.com/article.aspx?doi= . /jamapediatrics. . &utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ https://www.ncbi.nlm.nih.gov/pubmed/ http://www.jamapediatrics.com/?utm_campaign=articlepdf% utm_medium=articlepdflink% utm_source=articlepdf% utm_content=jamapediatrics. . revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . dogmática jurídica e controvérsias religiosas à luz de robert cover: a recusa de transfusão sanguínea por parte das testemunhas de jeová legal dogmatics and religious controversies in the light of robert cover: the blood transfusion refusal by jehovah’s witness mauricio pedroso flores josé rodrigo rodriguez resumo: o presente artigo se propõe a demonstrar como controvérsias judiciais envolvendo a recusa de transfusão sanguínea por parte das testemunhas de jeová podem ser compreendidas sob uma nova perspectiva se reconhecermos que a narrativa religiosa do grupo também comporta uma afirmação de direito. na primeira parte, após uma rápida descrição sobre as origens e características dessa religião, o artigo reconstitui a forma pela qual a doutrina e a jurisprudência brasileiras têm tratado as controvérsias suscitadas pela recusa de seus membros: como uma colisão normativa entre o direito à vida e a proteção da liberdade religiosa. em seguida, sugere que, a despeito da qualidade da prestação jurisdicional nesses casos, é possível pensar em uma argumentação mais inclusiva do ponto de vista de uma dogmática preocupada com questões de legitimidade do direito. para tanto, o artigo recorre às formulações teóricas do jurista norte-americano robert cover, especialmente o seu conceito de jurisgenesis, que sugere que a criação de significado jurídico transcende os limites do estado. ampliando o raciocínio dogmático aplicável ao caso com base no pensamento desse autor, passa-se a compreender a controvérsia das testemunhas de jeová como um choque de normatividades distintas – entre a narrativa religiosa do grupo e os preceitos estatais. por fim, sugere-se que a recusa das testemunhas de jeová pode ser compreendida como uma afirmação de significado jurídico, traçando-se um paralelo com as reflexões feitas por cover sobre o caso bob jones university vs. united states. palavras-chave: controvérsias religiosas; testemunhas de jeová; robert cover; jurisgenesis. mestre em direito público pela universidade do vale do rio dos sinos (unisinos). e-mail: mauriciopflores@gmail.com professor de graduação e do ppg (mestrado e doutorado) da universidade do vale do rio dos sinos (unisinos) e pesquisador permanente do cebrap ligado ao núcleo direito e democracia. e-mail: jrodrigorodriguez@gmail.com revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . abstract: this article aims to demonstrate how legal controversies raised by jehovah’s witness refusal of blood transfusion may be understood under a new perspective since we recognize that its religious narrative also holds a statement of law. first part of the article briefly describes origins and features of this religion and then shows how legal dogmatics and courts in brazil have been dealt with these controversies: as a normative collision between right to live and religious freedom protection. then, it suggests that – despite the quality of jurisdictional responses on these cases – is possible to think of a more inclusive argumentation from the viewpoint of a legal dogmatics worried about law legitimacy issues. therefore, the article employs the theoretical formulations of north-american jurist robert cover and especially his concept of jurisgenesis, which suggests that the creation of legal meaning takes place beyond state limits. by extending – based on this author – the dogmatic reasoning applicable to the case, we come to understand the controversy of jehovah's witnesses as a clash of distinct normativities – between the religious narrative of the group and the state precepts. finally, it is suggested that the refusal of jehovah's witnesses can be understood as a statement of legal meaning by drawing a parallel with cover's comments on bob jones university vs. united states case. keywords: religious controversies; jehovah’s witness; robert cover; jurisgenesis. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . dogmÁtica jurÍdica e controvÉrsias religiosas À luz de robert cover: a recusa de transfusÃo sanguÍnea por parte das testemunhas de jeovÁ artigo submetido em / / . introduÇÃo a instauração do regime republicano, em , representa um dos principais marcos para as discussões sobre liberdade religiosa no brasil. foi nesse momento histórico em que o estado brasileiro adotou a laicidade como princípio, separando-se da igreja católica. a primeira constituição republicana assumia a promessa secular de tolerância com as mais diferentes manifestações religiosas, em especial aquelas não católicas. essa promessa de liberdade religiosa deu início a um histórico de disputas em torno daquilo que poderia ser considerado como religião e, portanto, seria digno de proteção do estado. sob argumentos jurídicos-científicos, algumas religiões – nomeadamente católicas e protestantes – gozaram de plena liberdade de culto, enquanto outras, estigmatizadas por cientistas, juristas, jornalistas e políticos, trilharam um longo caminho pelo devido reconhecimento de suas práticas. as religiões espíritas e de matrizes africanas estiveram por muito tempo no centro desses debates: suas práticas eram classificadas como “mágicas” (em contraposição às religiões “verdadeiras”) e sancionadas pela legislação penal como atos de ameaça à ordem ou à saúde pública (montero et. al., ). após décadas de debates acerca da relação entre estado e religião, e com forte participação de representantes religiosos em seu processo de elaboração, a constituição federal de consagrou a liberdade religiosa como garantia fundamental em seu artigo º, inciso vi. esse dispositivo estabelece o atual marco normativo para discussões acerca da liberdade religiosa, ainda que esteja muito aquém de pretender solucionar eventuais conflitos entre práticas religiosas e preceitos estatais. ou seja, da mesma forma como aconteceu com boa parte das demandas sociais emergentes no cenário pós- , as controvérsias religiosas se transferiram gradativamente para a arena do judiciário. “[É] inviolável a liberdade de consciência e de crença, sendo assegurado o livre exercício dos cultos religiosos e garantida, na forma da lei, a proteção aos locais de culto e a suas liturgias”. (brasil, ). revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . uma dessas controvérsias envolve as testemunhas de jeová, uma denominação religiosa que possui milhões de adeptos no mundo todo. em razão de uma crença fortemente arraigada e difundida pelo grupo religioso, as testemunhas de jeová se negam a receber qualquer tipo de tratamento médico que envolva transfusão sanguínea, mesmo quando há risco de morte. mas essa recusa é confrontada por parte do estado, que visa proteger a vida de seus cidadãos – preservando-os inclusive de suas próprias escolhas quando entende necessário. essas duas atitudes, baseadas em princípios e textos distintos – recusa e dever de cuidado, lei bíblica e constituição estatal – refletem as posições antagônicas presentes na controvérsia que motiva esse artigo: pode o estado, no afã de proteger a vida, obrigar que uma pessoa realize um procedimento que contrarie sua crença religiosa? após uma breve exposição sobre o histórico de formação da organização religiosa, a primeira parte deste artigo mostra como essa controvérsia tem sido enfrentada pela doutrina jurídica e pelos tribunais brasileiros. nossa pretensão aqui não é exaustiva. recorremos a um número reduzido de decisões judiciais que apresentam as principais circunstâncias que tais casos podem envolver, tais como a possibilidade de tratamentos alternativos e a ausência de capacidade jurídica da pessoa afetada. nosso objetivo não é verificar a qualidade da prestação jurisdicional brasileira nessas questões, mas os argumentos que costumam ser invocados nas decisões ou, mais especificamente, a maneira pela qual o judiciário enquadra a questão. veremos que, salvo poucas exceções, as cortes – e também a doutrina – costumam colocar a controvérsia em termos de um conflito entre o direito à vida e a liberdade religiosa. mas quais seriam as consequências de uma crítica e eventual mudança de enquadramento destas decisões? faz sentido alterar o modo pelo qual a questão é tratada? e, caso haja uma boa justificativa para isso, como seria possível colocar a questão em outros termos? a segunda parte do artigo busca responder a essas questões. primeiro, procurando demostrar a utilidade de se ampliar a racionalidade das decisões, com base em uma reconsideração do papel da dogmática jurídica; segundo, apontando como alternativa a concepção de direito do jurista norte-americano robert cover ( - ). finalmente, a terceira parte do artigo discute como essa concepção poderia ser incorporada à controvérsia das testemunhas de jeová, traçando-se um paralelo com o caso bob jones university, discutido por cover em seu principal texto, nomos and narrative. se nosso raciocínio estiver correto, pretendemos demonstrar que a posição religiosa das de acordo com o site oficial das testemunhas de jeová, em o número de adeptos da religião ao redor do mundo chegava a cerca de oito milhões de pessoas. (jehovah's witness, ). revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . testemunhas de jeová, em sua recusa de transfusão sanguínea, implica também uma afirmação de direito. cumpre desde logo esclarecer que o objetivo de nossa posição é considerar uma argumentação que melhor reflita e respeite o ponto de vista das testemunhas de jeová no interior da dogmática jurídica, sem qualquer pretensão, em nenhum momento, de propor uma solução ou falar em nome da organização religiosa. a controvÉrsia jurÍdica das testemunhas de jeovÁ em , um comerciante chamado charles taze russell ( - ) fundou em pittsburgh, nos estados unidos, a instituição hoje conhecida como watch tower bible and tract society – nome oficial da organização das testemunhas de jeová. fascinado pela escatologia bíblica e insatisfeito com a teologia do cristianismo ortodoxo, russell enxergou em sua turbulenta época de conflitos sociais o cataclismo que precederia o retorno de cristo à terra, segundo uma profecia bíblica (holden, , p. ). a partir desse diagnóstico, deu origem a um movimento milenarista que até hoje reclama possuir a verdade sobre o destino do mundo. as testemunhas de jeová crêem firmemente em suas interpretações restritivas sobre a bíblia, rejeitando qualquer tipo de interação com as doutrinas de outras denominações religiosas, consideradas errôneas (holden, , p. ). em um mundo marcado por uma crescente secularização e uma multiplicidade de crenças e de identidades, as testemunhas de jeová resistem como um grupo admiravelmente coeso em torno da autoridade de seus preceitos, cuja ideia central é a de que a humanidade se encontra à beira do fim do mundo como o conhecemos, que se dará com a “segunda chegada” de cristo à terra. a despeito de ter sucessivamente falhado em sua profecia – ao menos cinco anos foram apontados como aqueles em que ela se concretizaria – a watch tower bible and tract society não deixou de conquistar adeptos ao longo dos anos, crescendo em média % ressalvamos aqui que nenhum de nós faz parte da religião das testemunhas de jeová ou compartilha de seus preceitos. acreditamos, porém que o objeto da controvérsia da transfusão sanguínea pode ser lido, sem prejuízo de nossa posição de observador externo ao grupo, como uma questão atinente à dogmática jurídica. nossas reflexões são dirigidas tão somente a esse campo, não refletindo atitudes valorativas ou pessoais em relação ao preceito religioso em si. importantes grupos religiosos heterodoxos como os mórmons, os adventistas do sétimo dia e a ciência cristã também se originaram nesse período de grande agitação social, entre as décadas de e . todos eles buscavam, de alguma forma, uma leitura messiânica da bíblia que apontasse para a superação das mazelas sociais da época. as doutrinas religiosas milenaristas são aquelas que, baseadas no livro bíblico do apocalipse, sustentam que jesus cristo irá retornar à terra para construir um reino cuja duração será de mil anos. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . por ano durante o século xx e chegando à marca de seis milhões de seguidores no começo do terceiro milênio (holden, , p. ). ao contrário de outras religiões que se manifestam sobre questões controversas (aborto, pesquisas com células-tronco etc.) ou que buscam reconhecimento perante a esfera pública, as testemunhas de jeová mantêm um envolvimento mínimo com a ampla sociedade, sendo proibidas pela administração da watch tower de tomar parte em atividades públicas (holden, , p. ). a sociedade secular é descartada por seus membros, segundo holden ( , p. ), como um lugar contaminado por práticas imorais. a despeito disso, porém, o grupo não vive em comunidades isoladas – ao contrário de grupos fechados como os amish norte-americanos, por exemplo. as testemunhas de jeová encontram-se assim em uma situação peculiar: renunciam ao mundo onde vivem, mas permanecem vivendo nele. em uma situação como essa, conflitos com as estruturas seculares nem sempre podem ser evitados. frequentemente os preceitos sagrados irão se chocar com a realidade circundante e, em especial, com as normas promulgadas pelo estado. nesse último caso, reportar-se à autoridade da doutrina teológica da organização, que separa simbolicamente seus membros do mundo exterior (holden, , p. ), pode não ser suficiente para evitar os conflitos reais entre o sistema de direitos e liberdades do estado, por um lado, e as escolhas existenciais do indivíduo, por outro. esse é o pano de fundo da controvérsia jurídica relacionada à recusa, por parte das testemunhas de jeová, de tratamentos médicos que necessitem de transfusão sanguínea. ao colocarem a autoridade de seus preceitos acima da autoridade do estado, as testemunhas de jeová não reivindicam que o poder legislativo delibere acerca de sua recusa, tida por seus membros como um dogma. do ponto de vista do grupo, sua visão é indiscutível – tanto mais que a discussão, nesse caso, extrapolará os limites simbólicos entre membros e não membros, envolvendo ambos. de acordo com o site oficial das testemunhas de jeová (jw.org), a bíblia – tanto no velho como no novo testamento (gênesis : ; levítico : ; deuteronômio : ; atos : , ) – ordena claramente a abstenção de sangue sob qualquer via. dado que o sangue representa a vida (levítico : ) , diz a organização religiosa, "nós evitamos tomar sangue segundo a tradução que consta no portal oficial das testemunhas de jeová: "pois a vida de todo tipo de criatura é seu sangue, porque a vida está no sangue. por isso eu disse aos israelitas: 'não comam o sangue de nenhuma criatura, porque a vida de todas as criaturas é seu sangue. quem o comer será eliminado.'" (jehovah's witness, ). revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . por qualquer via não só em obediência a deus, mas também por respeito a ele como dador da vida.” (jehovah's witness, ). por outro lado, o estado encontra-se na obrigação de proteger a vida de seus cidadãos e cidadãs, mesmo contra suas próprias vontades. na linguagem bioética, trata-se do reconhecimento da dimensão heterônoma do princípio da dignidade humana: busca-se proteger, de forma objetiva e por meio da imposição de padrões sociais exteriores, bens considerados indisponíveis à livre escolha dos indivíduos – sendo a vida o principal deles. concomitantemente, a bioética também engloba, no entanto, uma dimensão autônoma da dignidade, disposta a tutelar as possíveis escolhas dos indivíduos, notadamente as de caráter existencial – e, dentre elas, as que são envolvidas pelo manto da liberdade religiosa (barroso, , p. ). o atual ministro do supremo tribunal federal (stf) luis roberto barroso, quando instado a elaborar parecer sobre a questão da transfusão sanguínea em testemunha de jeová, salientou que a dignidade como autonomia possui preferência no ordenamento constitucional brasileiro, sendo também predominante no âmbito das decisões proferidas pelo stf (barroso, , p. ). ou seja, a liberdade religiosa costuma se sobrepor ao dever de cuidado do estado. com base nessa constatação, barroso desenvolve uma linha de argumentação que entrelaça os direitos e garantias fundamentais elencados pela constituição com os principais conceitos da bioética moderna, que substituiu o paradigma paternalista pelo da centralidade da autonomia do paciente. seu parecer busca responder à questão central da controvérsia religiosa que cerca as testemunhas de jeová: “pode o estado proteger um indivíduo em face de si próprio, para impedir que o exercício de sua liberdade religiosa lhe cause dano irreversível ou fatal?” (barroso, , p. ). na leitura de barroso, portanto, a questão se resume ao equacionamento entre dois polos: de um lado, a vida humana; de outro, a liberdade religiosa. a premissa da laicidade do estado pode, se mal compreendida, levar ao equívoco de se enxergar a questão como resolvida em favor do direito à vida. com efeito, é preciso ressaltar aqui que laicidade de forma alguma significa hostilidade à religião. não se deve esperar que o poder público seja indiferente às manifestações religiosas existentes, bem como nesse sentido, afirma barroso ( , p. ): “a ordem constitucional reconhece a religião como uma dimensão relevante da vida das pessoas, quer sejam crentes, quer ateias ou agnósticas. afinal, submeter um crente a práticas contrárias a sua religião é tão invasivo quanto determinar a um ateu que se ajuste a padrões religiosos. em qualquer dos casos haverá a imposição externa de valores existenciais e a conseqüente violação da dignidade como autonomia.”. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . a suas práticas específicas. em alguns casos, inclusive, caberá exigir uma postura ativa do estado a fim de protegê-las. isto porque, em um ambiente de multiculturalismo, as questões religiosas, como questões identitárias que são, tendem a comportar relações de poder que se tornam perigosas na medida em que o predomínio de uma crença represente risco para o exercício das demais (rothenburg, , p. ). assim, a defesa da liberdade religiosa – que inclui a recusa de transfusão sanguínea das testemunhas de jeová – não implica uma afronta ao princípio da laicidade, ainda que a proteção à vida humana esteja em jogo. mesmo em um caso que envolva um direito fundamental tido como indisponível, “o estado terá o ônus argumentativo de demonstrar que se trata de uma restrição legítima, e não uma violação à liberdade de escolha do indivíduo” (barroso, , p. ). em nome do exercício da liberdade religiosa, portanto, barroso ( , p. ) considera legítima a recusa de tratamento que envolva a transfusão de sangue por parte das testemunhas de jeová, devendo esta ser respeitada como escolha existencial que cada um tem o direito de fazer. quando instados a decidir sobre a controvérsia, os tribunais brasileiros também têm reconhecido a presença do dilema constitucional suscitado por barroso ( ). no entanto, a tarefa de considerar esse conflito normativo à luz de determinado caso concreto pode se revelar mais espinhosa do que os pareceres técnicos sugerem. se considerarmos a controvérsia do ponto de vista dos médicos, por exemplo, será preciso levar em conta a existência de um ordenamento próprio atinente a sua conduta, o que torna o problema um pouco mais complexo. de acordo com o artigo do código de Ética médica vigente no brasil, é vedado ao médico "deixar de obter consentimento do paciente ou de seu representante legal após esclarecê-lo sobre o procedimento a ser realizado, salvo em caso de risco iminente de morte." (conselho federal de medicina, , p. ). portanto, um médico que deixa de realizar um procedimento de transfusão de sangue em uma testemunha de jeová em estado terminal corre o risco de ser responsabilizado pelo conselho federal de medicina. mas casos como esse são tão recorrentes que o próprio conselho editou, em separado, a resolução cfm nº . / , que afirma expressamente: em caso de haver recusa em permitir a transfusão de sangue, o médico, obedecendo a seu código de Ética médica, deverá observar a seguinte conduta: º - se não houver iminente perigo de vida, o médico respeitará a vontade do paciente ou de seus responsáveis. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . º - se houver iminente perigo de vida, o médico praticará a transfusão de sangue, independentemente de consentimento do paciente ou de seus responsáveis. (conselho federal de medicina, ). o código e a resolução certamente são orientações válidas para uma equipe médica que se depara com esse tipo de situação. mas é evidente que seus termos gerais são incapazes de abranger todas as circunstâncias possíveis de cada caso. sempre que "houver iminente perigo de vida" o médico deverá obrigar o paciente, mesmo sem seu consentimento, a passar por tratamento que envolva transfusão de sangue? o risco de morte sempre justifica tal medida? questões como essas acabam por exigir do judiciário uma postura interventiva em assuntos clínicos, seja para obrigar pacientes a aderirem a tratamentos por eles rejeitados seja para deliberar acerca de sua necessidade. nem sempre, contudo, as instâncias jurisdicionais estarão dispostas a assumir tal postura. em , por exemplo, o tribunal de justiça do estado do rio grande do sul (tj/rs) considerou que o hospital que buscava obter provimento jurisdicional para submeter uma paciente à transfusão sanguínea carecia de interesse processual, uma vez que os profissionais de saúde tinham o dever de empreender todas as diligências necessárias ao tratamento da paciente, independentemente de seu consentimento (rio grande do sul, , p. ). evidentemente, afirmar que não há necessidade de intervenção judicial em assuntos como esse significa, na prática, apenas deslocar o polo da responsabilidade para os médicos envolvidos, que continuam tendo de enfrentar a questão sem um instrumento que legitime suas decisões – ou seja, sem qualquer garantia de que eles não serão acionados judicialmente no futuro. dentre os fatos a serem considerados pelos médicos em face da decisão de realizar ou não uma transfusão de sangue está, por exemplo, a existência ou não de tratamentos alternativos disponíveis. nesse sentido, a questão colocada pelas testemunhas de jeová tem levado inclusive a avanços na medicina, que dispõe cada vez mais de métodos de tratamento que dispensam a prática de transfusão sanguínea (the new yorker, ). mas nesse caso, havendo possibilidade de tratamento diferenciado sem a utilização de transfusão de sangue, o estado poderia ser compelido a financiá-lo? em decisão proferida em abril de , o tj/rs entendeu que não: a liberdade religiosa não garante o direito à prestação diferenciada no serviço público (rio grande do sul, ). mais recentemente, essa questão foi revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . levada ao stf, tendo reconhecida sua repercussão geral no recurso extraordinário nº , a ser julgado pela corte (supremo tribunal federal, ). mas cumpre atentar para outra circunstância extremamente relevante em um caso concreto: quando a controvérsia envolve paciente menor de idade – ou seja, juridicamente incapaz de manifestar sua escolha – cujos pais ou responsáveis recusam, em seu nome, tratamento que envolva transfusão de sangue. se os tribunais tendem a respeitar a recusa por parte de adultos capazes, a orientação nesses casos costuma ser diferente. em , o superior tribunal de justiça (stj) se deparou com um caso desse tipo, em que a recusa dos pais agravou a situação de sua filha de anos de idade, que veio a falecer (brasil, ). ambos os genitores foram acusados de homicídio, bem como um médico da comissão de ligação com hospitais das testemunhas de jeová (colih) que teria supostamente pressionado os médicos responsáveis a não realizar a transfusão. considerando, entre outras coisas, que o processo penal já se arrastava por duas décadas, o tribunal resolveu extinguir a ação penal sob o argumento de atipicidade do comportamento dos genitores. a principal razão apresentada foi a de que os médicos, independentemente do consentimento dos pais, deveriam ter tomado as medidas necessárias para salvar a vida da adolescente. ao levarem sua filha ao hospital, os pais teriam demonstrado que com ela se preocupavam, a despeito de sua crença religiosa não permitir a transfusão de sangue considerada necessária. ou seja, embora os pais tenham sido absolvidos no processo penal, o tribunal não entendeu que sua decisão em nome da filha devesse ser respeitada pelos médicos. em , ao ser confrontado com situação semelhante, o tribunal regional federal da ª região (trf- ) conseguiu intervir na controvérsia ainda em andamento, substituindo a vontade dos pais pela proteção do interesse do menor envolvido, que foi submetido a tratamento oncológico que incluía transfusão sanguínea (brasil, ). quando vista sob a perspectiva de um menor de idade que perdeu a vida antes de sequer poder expressar sua adesão ou não à religião, a controvérsia das testemunhas de jeová parece ganhar contornos dramáticos. certamente podemos nos perguntar até que ponto é válido que os pais tomem decisões desse tipo em nome de seus filhos. uma pessoa adulta pode decidir aceitar as consequências advindas da colisão dos preceitos de suas crenças com comissão composta por médicos praticantes da religião com o intuito de acompanhar e orientar testemunhas de jeová que se submetam a procedimentos médicos possivelmente incompatíveis com a sua crença. a literatura pode proporcionar uma boa noção desses contornos. sobre essa controvérsia em específico, ver mcewan, . revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . as estruturas da realidade jurisdicional; mas é razoável pensar que uma criança ou um adolescente também possa ser capaz de assumir tamanha responsabilidade, ou que outros possam falar por ela? segundo a interpretação que vem sendo adotada pelos tribunais brasileiros, não faz sentido respeitar a escolha dos pais quando o envolvido é menor incapaz, ainda mais na presença de risco iminente de morte ou quando não há alternativas para o devido tratamento que não envolvam a transfusão sanguínea. podemos dizer que, em linhas gerais, a prestação jurisdicional brasileira na controvérsia das testemunhas de jeová tem sido satisfatória. juízes e juízas reconhecem que a liberdade religiosa pode, em alguns casos, se equiparar ou até se sobrepor à proteção da vida – particularmente, da vida da própria pessoa que evoca razões religiosas. mas haveria outra argumentação possível nesses casos? e por que seria útil empregá-la, se os resultados provavelmente seriam os mesmos que a jurisdição tem alcançado? depois de descrever a controvérsia, passamos agora à parte propositiva do artigo, que busca responder a esses dois questionamentos, começando pela fundamentação teórica de nosso argumento, baseada em uma reconsideração do papel da dogmática jurídica e nos insights sobre o fenômeno jurídico inscritos na obra do jurista norte-americano robert cover. o nomos em conflito de robert cover a argumentação adotada pelos tribunais, como vimos acima, utiliza a linguagem tradicional do direito constitucional, que coloca a questão em termos de colisão de princípios – direito à vida de um lado e liberdade religiosa de outro – e mantém no centro da reflexão apenas as normas produzidas pelo estado. os conflitos têm sido resolvidos partindo-se do pressuposto de que estamos diante de indivíduos situados em uma controvérsia passível de solução mediante a modulação do alcance de determinados direitos, dentre os quais, a liberdade religiosa, que é tratada aqui como uma “crença”, como uma escolha individual, por assim dizer, fungível, equiparável a outras escolhas quaisquer. as “crenças” no catolicismo, no protestantismo, no islamismo e assim por diante merecem, aos olhos da lei, igual proteção. no entanto, parece razoável imaginar que estas mesmas questões podem ganhar um outro peso, um outro sentido, se imaginarmos que as testemunhas de jeová são uma comunidade religiosa com legitimidade para produzir o seu próprio direito, vigente em paralelo ao direito produzido pelo estado. neste caso, seria possível atingir os mesmos resultados dogmáticos, mas com fundamento em outros argumentos. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . assim, em vez de apresentar estes problemas como conflitos entre direitos individuais e resolvê-los por meio da modulação de seu âmbito de incidência, tendo em vista as características do caso concreto, seria possível apresentá-los como conflitos entre normas nascidas de fontes normativas diversas e, especialmente no caso da transfusão de sangue em incapazes, de conflitos entre indivíduos e comunidade religiosa. de um ponto de vista radicalmente operacional, ou seja, no que diz respeito aos resultados obtidos pelos raciocínios dogmáticos expostos acima, a discussão proposta neste artigo faz pouco sentido. o direito brasileiro tem produzido respostas razoáveis aos problemas jurídicos apresentados ao judiciário com fundamento no que se pode chamar de gramática das regras, ou seja, a gramática que gira em torno das normas e princípios positivados pelo estado. no entanto, discutir os conflitos relativos às testemunhas de jeová como conflitos entre indivíduos deixa em segundo plano uma dimensão fundamental destas questões, enfrentadas não apenas pelo direito brasileiro. se olharmos com mais cuidado os termos do debate, veremos que seu horizonte está para além da capacidade de produzir respostas aos litígios, mas aponta para uma questão importante sobre a própria legitimidade do direito. afinal, diante de pessoas capazes de colocar seu corpo em questão de forma tão radical, arriscando a própria vida em nome de sua religião, parece ser mais adequado apresentar tais conflitos de outra forma, dando expressão dogmática às práticas religiosas como um corpo normativo relativamente autônomo em relação ao direito estatal – e não apenas como um objeto fungível, passível de escolhas individuais “existenciais”. se pensarmos a atividade dogmática como um exercício da imaginação a respeito da igualdade (warat, ), ou como um espaço de conflitos sobre a igualdade (rodriguez, ), o exame das características da fundamentação das decisões judiciais passa a ocupar o primeiro plano do debate. assim, dar expressão dogmática à comunidade das testemunhas de jeová – em que pese sua própria recusa em participações mais diretas – pode contribuir para aumentar a legitimidade do direito e do estado brasileiro, mesmo que em certos casos suas normas não prevaleçam diante das normas estatais. ainda mais se levarmos em conta que esta não é a única comunidade vivendo nas fronteiras do brasil: basta lembrar das comunidades indígenas, cuja normatividade tem o potencial de criar conflitos do mesmo jaez. a centralidade que a narrativa bíblica sobre a abstenção de sangue possui no interior do espaço normativo habitado pelas testemunhas de jeová não pode ser ignorada se revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . quisermos compreender a real dimensão das crises de consciência pelas quais os fiéis desse grupo religioso passam e também se quisermos produzir uma dogmática capaz de oferecer boas soluções – operacionais e legítimas – para os problemas que este tema levanta. mas como dar expressão dogmática a um grupo que não pretende ser – ao menos diretamente – ouvido? É exatamente nesse ponto que os insights do jurista norte-americano robert cover parecem fundamentais para a controvérsia em questão. mas, antes de retomar este problema sob uma nova perspectiva, vejamos como robert cover pensa os problemas jurídicos e, em especial, como o autor enxerga as ligações e os conflitos entre a gramática das regras produzidas pelo estado e a gramática da regulação social, ou seja, as normas produzidas pela sociedade – neste caso, no âmbito das comunidades religiosas. robert cover escreveu nomos and narrative, seu mais famoso e debatido texto, no contexto da decisão tomada pela suprema corte norte-americana no caso bob jones university vs. united states, em . embora tenham servido como mote, as críticas direcionadas à argumentação dos julgadores – que ocupam a última parte do ensaio – são apenas o desfecho prático de um denso e elaborado raciocínio teórico. a icônica passagem inicial ilustra bem o desafio que cover estava lançando não apenas aos juízes da suprema corte, mas de modo geral a todos os teóricos que partem de concepções jurídicas fixadas na figura do estado: nós habitamos um nomos - um universo normativo. nós constantemente criamos e mantemos um mundo do certo e errado, do lícito e ilícito, do válido e inválido. o estudante de direito pode chegar a identificar o mundo normativo com a parafernália profissional do controle social. as regras e princípios de justiça, as instituições formais do direito e as convenções de uma ordem social são, de fato, importantes para esse mundo; contudo, elas são apenas uma pequena parte do universo normativo que deveria chamar nossa atenção. nenhum conjunto de instituições ou preceitos legais existe separado das narrativas que o situam e lhe dão sentido. (cover, , p. ). o primeiro alvo de cover nesse ensaio são as concepções de direito que se aferram a um modelo estatista. sua pretensão é a de que deixemos de lado por um instante a ideia de que o aparato jurídico do estado, com sua "parafernália profissional", represente tudo o que devemos tomar como direito. mais do que um conjunto de instituições ou um sistema de regras a ser observado, o direito dá vazão a um nomos, um universo normativo que habitamos todas as traduções do inglês são nossas. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . e que é, na visão do autor, "tão 'nosso mundo' quanto o universo físico de massa, energia e movimento". (cover, , p. ). para cover ( , p. ), os preceitos legais são mais do que imposições feitas a nós em nome do povo, da sociedade, do estado; são também signos pelos quais nos comunicamos com os outros. de acordo com essa ideia, o direito é visto como um "sistema de tensão ou uma ponte ligando um conceito da realidade a uma alternativa imaginada" (cover, , p. ). o nomos é assim constituído por um elo entre realidade e visão, que supõe a aplicação da vontade humana a um estado de coisas existente de onde se projetam, a partir de dispositivos narrativos, possibilidades de futuros alternativos. viver em um universo normativo implica saber não apenas o que "é" ou o que "deve ser", mas também o que "poderia ser" (cover, , p. ). um universo normativo é mantido, segundo cover ( , p. ), pela força de compromissos interpretativos – "alguns pequenos e privados, outros imensos e públicos" – que determinam o que o direito significa e o que ele deverá se tornar. isso quer dizer que o direito não é apenas uma questão de regras distintas: como o autor sugere, duas ordens jurídicas idênticas podem diferir em termos de significado se em uma das ordens os preceitos são universalmente aceitos, mas são considerados injustos na outra. para cover, a criação de significado jurídico – batizada por ele de jurisgenesis – implica não apenas a criação de novas regras, mas na instituição de novos mundos, universos normativos que, cumpre ressaltar, não são necessariamente criados pela ação do estado. em vez disso, a jurisgenesis é um processo coletivo ou social essencialmente enraizado em um meio cultural, o que implica na possibilidade de que grupos e comunidades articulem suas próprias criações jurídicas sem qualquer prejuízo interpretativo em relação às legislações estatais (cover, , p. ). a centralidade comumente atribuída ao estado na criação e manutenção do direito não decorre, segundo cover, de uma superioridade interpretativa, mas unicamente porque o estado é capaz de garantir, através da violência, o comprometimento necessário para a afirmação do significado jurídico. em outras palavras, o autor nega que o direito estatal seja qualitativamente melhor do que as normas criadas por grupos e comunidades com base em preceitos religiosos ou em identidades sociais. esta não é uma afirmação trivial no campo da teoria do direito. não se trata aqui de reconhecer que determinados grupos possuem reivindicações que possam ser traduzidas na gramática do direito e levadas às instâncias revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . jurisdicionais, mas de que esses grupos vivem seu próprio direito – muitas vezes em franco conflito com as normas estatais. cover mostra, por exemplo, de que forma comunidades religiosas como os amish e os menonitas tendem a constituir seus próprios nomoi baseados em textos fundamentais e escrituras sagradas, segundo um modelo batizado pelo autor como autonomia insular. nesses casos, as comunidades com pretensões de autorregulação estabelecem significados próprios para interpretar os princípios constitucionais dos estados onde se localizam. sempre que possível, tais comunidades procuram respeitar as normas do estado (no caso, a constituição americana), mas negam que ele detenha o monopólio da interpretação sobre o direito. nasce daí uma luta constante para definir e manter a independência de seus nomoi em relação ao estado, uma vez que este detém a violência necessária para destruí-los. do ponto de vista da legalidade estatal, é plenamente justificável que os estados não estejam facilmente inclinados a aceitar a afirmação de um nomos autônomo dentro de seus domínios. como afirma cover ( , p. - ), "cada grupo deve acomodar dentro de seu próprio mundo normativo a realidade objetiva do outro", o que costuma resultar em conflitos interpretativos ou mesmo sangrentos. as narrativas amish e menonita são, não por acaso, marcadas pela resistência e a necessidade de rápida adaptação a mudanças, dada a dificuldade da tarefa de procurar um lugar para poder viver conforme seus preceitos sem sofrer perseguições. excetuando-se o fato de que não vivem em comunidades isoladas, as testemunhas de jeová se parecem com as comunidades descritas por cover, podendo ser incluídas no rol de movimentos que afirmam o que o autor classifica como de autonomia insular. embora suas reivindicações não estejam frequentemente explícitas, elas acabam ficando em evidência, de uma forma ou de outra, nos casos levados ao judiciário sobre a recusa de transfusão sanguínea. uma primeira forma de se reconhecer as reivindicações desses grupos como legítimas é partir da doutrina estatal da liberdade religiosa, presente tanto no ordenamento constitucional brasileiro como no norte-americano, assim como na maioria das democracias ocidentais. como vimos, essa tende a ser a atitude não só na esfera judicial, mas também na sobre os usos da gramática estatal por parte de agentes sociais, ver rodriguez, . quando não há pretensão de constituir uma insularidade nômica, mas de transformar o significado constitucional dentro do próprio âmbito estatal, a jurisgenesis assume outra natureza, batizada por cover de constitucionalismo redentor. esse registro compreende grupos e instituições que procuram modificar o direito a partir de diferentes narrativas sobre a realidade, com objetivos integrativos, e não sectários. (cover, . p. ). revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . doutrina brasileira. mas isso, diria cover, significaria assumir uma perspectiva inteiramente estatista, cuja compreensão do fenômeno jurídico situado no interior das comunidades é limitada. para realmente fazer jus a esse modo de constituição de direito, com toda a complexidade que dele deriva, é antes preciso colocar as próprias narrativas desses grupos como o ponto sobre o qual se enxergam as demais manifestações de direito, sejam elas estatais ou não. apesar da previsão constitucional de liberdade religiosa ter sido indispensável para que o nomos amish, por exemplo, pudesse prosperar dentro do território norte- americano, o seu texto fundamental não é a constituição dos estados unidos, mas o novo testamento – é a partir dele que os membros da comunidade enxergam o mundo em sua forma normativa (cover, , p. - ). ao postularem um direito estranho ao do estado, comunidades como os amish ou as testemunhas de jeová não se darão por satisfeitas com uma decisão que permita que elas sejam ouvidas – sob o argumento da liberdade de expressão, por exemplo – mas que lhes negue a possibilidade de viver conforme suas próprias regras (cover, , p. ). aqui, cover inverte a perspectiva e mostra como o direito estatal, a quem se costuma pleitear para que reconheça outras manifestações de direito, pode ser ele próprio visto como um "outro", ao assumirmos o ponto de vista de um amish, de uma testemunha de jeová ou de um indígena, por exemplo. esta maneira de pôr o problema contribui para uma reflexão mais ampla sobre as alternativas institucionais disponíveis para regular problemas semelhantes. pensando a partir do texto “inverter o espelho: o direito ocidental em normatividades plurais” de josé rodrigo rodriguez (rodriguez, ), é possível imaginar alternativas institucionais que se destinem a homogeneizar as formas de vida sobre um determinado território, inclusive com o desenvolvimento de políticas ativas de integração de todas as pessoas a uma mesma forma de pensar e de viver – na terminologia de cover, como veremos a seguir, modelos de regulação imperial. também é possível imaginar alternativas institucionais que tratem esta diversidade de formas de vida como casos particulares de normas gerais, as quais sempre irão prevalecer em caso de conflito, deixando clara a posição dominante de uma determinada forma de vida e como vimos anteriormente, as testemunhas de jeová sequer têm interesse em “serem ouvidas”, ao contrário das comunidades religiosas descritas por cover que, embora não considerem isso como algo suficiente, não deixam de acreditar que é importante para os objetivos do grupo a circunstância de poder ter voz perante a esfera pública. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . marcando seu caráter igualmente imperial. finalmente, é possível imaginar modelos de regulação, por assim dizer, “multinormativos”, os quais admitam a presença de diversos nomoi e suas respectivas ordens jurídicas em um mesmo espaço social, com a previsão de regras para solucionar conflitos, ponto ao qual cover dedicou pouca atenção, diga-se de passagem. ora, a opção por um modelo “multinormativo” a partir de cover parece evidente em face do caráter radical que o pertencimento a uma ordem normativa pode assumir. negar reconhecimento a uma determinada ordem normativa, como é o caso do problema que estamos examinando, significa negar a identidade mesma das pessoas testemunhas de jeová que pensam a si mesmas como fazendo parte de outro mundo normativo autônomo. não passa despercebido a cover, porém, como antecipamos acima, o fato de que o nomos estatal apresenta notáveis diferenças quando comparado a qualquer um dos nomoi formados por grupos ou comunidades menores. pensando nisso, cover estabelece uma distinção entre dois modelos típicos de constituição de nomos. o primeiro modelo, denominado paideico, é responsável por "criar mundos"; acontece quando grupos ou comunidades criam seu próprio espaço normativo a partir de um corpo comum de preceitos geralmente provenientes de textos sagrados. nesse modelo, a possibilidade de novas interpretações encontra-se permanentemente aberta, e o direito assume um caráter pedagógico: os indivíduos são educados na lei (cover, , p. - ). já o modelo denominado imperial caracteriza-se, por sua vez, pela objetividade de seus preceitos – normas jurídicas abstratas e universais – e pelo fato de que sua aplicação fica a cargo de instituições devidamente constituídas para esse fim. em sua maior parte, é como o direito moderno funciona; é uma forma de "manter o mundo" diante de suas tendências plurais e potencialmente destrutivas, tornando possível a difícil tarefa de acomodá-las dentro de um mesmo espaço normativo (cover, , p. ). se a forma de organização social adotada pelos estados-nação modernos se aproxima bastante com o modelo imperial, as narrativas que preenchem os seus preceitos com diferentes significados remetem, por outro lado, ao modelo paideico (cover, , p. ). desse estado de coisas deriva, segundo cover ( , p. ), a conclusão de que "há uma dicotomia radical entre a organização social do direito como poder e a organização do direito como significado." tendo em vista essa distinção, é mais plausível conceber que um tribunal, ao proferir uma sentença que afete a constituição de um nomos, não está afirmando um direito revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . hermeneuticamente superior, mas apenas o poder que emana de sua autoridade acerca do sentido do direito no interior de seu território. entender que a produção do direito, ao menos como significado, encontra-se inteiramente aberta à multiplicidade de preceitos elaborados por grupos que adotam narrativas diferentes daquela do estado parece ser a grande contribuição que cover tem a nos oferecer sobre o tema das controvérsias religiosas. tal afirmação explica, em boa parte, a existência de soluções dogmáticas em conflito no campo do direito. essa ideia será retomada, a partir da discussão de cover sobre o caso bob jones university, para tratar da questão específica das testemunhas de jeová. em ambos os casos, percebe-se que acomodar diferentes reivindicações de nomoi próprios sob o manto de uma jurisdição que se pretende única não é, na maioria das vezes, uma tarefa simples. do caso bob jones às testemunhas de jeová o pano de fundo da controvérsia presente no caso bob jones university vs. united states é a decisão, tomada pelo internal revenue service (irs) , de retirar o benefício de isenção de impostos das instituições consideradas filantrópicas que mantinham qualquer forma de discriminação racial em suas práticas. até , a universidade bob jones sequer aceitava inscrições de pessoas negras. por conta das políticas anti-segregação adotadas a partir da década de , a instituição educacional passou a aceitar, em um segundo momento, pessoas negras casadas com outras de sua própria raça. somente em permitiu, por fim, a adesão de pessoas negras independentemente de seu estado civil, mas com uma ressalva: um regulamento disciplinar proibia relacionamentos interraciais e até mesmo participação em grupos que advogavam pelo casamento interracial, sob pena de expulsão dos estudantes envolvidos (cover, , p. ). insatisfeita com a retirada de seu status de isenção por parte do irs, a universidade bob jones recorreu da decisão administrativa, chegando o caso à apreciação da suprema corte. a despeito das questões pontuais que envolviam o caso, o que inicialmente chamou mais a atenção de cover no julgamento foi o significado contido na manifestação de alguns amicus curiae no decorrer do processo. entre eles, membros de comunidades anabatistas como os já mencionados amish e menonitas. instituição do governo norte-americano que executa função semelhante à da receita federal no brasil. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . o que suscitou as manifestações dos porta-vozes desses grupos em favor da universidade não foi, como sublinha cover, a perda da isenção de impostos em razão da discriminação com base em raça. senão todos, ao menos a grande maioria dos amicus curiae presentes no processo não praticavam qualquer tipo de discriminação racial. como explica cover ( , p. ), o que motivou esses grupos a intervir no processo foi a circunstância de que "uma mera 'política pública', ainda que admirável" pudesse triunfar diante da proteção especial à liberdade religiosa contida na primeira emenda à constituição norte-americana. sem prejuízo do que foi dito antes sobre a ausência de centralidade do direito estatal segundo a concepção de cover, a invocação de um texto constitucional em vez de uma norma específica do grupo – baseada em uma interpretação da bíblia, por exemplo – se justifica pelo fato de que a narrativa da liberdade religiosa, além de unir todas as comunidades envolvidas, é muito mais convincente e de reconhecimento obrigatório para o tribunal. ou seja, o fato de que o centro do universo normativo da comunidade amish seja o novo testamento não impede que, em face da jurisdição estatal, seu porta-voz recorra à gramática jurídica oficial – até porque, conforme já mencionado, as comunidades insulares que vivem nos estados unidos não têm problemas em aceitar os preceitos constitucionais vigentes no país na medida em que eles não entrem em conflito com o seu próprio nomos. no entanto, ainda que as comunidades religiosas abracem um princípio constitucionalmente vigente e o utilizem como uma salvaguarda contra possíveis violações de direito, não há como compreender suas reivindicações normativas inteiramente sob o viés do direito estatal. de maior importância para todos os grupos e comunidades religiosas mencionadas por cover é o fato de que, ao menos dentro de seus universos normativos, o respeito pela lei de deus encontra-se acima do respeito pelas leis humanas. É o que acontece com as testemunhas de jeová e também o que se verifica, por exemplo, no depoimento do amicus curiae que representou a comunidade menonita no caso bob jones: nossa fé e entendimento das escrituras nos impõem respeito e obediência pelos governos seculares sob os quais vivemos. nós os reconhecemos como instituições estabelecidas por deus para colocar ordem na sociedade. apenas por essa razão nós sempre exercemos completamente o papel de cumpridores da lei, sem a aflição adicional de sermos punidos ao falhar em seu cumprimento. nossas crenças religiosas, entretanto, são profundamente arraigadas. quando essas crenças colidem com as demandas da sociedade, a nossa mais alta submissão deve ser a deus, e nós devemos dizer com os homens de deus do passado: 'nós devemos obedecer a deus acima dos homens'; essas são as crises das quais nós gostaríamos de ser poupados. (cover, , p. ). revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . por melhores que sejam as intenções dessas comunidades, entretanto, as “crises” precipitadas por conflitos entre a lei de deus e os ordenamentos estatais são muitas vezes inevitáveis. nesses casos, o que se deve fazer? estimular a criação de ordenamentos próprios dentro do estado, respeitando as decisões tomadas por grupos autônomos baseados em narrativas próprias? ou reafirmar a autoridade do estado, via jurisdição, de impor a sua lei mesmo sobre aqueles que colocam o respeito a deus acima de qualquer coisa? as respostas nesses casos costumam ser difíceis, como o próprio cover atesta. no caso bob jones, por exemplo, o autor ficou insatisfeito com a decisão da suprema corte – não com o resultado em si, mas sim com o entendimento que foi adotado. o tribunal simplesmente reiterou o compromisso dos órgãos governamentais de erradicar prát icas de discriminação racial em ambientes educacionais, assegurando o cabimento da medida tomada pelo irs. o que a suprema corte fez, na verdade, foi afirmar que não havia nada de inconstitucional com a política pública adotada pelo irs. cover, no entanto, queria mais. caso levasse a sério as implicações contidas nas reivindicações de insularidade do grupo, o tribunal teria sido capaz de produzir uma narrativa redentora (narrative of redemption) que deixasse claro a todos os grupos ou comunidades que práticas de discriminação racial não seriam toleradas em um país realmente democrático como os estados unidos pretendiam se afirmar. tal posição, segundo ele, revelou uma fraqueza na interpretação adotada: [...] a força da interpretação da corte no caso bob jones university é muito débil. débil não por causa da forma do argumento, mas em razão da falta de comprometimento da corte - uma falta que se manifesta na designação de autoridade para a decisão. a corte assume uma posição que não coloca nada em risco e por meio da qual não faz nenhum gesto interpretativo em absoluto, salvo o gesto essencial aos cânones jurisdicionais: a declaração de que um exercício de autoridade política não era inconstitucional. o grande esforço nacional contra a discriminação não ganha status normativo na opinião da corte, salvo que ela significa que o irs não estava errado. (cover, , p. ). em vez de estabelecer uma narrativa redentora baseada na proibição de discriminação racial que agregasse as diferentes reivindicações de nomos existentes no interior dos estados o tribunal sempre pode escolher tolerar ou não normas provenientes de ordenamentos jurídicos não-estatais, mas não pode se furtar de fornecer narrativas que reintegrem esses ordenamentos no quadro geral de seu nomos: "as cortes podem muito bem se fixar sobre o painel jurisdicional e as regras de tolerância de modo a evitar matar o direito das comunidades insulares que habitam nossa paisagem normativa. mas elas não podem evitar a responsabilidade de aplicar ou se recusar a aplicar seu poder para preencher uma visão redentora." (cover, , p. ). revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . unidos, a interpretação da suprema corte se limitou a não interferir na discricionariedade que uma agência do governo possui no momento de colocar políticas públicas em prática. se a medida do irs foi aceita por "não estar errada", isso significa, pergunta cover ( , p. ), que as comunidades insulares como os amish e os menonitas estão à mercê de qualquer política pública que "não esteja errada", independentemente das disposições que constituem seus universos normativos? para que fique claro, cover não queria que o tribunal acatasse o pedido da universidade, reavendo a decisão tomada pelo irs. na verdade, o autor advoga por uma interpretação que afirmasse de forma mais enfática a necessidade de se proibir práticas discriminatórias nos estados unidos: uma decisão que transformasse a antidiscriminação em narrativa fundamental a ser seguida por todos os grupos e comunidades que compartilham o espaço normativo norte-americano; uma narrativa redentora que, considerando as implicações dos diversos nomos que se apresentam, não deixasse dúvidas acerca desse tema em específico. de outra parte, cover também critica a falta de comprometimento da própria universidade ao recuar em suas interpretações racistas diante da ameaça de um significativo corte em seu orçamento. se a interpretação das escrituras adotada pela instituição era contrária a relacionamentos interraciais, o que explica as constantes mudanças de posicionamento em relação ao tema, senão a postura cada vez menos tolerante do poder público com práticas de discriminação racial? sem levar suas interpretações à última instância, juízes e representantes da universidade fizeram do caso bob jones, segundo cover ( , p. ), "uma peça para - atores prudentes e cautelosos, alguma eloquência, mas nenhum comprometimento". o comprometimento com as regras a que um grupo se propõe seguir se mostram de profunda importância para a descrição que cover faz sobre a jurisgenesis. não basta simplesmente criar significado jurídico, como num passe de mágica: é preciso comprometer- se com ele. e por comprometimento entenda-se o ato de aceitar as exigências que a defesa de uma determinada interpretação legal implica na prática, ou seja, as consequências fáticas da transformação pretendida na realidade do nomos. mas, voltando a nosso assunto principal, é possível dizer que a ausência ou fraqueza de comprometimento apontada por cover em relação à universidade bob jones não encontra correspondência na controvérsia jurídica sobre as testemunhas de jeová. a recusa de "dado que o nomos não é mais do que um processo de ação humana tensionado entre a visão e a realidade, uma interpretação legal não pode ser válida se não se está preparado para viver de acordo com ela." (cover, , p. ). revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . transfusão de sangue, tida por esse grupo religioso como uma narrativa sagrada, é defendida de forma implacável por seus membros, a despeito de ser pouco usual entre as demais ordens cristãs existentes. ao menos nos casos sob julgamento, a recusa é levada às últimas consequências, sendo que muitas vezes as partes não recuam nem mesmo sob a ameaça de sanções criminais. esse é um ponto importante para se compreender quão dramáticas são as decisões proferidas nesse âmbito. se, como afirma judith resnik ( , p. ), "as apostas das comunidades insulares para viver conforme suas próprias leis mudam na medida em que o estado ou outros agentes poderosos lançam objeções", isso não parece se confirmar em relação às testemunhas de jeová. a narrativa nesse caso é – muitas vezes literalmente – de vida ou morte; e o objeto da narrativa é o próprio corpo dos indivíduos, que se colocam perante as ameaças do poder estatal com a firmeza de quem age simplesmente no cumprimento de um dever. se aceitarmos as testemunhas de jeová como um grupo que forma um nomos, temos de aceitar que a recusa de transfusão sanguínea é parte inseparável de seu universo normativo. mas isso significa que o estado deveria reconhecer integralmente um nomos com regras tão divergentes das suas? não há resposta óbvia a partir da leitura de cover. percebe-se, aliás, certa ambiguidade no pensamento do autor nesse ponto. do fato de que o estado deveria reconhecer outras ordens normativas – uma vez que não possui o monopólio do direito – não deriva a conclusão de que ele sempre deva buscar aceitar as regras elaboradas por grupos e comunidades. a jurisgenesis pode ser incontrolável, mas isso não implica que os tribunais devam se abster de controlar as possibilidades de significados jurídicos dentro de seu espaço normativo. para tanto, as cortes recorrem a um elemento que, segundo o autor, é indissociável da interpretação legal: a violência. "a intepretação legal", começa cover ( , p. ) em seu artigo chamado violence and the word, "tem lugar em um campo de dor e morte." em outras palavras, a interpretação legal sempre justifica uma violência que já ocorreu ou que está por acontecer. e se entenda violência aqui tanto em sentido físico – conter fisicamente os condenados e levá-los para a prisão, ordenar execuções – como em sentido epistêmico – o ato sobre a possível existência de relações de poder dentro do próprio grupo, ver resnik, , p. . revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . de afirmar determinado significado jurídico em detrimento de outros, o que significa eliminar interpretações que não sejam aceitas. nesse sentido, ao mesmo tempo em que estimula a criação e a proliferação de novos universos normativos , cover faz uma espécie de apologia ao trabalho "violento", mas aparentemente necessário, dos tribunais: [...] o princípio jurisgenerativo pelo qual o significado jurídico se prolifera em todas as comunidades nunca existe apartado da violência. a interpretação sempre tem lugar à sombra da coerção. e a partir desse fato podemos reconhecer um papel especial para as cortes. as cortes, ao menos as estatais, são caracteristicamente "jurispáticas". [...] É a multiplicidade de leis, a fecundidade do princípio jurisgenerativo, que cria o problema para o qual o tribunal e o estado são a solução. (cover, , p. ). essas duas tendências conflitantes sugerem a mensagem de que, sempre que possível, a violência deve ser contida, deixando que novos universos normativos floresçam; no entanto, não se deve esquecer que às vezes ela é necessária. o direito deve ser violento, isso é inevitável; mas deve ser o menos violento possível. o trabalho das cortes jurispáticas é fundamental para colocar uma ordem mínima dentro de universos normativos que, de outra forma, se desintegrariam. não há espaço aqui para desenvolver de forma mais elaborada a tensão entre violência e interpretação legal na obra de cover. de fato, este parece ser um ponto mal resolvido de sua teoria que poderia receber uma resposta mais adequada por meio de uma reflexão normativa que apresente critérios para que se possa escolher entre uma forma de vida social homogênea ou uma forma marcada pela multinormatividade. este ponto, diga- se, permitiria aproximar, inclusive, a obra de robert cover da teoria crítica. importa notar, entretanto, que o autor está atento às implicações, para o direito estatal, de se aceitar ou não a presença de novos significados jurídicos. os preceitos que denominamos direito são marcados "pelo controle social sobre sua origem, seu modo de articulação e seus efeitos" (cover, , p. ), e os tribunais não podem escapar da responsabilidade de assumir esse controle. sobre esses dois tipos de violência, e também para uma crítica ao peso que cover concederia à violência física, ver beckett, . como na conclusão de nomos and narrative: "o significado jurídico é um enriquecimento desafiador da vida social, e uma potencial restrição ao poder arbitrário e à violência. nós deveríamos parar de circunscrever o nomos; nós deveríamos encorajar novos mundos." (cover, , p. ). sobre essa interpretação, ver sarat e kearns, , p. . os autores entretanto negam que um balanço dessa natureza seja possível dentro da teoria de cover, denunciando a impossibilidade de reconciliar as lições de nomos and narrative (cover, ) com o diagnóstico de violence and the word (cover, ). para uma descrição mais completa, ver sarat e kearns, . para uma tentativa nesse sentido ver benhabib, . uma aproximação mais detalhada poderia caminhar no sentido de uma reflexão sobre o lugar do pensamento de cover em uma teoria da institucionalização democrática como transformação institucional permanente, no sentido de rodriguez, a. revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . essa constatação é bem exemplificada pelos casos levados ao judiciário brasileiro envolvendo testemunhas de jeová que recusaram, a si próprias ou aos filhos menores de idade, tratamentos médicos nos quais a transfusão de sangue é utilizada. com base nas reflexões de cover, é possível ler a controvérsia sob outro viés, procurando fazer jus ao peso que a organização religiosa e seus fiéis concedem à questão. antes, porém, uma ressalva: ao retomar o pensamento de cover, não pretendemos apresentar uma teoria estrangeira que possa (ou deva) ser aplicada ou incorporada no brasil, superando um suposto déficit de racionalidade em nossa jurisdição. isso porque, em pr imeiro lugar, não podemos dizer sequer com alguma certeza que cover possua uma teoria do direito propriamente dita – sua morte precoce, aos anos, limitou sua obra a alguns poucos textos, muito deles sem qualquer traço de continuidade. mas em segundo lugar, e mais importante, é preciso notar que seus conceitos mais importantes (nomos e jurisgenesis), bem como as análises possíveis a partir de cada um deles, não parecem implicar necessariamente a adoção de uma teoria do direito ou de uma teoria da decisão jurídica em específico. sua descrição sobre as fases do processo de criação jurídica – narrativa, interpretação, comprometimento, objetivação e, por fim, a violência – contempla uma multiplicidade de atores (estatais ou não), espaços (local, nacional, transnacional ) e tempos distintos (antiga lei judaica, estados unidos da década de oitenta ou mesmo a discussão no contexto brasileiro que estamos propondo). feita essa ressalva, voltemos à jurisdição brasileira como forma de demonstrar a pertinência de nosso argumento geral sobre como podemos entender a discussão dogmática, a partir de cover, sobre a controvérsia das testemunhas de jeová. em , o tj/rs reformou uma decisão de primeiro grau que autorizava a realização de transfusão de sangue em testemunha de jeová mesmo contra sua vontade expressa. o tribunal afirmou, na ocasião, a "inexistência do direito estatal de 'salvar a pessoa dela própria', quando sua escolha não implica violação de direitos sociais ou de terceiros." (rio grande do sul, , p. ). chama atenção nesse acórdão, porém, a seguinte consideração feita pelo relator, o desembargador cláudio baldino maciel: não se trata de singelamente ponderar qual direito fundamental deve ser preservado e qual deve sofrer limitação. violence and the word (cover, ) contém alguns esboços do que poderia ser uma teoria da decisão jurídica, mas de forma muito incipiente. para autores que leem as categorias de cover a partir dos regimes jurídicos transnacionais, ver benhabib, ; berman, ; dunoff, ; melissaris, . revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . a liberdade de crença expressada pela paciente, ora agravante, reveste sua vida de sentido, sentido este não compreendido, na sua verdadeira dimensão, por quem não vive e não comunga de tais valores. a dignidade que emana da sua escolha religiosa tem tamanha importância para ela que, entre correr o risco de perder a vida, mas permanecer íntegra em relação aos seus valores/ideais religiosos, e receber uma transfusão de sangue, tendo violados seus valores e sua dignidade de pessoa humana, esta escolheu manter-se íntegra em sua crença. (rio grande do sul, , p. ). constitucionalmente, é a liberdade religiosa que assegura à paciente em questão o direito de não se submeter a procedimentos médicos que contrariem suas convicções. mas a afirmação de “liberdade religiosa” é, nesse caso, apenas uma tentativa – ainda que válida – de traduzir convicções existenciais profundas para a linguagem principiológica do direito constitucional. É evidente que tal crença possa soar aos julgadores como absurda; contudo, o tribunal se furta de juízos de valor acerca da narrativa religiosa e do preceito dela extraído. ao atentar para a importância da narrativa religiosa situada no centro da controvérsia, o des. cláudio baldino executa um movimento interessante: afastando a linguagem do direito constitucional por um momento, o julgador reconhece a dignidade da pessoa afetada dentro da lógica colocada pela sua própria crença. ao fazer isso, em vez de direitos fundamentais em colisão, o desembargador vislumbra um dilema de vida ou morte – diante do qual a paciente escolheu firmemente manter-se de acordo com a sua crença. essa posição é bastante elogiável do ponto de vista da concepção de direito trazida por cover. a terminologia adotada pelo julgador não chega a deixar explícito aquilo que, para cover, é um fato evidente. não é por certo a “liberdade de crença”, como afirma o desembargador, que reveste a vida da paciente de sentido; mas sim a própria crença, manifestada na recusa. da mesma forma, o julgador coloca a defesa da escolha religiosa em termos de “dignidade” – o que é plenamente aceito tendo em vista ser um termo constitucional, habitualmente empregado por juízes e demais atores judiciais. a partir de cover, porém, poderíamos dizer que seria mais adequado falar nessa escolha religiosa como uma manifestação de direito: como a afirmação de um determinado significado jurídico. seria equivocado, no entanto, deduzir que essa posição implica na aceitação passiva de um direito “não-estatal”. não estamos propondo qualquer versão alternativa de direito, muito menos querendo afastar o direito constitucional das decisões judiciais. há uma grande diferença em reconhecer, por um lado, que uma posição de caráter moral ou religioso possua um significado jurídico e, por outro, entender que a narrativa das testemunhas de jeová ou de qualquer outra organização religiosa deva ser entendida como “norma” ou como “princípio” revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . legal que deva ser sopesado (ou combatido) pelas normas e princípios de direito estatal. no primeiro caso, trata-se de ampliar os limites da dogmática jurídica e da racionalidade das decisões judiciais. no segundo, tratar-se-ia de opor duas narrativas (a do estado e a da organização religiosa), dotadas de sentido e legitimidade distintos, por meio de uma instância decisória que, conforme se espera, se apoia em uma delas – a narrativa do direito estatal e da constituição. uma das circunstâncias que descrevemos na primeira parte ajuda a esclarecer esse ponto. quando a controvérsia envolve menores de idade, devemos aceitar a manifestação de seus pais ou responsáveis? pensamos que, nesse caso, a narrativa das testemunhas de jeová colide com outra estrutura do direito estatal, a da capacidade jurídica. o estado concede, através de suas normas e princípios, proteção especial a alguns indivíduos. É razoável que não se abra mão dessa proteção, salvo em raros casos. isso implica refutar o nosso argumento? pensamos que não – e a solução passa, uma vez mais, por robert cover. entendemos que, em um estado laico, a integridade da narrativa das testemunhas de jeová deveria, na medida do possível, ser preservada. isso está implicado na garantia do exercício da liberdade religiosa. ao mesmo tempo, não podemos fechar os olhos para o fato de que haverá conflitos entre essa narrativa e os preceitos estatais. as mesmas circunstâncias estavam presentes no caso bob jones university, segundo a leitura de cover: ao mesmo tempo em que garantiam a sobrevivência de suas práticas religiosas sob o manto da constituição norte-americana, grupos como os amish e os menonitas lutavam para que o mesmo estado que os protegia não os proibisse de viver conforme seus preceitos. mas cover entendeu que, nesse caso, a balança pendia a favor do estado e de seus esforços pelo fim da segregação racial. essa “narrativa redentora”, para utilizar os termos do autor, seria declarada como fundamental para a continuidade da ordem estatal e todos aqueles que habitam seu espaço normativo. sob a lógica do argumento, o estado reafirmaria os contornos de seu nomos e, consequentemente, dos diferentes nomoi que o integram. mas, o que é mais importante: tal narrativa redentora não implica na negação de um direito amish ou menonita. pensamos que uma narrativa redentora desse tipo seja possível no caso das testemunhas de jeová quando a recusa da transfusão envolve menores de idade. por meio dessa narrativa, o estado estaria afirmando um compromisso público e permanente com a defesa desses indivíduos em tais assuntos. seria, certamente, uma intromissão no espaço normativo das testemunhas de jeová; porém, visto que esse espaço é compartilhado com o revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . estado, algum grau de intromissão sempre vai haver – como demonstrado, aliás, na primeira parte do artigo. a diferença é que, nesse caso, as testemunhas de jeová não ficariam reféns de decisões judiciais pontuais – diferentemente de como os grupos e comunidades mencionados por cover ficaram, com a decisão da suprema corte, em relação a quaisquer políticas públicas que não fossem consideradas inconstitucionais. a narrativa das testemunhas de jeová, que inclui a proibição de transfusão sanguínea, é uma realidade objetiva para seus membros. ao manifestar sua recusa, mesmo em casos extremos, o indivíduo membro coloca seu próprio corpo à prova de forma a não comprometer a totalidade de sua visão de mundo. a linguagem do direito estatal pode, no máximo, proteger essa atitude sob o manto da liberdade religiosa; ao fazê-lo, inscreve a significação jurídica desse ato dentro de um rol de normas e princípios que devem ser balanceados. nada disso fará muito sentido para uma testemunha de jeová. por outro lado, se o que realmente importar, afinal, for o resultado, então devemos dizer que a jurisdição brasileira tem, na medida do possível, dado conta do problema – e, nesse caso, nada do que dissemos até aqui terá feito muito sentido. porém, um último ponto cumpre ser mencionado: mesmo que os resultados atingidos pela jurisdição sejam relativamente iguais, as razões para decidir podem variar. direito à vida e liberdade religiosa sempre estarão no cerne do conflito, mas uma maior ou menor importância pode ser dada a um ou outro princípio e, como diagnosticado por rodriguez ( b), a racionalidade jurisdicional brasileira parece ser guiada muito mais por argumentos de autoridade, que podem fazer a decisão pender para qualquer um dos lados, do que por raciocínios dogmáticos que incorporem decisões anteriores ou que procurem decidir casos semelhantes de forma igualmente semelhante. se for verdade que a dogmática, dentro de um estado de direito, é um exercício da imaginação a respeito da igualdade, como sugeriu warat ( ), então podemos afirmar que uma argumentação que reconheça a narrativa religiosa das testemunhas de jeová como contendo uma afirmação de significado jurídico coloca a questão da igualdade em primeiro plano – seja no sentido de não estabelecer hierarquias (ao menos do ponto de vista argumentativo) entre diferentes ordens normativas, seja no sentido de discutir casos semelhantes de acordo com um padrão de racionalidade comum. diante de casos como os que trouxemos à tona, os julgadores poderiam se perguntar: há comprometimento com uma ordem normativa que não a estatal? a pessoa está disposta a levar adiante o entendimento de seu revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . nomos? nesses casos, como a própria doutrina e jurisprudência têm reconhecido, a melhor escolha parece ser não invadir o espaço normativo do indivíduo, respeitando sua escolha. nos demais casos, onde pais ou responsáveis tomam a decisão em nome de menores de idade, é possível perguntar: a exigência de manutenção da integridade do nomos compartilhado pelos pais e responsáveis é suficiente para afastar a narrativa da capacidade jurídica, fixada pelo estado como forma de defender menores e incapazes? conforme a própria jurisprudência igualmente tem se manifestado, nesses casos a resposta tende a ser negativa: o dever de cuidado que o estado assumiu para si o compele a não abrir mão da capacidade jurídica em favor de outra ordem normativa, não obstante a reconheça enquanto tal. conclusÃo de acordo com nosso raciocínio, a controvérsia aqui debatida pode ser vista da seguinte maneira: as testemunhas de jeová recusam a transfusão sanguínea mesmo sob risco de morte, e tal recusa não é passível de discussão na esfera pública – antes de tudo porque, para as testemunhas de jeová, essa não é a “sua” esfera pública, mas a do mundo exterior. quando a controvérsia suscitada por esse preceito chega ao judiciário, a resposta jurisdicional costuma ser dada pelo equacionamento de dois polos: direito à vida e liberdade religiosa. sugerimos que essa linguagem constitucional, apesar de válida, não se aproxima do dilema efetivo que se coloca às testemunhas de jeová. com base no pensamento de robert cover, formulamos uma alternativa que aponta para o reconhecimento de um significado jurídico na escolha religiosa em questão. pensamos que, dessa forma, é possível dar maior legitimidade às decisões tomadas e, ao mesmo tempo, lidar com a impossibilidade de falar do ponto de vista de uma testemunha de jeová, para quem a narrativa religiosa é inquestionável e se sobrepõe a qualquer regra de direito estatal. dito isso, gostaríamos de esclarecer dois pontos que podem gerar confusões ou interpretações equivocadas. em primeiro lugar, não pretendemos de forma alguma subestimar a importância do princípio da liberdade religiosa: sem ele, uma discussão desse tipo sequer seria possível. o mesmo vale para a aqui mencionada “linguagem constitucional”. não cremos que haja algo de errado com ela. como linguagem normativa, é evidente que ela não é capaz de dar conta de situações reais de forma plena. É justamente por isso que pensamos ser possível ir além dela na controvérsia das testemunhas de jeová. se o pensamento dogmático revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . “organiza o material jurídico com o objetivo de fornecer balizas decisórias” (rodriguez, , p. ) à jurisdição, então reconhecer a existência de um significado jurídico na escolha religiosa das testemunhas de jeová representa a ampliação do raciocínio dogmático. em segundo lugar, seria possível questionar se nossa argumentação acarreta a conclusão de que todo preceito religioso deve ser visto também como uma afirmação de direito. não concordaríamos, de imediato, com essa conclusão; seria preciso analisar os elementos de cada controvérsia. além disso, reconhecemos que os limites poderiam não ser muito claros. acreditamos, porém, que em face de uma determinada concepção religiosa como a que foi aqui discutida – ou seja, uma concepção representativa dentro da sociedade, que é cultivada há um tempo razoável pelo grupo, e é tão profundamente arraigada que envolva, por exemplo, uma escolha de vida ou morte – seja verdadeiramente possível pensar dogmaticamente da forma como sugerimos, em termos não de colisão entre direitos, mas de diferentes normatividades. em suma, não estamos pensando aqui em afirmação de direito (ou melhor, de significado jurídico) como normalização ou adoção de princípio. não é preciso que o estado positive um “direito de recusar a transfusão sanguínea” para que essa recusa, que é real e efetiva para as testemunhas de jeová, possa ser reconhecida como um argumento válido para a dogmática jurídica. esse reconhecimento poderá, certamente, parecer algo distante de nossas práticas jurídicas. no entanto, manifestações como as contidas no voto do des. cláudio baldino maciel, ao enfatizarem a integridade da crença religiosa professada por uma das partes, parecem demonstrar que um reconhecimento desse tipo não é inimaginável. pelo contrário, e sem prejuízo da aplicação do direito estatal, pode se constituir até mesmo em um esforço desejável. em um contexto marcado não mais apenas pela secularização, mas também pelo retorno do fenômeno religioso sob diferentes matizes, com diferentes narrativas que expressam visões de mundo também distintas, resta saber até que ponto o direito e os tribunais brasileiros irão tolerar ou restringir a pluralidade de significados jurídicos contidos nessas manifestações religiosas. referÊncias barroso, luis roberto. legitimidade da recusa de transfusão de sangue por testemunhas de jeová. dignidade humana, liberdade religiosa e escolhas existenciais. parecer em processo administrativo da procuradoria-geral do estado do rio de janeiro. rio de janeiro, de abril revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . de . disponível em: <http://www.conjur.com.br/dl/testemunhas-jeova-sangue.pdf>. acesso em: fev. . berman, paul schiff. global legal pluralism. southern california law review, los angeles, v. , p. - , . benhabib, seyla, another cosmopolitanism: hospitality, sovereignty, and democratic iterations. oxford: oxford university press, . brasil. constituição ( ). constituição da república federativa do brasil de . disponível em: <http://www.planalto.gov.br/ccivil_ /constituicao/constituicaocompilado.htm>. acesso em: mar. . brasil. superior tribunal de justiça. habeas corpus nº . -sp. inteiro teor do acórdão. relatora: ministra maria thereza de assis moura. setembro, . brasil. tribunal regional federal da ª região. apelação cível rs . . . - . inteiro teor do acórdão. relatora: des. des. vânia hack de almeida. outubro, . conselho federal de medicina. código de ética médica: resolução cfm nº . , de de setembro de . brasília: conselho federal de medicina, . conselho federal de medicina. resolução cfm nº / , de de setembro de . disponível em: <http://www.portalmedico.org.br/resolucoes/cfm/ / _ .htm>. acesso em: jan. . cover, robert. violence and the word. yale law journal, v. , n. , p. - , . cover, robert. the supreme court, term - foreword: nomos and narrative. harvard law review, v. , n. , p. - , . dunoff, jeffrey. a new approach to regime interaction. in: young, margaret (ed.). regime interaction in international law: facing fragmentation. cambridge: university press, . holden, andrew. jehovah’s witnesses: portrait of a contemporary religious movement. nova york: routledge, . jehovah's witness. bíblia on-line: tradução do novo mundo da bíblia sagrada. . disponível em: <https://www.jw.org/pt/publicacoes/biblia/>. acesso em: fev. . jehovah's witness. jehovah’s witnesses reach membership milestones. . disponível em: <https://www.jw.org/en/jehovahs-witnesses/activities/ministry/how-many- jehovahs-witnesses- />. acesso em: fev. . revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . jehovah's witness. por que as testemunhas de jeová não aceitam transfusão de sangue? . disponível em: <https://www.jw.org/pt/testemunhas-de-jeova/perguntas- frequentes/por-que-testemunhas-jeova-nao-transfusao-sangue/#?insight[search_id]=acb f c- bf e- afd- c-c decc &insight[search_result_index]= >. acesso em: fev. . mcewan, ian. a balada de adam henry. são paulo: companhia das letras, . melissaris, emmanuel. the more the merrier? a new take on legal pluralism. social & legal studies, [s.i.], v. , n. , p. - , . montero, paula; sales, lilian; teixeira, jacqueline moraes. as relações entre estado e religião no brasil. in: silva, felipe gonçalves; rodriguez, josé rodrigo (coords.). manual de sociologia jurídica. ed. são paulo: saraiva, . resnik, judith. living their legal commitments: paideic communities, courts, and robert cover. yale journal of law & the humanities, v. , . rio grande do sul. tribunal de justiça do estado do rio grande do sul. agravo de instrumento nº . inteiro teor do acórdão. presidente e relatora: des.ª maria isabel de azevedo souza. abril, . rio grande do sul. tribunal de justiça do estado do rio grande do sul. agravo de instrumento nº . inteiro teor do acórdão. relator: des. cláudio baldino michel. março, . rio grande do sul. tribunal de justiça do estado do rio grande do sul. apelação cível nº . inteiro teor do acórdão. relator: des. umberto guaspari sudbrack. agosto, . rodriguez, josé rodrigo. a desintegração do status quo: direito e lutas sociais. novos estudos cebrap, v. , p. - , a. rodriguez, josé rodrigo. como decidem as cortes?: para uma crítica do direito (brasileiro). rio de janeiro: fgv, b. rodriguez, josé rodrigo. dogmática é conflito: a racionalidade jurídica entre sistema e problema. in: rodriguez, josé rodrigo; pÜschel, flavia portella; machado, marta rodriguez de assis (orgs.). dogmática é conflito: uma visão crítica da racionalidade jurídica. são paulo: saraiva, . rodriguez, josé rodrigo. inverter o espelho: o direito ocidental em normatividades plurais. in: rossana rocha reis. (org.). política de direitos humanos. são paulo: hucitec, p. - , . rothenburg, walter. liberdade religiosa no multiculturalismo. ius gentium, curitiba, v. , n. , p. - , jan./jun. . revista juris poiesis - rio de janeiro. vol. -n° , , pg. - . issn - rio de janeiro, de agosto de . sarat, austin; kearns, thomas. making peace with violence: robert cover on law and legal theory. in: sarat, austin (ed.). law, violence, and the possibility of justice. princeton, new jersey: university press, . supremo tribunal federal. stf vai decidir se liberdade religiosa justifica custeio de tratamento de saúde pelo estado. ago. . disponível em: <http://www.stf.jus.br/portal/cms/vernoticiadetalhe.asp?idconteudo= >. acesso em: mar. . the new yorker. how jehovah’s witnesses are changing medicine. ago. . disponível em: <http://www.newyorker.com/news/news-desk/how-jehovahs-witnesses-are- changing-medicine>. acesso em: mar. . warat, luis alberto. a dogmática jurídica e o estado de direito. in: warat, luis alberto. introdução geral ao direito, v. : o direito não estudado pela teoria moderna. porto alegre: sergio antonio fabris editor, . p. - . submetido em: / / aceito em: / / wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ ileal apical na+-dependent bile acid transporter asbt is upregulated in rats with diabetes mellitus induced by low doses of streptozotocin ileal apical na�-dependent bile acid transporter asbt is upregulated in rats with diabetes mellitus induced by low doses of streptozotocin fadi annaba, * ke ma, * pradeep kumar, amish k. dudeja, rhonda d. kineman, , benjamin l. shneider, seema saksena, ravinder k. gill, and waddah a. alrefai , section of digestive diseases and nutrition, section of endocrinology, diabetes & metabolism, department of medicine, university of illinois at chicago and jesse brown veterans affairs medical center, chicago, illinois; and pediatric gastroenterology, hepatology and nutrition, children’s hospital of the university of pittsburgh medical center, pittsburgh, pennsylvania submitted march ; accepted in final form july annaba f, ma k, kumar p, dudeja ak, kineman rd, shnei- der bl, saksena s, gill rk, alrefai wa. ileal apical na�-depen- dent bile acid transporter asbt is upregulated in rats with diabetes mellitus induced by low doses of streptozotocin. am j physiol gastrointest liver physiol : g –g , . first published july , ; doi: . /ajpgi. . .—increased intestinal bile acid absorption and expansion of the bile acid pool has been implicated in the hypercholesterolemia associated with diabetes mel- litus. however, the molecular basis of the increase in bile acid absorption in diabetes mellitus is not fully understood. the ileal apical na�-dependent bile acid transporter (asbt) is primarily responsible for active reabsorption of the majority of bile acids. current studies were designed to investigate the modulation of asbt function and expression in streptozotocin (stz)-induced diabetes mellitus in rats and to examine the effect of insulin on rat asbt promoter by insulin. diabetes mellitus was induced in sprague-dawley rats by intraperi- toneal injection of low doses of stz ( mg/kg body wt) on five consecutive days. human insulin ( u/day) was given to a group of diabetic rats for days before euthanasia. rna and protein were extracted from mucosa isolated from the small intestine and asbt expression was assessed by real-time quantitative rt-pcr and west- ern blotting. our data showed that asbt mrna and protein expres- sion were significantly elevated in diabetic rats. insulin treatment of diabetic rats reversed the increase in asbt protein expression to control levels. consistently, ileal na�-dependent [ h]taurocholic up- take in isolated intestinal epithelial cells was significantly increased in diabetic rats. in vitro studies utilizing intestinal epithelial caco- cells demonstrated that asbt expression and promoter activity were sig- nificantly decreased by insulin. these studies demonstrated that insu- lin directly influences asbt expression and promoter activity and that asbt function and expression are increased in rats with stz- induced diabetes mellitus. the increase in asbt expression may contribute to disturbances in cholesterol homeostasis associated with diabetes mellitus. hypercholesterolemia; insulinopenia; enterohepatic circulation diabetes mellitus is a complex disorder resulting from insulin imbalance and characterized by dyslipidemia and hypercholes- terolemia ( , ). high levels of plasma cholesterol in diabe- tes mellitus represent a major risk factor for atherosclerosis and cardiovascular diseases ( ). the imbalance in cholesterol homeostasis could result from either an increase in cholesterol synthesis and intake or a decrease in cholesterol removal from the body ( , ). a major route for eliminating cholesterol from the body occurs via the conversion of the insoluble molecules of cholesterol into detergent-like bile acids that are secreted in the intestine to emulsify dietary fat ( , ). the majority of the secreted bile acids are efficiently reabsorbed in the distal ileum and return back to the liver for resecretion in the intestine, whereas � – % of bile acids are excreted in the feces and compensated for by de novo synthesis, accounting for a major route of cholesterol removal from the body ( ). the circulating bile acids play important roles as signaling mole- cules and are critical modulators of lipid and cholesterol metabolism ( , ). under pathological conditions, an increase in the amount of bile acids returning to the liver causes more inhibition of cholesterol catabolism, leading to hypercholester- olemia ( , ). indeed, the size of the circulating pool of bile acids has been shown to be increased in diabetic patients and in animal models of diabetes mellitus ( , , ). however, the mechanism underlying this increase is not fully understood. intestinal absorption of bile acids has been shown to be the major determinant of the amount of bile acids circulating between the intestine and the liver ( , ). indeed, studies in animal models of diabetes mellitus have shown an increase in the intestinal absorption of bile acids ( ). moreover, recent clinical trials demonstrated the beneficial effects of blockers of bile acid absorption in improving the lipid and cholesterol profile in patients with diabetes mellitus ( , ). current therapy of inhibiting bile acid absorption is based on seques- tering them by using resins such as cholestyramine and colesevelam that bind bile acids in the intestinal lumen and prevent their absorption ( , ). however, poor patient com- pliance for cholestyramine and the need of high doses (several g/day) of bile acid sequestrants warrant the need for better and more efficient inhibitors of bile acid absorption ( , ). there- fore, further investigations are needed to delineate the modu- lation of bile acid absorption in diabetes mellitus at the mo- lecular level. the first and rate-limiting step of intestinal bile acid absorp- tion occurs via the ileal apical na�-dependent bile acid trans- porter (asbt) that is expressed on the brush-border membrane of ileal epithelial cells ( , ). the essential role of asbt in maintaining the recycling of bile acids is clear from studies showing the depletion of bile acid pool and the low level of plasma cholesterol in asbt knockout mice ( ). whether asbt expression is altered in diabetes mellitus is not known. diabetes mellitus is characterized by hormonal dysregulation, typically insulin imbalance. previous studies have shown that asbt function and expression are regulated by hormones such * f. annaba and k. ma equally contributed to this work. address for reprint requests and other correspondence: w. a. alrefai, univ. of illinois at chicago, jesse brown va medical center, medical research service ( / ), south damen ave., chicago, il (e-mail: walrefai@uic.edu). am j physiol gastrointest liver physiol : g –g , . first published july , ; doi: . /ajpgi. . . http://www.ajpgi.orgg downloaded from journals.physiology.org/journal/ajpgi at carnegie mellon univ ( . . . ) on april , . as glucocorticoids ( , , ). whether insulin modulates asbt expression via a mechanism that influences asbt expression in diabetes mellitus is not known. therefore, we investigated the modulation of asbt func- tion and expression in the widely used streptozotocin (stz)- induced model of diabetes mellitus in rats. to avoid confound- ing toxicity inflicted by the high dose of stz, we used the recently described model of inducing diabetes mellitus with multiple injections of low doses of stz ( ). our findings demonstrated an increase in asbt function and expression in the distal ileum of diabetic rats. the increase in asbt expres- sion reverted back to normal by insulin treatment. insulin decreased the promoter activity of rat asbt in intestinal caco- cells, suggesting that the lack of insulin contributes to the pathological increases in asbt expression in diabetic rats. our data suggest that asbt dysregulation induced by alter- ations in the level of insulin may underlie the increase in bile acid pool observed in diabetes mellitus. materials and methods animal studies. sprague-dawley rats (males, – wk of age, – g) were purchased from charles river laboratories (wil- mington, ma) and housed at the veterinary medical unit at the jesse brown veterans affairs medical center ( : -h light-dark cycle). animals had free access to food and water and were fed regular chow diet. diabetes mellitus was induced in the rats by low doses ( mg·kg body wt� ·day� ) of stz (sigma) administered by intraperitoneal injections on five consecutive days (stz was dissolved in citrate buffer, ph . ). control animals were injected with vehicle alone. blood samples were collected from tail snips, and the level of glucose was measured by a true-track glucometer (smart system). rats were killed wk after the last stz injection, and blood and small intestinal tissues were collected. human insulin (humalin; eli lilly) was given ( u/day) to a group of diabetic rats via subcutaneous osmotic mini pumps (osmotic pump model microl ; alzet) for days before death. the level of insulin in the plasma was evaluated using a rat/mouse insulin elisa kit (millipore, billerica, ma). each group of experimental animals contained at least three to six rats. immediately after euthanasia the intestine was removed and flushed with ice-cold � pbs, ph . . the small intestine was divided into three equal parts, and the mucosa from the first proximal part (jeju- num) and the distal part (ileum) was scraped, snap-frozen in liquid nitrogen, and stored at � °c for subsequent protein and rna extractions. small pieces of the intestine from different regions (jejunum, ileum, and colon) were cut and were immediately snap- frozen in optimal cutting temperature embedding medium (tissue- tek oct compound; sakura) for immunofluorescence analysis and laser capture microdissection. protocols for animal studies were ap- proved by animal care committees at the university of illinois at chicago and the jesse brown veterans affairs medical center. protein extraction and western blotting. frozen intestinal mucosa was cut, weighed ( – mg), and immediately transferred to ice-cold lysis buffer. the tissues were then homogenized by douncing in the lysis buffer containing mm tris-hepes, ph . , mm nacl, % triton x- , and protease cocktail inhibitors (roche). the homogenates were then centrifuged at , rpm to pellet the nuclei and other debris. the clear supernatant was removed, and the con- centration of protein was assessed by the method of bradford ( ). equal amounts of protein ( �g) were solubilized in laemmli sample buffer ( % sds, % glycerol, mm dithiothreitol, mm tris, ph . , . % bromphenol blue), separated on % tris-glycine sds polyacrylamide gel, and electrotransferred to nitrocellulose mem- branes. western blotting was performed by washing the nitrocellulose membranes three times and then blocking overnight in pbs contain- ing % nonfat dry milk. the blots were then incubated for h at room temperature or left overnight at °c with anti-rat asbt antibodies (santa cruz) diluted ( : ) in the blocking solution. blots were washed with pbs containing . % tween and then incubated with the same buffer containing donkey anti-goat antibodies (santa cruz). the bands were visualized by an enhanced chemiluminescence kit according to the manufacturer’s instructions (amersham, arlington heights, il). immunofluorescence analysis. snap-frozen tissues were sectioned ( �m) using cryostat, mounted on the slides, and preserved at � °c until further use. for immunostaining, the sections were fixed with % paraformaldehyde in pbs for min at room temperature followed by blocking in pbs containing % donkey serum and . % triton x- for min at room temperature. sections were then incubated with goat, anti-rat asbt antibodies (dilution : ) and rabbit, anti-villin ( : ) for h in the blocking buffer. after pbs washes, the sections were incubated with the secondary antibodies, alexa fluor - conjugated donkey anti-goat igg (green) and alexa fluor -conju- gated goat anti-rabbit igg (red) for min, and then washed and mounted with slow-fade dapi (blue, nuclei) by using cover slips. microscopy was performed with a � oil immersion objective of a zeiss immunofluorescence microscope (observer z ) equipped with deconvolution software (axiovision). laser capture microdissection. frozen intestinal tissue sections ( �m) were mounted on special stainless steel slide carrier pet foil (w. nuhsbaum, mchenry, il) suitable for laser capture microdissection. the tissues were stained with hematoxylin and eosin, and the cells were visualized with a leica laser capture microscopy system. epi- thelial cell size was estimated, and equal numbers of epithelial cells (� , cells) were cut from ileal tissues obtained from control and diabetic rats. total rna was then extracted, and the relative level of asbt mrna expression was evaluated by real-time rt-pcr as outlined below. rna extraction and quantitative real-time rt-pcr analysis. total rna was extracted from tissues and cells using the rneasy mini kit (qiagen) according to the manufacturer’s instructions. total rna was treated with dnase i to avoid genomic dna contamination. equal amounts of rna from both treated and control samples were reverse transcribed and amplified in a one-step reaction using a brilliant sybr green qrt-pcr master mix kit (stratagene). primers used for the current studies are mentioned in table . because the amplification efficiencies were approximately equal, the quantitation was expressed as a ratio of �ct(target) gene/ �ct(in- ternal control), where �ct represents the difference between the threshold cycle of amplification of rna from different experimental groups for target gene and internal control gene ( s or actin). isolation of intestinal epithelial cells and bile acid uptake. epithe- lial cells were isolated from the distal ileum as previously described ( ). briefly, intestinal fragment was washed with ice-cold pbs, table . primers used for real-time pcr primer rat asbt forward: =-ggttgcgcttgttattcctgt- = reverse: =-ggttcaatgatccaggcactt- = rat ost � forward: =-ccctcatacttaccaggaagaagctac- = reverse: =-ccatcaggaatgagaaacaggc- = rat ost � forward: =-tattccatcctggttctggcagt- = reverse: =-cgttgtcttgtggctgcttctt- = rat s forward: =-cccagtaagtgcgggtcataa- = reverse: =-gatccgagggcctcactaaac- = human asbt forward: =-gccccaaaaagcaaagatca- = reverse: =-gctatgagcacaatgaggatgg- = human actin forward: =-catgtttgagaccttcaacac- = reverse: =-ccaggaaggaaggctggaa- = asbt, apical na�-dependent bile acid transporter; ost, organic solute transporter. g high bile acid absorption in stz-induced diabetes mellitus ajp-gastrointest liver physiol • vol • october • www.ajpgi.org downloaded from journals.physiology.org/journal/ajpgi at carnegie mellon univ ( . . . ) on april , . opened, and sliced into small pieces (� cm). the intestinal pieces were then washed with hanks’ solution supplemented with % bsa and % glucose. the intestinal pieces were then incubated for min at °c with shaking in hanks’ solution containing . mm dithio- threitol and . mm edta to isolate the epithelial cells. the cells were collected with low-speed centrifugation at g for min, resuspended in the uptake buffer, and immediately used for assess- ment of bile acid uptake. cell viability was determined by trypan blue exclusion. a fraction of the same amount of cells was used for protein extraction and western blotting analysis to determine the amount of villin (the epithelial cell marker) per milligram protein of isolated cells. equal amounts of solution containing isolated intestinal epithe- lial cells were then used for na�-dependent [ h]taurocholic acid (tc) transport. cells were incubated at °c with buffer containing (in mm) nacl (with na�) or choline chloride (without na�), kcl, mgso , cacl , mannitol, and hepes (ph . ) as well as �m of [ h]tc (perkin elmer, boston, ma) for the designated period of time. the transport process was terminated by quick cen- trifugation followed by two washes with ice-cold pbs. cells were then solubilized with . n naoh for at least h. the protein concentration was measured by the method of bradford ( ), and the radioactivity was counted by a packard liquid scintillation analyzer tri-carb -tr (packard instrument, downers grove, il). na�- dependent tc uptake was expressed as picomoles per milligram protein, and the uptake values were normalized to the number of viable cells and the density of villin obtained by western blotting. transient transfection and luciferase assay. for in vitro studies, human intestinal caco- cells were obtained from american type culture collection and were cultured in minimum essential medium (eagle) adjusted to contain . g/l sodium bicarbonate, . mm nonessential amino acids, and . mm sodium pyruvate and supple- mented with fbs ( %). caco- cells were transiently cotransfected with the -kb rat asbt promoter construct ( ), pcmv� vector expressing �-galactosidase, and insulin receptor expression vector (generous gift from dr. michael quon from the national institutes of health, national center for complimentary and alternative medi- cine) by electroporation using amaxa technology (amaxa). briefly, � � cells were harvested and then were electroporated in �l of solution t (supplied by amaxa) along with �g of dna and then transferred to full media and plated on transwell inserts ( -well plate). cells were then incubated ( h after transfection) with insulin (humalin) for – h. cells were then washed with pbs and lysed in � of the reporter lysis buffer from promega. the activities of both firefly luciferase and �-galactosidase were measured by luminom- eter using kits from promega and clontech, respectively, according to the manufacturer’s instructions. the promoter activity was expressed as a ratio of luciferase to �-galactosidase activity in each sample. statistical analysis. results are expressed as means 	 se. stu- dent’s t-test was used in statistical analysis. p � . was considered statistically significant. results induction of diabetes mellitus by multiple low doses of stz. insulin-dependent diabetes mellitus in rats is commonly in- duced by a single high dose ( – mg/kg body wt) of intraperitoneal injection of stz. recent studies, however, indicated that high toxicity and severe weight loss caused by the high dose of stz might confound the results obtained from this model ( ). an alternative approach that uses multiple injections of low doses of stz ( mg/kg body wt) has been shown to be equally effective and less toxic ( ). to investi- gate alterations in asbt expression in diabetes mellitus, we used the rat model with multiple low doses of stz. because the results of stz injections could differ between different strains of rats, we first examined whether low-dose injections of stz ( mg/kg body wt) over five consecutive days are sufficient to produce diabetes mellitus with minimal toxic effects in sprague-dawley rats obtained from charles river laboratories international. rats ( – wk old) were injected (ip) with mg/kg body wt every day for five consecutive days. as shown in fig. a, the multiple low doses of stz induced hyperglycemia similar to that previously reported in the model with a high dose of stz. also, the level of insulin after days of the last injection was significantly decreased, further confirming insulinopenia in these rats (fig. b). how- ever, the diabetic rats maintained weight throughout the course of the experiments, as shown in fig. c, indicating that multiple low doses of stz are less toxic compared with fig. . multiple low doses of streptozotocin (stz) are not associated with weight loss. sprague-dawley rats obtained from charles river were given ip injections (Œ) with multiple low doses ( mg·kg body wt� ·day� ). control rats (�) were injected with vehicle alone (citrate buffer, ph . ). glucose was measured at the indicated day (a), and insulin level was assessed after the animals were euthanized (b). body weight was measured at the indicated day (c), demonstrating that the diabetic rats did not lose weight throughout the experiment. values are expressed as means 	 se from animals. *p � . compared with control. g high bile acid absorption in stz-induced diabetes mellitus ajp-gastrointest liver physiol • vol • october • www.ajpgi.org downloaded from journals.physiology.org/journal/ajpgi at carnegie mellon univ ( . . . ) on april , . previously reported weight loss caused by the high-dose model ( ). to further characterize the model of diabetes mellitus induced by multiple low doses of stz, we measured the levels of plasma cholesterol in control and experimental rats. our data showed that cholesterol levels were significantly increased in diabetic rats compared with control rats and that the high levels of cholesterol reverted back to normal with insulin treatment [ . 	 . mg/dl in control, . 	 . mg/dl in diabetic rats (p . ), and . 	 . mg/dl in diabetic rats with insulin treatment (p . )]. these data are consistent with previous studies ( ) showing an increase in plasma levels of cholesterol in rats with diabetes mellitus induced with a single high dose of stz. expression of intestinal bile acid transporter is increased in diabetic rats. previous studies have shown an increase in the bile acid pool in diabetic patients and rats with experimentally induced diabetes mellitus ( , , ). because intestinal bile acid absorption is one of the major processes that determine the amount of circulating bile acids ( ), we investigated the ex- pression of intestinal bile acid transporters. total rna was extracted from intestinal mucosal scrapings from diabetic and control rats, and the level of ileal asbt expression was assessed by real-time qrt-pcr. as shown in fig. a, the relative level of asbt mrna was significantly increased in diabetic rats compared with control. furthermore, the expres- sion of bile acid organic solute transporters (ost) � and �, responsible for the transport of bile acids across the basolateral membrane of intestinal epithelial cells, was also significantly increased in diabetes mellitus (fig. b). it has been previously reported that hyperplasia and hypertrophy might contribute to the increase in the absorptive processes observed in rats with diabetes mellitus induced by a high dose of stz ( ). in the current model of multiple low doses of stz-induced diabetes mellitus, the intestinal epithelia appear to be hy- perplastic with no sign of hypertrophy compared with con- trol rats (fig. c). we used the laser capture microdissec- tion technique to further determine that hyperplasia is not contributing to the observed increase in asbt mrna expression in diabetic rats. tissues harvested from the distal ileum of diabetic and control rats were embedded in optimal cutting temperature media, and frozen tissues were sec- tioned (� �m). hematoxylin- and eosin-stained tissues where then visualized under the microscope, and an equal number of epithelial cells were cut by the laser from tissues of diabetic and control rats. cells were then collected, and total rna was extracted. as depicted in fig. d, asbt mrna expression normalized to the same number of epi- thelial cells was significantly increased in ileum of diabetic rats compared with control. collectively, these data indicate that the expression of ileal asbt and other intestinal bile acid transporters is significantly elevated in rats with stz- induced diabetes mellitus. insulin treatment decreases the elevated expression of asbt in diabetic rats. to further examine the role of insulin defi- ciency in the observed increase in asbt expression, we treated a group of diabetic rats with human insulin supplied continuously from a subcutaneous osmotic pump. replacement of insulin for days from the osmotic pump at the rate of u/day was sufficient to decrease the high levels of glucose to almost normal levels. we next investigated the effect of insulin treatment on asbt protein expression in rats with stz- induced diabetes mellitus. total protein lysates were prepared from mucosal scrapings obtained from rat distal ileum and investigated for asbt protein expression by western blotting. as shown in fig. a, anti-asbt antibodies detected the expected size band in distal ileum but not in jejunum or colon, indicating their specificity. similar to the increase in asbt mrna, fig. b shows a significant increase in asbt protein expression in the distal ileum of diabetic compared with control rats. furthermore, asbt protein expression in diabetic rats was reverted back to control levels after days of insulin treatment. in addition, immunofluorescence analysis demon- strated an increase in asbt staining (green) in epithelial cells of the distal ileum from diabetic rats compared with asbt staining in tissues obtained from control and diabetic rats with insulin treatment (fig. c). these findings suggest that insulin may directly modulate asbt expression in intestinal epithelial cells. asbt function is increased in isolated ileal enterocytes. to examine changes in asbt function in diabetic rats, we mea- sured the na�-dependent transport of [ h]tc in isolated epi- thelial cells. intestinal epithelial cells were isolated from the distal ileum as previously described ( ). cells in suspension were then incubated with the uptake buffer containing �m [ h]tc in the presence and the absence of na�. cell viability was determined by trypan blue exclusion. the amount of villin (as a marker for epithelial cells) in each sample of isolated epithelial cells was estimated by western blotting to ensure equal loading of epithelial cells in each uptake sample per milligram of protein. as shown in fig. a, the na�-dependent uptake in isolated enterocytes was linear as a function of time. figure b shows na�-dependent uptake was significantly increased in cells isolated from diabetic rats compared with control animals. taken together, these data suggest that the insulin deficiency resulted in an increase in asbt function and expression. insulin decreases rat asbt promoter activity in intestinal epithelial cells. because insulin treatment to diabetic animals reduced the elevated level of asbt protein, we hypothesized that insulin directly modulates asbt expression in intestinal epithelial cells. therefore, the modulation of asbt mrna expression by insulin was investigated using caco- cells. cells were plated on transwell inserts and exposed to insulin from the basolateral side. as shown in fig. a, asbt mrna expression was significantly decreased by incubation with nm insulin. we next investigated the effects of insulin on the activity of a previously characterized kb of rat asbt pro- moter ( ). caco- cells were transiently cotransfected with rat asbt promoter by electroporation using amaxa technology along with mammalian expression vector for human insulin receptor. as shown in fig. b, incubation with nm insulin for h significantly decreased rat asbt promoter activity in caco- cells by � – %. insulin effects on rat asbt pro- moter activity were specific, since activation of insulin receptor in caco- cells did not alter the promoter activity of the slc a anion transporter (fig. b). also, the observed decrease was dependent on the amount of the expressed insulin receptor, as shown in fig. c. these data clearly indicate that insulin directly downregulates asbt expression and promoter activity in intestinal epithelial cells. g high bile acid absorption in stz-induced diabetes mellitus ajp-gastrointest liver physiol • vol • october • www.ajpgi.org downloaded from journals.physiology.org/journal/ajpgi at carnegie mellon univ ( . . . ) on april , . discussion diabetes mellitus is associated with a high risk of develop- ing cholesterol-related disorders such as atherosclerosis ( , , ). a major factor causing hypercholesterolemia has been suggested to be an increase in the circulating pool of bile acids reported in diabetic patients and animal models of diabetes mellitus ( , , ). however, the mechanism(s) involved in the expansion of the bile acid pool in diabetes mellitus is not fully defined. the current studies present novel data demonstrating an increase in the function and expression of ileal bile acid transporter asbt in rats with stz-induced diabetes mellitus. the increase in asbt expression was reversed by insulin fig. . apical na�-dependent bile acid transporter (asbt) expression is increased in diabetes mellitus. distal ileum was removed from control and diabetic animals, and mucosa was scraped as mentioned in materials and methods. total rna was extracted, and the relative expression of asbt mrna (a) and organic solute transporter (ost) � and � mrna (b) was assessed by real-time rt-pcr using sybr green. the level of mrna expression for each target gene was normalized to the expression of s mrna in the same sample. in c, immunofluorescence staining for actin (red) in optimal cutting temperature (oct)-embedded sections of rat ileum from control and diabetic rats showing long villi but no apparent change in the size of epithelial cells. in d, ileal segments were embedded in oct media, sectioned to �m thickness. the tissues were then mounted on special slides suitable for laser capture microdissection as mentioned in materials and methods. ileal sections were stained with hematoxylin-eosin (h&e), and epithelial cells were visualized under the microscope. equal number of cells was cut by laser from tissues from control and diabetic rats. cells were collected, and total rna was extracted. the relative expression of asbt mrna normalized to s mrna was evaluated by real-time rt-pcr. values represent means 	 se from at least – animals. *p � . compared with control. g high bile acid absorption in stz-induced diabetes mellitus ajp-gastrointest liver physiol • vol • october • www.ajpgi.org downloaded from journals.physiology.org/journal/ajpgi at carnegie mellon univ ( . . . ) on april , . treatment of diabetic rats. furthermore, we demonstrated that insulin directly modulated rat asbt promoter activity in intestinal epithelial cells. our data suggest that insulin defi- ciency is involved in the observed upregulation of asbt, which may underlie the associated enlargement in the circu- lating pool of bile acids in stz-induced diabetes mellitus. stz has been widely used to induce diabetes mellitus in rodents. stz is an antibiotic that destroys �-cells in the pancreas, causing insulinopenia, hyperglycemia, and a disease process similar to type diabetes mellitus ( , ). conven- tionally, diabetes mellitus in rats is induced by a single intra- peritoneal injection of a high dose of stz ( – mg/kg body wt) ( , ). a high dose of stz, however, has been shown to cause rapid and severe weight loss with other toxic effects influencing gastric motility and the function of gastrointestinal neuroendocrine cells ( , ). therefore, observations pertain- ing to the gastrointestinal tract obtained from the high-dose model of stz-induced diabetes mellitus should be interpreted with caution. on the contrary, recent studies using multiple low doses of stz have shown less toxicity and similar efficiency of induction of diabetes mellitus compared with the single high dose ( ). our current data confirmed these observations in sprague-dawley rats (obtained from charles river), demon- strating efficient induction of insulinopenia and hyperglycemia fig. . asbt protein expression is upregulated in diabetic rats. total protein extracts were prepared from mucosal scrapings from rat colon and small intestine. equal amounts of protein were separated on % sds-page and electrotransferred to nitrocellulose blots. blots were then probed with anti-rat asbt antibodies, and bands corresponding to asbt fusion proteins were visualized as described in materials and methods. blot in a shows that asbt expression is detected in rat ileum but not jejunum or colon. in b, a representative blot demonstrating an increase in asbt protein expression in diabetic rat ileum compared with control is shown. insulin treatment to diabetic rats reversed the increase in asbt protein expression to the level of control rats. in c, immunofluorescence staining for asbt (green) and villin (red) with blue-stained nuclei in oct-embedded sections of rat ileum from control, diabetic, and insulin-treated diabetic rats is shown. data are representative of different experiments. g high bile acid absorption in stz-induced diabetes mellitus ajp-gastrointest liver physiol • vol • october • www.ajpgi.org downloaded from journals.physiology.org/journal/ajpgi at carnegie mellon univ ( . . . ) on april , . by five consecutive injections with a low dose of stz ( mg·kg body wt� ·day� ). noticeably, diabetic rats did not lose weight as opposed to the drastic loss of weight associated with the high dose of stz. upon insulin treatment from subcuta- neous osmotic pumps, glucose levels were restored to normal (data not shown), indicating the efficiency of insulin treatment and suitability of the model to investigate the role of insulin in the modulation of intestinal bile acid absorption associated with insulin deficiency. intestinal bile acid absorption is a major determinant of the enterohepatic circulation of bile acids ( , ). the increase in bile acid absorption causes an enlargement in the circulating pool, whereas a reduction in the intestinal absorption leads to depletion of the bile acid pool ( , ). ileal asbt mediates the first and the rate-limiting step of intestinal bile acid absorption ( , ). bile acids are then transported by the intracellular ileal bile acid-binding protein and exit the cell across the basolateral membrane via the action of a heterodimer of ost � and � ( , ). studies in knockout mice provided compelling evidence fig. . rat asbt promoter activity is decreased by insulin. a: intestinal caco- cells were plated on transwell inserts and were exposed to nm insulin for h from the basolateral compartment. total rna was extracted, and asbt mrna expression was assessed by real-time rt-pcr normalized to actin mrna level (internal control). control cells were treated with vehicle only. b: caco- cells were transiently cotransfected by electroporation (amexa) with rat asbt or the promoter or human slc a transporter along with expression vector of human insulin receptor and cmv � for �-galacto- sidase to normalize for the transfection efficiency. cells were plated on transwell, treated with nm insulin for h, and harvested for firefly luciferase and �-galactosidase assays to assess the promoter activity. control cells are cells treated with vehicle only. c: caco- cells were transfected with rat asbt promoter along with different amounts of the expression vector of human insulin receptor. cells were plated on transwell, treated with nm insulin from h, and harvested for firefly luciferase and �-galactosidase assays. control cells are transfected with rat asbt promoter and �-galacto- sidase vectors only without insulin receptor expression vector. results are presented as % of control and represent means 	 se for – determinations performed on separate occasions. *p � . compared with control. fig. . asbt function is elevated in rats with diabetes mellitus. intestinal epithelial cells were isolated from ileal segments, washed, and immediately resuspended in uptake buffer containing �m of [ h]taurocholic acid (tc) in the presence or absence of na�. uptake was terminated at the indicated time point by two washes in ice-cold pbs buffer and was expressed as pmol/mg protein. na�-dependent [ h]tc uptake was linear up to min of incubation with uptake buffer (a). na�-dependent [ h]tc uptake was evaluated in isolated epithelial cells from control and diabetic rats (b). results are presented as means 	 se of uptake values obtained from different animals. where not shown, error bars are smaller than symbol. *p � . compared with control. g high bile acid absorption in stz-induced diabetes mellitus ajp-gastrointest liver physiol • vol • october • www.ajpgi.org downloaded from journals.physiology.org/journal/ajpgi at carnegie mellon univ ( . . . ) on april , . demonstrating that asbt represents the essential intestinal component of the enterohepatic circulation of bile acids ( ). furthermore, the fact that plasma cholesterol is reduced in asbt knockout mice strongly indicates the role of asbt in cholesterol homeostasis ( ). our findings showed an increase in asbt and ost � and � mrna expression in rats with stz-induced diabetes mellitus. it could be argued that the observed increase in intestinal bile acid transporters is a result of the associated hypertrophy and hyperplasia in the intestinal epithelia ( ). while histological examination of the intestinal epithelia in our model revealed an increase in the length of the villi, there were no apparent changes in the size of epithelial cells excluding hypertrophy. furthermore, the amount of dna extracted per milligram protein harvested from the distal ileum of diabetic and control rats showed no significant changes (data not shown), ruling out a possible epithelial hypertrophy. ad- ditionally, data obtained by laser capture microdissection from an equal number of ileal cells collected from diabetic and control rats show a significant increase in asbt mrna expression at the cellular level and therefore minimize a role of epithelial hyperplasia or hypertrophy in the observed changes. our data also show that asbt protein expression was significantly increased in diabetes mellitus as judged by the following two complementary approaches: western blotting and immunofluorescence. the specificity of rat asbt antibod- ies (obtained from santa cruz) is confirmed by the fact that the expected size band is detected in ileum but not in jejunum and colon similar to asbt pattern of expression. asbt staining was predominantly present on the apical membranes of epithe- lial cells as indicated by the colocalization with villin (the marker for the apical membrane of epithelial cells). it should be noted that the level of villin expression observed by immu- nostaining was not altered in diabetic rats compared with normal rats, indicting that the observed changes are specific for asbt. along with the increase in asbt mrna and protein expression, our findings showed that asbt activity was sig- nificantly increased in diabetes mellitus. asbt function in the current studies was examined as na�-dependent [ h]tc up- take in isolated intestinal epithelial cells. the increase in asbt activity in isolated epithelial cells further rules out the involve- ment of hypertrophy or hyperplasia in the observed increase in asbt function and expression in stz-induced diabetes mel- litus. insulin deficiency is the main characteristic of stz-induced diabetes mellitus in rats ( ). interestingly, the increase in asbt protein expression demonstrated both by western blot- ting and immunofluoresce was decreased back to the normal level by insulin treatment. this observation suggested the direct involvement of insulin in the regulation of asbt ex- pression. in this regard, the role of insulin in the regulation of a number of intestinal epithelial processes is well established. for example, insulin has been shown to stimulate the function of oligopeptide transporter, pept- ( ), to decrease the trans- epithelial resistance ( ), and to abrogate calcium-dependent chloride secretion in t intestinal epithelial cells ( ). addi- tionally, previous studies have shown that insulin acutely decreased glucose flux across rat jejunum and ileum ( ). indeed, our data showed that insulin directly downregulated asbt expression in an in vitro model using intestinal caco- cells. furthermore, our data demonstrate that insulin has a negative effect on rat asbt promoter activity, indicating alterations at the level of gene transcription. in this regard, insulin is known to stimulate several downstream effector molecules in various cells types. for example, pkc� isoform activation was shown to mediate the effects of insulin on the translocation of glut to the plasma membrane of rat adipo- cytes ( ). interestingly, the role of pkc� in the transcriptional and posttranscriptional regulation of asbt has been recently shown ( , ). therefore, future studies will focus on deter- mining the role of pkc� and other signaling molecules in the regulation of asbt expression and promoter activity by insu- lin. also, preliminary sequence analysis identified a potential binding site � , bp upstream of the transcription initiation site for the ccaat/enhancer binding protein that has been previously shown to mediate the effects of insulin on gene transcription ( ). future studies should focus on determining the role of this transcription factor in mediating the regulation of asbt expression and promoter activity by insulin. in summary, the current studies demonstrated that asbt function and expression are increased in a stz-induced rat model of diabetes mellitus. the increase in asbt expression may contribute to the enlargement in the circulating pool of bile acids and high plasma level of cholesterol occurring in diabetes mellitus. interestingly, recent clinical trials have shown the beneficial effects of bile acid sequestrants in im- proving lipid profile and hyperglycemia in patients with dia- betes mellitus. the use of bile acid sequestrants to block bile acid absorption, however, is problematic because of the un- pleasant nature of cholestyramine and the need of high dose (in grams) for effective treatment. the data described in the current studies provide a unique experimental tool to further investigate the impact of asbt inhibition on the associated hypercholesterolemia. grants these studies were supported by grants from the department of veteran affairs (w. a. alrefai and r. d. kineman), national institute of diabetes and digestive and kidney diseases grants f dk- (f. annaba), dk- (b. l. shneider), dk- (r. k. gill), and dk- (w. a. alrefai), and the crohn’s and colitis foundation of america ccfa ref. no. 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differential effects of streptozotocin- induced diabetes on expression of hepatic abc-transporters in rats. gastroenterology : – , . g high bile acid absorption in stz-induced diabetes mellitus ajp-gastrointest liver physiol • vol • october • www.ajpgi.org downloaded from journals.physiology.org/journal/ajpgi at carnegie mellon univ ( . . . ) on april , . extracellular vesicles, apoptotic bodies and mitochondria: stem cell bioproducts for organ regeneration cellular transplants (g orlando, section editor) extracellular vesicles, apoptotic bodies and mitochondria: stem cell bioproducts for organ regeneration natalia gebara & andrea rossi & renata skovronova & justine mariam aziz & amish asthana & benedetta bussolati , # the author(s) abstract purpose of review in the current work, we will present the characterization of the main different stem cell-derived vesicular bio- products with potential application in organ regeneration. recent findings the therapeutic effects of stem cell therapy in organ repair, specifically those utilizing mesenchymal stromal cells, are largely dependent on the cells’ release of different bio-products. among these bio-products, extracellular vesicles (evs) appear to play a major role due to their ability to carry and deliver bioactive material for modulation of cellular pathways in recipient cells. concurrently, mitochondria transfer emerged as a new mechanism of cell communication, in which the bioener- getics of a damaged cell are restored. finally, apoptotic bodies released by dying apoptotic stem cells contribute to stimulation of the tissue’s stem cells and modulation of the immune response. summary exploitation of isolated extracellular vesicles, mitochondria and apoptotic bodies in preclinical models of organ damage shows promising results. here, we describe the results of the pre-clinical applications of stem cell vesicular products, as well as the first clinical trials approaching artificial administration of extracellular vesicles and mitochondria in human subjects and their possible benefits and limitations. keywords msc . regenerative medicine . microrna . exosomes . microvesicles . mitochondrial transfer . apoptosis introduction organ failure is the most frequent cause of morbidity and mortality recorded in europe and in the united states in recent decades. organ dysfunction can be attributed to fibrosis, a pathological feature of many chronic inflammatory diseases, as its extensive remodelling of tissues leads to functional in- sufficiency [ ]. the burden associated with fibrosis is discon- certing, representing in the united states almost half, and in the industrialized world about %, of all deaths attributed to fibrotic heart, lung, kidney and liver diseases [ , ]. in addition, episodes of acute tissue injury, especially if severe and repeated, are closely associated to development of chronic organ disease [ ]. it has therefore become of increasing interest in regenerative medicine to limit the progression of fibrosis, promote restoration of organ function in chronic settings and support organ repair after acute injury to regain tissue integrity. in this context, increas- ing studies underline the role of stem cell bio-products, including secreted soluble factors and extracellular vesicles (evs), as pow- erful instruments in organ regeneration. evs, in particular, have been proposed as a new form of intracellular messaging through their ability to reach distant organs and deliver the active cargo necessary for reprogramming of the target cells. in addition, evs released by apoptotic cells, including apoptotic bodies (apobds), are recently emerging as part of the therapeutic and immune-modulating mechanisms of injected stem cells within injured tissues [ ] (fig. ). finally, stem cell therapy involves the transfer of mitochon- dria, the organelles responsible for cellular energy production, from stem cells to damaged cells. mesenchymal stromal cells natalia gebara, andrea rossi and renata skovronova contributed equally to this work. this article is part of the topical collection on cellular transplants * benedetta bussolati benedetta.bussolati@unito.it department of molecular biotechnology and health sciences, university of torino, torino, italy wake forest univ. school of medicine, winston-salem, nc, usa molecular biotechnology centre, university of torino, via nizza , torino, italy https://doi.org/ . /s - - - current transplantation reports ( ) : – april published online: http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf https://orcid.org/ - - - mailto:benedetta.bussolati@unito.it (mscs) are shown to transfer mitochondria to the recipient cells in different ways: encapsulated within evs [ ]; via cell- to-cell direct communication through tunnelling nanotubes; or through direct release of “naked” mitochondria into the extra- cellular microenvironment [ ]. the organelle incorporates in- to the endogenous mitochondrial network of the damaged recipient cell that needs to be rescued, restoring its bio- energetic profile and health [ ]. in this review, we will present the recent knowledge on mechanisms of action involved in the therapeutic effects of healthy and apoptotic evs, as well as of mitochondria transfer, and the exploitation of these bio-products in preclinical models of organ damage. finally, we will describe the first clinical trials approaching their use on human subjects and the possible benefits and limitations. extracellular vesicles since the ‘discovery’ of evs in blood plasma in by erwin chargaff and rudolph west [ ], interest in those cells-to-cell communicators has risen in almost all fields of biology and chemistry. evs have been proven to natu- rally occur in prokaryotes, eukaryotes, plants and cells. evs are membrane-bound, spherical particles enclosed in a lipid bilayer. in biological samples, evs originate from their parental cell, taking up their internal and exter- nal composition. guidelines from the international society for extracellular vesicles (isev) classify three main categories of evs; exosomes also named small evs (~ – nm), large evs or microvesicles (~ – nm) and apobds (> μm) [ ••]. small evs and microvesicles are released from metabolically active cells, whereas apobds are exclusively produced during cell apoptosis [ ] (fig. ). the content of an ev is dependent on its origin, size and the route of biogenesis. ev surface markers and cargo are specific to the three types of vesicles (table ) and are most commonly associated with the route of vesicle formation. the process of exosome formation begins with inward budding of early endosomes and formation of intraluminal vesicles; this involves the escort complex, alix and tumour suscepti- bility gene (tsg ), all of which are responsible for cargo sorting. intraluminal vesicles mature into multi- vesicular bodies, followed by fusion with the cell membrane and release of vesicles into the extracellular environment. alternatively, multi-vesicular bodies are degraded by lyso- somes and their components recycled. exosomes are distin- guished by the presence of all three tetraspanin markers re- sponsible for induction of membrane curvature (cd , cd and cd ). proteins that can be detected and are involved in exosome biogenesis include rab, gtpases, annexin, flotillin, d a b c apoptotic cell healthy cell a b c d fig. presentation of apoptotic and healthy cell secretome curr transpl rep ( ) : – alix, tsg , vps , heat shock protein (hsp ) and the escort complex [ ]. microvesicles are formed by direct budding from the cell plasma membrane with involvement of cytoskeleton compo- nents and fusion machinery, though the process is not yet fully understood. due to their biogenesis pathway, microvesicles are primarily composed of a plasma membrane and of cytosolic-associated proteins. other commonly found compo- nents include heat shock proteins, integrins, post- translationally modified proteins and rna species. several structural components are shared between exosomes and microvesicles due to their similar release pathways and origin [ ]. evs have been designated as novel cell-to-cell communi- cators due to their effect on cells on a paracrine and endocrine level, specifically through the direct stimulation of cell surface receptors and transfer of bioactive molecules. indeed, ev sur- face receptors may act as signalling complexes and directly stimulate target cells or, alternatively, transfer functionally ac- tive receptors from one cell to another. for instance, bystander b cells can acquire antigen receptors from activated b cells becoming specific activated antigen presenting cells for cd t cells [ ]. in addition, the presence of a complex cargo (mirna, rna, proteins, lipids, cytokines and mitochondria) within evs results in a multilevel modulation of cell functions in the recipient cells [ , ]. small rna species, including mirna, are present within evs and, interestingly, recent studies found that the overall pattern of mirna content of small and large evs appears similar but distinctly different from that of the originating cells [ ••], implying specific mechanisms of mirna packaging into evs. moreover, ex- tensive proteomic studies on evs originating from cell cul- tures, tissue cultures and isolated bio-fluids have shown a significant ev protein content. online databases created through the collaboration of ev research groups provide us with catalogued ev components, such as exocarta, (www. exocarta.org), evpedia (www.evpedia.info) and vesiclepedia (www.microvesicles.org). evs contain many common proteins involved in vesicle trafficking and serving as part of the cytoskeleton and the plasma membrane. furthermore, specific protein content reflects the ev mechanism of generation and origin, as well as the cellular state of the ev originating cell. finally, in addition to the structural functions of lipids in ev membranes, bioactive lipids such as eicosanoids, fatty acids and cholesterol are transferred by evs to recipient cells. for instance, sphingomyelin has been shown to regulate angiogenesis in vitro and in vivo in tumour derived evs [ ]. apoptotic evs apoptosis is commonly known as programmed cell death. an apoptotic cell undergoes several morphological changes: membrane blebbing, membrane protrusion formation and generation of apobds [ – ]. the membrane of apobds reflects the main changes occurring in the cell surface of the apoptotic cell. in particular, apoptotic cells express markers promoting their removal by surrounding cells or macrophages before the cell membrane ruptures [ ]. for instance, calreticulin, an “eat me” ligand is physiologically silenced by the cd “don’t eat me” ligand; and only expressed by cells and apobds when cd is downregulated [ ]. the size of apobds ranges from μm to μm [ , ]. this characteristic is similar to oncosomes (evs secreted by cancer cells), but the biogenesis of these vesicles differs [ ]. the number of apobds produced per cell was quantified as . ± . per hour [ ••]. in comparison, the average num- ber of released evs by mesenchymal stem cells was found be in the range of per cell, overnight [ ]. during apoptosis, apoptotic microvesicles . – μm in di- ameter and small exosome-like evs are released [ , ]. however, these vesicles are less characterized than the apobds. apobds are characterized by the presence of externalized phosphatidylserine and by a permeable membrane. as men- tioned above, they express phagocytosis-promoting signals, such as calreticulin [ ] and calnexin [ ]. in addition, apobds express chemokines and adhesion molecules, such as cx cl /fractalkine and icam , and mhc class ii table composition, size and biogenesis route of evs vesicle type origin size markers components exosomes endolysosomal pathway; fusion of mvb with cell membrane - nm cd , cd , cd escort components, tsg , flotillin, annexin mrna, mirna and other non-coding rnas; membrane and cytoplasmic proteins, lipids, receptors microvesicles cell membrane; bud off directly from cell surface - nm cd mrna, mirna and other non-coding rnas; membrane and cytoplasmic proteins, lipids, receptors apoptotic bodies cell membrane; membrane blebbing during apoptosis > μm phosphatidylserine, calreticulin, calnexin nuclear fragments and cell organelles curr transpl rep ( ) : – http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / molecules, allowing for direct antigen presentation to cd + t cells and activation of immunological memory [ ]. the car- go of apobds consists of cellular components enclosed dur- ing protrusion. due to this fact, the content of apoevs can be very diverse. indeed, apobds can contain micrornas, rna and dna. diversity of apobds content affects their physio- logical properties. apobds can be subdivided into two groups: dna-carrying apobds and cytoplasm-carrying apobds. ′ phosphorylated blunt-ended dna can be used as a distinctive marker of dna-carrying apobds because it is exclusively found in apobds, which undergo apoptosis and contain the dna fragments [ ]. mitochondria while mitochondria are widely considered the powerhouse of the cell, as they are responsible for atp production through oxidative phosphorylation, these organelles are also involved in several other pathways. they serve a role in pluripotent stem cell maintenance [ ], apoptosis and cell death regula- tion and proliferation capacity through complex interactions between p and reactive oxygen species (ros) production [ ]. energy deprivation and mitochondria dysfunction has been strictly associated with end stage organ disease [ ]. therefore, while metabolic patterns and mitochondria con- tents can strictly vary among the organs, it appears evident that mitochondrial alterations are closely correlated with most of the clinical conditions that lead to organ failure [ , ]. as mitochondria do not possess an efficient dna-repair system [ ], these organelles are typically recycled through mitophagy, a form of autophagy [ ]. moreover, the transfer of respiration-competent mitochondria from cell to cell emerged in the past few years [ ] as a mechanism of damage repair or cell reprogramming [ ]. the physiological mito- chondria transfer is a biological phenomenon in which the organelle from a healthy donor cell is relocated into a stressed recipient cell, resulting in repair and survival of the damaged cell. during this process, the mitochondria’s small size and plasticity allow it to be transported from donor to recipient cells through transporting mechanisms, such as tunneling nanotubes and microvesicles. the organelle is eventually in- corporated into the endogenous mitochondrial network of the recipient cell that needs to be rescued, restoring the cell to its bio-energetic profile and health [ ]. the incorporation of respiration-competent mitochondria within released microvesicles has been extensively studied in mscs, where, once internalized, mitochondria-containing microvesicles can rescue cells from injury or act as reprogramming factors [ , ••]. in , islam et al. demon- strated that mitochondria can be transferred in vivo through a mouse model of acute lung injury [ ]. firstly, mitochondria- labelled mscs were administered by injection into the damaged lungs. these mscs homed in the damaged tissue and produced microvesicles containing the labelled mitochon- dria h post-injection. the microvesicles were then directly transferred into the damage lung cells, resulting in the restora- tion of their atp concentrations and secretory responses [ ]. pmt has also been observed through the establishment of tunnelling nanotubes (tnts) [ ]. tnts are filamentous connec- tions formed by protrusions of a cell’s membrane and are used to share organelles and contents of the cell’s cytoplasm with other cells [ ]. the utilization of tnts in pmt has been observed in transfers between mscs and macrophages, which served to enhance macrocytosis and activate antimicrobial re- sponse [ ]. moreover, sinclair et al. demonstrated that the transfer of mitochondria via tnts is essential due to the regen- erative capacity exerted by mscs [ ]. however, the role and mechanism of pmt through the establishment of tnts is poor- ly understood. the last and least known mechanism of pmt is the direct release of mitochondria in the extracellular microen- vironment, usually in response to cell stress. “naked” mito- chondria can be encapsulated in a vacuole and then extruded by apoptotic cells, specifically hepatocytes [ ]. similarly, platelets can release respiration-competent mitochondria as a mediator of innate immune response [ ]. therapeutic effects of evs, apobds and mitochondria for organ regeneration ev reprogramming of injured tissue following the emerging interest of stem cell therapy, an in- creasing number of research and pharmaceutical groups are focusing on stem cell derived evs, specifically msc-derived evs, as a new form of therapeutic agents for organ regenera- tion and protection [ – ]. due to the ability of msc- derived evs to transfer therapeutic molecules, such as mrnas, mirnas and protein, and several of their regenerating effects to mscs, this source of evs is one of the most studied in regenerative medicine. moreover, mscs produce a higher number of evs in comparison with other stem cells [ ]. msc-derived evs were proven to mod- ulate the immune system and stimulate regeneration in a mul- titude of preclinical models, including graft-versus-host-dis- ease, lung, liver, kidney and cardiovascular injury [ ]. there are a significant number of studies describing the pro- regenerative effects of stem cell-evs for organ regeneration, but it is not within the scope of this review to detail all the different preclinical models of application. as detailed above, the therapeutic effect of stem cell-evs on organ repair is related to transfer of pro-regenerative proteins or micrornas. for instance, although numerous factors have demonstrated the therapeutic effects of different ev models and origins, not a single agent emerged as pivotal or curr transpl rep ( ) : – indispensable. therefore, it can be hypothesized that not a single factor, but rather a synergic and multi-target action of ev com- ponents is responsible for the therapeutic ev results. indeed, the common and characterizing action of therapeutic msc-evs can be described as a reprogramming activity on tissue expression profiles. for instance, in models of kidney and liver injury [ ], the expression profile of the ev-treated diseased organ, as assessed by rna sequencing, correlated with that of the normal tissue. moreover, in models of chronic tissue injury, an upregu- lation of anti-fibrotic genes and downregulation of pro-fibrotic genes was common to the different diseases as well as stem cell sources [ , ]. through modulation of their phenotype and subsequent secretomes, therapeutic utilization of evs may ben- efit from an in vitro stimulation or manipulation of the generat- ing cell source. for instance, ischemic or hormonal stimulation may ameliorate evactivities [ – ]. in addition, evadminis- tration for chronic diseases might require multiple administra- tions. the possible development of immune reactions in this setting has not yet been studied in depth. regenerative effect of apoptotic body phagocytosis due to the rapid clearance of damaged cells by immune cells, namely phagocytes, apobds play a major role in immune reg- ulation [ , ]. apobds are emerging as a pivotal tool in cell- to-cell communication between damaged and healthy cells, therefore modulating mechanisms of organ repair. indeed, apobds may stimulate proliferation of resident stem/progenitor cells, improving tissue regeneration and replac- ing damaged cells [ ••, ]. for instance, phagocytosis of the apobds by hepatic stellate cells can promote their differentia- tion and increase their cell survival [ ]. moreover, apobds’ engulfment may support msc homeostasis. in particular, sys- temic infusion of exogenous apobds was able to rescue apo- ptotic mscs by transferring rnf and mir- - p and ac- tivating the wnt/β-catenin signalling [ •]. in parallel, in zebrafish, dying epithelial stromal cells of the epidermis were observed to generate wnt a enriched apobds, supporting the hypothesis of apobds being biologically active vehicles in cell-to-cell communication [ ••]. neighbouring p -positive stromal cells engulfed the apobds, which caspase- dependently activated wnt signalling and stimulated cell pro- liferation and tissue homeostasis over h. in this model, inhi- bition of apoptosis significantly reduced the number of prolif- erating stromal cells. on the contrary, overexpression of the wnt pathway in combination with apoptosis induced a signifi- cant increase of stromal cell proliferation [ ••]. apobds can deliver micrornas, dna and other genetic material to target cells, resulting in a multitude of different effects. for example, mirna- , present in endothelial apobds, promoted chemokine cxcl expression in healthy endothelium, and repeated administration of those apobds in mice with atherosclerosis induced an athero- protective effect [ ]. although the use of apobds generated in culture as ther- apeutic has not yet been tested, their role appears of increasing interest in the field of regenerative medicine. artificial mitochondria transfer in recent years, artificial mitochondrial transfer (amt) emerged as a new possible therapeutic option for tissue repair. amt has been intensively investigated in cardiac disease models. in a rabbit model of cardiac iri, the injection of viable respiration-competent mitochondria, isolated from donor rabbit cardiac or muscular tissues, was able to significantly reduce the infarct size area, kinase mb, cardiac troponin-i and apoptosis in the regional ischemic zone [ , ]. amt has also been recent- ly tested in a mouse model of heterotopic heart transplantation: mitochondria isolated from gastrocnemius muscle were autologously administrated in the heart coronary ostium before and after the transplant. within h after transplant, necrosis and neutrophil infiltration were significantly decreased com- pared with the vehicle-treated group. moreover, the mitochon- drial treatment significantly enhanced the beating score after transplant [ ]. interestingly, these papers demonstrated both in vitro and in vivo that fully differentiated cells can be used as a source for the mitochondrial injection. moreover, transfer of mitochondria, mainly derived from mscs, has been proven effective in other pathologic models involving liver, brain and kidney. in a rat model of liver iri, the intra-splenic administration of msc mitochondria mitigat- ed the necrosis of hepatocytes as well as reduced the expres- sion of mitochondrial-induced apoptosis markers [ ]. in the kidney, the effectiveness of amtwas demonstrated by rescue of damaged renal proximal tubular cells. in vitro, the admin- istration of msc-derived mitochondria reduced ros produc- tion and increased the expression of the tubular marker megalin and mitochondrial superoxide dismutase , whereas in vivo, both the tubular basement membrane and brush bor- der were protected [ ]. moreover, in normal mice, the ad- ministration of mitochondria improved endurance during forced swimming test. finally, amt has also been used in vivo in a murine model of parkinson's disease and in vitro for the regeneration of damaged hippocampal cells [ , ]. although of great novelty, these therapeutic approaches have already started to be applied in the human setting. clinical trials involving evs and mitochondria transfer all the clinical trials concerning msc-evs and atm can be found at www.clinicaltrails.gov. although the majority of listed trials focus on the diagnostic properties of evs, there curr transpl rep ( ) : – http://creativecommons.org/licenses/by/ . / are five trials testing the therapeutic applications of mscs- evs and two proposing the use of atm (table ). a first trial is designed to test the anti-inflammatory proper- ties of umbilical cord derived mscs-evs to prevent the de- struction of pancreatic β-cell islets. the mscs-evs will be administered intravenously in two doses, the first dose of exosomes and, after seven days, the second dose of microvesicles (nct ). two other clinical trials using evs will involve allogeneic mscs-evs. one of them will ad- minister evs enriched by mir- for the treatment of acute ischemic stroke (nct ). the second clinical trial will attempt to treat lesions in patients affected by dystrophic epidermolysis bullosa (nct ). the last clinical trial using evs that is in the recruiting phase focuses on promoting the healing and recovery of refractory macular holes through direct injection of msc exosomes to the site of the injury (nct ). finally, the only concluded trail to date used umbilical cord derived mscs-evs to inhibit the progression of chronic kidney disease in patients with grade iii-iv ckd [ ]. the study showed stabilization of the disease progression, as confirmed by stable levels of glomerular filtration rate, serum creatinine and blood urea in treated patients, and an increased level of anti-inflammatory factors (tgf-β and il- ) in com- parison with the matching placebo group. the first clinical trial using administration of isolated mito- chondria for the treatment of myocardial iri has also been concluded with positive results [ ]. mitochondria were isolat- ed from non-ischemic skeletal muscles and injected in the myo- cardium of paediatric patients with myocardial iri. no adverse effects were detected after amt, and four out of five patients demonstrated an enhancement in ventricular function [ ]. other clinical trials using amt are focused on the im- provement of infertility treatments (table ). through autolo- gous micro-injection of mitochondria prior to intra- cytoplasmic sperm injection, the patients’ oocyte quality was enhanced. in the first trial, concluded in , mitochondria were isolated from autologous ovarian stem cells and directly injected into the oocytes themselves. to date, no results have been published. embryo quality has been quantified through the pregnancy rate after treatment and morphological evalua- tion of the treated embryos. in the second clinical trial, which is still ongoing, mitochondria will be isolated from autologous bone marrow-mscs and administrated immediately before intra-cytoplasmic sperm injection in the oocytes. live birth table current clinical trials concerning the use of evs or amt as therapeutic agents intervention n. pts follow up state location number/ref. diabetes mellitus type two doses of msc-evs months unknown sahel teaching hospital sahel, cairo, egypt nct chronic kidney disease two doses of umbilical cord msc-evs ( μg/kg/dose) year concluded sahel teaching hospital sahel, cairo, egypt nassar et al. molecular degeneration – mg of cord tissue msc-evs injected directly around macular hole weeks recruiting tianjin medical university hospital tianjin, china nct cerebrovascular disorders acute ischemic stroke allogeneic msc-evs enriched by mir- months not recruiting yet shahid beheshti university of medical sciences, tehran, iran nct dystrophic epidermolysis bullosa allogeneic msc-evs applied directly to lesions, blisters for days months not recruiting yet aegle therapeutics, miami, florida usa nct repetition failure clinical application of autologous mitochondria transfer for improving oocyte quality. - years not recruiting yet nct infertility amelioration of oocyte quality using autologous mitochondria transfer months concluded valencia, spain nct iri autologous mitochondrial transfer for dysfunction after ischemia-reperfusion injury days concluded boston, ma, usa emani et al. curr transpl rep ( ) : – rate, pregnancy rate, number of oocytes retrieved and fertility rate are going to be evaluated. conclusion numerous discoveries within the regenerative medicine field have highlighted the bioactivity of stem cell bio-products and their role in cell-to-cell communication. in particular, evs are the most advanced as potential therapeutic agents due to their ability to modulate the function of targeted cells. together with stem cell-evs, proven to be of therapeutic effect in a large variety of pre-clinical models, apobds and amt can be utilized for selected and specific applications. the major issue with use of evs in clinical practice is the standardization of evs isolation, usage and storage. however, once those issues can be overcome, using evs as therapeutic agents provides solutions to numerous complications caused by stem-cell therapy, including immune compatibility, maldifferentiation and tumourigenicity. ev therapy allows for dosage control, evaluation of safety and potency equiva- lent to pharmaceutical agents. in comparison to stem cell ther- apy, evs are potentially an easier option as they can be direct- ly obtained from the medium of cultured cells, massively pro- duced and stored without the application of toxic cryo- preservative agents and/or loss of ev potency [ ]. the bio- logical properties of ev allow for modification of the ev content to obtain desired cell-specific effects. encapsulation of effector molecules (nucleic acids, lipids and proteins) by evs allows delivery of its cargo without the risk of degrada- tion. genetically modified evs may offer a more effective and natural solution than usage of fda-approved lipid nanoparti- cles, which pose a low-dose toxicity upon cell entry [ ]. the application of apobds is still poorly explored, as several aspects still require further investigation. for in- stance, apoptotic vesicles are quite heterogeneous and may have different compositions and properties depend- ing on their size. while evs released by primary murine aortic endothelial apoptotic cells enhanced inflammation in mice transplanted with an mhc-incompatible graft, apobds did not show this behaviour [ ]. therefore, as increasing experimental evidence suggests, the therapeutic effects of stem cells are due to their clearance by the immune system [ , ]. for this reason, therapeutic utili- zation of these cell products appears of interest. finally, the use of mscs as a source of mitochondria for amt is a novel, therapeutic option that shows regenerative effects in the treatment of acute cell damage. however, more studies are required for better understanding of mitochondria internalization, their fate once inside the injured cells and how mitochondria can survive in the extracellular microenviron- ment or the blood flow during amt. acknowledgements we would like to thank lola buono for her contri- bution to the review by designing figure . funding this project has received funding from the european union’s horizon research and innovation programme under the marie sklodowska-curie grant agreements no. and . compliance with ethical standards conflict of interest all authors declare no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, pro- vide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation 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( ): . murphy de, et al. extracellular vesicle-based therapeutics : natural versus engineered targeting and trafficking. exp mol med. ; : – . vagnozzi rj, et al. an acute immune response underlies the benefit of cardiac stem-cell therapy. nature. ; : – . publisher’s note springer nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. curr transpl rep ( ) : – 	extracellular vesicles, apoptotic bodies and mitochondria: stem cell bioproducts for organ regeneration 	abstract 	abstract 	abstract 	abstract 	introduction 	extracellular vesicles 	apoptotic evs 	mitochondria 	therapeutic effects of evs, apobds and mitochondria for organ regeneration 	ev reprogramming of injured tissue 	regenerative effect of apoptotic body phagocytosis 	artificial mitochondria transfer 	clinical trials involving evs and mitochondria transfer 	conclusion 	references 	papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance games:science:science_magazine_ - :root:data:science_ - :pdf: _v _n : .pdf [ganino] ganino user tools 	register 	log in site tools search tools show pagesource old revisions backlinks recent changes media manager sitemap register log in > 	recent changes 	media manager 	sitemap trace: games:science:science_magazine_ - :root:data:science_ - :pdf: _v _n : .pdf this topic does not exist yet you've followed a link to a topic that doesn't exist yet. if permissions allow, you may create it by clicking on create this page. page tools 	show pagesource 	old revisions 	backlinks 	back to top not all animals are equal - farm living and allergy in upper bavaria not all animals are equal - farm living and allergy in upper bavaria short title: not all animals are equal matthias wjst institut für lungenbiologie (ilbd) helmholtz zentrum münchen german research center for environmental health (gmbh) ingolstaedter landstr. 
 neuherberg
 germany institut für medizinische informatik 
 statistik und epidemiologie 
 lehrstuhl für medizinische informatik 
 klinikum rechts der isar 
 grillparzerstr. 
 muenchen
 germany all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint note: this preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. https://doi.org/ . / abstract background: a lower allergy and asthma prevalence in farm children has been described three decades ago in switzerland. objective: after years of research into bacterial exposure at farms, the origin of the farm effect is still unknown. we now hypothesize, that there is no such an effect in large industrial cattle farms with slatted floors indoors but in small farms only where animals are grazing outdoors and are having a higher endoparasite load. methods: we re-analyze an earlier epidemiological study by record-linkage to later agricultural surveys. the asthma and allergy study in / was a cross-sectional study of ten year old children in villages covering ten different districts of upper bavaria. the farm effect is defined here as the association of number of cows per villager on lifetime prevalence of allergic rhinitis prevalence in the children of this village. results: the farm effect is restricted to small villages only. furthermore, districts with higher fasciola infection rates of cows, show a significant stronger farm effect than districts with lower infection rates. conclusions: the results warrant further research into human immune response to endoparasites in livestock. 
 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / introduction the lower allergy prevalence in the farming population has been described already in one of the very first allergy text books in ( ): „these statistics of the occupations of hay-fever patients bring out prominently the very curious circumstance that the persons who are most subjected to the action of pollen belong to a class which furnished the fewest cases of the disorder, namely, the farming class“. a direct allergy preventive effect by farming conditions remained unlikely as occupational asthma is frequently increased in farmers due to exposure to grain dust, animal dander and various chemicals ( ). in , however, the „farm effect“ was re-discovered by gassner in the canton of st. gallen in the swiss alps ( ), followed by studies of braun-fahrländer ( ) and several other groups ( ). we already noticed the effect in the asthma and allergy study in upper bavaria, germany ( ) but attributed it to rather trivial reasons. a healthy worker effect is likely in farmers, as allergy to grass or dust would leave to a drop-out of farming. not unexpected farm children have a much lower family history of „hay fever“ ( ) possibly related to genetic differences in the farming population ( ). farm children may have experienced some kind of auto- desensitization due to the extreme allergen exposure ( ). all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / furthermore, farmer’s children are known to supplement less vitamin d which means avoiding a known risk factor ( ). none of these objections have not been thoroughly addressed so far. while the „farm effect“ has had little value in explaining the temporal rise of allergy, it may represent, however, an interesting observation where indeed an environmental substance may modify the allergic reaction. it has been reported several times that dust collected from cow stables has suppressive effects on the allergic sensitization in laboratory animals ( ), ( ). although this could be an artifact as well either by the high allergen doses used or by the different immune reaction of mice, it raises the possibility that there is an indeed a protective factor of stable dust. as ige production in the human host is usually related to helminth exposure and not so much of bacterial load as being used as explanation ( - ), we now ask if cattle endoparasites could be responsible for an allergy modifying effect. participants, materials and methods data of the asthma and allergy study in upper bavaria / are used for an ecological re-analysis. methods have been reported elsewhere including informed consent and ethics procedures ( - ). briefly, we approached primary grade all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / schools of villages where schools could visited in a semi- random order. of children participated in the study ( . %). parents filled in a self-administered questionnaire, children were tested by several allergens on the skin along with baseline lung function and lung function after inhalation of cold air. for the current analysis we did not any exclude any children. geocodes data for school districts have been obtained from geoportal.bayern.de/bayernatlas. number of village inhabitants and assignment to districts was obtained of de.wikipedia.org/wiki/ oberbayern. the number of farm animals per village was imported from the official survey („agrarstrukturerhebung“, www.statistikdaten.bayern.de/genesis, tables -xxxx ) where all farms with an area > ha or > cows are being recorded. cow fasciola infection ratio by districts was taken of a german thesis who tested between and milk samples from farms in bavaria for being positive in an anti-f fasciola hepatica igg assay. altogether tank milk samples were examined and mean values calculated per district ( ). although most data here were acquired - years after the initial survey, farm size usually does not change and also conditions of wet farm ground with continuous re-infection remain unchanged. life expectancy of cattle is around years. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint http://geoportal.bayern.de/bayernatlas http://de.wikipedia.org/wiki/oberbayern http://de.wikipedia.org/wiki/oberbayern http://www.statistikdaten.bayern.de/genesis https://doi.org/ . / data were visualized using the r libraries ggplot , cross-tabulated and analyzed by logistic regression ( , ). results mean age of the children was years with an age range of - years. there were males, females, while in children sex was not known. lifetime prevalence of physician diagnosed allergic rhinitis was . % and of asthma . %. supplemental figure shows the geographical distribution of the villages. as school sizes ranged from to children, mean rhinitis prevalence by village may be misleading in particular when villages are small. village size ranged from to , residents, with a mean of , residents. cow numbers ranged from to , with a mean of , animals. the cows/resident ratio in the villages was found between to . mit a mean of . . allergic rhinitis prevalence decreased with increasing number of cows per village inhabitants, however, this effect was restricted to small villages (supplement figure ) leading to the known %- % reduction of allergic rhinitis. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / fasciola infection rate was found to be highly variable in the districts with a prevalence between % and %. the prevalence of allergic rhinitis decreased with increasing fasciola infection, however, only in those villages that have > . cows/resident (supplement figure ). in a last step we analyze the data using separate logistic regression equations for each district (table). fasciola infection was not significant on the p< . level in any district while in the total sample the cows/resident ratio was a significantly protective factor (p= . , figure). discussion in this ecological analysis we replicate the known negative association of cattle farming and allergic rhinitis and find some preliminary evidence that cow endoparasites may be involved in this association. as there were no effects by other farm animals like pigs, goats, horses and chicken (data not shown) it may indeed be a cow derived factor that could explain the farm effect. ecological studies are studies of risk-modifying factors on health defined either geographically or temporally. as risk-modifying factors and outcomes are averaged for the populations in each geographical unit and then compared using standard statistical all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / methods, this study is useful for quickly generating a hypotheses as it could use already existing data. as an ecological analysis it may fail, however, for reasons already delineated in the introduction, and will need further clinical and experimental support. at least it seems straightforward to relate ige biology to helminthic exposure rather than to bacterial load while fasciola may be just an indicator of mixed infections usually found in cattle. infected cattle rarely demonstrate clinical disease, while it is known that fasciola has numerous immunosuppressive functions in the host. ige is not always raised ( ) probably as fasciola can degrade human immunoglobulins ( ) and even induce eosinophil apoptosis ( ). so far, the possibility of any helminth effect has been excluded ( ) due to the fact, that farm children do not show raised eosinophils or high ige levels. if, however, already contact to eggs or other secreted protein is being sufficient as observed for example with schistosoma eggs ( ), this argument may not be conclusive. this may be also the case with another recent study ( ) that found remarkable differences in the asthma prevalence between amish and hutterite populations. the lifestyle of both communities is similar but their farming practice is distinct as the all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / amish follow a more traditional style of outdoor grazing whereas the hutterities use more industrialized farming practices. gene expression data in the amish children have been interpreted as „intense exposure to microbes“ because protection of experimental asthma by amish derived house dust was nearly abrogated in mice deficient for myd . but even here the argument for any bacterial effect is not convincing as for example schistosome egg antigen can activate both myd dependent and independent pathways ( ). interestingly the gene-expression profile in the amish children ( ) look very much like the profile obtained after fasciola infection ( ). this ecological study can not answer any of the critical questions in the introduction. it raises again the question of possible healthy worker effect as smaller herd size was a significant predictor of quitting farming ( ). but even if we believe in any farm effect, cow endoparasites provide a new research strategy as dna based identification of most helminths is now possible ( ). studies of helminth load would be needed in cattle, as well as in environmental dust, in experimental animal models using controlled exposures, as well as detailed immune profiling in humans. parasite effector molecules have been largely underrated so far with numerous candidates are being available for experimental all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / and clinical testing ( ). this may be even in line with the current „one health initiative“ of the who addressing the connections between health and the environment by multidisciplinary research. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / table: logistic regression analysis of allergic rhinitis in upper bavaria in children. separate models are given for each district using the ratio of cows/resident as dependent variable. the three districts with the lowest number of children examined (altötting n= , mühldorf n= and rosenheim n= children) were collapsed into one category. district % milk samples fasciola positive children n regression coefficient cows/ resident standard error cows/ resident p altötting/rosenheim/mühldorf - , , , garmisch-partenkirchen - , , , landsberg am lech , , , miesbach - , , , ostallgäu - , , , tölz-wolfratshausen - , , , traunstein , , , weilheim-schongau , , , total sample - , , , all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / figure: logistic regression analysis of allergic rhinitis in upper bavaria in children. regression coefficients from separate models for each district for the cows/resident ratio are plotted against the average percentage of milk samples with fasciola igg. 
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 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / author contribution i revised questionnaire and protocols, contacted school authorities, selected and visited all schools, examined children, supervised data entry, analyzed data, developed the hypothesis and wrote the paper. acknowledgments i wish to thank w. lehmacher, e. stiepel, r. frentzel-beyme-bauer, b. berbig, t. nicolai, e. von mutius, s. dold, e. von loeffelholz- colberg and k. winter for help with planning the study; p. reitmeir and g. röll for inital statistical analysis; s. braun, a. fuger from my team for examining the children; c. malin, m. von mutius, t. kreyssig, a. provelegios, r. berbig, and a. jensen for transporting our equipment; e. kienle for technical help with the lung function devices; c. bremer, j. rohe, s. funk, a. stankiewicz, l. pritscher, a. wulff and m. molette de morangier for data typing. we thank all head teachers, school secretaries, and class teachers who participated in the project. finally, we thank all the parents and children who took part. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / during the re-analysis, many people helped with advice: p. tschierse, c. prazeres da costa, t. stöger, d. andrade, h. smits, j. esser von bieren, m. scheuerle, l. gershwin, g. knubben- schweitzer, n. beyls, b. gottstein and j. charlier. data availability aggregated epidemiological data on village basis are available from the author. all other datasets are available directly online from the indicated webpages ( geoportal.bayern.de/bayernatlas, de.wikipedia.org/wiki/oberbayern, www.statistikdaten.bayern.de/ genesis and edoc.ub.uni-muenchen.de/ / /koch_sandra.pdf ) conflicts of interest i declare that i have no conflicts of interest related to this analysis. the study was conducted in / by order of the „bayerische staatsministerium für landesentwicklung und umweltfragen“ (now „bayerisches staatsministerium für wirtschaft, landesentwicklung und energie“) to “dr. von haunersches all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint http://geoportal.bayern.de/bayernatlas http://edoc.ub.uni-muenchen.de/ / /koch_sandra.pdf https://doi.org/ . / kinderspital“ (now „kinderklinik und kinderpoliklinik im dr. von haunerschen kinderspital“). all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / supplemental figure : study villages are located in the south of munich in upper bavaria. for color codes see legend to supplement figure . all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / supplemental figure : allergic rhinitis prevalence in study villages separated by low or high resident number. villages with allergic rhinitis > % (outlier) are removed from the regression. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / supplemental figure : allergic rhinitis prevalence in small sized study villages only by cattle population. villages with allergic rhinitis > % (outlier) are removed from the regression. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (whichthis version posted october , . ; https://doi.org/ . / doi: medrxiv preprint https://doi.org/ . / pathways to dewetting in hydrophobic confinement richard c. remsinga, , erte xia, , srivathsan vembanurb, sumit sharmac, pablo g. debenedettic, shekhar gardeb, and amish j. patela, adepartment of chemical & biomolecular engineering, university of pennsylvania, philadelphia, pa ; bhoward p. isermann department of chemical & biological engineering, and center for biotechnology and interdisciplinary studies, rensselaer polytechnic institute, troy, ny ; and cdepartment of chemical & biological engineering, princeton university, princeton, nj edited by ken a. dill, stony brook university, stony brook, ny, and approved may , (received for review february , ). liquid water can become metastable with respect to its vapor in hydrophobic confinement. the resulting dewetting transitions are often impeded by large kinetic barriers. according to macroscopic theory, such barriers arise from the free energy required to nucleate a critical vapor tube that spans the region between two hydrophobic surfaces—tubes with smaller radii collapse, whereas larger ones grow to dry the entire confined region. using exten- sive molecular simulations of water between two nanoscopic hydro- phobic surfaces, in conjunction with advanced sampling techniques, here we show that for intersurface separations that thermodynami- cally favor dewetting, the barrier to dewetting does not correspond to the formation of a (classical) critical vapor tube. instead, it corre- sponds to an abrupt transition from an isolated cavity adjacent to one of the confining surfaces to a gap-spanning vapor tube that is already larger than the critical vapor tube anticipated by macroscopic theory. correspondingly, the barrier to dewetting is also smaller than the classical expectation. we show that the peculiar nature of water density fluctuations adjacent to extended hydrophobic surfaces— namely, the enhanced likelihood of observing low-density fluctua- tions relative to gaussian statistics—facilitates this nonclassical behavior. by stabilizing isolated cavities relative to vapor tubes, enhanced water density fluctuations thus stabilize novel path- ways, which circumvent the classical barriers and offer diminished resistance to dewetting. our results thus suggest a key role for fluctuations in speeding up the kinetics of numerous phenomena ranging from cassie–wenzel transitions on superhydrophobic surfaces, to hydrophobically driven biomolecular folding and assembly. capillary evaporation | fluctuations | kinetic barriers | assembly the favorable interactions between two extended hydrophobicsurfaces drive numerous biomolecular and colloidal assem- blies ( – ), and have been the subject of several theoretical, computational, and experimental inquiries ( – ). examples include the association of small proteins to form multimeric protein com- plexes, of amphiphlic block copolymers, dendrimers, or proteins to form vesicular suprastructures, and of patchy colloidal particles into complex crystalline lattices ( – ). when two such hydrophobic surfaces approach each other, water between them becomes meta- stable with respect to its vapor at a critical separation, dc, that can be quite large ( , , – ). for nanometer-sized surfaces at ambient conditions, dc is proportional to the characteristic size of the hy- drophobic object, whereas for micron-sized and larger surfaces, dc ∼   μm ( , ). however, due to the presence of large kinetic barriers separating the metastable wet and the stable dry states, the system persists in the wet state, and a dewetting transition is trig- gered only at much smaller separations (∼ nm) ( , , , ). to uncover the mechanism of dewetting, a number of theoretical and simulation studies have focused on the thermodynamics as well as the kinetics of dewetting in the volume between two parallel hydrophobic surfaces that are separated by a fixed distance, d < dc ( , – , – ). these studies have highlighted that the bottle- neck to dewetting is the formation of a roughly cylindrical, critical vapor tube spanning the region between the surfaces ( , , ). a barrier in the free energetics of vapor tube formation as a function of tube radius is also supported by macroscopic interfacial ther- modynamics, wherein the barrier arises primarily from a competition between the favorable solid–vapor and unfavorable liquid–vapor surface energies (eq. and fig. ). thus, the classical mechanism for the dewetting transition prescribes that a vapor tube that spans the volume between the two surfaces must first be nucleated, and if the vapor tube is larger than a certain critical size, it will grow until the entire confined volume is dry ( ). although it has been recognized that water density fluctua- tions must play a crucial role in nucleating vapor tubes ( , ), the precise mechanism by which these tubes are formed is not clear. to understand how vapor tubes are formed and to investigate their role in the dewetting process, here we use molecular simula- tions in conjunction with enhanced sampling methods ( , ) to characterize the free energetics of water density fluctuations in the region between two nanoscopic hydrophobic surfaces. such a characterization of water density fluctuations in bulk water and at interfaces has already provided much insight into the physics of hydrophobic hydration and interactions ( , , – ). in particular, both simulations and theory have shown that the likelihood of observing low-density fluctuations adjacent to ex- tended hydrophobic surfaces is enhanced relative to gaussian statistics ( , , – , ). further, the intricate coupling be- tween enhanced solvent fluctuations and dewetting kinetics has been highlighted by both coarse-grained ( – ) and atomistic simulations ( – ). here we show that such enhanced water density fluctuations influence the pathways to dewetting in hydrophobic confinement by stabilizing isolated cavities adjacent to one of the confining surfaces with respect to vapor tubes. as the density in the con- fined region is decreased, the stability of isolated cavities relative to vapor tubes also decreases, and at a particular density, isolated significance dewetting in hydrophobic confinement plays an important role in diverse phenomena, ranging from protein folding and as- sembly, to the heterogeneous nucleation of vapor bubbles and superhydrophobicity. using molecular simulations, we find that dewetting proceeds through the formation of isolated cavities adjacent to one of the confining surfaces. these iso- lated cavities are stabilized by enhanced water density fluctu- ations, and their growth is uphill in free energy. upon growing to a certain size, the isolated cavities transition abruptly into supercritical vapor tubes that span the confined region, and grow spontaneously. consequently, this nonclassical pathway results in lower free energy barriers than anticipated by mac- roscopic theory, with important implications for the kinetics of dewetting and hence for water-mediated self-assembly. author contributions: s.v., s.s., p.g.d., s.g., and a.j.p. designed research; r.c.r., e.x., and a.j.p. performed research; r.c.r., e.x., and a.j.p. analyzed data; and r.c.r., e.x., p.g.d., s.g., and a.j.p. wrote the paper. the authors declare no conflict of interest. this article is a pnas direct submission. r.c.r. and e.x. contributed equally to this work. to whom correspondence should be addressed. email: amish.patel@seas.upenn.edu. this article contains supporting information online at www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental. www.pnas.org/cgi/doi/ . /pnas. pnas | july , | vol. | no. | – a p p li ed p h y si c a l sc ie n c es http://crossmark.crossref.org/dialog/?doi= . /pnas. &domain=pdf mailto:amish.patel@seas.upenn.edu http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental www.pnas.org/cgi/doi/ . /pnas. cavities abruptly transition to vapor tubes. surprisingly, for d k dc, that is, separations for which dewetting is thermodynamically favorable, we find that the nascent vapor tubes formed from the isolated cavities are already larger than the corresponding criti- cal vapor tubes predicted by classical theory. because the newly formed vapor tube is supercritical, it grows spontaneously. im- portantly, because the formation of this supercritical vapor tube involves a nonclassical pathway that circumvents the critical vapor tube altogether, the process entails a smaller free energetic cost. our results thus point to smaller kinetic barriers to dewetting than predicted by macroscopic theory. macroscopic theory according to classical interfacial thermodynamics, the free energy for creating a cylindrical vapor tube of radius r, which spans the volume between two surfaces separated by a distance d, is given by: Δgthðr; dÞ = π � r dΔp + rdγ + r γ cos θ + rλ � , [ ] where Δp is the difference between the system pressure and the saturation pressure, γ is the liquid–vapor surface tension, θ is the contact angle, and λ is the line tension. for nanoscopic surfaces, the pressure-volume contribution is negligible at ambient condi- tions ( , , ), whereas the line tension contribution can be important ( , ). the term containing cos θ is negative for hydrophobic surfaces and favors dewetting, whereas the term corresponding to formation of the vapor–liquid area is unfavor- able. the functional form of Δgthðr; dÞ given in eq. is illus- trated in fig. d for three d values. in each case, a barrier separates the liquid ðr = Þ and vapor (large r) basins, supporting the notion of dewetting mediated by the nucleation and growth of a vapor tube; both the critical vapor tube radius and the barrier height increase with increasing d. density-dependent free energy profiles feature a kink to investigate how water density fluctuations influence the mechanism of dewetting in hydrophobic confinement, here we perform molecular dynamics simulations of water confined be- tween two roughly square hydrophobic surfaces of size l = nm, separated by a distance, d, as shown in fig. a. water in the confined region is in equilibrium with a reservoir of water, which in turn is in coexistence with its vapor ðΔp = Þ ( , ). we characterize the statistics of water density fluctuations in the confined volume using indirect umbrella sampling (indus) ( , ); that is, we estimate the free energy, Δg, of observing n water molecules in that volume. the free energy, Δgðn; dÞ, thus estimated is shown in fig. a for a range of separations, d, with the free energy of the liquid basin, n = nliq, being set to zero in each case. over the entire range of separations considered, the free energy profile displays distinctive liquid (high n) and vapor (low n) basins with barriers separating them. interestingly, the free energy profiles also feature a kink, that is, an abrupt change in the slope of Δgðn; dÞ is observed at a particular value of n between the liquid and vapor basins; we refer to this value of n as nkink. this discontinuity in slope is seen more clearly in the derivatives of the free energy, shown in fig. b. small errors in Δg are amplified if simple finite differences are used to evaluate the derivatives; we therefore smooth the free energy profiles before evaluating the derivatives. details of the smoothing procedure as well as the unsmoothed derivatives are shown in the si appendix. kink separates the vapor tube and isolated cavity ensembles to investigate the significance of the kink in the free energy, we characterize configurations corresponding to n on either side of nkink. we do so by building upon the instantaneous interface method of willard and chandler ( ) to identify isosurfaces a c b d fig. . (a–c) simulation snapshots of water (shown in red/white) in confine- ment between two square hydrophobic surfaces (shown in cyan) of size l = nm that are separated by a distance of d = Å; configurations highlighting (a) the liquid basin, (b) a cylindrical vapor tube of radius, r, that spans the confined region, and (c) the vapor basin are shown. in the front views, only one of the confining surfaces is shown. (d) macroscopic theory predicts a free energetic barrier to vapor tube formation (eq. ), suggesting that a vapor tube larger than a critical size must be nucleated before dewetting can proceed. a b fig. . (a) the simulated free energy profiles, βΔgðn; dÞ, as a function of the number of water molecules between the surfaces, n, display marked kinks (highlighted by circles). here, β = =kbt, with kb being the boltzmann constant and t being the temperature. the size of the largest error bar is also shown for d = Å and n = . (b) the kinks are also apparent in the smoothed derivatives of the free energy profiles, which display a sharp decrease in the vicinity of nkink. | www.pnas.org/cgi/doi/ . /pnas. remsing et al. http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf www.pnas.org/cgi/doi/ . /pnas. that encompass the dewetted regions, that is, the regions from which water is absent. the details of the method are included in the si appendix. as illustrated in fig. a for d = Å and n = nkink − , characteristic configurations with n k nkink con- tain a vapor tube, that is, the dewetted region (in purple) clearly spans the confined volume between the two surfaces (side view, left). water molecules (not shown for clarity) occupy the entire re- gion between the surfaces not shown in purple and are also pre- sent outside the confinement region. in contrast, for configurations with n j nkink, isolated cavities are observed adjacent to one sur- face or the other; however, as seen in fig. b for n = nkink + , the cavities do not span the region between the surfaces to form vapor tubes. movies corresponding to the configurations shown in fig. a and b are included in the supporting information as movies s –s . the configurations shown in fig. a and b suggest that n = nkink marks the boundary between the vapor tube and iso- lated cavity ensembles. to put this notion on a quantitative footing, we define indicator functions, htube and hcav, which are if a given configuration has a vapor tube or an isolated cavity, respectively, and otherwise. the average value of the indicator function hhtubein, subject to the constraint that the number of water molecules in confinement is n, is shown in fig. c for the entire range of n values, and for several separations, d. because the configurations were generated in the presence of biasing potentials, care must be exercised in evaluating the averages shown in fig. c; details of the averaging procedure as well as the criteria used in the definition of the indicator functions can be found in the si appendix. for a given separation, hhtubein, which is the probability of observing a vapor tube conditional on the number of waters in confinement being n, is a sigmoidal function of n, decreasing sharply from at low n to for high n. we define ntube to be the value of n at which hhtubein undergoes a sharp transition; in particular, where hhtubein crosses . . as shown in fig. d, for the entire range of d values studied here ð   Å ≤ d ≤   ÅÞ, ntube is equal to nkink, formalizing the notion that the kink in Δgðn; dÞ demarcates configurations that display vapor tubes and those that do not. analogous to hhtubein, the conditional average hhcavin quan- tifies the fraction of configurations with n confined waters, which feature isolated cavities. for all separations, hhcavin dis- plays a sharp increase, followed by a gradual decrease; see fig. e. the sharp increase occurs in the vicinity of n = nkink as vapor tubes give way to isolated cavities, whereas the gradual decrease corresponds to a crossover from isolated cavities to uniform configurations. to specify the location of this crossover, we de- fine ncav to be the value of n where hhcavin crosses . (with a negative slope). despite these common features in the functional form of hhcavin, there are subtle differences in hhcavin at small and large separations, which nevertheless have interesting con- sequences. for the largest separations, a well-defined plateau at hhcavin =   separates the sharp increase in hhcavin and its gradual decrease; this plateau demarcates the range of n values, which reliably feature isolated cavities. in contrast, the plateau at hhcavin =   is absent for the smaller separations. instead, the sharp increase in hhcavin and its gradual decrease overlap in their range of n values. this overlap suggests that configurations corresponding to n = nkink are not limited to those with vapor tubes or isolated cavities, but could also be uniform. given that hhtubein = . at n = ntube by definition, a value of hhcavintube <   . would correspond to a nonzero likelihood of observing uniform configurations with n = ntube = nkink. as shown in fig. f, that is indeed the case for d ≤ Å, suggesting that although the nucleation of a vapor tube must proceed through the formation of isolated cavities for d > Å, vapor tubes may be nucleated directly from uniform configurations for smaller separations. free energetics of vapor tubes and isolated cavities given the abrupt change in the dewetted morphologies at n   =   nkink, we expect the functional form of the free energetics for n > nkink and n < nkink to be different. fig. c and d collectively show that configurations with n k nkink feature a va- por tube spanning the confined region, consistent with classical arguments. although the vapor tube undergoes extensive shape fluctuations, we find its average shape to be roughly cylindrical. coarse-grained density maps of select configurations reflecting the average vapor tube shape are included in the si appendix. to compare the free energetics of the vapor tubes obtained from our simulations to macroscopic theory, we first transform the number of waters in the confined region, n, to an approximate vapor tube radius, r, using the simple relation, πr =l = ðnliq − nÞ=nliq. the values of vapor tube radii thus obtained are consistent with the average radii of the vapor tubes observed in our simulations for a c d e f b fig. . instantaneous interfaces encompassing dewetted regions (shown in purple) between the hydrophobic surfaces (shown in cyan) separated by d = Å highlight the presence of (a) a vapor tube for n = nkink − , and (b) an isolated cavity for n = nkink + . water molecules not shown for clarity. (c) average of the binary vapor tube indicator function, htube, conditioned on the number of waters in confinement being n, displays a sharp transition from to as n is increased. the color scheme is the same as that in fig. . the value of n corresponding to hhtubein = . (dashed line) is defined as ntube. (d) ntube is identical to the location of the kink in the free energy profiles, nkink, as shown by the agreement between the simulation data and a straight line (dashed). this agreement confirms that the kink demarcates conformations with and without vapor tubes. (e) conditional average of the isolated cavity indicator function, hhcavin, shows a sharp increase in the vi- cinity of nkink (the square symbols correspond to ntube), followed by a gradual decrease at larger n values, and eventually vanishes around n = nliq. (f) for the larger d values, hhtubeintube = hhcavintube = . . however, for the smaller d values, hhcavintube < . , suggesting the possibility of direct vapor tube nucleation without isolated cavities as intermediates. remsing et al. pnas | july , | vol. | no. | a p p li ed p h y si c a l sc ie n c es http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=stxt http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf n < nkink, as shown in the si appendix. having a one-to-one re- lation between n and r allows us to transform the simulated free energies, Δgðn; dÞ, into r-dependent free energies, Δgðr; dÞ in the region rkink < r < l= ; the corresponding free energies are shown as symbols in fig. a. the lines are fits to the ΔgðrÞ data using the macroscopic expectation, ΔgfitðrÞ = ΔgthðrÞ + kbt lnð − r=lÞ, where the logarithmic term corresponds to the translational en- tropy of the vapor tube. ΔgðrÞ is fit separately for each d value and yields values of γ and λ that are reasonable. values of γ are in the range of . − .   kbt=nm , comparable to the reported value of .   kbt=nm for the water model that we use ( ). our fits yield −λ=γ in the . − .   Å range, in accord with a recently reported experimental value of λ = − pn ( ), which yields −λ=γ = .   Å. these agreements are remarkable considering the simplicity of the model that we use as well as the assumptions that we make (cylin- drical vapor tube shape, constant surface tension independent of vapor tube curvature, etc.), and suggest that the energetics of the vapor tube are well-described by classical macroscopic theory. fur- ther details of our fitting procedure, the values of the fit parameters for each d, as well as our attempts to fit the simulation data to other reasonable expressions of gthðrÞ can be found in the si appendix. to investigate the free energetics of the isolated cavity en- semble, in fig. b, we focus on Δgðn > nkink; dÞ. in the liquid basin, that is, in the vicinity of n = nliq, the free energy (symbols) is parabolic (solid lines), indicating that the underlying density fluctuations are gaussian. although Δg remains harmonic for n > nliq, it crosses over to being roughly linear (dashed lines) for n < nliq. such a crossover from parabolic to linear has also been observed adjacent to single extended hydrophobic surfaces ( , , ), and corresponds to the interfacial water undergoing a collective dewetting transition to expel water from a nanometer- sized cavity ( , , ). indeed, as shown in fig. b, the location of the crossover agrees well with the corresponding ncav values (squares) discussed in the previous section. interestingly, the slopes of the linear fat tails are similar for all d values (in the range of nkink + ≤ n ≤ nliq − ); the dashed lines shown in fig. b are linear fits with a slope of − .     kbt per water. additional details of the fitting procedure and the values of the parameters obtained are provided in the si appendix. the dif- ference between the values of nliq and the x intercepts of the linear fits is also approximately the same for all d values, and is equal to   ±   waters. thus, the free energy for forming an isolated cavity of a given size (as quantified by the number of waters displaced from the confined region, nliq − n), is independent of the separation between the surfaces; that is, the free energetics of isolated cavity formation adjacent to one hydrophobic surface are largely unaffected by the presence of the other confining surface. in contrast, the free energetics of vapor tube formation clearly depend on the intersurface separation, d. as a result, the location of the kink, where isolated cavities become metastable with respect to vapor tubes, also depends on d. nonclassical dewetting mechanism reduces barriers fig. a summarizes our findings for the dewetting mechanism presented thus far; in addition to the simulated Δgðn; dÞ for d =   Å, it highlights the metastable branches of the vapor tube and isolated cavity ensemble free energies, anticipated from the fits shown in fig. . it is clear that the system minimizes its free energy at all times by staying on the branch with the lower free energy; at n = nkink, where the two free energy profiles in- tersect, the system jumps from the isolated cavity to the vapor tube ensemble. importantly, the nonclassical path leading up to the formation of nascent vapor tubes ðn > nkinkÞ can result in smaller barriers to dewetting than anticipated by classical theory, as shown in fig. b. for d =   Å, the classical barrier (critical vapor tube) appears in the metastable segment of the free energy profile. the system thus circumvents the classical barrier, and instead adopts the path involving isolated cavities, which give way to vapor tubes only at n = nkink; these nascent vapor tubes are larger than the critical vapor tube, so their subsequent growth is downhill in energy. thus, the barrier to dewetting is the free energetic cost for forming these nascent, supercritical vapor tubes, which is clearly smaller than the classical barrier. in fig. c, we illustrate that the nascent vapor tubes are not supercritical for all separations; for d =   Å, the classical barrier appears in the stable segment of the free en- ergy profile. thus, although the kink in Δgðn; dÞ again marks the formation of a vapor tube for d =   Å, the vapor tube formed is smaller than the critical vapor tube, and must grow further in a process that is uphill in energy, before dewetting can proceed. as a result, the barrier to dewetting coincides with that predicted by macroscopic theory. to uncover the separation at which the system transitions from a supercritical to a subcritical nascent vapor tube, in fig. d, we plot nkink and nmax as a function of d. here, nmax cor- responds to the value of n between the liquid and vapor basins where Δgðn; dÞ is the highest. for small values of d, nmax = nkink, a b fig. . (a) βΔgðn ≤ nkink; dÞ is recast as βΔgðr; dÞ, the free energy to form a vapor tube of radius, r. the points were obtained from the simulated free energy profiles by using the relation πr =l = ðnliq − nÞ=nliq in the region rkink < r < l= , and the lines are fits to macroscopic theory. (b) the portion of the free energy corresponding to the liquid basin ðn ≥ nkinkÞ is parabolic at high n (gaussian fluctuations), but linear at low n (fat tails in water number distributions). the crossover is gradual and occurs in the vicinity of ncav (square symbols), that is, the value of n for which hhcavin is . with a negative slope. the linear regions have roughly the same slope for all separations. | www.pnas.org/cgi/doi/ . /pnas. remsing et al. http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf www.pnas.org/cgi/doi/ . /pnas. indicating that Δgðn; dÞ is the highest at the kink, consis- tent with the formation of supercritical nascent vapor tubes. for larger values of d, there is an additional maximum in Δg at nmax < nkink, suggesting that the newly formed vapor tubes are smaller than the corresponding critical vapor tubes. interestingly, the separation at which the system transitions from nonclassical to classical dewetting barriers is close to the separation, dc, at which there is coexistence between the liquid and vapor. thus, for the separations with a thermodynamically favorable driving force for dewetting, the mechanism for dewetting is manifestly nonclassical, corresponding to the formation of supercritical vapor tubes from isolated cavities, and requiring a smaller free energetic barrier than anticipated by macroscopic theory. discussion and outlook our results highlight that water density fluctuations can play a central role in determining the pathways to dewetting in hydro- phobic confinement. enhanced water density fluctuations in the vicinity of hydrophobic surfaces stabilize isolated cavities relative to vapor tubes for n > nkink; the system resides in the classical vapor tube ensemble only for n < nkink. our results thus high- light a breakdown of classical nucleation theory that stems from the stabilization of a nonclassical species (isolated cavities) and leads to an alternative pathway with a lower barrier. similar findings have been reported for crystal nucleation, wherein a nonclassical pathway is stabilized by liquidlike solute clusters, which results in a reduced barrier ( , ). although the free energetics of both the isolated cavity and vapor tube ensembles are well described by the number of waters in confinement, n, this simple order parameter may not be sufficient to describe the transition from one ensemble to the other. indeed, Δgðn; dÞ represents a projection of a complex free energy land- scape onto the single parameter, n, and a kink in Δgðn; dÞ strongly suggests that the transition from an isolated cavity to a vapor tube involves order parameter(s) that are orthogonal to n. although beyond the scope of the present work, it would be interesting to uncover these order parameters that define the transition state en- semble along with n. determining these additional parameters will require path sampling techniques in conjunction with a character- ization of commitment probabilities ( ); it is conceivable that ad- ditional barriers may present themselves in these order parameters. dewetting in nanoscopic hydrophobic confinement plays an im- portant role in biology; ranging from the assembly of multimeric proteins and the collapse of the hydrophobic protein core, to the vapor-lock gating of ion channels and the specific binding of ligands to hydrophobic grooves on their binding partners. in particular, recent work has highlighted the importance of including the solvent coordinate, n, in describing the kinetics of hydrophobically driven collapse and assembly ( – , ). our results show that water density fluctuations stabilize nonclassical pathways, which reduce the barriers along the n coordinate, and should therefore enhance the kinetics of dewetting-mediated biophysical phenomena. dewetting in hydrophobic confinement can also be important in a host of nonbiological phenomena, ranging from heteroge- neous nucleation of vapor bubbles and contact line pinning, to the cassie–wenzel transitions on textured surfaces ( ). these phenomena involve intricate confinement geometries, which could result in complex pathways involving one or more transi- tions between various dewetted morphologies, akin to the tran- sition between isolated cavities and vapor tubes observed here. fluctuation-mediated pathways ought to also reduce dewetting barriers associated with these diverse phenomena. materials and methods we simulate the spc/e (extended simple point charge) model of water in con- finement between two square hydrophobic surfaces of size l = nm, for a range of separations, d (fig. a), ranging from to Å, chosen to span the entire range of d values with both liquid and vapor basins. the surfaces are composed of , atoms each, which are arranged on a hexagonal lattice with a spacing of . Å. the surface atoms interact with the water oxygens through the len- nard–jones potential with the parameters, σ = . Å and e = . kj/mol (see refs. , for further details). as shown in the si appendix, this well depth was chosen so that a water droplet on the hydrophobic surface makes a contact angle, θ ≈ °, which is characteristic of alkyl-terminated self-assembled monolayer surfaces ( ). for water, we have chosen the spc/e model ( ) be- cause it adequately captures the experimentally known features of water such as surface tension, compressibility, and vapor–liquid equation of state near ambient conditions, which are important in the study of hydrophobic effects ( , ). our simulations contain roughly , – , water molecules and were performed in the canonical ensemble, thermostated at t = k using the canonical ve- locity rescaling thermostat ( ). we use a periodic simulation box with the hy- drophobic surfaces of interest fixed at the center of the box, and a buffering liquid–vapor interface nucleated at the top of the box with the help of a wall of purely repulsive particles. the buffering interface ensures that the system is at the saturation pressure of spc/e water at k ( , ); free energies obtained with such a construct have been shown to be nearly indistinguishable from those obtained in the npt ensemble at a pressure of bar ( ). short-ranged interactions were truncated at nm; whereas, long-ranged electrostatic interactions were computed using the particle mesh ewald method ( ). the bonds in water were constrained using the shake algorithm ( ). to study the free energetics of dewetting, we select the cuboid shaped ðl × l × dÞ observation volume between the hydrophobic surfaces, and estimate the free energies, Δgðn; dÞ, using the indus method ( , ). each biased simulation was run for ns and the first ns was discarded for equilibration. a b c d fig. . (a) the simulated βΔgðn; dÞ for d =   Å (solid) is shown along with the expected metastable branches of the free energies corresponding to the vapor tube (dot-dashed) and isolated cavity (dashed) ensembles. the metastable branches are anticipated from the fits shown in fig. . the system minimizes its free energy by localizing to the ensemble with the lower free energy. (b) for d = Å, the nascent vapor tube formed at the kink is larger than the critical vapor tube anticipated by macroscopic theory, and therefore grows spontaneously. as a result, the corresponding barrier to dewetting is smaller than that predicted by macroscopic theory. (c) for larger separations, here d = Å, the newly formed vapor tube is subcritical, and has to grow larger for dewetting to proceed. (d) comparison of the location of the kink, nkink, with the location of the barrier between the liquid and vapor basins, nmax. for small d, the barrier (point of highest Δg) occurs at the kink, so that nmax ≈ nkink. in contrast, for larger d values, the barrier occurs in the vapor tube segment of the simulated free energy profile and corresponds to the classical critical vapor tube, so that nmax < nkink. the transition from nonclassical to classical behavior occurs in the vicinity of the coexistence separation, dc. remsing et al. pnas | july , | vol. | no. | a p p li ed p h y si c a l sc ie n c es http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf acknowledgments. r.c.r. and a.j.p. were supported in part by financial support from the national science foundation (nsf) through a seed grant from the university of pennsylvania materials research science and engineering center (nsf upenn mrsec dmr - ). s.g. was supported by the nsf (cbet- ). p.g.d. gratefully acknowledges support from the nsf (cbet- and che- ). . tanford c ( ) the hydrophobic effect - formation of micelles and biological membranes (wiley interscience, new york). . kauzmann w ( ) some factors in the interpretation of protein denaturation. adv protein chem : – . . stillinger fh ( ) structure in aqueous solutions of nonpolar solutes from the standpoint of scaled-particle theory. j solution chem : – . . israelachvili jn ( ) intermolecular and surface forces (academic, waltham, ma), revised third edition. . chandler d ( ) interfaces and the driving force of hydrophobic assembly. nature ( ): – . . israelachvili j, pashley r ( ) the hydrophobic interaction is long range, decaying exponentially with distance. nature ( ): – . . christenson hk, claesson pm ( ) cavitation and the interaction between macro- scopic hydrophobic surfaces. science ( ): – . . bérard dr, attard p, patey gn ( ) cavitation of a lennard-jones fluid between hard walls, and the possible relevance to the attraction measured between hydrophobic sur- faces. j chem phys : – . . parker jl, claesson pm, attard p ( ) bubbles, cavities, and the long-ranged at- traction between hydrophobic surfaces. j phys chem : – . . wallqvist a, berne bj ( ) computer simulation of hydrophobic hydration forces on stacked plates at short range. j phys chem : – . . lum k, luzar a ( ) pathway to surface-induced phase transition of a confined fluid. phys rev e stat phys plasmas fluids relat interdiscip topics :r –r . . lum k, chandler d ( ) phase diagram and free energies of vapor films and tubes for a confined fluid. int j thermophys : – . . lum k, chandler d, weeks jd ( ) hydrophobicity at small and large length scales. j phys chem b : – . . bolhuis pg, chandler d ( ) transition path sampling of cavitation between mo- lecular scale solvophobic surfaces. j chem phys : – . . leung k, luzar a, bratko d ( ) dynamics of capillary drying in water. phys rev lett ( ): . . huang x, margulis cj, berne bj ( ) dewetting-induced collapse of hydrophobic particles. proc natl acad sci usa ( ): – . . urbic t, vlachy v, dill ka ( ) confined water: a mercedes-benz model study. j phys chem b ( ): – . . choudhury n, pettitt bm ( ) the dewetting transition and the hydrophobic effect. j am chem soc ( ): – . . xu l, molinero v ( ) liquid-vapor oscillations of water nanoconfined between hydrophobic disks: thermodynamics and kinetics. j phys chem b ( ): – . . sharma s, debenedetti pg ( ) evaporation rate of water in hydrophobic con- finement. proc natl acad sci usa ( ): – . . sharma s, debenedetti pg ( ) free energy barriers to evaporation of water in hydrophobic confinement. j phys chem b ( ): – . . mastropietro dj, ducker wa ( ) forces between hydrophobic solids in concen- trated aqueous salt solution. phys rev lett ( ): . . yu n, hagan mf ( ) simulations of hiv capsid protein dimerization reveal the effect of chemistry and topography on the mechanism of hydrophobic protein as- sociation. biophys j ( ): – . . discher bm, et al. 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( ) a smooth particle mesh ewald method. j chem phys : – . . ryckaert jp, ciccotti g, berendsen hjc ( ) numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. j comput phys : – . | www.pnas.org/cgi/doi/ . /pnas. remsing et al. www.pnas.org/cgi/doi/ . /pnas. pnas .. homozygous mutation in samhd gene causes cerebral vasculopathy and early onset stroke baozhong xina, stephen jonesb, erik g. puffenbergerc,d, claas hinzee, alicia brighta, haiyan tanf, aimin zhouf, guiyun wub, jilda vargus-adamse, dimitris agamanolisg, and heng wanga,h,i, addc clinic for special needs children, middlefield, oh ; bdepartment of radiology, cleveland clinic, cleveland, oh ; cthe clinic for special children, strasburg, pa ; ddepartment of biology, franklin and marshall college, lancaster, pa ; edepartment of pediatrics, cincinnati children’s hospital medical center, cincinnati, oh ; fdepartment of chemistry, cleveland state university, cleveland, oh ; gdepartment of pathology, akron children’s hospital, oh ; hdepartment of pediatrics, rainbow babies and children’s hospital, cleveland, oh ; and idepartment of molecular cardiology, cleveland clinic, cleveland, oh edited by c. thomas caskey, university of texas-houston health science center, houston, tx, and approved february , (received for review september , ) we describe an autosomal recessive condition characterized with cerebral vasculopathy and early onset of stroke in individuals in old order amish. the phenotype of the condition was highly het- erogeneous, ranging from severe developmental disability to nor- mal schooling. cerebral vasculopathy was a major hallmark of the condition with a common theme of multifocal stenoses and aneur- ysms in large arteries, accompanied by chronic ischemic changes, moyamoya morphology, and evidence of prior acute infarction and hemorrhage. early signs of the disease included mild intra- uterine growth restriction, infantile hypotonia, and irritability, fol- lowed by failure to thrive and short stature. acrocyanosis, raynaud’s phenomenon, chilblain lesions, low-pitch hoarse voice, glaucoma, migraine headache, and arthritis were frequently ob- served. the early onset or recurrence of strokes secondary to ce- rebral vasculopathy seems to always be associated with poor outcomes. the elevated erythrocyte sedimentation rate (esr), igg, neopterin, and tnf-α found in these patients suggested an immune disorder. through genomewide homozygosity mapping, we localized the disease gene to chromosome (chr) q . -q . candidate gene sequencing identified a homozygous mutation, c. – a > g, in the samhd gene, being associated with this condition. the mutation appeared at the splice-acceptor site of intron , resulted in the skipping of exon , and gave rise to an aberrant protein with in-frame deletion of amino acids. im- munoblotting analysis showed lack of mutant samhd protein expression in affected cell lines. the function of samhd remains unclear, but the inflammatory vasculopathies of the brain found in the patients with samhd mutation indicate its important roles in immunoregulation and cerebral vascular hemeostasis. genotyping | snp arrays | autozygosity cerebrovascular diseases and stroke are a leading cause ofdeath and disability in developed countries. although they are less common in children, pediatric cerebrovascular disorders are important causes of morbidity and mortality and are in- creasing in prevalence ( , ). because of the frequent need for life-long medical services and social supports in the survivors, the implications for healthcare and social resource utilization in pe- diatric patients are often greater; thus, the identification of young populations at risk, early diagnosis, and appropriate management of the diseases become more important. increasing evidence suggests that there is a significant genetic predisposition to stroke, with more convincing findings through several single gene disorders in young patients ( ). to identify these single genes and study the pathogenesis of cerebrovascular disorders caused by the alteration of these genes will not only help the disease diagnosis and treatment in the affected patients, but also potentially provide valuable information in understanding the pathophysiology of cerebrovascular diseases in general. here we describe a cohort of patients with cerebral vasculopathy and early onset of stroke in an extended old order amish pedigree. through a genomewide homozygosity mapping study and muta- tional analysis, we identified a genetic variation in the samhd gene associated with this autosomal recessive condition. results clinical phenotype. fourteen individuals ( males and four females), ranging from newborn to y in age, were identified with this condition. the phenotype was not observed in the parents or unaffected siblings. three full-term stillbirths, without knowledge of phenotype, were reported from two mothers. all patients demonstrated old order amish ancestry, and genealogical analyses revealed multiple lines of common descent between all parents of affected children although they resided in three different states (fig. ). all affected children were full term, born after an uneventful pregnancy and delivery. routine hematologic and metabolic screenings were all within normal ranges at birth. standard karyotype analysis was performed on at least two patients and reported as normal. although there were no significant dys- morphic features noted at birth, the affected newborns tended to be relatively smaller for their gestational age with out of weighing less than the th percentile. their average birth weight ( , ± g), length ( . ± . cm), and occipitofrontal cir- cumference (ofc) ( . ± . cm) were all in the low end of the normal range (table s ). thin and transparent underdeveloped skin, similar to what we might see in preterm infants, was noted in at least of these newborns. thirteen infants were thereafter included for further clinical phenotype description, whereas newborn identified through dna mutation analysis right before the end of this study was excluded because his phenotype might not be fully expressed. the overall clinical phenotype of this condition was very het- erogeneous with a wide range of clinical manifestations and considerably diverse clinical presentations, including cerebral palsy, stroke, developmental delay, failure to thrive, chilblains, and arthritis (tables and ). more than half of the affected children were hypotonic and severely irritable during their in- fancy (table ). failure to thrive, short stature, joint stiffness or arthritis, high-arched palate, and low-pitch hoarse voice were also observed in the majority of the affected individuals. the children tended to have poor tolerance to extreme environments, both cold and hot. acrocyanosis was often found on their hands, feet, and face, worsening during cold weather (raynaud’s phe- nomenon), and eight children had a history of chilblain lesions in acral locations during winter. migraine headache, seizure, hy- author contributions: b.x. and h.w. designed research; b.x., e.g.p., c.h., a.b., h.t., a.z., j.v.-a., and h.w. performed research; b.x., s.j., e.g.p., c.h., a.b., g.w., j.v.-a., d.a., and h.w. analyzed data; and b.x. and h.w. wrote the paper. the authors declare no conflict of interest. this article is a pnas direct submission. to whom correspondence should be addressed. e-mail: wang@ddcclinic.org. this article contains supporting information online at www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental. www.pnas.org/cgi/doi/ . /pnas. pnas early edition | of m ed ic a l sc ie n c es d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=st mailto:wang@ddcclinic.org http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental www.pnas.org/cgi/doi/ . /pnas. pothyroidism, and glaucoma, although less common, had been found in at least three patients (table ). the variations of cognitive and motor function development in these patients were striking. four severely affected children manifested with profound global developmental delays and were completely dependent on their caregivers, whereas five patients demonstrated typical development without intellectual disability and participated in regular school. three of them worked as i ii iii iv v vi vii viii ix x ?? ? ? ? fig. . partial pedigree of the family with samhd gene mutation associated with cerebral vasculopathy. filled symbols represent affected individuals, and open symbols represent unaffected individuals. circles and squares denote females and males, respectively. a double line identifies consanguinity. arrows indicate affected individuals included in the genetic mapping study and sequence analysis. table . clinical features of patients with the homozygous mutation in samhd gene features incidence, % or average age neonatal features mild intrauterine growth restriction (< th percentile) ( / ) underdeveloped (thin and transparent) skin at birth ( / ) severe infantile irritability ( / ) hypotonia ( / ) failure to thrive ( / ) short stature (< th percentile) ( / ) poor tolerance to extreme (cold and hot) environments ( / ) acrocyanosis of hands, feet and face ( / ) raynaud’s phenomenon ( / ) chilblain lesions in acral locations ( / ) age of onset y ( – ) low-pitch hoarse voice ( / ) age of onset y ( – ) high-arched palate ( / ) glaucoma ( / ) migraine headache ( / ) seizure ( / ) hypothyroidism ( / ) joint stiffness or arthritis ( / ) scoliosis ( / ) hemorrhagic stroke confirmed ( / ) suspected ( / ) cognitive development severely delayed ( / ) mildly delayed ( / ) normal ( / ) elevated erythrocyte sedimentation rate (esr) ( / ) hyperimmunoglobulin g ( / ) elevated neopterin ( / ) incidence is expressed as a percentage with the number of patients applied in parentheses. the numbers in parentheses behind average age are ranges. of | www.pnas.org/cgi/doi/ . /pnas. xin et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , www.pnas.org/cgi/doi/ . /pnas. farmers or carpenters when they reached their adulthood. it was noted that all eight patients with cognitive disability had a history of strokes, and for the four severely affected patients, the onset of stroke occurred during their early infancy. in fact, intracranial hemorrhagic strokes were documented in nine patients between the ages of mo and y, with five of them having recurrent cerebral events. the neurological outcomes were poor if the strokes occurred during early infancy as patients often became full-time wheelchair users with progressing spasticity and con- tracture of extremities over time. likewise, the recurrent strokes were often associated with severe outcomes as both deceased patients died from recurrent strokes (tables and ). neither hypertension nor significantly abnormal hepatic and renal func- tions were documented in these patients. abnormal neuroimaging findings were identified in all patients with available imaging studies such as magnetic reso- nance imaging (mri) and magnetic resonance angiogram (mra) and computed tomography (ct) and ct angiogram (cta) (fig. table . additional clinical features in individual patient with the homozygous mutation in samhd gene patient sex initial presentation age at study, y ht at study, cm (per) other significant clinical history neuroimaging study findings wm change aneurysms stenoses ischemic change volume loss age at imaging, y x- m ftt, chilblain (< ) recovered from aneurysmal rupture at y old, now farming mild–mod + + − − x- m ftt, chilblain (< ) normal schooling mild–mod − − − − x- m ftt, chilblain (< ) normal schooling mod − − − mild x- m ftt, mild dd , died (< ) glaucoma, died from recurrent strokes mod–severe − mm + mild x- f ftt, mild dd , died (< ) glaucoma, died from recurrent strokes severe + + − − x- m ftt, mild dd (< ) history of stroke mod − mm + mild–mod x- f cp, severe dd n/a history of possible stroke, severe spasticity x- m arthritis ( ) working as a carpenter − − + − − x- m arthritis ( ) working as a carpenter − − mm − mild x- f cp, severe dd (< ) history of possible stroke, glaucoma, spasticity mod n/a n/a − mild–mod x- m stroke (< ) stroke at mo old, recurrent strokes, spasticity mod–severe − mm + − . x- m stroke ( ) stroke at mo old mild–mod n/a n/a − mild x- f stroke (< ) stroke at mo old x- m none newborn ( ) found through requested dna testing per, percentile; ht, height; wm, white matter; m, male; f, female; ftt, failure to thrive; dd, developmental delay; cp, cerebral palsy; mod, moderate; mm, moyamoya pattern; +, present; −, negative finding; n/a, no data are available. fig. . spectrum of neuroimaging abnormalities as- sociated with the cerebral vasculopathy caused by samhd gene mutation. (a) axial flair image from x- shows moderate nonspecific white matter signal hyperintensity, which is abnormal for age. the terri- tory matches the internal–external watershed, which is vulnerable to chronic small vessel disease as often seen in elderly patients. (b) axial flair image from x- shows encephalomalacia from a prior right pa- rietal transcortical infarct (arrow), with subcortical gliosis. additional white matter change is present in the left periventricular regions, likely from chronic small vessel disease. (c) axial noncontrast ct in x- shows encephalomalacia in the left anterior temporal lobe (arrow) from a preceding hemorrhagic stroke. (d) coronal cta from x- shows a large aneurysm arising from the left mca bifurcation (arrow), pro- jecting inferiorly. (e) coronal maximal-intensity pro- jection (mip) from an mra from x- shows extensive stenoses, severe in the right mca territory with an acute cutoff (left arrow) and minimal distal flow- related enhancement. there are similar stenoses in the bilateral a segments and a moderate stenosis of the distal left internal carotid artery (right arrow). xin et al. pnas early edition | of m ed ic a l sc ie n c es d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , and table , and fig. s ). all patients showed some degree of chronic ischemic change, manifested as abnormally increased t / flair white matter signal. the next most common finding, ob- served in seven out of nine mras and ctas, was multifocal stenosis of the large intracranial arteries, namely the distal in- ternal carotid artery, proximal middle cerebral artery (mca), and anterior cerebral artery. four patients showed stenoses severe enough to suggest a frank moyamoya pattern. several studies revealed earlier frank acute infarction, both local and territorial. two patients showed saccular aneurysms arising from the large vessels, particularly the mca bifurcation. a marked increase in aneurysm size over an interval of mo was observed in one pa- tient. in addition, one patient showed encephalomalacia from a prior frank intracranial hemorrhage, and one showed subtle he- mosiderin from a prior petechial hemorrhage. postmortem ex- amination of patient x- in the pedigree of fig. suggested early cerebral atherosclerosis, intimal proliferation, mild leukoence- phalopathy, cerebellar hemorrrhage, and optic atrophy (fig. s ). elevated erythrocyte sedimentation rate (esr) ( ± mm/h) was observed in patients, and increased serum igg ( , ± mg/dl) was detected in patients. abnormally increased serum neopterin level was also noted in all patients tested ( . ± . nmol/l) (table and table s ). in addition, mildly elevated anticardiolipin antibodies and antithyroglobulin antibodies were found in patients ( / ), a positive rheumatoid factor was noted in patient ( / ), whereas several other serum markers, such as c-reactive protein and antinuclear antibody c and c , were in the normal range (table s ). furthermore, higher levels of serum tumor necrosis factor-α (tnf-α) were observed in affected indi- viduals ( . ± . pg/ml, n = ) compared with normal controls from the same community ( . ± . pg/ml, n = ) (p < . ). genotyping and mapping. to determine the genetic basis of this condition, we performed a genomewide autozygosity mapping study using affymetrix genechip mapping k snp arrays with six affected individuals from three different sibships in the large consanguineous pedigree (fig. ). this mapping study was more difficult than previous studies due to the paucity of snps in the disease gene interval. our initial analyses using all six patients failed to conclusively identify a single large homozygous region. the genotype data were then analyzed by assessing genotype identity (identity by state) between affected members within two families. four genomic regions (on chromosomes (chr) , , , and ) demonstrated identity by state between affected mem- bers within each family. on the basis of these data, homozygosity was assessed within the families with respect to the four blocks of genotype identity. in the first family with three affected indi- viduals, a single large shared homozygous region consisting of contiguous snps was identified on chromosome q . -q (fig. s a). this shared homozygous block was . mb in length and was bounded by snps rs and rs . in the sec- ond family with two affected individuals, multiple homozygous blocks were identified, one of which overlapped with the large block from the first family on chromosome . the overlapping interval, delimited by snps rs and rs , further narrowed the candidate region to . mb. finally, examination of the single patient from the third family revealed a modest shared homozygous block of snps in all patients (fig. s b). exami- nation of the minimal shared region, which was flanked by snps rs and rs , revealed known or predicted genes based on both the ncbi and celera annotations (table s ). mutation identification. assessment of the genes in the putative disease locus revealed were predicted or hypothetical genes and were pseudogenes. we prioritized sequence screening to the remaining genes with known function or with sequence information validated in genbank. candidate gene sequencing was performed to screen the coding region and associated intronic splice junctions using genomic dna from one patient. we sequenced five genes (adig, fam d, ghrh, ppp r b, and slc a ) before we identified a homozygous splice- acceptor site mutation (c. - a > g) in intron of the samhd gene (fig. a). the targeted sequencing of samhd intron and exon boundary revealed that all affected indi- viduals (n = ) were homozygous for the mutation, their parents were heterozygous, and no unaffected siblings (n = ) were homozygous for the change. we next screened healthy amish control samples (n = chromosomes) from the same geographic area where the patients were found and determined that none were homozygous, whereas were heterozygous for the c. - a > g mutation (estimated carrier frequency of . %). further mutation screening for the dna bank of the undiagnosed patients (n = ), mostly with developmental delay in our institution, identified more c. - a > g homozygotes, raising the total number of affected individuals to . review of their clinical history revealed a phenotype very similar to the patients initially identified, with two infant patients being hypo- tonic and severely irritable, and the third one ( y old) with a di- agnosis of cerebral palsy with abnormal brain mri in the past. rna analysis. to investigate the consequence of this splice site mutation at the transcript level, we performed rt-pcr using rna extracted from ebv-transformed lymphoblastoid cell lines with different c. - a > g genotypes. this showed an aber- rant shorter transcript from cell lines of affected individuals compared with the normal controls, whereas both normal and the aberrant transcripts were detected from heterozygous car- riers (fig. b). direct sequencing of the pcr products after gel extraction revealed skipping of exon ( bp) in the abnormal transcript, which led to an in-frame deletion of amino acids in translation (p.glu _asp del) (fig. c). protein analysis. immunoblotting assays were performed with a polyclonal antibody raised against the full-length samhd protein and cell lysates prepared from lymphoblastoid cell lines. a protein band corresponding to wild-type samhd (∼ kda) was detected from cell lines of a normal control and heterozygous carrier, but not the affected subject (fig. d). surprisingly, the mutant protein was barely detected from either the carrier or af- fected subject. even after a prolonged exposure, only a faint band corresponding to the mutant samhd (p.glu _asp del) was observed in the cell lysate from the affected subject (fig. d). meanwhile, a trace amount of degraded samhd product was also observed in the affected subject, but not from the normal control (fig. d). discussion here we describe an autosomal recessive condition manifesting with cerebral vasculopathy and early onset of stroke in an extended old order amish pedigree. the patients seem affected during the early stage of development, even before they are born. three full- term stillbirths are noted in two of the five affected families. the live newborns affected with this condition were relatively smaller, with thin, transparent, underdeveloped skin at birth, followed by hypotonia and severe irritability during early infancy. cerebral vasculopathy is a major hallmark found in all af- fected individuals with a common theme of stenoses and aneurysms. typical neuroimaging findings include chronic is- chemic changes, multifocal stenoses of the large intracranial arteries, including some with moyamoya morphology and evi- dence of prior acute infarction and hemorrhage. migraine headaches might result from cerebral vasculopathy, whereas seizures seem to be a consequence of strokes as no patient had epilepsy documented before stroke. ruptured aneurysms likely cause the most acute and severe outcomes. progressive stenoses likely cause more chronic changes, starting with white matter gliosis in the deep watershed regions of the brain, where lower physiologic perfusion causes increased susceptibility to micro- ischemia from any further reduction of perfusion pressure. with the progression of stenosis, the risk of catastrophic territorial of | www.pnas.org/cgi/doi/ . /pnas. xin et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=sf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=sf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=st http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=st http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=sf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=sf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=st www.pnas.org/cgi/doi/ . /pnas. infarction increases. indeed, it has been noted that the clinical outcomes in these patients seem largely dependent on the degree of the cerebral vasculopathy, particularly if a stroke has occurred before evaluation. the early onset or recurrence of strokes se- ems associated with poor outcomes and variable cognitive dis- ability, whereas patients without a history of stroke, although in a guarded condition, show fairly normal development without in- tellectual or motor disability. the phenotypic heterogeneity noted in this condition caused by the identical single mutation is remarkable, particularly considering the relatively uniform ge- netic background of the amish population. we speculate that the time and location of catastrophic cerebral events in affected individuals may be largely responsible for such phenotype vari- ation, implying a potential for proactive interventions. thus, periodic cerebral angiograms as routine follow-up seem war- ranted for these patients as some vascular stenoses and aneur- ysms may be highly treatable with surgical procedures. the clinical diagnosis of the disease remains challenging, particularly before the phenotype is described. this was well exemplified by unexpected diagnosis of three new patients through targeted sequencing of samhd intron and exon boundary among undiagnosed patients with developmental delay in our institution. indeed, the clinical features of this condition overlap with many assorted conditions such as congenital in- fection, cerebral palsy, moyamoya, vasculitis, lupus, rheumatoid arthritis, mixed connective-tissue disease, and primary angiitis of the central nervous system. it is noted that the pathological vascular changes are fairly organ specific, primarily notable in the brain and skin. other than the many clinical and radiological findings related to cerebral vasculopathy found in these patients, acrocyanosis, raynaud’s phenomenon, and chilblain lesions in hands, feet, and face during winter are fairly characteristic of this condition. these clinical features might be a reflection of patho- logical vascular changes in skin. interestingly, other essential organs are not notably affected as all patients have normal blood pressure and normal cardiac, hepatic, and renal functions. al- though the specific relationship between abnormal skin and cerebral vasculopathy remains to be understood, these rare der- matological manifestations seem fairly disease specific. we won- der whether these clinical features could potentially serve for identification of affected individuals in the general population. through genomewide homozygosity mapping and mutational analysis in affected individuals from a consanguineous pedigree, we have identified a pathogenic sequence variant, c. - a > g, in the samhd gene that is associated with the disease. this alteration has not been reported as a polymorphic variant in genbank. it cosegregates consistently with the disease pheno- type, as all affected individuals are homozygous, their parents are heterozygous, and no unaffected siblings or normal controls are homozygous for the mutation. the samhd gene consists of coding exons and encodes a protein of amino acids. the c. - a > g mutation, appeared at the consensus splice- acceptor site in intron , resulted in skipping of exon of mrna transcript, and gave rise to an aberrant protein with in- frame deletion of amino acids (p.glu _asp del). al- though rt-pcr showed robust expression of aberrant samhd mrna transcript in affected cell lines, the mutated form of samhd protein was barely detected by western blot from those cells, suggesting that the lack of samhd protein expression might be due to protein degradation or reflects impaired protein translation. the results further indicate that the disease patho- genesis likely reflects a loss of samhd protein rather than ex- pression of a partially functional protein. little is known about the function of samhd . smart se- quence analysis shows two functional domains in samhd protein: a sterile alpha motif (sam) (residuals – ) and an hd domain (residuals – ). the sam domain is a putative protein interaction module mediating interactions with other sam domain and non-sam domain-containing proteins ( ). in addition, sam domains also appear to possess the ability to bind rna ( ). the hd domain is found in a superfamily of enzymes with a predicted or known phosphohydrolase activity appearing to be involved in the nucleic acid metabolism ( ). this evidence suggests that samhd may act as a nuclease. mutations in the samhd gene have recently been identified as one of five causative agents for a rare genetic condition, aicardi-goutières syndrome ( , ), highlighting the presence of different patho- genic mutations in the samhd gene beyond the amish pop- ulation. however, the phenotype reported here is apparently incompatible with aicardi-goutières syndrome, which is a type of encephalopathy whose clinical features mimic those of ac- quired in utero viral infection. furthermore, none of our intron exon normal mutant n or m al - n or m al - ca rr ie r- ca rr ie r- af fe ct ed - af fe ct ed - d n a m ar ke r a b gattaaaagg gttatcaaca gaggactatg // tatagtggat // exon exon exon exon exon c d samhd no rm al ca rri er kd kd kd kd af fec ted β-ac�n fig. . identification of the disease-causing mu- tation in samhd gene. (a) sequence electro- pherograms showing the homozygous c. - a > g mutation from affected individuals compared with a normal control. (b) agarose gel electro- phoresis of rt-pcr products from lyphoblastoid cells showing a shorter abnormal transcript as a consequence of c. - a > g mutation. (c) se- quencing analysis of the rt-pcr products showing the splicing out of exon in the aberrant tran- script. (d) western blot analysis of samhd pro- tein expression in cell line samples. cell lysates of lymphoblastoid cell lines from a normal control, a carrier, and an affected individual were immu- noblotted with anti-samhd antibody. an arrow indicates the trace amount of mutant samhd detected. a diamond shows a smaller weak band suggesting protein degradation of the mutant samhd . β-actin levels were monitored by im- munoblotting as a control of protein loading. xin et al. pnas early edition | of m ed ic a l sc ie n c es d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , patients has been diagnosed with aicardi-goutières syndrome. the wide spectrum of the disease phenotype caused by samhd mutation reported here indicates a potentially much broader implication of this study. in fact, samhd was originally iden- tified in a human dendritic cell cdna library as an ortholog of the mouse ifn-γ–induced gene mg and even termed dendritic cell derived ifn-γ–induced protein (dcip) ( ). several lines of evidence implicate samhd in immune function as it is up- regulated in response to viral infections and may have a role in mediating tnf-α proinflammatory responses ( – ). a more recent study suggests that samhd may act as a negative reg- ulator of the immunostimulatory dna response and may have a protective role in preventing self-activation of innate immunity ( ). indeed, many clinical findings in our report, particularly abnormal laboratory findings (e.g., elevated esr, igg, neo- pterin, and tnf-α) in the patients support a particular role for samhd as an immunomodulator. it is noted that innate and adaptive immune processes are in- volved in medium- and large-vessel vasculitis ( – ). the vas- culopathy associated with samhd mutation in this study consistently targets certain organs while sparing others. remark- able target tissue tropisms with distinctive vascular territories have been observed in several other inflammatory vasculopathies re- lated to their vessel-specific toll-like receptor (tlr) profile ( – ). we speculate that samhd may play an important role in cerebral vasculopathy, and further studies of the role of samhd in blood vessel integrity and homeostasis of the brain will be very significant not only for this condition, but for a group of diseases related to cerebral vasculopathies, such as moyamoya ( ) and primary angiitis of the central nervous system ( ). materials and methods subjects and clinical assessment. the study was approved by the ddc clinic for special needs children institutional review board, and written informed consent was obtained from each participant or their legal guardian. a total of affected individuals from four separate old order amish settlements in ohio, kentucky, and tennessee were clinically evaluated. additional clinical information and neuroimaging studies obtained through the medical records were reviewed. the diagnosis was established on the basis of family history, physical examination, laboratory results and imaging studies, and later confirmed by dna mutation analysis. most parents and siblings of the patients also chose to participate in the study. plasma tnf-α, as a part of humanmap antigen panel, was measured using fully automated, bead- based multiplex sandwich immunofluorescence assays provided by rules- based medicine in nine affected individuals and normal controls of the same ethnic background and age range. the genealogical information obtained from the affected families was confirmed and expanded through the swiss anabaptist genealogical association. genotyping and mutation detection. dna isolation, genotyping, and mutation detection were performed as described previously ( , ). pcr primers were designed to amplify each of the protein-coding exons and their flanking intronic sequences of samhd . primer sequences are provided in table s . the genbank accession nos. of both genomic dna and mrna reference sequences used in this study are nt_ and nm_ , respectively. reverse transcription-pcr. total rna was isolated from ebv-transformed lymphoblastoid cell lines using qiaamp rna kit (qiagen) according to the manufacturer’s protocol. cdna was synthesized by priming of μg of total rna using superscript first-strand synthesis system (invitrogen) following the manufacturer’s protocol. pcr amplification of samhd cdna was per- formed using primers f and r located in exons and , re- spectively (table s , the base pair numbers of primers were determined according to the mrna sequence nm_ ). the amplicons were ana- lyzed by agarose gel electrophoresis, purified on qiaquick spin columns, and sequenced by big dye terminator cycle sequencing. protein analysis. lymphoblastoid cell lines from peripheral blood samples were prepared and maintained using standard techniques. total cell lysates were prepared by incubating cells at °c for min in the nonidet p- lysis buffer supplemented with protease inhibitors. lysates were centrifuged for min at , rpm at °c, and the clarified supernatants were then loaded onto sds polyacrylamide gels. after sds/page, proteins were transferred onto polyvinylidene difluoride membranes (millipore). mem- branes were blocked for h with % nonfat milk and incubated overnight at °c with a : dilution of mouse anti-samhd polyclonal antibody (sigma-aldrich). the following day, the blots were washed × min with pbs/ . % tween- and then incubated for h at room temperature with a : , dilution of horse antimouse igg horseradish peroxidase (cell sig- naling technology). after washing with pbs/ . % tween- , the protein bands were detected by ecl chemiluminescent assay (ge healthcare). statistical analysis. all data are presented as means ± sd. comparisons of the affected individuals and controls were made using student’s t test and p value < . was considered statistically significant. acknowledgments. we thank the families for their patience and support. we appreciate the physicians who provided outstanding and com- passionate care to the affected children through rainbow babies and children’s hospital, cincinnati children’s hospital medical center, kosair children’s hospital, vanderbilt university medical center, and shriners hos- pitals for children, and carol troyer, carol nelson, and betty presler who helped with the data collection. the study was supported in part by the elisabeth severance prentiss foundation, the reinberger foundation, and the leonard krieger fund of the cleveland foundation (l - ). . pappachan j, kirkham fj ( ) cerebrovascular disease and stroke. arch dis child : – . . seidman c, kirkham f, pavlakis s ( ) pediatric stroke: current developments. curr opin pediatr : – . . francis j, raghunathan s, khanna p ( ) the role of genetics in stroke. postgrad med j : – . . schultz j, ponting cp, hofmann k, bork p ( ) sam as a protein interaction domain involved in developmental regulation. protein sci : – . . kim ca, bowie ju ( ) sam domains: uniform structure, diversity of 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( ) homozygous frameshift mutation in tmco causes a syndrome with craniofacial dysmorphism, skeletal anomalies, and mental retardation. proc natl acad sci usa : – . of | www.pnas.org/cgi/doi/ . /pnas. xin et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=st http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. si.pdf?targetid=nameddest=st www.pnas.org/cgi/doi/ . /pnas. _ftp.pdf variable bone fragility associated with an amish col a variant and a knock-in mouse model ethan daley, elizabeth a streeten, john d sorkin, natalia kuznetsova, sue a shapses, stephanie m carleton, alan r shuldiner, , joan c marini, charlotte l phillips, steven a goldstein, sergey leikin, and daniel j mcbride jr orthopaedic research laboratories, department of orthopaedic surgery, university of michigan, ann arbor, mi, usa division of endocrinology, diabetes & nutrition, university of maryland baltimore, baltimore, md, usa division of gerontology, university of maryland baltimore and the baltimore va medical center, geriatric research, education and clinical center (grecc), baltimore, md, usa national institute of child health and human development, national institutes of health, bethesda, md, usa department of nutritional sciences, rutgers university, new brunswick, nj, usa department of biochemistry, university of missouri–columbia, columbia, mo, usa abstract osteogenesis imperfecta (oi) is a heritable form of bone fragility typically associated with a dominant col a or col a mutation. variable phenotype for oi patients with identical collagen mutations is well established, but phenotype variability is described using the qualitative sillence classification. patterning a new oi mouse model on a specific collagen mutation therefore has been hindered by the absence of an appropriate kindred with extensive quantitative phenotype data. we benefited from the large sibships of the old order amish (ooa) to define a wide range of oi phenotypes in individuals with the identical col a mutation. stratification of carrier spine (l – ) areal bone mineral density (abmd) z-scores demonstrated that % had moderate to severe disease (less than � ), % had mild disease (� to � ), and % were in the unaffected range (greater than � ). a line of knock-in mice was patterned on the ooa mutation. bone phenotype was evaluated in four f lines of knock-in mice that each shared approximately % of their genetic background. consistent with the human pedigree, these mice had reduced body mass, abmd, and bone strength. whole-bone fracture susceptibility was influenced by individual genomic factors that were reflected in size, shape, and possibly bone metabolic regulation. the results indicate that the g c oi (amish) knock-in mouse is a novel translational model to identify modifying genes that influence phenotype and for testing potential therapies for oi. � american society for bone and mineral research. key words: osteogenesis imperfecta; bone; collagen; knock-in; rodent introduction osteogenesis imperfecta (oi) is a heritable form of bonefragility with an overall spectrum of disease severity that ranges from a perinatal lethal form to mild disease that can remain clinically silent.( – ) multiple fractures is the principal clinical presentation that brings oi patients to medical attention. additional phenotype traits can include bone deformity (e.g., long bone bowing), short stature, blue sclerae, dentinogenesis imperfecta (di), and hearing loss. sillence originally classified oi into types i to iv on the basis of clinical presentation, radiographic features, and mode of inheritance.( , ) oi types i to iv typically are associated with heterozygous mutations in the genes (col a and col a ) that encode the a chains of type i procollagen molecules. currently, more than col a and col a mutations have been identified in oi patients, with many examples of recurrent mutations in unrelated individuals.( ) however, there are no large pedigrees with quantitative phenotype data for oi patients with the identical mutation. the predominant type i collagen isotype in the extracellular matrix (ecm) is a heterotrimeric molecule comprised of two a (i) chains and one a (i) chain. a homotrimeric isotype of type i collagen, comprised of three a (i) chains, is also found in tissues in small amounts in the nonpathologic state. the homotrimeric isotype is associated with autosomal recessive connective tissue disorders in humans when no a (i) chains or only nonfunctional a (i) chains are synthesized.( , ) the amino acid sequences of both a (i) and a (i) chains are homologous and have a characteristic [gly-x-y] repeat within their respective triple- helical domains. the small side chains of glycine residues original article jjbmr received in original form march , ; revised form may , ; accepted july , . published online july , . address correspondence to: daniel j mcbride, jr., montgomery woods drive, hockessin, de , usa. e-mail: dmcbride@udel.edu journal of bone and mineral research, vol. , no. , february , pp – doi: . /jbmr. � american society for bone and mineral research positioned at every third position of the repeat are sterically required for helix formation, and mutations in these glycine residues account for % of the known triple-helical mutations that cause moderately severe to lethal forms of oi. over the past two decades, several laboratories have developed murine oi models based on mutations in the proa (i) chain of type i collagen patterned after those described in human oi to extend our understanding of oi pathobiology. all the existing mouse models have a moderate-to-severe bone phenotype with impaired viability. the two early models of note are the col a null allele mov- mice( – ) and the ‘‘protein suicide’’ or collagen minigene model.( – ) the homozygous mov- mouse produces no type i collagen, and this is lethal. heterozygous mov- mice deposit reduced amounts of normal type i collagen in the ecm that is associated with an osteopenic phenotype and abnormal skeletal biomechanics. the minigene model construct expresses a version of the human col a gene that is missing the central exons that encode a significant portion of the triple-helical domain. the shortened human proa (i) chains associate with normal murine proa (i) and proa (i) chains, depleting the amount of normal type i collagen and altering ecm organization. most recently, the cre/lox recombination system was used to develop a col a g c substitution to reproduce a human oi type iv (moderately severe) phenotype termed brtliv.( – ) the brtliv mouse represented a major advance in oi mouse models because it is the only col a model with a triple-helical glycine mutation. the oi phenotype pattern of proa (i) glycine substitutions appears to be different from proa (i) substitution.( ) generally, proa (i) mutations are less severe. they also may represent an easier target for treatment because only % of collagen molecules are affected in col a heterozygotes versus % in col a heterozygotes. however, the only reported mouse model for oi with a col a mutation is osteogenesis imperfecta- murine (oim). homozygous oim mice exhibit skeletal disease with clinical and biochemical features of sillence type iii (severe progressive) oi. the oim mutation and its biologic conse- quences( – ) are strikingly similar to those found in a german child with type iii oi.( , , ) in both instances, a frame-shift mutation in the region of the col a gene that encodes the terminal portion of the proa (i) c-propeptide predicts the synthesis of nonfunctional proa (i) chains. despite being the only naturally occurring oi mouse model, the oim mouse has a rarely observed type of mutation and an atypical biochemical phenotype because only proa (i) homotrimeric molecules are formed. the absence of a col a murine oi model with autosomal dominant inheritance, mild to moderate disease, and phenotype variation represents a major gap in our tools to understand oi pathobiology and to develop effective treatment. defining the molecular basis of phenotype variation also has been limited by the absence of large numbers of oi patients carrying the identical causative mutation with quantitative phenotype data to use as a prototype for a new oi mouse model. here we report the clinical and biochemical phenotype of a col a gene variant found among a lancaster county, pennsylvania, old order amish (ooa) kindred. this col a variant is a g-to-t transversion at nucleotide that alters the gly- codon (ggt) to a cysteine (tgt) codon.( ) we also report the creation of the first glycine-substitution knock-in col a mouse that replicates the gene and protein phenotypes observed in the ooa kindred. the knock-in g c oi (amish) mouse represents a unique translational model for evaluating the molecular basis of phenotype variability and to test potential therapeutic strategies for oi. materials and methods ethical considerations all human subject research was reviewed and approved by the institutional review board of the university of maryland baltimore (umb). informed consent was obtained for all study participants. all animal procedures were reviewed and approved by the umb institutional animal care and use committee. recruitment of family members and phenotype assessment the study had three stages of informed consent: ( ) mutation screening, ( ) clinical assessment, and ( ) skin-biopsy harvest. the screening enrollment age requirement of years or older was waived if one parent was mutation-positive by sequence analysis. after obtaining informed consent (or assent from the children), buccal cells were harvested as a source of genomic dna (gdna) for genotyping. all gdna- and cdna-derived sequence data (see below) were analyzed, using sequencher software (genecodes, ann arbor, mi, usa). all volunteers verified by gdna sequence analysis to carry a single copy of the t allele (oi group) and their control (g allele) nuclear family members (age � years) were invited to participate in the clinical assessment and skin-biopsy phases. the phenotype assessment consisted of a medical and family history, physical examination, blood draws for serum procolla- gen type i c-terminal propeptide (picp) and crosslaps measurements (markers of bone formation and resorption), and measurement of areal bone mineral density (abmd). lumbar spine (l – ) and proximal femur (total and subregions) abmd was measured by dual-energy x-ray absorptiometry (dxa) on a qdr w instrument (hologic, bedford, ma, usa), and the results reviewed by a single clinician (eas) and another author (djm). the in vivo coefficient of variation for replicate abmd measurements was . % for the lumbar spine, . % for total hip, and . % for the femoral neck. the hologic pediatric reference curve option (hip: to years; spine: to years) and caucasian reference (age > years) were used to obtain age- specific z-scores. since oi is frequently associated with short stature and bone mineral content (bmc) and abmd are influenced by bone size, volumetric bone mass [bone mineral apparent density (bmad)] also was estimated to minimize the influence of small bone size.( , ) a subset of the phenotype assessment volunteers also provided mm dermal punch biopsies from the posterior upper arm from which fibroblasts were derived and stored at umb and/or submitted to the coriell institute (oi families , , , and ). journal of bone and mineral research daley et al. generation of knock-in mice knock-in mice were created using embryonic stem cells and cre/ lox p technology. a kb genomic clone containing the relevant segment of the murine col a gene was isolated from a l phage library constructed with gdna of the sv/ev taconic mouse strain. pcr site-directed mutagenesis was used to change the targeted codon from ggt to tgt. the col a g c-positive embryonic stem cells were injected into blastocysts of c bl/ j (b ) mice. founder mice that retain the neo targeting vector are termed neoþ or g c neo mice. progeny obtained by breeding a founder male with a female that expressed cre recombinase (jackson laboratory, bar harbor, me, usa, stock number ) are termed neo– or g c oi mice. the g c oi mouse line is available to the research community through the jackson laboratory mouse repository (jax stock number: ). four f strains of g c oi mice were used to determine the effect of genetic background on phenotype in -month-old male mice. incipient congenic g c oi b (� % b genetic background) male breeders were generated by six generations of backcrosses to jackson laboratory b mice (stock number ). experimental heterozygous b male breeders were crossed with a/j (stock number ), balb/cbyj (stock number ), c h/hej (stock number ), and fvb/nj (stock number ) females purchased from the jackson laboratory. progeny of these crosses are designated, respec- tively, as a.b , cby.b , c .b , and fvb.b . experimental mice were housed at umb in a single specific-pathogen-free room and were exposed to identical environmental conditions consisting of a hour light/dark cycle, an ambient temperature of c, and ad libitum access to water and laboratory mouse chow. genotype was assigned using a pcr assay that can discriminate the three possible g c oi mouse genotypes. the forward primer (tcc ctg ctt gcc cta gtc cca aag atc ctt) and the reverse primer (aag gta tag atc aga cag ctg gca cat cca) will generate a bp (wild type) or a and a bp (heterozygous) or a bp (homozygous) pcr product using g c oi mice gdna. all animals were euthanized by co asphyxiation. human and mouse collagen analysis pepsin-soluble collagen was purified from mouse tail tendons and human fibroblast culture medium for analysis by sds-page and differential scanning calorimetry (dsc). purified collagen samples were labeled by fluorescent monoreactive cy nhs ester (ge healthcare bio-sciences corp., piscataway, nj, usa) or double-labeled by cy and fluorescent bodipy-l-cys (molecular probes, invitrogen, inc., carlsbad, ca, usa).( ) labeled proteins were size-separated by sds/page on precast % to % gradient tris-acetate minigels (invitrogen). following electrophoresis, the gels were briefly rinsed in distilled water and scanned on a fuji fla fluorescence scanner (fuji medical systems, stamford, ct, usa) using a nm excitation laser for bodipy-l-cys and a nm laser for cy . for dsc, collagen was redissolved ( to mg/ml final collagen concentration) in mm hcl (ph . ) or in . m sodium phosphate/ . m glycerol (ph . ) and then dialyzed extensively against the same buffer to remove excess salt. dsc thermograms( ) of approximately . mg/ml of pepsin- treated collagen in mm hcl (ph . ) and in the phosphate/ glycerol buffer (ph . ) were recorded in a nano ii differential scanning calorimeter (calorimetry sciences corp. lindon, ut, usa) at several different scanning rates. the apparent melting temperature tm was defined at the maximum of the melting peak after baseline subtraction. total rna was extracted from fibroblast cultures, and cdna was synthesized using race [ -gat gga tcc tgc aga agc (t) - ]. an aliquot of cdna and forward exon primer and downstream reverse exon primer (selected to cross intron-exon) boundaries were used for second-strand synthesis and pcr amplification. pcr products of the expected size were confirmed by agarose gel size separation prior to purification for direct nucleotide sequencing. phenotype assessment of f g c oi mice abmd measurements the abmds of right femurs were measured by dxa (ge-lunar densitometer, piximus; software version; . , ge medical systems, wi, usa). femur measurements were obtained by placing an excised femur on a delrin block. the percent coefficient of variation (cv) was . % for bmc and . % for bmd. confocal raman microspectroscopy segments of cortical bone from the femur diaphysis were embedded in cryo-gel (instrumedics, inc., st. louis, mo, osa) and cut longitudinally normal to the bone surface on a cryo-cut one cryostat (vibratome, bannockburn, il, usa). the mm sections were examined with a senterra confocal raman microscope (bruker optics, billerica, ma, usa). raman spectra from to cm� were collected with to cm� resolution using a circularly polarized mw ( mw at the sample), nm laser beam, x / . na objective, and a mm confocal pinhole. the spectra were acquired at points visually selected outside osteocytes along the periosteal to endosteal axis in the midplane of the sections. the accumulation time for each point was minute. the mineral content of bone matrix (po :ch), collagen content of bone matrix (amide iii:ch), and mineral:collagen ratio (po :amide iii) were determined from the areas under n -po (integrated from to cm� ), amide iii ( to cm� ), and ch ( to cm� ) raman peaks. relative collagen content from pro:ch ratio ( to cm� ) was consistent with that evaluated from the amide iii:ch ratio but less accurate owing to low intensity of the pro peak. femur mct micro-computed tomography (mct) was used to quantify femoral geometry, morphology, and mineralization (ge medical systems, london, on, canada). femurs from f male -month- old mice were scanned over degrees of rotation and reconstructed on mm voxels. cortical and trabecular analyses included measures of bone mineral content and density, cortical and trabecular thickness, and trabecular spacing. cortical regions of interest (rois) consisted of cylindrical segments (radius . mm, height mm) of the middiaphyses. trabecular rois also consisted of cylindrical segments (radius . mm, height equal to % of total femur length) of the distal femoral metaphyses, amish oi mutation journal of bone and mineral research proximal to the growth plates. separate thresholds were applied to the cortical and trabecular rois ( and hu, respectively). ex vivo mechanical testing femurs were mechanically tested at room temperature using a four-point bending apparatus ( mini-bionix, mts, eden prairie, mn, usa) in a manner described previously by jepsen.( ) with posterior femoral surfaces kept in tension, displacement was applied at . mm/s. the upper and lower tines of the four- point support were separated by . and . mm, respectively. custom software was used to determine stiffness, yield, maxi- mum load, and pre- and postyield fracture energy. standard beam theory was used to calculate yield stress and young’s modulus based on the mechanical testing data and femoral geometry as measured by mct. statistical analysis statistical analysis was performed using instat and prism statistical software packages (graphpad, la jolla, ca, usa) or sas statistical software (sas institute, cary, nc, usa). a two-tailed p value of . or less for any single analysis was considered statistically significant. outliers (defined as values exceeding � s from the group mean) were removed from the mct and four- point bending data sets. results ooa kindred genotyping/pedigree structure putative founder couple identification a -year-old woman with a history of fracture, low hip and spine abmd, and a differential diagnosis of idiopathic osteoporosis or oi was identified in the amish family osteoporosis study (afos).( , ) a brother and a niece of the woman also were identified with a history of fracture and low abmd. gdna was extracted from blood of the proband’s brother for analysis of col a and col a genes.( – ) nucleotide sequencing identified a g-to-t substitution that converts the triple-helical codon for glycine- (ggt) to cysteine (tgt). sequence analysis confirmed that the proband, brother, and niece carried a single copy of the mutant t allele. three additional afos dna samples (a grandmother, mother, and granddaughter) were found subsequently to carry single copies of the variant t allele. the anabaptist genealogy database (agdb )( , ) was queried using pedhunter( ) software to link the six individuals with the mutant t allele to a putative founder couple (fig. ) born approximately years ago. agdb genealogic records listed approximately descendents from four of the putative founder couple’s children, of which approximately are estimated to be alive today. genotype status was obtained for study participants; were direct descendants of the putative founder couple, were direct-descendant married-in spouses, and were descendants of the putative founder couple’s siblings or more distant relatives. the variant t allele was identified in descendants in sibships ranging in age from weeks to years. sibships ranged from to individuals with an average of approximately individuals. the overall sibship genotype distribution exhibited a normal mendelian : ratio expected for offspring from one carrier parent and one wild-type parent. no homozygous individuals and no carrier � carrier couples were identified. ooa kindred phenotype standing height standing height as a function of age (fig. ) suggested that oi family members tended to be shorter than their unaffected family members. standing height was converted to z-scores to better assess the effect of the t allele across the wide age range of study participants. ooa-specific standing height z-scores were generated separately for women and men from height data obtained from females and males (age > years) enrolled in non-oi research studies (unpub- lished umb data). since ooa-specific data were unavailable for study participants to years of age, standing height z-scores were generated using the national health and nutrition examination survey (nhahes) data (https://web.emmes.com/ study/ped/resources/htwtcalc.htm). mean standing height z-score was significantly less (p < . ) for carriers of the t allele as compared with control family members but with appreciable overlap in z-scores between control and oi study participants (see fig. ). biomarker measurements picp values for oi family members were relatively constant as a function of age (see fig. ). however, picp for control family fig. . ooa col a g c putative founder couple pedigree. the col a variant is a g-to-t transversion at nucleotide that alters the gly- codon (ggt) to a cysteine (tgt) codon. the mutant t allele has been mapped in heterozygous descendants of the putative founder couple born approximately years ago. four of the founder couple’s offspring have an estimated living descendants, whereas the other five offspring produced no progeny. (control genotype ¼ gg; oi genotype ¼ gt.) journal of bone and mineral research daley et al. members younger than years of age were markedly higher compared with oi family members. genotype (p < . ) and age (p < . ) were found to make significant contributions in a multiple regression model using age, sex, and genotype as variables for picp. in contrast, only age (p < . ) was found to make a significant contribution to serum crosslaps. bmd measurements lumbar spine (l – ) and femoral neck (fn) bmad by age shown for males and females suggested that the oi group has reduced bmad compared with family controls. age, sex, genotype, and weight were used as predictor variables in multiple regression models for lumbar spine and fn bmad. sex (p < . ) and genotype (p < . ) made significant contributions to lumbar spine and fn bmad. weight (p < . ) also contributed to lumbar spine bmad, and age (p < . ) contributed to fn bmad. bmd assessed as z-scores for the spine and hip are shown in fig. . mean z-score differences between the oi and control groups at the lumbar spine and the fn were significant (p < . ). the mean z-score at the lumbar spine was � . � . for the oi group and � . � . for the control group. mean fn z-scores were � . � . for the oi group and . � . for the control group. production of the knock-in mice nucleotide gdna sequence (genbank accession identifier dq ) analysis demonstrated that f founder mice (male ¼ , female ¼ ) were heterozygous for the knock-in mutation in cola exon (equivalent to col a exon ). intron for the f founder knock-in allele [col a tm mcbr; mouse genome informatics (mgi) id ] contains nucleo- tides, whereas the wild-type b allele contains nucleotides. f founder mice cdna sequence analysis confirmed expression of mutant and wild-type mrna. offspring carrying one or two copies of the knock-in col a tm mcbr allele from f founder breeding survived without any detectable lethality at birth or beyond weaning and were fertile. genomic dna sequence analysis confirmed the cre-mediated excision of the floxed neo cassette in an f male mouse that was subsequently used to sire g c oi mice progeny on a b background. intron in g c oi mice is comprised of nucleotides, and its mgi allele designation is col a tm . mcbr (mgi accession identifier ). it differs in size from the wild-type b sequence by the addition of bp of residual targeting vector sequence that flanked the lox p sites and the loss of bp of b sequence (genbank accession identifier dq ). heterozygous � wild-type breeding produced litters with both expected genotypes. however, no viable homozygous offspring were obtained from heterozygous matings. genotype analysis of dead pups recovered within hours of birth did demonstrate the presence of pups homozygous for the col a tm . mcbr allele in the litters. human and mouse sds-page analysis double labeling with cy and bodipy-l-cys revealed intense cys staining of a (i) chains from all mutant animals and proband collagens (fig. a, b), indicating the presence of exposed reactive cys-sh residues in a (i) chains of type i collagen triple helices. interestingly, sds-page also revealed the presence of reducible a -cys-s-s-cys-a dimers in tendons of all mutant animals (see fig. c, d). since type i collagen triple helix contains only one a fig. . ooa kindred standing-height and serum biomarker measurements. height (left four panels): male (left) and female (right) carriers (gt) of the col a g c allele (oi) tend to have reduced standing height compared with control (gg) family members. standing height z-scores for individuals younger than years of age were calculated using nhanes normative data, whereas an ooa-specific z-score was calculated for individuals older than years of age. the mean oi z-score for standing height was significantly reduced (p < . ) compared with family controls for both age groups. biomarkers (right four panels): picp (left) and crosslaps (right) are serum biomarkers associated with collagen metabolism. picp reflects collagen biosynthesis in bone. picp values exhibited variations with age and disease status. the participants heterozygous for the g c col a allele (oi) who were younger than years of age exhibited significantly reduced pcip as compared with normal control family members. crosslaps levels reflect the degradation of bone collagen and can be considered a surrogate for bone resorption. no obvious association with age or disease status was observed for the crosslaps data. amish oi mutation journal of bone and mineral research chain, such dimers must form between two different molecules. the intermolecular dimers likely form prior to fibrillogenesis because in the fibrils the cys residues of adjacent triple helices are axially separated by at least one d period. dimer formation likely occurs in the golgi stack by nonspecific side-by-side interactions of procollagen molecules. surprisingly, the aberrant intermolecular dimers are incorporated into the tissues of mutant animals. from the intensities of the bands containing the s-s dimers and corresponding bands without the dimers, the estimated fraction of mutant chains involved in the dimers was approximately % in heterozygous neoþ, approximately . % in heterozygous neo–, and approximately % in homozygous neoþ animals (table ). sds-page analysis also demonstrated an abnormally high ratio of a (i):a (i) fluorescence intensities in neoþ (col a tm mcbr allele) animals, approximately : in heterozygous neoþ and approximately : in homozygous neoþ versus approximately : in wild-type and heterozygous neo– (col a tm . mcbr allele) animals. most likely this was caused by insufficient synthesis of mutant a (i) chains and formation of a (i) homotrimers [e.g., owing to partial degradation and/or slower transcription, splicing, and/or translation of the neoþ (col a tm mcbr) mrna containing the intron targeting vector]. based on this assumption, we estimated the homotrimer content as approxi- mately % and approximately % in tendons of heterozygous and homozygous neoþ animals correspondingly (see table ). human and mouse dsc analysis previous differentail scanning calorimetry (dsc) studies showed that the denaturation temperature tm of type i collagen homotrimers at acidic ph is approximately . c higher than that of normal heterotrimers.( , ) thus, to test whether the high a (i):a (i) ratio was consistent with homotrimer formation, we performed dsc scans of collagen from all mouse tendons and human fibroblast cultures in mm hcl (ph . ). in addition to the denaturation peak of normal type i heterotrimers, the dsc thermograms of collagen from neoþ animals (see fig. e) did reveal a low-temperature peak consistent with mutant hetero- trimers and a high-temperature peak. the high-temperature peak was identical to oim and artificial homotrimers measured at the same conditions.( , ) this peak was absent in collagen from neo– animals (see fig. f) and from human proband fibroblasts (see fig. g). deconvolution of the dsc thermograms suggested approximately % and approximately % content of homo- trimers in heterozygous and homozygous neoþ animals, respectively, in agreement with sds-page. to evaluate the effect of the g c substitution on the thermal stability of mutant collagen, we measured similar dsc thermograms in . m sodium phosphate and . m glycerol (ph . ), which can be used to evaluate the tm under physiologic conditions by subtraction of . c. from the buffer-corrected thermograms (see fig. h–j), we estimate that the incorporation of the mutant a (i) chain results in a tm reduction by . c in mouse and approximately c in human collagens correspondingly. f g c oi mouse phenotype breeding a total of experimental mice were obtained from litters. genotype was determined for the mice, except for one b .cby female mouse. mean litter sizes differed among the four f stains (one-way anova p ¼ . ). the mean litter sizes were . � . (fvb.b ), . � . (cby.b ), . � . (c .b ), and . � . (a.b ). fig. . ooa kindred bone mineral density (dxa) measurements. the principal quantitative measure of phenotype severity was abmd using dxa. bmad was calculated from l – spine (a, b) and femoral neck (c, d) dxa abmd measurements as an estimate of volumetric bone density. both male (left) and female (center) t allele individuals (oi) tended to have reduced bmad compared with family controls as a function of age. mean z-scores using hologic normative date are reduced in oi family members as compared with controls in both the spine (e) and hip ( f). journal of bone and mineral research daley et al. goodness-of-fit testing indicated a significant deviation from expected genotype ratios looking at data from all litters (p ¼ . ). however, no individual strains had a significant deviation from expected ratios. body weight there was a curvilinear relation between body weight (measured longitudinally on days to ) and age (fig. ; age and age squared both p < . ). wild-type animals were heavier than oi animals (difference . g, se . , p < . ). the a.b mice had the lowest and the fvb.b mice had the greatest body weight. fvb.b mice were significantly heavier than a.b mice (difference . g, p < . ), fvb.b mice were significantly heavier than cby.b mive (difference . g, p < . (both p values adjusted for multiple comparisons using tukey-kramer). there was no evidence of a strain-by-genotype interaction (strain � genotype p < . ). fig. . type i collagen analysis. type i collagen from human fibroblast cultures and from mouse tail tendons was analyzed by sds-page (a–d) and dsc (e– j). g c genotype status is represented as gg (control), gt (heterozygous), and tt (homozygous). neoþ represents the col a tm mcbr allele in g c mice, and neo– represents the g c oi mouse col a tm . mcbr allele. the gels labeled by cy and bodipy-cys are shown only in the bodipy-cys fluorescence (a, b). the cy fluorescence of these gels was used for identification of the bands and correction for nonspecific bodipy-cys labeling (the residual nonspecific labeling still can be observed in the dimer bands). dtt was included in the sample loading buffer in panel (c) and excluded from the sample buffer in panels (a), (b), and (d). normalized dsc thermograms of purified pepsin-treated collagen from g c (neoþ) and g c oi (neo–) mouse tail tendons and human fibroblasts were measured at ph . ( mm hcl; e–g) and ph . (measured in . m sodium phosphate and . m glycerol and corrected by . c to represent physiological conditions; h–j). each peak represents the heat of denaturation of a distinct molecular form of type i collagen. relative areas under the peaks on each thermogram represent the fraction of the corresponding molecules in the mixture. the superscript m identifies mutant a (i) chains. amish oi mutation journal of bone and mineral research abmd of g c oi mouse femurs g c oi mice had lower bmc (d ¼ . g, p < . ) and bmd (d ¼ . g/cm , p ¼ . ). mean abmd rank order of the g c oi mice by maternal background strain for was a.b < cby.b < fvb.b < c .b . pairwise comparisons of the g c oi mice using the scheffe multiple-comparisons adjustment procedure resulted in a statistically significantly difference between a.b oi and c .b oi (d ¼ . g/cm , p ¼ . ). a strain-by- genotype interaction was not significant for either bmc or bmd (p � . ). confocal raman microspectroscopy mineral content of bone matrix (po :ch) (fig. ) was found to be affected by genotype (p < . ) and mouse strain (p ¼ . ). a significant curvilinear relationship was observed between mineral content and bone location (p ¼ . ). the miner- al:collagen ratio (po :amide iii) also was found to have a significant genotype (p < . ) effect and a curvilinear relation- ship between mineral content and location (p ¼ . ) but no evidence of a strain effect (p ¼ . ). the curvilinear relationships for po :ch and po :amide iii had peak values located around the middle of the cortical bone. only genotype was found to have a significant (p < . ) effect for the collagen content of bone matrix (amide iii:ch). mouse strain-by-genotype interaction was not significant for any of the raman data. mct table shows mean values of selected trabecular and cortical bone parameters obtained from isolated femora. cortical shape and femur length were qualitatively very similar for each table . differential scanning calorimetry (dsc) and sds-page analysis of type i collagen sample genotype dsc sds-page a a , % a a m, %a a , % a , % reactive cys-sh a m–s–s–a m, %a mouse gg mouse neoþ gt � b � b � b � yes � . mouse neoþ tt � b � b � yes � mouse neo– gt � b � b yes . � . human gg no — human gt � b � b yes — athe superscript m identifies mutant chains. bestimated deconvolution error. fig. . f g c oi mouse growth curves. body-weight curves demonstrated a curvilinear relation between weight and age for all groups of mice. strain and genotype were independent predictors of weight. the wild-type (control; black squares) mice were . g heavier than the oi mice (heterozygous for the col a tm . mcbr allele; gray diamonds), with the greatest difference in body weight between the fvb.b and the a.b groups. journal of bone and mineral research daley et al. background strain control-oi pair (data not shown). overall, the mct trabecular and cortical data show that all g c oi mice carrying one copy of the col a tm . mcbr allele had less bone as measured by bone volume fraction. the trabecular bone was distinguished by fewer and more widely spaced trabeculae. except for the fvb.b mice, the mean cortical thickness and area were reduced by the presence of the col a tm . mcbr allele. the fvb.b control and oi groups had similar mean values for cortical thickness and area. all the oi femurs had reduced moment of inertia (iyy) compared with age-matched genetic background controls. the cortical volumetric tissue mineral content (vtmc) was lower in all oi mice relative to their genetic background controls, except for the fvb.b oi group, which was the same as the fvb.b control group. the cortical volumetric tissue mineral density (vtmd) was elevated in all mice carrying col a tm . mcbr allele relative to their genetic background controls. the trabecular vtmd was statistically the same for each genetic background pair. four-point bending biomechanics isolated femurs were loaded to failure in a four-point bend- testing apparatus to assess the impact of a single copy of the col a tm . mcbr allele and the role of genetic background on the structural phenotype (table ). specifically, femurs from mice with one copy of the variant allele were significantly weaker and more brittle than femurs from control mice. in addition, a genotype-by-genetic-background (strain) interaction was sig- nificant for failure load, stiffness, postyield ultimate displace- ment, and energy to failure. the presence of the col a tm . mcbr allele resulted in a striking reduction in failure load for all f oi mice compared with their strain-specific controls. the mean failure load range was greater for control mice ( n for a.b mice to n for c .b ) compared with oi mice ( n for a.b mice to n for c .b mice). the impact of the col a tm . mcbr allele on the magnitude of the intrastrain control-oi difference in stiffness was variable. the greatest intrastrain oi-control mean difference was found in the c .b group, where the col a tm . mcbr allele was associated with a % reduction in mean stiffness. the oi mice in the other three groups had mean stiffness values of % (a.b ), % (cby.b ), and % (fvb.b ) of their intrastrain controls. mean values for postyield ultimate displacement for oi mice were % (cby.b ), % (c .b ), and % (fvb.b ) of strain-matched control values. however, the mean value for a.b oi mice was % of the control value. failure energy ranged from . (cby.b ) to . n-mm (c .b ) for the oi mice and from . (a.b ) to . n-mm (fvb.b ) for controls. ultimate stress and young’s modulus had statistically significant genotype and strain differences, but the genotype- by-genetic-background interaction was not significant (see table ). mean ultimate stress was reduced for all oi groups relative to their genetic background controls and ranged from % (cby.b oi mice) to % (c .b oi mice). young’s modulus was increased across all oi groups carrying the col a tm . mcbr allele relative to their genetic background controls. the magnitude of the mean young’s modulus was not different between oi-control pairs but varied by genetic background of the strain. fig. . f g c oi mouse raman microspectroscopy. the collagen content of the oi (heterozygous for the col a tm . mcbr allele) femurs was reduced relative to wild-type (wt) control femurs, and the collagen was hypermineralized. a representative raman spectrum illustrates the peaks used for data analysis (upper left), and comparison of chemical-specific ratios in wt (black bars) and g c oi (gray bars) femurs determined from the raman spectra are shown in the balance of the figure. amish oi mutation journal of bone and mineral research t a b le . t ra b e cu la r a n d c o rt ic a l p a ra m e te rs d e ri v e d f ro m m c t m e a n s d a .b c b y .b f v b .b c .b a n c o v a p v a lu e w t o i w t o i w t o i w t o i in te ra ct io n g e n o ty p e s tr a in t ra b e cu la r b v /t v (% ) . . . . . . . . < . < . . . . . . . . . t ra b e cu la r n u m b e r . . . . . . . . < . < . . . . . . . . . t ra b e cu la r v b m d . . . . . . . . < . . . . . . . . . c o rt ic a l b o n e v o l. fr a ct io n (% ) . . . . . . . . . . . . . . . . . c o rt ic a l th ic k n e ss (m m ) . . . . . . . . . . . . . . . . . c o rt ic a l a re a (m m ) . . . . . . . . . . . . . . . . . c o rt ic a l i y y (m m ) . . . . . . . . < . < . . . . . . . . . c o rt ic a l v t m c (m g ) . . . . . . . . . . . . . . . . . c o rt ic a l v t m d (m g /m l ) . . . . . . . . . . . . . . . . . journal of bone and mineral research daley et al. discussion the ascertainment of related individuals harboring the mutant t allele represents the largest reported collection of oi patients with an identical collagen mutation. amino acid substitutions at invariant glycine residues typically result in clinically apparent phenotypes and make ascertainment more likely. while the original brother-sister-niece proband cluster presented with some of the most severe clinical signs and symptoms of oi in this kindred, our overall clinical data support a sillence type i (mild) to type iv (moderate) phenotype range. pedigree analysis suggests that the t allele was a de novo mutation that occurred in the putative founder couple born circa . the rapid ooa population growth from approximately individuals in to about , children and adults today,( ) combined with stochastic events, endogamy, and large sibships, likely contributed to the mutation’s expansion within the lancaster settlement. fracture history is the most relevant phenotypic trait used to make a clinical diagnosis of oi and to classify disease severity. however, since radiographic and medical record documentation of fractures were unavailable in the ooa kindred, the use of fracture history was problematic in defining the range of phenotypic severity associated with the t allele. the self- reported fracture number (not shown), nevertheless, indicated that the oi group was more prone to fracture and reinforced a clinical diagnosis of oi. instead, dxa measurements of abmd were used as a continuous quantitative trait to assess phenotypic severity that reflected the sillence oi type clinical spectrum. prior work suggests that mean abmd for children and adults with oi types iii and iv is significantly reduced compared with controls, but a significant number of oi type i patients had abmd values within the reference range.( – ) non-ooa oi patients often have l – spine z-scores in the � to � range for type i oi and the � to � range for type iv oi patients (jcm clinical experience). these stratification ranges demonstrate that the t allele was associated with a wide range of disease severity (table ) in the spine and a milder phenotype in the hip. skeletal growth deficiency is a cardinal secondary feature of oi that is reflected in the standing-height measurement. type i oi patients typically attain a subnormal to normal adult height, whereas type iv oi patients, with a greater propensity for fragility t a b le . s tr u ct u ra l a n d p re d ic te d m a te ri a l p a ra m e te rs d e ri v e d f ro m f o u r- p o in t b e n d in g m e a n s d a .b c b y .b f v b .b c .b a n c o v a p v a lu e w t o i w t o i w t o i w t o i in te ra ct io n g e n o ty p e s tr a in f a il u re lo a d (n ) . . . . . . . . . . . . . . . . . s ti ff n e ss (n /m m ) . . . . . . . . . . . . . . . . . p o st y ie ld d is p la ce m e n t (m m ) . . . . . . . . . . . . . . . . . u lt im a te st re ss (m p a ) . . . . . . . . < . . . . . . . . . . y o u n g ’s m o d u lu s . . . . . . . . . . . . . . . . . . y ie ld e n e rg y . . . . . . . . . . . . . . . . . f a il u re e n e rg y (n -m m ) . . . . . . . . . . . . . . . . . table . dxa z-score stratification sillence classificationa z-score range spine (l – ) femoral neck (fn) control (gg) oi (gt) control (gg) oi (gt) normal > � type i � to � type iv � to � type iii–iv < � total number astratification by sillence type is based on clinical experience (jcm) with non-ooa oi patients, amish oi mutation journal of bone and mineral research fracture, have a shorter final standing height in the range of pubertal children.( ) figure demonstrates a consistent reduction in standing height of t allele carriers compared with kindred controls (mean z-score difference of approximately . in both the < - and > -year-old groups). during childhood and adolescence, some % of skeletal mass is attained,( ) and peak stature normally is attained by approximately years of age. the cross-sectional serum biomarker data suggest impaired skeletal accretion and linear bone growth in t allele carriers; picp levels were relatively flat across the entire age spectrum for t allele carriers (see fig. ). in normal, healthy children, picp levels reflect collagen biosynthesis associated with skeletal growth and are known to change significantly with age and pubertal development stages.( – ) in contrast to the picp estimates of bone formation, bone resorption (serum crosslaps) had similar age and sex patterns for both the oi and control groups. this suggests an uncoupling between bone formation and resorption for the oi group during a major period of bone mass accretion that favors a net decrease in bone mass. the ooa kindred and the g c oi mouse share the classic oi features of dominant inheritance and a triple-helical glycine substitution. the biomedical significance of this kindred is its clinical and quantitative prototypic varied disease severity that is further enhanced by its rich resource of phenotype data, dna, and fibroblast cell lines. however, repeated access to kindred bone samples and other tissues for experimental uses are unlikely, thus necessitating creation of the g c oi mouse to aid future studies. establishing the validity of the g c oi mice as the first a (i) glycine substitution model required the following elements: ( ) duplication of the codon change, ( ) expression of mutant proa (i) transcript, ( ) deposition of mutant protein in the ecm, and ( ) confirmation of a bone phenotype. our initial evaluation of founder (neoþ) mice and their progeny carrying the col a tm mcbr allele demonstrated that the g codon was altered as planned and the mutant transcript was expressed. surprisingly, these mice secreted a significant fraction of type i collagen homotrimer in addition to g c- containing heterotrimeric collagen. the formation of homo- trimers appeared related to intron targeting vector retention because it was not detected in human patients and neo– mice. therefore, g c oi (or neo–) mice carrying the col a tm . mcbr allele actually model the human condition accurately at the gene, transcript, and protein levels. however, the neoþ animals are of interest and important to understand because g c (neoþ) mice carrying both one and two copies of the col a tm mcbr allele were viable yet had a very mild phenotype by torsional testing of femurs (data not shown). this altered phenotype associated with production of homotrimers suggests that some oi patients with a (i) mutations may benefit from treatments designed to shift the balance of the chain synthesis to favor homotrimer production. given that phenotype variation was a significant clinical feature observed among the ooa kindred, the impact of the genetic background on phenotype severity was tested in g c oi mice carrying the col a tm . mcbr allele. heterozy- gous � wild-type mouse crosses resulted in good reproductive fitness that matched the reproductive fitness of the ooa kindred and yielded f strains with approximately % interstrain genetic background identity. interestingly, reproductive fitness of heterozygous � heterozygous b matings was poor because trial matings failed to produce offspring homozygous for the col a tm . mcbr allele that survived beyond the perinatal period. growth curves (body weight) from weaning to months of age demonstrated impaired weight gain for all four f mouse strains carrying the col a tm . mcbr allele relative to their controls. this is likely a generalizable effect in mice caused by type i collagen gene mutations because it has been reported for the mov , oim, and brtliv oi models. body weight is a complex quantitative trait,( ) and different inbred mice are exemplified by characteristic patterns of weight gain with time (mgi database). it is likely that the different growth curves reflect the maternal contribution to the genetic background of the f mice because inbred mouse strains exhibit characteristic patterns of weight gain with age. since the degree and nature of bone matrix mineralization may be an important determinant of resistance to fracture, whole-femur mineral mass in f mice was assessed by abmd and mct measurements. piximus measurements (fig. ) demon- strated that all strains of mice carrying the col a tm . mcbr allele had a reduction in abmd compared with controls, and this is consistent with dxa data from the ooa kindred. however, mct measurement of increased cortical vtmd (see table ) better reflects the tissue effects of the variant col a allele. this apparent contradiction is explained by the d nature of abmd estimated by dxa versus the d data obtained by mct. the mct data suggest the g c oi mice have reduced total tissue mineral content but also have reduced total bone mass that results in greater bone mineralization compared with control mice. hypermineraliztion at the level of collagen fibers was confirmed by raman microspectroscopy and is consistent with higher mean mineralization density in oi patient bone samples measured by backscattered electron imaging.( ) structural integrity of femoral cortical bone was assessed using four-point bending, which is an ex vivo surrogate in mice for increased long bone fracture risk associated with oi. the structural (failure load) and material property (ultimate stress) measures of femur strength at months of age demonstrated that a single copy of the col a tm . mcbr allele reduced mechanical integrity regardless of genetic background. key fig. . f g c oi mouse dxa. oi mice demonstrated a reduction in mean whole-femoral bmc and abmd relative to their genetic background wild-type (wt) controls. the rank order of the oi (heterozygous for the col a tm . mcbr allele) mice by background strain for abmd means was a.b < cby.b < fvb.b j < c .b . journal of bone and mineral research daley et al. contributors to the structural soundness of bone include geometric parameters (i.e., size, shape) and inherent material properties such as the molecular integrity, quantity and organization of type i collagen, and degree and nature of collagen mineralization. while the mineral content of bone contributes substantially to its stiffness, collagen tends to dominate the properties of the postyield region of the load- deformation curve. it would appear from the yield energy:total energy ratio that once the bone starts to break, the presence of the mutated collagen offers less resistance to complete failure in g c oi mice. the failure of the abnormal collagen heterotimer to ‘‘toughen’’ the bone is likely due to a structural and/or organizational defect caused by the mutation and/or a general reduction in the amount of collagen per unit volume of bone. our human and mouse findings of reduced dsc denaturation temperature tm suggest that the global molecular (structural) stability of the mutant collagen heterotrimer is weakened. this reduction in tm is commonly associated with oi collagen mutations, although there is no simple relationship between the magnitude of tm decrease and oi disease severity. reduced bone matrix collagen content (measured by raman microspectro- scopy) also appears to contribute to loss of femoral structural integrity in g c oi mice. the variations in the murine bone biomechanical properties across the several experimental strains used in this study (genotype-by-strain interactions) provide additional insight about skeletal complications among human patients. the effect of the mutation on the estimated bone tissue properties was the same across all strains, each demonstrating significant and similar reductions in inherent tissue integrity. however, the magnitude of structural property alterations (such as failure load) appears to be dependent on genetic background (strain). taken together, these results suggest that the effect of the mutation on basic, inherent bone tissue integrity is similar in all ooa kindred patients (reduced modulus and increased brittleness). however, the consequence of the defect on whole- bone fracture susceptibility is influenced by individual genomic factors that are reflected in size, shape, and possibly bone metabolic regulation. mouse models have the advantage over human samples of tight control of genetic background and the ability to circumvent or minimize the role of environment on phenotype. the f g c oi screening crosses were successful in demonstrating that genetic background variation affected bone geometry and structural property phenotype. we speculate that additional backcrosses will further magnify interstrain phenotype differ- ences and enhance the probability of identifying major genetic modifiers using standard and novel backcross and intercross genetic mapping tools with proven success in the detection of human disease gene modifiers.( , ) bone trait modifiers identified in the g c oi mouse likely will include col a or col a mutation-specific modifiers, some that are generalizable for many oi mutation loci, and others that overlap with genes associated with common forms of osteoporosis. quantitative trait locus (qtl) investigation in the g c oi mouse also likely will confirm qtls found in prior osteoporosis studies, thus narrowing the critical qtl regions.( , ) the ooa oi kindred and afos data then can be used to confirm both sets of genes— those common to osteoporosis and those unique to ol. in summary, the ooa kindred is the largest known oi kindred with an identical type i collagen defect and extensive phenotype data. the triple-helical glycine substitution and dominant inheritance pattern are prototypic for most reported cases of oi. while all the affected individuals share the same mutation, they varied widely in phenotype, as quantified by abmd z-scores. the g c oi mouse, patterned on the ooa family, duplicates its gene and protein profile. the magnitude of structural property alterations owing to the mutation was dependent on genetic background (strain). the bone geometry and structural property gene � genetic background interactions suggest that noncolla- gen genes can modify the g c oi mouse oi phenotype. overall, these results demonstrate that g c oi mice represent a unique translational oi model to define the complex genetic architecture controlling phenotype and for testing of potential therapeutic approaches and treatment strategies for oi. disclosures the authors state that they have no conflicts of interest. acknowledgments this study was supported primarily by grants from the arthritis foundation (djm, eas, and ars), the charitable & research foundation (djm), and a university of maryland baltimore seed grant (djm, ars). other support includes nichd intramural funds (sl, jcm, djm), nih ro -ar (sag, ed), leda j. sears trust foundation (clp, sc), nih p -dk (jds, ars), and njaes- (ss). references . spotila ld, constantinou cd, sereda l, ganguly a, riggs bl, prockop dj. mutation in a gene for type i 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mya thu, christine rongey, james n. weiss, and miguel valderrábano cardiovascular research laboratory, departments of medicine (cardiology) and pediatrics, david geffen school of medicine, university of california-los angeles, los angeles, california submitted october ; accepted in final form november de diego c, chen f, xie lh, dave as, thu m, rongey c, weiss jn, valderrábano m. cardiac alternans in embryonic mouse ventricles. am j physiol heart circ physiol : h –h , . first published november , ; doi: . /ajpheart. . .—t-wave alter- nans, an important arrhythmogenic factor, has recently been described in human fetuses. here we sought to determine whether alternans can be induced in the embryonic mouse hearts, despite its underdeveloped sarcoplasmic reticulum (sr) and, if so, to analyze the response to pharmacological and autonomic interventions. immunohistochemistry confirmed minimal sarcoplasmic-endoplasmic reticulum ca-atpase a expression in embryonic mouse hearts at embryonic day (e) . to e . , compared with neonatal or adult mouse hearts. we optically mapped voltage and/or intracellular ca (cai) in embryonic mouse hearts (dual mapping in hearts) at these ages. under control conditions, ventricular action potential duration (apd) and cai tran- sient alternans occurred during rapid pacing at an average cycle length of � ms in % (n � / ) of e . -e . hearts. maximum apd restitution slope was steeper in hearts developing alternans than those that did not ( . � . vs. . � . ; p � . ). disabling sr cai cycling with thapsigargin plus ryanodine did not significantly reduce alternans incidence ( %, n � / , p � . ), whereas isoproterenol (n � ) increased the incidence to % (p � . ), coincident with steepening apd restitution slope. verapamil abol- ished cai transients (n � ). thapsigargin plus ryanodine had no major effects on cai-transient amplitude or its half time of recovery in e . hearts, but significantly depressed cai-transient amplitude (by � %) and prolonged its half time of recovery (by � %) in e . and older hearts. embryonic mouse ventricles can develop cardiac alternans, which generally is well correlated with apd resti- tution slope and does not depend on fully functional sr cai cycling. calcium cycling; cardiac development t-wave alternans has been recognized as an important risk marker of sudden cardiac death ( , ). at the cellular level, both action potential duration (apd) and intracellular ca (cai) transient amplitude typically alternate together due to their bidirectional coupling (i.e., apd directly affects cai transient amplitude, and the cai transient amplitude directly affects apd via ca-sensitive currents, such as the l-type ca current and electrogenic na/ca exchange) ( ). factors promoting cardiac alternans include steep apd restitution slope ( , ) and sarcoplasmic reticulum (sr) cai cycling dynamics ( , ). in adult ventricular muscle, current evidence ( , , ) suggests that, as heart rate increases, the cai cycling instability develops first and typically causes the onset of alternans ( , , , ), although the interaction with apd restitution properties is important in influencing the onset, amplitude, and pattern ( ), especially as the heart rate increases further. in embryonic heart, however, the cai transient is predominantly generated by transsarcolemmal ca entry through the voltage-dependent ca channels rather than sr ca release ( , , ). how quickly the sr matures and contributes to excitation-contraction coupling among different tissues in the embryonic heart remains con- troversial ( , , ). nevertheless, a recent human fetal study reported that t-wave alternans could be detected in utero and was related to ventricular arrhythmias ( ). to investigate prenatal alternans further, we studied embryonic mouse hearts using optical mapping to ) determine whether apd and cai transient alternans can be induced by rapid pacing; ) address its relationship to the development of sr cai cycling function; and ) evaluate its response to pharmacological interventions and autonomic factors during embryonic development, since we previously reported that both �-adrenergic and muscarinic receptors are present and functional in the embryonic mouse hearts at embryonic day (e) . or older ( ). methods dissection of embryonic mouse hearts. this study conformed to the guide for the care and use of laboratory animals, published by the national institutes of health (nih publication no. – , revised ), and protocols were approved by the chancellor’s animal research committee of the university of california at los angeles. pregnant mice with different embryonic age groups were first sedated by inhalation of isoflurane and then killed by cervical dislocation ( ). the uterus was dissected, and whole embryos were exposed. embry- onic mouse hearts were then isolated under a dissecting microscope, while bathed in modified oxygenated tyrode solution containing (mmol/l) nacl, . kcl, . cacl , . nah po , mgcl , hepes, and glucose, ph . . the same solution was used as the standard bath solution in experiments with . mmol/l cacl . stimulation protocol. unipolar stimuli were delivered at the ven- tricle at - to -v output using silver chloride electrodes and a grass stimulator triggered by computer-controlled pacing sequences. specimens were paced at hz, followed by a rapid pacing protocol, initially at -ms cycle length (cl), decremented by ms every eight beats until reaching -ms cl, and then incremented by ms every eight beats back to ms. optical mapping system. isolated e . –e . embryonic mouse hearts (total n � ; e . hearts, n � ; e . hearts, n � ; and e . hearts, n � ) were incubated with the ca indicator rhod- - acetoxymethyl ester ( �mol/l) and pluronic f- ( . %) dissolved in dimethyl sulfoxide for min, as previously described ( ). in embryonic mouse hearts, ca dye staining was followed by an addi- tional -min incubation with the voltage indicator rh- ( �mol/l). the embryonic mouse hearts were then washed in tyrode solution before transfer to the experimental chamber on a modified inverted microscope and superfused at °c with tyrode solution. green light * c. de diego and f. chen contributed equally to this work. address for reprint requests and other correspondence: j. n. weiss, division of cardiology, mrl bldg., david geffen school of medicine at ucla, los angeles, ca (e-mail: jweiss@mednet.ucla.edu). the costs of publication of this article were defrayed in part by the payment of page charges. the article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. am j physiol heart circ physiol : h –h , . first published november , ; doi: . /ajpheart. . . - / $ . copyright © the american physiological societyhttp://www.ajpheart.org h downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . ( � nm) from a high-intensity, light-emitting diode (luxeon, calgary, canada) delivered via the microscope lens was focused on the heart; fluorescence was collected through the lens and split with a -nm dichroic mirror, such that passed light (� nm) was filtered through a -nm long-pass filter (voltage signal) and reflected light (� nm) was filtered through a � nm filter (ca signal). all filters were obtained from chroma (rockingham, vt). ca and voltage signals were simultaneously recorded with two electron-multiplier charge-coupled device cameras operating at – frames/s (pho- tometrics cascade �, tucson, az) with a spatial resolution of � pixels (n � specimens). in some specimens, ca alone was recorded using a different charge-coupled device camera (model lcl k, watek america, las vegas, nv) at frames/s. specimens were optically mapped under control conditions and after the addition of one or more of following drugs to the tyrode solution: ) �-adrenergic agonist isoproterenol (iso; �mol/l, n � ); ) the muscarinic receptor agonist carbachol (cch; �mol/l, n � ); and ) ryanodine and thapsigargin (ry: �mol/l; and tg: nmol/l; n � ); and ) verapamil ( �mol/l, n � ). , - butanedione monoxime ( mmol/l) was used as an electromechan- ical uncoupler to reduce motion artifact. experiments were conducted at °c. data analysis. data were analyzed with custom software. after spatiotemporal filtering, apd at % repolarization (apd ) was measured as the time interval during which fluorescence exceeded % of the baseline diastolic value at each pixel. cai transient amplitude was measured as the increase in fluorescence (arbitrary units) from onset to peak (arbitrary units). to assess the effects of short-acting agents (iso, cch, and verapamil) on cai transient ampli- tude, we compared pre- and postdrug cai transient amplitudes di- rectly. however, because treatment with tg and/or ry required -min incubation (during which dye bleaching and other factors made comparison to the predrug cai transient amplitude unreliable), we assessed the effects of tg and ry on cai transient amplitude by dividing each batch of embryonic hearts on a given day into a control group and treated group. using identical loading and imaging condi- tions, cai transient amplitudes (as the change in absolute fluorescence units) were measured after min in the control and treated groups and compared. the half time of cai removal was measured from the peak of the cai transient to % of the return to baseline. apd alternans and cai alternans were quantified by subtracting apd [change in apd (	apd)] and cai transient amplitude (	cai), respec- tively, of consecutive beats. we considered apd and cai alternans to be present if alternation persisted until the pacing protocol was completed using thresholds of 	apd � ms and 	cai � % between successive beats. onset of alternans was defined as pacing cl at which alternans was first detected during the decreasing cl phase ( – ms) of the pacing protocol. offset of alternans was defined as the pacing cl at which alternans was not longer detectable during the increasing cl phase ( – ms) of the pacing protocol. spatial maps of 	cai and 	apd were generated for two consecutive beats using shades of red for positive 	apd (i.e., short-long apd) or 	cai (i.e., small-large ca amplitude), and shades of green for negative 	apd (i.e., long-short apd) or 	cai (i.e., large-small ca amplitude). apd restitution curves were generated by plotting apd against diastolic intervals (di) and fitting to a single exponential using origin (microcal) software. the maximum apd restitution slope was ob- tained from the first derivative of the monoexponential curve at the shortest di that elicited : capture. immunofluorescence labeling. as previously reported ( ), immu- nostaining of sarcoplasmic-endoplasmic reticulum ca atpase a (serca a) was performed on paraffin-embedded tissue sections. briefly, isolated hearts of different age groups (embryonic mouse ventricles at e . –e . ; -day-old neonate mouse ventricles, and -mo-old adult mouse ventricles) were fixed with % formalin, dehydrated with graded alcohol, cleared in xylene, and embedded in paraffin wax. a total of four heart sections were analyzed for each group. transmural sections of – �m were cut both perpendicular and parallel to the epicardium from a paraffin-embedded tissue and mounted onto slides. slides were incubated with protein block solu- tion, and then sections were incubated with primary antibody of serca a (affinity bioreagents, golden, co) for h and with secondary antibodies for min. cell membrane was stained using wheat germ agglutinin coupled to fluorescein isothiocyanate (vector laboratories, burlingame, ca) for min. finally, the section slides were stained with , -diamidino- -phenylindole (sigma, st. louis, mo; nuclear staining) for min, washed two times in normal tyrode solution, and then mounted on slides. control experiments were performed by exposing the tissue sections to the secondary antibody alone. all slides were viewed on an epifluorescence microscope and digitally photographed for analysis. to estimate quantitative differ- ences in serca a expression at different ages, the ratio of serca a fluorescence intensity to nuclear fluorescence intensity and the ratio of serca a fluorescence intensity to membrane fluorescence intensity were measured in each section, averaged, and compared between hearts at different ages. statistics. data are shown as means � sd. statistical tests included test and student’s t-test. p values � . were considered signif- icant. results sr function in developing embryonic mouse hearts. it has been suggested that the sr is neither fully developed ( , , ) nor functionally well coupled to the l-type ca current during embryonic development ( ). we performed immuno- histochemical and pharmacological studies to assess the status of sr ca cycling in developing embryonic mouse hearts. immunostaining for serca a showed that the cardiac sr ca uptake pump was detectable as early as e . –e . , but at much lower intensity relative to nuclear and membrane stain- ing than in neonatal or adult hearts (fig. ). using the identical labeling protocols and imaging settings, the ratios of serca a fluorescence intensity to either nuclear fluorescence intensity or membrane fluorescence intensity in the same cardiac section were measured. figure b shows that both ratios were much lower in e . and e . embryonic hearts than neonatal or adult hearts. to assess whether sr ca cycling is functional at these embryonic stages, we investigated the effects of blocking sr function with the serca a inhibitor tg ( �mol/l) plus the sr ca release channel inhibitor ry ( �mol/l). we also studied the effects of �-adrenergic activation with iso ( �mol/l) and l-type ca channel inhibition with verapamil ( �mol/l). at e . , the combination of tg�ry only mildly decreased cai transient amplitude to � % of the control value (p � . ) and did not significantly affect the half time for cai removal (fig. , c and d). iso increased cai transient ampli- tude (to � %, p � . ), as expected from augmenting the l-type ca current, but did not significantly shorten the half time of cai removal. in contrast, at e . , tg�ry significantly depressed cai transient amplitude to � % (fig. c) and prolonged the half time for cai removal ( � vs. � ms for control, p � . ) (fig. d). iso increased the ca transient amplitude (to � %, p � . ) (fig. c) and also shortened the half-time for cai removal (to � ms, p � . ) (fig. d), similar to adult hearts in which iso shortens the half time of cai removal due to enhanced serca a activity h alternans in embryonic mouse ventricles ajp-heart circ physiol • vol • january • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . ( ). at both e . and e . stages, verapamil abolished cai transients in all of the specimens (fig. c). these findings indicate that, in the embryonic mouse heart, the sr is largely nonfunctional at e . , but begins to con- tribute significantly to excitation-contraction coupling by e . . this is generally consistent with previous studies ( , , ), indicating that the contribution of sr ca cycling increases progressively during embryonic development. pacing-induced alternans under control conditions. the above observations indicate that the e . –e . stages pro- vide an optimal time window in which to evaluate the impor- tance of a functional sr in the development of cardiac alter- nans. figure a shows an example of simultaneous voltage and cai mapping in an embryonic mouse heart at e . . the two atria (right and left) and two ventricles (right and left) are separated by the atrioventricular ring. under control conditions during pacing at hz, the ventricular apd and cai transient duration averaged � and � ms, respectively. embryonic mouse ventricles could be paced to a cl of � ms before loss of : capture. the incidence of alternans did not differ between e . ( %, n � / ) vs. e . or older ventricles ( %, n � / ) (fig. b, solid bars). overall, alternans of both apd and the cai transient developed concur- rently at an average pacing cl of � ms, at which the di averaged � ms (fig. c). the pacing cl at the onset of alternans also did not differ between e . and e . or older ventricles ( � ms for e . vs. � ms for e . or older, p � . ), at which the di averaged � ms for e . vs. � ms for e . or older (p � . ) (fig. d). during alternans, the longer apd was accompanied by the larger cai transient and vice versa. the offset of alternans (the cl at which alternans resolved during the deceleration phase of the pacing protocol) was also simultaneous for apd and cai, occurring at an average pacing cl of � ms. thus there was minimal hysteresis between the onset and offset of alternans (only one specimen in the control group). the role of functional sr cai cycling in alternans in embry- onic mouse ventricles. we next investigated how disabling sr cai cycling with tg�ry affected alternans. after tg�ry, the ventricular apd during pacing at hz decreased modestly (from � to � ms, p � . ). for e . hearts, the incidence of alternans after tg�ry remained similar to control ( %, n � / vs. %, n � / ) (fig. b). for e . or older hearts, however, tg�ry tended to suppress alternans, but the difference did not reach statistical signifi- cance ( %, n � / vs. %, n � / , p � . ) (fig. b). the maximal amplitude of cai alternans after tg�ry also did not differ significantly from control at either e . ( � fig. . sarcoplasmic reticulum (sr) features in embryonic mouse hearts. a: immunostaining of sarcoplasmic-endoplasmic reticulum ca atpase (serca) a (red), cell membrane (green), and nuclei (blue) in embryonic day (e) . , e . , -day-old neonatal, and adult mouse ventricular tissue sections. e . –e . mouse ventricles show lower staining intensity of serca a compared with older hearts. b, left: the ratio of serca a fluorescence intensity to nuclear fluorescence intensity (solid bars), and the ratio of serca a fluorescence intensity to membrane fluorescence intensity (shaded bars) in sections at different heart ages. c and d: the effects of thapsigargin and ryanodine (tg�ry at e . and e . ), isoproterenol (iso), and verapamil (ver) on intracellular ca (cai) transient (cat) amplitude (c) and the half time (t . ) for cat recovery (d). values are means � sd for the no. of hearts indicated. *p � . . h alternans in embryonic mouse ventricles ajp-heart circ physiol • vol • january • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . vs. � %) or e . –e . ( � vs. � %) (fig. c). for e . , the average di at the onset of alternans was similar after tg�ry compared with control ( � vs. � ms for the control group, fig. d). at e . –e . , however, the average di at the onset of alternans decreased significantly after tg�ry ( � vs. � ms for the control group, p � . , fig. d). these results indicate that, in embryonic mouse hearts, alternans occurs whether the sr is fig. . cardiac alt in embryonic mouse ventricles after sr blockade. a: example of apd and cat alt after sr blockade with tg�ry at -ms pacing cl. l and s apd and cat are labeled. b–d: the corresponding incidences, amplitude, and diastolic interval (di) at onset of alt before and after tg�ry in e . vs. e . –e . hearts. bars indicate means � sd, for the no. of hearts indicated. p � . for e . vs. e . . fig. . action potential duration (apd) and cat alternans (alt) in embryonic mouse ventricles. a: ca fluorescence (fca) image of an e . mouse heart. the two atria [right (ra) and left (la)], the atrioventricular ring (avr), and two ventricles [right (rv) and left (lv)] are labeled. b: example of cardiac alt. consecutive fca snapshots (beats – ) exhibited alternation in the maximum fluorescence amplitude (large-small-large). isochronal map shows the propagation pattern across the ventricle. apd alt maps show apd differences (	) between consecutive beats (beat – and beat – ). red and green represent positive and negative differences, respectively. c: traces of voltage fluorescence (fv) and fca at the pixel indicated by the red asterisk in b (beat ) demonstrating cat and apd alt at -ms pacing cycle length (cl), in which the shorter apd is associated with a smaller cat amplitude (concordant apd-cai alt). long/large (l) and short/small (s) apd and cat are labeled. h alternans in embryonic mouse ventricles ajp-heart circ physiol • vol • january • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . essentially nonfunctional (e . ) or significantly functional (e . and older). thus alternans in the embryo does not appear to be critically dependent on a functioning sr. apd restitution properties and alternans in embryonic mouse hearts. if a functional sr is not essential for the development of alternans in the embryonic heart, then apd restitution slope is likely to play a key role ( ). to examine how apd restitution slope correlates with the development of alternans, we compared apd restitution curves from hearts that developed alternans with those that did not (see fig. a). as summarized in fig. b, left, the maximum apd restitution slope was significantly greater in both e . and e . –e . hearts that developed alternans (open bars) than in those that did not (solid bars), averaging . � . vs. . � . , respectively. for both age groups, the onset of alternans oc- curred at an average di of � ms, at which point the apd restitution slope averaged . � . (fig. b, right). in of hearts, however, alternans appeared when the slope of apd restitution was � . after tg�ry, the maximum apd restitution slope in hearts that developed alternans was not significantly different compared with control conditions, at either e . ( . � . vs. . � . for control, p � . ) or e . ( . � . vs. . � . for control, p � . ). the incidence of alternans was also not significantly different from control at either e . ( vs. % for control) or e . ( vs. % for control). in all hearts developing alternans after tg�ry, the maximum apd restitution slope exceeded one, averaging . � . , compared with . � . in e . –e . hearts without alternans (p � . ). the onset of alternans occurred at an average di of � ms, at which point the apd restitution slope was always � (fig. b, right). effects of autonomic modulation on alternans and apd restitution slope in embryonic mouse hearts. iso and cch were used to mimic for �-receptor and muscarinic receptor stimu- lation, respectively. in hearts studied, iso (n � ) did not significantly affect apd during pacing at hz ( � ms after iso vs. � ms before), but iso � cch decreased apd significantly to � ms (p � . ). iso increased the incidence of alternans to % in both e . and e . – e . hearts (fig. a and supplemental file movie , contained in the online version of this article). therefore, we combined both age groups for further analysis. one-to-one capture was lost at a pacing cl of � ms after iso and � ms after iso � cch. the onset of alternans after iso occurred at a significantly longer pacing cl compared with control ( � vs. � ms, p � . ) and at a significantly longer di ( � vs. � , p � . ). hysteresis between the onset and offset of alternans was uncommon (only in one specimen in the iso group). in the presence of iso, alternans was suppressed by the addition of cch in six hearts (fig. ). as shown in fig. , a and b, iso significantly increased the maximum apd restitution slope from . � . (n � ) to . � . (n � , p � . ), which was reversed by subsequent addition of cch. after iso, the onset of alternans occurred at a significantly longer di compared with control ( � vs. � ms, p � . ), where the slope of apd restitution averaged . � . . in of specimens, how- ever, alternans occurred at a di corresponding to an apd restitution slope � (fig. c). cch reversed these effects (fig. , b and c). fig. . apd restitution (apdr) in embryonic mouse ventricles under control conditions and after blocking sr function with tg�ry. a, left: representative apdr curves from two hearts: one that developed alt (e), and one that did not (f). nos. indicate the maximum slope of apdr obtained from monoexponential fits. right: apdr curves in two hearts after tg�ry, one that developed alt (e) and one that did not (f). b, left: average maximum apdr slopes in control conditions and after tg�ry in the hearts that developed alt (open bars) and in those that did not (no alt, solid bars). dashed line indicates slope � . nos. of hearts are shown. *p � . . right: for all hearts that devel- oped alt, the value of the apdr slope at the onset of alt, in control conditions and after tg�ry. h alternans in embryonic mouse ventricles ajp-heart circ physiol • vol • january • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . discussion the major findings of our study are as follows: ) in embryonic mouse ventricles, the sr develops significant func- tionality between e . and e . , a period that corresponds to – wk in human fetuses ( ); ) both apd and cai transient alternans can be induced by rapid pacing in early embryonic mouse ventricles, with a similar incidence both before and after sr ca cycling becomes functional; ) alternans in embryonic ventricles generally correlates well the apd restitution slope; ) �-adrenergic stimulation steepens apd restitution slope and promotes alternans in embryonic mouse ventricles; and ) muscarinic stimulation with cch reverses the effects of �-adrenergic stimulation on both apd restitution slope and alternans. fig. . autonomic regulation of cardiac alt in embry- onic mouse ventricles. a: example of apd and cat alt during pacing at -ms cl in the presence of iso. subsequent addition of carbachol (cch) in the same heart decreased alt, even at -ms pacing cl. l and s apd and cat are labeled. b: incidence of alt and the average pacing cl at the onset of alt under control conditions, after iso and after iso�cch. no. of hearts and p values are indicated above the bars. fig. . autonomic effects on apdr in embryonic mouse ventricles. a: apdr curves of a representative heart during iso, (e) and after iso�cch (f). nos. indicate the maximum apdr slopes obtained from the monoexponential fits. b: average maximum apdr slopes under control conditions, after iso and after iso�cch. no. of hearts are shown. *p � . . c: in all hearts that developed alt, the distribution of apdr slopes at the onset of alt under control conditions, after iso and after iso�cch. h alternans in embryonic mouse ventricles ajp-heart circ physiol • vol • january • www.ajpheart.org downloaded from journals.physiology.org/journal/ajpheart at carnegie mellon univ ( . . . ) on april , . sr function. previous histological and functional studies ( , , ) suggest that sr ca cycling system in mammalian hearts does not fully mature until well after birth, supporting the traditional view that transsarcolemmal ca influx through voltage-dependent ca channels comprises the main component of action potential-coupled cai transients before birth ( ). consistent with this view, liu et al. ( ) found that sr was not functional in mouse embryonic hearts at . days postconcep- tion. however, moorman et al. ( ) found that the sr ca cycling was functional in rat embryonic hearts at days postconception. moreover, it has also been shown that sr ca cycling plays an important role in pacemaking activity in the embryonic hearts ( ). therefore, how quickly the sr matures and contributes to excitation-contraction coupling among dif- ferent tissues in the embryonic heart remains controversial. consistent with a minimally functional sr in embryonic mouse ventricles at e . , we found that sr blockade with tg�ry decreased the amplitude and duration of cai transients by only � %. in contrast, the effect of tg�ry on the amplitude of cai transient became significant (� %) at e . . in addition, tg�ry significantly increased the half time of the cai removal at e . , as is expected if ca re-uptake by the sr is an important mechanism of cai removal, as in adult mammalian heart ( ). however, this gain of function in sr ca cycling at e . did not significantly enhance the inducibility of alternans compared with e . . we have previously shown that �-adrenergic receptors are functional at these embryonic heart stages ( ). at e . , iso decreased the half time of the cai removal, as is observed in adult heart due to iso-mediated serca a stimulation ( ). l-type ca channel inhibition with verapamil completely abol- ished cai transients and contraction, as expected, since it eliminates transsarcolemmal ca influx as well as the trigger for ca-induced ca release from the sr. the role of apd restitution and sr cai cycling in alternans in embryonic mouse hearts. in the adult heart, the underlying mechanism of alternans was originally attributed to steep apd restitution slope (so-called voltage-driven alternans) ( ). however, recent evidence ( , , ) supports the idea that, in adult mammalian heart, cai transient alternans, due to a dy- namic instability in sr ca cycling, also plays a key role, especially in the onset of apd alternans. however, since membrane voltage and cai cycling are bidirectionally coupled, overall apd stability depends on both factors and their cou- pling ( , ). recently, it has been reported that t-wave alternans can occur in utero in human fetuses at – wk of gestation ( ). here we found that both apd and cai transient alternans can be induced by pacing at an even earlier embryonic stage in embryonic mouse ventricles, since e . is equivalent to - to -wk gestation in humans. at this stage in the embryonic mouse heart, we have shown that sr ca cycling is not yet fully functional. thus, even if the sr is not yet functional at – wk in human fetuses, our findings suggest that apd alternans could still be explained by the apd restitution slope mecha- nism. moorman et al. ( ) detected serca a expression at as early as wk in human fetuses, but the functionality of sr ca cycling has not been well characterized during human fetal gestation. under control conditions, the development of alternans cor- related strongly with apd restitution slope � , although the correlation was not perfect. for example, a minority of embry- onic ventricles developed apd alternans at a di at which apd restitution slope was still � . this could be due to short-term memory effects, which are known to distort the relationship between the apd restitution slope � criteria and the onset of alternans ( ). in support of a predominant role of apd restitution, when sr function was suppressed by tg�ry, alternans exclusively developed at apd restitution slopes � . alternans occurred with similar incidence and magnitude, whether the sr ca cycling was essentially nonfunctional (e . ) or significantly functional (e . or older), indicating that alternans is not critically dependent on a functional sr at this stage of cardiac embryogenesis. a caveat, however, is that the interaction between apd restitution slope and sr cai cycling properties in producing alternans is highly interdepen- dent ( , , ), making it difficult to distinguish unequivocally their respective contributions, especially at e . or older. autonomic effects on cardiac alternans in embryonic mouse ventricles. in the adult heart, �-adrenergic stimulation has multiple effects on membrane currents and sr cai cycling. stimulation of the l-type ca current tends to promote alternans by steepening apd restitution slope and augmenting sr ca loading, but enhanced serca activity tends to suppress alter- nans ( ). depending on which of these effects predominate, �-adrenergic stimulation can either promote or inhibit alter- nans, and both effects have been observed experimentally in mammalian ventricles ( , ). in human ventricles, adrenergic stimulation steepens apd restitution ( ), and �-blockers have been shown to flatten the apd restitution slope ( a), decrease t-wave alternans ( ), and reduce sudden cardiac death in cardiomyopathy patients ( ). our laboratory previously reported that both �-adrenergic and muscarinic receptors are present and functional in the embryonic mouse hearts at e . or older ( ). consistent with a partly functional sr at this stage, we found that iso pro- moted, rather than suppressed, alternans in embryonic mouse ventricles. thus, unlike the adult heart in which �-adrenergic stimulation with iso may often suppress alternans by enhancing ca uptake by the sr, the major effect of iso in embryonic mouse ventricles is to steepen apd restitution slope and thereby potentiate alternans. muscarinic receptor stimulation by cch in the presence of iso reversed these effects. conclusions. embryonic mouse ventricles without signifi- cantly functional sr ca cycling can develop cardiac alternans, which is generally well correlated to apd restitution slope and is modulated by autonomic factors. acknowledgments this study was supported by grants from the american heart association ( y to c. de diego; y and y to m. valderrábano), the national heart, lung, and blood institute (p hl and p hl to j. n. weiss), and the laubisch and kawata endowments (to j. n. weiss). grants we thank lingqin pan for excellent technical assistance. references . 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institute of varanasi- , india. e-mail: schatterji.a bschool of material science & technology, hindu university), varanasi , india cdepartment of physics, banaras hindu uni dsolid state physics division, bhabha atomic eindus synchrotrons utilization division, technology, indore , india fcsir-national physical laboratory, dr. k.s. gdepartment of cmp & ms tata institute of india cite this: rsc adv., , , received th july accepted th september doi: . /c ra j www.rsc.org/advances | rsc adv., , , – tical properties of fe doped crednerite cumno kaushal k. shukla,a p. shahi,a gopal s.,b a. kumar,a a. k. ghosh,c ripandeep singh,d neetika sharma,d a. das,d a. k. sinha,e amish g. joshi,f a. k. nigamg and sandip chatterjee*a a geometrically frustrated magnetic cumno system has been investigated because of its rich magnetic properties. neutron diffraction, synchrotron x-ray, magnetic, x-ray photoemission spectroscopy (xps) and uv-visible spectroscopy measurements have been carried out on cumno and % fe doped cumno samples. fe doping reduces the distortion. moreover, fe doping induces the ferromagnetic coupling between ab planes. the value of magnetization is increased with fe doping but coercivity is decreased. these might be due to the direct mn–mn exchange and mn–o–cu–o–mn super–super exchange interactions. the uv-vis data indicate the appearance of new energy bands in these compounds. the xps study indicates that fe is in the + state. introduction geometrically frustrated magnetic systems have attracted much attention due to their interesting magnetic properties. – in these systems, spin ordering is suppressed by competing exchange interactions well below the curie temperature. abo -type triangular-lattice antiferromagnets in which not all the interac- tions are minimized simultaneously, are popular examples of geometrically frustrated systems. – crednerite cumno is an abo -type triangular-lattice antiferromagnet. the crystal struc- ture of cumno contains of isosceles-triangular lattices of mn. it has a monoclinic structure with c /m space group; it is distorted from the hexagonal delafossite structure because of jahn–teller effect of mn + ions that have crystal-eld splitting in eg orbitals due to d electronic conguration. moreover, different directions in the triangular ab plane become inequivalent: for each mn, two short and four long mn–mn distances exist. it is observed that the exchange interaction at short bonds is stronger, which, with the uniaxial magnetic anisotropy of mn + (spins are oriented predominantly along the long mn–o bonds), leaves the system frustrated. this degeneracy is lied below magnetic transition: technology (banaras hindu university), pp@iitbhu.ac.in indian institute of technology, (banaras versity, varanasi , india research centre, mumbai , india raja ramanna centre for advanced krishnan road, new delhi , india fundamental research, mumbai, , at t < tn ¼ k and the structure changes from monoclinic to triclinic (space group c ) due to magnetostriction. recently, ushakov et al. have shown that the presence of ferro-orbital ordering in cu +xmn �xo plays a very important role in deter- mining the exchange constants and the magnetic structure. furthermore, in nonstoichiometric crednerite with a small excess of copper (cu . mn . o ), the in plane magnetic ordering remains practically the same as for pure cumno , the interlayer exchange coupling changes from antiferromagnetic in cumno to ferromagnetic in cu . mn . o and vice versa. recent neutron diffraction studies have revealed the magnetic structure of cumno and the crystal structure deformation associated with the magnetic ordering. , the magnetic structure below tn ¼ k is the collinear one with the magnetic propagation vector k ¼ ( / $ / $ / ). in addition to k , they observed the other group of the magnetic bragg reections which are assigned by k ¼ ( / $ / $ ). while the intensity for k is dominant, that for k is speculated to be caused by a small amount of impurity of cu +xmn �xo . trari et al. reported the magnetic susceptibility of cu +xmn �xo (with x ¼ – . ), suggesting that the magnetic susceptibility is highly sensitive to the atomic disorders. however, the minor fraction for k has not been investigated thus far. the structural phase transition also occurs below tn from the monoclinic c /m to the triclinic c� in cumno . , the degen- eracy in the exchange interaction paths between base sites and apex sites in isosceles triangular lattice, j , is lied by the distortion. it is evident from these previous studies that inter- layer coupling plays a signicant role in the long range magnetic ordering in cumno , and that this can be characterised using neutron diffraction. there is some dispute, however, over the source of the k ordering as to whether it is intrinsic to the structure or a result of some impurity. in this study, we have thus this journal is © the royal society of chemistry http://crossmark.crossref.org/dialog/?doi= . /c ra j&domain=pdf&date_stamp= - - http://dx.doi.org/ . /c ra j http://pubs.rsc.org/en/journals/journal/ra http://pubs.rsc.org/en/journals/journal/ra?issueid=ra fig. rietveld refinement of synchrotron x-ray powder diffraction data of cumn . fe . o at room temperature. paper rsc advances p ub li sh ed o n o ct ob er . d ow nl oa de d by u ni ve rs it y of l et hb ri dg e on / / : : . view article online doped fe into the mn site in cumno so as to examine the effects on the inter-layer coupling and on this ordering. experimental the cumn �xfexo (with x ¼ . and . ) samples were prepared by solid state reaction in an evacuated quartz tube. powders cuo, mno and fe o were mixed in appropriate ratio and pressed into pellets. the pellets were then placed in an alumina crucible, sealed in quartz tube under high vacuum (� � mbar) and heated at �c for h. powder xrd data were recorded using adxrd beam line (on bending magnet port bl- ) of the indus- ( . gev, ma) synchrotron radi- ation (sr) source at raja ramanna centre for advanced tech- nology (rrcat), indore, india. the diffraction data were collected on a image plate (mar ) detector. the diffraction images were integrated using fit d program. wave length and sample to detector distance were accurately calibrated using xrd pattern of lab nist standard. neutron powder diffraction (npd) patterns were recorded on the pd diffractometer (l ¼ . å) at bhabha atomic research centre, mumbai, india. the observed xrd and neutron powder diffraction patterns were analyzed by rietveld method using the fullprof- k so- ware package. the basis vector for the magnetic renement was determined using basireps program. magnetic measurements were carried out using mpms, squid (quantum design) magnetometer with the bulk samples. data were collected during warm up cycle. the absorption spectra were measured in the range of – nm using uv-vis spectrometer. x-ray photoelectron spectroscopy (xps) experiments were per- formed using omicron nanotechnology uhv system equipped with a twin anode mg/al x-ray source (dar ), a mono- chromatic source (xm ) and a hemispherical electron energy analyzer (ea ). all the xps measurements were per- formed inside the analysis chamber under average base vacuum of . � � torr using monochromatized alka at kv and watt. the total energy resolution, estimated from the width of the fermi energy, was about mev for monochromatic alka line with photon energy . ev. during photoemission studies, small specimen charging was observed which was later calibrated by assigning the c s signal at . ev. resistivity measurements have been performed by four probe method. results and discussions structural characterization the results of the renements using synchrotron x-ray diffraction data and neutron diffraction data are shown in fig. and (room temperature), and tables and , respec- tively. these show good agreement with previously reported data for the crednerite structure of cumno . , the cell volume of cumn . fe . o ( . å ) is slightly larger than that of cumno ( . å ); it corresponds mainly to a decrease of a, an increase of the b angle, and a small increase of b and c. the changes in the b angle, a and b parameters will have effects on the exchange interactions between ab planes and in the basal plane, respectively. in the same way, the dilution on the mn site, this journal is © the royal society of chemistry because of the small substitution of fe for mn, induces a smaller (mn/fe–o) average distance . ( ) å compared to . ( ) å for cumno and a smaller jahn–teller distortion of the mno octahedra (by comparing the d ¼ d(mn–o)apical/ d(mn–o)equatorial). the irregularity in the triangular mn lattice in the basal plane also increases very slightly with the shortest mn–mn distance ( . ( ) å) which is slightly longer than in cumno ( . ( ) å) and also the two longest distances ( . ( ) å) are larger than in cumno ( . ( ) å). at room temperature, the cu–o distances also slightly vary by the substitution, close to . ( ) å in cumno and to . ( ) å in cumn . fe . o . we have also measured the neutron diffraction at k for both cumno and cumn . fe . o . in fig. we have shown the neutron diffraction pattern of cumn . fe . o . on lowering the sample temperature superlattice reections in both these compounds are observed indicating the antiferro- magnetic nature of these compounds. on lowering of temper- ature we do not observe the splitting of the ( ) reection (the splitting of which indicates the transition from monoclinic to triclinic structure). a marginal improvement in the t is obtained in the triclinic phase but we have analysed the diffraction in the monoclinic structure in c /m space group at k. the cell parameters of fe doped cumno (a ¼ . ( ) å, b ¼ . ( ) å, c ¼ . ( ) å, b ¼ . ( )), signicantly differ from those of cumno (a ¼ . ( ) å, b ¼ . ( ) å, c ¼ . ( ) å, b ¼ . ( )). compared to rt, the difference in the cell volume is more at low temperature (lt) in fe doped cumno . in this low temperature structure, the oxygen atoms occupy a general symmetry lattice site (x, y, z) and the oxygen position at low temperature also varies with fe doping. dealing with the triangular mn-array in the (a, b) plane, the mn–mn shortest edge of the triangle is slightly elongated (from . å along [ ] to . å along [ ]). in the mno octahedron at low temperature, the two long apical mn–o distances are . å, while the four equatorial distances are ( . å). while, for cumno in the mno octahedron at low temperature, the apical mn–o distances are . å, and the equatorial distances are . å indicating that fe doping decreases the distortion in mno octahedra. for nonstoichiometric cu . mn . o sample also the distortion in mno octahedra is decreased. therefore, no change in the chemical structure is rsc adv., , , – | http://dx.doi.org/ . /c ra j fig. rietveld refinement of neutron powder diffraction data of cumn . fe . o at room temperature. rsc advances paper p ub li sh ed o n o ct ob er . d ow nl oa de d by u ni ve rs it y of l et hb ri dg e on / / : : . view article online observed on fe doping, although the magnetic structure is found to be different on fe doping. the structural parameters obtained from the analysis are summarized in table . in the table rietveld refinement of room temperature synchrotron xrd of cumno and cumn . fe . o sample/parameters cumno , k cumn . fe . o , k space group c /m c /m cell parameters a (å) . ( ) . ( ) b (å) . ( ) . ( ) c (å) . ( ) . ( ) cell volume (å) . . a (deg) b (deg) . ( ) . ( ) g (deg) atomic positions o( i)x . . y z . . occupancy cu mn . fe . mn–mn . ( ) � . ( ) � . ( ) � . ( ) � cu–o . ( ) � . ( ) � bragg r factor . . rf . . rp . . rwp . . x . . | rsc adv., , , – case of cumno the superlattice reections were indexed using the two propagation vectors k ¼ ( / $ / $ / ) and k ¼ ( / $ / $ ). the intensities corresponding to k were very weak in agreement with damay et al., but not absent as reported previously in this compound. the magnetic structure described by k consists of antiferromagnetic chains in the (a, b) plane coupled antiferromagnetically along the c-axis. the components of the moment are . mb and . mb along a and c-axes corresponding to . mb. for k vector the magnetic table rietveld refinement of the neutron powder diffraction data for cumno and cumn . fe . o at k sample/parameters cumno , k cumn . fe . o , k space group c /m c /m cell parameter a (å) . ( ) . ( ) b (å) . ( ) . ( ) c (å) . ( ) . ( ) cell volume (å) . . a (deg) b (deg) . . ( ) g (deg) atomic positions o( i)x . ( ) . ( ) y z . ( ) . ( ) mn–o . (� ) . (� ) mn–o . (� ) . (� ) mn–mn . (� ) . (� ) . (� ) . (� ) this journal is © the royal society of chemistry http://dx.doi.org/ . /c ra j fig. rietveld refinement of neutron powder diffraction data of cumn . fe . o at k. paper rsc advances p ub li sh ed o n o ct ob er . d ow nl oa de d by u ni ve rs it y of l et hb ri dg e on / / : : . view article online moment is . ( ) mb and oriented along c-axis. the total moment is lower than the expected moment of mb for mn + in high spin state. fe is found to substitute at the mn site. it results in a large increase in the intensity of ( / $ / $ ) reection corresponding to k . the moment oriented along a and c are . mb and . mb, respectively leading to a total moment of . mb. the moment corresponding to k vector is . mb. a net increase in the moment ( . mb) is observed as expected for a mixture of mn + ( mb) and fe + ( mb), which is nearly equal to the satu- ration value of the cu(mn, fe)o sample. the appearance of k propagation vector indicates the ferromagnetic coupling between ab planes. optical properties we have also studied the electronic structure of cumno and cumn . fe . o using x-ray photoemission spectroscopy (xps). the purpose of this study was to investigate any role of the electronic structure on the magnetic properties of cumno . the xps core level spectra of cu p, mn p and fe p and o s are shown in fig. (a–d). fig. (a) shows high resolution spectra of cu p core level. two clear distinct states of cu( p / ) at ev and cu( p / ) at ev are separated by . ev and . ev for cumno and cumn . fe . o , respectively. fig. (b) shows core level spectra of mn p. two separate states of mn( p / ) and mn( p / ) observed at . ev and . ev for cumno and . ev and . ev for cumn . fe . o , respectively. fig. (c) exhibit fe p core level spectra of cumn . fe . o compound which shows the spin–orbit splitting of the fe p level, manifested as fe p / and fe p / . the difference between these two fe peaks is . ev, which conrms the presence of this journal is © the royal society of chemistry fe + state. these observed doubly states are due to the spin– orbit coupling. slight shiing in the states with fe doping occurs due to change in interaction energy between cu and transition metal ion. these data clearly suggest that cu is in + state and both mn and fe are in + state. fig. (d) shows the spectra of oxygen which has two peak structures. two peaks marked as x observed at . ev and . ev and y at . ev and . ev for cumno and cumn . fe . o , respec- tively. the rst peak marked as x, is characteristic peak of “o �” ions of the lattice oxygen, while peak y denotes o( s) lateral structure. this lateral peak corresponds to the ionizations of weakly adsorbed species and also the ionizations of oxygen ions with particular coordinates, more specically integrated in the subsurface. this suggests that the existence, in the subsurface of oxygen ions that bear lower electron density than the “o �” ions. normally, these oxide ions can be described as “o�” species or excess oxygen. when the density of lattice oxygen varies, the area ratio of these two peaks i.e. x and y also changes. valence level spectra of cumno and cumn . fe . - o samples are shown in fig. . four features (a, b, c, d) can be identied in the experimental spectra of both the samples. feature a relate to state of dominant cu d atomic character while b and c relate to the hybridization with cu d and mn d to o p. feature d is a tail like structure near the fermi level, ef. the electronic states near the fermi level are mostly responsible for the electronic properties. a comparison of the valence band spectra for the two samples reveals that the density of states is negligibly small but nite at ef which is clear from the inset of fig. . the density of states slightly increases with fe doping. therefore, conductivity increases slightly with fe doping. the xrd and neutron diffraction data also support rsc adv., , , – | http://dx.doi.org/ . /c ra j fig. valance-band xps spectra of cumno and cumn . fe . o [(c) blue dots for cumno and red dots (c) for cumn . fe . o ]. fig. xps core level spectra of (a) cu p (b) mn p (c) fe p and (d) o s [(c) blue dots for cumno and red dots (c) for cumn . fe . o ]. rsc advances paper p ub li sh ed o n o ct ob er . d ow nl oa de d by u ni ve rs it y of l et hb ri dg e on / / : : . view article online this. it is observed from table that on fe doping cu–o bond length increases and mn–o bond length decreases. this will lead to the increase of bandwidth and as a consequence band gap will decrease. moreover, it has been observed (table ) that on fe doping the c axis is elongated leading to weaker cu d–o p hybridization. in the valence band spectra (fig. ) also it is found that in cumn . fe . o the b and c features are reduced with respect to the a feature in accordance with the xrd and neutron diffraction data (table ). it is worthwhile to mention that the valence band spectrum of cumno differs from that of cucro (ref. ) in respect of non-existence of the shoulder at the upper part of the valence band indicating that mn d is located away from the fermi level. the most interesting feature is the shi of fermi level towards lower binding energy for both cumno and cumn . fe . o unlike cucro . , similar behavior is observed in k doped srcu o . this shi is related to low activation energy of cumno . furthermore, with fe doping it is observed that shi in fermi energy is slightly larger than that in undoped sample. this is close to the further change in activation energy. therefore, the photo emission spectroscopy measurements conrm the movement of fermi level towards the valence band edge on fe doping which is accompanied by corresponding shis in core level binding energies. similar kind of movement is observed in cucro with mg doping. in the present investigation the cu p / peak shis with fe doping from . ev to . ev. the absorption curves of cumno and fe doped cumno are shown in fig. . as the photon energy increases the absorption | rsc adv., , , – intensity increases and attains a maximum. for the undoped sample the maximum occurs at e ¼ . ev whereas for the fe doped sample it increases to . ev. the peak can be assigned to this journal is © the royal society of chemistry http://dx.doi.org/ . /c ra j paper rsc advances p ub li sh ed o n o ct ob er . d ow nl oa de d by u ni ve rs it y of l et hb ri dg e on / / : : . view article online an excitonic excitation from cu d + o p to cu d + s. , for the fe doped sample the peak intensity becomes negligibly small, the reason of which is not yet clear. it deserves further study. moreover, the optical band gaps result from the rela- tionship between the optical absorption coefficient and the photon energy (hn) can be expressed as: (ahn) ¼ a(hn � eg) where a is a constant and eg is the direct optical band gap of the material. the inset of fig. shows the optical band gap of the undoped and fe doped cumno . it is observed from the gure that both samples have two energy band gaps. the band gaps of the crednerite cumno ( . ev and . ev) and cumn . - fe . o ( . ev and . ev) are close to each other. therefore, for both the samples an additional energy level exists near the valence band edge, as also revealed by valence band spectra (fig. ). the difference between the new band and the conduc- tion band is . ev for both the samples. on the other hand, the new band resides . ev and . ev above the top of the valence band respectively for cumno and cumn . fe . o . similar, new band is observed in cuga . cr . o lm. appearance of new band might be the reason of the shiing of fermi level towards the valence band edge observed in valence band spectra (fig. ). moreover, observed band gaps of these systems come in the range of wide band gap semiconductors and these values are even more than the band gap of zno ( . ev) and gan ( . ev). – additionally, these systems show much better magnetic ordering and magnetic moment than any known diluted magnetic semiconductors, which might prove signicant in the application of these materials as magnetic semiconductors. electrical properties in order to understand the intrinsic characteristic, we have also studied the temperature dependence of resistivity for both cumno and cumn . fe . o compounds. the exponential decrease in resistivity with the increase in temperature indi- cates the semiconducting nature of both the samples. the plot of ln r versus /t (fig. ) shows that thermally activated band fig. absorption spectra of cumno and cumn . fe . o . inset: optical band gap from uv-visible spectroscopic measurement of cumno and cumn . fe . o . this journal is © the royal society of chemistry conduction is the dominant mechanism at high-temperature region. the thermally activated resistivity at high-temperature region follows the arrhenius law r(t) ¼ r exp[ea/kbt] ( ) where kb is the boltzmann's constant and ea is the activation energy. the deviation from the linear t indicates that thermal activation mechanism is not valid at lower temperature region. the variable-range-hopping (vrh) conduction of polarons has been found to dominate in this temperature region. the conduction mechanism due to the variable range hopping of polaron at low temperature can be described by the mott's equation r(t) ¼ r exp[t /t] / ( ) where r and t are constants and are given by r ¼ {[ pakbt/ n(ef)] / }/( e nph) and t ¼ a /[kn(ef)] where nph (� s� ) is the phonon frequency at debye temperature, n(ef) is the density of localized electron states at the fermi level, and a is the inverse localization length. for cumno two slopes are observed which can be tted with the eqn ( ). the estimated activation energy (ea) from the two slopes are . ev and . ev. the estimated activation energy (ea) for cumn . - fe . o using arrhenius law is . ev. moreover, we did not get proper tting with the eqn ( ). magnetic properties fig. shows the magnetization of cumno as a function of magnetic eld. at k, a clear hysteresis with a coercive eld of . koe is observed. the magnetization increases almost linearly with magnetic eld aer closing of the hysteresis. the magnetic properties of cumno are controversial. , our data match with the data in ref. . the data indicate the presence of the dominating afm order with some fm ordering. the afm fig. variation of resistivity versus temperature for cumn �xfexo (where x ¼ , . ). inset variation of ln r vs. /t for (x ¼ and . ) samples. rsc adv., , , – | http://dx.doi.org/ . /c ra j fig. magnetization curve m(h) of cumn . fe . o at k. fig. magnetization curve m(h) of cumno at k. rsc advances paper p ub li sh ed o n o ct ob er . d ow nl oa de d by u ni ve rs it y of l et hb ri dg e on / / : : . view article online ordering at k has also been reported recently by kurakawa et al. the increase of high eld magnetization along with the appearance of hc indicates the emergence of an uncompen- sated moment. when fe is doped (fig. ), the magnetization is increased but the coercivity is decreased. with increase of the magnetic eld the ferromagnetic correlation precipitates and the antiferromagnetic correlation is increased which is clear from the m(h) curve. the change in magnetic behavior with fe doping can be explained in terms of magnetic exchanges, direct mn–mn interactions may be considered as dominant in-plane but indirect exchanges (via oxygen) could also play a role, all mn–o–mn angles being close to �. the super–super exchange, along mn–o–cu–o–mn path ways, may also play the role for the d magnetic ordering. in fact super–super exchange via diamagnetic cation is quite common, as is observed in different oxides. the difference in magnetic behavior with fe doping might be due to the presence of k ¼ ( / $ / $ ) vector which has been observed from neutron diffraction measurement. as has been mentioned that the appearance of k ¼ ( / $ / $ ) vector is the indication of ferromagnetic coupling between ab planes implying that fe doping induces this ferromagnetic coupling. similar behavior is observed in the non- stoichiometric cu . mn . o . conclusion neutron diffraction, synchrotron x-ray diffraction, xps, magnetic and uv-visible spectroscopic measurements have been investigated on cumno and % fe doped cumno samples, with assumption that these measurements have | rsc adv., , , – complementary information on structural and magnetic behaviour of the samples. on fe doping, the apical mn–o distances decrease while the equatorial distances slightly increase, reducing the distortion in mno octahedra. moreover, when fe is doped along with k ¼ ( / $ / $ / ) the magnetic peaks can also be indexed with the propagation vector k ¼ ( / $ / $ ) indicating the appearance of ferromagnetic coupling between ab planes. value of magnetization is increased with fe doping but coercivity is decreased. these might be due to direct mn–mn exchange and mn–o–cu–o–mn super–super exchange interactions. the uv-vis data showed the increase in one of the two energy gaps, on fe doping, indicating the usefulness of these materials as wide band gap magnetic semiconductors. acknowledgements s.c. is grateful to brns, dae, india (grant no.: / p/ / brns) for providing nancial support. p.s. is grateful to csir, india for providing nancial support. authors are also grateful to d. budhikot for his help in magnetization measurement. references t. kimura, t. goto, h. shintani, k. ishizaka, t. arima and y. tokura, magnetic control of ferroelectric polarization, nature, , , – . n. hur, s. park, p. a. sharma, j. s. ahn, s. guha and s. w. cheong, electric polarization reversal and memory in a multiferroic material induced by magnetic elds, nature, , , – . t. lottermoser, t. lonkai, u. amann, d. hohlwein, j. ihringer and m. fiebig, magnetic phase control by an electric eld, nature, , , – . a. j. w. reitsma, l. f. feiner and a. m. oles, orbital and spin physics in linio and nanio , new j. phys., , , . f. ye, j. a. fernandez-baca, r. s. fishman, y. ren, h. j. kang, y. qiu and t. kimura, magnetic interactions in the geometrically frustrated triangular lattice antiferromagnet cufeo , phys. rev. lett., , , . d. j. singh, magnetic and electronic properties of limno , phys. rev. b: condens. matter mater. phys., , , – . t. jia, g. guo zhang and z. zeng, 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post-perovskite structure, phys. rev. b: condens. matter mater. phys., , , . g. gusmano, g. montesperelli, e. traversa and g. mattogno, microstructure and electrical properties of mgal o thin lms for humidity sensing, j. am. ceram. soc., , , – . d. shin, j. s. foord, d. j. payne, t. arnold, d. j. aston, r. g. egdell, k. g. godinho, d. o. scanlon, b. j. morgan, g. w. watson, e. mugnier, c. yaicle, a. rougier, l. colakerol, p. a. glans, l. f. j. piper and k. e. smith, comparative study of bandwidths in copper delafossites from x-ray emission spectroscopy, phys. rev. b: condens. matter mater. phys., , , . t. arnold, d. j. payne, a. bourlange, j. p. hu, r. g. egdell, l. f. j. piper, l. colakerol, a. d. masi, p. a. glans, this journal is © the royal society of chemistry t. learmonth, k. e. smith, d. o. scanlon, a. walsh, b. j. morgan and g. w. watson, x-ray spectroscopic study of the electronic structure of cucro , phys. rev. b: condens. matter mater. phys., , , . c. c. b. lynch, r. g. 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the crednerite cumno , eur. j. solid state inorg. chem., , , – . y. bessekhouad, y. gabes, a. bouguelia and m. trari, the physical and photo electrochemical characterization of the crednerite cumno , j. mater. sci., , , – . a. kurakawa, t. yanoh, s. yano and y. ichiyanagi, preparation and magnetic properties of multiferroic cumno nanoparticles, j. nanosci. nanotechnol., , , – . rsc adv., , , – | http://dx.doi.org/ . /c ra j 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno 	magnetic and optical properties of fe doped crednerite cumno cardiac arrest in pregnancy aha scientific statement abstract—this is the first scientific statement from the american heart association on maternal resuscitation. this document will provide readers with up-to-date and comprehensive information, guidelines, and recommendations for all aspects of maternal resuscitation. maternal resuscitation is an acute event that involves many subspecialties and allied health providers; this document will be relevant to all healthcare providers who are involved in resuscitation and specifically maternal resuscitation. (circulation. ; : - . doi: . /cir. .) key words: aha scientific statements ■ cardiopulmonary resuscitation ■ heart arrest ■ pregnancy © american heart association, inc. circulation is available at http://circ.ahajournals.org doi: . /cir. the american heart association makes every effort to avoid any actual or potential conflicts of interest that may arise as a result of an outside relationship or a personal, professional, or business interest of a member of the writing panel. specifically, all members of the writing group are required to complete and submit a disclosure questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest. this statement was approved by the american heart association science advisory and coordinating committee on july , , and the american heart association executive committee on august , . a copy of the document is available at http://my.americanheart.org/statements by selecting either the “by topic” link or the “by publication date” link. to purchase additional reprints, call - - or e-mail kelle.ramsay@wolterskluwer.com. the online-only data supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi: . /cir. /-/dc . the american heart association requests that this document be cited as follows: jeejeebhoy fm, zelop cm, lipman s, carvalho b, joglar j, mhyre jm, katz vl, lapinsky se, einav s, warnes ca, page rl, griffin re, jain a, dainty kn, arafeh j, windrim r, koren g, callaway cw; on behalf of the american heart association emergency cardiovascular care committee, council on cardiopulmonary, critical care, perioperative and resuscitation, council on cardiovascular diseases in the young, and council on clinical cardiology. cardiac arrest in pregnancy: a scientific statement from the american heart association. circulation. ; : – . expert peer review of aha scientific statements is conducted by the aha office of science operations. for more on aha statements and guidelines development, visit http://my.americanheart.org/statements and select the “policies and development” link. permissions: multiple copies, modification, alteration, enhancement, and/or distribution of this document are not permitted without the express permission of the american heart association. instructions for obtaining permission are located at http://www.heart.org/heartorg/general/copyright- permission-guidelines_ucm_ _article.jsp. a link to the “copyright permissions request form” appears on the right side of the page. cardiac arrest in pregnancy a scientific statement from the american heart association farida m. jeejeebhoy, md, chair; carolyn m. zelop, md; steve lipman, md; brendan carvalho, md; jose joglar, md; jill m. mhyre, md; vern l. katz, md; stephen e. lapinsky, mb bch, msc; sharon einav, md; carole a. warnes, md; richard l. page, md; russell e. griffin, lp, fp-c; amish jain, md; katie n. dainty, phd; julie arafeh, rn, ms; rory windrim, md; gideon koren, md; clifton w. callaway, md, phd; on behalf of the american heart association emergency cardiovascular care committee, council on cardiopulmonary, critical care, perioperative and resuscitation, council on cardiovascular diseases in the young, and council on clinical cardiology cardiac arrest in pregnancy is one of the most challenging clinical scenarios. although most features of resuscitating a pregnant woman are similar to standard adult resuscitation, several aspects and considerations are uniquely different. the most obvious difference is that there are patients, the mother and the fetus. caregivers must have a thorough understanding of maternal mortality to best prevent and treat cardiac arrest in pregnancy. maternal mortality is defined as the death of a woman during pregnancy and up to days after delivery or termination of pregnancy, provided that the cause of death is related to or aggravated by the pregnancy or its manage- ment. recent data from the us nationwide inpatient sample suggest that cardiac arrest occurs in : admissions for delivery. globally, maternal deaths occur daily. , maternal mortality trends in the united states as reported by the centers for disease control and prevention from to have documented a steady increase from . deaths per live births in to . deaths per live births in . however, maternal mortality rates are just a small representation of maternal critical events; maternal near-miss data should be considered. a maternal near miss is defined as “a woman who nearly died but survived a compli- cation that occurred during pregnancy, childbirth, or within days of termination of pregnancy.” data from the netherlands show an incidence of maternal near miss of : in delivery wards. among cases with severe maternal morbidity, there was an overall case fatality rate of : . knowledge deficits , and poor resuscitation skills could be major contributors to poor outcomes once cardiac arrest has occurred. despite these problems, recent data show that the rate of survival to hospi- tal discharge after maternal cardiac arrest may be as high as . %, far higher than most arrest populations, further jus- tifying appropriate training and preparation for such events despite their rarity. d ow nloaded from http://ahajournals.org by on a pril , http://my.americanheart.org/statements mailto:kelle.ramsay@wolterskluwer.com http://circ.ahajournals.org/lookup/suppl/doi: . /cir. /-/dc http://my.americanheart.org/statements http://www.heart.org/heartorg/general/copyright-permission-guidelines_ucm_ _article.jsp http://www.heart.org/heartorg/general/copyright-permission-guidelines_ucm_ _article.jsp circulation november , this scientific statement addresses all of the important factors related to maternal arrest, including maternal physi- ology as it relates to resuscitation, pre-event planning of the critically ill pregnant patient, risk stratification during preg- nancy, management of the unstable pregnant patient, basic life support (bls) in pregnancy, advanced cardiovascular life support (acls) in pregnancy, neonatal considerations, emer- gency medical service (ems) care, cause of maternal arrest (with a comprehensive discussion found in the online-only appendix), point-of-care instruments, immediate postarrest care, medicolegal considerations, and knowledge translation, training, and education recommendations. methods authors with expertise in maternal resuscitation and relevant areas of specialty were selected to contribute to this statement. selection of the writing group was performed in accordance with the american heart association’s (aha’s) conflict-of-interest management policy. relevant literature considered for inclusion in this statement was identified through an up-to-date search strat- egy of the process used for the international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations and the international liaison committee on resuscitation worksheets, pubmed, embase, and an aha master resuscitation reference library. the search also included a review of bibliographies and manual searches of key articles. participants volunteered to write sections relevant to their expertise and experience. drafts of each section were written and sent to the chair of the writing group for incorporation into a single document, which was then edited. the edited document was discussed during webinars in which par- ticipants provided feedback to the primary author of each section and discussed the document as a group. on the basis of these discussions and consensus, the sections were then edited accord- ingly by the primary author, returned to the chair, and reincorpo- rated into the single document. further edits were performed by the chair, and a final version of the document was produced. the final document was circulated among all contributors, and con- sensus was achieved. recommendations were generated from this process and then assigned a class of recommendation and level of evidence (table ). the final document was submitted for independent peer review and has been approved for publica- tion by the aha emergency cardiovascular care committee and science advisory and coordinating committee. important physiological changes in pregnancy fetal development and maternal maintenance of pregnancy require multiorgan physiological adaptations that are pertinent to the team responding to cardiopulmonary arrest during pregnancy. cardiac output rises % to % as a result of increased stroke volume and, to a lesser extent, increased maternal heart rate ( – bpm). , systemic vascular resistance decreases as a result of an increase in several endogenous vasodilators, including progesterone, estrogen, and nitric oxide, leading to a decrease in mean arterial pressure, reaching a nadir in the second trimester. the enlarging uterus can produce increased afterload through compression of the aorta and decreased cardiac return through compression of the inferior vena cava, starting at ≈ to weeks of gestational age. as a result, the supine position, which is most favorable for resuscitation, can lead to hypoten- sion. , a magnetic resonance imaging study comparing the maternal hemodynamics in the left lateral position with those in the supine position was performed. this study found that at weeks of gestational age, there was a significant increase in ejec- tion fraction of % and stroke volume of % in the left lateral position. at weeks, there was a significant increase in ejection fraction of %, in end-diastolic volume of %, in stroke vol- ume of %, and in cardiac output of % in the left lateral posi- tion. uteroplacental blood flow increases from to close to ml/min during pregnancy, receiving up to a maximum of % of maternal cardiac output at term. expanded intravascu- lar volume and a decrease in uterine vascular resistance facilitate sufficient uterine placental blood. overall, uterine vascular reac- tivity is altered, characterized by reduced tone, enhanced vasodi- lation, and blunted vasoconstriction. systemic hypotension can overwhelm the compensatory mechanisms, which attempt to maintain uterine blood flow. functional residual capacity decreases by % to % during pregnancy as the uterus enlarges and elevates the dia- phragm. increased ventilation (ie, an increase in tidal volume and minute ventilation) occurs, beginning in the first trimester, reaching a level % to % above baseline by term, mediated by the elevated serum progesterone levels. this produces a mild respiratory alkalosis with compensatory renal excretion of bicarbonate, resulting in an arterial carbon dioxide pressure of ≈ to mm hg ( . – . kpa) and a plasma bicarbonate level of to meq/l. oxygen consumption increases because of the demands of the fetus and maternal metabolic processes, reaching a level % to % above baseline by the third tri- mester. the reduced functional residual capacity reservoir and increased consumption of oxygen are responsible for the rapid development of hypoxemia in response to hypoventila- tion or apnea in the pregnant woman. the oxyhemoglobin dissociation curve is shifted to the right in the mother dur- ing pregnancy (p increases from to mm hg). a higher partial pressure of oxygen is therefore required to achieve the same maternal oxygen saturation. the same curve is shifted to the left in the fetus (p is mm hg), conferring relative resil- ience to hypoxic conditions. upper airway edema and friability occur as a result of hormonal effects and may reduce visualiza- tion during laryngoscopy and increase the risk of bleeding. pregnancy is characterized by glomerular hyperfiltration and increased renal blood flow by % to accommodate the maternal role of fetal detoxification of metabolic byproducts and maintenance of maternal osmoregulation in the face of increased circulatory intravascular volume. altered tubular function prevents wasting of glucose, amino acids, and pro- teins required by both maternal and fetal metabolisms. on balance, starling forces favor a narrowing of the oncotic pres- sure–wedge pressure gradient, increasing the tendency for pulmonary edema to develop. progesterone relaxes gastroesophageal sphincters and prolongs transit times throughout the intestinal tract during the second and third trimesters, , predisposing the patient to aspiration of stomach contents. drug metabolism is altered by several different mecha- nisms in pregnancy. a– d in addition to changes in renal d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy physiology, gastrointestinal absorption and gastrointestinal transit affect bioavailability. protein binding changes also alter the free fraction of the drug available. steroid-induced accel- eration of the hepatic p metabolism and increased renal clearance will also lower circulating drug levels. estimating gestational age management decisions made during a maternal cardiac arrest may require estimation of gestational age. symphysis fundal height is the measurement from the top of the maternal pubic bone to the top of the uterine fundus. in a singleton pregnancy, with the fetus in a longitudinal lie, this height in centimeters will approximately correspond to the gestational age in weeks when measured between and weeks of gestation. if a tape mea- sure is not available, finger breadths are usually used as a sur- rogate for the centimeters. classically accepted rule-of-thumb landmarks may also be used: gestational age is weeks if the uterus is palpable at above the pubic symphysis, weeks if the uterus is palpable at the level of the umbilicus, and weeks if the uterus is palpable at the level of the xiphisternum. however, the fundus can be a poor predictor of gestational age and may reach the umbilicus between and weeks of gestation. in the last month of pregnancy, after weeks of gestation, there may be diminution of the fundal height from down to ≈ cm as the fetal head engages into the pelvis. fundal height may also be skewed by other factors such as abdominal distention table . applying classification of recommendations and level of evidence a recommendation with level of evidence b or c does not imply that the recommendation is weak. many important clinical questions addressed in the guidelines do not lend themselves to clinical trials. although randomized trials are unavailable, there may be a very clear clinical consensus that a particular test or therapy is useful or effective. *data available from clinical trials or registries about the usefulness/efficacy in different subpopulations, such as sex, age, history of diabetes, history of prior myocardial infarction, history of heart failure, and prior aspirin use. †for comparative effectiveness recommendations (class i and iia; level of evidence a and b only), studies that support the use of comparator verbs should involve direct comparisons of the treatments or strategies being evaluated. d ow nloaded from http://ahajournals.org by on a pril , circulation november , and increased body mass index; therefore, fundal height may be a poor predictor of gestational age. recommendation . code team members with responsibility for preg- nant women should be familiar with the physiologi- cal changes of pregnancy that affect resuscitation technique and potential complications (class i; level of evidence c). the critically ill pregnant patient pre-event planning the critically ill pregnant patient may be managed in units not accustomed to managing obstetric patients such as the inten- sive care unit (icu), coronary care unit, or medical or surgical ward. teams on these units need to prepare for unexpected emergencies in these patients by covering the following important steps: . preparation for cardiac arrest: educate staff about the management of cardiac arrest in pregnancy. . preparation for perimortem cesarean delivery (pmcd): identify contact details or appropriate code calls to mobilize the entire maternal cardiac arrest response team, and ensure the availability of equipment for cesarean delivery and resuscitation of the neonate. in cultures that require consent for a pmcd, even in the event of a cardiac arrest, pre-event consent should be obtained. . preparation for management of obstetric complica- tions: stock drugs and equipment commonly available in obstetric units, including oxytocin and prostaglandin f α . pre-event planning for power of attorney related to healthcare decisions should be done for the critically ill patient. . decisions involving the resuscitation status of the neo- nate: decisions about fetal viability should be made in collaboration with the obstetrician, neonatologist, and family. the decision depends on the gestational age and, to a significant degree, the neonatal facilities available. this information should be clearly documented. severity of illness and early warning scores the british center for maternal and child enquiries report of ( – triennium) has stated that timely recog- nition of pregnant women at risk of potentially life-threat- ening conditions plays an important role in the appropriate institution of treatment. brief checklists are provided for the identification of a number of conditions, including sep- sis, respiratory distress, and neurological complications. the report also highlights the potential value of a modified early obstetric warning score. in a more recent publication, using a large british icu data set, carle et al described the evalua- tion of several preexisting obstetric early warning scores and the development and validation of a new obstetric score and demonstrated excellent discrimination between survivors and nonsurvivors for this new score (area under the receiver-oper- ating characteristic curve, . ). these scores can be used to monitor patients by clinical use of an early warning score chart (figure ) and can accurately identify patients at high risk of mortality, although not specifically mortality result- ing from cardiac arrest. therefore, they are of value in patient management and triage. recommendations . pregnant women who become ill should be risk stratified by the use of a validated obstetric early warning score (class i; level of evidence c). . hospital units with a pregnant woman in their care should ensure that proper pre-event planning has been instituted, including preparation for maternal cardiac arrest and neonatal resuscitation (class i; level of evidence c). management of the unstable pregnant patient rapid response to instability in the pregnant patient is essen- tial for the prevention of cardiac arrest. maternal hemody- namics must be optimized; hypoxemia must be treated; and intravenous access must be established. recommendations . the patient should be placed in a full left lateral decubitus position to relieve aortocaval compression (class i; level of evidence c). . administration of % oxygen by face mask to treat or prevent hypoxemia is recommended (class i; level of evidence c). . intravenous access should be established above the diaphragm to ensure that the intravenously admin- istered therapy is not obstructed by the gravid uterus (class i; level of evidence c). . precipitating factors should be investigated and treated (class i; level of evidence c). cardiac arrest management basic life support the cardiac arrest in pregnancy in-hospital bls algorithm should be used as a guide to management (figure ). first responders nurses are often first responders in cardiopulmonary arrest; however, any hospital staff member may witness or discover a patient in arrest and should be able to begin basic emergency care. basic emergency care is crucial. rapid mobilization of expert resuscitation teams and bls performed competently until the arrival of these teams give the woman the best chance for return of spontaneous circulation (rosc). accomplishing a coordinated, well-executed first response is challenging in patient care areas in which cardio- pulmonary arrest rarely occurs, including obstetric units. – the unique physiology of pregnancy renders the patient vulnerable to hypoxemia and hemodynamic disadvantage, given the rapid development of desaturation with apnea and the presence of aortocaval compression when the patient is unconscious and supine. therefore, all bls interventions are essential and should be initiated rapidly and simulta- neously once the rescuers arrive. first responders should d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy initiate the usual resuscitation measures simultaneously, including placement of the backboard and provision of chest compressions and appropriate airway management, defibril- lation when appropriate, and manual left uterine displace- ment (lud). to accomplish all tasks effectively, a minimum of bls responders should be present. chest compressions in pregnancy adult chest compression science as with all adult resuscitations, high-quality chest compres- sions are essential to maximize the patient’s chance of sur- vival. for high-quality chest compressions, the patient must be supine on a hard surface, the rescuer’s hands must be placed correctly, the correct rate and depth of compressions must be performed, and interruptions must be minimized. because hos- pital beds are typically not firm and some of the force intended to compress the chest results in mattress displacement rather than chest compression, we have traditionally recommended the use of a backboard despite insufficient evidence for or against the use of backboards during cardiopulmonary resus- citation (cpr). – if a backboard is used, care should be taken to avoid delays in the initiation of cpr, to minimize interrup- tions in cpr, and to avoid line/tube displacement. air-filled mattresses should be deflated when cpr is performed. – chest compression physiology has been best studied only with the patient in the supine position and has never been studied in a patient placed in a tilt. chest compression recommenda- tions for the pregnant patient are the same as the most current recommendations for adult resuscitation. recommendations . chest compressions should be performed at a rate of at least per minute at a depth of at least in ( cm), allowing full recoil before the next com- pression, with minimal interruptions, and at a compression-ventilation ratio of : (class iia; level of evidence c). . interruptions should be minimized and limited to seconds except for specific interventions such as insertion of an advanced airway or use of a defibril- lator (class iia; level of evidence c). . the patient should be placed supine for chest com- pressions (class i; level of evidence c). . there is no literature examining the use of mechani- cal chest compressions in pregnancy, and this is not advised at this time. factors affecting chest compressions in the pregnant patient aortocaval compression. in the pregnant patient, supine posi- tioning will result in aortocaval compression. relief of aorto- caval compression must be maintained continuously during resuscitative efforts and continued throughout postarrest care. manual lud should be used to relieve aortocaval compres- sion during resuscitation. historically, tilt has been used as an option to relieve aortocaval compression during resuscitation. rees and willis found that at > ° left lateral tilt, the manikin slid off the incline plane and that chest compression force was date time systolic bp < – – – – > respiratory rate < – – – > heart rate < – – > fio to keep sat > % room air %– % > % temperature < . – . . – . . – . > . consciousness alert (gcs= ) not alert (< ) figure . example of a color-coded early warning score chart based on the score developed by carle et al. a score ≥ should trigger a call for support from the intensive care unit or rapid response team and initiation of continuous monitoring of vital signs. bp indicates blood pressure; fio , fraction of inspired oxygen; gcs, glasgow coma scale score; and sat, saturation. d ow nloaded from http://ahajournals.org by on a pril , circulation november , reduced as the angle of inclination was increased. it has also been found that at > ° lateral tilt, inferior vena cava compres- sion can still occur. in addition, the heart has been shown to shift laterally during tilt compared with the supine position. therefore, chest compressions performed with the patient in a tilt could be significantly less effective than those performed with the patient in the usual supine position, and this could have a major impact on the chance of successful resuscitation. , in the nonarrest population, manual lud compared with ° left lateral tilt has been shown to result in less hypotension and a significantly lower ephedrine requirement during a cesarean delivery. additional benefits of manual lud over tilt include easier access for both airway management and defibrillation. while manual lud is performed, the patient can remain supine and receive usual resuscitative measures, including high-quality chest compressions without hindrance (figure ). manual lud can be performed from the left of the patient (figure ), where the uterus is cupped and lifted up and leftward off the maternal vessels, or from the right of the patient (figure ), where the uterus is pushed upward and leftward off the maternal vessels. the rescuer must be careful not to inadvertently push down, which would increase the amount of inferior vena cava com- pression and negatively affect maternal hemodynamics. recommendations . continuous manual lud should be performed on all pregnant women who are in cardiac arrest in which the uterus is palpated at or above the umbili- cus to relieve aortocaval compression during resus- citation (class i; level of evidence c). . if the uterus is difficult to assess (eg, in the morbidly obese), attempts should be made to perform man- ual lud if technically feasible (class iib; level of evidence c). positioning of hands during chest compressions. there is no scientific evidence to support changing the recommenda- tion for hand placement for chest compressions in the preg- nant patient compared with the nonpregnant patient. previous guidelines recommended placing the hands slightly higher on the sternum in the pregnant patient, but there are no scientific data to support this recommendation. figure . cardiac arrest in pregnancy in-hospital basic life support (bls) algorithm: simultaneous c-a-b-u (chest compressions/current- airway-breathing-uterine displacement). acls indicates advanced cardiovascular life support; aed, automated external defibrillator; cpr, cardiopulmonary resuscitation; lud, left uterine displacement; and pea, pulseless electric activity. d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy recommendation . the rescuer should place the heel of hand on the center (middle) of the victim’s chest (the lower half of the sternum) and the heel of the other hand on top of the first so that the hands overlap and are parallel (class iia; level of evidence c). transporting pregnant women during chest compressions. simulation of chest compressions on manikins has shown that the quality of cpr decreases during transport to the operating room. recommendation . because an immediate cesarean delivery may be the best way to optimize the condition of the mother and fetus (see section on “pmcd”), this operation should optimally occur at the site of the arrest. a pregnant patient with in-hospital cardiac arrest should not be transported for cesarean delivery. management should occur at the site of the arrest (class i; level of evidence c). transport to a facility that can perform a cesarean delivery may be required when indicated (eg, for out-of-hospital cardiac arrest or cardiac arrest that occurs in a hospital not capable of cesar- ean delivery). defibrillation issues during pregnancy prompt application of defibrillation in the setting of ventricu- lar fibrillation or pulseless ventricular tachycardia is critical to maximize the likelihood of survival. this is no different in the pregnant patient. transthoracic impedance remains unchanged during pregnancy compared with the nonpregnant state; there- fore, the energy required for defibrillation during cardiac arrest in pregnancy would be the same as the most current recom- mendations for the nonpregnant patient. application of defi- brillation and cardioversion shocks to the maternal chest would be expected to pass minimal energy to the fetus and is consid- ered safe in all stages of pregnancy. defibrillation would be unlikely to cause electric arcing to fetal monitors, and the pres- ence of fetal monitors should not deter providers from the use of rapid defibrillation when indicated. when indicated, defi- brillation should be performed in the pregnant patient without hesitation or delay. the risk to the mother in delaying appropri- ate defibrillation would outweigh any potential concern about defibrillation in the setting of fetal monitors. recommendations . the same currently recommended defibrillation protocol should be used in the pregnant patient as in the nonpregnant patient. there is no modification of the recommended application of electric shock dur- ing pregnancy (class i; level of evidence c). . the patient should be defibrillated with bipha- sic shock energy of to j (class i; level of evidence b) with subsequent escalation of energy output if the first shock is not effective and the device allows this option. . compressions should be resumed immediately delivery of the electric shock (class iia; level of evidence c). . for in-hospital settings where staff have no ecg rhythm recognition skills or where defibrillators are used infrequently such as in an obstetric unit, the use of an automated external defibrillator may be considered (class iib; level of evidence c). . anterolateral defibrillator pad placement is recom- mended as a reasonable default (class iia; level of evidence c). the lateral pad/paddle should be placed under the breast tissue, an important consid- eration in the pregnant patient. . the use of adhesive shock electrodes is recom- mended to allow consistent electrode placement (class iia; level of evidence c). airway and breathing hypoxemia develops more rapidly in the pregnant patient com- pared with the nonpregnant patient; therefore, rapid, high-quality, and effective airway and breathing interventions are essential. as discussed above, a higher partial pressure of oxygen is required to achieve the same maternal oxygen saturation, thus highlight- ing the importance of ensuring maternal oxygenation and ventila- tion concurrent with effective chest compressions in the pregnant figure . manual left uterine displacement by the -handed technique from the right of the patient during adult resuscitation. figure . manual left uterine displacement by the -handed technique from the left of the patient. d ow nloaded from http://ahajournals.org by on a pril , circulation november , patient. as a result, in the aha guidelines for cpr and emergency cardiovascular care (ecc), the recommendations for management of cardiac arrest in pregnancy note the impor- tance of early bag-mask ventilation with % oxygen. airway management should always be considered more difficult in the pregnant patient; therefore, appropriate airway algorithms for pregnancy should be instituted. for first responders with minimal airway experience, bag-mask ventilation with % oxygen is the most rapid noninvasive strategy to initiate ventilation. the standard initial compression-ventilation ratio of : will mini- mize interruptions in chest compressions. hyperventilation has been shown to decrease the likelihood of survival in nonpreg- nant arrest victims, particularly when it interrupts chest com- pressions. two-handed bag-mask ventilation is more effective than a single-handed technique and should be used as soon as a second provider is available to compress the self-inflating bag. if attempts at mask ventilation do not produce visible chest rise or fog within the face mask, the rescuer should try to reopen the airway and improve the seal of the mask on the patient’s face. an oral airway may help relieve airway obstruction in the hypophar- ynx. ideally, airway patency should be maintained continuously to optimize oxygen delivery. obesity, sleep apnea, and airway edema all increase the difficulty of face mask ventilation. bls recommendations (actions are simultaneous, not sequential) . rapid notification should be provided to the mater- nal cardiac arrest response team , – (class i; level of evidence c). . the time when pulselessness was confirmed should be documented (class i; level of evidence c). . high-quality cpr should be paired with uterine dis- placement, and a firm backboard should be used - (class i; level of evidence c). . rapid automated defibrillation should be provided whenever it is indicated as appropriate by rhythm analysis , (class i; level of evidence c). . appropriate bls airway management should be initiated. a. a member of the first responder team should perform bag-mask ventilation with % oxy- gen flowing to the bag at a rate of at least l/ min (class iib; level of evidence c). b. two-handed bag-mask ventilation is preferred (class iia; level of evidence c). . hospitals need to establish first-responder roles that satisfy all of the requirements for bls, includ- ing modifications recommended during pregnancy. a minimum of staff members should respond for bls resuscitation of the pregnant patient. all hospi- tal staff should be able to fulfill first-responder roles (class i; level of evidence c). advanced cardiovascular life support a fast and well-coordinated response to maternal cardiac arrest is important, and the cardiac arrest in pregnancy in-hospital acls algorithm should be used as a guide to management (figure ). the acls maternal cardiac arrest team will continue bls tasks and perform advanced airway management, insert an intravenous access above the dia- phragm, and administer the usual acls drugs and doses when indicated. with the arrival of the obstetric and neonatal teams, preparation for pmcd can begin. the acls algo- rithm includes pmcd as a treatment option for the mother who has not achieved rosc by minutes after the onset of cardiac arrest and in whom the uterus extends to or above the umbilicus. the cause of the arrest needs to be considered and addressed as necessary. the maternal cardiac arrest team activating and achieving prompt code team response is one of the most fundamental tasks to be completed during mater- nal cardiac arrest. each hospital must have a specific method to activate the maternal cardiac arrest team; for example, “maternal code blue” or “code blue maternal” could serve as a universal call to action. creating “bundled” emergency code call (eg, maternal code blue) to all necessary respond- ers simultaneously may save time, help prevent confusion, and reduce the risk of team members not being notified. the universal call to action should also prompt rescuers to bring the necessary specialized equipment (see section on “special equipment required for a maternal cardiac arrest”) to the scene of the arrest without delay. the composition of the code team must reflect the fact that critically ill patients (mother and fetus) must be resuscitated. recommendations . there should be call to action that activates the maternal cardiac arrest team, notifies all mem- bers, and ensures that specialized equipment is brought to the scene without delay (class i; level of evidence c). . the maternal cardiac arrest team would ideally be composed of the following (class i; level of evidence c): a. an adult resuscitation team (potentially com- posed of critical care physicians and nurses, and/or emergency physicians and nurses, and/ or internal medicine physicians and nurses, or other service lines such as general surgery and trauma, with respiratory therapy or equivalent [ie, nurse or physician] and pharmacy represen- tatives according to institutional policy, etc) b. obstetrics: obstetric nurse, obstetrician c. anesthesia care providers: obstetric anesthesiol- ogist if available or staff anesthesiologist; anes- thesia assistant or certified nurse anesthetist if available d. neonatology team: nurse, physician, neo- natal respiratory therapist or equivalent (ie, nurse or physician) e. in centers without obstetric/neonatology services, it is suggested that the cardiac arrest committee and hospital emergency services discuss contin- gency plans in the event of maternal cardiac arrest. . leadership during a maternal cardiac arrest is complicated, given the multiple teams involved. leadership will depend on where the arrest occurs and may be specific to institutional practices. in d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy general, there should be a team leader for adult resuscitation, a team leader for obstetric care, and a team leader for neonatal/fetal care. one approach to deal with multiple subspecialties is for the usual cardiac arrest team leader to delegate leadership for obstetric care, fetal/neonatal care, and airway/ventilatory management. all team leaders must communicate effectively together to make decisions about code management (class i; level of evidence c). special equipment required for a maternal cardiac arrest special equipment is required for a maternal cardiac arrest. emergency response committees must ensure that there is a process for delivering this specialized equipment to the code scene without delay if it is not already located on the code cart. options include either delegating a specific member of the code team to bring the equipment or locating it on the code cart. specialized equipment should include a pmcd tray (table ) but at a minimum must include a scalpel. in addition, equipment for a difficult airway (table ) may be required for the mother. neonatal resuscitation equip- ment will be required if the fetus is delivered and viable (table ). breathing and airway management in pregnancy management of hypoxia current guidelines for the management of cardiac arrest in adults stress that oxygen delivery to vital organs is limited by blood flow during cpr and that chest compressions should not initially be interrupted for ventilation or airway place- ment. the pregnant patient has a very limited oxygen reserve. furthermore, it should be noted that cardiac arrest secondary to hypoxia (eg, severe pneumonia, aspiration, amniotic fluid embolism, acute respiratory distress syndrome, narcotic therapy, high spinal block) requires early attention to airway and ventilation. although delayed endotracheal intubation combined with passive oxygen delivery and minimally inter- rupted chest compressions has been associated with a better figure . cardiac arrest in pregnancy in-hospital advanced cardiovascular life support (acls) algorithm. bls indicates basic life support; cpr, cardiopulmonary resuscitation; ett, endotracheal tube; iv, intravenous; io, intraosseous; lud, left uterine displacement; and rosc, return of spontaneous circulation. d ow nloaded from http://ahajournals.org by on a pril , circulation november , outcome in witnessed ventricular fibrillation arrest, this is not necessarily the case in the pregnant patient, particularly those with preexisting hypoxia. recommendation . hypoxemia should always be considered as a cause of cardiac arrest. oxygen reserves are lower and the metabolic demands are higher in the pregnant patient compared with the nonpregnant patient; thus, early ventilatory support may be necessary (class i; level of evidence c). airway management it is essential to be familiar with airway management algo- rithms in maternal cardiac arrest, given the high likelihood of a difficult airway in the pregnant patient. because endotracheal intubation is frequently more dif- ficult in pregnant patients compared with the nonpregnant surgical population , and is best achieved with minimal or no disruption in chest compressions, any intubation attempts should be undertaken by an experienced laryngoscopist. forceful laryngoscopy can lead to bleeding and airway edema that interferes with ventilation. therefore, regardless of pro- vider experience with laryngoscopy, optimally no more than attempts at either direct laryngoscopy or videolaryngoscopy should be made before insertion of a supraglottic airway. , the glottis in pregnancy is often smaller because of edema; therefore, starting with a smaller endotracheal tube (ett) may increase the likelihood of successful intubation. face mask ventilation between laryngoscopic attempts may pre- serve oxygenation; any difficulty in ventilation indicates the need to avoid further laryngoscopy and to select alternative methods of airway management. supraglottic airway place- ment is the preferred rescue strategy to facilitate ventilation after failed intubation. supraglottic airway devices with an esophageal drain provide access to the stomach to relieve air and stomach contents and may reduce the risk of regurgita- tion and aspiration pneumonitis. subsequent exchange with a definitive airway with fiberoptic guidance may be consid- ered for women with rosc. if oxygenation and ventilation are not successful with a supraglottic device or ett and mask ventilation is impossible, a “cannot intubate, cannot ventilate” situation has occurred. help (in-house general, trauma, or otolaryngology surgeons) must be called emergently, and the final pathway steps in the difficult airway algorithm must be followed for establishing emergency invasive airway access (eg, percutaneous cricothyroidotomy). pregnant women and those who are immediately postpartum are at increased risk of regurgitation and aspiration of stomach contents. despite these concerns, chest compressions, oxygenation, and relief of aortocaval compression are a higher priority than techniques to limit the risk of regurgitation (eg, cricoid pressure, rapid intubation) when caring for the obstetric victim of cardiopul- monary arrest. the aha guidelines for cpr and ecc do not recommend the use of cricoid pressure during resusci- tation of nonpregnant patients, and there are no data to sup- port its use in the management of pregnant patients during cardiopulmonary arrest. cricoid pressure may not be effec- tive at preventing aspiration and can impede ventilation and laryngoscopy. if cricoid pressure is used, it should be adjusted or released if ventilation is difficult or the laryngoscopic view is poor. in the event of regurgitation before intubation, suction may be used to remove gastric contents from the oropharynx during ongoing chest compressions. continuous capnography should be used if available to assess correct placement of the ett, the quality of chest com- pressions, and rosc. confirmation of endotracheal place- ment is complicated by the fact that end-tidal partial pressure of carbon dioxide (petco ) may decrease to almost zero dur- ing cardiac arrest and increase only after the onset of effective cpr. the presence of a decreasing or flat capnographic tracing should prompt the physician leading the code to ensure vig- orous chest compressions and lud, to re-evaluate the loca- tion of the airway device, and to consider obstructive causes of cardiopulmonary arrest (ie, massive pulmonary embolism, cardiac tamponade, or pneumothorax). rosc is more likely when petco can be sustained > mm hg; an abrupt increase in petco by ≈ mm hg is consistent with rosc. findings table . recommended airway and breathing equipment* equipment to be used by first responders equipment to be used by experts oxygen laryngoscope and assorted blades bag-valve-mask devices (eg, ambu bag with disk valve as opposed to duckbill valve preferred videolaryngoscope appropriate size face masks and oral airways cuffed tracheal tubes: size . - to . -mm inner diameter with a semirigid stylet and a range of backup sizes available stethoscope gum elastic bougie pulse oximeter airway exchange catheter qualitative carbon dioxide detector supraglottic airways in a range of sizes suction device flexible fiberoptic intubation equipment equipment suitable for emergency invasive airway access (eg, cricothyrotomy) exhaled carbon dioxide detector *the items listed in this table represent suggestions. the contents should be selected to meet the specific needs, preferences, and skills of the practitioner and healthcare facility. table . recommended equipment for perimortem cesarean section* equipment contents of the emergency cesarean delivery tray scalpel with no. blade lower end of a balfour retractor pack of sponges kelly clamps needle driver russian forceps sutures and suture scissors *the items listed in this table represent suggestions. the contents should be selected to meet the specific needs, preferences, and skills of the practitioner and healthcare facility. d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy suggestive of adequate chest compressions, rosc, or both include a rising petco level or levels > mm hg. – recommendations . endotracheal intubation should be performed by an experienced laryngoscopist (class i; level of evidence c). a. starting with an ett with a . - to . -mm inner diameter is recommended (class i; level of evidence c). b. optimally no more than laryngoscopy attempts should be made (class iia; level of evidence c). c. supraglottic airway placement is the preferred rescue strategy for failed intubation (class i; level of evidence c). d. if attempts at airway control fail and mask ven- tilation is not possible, current guidelines for emergency invasive airway access should be fol- lowed (call for help, obtain equipment). . prolonged intubation attempts should be avoided to prevent deoxygenation, prolonged interruption in chest compressions, airway trauma, and bleeding (class i; level of evidence c). . cricoid pressure is not routinely recommended (class iii; level of evidence c). . continuous waveform capnography, in addition to clinical assessment, is recommended as the most reliable method of confirming and monitoring correct placement of the ett (class i; level of evidence c) and is reasonable to consider in intu- bated patients to monitor cpr quality, to optimize chest compressions, and to detect rosc (class iib; level of evidence c). findings consistent with adequate chest compressions or rosc include a rising petco level or levels > mm hg (class iia; level of evidence c). . interruptions in chest compressions should be mini- mized during advanced airway placement (class i; level of evidence c). arrhythmia-specific therapy during cardiac arrest medical therapy during cardiac arrest is no different in the pregnant patient than in the nonpregnant patient. for patients with refractory (shock-resistant) ventricular fibrillation and tachycardia, the drug of choice is amiodarone; in separate randomized studies, amiodarone has been shown to improve survival to hospital admission compared with standard of care and lidocaine. , the us food and drug administration cat- egories of fetal risk of medications do not apply in the cardiac arrest scenario because fetal concerns are overshadowed by the arrest outcome. recommendations . for refractory (shock-resistant) ventricular fibril- lation and tachycardia, amiodarone mg rapid infusion should be administered with -mg doses repeated as needed (class iib; level of evidence c). . medication doses do not require alteration to accommodate the physiological changes of preg- nancy. although there are changes in the volume of distribution and clearance of medication during pregnancy, there are very few data to guide changes in current recommendations (class iib; level of evidence c). . in the setting of cardiac arrest, no medication should be withheld because of concerns about fetal terato- genicity (class iib; level of evidence c). table . recommended neonatal resuscitation equipment airway breathing circulation/drugs miscellaneous suction apparatus: suction bulb mechanical suction suction catheters ( f– f) meconium aspirator et intubation supplies: laryngoscope (blade size: for preterm and for term infants) extra bulbs and batteries et tubes (internal diameters: . , . , . , . mm) stylets et-securing device carbon dioxide detector tapes and scissors laryngeal mask airway size (for use when et intubation is not feasible) positive-pressure ventilation: device: t-piece resuscitator/flow- inflating bag/self-inflating bag with oxygen reservoir (least preferred) bag sizes – ml, fitted with pop-off valve mask sizes and (cushioned preferred) sources of compressed air and o o blender with a flowmeter (capacity up to l/min) plastic tubing iv access: sterile gloves antiseptic solution cord tie scalpel umbilical venous catheters ( . f– . f) -way stopcock suture for securing -gauge iv cannulas (for use if umbilical vessel access not feasible) io needle (rarely needed) drugs: epinephrine : ( . mg/ml) normal saline bags % dextrose bags . % sodium bicarbonate (rarely indicated) saline flushes temperature regulation: heat source (radiant warmer preferred) warm towels thermometer (servo-controlled preferred) clean plastic bag (for extremely premature neonates) monitoring: neonatal stethoscope neonatal cardiac leads pulse oximeter neonatal/infant oximeter probes clock (digital preferred) other: firm resuscitation surface (preferably height adjustable) good light source nasogastric tubes ( f– f) sterile syringes ( , , , ml) clean, sterile gloves sterile gauzes bottles to collect blood samples suitable for low-volume blood testing et indicates endotracheal; io, intraosseous; and iv, intravenous. d ow nloaded from http://ahajournals.org by on a pril , circulation november , . physiological changes in pregnancy may affect the pharmacology of medications, but there is no scien- tific evidence to guide a change in current recom- mendations. therefore, the usual drugs and doses are recommended during acls (class iib; level of evidence c). other drugs used during acls historically, in the setting of cardiac arrest, vasopressors such as epinephrine and vasopressin have been used with the goal of increasing myocardial and cerebral blood flow and improving patient outcomes. however, as stated in the aha guidelines for cpr and ecc, these drugs have not been shown to improve neurologically intact long-term survival. epinephrine, an α-adrenergic receptor stimulant that has been used for many years, has been shown to augment cerebral and myocardial perfusion during cardiac arrest in preclinical studies. however, data in support of clinical efficacy in humans are scant. the first randomized trial eval- uating this drug, involving patients assigned to acls with drug versus acls with no drug, demonstrated an improvement in rosc with epinephrine but no difference in survival to hospital discharge or long-term survival. these results are consistent with prior observations, and thus, in view of the short-term benefit, the aha guidelines for cpr and ecc state that it is reasonable to administer mg iv/io epinephrine every to minutes (class iib; level of evidence a). vasopressin is a nonadrenergic peripheral vasoconstrictor that was studied as an alternative to epinephrine in view of its powerful vasoconstrictive properties. however, in several randomized trials, vasopressin did not prove to be superior to epinephrine, either alone or in combination with epineph- rine. , because the clinical effect of vasopressin is regarded as equivalent to that of epinephrine, the aha guidelines for cpr and ecc recommend u iv/io vasopressin as an alternative to the first or second dose of epinephrine (class iib; level of evidence a). a new concept under investigation is the use of a combi- nation of drugs during vasopressor-requiring cardiac arrest. a randomized study of patients with in-hospital cardiac arrest demonstrated that the combination of vasopressin-epineph- rine and methylprednisolone during cpr and stress-dose hydrocortisone in postresuscitation shock led to improved survival to hospital discharge compared with epinephrine alone. despite the promising results, additional studies are needed before recommendations can be made about combined vasopressors. no data are available comparing the use of different vaso- pressors during arrest in pregnant patients, but because vaso- pressin can induce uterine contraction and both agents are considered equivalent in the nonpregnant patient, epinephrine is the preferred agent of the two. atropine was removed from the aha guidelines for cpr and ecc acls cardiac arrest algorithm in view of its lack of efficacy, and it is indicated only for cases of bradycardia. recommendations . administering mg epinephrine iv/io every to minutes during adult cardiac arrest should be considered. in view of the effects of vasopressin on the uterus and because both agents are considered equivalent, epinephrine should be the preferred agent (class iib; level of evidence c). . it is recommended that current acls drugs at rec- ommended doses be used without modifications (class iia; level of evidence c). fetal assessment during cardiac arrest during active cpr, the focus should remain on maternal resuscitation and restoration of maternal pulse and blood pres- sure with adequate oxygenation. during this time, evaluation of the fetal heart will not be helpful and carries the risk of inhibiting or delaying maternal resuscitation and monitoring. should the mother achieve rosc and her condition be sta- bilized, then fetal heart surveillance may be instituted when deemed appropriate. recommendations . fetal assessment should not be performed during resuscitation (class i; level of evidence c). . fetal monitors should be removed or detached as soon as possible to facilitate pmcd without delay or hindrance (class i; level of evidence c). delivery this section is written on the premise that the patient’s arrest occurred in an institution that has staff with the exper- tise to deliver a neonate. pmcd is defined as the birth of the fetus after maternal cardiac arrest, most commonly during resuscitation. birth is almost always accomplished through cesarean delivery. a review of all published cases of pmcd up to showed that pmcd led to a clear maternal sur- vival benefit in of cases ( . %), and there were no cases in which pmcd may have been deleterious to mater- nal survival. there may be situations during advanced pregnancy in which noninvasive relief of inferior vena cava compression with manual lud is not enough to provide a hemodynamic advantage to result in successful resuscitation. this is when pmcd needs to be considered as the definitive means to achieve complete relief of inferior vena cava compression and as a treatment option during acls measures for mater- nal cardiac arrest. the purpose of timely perimortem delivery is -fold. the first is facilitation of resuscitation. if cardiac output has not yet been effectively established, relieving aortocaval compression by emptying the uterus significantly improves resuscitative efforts. second, and of critical importance, early delivery of the baby, the second patient, is accomplished with a decreased risk of permanent neurological damage from anoxia. in situ- ations in which the mother is nonresuscitatable (eg, severe trauma is present), timely delivery of the fetus is essential. resuscitation team leaders should activate the protocol for a pmcd as soon as cardiac arrest is identified in a pregnant d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy woman whose uterus extends to or above the umbilicus. by the time the physician is ready to deliver the baby, stan- dard acls should be underway, and immediately reversible causes of cardiac arrest should have been ruled out. when the gravid uterus is large enough to cause maternal hemodynamic changes as a result of aortocaval compression, pmcd should be considered regardless of fetal viability. what defines a gravid uterus with the potential to cause aortocaval compression? several factors determine the weight of the gravid uterus, and these additive factors could result in a uterus that is heavy enough or positioned in such a manner to cause aortocaval compression. determinants of uterine weight include weight of the fetus, the number of fetuses, and the weight of the fluid (ie, polyhydramnios). other factors that may affect the degree of aortocaval compression include the size of the fetus, the rela- tionship of the fetus to the woman’s anatomy, and additional factors such as increased body mass index and morbid obesity. unfortunately, published evidence does not adequately address each of these contingencies. however, some general principles can be used to guide recommendations. a study found that maternal aortocaval compression can occur for singleton pregnancies at > weeks of gestational age. one review of pmcd in maternal cardiac arrest before the third trimester concluded that if the fundus extends above the level of the umbilicus, aortocaval compression can occur, and pmcd should be performed regardless of gestational age. two cases of maternal cardiac arrest in early preg- nancy of to weeks were reported in which the mother was resuscitated without pmcd being performed and the pregnancy continued to successful delivery of a live infant at term. , not every pregnant woman in cardiac arrest is a can- didate for pmcd; the decision depends on whether the gravid uterus is thought to interfere with maternal hemodynamics. why perform pmcd in cardiac arrest? several case reports of pmcd during a maternal cardiac arrest resulted in rosc or improvement in maternal hemodynamic status only after the uterus had been emptied. – in a case series of pmcds, of women for whom maternal out- come was recorded had rosc immediately after delivery. no cases of worsened maternal status after cesarean delivery were reported. the critical point to remember is that both mother and infant may die if the provider cannot restore blood flow to the mother’s heart. the importance of timing with pmcd the -minute window that providers have to determine whether cardiac arrest can be reversed by bls and acls was first described in and has been perpetuated in specialty guidelines. , a it was recommended that pmcd should begin at minutes to effect delivery at minutes after failed resus- citative efforts. this time interval was chosen to minimize the risks of neurological damage, which begins to develop after to minutes of anoxic cardiac arrest if there is no rosc. the rescue team was not required to wait minutes before initiating pmcd, and there are circumstances that support an earlier start. for instance, in an obvious nonsurvivable injury in which the maternal prognosis is grave and resus- citative efforts appear futile, moving straight to pmcd may be appropriate, especially if the fetus is viable. in the situa- tion of an unwitnessed arrest in which a prolonged period of pulselessness is suspected, the priority of pmcd comes to the forefront. many reports document long intervals between a decision for an urgent hysterotomy and actual delivery of the infant, far exceeding the obstetric guideline of minutes for patients not in arrest. , very few cases of pmcd fall within the previ- ously recommended -minute period. , , however, survival of the mother has been reported with pmcd performed up to minutes after the onset of maternal cardiac arrest. , – therefore, if pmcd could not be performed by the -minute mark, it was still advisable to prepare to evacuate the uterus while the resuscitation continued. at > to weeks of ges- tation, the best survival rate for the infant occurs when the infant is delivered no more than minutes after the mother’s heart stops beating. , – at gestational ages > weeks, infant survival has been seen even when delivery occurred > minutes from the onset of maternal cardiac arrest. in a recent retrospective cohort series, neonatal survival was documented even when delivery occurred up to minutes after the onset of maternal cardiac arrest. a systematic review of all pub- lished maternal cardiac arrests occurring before delivery after widespread adoption of resuscitation guidelines ( – ) demonstrated a good outcome in most cases; cerebral performance category was assessed to have been or in . % ( of ) of mothers and . % ( of ) of neo- nates after the event. in cases undergoing pmcd, the average time elapsing from arrest to delivery was significantly differ- ent between surviving ( of ) and nonsurviving ( of ) mothers ( . ± . minutes [median, minutes; range, – minutes and . ± . minutes [median, minutes; range, – minutes], respectively; p< . ; % confidence inter- val, . – . ). the area under the receiver-operating curve for the prediction of maternal death by the time that elapsed from arrest to delivery was . . for neonates, the time elapsing from arrest to delivery was described in only cases and was < minutes in only cases. mean times were ± minutes (median, minutes; range, – minutes) and ± min- utes (median, minutes; range, – minutes) in surviving and nonsurviving neonates, respectively (p= . ), and the area under the receiver-operating curve for the prediction of neonatal death by the time that elapsed from arrest to delivery was only . , reflecting the wide range of arrest-to-delivery survival times. pmcd technique the procedure should be performed at the site of the maternal resuscitation. time should not be wasted moving the patient. simulation with a manikin of maternal transport to the oper- ating room during cardiac arrest has found that transport increases the time to pmcd. additionally, time should not be wasted waiting for surgical equipment or doing abdominal preparation. if desired, antiseptic solution may be poured on the maternal abdomen. the only equipment needed to start a pmcd is a scalpel. d ow nloaded from http://ahajournals.org by on a pril , circulation november , resuscitative efforts should be continued during cesar- ean delivery, including manual lud. the position of the res- cuer performing the manual lud will need to accommodate the surgical field to allow access and to prevent injury to the rescuer. the technique used to perform the pmcd is at the dis- cretion of the physician performing the procedure. both the vertical incision and the pfannenstiel incision are accept- able. the vertical incision provides better visualization of the abdomen and pelvis and may prove beneficial in treat- ing the cause of the arrest. the vertical abdominal incision is also considered faster. however, the pfannenstiel incision may be preferred by a provider who is more comfortable with performing this technique. because successful pmcd has also been described with the pfannenstiel incision, it is a reasonable alternative. during pmcd, the fetus is delivered and given to the neo- natal resuscitation team. the placenta is delivered. the uterus should then be quickly wiped clean, and the uterine incision should be closed with a running locking stitch of absorbable suture. the abdomen is closed in the regular fashion. a foley catheter should be placed at this time if not already present. after the procedure, if maternal resuscitation has been suc- cessful, administration of antibiotics and oxytocin may be considered. however, oxytocin should be used with caution because it can precipitate rearrest (see the section on etiology in the online-only appendix). teams find it difficult to perform pmcd in a timely fashion. therefore, emergency preparedness is important for maternal cardiac arrest. institutions with limited resources or only staff member available (who, for example, may be involved in an operation when the cardiac arrest is called) should have staff respond as quickly as possible. response times may be dictated by the reality of staff numbers and availability. given the rarity of maternal cardiac arrest, it is not reasonable to allocate in-house obstetricians to be on call / solely for the purpose of responding to a mater- nal cardiac arrest if the institution otherwise has a more restricted staffing protocol. recommendations . during cardiac arrest, if the pregnant woman (with a fundus height at or above the umbilicus) has not achieved rosc with usual resuscitation measures with manual uterine displacement, it is advisable to prepare to evacuate the uterus while resuscitation continues (class i; level of evidence c). . decisions on the optimal timing of a pmcd for both the infant and mother are complex and require con- sideration of factors such as the cause of the arrest, maternal pathology and cardiac function, fetal ges- tational age, and resources (ie, may be delayed until qualified staff is available to perform this proce- dure). shorter arrest-to-delivery time is associated with better outcome (class i; level of evidence b). . pmcd should be strongly considered for every mother in whom rosc has not been achieved after ≈ minutes of resuscitative efforts (class iia; level of evidence c). . if maternal viability is not possible (through either fatal injury or prolonged pulselessness), the proce- dure should be started immediately; the team does not have to wait to begin the pmcd (class i; level of evidence c). . when pmcd is performed, the following are recommended: a. the woman should not be transported to an operating room for pmcd during the manage- ment of an in-hospital maternal cardiac arrest (class iia; level of evidence b). b. the team should not wait for surgical equip- ment to begin the procedure; only a scalpel is required (class iia; level of evidence c). c. the team should not spend time on lengthy anti- septic procedures. either a very abbreviated antiseptic pour should be performed, or the step should be eliminated entirely (class iia; level of evidence c). d. continuous manual lud should be performed throughout the pmcd until the fetus is deliv- ered (class iia; level of evidence c). care should be taken to avoid injury to the rescuer performing the manual lud during pmcd. . if the uterus is difficult to assess (eg, in the morbidly obese), then determining the size of the uterus may prove difficult. in this situation, pmcd should be considered at the discretion of the obstetrician by using his or her best assessment of the uterus. in these patients, bedside ultrasound may help guide decision making (class iia; level of evidence c). vaginal delivery during maternal cardiac arrest few published cases describe vaginal delivery during a car- diac arrest in pregnancy. obstetric caregivers involved in an intrapartum cardiac arrest resuscitation may conduct a vaginal examination, provided that cpr is being adequately performed by the medical team. if the cervix is found to be fully dilated and the fetal head is at an appropriately low sta- tion, immediate assisted vaginal delivery can be considered. this will allow resuscitation of the fetus and will facilitate the resuscitation of the mother because of the factors discussed above. recommendation . assisted vaginal delivery should be considered when the cervix is dilated and the fetal head is at an appropriately low station (class iib; level of evidence c). neonatal resuscitation considerations neonatal resuscitation team it is expected that each maternity hospital will have a desig- nated team for managing unexpected neonatal resuscitations. because of the high likelihood of delivering a depressed neo- nate after maternal arrest, the team attending delivery must anticipate and be prepared for an advanced resuscitation. this includes designating a team leader, checking equipment, and d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy preassigning specific roles to team members. team compo- sition optimally should include a neonatologist/pediatrician, neonatal nurses, and respiratory therapists who should be familiar with the local neonatal resuscitation algorithms. at least member of the team must be skilled in emergency neonatal endotracheal intubation. in some settings, this may require accepting the urgent assistance of other subspecialty professionals, for example, an anesthesiologist, an otolaryn- gologist, or emergency physicians. recommendations . the neonatal resuscitation team should be notified of the impending delivery and its circumstances as early as feasible to allow maximum preparatory time (class i; level of evidence c). . the following critical information should be pro- vided to the neonatal resuscitation team leader: gestational age, number of fetuses, and mode of delivery (class i; level of evidence c). . in the event of multiple pregnancies, it is recom- mended that each fetus be resuscitated by a separate resuscitation team (class i; level of evidence c). neonatal resuscitation equipment pmcd may be performed outside the maternity unit and will require the team to perform resuscitation in a relatively unfa- miliar environment that may lack optimal equipment. each hospital must have prestocked neonatal crash carts available, the locations of which should be clearly marked and known to the neonatal resuscitation team. alternatively, neonatal resus- citation equipment can be prestocked in easy-to-carry bags that can be taken to the area of need by the resuscitation team on notification of impending delivery. such carts/bags must be fully stocked, regularly checked, and accessible from all rel- evant clinical locations of the hospital. a comprehensive list of all equipment deemed necessary for neonatal resuscitation is presented in table . ems considerations maternal cardiac arrest that occurs out of hospital will likely have worse outcomes than cardiac arrest that occurs in hospital. therefore, a coordinated ems response to maternal cardiac arrest is of critical importance. if possible, additional prehospital providers should respond to the loca- tion of the maternal arrest to ensure that a sufficient num- ber of providers is available to provide bls (figure ) and acls care, including lud. prehospital providers should not be expected to perform a pmcd; however, transport- ing the mother in cardiac arrest to a location where pmcd can be performed in a timely manner is essential. fetal car- diac activity may be slow but present after many minutes of maternal pulselessness. , as a result, fetal survival can occur in cases when maternal vital signs are lost before arrival in the emergency department and when cpr fails to restore maternal pulses. immediate transport of the obviously pregnant patient, identified as the uterus extending to or above the umbilicus, should be initiated. this is justified because pmcd may be required to achieve rosc by relieving aortocaval compres- sion, decreased time to pmcd is associated with better fetal outcomes, resources to perform pmcd are usually lacking in the field, and multiple teams will be required to resusci- tate the neonate and the mother after pmcd. ems medical directors should identify appropriate receiving hospitals for obviously pregnant patients accord- ing to the resources available within the service area. choices should include whether a specialized obstetric cen- ter or a center with a neonatal icu is preferred over the closest destination. as a result of the geographical restric- tions of rural systems, the closest appropriate receiving hos- pital might be used regardless of the availability of obstetric or neonatal services. for pmcd to be used as a lifesaving procedure, it is extremely time dependent; delays as short as minutes affect fetal survivability. therefore, transport should be directed toward a center that is prepared to per- form pmcd rather than the closest facility, but optimally transport should not be prolonged by > minutes to reach a center with more capabilities (eg, neonatal icu). although it is possible that prehospital transport will take > min- utes and the likelihood of pmcd success will therefore be lower, there is still less advantage to transporting a patient to a facility where pmcd cannot be offered. when trauma is the proximate cause of maternal cardiac arrest, resuscita- tion including pmcd can be performed at a trauma center with early activation of the hospital maternal cardiac arrest team. bypassing the trauma bay or emergency department to arrive at the operating room is not advisable, given the evidence that cpr quality is impaired during transfer and that procedures do not occur faster in the operating room compared with other settings. ems providers in rural or other resource-limited settings may be faced with limited staff and equipment and extended transportation distances to the most appropriate receiving hospitals. ems systems that provide care in the rural setting should consider these factors during planning and attempt to optimize care through coordinated education and training with first-responder organizations. ems responders should use the cardiac arrest in preg- nancy out-of-hospital bls algorithm for healthcare pro- viders as the basis of care during a maternal cardiac arrest (figure ). recommendations . if resources are available, ems response to a mater- nal cardiac arrest should include the appropriate complement of staff to ensure that bls and acls actions can be performed, including chest com- pressions, lud, defibrillation when indicated, and management of the difficult airway (class i; level of evidence c). . if available, transport should be directed toward a center that is prepared to perform pmcd, but transport should not be prolonged by > minutes to reach a center with more capabilities (class iib; level of evidence c). d ow nloaded from http://ahajournals.org by on a pril , circulation november , . ems and the receiving emergency department must establish optimal communication and an action plan for the transport of a maternal cardiac arrest patient. the emergency department should be able to rapidly mobilize the maternal cardiac arrest team, and specialized equipment should be available from the time the patient arrives in the emergency department (class i; level of evidence c). cause of the cardiac arrest similar to the recommendations for adult (nonpregnant) acls, an understanding of the importance of diagnosing and treating the underlying cause or aggravating factors of the cardiac arrest is fundamental to the management of cardiac arrest in pregnancy. it is important to consider the cause of the cardiac arrest early in the management algorithm. specific therapy directed at the cause of the cardiac arrest can be lifesaving. it is important to under- stand the causes of maternal mortality to have an understanding of the unique pathogenic factors that may have precipitated the maternal cardiac arrest. the most common causes of maternal cardiac arrest and mortality are listed in table . caregivers not routinely involved in high-risk pregnancy may not have experience with the presentation or frequency in which specific diagnoses can result in maternal mortality and cardiac arrest. therefore, the online-only appendix includes a specific robust chapter that reviews the causes of maternal mortality and high- lights important diagnostic and treatment considerations. point-of-care instruments checklists may help individual responders access temporarily inaccessible cognitive information during periods of intense stress or task saturation. however, the use of checklists dur- ing medical emergencies is not consistent, despite the evidence of recurrent cognitive errors on the part of medical provid- ers. checklists seem particularly well suited to the obstet- ric domain. one study found that all critical actions for simulated obstetric cardiac arrest were performed only when a cognitive aid reader assisted the team leader. several groups figure . cardiac arrest in pregnancy out-of-hospital basic life support (bls) algorithm for healthcare providers. aed indicates automated external defibrillator; cpr, cardiopulmonary resuscitation; ems, emergency medical services; and lud, left uterine displacement. d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy have recommended and produced obstetric crisis−specific checklists. , a, one such checklist for maternal cardiac arrest is provided in figure . these checklists may include, but are not limited to, contact numbers, critical service lines (eg, transfusion services, neonatal/pediatric teams), locations of necessary equipment (eg, scalpel location), and critical steps in care (eg, prepare to make the incision for delivery). the key factors for optimal use of point-of-care instruments are that teams should be familiar with the content and use of check- lists in general and that the checklist should be written specifi- cally for the institution with input from providers who would respond and be involved in rendering care at that institution. recommendation . institutions should create point-of-care checklists to help guide and support critical interventions during obstetric crises (class i; level of evidence b). immediate postarrest care it is essential that a multidisciplinary team continue care in the postarrest period. as with all postarrest patients, the pregnant patient who is successfully resuscitated will require thorough assessment, monitoring, and treatment as complications arise. for example, as perfusion improves, bleeding may become a serious issue. if the patient is not delivered, aortocaval compression could precipitate hypo- tension and rearrest. recommendations . if the patient is still pregnant, she should be placed in the full left lateral decubitus position, provided that this does not interfere with additional manage- ment issues such as monitoring, airway control, and intravenous access. if the patient is not in full left lateral tilt, manual lud should be maintained con- tinuously (class i; level of evidence c). . the patient should be transferred to the icu unless an operation is required (class i; level of evidence c). . optimal pre-event planning should be ensured as discussed above (class i; level of evidence c). . multidisciplinary care must continue (class i; level of evidence c). . the cause of the arrest should continue to be considered and treated accordingly (class i; level of evidence c). antiarrhythmic therapy postarrest therapy for recurrent life-threatening arrhythmias includes consideration of placement of an implantable cardio- verter-defibrillator or medication therapy in the pregnant patient as in the nonpregnant patient (class i; level of evidence c). β-blockers are often used as first-line therapy for a diversity of arrhythmias; they are generally safe in pregnancy (meto- prolol is a preferred β-antagonist used in pregnancy) (class iia; level of evidence c). for long-qt syndrome, β-blockers were found to be effective in reducing the incidence of adverse events and therefore highly recommended during pregnancy and the postpartum period , (class iia; level of evidence c). in general, for recurrent primary ventricular tachycardia and ventricular fibrillation, amiodarone should be considered (class iib; level of evidence c). evaluation for reversible causes of cardiac arrhythmias should be routine. thyroid dys- function, adverse drug effects, electrolyte disturbances, cardiac ischemia, and heart failure should all be identified and cor- rected when possible (class i; level of evidence c). table . most common etiologies of maternal arrest and mortality letter cause etiology a anesthetic complications high neuraxial block hypotension loss of airway aspiration respiratory depression local anesthetic systemic toxicity accidents/trauma trauma suicide b bleeding coagulopathy uterine atony placenta accreta placental abruption placenta previa retained products of conception uterine rupture surgical transfusion reaction c cardiovascular causes myocardial infarction aortic dissection cardiomyopathy arrhythmias valve disease congenital heart disease d drugs oxytocin magnesium drug error illicit drugs opioids insulin anaphylaxis e embolic causes amniotic fluid embolus pulmonary embolus cerebrovascular event venous air embolism f fever sepsis infection g general h’s and t’s h hypertension preeclampsia eclampsia hellp syndrome, intracranial bleed hellp indicates hemolysis, elevated liver enzymes, and low platelet count. d ow nloaded from http://ahajournals.org by on a pril , circulation november , targeted temperature management/therapeutic hypothermia even when resuscitative efforts are successful in restoring spontaneous circulation, postarrest brain injury often limits the positive outcome for cardiac arrest. one apparent ben- eficial intervention has been mild postarrest-induced hypo- thermia, which is based on scientific plausibility and the positive results of randomized, clinical trials published in . , on the basis of these data, targeted temperature management to °c to °c ( . °f– . °f) for to hours has been recommended for nonpregnant comatose adult patients with rosc after out-of-hospital ventricu- lar fibrillation cardiac arrest (class i; level of evidence b). there are case reports with favorable maternal and fetal outcomes in which postarrest cooling was instituted in early pregnancy, the fetus was monitored, and emergency cesarean delivery was not necessary. , there is case report of fetal demise in the setting of targeted temperature management, but in this case, there was a period of unsuc- cessful resuscitation of minutes before resuscitation by ems was started. therefore, data for hypothermia in pregnancy are scant. pregnancy is thus not an absolute contraindication to targeted temperature management. however, given the lack of data on the use of targeted tem- perature management after pmcd and the risk of impaired coagulation during the lowering of systemic temperatures, targeted temperature management may be considered on an individual basis after cardiac arrest in a comatose pregnant patient. recommendations . targeted temperature management should be con- sidered in pregnancy on an individual basis (class iib; level of evidence c). . if used in pregnancy, targeted temperature man- agement should follow the same current protocol as for the nonpregnant patient (class iib; level of evidence c). . fetal monitoring should be performed throughout targeted temperature management (class i; level of evidence c). fetal risk of postresuscitation intervention a large number of medications may be used after rosc is achieved. unlike in the nonpregnant state, whether medications may adversely affect the fetus must be con- sidered, in addition to the potential compromise caused by circulatory failure, lack of adequate placental perfusion, and impaired oxygen and nutrient exchange between the mother and fetus. figure . cognitive aid checklist for cardiac arrest in pregnancy. aed indicates automated external defibrillator; bls, basic life support; cpr, cardiopulmonary resuscitation; ett, endotracheal tube; iv, intravenous; and pmcd, perimortem cesarean delivery. modified with permission from lipman et al. a copyright © , international anesthesia research society. d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy three major principles guide the decisions made by clini- cians at this stage: . maternal well-being is the overriding priority because maternal demise or unfavorable recovery never bodes well for the unborn baby. . embryogenesis is mostly complete by weeks of gestation; hence, even teratogenic drugs (eg, warfarin, phenytoin, corticosteroids) are unlikely to cause malfor- mations if the event occurs after the first trimester. . drugs may cause fetal toxicity rather than teratoge- nicity in late pregnancy (eg, angiotensin-converting enzyme inhibitors, which can cause fetal renal failure and oligohydramnios). in general, most therapeutic drugs have a molecular weight of < , allowing them to cross the human placenta from the maternal to the fetal circulation. the exceptions are large molecules such as heparin and low-molecular-weight heparin, insulin, and other proteins. however, after weeks of gesta- tion, all biologics that are igg can cross the placenta by using the fc transporters. the fact that a drug crosses the placenta is, per se, not a reason for concern because the concentrations of most of these agents do not inflict fetal damage. the risks and benefits of medication use in the postarrest period should be considered on an individual basis. assessment of the newborn the majority of neonates delivered by pmcd are likely to require active resuscitation ; the severity of perinatal depres- sion and the extent of resuscitation may vary. management of the neonate after pmcd should follow the most current aha guidelines. fetal assessment (if undelivered) in cases when maternal cardiac arrest is treated without delivery of the fetus and the pregnancy is considered potentially viable (minimum, weeks of gestation), continuous fetal heart rate monitoring with cardiotocography should be started as soon as feasible after maternal rosc and continued until clini- cal recovery of the mother. the goal for such monitoring is to assess for signs of nonreassuring fetal status (fetal tachycardia, bradycardia, loss of heart rate variability, variable or late decel- erations) and to monitor for uterine activity. , furthermore, because the fetus is considered to be more sensitive to changes in environment, nonreassuring fetal well-being could be the first sign of deterioration of the maternal clinical condition and may signify impending maternal decompensation. the presence of signs of nonreassuring fetal well-being on fetal heart rate moni- toring should prompt an urgent obstetric and medical review because an emergency cesarean delivery may be necessary. recommendations . postarrest assessment of the fetus should include continuous fetal heart rate monitoring (class i; level of evidence c). . signs of nonreassuring fetal status should prompt a thorough maternal and fetal reassessment (class i; level of evidence c). . delivery could be considered if signs of nonreassur- ing fetal status occur (class i; level of evidence c). medical-legal considerations maternal cardiac arrest and the death of the mother, the fetus, or both are traumatic and usually unexpected events. as high- lighted throughout this scientific statement, efforts of all those involved in the care of pregnant women should be directed at prevention, identification of high-risk patients, and refer- ral to specialized care for those at risk for adverse events. however, not all events can be prevented, and if cardiac arrest does occur, ems, institutions, and individual healthcare team members who would be involved in resuscitative efforts must be prepared. patient safety is strengthened through this type of proactive approach to pregnancy care. one such example of a system-wide approach to pregnancy care has been shown to result in reduced claims. once a cardiac arrest has occurred during pregnancy, important steps should be taken to review the care received by the patient to improve systems of care going forward. implementation of a quality incident notifica- tion system has shown that this type of program can help iden- tify avoidable adverse outcomes and can be used to improve practices of care. , , a the references highlighted above are useful tools for readers to review and potentially improve their own institutional practices. recommendations . all cases of cardiac arrest and maternal near miss should be reviewed by the maternal cardiac arrest com- mittee for the hospital (class i; level of evidence c). . identified deficiencies should be corrected (class i; level of evidence c). knowledge translation strategy knowledge translation, also referred to as dissemination and implementation, has been defined by the national center for the dissemination of disability research as “the collaborative and systematic review, assessment, identifica- tion, aggregation, and practical application of high-quality research by key stakeholders (consumers, researchers, practitioners, policy makers) for the purpose of improving the lives of individuals....” this statement represents an important step in the knowledge translation process: the col- laborative filtering of information by experts so that only the most valid and useful knowledge is left. clinicians are often faced with an unmanageable multitude of primary studies or information of variable quality. comprehensive syntheses presented in statements such as this one provide a trustworthy and applicable aggregation and appraisal of the existing knowledge. this scientific statement is specifically designed to increase the likelihood of translation and uptake by using reviews of the evidence and providing direct prac- tice recommendations. reading this statement cannot be where the knowledge translation process ends. an active knowledge translation strategy, which includes multidisciplinary involvement (cus- tomized information), barrier assessment, full leadership d ow nloaded from http://ahajournals.org by on a pril , circulation november , commitment and support, and a variety of ongoing dis- semination approaches, is crucial to ensure routine use of evidence-based practices. there is an entire body of literature on various approaches to ensure the routine use of knowledge, all with variable success, depending on the context and intensity with which they are applied. some approaches that may be specifically pertinent in the case of maternal cardiac arrest include the following: •	 instituting standardized order sets, a prefilled form of evidence-based orders that can be signed by the lead physician so that there is no confusion as to what needs to be done and no time is lost starting from blank orders. •	 developing a program of mock code drills. lifelike maternal cardiac arrest scenarios are simulated for all staff at regular intervals to make crisis situations feel more commonplace, to decrease fears and anxiety in the team, to improve communication, and to increase familiarity with resuscitation guidelines. this has been shown to directly improve resuscitation skills performance in both the adult and pediatric settings. recommendations by the confidential enquiries into maternal and child health of the united kingdom, the joint commission, and others emphasize the use of periodic emergency drills that involve both the obstetric and neonatal teams as a way to practice critical communication skills and to identify occult errors in the system. – •	 building in an audit and feedback mechanism to collect data during resuscitation situations and to provide con- structive feedback to the team to identify key areas of success and areas for improvement. •	 holding case debriefing sessions, which are similar to audit and feedback, to provide an opportunity for the team to talk about how the event went, what worked well and what did not work, and to offer a chance for others who may never have been part of a maternal resuscita- tion to learn from their colleagues’ experience. the key factor to successful knowledge translation is an active, multifaceted approach that attends to the multidis- ciplinary nature of health care. a unique consideration for maternal cardiac arrest is the relatively low volume of events any one clinician will be involved in treating; this can be detrimental to the impact of knowledge translation efforts. however, strategies such as case discussions and mock drills can be very effective in ensuring that the team is ready when a pregnant patient in cardiac arrest comes through the door. recommendation . all stakeholders/specialties involved in maternal resuscitation should form maternal cardiac arrest committees within each institution to ensure guide- line implementation, training, and institution of mock code drills (class i; level of evidence c). maternal resuscitation training an analysis of simulated maternal cardiac arrests involving participants trained in acls suggests that performance dur- ing an actual event may be suboptimal. aha acls courses do not routinely emphasize obstetric-specific interventions. in addition, cpr courses tend to stress knowledge and tech- nical skills over behavioral/communication skill sets. , the rarity of maternal cardiac arrest implies that participants in cpr courses could benefit from a review of obstetric- specific interventions. although many obstetric providers are not trained in acls, knowledge decay and gaps in fund of information specific to the obstetric domain exist even among those who are. , , , maternal cardiac arrest demands a multidisciplinary response that requires unique coordination among teams. such coordination is predicated on clear and succinct communica- tion. data from the joint commission suggest that commu- nication failures are the root cause of neonatal morbidity and mortality in % of cases that occur in the obstetric domain. in addition, in an analysis of preventable maternal mortalities, facility factors contributed significantly to the fatal outcome in % of cases. a this suggests that lack of institutional pre- paredness can decrease or even negate the ability of highly functioning staff to render optimal care. moreover, acls courses cannot hope to address the identification and correc- tion of institutional system issues; this suggests that multidis- ciplinary maternal cardiac drills are important components of institutional preparedness. maternally oriented cpr courses are likely more relevant to the learning needs and goals of obstetric staff; these courses have been developed by groups in the united kingdom and the united states. , , recommendations . periodic multidisciplinary drills may help institu- tions optimize safety systems (class iia; level of evidence c). . specific courses on maternal resuscitation should be available to staff if not available outside local institu- tions (class iia; level of evidence c). . the future goal should be to have national and inter- national programs in maternal resuscitation (class i; level of evidence c). future considerations the questions remaining unanswered in relation to both treat- ment and outcomes (both maternal and neonatal) should prompt the establishment of a central registry of cases of maternal near miss and cardiac arrest. maternal cardiac arrest represents the tip of the iceberg of near-miss maternal complications. understanding why in some cases the pregnant woman reached a state of arrest may require root-cause analysis. multiple issues related to maternal resuscitation remain. some could be addressed by simple analyses of database information, as mentioned above, for example, whether pmcd improves the rate of rosc in accordance with the theory of aortocaval decom- pression, the optimal timing of cesarean delivery for both mater- nal and neonatal outcomes, and whether these outcomes are as good as those suggested in the most recent literature. current guidelines advocate placement of the woman in the full supine position with manual uterine displacement to alleviate aortocaval compression. however, there are sparse data on the impact of the anterior and left lateral displacement d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy of the heart during pregnancy on the probability of hand place- ment on the cardiac apex, and data on the effect of manual uterine displacement or pelvic wedging on the position of the heart and upper back are also scant. finally, both maternal and neonatal long-term neurological status and functional status after resuscitation remain enigmas. recommendations . a central registry of cases of maternal near miss and cardiac arrest with documentation of both process and outcome should be established (class i; level of evidence c).* . a standardized training course in maternal resuscita- tion should be developed (class i; level of evidence c). conclusion maternal cardiac arrest is a complex clinical scenario. resuscitation of the pregnant woman involves multispecialties and complex care decisions. it is unlikely that prospective stud- ies on maternal resuscitation will provide additional data in the future, despite the fact that clinical equipoise remains for most treatments in this situation. although maternal cardiac arrest is rare, it appears to be increasing in frequency. the number of high-risk women undergoing pregnancy is on the rise, as is the rate of severe complications related to pregnancy (including cardiac arrest). the writing group acknowledges that scien- tific evidence for management of cardiac arrest in pregnancy is lacking. , the majority of the writing group’s recommen- dations are level of evidence c, which underscores the need for further research. this expert panel of authors has applied a multispecialty, expert approach to develop these recommen- dations through experience, previous publication of direct and indirect data, and expert opinion to reach consensus. the writ- ing group recognizes that without an organized approach to maternal cardiac arrest, chaos will likely ensue. therefore, the development and implementation of the recommendations con- tained in this document will be beneficial to maternal care. this scientific statement will help healthcare providers be prepared and provide the best possible care for a maternal cardiac arrest. the newly developed in-hospital and out-of-hospital bls and acls algorithms should be the backbone of the response plan to a maternal cardiac arrest. special attention should be paid to manual lud, the difficult airway, and appropriate use of pmcd. lifesaving interventions such as defibrillation and medications should not be withheld in the setting of pregnancy. the healthcare community must be proactively prepared to respond to a maternal cardiac arrest. a maternal cardiac arrest committee must be formed at every institution, and emergency response plans specific to each institution must be developed and implemented. the maternal cardiac arrest committee would link adult resuscitation teams with obstetrics, neonatol- ogy, intensive care, anesthesia, the emergency department, and ems and involve the allied healthcare teams, including nurs- ing, respiratory therapy, social work, and clergy, as available and necessary to implement guidelines and recommendations. training, mock code drills, and review of cases should become routine. this scientific statement has addressed all aspects of maternal resuscitation: prearrest care, bls, acls, and postar- rest care. in addition, the online-only appendix has a robust chapter on the causes of maternal mortality and cardiac arrest in pregnancy with specific therapies to consider. the statement has also provided readers with resources, point-of-care instruments, and algorithms that will be useful to consider when developing institutional response plans. *for those interested in joining the registry, please email us at eccscience@heart.org. d ow nloaded from http://ahajournals.org by on a pril , mailto:eccscience@heart.org circulation november , writing group disclosures writing group member employment research grant other research support speakers’ bureau/ honoraria expert witness ownership interest consultant/ advisory board other farida m. jeejeebhoy university of toronto, william osler health system rescu project grant (all funds provided through nonindustry funds)* none none none none none none julie arafeh stanford university none none none none none none none clifton w. callaway university of pittsburgh nhlbi (research outcome consortium)† none none none none none none brendan carvalho stanford university none none none none none none none katie n. dainty st. michael’s li ka shing knowledge institute none none none none none none none sharon einav shaare zedek medical center (jerusalem, israel) covidien*; zoll*; diasorin*; israel ministry of health†; national institute for health policy research† none none none patent on capnography in weaning from mechanical ventilation* none european society of anaesthesia*; american college of chest physicians* amish jain mount sinai hospital none none none none none none none jose joglar ut southwestern none none none none none none none vern l. katz oregon health sciences university none none none none none none none gideon koren university of toronto duchesnay inc† none none none none duchesnay inc*; novartis*; bayer* none stephen e. lapinsky university of toronto none none none none none none none steve lipman stanford university none none none none none none none jill m. mhyre university of arkansas for medical sciences none none none none none none none richard l. page university of wisconsin none none none none none none none carole a. warnes mayo clinic rochester none none none none none none none rory windrim university of toronto none none none none none none none carolyn m. zelop the valley hospital none none acog annual meeting* none none uptodate* none staff russell e. griffin american heart association none none none none none none none this table represents the relationships of writing group members that may be perceived as actual or reasonably perceived conflicts of interest as reported on the disclosure questionnaire, which all members of the writing group are required to complete and submit. a relationship is considered to be “significant” if (a) the person receives $ or more during any -month period, or % or more of the person’s gross income; or (b) the person owns % or more of the voting stock or share of the entity, or owns $ or more of the fair market value of the entity. a relationship is considered to be “modest” if it is less than “significant” under the preceding definition. *modest. †significant. disclosures d ow nloaded from http://ahajournals.org by on a pril , jeejeebhoy et al cardiac arrest in pregnancy references . mhyre jm, tsen lc, einav s, kuklina ev, leffert lr, bateman bt. cardiac arrest during hospitalization for delivery in the united states, - . anesthesiology. ; : – . doi: . / aln. . . say l, chou d, gemmill a, tunçalp Ö, moller ab, daniels j, gülmezoglu am, temmerman m, alkema l. global causes of maternal death: a who systematic analysis. lancet glob health. ; :e –e . doi: . 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arkadeb pal  · somnath roy  · amish g. joshi  · a. k. ghosh  · sandip chatterjee received: august / accepted: november / published online: november © springer nature switzerland ag abstract charge transfer across the interface of two materials in a composite can create reconstruction of bands near the inter- face which in turn brings multiple changes in physical properties of the materials. thus, investigation of band structure experimentally is of immense importance in studying composite materials to understand their physical properties. here, we have studied magnetoelectric multiferroic composite of two types of multiferroic (types i and ii) consisting of bifeo and tbmno for enhanced magnetic and transport properties. the band structure was investigated with the help of uv–visible absorption spectrum, the valence band x-ray photoemission spectra (xps), and ultraviolet photoemission spectra. the band structure thus obtained can successfully explain the magnetic and transport properties of the compos- ite. the insulating behavior of the system is understood from the reconstruction of the energy bands at the interface and subsequent decrease in the band gap which happens due to lattice mismatch of the two materials. the large coercivity and the increase in the magnetization value are understood to be due to superexchange interaction between different mn ions (mn +, mn +, and mn +). from the composition study of edxa and core-level xps, oxygen vacancy was found which in turn creates the mixed valence state of mn to maintain the charge neutrality. keywords bifeo  · tbmno  · band structure · magnetoelectric multiferroic introduction multiferroic materials have been attracting researchers recently for their interesting fundamentals as well as for the possibility of application of these materials in different spintronic devices [ – ]. in magnetoelectric multiferroic materials, ferroic (or anti-ferroic) magnetic and electric ordering coexist in a single phase giving rise to the pos- sibility of controlling the magnetization (intrinsic polariza- tion) with the application of electric field (magnetic field) [ ]. due to these coupling between the two properties, magnetoelectric multiferroic materials have become one of the most important materials of today [ ]. the reason for the limited number of multiferroic material is the mutual exclusive origin of the two ordering (empty d shell for fer- roelectricity and partially filled d shell for magnetic order- ing) [ – ]. bifeo is one of the most interesting and well-studied multiferroic as it is the only multiferroic material to show both the ordering (magnetic and ferroelectric) above the room temperature (ferroelectric curie temperature tn ~  k and neel temperature tn ~  k) [ – ]. it exhibits g-type canted antiferromagnetic ordering with a cycloid frequency of ~   nm [ ]. bifeo shows large sponta- neous polarization of order – µc/cm because of polar displacement of cations and anions relative to each other pointing along one of the eight pseudo-cubic [ ] axes [ – ]. the lone pair electron at the  s shell of bi is * sandip chatterjee, schatterji.app@iitbhu.ac.in | department of physics, indian institute of technology (bhu) varanasi, varanasi    , india. department of physics, banaras hindu university, varanasi    , india. csir-national physical laboratory, dr. k. s. krishnan road, new delhi    , india. http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf vol:.( ) research article sn applied sciences ( ) : | https://doi.org/ . /s - - - considered to be the main reason behind the observed ferroelectricity on bifeo while the partially filled d shell of fe is responsible for the canted antiferromagnetic order- ing [ – ]. bifeo has been found to be useful in different modern-day technologies including microwave synthe- sis [ , ]. in spite of having these features, bifeo is not considered suitable for many applications due to many reasons. structural instability, difficulty in synthesizing sin- gle phase, high leakage current, and low resistivity of the material due to the presence of fe + and oxygen vacancies are some of them [ – ]. to overcome the shortcomings, there have been numerous attempts involving doping of different transition metal at fe site and rare earth metals at bi sites, inducing chemical pressure or strain in the system [ – ]. an alternate option is to prepare composite of dif- ferent multiferroic materials with a similar structure involv- ing bifeo . there are reports on composite structures and superlattice structures of bifeo –batio , bifeo –pbtio , and bifeo –bimno , showing improvement in multi- ferroic properties [ – ]. yu et  al. [ ] have reported exchange bias and other enhanced magnetic properties in bifeo –la . sr . mno heterostructure due to charge transfer-assisted band reconstruction near the interface which in turn creates additional ferromagnetic and antifer- romagnetic exchange interactions. in this context, we have prepared a composite of bifeo and tbmno , belonging to a different type of mul- tiferroic. tbmno is type ii multiferroic material in which the ferroelectric ordering (tc ~  k) arises as a result of magnetic ordering (tn ~  k), and the material is known to possess strong magnetoelectric coupling [ , ]. it crystallizes in orthorhombic perovskite (pbnm) close to that of bifeo which has rhombohedral perovskite (r c) structure [ , ]. the lattice mismatch between them is expected to trigger band reconstruction near the inter- face [ ]. band reconstruction can influence many physi- cal properties of the composite including transport and magnetic properties. in one of our earlier reports, it was reported that the composite of bifeo and tbmno (in : ratio) . bfo– . tmo shows interesting properties like spin–phonon coupling, magnetodielectric coupling, high dielectric constant, etc. [ ]. it was also seen that charge accumulation and charge transfer at the interface of two materials of the composite in determining different properties of the composite. thus, it would be significant to study the band structure of the composite and band reconstruction if any due to the charge transfer. it is also interesting to study the changes in transport and mag- netic properties due to band reconstruction. in this report, we have studied the band structure through valence band x-ray photoemission spectroscopy (xps) and ultraviolet photoemission spectroscopy (ups) and uv–visible absorp- tion spectroscopy. the effect of band reconstruction on the transport and magnetic properties has also been studied. materials and methods the compounds bifeo , tbmno , and the composite of bifeo and tbmno (in : ratio), i.e., . bfo– . tmo, were prepared following a conventional solid-state reac- tion method by taking the precursors for all the constitu- ent elements i.e., bi o , fe o , mn o , and tb o (alfa aesar, usa) in proper stoichiometric ratio. at first, powders of tbmno were prepared by solid-state reaction of tb o and mn o in proper stoichiometric ratio. the mixture was calcined °c for  h. the calcined powders were pel- letized and sintered at and °c for  h. the pow- der was ground thoroughly in between. then the tbmno powder was mixed with bi o and fe o in stoichiometric ratio, and the mixture was ground with mortar pestle for  h. the mixture was calcined at °c for  h after which it was pelletized and sintered for  h at °c to get the final sample. the surface morphology and grain growth are studied from field emission scanning electron micro- scope (fesem, nova nano sem ). the composition of the samples was also studied from energy-dispersive x-ray analysis (team eds system with octane plus sdd detector) integrated with the sem. x-ray photoemission spectroscopy (xps) experiments were performed using omicron multi-probe® surface science system, gmbh, equipped with a dual-anode non-monochromatic mg/ al x-ray source (dar ), a monochromatic source (xm ), and a hemispherical electron energy analyzer (ea ). all the xps measurements were performed inside the analysis chamber under base vacuum of ~ . × − torr using monochoromatized alkα with a power of w. the total energy resolution for monochromatic alkα line with photon energy .  ev, estimated from the width of the fermi edge, was about .  ev. the pass energy for survey scan spectra and core level spectra was kept at  ev and  ev, respectively. ultraviolet photoemission spectroscopy (ups) and uv–visible absorption spectros- copy were employed to study valence band structure and electronic properties. to investigate the fine changes near fermi level, fermi edge ultraviolet photoemission spectra were collected using the non-monochromatic he i ( .   ev ) line at an average base pressure of . × − torr. the energy resolution of the analyzer and the step size were set at .  ev and .  ev, respectively. the magnetic measurements were carried out in magnetic property measurement system (squid-mpms, quantum design, usa). the temperature-dependent transport properties were measured in close cycle he cryostat vol.:( ) sn applied sciences ( ) : | https://doi.org/ . /s - - - research article (advanced research systems, inc.) using a keithley b electrometer. result and discussion in order to visualize surface morphology and microstruc- ture of the composite images of sintered . bfo– . tmo composite along with the pure bfo and tmo were recorded in the field emission scanning electron micro- scope (fesem) (shown in fig.  a–c). it can be observed that pure bfo pellet exhibits rectangular and non-homoge- nously distributed grains with smooth surfaces showing crystal growth of bfo. the average grain size was calcu- lated using imagej software with an average size of ~ .  μm. tmo microstructure consisted of non-homoge- nously distributed grains of an irregular shape with aver- age grain size of .  μm. the composite also shows similar morphology with irregular-shaped grains, but the grain size varies in broad range as compared to that of bfo and tmo grains. the average grain size also lies in between that of the two pure compounds ( .  μm) [ ]. in order to identify all the elements present in . bfo– . tmo composite, energy-dispersive x-ray analysis (edxa) analy- sis was carried out, which is shown in fig.  d. the obtained edxa result confirmed the homogenous mixing and phase formation of the composite. the edxa result also indicated the presence of oxygen vacancy in the system. mangan- ite systems sintered at a higher temperature tend to have oxygen vacancies which in turn can influence the mn ions present in the system to have multiple valence states in order to maintain the charge neutrality [ ]. fourier transform infrared (ftir) spectroscopy has been one of the most used techniques these days to analyze different functional groups and corresponding vibrational bands. the ftir spectrum of the composite . bfo– . tmo was recorded in the wavenumber range of –  cm− which is presented in fig.  . the vibra- tional bands of metal–oxygen bonds generally lie below  cm− . in the case of pure bifeo , the bending and stretching vibrations of the fe–o in feo octahedra and the vibrational band of bio octahedra lie in the range from to  cm− , which has been clearly observed in the spectrum (peaks denoted as a and b) [ , ]. the presence of tbmno perovskite phase in the composite is confirmed by identifying the stretching vibration of fig. a–c fesem images from fractured surfaces of the single- phase bfo, tmo, and composite . bfo– . tmo, respectively, and insets show the corresponding calculation of grains size bye image j software. d shows edxa graph of the composite . bfo– . tmo with its chemical composition in the insets vol:.( ) research article sn applied sciences ( ) : | https://doi.org/ . /s - - - mn–o–mn bond ~  cm− arising from mno octahedra of the compound (denoted as c in the spectrum). apart from these bands few other bands can be identified in the spectrum such as  cm− assigned to the bend- ing vibrations of h o which arises due to the presence of moistures in the powder [ , ]. the bands ~  cm− and  cm− are ascribed to the stretching vibrations of c=o and c–c, respectively, which appears due to the absorption of carbon during the exposure of the sample to air [ ]. x-ray photoemission spectroscopy (xps) is a promi- nent technique followed by many researchers to study the chemical composition and valence state of constitu- ent elements of material. we have studied the survey scan and detail scan of fe p, mn p, o s, bi f, and tb d spectra to gain detail knowledge of their electronic structure and composition in the composite bifeo –tbmno . all the peak positions were matched with the national institute of standards and technology (nist ) xps database. from the survey, it was confirmed that all the constituent elements (i.e., bi. fe, tb, mn, and o) were present in the composite (fig.  a). the absence of any foreign element in the com- pound was also confirmed from the spectrum. although a c s peak can be seen from the survey scan spectrum, the presence of carbon does not influence any physical property of the composite. in many systems, such surface absorbed/adsorbed carbon has been found which does not have any effect on the physical properties. the detail spectra were deconvoluted using the xpspeak software. in fig.  b, the deconvoluted spectrum of the fe p core level is shown, which is split into two broad peaks due to spin–orbit coupling. the characteristic peaks of fe p / and fe p / are observed around the binding energies . and .   ev, respectively, which correspond to fe + state in bfo [ – ]. in addition to the characteristic peaks, there is a satellite peak, which is observed at the binding energy position  ev. the presence of the satel- lite peak ~  ev confirms that trivalent oxidation state is the dominant state of iron in bfo which is expected for bifeo [ , ]. the xps analysis indicates that only pure bifeo phase is present without any type of impurities. figure  c shows the o s xps spectrum of the composite which is split into two parts revealing two kinds of chemi- cal state of oxygen present in the composite. the peaks at binding energy position ~ .  ev correspond to the oxygen situated at lattice denoted as ol and the peak positioned at ~ .  ev corresponds to the surface chem- isorbed oxygen denoted by ov. the surface chemisorp- tions of the oxygen ions occur due to the presence of oxy- gen vacancy in the system. in bifeo -based compounds, it t ra ns m it ta nc e % wavenumber (cm- ) a b c d e f fig. ftir spectrum of the composite . bfo– . tmo recorded at  k vol.:( ) sn applied sciences ( ) : | https://doi.org/ . /s - - - research article is common to find the oxygen vacancies which are created at the surface due to lattice defects [ , ]. moreover, our xps results conform with edx analysis which indicated the presence of oxygen vacancy in the system. in fig.  d, the deconvoluted mn p spectrum is pre- sented which is also found to be split into two charac- teristic spin–orbit peaks for mn p / and mn p / ~ . and .  ev, respectively. interestingly, the peaks were asymmetric in nature indicating multiple valence states of mn ions in the composite. the mn p / and peak were deconvoluted into three characteristic peaks at binding energies . , . , and .  ev, which correspond to mn + mn + and mn +, respectively. the deconvoluted peak positions for the mn +, mn +, and mn + species (a) (b) (c) (d) (e) (f) fig. a xps survey scan of . bfo– . tmo at  k. deconvoluted detail spectra of b fe p, c o s, d mn p of composite . bfo– . tmo. detail spectra of e tb d and f bi f vol:.( ) research article sn applied sciences ( ) : | https://doi.org/ . /s - - - were found at . , . , and .  ev, respectively. from the deconvoluted characteristic peaks, mn + was found to be more intense, indicating dominant presence of mn + which is obvious in case of tbmno . it is possible to estimate the fraction from the area under each curve representing a particular ion. the fractional composition is estimated using the relation: where sj is the relative sensitivity factor. the relative frac- tions have been found to be . %, . %, and . % for the mn +, mn +, and mn + respectively. the mixed phase of the mn ions evolves in the system to maintain the charge neutrality which is affected by the oxygen vacancy observed from the o  s detail spectrum. it has been observed that, in mn-based systems, charge transfer effect between the mn d orbitals and o p ligand orbit- als can give rise to the mixed phase of mn [ and refer- ences therein]. the creation of mn + and mn + species can also be due to the valence instability of mn + ions. the mix valence state of mn ions is known to modify the bulk magnetic and electrical properties of different manganite systems [ ]. in fig.  e, core level xps spectra of tb d element is pre- sented. the spectra show spin-orbit coupling peaks of tb d positioned at ~ .  ev and ~ .  ev for d / and d / , respectively. the peak positions and the doublet separation clearly suggest that tb is in + oxidation state [nist ]. the core-level spectra of bi f are also studied and are shown in fig.  f. the spin–orbit split peaks of bi f / and bi f / are observed to be present at ~ .  ev and ~ .  ev, which corroborate well with earlier reports of trivalent valence states of bi [ , ]. the presence of different charge states of tm ions at the interface due to the charge transfer between mn–mn ions may lead to the band reconstruction at the interface. as a result of the reconstruction, band gap of the com- posite is expected to decrease [ , ]. thus to confirm the band gap reduction, we have measured the absorp- tion spectrum in the uv and visible range. for studying the absorption characteristics of the composite, absorb- ance at different wavelengths (k) (range of –  nm) was recorded and the absorption coefficients (a) were calculated at corresponding wavelengths. as can be seen from the spectrum, the absorption band edge lies beyond the range of measurement. we have followed the tauc’s method to estimate the band gap of the composite from the absorption spectra [ ]. the photon energy (hν) and the band gap energy for a particular transition are related by the equation: %fraction = areaj � sj ∑ areaj � sj , where α is the absorption coefficient given by α = . (ab/t) (here, ab is absorbance and t is the thickness of the cuvette which is   cm) and k is the edge width param- eter. the value of n depends on the type of transition, i.e., allowed direct, allowed indirect, forbidden direct, and for- bidden indirect for which it can have values / , , / , and , respectively. bifeo is known to have a band gap of .  ev to .  ev in different forms of the material such as bulk, nanomaterials, or single crystalline material [ – ]. since bifeo and tbmno both are known to be direct band gap material, the band gap of the composite was determined from the linear fitting of the straight-line part of the (αhν) versus photon energy (hν) plot on the hν axis [ ]. from the tauc’s plot (fig.  ), it is evident that the band gap of the material lies ~ .  ev, which is low compared to other reported values for bifeo [ – ]. therefore the reduction in the band gap of the composite system con- firms the band reconstruction phenomenon due to the charge transfer between the tm ions in the composite. t h e x p s v a l e n c e b a n d ( v b ) s p e c t r a o f t h e . bfo– . tmo system has been recorded at room tem- perature (   k) to gain better insight into its detailed electronic structure (fig.  b). it is evident from the figure that near fermi level (ef =  ev), the spectral weight of the electronic states is extremely weak or merely absent which essentially suggests an insulating ground state of the system. moreover, it is relevant here to mention that the uv–visible spectroscopy study yielded a high band gap of ~ .  ev and the resistivity data exhibited an insulating ( )(�h�) = k ( h� − eg )n , fig. tauc plot for the determination of the optical band gap of the composite . bfo– . tmo at room temperature. the black dashed arrow is to guide the eye, showing the extracted bad gap. the inset shows the absorption spectrum of the composite from which the tauc plot has been estimated vol.:( ) sn applied sciences ( ) : | https://doi.org/ . /s - - - research article nature with high resistance (discussed later). hence, our vb spectra corroborate well with our earlier results. the energy position of the valence band maximum has been estimated to be ~  ev by making a linear extrapolation of the sharply rising feature immediately below the fermi level (as shown in inset of fig.  a) [ , ]. apart from this, knowing the band gap of the system (~ .  ev) and the valence band maximum energy position (~  ev), the posi- tion of the conduction band (cb) minimum can be readily determined to be ~ − .  ev. hence, a schematic diagram of its possible density of states (dos) (band structure) is depicted in fig.  a. furthermore, three main features of xps vb spectra below the ef can be observed at ~ .  ev,  ev, and .  ev, which are denoted as vb , vb , and vb respectively. the complete occupied vb spectra below ef (ranging from  ev to ~  ev) is mainly composed of hybridized states of tb f, extended mn d, fe d, and o p orbitals. since, for the present system, the mn and fe are in octahedral co-ordination with ligand oxygen ions, the crystal field effect causes the mn/fe d states to split into eg and t g levels. moreover, for such kind of co-ordination, the t g states always lie below the eg states. therefore, the spec- tral weight (in the range of  ev to ~  ev ) immediately below ef can be mainly attributed to the contributions from partially occupied mn d_eg and fe d_eg orbitals [ ]. however, the first shoulder like feature vb positioned at ~ .  ev has appeared mainly due to the hybridization of tb f, extended mn d_t g, and fe d_t g with o p orbit- als. however, a significant contribution to this feature vb is expected to be from tb f states as the other states (mn d_t g, fe d_t g) are extended over a long range [ ]. the most intense feature of the vb spectra at ~  ev (vb ) can be attributed mainly to the hybridization of the mn d_t g and fe d_t g orbitals with the o p states. on the other hand, the feature vb at the lowest energy ~ .  ev emanates from the hybridized states of o p - mn d_t g/ fe d_t g and other oxygen bonding states o p–mn/fe sp and o p–tb sd, etc., with significant contribution being from o p states [ ]. moreover, to probe how the vb spectral features get modified with higher resolution and also to augment with our previous results of a electronic structure near fermi level, we have carried out the ultraviolet photoemission spectroscopy (ups) measurement on the same system (fig.  c). it is interesting to note here that the spectral features in ups vb spectra immediately below the ef are of relatively low intensity as compared to those observed in xps vb spectra. this in turn predicts the fact that the region immediately below ef has significant contribu- tion from the mn/fe d states since the photo-ionization cross-section for the mn/fe d photoemission is consider- ably higher than that for the o p photoemission in the xps process. as a consequence, the low-energy ups is less sensitive to the heavier atoms and highly sensitive to the lighter atoms such as oxygen. hence, in the whole ups spectra, the predominant contribution comes from the (a) (b) (c) ∆ fig. a schematic diagram of the band structure. inset shows the xpas valence spectra of the composite . bfo– . tmo. the red line is to guide the eye showing the position of the edge of the valence band. b valence band xps spectrum of the composite at  k. c ups spectrum of the composite at room temperature vol:.( ) research article sn applied sciences ( ) : | https://doi.org/ . /s - - - o p states, while the contributions from the tb f, mn d, and fe d states get suppressed. irrespective of the above facts, the ups vb spectra agreed mostly with the xps vb spectra. the absence of the electronic states near the fermi level in the ups vb spectra supports our previous results, thus suggesting its insulating nature. moreover, a feature (vb ) near ~ .  ev (associated with the hybridized states of o p–mn/fe d and o p–tb f orbitals) can be vis- ible in the ups vb spectra which corroborate with the xps vb spectra. similarly, the second feature (vb ) which is the intense broad peak near ~  ev matches well with the most intense peak of the xps vb spectra. another weak feature (vb ) near ~ .  ev can also be visible which is seemingly associated with the o p–mn/fe d hybridized states with significant contribution coming from o p states. the posi- tion of the last feature differs by ~  ev with that of the vb feature observed in xps vb spectra. this can presumably be attributed to the possible charging effect due to the high resistivity of the system. in our earlier study and in other works, it has been found that the bifeo -based materials show high dielec- tric constant and magnetodielectric coupling [ , , ]. hence, it is significant to study the transport property of the composite. figure  shows the resistivity vs. tempera- ture plot of the composite measured in the temperature range –   k. the resistivity of the sample increases continuously with decreasing temperature indicating semiconducting nature of the composite. this also indi- cates the involvement of activation process in the trans- port mechanism. below  k, the resistivity of the sample increases sharply. the inset (a) of fig.  shows lnρ versus /t plot signifying arrhenius fitting which shows that our data poorly fit the arrhenius equation. hence, the mott’s variable-range hopping (vrh) mechanism was con- sidered to govern the transport process. the resistivity in case of vrh model can be written as: [ ] where tm is known as mott’s characteristic temperature and is expressed as: the most probable hopping distance (r) and the hop- ping energy (w) can be presented as: where /α is the localization length. the average ionic radii of the two transition metal ions (fe + and mn +) which are considered responsible for the conduction mechanism are taken as the localization length. inset (b) of fig.  shows the lnρ versus ( /t) / plot, which displays a good linear fit- ting thus supports the transport mechanism to be vrh. it is noteworthy to mention here that in disordered systems with random distribution of defects, vrh mechanism can generally be found to control the transport process [ ]. in our sample, there are site defects due to the presence of mixed valence of mn ions. the large strain produced at the interface of the two materials due to lattice mismatch would definitely create lattice defects in the composite system. moreover, the high resistivity of the sample is con- sistent with band gap ascribed from the uv–visible spec- trum and the band structure calculated from the vb xps and ups spectra. to gain knowledge about the effect of band reconstruc- tion and creation of different valence state of mn ions on the magnetic property of the composite, we have meas- ured the room-temperature magnetization (m) versus magnetic field (h) hysteresis loop of the composite and compared with that of pure bfo. enhancing the mag- netic property of bifeo is one of the most significant challenges in multiferroic material study as it is the only multiferroic whose ordering temperatures (both magnetic and electric) are above the room temperature. figure  presents the measured m–h loops for the magnetic field ± t from which it can be seen that the magnetization increases linearly with the applied field. this unsaturated behavior is consistent with the antiferromagnetic nature of bifeo at room temperature. a closer look around the zero field reveals that at low fields there is deviation from � = � exp [ ( tm t ) ] , tm = . � ∕kbn ( ef ) . r = [ ��n ( ef ) kbt / ]− , w = / �r n ( ef ) , fig. temperature-dependent resistivity of the composite . bfo– . tmo. inset a lnρ versus /t plot showing arrhenius plot. inset b lnρ versus ( /t) / showing the plot for variable-range hop- ping vol.:( ) sn applied sciences ( ) : | https://doi.org/ . /s - - - research article the linearity and the loop is wide open. this along with the loop opening even at high fields signifies ferromag- netic contribution in the magnetic property of the sample. there are reports on the room-temperature weak ferro- magnetism in the nanoparticles of bifeo [ ]. also bifeo is known to possess canted antiferromagnetic ordering with a cycloid frequency ~  nm which is governed by the dzyaloshinskii–moriya interaction [ , , ]. the weak fer- romagnetism can also arise from the canted antiferromag- netic ordering. inset in fig.  shows the m–h loop for the pure bfo sample. it can be seen that the magnetization of bfo also does not saturate showing its antiferromagnetic nature. interestingly, it can be noticed that the magnetiza- tion value increases by an order of two. the coercivity of the material is found to increase from that of pure bfo. the increase and the low field weak ferromagnetic behavior can be understood to be due to the possible ferromag- netic superexchange interaction between different mn ions (mn +, mn +, and mn + as seen from xps spectra) at the interface. according to goodenough–kanamori rules the superexchange interactions between fe +–o–mn +/ mn + are also expected to be ferromagnetic [ , , ]. it is difficult to estimate the contribution of different interac- tions which dominate the magnetic behavior. however, from the xps study, it is observed that the relative frac- tions are found to be . %, . %, and . % for the mn +, mn +, and mn + respectively. mn + dominates the fractions as expected, whereas mn + and mn + have also significant fractions. from the analysis, it can be concluded that fe + ions are present in the sample in the largest amount among the transition metal mn +, mn +, and mn + in decreasing order. clearly, the most dominating interac- tions would be fe +–o–fe +. among the ferromagnetic interactions, fe +–o–mn + would be the strongest, and similarly, we can say that the superexchange interaction of mn +–o–mn + would be the weakest. thus, the composite shows higher coercivity in comparison with pure bifeo . conclusion to summarize, we have prepared the bifeo –tbmno composite ( : ) via conventional solid-state reaction and studied the interface through different characterizations. morphological detail and the grain growth were studied from the sem images. chemical state and the compo- sition of the material were studied from xps and edxa which revealed that oxygen vacancy is there which in turn creates mix valence state of mn ions to maintain the charge neutrality. a remarkable decrease in band gap was observed from the uv–visible absorption spectra from that of bifeo . based on the band gap (~ .  ev) and the results from xps valence band spectra and ups spectra, the band structure of the material was drawn in which the conduction band edge was found ~ .   ev. in the valence band, three main features were observed at bind- ing energy positions ~ .  ev,  ev, and .  ev (vb , vb , vb , and vb ) which were composed of hybridized states of tb f, extended mn d, fe d, and o p orbitals. the most intense feature (vb ) was attributed to the hybridization of the mn d_t g and fe d_t g orbitals with the o p states, while the weak shoulder-like feature vb which close to the fermi energy is attributed to the tb f states. such band diagram and the reduction in the band graph are due to the reconstruction of the bands due to interfacial strain. moreover, the transport property was found to be dominated by variable-range hopping mechanism, and the high resistivity of the material was also found to be consistent with the band diagram. antiferromagnetic like non-saturating m–h loop was observed with weak ferro- magnetic features at low fields which was attributed to the superexchange interaction between different mn ions. acknowledgements authors would like to acknowledge the central instrument facility center of iit (bhu) for sem, edxa, and magnetic measurements. compliance with ethical standards conflict of interest on behalf of all authors, the corresponding au- thor 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band gap magnetic semiconductor (la . pr . ) . ca . mno nanocrystals in paramagnetic and fer- romagnetic phases. appl phys lett : . shklovskii bi, efros al ( ) electronic properties of doped semiconductors. springer, berlin . wang h, li g, zhao m, li l ( ) spin state transition and giant dielectric constant in pr . na . coo . appl. phys. lett. : . goodenough jb ( ) theory of the role of covalence in the perovskite-type manganites [la, m(ii)]mno . phys rev : – . kanamori j ( ) superexchange interaction and symmetry properties of electron orbitals. j phys chem solids : – publisher’s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 	study of band structure, transport and magnetic properties of bifeo –tbmno composite 	abstract 	 introduction 	 materials and methods 	 result and discussion 	 conclusion 	acknowledgements 	references [pdf] sustained therapeutic hypercapnia attenuates pulmonary arterial rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /ajpheart. . corpus id: sustained therapeutic hypercapnia attenuates pulmonary arterial rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats. @article{peng sustainedth, title={sustained therapeutic hypercapnia attenuates pulmonary arterial rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats.}, author={gary peng and j. ivanovska and crystal kantores and t. van vliet and d. engelberts and b. kavanagh and m. enomoto and j. belik and a. jain and p. mcnamara and r. jankov}, journal={american journal of physiology. heart and circulatory physiology}, year={ }, volume={ }, pages={ h - } } 	gary peng, j. ivanovska, + authors r. jankov 	published 	medicine 	american journal of physiology. heart and circulatory physiology sustained therapeutic hypercapnia prevents pulmonary hypertension in experimental animals, but its rescue effects on established disease have not been studied. therapies that inhibit rho-kinase (rock) and/or augment nitric oxide (no)-cyclic guanosine monophosphate (cgmp) signaling can reverse or prevent progression of chronic pulmonary hypertension. our objective in the present study was to determine whether sustained rescue treatment with inhaled co( ) (therapeutic hypercapnia) would improve… expand view on pubmed ajpheart.physiology.org save to library create alert cite launch research feed share this paper citationsbackground citations methods citations results citations view all figures, tables, and topics from this paper 	 table 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure view all figures & tables 	pulmonary hypertension 	hypoxia 	hypertensive disease 	cyclic gmp 	torr 	lung diseases 	nitric oxide synthase 	ventricular hypertrophy 	positron-emission tomography 	guanosine monophosphate 	myocytes, smooth muscle 	smooth muscle (tissue) 	structure of parenchyma of lung 	y 	systolic pressure 	guanosine tetraphosphate 	congenital abnormality citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency therapeutic hypercapnia prevents inhaled nitric oxide-induced right-ventricular systolic dysfunction in juvenile rats. 	kristyn dunlop, kiranjot gosal, + authors r. jankov 	medicine 	free radical biology & medicine 	 	 save alert research feed sodium nitrite augments lung s-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats. 	r. jankov, k. l. daniel, + authors a. jain 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	 save alert research feed rho kinase mediates right ventricular systolic dysfunction in rats with chronic neonatal pulmonary hypertension. 	kiranjot gosal, kristyn dunlop, + authors r. jankov 	medicine 	american journal of respiratory cell and molecular biology 	 	 save alert research feed hypercapnia downregulates hypoxia‐induced lysyl oxidase expression in pulmonary artery smooth muscle cells via inhibiting transforming growth factor β signalling 	xiao-dong xia, yanping peng, dan lei, wei-qian chen 	chemistry, medicine 	cell biochemistry and function 	 	 	view excerpts, cites results and background save alert research feed effect of fasudil on hypoxic pulmonary hypertension and right ventricular hypertrophy in rats. 	x. sun, s. li, xiang-yang tian, qing-quan wu 	medicine 	international journal of clinical and experimental pathology 	 	 	pdf 	view excerpt, cites background save alert research feed rho-kinase inhibitors. 	y. fukumoto, h. shimokawa 	medicine 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camprubí-rimblas, j. bringué, l. bos, m. schultz, a. artigas 	medicine 	intensive care medicine experimental 	 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency therapeutic hypercapnia prevents chronic hypoxia-induced pulmonary hypertension in the newborn rat. 	crystal kantores, p. mcnamara, + authors r. jankov 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf 	view excerpts, references methods and background save alert research feed acute vasodilator effects of rho-kinase inhibitors in neonatal rats with pulmonary hypertension unresponsive to nitric oxide. 	p. mcnamara, p. murthy, + authors r. jankov 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf 	view excerpts, references background and methods save alert research feed chronic hypercapnia downregulates arginase expression and activity and increases pulmonary arterial smooth muscle relaxation in the newborn rat. 	j. belik, danielle stevens, + authors r. jankov 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf save alert research feed chronic hypercapnia inhibits hypoxic pulmonary vascular remodeling. 	h. ooi, e. cadogan, m. sweeney, k. howell, r. o’regan, p. mcloughlin 	medicine 	american journal of physiology. heart and circulatory physiology 	 	 	pdf 	view excerpt, references background save alert research feed rescue treatment with a rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension. 	emily z xu, crystal kantores, + authors r. jankov 	medicine 	american journal of physiology. heart and circulatory physiology 	 	 	pdf 	view excerpts, references background and methods save alert research feed peroxynitrite mediates right-ventricular dysfunction in nitric oxide-exposed juvenile rats. 	r. jankov, p. lewis, + authors p. mcnamara 	biology, medicine 	free radical biology & medicine 	 	 	view excerpts, references methods save alert research feed therapeutic hypercapnia reduces pulmonary and systemic injury following in vivo lung reperfusion. 	j. laffey, m. tanaka, + authors b. kavanagh 	medicine 	american journal of respiratory and critical care medicine 	 	 	pdf 	view excerpt, references background save alert research feed hypercapnia attenuates hypoxic pulmonary hypertension by inhibiting lung radical injury. 	m. chovanec, j. novotná, j. wilhelm, v. hampl, m. vízek, j. herget 	medicine 	physiological research 	 	 	pdf 	view excerpts, references background save alert research feed pde a inhibition attenuates bleomycin-induced pulmonary fibrosis and pulmonary hypertension through inhibition of ros generation and rhoa/rho kinase activation. 	a. hemnes, a. zaiman, h. champion 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	view excerpt, references background save alert research feed therapeutic effects of hypercapnia on chronic lung injury and vascular remodeling in neonatal rats. 	azhar masood, m. yi, + authors a. k. tanswell 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf 	view excerpts, references background and methods save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue https://e-aair.org abstract allergic asthma is a public health problem that affects human health and socioeconomic development. studies have found that the prevalence of asthma has significantly increased in recent years, which has become particularly pronounced in developed countries. with rapid urbanization in china in the last decades, the prevalence of asthma has increased significantly in urban areas. as changes in genetic backgrounds of human populations are limited, environmental exposure may be a major factor that is responsible for the increased prevalence of asthma. this review focuses on environmental components of farms and rural areas that may have protective effects in reducing the development of asthma. farm and rural related microorganism- and pathogen-associated molecular patterns are considered to be important environmental factors that modulate host's innate and adaptive immune system to induce protection effects later in life. environmental microbial-related immunotherapy will also be discussed as the future research direction for the prevention of allergic asthma. keywords: asthma; epidemiology; hygiene hypothesis; environmental exposure; house dust mite introduction epidemiological studies have confirmed that allergic asthma becomes more prevalent around the world in the past few decades, and the prevalence of asthma is higher in urban areas than in rural areas. the increased occurrence of asthma is often found to be associated with improvement in hygiene conditions of humans. thirty years ago, the classic “hygiene hypothesis” stated that crowded and unhygienic living conditions will lead to a decrease in the prevalence of asthma as well as in other allergic disorders including eczema and hay fever. the unhygienic environment increases the opportunities for children to contact microorganisms at the early stages of life. the advent of this hypothesis has increased research on the role of environmental factors in allergic diseases, especially asthma. currently, reports on asthma-protective factors in the environment have yielded inconsistent findings. endotoxin has been found to be associated with protection against asthma as a allergy asthma immunol res. nov; ( ): - https://doi.org/ . /aair. . . . pissn - ·eissn - review received: nov , revised: apr , accepted: may , correspondence to jing li, md department of allergy and clinical immunology, guangzhou institute of respiratory health, state key laboratory of respiratory disease, the first affiliated hospital of guangzhou medical university, guangzhou , china. tel: + - - fax: + - - e-mail: lijing@gird.cn zhong su, phd guangzhou institute of biomedicine and health, chinese academy of sciences, kai yuan road, guangzhou , china. tel: + - - e-mail: su_zhong@gibh.ac.cn copyright © the korean academy of asthma, allergy and clinical immunology • the korean academy of pediatric allergy and respiratory disease this is an open access article distributed under the terms of the creative commons attribution non-commercial license (https:// creativecommons.org/licenses/by-nc/ . /) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. xinliu lin , xia ren, xiaojun xiao , zhaowei yang , , siyang yao , gary wk wong , zhigang liu, charles wang, , zhong su , * jing li * department of allergy and clinical immunology, guangzhou institute of respiratory health, state key laboratory of respiratory disease, the first affiliated hospital of guangzhou medical university, guangzhou, china institute of allergy and immunology, school of medicine, shenzhen university, shenzhen, china departments of pediatrics, prince of wales hospital, the chinese university of hong kong, china guangzhou institute of biomedicine and health, chinese academy of sciences, guangzhou, china center for genomics, loma linda university school of medicine, loma linda, ca, usa important role of immunological responses to environmental exposure in the development of allergic asthma https://creativecommons.org/licenses/by-nc/ . / https://creativecommons.org/licenses/by-nc/ . / https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - x https://orcid.org/ - - - https://orcid.org/ - - - http://crossmark.crossref.org/dialog/?doi= . /aair. . . . &domain=pdf&date_stamp= - - orcid ids xinliu lin https://orcid.org/ - - - xiaojun xiao https://orcid.org/ - - - zhaowei yang https://orcid.org/ - - - siyang yao https://orcid.org/ - - - gary wk wong https://orcid.org/ - - - x zhong su https://orcid.org/ - - - jing li https://orcid.org/ - - - disclosure there are no financial or other issues that might lead to conflict of interest. recent study reported that the prevalence of asthma was lower in amish children in a high endotoxin environment. the mechanisms by which environmental factors regulate allergic asthma have been extensively investigated and the findings further expand our knowledge of the interaction between environmental factors and allergic asthma. the aim of the present review is to summarize the research works in china as well as the findings of research groups from other countries in epidemiological surveys on asthma, environmental microorganism factors, immunoregulation mechanisms, and microbial-related immunotherapy. new research directions on relations between environmental factors and allergic asthma are proposed which are expected to guide future studies. epidemiological trends of allergic asthma trends in the prevalence of allergic asthma since the s, the global prevalence of allergic asthma has been increasing. the international study of asthma and allergies in childhood analyzed the multicenter survey data on allergic asthma, allergic rhinoconjunctivitis, and eczema at -year intervals on average, and the results showed increases in all allergic diseases. generally, the prevalence of asthma is higher in developed countries, while its increasing trend has slowed down in recent years. however, the asthma prevalence showed remarkably increased in the developing countries with rapid economic growth and urbanization. in the usa, the prevalence of asthma increased at an annual rate of . % up to , and reached . % in . however, it has shown a steady trend since , with . % in and . % in , according to the national survey data. , the prevalence in the uk had also stabilized in recent years ( . % in and . % in ). , a high asthma prevalence was found in canada from . % in to % in . the rate of school-age children with asthma in sweden was . %, . %, and . % in , , and , respectively. in , . % of children aged – years in australia had asthma. in melbourne, the prevalence in -year-old children remained high at . % in . singapore showed significantly increased prevalence trend from . % in , to . % in , and % in , , and the prevalence continuously went up to . % in to . % in for - to -year-old children. data from national health insurance sharing service of korea revealed a . -fold increase from . % to . % between and for asthma prevalence, which was more common in elderly population aged ≥ years. in china, the prevalence rates of asthma and other allergic diseases have been increasing each year. pediatric asthma surveys that were conducted in , , and showed a substantial rise in asthma prevalence in most cities. - for example, in beijing, prevalence of asthma in children increased from . % in , to . % in , and to . % in . - shanghai children also demonstrated a similar increasing trend in asthma prevalence from . %, to . %, and to . %, respectively. - a survey in school children from guangzhou also showed increasing prevalence of asthma from . % in , to . % in , and to . % in . in , and , taiwanese showed their prevalence of asthma at . %, . %, and . %, respectively, , while the prevalence of doctor diagnosed asthma increased dramatically years later at . % in and . % in . in hong kong, however, the prevalence of asthma in - to -year-old children did not change significantly between ( . %) and ( . %). , https://e-aair.org https://doi.org/ . /aair. . . . environment and asthma https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - x https://orcid.org/ - - - x https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - difference in the prevalence of asthma between urban and rural populations in contrast to the high prevalence of asthma in urban areas, asthma is less prevalent in rural regions. farming and rural environment are proposed to be classic protective factors against asthma in europe. , stein et al. compared matched amish children from traditional farms and hutterite children from modern farms, and found that although both groups had similar genetic backgrounds and lifestyle habits, the prevalence of asthma was to times higher in the hutterite population than in the amish population who were exposed to the high endotoxin levels in house dust. our study showed that the prevalence of asthma in - to -year-old children from rural conghua was significantly lower than that in those from urban guangzhou ( . % vs. . %). the prevalence rated of allergic rhinitis and eczema in the groups of children also showed similar rural-urban differences. similar to the situation observed in southern china, the prevalence of asthma in children from rural regions in northern china was significantly lower than in beijing city ( . % vs. . %). environmental and microbial exposure environmental exposure and asthma asthma pathogenesis is multifactorial and the most important factors are genetics and environmental factors. since significant changes in genetic background of humans is less likely to occur within several decades, environmental factors might be the major determinants modulating the prevalence of asthma. the “hygiene hypothesis” proposed in by strachan suggested that the rapid increase in the prevalence of allergies and autoimmune diseases in humans is due to an overly clean environment. following the initial “hygiene hypothesis,” many epidemiological studies provided direct evidence for the correlation between the prevalence of asthma and living environmental condition in children and explored the underlying immunological and molecular mechanisms. - in , braun- fahrländer et al. first found that the risk of atopic constitution and allergies in children who grew up on farms was significantly lower than those who grew up in non-farm conditions. subsequently, many epidemiological studies that investigated children asthma on european farms, - traditional and modern farms in the usa, as well as the studies comparing urban and rural environment , - have validated the protective effect of a farm environment so called “farm effect.” in the southern and northern regions of china, we have also found that the agricultural environment and rural house dust endotoxin have protective effects on childhood asthma. , it is generally believed that exposure to the farm environment (particularly traditional farms) early in life has significantly reduced the prevalence of asthma in the children compared with those who are not exposed the farm environment. due to environment diversity, multiple protective environmental factors were found in different studies. the contact factors included livestock, pets, farm crops, unpasteurized milk, - breastfeeding, farm or village cultivation activities, , and fishing. the major findings from these studies are summarized in table. , , , - microbial exposure and asthma among the complex and diverse factors of the living environment that we are exposed, microbes and their chemical compounds might be the key elements that play roles in the modulation of immune responses and the pathogenesis of allergic diseases like asthma. acinetobacter lwoffii and lactococcus lactis from farms that can induce t-helper cell (th ) differentiation and mediate protective effects against allergic airway inflammation. ege et al. reported that bacterial and fungal derivatives in house dust had a significant negative https://e-aair.org https://doi.org/ . /aair. . . . environment and asthma correlation with the incidence of asthma in exposed children. in one finland study, children who grew up in non-farm families showed decreased risk of asthma, but their family bacterial microbiota composition is similar to that of farm families, suggesting that the microbes are accountable for the farm environment-associated protection against development of asthma. in addition, high levels of endotoxin in farm and rural environments is related to a low prevalence of asthma and other allergic diseases. - endotoxin is a component of the cell wall of gram-negative bacteria and cyanobacteria. a previous study found that the protective effects of endotoxins might be related to the induction of a expression in lung epithelial cells. another investigation revealed that endotoxin levels were . times higher in traditional amish farms than in modern hutterite farms and may regulate innate immune pathways in children to prevent the development of asthma. in addition to the endotoxins, many pathogen-associated molecular patterns (pamps), such as extracellular polysaccharides, muramic acid, and glucans, have been shown to be closely associated with the protective effects of farms. https://e-aair.org https://doi.org/ . /aair. . . . environment and asthma table. studies on environmental exposure and allergies in different countries and regions region exposure factor sample size (no.) sample age (yr) main findings outcomes (onset of allergy) ref. asia-beijing, china farm livestock and farming behavior , – contact with farming and livestock has protective effects. reduced oceania-new zealand traffic at place of residence, drugs, and farm foodstuffs , – ; – truck traffic, antibiotics or paracetamol exposure during early life, were positively correlated with eczema. consumption of milk, seafood, eggs, and have a dog in home, were negatively correlated with eczema. reduced europe-turku, finland and neighboring regions indoor pet exposure during the perinatal period – fecal bifidobacterium longum counts in non-wheezing infants who were exposed to pets were significantly higher than those of wheezing infants who were not exposed to pets. reduced central europe-silesia, poland unpasteurized dairy products and activities related to livestock , > agriculture-related contact significantly decreased in silesia and the prevalence of allergies drastically increased within a short period of nine years. reduced northern europe livestock , mean age: subjects who grew up in livestock farms had a lower incidence of asthma ( %) compared to those who grew up in inner cities ( %). reduced asia-guangzhou, china farming environment and endotoxin levels , – early contact with crops and high levels of environmental endotoxins may protect children from the effects of asthma. reduced north america-usa endotoxin levels in homes – high endotoxin levels in traditional amish farms was a protective factor for asthma, and innate immunity also played an important role. reduced north america-canada farming environment , – the cumulative -year asthma incidence in children living in a farming environment was significantly lower than live in non- rural and rural non-farming environments. reduced south america-cordoba rural areas contact with livestock, such as dairy farms , – residency on dairy farms, including periodic livestock contact reduced allergic rhino-conjunctivitis. reduced africa-cape town and eastern cape province farming exposure, sunlight exposure, pet, antibiotic and probiotic exposure, antihelminth exposure, cigarette smoke and fossil fuel exposure. , – farm animal exposure but not unpasteurized milk is the strongest factor to against allergy. fermented milk produces has a significant effect in urban cohort but not in rural. reduced antibiotic exposure during maternal pregnancy or early life - and cesarean delivery , , can increase the prevalence of asthma in children by affecting the gut microbiota and other mechanisms. anti-parasitic therapy is also shown to be associated with increased prevalence of asthma. , it was also noted that the prevalence of eczema in children would increase when their mothers accepted deworming treatment during pregnancy, while anthelminthic in children had no significant effect on eczema, suggesting that the protective effect of allergies induced by parasite infection might begin in the mother's uterus. however, it is worthy of noting that some types of microorganisms may have opposite effects on development of asthma. for example, infection with bacterial species like streptococcus pneumoniae, haemophilus influenzae and moraxella catarrhalis and some virus often aggravates asthma. , environmental factors and immunological regulation of asthma there is still an insufficient understanding of the immunological relationship between the dramatic increase in allergy prevalence and environmental changes. however, in recent years, it has become clear that asthma is a heterogeneous disease in which both innate and acquired immunities are involved. common factors that promote or inhibit the onset of asthma include allergen exposure and microbial/parasitic infection, oxidative stress as well as environmental and microbial related metabolites. cells involved in this process include eosinophils, basophils, neutrophils, mast cells, macrophages, dendritic cells (dcs), epithelial cells, natural killer t (nkt) cells, natural helper cells, th cells, t-helper (th ) cells, group innate lymphoid cells (ilc ), b cells, regulatory t cells (tregs), and regulatory b cells (bregs). in figure, we illustrate the environmental factors and immunological regulatory mechanisms involved in the development and prevention of asthma. innate immune mechanisms during human evolution, our immune system is exposed to and interacted with symbiotic surrounding pathogens. the pamps on the gut or respiratory microbiota and parasitic worms might be recognized by the pattern recognition receptors on innate immune cells to initiate the immune responses. study by lauener et al. found that compared with the unexposed children, children with farm exposure had increased cd /toll-like receptor (tlr) expression levels and that exposure to a farm environment early in life correlates to important regulatory effects on asthma through tlrs. a subsequent study showed that maternal exposure to a microbe-rich environment before delivery resulted in increasing levels of genes expression for the receptors of the innate immune system (i.e. tlr , tlr , and cd ) in their children. the underlying mechanism for this phenomenon may be related to the intrauterine environment and/or epigenetic changes. tlrs are considered to be key systems in immune response regulation and epithelial cells and dcs play a vital role in linking innate immunity to adaptive immunity. exposure to appropriate doses of lipopolysaccharide (lps) weakens signaling pathways required for the production of allergy-related cytokine while retaining double-stranded rna/dc pathways, thereby activating dcs to promote production of th cytokines but not th . in addition, lps exposure will increase the synthesis of the zinc finger protein a , thereby inhibiting airway allergic responses to dermatophagoides pteronyssinus. a prospective study showed that maternal exposure to livestock before delivery resulted in significant increases in tlr and tlr in umbilical cord blood in children while the risk of atopic dermatitis was significantly reduced. stein et al. compared the peripheral https://e-aair.org https://doi.org/ . /aair. . . . environment and asthma blood cell compositions and the phenotypes of traditional farm and modern farm children, and observed that the percentage of eosinophils was decreased in the traditional farm children and that monocytes exhibited an inhibitory phenotype (lower levels of the human leukocyte antigens dr and hla-dr as well as higher levels of immunoglobulin-like transcript , ilt ). in addition, traditional farm dust extracts had significant protective effects in animal models of asthma. in myeloid differentiation primary response gene (myd )-deficient mice, this protective effect was drastically reduced. in myd - and toll/interleukin- receptor domain- containing adaptor inducing ifn-β (trif)-deficient mice, this protective effect was completely abolished. these molecules are located at the convergence of multiple immune signaling https://e-aair.org https://doi.org/ . /aair. . . . environment and asthma environmental exposure destructive factors protective factors microbial molecules, metabolites, toxicant air-lung axis normal airway bronchial epithelial cells smooth muscle cells fibroblasts ecm innate immune pathway & adaptive immune pathway gut-lung axis helpful microbe contact farm exposure gut microbiome & parasites harmful microbe contact vehicle exhaust industrial waste gases innate cells - dc - basophils - m macrophages - ilc il- il- il- il- il- il- ige il- tgf-β igg iga a th il- , il- , il- th b cell ige foxp , il- , tgf-β treg il- breg igg , iga b cell nkt ifn-γ allergic disease asthmatic airway goblet cell eosinophil figure. potential immunological mechanisms by which environmental factors affect allergic asthma. environmental microbial molecules and metabolites act on innate and adaptive immune pathways through the air-lung axis and/or the gut-lung axis to exert their immunoregulatory effects. harmful bacteria, viruses, and industrial waste gases, as well as automobile exhaust, can promote airway inflammation through induction of il- , il- , il- , and il- secretion by innate immune cells (e.g., ilc , airway epithelial cells, dcs, macrophages, and basophils) and adaptive immune cells (e.g., th cells, th cells, and b cells), resulting in airway mucosal damage, smooth muscle hyperplasia, and fibrotic changes. conversely, protective environmental factors, such as early life contact with helpful microorganisms, gut parasitic worm infection, gut microbiota, and farm exposure, can result in the production of il- , tgf-β, ifn-γ, iga, and igg by innate (toll-like receptors, airway epithelium, dcs, natural killer t cells), and adaptive immune cells (tregs, bregs, and b cells) to protect against allergic asthma. dc, dendritic cell; ilc , group innate lymphoid cells; ecm, extracellular matrix; il, interleukin; tgf-β, transformation and growth factor β; ig, immunoglobulin; ifn-γ, interferon-γ. pathways. these results suggest that environment microbial factors modulate immune response and induce immune suppression allergic asthma through innate immunity. it is known that, in addition to endotoxins, diverse microbial products in the environment are effective stimuli for innate immunity. , invariant nkt (inkt) cells are able to recognize glycolipid antigens and some pamps may directly activate inkt cells independently of tlrs. house dust extract (hde) could promote ova-induced airway inflammation in asthmatic mice through inkt cells, but the source of inkt-related antigens in hde has not yet been identified. however, a previous study found that influenza a infection in early life could protect adult mice from allergen-induced airway hyperreactivity (ahr), which was associated with a population of nkt cells, enriched for a cd -cd -(dn), t-box transcription factor expressed in t-cells (t-bet)-dependent, and interferon-γ (inf-γ)-secreting subset. chuang et al. proposed that early/neonatal infection or antigen stimulation could induce certain nkt subsets, such as cd hidn nkt cells, in the lung to produce ifn-γ but not il- , il- , or il- , demonstrating that this nkt subset could directly contact and inhibit cd + t cell proliferation, thereby blocking allergen-induced ahr. acquired immune mechanisms study by gereda et al. demonstrated that persistent environmental endotoxin exposure could induce th immune responses resulting in change in the th /th equilibrium, thereby reducing allergen sensitivity and protection of infants from allergen sensitization. asthmatic mice infected with hookworms or whipworms secrete anti-inflammatory protein- (aip- ) and protein p that inhibited costimulatory molecule expression in dcs and th response by binding to il- . , regulatory cd + t cells (tregs) play a vital role at the sensitization stage in the pathogenesis of allergic asthma and contribute to balancing t-cell differentiation and maturation. the functions of effector cd + t cells, including th , th , and th cells, can be regulated by tregs, which play an important role in the immunoregulation and suppression of asthma. many studies have found a correlation between environmental microbial exposure and treg differentiation/ development. mcguirk and mills and wilson et al. reported that bacteria and helminth infection could facilitate the development of tregs. schaub et al. found that maternal farm exposure could regulate the immune systems of children, increased the number and functions of tregs in umbilical-cord blood in newborns, decreased the proliferation of th lymphocytes, and reduced th cytokine levels. they also found that consumption of farm milk by mothers could increase forkhead box p (foxp ) demethylation in their children, thereby reducing their children's risk of asthma during childhood and adulthood. lluis et al. , reported that during early childhood, farm milk exposure increase foxp demethylation and treg cell counts through at least different signaling pathways. our group used a murine gut parasite, heligmosomoides polygyrus, to investigate the immunoregulation mechanism of gut parasites infection against allergic airway inflammation. it was observed that infection with h. polygyrus induced prominent response of il- -producing regulatory b cells (il- + bregs). these breg cells were able to promote differentiation and proliferation of il- -producing and foxp regulatory cd + t cells. cell transfer and depletion experiments demonstrated that il- +breg, il- +treg and foxp +treg cells participated in the parasite-induced immunosuppression of allergic airway inflammation. a birth cohort study showed that contact with livestock or cats during pregnancy could increase secretory immunoglobulin (siga) levels in breast milk and reduced atopic dermatitis in their children. many studies have shown that elevated siga and transforming growth factor-β (tgf-β) levels in breast milk have protective effects against asthma in children. , https://e-aair.org https://doi.org/ . /aair. . . . environment and asthma tgf-β secreted by treg cells is a key factor for survival of iga+ b cells. iga limits the responses of microorganisms to host mucosal immunity. these two effects jointly maintain symbiosis with microorganisms in the body, which may be an indirect protective mechanism against allergies. animal experiments have shown that soluble extracts from nematodes can increase il- and tgf-β levels, thereby inhibiting airway inflammation in asthma. future research directions epidemiological observations provide a mountain of evidence in proving the link between asthma and environmental microbial and parasite exposure. , - human fecal bacteria transplantation, probiotic therapy , and parasite immunotherapy , used in clinical trials, and mouse model research , have enhanced our understanding of the interconnection between environmental microbes, symbionts, - and asthma. the available data support the negative correlation between exposure to farms and rural environmental microorganisms in early life and that the incidence of allergic asthma and the specific exposure time window could be critical. , to determine the “best time window” for microbial exposure and to identify the protective microbiome, more cohort observations are needed in the future. although observational studies help understand the importance of environmental factors in the modulation of allergic asthma, more intervention studies are needed to further elucidate the molecular mechanisms involved in asthma pathogenesis. animal models are also powerful tolls for the mechanism study of asthma pathogenesis regulated by environmental microorganisms. furthermore, microbial immunotherapy has been tested as the potential means to protect against allergic asthma; therefor, in-depth studies are needed to characterize the derivatives of symbiotic or infective microorganisms for rational and safe use to control asthma and other allergic diseases. summary it is well established that environmental factors strongly influence the onset and development of asthma and other allergic diseases. the external microorganisms and internal microbiota, and their derivatives have been identified as major components that modulate the immune responses of humans and, in most cases, induce immunosuppression to prevent or alleviate asthma pathogenesis. research findings have revealed that multiple facets of the immune system are involved in the complex interaction between environmental factors and the prevention of asthma. further studies on the pathogenesis of allergic asthma, the nature of microorganism derivatives, and immune responses to the bacterial components may result in novel therapies to control and prevent occurrence of allergic asthma in human populations. acknowledgments this study was supported by the following projects: key project of national natural science foundation of china-guangdong joint fund (u ); special funding program for science and technology development of guangdong province (frontier and key technology innovation direction - major science and technology special project) ( b ); precision medicine research program of national key research and development project of china ( yfc ); key program of science & technology specific project of guangzhou science and innovation bureau ( ). https://e-aair.org https://doi.org/ . /aair. . . . environment and asthma references . strachan dp. hay fever, hygiene, and household size. bmj ; : - . pubmed | crossref . stein mm, hrusch cl, gozdz j, igartua c, pivniouk v, murray se, et al. innate immunity and asthma risk in amish and hutterite farm children. n engl j med ; : - . pubmed | crossref . robertson cf, roberts mf, kappers jh. asthma prevalence in melbourne schoolchildren: have we reached the peak? med j aust ; : - . pubmed | crossref . asher mi, montefort s, björkstén b, lai ck, strachan dp, weiland sk, et al. worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: isaac phases one and three repeat multicountry cross-sectional 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populations 	environmental and microbial exposure 	microbial exposure and asthma 	environmental factors and immunological regulation of asthma 	innate immune mechanisms 	acquired immune mechanisms 	future research directions 	summary 	references drivers of decoupling and recoupling of crop and livestock systems at farm and territorial scales hal id: hal- https://hal.inrae.fr/hal- submitted on jun hal is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. the documents may come from teaching and research institutions in france or abroad, or from public or private research centers. l’archive ouverte pluridisciplinaire hal, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. distributed under a creative commons attribution - noncommercial| . international license drivers of decoupling and recoupling of crop and livestock systems at farm and territorial scales rachael garrett, julie ryschawy, lindsay bell, owen cortner, joice ferreira, anna victoria n. garik, juliana gil, laurens klerkx, marc moraine, caitlin peterson, et al. to cite this version: rachael garrett, julie ryschawy, lindsay bell, owen cortner, joice ferreira, et al.. drivers of decoupling and recoupling of crop and livestock systems at farm and territorial scales. ecology and society, resilience alliance, , ( ), � . /es- - �. �hal- � https://hal.inrae.fr/hal- http://creativecommons.org/licenses/by-nc/ . / http://creativecommons.org/licenses/by-nc/ . / http://creativecommons.org/licenses/by-nc/ . / https://hal.archives-ouvertes.fr copyright © by the author(s). published here under license by the resilience alliance. garrett, r. d., j. ryschawy, l. w. bell, o. cortner, j. ferreira, a. v. n. garik, j. d. b. gil, l. klerkx, m. moraine, c. a. peterson, j. c. dos reis, and j. f. valentim. . drivers of decoupling and recoupling of crop and livestock systems at farm and territorial scales. ecology and society ( ): . https://doi.org/ . /es- - synthesis drivers of decoupling and recoupling of crop and livestock systems at farm and territorial scales rachael d. garrett , , julie ryschawy , lindsay w. bell , owen cortner , , joice ferreira , anna victoria n. garik , juliana d. b. gil , laurens klerkx , marc moraine , caitlin a. peterson , júlio césar dos reis and judson f. valentim abstract. crop and livestock production have become spatially decoupled in existing commercial agricultural regimes throughout the world. these segregated high input production systems contribute to some of the world’s most pressing sustainability challenges, including climate change, nutrient imbalances, water pollution, biodiversity decline, and increasingly precarious rural livelihoods. there is substantial evidence that by closing the loop in nutrient and energy cycles, recoupling crop and livestock systems at farm and territorial scales can help reduce the environmental externalities associated with conventional commercial farming without declines in profitability or yields. yet such “integrated” crop and livestock systems remain rare as a proportion of global agricultural area. based on an interdisciplinary workshop and additional literature review, we provide a comprehensive historical and international perspective on why integrated crop and livestock systems have declined in most regions and what conditions have fostered their persistence and reemergence in others. we also identify levers for encouraging the reemergence of integrated crop and livestock systems worldwide. we conclude that a major disruption of the current regime would be needed to foster crop-livestock reintegration, including a redesign of research programs, credit systems, payments for ecosystem services, insurance programs, and food safety regulations to focus on whole farm outcomes and the creation of a circular economy. an expansion of the number of integrated crop and livestock systems field trials and demonstrations and efforts to brand integrated crop and livestock systems as a form of sustainable agriculture through the development of eco-labels could also improve adoption, but would likely be unsuccessful at encouraging wide-scale change without a more radical transformation of the research and policy landscape. key words: innovation; integrated crop livestock systems; mixed farming systems; socio-technical transitions; sustainable agriculture; technology adoption introduction crop and livestock production is essential to human well-being, yet contributes to numerous global sustainability challenges. it is the largest land and fresh water user on the planet, largest source of ghg emissions and water pollution in many countries, and a major driver of global biodiversity loss (vitousek et al. , tilman et al. , foley et al. , henders et al. ). many of the undesirable social and ecological outcomes of agriculture can be traced to the decoupling of global crop and livestock systems and increased adoption of specialized, continuous cropping or livestock operations (naylor et al. ). the origins of these systems can, in turn, be traced to shifts in the political economy of global food systems, including an orientation toward capitalist logic, including surplus production, liberalization of trade, technological supremacy, and financialization (mcmichael ). specialized systems are characterized on the cropping side by frequent tillage and synthetic input use to reduce pest and weed pressure and manage nutrient availability, leading to erosion, pollution, and rising costs of production, and on the livestock side by waste accumulation, leading to high pollution and greenhouse gas emissions (pimentel et al. , tilman et al. , pimentel , chadwick et al. ). this specialization of agricultural management tends to make farmers more exposed to weather and market variability risks and creates an ecological and technological treadmill of production, such that each solution only creates additional challenges (ward ). this treadmill, though seemingly paradoxical from a farmer perspective, has clear benefits for multinational agribusiness firms by creating demand for their goods and services. innovative solutions are needed to tackle these broad ranging challenges at their root cause, i.e., considering sustainability and resilience throughout the conception, implementation, and management of food systems (and associated landscapes), instead of focusing on economic and production outcomes (therond et al. ). systems that increase the diversity of agricultural production activities in time and space have been proposed as a mechanism to improve sustainability and resilience. examples include permaculture, diversified cropping systems, and integrated crop, livestock, and forestry systems (agricultural systems that integrate crops, livestock, and/or trees on the same area, for example, via intercropping or rotations). here we focus on integrated crop and livestock systems (icls), systems that specifically recouple the crop and livestock dimensions of farming, as a potential solution. various case studies throughout the world have shown that icls in commercial agricultural environmental policy group, departments of environmental systems science and humanities, social, and political sciences, eth zürich, zürich, switzerland, department of earth and environment, boston university, ma, usa, agir, université de toulouse, inpt, inrae, france, csiro agriculture and food, toowoomba qld, australia, embrapa amazônia oriental, belém, brazil, plant production systems, wageningen university, wageningen, the netherlands, knowledge, technology and innovation group, wageningen university, the netherlands, umr innovation, inrae, cirad, montpellier supagro, montpellier, france, department of plant sciences, university of california, davis, ca, usa, embrapa agrossilvipastoril, sinop, mato grosso, brazil, agroforestry research center of acre, embrapa acre, rio branco, acre, brazil https://doi.org/ . /es- - mailto:rgarrett@ethz.ch mailto:rgarrett@ethz.ch mailto:julie.ryschawy@inra.fr mailto:julie.ryschawy@inra.fr mailto:lindsay.bell@csiro.au mailto:lindsay.bell@csiro.au mailto:ocortner@ethz.ch mailto:ocortner@ethz.ch mailto:joice.ferreira@embrapa.br mailto:joice.ferreira@embrapa.br mailto:avgarik@bu.edu mailto:avgarik@bu.edu mailto:judbgil@gmail.com mailto:judbgil@gmail.com mailto:judbgil@gmail.com mailto:laurens.klerkx@wur.nl mailto:laurens.klerkx@wur.nl mailto:marc.moraine@inra.fr mailto:marc.moraine@inra.fr mailto:capeterson@ucdavis.edu mailto:capeterson@ucdavis.edu mailto:julio.reis@embrapa.br mailto:julio.reis@embrapa.br mailto:judson.valentim@embrapa.br mailto:judson.valentim@embrapa.br ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / landscapes are associated with lower environmental externalities than conventional farming, without declines in profitability or yields (lemaire et al. , franzluebbers et al. , garrett et al. a, ryschawy et al. , dos reis et al. ). icls may also enhance ecosystem services both on- and off-farm, for example, erosion control and nutrient cycling, and increase farm productivity as a function of all inputs, i.e., total factor productivity (coomes et al. ). despite substantial research on their environmental and economic performance, surprisingly little is known about the prevalence and trajectories of icls globally. why have icls been retired in some regions, and persisted or reemerged in others (garrett et al. a)? why have specialized (nonintegrated) systems come to dominate most commercial agricultural production despite their numerous negative impacts? past studies have examined the importance of mixed crop and livestock systems in smallholder subsistence production, largely in sub- saharan africa and asia (thornton et al. , thornton and herrero ). however, the drivers for decoupling and reintegration of crop and livestock in commercial systems, e.g., farms that primarily sell, rather than consume their own production, particularly in north and south america, europe, and oceania, remain poorly understood (garrett et al. a). given the complex distal and multilevel interactions influencing agricultural systems (hull and liu , garrett and rueda ), an international and historical perspective on the drivers of crop and livestock decoupling and conditions for their persistence and reemergence is urgently needed. based on a workshop involving international icls scientists and practitioners we analyzed secondary data and case studies to draw general conclusions on the factors influencing icls prevalence and trajectories within commercial agricultural systems. we ask the following: ( ) what are current trends in icls adoption or retirement? ( ) what are the causes of icls persistence, adoption, or retirement? ( ) what policy levers could contribute to greater adoption of icls? we use a multilevel perspective of system innovations, i.e., a means of explaining how technological transitions come about in a given multilevel context, (geels ) to understand current icls prevalence and trajectories. types of icls understanding variation in crop and livestock system types across gradients of integration is useful to clarify the expected benefits, trade-offs, and barriers to adoption. previous work has classified integrated systems according to their reliance on inputs, capital and labor (schiere et al. ), space, time, ownership, and management (bell and moore ), and the level of interactions between crops, livestock, and animals (moraine et al. ). building on these typologies, we define systems with the lowest level of integration as segregated high input agriculture, or segregated-hia (fig. ), where crop and livestock units interact primarily through the market. segregated hia crop systems often produce very high yields, but rely on high levels of synthetic fertilizer and pesticides. segregated hia livestock systems also rely heavily on off-farm feed sources and aggregate high densities of livestock on a small land area, leading to the production of manure volumes that tend to exceed the assimilative capacity of the land that they occupy. fig. . types of integrated systems according to the level of integration between livestock and crops and the levels of inputs used. crop systems are indicated by (c), while livestock systems are disaggregated into animals (a) and pastures (p). the size of the arrows represents differences in volumes of inputs (e.g., feed, fertilizer, pesticides, etc.), outputs (e.g., food, biomass, or nutrients with commercial value), and losses (nutrient losses, greenhouse gases emissions). the size of the bubbles is meant to represent productivity of the crop, pasture, and animal components relative to inputs and outputs. in segregated-hia (high input agriculture) and semi-icls (integrated crop and livestock systems) animals are present, but there is no pasture component (they are fed harvested forages, crops, and crop residues). traditional-icls involve grazing on pastures, but often have low levels of external inputs and/or poor management, leading to an underutilization of potential synergies between systems. new-icls (on the right) differ from traditional-icls, segregated-hia, and semi-icls (on the left) in that they seek to maintain high outputs while reducing external inputs and increasing input efficiency through synergies between crop and livestock systems, thereby maintaining or increasing economic competitiveness. territorial icls occur at the landscape level, i.e., through local exchanges occurring between neighboring farms (lower right side). given greater synergies in space and time between functional agrobiodiversity (crops, pastures, and animals), sustainability and resilience is expected to increase as one moves from segregated-hia practices to semi-icl to territorial-icl to new-icl systems. at the other end of the integration spectrum, crops and livestock can be highly coupled through crop-pasture rotation and in situ animal grazing, which increases nutrient availability for crops and can improve soil structure, if kept at low to moderate grazing intensities (schiere et al. , garrett et al. a). we refer to all of these systems as icls, distinguishing between traditional-, new-, and territorial-icls. traditional-icls, which have been around for centuries, have no or very low external resource inputs. they rely almost entirely on on-farm resources to manage nutrient supply and weed pressure, rather than external inputs of fertilizers and chemicals. new-icls are a form of “retro-innovation” https://www.ecologyandsociety.org/vol /iss /art / ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / (stuiver , sixt et al. ), combining traditional-icls practices with modern advances to maximize the economic and ecological benefits linked to greater levels of integration and support agricultural regime change. territorial-icls are direct exchanges of crop and livestock products among farmers. the system of organization linking these farmers often occurs at the landscape level, e.g., a watershed, community, island, or complementary plain and mountain areas. somewhere in the middle of segregated-hia and icls are semi-icls, systems that have only minimal levels of integration. crops grown on the farm are harvested and may be used to supplement livestock on another part of the farm, rather than directly grazed, and/or excess manure is collected and distributed as fertilizer, rather than directly deposited by animals. the logic of these systems is (i) to keep costs down by maintaining economies of scale via specialized land use and producing one’s own feed supply for livestock, and (ii) like all integrated systems, to diversify income streams. the farms participating in territorial-icls systems may be semi-icls or segregated-hia systems (moraine et al. ). a common conclusion of several existing farm system typologies (schiere et al. , bell and moore , moraine et al. ) is that more integrated farms are likely to be more sustainable and resilient because of synergies in space and time between functional agrobiodiversity, i.e., crops, pastures, and animals (stuiver , sixt et al. ; fig. ). methods we used a multilevel perspective (geels ) to understand the prevalence and trajectory of different types of icls within countries, following gaitán-cremaschi et al. ( ) who used it as a food system diagnostic and classification tool. this perspective focuses on three levels to explain how socio-technical transitions occur and how social, technological, and institutional aspects coevolve: (i) landscapes: the external factors influencing the whole agricultural system, such as socio-technical trends (e.g., globalization) or climate change (e.g., reductions or changes in the distribution of rainfall) that may put pressure on agricultural regimes and create windows of opportunity for niches (wigboldus et al. ), (ii) agricultural regimes: the dominant modes of production, sourcing, value accumulation, and consumption in agricultural supply chains, evolving product markets and market demands, the focus of the policy setting, and scientific and technological paradigms (mcmichael , gaitán-cremaschi et al. ), and (iii) niches: networks in which novel systems are developed that propose an alternative to the current agricultural regime that may come both from deviant or change-oriented actors within the regime and from grassroots innovation movements (tittonell et al. ). using this perspective, we can assess to what extent icls may originate from within existing agricultural regimes or as niche systems outside existing regimes. data from agricultural censuses (outlined in appendix ) were synthesized to describe the status of icls in each region. the definitions used to identify icls farms in these censuses vary across countries and icls data were not available for all regions (appendix ). we excluded forestry from our analysis because of limited data and research on systems that also integrate trees. a review of the existing literature and a consultative process with icls and agricultural innovation experts within each of the major focal regions were used to trace the reasons for the relative abundance and trajectory (decline, stability, or resurgence) of icls across regions. identification of factors influencing icls adoption and retirement began with a two-day workshop with international icls scientists and practitioners and agricultural innovation experts from australia, brazil, europe, and the united states as part of a national science foundation “science, engineering, and education for sustainability” grant (# ). the meeting took place in august in belém, brazil, through a partnership between boston university and embrapa (more details included in appendix ). this workshop was supplemented with an extensive literature review of both the limited icls literature and broader research on farm structural changes, farm diversity, and sustainable agriculture. more details on our qualitative characterization of these conditions can be found in appendix . explaining icls abundance and trajectories in commercial agriculture status of icls in regions where data are available icls were once highly abundant in north america, but have been decreasing as a proportion of both agricultural area and farms since at least (fig. a-b). most farms are now best described as segregated-hia. from to , u.s. farms went from producing (on average) five agricultural commodities per farm to just a single commodity per farm (dimitri et al. ). nevertheless, icls remain moderately abundant in north america, compared to other commercial production regions (fig. c). canada has one of the highest levels of mixed crop and livestock production in terms of both area ( %) and farms ( %) across the commercial production regions for which there are data. in the u.s., traditional-icls have been maintained in amish country and various forms of new-icls have been documented at low levels in new england, the great plains, and the high plains, including grazing of cover crops and crop stubble, sometimes in association with permaculture, organic, and biodynamic farms (warren , cunfer , allen et al. , lovell et al. , faust et al. ). levels of integration did not change as precipitously in western europe after because they were already quite low (fig. b). traditional-icls in europe have been mostly maintained in less- favored areas (regions with inferior market access or soil and climatic conditions, such as mountainous regions), particularly in association with dairy production (entz et al. , veysset et al. ). segregated-hia have become the dominant form of agriculture in most other regions. several countries in central, e.g., poland, slovakia, and baltic europe, e.g., lithuania, latvia, show a slightly different story for the years that data are available. in poland and slovakia, icls levels remained fairly steady between and , but higher than western europe, at around % of the farms. in lithuania and latvia, icls levels declined quickly after , but are still more than double most western european countries. in contrast to other regions, australia, brazil, uruguay, and most recently new zealand, have all experienced a combination of resurgence and persistence in icls. in australia, farms maintaining a mix of crops and livestock that are integrated to varying degrees remain common, covering roughly % of the agricultural area (roughly million hectares), excluding the https://www.ecologyandsociety.org/vol /iss /art / ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / fig. . integrated crop and livestock systems (icls) as percentage of agricultural area or farms by country. nz = new zealand, uk = united kingdom. us= united states. countries for which there at least years of data for icls area were included only in figure a and not repeated in figure b even if they also contained data on farm numbers. in figure c “the latest available year” is as follows: for argentina, for brazil, for iceland, montenegro, and switzerland, for uruguay, for us and nz, for japan and saudia arabia, for canada, for australia, and for all other countries. see table a. for icls definitions and calculations and years of data availability by country. extensive pastoral grazing regions of the interior (fig. a). these farms include both semi- and new-icls with trends of increasing intensity and specialization to cropping (garrett et al. a), but still % of the cropped area on these farms involve new-icls practices. in new zealand, there are high levels of integration of sheep and beef cattle in grain and viticulture cropping regions. as of , % of grain cropping area and % of grain farms pursued integrated grain-beef or grain-sheep production (statistics new zealand ). yet, levels of integration remain low as a proportion of the total agricultural land area in new zealand (only , hectares) because there are still > , pastoral farms, comprising > million hectares that pursue specialized beef and/or sheep production in the hilly grasslands (statistics new zealand ). in brazil, many smaller scale family farms have maintained traditional-icls, while new-icls is increasing on larger, commercial farms in the form of integrated grain-beef production (balbino et al. , carvalho et al. , gil et al. , vicente ). between and , the area occupied by icls increased from . to . million hectares (embrapa ). in uruguay, icls remains common in all forms of agriculture, including rice, beef cattle, and dairy cattle production, totaling . million hectares in . very little data on icls was available for asia or the middle east. icls occupies < % of the agricultural area in japan and < % of the farms in saudi arabia are icls (fig. c). in japan, because of the limited agricultural area, pastures have all but disappeared and most livestock are produced in confinement systems (obara et al. ). in saudi arabia growing consumption of meat has made the country a growing livestock producer, but a lack of domestic water resources has led to a reliance on confinement systems and large imports of feed grains and live cattle for finishing (fao ). nevertheless, it is likely that icls remain common in small-scale commercial production systems throughout much of asia (devendra and thomas ). in summary, while once a “regime practice” (pre- ), icls has become a niche in many commercial agricultural systems in much of the global north, occupying < % of the agricultural area https://www.ecologyandsociety.org/vol /iss /art / ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / and number of farms in most of northern, central, and western europe, and the united states (fig. c). in brazil and central and baltic europe roughly – % of all farms are integrated, while % of all farms in canada, – % of nonpastoral farms in australia and new zealand, and – % of all farms in argentina and uruguay are integrated. these trends are summarized in figure , which illustrates a general global timeline for traditional-icls retirement in most countries since at least , followed by reemergence of new- icls in some regions after from various starting points. in regions where traditional-icls were retired, new-icls has emerged from specialized-hia or semi-icls systems, e.g., new zealand, australia. in other regions new-icls have evolved from the persistence of traditional-icls, e.g., brazil. the region- specific trajectories and transitions of icls adoption and retirement, and their associated causes, are discussed in more detail below. fig. . trajectories of agricultural decoupling or integration of crop and livestock components. historically, most traditional agricultural systems included both crops and livestock, i.e., traditional-icls (integrated crop and livestock systems). since , integrated systems declined in favor of specialized-hia (high input agriculture) cropping or livestock production and semi-icls in commercial agricultural systems (a), though in some regions traditional-icls have persisted (b). new-icls have emerged as an alternative to traditional-icls, segregated- hia, and semi-icls since (c). yet, in most regions in the global north they remain rare because of a lack of landscape pull factors (unsupportive global market, low remuneration of labor, habits of specialization, and policies leading to political and economic lock-in), despite some niche push factors (successful new-icls development, consumer awareness for environmental aspects, quality-labeled products) (d). decline of icls because of large landscape changes the nearly universal decline of integrated crop-livestock systems in commercial agriculture is linked to several major structural landscape changes that occurred from to . major influences include globalization, industrial development, and the financialization of agriculture, which influenced the market integration of farms, relative input prices, and demands to compete on the world market (barbieri et al. , mcmichael , ryschawy et al. ). liberalization of trade throughout the latter half of the th century forced farms to compete globally, increasing incentives to specialize to enhance economies of scale and adopt technologies that reduced costs or increased yields (entz et al. , vicente ). to gain global market share and protect farmers from international competition, many countries developed agricultural subsidy policies, such as the eu common agricultural policy in and the u.s. farm bill of - (garrett et al. b). these policies tended to focus on commodity crops, thereby increasing the profitability of specialized-hia cropping versus more diversified cropping systems or icls (garrett et al. b). given the increasing economic risks associated with the high costs and low diversity of these specialized production systems, some regions (including the u.s. with the farm bill) also developed insurance systems to protect farmers from climate and market fluctuations. large-scale low-cost nitrogen fertilizer production further reduced farmers’ reliance on livestock as a fertilizer source, allowing segregated- hia cropland area to increase (smil ). similarly, the growing availability of labor-saving farm equipment and increasing costs of labor from demographic change and structural transformations in the economy (toward manufacturing and services) increased farmers’ incentives to adopt more specialized agricultural systems that could employ mechanization to increase returns to labor. once a competitive advantage for an individual crop or livestock product was established, agglomeration economies, i.e., clusters of related agribusinesses, developed, leading to economies of scale and expansion of area devoted to individual crop or livestock products (sulc and tracy , garrett et al. ). in the context of globalization, multinational agribusiness companies assumed more power globally and in domestic politics (kearney ). such actors lobbied for continued market liberalization for a handful of crops that could be produced cheaply but increased in value via processing activities (mcmichael ). a single product focus in agriculture was further cemented by the changes in the orientation of most agricultural research agencies and grant programs toward global competitiveness and biotechnology, rather than holistic farm outcomes, such as health, efficiency, and sustainability (balbino et al. , bonaudo et al. ). specialization was compounded by a more incremental technology transfer model of agricultural innovation for existing systems, rather than a reflexive, adaptive management approach to optimize farm and landscape ecosystems services (moore ). all of these changes created path dependencies toward specialization and barriers to diversification. beginning in the s, many countries reduced producer supports, e.g., subsidies, price supports, etc., in compliance with changes in world trade regulations (imf ). simultaneously, many countries, including the u.s., new zealand, and the eu, established increasing environmental and soil conservation programs, e.g., u.s. soil conservation service (later nrcs) in ; european society for soil conservation in ; nz landcare trust in ; nz soil conservation and rivers control act in . reductions in producer supports and increasing attention to environmental outcomes should have promoted greater input efficiency and diversification as alternative means to reduce costs, risk, and environmental impact (bradshaw , garrett et al. b). however, nonlabor input costs, e.g., fertilizers, pesticides, have remained relatively low across most countries as the environmental impacts of these products have not yet been captured by markets (peyraud et al. ). consequently, pressures to reduce economic risk and increase household income have tended to result in diversification of income via off-farm income opportunities, rather than on-farm production diversity and/or input reduction (lobao and meyer , bradshaw ). https://www.ecologyandsociety.org/vol /iss /art / ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / persistence of niches of traditional-icls within the dominant agricultural regime traditional-icls persistence is often linked to cultural and economic factors (table ). such systems could be a source of inspiration or “retro-innovation” to recreate connections between crops, grasslands, and animals within new-icls. as a potentially self-sufficient livelihood, farming has long been driven by a desire to pursue an independent lifestyle, free from dependence on markets and governments (van der ploeg ). traditional-icls practices enable this self-sufficiency and autonomy by producing all of the necessary inputs to production, as well as a diversity of food sources (ryschawy et al. , coquil et al. ). in the u.s., old order amish farmers continue to pursue traditional-icls because of social controls on the introduction of new technologies, e.g., synthetic inputs or heavy machinery, and a refusal of government assistance, such as subsidized insurance (stinner et al. ). similar trends can be found for anabaptist farming communities throughout the world. in some cases, self-sufficiency has been forced upon communities because of economic conditions. even within commercial agricultural production regions, farmers with fewer assets and less access to government resources or markets maintain traditional-icls as a closed loop farming system to provide sufficient food for household consumption and avoid input purchases. livestock, in particular, ensure a source of fertilization for crop production and serve as a savings account for times of crisis (herrero et al. , garrett et al. c). biophysical conditions can reinforce cultural tendencies. traditional-icls are often maintained in less-favored areas to overcome the resource constraints that inhibit specialization (schiere et al. , ryschawy et al. ). in europe and the u. s., farmers operating in more marginal areas have maintained traditional-icls often because they have no other choice (ryschawy et al. ). in south asia, water scarcity from climate change is creating pressure to transition from rice and wheat systems to icls with cattle or buffalo (herrero et al. ). at a smaller scale, higher within-farm heterogeneity, e.g., the presence of steep slopes, poorly drained soils, and wetlands, etc., supports the use of traditional-icls to take advantage of the entire landscape and manage variability (ruben and pender ). this heterogeneity has also supported the reemergence of new-icls in other regions, as noted below. opportunities for new-icls emergence within the dominant agricultural regime rising environmental awareness, changes in agricultural policy, and changes in input and product markets have created opportunities for new-icls to increase globally (bell and moore , gil et al. , garrett et al. b, cortner et al. ). these opportunities could be considered as induced from grassroots niches. they are constrained by the relative prevalence of livestock in a region, local biophysical conditions (e.g., water scarcity, topography), the profitability of monoculture systems during high price periods, and cultural preferences (bonaudo et al. , garrett et al. b; table ). an additional stimulus for new-icls adoption comes from the rise of peasant movements calling for self-sufficiency and autonomy in reaction to globalization, such as la via campesina and fédération associative pour le développement de l'emploi agricole et rural (fedear; dumont et al. ). in seeking self-sufficiency for cost-savings and autonomy, these social movements often promote more holistic and agro-ecological farm-management approaches that reduce reliance on external inputs, including types of new-icls (bonaudo et al. , dumont et al. ). other opportunities are coming from the regime itself, promoted by institutionalized actors. in the eu, the second pillar of the common agriculture policy (cap) has been supporting agro- environmental practices, in particular, the maintenance of grasslands and seminatural areas. the second pillar has thus encouraged the persistence of grazing systems in less favored areas and the emergence of new-icls. since , % of the eu budget for cap direct subsidies has been allocated to environmental-friendly practices such as crop diversification through the greening of the cap (european commission a), but this subsidy does not take into account the level of integration between crops and animals. in france, the / initiative, an effort to increase soil carbon stocks by . % per year (https:// www. p .org/) and the food and agriculture modernization law (bellon and ollivier ) both support more multifunctional practices and agroecology, in particular the improvement of soil quality through legume-based diversified rotations and reintegration of livestock into cropping systems. in european agricultural research agencies, new participatory design efforts involving farmers and advisers are also attempting to foster greener agriculture (martin et al. ). in the netherlands there has been a movement toward “circular agriculture,” emphasizing the use of residuals of agricultural biomass and food processing within the food system to reduce dependency on chemical fertilizers and remote livestock feeds (thigssen ). feeding nonedible crop by-products to animals has also been proposed in other places as an option to limit feed-food competition (van zanten et al. ). in brazil, new-icls is being promoted by the government’s low carbon agriculture (abc) plan and increasing restrictions on native vegetation clearing that are linked to brazil’s broader international commitment to reduce national greenhouse gas emissions (gil et al. ). the abc program provides subsidized loans for the adoption of icls to combat soil degradation and recuperate degraded pastures, thereby improving animal performance and reducing the amount of time it takes to get cattle to slaughter weight and thus emissions per unit of food produced (observatorio abc ). restrictions on forest clearing have incentivized the adoption of icls to increase productivity on the existing land area (garrett et al. , cortner et al. ). in new zealand, beef and sheep are highly abundant relative to cropping because of biophysical limitations in the landscape, such as unfavorable soils with low water holding capacity. yet, these same soil and water constraints also limit forage production, creating incentives for beef and sheep farmers to seek out additional grazing areas to supplement their livestock, i.e., rows between grape vines or stubble of cover and forage crops. changes in nutrient emission policies and gradual recognition of the economic and environmental benefits of such practices in improving nutrient management have helped foster this integration (niles et al. ). in australia, various research and adoption programs, e.g., grain and graze, have aimed to increase integration of beef and sheep https://www.ecologyandsociety.org/vol /iss /art / https://www. p .org/ https://www. p .org/ ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / table . local context factors supporting or inhibiting for persistence of traditional-icls (integrated crop and livestock systems) or reemergence of new-icls across different countries (justifications for categories and rankings provided in appendix ). traditional-icls persistence new-icls reemergence cases amish farms in united states family farms in brazil pastures and croplands in brazil nonpastoral areas in australia nonpastoral areas in new zealand agro-ecological farms france carbon farming in united states main objectives maintain autonomy respect for community values and norms control or avoid new technology reduce cost and economic risk household consumption reduce deforestation increase productivity and profits reduce costs maintain social status resilience reduce costs increase profits reduce pollution reduce costs self-sufficiency at farm or territorial levels reduce costs and variability of inputs cost reduce pollution climate resilience reduce costs reduce pollution factors supporting icls pull factors coming from the landscape, i.e., landscape opportunities preference for autonomy ++++ +++ ++ +++ +++ +++ ++ targeted by climate or pollution mitigation policy + ++ +++ ++ ++ +++ ++ (occurs at state level) highly variable topography, climate, or soil ++ ++ ++ +++ ++ ++ + agricultural research focused on sustainability and climate + + +++ ++ ++ ++ ++ push factors coming from the niches, i.e., niche opportunities industry and civil society initiatives promoting icls +++ + ++ ++ ++ + ++ differentiated value chains and eco- or social labels ++ + + + ++ ++ +++ factors inhibiting icls absence of pull factors coming from the landscape, i.e., landscape opportunities lack of supply chain infrastructure or marketing opportunities ++ +++ +++ ++ + +++ ++ high cost of labor + + ++ +++ +++ +++ ++ high prices of synthetic inputs and fuel or feed + + ++ +++ ++ +++ +++ protectionist policies (e.g., insurance, subsidies) + + + + + +++ +++ food safety regulations restricting integration ++ + + + + ++ +++ absence of push factors coming from the niches, i.e., niche opportunities lack of icls farm trials or demonstrations + ++ ++ ++ +++ +++ +++ lack of farmer networks to share knowledge on icls + + ++ ++ + ++ +++ average inhibiting factor score ++ ++ +++ +++ +++ +++ ++ average supporting factor score ++ + ++ ++ ++ +++ +++ note: low = +, moderate = ++, high = +++, and very high = ++++. https://www.ecologyandsociety.org/vol /iss /art / ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / with crop production by improving the profits, reducing environmental impacts, and building social capital in icls via the adoption of best management practices (price and hacker ). a major objective of this program was to improve “whole farm knowledge” and promote researcher-to-farmer knowledge networks via annual research and extension forums (hacker et al. ). the program is credited with the successful adoption of new-icls practices, e.g., dual-purpose crops, improved forage systems, and pasture rotations (price and hacker ). as with new zealand, landscape heterogeneity has also been a major impetus for adoption of new-icls, leading to the incorporation of livestock areas to take advantage of topographical and soil features that are not suitable for cropping (lacoste et al. ). given the technical, labor, and organizational barriers to the adoption of new-icls on individual farms, as well as regime- induced institutional barriers such as limited marketing channels (ipes food , martin et al. ), groups of farmers are now developing localized exchanges of crops and grazing services, i.e., territorial-icls (meynard et al. , magrini et al. ). niches of this type have been observed in france (ryschawy et al. ), the netherlands, finland (hacker et al. ), the u.s., and new zealand. for example, wine grape growers in the u.s. contract sheep grazers to reduce their mowing and herbicide usage (j. ryschawy, personal observation), while sheep producers in new zealand pay wine-grape growers to graze their herd in the vineyard to manage forage scarcity (niles et al. ). regime factors inhibiting new-icls emergence many of the same regime factors that encouraged traditional- icls retirement have restricted farmers’ incentives and ability to adopt new-icls (table ). price and income supports for specialized agricultural production, biofuels mandates for agricultural crops, and subsidized insurance programs that shield farmers from ecological, climate, and market risks all disincentivize new-icls adoption (o'donoghue et al. , de gorter et al. , lark et al. ). food safety restrictions prohibiting animals in cropping areas make it illegal to practice certain forms of icls (garrett et al. b). likewise, segregated-hia systems are favored by easy access to synthetic nitrogen in most developed regions and market failures (environmental costs are not accounted for) that keep production costs artificially low. additional regime factors arising from decades of retirement including knowledge gaps, supply chain lock-in, and habits of specialization further constrain new-icls adoption (table ). icls are often perceived to have lower profitability and involve higher (and more skilled) labor requirements and upfront costs than specialized systems (cortner et al. ), even though returns on investment have been shown to be faster and higher than investments in specialized systems (dos reis et al. ). in more remote agricultural regions, both perceived and actual gaps for marketing diversified products limit icls adoption (gil et al. , eip-agri , ryschawy et al. , cortner et al. ). many farmers have reported that they lack technical knowledge or experience to adopt new-icls appropriate to their context (allen et al. , sulc and franzluebbers , eip-agri ). the exchange or hiring of labor is challenged by a lack of cross- training of individuals with both livestock grazing and crop expertise (garnett et al. ). furthermore, the specialized nature of many research and advisory systems centered around individual crop or livestock commodities fails to provide adequate extension services to train farmers for new-icls management. similarly, regulations, credit mechanisms, and supply-chains are often focused on single commodities, making financing and marketing of icls challenging (gil et al. , cortner et al. ), though direct marketing can help overcome this challenge for some products, such as fresh produce, meat, and wine (e.g., vidal ). in terms of culture, lifestyle preferences for either crop or livestock management based on family experience and seasonal labor requirements and links between personal identity and current farming systems, may limit adoption among certain individuals (garrett et al. c, cortner et al. ). higher perceived managerial intensity reduces farmers’ incentives to cooperate for exchanges to achieve territorial-icls (eip- agri ). planning, operational and monitoring costs limit the feasibility of such exchanges around the world (asai et al. ). organizing exchanges requires trust among partners, overcoming legal constraints (e.g., taxes on transactions, manure transportation norms), and organizing appropriate governance to face uncertainties (e.g., variability in feed quality or quantity, animal management in partners’ fields). the identification of cost-benefit trade-offs between individual and collective levels is of primary importance and requires tailored support from research or extension services (ryschawy et al. ). levers for reintegration of crop and livestock systems worldwide levers for furthering the adoption of various types of innovative icls include both pull and push factors that could disrupt the existing agricultural regime and destabilize locked-in specialized- hia practices and associated value chains (fig. ). pull factors are conditions and changes in the landscape and regime that create a need for innovation (top-down processes). push factors are bottom-up processes that emerge from the niche context, i.e., local knowledge, social, and institutional changes, to supply and support new technologies and can address top-down needs. push factors alone are often not enough to disrupt the socio-technical regime and bring about practice change, but are often critical in testing and refining technologies to be ready for adoption should other drivers sufficiently favor a shift in practice (turnheim and geels ). system transformation is most likely when policy mixes include both push and pull levers that reinforce each other, and when promotion of a new system is coupled with creative disruption of the old regime (kivimaa and kern ). pull factors for icls existing agricultural research paradigms often prioritize yields over whole farm outcomes, such as economic risk reduction, resilience, production diversity, cost minimization, and input efficiency. this yield-centric approach downplays objectives that are important to farmers and society and ignores externalities. participatory design would allow the exchange of knowledge on technical, social, and policy issues to regionally appropriate models of icls. such interactive and multiactor design approaches are currently favored by the european commission under the european partnership innovation through grants for projects that are supposed to involve multiple stakeholders in a local area over several years (european commission b). research programs should be redesigned to focus more on whole farm outcomes and participatory design (meynard et al. ). https://www.ecologyandsociety.org/vol /iss /art / ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / fig. . pull and push factors that could disrupt or reform the existing agricultural regime and promote reemergence of icls (integrated crop and livestock systems). the size of the arrows reflects their relative individual strength. for example, some push factors, such as field trials, are individually small in influence, but collectively gain force when embedded into farmer knowledge systems to help promote adoption of new-icls. hia, high input agriculture. credit mechanisms that only cover planting costs or the purchase of new machinery and stock may be inadequate for transforming farming practices from specialized systems to new-icls because of inadequate funding levels, high interest rates, and short-term payback periods (garrett et al. ). existing funding lines prioritize annual profit outcomes over risk reduction or have inadequate data about the financial returns for icls. additionally, new credit sources are often inadequately linked to the provision of technical assistance to promote adoption. this assistance is necessary since the relevant knowledge and skills for integration are often absent after decades of specialization (price and hacker ). these shortcomings have been documented even in brazil, where credit programs are targeted at improving new-icls adoption (gil et al. , observatorio abc , cortner et al. ). credit systems should be adjusted to take into account a longer term view of whole farm outcomes with system transformation, including a reduction of economic risk and negative social externalities relative to private returns. credit lines should be developed to encourage territorial-icls through collective subsidies. profit margin and yield insurance programs serve an important social role in reducing farmers’ vulnerability to market and weather variability. however, in regions such as the u.s., farm insurance programs offer payouts for only a limited number of crops and do not base premiums on diversification or management changes that reduce risk of yield or income variability (nsac ). in contrast, in australia and new zealand, farmers are exposed to high levels of climate and commodity price variability, but do not have the benefit of a federal crop insurance system or other substantial market protections. thus, the income diversification and reduced input costs that icls systems provide serve as a critical risk mitigation strategy for farmers. in the european union, crop premiums have enabled productivity increases, but in some cases led to perverse incentives, e.g., crops could be planted but left unharvested in the interest of earning a premium. crop insurance programs should be improved to better take into account farm diversity and risk profiles and or eliminated to reduce perverse outcomes. in some regions, regulations inhibit the trade and transportation of crop by-products for use as animal feed, the use of animal waste for fertilization, or the presence of animals grazing in areas used to grow crops (garrett et al. b). even where integration is legal, the fear of liability discourages experimentation with most types of icls (garrett et al. b). in the eu, the use of food waste as livestock feed is prohibited. this is a legacy of foot-and- mouth disease outbreaks, which were thought to be caused by feeding pigs uncooked food waste, even though cooked food waste has been proven to be a safe and efficient source of feed (gaudré et al. , zu ermgassen et al. , dumont et al. ). in the u.s., food safety regulations prohibit the use of raw manure or the presence/grazing of animals in areas used to produce foods that are consumed directly by humans within – days of the harvest (fda ). although the purpose of these regulations is to protect human health, they conflate practices with different https://www.ecologyandsociety.org/vol /iss /art / ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / risks, e.g., using massive quantities of raw chicken manure on spinach fields is likely riskier than grazing sheep in the understories of fruit trees or feeding pigs with treated biscuit waste. regulations that inhibit a circular economy should be adjusted to allow increased use of farm outputs, while respecting health and environment issues. finally, current agricultural transitions are impeded by deeply embedded habits of specialization, farm income, or firm profit maximization priorities, and a lack of awareness about whole farm management and the environmental impacts of existing production systems. as farming systems experience ownership transitions, facilitating land purchases by young farmers could help modify the habits, priorities, and knowledge gaps of current farming cultures. push factors successful practices to combine crops and livestock in a sustainable way have been studied by researchers in case studies and experimental plots and farms (bell and moore , sulc and franzluebbers ), yet there is limited information on exactly which locations and practices (types of crops and animals and levels of integration) may be more suitable for new-icls. greater qualitative and quantitative assessment of both successful and unsuccessful examples of new-icls on actual farms could provide improved understanding of the conditions underpinning their viability. demonstration plots on existing farms and field days would also be a powerful tool for motivating adoption. for example, in brazil, uptake of new-icls was substantially higher near locations of icls experiments (gil et al. ). technical assistance at the field scale must be coupled with better training of rural extension workers, farmers, and farm workers. agricultural research organizations should increase gatherings, organization and knowledge exchange on successful farms that have already adopted icls, and work jointly with farmers to develop and disseminate successful forms of new-icls, for example via demonstration plots and field days. farmers’ networks have already proved to be influential at altering perceptions of specific technologies (prokopy et al. , lubell et al. ). still, development of multiactor and cross-sectoral groups could be a further step in supporting innovation niches (pigford et al. ). for instance, recent participatory research has allowed farmers to build scenarios of territorial-icls in two areas of southwestern france (moraine et al. , ryschawy et al. ). crop farmers and livestock farmers wanted to decrease their dependence on external inputs by buying feed and fertilizer from their neighbors. win-win economic, environmental, and social scenarios were identified through collective organization and trust building (asai et al. ). power asymmetries between participants were addressed through the use of tools such as roundtables and the comanagement of the project between a farmer, an adviser, and a researcher, although it remains a significant challenge for such exercises. to improve the success of participative design approaches, further work on farmers’ motivations to manage new-icls or territorial-icls should be developed (ryschawy et al. ). connecting farmers and stakeholders, e.g., cooperative leaders, policy makers, and consumers, could also have the additional benefit of improving lobbying for policies and regulations that favor new-icls or territorial-icls, as exemplified by lobbying for grass-fed beef (soil carbon cowboys), agroforestry (arbores et paysages), and conservation agriculture (goulet and vinck ). cooperatives could play a role as change agents in the organization of local exchanges among farmers and broader diversification of products by identifying new markets (yang et al. ). agricultural researchers and practitioners should foster knowledge exchange regarding new-icls between farmers and other cross-sectoral stakeholders. the possibility to market new-icls products as local and green, leading to the creation of a differentiated market in crop and livestock value chains could be a powerful push factor in regions where consumers already have strong environment or local food preferences. such niche systems have emerged in finland, france, the u.s., and new zealand, including the marketing of sheep in vineyards as sustainable viticulture in the latter two countries (eip-agri , niles et al. ). however, these marketing campaigns may risk oversupply and falling prices without commensurate increases in demand. value chain upgrading into differentiated markets should be encouraged to incentivize new- icls practices, create a territorial identity for associated products, and create new marketing opportunities for value chain firms that lead in this area. labeling programs and certifications could help with this effort. conclusion there is substantial evidence that icls can help reduce the environmental externalities associated with conventional commercial farming, without declines in profitability or yields, by making progress toward closing the loop in nutrient and energy cycles (lemaire et al. , franzluebbers et al. , garrett et al. a, ryschawy et al. ). yet across most regions they have been declining for several decades and remain rare as a proportion of global agricultural area. this paper provides a historical and international perspective on multilevel factors that have contributed to the decline of icls within major commercial production regions. we also synthesize conditions that have fostered their persistence and reemergence in some areas, which provides a basis for understanding how to overcome barriers to reintegration and facilitate a socio-political environment where icls are supported. using a multilevel perspective, we identified one dominant global trajectory of icls retirement and two niches: traditional-icls persistence and new-icls emergence from both grassroots efforts and institutionalized regime players that could be leveraged into wider adoption. the dominant trajectory of icls retirement in commercial agricultural systems is linked to global and national landscape factors: a lessening of trade barriers, declining agricultural prices relative to wages, artificially low prices for synthetic inputs, e.g., fuel, fertilizers, and pesticides, and agricultural subsidies oriented toward specialized systems. these processes have favored the development of segregated-hia, and semi-icls, where only minimal integration occurs. amid this broader agricultural regime, traditional-icls have persisted in regions where unique cultural considerations, i.e., religious beliefs spurning technological change, have offset incentives to compete globally in specialized segregated-hia production or where protectionist government policies in the global north have buffered farmers from market changes, especially in less-favored soil-climatic contexts. in other regions, https://www.ecologyandsociety.org/vol /iss /art / ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / new-icls have emerged with two different pathways: (i) adoption of commercial cropping systems in regions with a large livestock sector and a legacy of traditional-icls, and (ii) adoption of new- icls in regions where environmental concerns and changing environmental policies are encouraging more sustainable agriculture via diversification or sustainable intensification. the first pathway tends to occur in regions with less government protections for farmers and difficult livestock grazing conditions that require additional feed sources, e.g., australia and new zealand, leading to seasonal integration of beef and sheep farming within arable or viticulture cropland. the latter pathway is occurring for a large diversity of systems, where environmental- friendly practices are being encouraged by both institutionalized regime players and grassroots movements, e.g. brazil, europe, and the u.s.). our study is limited by data availability on agricultural management practices, the geographical scale at which global data on icls and agricultural management are available, and the local knowledge of participants in our workshop and analysis, which spans australia, brazil, france, netherlands, new zealand, the u.s., and canada, but lacks representation from other major commercial production regions (eip-agri ). because of a focus on national-scale aggregate trends, major knowledge gaps remain about the precise locations of icls and levels of integration between the crop and livestock components. this knowledge gap limits our understanding of the social and ecological drivers of icls and their sustainability outcomes. our international approach underestimates the importance of very local factors and may have failed to capture potentially insightful outlier cases that could have illuminated additional important push and pull factors. nevertheless, our diagnosis and synthesis of existing cases from a multilevel perspective highlights several key problems with the social and ecological landscape that encourage locked-in agricultural regimes and inhibit new-icls or territorial-icls adoption and identifies leverage points that could be used to improve the adoption and sustainability of icls. this approach is a much needed complement to continued case study work and participatory design efforts, to identify worldwide opportunities and barriers to recoupling crop and livestock systems that emerge from complex multilevel interactions and historical legacies. to increase the level of icls adoption and improve the sustainability of existing agricultural regimes in commercial agricultural production systems, we suggest a combination of major structural changes that can provide top-down impetus to adjust agricultural management and encourage a “creative disruption” of the existing regime (kivimaa and kern ). this should be coupled with more small-scale, bottom-up efforts that can help support wider scale adoption of existing niche icls reemergence. on the structural side (pull factors), we encourage a redesign of research programs, credit systems, and insurance programs to focus more on whole farm outcomes over longer time horizons in a way that ensures farmers’ own their risk minimization efforts. we also suggest that current regulations be adjusted to focus more on a circular economy, with greater flexibility in addressing food safety concerns, rather than outright prohibitions on integration and material reuse. to disrupt current practices, governance changes would need to be ambitious, leading to the abolishment or replacement of existing policies, rather than just layering new incentives on existing policies that continue to support lock-in (kivimaa and kern ). from a bottom-up perspective, we suggest that agricultural research organizations expand the number of field trials and demonstration farms and make more of an effort to gather, organize, synthesize, and disseminate information on successful icls outcomes in existing farms through knowledge exchange networks between farmers and other stakeholders’ efforts. we also encourage researchers and practitioners to engage with farmers about design approaches to implement successful new-icls. greater effort should be made to brand icls as sustainable agriculture and educate consumers through the development of new sourcing standards and social- and eco-labels and to quantify the sustainability credentials required to garner market support for differentiated products. finally, there is an urgent need to improve government data collection and remote sensing efforts to characterize and assess the management of global pasture and livestock areas (garrett et al. a). these data are needed to better understand current levels of icls adoption, their drivers, and their ecological outcomes (manabe et al. ). given the power of entrenched interests within the existing regime, it is unlikely that these levers will be easy to establish because they involve value trade-offs and changes in the distribution of costs and benefits associated with global food systems (ipes food ). indeed few examples of policy replacement for food system transformation can be found in recent history (kivimaa and kern ). yet, climate change, increasing market volatility, global geopolitical restructurings associated with income growth, and changing demand may create new opportunities for change by encouraging practices, including icls, that provide farmers with greater resilience to all types of external shocks (garrett et al. a). policy makers and practitioners should proactively address innovation system reform by pursuing the above described pull and push levers to seize this opportunity for improved sustainability. author contributions rachael garrett and julie ryschawy contributed equally to the article conception, figures, and writing. rachael garrett led the international workshop underpinning the article. all other authors contributed to the development of the ideas and specific case studies included the study, as well as the writing and editing of the manuscript. responses to this article can be read online at: http://www.ecologyandsociety.org/issues/responses. php/ acknowledgments: this study was supported by the united states national science foundation grant no. ; the thomas jefferson fund make the planet great again program, the sustainability science program at harvard university; and the italian ministry for environment, land and sea. this work and the workshop that supported it was made possible through close cooperation and https://www.ecologyandsociety.org/vol /iss /art / http://www.ecologyandsociety.org/issues/responses.php/ http://www.ecologyandsociety.org/issues/responses.php/ ecology and society ( ): https://www.ecologyandsociety.org/vol /iss /art / collaboration with the brazilian agricultural research corporation (embrapa). we would also like to thank the global development policy center at boston university for supporting the students who worked on this project. literature cited allen, v. g., m. t. baker, e. segarra, and c. p. brown. . integrated irrigated crop-livestock systems in dry climates. agronomy journal : - . https://doi.org/ . /agronj . allen, v. g., c. p. brown, r. kellison, e. segarra, t. wheeler, p. a. dotray, j. c. conkwright, c. j. grenn, and v. acosta-martinez. . integrating cotton and beef production to reduce water withdrawal from the ogallala aquifer in the 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https://doi.org/ . /j.foodpol. . . https://doi.org/ . /j.foodpol. . . https://www.ecologyandsociety.org/vol /iss /art / appendix table a . : data sources and definitions for integrated crop livestock system (icls) estimates country icls definition icls % calculation years available source argentina commercial farms in mixed production (livestock and agriculture) as a proportion of all livestock and agriculture farms “la agricultura familiar en la argentina diferentes abordajes para su studio” – ministry of agriculture, livestock, and fishing australia mixed livestock area (farms that receive < % of their receipts from a single enterprise + wheat farms that integrate crops and livestock) as a proportion of all non-pastoral area all years from - australian bureau of agricutural and resource economics (abare) brazil area or farms in “mixed” crop livestock use (farms that have crop production combined with the rearing of animals in a joint activity, with a percentage of specialization in each less than %). as a proportion of all livestock and crop farms or area , , , censo agropecuario - brazilian institute of geography and statistics area where crops are used to reform or renovate or recuperate pastures + area where animal manure is used on crop area as a proportion of all livestock and crop area censo agropecuario - brazilian institute of geography and statistics canada - : mixed farms (the mixed farm operation is any farm that raises livestock and has some area devoted to crops for food) as a proportion of all livestock and crop farms or area , , , , , , , , , census of agriculture – statistics canada europe - & - : mixed farming (an agricultural holding where neither livestock nor crop production is the dominant activity; an activity is called dominant if it provides at least / of the production or the business size of an agricultural holding.) as a proportion of all livestock and crop farms or area , / , , , / , , , , , , , , - : eurostat farm structure historical results; : eurostat agriculture statistical yearbook data; : eurostat pocketbooks agricultural statistics main results; : eurostat pocketbooks agriculture, forestry and fishery statistics; : eurostat file: agricultural holdings, by farm type, by country - : "field crops-grazing" and "various crops and livestock combined" as a proportion of all farms / , eurostat statistics in focus structure of agricultural holdings japan area of dairy farms which own cultivated land (crops) + area of breeding cattle farms which own cultivated land (crops) as a proportion of dairy and breeding cattle farms , , , census of agriculture and forestry - statistics of japan new zealand - : area of cropping with sheep + area of sheep farming with cropping all land: as a proportion of all grain, sheep, and beef areas or non- pastoral land: as a proportion of “cropping” + cropping with sheep + sheep farming with cropping areas , , , , agricultural production statistics - statistics new zealand - : area of grain-sheep farming + area of grain-beef farming all land: as a proportion of all grain growing, sheep, and beef areas or non-pastoral land: as a proportion of “grain growing” + grain-sheep farming + of grain-beef farming area , , agricultural production statistics - statistics new zealand saudia arabia : mixed area/mixed holdings all agricultural census united states - : cropland used only for pasture or grazing (farms and area) as a proportion of all cropland (farms and area) , , , , , , , , , , , , census of agriculture – land use – us national agricultural statistics service : renamed “other pasture and grazing land that could have been used for crops without additional improvement,” but same meaning as past definition as you can see from data in table as a proportion of all cropland (farms and area) census of agriculture – land use – us national agricultural statistics service uruguay rice-livestock; livestock over rice as a proportion of rice area + livestock area , , agricultural statistical yearbook notes: brazilian data not used: farms or area in diversified use - if the sum of the production value of a single product has not reached % of the total production value (because the uses were not specified) or - mixed farms from cnae because methodology of farms included not clear; years available for each country: belgium, denmark, france, germany, ireland, italy, luxemberg, netherlands, spain, united kingdom = , / , , , / , , , , , , , , ; spain, portugal = , / , , , , , , , , greece = , , , , , , , ; austria, finland, sweden = , , , , , , ; bulgaria, cyprus, czech republic, estonia, hungary, latvia, lithuania, malta, poland, romania, slovakia, slovenia ( , , , ); croatia, norway = , , iceland, montenegro, switzerland = appendix additional information on the workshop held in belÉm - “understanding barriers to the wide-scale adoption of integrated crop, livestock and forestry systems and potential impacts” . goals of the workshop in this workshop we sought to better understand barriers to the adoption of integrated systems, the socioeconomic outcomes of adoption, and farmers’ perceptions of these systems. of particular interest were differences in motivations and outcomes between actors and regions, including differences between: i) subsistence and commercial agricultural systems, ii) farmers of different cultural backgrounds and genders, iii) frontier areas and long-established agricultural regions, and iv) national policy contexts. variations in integration possibilities across different markets and supply chain were another area of interest. from an academic perspective, the specific objectives of this workshop were to: ) share our current understanding of farmers’ barriers, motivations, and outcomes related to icls across and within regions based on existing work, ) assess the generalizability of regional findings, ) identify major research gaps, and ) develop new ideas for collaborative and comparative work across regions to address research gaps. based on this meeting and in cooperation with a handful of other researchers that could not attend, we aimed to produce a synthetic paper regarding the theoretical and empirical evidence base for assessing barriers to wide-scale adoption of integrated systems and potential outcomes of wide-scale adoption. from an applied perspective, a primary objective of this workshop is to help facilitate knowledge transfer between the international agricultural social science community and embrapa to help inform their future research and extension activities related to icls. early in the course of the workshop it became apparent that a broader focus beyond understanding barriers and motivations to adoption of new versions of icls was needed. we thought it was necessary to also examine the reason icls were retired in the first place. this was added to the discussions on the second day. . structure of the workshop days & - optional • field trips to paragominas to see different types of integrated and non-integrated grain and pasture systems in the brazilian amazon and talk to farmers about why they adopted/use these systems. day • overview & introductions • presentations – the challenge of sustainable development in the brazilian amazon: what is the role of integrated systems in meeting this challenge? what is embrapa doing in this area • presentations – farm level integration existing theoretical frameworks for understanding adoption and outcomes of integrated systems given the social and economic contexts in which agricultural systems are embedded. special attention to: i) motivations and competences in subsistence vs. commercial agricultural systems, ii) heterogeneity in preferences across culture and gender, iii) resources in frontier areas vs. long established agricultural regions, iv) differing incentives provided by national policy structures. • presentations – beyond farm integration and diffusion of integration existing theoretical frameworks for understanding cooperation between farmers to achieve integration of cropping and livestock systems and the role of institutions and innovation systems in promoting change. with specific attention to areas of interest mentioned above, as well as differences in scale, market attributes, and the structure of food chains. • small group work & report out: are there a common set of theories regarding adoption and diffusion that we can agree on within certain contexts? what theories do we lack? day • recap of day • presentations: empirical results from different regions: brazil, france, australia, us, and new zealand • group work & report out: what are the points of convergence and divergence between regions? what findings are generalizable? where are the gaps in empirical analysis? -feel free to draw from other literature. • group work & report out: developing frameworks to understand trajectories of integrated systems across regions and mapping out the differences between regions. • full discussion: synthesis of insights from the workshop and future directions for research and collaboration to address research gaps . how were participant chosen and expertise of those who were able to attend researchers and practitioners that were known to have written about the socioeconomic and policy dimensions of icls based on a literature review were invited. we aimed to have the widest swath of countries with primarily commercial agricultural systems represented, but not all researchers could attend. attendees had regional expertise on icls in australia, brazil, china, france, us, new zealand, netherlands, and disciplinary expertise in: agroecology, agronomy, economics, geography, innovation studies, and policy analysis, as well as methodological expertise in quantitative and qualitative methods, interviews, experiments, and synthesis. researchers with expertise on commercial systems in southeast asia and sub-saharan africa were invited, but were not able to attend. appendix additional information on definitions of supporting and inhibiting factors . supporting factors (opportunities arising from landscape changes or niche developments) a. cultural preference for autonomy: as a potentially self-sufficient livelihood, farming has long been driven by a desire to pursue an independent lifestyle, free from dependence on markets and governments (ploeg, ). traditional-icls practices enable this self-sufficiency and autonomy by producing all of the necessary inputs to production, as well as a diversity of food sources. while landscape changes and agricultural regime shifts have generally reduced the autonomy of individual farms, certain communities, have withstood pressures to change their systems to prioritize their autonomy due to cultural, religious, and political reasons. b. targeted by climate or pollution mitigation policy: policies that create taxes or fines for carbon, nitrogen, or phosphorus emissions or soil erosion force farmers to internalize the costs of pollution and land degradation to society, incentivizing more environmentally responsible production (zilberman et al., ). since icls tend to promote soil conservation and reduce the carbon and nutrient emissions associated with agricultural production (balbino et al., ; lemaire and franzluebbers, ), the introduction of taxes or fines on greenhouse gas and nitrate pollution should encourage their adoption vis-à-vis both continuous cropping and animal confinement systems. conversely, any public policy that pays farmers for the environmental services provided by their farm will have a similar impact on icls adoption, albeit by shifting the burden of payment to taxpayers rather than farmers. c. highly variable topography, climate, or soil: soil and water constraints can limit crop and forage production. this works to incentivize icls from two directions: livestock farmers seek out additional grazing areas (e.g., on neighboring farms) to supplement their livestock (i.e., rows between grape vines, stubble of cover and forage crops), while would-be crop farmers raise livestock in areas with topographical and soil features that are not suitable for cropping (lacoste et al., ). d. creation of agricultural research and development programs focused on sustainability and climate adaptation : federally supported research and extension directly focused on icls can help incentivize the adoption of these management systems (fao, ). long term agronomic and animal health research can help improve the production, animal welfare, and environmental outcomes of these systems (fao, ), while economic research can help identify which systems are most efficient. e. industry and civil society initiatives promoting icls: icls is often implicitly promoted by permaculture, organic, and biodynamic civil society networks (allen et al., ; cunfer, ; faust et al., ; lovell et al., ). an additional stimulus for new-icls adoption comes from the rise of peasant movements calling for self-sufficiency and autonomy in reaction to globalization, such as la via campesina and fédération associative pour le développement de l'emploi agricole et rural (fedear) (dumont et al., ). in seeking self-sufficiency for cost-savings and autonomy, these social movements often promote more holistic and agro-ecological farm-management approaches that reduce reliance on external inputs, including types of new-icls (bonaudo et al., ; dumont et al., ). f. differentiated value chains and eco- or social labels: the possibility to market icls products as “local” and “green”, leading to the creation of a differentiated market in crop and livestock value chains could help incentivize adoption in regions where consumers already have strong environment or local food preferences. . inhibiting factors (barriers arising from the agricultural regime or a lack of niche developments needed to push changes) a. lack of supply chain infrastructure or marketing opportunities: icls require a diverse union of supply chain infrastructure that enable access to markets for multiple products (gil et al., ). in some agricultural regions, where economies of scale have favored the specialization of agribusiness around a single product, there may be limited supply chain infrastructure or marketing channels for diverse products (garrett et al., a, b). b. high labor prices: livestock production tends to be more labor intensive than cropping (at least specialized, mechanized cropping) and most forms of crop-livestock integration require greater management attention (bell and moore, ). c. low prices of synthetic inputs and fuel or feed: the lower the prices for synthetic inputs and fuel or feed, the fewer incentives farmers have to produce these products on their own farm via icls (garrett et al., b). d. protectionist policies (e.g., insurance, subsidies): as a more diversified form of production vis-à-vis continuous crop monocultures or single animal systems, icls can be an important mechanism for reducing farmers’ risk (bowman and zilberman, ; o’donoghue et al., ). therefore, policies that protect policies via price subsidies, minimum price floors, or subsidized insurance on margin or production losses will also reduce incentives for icls and encourage specialization. e. food safety regulations restricting integration: policies that create restrictions and fines regarding the presence of animals or animal excrement in cropland areas will disincentivize many forms of icls (garrett et al., b). the impact of these food safety restrictions will depend on the types of crops they apply to (typically non-food crops and crops that are processed or intended for home consumption are excluded) and the minimum exclusion time between animal grazing or manure application and planting. f. lack of icls farm trials/demonstration: demonstration farms, farm trials, and extension programs can help spread information about the potential benefits of icls and technical details about how to operate such systems (gil et al., ). in regions where these don’t exist farmers may not have sufficient information about how to adopt icls. g. lack of farmer networks to share knowledge on icls: farmers’ networks have proved to be influential on perceptions of specific technologies (lubell et al., ; prokopy et al., ). creation of multi-actor and cross-sectoral groups can be a further step in supporting icls (pigford et al., ), especially when these groups are supported by participatory scenario building (ryschawy et al., ). additional information on rankings of supporting and inhibiting factors we used a combination of our expert knowledge and additional literature review of each case to develop the qualitative rankings that are presented below (and summarized in table ). . traditional-icls persistence a. amish farms in united states supporting factor - cultural preference for autonomy - very high supporting factor - industry or civil society networks promoting icls - high inhibiting factor - lack of icls farm trials/demonstration - low inhibiting factor - lack of farmers’ knowledge networks on icls - low the religious/cultural beliefs of amish farming groups include controls on new technologies that would threaten their christian values (brock and barham, ). for some orders this includes an avoidance of modern technology (e.g., synthetic inputs, heavy machinery) and a refusal of government assistance, such as subsidized insurance (stinner et al., ). amish tradition encourages “the purposeful stability of cultural practices and ideas with controlled introductions of new forms,” (parker, ). these traditions helped lock-in traditional-icls as a preferred model of farming. given that most old order amish farmers are surrounded by other farmers pursuing the same practices, there is no shortage of successful demonstration farms. supporting factor - targeted by climate or pollution mitigation policy - low supporting factor - creation of agricultural r&d programs focused on sustainability and climate adaptation - low inhibiting factor - low prices of synthetic inputs and fuel - low inhibiting factor - protectionist policies (e.g., insurance, subsidies) - low old order amish farmers tend to be influenced more by tradition and their cultural model of good stewardship, rather than external policies and research or market prices (parker, ). the emphasis on autonomy and control over ideas is associated with a rejection of government interference/support. since religious views explicitly prohibit the use of synthetic inputs, the overall low prices of these inputs in the us would not play a large role in the broader choice to pursue icls. inhibiting factor - food safety regulations restricting integration - moderate the food safety modernization act of provides standards for the safe production and harvesting of food crops. this act has the potential to impact certain forms of icls adoption through rules limiting the presence of animals and use of animal excrement on cropland. while these regulations provide potential benefits for public health, they could discourage the integration of animals into cropping systems. yet, foods destined for home consumption and foods that will be processed before consumption are exempt from the policy. this limits their impact on amish farming systems. supporting factor - highly variable topography, climate, or soil - moderate ohio has the largest amish settlement in the world and a majority of this group reside in sugar creek, ohio (parker et al., ). this region is characterized by a high degree of variability in topography, geology and soil, and ecosystems. yet, this variability is not necessarily high within individual farms, and given the cultural considerations above, not the major driver of icls persistence. supporting factor - differentiated value chains and eco- or social labels - moderate inhibiting factor - lack of supply chain infrastructure or marketing opportunities - moderate amish farmers have found a strong and stable niche in marketing their products via organic dairies, either via cooperatives or directly trading to individual cheese brands (brock and barham, ). high cost of labor - low traditional old order amish discontinue education after primary school as labor is directed at the farming unit. furthermore, labor is fairly abundant (parker et al., ) - population growth in amish communities has led to high increased migration out of existing regions. b. family farms (smallholders*) in brazil *in brazil family farms are defined as farms under a certain size that primarily rely on household labor. supporting factor - cultural preference for autonomy - moderate generally speaking, small “peasant” farmers are thought to have a high preference for autonomy (ploeg, ). yet smallholder autonomy in brazil has also developed out of a “lack of options”. that is farmers cannot necessarily rely on the government for support and have to be self-sufficient. supporting factor - targeted by climate or pollution mitigation policy - moderate brazil implemented many aggressive climate change policies in brazil during the s (see “pastures and croplands in brazil”). however, in practice, family farmers are much less targeted by these policies than large farmers. supporting factor - highly variable topography, climate, or soil - moderate even small farms in brazil can be fairly large (depending on the region), so there can be large variety of soil types within a single geography. however, outside of the mountainous regions in minas gerais there is not normally substantial variation in topography within single farms. supporting factor - creation of agricultural r&d programs focused on sustainability and climate adaptation - low supporting factor - industry or civil society networks promoting icls - low inhibiting factor - lack of icls farm trials/demonstration - moderate inhibiting factor - lack of farmers’ knowledge networks on icls - low despite large r&d efforts for new-icls adoption in brazil, including cooperation with agribusinesses to support new-icls technologies (see “pastures and croplands in brazil”), federal research and extension programs often fail to reach small and medium farmers (cortner et al., ). additionally smallholders are not likely to interact with major agribusinesses (garrett et al., a). nevertheless, traditional forms of icls have persisted and are socially embedded, so there is general community knowledge about how to pursue these types of systems. supporting factor - differentiated value chains and eco- or social labels - low inhibiting factor - lack of supply chain infrastructure or marketing opportunities - high inhibiting factor - low prices of synthetic inputs and fuel - low many agricultural regions have limited agribusiness infrastructure (garrett et al., b), which constrain farmers’ ability to adopt newer forms of icls (gil et al., ). because of these infrastructure limitations, the prices of synthetic inputs are higher in more remote regions where smallholders typically reside. additionally, these inputs tend to be financially out of reach for smallholders who have little cash or credit available. there are very few channels for marketing higher value crops or niche products that could support smallholders in leveraging their traditional-icls systems toward differentiated value chains (garrett et al., a). inhibiting factor - high cost of labor - low as mentioned above, in brazil family farms are defined as farms under a certain size that primarily household labor. thus, relative to their size, labor tends to be abundant and low cost (since off-farm job opportunities are low) (garrett et al., a). inhibiting factor - protectionist policies (e.g., insurance, subsidies) - low family farmers are required to purchase coverage through the insurance for family farmers (seaf) program when they access subsidized loans through the program for strengthening family agriculture. however, currently this program reaches less than % of farmers (canal do produtor, ; ibge, ). inhibiting factor - food safety regulations restricting integration - low food safety laws in brazil are geared toward processing facilities and complying with the sanitary and phytosanitary regulations of importing countries (salay and caswell, ). brazil does not have restrictions on the use of animal grazing or manure in food crop areas (garrett et al., b). . new-icls reemergence a. pastures and croplands in brazil supporting factor - cultural preference for autonomy - moderate farmers in brazil tend to be open to government support in the sense that they receive substantial subsidies via low interest loans and are willing to engage in complex agribusiness markets (garrett et al., b). however, many farmers are averse to government interventions that constrain their behaviors or encourage them to adopt more pro-environmental behaviors (cortner et al., ). supporting factor - targeted by climate or pollution mitigation policy - high in brazil, new-icls is being promoted by the government’s low carbon agriculture (abc) plan and increasing restrictions on native vegetation clearing that are linked to brazil’s broader international commitment to reduce national greenhouse gas emissions (gil et al., ). the abc program provides subsidized loans for adoption of integrated systems to combat soil degradation and recuperate pastures through the introduction of crop species, thereby shortening the cattle life cycle and reducing emissions per unit of food produced (observatorio abc, ). restrictions on forest clearing have incentivized the adoption of icls to increase productivity on the existing land area (cortner et al., ; garrett et al., ). supporting factor - highly variable topography, climate, or soil - moderate there is a large variety of soil types within a single farm. however, outside of the mountainous regions in brazil there is not substantial variation in topography in the major agricultural regions. supporting factor - creation of agricultural r&d programs focused on sustainability and climate adaptation - high supporting factor - industry or civil society networks promoting icls - moderate inhibiting factor - lack of icls farm trials/demonstration - moderate inhibiting factor - lack of farmers’ knowledge networks on icls - moderate since the s, the brazilian agricultural research corporation (embrapa) has been doing research on icls in beef cattle systems as a mechanism to restore degraded pastures. in the early s, six existing embrapa state research units in the north region were transformed in agroforestry research centers. this restructuring process strengthened r&d on agroforestry and integrated crop, livestock, and forestry systems in deforested areas (flores, ). federal research on icls increased substantially during the s and resulted in the development of icls farm trials and demonstration site (embrapa, ). recent research has shown that icls adoption is significantly higher near these sites (gil et al., ). these efforts have included cooperation with agribusinesses to support new-icls technologies (i.e., the “rede-ilpf” [icls-network], https://www.embrapa.br/web/rede-ilpf/rede-ilpf ). yet, the engagement of retailers is only starting and few farmer associations have icls on their agenda. in a study of icls adopters and non-adopters the brazilian amazon, most adopters had high awareness of the technical aspects and benefits of icls, and were well connected to social groups supporting these systems. yet, non-adopters were less well connected and cited a lack of information as one barrier to adoption (cortner et al., ). inhibiting factor - lack of supply chain infrastructure or marketing opportunities - high inhibiting factor - low prices of synthetic inputs and fuel - moderate many agricultural regions have limited agribusiness infrastructure (garrett et al., b), which constrains farmers’ ability to adopt newer forms of icls (gil et al., ). because of these infrastructure limitations, the prices of synthetic inputs in brazil are higher in more remote regions. supporting factor - differentiated value chains and eco- or social labels - low there are very few channels for marketing higher value crops or niche products (garrett et al., a). one exception is the higher meat grading for cattle that have higher fat content, which can be linked to icls (cortner et al., ). inhibiting factor - high cost of labor - moderate while wages are not as high as developed countries, they are increasing and labor trained to work with machinery or advanced livestock management tends to be scarce. farmers’ perceive skilled labor scarcity as a major barrier to adopting icls (cortner et al., ). inhibiting factor - protectionist policies (e.g., insurance, subsidies) - low in , all public and private mechanisms for mitigating risks in agriculture were accessed by . million farmers, covering % of the agricultural area in brazil (mbagro, ). inhibiting factor - food safety regulations restricting integration - low food safety laws in brazil are geared toward processing facilities and complying with the sanitary and phytosanitary regulations of importing countries (salay and caswell, ). brazil does not have restrictions on the use of animal grazing or manure in food crop areas (garrett et al., b). b. non-pastoral areas in australia supporting factor - cultural preference for autonomy - high the concept of autonomy is more commonly expressed as “self-reliance” in the australian literature and is highly valued as an objective of farming (waters et al., ). indeed, much of the agricultural policy is built around the concept of farmer self-reliance - that is, policy interventions should not create dependency, but rather leave farmers in charge of their own destiny (kiem, ). supporting factor - targeted by climate or pollution mitigation policy - moderate in australia passed the carbon credits act (c c ). this policy provides carbon credits to farmers and land managers through eligible carbon abatement activities which store or reduce greenhouse gas (ghg) emissions on land. this could include icls practices. supporting factor - highly variable topography, climate, or soil - high in australia farms are very large. landscape heterogeneity has been a major impetus for adoption of new-icls (bell and moore, ), leading to the incorporation of livestock areas to take advantage of topographical and soil features that are not suitable for cropping (lacoste et al., ). supporting factor - creation of agricultural r&d programs focused on sustainability and climate adaptation - high supporting factor - industry or civil society networks promoting icls - moderate inhibiting factor - lack of icls farm trials/demonstration - moderate inhibiting factor - lack of farmers’ knowledge networks on icls - moderate the grain and graze program aimed to increase integration of beef and sheep with crop production by improving the profits, reducing environmental impacts, and building social capital in icls via the adoption of best management practices (price and hacker, ). a major objective was to improve “whole-farm knowledge” and promote researcher-to-farmer knowledge networks via annual research and extension forums (hacker et al., ). the program is credited with the adoption of new-icls practices by farmers over five years despite unfavorable climatic conditions (price and hacker, ). nevertheless, knowledge gaps and extension remain an important challenge for scaling up icls (price, ). supporting factor - differentiated value chains and eco- or social labels - low the development of value chains for more sustainable food products has been slow to take off in australia due to consumer skepticism (bhaskaran et al., ). while international markets for sustainable product are growing, most of the beef exported from australia is still sent to commodity markets that differentiate only on meat grade (lawrence, ). inhibiting factor - lack of supply chain infrastructure or marketing opportunities - moderate most of the grains produced via icls are intended to overcome seasonal forage production shortfalls and are consumed locally rather than marketed (hacker et al., ). further development of cropping systems could be constrained by limited infrastructure. inhibiting factor - low prices of synthetic inputs and fuel - high low and declining input prices have favored cropping over icls systems in australia (bell and moore, ). inhibiting factor - high cost of labor - high studies of australian farms have shown that labor costs for integrated systems are significantly higher than specialized cropping systems and a likely factor inhibiting adoption/encouraging retirement of new-icls (bell and moore, ). inhibiting factor - protectionist policies (e.g., insurance, subsidies) - low australia dismantled most supports for farmers in the s. most industries receive little assistance and it is mainly in the form of adjustment assistance, r&d support, drought relief and tax concessions (productivity commission, ). inhibiting factor - food safety regulations restricting integration - low none of australia’s food safety policies prohibit the presence or use of animals or manure on cropland area. c. non-pastoral areas in new zealand supporting factor - cultural preference for autonomy - high in a survey % of new zealand dairy farmers stated autonomy or “being one’s own boss” was their primary motivation for farming (pangborn, ). additionally, the economic reforms in the s have nearly completely deregulated farming, leaving them as economic entrepreneurs (stock and forney, ). supporting factor - targeted by climate or pollution mitigation policy - moderate the resource management act (rma) of (public law no ) stipulates that the use of land must be consistent with “national environmental standards, regional rules, or district rules”. the rma and other environmental regulations are administered by regional councils, who are tasked with issuing permits for resource consents (activities that may influence environmental quality, including agriculture). the national policy statement for freshwater management reinforces the responsibilities of regional councils for dealing with these issues, clarifying their responsibility under the rma for decision-making and management planning. the policy statement emphasizes responsible use of water resources with respect to climate change, prohibits the over-allocation of water, and charges regional councils with mitigating adverse effects. the most significant aspect of the regulation was to establish a minimal acceptable condition for freshwater across a variety of contaminant parameters. regional councils with particularly acute water quality issues have already or are currently implementing regional policies related to nitrogen management through cap and trade programs. supporting factor - highly variable topography, climate, or soil - moderate-high given the rolling topography of many of the agricultural regions, there is substantial variation in climate and soils in new zealand farming systems. soil and water constraints limit forage production, creating incentives for beef and sheep farmers to seek out additional grazing areas to supplement their livestock (i.e., rows between grape vines, stubble of cover and forage crops). in some regions, however, this variation serves as a barrier for icls because the soil type and water accessibility are insufficient for cropping systems (garrett et al., b; minneé et al., ). supporting factor - creation of agricultural r&d programs focused on sustainability and climate adaptation - moderate supporting factor - industry or civil society networks promoting icls - moderate inhibiting factor - lack of icls farm trials/demonstration - high inhibiting factor - lack of farmers’ knowledge networks on icls - low there is some focus within new zealand crown research institutes (cris), to conduct research on icls. plant and food work on integrating sheep into vineyards, while agresearch works on crop, forage, pasture, and sheep/beef and dairy integration. the new zealand agriculture greenhouse gas research center pursues a research agenda of reducing greenhouse gas emissions across sectors by partnering with the industry group dairynz and the cri agresearch to conduct analysis of integrated systems (nzagrc, ). the sustainable farming fund invests up to $ million per year in research and extension programs led directly by farmers to fill gaps in industry-funded research by opening a grass-roots award mechanism focused on sustainability to farmers. several of the funded projects in sff’s portfolio are explicitly directed toward integration (mpi, ). despite all this funding there are still few icls specific farm trails. supporting factor - differentiated value chains and eco- or social labels - moderate inhibiting factor - lack of supply chain infrastructure or marketing opportunities - low due to the small size of new zealand and the presence of both crop and livestock farming throughout all major production regions, supply chain infrastructure for marketing conventional icls products is not a major issue. however, given the small population size and large distance to international markets, reaching differentiated markets can be somewhat of a challenge. still, grass-fed lamb labels and integrated sheep- viticulture systems have made their way onto labels and differentiated markets (niles et al., ). more generally new zealand has been working on branding its wine in terms of environmental sustainability. inhibiting factor - low prices of synthetic inputs and fuel - moderate new zealand has no tariff on fertilizer imports and a % tariff on imported animal feeds (new zealand ministry for foreign affairs and trade (mfat), ). however, five separate biosecurity acts and standards regulate the import of animal feeds, including the import health standard, animal products act ( ), agricultural compounds and veterinary medicines act ( ), biosecurity (ruminant protein) regulations ( ), and the biosecurity act ( ). these acts make it relatively expensive to import feed or feed components (new zealand government, ). fonterra, the largest dairy company in the world, and virtual monopoly in new zealand, encouraged farmers to keep palm kernel rations (an imported feed source) at kg per animal per day in (fonterra, ). nevertheless, fertilizer and feed imports (particularly for dairy) are high in new zealand and have continued to increase since policy reforms in the s (macleod and moller, ). inhibiting factor - high cost of labor - high labor is frequently mentioned as a limiting factor for new zealand agriculture, as they rely heavily on seasonal migration for labor intensive agricultural industries. labor savings are cited as one of the major benefits of adopting icls (niles et al., ). inhibiting factor - protectionist policies (e.g., insurance, subsidies) - low farmers in new zealand receive no support through minimum prices or direct payments (macleod and moller, ) - all agricultural subsidies were removed in . as a result, new zealand has the lowest support for agriculture of any oecd country (producer supports are estimated at . % of gross farm receipts) (organisation for economic development (oecd), ). crop insurance in new zealand is voluntary and unsubsidized. these factors require farmers to manage their own risks, often by diversifying production (evans et al., ). inhibiting factor - food safety regulations restricting integration - low none of new zealand’s food safety acts prohibit the presence or use of animals or manure on cropland area (ministry for primary industries, ). d. agroecological farms in france supporting factor - cultural preference for autonomy - high recent studies have highlighted the autonomy as a major motivation for icls in france, especially limiting reliance on external markets for crop inputs and livestock feed (coquil et al., ; ryschawy et al., ). this autonomy is not necessarily limited to the farm level, but also may be supported via territorial synergies, e.g. exchanges within groups of - neighboring farmers (moraine et al., ; ryschawy et al., ). supporting factor - targeted by climate or pollution mitigation policy - high icls and other forms of agro-ecological farming systems have been promoted via the agro-ecological plan launched in by the french ministry of agriculture. a specific program called / was implemented to encourage carbon storage in soils through agroecological practices ( / pertains to a target of growing soil carbon by . % per year). self-sufficiency and in particular icls were particularly encouraged at farm or collective levels through this policy and through the european commission. at the collective level, funding was given to groups of farmers engaged in agro-ecological activities, including territorial-icls (alim’agri, ) supporting factor - highly variable topography, climate, or soil - moderate in france traditional-icls and new-icls tend to occur in less-favored areas where crops cannot be grown (e.g., steep slopes or wetlands), or where cropping is less profitable (ryschawy et al., ; schiere et al., ). supporting factor - creation of agricultural r&d programs focused on sustainability and climate adaptation - moderate the european commission has launched a european innovation partnership (eip) focus group of experts to build state of the art icls (eip- agri, ). operational groups have been funding field projects across europe where research, advising, marketing and farmers are brought together to develop agro-ecological forms of icls and new skills for farmers (eip-agri, ). supporting factor - industry or civil society networks promoting icls - low most industry marketing channels remain highly focused on either the crop or livestock sector independently (eip-agri, ). civil society is generally not informed of the challenges of icls and more concerned by animal welfare, quality labels or local food chains (dumont et al., ). supporting factor - differentiated value chains and eco- or social labels - moderate in specific areas, there are well-established quality-label products ensuring remuneration to farmers for products based on locally-sourced feeds, good image, and contributions to territorial vitality, but those are mostly linked to livestock products and not icls specifically (beudou et al., ). the recent consumer interest in decreasing food waste could be seen as an opportunity for icls (dumont et al., ). inhibiting factor - lack of supply chain infrastructure or marketing opportunities - high agglomeration economies have favored the regional concentration of supply chain infrastructure for certain crops or livestock (gaigne, ). in general, there is a lack of marketing options for livestock by-products (wool, manure, …). inhibiting factor - low prices of synthetic inputs and fuel - high inhibiting factor - high cost of labor - high the labor supply available in agriculture across europe has shrunk over recent decades and there has also been a loss of skills and motivation to manage both crops and livestock (peyraud et al., ). in france, the large decrease of icls has been linked explicitly to the lack of workforce to manage both crop and livestock and the high cost of labor relative to input and fuel costs (ryschawy et al., ; veysset et al., ). inhibiting factor - protectionist policies (e.g., insurance, subsidies) - high livestock has suffered from strong competition with crops as a result of protectionist policies. since the st pillar of cap has provided high subsidies for crops, which has led to a general process of specialization and modernization of local agriculture (veysset et al., ). inhibiting factor - food safety regulations restricting integration - moderate food safety regulations are not limiting icls at the farm level directly, but they are limiting the recycling of waste between farms for animal feed or manure use (dumont et al., ). specific regulations around direct sales of products between farmers are limiting the development of icls beyond farm level in france (ryschawy et al., ). inhibiting factor - lack of icls farm trials/demonstration - high research and advising have been mostly focused on either crop or livestock management (eip-agri, ). there are few examples of farm demonstrations for icls. at inra (french national institute for research in agronomy), only three demonstration farms are considering icls. in general, advising systems are implementing trials on specific crops and not considering the effects of crop-livestock integration. inhibiting factor - lack of farmers’ knowledge networks on icls - moderate a lack of farmers’ skills to manage icls has been underlined by the eip focus group around icls (eip-agri, ). still, some farmers’ knowledge networks that were not focusing on icls are now considering these systems in their discussions, such as groups around conservation agriculture or livestock feed autonomy through pasture use e. carbon farming in the united states supporting factor - cultural preference for autonomy - high the value of autonomy in american agriculture is linked to jeffersonian agrarian values and the concept “rugged individualism” (sullivan et al., ). in a survey of both conventional and organic crop farmers in the us, independence was listed as the major benefit of being a farmer (whereas low financial return was listed as the biggest drawback) (sullivan et al., ). even in the hog industry, independence has been shown to be an important driver of behavior - farmers will avoid contractual arrangements that could reduce risk to maintain autonomy (key, ). inhibiting factor - protectionist policies (e.g., insurance, subsidies) - high farmers receive very high levels of support, though the type of support they receive has shifted substantially over time from price supports to income supports based on current production (after ), to income supports based on historical production (after ), to insurance for yield and revenue losses (after ) (dimitri et al., ; garrett et al., b). uptake of insurance programs is fairly widespread (farrin et al., ; usda, ). in areas where there are no available crop insurance options, the noninsured crop assistance program provides coverage for losses due to weather. supporting factor - targeted by climate or pollution mitigation policy - moderate the clean water act aims to mitigate the pollution of water through the approval of discharge permits ( u.s.c. § ; ), many of which are required of confined animal feeding operations (cafos) if they propose to discharge to water. the safe drinking water act protects underground sources of drinking water by regulating how farms handle both liquid waste and wastewater and requires regular sampling of drinking water to identify microbial contamination ( u.s.c. § f; ). however, farmers are often provided with exemptions to these rules and violations are rarely enforced (dowd et al., ). since the us farm bill has included additional environmental considerations, including the environmental benefits index (ebi), which helps prioritize land for conservation across multiple environmental attributes, and the environmental quality incentives program (eqip), which provides financial and technical assistance for investments in environmental protection. the farm bill’s conservation stewardship program (csp) and eqip both provide payments to farmers for several icls related behaviors such as not burning crop residue, intensive rotational grazing, transition to organic cropping systems, and nutrient and feed management. there is currently no climate policy addressing agriculture. supporting factor - highly variable topography, climate, or soil - low we consider biophysical and climate variability to be of low importance since to date it is not mentioned in any of the us literature as a motivation for icls adoption. supporting factor - creation of agricultural r&d programs focused on sustainability and climate adaptation - moderate inhibiting factor - lack of icls farm trials/demonstration - high inhibiting factor - lack of farmers’ knowledge networks on icls - high agricultural research is mainly supported through the farm bill and the national institute of food and agriculture (nifa). nifa funds several programs that are salient to icls, including programs on sustainable agriculture, organic agriculture, soil health, manure and nutrient management, and risk management education (nifa, ). recently nifa has developed several grant programs that support research on icls (nifa, ; usda, ). nevertheless, allocations to icls comprise only % of the $ million in agricultural research funding that is provided by the farm bill per year (nifa, ; usda, ). farm trials and demonstrations are rare, but do occur at some nifa-funded land grant colleges (see garrett et al., b for specifics). supporting factor - industry or civil society networks promoting icls - moderate in the us icls has been promoted via permaculture, organic, and biodynamic civil society networks, e.g. “soil carbon cowboys” (allen et al., ; cunfer, ; faust et al., ; lovell et al., ). supporting factor - differentiated value chains and eco- or social labels - high consumer markets for local and sustainable products (e.g., high end retailers, farmers markets, etc.) have grown substantially in recent decades (dimitri and greene, ). besides differentiation via organic and biodynamic labels, more specific marketing opportunities have been identified for grass-fed livestock (gwin, ) and integrated sheep-viticulture (ryschawy et al., in review). inhibiting factor - lack of supply chain infrastructure or marketing opportunities - moderate limited meat processing infrastructure, specifically for small livestock, has been noted as a challenge for icls in the us (hilimire, ). inhibiting factor - low prices of synthetic inputs and fuel - high inhibiting factor - high cost of labor - moderate low prices of fertilizers and fuel explain structural changes in us agriculture, including less diversification (dimitri et al., ). specialization in the us has also been fostered via mechanization, which helped reduce the costs of labor (sulc and tracy, ). nevertheless, the us has greater abundance to cheap agricultural labor relative to many other regions due to seasonal migration from mexico. inhibiting factor - food safety regulations restricting integration - high the food safety modernization act of provides standards for the safe production and harvesting of food crops and has the potential to impact certain forms of icls adoption through rules related to the presence of animals and use of animal excrement on cropland that produces food for human consumption. while these regulations provide potential benefits for public health, they could 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self. journal of rural studies , – . sulc, r.m., tracy, b.f., . integrated crop–livestock systems in the u.s. corn belt. agronomy journal , – . sullivan, s., mccann, e., de young, r., erickson, d., . farmers’ attitudes about farming and the environment: a survey of conventional and organic farmers. journal of agricultural and environmental ethics , – . https://doi.org/ . /bf usda, . usda nifa integrated research, education, and extension competitive grants program – organic transitions. usda, . about the risk management agency, a risk management agency fact sheet. veysset, p., bébin, d., lherm, m., . adaptation to agenda (cap reform) and optimisation of the farming system of french suckler cattle farms in the charolais area: a model-based study. agricultural systems , – . waters, w., thomson, d., nettle, r., . derived attitudinal farmer segments: a method for understanding and working with the diversity of australian dairy farmers. extension farming systems journal , . zilberman, d., templeton, s.r., khanna, m., . agriculture and the environment: an economic perspective with implications for nutrition. food policy , – . https://doi.org/ . /s - ( ) - 	title 	abstract 	introduction 	types of icls 	methods 	explaining icls abundance and trajectories in commercial agriculture 	status of icls in regions where data are available 	decline of icls because of large landscape changes 	persistence of niches of traditional-icls within the dominant agricultural regime 	opportunities for new-icls emergence within the dominant agricultural regime 	regime factors inhibiting new-icls emergence 	levers for reintegration of crop and livestock systems worldwide 	pull factors for icls 	push factors 	conclusion 	responses to this article 	acknowledgments 	literature cited 	figure 	figure 	figure 	figure 	table 	appendix 	appendix 	appendix wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === 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http://europepmc.org/abstract/med/ imaging research center | uc davis school of medicine 		 	 	 			 		 		skip to main content 		 		 					 			 		 				 news | careers | giving | uc davis health 			 					 			 	 			 	 		 	 				 				 					 					 						 toggle navigation 					imaging research center 			 	 getting started 					 					 									 begin a new study 	 pilot program 	 scheduling and billing 	 safety training and operator training 	 computing 	 resources 	 documents 	 faqs 	 facilities 					 					 									 imaging research center in sacramento 	 imaging research center in davis 	 people 					 					 									 faculty 	 staff 	 executive advisory committee 	 research partners 	 participate 					 					 									 information for researchers 	 information for volunteers 	 mri methods 	 contact us 					 					 									 irc in sacramento 	 irc in davis 	 					 					 				 				 				 		 		 	 	 	 								 								 								 									 										imaging research center 																				 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	 	 	 	 		 . masters sl, simon a, aksentijevich i, kastner dl: horror autoinflam- maticus: the molecular pathophysiology of autoinflammatory disease. annu rev immunol : – , . zheng l, sinniah r, hsu si: in situ glomerular expression of activated nf-kappab in human lupus nephritis and other non-proliferative pro- teinuric glomerulopathy. virchows arch : – , . fujihara ck, antunes gr, mattar al, malheiros dm, vieira jm jr, zatz r: chronic inhibition of nuclear factor-kappab attenuates renal injury in the / renal ablation model. am j physiol renal physiol : f –f , . mattar al, machado fg, fujihara ck, malheiros dm, zatz r: persistent hypertension and progressive renal injury induced by salt overload after short term nitric oxide inhibition. clinics (sao paulo) : – , . panzer u, steinmetz om, turner je, meyer-schwesinger c, von ruffer c, meyer tn, zahner g, gomez-guerrero c, schmid rm, helmchen u, moeckel gw, wolf g, stahl ra, thaiss f: resolution of renal inflam- mation: a new role for nf-kappa b (p ) in inflammatory kidney diseases. am j physiol renal physiol may , [epub ahead of print] . oakley f, mann j, nailard s, smart de, mungalsingh n, constandinou c, ali s, wilson sj, millward-sadler h, iredale jp, mann da: nuclear factor-kappab (p ) limits the inflammatory and fibrogenic responses to chronic injury. am j pathol : – , . hussain s, romio l, saleem m, mathieson p, serrano m, moscat j, diaz-meco m, scambler p, koziell a: nephrin deficiency activates nf-kappab and promotes glomerular injury. j am soc nephrol : – , . chuang py, he jc: signaling in regulation of podocyte phenotypes. nephron physiol : – , . faul c, donnelly m, merscher-gomez s, chang yh, franz s, delf- gaauw j, chang jm, choi hy, campbell kn, kim k, reiser j, mundel p: the actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine a. nat med : – , see related article, “nephrin deficiency activates nf-�b and promotes glo- merular injury,” on pages – . low calcidiol levels and coronary artery calcification: true, true, and related? michal l. melamed* and ravi thadhani† *departments of medicine and epidemiology and population health, albert einstein college of medicine, bronx, new york; and †department of medicine and renal unit, massachusetts general hospital, boston, massachusetts j am soc nephrol : – , . doi: . /asn. there is increasing interest in the role of vitamin d in health and disease. from early articles showing associations between use of activated vitamin d and improved survival on dialysis to more recent analyses showing that low -hydroxyvitamin d (calcidiol) and , -dihydroxyvitamin d (calcitriol) levels are associated with mortality in dialysis patients, the observa- tional findings have been, for the most part, consistent. the associations between calcitriol use and survival have been ex- tended to the predialysis chronic kidney disease (ckd) popu- lation, as have associations between low calcidiol levels and mortality. it is true that patients with renal disease represent an extreme population with profound deficiencies of both cal- cidiol and calcitriol. low calcidiol levels associate with all- cause mortality in the general population. the elevated mor- tality risk is perhaps due to an increased hazard for cardiovascular events. low calcidiol levels link with incident cardiovascular disease (cvd) and myocardial infarctions. studies of vitamin d deficiency and outcomes in the general population require larger sample sizes than studies of patients with renal disease because less profound deficiency is found in the former population. the mechanisms underlying the benefits of adequate levels of vitamin d have not been fully elucidated. low calcidiol lev- els associate with diabetes and hypertension and therefore may link to cvd by predisposing people with vitamin d defi- ciency to two diseases that place them at high risk. vitamin d regulates the renin-angiotensin system and may exert cardio- protection through this action. one of these effects may be protection against left ventricular hypertrophy. activated vita- min d therapy prevents the progression of cardiac hypertro- phy in the dahl salt-sensitive rat model of heart failure. the effect of calcitriol on cardiac structure and function has also been noted by other studies. , another area where vitamin d may play a role is in vascular calcification. one of the earliest models of atherosclerosis was an animal fed a high-cholesterol and high–vitamin d diet. these rats were fed . million u/kg vitamin d and developed aortic atherosclerosis. recent experiments by hruska and colleagues showed that high dosages of calcitriol led to aortic calcification in a mouse model of ckd; however, low-dosage calcitriol, at dosages sufficient to treat secondary hyperpara- thyroidism, were protective against calcification. thus, there may be an optimal level of activated vitamin d that is neither too high nor too low. human studies evaluating an association between vitamin d levels and coronary artery calcification (cac) have shown conflicting results. an analysis of amish individuals did not find an association between calcidiol levels and prevalent cac. an analysis of individuals at high risk for coronary artery disease found an inverse correlation between calcitriol levels and vascular calcification. an analysis of children on dialysis revealed that both high and low calcitriol levels associ- ate with higher calcification scores. this latter study poten- tially suggests an optimal level of vitamin d exists that is nei- ther too high nor too low. in this issue of jasn, de boer et al. report an analysis from the multi-ethnic study of atherosclerosis (mesa) suggesting published online ahead of print. publication date available at www.jasn.org. correspondence: dr. ravi thadhani, bullfinch , massachusetts general hospital, boston, ma . phone: - - ; fax: - - ; e-mail: thadhani.r@mgh.harvard.edu copyright � by the american society of nephrology editorialswww.jasn.org j am soc nephrol : – , editorials that low calcidiol levels associate with subsequent develop- ment of cac. in this well-analyzed study of participants (mean age . yr; % white, % black, % chinese, and % hispanic; % with estimated gfr � ml/min per . m ; % with calcidiol levels � ng/ml; all free from cac at baseline), participants developed incident cac during yr of follow-up. low calcidiol levels were independently associ- ated with a higher risk for developing cac. comparing those with calcidiol levels � ng/ml to those with levels � ng/ml, the risk was . ( % confidence interval [ci] . to . ) for developing incident cac, but for each ng/ml lower cal- cidiol level, the risk was . ( % ci . to . ; p � . ). interestingly, the authors did not find an association between high calcidiol levels and cac, either because one does not exist, or possibly there were not enough participants with higher levels of calcidiol. the association and the magnitude of the effect of low calcidiol levels and risk for cac also did not seem as strong as those found for other cardiovascular risk factors in mesa. for example, the multivariable adjusted relative risk for incident cac for those with untreated diabetes was . ( % ci . to . ; p � . ) and among men was . ( % ci . to . ; p � . ). strengths of this study include a well-characterized study population, free of clinical cvd at baseline but not free of subclinical cvd, such as cac. an additional strength is the temporality of the association: participants with low calcidiol levels at baseline subsequently developed cac. whereas most previous studies linking vitamin d and vascular calcification used a cross-sectional design, the study by de boer et al. is unique because of its prospective data. a few potential limita- tions of the study include that, in secondary analyses, calcidiol levels did not associate with the progression of plaque or sever- ity of plaque. in other analyses of mesa, cardiovascular risk factors including age, male gender, white race, body mass in- dex, elevated bp, and diabetes all were independently associ- ated with progression of cac. like most studies using rich databases, multiple comparisons without adjustment for sta- tistically significant results are made in this analysis. it is true that cacs associate with coronary events ; how- ever, to be an ideal surrogate end point, regression of cac needs association with a lowering of coronary events. this step has not been proved. notably, studies of sevelamer hydrochlo- ride showed less progression of calcification compared with calcium-based binders, but no randomized clinical trial showed a survival benefit to sevelamer in intention-to-treat analyses ; therefore, although cac associates with coronary events and is considered a measure of subclinical atherosclero- sis, it is unclear whether regression or less progression of cac links with improved outcomes. it may be that cac is yet an- other critical cardiovascular risk factor, which, like diabetes and hypertension, is now related to low calcidiol levels. what does this study mean for patients with ckd and esrd? in patients with esrd, coronary events are not likely the primary cause of cvd deaths; instead, sudden cardiac death from arrhythmias are probably the most common causes of death, as recently suggested by large clinical trials. poten- tially, cac associates with calcification in other vascular beds, which then leads to poor vascular compliance, left ventricular hypertrophy, and arrhythmias with sudden cardiac death. cal- cification may be a marker of overall cvd status, rather than a specific risk factor for a unique cardiac event. consequently, vitamin d effects on vascular calcification may be another mechanism whereby activated vitamin d plays a protective role in ckd, where a profound deficiency of this important hormone is highly prevalent. what does this study mean for the general population? these observational studies, like those published in the past, require confirmation by trials. although the general popula- tion does not have as profound a vitamin d deficiency as those with ckd, a significant portion of the population in the united states have calcidiol levels � ng/ml. this deficiency may put them at risk for the development of diabetes, hyper- tension, and possibly cac. it is important to remember that high levels of vitamin d may also have deleterious effects; therefore, exuberant use of this therapy should not be encour- aged. randomized clinical trials of vitamin d supplementation are needed to evaluate whether the associations seen in these observational studies will translate into true effects or just rep- resent confounding as in many classic examples. these studies may be better performed in patients with ckd, in whom there is more profound deficiency and thus a larger effect (if one exists) may be seen. the challenge, of course, is when we will obtain these definitive results. acknowledgments m.l.m. is supported by k dk from the national institutes of health and by an american heart association clinically applied research award; r.t. is supported by grant hl from the na- tional institutes of health. disclosures m.l.m. has received honoraria from medscape and the american society of nephrology; r.t. has received grant support from abbott laboratories and speaking honoraria from abbott and genzyme. references . teng m, wolf m, ofsthun mn, lazarus jm, hernan ma, camargo ca jr, thadhani r: activated injectable vitamin d and hemodialysis sur- vival: a historical cohort study. j am soc nephrol : – , . wolf m, shah a, gutierrez o, ankers e, monroy m, tamez h, steele d, chang y, camargo ca jr, tonelli m, thadhani r: vitamin d levels and early mortality among incident hemodialysis patients. kidney int : – , . shoben ab, rudser kd, de boer ih, young b, kestenbaum b: asso- ciation of oral calcitriol with improved survival in nondialyzed ckd. j am soc nephrol : – , editorials www.jasn.org journal of the american society of nephrology j am soc nephrol : – , . ravani p, malberti f, tripepi g, pecchini p, cutrupi s, pizzini p, mallamaci f, zoccali c: vitamin d levels and patient outcome in chronic kidney disease. kidney int : – , . melamed ml, michos ed, post w, astor b: -hydroxyvitamin d levels and the risk of mortality in the general population. arch intern med : – , . wang tj, pencina mj, booth sl, jacques pf, ingelsson e, lanier k, benjamin ej, d’agostino rb, wolf m, vasan rs: vitamin d deficiency and risk of cardiovascular disease. circulation : – , . giovannucci e, liu y, hollis bw, rimm eb: -hydroxyvitamin d and risk of myocardial infarction in men: a prospective study. arch intern med : – , . pittas ag, harris ss, stark pc, dawson-hughes b: the effects of calcium and vitamin d supplementation on blood glucose and mark- ers of inflammation in nondiabetic adults. diabetes care : – , . forman jp, giovannucci e, holmes md, bischoff-ferrari ha, tworoger ss, willett wc, curhan gc: plasma -hydroxyvitamin d levels and risk of incident hypertension. hypertension : – , . li yc, kong j, wei m, chen zf, liu sq, cao lp: , -dihydroxyvitamin d( ) is a negative endocrine regulator of the renin-angiotensin system. j clin invest : – , . thadhani r: activated vitamin d regresses cardiac hypertrophy and attenuates progression to heart failure in dahl salt-sensitive rats [ab- stract]. j am soc nephrol : f-fc , . weishaar re, simpson ru: vitamin d and cardiovascular function in rats. j clin invest : – , . xiang w, kong j, chen s, cao lp, qiao g, zheng w, liu w, li x, gardner dg, li yc: cardiac hypertrophy in vitamin d receptor knock- out mice: role of the systemic and cardiac renin-angiotensin systems. am j physiol endocrinol metab : e –e , . kunitomo m, kinoshita k, bando y: experimental atherosclerosis in rats fed a vitamin d, cholesterol-rich diet. j pharmacobiodyn : – , . mathew s, lund rj, chaudhary lr, geurs t, hruska ka: vitamin d receptor activators can protect against vascular calcification. j am soc nephrol : – , . michos ed, streeten ea, ryan ka, rampersaud e, peyser pa, bielak lf, shuldiner ar, mitchell bd, post w: serum -hydroxyvitamin d levels are not associated with subclinical vascular disease or c-reactive protein in the old order amish. calcif tissue int : – , . watson ke, abrolat ml, malone ll, hoeg jm, doherty t, detrano r, demer ll: active serum vitamin d levels are inversely correlated with coronary calcification. circulation : – , . shroff r, egerton m, bridel m, shah v, donald ae, cole tj, hiorns mp, deanfield je, rees l: a bimodal association of vitamin d levels and vascular disease in children on dialysis. j am soc nephrol : – , . de boer ih, kestenbaum b, shoben ab, michos ed, sarnak mj, siscovick ds: -hydroxyvitamin d levels inversely associate with risk for developing coronary artery calcification. j am soc nephrol : – , . kronmal ra, mcclelland rl, detrano r, shea s, lima ja, cushman m, bild de, burke gl: risk factors for the progression of coronary artery calcification in asymptomatic subjects: results from the multi-ethnic study of atherosclerosis (mesa). circulation : – , . detrano r, guerci ad, carr jj, bild de, burke g, folsom ar, liu k, shea s, szklo m, bluemke da, o’leary dh, tracy r, watson k, wong nd, kronmal ra: coronary calcium as a predictor of coronary events in four racial or ethnic groups. n engl j med : – , . chertow gm, raggi p, mccarthy jt, schulman g, silberzweig j, kuhlik a, goodman wg, boulay a, burke sk, toto rd: the effects of sevel- amer and calcium acetate on proxies of atherosclerotic and arterio- sclerotic vascular disease in hemodialysis patients. am j nephrol : – , . fellstrom bc, jardine ag, schmieder re, holdaas h, bannister k, beutler j, chae dw, chevaile a, cobbe sm, gronhagen-riska c, de lima jj, lins r, mayer g, mcmahon aw, parving hh, remuzzi g, samuelsson o, sonkodi s, sci d, suleymanlar g, tsakiris d, tesar v, todorov v, wiecek a, wuthrich rp, gottlow m, johnsson e, zannad f: rosuvastatin and cardiovascular events in patients undergoing hemo- dialysis. n engl j med : – , . looker ac, pfeiffer cm, lacher da, schleicher rl, picciano mf, yetley ea: serum -hydroxyvitamin d status of the us population: – compared with – . am j clin nutr : – , see related article, “ -hydroxyvitamin d levels inversely associate with risk for developing coronary artery calcification,” on pages – . editorialswww.jasn.org j am soc nephrol : – , editorials wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ drusen and rpe atrophy automated quantification by optical coherence tomography in an elderly population drusen and rpe atrophy automated quantification by optical coherence tomography in an elderly population this article has been corrected since advance online publication and a corrigendum is also printed in this issue. b diniz ; , dc rodger ; , vr chavali , t mackay , sy lee ; , d stambolian and svr sadda ; abstract purpose correlate oct-derived measures of drusen and retinal pigment epithelium (rpe) atrophy areas (ras) with demographic features in an elderly population. patients and methods subjects aged years and older underwent cirrus oct scanning. drusen area and volume were obtained from the macula within a central circle (cc) of mm and a surrounding perifoveal ring (pr) of – mm, using the rpe analysis software ( . ). ra measurements were generated for the � mm retinal area. gender, age, smoking status, and systolic blood pressure (sbp) were considered. results a total of eyes were included. ra was larger in women ( . ± . vs . ± . mm , p¼ . ) and with increasing age. the pr drusen area increased with increasing age (po . ), whereas the cc drusen area remained stable after the age of years ( . ± . mm for ages – years and . ± . mm for ages years). drusen volume in the cc was smaller after the age of years ( . ± . mm ) compared with the - to -year-old group ( . ± . mm ). drusen measurements were similar between smokers and nonsmokers, but the pr drusen area ( . mm , p¼ . ) and volume ( . mm , p¼ . ) were correlated with years smoked. ra ( . mm , p¼ . ), pr drusen area ( . mm , p¼ . ), and volume ( . mm , p¼ . ) were found to be directly associated with sbp. there was a high correlation between the eyes of the same subject. conclusion oct-based automated algorithms can be used to analyze and describe drusen and geographic atrophy burden in such population-based studies of elderly patients. eye ( ) , – ; doi: . /eye. . ; published online november introduction age-related macular degeneration (amd) is the leading cause of irreversible, severe visual loss in developed countries among people of age years and older. several studies have identified a multitude of potential risk factors associated with amd, but the results have not always been conclusive. aside from age and cigarette smoking, previous findings of associations between amd and other risk factors, such as hypertension, alcohol consumption, systolic blood pressure (sbp), sunlight exposure, and cardiovascular disease, have been inconsistent. drusen—in particular the large drusen associated with pigment epithelium abnormalities—are a known risk factor for progression to advanced amd. drusens are extracellular deposits that accumulate between the retinal pigment epithelium (rpe) and the inner collagenous layer of bruch’s membrane. geographic atrophy (ga) occurs in the late stage of non-neovascular amd and is the second most common cause of severe visual loss due to amd, the most common being choroidal neovascularization. ga is characterized by the development and gradual enlargement of atrophic patches that involve the rpe, the overlying neurosensory retina, and the underlying choriocapillaris. bilateral involvement of drusen and late-stage amd have long been recognized, – and concordance or symmetry between eyes has been demonstrated for the individual phenotypes. those studies were based on doheny eye institute, los angeles, ca, usa department of ophthalmology, universidade federal de são paulo, são paulo, brazil department of ophthalmology, keck school of medicine of the university of southern california, los angeles, ca, usa department of ophthalmology, university of pennsylvania, philadelphia, pa, usa correspondence: b diniz, vistta ophthalmology, al. ricardo paranhos n , goiania-go , brazil tel: + ; fax: + . e-mail: drbrunodiniz@ gmail.com received: january accepted in revised form: september published online: november c l in ic a l s t u d y eye ( ) , – & macmillan publishers limited all rights reserved - x/ www.nature.com/eye http://dx.doi.org/ . /eye. . mailto:drbrunodiniz@gmail.com mailto:drbrunodiniz@gmail.com http://www.nature.com/eye clinical examination or postmortem hitopathological analysis. until recently, color fundus photography was the only imaging modality used to evaluate the severity and progression of amd in major epidemiologic eye disease studies. however, this imaging technique has limitations with regard to the measurement of the geometric area of rpe atrophy and to the precise quantification of drusen boundaries. measurement of these types of data is confounded on color fundus photography by interpatient variability of fundus pigmentation and media opacities and by the exposure settings used. optical coherence tomography (oct) provides the opportunity to assess the effect of drusen and rpe atrophy area (ra) on the geometry of the rpe in vivo. segmentation algorithms have been developed to extract quantitative information from the new spectral-domain (sd)-oct data. quantification of drusen and ra has been demonstrated with both manual and automated strategies, and recently automated quantification techniques have become available in commercial sd-oct (carl zeiss meditec inc, dublin, ca, usa) software. validation of the accuracy and reproducibility , , of these fully automated algorithms has created an opportunity to study amd lesions in population studies. to our knowledge, the epidemiologic features of amd have not previously been associated with automated oct quantification data. herein, we describe oct-derived measures of drusen and ra in an elderly population and correlate these oct findings with other demographic features. methods subjects an ongoing study to identify multiple eye diseases in the old order amish community living in lancaster and franklin counties in pennsylvania has identified a large number of amish individuals with early–late-onset amd. details of the epidemiology of amd in this population are being isolated in an attempt to elucidate genetic factors that may confer susceptibility and predispose some amish individuals and their families to developing amd. the current study, approved by the institutional review board of the university of pennsylvania, was instituted as an investigation to assess the value of the oct images and quantify changes related to amd in the macula. study participants, all aged years and older, were asked to complete a brief questionnaire about personal exposures. present or past cigarette smoking history and number of years smoked were self-reported. a qualified examiner obtained sbp on the day of the ophthalmic evaluation. all study participants who agreed to enroll and provide informed consent had digital stereo images of the macula, optical disc, and macular oct scans. optical coherence tomography acquisition a cirrus hd-oct instrument (carl zeiss meditec inc) was used for this study. each eye was imaged in a single session using a volume scan protocol ( � mm ) whose repeatability for drusen quantification has been well established and described in the previous publication. each scan covered a retinal area of � mm , centered on the fovea, and was obtained after dilation. the scans were assessed after acquisition and repeated if necessary to achieve optimal image quality (signal strength ). quantification of drusen and ra the raw oct datasets were exported to a computer for analysis with an algorithm developed by zeiss used to segment the rpe ( . ; carl zeiss meditec inc), cleared by the fda and now commercially available. this algorithm, which has been recently validated by comparison with manual segmentation, , uses the rpe geometry and compares this segmentation map with a virtual map of the rpe free of deformations (rpe floor) (figure ). using these two maps, the algorithm creates an elevation map that permits measurement of drusen area and volume. elevations of o mm are not included. areas of atrophy are detected by the algorithm as contiguous or geographic areas of hyperintensity. these areas of hyperintensity are evident in partial oct- projection maps obtained by summing a-scans from a choroidal ‘slab’ below the rpe (loss of rpe in lesions resulted in increased choroidal reflectivity). eyes with poor quality scans (signal strength o ) or features of neovascular amd, such as subretinal fluid, intraretinal edema, subretinal tissue, or serous pigment epithelial detachments, in the oct b-scans were excluded from subsequent quantitative lesion analysis. because an important component of the planned analyses was to look for associations between the eyes, if one eye was ineligible (owing to missing scans, poor quality scans, or neovascular amd) both the eyes of that subject were removed from subsequent analysis. the cirrus automated algorithm provided quantitative measurements of macular drusen area and volume within a central circle (cc) of mm diameter and a surrounding perifoveal ring (pr) of – mm diameter, both of which were centered on the fovea. the ra measurements were provided for the entire � mm by the software. amd automated quantification by optical coherence tomography b diniz et al eye statistical methods the variables considered in this analysis included age (continuous and separated into four categories: – , – , – , and years of age), gender, past or present smoking history, years smoked (continuous and three categories: o , – , and years), sbp (continuous and separated into three categories: o , – , and mm hg), and the various quantitative measurements of macular drusen area, drusen volume, and ra for the right and left eyes. comparison between the groups was done by analysis of variance. demographic comparisons between males and females used an independent samples t-test for mean age and w -square tests for frequencies by age groups and smoking status. because these data included measures for both the eyes for all subjects, the analyses that tested the associations with oct areas and volume used generalized estimating equations (gee), which adjusted the correlation between the eyes. paired t-tests were used to compare mean oct measurements between the right and left eyes, and spearman correlations were used to test the association of continuous age, years smoked (adjusted for age), and sbp with subject-averaged oct measures. sas v . (sas inst., cary nc, usa) software was used for all analyses; the accepted level of significance was po . . results a total of subjects were recruited for this study, but subjects were excluded owing to poor quality scans, missing scans, or features of neovascular amd on oct in at least one eye. thus, eyes of consecutive subjects were included in this analysis. the sample included women and men with a mean age of ± . years (range – years). the majority of the population studied was between and years of age ( . %). there was no significant difference in age distribution (p ¼ . ) between male and female groups. twenty-three percent of the population had a history of smoking, with a much higher prevalence in the male population ( . % vs . %, po . ). age distribution and smoking status are shown in table . the drusen area and volume measurements were similar between males and females, but the ra found by oct was significantly larger in women ( . ± . vs . ± . mm , p ¼ . ). for both males and females, the average drusen area and volume were larger in the cc than in the pr (table ). one-way analysis of variance determined that the drusen area, drusen volume, and ra differed among the various age groups (po . , for all quantitative measurements). the perifoveal drusen area and volume, as well as the ra, were larger in older patients. the drusen area of the cc, in contrast, was relatively similar in patients of years of age or more, with an area of . ± . mm for the – -year age group and . ± . mm for the -year age group, and the cc drusen volume was smaller in the age -year age group ( . ± . mm ) when compared with the subjects in the – -year age group ( . ± . mm ). figure the cirrus automated algorithm provides quantitative measurements of macular drusen area and volume within a mm cc and a – mm surrounding pr. the ra measurements are provided for the entire � mm area. it uses the rpe geometry (black line) and compares this segmentation map with a virtual map of the rpe free of deformations (pink line). using these two maps, the algorithm creates an elevation map that permits measurement of drusen area and volume. amd automated quantification by optical coherence tomography b diniz et al eye in the older subjects (the -year age group), the perifoveal drusen area ( . ± . mm ) and volume ( . ± . mm ) were slightly larger than those of the cc ( . ± . mm and . ± . mm ). pr and cc drusen area and volume and ra measurements were similar between the nonsmoking and smoking groups. in the smoking group, however, the perifoveal drusen area (p ¼ . ) and volume (p ¼ . ) had an increasing correlation with the number of years smoked (table ) when adjusted for age. no significant difference was noted between the groups with different smoking duration. the oct findings were associated with sbp (table ). larger drusen area and volume in the pr, as well as ra, were correlated with increase in sbp. the drusen area in the cc differed significantly among the sbp groups (p ¼ . ). measurements were . ± . mm for the o mm hg group, . ± . mm for the – mm hg group, and . ± . mm for the mm hg group. a significant correlation (po . ) was found between the right and left eyes with respect to drusen area, drusen volume, and ra, as determined by oct. cc drusen area and volume showed a stronger correlation between eyes compared with the pr (for area r ¼ . vs . and volume r ¼ . vs . , po . for all correlations). the correlation coefficient for ra between eyes was . . table demonstrates the relationship between drusen measurements in the cc and pr, as well as their relationship with ra in the same eye. the volume measured appeared to show a similar correlation compared with area (r ¼ . vs . ). the larger ra was also correlated with larger drusen area in the pr. discussion segmentation of the retinal layers and measurement of drusen area, drusen volume, and ra would appear to be critical for quantitative amd studies. automated algorithms to segment the oct images accurately and table demographic characteristics of participants number (%) total male female p-valuea characteristics ( . ) ( . ) age, years . t mean . . range – – – age group . c – ( . ) ( . ) ( . ) – ( . ) ( . ) ( . ) – ( . ) ( . ) ( . ) z ( . ) ( . ) ( . ) unknown smoking status o . c unknown ( . ) ( . ) ( . ) non-smoking ( . ) ( . ) ( . ) smoker ( . ) ( . ) ( . ) years smoked mean . range – r – z unknown a t, independent samples t-test; c, w test. table association of age and gender with oct areas and volumes area cc area pr volume cc volume pr ra sex, mean±sd male (n ¼ ) . ± . . ± . . ± . . ± . . ± . female (n ¼ ) . ± . . ± . . ± . . ± . . ± . p-valuea . . . . . age group (years) – (n ¼ ) . ± . . ± . . ± . . ± . . ± . – (n ¼ ) . ± . . ± . . ± . . ± . . ± . – (n ¼ ) . ± . . ± . . ± . . ± . . ± . z (n ¼ ) . ± . . ± . . ± . . ± . . ± . p-valuea o . o . o . o . o . spearman correlation with continuous age . , p ¼ . . , po . . , po . . , po . . , po . abbreviations: cc, central circle; pr, perifoveal ring; ra, retinal pigment epithelium atrophy. a generalized estimating equations (gee). amd automated quantification by optical coherence tomography b diniz et al eye reproducibly have been described, – and, importantly, have now become available in commercial devices. in the present study, we demonstrate that these algorithms may be used to study and describe drusen and ra burden in a population-based study of elderly patients. our analysis confirms that, in general, larger and more abundant drusen are found with increasing age, but in the cc, after the age of years, the drusen volume and area appear to stabilize and are eventually smaller after the age of years. although this correlation between age and drusen area/volume is statistically significant, the strength of the association is relatively weak (r ¼ . – . ), suggesting that many other factors (eg, genetic and environment) may contribute to the variability in drusen burden. ra, in contrast, is progressively larger with advanced age when analyzed using this segmentation algorithm. although the ra algorithm measures the entire � mm and not the cc alone, one suspects that the increase in the ra in the central area may account for some of the apparent decrease in the cc drusen measurements in the -year age group. these findings may be consistent with the regression of soft macular drusen and evolution of atrophy that has been described in clinical and hitopathological studies. gass first noted that drusen could fade and disappear, leaving only an irregular mottling of the rpe. he also observed that most cases of ga followed the fading of drusen, resulting in degeneration and atrophy of the rpe and photoreceptors. our observations agree with the clinical impression that ga is rare before the age of years and that it becomes more common in individuals aged years or older. drusen area and volume were similar in males and females, but interestingly, ra was significantly more common in women. according to one study, the diagnosis of late amd has been reported to be slightly more frequent in women than in men. in this study a higher prevalence of neovascular amd and no significant difference in ga was observed. other studies show a similar prevalence of any amd stage in males and females. table association of smoking and sbp with oct areas and volumes area cc area pr volume cc volume pr ra smoking status, mean±sd nonsmokers (n ¼ ) . ± . . ± . . ± . . ± . . ± . smokers (n ¼ ) . ± . . ± . . ± . . ± . . ± . p-valuea . . . . . years smoked r (n ¼ ) . ± . . ± . . ± . . ± . . ± . – (n ¼ ) . ± . . ± . . ± . . ± . . ± . z (n ¼ ) . ± . . ± . . ± . . ± . . ± . p-valuea . . . . . sbp, mm hg o (n ¼ ) . ± . . ± . . ± . . ± . . ± . – (n ¼ ) . ± . . ± . . ± . . ± . . ± . z (n ¼ ) . ± . . ± . . ± . . ± . . ± . p-valuea . . . . . spearman correlation with continuous years smoked, adjusted for age . , p ¼ . . , p ¼ . . , p ¼ . . , p ¼ . . , p ¼ . spearman correlation with continuous sbp . , p ¼ . . , p ¼ . . , p ¼ . . , p ¼ . . , p ¼ . abbreviations: cc, central circle; oct, optical coherence tomography; pr, perifoveal ring; ra, retinal pigment epithelium atrophy; sbp, systolic blood pressure. a generalized estimating equations (gee). table correlations between rpe atrophy area and drusen area and volume in the cc and pr variable variable spearman correlation coefficient p-value area cc area pr . o . volume cc volume pr . o . area cc ra . o . area pr ra . o . volume cc ra . o . volume pr ra . o . abbreviations: cc, central area; pr, perifoveal ring; ra, retinal pigment epithelium atrophy. amd automated quantification by optical coherence tomography b diniz et al eye cigarette smoking has been found to be related to late age-related maculopathy in some, – but not all, – epidemiologic studies. a smoking history was neither associated with larger drusen area and volume, nor with ra in our population. in part, the small number of cases, particularly of women smokers, may explain this. we did not make any distinction between current past smoking history, and possibly some of our subjects were former smokers. it has been reported that, although previous smoking history also increases the progression of amd, current smoking is more likely to increase the incidence of amd lesions. mccarty et al argued that the total duration of smoking history was the most significant factor for development of amd rather than pack years, current, or former smoking status. in other studies, the pack years of cigarette smoking has proven to be a better correlate than the number of years of smoking, and it was the most significant risk factor for amd. a dose– response effect has been demonstrated: the risk of developing amd increases as the intensity of smoking increases. in our amish population, an increased duration of smoking did appear to be associated with a larger drusen area and volume in the pr. in the present study, an increase in sbp was directly correlated with larger drusen area and volume in the perifoveal zone and with larger ra. the relationship between blood pressure and amd has proven to be inconsistent in epidemiologic population-based studies, with some studies suggesting an increased risk of developing amd with increased pulse pressure. in the beaver dam study, sbp, hypertension, and pulse pressure were significantly associated with rpe depigmentation, progression of amd, and incidence of neovascular amd, but not ga. the mechanism by which hypertension can increase the risk of amd is not immediately apparent. however, age-related degenerative changes in collagen and elastin are known to produce a decrease in the distensibility of blood vessels. this in turn results in higher sbp and lower diastolic blood pressure and widening of the pulse pressure. klein et al postulated that a wide pulse pressure may be a marker of similar degenerative changes also occurring in the bruch membrane of these individuals’ eyes, thus increasing the risk of development and progression of amd. although we found a significant correlation, our conclusions are limited as the blood pressure reading was acquired just once and may not reflect a chronic systemic hypertension diagnosis. the right and left eyes of subjects demonstrate a high correlation in drusen and ra measurements using the automated oct algorithms. this finding is in agreement with a previous analysis that demonstrated good to excellent symmetry for all ophthalmoscopic and fluorescein angiographic characteristics of drusen in the central and peripheral areas of the posterior pole between both the eyes of subjects with amd. sunness et al described a high correlation in the size and progression of ga between both eyes. studies suggest that – % of prevalent ga cases show bilateral involvement. thus, our automated oct observations of ra are consistent with previous observations of bilateral incidence and with ophthalmoscopic assessment of amd symmetry. , despite these apparent observations and associations, our study is not without limitations. first, although this was a population study, we only studied individuals aged years or older. thus, information about oct- quantified drusen burden in younger individuals is unknown. our study does demonstrate that oct-based assessment can be applied to population analyses. furthermore, our oct data may be correlated with future genetic information that may be forthcoming from this population. second, our analysis only used the automated quantitative data provided by the instrument. we did not perform additional qualitative analysis of drusen morphology or other oct features of amd, such as the presence of intraretinal hyperreflective foci (known to correlate with areas of rpe migration), nor did we correct subtle segmentation errors. on the other hand, by using commercial algorithms that had been fda-cleared and previously validated for reliability and accuracy, the data considered in our analysis can easily be compared with the data obtained in future studies in other and larger populations. third, the commercial drusen segmentation algorithm only includes rpe elevations that exceed a threshold of mm. although very shallow drusen are likely to be ignored by this analysis, they would probably only contribute a small amount to the total area or volume in eyes with significant numbers of larger drusen even if they were to be included. fourth, color fundus photographs were not classified or compared with the drusen and ra segmented by the software. color fundus photographs offer potentially complementary information to sd-oct and both may have an important role in assessing drusen and ga in patients with non-exudative amd. while this would have been an interesting analysis, this has been evaluated in other studies. finally, the commercial ra segmentation algorithm was designed to include all contiguous areas that demonstrated significant transmission of light into the choroid with associated increased choroidal reflectivity. although these measurements have been shown to compare favorably with fundus autofluorescence-based measures of ra, areas of severe depigmentation of the rpe without actual loss of the rpe may produce a similar appearance on oct and thus may be inadvertently included in the oct-derived ra measurements. amd automated quantification by optical coherence tomography b diniz et al eye polarization-sensitive sd-oct combines the advantage of sd-oct imaging with a selective identification of the polarization state of backscattered light. because the melanosomes of the rpe change the polarization state in a random manner, ps-oct is capable of precisely depicting its exact location and configuration. this selectivity can be used for a reliable automated segmentation of alterations at the retina–rpe boundary. further studies are needed to evaluate the differences between the two methods. despite these limitations, the oct-derived automated analyses in the amish population suggest that increasing age appears to be an important risk factor for increased oct drusen area and volume in the cc and pr although this increase in drusen area in the cc does not continue after the age of years. drusen and ra measurements are highly correlated and similar between the eyes of the same individual. the number of continuous years smoked and sbp are also associated with an increased area and volume of perifoveal drusen. female gender, age, and sbp are all associated with ra to a greater extent. these findings may be useful as a starting point in guiding future, larger, population- based studies on the prevalence of features of non- neovascular amd, and in better phenotyping and characterization of populations for genetic association studies. summary what was known before k color fundus photography was the only imaging modality used to evaluate the severity and progression of amd in major epidemiologic eye disease studies. k this imaging technique has limitations with regard to the measurement of rpe atrophy and to the precise quantification of drusen boundaries. k oct provides the opportunity to assess the effect of drusen and ra on the geometry of the rpe in vivo. what this study adds k automated oct-based assessment of lesions associated with non-neovascular amd can be applied to population analyses and may allow for more complete and quantitative assessment of patients for longitudinal assessments. conflict of interest dr sadda has previously shared in royalties from intellectual property licensed by the doheny eye institute to topcon medical systems. he has served in the past as a consultant to heidelberg engineering and receives research support from optos, carl zeiss meditec, and optovue. the remaining authors declare no conflict of interest. acknowledgements we thank laurie dustin for statistical analysis and susan clarke for text review. this work was supported in part by nei core grant ey , the beckman institute for 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bird ac. bilaterality of drusen. br j ophthalmol ; : – . jain n, farsiu s, khanifar a, bearelly s, smith rt, izatt ja et al. quantitative comparison of drusen segmented on sd oct versus drusen delineated on color fundus photographs. invest ophthalmol vis sci ; : – . yehoshua z, rosenfeld pj, gregori g, feuer wj, falcão m, lujan bj et al. progression of geographic atrophy in age-related macular degeneration imaged with spectral domain optical coherence tomography. ophthalmology ; : – . schlanitz fg, baumann b, spalek t, schütze c, ahlers c, pircher m et al. performance of automated drusen detection by polarization-sensitive optical coherence tomography. invest ophthalmol vis sci ; : – . amd automated quantification by optical coherence tomography b diniz et al eye 	drusen and rpe atrophy automated quantification by optical coherence tomography in an elderly population 	introduction 	methods 	subjects 	optical coherence tomography acquisition 	quantification of drusen and ra 	statistical methods 	results 	discussion 	acknowledgements 	references diabetes care, volume , number , january o b s e rvat i o n s diabetes scre e n i n g practices among i n d i v i d u a l s a g e d ye a r s a nd o l d e r i n , the american diabetes associa- tion (ada) adopted new re c o m m e n d a- tions for screening the general popula- tion aged years for diabetes every years with an emphasis on those at high risk for undiagnosed diabetes ( ). few studies have examined the extent to which this screening has been adopted. this re p o rt describes the results of a telephone s u rvey of montana residents aged years to assess the diabetes screening prac- tices in this population. f rom october to december , the montana department of public health and human services conducted a random household telephone survey of montana residents aged years living in counties. respondents indicated whether they ever had been told by a physician that they had diabetes, the number of visits they made to a health care provider during the past year, their family history of dia- betes, whether they had ever been told they had high cholesterol and/or high blood pre s s u re, and their height and weight. respondents were asked the fol- lowing question to identify whether they had ever been screened for diabetes: “glu- cose or sugar is a substance found in your blood. have you ever had your blood glu- cose or sugar checked to see if you have diabetes?” when respondents re s p o n d e d “yes” to this question, they were asked to identify when screening was completed (“when was the last time your blood glu- cose or sugar level was measured by a health care professional?”). the re s p o n s e categories for this question included within the past year, within the past years, years ago, do not know/not sure, and refused to answer. pearson tests were used to assess associations between dia- betes screening and risk factors for dia- betes. logistical re g ression analyses were conducted to identify independent vari- ables associated with screening for diabetes during the past year. odds ratios ( % cis) were calculated. of the , respondents, ( . %) re p o rted that they had diagnosed diabetes. the remaining , respondents re p o rt e d that they did not have diagnosed diabetes and are included in the following analyses. of the respondents, % re p o rted a family h i s t o ry of diabetes, % re p o rted a bmi kg/m , % re p o rted having hyper- tension, and % re p o rted having high c h o l e s t e rol. excluding age, % of re s p o n- dents had one risk factor for diabetes, and % had two or more risk factors. of the , respondents without diagnosed dia- betes, % re p o rted that they had been s c reened for diabetes during the past year, % re p o rted screening from to years ago, and % re p o rted screening years ago or having never been scre e n e d . respondents who re p o rted being s c reened for diabetes during the past year w e re more likely to be age years and to have a family history of diabetes, two or m o re visits to a health care provider dur- ing the past year, hypertension, and high c h o l e s t e rol levels (table ). we found no association between recent screening and sex ( % men vs. % women), ameri- can indian ancestry ( % yes vs. % no), or bmi ( % kg/m vs. % kg/m ). respondents with three or m o re risk factors (e.g., aged years, american indian ancestry, family history of diabetes, hypertension, high choles- t e rol, or bmi kg/m ) were more likely to be screened for diabetes compared with respondents with only one risk factor ( vs. %, respectively). however, % of individuals with two risk factors for dia- betes and % of individuals with more than three risk factors had not been s c reened during the past years. based on logistical re g ression analysis, t h ree factors were associated with scre e n i n g for diabetes during the past year: two or m o re visits to a health care provider during the past year ( . [ % ci . – . ]), high cholesterol level ( . [ . – . ]), and family history of diabetes ( . [ . – . ]). respondents aged – years were less likely to re p o rt re c e n t s c reening than those aged years ( . [ . – . ] ) . a limitation of this assessment is that these data are self-re p o rted. pre v i o u s studies, however, have found that self- re p o rts of conditions such as diabetes and h y p e rtension are reliable ( , ). in addi- tion, the survey was conducted by tele- phone and does not re flect the experience of individuals in montana homes without t e l e p h o n e s . the findings suggest that diabetes s c reening is being adopted by physicians for individuals aged years at risk for diabetes and that the ada re c o m m e n d a- tions are being implemented in the general c o m m u n i t y. however, these data also indi- cate a need to develop strategies to encour- l e t t e r s table —characteristics of respondents aged years reporting screening for diabetes in montana in s c reening for diabetes past year – years years or never n age (years) – ( ) ( ) ( ) – ( ) ( ) ( ) ( )* ( ) ( ) family history of diabetes ye s ( )* ( ) ( ) n o ( ) ( ) ( ) visits to a health care provider during the past year ( )* ( ) ( ) ( ) ( ) ( ) h y p e rt e n s i o n ye s ( )* ( ) ( ) n o ( ) ( ) ( ) high cholestero l ye s ( )* ( ) ( ) n o ( ) ( ) ( ) data are n (%). *p . . diabetes care, volume , number , january letters age screening among all individuals at high risk for diabetes. todd s. harwell, mph jane g. smilie, ba janet m. mcdowall, rn, bsn steven d. helgerson, md, mph dorothy gohdes, md f rom the montana diabetes project, montana d e p a rtment of public health and human serv i c e s , helena, montana. a d d ress correspondence to todd s. harw e l l , mph, montana department of public health and human services, cogswell building, c- , p. o . box , helena, mt - . e-mail: t h a rw e l l @ s t a t e . m t . u s . a c k n o w l e d g m e n t s — this project was sup- p o rted through a cooperative agreement (u- / ccu- - ) with the centers for disease c o n t rol and prevention, division of diabetes translation, atlanta, georg i a . we thank linda priest and the staff mem- bers at northwest resource consultants for their work on the telephone surv e y. the contents of this letter are solely the responsibility of the authors and do not neces- sarily re p resent the official views of the centers for disease control and pre v e n t i o n . r e f e re n c e s . american diabetes association: screening for type diabetes. diabetes care (suppl. ): s –s , . kehoe r, wu sy, leske mc, chylack lt jr: comparing self-re p o rted and physician- re p o rted medical history. am j epidemiol : – , . jackson c, jatulis de, fortmann sp: the behavioral risk factor survey and the stan- f o rd five-city project survey: a comparison of cardiovascular risk behavior estimates. am j public health : – , h b a c is not recommended as a s c reening test for diabetes in cystic fibro s i s i n the june issue of diabetes care, h u n k e rt et al. ( ) recommend the use of h b a c for early detection of cystic fib ro- s i s – related diabetes (cfrd). their re c o m- mendation is based on the finding that mean hba c is slightly higher in cystic fib rosis (cf) patients requiring insulin t h e r a p y, compared with cf patients with i m p a i red or normal glucose tolerance. h o w e v e r, no data on the validity of this a p p roach for the diagnosis of asympto- matic diabetes in patients with cystic fib ro- sis are pre s e n t e d . our group has a long-standing intere s t in the early diagnosis of cfrd, comparing fasting blood glucose levels with oral glu- cose tolerance test results ( ). in our series, we have now cf patients with newly diagnosed diabetes based on a -h venous plasma glucose value mg/dl ( . mmol/l), and simultaneous determ i n a t i o n of hba c ( h i g h - p e rf o rmance liquid chro- matography method [pharmacia, erlan- gen, germany], normal range . – . %). only out of cf patients ( %) diag- nosed as diabetic according to the ameri- can diabetes association and world health o rganization criteria ( ) had an hba c value above the normal range (individual values . , . , . , and . %). in nine diabetic cf patients with normal hba c, values between . and . % were e n c o u n t e red (mean ± sd, . ± . %). the mean -h blood glucose value after inges- tion of oral glucose was not signific a n t l y d i ff e rent between diabetic cf patients with n o rmal hba c ( ± mg/dl, mean ± sd) and diabetic cf patients with elevated h b a c ( ± mg/dl, student’s t t e s t ) . these data clearly demonstrate that the d e t e rmination of hba c is not able to sub- stitute for the oral glucose tolerance test in the early diagnosis of cfrd. our fin d i n g s a re in agreement with several re p o rts in the l i t e r a t u re ( – ) as well as the consen- sus conference on cfrd ( ). in addition to its low sensitivity when used as a diagnos- tic tool for the detection of cfrd, the mea- s u rement of hba c has the disadvantage of considerable interassay variability and lack of standardization. there f o re, we stro n g l y advise against the use of glycosylated hemoglobin as a screening test for the early diagnosis of diabetes in patients with cystic fib ro s i s . reinhard w. holl, md christian buck, md christine babka, md anna wolf, md angelika thon, md f rom the department of pediatrics (r.w.h.), uni- versity of giessen, giessen; the department of pedi- atrics (c.bu., c.ba., a.w.), the university of ulm, ulm; and the medical school hannover (a.t. ) , h a n n o v e r, germ a n y. a d d ress correspondence to pd dr. reinhard w. holl, universitätskinderklinik giessen, feulgenstr. , d- giessen, germ a n y. r e f e re n c e s . h u n k e rt f, lietz t, stach b, kiess w: potential impact of hba c d e t e rm i n a t i o n on clinical decision making in patients with cystic fib ro s i s – related diabetes (let- ter). diabetes care : – , . holl rw, buck c, cario h, wolf a, thon a, heinze e, kohne e, debatin k-m: diag- nosis of diabetes in cystic fib rosis and tha- lassemia major. diabetes care : – , . the expert committee on the diagnosis and classification of diabetes mellitus: r e p o rt of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care : – , . lanng s, hansen a, thorsteinsson b, n e rup j, koch c: glucose tolerance in patients with cystic fib rosis: five year p rospective study. b m j : – , . deluca f, arrigo t, nibali sc, sferlazzas c, gigante a, dicesare e, cucinotta d: insulin secretion, glycosylated haemoglo- bin and islet cell antibodies in cystic fib ro- sis children and adolescents with diff e re n t d e g rees of glucose tolerance. h o rm metab r e s : – , . moran a, doherty l, wang x, thomas w: a b n o rmal glucose metabolism in cystic fib rosis. j pediatr : – , . moran a: highlights of the febru a ry consensus conference on cfrd. bonn, g e rm a n y, cystic fibrosis foundation, p ro gln p e roxisome p ro l i f e r a t o r- a c t i v a t e d r e c e p t o r- and o b e s i t y r istow et al. ( ) re p o rted an activating mutation in the peroxisome pro l i f- e r a t o r-activated re c e p t o r- g e n e ( p ro gln ppa r - ), which was pre s e n t in % ( of ) of obese and % ( of ) of nonobese german caucasians. these findings may have profound implica- tions, particularly if the presence of this variant and its association with obesity are c o n firmed in other populations. we perf o rmed polymerase chain re a c- t i o n – restriction fragment length polymor- phism analysis for the pro gln ppa r - variant as described ( ) on dna samples f rom several independent populations, including lean and obese caucasians fro m the baltimore, maryland, region; african- diabetes care, volume , number , january letters americans from jackson, mississippi, and forsyth county, north carolina; pima indians from arizona; and old ord e r amish from lancaster county, pennsylva- nia (table ). a pcr fragment corre s p o n d- ing to gastric insulinotropic peptide, which has two known restriction sites for h i n dii, was mixed with each sample as a positive control. among a total of sub- jects ( , alleles), the pro gln variant was not detected in a single subject. these findings were unexpected because there is substantial overlap of gene pools of caucasians from central e u rope and the baltimore and amish caucasians studied ( ). the germ a n caucasians studied by ristow et al. were said to be unrelated and re c ruited fro m the nord rh e i n - westfalen region, but they may be a genetic isolate whose gene pool does not re flect that of other caucasian populations. altern a t i v e l y, if the individ- uals carrying the mutation were re l a t e d , the true frequency of the pro g l n p pa r - variant may have been overe s t i- mated. the absence or very low fre- quency of this variant has also been doc- umented in danish ( ) and german ( ) populations. this study is the first, to our knowledge, to examine american popu- lations for this variant. in summary, the study by ristow et al. demonstrating that the pro g l n p pa r - variant is activating and can influence body weight in people is important. however, this variant appears to be absent or very r a re in the american populations studied. additional studies are re q u i red in other regions of europe and the u.s. to furt h e r d e fine the relevance of this intere s t i n g genetic variant to susceptibility to obesity. alan r. shuldiner, md william nguyen, bs w.h. linda kao, phd brock a. beamer, md ross e. andersen, phd richard pratley, md frederick l. brancati, md, phd f rom the department of medicine (a.r.s., w. n . ) , university of maryland school of medicine; the d e p a rtments of medicine (b.a.b., f.l.b.) and epi- demiology (w.h.l.k.), johns hopkins university school of medicine, baltimore, maryland; and the national institute of diabetes and digestive and kidney diseases (r.p.), national institutes of health, phoenix, arizona. a d d ress correspondence to alan r. shuldiner, md, professor and head, division of endocrinol- o g y, diabetes, and nutrition, department of medi- cine, university of maryland school of medicine, w. lombard st., room s- , baltimore, md . e-mail:ashuldin@medicine.umary l a n d . e d u . a c k n o w l e d g m e n t s — this study was sup- p o rted by nih r dk- , the american diabetes association, glaxowellcome, the b a l t i m o re geriatrics research and education clinical center of the baltimore ve t e r a n s administration medical center. r e f e re n c e s . ristow m, muller- wieland d, pfeiffer a, k rone w, kahn cr: obesity associated with a mutation in a genetic regulator of adipocyte diff e rentiation. n engl j med : – , . c a v a l l i - s f o rza ll, menozzi p, piazza a: t h e h i s t o ry of human genes. princeton univer- sity press, princeton, nj, . elk j, urhammer sa, sorensen ti, ander- sen t, auwerx j, pedersen o: homozygosity of the pro ala variant of the pero x i s o m e p roliferation-activated re c e p t o r- g a m m a ( p par-gamma ): divergent modulating e ffects on body mass index in obese and lean caucasian men. diabetologia : – , . mamann a, munzberg h, buttron p, busing b, hinney a, mayer h, siegfried w, hebe- brand j, greten h: missense variants in the human peroxisome pro l i f e r a t o r- a c t i v a t e d re c e p t o r-gamma gene in lean and obese subjects. eur j endocrinol : – , insulin secre t i o n , insulin sensitivity, and glucose e ffectiveness in nonobese individuals with va ry i n g d e g rees of glucose to l e r a n c e a lthough it is well known that insulin s e c retion, insulin sensitivity, and glu- cose effectiveness are impaired in type diabetic patients ( – ), little is known about the role of each of these fac- tors individually on the evolution of type diabetes. in this context, a major issue is that hyperglycemia per se impairs insulin s e c retion and insulin sensitivity and that obesity observed in type diabetic patients per se causes insulin resistance ( , ). to o v e rcome this problem, we studied u n t reated nonobese subjects classified as having normal glucose tolerance (ngt) (n = ; bmi . ± . kg/m [ r a n g e . – . ], mean ± sem), impaired glu- cose tolerance (igt) (n = ; bmi . ± . kg/m [ . – . ]), and type dia- betes (n = ; fetal bovine serum . ± . mmol/l [range . – . ], bmi . ± . k g / m [ . – . ]), based on the criteria of the world health organization ( ). they were normotensive and had norm a l renal, hepatic, and thyroid function. insulin sensitivity and glucose eff e c t i v e n e s s w e re estimated by the minimal model a p p roach ( – ). insulin secretion was e x p ressed as the area under the insulin c u rve between and min after an intra- venous glucose injection ( ). after the data w e re analyzed by one-way analysis of vari- ance, bonferroni correction was used to evaluate the diff e rences between any two of the three groups we studied ( ). no signifi- cant difference was observed in bmi among the three groups. compared with the subjects with ngt, the subjects with table —characteristics of subjects screened for pro gln ppa r - n ( a l l e l e s age ± sd f e m a l e bmi ± sd p o p u l a t i o n t y p e d ) ( y e a r s ) ( % ) ( k g / m ) c a u c a s i a n s b a l t i m o re longitudinal ( ) . ± . . . ± . study on aging johns hopkins we i g h t ( ) . ± . . . ± . management center amish, lancaster, pa ( ) . ± . . . ± . a f r i c a n - a m e r i c a n s a t h e ro s c l e rosis risk in ( ) . ± . . . ± . communities study pima indians a r i z o n a ( ) . ± . — . ± . all non-amish subjects were unrelated; amish subjects were not fir s t - d e g ree relatives of each other. diabetes care, volume , number , january letters igt had significantly lower insulin secre- tion ( , ± vs. , ± pmol l m i n , p = . ) and glucose eff e c- tiveness ( . ± . vs. . ± . m i n , p . ). insulin sensitivity index was lower in subjects with igt ( . ± . m i n pmol l) than in those with ngt ( . ± . min pmol l ) , but was not statistically significant (p = . ). in con- trast, disposition index calculated by the p roduct of insulin secretion and insulin sensitivity was significantly lower in sub- jects with igt ( , ± ) than in those with ngt ( , ± , p = . ). on the other hand, patients with type dia- betes had significantly lower insulin secre- tion ( ± pmol l m i n , p = . ) compared with subjects with igt. although no significant diff e rence was observed in insulin sensitivity index between subjects with type diabetes and igt ( . ± . vs. . ± . min pmol l, p = . ), disposition index was s i g n i ficantly diminished in type diabetic patients as compared with subjects with igt ( ± vs. , ± , p = . ). glucose effectiveness in type diabetic patients ( . ± . min ) was similar to that in subjects with igt ( . ± . m i n , p = . ) but was signific a n t l y lower than that in the subjects with ngt (p . ). from these results, the fol- lowing may be hypothesized: ) impair- ments in insulin secretion and disposition index and decreased glucose eff e c t i v e n e s s , but not insulin resistance, seem to consti- tute the basic characteristics of patients with igt or type diabetes in nonobese japanese populations. ) risk factors wors- ening to type diabetes in subjects with igt are associated with further impair- ments in insulin secretion and disposition index, but not associated with furt h e r derangement in glucose effectiveness in japanese populations. ataru taniguchi, md mitsuo fukushima, md masahiko sakai, md itaru nagata, md kentaro doi, md shoichiro nagasaka, md kumpei tokuyama, md yoshikatsu nakai, md f rom the first department of internal medicine ( a . t., m.s., i.n.), kansai-denryoku hospital, and the department of internal medicine, hoshida- minami hospital (m.f.), osaka; the second depart- ment of internal medicine (k.d.) and the college of medical technology (y.n.), kyoto university, kyoto; the department of internal medicine (s.n.), jichi medical college, tochigi; and the laboratory of biochemistry of exercise and nutrition (k.t. ) , tsukuba university, tsukuba, japan. a d d ress correspondence to ataru ta n i g u c h i , md, first department of internal medicine, kansai- d e n ryoku hospital, - - , fukushima-ku, fuku- shima, osaka city, osaka - , japan. e-mail: k @ k e p c o . c o . j p . r e f e re n c e s . b e rgman rn: to w a rd physiological under- standing of glucose tolerance: minimal- model approach. d i a b e t e s : – , . welch s, gebhart ssp, bergman rn, phillips ls: minimal model analysis of intravenous glucose tolerance test-derived insulin sensitivity in diabetic subjects. j c l i n endocrinol metab : – , . taniguchi a, nakai y, fukushima m, kawamura h, imura h, nagata i, tokuyama k: pathogenic factors re s p o n s i- ble for glucose tolerance in patients with niddm. d i a b e t e s : – , . nagasaka s, tokuyama k, kusaka i, hayashi h, rokkaku k, nakamura t, kawakami a, higashiyama m, ishikawa s, saito t: endogenous glucose pro d u c t i o n and glucose effectiveness in type diabetic subjects derived from stable-labeled mini- mal model approach. d i a b e t e s : – , . best jd, kahn se, ader m, watanabe rm, ni t-c, bergman rn: role of glucose eff e c- tiveness in the determination of glucose tol- erance. diabetes care : – , . rossetti l, giaccari a, defronzo ra: glu- cose toxicity. diabetes care : – , . taniguchi a, nakai y, doi k, fukuzawa h, fukushima m, kawamura h, to k u y a m a k, suzuki m, fujitani j, tanaka h, nagata i: insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis. m e t a b o l i s m : – , . world health organization: diabetes melli - tus: report of a who study gro u p . g e n e v a , world health org., (tech. rep. ser. , no. ) . winer bj: statistical principles in experimen - tal design. nd ed. new york, mcgraw-hill, , p. – late-onset tro g l i t a z o n e - i n d u c e d hepatic dysfunction r e c e n t l y, iwase et al. ( ) re p o rted a case of liver dysfunction occurring after months of troglitazone therapy. because it was thought before this re p o rt that the risk of liver dysfunction with tro g l i- tazone after months was negligible, we wish to re p o rt another patient who took t roglitazone intermittently and developed hepatic dysfunction after months. a -year-old white man with type diabetes, ischemic heart disease (post angioplasty and stent placement), hyper- tension, degenerative joint disease, benign p rostatic hypert ro p h y, dyslipidemia, and g a s t roesophageal re flux disease had tro g l i- tazone mg daily added to his re g i m e n of glimeperide mg daily and metform i n mg b.i.d. because of poor glycemic c o n t rol (hba c . % [normal – %]). the other medicines he used were aspirin and pravastatin. after months of triple oral therapy, his hba c level dropped to . %, and, after months, to . %. after months, the patient’s hba c began to rise: . % at months, . % at year, and . % at months. liver function tests were normal until months, when his aspartate amino- transferase (ast) was found to be (nor- mal – u/l) and alanine aminotrans- ferase (alt) (normal – u/l). the t roglitazone regimen was discontinued, and testing for hepatitis b and c, h e m a c h romatosis, autoimmune liver dis- ease, and gallbladder disease were nega- tive. two months after discontinuing t roglitazone, the patient’s ast and alt had decreased to and u/l, and, after months, had re t u rned to normal at and u/l, re s p e c t i v e l y. his ast and alt have remained normal since then and he has continued to take pravastatin, aspirin, m e t f o rmin, and glimeperide. when the patient was told to discon- tinue troglitazone, he admitted that he had been taking it only interm i t t e n t l y. he esti- mated that he took the drug regularly at first, but after the first months, he took the drug only once or twice weekly on aver- age. he gave the following three reasons for his lack of compliance: a lack of funds, a fear of liver disease, and a tendency to avoid taking drugs whenever possible. this case, like the case described by iwase et al., illustrates that the hepatic dysfunction caused by troglitazone can occur after months and further sup- p o rts the u.s. food and drug administra- t i o n ’s current recommendation that quar- terly liver function tests should be obtained when troglitazone utilization extends beyond year ( ). in this case, could the onset of hepatic dysfunction have been delayed because the diabetes care, volume , number , january letters d rug was being taken only interm i t t e n t l y after the first months, and the estimated total load presented to the liver would be equivalent to the exposure at months in a compliant patient? we doubt this, since t ro g l i t a z o n e ’s hepatic effects are thought to be idiosyncratic and there f o re the total e x p o s u re should be irre l e v a n t . david s.h. bell, mb fernando ovalle, md f rom the division of endocrinology and metabo- lism, department of medicine, school of medicine, university of alabama, birmingham, alabama. a d d ress correspondence to david s.h. bell, mb, th ave. s., birmingham, al . d.s.h.b. and f.o. have served on an advisory panel for sankyo parke-davis and have received con- sulting fees, re s e a rch grant support, and honoraria for speaking engagements from sankyo parke-davis. r e f e re n c e s . iwase m, yamaguchi m, yoshinari m, oka- mura c, hirahashi t, tsuji h, fujishima m: a japanese case of liver dysfunction after months of troglitazone tre a t m e n t . diabetes care : – , . parke-davis, division of wa rn e r- l a m b e rt : rezulin package insert. morris plains, nj, g l y b u r i d e - i n d u c e d hemolysis in m y e l o d y s p l a s t i c s y n d ro m e g lyburide, also known as gliben- clamide, is a widely used sulfonylure a to treat patients with type diabetes. hemolytic anemia is an extremely rare side e ffect of which there have been only a few re p o rts ( – ). we describe a patient with myelodysplastic syndrome who pre s e n t e d with glyburide-induced hemolysis. a -year-old man with a long history of type diabetes presented with left foot cellulitis of week’s duration. this patient was known to have slowly pro g re s s i v e pancytopenia for years, for which no work-ups had been perf o rmed. his med- ications included the following: glyburide, mg per day, which he had taken for m o re than year; buformine, mg per day; and boglibose, . mg per day. he was afebrile, and the physical examination was normal, except for localized cellulitis on his left foot, for which he was start e d on intravenous antibiotics. the laboratory studies revealed a white blood cell count of . / l , hemoglobin . g/dl, platelet count /l, reticulocyte count . %, mean cor- puscle volume fl, moderate anisocyto- sis, fasting plasma glucose mg/dl, h b a c . %, lactate dehydrogenase iu/l, total bilirubin . mg/dl, and hapto- globin mg/dl. red cell glucose- - phosphate dehydrogenase level was ade- quate. cold agglutinin test, ham’s test, and sugar water test were normal. both dire c t and indirect coombs’ tests were negative. red cell resistance to osmolarity was mildly low (parpart ’s method). urinalysis demonstrated no urobilinogen. endo- scopic studies did not reveal gastro i n t e s t i- nal bleeding. ultrasonography of the abdomen showed no splenomegaly. the result of bone marrow aspiration was equivocal. on the basis of pre s u m p t i v e glyburide-induced hemolysis, glyburide was discontinued and the patient was switched to subcutaneous insulin on the seventh day. there a f t e r, his hemoglobin level increased to . g/dl, re t i c u l o c y t e count decreased to . %, and anisocytosis d i s a p p e a red pro m p t l y. he was discharg e d with insulin therapy after month in the hospital, which is when the cellulitis re s o l v e d . t h ree months later, his hemoglobin level was . g/dl and his haptoglobin remained low. repeated bone marro w aspiration confirmed the diagnosis of myelodysplastic syndro m e . we conclude that this patient devel- oped glyburide-induced hemolysis super- imposed on red cell fragility secondary to an underlying bone marrow disord e r. t h e re have been several re p o rts of hemoly- sis caused by sulfonylureas, most of which have been considered immune-mediated ( , , ). our case points to the possibility that glyburide could cause hemolysis by a non–immune-medicated mechanism. it is i m p o rtant to be aware of this potential side e ffect of glyburide in light of this medica- t i o n ’s widespread prescription, even though such a side effect is rare . hiroshi noto, md kazuhisa tsukamoto, md satoshi kimura, md f rom the department of diabetes and metabolism, tokyo university hospital, tokyo, japan. a d d ress correspondence to hiroshi noto, md, d e p a rtment of diabetes and metabolism, tokyo uni- versity hospital, - - hongo, bunkyo-ku, to k y o - , japan. e-mail: noto-tky@umin.ac.jp. r e f e re n c e s . nataas ob, nesthus i: immune haemolytic anaemia induced by glibenclamide in selective iga defic i e n c y. b m j : – , . abbate sl, hoogwerf bj: hemolytic ane- mia associated with sulfonylurea use. d i a - betes care : – , . meloni g, meloni t: glyburide-induced acute haemolysis in a g pd-defic i e n t patient with niddm. br j haematol : – , . kopicky ja, packman ch: the mechanism of sulfonylurea-induced immune hemoly- sis: case re p o rt and review of the literature . am j hematol : – , e ffects of exposure at an altitude of , m on p e rf o rmance of glucose meters s elf-monitoring of blood glucose is m a n d a t o ry for type diabetic p a t i e n t s who participate in sports to adjust insulin dose and carbohydrate ingestion ( ). sports also include activities p e rf o rm e d at moderately high altitudes, such as hiking or skiing. capillary blood glucose monitors (bgms) have been shown to underestimate blood glucose values at an altitude of , m ( ) and at a simulated altitude of , m with t e m p e r a t u re and humidity kept constant ( ). the aim of the present study was to assess the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and p o can result in errors in blood glucose d e t e rmination ( ). two bgms, the lifescan one touch ii (ot) (ortho diagnostics, milpitas, ca) and the glucometer elite ii (ge) (bayer diagnostics, brussels, belgium), were tested during a study on the effects of acute exposure at an altitude of , m and exercise on blood pre s s u re and albu- min excretion rate in six type diabetic p a t i e n t s . all subjects (four men and two women) were free of disease-related com- plications and in good and stable glycemic control (ghb . ± . %). all subjects gave their informed and written consent to participate in the study pro t o- col. all subjects were investigated both at diabetes care, volume , number , january letters sea level and after ascent by car and cable car to the angelo mosso institute at col d’olen ( , m altitude), gressoney la trinité, italy. at sea level and at a moderately high altitude, bgm reliability at diff e rent blood glucose levels was tested, and blood glu- cose was assessed in fasting and re s t i n g conditions at : a.m.; at : a.m. b e f o re an in-field exercise test; and imme- d i a t e l y, min, and min after the exer- cise stopped. capillary glucose was simul- taneously assessed with the ot and the ge. both of these bgms measure capillary blood glucose through the glucose oxi- d a s e - p e roxidase reaction. bgms were cali- brated at the beginning of each test ses- sion. a venous blood sample was simulta- neously drawn from the contralateral antecubital vein in a sodium fluoride tube, centrifuged, and stored at °c. plasma glucose was assayed with the glucose oxi- dase method (go) within days. this last assessment was taken as a re f e re n c e method. statistical analysis compare d bgm capillary glucose values and go plasma glucose values for each blood col- lection time. measurement linearity was tested with pearson’s correlation coeff i- cient. the mean of the diff e rences between the bgm and go results re p resents the mean bias between the methods with accuracy expressed as percent error (pe): pe (%) = bmg – go % g o the level of statistical significance was c o n s i d e red to be p . . the ge and ot measurements had a good correlation with plasma glucose both at moderately high altitude and at sea level. p e a r s o n ’s correlation coefficients were . and . for the ge and . and . for the ot at sea level and at moderately high altitude, re s p e c t i v e l y. biases between plasma glucose and bgm measure m e n t s w e re as follows: for the ge, . ± . at sea level and . ± . at moderately high altitude; for the ot, . ± . at sea level and . ± . at moderately high alti- tude. at sea level, both the ge and the ot tended to underestimate glucose values (ns); at moderately high altitude, the ge tended to overestimate and the ot tended to underestimate glucose values (ns). mean pes between plasma glucose and bgm m e a s u rements were . (ot) and . (ge) at sea level and . (ot) and . (ge) at moderately high altitude. pe tended to be higher for both bgms at moderately high altitude (ns). figures and show the bias between the single measurements with both devices at sea level and at moderately high altitude, re s p e c t i v e l y. at moderately high altitude (fig. ), the tendency of the ge to o v e restimate was more evident for low ( mg/dl) and intermediate ( – mg/dl) blood glucose values, whereas the ot tended to underestimate mainly high blood glucose values (ns). in our study, bgm perf o rmance was similar and good at sea level. at a moder- ately high altitude, a tendency to overe s t i- mate blood glucose for the ge and to u n d e restimate for the ot was observ e d . the overestimation for the ge involved mainly low ( mg/dl) and interm e d i- ate ( – mg/dl) blood glucose val- ues. this could present a problem in the p resence of symptoms suggesting hypo- figure —relationship between plasma and capillary glucose at sea level. figure —relationship between plasma and capillary glucose at moderately high altitude. lifescan one touch ii glucometer elite ii lifescan one touch ii glucometer elite ii diabetes care, volume , number , january letters glycemia and normal blood glucose val- ues. the ot tended to undere s t i m a t e mainly high blood glucose values, although its perf o rmance with low to i n t e rmediate values was good. the pre s- ent study assessed the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and po can result in errors in blood glu- cose determination ( ). our results are consistent with previous studies ( , ). the decrease in po could alter the sec- ond phase of the chromogen reaction and u n d e restimate blood glucose values ( ); on the other hand, an increase in atmos- pheric pre s s u re could overestimate blood glucose values ( ). in our study, minimal o v e restimation by the ge at low interm e- diate blood glucose values at moderately high altitude cannot be explained by the a l t e red po . an increase in hematocrit, which is known to alter blood glucose m e a s u rements with bgms ( ), may also occur after prolonged exposure to high altitude or as a consequence of dehydra- tion. although our study did not deter- mine hematocrit, the exercise test was s h o rt, and the patients were instructed to drink according to their thirst during the , -m exposure; there f o re, dehydration was not likely to have occurred. in con- clusion, bgm perf o rmance is similar and good at sea level. at a moderately high altitude similar to that experienced during winter skiing or summer hiking, a ten- dency to overestimate low to norm a l blood glucose values for the ge and to u n d e restimate high blood glucose values for the ot was observed. the bias is not clinically meaningful for either bgm, both of which can be safely used by dia- betic patients during exposure to moder- ately high altitudes. some care in the eval- uation of low and intermediate blood glu- cose values measured with the ge is n e v e rtheless re c o m m e n d e d . oriana pecchio, md simona maule, md marco migliardi, md marina trento, bsc massimo veglio, md f rom the italian alpine club medical commission ( o . p.); the department of internal medicine (m.t. ) , university of turin; the s. giovanni battista hospital (s.m.); and the department of endocrinology (m.m., m . v.), mauriziano umberto i hospital, turin, italy. a d d ress correspondence to dr. m. veglio, via mancini , torino, italy. e-mail: veglio@ o n w. n e t . r e f e re n c e s . h o rton es: role and management of exer- cise in diabetes mellitus. diabetes care : – , . g i o rdano bp, trash w, hollenbaugh l, dube wp: perf o rmance of seven blood glucose testing systems at high altitude. diabetes educ : – , . gautier jf, bigard ax, douce p, duvallet a, cathelinau g: influence of simulated alti- tude on the perf o rmance of five blood glu- cose meters. diabetes care : – , . b a rnett c, ryan f, ballonoff l: effect of altitude on the self monitoring of blood glucose (smbg) (abstract). diabetes (suppl.): a, . piepmeier eh, hammett-stabler c, price me, kemper gb, davis mg: atmospheric p re s s u re effects on glucose monitoring devices (letter). diabetes care : – , . b a rreau pb, buttery je: effect of hematocrit concentration on blood glucose value d e t e rmined on glucometer ii. diabetes care : – , c o m m e n t s a n d r e s p o n s e s deterioration of glycemic contro l after long-te rm treatment wi t h troglitazone in nonobese type diabetic patients t roglitazone is an oral antidiabetic d rug used to treat type diabetic patients with insulin re s i s t a n c e . troglitazone improves overall insulin sen- sitivity in the liver and skeletal muscles, which are the largest consumers and metabolizers of glucose in the body ( – ). recent re p o rts showed that troglitazone is also effective in nonobese type diabetic patients whose hyperglycemia could not be controlled with sulfonylurea therapy ( , ). however, we aware that in some patients in whom adequate glycemic con- t rol is obtained during the first several months of troglitazone treatment, their glycemic control deteriorates several months later. we assume that two distinct g roups of type diabetic patients exist who respond diff e rently to long-term administration of troglitazone, one gro u p that maintains a steady response and another group that has a decre a s i n g response after certain periods. in this s t u d y, we re t rospectively examined patients with type diabetes who were t reated with troglitazone for months and whose hba c levels had improved by % with troglitazone by month . in of the patients ( %), hba c levels increased by . % after – months despite continuous tro g l i t a z o n e t reatment (group p). in contrast, the rest of the patients experienced steady glycemic c o n t rol with . % of hba c flu c t u a t i o n ( g roup g). during the first months, h b a c levels decreased from means ± sem . ± . to . ± . % in group p and fro m . ± . to . ± . % in group g, re s p e c- t i v e l y. no significant diff e rences were evi- dent between the two groups re g a rding the d e c rease in hba c during the first months (fig. ). from month onward, hba c l e v- els in group p climbed gradually by . % a month up to the baseline level at month , but hba c levels were stable in group g t h roughout the treatment period. a signifi- cant diff e rence in hba c levels was evident during months – (p . ) . among clinical characteristics, gro u p p had a significantly lower bmi ( . ± . vs. . ± . kg/m , p . ) and s i g n i ficantly lower fasting insulin levels ( . ± . vs. . ± . µu/ml, p . ). of the patients with a bmi of kg/m ( %), exhibited deteriora- tion of glycemic contro l . in this study, we re p o rt a group of patients who showed a renewed decline in glycemic control after long-term tre a t m e n t with troglitazone. these results seemed to suggest a secondary failure of tro g l i t a z o n e . our study demonstrates that this drug is indeed useful for a long-standing obese i n s u l i n - resistant diabetes but not for a nonobese type diabetes. yuko murase, md takanobu wakasugi, md kunimasa yagi, md hiroshi mabuchi, md f rom the department of internal medicine (y. m . , t. w.), fukui perfectural hospital; and the second d e p a rtment of internal medicine (k.y., h.m.), kanazawa university, ishikawa, japan. a d d ress correspondence to yuko murase, md, the second department of internal medicine, kanazawa university, - takara-machi, kanazawa, ishikawa - , japan. e-mail: diabe@med. k a n a z a w a - u . a c . j p . diabetes care, volume , number , january letters r e f e re n c e s . suter sl, nolan jj, wallace p, gumbiner b, olefsky jm: metabolic effects of new oral hypoglycemic agent cs- in niddm subjects. diabetes care : – , . o’rourke cm, davis ja, saltiel ar, corn i- celli ja: metabolic effects of troglitazone in the goto-kakizaki rat, a non-obese and n o rmolipidemic rodent model of nonin- sulin-dependent diabetes mellitus. m e t a b - o l i s m : – , . troglitazone study group: the metabolic e ffects of troglitazone in non-insulin dependent diabetes (abstract). d i a b e t e s (suppl. ): a, . mori k: the effect of troglitazone in combi- nation with sulfonylurea in non-insulin dependent diabetes mellitus (abstract). j japan diabetes soc (suppl. ): , . h o rton es, venable tc, whitehouse f, the troglitazone study group, ghazzi mn, whitcomb rw: troglitazone in combina- tion with sulfonylurea re s t o res glycemic c o n t rol in patients with type diabetes. diabetes care : – , figure —change from baseline in hba c. values are means ± sem. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ [pdf] measurement without theory, once again. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /dem. . corpus id: measurement without theory, once again. @article{greenwood measurementwt, title={measurement without theory, once again.}, author={j. greenwood and a. seshadri and guillaume vandenbroucke}, journal={journal of demographic economics}, year={ }, volume={ }, pages={ - } } 	j. greenwood, a. seshadri, guillaume vandenbroucke 	published 	psychology, medicine 	journal of demographic economics bailey and collins ( ) argue that greenwood, seshadri, and vandenbroucke's ( ) hypothesis that the baby boom was partly due to a burst of productivity in the household sector is not supported by evidence. this conclusion is based on regression results showing that appliance ownership is negatively correlated with fertility. they also argue that the amish, who limit the use of modern technology, had a baby boom. first, it is demonstrated that a negative correlation between appliance… expand view on pubmed cambridge.org save to library create alert cite launch research feed share this paper citationsbackground citations view all figures, tables, and topics from this paper 	 table 	 figure 	 table 	 table 	 table view all figures & tables 	phobic anxiety disorder citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency discussion paper no 	n. dao, j. dávila, angela greulich 	 	pdf save alert research feed family economics writ large 	j. greenwood, n. guner, guillaume vandenbroucke 	sociology, geography 	 	 	pdf save alert research feed economic uncertainty and fertility cycles: the case of the post-wwii baby boom 	bastien chabé-ferret, paula e. gobbi 	economics 	 	 	pdf 	view excerpt, cites background save alert research feed references showing - of references sort byrelevance most influenced papers recency the baby boom and baby bust 	j. 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polinsky, s. shavell 	 	 , 	pdf 	view excerpt, references background save alert research feed are structural vars with long-run restrictions useful in developing business cycle theory? 	v. chari, p. kehoe, ellen r. mcgrattan 	economics 	 	 	pdf 	view excerpt, references methods save alert research feed the career decisions of young men 	m. keane, k. wolpin 	economics 	journal of political economy 	 	 , 	pdf save alert research feed measuring trends in leisure: the allocation of time over five decades 	erik hurst, mark aguiar 	economics 	 	 	pdf 	view excerpt, references background save alert research feed the riddle of amish culture. 	r. c. buck, donald b. kraybill 	art, sociology 	 	 save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute 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(url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ microsoft word - bailey_collins_ _ c.docx did improvements in household technology cause the baby boom? evidence from electrification, appliance diffusion, and the amish martha j. bailey and william j. collins first draft: march this draft: december abstract: more than a half century after its peak, the baby boom’s causes remain a puzzle. a novel argument posits that rapid changes in household technology from to account for this large increase in fertility. we present new empirical evidence that is inconsistent with this claim. rapid advances in household technology began long before while fertility declined, and differences and changes in appliance ownership and electrification in u.s. counties are negatively correlated with fertility rates from to . finally, the amish, a group strictly limiting the use of modern technologies, experienced a coincident and sizable baby boom. contact information: martha bailey, university of michigan, department of economics, tappan street, ann arbor, michigan - ; email: baileymj@umich.edu; website: www- personal.umich.edu/~baileymj. william collins, department of economics, box -b, vanderbilt university, nashville, tn ; email: william.collins@vanderbilt.edu. bailey is an assistant professor of economics and a faculty affiliate at the population studies and national poverty centers at the university of michigan, and a faculty research fellow at the national bureau of economic research. collins is a professor of economics at vanderbilt university and a research associate of the national bureau of economic research. we are grateful to soren anderson, john bound, alison buttenheim, charlie brown, robert driskill, richard easterlin, daniel eisenberg, chris house, miles kimball, david lam, robert margo, john dinardo, elyce rotella, katherine shester, matthew shapiro, jeff smith, and gary solon for useful comments and suggestions. we also thank participants in seminars at clemson university, the cswep junior faculty mentoring workshop, the economic history association meetings, lehigh university, mcgill university, the national bureau of economic research, the population association of america’s meetings, queen’s university, the university of michigan (economic history, macroeconomics, and population studies), university of north carolina-greensboro, and the university of toronto. emily boleman, emily gray, and brad hershbein provided outstanding research assistance. we gratefully acknowledge financial support from the national science foundation ( ) and the robert wood johnson foundation and helpful comments from ananth seshadri and guillaume vandenbroucke. . introduction after at least years of secular decline, births per , women (ages to ) in the united states increased by more than percent between and (see figure ). this remarkable departure from longer-term trends, often called the “baby boom,” was not a short- lived, statistical aberration reflecting postponed births from the depression or world war ii. rather, it stretched over two decades and was driven by earlier marriage and childbearing, shorter birth intervals, and increases in completed childbearing (ryder , rogers and o’connell ). these features of the american baby boom present a fascinating challenge to scholars, especially because the rise in fertility took place against a backdrop of increasing income, urbanization, educational attainment, and women’s labor force participation—all trends typically associated with declining fertility. more than a half century after its peak, the ultimate causes of the baby boom remain one of the twentieth century’s great puzzles. a recent article by greenwood, seshadri, and vandenbroucke ( , henceforth gsv) proposes a novel neoclassical explanation of the baby boom. using an overlapping-generations model that integrates fertility decisions, advances in household technology, and changes in wage rates, they simulate both the secular decline in u.s. fertility, which is driven by rising wages (consistent with a rising opportunity cost of childrearing), and the baby boom, which is driven by a burst in the productivity of household technology between and . in their words, “…technological advance in the household sector, due to the introduction of electricity and the development of associated household products such as appliances and frozen foods, reduced the need for labor in the child-rearing process. this lowered the cost of having children and should have caused an increase in fertility, other things equal. this led to the baby boom” ( ). thus, economic theory elegantly solves the puzzle of the baby boom, while also accommodating and explaining the long-run decline in fertility. if correct, this household-technology centered explanation of the baby boom is a great achievement for fertility models that highlight changes in technology and prices. it also represents a formidable challenge to arguments from richard easterlin’s point of view, which completed cohort fertility is plotted with period fertility in figure . women born during the s, the mothers of the “baby boomers,” had completed fertility rates as high as women born in the late nineteenth century. emphasize an independent and causal role for changes in preferences (easterlin , ; fernandez ). indeed, the ascendance of the household-technology centered explanation of the baby boom would entail a fundamental shift in standard narratives of american demographic history (see inter alia montgomery and trussell , haines ). our analysis subjects this important hypothesis to closer historical, empirical, and theoretical scrutiny. first, we examine the historical validity of key assumptions that underpin gsv’s quantitative simulation and conclude that the central claim of an exceptional and sudden “burst” of household productivity relative to productivity growth in other sectors between and is questionable. second, newly-encoded county-level information on household technology, combined with existing county-level census data on fertility and other household characteristics, provides further evidence that is inconsistent with the household-productivity hypothesis. this is true of both cross-sectional and first-differenced, regression-adjusted estimates of the relationship between fertility rates and the diffusion of electrical service and modern appliances. moreover, individual-level data on completed fertility reveal no evidence that women who had greater exposure to electrical service in early adulthood had more children, even after conditioning on education, race, and state and cohort fixed effects. finally, we document that the old order amish, a group that limited its use of modern appliances on religious grounds, had a baby boom that began at nearly the same time, lasted just as long, and was approximately as large as the baby boom in the rest of the u.s. population. our findings and conclusions are consistent with economic theory as long as the quantity of children is not restricted to be a normal good or consumers are allowed to value other home- produced goods. we demonstrate that incorporating other household commodities into the utility function and allowing their prices to fall with improvements in household technology produces a theoretically ambiguous relationship between the number of children and technological progress in the household. in sum, this paper’s historical and econometric easterlin emphasizes the importance of a cohort’s perceived “earnings potential” relative to its “material aspirations.” children who grew up in the depression, for example, may have formed material aspirations that were far exceeded by their actual experience as young adults in the later s and s, and they may have responded to their surprisingly good fortune by having more children. see macunovich ( ) for a review of the empirical literature that the easterlin hypothesis spawned. see doepke, hazan, and maoz ( ) for an even more recent view based on world war ii’s effect on women’s labor force experience. evidence calls scholars to look beyond the diffusion of modern household technology for the causes of the u.s. baby boom. . a simple framework for understanding the impact of household technology the gsv model of fertility yields an unambiguous prediction that the number of children rises when the productivity of household technology increases. this result provides the theoretical basis for the claim that a burst of household technology caused the baby boom. this section demonstrates that relaxing critical assumptions changes this unambiguous theoretical result to an ambiguous one. without loss of generality, consider a static framework where households maximize utility, , , defined over the number of children, , and a composite non-child commodity, . the price of the non-child composite is normalized to , and the price of children, , is a shadow price (or marginal cost of a child) and defined relative to the price of . as in the gsv framework, we assume that falls as progress is made in household technology, which is represented in the exogenous parameter . in this framework, the demand for children is a function of the relative price and income, ; that is, , . using the slutsky equation and the assumption that the relative price of falls with a, the impact of technological progress in the household on the demand for children should be nonnegative, , · , , where is the compensated price effect. consistent with the properties of the slutsky matrix, the own-price effect should be nonpositive, . moreover, the income effect should be the implications of the gsv model for childbearing remain in the one-period case, because they assume that childbearing decisions occur only in the first period of adulthood. we additionally assume that is continuous and represents a locally nonsatiated and strictly convex preference relation. to this end, gsv employ a simple production function for children, , where is the amount of labor input and . nonnegative, , if children are a normal good. these properties combined with the assumption that household technological progress lowers the relative price of children, lead to a straightforward result: both substitution and income effects encourage the production of more children when household productivity rises. the simple intuition from this general formulation is exactly what drives the results in the basic gsv model. one potential problem with the assumption that child quantity is a normal good is that empirical studies find that the number of children tends to fall with income. becker ( ) argues that one important reason for this is that increases in income may induce much larger increases in child quality than child quantity. becker and lewis ( ) and willis ( ) formalize this argument by introducing a nonlinear budget constraint, in which the shadow prices of quality and quantity are jointly and endogenously determined. the well-known result, and therefore not demonstrated here, is that nonlinearities in these models’ budget constraints allow the number of children to fall as income increases. another critical assumption in this formulation is that an increase in household productivity does not alter the relative price of other consumption goods. to see the importance of this, consider the addition of another good, , to the model, and assume that its shadow price, , falls with a as well. some examples of the goods in might include listening to music (produced with a combination of time and a record player or radio); cleaning one’s home or clothing (produced with time and a washing machine or other appliances); or making or ironing one’s clothes (produced with time and a sewing machine or iron). alternatively, might be conceptualized as “child quality” as considered in becker ( ), becker and lewis ( ), and willis ( ), if child quality includes commodities like healthy meals, which may require fresh produce or meat to be refrigerated, or dance lessons, which could require automobile transportation to schools or studios. following the notation and logic of ( ), we now have this assumption is considered in several paragraphs. the specific example is shown in the theory appendix to this paper. see also gsv’s section i ( ) and section iv ( ), which considers an extension of their model that incorporates child quality. this well-known empirical regularity has been documented using inter-temporal and cross-sectional variation in observed income (becker , hotz et al. ). this could be achieved by using a parallel non-child, home goods production function, , where is the amount of labor used and . , , , , , , . in this case the sign of the second term is theoretically ambiguous. for instance, if and are gross substitutes (implying that , , ), then the sign of the second term would be nonpositive (because by assumption). the addition of a potentially substitutable, home-produced commodity, , and the relaxation of the assumption that children are a normal good allow for the possibility that the number of children might increase or decrease in response to an increase in . the point of this simple analysis is to show that—independent of specific functional form assumptions— basic economic theory predicts that advances in household technology may lead to a rise or fall in the number of children born. in summary, both the sign and the magnitude of the impact of rapid advances in household technology on child quantity depends upon whether child quantity is a normal good and how the shadow prices of other consumption commodities, such as home-produced goods or child quality, are affected. finally, even if the number of children did increase on net with advances in household technology, reductions in the price of other home-produced goods and, perhaps, substitution toward child quality (away from quantity) would temper the magnitude of this positive fertility response, making it a less likely candidate to account for the enormous increase in fertility rates during the baby boom. the next sections examine whether changes in appliance ownership and electrification between and were sufficiently large and correlated with fertility changes to represent an empirically plausible explanation of the u.s. baby boom. . the diffusion of household technology in the first half of the twentieth century the main evidence supporting the household-productivity hypothesis is a calibration exercise, which can mimic the actual time-series of the u.s. fertility rate up to , including the baby boom (gsv : - ). the calibrated model’s key parameter is household productivity, but its time path is neither measured directly nor easy to compute using available data. instead, values of the household productivity parameter that best fit the fertility time series are selected within a constrained minimization problem, where household productivity is constrained to be constant from to and from to . to is the only period in which household productivity is allowed to accelerate. the imposition of these constraints is intended to correspond to a “burst of technological progress in the household sector” between and (gsv : , ). when combined with the model’s assumptions that dictate a positive fertility response (discussed above), this sudden increase in household productivity yields the baby boom. this burst of household technological progress at mid-century, however, is historically questionable. scholarship on the history of home production reveals significant technological progress that long predates the baby boom. for example, improvements in stoves and the distribution of processed foods, including canned goods, refrigerated and preserved meat, and ready-to-eat cereals, transformed meal preparation during the nineteenth and early twentieth centuries (giedion , strasser , cowan ). indoor running water, a time-saving improvement rivaled by few others, became increasingly common in cities in the late s and proliferated thereafter. sewing machines diffused widely after (godley ). christine frederick’s widely circulated writings ( ) on how to improve efficiency in household work were published decades before the baby boom and were a continuation of a literature that dates to the mid-nineteenth century work of catharine beecher ( ). for quantitative evidence on the speed of technological progress in the household, we focus on the diffusion of household technologies. rather than combining separate series into an index, figure plots them separately to highlight the timing of diffusion for different three parameters determine fertility trends in the gsv model ( : ): ( ) the state of household technology, ( ) the market wage rate, and ( ) the time price of home production technology. ( ) and ( ) are inherently difficult to measure. in the gsv calibration, the state of household technology is inferred as described above and the time price of new technology is simply set to zero for all periods ( : fn). this means that changes in ( ) and ( ) are primarily responsible for the timeseries generated in the calibration exercise. we concentrate on measures of appliance diffusion and electrification because these are the hallmarks of twentieth-century change in household production techniques, and they are specifically cited as the instruments that reduced the need for labor in the child- rearing process ( and section v). cowan writes, “by the turn of the century, canned goods were a standard feature of the american diet: women’s magazines contained advertisements for them on nearly every page, standard recipes routinely called for them, and the weekly food expenditures of even the poorest urban families regularly included them” ( : ). some of these early developments are also cited in gsv’s description of the history. we have found little, quality-adjusted information on national price changes for the pre- period and focus, therefore, on the actual diffusion of technology to households. another issue that arises with price data is that the magnitudes of household responses to changes in price are difficult to quantify. using information on ownership rather than price has the advantage of reflecting actual changes in behavior. technologies. these series provide no support for the idea that advances in household technology accelerated from to , let alone that advances were negligible beforehand. the pre- diffusion of modern conveniences was significant and rivaled the changes that unfolded between and . in , only percent of homes had running water and percent had flush toilets; only percent of homes had electrical service in , and essentially none had mechanical refrigerators, washing machines, or vacuum cleaners (lebergott : , , ). by —before the baby boom—the census reported that approximately percent of households had running water, percent had a private flush toilet, percent had electrical lighting (and nearly as many had electric irons), percent had mechanical refrigerators, percent had gas or electric stoves (rather than wood, coal, or kerosene), and percent had central heating (brunsman and lowery ). we estimate that between and percent had power-driven washing machines in . the overwhelming impression from figure is that a strong trend of household technological change preceded the baby boom by at least three decades. contrary to the hypothesized positive link between modern appliances and fertility, the u.s. fertility rate declined sharply in the context of rapid increases in home production technology between and . then, as home production technologies continued to improve (but as domestic help became increasingly expensive), this relationship reversed and fertility increased sharply from to . the calibrated model’s assumption that the acceleration in the advance of household lebergott estimates that percent of homes owned electric irons in , whereas about percent had electrical lighting in ( : , ). the higher estimate ( percent) is from multiplying figures for clothes washer ownership in among “wired” households ( percent) from bowden and offer ( : ) by the proportion of occupied dwellings with electrical lighting ( percent) from the u.s. census (as reported in brunsman and lowery : ). the lower estimate ( percent) is from using an alternative measure of “wired” houses ( percent) from bowden and offer ( ). george stigler noted that “in there were as many domestic servants as employees of the railroads, coal mines, and automobile industry combined” ( : ). but as the baby boom began, the proportion of the labor force working in domestic service fell by over percent. because this shift was accompanied by an increase in the wages of female domestic servants relative to other occupations (such as manufacturing operatives or clerical workers), bailey and collins ( ) argue that the pattern reflects increasing demand for women’s labor in other sectors, as opposed to decreasing demand for household service. along these lines, cowan suggests that “twentieth-century housewives may have wished to trade in their vacuum cleaners for a ‘good old-fashioned maid’, but could not do it because the good old-fashioned maids preferred positions on the assembly lines to positions in the parlor” ( : ). these trends would tend to increase, rather than decrease, the shadow price associated with childrearing during the baby boom. production technology slowed in may also be inconsistent with history. after , automatic dishwashers, clothes dryers, air-conditioning, and microwaves were recasting patterns of housework yet again (cox and alm : ). moreover, television entertainment and expanding pre-school and kindergarten programs may have lessened children’s demands on parents’ time and energy. we do not focus on this in our discussion, however, because gsv emphasize the calibrated model’s ability to explain the baby boom between and and acknowledge that it fails to match the post- baby bust. in sum, these facts present difficult challenges for the household-technology centered explanation of the baby boom. a sudden and unprecedented acceleration in household productivity between and is essential for the calibration exercise to produce a baby boom, yet history questions this characterization of technological change and, instead, suggests a much longer period of transition. during much of this transition, fertility rates declined rapidly. in light of these time-series patterns, we turn to more disaggregated evidence to test the model’s central, causal claim that modern appliances positively affected fertility levels after . . appliance ownership and the general fertility rate this section uses a newly-compiled, county-level panel dataset for the united states to subject the household-technology hypothesis to direct empirical scrutiny. this analysis is akin to a set of unadjusted, cross-national correlations presented in gsv, but our dataset is much larger, contains more covariates and, therefore, has several advantages. approximately percent of - year olds were enrolled in school in , but percent were enrolled in (goldin : - ). from to , the average public school student attended additional days of school ( - ). the gsv simulation faces another serious critique, which is not discussed in detail in this paper. the results predict a large decline in time spent in household production, which gsv point out is consistent with the lebergott ( ) estimates. however, ramey and francis ( ) and ramey ( ) report that lebergott’s estimates are due to a mistake in reading the primary data source. in addition, the claim does not square with a large literature on household-level time-use studies, which suggests that there were small (if any) declines in time spent on housework before the mid- s (see, inter alia, vanek , cowan , bryant , ramey and francis , ramey ). in short, the gsv simulation predicts a large change in time use that apparently did not actually occur. gsv graphically compare (on one axis) baby-boom magnitude or the year-of-boom onset and (on the other axis) income per capita in in oecd countries or an index of appliance ownership in countries ( : - ). the interpretation of the gdp per capita comparisons hinges on the idea that households in rich countries were more likely to own labor-saving appliances. of course, this correlation is also consistent with the fundamentally different interpretation of easterlin ( ) and is vulnerable to omitted variable biases. to create this dataset, we entered county-level appliance ownership counts from the census volumes for the to period. we supplemented these data with publicly- available, county-level economic and demographic information collected by michael haines ( ). every county with available information (excluding those in hawaii and alaska) is in our dataset, yielding approximately , county-level observations per decade. our proxy for the general fertility rate is the number of infants (under age one) per , women of ages to . the level of household technology is measured as the proportion of housing units with refrigerators, washing machines, modern stoves (e.g., fueled by electricity or gas, rather than coal, wood, or kerosene), and electrical service. the census data provide a geographically detailed view of the diffusion of modern appliances during the baby-boom period, but the census did not collect information on every appliance in each year. electrical service and refrigerators were reported in and , washing machines only in , and cooking fuel (an indicator of modern stoves) in , , and . additional information about variable definitions and the construction of our measures appears in the data appendix. table reports means and standard deviations for the household technology variables and the number of infants per , women ages to . the diffusion of electrical service and modern appliances was widespread, as reflected in the large increases in average rates of appliance ownership, but the levels and changes in appliance ownership were highly uneven across counties. for instance, the increase in electrical service ( to ) ranges from . percentage points at the th percentile to . at the th percentile, and the rise in modern stoves ( to ) ranges from . percentage points at the th percentile to . at the th percentile. a pervasive-but-uneven pattern of increase is also evident in the general fertility rate. from to , more than percent of counties recorded a rise in infants per , women age to , and the average ratio increased from to . over the same period, changes in the ratio ranged from . at the th percentile to . at the th percentile. our analysis exploits the intertemporal and geographic variation in these measures to describe the relationship between fertility and the state of household technology while accounting for other relevant demographic and economic characteristics in the following linear consistent data on household appliances are unavailable in the micro-level ipums samples. regression framework, ( ) ∑ , where f is the fertility rate in county j and state s, a is our measure of the state of household technology, and ( ) is a state dummy variable. to account for county-level characteristics that may affect both fertility rates and the state of household technology, x includes median years of schooling for those over age , log of median property value for owner-occupied housing, log of median family income (in and only due to data constraints), racial composition, measures of local economic development (the proportion working in agriculture, the proportion working in manufacturing, the urban proportion of the county’s current population, and log population density), and a correlate of the opportunity cost of childrearing (the proportion of women in the labor force). testing whether is one way to evaluate statistically the hypothesized positive association between household technology and fertility. table reports least-squares estimates of τ separately for , and . each point estimate is from a separate, unweighted regression of the general fertility rate on appliance ownership. heteroskedasticity-robust standard errors are corrected for correlation within states and presented in brackets below each estimate. the specification in column includes no covariates or fixed effects, and the unadjusted correlation between fertility and appliance ownership (refrigerators, modern stoves, and washing machines) is strongly negative. this is consistent with the observation that the level of workers’ labor market productivity is positively correlated with appliance ownership and negatively correlated with fertility (as in the gsv model). to reduce the scope for this source of omitted variable bias, column includes state fixed effects and column adds the full set of county-level demographic and economic covariates described above. identifying from within-state variation in appliance ownership and fertility (column ) tends to increase the magnitude of the point estimates slightly. adding controls for county-level characteristics (column ) results in point estimates that are considerably smaller in magnitude, but none of the six is positive and statistically significant. moreover, the largest point estimate ( . on washing machines in ) in conjunction with a results from quantile regressions at the median, which are less sensitive to outliers than ols, yield results that are qualitatively similar to the ols estimates reported in tables , and . percentage point increase in washing machine ownership (the change in national average in figure ) implies an increase of . infants per , women ages to . even using the upper bound of the percent confidence interval implies an increase of only . infants. because the baby boom entailed an increase of about infants per , women, the magnitude of this relatively large point estimate, even at the outer reaches of its confidence interval, is not consistent with the hypothesis that appliance diffusion caused the baby boom. one problem with the cross-sectional regressions is that unobserved differences in factors that influence both appliance ownership and fertility outcomes, such as unobserved differences in labor market opportunities for women, might bias estimates of downward. with this in mind, we use the following first-differenced specification to eliminate time-invariant, unobservable county-level differences, ( ) ∆ ∆ ∆ ∑ , where Δ denotes county-level changes in the variable over a or year period (either from to , to , or to ), and the remaining notation is as previously described. in this specification, observed, time-varying county characteristics (including changes in women’s labor market participation rates) are captured in ∆ ; and the effects of unobserved, fixed differences are differenced out by design. finally, state fixed effects now absorb unobserved changes at the state level, such as changes in perceived prosperity, policy, or other relevant conditions that may otherwise bias the point estimates. in effect, county-level, unobservable shocks that are correlated with changes in appliance ownership and fertility are the primary source of potential bias for the coefficient of interest. table reports unweighted, least-squares estimates of for pairs of census years with information on the same appliance measures ( to , to , or to ). the we concentrate on potential bias against the gsv hypothesis, but one can also imagine scenarios in which the coefficient is positively biased in favor of the gsv hypothesis (e.g., reverse causality would lead families with more children to buy more appliances to keep up with added housework). because median family income is unobservable in , differenced regressions with cannot include the change in log median family income as a control variable. however, it can be included in panel c of table ( - difference), in which case it slightly strengthens the negative coefficient (- . , s.e. = . ). omitting the control variable for observed changes in women’s labor market participation has a small effect on the coefficients of interest in table . this suggests that the scope for bias from unobservable changes in women’s market opportunities is small relative to the size of the coefficient one would need to account for the baby boom. sequence of specifications across columns corresponds to those in table . as before, heteroskedasticity-robust standard errors are computed and corrected for correlation within states for all specifications reported. the unadjusted correlations in appliance and fertility changes are negative (column ). when we account for observable changes in county-level covariates (including women’s labor-force participation), unobservable state-level changes, and time- invariant county-level unobservable characteristics (column ), the coefficient estimates are still negative. if anything, the first-differenced regressions provide evidence that counties with greater growth in appliance ownership experienced smaller changes in their fertility rate. in light of the theoretical framework presented in section , these results are consistent with appliances enabling the substitution toward child quality or other home produced goods. . electricity, the general fertility rate, and completed fertility a potential concern with the appliance regressions in tables and is that appliance ownership might be a poor measure of the “state of household technology.” from the perspective of households, access to electrical service may be a better indicator of access to current technology than ownership of specific appliances: few families that had electrical service available to them declined to have lights (or electric irons), whereas the decision to purchase specific, large consumer durable goods might have reflected a variety of household- level differences in preferences, prices, and plans. another advantage of using electrical service is that it is a relatively homogenous and easily comparable product across locations and time. our data on county-level electrification come from the published volumes of the census of housing in and from the haines ( ) county files for . panels a and b of table for brevity, we omit one set of results ( - change in modern stoves) in which the estimate in column is positive but statistically insignificant ( . , s.e. . ). as before, this estimate is too small to account for a substantial portion of the baby boom. it should also be considered in light of the to (panel b) and the to (panel c) regressions which exhibit negative point estimates. the census does not report electric lighting in , presumably because it was nearly universal. in more than percent of homes had electrical service (united states department of commerce ). it is also important to note that the county-level electrification rate is a relevant but invalid instrument for appliance ownership. although it is true that electric lighting was strongly, positively correlated with the level of refrigerator and modern stove ownership in , electrification is also highly correlated with a host of other county-level measures of economic development. therefore, electrification does not provide a valid instrument for estimating the causal effect of appliance ownership on fertility. report estimates from separate, unweighted, least-squares regressions using equation , and panel c reports estimates from a regression of the change in the fertility rate on the change in electrical service between and using equation . overall, the cross-sectional and first- differenced results provide no evidence of a positive relationship between electrification and fertility. in fact, the point estimates indicate that electrification is significantly and negatively correlated with county fertility rates even after accounting for economic and demographic differences and changes in column . taken together, the results provide no suggestive evidence consistent with improvements in household technology causing the baby boom. as suggested before, it appears that whatever caused the baby boom worked against secular economic forces that tended to reduce u.s. fertility rates over this period. a separate matter of concern is that changes in period fertility rates might not adequately reflect changes in completed fertility, the key outcome variable in the gsv model. although period rates are used as evidence by gsv and period and completed fertility are highly correlated (see figure ), we have assembled a new dataset that allows for a test based on cohorts’ completed fertility rates. these completed fertility rates are constructed from the integrated public use microdata series (ipums, ruggles et al. ) for women ages to aggregated into birth-state and year-of-birth cells. we link estimates of completed fertility with newly-entered data from archived issues of the edison electrical institute’s statistical bulletin, which contain annual, state-level information on the number of residential electrical service customers from to . with this information, each woman can be assigned an “electrical service exposure index” based on her year of birth and state of birth, which is the average ratio of electric customers over total households in that state during the time in which the woman would have been at the peak of her childbearing years (ages to ). this index is a rough measure we divided the eei customer counts by the census of housing counts of families ( and ) or occupied dwelling units ( - ) in each state to estimate the proportion of families with electrical service. we interpolated the housing counts between census dates. this choice of denominator is consistent with the housing unit counts in historical statistics of the united states ( ). see kenneth snowden’s discussion (volume : - and - ): “before the census enumerated ‘families’ and not housing unit[s]…. however, the two concepts are closely related: a census family was defined in as a single person living alone, a small group of unrelated persons sharing living accommodations, or, more normally, a group of related persons who live together as one household. despite differences in terminology, therefore, the basic notion of a family, dwelling unit, or housing unit has provided essentially comparable measures of the residential housing stock since .” of the probability of having access to electricity during the main years of family formation. it corresponds to the spirit of the gsv model, which assumes that households make decisions about the number of children in the first period of adulthood based upon the current “state of household technology.” the limitation of this definition, given the available data, is that the index can only be constructed for women born from (ages to from to ) to (ages to from to ). as a result, the final sample consists of women born in the coterminous u.s. between and in the to ipums. the positive correlation between completed fertility and advances in household technology can be tested by exploiting variation in the timing of electrification in women’s state of birth using the following linear regression framework, ( ) ∑ ∑ , where y denotes the birth cohort (inferred from age and year of observation) and b denotes the state of birth; n is the mean self-reported number of children ever born (excluding miscarriages and still-births); exp is the “exposure to electricity” index; and and capture year-of-birth invariant differences across birth states and state-of-birth invariant differences across birth cohorts, respectively. the set of demographic controls is limited, because measures of income, place of residence, and other life circumstances at the time of observation in to are poor proxies for circumstances in early adulthood. as a result, includes a constant and characteristics that should vary little over the life-course but are strongly correlated with differences in lifetime income and socio-economic status: the proportion of the cohort that is “nonwhite”; mean educational attainment; and, in one specification, the mean of husbands’ educational attainment and proportion of husbands who are nonwhite, which are only observed for women residing with their husbands at the time of the census. table presents population weighted least-squares estimates from specifications that add fixed effects and control variables sequentially. heteroskedasticity-robust standard errors are corrected for correlation within states the median age of last birth for these cohorts was approximately . completed fertility peaked around the birth cohorts of . our birth cohorts cover most of the increase in completed fertility for the cohorts born between and as well as years of cohorts prior to the increase. in the eei data, maryland and washington dc customers are always counted together, and north carolina and south carolina customers are often counted together. for consistency we have used these larger units of aggregation for all years, which yields birth state groups. and are reported in parentheses beneath the point estimates. column presents the unadjusted correlation between children-ever-born and exposure to electricity. column adds state- and year-of-birth fixed effects. column includes controls for the woman’s race and education level, and column includes her husband’s education level (this reduces the sample to women who are currently married at the time of observation). we prefer the specifications that include birth-state and birth-year fixed effects (columns to ), as they rely upon within-state variation in the speed of electrification to estimate . in contrast to the hypothesized positive link between the state of household technology and completed fertility, cohorts born into states with higher rates of electrification in their early adult years had fewer children on average. regardless of specification, the inclusion of covariates has a negligible impact on the magnitude of the point estimates of interest. overall, the estimates in table provide compelling evidence that the “state of household technology,” as embodied in electrification, is not positively associated with completed fertility. in summary, empirical tests using disaggregated data with the best available measures of appliance ownership, electrification, and the general fertility rate provide no evidence that advances in household technology caused the baby boom. after controlling for a host of observable characteristics and fixed effects, the point estimates are negative or small in magnitude (table , , and ). moreover, the negative relationship between exposure to electricity in young adulthood and completed fertility—the closest empirical test of the mechanism proposed in gsv’s model—also fails to support the claim that advances in household technology caused the baby boom (table ). . outen the lights: the amish baby boom a final test of the household-productivity hypothesis investigates fertility change among a group that limited its use of modern household conveniences and generally refused to adopt appliances powered by electricity: the old order amish. in sociologist john hostetler wrote, “custom is regional and therefore not strictly uniform. the most universal of all amish the amish began settling in pennsylvania in the early s and later settled in parts of ohio, new york, indiana, illinois, and ontario. for background on the amish, see hostetler ( ) or nolt ( ). norms across the united states and canada are the following: no electricity, telephones, central- heating systems, automobiles, or tractors with pneumatic tires…” ( ). although he also notes that some amish had started to use gas-powered kitchen and farm equipment ( ), it is clear that the amish uptake of modern appliances was very limited and late in comparison to the u.s. farm and general populations. if rapid improvements in the “state of household technology” were the main cause of the midcentury baby boom, and the amish were not “treated” with these improvements, then one would expect an absence of the baby boom among the amish. in this sense, amish demographic history provides an independent test of the household-technology centered explanation of the baby boom. using information on the language spoken at home (pennsylvania dutch) in the and ipums (the only census years with information on language spoken at home and children ever born), we document changes in completed fertility among the amish. the percent and ipums samples contain information on , women ages to at the time of observation who spoke pennsylvania dutch at home. it is likely that most women speaking pennsylvania dutch at home in the late twentieth century were amish, but we cannot observe religion directly in the census data and, therefore, we call this sample the “likely amish.” in addition, we generate a sample of “likely old order amish” by excluding respondents who had telephones in their residence in or . this conservative approach also excludes women who followed the practices of the old order amish during the to period (i.e., did not adopt new home production technologies) but who subsequently acquired a phone. this additional filter yields even smaller sample sizes (see data appendix table ) and, therefore, the completed fertility of likely old order amish can only be tracked in the census data for the most recent birth cohorts. for comparison groups, we create samples of ever-married women in the same birth cohorts who were residing on farms in indiana, ohio, and pennsylvania (states with the largest amish populations) and u.s. women not residing on farms. unfortunately, the hostetler explains, “the social organization of the amish community has little facility for dealing with change. the general effort to preserve the old and degrade the new is so pervasive that change must occur slowly…” ( : ). the old order amish remain more conservative than some other anabaptist groups who may also speak pennsylvania dutch. see kraybill ( ) for discussion of splits within the amish and differences across groups in appliance use in the s in holmes county, ohio; see umble ( ) for a discussion of phone use. census did not ask about the number of children ever born, so the estimates for each of these groups only capture the upswing in fertility rates during the baby boom and very little of the baby bust. figure plots the mean number of children ever born by birth cohort for these four groups. for each birth cohort, u.s. native, nonfarm residents have the lowest completed fertility rate, followed by women on farms in indiana, ohio, and pennsylvania. the likely amish women had higher levels of completed fertility than the non-farm population, as one might expect in an agrarian population. the likely old order amish had the highest levels of completed fertility, having an average of almost two more children over their lifetimes. the key result of this analysis is that the fertility trends of the likely amish track those in the two comparison groups quite closely. in a reversal of a long period of fertility decline, each of the groups in figure – including the amish – had large increases in completed fertility among the cohorts of women born after . in the u.s. nonfarm sample and in the indiana, ohio and pennsylvania farm sample, completed fertility increased by roughly one birth per woman from trough ( - ) to peak cohorts ( - ). the likely amish group had an increase of . births from - to the peak - cohort, and the likely old order amish had an increase of . births from - to the peak - cohort (and a rise of . births between the - and - cohorts). in short, the amish had a baby boom that roughly began at the same time, lasted as long as, and matched the magnitude of the boom in the rest of the u.s. population. these findings are not unique to the census data or due to problems with distinguishing the amish population. demographic studies of the old order amish based on non-census sources (typically directories with genealogical information) corroborate these findings. ericksen et al. ( ) compiled data from the four largest amish settlements in the united states: lancaster, pennsylvania; elkhart, indiana; and holmes and geauga counties, ohio. between the - birth cohort of amish women and the - cohort, they document a decline in the proportion of childless women, a rise in age-specific marital fertility for - and - year olds, and a rise in cumulative marital fertility by about . children at age ( - ). these findings correspond fairly closely to our census-based estimates. markle and pasco ( ) relied on the indiana amish directory from to calculate period fertility rates. between - and - , the average age at marriage for women in their sample fell from . to . years, and the average time between marriage and first birth declined. between - and - , they also document large increases in birthrates among women in their s ( , figure ). most recently, greska ( ) compiled data from a directory for the amish settlement in geauga, ohio, including , women. consistent with the studies above, he finds a dip in age at first marriage and age at first birth for the - birth cohorts of women, and he finds a jump in fertility rates for women in their s from the - birth cohorts relative to previous cohorts. consistent with our estimates, he reports that the - cohorts had a cumulative fertility rate that was . higher than the - cohorts, and . higher than the - cohorts ( - ). in summary, our census-based estimates are broadly consistent with an independent demographic literature, which also finds signs of a sizable increase in fertility among the old order amish that is coincident with the general population’s baby boom. . conclusions the mid-twentieth-century rise in fertility is a compelling puzzle not only because it was a dramatic departure from the previous years of american demographic history, but also because it unfolded against a background of rising income, urbanization, educational attainment, infant health, and women’s labor force participation—many of the factors that economists and demographers typically associate with declining fertility. women who reached their childbearing years in the s and s (women born between and ) got married younger, bore their first child sooner, and had more children over their lifetimes than women born earlier in the century. richard easterlin’s longstanding view of the baby boom ( , ), which emphasizes the contrast between pre-war penury and post-war prosperity, has anchored the literature for decades, but there is no scholarly consensus about the baby boom’s ultimate causes. markle and pasco do not report the exact birthrate figures, but rather present a graph ( : , figure ). from their figure, we infer that the birthrate (divided by ) for women aged - increased from approximately . to . , and for women - it increased from about . to . . in earlier work, smith ( ) studied the amish in rural southeastern pennsylvania. he also reports that birth spacing was significantly shorter for amish women who married in the s and s compared to earlier cohorts ( ). our goal in this paper is to provide a comprehensive evaluation of a novel household- productivity centered explanation of the baby boom (greenwood, seshadri, and vandenbroucke ). this explanation uses the formal structure of modern, macroeconomic theory and calibration techniques to model the american baby boom within the context of the secular decline in fertility. our analysis of a newly-compiled dataset for approximately , u.s. counties provides scant evidence that advances in household technology contributed to the baby boom. this does not appear to be an artifact of poor measurement of key variables such as the “state of household technology” or completed fertility. in fact, in a sample of over , women, within year-of-birth and state-of-birth cohort variation in exposure to electricity (a broad proxy for the “state of household technology”) is negatively related to completed fertility, even after adjusting for differences in education and race (strong correlates of lifetime income). these findings are consistent with basic economic theory as long as child quantity is not assumed to be a normal good or advances of household technology are permitted to impact the demand for other home produced goods. one final piece of evidence is that the mid-century increase in fertility among the amish suggests that the baby boom occurred among populations that were relatively unaffected by changes in electrification, modern appliances, the availability of frozen foods, and other advances in household technology. in combination with the paper’s previous sections, which argue that neither theory nor history lend unqualified support for the household- productivity-centered view, this new empirical evidence calls scholars to look elsewhere for the baby boom’s causes. although this paper concentrates on a single specific hypothesis, insights emerge that can inform broader research on demographic history and the baby boom. first, whatever factors explain the american baby boom must account for its occurrence in urban and rural areas, among different educational and racial groups, and in all regions. it was a remarkably pervasive event, and scholars should endeavor to explain the near simultaneity of baby booms in places and populations that varied widely in their social and economic circumstances. in this context, explanations centered on mass mobilization during world war ii, on rapid suburbanization, or on changing norms and culture may not satisfactorily explain the nearly simultaneous occurrence of the baby boom among the amish nor, perhaps, in several other countries. at the same time, we find that although the baby boom was widespread, the boom was not evenly spread, and the variation in changes in fertility invites analyses based on detailed cross-place and cross- household data. both easterlin’s pioneering work and gsv’s ambitious argument are based on national-level time-series patterns in the united states, but more disaggregated views may prove extremely valuable, and indeed necessary, for discerning among the many potential causes. in formulating new causal hypotheses, identifying “untreated” populations that provide effective falsification tests should prove useful. based on the evidence we have seen, it seems unlikely that any monocausal explanation for the baby boom will suffice, but the question remains open. references bailey, martha j. and william j. collins. . “the wage gains of african-american women in the s.”journal of economic history , : - 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[originally ]. never done: a history of american housework. new york: henry holt and company. stigler, george j. . “domestic servants in the united states, - .” new york: nber occasional paper . umble, diane zimmerman. . “amish on the line: the telephone debates.” in kraybill and olshan, eds., the amish struggle with modernity. hanover, nh: university press of new england: - . u.s. department of commerce. . historical statistics of the united states: colonial times to . washington dc: gpo. u.s. department of commerce, bureau of the census. . sixteenth census of the united states: . housing, volume ii: general characteristics. washington, dc: government printing office. u.s. department of commerce, bureau of the census. . census of housing: . volume i: general characteristics. washington, dc: government printing office. u.s. department of commerce, bureau of the census. . census of housing, . volume i: states and small areas. washington, dc: government printing office. vanek, joann. . “time spent in housework.” scientific american : - .willis, robert j. . “a new approach to the economic theory of fertility behavior.” journal of political economy , : s -s . willis, robert j. . “a new approach to the economic theory of fertility behavior.” journal of political economy , : s -s . table . summary statistics, u.s. county-level data infants per , women of age to . ( . ) . ( . ) . ( . ) proportion of housing units with electric lights . ( . ) . ( . ) --- proportion of housing units with a mechanical refrigerator . ( . ) . ( . ) --- proportion of housing units with a modern stove (using gas or electricity as fuel) . ( . ) . ( . ) . ( . ) proportion of housing units with a power-driven washing machine --- --- . ( . ) notes: the table reports unweighted averages across u.s. counties (excluding hawaii and alaska). standard deviations are in parentheses. counties that are omitted from table ’s regressions are also excluded in this table, but this has little effect on the reported figures. sources: infants per woman, proportion of homes with lights (in ), refrigerators, and washing machines are from haines ( ). we compiled data on electric lighting in and stoves in all years from the published volumes of the census of housing (u.s. department of commerce , , ). see data appendix for more information about the dataset. table . cross-sectional regressions of fertility on appliances in u.s. counties, - dependent variable: general fertility rate ( ) ( ) ( ) panel a: percent with refrigerator - . [ . ] - . [ . ] . [ . ] observations r-squared . . . percent with modern stove - . [ . ] - . [ . ] . [ . ] observations r-squared . . . panel b: percent with refrigerator - . [ . ] - . [ . ] - . [ . ] observations r-squared . . . percent with modern stove - . [ . ] - . [ . ] . [ . ] observations r-squared . . . panel c: percent with washing machine - . [ . ] - . [ . ] . [ . ] observations r-squared . . . percent with modern stove - . [ . ] - . [ . ] - . [ . ] observations r-squared . . . state fixed effects no yes yes economic and demographic controls no no yes notes: each point estimate is from a separate regression corresponding to equation . the dependent variable is the number of infants (under year) per thousand women ages to . a “modern stove” is defined to use electricity or gas (not wood, coal, or kerosene). the unit of observation is a county. heteroskedasticity-robust standard errors have been corrected for correlation at the state level and are reported in brackets. the covariates in column include the urban proportion of the county’s population, log population density, nonwhite proportion of the county’s population, proportion of employment in agriculture and manufacturing (separately), median years of schooling for those over age , log of median property value, and the proportion of women in the labor force. the and specifications also control for log median family income (this variable is unavailable in ). the urban variable generally measures the proportion of the population residing in incorporated places with more than , residents. the density measure is the log of residents per square mile. nonwhite includes black and “other” racial categories. the proportion of workers employed in agricultural and manufacturing industries are expressed relative to total employment. the percent of women in the labor force is the ratio of all women in the labor force divided by the number of women over age . the median schooling variable in the table is for women, whereas in and it is for both men and women. observations with missing values for any economic or demographic control variable are dropped to maintain a consistent sample across specifications. sources: data for refrigerators, washing machines, and covariates are from haines ( ). data on the type of cooking fuel, which are used to define “modern stoves,” were entered from the published census of housing volumes (u.s. department of commerce , , ). table . differenced regressions of fertility on appliances in u.s. counties, - dependent variable: change in general fertility rate ( ) ( ) ( ) panel a: refrigerators - Δ percent with refrigerator - . [ . ] - . [ . ] - . [ . ] observations r-squared . . . panel b: modern stoves, - Δ percent with modern stove - . [ . ] - . [ . ] - . [ . ] observations r-squared . . . panel c: modern stoves, - Δ percent with modern stove - . [ . ] - . [ . ] - . [ . ] observations r-squared . . . state fixed effects no yes yes economic and demographic controls no no yes notes: each point estimate is from a separate regression corresponding to equation . the dependent variable is the change in the number of infants (under year) per thousand women ages to between two census years at the county-level. heteroskedasticity-robust standard errors have been corrected for correlation at the state level and are reported in brackets. the covariates in column include the change in urban proportion of the county’s population, the change in log population density, the change in nonwhite proportion of the county’s population, the change in proportion of employment in agriculture and manufacturing (separately), the change in median years of schooling for those over age , the change in log of median property value, and the change in the proportion of women in the labor force. urban, density, nonwhite, employment and labor force variables are defined as in table ’s notes. in this table, for better comparability with the variables available in and (which include both men and women), the schooling variable in is the average of the median schooling values for men and women. when necessary, observations with missing values are dropped to maintain a consistent sample across specifications. sources: data for refrigerators, washing machines, and covariates are from haines ( ). data on the type of cooking fuel were entered from the published census volumes in each year as described in the data appendix. table . regressions of period fertility on electrical service, - dependent variable: general fertility rate ( ) ( ) ( ) panel a: fertility cross section, percent with electric lights - . [ . ] - . [ . ] - . [ . ] observations r-squared . . . panel b: fertility cross section, percent with electric lights - . [ . ] - . [ . ] - . [ . ] observations r-squared . . . panel c: fertility change, - Δ percent with electric lights - . [ . ] - . [ . ] - . [ . ] observations r-squared . . . state fixed effects no yes yes economic and demographic controls no no yes notes and sources: see table . we compiled the data for electric lights in from the published volumes of the census of housing (u.s. department of commerce ); the electric light data are from haines ( ). table . regressions of children-ever-born on exposure to electrical service dependent variable: children ever born ( ) ( ) ( ) ( ) exposure to electricity x - . - . - . - . [ . ] [ . ] [ . ] [ . ] state of birth f.e. no yes yes yes year of birth f.e. no yes yes yes race and education no no yes yes husband’s education no no no yes observations r-squared . . . . notes: the dependent variable is the mean self-reported children-ever-born to ever married women. observations are birth state-birth year cells. heteroskedasticity-robust standard errors have been corrected for correlation by birth state and are reported in brackets. the numerator for “mean exposure to electricity” in a given state-year cell is constructed from edison electrical institute (eei) statistical bulletin which contains annual state-level information on the number of residential electrical customers from to . in the eei data, maryland and washington dc customers are always counted together. north carolina and south carolina customers are often counted together. for consistency we have used these larger units of aggregation for all years. to calculate the denominator, we use the housing unit count from the census (interpolated between dates). “exposure to electricity” is calculated as the mean of this proportion over the peak child-bearing years ( to ) for each year-of-birth and birth state cohort (and multiplied by ). the sample includes women born from to ( cohorts) and geographic units for observations. sources: edison electric institute statistical bulletin (various years) and - ipums (ruggles et al. ). figure . u.s. general fertility rate and children ever born from to notes: the outcome variables are the period fertility rate and the mean self-reported number of children by birth cohort. birth cohorts are indexed to year of birth and increased by years. for instance, the birth cohort of corresponds to the year on the graph’s horizontal axis. computations using the ipums use population weights. sources: annual fertility rates are calculated using historical statistics, http://www.cdc.gov/nchs/data/statab/t x .pdf. the mean number of children ever born per woman is calculated using a sample of ever-married women ages to in the , , , and ipums (ruggles et al. ) . . fertility rate (left axis) white fertility rate mean children ever born (right axis) figure . proportion of households with modern household technology, - note: dashed lines indicate linear interpolation between data points. source: lebergott ( : - ). electric lighting washing machines running water indoor toilet central heating mechanical refrigerators vacuum fig notes: t or ten-ye informati observati pennsylv likely am order am axis. see source: ure . mean the horizonta ear categorie ion). the sa ion. the “lik vania dutch mish who do mish” are plo e text and da - ip n children b al axis repre s to maintain ample is com kely amish” at home. th o not have ph otted on the ata appendix ipums (rug born to am esents the bir n informativ mprised of ev ” sample con he “likely ol hones in thei right vertica x for more in ggles et al. mish and oth rth-cohort of ve samples si ver-married w nsists of wom ld order am ir residence al axis; the o formation. ). her women f the women izes (see dat women ages men reportin mish” sample ( - other series a n born from n, which are g a appendix s to at ng that they s e is the subsa ipums). t are plotted on m - grouped into for more the time of speak ample of the the “likely o n the left ver o five e old rtical theory appendix consider a static version of the gsv model’s maximization problem, where households enjoy utility from consuming children, , and a composite of other goods purchased on the market, (price normalized to ). nonlabor income is given by , and is the market wage. children are produced using some fraction of an individual’s time and their shadow price is given by . thus, advances in household technology increase and reduce the shadow price of children, . in this framework, the household’s problem can be written as , : , and optimality implies that ′ ′ . under the common assumptions that ′′ and ′′ , differentiating (a ) with respect to (while holding the relative price of children fixed), ′′ ′′ , shows that this framework implicitly assumes that child quantity is a normal good. it follows from the logic in equation ( ) of the paper that a change in the state of household technology, , which induces a reinforcing substitution and (because children are a normal good) income effect, has an unambiguously positive impact on the number of children. this can be shown directly by differentiating (a ) and reorganizing, ′ ′′ ′′ ′′ . data appendix county-level data data for infants per woman aged to and for the proportions of homes with lights (in ), refrigerators (in and ), and washing machines ( ) are from the files compiled by michael haines ( ). specifically, files “ : census i”, “ : census ii”, and “ : county data book” provide demographic and economic data for . files “ : census i”, “ : census ii”, “ : county data book” and “ : county data book” provide data for . files “ : census ii”, “ : census iii”, “ : county data book”, and “ : county data book” provide data for . in and the housing appliance data are based on subsamples of the full population. we typed in the data for electric lights in and for cooking fuel in , , and from the published volumes of the census of housing, and combined that information with the data from haines ( ) described above. for , the cooking fuel figures are from volume , table (for each state) of the census of housing. for , the lighting and cooking fuel figures are from volume , table (for each state) of the census of housing. for , the cooking fuel figures are from volume , tables and of the census of housing. the proportion of homes with “modern stoves” is the ratio of the number using electricity, utility gas, or bottled gas for the principal cooking fuel divided by the total number of units that report the cooking fuel variable; implicitly, we define those using wood, coal, kerosene, “other”, or no fuel as “not modern.” the “mechanical refrigerator” variable pertains to any type of refrigeration equipment powered by electricity, gas, kerosene, or gasoline; this is distinct from an “ice box.” the “washing machine” variable that is reported in the haines files for includes “automatic and semi-automatic” washing machines that wash, rinse, and damp dry the laundry; “washer-dryer combination” machines that wash, rinse, and fully dry the clothes in the same tub; and power-operated “wringer or spinner” machines. we made the following adjustments to the data from haines ( ): • in , the proportion of housing units with refrigerators in raleigh county, west virginia should be . percent, and the proportion in washington, dc should be . percent. the median years of schooling for women and men in cooke county, texas should be . and . respectively. the county code for warwick, virginia is adjusted in to facilitate merger across datasets. • in , approximately counties with missing values for refrigerators in the county data book (underlying the haines data) are listed as zeros in the haines files. we referred back to the original census volumes to fill in the correct figures when possible, or to set the value to “missing” if unavailable in the census (replacing zero). separately, the proportion of housing units with refrigerators in washington, dc should be . . • in , the figure for washing machines in lee county, kentucky should be . percent according to the county and city data book. the median property value in milam county, texas should be $ , . • for , we combined the counts for infants and women in fulton, milton, and campbell counties in georgia to be comparable with subsequent years ( fertility is used as a control variable in some regressions). • partial county entries for yellowstone national park are dropped from the analysis, as are counties/territories in hawaii and alaska. matching counties over time is imperfect due to occasional mergers and changes in boundaries. excluding counties with reported changes of more than square miles does not change the qualitative results from tables and . the coefficient in panel a, column (refrigerators, - ) increases in magnitude from - . (s.e.= . ) to - . (s.e.= . ); the coefficient in panel b, column (stoves - ) falls in magnitude from - . (s.e.= . ) to - . (s.e.= . ). the coefficient change for electric lighting ( - ) in table is from - . to - . (s.e.= . in both cases). in , approximately percent of counties have a bottom code for median property values of $ , in the census data. the results in the text are not sensitive to resetting these observations to $ , ( % of , ). in table , panel c, column , the coefficient on washing machines falls from . to . (s.e.= . in both cases); the coefficient on modern stoves falls from - . (s.e.= . ) to - . (s.e.= . ). in table , panel b, column , the coefficient on modern stoves ( - ) falls from - . to - . (s.e. = . in both cases); in panel c, column , the coefficient on modern stoves ( - ) increases from - . to - . (s.e. = . in both cases). state-level, annual electricity data the numerator for the “mean exposure to electricity” variable is constructed from the edison electrical institute (eei) publication, statistical bulletin. the bulletin provides annual state-level reports of the number of residential electrical customers from to . in the eei data, maryland and washington dc customers are always counted together. north carolina and south carolina customers are often counted together, and for consistency we used these larger units of aggregation for all years. to calculate the denominator, we used the housing unit counts from the census, which we interpolated between dates with constant growth rates. then, we divided the eei customer counts by the census of housing counts of families (in and ) or occupied dwelling units (in , , and ) in each state to estimate the proportion of families with electrical service. the figures for the denominator are consistent with the housing unit counts in historical statistics of the united states (carter et al., ). kenneth snowden discusses the comparability of housing count data across census years in volume of historical statistics ( - and - ): “before the census enumerated “families” and not housing unit…. however, the two concepts are closely related: a census family was defined in as a single person living alone, a small group of unrelated persons sharing living accommodations, or, more normally, a group of related persons who live together as one household. despite differences in terminology, therefore, the basic notion of a family, dwelling unit, or housing unit has provided essentially comparable measures of the residential housing stock since .” on occasion, the ratio of residential customers from eei to housing units from the census slightly exceeds unity (in approximately percent of state-year cells from to ). nearly all such cases ( percent) occur between and when the true rate of electrification is likely to be close to percent for some states. we have left these values in place rather than making ad hoc adjustments to the underlying data. data appendix table : summary statistics for the likely amish samples birth cohort likely amish likely old order amish farm residents in in, oh, pa non-farm u.s. residents - . . . [ ] [ ] [ , ] - . . . [ ] [ ] [ , ] - . . . [ ] [ ] [ , ] - . . * . . [ ] [ ] [ ] [ , ] - . . . [ ] [ , ] [ , ] - . . . . [ ] [ ] [ , ] [ , ] - . . . . [ ] [ ] [ , ] [ , ] - . . . [ ] [ , ] [ , ] - . . . . [ ] [ ] [ , ] [ , ] - . . . [ ] [ , ] [ , ] - . . . . [ ] [ ] [ , ] [ , ] - . . . . [ ] [ ] [ , ] [ , ] - . . . . [ ] [ ] [ ] [ , ] column total . . . . [ , ] [ ] [ , ] [ , , ] notes: the table entries represent the mean number of children ever born, and the figures in brackets are the cell sample sizes. birth cohorts are grouped into five or ten-year categories to maintain informative samples sizes. *the cohort labeled - for the likely old order amish corresponds to the to cohort. the younger cohorts of the likely old older amish are grouped into ten year cohorts, so the cohorts labeled - and - correspond to the ten- year groupings - and - , respectively. for sample definitions and source information, see text and figure notes. the hydrophobic effect, and fluctuations: the long and the short of it commentary the hydrophobic effect, and fluctuations: the long and the short of it erte xia and amish j. patela, the hydrophobic effect, which is used to describe the aversion of oil for water or the affinity of oily objects for one another in water, plays an important role in diverse disciplines ( ). for example, by segregating to the oil–water interface, amphiphilic molecules that possess both hydrophobic and hydrophilic groups can mitigate unfavorable oil–water interactions, thereby stabilizing emulsions and facilitating detergency. be- cause roughly half the amino acids, which form the basic building blocks of proteins, are hydrophobic, the hydrophobic effect also plays a central role in bio- physics. owing to its ubiquity and its multifaceted nature ( , ), being able to accurately model the hydrophobic effect is both important and challenging. in pnas, vaikuntanathan et al. ( ) provide important insights into the essential ingredients required in a minimal model of the hydrophobic effect. the hydrophobic effect characteristically mani- fests itself in very different ways at microscopic and macroscopic length scales ( ). macroscopically, the aversion of oil and water for one another is captured by the large surface tension associated with the oil– water interface. at this scale, the hydrophobic effect drives the minimization of the unfavorable interfacial area (for example, by the coalescence of oil droplets in water). although the macroscopic hydrophobic effect is governed by the relatively straightforward physics of interfaces, it can nevertheless lead to complex phe- nomena, such as the nanobubble-mediated long- ranged forces between extended hydrophobic sur- faces ( ), or the assembly of anisotropic particles at curved interfaces ( ). interestingly, the thermody- namic driving force for such hydrophobic assembly, which is dictated by interfacial tension, decreases with increasing temperature. in contrast, at the molecular scale, the driving force for hydrophobic assembly famously increases with increasing temperature ( ). in fact, whenever biomo- lecular assembly occurs upon increasing temperature, in seeming disregard for entropic considerations, the hydrophobic effect is often the first suspect. a classic example is the cold denaturation of proteins. whereas the denaturation or unfolding of proteins upon heating is common, and is ascribed to the configura- tional entropy of the macromolecule prevailing over the favorable energy of the folded state, certain pro- teins also denature upon cooling. such cold denaturation is explained by the fact that the molecular hydropho- bic effect, which stabilizes the folded state of the pro- tein, is weakened upon cooling, causing the protein to unravel ( ). to understand these contrasting manifestations of the hydrophobic effect, it is instructive to consider how water structure is perturbed near small (molecu- lar) and large (macroscopic) hydrophobic solutes. near a macroscopic hydrophobic object, such as an oil droplet, interfacial water molecules are unable to participate in four hydrogen bonds like their bulk counterparts ( ). this energetically unfavorable sce- nario is responsible for the large oil–water surface ten- sion and drives the minimization of interfacial area. in the vicinity of molecular hydrophobes such as meth- ane, the hydration waters are able to maintain all their hydrogen bonds, but accommodating the hydro- phobe severely constrains the hydrogen bonding net- work of water ( ). this strain on the hydrogen bonding network is reflected in a large negative entropy of hydration; it is this entropic penalty that causes molec- ular hydrophobes to assemble more strongly as tem- perature is increased. thus, hydrophobic hydration and assembly at the molecular level are not governed by interfacial physics, but are fundamentally different. given these differences in hydrophobic hydration in the molecular and macroscopic limits, at what size do we cross over from one regime to the other? theory, simulation, and experiment all agree that the cross-over occurs for solutes that are about nm in size ( , – ). this length scale emerges from a prod- uct of the surface tension of water and its isothermal compressibility, properties that respectively influence the hydration of large and small solutes ( , , ). the -nm length scale also plays a key role in numerous molecular assemblies; for example, most amino acid side chain residues are smaller than nm, but folded proteins are larger than nm. being able to quantita- tively capture the length scale cross-over in hydrophobic adepartment of chemical and biomolecular engineering, university of pennsylvania, philadelphia, pa author contributions: e.x. and a.j.p. wrote the paper. the authors declare no conflict of interest. see companion article on page e in issue of volume . to whom correspondence should be addressed. email: amish.patel@seas.upenn.edu. www.pnas.org/cgi/doi/ . /pnas. pnas | april , | vol. | no. | – c o m m e n t a r y d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://crossmark.crossref.org/dialog/?doi= . /pnas. &domain=pdf mailto:amish.patel@seas.upenn.edu www.pnas.org/cgi/doi/ . /pnas. hydration is thus critical for understanding biophysical phenom- ena such as protein folding and aggregation, and must be a central requirement of any comprehensive theory of the hydrophobic effect. in a seminal contribution, lum, chandler, and weeks (lcw) introduced the key theoretical principles necessary for reconciling hydrophobic hydration at small and large length scales ( ). the lcw theory of hydrophobicity prescribed decomposing the spa- tial variations in water density, which arise from the presence of a hydrophobic solute, into two separate components. the first com- ponent, ρlðrÞ, is governed by interfacial physics and displays only gentle variations over molecular length scales; lcw used a landau– ginzburg density functional to describe the free energetics of this component. the second component, ρsðrÞ, incorporates the small-amplitude, short-wavelength fluctuations in molecular density. importantly, the statistics of such small fluctuations in water density are gaussian, a fact that underpins liquid-state theories ( , ) and has been confirmed by molecular simulations ( ). lcw theory leverages the gaussian statistics of the small length scale fluctuations to integrate out ρsðrÞ, thereby providing a renormalized and coarse-grained description of the free ener- getics of ρlðrÞ. in particular, ρlðrÞ remains close to its bulk liquid value, ρbulk, within and near molecular solutes, because the small ρsðrÞ fluctuations are sufficient to vacate the region occupied by the solute. in contrast, vacating waters from a region larger than nm (for the solute to occupy) requires nucleation of an interface, with ρlðrÞ vanishing within the solute region and approaching ρbulk outside. the original lcw paper used a variational formalism to evaluate ρlðrÞ in the mean-field limit ( ). to incorporate fluctua- tions in ρlðrÞ, subsequent lcw-inspired treatments ( , ) used simple models capable of capturing interfacial physics, such as the lattice gas model. in the lattice gas model, variations in density between adjacent lattice sites carry an energetic penalty, «, which along with the lattice spacing, δ, determines the interfacial tension, γ. however, the formation of a water–vapor interface at ambient conditions is associated not only with an energetic penalty, but also with entropically favorable interfacial fluctuations (fig. a); indeed, the statistics of such long-wavelength capillary wave fluc- tuations are well understood ( ). vaikuntanathan and geissler ( ) recognized that for the lattice gas model to appropriately capture capillary wave fluctuations, only a small range of « values could be used. if « is too small, we approach the critical point, and the bulk liquid and vapor phases display large density fluctua- tions; however, if « is too large, interfacial fluctuations are sup- pressed. with the optimally parameterized lattice gas for ρlðrÞ, the authors’ lcw-inspired model was able to quantitatively capture, without any adjustable parameters, the length scale cross-over in the hydration free energies of idealized hydrophobes of various shapes. remarkably, their model was also able to capture subtle features of the interfacial free energetics, such as a preference for droplets over bubbles ( , ), highlighting the importance of capillary fluctua- tions even for nanoscale interfaces (fig. b and c). in their recent study, vaikuntanathan et al. ( ) subject their lcw-inspired model to another stringent test, that of quantifying the statistics of water density fluctuations; such fluctuations, char- acterized by the probability, pvðnÞ, of observing n waters in a probe volume, v, have provided numerous insights into the mul- tifaceted nature of the hydrophobic effect ( , , , ). for example, low-n fluctuations are significantly enhanced near a hy- drophobic surface, situating the interfacial waters at the edge of dewetting transition (fig. d) and making them sensitive to pertur- bations ( , ). using their lcw-inspired model, vaikuntanathan et al. ( ) obtain not just qualitative, but also quantitative, agreement with molecular simulations, in their estimates of pvðnÞ for probe volumes in bulk water, at interfaces, and in hydrophobic confinement. furthermore, this quantitative agree- ment is lost if either the small length-scale gaussian fluctuations or the large length-scale interfacial fluctuations are not included in the model. by identifying capillary fluctuations as an essential ingredient of a minimal model of the hydrophobic effect, vaikuntanathan et al. ( ) have laid the groundwork for studying aqueous systems using a coarse-grained representation of water, which has the accuracy of molecular simulations but exacts a tiny fraction of the computational cost. given the importance of solvent fluctua- tions in modulating dewetting barriers and the kinetics of hydro- phobic interactions ( – ), their model could conceivably be used to study not only the thermodynamics, but also the kinetics of complex self-assemblies. another important application is the study of biomolecules, which are too large to model using explicit water. along with hydrophobic interactions, electrostatic interac- tions also play an important role in the biomolecular context. we hope that the success of lcw-inspired models motivates the de- velopment of minimal models that efficiently incorporate fluctua- tions in both the short- and long-wavelength water polarization response to charged moieties ( , ). acknowledgments this work was supported by national science foundation grants upenn mrsec dmr - and cbet , and the charles e. kaufman foundation grant ka - . a b c d fig. . long-wavelength interfacial fluctuations can be seen in simulation snapshots of (a) the liquid–vapor interface, (b and c) nanoscopic vapor bubbles in bulk water, and (d) at a hydrophobic surface. the work of vaikuntanathan et al. ( ) and vaikuntanathan and geissler ( ) highlights the importance of such fluctuations and provides a way to incorporate them into a minimal model of the hydrophobic effect. the average interface in each case is either flat (a and d) or spherical (b and c), and the color represents deviation away from the average interface toward (red) or away (blue) from water (cyan). the instantaneous interfaces were computed using the algorithm outlined in ref. . | www.pnas.org/cgi/doi/ . /pnas. xi and patel d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , www.pnas.org/cgi/doi/ . /pnas. chandler d ( ) interfaces and the driving force of hydrophobic assembly. nature ( ): – . lum k, chandler d, weeks jd ( ) hydrophobicity at small and large length scales. j phys chem b : – . patel aj, et al. ( ) extended surfaces modulate hydrophobic interactions of neighboring solutes. proc natl acad sci usa ( ): – . vaikuntanathan s, rotskoff g, hudson a, geissler pl ( ) necessity of capillary modes in a minimal model of nanoscale hydrophobic solvation. proc natl acad sci usa ( ):e –e . ducker wa, mastropietro d ( ) forces between extended hydrophobic solids: is there a long-range hydrophobic force? curr opin colloid interface sci : – . cavallaro m, jr, botto l, lewandowski ep, wang m, stebe kj ( ) curvature-driven capillary migration and assembly of rod-like particles. proc natl acad sci usa ( ): – . matysiak s, debenedetti pg, rossky pj ( ) role of hydrophobic hydration in protein stability: a d water-explicit protein model exhibiting cold and heat denaturation. j phys chem b ( ): – . lee cy, mccammon ja, rossky pj ( ) the structure of liquid water at an extended hydrophobic surface. j chem phys : – . stillinger fh ( ) structure in aqueous solutions of nonpolar solutes from the standpoint of scaled-particle theory. j solution chem : – . ashbaugh hs, pratt lr ( ) colloquium: scaled particle theory and the length scales of hydrophobicity. rev mod phys : . southall nt, dill ka ( ) the mechanism of hydrophobic solvation depends on solute radius. j phys chem b : – . rajamani s, truskett tm, garde s ( ) hydrophobic hydration from small to large lengthscales: understanding and manipulating the crossover. proc natl acad sci usa ( ): – . li it, walker gc ( ) signature of hydrophobic hydration in a single polymer. proc natl acad sci usa ( ): – . pratt lr, chandler d ( ) theory of the hydrophobic effect. j chem phys : – . chandler d ( ) gaussian field model of fluids with an application to polymeric fluids. phys rev e stat phys plasmas fluids relat interdiscip topics ( ): – . hummer g, garde s, garcía ae, pohorille a, pratt lr ( ) an information theory model of hydrophobic interactions. proc natl acad sci usa ( ): – . ten wolde pr, sun sx, chandler d ( ) model of a fluid at small and large length scales and the hydrophobic effect. phys rev e stat nonlin soft matter phys ( pt ): . varilly p, patel aj, chandler d ( ) an improved coarse-grained model of solvation and the hydrophobic effect. j chem phys ( ): . weeks jd ( ) structure and thermodynamics of the liquid–vapor interface. j chem phys : – . vaikuntanathan s, geissler pl ( ) putting water on a lattice: the importance of long wavelength density fluctuations in theories of hydrophobic and interfacial phenomena. phys rev lett ( ): . sedlmeier f, netz rr ( ) the spontaneous curvature of the water-hydrophobe interface. j chem phys ( ): . willard ap, chandler d ( ) instantaneous liquid interfaces. j phys chem b ( ): – . godawat r, jamadagni sn, garde s ( ) characterizing hydrophobicity of interfaces by using cavity formation, solute binding, and water correlations. proc natl acad sci usa ( ): – . patel aj, et al. ( ) sitting at the edge: how biomolecules use hydrophobicity to tune their interactions and function. j phys chem b ( ): – . setny p, baron r, michael kekenes-huskey p, mccammon ja, dzubiella j ( ) solvent fluctuations in hydrophobic cavity-ligand binding kinetics. proc natl acad sci usa ( ): – . remsing rc, et al. ( ) pathways to dewetting in hydrophobic confinement. proc natl acad sci usa ( ): – . tiwary p, mondal j, morrone ja, berne bj ( ) role of water and steric constraints in the kinetics of cavity-ligand unbinding. proc natl acad sci usa ( ): – . hummer g, pratt lr, garcía ae ( ) multistate gaussian model for electrostatic solvation free energies. j am chem soc : – . remsing rc, liu s, weeks jd ( ) long-ranged contributions to solvation free energies from theory and short-ranged models. proc natl acad sci usa : – . xi and patel pnas | april , | vol. | no. | d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // 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dog horse , macaca mulatta (rhesus monkey) mouse , , , , , , oryzias latipes (medaka) pan troglodytes (chimpanzee) papio hamadryas (baboon) , papio papio (baboon) pig , river buffalo banded chromosome analysis baboon(dapi) , baboon(gtg) , chimpanzee(dapi) horse(r) , human(dapi) , , , , , human(g) , , , , human(q) , , human(r) , , , , , , marsupial(g) monkey(dapi) mouse(dapi) , , , river buffalo(r) cell cycle chromosome position gene expression replication timing chromosome aberration breakpoint centric fusion , deletions , , , duplication/deletion isochromosome , , pericentric inversion , , rearrangements , , , translocations , , chromosome mapping workshops chromosome chromosome structure chromatin organization chromosome stretching interphase nuclei comparative gene mapping baboon , , bovine horse , mouse , , , , , , , , , primates , , river buffalo ests electron transport complex i genes fad region q . evolution ancestral gene , , chromosome robertsonian fusions fluorescent in situ hybridization (fish) chromosome painting , , , , , , , comparative genomic hybridization , d/c r-banding direct r banding dna breakage detection (dbd) fiber-fish interphase fish , , multicolor fish , , , , , , , , multiplex (m)-fish quantitative image analysis replication timing spectral karyotyping , zoo-fish framework map q . chromosome p chromosome pig chromosome gene mapping baboon fcgr chimpanzee fcgr horse esr , pgr got , kit human adprt, fra h, s rrna bcyrn casp copb , rbp dnch dnfa dnmt dusp e f elanh , pi , pi ftf galgt, kif a glclr ikbkb impdh mafg, mafk mc r ndufa , a , a , b , s ndufa , a , ab , b , b , b , b , b , b , c , s , s , s , s , v nfkbib olr pld pld shox stk tcfl tp wbscr xylb medaka sl abc fax + © s. karger ag, basele-mail karger@karger.ch www.karger. com accessible online at: http://biomednet.com/ karger monkey fcgr mouse casp cenpe, incenp cenpf lims l pkch ptprn sptlc pig ggt genetic damage breakage detection canine mammary carcinoma diagnosis accuracy gene organization cyp dusp ewsr -etv breakpoint region glclr mafg, mafk serpin gene cluster wbscr xylb human disorders alzheimer’s disease , atherosclerosis attention deficit hyperactivity disorder (adhd) b-cell chronic lymphocytic leukemia blepharophimosis syndrome , breast cancer cornelia de lange syndrome deafness , diabetes mellitus ewings sarcoma gliomas glutathionuria gonadal dysgenesis gonadoblastoma male fertility , malignant mesothelioma mental retardation with good memory mesenchymal tumors myotonic dystrophy nephroblastoma neuroblastoma pancreatic cancer peripheral neuroectodermal tumors retinal degeneration turners syndrome williams syndrome williams-beuren syndrome wilm’s tumor hybrids pig-hamster radiation hybrid panel , , , , , , , , somatic cell hybrid panel sperm-hamster oocyte immunohistochemical staining b- and t-cell lineage differentiation gene activation primary spermatocytes karyotype automated analysis , consistent changes in soft tissue tumors marsupial evolution xy mosaicism linkage hearing loss on chr interspecific backcross analysis , , old order amish and bcyrn sex and body color meiosis segregation of ccrs minireview cytogenetic characterization of soft tissue tumors physical mapping p . q . p . q . p . q p and q homology bovine cyp locus chromosome dmpk region pig radiation hybrid panel serpin genes on p wbs deletion region repeats (ca)n polymorphisms , alu repeat in breakpoint region loh detected by strp markers repeat expansion telomere sequence bcyrn ewsr -etv fusion gene ftf glclr mafg, mafk sl stk tcfl wbscr xylb sex chromosomes abnormal baboon x barr body location freshwater fish (medaka) x inactivation x rearrangements xy mosaicism in gonads y role in gonad development spermatogenesis pachytene stage techniques d/c r-banding dna damage analysis at specific regions mitogen optimization multiplex-fish rh mapping on non-exon sequences cytogenet cell genet vol. , nov.dec.edits.indd a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r primary care workforce expansion i n this issue, an analysis of the primary care work- force predicts that the united states will need , more primary care physicians by . population growth will be the single, most important driver, -fold more than expansion of insurance cov- erage—but insurance expansion will occur soonest and most abruptly. the new estimate recognizes that not all primary care physicians practice full time in the offi ce; it is based on the current average across all primary care physicians of offi ce visits with patients per week (rather than visits per week for a physician in full-time offi ce practice). clinically relevant research as a family physician, i am fascinated by the variety of clinically relevant articles in this issue. because almost all of this research was conducted—and much of it generated—in primary care, it can directly help us to understand and improve what we do. • the systematic review and meta-analysis by john- son et al compares more- and less-effective ways to increase infl uenza and pneumococcal immuni- zation rates, which are currently below national targets. • a -country study reveals the prevalence of undiagnosed asthma or chronic obstructive pul- monary disease in unselected patients with acute cough. • a companion article shows a low yield of action- able incidental fi ndings on chest radiographs of patients with acute cough in primary care. • systematically asking women’s pregnancy inten- tions and contraceptive method as a vital sign increases documentation. one goal is to pre- vent prescribing of teratogenic medications (eg, statins, angiotensin-converting enzyme inhibi- tors) to fertile women. including men in this vital sign might further enhance the vital preventive effort to implement effective contraception for everyone who wants or needs it. • karaca describes a method for treating ingrown toenails that prevents recurrences. the annals editors thought that, were we to adopt this pro- cedure, we would probably substitute local anes- thetic without a vasoconstrictor, recognizing that it is common practice in the united states not to use epinephrine in digital blocks. • a placebo-controlled trial among vitamin d-defi - cient people found vitamin d helpful for nonspe- cifi c muscular aches and pains. does this agree with your clinical experience? • a birthing center located in a rural family prac- tice serving amish women offers childbirth care tailored to the community—and “an opportunity to look at the effects of local culture and prac- tices that support vaginal birth and [successful] tolac [trial of birth after cesarean].” these studies range from case series to random- ized controlled trials, with many different research techniques. to further develop research capacity, peterson et al report that they have defi ned research architecture, processes, and requirements of software to support community practice-based translational research: eg, recruitment of participants, collection of aggregated anonymous data, and retrieval of identifi - able data from previously consented adults across hun- dreds of practices. prevention ‘numeracy’ in this issue you will fi nd a research study, an essay, and a guest editorial on screening. in their essay hoffman and colleagues caution guideline makers to “avoid distracting primary care clinicians from providing services with proven benefi t and value for editorial in this issue: through the lens of a clinician louise s. acheson, md, ms, associate editor department of family medicine and community health, case western reserve university, cleveland, ohio ann fam med ; : - . doi: . /afm. a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r e d i t o r i a l s patients.” indeed, many preventive interventions have proven benefi t. yet hudson et al present the quandary that many patients appear willing to undergo preven- tive care on the basis of “overly optimistic expectations of the benefi ts of preventive interventions and screen- ing.” are they innumerate or overly optimistic? what about policy makers? what about clinicians? we hope you will share your thoughts about the articles in this issue. join the discussion at http://www. annfammed.org. references . petterson sm, liaw wr, phillips rl jr, rabin dl, meyers ds, baze- more aw. projecting us primary care physician workforce needs: - . ann fam med. ; ( ): - . . lau d, hu j, majumdar sr, storie da, rees se, johnson ja. inter- ventions to improve infl uenza and pneumococcal vaccination rates among community-dwelling adults: a systematic review and meta- analysis. ann fam med. ; ( ): - . . van vugt s, broekhuizen l, zuithoff n, et al. airway obstruction and bronchodilator responsiveness in adults with acute cough. ann fam med. ; ( ): - . . van vugt s, broekhuizen l, zuithoff n, et al. incidental chest radio- graphic fi ndings in adult patients with acute cough. ann fam med. ; ( ): - . . schwarz eb, parisi sm, williams sl, shevchik gj, hess r. pro- moting safe prescribing in primary care with a contraceptive vital sign: a cluster-randomized controlled trial. ann fam med. ; ( ): - . . karaca n, dereli t. treatment of ingrown toenail with proximolat- eral matrix partial excision and matrix phenolization. ann fam med. ; ( ): - . . schreuder f, bernsen rmd, van der wouden jc. vitamin d supple- mentation for nonspecifi c musculoskeletal pain in non-western immigrants: a randomized controlled trial. ann fam med. ; ( ): - . . deline j, varnes-epstein l, dresang lt, gideonsen m, lynch l, frey jj iii. low primary cesarean rate and high vbac rate with good outcomes in an amish birthing center. ann fam med. ; ( ): - . . peterson ka, delaney bc, arvanitis t, et al. a model for the elec- tronic support of practice-based research networks. ann fam med. ; ( ): - . . hudson b, zarifeh a, young l, wells je. patients’ expectations of screening and preventive treatments. ann fam med. ; ( ): - . . hoffman rm, barry mj, roberts rg, sox hc. reconciling primary care and specialist perspectives on prostate cancer screening. ann fam med. ; ( ): - . . woolf sh. the price of false beliefs: unrealistic expectations as a contributor to the health care crisis. ann fam med. ; ( ): - . editorial the price of false beliefs: unrealistic expectations as a contributor to the health care crisis steven h. woolf, md, mph department of family medicine, school of medicine, virginia commonwealth university, richmond, virginia ann fam med ; : - . doi: . /afm. . t he alarming rise in health care costs haunts our society. the united states now spends $ . trillion per year on health care, and the spiral- ing costs are placing unsustainable burdens on employ- ers and workers, medicare and medicaid, state and local governments, and american families. a growing proportion of americans are now foregoing health care to pay for other household needs or are facing bank- ruptcy. a variety of strategies have been proposed to slow medical cost infl ation, such as realigning fi nancial incentives to discourage costly procedures, account- confl icts of interest: none reported. corresponding address steven h. woolf, md, mph department of family medicine school of medicine virginia commonwealth university east broad st po box richmond, va - swoolf@vcu.edu a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r e d i t o r i a l s able care organizations, the patient-centered medical home, and malpractice reforms. evidence that any of these ideas will bend the cost curve remains limited. a more basic but possibly neglected strategy for reducing demand for health services is to confront unrealistic beliefs about their benefi ts. health care expenditures ultimately begin with a decision to use the service, a decision that may rest on false expecta- tions—among patients, clinicians, or both. removing the need for the service by correcting such misper- ceptions is a potentially more effective way to curb costs than many current reforms can achieve. financial incentives are important, but they are weak when pit- ted against core beliefs. if patients and clinicians widely hold that a procedure is life-saving and harmless, any reform is unlikely to curb demand until those miscon- ceptions are addressed. studies suggest that patients, clinicians, and soci- ety often hold unrealistic expectations about the effectiveness of tests and treatments. two articles in this issue add to that literature. in new zealand, hudson et al surveyed primary care patients and found that many overestimated the benefi ts of cancer screening and chemopreventive medications. the min- imum benefi t from screening that respondents deemed acceptable was less than their known benefi t. the survey had a modest sample size and low response rate ( %), and its fi ndings might not be fully applicable to other countries, but us studies have reported a similar problem. for example, a variety of studies document americans’ appetite for procedures of dubious effec- tiveness and their overestimation of benefi ts. , many americans underestimate the probability of harms and are quite willing to receive false-positive results and unnecessary biopsies for the chance to detect can- cer. , public complacency about the safety of health care is only occasionally shaken, as when a conspicu- ous tragedy or disclosures of industry wrongdoing draw attention to specifi c dangers. physicians are not immune to false beliefs about clinical effi cacy or complication rates. correcting such misperceptions has always been part of the impe- tus for the evidence-based medicine movement and its promulgation of systematic evidence reviews, prac- tice guidelines, and other tools that present the facts on benefi ts, safety, and scientifi c uncertainties. even these tools, however, can refl ect the misconceptions of those who produce them. the specialists who serve on expert panels derive much of their clinical case knowl- edge from the patients with advanced disease who fi ll their clinics. having seen the worst of the worst, they are less sympathetic to expressions of concern about the potential harms of interventions or imperfections in effi cacy studies. whereas epidemiologists consider the population denominator to put the numerator in perspective, the world of specialists is confi ned to the numerator, giving them a skewed basis for judging the population prevalence of diseases or benefi t-risk ratios. were this not enough, the preeminent scientists who often serve on guideline panels bring additional biases, such as being the authors of key studies under review or having fi nancial ties to industry. guideline panels composed of generalists tend to produce recommendations that are more conservative than those dominated by experts, , in part because they are chosen for their skills in critical appraisal and because they have little to gain from the recommenda- tions. in an essay in this issue, hoffman et al cite this phenomenon in explaining why guideline panels domi- nated by cancer specialists advocate prostate cancer screening beginning at age years, despite evidence that the lifetime benefi t of an earlier starting age is averted death per , men. even guideline bodies harbor unrealistic expectations of effi cacy. a seemingly simple solution is to arm patients and clinicians with more realistic data, the very motive behind the production of evidence-based decision sup- port tools for clinicians and decision aids for patients. large initiatives in comparative effectiveness research are now underway to assemble such data for patients, and research in decision science and risk communication is seeking the best formats and framing for explaining likely outcomes and scientifi c uncertainty. information technology and innovative infographics are helping to address challenges with health and numeric literacy. these important efforts can help only to the extent that people make choices through the cognitive act of weighing benefi ts, risks, and scientifi c uncer- tainty. in real life, decisions are shaped by affective infl uences: beliefs and fears; vulnerability; faith and trust; long-standing routines; personal experiences; messages conveyed by advertising and media; and the advice, testimonials, and transmitted knowledge imparted by trusted sources. patients’ explanatory models of illness may clash with scientifi c data but represent a form of “evidence” that must be respected. fact sheets and bar charts exert marginal infl uence if they ignore this larger context. if people are widely convinced that a screening test or drug is benefi cial, confronting these beliefs can, if anything, engender suspicions about one’s veracity and motives. whether the messenger is one’s physician, a health plan, or a government task force, attempts to set more realistic expectations about benefi ts, risks, and scientifi c validity are often taken as insensitivity to suffering, discrimination, or a pretext for cutting costs, rationing health care, or threatening personal autonomy. in today’s media environment, the political a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r e d i t o r i a l s narrative these ideas feed allows for viral dissemination of distorted characterizations by websites, talk shows, blogs, and social networks. ours is an era of “death panel” debates in which facts are swept aside by politi- cal agendas and talking points. it is an increasingly dif- fi cult environment for the american public to receive, let alone absorb, undistorted scientifi c information from reputable bodies. unrealistic expectations therefore persist, surviv- ing not only on misinformation but also by serving other purposes. for example, false beliefs meet the psychological needs of patients for hope and safety, as well as for action, agency, and a sense of control. they enable clinicians to feel they are making a differ- ence; even physicians who know better order unneces- sary tests to please their patients. false expectations fuel market demand for products, industries, and health delivery systems and can be fomented by mis- leading advertising. confronting these expectations can not only dash hopes but potentially threaten prof- its, shareholders, clinical practices, industries, legisla- tion, and political careers. but good news on the horizon hints at a shift in societal attitudes. increasingly, overutilization of medical services, overdiagnosis, and profl igate use of screening tests are being covered by major newspa- pers and magazines - and are the subject of books in the popular press. , the american cancer society has adopted more rigorous methods for developing screening guidelines, and in broadcast appearances its chief medical offi cer has openly discussed the limita- tions of screening. , although the us preventive services task force recommendations about the start- ing age for mammography sparked infamous outrage in , the same group’s recommendations against prostate-specifi c antigen screening met with softer criticism when proposed in , and its recom- mendation to delay the starting age and reduce the frequency of cervical cancer screening—fi rst issued by the american college of obstetricians and gyne- cologists —raised no tempest. equally encouraging is the choosing wisely cam- paign, organized by the american board of internal medicine foundation. in april , medical specialty societies—from primary care to oncol- ogy and nuclear cardiology—each released a list of tests or procedures that their specialists commonly use and “whose necessity should be questioned and discussed.” consumer reports and other organi- zations are helping these medical groups relay these messages to large audiences in consumer-friendly language. for example, the material on antibiotics for sinusitis, cobranded by consumer reports and the american academy of family physicians, uses plain-spoken headings: “the drugs usually don’t help,” “they can pose risks,” and “they’re usually a waste of money.” organized medicine appears to be embrac- ing this movement: the foundation’s website now lists specialty societies that have joined the initiative and will be releasing their own lists of questionable proce- dures in late or . time will tell whether such efforts succeed and whether the medical profession will emerge as the change agent that brings more realistic expectations to patient care. regardless of whether physicians or other stakeholders ultimately take the lead, the power of this strategy should not be overlooked by government, businesses, or others who urgently seek solutions to the health care crisis. the best way to reduce wasteful spending is to convince the purchaser that the product is not worth buying. it is a straightforward economic argument, but it can also save lives. to read or post commentaries in response to this article, see it online at http://www.annfammed.org/content/ / / . key words: decision making; diagnosis; screening submitted september , ; accepted september , . references . martin ab, lassman d, washington b, catlin a; national health expenditure accounts team. growth in us health spending remained slow in ; health share of gross domestic product was unchanged from . health aff (millwood). ; ( ): - . . cohen ra, gindi rm, kirzinger wk. burden of medical care cost: early release of estimates from the national health interview sur- vey, january–june . national center for health statistics. http:// www.cdc.gov/nchs/nhis/releases.htm. accessed jun , . . hudson b, zarifeh a, young l, wells je. patients’ expecta- tions of screening and preventive treatments. ann fam med. ; ( ): - . . domenighetti g, d’avanzo b, egger m, et al. women’s perception of the benefi ts of mammography screening: population-based sur- vey in four countries. int j epidemiol. ; ( ): - . . herndon mb, schwartz lm, woloshin s, et al. older patients per- ceptions of “unnecessary” tests and referrals: a national survey of medicare benefi ciaries. j gen intern med. ; ( ): - . epub jul . . schwartz lm, woloshin s, sox hc, fischhoff b, welch hg. us wom- en’s attitudes to false positive mammography results and detection of ductal carcinoma in situ: cross sectional survey. bmj. ; ( ): - . . schwartz lm, woloshin s, fowler fj jr, welch hg. enthusiasm for cancer screening in the united states. jama. ; ( ): - . . wegwarth o, schwartz lm, woloshin s, gaissmaier w, gigerenzer g. do physicians understand cancer screening statistics? a national survey of primary care physicians in the united states. ann intern med. ; ( ): - . . eccles mp, grimshaw jm, shekelle p, schünemann hj, woolf s. developing clinical practice guidelines: target audiences, identify- ing topics for guidelines, guideline group composition and func- tioning and confl icts of interest. implement sci. jul ; ( ): . [epub ahead of print]. a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r e d i t o r i a l s . institute of medicine. clinical practice guidelines we can trust. wash- ington, dc: national academies press; . . kahan jp, park re, leape ll, et al. variations by specialty in physi- cian ratings of the appropriateness and necessity of indications for procedures. med care. ; ( ): - . . hutchings a, raine r. a systematic review of factors affecting the judgments produced by formal consensus development methods in health care. j health serv res policy. ; ( ): - . . hoffman rm, barry mj, roberts rg, sox hc. reconciling primary care and specialist perspectives on prostate cancer screening. ann fam med. ; ( ): - . . selby jv, beal ac, frank l. the patient-centered outcomes research institute (pcori) national priorities for research and initial research agenda. jama. ; ( ): - . . woloshin s, schwartz lm. communicating data about the ben- efi ts and harms of treatment: a randomized trial. ann intern med. ; ( ): - . . campbell eg, regan s, gruen rl, et al. professionalism in medi- cine: results of a national survey of physicians. ann intern med. ; ( ): - . . jain m. “are we relying too much on cancer screening?” washington post. november , :e . . rosenthal e. “let’s (not) get physicals.” new york times sunday review. june , . . “false promises on ovarian cancer” (editorial). new york times, sep- tember , . . the business of healing hearts: cardiac care is a money-making machine that too often favors profi t over science. consum rep. ; ( ): - . . brownlee s. overtreated: why too much medicine is making us sicker and poorer. new york, ny: bloomsbury; . . brawley ow, goldberg p. how we do harm: a doctor breaks ranks about being sick in america. new york, nt: st. martin’s press; . . brawley o, byers t, chen a, et al. new american cancer society process for creating trustworthy cancer screening guidelines. jama. ; ( ): - . . brawley ow. prostate cancer screening may do more harm than good. cnn. http://www.cnn.com/ / / /opinion/brawley- prostate-cancer-screening/index.html. accessed dec , . . acog committee on practice bulletins—gynecology. acog prac- tice bulletin no. : cervical cytology screening. obstet gynecol. ; ( ): - . . cassel ck, guest ja. choosing wisely: helping physicians and patients make smart decisions about their care. jama. ; ( ): - . . brody h. medicine’s ethical responsibility for health care reform— the top fi ve list. n engl j med. ; ( ): - . . when to say ‘whoa!’ to doctors: a guide to common tests and treatments you probably don’t need. consumer reports health, . http://consumerhealthchoices.org/wp-content/uploads/ / / choosingwiselywhoapkg.pdf. accessed sep , . . treating sinusitis: don’t rush to antibiotics. consumer reports health, . http://consumerhealthchoices.org/wp-content/ uploads/ / /choosingwiselysinusitisaafp.pdf. accessed sep , . [pdf] evidence hdac genetic variant associated with ischemic stroke increases risk via promoting carotid atherosclerosis | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /strokeaha. . corpus id: evidence hdac genetic variant associated with ischemic stroke increases risk via promoting carotid atherosclerosis @article{markus evidencehg, title={evidence hdac genetic variant associated with ischemic stroke increases risk via promoting carotid atherosclerosis}, author={h. markus and kari-matti m{\"a}kel{\"a} and s. bevan and e. raitoharju and n. oksala and j. bis and c. o'donnell and a. hainsworth and t. lehtim{\"a}ki}, journal={stroke}, year={ }, volume={ }, pages={ – } } 	h. markus, kari-matti mäkelä, + authors t. lehtimäki 	published 	medicine 	stroke background and purpose— a novel association between a single nucleotide polymorphism on chromosome p . and large-vessel ischemic stroke was recently identified. the most likely underlying gene is histone deacetylase (hdac ). the mechanism by which hdac increases stroke risk is not clear; both vascular and neuronal mechanisms have been proposed. methods— we determined whether the lead single nucleotide polymorphisms were associated with asymptomatic carotid plaque (n= ) and carotid… expand view on wolters kluwer ahajournals.org save to library create alert cite launch research feed share this paper citationshighly influential citations background citations view all figures, tables, and topics from this paper 	 table 	 figure 	 table 	 figure 	 table 	 table view all figures & tables 	atherosclerosis 	cerebral infarction 	hdac gene 	senile plaques 	cerebrovascular accident 	myocytes, smooth muscle 	promotion (action) 	smooth muscle (tissue) 	muscle cells 	chest 	plaque, atherosclerotic citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency deficiency of the stroke relevant hdac gene attenuates atherosclerosis in accord with allele-specific effects at p . 	sepiede azghandi, c. prell, + authors m. dichgans 	medicine 	stroke 	 	 	pdf 	view excerpts, cites background save alert research feed polymorphism of hdac gene is associated with increased risk of acute coronary syndrome in chinese han population 	zhenhua han, x. dong, chaoying zhang, y. wu, z. yuan, x. wang 	medicine, biology 	biomed research international 	 	 	pdf save alert research feed hdac variant rs modifies susceptibility to coronary artery disease and the severity of coronary atherosclerosis in a chinese han population 	x. wang, ya-di han, + authors fang zheng 	medicine 	plos one 	 	 	pdf save alert research feed association between rs hdac gene polymorphism and advanced carotid atherosclerosis in the slovenian cohort 	emin grbić, nataša gorkič, + authors z. fras 	medicine 	lipids in health and disease 	 	 	 	view excerpts, cites background save alert research feed association between the gene polymorphisms of hdac and the risk of atherosclerosis and ischemic stroke 	guo qingxu, zhang yan, xu jiannan, liu yun-long 	medicine 	pathology & oncology research 	 	 	view excerpt, cites background save alert research feed hdac is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype 	r. malhotra, a. mauer, + authors c. o’donnell 	biology, medicine 	nature genetics 	 	 	 save alert research feed hdac polymorphisms predict susceptibility, severity, and short-term outcome of large artery atherosclerotic stroke in chinese population 	m. wang, mengmeng gu, + authors x. liu 	medicine 	journal of molecular neuroscience 	 	 	view excerpt save alert research feed hdac polymorphism alters blood gene expression in patients with large vessel atherosclerotic stroke 	natasha shroff, b. ander, + authors g. jickling 	medicine 	translational stroke research 	 	 	 save alert research feed the association between hdac gene polymorphisms and stroke risk in the chinese population: a meta-analysis 	x. zhou, tangming guan, + authors qiuhong ji 	medicine 	scientific reports 	 	 	pdf save alert research feed the atherosclerosis risk variant rs mediates allele-specific transcriptional regulation of hdac via e f and rb . 	m. prestel, caroline prell-schicker, + authors m. dichgans 	medicine 	stroke 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency genome-wide association study identifies a variant in hdac associated with large vessel ischemic stroke 	c. bellenguez, s. bevan, + authors h. markus 	biology, medicine 	nature genetics 	 	 	pdf 	view excerpts, references background and methods save alert research feed genes involved in systemic and arterial bed dependent atherosclerosis - tampere vascular study 	m. levula, n. oksala, + authors t. lehtimäki 	medicine 	plos one 	 	 	pdf 	view excerpts, references methods save alert research feed meta-analysis of genome-wide association studies from the charge consortium identifies common variants associated with carotid intima media thickness and plaque 	j. bis, m. kavousi, + authors c. o’donnell 	medicine, biology 	nature genetics 	 	 	pdf 	view excerpts, references methods save alert research feed genetic risk factors for ischaemic stroke and its subtypes (the metastroke collaboration): a meta-analysis of genome-wide association studies 	m. traylor, m. farrall, + authors h. markus 	medicine 	the lancet neurology 	 	 	pdf save alert research feed prediction of clinical cardiovascular events with carotid intima-media thickness: a systematic review and meta-analysis 	m. lorenz, h. markus, m. bots, m. rosvall, m. sitzer 	medicine 	circulation 	 	 , 	pdf 	view excerpt, references background save alert research feed large-scale association analysis identifies new risk loci for coronary artery disease 	p. deloukas, s. kanoni, + authors n. samani 	biology, medicine 	nature genetics 	 	 , 	pdf save alert research feed internal carotid artery angle of origin: a novel risk factor for early carotid atherosclerosis 	m. sitzer, damir puac, + authors h. steinmetz 	medicine 	stroke 	 	 	 	view excerpt, references results save alert research feed ultrasonographic measurements of subclinical carotid atherosclerosis in prediction of ischemic stroke 	e. mathiesen, s. johnsen 	medicine 	acta neurologica scandinavica. supplementum 	 	 	view excerpt, references background save alert research feed targeting histone deacetylases as a multifaceted approach to treat the diverse outcomes of stroke. 	b. langley, c. brochier, mark a. rivieccio 	medicine 	stroke 	 	 	pdf 	view excerpts, references background save alert research feed targeting inflammation in heart failure with histone deacetylase inhibitors 	t. mckinsey 	medicine 	molecular medicine 	 	 	highly influential 	view excerpts, references background save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ doi: . /j.bbabio. . . p/ import and assembly of mitochondrial proteins nikolaus pfanner institute for biochemistry and molecular biology, university of freiburg, germany e-mail: nikolaus.pfanner@biochemie.uni-freiburg.de mitochondria contain about different proteins. % of the proteins are synthesized as precursors on cytosolic ribosomes. the precursors are imported via the translocase of the outer mitochon- drial membrane (tom complex) and are subsequently sorted into the four mitochondrial subcompartments, outer membrane, inter- membrane space, inner membrane and matrix. (i) cleavable preproteins are transported from the tom complex to the presequence translocase of the inner membrane (tim complex). the presequence translocase-associated motor (pam) drives trans- location into the matrix. (ii) hydrophobic inner membrane proteins are transferred through the intermembrane space by a chaperone complex (small tim proteins) and inserted into the inner membrane by the tim complex. (iii) the mitochondrial import and assembly machinery (mia) directs small proteins into the intermembrane space and promotes the formation of disulfide bonds. (iv) beta- barrel proteins are transported from the tom complex to the sorting and assembly machinery of the outer membrane (sam complex). doi: . /j.bbabio. . . p/ new functions for novel mitochondrial transporters ferdinando palmieri university of bari, italy e-mail: fpalm@farmbiol.uniba.it a strikingly large number of mitochondrial dna (mtdna) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. these mitochondrial dis- orders were the first to be investigated after the small mtdna had been sequenced in the 's. only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, hhh syndrome, aspartate/glutamate isoform deficiency, amish microce- phaly and neonatal myoclonic epilepsy; these disorders are characterised by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabo- lism. defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phos- phate and adp, are responsible for diseases characterised by defective energy production. herein, all the mitochondrial carrier- associated diseases known to date are reviewed for the first time. particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders. doi: . /j.bbabio. . . p/ the water oxidizing enzyme a. william rutherford ibitec-s, ura cnrs, cea saclay, gif-sur-yvette, france e-mail: alfred.rutherford@cea.fr photosystem ii, the water oxidising enzyme of photosynthesis, put the energy (or at least a major fraction of it) into the biosphere and the oxygen into the atmosphere. it is certainly one the most influential and important enzymes on the planet. the aim of our research is to understand how this enzyme works as ) a solar energy converter and ) the only known thermodynamically efficient catalyst for oxidizing water. the information obtained is used in the design of artificial catalysts and photocatalysts. a chemical catalyst that has the thermodynamic efficiency of the enzyme could greatly improve the efficiency of ) water electrolysis and photolysis for fuel (e.g. h ) production and ) the reverse reaction, oxygen reduction, in fuel cells. there is therefore a great interest in understanding the mechanism of this enzyme and in reproducing aspects of its function in artificial systems. i will describe our current knowledge of photosystem ii, including some recent experimental studies, as well as recent efforts in our joint saclay/orsay program aimed at producing bio-inspired water oxidizing catalysts. doi: . /j.bbabio. . . p/ the structure of purple bacterial antenna complexes: from single molecules to native membranes richard j. cogdella, alastair t. gardinera, mads gabrielsena, aleks w. roszaka, june southalla, tatas brotosudarmoa, neil w. isaccsa, hideki hashimotob, juergen baierc, silke oellerichc, martin richterc, juergen koehlerc, francesco franciad, giovanni venturolid, dieter oesterhelte adivision of biochemistry and molecular biology, ibls and department of chemistry, university of glasgow, glasgow g qq, uk bcrest-jst and department of physics, graduate school of science, osaka city university, - - sugimoto, sumiyoshi-ku, osaka - , japan cexperimental physics iv, university of bayreuth, d- bayreuth, germany ddepartment of biology, university of bologna, bologna, italy edepartment of membrane biochemistry, max-planck institute for biochemistry, martinsried, germany e-mail: r.cogdell@bio.gla.ac.uk the photosynthetic unit of purple photosynthetic bacteria typically contains two types of light-harvesting complexes, called lh and lh . these antenna complexes are constructed on a modular principle. they are circular or elliptical oligomers of dimers of two low-molecular weight, hydrophobic apoproteins, called a and b, that bind bacteriochlorophylls and carotenoids non-covalently. the lh complex surrounds the reaction centre and, depending on the species, is either a monomer or a dimer. the lh complexes are arranged around the lh -rc complexes. this plenary lecture will present the current status of structural studies on these pigment- protein complexes, based upon a combination of x-ray crystal- lography and single molecule spectroscopy. then an overall view of how they are arranged in their native photosynthetic membranes will be presented. doi: . /j.bbabio. . . p/ catalysis of substrate conversion and electron transfer by mitochondrial complex i judy hirst medical research council dunn human nutrition unit, cambridge, cb xy, uk e-mail: jh@mrc-dunn.cam.ac.uk s abstracts / biochimica et biophysica acta ( ) s –s http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . mailto:nikolaus.pfanner@biochemie.uni-freiburg.de mailto:fpalm@farmbiol.uniba.it mailto:alfred.rutherford@cea.fr mailto:r.cogdell@bio.gla.ac.uk mailto:jh@mrc-dunn.cam.ac.uk complex i (nadh:ubiquinone oxidoreductase) is the first enzyme of the respiratory electron transport chain in mitochondria. it catalyses the reduction of ubiquinone by nadh, coupled to the translocation of four protons across the inner-mitochondrial mem- brane, and it is a significant source of reactive oxygen species, linked to neuromuscular diseases and ageing. in bovine mitochondria complex i comprises different subunits, a flavin mononucleotide at the active site where nadh is oxidised, and eight iron sulphur clusters. this talk will focus on the mechanism of the redox reaction in the enzyme (the mechanism of proton translocation remains unknown). the structure of the hydrophilic domain of complex i from thermus thermophilus has provided a framework for under- standing the redox reaction, and it makes the pathway that the electrons take through the enzyme easy to visualise. however, it does not identify rate limiting steps, or describe the reaction intermediates formed and the free energy changes as the reaction progresses. a mechanistic understanding at this level is crucial for understanding how complex i conserves the potential difference between nadh and ubiquinone as a proton motive force so effectively, for defining how electron and proton transfer are coupled, and for understanding the formation of reactive oxygen species. this talk will describe current understanding and new information about the mechanism of the redox reaction in complex i. doi: . /j.bbabio. . . p/ structural and functional insight into mitochondrial complex i ulrich brandt johann wolfgang goethe-universität, fachbereich medizin, zentrum der bi o lo g i sche n chemie, molekulare bioenergetik, frank fu r t a m m ain, germany e-mail: brandt@zbc.kgu.de the molecular mechanism how complex i (nadh:ubiquinone oxidoreductase) links electron transfer to proton translocation is still elusive. we have established the strictly aerobic yeast yarrowia lipolytica as a powerful genetic system to study structure, function and biogenesis of this mitochondrial complex i. the ubiquinone reducing catalytic core of complex i resides at the interface between the -kda and the psst subunit of the peripheral arm where iron–sulfur cluster n serves as the immediate reductant of ubiquinone. in an extensive mutagenesis study based on the recently published partial structure of bacterial complex i, we have identified the entry pathway for ubiquinone and domains interacting with hydrophobic complex i. single particle analysis of antibody decorated complex i indicated that the -kda subunit is located surprisingly far away from the membrane arm. this unexpected result was confirmed by further structural studies with a subcomplex of complex i lacking the -kda and -kda subunits. electron microscopic d reconstructions of this subcom- plex allowed positioning the partial structure of the bacterial complex within y. lipolytica complex i. therefore, we propose that ubiquinone reaches its site of reduction via a hydrophobic ramp or channel within complex i. we propose a two-state mechanism of energy conservation for complex i that is based on long range conformational changes of the enzyme driven by stabilization changes of ubiquinone intermediates. doi: . /j.bbabio. . . p/ the genetics and pathophysiology of mitochondrial disease douglas c. wallace ce nter fo r molecular and mitochondria l medicine and g enetics, university of california, irvine, irvine, ca - , usa e-mail: dwallace@uci.edu mitochondrial diseases are genetically and phenotypically hetero- geneous. the human mitochondrial dna (mtdna) trnaleu(uur) g a mutation causes diabetes at low heteroplasmy but melas at high heteroplasmy, the two phenotypes associated with differences in nuclear dna (ndna) and mtdna gene mitochip gene expression profiles. inactivation of the mouse heart–muscle–brain isoform of the adenine nucleotide translocator (ant ) results in mitochondrial myopathy and cardiomyopathy associated with defects in mitochon- drial adp–atp exchange and increased mitochondrial reactive oxygen species (ros) production. mitochip expression analysis of ant - deficient skeletal muscle revealed the coordinate up-regulation of mtdna and ndna oxidative phosphorylation, antioxidant and anti- apoptotic genes and the down-regulation of glycolytic and pro- apoptotic genes, all in association with increased protein levels for pgc- α, nrf , myogenin, and tfam but reduced levels of c-myc. neurons from ant -deficient mice were more resistant to glutamate and etoposide induced apoptosis and ant -deficient mice were less sensitive to kainic acid excitotoxicity. mice harboring a heteroplasmic mtdna nd frameshift mutation (nt insc) showed directional loss of the mutant mtdnas from the female germline in subsequent liters and generations implying intra-ovarian selection against severely deleterious mtdna mutations. by contrast, a coi nt t c missence mutation (v a) was retained and resulted in the development of mitochondrial myopathy and cardiomyopathy and neuronal cell loss. therefore, mutations in different mitochondrial genes can produce similar tissue phenotypes, perhaps reflecting similar gene expression profiles. doi: . /j.bbabio. . . p/ quantifi cation of the electrochemical proton gradient and activation of atp synthase in leaves pierre joliot institut de biologie physico-chimique, paris, france e-mail: pierre.joliot@ibpc.fr we have developed a new method to quantify the transmem- brane electrochemical proton gradient present in chloroplasts of dark-adapted leaves. when a leaf is illuminated by a short pulse of in ten s e l ig h t, we ob se rved that the light-induced membrane potential change reaches a maximum value (~ mv) determined by ion leaks that occur above a threshold level of the electro- chemical proton gradient. after the light-pulse, the decay of the membrane potential displays a marked slowdown, which reflects the switch from an activated to an inactivated state of the atp synthase that occurs at ~ mv. we have estimated the Δ ~ µh + level that preexists in the dark ( to mv), which collapses upon addition of inhibitors of the respiratory chain. thus, it shows that it results from the hydrolysis of atp of mitochondrial origin. illumination of the leaf induces a Δ ~ µh + increase (up to ~ mv) that reflects the light-induced increase in atp concentration. following the illumination, Δ ~ µh + relaxes to its dark-adapted value according to a multiphasic kinetics completed in more than h. in mature leaf, the deactivation of the benson–calvin cycle follows similar kinetics as Δ ~ µh + decay, showing that its state of activation is mainly controlled by atp concentration. doi: . /j.bbabio. . . s abstract s / biochimica et biophysica acta ( ) s –s http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . mailto:dwallace@uci.edu mailto:brandt@zbc.kgu.de mailto:pierre.joliot@ibpc.fr diabetes care, volume , number , january o b s e rvat i o n s diabetes scre e n i n g practices among i n d i v i d u a l s a g e d ye a r s a nd o l d e r i n , the american diabetes associa- tion (ada) adopted new re c o m m e n d a- tions for screening the general popula- tion aged years for diabetes every years with an emphasis on those at high risk for undiagnosed diabetes ( ). few studies have examined the extent to which this screening has been adopted. this re p o rt describes the results of a telephone s u rvey of montana residents aged years to assess the diabetes screening prac- tices in this population. f rom october to december , the montana department of public health and human services conducted a random household telephone survey of montana residents aged years living in counties. respondents indicated whether they ever had been told by a physician that they had diabetes, the number of visits they made to a health care provider during the past year, their family history of dia- betes, whether they had ever been told they had high cholesterol and/or high blood pre s s u re, and their height and weight. respondents were asked the fol- lowing question to identify whether they had ever been screened for diabetes: “glu- cose or sugar is a substance found in your blood. have you ever had your blood glu- cose or sugar checked to see if you have diabetes?” when respondents re s p o n d e d “yes” to this question, they were asked to identify when screening was completed (“when was the last time your blood glu- cose or sugar level was measured by a health care professional?”). the re s p o n s e categories for this question included within the past year, within the past years, years ago, do not know/not sure, and refused to answer. pearson tests were used to assess associations between dia- betes screening and risk factors for dia- betes. logistical re g ression analyses were conducted to identify independent vari- ables associated with screening for diabetes during the past year. odds ratios ( % cis) were calculated. of the , respondents, ( . %) re p o rted that they had diagnosed diabetes. the remaining , respondents re p o rt e d that they did not have diagnosed diabetes and are included in the following analyses. of the respondents, % re p o rted a family h i s t o ry of diabetes, % re p o rted a bmi kg/m , % re p o rted having hyper- tension, and % re p o rted having high c h o l e s t e rol. excluding age, % of re s p o n- dents had one risk factor for diabetes, and % had two or more risk factors. of the , respondents without diagnosed dia- betes, % re p o rted that they had been s c reened for diabetes during the past year, % re p o rted screening from to years ago, and % re p o rted screening years ago or having never been scre e n e d . respondents who re p o rted being s c reened for diabetes during the past year w e re more likely to be age years and to have a family history of diabetes, two or m o re visits to a health care provider dur- ing the past year, hypertension, and high c h o l e s t e rol levels (table ). we found no association between recent screening and sex ( % men vs. % women), ameri- can indian ancestry ( % yes vs. % no), or bmi ( % kg/m vs. % kg/m ). respondents with three or m o re risk factors (e.g., aged years, american indian ancestry, family history of diabetes, hypertension, high choles- t e rol, or bmi kg/m ) were more likely to be screened for diabetes compared with respondents with only one risk factor ( vs. %, respectively). however, % of individuals with two risk factors for dia- betes and % of individuals with more than three risk factors had not been s c reened during the past years. based on logistical re g ression analysis, t h ree factors were associated with scre e n i n g for diabetes during the past year: two or m o re visits to a health care provider during the past year ( . [ % ci . – . ]), high cholesterol level ( . [ . – . ]), and family history of diabetes ( . [ . – . ]). respondents aged – years were less likely to re p o rt re c e n t s c reening than those aged years ( . [ . – . ] ) . a limitation of this assessment is that these data are self-re p o rted. pre v i o u s studies, however, have found that self- re p o rts of conditions such as diabetes and h y p e rtension are reliable ( , ). in addi- tion, the survey was conducted by tele- phone and does not re flect the experience of individuals in montana homes without t e l e p h o n e s . the findings suggest that diabetes s c reening is being adopted by physicians for individuals aged years at risk for diabetes and that the ada re c o m m e n d a- tions are being implemented in the general c o m m u n i t y. however, these data also indi- cate a need to develop strategies to encour- l e t t e r s table —characteristics of respondents aged years reporting screening for diabetes in montana in s c reening for diabetes past year – years years or never n age (years) – ( ) ( ) ( ) – ( ) ( ) ( ) ( )* ( ) ( ) family history of diabetes ye s ( )* ( ) ( ) n o ( ) ( ) ( ) visits to a health care provider during the past year ( )* ( ) ( ) ( ) ( ) ( ) h y p e rt e n s i o n ye s ( )* ( ) ( ) n o ( ) ( ) ( ) high cholestero l ye s ( )* ( ) ( ) n o ( ) ( ) ( ) data are n (%). *p . . diabetes care, volume , number , january letters age screening among all individuals at high risk for diabetes. todd s. harwell, mph jane g. smilie, ba janet m. mcdowall, rn, bsn steven d. helgerson, md, mph dorothy gohdes, md f rom the montana diabetes project, montana d e p a rtment of public health and human serv i c e s , helena, montana. a d d ress correspondence to todd s. harw e l l , mph, montana department of public health and human services, cogswell building, c- , p. o . box , helena, mt - . e-mail: t h a rw e l l @ s t a t e . m t . u s . a c k n o w l e d g m e n t s — this project was sup- p o rted through a cooperative agreement (u- / ccu- - ) with the centers for disease c o n t rol and prevention, division of diabetes translation, atlanta, georg i a . we thank linda priest and the staff mem- bers at northwest resource consultants for their work on the telephone surv e y. the contents of this letter are solely the responsibility of the authors and do not neces- sarily re p resent the official views of the centers for disease control and pre v e n t i o n . r e f e re n c e s . american diabetes association: screening for type diabetes. diabetes care (suppl. ): s –s , . kehoe r, wu sy, leske mc, chylack lt jr: comparing self-re p o rted and physician- re p o rted medical history. am j epidemiol : – , . jackson c, jatulis de, fortmann sp: the behavioral risk factor survey and the stan- f o rd five-city project survey: a comparison of cardiovascular risk behavior estimates. am j public health : – , h b a c is not recommended as a s c reening test for diabetes in cystic fibro s i s i n the june issue of diabetes care, h u n k e rt et al. ( ) recommend the use of h b a c for early detection of cystic fib ro- s i s – related diabetes (cfrd). their re c o m- mendation is based on the finding that mean hba c is slightly higher in cystic fib rosis (cf) patients requiring insulin t h e r a p y, compared with cf patients with i m p a i red or normal glucose tolerance. h o w e v e r, no data on the validity of this a p p roach for the diagnosis of asympto- matic diabetes in patients with cystic fib ro- sis are pre s e n t e d . our group has a long-standing intere s t in the early diagnosis of cfrd, comparing fasting blood glucose levels with oral glu- cose tolerance test results ( ). in our series, we have now cf patients with newly diagnosed diabetes based on a -h venous plasma glucose value mg/dl ( . mmol/l), and simultaneous determ i n a t i o n of hba c ( h i g h - p e rf o rmance liquid chro- matography method [pharmacia, erlan- gen, germany], normal range . – . %). only out of cf patients ( %) diag- nosed as diabetic according to the ameri- can diabetes association and world health o rganization criteria ( ) had an hba c value above the normal range (individual values . , . , . , and . %). in nine diabetic cf patients with normal hba c, values between . and . % were e n c o u n t e red (mean ± sd, . ± . %). the mean -h blood glucose value after inges- tion of oral glucose was not signific a n t l y d i ff e rent between diabetic cf patients with n o rmal hba c ( ± mg/dl, mean ± sd) and diabetic cf patients with elevated h b a c ( ± mg/dl, student’s t t e s t ) . these data clearly demonstrate that the d e t e rmination of hba c is not able to sub- stitute for the oral glucose tolerance test in the early diagnosis of cfrd. our fin d i n g s a re in agreement with several re p o rts in the l i t e r a t u re ( – ) as well as the consen- sus conference on cfrd ( ). in addition to its low sensitivity when used as a diagnos- tic tool for the detection of cfrd, the mea- s u rement of hba c has the disadvantage of considerable interassay variability and lack of standardization. there f o re, we stro n g l y advise against the use of glycosylated hemoglobin as a screening test for the early diagnosis of diabetes in patients with cystic fib ro s i s . reinhard w. holl, md christian buck, md christine babka, md anna wolf, md angelika thon, md f rom the department of pediatrics (r.w.h.), uni- versity of giessen, giessen; the department of pedi- atrics (c.bu., c.ba., a.w.), the university of ulm, ulm; and the medical school hannover (a.t. ) , h a n n o v e r, germ a n y. a d d ress correspondence to pd dr. reinhard w. holl, universitätskinderklinik giessen, feulgenstr. , d- giessen, germ a n y. r e f e re n c e s . h u n k e rt f, lietz t, stach b, kiess w: potential impact of hba c d e t e rm i n a t i o n on clinical decision making in patients with cystic fib ro s i s – related diabetes (let- ter). diabetes care : – , . holl rw, buck c, cario h, wolf a, thon a, heinze e, kohne e, debatin k-m: diag- nosis of diabetes in cystic fib rosis and tha- lassemia major. diabetes care : – , . the expert committee on the diagnosis and classification of diabetes mellitus: r e p o rt of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care : – , . lanng s, hansen a, thorsteinsson b, n e rup j, koch c: glucose tolerance in patients with cystic fib rosis: five year p rospective study. b m j : – , . deluca f, arrigo t, nibali sc, sferlazzas c, gigante a, dicesare e, cucinotta d: insulin secretion, glycosylated haemoglo- bin and islet cell antibodies in cystic fib ro- sis children and adolescents with diff e re n t d e g rees of glucose tolerance. h o rm metab r e s : – , . moran a, doherty l, wang x, thomas w: a b n o rmal glucose metabolism in cystic fib rosis. j pediatr : – , . moran a: highlights of the febru a ry consensus conference on cfrd. bonn, g e rm a n y, cystic fibrosis foundation, p ro gln p e roxisome p ro l i f e r a t o r- a c t i v a t e d r e c e p t o r- and o b e s i t y r istow et al. ( ) re p o rted an activating mutation in the peroxisome pro l i f- e r a t o r-activated re c e p t o r- g e n e ( p ro gln ppa r - ), which was pre s e n t in % ( of ) of obese and % ( of ) of nonobese german caucasians. these findings may have profound implica- tions, particularly if the presence of this variant and its association with obesity are c o n firmed in other populations. we perf o rmed polymerase chain re a c- t i o n – restriction fragment length polymor- phism analysis for the pro gln ppa r - variant as described ( ) on dna samples f rom several independent populations, including lean and obese caucasians fro m the baltimore, maryland, region; african- diabetes care, volume , number , january letters americans from jackson, mississippi, and forsyth county, north carolina; pima indians from arizona; and old ord e r amish from lancaster county, pennsylva- nia (table ). a pcr fragment corre s p o n d- ing to gastric insulinotropic peptide, which has two known restriction sites for h i n dii, was mixed with each sample as a positive control. among a total of sub- jects ( , alleles), the pro gln variant was not detected in a single subject. these findings were unexpected because there is substantial overlap of gene pools of caucasians from central e u rope and the baltimore and amish caucasians studied ( ). the germ a n caucasians studied by ristow et al. were said to be unrelated and re c ruited fro m the nord rh e i n - westfalen region, but they may be a genetic isolate whose gene pool does not re flect that of other caucasian populations. altern a t i v e l y, if the individ- uals carrying the mutation were re l a t e d , the true frequency of the pro g l n p pa r - variant may have been overe s t i- mated. the absence or very low fre- quency of this variant has also been doc- umented in danish ( ) and german ( ) populations. this study is the first, to our knowledge, to examine american popu- lations for this variant. in summary, the study by ristow et al. demonstrating that the pro g l n p pa r - variant is activating and can influence body weight in people is important. however, this variant appears to be absent or very r a re in the american populations studied. additional studies are re q u i red in other regions of europe and the u.s. to furt h e r d e fine the relevance of this intere s t i n g genetic variant to susceptibility to obesity. alan r. shuldiner, md william nguyen, bs w.h. linda kao, phd brock a. beamer, md ross e. andersen, phd richard pratley, md frederick l. brancati, md, phd f rom the department of medicine (a.r.s., w. n . ) , university of maryland school of medicine; the d e p a rtments of medicine (b.a.b., f.l.b.) and epi- demiology (w.h.l.k.), johns hopkins university school of medicine, baltimore, maryland; and the national institute of diabetes and digestive and kidney diseases (r.p.), national institutes of health, phoenix, arizona. a d d ress correspondence to alan r. shuldiner, md, professor and head, division of endocrinol- o g y, diabetes, and nutrition, department of medi- cine, university of maryland school of medicine, w. lombard st., room s- , baltimore, md . e-mail:ashuldin@medicine.umary l a n d . e d u . a c k n o w l e d g m e n t s — this study was sup- p o rted by nih r dk- , the american diabetes association, glaxowellcome, the b a l t i m o re geriatrics research and education clinical center of the baltimore ve t e r a n s administration medical center. r e f e re n c e s . ristow m, muller- wieland d, pfeiffer a, k rone w, kahn cr: obesity associated with a mutation in a genetic regulator of adipocyte diff e rentiation. n engl j med : – , . c a v a l l i - s f o rza ll, menozzi p, piazza a: t h e h i s t o ry of human genes. princeton univer- sity press, princeton, nj, . elk j, urhammer sa, sorensen ti, ander- sen t, auwerx j, pedersen o: homozygosity of the pro ala variant of the pero x i s o m e p roliferation-activated re c e p t o r- g a m m a ( p par-gamma ): divergent modulating e ffects on body mass index in obese and lean caucasian men. diabetologia : – , . mamann a, munzberg h, buttron p, busing b, hinney a, mayer h, siegfried w, hebe- brand j, greten h: missense variants in the human peroxisome pro l i f e r a t o r- a c t i v a t e d re c e p t o r-gamma gene in lean and obese subjects. eur j endocrinol : – , insulin secre t i o n , insulin sensitivity, and glucose e ffectiveness in nonobese individuals with va ry i n g d e g rees of glucose to l e r a n c e a lthough it is well known that insulin s e c retion, insulin sensitivity, and glu- cose effectiveness are impaired in type diabetic patients ( – ), little is known about the role of each of these fac- tors individually on the evolution of type diabetes. in this context, a major issue is that hyperglycemia per se impairs insulin s e c retion and insulin sensitivity and that obesity observed in type diabetic patients per se causes insulin resistance ( , ). to o v e rcome this problem, we studied u n t reated nonobese subjects classified as having normal glucose tolerance (ngt) (n = ; bmi . ± . kg/m [ r a n g e . – . ], mean ± sem), impaired glu- cose tolerance (igt) (n = ; bmi . ± . kg/m [ . – . ]), and type dia- betes (n = ; fetal bovine serum . ± . mmol/l [range . – . ], bmi . ± . k g / m [ . – . ]), based on the criteria of the world health organization ( ). they were normotensive and had norm a l renal, hepatic, and thyroid function. insulin sensitivity and glucose eff e c t i v e n e s s w e re estimated by the minimal model a p p roach ( – ). insulin secretion was e x p ressed as the area under the insulin c u rve between and min after an intra- venous glucose injection ( ). after the data w e re analyzed by one-way analysis of vari- ance, bonferroni correction was used to evaluate the diff e rences between any two of the three groups we studied ( ). no signifi- cant difference was observed in bmi among the three groups. compared with the subjects with ngt, the subjects with table —characteristics of subjects screened for pro gln ppa r - n ( a l l e l e s age ± sd f e m a l e bmi ± sd p o p u l a t i o n t y p e d ) ( y e a r s ) ( % ) ( k g / m ) c a u c a s i a n s b a l t i m o re longitudinal ( ) . ± . . . ± . study on aging johns hopkins we i g h t ( ) . ± . . . ± . management center amish, lancaster, pa ( ) . ± . . . ± . a f r i c a n - a m e r i c a n s a t h e ro s c l e rosis risk in ( ) . ± . . . ± . communities study pima indians a r i z o n a ( ) . ± . — . ± . all non-amish subjects were unrelated; amish subjects were not fir s t - d e g ree relatives of each other. diabetes care, volume , number , january letters igt had significantly lower insulin secre- tion ( , ± vs. , ± pmol l m i n , p = . ) and glucose eff e c- tiveness ( . ± . vs. . ± . m i n , p . ). insulin sensitivity index was lower in subjects with igt ( . ± . m i n pmol l) than in those with ngt ( . ± . min pmol l ) , but was not statistically significant (p = . ). in con- trast, disposition index calculated by the p roduct of insulin secretion and insulin sensitivity was significantly lower in sub- jects with igt ( , ± ) than in those with ngt ( , ± , p = . ). on the other hand, patients with type dia- betes had significantly lower insulin secre- tion ( ± pmol l m i n , p = . ) compared with subjects with igt. although no significant diff e rence was observed in insulin sensitivity index between subjects with type diabetes and igt ( . ± . vs. . ± . min pmol l, p = . ), disposition index was s i g n i ficantly diminished in type diabetic patients as compared with subjects with igt ( ± vs. , ± , p = . ). glucose effectiveness in type diabetic patients ( . ± . min ) was similar to that in subjects with igt ( . ± . m i n , p = . ) but was signific a n t l y lower than that in the subjects with ngt (p . ). from these results, the fol- lowing may be hypothesized: ) impair- ments in insulin secretion and disposition index and decreased glucose eff e c t i v e n e s s , but not insulin resistance, seem to consti- tute the basic characteristics of patients with igt or type diabetes in nonobese japanese populations. ) risk factors wors- ening to type diabetes in subjects with igt are associated with further impair- ments in insulin secretion and disposition index, but not associated with furt h e r derangement in glucose effectiveness in japanese populations. ataru taniguchi, md mitsuo fukushima, md masahiko sakai, md itaru nagata, md kentaro doi, md shoichiro nagasaka, md kumpei tokuyama, md yoshikatsu nakai, md f rom the first department of internal medicine ( a . t., m.s., i.n.), kansai-denryoku hospital, and the department of internal medicine, hoshida- minami hospital (m.f.), osaka; the second depart- ment of internal medicine (k.d.) and the college of medical technology (y.n.), kyoto university, kyoto; the department of internal medicine (s.n.), jichi medical college, tochigi; and the laboratory of biochemistry of exercise and nutrition (k.t. ) , tsukuba university, tsukuba, japan. a d d ress correspondence to ataru ta n i g u c h i , md, first department of internal medicine, kansai- d e n ryoku hospital, - - , fukushima-ku, fuku- shima, osaka city, osaka - , japan. e-mail: k @ k e p c o . c o . j p . r e f e re n c e s . b e rgman rn: to w a rd physiological under- standing of glucose tolerance: minimal- model approach. d i a b e t e s : – , . welch s, gebhart ssp, bergman rn, phillips ls: minimal model analysis of intravenous glucose tolerance test-derived insulin sensitivity in diabetic subjects. j c l i n endocrinol metab : – , . taniguchi a, nakai y, fukushima m, kawamura h, imura h, nagata i, tokuyama k: pathogenic factors re s p o n s i- ble for glucose tolerance in patients with niddm. d i a b e t e s : – , . nagasaka s, tokuyama k, kusaka i, hayashi h, rokkaku k, nakamura t, kawakami a, higashiyama m, ishikawa s, saito t: endogenous glucose pro d u c t i o n and glucose effectiveness in type diabetic subjects derived from stable-labeled mini- mal model approach. d i a b e t e s : – , . best jd, kahn se, ader m, watanabe rm, ni t-c, bergman rn: role of glucose eff e c- tiveness in the determination of glucose tol- erance. diabetes care : – , . rossetti l, giaccari a, defronzo ra: glu- cose toxicity. diabetes care : – , . taniguchi a, nakai y, doi k, fukuzawa h, fukushima m, kawamura h, to k u y a m a k, suzuki m, fujitani j, tanaka h, nagata i: insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis. m e t a b o l i s m : – , . world health organization: diabetes melli - tus: report of a who study gro u p . g e n e v a , world health org., (tech. rep. ser. , no. ) . winer bj: statistical principles in experimen - tal design. nd ed. new york, mcgraw-hill, , p. – late-onset tro g l i t a z o n e - i n d u c e d hepatic dysfunction r e c e n t l y, iwase et al. ( ) re p o rted a case of liver dysfunction occurring after months of troglitazone therapy. because it was thought before this re p o rt that the risk of liver dysfunction with tro g l i- tazone after months was negligible, we wish to re p o rt another patient who took t roglitazone intermittently and developed hepatic dysfunction after months. a -year-old white man with type diabetes, ischemic heart disease (post angioplasty and stent placement), hyper- tension, degenerative joint disease, benign p rostatic hypert ro p h y, dyslipidemia, and g a s t roesophageal re flux disease had tro g l i- tazone mg daily added to his re g i m e n of glimeperide mg daily and metform i n mg b.i.d. because of poor glycemic c o n t rol (hba c . % [normal – %]). the other medicines he used were aspirin and pravastatin. after months of triple oral therapy, his hba c level dropped to . %, and, after months, to . %. after months, the patient’s hba c began to rise: . % at months, . % at year, and . % at months. liver function tests were normal until months, when his aspartate amino- transferase (ast) was found to be (nor- mal – u/l) and alanine aminotrans- ferase (alt) (normal – u/l). the t roglitazone regimen was discontinued, and testing for hepatitis b and c, h e m a c h romatosis, autoimmune liver dis- ease, and gallbladder disease were nega- tive. two months after discontinuing t roglitazone, the patient’s ast and alt had decreased to and u/l, and, after months, had re t u rned to normal at and u/l, re s p e c t i v e l y. his ast and alt have remained normal since then and he has continued to take pravastatin, aspirin, m e t f o rmin, and glimeperide. when the patient was told to discon- tinue troglitazone, he admitted that he had been taking it only interm i t t e n t l y. he esti- mated that he took the drug regularly at first, but after the first months, he took the drug only once or twice weekly on aver- age. he gave the following three reasons for his lack of compliance: a lack of funds, a fear of liver disease, and a tendency to avoid taking drugs whenever possible. this case, like the case described by iwase et al., illustrates that the hepatic dysfunction caused by troglitazone can occur after months and further sup- p o rts the u.s. food and drug administra- t i o n ’s current recommendation that quar- terly liver function tests should be obtained when troglitazone utilization extends beyond year ( ). in this case, could the onset of hepatic dysfunction have been delayed because the diabetes care, volume , number , january letters d rug was being taken only interm i t t e n t l y after the first months, and the estimated total load presented to the liver would be equivalent to the exposure at months in a compliant patient? we doubt this, since t ro g l i t a z o n e ’s hepatic effects are thought to be idiosyncratic and there f o re the total e x p o s u re should be irre l e v a n t . david s.h. bell, mb fernando ovalle, md f rom the division of endocrinology and metabo- lism, department of medicine, school of medicine, university of alabama, birmingham, alabama. a d d ress correspondence to david s.h. bell, mb, th ave. s., birmingham, al . d.s.h.b. and f.o. have served on an advisory panel for sankyo parke-davis and have received con- sulting fees, re s e a rch grant support, and honoraria for speaking engagements from sankyo parke-davis. r e f e re n c e s . iwase m, yamaguchi m, yoshinari m, oka- mura c, hirahashi t, tsuji h, fujishima m: a japanese case of liver dysfunction after months of troglitazone tre a t m e n t . diabetes care : – , . parke-davis, division of wa rn e r- l a m b e rt : rezulin package insert. morris plains, nj, g l y b u r i d e - i n d u c e d hemolysis in m y e l o d y s p l a s t i c s y n d ro m e g lyburide, also known as gliben- clamide, is a widely used sulfonylure a to treat patients with type diabetes. hemolytic anemia is an extremely rare side e ffect of which there have been only a few re p o rts ( – ). we describe a patient with myelodysplastic syndrome who pre s e n t e d with glyburide-induced hemolysis. a -year-old man with a long history of type diabetes presented with left foot cellulitis of week’s duration. this patient was known to have slowly pro g re s s i v e pancytopenia for years, for which no work-ups had been perf o rmed. his med- ications included the following: glyburide, mg per day, which he had taken for m o re than year; buformine, mg per day; and boglibose, . mg per day. he was afebrile, and the physical examination was normal, except for localized cellulitis on his left foot, for which he was start e d on intravenous antibiotics. the laboratory studies revealed a white blood cell count of . / l , hemoglobin . g/dl, platelet count /l, reticulocyte count . %, mean cor- puscle volume fl, moderate anisocyto- sis, fasting plasma glucose mg/dl, h b a c . %, lactate dehydrogenase iu/l, total bilirubin . mg/dl, and hapto- globin mg/dl. red cell glucose- - phosphate dehydrogenase level was ade- quate. cold agglutinin test, ham’s test, and sugar water test were normal. both dire c t and indirect coombs’ tests were negative. red cell resistance to osmolarity was mildly low (parpart ’s method). urinalysis demonstrated no urobilinogen. endo- scopic studies did not reveal gastro i n t e s t i- nal bleeding. ultrasonography of the abdomen showed no splenomegaly. the result of bone marrow aspiration was equivocal. on the basis of pre s u m p t i v e glyburide-induced hemolysis, glyburide was discontinued and the patient was switched to subcutaneous insulin on the seventh day. there a f t e r, his hemoglobin level increased to . g/dl, re t i c u l o c y t e count decreased to . %, and anisocytosis d i s a p p e a red pro m p t l y. he was discharg e d with insulin therapy after month in the hospital, which is when the cellulitis re s o l v e d . t h ree months later, his hemoglobin level was . g/dl and his haptoglobin remained low. repeated bone marro w aspiration confirmed the diagnosis of myelodysplastic syndro m e . we conclude that this patient devel- oped glyburide-induced hemolysis super- imposed on red cell fragility secondary to an underlying bone marrow disord e r. t h e re have been several re p o rts of hemoly- sis caused by sulfonylureas, most of which have been considered immune-mediated ( , , ). our case points to the possibility that glyburide could cause hemolysis by a non–immune-medicated mechanism. it is i m p o rtant to be aware of this potential side e ffect of glyburide in light of this medica- t i o n ’s widespread prescription, even though such a side effect is rare . hiroshi noto, md kazuhisa tsukamoto, md satoshi kimura, md f rom the department of diabetes and metabolism, tokyo university hospital, tokyo, japan. a d d ress correspondence to hiroshi noto, md, d e p a rtment of diabetes and metabolism, tokyo uni- versity hospital, - - hongo, bunkyo-ku, to k y o - , japan. e-mail: noto-tky@umin.ac.jp. r e f e re n c e s . nataas ob, nesthus i: immune haemolytic anaemia induced by glibenclamide in selective iga defic i e n c y. b m j : – , . abbate sl, hoogwerf bj: hemolytic ane- mia associated with sulfonylurea use. d i a - betes care : – , . meloni g, meloni t: glyburide-induced acute haemolysis in a g pd-defic i e n t patient with niddm. br j haematol : – , . kopicky ja, packman ch: the mechanism of sulfonylurea-induced immune hemoly- sis: case re p o rt and review of the literature . am j hematol : – , e ffects of exposure at an altitude of , m on p e rf o rmance of glucose meters s elf-monitoring of blood glucose is m a n d a t o ry for type diabetic p a t i e n t s who participate in sports to adjust insulin dose and carbohydrate ingestion ( ). sports also include activities p e rf o rm e d at moderately high altitudes, such as hiking or skiing. capillary blood glucose monitors (bgms) have been shown to underestimate blood glucose values at an altitude of , m ( ) and at a simulated altitude of , m with t e m p e r a t u re and humidity kept constant ( ). the aim of the present study was to assess the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and p o can result in errors in blood glucose d e t e rmination ( ). two bgms, the lifescan one touch ii (ot) (ortho diagnostics, milpitas, ca) and the glucometer elite ii (ge) (bayer diagnostics, brussels, belgium), were tested during a study on the effects of acute exposure at an altitude of , m and exercise on blood pre s s u re and albu- min excretion rate in six type diabetic p a t i e n t s . all subjects (four men and two women) were free of disease-related com- plications and in good and stable glycemic control (ghb . ± . %). all subjects gave their informed and written consent to participate in the study pro t o- col. all subjects were investigated both at diabetes care, volume , number , january letters sea level and after ascent by car and cable car to the angelo mosso institute at col d’olen ( , m altitude), gressoney la trinité, italy. at sea level and at a moderately high altitude, bgm reliability at diff e rent blood glucose levels was tested, and blood glu- cose was assessed in fasting and re s t i n g conditions at : a.m.; at : a.m. b e f o re an in-field exercise test; and imme- d i a t e l y, min, and min after the exer- cise stopped. capillary glucose was simul- taneously assessed with the ot and the ge. both of these bgms measure capillary blood glucose through the glucose oxi- d a s e - p e roxidase reaction. bgms were cali- brated at the beginning of each test ses- sion. a venous blood sample was simulta- neously drawn from the contralateral antecubital vein in a sodium fluoride tube, centrifuged, and stored at °c. plasma glucose was assayed with the glucose oxi- dase method (go) within days. this last assessment was taken as a re f e re n c e method. statistical analysis compare d bgm capillary glucose values and go plasma glucose values for each blood col- lection time. measurement linearity was tested with pearson’s correlation coeff i- cient. the mean of the diff e rences between the bgm and go results re p resents the mean bias between the methods with accuracy expressed as percent error (pe): pe (%) = bmg – go % g o the level of statistical significance was c o n s i d e red to be p . . the ge and ot measurements had a good correlation with plasma glucose both at moderately high altitude and at sea level. p e a r s o n ’s correlation coefficients were . and . for the ge and . and . for the ot at sea level and at moderately high altitude, re s p e c t i v e l y. biases between plasma glucose and bgm measure m e n t s w e re as follows: for the ge, . ± . at sea level and . ± . at moderately high altitude; for the ot, . ± . at sea level and . ± . at moderately high alti- tude. at sea level, both the ge and the ot tended to underestimate glucose values (ns); at moderately high altitude, the ge tended to overestimate and the ot tended to underestimate glucose values (ns). mean pes between plasma glucose and bgm m e a s u rements were . (ot) and . (ge) at sea level and . (ot) and . (ge) at moderately high altitude. pe tended to be higher for both bgms at moderately high altitude (ns). figures and show the bias between the single measurements with both devices at sea level and at moderately high altitude, re s p e c t i v e l y. at moderately high altitude (fig. ), the tendency of the ge to o v e restimate was more evident for low ( mg/dl) and intermediate ( – mg/dl) blood glucose values, whereas the ot tended to underestimate mainly high blood glucose values (ns). in our study, bgm perf o rmance was similar and good at sea level. at a moder- ately high altitude, a tendency to overe s t i- mate blood glucose for the ge and to u n d e restimate for the ot was observ e d . the overestimation for the ge involved mainly low ( mg/dl) and interm e d i- ate ( – mg/dl) blood glucose val- ues. this could present a problem in the p resence of symptoms suggesting hypo- figure —relationship between plasma and capillary glucose at sea level. figure —relationship between plasma and capillary glucose at moderately high altitude. lifescan one touch ii glucometer elite ii lifescan one touch ii glucometer elite ii diabetes care, volume , number , january letters glycemia and normal blood glucose val- ues. the ot tended to undere s t i m a t e mainly high blood glucose values, although its perf o rmance with low to i n t e rmediate values was good. the pre s- ent study assessed the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and po can result in errors in blood glu- cose determination ( ). our results are consistent with previous studies ( , ). the decrease in po could alter the sec- ond phase of the chromogen reaction and u n d e restimate blood glucose values ( ); on the other hand, an increase in atmos- pheric pre s s u re could overestimate blood glucose values ( ). in our study, minimal o v e restimation by the ge at low interm e- diate blood glucose values at moderately high altitude cannot be explained by the a l t e red po . an increase in hematocrit, which is known to alter blood glucose m e a s u rements with bgms ( ), may also occur after prolonged exposure to high altitude or as a consequence of dehydra- tion. although our study did not deter- mine hematocrit, the exercise test was s h o rt, and the patients were instructed to drink according to their thirst during the , -m exposure; there f o re, dehydration was not likely to have occurred. in con- clusion, bgm perf o rmance is similar and good at sea level. at a moderately high altitude similar to that experienced during winter skiing or summer hiking, a ten- dency to overestimate low to norm a l blood glucose values for the ge and to u n d e restimate high blood glucose values for the ot was observed. the bias is not clinically meaningful for either bgm, both of which can be safely used by dia- betic patients during exposure to moder- ately high altitudes. some care in the eval- uation of low and intermediate blood glu- cose values measured with the ge is n e v e rtheless re c o m m e n d e d . oriana pecchio, md simona maule, md marco migliardi, md marina trento, bsc massimo veglio, md f rom the italian alpine club medical commission ( o . p.); the department of internal medicine (m.t. ) , university of turin; the s. giovanni battista hospital (s.m.); and the department of endocrinology (m.m., m . v.), mauriziano umberto i hospital, turin, italy. a d d ress correspondence to dr. m. veglio, via mancini , torino, italy. e-mail: veglio@ o n w. n e t . r e f e re n c e s . h o rton es: role and management of exer- cise in diabetes mellitus. diabetes care : – , . g i o rdano bp, trash w, hollenbaugh l, dube wp: perf o rmance of seven blood glucose testing systems at high altitude. diabetes educ : – , . gautier jf, bigard ax, douce p, duvallet a, cathelinau g: influence of simulated alti- tude on the perf o rmance of five blood glu- cose meters. diabetes care : – , . b a rnett c, ryan f, ballonoff l: effect of altitude on the self monitoring of blood glucose (smbg) (abstract). diabetes (suppl.): a, . piepmeier eh, hammett-stabler c, price me, kemper gb, davis mg: atmospheric p re s s u re effects on glucose monitoring devices (letter). diabetes care : – , . b a rreau pb, buttery je: effect of hematocrit concentration on blood glucose value d e t e rmined on glucometer ii. diabetes care : – , c o m m e n t s a n d r e s p o n s e s deterioration of glycemic contro l after long-te rm treatment wi t h troglitazone in nonobese type diabetic patients t roglitazone is an oral antidiabetic d rug used to treat type diabetic patients with insulin re s i s t a n c e . troglitazone improves overall insulin sen- sitivity in the liver and skeletal muscles, which are the largest consumers and metabolizers of glucose in the body ( – ). recent re p o rts showed that troglitazone is also effective in nonobese type diabetic patients whose hyperglycemia could not be controlled with sulfonylurea therapy ( , ). however, we aware that in some patients in whom adequate glycemic con- t rol is obtained during the first several months of troglitazone treatment, their glycemic control deteriorates several months later. we assume that two distinct g roups of type diabetic patients exist who respond diff e rently to long-term administration of troglitazone, one gro u p that maintains a steady response and another group that has a decre a s i n g response after certain periods. in this s t u d y, we re t rospectively examined patients with type diabetes who were t reated with troglitazone for months and whose hba c levels had improved by % with troglitazone by month . in of the patients ( %), hba c levels increased by . % after – months despite continuous tro g l i t a z o n e t reatment (group p). in contrast, the rest of the patients experienced steady glycemic c o n t rol with . % of hba c flu c t u a t i o n ( g roup g). during the first months, h b a c levels decreased from means ± sem . ± . to . ± . % in group p and fro m . ± . to . ± . % in group g, re s p e c- t i v e l y. no significant diff e rences were evi- dent between the two groups re g a rding the d e c rease in hba c during the first months (fig. ). from month onward, hba c l e v- els in group p climbed gradually by . % a month up to the baseline level at month , but hba c levels were stable in group g t h roughout the treatment period. a signifi- cant diff e rence in hba c levels was evident during months – (p . ) . among clinical characteristics, gro u p p had a significantly lower bmi ( . ± . vs. . ± . kg/m , p . ) and s i g n i ficantly lower fasting insulin levels ( . ± . vs. . ± . µu/ml, p . ). of the patients with a bmi of kg/m ( %), exhibited deteriora- tion of glycemic contro l . in this study, we re p o rt a group of patients who showed a renewed decline in glycemic control after long-term tre a t m e n t with troglitazone. these results seemed to suggest a secondary failure of tro g l i t a z o n e . our study demonstrates that this drug is indeed useful for a long-standing obese i n s u l i n - resistant diabetes but not for a nonobese type diabetes. yuko murase, md takanobu wakasugi, md kunimasa yagi, md hiroshi mabuchi, md f rom the department of internal medicine (y. m . , t. w.), fukui perfectural hospital; and the second d e p a rtment of internal medicine (k.y., h.m.), kanazawa university, ishikawa, japan. a d d ress correspondence to yuko murase, md, the second department of internal medicine, kanazawa university, - takara-machi, kanazawa, ishikawa - , japan. e-mail: diabe@med. k a n a z a w a - u . a c . j p . diabetes care, volume , number , january letters r e f e re n c e s . suter sl, nolan jj, wallace p, gumbiner b, olefsky jm: metabolic effects of new oral hypoglycemic agent cs- in niddm subjects. diabetes care : – , . o’rourke cm, davis ja, saltiel ar, corn i- celli ja: metabolic effects of troglitazone in the goto-kakizaki rat, a non-obese and n o rmolipidemic rodent model of nonin- sulin-dependent diabetes mellitus. m e t a b - o l i s m : – , . troglitazone study group: the metabolic e ffects of troglitazone in non-insulin dependent diabetes (abstract). d i a b e t e s (suppl. ): a, . mori k: the effect of troglitazone in combi- nation with sulfonylurea in non-insulin dependent diabetes mellitus (abstract). j japan diabetes soc (suppl. ): , . h o rton es, venable tc, whitehouse f, the troglitazone study group, ghazzi mn, whitcomb rw: troglitazone in combina- tion with sulfonylurea re s t o res glycemic c o n t rol in patients with type diabetes. diabetes care : – , figure —change from baseline in hba c. values are means ± sem. doi: . /j.bbabio. . . p/ import and assembly of mitochondrial proteins nikolaus pfanner institute for biochemistry and molecular biology, university of freiburg, germany e-mail: nikolaus.pfanner@biochemie.uni-freiburg.de mitochondria contain about different proteins. % of the proteins are synthesized as precursors on cytosolic ribosomes. the precursors are imported via the translocase of the outer mitochon- drial membrane (tom complex) and are subsequently sorted into the four mitochondrial subcompartments, outer membrane, inter- membrane space, inner membrane and matrix. (i) cleavable preproteins are transported from the tom complex to the presequence translocase of the inner membrane (tim complex). the presequence translocase-associated motor (pam) drives trans- location into the matrix. (ii) hydrophobic inner membrane proteins are transferred through the intermembrane space by a chaperone complex (small tim proteins) and inserted into the inner membrane by the tim complex. (iii) the mitochondrial import and assembly machinery (mia) directs small proteins into the intermembrane space and promotes the formation of disulfide bonds. (iv) beta- barrel proteins are transported from the tom complex to the sorting and assembly machinery of the outer membrane (sam complex). doi: . /j.bbabio. . . p/ new functions for novel mitochondrial transporters ferdinando palmieri university of bari, italy e-mail: fpalm@farmbiol.uniba.it a strikingly large number of mitochondrial dna (mtdna) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. these mitochondrial dis- orders were the first to be investigated after the small mtdna had been sequenced in the 's. only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, hhh syndrome, aspartate/glutamate isoform deficiency, amish microce- phaly and neonatal myoclonic epilepsy; these disorders are characterised by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabo- lism. defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phos- phate and adp, are responsible for diseases characterised by defective energy production. herein, all the mitochondrial carrier- associated diseases known to date are reviewed for the first time. particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders. doi: . /j.bbabio. . . p/ the water oxidizing enzyme a. william rutherford ibitec-s, ura cnrs, cea saclay, gif-sur-yvette, france e-mail: alfred.rutherford@cea.fr photosystem ii, the water oxidising enzyme of photosynthesis, put the energy (or at least a major fraction of it) into the biosphere and the oxygen into the atmosphere. it is certainly one the most influential and important enzymes on the planet. the aim of our research is to understand how this enzyme works as ) a solar energy converter and ) the only known thermodynamically efficient catalyst for oxidizing water. the information obtained is used in the design of artificial catalysts and photocatalysts. a chemical catalyst that has the thermodynamic efficiency of the enzyme could greatly improve the efficiency of ) water electrolysis and photolysis for fuel (e.g. h ) production and ) the reverse reaction, oxygen reduction, in fuel cells. there is therefore a great interest in understanding the mechanism of this enzyme and in reproducing aspects of its function in artificial systems. i will describe our current knowledge of photosystem ii, including some recent experimental studies, as well as recent efforts in our joint saclay/orsay program aimed at producing bio-inspired water oxidizing catalysts. doi: . /j.bbabio. . . p/ the structure of purple bacterial antenna complexes: from single molecules to native membranes richard j. cogdella, alastair t. gardinera, mads gabrielsena, aleks w. roszaka, june southalla, tatas brotosudarmoa, neil w. isaccsa, hideki hashimotob, juergen baierc, silke oellerichc, martin richterc, juergen koehlerc, francesco franciad, giovanni venturolid, dieter oesterhelte adivision of biochemistry and molecular biology, ibls and department of chemistry, university of glasgow, glasgow g qq, uk bcrest-jst and department of physics, graduate school of science, osaka city university, - - sugimoto, sumiyoshi-ku, osaka - , japan cexperimental physics iv, university of bayreuth, d- bayreuth, germany ddepartment of biology, university of bologna, bologna, italy edepartment of membrane biochemistry, max-planck institute for biochemistry, martinsried, germany e-mail: r.cogdell@bio.gla.ac.uk the photosynthetic unit of purple photosynthetic bacteria typically contains two types of light-harvesting complexes, called lh and lh . these antenna complexes are constructed on a modular principle. they are circular or elliptical oligomers of dimers of two low-molecular weight, hydrophobic apoproteins, called a and b, that bind bacteriochlorophylls and carotenoids non-covalently. the lh complex surrounds the reaction centre and, depending on the species, is either a monomer or a dimer. the lh complexes are arranged around the lh -rc complexes. this plenary lecture will present the current status of structural studies on these pigment- protein complexes, based upon a combination of x-ray crystal- lography and single molecule spectroscopy. then an overall view of how they are arranged in their native photosynthetic membranes will be presented. doi: . /j.bbabio. . . p/ catalysis of substrate conversion and electron transfer by mitochondrial complex i judy hirst medical research council dunn human nutrition unit, cambridge, cb xy, uk e-mail: jh@mrc-dunn.cam.ac.uk s abstracts / biochimica et biophysica acta ( ) s –s http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . mailto:nikolaus.pfanner@biochemie.uni-freiburg.de mailto:fpalm@farmbiol.uniba.it mailto:alfred.rutherford@cea.fr mailto:r.cogdell@bio.gla.ac.uk mailto:jh@mrc-dunn.cam.ac.uk letter to the editors rebuttal to “ezetimibe treatment should be considered for patients with sitosterolemia” elif erkan received: april /revised: april /accepted: april /published online: may # ipna we have presented an adolescent of amish descent with atherosclerotic renal artery stenosis as a cause for hyperten- sion. he was subsequently diagnosed with sitosterolemia, a rare autosomal recessive genetic disorder characterized by the retention of both plant sterols and cholesterol in affected individuals. determination of his serum sterol profile by gas chromatography revealed . % cholesterol (normal > %), with elevated plant sterol levels (normal < %) at . % campesterol, . % beta-sitosterol and . % stigmasterol; these findings were consistent with sitosterolemia [ ]. his genetic testing has not yet been completed, but the clinical picture of the patient and elevated serum plant sterols support the diagnosis of sitosterolemia. the patient was initially placed on atorvastatin. after he was diagnosed with sitosterolemia he was started on ezetimibe and resin colestid. we agree with hu et al. that ezetimibe is the drug of choice for sitosterolemia [ ]. ezetimibe binds directly to niemen pick c -like (npc l ) and inhibits absorption of sitosterols from the intestinal epithelium. in a multicenter study patients were randomized to receive placebo (n= ) versus ezetimibe (n= ) for weeks. ezetimibe treatment resulted in a significant reduction in sitosterol and campesterol levels [ ]. we believe a multidrug approach is warranted to treat sitosterolemia in order to prevent premature atherosclerotic cardiovascular disease. although there is no doubt that ezetimibe is beneficial for patients with sitosterolemia, close monitoring for future side effects is warranted particularly in children, considering their longer life span. references . webb tn, ramratnam m, pacella j, erkan e ( ) atherosclerotic renal artery stenosis as a cause of hypertension in an adolescent patient. pediatr nephrol. doi: . /s - - -y . hu m, tomlinson b ( ) ezetimibe treatment should be considered for patients with sitosterolemia. pediatr nephrol. doi: . /s - - - . salen g, von bergmann k, lutjohann d, kwiterovich p, kane j, patel sb, musliner t, stein p, musser b, and the multicenter sitosterolemia study group ( ) ezetimibe effectively reduces plasma plant sterols in patients with sitosterolemia. circulation : – e. erkan (*) pediatrics, division of nephrology, cincinnati children’s hospital, cincinnati, oh, usa e-mail: elif.erkan@cchmc.org pediatr nephrol ( ) : doi . /s - - - http://dx.doi.org/ . /s - - -y http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - 	rebuttal to “ezetimibe treatment should be considered for patients with sitosterolemia” 	references _ _ .book(tai _fm.fm) mja vol august notable cases the medical journal of australia issn: - x august - ©the medical journal of australia www.mja.com.au notable cases a preterm infant born to a woman with chronic lead poisoning was found to have the highest blood lead level recorded for a surviving neonate. parenteral calcium disodium edetate, but not oral succimer, was effective in reducing the infant’s lead burden in the neonatal period. an exposure assessment revealed the mother’s long-term ingestion of lead-contaminated herbal tablets as the source. (mja ; : - ) chronic lead poisoning may manifest clinically as abdominal pain, constipation, proteinuria, haematuria, peripheral neuropathy and muscle weakness. with more significant lead poisoning, encephalopathy may occur. sus- tained blood lead levels of over . �mol/l in early child- hood are likely to be associated with intellectual underperformance. however, congenital lead poisoning and its subsequent management has rarely been reported in preterm infants. treatment is with chelating agents, which reduce lead concentrations in the blood and tissues by forming water- soluble complexes that are cleared by the kidneys. we report a case in which the neonatal blood lead level was the highest recorded for a surviving infant. clinical record a -year-old pregnant woman, who had recently emigrated from india, was found to have a haemoglobin level of g/l at weeks’ gestation. at that time it was noted that she was a vegan and had a normal blood film and iron studies. at weeks’ gestation, she presented with abdominal pain and a progressive confusional state culminating in seizures. the blood film now showed basophilic stippling, a typical sign of lead poisoning (box ), and subsequent testing showed she had a blood lead concentration of . �mol/l (the national health and medical research council’s public health goal is a level of � . �mol/l). chelating therapy was initiated with intramuscular dimer- caprol and intravenous calcium disodium edetate (cana edta). thirty-six hours later the woman had an antepartum haemorrhage, and, after induction of labour, she gave birth to a . kg ( th percentile) female baby by vaginal delivery. apgar scores were and . the infant was flaccid and areflexic and did not move in response to noxious stimuli, although spontaneous ocular movements were present. as she had emerging alveolar hypoventilation and no gag reflex, she was intubated and ventilated. the clinical suspicion of bilateral diaphragmatic palsy was later confirmed by fluoroscopy. basophilic stippling was not seen on the infant’s blood films, although heinz bodies were present. lead concentra- tion in the cord blood was . �mol/l ( hours before birth, maternal blood lead concentration had been . �mol/l). the concentration of erythrocyte porphyrins was . �mol/l (normal range, . – . �mol/l), and radiographs of the long bones showed an increase in the bone density adjacent to the metaphyses (both signs of lead poisoning). within hours of birth, the infant was commenced on chelation therapy (intramuscular dimercaprol mg/kg/dose every four hours and intravenous cana edta mg/kg/ day, given after the second dose of dimercaprol). the blood lead concentration initially rose to . �mol/l within the first hours of therapy, then fell rapidly over the next few days. the high urinary lead concentration of �mol/l on day was an indicator that lead was being excreted. the time course of blood and urinary lead concentrations for the first weeks post partum is shown in box . on day , it was judged appropriate to cease intravenous chelating ther- apy. succimer (an oral chelating agent) mg/kg/day was commenced three days later. at that time, the urinary lead concentration had fallen to . �mol/l. over the three-week course of succimer, urinary lead concentrations fell further, while blood lead concentrations remained relatively con- stant. as the succimer appeared to be having little effect, it was discontinued on day , and intravenous cana edta was recommenced hours later. a rise in urinary lead excretion was observed after this course and subsequent courses of parenteral therapy. by day , facial, bulbar, proximal-limb and diaphrag- matic muscle activity had improved sufficiently to allow successful extubation. by day , the blood lead concentra- tion appeared to have fallen to a satisfactory level, and an attempt at maintenance therapy using oral succimer at the higher dose of mg/kg/day was commenced. , however, severe congenital lead poisoning in a preterm infant due to a herbal remedy paul a tait, amish vora, simon james, d james fitzgerald and beverly a pester pharmacy department, women’s and children’s hospital, north adelaide, sa. paul a tait, bpharm, clinical pharmacist and lecturer. neonatal unit, flinders medical centre, bedford park, sa. amish vora, mb bs, neonatal registrar; simon james, fracp, paediatrician. environmental health branch, department of human services, adelaide, sa. d james fitzgerald, phd, principal toxicologist; beverly a pester, ba ma, scientific officer. reprints will not be available from the authors. correspondence: dr d james fitzgerald, environmental health branch, department of human services, po box , rundle mall, adelaide, sa . jim.fitzgerald@dhs.sa.gov.au notable cases mja vol august notable cases the blood lead concentration increased during the second course of succimer, with a corresponding decrease in the urinary lead concentration. intravenous cana edta was recommenced. when chelation therapy was subsequently discontinued for two weeks, the blood lead concentration again rose, and a fifth course of cana edta was initiated. progress of infant after first weeks. by three months’ corrected age (ie, about ½ months after birth) the infant was able to fully feed by sucking, although there was significant gastroesophageal reflux. her peripheral weakness had almost resolved, but bilateral wrist drop and poor head control persisted. blood lead concentration had fallen to . �mol/l. it was decided to reintroduce oral succimer at mg/kg/day. this appeared to be effective, as the lead levels not only fell in the blood but increased in the urine. a brainstem auditory response at four months’ corrected age showed right sensorineural deafness. (a magnetic reso- nance image of the brain at three weeks of age had been normal.) throughout the hospital stay her serum calcium, zinc, iron, renal and liver function tests were normal. the infant was discharged home, on succimer, at five months’ corrected age with a blood lead concentration of . �mol/l. a neurodevelopmental examination at the time revealed a two-month delay. her two siblings, aged two and four years, were found to have blood lead concentra- tions of . and . �mol/l, respectively. lead source identification. an environmental audit of the home and the local sikh community kitchen revealed no obvious source of lead. interviews conducted with the mother established that she had been taking several tablets, prescribed by an ayurvedic doctor in india for treatment of a gastrointestinal complaint, periodically over the course of the past nine years. analytical results for the various tablets are presented in box , the most notable being the high lead content of two of the tablet types ( . %– . % lead). the mother’s tablet use in the nine months before the child’s birth represented a lead intake of at least times the average weekly lead intake of western populations. , correspondence with the ayurvedic doctor in india, who prescribed the brown (“hsy- ”) tablets in this case, has not yet provided any clues as to the origin of the lead in these tablets. discussion clinical considerations the diagnosis of intrauterine lead intoxication was made on the basis of a maternal encephalopathy with a high blood lead concentration, maternal anaemia with basophilic stip- pling, and a high cord-blood lead concentration. infant blood lead levels of a third to a half the peak level reported here are usually associated with severe cerebral oedema and death. in this instance, the infant was encephalopathic and profoundly weak and had diaphragmatic palsy at birth. the limb weakness clinically appeared to be predominantly neuropathic. several electrophysiological attempts to clarify : infant blood and urinary lead concentrations from birth to weeks of age* *horizontal lines across top of graph indicate periods of parenteral therapy with calcium disodium edetate and oral succimer. dotted horizontal line shows national health and medical research council public health goal for maximum lead concentration in blood ( . �mol/l). b lo o d le a d c o n ce n tr a tio n ( � m o l/l ) u rin a ry le a d co n ce n tra tio n (� m o l/l ) postnatal age (days) parenteral therapy oral succimer blood lead concentration urinary lead concentration nhmrc goal : metal analysis of tablets used by the patient* tablet colour lead per tablet (mg) (%) mercury per tablet (�g) (%) brown (“hsy- ”) ( . %) ( . %) red ( . %) na pink ( . %) ( . %) green ( . %) ( . %) *analysis by imvs laboratories, adelaide. na = not analysed. : mother’s blood film just before parturition, showing basophilic stippling of red cells mja vol august notable cases the presence of either a neuropathy or a myopathy failed owing to technical and interpretive difficulties. although lead-induced peripheral neuropathy is well described, dia- phragmatic palsy is unreported. there was no evidence of renal involvement or proteinuria, possibly due to the infant’s renal immaturity. there was no basophilic stippling in any neonatal blood film. this relative paradox — the absence of basophilic stippling despite lead intoxication — has been noted previously. the unilateral sensorineural deafness is likely to be due to lead toxicity. previous reports of congenital lead intoxication indicate that maternal and cord blood lead levels correlate. the marked discrepancy in this case ( . �mol/l [mother] v . �mol/l [cord]) could be due to the possibility that at high maternal blood lead concentrations the lead-binding capacity of the erythrocytes becomes saturated and more lead is available for transfer to the fetus. alternatively, the discrepancy could be due to in-utero mobilisation of lead from fetal tissues without adequate maternal clearance. this latter consideration indicates a potential detrimental effect on the fetus of chelation therapy in pregnancy. as chelating agents bind lead into complexes that are cleared by the kidneys, an increase in urinary lead concen- tration was expected after administration of each of the chelating agents. this phenomenon was consistently observed after courses of dimercaprol and cana edta. however, variable doses of oral succimer during the preterm period did not lead to corresponding increases in urinary lead concentration, despite raising the dose to a level higher than those recommended in the literature. - only when oral succimer was recommenced at three months’ corrected age did the predicted response occur. this apparent early ineffectiveness of succimer could be due to absorptive failure from the infant’s immature gut. furthermore, succi- mer is a prodrug that requires cysteine for activation. it is unclear whether or not this enzymatic activation process is mature in the premature infant. public health considerations while lead was the principal concern in this case, mercury was also detected in some of the tablets (box ). weekly ingestion of one of the tablets containing �g would exceed the australian average weekly mercury intake by at least fourfold. mercury poisoning was of secondary con- cern in the current circumstance, and chelation therapy was expected to remove the mercury along with the lead. this does, however, illustrate that herbal medicines may contain more than one potentially toxic component. in many countries, including australia, it is legal to import herbal remedies for personal use, although some restrictions apply. in addition, testing programs for con- taminants in herbal remedies are virtually non-existent. acute lead poisoning has been reported from traditional and herbal medicines and cosmetics obtained from india, - the middle east, - mexico and asia. - this should be a matter of grave concern to patients who take such remedies, and to health professionals and health authorities in these countries and in australia. acknowledgements the authors gratefully acknowledge dr stephen rothenberg, of charles drew univer- sity, los angeles, for discussion of fetal and maternal blood lead levels; mike stevenson, of berri/barmera council, for the environmental audit; sam mangas, for assistance with the source investigation; and bill dollman and dr brian priestly, for discussions on the regulation of herbal remedies in australia. competing interests none identified. references . fuhrman bp. the acutely ill child. in: nelson’s essentials of paediatrics. nd ed. philadelphia: wb saunders, : - . . klaassen cd. heavy metals and heavy-metal antagonists. in: goodman and gilman’s the pharmacological basis of therapeutics. th ed. new york: mcgraw hill, : - . . statement issued by th session of the national health and medical research council, june . revision of the australian guidelines for lead in blood and lead in ambient air. available at: <http://www.health.gov.au/nhmrc/publications/ pdf/ th.pdf>, pp - . accessed july . . parfitt k, editor. martindale: the complete drug reference. nd ed. london: pharmaceutical press, . . treatment guidelines for lead exposure in children (re ). american academy of pediatrics committee on drugs. pediatrics ; : - . . graziano jh, lolacono nj, meyer p. dose-response study of oral , - dimercaptosuccinic acid in children with elevated blood lead concentrations. j pediatr ; : - . . graziano jh, lolacono nj, moulton t, et al. controlled study of , -dimercapto- succinic acid for management of childhood lead intoxication. j pediatr ; : - . . th australian total diet survey. a total diet survey of pesticide residues and contaminants. canberra: australian new zealand food authority, . . howard h. heavy metal poisoning. in: harrison’s principles of internal medicine. th ed. new york: mcgraw-hill; : - . . timpo ae, amin js, casalino mb, yuceoglu am. congenital lead intoxication. j pediatr ; : - . . knecht cd, crabtree j, katherman a. clinical, clinicopathologic, and electroen- cephalographic features of lead poisoning in dogs. j am vet med assoc ; : - . . goyer ra. transplacental transport of lead. environ health perspect ; : - . . marcus ah. multicompartment kinetic model for lead. iii. lead in blood plasma and erythrocytes. environ res ; : - . . therapeutic goods administration. australian department of health and age- ing. . available at: <http://www.health.gov.au/tga/cm/cm.htm>. accessed july . . mcelvaine md, harder em, johnson l, et al. lead poisoning from the use of indian folk medicines. jama ; : - . . dunbabin dw, tallis ga, popplewell py, lee ra. lead poisoning from indian herbal medicine (ayurveda). med j aust ; : - . . van vonderen mga, klinkenberg-knol ec, craanen me, et al. severe gastroin- testinal symptoms due to lead poisoning from indian traditional medicine. am j gastroenterol ; : - . . shaltout a, yaish sa, fernando n. lead encephalopathy in infants in kuwait. ann trop paed ; : - . . rahman h, al khayat a, menon n. lead poisoning in infancy – unusual causes in the u.a.e. ann trop paediatr ; : - . . lead poisoning associated with use of traditional ethnic remedies — california, – . mmwr morb mortal wkly rep ; : - . . adult lead poisoning from an asian remedy for menstrual cramps — connecti- cut, . mmwr morb mortal wkly rep ; : - . (received jan , accepted jun ) ❏ knockout of slc a causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, cns malformations, and anemia marjorie j. lindhurst*†, giuseppe fiermonte‡, shiwei song§, eduard struys¶, francesco de leonardis‡, pamela l. schwartzberg*, amy chen*, alessandra castegna‡, nanda verhoeven¶, christopher k. mathews§, ferdinando palmieri‡�, and leslie g. biesecker* *national human genome research institute, national institutes of health, bethesda, md ; ‡department of pharmaco-biology, laboratory of biochemistry and molecular biology, university of bari, bari, italy; §department of biochemistry and biophysics, oregon state university, corvallis, or ; and ¶department of clinical chemistry, vu university medical center, hv, amsterdam, the netherlands communicated by victor a. mckusick, johns hopkins university school of medicine, baltimore, md, september , (received for review may , ) slc a mutations cause amish lethal microcephaly (mcpha), which markedly retards brain development and leads to �-keto- glutaric aciduria. previous data suggested that slc a , also called dnc, is a mitochondrial deoxyribonucleotide transporter. we generated a knockout mouse model of slc a . these animals had % prenatal lethality by embryonic day . affected em- bryos at embryonic day . have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated �-ketoglutarate in the amniotic fluid. we found that these animals have normal mitochondrial ribo- and deoxyribo- nucleoside triphosphate levels, suggesting that transport of these molecules is not the primary role of slc a . we identified thiamine pyrophosphate (thpp) transport as a candidate function of slc a through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein. the mitochondria of slc a �/� and mcpha cells have undetect- able and markedly reduced thpp content, respectively. the reduc- tion of thpp levels causes dysfunction of the �-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in mcpha and suggests that mitochondrial thpp transport is important for cns development. development � mitochondrial transporter � mouse model � thiamine pyrophosphate deficiency � neural tube defect microcephaly can be caused by many genetic and environ-mental factors. amish lethal microcephaly (mcpha) [online mendelian inheritance in man (omim) ; www. ncbi.nlm.nih.gov�entrez�query.fcgi?db�omim] is inherited in an autosomal recessive pattern and includes severe congenital microcephaly (head circumference � sd below the mean), elevated levels ( – times) of �-ketoglutaric acid (akguria), and death, usually by months of age ( ). previously, we found a causative mutation in patients with mcpha (c. g�c which predicts p.g a) in slc a , which encodes a mitochondrial membrane carrier superfamily member (omim ) ( ). the slc a gene also is called dnc, which stands for deoxynucleotide carrier (genbank acces- sion no. nm� ) ( ). slc a contains three typical mito- chondrial inner mitochondrial membrane carrier motifs ( ). the p.g a substitution was absent in control chromosomes. in addition, this glycine was conserved in of sequences in the conserved domain database (www.ncbi.nlm.nih.gov�structure� cdd�cdd.shtml). functional data showed that slc a trans- ported deoxynucleotides across membranes in an in vitro assay system ( ). finally, we showed that g a slc a had no activity in this assay ( ), indicating that mcpha is associated with a slc a loss-of-function mutation. these data did not prove that slc a mutations caused mcpha, because it was possible that there was a mutation in another gene on the mutant amish mcpha haplotype. although it has been demonstrated that slc a transported ribo- and deoxyribonucleotides in vitro, it was not known whether that was its primary function. therefore, we designed experiments to address the issue of causation and the function of slc a . the results show that knockout of slc a causes cns defects and akguria and that the pathogenic mech- anism of slc a mutations in mcpha may be a decrease in mitochondrial thiamine pyrophosphate (thpp) levels. results the strategy used to generate slc a �/� mice is shown in fig. . homologous recombination of the targeting vector replaced exons – of slc a with a neor gene. if this transcript was stable, the slc a protein would be missing amino acids – and should be nonfunctional. southern blot analysis showed that of es cell lines underwent proper recombination (data not shown), and was chosen for blastocyst injection. three chimeric mice showed germ-line transmission of the mutant allele. the chimeras were mated to black swiss and svev female mice. no genetic background differences were noted. slc a �/� animals appeared normal. no homozygous mutant pups were found among pups from slc a �/� intercrosses, suggesting embryonic lethality. embryos were collected from timed matings, examined, and genotyped. the optimal timing for embryo evaluations was embryonic day (e) . ; however, mutants were recognizable at e . . no slc a �/� embryos were found after e . . most e . mutants were necrotic, indicating death by e . fewer than one-quarter of the e . embryos were ���, suggest- ing earlier mortality. the open neural tube was the most striking feature of the slc a �/� embryos (fig. and fig. , which is published as supporting information on the pnas web site). the exencephaly varied but commonly involved the entire brain. the edges of the open neural tube were ruff led and fused at the hindbrain (figs. and ). in the trunk, the fused neural tube was convoluted, suggesting irregular relative growth rates of neural to nonneural author contributions: m.j.l. and g.f. contributed equally to this work. m.j.l., g.f., e.s., p.l.s., n.v., f.p., and l.g.b. designed research; m.j.l., g.f., s.s., e.s., f.d.l., a. chen, and a. castegna performed research; m.j.l., g.f., f.d.l., a. chen, a. castegna, and c.k.m. con- tributed new reagents�analytic tools; m.j.l., g.f., s.s., e.s., f.d.l., p.l.s., a. castegna, n.v., c.k.m., f.p., and l.g.b. analyzed data; and m.j.l., g.f., s.s., f.p., and l.g.b. wrote the paper. the authors declare no conflict of interest. freely available online through the pnas open access option. abbreviations: mcpha, amish lethal microcephaly; en, embryonic day n; akg, �-ketoglu- tarate; thpp, thiamine pyrophosphate; akguria, �-ketoglutaric acid; mef, murine embry- onic fibroblast; thmp, thiamine monophosphate; kgdh, akg dehydrogenase; pdh, pyru- vate dehydrogenase. †to whom correspondence may be addressed at: national institutes of health, building , room c , bethesda, md - . e-mail: marjr@mail.nih.gov. �to whom correspondence may be addressed at: dipartimento farmaco-biologico, univer- sità di bari, via e. orabona , bari, italy. e-mail: fpalm@farmbiol.uniba.it. © by the national academy of sciences of the usa www.pnas.org�cgi�doi� . �pnas. pnas � october , � vol. � no. � – g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , tissue. mutant embryos were smaller than unaffected littermates (fig. a and b). no malformations were noted in the trunk or tail region. some slc a �/� embryos apparently arrested earlier in development, resulting in amorphic embryos, but this was not further evaluated (fig. d). serial sections showed that loss of slc a primarily affects anterior structures. neural ectoderm was present on the outer edges of the forebrain and midbrain regions with no evidence of ventricle formation (fig. , which is published as supporting information on the pnas web site). coronal sections show that the neural tube was not fully fused at the level of the heart (fig. and fig. , which is published as supporting information on the pnas web site). we conclude that a slc a loss-of-function mutation causes severe midgestation cns defects and, occasionally, earlier developmental arrest. sections through the heart (fig. ) showed very few erythrocytes in the slc a �/� embryos. blood smears from the yolk sacs of e . embryos showed that unaffected embryos had many normal erythrocytes, whereas mutants had few erythrocytes and few of those were normal (fig. , which is published as supporting information on the pnas web site). to determine whether the mutants had elevated �-ketoglutarate (akg) as found in humans with mcpha, we devised techniques to sample amniotic f luid from midgestation mouse embryos. am- niotic f luid akg was elevated � in mutants, compared with controls (fig. ). this finding shows that the two major findings in b bn e eb nn n bee m bm b nn n ee bm ns/n m e/msbe ns m e ns/n m e/msb e ʼ ʼ endogenous knockout construct homologous recombinant neor neor neo fig. . diagram of the slc a knockout strategy. a . -kb ecori (e)�bamhi (b) fragment ( �) and a . -kb spei (s)�ecori ( �) fragment were cloned into ppnt-far. the construct is missing exons – . transfected es cell clones were digested with either bamhi (b) ( � and neo probes) or nhei (n) ( � probe) to test for homologous recombination. thick lines represent mouse sequence, thin lines represent vector sequence, and the arrow represents the neor cassette. the stippled boxes represent the southern blot analysis probes. black numbered boxes are exons (the translation start site is in exon ). x a x b x c x d fig. . examples of unaffected and mutant embryos at e . . (a) unaffected slc a �/� embryo. (b) slc a �/� embryo taken at the same magnification as in a. (c) higher magnification of embryo in b. note the ruffling along the open neural tube (yellow arrowheads), point of fusion of neural tube (green arrowhead), and lack of color in heart and blood vessels. (d) example of an amorphous embryo arrested early in development. slc a +/+ slc a -/- fig. . coronal sections of unaffected (left) and mutant (right) e . embryos. these images are higher magnifications of the last of the serial sections shown in fig. ( and , respectively). yellow arrowheads mark the erythrocytes in the heart and vessels in the section from the unaffected embryo. the rostral neural tube is not fused (green arrows) but is fused caudally (red arrow) in the mutant embryo. � www.pnas.org�cgi�doi� . �pnas. lindhurst et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , human mcpha, cns defects and akguria, are caused by a knockout mutation of slc a . to study the cellular phenotype, murine embryonic fibroblasts (mefs) were derived from e . embryos and stained with anti- bodies to mitochondrial hsp and subunit i of the cytochrome c oxidase complex to determine whether there were any gross changes in mitochondrial numbers. no differences were observed (data not shown). as slc a was known to transport deoxynucleotides ( ), we hypothesized that the knockout would disrupt the mitochondrial nucleotide pool and cause depletion or deletion of mitochondrial dna. we used quantitative pcr to detect mitochondrial dna depletion in whole embryos, yolk sacs, and mefs. no consistent differences in the ratio of mitochondrial to nuclear dna were found (data not shown). we also used southern blot analysis to screen for mitochondrial deletions, but the results were normal (data not shown). we conclude that there was no evidence for mitochondrial genomic abnormalities in slc a �/� animals. we then speculated that disruption of nucleotide transport might alter mitochondrial function without affecting dna synthesis or integrity. to test this idea, mitochondria from wild-type and mutant mefs were assayed directly for dntp levels (fig. ). no significant differences were found in the datp, dctp, and dgtp levels. surprisingly, the dttp level was slightly higher in the mutant mitochondria. similar results were obtained with mitochondria isolated from human lymphoblast cell cultures derived from two children with mcpha and two controls. slc a previously has been shown to transport ribonucleotides ( ), so measurements of mitochondrial rntp pools from these lymphoblasts also were performed (fig. , which is published as supporting information on the pnas web site). because of the limited sensitivity of this assay, only levels of atp and gtp were obtained. again, no differences in the level of these nucleotides in patient samples compared with control samples were seen. we conclude that loss-of-function mutations in slc a do not disrupt mitochon- drial deoxynucleotide pools in mice or in humans. while this study was underway, a novel mitochondrial trans- porter, tpc p, was discovered in saccharomyces cerevisiae ( ). this protein transports thpp into the mitochondria in exchange for thiamine monophosphate (thmp) and is the yeast protein most similar to human slc a . because of the similarity of slc a to this yeast protein ( % amino acid identity and % similarity), the ability of slc a to transport thpp and thmp was tested by using phospholipid vesicles reconstituted with recom- binant wild-type and g a mutant human slc a . because slc a catalyzes only exchange and not uniport of substrates ( ), and because thpp and thmp are not available in radioactive form, their transport was tested by measuring the uptake of �- s-datp into liposomes reconstituted with slc a that had been preloaded with thpp or thmp (fig. ). with the wild-type protein, similar �- s-datp uptake was found with internal thpp as with internal datp. �- s-datp also exchanged with internal − α ) l/l o m µ( et ar at ul g ot e k unaffected affected (slc a +/+) (slc a +/-) (slc a -/-) genotype fig. . akg levels in amniotic fluid from embryos at e . . akg was measured by using tandem ms and an internal standard. five embryos were slc a �/� (triangles), embryos were slc a �/� (squares), and embryos were slc a �/� (circles). the samples represented by each color were measured during the same experiment. the mean values were . � . �mol�liter for the unaffected and . � . �mol�liter for the mutants (p � . mann– whitney). the data also were dichotomized by using the mean of the entire data set ( . ) and tested with fisher’s exact test (p � . ). the t test could not be used because the data were not normally distributed. dttp datp dctp dgtp ni et or p l ai r d n o h c oti m g m/ p t n d l o m p wild-type mef mutant mef control lymphoblasts mcpha lymphoblasts fig. . mitochondrial dntp levels in wild-type and mutant mefs and human lymphoblasts. for the mefs, mitochondria were isolated from a wild-type (blue bars) or mutant (red bars) culture on two occasions. dntp levels were measured in duplicate and averaged (�sd). lymphoblast nucleotide levels were deter- mined from mitochondria isolated from two control (yellow bars) or patient (green bars) cultures and averaged (�sd) according to affection status. fig. . transport assays of wild-type and mutant slc a . proteoliposomes reconstituted with recombinant wild-type (gray bars) and g a mutant (white bars) slc a were preloaded internally with �m each datp, thpp, thmp, or thiamine. transport was started with �m �- s-datp and terminated at min. the values are means �sd of at least three experiments. lindhurst et al. pnas � october , � vol. � no. � g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , thmp, although to a lower extent. in contrast, thiamine was not exchanged. the g a mutant diminished transport by � % compared with wild type. partial reduction of the g a mutant activity, as shown in fig. , was observed when using proteins overexpressed in escherichia coli at °c (see materials and meth- ods), and a complete inhibition was observed when expression was done at °c (data not shown and ref. ). to test whether the loss of slc a caused changes in levels of thpp and thmp, mitochondrial and cytosolic fractions from wild-type and mutant human and mouse cells were isolated. thpp and thmp levels were assayed by ms and compared with total cellular protein (table ). no thpp or thmp was detected in the mitochondrial fraction of slc a �/� mefs. in the cytosolic fraction, a . � and . � increase in thpp and thmp compared with wild-type mefs was seen, respectively. in the mitochondrial fraction of human lymphoblasts, thpp and thmp levels were decreased � and �, respectively, compared with controls. the thpp and thmp were increased . � and . � in the cytosol compared with controls. to assess the consequences of lack of thpp in the mitochondria, the activities of the thpp-requiring enzyme complexes akg dehydrogenase (kgdh) and pyruvate dehydrogenase (pdh) from mutant and wild-type mitochondria were assayed directly in an in vitro system. kdgh and pdh complex activities were severely reduced in mutant fibroblasts (fig. a and c). addition of thpp to the assay resulted in full recovery of activity. pdh complex activity also was measured indirectly by acetylcarnitine formation, which confirmed the results of the direct measurement (data not shown). kgdh and pdh complex activities in patient mitochon- dria also were decreased but less severely (fig. b and d). the lactate level in the medium was measured in both mefs and lymphoblasts. in mutant mefs, a . � increase and in patient lymphoblasts, a . � increase in lactate was seen (fig. , which is published as supporting information on the pnas web site). we conclude that slc a transports thpp. mitochondrial thpp levels were undetectable in mutant cells from slc a �/� mice and markedly reduced in human cells with a point mutation. the data suggest that reduced mitochondrial levels of thpp contribute to the metabolic abnormalities in human mcpha. discussion these data show that slc a is essential for murine development. all mice homozygous for this knockout mutation die before e . the homozygous animals are growth-retarded and have abnormal cns development with an open and convoluted neural tube. the animals also manifest erythropoietic failure, being nearly devoid of erythrocytes at e . . in addition, the akg levels in the amniotic f luid in of affected embryos were elevated in comparison to heterozygous and homozygous wild-type littermates. that the amniotic f luid akg elevation is less dramatic than the akguria in the mcpha patients is expected because the mouse embryos die before kidney formation and therefore do not concentrate the metabolites. finally, a subset of embryos appears to undergo an earlier gestational demise because of an unknown mechanism. previous data suggested that the function of slc a was to transport deoxynucleotides across the inner mitochondrial mem- brane ( ). we hypothesized that a loss of this function would lead to abnormally low levels of mitochondrial matrix deoxyribonucle- otides and depletion or deletions in mitochondrial dna ( ), with abnormal mitochondria and reduced respiratory function ( , ). however, there were no gross changes in the number or morphol- ogy of the mitochondria, nor did we detect mitochondrial deletions in the knockout mice, as would be expected from abnormal nucleotide levels. although slc a may be able to transport dntps in vivo, this function is not critical because loss of this gene does not alter dntp levels in mitochondria of mutant cells. that the physiological role of slc a is something other than dntp transport is further supported by a recent article by lam et al. ( ). they showed that slc a is not associated with mitochondrial dna depletion caused by dideoxynucleoside analogs used in antiretroviral therapy or with mitochondrial dntp uptake. table . levels of thpp and thmp mitochondria postmitochondrial supernatant cell type thpp* thmp* thpp* thmp* wild-type mefs . � . . � . . � . . � . mutant mefs not detectable not detectable . � . † . � . † control lymphoblasts . � . . � . . � . . � . patient lymphoblasts . � . † . � . † . � . † . � . † data represent the mean � sem of three independent experiments. *data are presented as ng�mg of total cellular protein. †values are significantly different from control (p 	 . ). fig. . kgdh and pdh complex assays. the kgdh (a and b) and pdh (c and d) complex activities were assayed by measuring nadh formed in the pres- ence or absence of thpp. (a and c) wild-type (white bar) and knockout (gray bar) mefs. (b and d) human lymphoblasts from a normal individual (white bar) and a mcpha patient (cross-hatched bar). the data represent the mean � sem of three independent experiments in duplicate. anova analysis (two-way) was applied to compare the two groups, with and without thpp, and ** denotes significance compared with control (p 	 . ). � www.pnas.org�cgi�doi� . �pnas. lindhurst et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , the recent discovery of the tpc p gene ( ) in yeast (swiss-prot entry p , ygr w) suggested another function for slc a . tpc p transports thpp produced in the cytosol into the mitochondria in exchange for mitochondrially generated thmp, which then is metabolized and converted back to thpp ( ). in an in vitro exchange assay, we found that a protein containing the g a mutation reduced thpp and thmp transport by � % compared with wild-type protein. mitochondrial thpp was not detectable in mefs from knockout mice and markedly reduced in lymphoblasts from patients with mcpha. we also showed that the mitochondrial thpp-dependent enzyme complex pdh and kgdh activities were lower in slc a mutant cells than in controls. the addition of thpp to the pdh and kgdh complex assay mixtures restored full activity in both the mouse and human cell lines. we recognized differences comparing the murine slc a knockout to the human mcpha phenotype. the embryonic lethality of the slc a �/� animals illustrates the increased severity of the mouse model. humans with mcpha are viable and appear to be born at ratios (to unaffected children) that show that the amish slc a mutation that causes mcpha does not cause pregnancy loss at an appreciable rate. the neural tube defect and arrest of brain formation in the mouse as compared with the microcephaly in mcpha patients also illustrates the increased severity in the mouse model. the murine erythropoietic defect does not mirror a recognized aspect of human mcpha [patients with mcpha have normal blood counts (r. i. kelley, personal communication)]. a likely explanation for the differences in the mouse and human phenotypes is that the human point mutation retains some thpp transport activity, whereas the murine null mutation does not. that the mitochondrial thpp levels and activities of mitochondrial thpp-requiring enzymes were less severely reduced in cells from mcpha patients than in knockout mefs, and that the g a mutant protein retained some activity when expressed at °c and not at °c, supports this hypothesis. however, alternative expla- nations must be considered. first, another transporter may have overlapping functions with slc a , and there may be differ- ences in the functional overlap in the two species. it also is possible that slc a has unrecognized functions in addition to nucleo- tide and thpp transport, and that these functions are lost in the knockout mouse but retained in the human mcpha mutation. finally, it is possible that the mouse model is not ideal for these experiments. to address these issues, future studies will need to involve knockin constructs that model the human mutation. oxidative phosphorylation is important in early embryogenesis ( – ). blocking the tricarboxylic acid cycle by targeted disruption of the murine dihydrolipoamide dehydrogenase (dld) gene [en- coding the e component of pdh, kgdh, and the branched-chain �-ketoacid dehydrogenase (bckdh) complexes], or the gene for pdh e � subunit (pdha ), causes developmental arrest and le- thality by e . or e . , respectively ( , ). we speculate that the growth retardation of the slc a �/� embryos is caused by de- creased f lux through the pdh and kgdh complex reactions. the structural brain malformations of slc a �/� embryos support the hypothesis that the developing brain has a higher need for oxidative metabolism than other organs do ( ). in addition to the reduction of energy produced by oxidative metabolism in the slc a �/� embryos and mcpha patients caused by reduced pdh and kgdh complex activities, reduced krebs cycle intermediates also could play a role in the phenotype of the mutants. in fact, the lower level of succinyl-coa in the mitochondria, a product of the kgdh complex, may be the cause of erythropoietic failure because heme biosynthesis requires condensation of succinyl-coa and glycine. heme deficiency caused by disruption of the murine gene for this enzyme in erythroid cells (alas-e) caused anemia and death by e . ( ). in contrast to the slc a �/� mutant, the alas-e-null embryos had some immature primitive nonhemoglobinized eryth- rocytes, suggesting that reduction of heme synthesis cannot solely be responsible for the lack of red cell development in the slc a �/� embryos. this issue will need to be addressed by future studies. in summary, we have made a mouse model for human amish microcephaly by knocking out the murine slc a gene, an ortholog of human slc a or dnc. the mutant has a cns and metabolic phenotype that is similar to the human disease. loss of murine slc a function does not cause mitochondrial dntp pool reduction, as previously predicted, but instead reduces mito- chondrial thpp. the reduction in thpp inhibits the activity of key metabolic enzymes, including the kgdh complex, which explains the akguria in mcpha and emphasizes the role of oxidative metabolism in early embryogenesis. materials and methods generation of slc a �/� animals. the ppntdnc targeting con- struct (fig. ) was linearized with noti. tc cells ( � ) were transfected with �g of linear dna in ml of pbs with a gene pulser (bio-rad, hercules, ca). cells were changed at h to medium with g and -iodo- �-f luoro- �deoxy- -�-d-arabino- furanosyl-uracil (fiau). seven days later, colonies were selected and expanded for freezing and dna analysis. approximately targeted es cells were injected into each c b �j blastocyst. the blastocysts were implanted into . -days postcoitum pseudopreg- nant nih swiss–webster mice. chimeras were mated to black swiss mice at weeks of age to assess germ-line transmission. embryo procedures. embryos were harvested at e . –e . , and yolk sacs were collected for genotyping. most embryos were fixed in % paraformaldehyde overnight at °c, transferred to % methanol, and stored at � °c. paraffin embedding, sectioning, and hematoxylin and eosin staining of embryos were performed by histoserve (germantown, md). to collect fetal blood, an embryo in its yolk sac was put in a -well culture dish with ml of pbs. the yolk sac and amnion were removed, allowing the yolk sac vessel blood to spill into the pbs. cells were centrifuged, spread on slides, stained with wright– giemsa, and examined by microscopy. to collect amniotic f luid, e . embryos were dissected with the yolk sac intact. embryos were transferred to a dry dish and rolled to remove pbs to avoid diluting the amniotic f luid. amniotic f luid was collected with a -ml syringe and a -gauge needle. to minimize clogging, the needle was inserted into the amniotic cavity with the opening away from the embryo, and f luid was slowly drawn into the syringe. cell culture. mefs were collected from e . embryos, and yolk sacs were used for genotyping. embryos were minced with forceps and put in a -well cell dish with dmem with % fbs, penicillin�streptomycin, and glutamine. adherent cells were ex- panded by using standard techniques. dna was isolated periodi- cally and genotyped. lymphoblasts from two mcpha patients (provided by r. i. kelley, kennedy krieger institute, baltimore, md) were grown in rpmi medium with % fbs, penicillin� streptomycin, and glutamine. akg assay. aliquots ( – �l) of mouse amniotic f luid were pipetted in a glass tube, followed by . ml of water and . nmol of h -akg (eurisotop, saint-aubin cedex, france), for an internal standard. akg was derivatized by adding �l of m hcl and �l of mg�ml pentaf luorobenzylhydroxylamine for min at °c. the derivatives were extracted by using a solid-phase extrac- tion cartridge and eluted with . ml of methanol in an hplc vial. ten microliters of eluate was injected on a liquid chromatography- tandem ms system (api ; applied biosystems, foster city, ca) with a c analytical column, and elution was done with a binary linear gradient. the tandem ms was used in the negative lindhurst et al. pnas � october , � vol. � no. � g en et ic s d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , mode, and akg and the internal standards were measured in the multiple-reaction mode. nucleotide levels. mitochondrial nucleotide pools were prepared as per song et al. ( ). brief ly, mefs were harvested in -mm dishes by trypsinization. human lymphoblasts were collected from suspension culture in five t f lasks. mitochondrial isolation and preparation of extracts were as described in ref. . rntp pools were determined by hplc using a hewlett–packard system with -nm detection. extracts were injected into an altex partisil sax ion-exchange column equilibrated in mm nh po (ph . ). rntps were eluted with a nh po gradient buffer ranging from . m to . m (ph . ) at . ml�min. the ribonucleotides were identified by retention times and quantitated by peak area in comparison to authentic standards. overexpression and purification of slc a and slc a mutant. wild-type and g a mutant slc a were produced as inclu- sion bodies in e. coli bl (de ) as described in ref. , except that induction was carried out for h at °c. inclusion bodies were purified as described in ref. , except that the triton buffer contained hepes, ph . (instead of pipes). reconstitution of slc a proteins into liposomes and transport measurements. the recombinant proteins were reconstituted into liposomes in the presence of substrates, as described in ref. . external substrate was removed from proteoliposomes on seph- adex g- columns and preequilibrated with mm nacl and mm pipes at ph . . transport was started by adding s-datp (perkinelmer, wellesley, ma) to proteoliposomes and terminated by addition of . mm p-chloromercuribenzene sulfonate (the ‘‘inhibitor-stop’’ method; see ref. ). the amount of wild-type and slc a mutant proteins incorporated into liposomes varied from % to % of the protein added to the reconstitution mixture. thpp and thmp determination by ms. thpp and thmp extraction from mitochondria and postmitochondrial supernatant was per- formed essentially as described by pontarin et al. ( ). cells were suspended in . m mannitol� . m sucrose� . mm egta� . % bsa� mm tris�hcl (ph . ) and homogenized and centrifuged as described in ref. . the mitochondrial extract was lyophilized overnight and frozen. the postmitochondrial superna- tant was treated with % methanol, centrifuged, lyophilized overnight, and frozen. the samples were dissolved in water, filtered, and subjected to liquid chromatography-ms analysis. a quattro premier mass spectrometer with an alliance hplc system (waters, milford, ma) was used for electrospray- ionization liquid chromatography-tandem ms analysis of thpp and thmp in the positive-ion mode. the multiple reaction monitoring transition monitored for thpp was m�z . � . and for thmp was m�z . � . . chromatographic resolution of thpp and thmp was achieved by using a hypercarb porous graphitic carbon column ( . � mm, -�m particle size; thermo electron, waltham, ma) eluted with a linear gradient from % water with . % tfa (initial phase) to % water containing . % tfa� % acetonitrile. hplc f low was ml�min and split to �l�min before entering the ms source. calibration curves were set at four concentrations by using standards processed under the same conditions as the samples. the best fit was determined by using regression analysis of the peak analyte area. pdh and kgdh complex assays. mitochondria were isolated from human lymphoblasts and murine fibroblasts by using a kit (pierce, rockford, il) with the halt protease inhibitor mixture (pierce, product no. ) according to the manufacturer’s instructions. pdh and kgdh complex activities were assayed essentially as by munujos et al. ( ). lymphoblast and fibroblast mitochondria were preincubated in mm tris�hcl at ph . for min at °c, solubilized with . % (wt�vol) triton x- , and centrifuged at , � g for min. seventy microliters of supernatant ( – �g of protein) was added to �l of a mixture containing mm tris�hcl at ph . , mm coa, mm nad�, and �m rotenone with or without mm thpp. the assay was started with mm akg or mm pyruvate, and the formation of nadh followed at nm. lactate release in extracellular medium. a total of � human lymphoblasts were grown in ml of medium for h, then spun down at , � g for min. then, � mouse embryo fibroblasts were grown in -cm f lasks for h (close to conf lu- ence); the medium was collected and centrifuged to remove cell debris. the lactate in the supernatants from both cell types was assayed as per gutmann and wahlefeld ( ). we thank g. elliot, c. rivas, r. nussbaum, d. bodine, l. wheeler, w. pavan, c. yang, s. loftus, d. larson, j. fekecs, and r. i. kelley for their technical expertise, advice, and encouragement. this work was sup- ported by funds from intramural research program of the national human genome research institute grant ls- from the army research office (to c.k.m.), the ministero dell’università e della ricerca, ministero della salute, ministero dell’università e della ricerca grant l. � (cluster ), and european commission contract lshm-ct- - . . kelley ri, robinson d, puffenberger eg, strauss ka, morton dh ( ) am j med genet : – . . rosenberg mj, agarwala r, bouffard g, davis j, fiermonte g, hilliard ms, koch t, kalikin lm, makalowska i, morton dh, et al. ( ) nat genet : – . . dolce v, fiermonte g, runswick mj, palmieri f, walker je ( ) proc natl acad sci usa : – . . palmieri f ( ) pflügers arch : – . . marobbio cm, vozza a, harding m, bisaccia f, palmieri f, walker je ( ) embo j : – . . song s, wheeler lj, mathews ck ( ) j biol chem : – . . holmuhamedov e, jahangir a, bienengraeber m, lewis ld, terzic a ( ) mitochondrion : – . . miranda s, foncea r, guerrero j, leighton f ( ) biochem biophys res commun : – . . lam w, chen c, ruan s, leung ch, cheng yc ( ) mol pharmacol : – . . johnson mt, mahmood s, patel ms ( ) j biol chem : – . . hance n, ekstrand mi, trifunov ic a ( ) hum mol genet : – . . larsson ng, wang j, wilhelmsson h, oldfors a, rustin p, lewandoski m, barsh gs, clayton da ( ) nat genet : – . . li k, li y, shelton jm, richardson ja, spencer e, chen zj, wang x, williams rs ( ) cell : – . . johnson mt, yang hs, magnuson t, patel ms ( ) proc natl acad sci usa : – . . johnson mt, mahmood s, hyatt sl, yang hs, soloway pd, hanson rw, patel ms ( ) mol genet metab : – . . brown gk, otero lj, legris m, brown rm ( ) j med genet : – . . nakajima o, takahashi s, harigae h, furuyama k, hayashi n, sassa s, yamamoto m ( ) embo j : – . . fiermonte g, walker je, palmieri f ( ) biochem j : – . . palmieri f, indiveri c, bisaccia f, iacobazzi v ( ) methods enzymol : – . . pontarin g, gallinaro l, ferraro p, reichard p, bianchi v ( ) proc natl acad sci usa : – . . munujos p, coll-canti j, beleta j, gonzalez-sastre f, gella fj ( ) clin chim acta : – . . gutmann i, wahlefeld a ( ) in methods in enzymatic analysis, ed bergmeyer hu (academic, new york), pp – . � www.pnas.org�cgi�doi� . �pnas. lindhurst et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , clinical and genetic heterogeneity in nemaline myopathy--a disease of skeletal muscle thin filaments. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /s - ( ) - corpus id: clinical and genetic heterogeneity in nemaline myopathy--a disease of skeletal muscle thin filaments. @article{sanoudou clinicalag, title={clinical and genetic heterogeneity in nemaline myopathy--a disease of skeletal muscle thin filaments.}, author={d. sanoudou and a. beggs}, journal={trends in molecular medicine}, year={ }, volume={ }, pages={ - } } 	d. sanoudou, a. beggs 	published 	biology, medicine 	trends in molecular medicine the term nemaline myopathy (nm) encompasses a heterogeneous group of disorders of primary skeletal muscle weakness characterized by the presence of nemaline rods in muscles of affected individuals. disease severity is variable and unpredictable, with prognosis ranging from neonatal death to almost normal motor function. recent advances in the identification of nm disease genes demonstrate that nm is a disease of the skeletal muscle sarcomere and, in particular, of the thin filaments. these… expand view on pubmed childrenshospital.org save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all figures, tables, and topics from this paper 	 table 	 figure 	 figure 	 figure 	myopathy 	myopathies, nemaline 	muscle weakness 	cessation of life 	hereditary diseases 	sarcomeres 	rod photoreceptors citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency heterogeneity of nemaline myopathy cases with skeletal muscle α‐actin gene mutations 	p. agrawal, c. d. strickland, + authors a. beggs 	biology 	 	 	view excerpts, cites background save alert research feed molecular diagnosis of myopathies. 	andrew gómez-vargas, s. baker 	medicine 	rheumatic diseases clinics of north america 	 	 save alert research feed craniofacial manifestations in severe nemaline myopathy 	yunfeng xue, p. magoulas, j. wirthlin, e. buchanan 	medicine 	the journal of craniofacial surgery 	 	 	view excerpts, cites background save alert research feed molecular classification of nemaline myopathies: “nontyping” specimens exhibit unique patterns of gene expression 	d. sanoudou, leslie a frieden, j. n. haslett, a. kho, a. beggs 	biology, medicine 	neurobiology of disease 	 	 	view excerpt, cites background save alert research feed sarcomere dysfunction in nemaline myopathy 	j. d. de winter, c. ottenheijm 	biology, medicine 	journal of neuromuscular diseases 	 	 	pdf 	view excerpt, cites background save alert research feed [nemaline rod myopathy treated with l-tyrosine to relieve symptoms in a neonate]. 	s. Şahin, m. oncel, d. bidev, n. okur, beril talim, s. oguz 	medicine 	archivos argentinos de pediatria 	 	pdf save alert research feed clinical and pathologic aspects of congenital myopathies 	i. nonaka 	biology 	 	 save alert research feed autosomal dominant nemaline myopathy: a new phenotype unlinked to previously known genetic loci 	p. jeannet, l. mittaz, m. dunand, j. lobrinus, t. kuntzer 	biology, medicine 	neuromuscular disorders 	 	 	highly influenced 	view excerpts, cites background save alert research feed clinical and pathologic aspects of congenitalmyopathies 	i. nonaka 	 	pdf save alert research feed clinical and pathological features of patients with nemaline myopathy. 	x. yin, c. q. pu, q. wang, j. liu, y. l. mao 	biology, medicine 	molecular medicine reports 	 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency nemaline myopathy caused by mutations in the muscle α-skeletal-actin gene 	b. ilkovski, s. cooper, + authors k. north 	biology 	 	 	 save alert research feed adult-onset nemaline myopathy: a case report and review of the literature. 	k. gyure, r. prayson, m. estes 	medicine 	archives of pathology & laboratory medicine 	 	 save alert research feed nemaline myopathy: comparative muscle histochemistry in the severe neonatal, moderate congenital, and adult-onset forms. 	c. shimomura, i. nonaka 	biology, medicine 	pediatric neurology 	 	 save alert research feed nemaline myopathy : current concepts 	k. north, c. wallgren-pettersson 	 	pdf save alert research feed nemaline myopathy appearing in adults as cardiomyopathy. a clinicopathologic study. 	c. meier, w. voellmy, m. gertsch, a. zimmermann, j. geissbühler 	medicine 	archives of neurology 	 	 save alert research feed a mutation in alpha-tropomyosin(slow) affects muscle strength, maturation and hypertrophy in a mouse model for nemaline myopathy. 	m. corbett, c. s. robinson, + authors e. hardeman 	biology, medicine 	human molecular genetics 	 	 	pdf save alert research feed intranuclear rods in severe congenital nemaline myopathy 	z. rifai, a. m. kazee, c. kamp, r. griggs 	biology, medicine 	neurology 	 	 save alert research feed congenital nemaline myopathy. i. defective organization of α‐actinin is restricted to muscle 	f. jennekens, john j. roord, h. veldman, j. willemse, b. jockusch 	medicine 	muscle & nerve 	 	 save alert research feed a novel nemaline myopathy in the amish caused by a mutation in troponin t . 	j. johnston, r. kelley, + authors l. biesecker 	biology, medicine 	american journal of human genetics 	 	 	pdf save alert research feed mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. 	k. pelin, p. hilpelä, + authors c. wallgren‐pettersson 	biology, medicine 	proceedings of the national academy of sciences of the united states of america 	 	 	pdf save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue a multiplex human syndrome implicates a key role for intestinal cell kinase in development of central nervous, skeletal, and endocrine systems article a multiplex human syndrome implicates a key role for intestinal cell kinase in development of central nervous, skeletal, and endocrine systems piya lahiry, , jian wang, john f. robinson, jacob p. turowec, david w. litchfield, matthew b. lanktree, , gregory b. gloor, erik g. puffenberger, kevin a. strauss, mildred b. martens, david a. ramsay, c. anthony rupar, , , victoria siu, , , and robert a. hegele , ,* six infants in an old order amish pedigree were observed to be affected with endocrine-cerebro-osteodysplasia (eco). eco is a previ- ously unidentified neonatal lethal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal systems. autozygosity mapping and sequencing identified a previously unknown missense mutation, r q, in ick, encoding intestinal cell kinase (ick). our results established that r is conserved across species and among ethnicities, and three-dimensional analysis of the protein structure suggests protein instability due to the r q mutation. we also demonstrate that the r q mutant fails to localize at the nucleus and has diminished kinase activity. these findings suggest that ick plays a key role in the development of multiple organ systems. introduction protein kinases belong to one of the largest and most func- tionally diverse gene families in eukaryotes, constituting approximately . % of all human genes. by phosphory- lating substrates, kinases can direct activity, localization, and overall function of numerous target proteins. kinases are particularly important for signal transduction and coor- dination of complex cellular functions, as seen with the mitogen-activated protein (map) kinases and cyclin-depen- dent kinases (cdks), which play a central role in regulating mammalian cell proliferation and division. altered kinase activity or abnormal substrate phosphorylation has been implicated in monogenic diseases—‘‘kinasopathies’’— including endocrine disorders, cancers, immunodefi- ciencies, and cardiovascular diseases. inherited skeletal dysplasias or osteochondrodysplasias are characterized by abnormal development, growth, and maintenance of the skeleton. manifestations of skeletal dysplasias range from clinically undetectable to severe deformities and lethality. a group of lethal autosomal- recessive skeletal dysplasias are the short rib-polydactyly (srp) syndromes, which include srp type ii or majewski syndrome (mim: ). herein we report a new syndrome, to our knowledge, comprising osteodysplasia, cerebral anomalies, and endo- crine gland hypoplasia. a pedigree from an old order amish community was identified as having six affected infants with this previously unreported multisystem, neonatal lethal condition, designated here as the endocrine-cere- bro-osteodysplasia (eco) syndrome. because the old order the american journal of human genetics , – , februar amish population reportedly have a high degree of consan- guinity, this pedigree was ideal for autozygosity mapping of the putative molecular defect. , we delineate the clinical features of eco and report the first mutation, r q (c. g/a), within ick, encod- ing intestinal cell kinase (ick). biochemical and immuno- cytochemical studies indicate function and localization deficits, implicating ick as a key player in development of the central nervous, skeletal, and endocrine systems. material and methods patients and biological materials two families from an old order amish community were referred for genetic assessment and counseling, and all six affected infants were examined by one of the authors (v.s.). photos, blood, and tissue samples were provided for research purposes, with ethics approval (from the office of research ethics at the university of western ontario) and informed consent from participating parents. peripheral blood and skin biopsy for dna extraction was collected from five affected children, three parents, and two unaf- fected siblings and used for autozygosity mapping. the genealogy of the families affected with the disease was prepared through inter- views and local amish community records. dna from umbilical cord blood and buccal swabs was extracted from eco-unaf- fected individuals born into the old order amish community for determining the mutant-allele frequency in the community. autopsies were performed on three patients (iv- , iv- , and iv- from pedigree) after , , and weeks of gestation, respectively. histology routine tissue samples including liver, kidney, and central nervous system (cns) from each of the three autopsy patients were collected robarts research institute, london, ontario n a k , canada; department of biochemistry, university of western ontario, london, ontario n a c , canada; clinic for special children, strasburg, pa , usa; department of pathology, london health sciences centre, london, ontario n a a , canada; medical genetics program, london health sciences centre, london, ontario n c v , canada; children’s health research institute, london, ontario n c v , canada *correspondence: hegele@robarts.ca doi . /j.ajhg. . . . ª by the american society of human genetics. all rights reserved. y , mailto:hegele@robarts.ca for histopathological examination, formaldehyde fixed, paraffin embedded, sectioned, and then stained with hemolysin and eosin. neuropathological examination was performed on formalin-fixed brain and spinal cord of each of the three autopsy patients. genotyping dna from five affected infants and five unaffected parents and siblings were genotyped for single-nucleotide polymorphisms (snps) with genechip mapping k array set (affymetrix, santa clara, ca, usa) at the london regional genomics centre. ng of double-stranded genomic dna was digested with either nsp or sty, followed by adaptor ligation and pcr amplification with generic primers. pcr products were then purified, fragmented with dnasei, labeled with terminal deoxytransferase, and finally hybridized to the mapping k nsp or sty genechips. snp geno- types were determined with the b-rlmm algorithm implemented in affymetrix gtype software. autozygosity mapping autozygosity (homozygosity) mapping was performed with agi- lent gt v . (agilent technologies, santa clara, ca, usa), which scans the genome for regions that are identical by descent. snp allele frequencies from controls of european descent were used for estimating the two-point logarithm of the odds (lod) scores for each snp. location scores, which are the summation of two- point lod scores for a block of homozygous snps, were then calculated for providing a relative measure of likelihood that the region harbors the disease gene. mutation analysis eleven of the positional candidate genes in the linked region on chromosome p were screened by genomic dna sequencing in an affected individual, a parent, an unaffected sibling, and a non-amish control. candidate genes from genomic dna were pcr amplified with primer pairs designed for all coding exons and intron-exon boundaries. all amplicons were then sequenced with the sequencing platform of the london regional genomics centre sequencing facility. cosegregation of the exon ick mutant with disease in the pedi- gree was demonstrated with direct sequence analysis of all available family members. the target sequence ( base pairs [bp]) was amplified with primers ctc att cca tac agt gcc aca and gaa tta cat gcc aat ttt caa ag, followed by electropho- resis purification on a . % agarose gel and analysis on a abi dna sequencer (applied biosystems, mississauga, on, canada). a total of family members, including the affected individuals, were studied. snapshot and taqman assays were used for identifying allele frequency of the ick variation in exon , c. g/a (gi: ), within old order amish controls and ethnically diverse and healthy non-amish controls, respectively. for snapshot, which is a rapid allele-specific genotyping method, the purified bp amplicon (with the above primers) was sub- jected to ddntp extension (snapshot, applied biosystems) with primer cag tgg gat ccc aag aaa c and analyzed by abi dna sequencer. taqman quantitative real-time pcr assays were performed with an abi sequence detection system (applied biosystems) for providing allele discrimination with pcr primers (forward primer: gct cct gag aga cat gct tca; reverse primer: aag aaa atg gaa gaa aac ctg act agc t) and two allele-specific taqman probes synthesized for detecting the ick variation (allele g: vic-ccc aag aaa cga cca ac and mutant allele a: fam-cca aga aac aac caa c). the americ in silico analysis conservation of the ick protein across species was determined with clustalw, which is a multiple-sequence-alignment computer program, by initially creating a phylogenetic tree of the query sequence. impact of the amino acid mutation (r/q at residue ) on ick protein structure, function, and pathological implica- tion was predicted with four online tools, namely pmut, polyphen, snps d, and sift. the crystal structure of human cdk in complex with isopente- nyladenine (pdb id: exm), solved by schulze-gahmen et al. , was used as a basis for modeling the ick with and without the r/q mutation at residue . for mimicking ick, exm was substituted at a p and i v. the resulting structure was visual- ized in the program pymol (v . , delano scientific, san fran- cisco, ca, usa). with the rosetta design program, used for approximating the change in potential energy (in kilocalories) of the ick structure with the r/q mutation, side chains of nearby contacting amino acids were allowed to vary in conformation. change in energy values (in kilocalories) was replicated in eris server, which is a protein-stability prediction server that calculates the change in protein stability caused by mutations. eris server has the added feature of allowing backbone motion of the amino acids, which is crucial for protein-stability estimation of small-to- large mutations. plasmids and cell culture the ultimate orf clone of human ick cdna (clone id: ioh ) was provided in the gateway entry vector, pentr , containing a kanamycin-resistance cassette (invitrogen, carlsbad, ca, usa). the r/q mutation was introduced into the wild-type ick clone within pent in vitro with the genetailor site-directed muta- genesis system (invitrogen). with clonase ii (invitrogen) for aiding homologous recombination, the wild-type and mutant ick cdna was cloned directionally into the gateway destination vector, pcdna-dest , containing an n-terminal green fluorescent protein (gfp) tag and neomycin-resistance cassette. all clones were sequence verified. the plasmid pcdna/gw- /cat, which contained an n-terminal gfp tag, neomycin-resistance cassette, and chloramphenicol-acetyltransferase (cat) cassette, was pro- vided as a vector control. hek cells were maintained at �c and % co in dulbecco’s modified eagle’s medium (gibco, carls- bad, ca, usa) supplemented with % fetal bovine serum. immunocytochemistry for assessing nuclear localization of ick constructs, hek cells were grown on coverslips in six-well mm dishes to %– % con- fluency, followed by transfection with either wild-type (wt), r q mutant ick-expression plasmid, or control vector containing a cat cassette ( mg dna) by a calcium-phosphate-based method. hr after transfection, cells were washed two times with pbs, fixed with % paraformaldehyde, and stained with hoechst dye ( . mg/ml in pbs) (sigma-aldrich, oakville, on, canada) on ice for min. cells were then washed three times with pbs and mounted on glass slides with permafluor aqueous mounting medium (fisher, markham, on, canada). images were captured with fitc and uv filter sets and objective with a leica (deerfield, il, usa) dmi b inverted fluorescence microscope, followed by image acquisition with the leica application suite (las v. . . ). protein quantification hek cells were grown in cm flasks until %– % conflu- ency was reached, followed by transfection with gfp-tagged an journal of human genetics , – , february , expression constructs of either wt, r q mutant, or control vector containing a cat cassette ( mg dna) by a calcium-phos- phate-based method. hr after transfection, cells were harvested in ice-cold pbs and lysed in lysis buffer ( mmol/l tris-hcl [ph ¼ . ], mmol/l nacl, mmol/l edta, mmol/l egta, % triton x- , mmol/l naf supplemented with phosphatase and protease inhibitors). the lysate was cleared by centrifugation. cell lysates were precleared with immobilized protein-g beads (fisher) for hr at �c and then incubated with anti-gfp ( mg) for hr at �c, followed by incubation with immobilized protein-g beads (fisher) overnight at �c. the beads were washed extensively with lysis buffer and then divided for immunoblotting and kinase assay. gfp-immunoprecipitated beads of all three constructs were boiled in the sds loading buffer for min. proteins were then resolved by % sds-page and transferred onto polyvinylidene difluoride membranes (invitrogen). the membranes were blocked in tbs containing . % tween- and % fat-free dry milk for hr and then incubated with anti-ick ( : , santa cruz biotechnology, santa cruz, ca, usa) overnight at �c. the membrane was then incubated with horseradish peroxidase- conjugated anti-goat secondary antibodies ( : , , santa cruz biotechnology) for hr, followed by ick protein visualiza- tion with enhanced chemiluminescence-detection luminol reagent according to the manufacturer’s instructions (santa cruz biotechnology). kinase assays for assessing kinase activity, gfp-immunoprecipitated wt ick, r q mutant ick, and vector control were washed extensively in kinase assay buffer ( mm hepes [ph . ], mm mgcl , sup- plemented with mm dtt, protease inhibitors and phosphatase inhibitors). the samples were then incubated with mci [g- p] atp, mm atp, and mg purified myelin basic protein (mbp) (millipore, billerica, ma, usa) at �c for min in ml kinase assay buffer. as a positive control, active mapk (millipore) was incubated with mci [g- p] atp, mm atp, and mg mbp at �c for min in ml kinase assay buffer. proteins in the reac- tion were separated by % sds-page. the gels were dried, and p was detected by autoradiography. statistical analysis immunofluorescence localization data were analyzed with pear- son’s chi-square test with sas v . (sas institute, cary, nc, usa). results clinical and pathological features of eco three affected individuals from two old order amish fami- lies were originally reported as having a majewski-hydrole- thalus phenotype in (s. bakker and v. siu, , am. soc. hum. genet., abstract). three further infants were subsequently born, and the phenotype was further charac- terized (figure a and table ). the infants have had a high birth weight ( th percen- tile) in four out of six times that this was measured. some of the excess weight may be attributed to the excess fluid associated with severe hydrocephalus. in all of the pregnancies in which antenatal ultrasound was performed, the american journal of human genetics , – , februar multiple anomalies including ventriculomegaly, cleft lip, and shortened limbs were observed in the second trimester. polyhydramnios occurred in one pregnancy. spontaneous onset of labor occurred in two of the preg- nancies at – weeks gestation. one infant (iv- ) showed no spontaneous respirations at birth, was intu- bated for a day, then extubated and taken home for pallia- tive care. he died on day . the second infant (iv- ) had severe hydrocephalus, requiring drainage of ml of cere- brospinal fluid from the ventricles to enable vaginal delivery. the baby died during birth. the other pregnan- cies were induced between and weeks gestation because of the severity of the malformations. integrated prenatal screening was undertaken in one pregnancy and was screen positive for increased risk of trisomy- , with a low unconjugated estriol of . mom and a low alpha- fetoprotein of . mom. adrenal hypoplasia found in this infant (iv- ) may potentially account for the low prenatal estriol levels. the face shows a wide nasal bridge and flattened nasal tip with median cleft lip in four of the six cases and a small premaxilla with bilateral cleft lip and palate in the remain- ing two cases. there is swelling of the tissue derived from the maxillary prominences. the eyes are small and sunken, with a cystic component observed in two cases. as expected, the eyelids were fused in the three infants born between and weeks gestation, but they were also fused in the infant born at weeks (eyelids normally remain fused until approximately weeks gestation) . ears show varying degrees of dysplasia. the lower lip is deficient later- ally, and the chin is small. one infant (iv- ) had two congenital supernumerary teeth at weeks gestation. the upper limbs are markedly shortened, with a charac- teristic bowing of the forearms, ulnar deviation of the hands, and postaxial polydactyly. x-rays reveal angulation of the diaphyses. palmar creases are abnormal. usually the index fingers have only one interphalangeal crease, whereas the other digits lack at least one, if not both, inter- phalangeal creases. there is severe brachydactyly of the digits on the hands, as well as syndactyly involving various combinations of digits two to six. the hips are abducted, with the thighs held at degrees to the lower legs. there is a very wide gap between the halluces and the second toes, reminiscent of atelosteogenesis type . on x-rays, the proximal metaphyses of the femur and tibia are widened. overall, the radiologic findings are most consis- tent with a diagnosis of majewski syndrome. chest tends to be broad, with widely spaced nipples only in patient iv- . no congenital heart defect was found in any of the three autopsies. the adrenal glands were hypo- plastic in two cases and absent in one case. four genotypic males have all had varying degrees of abnormal differentiation of the external genitalia. patient iv- , born at weeks, had apparent sex reversal, with unfused urogenital folds and microphallus. position of the urethral orifice was not determined, and no autopsy was done for assessing for testes. patients iv- , iv- , and y , figure . clinical and pathological findings in eco patients (a) serial photographs of all six affected individuals (left to right from pedigree) showing full body with polydactyly and micromelia, craniofacial abnormalities including cleft lip and palate, bowed forearms, wide-gapped hallux (not shown for iv- and iv- ), and karyo- type (top to bottom). (b) kidney cortex from patient iv- (top left) and medulla from patient iv- (top right) show cystically dilated tubules (hematoxylin and eosin staining [h&e], magnification). liver sections from the -week-gestated patient iv- (bottom left) illustrate the persistence of circular ductal plates ( magnification). squamous metaplasia is seen in the intraprostatic urethra from patient iv- (bottom right) (h&e, magnification). (c) representative gross coronal view of the brain of patient iv- , presenting with absent septum pellucidum, secondary to ventricular hydrocephalus, and fused thalami due to a lack of the third ventricle. iv- had microphallus, hypoplastic scrotum with no rugae, prominent scrotal raphe, and bilateral cryptorchidism. although the cryptorchidism may not be significant in the context of prematurity (two were delivered at and weeks), iv- was delivered at weeks gestation and had first-degree hypospadias. the urethral opening was present at the tip of the microphallus in the other two infants. the two genotypic females had unfused urogenital folds. born at weeks gestation, iv- had unusually prominent labia majora, whereas iv- ( weeks gestation) had normal external female genitalia. tissue specimens examined from autopsies of patients iv- , iv- , and iv- (figure b) showed cystically dilated tubules to a variable extent in both the medulla and the cortex of the kidney, as well as persistence of circular ductal plates in the liver. in addition, patient iv- had squamous metaplasia of the urethra and periurethral glands in the prostate. the america neuropathology findings were extensive, with the main features being evidence of holoprosencephaly, hypoplastic or absent corpus callosum, agenesis of the pituitary, and cerebral cortex malformations. patients iv- (figure c) and iv- had hydrocephalus in the form of ventricular dila- tation, absence of the third ventricle, dysmorphic septum pellucidum, and malformed diencephalic elements. it is conceivable that the apparent overgrowth of the medial diencephalic structures led to the obliteration of the third ventricle, initiating secondary hydrocephalus, followed by the rupture or absence of the septum pellucidum. in patient iv- , there is a development of a semilobar holoprosence- phaly, which indicates an earlier initiation of the genetic defect resulting in hypertrophic diencephalic elements. overall, affected individuals had ventricular hydroceph- alus, midline cleft lip and palate, abnormal bone develop- ment manifesting as micromelia, bowing of the long bones, postaxial polydactyly, hypoplastic adrenal and pituitary n journal of human genetics , – , february , table . clinical and pathological descriptiona clinical features affected individuals (year of birth) iv- ( ) iv- ( ) iv- ( ) iv- ( ) iv- ( ) iv- ( ) age at delivery (weeks) sex male male female female male male karyotype , xy , xy , xx , xx , xy , xy height cm ( rd percentile) . cm ( th percentile) cm ( th percentile) . cm ( th percentile) . cm ( th percentile) weight grams ( th percentile) grams ( th percentile) grams ( th percentile) grams ( th percentile) grams ( th percentile) grams ( th percentile) head circumference . cm (> th percentile) . cm (> th percentile) cm ( th percentile) cm ( rd percentile) . cm ( th percentile) autopsy þ þ � � � þ oral cleft palate midline midline notch in alveolar ridge midline midline midline cleft lip bilateral median median bilateral median median presence of premaxilla þ � � þ, tiny � � prominent upper lip region � þ � þ � � hypoplastic/absent epiglottis þ � þ hypoplastic/absent larynx þ � þ facial midface hypoplasia þ þ þ þ þ þ hypoplastic eyes þ þ � þ present; right: hypoplastic, left: cystic cystic retinal dysplasia � � þ, cataracts deep-set eyes þ þ þ þ þ fused eyelids þ þ þ þ hypotelorism þ flat and wide nasal bridge þ þ þ þ þ þ dysplastic and low-set ears þ þ dysplastic þ þ micrognathia þ þ þ þ þ þ excess skin below chin þ þ � � þ þ teeth � two in lower jaw � � � � skeletal dolicocephalic � þ � � þ þ prominent xyphoid � þ � � � � polydactyly (postaxial) limbs limbs limbs limbs limbs limbs syndactyly þ þ þ þ þ þ brachydactyly þ þ þ þ þ þ single transverse palmar crease unilateral bilateral unilateral ulnar deviation of hands þ þ þ þ þ þ bowing of forearms (radius and ulna) þ þ þ þ þ þ bowing of lower legs (fibula and tibia) þ � � � � þ hitch-hikers’ thumbs � � � � � unilateral abducted hips þ þ þ þ þ þ wide gap between first and second toe þ þ unilateral-right þ þ þ talipes equinovaris � � � � � � chest width broad with wide-spaced nipples � � � narrow � the american journal of human genetics , – , february , table . continued clinical features affected individuals (year of birth) iv- ( ) iv- ( ) iv- ( ) iv- ( ) iv- ( ) iv- ( ) micromelia þ þ þ þ þ þ b radiography abnormal long bones (radius, ulna, tibia, fibula) short diaphysis short and incurved ulnae þ short and ovoid tibiae þ abnormal humerus short diaphysis abnormal femur short and ovoid abnormal ilium þ abnormal/ hypoplastic acetabular roof þ central nervous system evidence of holoprosencephaly failure of separation of diencephalic elements semilobar, monoventricular, fusion of basal ganglia and thalami diencephalic agenesis corpus callosum þ agenesis hypoplastic absence of septum pellucidum þ þ hydrocephalus (ventriculomegaly) communicating þ dysmorphic cerebral aqueduct þ stenosis olfactory bulbs þ absent cerebral cortex malformation frontal and occipital regions focal polymicrogyria rudimentary sulcation brainstem malformation þ þ small brainstem cerebellar abnormalities hemorrhagic, dysmorphic peduncles small cerebellum hippocampus agenesis þ þ leptomeningeal glioneruronal heterotopia þ spinal cord malformation þ � endocrine system pituitary gland absent not identified adrenal glands hypoplastic absent hypoplastic other pulmonary hypoplasia þ gastrointestinal anomalies tube-like stomach small stomach � squamous metaplasia of bladder þ � � external genitalia microphallus, hypoplastic scrotum microphallus, hypoplastic scrotum normal normal sex reversal; hypoplastic labia majora present, microphallus cryptorchidism � bilateral � � � � polyhydramnios � � � � present � note: blank cells indicate that information was unavailable. a information gathered from attending-physician reports. b report from international skeletal dysplasia registry (cedars-sinai medical center) for patient iv- . the american journal of human genetics , – , february , glands, and ambiguous genitalia. many of the malforma- tions observed involve a defect of apoptosis, especially the cleft lip and palate, syndactyly, prolonged persistence of fusion of the eyelids, and unfused urogenital folds. autozygosity mapping of a candidate locus on chromosome p pedigree analysis (figure ) of affected individuals revealed that the syndrome followed an autosomal-recessive inher- itance pattern, with two parental consanguineous matings (iii- with iii- and iii- with iii- ). genome-wide auto- zygosity mapping performed on five eco-affected infants and three of the four parents from the pedigree unambig- uously mapped eco to p . –p with a location score of , the summation of two-point lod data, , which spans over . mb of the chromosome (figure a). this region in chromosome p is bound by snps rs and rs and is comprised of genotyped figure . snp-based genotyping of eco-affected pedigree identified a homozygous region in chromosome p eco in a consanguineous old order amish pedigree. the inheritance of the disease follows an autosomal-recessive mode of transmission. affected individuals are shown as blackened squares (male) and circles (female). diagonal lines across symbols indicate deceased indi- viduals. a consanguineous marriage is shown by a double line between two individuals. confirmation of autozygosity-based linkage was performed by observing the snp-based genotyping within the candidate region on p . –p . a portion of the homozygous region within patients is shown in boxes. the ick variation at the coding sequence (g/a, accession number: nm_ ) that causes a r/q change at residue in the protein was analyzed (see figure c for more details) and highlighted in red. horizontal dashes above symbols indicate individuals who underwent dna analysis, and hyphens indicate nongenotyped markers. the american journal of human genetics , – , february , contiguous snps (figure b). autozygosity-based linkage was confirmed by haplotype analysis of the snp genotypes within the candidate region at chromosome p (figure ). identification of the causative mutation, r q, in ick the candidate interval harbored a total of known and hypothetical genes. we prioritized sequencing of candidate genes on the basis of the following criteria: ( ) gene involve- ment in other single-gene disorders, ( ) the role and tissue specificity of the encoded protein, and ( ) the number of exons per gene, for feasibility reasons. on the basis of this priority list, exons of candidate genes were directly sequenced from the genomic dna of one of each of the following: affected individuals, unaffected siblings, parents, and non-amish controls. this sequencing led to the discovery of numerous dna sequence variations (table s available online). in an autosomal-recessive mode of inheritance, the disease causing the variation would be due to a homozygous genotype observed only in the affected individual, and the parent would be an obligate carrier of the causative allele. the only such variation that also affected the gene and/or its gene product was in ick. the ick gene, composed of exons, had a nonsynony- mous nucleotide change c. g/a in exon (figure c) in the dna of the affected individual. this nucleotide change results in an amino acid change from arginine to glutamine at residue (r q), which lies within the nuclear-localization-signal domain of ick (figure d). dna sequencing further demonstrated that this alteration was homozygous in all five affected individuals, whereas the phenotypically unaffected parents and siblings were heterozygotes, consistent with the predicted autosomal- recessive pattern of inheritance. complete linkage of eco to the r q mutation (at chromosome p) within the pedigree was observed, with a two-point linkage lod score of . (at recombination fraction ¼ ). in silico analysis demonstrates r to be conserved and required for protein stability the ick protein belongs to the cdk subfamily, which is within the cmgc serine-threonine protein kinase family. r in human ick is conserved in the cmgc group of kinases, which includes the cdk, map kinase, glycogen synthase kinase (gsk ), and cdc-like kinase (clk) fami- lies. ick homologs were identified across phylogeny, ranging from homo sapiens to candida albicans. using clustalw protein sequence alignment, we observed r to be conserved in all known orthologous ick homologs (a representative set is shown in figure a). the conserva- tion of r suggests that mutations in this amino acid are not well tolerated, given that it may be involved in crucial aspects of structure and function. a previous study of ick that mutagenized several residues, including r , suggested that the arginine is required to create a function- ally and structurally stable conformation by forming ionic bonds with glutamic acid at residue . there is pre- the americ sently no three-dimensional ( d) model of ick because it has not yet been crystallized. however, cdk and ick share very high amino acid homology within the critical func- tional domain of interest. for homology-based modeling of ick in pymol, the d region harboring the nuclear- localization signal of cdk was substituted at only of the residues (residues and ). as visualized in figure b, when arginine is mutated to glutamine, the bond with glutamic acid is disrupted such that the glutamic acid rotates its side chain so that it is no longer buried in the protein but becomes surface exposed, suggesting a cause for protein instability. instability was confirmed with thermo- dynamic values acquired from rosetta and eris, two discrete protein-structure programs. both rosetta and eris, which predict protein-packing energy changes, predicted that the r q mutant protein was less stable (by . and . kcal/mol, respectively) compared to the wild-type protein. in addition, four different bioinformatic programs predicted a deleterious effect of r q mutation on the ick protein (‘‘pathological,’’ ‘‘probably damaging,’’ ‘‘affected protein function,’’ and ‘‘deleterious’’ from pmut, poly- phen, sift, and snps d, respectively). mutation screening in amish and non-amish populations suggests r q to be a private mutation the frequency of the a allele in ick at exon , c. g/a, was assessed within members in the old order amish community as well as individuals from other ethnic groups. genotype analysis of healthy old order amish controls demonstrated no aa homozygote but identified five heterozygote ga carriers outside the eco- affected family. the a allele frequency in the old order amish community was . %; thus, ~ in , old order amish subjects would be predicted to have the aa genotype. taqman-based genotyping of the ick muta- tion in an additional individuals from six ethnic groups demonstrated the complete absence of the a allele, indicating that this mutation was specific to the amish. nuclear localization affected by ick r q mutation gfp-tagged ick wild-type overexpressed in hek cells localized predominately to the nucleus, whereas the gfp- tagged ick r q mutant localized predominantly to the cytoplasm (figure a). therefore, this single r q point mutation was sufficient to cause a loss of nuclear localization. statistical evidence in a bar graph is provided in figure s . loss of kinase activity of ick with r q mutation we studied kinase activity in the presence of the r q mutation. gfp-labeled ick wild-type and r q mutant were overexpressed in hek cells prior to isolation via immunoprecipitation, and subsequent kinase-activity assays were performed for phosphotransferase activity. incorporation of p into the exogenous substrate, mbp, was catalyzed by ick wild-type, but no such incorporation was detected in the presence of the ick r q mutant (figure b). the lower panel for figure b indicates that an journal of human genetics , – , february , figure . autozygosity mapping of eco-affected pedigree identified an amino acid change, r q, in ick (a) autozygosity mapping with snp genotypes from , snp microarrays across the chromosomes (x axis) yielded snp haplotype location scores (y axis), with the highest peak on chromosome . location score is the summation of the point lod scores representing the likelihood of observing contiguous homozygosity in all five affected individuals of each snp within the region of homozygosity. (b) expanded view of the candidate locus shows the peak to be on chromosome p . –p (indicated by a star), which is defined by snps rs and rs and consists of candidate genes, including ick (indicated in red with a star), distributed over . mb of the genome. transcriptional direction is indicated by arrowheads. (c) the genomic structure of ick gene consists of coding exons with a nonsynonymous nucleotide change in exon (indicated in red with a star) that alters the amino acid of arginine to glutamine at residue (r q). dna sequence analysis of ick exon from genomic dna of a normal (i, top left tracing) individual, an affected (ii, top right tracing) individual, and a r q heterozygote (bottom middle tracing). for each tracing, a normal nucleotide sequence is shown in the top line of letters, with single-letter amino acid codes and codon numbers beneath. the position of the mutated nucleotides is indicated by the star. the american journal of human genetics , – , february , figure . ick protein analysis demonstrates that r is highly conserved and that the r q mutation alters protein structure (a) multiple alignments demonstrate that r residue is highly conserved across a representative set of species-specific ick homologs. a clustalw analysis of the ick region encompassing the mutation site at residue (highlighted in red) in aligned homologs with multiple divergent sequences is shown. the residues shaded in blue indicate amino acids that are similar between homologs. (b) a magnified look at the d region surrounding residue in ick according to pymol modeling, such that the normal (i) protein is superimposed on the r q mutant (ii) protein. this modeling predicts that the arginine (arg, in red) and glutamic acid (glu, in blue) form an ionic pair because of close proximity, in normal or wild-type protein (indicated by i). by comparison, the r q mutant (indi- cated by ii) is a basic polar to neutral polar substitution and predicts a change in structure such that the glutamine (gln, in orange) and glutamic acid (glu, in orange) can no longer ion pair, leaving the glutamic acid exposed to the surface of the protein rather than buried within the d structure. equal amounts of ick protein were assayed for kinase activity. bands for the ick constructs were observed specifi- cally, providing solid evidence for specificity of the ick anti- body. overall, the assay for p incorporation suggests that the r q mutation significantly impairs kinase activity. discussion rare congenital disorders provide new information on the biological pathways in human organogenesis. we report the america in an old order amish community a previously unidenti- fied and phenotypically distinct syndrome, eco, whose clinical manifestations occur early in development and include dysplasias of skeletal, cerebral, and endocrine systems resulting in neonatal mortality. in affected patients, we identified a homozygous missense mutation, r q, in the kinase ick. r was highly conserved across species and kinase family members. moreover, the protein carrying the mutation was predicted to change the conformation of the structure, as visualized in pymol, and to cause protein instability, as predicted by the (d) the domain structure of ick protein consists of the protein serine-threonine kinase catalytic domain, a catalytic loop, two activation loops, two nuclear-localization-signal sites, and a proline-rich region, from the n-terminal to c-terminal end. the amino acid (aa) (indicated in red) lies within the nuclear-localization signal (indicated with a star). n journal of human genetics , – , february , figure . altered subcellular localiza- tion and protein activity of the r q ick mutation (a) subcellular localization of wild-type and mutant ick is mainly nuclear and cyto- plasmic, respectively. hek cells were transfected for hr with wild-type ick (ick wt), mutant ick (ick r q), or vector control tagged with gfp and analyzed by immunofluorescence micros- copy at magnification. transfected cells were identified by gfp fluorescence (green), and nuclei were stained with dapi (blue). the signals obtained for dapi and gfp are shown separately (rows and ), and an overlay of both fluores- cence signals with differential interference contrast (dic) is shown in row . we counted , , and transfected cells per construct of wild-type ick, mutant ick, and control, respectively. the differ- ence in subcellular localization is statisti- cally significant (chi-square p value ¼ . � ), such that the wild-type and the mutant ick protein localizes in the nucleus of . % and . % of the trans- fected cells, respectively. in addition, blinded counting was performed in a small subset of the whole, and the overall results of the much larger cell numbers were repre- sentative of this blinded sample. (b) in vitro kinase assays were performed with mbp as substrate with anti-gfp immu- noprecipates obtained from hek trans- fected cells and active-purified erk as a positive control (top panel). hek cells were transfected for hr with gfp- tagged ick wt, ick r q, or vector control followed by anti-gfp immunopre- cipitation. bar graphs indicate means standard deviations from three sets of experiments, showing the relative kinase activities normalized to vector control quantitated by densitometry. the autoradiograph shows results of one experiment demonstrating incorporation of p into mbp. the upper blot shows results of a representative experiment demonstrating the levels of mbp phosphorylation. the lower blot, developed with anti-ick, demonstrates equal amounts of ick wt and ick r q. change in energy values in the programs rosetta design and eris. the r q mutation impairs nuclear localiza- tion and kinase activity. the ick r q mutation under- lying the eco syndrome implicates intestinal cell kinase as central to the development of several organ systems in humans. although eco is, to our knowledge, a distinct new syndrome, it does share some clinical features with previ- ously reported syndromes, such as majewski syndrome and hydrolethalus. majewski syndrome, described in , maps to chromosome and is a lethal form of neonatal dwarfism characterized by short ribs, microme- lia, polysyndactyly, median cleft lip, polycystic kidneys, ambiguous genitalia, and hypoplastic epiglottis, larynx, and lungs. hydrolethalus, described in , maps to the american journal of human genetics , – , februar chromosome q . and is a neonatal lethal auto- somal-recessive syndrome characterized by multiple congenital anomalies including hydramnios, hydrocephalus, a ‘‘keyhole’’ defect of the occipital bone, low-set ears, and midline malformations, such as heart and brain defects, cleft lip or palate, an abnormally shaped nose or jaw, incomplete lung development, and abnormal genitalia. the causative genes for both majewski syndrome and hydrolethalus have yet to be identified, but neither locus overlaps with p . –p harboring ick. a transitional clinical condition, pseudotrisomy (mim ), consists of characteristics found in both majewski syndrome and hydrolethalus, including holo- prosencephaly, hydrocephalus, polydactyly, heart defects, and facial anomalies consistent with the trisomy y , figure . schematic of overlapping features between six disorders, including eco the grid shows organ-system involvement, with darkened cells indicating the presence of the subphenotype for each disease. prevalence of disorder is in a white-to-black gradient system in which a frequency of % is white, % is gray, and % is black. the reordering of the disorders was the result of hierarchical cluster analysis that clustered the disease on the basis of similarities in organ-system involve- ment. the diseases included are (from top to bottom) hydrolethalus, pseudotrisomy , majewski hydrolethalus, mohr-majewski, majew- ski syndrome, and eco (highlighted in red). condition without any chromosomal defects. another clinical condition that bears some similarity to majewski syndrome and hydrolethalus was reported in , uniting two distinct genetic conditions such that the two conditions are suspected to be causally related. ‘‘ma- jewski hydrolethalus’’ was characterized by short limbs and ribs and abnormal tibiae consistent with majewski syndrome as well as hydraminos, hydrocephalus, and keyhole-shaped occipital bone consistent with hydrole- thalus syndrome. again, the causative gene is unknown. another distinct yet related condition with an unknown causative gene is the autosomal-recessive mohr-majewski syndrome (mim: ). hierarchical cluster analysis reordered these six related diseases on the basis of clinical features such that eco had the closest clinical description to the majewski and mohr-majewski syndromes (figure ). however, majewski and mohr-majewski have the presence of short ribs and cardiac defects, respectively, which are features that are not observed in eco-affected infants. despite the absence of overlap of loci from mapping studies, dna sequencing of ick in individuals with majewski and hydrolethalus syndromes might help to establish whether these disorders are allelic or whether eco truly represents a distinct and unique disorder. ick was initially cloned from the human intestinal crypt with degenerate primers specific for map kinases. , the designation ick appears to be a misnomer, given that ick is ubiquitously expressed in adult human tissues. , alternatively, this protein has sometimes been called mak-related kinase (mrk) in previous reports. , ick has a variant, ickb (accession number aah ); however, ickb is amino acids shorter with an alterna- tive c terminus, such that ickb is predominantly in the cytoplasm. ick shares % to % identity to the cata- lytic domains of cdks, which are involved in cell-cycle transition, and map kinases, regulators of cell-cycle entry. ick is activated by dual phophorylation of the the americ tdy motif (y- and t- ), such that it autophosphory- lates at y- and can be phosphorylated by human kinase cell-cycle-related kinase (ccrk), while being deactivated by protein phosphatase (pp ). it may be speculated that because the residue mutation of ick prevents nuclear localization, it hinders phosphorylation of the ick by the nuclear-residing ccrk, which is ultimately required for kinase activity of ick. a previous in vitro study of ick coincidentally mutagen- ized residue from arginine to alanine (r a). like r q, this non-naturally occurring r a mutant showed impaired nuclear localization and kinase activity. as determined by in vitro studies, ick can phosphorylate exogenous substrates, such as myelin basic protein , and scythe, an antiapoptotic protein required during mammalian development. scythe (bat ) is a nuclear protein that is implicated in apoptosis. scythe knockout mice display brain heteropia (abnormal migration), hydro- cephalus, and dilated and hypoplastic kidneys as well as lung abnormalities, leading to perinatal death. in addi- tion to endocrine hypoplasia, syndactyly, cleft lip, and cleft palate, eco-affected infants also develop phenotypes observed with scythe deficiency. because ick is involved in phosphorylation and activation of scythe, it is presum- able that an ick mutation would include defects observed with scythe inactivation. overall, ick seems to be involved in cell-cycle regulation and apoptosis during mammalian development. other evidence that ick has an effect on embryological development is the observed presence of ick mrna in placental tissue, although specific experi- ments will need to be performed for confirmation. previous work has suggested that ick has a role in development of the myocardium, but because eco syndrome is a multior- gan disorder, this kinase must have a role in development of other tissues and organ systems. interestingly, no cardiac anomalies have been observed in eco syndrome patients. thus, we have characterized a previously unreported human syndrome—eco—in which mutated ick is an journal of human genetics , – , february , functionally impaired. this single point mutation at residue not only affects structure, but also function, such that it can no longer localize properly, which has detrimental downstream effects and ultimately leads to abnormal fetal development. these findings suggest that ick plays a key role in the development of multiple organ systems. supplemental data supplemental data include one table and one figure and can be found with this article online at http://www.ajhg.org/. acknowledgments we acknowledge the generous cooperation of the families partici- pating in this study. we acknowledge the excellent technical assis- tance of rebecca provost, rachel rollings, caroline o’neil, and roger dewar. r.a.h. is a career investigator of the heart and stroke foundation of ontario and holds the edith schulich vinet canada research chair (tier i) in human genetics and the jacob j. wolfe distinguished medical research chair. p.l. was supported by the canadian institutes of health research (cihr) scriver family md/phd studentship award and the hsfo vascular training program. this work was supported by a team grant from the cihr (ctp- , mop- ), the heart and stroke foundation of ontario, genome canada through the ontario genomics institute, and grants from the children’s health research institute, the lawson health research institute, and the garrod association. received: november , revised: december , accepted: december , published online: january , web resources the urls for data presented herein are as follows: clustalw alignments, http://www.clustal.org eris server, http://troll.med.unc.edu/eris/login.php london regional genomics centre, http://www.lrgc.ca online mendelian inheritance in man (omim), http://www.ncbi. nlm.nih.gov/omim pmut, http://mmb .pcb.ub.es: /pmut polyphen, http://coot.embl.de/polyphen/ pymol, http://pymol.sourceforge.net rosetta design, http://rosettadesign.med.unc.edu sift, http://blocks.fhcrc.org/sift/sift.html snps d, http://www.snps d.org references . manning, g., whyte, d.b., martinez, r., hunter, t., and sudar- sanam, s. 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( ). mutations in the pericentrin (pcnt) gene cause primordial dwarfism. science , – . . puffenberger, e.g., hu-lince, d., parod, j.m., craig, d.w., dobrin, s.e., conway, a.r., donarum, e.a., strauss, k.a., dunckley, t., cardenas, j.f., et al. ( ). mapping of sudden infant death with dysgenesis of the testes syndrome (siddt) by a snp genome scan and identification of tspyl loss of function. proc. natl. acad. sci. usa , – . . higgins, d., thompson, j., and gibson, t. ( ). clustal: multiple sequence alignment (http://www.clustal.org). . ferrer-costa, c., gelpi, j.l., zamakola, l., parraga, i., de la cruz, x., and orozco, m. ( ). pmut: a web-based tool for the annotation of pathological mutations on proteins. bioinformatics , – . . ramensky, v., bork, p., and sunyaev, s. ( ). human non- synonymous snps: server and survey. nucleic acids res. , – . . yue, p., melamud, e., and moult, j. 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( ). multiple congenital malformations in two sibs reminiscent of hydrolethalus and pseudotrisomy syndromes. am. j. med. genet. , – . . neri, g., gurrieri, f., and genuardi, m. ( ). oral-facial- skeletal syndromes. am. j. med. genet. , – . . sharma, a.k., phadke, s., chandra, k., upreti, m., khan, e.m., naveed, m., and agarwal, s.s. ( ). overlap between the america majewski and hydrolethalus syndromes: a report of two cases. am. j. med. genet. , – . . rosing, b., kempe, a., berg, c., kahl, p., knopfle, g., gembruch, u., and geipel, a. ( ). orofaciodigital syn- drome type iv (mohr-majewski): early prenatal diagnosis in siblings. ultrasound obstet. gynecol. , – . . togawa, k., yan, y.x., inomoto, t., slaugenhaupt, s., and rustgi, a.k. ( ). intestinal cell kinase (ick) localizes to the crypt region and requires a dual phosphorylation site found in map kinases. j. cell. physiol. , – . . yang, t., jiang, y., and chen, j. ( ). the identification and subcellular localization of human mrk. biomol. eng. , – . . abe, s., yagi, t., ishiyama, s., hiroe, m., marumo, f., and ikawa, y. ( ). molecular cloning of a novel serine/threo- nine kinase, mrk, possibly involved in cardiac development. oncogene , – . . fu, z., larson, k.a., chitta, r.k., parker, s.a., turk, b.e., law- rence, m.w., kaldis, p., galaktionov, k., cohn, s.m., shabano- witz, j., et al. ( ). identification of yin-yang regulators and a phosphorylation consensus for male germ cell-associated kinase (mak)-related kinase. mol. cell. biol. , – . . gardina, p.j., clark, t.a., shimada, b., staples, m.k., yang, q., veitch, j., schweitzer, a., awad, t., sugnet, c., dee, s., et al. ( ). alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. bmc genomics , . . desmots, f., russell, h.r., lee, y., boyd, k., and mckinnon, p.j. ( ). the reaper-binding protein scythe modulates apoptosis and proliferation during mammalian development. mol. cell. biol. , – . . fenton, t.r. ( ). a new growth chart for preterm babies: babson and benda’s chart updated with recent data and a new format. bmc pediatr. , . n journal of human genetics , – , february , 	a multiplex human syndrome implicates a key role for intestinal cell kinase in development of central nervous, skeletal, and endocrine systems 	introduction 	material and methods 	patients and biological materials 	histology 	genotyping 	autozygosity mapping 	mutation analysis 	in silico analysis 	plasmids and cell culture 	immunocytochemistry 	protein quantification 	kinase assays 	statistical analysis 	results 	clinical and pathological features of eco 	autozygosity mapping of a candidate locus on chromosome p 	identification of the causative mutation, r q, in ick 	in silico analysis demonstrates r to be conserved and required for protein stability 	mutation screening in amish and non-amish populations suggests r q to be a private mutation 	nuclear localization affected by ick r q mutation 	loss of kinase activity of ick with r q mutation 	discussion 	acknowledgments 	web resources 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-α therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-� a. charlotte p. sewing, crystal kantores, julijana ivanovska, alvin h. lee, azhar masood, , amish jain, , , patrick j. mcnamara, , , a. keith tanswell, , , and robert p. jankov , , , physiology and experimental medicine program, hospital for sick children research institute, toronto, ontario, canada; heart and stroke richard lewar centre of excellence, university of toronto, toronto, ontario, canada; department of physiology, university of toronto, toronto, ontario, canada; and division of neonatology, department of paediatrics, university of toronto, toronto, ontario, canada submitted february ; accepted in final form may sewing ac, kantores c, ivanovska j, lee ah, masood a, jain a, mcnamara pj, tanswell ak, jankov rp. therapeutic hyper- capnia prevents bleomycin-induced pulmonary hypertension in neo- natal rats by limiting macrophage-derived tumor necrosis factor-�. am j physiol lung cell mol physiol : l –l , . first published may , ; doi: . /ajplung. . .—bleomy- cin-induced lung injury is characterized in the neonatal rat by inflam- mation, arrested lung growth, and pulmonary hypertension (pht), as observed in human infants with severe bronchopulmonary dysplasia. inhalation of co (therapeutic hypercapnia) has been described to limit cytokine production and to have anti-inflammatory effects on the injured lung; we therefore hypothesized that therapeutic hypercapnia would prevent bleomycin-induced lung injury. spontaneously breath- ing rat pups were treated with bleomycin ( mg/kg/d ip) or saline vehicle from postnatal days – while being continuously exposed to % co (paco elevated by – mmhg), % co (paco elevated by mmhg), or normocapnia. bleomycin-treated animals exposed to %, but not %, co , had significantly attenuated lung tissue macrophage influx and pht, as evidenced by normalized pulmonary vascular resistance and right ventricular systolic function, decreased right ventricular hypertrophy, and attenuated remodeling of pulmo- nary resistance arteries. the level of co neither prevented increased tissue neutrophil influx nor led to improvements in decreased lung weight, septal thinning, impaired alveolarization, or decreased num- bers of peripheral arteries. bleomycin led to increased expression and content of lung tumor necrosis factor (tnf)-�, which was found to colocalize with tissue macrophages and to be attenuated by exposure to % co . inhibition of tnf-� signaling with the soluble tnf- receptor etanercept ( . mg/kg ip from days – on alternate days) prevented bleomycin-induced pht without decreasing tissue macro- phages and, similar to co , had no effect on arrested alveolar development. our findings are consistent with a preventive effect of therapeutic hypercapnia with % co on bleomycin-induced pht via attenuation of macrophage-derived tnf-�. neither tissue macro- phages nor tnf-� appeared to contribute to arrested lung develop- ment induced by bleomycin. that % co normalized pulmonary vascular resistance and right ventricular function without improving inhibited airway and vascular development suggests that vascular hypoplasia does not contribute significantly to functional changes of pht in this model. carbon dioxide; inflammation; neonatal lung injury bronchopulmonary dysplasia (bpd) is a chronic pneumopathy affecting extremely premature infants who frequently require supplemental oxygen and/or mechanical ventilation. the inci- dence of bpd is inversely proportional to the gestational age at which infants are born, with an incidence approaching % in the smallest survivors ( ). the cardinal feature of bpd, as observed in the current era, is an inhibition or arrest of alveolar and vascular growth ( ). the underlying pathogenesis of bpd appears to be multifactorial, but upregulation of inflammatory mediators leading to, or caused by, infiltration of inflammatory cells, including macrophages and neutrophils, is believed to play a major role ( , , ). pulmonary hypertension (pht) is common in those infants that are most severely affected ( , , ), which signals greatly increased morbidity and mortal- ity related, in major part, to consequent right heart failure ( ). putative factors contributing to progressive and fatal pht in bpd include sustained vasoconstriction, arterial wall remod- eling, and pruning of peripheral resistance arteries ( ), but the upstream mediators and relative contributions of these changes to chronically increased pulmonary vascular resistance (pvr) and right ventricular dysfunction remain poorly understood. management strategies employed in an attempt to prevent or ameliorate bpd have thus far met with limited success but include avoidance or minimization of mechanical ventilation and high inspired o concentrations to reduce the potential for volutrauma and oxygen toxicity ( ). with avoidance of exces- sive mechanical ventilation has come a necessary shift in clinical practice towards “permissive hypercapnia” (acceptance of “higher-than-normal” paco levels) becoming the norm ( ), although evidence of the effectiveness in reducing the inci- dence and severity of bpd remains lacking ( ). possible explanations for a lack of benefit in clinical trials ( ) may be that the target paco was less than required or that harmful effects of hypoventilation and small tidal volumes in the suboptimally recruited lung may have counteracted any bene- fit. despite ongoing uncertainty about the utility of permissive hypercapnia in the neonate ( ), there is growing experimental evidence to indicate that inhaled or exogenous co (so-called “therapeutic hypercapnia”) may protect the lung from diverse injuries, including those secondary to systemic ischemia rep- erfusion ( ), endotoxin ( , ), high tidal volume mechani- cal ventilation ( , , ), and chronic exposure to hyperoxia ( ) or hypoxia ( , ). putative mediators of lung injury shown to be attenuated by therapeutic hypercapnia in these models have included inflammatory cell influx ( , , ), proinflammatory cytokines ( , , ), and oxidative stress ( , , , ). however, several studies have also indicated the potential for hypercapnia to worsen lung injury ( , , ) and to potentially cause an increase in inflammation in the address for reprint requests and other correspondence: r. p. jankov, physiology and experimental medicine program, hospital for sick children research institute, univ. ave, toronto, ontario, canada m g x (e-mail: robert.jankov@sickkids.ca). am j physiol lung cell mol physiol : l –l , . first published may , ; doi: . /ajplung. . . - / copyright © the american physiological societyhttp://www.ajplung.org l downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . noninjured lung ( , ), highlighting the need for further study. our aim therefore was to examine effects of therapeutic hy- percapnia in a new rat model with similarities to human bpd ( ), secondary to bleomycin exposure. bleomycin sulfate is a chemotherapeutic agent that produces a dose-dependent pulmonary inflammatory and fibrotic re- sponse in humans when administered systemically and in adult rodents when instilled as a single intratracheal dose ( ) or by repeated intraperitoneal injection ( ). the early phases of bleomycin-induced lung injury are characterized by increased expression of proinflammatory cytokines ( ), a marked in- flammatory cell influx that is dominated by macrophages ( ), increased apoptosis ( ), changes in growth factors, such as transforming growth factor-� ( , ), “emphysematous” lung morphology, and severe pht ( , ). in neonatal rats, selec- tive decrease in lung growth, arrested alveolarization and vascular hypoplasia are prominent features ( , ). herein, we report that exposure to co led to dose-dependent preventive effects on bleomycin-induced pht without af- fecting lung growth, arrested alveolarization, or vascular hypoplasia. tumor necrosis factor (tnf)-� was found to be increased by bleomycin and to colocalize with tissue mac- rophages, both of which were attenuated by % co . inhibition of tnf-� signaling with a soluble tnf- recep- tor, etanercept, also prevented the hemodynamic and struc- tural changes of chronic pht, suggesting that the benefits of co on the pulmonary vasculature were mediated, in major part, via this pathway. materials and methods materials. bleomycin sulfate was purchased from calbiochem (san diego, ca). etanercept (enbrel) was from amgen (thousand oaks, ca). plexiglas animal exposure chambers and automated o / co controllers were from biospherix (lacona, ny). acids, alcohols, organic solvents, paraformaldehyde, permount, and superfrost/plus microscope slides were from fisher scientific (whitby, on, canada). dmem, trypsin, and fbs were from wisent bioproducts (st-bruno, quebec, canada). avidin-biotin-peroxidase complex immunohisto- chemistry kits, , =-diaminobenzidine staining kits, dapi fluorescent mounting medium, and normal goat serum were from vector labo- ratories (burlingame, ca). weigert’s resorcin-fuchsin stain was from rowley biochemical (danvers, ma). terminal deoxyuridine triphos- phate (dutp) nick-end labeling (tunel) assay kits were from roche (laval, québec, canada). alexa fluor -conjugated isolectin b (cat no. i ), derived from griffonia simplicifolia, alexa fluor -conjugated goat anti-mouse (cat no. a- ), and alexafluor -conjugated anti-goat secondary antibody (cat no. a- ) were from life technologies (burlington, on, canada). anti-glyceralde- hyde- -phosphate dehydrogenase (gapdh; cat no. sc- ), anti- tumor necrosis factor (tnf)-� (cat no. sc- , used for fluorescent immunostaining), and goat anti-rabbit and -mouse igg-biotin antibod- ies were from santa cruz biotechnology (santa cruz, ca). anti- cluster of differentiation (cd) (cat no. mca r) was from serotec (raleigh, nc). anti-myeloperoxidase (mpo; cat no. a ) was from dakocytomation (mississauga, on, canada). anti- tnf-� (cat no. hp , used for western blotting) was from hycult biotech (uden, the netherlands). goat anti-rabbit and anti-mouse igg-peroxidase antibodies were from cell signaling technology (beverly, ma). unless otherwise specified, all other chemicals and reagents were from bioshop canada (burlington, on, canada). animal exposures and interventions. all procedures involving animals were performed in accordance with the standards of the canadian council on animal care and were approved by the animal care committee of the hospital for sick children research institute. commencing on the day after birth, pups received bleomycin sulfate mg/kg in . % saline ( �l/g body wt by -g needle in the right iliac fossa) or . % saline (vehicle control) daily intraperitoneally for days, as previously described ( ), while being concurrently exposed to either normocapnia (� . % environmental co ) or % or % co (o %, balance n ) ( ). in separate experiments, bleo- mycin-exposed pups received an additional intraperitoneal injection of etanercept ( . mg/kg in . % saline; �l/g body wt ip) on alternate days commencing on postnatal day . the dose of etanercept used was the same as that recently described to be effective in preventing pht in monocrotaline-exposed adult rats ( ). each litter was maintained at n � – pups to control for nutritional effects. at the end of each -day exposure period, pups were either killed by pentobarbital overdose or exsanguinated after anesthesia. arterial blood gas measurement. pups were lightly anesthetized with ketamine ( mg/kg ip) and xylazine ( mg/kg ip), and the neck was dissected to expose the external carotid artery. after a -min recovery, while the animals breathed the appropriate concentrations of co , the artery was transected and blood was immediately collected with a heparinized capillary tube and analyzed (abl ; radiometer, copenhagen, denmark). cardiac ventricular weights, lung wet/dry weights, and brain weight. right ventricular hypertrophy (rvh) was quantified by mea- suring the right ventricle (rv) to left ventricle and septum (lv � s) weight ratio (fulton index), as previously described ( ). lung wet-to-dry weight ratio was determined by measuring left lung weight immediately upon harvest and again following baking at °c for h. brain wet weight included the cortex and cerebellum only after removal of the brainstem and spinal cord. two-dimensional echocardiography-derived measurements of pul- monary hemodynamics. pulmonary hemodynamics were evaluated noninvasively using two-dimensional echocardiography and doppler ultrasound (vivid cardiac ultrasound system and i l linear probe; ge medical systems, milwaukee, wi), as previously described in detail ( , ). animals were lightly anesthetized with ketamine/ xylazine and spontaneously breathing at the time of the study. for estimation of pvr, a short axis view at the level of the aortic valve was obtained and the pulmonary artery was identified by color flow doppler. the pulmonary arterial acceleration time (paat) was mea- sured as the time from the onset of systolic flow to peak pulmonary outflow velocity and the rv ejection time (rvet) as the time from onset to completion of systolic pulmonary flow. pulmonary vascular resistance was estimated using the formula: rvet/paat. for esti- mation of rv stroke volume, the pa diameter was measured by color flow doppler at the hinge point of the pulmonary valve leaflets. from the same doppler interrogation of the pulmonary artery used to measure paat and rvet, rv output was calculated using the formula: (pa diameter/ ) � . � pa velocity time integral � heart rate (beats/min). the pa velocity time integral was measured by tracing the leading edge of the velocity time graph from the onset to completion of systolic pulmonary flow. rv output (ml/min) was corrected for body weight to derive a rv performance index (rvi; ml·min� ·kg� ). left ventricular (lv) shortening fraction was mea- sured from the parasternal short axis view according to the formula: [(lv end-diastolic diameter � lv systolic diameter)/lv end-dia- stolic diameter] � . histological studies. lungs from four animals from each group ( from each of separate litters) were air inflated and perfusion fixed at a constant pressure, embedded in paraffin, sectioned, immunostained for cd (to identify macrophages) and myeloperoxidase (mpo; to identify neutrophils), and stained with hematoxylin and eosin, or for elastin, as previously described ( , , ). paraffin-embedded cortical brain sections ( �m) were labeled for apoptotic nuclei using a commercially available fluorescein tunel enzymatic assay kit (roche). l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . morphometric analyses. for all analyses, measurements were car- ried out on four left lung sections per animal and four animals (representing litters) per treatment group by observers blinded to group identity. for assessment of percentage arterial medial wall thickness (%mwt), pulmonary arteries were identified by the pres- ence of both inner and outer elastic lamina using hart’s elastin stain, as previously described in detail ( ). analyses of tissue macrophage (cd -positive) and neutrophil (mpo-positive) cell numbers and distal airway structure, including mean linear intercept (mli; using hematoxylin and eosin-stained sections), tissue fraction (hematoxylin and eosin), secondary crest numbers (identified by positive staining for elastin at their tips), and counts of peripheral arteries (identified as vessels of external diameter between and �m with both internal and external elastic laminae visible) were conducted as previously described in detail ( , , ) from random nonoverlapping fields captured from each section. staining of frozen sections. lungs were inflated and snap frozen in optimum cutting temperature compound, as previously described ( ) and then cut by cryostat in to -�m sections, which were mounted and stored at � °c until analysis. slides were fixed in ice-cold acetone and incubated with isolectin b (diluted : ), according to the manufacturer’s instructions, known to label “stimulated” or clas- sically activated macrophages by binding to cell membrane glycocon- jugates bearing terminal �-d-galactose ( ). for colabeling studies, anti-cd and -tnf-� were applied (both diluted : , h at room temperature), followed by appropriate secondary antibodies (each diluted to : with blocking solution, at room temperature in the dark for min), before aqueous mounting with dapi. images were digitally captured using an epifluorescent microscope with appropriate filter sets. identical images acquired with different filter sets were merged using image-pro plus software (version . , media cybernetics, bethesda, md). quantitative pcr. rna was extracted (absolutely rna miniprep kit; agilent/stratagene, la jolla, ca) and reverse transcribed (affini- tyscript; agilent) from lung tissue samples stored in rnalater (applied biosystems, streetsville, on, canada). quantitative (q)pcr was performed on a stratagene mx p qpcr system using sybr green qpcr master mix (sa biosciences, frederick, md). cycling conditions were °c for min followed by cycles of °c for s and °c for s. a standard curve for each primer set was run, using a rat lung cdna standard (agilent), to ensure that reaction efficiency was equivalent to primers for housekeeping genes. primer sequences are listed in table . a dissociation curve was run for each set of samples to exclude nonspecific product formation and reaction contamination. samples were run in duplicate, and expression of the gene of interest, where reported, was normalized to �-actin and gapdh. fold or fraction change in expression relative to control samples was calculated by the �		ct method using stratagene mxpro software (v . ). western blot analyses. lung tissues from four animals per group ( from each of separate litters) were lysed in ripa buffer containing protease inhibitors, fractionated by sds-page, transferred to poly- table . rat primer sequences for quantitative pcr gene (refseq accession no.) forward =- = reverse =- = �-actin (nm_ ) ctgggtatggaatcctgtgg tagagccaccaatccacaca ccl (mcp- ) (nm_ ) ctgtagcatccacgtgctgt tgaggtggttgtggaaaaga ccl (mip- �) (nm_ ) ccaccgctgcccttgctgtt cacccggctgggagcaaagg ccl (mip- �) (nm_ ) ctctctcctcctgcttgtgg cacagatttgcctgcctttt ccl (rantes) (nm_ ) ctgctgctttgcctacctct cgagtgacaaagacgactgc ccl (mcp- ) (nm_ ) aaccagatgggaccaattca cacagacttccatgcccttt ccr (nm_ ) acctgttcaacctggctgtc agggaaaacactgcatggac ccr (nm_ ) ctgcccctacttgtcatggt ggcctggtctaagtgcatgt ccr (nm_ ) cagcagagcatacacctgga cgccaggaaggaatgaaata ccr (nm_ ) aaagtctggcaatggtgagc ctcccagtaaacctcccaca cxcl (cinc- ) (nm_ ) agacagtggcagggattcac ggggacaccctttagcatct cxcl (mip- �) (nm_ ) accaaccatcagggtacagg ggcttcagggttgagacaaa cxcr (nm_ ) gctatgaggtcctgggtgaa gagtgtccgagagcagaacc cxcr (nm_ ) gattcttggcttcctccaca ggaggtgttcgctgaagaag gapdh (nm_ ) ccatgtttgtgatgggtgtg ggcatggactgtggtcatga interleukin- � (nm_ ) tcgggaggagacgactctaa gaaagctgcggatgtgaagt interleukin- � (nm_ ) ctgtgactcgtgggatgatg gggattttgtcgttgcttgt interleukin- receptor antagonist (nm- ) gaaaagaccctgcaagatgc gatgcccaagaacacattcc interleukin- (nm_ ) gccagagtcattcagagcaa ggtttgccgagtagacctca tumor necrosis factor-� (nm_ ) cagcagatgggctgtacctt ctggaagactcctcccaggt tumor necrosis factor receptor- (nm_ ) gtcaaagaggtggagggtga ttacaggtggcacgaagttg tumor necrosis factor receptor- (nm_ ) gtcatccccaagcaagagtc catcctttggagaccctgaa table . body weight, lung weight and arterial blood gas values on day parameter/group vehicle � normocapnia bleomycin � normocapnia vehicle � % co bleomycin � % co vehicle � % co bleomycin � % co bw, g . . . . . . . . . . . . lw, mg * * * lw/bw � . . . . * . . . . * . . . . * ph . . . . . . . . . . † . . † paco , mmhg . . . . . . . . † . . † . . † pao , mmhg . . . . . . † . . † . † . . † hco �, mmol/l . . . . . . † . . ‡ . . † . . † values represent means sd; n � – animals per group for weight data and – animals per group for blood gas data. lw, lung weight; bw, body weight. *p � . , by anova, compared with respective vehicle-treated group. †p � . , by anova, compared with normocapnia-exposed groups. ‡p � . , by anova, compared with all other groups. l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . vinylidene difluoride membranes, and blotted, and band densities were measured as previously described ( ). differences in protein loading were compensated for by reblotting for gapdh, the expres- sion of which was found, in preliminary studies, to be unaffected by chronic exposure to bleomycin or to co . dilutions of primary antisera were : for tnf-� and : , for gapdh. protein bands were identified using enhanced chemiluminescent substrate (immobilon; millipore), and images were digitally captured using a microchemi chemiluminescent image analysis system (dnr bio- imaging systems, jerusalem, israel). bands were quantified by digital densitometry of nonsaturated images with background density re- moved (imagej, nih, bethesda, md). data presentation and analysis. all values are expressed as means se. statistical significance (p � . ) was determined by one-way anova followed by pair-wise multiple comparisons using the tukey test (sigmastat; systat software, san jose, ca). fig. . exposure to % co prevents bleomycin- induced pulmonary hypertension. pups were treated from postnatal days – with vehicle (open bars) or bleomycin ( mg·kg� ·day� ; closed bars) while receiving concurrent exposure to normocapnia (� . %) or % or % co . all values represent means se. a: pulmonary vascular resistance (pvr; left), as estimated by the right ventricular ejection time (rvet)/pul- monary arterial acceleration time (paat) ratio and right ventricular index (right), as a marker of right-ventricular systolic function (n � – ani- mals/group). b: right ventricle (rv)/left ventricle � septum (lv � s) dry weight ratios as a marker of rv hypertrophy (n � – animals/group) and percent arterial medial wall thickness (n � animals/group) as a marker of pulmonary arterial remodeling. *p � . , by anova, compared with bleomycin-treated normocapnia-exposed group. #p � . , by anova, compared with vehicle-treated groups. c: representative photo- micrographs of elastin staining (dark brown inner and outer elastic laminae delineating the medial vascular wall; bar length � �m) demonstrat- ing medial wall thickening in bleomycin-exposed animals (bleomycin), which was largely pre- vented by concurrent exposure to % co (bleo- mycin � % co ). l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . fig. . exposure to co does not improve abnormal distal airway morphology. pups were treated from postnatal days – with vehicle (open bars) or bleomycin ( mg·kg� ·day� ; closed bars) while receiving concurrent exposure to normocapnia (� . %) or % or % co . representative low-power photomicrographs of hematoxylin and eosin-stained sections (a) demonstrating marked distal airway simplification and septal thinning in bleomycin-treated animals and elastin-stained sections (b) demonstrating a marked decrease in numbers of small peripheral arteries (outlined by dark brown stain for elastin) in bleomycin-treated animals, both of which were unaffected by exposure to co . bar lengths � �m. c: morphometric analyses of mean linear intercept (lm), tissue fraction, and secondary crests or peripheral arteries per field, corrected for tissue fraction. values represent means se for n � animals/group. *p � . , by one-way anova, compared with respective vehicle-treated groups. #p � . , by one-way anova, compared with respective vehicle-treated group. l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . results body weights, lung weights, and arterial blood gas measurements. values are shown in table . neither treatment with bleomycin nor exposure to either level of co for days had any significant effect on body weight, relative to vehicle-treated or normocapnia-exposed controls. in contrast, lung weights and lung weight-to-body weight ratios were significantly decreased in bleomycin-treated animals, whether or not they were ex- posed to co . treatment with bleomycin and/or exposure to co had no significant effects on lung wet/dry weight ratios (p � . ; data not shown). exposure of bleomycin-treated animals to % co increased paco by � – mmhg and exposure to % co by � mmhg above levels measured in normocapnia-exposed controls. only exposure to % co caused significant persistent acidosis at days due to partial metabolic correction, as reflected in lower ph and higher hco �hco levels than normocapnic controls, whereas ani- mals exposed to % co had evidence of complete metabolic correction by day . interestingly, and as previously reported in hypoxia-exposed animals ( ), exposure to either level of co significantly increased pao in both vehicle- and bleomy- cin-treated animals. effects of co on abnormal pulmonary hemodynamics. to distinguish acute from chronic effects of hypercapnia, prelim- inary measurements were first obtained while animals contin- ued to breathe co and were repeated following a -min period of recovery in room air. pvr index was found to be unchanged (p � . by anova; data not shown) by removal from co ; therefore, all reported data were obtained while animals were breathing room air. as previously reported ( ), treatment with bleomycin for days led to a significantly increased the pvr index (fig. a, left) and decreased rvi (fig. a, right), relative to vehicle-treated controls. bleomy- cin-induced increase in pvr index and decrease in rvi were normalized by exposure to % co (fig. a), whereas expo- sure to % co had no significant effect (p � . ). neither treatment with bleomycin, nor exposure to co , caused any significant change in lv fractional shortening (p � . by anova; data not shown). effects of co on structural changes of pulmonary hypertension. as previously reported ( ), treatment with bleomycin for days led to significant rvh (fig. b) and increased %mwt (fig. b) in pulmonary resistance arteries. exposure to % co significantly (p � . ) attenuated rvh (fig. a) and %mwt in bleomycin-treated animals (fig. b). exposure to % co caused no change in rvh (p � . ) and a smaller, but significant (p � . ) decrease in %mwt (fig. b). relative differences in medial wall thickness between groups are illustrated by high-power images of elastin-stained pulmo- nary arteries (fig. c). changes in distal airway morphology secondary to bleomycin. as shown in representative low-power hematoxylin and eosin- stained sections (fig. a) and by low-power elastin-stained sections (fig. b), the lung structure of bleomycin-treated animals was characterized by septal thinning, arrested alveo- larization (manifesting as “emphysematous” distal airspaces), and vascular rarefaction as quantified by increased mli and by decreased tissue fraction, secondary crest counts and peripheral artery counts (fig. c). neither level of co had any effects on these changes in either vehicle- or bleomycin-treated animals (fig. c). effects of co on tissue inflammatory cells. as illustrated by cd immunostaining (fig. a) and by isolectin b binding (fig. b), large classically activated macrophages were present in greatly increased numbers in the lungs of animals treated with bleomycin for days (fig. c). concurrent exposure to % co significantly (p � . ) attenuated macrophage num- bers in the bleomycin-treated lung, whereas % co had no significant effect (p � . ; fig. c). as also shown in fig. c, treatment with bleomycin led to significantly (p � . ) increased numbers of mpo-positive neutrophils, which were unaffected by exposure to either level of co . changes in expression of proinflammatory cytokines and chemokines (listed in table ) were screened by qpcr on day . genes for which significant (p � . ) changes were found are listed in table . the neutrophil chemokine cxcl (also known as cinc- ) and its receptor, cxcr , were greatly increased by bleomycin but were unaffected by exposure to % co (table ). tnf-�, a proinflammatory cytokine that may recruit mac- rophages to the lung ( , ) or be tissue macrophage derived ( ), was increased in bleomycin-exposed lungs and was normalized by concurrent exposure to % co (table ). similar to findings in qpcr, lung tnf-� protein content was significantly (p � . ) increased by treatment with bleomycin and completely normalized by exposure to %, but not by %, co (fig. d). double labeling for immunoreactive cd and tnf-� in lungs of bleomycin-treated animals revealed that tnf-� colocalized with macrophages (fig. e), implicating fig. . exposure to % co normalizes lung tissue macrophage number and tumor necrosis factor (tnf)-� content. pups were treated from postnatal days – with vehicle (open bars) or bleomycin ( mg·kg� ·day� ; closed bars) while receiving concurrent exposure to normocapnia (� . %) or % or % co . representative medium-power photomicrographs of cd -immunostaining (large dark brown cells highlighted by arrows; a) and isolectin b -labeled frozen sections (green fluorescent-labeled cells highlighted by arrows; b) demonstrating increased numbers of tissue macrophages in bleomycin-exposed animals (bleomycin), which was largely prevented by concurrent exposure to % co (bleomycin � % co ). bar lengths � �m. c: tissue macrophage and neutrophil counts per field normalized to tissue fraction. values represent means se for n � animals/group. d: western blot analyses of lung tnf-� content normalized to gapdh. representative immunoblots are shown with noncontiguous gel lanes demarcated by black lines. values represent means se for n � samples/group. *p � . , by one-way anova, compared with vehicle-treated groups. #p � . , by one-way anova, compared with bleomycin-treated normocapnia-exposed group. e: representative photomicrographs of fluorescent immunolabeling for cd and tnf-�, demonstrating colocalization with tissue macrophages (bar length � �m). table . changes in mrna expression secondary to bleomycin gene bleomycin vehicle � % co bleomycin � % co cxcl . . * . . * . . * cxcr . . * . . * . . tumor necrosis factor-� . . * . . . . values are means se of samples per group relative to control (vehicle-treated normocapnia-exposed) group, which was assigned a value of . *p � . , by anova, compared with control. l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . this cell type as the major source of tnf-� in the bleomycin- exposed lung. effects of tnf-� inhibition on bleomycin-induced pulmo- nary hypertension, macrophage influx, and abnormal distal lung morphology. bleomycin-induced increases in pvr index, rvh and %mwt in pulmonary resistance arteries were all normalized by treatment with etanercept (figs. , a and b), in contrast, treatment with etanercept had no effect on bleomycin- induced macrophage influx (fig. c), indicating that upregu- lated tnf-� signaling does not contribute to increased num- bers of activated tissue macrophages in the bleomycin-exposed lung. as shown in representative low-power hematoxylin and eosin-stained sections (fig. a), abnormal lung morphology in bleomycin-treated animals was unaffected by treatment with etanercept, as evidenced by mli, tissue fraction, secondary crest counts, and peripheral artery counts that did not differ (p � . ) from animals treated with bleomycin and vehicle (fig. b). effects of bleomycin and/or % co on brain, liver, and kidney weights and on apoptosis in brain cortex. as shown in table , exposure to bleomycin led to no change in brain or liver weight, while causing a small, but statistically significant (p � . ), decrease in combined kidney weight. exposure to % co caused no change in brain, liver, or kidney weight in either vehicle- or bleomycin-treated animals (table ). in support of a lack of effect of a -day exposure to % co on brain growth, no apparent increase in tunel-labeled nuclei was observed compared with normocapnic controls (fig. ). discussion the effects of chronic co exposure on the neonatal lung and pulmonary vasculature have received limited attention in experimental models despite the widespread use of permissive hypercapnia in the newborn intensive care unit in the hope of limiting lung injury. we have previously shown that chronic inhalation of co prevented pht in chronic hypoxia-exposed neonatal rats ( ), a model in which pulmonary inflammation is not apparent ( ). given the major putative role for lung inflammation in the pathogenesis of bpd ( , , ) and evidence for anti-inflammatory effects of inhaled co ( , , , ), we were interested in examining the effects of thera- peutic hypercapnia on an alternative rat model in which in- flammation is a prominent early feature. the concentrations of co examined in bleomycin-exposed animals were intended to reproduce what are generally considered, in the clinical setting, to represent moderate ( % co ; mean elevation of paco – mmhg) and severe ( % co ; mean elevation of paco mmhg) levels of hypercapnia. our findings were that % co attenuated the hemodynamic and structural indexes of pht secondary to bleomycin, whereas % co had lesser or no effects on these parameters. these changes were associated with attenuated tissue macrophage influx and tnf-� expres- sion and content in the bleomycin-exposed lung. our findings, which suggest a major role for tissue macrophages in the pathogenesis of chronic neonatal pht, are in agreement with previous work from our group using a neonatal rat model of fig. . tnf-� inhibition prevents bleomycin-induced pulmonary hypertension without affecting tissue macrophage number. pups were treated from postnatal days – with . % saline vehicle (control; open bars), bleomycin alone ( mg·kg� ·day� ; closed bars) or bleomycin and etanercept ( . mg/kg ip alternate days; grey bars). all values represent means se. a, left: pvr, as estimated by the rvet/paat ratio (n � animals/group). a, middle: rv/lv � s dry weight ratios as a marker of right-ventricular hypertrophy (n � animals/group). a, right: percentage arterial medial wall thickness as a marker of pulmonary arterial remodeling (n � – animals/group). *p � . , by anova, compared with all other groups. b: representative photomicrographs of elastin staining (dark brown inner and outer elastic laminae delineating the medial vascular wall; bar length � �m) demonstrating medial wall thickening in bleomycin-exposed animals (bleomycin), which was prevented by treatment with etanercept (bleomycin � etanercept). c: tissue macrophage counts per field normalized to tissue fraction. values represent means se for n � samples or animals/group. *p � . , by anova, compared with other groups. l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . bpd secondary to hyperoxia ( ), in which chronic inhalation of co was also shown to limit macrophage influx and to prevent the structural changes of pht ( ). examination of arterial acid-base status revealed that a -day exposure to % co caused significant acidosis, whereas % co did not, due in part to differing degrees of metabolic correction. a possible implication of these observa- tions is that benefits of co were related to acidosis, rather than to hypercapnia, in keeping with recent findings in adult animals by christou et al. ( ). furthermore, in short-term lung injury models, normalizing ph by buffering has been shown to worsen injury ( ) where inhaled co has otherwise been protective ( , , ). it was not possible to separate the effects of acidosis from hypercapnia by buffering in this chronic model, leaving the relative contributions of hypercap- nia and acidosis an open question. interestingly, both levels of hypercapnia increased pao levels, in keeping with previously reported observations made by our group ( ) and others ( , ). possible mechanisms for this effect include increased cardiac output, improved ventilation-perfusion matching, and reduced tissue metabolic activity and o consumption ( ). macrophages are differentiated mononuclear phagocytes that may reside in tissues for several months or be recruited to injured tissue from circulating monocytes, which then change phenotype. although macrophages are essential for tissue re- modeling and wound healing, when activated and present in fig. . tnf-� inhibition does not improve abnormal distal airway morphology. pups were treated from postnatal days – with . % saline vehicle (control; open bars), bleomycin alone ( mg·kg� ·day� ; closed bars), or bleomycin and etanercept ( . mg/kg ip alternate days; grey bars). a: representative low-power photomicrographs of hematoxylin and eosin-stained sections demonstrating marked distal airway simplification and septal thinning in bleomycin-treated animals, which was unaffected by treatment with etanercept. bar length � �m. b: morphometric analyses of mean linear intercept (lm), tissue fraction, and secondary crests or peripheral arteries per field, corrected for tissue fraction. values represent means se for n � animals/group. *p � . , by one-way anova, compared with other groups. #p � . , by one-way anova, compared with other groups. l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . exaggerated numbers, they may produce large quantities of reactive oxygen ( ) and nitrogen ( , ) species, bioactive lipid peroxidation products ( ), and cytokines ( ). in human preterm infants with respiratory distress, macrophage numbers in bronchoalveolar lavage fluid increase early in the second week of life ( ), remain elevated in infants who later develop clinical and radiological features of bpd, and decline in those who do not ( ). pulmonary macrophages are also central to the pathogenesis of pht induced by monocrotaline injection in rats ( , ) and in anti-platelet serum-induced pht in sheep ( ). we remain uncertain whether increased tissue macro- phages in the present model result from recruitment of circu- lating monocytes to the lung or whether they result from expansion of resident macrophages. however, our observation that expression of classical macrophage chemokines, including ccl (also known as monocyte chemoattractant protein- ), ccl , and ccl , were not increased in bleomycin-exposed animals suggests the latter possibility. of a number of cytokines screened to determine whether a causative role may exist, tnf-� was the only candidate found to be upregulated by bleomycin at the time point (day ) examined. that both macrophage numbers and tnf-� expres- sion/content were found to be attenuated by concurrent expo- sure to % co suggested a causative role in pht, with a lesser effect of % co and colocalization indicating that macrophages were the major source of tnf-�. lang et al. ( ) have described inhibition of tnf-� secretion by co in pulmonary macrophages activated by lps in vitro. exogenous administration of tnf-� is also known to increase pulmonary vascular reactivity in isolated rat lungs ( ), and its overex- pression has been described to cause emphysematous lung structure and pht in mice ( , ), similar to bleomycin- induced lung injury. furthermore, sutendra et al. ( ) recently reported that sustained inhibition of tnf-� signaling both prevented and reversed chronic pht in monocrotaline-exposed rats. similarly, a critical role for increased tnf-� signaling in pht was confirmed in the present model using a soluble tnf- receptor/igg fusion protein, etanercept, which prevents binding of the endogenous ligand to its receptors. our group has used this approach successfully in the past to explore the roles of various growth factors in lung development and injury ( , ). our data do not provide insight into the downstream mediators of upregulated tnf-� signaling that led to pht, but candidates implicated in other models include upregulation of endothelin- ( ) and activation of rho-kinase ( ). further, the upstream mechanisms causing a decrease in tissue macro- phages by % co were also not elucidated in the present study; however, our data suggest there is no relationship table . brain, liver, and kidney weights on day parameter/group vehicle � normocapnia bleomycin � normocapnia vehicle � % co bleomycin � % co brain weight, mg , , , , brain weight/body weight � . . . . . . . . liver weight, mg liver weight/body weight � . . . . . . . . kidney weights, mg * kidney weights/body weight � . . . . . . . . * values represent means sd; n � – animals per group. *p � . , by anova, compared with vehicle-treated % co -exposed group. fig. . neither bleomycin nor exposure to % co increases apoptosis in brain cortex. rep- resentative photomicrographs of terminal de- oxyuridine triphosphate nick-end-labeled sec- tions of brain cortex demonstrating no differ- ences in numbers of apoptotic nuclei between groups. inset (top left): dnase i-treated pos- itive control section. bar length � �m. l co prevents bleomycin-induced pht ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . between inhibition of tnf-� and decrease in recruitment or activation of macrophages in the bleomycin-exposed lung. in contrast with our previous findings in % o -exposed neonatal rats ( ), therapeutic hypercapnia did not improve abnormal lung structure in the present model. tourneux et al. ( ) reported in bleomycin-exposed neonatal rats that inhaled nitric oxide prevented pht and partially improved vascular rarefaction and lung structure, suggesting a major role for decreased endogenous no. our observation that pvr was normalized by % co , while arterial rarefaction was not, suggests that vascular hypoplasia was not the major factor accounting for raised pvr. these findings are in keeping with our previous work ( ) that demonstrated that acute treatment with a potent vasodilator (rho-kinase inhibitor) also normal- ized pvr, thus pointing to sustained vasoconstriction as the major contributor to raised pvr in bleomycin-exposed ani- mals, as described in other chronic pht models ( ). we observed that neutrophils, which were greatly increased in number in the lung tissue of bleomycin-exposed animals, were unaffected by exposure to co . these findings suggest that neutrophils alone are unlikely to be contributory to chronic pht and that macrophages may not recruit neutrophils to the bleomycin-exposed lung. indeed, work by others ( ) suggests that neutrophils do not play a major role in bleomycin-induced lung injury, as least in adult animals, but our current observa- tions leave open the question of their involvement in arrested lung development in the bleomycin-exposed neonatal rat. al- though there are many other factors in the bleomycin model that could contribute to arrested development, neutrophils have been suggested to play a part by producing proteases that disrupt elastin deposition, which is essential to secondary septation ( ). we examined the effects of co on inflammation and lung structure in control (vehicle-treated) animals and observed small, but nonstatistically significant, trends toward increased inflammatory cells, decreased mli, and decreased secondary crest numbers with % co . in neonatal mice, chronic expo- sure to % co from birth has been reported to induce changes in lung morphology consistent with accelerated maturation, including thinning of alveolar walls and increased secondary septation ( ). taken together, these observations sound a note of caution that the risks vs. benefits of chronic hypercapnia for long-term lung development, function, and susceptibility to lung injury remain unclear and require further study. in addi- tion, despite our present findings suggesting a lack of effect of % co on brain growth, there remains the potential for harmful effects of hypercapnia, or hypercapnic acidosis, on the immature brain ( , ). there are a number of limitations to this study. first, a major feature of bleomycin-induced lung injury is collagen deposi- tion ( ), which is not a feature of modern bpd ( ). our use of this model for the present studies was based on the striking degree of arrested lung growth and development, which is disproportionate to effects on other organs despite systemic administration, and the fact that macrophage influx, known to be involved in the pathogenesis of pulmonary hypertension, is a prominent early feature. second, our estimation of arterial blood gases in anesthetized animals introduced the likelihood of respiratory suppression, which may have confounded data on paco , although we anticipate that all groups would have been affected equally. our purpose in providing these data was to estimate the degree of change in paco and acid-base status secondary to provide some clinical context. in conclusion, our current findings indicate that therapeutic hypercapnia has a dose-dependent preventive effect on chronic bleomycin-induced pht relating to inhibitory effects on mac- rophage influx and subsequent attenuation of tnf-�. we propose that bleomycin-induced lung injury may represent a potentially useful new model for mechanistic studies relating to arrested development and chronic pht in the neonatal lung. grants this work was supported by operating funding from the canadian institutes of health research (cihr; mop- to r. p. jankov and mop- to a. k. tanswell) and by infrastructure funding from the canada foundation for innovation (to r. p. jankov). r. p. jankov is a cihr new investigator. disclosures no conflicts of interest, financial or otherwise are declared by the author(s). author contributions author contributions: a.c.p.s., a.k.t., and r.p.j. conception and design of research; a.c.p.s., c.k., j.i., a.h.l., a.m., and a.j. performed experiments; a.c.p.s., c.k., j.i., a.h.l., a.j., p.j.m., and r.p.j. analyzed data; a.c.p.s., a.j., p.j.m., a.k.t., and r.p.j. interpreted results of experiments; a.c.p.s., a.m., p.j.m., and r.p.j. prepared figures; a.c.p.s. drafted manuscript; a.c.p.s., c.k., j.i., a.h.l., a.m., a.j., p.j.m., a.k.t., and r.p.j. edited and revised manuscript; a.c.p.s., c.k., j.i., a.h.l., a.m., a.j., p.j.m., a.k.t., and r.p.j. approved final version of manuscript. references . abman sh, groothius jr. pathophysiology and treatment of broncho- pulmonary dysplasia. current issues. pediatr clin north am : – ., 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criteria – were programmed to autopopulate the ordering screen, including laxative name and time administered if given within hours. any contraindication to testing resulted in a “hard stop” prompt instructing prescribers to either exit the order or to submit the name of an approving infectious diseases (id) or gastrointestinal (gi) physician to override hospital protocol (see supplemental figure ). to ensure adherence, infection preventionists reviewed overrides weekly. approving id and/or gi physician names were verified, and physicians placing orders without appropriate approval received a warning e-mail signed by id and/or gi leadership and the chief medical officer (cmo) that reiterated protocol criteria and reminded physicians that orders without approval are being monitored. an e-mail with the following text was sent to physicians who did not seek proper approval for c. difficile testing when ordering criteria were not met: “we received notification that you have input false or non-id/gi physician names for approval of c. difficile testing in patients who were either ( ) already tested within days or ( ) had received laxatives within hours. testing outside of these parameters requires careful clinical consideration and approval from id/gi specialists. ordering without approval is being monitored. repeat inappropriate orders will be reported to your division chief, department chair, and chief medical officer.” we evaluated the following: ( ) national healthcare safety network (nhsn) case counts per , patient days and standardized infection ratios (sirs), ( ) tests ordered in patients receiving laxatives within hours, ( ) repeat testing within days, and ( ) protocol overrides. we used χ tests to compare changes in cdi testing and rates preintervention versus post intervention; quarterly sirs were compared using t tests. results the baseline cdi testing rate decreased from per , patient days preintervention to per , patient days postintervention (p = . ). the cdi testing in the hospitalonset (ho) period decreased % postintervention, from per , patient days preintervention to tests per , patient days postintervention (p < . ). at baseline, % of cdi tests were for patients receiving laxatives within hours, and % were ordered despite prior results available within days. testing while on laxatives decreased by %, from per , patient days preintervention to per , patient days postintervention (p < . ) (figure b). the number of cdi tests reordered within days also decreased by %, from per , patient days preintervention to per , patient days postintervention (p < . ). hospital-onset cdi rates decreased %, from per , patient days preintervention to cases per , patient days postintervention (p < . ), resulting in a % reduction in the average quarterly ho sir, from . preintervention to . postintervention (p < . ) (figure b). improved testing protocol compliance was tied to monitoring and feedback with a templated cmo response to physicians bypassing the protocol without approval. in the first month of implementation, there were unauthorized overrides, but these incidents decreased to zero by the end of the study period. figure . hospital-onset c. difficile infection (cdi) orders decreased after launch of the automated real-time intervention, while community-onset orders were unchanged. the number of orders placed for patients receiving laxatives decreased sharply after a real-time computer physician order entry (cpoe) system was launched. discussion proactive approaches to clinically appropriate diagnostic testing can be important for high-sensitivity tests, such as the c. difficile pcr test, which can identify colonization and can lead to unnecessary treatment and concern. – our real-time cpoe criteria-based testing protocol reduced inappropriate testing by % and ho c. difficile rates by % without changing the cdi testing method. electronic health record (ehr) strategies using passive alerts with information alone run the risk of being ignored over time and can be met with variable compliance. , our smart prompt provided clinicians with actionable data and also used a “hard stop” when testing criteria were not met. to address the rare but important possibility of cdi in complicated or high-risk patients not meeting testing criteria (eg, icu patient on daily laxatives who develops abdominal distention and leukocytosis), physicians could override the protocol with id or gi physician approval. this strategy encouraged thoughtful testing and provided an opportunity for specialist-level education of frontline physicians. electronic algorithms and protocols can often be circumvented; compliance monitoring and timely feedback are needed to achieve meaningful and sustainable changes. in our case, noncompliant physicians were sent e-mail warnings signed by our cmo, sending a clear message that appropriate testing was an institutional priority while also educating physicians. an important limitation of this intervention was the inability to capture the number of times a cdi test order was initiated but then cancelled due to the protocol, which limited our ability to describe the learning curve associated with this cpoe strategy. nevertheless, the sustained decreases in overall testing strongly suggest decreases in order initiation. data on the harmful effects antibacterial agents on the gut microbiome are mounting, and treatment of asymptomatic c. difficile colonization has been shown to increase future risk of colitis and recurrent disease. , in addition, oral vancomycin use increases the carriage rate of vancomycin-resistant enterococci, a drug-resistant organism associated with healthcare-associated infections. as data showing the harms of overtesting and overtreatment for cdi emerge, cpoe strategies can be an effective training tool to improve use and stewardship of diagnostic tests. , our electronic solution to enforce clinically appropriate cdi testing is an example of a strategy that integrates real- time cpoe alerts, specialist review, compliance monitoring and feedback, and leadership-level enforcement. acknowledgments financial support: no financial support was provided relevant to this article. potential conflicts of interest: all authors report no conflicts of interest relevant to this article. kathleen a. quan, rn, msn; ,a jennifer yim, rn, bsn; ,a doug merrill, md, mba, ma, fasa; usme khusbu, ms; keith madey, mafis, bba; linda dickey, rn, mph; amish a. dangodara, md; , scott e. rudkin, md, mba; , margaret o’brien, rn, bsn; daniel thompson, mafis; nimisha parekh, md, mph, facg, agaf; , c. gregory albers, md, facg; , william c. wilson, md; , , lauri thrupp, md; , cassiana e. bittencourt, md; susan s. huang, md, mph; , shruti k. gohil, md, mph , affiliations: . epidemiology and infection prevention, university of california irvine health, orange, california; . division of infectious diseases and health policy research institute, school of medicine, university of california irvine, irvine, california; . renown health, reno school of medicine, university of nevada, reno, nevada; . department of medicine, school of medicine, university of california irvine, irvine, california; . department of emergency medicine, school of medicine, university of california irvine, irvine, california; . health affairs information services, school of medicine, university of california irvine, irvine, california; . division of gastroenterology, school of medicine, university of california irvine, irvine, california; . department of anesthesiology, school of medicine, university of california irvine, irvine, california; . department of surgery, school of medicine, university of california irvine, irvine, california; . department of pathology, school of medicine, university of california irvine, irvine, california. supplementary material to view supplementary material for this article, please visit https://doi.org/ . /ice. . address correspondence to shruti k. gohil, md, mph, assistant professor, university of california, irvine, school of medicine, uc irvine health, the city drive, bldg , suite , rte , orange, ca (skgohil@uci.edu). aco-authors of equal contribution. received december , ; accepted: january , . infect control hosp epidemiol ; – © by the society for healthcare epidemiology of america. all rights reserved. doi: . /ice. . references . lessa fc, mu y, bamberg wm, et al. burden of clostridium difficile infection in the united states. n engl j med feb ; 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: – . . isaac s, scher ju, djukovic a, et al. shortand long-term effects of oral vancomycin on the human intestinal microbiota. j antimicrob chemother ; : – . . vrieze a, out c, fuentes s, et al. impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity. j hepatol apr ; : – . . al-nassir wn, sethi ak, li y, pultz mj, riggs mm, donskey cj. both oral metronidazole and oral vancomycin promote persistent overgrowth of vancomycin-resistant enterococci during treatment of clostridium difficile–associated disease. antimicrob agents chemother jul ; : – . corresponding author at: department of epidemiology, university of california irvine, irvine, ca - , united states. e-mail address: lhjiang@uci.edu (l. jiang). grant programs participating in the special diabetes program for indians diabetes prevention program: confederated tribes of the chehalis reservation, cherokee nation, cheyenne river sioux tribe, the chickasaw nation, coeur d'alene tribe, colorado river indian tribes, colville confederated tribes, cow creek band of umpqua tribe, fond du lac reservation, gila river health care, haskell health center, ho-chunk nation, indian health board of minneapolis, urban native diabetes prevention consortium, kenaitze indian tribe ira, lawton ihs service unit, menominee indian tribe of wisconsin, mississippi band of choctaw indians, norton sound health corporation, pine ridge ihs service unit, pueblo of san felipe, quinault indian nation, rapid city ihs diabetes program, red lake comprehensive health services, rocky boy health board, seneca nation of indians, sonoma county indian health project, south east alaska regional health consortium, southcentral foundation, trenton indian service area, tuba city regional health care corporation, united american indian involvement, inc., united indian health services, inc., warm springs health & wellness center, winnebago tribe of nebraska, zuni pueblo. turismo no espaço rural: uma reflexão acerca da autenticidade dos atrativos marcos luiz filippim * valmir emil hoffmann ** resumo a discussão proposta neste artigo consiste em analisar o caráter de autenticidade e singularidade dos atrativos turísticos, em especial no meio rural e, particularmente, nas chamadas fazendas-hotel, em contraponto ao processo de artificialização e espetacularização do turismo. o presente estudo apresenta resultados parciais de uma pesquisa que analisa as características do turismo rural no meio- oeste e planalto serrano de santa catarina, regiões onde surgiram as primeiras iniciativas dessa modalidade de turismo no brasil, em meados dos anos do século xx. a pesquisa trata de um estudo descritivo de ordem transversal e utilizou uma composição de mé- todos quali-quantitativos como instrumentos de coleta de dados e análise. os resultados apontam para uma tendência de adaptação dos espaços rurais destinados à visitação turística e apresentação de manifestações culturais tradicionais, sob forma de encenações alegóricas direcionadas aos visitantes. palavras-chave: turismo rural. autenticidade de atrativos turísticos. fazenda-hotel. artificialização do turismo. * mestre em turismo pela univali, professor no curso de turismo e administração da universidade do oeste de santa catarina – unoesc, campus de joaçaba, sc. ** mestre em turismo pela univali, professor no curso de turismo e administração da universida- de do oeste de santa catarina – unoesc, campus de joaçaba, sc. race, unoesc, v. , n. , p. - , introduÇÃo este artigo apresenta resultados parciais de uma pesquisa que aborda as características do turismo rural no planalto serrano e meio-oeste de santa catarina, realizada pelo programa de mestrado em turismo da universidade do vale do itajaí – univali. o presente estudo discute o caráter de autenticidade e singulari- dade dos atrativos turísticos de fazendas-hotel, situa- das na região de abrangência da pesquisa. conforme tulik ( ), o município de lages, situado no planalto serrano catarinense, foi o pionei- ro na exploração do turismo rural no brasil. segundo a autora, a partir de meados dos anos ; século xx, passou-se a buscar novas alternativas de renda para as tradicionais fazendas de gado da região, já que o turismo apresentou-se como uma opção adequada, pois os recursos paisagísticos, aliados ao clima típi- co da serra, onde se registram temperaturas negati- vas, ocorrência de neve e geadas durante o inverno, representavam potenciais atrativos para o mercado turístico emergente. além disso, as sedes de fazenda dispõem de aspectos histórico-culturais que também funcionam como atrativos, tais como: construções centenárias e manutenção de práticas tradicionais que podem ser presenciadas pelos visitantes. por essa razão, os empreendimentos estudados são qualificados como “fazendas-hotel”, já que man- têm suas atividades produtivas originais, que eram exploradas antes de passarem a operar com o turismo. assim, optou-se pelo uso desse termo, como alter- nativa aos chamados “hotéis-fazenda”, que, para os efeitos desta pesquisa, são empreendimentos com as mesmas características dos hotéis urbanos convencio- nais, mas que se situam em áreas rurais. nota-se, no entanto, que o senso comum e o material publicitário de alguns destinos qualificam genericamente como “hotel-fazenda” quaisquer estabelecimentos de hos- pedagem no meio rural. autores como harvey ( ) e connor ( ) sustentam que a humanidade está passando por um conjunto de transformações, especialmente no campo da cultura, que originou o que se convencionou cha- mar de pós-modernidade. postula-se que a fronteira entre o real e o virtual está se tornando paulatinamen- te mais tênue; dada a possibilidade técnica de desen- volvimento de universos paralelos e espaços virtuais. para baudrillard ( ), a humanidade é caracteri- zada por um grande simulacro, em que o verdadeiro cede lugar ao verossímil. ainda no espectro cultural, é importante mencionar uma tendência paradoxal: ao mesmo tempo em que se uniformizam procedimen- tos e práticas sociais, em decorrência do processo de globalização, também se observa uma tendência de valorização das culturas locais, talvez até como mo- vimento de resistência a esse fenômeno de homoge- neização. a intenção desta pesquisa é justamente investi- gar as implicações desse novo cenário sobre o objeto de estudo em questão; a experiência do turismo rural. na era da globalização, da vivência de não lugares, há pessoas (turistas) que idealizam um lugar onde passar as férias ou o final de semana, concedendo a esse lugar uma identidade, com atributos singulares de vivência local. a opção metodológica deste trabalho seguiu uma abordagem descritiva de ordem transversal. op- tou-se por uma composição entre as análises quali- quantitativa, uma vez que foram realizadas entrevis- tas com turistas e proprietários de empreendimentos, assim como um questionário com perguntas abertas e fechadas de múltipla escolha, que foi aplicado tanto nos destinos quanto em áreas de emissão dos turistas. neste artigo, em particular, foram considerados, espe- cialmente, os resultados obtidos na revisão teórica e nas entrevistas realizadas. a pesquisa foi realizada em três municípios do estado de santa catarina: Água doce e joaçaba, si- tuados no meio-oeste do estado, e lages, situada no planalto serrano. turismo rural o caráter relativamente recente da exploração e, especialmente, do estudo do turismo rural, confere à tarefa de defini-lo conceitualmente em um alto grau de dificuldade. a literatura da área remete a várias mo- dalidades de turismo que, por vezes, são englobadas por alguns estudos teóricos como rural, embora haja sérias dissensões entre os autores. o principal ponto discutido trata da definição de rural e urbano, ou seja, se o turismo praticado em áreas rurais pode ser deno- race, unoesc, v. , n. , p. - , minado como turismo rural apenas por ocorrer fora da zona urbana, embora seja desenvolvido em moldes idênticos àqueles praticados nas cidades ou se, por outro lado, deva estar associado à participação do vi- sitante em rotinas típicas do campo, assim como man- ter características socioculturais desse entorno. tulik ( , p. ) apresenta a seguinte reflexão: “turismo rural, no estrito senso, deve estar ligado às caracterís- ticas próprias do meio rural, excluindo-se desse rótu- lo outras formas que nada têm a ver com a prática e o conteúdo rural; deve estar ligado à paisagem rural, ao estilo de vida e à cultura rural.” considerando o interesse em prospectar a experiência turística em um espaço com características típicas e singulares, utili- za-se como base conceitual a premissa de que o tu- rismo rural deve estar necessariamente associado aos valores culturais, ao estilo de vida, ao imaginário e às práticas laborais do campo. assim, vale ressaltar o conceito apresentado no glossário do programa de mestrado em turismo e ho- telaria da universidade do vale do itajaí, pois conver- ge aos objetivos da pesquisa: “turismo rural – viagem que envolve vivência em propriedades rurais, usual- mente com o acompanhamento da rotina de trabalho destas.” (turismo: visÃo e aÇÃo, , p. ). as divergências terminológicas, em relação ao turismo rural, apresentam desdobramentos afetos a outros aspectos do setor, como os conceitos de fazenda-hotel e hotel-fazenda. conforme oliveira ( ), o segundo caso tem como característica o fato de ser uma estrutura hoteleira com os serviços oferecidos por esse tipo de empreendimento em qualquer lugar, situada em área rural, enquanto que o primeiro caso “é uma propriedade tipicamente rural, que mantém suas atividades primárias e possibilita o real contato entre a vida rural e os visitantes.” (oliveira, , p. ). a natureza desta investigação evidencia a op- ção pelo uso do termo fazenda-hotel, uma vez que as fazendas que constituem o objeto do estudo em ques- tão mantêm atividades produtivas tradicionais, sendo que a exploração turística consiste em uma alternati- va complementar de renda (em maior ou menor grau, conforme o empreendimento) e foram feitas apenas alterações para a recepção dos visitantes, buscando-se manter as características preexistentes. o turismo rural também representa uma alter- nativa ao turismo de massa. barreto ( , p. ), aponta como tendência a busca por hotéis e pousadas familiares situados em locais isolados, onde o turista convive com os proprietários e participa do cotidiano da vida no campo. ao mesmo tempo em que o turismo rural des- ponta como atividade que pode resgatar áreas econo- micamente estagnadas, ele também pode representar um alento à preservação de ambientes naturais. para talavera ( , p. ) “o turismo rural [...] é hoje contemplado como ferramenta estratégica na conser- vação do meio ambiente e na preservação das culturas locais.” o simulacro no turismo contemporÂneo a evolução tecnológica possibilitou a chamada “compressão do tempo-espaço”, como é qualificada por harvey ( ). esse autor pondera que o desen- volvimento de novas tecnologias de transporte e co- municações está promovendo o “encolhimento” do mundo. há algumas décadas, era necessário sema- nas para realizar a travessia do atlântico; aventura que, atualmente, não demanda mais que algumas horas. pelo mesmo viés, a internet e outras novas mídias permitiram a ampla circulação de informa- ções em tempo real, acessíveis de qualquer ponto do planeta. no âmbito do turismo, é possível as- sinalar como corolários dessa sofisticação os pro- cessos de desterritorialização e espetacularização. nesse campo, a artificialização do espaço turístico vem sendo amplamente discutida. moesh ( ), por exemplo, descreve a reprodução de ambientes afri- canos e tempos jurássicos em plena flórida, no walt disney world. a capacidade técnica de reprodutibilidade cêni- ca conduz ao simulacro. a virtualidade dos ambientes em alguns atrativos turísticos guarda estreita seme- lhança com as novas mídias, em que ambos podem caracterizar o advento da pós-modernidade; um tem- po em que se busca a satisfação das necessidades hu- manas e a fruição do lazer em espaços virtuais. mac- cannell (apud luchiari, , p. ) afirma que “a autenticidade encenada é construída pelas relações race, unoesc, v. , n. , p. - , sociais do turismo.” assim, a atividade turística seria considerada apenas um jogo, uma parte da cultura po- pular pós-moderna, em que as pessoas são encoraja- das a agir como turistas. na obra “viagem na irrealidade cotidiana”, eco ( ) descreve impressões de viagens que realizou pelas américas. o semiólogo italiano oferece um pa- norama da cultura kitsch quando examina os exage- ros dos museus de cera americanos, na tentativa de reproduzir fielmente obras de arte ou personalidades do passado. o leitor é levado a perceber, progressi- vamente, a situação de liminaridade entre o real e o ilusório a que se sujeita o visitante desses museus, o que parece ser o objetivo primaz do autor: “eis a ra- zão desta nossa viagem pela hiper-realidade, à procu- ra dos casos em que a imaginação [...] deseja a coisa verdadeira e para atingi-la deve realizar o falso abso- luto.” (eco, , p. ). a capacidade de reprodução já havia sido apon- tada por benjamin em sua proposição sobre a perda da aura das obras de arte a partir da possibilidade téc- nica de sua clonagem em série. ao analisar esse fenô- meno, urry ( , p. ) escreve que: afirmar que determinado fenômeno cultural tinha uma aura é dizer que ele era radicalmente separado do social, proclamava sua própria originalidade e singularidade, o fato de que era único, e baseava-se em um discurso de unidade orgânica formal e criatividade artística. a cultura pós-moderna é anti-aurática. tais formas não proclamam que são únicas, mas são mecânica e eletronicamente reproduzidas. existe uma negação da separação entre o estético e o social e do ponto de vista segundo o qual a arte pertence a uma ordem diferente que a da vida. o valor atribuído à unidade do trabalho artístico é desafiado por meio de uma ênfase no pastiche, na colagem, na alegoria, etc. as formas culturais pós-modernas não são consumidas em um estado de contemplação, a exemplo do que ocorre em um concerto clássico, mas de distração. a cultura pós-moderna afeta os espectadores através de seu impacto imediato, por meio daquilo que ela faz por alguém, através de regimes de prazer e não das propriedades formais do material estético. e se propusermos que as cidades ou os desti- nos turísticos também podem ser vistos como fenô- menos culturais pós-modernos ou antiauráticos? nas palavras de eco ( , p. ): “[...] a flórida se apre- senta como região artificial, continuum ininterrupto de centros citadinos [...], cidades artificiais dedicadas à diversão.” para carlos ( , p. - ), “cidades inteiras se transformam com o objetivo precípuo de atrair turistas, e esse processo provoca de um lado o sentimento de estranhamento [...] e de outro transforma tudo em espetáculo e o turista em espectador passivo.” em que proporção o turismo em espaços rurais insere-se no contexto descrito por urry ( ), eco ( ) ou carlos ( )? o pastiche e o impacto hip- nótico não estariam presentes nos próprios atrativos, sejam eles naturais ou construídos? o turista de fa- zendas-hotel percebe a artificialização do espaço rural para atender suas expectativas, quando presume-se; procura uma experiência genuína, de raízes? essas são algumas das questões que justificam este estudo e sobre as quais se deseja erigir uma reflexão. experiÊncia turÍstica no espaÇo rural: cultura local e autenticidade uma das motivações apontadas de forma mais recorrente para explicar o fenômeno turístico consiste no desejo de conhecer outras culturas. assim, parece razoável que os operadores de equipamentos turísti- cos realizem um esforço no sentido de evidenciar as especificidades dos destinos turísticos onde estão ins- talados; sejam estas de ordem física ou natural, histó- rica, arquitetônica, ou ainda, manifestações artísticas ou culturais. tais esforços, no sentido da caracterização da tipicidade dos destinos turísticos, podem levar, em alguns casos, a uma artificialização dos atrativos ou à espetacularização do turismo. esse fenômeno tem sido objeto de análise de vários estudiosos, tais como moesh ( ), luchiari ( ), yázigi ( ), entre outros. no que se refere ao meio rural, onde a popula- ção, especialmente a jovem, não constitui exceção à forte influência do processo de urbanização e mun- dialização da cultura, que lhes impõe padrões globais race, unoesc, v. , n. , p. - , de comportamento, a emergente demanda do turista pelas manifestações típicas pode significar a recupe- ração de práticas que estavam fadadas ao desapareci- mento. a valorização das práticas tradicionais e das ca- racterísticas de um determinado entorno espacial tam- bém podem revitalizar o sentimento de orgulho em relação ao local onde se vive e onde as experiências de convivência foram realizadas. tuan ( ) deno- mina esse fenômeno como topofilia. esse sentimento de pertencimento origina-se, assim, na experiência de vida, ou seja, na construção de uma existência em tor- no de um determinado ambiente físico e com a con- corrência de outras pessoas, com as quais se compar- tilha não apenas o espaço, mas também, um modus vivendi, uma praxis social. assim, as modificações produzidas em um am- biente natural a partir de sua antropização também podem ser entendidas como recursos turísticos, pois podem representar singularidades da ocupação que, freqüentemente, são reveladoras acerca da cultura dos grupos humanos que a promoveram. pires ( , p. , grifo do autor) afirma: a paisagem rural, situada numa posição intermediária na escala de modificações do ambiente natural perde, em tese, va- lor de naturalidade – um dos critérios adotados na valoração da qualidade visual da paisagem. porém, essa perda relativa pode ser compensada, inclusi- ve com vantagem, por outros atributos igualmente definidores da qualidade vi- sual como diversidade e singularidade, não raro encontrados no meio rural. a diversidade mencionada por pires ( ) diz respeito à ocorrência simultânea de variada gama de componentes visuais, tais como: relevo, água, vegetação, atividades humanas e outros fatores que tornam o cenário atraente em função de sua profusão. a singularidade seria dada pela “[...] presença de elementos ou da ocorrência de manifestações [...] impregnados de significados e valores captados pelo turista-usuário da paisagem e codificados pelos filtros perceptivos – pessoais, sensitivos, emocionais, sociais, culturais e educativos – de que é dotado [...].” (pires, , p. ). tuan ( ), ao analisar o fenômeno de iden- tificação do homem do campo com a terra, observa que, a topofilia do agricultor está formada dessa in- timidade física, da dependência material e do fato de que a terra é um repositório de lembrança e mantém a esperança. a apreciação estética está presente mas raramente é expressada (tuan, , p. ). o chamado repositório de lembrança será de- nominado por nora (apud augÉ, ) como lu- gares de memória e por augé ( ) como lugares antropológicos. o que caracteriza esses espaços é o fato de lhes ser atribuído um sentido vital por seus ocupantes, fruto da vivência neles. assim, a topofilia de tuan ( ), caracterizada pela afeição que une o homem a seu ambiente natural, seria necessariamente histórica, no sentido que “[...] aqueles que nele vivem podem aí reconhecer marcos que não têm que ser ob- jetos de conhecimento. o lugar antropológico, para eles, é histórico na exata proporção em que escapa à história como ciência.” (augÉ, , p. ). em outros termos, não se trata da história que se registra ou mesmo que se conta, mas da história que se vive. a idéia de um lugar marcado pela história pes- soal e social de seus habitantes opõe-se aos espaços especialmente construídos para o turismo que, con- forme carlos ( ), carecem de identidade, pois representam ambientes artificiais. assim, “o espaço produzido pela indústria do turismo perde o sentido, é o presente sem espessura, quer dizer, sem história, sem identidade; neste sentido é o espaço do vazio.” (carlos, , p. ). então, à medida que o tu- rismo rural proporciona um elemento novo na con- vivência das populações autóctones com seu meio, é natural que essa relação passe por transformações. essa reflexão suscita duas questões sobre as quais convém discorrer: como ocorre o processo de apropriação, pelo turista, da cultura ou ambiente lo- cal? os efeitos da ação ou presença desse novo perso- nagem transformam o modo de relação dos habitantes locais com seu meio? tomando as questões pela or- dem em que são apresentadas, pode-se imaginar um movimento de reação ao processo de homogeneiza- ção da cultura que a internacionalização da economia proporciona. esse movimento parece caracterizar-se, no que tange aos turistas, pela busca de atrativos e destinos autênticos, que proporcionam o contato com race, unoesc, v. , n. , p. - , uma história e cultura próprias e, ao lado dos habitan- tes locais, pode representar ênfase aos regionalismos. analisando a relação entre turismo rural e cultu- ra local, donnermeyer ( ) descreve a experiência dos amish, uma cultura religiosa e étnica tradicional, localizada no nordeste do estado de ohio, eua. se- gundo esse autor, onde quer que haja um povoado de famílias amish, há turistas que “desenvolveram um interesse ávido por culturas tradicionais e grupos sub- culturais únicos.” (donnermeyer, , p. ). o autor observa que o turismo provoca alterações no cotidiano dos amish, criando novas ocupações até en- tão inexistentes, embora não veja o fenômeno como uma ameaça à cultura local, pois houve uma acomo- dação quanto as condições impostas pela sociedade, a fim de manter suas tradições; também eles desen- volveram habilidades de lidar com o turismo que lhes permitissem a sobrevivência. por outro lado, donnermeyer conclui que “o tráfego é mais um incômodo que uma ameaça, e não atinge o tecido da cultura amish. porém, o preço da terra ascendente limita severamente a habilidade dos jovens amish de se tornarem agricultores e assim criarem suas famílias no estilo historicamente tradi- cional.” (donnermeyer, , p. ). no que se refere às razões para o sucesso dos amish, em sua relação com o turismo, aparece a questão do modelo desse relacionamento: a [...] razão para o sucesso dos amish em lidar com o turismo, e para a pro- vável continuação desse sucesso, é que seu limite com os turistas é protegido, em parte, por outras pessoas que vivem na região [...]. esses indivíduos são os que estabelecem empresas que suprem os turistas. isso serve para impedir que os turistas tenham contato direto com os amish, contudo proporciona para os turistas uma falsa sensação de ter ex- perimentado a “sociedade amish”, com seu estilo “tradicional e rústico.” (don- nermeyer, , p. ). a falsa sensação de ter conhecido uma cultu- ra, aludida pelo autor, é reforçada por krippendorf ( ), para quem “nem o turista, nem o autóctone sabem como é, de fato, o universo do outro. eis por que a viagem geralmente conduz à confirmação recí- proca dos clichês [...]. estamos longe de um encontro verdadeiro entre os turistas e os autóctones. muito longe, de fato.” (krippendorf, , p. ). as- sim, no entendimento do pensador suíço, as viagens contemporâneas não contribuem para o conhecimen- to entre os povos, mas antes, reforçam preconceitos mútuos acerca do estilo de vida do outro. diante de tais assertivas, retorna-se à questão proposta anteriormente: como explicar o interesse em conhecer novas culturas, freqüentemente apon- tado como causa da viagem pelos turistas? para ta- lavera ( , p. ), “o turista rural vê a si mesmo como único, como aventureiro explorador e aluno de culturas e natureza, às vezes vistas como a sua própria em outra dimensão temporal.” o pressupos- to de revisitar a própria cultura em outro estágio de desenvolvimento revela uma idéia de atraso tecno- lógico das áreas rurais, como se essas permaneces- sem incólumes ao processo de desenvolvimento. o mesmo autor sustenta que esses aspectos deter- minam a necessidade de que os moradores locais sejam treinados para atender as expectativas dos vi- sitantes, sendo necessário “reconstruir sua apresen- tação ante os outros [...] [pois] os turistas passam a ter entidade cultural para os moradores e, com isso, as modificações realizadas, para satisfazer a deman- da, em sua própria cultura.” (talavera, , p. - ). como se vê, a premissa de krippendorf acerca da criação dos clichês e preconceitos está confirmada. winkin ( ) propõe que a relação turística caracteriza-se pela mediação entre o visitante e os autóctones. em seu ensaio “o turista e seu duplo”, ele progressivamente busca convencer de que ocorre uma intermediação entre o turista e o local visitado, feita pelo guia ou por algum preposto responsável pela recepção. assim, retomando a questão da au- tenticidade, esse autor propõe que há uma distinção entre as figuras do viajante e do turista; o primeiro buscaria um contato direto, não mediado, com os lo- cais que visita e, o segundo, busca somente diversão. no entanto, o próprio winkin reconhece a impossi- bilidade da relação pura: “ou ele se imobiliza, quer ficando em casa, quer sedentarizando-se na casa do habitante – e então ele já não é um viajante. ou então viaja: ‘corre para os lugares não-turísticos’, e sua mera race, unoesc, v. , n. , p. - , presença transforma esses lugares em lugares turísti- cos e ele num turista.” (winkin, , p. ). a pretensa divisão entre viajante e turista, atri- buindo a cada qual um estatuto distinto, ultrapassa a questão da autenticidade dos atrativos, pois remete à noção de que há um movimento voluntário dos agentes receptivos no sentido de espetacularizar determinadas práticas culturais ou produtivas e ofe- recê-las como um produto turístico. assim, o inte- ressado em conhecer manifestações autênticas não poderia fazê-lo porque o modelo de relação entre os autóctones e seu meio foi alterado para vendê-lo aos visitantes. talavera ( ) também discute esse fenômeno: o ente patrimonial [...] deve ser fre- qüentemente recriado – ou construído ex-novo –, acompanhado de uma ceno- grafia apropriada e, de maneira espo- rádica e conforme requeira o mercado, espetacularizado. ao entrar na dinâmica do mercado e da cultura, o sentimento de pertença, de origem, mais ou menos sacralizado, e de renovação do grupo, é geralmente prefixado pelos intermediá- rios da viagem como mostras de auten- ticidade do lugar. (talavera, , p. - ). winkin ressente-se da impossibilidade de co- nhecer um determinado local e seu povo como ele realmente é (ou era), pois “sei que não posso fugir à minha condição de turista” (winkin, , p. ). para o autor, a preparação de um ambiente ou a ence- nação de um ritual com o intuito de que seja assistido pelo visitante quebra o encantamento. em outro mo- mento, ele menciona uma ocasião em que visita um local ainda não “contaminado”, onde presencia uma apresentação de dança típica: [...] “eles não dançam para nós; eles con- tinuam a dançar quando vamos deitar”. imediatamente nos animamos: as danças parecem-nos “autênticas”; não somos considerados vulgares turistas que têm que ser divertidos. não nos remetendo à nossa condição objetiva, a equipe local consegue, menos por sutileza que por ingenuidade, ganhar nossa confiança e preservar o encantamento. (winkin, , p. , gripo do autor). a busca, então, pela autenticidade do lugar re- flete-se na tentativa de apropriar-se da cultura que se está conhecendo por meio do consumo de objetos ou bens que possam representar um souvenir válido e re- presentativo do modus vivendi local. para talavera ( ), há uma preferência pelos bens usados ou em uso que não possam ser confundidos com produtos turísticos. o turista rural [...] interessado pela cul- tura, pede da área visitada um elemen- to diferencial que não seja confundível com o objeto turístico, que possa ser identificado como próprio e exclusivo, como exótico e, se possível, como usa- do. o esteticamente atrativo do souvenir pode ser substituído pela qualidade ex- pressa de constituir uma representação étnica ou de estilos de vida diferentes do seu. esse turista sente, ao menos, curio- sidade pelo estilo e pela forma, pelo que representa e expressa para os morado- res, pela história real ou imaginária do objeto que, apesar de tudo, deve cumprir para ele sua missão recordatória e suntu- ária. (talavera, , p. ). a demanda por objetos em uso ou usados en- contra o obstáculo do envelhecimento da população e substituição das práticas produtivas. assim, talavera ( ) observa que esse fenômeno irá contribuir para a recuperação da atividade artesanal com o objetivo de recriar objetos que tiveram uma função importante em processos laborais já abandonados ou em fase de substituição. É interessante observar que esses bens são agora produzidos com uma nova função, qual seja, servir como prova ou souvenir de uma experi- ência turística. segundo talavera ( , p. , grifo do autor), “o bem ‘usado’ chega a representar, para muitos compradores, um objeto de culto: é exposto e mostrado como tal, sendo considerado a representação mesma do passado e dos estilos de vida”. assim, o turismo pode representar a transformação de espaços de trabalho em espaços de lazer e de objetos e imagens de práticas produtivas em ícones de um modelo existencial experienciado pelo turista. a tentativa de conseguir um objeto usado in- dica a preocupação com a autenticidade do souvenir, race, unoesc, v. , n. , p. - , buscando diferenciá-lo do objeto turístico, este pro- duzido especialmente para o consumo do turista. as- sim, o visitante efetivamente interessado pela cultura local, normalmente avesso aos pacotes turísticos e aos atrativos de massa – seguindo a tipologia de plog para os turistas alocêntricos – tende a sentir-se usado pela “indústria” do turismo, que o reduz a consumidor pas- sivo de espaços turísticos encenados para diverti-lo e atraí-lo ao consumo. descrevendo uma viagem que realizou pelo havaí, carlos ( ) explica como se sentiu: a indústria do turismo transforma tudo o que toca em artificial, cria um mundo fictício e mistificado de lazer, ilusório, onde o espaço se transforma em cenário para o “espetáculo” para uma multidão amorfa mediante a criação de uma série de atividades que conduzem à passividade, produzindo apenas a ilusão da evasão, e, desse modo, o real é metamorfoseado, transfigurado, para seduzir e fascinar [...]. sensação que se é parte de um cenário, a sensação de que tudo é controlado, que cada passo seu ou mesmo cada gesto é esperado, cada atitude predeterminada [...]. o show de “hula” dançada em cenários cinematográficos com scripts bem ensaiados e pausas para fotografias no meio e no final do espetáculo quando os espectadores são convidados a se somarem às dançarinas para tirar fotos, como parte intrínseca do show. (carlos, , p. - , grifo do autor). nesse trecho, a autora evidencia as diferen- ças de nuances no perfil do turista, o que é par- ticularmente importante aos empreendedores do turismo que, na tentativa de agradar a um público de massa, podem estar relevando ao descrédito o próprio negócio, à medida que uma parcela dos visitantes enxerga evidências de simulação nos atrativos. a questão de fundo deste estudo preocupa-se com a natureza da experiência que os turistas de fa- zenda-hotel vivenciam e esperam desse tipo de des- tino. assim, o trecho do depoimento de um hóspede, (informante ) de fazenda-hotel, evidencia como se sente em relação a sua visita ao imóvel e caracteriza sua percepção acerca da experiência: o que me chamou atenção é que ele, como posso dizer... devia ser uma casa de uma família um pouco grande e eles transformaram num hotel, né. então, você tem os dormitórios que tinha anti- gamente, foi feito um ou dois banheiros, como posso dizer, não tem como chegar em um hotel onde tem uma suíte com ar condicionado e tal, é uma coisa mais rústica [...] eu acho que faz parte, é as- sim que tem que ser. poderia até ter, uma coisa reservada, uma suíte com ar con- dicionado, mas eu acho que no turismo rural tem que funcionar desse jeito [...]. você sente como uma visita dentro de uma casa de família [...] não é serviço de empregado... você se mistura junto com a família. o depoimento do turista aponta para uma per- cepção diferenciada do que ele considera turismo e “uma visita dentro de uma casa de família”, aparen- temente colocados em campos opostos. a apologia à rusticidade do ambiente denota o interesse em encon- trar um espaço não modificado para receber o visitan- te, que conserve suas características naturais, ainda que isso implique em um nível de conforto menor. parece significativo que os turistas entrevistados não percebam como problema as condições de acesso a um dos empreendimentos, ainda que sejam precárias. quando questionados sobre dificuldades para chegar ao hotel, os informantes normalmente apontam falhas na sinalização, porém não mencionam como obstá- culo as condições da estrada. sobre esse aspecto, um dos hóspedes (informante ) afirma: [...] foi tranqüilo, né, claro que um car- ro mais sofisticado sofre um pouquinho, porque é chão, um pouco de pedra, mas não tem problema de barro, não tem pro- blema de ficar na estrada... é precário... é um acesso bem... mas também não faz mal...quer dizer o estilo hotel-fazenda, você tem um pouquinho de dificuldade pra chegar, né? o depoimento faz alusão ao “estilo hotel-fazenda”, encarando com naturalidade as dificuldades de aces- so, como se estas compusessem o cenário rural. ocor- re novamente a busca de um espaço real, não artificia- lizado para a recepção do turista, que deseja conhecer race, unoesc, v. , n. , p. - , o ambiente rural como ele realmente é, ou como o idealiza. concorre também para confirmação dessa tese, a explicação do proprietário de um dos empreen- dimentos sobre o fato de manter na estrada de acesso inúmeras porteiras. segundo (informante ) , os hós- pedes gostam de ser fotografados abrindo as porteiras porque isso faz parte da vida na fazenda. depreende- se, a partir disso, a construção de uma representação do que seja a vida no campo. essa representação compõe o imaginário do turista que aparentemente aspira a participar efetiva- mente das atividades ali desenvolvidas, passando de mero visitante a partícipe interativo do momento que está presenciando. as alusões à exuberância da natureza e à tran- qüilidade do lugar são freqüentes nas manifestações dos hóspedes. uma das fazendas dispõe de um livro, em que são registradas as impressões dos visitantes. ali se encontram frases como as que foram transcritas a seguir: “recanto da paz”; “beleza, energia e acon- chego”; “local para se recarregar as energias”. as impressões aqui transcritas identificam o caráter ou propriedade terapêutica que os visitantes atribuem ao local, como se a natureza tivesse a condição de supe- rar o estresse provocado pela vida moderna. em de- poimentos orais dos turistas, esse fenômeno também aparece. uma vez questionado sobre o que procura na fazenda-hotel, o informante menciona: primeiro descansar, relaxar, né? como aqui tem trilhas, a gente dá aquela... fazer um pouco de exercício, a questão de cachoeira que relaxa muito ficar em contato com a água, então é relaxamen- to, mais tirar um pouco do stress do dia- a-dia e também passear um pouco, dar essa saída para um ambiente novo do normal, de cidade em si. a natureza da descrição do hóspede remete a uma idealização da natureza, como se fossem atribu- ídas virtudes mágicas ao contato com o meio natural, colocando-o em contraponto ao espaço urbano, este visto como algo quase pernicioso. a contraposição do urbano versus rural também parece ocorrer quando se analisa a questão do nível de desenvolvimento tecnológico que os hóspedes esperam encontrar na fazenda-hotel. a idéia de um espaço que permanece alheio à evolução tecnoló- gica parece compor o imaginário dos visitantes; também freqüente em manifestações artísticas po- pulares. em uma das entrevistas, o segundo informante, ponderando sobre os equipamentos necessários a uma fazenda-hotel, menciona: “não precisa ter piscina, não faz falta; telefone não faz falta [...]; a televisão tinha, mas se não tivesse não faria falta, não tem necessida- de”. a natureza desse depoimento parece remeter a uma tentativa de afastamento das rotinas do cotidia- no, tipicamente urbano. o que parece particularmente diferenciado no turismo em fazendas-hotel consiste na busca do atrati- vo original, supostamente não preparado para receber o turista. esse fenômeno sustenta-se em parte; porque uma parcela significativa dos praticantes desse tipo de turismo conhece ou teve alguma espécie de vivência em áreas rurais ou no seu entorno, conforme eviden- ciou o resultado da pesquisa. É oportuno observar aqui que os depoimentos dos turistas apontam para uma idealização do que es- peram encontrar na fazenda. essa projeção baseia-se em dois aspectos: sua experiência vivencial anterior, ou os chamados lugares de memória (nora apud augÉ, ) e em informações que são obtidas na imprensa, cinema, material publicitário ou outras mí- dias. ocorre, então, a midiatização do turismo – como de resto de qualquer atividade humana. a interativi- dade e a virtualidade, emblemas onipresentes nos processos comunicacionais contemporâneos, apre- sentam suas faces também na produção do turismo. “a interatividade nos ameaça de toda a parte”, dirá baudrillard ( , p. ), analisando o fenômeno. há, então, maiores possibilidades de escolha? o pró- prio autor conclui por outro viés, contrapondo a essa aparente autonomia do turista ou do receptor de men- sagens na comunicação, a idéia de que tudo consiste num grande simulacro; “a demanda não é mais que a resposta à solicitação do modelo” (baudrillard, , p. ). constata-se, portanto, a perda do direi- to, ou mesmo da possibilidade de opção, a partir de um comportamento reativo, ou seja, o turista encon- tra um cenário organizado nos moldes do que lhe foi anteriormente apresentado no material publicitário do destino turístico. race, unoesc, v. , n. , p. - , a busca da autenticidade revela-se no esfor- ço de encená-la da parte dos promotores do turismo. nesse sentido, há uma nítida ênfase na manutenção de equipamentos antigos e na resistência à adoção de no- vas tecnologias nos processos laborais ou mesmo nas acomodações dos hóspedes. um dos proprietários, (informante ) relata que muitos hóspedes preferem que não haja aparelhos de televisão em seus quartos. o local também não dispõe de serviço de telefonia celular, mas isso sintomaticamente não está entre as preocupações do proprietário. no mesmo empreen- dimento, a casa centenária onde funciona a pousada foi ampliada, mas tentou-se manter suas característi- cas arquitetônicas originais, conforme depoimento do proprietário. o contexto descrito contribui para a inferência de que o turista de fazenda-hotel privilegia o espaço original, não preparado para recebê-lo. no entanto, muitas vezes não percebe que ocorre uma maquiagem desse espaço no intuito de mantê-la artificialmente com as características originais, por mais contraditó- ria que pareça essa assertiva. assim, as porteiras não são substituídas por mata-burros (espécie de pontilhão com espaços vazados em linhas horizontais, o que permite a passagem de pessoas e veículos mas não do gado) porque esse equipamento, embora mais prático e eficiente, não compõe o imaginário do que se espera encontrar no campo. a velha charrete também é pre- ferida em relação ao transporte em automóveis. conclusÃo os resultados obtidos na pesquisa indicam que a maioria dos informantes prefere espaços e atrativos que sejam adaptados para o turismo, a fim de garan- tir a comodidade e segurança dos usuários, embora considere que devam ser mantidas as características originais desses atrativos. o acompanhamento de práticas tradicionais do entorno visitado e os contatos com a cultura lo- cal não são prioritários e acontecem sob o formato de shows ou espetáculos para serem presenciados desde uma “bolha” de segurança, o que torna esse conta- to intercultural praticamente impossível. em outros termos, o que o turista vê, efetivamente, não são as comunidades visitadas e a população autóctone em seu modus vivendi cotidiano, mas uma encenação de natureza alegórica, cuidadosamente preparada para o consumo dos visitantes sob um caráter meramente contemplativo. essa produção artificial de rusticidade e de prá- ticas locais encoraja a chamada “turistificação” dos lugares e a espetacularização do turismo, movimento já observado por silva ( ), que se caracteriza pela conversão dos espaços para que os turistas possam consumi-los, ou seja, transformar deliberadamente esses espaços em bens mercantilizáveis. esse fenô- meno, embora mais característico de áreas urbanas, balneários e parques temáticos, parece estar também atingindo o meio rural. baudrillard ( ) afirma que a humanidade vive na cultura do simulacro, en- quanto maccannell ( apud luchiari, , p. ) chama esse processo de “autenticidade encenada [que] é construída pelas relações sociais do turismo.” cabe, no entanto, ponderar que a produção de espaços – artificiais ou não – destinados ao consumo turístico, não constitui demérito para os operadores do turismo, uma vez que estes atendem a uma deman- da que busca formas de lazer e entretenimento para se distanciar das pressões do cotidiano e, mesmo que esses destinos ou práticas compõem um simulacro, também é verdadeiro que oferecem segurança e co- modidade aos turistas e proporcionam oportunidades de emprego e renda às comunidades receptoras. assim, pode-se mencionar uma divisa acerca das modalidades mais alternativas ou exóticas de tu- rismo que se atribui a ruschmann ( ), por mais rústica ou alternativa que seja a proposta de um des- tino, no final do dia os turistas querem três coisas: um banho quente, uma cerveja gelada e uma cama confortável. os resultados desta investigação indicam que esse fenômeno tende a acontecer no turismo rural dirigido às fazendas-hotel da região estudada e, tal- vez, por um processo de indução, possam ser conva- lidados para empreendimentos desse gênero situados em outros lugares. race, unoesc, v. , n. , p. - , tourism in the agricultural area: a reflection concerning the authenticity of the attractions abstract the proposed discussion in this article consists of analyzing the authenticity and singularity character of the tourist attractions, especially in the agricultural area and particularly in the “farm-hotels”, besides the process of making this kind of tourism artificial and as a spectacle. the present study presents partial results of a research that analyzes the characteristics of the agricultural tourism in the western region of santa catarina state, where the first initiatives of this modality of tourism in brazil had appeared, in the xx century ’s. the research is a descriptive study of transverse cut and it was used a composition of qualitative and quantitative methods as instruments of data collection and analysis. the results point to a trend of the agricultural spaces adaptation des- tined to the tourist visitation and presentation of traditional cultural manifestations under the form of allegorical stages destined to the visitors’ consumption. keywords: agricultural tourism. authenticity of tourist attractions. farm-hotel. artificial tourism. notas explicativas depoimento fornecido a marcos luiz filippim por informante em . depoimento fornecido a marcos luiz filippim por informante em . depoimento fornecido a marcos luiz filippim por informante em . referÊncias augÉ, m. não-lugares: introdução a uma antropologia da supermodernidade. campinas: papirus, . barretto, m. manual de iniciação ao estudo do turismo. . ed. campinas: papirus, . baudrillard, j. tela total: mito-ironias da era do virtual e da imagem. porto alegre: sulina, . carlos, a. f. a. o turismo e a produção do não-lugar. in: yÁzigi, e.; carlos, a. f. a.; cruz, r. de c. a. da. turismo: espaço, paisagem e cultura. . ed. são paulo: hucitec, . parte i, cap. , p. - . connor, s. cultura pós-moderna: introdução às teorias do contemporâneo. . ed. são paulo: loyola, . donnermeyer, j. f. turismo rural e cultura local: a experiência amish. in: almeida, j. a.; riedl, m. (org.). turismo rural: ecologia, lazer e desenvolvimento. bauru: edusc, . cap. , p. - . eco, u. viagem na irrealidade cotidiana. . ed. rio de janeiro: nova fronteira, . harvey, d. condição pós-moderna. são paulo: loyola, . krippendorf, j. sociologia do turismo: para uma nova compreensão do lazer e das viagens. . ed. são paulo: aleph, . race, unoesc, v. , n. , p. - , luchiari, m. t. d. p. urbanização turística: um novo nexo entre o lugar e o mundo. in: serrano, c.; bruhns, h. t.; luchirari, m.t. d. p. olhares contemporâneos sobre o turismo. . ed. campinas: papirus, . cap. , p. - . moesch, m. a produção do saber turístico. são paulo: contexto, . oliveira, c. g. de s. viabilidade e sustentabilidade do turismo rural. brasília: sescoop, . disponível em: <http://www.turismocooperativo.coop.br>. acesso em: fev. . pires, p. dos s. a paisagem rural como recurso turístico. in: rodrigues, a. b. (org.). turismo rural. são paulo: contexto, . parte , p. - . rodrigues, a. b. turismo rural no brasil: ensaio de uma tipologia. in: _____ (org.). turismo rural: práticas e perspectivas. são paulo: contexto, . p. - . ruschmann, d. van de m. turismo e desenvolvimento sustentável: a proteção do meio ambiente. . ed. campinas: papirus, . ______ . planejamento e gestão sustentável dos espaços para o turismo. aula proferida no programa de mestrado em turismo e hotelaria da universidade do vale do itajaí. balneário camboriú: univali, . silva, m. da g. l. da. cidades turísticas: identidades e cenários de lazer. são paulo: aleph, . talavera, a. s. o rural como produto turístico: algo de novo brilha sob o sol? in: serrano, c.; bruhns, h. t.; luchirari, m.t. d. p. olhares contemporâneos sobre o turismo. . ed. campinas: papirus, . cap. , p. - . tuan, y. f. espaço e lugar: a perspectiva da experiência. são paulo: difel, . tulik, o. turismo rural. são paulo: aleph, . turismo visÃo e aÇÃo. universidade do vale do itajaí, curso de mestrado em turismo e hotelaria. ano , n. . itajaí: univali, fev. . semestral. issn - . urry, j. o olhar do turista: lazer e viagens nas sociedades contemporâneas. . ed. são paulo: sesc, . winkin, y. o turista e seu duplo. in:______. a nova comunicação: da teoria ao campo de trabalho. campinas: papirus, . cap. , p. - . yÁzigi, e. a alma do lugar: turismo, planejamento e cotidiano em litorais e montanhas. são paulo: contexto, . race, unoesc, v. , n. , p. - , in this issue kidney international ( ) http://www.kidney-international.org © international society of nephrology i n t h i s i s s u e kidney international ( ) , . doi: . /ki. . it’s now cheaper to print color figures in ki have you been concerned in the past about the cost of print- ing color figures in kidney international (ki)? ki has listened to your concerns and is now offering permanently reduced pricing for color figures—a flat fee of $ per printed page, no matter how many color figures are on a page. this pricing is more affordable for authors and competitive with other top scientific journals. we want to make it easier for authors to publish the color figures that demonstrate important scien- tific findings, and our new pricing reflects our commitment to achieving this goal. chronic kidney disease following acute kidney injury tradit iona l t houg ht has held t hat acute kidney injury (aki) is revers- ible. however, as the population ages, and patients with more serious dis- eases survive, the issue of whether aki actually leads to de novo chronic kid- ney disease (ckd) has emerged as an important epidemiological question. as they report in this issue, bucaloiu et al. examined the records from a single hospital and found that of those patients who were discharged and alive months later, had aki that resolved. a cohort of formed the control group and had no aki. follow-up of these two groups of patients for more than years showed that the risk of death associated with reversible aki was significant. reversible aki was associated with a significant risk of de novo ckd. these studies demonstrate that patients who develop aki need to be followed up for appearance of ckd. see page . hyperfiltration and progression in diabetes to investigate the consequences of hyperfiltration in diabetes, moriya et al. studied patients with type diabe- tes. they measured glomerular filtration rate (gfr), by the iohexol clearance, and estimated the filtration surface in glomeruli determined by renal biopsy and morphometr y. they found that gfr correlated with only the filtration surface area among other morpho- logical measurements. the filtration area was positively correlated with the decrease in gfr in the first year of fol- low-up but not later. gfr decreased in nine of the patients followed, while in patients, there was no change in gfr. those that had hyperfiltration were more likely to have a subsequent decline in gfr. see page . uric acid and blood pressure there is increasing evidence for the association of uric acid levels and hypertension. parsa et al. studied the relationship between blood pressure and single-nucleotide polymorphisms of glut , a uric acid transporter, in an amish community. all subjects were untreated and were placed on known diets with high or low salt intake. the investigators found that each copy of the glut minor ile allele conferred a sig- nificant . -mg/dl reduction in serum uric acid, which was associated with a significant mean decrease in the systolic blood pressure on the high- and low- sodium diets. this result shows in the mendelian randomization cohort that reduced uric acid lowered blood pres- sure. see page . http://www.kidney-international.org 	chronic kidney disease following acute kidney injury 	hyperfiltration and progression in diabetes 	uric acid and blood pressure abstract .. abstracts st annual meeting . american society of preventive oncology, houston, texas . march – , the following are the highest-scoring abstracts of those submitted for presentation at the st annual aspo meeting to be held march – , in houston, texas. cytokine polymorphisms and pain in lung cancer reyes-gibby c, spitz m, wu x, merriman k, etzel c, kurzrock r, shete s purpose: cytokines, aberrantly produced by cancer cells, have recently been implicated in the severity of cancer- related pain. we hypothesize that functional variations in cytokine genes could explain the variability in cancer- related pain. methods: pain, clinical and demographic variables were assessed at presentation and prior to initiating any cancer treatment in patients with non – small cell lung cancer. using the taqman method, we genotyped single nucleotide polymorphisms in interleukin (il)- (� gc), il- (� ta), and tumor necrosis factor-a (tnf-a; � ga), and determined their associations with pain severity in newly diagnosed early and advanced stage lung cancer. results: white caucasians with early (n = ) and advanced stage (n = ) non – small cell lung cancer comprised the sample. pain severity predictably varied by stage of disease, sex, depressed mood, age, and genotype groups. linear regression analyses showed tnf- � ga (coeff = . ; p = . ); sex (coeff = . ; p = . ), and age (coeff = � . ; p = . ) as significant predictors for pain severity in early stage lung cancer. among those with advanced stage lung cancer, we observed statistical- ly significant main effects for il- � ta (coeff = . ; p < . ) and significant joint effects of il- � ta and age (coeff = � . ; p < . ) and tnf- � ga and age (coeff = . ; p < . ) on pain severity. classification and regression tree analyses showed the same distinct patterns for early and advanced stage lung cancer. conclusion: variations in individual inflammatory responses could partly explain the variability in cancer- related pain among patients with early and advanced stage lung cancer. long-term follow-up of women’s decisions to share brca / test results with first-degree relatives tercyak kp, graves kd, peshkin bn, gell ce, hecker sl, schwartz md purpose: we investigated the prevalence of disclosure of genetic test results to first-degree relatives among women who had participated in brca / testing to + years previously. we also assessed women’s closeness to each of these relatives at the time they underwent testing, and examined disclosure-closeness relationships. methods: interviews were conducted by telephone with women—all of whom were the first members of their families to be tested for brca / mutations. respondents were asked if they had disclosed (yes/no) to their mothers, fathers, sisters, brothers, spouses, and children (as applicable). results: the frequency of disclosure was . % to mothers, . % to fathers, . % to sisters, . % to brothers, . % to spouses, and . % to children (reported as a percentage of those who disclosed/presence of that relative category in the family). after controlling for the effect of positive genetic test results, women who felt more personally connected to their adult relatives were more likely to have disclosed to their mothers (r = . , p = . ), fathers (r = . , p = . ), brothers (r = . , p = . ), and spouses (r = . , p = . ); closeness was unrelated to disclosure to sisters and children, although older children (age > ) were more likely to be informed than younger children (r = . , p = . ). summary: these data suggest that the majority of first- degree relatives were informed of women’s test results. in addition to age and gender, family dynamics seem to be related to disclosure decisions. to promote cascade testing, open communication, and social support, novel counseling strategies may be warranted. antioxidant nutrients and oxidative dna damage in healthy african-american and white adults watters jl, satia ja purpose: to examine potential racial differences in: (a) dietary intakes and plasma concentrations of vitamin c, vitamin e, and carotenoid and oxidative dna damage (odd) levels, and (b) associations between plasma antiox- idants and odd. methods: data were from the diet, supplements, and health study, a cross-sectional study of generally healthy nonsmoking african-americans and whites in north carolina ages to . participants completed a demographic and health questionnaire, a newly developed antioxidant food frequency questionnaire, four -h dietary recalls, and a dietary supplement inventory, had height and weight measured, and provided a semifasting blood sample. results: african-americans had statistically significantly lower plasma concentrations of vitamin a, vitamin e, a- and h-carotene, and lutein + zeaxanthin than whites, as well as lower self-reported intake of most antioxidants. levels of odd, measured using the alkaline comet assay, were lower in african-americans than whites. an inverse association between lycopene and odd (pearson’s r = � . , p = . ) was found in the combined study population after controlling for sex, age, body mass index, passive smoke exposure, physical activity, educa- tion, income, and alcohol intake. there was also a significant positive association of a-tocopherol with odd in the total population (r = . , p = . ) and in african-american men (r = . , p = . ) after adjusting for covariates. conclusions: this study is among the first to examine the associations of antioxidants and odd in a sample of healthy adults with an adequate representation of african- americans. given the higher cancer burden among african-americans, identifying modifiable factors, such as diet, and possible mechanisms of carcinogenesis, are critical components of cancer prevention initiatives. cancer epidemiol biomarkers prev ; ( ). february on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ physical activity levels among the amish and non- amish living in ohio appalachia ml katz, a ferketich, ed paskett, a harley, s lemeshow, s clinton, bloomfield cd purpose: we hypothesize that a lower cancer incidence among amish adults is possibly due to diet and lifestyle factors. this study examines the physical activity (pa) levels between amish and non-amish adults living in ohio appalachia. methods: amish (n = ) and non-amish (n = ) adults completed interviews as part of a lifestyle study. self-report of pa level was measured by the international physical activity questionnaire (ipaq) and by a diary of steps per day (pedometer: digi-walker sw- ). total metabolic equiv- alent tasks minutes were calculated from the ipaq and average number of steps per day from the pedometer diary. results: the amish men walked more steps per day ( , + versus , + ; p < . ) and had a higher ipaq score (metabolic equivalent tasks min/wk; , + versus , + ; p = . ) than non-amish men. in addition, amish farmers walked more steps per day than amish non-farmers ( , + , versus , + ; p = . ). amish women walked more steps per day ( , + versus , + ; p = . ) and had higher ipaq scores ( , + versus , + ; p = . ) compared with non-amish women. conclusions: two measures of pa showed a higher pa level among amish men, especially farmers, and a trend for higher pa level among amish women. higher levels of pa warrants further investigation as one factor contributing to reduced cancer incidence rates among the amish. undertreatment of breast cancer in older women increases risk of death from breast cancer ulcickas yood m, owusu c, buist d, geiger a, field t, silliman ra, for bow investigators background: older women with breast cancer ( +) are underrepresented in clinical trials and bear the breast cancer mortality burden. we compared survival in older women receiving mastectomy, and breast-conserving sur- gery (bcs) with/without radiation therapy (rt), and the effect of tamoxifen exposure duration. methods: six cancer research network sites participated: group health, kaiser permanente (southern california), lovelace, henry ford, healthpartners, and fallon. sites identified women + years receiving mastectomy or bcs for stage i/ii breast cancer ( - ), used medical record review to obtain clinical data, and national death index for mortality ( years follow-up). cox proportional hazards models estimated hazard ratios (hr) and % confidence intervals (ci) comparing the effect of treatment on mortality. results: among , women, % were + years, % received bcs only, % received bcs + rt, and % had undergone mastectomy. adjusting for site, age, race, baseline charlson comorbidity, tumor size, nodes, receptor status, and grade, compared with mastectomy, the chance of breast cancer death was two times greater in women receiving bcs only (hr, . ; % ci, . - . ) and was equivalent to mastectomy in those receiving bcs + rt (hr, . ; % ci, . - . ). patients who underwent rt did not have elevated all-cause or other cause mortality. in tamoxifen-eligible women, decreasing exposure durations increased the chance of death, with the greatest risk for breast cancer mortality in those treated < years (hr, . ; % ci, . - . for < year and hr, . ; % ci, . - . for - years). conclusions: bcs without rt and < years tamoxifen were associated with an increased risk of breast cancer mortality. provision of standard therapies to older women may reduce the disproportionate burden of breast cancer mortality. cervical cancer screening in the time of the human papillomavirus vaccine sheinfeld gorin s, franco r, hajiani f, westhoff c, nypac study group despite the effectiveness of the pap smear, testing for human papillomavirus (hpv)-dna, and the approval of the hpv vaccine, cervical cancer remains a major cause of mortality among black and hispanic women both in the u.s. and worldwide. physician recommendation is key to the uptake of screening. no studies have yet examined physician cervical cancer screening in urban minority communities, alongside new screening protocols and the hpv vaccine. with the baseline findings from primary care physicians who were enrolled in a three-arm rando- mized controlled trial of an educational intervention, we used multilevel structural equation modeling to identify screening predictors and mediators including: access, medical practice barriers, patient and provider sociodemo- graphics, and attitudes and beliefs toward screening (measured as pap smear and appropriate hpv-dna testing). the model fit the data well [m ( ), . ; p, . ; cfi, . ; and rmsea, . ]. structural equation modeling confirmed that practice barriers (b = � . ) had a significant direct negative effect on screening. practice barriers also had a strong positive effect (b = . ) on perceived behavioral control over uncertainty in patient care, and a strong negative effect on counseling self-efficacy (b = � . ), although neither had significant direct effects on screening. stage of change (b = . ) showed a trend toward direct effects on screening. mutable office practice barriers, such as limited systems for tracking and remind- ing patients, and provider attitudes and beliefs may contribute to less effective cervical cancer screening, increasing morbidity and mortality. association of dietary magnesium and dna repair capacity with lung cancer risk mahabir s, wei q, spitz mr, forman m magnesium is an essential nutrient for humans because it is required for the maintenance of genomic stability and dna repair. because the associations between dietary magnesium (mg) intake and lung cancer risk have not been reported, we examined the relationship of mg intake and dna repair capacity (drc) on lung cancer risk in an ongoing case-control study. a total of , incident lung cancer cases and , matched healthy controls with data on drc were used to achieve our objective. a modified national cancer institute-block food frequency question- naire and a lifestyle questionnaire were interviewer- administered to each participant. cellular drc was assessed by the host-cell reactivation assay, which mea- sured nucleotide excision repair capacity in peripheral blood lymphocyte cultures. mg intake in our control population was comparable to mg intake in the national health and nutrition examination survey ( - ). after adjustment for recognized confounding factors, the odds ratios (or) and % confidence intervals (ci) of lung cancer according to increasing quartiles of dietary magne- sium intake for all subjects were: . , . ( . - . ), . ( . - . ), . ( . - . ), respectively (p trend < . ). similar ors and trends were observed in men and women. in stratified analysis, using those with high dietary mg and proficient drc as the referent group, the or for all subjects were: . , . ( . - . ) for high dietary mg and suboptimal drc, . ( . - . ) for low dietary mg and proficient drc, and . ( . - . ) for low dietary mg and suboptimal drc, respectively (p trend < . ). these associations were generally similar in men and women. our results suggest that low dietary mg intake was associated with increased risk of lung cancer, and there may be joint effects between mg intake and drc on lung cancer risk. cancer epidemiol biomarkers prev ; ( ). february on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ long-term prophylactic surgery outcomes following brca / genetic testing graves kd, gell ce, hecker sl, peshkin bn, taylor kl, schwartz md purpose: women with a brca / mutation may choose to reduce their breast and ovarian cancer risk through prophylactic mastectomy (pm) and/or prophylactic oopho- rectomy (po). we assessed the long-term rates and predictors of pm and po. methods: participants were women with z % probability of carrying a brca / mutation who received genetic testing to years earlier. for pm analyses, women (n = ) had no history of breast cancer or had unilateral breast cancer. for po analyses, women (n = ) had no history of ovarian cancer. women completed assessments before and to years (m = . years) after testing. results: among women without pm before testing ( % had prior pm), % with positive test results ultimately had pm. among women without oophorectomy before testing ( % had prior po), % of positives ultimately had po. most women opting for pm did so within year of testing ( %), whereas only % of those with po had it within a year. pm was predicted by positive test results [odds ratios (or), . ; % confidence intervals ( % ci), . - . ] and being affected with unilateral breast cancer (or, . ; % ci, . - . ). po was predicted by age z (or, . ; % ci, . - . ), positive test results (or, . ; % ci, . - . ), and having pm (or, . ; % ci, . - . ). at follow-up, women with prophylactic surgery did not differ from women without surgery on quality of life or distress. summary: prophylactic surgery is being appropriately used by women at the highest risk of hereditary breast and ovarian cancer and does not seem to adversely affect long- term psychological outcomes. two-modality mammography may confer an advantage over either full-field digital mammography or screen- film mammography glueck dh, lamb mm, lewin jm, pisano ed purpose: to compare the cancer detection rate and receiver operating characteristics (roc) area under the curve of full-field digital mammography, screen-film mammography, and a combined technique that allowed diagnosis if a finding was suspicious on film, on digital, or on both. methods: we used the data originally analyzed in lewin et al.’s trial, in which , paired full-field and digital mammograms were done in , women. we used parametric and nonparametric tests to compare the area under the curve for roc scores of film-screen only, digital mammography only, and the combined test. we used mcnemar’s test for paired proportions to compare the cancer detection rates. results: with the parametric test, neither the difference in the area under the curve between the film and combined, nor the difference between the digital and combined roc curves was significant at the bonferroni-corrected . a level (film versus combined difference = . , p = . ; digital versus combined difference = . , p = . ). the nonparametric test showed that there was a significant difference between both film and combined (difference = . , p = . ) and digital versus combined roc curves (difference = . , p = . ). the continuity- corrected mcnemar’s test showed a significant increase in the proportion of cancers detected by the combined modality over film mammography (m = . , df = , p = . ), and over digital mammography (m = . , df = , p = . ). conclusions: using two mammograms, one film and one digital, significantly increases the detection of breast cancer. exploring the needs of men in brca / families daly m male carriers of deleterious brca / mutations have a % lifetime risk of male breast cancer, and a % to % lifetime risk of prostate cancer. despite these health implications, we have found a lack of understanding of genetic test results among the men in these families. as part of a larger study to explore family communication patterns of genetic risk, we identified male first-degree relatives whose proband received a true positive test result and reported telling the result to her male relatives. six ( %) of these male relatives reported that they hadn’t received the results or forgot it. of the remaining ( %) who did report receiving the result, two ( %) reported that it was a negative result. only two ( %) reported any level of difficulty in understanding the test results, or indicated that they would like more information. how- ever, only five ( %) could correctly identify their chance of being a mutation carrier. seven ( %) did not believe that the test results increased their own risk for cancer, reflected in a relatively low level ( %) of interest in genetic testing. of the six men who did express interest, half expressed interest primarily for their children’s sake. and finally, of the men who expressed any level of concern about the meaning of the test result, ( %) directed their concern toward other family members, primarily daughters and sisters. this limited experience tends to confirm a level of cognitive and emotional distance that men experience from the genetic testing process as it applies to them. dietary intake of g- and a-tocopherol reduces lung cancer risk schendel k, mahabir s, swartz m, barrera s, spitz mr, forman mr because epidemiologic data on vitamin e and lung cancer risk is controversial and limited in terms of g-tocopherol, we investigated the association between dietary intake of a- and g-tocopherol and lung cancer risk in a case-control study. cases were newly diagnosed, previously untreated, histologically confirmed lung cancer patients recruited from the university of texas m.d. anderson cancer center; controls were healthy patients from a health maintenance organization. a- and g-tocopherol values were updated using release of the u.s. department of agriculture national nutrient database for standard reference. compared with those in the lowest quartile of intake for a-tocopherol, the odds ratios (or) for lung cancer decreased in a monotonic fashion [(q ) or, . ; % confidence intervals ( % ci), . - . ; (q ) or, . ; % ci, . - . ; and (q ) or, . ; % ci, . - . ]. the trend was significant (p < . ) with limited variation by gender. compared with those in the lowest quartile of intake for g-tocopherol, individuals in the two highest quartiles had significantly reduced odds of lung cancer [(q ) or, . ; % ci, . - . ; and (q ) or, . ; % ci, . - . , p trend = . ]. similar associations became apparent in women, but not in men. we present, for the first time, an association of dietary intake of g- tocopherol and lung cancer in the united states. given the antioxidant and immune functions of tocopherols and the limitations of our study design, further research in the chemopreventive potential of both tocopherol metabolites is warranted. cancer epidemiology, biomarkers & prevention cancer epidemiol biomarkers prev ; ( ). february on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ choice of survey completion method differs by participant characteristics in african-americans satia j, galanko j objective: to describe participant characteristics associated with choice of completion method of a -item cancer risk behavior survey in african-americans. methods: african-americans in north carolina (n = , ), to years of age, were randomly selected from department of motor vehicle rosters and given the choice of completing the survey by one of three methods: mail, internet, or telephone. everyone received the mailed survey and information on how to participate via web or phone. results: among eligible respondents, completion rates were . % by mail, . % by internet, and . % by phone (p < . ). the respondent mean age was . years, % were male, and % were college graduates. choice of method differed by age, education, marital status, and internet access (p < . ). respondents who completed mailed surveys were more often high school graduates and married, whereas internet responders were younger, college graduates, and % had easy web access; there were no differences according to sex, body mass index, physical activity, or rural versus urban residence. the median number of missing items was highest for telephone ( . ) relative to mail ( . ) and web ( . ) surveys (p < . ), and for older compared with younger respondents (p < . ). conclusions: choice of completion method differed by participant characteristics, and telephone surveys had the highest number of missing responses. this information can be applied in the design of population-based cancer risk behavior surveillance for african-americans. obesity predicts differential response to a cancer prevention intervention among african-americans leone l, james a, hudson m, campbell m purpose: the purpose of this analysis was to determine if the effectiveness of a colorectal cancer (crc) prevention intervention promoting screening and physical activity was affected by weight group (normal weight, overweight, obese i, obese ii), and if certain weight groups responded better to certain interventions. methods: the watch project, a randomized controlled trial in african-american churches, tested the effects of a tai- lored print and video and/or a lay health advisor interven- tion to promote multiple behavior changes for crc prevention. a telephone survey was given at baseline and months. recreational physical activity (rpa) was calcu- lated as metabolic equivalent task hours per week. the crc screening outcome was test completion (fecal occult blood test, flexible sigmoidoscopy, double contrast barium enema, or colonoscopy) within the past year and was limited to participants ages and older. analyses controlled for baseline levels of screening/rpa, age, education, gender, and church cluster. results: analyses revealed a significant interaction effect (p = . ) of weight group and intervention condition on rpa metabolic equivalent tasks at follow-up, but not for weight or condition alone. normal and overweight indi- viduals receiving the lay health advisor intervention increased rpa more, whereas obese i and ii responded more to the tailored print and video intervention. for crc screening, the interaction was not significant; only weight remained related to past year of screening at follow-up (n = , p = . ), with obese individuals reporting fewer crc screenings. conclusion: results suggest that, at least for physical activity, interventions may be differentially effective based on obesity status. targeting and tailoring of cancer prevention interventions based on weight may be a promising strategy for reaching out to risk groups. relative weight at age and risk of postmenopausal breast cancer bardia a, vierkant ra, hartmann lc, vachon cm, wang ah, olson je, sellers ta, cerhan jr background: early adolescent weight may affect the risk of postmenopausal breast cancer, however, this has not been well studied. methods: the iowa women’s health study is a prospective cohort study of postmenopausal women. relative weight at age (above, below, or average weight compared with female peers) and family history of cancer were ascertained using a mailed questionnaire ( ). breast cancer incidence were identified using the iowa surveillance, epidemiology, and end results cancer registry. relative risks (rr) and % confidence intervals ( % ci), were estimated using cox proportional hazards regression. results: throughout , , breast cancers were identified among , women. compared with women with average weight at age , there was no association of below average weight with breast cancer risk (rr, . ; % ci, . , . ), whereas women with above average weight had lower risk (rr, . ; % ci, . , . ). the latter association was observed for all er and pr subtypes, but was strongest for pr� tumors (rr, . ; % ci, . - . ). there was no evidence of an interaction between weight at age and family history (p = . ). conclusion: above average weight at age was inversely associated with risk of postmenopausal breast cancer, and was similar in subtype analyses defined by er/pr status, and family history. the study facilitates a mechanistic understanding of the effects of early adolescent weight on breast cancer risk and may help identify new prevention strategies. familial aggregation of cancer among first-degree relatives of brain tumor patients scheurer me, etzel cj, lu m, el-zein r, bondy ml purpose: brain tumors affect members of the same family, but the specific cancer risk for relatives of patients with brain tumors has been inconclusive. methods: we obtained family history information on , first-degree relatives (fdr) from , white glioma patients registered at m.d. anderson cancer center between june and june . standardized incidence ratios (sir) were computed using the age, sex, and time period – specific rates from the surveillance, epidemiology, and end results program. results: the mean age of the probands was . years, and % were male. fifteen percent of probands had two or more fdrs with cancer, and % had one or more fdrs with a brain tumor. the sir was significantly elevated for all malignancies (sir = . ) and was higher for siblings (sir = . ) and offspring (sir = . ). sirs were significantly higher for brain (sir = . ) and bone (sir = . ) cancer and melanoma (sir = . ) among fdrs of male probands. sirs were significantly higher for brain (sir = . ) and bone (sir = . ) cancer among fdrs of female probands. there was a trend of decreasing sirs with increasing age of the proband. there was an increase in the sir among fdrs who were < years old at diagnosis, suggesting a genetic component for cancer in these families. conclusions: we found a -fold increase in brain tumors and a % increase in expected cases of all cancers among the fdrs of glioma probands. the excess of brain tumor and melanoma cases supports previous reports; however, an increase in bone cancer has not been previously reported. we also found a borderline increase in pancreatic cancer, likely due to p mutations among these families. cancer epidemiol biomarkers prev ; ( ). february on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ time to breast cancer diagnosis and treatment in the national breast and cervical cancer early detection program richardson lc, royalty j, howe w, helsel w, kammerer w, benard vb purpose: to examine the intervals between breast cancer screening, diagnosis, and treatment initiation among low- income and uninsured women screened in the national breast and cervical cancer early detection program (nbccedp) during two time periods. methods: we examined diagnostic and treatment intervals for the time periods to and to . the intervals were defined as: (a) time between mammography and final diagnosis for all women receiving an abnormal mammogram result (including suspicious abnormality, highly suspicious for malignancy, and assessment is incomplete) or abnormal clinical breast exam result and; (b) time between diagnosis and treatment initiation among those diagnosed with cancer. results: for the study period, , women screened for breast cancer through the nbccedp had abnormal screen results. for to and to , the median interval to diagnosis after screening was and days, respectively. for , women found to have cancer, the median interval from diagnosis to treatment increased from to days in the later time period. overall, time to diagnosis improved in to compared with to . in the second time period, % of women were treated within days. summary: the goal of the nbccedp is to assure that women receive quality breast cancer screening and diag- nostic services, and initiate treatment. our data show that the program is meeting this goal. disparities in breast cancer outcomes should diminish once all women have timely work-up and treatment of breast problems. postmenopausal hormone use and mortality after breast cancer newcomb pa, egan km, trentham dietz a, titus ernstoff l, baron ja, hampton jm, wong ey, stampfer mj, willett wc some prior small studies have observed reduced breast cancer mortality in women who used postmenopausal hormones (pmh) prior to diagnosis. to evaluate the influence of pmh use on breast cancer mortality, we analyzed data from a prospective cohort of , women with incident invasive breast cancer at least years of age and residents of wisconsin, massachusetts, or new hampshire. women were enrolled in three phases between and , and followed for death until december , using the national death index. a total of , deaths from breast cancer were documented during an average . years of follow-up. hazard rate ratios and % confidence intervals (ci) were estimated using cox proportional hazards regression. breast cancer survival varied by duration of hormone use prior to diagnosis, with the lowest cumulative mortality among pmh users. compared with women who had never used pmh, we observed a reduced risk of death from breast cancer among users of estrogen-progestin preparations at the time of diagnosis (adjusted hazard rate ratio, . ; % ci, . - . ) and among users for z years ( . ; % ci, . - . ). no association was observed for women who had formerly used these preparations or for former or current users of estrogen-only preparations. however, among women with lobular breast cancer, those who were using estrogen-only preparations at diagnosis expe- rienced a halving in breast cancer mortality ( . ; % ci, . - . ). in this large population-based cohort of women with breast cancer, recent use of pmh was associated with decreased breast cancer – specific mortality compared with never users of these preparations. survival was best among current and long-term users of combined estro- gen-progestin therapy. duration and type of postmenopausal hormone use and ovarian cancer wernli kj, newcomb pa, trentham dietz a, hampton jm, wong ey, egan km although long-term use of oral contraceptives is well- established to reduce ovarian cancer risk, the influence of postmenopausal hormones (pmh) is less clear. long-term use or pmh preparation type may be of importance. we explored this association in a population-based case- control study of ovarian cancer conducted in massachu- setts and wisconsin during to and to . information on pmh use and other potential risk factors for ovarian cancer were obtained via telephone interview. exposures histories were compared in a total of incident invasive epithelial ovarian cancers identified from statewide cancer registries and , similarly aged population controls randomly selected from lists of licensed drivers and medicare beneficiaries. the relative risk and % confidence intervals (ci) for pmh use was estimated using multivariable logistic regression. women that ever used pmh had a . -fold increase in risk ( % ci, . - . ) when compared with never users of pmh. these elevated risk estimates were observed with use of estrogen-only preparations ( . ; % ci, . - . ) but not with combined estrogen-progestin preparations ( . ; % ci, . - . ), with evidence of significant hetero- geneity by pmh type (p < . ). long-term users of estrogen-only preparations also experienced elevations in risk. these data suggest that use of estrogen-only pmh preparations may modestly increase ovarian cancer risk. genetic variants in the promoter region of h ax are associated with risk of sporadic breast cancer in non- hispanic white women aged v years lu j, wei q, bondy ml, brewster am, bevers tb, yu tk, buchholz ta, meric-bernstam f, hunt kk, singletary se, wang l-e in this case-control study, we genotyped four common single nucleotide polymorphisms (i.e., � a>g, � g>a, and � t>c in the promoter and c>t in the ¶ untranslated regions) in non-hispanic white patients with sporadic breast cancer and cancer-free controls, all of whom were v years old and frequency- matched by age (f years). in the individual single nucleotide polymorphism analysis, only � a>g was significantly associated with increased risk of breast cancer [adjusted odds ratio (or) . ; % confidence interval (ci), . - . for � ag; and or, . ; % ci, . - . for � gg compared with the � aa genotype]; how- ever, the number of variant (risk) alleles of � g, � a, and � c were associated with increased risk of breast cancer in a dose-response manner (ptrend = . ). there was evidence of an interaction between the number of variants and age (pinteraction = . ) and alcohol use (pinteraction = . ). haplotypes derived from the observed genotypes were also significantly associated with risk in an allele dose-response manner compared with haplotypes with no variant allele (or, . ; % ci, . - . for one variant allele; or, . ; % ci, . - . for two variant alleles; and or, . ; % ci, . - . for three variant alleles; ptrend = . ). these findings suggest that h ax promoter polymorphisms contribute to the etiology of sporadic breast cancer in young non-hispanic white women. larger studies are warranted to confirm these findings. cancer epidemiology, biomarkers & prevention cancer epidemiol biomarkers prev ; ( ). february on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ susceptibility to smoking in mexican origin youth in houston, texas wilkinson a, spelman a, prokhorov a, bondy m, spitz m purpose: to examine the relationship between cognitive sus- ceptibility to smoking and well-known risk factors for smok- ing initiation among - to -year-olds of mexican origin. methods: participants in this study were drawn from an infrastructure of population-based households created by the department of epidemiology at m.d. anderson cancer center. households with age-eligible participants were iden- tified from the cohort database; > % of all parents who were contacted agreed to enroll their child in the study. after obtaining informed consent, survey data were collected on smoking susceptibility and smoking status, acculturation, demographics, peer and family influences, school and neigh- borhood characteristics, and attitudes towards smoking. results: bivariate associations between susceptibility and risk factors were predominantly significant and in the expected direction. significant predictors were included in stepwise and additive effects logistic regression models. the stepwise model revealed that each additional positive smoking expectation was associated with a -fold in- creased chance of being susceptible. susceptibles were more likely to report that most friends smoke, have a mother who smokes, believe that peer norms strongly support smoking, were years old, male, perceived more temptations to smoke, have more acculturated parents, and report lower subjective social status than their nonsusceptible peers. the additive effects model noted increasing risk of susceptibility with increasing number of risk factors (ptrend < . ). participants reporting three risk factors were . times and those with six or more risk factors were . times more likely to be susceptible to smoking, compared with participants reporting one or no risk factors. summary: our findings suggest a need to develop both family- and school-based primary prevention programs. cancer epidemiol biomarkers prev ; ( ). february on april , . © american association for cancer research. cebp.aacrjournals.org downloaded from http://cebp.aacrjournals.org/ ; : - . cancer epidemiol biomarkers prev , − march •oncology, houston, texas american society of preventive• st annual meeting updated version http://cebp.aacrjournals.org/content/ / / access the most recent version of this article at: e-mail alerts related to this article or journal.sign up to receive free email-alerts subscriptions reprints and .pubs@aacr.orgdepartment at to order reprints of this article or to subscribe to the journal, contact the aacr publications permissions rightslink site. 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left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ psychiatrie bulletin ( ), , conference report first world congress on psychiatrie genetics cambridge, - august michaelmorris,research senior registrar, institute of medical genetics, university of wales college of medicine, cardiff cf xn; and s. j. adams,lecturer, academic department of psychiatry, st bartholomew's hospital, london ec a be this conference was organised by the biological psy chiatry group of the college under the auspices of the world federation of societies of biological psy chiatry with dr t. j. crow as chairman of the organ ising committee. the meeting was attended by participants from countries. the oral and pos ter presentations, many of which reported on the ap plication of recombinant dna techniques to psy chiatry, reflected a growing field of intensive research. the main focus of the meeting was on the genetics of depression and schizophrenia. it is well-known that a locus for bipolar affective disorder has been implicated on chromosome in one amish family in a study by professor janice egeland et al. however, re-analysis of the data, updated psychiatric status and extensions of the core pedigree have shown that genetic linkage is not as close as was originally reported. other centres have not replicated the original finding of linkage to chromosome p . dr hugh gurling presented data showing that a schizophrenia susceptibility locus may lie on the proximal long arm of chromosome ( q . - . ) in seven british and icelandic families. however, nega tive studies from various parts of the world were reported (scotland, wales, ireland, usa, canada, italy). evidence supportinga locus for psychosis in the pseudo-autosomal region of the sex chromosomes was presented by the northwick park hospital group. the d -dopamine receptor gene has been recently cloned and this gene has been linked to chromosome . however, strong evidence against linkage of the d gene to schizophrenia was presented in a large swedish kindred. the great difficulties of research in psychiatric genetics were debated. segregation studies have suggested that genetic models for psychiatric dis orders such as schizophrenia are complex and the pattern of inheritance is often unknown. there fore lod scores, which are usually applied to single locus models, must be interpreted cautiously. other problems are genetic heterogeneity, the presence of phenocopies (non-genetic alterations of the phenotype), diagnostic validity and non- random mating (for example, schizophrenic suf ferers differentially mate with 'alcoholics'). yet more problems are reduced penetrance, variable age of onset and environmental factors such as life events. many strategies for future research were proposed. there should be a high threshold for definition of a case. more families should be recruited and other areas of the genome should be examined for linkage. new linkage programmes should be developed to allow for non-mendelian forms of inheritance. in the affected sib-pair method of analysis, for example, no model of inheritance is specified. several other neuropsychiatrie diseases were dis cussed. closer probes than g have been discovered to be linked to huntington's disease and preliminary reports of the clinical application of such probes in predictive testing were presented by several centres (baltimore, cardiff, manchester). regarding tourette syndrome, pedigrees of the disorder from the middlesex hospital are consistent with autosomal dominant inheritance and a locus on chromosome is under investigation. st george-hyslop's finding that the genetic defect causing familial alzheimer's disease maps on chromosome has been replicated at st mary's hospital, london. mental handicap and the molecular study of a fragile site at xq . are sources of vigorous investigation. the gene for arylsulfatase a (deficiency of which leads to meta- chromatic leucodystrophy, a recognised cause of psychosis and mental retardation) has been cloned in germany and this should facilitate the study of genotype-phenotype relationships. data on the genetics of narcolepsy showed that it has the tightest hla linkage of any disease ( % of all subjects with narcolepsy and cataplexy are dr positive). there is suggestive evidence that there is linkage between al coholism and the mns blood group on chromosome . current research indicates that infantile autism is genetically heterogeneous. professor andreas rett, who described the eponymous rett's syndrome in , gave an account of the profound mental and physical handicaps associated with this disorder. an important session was held on the ethical and social implications of molecular and clinical psychi atric genetics. these include problems associated with confidentiality and informed consent. the contro versial subject of carrier detection was debated but the possibility of applying presymptomatic and prenatal tests in disorders such as schizophrenia is remote. epigenetic modulation of intestinal cholesterol transporter niemann-pick c -like (npc l ) gene expression by dna methylation* epigenetic modulation of intestinal cholesterol transporter niemann-pick c -like (npc l ) gene expression by dna methylation* received for publication, december , , and in revised form, june , published, jbc papers in press, june , doi . /jbc.m . pooja malhotra‡, vinay soni‡, anoop kumar‡, arivarasu n. anbazhagan‡, amish dudeja‡, seema saksena‡, ravinder k. gill‡, pradeep k. dudeja‡§, and waddah a. alrefai‡§ from the §jesse brown veterans affairs medical center, ‡division of gastroenterology and hepatology, department of medicine, university of illinois at chicago, chicago, illinois background: cholesterol transporter npc l is expressed in small intestine but not in colon. results: dna in the mouse npc l gene is hypermethylated in colon as compared with small intestine. dna methylation decreases the promoter activity of npc l . conclusion: dna hypermethylation may be responsible for silencing npc l expression in the colon. significance: altering dna methylation may represent a novel mechanism to modulate npc l expression and cholesterol absorption. intestinal npc l transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. npc l is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared with the small intestine. this study was undertaken to examine whether dna methylation was responsible for segment-specific expression of npc l . treatment of mice with -azacytidine (i.p.) resulted in a significant dose-dependent increase in npc l mrna expression in the colon. the lack of expression of npc l in the normal colon was associated with high levels of methylation in the area flanking the -kb fragment upstream of the initiation site of the mouse npc l gene in mouse colon as analyzed by epityper� mass- array�. the high level of methylation in the colon was observed in specific cpg dinucleotides and was significantly decreased in response to -azacytidine. similar to mouse npc l , -azacy- tidine treatment also increased the level of human npc l mrna expression in the intestinal hutu- cell line in a dose- and time- dependent manner. silencing the expression of dna methyltrans- ferase dnmt , - , - a, and - b alone by sirna did not affect npc l expression in hutu- cells. however, the simultaneous attenuation of dnmt and - b expression caused a significant increase in npc l mrna expression as compared with control. also, in vitro methylation of the human npc l promoter signif- icantly decreased npc l promoter activity in human intestinal caco cells. in conclusion, our data demonstrated for the first time that dna methylation in the promoter region of the npc l gene appears to be a major mechanism underlying differential expres- sion of npc l along the length of the gastrointestinal tract. intestinal cholesterol absorption occurs mainly in the proximal small intestine and positively correlates with levels of plasma cholesterol ( , ). the niemann-pick c -like (npc l ) protein, localized to the brush border membrane of intestinal epithelial cells, is shown to be essential for cholesterol absorption ( ). the loss of npc l in mice causes a remarkable decrease in cholesterol absorption, and npc l protein has been shown to be the molecular target for ezetimibe ( ). also, studies have shown that the knock-out of npc l prevented atherosclerosis in apoe knock-out mice ( ). these observations suggest that npc l is an attractive target for the treatment of hypercholesterolemia and the prevention of atherosclerosis ( ). the expression of npc l exhibits species- and tissue-spe- cific patterns. for example, npc l has been shown to be expressed in the liver of humans but not in mice and rats ( ). also, the expression of npc l mrna in humans, mice, and rats is significantly higher in the small intestine as compared with the colon demonstrating differential expression along the length of the gastrointestinal tract ( ). nevertheless, the mech- anisms underlying the segment-specific expression of npc l have not yet been investigated. tissue-specific expression of genes is determined by differ- ent mechanisms, including differential expression of certain transcription factors or by involvement of epigenetic mecha- nisms ( ). in this regard, npc l expression has been shown to be modulated at the transcriptional level. for example, srebp and hnf � transcription factors have been shown to stimulate npc l promoter activity and increase its mrna expression ( , ). because the expression of these transcription factors is not intestinal segment-specific ( , ), their effects on gene transcription may not explain the region-specific expression of intestinal npc l mrna. this study investigated the role of epigenetic mechanisms such as dna methylation in dictating region-specific expression of npc l . dna methylation is a process mediated by dna methyltransferases by which methyl groups are covalently added to the �-position of cytosine in the cpg dinucleotide ( ). dna methylation is often associated * this work was supported, in whole or in part, by national institutes of health grants dk , dk , and dk (to p. k. d.), dk (to r. k. g.), dk (to s. s.), and dk (to w. a. a.) from niddk. this work was also supported by the department of veterans affairs (to w. a. a. and p. k. d.). to whom correspondence should be addressed: university of illinois at chi- cago, jesse brown va medical center, medical research service ( / ) south damen ave., chicago, il . tel.: - - ; fax: - - ; e-mail: walrefai@uic.edu. the journal of biological chemistry vol. , no. , pp. – , august , published in the u.s.a. journal of biological chemistry volume • number • august , this is an open access article under the cc by license. http://creativecommons.org/licenses/by/ . / with suppression of gene expression ( ). the modification of dna by methylation alters gene transcription by either block- ing the access of certain transcription factors to their consensus sequences on the promoter region ( ) or by allowing the bind- ing of methyl-cpg-binding proteins that recognize methylated dna and recruit protein partners to suppress gene expression ( ). in this regard, previous studies have shown that the knock- out of the methyl dna-binding protein mbd induced the expression of certain genes in the colon that are specifically expressed in the duodenum and pancreas ( ). these observa- tions indicated that dna methylation might play a role in sup- pressing the colonic expression of genes that are usually expressed in the small intestine. our data show for the first time that the � upstream regula- tory region of mouse npc l was hypermethylated in the colon as compared with the small intestine. treatment with the hypomethylating agent -azacytidine significantly increased npc l expression in the colon concomitant with a decrease in dna methylation at specific cpg dinucleotides in the npc l gene. furthermore, in vitro methylation of the human npc l promoter caused a decrease in the promoter activity in the human intestinal caco cells. these findings clearly indi- cate the essential role of dna methylation in controlling the expression of npc l along the length of the gastrointestinal tract. harnessing alterations in dna methylation by dietary manipulations or environmental stimuli may prove to be of potential benefit in decreasing cholesterol absorption and pre- venting atherosclerosis. experimental procedures cell culture and materials—human duodenal hutu- and human intestinal caco- cell lines were obtained from the atcc and grown routinely in t- -cm plastic flasks at °c in a % co , % air environment. the hutu- cells were cultured in minimum essential medium (eagle’s) containing mm l-glutamine, earle’s balanced salt solution adjusted to con- tain . g/liter sodium bicarbonate, . mm nonessential amino acids, and . mm sodium pyruvate and supplemented with % fetal bovine serum, units/ml penicillin, and mg/ml gen- tamicin (invitrogen). caco- cells were grown in the same min- imum essential medium supplemented with % fbs. -azacy- tidine, inhibitor of dna methylation, was purchased from sigma. all the sirnas were obtained from qiagen (valencia, ca). dnmt antibodies were obtained from cell signaling; antibodies for dnmt b were obtained from abcam, and anti- bodies for dnmt a were from santa cruz biotechnology. all chemicals were of reagent grade and obtained from commercial sources. inhibition of dna methylation and rna extraction— hutu- cells and caco cells were seeded � cells/well in -well plates (transwell inserts for caco ), and cells were treated with different concentrations ( , , and �m) of the dna methylation inhibitor ( -azacytidine) for and h. rna was isolated by using qiagen rneasy mini kit (qiagen) according to the manufacturer’s instructions. real time pcr analysis—equal amounts of rna from con- trol and treated samples were reverse-transcribed and ampli- fied in a one-step reaction utilizing brilliant sybr green qrt- pcr master mix kit (stratagene, clara, ca). human npc l was amplified with gene-specific primers as follows: sense primer, �-tatcttccctggttcctgaacgac- �, and antisense primer: �-ccgcagagcttctgtgtaatcc- �. gapdh was amplified as an internal control utilizing gene- specific primers as follows: sense primer, �-gaaatcccat- caccatctt- �, and antisense primer, �-aaatgagccc- cagccttct- �. the quantification was expressed as a ratio of �ct-npc l / �ct-gapdh, where �ct-npc l and �ct- gapdh represent the difference between the threshold cycle of amplification of treated and control rna for npc l and gapdh, respectively. all real time qpcrs were performed in triplicate. cloning and in vitro methylation of pcpg free-l vector— for the construction of pcpg free l vector, the dna fragment containing � /� region of the human npc l pro- moter was amplified using forward -atcgatgcgagtac- ttggactctatctctctgtgg- � and reverse -atcgat- gcgaagcttcccaggtctgggaaggggtca- � primers, and the amplified promoter fragments were then inserted into a promoterless cpg free vector (pcpg free basic lucia, invivo- gen, san diego) upstream of the lucia reporter gene. cloned vector was then methylated in vitro utilizing ss methylases (new england biolabs, frankfurt, germany) according to the manufacturer’s instruction. briefly, �g of plasmid dna was added to a reaction containing cpg methyltransferase (sss , units/�l) in the presence of �m s-adenosylmethionine (new england biolabs) and incubated for h at °c, and s- adenosylmethionine was replenished after every h. unmethy- lated control reaction contained the npc l promoter con- struct and methylases but not s-adenosylmethionine. plasmid dna was then purified by using qiagen miniprep kit and quan- tified using a spectrophotometer. transient transfections and luciferase assay—caco cells were seeded at a density of . � cells/well on -well plates and cotransfected while still in suspension, with the npc l promoter in the pcpg free vector along with �-galactosidase mammalian expression vector (to control for transfection effi- ciency) utilizing lipofectamine reagent (invitrogen). a pcpg free lucia vector with ef promoter (invivogen, san diego) was used as a control for the methylation experiment. after h of transfection, cells were washed with phosphate- buffered saline and lysed using a passive lysis buffer from pro- mega (madison, wi). the activities of both lucia and �-galac- tosidase were measured using quanti-luc (lucia assay reagent) from invivogen and �-galactosidase luminescent detection system from clontech, respectively, according to the manufacturer’s instructions in a luminometer (promega). the promoter activity was expressed as a ratio of lucia to �-galacto- sidase activity in each sample. sirna transfection—expression of dnmts (dnmt , - , - a, and - b) in hutu- cells was selectively silenced utilizing specific sirnas (qiagen). scrambled sirna was used as a neg- ative control. hutu- cells were plated in -well culture plates, and the next day cells were transiently transfected with the abbreviations used are: dnmt, dna methyltransferase; q-rt, quantita- tive rt. epigenetic regulation of npc l august , • volume • number journal of biological chemistry pmol of sirna duplexes using lipofectamine (invitrogen). cells were harvested – h after the transfection. silencing was validated by real time pcr utilizing dnmt-specific prim- ers as well as by western blotting using dnmt-specific antibodies. western blotting—control or sirna-transfected hutu- cells were first rinsed with ice-cold � pbs and then lysed with a cell lysis buffer containing mm tris-hcl, ph . , mm nacl, % triton x- , mm edta, mm egta, � protease inhibitor mixture, and . % sds. the cells were incubated with this mixture for – min on ice and further lysed by sonica- tion. the lysate was centrifuged at rpm for min at °c, and supernatant was collected. �g of cell lysates were loaded on sds-polyacrylamide gels and transferred to nitrocellulose membranes. membranes were blocked with % nonfat dry milk blocking buffer in � pbs for h and then probed with specific dnmt antibodies ( : dilution) or gapdh anti- body ( : dilution) in % nonfat dry milk in � pbs over- night at °c. the membranes were washed four times for min with the wash buffer containing � pbs and . % tween . furthermore, membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit igg antibody for h, and the bands were visualized with enhanced chemiluminescence detection reagents. animal studies—animal studies were approved by the insti- tutional animal care and use committee (iacuc) at the jesse brown veterans affairs medical center. -week-old c bl /j mice were obtained from the jackson laboratory (bar harbor, me) and were acclimatized for days with free access to food and water and : h light/dark cycles. mice were then divided into different groups ( – animals/group) as follows: (i) con- trol group that received intraperitoneal injection of vehicle; (ii) treatment groups received i.p. injections of either or mg/kg body weight of -azacytidine. the injections were given to the animals three times/week for weeks, and the animals were sacrificed, and the intestine and colon were harvested. intesti- nal mucosa from jejunum, ileum, and colon were scraped and stored frozen at � °c. total rna was extracted utilizing qia- gen rneasy mini kit (qiagen), and the expression of npc l was assessed by real time pcr. the sequences for the primers are as follows: mouse npc l forward primer, �-tggact- ggaaggaccatttcc- �, and reverse primer, �-gacag- gtgccccgtagtca- �. mouse gapdh was amplified as an internal control utilizing gene-specific primers �-tgtgt- ccgtcgtggatctga- � (forward), and �-cctgcttca- ccaccttcttgat- � (reverse). dna methylation analysis by epityper� massarray�— the epityper� massarray� from sequenom was utilized to measure the levels of the dna methylation in mouse npc l gene in jejunum, ileum, and colon. genomic dna was isolated from different regions of the gastrointestinal tract utilizing the dneasy blood and tissue kit (qiagen). the epityper� mass- array� assay was performed by sequenom inc. (san diego). we have focused on a -kb fragment of mouse npc l gene upstream of the translation initiation site (that was considered as � ). briefly, the dna was treated with bisulfite, and five fragments were then amplified from the -kb npc l frag- ment using bisulfite-specific pcr primers (table ). the reverse pcr primers contained a -mer tag, and the forward primers contained a t promoter tag for the subsequent in vitro transcription. the products of the in vitro transcription were then cleaved by ribonuclease, and mass spectra were then acquired to measure the different levels of guanine and adenine in the cleavage products reflecting the level of dna methyl- ation. the investigated -kb region of the npc l gene con- tains cpg dinucleotides, and the assay generated successful data for cpg sites. the assay, however, failed to generate successful data regarding the methylation of seven cpg sites due to either low mass or high mass of the cleavage products. statistical analysis—results are expressed as mean � s.e. student’s t test was utilized for statistical analysis. comparisons of multiple treatment conditions with controls were analyzed by one-way analysis of variance. a p value of . or less was considered statistically significant. results dna demethylation increases npc l expression in mouse colon—npc l protein is expressed in the small intestine but not in the colon ( ). to determine whether the expression profile of npc l mrna is similar to the protein expression, we examined the relative abundance of npc l mrna along the length of the mouse small intestine and colon. as shown in fig. a, npc l mrna expression was about -fold more in the ileum as compared with jejunum. the level of npc l mrna in the colon was remarkably less (� -fold) as com- pared with jejunum and ileum. to examine the possible role of dna methylation in suppressing the expression of npc l mrna in colon, we treated mice with the dna methyltrans- ferase inhibitor -azacytidine. fig. b shows that the intraperi- toneal injections of -azacytidine (three times/week for weeks) led to a remarkable increase in colonic npc l expres- sion in a dose-dependent manner (� - and -fold at and mg/kg body weight, respectively). the levels of npc l mrna expression in the jejunum and ileum remained unaltered by -azacytidine (fig. c). these findings suggest that the low lev- els of npc l mrna expression may be associated with hypermethylation of the npc l gene in the colon resulting in low expression of npc l in the colon. we have also examined the relative expression of dna methyltransferases (dnmt , dnmt a, and dnmt b) along the length of the gastrointes- table primer sequences for epityper� massarray fragment sense primer antisense primer primer gtttgtttggttgttatggattttt ctctctattctccaacaacattcaa primer tttgaatgttgttggagaatagaga cacaaaccaaacatcaataatacca primer gaggatatgttttagatataaaggggtt aaacattaaaccctaccttctccac primer gatttgtaagtgtggatggtgttat tactaaattccaccaatatcccaaa primer ggatttaaggaatttttggttttgt catcaaataataccattccatctcc epigenetic regulation of npc l journal of biological chemistry volume • number • august , tinal tract. data presented in fig. , d–f, show that these enzymes responsible for dna methylation are differentially expressed in jejunum, ileum, and colon. assessment of dna methylation of mouse npc l promoter in colon—dna methylation in the promoter region of genes usually suppresses their expression ( ). we sought to investi- gate the relative methylation of � upstream regulatory region of the mouse npc l gene in colon as compared with small intestine. in silico analysis of the -bp fragment upstream of the translation initiation site of the mouse npc l promoter revealed the presence of cpg dinucleotides that are poten- tial targets for methylation (fig. a). the methylation of these cpg sites in the mouse npc l promoter was analyzed by the epityper� massarray� (sequenom). because of inherent limitations of the assay ( ), of the cpg dinucleotides were not analyzed by this approach, including � , � , � , figure . -azacytidine increases npc l expression in mouse colon. a, -week-old c bl mice were sacrificed, and the mucosa was scraped from the jejunum, ileum, and colon. total rna was extracted from the mucosal scrapings, and q-rt-pcr was used to assess the mrna expression of npc l relative to gapdh mrna expression. data are means � s.e. of each animal group (n 	 animals/group) and presented as arbitrary units. *, p . as compared with jejunum. b and c, two doses of -azacytidine ( and mg/kg body weight) were administered to mice by intraperitoneal injections (three injections/week for weeks), and colon and intestine were harvested, and total rna was extracted. relative levels of npc l mrna expression were evaluated by q-rt-pcr in mouse colon (b) and small intestine (c) using gapdh as an internal control. data are mean � s.e. of each animal group (n 	 – animals/group) and presented as arbitrary units. *, p . as compared with untreated mice; #, p . as compared with mice treated with mg/kg body weight. d–f, – -week-old c bl mice were sacrificed, and the mucosa was scraped from the jejunum, ileum, and colon. total rna was extracted from the mucosal scrapings, and mrna expression of dnmt (d), dnmt a (e), and dnmt b (f) was assessed by q-rt-pcr relative to gapdh mrna expression. data are mean � s.e. of each animal group (n 	 animals/group) and presented as arbitrary units. *, p . as compared with jejunum. epigenetic regulation of npc l august , • volume • number journal of biological chemistry � , � , � , and � (the translation initiation site is designated as � ). the percent of methylation was examined in the jejunum, ileum, and colon, both in control and -azacyti- dine-treated mice ( mg/kg body weight). the results showed that the overall dna methylation level is higher in colon as compared with jejunum and ileum. the difference in dna methylation from the available data reached statistical signifi- cance with higher levels in the colon as compared with jejunum alone or jejunum and ileum at cpg sites as follows: � , � , and � (fig. b); � , � , and � (fig. c); � (fig. d); � and � (fig. f); � , � , and � (fig. g); and � and � (fig. h). the methyl- ation of cpg sites � and � (fig. h) was more in the colon as compared with ileum and jejunum, although the dif- ference did not reach statistical significance. also, there were cpg sites that exhibited equal levels of methylation in the colon, ileum, and jejunum, including the following: � , � , and � (fig. d); � , � , � , and � figure . dna methylation of mouse npc l promoter in the small intestine and colon. a, schematic representation of the -bp fragment of mouse npc l promoter region (� represents the translation initiation site). the cpg dinucleotides are indicated as dark circles. b–h, genomic dna was isolated from jejunum (jej), ileum (ile), and colon (col) of control and -azacytidine-treated mice. dna methylation was investigated by epityper� massarray� assay in a -bp fragment of mouse npc l gene. the % of methylation of each of the cpg sites that were covered by the assay is shown in the figures. the st bar for each intestinal segment represents control mice, and the nd bar represents mice treated with -azacytidine. data are presented as mean � s.e. from three samples of each group. *, p . as compared with jejunum; #, p . as compared with ileum; $, p . as compared with colon from untreated mice; ##, p . as compared with jejunum of untreated mice; $$, p . as compared with ileum of untreated mice. epigenetic regulation of npc l journal of biological chemistry volume • number • august , (fig. e); and � and � (fig. f). dna methylation was significantly decreased by treatment with -azacytidine in all the regions (colon, ileum, and jejunum) at cpg sites as fol- lows: � , � , and � (fig. e); � , � , and � (fig. d); and � , � , and � (fig. c). more- over, treatment with -azacytidine significantly decreased the methylation in the colon but not in jejunum and ileum in the cpg sites � (fig. b), � (fig. d), and � (fig. h). overall, these data indicate that mouse npc l promoter region is hypermethylated at specific cpg dinucleotides in the colon as compared with ileum and jejunum, and the treatment with -azacytidine decreases dna methylation in these sites. -azacytidine increases the expression of human npc l — we next examined the effect of dna demethylation on the expression of human npc l mrna in an in vitro cell culture model. we utilized the hutu- human intestinal cell line as a cellular model because npc l mrna is minimally expressed in these cells. as shown in fig. a, treatment with -azacytidine significantly increased the expression of human npc l mrna in a dose-dependent manner. moreover, the increase in npc l mrna expression in hutu- cells was time-depen- dent with almost -fold stimulation occurring after h of incubation with �m -azacytidine (fig. a). we have also examined the effect of -azacytidine in human intestinal caco cells that have high levels of npc l mrna expression. data depicted in fig. b show that -azacytidine also increases npc l expression in caco cells. these results indicate that human npc l mrna is also sensitive to dna demethylation suggesting that dna methylation plays a role in controlling the mrna expression of human npc l in hutu- and caco cells. to further investigate the effect of inhibiting dna methyl- ation on npc l expression in hutu- cells, we attenuated the expression of dnmts, which catalyze the process of dna methylation in mammalian cells utilizing sirna. four dnmt isoforms have previously been identified as follows: dnmt , dnmt , dnmt a, and dnmt b ( ). fig. , a and b, shows that sirna transfection caused a significant decrease in the mrna and protein expression of respective dnmt in hutu- cells. the expression of npc l was not altered in response to the attenuation of dnmt and dnmt b expres- sion (fig. c). however, the simultaneous suppression of dnmt and dnmt b by sirna knockdown caused a signif- icant increase in npc l mrna expression, further indicating the essential role of both dnmt and - b in maintaining dna methylation status of human npc l (fig. d). dna methylation decreases npc l promoter activity— we next investigated the direct effects of dna methylation on the promoter activity of human npc l . we have previously cloned and characterized a promoter fragment flanking the region between � and � (� is the transcription initi- ation site) of the human npc l gene ( ). to examine the effects of dna methylation, the npc l promoter fragment was subcloned into the cpg-free-basic lucia vector and then subjected to in vitro dna methylation as described previously ( ). the methylated construct of npc l promoter along with the expression vector for �-galactosidase (control for transfection efficiency) were transfected into human caco cells, and the promoter activity was evaluated by assessing the ratio of lucia to �-galactosidase activities. as shown in fig. a, npc l promoter activity was significantly higher (� -fold) than the activity of empty vector alone. fig. a also shows that in vitro dna methylation before transfection significantly reduced npc l promoter activity to the levels of empty vec- tor. the activity of the control promoter (ef ) in the same cpg-free vector was not affected by in vitro dna methylation indicating that the decrease observed in npc l promoter activity by in vitro dna methylation was specific (fig. b). these findings clearly show that the promoter activity of human npc l gene is suppressed by dna methylation. discussion npc l mrna and protein are differentially expressed along the length of the gastrointestinal tract. indeed, the npc l protein is absent in the colon, and the mrna is min- imally expressed. our data showing that the level of npc l mrna is remarkably lower in the mouse colon by � -fold as compared with ileum and jejunum are in agreement with the recent findings reported by xie et al. ( ). this segment-spe- cific expression raises the following important physiological question. what is the mechanism responsible for the lack of npc l expression in colon? figure . -azacytidine increases npc l expression in hutu- and caco cells. a, hutu- cells were treated with different concentrations of -aza- cytidine ( , , and �m) for h (white bars) and h (dark bars). b, caco cells grown on filter support were treated with -azacytidine for h. cells were then harvested, and rna was isolated. npc l mrna expression relative to gapdh mrna expression was assessed by q-rt-pcr. results are presented as fold increase as compared with untreated control that is set as . data represent mean � s.e. of three different experiments. *, p . treated versus control untreated cells. epigenetic regulation of npc l august , • volume • number journal of biological chemistry intestinal segment-specific patterns of gene expression have long been investigated, and a number of mechanisms have been suggested. different levels of expression of transcription factors (stimulator or suppressor) may be involved, as is the case for gata- , which recently has been shown to be essential for defining the jejunal phenotype in the small intestine ( , ). epigenetic mechanisms, including histone modifications and/or dna methylation or regulation by mirna, may also be involved ( – ). indeed, in earlier studies a possible role of dna methylation was suggested based upon the fact that the lack of the methyl dna-binding protein mbd in knock-out mice resulted in stimulation of expression of a set of pancreatic and duodenal genes in the colon ( ). it is also interesting to note that the genes induced in the colon of these mice were generally involved in nutrient digestion and absorption in the small intestine ( ). this report suggests that dna methylation represents one of the major mechanisms responsible for sup- pressing nutrient digestion and absorption processes in the colon. because npc l mediates cholesterol absorption, it is, therefore, logical to consider dna methylation as one of the potential mechanisms responsible for the suppression of its expression in the colon. indeed, our data showed that npc l figure . sirna-mediated knockdown of dnmts increases npc l expression. hutu- cells were transfected with sirnas specific for different isoforms of dnmts or control (ct) scramble sirna. cells were then harvested h post-transfection, and total rna and protein were isolated. mrna expression of each dnmt in respective dnmt silencing was assessed by q-rt-pcr relative to gapdh expression (a). data are presented as mean � s.e. from three different experiments. *, p . as compared with respective control. protein expression of each dnmt in respective dnmt silencing was assessed by western blotting (wb) relative to gapdh expression (b). npc l mrna expression in respect to silencing of respective dnmts was assessed by q-rt-pcr relative to gapdh expression (c). relative expression of npc l mrna was evaluated after simultaneous attenuation of dnmt and - b expression in hutu- cells (d). data are presented as mean � s.e. from three different experiments. *, p . as compared with control. figure . effect of dna methylation on npc l promoter activity. human npc l promoter region flanking the area between � and � (� is the transcription initiation site) was amplified by pcr and cloned into a promoterless cpg free vector (pcpg free basic lucia) upstream of the lucia gene. the npc l promoter construct as well as the empty vector were then subjected to in vitro methylation. the methylated and unmethylated empty vector (pev) and npc l promoter construct (l p) were transiently transfected into caco cells along with the mammalian expression vector of �-galactosidase. h post- transfection, cells were harvested, and lucia activity and �-galactosidase activity were measured. the relative promoter activity was determined as a ratio of lucia to �-galactosidase (a). data are presented as fold increase compared with unmethylated empty vector and represent mean � s.e. of three independent experiments performed on separate occasions. *, p . compared with empty vector pev. cpg free ef promoter lucia vector (pcpg free lucia) was used as control for methylation as described under “results” (b). data are presented as fold change relative to unmethylated vector. epigenetic regulation of npc l journal of biological chemistry volume • number • august , mrna expression in mouse colon is remarkably induced by the dna methyltransferase inhibitor -azacytidine. the -azacy- tidine is an aza-nucleoside that enters the cells and is subse- quently converted to -aza-dctp and incorporated into the dna ( ). the incorporated aza-cytosines in the cpg dinucle- otides are recognized as natural substrates for the dna meth- yltransferases. the binding of the methyltransferases to the aza-cytosine is covalent resulting in degradation of the enzyme and loss of the dna methylation mark ( ). the dose and duration of -azacytidine treatment utilized in current studies in mice have been previously shown to be effective in decreasing dna methylation and stimulating gene expression ( ). the fact that -azacytidine signifi- cantly increased colonic expression of npc l mrna in mice strongly suggests that npc l gene expression is silenced in the colon by dna hypermethylation. the mapping of methylation sites in the npc l gene in mouse intestine revealed valuable information supporting the role of dna methylation in controlling segment-specific expression of mouse npc l . our data showed that the overall level of dna methylation of cpg dinucleotides present in the -kb region of the npc l gene upstream of the translation ini- tiation site was similar between ileum and jejunum and signif- icantly higher in the colon as compared with both ileum and jejunum. it is important to note that methylation of some of the cpg sites analyzed were similar between the colon and the small intestine suggesting that the methylation of specific cpg dinucleotides in the npc l promoter appears to be crucial for suppressing the expression of npc l . the methylation of these sites with higher levels in the colon was significantly decreased by treatment with -azacytidine. the decrease in dna methylation in a number of cpg sites by -azacytidine also occurred in a number of cpg dinucleotides in ileum and jejunum as well, but it was not associated with any increase in npc l expression in these regions. only three cpg dinucle- otides, i.e. � , � , and � (relative to the translation initiation site), in the npc l gene showed a statistically sig- nificant decrease in dna methylation by -azacytidine that was concurrent with the increase in npc l mrna expression in the colon. altogether, our initial mapping of dna methylation sites on the mouse npc l promoter demonstrated for the first time a strong correlation between the methylation of spe- cific cpg sites and npc l mrna expression. furthermore, it appears that the high levels of dna methylation of npc l promoter are strongly associated with the suppression of mrna expression in the colon. the level of dna methyltrans- ferase dnmt is significantly less in the colon as compared with the small intestine. however, the mrna expression of dnmt a and dnmt b is significantly higher in the colon as compared with the jejunum. it should be noted, however, that the levels of dnmt a and - b are similar in both the ileum and colon. these data suggest that dna methylation of the npc l promoter does not correlate with the expression of dnmts. the findings suggest that npc l gene methylation may be dependent on other factors beside dnmts such as dna demethylases. similar to mouse npc l , dna methylation appears to play a critical role in the expression of human npc l mrna according to our findings in human intestinal hutu- cells. dna methylation is mediated by dnmts that catalyze the transfer of methyl groups from s-adenosylmethionine to the cytosine residue in the cpg dinucleotides ( ). three distinct families of dnmts, dnmt , dnmt , and dnmt , have been identified in mammals. dnmt , known as the mainte- nance dnmt, mediates the methylation of the hemimethyl- ated double-stranded dna. dnmt is responsible for the de novo methylation, as it methylates the unmethylated and hemi- methylated double-stranded dna. dnmt family consists of three members as follows: dnmt a, dnmt b, and dnmt l. dnmt l lacks the cytosine methylation activity and functions as a regulatory factor in germ cells ( ). the func- tional roles of dnmt are slowly emerging, and recent studies suggested its involvement in trna methylation ( ). our find- ings show that the attenuation of either dnmt or dnmt b alone did not affect npc l mrna expression. this is not surprising as previous studies showed functional redundancy between these different isoforms of dnmts and that dnmt and dnmt b cooperate to maintain dna methylation in human cancer cell lines ( – ). indeed, the expression of npc l was significantly induced only when the expression of both dnmt and dnmt b was attenuated in hutu- cells. moreover, we have provided novel experimental evidence clearly showing that the in vitro methylation significantly decreased npc l promoter activity in caco cells. overall, our results show for the first time that dna methylation sup- presses npc l promoter activity and decreases npc l mrna expression. it is, however, important to note that the contribution of other pathways beside dna methylation in dic- tating the differential expression of intestinal npc l is also possible. for example, various transcription factors such as srebp and hnf � are known to modulate npc l expres- sion ( , ). these and other transcription factors may play a role in its distribution along the duodenal-colonic axis. the data from this study not only unravel a mechanism involved in the segment-specific expression of intestinal npc l , but also demonstrate a novel means to modulate its expression. in this regard, the increase in npc l expression has been shown in diseases associated with hypercholesterol- emia such as diabetes mellitus ( ). also, the inhibition of npc l expression was shown to be beneficial in decreasing the levels of blood cholesterol ( ). moreover, hepatic npc l was recently shown to be involved in infection with hepatitis c virus and other hepatic disorders such as steatosis ( ). there- fore, npc l appears to be an attractive target for inhibition in the treatment of a number of disorders, including infection with hepatitis c virus and hypercholesterolemia. dna meth- ylation was recently shown to be a dynamic process that is altered in somatic adult cells in response to environmental fac- tors such as exercise ( ). also, recent investigations have shown that dietary supplements containing betaine, metafo- line, creatine, and vitamin b could alter dna methylation ( ). future studies will aim at examining the effects of these types of dietary supplements on dna methylation of the npc l gene and its expression in the intestine and liver. in conclusion, our current findings suggest that increasing dna methylation of the npc l gene may suppress npc l epigenetic regulation of npc l august , • volume • number journal of biological chemistry expression in the small intestine and liver. the increase in dna methylation of npc l by dietary manipulation may therefore provide a novel therapeutic means for the treatment of a num- ber of disorders such as hypercholesterolemia, hepatic hepatitis c virus infection, and hepatic steatosis. acknowledgment—we thank dr. k. ramaswamy for the critical review of the manuscript and helpful discussion. references . van der wulp, m. y., verkade, h. j., and groen, a. k. 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( ) a therapeutic trial of pro-methylation dietary supplements in angelman syndrome. am. j. med. genet. a a, – epigenetic regulation of npc l journal of biological chemistry volume • number • august , 	epigenetic modulation of intestinal cholesterol transporter niemann-pick c -like (npc l ) gene expression by dna methylation* 	experimental procedures 	results 	discussion 	acknowledgment 	references journal of neurology, neurosurgery, and psychiatry ; : - letters to the editor insulinoma complicating tuberous sclerosis a young man, known to have tuberous sclerosis, recently presented to us with tired- ness and recurrent seizures after being fit free for fifteen years. removal of an insulinoma led to complete relief of symptoms which had for some time been attributed to the tuberous sclerosis. the patient was a year old man. diagnosis of tuberous sclerosis was made in childhood. he had adenoma sebaceum and mild mental retardation. he had had gener- alised seizures up to the age of seven years and had then been fit free until months before presentation. seizures continued despite therapeutic levels of carbamazepine and primidone. he also complained of sleep- iness after exertion and an increased appetite for sweet foods. there was no family history of tuberous sclerosis. apart from the typical skin changes and retardation there were no abnormal physical findings. his weight was normal for his height and there had been no change in weight. ct scanning of the brain showed the typical intracerebral masses of tuberous sclerosis. he was referred for investigation when a plasma glucose of - mmol/l (normal range - mmol/l) was noted following a sei- zure. excess insulin excretion was demon- strated hours into a fast when he suffered a generalised seizure with plasma glucose - mmol/l and insulin miu/l (expected < in presence of hypoglycaemia). whilst ct of the pancreas was unremarkable, coeliac angiography demonstrated a cm blush in the inferior portion of the pancreatic head. a benign islet cell tumour was subsequently removed. he has remained seizure free and maintains a normal blood sugar without excess carbohydrates. there is only one other reported case of insulinoma complicating tuberous sclerosis and as in our patient the diagnosis was delayed.' a second patient has been reported with a non-functioning islet-cell tumour found at necropsy. she also had hyper- parathyroidism as part of a multiple endo- crine neoplasia. her mother had a parathyroid adenoma and adenoma seba- ceum, probably representing a forme fruste of tuberous sclerosis. there may be a more than chance associa- tion between these two rare conditions. the incidence of insulinoma is estimated at one case per million per year " and the point prevalence of tuberous sclerosis at - per persons in a community based study.' insulinoma should be considered in patients with tuberous sclerosis who present with recurrent or uncontrolled fitting. pm davoren mt epstein royal newcastle hospital, newcastle, nsw australia gutman a, leffkowitz m. tuberous sclerosis associated with spontaneous hypoglycaemia. bmjt ;i: - . ilgren eb, westmoreland d. tuberous sclerosis: unusual associations in four cases. j clin pathol ; : - . kavlie h, white tt. pancreatic islet beta cell tumours and hyperplasia: experience in seattle hospitals. ann surg ; : - . buchanan kd, johnston cf, o'hare mmt, et al. neuroendocrine tumours; a european review. am med ; (suppl b): - . wiederholt wc, gomez mr, kurland lt. incidence and prevalence of tuberous sclero- sis in rochester, minnesota, through . neurology ; : - . the influence of head position upon head tremor an alteration in head position influences hemiparetic limb posture,' torticollis and the amplitude of head tremor. there is some conjecture as to whether this is due to: ) a muscle spindle effect; ) a consequence of altered muscle tone due to a change in loading of neck muscles when the head is in a dependent position; ) a cns effect related to the execution of a motor programme, as suggested in writing tremor or ) whether it is dependent upon vestibular mechanisms, with an alteration in the tonic discharge of the otolith receptors in response to gravity,' for example, in the modulation of downbeat nystagmus by head position.' head tremor may occur in a variety of conditions including essential tremor, dys- tonia and cerebellar disease, though mecha- nisms underlying such head tremors are poorly understood. in some patients, the amplitude of head tremor changes con- siderably with head position. we describe in detail one of four such patients in whom this effect appears to be due to factors other than a vestibular mechanism. a year old woman developed increasing head tremor over a period of years. it had first been noticeable on eating and drinking. over the past three years tremor had affected her voice and arms. there was some improve- ment of tremor with alcohol but no family history of tremor in first degree relatives. on examination, with the head upright, there was a marked tremor of the head in the planes of yaw (no-no) and pitch (yes-yes) which increased in amplitude on neck flex- ion. her speech was interrupted by tremor though this did not affect the tongue at rest. eye movements were normal. there was a variable head tilt and slight rotation of the head on attempted drinking. there was a postural and action tremor of the out- stretched arms at - - hz, similar to that of the head. deep tendon reflexes were brisk with bilateral extensor plantars. she was mildly ataxic with poor heel-to-toe walking. there was no dystonic posturing of the limbs. cortical somatosensory evoked poten- tials were delayed and ct scanning showed mild cerebral and cerebellar atrophy. csf analysis was normal with no oligoclonal bands. there were no prolonged spasms or long bursts ofemg activity on surface emg recording of spleni and sternomastoids, as may be typically seen in dystonia. influence of head position upon head tremor the amplitude of head tremor was meas- ured using an angular accelerometer (schae- vitz, asamp- ), with the sensitive axis orientated in the horizontal plane and with the patient sitting upright with the head in the neutral position and separately with the neck flexed and extended. tremor amplitude during neck extension was unchanged from that in the neutral position. with neck flex- ion, tremor amplitude increased in magni- tude eight-fold though the frequency did not change (table). this effect could be attrib- uted to a change in either proprioception, muscle tone, the motor programme or vestib- ular input. to distinguish between these possibilities tremor was assessed with the patient lying prone and supine on a firm mattress. in this way the muscle spindle input from neck flexion and extension was reduced, the loading on neck musculature was reduced and the motor programme of the cns was changed-thereby allowing us to assess any otolith effect. with the patient lying prone (and with the face in the same position in relation to gravity as with the neck flexed) the amplitude of the tremor decreased slightly compared with head neutral (table). with the patient lying supine, the amplitude of head tremor was little changed, despite the head being sim- ilarly supported by the mattress (table). this argues against the criticism that the absence of an increase in amplitude ofhead tremor on lying prone was due to the head being partially supported on the mattress. thus the marked change in amplitude of head tremor between neck flexion and the patient lying prone, with a constant level of otolith input, implies that the head tremor was not influ- enced by otolith function, whose tonic firing is dependent upon their orientation to the gravity vector. further, the absence of a major change in the amplitude of the head tremor while prone and supine also provides evidence against the head tremor being influ- enced by the otoliths in this patient. nystagmus that appears with the head in a static position, that is, otolith-dependent static positional nystagmus, may be accen- tuated with one ear down and lessened with the opposite ear down, reflecting the influ- ence of gravity upon static otolith receptors. using the same analogy, as tremor was maximal with the neck flexed, with "face down", it would be expected that tremor would decrease in "face-up" positions (neck extension or supine) if otolith mechanisms were involved. this was not the case, which suggests, that in this patient, the effect of head position in altering head tremor is likely to be due to an alteration in muscle spindle input, with altered stretch of muscles and/or altered muscle tone, with a change in the contractile force of different muscles when the head is in a different position and/or related to the execution of a motor pro- patient sitting patient lying head neutral neck extended neck flexed prone supine tremor amplitude variables influencing tremor amplitude muscle spindle + + - - muscle tone + + vestibular t i t motor programme + + + increased or changed with respect to head neutral - decreased or changed with respect to head neutral i t similar or opposing directions of otolith input tremor amplitude refers to peak amplitude in degrees tremor frequency was similar in all instances ( - - hz). o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn n p .b m j.co m / j n e u ro l n e u ro su rg p sych ia try: first p u b lish e d a s . /jn n p . . . -a o n d e ce m b e r . d o w n lo a d e d fro m http://jnnp.bmj.com/ letters to the editor gramme. this is in keeping with the tremor of other body parts, for example the wing- beating arm tremor of wilson's disease and task-specific tremors such as writing and other occupational tremors. we have examined clinically, three other patients with clear dystonic head tremor in whom head tremor was altered by the posi- tion of the head in the anterior-posterior plane. in none of the others could we demonstrate a vestibular (otolith) mecha- nism. despite these negative observations for a clear otolith influence upon head tremor electrophysiological measurements have shown that otolith spinal reflexes may be modulated by both head and body position. thus there still remains the possibility that a change in head or body position may in itself modify vestibular influences upon body movement. s mossman l cleeves l findley mrc human movement and balance unit, section of neuro-otology, national hospital for neurology and neurosurgery, london, uk correspondence to: dr mossman, department of neurology, wellington hospital, private bag, wel- lington south, wellington, new zealand. denny-brown d. clinical symptomatology of diseases of the basal ganglia. in: vinken p, bruyn g, eds. handbook of clinical neurology, vol . amsterdam: north holland publishing co, : - . bronstein a, rudge p, beechey a. spasmodic torticollis following unilateral viii nerve le- sions: neck emg modulation in response to vestibular stimuli. j neurol neurosurg psy- chiatry ; : - . cleeves l, findley l, marsden d. odd tremors. in: marsden d, fahn s, eds. movement disorders . butterworths (in press). podivinsky f. torticollis. in: vinken p, bruyn g, eds. handbook of clinical neurology, vol . amsterdam: north holland. : - . gresty m, barratt h, page n, rudge p. analysis of downbeat nystagmus. otolith vs semi- circular canal influences. arch neurol ; : - . dieterich m, brandt t, fries w otolith func- tion in man. brain ; : - . probable cases of mast syndrome in a non-amish family complicated forms of hereditary spastic paraplegia are rare. a year old flemish woman was admitted with a clinical picture of slowly progressive spastic paraplegia, dys- arthria, presenile dementia and mild atheto- sis. at the age of her gait became shuffling. before the age of she used a walking stick, since the age of years she has needed a walking frame and from the age of she has become more wheelchair bound. during the third decade (maybe earlier) dysarthria, apa- thy and negativism appeared. towards her th year, urinary incontinence began. on admission, aged , significant bradyphrenia and comprehension difficulties were noted and during the following years she presented a further mental deterioration. she is now bedridden and her speech restricted to rare, usually inappropriate, single syllable answers, which are sometimes repeated. she has diffi- culty swallowing fluids. she often shows spontaneous repeated slow turning of the head to the right and left and mild tortuous movements of the shoulders. except for a divergent strabismus there are neither oculo- motor abnormalities, nor fundoscopic anomalies. there is a slight bilateral facial weakness. the fine motor hand skills are lost , = proband; * = birth order in this sibship is uncertain figure pedigree without clear paresis of the upper limbs. the strength of leg muscles measures about to / . there were neither sensory deficits nor cerebellar signs. deep tendon reflexes in the upper limbs were increased slightly. knee jerks were unusually brisk and without clo- nus; ankle jerks were decreased. plantar responses were extensor. snout reflex and bilateral palmomental reflexes were present whilst corneomandibular reflexes were absent. the following laboratory investigations showed no significant abnormalities: routine blood examination (except for intermittent elevation of glucose with normal haemoglo- bin a c ), creatine kinase, copper, lipids, very long chain fatty acids ratios c /c and c /c , vitamin e, vitamin b , folate, cortisol, adrenocorticotropic hor- mone, thyroid hormones, arylsulfatase a and hexosaminidase a + b; the csf protein was mg/l with normal electrophoretic pat- tern. the eeg showed mild general slowing. nerve conduction studies and needle electro- myography showed a mild axonal polyneur- opathy. the somato-sensory evoked potentials demonstrated a slightly prolonged central conduction time. an electrocardio- gram was normal. mri of the brain showed diffuse cortico-subcortical atrophy, periven- tricular hyperintensities, thin corpus callo- sum and less marked atrophy of the brainstem and cerebellum. light microscopic and electron microscopic examination of conjunctiva and skin showed some mem- branous cytoplasmic body-like inclusions (professor j j martin, dr c ceuterick-de groote, university hospital antwerp). the family history revealed two similar cases (figure). there was no known con- sanguinity. the monozygotic twin has an almost identical medical history and clinical picture. she is able to stammer a few simple words. her answers are sometimes slightly more appropriate, particularly for old memories. her knee jerks and ankle jerks are both unusually brisk, and the plantar responses are extensor. one brother died at the age of . the medical records and relatives des- cribed difficulties with walking from the age of (maybe earlier). during the following decades he presented a progressive spastic paraparesis, dysarthria, mental deterioration and urinary and faecal incontinence; no deficits of sensation or coordination were demonstrated. there was dysphagia in his last years. death was due to pneumonia. the pedigree suggests an autosomal reces- sive inheritance. the neurodegenerative syn- drome in this family seems fully comparable to the mast syndrome, described in in an ohio amish isolate by cross and mcku- sick.`'- there appears to be no similar cases that have been described outside the amish population. marc d'hooghe department of neurology, algemeen ziekenhuis st j an, ruddershove , brugge, belgium cross he, mckusickva. the mast syndrome: a recessively inherited form of presenile de- mentia with motor disturbances. arch neurol (chic) ; : - . harding ae. classification of hereditary ataxias and paraplegias. lancet ;i: - . mckusick va. mendelian inheritance in man. baltimore: the johns hopkins university press, : . ultrasensitive tsh assay and anti-par- kinsonian treatment with levodopa we have recently reported the association of parkinson's disease and hyperthyroidism in a group of patients.' in that report, symp- toms of parkinson's disease were always significantly exacerbated by the development of hyperthyroidism and improved by its successful treatment.' we proposed that hyperthyroidism should be suspected in all parkinsonian patients when their condition deteriorates.' since clinical diagnosis of thy- rotoxicosis is difficult in parkinsonian patients, they should have a comprehensive thyroid examination and, if there is the slightest suspicion of hyperthyroidism, a hor- monal evaluation of thyroid function (free t , ultrasensitive tsh). thyroid hormone levels (t and t ) have been found to be normal in parkinsonian patients untreated or treated with levodopa. however, a decreased response of thyro- tropin (tsh) after stimulation by trh (thyrotropin releasing hormone) has been reported in parkinsonian patients treated with levadopa. such a decreased tsh response after trh stimulation is observed during hyperthyroidism, and is sometimes the only hormonal abnormality, especially in elderly patients with autonomous thyroid nodules. thus the decreased tsh response after trh-stimulation in patients treated with levodopa could be responsible for a false diagnosis of hyperthyroidism. these results, however, were obtained before the ultra- sensitive tsh determination with a mono- clonal antibody assay was available. a low ultrasensitive tsh level has the same sig- nificance as a decreased tsh response to trh, indicating an increased negative feed- o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn n p .b m j.co m / j n e u ro l n e u ro su rg p sych ia try: first p u b lish e d a s . /jn n p . . . -a o n d e ce m b e r . d o w n lo a d e d fro m http://jnnp.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ revista chilena de pediatría - septiembre - octubre abstract ellis van creveld. case report introduction: ellis-van creveld (evc) (omim # ) syndrome is a rare skeletal dysplasia disorder trans- mitted by autosomal recessive inheritance. the diagnosis is made based on phenotypic characteristics such as chondrodysplasia, heart defects and polydactyly. the prognosis depends mainly on the severity of the disease, diagnosis and comprehensive management of the condition. objective: to describe a patient diagnosed with evc syndrome. case report: newborn diagnosed with evc syndrome who presented dysmorphic facies, shortened long bones, rhizomelic shortening, small hands, brachydactyly, single transverse palmar crease, postaxial polydactyly in the upper limbs, bilateral preaxial polysyndactyly in lower limbs and hypoplastic nails, complex heart defects and narrow thorax. the evolution was unfavorable; the patient died weeks after birth from complications due to heart defects. conclusions: evc syndrome is rare and unknown; therefore, it is important to spread its characteristics within the pediatric community, emphasizing that it affects multiple organ systems and requires a multidisciplinary approach to treat individually each patient, to provide genetic and reproductive counseling to couples and to give information regarding child development expectations. (key words: ellis–van creveld syndrome, chondroectodermal dysplasia, polydactyly, congenital anomalies). rev chil pediatr ; ( ): - resumen introducción: el síndrome ellis-van creveld (evc) (omim # ) es una displasia esquelética rara de he- rencia autosómica recesiva, cuyo diagnóstico se realiza por sus características fenotípicas como la condrodis- casos clínicos rev chil pediatr ; ( ): - doi: . /s - ellis van creveld: reporte de caso m. cruz-perea • d. luna • j. ramírez-cheyne • w. saldarriaga • c. isaza recibido el de enero de , última versión aceptada el de octubre de . manuel cruz-perea, dayhanna luna balcázar estudiante de pregrado, escuela de medicina, universidad del valle. cali, colombia. julián ramírez-cheyne md. (*) médico, escuela de ciencias básicas médicas, universidad del valle. cali, colombia. e-mail: juracheyne@gmail.com wilmar saldarriaga gil md., msc. médico, ginecólogo y obstetra, magíster en ciencias básicas medicas, embriología y genética. profesor asociado, escuela de ciencias básicas médicas, universidad del valle. cali, colombia. carolina isaza md., msc. médico, magíster en ciencias básicas médicas, embriología y genética. profesor titular, escuela de ciencias básicas médicas, universidad del valle. cali, colombia. volumen - número introducción el síndrome ellis-van creveld (evc) (omim # ) es una displasia condroecto- dérmica que se caracteriza por costillas cortas, polidactilia, retraso del crecimiento, defectos ectodérmicos y anomalías cardíacas; produci- das por mutaciones en los genes evc y evc (ellis van creveld y ellis van creveld ) lo- calizados en p y que tienen un patrón de herencia autosómica recesiva . las mutaciones en evc se describieron ini- cialmente en las genealogías amish y en bra- sil, aunque estas sólo representan una pequeña proporción de la totalidad de afectados . pos- teriormente, se describió la mutación en el gen evc en un niño asquenazí que presentaba este síndrome . los pacientes con mutaciones en evc o evc son fenotípicamente indistin- guibles . es una enfermedad poco frecuente, de pre- valencia estimada en por cada . . de habitantes en población general , sin embargo, es conocido que los trastornos recesivos infre- cuentes pueden tener frecuencias elevadas en grupos genéticamente aislados o con efecto fundador, como es el caso de la frecuencia de evc en el grupo etno-religioso amish . uno de los mayores asentamientos de este grupo se ubica en el condado de lancaster, pennsyl- vania, y presenta una prevalencia de evc es- timada en por . nacidos vivos, siendo esta la más alta conocida . la frecuencia de portadores en esta población puede ser hasta de un % . para este reporte se atendieron las respectivas consideraciones éticas, dentro de las que realizó un consentimiento informado a los padres del paciente. objetivo caracterizar un caso de paciente con diag- nóstico clínico de síndrome de evc. se hace la discusión del caso con una breve revisión de la literatura, dando información a la co- munidad pediátrica de un síndrome poco co- nocido. caso clínico recién nacido de sexo masculino, cesárea a las semanas de edad gestacional debido a una cardiopatía compleja; peso de nacimiento . g (p ), longitud cm (p ), perímetro cefálico cm (p ). padres no consanguíneos sin antecedentes personales ni familiares de importancia, madre primigesta de años. a las semanas de gestación se encontró translucencia nucal mayor de p , por lo que se realizó un cariotipo en biopsia de vellosida- des coriales que mostró un resultado , xy; a las semanas una ecografía evidenció car- diopatía compleja, con un canal aurículoven- tricular completo, mientras que en una eco- plasia, cardiopatía y polidactilia. el pronóstico depende fundamentalmente de la severidad de la cardiopatía, al igual que del diagnóstico y manejo integral oportunos. objetivo: caracterizar un paciente con diagnóstico clínico de síndrome de evc, cuya baja frecuencia dificulta el correcto diagnóstico en pediatría. caso clíni- co: recién nacido con facies dismórfica, extremidades con huesos largos cortos, acortamiento rizomélico, manos pequeñas, braquidactilia, pliegue palmar único, polidactilia post axial en miembros superiores, polisin- dactilia preaxial bilateral en miembros inferiores y uñas hipoplásicas, cardiopatía compleja y tórax estrecho, en el que se concluyó un diagnóstico clínico de evc. la evolución fue desfavorable, falleciendo a las semanas de nacimiento por complicaciones secundarias a la cardiopatía. conclusiones: el síndrome de evc es de baja frecuencia y poco conocido, por lo que es importante difundir sus características en la comunidad pediátrica, haciendo énfasis en que al afectar múltiples sistemas y órganos, requiere un manejo multidisciplinario con el objetivo de intervenir en la patología individualizando cada paciente; además de consejería genética y repro- ductiva a las parejas, e información de las expectativas del desarrollo del niño. (palabras clave: síndrome ellis-van creveld, displasia condroectodérmica, polidactilia, anomalías congé- nitas). rev chil pediatr ; ( ): - ellis van creveld revista chilena de pediatría - septiembre - octubre cruz-perea p. y cols. figura . foto de acercamiento facial y vista de cabeza, tronco y extremidades parcialmen- te. se observa orejas de implantación baja con rotación hacia atrás y puente nasal deprimido. además acortamiento de todas las extremidades al comparar con el tronco; tórax estrecho, mos- trando desproporción con el abdomen. figura : foto de mano y pie, se observa polidactilia post axial en mano y preaxial en miembro inferior; además uñas hipoplásicas. grafía de tercer trimestre los huesos largos se encontraban en p . al nacimiento fue hospitalizado en unidad de cuidados intensivos neonatal. evaluado por genetista clínico, quien al examen dismorfoló- gico describió: microcefalia, orejas de implan- tación baja con rotación hacia atrás, puente na- sal deprimido, pliegue epicántico interno, es- trabismo convergente, en boca labio superior corto unido por frenillos al reborde alveolar, extremidades con huesos largos cortos, rizo- melia (figura ), manos pequeñas, braquidac- tilia, pliegue palmar único, polidactilia post axial en miembros superiores, polisindactilia preaxial bilateral en miembros inferiores y uñas hipoplásicas (figura ). el ecocardiograma mostró canal aurículo- ventricular, estenosis aórtica, hipoplasia del arco aórtico, ductus arterioso e hipoplasia leve de arteria pulmonar derecha, también se rea- lizó ecografía transfontanelar y renal que re- sultaron normales; radiografías de miembros inferiores mostraron fémur y la tibia cortos con adecuada osificación, en radiografía tora- coabdominal se observó un tórax estrecho al comparar con el abdomen y una silueta cardía- ca aumentada de tamaño al comparar con el tó- rax (figura ); en la columna no se observaron alteraciones vertebrales (figura ). la suma de hallazgos permitió diagnosticar el síndrome de ellis van creveld (tabla ). el paciente tuvo una evolución desfavorable fa- lleciendo a las semanas de nacido, por pro- blemas secundarios a su cardiopatía compleja. discusión desde la descripción del síndrome de evc en el año , con el nombre de displasia condroectodérmica, se han reportado en la li- teratura aproximadamente casos . el caso volumen - número ellis van creveld tabla . se comparan características fenotípicas reportadas (omim ) para el síndrome de ellis van creveld (evc) y las encontradas en el paciente características reportadas en la literatura características presentadas por el paciente talla baja con extremidades cortas al nacimiento si normocefalia no (microcefalia) facies normales (con excepción del labio superior) pliegue epicantico interno, puente nasal deprimido anomalías del labio superior si (labio superior corto) labio fisurado no defectos de cresta alveolar si (sinequia labio-encía) dientes neonatales no hipodontia, erupción retrasada no aplica defecto septal atrial, atrio único si canal aurículoventricular si defecto septal ventricular si ductus arterioso persistente si otros defectos cardíacos si tórax estrecho si pectus carinatum no costillas cortas, poco desarrolladas si anomalías génito urinarias no rizomelia si braquidactilia si polidactilia en manos y pies si epífisis en cono de las falanges a de las manos no evaluado pie equinovaro no sindactilia en dedos de pies si displasia de uñas si déficit cognitivo no aplica figura : radiografías tóracoabdomi- nal y de miembros inferiores, se observa tórax estrecho al comparar con el abdo- men, costillas cortas; silueta cardíaca aumentada de tamaño al comparar con el tórax; fémur y la tibia cortos, ensancha- miento anormal de porción proximal de humeros, con adecuada osificación. revista chilena de pediatría - septiembre - octubre aquí presentado sería el segundo reportado en colombia . este síndrome tiene una expresión fenotí- pica variable, observable en el período fetal a través de ecografía obstétrica, lo cual permi- te sospechar prenatalmente su presencia , , al encontrar hallazgos de aumento de la translu- cencia nucal y cardiopatía , como ocurrió en el caso reportado. los pacientes con evc presentan talla me- nor al percentil , con extremidades despro- porcionadas con respecto del tronco, anoma- lías esqueléticas, polidactilia principalmente en manos, generalmente bilateral, ocasional- mente en los pies; cardiopatías como: aurícula única, defectos de la válvula mitral y tricúspi- de, ducto arterioso persistente, comunicación interventricular, comunicación interauricular e hipoplasia cardíaca izquierda ; displasia de uñas, cabello quebradizo y erupción temprana de dientes . su perímetro cefálico suele ser normal y usualmente no se presentan anoma- lías del sistema nervioso central por lo que el desarrollo cognitivo es normal . el paciente presentado mostró todas las características del síndrome de evc al nacimiento excepto la erupción temprana de dientes. la condrodistrofia es un rasgo caracterís- tico y se manifiesta con retardo en la madu- ración ósea, fusión de los huesos del carpo y metacarpo, depresión proximal y lateral de la tibia, luxación congénita de rótula, cubito val- go, hipoplasia cubital, genu valgo, clinodacti- lia del quinto dedo y alteración del modelado óseo de los metacarpianos y falanges. el tórax presenta frecuentemente costillas cortas y es- trechas, con pectus excavatum y lordosis lum- bar aumentada , . hasta en un % de los casos se presenta displasia ectodérmica hidrótica caracteriza- da por uñas friables, distróficas, hipoplásicas o incluso completamente ausentes; el cabello puede ser escaso o quebradizo; y las anomalías dentarias incluyen presencia neonatal de dien- tes, anodoncia parcial, hipoplasia del esmalte que se manifiesta con dientes pequeños anor- malmente formados, mal oclusión o retraso en la erupción , , . pueden encontrarse pacientes con evc que presenten agenesia o displasia renal, megauré- ter, epi o hipospadias, en un % de los casos ; en menor proporción estrabismo, malforma- ciones pulmonares, de la pared torácica , , y sólo un caso en la literatura reporta alteracio- nes hematológicas (leucemia mieloblástica pe- rinatal) . el síndrome evc hace parte un grupo de- nominado como “síndromes de costillas cor- tas-polidactilia”, donde también se encuentran el síndrome de saldino-noonan (tipo i), el sín- drome de majewski (tipo ii), el síndrome de verma-naumoff (tipo iii), el síndrome de bee- mer-langer (tipo iv) y distrofia de jeune . las características fenotípicas comunes de estos síndromes pueden dificultar su dife- renciación, aunque existen indicios que per- miten aclarar el diagnóstico: paladar hendido en majewski; onfalocele y fémur curveado en beemer-langer ; distrofia torácica, reducción de las extremidades y displasia ósea generali- zada en jeune ; prevalencia mayor de cardio- patías, condrodisplasia y polidactilia postaxial en evc . además en el paciente reportado se diferenció del síndrome de saldino-noonan, por no observarse en las radiografías las cos- tillas deformadas. se debe sospechar el síndrome de evc en casos de cardiopatías complejas principalmen- te aurícula única, facies dismórficas, tórax es- trecho, acortamiento rizomélico, polidactilia; en estos casos se deben realizar radiografías del tórax observando detenidamente las costillas; y de extremidades buscando condrodistrofias; ecografía renal con el objeto de descartar age- nesia y alteraciones de la vía urinaria; además se deben descartar alteraciones cromosómicas numéricas o estructurales con cariotipo con bandeo g, e idealmente realizar la prueba mo- lecular para evc y evc , teniendo en cuenta que en un tercio de los pacientes estas pueden no mostrar alteraciones . el manejo integral de un paciente con evc se debe realizar por un grupo multidisciplina- rio, idealmente desde la etapa prenatal, iden- tificando características fenotípicas sugestivas del síndrome, permitiendo preparar a la familia y particularmente al equipo de atención neona- tal, para tratar adecuada y oportunamente los problemas respiratorios y cardíacos, ya que si estos se superan mejora el pronóstico. el desa- cruz-perea p. y cols. volumen - número rrollo motor de los pacientes que logran supe- rar la etapa neonatal depende de las alteracio- nes y deformidades óseas que ellos presenten. además se debe realizar consejería genética y reproductiva a los padres dentro de dicho ma- nejo integral; la probabilidad de recurrencia del evento es % en cada embarazo. conclusión el síndrome de evc es un síndrome de baja frecuencia, poco conocido, por lo que es importante difundir sus características en la comunidad pediátrica. debido a que en este síndrome se afectan múltiples sistemas y ór- ganos, requiere un manejo multidisciplinario que incluye un genetista clínico, con el obje- tivo de intervenir adecuadamente en la pato- logía, individualizando cada paciente; además de brindar consejería genética y reproductiva a las parejas y dar adecuada información de las expectativas del desarrollo del niño. potenciales conflictos de interés: este trabajo cumple con los requisitos sobre consentimiento/asentimiento informado, comité de ética, financiamiento, estudios animales y sobre la ausencia de conflictos de intereses según corresponda. referencias .- baujat g, le merrer m: ellis-van creveld syndrome. orphanet j rare dis ; : . .- ruiz-pérez vl, blair hj, rodríguez-andrés me, blanco mj, wilson a, liu yn: evc is a positive mediator of ihh-regulated bone growth that localises at the base of chondrocyte cilia. development ; ( ): - . .- galdzicka m, patnala s, hirshman mg, cai jf, ni- towsky h, egeland ja: a new gene, evc , is mutated in ellis-van creveld syndrome. mol genet metab ; ( ): - . .- ruiz-pérez vl, ide se, strom tm, et al: mutations in a new gene in ellis-van creveld syndrome and weyers acrodental dysostosis. nature genet ; : - . .- zeng z, kun b, jin-wei h, wen-zhen f, chang-qing z, zhen-lin z: identification of one novel mutation in the evc gene in a chinese family with ellis–van creveld syndrome, gene ; : - . .- thompson mw, mclnnes rr, willard hf: genética en medicina. a ed. barcelona, masson, s. a. . .- mckusick va: ellis-van creveld syndrome and the amish. nat genet. ; ( ): - . .- chen h: ellis van creveld syndrome. medscape. com. http://emedicine.medscape.com/article/ - overview#showall . updated: aug , . .- estrada t, montoya j, cortés-yepes h: diagnóstico prenatal del síndrome ellis van creveld: reporte de caso y revisión de la literatura. revista colombiana de obstetricia y ginecología ; ( ): - . .- elçioglu nh, hall cm: diagnostic dilemmas in the short rib-polydactyly syndrome group. am j med genet ; : - . .- venkat-raman n, sebire nj, murphy kw, carvalho js, hall cm: increased first-trimester fetal nucal translu- cency thickness in association with chondroectodermal dysplasia (ellis-van creveld syndrome). ultrasound obstet gynecol ; : - . .- slavkin hc: what the future holds for ectodermal dys- plasias: future research and treatment directions. am j med genet a ; : - . .- scurlock d, ostler d, nguyen a, wahed a: ellis-van creveld syndrome and dyserythropoiesis. arch pathol lab med ; : - . .- tompson sw, ruiz-pérez vl, blair hj, et al: sequen- cing evc and evc identifies mutations in two-thirds of ellis-van creveld syndrome patients. hum genet ; ( ): - . ellis van creveld http://emedicine.medscape.com/article/ -overview#showall http://emedicine.medscape.com/article/ -overview#showall brief genetics report calsquestrin (casq ) gene polymorphisms under chromosome q linkage peak are associated with type diabetes in northern european caucasians swapan kumar das, winston chu, zhengxian zhang, sandra j. hasstedt, and steven c. elbein , genome-wide scans in multiple populations have iden- tified chromosome q -q as one susceptibility region for type diabetes. to map the susceptibility genes, we first placed a dense single nucleotide polymorphism (snp) map across the linked region. we identified two snps that showed strong associations, and both mapped to within intron of the calsequestrin (casq ) gene. we tested the hypothesis that sequence variation in or near casq contributed to type diabetes susceptibil- ity in northern european caucasians by identifying additional snps from the public database and by screen- ing the casq gene for additional variation. in addition to known snps in this region, we found new snps, of which were in exons. a single rare nonsynonymous snp in exon (a v) was not associated with type diabetes. the associated snps were localized to the region between � , in the � flanking region and , in intron (p � . to p � . ). no snp � to intron , including the adjacent gene pea , showed an association. the strongest associations were restricted to individuals of northern european ancestry ascer- tained in utah. a six-marker haplotype was also associ- ated with type diabetes (p � . ), but neither transmission disequilibrium test nor family-based asso- ciation studies were significant for the most strongly associated snp in intron (snp casq ). an inde- pendent association of snps in introns and with type diabetes is reported in amish families with linkage to chromosome q -q . our findings suggest that non- coding snps in casq alter diabetes susceptibility, either by a direct effect on casq gene expression or perhaps by regulating a nearby gene such as pea . diabetes : – , c onsiderable data support a genetic etiology for type diabetes, but multiple susceptibility loci on different chromosomes are likely involved ( ). we previously mapped one type diabetes susceptibility locus in extended north european cauca- sian families to chromosome q -q ( ), a region that is now well replicated in old order amish, pima indian, chinese, french, and british families ( ). recently, we completed a dense linkage map and identified two linkage peaks centered on locations and mb ( ). addition- ally, we have previously reported the association of poly- morphisms of the liver pyruvate kinase enzyme with type diabetes, located centromeric to the first linkage peak ( ). however, analysis of many strong candidate genes under the linkage peaks have not yet identified an associ- ation with type diabetes that could explain the linkage. to further map the susceptibility loci within the region of linkage, we collaboratively placed single nucleotide polymorphisms (snps) over a -mb region that encom- passed both linkage peaks using massarray maldi-tof mass spectrometry (sequenome) ( , ). we typed dna pools constructed from individual samples in three popu- lations: utah caucasians, amish caucasians, and pima indians. of snps that showed evidence for association in the original sample, we selected snps to test in individual samples based on significance on replication in pools (p � . ). two adjacent snps were highly signifi- cant in pools (p � . and . ), subsequently confirmed in individual samples by a different method, and mapped to the first and larger of the two linkage peaks ( mb). the two snps were localized to intron of the calsquestrin (casq ) gene in a region that was also associated with type diabetes in the amish family diabetes study (afds) ( ) and that also included the gene pea , which was reported to show increased expression in tissues from diabetic subjects ( ). to test the hypothesis that sequence variants in or near the casq gene increase susceptibility to type diabetes and to define the region of association, we undertook a two-stage study. we first evaluated snps reported in the public database and extending � and � of casq , includ- ing adjacent genes. subsequently, we screened the exons, untranslated regions, � and � flanking regions, and all of intron of the casq gene for additional sequence from the department of medicine, university of arkansas for medical sciences, little rock, arkansas; the department of human genetics, univer- sity of utah health sciences center, salt lake city, utah; and the department of endocrinology, central arkanasas veterans healthcare system, little rock, arkansas. address correspondence and reprint requests to steven c. elbein, md, professor of medicine, university of arkansas for medical sciences, endocri- nology j- /lr, john l. mcclellan memorial veterans hospital, w. th st., little rock, ar . e-mail: elbeinstevenc@uams.edu. received for publication may and accepted in revised form september . additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. afds, amish family diabetes study; casq, calsquestrin; ld, linkage disequilibrium; snp, single nucleotide polymorphism. © by the american diabetes association. diabetes, vol. , december variation in north european caucasian subjects. we tested each snp for an association with type diabetes. the most promising snps were tested for family-based associations with glucose levels and type diabetes. research design and methods casq gene variation was detected in unrelated caucasian individuals from utah families linked to the q region, including nondiabetic family members and individuals with type diabetes. initial studies to follow up on the pooled associations were conducted in individuals with type diabetes and control individuals from utah that overlapped with but slightly expanded the sample constructed for the original pooling experiment. most individuals were selected from the families used in the linkage study. we subsequently sought to expand the sample size. because insufficient control subjects were available from utah, we examined two sample sets. the first set comprised diabetic individuals and control individuals, all ascertained in utah for northern european ancestry and including the case and control subjects used in the first follow-up studies. to improve our power, albeit at the expense of maintaining homogeneity, we also examined an expanded caucasian sample of caucasian individuals with diabetes and a first-degree relative with diabetes and control individuals with a normal glucose tolerance test or a fasting or random glucose � . mmol/l and no family history of diabetes in a first-degree relative. the expanded sample included additional similarly ascertained individuals from arkansas with mixed caucasian ancestry ( diabetic subject and nondiabetic control subjects). of the diabetic subjects for both analyses, individuals were ascertained from utah families used in the linkage studies and the remainder were ascertained for diabetes by history and medication or documented by oral glucose tolerance test using world health organization criteria. all individuals for the case-control studies were unrelated. because some sam- ples were no longer available or were of poor quality, some case and control samples from utah in the original association study were not included in the expanded sample. transmission disequilibrium test and family-based association studies were conducted on members of families from utah and of northern european ancestry, of whom were considered affected, as previously described ( , ). subjects ascertained in utah provided written informed consent under a protocol approved by the university of utah institutional review board. subjects studied in arkansas provided written informed consent under protocols approved by the university of arkansas for medical sciences human research advisory committee. genotypic analysis and marker selection. additional markers in the casq region were chosen from dbsnp ( ) for the region from kb upstream of casq to kb downstream of the gene. all snps except rs were typed by pyrosequencing using the psq- (pyrosquencing, uppsala, sweden) according to the manufacturer methods but modified to use a universal biotinylated primer (sequences available from authors). snp rs was typed by using oligonucleotide ligation assay with radioactive labeling and detection with a storm optical scanner (molecular dynamics, sunnyvale, ca), as described previously ( ). the insertion/deletion variant rs was typed using infrared dye–labeled m primers on a li-cor gr sequencer and scored using gene imagir software (version . . ; scanalytics, fairfax, va). all variants were in hardy-weinberg equilibrium. detection of casq sequence variants. we screened a total of , bp of sequence, including , bp of the � flanking sequence ( , bp upstream of atg start site), bp of the � flanking sequence, the � and � untranslated regions, all exons, and – bp of the sequence flanking each exon, for mutations using sets of primers (sequences available from authors). amplicons were designed using wavemaker software (version . ; trans- genomic, omaha, nebraska), with sizes of – bp, and were screened at up to three denaturing temperatures using denaturing high-pressure liquid chromatography on a waveht dna fragment analysis system (trans- genomic). both strands of fragments showing altered migration were sequenced using the ceq genetic analysis system (beckman-coulter, fullerton, ca). additionally, we directly sequenced intron ( bp) of casq in two overlapping fragments to catalog additional variation near the associated markers in six individuals selected to represent each of the three observed haplotypes constructed from the two intron snps, rs and rs . putative transcription factor binding sites in intron were detected us- ing tfsearch (version . ; available at http://www.cbrc.jp/research/db/ tfsearch.html) using the transfac database ( ). statistical analysis. allele frequencies were compared using the fisher’s exact test. for all statistical tests, we considered p � . to be evidence of significance. we report the p values without bonferroni correction. secondary analyses of genotypic association were computed using pearson’s � test under additive, dominant, and recessive models. pairwise haplotypes were estimated from the combined case and control population data using the expectation maximization algorithm, from which two linkage disequilibrium (ld) statistics, d� and r , were calculated. extended haplotypes were pre- dicted using the expectation maximization algorithm, as implemented in the arlequin program. because snps casq � , , , , , and all showed an association or a trend to an association with type diabetes in our utah caucasian sample set, we compared the frequency of this six-marker haplotype among case and control subjects. studies of fasting and -, -, -, and -min postchallenge glucose levels were performed on the full set of families included in the linkage study ( ), including families not showing linkage to chromosome q , using the pedigree analysis package ( ). the total number of subjects was for fasting glucose, for -min glucose, for -min glucose, for -min glucose, and for -min glucose. the number of unaffected individuals was for fasting glucose, for -min glucose, for -min glucose, for -min glucose, and for -min glucose. excess transmission of alleles from parents to affected offspring was tested by a maximum likelihood implementation of the trans- mission disequilibrium test, as previously described ( , ). genotype associ- ations with type diabetes were tested using the logit of the probability of type diabetes as the dependent variable. the genotype at each snp was coded as , , or (homozygous common allele, heterozygous rare allele, and homozygous rare allele, respectively) for the independent variable. the logit of the probability of type diabetes was assumed to equal �i � � (age � ) � 	 (bmi � ), where i is , , or . nonindependence of observations was accounted for by a polygenic component, and penetrance at age years and a bmi of kg/m was estimated for each genotype as fi � e �i/( � e �i). snp effects on type diabetes were tested as the difference in penetrance between genotypes ( degrees of freedom). results we initially evaluated snps from the public database over the -kb region that surrounded the initial obser- vation, encompassing genes atp a , casq , pea , and h . of these, snps and one insertion/deletion variant were both polymorphic and able to be assayed, spanning the region from � , to , bp relative to the atg start of casq (table ), for an average interval between polymorphic markers of . kb. the only associations were identified within casq ; hence, we searched for additional variants by screening the exons, the immediate � flanking region, and the immediate � region. we con- firmed the seven snps previously selected and identified seven new variants (table and fig. ). we failed to confirm one database snp (rs , intron ). we iden- tified rare (frequency � %) synonymous variants in exons and and a rare single nonsynonymous snp in exon (casq ; a v) that was not associated with type diabetes. we were unable to develop assays for two of the new snps identified, and three of these were rare and not typed in the full sample (table ). the results are summarized in table and fig. . significant association or trends to an association were limited to intron and to snps in the proximal � flanking region and were therefore most consistent with an asso- ciation in casq rather than the neighboring genes atp a or pea . we found the most significant associ- ations in samples used to confirm the initial pooled findings, for which snps � , , , and all showed nominal significance (p � . – . ). the re- sults when all available utah samples were tested, includ- ing additional case and control subjects, are shown in table . when additional caucasian samples of non– northern european ancestry were included, the significant allelic association was limited to snp casq- in the � flanking region. based on the concentration of associated s.k. das and associates diabetes, vol. , december t a b l e c a s q regio n variatio n an d asso ciatio n w ith typ e d iab etes l ab n am e rs n u m b er l o catio n p o sitio n (b p ) v arian t f requ en cy, u tah cases f requ en cy, u tah co n tro l su b jects p f requ en cy, all cases f requ en cy, all co n tro l su b jects p c a s q � rs ’ fl an k � c /g . . . . . . c a s q � rs ’ fl an k � t /c . . n s . . n s c a s q � rs ’ fl an k � a /g . . n s . . n s c a s q � rs ’ fl an k � g /c . . n s . . n s c a s q � rs ’ fl an k � t /c . . n s . . n s c a s q � rs ’ fl an k � g /t . . n s . . n s c a s q � rs ’ fl an k � a /g . . n s . . n s c a s q � rs ’ fl an k � c /a . . n s . . n s c a s q � rs ’ fl an k � c /t . . . . . . c a s q � n ew ’ fl an k � g /a n d * n d — n d n d — c a s q � n ew ’ fl an k � g /a n d † n d — n d n d — c a s q rs in tro n d el/in s . . . . . n s c a s q rs in tro n a /g . . . . . n s c a s q n ew in tro n g /a . . . . . . c a s q rs in tro n g /a . . . . . n s c a s q rs in tro n c /t . . . . . n s c a s q n ew e x o n c /t n d † n d — n d n d — c a s q n ew e x o n g /a n d † n d — n d n d — c a s q rs in tro n a /c . . . . . n s c a s q n ew in tro n c /t n d * n d — n d n d — c a s q rs in tro n g /a . . . . . n s c a s q rs in tro n c /t . . n s . . n s c a s q n ew e x o n c /t a v . . n s . . n s c a s q rs ’ fl an k t /a . . . . . n s c a s q n ew ’ fl an k a /c . . n s . . n s c a s q rs ’ fl an k c /t . . . . . n s s h o w n are th e s n p s fl an k in g an d in th e c a s q gen e,id en tifi ed eith er fro m th e p u b lic d atab ase an d co n fi rm ed o r fro m screen in g.l ab n am e refers to f ig. ,rs n u m b er is th e d b s n p catalo g n u m b er, an d p o sitio n is relative to th e a t g start o f c a s q . f requ en cy is p ro vid ed fo r th e sam p les strictly ascertain ed fro m u tah , in clu d in g case an d co n tro l su b jects, an d fo r th e case an d co n tro l su b jects, in clu d in g ad d itio n al sam p les m o re b ro ad ly ascertain ed . d el/in s, d eletio n /in sertio n ; n d , n o t d eterm in ed ; n s , n o t sign ifi can t ( p . ). *n o t d eterm in ed d u e to ex ten d ed rep etitive sequ en ces an d assay d esign p ro b lem s; †n o t typ ed d u e to lo w allele frequ en cy (� . ). a ll varian ts are listed w ith th e co m m o n allele fi rst, an d frequ en cies are rep o rted fo r th e m in o r allele. casq is associated with type diabetes diabetes, vol. , december f i g . . m a p o f c a s q g e n e . t h e m a p s h o w s t h e lo c a t io n o f t h e s n p s a n d e x o n s f o r t h e c a s q g e n e . t h e lo c a t io n o f e a c h s n p is s h o w n b e lo w t h e fi g u r e a s a b a r if t h e s n p w a s t y p e d o r in d ic a t e d b y # if t h e s n p w a s n o t t y p e d in t h e f u ll c a s e -c o n t r o l p o p u la t io n . s n p s t y p e d o n ly t o e s t a b li s h l d a r e s h o w n in it a li c s , a n d t h o s e f o r w h ic h a s s a y s c o u ld n o t b e d e s ig n e d a r e s h o w n in g r a y . e x o n s a r e s h o w n a s b o x e s , w it h t r a n s la t e d r e g io n s s h o w n in b la c k a n d u n t r a n s la t e d r e g io n s s h o w n in li g h t g r a y . s n p s t h a t a r e s ig n ifi c a n t ( p < . ) in b o t h u t a h a n d a m is h p o p u la t io n s a r e in d ic a t e d b y * ; s n p s s ig n ifi c a n t in t h e a m is h b u t n o t u t a h p o p u la t io n a r e in d ic a t e d b y ¶ . s.k. das and associates diabetes, vol. , december snps in intron , which was not originally screened, we resquenced this intron in six individuals chosen for each of the observed haplotypes. one additional snp (snp casq ) was identified (table ) that also showed a trend to an association (p � . ), with the minor allele overrep- resented among cases. the haplotype distribution for six snps from the � flanking region (� , ) to intron ( , ) was significantly different in case and control subjects (p � . ); the difference remained significant, albeit less so, among the full sample set (p � . ) (table ). analysis of genotype associations for individual snps was most consistent with a dominant or additive model, with the minor allele as the risk allele for snps casq � , , , , , and (data not shown). we typed snp casq (rs ), which was associ- ated with type diabetes in the case-control study, in the full family sample. no allele was transmitted in excess from parents to affected offspring, and diabetes pene- trance did not differ among genotypes, whether in all families or in only those families linked to this region. the pairwise ld statistics across snps from � to � is shown in figs. and in the online appendix (available at http://diabetes.diabetesjournals.org). when haplotype blocks were defined using d� and the methods of gabriel et al. ( ), snps (in intron ) through (just upstream of exon ) were in a single haplotype block (fig. a, online appendix) comprising only three common hap- lotypes (fig. b, online appendix), whereas associated snp � , fell � to this block. using the r statistic, which best indicates the similarity in the information about association between two snps, only four clusters of snps had r values . : � /� , / , / / , and / / . using the ldselect program ( ), snps would be required to capture the haplotypes at r � . and snps at r � . (table , online appendix). notably, the snps associated with type diabetes or showing a trend to an association in this study fell into three bins by r , bin (� ), bin ( / / ), and bin ( / ) (table , online appendix), but only two haplotype blocks (fig. , online appendix). combined, these six snps form the highest- risk haplotype (table ). surprisingly, analysis of snp � and the block tag snps and dropped the haplotype significance to . from . for the six-marker combination among northern european caucasians. among the amish, casq snps were associated with elevated glucose. we tested for a similar association with fasting and -, -, -, and -h postchallenge glucose lev- els, both in all family members and in nondiabetic family members only. we found no significant association with snp casq for any glucose measure in either group. discussion chromosome q is among the best replicated regions linked to type diabetes. the region is also linked to other potential elements of the metabolic syndrome: hyperten- sion ( ) and familial combined hyperlipidemia ( , ). work from our laboratory suggests that this region har- bors at least three loci based on association ( ) and linkage ( ) studies. the current analyses were initiated based on a snp map that encompassed both linkage peaks and which showed an association of two snps in this region. the broadly defined region of association includes three genes, atp a , casq , and pea . pea is over- expressed in fibroblasts, adipose, and skeletal muscle from diabetic subjects ( ), is a substrate from protein kinase c, alters glucose transport ( ), and is thus a strong candidate gene. however, a previous study of pea in pima indians found no coding snps and no association with type dia- betes ( ), and our association clearly does not extend � of casq into the pea region. instead, our data restrict the association to the six snp haplotypes that extend from the � flanking region through intron of casq : � , , , , , , and . nonetheless, we cannot exclude the possibility that these associated variants mod- ulate pea expression. the associated snps belong to three noncontiguous bins based on the r ld statistic. based on d� values, snp � is not in the block defined by snps – (figs. and , online appendix). hence, the observed associations are partially independent and not related exclusively to strong ld between closely spaced snps. this observation, along with data from fu et al. ( ) from the afds, suggests that multiple casq snps may con- tribute to disease susceptibility. like calpain and many other recently described complex disease genes ( – ), no associated variant alters the coding sequence and no single variant is likely to explain the association. however, we did not observe a much stronger association of pre- dicted haplotypes than of individual snps. additional support for casq as a candidate comes from the independent association of snps within casq in the afds ( , ). that study also found an association at snp (rs ), which in both the amish and utah populations is in strong ld with snp that showed the strongest initial association in our studies. however, much of the association in the amish study maps to intron (snp in our nomenclature; rs ), where we have not found any evidence for association. notably, snp is not in ld with snps in intron . in contrast to the afds, where the haplotype / was significantly associated with type diabetes, this combination showed table haplotype frequencies of six-marker haplotype in case and control subjects haplotype � , , , , , utah control subjects utah case subjects all control subjects all case subjects c del g c a t . . . . c del a c g c . . . . c ins a c g c . . . . c del a t g c . . . . c del g t a t . . . . c ins a t g c . . . . t del a c g c . . . . t ins a c g c . . . . t del g c a t . . . . global p value . . shown are the haplotype frequencies for the six-marker haplotype covering � to in both northern european caucasians ascertained in utah and in all samples tested. note that the case samples are nearly identical in the two analyses. snps selected as tagsnps from haploview are marked in bold. snp is a -base insertion/deletion; the larger allele is marked ins and the smaller del in the haplotypes. casq is associated with type diabetes diabetes, vol. , december no association with type diabetes in either our restricted population (utah only) or our full case-control study (global p value . ). despite the evidence for replicated association of snps in the casq gene with type diabetes, as has been typical for complex disease genes, the significance of the associations for individual snps is modest. the predicted odds ratios are in the range of . – . . additionally, as has also been observed for other type diabetes susceptibility genes, the snps associated in the two populations show only partial overlap. these differences may result from multiple susceptibility alleles, differing ld between founder (amish) and outbred (utah) populations, or spu- rious associations in one or both populations. finally, where the same snps were associated in both amish and utah samples (intron ), we show opposite associations (minor allele is the risk allele in utah, major allele is the risk allele in the amish). possible explanations for this paradox include different susceptibility snps at some distance from intron that were not detected in this study, different gene-gene interactions in the two populations, or an effect of gene-environment interactions in populations with different lifestyles. in our studies, the differences between case and control subjects were observed primarily when we used the restricted population of case and control subjects ascer- tained in utah. the two association studies differ primarily in the control populations. the utah population is exclu- sively of northern european origin and maps most closely to the u.k. and scandinavia. the exact origins of our arkansas caucasian population are broad and may contain more native-american and african-american admixture, although known admixture was avoided. whether the differences observed between these populations represent true differences in allele frequency, the effects of increas- ing the sample size or more complex underlying gene- environment interactions are unknown. in general, utah and arkansas populations show similar allele frequencies. we were unable to confirm the association with type diabetes in our utah families using family-based ap- proaches, and unlike the afds, we did not find evidence that those snps associated with type diabetes also influenced fasting or postchallenge glucose levels among either all family members or nondiabetic family members only. these failures most likely relate to the low power of family- as opposed to population-based approaches, al- though we cannot formally exclude population stratifica- tion or a type error in the original observation. the number of subjects included in our analysis of glucose levels was considerably lower than that analyzed in the amish, in part due to the larger number of diabetic family members in our study and the slightly smaller overall study size. despite these uncertainties, the replication by nearby snps not in ld with the initial observation and independent observations in a second population both support a role for snps within casq in the pathogenesis of type diabetes. casqs are major calcium binding proteins localized in the terminal cisternae of the sarcoplasmic reticulum. casq , the primary skeletal muscle casq, binds calcium with high capacity but moderate affinity ( ) and modu- lates activity of the ryanodine receptor to control muscle calcium homeostasis and thus excitation-contraction cou- pling ( ). inactivating mutations of casq , the cardiac casq, are known to result in cardiac arrhythmias, and overexpression of casq in mice causes cardiac hyper- trophy and cardiomyopathy ( ), but little is known about genetic variation in casq . nonetheless, the regulation of muscle calcium may have implications for insulin action. casq is upregulated in the streptazotocin-induced dia- betic rat skeletal muscle ( ). glut can be recruited by either of two pathways, one proceeding from insulin through phosphatidylinositol kinase and the other through mus- cle contraction, exercise, calcium release, and hyperglyce- mia ( , ). hence, variation in casq expression might easily alter myocellular calcium homeostasis, glut re- cruitment, and insulin sensitivity. the snps in intron reside in a region of marked dna conservation between mouse and human. such conserved nongenic sequences were recently proposed as additional regulatory elements ( ). indeed, snps , , and alter putative binding sites for transcription factors tata, mzf and p , and sp , respectively (fig. , online ap- pendix). of these, snp is predicted to abolish binding for mzf and p and snp is predicted to abolish binding for sp transcription factors. thus, this region might alter casq or perhaps pea regulation and con- tribute to diabetes risk. interestingly, considerable data have implicated sp in the downstream actions of insulin, although the role of sp in intronic regions is unknown ( ). additional studies are needed to explore gene ex- pression in muscle among individuals with the high-risk haplotype in order to determine whether insulin sensitivity is altered among carriers of high-risk snps and to confirm the association of snps in this region with type diabetes in additional populations with linkage to q . acknowledgments this work was supported by grants dk from the national institutes of health (nih)/national institute of diabetes and digestive and kidney diseases, by the re- search service of the department of veterans affairs, by grant support from the american diabetes associa- tion, and by general clinical research center grant m rr from the national center for research re- sources (nih) to the university of arkansas for medical sciences. references . mccarthy mi: growing evidence for diabetes susceptibility genes from genome scan data. curr diab rep : – , . elbein sc, hoffman md, teng k, leppert mf, hasstedt sj: a genome-wide search for type diabetes susceptibility genes in utah caucasians. diabetes : – , . das sk, hasstedt sj, zhang z, elbein sc: linkage and association mapping of a chromosome q – q type diabetes susceptibility locus in northern 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eur j biochem : – , . howarth fc, glover l, culligan k, qureshi ma, ohlendieck k: calseques- trin expression and calcium binding is increased in streptozotocin-induced diabetic rat skeletal muscle though not in cardiac muscle. pflugers arch : – , . hayashi t, wojtaszewski jf, goodyear lj: exercise regulation of glucose transport in skeletal muscle. am j physiol :e –e , . nolte la, rincon j, wahlstrom eo, craig bw, zierath jr, wallberg- henriksson h: hyperglycemia activates glucose transport in rat skeletal muscle via a ca �-dependent mechanism. diabetes : – , . dermitzakis et, reymond a, scamuffa n, ucla c, kirkness e, rossier c, antonarakis se: evolutionary discrimination of mammalian conserved non-genic sequences (cngs). science : – , . samson sl, wong nc: role of sp in insulin regulation of gene expression. j mol endocrinol : – , casq is associated with type diabetes diabetes, vol. , december wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty 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overcoming bet inhibitor resistance in malignant peripheral nerve sheath tumors cooper and patel, et al. page overcoming bet inhibitor resistance in malignant peripheral nerve sheath tumors jonathan m. cooper , , amish j. patel , , , zhiguo chen , , chung-ping liao , , kun chen , , juan mo , yong wang , lu q. le , , , department of dermatology, cancer biology graduate program, simmons comprehensive cancer center, utsw comprehensive neurofibromatosis clinic, hamon center for regenerative science and medicine, university of texas southwestern medical center at dallas, dallas, texas, - , usa co-first authors. these authors contributed equally. author for correspondence: lu q. le, m.d., ph.d. associate professor department of dermatology simmons comprehensive cancer center ut southwestern medical center phone: ( ) - fax: ( ) - e-mail: lu.le@utsouthwestern.edu running title: cancer cell sensitivity and resistance to bet inhibitors keywords: neurofibromatosis type , nf , malignant peripheral nerve sheath tumor, mpnst, neurofibrosarcomas, jq , brd levels, bet inhibitor sensitivity and resistance, bet bromodomain, protac, arv the authors have declared that no conflict of interest exists. on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page statement of translational relevance malignant peripheral nerve sheath tumors (mpnst) are aggressive sarcomas with no effective therapies. this study reveals a targetable requirement of brd protein level for mpnst survival in the presence of bet inhibitors as genetic depletion of brd synergistically sensitized mpnst cells to diverse bet inhibitors in culture and in vivo. this nominates mpnst with high levels of brd for the investigation of emerging therapeutic interventions such as proteolysis- targeting chimeras (protacs) that simultaneously target bromodomain activity and bet protein abundance. on the other hand, brd -low tumors could be predicted to respond best to strategies using direct bet inhibition alone or in combination with other anti-cancer agents. these strategies could then be employed in a data-driven manner to provide rational approaches to develop breakthrough treatments for currently therapy-refractory mpnst patients on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page abstract purpose: bet bromodomain inhibitors have emerged as a promising therapy for numerous cancer types in pre-clinical studies, including neurofibromatosis type (nf )-associated malignant peripheral nerve sheath tumor (mpnst). however, potential mechanisms underlying resistance to these inhibitors in different cancers are not completely understood. in this study, we explore new strategy to overcome bet inhibitor resistance in mpnst. experimental design: through modeling tumor evolution by studying genetic changes underlying the development of mpnst, a lethal sarcoma with no effective medical treatment, we identified a targetable addiction to bet bromodomain family member brd in mpnst. this served as a controlled model system to delineate mechanisms of sensitivity and resistance to bet bromodomain inhibitors in this disease. results: here, we show that a malignant progression-associated increase in brd protein levels corresponds to partial sensitivity to bet inhibition in mpnst. strikingly, genetic depletion of brd protein levels synergistically sensitized mpnst cells to diverse bet inhibitors in culture and in vivo. conclusions: collectively, mpnst sensitivity to combination genetic and pharmacological inhibition of brd revealed the presence of a unique addiction to brd in mpnst. our discovery that a synthetic lethality exists between bet inhibition and reduced brd protein levels nominates mpnst for the investigation of emerging therapeutic interventions such as proteolysis-targeting chimeras (protacs) that simultaneously target bromodomain activity and bet protein abundance on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page introduction the discovery of oncogenes, tumor suppressors, and more recently mutational landscapes across human tumors has provided unprecedented knowledge leading to the identification of numerous therapeutic targets against cancer ( - ) (the cancer genome atlas research network). nonetheless, traditional surgery, chemotherapy, and radiotherapy remain as frontline therapeutic strategies for many cancer patients today (pdq ® - national cancer institute). however, toxicity of chemotherapy and radiotherapy on normal tissues has spurred interest in the development of cancer tissue-specific therapies ( , ). in recent decades, targeted therapy against cancer-specific kinase dependencies has emerged as a promising treatment modality ( - ). however, variable resistance mechanisms have also hindered the therapeutic success of targeted therapies against specific cancer dependencies on kinases such as bcr-abl, mek, braf, egfr, and smo ( , ). furthermore, recent awareness of mutational heterogeneity in human tumors through cancer genomic sequencing studies has suggested the need for an expanded arsenal of targeted therapeutics for use either alone or in combination to improve survival of cancer patients ( , ). in contrast, targeted therapy against epigenetic or chromatin regulators has emerged as an attractive alternative strategy against cancer, due in part to the fact that these proteins can maintain tumorigenesis through regulation of gene expression programs downstream of both oncogenes and tumor suppressors, and these proteins are druggable ( - ). epigenetic writers, on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page erasers, and readers are three categories of chromatin regulators for which drug targets and small molecule inhibitors have been developed and shown to be promising targets in either pre-clinical mouse tumor models or in clinical trials ( - ). of these, bet bromodomain protein brd has recently emerged as an important chromatin regulatory protein across multiple cancer types ( - ). brd is critical member of the broader bromodomain and extra-terminal domain (bet) family of epigenetic reader proteins. these include the ubiquitously expressed isoforms brd , brd , brd , and testis- and ovary-specific isoform brdt ( ). bet proteins are characterized by two tandem bromodomains (bd), which bind acetylated histones to support the recruitment of transcription elongation factor machinery to open chromatin regions, and an extra-terminal domain (et) that mediates protein-protein interactions ( ). specifically, bet proteins often interact with and support the activity of key transcription factors such as c-myc, c-jun, tp , and ptefb ( , ). members of the bet protein family, including brd , brd , and brd can be potently inhibited pharmacologically with pan-bet bromodomain inhibitors (bet inhibitors) including jq , i-bet , cpi- , or otx- , which competitively bind both bromodomains, perturbing bet protein association with histones and subsequent regulation of gene expression ( - ) bet inhibitors show great potential as selective, anti-cancer therapeutics, but the mechanisms underlying sensitivity and resistance to apoptosis in the presence of these inhibitors is less clear. some recent reports have indicated cancer-specific modes of resistance can occur, such as compensatory kinase signaling in ovarian cancer ( ); bromodomain-independent recruitment of bet proteins to chromatin in triple-negative breast cancer ( ); and engagement of wnt/beta-catenin signaling in acute myeloid leukemia (aml) ( , ). it has also been on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page reported that brd regulates distinct super-enhancer-associated oncogenes in specific tumor types (e.g. myc in leukemia and multiple myeloma) ( - ). furthermore, within specific tumor subtypes, there are varying responses to bet inhibitors in pre-clinical studies and in on-going phase i clinical trials ( , ). although suppression of myc expression has been demonstrated as a mechanism of growth suppression via bet inhibitors, it is not always a key mechanism of action in other tumor types ( , , , ). given these pressing issues, the elucidation of mechanisms governing bet inhibitor sensitivity or resistance would serve as a valuable platform to better understand these inhibitors and develop diagnostic biomarkers for their usage in cancer patients to maintain the long-term success of this epigenetic therapy. recently, we reported that brd plays a critical role in the tumorigenesis of neurofibromatosis type i (nf )-associated malignant peripheral nerve sheath tumors (mpnsts), and that pharmacological inhibition with bet inhibitor jq is effective in pre- clinical in vivo mpnst tumor studies ( ). mpnsts are highly aggressive sarcomas that develop sporadically or in nf patients. there is no effective treatment for mpnsts and they are typically fatal. we identified a brd dependency in mpnst while studying tumor initiation and progression through step-wise loss of tumor suppressor genes nf and tp in neural crest- related skin-derived precursor cells (skps), which we recently established as an ex-vivo transplantable model of mpnst development ( , , ). thus, we reasoned that this model would serve as a controlled system to delineate genetic mechanisms of sensitivity or resistance to jq in mpnst. on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page shortly after our study was published, three independent groups reported that loss of function in members of the polycomb repressor complex (prc ) is a characteristic of mpnst ( , ) and even promotes sensitivity to bet inhibition ( ). prc loss of function results in reduced h k methylation and allows for subsequent h k acetylation, which mediates brd recruitment to chromatin. these studies suggest that, in mpnst, prc loss of function allows for histone priming for brd recruitment and activation, facilitating its role in maintaining mpnst survival and nominating its use as a therapeutic target. importantly, these observations illustrate how brd dependency is possible even in the absence of mutations in brd -interator tp , as tp mutations and loss of function mutations in prc members are often non-redundant in mpnst patient tumor samples ( ). however, prc loss of function may not confer sensitivity to bet inhibition in all cancer contexts, as it can actually promote bet inhibitor resistance in aml ( ). importantly, however, we also observed in mpnst that brd inhibition with jq or rnai promotes active engagement of apoptosis through upregulation of pro-apoptotic bim and downregulation of anti-apoptotic bcl ( ). this is consistent with the observation that the preclinical anti-tumor efficacy of bet inhibition is in part dependent on the degree to which their administration engages apoptosis ( ). it was in light of these observations that we chose to explore modes of bet inhibition sensitivity and resistance in mpnst and in so doing uncovered a targetable brd protein addiction in this disease. materials and methods cells and reagents primary mouse mpnst (mmpnst) cells were generated via a mouse mpnst model as described previously ( , , ). human s mpnst cells were a kind gift from karen on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page cichowski (harvard medical school). human mpnst cells were authenticated with human- specific pcr primers on / / to confirm the absence of mouse tumor cell contamination. all leukemia cell lines were a kind gift from dr. chengcheng zhang (ut southwestern, dallas, tx). routine mycoplasma testing of the cell lines was not performed. all cells were cultured in dmem ( % fbs, % l-glutamine, % sodium pyruvate, % penicillin-streptomycin). drugs used: jq (cayman chemical and medchem express), otx- (cayman chemical), cpi- (cayman chemical), cpi- (axon medchem), arv- and arv- (medchem express). animal studies all mice were housed in the animal facility at the university of texas southwestern medical center at dallas (utsw). animal care and use were approved by the institutional animal care and use committee (iacuc) at utsw. female athymic nude mice (≥ -week old) obtained from the jackson laboratory (bar harbor, me) were used for tumor studies. in vivo induction of shrnas in mmpnst-ptripz tumors, jq dosing, and tumor bioluminescence imaging were all carried out as described ( ). in vivo administration of arv bet protac was conducted using a modified treatment regimen from ( - ). briefly, tumorigenic clones of s were subcutaneously injected into the right and left hind flank of mice per treatment group ( x cells per tumor). xenograft tumors were allowed to form until palpable ( weeks). tumors were measured by electronic caliper on treatment experiment day (volume = l x w x . ) and mice were divided between treatment groups (n = mice) so that the average tumor size per group reach approximately mm . vehicle ( % sorbitol hs and % etoh in d w) or mg/kg arv were subcutaneously injected daily (beginning on experiment day ) for the length of treatment period. mouse weight changes (to evaluate treatment toxicity) and tumor on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page volume measurements were routinely collected throughout the treatment period. as in ( - ), all mice received a drug holiday of - days during the course of the treatment. on day of the treatment period ( day after the final dose), final measurements of tumor volume and tumor mass were conducted. brd knockout by crispr/cas genomic editing doxycycline inducible cas cdna lentiviral vector (addgene plasmid # = pcw-cas ) and additional lentivector constitutively expressing aavs -targeting sgrna (sgcon) (addgene plasmid # = plx-sgrna) or sgbrd . lentivector (plx-sgrna vector with aavs -sgrna+pam_sequence replaced with the following brd -targeting sgrna+pam_sequence: gttcagcttgacggcatcca) were packaged into lentiviral particles that were used to infect, and select for transduced cells. brd sgrna was designed using e-crisp software (http://www.e-crisp.org/e-crisp). for genomic editing, stably infected cells were plated as single cell clones, followed by cas induction for days with doxycycline. single cell clone outgrowths were expanded, and screened as described previously ( , ). to determine the efficacy of crispr/cas gene editing, genomic dna was extracted from mouse mpnst cells (parental, sgcon clones and , and sgbrd . clones , , and ) and the genomic region surrounding the brd sgrna-targeted sequence was amplified by pcr (primers used: ’ – ’ [f : ctaacaagcccaagagacag, r : ccaactttacccttctgcag]). the amplified pcr product was submitted for sanger sequencing by the mcdermott center sequencing core facility at utsw. the pcr products of sgbrd . _clone and sgbrd . _clone were further sub-cloned into pgem-t easy (promega) cloning plasmids for on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page additional sanger sequencing via t primers. sequence similarity was assessed using the basic local alignment search tool (blast) provided by the ncbi. lentiviral constructs mouse/human brd shrnas were generated as described previously ( ). human brd shrnas were generated by cloning of the following -mer sequences (shbrd .a: ccaaggaaatgtctcggatat, shbrd .b: gctgatgttctcgaattgcta, shbrd .c: cccaagaggaagttgaattat) into the plko. -puro empty backbone lentiviral vector. in vitro growth measurements atp celltiter glo assay (promega) was carried out according to modified manufacturer recommendations. luminescence was quantified via synergy|ht -well plate reader (biotek). quantification of cellular apoptosis for analysis of cellular apoptosis/death, fitc-annexinv kit (miltenyi biotec) or apc- annexinv kit (bio legend) was used per modified manufacturer’s instructions. the facscalibur, facscanto, and facslyric flow cytometers (bd biosciences) at the utsw flow cytometry core facility and the moody foundation flow cytometry facility of the children’s research institute (cri) at utsw were used for cellular analyses. cytobank and flowjo software (tree star) were utilized for data visualization and analyses. rna isolation, cdna synthesis, qrt-pcr on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page all procedures were performed as descried ( ). data quantified by Δct method, and normalized relative to gapdh (housekeeping gene). primers used ( ’-- ’): (hbrd _f: agtttgcatggcctttcc, hbrd _r: cctgagcattccagtaatagttg, hbrd _f: gaaggccaacagcacgac, hbrd _r: ccctcctcctcttcctctga) western blot western blot procedures were carried out as described previously ( , ). antibodies used: brd (bethyl labs); gapdh, brd , brd (santa cruz biotechnology), alpha/beta tubulin (a,b-tubulin), bim, cleaved-caspase , cleaved-parp, histone (cell signaling technology). statistical analyses data are displayed as the mean +/- s.e.m. two-tailed unpaired student’s t test was used to evaluate statistical significance (p < . was deemed statistically significant). on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page results brd levels underlie resistance to bet inhibitor-induced death in mpnst cells previously, we observed that malignant progression of normal skps to mpnst via inactivation of both nf and tp was associated with increased levels of brd ( ). interestingly, it has been reported that increased levels of brd are associated with increased sensitivity to bet inhibitor jq in the context of notch inhibitor-resistant t cell leukemia ( ). as mpnst cells expressing elevated brd exhibit enhanced sensitivity to jq over pre- tumorigenic, brd -low skps ( ), we hypothesized that increased levels of brd confer jq sensitivity in this context. we suspected that jq resistance would arise from mpnst cells with lower brd levels that are insensitive to jq inhibition. thus, we reasoned that stable suppression of brd expression would de-sensitize brd -high mpnsts to jq . through crispr/cas -based genome editing (figure a, s a-f) and the heterogeneity of endogenous brd expression levels in mpnst cells, we generated brd -depleted mouse mpnst (mmpnst) cell clones that offer different levels of brd expression for us to model jq sensitivity as a function of brd levels through functional assays (figure b). we employed targeted sequencing of representative sgcontrol and sgbrd clones to determine the exact mutations in each of the sgbrd -targeted lines. as expected, parental mmpnst cells and both sgcontrol clones (c and c ) have no brd mutation (figure s a-c). sgbrd clone c also has no brd mutation (figure s d); and accordingly maintains brd protein expression (figure b). most importantly, we found that a single base pair insertion in one allele and a -base pair deletion in the other allele in the sgbrd -targeted clone c (figure s e), as well as a two base pair insertion in one allele and a -base pair deletion in the other allele in the sgbrd -targeted clone c (figure s f), each resulted in loss of brd protein expression (figures b). when on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page these cells along with control cells were monitored for growth and apoptosis after jq treatment, we observed hyper-sensitivity to jq in brd -knockout mpnst cells (figures c-d, s a), which was unexpected given our initial hypothesis. interestingly, mpnst cells maintaining brd expression (e.g. c , c , c ), showed intermediate levels of cell death with jq , while cells with brd knockout (e.g. c ) displayed near-maximal cell death (figure d). together, these findings led us to refine our model in which we suggest that the persistence of higher brd levels post-treatment can mediate resistance in bet inhibitor-treated mpnst cells. brd depletion overcomes resistance to bet inhibitor-induced cell death in mpnst. to exclude the possibility that these results were due to the selection of single cell clones with pre-existing sensitivities to jq , we utilized potent doxycycline-inducible shrna against brd . upon doxycycline addition, we observed almost complete loss of brd protein levels (similar to a knockout) (figure e) compared to scrambled control (shcontrol). these phenotypes were verified with multiple shrnas in our previous studies ( ). we utilized this sensitive yet rapid system to acutely knockdown brd and monitor survival of mpnst cells with or without jq treatment (figure f). we observed massive cell death ( - % apoptosis) in as few as days when doxycycline-induced brd knockdown was combined with jq , while cell death occurred to a far lesser extent in controls as expected (figure f). using this system, we observed that low doses of jq were also sufficient to cooperate with acute brd depletion to kill most mpnst cells in culture by days, while control cells remained largely viable, but growth inhibited as expected (figures g, s b). we have previously shown that apoptotic cell death induced upon brd inhibition by shrna or small molecule bet inhibitors in mpnst cells occurs through upregulation of pro-apoptotic signaling protein bim and down regulation of anti- on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page apoptotic proteins such as bcl- ( ). consistent with our previous findings, apoptotic induction upon jq treatment was enhanced when mpnst cells were co-treated with jq and bcl- inhibitor abt (figure s c). co-treatment with jq and abt also abrogated cellular proliferation more than either inhibitor alone (figures s d-e). bcl- inhibition exhibited a smaller additive effect on apoptosis and cell proliferation in shbrd cells treated with jq than in jq -treated shcontrol cells, indicative that jq treatment in the context of brd depletion already approached maximal apoptosis engagement and cell proliferation inhibition. (figures s c-e). given that bet inhibitors (e.g. jq ) can inhibit multiple bet proteins in humans, we investigated if the depletion of bet bromodomain proteins in human mpnst cells would phenocopy what we observed in mouse mmpnst cells. consistent with our data thus far, we observed that shrna-mediated constitutive depletion of brd (figures h-i) conferred extreme sensitivity to jq -induced cell death compared to jq -treated shcontrol cells (figure j). similarly, we found that knockdown of bet family member brd was slightly growth inhibitory and toxic to mpnst cells, but it conferred acute sensitivity to jq co-treatment (figures s a-c), though to a smaller extent than brd knockdown. these data reveal that both brd and brd can support mpnst cell growth and that depletion of either one of them can sensitize human mpnst cells to bet inhibitor-induced death, suggesting bet bromodomain family members may possibly play coordinate roles in maintaining mpnst cell growth and survival. since persistent genetic deletion of brd did not markedly alter the protein levels of brd or brd (figure s a), it is likely any coordinate roles in this system would occur at the level of brd / / function, rather than expression. on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page genetic inhibition of brd overcomes mpnst cell resistance to diverse bet inhibitors having found that genetic depletion or knockout of brd rendered extreme sensitivity to bet inhibitor jq in mpnst cells, we expanded our investigation to include alternative bet inhibitors. to exclude the possibility that our observations were biased by the polypharmacology of jq , we additionally utilized otx- , cpi- , and cpi- , which are broad-spectrum bet inhibitors previously shown to inhibit brd , brd , and brd ( - ). comparative dose response analysis of jq , otx- , and cpi- revealed similar growth-inhibitory effects of these three independent bet inhibitors on both mouse and human mpnst cells (figure a-b). upon cell death analysis (figure c), a µm dose consistently led to about % cell death with each inhibitor, and higher doses (up to µm) of either jq or otx- led to further enhanced cell death in a dose-dependent manner. in contrast, mpnst cells were relatively resistant to higher doses of cpi- (figure c). remarkably, when mpnst cells were brd -depleted, co- treatment with a µm dose of cpi- led to massive cell death that was comparable to the same dose of jq or otx- (figure d). cpi- displayed similar, though less potent, inhibition of mpnst cell viability as jq in shcontrol cells; however, depletion of brd was sufficient to sensitize mpnst cells to bet inhibition by cpi- , particularly as the dose was increased (figures e-f). these comparative analyses reveal that genetic inhibition of brd can overcome mpnst resistance to a spectrum of bet inhibitors with differing intrinsic potencies. brd -high mpnst cells are sensitive to titrated brd depletion by proteolysis-targeting chimeras (protacs) on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page our studies to this point suggest that brd -addicted tumors would likely harbor resistance to bet inhibition alone, but could be sensitized by targeted depletion of brd . while this is relatively straightforward in vitro, genetic depletion of brd by rnai or crispr in humans is fraught with challenges making it not presently therapeutically viable. recent developments in chemical biology have sought to address the problem of achieving depletion of specific proteins in cells and in vivo without the use of oligonucleotides through the development of proteolysis-targeting chimeras (protacs) ( ). protacs contain a binding moiety against a target of interest, a linker region, and a separate binding moiety for an e ubiquitin ligase. the linker region allows for the specific localized ubiquitination of the target protein of interest for degradation by the e ligase that has been brought into its unique proximity by the protac. to assess whether selective degradation of brd is achievable in the context of mpnst, we employed two protac molecules, arv , which employs a cereblon e ligase-mediated degradation of brd ( ), and arv , which utilizes the von hippel-lindau (vhl) e ligase to degrade brd ( ). in human mpnst cells, both protacs elicited dose-dependent, persistent degradation of brd following days of treatment (figure a). arv was slightly more potent in this context than arv , as µm (the most effective dose of either protac) arv produced near undetectable levels of brd , while µm of arv allowed for noticeable residual brd expression (figure a). the degradation of brd corresponded to dose-dependent upregulation of cell death markers cleaved-parp and cleaved-caspase , and also induced apoptotic cell death (figures a-b). in addition, both protac molecules displayed a near -fold reduction of ic for cell viability compared to jq (figure c) in human mpnst cells. on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page while arv and arv were originally designed to target human brd , we also confirmed that they exhibit dose-dependent depletion of brd and engagement of apoptosis in mouse mmpnst cells (figures d-e). in mmpnst cells, arv was more potent than arv in inducing apoptosis, particularly at µm, which corresponded to greater brd depletion by arv at the same doses (figures d-e). additionally, in mmpnst cells, both protacs inhibited cell viability more potently than jq at their highest dose (figure f). in both mouse and human cells, we observed that the levels of brd began to rise between µm and µm doses (figures a,d). this is likely an example of a “hook effect” that has been observed for protacs, as increasing compound concentrations begin to out-compete themselves for binding to the target of interest, which results in reduced target degradation ( ). co-treatment of the protacs ( µm) with jq ( µm) produced little to no additional induction of apoptosis compared to either protac alone (figures b,e), indicating that at this dose protac treatment has maximally engaged the apoptotic response possible for brd inhibition. in addition, co-treatment of jq with either protac molecule produced less brd protein depletion than protac treatment alone (figures a,d). since the brd -binding moiety in the protac targets the same binding site on brd as jq , this blunting of brd depletion suggests that the addition of jq may have lowered the effective concentration of the protac, as it competed with protac for binding to brd . together, these results are proof- of-principle experiments that show a protac-mediated strategy for both brd depletion and bet inhibition may be a viable avenue to target brd -addicted mpnsts in human patients via a single cell-permeable small molecule. on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page protac-mediated brd depletion can bypass brd -high leukemia cell resistance to bet inhibitors given previous reports that bet bromodomain family members such as brd support the growth and proliferation of hematopoietic cancers such as aml and cml, we sought to validate whether the synthetic lethality we observed between brd depletion and bet pharmacological inhibition applied to broader cancer contexts beyond mpnst. we focused on a panel of human leukemia cells that possessed distinct oncogenic mutations and had been previously found to have differential sensitivities to jq ( , ). baseline analysis of brd expression in the four leukemia lines examined revealed that k- (cml) and kasumi- (aml) displayed relatively higher levels of brd , while both hl- (aml) and thp- (aml) showed relatively lower levels of brd (figure a). importantly, k- cells, which displayed the highest levels of baseline brd , are known to harbor resistance to bet inhibitors ( , ). these cells displayed no noticeable apoptotic induction and limited perturbation of cell viability when treated with jq , compared to the other cell lines tested (figure b-c). strikingly, both hl- and thp- cells, which both exhibited relatively lower levels of brd , were extremely sensitive to apoptosis induction upon jq treatment (figure b). interestingly, we found that these differences among the leukemia cell lines extended to a cell viability assay in which lower- expressing brd cells (hl- and thp- ) were most potently sensitive to jq in a dose- dependent manner (figure c). we then examined whether leukemia cell lines expressing higher levels of brd would be more sensitive to brd inhibition by protac than to bet- bromodomain antagonists. similar to our observations in mpnst, -day treatment of k- or kasumi- cells with arv potently mediated brd depletion to nearly undetectable levels, and this corresponded to a marked induction of apoptotic cell death compared to bet inhibitors on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page jq , otx , and cpi- (figures d-e). interestingly, treatment of kasumi- cells with each of the bet inhibitors, but not the protac, corresponded to an increase of brd levels after treatment, compared to vehicle control (figure e). as brd upregulation in response to bet bromodomain antagonists has been previously reported ( ) and may serve as a mechanism of resistance, we inquired whether bet inhibitors were less able to inhibit kasumi- cell viability than a bet protac, which suppressed brd levels over the course of treatment. protac treatment displayed a greater than -fold decrease in ic compared to all bet inhibitors assayed for effects on cell viability in brd -high kasumi- cells (figure f). interestingly, combination administration of jq and arv in k- , kasumi- and human mpnst cells displayed little to no added effect above single agent treatment alone, with arv -mediated apoptosis induction being the consistently dominant phenotype (figure b, s a-d), even at doses below µm. this is likely explained by the fact that both jq and arv are compensative inhibitors of the same sites on brd and the other bet proteins ( ), and thus would likely compete with each other for target binding if combined, as mentioned above. these data suggest that brd -high cancer cells that display relative resistance to small molecule bet-inhibitors retain a vulnerability to targeted degradation of brd proteins by a protac therapeutic strategy. genetic inhibition of brd improves bet inhibitor therapeutic efficacy against mpnst tumors in vivo we next investigated whether suppression of brd expression in brd -high/addicted mpnst tumors could improve or enhance the therapeutic efficacy of bet inhibition in vivo. toward this end, we utilized nf /tp -inactivated mmpnst cells (luciferase-expressing) that on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page possess both high baseline levels of brd and doxycycline (dox)-inducible shrnas (shcontrol or shbrd ). we implanted these tumor cells in nude mice and allowed solid tumors (< mm ) to form. this was followed by activation of shbrd or shcontrol with doxycycline in all tumors in combination with daily vehicle or jq administration to the mice for days (figure a). through measurement of palpable tumor volume and in vivo bioluminescence imaging at multiple points during this experiment, we observed a remarkable and significant therapeutic improvement when brd knockdown was combined with jq (figures b-f), whereas brd knockdown or jq treatment alone showed far lesser efficacy. during this treatment period, we also observed that, consistent with our previous results, jq - treated tumors underwent maximal tumor regression after days followed by a rapid relapse of these tumors at a slower growth rate compared to vehicle-treated tumors (figure d). in contrast, tumors with brd knockdown plus jq treatment had more striking tumor regression within the first days followed by a suppression of tumor relapse (figure d). even at days post- treatment, over half of the tumors in the jq plus shbrd group exhibited greater than % tumor regression (figure e). analysis of final tumor weight after days of treatment was consistent with these observations (figure f). these data collectively point to an in vivo synthetic lethality between brd depletion and bet inhibitor treatment in mpnst. as rnai-mediated targeting of brd in human patients is not a presently viable therapeutic modality, we examined whether single-agent bet protac treatment could translate our murine mpnst model results into a human mpnst xenograft model for the treatment of human mpnsts. we established an s -xenograft derived cell line (s - l), which possessed heightened tumorigenic potential for further xenograft studies and validated that on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page it maintained in vitro sensitivity to bet-inhibitor-induced inhibition of cell viability and induction of apoptosis (figure s a-b). mice bearing xenograft tumors were then treated daily (including a - day drug holiday per mouse) with subcutaneous administration of vehicle or mg/kg arv , a bet protac with confirmed in vivo efficacy ( - ). we observed that this treatment regimen was well-tolerated in both cohorts (figure s c). consistent with our in vitro data, tumors treated with arv experienced less per-tumor growth over the course of the nearly -week treatment (figure s d-e). these results confirm that arv can attenuate human mpnst cell growth in in vivo, as well as in vitro, and nominate bet protacs for further investigation to improve their pharmacokinetic and pharmacodynamic profile as clinically translatable therapeutic strategies for mpnst patients with no currently effective treatment options. discussion advances in therapeutic oncology have enriched cancer patients with multiple options for personalized targeted therapies, but resistance remains a challenge ( , ). pathway reactivation is a leading mechanism of resistance to targeted therapies (e.g. inhibitors of bcr-abl, braf v e , mek, or smo) ( , - ). from these studies emerges a concept that broadly acknowledges the ability of cancer cells to rewire, acquire, or hijack alternative signal transduction pathways to converge on common downstream signaling nodes or oncogenic addictions to sustain tumorigenesis and survival ( - ). consequently, novel targets or bottlenecks are being actively sought after as alternatives in therapeutic oncology. among alternative novel approaches, inhibition of chromatin/epigenetic/transcriptional regulators has recently emerged as a promising therapeutic strategy to disarm transcriptional events on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page downstream of oncogenic cell signaling networks in cancer ( - ). nevertheless, resistance also plagues the promise of this therapeutic strategy. the mechanisms underlying resistance to epigenetic inhibitors, however, are not currently well characterized. in our studies presented herein, through modeling tumor evolution by studying genetic lesions underlying the development of nf -associated mpnsts, we reveal that brd upregulation/addiction is associated with limited sensitivity to bet inhibitors. however, given the requirement for high-dose, single-agent bet inhibitors to efficiently trigger substantial cell death in mpnsts in vitro, and that such high doses are presently difficult to sustain in vivo, it is expected that bet inhibitor efficacy will remain limited in vivo compared to in vitro for many different cancer subtypes under current evaluation in the field. here, we show that genetic inhibition of brd overcomes resistance to bet inhibitors, thus instigating synthetic lethality in vitro and markedly restraining tumor relapse in vivo in mpnst. these findings suggest a working model in which strategies to suppress brd expression may lower the dosage of bet inhibitor necessary for in vivo mpnst treatment, and therefore improve its therapeutic efficacy. alternatively, our finding that bet-protein protac treatment surpasses the efficacy of bet inhibitors in cell culture models of mpnst inhibition suggests some brd -high tumors may be vulnerable to a protac-based therapeutic strategy to reduce brd levels in mpnst patient tumors. although unknown at this time, it may be plausible that bet inhibitor jq exerts off- target effects when brd is depleted. however, several data suggest this is likely not a factor in instigating lethality. first, we observed extreme sensitivity to cell death in brd -depleted on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page mpnst cells at low doses of jq (figure g). second, additional bet inhibitors including otx- , cpi- , and cpi- had potent effects on brd depleted mpnst cells (similar to jq ) (figure ). third, although high doses of both jq and otx- ( µm) induced massive cell death, cpi- was unable to do so. yet, cpi- , and cpi- , was much more potent when brd was depleted in mpnst cells. altogether, these observations suggest that lethality instigated by brd depletion is not likely to be due to off-target effects. instead, our data indicates that bet inhibitor therapeutic effects on mpnst cells may be attributed to synthetic lethality caused by additional depletion of bet protein levels. there are several possible mechanisms whereby this may take place. first, depletion of brd by crispr or shrna may not completely eliminate all brd proteins from the cell. in this scenario, the remaining brd may then be more acutely sensitive to bet inhibitors, since the inhibitor-to-target stoichiometry has been drastically perturbed. a second possible mechanism for the synthetic lethality we observed is that genetic loss of brd results in a compensatory response by other bet-bromodomain family members such as brd and brd , which can also be inhibited by bet inhibitors such as jq . here, depletion of brd would increase cells’ dependence on brd or brd function, which subsequent treatment with bet inhibitors would overcome to induce cell death. the possibility of bet-bromodomain family members compensating for one another may explain our observation of a synthetic lethality between shbrd and jq , which displayed a similar, though less potent, phenotype compared to the combination of jq and shbrd . this mode of compensation would likely occur by increased brd / function or relative recruitment to acetylated histones rather than by increased brd / expression, as brd depletion did not correspond to a concomitant increase in brd on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page or brd at the protein level (figure s a). a third explanation for the synthetic lethality between bet inhibitors and loss of brd could arise from reported bet-bromodomain dependent and independent functions of brd ( ). in this case, our data would reflect an mpnst cell addiction to the specific activity of the bet bromodomain (sensitive to bet inhibitors) as well as to bet domain-independent functions of brd proteins (sensitive to brd depletion). lastly, brd depletion may counteract possible bet inhibitor-induced feedback upregulation of brd levels or function. if this explanation underlies our data, then the use of protacs, which inhibit bet domain binding to acetylated proteins while simultaneously promoting brd degradation, would be preferable to the use of bet inhibitors alone as a therapeutic strategy. in conclusion, the elucidation of a link between bet inhibitor efficacy and the cellular level of wild-type bet proteins in mpnst cells provides an important insight into bet inhibitor sensitivity and resistance in cancer cells. this may then provide a framework for developing next-generation inhibitors to overcome resistance and improve the clinical efficacy of this epigenetic therapy. current and future clinical trials of bet inhibitors in mpnst and other bet-implicated cancers should consider the type of addiction to bet family member proteins such as brd that we report here in assessing future study efficacy. according to our data, we hypothesize that brd -high, and accordingly addicted, tumors could benefit from the recent development of small molecule targeted protein degraders such as protacs or future advances in in vivo gene targeting techniques using rnai. brd -low tumors, on the other hand, could be predicted to respond best to strategies using direct bet inhibition alone or in combination with other anti-cancer agents. these strategies could then be employed in a data-driven manner to on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page provide rational approaches to develop breakthrough treatments for currently therapy-refractory mpnst patients. author’s contributions l.q.l.: conception and experimental designs, data collection and/or assembly, data analysis and interpretation, manuscript writing and final approval of manuscript; j.m.c.: conception and experimental design, data collection and/or assembly, data analysis and interpretation, manuscript writing; a.p.: conception and experimental designs, data collection and/or assembly, data analysis and interpretation, manuscript writing; z.c.: data collection; c.p.l.: data collection; k. c.: data collection; y.w.: data collection. acknowledgments we thank all members of the le lab for helpful suggestions and discussions. we would also like to thank lili tao (utsw) and craig crews (yale university) for sharing reagents and methodological insights, respectively. a.j. patel and c.p. liao were recipients of the young investigator awards from children’s tumor foundation. c.p. liao also receives a career development award from dermatology foundation. j.m. cooper is funded by the dermatology research training program t grant t ar . l.q. le holds a career award for medical scientists from the burroughs wellcome fund. this work was supported by funding from the elisabeth reed wagner fund for research and clinical care in neurofibromatosis and cardiothoracic surgery, the texas neurofibromatosis foundation, u.s. department of defense grant number w xwh- - - , the national cancer institute of the nih grant number r on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page ca and specialized programs of research excellence (spore) grant number u ca to l.q. le. on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page figure legends figure . brd depletion overcomes resistance to bet inhibitor-induced cell death in mpnst (a) diagram illustrating the generation of mouse mpnst cells (mmpnst) with brd knockout via crispr-cas -based genomic editing. (b) western blot analysis of brd protein expression in mmpnst cell clones isolated after induction of crispr-cas genomic editing with sgrnas (sgcontrol or sgbrd . ) relative to parental mmpnst cells. (c) mmpnst cells with or without brd knockout were treated with vehicle or µm jq followed by cell viability analysis via atp celltiter-glo assay at the indicated time points. (d) mmpnst cells with or without brd knockout were treated with vehicle or µm jq for days followed by flow cytometry analysis for annexin v (+) apoptotic cells. (e) western blot validation of doxycycline (dox)-inducible shrna-mediated knockdown of brd in mmpnst cells ( days after dox treatment). (f) mmpnst cells were treated with doxycycline (to induce shcontrol or shbrd . ) in tandem with vehicle or µm jq for days followed by flow cytometry analysis for annexin v (+) apoptotic cells. (g) mmpnst cells were treated with or without doxycycline (to induce shbrd . ) in tandem with vehicle or jq at the indicated doses followed by cell viability analysis via phase contrast microscopy after days. (h) validation of brd knockdown in human s mpnst cells by qrt-pcr. on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page (i) western blot validation of constitutive brd protein knockdown in s mpnst cells with shrnas as listed. (j) s mpnst cells with or without constitutive brd knockdown were treated with vehicle or µm jq for days followed by flow cytometry analysis for annexin v (+) apoptotic cells. all error bars and statistics are represented as the mean +/- sem (*p < . , **p < . , ***p < . , ****p < . ). figure . genetic inhibition of brd overcomes mpnst cell resistance to diverse bet inhibitors. (a-b) (a) mmpnst and (b) s mpnst cells were treated bet inhibitors cpi- , jq , or otx at the indicated concentrations for days followed by cell viability analysis via atp celltiter-glo assay. (c) mmpnst cells were treated with vehicle or bet inhibitors cpi- , jq , or otx at the indicated concentrations for days, followed by flow cytometry for annexin v (+) apoptotic cells. (d) mmpnst cells with or without brd depletion were treatment with vehicle or µm of the indicated bet inhibitors followed by flow cytometry for annexin v (+) apoptotic cells after days (inset: western blot validation of shrna mediated brd depletion in mmpnst cells). (e-f) comparative analysis of pan-bet inhibitors jq and cpi- on mmpnst cell viability. shcontrol and shbrd cells were treated with doxycycline and jq or cpi- for days followed by atp celltiter-glo assay. data are plotted (e) as multipoint dose response curves relative to vehicle (dmso) and (f) as individual treatment points relative to vehicle (dmso). on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page all error bars and statistics are represented as the mean +/- sem (*p < . , **p < . , ***p < . , ****p < . ). figure . brd -high mpnst cells are sensitive to titrated brd depletion by proteolysis- targeting chimeras (protacs). (a-b) anti-brd protacs produce dose-dependent depletion of brd and induction of apoptosis in human s mpnst cells. (a) western blot analysis of brd and apoptosis induction markers following day treatment with dmso, µm jq , arv ( ), arv ( ), µm jq + µm , or µm jq + µm . densitometry percentages for brd (brd /a,b-tubulin) were calculated via imagej and are listed relative to dmso. (b) s mpnst cell death induction under the same treatment concentrations as (a) for days, followed by flow cytometry for annexin v (+) apoptotic cells (n= per treatment). (c) comparative analysis of jq , arv , and arv on human s mpnst cell viability. shcontrol s cells were treated with compounds as listed for days followed by atp celltiter-glo assay. data are plotted as multipoint dose response curves (n= per treatment) normalized to the lowest treated dose ( pm). (e-f) anti-brd protacs produce dose-dependent depletion of brd and induction of apoptosis in mmpnst cells. (e) western blot analysis of brd and apoptosis induction markers following day treatment as in figure a. densitometry percentages for brd (brd /a,b- tubulin) were calculated via imagej and are listed relative to dmso. (f) mmpnst cell death induction under the same treatment concentrations as (e) for days, followed by flow cytometry for annexin v (+) apoptotic cells (n= per treatment). on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page (g) comparative analysis of jq , arv , and arv on mmpnst cell viability. shcontrol mmpnst cells (no dox) were treated with compounds as listed for days followed by atp celltiter-glo assay. data are plotted as multipoint dose response curves (n= per treatment) normalized to the lowest treated dose ( pm). all error bars are represented as the mean +/- sem figure . protac-mediated brd depletion can bypass brd -high leukemia cell resistance to bet inhibitors. (a) western blot analysis of relative baseline brd protein expression in leukemia cell lines. densitometry percentages for brd (brd /gapdh) were calculated via imagej and are listed relative to k- cells. (b) leukemia cell lines were treated with vehicle or µm jq for days followed by flow cytometry analysis for annexin v (+) apoptotic cells. (c) leukemia cell lines were treated with vehicle or jq at the indicated concentrations for days followed by cell viability analysis via atp celltiter-glo assay. (d-e) effect of protac-mediated brd depletion versus bet inhibitor treatment on apoptosis induction in k- (d) or kasumi- (e) leukemia cells, as assessed by flow cytometry for annexin v (+) cells (n= per treatment) and by western blotting for brd expression ( days after treatment). densitometry percentages for brd (brd /a,b-tubulin) were calculated via imagej and are listed relative to vehicle (dmso). (f) comparative analysis of bet inhibitor or protac treatment on kasumi- cell viability. shcontrol kasumi- cells were treated with compounds as listed for days followed by atp on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page celltiter-glo assay. data are plotted as multipoint dose response curves (n= per concentration) normalized to the lowest treated dose ( pm). all error bars and statistics are represented as the mean +/- sem (*p < . , **p < . , ***p < . , ****p < . ). figure . genetic inhibition of brd improves bet inhibitor therapeutic efficacy against mpnst tumors in vivo. (a) flowchart diagram illustrating experimental outline for genetic and pharmacological inhibition of brd in mmpnst allograft tumors in vivo. (b) tumor growth curves of raw bioluminescence values from luciferase-expressing mmpnst allograft tumors in vivo. (c) tumor growth curves of mmpnst allograft tumor volume (mm ). (d) tumor growth or regression assessed by percent change in bioluminescence of luciferase- expressing mmpnst allograft tumors. (e) waterfall plot of final percent change (at days post-treatment) in bioluminescence of each luciferase-expressing mmpnst allograft tumor per treatment group. (f) photographs of mmpnst tumors isolated from mice treated with the indicated treatment regimen for days (left panel), and average final weight of tumors from each treatment group (right panel). all error bars and statistics are represented as the mean +/- sem (*p < . , **p < . , ***p < . , ****p < . ). on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page references . muller paj, and vousden kh. p mutations in cancer. nat cell biol. ; ( ): - . . parada lf, tabin cj, shih c, and weinberg ra. human ej bladder carcinoma oncogene is homologue of harvey sarcoma virus ras gene. nature. ; ( ): - . . kufe dwh, james f; frei, emil, iii; american cancer society. cancer medicine. hamilton (on): bc decker; . . choi g, huang b, pinarbasi e, braunstein se, horvai ae, kogan s, et al. genetically mediated nf loss in mice promotes diverse radiation-induced tumors modeling second malignant neoplasms. cancer research. ; ( ): - . . ris md, packer r, goldwein j, jones-wallace d, and boyett jm. intellectual outcome after reduced-dose radiation therapy plus adjuvant chemotherapy for medulloblastoma: a children’s cancer group study. journal of clinical oncology. ; ( ): - . . nazarian r, shi h, wang q, kong x, koya rc, lee h, et al. melanomas acquire resistance to b-raf(v e) inhibition by rtk or n-ras upregulation. nature. ; ( ): - . . poulikakos pi, persaud y, janakiraman m, kong x, ng c, moriceau g, et al. raf inhibitor resistance is mediated by dimerization of aberrantly spliced braf(v e). nature. ; ( ): - . . shi h, moriceau g, kong x, lee m-k, lee h, koya rc, et al. melanoma whole-exome sequencing identifies v eb-raf amplification-mediated acquired b-raf inhibitor resistance. nat commun. ; : . . gschwind a, fischer om, and ullrich a. the discovery of receptor tyrosine kinases: targets for cancer therapy. nat rev cancer. ; ( ): - . . sawyers c. targeted cancer therapy. nature. ; ( ): - . . dijkgraaf gjp, alicke b, weinmann l, januario t, west k, modrusan z, et al. small molecule inhibition of gdc- refractory smoothened mutants and downstream mechanisms of drug resistance. cancer research. ; ( ): - . . johannessen cm, boehm js, kim sy, thomas sr, wardwell l, johnson la, et al. cot drives resistance to raf inhibition through map kinase pathway reactivation. nature. ; ( ): - . on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ cooper and patel, et al. page . eskiocak u, kim sb, ly p, roig ai, biglione s, komurov k, et al. functional parsing of driver mutations in the colorectal cancer genome reveals numerous suppressors of anchorage-independent growth. cancer research. ; ( ): - . . wood ld, parsons dw, jones s, lin j, sjöblom t, leary rj, et al. the genomic landscapes of human breast and colorectal cancers. science. ; ( ): - . . dawson mark a, and kouzarides t. cancer epigenetics: from mechanism to therapy. cell. ; ( ): - . . rius m, and lyko f. epigenetic cancer therapy: rationales, targets and drugs. oncogene. ; ( ): - . . rodriguez-paredes m, and esteller m. cancer epigenetics reaches mainstream oncology. nat med. : - . . shortt j, ott cj, johnstone rw, and bradner je. a chemical probe toolbox for dissecting the cancer epigenome. nat rev cancer. ; ( ): - . . fujisawa t, and filippakopoulos p. functions of bromodomain-containing proteins and their roles in homeostasis and cancer. nat rev mol cell biol. ; ( ): - . . stathis a, and bertoni f. bet proteins as targets for anticancer treatment. cancer discov. ; ( ): - . . de carvalho daniel d, sharma s, you jueng s, su s-f, taberlay phillippa c, kelly theresa k, et al. dna methylation screening identifies driver epigenetic events of cancer cell survival. cancer cell. ; ( ): - . . hu x, feng y, zhang d, zhao sihai d, hu z, greshock j, et al. a functional genomic approach identifies fal as an oncogenic long noncoding rna that associates with bmi and represses p expression in cancer. cancer cell. ; ( ): - . . lee w, teckie s, wiesner t, ran l, prieto granada cn, lin m, et al. prc is recurrently inactivated through eed or suz loss in malignant peripheral nerve sheath tumors. nat genet. ; ( ): - . . lian christine g, xu y, ceol c, wu f, larson a, dresser k, et al. loss of - hydroxymethylcytosine is an epigenetic hallmark of melanoma. cell. ; ( ): - . . wang gg, allis cd, and chi p. chromatin remodeling and cancer, part i: covalent histone modifications. trends in molecular medicine. 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american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/ published onlinefirst february , .clin cancer res lu q. le, jonathan m. cooper, amish j. patel, et al. nerve sheath tumors overcoming bet inhibitor resistance in malignant peripheral updated version . / - .ccr- - doi: access the most recent version of this article at: material supplementary http://clincancerres.aacrjournals.org/content/suppl/ / / / - .ccr- - .dc access the most recent supplemental material at: manuscript author edited. author manuscripts have been peer reviewed and accepted for publication but have not yet been e-mail alerts related to this article or journal.sign up to receive free email-alerts subscriptions reprints and .pubs@aacr.orgdepartment at to order reprints of this article or to subscribe to the journal, contact the aacr publications permissions rightslink site. click on "request permissions" which will take you to the copyright clearance center's (ccc) .http://clincancerres.aacrjournals.org/content/early/ / / / - .ccr- - to request permission to re-use all or part of this article, use this link on april , . © american association for cancer research.clincancerres.aacrjournals.org downloaded from author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. author manuscript published onlinefirst on february , ; doi: . / - .ccr- - http://clincancerres.aacrjournals.org/lookup/doi/ . / - .ccr- - http://clincancerres.aacrjournals.org/content/suppl/ / / / - .ccr- - .dc http://clincancerres.aacrjournals.org/cgi/alerts mailto:pubs@aacr.org http://clincancerres.aacrjournals.org/content/early/ / / / - .ccr- - http://clincancerres.aacrjournals.org/ 	article file 	figure 	figure 	figure 	figure 	figure journal of medical genetics , , - hypertrichosis cubiti (hairy elbows) and short stature: a recognisable association k d macdermot*, m a pattont, m j h williamst, and r m winter* from *the kennedy galton centre, northwick park hospital, watford road, harrow, middlesex hal uj, tst george's hospital medical school, cranmer terrace, london sw ore; and :watford genera, hospital, vicarage road, watford, herts. summary we report four patients with hypertrichosis cubiti who were referred for investigation of short stature. two males, whose height was on and just below the rd centile respectively, were sporadic cases and two females with disproportionate short stature were mother and daughter. radiological changes present in the familial cases were non-specific and biochemical investigations were normal. of the four other published cases, two were sporadic and of normal height. the other two were sibs with short stature and their parents were heterozygous for the weill-marchesani syndrome. we were unable to ascertain whether hypertrichosis cubiti cosegregates with the same type of skeletal dysplasia or elucidate the type of genetic transmissior of hypertrichosis cubiti alone. hypertrichosis cubiti was first reported by beighton,l who described a localised hypertrichosis confined to the extensor surface of the elbow regions in two sibs from an inbred amish family. they had normal development and clinical abnormalities were confined to short stature and foreshortened, but not dysplastic, nails. their parents were short and both were heterozygous for the weill-marchesani syndrome. the short stature of the affected sibs was not investigated. subsequently, rudolph and andrev and stansky each reported a sporadic case of hypertrichosis cubiti without other clinical abnormalities. sighting of a case in the local parish a church congregation was reported by warner and a few cases were apparently observed, but not published. we have had the opportunity to examine four previously unreported patients with hypertrichosis cubiti, all of whom were referred for investigation of short stature. case reports two males and two females presented with a history of excessive hair growth over the lateral aspects of the lower third of the upper arm and upper third of the forearm observed soon after birth. in early childhood the hair became coarser, sometimes fig case . sporadic case of hypertrichosis cubiti received for publication october . with the height just below the rd centile. positional revised version accepted for publication january . elevation of the shoulders. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ hypertrichosis cubiti (hairy elbows) and short stature: a recognisable association curly, pigmented, and lengthened to to cm. the underlying skin was normal. they had a normal amount of body hair and no other areas of hyper- trichosis. the only adult patient seen reported abatement of this localised excessive hair growth around puberty and, when examined, it was no longer apparent. all patients had normal birth weight, psychomotor development, and clinical systems examination. they were in good health and took no medication. case (fig ) was a / year old male. his height was cm (< rd centile), with growth velocity just below the rd centile for the last three years. mid parental height could not be estimated as he was adopted. however, his foster parents, who also adopted his brother and sister, reported that his parents and sibs have normal stature. the skeletal survey was normal. there was no family history of hypertrichosis. case (figs and ) was a seven year old male with a height of cm ( rd centile), compatible with the predicted height from mid parental value. the height of his three and a half year old brother was also on the rd centile. the skeletal survey was fig case . hypertrichosis cubiti. normal. he had an antimongoloid slant of his eyes, mild hypertelorism, and a short nose with thick alae. there was no family history of hypertrichosis. cases and (fig ) are mother and daughter, respectively, with marked shortness of stature. neither of them had any sibs. in the pedigree, there were no other persons with short stature or hyper- trichosis. case was noted to be short at months but the measurement obtained is not available. at the age a i t i ! ffiof eight years three months her height was cm (> sd below the mean). on examination she appeared to have rhizomelic shortening of the extremities. skeletal survey showed a number of minor abnormalities. she had delayed bone age which was five years nine months at the age of eight years three months, some shortening of her meta- carpals, and clinodactyly. metaphyses were flared and bone modelling was normal. there was acetabu- lar dysplasia and the symphysis pubis was a little wide with short ischia. the capital femoral epiph- yses were small and the vertebral bodies slightly fig case . antimongoloid slant of the eyes, mild oval. the distal radial epiphyses sloped. the skull hypertelorism, and foreshortened nose. vault appeared relatively large. she had a round face o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ k d macdermot, m a patton, m j h williams, and r m winter fig cases and . mother and daughter with hypertrichosis cubiti, short stature, rhizomelic shortening of the extremities, round facies, and a heavy jaw. with epicanthic folds, mild hypertelorism, a low nasal root, and a foreshortened nose. case was noted to be short in early childhood and her adult height was cm (> sd below the mean). rhizomelic limb shortening was apparent. skeletal survey showed normal bone modelling and minor abnormalities confined to the spine. she had narrow interpedicular distances in the lumbar region and short pedicles. mild scoliosis with some vertebral end palate irregularity was present in the dorsal region. she had a round face with prominent jaw. the following investigations were normal in all cases: chromosome karyotype, routine haematology and biochemical profile, total t , thbi, free t index, and tsh. all patients except case (not measured) had normal trh and hypoglycaemia stimulated growth hormone assay. discussion generalised hypertrichosis is most commonly seen in certain racial groups and falls within the normal range of hair distribution. pathological generalised hypertrichosis with predilection for certain sites is associated with hyperactivity of the adrenal cortex or tumours producing excess steroids or androgens. drugs (valproic acid) and metabolic disorders (por- phyria, mucopolysaccharidosis) produce hypertrich- osis by an as yet unknown mechanism. generalised congenital hypertrichosis lanuginosa results from a rare autosomal dominant gene. localised hyper- trichosis is commonly seen over a naevus or spina bifida. the only biopsy report of the hairy site in hypertrichosis cubiti showed no structural abnor- malities of the hair follicles. the faster than normal hair growth was reflected by the finding of % of the hair follicles being in the anagen phase, % in the telogen, and % in the catagen phase. andreev and stransky concluded that it probably repre- sented a naevoid condition of the hair follicles. urinary -ketosteroids and -hydrocortisone, plasma cortisol, and testosterone were measured in one case and were normal. all patients so far reported, including our cases, simply improved their cosmetic appearance by regular cutting of the excess hair. the unresolved questions regarding hypertrich- osis cubiti are its type of genetic transmission and a possible association with short stature. the four familial cases have severe shortening of stature, while the height of the three sporadic cases was within the normal range and just below the rd centile in the fourth case. the aetiology of short stature in our cases and is unclear. hypochon- droplasia and dyschondrosteosis were considered and excluded. round facies, obesity, and short stature in mother and daughter are suggestive of albright's hereditary osteodystrophy but x ray findings do not support this diagnosis. the two affected sibs reported by beighton had brachy- dactyly and proportionate short stature, seen in weill-marchesani syndrome, but the results of a skeletal survey were not published. it is uncertain if skeletal changes are comparable in these two families or coincidental findings. genetic heterogeneity in the type of transmission of hypertrichosis cubiti may exist. autosomal domi- nant inheritance is most probable in cases and (mother and daughter). in the two sibs reported by beighton from the amish community, autosomal recessive inheritance is more likely. we are grateful to dr s greene for referring cases and and to mrs j bourne for typing this manu- script. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ hypertrichosis cubiti (hairy elbows) and short stature: a recognisable association references ' beighton p. familial hypertrichosis cubiti: hairy elbows syn- drome. j med genet ; : - . rudolph ri. hairy elbows. cutis ; : . andreev vc, stransky l. hairy elbows. arch dermatol ; : . warner tfcs. hairy elbows. arch dermatol ; : . correspondence to dr k d macdermot, kennedy galton centre, northwick park hospital, watford road, harrow, middlesex hal uj. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ i med genet ; : - recessive metaphyseal dysplasia without hypotrichosis. a syndrome clinically distinct from mckusick cartilage-hair hypoplasia alain verloes, gerald e pierard, martine le merrer, pierre maroteaux abstract among children with recessive metaphyseal dysplasia involving the knees and extremities, two types can be distinguished. in true cartilage-hair hypoplasia, as described by mckusick, many patients show clinical hair involvement and variable immunodeficiency. we present a series of six patients with the same radiological changes, but without apparent hypotrichosis. we suggest that they should be considered as having a variant form of cartilage-hair hypoplasia, with a clinicaily distinct phenotype, which could be as common as 'true' cartilage-hair hypoplasia among non-amish populations. microscopic examination of the hair may show reduction in the diameter of the hair shaft. this form of metaphyseal dysplasia may result from allelic heterogeneity. in the early s, one of us described five children with a metaphyseal chondrodysplasia mainly affecting the bones of the extremities, but sparing the femoral necks, under the name 'forme partielle de la dyso- stose metaphysaire'.' two cases had fine, sparse hair together with the typical metaphyseal changes. they were obviously affected with the metaphyseal dysplasia described two years later by mckusick as 'cartilage- hair hypoplasia' (chh). however, our first three cases (a boy and his two sisters) had perfectly normal hair (fig ), although their bone dysplasia was exactly the same as in chh. since that time, we have had the opportunity to investigate seven other children with a chh-like bone dysplasia without any clinical hair involvement. we report here three of them, of whom two are sibs. case reports case this boy was born at term to non-consanguineous parents originating from the same village. his father was and his mother years old. birth weight was g, length cm. his height fell to less than sd by ½ years, and less than sd at ½/ years. when we first saw him at the age of years centre for human genetics, liege university, belgium. a verloes department of dermatopathology, liege university, belgium. g e pierard clinique de genetique medicale et unite inserm u , hopital des enfants malades, paris, france. m le merrer, p maroteaux correspondence to dr verloes, centre de genetique humaine, pathologie b , chu sart tilman, b- liege, belgium. figure i unpublished picture ofthe original patient of maroteaux et al' at years. slight varus deformity ofthe legs and normal dark hair. received for publication february . revised version accepted for publication may . co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n n o ve m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ verloes, pierard, le merrer, maroteaux figure case i at '/z years showing dark, thick hair. months, he was cm tall and weighed - kg. he had obvious micromelia and short, stubby hands. there was a slight varus deformity of the legs. joint mobility was not restricted. his hair was dark and thick and growth was normal (fig ). calcium- phosphorus balance and gh releasing tests gave normal results. skull and spine radiographs were strictly normal. figure case i at ½ years showing slight metaphyseal irregularities and shortened bones. [ he long bones were shortened, but the temoral necks and heads were not affected. the lower femoral metaphyses, upper and lower tibial metaphyses, and lower radial and ulnar metaphyses were somewhat irregular. the bones of the feet and hands were shortened and stubby, with irregular and slightly cupped metaphyses (fig ). two brothers were born later and were unaffected. case this girl was born to non-consanguineous, caucasian parents; her father was and her mother years old. length was only cm at term and at year she was cm tall (- sd). when we saw her at the age of months, she was cm tall and weighed - kg. she had slight micromelia and varus deformities of the legs. her spine was straight and her hair and facies looked normal. she had a cafe au lait spot, cm wide, in the middle of her back. radiographs showed shortened long bones and severe deformation of the lower femoral metaphyses, which were slightly enlarged, very irregular, and had an inverted v shape (fig ). tibial and peroneal metaphyses were similarly affected, as well as the lower extremities of the ulna and cubitus. the pelvis, figure case at months. lower metaphysis ofthefemur and tibial metaphyses are enlarged and very irregular. co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n n o ve m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ recessive metaphyseal dysplasia without hypotrichosis. a syndrome clinically distinctfrom mckusick cartilage-hair hypoplasia figure s case at ½ years. femoral neck, and spine were normal. the metaphyses of the hand bones were shortened and concave. subsequent follow up was unremarkable. at years months (fig ), she was an intelligent girl, - cm tall (- to - sd), with moderate knock knees. joint movements were not restricted. meta- physeal deformities had improved, but irregularities were still severe in the hands, the bones of which were very short and stubby. examination of the hair was unremarkable under figure case at years showing normal hair. normal and polarised light, but the mean hair shaft diameter was abnormal: - im± - (controls - ilm± - between and years). case case is the older brother of case . he was only cm long at birth. we first saw him at the age of years months, when the diagnosis of md was established for his sister. at that time, he was cm tall (- sd) and had normal hair (fig ). radiological investigations showed severe metaphyseal alterations, very similar to those observed in his sister. the most prominent changes concerned the lower femora, which had a pseudoenchondromatous appearance (fig ). the bones of the hand were short and stubby (fig ). we saw him for the last time in , at the age of i years; he was cm tall (mean) and weighed kg. this apparent catch up in height was the result of a slightly premature pubertal growth spurt. the spine and legs were not deformed, but the hands were very short. all the metaphyseal alterations were less striking than during previous examinations. examination of hair structure was unremarkable, and the mean shaft diameter was - tm± - (normal). discussion these three cases, three children previously published, and fourother unpublished cases fromour files show the same pattern of defects. the clinical hallmarks are stunted growth, owing to disproportionate limb shortening, short hands and feet, and normal face and hair. although moderate dwarfism does exist from birth (birth length to cm), diagnosis is usually made later. growth retardation is usually severe, but intrafamilial variation exists, as illustrated in our cases and . the long bones are shortened. the distinctive radiological features are restricted to the metaphyses close to the knees and to the distal metaphyses of the tibia, ulna, and cubitus. hand bones are always involved: they are short and stubby, and their metaphyses look concave and irregular. the meta- physeal irregularities are more prominent in the first years, but they fade with time and are not apparent after the disappearance of the growth cartilage. the femoral necks, spine, and pelvis remain unaffected. the metaphyses of the ribs are sometimes enlarged and irregular. the recurrence of this dysplasia in a sibship with unaffected parents suggests autosomal recessive inheritance, although parental consanguinity has not been observed. both sexes are equally affected: five boys and five girls in our series. the general build and radiological features of this metaphyseal dysplasia suggest the diagnosis of "- el i f. "w' ip: k ol iwilk -, sa, qi co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n n o ve m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ verloes, pierard, le merrer, maroteaux figure case at years showing metaphyseal irregularities ofthe knee. figure case at years showing shortness ofhand bones with metaphyseal irregularities. mckusick cartilage-hair hypoplasia. the major difference is the apparent lack of hair involvement. none ofour cases had sparse or fair hair. nevertheless, measurement of hair diameter showed a significant reduction in one of them. however, this reduction was less than the diameters given by mckusick et a : of patients studied had a mean hair diameter between and rim. none of our patients had immunodeficiency. our experience is that this form of md without clinical hair dysplasia could be almost as common as typical chh in non-amish populations. linkage studies are required to determine if chh and recessive md are the result of defective expression of genes at distinct loci, or manifestations of allelic heterogeneity for the same gene. we are very grateful to dr p garnier and professor j c job for referring case and to dr sorin for referring cases and . maroteaux p, savart p, lefebvre j, royer p. les formes partielles de la dysostose metaphysaire. presse med ; : - . mckusick va, eldridge r, hostetler ja, ruangwit u, engeland ja. dwarfism in the amish. ii. cartilage hair hypoplasia. bull johns hopkins hosp % ; : - . ray hc, dorst jp. cartilage-hair hypoplasia. in: intrinsic diseases of bone. progress in pediatric radiology vol . basel: karger, : - . pierce cf, polmar sh. lymphocyte dysfunction in cartilage-hair hypoplasia. evidence for an intrinsic defect in cellular prolifera- tion. j immunol % ; : - . co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n n o ve m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ the founder effect and deleterious genes frank b. lningstone d e p a r t m e n t of a n t h r o p o b g y , u n i v e r s i t y of michigan abstract during the rapid growth of a population from a few founders, a single deleterious gene i n a founder can attain an appreciable frequency i n later generations. a computer simulation, which has the population double itself in early generations, indicates a lethal could attain a frequency of . . since deleterious recessive genes are eliminated from large populations a t a very slow rate, variations in their frequencies in present major human populations may be due to the founder effect during earlier rapid expansion. many distinctive human populations are characterized by the presence of one or more lethal or severely deleterious genes in frequencies which would be defined as polymorphic according to ford's (' ) fa- mous definition. the particular genetic disorder, however, varies. the old order amish of lancaster county, pennsylvania have a gene frequency of . for the re- cessive ellis-van creveld syndrome, while the amish as a whole have a frequency of about . of the recessive cartilage-hair hypoplasia syndrome ( mckusick et al., ' ). many of the tri-racial isolates of east- ern united states also have a high fre- quency of a deleterious gene (witkop et al., ' ). although such populations are fre- quently defined by religious or ethnic cri- teria, there are others not so defined. several island populations in the nand archipelago have a gene frequency of greater than . for von willebrand's dis- ease (eriksson, ' ), and the boer popula- tion of south africa and some populations of northern sweden have frequencies of porphyria much greater than those of other populations (dean, ' ; waldenstrom and haeger-aronsen, ' ). however, these con- ditions are dominant and do not have the very severe effects of other hereditary dis- orders found in high frequencies. on the other hand the population of the chicou- timi district of quebec has recently been found to have a gene frequency of about . for tyrosinemia, which is a lethal re- cessive (laberge and dallaire, ' ). in most of these cases the population in question has undergone a rapid increase in recent years, and the question arises as to whether this rapid expansion and the am. j. p h y s . antxrop., : - . original small size of the isolate could ac- count for the high frequency of the dele- terious gene. such an explanation by the founder effect seems obviously to apply to most of the cases cited above, but the founder effect may well be a more general explanation of human gene frequency dif- ferences. it is now becoming apparent that the major populations of mankind vary sig- nificantly in their frequencies of deleteri- ous genes and that many large populations such as eastern european jews have high frequencies of deleterious genes which are found in low frequencies in other popula- tions (mckusick, ' ). there have been many attempts to determine how such genes could be polymorphic, for example, anderson et al. (' ) and knudson et al. (' ) have discussed cystic fibrosis and myrianthopoulos and aronson ( ' ) , tay- sachs disease. the purpose of this paper js to attempt to determine the extent to which the founder effect can cause high frequencies of deleterious genes with vari- ous models of population expansion. the occurrence which initiated this re- search is the gene for sickle cell hemoglo- bin in the brandywine isolate of southeast maryland. at present the sickle cell gene frequency in this isolate is about . (rucknagel, ' ). the high frequencies of this gene in many parts of africa, india, and the middle east are now well-accepted as being due to a relative resistance of the sickle cell heterozygote to falciparum ma- laria. the high frequency i n the brandy- wine isolate may have a similar explana- tion, but the surrounding negro population does not have such a high frequency. and although the endemicity of falciparum f r a n k b . l i v i n g s t o n e malaria in southeast maryland in the last century is not known in any detail, it would not appear to have been great enough to explain the high sickle cell fre- quency in the brandywine isolate. the iso- late also has many other deleterious genes in high frequency (witkop e t al., ’ ). the brandywine isolate seems to have had its beginning in the early eighteenth century when laws were passed to prohibit co-habitation and marriage among races, which prior to then were presumably fre- quent or at least known. up to there were several prosecutions under these laws of individuals with surnames currently present in the isolate (harte, ’ ). harte (’ ) has maintained that the brandywine isolate is derived from these illegal unions, and witkop et al. (‘ ) show that the most common surname came from such a union. in the first united states cen- sus recorded persons with the group’s surnames as “other free people,” and since then over % of the recorded marriages have been endogamous or between individ- uals with surnames within the group (harte, ’ ). according to harte (’ ) there are six “core” surnames which have been associated with the group since its founding and comprise % of the popu- lation and another ten surnames which entered the group after the civil war, but witkop et al. (‘ ) list seven core surnames and eight marginal ones. the total popula- tion of the isolate is now estimated to be , (witkop et al., ’ ), and the statis- tics do indicate rapid, if erratic, growth (gilbert, ’ ; harte, ’ ). in order to simulate gene dynamics the population has been assumed to have doubled itself i n early generations, and then after slower growth to have ap- proached a doubling in recent generations. the simulation was run for generations with the following numbers in succeeding generations: , , , , , , , , , . this approximates the early demographic history of the bran- dywine isolate, but the isolate is much larger today. however, gene frequency change in later generations with a large population is very small. the simulation model randomly selects two parents from the initial population which has been assumed to have either one or two sickle cell heterozygotes among the founders. a family size is randomly determined, the offspring generated and then selected out or stored with no com- pensation for those not surviving ( a copy of the program is available on request). since the population is increasing rapidly, the family size distribution approximates that recorded by roberts (’ ) for a popu- lation in tanzania which has about . surviving offspring per female. the found- er population can actually vary in size, however. the size of the offspring genera- tion is the number which is set; but with an average of . offspring per marriage and offspring, the founder population would be expected to consist of ten mar- riages or individuals. figure shows the distribution of the deleterious gene frequencies after ten gen- erations for two sets of runs each with different initial conditions and different fit- nesses for the genotypes. with a gene fre- i- < d p - ll ct w m s = z waa = . was = . wss =o.o l t k . . . . .lo gene frequency gene frequency s t a r t . start . fig. the distributions of the frequency of the sickle cell gene ( s ) after ten generations of expansion for two sets of runs each with dif- ferent initial gene frequencies and different fit- nesses for the genotypes (w’s). note: the popu- lations in which the s gene has been completely eliminated are separated in the left column from those in which i t is still present. founder effect and deleterious genes - - a - l- - => p p - = - u. w m s z - u- quency of . , which is comparable to having two sickle cell heterozygotes among the founders, the gene is present at a fre- quency of greater than . in almost % of the populations, while for a starting gene frequency of . or one founder with the sickle cell trait, % of the popu- lations have the gene at a frequency of greater than . . with two founders there were runs which resulted in a gene fre- quency as high as that of the sickle cell gene in the brandywine isolate, but with one founder there were none as high. how- ever, there were many frequencies close to it, so that such an outcome is possible if not probable. hence there seems to be no necessity to postulate a selective ad- vantage for the sickle cell in the brandy- wine isolate. it should be pointed out that this simulation and further ones assume the population is closed. gene flow from other populations would tend to decrease the frequency of the deleterious gene, but if most of the population’s expansion is due to natural increase, then the founder effect would be most important. in order to determine whether such high frequencies could occur in a population with a greater number of founders, a simi- lar program was run with and founders. the results are shown in fig- ure . these runs were started with one carrier of the lethal gene and the popula- tion doubled itself for five generations, so that it ended with , and , indi- viduals, respectively. although the lethal gene is not present in high frequencies in as many populations, it is still present in about % in a frequency greater than . . the fact that populations begun with a few founders should have such high fre- quencies of lethal genes seems to indicate that they can contribute to the problem of the genetic load. according to morton (‘ ), the average individual has the equiv- alent of four recessive lethals in the hetero- zygous state. for a population with founders this would imply over lethal or deleterious genes among the founders, so that several would be expected to attain high frequencies. the fact that the num- ber of deleterious genes in small popula- tions started by a few founders seems to average much less may jndicate a lower genetic load, but the brandywine isolate, - the amish, and the eastern european jews do have several deleterious genes in high frequencies. in any case, it seems to be a possible way to study the problem. the most recent population expansion which seems to have increased the fre- quency of a lethal gene is the peopling of the saguenay river and lake st. john re- gion by french canadians. settlement of the upper saguenay did not begin until the ’ ~ ~ and chicoutimi was founded in by individuals from la malbaie, from eboulements, and from baie st. paul (buies, ). in the popu- lation of chicoutimi was , and in it rose to , . much of this in- crease was undoubtedly due to immigra- tion, but given the enormous rate of in- crease of the french canadian population as a whole (henripin, ’ ), the expansion of the population of chicoutimi was due to a great extent to natural increase. under these conditions a lethal gene frequency of . would not be unlikely and seems - ol . . - . .i d waa = . was = . wss = . l . . . . .i gene frequfncy gene frequency start . start. fig. the distributions of the frequency of a lethal recessive gene ( s ) after five generations of expansion for two sets of runs each with different initial gene frequencies which repre- sent one carrier of the lethal gene in a founder population of ( . ) and one carrier in a population of ( . ). note: the populations in which the lethal gene has been completely eliminated are separated in the left column from those in which it is still present. frank b. livingstone to agree with our simulation model. the fact that the entire french canadian pop- ulation stems from about , original settlers (henripin, ’ ) may have led to this population having its own set of lethal genes. the high frequencies of deleterious genes in the eastern european jewish pop- ulations of lithuania and eastern poland may have a similar explanation, although there is disagreement about this possibility (mckusick, ’ ). myrianthopoulos and aronson (’ ) do not consider such an explanation likely for the tay-sachs gene. instead they propose a slight selective ad- vantage for the heterozygote. they have to postulate the operation of this selective ad- vantage for generations which is longer than the population has been there. it is also much longer than the factor thought to confer the selective advantage, typhoid fever in the ghettos (aronson, ’ ), seems to have been present as a serious disease. the tay-sachs gene attains its highest fre- quencies in the jewish populations of southern lithuania and northeast poland, which were founded in the twelfth cen- tury after the crusades led to the persecu- tion of the jews in germany. although the jewish settlements in lithuania were founded by refugees from the west, accord- ing to herzog (’ ) they preceded by two or three centuries the jewish settlements in mazovia to the west in poland. thus, these colonies were isolated for some time and were actually expanding to the west into northern poland when the jews were expelled from lithuania in . most moved to adjacent territories but then moved back to lithuania in . hence, the population history of these jewish groups seems to be one of expansion from a few founders. in any case by the time of the flowering of eastern jewish culture in the sixteenth century, the population was very large and continued to expand up to the twentieth century. when a population size of , or more is attained, the change in gene frequency is approximated by deterministic equa- tions. for a lethal recessive the frequency after n generations is: qn = z + nqa , where q. is the initial gene frequency. a lethal gene in a large population is thus eliminated at a very slow rate, particularly when it occurs in a very low frequency. if the tay-sachs gene increased to . in the early generations before the popula- tion became large, in the approximately generations since then, the gene would have decreased to o s o which is about the frequency today in eastern european jews. the fact that lethal genes are eliminated at such a slow rate in large populations would make it possible for them to have “polymorphic” frequencies long after the original expansion. since most of the worlds populations have expanded rap- idly in the last , years, much of the variability in the frequencies of lethal genes (or non-lethals for that matter) could be a consequence of the original expansions of the major populations. as an example, it is suggested that this effect may explain the high frequencies of cystic fibrosis in the populations of europe, which range around a gene frequency of . . for the non-caucasian populations on hawaii wright and morton (’ ) have estimated the gene frequency for cystic fibrosis to be . , which presumably is close to the equilibrium frequency due to a balance of selection and mutation. given this fre- quency, over % of a set of founder pop- ulations of size would be expected to have a carrier of this lethal. with the sud- den expansion of such a set of founder populations it seems possible that such a lethal could attain a frequency of . for the entire population. a more precise mathematical expression of the problem seems possible and could perhaps yield a solution. + ( . ) ‘ ’ literature cited anderson, c. m., j. allan and p. g. johansen comments on the possible existence and nature of a heterozygote advantage in cystic fibrosis. in: cystic fibrosis. e. rossi and e. stoll, eds. bibliotheca paediatrica, no. . s. karger, new york, pp. - . aronson, s. m. epidemiology. in: tay- sachs’ disease. b. w. volk, ed. grune and stratton, new york, pp. - . buies, a. le saguenay et le bassin du lac saint-jean. irger brousseau, qukbec, rd edition. dean, g. the prevalence of the porphy- rias. so. afr. j. lab. clin. med., : - . founder effect and deleterious genes eriksson, a. w. eine neue blutersippe mit v. willebrand-jiirgens' scher krankheit (er- bliche thrompathie) auf aland (finnland). acta genet. med. gemell., : - . ford, e. b. polymorphism and taxonomy. in: the new systematics. j. s. huxley, ed. clarendon press, oxford, pp. - . gilbert, w. h. the wesorts of maryland; an outcasted group. jour. wash. acad. sci., : - . harte, t. j. trends in mate selection in a tri-racial isolate. social forces, : - . social origins of the brandywine population. phylon, : - . henripin, j. la population canadienne au dbbut d u xviiie sihcle. institut national d'ctudes dkmographiques, paris, cahier no. . herzog, m. i. the yiddish language in northern poland: its geography and history. indiana university research center in anthro- pology, folklore and linguistics, pub. no. . knudson, a. g., l. wayne and w. y. hallett on the selective advantage of cystic - brosis heterozygotes. amer. j. hum. genet., : - . laberge, c., and l. dallaire genetic as- pects of tyrosinemia in the chicoutimi region. canad. med. assoc. j., : - . mckusick, v. clinical genetics at a pop- ulation level. the ethnicity of disease in the united states. alabama j. med. sci., : - . mckusick, v., j. a. hostetler, j. a. egeland and r. eldridge the distribution of certain genes in the old order amish. cold spring harbor symp. quant. biol., : - . morton, n. e. the mutational load due to detrimental genes in man. amer. j. hum. genet., : - . myrianthopoulos, n. c., and s. m. aronson population dynamics of tay-sachs dis- ease. i. reproductive fitness and selection. amer. j. hum. genet., : - . roberts, d. f. assumption and fact in anthropological genetics. jour. roy. anthrop. inst., : - . rucknagel, d. l. the gene for sickle cell hemoglobin in the wesorts. thesis, uni- versity of michigan, ann arbor. waldenstrom, j., and b. haeger-aronsen the porphyrias: a genetic problem. in: prog- ress in medical genetics. volume v. a. g. steinberg and a. g. bearn, eds. grune and stratton, new york, pp. - . witkop, c. j., c. j. maclean, p. j. schmidt and j. l. henry medical and dental find- ings in the brandywine isolate. alabama j. med. sci., : - . . surgical neurology international editor:james i. ausman, md, phd university of california, los angeles, ca, usa open access for entire editorial board visit : http://www.surgicalneurologyint.com case report paradoxical evolution of a cerebellar tuberculosis abscess after surgical drainage and antibiotic therapy vivek joshi, isabelle germano , rana meenakshi , amish doshi departments of radiology, neurosurgery, infectious disease, mount sinai school of medicine, new york, ny, usa e‑mail: *vivek joshi ‑ vjoshi @gmail.com; isabelle germano ‑ isabelle.germano@mountsinai.org; rana meenakshi ‑ rana.meenakshi@mountsinai.org; amish doshi ‑ amish.doshi@mountsinai.org *corresponding author received: may accepted: july published: september access this article online website: www.surgicalneurologyint.com doi: . / - . quick response code: abstract background: involvement of the central nervous system (cns) by a tuberculosis abscess is a rare form of extra‑pulmonary tuberculosis. with proper treatment, the abscess most commonly follows a pattern of continued reduction in size. case description: a ‑year‑old male with a past medical history of kidney transplant on immunosuppressive therapy, presented to the hospital with a ‑day history of headache. on physical examination, the patient had no focal neurological symptoms. initial laboratory reports were unremarkable. contrast enhanced magnetic resonance imaging (mri) was performed, which showed a ring enhancing mass and perilesional edema in the left cerebellar hemisphere. the patient underwent a left posterior fossa biopsy and drainage. the lesion was encapsulated with a purulent center. cultures revealed pan‑sensitive mycobacterium tuberculosis and the patient was started on rifampicin, isoniazid, pyrazinamide, ethambutol, and b . the patient was monitored carefully and brain mris were obtained at , , , , and months. it was noted that the tuberculosis abscess had grown in size from month to month of treatment. since the patient’s neurologic examination and symptoms were stable at that time, the drug regimen was not changed. the ‑month follow up mri showed that the abscess had nearly resolved. conclusion: rarely, the pattern of cns tuberculosis abscess evolution may include growth, even with proper treatment. this pattern does not necessarily signify treatment failure, as our abscess resolved without change in treatment. given the possibility of asymptomatic abscess enlargement, close clinical and imaging follow up are crucial in management of these cases. key words: central nervous system, cerebellum, tuberculosis abscess introduction central nervous system (cns) tuberculosis abscess is a rare form of extra pulmonary tuberculosis with high morbidity and mortality rates internationally.[ ] a tuberculosis abscess is a focal collection of pus surrounded by a dense capsule containing acid fast bacilli compatible with mycobacterium tuberculosis.[ ] with proper treatment, the most common pattern of abscess evolution is gradual reduction in size.[ ] we present a rare case of a pan‑sensitive cns tuberculosis abscess affecting the cerebellum of an adult, with paradoxical enlargement this article may be cited as: joshi v, germano i, meenakshi r, doshi a. paradoxical evolution of a cerebellar tuberculosis abscess after surgical drainage and antibiotic therapy. surg neurol int ; : . available free in open access from: http://www.surgicalneurologyint.com/text.asp? / / / / copyright: © joshi v. this is an open‑access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. surgical neurology international , : http://www.surgicalneurologyint.com/content/ / / upon treatment and follow‑up imaging, and eventual resolution without change in treatment. case report a ‑year‑old male with a past medical history of kidney transplant on immunosuppressive therapy, diabetes, and hypertension presented to the hospital with a ‑day history of headache, nausea, and vomiting. on physical examination, the patient had no focal neurological symptoms, and normal blood pressure . initial laboratory reports showed an unremarkable cell blood count. basic metabolic panel, liver function tests, and coagulation panel parameters were all within normal limits. blood cultures and urine analysis were both negative. prior purified protein derivative (ppd) tuberculin test and human immunodeficiency virus (hiv) results were negative. chest x‑ray demonstrated a stable right upper lobe cavitary lesion that had been biopsied twice previously with nondiagnostic results. a head computer tomography (ct) was obtained, which demonstrated a cystic mass in the left cerebellum with perilesional edema [figure ]. further evaluation with contrast enhanced mri was performed that showed a well circumscribed left cerebellar hemisphere ring enhancing mass on t sequences with restricted diffusion, mass effect, and perilesional edema [figure ]. differential diagnosis included pyogenic abscess (bacterial or fungal), neurocysticercosis, cystic glioma, or other cystic malignant/metastatic process. the patient underwent a left posterior fossa biopsy and drainage. the lesion was well encapsulated with a frankly purulent center.numerous acid fast bacilli were seen on acid fast stain, and cultures returned positive for mycobacterium tuberculosis [figure ]. cerebrospinal fluid (csf) cultures were also obtained, which were negative. microbiology revealed pan sensitive mycobacterium tuberculosis and the patient was started on rifampicin, isoniazid, pyrazinamide, ethambutol (ripe), and b . follow‑up mris were obtained , , , , and months [figures and ]. during this time, the patient was seen monthly, and had continued to show clinical improvement with treatment. after months, ethambutol had been removed from the treatment regimen. treatment with rifampicin, isoniazid, and pyrazinamide was continued in order to maximize cns penetration, and it was noted on the ‑month mri that the tuberculosis abscess had grown in size from . × . × . to . × . × . cm [figure ]. while the abscess had grown in size at months, the amount of perilesional edema had decreased [figure b]. at this time the patient’s neurologic examination and symptoms were stable, and given the continued clinical improvement, and pan sensitive cultures, the drug regimen was not changed. due to the increase in size, vigilant surveillance with frequent clinic visits was pursued. the ‑month follow up mri showed that the abscess size was decreased to . × . × . cm, and on month , the abscess had nearly resolved [figure ]. discussion cns tubercular abscess is a rare form of extra‑pulmonary tuberculosis in which the typical granulomatous reaction associated with tuberculosis is not present.[ ] this case fits the criteria for an abscess by having positive macroscopic evidence of abscess formation with frank pus centrally and cavity formation, and positive mycobacterial tuberculosis cultures and acid fast organism staining.[ ]cns tuberculous abscesses are generally seen in immunocompromised individuals and generally occur in the supratentorial compartment.[ ] tb abscesses, less commonly, as in our case, occur within cerebellum.[ ] there are few reports of true cerebellar abscesses, and figure : (a) noncontrast head ct status post abscess drainage shows improvement in abscess size and mass affect. there is minimal residual hypodensity present. (b) acid fast stain shows numerous stained bacilli, which correlated with culture showing pan sensitive mycobacterium tuberculousis figure : (a) noncontrast head ct shows a cavitary lesion with perilesional edema and mass effect upon the th ventricle. (b) contrast enhanced mr t sequence shows a well circumscribed left cerebellar hemispheric lesion with prominent rim enhancement. there is associated vasogenic edema compressing both the fourth ventricle and medulla ba ba surgical neurology international , : http://www.surgicalneurologyint.com/content/ / / those cases mostly involved younger patients, as opposed to the ‑year‑old in our case. due to the rarity of cns tuberculosis abscess, more cases need to be evaluated to further assess if there is a statistically significant geographic distribution. the radiologic appearance of cns tb abscess on ct generally demonstrates a ring enhancing cystic lesion with perilesional edema, while mri demonstrates a hyperintense central area with a hypontense rim on t ‑weighted images, and hypointense central area with peripheral rim enhancement on t sequences.[ ] our case had the aforementioned characteristics. these lesions can be difficult to distinguish from pyogenic abscesses based on radiological appearance. the consequences of untreated neurotuberculosis are devastating, and choosing the proper treatment regimen is imperative.[ ] one of the most important components of treatment consists of long‑term antituberculosis drug therapy.[ ] surgical drainage and excision, as in our case, can be used to obtain a microbiological diagnosis and to relieve mass effect caused by large lesions.[ , ] most commonly, treatment of tuberculosis lesions involves a pattern of decreasing abscess size on anti‑tb drug treatment.[ ] rarely reported are enlarging tuberculomas of the brain, and very rarely, as in our case, has there been reports of enlarging tuberculous abscess after both surgical drainage and ripe treatment in an adult.[ ] the enlarging tubercular abscess can present a dilemma for the treating clinician as this may potentially signify a failure of treatment/drug resistance, infection with a secondary organism, underlying neoplasm, or simply inflammatory changes.[ ] prior review of enlarging cns tuberculosis lesions has shown the time to enlargement to occur up to months, however, in most cases the enlargement occurred at months from the start of therapy.[ ] in our particular case, the abscess evolved during treatment from no change in size but increasing wall thickness and nodularity, to a paradoxically enlarging thin walled abscess at months, to decrease in size at months and subsequent near resolution at months. it is interesting to note that although the abscess had increased in size at months, the amount of perilesional edema had decreased from prior study [figure ], which may have been a sign that the abscess was following a pattern of resolution. clinically, the patient remained stable with no focal neurological deficits and symptoms, and no mass effect on imaging, therefore treatment was not changed. most cases of enlarging cns tuberculosis lesions, resolve with no change in anti‑tb drug therapy, however, some cases may require the addition of steroids, change in antituberculous drug therapy, and, in rare instances, surgical decompression.[ , ] of the cns tuberculosis lesions, cns abscesses have been notoriously difficult to treat.[ ] while there is no agreed upon explanation as to the cause of tuberculosis abscess enlargement, several theories exist.[ , ] one theory suggested is the restoration of the blood–brain barrier during treatment possibly reduces drug penetration, allowing latent foci to reactivate.[ ] it is unclear whether the cerebellar location of the abscess, the size of the lesion, or the patient’s immune status affected the response to treatment. another explanation is the possibility of the host immune response eliciting a delayed hypersensitivity/inflammatory response to mycobacterial products resulting in an enlarging abscess.[ , ] it is also figure : evolution of tb cerebellar abscess. (a) contrast enhanced axial t at -month follow up shows enhanced thickened and nodular cavity wall, which was unchanged in size or appearance from the postoperative month follow up mr (not shown). (b) axial t flair at -month follow up showed increased cavitation size, but with decreased capsule thickness and nodularity. (c) contrast enhanced axial t at -month follow up showed decreased size of the lesion, without change in treatment. (d) contrast enhanced axial t at -month follow up shows significant reduction in size of previously noted cavitary lesion dc ba figure : evolution of tb cerebellar abscess on t flair imaging. (a) contrast enhanced axial t flair at -month follow up shows significant perilesional edema. (b) contrast enhanced axial t flair at -month follow up showed residual perilesional edema, which had decreased since prior study, even though the cavitation size had increased. (c) contrast enhanced axial t flair at -month follow up and month follow up (d) showed minimal to no perilesional edema ba dc surgical neurology international , : http://www.surgicalneurologyint.com/content/ / / possible that the abscess had become aseptic with permanence and even increment of pus and debris within the cavity. both corticosteroids and thalidomide (tumor necrosis factor modulating drug) are being researched as possible adjuncts to treatment for enlarging abscesses.[ ] it remains unclear why some tuberculosis abscesses enlarge, as such, close clinical and imaging follow up are strongly emphasized in these patients. conclusion in conclusion, we present a rare case of tuberculosis abscess of the cerebellum in an adult, which enlarged after treatment with both antituberculosis drugs and surgical drainage and reached near resolution with no change in antitubercular therapy. this case highlights the importance of close clinical and imaging follow up, as one of the uncommon patterns of abscess evolution may include growth even on proper treatment. this pattern does not necessarily signify treatment failure, as our abscess resolved without change in treatment. additional research is needed to determine factors that predispose to enlarging abscesses. references . afghani b, lieberman jm. paradoxical enlargement or development of intracranial tuberculomas during therapy: case report and review. clin infect dis ; : ‑ . . chambers st, hendrickse wa, record c, rudge p, smith h. paradoxical expansion of tuberculomas during chemotherapy. lancet; ; : ‑ . . ersoy y, ates o, onal c, but ad, cayli sr, bayindir y, et al. cerebellar abscess and syringomyelia due to isoniazid‑resistant mycobacterium tuberculosis. j clin neurosci ; : ‑ . . gurjar hk, joshua sp, agrawal d, mahapatra ak. large pontine tubercular abscess treated surgically. br j neurosurg ; : ‑ . . kumar r, prakash m, jha s. paradoxical response to chemotherapy in neurotuberculosis. pediatr neurosurg ; : ‑ . . menon s, bharadwaj r, chowdhary a, kaundinya d, palande d. tuberculous brain abscesses: case series and review of literature. j neurosci rural pract ; : ‑ . . roopesh kumar vr, gundamaneni sk, biswas r, madhugiri vs. tuberculous cerebellar abscess in immunocompetent individuals. bmj case rep ( ): ‑ . . schoeman jk, fieggen g, seller n, mendelson m, hartzenberg b. intractable intracranial tuberculous infection responsive to thalidomide: report of four cases. j child neurol ; : ‑ . ijc_jan_march_ .pmd cmyk original primary gastrointestinal non hodgkin’s article lymphoma chemotherapy alone an effective treatment modality: experience from a single centre in india raina vinod, sharma atul, vora amish, shukla nk*, deo svs*, dawar r** departments of medical oncology, *surgical oncology and **pathology, institute rotary cancer hospital, all india institute of medical sciences, new delhi, india correspondence to: atul sharma, e-mail: atul @hotmail.com abstract background: gastrointestinal tract (gi) is the most frequently involved extra nodal site in non-hodgkin’s lymphoma (nhl). surgery, radiotherapy and chemotherapy (ct) have been used mostly in various combinations, but lately chemotherapy alone has emerged as an effective option. the purpose of this study is to evaluate efficacy of ct alone in treatment of primary gi-nhl and to compare the results with combined ct + surgery. setting and design: retrospective analysis of case records of gi nhl patients. materials and methods: over a -year period ( - ), new cases of primary gi-nhl were registered at our center. gi-nhl was defined according to standard criteria. all patients received chemotherapy. results: the median age was years (range - ). endoscopy / ct guided biopsies were performed in % ( ) of patients for the purpose of diagnosis. laparotomy was done in % ( ) of patients to establish a diagnosis or as primary or debulking treatment. stomach and intestines were involved in % ( ) and % ( ) patients respectively. early stage disease was present in % ( ). seventy eight percent of tumors were intermediate to high grade, % ( ) received only ct while % ( ) received ct + surgery. five years efs and os were: % and % for all patients; % and % for ct only group; % and % for ct + surgery group (p= . ). four patients died of neutropenic infection. conclusion: organ- preservation strategy using chemotherapy alone (ct) can be successfully employed in a significant number of patients with primary gi-nhl. key words: primary non-hodgkin’s lymphoma of gastrointestinal tract, chemotherapy. introduction the incidence of non-hodgkin’s lymphoma (nhl) has been increasing over the last three decades. during the same period an increase in incidence of extra nodal nhl has also been noted.[ ] gastrointestinal tract represents the most frequent extra nodal site and it accounts for % of all gastrointestinal malignancies. although many large series of primary gi-nhl describing patterns of presentation and outcome have been published the consensus over the ideal treatment for gi-nhl still remains the subject of debate.[ - ] interpretation of the outcome data of these studies is hampered by differences in case selection, staging system, pathological classification and therapy. surgery, radiotherapy (rt) and chemotherapy (ct) have all been used either alone or in combinations. superiority of either single or combined modality over each others is still not proven. surgery has traditionally remained the treatment of choice and chemotherapy is often used after surgery. adjuvant radiotherapy is also practiced in some cases. nevertheless surgery and radiotherapy are not without significant morbidity. nhl being a highly indian journal of cancer | january–march | volume | issue mailto:atul @hotmail.com cmyk � � � � � � raina et al: gastrointestinal non hodgkin’s lymphoma chemotherapy chemo sensitive disease it is now highly questionable whether radical or mutilating surgery is still necessary. advances in endoscopic techniques for obtaining tissue diagnosis, refinements in radiological methods such as ct scans and guided biopsies and efficacy of chemotherapy have created a new option in the treatment of gi-nhl. chemotherapy has the advantage of organ preservation; in addition it is effective for micro-metastases and hence takes care of systemic disease. in the older studies of high grade gi-nhl, chemotherapy was employed either as an adjunct to surgery or in combination with radiotherapy. however more recent studies suggests that chemotherapy alone may be as effective particularly in primary gastric lymphomas.[ - ] materials and methods in this study we have analyzed patients of primary gi-nhl treated at our center over the last years ( - ). this review includes clinical features, histopathological classification, site of involvement, treatment outcome and prognostic factors. some of our patients were operated in other hospitals before being referred to us. this gave us the opportunity to compare surgery + ct vs. ct alone group. primary gi nhl was defined according to lewin et al i.e. patients had to present with gi symptoms or have predominant lesions in the gi tract.[ ] the initial evaluation of all patients included a complete history and physical examination, complete blood count, liver and renal function tests, chest x-ray and computed tomographic scans. all patients where primary surgery was not performed underwent upper gastrointestinal endoscopic or colonoscopic studies or guided biopsies for obtaining tissue diagnosis. bone marrow aspiration and biopsy was done in all patients. patients were staged according to the ann-arbor classification as modified by musshoff.[ ] the histopathology specimens of all patients were reviewed and classified according to the international working formulation, which was the classification followed in our institute during study period.[ ] all patients, irrespective of stage were administered chemotherapy. for patients with diffuse large b cell lymphoma and lymphoma of indeterminate histology, six cycles of chop (cyclophosphamide mg/m , doxorubicin mg/m , vincristine . mg/m with maximum of mg, prednisolone mg for days every days) were given. for burkitt’s lymphoma, eight cycles of dose intensive mcp was administered.[ ] all patients who were operated outside were also given chemotherapy as above. response criteria and end points were reported according to published guidelines.[ ] re-evaluation after completion of treatment included endoscopy, ct-scans and complete blood count and biochemistry in addition to clinical examination. patients were examined every three months for one year and six monthly thereafter. statistical analysis the date of analysis was july , . event free survival (efs) was determined from the time patients entered into complete or partial remission until recurrence or death from any cause, overall survival (os) as time from diagnosis until death from any cause or till the last follow up. patients in remission or alive were censored at the last known date of follow up evaluation. os and efs were calculated using kaplan meier method. for univariate analysis, log-rank test was performed using spss . version. results the clinical features and patient characteristics are given in [table ]. pain was the commonest symptom ( %­ patients); fever and weight loss were the other frequent symptoms, either of them being present in . % ( ) of patients. forty eight percent of patients presented with nausea, vomiting, constipation associated with abdominal pain. only % of patients presented with either hemetemesis or malena. seventy eight percent ( ) of patients had either intermediate or high grade nhl, none of our patients had low grade nhl. four patients had burkitt’s lymphoma. seventeen patients ( %) could not be classified further into intermediate or high grade, as slides available were not of good quality. stage wise, . % ( ) and . % ( ) of patients had early disease (i.e. stage i and stage table : clinical features and patient characteristics median age (range in years) ( - ) male to female ratio . : clinical features b- symptoms present ( . %) median duration of symptoms months pain ( %) hemetemesis ( . %) malena ( %) subacute intestinal obstruction ( . %) indian journal of cancer | january–march | volume | issue cmyk raina et al: gastrointestinal non hodgkin’s lymphoma chemotherapy ii respectively), while . % and . % had disseminated disease i.e. stage ii and stage iv respectively. seven ( %) patients could not be staged in the absence of the complete information. the diagnosis of lymphoma was established by endoscopy in ( . %) patients, by usg/ ct guided biopsy techniques in ( . %) patients. diagnostic laparotomy was performed in ( . %) patients. as shown in [table ], ulceroproliferative type of growth was seen in ( . %) patients. nodular, infiltrative and polypoidal pattern was seen in . % and % of patients respectively and mixed pattern was noted in . % of patients. stomach was the most common organ involved ( . %- ) as shown in [figure ]. median age for small intestine nhl was much lower ( years) compared to stomach and large intestinal nhl. b symptoms and abdominal pain was present in the majority of patients irrespective of site of involvement. there was equal distribution of patients as far as histology type of lesion and stage were concerned. clinical features, histology, stage and site distribution has been shown in [table ]. the median follow up of the cohort was months (range to months). main side effects of chemotherapy were myelosuppression, vomiting and diarrhoea. no patient had perforation or hemorrhage following chemotherapy. four patients died of neutropenic fever. the os and efs of the patients with primary gi-nhl were % and % respectively [figures and ]. we had three groups of patients depending on the treatment they received; . surgery (complete resection) + chemotherapy, . surgery (partial resection) + chemotherapy, . only chemotherapy. survival of different treatment has been table : type of lesion type of lesion n = (%) ulceroproliferative ( . ) nodular ( . ) infiltrative ( . ) polypoidal ( ) mixed ( . ) figure : shows distribution of gi-lymphoma according to site involved table : shows clinical features, histology type of lesion and stage wise distribution in gi­ lymphoma according to the site involved features stomach si l. intestine · number ( . ) ( . ) ( ) · b-symptoms ( ) ( . ) ( . ) histology intermediate grade ( . ) ( . ) ( . ) high grade ( . ) ( . ) ( . ) mixed — ( . ) ( . ) burkitt’s ( . ) ( . ) — unclassified ( . ) ( . ) ( . ) stage wise* stage i ( . ) ( . ) ( . ) stage ii ( ) ( . ) ( . ) stage ii ( . ) ( . ) ( . ) stage iv ( . ) ( . ) ( . ) resection complete ( . ) ( . ) ( . ) incomplete ( ) ( . ) ( . ) no surgery ( . ) ( ) ( . ) figurss in the parenthesis are in percentage shown in table . a total of deaths were recorded during the study period. eight patients died of disease progression after relapse, four of neutropenic infection, nine patients did not achieve cr and died, two died in cr due to causes unrelated to the disease. patients who did not achieve cr were treated with salvage protocols like imvp- (ifosfamide, methotrexate and vp- ) or mine (mitoxanterone, ifosfamide and vp - ). prognostic variables we analyzed prognostic factors using univariate and multivariate analysis. we did not find any correlation between b symptoms, age, sex, stage, histopathological grade, site of involvement and survival. patient who received chemotherapy alone had year survival of % compared to - % in group who had surgery and chemotherapy (p= . ). discussion primary gi-nhl represents a heterogeneous disease with regard to various characteristics like stage, site of involvement, histological subtypes and treatment offered. the commonest presenting symptom in gi-nhl is indian journal of cancer | january–march | volume | issue cmyk no at risk survival function figure : overall survival in months no at risk survival function figure : evemt free survival in months table : efs and os of various treatment groups type of treatment n efs os ( months) ( months) complete resection + ct % % partial resection + ct % % only chemotherapy % % all group % % abdominal pain.[ ] in our series, % of patients had abdominal pain as presenting feature. more than half ( . %) of our patients had b symptoms, which is higher than western data.[ , - ] we have noted equal distribution of gastric and intestinal lymphomas, gastric: . %, intestinal . %. [table ] summarizes anatomic location of the initial disease in the gi tract reported in recent studies.[ , , , , , ] in the majority of the studies, stomach is the commonest site of involvement. with the introduction of the entity malt lymphoma by issacson, more and more gastric malt lymphomas are diagnosed.[ ] as reported in recent series by peter et al, % of stomach nhl were of raina et al: gastrointestinal non hodgkin’s lymphoma chemotherapy table : sites of involvement in primary gi-nhl: different studies first author no. of distribution subjects raina et al (present study) stomach small intestine large intestine peter koch[ ] stomach small intestinal theocecal region multiple sites d’ amore[ ] stomach intestine both radaskiewicz[ ] stomach intestine gurney[ ] stomach intestine liang[ ] stomach intestine morton[ ] stomach intestine malt type.[ ] in our series, there was no patient with malt lymphoma. fifty two percent of our patients had disseminated disease at the time of diagnosis (stage ii e + stage ive) in contrast to the western reports of %­ %.[ , ] surgery with or without chemoradiotherapy has been the mainstay in the treatment of gi-nhl but this may be questioned. in a report by brigitte et al % of cases underwent surgery followed by chemotherapy with % os at years. [ ] two important large prospective studies have been reported recently. in german trial, of patients with primary gi-nhl % had intermediate to high-grade lymphoma; received surgery + chemotherapy and had year overall survival of %. [ ] in a study on gastric lymphoma from mexico patients were randomized to either surgery (s) with or without radiotherapy (srt) and chemotherapy (sct) and chemotherapy (ct) alone, actuarial curves at years showed that overall survivals (os) were: s: %; srt: %; sct: %; ct: % (p< . ). late toxicity was more frequent and severe in patients who underwent surgery.[ ] we believe that advanced primary gi-nhl requires a systemic rather than a local approach and therefore we administered chemotherapy in all patients. many of our patients underwent surgery ( %) before coming to us and this gave us an opportunity to indian journal of cancer | january–march | volume | issue cmyk raina et al: gastrointestinal non hodgkin’s lymphoma chemotherapy table : four large series of gi-nhl reported from india features raina ramesh chandran singh pandey (present study) et al[ ] et al[ ] et al[ ] median number (n) age (yrs) (mean) (mean) m: f ratio . : . : . : . : b symptoms (%) . . predominant symptoms abdominal pain abdominal pain abdominal pain vomiting stage wise distribution (%) ie . not specific ii e . not specific ii e . . (stageiii) not specific iv . not specific histopathology (%) diffuse large cell high grade diffuse histiocytic (commonest) (working) lymphoblastic (working) and diffuse poorly large cell differentiated (rappaport’s) os stomach % % (all patients) % % si % % % compare the results in two groups. we had % survival at years in patients receiving only chemotherapy as compared to - % in surgery + ct arm (incomplete surgery, chemotherapy and complete surgery and chemotherapy respectively), which had borderline statistical significance (p= . ). presentation of gi-nhl in india is different from that in developed world. different features of indian studies are summarized in [table ]. all the studies reported from india are retrospective and have small number of patients. survival with surgery and rt was reported to be % in stomach & % in intestinal nhl which is lower than surgery and chemotherapy ( % for stomach and % for intestinal type).[ , ] patients in the study by chandran et al had os of % at years and the treatment modalities used were surgery + rt, surgery + ct, surgery + ct + rt and ct + rt. [ ] os in our study was % for stomach and % for intestinal type at years. this study questions the need for surgical intervention in diagnosis and treatment of gi-nhl except in emergency situations. there is a need to have prospective studies in this country by various centers to come out with definitive treatment guidelines. conclusion treatment of primary gi nhl is changing and chemotherapy alone is emerging as an attractive option. this has been brought about by: ) advances in endoscopic diagnosis whereby a laparotomy is no longer necessary; ) very high efficacy of chemotherapy; ) studies showing that extensive resection may not be necessary with emphasis shifting to organ preservation and finally the concept that; ) nhl being a systemic disease, a systemic approach like chemotherapy would be more appropriate. our study indicates that results of chemotherapy alone are not inferior to resection with chemotherapy. references .	 devassa ss, fears t. non-hodgkin’s lymphoma time trends: 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al. prognostic factors for primary gastrointestinal lymphoma. hematol oncol ; : - . .	 isaacson p, wright dh. extranodal malignant lymphoma arising from mucosa associated lymphoid tissue. cancer ; : - . .	 singh dp, sharma ss, sandhu ap, goenka mk, kochhar r, nagi b, et al. primary gastrointestinal lymphoma - disease spectrum and management: a -year review from north india. indian j gastrointerol ; : - . .	 pandey m, wadhwa mk, patel hp, kpthari kc, shah m, patel dd. malignant lymphoma of the gastrointestinal tract. eur j surg oncol ; : - . indian journal of cancer | january–march | volume | issue ‘milk is milk’: organic dairy adoption decisions and bounded rationality sustainability , , - ; doi: . /su sustainability issn - www.mdpi.com/journal/sustainability article ‘milk is milk’: organic dairy adoption decisions and bounded rationality caroline c. brock , * and bradford l. barham department of rural sociology, university of missouri, columbia, mo , usa department of agricultural and applied economics, university of wisconsin, madison, wi - , usa; e-mail: barham@mailplus.wisc.edu * author to whom correspondence should be addressed; e-mail: brockcc@misssouri.edu; tel.: + - - - ; fax: + - - - . received: september ; in revised form: november / accepted: november / published: december abstract: bounded rationality is an especially appropriate framework for organic dairy adoption decisions as it recognizes internal and external constraints which are critical in understanding complex farm decision making. farmers use of, and access to, information is examined using interview data gathered from organic, conventional, managed graziers, and amish dairy farmers in southwestern wisconsin at a time when organic milk prices offered a % premium over conventional prices. focusing on certain aspects and impressions of organic dairy, such as the sentiment that ―milk is milk‖, may lead to information satisficing where farmers do not take full advantage of the information available to them. organic farmer interviews reveal the challenges they faced with bounded rationality constraints and how they countered these challenges with the help of social networks, as well as how situational factors such as economic and health crises may have motivated them to adopt organic dairy. the interview data from organic and conventional farmers alike also reveals how many conventional dairy farmers utilized information strategies which did not fully consider the pros and cons of the organic system. a bounded rationality framework could enlighten policy makers and educators as they tailor sustainable agricultural policy design and information dissemination strategies to serve the diversity of farmers on the landscape. keywords: organic; bounded rationality; decision making; dairy; managed grazing; amish open access sustainability , “aren’t you going to get on a [soap] box and tell her what a crock this organic is?” says mrs. teiner; to which mr. teiner replies, “you could take two glasses of milk and there’s nothing different. it’s like buying premier jeans. the jeans are probably the same material…but just twice the price, just because of the name [so]…why [do] people pay for it?” mr. teiner, conventional dairy farmer in southwest wisconsin . introduction ―milk is milk‖ was a common sentiment depicted in the quote above among dairy farmers in southwestern wisconsin at a time ( – ) when the adoption of organic dairy farming was accelerating rapidly in the region, and organic dairy farmers were reportedly prospering relative to other types of dairy farmers. ―milk is milk‖ reflects the belief that all milk is wholesome and nutritious regardless of management system, and more specifically that organic is no different from conventional milk in quality as illustrated by the comparison with premier jeans in the leading quote. this sentiment focused the comparison on whether a ‗composition‘ test would show organic milk components to be different in any way from conventional milk, and if not then what was the big deal about organic. ―milk is milk‖ was one of a number of responses given by non-organic dairy farmers when they were asked about adopting organic dairy farming practices which may have indicated that they were not fully considering organic dairy as a viable option. in the years leading up to this interview study, organic dairy farmers in wisconsin were more highly satisfied with income and overall quality of life than conventional dairy farmers according to statewide survey research [ , ]. at the time of the interview study, the growth in demand for organic milk was outpacing the growth in supply. there was also a sustained price premium for organic milk and organic milk pay prices that the farmer received were much more stable than conventional milk pay prices that farmers received. moreover, many dairy farms in southwestern wisconsin were well suited to convert based on their scale, biophysical features, and ongoing use of pasture as a significant source of forage for their cows [ ]. a ―help wanted: organic farmers‖ campaign had been launched in february of , and was supported actively by several commercial and non-profit organizations. organic valley, the nation‘s largest organic dairy cooperative, with its headquarters in the region, co-led the campaign as they were building on their leading role in marketing organic dairy products in the united states [ ]. at the time, pay prices to organic valley members had been stable and rising for almost a decade, providing on average about a % premium over conventional milk prices (for more detailed information on conventional and organic milk prices, please refer to figure in brock and barham, [ ]). later, in the subsequent deep recession of – when conventional dairy prices plummeted by almost percent, organic valley‘s pay prices declined by less than percent, thanks in part to the cooperative‘s choice to pursue private sector supply management as a price maintenance strategy [ , ]. thus, while net incomes of non-organic dairy farmers mostly turned strongly negative, organic dairy farmers‘ finances stayed mostly in the black. this outcome, ironically enough, hinged to a significant degree on the fact that organic dairy farmers were part of a segmented and more stable market (‗milk was not milk‘), within which consumer preferences helped to sustain large price sustainability , differences for reasons that may or may not have been based on their perceptions about real differences in organic milk‘s component composition. against that recent background, this article explores why it seemed that many farmers in the study region were not really considering the organic dairy adoption choice in the years preceding the recession ( – ). this period was a time when many farmers in southwestern wisconsin did choose to adopt ‗organic‘ and this growth led wisconsin to become the second largest organic dairy producing state in the country [ ]. however, there were far more farmers who seemed well set up to go organic but did not seem to be considering it seriously as a relevant management strategy. through in-depth, qualitative interviews, we explored the question of organic adoption within the broader framework of how farming system choice matched farmers‘ goals and aspirations in four domains: economic, ecological, social, and spiritual (this framework will be denoted by the greek work ‗oikonomia‘ which is related to the origins of economics. this oikonomia theme is explored more extensively in brock and barham, forthcoming [ ]). this perspective built on the work of other researchers who had found that farmers have diverse economic, social/spiritual and ecological values that motivate them to farm and choose distinctive management systems (e.g., [ , ]). the primary theme of this article is that the organic adoption choices of southwestern wisconsin dairy farmers appear to be shaped in many cases by bounded rationality heuristics rather than informed comparisons between options. this argument is illustrated here by the ―milk is milk‖ idea, but it is explored more deeply in the remainder of the article using a rich portrayal of distinctive bounded rationality approaches that were developed using the words of the farmers and our interpretation of them in relationship to their farm context. we interviewed a diverse set of small and moderate-scale farmers including conventional, amish, and even some managed graziers whose farm management practices overlapped notably with organic farms. as first explained by simon ( ), bounded rationality refers to situations when decision makers face choices in which they lack access to the full information about the problem in question, and/or the time and cognitive capacity to systematically weigh advantages and disadvantages [ ]. in contrast, full rationality assumes complete access to information and the time and human capacity to wade through and process the available information effectively [ ]. most adoption analyses assume fully informed rational decisions based on careful consideration of all the information available about a new technology. in this article, we define bounded rationality as a behavioral approach that recognizes that internal constraints (i.e., cognitive limitations and biases) and external constraints (i.e., uncertainty, limited time and resources) are both critical elements that help explain complex farm decision making, such as the decision to adopt organic practices. we view bounded rationality to be an especially appropriate framework for organic dairy adoption decisions due to the complexity associated with shifting all of the major farm management systems (crops, forages, and animal feeding and healthcare) and the ongoing challenge of accessing reliable information and support around organic dairy farming. in the process of examining the role of bounded rationality, we also consider how social networks can help farmers to overcome information constraints and learning challenges, or, in other instances reinforce biases and shape relatively information-poor decision-making processes. the organic adoption decision is described with an emphasis on farmer experiences and/or potential concerns with the shift, the informational constraints they perceived around this new approach to farming, and the role of social networks in their information acquisition and processing. sustainability , our exploration of organic adoption decisions is based on narratives that were constructed directly from the ideas, experiences, and decisions of farmers who were interviewed between august of and june of using semi-structured methods. almost all of the dairy farmer interviews in southwestern wisconsin were selected from a larger base of respondents from two random-sample surveys [ ]. nearly a third of the respondents were already farming organically, and most of the others had similar operations in terms of landscapes, scale of operation, and reliance on pastures for a significant share of the feedstock. thus, the objective differences across many of the farms were relatively small. in fact, only one or two of the conventional dairy farmers in the sample of interviewed were so far down the conventional path that the ‗sunk costs‘ of equipment, management strategies, and information investments were significant barriers to going organic. for the remainder of the sample, the farmer‘s explanations for not adopting organic were considered relative to their preferences and values, the informational barriers and concerns they identified, the social networks they described, and the biases they displayed. to the limited extent that previous researchers have attempted to develop empirical portrayals of bounded rationality behavior, it is generally done in the laboratory with highly controlled experiments involving games with payoffs under uncertain conditions [ , ]. by contrast, our discussion of bounded rationality behavior relies mostly on narratives, which for the sake of brevity are documented in an on-line appendix and selectively exploited in the text. in this regard, our bounded rationality research is unique because there are very few agricultural studies on adoption of any technology where bounded rationality issues play a prominent role in the analysis of farmer decisions. at a deeper epistemological level, we face the challenge that it is difficult–if not impossible–to know what farmers know and do not know about all of the farming system options and to apply that knowledge to their particular farming situation. thus, as researchers our interpretation of the on-farm interviews was also bounded by significant informational constraints. the best we can offer is to illustrate the bounded rationality behaviors that appear to guide the farmers‘ choices. perhaps this illustration will encourage other researchers to consider incorporating similar concerns into their analyses of farming system choices, particularly new ones that involve significant changes and uncertainties, where what farmers and researchers do, and can, know about the options is likely to be constrained by real informational limitations. the structure of the article is as follows. the next section develops our bounded rationality framework for studying the organic adoption decisions of dairy farmers, and relates it initially to the applied social science literature on local knowledge and networking with respect to farm management choices. our integrated bounded rationality framework centers on the concept of information satisficing. decision makers who exhibit minimal information satisficing will exhibit reasonable efforts to overcome objective constraints on time and information processing. those farmers who exhibit a lot of information satisficing will oversimplify a complex decision into one that may be characterized by such narrowly construed views as ‗milk is milk‘ or some other singular line of argument. section elaborates on our field methods and the article‘s use of the narrative form and semi-structured interviews as a way of developing the empirical analysis of bounded rationality behaviors or attitudes. section . explores the experiences of organic adopters. organic farmers confronted and overcame bounded rationality challenges to become early (or later) adopters. organic farmers also discussed their own observations on how non-adopters were ‗information satisficing‘ in a variety of ways sustainability , including using statements or biases that may have not been pertinent to the potential viability of the organic management system to guide their choices. the remainder of the empirical analysis (section . ) explores experiences of non-adopters and how their explanations for non-adopting relate to a variety of information gathering themes. because non- adopters often state multiple rationales that include their values and goals and a range of concerns with organic dairy farming and how it might work, this empirical analysis proves to be more illustrative than conclusive about the role of bounded rationality in any particular decision. it also features a comparison of two neighboring amish communities and how bounded rationality behaviors in one of them constrain the organic adoption opportunities of the other. overall, it is our view that the use of a framework of bounded rationality behaviors and values improves our capacity to understand adoption choices of a diverse array of farmers and the various communities that surround them. and, we hope that this approach can help to inform private and public strategies aimed at overcoming information constraints and biases especially related to emerging alternative farming systems. . bounded rationality in organic dairy adoption decisions this article‘s emphasis on bounded rationality and the social dimension of knowledge builds on previous household farm decision literature that discusses the role of information access (e.g., [ – ] and the adoption of alternative agricultural methods (e.g., [ , – ]. the social dimension of knowledge acquisition is highly interconnected with bounded rationality issues given the distinctive and complex nature of information required to manage organic systems (much of which is tacit or local knowledge) and the limits to acquiring information on organic farming [ ]. bell‘s work discusses how bounded rationality issues impact adoption decisions of sustainable agriculture without using the ‗bounded rationality‘ term explicitly. in his view, farmers have to negotiate a lot of uncertainty, and this may explain a reluctance to try different strategies. as bell states, ―farmers can‘t spend all day reading farming magazines, cruising the internet, talking to sales people and having coffee with locals at the café in town‖ when they are making decisions about their farm. social networks like practical farmers of iowa can help counter real information constraints [ ]. holistic organic dairy adoption research also emphasizes the unique factors involved with a comprehensive system shift into organic dairy management ([ , ]). they too combined data from interviews and surveys to understand the information gathering processes involved in the choices. lunneryd‘s work focuses particularly on information use in organic dairy adoption and his discussion refers to bounded rationality, but it is not a central theme in his work as it is here ([ ]). as mentioned above, our household decision-making framework integrates (economic, social, ecological, and spiritual value dimensions) and bounded rationality. we adopted a bounded rationality framework, because we perceived both actual information and knowledge transfer constraints associated with complex farm decisions, and it helped to make sense of the wide range of decision-making heuristics and approaches expressed in the farmer interviews. the bounded rationality framework emerged from an analysis of a substantial number of early interviews in the spirit of grounded theory [ ]. it helped to elucidate the divergent perspectives and arguments offered by farmers on common issues that seemed to reach beyond values into what they were willing or able to understand about the options facing them. since simon‘s seminal work ([ ]), the potential importance of bounded sustainability , rationality in new or highly uncertain situations has been a reoccurring theme but has generally not been included in behavioral models of technology adoption. in those cases when bounded rationality concerns are explicit, information satisficing strategies are often viewed as a principal way that decision-makers overcome real or perceived time limits, lack of capacity or information to address uncertainties, or other informational constraints [ , ]. in this article, we use ‗information satisficing‘ to describe the overarching bounded rationality behavior that guides decision-makers in uncertain situations with significant information gathering and processing costs. different types of information satisficing can lead to status quo bias where decision makers give automatic preference to their current situation/management practices since they are more concerned about their past investments then future possibilities [ , ]. we differentiate along a continuum of behaviors that involves distinctive approaches to both information gathering and criteria that guide those decisions especially non-adoption. ambiguity aversion, an example of a low level of bounded rationality, occurs when information that can help to address uncertainty is difficult to obtain [ ]. some agents may be averse to change as a result of the ambiguity or uncertainty [ ]. ambiguity-averse decision makers may not be able to estimate probabilities of different events based on their decisions, and this information barrier may discourage these decision makers from fully considering systematic management changes. for this particular article, ambiguity aversion will be used primarily to describe those farmers who explicitly state that there are elements of the organic systems where they have difficulty understanding the risks and returns in play, but that they were otherwise open to considering the options. anchoring is a heuristic method in which decision makers focus on a limited component of a problem which may or may not be highly relevant as a guide to comprehensive decision-making [ – ]. because decision makers tend to adjust their choices based on initial experiences or pieces of information under experimental conditions [ ], anchoring can also be the result of an incomplete computation [ ]. anchoring can also be viewed as ―one-reason decision making‖ where agents do not necessarily optimize over a range of aspirations that might otherwise be present in typical decisions [ ]. anchors are attractive as they are often time-saving and serve to simplify complex decisions. given that everyone operates with limited time and cognitive ability, it is no wonder that many of us use anchors as a way to navigate the complex world. examples of issues that conventional dairy farmers might anchor on include concrete sub-goals like crop prices and the cost of other organic inputs, rather than looking at the profitability of the whole farm system. organic farmers can also operate in a bounded rationality framework with regards to conventional farming. there are some obvious technologies that organic farmers will not explore like the use of rbst and many synthetic chemicals because they are not allowed within the organic system. therefore, organic farmers will information satisfice about these choices at a fairly high level, but may also satisfice on other more ‗conventional‘ practices that could crossover. anchoring can limit information gathering about the larger context and thwart fuller consideration of an important choice such as a farming system change. some anchors are so limiting that they do not seem to be of much relevance to the decision making process. this phenomenon will be denoted here as extremetization and this tends to arise when a decision maker anchors on a particular issue and makes sweeping generalizations out of extreme and potentially complex cases. a key example of extremetization in this study is the frequent use of the idea of ―milk is milk‖ as an anchor for deciding about organic dairy adoption. this sentiment explicitly sustainability , bypasses farm profitability comparisons, farmer satisfaction with the management system, and/or an open-minded view of factors shaping consumer preferences. by contrast, this sentiment seems to reflect views such as organic milk is part of a marketing scam, or it is a reaction against the idea of the inferiority of conventional milk and those who produce it. other examples of extremetization with regards to organic adoption could include focusing on stereotypes of organic farmers being hippies and/or old-fashioned. these stereotypes, like the idea of ―milk is milk‖, should have no bearing on whether the management system is a viable option for the farmers in question. we close this conceptual section by highlighting how social networks can influence access and exposure to information in ways that may encourage or discourage consideration of alternative management systems. if farmers are subject to a certain kind of bounded rationality, this may lead them to choose to interact with other farmers who think like them which may further perpetuate these bounded rationality constraints. farmer adoption decisions especially regarding complex systems such as organic dairy are embedded within networks of existing farmers [ , , – ]. social networks in sustainable agriculture knowledge transfer are often depicted in ways that encourage consideration of alternative adoption choices [ , , – ] as social networks can help farmers overcome the challenges of limited information and perceptions on organic dairy. social networks can also reinforce extreme types of information satisficing behavior which can also thwart self-critical reflection [ ]. socially specific satisficing behavior may be especially crucial to the adoption of organic systems given that a significant amount of information is transferred in social settings [ , , ]. our particular study area, southwest wisconsin, had a disproportionate share of the state‘s organic dairy farms. moreover, even within this area, clustered patterns of adoption were evident on the landscape, that seem to grow out of farmer-to-farmer interactions in the kickapoo valley region of this study [ ]. this pattern of clustering of organic dairy farmers was also evident in other geographic areas [ , ]. in this particular region, there is a growing proportion of dairy farms being owned by amish farmers (about % of the state‘s dairy) [ ]. amish farmers would seem to be particularly well situated to convert to organic considering certain aspects of their operations (small size, labor intensity, low cash inputs). as will be discussed below, the amish will be helpful to explore a particular type of information satisficing which will be denoted as principled satisficing [ ] where values can interact with bounded rationality issues to constrain information gathering by agents [ ]. . methods–field work, narratives, and empirical illustrations farm decisions related to organic dairy adoption were explored using approximately semi-structured farm interviews within a mile radius of organic valley. the majority of participants were originally survey respondents in a random statewide sample of conventional and grazing farmers in , a state-wide census of organic dairy farmers, and a census of amish dairy farmers who sell milk to an amish canned milk co-op in southwest wisconsin. the majority of interviews on farm management strategies were conducted between august of and june of , solely by the lead author. they, too, were mostly randomly selected, though in a stratified manner to be representative of the diversity of dairy farming strategies in the region [ ] [farmers were categorized by the farm management type indicated on the surveys (organic, conventional or managed grazing), though a number of the farmers sustainability , had since switched management systems between the time of survey completion and when the interview was conducted]. interviews lasted approximately one and a half hours but varied in length depending on time availability and conversation style of the farmer (introductory letters were sent to potential interviewees explaining the project with a list of the general topics that would be covered. the introductory letter was followed up with a phone call a week later. a return postcard was included in the letter for the amish who do not have phone access for the purposes of scheduling a time range for interviewing. interview participants completed a consent form). the number and types of farmer informant interviews were based primarily on the goal of gathering a diversity of farmer opinions on organic dairy adoption decisions. (in addition, key informants were interviewed to expand upon themes and offer further background and perspective at a more aggregate level than the individual. two of the grazing network leaders had grazing cow dairy operations, and all of the grazing network leaders had some kind of grazing livestock on their farm. six grazing network leaders were also interviewed toward the end of the interview process to insure a perspective from more intensive graziers. grazing networks are groups of farmers who exchange information about pasture management which are localized within regions. they often will organize pasture walks on each other‘s farms so the learning is very hands-on and social. generally farmers coordinate grazing networks but more recently, they also receive help from organizations like cooperative extension which will provide some infrastructure and administration so these groups can have mailings [ ]. a couple of organic valley staff, an organic educator, a few conservation agents, nonprofit representatives and amish cheese company representatives were also interviewed to gather additional perspectives on the challenges and opportunities in terms of the future of organic, grazing and amish dairy farming in the state). in total, farmers participated including organic farmers and graziers (non-organic) (the managed grazing farm strategy is a combination of a very old technique involving heavy reliance on pasture [ ] with more recent management knowledge aimed at managed intensive use of the pastures. technically, we define a farmer as a managed grazier based on their reliance on pastures as a source of feed during the grazing season and by their movement of cows to a new paddock about once a week. however, many of the graziers in this sample rotated their cows more than that the minimum of about once a week.), conventional farmers [all farmer participants (except the amish) consented to have their interviews taped. initial exploration revealed that it was culturally appropriate and respectful to not ask amish farmers to tape record their interviews. many other issues relating to farm decision making and viability were raised in the interviews themselves, and they were transcribed using open and closed coding. the two largest amish settlements in the kickapoo valley are hillsboro and cashton. roughly half of the amish sample was selected from each of the settlements] and amish farmers from two settlements. the interviews covered a number of topics relevant to farm decision making in the agro-ecological, economic, social, and religious/spiritual realms. please refer to the online supplementary appendix for an interview guide. the flexible approach helped to advance conversation about controversial issues around the organic adoption decision. bounded rationality emerged as a framework which enabled us to better understand the adoption choices because it offered a way to explain the diverse uses of distinct ideas and information sources. statements were coded and organized into bounded rationality categories that could indicate ambiguity aversion, anchoring, extremetizing, or some other form of sustainability , information satisficing such as principled satisficing. for example, when farmers were asked about the option of adopting organic farming, a primary response of ‗milk is milk‘, was categorized as extremetization. however, farmer statements were also considered within the broader construct of the larger interview. sometimes, these reactionary statements need to be tempered by the full context of their farm choices, and other times such statements appeared to be a good reflection of where farmers were with respect to decision making about organics. to help demonstrate our analysis of the role of bounded rationality behaviors in adoption decisions, we select and develop several farmer narratives with pseudonyms (we offer more narratives in the online supplementary appendix: four conventional, four graziers and six organic. more organic farmers were presented in part to capture the diversity of organic farmers especially related to the time of conversation). the narratives provide the reader with an account of the respondent‘s farm size and system, social networks, information sources, and decision making approach. these narratives are presented in an on-line appendix and offer a sense of the diversity of perspectives and experiences. additional farmer narratives, not explicitly mentioned in the next sections, are available there as well. these narratives provide a fuller context for our interpretation of the role and type of bounded rationality behavior than we can provide in the body of the article. the rest of this article draws closely on these narratives to construct arguments about the ways in which bounded rationality behaviors and social networks shape organic adoption decisions. we start with organic farmers because they describe well the uncertainties encountered, the role social networks played, and some of the precipitant factors that induced them to give organic a try despite the uncertainties and information constraints they faced. for the non-organic farmers, we organize the discussion of the narratives around information access and use themes which vary from limited information access to systematic review of information. for the amish in particular, it is more appropriate to discuss decision making on organic dairy adoption at the level of the community. for that discussion we draw on a comparison between two settlements, cashton and hillsboro, which will be briefly sketched below but developed in greater detail in another article [ ]. in all of the cases, it is important to know that our interviews occurred at a time when the market structure was wide open for those wanting to convert to organic. organic valley was recruiting actively and so were other organic processors. milk marketing was not constrained by contracts, and switching buyers was common among conventional growers. . results and discussion . . in the words of organic farmers–overcoming bounded rationality issues organic farmer narratives illustrate bounded rationality issues associated with adoption decisions on a continuum from ambiguity aversion to extremetization. this continuum may be based on the farmer‘s level of information satisficing and the context in which they confronted the adoption decision. given the dearth of scientific literature and well-developed markets on some aspects of organic dairying [ ] especially in its early years, farmers faced real uncertainty about certain relevant issues which address the question of discerning if organic dairy is a viable option for them. the issues were at the most basic level such as the availability of a market offering a better price for their milk or the accessibility of reliable supplies of organic grain and forage. however, in addition to these issues, sustainability , the organic farmers we interviewed also frequently discussed initial perceptions of organic that went beyond the basic features of the production, marketing, and management systems. concerns like ―milk is milk‖, or that some organic farmers cheat, arose for some of them as well, even though they do not seem as germane to their potential success in the organic management system. as we show below, social networks helped many of these farmers navigate the core management issues that directly affect the viability of an alternative farming system and other concerns which may not seem as pertinent. at the most basic level, these networks demonstrated that organic strategies could work on farms similar to those of the farmer in question, and provided a consistent source of information on how to manage the multiple changes required. for the less direct concerns (i.e., concerns not related directly to production, marketing, and management), it seemed to have helped that organic systems were presented in ways by other farmers that did not reinforce the negative stereotypes of organic farming that many farmers held initially. other factors which helped farmers transcend bounded rationality include their values, approach to learning, and an experimental nature. a context of economic or personal crisis at times served to reshape values and thus farmer information seeking strategies related to organics. organic farmers like matt drake described their previous financial situation when he was conventional as ―just trying to make ends meet,‖ whereas now they are in a much more comfortable situation. kerry martin emphasized the importance of a stable milk price for a beginning farmer when he stated ―there‘s no way that we could have gotten started financially with the conventional market...because organically you know pretty close to what you‘re going to get for your milk, and that conventional roller coaster would be pretty hard when you first start.‖ each farmer narrative depicts different bounded rationality issues and the ways they confronted these issues. the organic farmer narratives also include the bounded rationality issues that they view as pivotal to constraining the adoption of organics by conventional and grazing farmers. for organic farmers, the narratives are presented in a way that roughly depicts the organic story in wisconsin with the earliest adopters at one end of the spectrum in the s who had very little available information and supporting social networks to the other end with relatively recent adopters who were still transitioning at the time of the interview (in ). these narratives lead into section . ‘s discussion of how conventional and grazier farmers access and process information in ways that may lead them to view organic systems in less than a comprehensive manner. the smiths, who started practicing organic around , were among the very first organic dairy farmers in wisconsin. they farmed about years conventionally prior to practicing organic. codified information on alternative agriculture especially organic farming was scarce, and extant knowledge was transferred farmer-to-farmer. the first major organic milk marketing buyer, organic valley (ov) (at its inception, organic valley was referred to as coulee regional organic produce pool (cropp). cooperative, started in about eight years after the smiths began managing their farm organically. for the first eight years of organic production, the smiths did not have access to a distinct marketing channel for organic milk. there were a scattered number of farmers managing their vegetable farms organically and also crop and livestock farmers who practiced ―biological farming‖ as encouraged by midwest bioag, a company founded in . biological farming relies more on using minerals and rotations to manage the biology of the farm system rather than on the use of synthetic inputs, and it focuses especially on soil health (midwest bio ag, a biological agriculture input supplier, is an information source that appeals to different kinds of farmers). as such, many of its principles intersect sustainability , very closely with organic management practices, and provided a strong platform for farmers considering the organic option. managed grazing, another alternative farming practice which overlaps with organic, did not really take off until the mid- s [ ]. in this context, the smiths had to go further afield to find out about some of the main production practices that make organic dairy farming feasible. they reported making trips to germany to learn organic methods, as well as having a conversation with a farmer in minnesota who had been farming organically for years. mr. smith also had experience with organic farming because his father was old enough that he managed their farm in effect using organic methods like most farmers prior to the s. the smiths describe how their decision to go organic and eventually sell their milk to organic valley was primarily motivated by economic and ecological reasons. even though their decision well preceded the organic price premium, the smiths felt that they could make more money because organics was a low cost approach and that worked well. indeed, mr. smith observed that organic ―was the best thing for the cows, the best thing for the land, and it didn‘t matter that [we] didn‘t get any more for our milk.‖ local knowledge, learning by doing and tight social networks played a big role in the lives of early organic farmers as there was minimal support from academic researchers, extension agents, and industry professionals. the smiths were one of the early adopters who helped to build up this social information infrastructure. as mr. smith stated, ―it was a group of same-minded people…it was fun and exciting.‖ some of the neighbors got into organic farming after the smiths, and they had visitors come to their farm from long distances to learn about organic farming. as the smiths explained, ―we learned by our own mistakes and we learned from our neighbors... everybody was doing their own research.‖ they spoke of their ability to show that both significant yields and nutrient balance was possible with organic management. this may have helped others to address uncertainty about the feasibility of organics. overall, learning by doing played a big role in their experience. and, it was clear that ambiguity aversion did not hold them back from experimenting with something new and uncertain. to the contrary, mr. smith seemed to enjoy the challenge of discovery as he had an inquisitive, experimental nature, which was illustrated when his wife reported one of his favorite statements, that he‘d ―quit farming when [he] got the far end of the field to produce as much as the front-end of the field.‖ ben and dan crank‘s (two brothers) conversion to organic was primarily motivated by ben‘s health concerns that arose in when he was diagnosed with cancer. ―i wanted to get away from using the chemicals and the harsh fertilizers and stuff like that.‖ ben recalled that he sprayed harsh chemicals like -d without a shirt on when he was growing up, and felt that there was a connection between this pesticide exposure and his cancer. by , ben crank had converted fully to organic, only five years after organic valley formed, and then both brothers were shipping their milk to organic valley by the early s. ben crank recalled that there was not much information available about organics when he was getting started, but he also attended midwest bioag meetings, where he learned helpful techniques and principles. dan expressed how he experienced ambiguity aversion initially about organic as he wondered, ―what is organic, and why is it better?‖ dan crank did not go organic until well after ben. his brother, ben, was able to eventually persuade dan to adopt an organic system, perhaps by helping him to answer some fundamental questions with direct information. ben commented that having other farmers sustainability , demonstrate that organic farming works is ―what changes people‘s minds. if you can [get] them to try things, then they can see some results,‖ and social networks are instrumental for making that happen especially when your brother is leading the way. the cranks and other organic dairy farmers offered substantive insights on the ambiguity aversion issues that farmers face and how they navigate through these challenges in the organic adoption choice. according to interview results, after farmers adjusted to the organic system, they had comparable and/or somewhat lowered production but even with lowered production, the stability and increased pay prices of organic milk usually more than compensated for any production losses. [organic dairy research indicates that overall there are generally lower milk yields on organic dairy farms compared to conventional farms and similar yields compared to managed grazing farms with a few exceptions (e.g., [ – ]). for example, according to the wisconsin arms data, organic dairy yields ( , lbs per year) were % less than conventional farmers ( , lbs per year). it is possible that part of the higher milk yields on conventional farms are in part due to higher concentrated feeds on these farms as conventional farmers, the cranks had been strongly focused on herd productivity as a goal, and reduced production levels were a major concern for them during the transition period as they are frequently for other organic farmers. ―there was that pride with having that high producing herd; that‘s what‘s important.‖ the transition went well, and the cranks stated ―we are at , lb herd average...we have more livestock numbers than we ever had.‖ indeed, for the region, the cranks were at the upper end of both the organic and conventional productivity spectrum, and they may have achieved these high production levels because it remained a relatively high priority for them (many organic farmers are not as focused on the goal of a high producing herd as a goal which is independent of other endeavors like profitability. however, some farmers like the cranks are more interested in the goal of production than other organic farmers and may tailor their operation so they achieve that high production goal). in addition to the production concerns that the crank‘s discussed, conventional farmers are often concerned about organic herd health issues and at times they may express extreme views about the risks. the cranks stated that organic herd health management ―was the big scare with many of my friends that have converted to organic… [they proclaimed] ‗you can‘t do it. you can‘t control mastitis.‖ some of this concern is real, because once you have an outbreak it can be challenging. at the same time, one organic farmer stated ―the key to organics is prevention.‖ how rare and severe these events are remains poorly understood, but the cranks, like other organic farmers, felt that their herd health improved markedly after they went organic and they stopped pushing the high production conventional model. now that ben crank‘s herd health has improved, he exclaimed that he gets a kick out of the fact that when his friends ask him about mastitis, he can respond, ―i have virtually none. it is fun.‘‘ organic farmers acknowledged that overall herd health, milk production and feed provision can be significant challenges especially in the transition period and that organic may not be the best fit for certain farmers. organic feed provision can be a preventing factor for farmers who will struggle based on land access or other production constraints to be feed and/or forage self-sufficient. most organic farmers agreed that for farmers who do not raise their own feed, accessing affordable organic grain and forage could be a real challenge. organic valley has begun recently to help farmers source feed inputs and herd health consultation in order to alleviate concerns. this demonstrates that there can be sustainability , significant search costs for some farmers associated with organic management ([ ]) involving sourcing organic feeds and information on herd heath. organic farmers also pointed out how for the most part many conventional and grazing farmers do not consider the option deeply enough to even articulate specific concerns which would impact viability. as stated earlier, conventional farmers can have bounded rationality issues that go beyond these agronomic concerns. the crank brothers said that some of the conventional farmers were envious about the high organic pay prices, but ben always responded to this sentiment by stating, ―the only one stopping you from getting that price is yourself...it‘s a free world.‖ however, ben went onto to say, ―there are some that say, ‗i‘ll quit farming before i go organic.‘ they‘ll say that to your face,‖ and so clearly they not do not seem interested in learning from them about organics. this lack of serious consideration of the pros and cons of organic vs. the farmers‘ current management system may be reflected in the frequency of organic farmers who stated how issues such as social pressures may have a role in the decision making process. ben crank depicted the negative stigma against organic when he described how he overheard conventional farmers ―talking about so and so and they are organic. sometimes you feel like what a black person would have felt like in the s and s.‖ his wife said with a laugh, ―maybe not quite that bad.‖ but ben interjected, ―but they are kind of shunning ya‘…‖ the smiths also talked about how people laughed at them, and insinuated that they were going back in time by managing their farm organically. matt drake, who converted in the early s, said he had a lot of initial hesitations based on negative stereotypes associated with organics which depicted ―guys in the ponytails and the flip flops and the ripped shorts.‖ initially, he was not comfortable with socializing with organic people as he stated when ―i heard the o word, [then]…i joined the other table…‖ but, matt warmed up to the idea of organic farming by learning about biological farming from midwest bio ag. he said there is less baggage associated with ―biological farming‖ than with organic. for him, it was an ―an easy sell going from conventional to biological.‖ then once matt was biological for a while, he joined the organic table at the midwest bio ag meetings and ―got a wealth of information.‖ in fact, ―middle of the road‖ management systems, such as biological farming and rotational grazing appear to have played a key role in helping many dairy farmers in our interviews consider organic agriculture. the most recent converter to organic dairying in the narratives is the sert family. their account includes legitimate concerns with organic relative to their previous management system, but it also highlights the importance of issues which may not be as pertinent to potential viability. the influence of social networks can encourage or discourage organic dairy adoption. understandably, the serts were reluctant to go organic because they were deeply invested in a conventional dairy system with modern technology and at over cows were operating at a much larger size than most organic farm operations. moreover, they had no experience managing their cows on pasture. but eventually, they decided that they had had enough of the low and volatile prices and some of the other demands of the high production conventional management structure. as jerry stated, they decided that some of the conventional stuff ―wasn‘t so good. it [involves] shooting the cows all the time (injecting rbst and pushing high energy feeds)... i was shipping the most milk i ever shipped and i wasn‘t making any money…all you are doing is flooding the market and the farmers are not making any money with it.‖ indeed, the serts, like many recent organic adopters, report being mostly attracted to the organic price premium and stability as well as to the potential ecological benefits of organic farming. sustainability , although the serts had initial ambiguity concerns about production levels under organic management, they also expressed concerns that revolved around issues which were not relevant to potential viability that were more on the extremetization end of the bounded rationality spectrum. tom kept bringing up the organic possibility to his wife, and the conversation would often gravitate and get stuck on the idea if organic milk was really better than conventional milk. tom responded to his wife‘s concern by stating (paraphrased) ―let‘s have our organic friends over and ask them about it‖ to try and resolve the issue. tom‘s wife ―loved everything they had to say and she said the final question is, ‗is your milk better than ours?‘‖ when their organic friends said that organic milk was not any better, laura felt okay about adopting organic practices. this sentiment of ―milk is milk‖ may reflect the widespread idea among farmers that all milk is wholesome and the associated negative perceptions about marketing organic milk as superior. the serts were able to navigate their bounded rationality issues because of the social infrastructure and knowledge exchange that the smiths and other earlier farmers helped to create. many conventional farmers who exhibit extremetization do not get to the stage in reflective conversations that the serts did with organic farmers/friends. some farmers like the serts find that they have to be willing to move beyond their conventional-oriented networks and friends and create new bonds with other farmers who were practicing organic dairy [ ] partly because of the negative attitudes about organics in conventional circles. farming friends played a pivotal role in the serts‘ adoption decision process, and their encouragement was helpful in getting them through the challenging transition process especially given the intensity and size of their conventional operation. the serts report being thrilled to be participating in organic social networks and to be separated from the negativity about organics that is prevalent within many conventional farming networks. organic farmers consistently stated that other organic farmers as well as social networks created by organizations like organic valley and midwest bioag have been helpful for information exchange and encouragement. there were definitely ―keystone‖ organic farmers who had influenced a number of other farmers to go organic in the interviews. for example, one farmer, devin dooley, thought that organic valley focused on recruiting him to convert to organics because he was particularly well respected in the conventional community and his family had received production related awards in the past. if so, this is an example of how organic valley understood some of the direct challenges and bounded rationality issues associated with adoption decisions in a practical way and used the power of farmer networking. in fact, they rely on farmer members for a lot of their recruiting, as one organic valley representative stated if ―[farmers] see it themselves or hear it from other farmers‖ than ―that is when the light bulb goes on because they trust other dairy farmers more than any (other) industry person.‖ . . information satisficing among non-organic farmers in this section, we organize the discussion around information satisficing themes that conventional and grazing farmers expressed as well as the continuum of bounded rationality issues that the organic farmers discussed above. farmers who appeared ambiguity averse focused on the technical challenges with the conversion to organic dairy [ ] and were able to articulate those issues at some level. in contrast, farmers who exhibited bounded rationality at a more extreme level may not have considered sustainability , organic dairy enough to even articulate concrete reasons for not adopting. sometimes these reasons were based on narrowly defined views such as ―milk is milk‖. our presentation of the bounded rationality issues associated with non-adopters is not clearly divided into a clear linear progression along a spectrum, in part because some farmers exhibited multiple levels of bounded rationality at different times in the same interview. this made it difficult to categorize farmers. instead, we use the spectrum as a way to illustrate the different types of bounded rationality issues farmers expressed in their response to the organic dairy adoption question. we highlight four information-access and use themes that non-organic farmers exhibited, and each of them reflects multiple levels of information satisficing. the first theme illustrates how a community can put explicit limits on its members‘ information access and processing because of how they combine commonly held values and bounded rationality issues. by comparing two amish communities, we see meaningful differences in how information satisficing behavior can play out. next, a common way that people information satisfice is by getting information for general background purposes only. this can be demonstrated in a somewhat open way where the decision maker has neutral views of other production practices, but does not explore these options enough to learn very much about them. information satisficing can also be expressed at a more extreme level where negative ideas around issues that are not relevant to potential viability of the organic system persist because farmers are not acquiring much outside information. a third information theme is illustrated by farmers who may actually get a lot of information, but their values lead them to channel their efforts towards a clear and focused end goal like high production using conventional methods. this singular focus can in turn cause farmers to dismiss information about organics especially if they are satisfied with their current management system. at some level, all decision makers exhibited this kind of behavior when it comes to paths not taken. we cannot know everything about all of the alternatives. however, at an extreme level, this lack of information gathering could be typified as ―anchoring‖ or extremetizing especially when farmers focus almost exclusively on issues which are not relevant to the potential viability of the alternative management systems and do not provide meaningful contrasts with their current approach. finally, some farmers processed information in a systems-based way but have distinct reasons why they choose not to adopt organic dairy. these farmers were not information satisficing until after they appeared to have acquired a lot of information about the system, which in our view suggested that bounded rationality was at most secondary in their decision not to adopt organic methods. . . . explicit and implicit information limits based on community values social networks can cause decision-makers to place explicit and/or implicit limits on information access and use, and the amish provide a good example of this phenomenon in the organic dairy adoption context. the combination of strict traditional values and bounded rationality frameworks helps to explain how the amish constrain gathering and processing of information related to organics at the community level which then constrains individual farmers as members of that community. it is worth reiterating the assertion that in many ways organic farming seemed as if it would be an especially good fit for the amish, because they operate smaller farms which use pasture as a source of feed. they also prefer labor intensive strategies over technology intensive ones because working together sustainability , on the farm is viewed as pivotal to actualizing family and community values on a day-in-day-out basis. however, most amish farmers in this area did not practice organic dairy, and in percentage terms there were only a few more organic farmers proportionally captured in this sample than there were amongst the non-amish in the same region. a slower than expected adoption rate of organic amongst the amish may be explained by principled satisficing. principled satisficing is a bounded rationality behavior under which decision makers may acquire and use less information than they would under the usual assumptions of rational utility maximization for distinct value-based reasons [ , ]. in this context, decision makers, ―satisfice‖ or ―optimize‖ with the information they know and understand [ , ], and may even actively resist knowledge acquisition. (satisficing may in part explain why decision-makers stop their thinking or information gathering early on, based on an attitude that continued searching ―seems like a waste of time‖[ ]) in the case of the amish, bi-annually they consider limited reforms to the ordnung (ordinance), or church rules, which are an expression of the values and guiding principles of each amish community. these rules tend to change very slowly over the years [ ] as ―changes are clearly not encouraged, vocalized or rewarded in amish society‖ [ ]. as part of the ordnung, intellectual pursuits are not encouraged as they could lead one to be become worldly and proud. hand labor is viewed as ideal christian work, and that is reflected in the explicit limit on formal schooling beyond the th grade [ ]. likewise, amish community members are also generally not encouraged to attend large conferences and workshops with outsiders in order to avoid worldly entanglements, which in turn would make it difficult to learn about organics through social networks. as a result, efforts to gather information about alternative farming practices are generally not encouraged, and may be explicitly or implicitly discouraged. this asocial constraint functions as a form of principled satisficing, especially for a change as information intensive as a full system switch to organic dairying. recent management decisions of an amish cooperative, known as old country cheese co-op, also illustrated how principled satisficing at an organizational level constrained individual amish farmers in cashton and hillsboro from making a fully informed decision around organic dairy. old country cheese was started in by a group of cashton amish who were committed because of adherence to the local ordnung to making a market for canned milk, i.e., non-refrigerated milk collected in cans from amish farms (some other settlements in eastern states adopted bulk tanks as early as [ ]). in fact, in the study region, old country cheese was the only marketing outlet for canned milk as all other milk buyers in the area required farmers to acquire a bulk tank. as a result, at the time of the survey, old country cheese had monopsony control over major decisions around establishing and maintaining an organic line both at the community and the individual level, and they had not kept a consistent commitment to the organic line in previous years. this lack of consistency was partly due to the negative attitudes about organic among some cashton amish elders who had influence on the board. organic dairy was viewed as a potential threat to amish values among some leaders in the cashton amish church. based on the notion the amish have about the critical importance of unity or ―being one body‖ in a scriptural sense, these elders worried that some members of the community would receive higher pay prices for organic milk than the non-adopters. this differential could then translate into a sense that not all community members were being treated equally. in fact, some of the cashton elders and farmers stated the common refrain that ―milk is milk‖, but often it seemed based on an intention to avoid favoring one co-op/community member over another. value-based hesitations sustainability , specific to allowing a new practice which might divide the community seemed to underlie principled satisficing type of behavior with respect to organic dairy. in general, the hillsboro community held more positive views about organic and as such show that the interplay between amish values and bounded rationality depended on the context of the community. in hillsboro, some of the early settlers to the community wanted to avoid pesticides, and they had a positive view overall of organic from their previous farming experiences prior to migrating to wisconsin. due to the close social networks within their community, organic agriculture spread more quickly than it might have in the non-amish world. moreover, in the hillsboro community, local norms were not as strong as they were in cashton in dissuading farmers from acquiring information related to organics and other alternative practices like managed grazing. yet, the hillsboro farmers understandably expressed ambiguity concerns with respect to the benefits of going organic when they noted that the local cooperative, the cashton-controlled one, had not been totally committed to maintaining that product line. given the importance of values and social networks in the organic adoption process for many other non-amish farmers described in section . , the contrasting experiences of these two amish communities make it clear that influences at the community level can also be important in shaping the context for adoption decisions. principled satisficing may also apply to the broader landscape though perhaps less starkly than within the confines of these two amish settlements [ ]. for example, the emphasis on a high producing herd could be viewed as a value in a sense at the community and individual level which influenced information satisficing about an alternative production system like organic. . . . open to general background information but little more many non-adopters seemed to rely mostly on an information-gathering strategy where they read and interacted with other farmers for general background, but then did not seriously consider alternative management-systems. this particular kind of information satisficing could lead to more or less extreme status quo bounded rationality behaviors depending on the personality and the context of the situation. the theme of getting information mainly for general background is illustrated with two narratives, those of randy rod and andy son. part of the reason for the frequent expression of this information access theme is that farmers tended to have values which were similar to the amish in that they value working with their hands and getting things ―done‖ on their farm more than getting information about different possibilities. consider first randy rod, who was relatively open minded, gathered information widely, but did not openly wrestle with systems level changes. as a mid-sized conventional producer (about cows), randy farmed pretty much like his dad but did experiment with reducing his pesticide and fertilizer usage because this saved him money and also had environmental benefits. similar to several other conventional farmers, he would review alternative strategies somewhat objectively at a distance, but choose not to immerse himself in details. randy rod didn‘t express any kind of strong negative attitudes against organic but also could not explain in very much detail why it would not work for him. (for example, randy rod doesn‘t grow his own feed which could be an extra challenge for organic but he didn‘t get into that level of detail in the interview). when asked if he had thought about other sustainability , strategies like going organic or using pastures more intensively or getting bigger, he stated that his current approach: “works so you kind of stick with it…and the thing is that i would hate to upset the apple cart, you go with some other strategy and you say hey this ain’t working; now you’ve lost time and money fiddling with it, and to go back is going be costly too… if it’s not broke, don’t fix it.” the apple cart for randy was in a sense the status quo. this statement reflects a common and sensible attitude among many farmers that what is right for one person may not be right for another. farmers like randy rod stated that they were doing fine with where they were at so they did not see any need for change. in his view, as long as different strategies worked for different farmers, he was neutral and open minded about all of the strategies; he just felt that his current strategy worked for him so why bother to change. in all fairness, it was hard to dispute his claim, considering that he did not work off of the farm and neither did his wife. they appeared to have a farming strategy that kept them economically afloat in challenging times. andy son illustrates a more extreme version of limited information gathering, in which he did not generally consider outside information and expressed feelings of social isolation. he had only cows and used minimal modern technology. he called his farming system ‗high tech amish‘ as a way of slighting his relatively small-scale conventional operation. andy seemed to view himself as a victim of changing milk and fuel prices, environmental regulations and isolated social conditions, and it was clear that he did not get a lot of outside information. he seemed to take a very passive role in gathering and assessing the information. he was frustrated with his situation and expressed a victim mentality. however, it didn‘t seem that he had actively considered other options like organic that were garnering higher and steadier prices for operators whose farms were much like his in size, structure, and basic management practices prior to their organic conversion. andy did not exude randy‘s attitude that conventional and organic were both fine options that fit the needs of different kinds of producers. instead, he had more distinctive opinions about both organic and larger conventional production systems. on the one hand, andy son‘s statements seemed to reflect jealously that organic farmers got a higher pay price, as he felt like organic farmers were getting paid more to do the same thing he was, in other words a ―milk is milk‖ sentiment. along these same lines, he was skeptical that organic farmers were complying with the organic rules. on the other end of the continuum, he also was disdainful of larger conventional systems who ―spray, spray, spray‖. andy son seemed to be representative of the economic and quality of life dissatisfaction widely expressed by many small-scale conventional farmers at that time in the aforementioned large-scale survey [ ]. he lived with his parents, and his mother worked outside the home, perhaps providing the majority of the family earnings. it seems as if he felt isolated amongst larger scale conventional farmers, and he felt that he was the last young farmer amongst an aging and disappearing farm population. . . . using information selectively and potentially anchoring on narrow goals some farmers avidly pursue information to guide their farm management decision making, but seemed highly focused on narrow objectives. larry wagner, who milked over cows in a conventional confinement system, exemplifies this type. his primary focus was on increasing herd productivity sustainability , which is a commonly understood way that dairy farmers deal with price-cost pressures to improve returns per cow. in turn, they may get as much information as possible to support that goal. in that regard, larry was very progressive in terms of getting information tied to extension and the university, and was deeply embedded in a variety of networks that focused on improving herd productivity within conventional confinement systems. larry focused on production over other dimensions of the farm management system. larry, at times, cited purely structural issues as to why it would not make sense for him to consider organic because he already had invested so much in a high input system. however, he also discussed issues which did not seem pertinent to the potential viability of an organic system. these issues go beyond structural reasons and could be categorized more as anchoring and extremetization. larry wagner said that organic ―milk isn‘t any better‖ than conventional milk. he stated there may be an issue with chemicals on fresh produce but with ―the way a cow purifies the milk, i don‘t think that it‘s any better. what really gets me is [the] organic producer will come out and say in the paper that their product is so much better. there‘s no proof to that.‖ so larry seemed to imply that organic producers and consumers may be overly focused on the risks of pesticides. larry expressed similar sentiments about cheating as andy son as he thought a number of organic farmers actually use synthetic chemicals and hormones not allowed within organic standards; so he concluded, ―i‘m not going to lie or cheat.‖ the point is that ideas of all milk being the same (―milk is milk‖) and organic farmers cheating are arguably not relevant to if organic farming is the best fit for a conventional farmer considering profitability and other objective criteria. nonetheless, larry seemed content with his current management system. it is arguably true that his focus on productivity was highly profitable, and as such provided support for this particular case. in this way, larry typifies evidence that many larger scale confinement farmers are quite content with their farming and quality of life situation [ ]. the same conclusion might not be true for other producers who focus on narrow aspects of their farming approach. for example, the serts, who previously operated with a high herd productivity focus, decided that this approach was not the best for them and so adopted an organic system. . . . acquiring information systematically but still not going organic some farmers appeared to have examined the available information on alternative systems but had chosen not to convert to organic. these farmers gave specific ambiguity aversion reasons related to herd health, organic feed access, and/or other production-related reasons. many of the farmers who exhibited this systems based approach were managed graziers, and in part they tended to know more about organics because their emphasis on pastures creates overlapping social networks with organic producers. for example, one managed grazier, paul flecker, liked to ―keep experimenting‖ with different farm management techniques. he went to a lot of pasture walks and worked with extension folks closely. paul had the ability to encounter obstacles and gather information with respect to alternative management types, and he gave reasoned answers related to conversion challenges for his decision not to adopt organic. one of his main reasons for not going organic was that he was not going to be in dairy long enough to make the transition period worth it. he was hesitant to convert given the short time horizon he envisioned for himself as a dairy farmer. he acknowledged that with organic he sustainability , would have to think harder about weed and herd management. as paul said, ―it can also be so easy to let the weeds take over. i don‘t disparage the people that do it and do it successfully. they‘ve got it going on.‖ he also stated that organic dairy ―presents some challenges as far as herd health...those challenges can be overcome because obviously there are a lot of people doing organic dairy. but i just haven‘t.‖ overall, paul was very pleased with his managed grazing system and seemed to be achieving his goals. paul‘s information satisficing with respect to organics was at a much more advanced level than many other farmers as he seemed to interact readily with organic farmers and commended them for their skills in managing weeds and herd health. all decision-makers‘ information satisfice at some level given the constraints on time and human ability to process information. one could argue that paul had good reason to information satisfice at a certain level about organics given his short expected tenure as a dairy farmer. other informed graziers also exhibited ambiguity aversion about organic feed sourcing as it could be challenging to secure reliable organic feed supplies at good prices. for some of the managed graziers, they had preferences for pasture management over crop production and thus purchased all of their own grains rather than raising it themselves. in that sense, they were especially vulnerable to challenges accessing affordable organic feeds if they were to convert to organic production. a few intensive grazing farmers also had some sophisticated critiques with regards to managing invasive species without some minimal use of herbicides which would not be acceptable under organic standards. one of the managed grazing farmers in the sample changed his mind about the relevant importance of these constraints as he decided to adopt organic practices a couple of years after the interview illustrating that these uncertainties were not necessarily permanent constraints on adoption especially when bounded rationality behaviors are less extreme or almost non-existent. . conclusions bounded rationality appears to play a significant role in shaping adoption decisions around organic dairy farming in wisconsin. it is not always a binding constraint, and it may decline as information and knowledge spread, but it can be a major barrier for farmers to overcome in the early stages of a successful alternative management system or technology. bounded rationality can also be an ongoing constraint if farmers information satisfice in ways that might prevent them from taking viable alternatives seriously. organic farmers shared inspiring adoption histories and thought that organic dairy was a viable option that other similar farmers should consider. the historical evolution of their adoption experiences also shows how information challenges diminished as supporting social networks were built and information exchange opportunities expanded. overall, organic dairy farmers faced many of the same information constraints and social pressures as conventional farmers yet they overcame these issues through a range of motivations to consider organic dairy and their willingness to seek out information and social networks around organic. strong economic, ecological and health related motivations inspired the farmers to consider other alternatives. the preferences and bounded rationality dynamics of an individual‘s adoption decision may shift through time. in addition to the farmers‘ existing farm set up, bounded rationality and the farmer‘s personality and values/preferences, their context including personal crises and social factors were also sustainability , important in determining adoption decisions. as organic pioneers, the smiths faced the most information challenges, with no real price incentive for years. in contrast, as we saw in the case of the serts, who were embedded in a conventional confinement model, it took a significant price premium combined with their challenging economic situation as well as an encouraging group of friends to induce them to adopt organics. ben crank decided to stop using chemicals after he was diagnosed with cancer, and his brother followed once he saw that the organic system worked for his brother. matt, like devin and the cranks, had a shift in his preferences towards organics related to economics and experiencing the benefits of biological farming. some organic farmers expressed that they also originally had hesitations to convert to organic which involved anchoring and extremetization just like some of the conventional farmers. prior to the cancer diagnoses, the cranks were on a high production mindset and thus were probably not seriously considering alternatives like organic. the serts exhibited anchoring on all milk being the same as a reason to not convert to organic. social networks were particularly important for enabling shifts in preferences and bounded rationality thinking or in confirming current strategies. a number of the organic farmers highlighted the exchange of information and camaraderie that they have experienced in their farm networks. knowledgeable friends and relatives can determine how open a farmer may be to alternative management systems. the sert‘s friends convinced them to go organic and they were excited and joked about the ―organic clique‖ of farmers who helped each other out and exchanged information about farming. there had also been some peer pressure inhibiting the spread of organics which could serve as a challenge for some farmers to consider these systems. most conventional farmers in this area appeared more likely to make changes at the margin regarding management practices rather than to consider information about other alternatives which may be appropriate for them. they may be getting information and talking to other farmers, but it was mainly for general background instead of as a means to challenge the way they already do things as demonstrated by andy son and randy rod. there seemed to be an attitude amongst farmers that they just need to make sure the basic farm management demands are covered so they do not need to keep themselves open to different farm management possibilities. this approach was not necessarily reflective of their financial state and contentment with their quality of life as seen with andy son, as he was not satisfied with his current situation. as discussed above, information satisficing can dissuade farmers from fully considering organic dairy or any new system as a viable option. andy son revealed that he hadn‘t thought much about the details of organic beyond the possibility of cheating and that organic milk was not really any different and thus it would seem as if he had anchored/extremetized on these issues. neither of these issues were factors which would have much impact on andy‘s success as an organic dairy farmer. larry had acquired and utilized a lot of information but this was channeled mainly towards a high productivity goal which kept him invested in a high-input conventional form of production. in that sense, the ―fixation on productivity‖ evoked by larry wagner may be a form of anchoring that dissuades conventional operators from analyzing the benefits of organic management at a systems level. and it was also clear that he held strong negative emotions about organic that cannot be fully explained by this logic. like andy, larry also exhibited extremetization about some farmers being cheaters and he also was focused on how all ―milk is milk‖. sustainability , farmers who were more likely to access and consider information in a systems-based way were less likely to have more extreme bounded rationality issues with regards to systems like organic. paul flecker, and other managed graziers, seemed committed and proactive about information seeking on a broader range of alternatives than larry was with his production focus. farmers like, paul, had less reservations about organic, and their hesitations were more closely tied to ambiguity aversion. also, he was concerned about issues like the three year transition period which many conventional farmers were not even aware of because they had not acquired enough information on organics to know about it. farmers who are ambiguity averse may be focused on the technical challenges with organics and necessary changes with conversion [ ]. paul and other managed graziers also had preferences for managing pasture and so may not have wanted to venture into crop production. some organic farmers say that on-farm crop production is necessary given the challenges of accessing affordable organic grains and forages. in summary, the complexities of information access and processing related to organic adoption suggests the need for a multifaceted approach to understanding the ways in which bounded rationality contributes to farm system decisions. this article only highlights a few bounded rationality issues and information access themes; there are others that could be explored around these ideas. a bounded rationality framework mixed with values and preferences could be used to gain insights into complex farm decision making as was briefly depicted with the amish communities case study and is discussed further in brock and barham, forthcoming ([ ]). values can also evolve over time as we saw in dramatic ways in some of the organic farmer stories like ben crank‘s diagnosis of cancer. one could explore what common values farmers hold, and how these values may vary amongst and within farm management systems. these frameworks could be refined and honed for different complex systems based decisions outside of farming. finally, we hope that this view of farm decision making can help policy makers and educators respect and begin to understand the complex nature of adoption decisions and the role that information use themes play in decision making especially for emerging, alternative farming systems. information may need to be tailored to the specific needs of different communities and types of decision-makers. for example, perhaps fuller discussions are needed around the ―milk is milk‖ idea so that farmers might think more reflectively about what lies behind these extreme statements and have a more open and engaging dialogue similar to the one reported between the serts and their organic farmer friends. those types of discussions would allow farmers to more carefully consider new options, such as organic dairy farming, with a deeper view of the pros and cons. and in so doing, remain open to the prospect of discovering ways to improve their farm performance and achieve family goals in the realms that matter most to them. acknowledgments we are grateful to the dairy farmers who provided invaluable information on decision making and anthony austin who spent many hours assisting in transcription of data. some fraction of the financial support for this research was provided by sustainable agriculture research & education (a division of the usda) and through a grazing lands conservation initiative (glci) grant and by an nsf grant on spatial patterns of technology adoption. sustainability , institutional review board/farmer anonymity this research was approved by the institutional review board of the university of wisconsin-madison. as described in the methods section, the farmers‘ identities are fully protected. if the farmer was described in a narrative, they were given a pseudonym. conflicts of interest the authors declare no conflict of interest. references . barham, b.; brock, c.; foltz, j. organic dairy farms in wisconsin: prosperous, modern, and expansive; ; program on agricultural technology studies, university of wisconsin – madison: madison, wi, usa, . . brock, c.; barham, b., farm structural change of a different kind: alternative dairy farms in wisconsin - graziers, organic and amish. renew. agric. food syst. , , – . . help wanted: organic farmers. in farm progress. available online: http://farmprogress.com/ story-help-wanted-organic-farmers-campaign-introduced- - (accessed december ). . barham, b. price stability in an era of roller-coaster rides; department of agricultural and 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hopkins press: baltimore, my, usa, . . hostetler, j. amish society, th ed.; john hopkins press: baltimore, md, usa, . . kraybill, d. the riddle of amish culture; john hopkins press: baltimore, md, usa, . . kraybill, d.; olshan, m. the amish struggle with modernity; university press of new england: hanover, nh, usa, ; p. . . lloyd, s.; bell, m.; stevenson, s. milking more than profit: life satisfaction on wisconsin dairy farms; center for integrated agricultural systems, university of wisconsin-madison: madison, wisconsin, usa, . © by the authors; licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution license (http://creativecommons.org/licenses/by/ . /). wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . 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sabra, toni i. pollin, sandra h. ott, jian wang, michael j. garant, jeffrey r. o’connell, braxton d. mitchell, and alan r. shuldiner , calsequestrin (casq) is involved in intracellular stor- age and release of calcium, a process that has been shown to mediate glucose transport in muscle. its gene, casq , is encoded on chromosome q , a region that has been linked to type diabetes in the amish and several other populations. we screened all exons, exon-intron junctions, and the proximal regulatory re- gion of casq for mutations. we detected four novel single nucleotide polymorphisms (snps) (� c t, � delg, � insg, and c t). ten informative snps within casq were genotyped in amish subjects with type diabetes (n � ), impaired glucose toler- ance (n � ), and normal glucose tolerance (n � ). rs and rs in introns and were signif- icantly associated with type diabetes (p � . and . , respectively); three other snps showed borderline evidence for association to type diabetes (p � . – . ). furthermore, in nondiabetic subjects (n � ), both rs and rs were significantly associ- ated with glucose area under the curve during an oral glucose tolerance test (p � . and . , respec- tively). haplotype analysis suggested that no haplotype could explain these associations better than rs . these findings, coupled with similar findings in utah caucasians, suggest that sequence variation in casq may influence risk of type diabetes. diabetes : – , t ype diabetes is a common heterogeneous dis- order in which genetic and nongenetic factors (i.e., excess caloric intake and physical inactiv- ity) potently influence risk ( , ). genome-wide scans have led to the chromosomal localization of suscep- tibility loci for type diabetes, with the most promising and replicated findings on chromosomes q -q ( – ), q ( ), q ( – ), and chromosome ( – ). consistent with current thinking, multiple linked regions suggest that several susceptibility genes contribute to the development of type diabetes ( ). to date, positional cloning has identified variants in calpain (capn ) on chromosome q ( ) and hepatocyte nuclear factor � (hnf a) on chromosome q -q . ( , ) as type diabetes susceptibility genes in some populations. we previously reported evidence for linkage of type diabetes and impaired glucose homeostasis to a locus on chromosome q -q (logarithm of odds . , p � . ) in the old order amish ( ). linkage of this region on chromosome to type diabetes has been reported in at least five other populations, including pima indians ( ), utah caucasians ( ), french caucasians ( ), u.k. cauca- sians ( ), and chinese ( ); linkages of diabetes-related traits to this region have also been reported ( , ). such robust replication provides strong evidence for a shared type diabetes susceptibility locus in this chromosomal interval. however, this region contains at least genes that include several excellent positional candidate genes, introducing the possibility that more than one gene variant in the region may influence diabetes risk. a number of positional candidate genes within this region have been studied to date, with variation in potassium inwardly rectifying channel, subfamily j, members and (kcnj / ) ( , ), liver-specific pyruvate kinase (pklr) ( ), c-reactive protein (crp) ( ), lamin a/c (lmna) ( ), and omentin (itln) ( ) showing modest evidence for association with type diabetes or related traits. to further refine this interval with the aim of position- ally cloning the type diabetes gene(s) in this region, we embarked upon linkage disequilibrium (ld) mapping by performing genotype and association analysis of single nucleotide polymorphisms (snps) in amish type dia- betic cases and nondiabetic control subjects. to date, � snps over the . -mb region from . to mb (ncbi [national cancer for biotechnology] build . ) from the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland; the division of endocri- nology, second affiliated hospital, sun yat-sen university, guangzhou, china; and the geriatric research and education clinical center, veterans admin- istration medical center, baltimore, maryland. address correspondence and reprint requests to alan r. shuldiner, md, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood st., room , baltimore, md . e-mail: ashuldin@medicine.umaryland.edu. received for publication may and accepted in revised form september . additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. afds, amish family diabetes study; auc, area under the curve; casq, calsequestrin; homa, homeostasis model assessment; homa-ir, homa of insulin resistance; igt, impaired glucose tolerance; ld, linkage disequilib- rium; ngt, normal glucose tolerance; ogtt, oral glucose tolerance test; snp, single nucleotide polymorphism. © by the american diabetes association. diabetes, vol. , december have been genotyped. multiple snps associated with type diabetes were identified within the interval between � and . mb, including rs , located within the calsequestrin (casq ) gene. casq is a calcium storage protein localized in the sarcoplasmic reticulum of fast- twitch skeletal muscle cells. it is a constituent of the protein backbone of the luminal ca � reservoir and plays a pivotal role in calcium flux through regulation of calcium channel activity and interaction with other proteins present in the sarcoplasmic reticulum ( ). calcium re- lease from the sarcoplasmic reticulum into the cytosol has been shown to regulate glut expression ( ) and glu- cose transport in muscle ( ). furthermore, howarth et al. ( ) reported that casq expression and calcium binding is increased in streptozotocin-induced diabetic rat skeletal muscle. we hypothesized that variation in casq may affect insulin action on glucose uptake and glycogen synthesis in skeletal muscle and thus type diabetes susceptibility. to examine this positional candidate gene further, we screened casq for mutations and deter- mined whether the observed sequence variation was asso- ciated with type diabetes and related traits in the old order amish. research design and methods the old order amish are a genetically well-defined caucasian founder population who live in a relatively homogeneous environment and have large sibships. nearly all of these individuals share common ancestors; � % of the chromosomal material of the amish community of lancaster county (now numbering � , individuals) appears to have descended from � founders who emigrated to this area in the mid s ( – generations) ( , ). the amish family diabetes study (afds) was begun in to identify susceptibility genes for type diabetes and related traits. probands with diabetes onset between and years and all willing first- and second-degree family members � years of age were invited to participate. dna from non–first-degree relatives with type diabetes ( alleles) from families providing evidence for linkage of diabetes to chromosome q -q was used for sequence analysis of casq . to define ld and haplotype structure of casq , all snps were genotyped in a limited set of amish subjects chosen to be relatively unrelated (non–first-degree relatives) to each other. next, to examine the relationship between haplotype-tagging polymor- phisms in casq and diabetes, we genotyped dna from subjects with type diabetes (n � ), impaired glucose tolerance (igt) (n � ), and normal glucose tolerance (ngt) (n � ) for case-control association analysis. ngt subjects included in this analysis were required to be at least years of age at the time of examination. for association analysis of quantitative traits (e.g., glucose and insulin), a larger set of nondiabetic subjects (including the ngt and igt subjects described above) was studied. informed consent was obtained from all afds participants, and the study protocol was approved by the institutional review board at the university of maryland school of medicine. trait measurements. details of methods for phenotyping of afds subjects have been previously described ( , ). briefly, body height and weight were measured in all subjects using standard protocols. after a -h overnight fast, a -g oral glucose tolerance test (ogtt) was administered, with venous blood samples obtained at , , , , , , and min for glucose and insulin measurements. the total glucose and insulin areas under the curve (aucs) during the -h ogtt were calculated using the trapezoid method. insulin resistance was estimated from fasting blood glucose and insulin levels with the homeostasis model assessment (homa) (homa of insulin resistance [homa-ir] � [fasting insulin {�u/ml} � fasting glucose {mmol/l}]/ . ). diabetes was defined by fasting plasma glucose level (� mmol/l), -h ogtt plasma glucose level (� . mmol/l), random plasma glucose level (� . mmol/l), the use of insulin or oral glucose-lowering agents, or a diagnosis of diabetes, with age of onset between and years, documented by a physician. igt was diagnosed based on ogtt plasma glucose levels ( -h ogtt plasma glucose level between . and . mmol/l). ngt was defined based on fasting plasma glucose level (� . mmol/l) and -h ogtt plasma glucose level (� . mmol/l). mutation screening. primers were designed to specifically amplify casq exons, splice junctions, and , bp of the 	 flanking sequence using primer t a b l e f re qu en ci es o f c a s q s n p s in su b je ct s w it h ty p e d ia b et es , ig t , an d n g t m in o r al le le fr eq u en cy t yp e d ia b et es vs . n g t * t yp e d ia b et es � ig t vs . n g t * s n p n am e l o ca ti o n p o si ti o n † m aj o r/ m in o r al el e t yp e d ia b et es (n � ) ig t (n � ) n g t (n � ) o r ( % c i) p o r ( % c i) p c as qs n p ’ fl an k c /t . . . . ( . – . ) . . ( . – . ) . r s ’ fl an k c /t . . . . ( . – . ) . . ( . – . ) . r s in tr o n � /g gc at tc ag at ag gc ct . . . . ( . – . ) . . ( . – . ) . r s in tr o n � a /g . . . . ( . – . ) . . ( . – . ) . r s in tr o n � g /a . . . . ( . – . ) . . ( . – . ) . r s in tr o n � g /c . . . . ( . – . ) . . ( . – . ) . c as qs n p e x o n � c /t . . . . ( . – . ) . . ( . – . ) . r s in tr o n � c /a . . . . ( . – . ) . . ( . – . ) . r s in tr o n � g /a . . . . ( . – . ) . . ( . – . ) . r s in tr o n � g /a . . . . ( . – . ) . . ( . – . ) . *p va lu es ar e b as ed o n ge n o ty p e fr eq u en ci es an d o r s re fl ec t th e o d d s o f d is ea se as so ci at ed w it h h av in g tw o co p ie s o f th e m in o r al le le ve rs u s th e o d d s o f d is ea se as so ci at ed w it h h av in g tw o co p ie s o f th e m aj o r al le le . f o r rs an d rs , th e o r s fo r ty p e d ia b et es o f th e m aj o r al le le s ar e . ( % c i . – . ) an d . ( . – . ), re sp ec ti ve ly . p va lu es � . ar e sh o w n in b o ld . †t h e n u cl eo ti d e p o si ti o n o f ea ch p o ly m o rp h is m is co u n te d fr o m th e a o f th e a t g st ar t co d o n , w h ic h w as d es ig n at ed as p o si ti o n � . m. fu and associates diabetes, vol. , december (available at http//www-genome.wi.mit.edu/cgi-bin/primer/primer _www. cgi) (see table of online appendix [available at http://diabetes.diabetesjour- nals.org]). pcr conditions were °c for min, followed by cycles of °c for s, °c for s, and °c for min, with a final extension at °c for min. both strands were sequenced from dna of the subjects on an abi dna sequencer and analyzed with sequence analysis . software (applied biosystems division, perkinelmer, foster city, ca). genotyping. the c t polymorphism in exon (casqsnp ) was ampli- fied and detected by sequence analysis on an abi dna sequencer (a of atg start codon of genbank accession no. nm_ , designated as position � ). casqsnp ( c t), rs , rs , and rs were typed using the acycloprime-fp snp detection system (perkinelmer life sciences, boston, ma), which determines the base at a snp location by template- directed dye-terminator incorporation followed by detection with fluores- cence polarization ( ). rs , rs , rs , rs , rs , and rs were typed using the snpstream uht genotyping system (beckman coulter, fullerton, ca) ( ). snp delg (casqsnp ), rs , and rs were genotyped by pyrosequencing using a psq hs a pyrosequencer according to the manufacturer’s protocol (pyrosequenc- ing, uppsala, sweden). in addition to snps within casq , we also genotyped snps flanking the gene to determine whether associated snps within casq were in ld with extragenic snps. these included rs , rs , rs , rs , rs , rs , and rs , which are located . – . kb upstream of casq , and rs , rs , and rs , which are located . – . kb downstream of casq , all performed by snpstream uht genotyping. all typing included at least % duplicate samples to determine mistyping rates, which were – %. statistical analysis. before analysis, all snps were checked for mendel errors using the pedcheck software program ( ) in the extended amish pedigree. a small number of mendel errors were either resolved or genotypes removed before analysis. allele frequencies of polymorphisms were deter- mined by gene counting. because of the extensive relatedness of study subjects in our amish pedigrees, we tested for associations of snp genotypes while accounting for the pedigree structure. briefly, we used a variance component approach in which we modeled the probability that the subject was a case or control as a function of the individual’s age, sex, and genotype, conditional on the correlations in phenotype among relative pairs. for the primary analysis, we considered an additive genetic model in which the genotype was coded as , , or , depending on whether the subject was homozygous for the minor allele (genotype � ), heterozygous (genotype � ), or homozygous for the major allele (genotype � ). statistical testing was performed by testing the likelihood of the data under a model in which the genotype effect was estimated against the likelihood of a nested model in which the genotype effect was constrained to be zero. under the likelihood ratio test, two times the difference in minus log base likelihoods is distributed as a � statistic with degrees of freedom equal to the difference in number of parameters between the models (in this instance, one). secondary analyses were carried out assuming dominant and recessive genetic models, in which instance the genotype was coded as or depending on whether the subject carried the minor allele (genotype � if minor allele carrier, if not [dominant model]; genotype � if homozygous for minor allele, if not, [recessive model]). parameter estimates (i.e., �-coefficients) were obtained by maximum likelihood, and odds ratios (ors) were obtained by taking the inverse log of the �-coefficient. the or for the additive model was scaled to reflect the odds that a case was homozygous for the minor allele versus the odds that the case was homozygous for the major allele. the variance components analysis was carried out using the solar software program ( ). in similar fashion, we also evaluated the effect of genotype on levels of quantitative traits (e.g., glucose and insulin levels) by comparing mean trait levels across genotypes. we compared the likelihood of a model in which the trait values were allowed to vary by genotype (unconstrained model) with that in which the genotype effects were constrained to be zero. as before, the model likelihoods and parameter estimates were computed conditional on the pedigree structure, and likelihoods obtained from competing models were compared by likelihoods ratio test. again, the additive model was used as our primary analysis, and dominant and recessive models were used as secondary analyses. the quantitative trait analyses were conducted in nondiabetic (ngt and igt) individuals only. the disequilibrium statistics r and �d	� between each pair of snps were calculated using zaplo ( ). for snp pairs with r � . , only one of the two snps was genotyped in the full sample set. to determine the haplotype structure of snps with casq , we used haploview ( ). we used haploscore ( ), which uses efficient score statistics within a general linear model framework, to test for haplotype associations between case and control subjects. haploscore computes empirical p values by simulation. for all analyses, p values � . were considered statistically significant, while p values between . and . were considered of borderline statistical signifi- cance. results sequencing of all exons, exon-intron boundaries, and the proximal regulatory region of casq revealed a total of six polymorphisms. these include three novel snps up- stream of the atg start codon, t c (casqsnp ), delg (casqsnp ), and insg (casqsnp ), and one novel snp in exon , c t (casqsnp ), which was silent (genbank accession no. nm_ ) (fig. ). in addition, we detected two snps previously reported in the dbsnp database (rs and rs ), located at , bp upstream of the atg start codon and in intron , respectively. of the snps in and immediately flanking this gene reported in the public databases at the time of this study (including rs and rs ), we con- firmed by genotype analysis; the other were not polymorphic (or had very low allele frequency) in the amish (fig. ). in total, there were snps in casq ; one occurred in the coding region and did not predict alter- fig. . schematic of casq and snps. exons are shown as boxes and horizontal lines as introns, regulatory region or flanking segments. novel snps discovered by sequence analysis are underlined, and dbsnps are designated by the “rs ” numbers; those that were informative are above, while those that were not polymorphic in the amish are below. snps with asterisks next to them were designated as informative haplotype- tagging polymorphisms and genotyped in the full sample set (see text for details). note exon and intron sizes are not strictly drawn to scale. casq and type diabetes in the amish diabetes, vol. , december ations in the amino acid sequence, four were in the 	 flanking region, one was in the 	 flanking region, and all others were in introns and did not predict any obvious alterations in rna splicing. based on our initial genotype and ld analysis per- formed in relatively unrelated amish subjects, casqsnp and casqsnp , located only bp apart in the 	 flanking region, were in complete ld with one another; therefore, only casqsnp was genotyped in the full sam- ple. similarly, strong ld was observed between rs and rs (r � . ) and rs and rs (r � . ); thus, only rs and rs are reported in the full sample. in addition, casqsnp and rs were found to be rare (allele frequencies . and . , respectively) in the amish and not pursued further. we typed the remaining informative haplotype-tagging polymorphisms in type diabetic individuals, individuals with igt, and individuals with ngt and tested for association between casq polymorphism and diabetes. using an additive model, genotype frequencies of rs and rs in introns and of casq differed significantly between subjects with type diabe- tes and those with ngt (p � . and . , respectively), with the more common allele being the at-risk allele for type diabetes (or for common alleles . [ % ci . – . ] and . [ . – . ], respectively) (table ). three other snps spanning from intron to the immediate 	 flanking region showed borderline evidence for associ- ation to type diabetes (p � . – . ). rs was also associated with the combined trait type diabetes/ igt (or for common allele . [ . – . ], p � . ), as were the common alleles of rs ( . [ . – . ], p � . ) and rs ( . [ . – . ], p � . ), both in intron , and rs in intron ( . [ . – . ], p � . ). three other polymorphisms showed borderline association with type diabetes/igt (p � . – . ). genotypic associations estimated under recessive and dominant genetic models did not provide a better fit to the data than that estimated under the additive model (data not shown). eight snps lying . – kb upstream and three snps lying . – . kb downstream of casq were not significantly associated with type diabetes (data not shown). we assessed the relationship between the snps and quantitative traits in an expanded set of nondiabetic members of the afds. three snps (rs , rs , and rs ) were significantly associated with glucose auc during the ogtt (p � . ) (fig. ). these findings in nondiabetic subjects provide additional support for an effect of casq polymorphism on glucose homeostasis. fig. . association of casq snps rs and rs with glucose and insulin levels during a -h ogtt in nondiabetic individuals. both snps were associated with total ogtt glucose auc (p � . and . , addidtive model for rs and rs , respectively). the a allele of rs appeared to better fit a recessive genetic model, with aa homozygotes having significantly higher glucose auc than heterozygotes and gg homozygotes (p � . ). by contrast, the c allele of appeared to better fit a dominant genetic model, with cc homozygotes and heteroyzgotes having higher glucose auc than aa homozygotes (p � . ). *p < . between genotypes for a given time point. rs was also significantly associated with glucose auc (p � . ) (data not shown). there was no evidence for association of rs or rs with ogtt insulin levels. m. fu and associates diabetes, vol. , december there was no evidence for association of any of the snps with bmi, insulin levels (fig. ), or homa-ir. the two snps associated with type diabetes, rs and rs , are in introns and , and are located only , bp apart. we thus sought to further define the haplotype structure of casq to determine whether these two snps might be tagging a single haplotype that is associated with type diabetes or whether they may be defining distinct haplotypes, both of which are associated with type diabetes. using the hapview program ( ), the casq region formed a complex pattern of haplotype structure. there were four main blocks (or bins): bin (rs , rs , and rs ; promoter), bin (casqsnp and rs ; promoter), bin (rs , rs , rs , rs , and rs ; intron – ), and bin (rs , rs , , rs , and rs ; intron to 	 flank). rs in intron and several snps in bin (e.g., rs , rs , rs , and rs from intron to the 	 flanking region) appeared to be in partial ld with bin snps (fig. ) (pairwise r and �d	� for all snp pairs also reported in table in the online appendix). although the two snps most significantly associated with type diabetes, rs and rs , are not assigned to the same bin, they are moderately correlated (�d	� � . , r � . ). in fact, the borderline-significant p value of . for association of rs with type diabetes can be accounted for by this correlation by examining the haplotype analysis results in table . these results indicate that the more common c allele at rs is associated with increased risk of diabetes (as indicated by a positive haploscore) and that the a allele at rs is also associated with increased risk primarily because it tracks with the rs c allele. in other words, the rs at-risk a allele association with type diabetes is derived from its association with the common a–c haplotype containing the rs at-risk c allele. furthermore, examination of three-, four-, and five-snp haplotypes did not provide evidence for association with fig. . haplotype structure of casq and flanking regions. shown is the haplotype structure of casq , as estimated by haploview ( ), on genotypes from amish subjects. �d�� values � are shown in the boxes, with empty boxes being (or �d�� � ). not shown are casqsnp , which was in complete ld with casqsnp , and casqsnp and rs , which were both rare in the amish (allele frequency . and . , respectively). snps showing significant association to type diabetes are underlined, while snps showing borderline significant association to type diabetes are shown in italics. table haploscore results comparing type diabetic cases with ngt control subjects using snps rs and rs rs rs frequency haploscore p* g a . . . a a . . . g c . . . a c . . . *global p value � . . casq and type diabetes in the amish diabetes, vol. , december type diabetes that was any greater than that of rs alone (data not shown). discussion chromosome q -q has perhaps the best replicated linkage to type diabetes in diverse populations, including caucasians, native americans, and chinese ( – ). thus, there is a high likelihood that at least one type diabetes susceptibility gene resides in this region. we and others have embarked upon a systematic ld mapping and posi- tional candidate gene effort toward the positional cloning of the gene(s). our ld mapping in amish samples, as well as finer linkage analysis in utah caucasians ( ), suggest at least two distinct type diabetes loci within the q -q interval. one of these regions, at � – . mb, contains a number of good positional candidate genes, including casq . calcium release from the sarcoplasmic reticulum into the cytosol has been shown to regulate glut expression ( ) and glucose transport in muscle ( ). hayashi et al. ( ) showed that an increase in cytoplasmic calcium is a key step in the activation of intracellular signaling cas- cades that mediate the immediate and prolonged effect of exercise on glucose transport. casq is present in muscle cells and located in the lumen of the sarcoplasmic reticu- lum. the major casq isoforms in skeletal (casq ) and cardiac (casq ) muscle exist as predominantly fast and slow isoforms, respectively ( ). casq binds and re- leases large quantities of ca � rapidly through its high capacity ( – ca � per casq molecule) and relatively low-affinity interactions with ca � ( , ). howarth et al. ( ) investigated the expression of casq and ca � bind- ing in cardiac and skeletal muscle from the streptozotocin- induced diabetic rat and found no significant changes in heart but an increase in the relative abundance of casq and casq-like proteins in skeletal muscle. together, these data suggest that casq may play an important role in insulin- and/or contraction-stimulated glucose transport. we thus hypothesized that sequence variants in casq that alter expression or function may change intracellular ca � flux and decrease glucose transport leading to glu- cose intolerance and/or diabetes. we have extensively screened casq for variants in amish subjects with type diabetes. we identified four novel snps (three in the promoter and one in exon ), none of which encode variants that would be expected to alter the protein’s structure. although it is possible that the snps in the promoter may alter casq expression, none were associated with type diabetes. genotyping and association analysis of informative polymorphisms re- vealed that two were significantly associated with type diabetes and three others showed borderline evidence for association with type diabetes. the two most signifi- cantly associated snps, rs and rs , are lo- cated in introns and , respectively. interestingly, the more common alleles of rs and rs were the at-risk alleles, which is in line with the common disease, common variant hypothesis and with findings for other type diabetes susceptibility genes, e.g., calpain and pro ala persoxisome proliferator–activated receptor 
 . supportive evidence that one or more of these variants influence glucose homeostasis is that allele frequencies of type diabetes–associated snps were intermediate be- tween type diabetes and ngt in subjects with igt (table ). furthermore, rs and rs were associated with glucose levels in nondiabetic subjects (fig. ). unfortunately, since our original linkage was obtained from large multiplex amish families, determining how much of the original linkage can be accounted for by the casq snps is not straightforward. however, given the relatively modest evidence for association of casq snps with type diabetes, it is unlikely that these snps (or haplotypes) can explain our original linkage result. indeed, two-point linkage analysis with casq snps showed little evidence for linkage to type diabetes or type diabetes/ igt. might these associations represent false-positive results due to multiple comparisons? for association studies such as these, this is always a possibility. indeed a full bonfer- roni correction for multiple comparisons would render our associations nonsignificant (at the p � . level). how- ever, we believe that correction for multiple comparisons is overly conservative, since some of our traits are corre- lated with each other. furthermore, we have evidence for linkage in this region not only in the amish but also in several other populations, which led to this hypothesis- driven ld mapping and positional candidate gene effort. further reducing concern regarding false-positive results are two internal consistencies. first, the same snps showed association to type diabetes and glucose traits in nondiabetic subjects. second, for of the snps, the allele frequencies for the igt cases were intermediate between those for the type diabetes cases and the ngt control subjects. perhaps most compelling is that our findings are similar to studies conducted in an independent study of utah caucasians showing association of casq polymorphism with type diabetes ( ). the region of association with type diabetes that overlaps in the amish and the more outbred utah caucasian population appears to be within intron , specifically rs and rs in utah cau- casians and rs in the amish (although this snp was not the most significantly associated snp in the amish). rs and rs are only bp from one another and in moderate ld (d	 � . , r � . ) in the amish. however, contrary to our findings in the amish, in which the rs a allele was the at-risk allele for type diabetes, the frequency of the g allele was significantly higher in utah caucasian subjects with type diabetes. this discrepancy between the two populations may indi- cate that this snp is not the functional snp but is marking an at-risk haplotype that differs between the amish and utah caucasians. alternatively, these discrepancies be- tween populations could represent false-positive or false- negative results. the findings reported here do not provide insights into possible functional consequences of the variants in casq . it is possible that variants in introns may affect mrna splicing and/or expression. however, we cannot rule out the possibility that other variants in casq or other nearby genes in ld with casq snps might be the responsible functional variants. we have observed evidence for association of other q -q positional candidate genes with type diabetes m. fu and associates diabetes, vol. , december or related traits, namely lamin a (lmna) ( ), guanine exchange factor- (arhgef ) (m.s., a.r.s., unpub- lished observations), and omentin (itln ) ( ). while all of these genes reside under our linkage peak, they are . , . , and . mb from casq , respectively. there does not appear to be any significant ld for the two casq type diabetes–associated snps, rs and rs , with snps in lmna, arhgef , or itln , suggesting that the associations of variation in these genes with type diabetes and related traits are independent of one another. these findings, coupled with more than one association signal in pima indians and utah caucasians, suggest that there are likely to be more than one gene (possibly several genes) responsible for the linkage at q -q . in summary, we have reported a systematic search for polymorphism in casq on chromosome q and iden- tified four novel snps. none predict alterations in the protein sequence. we describe for the first time a signifi- cant association between casq polymorphism and type diabetes, possibly suggesting a new susceptibility gene for type diabetes. further analysis in other populations, as well as functional studies, will be necessary to further elucidate the role of polymorphism in casq in the pathogenesis of type diabetes. acknowledgments this work was supported by research grants r - dk , k -dk , u dk , and k -ca ; the university of maryland general clinical research center grant m rr ; the general clinical research centers program, the national center for research re- sources; the national institutes of health; and the 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promoter region of the hepatocyte nuclear factor- � gene on chromosome q is associated with type diabetes and appears to contribute to the evidence for linkage in an ashkenazi jewish population. diabetes : – , . silander k, mohlke kl, scott lj, peck ec, hollstein p, skol ad, jackson au, deloukas p, hunt s, stavrides g, chines ps, erdos mr, narisu n, conneely kn, li c, fingerlin te, dhanjal sk, valle tt, bergman rn, tuomilehto j, watanabe rm, boehnke m, collins fs: genetic variation casq and type diabetes in the amish diabetes, vol. , december near the hepatocyte nuclear factor- � gene predicts susceptibility to type diabetes. diabetes : – , . wolford jk, hanson rl, kobes s, bogardus c, prochazka m: analysis of linkage disequilibrium between polymorphisms in the kcnj gene with type diabetes mellitus in pima indians. mol genet metab : – , . farook vs, hanson rl, wolford jk, bogardus c, prochazka m: molecular analysis of kcnj on q as a candidate gene for type diabetes in pima indians. diabetes : – , . wang h, chu w, das sk, ren q, hasstedt sj, elbein sc: liver pyruvate kinase polymorphisms are associated with type diabetes in northern european caucasians. diabetes : – , . wolford jk, gruber jd, ossowski vm, vozarova b, antonio tataranni p, bogardus c, hanson rl: a c-reactive protein promoter polymorphism is associated with type diabetes mellitus in pima indians. mol genet metab : – , . steinle ni, kazlauskaite r, imumorin ig, hsueh wc, pollin ti, o’connell jr, mitchell bd, shuldiner ar: variation in the lamin a/c gene: associa- tions with metabolic syndrome. arterioscler thromb vasc : – , . fu m, gong d-w, damcott c, sabra m, yang r, pollin ti, tanner k, ott s, mclenithan jc, fried s, o’connell jr, mitchell bd, shuldiner ar: sys- temic analysis of omentin and omentin on q as candidate genes for type diabetes in the old order amish (abstract). diabetes (suppl. ):a , . shin dw, pan z, kim ek, lee jm, bhat mb, parness j, kim dh, ma j: a retrograde signal from calsequestrin for the regulation of store-operated ca � entry in skeletal muscle. j biol chem : – , . ojuka eo, jones te, notlte la, chen m, wamhoff br, sturek m, holloszy jo: regulation of glut biogenesis in muscle: evidence for involvement of ampk and ca. am j physiol endocrinol metab :e –e , . young jh, gulve ea, holloszy jo: calcium stimulates glucose transport in skeletal muscle by a pathway independent of contraction. am j physiol cell physiol :c –c , . howarth fc, glover l, culligan k, qureshi ma, ohlendieck k: caseques- trin expression and calcium binding is increased in streptozotocin-induced diabetic rat skeletal muscle though not in cardiac muscle. pflugers arch : – , . agarwala r, biesecker lg, hopkins ka, francomano ca, schaffer aa: software for constructing and verifying pedigrees within large genealogies and an application to the old older amish of lancaster county. genome res : – , . pollin ti, agarwala r, schaffer aa, shuldiner ar, mitchell bd, o’connell jr: investigations of male founder structure and the y chromosome in the old order amish (abstract). am j hum genet (suppl.): , . hsueh wc, mitchell bd, aburomia r, pollin t, sakul h, gelderehm m, michelsen bk, wagner mj, st jean pl, knowler wc, burns dk, bell cj, shuldiner ar: diabetes in the old older amish: characterization and heritability analysis of the amish family diabetes study. diabetes care : – , . hsu tm, chen x, duan s, miller rd, kwok p-y: universal snp genotyping assay with fluorescence polarization detection. biotechniques : – , . bell pa, chaturvedi s, gelfand ca, huang cy, kochersperger m, kopla r, modica f, pohl m, varde s, zhao r, zhao x, boyce-jacino mt: snpstream uht: ultra-high throughput snp genotyping for pharmacogenomics and drug discovery. biotechniques (suppl.): – , , – , [erratum in biotechniques : , ] . o’connell jr, weeks de: pedcheck: a program for identifying genotype incompatibilities in linkage analysis. am j hum genet : – , . almasy l, blangero j: multipoint quantitative-trait linkage analysis in general pedigrees. am j hum genet : – , . o’connell jr: zero-recombinant haplotyping: applications to fine mapping using snps. genet epidemiol (suppl. ):s –s , . daly mj, rioux jd, schaffner sf, hudson tj, lander es: high-resolution haplotype structure in the human genome. nat genet : – , . schaid dj, rowland cm, tines de, jacobson rm, poland ga: score tests for association between traits and haplotypes when linkage phase is ambiguous. am j hum genet : – , . das sk, hasstedt sj, zhang z, elbein sc: linkage and association mapping of a chromosome q – q type diabetes susceptibility locus in northern european caucasians. diabetes : – , . hayashi t, wojtaszewski jfp, goodyear lj: exercise regulation of glucose transport in skeletal muscle. am j physiol :e –e , . fliegel l, ohnishi m, carpenter mr, khanna vk, reithmeier ra, maclen- nan dh: amino acid sequence of rabbit fast-twitch skeletal muscle calsequestrin deduced from cdna and peptide sequencing. proc natl acad sci u s a : – , . fliegel l, leberer e, green nm, maclennan dh: the fast-twitch muscle calsequestrin isoform predominates in rabbit slow-twitch soleus muscle. febs lett : – , . das sk, chu w, zhang z, hasstedt sj, elbein sc: calsquestrin (casq ) gene polymorphisms under chromosome q linkage peak are associated with type diabetes in northern european caucasians. diabetes : – , m. fu and associates diabetes, vol. , december pteridines € eur - euro £ gbp - pound $ usd - dollar en english deutsch × your purchase has been completed. your documents are now available to view. × changing the currency will empty your shopping cart. confirm cancel pteridines official journal of the international society of pteridinology issn: - first published: april , editor-in-chief: dietmar fuchs impact factor: . 	overview 	latest issue 	issues 	ranking 	submit 	editorial about this journal objective pteridines is an open acess international quarterly journal dealing with all aspects of pteridine research. pteridines are heterocyclic fused ring compounds involved in a wide range of biological functions from the color on butterfly wings to cofactors in enzyme catalysis to essential vitamins. of the pteridines, , , , -tetrahydrobiopterin is the necessary cofactor of several aromatic amino acid monoxygenases, the nitric oxide synthases and glyceryl ether monoxygenase (gemo). neopterin plays an essential role in the immune system and is an important biomarker in laboratory medicine for diseases such as hiv, cardiovascular disease, malignant tumors, among others. topics 	neopterin, dihydroneopterin, monapterin 	biopterin, tetrahydrobiopterin 	folates, antifolates, riboflavin 	phenylalanine, tyrosine, phenylketonuria, serotonin, adrenalin, noradrenalin, l-dopa, dopamine, related biogenic amines 	phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, nitric oxide synthases (inos), alkylglycerol monooxygenase (agmo), dihydropterin reductase, sepiapterin reductase 	homocysteine, mediators of inflammation, redox systems, iron article formats original articles, reviews, mini reviews, commentaries, opinions on controversial subjects > information on submission process your benefits your benefits: 	unique interdisciplinary forum devoted to conjugated and unconjugated pteridines 	basic and clinical approach to the topic combining the fields of chemistry, biochemistry, clinical chemistry, and laboratory medicine 	international, authoritative editors and reviewers boosting the prestige of published papers 	free language-correction services for authors from non-english speaking regions 	worldwide dissemination and unrestricted access to all published articles 	promotion of each published article 	secure archiving by de gruyter and the independent archiving service portico 	no submission charges pteridines is the official journal of the international society of pteridinology (isp). history pteridines was founded in and has been continued at de gruyter since . abstracting and indexing pteridines is covered by the following services: 	baidu scholar 	biobase 	cabells journalytics 	case 	chemical abstracts service (cas) - caplus 	chemical abstracts service (cas) - scifinder 	chronos hub 	cnki scholar (china national knowledge infrastructure) 	cnpiec - cnplinker 	dimensions 	doaj (directory of open access journals) 	ebsco (relevant databases) 	ebsco discovery service 	embase 	genamics journalseek 	gooa 	google scholar 	index copernicus 	j-gate 	journal citation reports/science edition 	journalguide 	journaltocs 	kesli-ndsl (korean national discovery for science leaders) 	meta 	microsoft academic 	mysciencework 	naver academic 	naviga (softweco) 	primo central (exlibris) 	proquest (relevant databases) 	publons 	qoam (quality open access market) 	readcube 	reaxys 	scimago (sjr) 	scopus 	semantic scholar 	sherpa/romeo 	summon (proquest) 	tdnet 	text mining 	ulrich's periodicals directory/ulrichsweb 	wanfang data 	web of science - science citation index expanded 	worldcat (oclc) 	yewno discover supplementary materials 	 author''s ethical statements 	 editorial policy 	 data sharing policy 	 article processing charges 	 instruction for authors 	 privacy statement 	 guidelines for reviewers 	 open access license 	 publication ethics statement topics 	biochemistry 	human biology 	life sciences 	molecular biology external links 	journal archive 	open access statement 	crosscheck plagiarism screening 	online submission of manuscripts 	open access statement 	crosscheck plagiarism screening 	privacy statement volume issue january unable to retrieve citations for this document × retrieving citations for document... laboratory diagnostic value of neopterin measurements in patients with covid- infection dietmar fuchs, magnus gisslen january , page range: - more cite open access pdf pdf abstract the new coronavirus sars-cov- was identified to be responsible for the covid- pandemic. there are striking differences in the response to infection, some people develop no or mild symptoms, while other outcomes are severe of even fatal. for those covid- patients who require hospitalization, prognostic markers could help clinicians to identify patients with a poor outcome early. the serum levels of the immune activation marker neopterin have already been shown to be of prognostic value in patients with sars-cov- and a similar pattern can be observed for sars-cov- . this comment discusses the biochemical circuits that support the clinical value of neopterin measurements in covid- patients. unable to retrieve citations for this document × retrieving citations for document... norepinephrine was superior in death risk reducing and hemodynamics compared to dopamine in treatment of patients with septic shock xudong lu, xianghua xu, yueying wu february , page range: - more cite open access pdf pdf abstract background to investigate the clinical effects of norepinephrine versus dopamine in treatment of septic shock by pooling the data form open published clinical trials. material and methods the clinical trials relevant to norepinephrine versus dopamine in treatment of septic shock were electronically searched in the databases of pubmed, embase, the cochrane library, web of science, google scholar and cnki. the original data related to the treatment effects such as death risk, oxygen metabolism and hemodynamics index were extracted from the included original studies. the death risk was pooled by the effect size of relative risk (rr), the oxygen metabolism and hemodynamics index were pooled by standard mean difference (smd) and the corresponding % confidence interval ( %ci). the publication bias was evaluated by begg's funnel plot and egger's line regression test. results thirteen clinical trials were included in the meta-analysis. the pooled results demonstrated the death risk was significantly decreased (rr= . , %ci: . to . , p= . ) in septic shock patients who received norepinephrine compared to those receiving dopamine. the hr (smd=− . , %ci: − . to − . , p< . ) and cardiac index (smd=− . , %ci: − . to − . , p< . ) were lower in norepinephrine group compared to dopamine group. the systemic vascular resistance index (smd= . , %ci: . to . , p< . ) in norepinephrine group was higher than those of dopamine group with statistical difference. the begg's funnel plot and egger's line regression test (t=− . , p= . ) showed no publication bias. conclusions based on the present evidence, norepinephrine was superior to dopamine in the aspects of death risk reducing and hemodynamics. unable to retrieve citations for this document × retrieving citations for document... clinical significance of serum homocysteine as a biomarker for early diagnosis of diabetic nephropathy in type diabetes mellitus patients bin ye, xiangying zhu, zhifu zeng, xiaozhen ji, meixia ji march , page range: - more cite open access pdf pdf abstract objective the aim of this study was to investigate the clinical significance of serum homocysteine (hcy) as a biomarker for early diagnosis of diabetic nephropathy (dn) in type diabetes mellitus (t dm) patients. methods fifty-five t dm patients with dn and t dm patients without dn were prospectively recruited from january to may in our hospital. the serum hcy was tested by electrochemiluminescence assay in dn and t dm groups and compared. the diagnostic efficacy of serum hcy as a biomarker for early diagnosis of dn was evaluated by calculating the diagnostic sensitivity, specificity and area under the roc curve (auc). results the serum levels of hcy were . ± . and . ± .  μmol/l for dn and t dm patients, respectively, with statistical difference ( t = . , p < . ). in the dn group, the serum hcy levels for patients with hyperfiltration, intermittent proteinuria, microalbuminuria, macroalbuminuria and uremic were . ± . , . ± . , . ± . , . ± . and . ± .  μmol/l, respectively, which indicated that serum hcy levels in dn were higher than those of t dm patients and correlated with patient’s renal damage. using the serum hcy level as the reference, the diagnostic sensitivity, specificity and auc were . ( . – . )%, . ( . – . )% and . ( . – . )%, respectively, with the cutoff value of . between dn and t dm. the serum hcy also had relatively good differential diagnostic efficacy between different dn stages with high sensitivity, specificity and auc. conclusion serum hcy was obviously elevated in dn compared to t md and correlated with the renal damage severity, which can be applied as a potential serological marker for early diagnosis of dn. unable to retrieve citations for this document × retrieving citations for document... the urinary biopterin in autism spectrum disorder aleksandra waligóra, aleksandra damasiewicz-bodzek, piotr gorczyca, sławomir waligóra, krystyna tyrpień-golder march , page range: - more cite open access pdf pdf abstract objective the aim of the study was to determine whether biopterin is present in significantly lower quantities in urine samples of patients with autism spectrum disorder (asd) compared to healthy individuals. methods the concentration of biopterin in urine samples was measured by elisa using commercially available kit. the study involved children aged – years with asd and healthy children aged – years. results significantly lower biopterin concentration was observed in autistic patients compared to the control group. however, no significant difference was observed between mild, moderate, and severe asd. conclusion one of the potential causes of decrease in urinary biopterin levels may be tetrahydrobiopterin (bh ) deficiency, which has extensive and serious health consequences for the nervous system. the results of measuring biopterin as a fully oxidized form of bh may suggest that biosynthesis or regeneration of bh may be decreased in children with asd. on the other hand, decreased urinary biopterin levels in children with asd may be due to bh overuse, a good regeneration process, and decreased urinary excretion; and abnormalities in bh metabolism appear to be related to the aetiology of asd or may be due to asd. volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue - 	 issue 	 issue volume ( ) 	 issue - 	 issue - volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue - 	 issue 	 issue volume ( ) 	 issue - 	 issue 	 issue special issue: th international symposium on pteridines and folates, antalya, turkey, may – , volume ( ) 	 issue - 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue - volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 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(tianjin lung cancer institute, tianjin medical university general hospital, tianjin, china) editorial office publisher de gruyter poland bogumiła zuga a str. - warsaw, poland t: + editorial contact jędrzej daszkiewicz, managing editor jedrzej.daszkiewicz@degruyter.com​ open access details 	online issn: - 	 type: journal 	 language: english 	 publisher: de gruyter 	 first published: april , 	 publication frequency: issue per year 	 audience: researchers in the fields of chemistry, biochemistry, clinical biochemistry, metabolic disorders, clinical chemistry, clinical immunology, immunology, laboratory medicine, biology, molecular biology, molecular medicine, nutrition, phytomedicine 	contact us 	customer service 	human resources 	press 	contacts for authors 	career 	how to join us 	current vacancies 	working at de gruyter 	open access 	articles 	books 	funding & support 	for authors 	publish your book 	publish your journal article 	abstracting & indexing 	for libraries & trade partners 	electronic journals 	ebooks 	databases & online reference 	metadata 	our partner publishers 	rights & permissons 	repository policy 	free access policy 	about de gruyter 	de gruyter foundation 	our locations 	help/faq 	privacy policy 	terms & conditions 	legal notice © walter de gruyter gmbh defective mitochondrial mrna maturation is associated with spastic ataxia report defective mitochondrial mrna maturation is associated with spastic ataxia andrew h. crosby, , ,* heema patel, , barry a. chioza, christos proukakis, , kay gurtz, michael a. patton, reza sharifi, gaurav harlalka, michael a. simpson, katherine dick, johanna a. reed, ali al-memar, zofia m.a. chrzanowska-lightowlers, harold e. cross, and robert n. lightowlers in human mitochondria, polyadenylation of mrna, undertaken by the nuclear-encoded mitochondrial poly(a) rna polymerase, is essential for maintaining mitochondrial gene expression. our molecular investigation of an autosomal-recessive spastic ataxia with optic atrophy, present among the old order amish, identified a mutation of mtpap associated with the disease phenotype. when subjected to poly(a) tail-length assays, mitochondrial mrnas from affected individuals were shown to have severely truncated poly(a) tails. although defective mitochondrial dna maintenance underlies a well-described group of clinical disorders, our findings reveal a defect of mitochondrial mrna maturation associated with human disease and imply that this disease mechanism should be considered in other complex neurodegenerative disorders. in the current study, we investigated an extensive old order amish family in which multiple children were affected by a slowly progressive autosomal-recessive neurodegenerative condition, the key features of which are cerebellar ataxia (limb and truncal), spastic paraparesis (increased lower limb tone with brisk knee jerks and extensor plantars, but no weakness), dysarthria (mixture of cerebellar and spastic), optic atrophy, learning difficul- ties, and, in some, emotional liability. the clinical features found in the affected individuals are shown in detail in table . speech and walking are usually delayed and abnormal from the onset. the two younger cases (ix- and ix- ) have predominantly cerebellar gait and speech disturbance. in the four older cases (ix- to ix- ), tongue movements are slow and spastic, jaw jerk is brisk, and two have increased tone in the upper limbs. their parents report a clear progressive decline in function, although all are still independently mobile with a spastic ataxic gait, with no obvious intellectual deterioration, needing only mild assistance for most self-care. the most severely ataxic is ix- , whose oromandibular coordination has become so poor that, since the age of , she has had to use a straw for drinking. in addition to the parents’ accounts, the relative severity of the clinical picture in the older individuals compared to the much younger ix- and ix- also supports the impression that this is a progressive condi- tion. diminished or absent upper limb and ankle reflexes in older cases, which were said to have been present in pediatric assessments, suggest additional progressive lower motor neuron involvement. older individuals attended school until the age of (the traditional limit of amish centre for medical genetics, st. george’s university london, cranmer terrace neurology, university college london, london nw pf, uk; windows o neurology, atkinson morley wing, st. george’s hospital, tooting, london sw newcastle university medical school, newcastle upon tyne ne hh, uk; d north alveron way, tuscon, az , usa these authors contributed equally to this work *correspondence: acrosby@sgul.ac.uk doi . /j.ajhg. . . . � by the american society of human the american education), but special tuition was required from early on. individual ix- had a full iq estimate at age of only (verbal , performance ). all patients live with their parents, none are married, and older individuals either attend a special workshop or help with basic house- hold tasks. assuming that a founder mutation was responsible, we undertook a genome-wide microarray scan with dna ex- tracted from blood samples of family members obtained with informed consent and following approved institu- tional review board protocols. the genome-wide screen was performed with affymetrix k snp nspi arrays (gen- eservice) by using dna from the four affected cases initially available (ix- , ix- , ix- , ix- ; figure a). homozygosity analysis identified a single autozygous region of . mb on chromosome p . , delimited by markers rs and rs , likely to correspond to the disease locus; no other notable regions of autozygosity were observed. auto- zygosity across this interval was corroborated by microsatellite marker analysis, determined by pcr amplifi- cation, and subsequent analysis by polyacrylamide gel elec- trophoresis, visualized by silver staining via standard meth- odologies (figure b). multipoint lod scores were calculated across the microsatellite markers with simwalk version . , , revealing a lodmax score of . , assuming an autosomal-recessive mode of inheritance, complete penetrance, and a disease gene frequency of . %. the putative disease locus is predicted to contain genes, of which are hypothetical and are pseudogenes. while screening the remaining genes, a nonsynonymous base change was identified in the nuclear-encoded , london sw re, uk; department of clinical neurosciences, institute of f hope genetic study, geauga county, oh , usa; department of re, uk; mitochondrial research group, institute for ageing and health, epartment of ophthalmology, university of arizona school of medicine, genetics. all rights reserved. journal of human genetics , – , november , mailto:acrosby@sgul.ac.uk http://dx.doi.org/ . /j.ajhg. . . table . summary of clinical features of affected cases patient ix- ix- ix- ix- ix- ix- age when seen (years) age at which walked (months) (tiptoed until age ) (with falls) (abnormal) (with falls) age at which talked (years) > speech normal at onset? n n y n n n optic atrophy y y y y nk nk nystagmus n y (horizontal) y (vertical) n n n emotional liability y n y y n y dysarthria y y y y y y tone in upper limbs increased normal increased normal normal normal tone in lower limbs increased increased increased increased normal normal power in limbs normal normal normal normal normal normal reflexes: biceps þ þ � � þþ þ reflexes: supinator þ þ � � þþ þ reflexes: triceps þ þ � � þþ þ reflexes: knee þþþ þþþ � þþþ þþþ þþþ reflexes: ankle � � � � þþ þþ plantars mute extensor extensor extensor extensor extensor limb ataxia y y y y n n gait ataxia y y y y y y the following abbreviations are used: y, yes; n, no; nk, not known; reflexes: �, absent; þ, diminished; þþ, normal; þþþ, pathologically brisk. mitochondrial poly(a) rna polymerase gene mtpap (c. a>g; figure c). the c. a>g variant in exon of papd creates a bamhi restriction endonuclease recognition site. exon pcr products generated from control and patient samples were subsequently digested with one unit of bamhi and were visualized on agarose gels. this sequence variant cosegregated with the disease phenotype and was not detected in control chromo- somes of unaffected individuals of european ancestry. analysis of control chromosomes from unaffected individuals from the same ohio amish community identi- fied a single heterozygous carrier of the sequence variant, which is not unexpected in this endogamous community, in which founder mutations are commonly associated with rare inherited diseases in multiple families. – the resultant predicted p.n d substitution occurs within an n-p-f-e sequence (figure d) located within one of the most highly conserved regions of the molecule in all species examined. in order to confirm that the c. a>g alteration is pathogenic and to determine its functional consequence, we undertook mitochondrial poly(a) tail (mpat) assays of representative mitochondrial-encoded transcripts (rna and mtco [mim ]) of the amish family with the putative mutation. these assays allowed us to visualize the heterogeneous poly(a) tails of the various species generated by mitochondrial poly(a) rna poly- the american journal of human genetics , – , novemb merase (mtpap). rna ( . mg) was extracted from whole blood (paxgene blood rna kit, qiagen) according to the manufacturer’s instructions. linker primers were ligated with neb t rna ligase. all subsequent steps were exactly as described in temperley et al., including primer sequences for rna and anti-ligation primer. for mtco , first-round pcr primer was -catattcatc ggcgtaaatc- and labeled second-round pcr primer was -caaccccatggcctcca- . pcr profiles were as described. gels were exposed to phosphorimage screens and imagequant . software used for visualization and densi- tometric analyses. relative fraction of oligoadenylated species was calculated as oligo/(oligo þ poly) and pre- sented as a percentage. oligoadenylated species were defined as > nt extension from the deadenylated species and were measured against the size markers. for polyade- nylated species, rna was defined as – nt exten- sions, and mtco was defined as – nt extensions. as shown in figure , oligoadenylated species were present in all samples, consistent with previous reports in which mtpap had been depleted by small interfering rna treat- ment. , polyadenylation in both carrier parents appeared to be normal, but the mpat assay revealed a profound effect on polyadenylation associated with homozygosity of the sequence variant in all affected cases. whereas the percentage of oligoadenylated (> nt) versus er , figure . identification of mtpap mutation in amish pedigree (a) pedigree diagram of amish family. (b) marker genotypings across the region of homozygosity identified by whole-genome snp analysis. initial genome-wide screen was undertaken in individuals ix- , ix- , ix- , and ix- for which samples were available at the time. the region of autozygosity defined was delimited by snps rs and rs . the mutation (nc_ . : g. t>c; nm_ . : c. a>g) is shown in red. all affected individuals, including ix- and ix- , were subsequently found to be homozygous for the c. a>g variant, whereas parental samples and the unaffected sibling were carriers. (c) electropherograms showing mtpap exon sequence encompassing the nm_ . : c. a>g variant in a wild-type (wt) control, a heterozygous carrier parent, and a homozygous affected individual. (d) clustalw alignment of the region encompassing the pap/ a-associated domain of mtpap from various species. amino acid altered by the c. a>g substitution (p.n d) is indicated with the red arrow. polyadenylated (> nt extension) rna transcript was less than % in the unaffected parent carriers (figure , bottom, lanes and ), a dramatic reversal in this ratio was apparent in all patients homozygous for the c. a>g mutation ( %– %, lanes – ). a second transcript, mtco , was also assessed (figure , top), with similar results (unaffected parents %– %, affected siblings %– %). together with the genetic data, these findings strongly suggest that the p.n d mtpap is responsible for the condition in this family. what is the function of polyadenylation in the mamma- lian mitochondrion? the answer is complex and, to date, has not been fully resolved. terminal oligoadenylation of mitochondrial mrnas is clearly essential, because pro- the american cessing of several mitochondrial open reading frames from the primary polycistronic transcript leaves a trun- cated terminal codon that requires adenylation for com- pletion. polyadenylation of mrna is, however, crucial for maintaining global mrna expression in mammalian mitochondria. , , depletion of mtpap clearly causes instability of some transcripts, but not others. , , indeed, enzymatic removal of the poly(a) tail from mito- chondrial transcripts in intact cells can actually promote stability of a subset of mrnas. in contrast, occlusion of the poly(a) extensions clearly leads to a decrease in trans- lation efficiency. although the exact role or roles of mito- chondrial mrna polyadenylation remain unclear, deple- tion of mtpap has a profound effect on mitochondrial journal of human genetics , – , november , figure . poly(a) status of representative mitochondrial mrnas from patient and control samples available rna ( . mg) from each individual was ligated to linker and subjected to the mitochondrial poly(a) tail-length assay as described in temperley et al. radiolabeled products were separated through a % denaturing polyacrylamide gel, and lengths were calculated by comparison against size markers (p end-labeled synthetic oligomers of and nt). oligoadenylated (< nt extension from processed transcript) or polyadenylated ( – nt extension rna ; – nt mtco ) species were quantified following densitometric scans (imagequant software). lane denotes the mother, lane denotes the father, and lanes – denote the affected children. the following abbreviations are used: h, hek t control cell line; c and c , negative amplification controls from the first and second rounds of pcr; m, size marker; f, control fibroblast cell line. top: mtco transcript; bottom: rna transcript. the american journal of human genetics , – , november , function and is evidenced by the severe neurodegenera- tive disorder found in this family. inherited mitochondrial disorders commonly affect postmitotic tissues, such as muscle and the central and peripheral nervous system, and are widely acknowledged as an important cause of neurological disease, with cere- bellar ataxia a frequent feature and occasionally the main one (e.g., the mitochondrial recessive ataxia syndrome). adult-onset mitochondrial diseases are typically caused by mutations within the mitochondrial genome, whereas mutations in nuclear-encoded mitochondrial proteins usually present with more severe childhood phenotypes and multisystem disorders, in addition to neurological involvement (reviewed in rahman and hanna ). these childhood disorders are often associated with instability of the mitochondrial genome, resulting in mtdna dele- tions (e.g., polg [mim ], polg [mim ], ant [mim ], peo [mim ], opa [mim ], tymp [mim ]) or mtdna depletion (dguok [mim ], sucla [mim ], suclg [mim ], rrm b [mim ], mpv [mim ], tk [mim ]). in contrast, one category of mitochondrial disorders that is becoming increasingly diagnosed is that of nuclear gene mutations that cause mitochondrial protein synthesis disorders with no evidence of mtdna involvement. this highly heterogeneous group mostly shares a combined disorder of respiratory chain complexes. to date, the underlying genetic defects encode mt-trna modifying and aminoacylating enzymes (pus , trmu, dars , rars , yars ), an mrna stability factor and translational activator (lrpprc, taco ), translation factors (gfm , tufm, tsfm, c orf ), components of the mitochon- drial ribosome (mrps / ), and factors that mediate mitochondrial ribosome assembly (spg , afg l ). clin- ical presentations are diverse, but, other than the mt- trna modifying defects, almost all patients have neurolog- ical deficits. most strikingly, mutations in two components of the mitochondrial maaa protease that are essential for mitochondrial ribosome maturation, paraplegin and afg -like protein, cause hereditary spastic paraplegia (spg ) and cerebellar ataxia (sca ), respectively. both presentations are similar to the clinical disorder that we have identified in patients with mtpap defects. the spg phenotype can include cerebellar ataxia and optic atrophy in addition to spastic paraparesis, and sca patients have pyramidal signs in addition to ataxia. similarly, friedreich’s ataxia, the most common autosomal-recessive ataxia, may display a comparable phenotype to the patients described here with spasticity, areflexia, and optic atrophy. the mutated protein, frataxin, is also a mitochondrial protein, and the latest evidence suggests that it is involved in synthesis of iron-sulfur clusters whose diverse functions include electron transfer in the mitochondrial respiratory complex. although a number of diverse molecular mechanisms lead to auto- somal-recessive ataxias, our findings provide evidence the american of a new mitochondrial abnormality that may lead to a complex neurodegenerative disorder with ataxia as the prominent component. acknowledgments this work was supported by the birth defects foundation (uk)/ newlife foundation for disabled children. we would like to thank the amish families for participating in this study. sequencing was done in the medical biomics (st. george’s, university of london). r.n.l. and z.m.a.c.-l. would like to thank the wellcome trust ( /z/ /z) and the biotechnology and biological sciences research council (bb/f / ) for continuing support. received: august , revised: september , accepted: september , published online: october , web resources the urls for data presented herein are as follows: clustalw , http://www.ebi.ac.uk/tools/clustalw ncbi national center for biotechnology information, http:// www.ncbi.nlm.nih.gov/ online mendelian inheritance in man (omim), http://www.ncbi. nlm.nih.gov/omim ucsc genome browser, http://genome.ucsc.edu/ accession numbers the genbank accession numbers for papd are nm_ . and np_ . . references . sobel, e., and lange, k. ( ). descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker-sharing statistics. am. j. hum. genet. , – . . sobel, e., sengul, h., and weeks, d.e. ( ). multipoint estimation of identity-by-descent probabilities at arbitrary positions among marker loci on general pedigrees. hum. hered. , – . . patel, h., cross, h., proukakis, c., hershberger, r., bork, p., ciccarelli, f.d., patton, m.a., mckusick, v.a., and crosby, a.h. ( ). spg is mutated in troyer syndrome, an hereditary spastic paraplegia. nat. genet. , – . . simpson, m.a., cross, h., proukakis, c., pryde, a., hersh- berger, r., chatonnet, a., patton, m.a., and crosby, a.h. ( ). maspardin is mutated in mast syndrome, a compli- cated form of hereditary spastic paraplegia associated with dementia. am. j. hum. genet. , – . . simpson, m.a.,cross, h.,proukakis, c., priestman,d.a.,neville, d.c., reinkensmeier, g., wang, h., wiznitzer, m., gurtz, k., verganelaki, a., et al. ( ). infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of gm synthase. nat. genet. , – . . tomecki, r., dmochowska, a., gewartowski, k., dziembowski, a., and stepien, p.p. ( ). identification of a novel human nuclear-encoded mitochondrial poly(a) polymerase. nucleic acids res. , – . journal of human genetics , – , november , http://www.ebi.ac.uk/tools/clustalw http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/omim http://www.ncbi.nlm.nih.gov/omim http://genome.ucsc.edu/ . temperley, r.j., seneca, s.h., tonska, k., bartnik, e., bindoff, l.a., lightowlers, r.n., and chrzanowska-lightowlers, z.m. ( ). investigation of a pathogenic mtdna microdeletion reveals a translation-dependent deadenylation decay pathway in human mitochondria. hum. mol. genet. , – . . nagaike, t., suzuki, t., katoh, t., and ueda, t. ( ). human mitochondrial mrnas are stabilized with polyadenylation regulated by mitochondria-specific poly(a) polymerase and polynucleotide phosphorylase. j. biol. chem. , – . . ojala, d., crews, s., montoya, j., gelfand, r., and attardi, g. ( ).asmallpolyadenylatedrna( srna),containingaputa- tive ribosome attachment site, maps near the origin of human mitochondrial dna replication. j. mol. biol. , – . . slomovic, s., and schuster, g. ( ). stable pnpase rnai silencing: its effect on the processing and adenylation of human mitochondrial rna. rna , – . . wydro, m., bobrowicz, a., temperley, r.j., lightowlers, r.n., and chrzanowska-lightowlers, z.m. ( ). targeting of the cytosolic poly(a) binding protein pabpc to mitochondria causes mitochondrial translation inhibition. nucleic acids res. , – . . rahman, s., and hanna, m.g. ( ). diagnosis and therapy in neuromuscular disorders: diagnosis and new treatments the american journal of human genetics , – , novemb in mitochondrial diseases. j. neurol. neurosurg. psychiatry , – . . casari, g., de fusco, m., ciarmatori, s., zeviani, m., mora, m., fernandez, p., de michele, g., filla, a., cocozza, s., marconi, r., et al. ( ). spastic paraplegia and oxphos impairment caused by mutations in paraplegin, a nuclear-encoded mito- chondrial metalloprotease. cell , – . . di bella, d., lazzaro, f., brusco, a., plumari, m., battaglia, g., pastore, a., finardi, a., cagnoli, c., tempia, f., frontali, m., et al. ( ). mutations in the mitochondrial protease gene afg l cause dominant hereditary ataxia sca . nat. genet. , – . . wilkinson, p.a., crosby, a.h., turner, c., bradley, l.j., ginsberg, l., wood, n.w., schapira, a.h., and warner, t.t. ( ). a clinical, genetic and biochemical study of spg mutationsin hereditary spasticparaplegia.brain , – . . mariotti, c., brusco, a., di bella, d., cagnoli, c., seri, m., gellera, c., di donato, s., and taroni, f. ( ). spinocerebel- lar ataxia type : a novel autosomal dominant cerebellar ataxia characterized by slow progression and ophthalmopare- sis. cerebellum , – . . stemmler, t.l., lesuisse, e., pain, d., and dancis, a. ( ). frataxin and mitochondrial fes cluster biogenesis. j. biol. chem. , – . er , 	defective mitochondrial mrna maturation is associated with spastic ataxia 	acknowledgments 	web resources 	accession numbers 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ physical activity profile of old order amish, mennonite, and contemporary children browse explore more content msse-d- - (institutional repository).pdf ( . kb) physical activity profile of old order amish, mennonite, and contemporary children citedownload ( . kb)shareembed journal contribution posted on . . , : by dale esliger, mark s. tremblay, jennifer l. copeland, joel d. barnes, gertrude e. huntington, david r. bassett jr purpose: this study explored the influence of modernity on the physical activity behaviors (e.g., intensity and timing) of children. methods: children aged - yr living a traditional lifestyle (old order amish [ooa], n = ; old order mennonite [oom], n = ) were compared with children living a contemporary lifestyle (rural saskatchewan [rsk], n = ; urban saskatchewan [usk], n = ). physical activity was objectively assessed for seven consecutive days using actigraph accelerometers. custom software was used to reduce the raw accelerometer data into standardized outcome variables. results: on weekdays, there were group differences in moderate physical activity between all lifestyle groups (ooa > oom > usk > rsk). on the weekend, the group differences in moderate physical activity persisted between, but not within, lifestyle groups (ooa = oom > usk = rsk). during school hours, all groups had similar activity and inactivity periods; however, they differed in magnitude, with the ooa and oom being both more sedentary and more active. in comparison with the children in school, the ooa and the oom children had % lower sedentary time out of school compared with only % lower for rsk and usk children. conclusions: although cross sectional, these data suggest that contemporary/modern living is associated with lower levels of moderate-and vigorous-intensity physical activity compared with lifestyles representative of earlier generations. analyzing the physical activity and inactivity patterns of traditional lifestyle groups such as the ooa and the oom can provide valuable insight into the quantity and quality of physical activity necessary to promote health. copyright © by the american college of sports medicine. categories 	medical and health sciences not elsewhere classified keywords modernitytechnologysedentaryenergy expenditureobesityaccelerometry funding this research was supported by the canadian population health initiative of the canadian institute for health information and the charlie and mai coffey endowment in exercise science at the university of tennessee. history school 	sport, exercise and health sciences published in medicine and science in sports and exercise volume issue pages - citation esliger, d.w. ... et al., . physical activity profile of old order amish, mennonite, and contemporary children. medicine and science in sports and exercise, ( ), pp. - . publisher lippincott, williams & wilkins (©the american college of sports medicine) version am (accepted manuscript) publication date notes this is a non-final version of an article published in final form in medicine and science in sports and exercise, ( ), pp. - at: http://dx.doi.org/ . /mss. b e b afd doi 	https://doi.org/ . /mss. b e b afd issn - eissn - publisher version 	http://dx.doi.org/ . /mss. b e b afd language en administrator link 	https://repository.lboro.ac.uk/account/articles/ licence cc by-nc-nd . exports select an optionrefworksbibtexref. managerendnotedatacitenlmdc categories 	medical and health sciences not elsewhere classified keywords modernitytechnologysedentaryenergy expenditureobesityaccelerometry licence cc by-nc-nd . exports select an optionrefworksbibtexref. managerendnotedatacitenlmdc hide footeraboutfeaturestoolsblogambassadorscontactfaqprivacy policycookie policyt&csaccessibility statementdisclaimersitemap figshare. credit for all your research. cost-effectiveness of pulse oximetry screening for critical congenital heart defects in ontario amit mukerji, md a thesis submitted in conformity with the requirements for the degree of master of science institute of health policy, management and evaluation dalla lana school of public health university of toronto ©copyright by amit mukerji cost-effectiveness of pulse oximetry screening for critical congenital heart defects in ontario amit mukerji master of science institute of health policy, management and evaluation dalla lana school of public health university of toronto ii abstract background: pulse oximetry screening (pos) for critical congenital heart defects (cchds) is being increasingly adopted, but sparsely populated regions in ontario present unique challenges. objective: to estimate cost-effectiveness of pos for cchd in ontario, canada. methods: a cost-effectiveness analysis using a markov model was conducted from a healthcare payer perspective over a life-time horizon. outcome measures, discounted . %, were quality- adjusted life months (qalms), lifetime costs, and incremental cost-effectiveness ratios (icer) [Δcost / Δqalms]. an icer threshold of cad$ , . per qalm was used. probabilistic sensitivity analysis was conducted within expected variable ranges. results: pos is expected to lead to timely diagnosis of cchd cases annually. the estimated icer of cad$ , . was well below the threshold. probabilistic sensitivity analysis estimated a % chance of pos being cost-effective. conclusion: routine implementation of pos for cchd in ontario is expected to be cost- effective. further validation of this model may be conducted following implementation. iii acknowledgements i would like to thank my thesis supervisor dr. vibhuti shah, and members of the thesis committee drs. beate sander, amish jain, eyal cohen and prakeshkumar shah for their support and guidance. i would like to acknowledge the division of neonatology and department of pediatrics at mcmaster university to allowing me the time to work on this while retaining a full- time staff position, in particular i would like to thank our division chief dr. salhab el helou for his support. finally, i would like to thank my family for their help and support during this endeavour. iv table of contents acknowledgements .................................................................................................................... iii table of contents ........................................................................................................................ iv abbreviations .................................................................................................................................. vi . background: clinical problem ...................................................................................... . critical congenital heart disease lesions and clinical impact .............................................................. . pulse oximetry screening for cchd .................................................................................................. . pulse oximetry screening for cchds and cost-effectiveness ............................................................. . background: medical decision models ................................................................... . decision analysis ............................................................................................................................ . markov models ................................................................................................................................ . cost effectiveness analysis ............................................................................................................... . decision model for cchd screening in ontario ................................................ . the base case .................................................................................................................................. . diagnostic strategies ....................................................................................................................... . model structure................................................................................................................................ . parameter values .............................................................................................................................. . outcomes ......................................................................................................................................... . sensitivity analyses ......................................................................................................................... . model validity ................................................................................................................................. . variables and search strategies ............................................................................... . probability variables ........................................................................................................................ . utility variables ................................................................................................................................ . cost variables ................................................................................................................................... . results of decision model ............................................................................................... . base case analysis ............................................................................................................................ . one-way sensitivity analyses ........................................................................................................... . threshold analyses ........................................................................................................................... . probabilistic sensitivity analysis ...................................................................................................... . model validation .............................................................................................................................. . discussion .................................................................................................................................... . summary of results ......................................................................................................................... v . interpretation of findings ................................................................................................................. . assumptions in the model ................................................................................................................ . sensitivity analyses and interpretation ............................................................................................. . comparison to previous studies ....................................................................................................... . strengths and weaknesses ................................................................................................................ . clinical and policy implications ...................................................................................................... . conclusions and next steps .............................................................................................................. . references .................................................................................................................................. appendix a: ....................................................................................................................................... appendix b: ....................................................................................................................................... appendix c: ....................................................................................................................................... appendix d: ....................................................................................................................................... appendix e: ....................................................................................................................................... appendix f: ....................................................................................................................................... vi abbreviations aap – american academy of pediatrics ac – ambulatory care adrql – alzheimer’s disease related quality of life aos – aortic stenosis born – better outcomes registry and network cchd – critical congenital heart disease cet – cost-effectiveness threshold chq – child health questionannaire cihi – canadian institute for health information coa – coarctation of aorta dorv – double outlet right ventricle echo – echocardiogram ekg – electrocardiogram gerd – gastroesophageal reflux disease hlhs – hypoplastic left heart syndrome icer – incremental cost-effectiveness ratio ices – institute for clinical evaluative sciences ip – inpatient lhin – local health integrated network mesh – medical subject heading moh – ministry of health mohltc – ministry of health and long term care occi – ontario case costing initiative pa+ivs – pulmonary atresia with intact ventricular septum pa+vsd – pulmonary atresia with a ventricular septal defect pedsqol – pediatric quality of life scale pos – pulse oximetry screening qalm – quality-adjusted life month qaly – quality-adjusted life year qol-ad – quality of life – alzheimer’s disease sd – standard deviation sf- – short form- spo – oxygen saturation level tacqol – tno-azl child quality of life tga (d-tga/l-tga) – transposition of great arteries (dextro- or levo-) tof – tetralogy of fallot uk – united kingdom usa – united states of america whoqol – world health organization quality of life . background: clinical problem . critical congenital heart disease lesions and clinical impact congenital heart diseases are structural cardiac malformations during fetal development and occur in approximately - per , live births.( , ) of these, up to % are considered “critical” congenital heart disease (cchd) lesions – which are severe lesions that typically require invasive intervention (catheter-based, surgery or a combination of both) within the first months of life.( , ) broadly, cchd lesions are categorized into those that result in mixing of blood carrying deoxygenated and oxygenated hemoglobin (e.g. transposition of the great arteries, truncus arteriosus), and those that result in obstruction of flow to either the lungs and/or to the body (e.g. aortic stenosis, interrupted aortic arch). both these groups of lesions result in suboptimal delivery of oxygen to tissues throughout the body including vital organs such as the brain. as such, these lesions are associated with significant morbidity and mortality, and are responsible for more deaths than any other type of congenital malformation.( , ) early detection of cchds allows for interventions that minimize the duration of impaired tissue oxygenation and is therefore important in order to improve survival, minimize morbidity and improve post-surgical outcomes.( , ) there is a spectrum of severity among various types of cchd lesions. a newborn with transposition of the great arteries with an intact ventricular septum will present soon after birth with symptoms of cyanosis whereas coarctation of the aorta in a newborn may not be clinically apparent for hours or even days after delivery. there may also be differences in the severity and timing of onset of symptoms depending on the degree of abnormality within the same lesion. for example, a tetralogy of fallot with severe obstruction of blood flow to the lungs may be apparent shortly after birth, whereas the same lesion but less severe obstruction may not become apparent for days after birth. additionally, clinical presentation in many cchd lesions is masked by the presence of a patent ductus arteriosus, which is a conduit between the right and left-sided circulations that is essential during fetal life. the presence of the patent ductus arteriosus allows for mixing of blood as well as flow of blood to the body in various cchds, and its closure – normally occurring at - hours of age – often heralds the onset of symptoms. this variability in presentation and specifically, the lack of clinical symptoms and signs in early hours of life results in the lack of detection of many cases of cchd prior to discharge home post-delivery. it has been estimated that - % of cchds are missed by routine physical examination.( , ) cyanosis, the physical sign indicative of tissue hypoxemia and hallmark feature of mixing type cchds, is commonly missed on physical examination, particularly in newborns.( ) routine antenatal ultrasound scans for fetal anatomy detect many cchds, but many cases still remain undetected by the time of delivery.( , ) various factors have been postulated as reasons for lack of detection of all cchd lesions by routine antenatal anatomy ultrasound, including limitations in access in certain rural areas, variability of skill of technicians and subtleties of certain cchd lesions that preclude antenatal detection.( ) nevertheless, cchd lesions remaining undetected prior to discharge home remains a significant burden of illness. a recent review of data from ontario revealed that in a year period from april to march , % of all cchd lesions from hospital births were undetected prior to discharge home.( ) . pulse oximetry screening for cchd pulse oximetry is a simple, reliable, point-or-care technique which is now used routinely in medical practice for assessment of hypoxemia. it employs the use of spectrophotometric methodology, by illuminating the skin and measuring changes in differential light absorption of deoxygenated and oxygenated hemoglobin.( ) this yields the pulse oximetry measured hemoglobin oxygen saturation level (spo ), which has been found to be a reliable estimate of the measured arterial saturation of oxygen (sao ).( , ) pulse oximeters are readily available, and are in widespread use in hospitals – emergency rooms, inpatient settings including intensive care as well as in emergency services such as ambulances. its ubiquity and relative ease of use, along with its reliability in estimating oxygen saturation of hemoglobin, sparked interest in its use as a screening tool for detection of cchds.( ) as described earlier, many cchds that are undetected clinically do have underlying hypoxemia which is not reliably determined on clinical exam, making pulse oximetry an attractive screening tool to help reduce morbidity and mortality associated with missed cchd. a low spo detected on pulse oximetry would trigger a detailed evaluation for the presence of cchd, which may include an echocardiogram. initial studies on the use of pulse oximetry screening (pos) for detection of cchd were conducted in the early s and were seen as largely “proof of concept”.( , ) the largest of these were by koppel et al, a study of more than , well appearing newborns, which found a sensitivity of . %, specificity of . % and false positive rate of . %.( ) since that time, numerous studies have confirmed these findings,( - ) and a systematic review of this literature published in ( studies representing , babies) found an overall sensitivity of . % for detection of cchd, overall specificity of . % with a false positive rate of . %.( ) the sensitivity of the screening tool appears rather low compared to other screening tools, but is due to the fact that some cchds do not present with hypoxemia until closure of the patent ductus arteriosus, which may occur at a time following the conduct of the screening test. as such, it is known and accepted that not all cchds will be detected by pos.( ) nevertheless, based on the accumulating evidence on the effectiveness of pos as a screening tool for cchd in comparison to clinical examination alone, in the united states secretary of health and human services recommended the inclusion of cchd to the panel of conditions routinely screened for in the newborn period.( ) this view was subsequently also endorsed by the american academy of pediatrics (aap).( ) implementation of pos for cchd in the usa has been rapid, with states and the district of columbia incorporating this practice into routine newborn screening within years of the endorsement by the aap.( ) many centres in the usa and uk have also published their local experience with the implementation of pos for cchd, and have reported detecting cchd cases that might have been missed prior to initial hospital discharge.( - ) however, there are other common themes from these studies, namely: (a) the detection of other non-cchd conditions among the “false-positive” cases including sepsis, respiratory illnesses, non-critical cchds; (b) re-affirmation that not all cchds will be detected by pos; and (c) feasibility of pos implementation even in centres that do not have access to immediate definitive testing with echocardiography. some other important points were that (a) there is still variation in the list of congenital cardiac lesions that are labeled as cchds; and (b) there are discrepancies in the algorithm of pos testing (including cut-off values for what is considered a “positive” screen).( ) finally, most centres in the us and uk who report on pos implementation have relatively quick access to confirmatory echocardiogram, even if it is not in the centre where baby is delivered. for instance, in the relatively sparsely populated state of wisconsin, usa, same- day neonatal echocardiography service was available for approximately % of births, and for hospitals without this service, the nearest regional centre was on average only miles ( km) away.( ) in summary, in settings where implementation of pos for cchd has been reported, it appears to be feasible and has led to the detection of cases that would have otherwise been missed. in recent months, the canadian cardiovascular society, canadian pediatric cardiology association and the canadian pediatric society have all endorsed implementation of routine pulse oximetry screening for well appearing newborns.( ) however, this has not been implemented in all hospitals in ontario, and as described in the subsequent sections, it remains unknown whether this will be a cost-effective endeavour in ontario. . pulse oximetry screening for cchds and cost-effectiveness unlike most other screening tests, pos is a point-of-care test that requires real-time interpretation and in cases of positive result – immediate action to provide timely diagnosis and appropriate management. while there have been various algorithms used for pos, the most commonly employed is the algorithm proposed by the aap (appendix a). this algorithm indicates that screening should be performed at > hours of age (or before discharge if discharge is at < hours) on the right hand (“pre-ductal”) and either foot (“post-ductal”). if either reading is < %, the result is considered a positive screening result. if either reading is ≥ % and the difference between the readings is ≤ %, the result is considered a negative. results outside of these scenarios results in repeat testing after hour for up to additional tests.( ) unless another reason for the hypoxemia is identified in the ensuing investigations following a positive screen, an echocardiogram must be performed, which may require transfer to another centre where neonatal echocardiography is available. due to the resource implications, particularly the lack of availability of neonatal echocardiography at all centres, as well as the burden of a potentially large number of false positive results; there have been a number of cost-effectiveness analyses of pos for cchd.( , - ) a summary of the cost-effectiveness analyses published to date is shown in table . below. table . : cost-effectiveness analyses of pos for cchd author, year study characteristics summary of findings knowles, ( )  uk-based study on cost- effectiveness of pos (icer per diagnosis of cchd)  time horizon: until diagnosis of cchd established  perspective: national health service (uk)  icer was £ , per additional cchd diagnosed  sensitivity analysis showed % chance of being cost- effective at cost- effectiveness threshold of £ , griebsch, ( )  uk-based study on cost- effectiveness of pos (icer per diagnosis of cchd)  time horizon: until diagnosis of cchd established  perspective: national health service (uk)  icer was £ , per additional cchd diagnosed  deemed “likely” to be cost- effective de-wahl granelli, ( )  used model created by griebsch et al ( ) and employed swedish- based population data  perspective: not specified  icer was estimated at £ , per timely diagnosis of cchd  estimated to be “at a minimum” cost neutral ewer, ( )  uk-based study on cost- effectiveness of pos (icer per diagnosis of cchd)  time horizon: until diagnosis of cchd established  perspective: national health service (uk)  icer was £ , per additional diagnosis of cchd  deemed “likely” to be cost- effective roberts, ( )  uk-based study on cost- effectiveness of pos (icer per diagnosis of cchd)  time horizon: until diagnosis of cchd established  perspective: national health service (uk)  expected to lead to timely diagnosis of additional cases of chd per , births  icer was approximately £ , per additional timely diagnosis of cchd  sensitivity analysis showed % chance of being cost- effective at a cost- effectiveness threshold of £ , peterson, ( )  us-based study on cost- effectiveness of pos (icer per case identified and per life-year gained)  time horizon: infancy (< year)  perspective: us healthcare sector  icer was $ , per case identified and $ , per life year gained  sensitivity analysis indicated a % and % chance of cost-effectiveness at a threshold of <$ , and < $ , per life year gained, respectively abbreviations: cchd – critical congenital heart disease; icer – incremental cost-effectiveness ratio; pos – pulse oximetry screening . knowledge gap on cost -effectiveness in ontario all of the above models suggest that screening for cchd using pulse oximetry is likely to be cost-effective. however, there are some important considerations from these analyses. with the exception of the swedish model by de-wahl granelli et al and the us analysis by peterson et al, all others were from the uk, where the availability and distribution of clinical resources is much different than in ontario. for instance, in the analysis by peterson et al, they estimated that approximately % of infants would require transport to another facility to complete a confirmatory echocardiogram.( ) this figure is expected to be much higher in ontario, as only . % of all deliveries occur at a level institution with immediate access to echocardiograms, and it is estimated that only up to % of level hospitals have intermittent access to echocardiography and often only during regular office hours (described in section ). in the uk studies (which used similar data as identified by knowles et al), it was estimated that the cost of transport would be £ , which is a reflection of a relatively densely populated region with relatively quick access to tertiary level care centres.( ) on the other hand, in a geographical distribution such as that of ontario whereby densely populated regions are intermixed with remote locations that are very far from regional centres, even a relatively low percentage of deliveries in the latter regions may incur significantly more costs. even among deliveries in the densely populated regions, the majority of babies will still require transfer to another centre where an echocardiogram can be performed. (appendix b contains a description of levels of care on newborns in ontario and the geographical based local health integration networks (lhins) which plan, integrate and fund local health care, as it relates to pos screening for cchd). another limitation of the previous studies is that the cost effectiveness models represent the time until a diagnosis is made, with the exception of the study by peterson et al where the time horizon spanned year.( ) it may be argued that if pos is likely to be cost-effective until a diagnosis is made, it is even more likely to be cost-effectiveness over a longer time horizon. however, one factor that has not been included in previous studies is the quality of life associated with cchds and particularly in cchd cases where there may be associated morbidity, which may have an impact on cost-effectiveness. in light of the aforementioned limitations as well as in consideration of some of the unique logistical challenges towards the implementation of pos for cchd in ontario as described above, we conducted a cost-effectiveness analysis of the implementation of this screening program. this was a model based economic evaluation run over a life-time horizon, incorporating quality of life indicators, and conducted from the perspective of the ontario healthcare payer. the details of the decision model and its structure are delineated in section . . background: medical decision models . decision analysis originally developed in the field of economics, decision analysis has been applied to the field of medicine in order to rationalize often complex medical decisions,( ) both at an individual patient level (e.g. with regards to choice of a particular treatment strategy), as well as at an institutional or policy level (e.g. implementation of routine publicly funded vaccination programs). inherent in such complex decisions is a large degree of uncertainty as to what is the “optimal choice”, which often cannot be rationalized in an unstructured approach.( ) medical decision analysis is a structured, explicit and systematic approach to rational decision making incorporating probabilities of events occurring and the values (also known as “utilities”) associated with various health states or conditions.( ) medical decision analysis is conceptualized in the form of a decision tree, whereby a decision is divided into two main “stems” representing the two choices, each in turn followed by a series of chance occurrences (based on probabilities of events occurring depending on the medical condition under evaluation, and outcomes of interventions that may follow). the various possible health outcomes are associated with a “utility” value. patient utility is assigned an index value between (death) and (perfect health), which incorporates individuals’ assessment of and value associated with a particular health state, representing the quality of life.( ) the utilities associated with various health states are multiplied by the chances of each health state occurring (based on the probabilities), the sum of which yields the expected utility resulting from each of the decision choices. the decision with the higher expected utility is regarded as the “optimal” decision. the following example illustrates the concepts described above. in this hypothetical situation, medical “condition x” (with a quality of life value or utility score of . ) has a treatment available, which cures the condition but also has the possibility ( %) of a significant side effect (associated with a quality of life value or utility score of . ). on the other hand, not treating the condition results in a very small possibility ( %) of death. without a structured approach, it may not be clear which option is the most optimal approach for an individual patient. however, this can be turned into a structured rational approach by use of a decision analysis tree, as shown in figure . below. by convention, a decision is indicated by a square node, a round node represents a chance occurrence (based on known probabilities), and a triangular node represents the end of the decision tree, usually culminating in a particular health state. figure . : an example of a simple medical decision analysis tree regarding whether to treat a hypothetical condition x in this above example, the expected utility of each decision option (i.e. treating vs. not treating) will be calculated by multiplying the probability of health outcome by its utility and adding together the expected utilities of each health state resulting from each choice. for treatment, this would yield: ( . x . ) + ( . x . ) = . estimated utility. on the other hand, for no treatment, the estimated utility will be: ( . x . ) + ( . x ) = . . therefore, in this example, based on the utilities assigned to each health state, treatment of condition x would be the “rational” and preferable choice, despite the possibility of a severe complication of treatment. . markov models a simple decision analysis tree, as described above, has several practical limitations. one of the most important limitation is having to identify a finite time-horizon over which the model is applicable. this has limitations, as many of the health states described at the end of model may not be the “final” outcomes, and that individuals may have variable life spans. in the example of condition x above, one cannot explore condition x over time. presumably, remaining in condition x over the years will shorten the life expectancy, but due to a set time- frame this cannot be evaluated. one option is to design a “recursive” tree, whereby condition x (with a probability of . of death each year) is run over a chance node repeatedly. however, this approach becomes impractical in complex decision trees. another related limitation of regular decision analysis trees is the inability to change probabilities over time. what if condition x has varying probability of death (e.g. as a person ages, the probability of death increases)? regular decision trees cannot easily incorporate such variations in probabilities over time. finally, one of the important outcome measures in medical decision modeling is determining “quality-adjusted life spans” resulting from medical decisions or policy implementations (to be discussed in section . ). while these can be derived in regular decision models, such outcome measures are very readily derived by employing markov models.( ) also known as health state transition models, markov models place simulated individuals or a cohort into one of a number of exhaustive and mutually exclusive health states. the model is run over time, wherein during each time cycle, a simulated individual (or a certain fraction of the cohort) in the model may “transition” from one health state to another, or remain in the same health state as in the previous cycle.( ) cycles are run repeatedly in this model, with health state transitions occurring with each cycle, until either: (a) all simulated individuals are transitioned to the absorbing health state (usually “death”); (b) a predetermined number of cycles have run; or (c) the “incremental” utility (i.e. the utility added by running another cycle of the model) is lower than a predetermined threshold. a simple example of a markov model is depicted in figure . below. figure . : a simple markov (health state transition) model. in the example shown in figure . above, simulated individuals in the model are placed in of mutually exclusive health states – alive or dead. at time , all individuals are alive. with each cycle of the model, a certain fraction of alive individuals “transition” to the death state – determined by the “transitional probability”, while the remainder stay in the same health state of alive. the transition probability can be made to vary over time if needed. a markov model may have any number of health states, so long as they are mutually exclusive (i.e. there can be no ambiguity about which health state an individual simulated patient can belong to) and exhaustive (i.e. an individual simulated patient must be able to find a “home”). each cycle of a markov model yields “incremental” expected utility for each cycle – determined by the sum of the proportion of simulated individuals in each health state multiplied by the utility values associated with the respective health state during the given cycle. as will be described in the following section . , the cost associated with each health state may also be incorporated to yield an “incremental” total cost associated with each cycle of a markov model. . cost effectiveness analysis when applying medical decision analysis at the level of an individual patient, cost may not be an important factor in most cases – particularly in settings where the cost is incurred by a third party (e.g. insurance company or public funds). this is because both the health care practitioner and the patient have the interest of the individual patient as their primary focus.( ) however, when applying medical decision analysis at the policy level – e.g. whether to implement a publically funded vaccination program for influenza – cost is of paramount importance.( ) once it is established that a particular medical strategy is clinically effective, feasible, and does not lead to significant harm or side effects, cost becomes an important factor in determining whether widespread implementation of such a strategy is in the interest of the population as a whole (as compared to another initiative where the funds may be better suited – or more “cost effective”). as such, cost-effectiveness analyses are an important piece of evidence that helps guide the most effective allocation of resources. a cost-effectiveness analysis compares the relative costs and outcomes of two or more interventions or course of action (e.g. implementation of a vaccination program vs. not implementing the vaccination program, or medical treatment vs. surgical treatment for medical condition “x”). a cost-effective analysis may be performed from a healthcare payer perspective or from a societal perspective, and may be run over varying time horizons depending on the nature of the question. the effectiveness refers to the outcome being evaluated, which may incorporate quality of life indicators (in such a case the analysis is more correctly termed a cost- “utility” analysis), yielding what is known as “quality adjusted life year (qaly)” (or any other arbitrary unit of time). when one treatment or intervention is more effective with respect to the outcome at a lower cost, the decision is simple. however, the decision becomes more complex when one intervention is more effective but also costs more. to help standardize this in order to allow for comparisons with another intervention for the same condition (or whether to use the limited available resources to fund a different intervention for another medical condition entirely), the concept of incremental cost-effectiveness ratio (icer) is used.( ) the icer is the ratio of the “incremental” costs incurred by a certain medical intervention divided by the “incremental” qalys achieved. in effect, this yields the how much it would cost to gain a unit of qaly (or any other unit of quality-adjusted life). this icer may then be compared against a pre-determine icer “threshold”, below which an intervention is deemed cost-effectiveness. this icer threshold is also known as the “cost-effectiveness” threshold (sometimes also referred to as the “willingness to pay” threshold).( ) any intervention icer that falls below the cost-effectiveness threshold (cet) threshold would be cost-effective, whereas any intervention with an icer above the cet threshold deemed not. in canada, an icer threshold of $ , per qaly is commonly considered to be cost-effective.( ) figure . below illustrates the concept of icer and cet thresholds. figure . : plot of incremental cost vs. incremental effectiveness in the hypothetical example shown above in figure . of various interventions, intervention a costs less and leads to more effectiveness and as such is an easy choice for implementation. similarly, intervention b costs more and is less effective, therefore is an obvious decision to not be implemented. interventions c and d are in the quadrant where the cost is greater but so is effectiveness. however, intervention d is below the cet (therefore would be deemed cost effective), whereas intervention c lies above the threshold and would not be considered cost-effective. any intervention for which the incremental cost and incremental effectiveness plots “below” the cet slope is deemed to be cost-effective. . decision model for cchd screening in ontario the following section delineates the development of the medical decision model structure employing markov cycles for the implementation of cchd screening using pos in ontario, canada. a cost-effectiveness (utility) analysis was developed following the canadian agency for drugs and technologies in health guidelines,( ) and using ontario-specific data wherever possible. . the base case the base case subject was a well newborn infant ~ hours old born in ontario. the newborn could be born at home or in hospital but must be born in the presence of a qualified attendant (midwife, family doctor, or obstetrician) capable of performing pos. following birth, the infant had to have been asymptomatic of cchd and not requiring any medical attention beyond routine care for the first hours of life. . diagnostic strategies we compared two diagnostic strategies: pulse oximetry screening and no pulse oximetry screening (figure . ). our perspective was that of the ontario ministry of health (healthcare payer) figure . : base case and diagnostic strategies . model structure treeage pro software . . . -v (treeage software inc., williamstown, ma, usa) was used to create a markov decision model with one-month cycles, allowing lifetime follow-up of the simulated study cohort. the following exhaustive and mutually exclusive health states were created: ) cchd (pre-op); ) no cchd (< month); ) post-op cchd (with morbidity); ) post-op cchd (no morbidity); ) no cchd (> month), and ) death. at any given point in time, a simulated individual could only be in of the aforementioned mutually exclusive health states. at the beginning of cycle , all simulated individuals could only be in either one of the following two health states: ) cchd (pre-op); or ) no cchd (< month). an individual with (undiagnosed) cchd at the time of hours could have the screening performed and yield either a (true) positive result or a (false) negative result, latter being a case of missed cchd. similarly, an individual with no cchd could have a (false) positive or a (true) negative result. all positive results (regardless of whether true or false) were designed to require follow-up which may or may not have included a series of transfers to a tertiary level care facility. a brief overview of this initial portion of the model schematic for both health states is as outlined in figure . below. figure . : brief overview of results from pos screening in first markov cycle. panels a and b depict the diagnostic possibilities in an individual with cchd and without cchd, respectively in the initial markov cycle, the model depicted in detail the possibilities of patient location and requirement of transfers (as outlined in figure . ), and in cases of confirmed cchd, the possible outcomes from a cchd surgery (figure . ). the model also depicted the possible outcomes in the event of a missed cchd sent home after birth hospitalization (figure . ). in the figures below, the green circles represent a “chance” node with a certain probability associated with either arm emanating from that node being chosen (determined by the values for probabilities inputted into the model) while the red triangles represent a “terminal” node, culminating in the transition to another health state. figure . : illustration of decision tree during first markov cycle and various possible outcomes, in a simulated individual who has cchd figure . : possible outcomes of cchd surgery incorporated into first cycle of markov model figure . : illustration of possible outcomes after missed cchd incorporated into first cycle of markov decision model the end of the first markov cycle resulted with a simulated individual transitioning to of the following remaining mutually exclusive health states: ) post-op cchd (no morbidity); ) post-op cchd (with morbidity); ) no cchd (> month); or ) death. for the purpose of this model, morbidity referred to any neurodevelopmental impairment (section ). in subsequent markov cycles, a simulated individual could stay in the same health state, or transition to another health state. these probabilities varied over time (section ). the markov cycles continued until all individuals transitioned to the health state of “death” in keeping with a life-time horizon of the model. possible transitions amongst health states in these subsequent markov cycles are depicted in figure . below. figure . : illustration of the possible transitions (denoted by arrows) amongst the health states in subsequent markov cycles . parameter values three major categories of variables were used the decision model: (a) probabilities; (b) utilities; and (c) costs. probabilities determined the path through the decision tree that any simulated individual took during the first markov cycle, and the transitions amongst health states in the subsequent cycles. utilities were an indicator of the quality of life associated with being in any particular health state, as well as “transitional utilities” associated with temporary phenomenon such as transfers, having pos performed and having surgical procedures. these utility values were used to determine the expected number of “quality adjusted life months” with either diagnostic strategy (i.e. implementation of pos vs. no pos), and are described in further detail below. finally, costs associated with all procedures (including pos, echocardiograms), transfers, surgical procedures and being in health states were inputted to identify the comparative lifetime costs of either diagnostic strategy. given the life-time horizon of the model, all costs were discounted . % annually to adjust for inflation, as per canadian guidelines.( ) it is also acknowledged that for utilities and costs, spillover (caregiver) effects were not considered as part of this model, and values for these variables represented the individual patient. the only exception to this was the group of utility variables designated as “dis-utilites” associated with pos screening itself, transfer and surgery – which represented parental disutility associated with these action steps. . outcomes . . quality adjusted life months the quality of life in each cycle ( month) in a given health state was represented by a corresponding incremental utility score associated with that health state. the utilities could range from (death) to (perfect health/no cchd). each cycle of the model used the incremental utility to determine the quality adjusted life month (qalm) for that given health state. run over a lifetime horizon, the model yielded the expected per patient qalms with pos and per patient qalms without pos implementation. . . costs the per cycle incremental costs of being in a given health state included medical costs to the public health care system. in addition, costs of pos, transports, echocardiograms and hospitalizations were incorporated into the model. the model yielded the lifetime cost per individual with either diagnostic strategy (i.e. pos implementation vs. without pos implementation.) . . cost-effectiveness (utility) analysis incremental cost-effectiveness (utility) ratio (icer) expressed as added cost per qalm gained with pos (versus no pos) were calculated from the above outcomes. in order to determine the value of pos from a health care perspective, a cost-effectiveness threshold of cad$ , per qalm (equivalent to cad$ , per qaly, a commonly used cost- effectiveness threshold)( ) was used and our icer result was compared to this threshold. all outcomes were discounted at . % annually, as mentioned previously. . sensitivity analyses one-way sensitivity analyses were performed for each parameter value used in order to assess parameter uncertainty. in these analyses the model was run at various pre-specified intervals for each included variable within their plausible ranges. the model was considered robust if the overall icer result at any of the interval values did not change from the main analysis using point estimate values for each variable. the plausible ranges for each value were derived from existing literature where available. in cases when a plausible range was not available from the literature, a monte-carlo simulation with beta-distributions was run using the raw numbers (events over total number) to estimate the range – this strategy was employed for probability and utility values. for cost variables without a plausible range from the literature, a % reduction and increase in the estimate was used to determine the lower and upper ranges, respectively. the derivation of these ranges for all variables is described in detail in section . in addition to the one way sensitivity analysis, for select variables (either due to significant uncertainly regarding their plausible ranges, or their importance in the decision model), an analysis of extreme thresholds was run, where the range was extended to extremes and one-way sensitivity analyses were run. finally, a probabilistic sensitivity analysis was conducted in which multiple simulations were run where selected variables were varied simultaneously. this was used to generate an icer scatter plot as well as a cost-effectiveness acceptability curve (section ). . model validity validity of the model generated was assessed via face validity as the extent to which the model and its assumptions and applications correspond to current science and evidence.( ) verification of the model structure was performed by b.s., one of the thesis committee members with expertise in medical decision modeling, as well as other committee members with significant clinical experience and expertise in the areas of neonatology and neonatal cardiology. an external validation was deemed to be beyond the scope of the current study, but recognized that following implementation of pos screening – there will be adequate data available from the province to conduct this validation at a later time. . variables and search strategies . probability variables . . baseline health state probabilities . . . probability of a well appearing individual at hours age (base case) having cchd the incidence of critical congenital heart disease (cchd) is estimated to be . %.( ) a recent report from the institute for clinical evaluative sciences (ices) reported that over a year period (april to march ) in ontario, % of patients with cchd were missed and sent home from hospital prior to establishing the diagnosis.( ) these are the individuals with cchd who are likely to represent the base case of a well appearing baby, and as such the probability of cchd in a well appearing baby was estimated to be % of . % = . . to estimate the range, a monte-carlo simulation with a beta-distribution was run ( samples) which yielded an estimate of . for the . th%ile and . for the . th%ile. . . . probability of a well appearing individual at hours age (base case) not having cchd an individual without cchd at hours of age was designated in this (only other) alternate health state, yielding a point estimate probability value of . . a monte-carlo simulation with beta-distribution was run ( samples) to estimate the range of . to . . . . transitional probabilities – mortality a detailed literature search was conducted on medline with the help of a reference librarian (ms. laura banfield, health sciences library, mcmaster university) for mortality related to cchd (appendix c). there were , records identified from this search (after elimination of duplicate records). after screening titles and abstracts, records were reviewed in detail for data on short-term mortality related to cchd (within st month of life). for long- term mortality rates associated with cchd (beyond first month of life), records were reviewed in detail. after eliminating non-western european/non-north american studies, studies related to specific lesions/procedures, review articles, case reports (or series with < patients), and studies deemed not relevant including those that did not provide raw data of mortality or survival rates, a final of articles provided data for short-term mortality, whereas articles provided data for long-term mortality. . . . probability of early mortality ( st month) if individual has cchd from the search strategy outlined in appendix c, articles ( - ) were included in the analysis to determine the point estimate of the probability of early mortality with cchd. data from these studies (individual proportions) were meta-analyzed using a binary random effects model (meta-analyst software, brown university, usa), yielding a probability point estimate of . (with a % confidence interval of . to . ), as shown in figure . below. figure . : forest plot of proportions of early mortality with cchd . . . relative risk of early mortality ( st month) if cchd is detected late the search strategy for mortality and complications did not yield any suitable records for this variable. therefore, the following targeted search strategy was conducted on medline on august , : "mortality"[title/abstract] and "congenital heart disease"[title/abstract] and "hospitalization"[title/abstract] and "late"[title/abstract]. this search yielded articles, only of which (peterson et al. )( ) was reviewed in detail (full text) after screening of titles and abstracts. in this study, the mortality rate with early detection (prior to initial hospital discharge) was . % whereas it was . % with late detection. this was counter to the intuitive hypothesis, and is assumed to be due to the fact that more severe lesions were probably picked up clinically prior to discharge (and not in the scope of the base case our model is targeting), while the indolent and less severe lesions were discharged home, and likely to have less impact on mortality. additionally, since the lesions detected prior to discharge home in this study were detected clinically, rather than detected via a screening program, it is once again not generalizable to our context. hence this data was not used in the model. based on author consensus, it was agreed that a point estimate relative risk of neonatal mortality of . would be used for late identification of a cchd (with an estimated range of to ). . . . probability of all-cause neonatal ( st month) mortality this data was obtained from statistics canada report ( ) on mortality ( ) when there were neonatal deaths in ontario out of , births, yielding a probability of . . because some neonatal mortality in our model is expected to include undiagnosed cchd cases, and also due to the relatively low burden of cchd as a cause of mortality compared to all causes, we did not alter this number. using a monte-carlo simulation with beta distribution, a . % to . % range of . to . was estimated. . . . probability of a home death ( st month) if have cchd the following targeted search strategy was conducted on medline on august , : "mortality"[title/abstract] and "home"[title/abstract] and "missed"[title/abstract] and "congenital heart disease"[title/abstract]. this search yielded only article (de-wahl granelli et al ),( ) in which study out of patients who were discharged with a missed cchd died at home, yielding a probability of . . a monte-carlo simulation with beta distribution resulted in an estimated range of . % to . % of . to . , this large range reflecting the small sample size in original data and thus large degree of uncertainty around this parameter. . . . probability of death per cycle if individual does not have cchd the probabilities (annual) of mortality for all ages from to were obtained from statistics canada – data available at: http://www.statcan.gc.ca/pub/ - - x/ /article/ /fig/desc/desc -eng.htm (accessed on august , ). the annual probabilities of mortality were converted to monthly probabilities of mortality by the following formula: monthly mortality = – ([ – (average annual mortality probability)]^( / )) each point estimate was then multiplied by . and . to yield an estimate of the low and high ranges, respectively. for ages - years where the average monthly mortality rate was , the upper range for ages and were used. this data is shown in appendix d. . . . probability of death per cycle if individual is post-cchd repair without any morbidity from the detailed literature search for mortality as described above, articles provided data for long-term mortality rates related to cchd.( , , , , , - ) detailed characteristics and outcome data from these studies are as shown in table . below. table . : characteristics of studies reporting on long-term mortality associated with cchd http://www.statcan.gc.ca/pub/ - -x/ /article/ /fig/desc/desc -eng.htm http://www.statcan.gc.ca/pub/ - -x/ /article/ /fig/desc/desc -eng.htm author, year age at assessment cchd lesion survival rate alexiou years tof / ( %) amark years pa+vsd / ( %) bove year various / ( . %) brown years aos / ( %) cleves year various / ( . %) cross year hlhs / ( . %) daubeney years pa-ivs / ( . %) dyamenahalli years pa-ivs / ( %) fixler years various / ( . %) frike years tga / ( . %) ghanayem year hlhs / ( . %) hirsch year hlhs / ( %) lee years coa / ( %) losay years tga / ( %) oster year various / ( . %) prandstetter years tga / ( . %) turon-vinas years tga / ( . %) wong years tga/dorv / ( %) studies that reported on probability of survival at year’s age were meta-analyzed together, resulting in a pooled point estimate probability of survival of . ( % ci . to . ), as shown in figure . below. this was converted to monthly probability of survival over - months of life (excluding neonatal mortality period which was already ascertained previously), by solving for x in the following equation x = . . this yielded a monthly probability of survival of . . similar calculations were performed for the % cis, yielding . to . . finally, these values were converted to monthly probabilities of mortality (by subtracting from ) yielding final point estimates and % cis over months - of: . ( % ci: . to . ). figure . : forest plot of studies reporting on survival associated with cchd in st year of life the remaining studies that reported on survival rates beyond year of age were pooled separately, as shown in figure . below, yielding a pooled point estimate of survival of . ( % ci . to . ). a similar procedure described for conversion to monthly mortality as for months - was performed, assuming a constant cumulative mortality rate from age to years ( years x months = months). a similar process as described earlier was used to derive estimates of monthly mortality rates and % ci, yielding transitional monthly probabilities of death of . ( % ci . to . ). although there was scarcity of data that fit the criteria for study inclusion beyond age years, it was assumed that a similar rate of mortality would continue beyond years’ age. figure . : forest plot of studies reporting on survival associated with cchd beyond the st year of life . . . probability of death per cycle if individual is post-cchd repair with morbidity no data specific to this health state transition was available from the detailed literature search. it was assumed that some individuals in the studies for mortality rates indeed had cchd associated morbidity. as such, the same probability estimates as for the probability of death per cycle for individuals post-cchd repair without morbidity were used for this set of transitional probabilities, although acknowledged that in reality it may be higher. . . transitional probabilities - morbidity a detailed literature search was conducted on medline with the help of a reference librarian (ms. laura banfield, health sciences library, mcmaster university) for morbidity related to cchd (appendix e). this search yielded , articles after exclusion of duplicates, and after initial screen of titles and abstracts, articles were evaluated in detail (full text). out of these, articles met pre-defined eligibility criteria (after exclusion of studies that were non- western europe/non-north american in origin, studies that related to a very specific lesion/procedure, case/report or series of < subjects, or articles deemed not relevant). the articles, their characteristics and outcomes are as listed in table . below.( - ) table . : study characteristics and data for morbidity related to cchd author, year age range cchd lesion morbidity evaluation morbidity probability bellinger years d-tga wechsler iq > sd below norm / ( %) bellinger years d-tga executive function above cut-off for clinical concern (by parental report) using brief tool / ( %) fuller years various wechsler iq > sd below norm / ( . %) gaynor year various bayley mdi > sd below norm / ( %) jonas year various bayley mdi > sd below norm / ( . %) neuberger year various bayley mdi > sd below norm / ( . %) wernovsky years hlhs weshcler iq > sd below norm / ( . %) abbreviations: d-tga – dextro-transposition of the great arteries; hlhs – hypoplastic left heart syndrome . . . probability of early morbidity ( st month) if individual has cchd the studies reporting on probabilities morbidity over the first year were meta-analyzed using meta-analyst software (brown university, usa), which yielded a pooled probability estimate of . ( % ci . - . ), as shown in figure . . this was taken to be the probability at the year mark, which was then converted to monthly probabilities by solving for x in the following equation x = . . this yielded a monthly probability of developing cchd associated morbidity of . over the first year of life. similarly, the monthly % cis for the probabilities were estimated to be . to . . these transitional probability values were used for the first month, as well as the subsequent months of life. figure . : forest plot of pooled estimate of cchd associated morbidity at year of age . . . relative risk of early morbidity ( st month) if cchd is detected late none of the articles included from the detailed search addressed increase in morbidity based on late detection of cchd. therefore, a targeted search on medline was conducted on september , using the following search terms: "morbidity"[title/abstract] and "congenital heart disease"[title/abstract] and "hospitalization"[title/abstract] and "late"[title/abstract]. this search yielded articles, neither of which were relevant based on review of titles and abstracts. as such, a relative risk of . was assumed after consensus amongst authors, with an estimated range of to . . . . probability of an individual post-cchd repair without morbidity developing cchd- associated morbidity the probabilities of morbidity related to cchd from remaining studies identified in table . above were similarly meta-analyzed to yield an overall pooled point estimate probability of . ( % ci . to . ), as shown in figure . . this probability was assumed to represent a cumulative occurrence over ages year to years (representing x = months). this pooled probability was then converted to monthly probabilities by solving the following for x: x = . , yielding monthly probability of cchd associated morbidity of . . similar conversion of the % cis yielded a range of . to . . there probability estimates were used until age . after that stage no data were available. it was assumed and agreed upon by authors based on consensus that development of new morbidities after that age would be exceedingly rare, and as such, probability values of were used thereafter. figure . : forest plot of pooled estimate of cchd associated morbidity at ages - years . . population distribution and levels of care . . . probability that an individual patient will deliver at a level facility a customized request was made to better outcomes registry & network (born) ontario whereby all deliveries in ontario between april , and march , categorized by level of care were provided. out of , ontario deliveries in this time period, , occurred at level facilities, yielding a probability of . . a monte-carlo simulation with beta-distribution ( samples) yielded a . % to . % range of . to . . . . . probability that an individual in level or level catchment area delivers at a level facility a customized request was made to born ontario whereby all deliveries in ontario between april , and march , categorized by level of care were provided. there were , births in level facilities while there were , births at a level facility, yielding a probability of . ( , /[ , + , ]). a monte-carlo simulation with beta-distribution yielded a . % to . % range of . to . . . . . probability that an individual patient’s home hospital is a level facility a customized request was made to born ontario whereby all deliveries in ontario between april , and march , categorized by level of care were provided. there were , births at a level facility, out of a total of , ontario births, yielding a probability of . ( , / , ). a monte-carlo simulation with beta-distribution yielded a . % to . % range of . to . . . . . probability that an individual belongs to sickkids lhin this data was extracted from the born ontario annual reports from - and - . all births in ontario were categorized by lhin in these reports. lhins , , , , are served completely by sickkids region, while lhins and are shared with another centre; for the latter it was assumed that half the deliveries belonged to the sickkids region. after eliminating individuals with missing data, there were , births in ontario registered in born, out of which , deliveries occurred in the sickkids catchment area, yielding a probability of being born in sickkids region of . . a monte-carlo beta distribution simulation yielded a range of . to . . . . . probability that an individual patient is from the northern region that requires air transport it was assumed that a patient born in lhins or would require air transport due to the remote nature of these locations and distance from the nearest level facility. as such, from the born ontario annual reports for / and / , all deliveries in these lhins ( , ) were divided by the total number of births in ontario ( , , after eliminating births with unknown lhin). this yielded a probability of . , with a . % to . % range of . to . by way of monte-carlo simulation using beta distribution ( samples). . . techniques and detection rates . . . probability that a level facility has pediatric echocardiography capability for each level neonatal intensive care unit, the proportion of their regional level nurseries with the capability to perform pediatric echocardiograms was ascertained by way of personal communication (mcmaster university: dr. michael marrin, transport director, neonatology; university of ottawa: dr. stephanie redpath, transport director, neonatology; western university: dr. henry roukema, transport director, neonatology; university of toronto: dr. catalina tomayo, former clinical fellow, pediatric cardiology). it was estimated that out of the level facilities have access to pediatric cardiology and echocardiogram (albeit not / coverage). this yielded a probability of . and a monte-carlo simulation with beta-distribution yielded and . % to . % range of . to . . . . . probability of a false negative echocardiogram result at a level facility the following search strategy was conducted on medline on july , : "congenital heart"[ti] and ("echocardiography"[all fields] or "echocardiogram"[all fields]) and ("accuracy"[all fields] or "sensitivity"[all fields] or "precision"[all fields] or "miss*"[all fields]). this search yielded articles, and after screening of titles/abstracts – were reviewed in detail and were included.( , ) combining data from these studies, it was noted that out of , cases – there were missed cases, yielding a probability of . . the missed cases were identified by way of "sources of diagnostic error case discovery included information obtained from other tests (e.g., cardiac catheterization, magnetic resonance imaging), operative observations, subsequent echocardiographic examinations, and autopsy" in the former and "( ) results of subsequent imaging tests, consisting of either repeat echocardiograms or cardiac mri; ( ) cardiac catheterization; ( ) surgery; ( ) clinical follow-up; and ( ) autopsy" in the latter study. a monte-carlo simulation with beta-distribution yielded a . % to . % range of . to . . . . . probability of a false negative echocardiogram result at a level facility no such data specifically for level pediatric echocardiograms were available in the aforementioned literature search (which was not specific to level of care of facility). it was assumed that the false negative rate would be similar at a level facility and similar values as for level facilities were used in the model. . . . probability of a false positive echocardiogram result at a level facility a medline search was conducted on july , using the following strategy: "congenital heart"[ti] and ("echocardiography"[all fields] or "echocardiogram"[all fields]) and "false positive"[all fields]. this search yielded articles; however, after review of titles/abstracts, none of them were suitable for the specific question. after discussion amongst authors, it was agreed that a probability of for a false positive echocardiogram for cchd at a level facility would be a reasonable estimate, with a proposed range of to . . . . . probability of a false positive echocardiogram result at a level facility as for level false positives, no data was found for level false positive rates for cchd. after extensive discussion amongst committee members, a point estimate of . was chosen with a range of to . (this was largely based on the collective anecdotal experience of committee members, most of whom are neonatologists who receive such calls from level facilities). . . . probability of pos occurring with moh approval no ontario-specific data were available for this parameter, as pos has not yet been implemented. it was agreed upon by the authors to use a point estimate probability of . (with an assumed range of . to . ) of pos occurring for any individual baby if pos for cchd is approved by the ministry of health in ontario. . . . probability of pos occurring without moh approval similarly, it was assumed that an individual patient would have a probability of . (with an estimated range of to . ) of receiving pos, if the screening were not to be approved by the ministry of health. . . . probability of a pos screen being positive if individual has cchd the following targeted search was conducted on medline (july , ): ("pulse oximetry screening"[title/abstract]) and ("congenital heart"[title/abstract] or "cardiac disorder"[title/abstract]), which yielded articles. after screening of titles and abstracts, the following articles (listed in table . ) were included to provide data on true positives as well as false positive results, to provide estimates of overall accuracy of pos screening.( - , , - ) table . : studies reporting on accuracy of pulse oximetry screen for cchd study name how was a positive screen determined? true pos false pos false neg true neg disease n diseas e free n bhola post-ductal saturation < % on two readings (second reading only if necessary at least - hours later); or if < % at any time ewer saturation < % or and pre-post gradient > %; followed by expedited physical exam. if abnormal  confirmed positive, if exam normal  repeat test with same criteria - hours later and if still same results  confirmed positive koppel any spo < %  followed by echo meberg spo < % x or after st if obvious clinical signs (repeated once - hours later if needed) bakr spo < % ( reading)  followed by echo. to < % repeated thrice  echo arlettaz spo < %  echo, if to < % - repeat - hours later and if still < %  echo sendelbach < %  repeated after repositioning probe and warming up foot - if still < %  echo reich < %  repeated contra-diaphragm x if between to and if persisted  echo. echo also if single reading < % and if > % difference in contra-diaphragmatic values rosati ≤ % at > hours  echo richmond < %  clinical exam (if normal, spo repeated - hours later) if either exam or repeat spo abnormal  echo de wahl granelli < % in both pre and postductal or > % gradient  repeated thrice. if < % any time  echo riede ≤ %  repeated once - hours later  then echo kawalec unknown hoke % or more gradient, or postductal spo < % kochilas if spo < either site  fail; if between - or arm-foot gradient > : repeat  if still same  fail ruangritnamch ai if spo < %  echo singh if spo < % in either limb, or difference > % between limbs  clinical exam and if normal  repeated. it still same findings or initial clinical exam abnormal  admitted to nicu for detailed assessment tautz if between - %  repeat - h later and if still abnormal  echo. if initial < %  immediate echo turska-kmiec if spo < %, repeated after h  if persisted  standard protocol for suspected congenital heart disease (saturation measurement on upper extremity, hyperoxia test) zhao if any spo measurement < %; if between - % on both separated by hours (repeat measurement) or greater that % difference (also repeated, separated by hours) zuppa if measurements < % ( - min apart)  ekg and echo overall abbreviations: cchd – critical congenital heart disease; echo – echocardiogram; ekg – electrocardiogram; spo – oxygen saturation based on the overall numbers from the studies listed in table . , the probability of pos being positive if an individual has cchd was determined by dividing the true positive number ( ) by total number of cchd cases included in these studies ( ) to yield a probability of . . a monte-carlo simulation with beta distribution yielded a . % to . % range of . to . . . . . probability of a pos screen being positive if individual does not have cchd from the data in table . above, this probability was determined by dividing the false positives ( , ) by the number of individuals without cchd ( , ) to yield a probability of . , and . % to . % range of . to . . . . summary of all probabilities and their ranges table . : all probability variables used in the mode with point estimates and ranges variable point estimate low range high range probability of a well appearing individual at hours age (base case) having cchd* . . . probability of a well appearing individual at hours age (base case) not having cchd* . . . probability of a well appearing individual at hours age (base case) being dead probability of a well appearing individual at hours age (base case) being post-cchd with morbidity probability of a well appearing individual at hours age (base case) being post-cchd without morbidity probability of a well appearing individual at hours age (base case) being non-cchd (> month) probability of early mortality ( st month) if individual has cchd* . . . relative risk of early mortality ( st month) if cchd is detected late*† . probability of non-cchd related neonatal ( st month) mortality* . . . probability of a home death ( st month) if have cchd* . . . probability of death per cycle if individual does not have cchd as shown in appendix c probability of death per cycle if individual is post-cchd repair without any morbidity year . . . subsequent years . . . probability of death per cycle if individual is post-cchd repair with morbidity year . . . subsequent years . . . probability of early morbidity ( st month) if individual has cchd* . . . relative risk of early morbidity ( st month) if cchd is detected late*† . probability of an individual post-cchd repair without morbidity developing cchd- associated morbidity year . . . years - . . . years and above probability that an individual patient will deliver at a level facility* . . . probability that an individual in level or level catchment area delivers at a level facility* . . . probability that an individual patient’s home hospital is a level facility* . . . probability that an individual belongs to sickkids lhin* . . . probability that an individual patient is from the northern region that requires air transport* . . . probability that a level facility has pediatric echocardiography capability* . . . probability of a false negative echocardiogram result at a level facility* . . . probability of a false negative echocardiogram result at a level facility* . . . probability of a false positive echocardiogram result at a level facility† . probability of a false positive echocardiogram result at a level facility*† . . probability of pos occurring with moh approval*† . . probability of pos occurring without moh approval*† . . probability of a pos screen being positive if individual has cchd* . . . probability of a pos screen being positive if individual does not have cchd* . . . abbreviations: cchd – critical congenital heart disease; lhin – local health integrated network; moh – ministry of health; pos – pulse oximetry screening *variables included in probabilistic sensitivity analysis †variables for which point estimate and ranges were estimated by author consensus . utility variables . . utilities associated with health states utilities (quality of life indicator) for the following mutually exclusive health states were ascertained for use in the model. . . . utility of cchd the utility of cchd referred to that of the first month of life of an individual with cchd. the search strategy, screening and determination of point estimates and ranges were the same as for “utility of cchd post-repair without morbidity” – as such, please refer to section . . . for details. . . . utility of no cchd the utility of a no cchd health state was assumed to be , and no further formal literature search was conducted. . . . utility of cchd post repair without morbidity this utility referred to quality of life living with cchd following repair without any additional morbidity. note that the same search strategy and determination of point estimates was conducted for utility for cchd in the first month of life. a comprehensive literature search was conducted on medline with help of a reference librarian (ms. laura banfield, health sciences library, mcmaster university). the search strategy (conducted on september , ) is shown in appendix f. the search strategy for cchd yielded , studies, of which were screened in after review of titles and abstracts. of these, studies were ultimately included.( - ) reasons for exclusion were (a) lack of relevance (no utility data); (b) review article (without utility data); (c) relating to a very specific (or minor chd), surgery, therapy, assessment tool; (d) non- english article, or non-north america/non-western european source; (e) surgery or disease onset in adulthood (mean/median age years or higher); (f) studies with repeat patients already assessed previously (whether excluded or included); (g) lack of control group/data; and (h) other miscellaneous reasons (including studies with fewer than patients). of the included studies, provided data for the pediatric age range (< years’ age) while studies provided data only for adult age range (≥ years’ age). two studies provided extractable such data for both age ranges. the details of studies contributing to these two age ranges are as shown in tables . and . below, including age range of subjects, tool used to assess quality of life and cchd lesions involved. quality scores relating to mental and physical health (or closest equivalent based on tool used in study) were gathered, and general health scores – when available – were also taken. table . : studies contributing utility for cchd post-repair without morbidity (age < years) cchd control author year age range (years) tool lesion measure n mean sd n mean sd culbert - chq tga repair physical . . . general health . . . . mental health . . . summary . . . . de koning - tacqo l tga repaired motor functioning . . . . cognitive function . . . summary . . . . frigiola - pedsqo l tof repaired total . . . . summary . . . . idorn - pedsql univen- tricular lesion physical health . . . psychosoci al health . . . summary . . . - pedsql univen- tricular lesion physical health . . psychosoci al health . . summary . . . . neal - chq tof repair psychosoci al . . . . physical . . . . summary . . . . abbreviations: chq – child health questionnaire; pedsql – pediatric quality of life scale; tacqol – tno-azl child quality of life; tga – transposition of the great arteries; tof – tetralogy of fallot table . : studies contributing utility for cchd post-repair without morbidity (age ≥ years) cchd control author year age range (years) tool lesion measure n mean sd n mean sd bygstad - sf- tof repaired male physical male mental summary . . . cotts - sf- tga physical component summary . . . . mental component summary . . . . summary . . . . daliento - sf- tof repaired physical . . . . general health . . . . mental health . . . . summary . . . . dulfer - sf- tof/sin gle ventricle physical functioning . . . . general health . . . . mental health . . . . summary . . . . physical functioning . . . general health . . . mental health . . . summary . . . frigiola - whoqo l tof repaired physical . . . psychosocial . . . . summary . . . . idorn > sf- univen- tricular lesion physical component score . . . mental component score . . summary . . . . irtel - rand- tof/tg a repair tof general health perception summary tga general health perception summary abbreviations: sf- – short form- questionnaire; tga – transposition of the great arteries; tof – tetralogy of fallot; whoqol – world health organization quality of life scale where a study provided more than component of utility – a “summary” utility score for that study was determined by taking the average of the individual score components. the standard deviations were combined using the following formula: sd = √[∑ ((sdk)^ )/n] 𝑛 𝑘= where n is the number of individual component scores provided, to yield the standard deviation of the summary utility score for each study. next these summary means and their standard deviations for the cases (cchd) and controls/reference ranges from each study were meta- analyzed using the “ratio of means” technique, as described by friedrich et al( ) to yield a composite ratio of utility values (with % confidence intervals) for cchd post repair without morbidity compared to controls (no cchd). these values are shown in the summary table . below. . . . utility of cchd post repair with morbidity for post-cchd repair with morbidity, a targeted literature search on medline was conducted for childhood developmental disorder (representative of morbidity in post-cchd repair) using the following search terms: ("neurodevelopmental impairment"[title] or "neurodevelopmental disorder"[title] or "developmental disorder"[title] or "neurological disorder"[title] or "neurological impairment"[title] or "mental retardation"[title] or "cognitive impairment"[title]) and (utility[ti] or quality[ti]). the search strategy yielded articles, and after initial screening based on article titles that eliminated studies that evaluated morbidity states with adult/late adult onset (n= ), studies on very specific populations where it was deemed that baseline measures of quality of life may already be low before adding on the effects of neurodevelopmental morbidity (n= ), and non-north american/non-western european/non-australasian studies (n= ), and studies deemed not relevant (n= ), were evaluated in detail (full article). of these studies contributed utility data to adult age range,( , ) while study contributed utility data for the pediatric age range.( ) the details of these studies are shown in table . . table . : studies contributing data towards utility of neurological morbidity author year age range condition tool n mean sd n [control] mean [control] sd [control] adult values barrios . years ( . ) mild cognitive impairment qol-ad question- naire . . . . * . . . . summary . . . . missotten . years ( . ) mild cognitive impairment adrql . . . . pediatric values srivastava . years neurological impairment with gerd (and fundo) sf- . . . . abbreviations: adqrl – alzheimer’s disease related quality of life; gerd – gastroesophageal reflux disease; qol-ad – quality of life – alzheimer’s disease; sf- – short form- questionnaire *second are “informants” as before, the range of means meta-analysis technique described by friedrich et al was utilized to determine the utility (and respective % confidence intervals) associated with neurological morbidity in both pediatric age as well as in adulthood.( ) subsequently, these utility values were combined with the utility values for health state post-cchd repair without morbidity (multiplying the two utilities and their respective % confidence intervals) to yield a composite utility for the health state of post-cchd repair with morbidity. these final values are as shown in the summary table . below. . . . utility of death the utility of death was assumed to be , and no further formal literature search was conducted. . . summary table of utilities of health states table . : utilities associated with health states used in the model health state utility estimate minimum maximum cchd ( st month)* . . . no cchd death post cchd repair no morbidity < years . . . ≥ years . . . post cchd repair with morbidity < years . . . ≥ years . . . abbreviations: cchd – critical congenital heart disease *variable(s) included in probabilistic sensitivity analysis . . utilities unrelated to health states in the model, transitional “dis-utilities” were included. these were (a) disutility related to performing pulse oximetry screening; (b) disutility related to transfer of patient to higher care level facility (e.g. if pos is positive); and (c) disutility related to conduct of surgery for cchd. these largely reflected “spillover” effect into the parents as well as their perception of the inconvenience/discomfort associated with the aforementioned interventions and their impact on the quality of life. . . . disutility of having pos performed the following search strategy was conducted on medline on july , : ("utility"[ti] or "disutility"[ti] or "dis-utility"[ti]) and ("screening"[ti] or "pulse oximetry screening"[ti] or "oximetry screening"[ti] or "screen"[ti]) and "infant, newborn"[mesh terms]. the search strategy yielded articles, and upon screening of titles (and abstracts when deemed needed) – none of the studies were relevant for inclusion. given the lack of adequate data, a consensus was reached amongst authors to assume this transitional disutility value to be - . (with a range of - . and - . ). . . . disutility of transfer (due to positive screen or for definitive surgical repair) the following search strategy was conducted on medline on july , : ("utility"[ti] or "disutility"[ti] or "dis-utility"[ti]) and ("transport"[ti] or "transfer"[ti]) and "infant, newborn"[mesh terms]. the search strategy yielded articles, neither of which were relevant for inclusion. given the lack of adequate data, a consensus was reached amongst authors to assume this transitional disutility value to be - . (with a range of - . and - . ). . . . disutility of having surgery the following search strategy was conducted on medline on july , : ("utility"[ti] or "disutility"[ti] or "dis-utility"[ti]) and ("surgery"[ti] or "surgical"[ti] or "operative"[ti] or "operation"[ti]) and ("infant, newborn"[mesh terms] or "infant"[mesh terms]). the search strategy yielded articles, and upon screening of titles (and abstracts when deemed needed) – none of the studies were relevant for inclusion. given the lack of adequate data, a consensus was reached amongst authors to assume this transitional disutility value to be , as no parent would conceivably relay a lower quality of state related directly to the conduct of a potentially life-saving surgery. . . summary of utilities unrelated to health states the following table . summarizes the point estimates and ranges of disutilities used in the model. the point estimates were used for the base case while the ranges were used to conduct individual one-way sensitivity analyses for each variable, where applicable. table . : estimates of disutilities and ranges model variable point estimate minimum maximum disutility of pos*† - . - . - . disutility of transfer*† - . - . - . disutility of cchd surgery† abbreviations: cchd – critical congenital heart disease; pos – pulse oximetry screening *variable(s) included in probabilistic sensitivity analysis †variable(s) for which point estimates and ranges were estimated based on author consensus . cost variables . . cost of health states the following sections describe how the monthly costs for all the health states included in the model were obtained. . . . cost of cchd the monthly cost of having cchd in the first markov cycle (i.e. the first month of life) was obtained from the canadian institute for health information’s financial standards and information department through a customized request. the acute typical inpatient cost in the first month of life was $ , . . no standard deviations were available, and therefore this cost was multiplied by . and . respectively, to yield the lower and upper limits of the estimates of the cost range. . . . cost of no cchd this health state represents those individuals without cchd. this monthly cost was obtained from the canadian institute for health information’s national health expenditure database: https://www.cihi.ca/en/national-health-expenditure-trends. for ontario, the annual health expenditure was $ , . yielding a monthly cost of $ . . no standard deviations were available for the per capita health expenditure, therefore the final monthly cost was multiplies by . and . to estimate the lower and upper limits of the cost range, respectively. . . . cost of cchd post repair without morbidity this health state represents individuals with cchd who survived surgery and did not develop any long term morbidity. ontario’s ministry of health and long term care (mohltc) https://www.cihi.ca/en/national-health-expenditure-trends ontario case costing initiative (occi) tool (available at: https://hsim.health.gov.on.ca/hdbportal/) was used to determine annual average costs for case mix group labeled “congenital cardiac disorder” for fiscal year / for both inpatient and ambulatory costs, with their respective standard deviations. each of these costs was in turn provided by age groups, as shown in the table . below. table . : inpatient and ambulatory costs for congenital cardiac disorders from occi age group (years) ip cost ip cost sd ac cost ac cost sd total cost total cost sd monthly total cost monthly total cost sd - . . . - . + n/a n/a . . abbreviations: ac – ambulatory care; ip – inpatient; occi – ontario case costing initiative; sd – standard deviation the total cost was determined by adding the inpatient and ambulatory costs and the total cost standard deviation was obtained by the following formula: total cost sd = √[((ip cost sd) + (ac cost sd) )/ ]. the total cost and the standard deviation were both divided by to yield the monthly cost and monthly standard deviation, as shown in the table. finally, the monthly standard deviations were multiplied by two then subtracted from and added to the monthly cost to estimate the lower and upper limits of the cost ranges for each age group. . . . cost of cchd post repair with morbidity this health state represents those individuals who have had cchd repaired and are living with associated morbidity. for the purposes of this model, as described earlier, morbidity was limited to neurological morbidity and for the purposes of this cost estimate, childhood/adolescent development disorder (case mix group ) as defined by canadian institute for health information was used. data was obtained through a customized request for https://hsim.health.gov.on.ca/hdbportal/ inpatient, day surgery and emergency visits related to this condition through the financial standards and information department of cihi, yielding total monthly costs per individual in age groups similar to ones described above. these costs were added to the monthly costs of cchd health state without morbidity to yield total monthly health costs. no standard deviations were available, and ranges were estimated by multiplying the point estimates for each age group by . and . , as described above. . . . cost of death the cost associated with this health state was assumed to be ; no specific searches for cost estimates were conducted in this regard. . . summary table of costs of health states the final point estimates and ranges of the costs of the aforementioned health states are as shown in table . below. the point estimates were used in the markov model and the ranges were used to conduct individual one-way sensitivity analyses. table . : estimates of costs and ranges for health states used in markov model health state monthly cost estimate ($) estimated minimum ($) estimated maximum ($) cchd ( st month)* , . , . , . no cchd* . . . cchd post repair (no morbidity) - years . . . - years . . years and above . . . cchd post-repair (with morbidity) - years , . . , . - years , . . , . years and above , . . , . death abbreviations: cchd – critical congenital heart disease *variable(s) used in probabilistic sensitivity analysis . . costs unrelated to health states . . . cost of pulse oximetry screening the cost of each time a screening is performed was estimated by the cost of the disposable components of measuring oxygen saturation. it was assumed that the saturation checks will occur simultaneously in upper and lower limbs. the cost of nursing time was not factored in due to the short time it takes to conduct the screening. the cost of disposable components of each saturation measurement was obtained from the administrative coordinator at mcmaster children’s hospital (representing the clinical manager at the time, ms. lynda aliberti – personal communication date april , ). the final cost of each screening was estimated to be $ . ($ . per disposable unit x ). the range was obtained by multiplying the estimate by . and . to obtain lower and upper limits. . . . cost of performing echocardiogram the cost of echocardiogram was obtained via personal communication by two independent sources (ms. amuna yacob – administrative assistant, division of neonatology, university of toronto – data obtained from ms. julie alyssandratos-amatuzio, billing clerk for the department of pediatrics, communication date march , ; as well as dr. tapas mondal, pediatric cardiologist, mcmaster children’s hospital, personal communication date: april , ). the cost included technical/technician cost per echocardiogram ($ . ) as well as billing costs ($ . ) yielding a total cost per echocardiogram of $ . . range was estimated by multiplying the point estimate by . and . . . . . cost of surgery the cost of performing surgery for cchd was obtained through the mohltc’s occi (available at: https://hsim.health.gov.on.ca/hdbportal/). a manual search for all procedures among inpatients in ontario for fiscal year / was conducted and any procedure related to the repair of a critical congenital heart disease was selected. this yielded a total cost of $ , per surgery (with a minimum cost of $ , and a maximum cost of $ , , which were used for the range of costs). . . . cost of transfer the cost of land transport was estimated from local data at mcmaster children’s hospital (hamilton, ontario). the total budget for transport ($ , , ) was divided by the number of transports ( for / fiscal year) to yield an average cost per land transport of $ , – which was then multiplied by . and . to yield the range of costs. all transports were assumed to be land transports for the purposes of this calculation due to the rarity of air transports at this centre (< per year, personal communication dr. michael marrin, neonatologist, neonatal transport director, mcmaster children’s hospital, communication date: september , ). in addition, the cost at mcmaster was assumed to be representative of all land transports across the province. air transport costs were an estimate based on personal communication with dr. michael castaldo, neonatal transport director at british columbia children’s hospital – a centre where there is a heavy reliance on air transports, communication date: august , ). the average cost per air transport was estimated to be $ , (with an estimated range of $ , to $ , per air transport). https://hsim.health.gov.on.ca/hdbportal/ . . summary of costs unrelated to health states the following table . summarizes all costs (and respective ranges) unrelated to health states that were used in the model. table . : estimates of costs and ranges unrelated to health states used in model model variable cost estimate ($) estimated minimum ($) estimated maximum ($) cost of pulse oximetry screening* cost of echocardiogram* . . . cost of surgery for cchd* , , , cost of transport land transport* , , , air transport*† , , , abbreviations: cchd – critical congenital heart disease; pos – pulse oximetry screening *variable(s) included in probabilistic sensitivity analysis †variable(s) for which point estimates and ranges were estimated based on author consensus . results of decision model . base case analysis in our base case of a well appearing newborn, performing pos at hours of life as a screening measure to detect cchd was superior to a no screening strategy. there are approximately , births province-wide annually (canadian institute for health information; https://www.cihi.ca/en). based on incidence of missed cchd of . and probability of pos detecting . of all cchds, it is estimated that an additional cases of cchd will be detected in a timely fashion annually ( , x . x . ) with implementation of routine pos. as shown in table . , the incremental cost of performing pos was $ . per patient ($ , . – $ , . ), with a gain of . quality adjusted life months (qalms) per patient ( . – . ). based on an estimated , births per year in the province, this would lead to a gain of , qalms ( , x . ) or quality adjusted life years (qalys). the incremental cost and qalms yielded an estimated incremental cost-effectiveness ratio (icer) of $ , . [Δ cost / Δ qalms]. this was below the aforementioned a priori cost-effectiveness threshold of $ , . per qalm. table . : cost and adjusted life months with/without pos implementation strategy cost* quality-adjusted life months* pulse oximetry screening , . . no pulse oximetry screening , . . *accounting for a “discounting rate” of . % similar analysis as above without discounting yielded results as shown in table . , leading to an estimated incremental cost of cad$ . per patient, with incremental gain in qalm of . , for an icer of $ . per qalm gained. rest of results, however, will continue to incorporate a . % discount rate. table . : cost and adjusted life months with/without pos implementation (no discounting) strategy cost quality-adjusted life months pulse oximetry screening . . no pulse oximetry screening . . . one-way sensitivity analyses these analyses were conducted for all variables included in the model over their respective ranges. as described previously in section , for variables that varied over time, these analyses were conducted by running the decision model for the lower range as well as the higher range values for each variable at a time. the model was not sensitive to any variable in one-way sensitivity analyses, i.e. the the implementation of pos was superior to no pos implementation for all variables across their plausible reference ranges. . threshold analyses due to the importance and/or uncertainly around the point estimates/ranges, certain variables were tested for threshold values even outside the estimated plausible ranges based on group consensus amongst the committee members. these variables are indicated in table . below, and the thresholds (where identified) above or below which pos implementation is no longer expected to be cost effective for these variables are indicated, along with the base case values and original plausible estimated ranges. all probability variables were tested between values of and , while the ranges tested for cost variables are indicated in footnote of table . . table . : determination of threshold values for a limited set of variables variable base case value lower range higher range threshold value* probability that an individual patient is from the northern region that requires air transport . . . n/a probability that a level facility has pediatric echocardiography capability . . . n/a probability of a false negative echocardiogram result at a level facility . . . n/a probability of a pos screen being positive if individual has cchd . . . < . probability of a pos screen being positive if individual does not have cchd . . . > . probability home death with cchd in st month . . . < . probability of cchd . . . < . cost of echocardiogram† . . . n/a air transport‡ , , , n/a *variables with n/a did not have any threshold above or below which implementation of pos would no longer be cost effective. †tested from cad$ to cad$ , ‡tested from cad$ to cad$ , it was predicted that province-wide implementation of pos would not be cost-effective under the following conditions: (a) pos detects < . % of cchd lesions (well below the plausible lower limit of . %); (b) pos is (falsely) positive if an individual does not have cchd > . % of the time (well above the estimated higher range of . %; (c) if the probability of death at home in case of a missed cchd is < . %, below the estimated lower range value of . %; or (d) incidence of cchd in base case is less than . (just below the estimated lower limit of . , but well below the point estimate value of . ). . probabilistic sensitivity analysis as described previously in section , in a probabilistic sensitivity analysis, multiple simulations of the decision model are run with a value for each variable chosen at random from within the prescribed range. ten thousand simulations for the model were run as part of this probabilistic sensitivity analysis; most variables from the model were included in this analysis, and are indicated in section . time-varying variables (already found not to have impact model output in one-way sensitivity analyses) were not included. it was found that at the predetermined cost-effectiveness threshold of $ , . , implementation of pos would be cost effective . % of the time. figure . below shows the incremental cost-effectiveness scatter plot. each blue dot represents the incremental cost and incremental effectiveness (qalm) from each simulation, and all simulation values “below” the line of cost-effectiveness threshold are deemed to be cost- effectiveness, where the ones above the threshold line represent simulations where icer for pos implementation was above the pre-determined threshold value. figure . : cost-effectiveness scatter plot figure . : cost effectiveness acceptability at various cost-effectiveness (or “willingness to pay”) thresholds from this probabilistic sensitivity analysis, a cost-effectiveness acceptability curve was generated, as shown in figure . above. as mentioned above, at a cost-effectiveness threshold of $ . , pos implementation was estimated to be cost effectiveness in . % of the simulations, whereas if the cost-effectiveness threshold were doubled to $ , . , it is estimated that pos implementation has a . % chance of being cost-effective. the acceptability curve also indicated that pos implementation is more likely to be cost-effective than lack of implementation (i.e. > % chance) at a cost-effectiveness threshold value as low as $ , . . model validation the model developed for the clinical problem underwent face validity by the authors including neonatologists with expertise in neonatal cardiology as well as an expert in medical decision modeling. furthermore, the model – when run without any discounting – yielded qalm values of approximately . and . (in pos and no pos arms, respectively) – equivalent to years of life, which is in keeping with current canadian life expectancy values, lending further credence to the validity of the model as structured. . discussion . summary of results a province-wide implementation of pos for cchd in ontario appears to be a cost- effective endeavour. implementation of pos is expected to be associated with detection of an additional cases of cchd that would’ve been otherwise missed, with an icer of cad$ , . per qalm gained (below the predetermined cost-effectiveness threshold of cad$ , . per qalm). pos implementation was noted to have a . % chance of being cost-effective at a cost-effectiveness threshold of cad$ , . per qalm, whereas at a threshold of cad$ , . per qalm – it was estimated to have a . % chance of being cost- effective. these results are similar to those previously reported in the literature.( , - ) . interpretation of findings the findings of this model are consistent with what might be considered in the context of biological plausibility. patients with delayed diagnosis of a given cchd lesion are more likely to experience hemodynamic compromise, resulting in prolonged decreased oxygen supply to tissues in vital organs including the brain. they are more likely to not survive, as well as have a higher chance of morbidity.( ) this results in increased health care costs, as well as lower quality of life. our model’s primary aim was to determine whether the implementation of pos is cost-effective. the actual change in incremental cost between the two diagnostic strategy (i.e. implementation of pos vs. no implementation) was $ . (the additional dollar spending per individual) while the gain in qalms was . per individual, equivalent to an estimated addition of qalys over lifetime with each year of conducting pos. it is important to note that all values were discounted by . % per year, as both values of cost and qalms are deemed to have a higher value in the present than in the future.( ) we employed a markov modeling technique to determine the cost-effectiveness of pos over a life-time horizon. the advantage of this technique was that it allowed for transitional probabilities to vary over time, more accurately reflecting a life-time natural history of cchd. simulated individuals were allowed to transition among health states with each monthly cycle, which allowed for a realistic expression of the natural course as well as care pathways. the first cycle of the model was designed in great detail to be representative of the actual sequence of events from the time of a positive screen to the confirmation of diagnosis and surgical repair, as well as a detailed depiction of plausible events if a cchd were to be missed despite screening. as such, the model was very representative of the likely clinical scenario that will take place following implementation of such a program. . assumptions in the model a number of assumptions were made in the model structure itself, as well as with respect to values and ranges assigned to many variables due to lack of available data, all of which will be described in this section. a) it was assumed that a simulated individual with cchd and a positive pos would remain stable and asymptomatic until confirmation of diagnosis by echocardiogram. this was a reasonable assumption given the model was constructed to ensure a confirmatory echocardiogram is performed after only a single transfer. b) it was also assumed that all cchds (whether they were identified or missed, and regardless of what specific lesion it was) would receive surgery upon confirmation in the first markov cycle. this was assumed for simplicity, but it is recognized that not only can some conditions have a delay in the first surgery, but that some conditions require a series of surgeries.( ) however to build all possible permutations of timings of surgeries, or categorizing by specific lesion type, would add a significant level of complexity to this model without any conceivable difference in the model outcome. c) another important assumption was that morbidity related to cchd repair consists of neurological impairment. however, there may be many other forms of morbidity including cardiorespiratory compromise, and other issues that may impact on the quality of life including scarring from surgeries, need for pacemakers, missed school and time from work among many others.( - ) while it is recognized that limiting to neurological impairment as morbidity may be an oversimplification of an otherwise complex array of morbidities that impact quality of life, it is acknowledged that presence of other cchd-related morbidities amongst the survivors may decrease the quality of life further, and as such our model may be overestimating the benefit of implementation of pos. d) another assumption related to the issue of morbidity was that anyone that developed cchd-associated morbidity would have that over their lifetime. this was felt to be a reasonable assumption given that we limited the scope of morbidity to that of neurodevelopmental impairment, which are generally chronic conditions.( ) e) it was assumed that health care expenditure in an individual without cchd would be constant throughout their life, whereas in reality the costs are expected to change over lifetime. f) we assumed that all babies delivered in lhins and (representing northern ontario, appendix b) – whether born at a level or level facility – would require air transport to the nearest level centre if they screened positive on pos. this is a reasonable assumption given the sparse population distribution over a significant land mass in this part of ontario. g) the point estimates for a number of variables (and as a result, their ranges) were estimated due to a lack of relevant data. these have been indicated previously in section , but are listed here once again. for all these variables, the range was selected to be reasonably wide and all these variables underwent one-way sensitivity analyses to detect the threshold (if present) above or below which the model result was no longer robust. a. relative risk of neonatal mortality with late detection was assumed to be . (with an assigned range of - ) b. similarly, the relative risk of neonatal morbidity with late detection was assumed to be . (with an assigned ranged of - ) c. transitional monthly mortality rates among the group of individuals with cchd remains constant during adolescence and beyond d. transitional monthly mortality rates are same for cchd individuals with and without cchd-associated morbidity e. transitional rates of development of cchd-associated morbidity would be constant for the first year then again between ages - , and that no new morbidity would arise after that age f. probability of false negative results of an echocardiogram at a level facility was assumed to be the same as that of a level institution g. probability of false positive results at a level institution was assumed to be , while that at a level facility was assumed to be . h. probability of pos occurring in event of province-wide implementation was assumed to be . (range . to . ) whereas the probability was assumed to . (range to . ) without province-wide implementation i. disutility associated with the conduct of pos was assumed to be - . (range - . to - . ), and transfer assumed to be - . (range - . to - . ) while disutility with conduct of cchd surgery was assumed to be , as no parent would conceivably have any objections to a potentially life-saving surgery j. cost of air transport was estimated based on personal communication with the transport director at bc children’s hospital and assumed to be $ , (with an estimated range of $ , to $ , . . sensitivity analyses and interpretation on one-way sensitivity analyses limited to the estimated plausible range for each variable, model was not sensitive to any of the included variables. however, in addition to the aforementioned sensitivity analyses, analyses of extreme values (beyond the predetermined range) was performed for certain key variables, in particular variables for which there was low confidence in the range and/or variables of importance for the model. these variables have been listed earlier in table . . it was found that if probability of pos screen being positive in individuals with cchd is < . % then the implementation would no longer be cost-effective, which relates to increased costs yet diminishing benefit from pos. in addition, false positive rate > . % was a threshold above which pos would not be cost- effective, which intuitively also is understandable as far too many infants would require transport and evaluation. however, this model did not take into account the fact that many cases of false positive results are later identified to have some other non-cardiac etiology such as transient tachypnea of the newborn or sepsis.( , ) in any case, both of these thresholds were far beyond the expected range for these variables. it was also estimated that pos implementation would not be cost effective if the probability of death at home with missed cchd was less than . %, and also if the incidence of cchd in a well appearing newborn that has not been antenatally or postnatally detected fell below a threshold of . ( . %), indicating that the burden of illness in such an event would be far too low for pos to remain cost-effective. importantly, there was no threshold for air transport (checked up to an upper range of cad$ , ), which likely relates to the fact that air transport will be utilized in both decision arms, and is expected to serve a relatively small fraction of the population. the above analyses vary only variable at a time, while the probabilistic sensitivity analysis varied multiple variables at once, run over , simulations of the model. at the cost- effectiveness threshold of cad$ , . per qalm – it was estimated that pos had a . % probability of being cost-effective, while the chances increased to . % if the cost-effectiveness threshold was doubled. this analysis represents a more “wholesome” sensitivity analysis, as all variables placed in the model may in reality be different that those used for “base-case” analysis. the results of the probabilistic sensitivity analysis, depicted as chances of cost-effectiveness, are also more meaningful for the audience. finally, the probabilistic analysis also allowed for determination of the cost-effectiveness threshold below which it was less likely than not that pos would be cost-effective (cad$ , ). . comparison to previous studies a number of studies have previously assessed the cost-effectiveness of pos implementation. most such studies have been done in the uk,( - ) but also in the us and sweden.( , ) previous studies have evaluated a very limited time horizon, with all being until the point of diagnosis of cchd, with the exception of the study by peterson et al, which employed a one year time horizon.( ) the icer values for these studies varied from as low as £ , to as high as £ , per additional case of cchd diagnosed. the reason for the wide variability in the costs certainly relate to the nature of the models created and the variables put in, but all suggested that pos was likely to be cost-effective. the study by peterson et al determined the icer to be $ , per life year gained (which converts to $ , . per month gained in us dollars). it should be noted that, unlike our model, none of the aforementioned studies employed utilities to account for morbidities that may be associated with cchd and its outcomes (i.e. the various possible health states) – and as such do not present the costs for quality-adjusted life units gained and therefore, cannot truly evaluate cost-effectiveness. in addition, our study provides a life-time horizon which provides a clear sense of total lifetime costs and quality adjusted life units to be gained by the implementation of pos, after accounting for a discount rate of . % per year. in addition, the model structure of the aforementioned studies was rather simple, which did not take into account the various possible pathways to the ultimate diagnosis of cchd following a positive screen. finally, we employed a markov model to enable a more realistic modeling of a lifetime time horizon. despite these differences from our study, the aforementioned studies affirmed the cost-effectiveness of pos, and our study was consistent with this in the canadian context employing a lifetime horizon and associated utilities of various health states. . strengths and weaknesses . . model framework the most important strength of the model is the detailed framework (outlined in figures . , . and . ) that represents a realistic pathway from screening to diagnosis to outcomes in the first markov cycle. another important strength is the employment of time varying variables that allow the values to change over time for the transitional probabilities. this is close to the realistic progression through life following cchd. the limitations of the model framework itself relate largely to the assumptions made, and have been previously discussed in section . . another important limitation is the decreasing confidence in the point estimates of many variables the farther out the time horizon gets, due to lack of available data. additionally, evaluating a life-time span, particularly when accounting for an annual discount rate, may contribute to diluting the incremental gain in qalms. finally, no spillover (caregiver) effects were considered as it relates to the utility and cost variables (with a few exceptions indicated previously), although this makes our analysis conservative (i.e. if including spillover effects, it would have been expected that the icer would be lower still). . . variables and data the variables entered into the model underwent a comprehensive, targeted search from the literature where appropriate, and canadian specific databases included born, cihi and occi. canadian and ontario specific data were used for most of the model variables, and as such the model output truly reflects the canadian context. however, for many variables no data were available and the point estimates for the base case as well as the ranges for some values were estimated based on author consensus. additionally, for some variables only the point estimate was available. for probabilities and utilities for which this was the case, the range was estimated using the raw data and performing a monte carlo simulation with beta-distribution to derive an estimate of the range. for cost variables, when no range was available from the literature, % below and above the point estimate were used as the range. lack of available data and/or ranges for certain variables does inject a degree of uncertainty around the base case icer. however, these values and their ranges were estimated based on group consensus. finally, it is important to acknowledge the significant clinical and statistical heterogeneity of many of the meta-analyses, largely due to differences in study design, cchd lesion being studied, population and time-frame of follow-up. however, we employed a random effects model when meta-analyzing recognizing the degree of heterogeneity, and recognize the limitations in the resultant point estimates for many of these variables. this placed additional importance to the sensitivity analyses conducted utilizing the ranges derived from these meta- analyses, recognizing the tremendous heterogeneity in the types of lesions and clinical course that patients with cchd are expected to experience. . . analyses multiple sensitivity analyses were conducted, which is an important strength of the model. one-way sensitivity analyses were conducted for each variable in the model, including time-varying variables. for select variables extreme threshold analyses were conducted. finally, probabilistic sensitivity analysis was conducted with most variables. as such, despite the limitations of lack of data for point estimates and/or available ranges, these sensitivity analyses have largely shown the model to be robust. . clinical and policy implications the likelihood of pos being cost-effective, along with its safe and non-invasive method, make it a suitable screening tool for the early diagnosis of cchd, a condition with clear benefit from being diagnosed early. these criteria constitute the tenets of the wilson and jungner screening criteria.( ) our model also shows that despite many cases being detected antenatally and postnatally prior to hours of age, pos still remains cost-effective despite the relative rarity of otherwise “missed cchd”. in order to maximize the effectiveness of pos, it would be prudent for policy makers to create standardized guidelines for pos including recommended measurements techniques and standard procedures for follow-up (e.g. the appropriate lhin for transport and the recommended method of transportation, including contingency recommendations if a baby becomes symptomatic prior to diagnostic confirmation). these procedures would minimize the cost to the health care system by minimizing the effect of ambiguity in steps that need to be taken and would maximize the effect of pos. close collaboration between the various centres will be required for effective implementation of such a screening program. with the advent of telemedicine technology, it is conceivable that many infants with positive pos screens will have confirmatory echocardiograms performed remotely. although our model does not account for the costs associated with the investment required in implementation of such technology, it is reasonable to infer than this will render pos implementation even more cost-effective than our model suggests. . conclusions and next steps in conclusion, pos implementation in ontario is likely to be a cost-effective endeavour, with an estimated icer of cad$ , . per qalm gained. despite the creation of a realistic model framework, the limitations in available data mean that the robustness of this analysis could be enhanced by incorporating more local data. this would serve to confirm the findings on the model, and may present a more realistic estimate of incremental costs and quality-adjusted life units gained. this could be performed by incorporating data following implementation of pos into the current model structure. however, gathering such data – especially when considering the life-time horizon of the model – may be both resource and time intensive, and may not be justified given the high level of probability of cost-effectiveness based on our model. nevertheless, it will remain important to be mindful of the thresholds for variables presented at which pos is no longer estimated to be cost-effective, and monitoring of these values over time will be important following to ensure pos remains a cost-effective endeavour. . references . hoffman ji, kaplan s. the incidence of congenital heart disease. j am coll cardiol. ; ( ): - . . van der linde d, konings ee, slager ma, witsenburg m, helbing wa, takkenberg jj, et al. birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis. j am coll cardiol. ; ( ): - . . talner cn. 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[principles and practice of mass screening for disease]. bol oficina sanit panam. ; ( ): - . appendices appendix a: algorithm for pulse oximetry screening proposed by the american academy of pediatrics( ) appendix b: levels of newborn care in ontario and geographical distribution in local health integration networks the provincial council for maternal and child health (pcmch) in ontario has assigned a designation (and thereby the appropriate resources) to all hospitals that provide maternal and newborn care. the levels of care are fully delineated at their website as follows: http://www.pcmch.on.ca/health-care-providers/maternity-care/pcmch-strategies-and- initiatives/loc/. a brief summary of the capabilities of each level of care (as it relates to caring for a patient with suspected cchd) is summarized in table b. below. table b. : summary of hospital capabilities by levels of neonatal care designated by pcmch level  evaluation and postnatal care of healthy newborn infants who are predominantly cared for in a mother-baby dyad model (rooming-in)  resuscitation and stabilization of ill infants before transfer to an appropriate care facility  no echocardiographic capability level  moderately ill newborns with problems expected to resolve within a week or who are convalescing after intensive care  no echocardiographic capability officially, although some centres may have this available on an intermittent basis level  care of acutely unwell infants as well as extremely preterm neonates including mechanical ventilation support for as long as required  timely access to a comprehensive range of subspecialty consultants, including pediatric echocardiography  note: only the hospital for sick children (sickkids) has access to cardiovascular surgery for cchd in the province in addition, delivery of healthcare in ontario is distributed according to local health integration networks (lhins). each lhin constitutes a number of hospitals with varying levels of neonatal care, and each lhin, or certain hospitals within a lhin, are supported by one of the http://www.pcmch.on.ca/health-care-providers/maternity-care/pcmch-strategies-and-initiatives/loc/ http://www.pcmch.on.ca/health-care-providers/maternity-care/pcmch-strategies-and-initiatives/loc/ five “level ” centre clusters in ontario (toronto has three level hospitals, but for the purpose of this discussion are considered one cluster), which includes dedicated transport teams who would be responsible for moving such babies from the community level or level hospitals to the nearest level institution. a map of the lhins in ontario is shown below in figure b. . figure b. : geographical depiction of distribution of lhins in ontario appendix c: literature search strategy on medline for mortality related to cchd . aortic coarctation/ or ebstein anomaly/ or hypoplastic left heart syndrome/ or "tetralogy of fallot"/ or "transposition of great vessels"/ or double outlet right ventricle/ or tricuspid atresia/ or "trilogy of fallot"/ . pulmonary atresia/ . aortic valve stenosis/ . pulmonary valve stenosis/ . exp pulmonary valve stenosis/ . truncus arteriosus, persistent/ or truncus arteriosus/ . (truncus arteriosus or transposition of the great arteries or transposition of great arteries or transposition of the great vessels or transposition of great vessels or (tetralogy adj fallot*) or (tricuspid adj atresia*) or absent right atrioventricular connection or (pulmonary adj atresia*) or (aortic adj stenos?s) or (aort* adj coarctation*) or (pulmon* adj stenos?s) or (hypoplastic adj heart syndrom*) or heart hypoplasia syndrom* or taussig-bing anomal* or taussig bing anomal* or double outlet right ventricle or double-outlet right ventricle or (ebstein* adj (anomal* or malformation*))).ti,ab,kf. . total anomalous pulmonary venous drain*.ti,ab,kf. . total anomalous pulmonary venous drain*.mp. . total anomalous pulmonary venous return*.ti,ab,kf. . aortic arch atresia*.ti,ab,kf. . aortic arch atresia*.mp. . (aort* adj atresia*).ti,ab,kf. . or/ - . (critical congenital heart adj (disease* or defect*)).ti,ab,kf. . (cyanotic congenital heart adj (disease* or defect*)).ti,ab,kf. . (critical heart adj (disease* or defect*)).ti,ab,kf. . (cyanotic heart adj (disease* or defect*)).ti,ab,kf. . or/ - . or . exp infant, newborn/ . (neonate* or neo nate* or newborn*).ti,ab,kf. . or . and . exp mortality/ . mortalit*.ti,ab,kf. . death*.ti,ab,kf. . or or . fetal mortality/ or infant mortality/ or mortality, premature/ or perinatal mortality/ . ((f?etal or newborn* or neonat* or perinatal) adj (death* or mortalit*)).ti,ab,kf. . or . morbidity/ or incidence/ or prevalence/ . morbidit*.ti,ab,kf. . (incidence or prevalence).ti,ab,kf. . or/ - . or . or . and (( and ) or ) . and . and . survival rate/ . and . exp human development/ . exp intellectual disability/ or exp psychomotor disorders/ . motor disorders/ . exp neurodevelopmental disorders/ . cerebral palsy/ . or or or or . and . and . and . and . (burden adj (illness* or disease*)).ti,ab,kf. . and appendix d: monthly mortality probabilities with estimated ranges (adapted from statistics canada data) age (years) males females average monthly average low range high range statistics canada data derived data . . . . . . . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . e- . e- . e- . . . e- . e- . e- . . . e- . e- . e- . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . e- . . . . e- . e- . . . . . e- . e- . . . . . e- . e- . . . . . e- . e- . . . . . e- . e- . . . . . e- . e- . . . . . . e- . . . . . . e- . . . . . . e- . . . . . . e- . . . . . . e- . . . . . . e- . . . . . . e- . . . . . . e- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . appendix e: detailed search strategy for mortality and complications related to cchd . aortic coarctation/ or ebstein anomaly/ or hypoplastic left heart syndrome/ or "tetralogy of fallot"/ or "transposition of great vessels"/ or double outlet right ventricle/ or tricuspid atresia/ or "trilogy of fallot"/ . pulmonary atresia/ . aortic valve stenosis/ . pulmonary valve stenosis/ . exp pulmonary valve stenosis/ . truncus arteriosus, persistent/ or truncus arteriosus/ . (truncus arteriosus or transposition of the great arteries or transposition of great arteries or transposition of the great vessels or transposition of great vessels or (tetralogy adj fallot*) or (tricuspid adj atresia*) or absent right atrioventricular connection or (pulmonary adj atresia*) or (aortic adj stenos?s) or (aort* adj coarctation*) or (pulmon* adj stenos?s) or (hypoplastic adj heart syndrom*) or heart hypoplasia syndrom* or taussig- bing anomal* or taussig bing anomal* or double outlet right ventricle or double-outlet right ventricle or (ebstein* adj (anomal* or malformation*))).ti,ab,kf. . total anomalous pulmonary venous drain*.ti,ab,kf. . total anomalous pulmonary venous drain*.mp. . total anomalous pulmonary venous return*.ti,ab,kf. . aortic arch atresia*.ti,ab,kf. . aortic arch atresia*.mp. . (aort* adj atresia*).ti,ab,kf. . or/ - . (critical congenital heart adj (disease* or defect*)).ti,ab,kf. . (cyanotic congenital heart adj (disease* or defect*)).ti,ab,kf. . (critical heart adj (disease* or defect*)).ti,ab,kf. . (cyanotic heart adj (disease* or defect*)).ti,ab,kf. . or/ - . or . obstetric labor, premature/ or premature birth/ . infant, premature/ or infant, extremely premature/ . infant, low birth weight/ or infant, small for gestational age/ or infant, very low birth weight/ or infant, extremely low birth weight/ . ((prematur* or preterm*) adj (labo?r or infant* or newborn* or bab* or birth* or born)).ti,ab,kf. . (low adj (birthweight* or birth weight*)).ti,ab,kf. . (preemie* or premie*).ti,ab,kf. . or/ - [preterm birth and low birth weight] . multiple birth offspring/ or quadruplets/ or quintuplets/ or triplets/ or twins/ or twins, dizygotic/ or twins, monozygotic/ . pregnancy, multiple/ or pregnancy, quadruplet/ or pregnancy, quintuplet/ or pregnancy, triplet/ or pregnancy, twin/ or superfetation/ . ((birth or pregnan* or born) adj (twin* or triplets or quadruplets or quintuplet* or multiple*)).ti,ab,kf. . or/ - [multiples] . human development/ or adolescent development/ or child development/ or language development/ or child language/ or crying/ . intellectual disability/ or auditory perceptual disorders/ . exp mental disorders/ . and . (infant* or neonate*).mp. [mp=title, abstract, original title, name of substance word, subject heading word, keyword heading word, protocol supplementary concept word, rare disease supplementary concept word, unique identifier] . and . exp neurocognitive disorders/ or exp neurodevelopmental disorders/ . growth disorders/ or fetal growth retardation/ . exp infant, premature, diseases/ . child development disorders, pervasive/ or asperger syndrome/ or autism spectrum disorder/ or autistic disorder/ . exp hearing disorders/ or exp vision disorders/ . cognition disorders/ or mild cognitive impairment/ . cerebral palsy/ . mental disorders/ or neurocognitive disorders/ or neurodevelopmental disorders/ . neurodevelopmental disorders/ or communication disorders/ or developmental disabilities/ or intellectual disability/ or learning disorders/ or motor skills disorders/ . (vision or hearing disorders).mp. [mp=title, abstract, original title, name of substance word, subject heading word, keyword heading word, protocol supplementary concept word, rare disease supplementary concept word, unique identifier] . and [scope of neurodevelopmental disorders] . breast neoplasms/ and utility.mp. . health status indicators/ and "quality of life"/ . and . oximetry/ . pulse oximetry screening.ti,ab,kf. . exp "sensitivity and specificity"/ . and . remove duplicates from [pos and sensitivity] . echocardiography/ or echocardiography, doppler/ or echocardiography, doppler, color/ or echocardiography, doppler, pulsed/ or echocardiography, three-dimensional/ . and . infant, newborn/ . (newborn* or neonate* or neo nate*).ti,ab,kf. . or . and . diagnostic errors/ or false negative reactions/ or false positive reactions/ . ( or ) and . and . remove duplicates from . and . remove duplicates from [echo cchd infant sens and spec] appendix f: detailed search strategy for utility related to cchd . disutilit*.ti,ab,kf. . standard gamble*.ti,ab,kf. . time tradeoff.ti,ab,kf. . time trade off*.ti,ab,kf. . tto.ti,ab,kf. . (hui or hui or hui or hui ).ti,ab,kf. . (eq or euroquol or euro qol or eq d or eq d or euroqual or euro qual).ti,ab,kf. . (utilit* adj (valu* or measur* or health or life or estimat* or elicit* or disease* or score* or weight*)).ti,ab,kf. . (burden* adj (illness* or disease*)).ti,ab,kf. . "quality of life"/ . (quality adj life).ti,ab,kf. . value*.ti. . (quality adjusted or daily adjusted or qaly or daly).ti. . health stat* value*.ti,ab,kf. . (patient* adj (perspective* or perce*)).ti,ab,kf. . decision support techniques/ . gamble*.ti,ab,kf. . prospect theory.ti,ab,kf. . preference score*.ti,ab,kf. . preference elicitation*.ti,ab,kf. . (elicit* adj preference*).ti,ab,kf. . health state.ti,ab,kf. . feeling thermomet*.ti,ab,kf. . best-worst scal*.ti,ab,kf. . best worst.ti,ab,kf. . probability tradeoff*.ti,ab,kf. . probability trade off*.ti,ab,kf. . preference based.ti,ab,kf. . multiattribute*.ti,ab,kf. . multi attribute*.ti,ab,kf. . (euroq?l d or eq d or sf d or sf d or hui or d).ti,ab,kf. . (sf or sf or sf or sf or hrqol or qol).ti,ab,kf. . or/ - . aortic coarctation/ or ebstein anomaly/ or hypoplastic left heart syndrome/ or "tetralogy of fallot"/ or "transposition of great vessels"/ or double outlet right ventricle/ or tricuspid atresia/ or "trilogy of fallot"/ . pulmonary atresia/ . aortic valve stenosis/ . pulmonary valve stenosis/ . exp pulmonary valve stenosis/ . truncus arteriosus, persistent/ or truncus arteriosus/ . (truncus arteriosus or transposition of the great arteries or transposition of great arteries or transposition of the great vessels or transposition of great vessels or (tetralogy adj fallot*) or (tricuspid adj atresia*) or absent right atrioventricular connection or (pulmonary adj atresia*) or (aortic adj stenos?s) or (aort* adj coarctation*) or (pulmon* adj stenos?s) or (hypoplastic adj heart syndrom*) or heart hypoplasia syndrom* or taussig-bing anomal* or taussig bing anomal* or double outlet right ventricle or double-outlet right ventricle or (ebstein* adj (anomal* or malformation*))).ti,ab,kf. . total anomalous pulmonary venous drain*.ti,ab,kf. . total anomalous pulmonary venous drain*.mp. . total anomalous pulmonary venous return*.ti,ab,kf. . aortic arch atresia*.ti,ab,kf. . aortic arch atresia*.mp. . (aort* adj atresia*).ti,ab,kf. . or/ - . (critical congenital heart adj (disease* or defect*)).ti,ab,kf. . (cyanotic congenital heart adj (disease* or defect*)).ti,ab,kf. . (critical heart adj (disease* or defect*)).ti,ab,kf. . (cyanotic heart adj (disease* or defect*)).ti,ab,kf. . or/ - . or . and . remove duplicates from bmj london, saturday october affective disorder tends to run in families. the risk to relatives depends on whether the proband suffers from bipolar or unipolar illness-that is, manic depression or recurrent depression; the average lifetime risk of affective disorder in the first degree relatives of a patient with a unipolar illness is about % and roughly double that when the patient has a bipolar illness. the higher risk to the relatives of patients with bipolar illness implies that bipolar illness is under greater genetic influence than unipolar illness, and this has been confirmed by studies in twins' and in the adopted- away children of manic-depressive patients. it was therefore logical to apply the techniques ofmolecular genetics to bipolar disease and to study the interaction between genes and environment in patients with unipolar illness. both these approaches have recently born exciting if unexpected fruit. several groups have carried out linkage studies in those families of patients with bipolar illness that contain many other cases. the breakthrough came from research among the old order amish, a religious community in pennsylvania that is cut off from the outside world and in which families could be examined back through several generations. in one pedigree of members linkage was found between the occurrence of illness (in people) and two markers on chromosome , the insulin gene, and the cellular oncogene c-harvey-ras- . - this suggests that in this pedigree at least a dominant gene that confers a strong predisposition to bipolar disorder lies on the tip of the short arm of chromosome . the structural gene for tyrosine hydroxylase is located in this area and is a possible candidate for the proposed locus for the disease, but o'malley and rotwein suggest that it may not be in precisely the right position. the gene on chromosome does not, however, seem to account for most cases of bipolar disorder because two studies -one of north american and the other of icelandic pedigrees -have ruled out linkage of the insulin and c- harvey-ras- genes to bipolar disorder. further evidence for genetic heterogeneity has come from recent studies showing linkage between bipolar disorder and markers on the x chromosome in several israeli and belgian families. ' yet x linkage was ruled out by father to son transmission in the american and icelandic pedigrees, so possibly at least three genes may predispose to bipolar disorder: one on the x chromosome, one on chromosome , and one or more elsewhere. the priorities now must be to establish the prevalence ofx linked bipolar illness and whether the chromosome linkage may be replicated outside the genetically isolated amish community. methodological hazards await the molecular geneticist who strays into the psychiatric arena," but most militate against finding linkage. the greatest pitfall is defining the affected phenotype-for example, in the amish study relatives without bipolar disorder were scored as affected if they had suffered from schizoaffective disorder, atypical psychosis, or major depression. a good case can be made for including these disorders because of their genetic relation to bipolar illness. indeed, linkage studies may illuminate such relations by helping us to understand the range of phenotypic abnormality that can result from defects at a single locus. this may eventually mean that psychiatric disorders can be defined aetiologically rather than phenomenologically. in the short term, however, investigators must beware of generating spuriously inflated estimates of linkage by manipulating the definition of an affected case. in ordinary unipolar depression genetic transmission is likely to be polygenic and the social environment at least as important as the genetics. you might suppose that people with a strong family diathesis for affective disorder would need fewer adverse environmental events to succumb to depression than those without affected relatives, but the recently completed camberwell collaborative depression study has disproved this prediction.' indeed, the relatives of probands whose depression followed life events or long term difficulties had slightly higher rates of depression in their lifetimes than the relatives of probands whose illness began without associated adversity. an even more unexpected finding was that the relatives of depressed probands were significantly more likely to have suffered threatening life events in the period before the interviews than a sample from the community. these events did not follow the turmoil caused by the proband's illness. the intriguing possibility is thus raised that some people may either inherit or learn from their parents the propensity to encounter adversity. the blues singer memphis slim used to sing: my mama had them; her mama had them; now i've got them too; folks, you've got to inherit the blues. he was right about bipolar disorder, for which there seems to be at least three different "blue" genes. in people who bmj volume october blue genes three genes linked to bipolar affective disorder o n a p ril b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://w w w .b m j.co m / b m j: first p u b lish e d a s . /b m j. . . o n o cto b e r . d o w n lo a d e d fro m http://www.bmj.com/ suffer from unipolar depression the cause is more complicated: their parents may have passed on to them a tendency to bring troubles down on themselves. michael j owen medical research council research fellow, department of biochemistry and molecular genetics, st mary's hospital, london w lny robin m murray dean, institute of psychiatry, london se af i bertelsen a, harsald b, hauge m. a danish twin study of manic-depressive disorders. br j psvchiatrv ; : - . nmendelewicz j, rainer jd. adoption study suppyrting genctic transmission in manic-deprcssivc illness. nature ; : - . (ierhard d, egeland ja, pauls d)l, et al. is a gene for affective disorder located on the short arm of' chromosome ii ? .am] hum genet ; : . (;erhard d, egeland ja, pauls di, housman de. search for a gene that predisposes individuals to bipolar disorder. ] psvchiatr res ; : - . egeland ja, gerhard ds, pauls dl, et al. bipolar affective disorders linked t(o dna markers on chromosome . nature ; : - . o'mallev k, rotwein p. human tvrosine hydroxylase and insulin genes are contiguous on chromosome . nucltic acids res ; : - . detera-wadleigh sd, berrettini wh, goldin lr, boorman d, anderson s, gershon es. close linkage of c-harsev-ras- and the insulin gene to affective disorder is ruled out in three north american pedigrees. nature ; : - . hodgkinson s, sherrington r, gurling hmd, et al. molecular genetic evidence for heterogeneity in manic depression. nature ; : - . baron ml, risch n, hamburger r, et al. genetic linkage between x-chromosome markers and bipolar affective illness. nature ; : - . miendelwicz j, simon p, sevs s, et al. polymorphic dna markers on x chromosome and manic- depression. ianet ;i: - . sturt h. icguffin p. (,an linkage and marker association resolve the genetic aetiology of psychiatric disorders? psvchol aed ; : - . blehar mc, weismann mm, gershon es, hirschfield rma. family and genetic studies of affective disorders. . rch fen psvchtat-, ; : - . mcguiffin p, katz r, bebbington p. 'i'he camberwell collaborative depression study iii: depression and adversits in the relatives of depressed probands. br] psychiatry ; : - . anderson-fabry disease a commonly missed diagnosis angiokeratomata, the vascular skin lesions characteristic of anderson-fabry disease, an unusual x linked lysosomal storage disorder, were first described in . deficient activity of the lysosomal hydrolase ut-galactosidase a results in the progressive deposition of uncleaved, neutral glyco- sphingolipids (predominantly (a-galactosyl-lactosyl ceramide (trihexosyl ceramide)) within the lysosomes of endothelial, perithelial, and smooth muscle cells, producing the clinical manifestations of the disease.' skin lesions cluster in the "bathing trunk" area and are often overlooked. affected males have pain and paraesthesiae in the extremities and may be labelled as neurotic. diagnosis is often delayed until renal failure or the cerebrovascular complications of the disease develop. the mean age at diagnosis in index cases from large pedigrees known to us was years. one third presented with proteinuria and a diagnosis based on renal histopatho- logical examination. most women who are carriers are without symptoms, but they often have clinical evidence of the disease; in a few this may be as severe as in men. the carrier state may be diagnosed by detecting a corneal dystrophy (cornea verticil- lata) on examination with a slit lamp and by showing reduced leucocyte a(-galactosidase a activity, although there is con- siderable overlap with the lower end of the normal range. genetic counselling remains the cornerstone of management supported by biochemical prenatal diagnosis on amniocytes or chorionic villi. the gene responsible for expressing (c-galactosidase a has been localised to the middle of the long arm of the x chromosome. x studies of linkage have identified cdna markers that could provide a simple method of diagnosing the disease in carriers or affected fetuses in informative pedigrees. - there is no specific treatment for anderson-fabry disease, and the average life expectancy of affected men was years before renal replacement treatment became available. treat- ment is symptomatic, and painful crises often respond to conventional doses of phenytoin or carbamazepine, or both. deposition of sphingolipid in vascular endothelium activates platelets, contributing to embolic or thrombotic cerebrovascular events.' the early use of antiplatelet agents, such as aspirin, seems a rational prophylactic approach. cardiac abnormalities, including disturbances in conduction and valvular disease, are common,' "' and we recommend that affected patients and carriers should be assessed by echocardiography if a murmur is detected clinically. standard advice on dental and surgical antibiotic prophylaxis should be given. renal transplantation is appropriate when end stage renal failure develops, although maizel et al have reported a high incidence of sepsis and failure after transplantation.' anecdotal reports of improvement in symptoms and transient increases in plasma ct-galactosidase a activity after trans- plantation are numerous but not substantiated in most patients; and reaccumulation of ceramide has been observed in the transplanted kidney. ' enzyme replacement treat- ment has been explored with human plasma or a-galactosidase a extracted from spleen or placenta, but the results have been unsatisfactory. fetal liver transplantation has been similarly unrewarding in three adult patients followed long term. a complementary dna that encodes mature a-galacto- sidase a has been cloned and sequenced. this should facilitate analysis of the structure, organisation, and expres- sion of the normal and mutant gene and help delineate the nature of lysosomal enzyme biosynthesis and molecular recognition of events that are part of lysosomal intracellular processing. this basic scientific work should allow the eventual development of gene therapy with somatic cells. meanwhile, early recognition of this metabolic disorder would prevent much personal distress and loss of time from school and work. it would also facilitate early referral for genetic counselling and avoid regretted decisions in family planning. stephen h morgan medical research council clinical scientist and honorary senior registrar in medicine, division of inherited metabolic diseases, clinical research centre, middlesex hai uj martin d'a crawfurd consultant clinical geneticist, kennedy galton centre, northwick park hospital, and division of inherited metabolic diseases, clinical research centre, middlesex hai uj bmj volume october o n a p ril b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://w w w .b m j.co m / b m j: first p u b lish e d a s . /b m j. . . o n o cto b e r . d o w n lo a d e d fro m http://www.bmj.com/ doi: . /j.acra. . . real-time, interactive mri for cardiovascular interventions elliot r. mcveigh, phd, michael a. guttman, ms, peter kellman, phd, amish n. raval, md, robert j. lederman, md key words. mri; intervention; real-time; therapy over the past decade, there has been a small contingent of laboratories developing magnetic resonance imaging (mri)-guided intravascular techniques and applications. while these efforts have followed in the footsteps of mri-guided surgical technologies ( , ), intravascular techniques do not carry the requirement for an open ac- cess scanner, and hence higher imaging performance dur- ing procedures can be achieved. the concept of precise real-time tracking of an active catheter in a standard mri scanner was fully realized more than years ago by du- moulin and colleagues ( ). interventional mri has subse- quently developed into the obvious method for delivery of numerous therapies. this review addresses the recent de- velopments and state-of-the-art of a number of aspects of interventional cardiovascular mri. real-time imaging one of the principal enabling technologies for guiding intravascular procedures is real-time imaging. when cath- eter tracking was first implemented, special pulse se- quences were required to obtain the catheter position from a few rapid projections; for some applications, this is now unnecessary due to the ability of modern scanners to pro- vide up to frames per second. there is obviously a acad radiol ; : – from laboratory of cardiac energetics (e.r.m., m.a.g., p.k.) and cardiol- ogy branch (a.n.r., r.j.l.), national heart, lung, and blood institute, na- tional institutes of health, building , room b d , bethesda, md - . received january , ; accepted february , . the authors are supported through the intramural research program of the na- tional institutes of health, department of health and human services. ad- dress correspondence to e.r.m. e-mail: emcveigh@nih.gov © aur, doi: . /j.acra. . . tradeoff between spatial resolution and temporal resolu- tion, but this is fully adjustable in an interactive system. one of the significant developments for real-time imaging is the use of multiple receiver systems used in conjunc- tion with specially designed coil arrays to enable parallel imaging techniques. in this next section, some of these techniques are briefly described. parallel imaging methods parallel imaging is a rapid imaging method that can be applied to real-time imaging. parallel imaging exploits the difference in sensitivity profiles between individual coil elements in a receive array to reduce the number of gradi- ent encoding steps required for imaging. parallel imaging uses r-fold k-space undersampling to achieve an acceler- ation factor of rate r. the alias artifacts (r uniformly spaced ghosts) caused by k-space undersampling are then cancelled by the parallel image reconstruction algorithm. parallel image reconstruction may be performed in the k-space domain, for example, by using simultaneous ac- quisition of spatial harmonics (smash) ( ), or in the image domain, for example, by using the sensitivity en- coding method (sense) ( ). the acceleration factor is limited in practice by the available snr and the ability to cancel the alias ghost images caused by undersampling. parallel image recon- struction is based on solving a system of linear equations in the least-squares sense to separate the r-ghost alias images with signals from n receiver coils. the perfor- mance and robustness of the reconstruction algorithm are improved with r��n (overdetermined set of equations). mr surface coil array and digital receiver technology have improved dramatically, and mr system products are now available with up to n � channels, enabling ro- r � rame mcveigh et al academic radiology, vol , no , september bust performance with acceleration rate r � in a single dimension. the parallel imaging reconstruction assumes that the coil sensitivity profiles are known or can be estimated. a key area of current research has been on autocalibration methods for estimating the in vivo coil sensitivities ( – ). for real-time dynamic imaging applications, the adaptive tsense method ( ) provides a means of automatic up- date which is useful for interventional mr application, which are free-breathing. in the tsense method, the k-space undersampling is varied cyclically in time such that after r-frames, all of k-space is acquired in a fashion similar to viewsharing or unfold ( ). lower temporal resolution reference images may be reconstructed in order to calculate the coil sensitivities (b maps) used for parallel imaging solution. parallel imaging image reconstruction has been implemented in real-time with low latency using a software-based multithreaded implementation ( ). example real-time images acquired at approximately fps with sense rate are shown in figure (consec- utive time frames). a true-fisp (ssfp) sequence pro- vides high contrast between blood and myocardium. the imaging matrix in this example is � correspond- ing to . � m in-plane spatial resolution for the spec- ified fov. the autocalibrating tsense method is used; figure . example real-time images of short axis slice using rate figure . example real-time volumetric images for a single time f imaging was performed using a . -t siemens sonata with a custom eight-element linear array (nova medical, inc., wilmington, ma). real-time volumetric imaging of the entire heart was performed at volumes per second using high accelera- tion rate parallel imaging. in three-dimensional imaging applications using two phase encode dimensions, it is preferable to perform accelerated imaging (k-space under- sampling) in each of the two phase encode directions rather than a higher rate along a single direction. this has been referred to as d-sense ( ). example real-time, free-breathing volume images with a true-fisp sequence acquired at approximately volumes per second with sense rate � � are shown in figure (single volume). the imaging matrix in this example is � � corresponding to . � . � m resolution for the specified fov. the autocalibrating tsense method is used; imaging was performed using a -channel . -t siemens avanto with a prototype -element surface coil array from rapid biomedical (wurzburg, germany). real-time interactive scanner control one of the uniquely useful features of mri is that the sense at frames per second. using rate r � � sense at volumes per seconds. image contrast, the imaging planes, and the spatial and academic radiology, vol , no , september interactive mri for cardiovascular interventions temporal resolution can be changed interactively during imaging. saturation pulses can be played between excita- tions, timings within the sequence can be altered, gradient amplitudes can be modified, or k-space trajectories can be changed. image reconstruction and display can have many interactive features, ranging from changing reconstruction methods and parameters, to color highlighting, three-di- mensional rendering, and device tracking. the ability to change these imaging and display pa- rameters during a scan may be used as the physician ad- vances from one stage to another during an interventional procedure. for example, in a procedure requiring catheter- based delivery of a therapeutic agent to a precise target, one might start imaging with a large fov as a catheter is navigated through the major vessels, color-highlight im- ages using catheter coil signals, intermittently turn off slice selection to see the whole catheter (including por- tions that are outside the imaging plane), and then reduce the fov when nearing the target. saturation or other con- trast changes may be turned on to visualize delivery of the agent, and high-resolution images can be obtained to visualize the result of the delivery (ie, the shape of a burn or shape of an injection). multiple imaging planes may be imaged and interactively adjusted during the scan to allow simultaneous views of the current catheter position and the target. during the past several years, a number of research groups have developed systems that attach to an mr scanner and provide graphic user interfaces (guis) for real-time interactive imaging. the first of these systems allowed basic real-time imaging with a single adjustable imaging plane ( , ), then adding many of the features described earlier ( – ). more recently, there have been attempts to automate the changing of image parameters in response to motion of a device being tracked ( , ). some mr scanner manufacturers provide an interac- tive interface for adjusting imaging planes and some pa- rameters during a real-time scan. these products also typ- ically provide features for saving streams of images, book marking, pausing, and limited changes to image contrast, such as turning on a saturation pulse. in some cases, de- vice tracking is also available. many of the research implementations use a high-per- formance computer attached to the mr scanner to obtain echo (or “raw”) data or reconstructed images as they be- come available. it is important to complete all image re- constructions and renderings no more than one-fourth sec- ond after the data are available; otherwise, manual tasks become increasingly difficult to perform using image guidance. typically, a bus adapter or high-speed network connection is used for rapid data transfer. the computer can then perform reconstructions and graphic operations as necessary for the given procedure. commands can also be sent from the computer to the scanner to “close the loop” and perform all scanner operations using custom software. one such implementation at the national insti- tutes of health ( ) uses a workstation with multiple - bit cpus and high-performance graphics attached by gi- gabit ethernet to the reconstruction computer of a sie- mens sonata or avanto . -t scanner. the displays of the mr scanner, external computer, and hemodynamic moni- tor are rear-projected in the magnet room as shown in figure . interventional devices passive devices catheters can be located in the imaging volume using the contrast obtained from the distortion or loss of signal caused by the catheter ( ); however, many things pro- duce dark spots in mr images. if the imaging field of view containing the catheter is particularly cluttered, as is often the case in vascular areas around the heart, the de- vice can be lost. catheters filled with contrast agent such as gd-dtpa can highlight the device with bright signal ( ), but the device can still exit the imaging plane, caus- ing the loss of the location of the tip. the principal ad- vantage of passive devices is the fact that there is no con- cern about generating unwanted heating. a very success- ful application of a passive device is the co -filled balloon used by razavi and colleagues ( ), which was also used by kuehne and colleagues ( ) to obtain right ventricular pv loops. a recent passive device is a catheter filled with hyper- polarized c ( ). while this requires a second transmit/ receive system for the scanner (the resonant frequency of c is approximately one-fourth that of protons), the inde- pendent frequency is of great benefit in separating the device from the proton background image. in this way, it is conceivable that the device could be simultaneously imaged in a three-dimensional projection view, while si- multaneous imaging of the tissue with protons. active devices electrically connected devices.—the concept of incor- porating small locator coils into a catheter for tracking position, which was demonstrated more than a decade ago mcveigh et al academic radiology, vol , no , september figure . (a) picture during an interventional procedure at the national institutes of health (nih). the rear projection screen shows monitors from the mr scanner, an ex- ternal computer, and the hemodynamics monitor. (b) real-time multiple slice imaging with active invasive devices and three-dimensional rendering on the custom reconstruc- tion computer at the nih. procedure shown is experimental placement of an endograft in an abdominal aortic aneurysm in a pig. image data from coils in the guiding catheter are highlighted green; image data from coils just distal to the endograft are highlighted red. academic radiology, vol , no , september interactive mri for cardiovascular interventions by dumoulin and colleagues ( ), has been well estab- lished ( ). serfaty and colleagues ( ) demonstrated that active guidewires could be used to position devices under mri guidance and projection angiography could be performed ( ), and recently omary and colleagues ( ) demon- strated performing coronary catheterization on of swine with an active guidewire. a stelleto (boston scientific, natick, ma) injection catheter has been modified to act as two separate coils: one in the shaft of the catheter, and the other localized to the tip. these two coils can be connected to their own channels in the receiver. this device was used to target the injection of mesenchymal stem cells in the border zone of infarcts ( ). a similar injection cathe- ter design has been implemented by karmarkar and colleagues ( ). zuehlsdorff and colleagues ( ) made an active catheter with the ability to switch between a set of loops on the shaft of a catheter and a single small coil located at the tip. safety is an issue with any device that is electrically active; a considerable effort has been focused on the po- tential of conducting devices to generate unwanted heat- ing in the tissue ( – ). for widespread use in humans, electrically active devices will need to incorporate cables in which the currents are eliminated by radiofrequency (rf) chokes incorporated into the cable ( , ). inductively coupled devices.—quick and colleagues ( ) implemented a catheter device in which a resonant coil is imbedded, but this coil is not electronically con- nected to the scanner. the signal amplitude is amplified around the imbedded coil by inductive coupling between the imbedded coil and the receiver coil on the body sur- face. also, the effective tip angle around the imbedded coil is amplified, causing very bright signal for low tip angle images. these devices do not need to connected to a receiver, so there is no need to have a connector on the proximal end of the catheter; this makes it simpler. interventional procedures a host of applications are currently being developed with real-time mri guidance; this section lists a few of these applications. real-time display of the catheter position on three- dimensional mri has been shown to be useful for ana- tomically targeted catheter navigation and subsequent rf ablation in the inferior vena cava, the fossa ovalis, and the left atrium ( ). preliminary catheter tracking with acquisition of filtered local electrograms has been re- ported ( ), as has mri characterization of ablated myo- cardium ( ). the use of real-time interactive mri for full real-time guidance of these procedures will also allow the physician to monitor the size of the lesion immedi- ately after rf application. this will make the procedure faster and safer. schalla and colleagues ( ) demonstrated transvenous and transarterial cardiac catheterization in a porcine model of atrial septal defect wholly using ssfp rtmri and tracking receiver microcoils to mark the catheter tips. percutaneous transcatheter myocardial injection of gado- linium injectate ( ) and the targeted delivery of iron-labeled mesenchymal stem cells to specific myocardial infarct targets ( ) have both been reported. these injection applications used multiple active intravascular devices with three-di- mensional volume rendering of multislice acquisitions, with color-highlighting of catheter-related signal. also, retrograde transaortic access has been used to perform image-guided myocardial injections ( , ). mri-guided transcatheter aortic valve replacement in swine using passive nitinol devices was achieved by kuehne and colleagues ( ). this application is attractive because of the critical importance of image-guided place- ment of the stent-valve in relation to the coronary arteries and aortic root. several groups ( , ) performed percutaneous coro- nary artery intervention in healthy animals and demon- strated images of intracoronary stent artifacts ( ). extracardiac several groups have conducted angioplasty ( – ) and stenting ( – ) (telep, unpublished data, ) us- ing passive and active catheter techniques in animal mod- els of arterial stenosis. also, aortic aneurysm endografts ( , ) and inferior vena cava filter ( – ) devices have been placed, and embolization of renal artery segments ( , ) has been achieved. recently, ravel and colleagues ( ) recanalized long seg- ments of chronic total arterial occlusions in an animal model. using a custom catheter and guidewire coils, they were able to traverse long segments of occlusion while keep- ing within arterial adventitial borders, an important clinical challenge. weiss and colleagues ( ) made a transcatheter “mesocaval shunt,” or extrahepatic connection between portal and venous circulations, first using a septostomy mcveigh et al academic radiology, vol , no , september needle and later using a novel custom vascular connector. kee and colleagues integrated a flat-panel xrf system in a double-doughnut operative mri system and conducted multimodal transjugular intrahepatic portosystemic shunt- ing (tips) in animals ( ) and in patients ( ), showed a significant reduction in number of punctures required. early human experience a few human rtmri procedures have been conducted. razavi and colleagues ( ) reported a landmark series of cardiac catheterization performed successfully in children using a combined xmr environment. the regensburg team conducted high-quality selective intra-arterial mr angiography ( ) and reported some preliminary revascu- larization procedures using passive devices in the iliac ( ) and femoral ( ) arteries. as described earlier, the stanford team has conducted rtmri-assisted tips proce- dures in patients ( ). future directions the procedures that will benefit the most from mri guid- ance are those that are improved by immediate visualization of the effect of treatment. this is particularly obvious for ablation techniques, targeted injections, and vascular treat- ments in which the nature of the flow in a vessel is vital information. the technologies that are in critical need at this time are catheter-based instrumentation which is mri compatible. to date, there has been little interest from catheter manufacturers in developing these tools for the simple reason that they will not sell very many in the next few years. this means that demonstration of the ben- efits from these technologies and procedures will be prin- cipally due to the efforts of independent laboratories. references . jolesz fa, talos if, schwartz rb, et al. intraoperative magnetic reso- nance imaging and magnetic resonance imaging-guided therapy for brain tumors. neuroimaging clin n am ; 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interventions 	real-time imaging 	parallel imaging methods 	real-time interactive scanner control 	interventional devices 	passive devices 	active devices 	electrically connected devices 	inductively coupled devices 	interventional procedures 	extracardiac 	early human experience 	future directions 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ association of oct-derived drusen measurements with amd-associated genotypic snps in the amish population j. clin. med. , , - ; doi: . /jcm journal of clinical medicine issn - www.mdpi.com/journal/jcm article association of oct-derived drusen measurements with amd-associated genotypic snps in the amish population venkata ramana murthy chavali , bruno diniz , , jiayan huang , gui-shuang ying , srinivas r. sadda , and dwight stambolian ,* department of ophthalmology, university of pennsylvania, b stellar-chance labs, curie blvd., philadelphia, pa , usa; e-mail: vchavali@mail.med.upenn.edu doheny eye institute, los angeles, ca , usa; e-mails: bdinizlt@hotmail.com (b.d.); vassadda@gmail.com (s.r.s.) department of ophthalmology, universidade federal de são paulo, são paulo , brazil center for preventive ophthalmology and biostatistics, department of ophthalmology, university of pennsylvania, philadelphia, pa , usa; e-mails: huangjiayan@gmail.com (j.h.); gsying@mail.med.upenn.edu (g.-s.y.) department of ophthalmology, keck school of medicine of the university of southern california, los angeles, ca , usa * author to whom correspondence should be addressed; e-mail: stamboli@mail.med.upenn.edu; tel.: + - - - ; fax: + - - - . academic editors: lindsay farrer and margaret deangelis received: december / accepted: january / published: february abstract: purpose: to investigate the association of optical coherence tomography (oct)-derived drusen measures in amish age-related macular degeneration (amd) patients with known loci for macular degeneration. methods: members of the old order amish community in pennsylvania ages and older were assessed for drusen area, volume and regions of retinal pigment epithelium (rpe) atrophy using a cirrus high-definition oct. measurements were obtained in the macula region within a central circle (cc) of mm in diameter and a surrounding perifoveal ring (pr) of to mm in diameter using the cirrus oct rpe analysis software. other demographic information, including age, gender and smoking status, were collected. study subjects were further genotyped to determine their risk for the amd-associated snps in the syn , lipc, arms , c , cfb, cetp, cfi and cfh genes using taqman genotyping assays. the association of genotypes with oct measures were assessed using linear trend p-values calculated from univariate and open access j. clin. med. , multivariate generalized linear models. results: eyes were included in the analysis. multivariate analysis (adjusted by age, gender and smoking status) confirmed the known significant association between amd and macular drusen with the number of cfh risk alleles for the drusen area (the area increased . mm for a risk allele increase, p < . ), drusen volume (the volume increased . mm for a risk allele increase, p ≤ . ) and the area of rpe atrophy (the area increased . mm for a risk allele increase, p = . ). syn risk allele g is significantly associated with larger area pr (the area increased . mm for a risk allele increase, p = . ) and larger drusen volume in the central circle (the volume increased . mm for a risk allele increase, p = . ). conclusion: among the genotyped snps tested, the cfh risk genotype appears to play a major role in determining the drusen phenotype in the amish amd population. keyword: age-related macular degeneration; amd; older order amish; cfh; syn ; oct; drusen area; drusen volume; rpe atrophy; cirrus hd-oct . introduction age-related macular degeneration (amd) is a leading cause of vision loss and is influenced by genetic, environmental and dietary factors [ ]. it is estimated that in the united states alone, the number of advanced amd cases may reach up to three million by the year [ ]. clinical features of amd include the death of photoreceptors and retinal pigment epithelium (rpe). drusen are hallmark deposits associated with early amd and considered to be a major risk factor for the progression of amd [ – ]. the late stages of amd are classically divided into two forms: non-neovascular (“dry”) amd/atrophic amd and neovascular (“wet”) amd. several studies have identified a variety of potential risk factors associated with amd [ ]. in recent years, significant advances have been made in our understanding of the genetic basis of amd. gene variants or single-nucleotide polymorphisms (snps) are routinely used to test the association with disease phenotypes. snps that increase risk or are protective have been identified for several complement-related genes: factor h (cfh) [ – ], the complement factor b region (cfb) [ , ], complement factor i [ ], complement c [ , ] and the arms /serine protease htra [ – ]. a variant in the hepatic lipase gene, lipc, was reported to decrease the risk of advanced amd in a genome-wide association study (gwas) [ , ]. other genes in the hdl pathway and cholesterol transport, including cetp, are hypothesized to be involved in the etiology of amd [ ]. the risk of amd progression has been positively correlated with total drusen area and the drusen size using fundus photography [ , ]. color fundus photographs (cfps) are the most widely-used method for the assessment of amd. however, reliable quantitation of drusen using cfps is challenging due to the macular background from rpe and choroidal pigmentation [ ]. furthermore, most published methods for grading cfps for drusen feature a qualitative or semi-quantitative approach and are thus less sensitive to small changes in drusen number in early disease. furthermore, as cfps are two-dimensional, only the drusen area can be estimated, while drusen thickness and volume cannot be measured. we had earlier developed an automated drusen detection system for classifying age-related macular degeneration (amd) from color j. clin. med. , fundus photographs (cfps) to overcome a few of these limitations [ ]. more recently, optical coherence tomography (oct) has been advocated as a potentially useful technique for in vivo cross-sectional imaging of drusen and quantitative evaluation of retinal structure. development of higher speed and higher sensitivity spectral domain oct systems (sd-oct) has further enhanced our ability to obtain high resolution imaging of the rpe and drusen [ , ]. in the current study, we describe the correlation of oct-derived measures of drusen and rpe atrophy in an elderly amish population with snp genotypes in the cfh, cfi, cfb, cetp, c , arms , lipc and syn genes. . methods . . subjects amish individuals living in lancaster county, pennsylvania, with early amd were selected for inclusion in this study. these individuals were discovered as part of a larger population-based study in the old order amish. all subjects were given a complete eye examination with pupillary dilation. all of the study participants, aged years and older, were asked to complete a brief questionnaire about present or past cigarette smoking history and the number of years of smoking. simultaneously, ml of blood were drawn from each patient by a certified phlebotomist. digital stereo images of the macula, optic disc and macular oct scans were obtained from all study participants using a zeiss cirrus oct (carl zeiss meditec, dublin, ca, usa). the study was approved by the institutional review boards of the university of pennsylvania and university of southern california and is consistent with the tenets set forth in the declaration of helsinki. . . genotyping dna was extracted from peripheral blood leukocytes using standard protocols (qiaamp dna blood mini kit, cat# ). we genotyped a total of single-nucleotide polymorphisms for the following snps: syn (rs ), lipc (rs ), arms /htra (rs ), c (rs ), cfb (rs ), cetp (rs ), cfi (rs ) and cfh (rs ) genes. the genotyping was done using taqman snp genotyping assays (life technologies, grand island, ny, usa) following the manufacturer’s instructions. briefly, all pcr amplifications were performed with the following thermal cycling conditions: °c for min followed by cycles of °c for s and °c for min. pcr reactions were performed with taqman genotyping master mix (life technologies, ny, usa) on a ht fast real-time pcr system (life technologies, ny, usa). all pre- and post-pcr plate readings were performed on a ht fast real-time pcr system, and the allele types were confirmed by the system’s software ( ht fast real-time pcr system sds software version . ; life technologies). . . optical coherence tomography acquisition octs were obtained using the cirrus oct with a raster scan protocol ( × ). this acquisition protocol has been well established for gathering quantitative measurements of drusen area and drusen volume [ ]. scans of each eye were obtained after dilation and covered a retinal area of j. clin. med. , × mm, centered on the fovea. after acquisition, scans were immediately assessed by the operator for image quality (signal strength > ) and motion artifacts, and scans were repeated multiple times if necessary in order to obtain the best possible quality scan for subsequent analysis. . . quantification of drusen and areas of rpe atrophy drusen and rpe analyses were performed using the fda-cleared cirrus version . advanced rpe analysis algorithm. the reproducibility of this algorithm has been previously published [ – ], and we have demonstrated good agreement with manual segmentation of drusen by expert reading center graders [ , ]. the algorithm computes drusen area and volume by estimating the original rpe floor and subtracting it from the detected rpe surface (elevated by the drusen) [ ]. the drusen area and volume are reported within a -mm diameter circle centered on the fovea, as well as within a surrounding perifoveal ring (outer ring diameter of mm). areas of rpe atrophy are also detected and quantified (within the × -mm scanning square) automatically by the software based on identification of increased transmission of light into the choroid. two hundred sixty eight subjects were recruited for this study, but subjects were excluded from subsequent quantitative analysis due to poor quality scans, missing scans or features of neovascular amd, such as sub-retinal fluid, intra-retinal edema, sub-retinal tissue or serous pigment epithelial detachments in the oct b-scans in at least one eye. one subject was excluded due to poor genotyping quality. thus, eyes of consecutive subjects were included in this analysis. as one of our overall study objectives was to evaluate the correlation between eyes [ ], only subjects with good quality scans for both eyes were considered for subsequent analysis. . . statistical methods to determine if there was an association among genotypes of each snp and drusen area/volume or area of rpe atrophy, the number of risk alleles for each genotype was counted as either , or , and genotype association with each oct measure from an eye was evaluated using tests of linear trend from generalized linear models with inter-eye correlation accounted for using generalized estimating equations (gee). for the snp of syn , only two genotypes (gg and cg) were observed in the study participants, and the comparison between two genotypes were performed using analysis of variance, with inter-eye correlation accounted for using gee. the effect for each risk allele on the drusen area/volume was estimated based on the slope of generalized linear regression models. these evaluations were evaluated using univariate analysis (without adjustment of risk factors of amd) and multivariate analysis (with adjustment of age, gender and smoking status). to account for multiple comparisons from multiple snps and multiple oct measurements, we adjusted p-values using the global false discovery rate [ ]. all analyses were performed in sas v . (sas institute inc., cary, nc, usa) software; p < . was considered to be statistically significant. . results a total of subjects were recruited for our study; subjects were excluded either due to poor quality scans or features of neovascular amd on oct in at least one eye, and one additional subject was removed due j. clin. med. , to poor quality genotypes. thus, eyes of subjects were included for analysis. snp genotyping for eight snps was completed on all subjects. our sample included a total of women and men with a mean age (±standard deviation) of ± . years (range: – ). the majority of the subjects was between and years of age ( . %), and ( . %) had a history of smoking (table ). table . characteristics of study subjects (n = ). age (years) – ( . %) – ( . %) – ( . %) ≥ ( . %) unknown ( . %) mean (standard deviation) . ( . ) gender female ( . %) male ( . %) ever smoked yes ( . %) no ( . %) unknown ( . %) table shows the comparison of drusen areas/volumes and areas of rpe atrophy with genotype. in univariate analysis, significant associations of the drusen area in the perifoveal ring (pr, p = . ) and drusen area in the central circle (cc, p = . ) were observed for the risk allele gg in syn (snp, rs ). the drusen volume cc (p = . ) and drusen volume pr (p = . ) were also significantly associated with syn . the association of the area of rpe atrophy with syn showed a trend, but did not reach statistical significance (p = . ). the associations between drusen area pr (p = . ) and drusen volume cc (p = . ) with syn remained even after adjustment for age, gender and smoking status. however, these associations become non-significant after multiple comparison adjustment for the total of tests in this study. it is interesting to observe that the majority of the subjects have the syn gg risk alleles ( %). this high percentage of gg risk alleles could be attributed to interbreeding and being isolated in terms of lifestyle and culture. the homozygous cc genotype was not observed in our study population. in univariate analysis, we observed a significant association of the cfh risk allele gg for snp, rs , with drusen area cc (p = . ), drusen area pr (p = . ), drusen volume cc (p = . ), drusen volume pr (p = . ) and area of rpe atrophy (p = . ); and these associations remained after adjustment for age, gender and smoking status (table ). even after multiple comparison adjustment, the association between cfh with drusen area pr and drusen volume cc (p = . , respectively, table ) remained significant. our results indicate that snps in the cfh and syn genes are important influences for drusen size and volume in the macula and perifoveal region, a hallmark of amd. the drusen area, drusen volume and regions of rpe atrophy did not show any significant association with snps in the other genes that were tested (table ). j. clin. med. , table . genotypic associations with optical coherence tomography (oct) drusen area, drusen volume and area of retinal pigment epithelium (rpe) atrophy from univariate analysis and multivariate analysis § ( subjects, eyes). snp genotype n drusen area in mm : mean (se) drusen volume in mm : mean (se) area of rpe atrophy in mm : mean (se) central circle perifoveal ring central circle perifoveal ring normal/(risk) syn c/(g) rs gg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) cg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) cc - - - - - univariate p * . . . . . adjusted slope (se) † . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) multivariate p † . . . . . multiple comparison adjusted p by global fdr ‡ . . . . . (risk)/normal lipc (c)/g rs cc . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) cg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) gg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) univariate linear trend p * . . . . . adjusted slope (se) † . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) multivariate linear trend p † . . . . . multiple comparison adjusted p by global fdr ‡ . . . . . normal/(risk) arms g/(t) rs tt . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) gt . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) gg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) univariate linear trend p * . . . . . adjusted slope (se) † . ( . ) . ( . ) − . ( . ) − . ( . ) . ( . ) multivariate linear trend p † . . . . . multiple comparison adjusted p by global fdr ‡ . . . . . j. clin. med. , table . cont. snp genotype n drusen area in mm : mean (se) drusen volume in mm : mean (se) area of rpe atrophy in mm : mean (se) central circle perifoveal ring central circle perifoveal ring (risk)/normal c (c)/g rs cc . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) cg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) gg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) univariate linear trend p * . . . . . adjusted slope (se) † . ( . ) . ( . ) − . ( . ) − . ( . ) . ( . ) multivariate linear trend p † . . . . . multiple comparison adjusted p by global fdr ‡ . . . . . normal/(risk) cfb a/(g) rs gg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) ag . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) aa . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) univariate linear trend p * . . . . . adjusted slope (se) † . ( . ) . ( . ) − . ( . ) . ( . ) . ( . ) multivariate linear trend p † . . . . . multiple comparison adjusted p by global fdr ‡ . . . . . (risk)/normal cetp (a)/c rs aa . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) ac . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) cc . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) univariate linear trend p * . . . . . adjusted slope (se) † . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) multivariate linear trend p † . . . . . multiple comparison adjusted p by global fdr ‡ . . . . . j. clin. med. , table . cont. snp genotype n drusen area in mm : mean (se) drusen volume in mm : mean (se) area of rpe atrophy in mm : mean (se) central circle perifoveal ring central circle perifoveal ring normal/(risk) cfi c/(t) rs tt . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) ct . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) cc . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) univariate linear trend p * . . . . . adjusted slope (se) † − . ( . ) − . ( . ) − . ( . ) − . ( . ) . ( . ) multivariate linear trend p † . . . . . multiple comparison adjusted p by global fdr ‡ . . . . . normal/(risk) cfh a/(g) rs gg . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) ag . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) aa . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) univariate linear trend p * . . . . . adjusted slope (se) † . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) multivariate linear trend p † . . . . . multiple comparison adjusted p by global fdr ‡ . . . . . § adjusted by age, gender and smoking status; * from univariate analysis (without adjustment by any other risk factors of age-related macular degeneration (amd)) for testing whether oct measures were associated with the number of risk alleles; † from multivariate analysis (with adjustment by age, gender and smoking status) for testing whether oct measures were associated with the number of risk alleles; ‡ adjusted using global fdr (false discovery rate) for multiple comparisons from eight snps and five oct measurements (i.e., adjustment for a total of tests) for p-values from multivariate analysis. j. clin. med. , . discussion we have examined the association of eight known amd genetic variants with drusen phenotypes assessed by the cirrus oct. there was a strong association between drusen area, volume and rpe atrophy in the macula with risk alleles in cfh (rs ) and syn (rs ). these eight snps have been reported to be highly associated with amd [ ]. the role of genetic variants in drusen accumulation and early amd has received little attention [ – ]. hdl pathway genes are reported to be associated with the early stages of amd, whereas arms /htra and genes in the complement pathway are associated with more advanced stages [ ]. cfh, arms /htra and c genes have been reported to increase the risk of progression from intermediate drusen to large drusen and from large drusen to geographic atrophy and neovascularization. the t allele of rs in abca is associated with a decreased risk of intermediate drusen, large drusen, geographic atrophy (ga) and neovascularization [ ], whereas the c allele of col a is protective of the transition from intermediate to large drusen [ ]. significant association of drusen progression with cfh has been reported in age-related eye disease study (areds) and familial amd cohorts [ ]. smoking is strongly associated with a . - to . -fold increased risk for late amd [ ], and its risk is known to increase – -fold with the presence of the cfh polymorphism [ ]. the cfh y h is reported to act synergistically with smoking to increase the risk of wet amd [ ]. we have observed a strong association of the cfh snp rs with macular drusen area and volume in our amish amd cohort, thus supporting the hypothesis that this variant is relevant to drusen formation. variants in the syn gene are reported to influence susceptibility to amd [ ]. the syn gene is a member of the synapsin gene family and is known to play a role in synaptogenesis and in the modulation of neurotransmitter release. the associated syn snp (rs ) with amd is located in the syn intron . this snp could be influencing the expression of syn or a nearby gene, timp , which is located within the same intron. further screening of amish cohorts with snp, rs , in timp could elucidate its role in amd [ – ]. to our knowledge, this is the first report to demonstrate an association of syn with quantitative macular drusen burden in amd. additional snps for the syn gene need to be screened to further understand the role of this gene in influencing drusen formation. apart from cfh and syn , the other genotyped genes, arms , c , cfb, lipc, cetp and cfi genes, were not associated with our drusen phenotypes. this could be due to insufficient power in our study to detect small effects. additional studies of these snps in different populations are needed to establish haplotypes for drusen burden in amd. our study was limited to subjects older than years. therefore, we cannot determine the effect of these snps on the development of drusen at a younger age. although analysis of the amish cohort has identified strong associations between cfh, syn and drusen area and volume, both false positive and false negatives may be present due to the overall small sample size. the stringency of our inclusion criteria, the use of a validated fda-cleared oct algorithm for drusen measurements and our reproducible genotyping are the strengths of our study. our results also provide a foundation to further evaluate other snps in the known amd genes for association with drusen phenotypes. color fundus photographs (cfps) offer complementary information to sd-oct [ ]. both methods play an important role in assessing drusen and rpe atrophy in patients with non-exudative amd [ , ]. j. clin. med. , however, issues with the cfps’ reproducibility due to fundus pigmentation variability and drusen appearance and media opacity negatively impacting photograph quality make sd-oct a promising alternative for imaging drusen. sd-oct provides information regarding drusen ultrastructure in vivo and quantifies the thickness of the drusen, as well as the photoreceptor layer above the drusen [ – ]. future genetic correlation studies, however, may consider incorporating and integrating phenotypic information derived from multiple imaging modalities (e.g., color photographs, fundus autofluorescence, and oct) to yield a more precise description of the disease phenotype. . conclusions our data suggest that cfh and syn risk alleles play a role in determining the drusen phenotype in the amish population. this approach of stratifying drusen phenotypes by genotype has the potential to separate amd patients into homogenous groups for preventive and therapeutic studies. of course, more snps will need to be evaluated to deliver such a genotype-phenotype classification. acknowledgments this work is supported in part by nih grant ro ey to stambolian and sadda. author contributions venkata ramana murthy chavali, bruno diniz and jiayan huang wrote the initial draft based on initial discussions and suggestions from dwight stambolian, gui-shuang ying and srinivas r. sadda. gui-shuang ying and jiayan huang contributed the tables and data analysis. dwight stambolian, srinivas r. sadda and gui-shuang ying contributed to the critical evaluation and proof reading of the 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article distributed under the terms and conditions of the creative commons attribution license (http://creativecommons.org/licenses/by/ . /). serveur académique lausannois serval serval.unil.ch author manuscript faculty of biology and medicine publication this paper has been peer-reviewed but dos not include the final publisher proof-corrections or journal pagination. published in final edited form as: in the absence of a copyright statement, users should assume that standard copyright protection applies, unless the article c ontains an explicit statement to the contrary. in case of doubt, contact the journal publisher to verify the copyright status of an article. title: genetic variation in gipr influences the glucose and insulin responses to an oral glucose challenge. authors: saxena r, hivert mf, langenberg c, tanaka t, pankow js, vollenweider p, lyssenko v, bouatia-naji n, dupuis j, jackson au, kao wh, li m, glazer nl, manning ak, luan j, stringham hm, prokopenko i, johnson t, grarup n, boesgaard tw, lecoeur c, shrader p, o'connell j, ingelsson e, couper dj, rice k, song k, andreasen ch, dina c, köttgen a, le bacquer o, pattou f, taneera j, steinthorsdottir v, rybin d, ardlie k, sampson m, qi l, van hoek m, weedon mn, aulchenko ys, voight bf, grallert h, balkau b, bergman rn, bielinski sj, bonnefond a, bonnycastle ll, borch-johnsen k, böttcher y, brunner e, buchanan ta, bumpstead sj, cavalcanti-proença c, charpentier g, chen yd, chines ps, collins fs, cornelis m, j crawford g, delplanque j, doney a, egan jm, erdos mr, firmann m, forouhi ng, fox cs, goodarzi mo, graessler j, hingorani a, isomaa b, jørgensen t, kivimaki m, kovacs p, krohn k, kumari m, lauritzen t, lévy-marchal c, mayor v, mcateer jb, meyre d, mitchell bd, mohlke kl, morken ma, narisu n, palmer cn, pakyz r, pascoe l, payne f, pearson d, rathmann w, sandbaek a, sayer aa, scott lj, sharp sj, sijbrands e, singleton a, siscovick ds, smith nl, sparsø t, swift aj, syddall h, thorleifsson g, tönjes a, tuomi t, tuomilehto j, valle tt, waeber g, walley a, waterworth dm, genetic variation in gipr influences the glucose and insulin responses to an oral glucose challenge richa saxena , , , marie-france hivert , , , claudia langenberg , toshiko tanaka , , james s pankow , peter vollenweider , valeriya lyssenko , nabila bouatia-naji , josée dupuis , anne u jackson , w h linda kao , , , man li , nicole l glazer , alisa k manning , jian’an luan , heather m stringham , inga prokopenko , , toby johnson , niels grarup , trine w boesgaard , cécile lecoeur , peter shrader , jeffrey o’connell , erik ingelsson , , david j couper , kenneth rice , kijoung song , camilla h andreasen , christian dina , anna köttgen , olivier le bacquer , full membership list of the giant consortium is provided in the supplementary note. © nature america, inc. all rights reserved. correspondence should be addressed to r.m.w. (rwatanab@usc.edu), n.j.w. (nick.wareham@mrc-epid.cam.ac.uk), l.g. (leif.groop@med.lu.se) or j.m. (jmeigs@partners.org). these authors contributed equally to this work. note: supplementary information is available on the nature genetics website. author contributions writing group: r. saxena, m.-f.h., c. langenberg., t. tanaka, j.s.p., p.v., v.l., n.b.-n., j.c.f., m.i.m., m.b., i.b., r. sladek, p.f., j.b.m., l.g., n.j.w., r.m.w. project design, management and coordination: (amish) b.d.m., a.r.s.; (aric) j.s.p., w.h.l.k., s.j. bielinski, e. boerwinkle; (blsa) a. singleton, l.f.; (botniappp) l.g., t. tuomi., b.i.; (chs) n.l.g., k.r., n.l.s., b.m.p., j.i.r.; (colaus) p.v., m.f., v. mayor, g.w., d.m.w., v. mooser; (danish) k.b.j., a.s., t. jørgensen, t.l., t.h., o.p.; (diagen) p. schwartz, s.r.b.; (dgi) r. saxena, d.a., l.g.; (ely) c. langenberg, n.j.w.; (fenland) c. langenberg, n.g.f., r.j.f.l., n.j.w.; (fhs) j.d., j.b.m.; (french) n.b.-n., p.f.; (fusion) r.n.b., f.s.c., k.l.m., l.j.s., j. tuomilehto, m.b., r.m.w.; (hertfordshire) a.a.s., h.s., c.c.; (metsim) j.k., m.la.; (mpp) p.n.; (partners/roche) j.b.m., d.m.n., g.h.w.; (risc) m.w., l.p.; (sorbs) a.t., m.s.; (ulsam) e.i.; (whitehall ii) e. brunner, a.h., m. kivimaki, m. kumari, m.m. sample collection and phenotyping: (amish) a.r.s.; (blsa) j.m.e.; (botniappp) l.g., v.l., b.i., t. tuomi; (chs) b.m.p., d.s.s, n.l.s.; (colaus) p.v., g.w.; (danish) t.w.b., k.b.j, a.s., t. jørgensen, t.l., t.h., o.p.; (diagen) j.g., p. schwartz; (dgi) l.g., v.l., b.i., t. tuomi; (ely) n.j.w.; (fenland) n.g.f., r.j.f.l., n.j.w.; (french) p.f., d.m., b.b., c.l.-m., g.c., f. pattou; (fhs) j.b.m., c.s.f.; (fusion) r.n.b., t.a.b., j. tuomilehto, t.t.v.; (hertfordshire) a.a.s., h.s., c.c.; (metsim) j.k., m. laakso; (partners/roche) j.b.m., d.m.n., g.h.w.; (sorbs) p.k., a.t.; (whitehall ii) e. brunner, m. kumari, m.m. genotyping: (amish) r.p.; (aric) e. boerwinkle.; (blsa) a. singleton; (botniappp) g.j.c.; (chs) y.-d.i.c., m.o.g., j.i.r.; (colaus) v. mooser, d.m.w.; (danish) t.h., t.s., c.h.a., n.g., o.p.; (dgi) d.a., v.l., r. saxena; (diagen) d.p., a.j.s.; (ely) i.b., s.j. bumpstead, f. payne, n.j.w.; (fenland) r.j.f.l., n.j.w.; (fhs) j.c.f., j.b.m.; (french) n.b.-n., j.d., r. sladek, d.m., a.w.; (fusion) l.l.b., m.r.e., p.s.c.; (fusion stage ) p.s.c., a.j.s.; (hertfordshire) i.b., s.j. bumpstead, f. payne, n.j.w.; (metsim) m.a.m., n.n.; (partners/roche) j.c.f., j.b.m.; (sorbs) y.b., p.k., k.k.; (ulsam) a.-c.s.; (whitehall ii) m. kumari, c. langenberg, n.j.w. statistical analysis: (meta-analyses) r. saxena, j.d., d.r., w.h.l.k., a.u.j.; (amish) j.o.; (aric) w.h.l.k., m.l., a.k., d.j.c.; (blsa) t. tanaka; (botniappp) v.l.; (chs) n.l.g., k.r.; (colaus) t. johnson, k. song; (danish) t.s., c.h.a., t.w.b., n.g.; (dgi) r. saxena; (ely) c. langenberg, s.j.s.; (fenland) c. langenberg, j.l., j.h.z.; (french) n.-b.n, c. lecoeur, c.c-p., a.b., c.d.; (fhs) j.d., a.k.m., d.r., p. shrader; (fusion) a.u.j., h.m.s.; (fusion stage ) a.u.j., h.m.s.; (hertfordshire) c. langenberg, s.j.s.; (partners/roche) p.shr.; (risc) c. langenberg, s.j.s.; (sorbs) i.p.; (ulsam) e.i.; (whitehall ii) c. langenberg. expression analysis: (malmo) j. taneera, v.l., l.g; (french) n.b.-n., o.l.b., f. patou, p.f. type diabetes association: (dgi) d.a., l.g., r. saxena, b.f.v., k.a.; (decode) v.s., g.t., u.t., k. stefansson; (eurospan) y.s.a., j.f.w., m.v.h., e.s., c.v.d.; (french) n.b.-n., j. deplanque, c. lecoeur, g.c., p.f.; (addition-ely) c. langenberg, f. payne, s.j. bumpstead, i.b., n.j.w.; (norfolk diabetes case-control study) c. langenberg, f. payne, s.j. bumpstead, i.b., m.s., n.j.w.; (cambridgeshire case-control study) c. langenberg, f. payne, s.j. bumpstead, i.b., n.j.w.; (kora) h.g., w.r., t.i., h.e.w.; (mpp) p.n., v.l., l.g.; (nhs/hpfs) f.b.h. l.q., m.c.c.; (ukt d/ bc/oxgn) a.d., c.n.a.p., a.t.h., a.d.m.; t.m.f., m.i.m.; (wtccc-ukt d) m.n.w., e.z. competing interests statement the authors declare competing financial interests: details accompany the full-text html version of the paper at http://www.nature.com/naturegenetics/. reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. nih public access author manuscript nat genet. author manuscript; available in pmc august . published in final edited form as: nat genet. february ; ( ): – . doi: . /ng. . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript http://www.nature.com/naturegenetics/ http://npg.nature.com/reprintsandpermissions/ françois pattou , , jalal taneera , valgerdur steinthorsdottir , denis rybin , kristin ardlie , michael sampson , lu qi , mandy van hoek , michael n weedon , yurii s aulchenko , benjamin f voight , , harald grallert , beverley balkau , richard n bergman , suzette j bielinski , amelie bonnefond , lori l bonnycastle , knut borch-johnsen , yvonne böttcher , eric brunner , thomas a buchanan , , suzannah j bumpstead , christine cavalcanti-proença , guillaume charpentier , yii- der ida chen , peter s chines , francis s collins , marilyn cornelis , gabriel j crawford , jerome delplanque , alex doney , josephine m egan , michael r erdos , mathieu firmann , nita g forouhi , caroline s fox , , mark o goodarzi , jürgen graessler , aroon hingorani , bo isomaa , , torben jørgensen , , mika kivimaki , peter kovacs , knut krohn , meena kumari , torsten lauritzen , claire lévy-marchal , , vladimir mayor , jarred b mcateer , , , david meyre , braxton d mitchell , karen l mohlke , mario a morken , narisu narisu , colin n a palmer , ruth pakyz , laura pascoe , felicity payne , daniel pearson , wolfgang rathmann , annelli sandbaek , avan aihie sayer , laura j scott , stephen j sharp , eric sijbrands , andrew singleton , david s siscovick , nicholas l smith , , thomas sparsø , amy j swift , holly syddall , gudmar thorleifsson , anke tönjes , , tiinamaija tuomi , , jaakko tuomilehto , , timo t valle , gérard waeber , andrew walley , dawn m waterworth , eleftheria zeggini , jing hua zhao , giant consortium , thomas illig , h erich wichmann , , , james f wilson , cornelia van duijn , frank b hu , , andrew d morris , timothy m frayling , , andrew t hattersley , , unnur thorsteinsdottir , , kari stefansson , , peter nilsson , ann-christine syvänen , alan r shuldiner , , mark walker , stefan r bornstein , peter schwarz , gordon h williams , , david m nathan , , johanna kuusisto , markku laakso , cyrus cooper , michael marmot , luigi ferrucci , vincent mooser , michael stumvoll , ruth j f loos , david altshuler , , bruce m psaty , jerome i rotter , eric boerwinkle , torben hansen , , oluf pedersen , , jose c florez , , , , mark i mccarthy , , , michael boehnke , inês barroso , robert sladek , , philippe froguel , , james b meigs , , leif groop , nicholas j wareham , and richard m watanabe [on behalf of for the magic investigators] , program in medical and population genetics, broad institute of harvard and massachusetts institute of technology, cambridge, massachusetts, usa. center for human genetic research, massachusetts general hospital, boston, massachusetts, usa. general medicine division, massachusetts general hospital, boston, massachusetts, usa. department of medicine, harvard medical school, boston, massachusetts, usa. medical research council epidemiology unit, institute of metabolic science, addenbrooke’s hospital, cambridge, uk. medstar research institute, baltimore, maryland, usa. clinical research branch, national institute on aging, baltimore, maryland, usa. division of epidemiology and community health, school of public health, university of minnesota, minneapolis, minnesota, usa. department of internal medicine, centre hospitalier universitaire vaudois, lausanne, switzerland. department of clinical sciences, diabetes and endocrinology, lund university, university hospital malmo, malmo, sweden. centre national de la recherche scientifique–unité mixte de recherche , pasteur institute, lille -droit et santé university, lille, france. department of biostatistics, boston university school of public health, boston, massachusetts, usa. center for statistical genetics, department of biostatistics, university of michigan school of public health, ann arbor, michigan, usa. department of epidemiology, bloomberg school of public health, johns hopkins university, baltimore, maryland, usa. department of medicine, school of medicine and bloomberg school of public health, johns hopkins university, baltimore, maryland, usa. the welch center for prevention, epidemiology and clinical research, school of medicine and bloomberg school of public health, johns hopkins university, baltimore, maryland, usa. cardiovascular health research unit and department of medicine, university of washington, saxena et al. page nat genet. author manuscript; 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available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript department of internal medicine, erasmus university medical center, rotterdam, the netherlands. laboratory of neurogenetics, national institute on aging, bethesda, maryland, usa. departments of medicine and epidemiology, university of washington, seattle, washington, usa. department of epidemiology, university of washington, seattle, washington, usa. epidemiologic research and information center of the department of veterans affairs office of research and development, seattle, washington, usa. coordination centre for clinical trials, university of leipzig, leipzig, germany. department of medicine, helsinki university hospital, university of helsinki, helsinki, finland. diabetes unit, department of health promotion and chronic disease prevention, national public health institute, helsinki, finland. department of epidemiology and health promotion, national public health institute, helsinki, finland. genomic medicine, imperial college london, hammersmith hospital, london, uk. institute of medical informatics, biometry and epidemiology, ludwig-maximilians- universität, munich, germany. klinikum grosshadern, munich, germany. centre for population health sciences, university of edinburgh, teviot place, edinburgh, uk. channing laboratory, brigham and women’s hospital and harvard medical school boston, massachusetts, usa. diabetes genetics, institute of biomedical and clinical science, peninsula medical school, university of exeter, exeter, uk. genetics of complex traits, institute of biomedical and clinical science, peninsula medical school, university of exeter, exeter, uk. faculty of medicine, university of iceland, reykjavik, iceland. department of clinical sciences, medicine, lund university, university hospital malmo, malmo, sweden. department of medical sciences, uppsala university, uppsala, sweden. geriatric research and education clinical center, veterans administration medical center, baltimore, maryland, usa. department of medicine, university of kuopio and kuopio university hospital, kuopio, finland. departments of medicine, epidemiology, and health services, university of washington, seattle, washington, usa. the human genetics center and institute of molecular medicine, university of texas health science center, houston, texas, usa. faculty of health science, university of southern denmark, odense, denmark. institute of biomedicine, faculty of health science, university of copenhagen and faculty of health science, university of aarhus, denmark. oxford national institute of health research biomedical research centre, churchill hospital, oxford, uk. department of human genetics, faculty of medicine, mcgill university, montreal, quebec, canada. genome quebec innovation centre, montreal, quebec, canada. department of preventive medicine, keck school of medicine, university of southern california, los angeles, california, usa. abstract glucose levels h after an oral glucose challenge are a clinical measure of glucose tolerance used in the diagnosis of type diabetes. we report a meta-analysis of nine genome-wide association studies (n = , nondiabetic individuals) and a follow-up of independent loci (n = , – , ). we identify variants at the gipr locus associated with -h glucose level (rs , β (s.e.m.) = . ( . ) mmol/l per a allele, p = . × − ). the gipr a-allele carriers also showed decreased insulin secretion (n = , ; insulinogenic index, p = . × − ; ratio of insulin to glucose area under the curve, p = . × − ) and diminished incretin effect (n = ; p = . × − ). we also identified variants at adcy (rs , p = . × − ), vps c (rs , p = . × − ), gckr (rs , p = . × − ) and tcf l (rs , p = . × − ) associated with -h glucose. of the three newly implicated loci (gipr, adcy and vps c), only adcy was found to be associated with type diabetes in collaborating studies (n = , cases, , controls, or = . , % ci . – . , p = . × − ). type diabetes (t d) is defined as a state of chronic hyperglycemia defined as elevated glucose levels measured either when fasting or h after glucose challenge ( -h glucose) saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript during an oral glucose tolerance test (ogtt). gwas have contributed to the identification of many established t d-associated loci . more recently, collaborative efforts of the meta- analysis of glucose and insulin-related traits consortium (magic) and other investigators have led to the discovery of genetic variation associated with fasting glucose levels in nondiabetic individuals, with mtnr b additionally conferring risk of t d – . not all loci associated with fasting glucose showed association with t d , , suggesting that gwas of quantitative traits related to diabetes can also identify physiological loci that provide mechanistic insights into normal trait variation. an accompanying study by magic has identified loci associated with fasting glucose or fasting insulin in a gwas-based meta- analysis; of these loci are newly identified, and also show evidence for association with t d . although there are common mechanisms, such as insulin secretion, that regulate fasting and stimulated glucose levels, there are distinct mechanisms regulating glucose levels after an oral glucose challenge. for example, oral glucose intake engenders the incretin effect, in which intestinal cells release insulin secretagogues, mainly glucagon-like peptide (glp ) and gastric inhibitory polypeptide (gip), leading to a higher insulin response compared to that from a matched intravenous glucose stimulation. additionally, numerous epidemiological studies have shown that ogtt -h glucose levels predict cardiovascular disease morbidity and mortality , even in the nondiabetic range of hyperglycemia and independently of fasting glucose levels . two-hour glucose level is a heritable quantitative trait (heritability (h ) = . ) that has been associated with diabetes, and assessing the genetic contribution to variability in -h glucose provides an opportunity to identify genetic variation underlying this trait in nondiabetic individuals and to test the secondary hypothesis that these loci may also contribute to t d susceptibility. here we performed a meta-analysis of several -h glucose gwas to expand our understanding of post–oral glucose challenge physiology in nondiabetic individuals. a meta-analysis combining discovery gwas (n = , ) and replication stages with up to snps in studies comprising up to , individuals of european descent revealed loci associated with -h glucose at genome-wide significance (p = × − ; see online methods, table , fig. , supplementary fig. and supplementary tables and ). three loci were newly associated with -h glucose in an analysis adjusted for age, sex, bmi and study-specific covariates: gipr (gastric inhibitory polypeptide receptor, rs , β (s.e.m.) = . ( . ) mmol/l per a allele, p = . × − ), vps c (vacuolar protein sorting homolog c, rs , β (s.e.m.) = . ( . ) mmol/l per g allele, p = . × − ) and adcy (adenylate cyclase, rs , β (s.e.m.) = . ( . ) mmol/l per c allele, p = . × − ). the adcy locus was also identified by an accompanying study reporting meta-analysis in magic for fasting glucose levels (r = . to the most significant fasting glucose snp rs ) . the remaining loci identified here included the previously published fasting glucose–associated gene gckr (glucokinase (hexokinase ) regulator, missense snp rs , p = . × − ) and the established t d- associated gene tcf l (transcription factor -like , rs with r = . to most significant t d snp rs , p = . × − ) . to determine whether these associations reflected differences in fasting glucose levels or whether they primarily influenced the incremental response to a glucose challenge, we repeated our association analysis including fasting glucose as a covariate (table and supplementary table ). adjusting for fasting glucose resulted in increased effect size for the gckr, gipr and vps c loci and supported their specific role in post-challenge glucose regulation. in contrast, adjusting for fasting glucose slightly decreased the effect for saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript the adcy and tcf l loci, which suggested that the risk alleles in both genes increase glucose levels both in the fasting and post-challenge state. in meta-analyses available from magic , fasting glycemic traits variants at the gipr, vps c and adcy loci were not associated with fasting insulin or insulin resistance as measured by homeostasis model assessment , which may reflect the inadequacy of the crude measures used here or may reflect a lack of power to detect small effects (supplementary table ). associations of risk alleles in gckr and tcf l with fasting glycemic traits have been reported previously . in a large swedish meta-analysis (n = , ), the gipr rs -h glucose–raising allele was significantly associated with lower bmi (pmeta = . × − , v.l. and l.g., unpublished data). gip is one of the two incretin hormones that stimulate insulin response after an oral glucose challenge. it has been shown that the incretin effect is impaired in individuals with t d ; specifically, in individuals with t d, stimulated gip secretion appears normal and their insulinotropic response to gip is reduced . gipr is therefore a biologically plausible candidate for mediating insulin secretion after oral glucose challenge. we tested associations of gipr variants with indices of oral glucose–stimulated insulin secretion in up to studies with samples measured at multiple times during the ogtt (table and supplementary table ). the rs a allele associated with increased -h glucose was also associated with lower insulinogenic index (β (s.e.m.) = − . ( . ) µu/mmol, p = . × − ), which represents a reduction in the early phase of insulin secretion . the rs a allele was also associated with a lower ratio of insulin to glucose area under the curve (auc ins/gluc, β (s.e.m.) = − . ( . ) pmol/mmol, p = . × − ), which is an integrated measure of insulin response over the -h ogtt . furthermore, the rs a allele was associated with lower -h insulin level (adjusted for -h glucose, β (s.e.m.) = − . ( . ) pmol/l, p = . × − ). because gip is involved in the insulin response specific to an oral glucose challenge, gipr variation was not expected to influence the insulin response to an intravenous glucose load. we tested the insulin response in , nondiabetic participants from four studies who underwent an intravenous glucose tolerance test (ivgtt). no association was observed with measures of acute insulin response (air) from the ivgtt (p = . ; supplementary table ), even though the study had > % power to detect an effect explaining % trait variance (α = . ). we also derived an estimate of the incretin effect by comparing the insulin response to oral versus intravenous glucose administered to the same nondiabetic individuals from the botnia , denmark and eugene -kuopio studies . individuals carrying the a risk allele of rs in gipr showed a significantly lower incretin effect (β (s.e.m.) = − . ( . ), p = . × − ; fig. and supplementary table ). our results are consistent with animal studies, in which mice with targeted deletion of gipr showed mild glucose intolerance and reduced insulin secretion in response to an oral glucose challenge but showed normal fasting glucose and normal insulin secretion in response to an intraperitoneal glucose challenge . the variant in gipr most significantly associated with -h glucose (rs ) is an intronic snp with no known function based on fastsnp (see url section). notably, rs is in strong linkage disequilibrium (r = . ) with a missense mutation (at rs , resulting in the substitution e q). some groups have explored the e q substitution as a candidate for association with t d. one study showed that people homozygous for the gln -encoding allele of this gene had lower fasting and post oral- load c-peptide levels, suggesting a role for gipr in insulin secretion ; this is in line with our observations. in small t d case-control studies, no association has been observed at gipr – . we performed a meta-analysis of t d association studies (n = , saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript diabetic individuals (cases), , nondiabetic individuals) and found that the rs a allele was moderately associated with increased risk of t d (or = . , % ci . – . ; p = . × − ; table and supplementary table ). this result, although suggestive of association, highlights the challenge of genetic approaches to complex diseases, whereby important genes involved in pathophysiology might be difficult to identify even in large case-control collections due to small individual odds ratios . we assessed the mrna expression patterns of gipr and the nearest upstream (eml ) and downstream (snrpd ) genes in a human tissue panel (fig. ). all three genes were expressed in the pancreas, but only gipr had strong specific mrna expression in the sorted pancreatic beta cells, supporting the implication of gipr in insulin secretion. no significant difference in gipr, eml or snrpd mrna expression in pancreatic islets was seen based on the rs genotype (for gipr p = . , n = ; supplementary note). as adenylate cyclases have been implicated in the camp pathway of glp- and gip- induced insulin release by beta cells , , we also tested for association of the most significant adcy variant with measures of insulin response and risk of t d. the -h glucose-raising c allele of rs was associated with lower -h insulin (p = . × − ) but was not associated with aucins/gluc (p = . ) or with the insulinogenic index (p = . ; table and supplementary table ). the lack of association with the two latter indices suggests that acdy is unlikely to be directly involved in insulin secretion in response to an oral glucose challenge and may not operate in the same pathway as gipr. in support of our observations, the mrna expression pattern of adcy reported in the recent magic study on fasting glucose traits shows that adcy is most highly expressed in heart and brain tissues, with weaker expression in the pancreas, islets and sorted beta cells. finally, we found that the rs c allele was also associated with increased risk of t d (or = . , % ci . – . , p = . × − ) in a separate meta-analysis of association studies (total n = , cases, , controls; table and supplementary table ) and was associated with increased risk of developing future t d in , individuals from the malmo preventive project (or = . , % ci . – . , p = . × − ; see supplementary note). taken together, our results do not support a role for adcy in early insulin secretion in response to an oral glucose load, but it remains to be determined how it (or another causal gene at the locus) contributes to risk for t d. we tested association of the vps c variant with insulin secretion indices because of its novelty and unknown function (table and supplementary table ). the risk allele g of rs associated with higher -h glucose was also associated with lower -h insulin (p = . × − ). rs showed no association with aucins/gluc (p = . ) but was nominally associated with insulinogenic index (p = . ). the vps c variant was not associated with t d (or = . , % ci . – . , p = . ) (table and supplementary table ), suggesting that it may contribute to normal variation in -h glucose but not susceptibility to t d. investigation of the mrna expression profiles of vps c revealed the presence of transcripts in several organs including brain, adipose tissue, liver, pancreas, and, most strongly, in sorted beta cells (fig. ). analysis of the neighboring gene fam a indicated a pancreatic tissue-specific mrna expression profile, mainly in beta cells (fig. ); however, its expression was not altered by vps c genotype in pancreatic islets (p = . , n = ; supplementary note). vps c spans kb on chromosome and encodes a protein homolog of the yeast vacuolar protein sorting . this family of proteins is involved in trafficking of membrane proteins between the trans-golgi network and the prevacuolar compartment . rs , identified by the -h glucose meta-analysis, is kb from the fam b association signal (rs ) identified by the magic fasting glucose meta-analysis , but could represent saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript an independent signal, as rs is weakly correlated with rs (r = . in hapmap ceu, p -h glucose = . ). detailed fine-mapping and functional analyses will be needed to definitively establish the causal gene and variant(s) at this locus. in conclusion, we report a gwas for glucose levels h after an oral glucose challenge, and we have investigated the role of newly discovered -h glucose variants in influencing normal physiology and potentially influencing risk of t d. we identified five loci associated with -h glucose, in gipr, vps c, adcy , gckr and tcf l . as the physiological roles of gckr and tcf l variants have been examined in detail previously , , we focused on the three newly identified associated loci. adcy variants are associated with fasting and -h glucose levels and with an increased risk of t d, highlighting the fact that investigation of diabetes-related quantitative traits can lead to identification of additional t d-associated loci. vps c variants may contribute to normal variation in -h glucose, but their effect on t d pathogenesis is unclear. our association results suggest a role for gipr in the incretin effect and in early pathophysiologic pathways that could lead to impaired glucose tolerance and t d in humans. previously, it was hypothesized that patients with t d might express a smaller amount of gipr or defective gipr . meier et al. observed that individuals with t d and a subgroup of the first-degree relatives of these individuals had a blunted insulin response to gip, supporting the hypothesis that a defect of the gipr could be part of the t d pathophysiology . future studies should examine how gipr variants may modify response to treatments targeting the enteroinsular axis. methods methods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/. supplementary material refer to web version on pubmed central for supplementary material. acknowledgments the authors would like to thank the many colleagues who contributed to collection and phenotypic characterization of the clinical samples, as well as genotyping and analysis of the gwa data. we gratefully acknowledge those who agreed to participate in these studies. a full list of acknowledgments and funding support for each study is described in the supplementary note. references . prokopenko i, mccarthy mi, lindgren cm. type diabetes: new genes, new understanding. trends genet ; 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[pubmed: ] . almind k, et al. discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (gip) receptor: the impact on the pancreatic beta cell responses and functional expression studies in chinese hamster fibroblast cells. diabetologia ; : – . [pubmed: ] . kubota a, et al. identification of two missense mutations in the gip receptor gene: a functional study and association analysis with niddm: no evidence of association with japanese niddm subjects. diabetes ; : – . [pubmed: ] . nitz i, et al. association analyses of gip and gipr polymorphisms with traits of the metabolic syndrome. mol. nutr. food res ; : – . [pubmed: ] . hardy j, singleton a. genomewide association studies and human disease. n. engl. j. med ; : – . [pubmed: ] . drucker dj. the role of gut hormones in glucose homeostasis. j. clin. invest ; : – . [pubmed: ] . leech ca, castonguay ma, habener jf. expression of adenylyl cyclase subtypes in pancreatic beta-cells. biochem. biophys. res. commun ; : – . [pubmed: ] saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript . velayos-baeza a, vettori a, copley rr, dobson-stone c, monaco ap. analysis of the human vps gene family. genomics ; : – . [pubmed: ] . sparso t, et al. the gckr rs polymorphism is associated with elevated fasting serum triacylglycerol, reduced fasting and ogtt-related insulinaemia, and reduced risk of type diabetes. diabetologia ; : – . [pubmed: ] . holst jj, gromada j, nauck ma. the pathogenesis of niddm involves a defective expression of the gip receptor. diabetologia ; : – . [pubmed: ] . meier jj, et al. reduced insulinotropic effect of gastric inhibitory polypeptide in first-degree relatives of patients with type diabetes. diabetes ; : – . [pubmed: ] saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript figure . regional plots of five genome-wide significant associations for hour glucose based on hour glucose discovery analysis adjusted for age, sex, bmi and study-specific covariates. (a–e) for each of the gckr (a), adcy (b), tcf l (c), vps c (d) and gipr (e) regions, directly genotyped and imputed snps are plotted with their meta-analysis p values (as −log values) as a function of genomic position (ncbi build ; hg ). in each panel, the snp taken forward for replication (large red diamond) and joint discovery and replication p value (blue diamond) are shown. estimated recombination rates (hapmap) are plotted to reflect the local linkage disequilibrium structure around the associated snps and their correlated proxies ( < r < , represented on a white to red scale, based on pairwise r values from hapmap ceu). gene annotations were taken from the ucsc genome browser. saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript figure . percent incretin effect in the botnia, denmark and eugene -kuopio studies of nondiabetic individuals (n = ) by gipr rs genotype. mean and s.d. for each study are displayed by genotype (see supplementary table for details). incretin effect was adjusted for age, sex and bmi and study-specific covariates. saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript figure . mrna expression in human tissues of the genes located in the gipr (a) and vps c (b) regions. expression data is relative expression levels measured by quantitative rt-pcr. all samples were run in triplicate and normalized to the gapdh relative expression level. au, arbitrary units. saxena et al. page nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript saxena et al. page ta bl e g en om e- w id e si gn if ic an t l oc i f or -h g lu co se d ur in g an o g t t fr om st ud ie s in n on di ab et ic in di vi du al s d is co ve ry r ep lic at io n d is co ve ry a nd r ep lic at io n d is co ve ry a nd r ep lic at io n (f g a dj ) sn p c hr p os it io n (b p) n ea re st ge ne a lle le s (+ /− ) f re q (+ ) e ff ec t ( s. e. m .) m m ol /l p v al ue e ff ec t ( s. e. m .) m m ol /l p v al ue e ff ec t ( s. e. m .) m m ol /l p v al ue p v al ue (n o b m i) e ff ec t ( s. e. m .) m m ol /l p v al ue p v al ue (n o b m i) rs g c k r t /c . . ( . ) . × − . ( . ) . × − . ( . ) . × − . × − . ( . ) . × − . × − rs a d c y c /t . . ( . ) . × − . ( . ) . × − . ( . ) . × − . × − . ( . ) . × − . × − rs tc f l c /t . . ( . ) . × − . ( . ) . × − . ( . ) . × − . × − . ( . ) . × − . × − rs v p s c g /a . . ( . ) . × − . ( . ) . × − . ( . ) . × − . × − . ( . ) . × − . × − rs g ip r a /t . . ( . ) . × − . ( . ) . × − . ( . ) . × − . × − . ( . ) . × − . × − n , – , , – , , – , , – , r es ul ts fr om fi xe d ef fe ct s, in ve rs e va ri an ce m et a- an al ys is o f g w a (a r ic , b l sa , c h ss ta ge & , c ol au s, d g i, fe nl an d, f h s, f u si o n , s or bs ) a nd fo llo w -u p st ud ie s (a m is h, b ot ni ap pp , c h ss ta ge , d ia g e n , e l y , f re nc hf am ily m em be rs , f re nc hh ag ue na u, f re nc ho be se a du lts , f u si o n st ag e , h er tf or ds hi re , i nt er , m e t si m , n h a n e s, r is c , r oc he , u l sa m , w hi te ha ll ii ) w ith a dj us tm en t f or a ge , se x an d b m i. po si tio n ba se d on h g , n c b i b ui ld . c om bi ne d di sc ov er y an d re pl ic at io n p v al ue s fo r -h g lu co se a dj us te d fo r a ge a nd s ex (n o b m i) , a nd fu rt he r a dj us te d fo r f as tin g gl uc os e ar e al so pr es en te d. r ep lic at io n m et a- an al ys is re su lts a nd jo in t d is co ve ry a nd re pl ic at io n m et a- an al ys is re su lts in cl ud e pr ox y sn ps w ith r > . in h ap m ap c e u . a lle le fr eq ue nc ie s ba se d on h ap m ap p ha se ii c e u s am pl e. f g a dj , a dj us te d fo r f as tin g gl uc os e in a dd iti on to a ge , s ex , b m i a nd s tu dy -s pe ci fi c co va ri at es (c en te r) . nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript saxena et al. page ta bl e e ff ec t o f a d c y , v p s c a nd g ip r v ar ia nt s on in di ce s of in su lin re sp on se d ur in g an o g t t in su lin og en ic in de x a u c in s/ gl uc - h in su lin , a dj us te d fo r - h gl uc os e sn p c hr n ea re st ge ne e ff ec t al le le n e ff ec t ( s. e. m .) µu /m m ol (b m i- ad j) p v al ue (b m i- ad j) p v al ue n e ff ec t ( s. e. m .) pm ol /m m ol (b m i- ad j) p v al ue (b m i- ad j) p v al ue n e ff ec t ( s. e. m .) pm ol /l (b m i- ad j) p v al ue (b m i- ad j) p v al ue rs a d c y c , − . ( . ) . . , – . ( . ) . . , − . ( . ) . × − . × − rs v p s c g , . ( . ) . . , – . ( . ) . . , − . ( . ) . × − . × − rs g ip r a , − . ( . ) . × − . × − , − . ( . ) . × − . × − , – . ( . ) . × − . × − a u c in s/ gl uc , a re a un de r t he c ur ve fo r i ns ul in d iv id ed b y ar ea u nd er th e cu rv e fo r g lu co se . nat genet. author manuscript; available in pmc august . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript saxena et al. page ta bl e m et a- an al ys is o f t d a ss oc ia tio n st ud ie s fo r s n ps a t p re vi ou sl y un kn ow n - h gl uc os e– as so ci at ed lo ci t d fi xe d ef fe ct s t d r an do m e ff ec ts sn p c hr n ea re st g en e e ff ec t a lle le n st ud ie s n ca se s n co nt ro ls o r ( % c i) p v al ue i (% ) o r ( % c i) p v al ue rs a d c y c , , . ( . – . ) . × − . ( – . ) . ( . – . ) . × − rs v p s c g , , . ( . – . ) . . ( – . ) . ( . – . ) . rs g ip r a , , . ( . – . ) . × − . ( – . ) . ( . – . ) . × − pr ox ie s rs w ith r = . in h m c e u to rs us ed in e ig ht s tu di es ; r s w ith r = . in h m c e u u se d in tw o st ud ie s. p ro xy rs w ith r = . in h m c e u to rs us ed in tw o st ud ie s. pr ox y rs w ith r = . in h m c e u to rs u se d in th re e st ud ie s. nat genet. author manuscript; available in pmc august . 	serveur académique lausannois serval serval.unil.ch 	author manuscript 	faculty of biology and medicine publication 	published in final edited form as: wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ real-time magnetic resonance-guided endovascular repair of experimental abdominal aortic aneurysm in swine p r e a v a b r r b e e r l t r e d i b s d i c d b s w a journal of the american college of cardiology vol. , no. , © by the american college of cardiology foundation issn - / /$ . p reclinical research eal-time magnetic resonance-guided ndovascular repair of experimental bdominal aortic aneurysm in swine enkatesh k. raman, md,* parag v. karmarkar, msc,*‡ michael a. guttman, msc,† lexander j. dick, md,* dana c. peters, phd,† cengizhan ozturk, md, phd,* reno s. s. pessanha, md,* richard b. thompson, phd,† amish n. raval, md,* anil desilva, mbbs, phd,* ronnier j. aviles, md,* ergin atalar, phd,‡ elliot r. mcveigh, phd,† obert j. lederman, md* ethesda and baltimore, maryland objectives this study tested the hypotheses that endografts can be visualized and navigated in vivo solely under real-time magnetic resonance imaging (rtmri) guidance to repair experimental abdominal aortic aneurysms (aaa) in swine, and that mri can provide immediate assessment of endograft apposition and aneurysm exclusion. background endovascular repair for aaa is limited by endoleak caused by inflow or outflow malappo- sition. the ability of rtmri to image soft tissue and flow may improve on x-ray guidance of this procedure. methods infrarenal aaa was created in swine by balloon overstretch. we used one passive commercial endograft, imaged based on metal-induced mri artifacts, and several types of homemade active endografts, incorporating mri receiver coils (antennae). custom interactive rtmri features included color coding the catheter-antenna signals individually, simultaneous multislice imaging, and real-time three-dimensional rendering. results eleven repairs were performed solely using rtmri, simultaneously depicting the device and soft-tissue pathology during endograft deployment. active devices proved most useful. intrapro- cedural mri provided anatomic confirmation of stent strut apposition and functional corrobo- ration of aneurysm exclusion and restoration of laminar flow in successful cases. in two cases, there was clear evidence of contrast accumulation in the aneurysm sac, denoting endoleak. conclusions endovascular aaa repair is feasible under rtmri guidance. active endografts facilitate device visualization and complement the soft tissue contrast afforded by mri for precise positioning and deployment. magnetic resonance imaging also permits immediate post- procedural anatomic and functional evaluation of successful aneurysm exclusion. (j am coll ublished by elsevier inc. doi: . /j.jacc. . . cardiol ; : – ) © by the american college of cardiology foundation t s m p c p n e f d r p g c e m a ndovascular repair is an alternative to open surgery that is merging as an elective treatment for abdominal aortic aneu- ysm (aaa) ( – ). an important complication of endovascu- ar aaa repair is endoleak, a persistent systemic communica- ion with the aneurysm sac that risks continued expansion and upture. of the types described by white et al. ( ), type i ndoleak results from an incomplete seal at the proximal or istal attachment site of the endograft. this has been reported n up to % of cases ( – ) in older series, and may be caused y device misposition, stent malapposition, and device under- izing ( ) or oversizing ( ). better soft tissue visualization and epiction of complex three-dimensional ( d) anatomy by nteractive magnetic resonance imaging (mri) may limit this omplication. from the *cardiovascular branch and the †laboratory of cardiac energetics, ivision of intramural research, national heart, lung, and blood institute, ethesda, maryland; and the ‡department of radiology, johns hopkins university chool of medicine, baltimore, maryland. supported by nih z -hl - cvb (to dr. lederman). drs. raman and karmarkar contributed equally to this ork. c manuscript received may , ; revised manuscript received february , , ccepted march , . magnetic resonance imaging may be equivalent or superior o x-ray computed tomography for procedure planning and urveillance after endograft placement ( – ). real-time ri (rtmri) can guide clinical invasive procedures ( ) and re-clinical interventions, such as transcatheter repair of intra- ardiac shunt ( , ) and endomyocardial injection of thera- eutic agents ( ). an mri permits d tissue and hemody- amic characterization, creating opportunities to improve ndovascular aneurysm treatment and to limit procedural ailure from type i endoleak. we hypothesize that: ) en- ografts can be visualized and navigated in vivo solely under tmri to repair experimental aaa in swine; ) mri can rovide useful intraprocedural information about anatomy and uide device placement; and ) mri can provide immediate onfirmation of successful endograft apposition and aneurysm xclusion. ethods nimal preparation and aneurysm model. animal proto- ols were approved by the national heart, lung, and blood i s v t p a t ( p a v u o s i a r r c e p r ( e r i a i l s i t t f d a r y n p d d t m o e x l t r ( fi l t ( s e a e a a i s [ fi q i f f e t d s ( raman et al. jacc vol. , no. , real-time mri aaa repair june , : – nstitute animal care and use committee. eleven york- hire swine (animal biotech industries, danboro, pennsyl- ania) or national institutes of health mini-swine (na- ional institutes of health veterinary resource program, oolesville, maryland) weighing to kg were studied. nesthesia was induced with ketamine/xylazine and main- ained with inhaled isoflurane. nonferrous -f sheaths check flo ii, cook, bloomington, indiana) were placed ercutaneously in the femoral artery. animals underwent nticoagulation with a heparin -iu/kg bolus dose and a - to -iu/kg/h infusion. we modified an acute nonsurgical model of aaa ( ). essels were sized by x-ray digital subtraction aortography sing a marker pigtail catheter. single, double, or triple verlapping balloons (xxl, - to -mm diameter � -mm length, boston scientific/medi-tech, natick, mas- achusetts; and agiltrac, - to -mm diameter � -mm length, guidant, menlo park, california) were nflated for up to min within the infrarenal aorta to chieve an overstretch ratio of at a least : . this was epeated until the dilated segment was at least . times the eference diameter or until dissection or rupture. onstruction of endograft devices. different self- xpanding endograft designs were tested, including one assive device and three different active designs. passive efers to device visibility based on susceptibility artifacts dark spots on mri) generated by intrinsic magnetic prop- rties of the device. active refers to incorporation of an mri eceiver coil (antenna, electrically connected to the scanner) nto the catheter, which is sensitive to signal only from djacent tissue and is used to create bright spots on the mr mages. the passive device was an unmodified � mm iliac imb of a commercial nitinol endograft (vanguard, boston cientific, natick, massachusetts). a quarter-wave . - nch active guidewire was used to enhance visualization of he passive endograft. all active devices were built by one of he investigators (p.v.k.). endografts were constructed rom . -inch gold-plated nitinol wire in a z-stent esign, lined with thin expanded polytetrafluoroethylene nd having diameters of to mm and lengths of to mm. endografts were mounted on -f -cm nonfer- ous kumpe catheters (angiodynamics, queensbury, new ork), acting as delivery shafts, and crimped inside -f abbreviations and acronyms d � three dimensional aaa � abdominal aortic aneurysm mra � magnetic resonance angiography mri � magnetic resonance imaging rfov � reduced field of view ssfp � steady state free precession mri te � echo time tr � repetition time ylon sheaths (fast-cath, daig, minnetonka, minnesota). t olyimide tubing improved the stiffness of the kumpe elivery shaft and the ease of sheath retraction for endograft eployment (fig. ). schematics for the active devices are shown in figure . he passive endograft is depicted in figure a. an active- arker/passive-stent design (fig. b) incorporated two pposed solenoid coils built into the delivery shaft at each dge of the stent, analogous to radiopaque end markers on -ray systems. another active-stent design incorporated a oopless antenna. the delivery shaft acted as ground, and he z-stent served as antenna whip (region of antenna eceiving signal), connected to the shaft by a wire ( – ) fig. c) and designed to detach after deployment. the nal active-stent/active-marker system combined the loop- ess coil endograft with a separate loop coil wound around he delivery device’s distal cone just beyond the stent itself fig. d). decoupling circuitry was housed in external hielded boxes. ndograft phantom, heating, and in vivo testing. all ctive devices were imaged in saline phantoms before in vivo xperiments to confirm signal and appearance. heating of the endograft was tested in vitro in a poly- crylamide gel phantom and in vivo in one additional nimal. conditions were intended to exaggerate heating, ncluding continuous steady-state free precession (ssfp) canning with a high flip angle (alpha � °, repetition time tr] � . ms). temperature was measured using four beroptic thermistor probes (umi- , fiso technologies, uebec, canada) placed along the length of the device, ncluding the tip. unctional and anatomic mri techniques. func- igure . active-stent endograft. (a) components of homemade active ndograft, constructed from . -inch nitinol wire and expanded poly- etrafluoroethylene graft material. (b) completed one-channel active-stent evice mounted on a -f catheter and constrained within a -f nylon heath (arrowhead). matching tuning circuitry is housed in a separate box arrow). ional mri techniques included dynamic magnetic reso- n i f g m fi d j ( r u t � v o a w p d b s h ( a m s l s r fl e t s r c m v r r t f h d s d jacc vol. , no. , raman et al. june , : – real-time mri aaa repair ance angiography (mra) and phase-contrast imaging. mmediately after aneurysm creation, animals were trans- erred to a co-located . -t mri scanner (signa cv/i, eneral electric, waukesha, wisconsin, or sonata, sie- ens, erlangen, germany) for imaging using - or -channel phased-array surface coils (novamedical, wake- eld, massachusetts, or siemens). contrast-enhanced igital-subtraction mra was performed with systemic in- ection of . to . mmol/kg gadopentate dimeglumine magnevist, berlex, wayne, new jersey) with a d adiofrequency-spoiled gradient echo (spgr) acquisition sing the following parameters: repetition time (tr)/echo ime (te) . / . ms, flip angle °, matrix � � , field-of-view � � . cm, receiver bandwidth . khz, voxel size . � . � . mm. mask, arterial, enous (after s), and late phases (after min) were btained to identify slow contrast accumulation in the neurysm sac. a d, low-flip-angle fast-gradient-echo scan ith and without fat saturation was run with thin axial artitions delineating aortic anatomy before and after en- ograft deployment to assess stent strut apposition (tr/te . / . ms, flip angle °, matrix � � , andwidth hz/pixel, resolution . � . � mm). tent strut apposition was also confirmed on axial cuts using igure . schematic of endograft designs. (a) unmodified commercial de omemade endograft device with active opposed loop solenoid coils as m evice with active stent connected to delivery system shaft by a detachable tent inactive. (d) homemade endograft device with active stent as desc elivery shaft just beyond the distal stent edge. igh-resolution rectilinear trajectory ssfp (matrix � t , field-of-view � cm, in-plane resolution . � . mm, slice thickness mm) and reduced field-of-view rfov) radial trajectory ssfp ( ) (tr/te . / . ms, flip ngle °, projections with interleaves, regridded to atrix � , field-of-view � cm, slice thickness mm, bandwidth hz/pixel), as well as black-blood fast pin echo (tr/te / ms, flip angle °, echo train ength , matrix � , field-of-view � cm, lice thickness mm, bandwidth � . khz, in-plane esolution . � . mm). phase-contrast imaging was used qualitatively to assess ow perturbations within the aneurysm before and after ndograft deployment. flow velocities were assessed along hree axes using in-plane and through-plane phase-contrast cans (tr/te . / . ms, matrix � , field-of-view � cm, slice thickness mm, bandwidth � . khz, esolution . � . mm, through-plane velocity encoding cm/s, in-plane velocity encoding cm/s). phase- ontrast data were represented with through-plane flow apped as color, and in-plane flow was mapped as velocity ectors (matlab, mathworks, natick, massachusetts) ( ). eal-time mri and interventional procedure. the tmri for procedural guidance required several modifica- ions to commercial hardware and software, including ex- imaged on the basis of intrinsic magnetic susceptibility (signal void). (b) s delineating proximal and distal stent edges. (c) homemade endograft e that, after deployment and removal of the delivery catheter, renders the in (c) and second active marker composed of a multilooped coil on the vice arker cabl ribed ernal image reconstruction and in-room display, as previ- o w f c i s a t p s s m u n s a r p a b a c u i i o s r s c d t s n i r a f p s p t p u e h e d r t s a l m a t t c h f e r t l s c p a t s d s a g t w a v ( d p i i t n p r p c a m d i l d t r t t d a raman et al. jacc vol. , no. , real-time mri aaa repair june , : – usly described ( , , ). interactive rtmri user interfaces ere customized with the addition of several useful features or rectilinear ssfp imaging ( , ): individual receiver hannel gain, coloring, and highlighting for use with active ntravascular devices; preparatory saturation pulses to negate ignal from fat or specific spatial regions; simultaneous cquisition and display of multiple image slices; and real- ime d rendering of multislice data ( ). an interactive oint marking system was implemented allowing user- elected reference points on individual slices to be repre- ented on the d rendering. this was useful, for example, to ark visceral artery ostia. a parallel real-time environment sed radial k-space trajectories, a data-undersampling tech- ique that optimizes temporal resolution without sacrificing patial resolution ( ). this interface was also customized to llow interactive overlay of previously acquired angiographic oadmaps. typical real-time ssfp imaging used the following arameters: tr/te . / . ms, matrix � , flip ngle °, slice thickness mm, field-of-view � cm, andwidth � to khz. image position could be djusted interactively by drawing new prescriptions on the urrent image, pushing or pulling through parallel planes by ser-specified gap thickness, and rotating vertically or hor- zontally around the image center by user-specified angle ncrements. these yielded four to eight frames/s, depending n whether image acceleration techniques such as echo haring were used. the imaging latency, or time to acquire, econstruct, and display images to the operator using this ystem is approximately ms. orroboration of aneurysm exclusion. after endograft eployment and post-procedural mr anatomic and func- ional imaging as described for baseline studies, x-ray ubtraction angiography was repeated. animals were eutha- ized, and the abdominal aorta was excised for visual nspection in six. esults neurysm model. balloon overstretch dilation of the in- rarenal aorta was performed successfully in all animals. erforation/rupture of the aorta occurred in two, but both urvived for the duration of the nonsurvival experimental rotocol. at the widest, dilated segments were up to twice he reference aortic diameters. in all cases, aneurysms ersisted throughout the experimental protocol, up to h, sually without significant recoil. ndograft testing. in vitro all active devices showed en- anced signal in the immediately surrounding region. how- ver, the receiver coil on the active-marker/passive-stent evice (fig. b) coupled inductively with the nitinol stent, esulting in increased signal along the entire device rather han discretely at each end-marker coil. there was marked ignal drop-off within to mm of the distal end of the ctive-stent devices (fig. c), a known limitation of the oopless antenna design ( ). the active-stent/active- arker design (fig. d) overcame this limitation by placing loop coil ( ) to act as an edge marker distal to the stent. heating was only noted at the tip. the maximum emperature increase was . °c in the in vitro static phan- om, and . °c in vivo. signal-to-noise profiles of the final endograft design, ombining an active stent with active edge markers, was igher than for all previous designs, and contrast-to-noise or the final device was different primarily at the endograft dges (data not shown). eal-time mr guidance of endograft procedures. real- ime imaging with ssfp provided adequate temporal reso- ution for device navigation, positioning, and deployment. patial resolution was sufficient to visualize important vis- eral and branch artery origins necessary for precise device lacement. in one of the animals with aortic rupture, the rapidly ccumulating retroperitoneal hematoma obscured impor- ant anatomy. this problem was circumvented by thick- lice, real-time angiography with a hand injection of ilute ( mm) gadolinium contrast during image acqui- ition. active devices were easily visualized within the orta using color highlighting and individual channel ain adjustment. multislice imaging provided reference coronal and sagit- al slices of the infrarenal aorta with renal and iliac vessels hile allowing interactive adjustment of axial slices to verify nd adjust device position. the slices were displayed indi- idually and in combination after real-time d rendering figs. a and b). a point marking system allowed precise elineation of important anatomic references, including roximal and distal aneurysm extent, with display on the d mage to guide device positioning and deployment (fig. a). the balance between spatial resolution, the number of mage slices, and temporal resolution was adjusted itera- ively, in combination with temporal filtering and multipla- ar imaging. optimal anatomic guidance seemed to be rovided by multiple ( – ) non-orthogonal slices, each fully efreshed only approximately once per second. erformance of different mri-endograft designs. the ommercial endograft system (fig. a, one tested) was visu- lized passively by its marked susceptibility artifact, but this ade it very difficult to differentiate the stent itself from the elivery shaft. we attempted to improve device visualization by nserting a quarter-wave active antenna-guidewire through the umen of the endograft system. this enhanced local signal but id not satisfactorily delineate the device. the active-marker/passive-stent device (fig. b, one ested) showed bright signal along the length of the device ather than discretely at the markers only. the distal edge of he stent could not definitively be identified. additionally, he tip of the delivery system, extending cm beyond the istal active marker, was not conspicuous because of volume veraging. the loopless coil design of the active-stent device (fig. c, two tested) provided good signal except along the distal s t a c m o t t o u w m d t a t s r s e r p l l o c a c t l f i o b “ , i � i jacc vol. , no. , raman et al. june , : – real-time mri aaa repair everal millimeters of the actual endograft, reducing opera- or confidence during positioning and deployment. the final device iteration combined an active stent with n active distal loop coil at the distal tip of the delivery atheter (fig. d, seven tested). this design produced the ost reproducible signal pattern and provided satisfactory perator confidence in device position. the two procedural failures were attributed to the fragility of hese homemade prototypes, built primarily for imaging rather han mechanical characteristics. of the two procedural failures, ne was related to shifting of the self-expanding endograft during nsheathing, and the other was related to migration during ithdrawal of the detachable antenna connection. r assessment of procedural success. nine of en- ograft procedures were successful under rtmri guidance. he two failures were identified using first-pass mra. no ttempt was made to correct acute endoleaks using adjunc- igure . real-time multislice imaging and three-dimensional rendering d maging multislice axial, sagittal, and coronal images shown simultaneously n the right integrates multislice information. positioning three axial slice ifurcation, respectively, allows simultaneous capture of the most important bird’s eye” view of the aorta. orientation markers indicate: s � superior ndicates aneurysm. ive balloon or stent devices. c stent strut apposition at the proximal and distal target egments of the aorta was convincingly shown by high- esolution axial ssfp, fast spin echo, and d gradient echo cans (fig. ). repeat mra in of cases showed both aneurysm xclusion by the endograft (fig. ) and patency of the enal arteries. iliac arteries were also patent by angiogra- hy. successful exclusion was further corroborated by ack of contrast accumulation in the aneurysm sac during ate-phase angiographic and real-time ssfp scans. in the ther two cases, mr contrast-angiography revealed pro- edural failure with evidence of gadolinium within the neurysm sac. high-resolution phase-contrast studies with vector- and olor-flow mapping showed reduction of in-plane and hrough-plane turbulence consistent with restoration of aminar flow (fig. ). in the two procedural failures, phase endograft positioning. in the left column, real-time magnetic resonance tate precise device positioning. concomitant three-dimensional rendering he caudal renal artery origin, the middle of the aneurysm, and the aortic my for device placement. the coronal and sagittal slices provide an overall inferior, a � anterior, p � posterior, r � right, l � left. blue arrow uring facili s at t anato ontrast imaging was not specifically conducted to iden- t c x a d p f i f tent f exclu f m n raman et al. jacc vol. , no. , real-time mri aaa repair june , : – ify flow jets associated with endoleaks identified by ontrast mra. -ray angiography and pathology. digital subtraction ngiography corroborated mra findings in all nine cases igure . stent strut apposition. (a) fast spin echo image shows nitinol s rom stent struts). (b) spin-echo axial image at level of aneurysm, showing igure . magnetic resonance angiogram (maximum-intensity projectio agnetic resonance angiography shows infrarenal abdominal aortic aneurysm a itinol endograft (causing luminal artifact). orientation markers as in figure . eemed successful by mri. direct inspection and alpation of the resected abdominal aorta in six success- ul cases confirmed that the renal arteries were not nvolved. well apposed to target proximal infrarenal aorta (arrows show signal void ded sac (dashed outline). orientation markers as in figure . fore and after endograft delivery. (a) conventional contrast-enhanced n) be fter balloon overstretch. (b) abdominal aortic aneurysm is excluded by d w p s m a s e e t e s d d a b i t p i c s m c d c o a p p u r t r ( i c t t r w a g o t r c a e c c a s e s c m m s n i u c e p d m t l r g t f d p e fl c d i a jacc vol. , no. , raman et al. june , : – real-time mri aaa repair iscussion e showed the feasibility of endovascular repair of aaa erformed entirely under real-time mri guidance in a wine model. functional imaging with dynamic contrast ra and phase-contrast techniques complemented the natomic imaging for immediate assessment of procedural uccess. ndograft devices and mri. our approach to devices ncompassed both passive and active designs for visualiza- ion and tracking. the unmodified, commercial passive ndograft was difficult to differentiate from the delivery ystem, limiting operator confidence in positioning and eployment. moreover, signal voids generated by passive evices cannot be readily distinguished from signal attenu- tion of other causes or from volume averaging ( ). ecause active devices function as receiver coils, the result- ng local signal enhancement markedly facilitates visualiza- ion (figs. and ) ( , , , ). mahnken et al. ( ) ositioned passive commercial endografts below renal arter- a p rl a p rl igure . phase-contrast flow assessments before and after endograft eployment. axial overlays of in-plane (vector flow map) and through- lane (color map) flow within ruptured experimental aneurysm. before ndovascular repair, there is marked turbulence and evidence of retrograde ow (blue) within the aneurysm (dashed outline). the vena cava is ollapsed in this hemorrhagic state. laminar flow is restored after en- ograft (dashed outline) deployment. solid lines border vena cava, dentified on cine loops by constant nonpulsatile flow. orientation markers s in figure . es in healthy swine under mri guidance, but also con- c luded that dedicated mri endograft designs might be uperior. we constructed several different types of home- ade active endograft systems, of which the final two- hannel conductively coupled active-marker/active-stent evice provided the most useful signal profile. the rtmri with ssfp provided excellent intrinsic blood ontrast within the aorta and its branch vessels, allowing ptimal slice selection for the procedure. unlike coronary nd intracardiac procedures, in which ultrafast imaging is a rerequisite because of cardiac and respiratory motion, eripheral endovascular repair under mr guidance may be ndertaken with slower frame rates that allow better spatial esolution. radial k-space acquisition seems well suited to ranscatheter aneurysm therapy because of advantages in fov imaging and because of intrinsic edge enhancement ). customized features on the interactive real-time nterface proved very useful during the interventional pro- edure, particularly in channel coloring and gain adjustment o highlight and contrast the device from surrounding issue. multislice imaging with simultaneous real-time d endering facilitated device positioning and deployment ith complementary orthogonal and/or oblique views. our bility to precisely position and deploy the endografts was ood and was limited only by the mechanical shortcomings f our homemade devices. post-deployment scanning combined anatomic and func- ional assessment of procedural success. high-resolution, fov ssfp, fast spin echo and d gradient echo scans onvincingly showed stent strut apposition to the proximal nd distal target aortic segments. dynamic contrast- nhanced mra during arterial, venous, and late phases orroborated aneurysm exclusion in successful cases and learly showed contrast accumulation in the aneurysm sac fter procedural failures. phase-contrast scans visually howed restoration of laminar flow in the grafted segment. these studies were readily completed within minutes of ndograft delivery. tudy limitations and clinical implications for endovas- ular repair. although we successfully excluded aneurys- al segments immediately in all cases in which the home- ade device functioned appropriately, our experience hould be qualified. we used a model of aaa in healthy, onatherosclerotic swine. even after balloon overstretch njury, the course of the infrarenal aorta remained essentially nchanged. this and more complicated surgical models annot represent the complex d anatomy and tortuosity ncountered clinically in degenerative arterial segments in atients with aaa, in whom the expected benefits of etailed soft tissue contrast and d representation by mri ay be more dramatic. in particular, safe traversal of ortuous iliac artery segments, one of the procedural chal- enges of aaa endografting, might be simplified by using tmri to visualize device-related anatomic distortion and to uide operator adjustments. furthermore, we used a simple ubular endograft, although most clinical devices are bifur- ated to cover the aorta and both iliac limbs. w d s w m r t r a p d t d s m d p i c b g a c f a h g a b m c i m s fi a c t o u d t a d r g r c i n t r raman et al. jacc vol. , no. , real-time mri aaa repair june , : – our experience suggests that an active endograft design ould be most appropriate for further clinical development. espite iterative prototype testing, the optimal device de- ign remains undetermined. additional issues include hether an endograft system with associated invasive equip- ent (e.g., guidewires) made from nonferrous materials etains adequate mechanical characteristics to traverse tor- uous and/or calcified iliac arteries. inductive heating during adiofrequency excitation is an important safety consider- tion for long conductive catheter devices in mri. our rototype devices showed minimal local heating because of ecoupling circuitry to limit radiofrequency energy deposi- ion. this is amenable to further optimization ( , ). although aaa endografting has emerged over the past ecade as a viable alternative to open surgery, failure modes uch as malapposition could conceivably be improved using ri for on-line planning, procedural guidance, and imme- iate post-procedural assessment. the rtmri might im- rove procedural success by improving soft-tissue and d maging during the procedure. the ssfp provides intrinsic ontrast for excellent visualization of the aorta and major ranch vessels without exogenous agents. substituting adolinium-based mr contrast for iodinated radiocontrast gents might reduce contrast nephropathy in this at-risk linical population ( ). magnetic resonance imaging may acilitate device selection by improving the measurement ccuracy of aneurysm neck dimension, in which oversizing as been associated with endograft displacement and mi- ration ( ). furthermore, aneurysm anatomy may be ltered by the use of a stiff guidewire and introduction of a ulky, high-profile endograft delivery system. interactive ri may offer better delineation of this complex intrapro- edural anatomy than conventional x-ray projection imag- ng. finally, combined on-line anatomic and functional ri may be useful for guiding and evaluating procedural uccess. the chief obstacle to clinical translation of our ndings is the unavailability of clinical-grade commercial ctive mri endografts. onclusions hese experiments show that successful endovascular repair f experimental aaa in swine can be conducted solely nder rtmri. endografts can be built to be conspicuous uring the procedure. anatomic and complementary func- ional mri can guide graft deployment and can immedi- tely assess procedural success. although significant future evelopment of active devices is required, we believe this epresents an attractive application for cardiovascular mr- uided intervention. eprint requests and correspondence: dr. robert j. lederman, ardiovascular branch, clinical research program, division of ntramural research, national heart, lung, and blood institute, ational institutes of health, building , room c , be- hesda, maryland - . e-mail: lederman@nih.gov. eferences . buth j. endovascular repair of abdominal aortic aneurysms. results from the eurostar registry. european collaborators on stent- graft techniques for abdominal aortic aneurysm repair. semin interv cardiol ; : – . . matsumura js, brewster dc, makaroun ms, naftel dc. a multi- center controlled clinical trial of open versus endovascular treatment of abdominal aortic aneurysm. j vasc surg ; : – . . zarins ck, white ra, moll fl, et al. the aneurx stent graft: four-year results and worldwide experience . j vasc surg ; s : – . . white gh, yu w, may j, chaufour x, stephen ms. endoleak as a complication of endoluminal grafting of abdominal aortic aneurysms: classification, incidence, diagnosis, and management. j endovasc surg ; : – . . may j, white gh, waugh r, et al. adverse events after endoluminal repair of abdominal aortic aneurysms: a comparison during two successive periods of time. j vasc surg ; : – ; discussion – . . veith fj, baum ra, ohki t, et al. nature and significance of endoleaks and endotension: summary of opinions expressed at an international conference. j vasc surg ; : – . . alric p, hinchliffe rj, wenham pw, whitaker sc, chuter ta, hopkinson br. lessons learned from the long-term follow-up of a first-generation aortic stent graft. j vasc surg ; : – . . tzortzis e, hinchliffe rj, hopkinson br. adjunctive procedures for the treatment of proximal type i endoleak: the role of peri-aortic ligatures and palmaz stenting. j endovasc ther ; : – . . tummala s, powell a. imaging of endoleaks. tech vasc interv radiol ; : – . . badran mf, gould da, raza i, et al. aneurysm neck diameter after endovascular repair of abdominal aortic aneurysms. j vasc interv radiol ; : – . . thurnher sa, dorffner r, thurnher mm, et al. evaluation of abdominal aortic aneurysm for stent-graft placement: comparison of gadolinium-enhanced mr angiography versus helical ct angiography and digital subtraction angiography. radiology ; : – . . ludman cn, yusuf sw, whitaker sc, gregson rh, walker s, hopkinson br. feasibility of using dynamic contrast-enhanced mag- netic resonance angiography as the sole imaging modality prior to endovascular repair of abdominal aortic aneurysms. eur j vasc endo- vasc surg ; : – . . cejna m, loewe c, schoder m, et al. mr angiography vs ct angiography in the follow-up of nitinol stent grafts in endoluminally treated aortic aneurysms. eur radiol ; : – . . razavi r, hill dl, keevil sf, et al. cardiac catheterisation guided by mri in children and adults with congenital heart disease. lancet ; : – . . buecker a, spuentrup e, grabitz r, et al. magnetic resonance-guided placement of atrial septal closure device in animal model of patent foramen ovale. circulation ; : – . . rickers c, jerosch-herold m, hu x, et al. magnetic resonance image-guided transcatheter closure of atrial septal defects. circulation ; : – . . dick aj, guttman ma, raman vk, et al. magnetic resonance fluoroscopy allows targeted delivery of mesenchymal stem cells to infarct borders in swine. circulation ; : – . . hallisey mj. scvir gary j. becker young investigator award paper. a transluminally created abdominal aortic aneurysm model. j vasc interv radiol ; : – . . quick hh, ladd me, nanz d, mikolajczyk kp, debatin jf. vascular stents as rf antennas for intravascular mr guidance and imaging. magn reson med ; : – . . ocali o, atalar e. intravascular magnetic resonance imaging using a loopless catheter antenna. magn reson med ; : – . . ladd me, zimmermann gg, quick hh, et al. active mr visual- ization of a vascular guidewire in vivo. j magn reson imaging ; : – . . peters dc, lederman rj, dick aj, et al. undersampled projection reconstruction for active catheter imaging with adaptable temporal reso- lution and catheter-only views. magn reson med ; : – . . thompson rb, mcveigh er. high temporal resolution phase con- trast mri with multiecho acquisitions. magn reson med ; : – . jacc vol. , no. , raman et al. june , : – real-time mri aaa repair . guttman ma, mcveigh er. techniques for fast stereoscopic mri. magn reson med ; : – . . guttman ma, lederman rj, sorger jm, mcveigh er. real-time volume rendered mri for interventional guidance. j cardiovasc magn reson ; : – . . lederman rj, guttman ma, peters dc, et al. catheter-based endomyocardial injection with real-time magnetic resonance imaging. circulation ; : – . . serfaty jm, yang x, aksit p, quick hh, solaiyappan m, atalar e. toward mri-guided coronary catheterization: visualization of guiding catheters, guidewires, and anatomy in real time. j magn reson imaging ; : – . . bakker cj, bos c, weinmann hj. passive tracking of catheters and guidewires by contrast-enhanced mr fluoroscopy. magn reson med ; : – . . atalar e, bottomley pa, ocali o, et al. high resolution intravascular mri and mrs by using a catheter receiver coil. magn reson med ; : – . . mahnken ah, chalabi k, jalali f, gunther rw, buecker a. magnetic resonance-guided placement of aortic stents grafts: feasibil- ity with real-time magnetic resonance fluoroscopy. j vasc interv radiol ; : – . . yeung cj, susil rc, atalar e. rf heating due to conductive wires during mri depends on the phase distribution of the transmit field. magn reson med ; : – . . susil rc, yeung cj, atalar e. intravascular extended sensitivity (ives) mri antennas. magn reson med ; : – . . spinosa dj, kaufmann ja, hartwell gd. gadolinium chelates in angiog- raphy and interventional radiology: a useful alternative to iodinated contrast media for angiography. radiology ; : – ; discussion – . 	real-time magnetic resonance-guided endovascular repair of experimental abdominal aortic aneurysm in swine 	methods 	animal preparation and aneurysm model 	construction of endograft devices 	endograft phantom, heating, and in vivo testing 	functional and anatomic mri techniques 	real-time mri and interventional procedure 	corroboration of aneurysm exclusion 	results 	aneurysm model 	endograft testing 	real-time mr guidance of endograft procedures 	performance of different mri-endograft designs 	mr assessment of procedural success 	x-ray angiography and pathology 	discussion 	endograft devices and mri 	study limitations and clinical implications for endovascular repair 	conclusions 	references psychiatr & psychobio/ ( ) , - © elsevier, paris editorial genetic research in psychiatry: update from the society of biological psychiatry and the american psychiatrie association, new york, may ma crocq, f duval, jp mâcher centre hospitalier spécialisé, r ouffach, france summary — a stimulating variety of papers on genetic and elinical research in psychiatry was discussed at the latest meeting of the american psychiatrie association and the society of biological psychiatry in may in new york. conflicting results point out that extreme caution must be taken in interpreting linkage studies of psychiatrie disorders. difficulties stem from complex rnodels on inheritance as well as the genetic heterogeneity of psychiatrie disorders. progress lias been made in the approach to the régulation of receptor genes that hâve been implicated in the pathogenesis of psychiatrie disorders. in some cases, gene régulation may be tissue- dependent, as is suggested by the alternative splicing of d, receptor mrna. genetics / linkage / manic-depressive illness / amish / alzheimer’s diseuse / schizophrenia / d receptors / american psychiatrie as­ sociation / society of biological psychiatry résume - recherche génétique en psychiatrie : mise à jour de la société américaine de psychiatry biologique et de l’association améri­ caine de psychiatrie. les congrès annuels de l'association américaine de psychiatrie et de ta société américaine de psychiatrie biologi­ que se sont tenus en mai à new york. les recherches génétiques (études de liaison, études chez des jum eaux, biologie moléculaire des gènes des récepteurs impliqués dans les maladies mentales) ont occupé une place de choix. les controverses se poursuivent autour des liaisons qui ont été rapportées antérieurement entre la psychose maniaco-dépressive, la schizophrénie, la maladie d ’alzheimer, et des marqueurs génétiques. ces difficultés sont expliquées par des caractères en partie propres à la psychiatrie, tels que la complexité des modes de transmission génétique, l ’hétérogénéité génétique et clinique des troubles, la difficulté de définir les phénotypes et la révélation tardive de certaines affections. la connaissance de la régulation des gènes impliqués dans les maladies mentales, notam ­ m ent celle des gènes des récepteurs d ,, a aussi progressé notablement. génétique / liaison / psychose maniaco-dépressive / amish / maladie d’alzheimer / schizophrénie / récepteurs ), / association amé­ ricaine de psychiatrie / société américaine de psychiatrie biologique introduction genetics has emerged as a highly fruitful area of research in psychiatry. it has contributed to the évo­ lution of psychiatrie nosology by indicating the étio­ logie heterogeneity of seemingly identical elinical conditions, and conversely by suggesting the com- mon nature of apparently unrelated disorders. research strategies hâve varied over the years. they hâve relied on twin and adoption designs, familial ségrégation analyses and chromosomal studies. more recently, linkage analyses were made possible by the identification of polymorphie dna fragments (restriction fragment length polymor- phisms). finally, today’s molecular biology aims at identifying receptor genes and unravelling the régu­ lation of their expression in the brain. the sheer number of présentations at the latest annual meetings of the society o f biological psy­ chiatry and the american psychiatrie association in may in new york confirms that genetic research is an active field. no particular research strategy is outdated ; each one is particularly suited to approaching certain questions. regardless of the method used, thc field of psychiatry lias its own ad- ditional complexities, such as thc difficulty of iden- tifying phenotypes and cases. the l'ata morgana of linkage in manic-depressive illness manic-depressive illness has been linked with the x chromosome in independent samples in thc usa, belgium, israël and sardinia (winokur et al, ; mendlewicz et al, ; baron et al, ; delzom- po, ). linkage has been established notably with markers o f thc terminal fragment ( ) of the long arm (q) of the x chromosome, including pro­ tan and deutan color blindness, and g pd deficien- cy. a report by egeland et al ( ) supported the existence of a gene conferring a strong prédisposi­ tion to bipolar affective disorder linked to two loci on the short arm of chromosome ( p ), the harvey-ras- oncogene locus (hras ) and the insulin (ins) locus. these studies had a noticeable impact on psy­ chiatrie thinking. they came as thc logical molecu- lar biology sequel to the adoption and twin studies of the previous décades and suggested that the iden­ tification of m ajor susceptibility genes was just around the corner. however, linkage studies ol psy­ chiatrie disorders hâve had notorious difficulties withstanding the trial o f time. controversies were revivcd by the re-analysis of the linkage between chromosome lp loci and affective disorders in thc old order amish, presented at the apa meeting by kelsoe ( ), in the wakc of thc article published in the november issue o f nature. as far as is known, the chromosome linkage has not been confirmed. the initial results of egeland ( ) were based on restriction fragment length polymorphism (rflp) studies at the hras- and ins loci in an -member pedigree (pedigree ) with ill pro- bands front thc old order amish community in southeastern pennsylvania. the amish are an ex- ceptional study environment because their commu­ nity is extremcly endogamous; they arc a genetic isolate with minimal heterogeneity. kelsoe presented results on an extended version of pedigree . in addition to the members o f the original core pedigree, the new pedigree includes a -member ( affected) “ right” extension and a smaller six- member ( affected) “ left extension” . furthermorc, two members o f the core pedigree had their clini- cal status changed front unaffected to affected, and thc genotyping of unaffected individuals of the core pedigree was completed. with this new data, linkage to an affective disorder locus can be ex- cluded to centimorgans foreither hras or ins, in the total -member pedigree and in the right extension alone. the new data, notably the présence of obligate recombinants in the core pedigree, has causcd thc lod score to drop front an original value ol . to non-significance (at = in the two-point analysis between hras and bipolar affective disorder). besides the importance o f diagnostic follow-up and ol at least yearly visits of ail the pedigree mem­ bers, what particular or general conclusions can be drawn iront the amish study? first, soute factors are related to the genetic paradignts that are pur- sued. it is important to bear in mind that linkage norntally occurs in relation to a putative m ajor sus- ceptibility gene. linkage is a strategy directed toward the search for a major mendelian suscepti- bility locus, as opposed to additive “ g altonian” polygénie effects. lack of réplication in large popu­ lations casts doubt on the universal validity of a single-gene etiology. secondly, other factors such as the problents of genetic and clinical heterogeneity, and définition of penetrance and caseness, are closely dépendent upon the particular psychiatrie disorder that is studied. there are scveral possible interprétations of the non- rcplication in the amish group: either the initial report is simply not reproducible, or else there is genetic heterogeneity, which means that at least two different genes may be rcsponsible for affective ill­ ness in the pedigree. it has been suggested by egeland et al ( ) that several genes may be rcsponsible for affective disorder within the am­ ish population itself, as several of the founding cou­ ples had mental illness among their first and second génération descendants. thirdly, questions arise concerning thc exception- al genetic isolate that is studied and the gcneraliza- tion of linkage results beyond this highly inbred community. the amish are descended frorn the swiss anabaptists who appeared in the wake o f the th century religious reformation and later emigrated to southern gcrmany and alsace to flee persécution. it would be interesting to investigate whether (lie chromosome linkage could be repli- cated in thc european parent population, which is considerably less inbred. it may be noted that the american old order amish population is exception- ally inbred; genes l'iow out when subjects marry out- side the community, but almost no genes flow in. the very closcd american amish community which is reported herc might be expected to differ from its european forefathers by the combined resuit of two effects: the “ founder effects” (oniy thosc who emigrated contributed to the new genetic pool) and the “ genetic drift” (the genetic pool of the new com- munity evolved due to different fertility rates). three paths to linkage ln general, the search for linkage can be guided by three possible strategies (gershon, ). the first strategy is to investigate linkage with “ candidate genes” , such as those involved in neurotransmis­ sion or neuroreceptors. however, this approach has so far failed to show a m ajor susceptibility locus. for instance, linkage with the d receptor région ( q - ) has been excluded in bipolar and schizophrénie pedigrees. similarly, lentes et ai ( ) failed to show linkage with beta- (chromo­ some q ) and beta- (chromosome q ) adrenergic receptor genes in manie dépressive pedigrees. the second strategy is the exploration of markers in the région of a rare cytogenetic event found in rare pedigrees. this has been the case in schizophre- nia where a report of a trisomy implicating the prox­ imal long arm of chromosome ( q - ) inherited with schizophrenia (basset et al, ) was followed by the finding that schizophrenia might be linked to a gene locus with a dom inant susceptibility al- lele on chromosome q in british and icelandic fa- milies (sherrington et al, ). however, négative findings were published at the same time by other researchers in a swedish population (kennedy et al, ). kennedy et al ( ) presented additional results at the american psychiatrie association meeting, and confirmed the absence of linkage to schizophrenia of the genes for tyrosine hydroxylase and the d receptor (chromosome ); other areas of interest were also excluded for linkage in the same swedish population : the genes for nerve growth fac­ tor, pro-opiomelanocortin, and the pseudoau- tosomal boundary (mic ) of the sex chromosomes. the third strategy is systematic mapping, and several papers based on this approach were present­ ed in new york (berrettini et al, ; byerley et al, ; polymeropoulos et al, ). systematic mapping, leaving no région unexplored, is now pos­ sible, mainly because of the availability of poly­ m o rp h ie in fo rm a tiv e m a rk e rs d is trib u te d throughout the genome and the ability to screen the whole genome in a less costly and more automa- tized manner. it had been estimated as carly as (botstein) that evenly spaced markers would en- sure that no disease locus could be more than a % recombination distance from a marker locus. preliminary results on systematic mapping of genes for manic-depressive disease were reported in may in new york. berrettini et al ( ) studied a sériés of bipolar pedigrees consisting of inform a­ tive subjects ( bipolars, unipolars or schizoaffec- tives) under the assumption of a dominant gene, % frequency and % penetrance. results with dna markers exclude linkage to p (short arm of chromosome ), q ll- q l , q - , p (région reported to contain the susceptibility gene for bipolar disease in the old order amish study) and llq - (d receptor région). in the same line, byerley et al ( ) conducted a systemat- ic genomic mapping study using over dna mar­ kers and also reported absence of linkage on chromosome , and in eight manic-depressive kindreds and eight schizophrénie families. late onset of alzheiiner’s disease. hinders linkage several iinkages hâve also been reported for al- zheimer’s disease (ad). a number of authors exa- mined chromosome because alzheimer-like neuropathology develops among patients with down’s syndrome. st george-hyslop et al ( ) reported linkage o f the rare disorder familial al­ zheimer disease (fad) to anonymous polymorph­ ie dna sequences mapping to the proximal long arm of chromosome . positive lod scores on q were also reported in fad families in england by goate et al ( ). however, some authors could not confirm these results ; others even excluded link­ age to the same markers by mapping other pedigrees (pericak-vance et al, ; schellenberg et al, ). more recently, roses et al ( ) reported positive lod scores between ad and several m ar­ kers on the long arm of chromosome in an en- larged set of families. in this case too, genetic heterogeneity, ie multiple predisposing genes result- ing in indistinguishable phenotypes, has been sug- gested to account for these discrepant or négative linkage results. in addition, clinieal heterogeneity has been suggested. one form o f the illness may be caused by a genetic del'ect on chromosome and show early onset, typically in the fifth decade, while another more common form o f the disorder may be a different genetic defect and show later onset, typically around the âge of . however, proving genetic etiology and showing linkage mcet with considerably more difficulties in ad than in manic-depressive illness or schizophre­ nia. it has been said that a large proportion of sporadic cases argue against the presence of genet­ ic causes in most cases of ad. however, the dis­ cussion of results must take into account the particular characteristics of the disorder that is studied. breitner et al ( , , , and un- published data) hâve emphasized that the very late onset of ad may preclude the occurrence of disease in many relatives whose earlier death has censored the expression of any inherited disease tendency. a characteristic of ad is age-dependent expression and variable âge of onset. in the m ajority o f cases, onset occurs in the eighth and ninth decade and therefore only one or two family members are af- fected. such a limited number o f affected subjects precludes the démonstration of linkage. it has been suggested that most ad cases may in fact be shown to hâve genetic causes. when appropriate allowance is made for censorship of disease tendency in rela­ tives by other causes of death, the risk of alzheimer- like dementia in first degree relatives of unselected clinically diagnosed ad probands increases with âge and is about % over a lifespan o f years. one cannot help noticing that this figure is the risk ex- pected in an autosomal dominant mendelian illness. because of the late onset of ad, other methods such as twin studies may prove particularly useful. a pi­ lot study in the national academy of sciences registry of aging twin vétérans was reported by breitner et al ( ). the study o f a sample of pairs ( subjects) suggests a % concordance rate for alzheimer’s disease in monozygotic twin pairs. this concordance rate will likely increase with on- going longitudinal observation and exceeds prior estimâtes. new approachcs to d receptor genes our understanding of neurotransm itter action has been refined by molecular biology studies of recep­ tor genes expressed in the brain. this is particular­ ly the case with d receptors, which hâve been implicated in the pathogenesis of schizophrenia. some data suggested an increased number of d receptors in the brains of schizophrénies. two main types o f dopamine receptors, d, and d , hâve been identified by pharm acological studies. bunzow et al ( ) hâve reported the clon- ing of a rat cdna (complementary dna copied from mrna by the enzyme reverse transcriptase) with the expression characteristics o f a d recep­ tor. todd et al ( ) reported that the overall or- ganization of this d gene is similar in rats, mice and man. it contains exons (expressed dna sequences), a very long first intron (intervening dna sequence that is not translated in the poly­ peptide gene product), and spans at least kilo- bases in the rat. in contrast to the genomic organization of most g-protein-coupled receptors, this d gene contains multiple introns. the in- teresting finding was that two forms of d mrna were detected in various brain régions, and it has been suggested that the d receptor gene identified by bunzow gives rise to at least two m ajor d receptor mrna subtypes (todd et al, , ). the two forms are generated by alternative splic- ing of an additional exon (exon ) within intron ; consequently the two resulting d proteins differ by the insertion of a amino acid sequence. splic- ing refers to the stage o f mrna processing where- by introns are removed. the relative proportion of the two alternative transcripts varies -fold in different brain tissues. the exon containing mrna predom inated in areas where d receptor number is highest-basal ganglia, anterior pituitary, and olfactory bulb. these findings imply the presence of tissue-specific factors that control not only d gene transcription, but also alternative splicing of the pre-mrna. knowledge of the genetic organization o f d receptors is of obvious im por­ tance in schizophrenia and neuroleptic drug treatments. new methods for the approach to neurons ex­ pressing d receptors were also discussed. in situ hybridization of d receptor mrna was reported in the prim ate brain by m eador-w oodruff et al ( ) after being reported in rats a short time ago (m eador-w oodruff et al, ). this technique compléments studies using receptor autoradiogra- phy and pet ; it reveals cell bodies and allows the study of earlier events in receptor synthesis. régu­ lation at the gene transcription or translation level might thus be explored. conclusion no one doubts that genetics is a promising field in psychiatry. from pedigree studies to the molecular biology of receptor genes expressed in the brain, no particular approach is outdated. each one is ideal- ly suited tow ard investigating spécifie questions. some results that were first presented a few years ago become the object of renewed controversy as recent findings emerge. particular caution must be taken in interpreting the results of linkage studies until models o f inheritance can be delineated with more clarity. acknowledgments the authors are indebted to ms hein who kindly read the original manuscript. référencés berrettini w h, detera-wadleigh s, goldin l, martinez m, hsieh w t, gershon e ( ) systematic genomic mapping o f genes for manic-depressive disease : stu- dies o f chromosomes and . biol psychiatry suppl, , a, p a breitner jcs, m urphy ea, silverman jm , mohs rc, davis kl ( ) age-dependent expression o f fami­ lial risk in alzheimer’s disease. a m j epidemiol : , - breitner jcs, m agruder-habib km ( ) criteria for onset critically influence the estimation o f familial risk in alzheimer’s disease. genet epidem iol , - breitner jcs, welsh k, magruder-habib k, churchill c, robinette cd, folstein m, priolo cc, brandt j ( ) pilot studies suggest % concordance for alzheimer’s disease in mz twin pairs. biol psychiatry suppl, , a, p a bunzow jr, van toi hhm , grandy dk, albert p, salon j, christie m, machida ca, neve ka, civelli o ( ) cloning and expression o f a rat d dopamine recep- to r cdna. n ature , - byerley w f, leppert m, holik j, lubbers am, jensen s, white r ( ) mapping genes for manie déprés­ sion and schizophrenia. a p a rd artnual meeting. n ew research abstracts. nr , p egeland ja , gerhard ds, pauls dl, sussex jn , kidd kk, allen cr, h ostetter am , housem an de ( ) bipolar affective disorder linked to dna markers on chromosome . nature , — gershon es ( ) genetics. in: manic-depressive iii- ness (goodwin fk, jam ison kr, eds) oxford univer- sity press, pp - goate am, owen m j, james la, mullan m j, rossor mn, haynes ar, farrall m, lai lyc, roques p, wil­ liamson r, hardy j ( ) predisposing locus for alz­ heimer’s disease on chromosome . lancet i, - gurling h ( ) genetic linkage and psychiatrie disease. nature , haroutuninan v, braider s, snyder l, sealfon s, roberts jl, davis kl ( ) régulation o f dopamine d recep- tor mrna by chronic haloperidol treatm ent. biol psychiatry suppl, , a, p a kelsoe jr , ginns e l, egeland ja , gerhard ds, golds­ tein am , baie sj, pauls dl, long rt, kidd kk, conte g, housman de, paul sm ( ) re-evaluation o f the linkage relationship between chromosome l l p loci and the gene for bipolar affective disorder in the old order amish. nature , - kelsoe jr , ginns e l, egeland ja , goldstein am, baie sj, pauls dl, long rt, conte g, gerhard ds, hous­ man de, paul sm ( ) bipolar affective disorder in the old order amish in: proceedings, a p a rd a n n u a l m eeting, b, p kennedy jl , g iuffra la, moises hw , cavalli-sforza ll, pakstis a j, kidd jr , castiglione cm, sjogren b, wetterberg l, kidd kk ( ) evidence against linkage o f schizophrenia to m arkers on chrom o­ some in a northern swedish pedigree. nature , - kennedy jl, giuffra l, wetterberg l, sjogren b, cavalli- sforza ll, moises hw, gelernter je, pakstis aj, cas­ tiglione cm, kidd kk, kidd jr ( ) genetic linkage studies o f schizophrenia. in : proceedings, a p a i rd a n n u a l meeting, d, p lentes ku, hôhe mr, berrettini w h, goldin lr, gers­ hon es ( ) eine kopplungsanalyse beta-adrenerger rezeptoren bei manisch-depressiver krankheit mit molekulargenetischen methoden. biologische psychia­ trie - meador-woodruff jh , mansour a, bunzow jr, van toi hhm, watson sj, civelli o ( ) distribution of d receptor mrna in rat brain. proc n atl a ca d sci usa , - meador-w oodruff jh , mansour a, healy dj, civelli o, w atson sj ( ) dopamine d receptor mrna in brain. in : procedings, a p a i rd a n n u a l meeting, e, p m eador-w oodruff jh , m ansour a, healy d j, watson sj ( ) distribution o f dopamine d receptor mrna in primate brain : an in-situ hybridization study. biol psychiatry suppl , a, p a pericak-vance ma, yamaoka lh, haynes cs, speer mc, haines jl , gaskell pc, hung wy, clark cm, heyman al, t rofatter ja , eisenmenger jp , gilbert jr, lee je, alberts mj, dawson dv, bartlett rj, earl nl, siddique t, vance jm , conneally pm , roses ad ( ) genetic linkage studies in alzheimer’s disease families. exp n eurol , - polymeropoulos mh, xiao h, delisi l, weber lj, merril rc ( ) use o f (ac)n repeat polymorphisms in schi­ zophrenia. a p a i rd a nnual meeting, new research abstracts. nr , p robakis nk, m ohamadi m, fu d, refolo lm ( ) cloning and characterization o f two cdna’s encoding the human d receptor. biol psychiatry suppl, , a, p a roses ad, bebout j, yamaoka l, gaskell pc, hung wy, alberts m j, clark c, welsh k, earl n, heyman a ( ) linkage studies in familial alzheimer’s disease: application o f the affected pedigree mentber m ethod. neurology (suppl ) st george-hyslop ph , tanzi re, polinsky rj ( ) the genetic defect causing familial alzheimer’s disease maps on chromosome . science , - schellenberg gd, bird td, wijsman em ( ) absence o f linkage o f chromosome q markers to familial alzheimer’s disease. science , - sherrington r, brynjolfsson j, petursson h, potter m, dudleston k, barraclough b, w asmulh j, dods j, gurling h ( ). localization of a susceptibility locus for schizophrenia on chromosome . nature , - todd rd, mack k, gandelman k-y, o ’malley kl ( ) organization and expression o f a dopam ine d recep­ tor gene. biol psychiatry suppl, , a, p a welsch k, breitner jcs, magruder-habib km, churchill cm, robinette cd, folstein mf ( ) sixty percent concordance for alzheimer’s disease in monozygotic twin pairs. a p a i rd a nnual meeting, new research abstracts. nr , p management of patients on non–vitamin k antagonist oral anticoagulants in the acute care and periprocedural setting: a scientific statement from the american heart association e march , circulation. ; :e –e . doi: . /cir. abstract: non–vitamin k oral anticoagulants (noacs) are now widely used as alternatives to warfarin for stroke prevention in atrial fibrillation and management of venous thromboembolism. in clinical practice, there is still widespread uncertainty on how to manage patients on noacs who bleed or who are at risk for bleeding. clinical trial data related to noac reversal for bleeding and perioperative management are sparse, and recommendations are largely derived from expert opinion. knowledge of time of last ingestion of the noac and renal function is critical to managing these patients given that laboratory measurement is challenging because of the lack of commercially available assays in the united states. idarucizumab is available as an antidote to rapidly reverse the effects of dabigatran. at present, there is no specific antidote available in the united states for the oral factor xa inhibitors. prothrombin concentrate may be considered in life-threatening bleeding. healthcare institutions should adopt a noac reversal and perioperative management protocol developed with multidisciplinary input. a s the us population ages, the burden of atrial fibrillation (af) and venous throm- boembolic disease is expected to increase, and prescriptions for long-term anticoagulation will climb. anticoagulated patients are vulnerable to spontane- ous, traumatic and perioperative bleeding. warfarin is a vitamin k antagonist (vka) that has been used for decades to prevent and treat arterial and venous thromboem- bolism (vte). more recently, non–vitamin k antagonist oral anticoagulants (noacs) have been approved in the united states as alternatives to warfarin for prevention of stroke resulting from nonvalvular af (nvaf), and prevention and treatment of vte. these are dabigatran etexilate (pradaxa, boehringer ingelheim, germany); rivaroxa- ban (xarelto, bayer healthcare ag, leverkusen, germany), apixaban (eliquis, pfizer and bristol-myers squibb, new york, ny) and edoxaban (savaysa, daiichi sankyo, tokyo, japan). direct oral anticoagulants has been proposed as alternative nomen- clature for these class of agents. noacs are associated with comparable or lower risk of stroke, systemic embolism, major bleeding, and death compared with warfa- rin for nvaf. – in contrast with warfarin, noacs have a more predictable therapeutic effect, do not require routine monitoring, have fewer potential drug-drug interactions and no restriction on dietary consumption of vitamin k–containing food. however, universal adoption of noacs has been stunted by the lack of specific antidotes and measurement assays. this scientific statement reviews the literature and offers practical suggestions for providers who manage patients who are actively bleeding and who are at risk for bleeding in the acute care and periprocedural setting. this statement focuses on interpreting available data rather than providing specific man- amish n. raval, md, faha, chair joaquin e. cigarroa, md mina k. chung, md, faha larry j. diaz-sandoval, md, faha deborah diercks, md jonathan p. piccini, md, mhs, faha hee soo jung, md jeffrey b. washam, pharmd, faha babu g. welch, md allyson r. zazulia, md, faha sean p. collins, md, msc, faha, co-chair on behalf of the ameri- can heart association clinical pharmacology subcommittee of the acute cardiac care and general cardiology committee of the coun- cil on clinical cardiol- ogy; council on car- diovascular disease in the young; and council on quality of care and outcomes research management of patients on non–vitamin k antagonist oral anticoagulants in the acute care and periprocedural setting a scientific statement from the american heart association © american heart association, inc. key words: aha scientific statements ◼ acute care ◼ anticoagulants ◼ non–vitamin k antagonist ◼ periprocedural aha scientific statement d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines agement recommendations in under-studied populations such as oncology patients. members of this american heart association (aha) writing group were selected for their diverse expertise in cardiovascular medicine, emergency medicine, critical care, neurology, surgery, and pharmacology. a system- atic search of the literature for each subtopic was per- formed in pubmed and ovid and was supplemented by review of bibliographies as well as manual searches of key articles. each of the following search terms were in- cluded individually and in combination: dabigatran, apix- aban, rivaroxaban, edoxaban, anticoagulation, reversal, antidote, atrial fibrillation, venous thromboembolism, bleeding, intracranial, cardioversion, catheterization, cardiac implantable devices, kidney injury, transition, switching, pharmacology, andexanet alfa, idarucizumab, ciraparantag, gastrointestinal, trauma, surgery, percu- taneous coronary intervention, neuraxial anesthesia, stroke, and overdose. writing group members were instructed to write subtopic sections aligned with their experience. members were instructed to cite contempo- rary guidelines and scientific statements where appropri- ate. the writing group did not assign formal classes of recommendation/level of evidence per the aha scien- tific document development process recommendation that went into effect september , . sections were then reviewed by another writing group member. sec- tion drafts were submitted to the writing group chair and co-chair and compiled into a single document. web and teleconferences were convened to review and edit the full draft. the final document was submitted for indepen- dent peer review and approved for publication by the aha manuscript oversight committee on april , . pharmacology of noacs noacs act through direct inhibition of thrombin or inhi- bition of factor xa (figure ). dabigatran etexilate me- sylate is a competitive direct thrombin inhibitor. rivar- oxaban, apixaban, and edoxaban inhibit factor xa and prothrombinase activity, thus inhibiting the conversion of prothrombin to thrombin. thrombin catalyzes the con- version of fibrinogen to fibrin; activates factors v, viii, xi, and xiii; and activates platelets. therefore, inhibiting thrombin decreases thrombus formation. in contrast with warfarin, noacs have a rapid onset of action, a shorter half-life, and more predictable pharmacokinet- ics. routine therapeutic monitoring was not done in the major noac efficacy trials and is at present not recom- mended in usual clinical practice. information pertaining to noac dose, time to peak effect, and time to offset of effect is outlined in table . noacs are substrates for p-glycoprotein (p-gp) trans- port and apixaban and rivaroxaban are substrates for cyp a metabolism. therefore, concomitant medica- tions that are inducers or inhibitors of these pathways should be evaluated for the potential to interact (table ). macrolides and nondihydropyridine calcium channel blockers are commonly prescribed classes of medica- tions that impact therapeutic levels of noacs, although a post hoc analysis of rocket af (rivaroxaban once daily oral direct factor xa inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation) showed no evidence of differential outcomes between rivaroxaban and warfarin in patients treated with ≥ combined p-gp and cyp a inhibitors. edoxaban exists in a predominantly unchanged form figure . clotting cascade and anticoagulants. vka indicates vitamin k antagonist. d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e table . comparison among noacs dabigatran rivaroxaban apixaban edoxaban approved indications nonvalular af ↓ risk of stroke and systemic embolism nonvalular af ↓ risk of stroke and systemic embolism nonvalular af ↓ risk of stroke and systemic embolism nonvalular af ↓ risk of stroke and systemic embolism. limitation: should not use in patients with crcl > ml/min as a result of ↑ risk of ischemic stroke compared with warfarin at mg dvt, pe treatment after – d parenteral ac ↓ recurrence prophylaxis after hip replacement dvt, pe treatment ↓ recurrence prophylaxis after hip or knee replacement dvt, pe treatment ↓ recurrence prophylaxis after hip replacement dvt, pe ↓ recurrence treatment after – d initial parenteral ac mechanism of action direct thrombin inhibitor factor xa inhibitor factor xa inhibitor factor xa inhibitor time to peak h; delayed to h with food – h – h – h bioavailability %– % -mg dose: %– % ~ % % -mg dose: % ↑ with food plasma protein binding % %– % ~ % % volume of distribution – l l l l plasma t / – h – h ~ h ( – h) – h elderly – h elderly – h mild to moderate renal impairment – h severe renal impairment h metabolism hepatic and plasma hydrolysis to active dabigatran hepatic: oxidation by cyp a / , cyp j ; hydrolysis to inactive metabolites ( %) hepatic: % mainly by cyp a / ; lesser by cyp a , cyp c , cyp c , cyp c , cyp j ; o-demethylation and hydroxylation minimal cyp a hydrolysis, conjugation, oxidation hepatic glucuronidation to active metabolites (< %) p-gp substrate no active circulating metabolites active metabolite (m- , < % of parent) p-gp substrate no major or active circulating metabolites substrate of cyp a , p-gp, bcrp p-gp substrate substrate of p-gp and abcg (bcrp) excretion renal (~ %) after iv administration renal ( %): % active, % inactive metabolites renal ( %) renal (~ %): primarily as unchanged drug after oral, % recovered in urine, % in feces feces ( %): % active, % inactive metabolites biliary and direct intestinal excretion metabolism and biliary/ intestinal excretion accounts for the rest (continued ) d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines dosing nonvalvular af crcl > ml/min: mg bid crcl > ml/min: mg daily with evening meal mg bid crcl > to ≤ ml/min: mg daily crcl – ml/min: mg bid crcl – ml/min: mg daily with evening meal . mg bid, if of characteristics: cr ≥ . mg/dl, age ≥ y, weight ≤ kg crcl – ml/min: mg daily crcl < ml/min or on dialysis: not recommended not recommended for crcl < ml/min or on dialysis in patients with af not recommended for crcl > ml/min crcl – ml/min with concomitant p-gp inhibitors: mg bid crcl < ml/min with concomitant p-gp inhibitors: avoid coadministration dvt or pe treatment crcl > ml/min: mg bid after - d parenteral anticoagulation mg bid with food first d for initial treatment, then mg once daily with food mg bid x d, then mg bid mg once daily crcl ≤ ml/min or on dialysis: not recommended not recommended for crcl < ml/min in patients with dvt or pe crcl – ml/min or weight ≤ kg or on certain p-gp inhibitors: mg once daily ↓ in recurrent dvt/pe crcl > ml/min: mg bid after – d parenteral anticoagulation mg daily with food . mg bid crcl ≤ ml/min or on dialysis: not recommended dvt, pe prophylaxis after hip or knee replacement after hip replacement surgery: crcl > ml/min after achievement of hemostasis: if given day of surgery, mg – h postop; after day of surgery mg once daily x – d crcl ≤ ml/min or on dialysis: not recommended crcl < ml/min with concomitant p-gp inhibitors: avoid coadministration initial dose – h after surgery provided hemostasis established . mg bid x d after hip replacement surgery or x d after knee replacement surgery mg daily with or without food x d for hip replacement, x d for knee replacement additional dosing comments avoid use with patients with moderate-severe hepatic impairment (child- pugh class b/c) or hepatic disease with coagulopathy not recommended in patients with severe hepatic impairment (child-pugh class c) not recommended with crcl < ml/min - mg taken with food; mg with or without food not recommended in patients with moderate-severe hepatic impairment (child-pugh class b/c) table . continued dabigatran rivaroxaban apixaban edoxaban (continued ) d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e in plasma with minimal metabolism through hydrolysis, conjugation, and oxidation by cyp a . laboratory measurement of noac effect one advantage of noacs over warfarin is more rapid on- set and offset of action with predictable pharmacokinet- ics and anticoagulant effect. this eliminates the necessi- ty for routine therapeutic monitoring except for periodic assessment of renal function. laboratory measurement of noac level or effect may be necessary in certain acute care or perioperative settings, particularly when there is uncertainty about the timing of last ingestion, renal function, and gastrointestinal absorption. however, the lack of us food and drug administration–approved noac laboratory assays complicates the management of noac overdose, noac-associated life-threatening bleeding, and the scheduling of urgent surgical proce- dures. all noac agents affect routine coagulation tests but not in a manner that allows for a predictable and quantitative measurement of anticoagulation effect. spe- cific noac agents are subsequently discussed. dabigatran this agent is known to prolong the activated partial thromboplastin time, prothrombin time, and thrombin time. the package insert recommends using partial thromboplastin time for measurement; however, there is no defined partial thromboplastin time therapeutic range for dabigatran and the assay is relatively insensitive to different plasma concentrations of direct thrombin inhibi- tors. furthermore, the partial thromboplastin time can- not be used in patients with lupus anticoagulant or an intrinsic clotting factor deficiency because its prolonga- tion from these conditions would mask the anticoagulant effect of dabigatran. thrombin time is far more sensi- tive, and prothrombin time is less sensitive to dabigatran. a normal partial thromboplastin time or thrombin time most likely excludes therapeutic levels of dabigatran, whereas a normal prothrombin time may not. quantita- tive assessments of dabigatran levels can be obtained with the dilute thrombin time, the ecarin clotting time, or the ecarin chromogenic assay. thrombin time and ecarin- based assays show excellent linearity across on-therapy drug concentrations and may be used for drug quantifica- tion. however, the us food and drug administration has not approved these latter assays for measuring levels of dabigatran or other direct thrombin inhibitors. rivaroxaban, apixaban, edoxaban at present, there are no us food and drug administra- tion–approved assays or calibration reagents to measure the effect of direct oral factor xa inhibitors. rivaroxaban and apixaban affect activated clotting time and chro- mogenic anti–factor xa assay; however, no therapeutic range exists. prothrombin time is less sensitive (espe- cially for apixaban), and a normal prothrombin time may not exclude clinically relevant levels. partial thromboplas- tin time demonstrates insufficient sensitivity and linearity for quantification. studies using spiked plasma samples suggest using prothrombin time for a qualitative assess- ment of direct oral factor xa inhibitors or chromogenic anti–factor xa assay for a quantitative assessment of direct oral factor xa inhibitors. – anti-xa activity is linear over a wide range of drug levels and may be used for drug quantification. undetectable anti-xa activity likely excludes clinically relevant drug concentrations. in summary, although routine noac monitoring is unnecessary, measurement of noac effect may assist clinical management in certain acute care and peripro- cedural settings. in most situations, the time of last drug ingestion combined with a recent assessment of creati- nine clearance (crcl) should enable appropriate clinical decision making. therapeutic measurement routine not required routine not required routine not required routine not required to detect presence: aptt, ect (if available), tt to detect presence: pt, aptt, antifactor xa activity to detect presence: pt, aptt, antifactor xa activity prolongs pt, aptt, antifactor xa activity aptt > . times control may indicate overanticoagulation renal function, cbc periodically, at least annually; hepatic function renal function, cbc periodically, at least annually renal function, cbc periodically, at least annually renal function, cbc periodically, at least annually ac indicates anticoagulant; af, atrial fibrillation; aptt, activated partial thromboplastin time; bid, twice daily; cbc, complete blood count; crcl, creatinine clearance; dvt, deep vein thrombosis; ect, ecarin clotting time; iv, intravenous; noacs, non–vitamin k antagonist oral anticoagulants; pe, pulmonary embolism; p-gp, p-glycoprotein; pt, prothrombin time; and tt, thrombin time. table . continued dabigatran rivaroxaban apixaban edoxaban d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines table . noac drug interactions noac interacting medications effect on noac labeled guidance; comments dabigatran p-gp inducer: rifampin ↓ dabigatran exposure concomitant use should generally be avoided. p-gp inhibitors: ketoconazole, dronedarone ↑ dabigatran exposure if concomitant moderate renal impairment if moderate renal impairment (crcl – ml/min) ↓ to mg bid during concomitant use p-gp inhibitors: ketoconazole, dronedarone, verapamil, amiodarone, quinidine, clarithromycin, ticagrelor ↑ dabigatran exposure if concomitant severe renal impairment if severe renal impairment (crcl – ml/ min) avoid concomitant use apixaban strong dual p-gp and cyp a inducers: rifampin, carbamazepine, phenytoin, st. john’s wort ↓ apixaban exposure avoid concomitant use strong dual p-gp and cyp a inhibitors: ketoconazole, itraconazole, ritonavir, clarithromycin ↑ apixaban exposure in patients on mg or mg bid, ↓ dose by % when coadministered avoid coadministration on . mg bid rivaroxaban combined p-gp and strong cyp a inducers: rifampin, carbamazepine, phenytoin, st. john’s wort ↓ rivaroxaban exposure avoid concomitant use; may decrease rivaroxaban efficacy combined p-gp and strong cyp a inhibitors: ketoconazole, itraconazole, hiv protease inhibitors (ritonavir, lopinavir/ ritonavir, indinavir), conivaptan ↑ rivaroxaban exposure avoid concomitant use combined p-gp and moderate cyp a inhibitors: diltiazem, verapamil, amiodarone, dronedarone, erythromycin ↑ rivaroxaban exposure in patients with renal impairment in patients with crcl to < ml/min, rivaroxaban should not be used concomitantly unless the potential benefit justifies the potential risks no evidence of interaction observed in rocket af between treatment assignment and outcomes in patients using ≥ combined p-gp and moderate a inhibitors (including amiodarone, diltiazem, and verapamil) edoxaban p-gp inducer: rifampin ↓ edoxaban exposure avoid concomitant use strong p-gp inhibitors: ritonavir, nelfinavir, saquinavir, indinavir, cyclosporine ↑ edoxaban exposure avoid concomitant use in patients taking edoxaban for treatment of vte p-gp inhibitors: verapamil, quinidine, azithromycin, clarithromycin, itraconazole, ketoconazole ↑ edoxaban exposure ↓ to mg daily during concomitant administration for patients taking edoxaban for the treatment of vte dose reduction is not recommended for af indications in engage af, a ↓ dose of edoxaban as a result of concomitant p-gp inhibitor use (verapamil, quinidine, dronedarone) was associated with ↓ edoxaban exposure and a relative ↑ in risk of stroke or systemic embolism with edoxaban relative to warfarin af, atrial fibrillation; bid, twice daily; crcl, creatinine clearance; engage af, effective anticoagulation with factor xa next generation in atrial fibrillation trial; noac, non–vitamin k antagonist oral anticoagulant; p-gp, p-glycoprotein; rocket af, rivaroxaban once daily oral direct factor xa inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation; and vte, venous thromboembolism. d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e noac reversal this aha writing group suggests hospital systems adopt anticoagulation reversal protocols with multidisciplinary representation from emergency medicine, critical care, cardiology, hematology, gastroenterology, neurology, neurosurgery, trauma, acute care surgery, cardiotho- racic surgery, vascular surgery, pharmacy, and nursing. an example of a noac reversal protocol is shown in figure . dabigatran for minor bleeding, supportive care and careful obser- vation are suggested. for major bleeding, intravenous idarucizumab (praxbind, boehringer-ingelheim, germa- ny) at a dose of grams ( consecutive intravenous infu- sions of . g each) will reverse the anticoagulant effect of dabigatran within minutes. idarucizumab is a mono- clonal antibody fragment that binds dabigatran with an affinity times that of thrombin. the re-verse ad (reversal effects of idarucizumab on active dabigatran) was a prospective cohort study that showed that ida- rucizumab administration reversed anticoagulation as evidenced by the normalization of the dilute thrombin time and ecarin clotting time within minutes among sub- jects suffering a serious hemorrhage or who required an urgent procedure. early hemostasis was achieved in bleeding subjects, and a low rate of perioperative bleeding events was observed in subjects undergoing urgent surgery. however, the strength of these clinical observations is limited by the nonrandomized nature of this study. several studies have investigated the efficacy of pro- thrombin complex concentrates (pccs), recombinant fac- tor vii activated, and fresh frozen plasma (ffp) in animal models; however, human data are mixed. one random- ized, placebo-controlled trial in healthy men treated with dabigatran showed that -factor pcc did not reverse the dabigatran effect on partial thromboplastin time, endoge- nous thrombin potential lag time, thrombin time, or ecarin clotting time. case reports of patients with life-threat- ening bleeding associated with dabigatran therapy have demonstrated mixed results with the use of ffp, recombi- nant factor vii activated, pccs, fibrinogen, and platelets. hemodialysis may remove % to % of dabigatran within hours given that the drug is only % bound to plasma proteins. , hemodialysis may be considered if the crcl is chronically below ml/min or in acute kidney injury. for major ingestion, there is some evi- dence to support the use of activated charcoal therapy if dabigatran was consumed within to hours; however, care must be taken to prevent aspiration in patients with decreased level of consciousness. , furthermore, acti- vated charcoal induced vomiting could have deleterious effects by increasing intracranial pressure in patients with intracranial hemorrhage (ich). in summary, the aha writing group suggests com- pression when possible, supportive measures, and up- front idarucizumab in the event of dabigatran-associated major bleeding. rivaroxaban and apixaban similar to dabigatran, activated charcoal may prevent absorption of rivaroxaban and apixaban if administered figure . example of a “serious bleeding on a noac” protocol. bun indicates blood urea nitrogen; cbc, complete blood count; iu, international units; iv, intravenous; noac, non–vitamin k antagonist oral anticoagulants; pcc, prothrombin complex concentrate; prbc, packed red blood cells; ptt, partial thrombo- plastin time; and tt, thrombin time. d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines within to hours after noac ingestion. rivaroxaban and apixaban are highly bound to plasma proteins; therefore, dialysis is ineffective in clearing these drugs. andexanet alfa is a recombinant modified human fac- tor xa decoy protein that serves as a specific reversal agent to neutralize the anticoagulant effects of direct and indirect factor xa inhibitors. this drug is admin- istered as an initial intravenous bolus followed by an infusion for up to hours. a recent study revealed that andexanet alpha reversed the laboratory assessed anticoagulant activity of rivaroxaban and apixaban in older healthy individuals within minutes of administra- tion. at present, the single arm, open-label annexa- (andexanet alfa for acute major bleeding associated with factor xa inhibitors) trial is under way to confirm the clinical benefit of this drug in patients on apixaban, rivaroxaban, edoxaban, or enoxaparin who present with an acute major hemorrhage. an interim analysis of patients revealed an % and % reduction in anti– factor xa activity for those on rivaroxaban and apixaban respectively. of the entire cohort, patients were followed for clinical hemostasis. of these, ( %; % confidence interval [ci], – ) were adjudicated as having excellent or good clinical hemostasis. the dosing protocol in this study was as follows: ( ) for patients who had taken apixaban, or rivaroxaban > hours prior, andexanet alfa was given as a bolus dose of mg followed by an infusion of mg over hours; and ( ) for patients who had enoxaparin, edoxa- ban, or rivaroxaban < hours prior or at an unknown time, the bolus dose and infusion dose amount was doubled ( -mg bolus, -mg infusion over hours). at present, andexanet alfa is not approved in the united states or elsewhere. a randomized placebo-controlled study of young, healthy volunteers treated with mg of rivaroxaban dosed twice daily found that administration of a -factor pcc led to normalization of the prothrombin time and the endogenous thrombin potential. in contrast, an in vitro study using human plasma obtained from healthy donors found that recombinant factor vii activated was superior to a -factor pcc at normalizing laboratory co- agulation studies. case reports of using ffp or pcc to treat excess rivaroxaban ingestion have shown mod- est success in improving laboratory coagulation param- eters. – however, the correction of coagulation tests by pcc, ffp, or recombinant factor vii activated does not imply the reversal of the clinical anticoagulation ef- fect of the drug. there is no evidence that ffp or pcc controls noac-associated bleeding in humans. edoxaban four-factor pcc showed dose-dependent reversal of edoxaban effect with complete reversal of bleeding duration after skin punch biopsy in volunteers and par- tial reversal of prothrombin time after a -iu/kg dose administration. however, the clinical relevance of this finding is uncertain. ciraparantag (per ) is a small synthetic, water- soluble, cationic molecule designed to specifically bind to unfractionated heparin and low-molecular-weight hep- arin through noncovalent hydrogen bonding and charge- charge interactions. it also binds in a similar way to di- rect xa inhibitors and direct thrombin inhibitors. it has been shown to normalize whole blood clot time within to minutes of administration. , ciraparantag is still being investigated in early clinical trials as an antidote for edoxaban associated bleeding. it remains unknown whether andexanet alfa will have greater, equal, or less- er clinical efficacy for edoxaban reversal compared with ciraparantag. management of life-threatening bleeding all patients with life-threatening bleeding should be man- aged with similar basic resuscitation principals, irrespec- tive of what type of anticoagulant they may be on. imme- diate management of the patient’s airway, breathing, and circulation with attempts to control hemorrhage is vital. when life-threatening bleeding occurs in a compressible area of the body, direct pressure along with selective use of tourniquets can be life-saving. similarly, immedi- ate resuscitation and stabilization with intravenous flu- ids, packed red blood cells and plasma may be required in the unstable patient. noac reversal as indicated in noac reversal should be considered. these concepts apply to blunt and penetrating trauma, massive gastro- intestinal, retroperitoneal, pericardial hemorrhage, and other forms of major bleeding. specific scenario: ich a meta-analysis of studies that have tested noacs for ischemic stroke prevention in nvaf have estimated a pooled incidence of hemorrhagic stroke of . %. – , overall, this represents a > % relative reduction in ich rate from the . % observed with warfarin. past vka studies suggest that ich is times more likely to result in mortality compared with extracranial hemorrhage. the reduction in ich rate coupled with consistent non- inferiority compared with vkas in preventing thrombotic events has produced a steady increase in the use of noacs to prevent stroke in patients with nvaf. uniform recommendations do not exist regarding man- agement of patients on noacs who suffer ich primarily because no consistent approach to their management was undertaken in the noac trials. factors to consider include availability of reversal agents, the timing of urgent neurosur- gery, risk of thromboembolic events during the period off d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e the anticoagulant, and reinstitution of anticoagulant therapy after the ich event or after surgery. the presence of ich creates a unique circumstance because of the noncom- pressible location of the hemorrhage and poor tolerance of the brain to continued bleeding. the aha/american stroke association “guidelines for the management of spontane- ous intracerebral hemorrhage” recommends prompt cre- ation of a hemostatic environment to limit extension of the hemorrhage and before surgical treatment. any acute neurological change in a patient on noac therapy should be presumed to be vascular in origin. a baseline severity score should be performed as part of the initial evaluation. , computed tomography (ct) is widely available, detects acute hemorrhage with high sensitivity, and defines the extent of the injury on the sur- rounding parenchyma. contrast-enhanced ct may iden- tify patients at high risk of ich expansion on the basis of the presence of contrast within the hematoma, also known as the spot sign. , detailed vascular imaging may identify predisposing vascular lesions such as an- eurysm, arteriovenous malformation, and dural fistula. concurrent with reversing the noac effect, blood pressure needs to be intensively managed. many studies associate elevated systolic blood pressure with greater hematoma expansion, neurological deterioration, and death and dependency after ich. , the interact tri- al (intensive blood pressure reduction in acute cerebral hemorrhage trial ) showed that acute blood pressure reduction to < mm hg systolic was safe and resulted in a trend toward improvement in functional recovery de- spite no significant reduction in the rate of hematoma growth. no patients with noac use were included in this trial. however, recent results from atach- (antihy- pertensive treatment of acute cerebral hemorrhage-ii) suggest aggressive lowering of systolic blood pressure to to mm hg may not confer benefit. a the safety of resuming a noac regimen after ich is a common clinical dilemma. decisions about whether to resume anticoagulation after ich must take into account the patient’s underlying thromboembolic risk and the risk for ich recurrence. embolic stroke risk versus bleeding risk stratification schemes such as the cha ds -vasc and has-bled scores may help guide treatment after ich. , the has-bled score has been validated in a wide range of patients (af and non-af, vka and non-vka) and is the only bleeding risk scheme that is predictive of ich. however, a high has-bled score should not be the sole consideration in clinical management. the presence of a recent ich should prompt closer evalua- tion of other factors related to ich reoccurrence. , , factors that are suggested to increase ich risk include older age, poor blood pressure control, lobar ich loca- tion, presence of microbleeds on gradient echo magnetic resonance imaging, concurrent aspirin use, and the pres- ence of apolipoprotein e ε or ε alleles. aha/american stroke association guidelines provide a class iib recom- mendation for anticoagulation to be considered only after nonlobar ich; however, this recommendation is based on warfarin-associated ich data. whether noacs can be safely administered in this population is still unknown. there is no clinical trial evidence to guide the man- agement of patients with traumatic brain injury while on anticoagulants. an initial head ct is typical; however, the role of repeated ct or inpatient observation with neuro- logical assessment remains controversial when the initial head ct is negative. until further data become available, noac reversal for traumatic ich should be considered similar to nontraumatic ich. in summary, the aha writing group suggests that trau- matic and nontraumatic ich patients on dabigatran who re- quire noac reversal receive idarucizumab. ich patients on rivaroxaban, apixaban, or edoxaban should receive pcc until more specific antidotes become available. specific scenario: trauma the prevalence of noac use in the trauma population is unknown. to compare, the prevalence of warfarin use in the trauma population in was % with a . % absolute rate increase over the previous years. pa- tients should be encouraged to carry information cards or bracelets that would alert emergency medical provid- ers regarding oral anticoagulation use. apart from a few case reports, there are limited data to guide the management of noacs in the setting of trauma. – the american college of surgeons advance trauma life support course recommends obtaining a brief, focused history during the initial evaluation of traumatically injured patients. this should include iden- tifying the specific noac, timing of last ingestion, and the underlying reason for noac use. laboratory testing of renal function and coagulation parameters described in laboratory measurement of noac effect may help with treatment decisions. thromboelastography and rotational thromboelastometry to detect noac activity in isolated trauma cases has been reported; however, routine use cannot be recommended until further data becomes available. , noacs may be held during the period of clinic as- sessment or until hemostasis has been achieved in trauma patients without bleeding and with mild bleeding, or bleeding from easily controllable foci. maintaining ade- quate urine output and specific noac reversal strategies (noac reversal) should be considered in trauma patients with moderate or severe bleeding, or suspected bleed- ing that requires further evaluation. specific scenario: gastrointestinal bleeding in major trials, dabigatran mg twice daily, rivaroxa- ban, and edoxaban mg once daily were associated with a . -fold increased risk of gastrointestinal bleeding d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines compared with warfarin; apixaban and dabigatran mg twice daily had similar gastrointestinal bleeding risk; and edoxaban mg once daily had significantly lower risk. , , factors associated with gastrointestinal bleed- ing with noac use are anemia, previous gastrointestinal bleeding, long term aspirin use or baseline nonaspirin antiplatelet use, age, diastolic hypertension, smoking, sleep apnea, chronic obstructive pulmonary disease, pre- vious proton pump inhibitor use, renal dysfunction, and male sex. although gastrointestinal bleeding accounts for nearly % of major extracranial hemorrhages in nvaf patients on therapeutic anticoagulation, clinical data specifically pertaining to noac reversal are lacking. of the . % of patients in the dresden registry who expe- rienced major bleeding while on rivaroxaban, the majority of patients were managed conservatively without requir- ing surgery. as in the case of trauma, general resuscita- tion principles of airway, intravenous fluid, blood transfu- sion, and maintaining adequate urine output should be applied. a blakemore tube for bleeding from esophageal varices may be considered. immediate noac reversal should be considered in the unstable patient. reinitiating noac therapy after gastrointestinal bleed- ing should take into account the patient’s underlying risk of bleeding and thrombosis risk. in a retrospective study of > patients with nvaf who suffered gastrointes- tinal bleeding on anticoagulation (primarily warfarin), re- sumption of a single anticoagulant was associated with the lowest risk of mortality and thromboembolism com- pared with nonresumption of antithrombotic treatment. the risk of recurrent gastrointestinal bleeding was also low in the anticoagulated patients. patients on noacs comprised a very small subset of the entire cohort; therefore, it remains uncertain whether noac resump- tion after gastrointestinal bleeding would be similarly linked to these favorable outcomes. management of patients on noacs who are at risk for bleeding management of patients who overdose on noacs data regarding the prevalence of overdoses or unpre- scribed exposures to noacs are largely based on ob- servational data from poison control centers and case reports. , , , , – stevenson et al reported that be- tween january and july , there were calls to a single poison control center regarding dabigatran and rivaroxaban. of these, only cases were a result of self-harm, and only mild bleeding was reported in case. the majority of bleeding events were noted in pa- tients on long-term treatment and not acute ingestions, and there was no association with coagulation abnor- malities and risk of bleeding. conway et al reported dab- igatran exposures from a national poison control center and noted that adverse outcomes occurred in only % of all calls, and only . % were considered intentional. an observational study from poison control centers in states showed that among noac exposure calls related to rivaroxaban and apixaban ingestions, % had abnormal coagulation studies and no patient had bleeding. unfortunately, there is limited information to guide management of patients with noac overdose with and without bleeding. collection of information on the type of noac, the ingested dose, time of ingestion, concomitant renal/liver disease, and relevant medica- tion coingestion is critically important in the acute pe- riod. therapeutic management strategies in the acute care setting have largely been developed on the basis of clinical experience and an understanding of the phar- macology rather than trial data. management of patients with acute kidney injury on noacs the risk of acute kidney injury is high in the patient pop- ulation who are frequently prescribed noacs. andreu- cayuelas et al performed an observational study of patients with nvaf after hospitalization for acute heart failure. creatinine was measured during follow-up to de- termine the need for dose adjustment of the hypotheti- cal noacs. the investigators reported % of patients would have needed dabigatran dosage adjustment, % would have needed rivaroxaban adjustment, and % would have needed apixaban dosage adjustment. the patients with a baseline crcl of < ml/min or age > years were at greatest risk of needing a dose adjust- ment during follow-up. vka-associated nephropathy has recently been de- scribed as acute kidney injury with supratherapeutic in- ternational normalized ratio (inr) values with and without hematuria. – alternatively, noacs do not appear to be associated with kidney injury. in a meta-analysis conduct- ed by caldeira et al, noacs did not increase the risk of renal failure (relative risk [rr], . ; % ci, . – . ; i = . %; randomized controlled trials) when com- pared with a vka. a recent analysis of rocket af re- vealed a small but statistically significant decline in mean crcl± standard deviation among patients receiving war- farin (− . ± . ml/min) compared with patients re- ceiving rivaroxaban (− . ± . ml/min; p< . ). a post hoc analysis of the re-ly (randomized evaluation of long-term anticoagulation therapy) trial similarly re- vealed greater declines in crcl with warfarin compared with dabigatran. administering a noac in a patient with acute kidney injury increases the risk of bleeding. all noacs should be used with caution in patients on hemodialysis, given the limited data available. although a dosing recommen- dation for apixaban is provided for such patients in the product monograph, this recommendation is based on d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e pharmacokinetic data in fewer than patients. there are no efficacy or safety data in this patient population. until these data become available, close measurement or switching to an alternative anticoagulant is suggested for patients who develop acute kidney injury as a result of acute illness or injury. management of patients with ischemic stroke on noacs whereas noacs represent a major advance in stroke prevention, it is still anticipated that acute ischemic stroke (ais) will occur in % to % of individuals with nvaf treated with these agents each year. – their use presents a number of challenges for clinicians managing patients with ais, including appropriate measurement of anticoagulant activity in neurovascular emergencies, the role of thrombolysis and endovascular therapy in ais, and timing of reinstitution of oral anticoagulation after ais. thrombolytic therapy with intravenous recombinant tissue-type plasminogen activator within . hours of symptom onset is an established treatment in ais , but is associated with a > -fold increase in the rate of ich. because of the danger of further increasing ich, therapeutic anticoagulation is considered a contraindica- tion to thrombolytic therapy in ais. aha guidelines and observational data support intravenous thrombolysis in warfarin-treated patients provided the inr is no greater than . . , the data on safety of thrombolysis in the presence of low levels of anticoagulation with warfarin raises hope that the same may apply to noacs. determining appropriate treatment for ais patients re- ceiving noacs must balance the anticoagulant effect of these agents and the ich risk associated with reperfusion strategies. as has been mentioned previously, routinely performed blood coagulation studies do not reliably ex- clude a significant plasma concentration of the noacs. another difficulty in a time-sensitive setting is that the more sensitive blood tests are either not routinely avail- able or have an unacceptably long delay to results. in experimental studies, pretreatment with dabigatran or rivaroxaban did not increase the rate of thrombolysis- associated ich. , data on the safety and efficacy of intravenous thrombolysis in ais patients receiving no- acs are limited to approximately dozen case reports and a retrospective multicenter cohort study. among the case reports, ich and poor outcome were rarely report- ed when recombinant tissue-type plasminogen activator was administered minutes to hours after the last an- ticoagulant dose. , the cohort study comprised noac-treated patients undergoing intravenous throm- bolysis or intra-arterial therapy a median of hours after the last noac dose compared with warfarin- treated patients and on no anticoagulants. after propensity score matching, there was no significant dif- ference in rate of any ich, symptomatic ich, or death among the groups. in the absence of immediately avail- able blood tests sensitive to the presence of noacs, determining which patients taking these agents might be appropriate candidates for thrombolysis requires con- sideration of time from last dose, half-life of the agent used, and presence of impaired renal function that may reduce drug clearance. a new recommendation in the aha “guidelines for the early management of patients with acute ischemic stroke” is that recombinant tissue- type plasminogen activator should not be administered to patients who take noacs unless sensitive laboratory tests are normal or the patient has not received a dose of these agents for > hours. data guiding the use of endovascular therapy in ais patients who take noacs are even more limited. among the pivotal trials that established the safety and efficacy of mechanical thrombectomy in patients with ais and large vessel occlusion, patients receiving noacs were either excluded or not specifically reported. – a handful of case reports suggest safety of endovascular therapy in patients on dabigatran and rivaroxaban even in the setting of abnormal coagulation studies. – in the previously described cohort study, none of the pa- tients who underwent endovascular therapy with or with- out intravenous thrombolysis experienced a symptom- atic ich. reflecting the paucity of data in this area, the aha’s guidelines provide no recommendations regarding mechanical thrombectomy in patients whose use of anti- coagulant medications excludes them from intravenous thrombolysis. the optimal timing of restarting anticoagulation after ais presents another challenge to healthcare profession- als managing this population. meta-analysis of trials of parenteral anticoagulation started within hours of cardioembolic ischemic stroke and systematic review of trials involving participants with ais test- ing various parenteral and oral anticoagulants each con- cluded that while early anticoagulation is associated with a reduced risk of recurrent ischemic stroke, this benefit is entirely offset by an increased risk of symptomatic ich with no reduction in risk of death or dependency. the decision of when to restart oral anticoagulation must balance the competing risks of recurrent thrombo- embolic events and of hemorrhagic transformation. con- sideration is given to the type of event (transient isch- emic attack versus cerebral infarct), time from stroke onset, and presence of factors associated with increased hemorrhage risk (large infarct size, uncontrolled blood pressure, hyperglycemia, thrombocytopenia, previous hemorrhagic stroke, and thrombolytic treatment). , hemorrhagic transformation of ischemic brain tissue is a relatively common occurrence that is often asymp- tomatic or minimally symptomatic and uncommonly pro- gresses in extent in the absence of predisposing fac- tors. , assuming the hemorrhagic transformation is asymptomatic and remains stable, case series support d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines the safety of starting or continuing warfarin in carefully selected patients with a compelling indication. whether optimal timing of resumption of oral anticoagulation with noacs should follow similar recommendations is un- known. differences in the pharmacological properties of warfarin and the noac must be considered, notably the more rapid time to anticoagulant effect with the noacs (a few hours compared with to days for warfarin). in experimental models of ischemic stroke, neither dabiga- tran pretreatment nor continued administration of dabi- gatran after stroke onset significantly increased the risk or volume of hemorrhagic transformation after middle cerebral artery occlusion. , clinical data are anecdotal only. the phase iii trials establishing the role of noacs for stroke prevention in nvaf excluded patients within to days of stroke. – , in general, guidelines support withholding oral anticoagulation until to weeks after stroke among individuals with nvaf, with shorter times for those with transient ischemic attack or small, nondis- abling strokes and longer times for moderate to severe strokes. – in noac-treated patients who have an ais, compliance with noac therapy should be established and alternative causes for the stroke investigated. there are no data to indicate that increasing the intensity of anticoagulation, adding an antiplatelet agent, or switch- ing to another oral anticoagulant provides additional pro- tection against future ischemic events. because of the short half-lives of noacs and rapid decline of protective anticoagulation that occurs with missed doses, patients with poor compliance might be more appropriately man- aged with the longer-acting warfarin. transitioning between noacs and other anticoagulants in the acute care setting indications that require considerations for the transition- ing of anticoagulants in the acute care setting include the occurrence of a new clinical event (eg, myocardial infarction) in patients on established oral anticoagulant regimens, the development of a new or worsening co- morbid medical condition (eg, renal failure) that neces- sitates an anticoagulant transition and the need for an invasive procedure. in the united states, the current la- beled prescribing information for each noac provides guidance for the transition to and from noac agents to other anticoagulants; however, these suggestions are not specific for patients in the acute care setting (table ). , , , temporary interruptions in oral anticoagulation are commonly encountered in the acute care setting. on the basis of trial observations from noac agents in patients with af, approximately one third of af patients will ex- perience the need for a temporary interruption over the course of years. – the association of temporary interruptions in oral anticoagulant therapy with the risk for clinical events has been reported in of the clinical trials comparing noac agents to vkas in patients with af. – in addition, a meta-analysis using data from trials comparing the risk of thromboembolic events as- sociated with temporary discontinuation found no sta- tistically significant differences in the noac versus vka randomized groups (rr, . ; % ci, . – . ). whereas the majority of the temporary interruptions in the trials were around procedures, the use of peripro- cedural bridging regimens varied on the basis of pa- tient characteristics and trial protocols. only % and . % of patients with temporary oral anticoagulation interruption received bridging in rocket af and the aristotle trial (apixaban for reduction in stroke and other thromboembolic events in atrial fibrillation), re- spectively. , much of the clinical outcome data regarding the switching or transitioning between noac agents and other anticoagulants comes from the clinical trials in pa- tients with nvaf. observations from trials reported the risk of embolic and bleeding events in the noac and vka treatment groups associated with the transitions at the beginning and end of the trials have been published. a post hoc analysis of rocket af reported an increased risk of stroke in the rivaroxaban treatment group during the end-of-study transition to the open-label therapy pe- riod. patients who received rivaroxaban compared with those who received warfarin were observed to have an increased incidence of stroke during the period of transi- tion ( to days after the end of the study) to open- label therapy (n= versus n= ; hazard ratio [hr], . ; % ci, . – . ) as well as a greater proportion of major bleeding events (n= versus n= ; hr, . ; % ci, . – . ). , in addition, during the end-of-study transition period, the median time to first therapeutic inr was days in the warfarin treatment group compared with days in the rivaroxaban treatment group. simi- lar observations of an increased risk of clinical events in those assigned to noac therapy have been reported from the aristotle trial end-of-study open-label transi- tion period. at the end of aristotle, a -day bridging period with apixaban or apixaban placebo was recom- mended during the initiation of open-label vkas. during the first days after stopping blinded study drug, stroke or systemic embolism events were noted in the apixaban group versus in the warfarin group (adjust- ed hr, . ; % ci, . – . ). an excess in major bleeding events was also observed during this period in the apixaban versus warfarin groups (n= versus n= ; adjusted hr, . ; % ci, . – . ). on the basis of these observations, an end-of-study transition plan was designed for patients enrolled in the engage af (effec- tive anticoagulation with factor xa next generation in atrial fibrillation) trial. in brief, for patients who were planned to transition to open-label noac therapy, mea- d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e surement of inr was conducted and the open-label noac was initiated when the inr was < . . for patients transi- tioning to a vka, a -day kit was provided that included a vka algorithm and a modified dose of edoxaban, which was to be continued until day or an open-label inr ≥ . , whichever occurred first. within days of study drug discontinuation, strokes were observed to occur in patients in each of the study treatment groups with major bleeding events noted in patients in the warfarin group, patients in the edoxaban high-dose table . overview of us-labeled guidance for noac anticoagulant transitions noac vka intravenous anticoagulant lmwh/other noac apixaban apixaban→warfarin: discontinue apixaban and begin a parenteral anticoagulant and warfarin at the time the next scheduled apixaban dose would have been taken apixaban→parenteral anticoagulant: discontinue apixaban and begin the new anticoagulant at the usual time of the next dose of apixaban apixaban→lmwh/other noac: discontinue apixaban and begin the lmwh/other noac at the usual time of the next dose of apixaban warfarin→apixaban: discontinue warfarin and start apixaban when inr < . lmwh/other noac→apixaban: discontinue current noac/lmwh and begin apixaban at the usual time of the next dose of the other noac/lmwh dabigatran dabigatran→warfarin: for crcl ≥ ml/min, start warfarin d before discontinuing dabigatran for crcl – ml/min, start warfarin d before discontinuing dabigatran for crcl – ml/min, start warfarin d before discontinuing dabigatran dabigatran→parenteral anticoagulant: wait h (crcl ≥ ml/min) or h (crcl < ml/min) after last dabigatran dose before initiating a parenteral anticoagulant dabigatran→lmwh: wait h (crcl > ml/min) or h (crcl < ml/min) after last dabigatran dose before initiating a parenteral anticoagulant warfarin→dabigatran: discontinue warfarin and start dabigatran when inr < . ufh→dabigatran: start dabigatran at the time of continuous infusion discontinuation lmwh→dabigatran: start dabigatran – h before the time that the next lmwh dose would have been given edoxaban edoxaban→warfarin: oral option: reduce daily edoxaban dose by % and begin taking warfarin concomitantly. measure inr at least weekly just before daily edoxaban dose. once a stable inr ≥ . is achieved, discontinue edoxaban and continue warfarin parenteral option: discontinue edoxaban and administer a parenteral anticoagulant and warfarin at the time of the next scheduled edoxaban dose. edoxaban→parenteral anticoagulant: discontinue edoxaban and start the parenteral anticoagulant at the time of the next scheduled dose of edoxaban edoxaban→lmwh/other noac: discontinue edoxaban and start the lmwh/other noac at the time of the next scheduled dose of edoxaban warfarin→edoxaban: discontinue warfarin and start edoxaban when the inr is < . ufh→edoxaban: discontinue ufh infusion and start edoxaban h later lmwh/other noac→edoxaban: discontinue current noac/lmwh and start edoxaban at the time of the next scheduled other noac/lmwh dose rivaroxaban rivaroxaban→warfarin: discontinue rivaroxaban and begin a parenteral anticoagulant and warfarin at the time the next scheduled rivaroxaban dose would have been taken rivaroxaban→ufh: discontinue rivaroxaban and initiate the parenteral anticoagulant at the time the next rivaroxaban dose would have been taken rivaroxaban→lmwh/other noac: discontinue rivaroxaban and start the lmwh/other noac at the time of the next scheduled dose of rivaroxaban warfarin→rivaroxaban: discontinue warfarin and start rivaroxaban as soon as inr < . ufh→rivaroxaban: stop ufh infusion and administer rivaroxaban at the same time lmwh/other noac→rivaroxaban: start rivaroxaban – h before the next scheduled evening lmwh/other noac dose and omit administration of the lmwh/other noac crcl indicates creatinine clearance; inr, international normalized ratio; lmwh, low-molecular-weight heparin; noac, non–vitamin k antagonist oral anticoagulant; ufh, unfractionated heparin; and vka, vitamin k antagonist. d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines group, and patients in the edoxaban low-dose group. no statistically significant differences were observed in primary efficacy or safety events among the treatment groups in patients transitioning to open-label vkas or in those transitioning to open-label noacs. it is notable that in patients transitioning to open-label vkas, % had at least inr ≥ by day . registry data on the outcomes of ambulatory af pa- tients transitioning from a vka to a noac (dabigatran or rivaroxaban) have also been published. , in a matched-cohort study of af patients, there was no as- sociation of transitioning from a vka to either dabigatran or rivaroxaban compared with remaining on vka therapy for embolic or bleeding events at a median follow-up of months. data from a large regional prospective registry showed clinical events were relatively infrequent in the -day period after vka to noac transitions de- spite only % of patients having an inr measurement before noac initiation. although clinical decisions regarding the transition between anticoagulants in the acute care setting are likely to be affected by a number of factors, careful consideration should be given to strategies that mini- mize prolonged durations of both subtherapeutic and excessive anticoagulation during the transition periods. given the relatively infrequent use of periprocedural bridging strategies during temporary interruptions in the clinical trials, clinical consideration should be given to managing patients experiencing temporary inter- ruptions without bridging, as outlined in the individual noac trials. periprocedural management of patients who take noacs each year, ≈ % of patients on any long-term oral antico- agulation require surgery or other invasive procedures. approximately % of patients on warfarin undergo sur- gery that has an extremely low risk of bleeding such as mi- nor dental, dermatologic, or ophthalmologic procedures where anticoagulation may be safely continued without interruption. it is recommended that warfarin be held for days before surgery when significant bleeding is anticipated and then reinitiated postoperatively when he- mostasis is secured. pre- and postoperative bridging using low-molecular-weight heparin is recommended for those patients with high thrombosis risk, such as those with certain mechanical valve prostheses or recent pul- monary embolism. in patients at low to intermediate risk of thrombosis, bridging low-molecular-weight heparin does not prevent thrombotic events and increases bleed- ing events (figure ). therefore, bridging anticoagula- tion is not necessary in this subgroup of patients. the limited data available pertaining to patients on noac therapy who require surgery suggest that the perioperative bleeding risk is low for nonurgent sur- gery. the dresden noac registry prospectively evalu- ated patients taking noacs, of which pa- tients ( . %) underwent invasive procedures; most were not urgent. invasive procedures were cat- egorized as major or minor, and a bleeding event was categorized as major, clinically relevant nonmajor, or minor per the international society of thrombosis and figure . periprocedural management of patients on noacs (non–vitamin k antagonist oral anticoagulants). crcl indicates creatinine clearance; icd, implantable cardioverter-defibrillator; pt, prothrombin time; svt, supraventricular tachy- cardia; te, thromboembolic event; tia, transient ischemic attack; and vte, venous thromboembolism. *bridging may be considered in patients with a history of systemic embolus in the last weeks. a d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e haemostasis definition. of the entire cohort, only patients ( . %) experienced any bleeding complication up to ± days after the procedure. major bleeding occurred in of ( . %) procedures. clinically relevant nonmajor bleeding occurred in patients ( . %) and minor bleeding occurred in only patients ( . %). periprocedural bleeding was studied in a sub- group analysis of the rely trial which compared war- farin to dabigatran for stroke prevention in nvaf. procedures were classified as being associated with a low (coronary angiography, defibrillator implantation) or high risk of bleeding (cardiac, abdominal, and neu- rosurgery, or procedures requiring spinal anesthesia). there was no significant difference in the rates of peri- procedural major bleeding between patients who re- ceived dabigatran mg ( . %), dabigatran mg ( . %), or warfarin ( . %); dabigatran mg versus warfarin: rr, . ; % ci, . to . ; p= . ; dabigatran mg versus warfarin: rr, . ; % ci, . to . ; p= . . among patients who had ur- gent surgery, major bleeding was increased, occurring in . % with dabigatran mg, . % with dabi- gatran mg, and . % with warfarin: dabigatran mg: rr, . ; % ci, . to . ; p= . ; dabigatran mg: rr, . ; % ci, . to . ; p= . . tailoring periprocedural noac management to the type of invasive procedure may mitigate against bleeding. common clinical scenarios are subsequently discussed. cardiac catheterization and percutaneous coronary intervention patients with af commonly have coexisting coronary artery disease with an estimated % requiring per- cutaneous coronary intervention (pci). the american college of cardiology/society for cardovas- cular angiography and interventions consensus docu- ment recommends that elective coronary angiography for patients on long-term warfarin be deferred until the inr is . for femoral artery access or < . for radial artery access. unfortunately, there are very limited data that address the management of patients on a noac who require cardiac catheterization or pci. pre-, peri-, and postprocedural considerations are subse- quently discussed. preprocedural considerations patients with stable ischemic heart disease with ischemic symptoms despite medical therapy or with intermediate- or high-risk features on stress testing are often referred for coronary angiography and possible pci. patients with stable ischemic heart disease on a noac and who are not at high thrombosis risk should have the noac held until the antico- agulation effect is dissipated before undergoing coronary angiography and pci. from the prescribing information, dabigatran should be held for at least hours if crcl ≥ ml/min; for at least hours if crcl < ml/min; rivar- oxaban, apixaban and edoxaban should be held for at least hours. , , , in the absence of high risk features, patients should not be bridged with a heparin before or after the proce- dure. the decision to resume antithrombotic therapy after the procedure should be guided by the throm- boembolic risk as assessed by the cha ds -vasc score. clinicians need to consider which antithrom- botic and antiplatelet agents to resume and the du- ration of antiplatelet therapy, balancing ischemic and thrombotic events while minimizing the hemorrhagic complications. patients presenting with an acute coronary syn- drome (acs) often undergo coronary angiography and revascularization to reduce their risk of recurrent events, especially if they have an elevated thromboly- sis in myocardial infarction (timi) risk score. where- as patients with unstable angina or a non–st-segment elevation myocardial infarction do not require im- mediate angiography, patients presenting with a st- segment elevation myocardial infarction require emer- gency coronary angiography and revascularization of the infarct related artery. for the unstable angina/ non–st-segment elevation myocardial infarction pa- tient, appropriate dual antiplatelet therapy (dapt) and heparin therapy should be started upstream, the noac should be discontinued and the patient should be scheduled for an urgent catheterization. in the ab- sence of electrical or hemodynamic instability, it is reasonable to wait for the effects of the noac to dissi- pate and then perform the procedure through a radial artery approach. periprocedural considerations patients on noacs undergoing coronary angiography or pci will have an increased risk of hemorrhagic com- plications, and therefore, careful attention should be made to choice of vascular access site and use of ad- junctive anticoagulants. patients should undergo radial artery access, unless there is a contraindication, be- cause the risk of bleeding and vascular complications is reduced as compared with a femoral approach. if a femoral approach is required, one should consider using ultrasonography and fluoroscopy to guide vas- cular access. a micropuncture needle technique may decrease the probability of a retroperitoneal bleed. although no data exist, it may be reasonable to use a vascular closure device to assist with postproce- dure hemostasis if the patient has amenable vascular anatomy. venous access should be avoided unless absolutely required. all patients undergoing pci re- quire antiplatelet therapy coupled with either heparin or bivalirudin to reduce the periprocedural thrombotic complication rates, irrespective of background use of d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines vkas or noacs. the use of intravenous glycoprotein agents should be discouraged and reserved for bailout scenarios. for patients who receive intravenous hepa- rin, one should use low-dose heparin regimens with an activated clotting time goal of ≈ seconds to re- duce hemorrhagic complications. postprocedural considerations the clinician should consider the patient’s risk of recur- rent myocardial infarction, stent thrombosis, thromboem- bolic risk, and hemorrhagic complications when selecting anticoagulants. it is helpful to use the cha ds -vasc risk score to estimate the thromboembolic risk and the has- bled risk score to estimate the hemorrhagic risk and in- clude the patient in a shared decision regarding the selec- tion of dapt versus triple therapy as well as the duration of therapy. several themes have emerged. the standard of care to reduce coronary ischemic events post-pci and post-acs is dapt. the duration of dapt is directly impact- ed by the stent type (bare metal stent versus drug-eluting stent) and whether the patient underwent pci for stable ischemic heart disease or acs. however, oral anti- thrombotic agents (not antiplatelet agents) are required to prevent nvaf related stroke or vte. therefore, the clini- cian is faced with the consideration of dapt, dapt plus warfarin (triple therapy), dapt plus a noac (triple therapy) or warfarin plus single antiplatelet therapy. in a phase ii study, triple therapy with dabigatran in patients with acs was associated with an increased risk of bleeding complications and planned phase iii trials were not pursued. in a randomized clinical trial of patients with acs, apixaban increased bleeding without reducing ischemic event in patients on either dapt or aspirin alone. intracranial bleed rates were in- creased in patients treated with apixaban. because of concerns regarding safety without a signal of efficacy, the trial was terminated. rivaroxaban was studied in the atlas acs-timi (anti-xa therapy to lower cardiovascular events in addition to aspirin with or without thienopyridine therapy in subjects with acute coronary syndrome—thrombolysis in myocardial in- farction) trial, which compared rivaroxaban or placebo in addition to standard acs therapies. compared with placebo, rivaroxaban ( . mg twice daily and . mg twice daily) decreased the rates of the composite pri- mary end point including cardiovascular death, myo- cardial infarction or stroke ( . % versus . %) while increasing the rates of bleeding (non–coronary artery bypass graft surgery) and ich. only rivaroxaban cou- pled with dapt has been demonstrated to reduce isch- emic events at a cost of increased bleeding. however, the studied doses of rivaroxaban are not the doses proven to reduce the risk of thromboembolic events secondary to af. european and canadian guidelines suggest noacs are preferred over warfarin when it comes to triple ther- apy. however, these recommendations are based on observational data and post hoc analysis of warfarin vs. noac studies with limited number of patients. for ex- ample, in rocket af, only % of patients underwent pci during the trial. until further prospective, randomized trial data become available on the subject, the aha writ- ing group suggests that clinicians use good judgment, weighing the risk/benefits of noacs in the context of triple therapy for their patients. proton pump inhibitors decrease the rates of upper gastrointestinal bleeding in patients with dapt and in pa- tients with dapt and antithrombotic therapy. patients should be advised to avoid nonsteroidal anti-inflammato- ry medications as the risks of myocardial infarction and hemorrhagic complications are increased. ongoing ran- domized trials (pioneer af-pci [open-label, randomized, controlled, multicenter study exploring two treatment strategies of rivaroxaban and a dose-adjusted oral vi- tamin k antagonist treatment strategy in subjects with atrial fibrillation who undergo percutaneous coronary intervention], re-dual pci [evaluation of dual therapy with dabigatran vs. triple therapy with warfarin in pa- tients with af that undergo a pci with stenting], rt-af [rivaroxaban in patients with atrial fibrillation and coro- nary artery disease undergoing percutaneous coronary intervention], safe-a [safety and effectiveness trial of apixaban use in association with dual antiplatelet thera- py in patients with atrial fibrillation undergoing percuta- neous coronary intervention] and augustus [a study of apixaban in patients with atrial fibrillation, not caused by a heart valve problem, who are at risk for thrombo- sis (blood clots) due to having had a recent coronary event, such as a heart attack or a procedure to open the vessels of the heart]) will assess the efficacy of a noac coupled with antiplatelet therapy in patients undergoing pci. until these trials are completed and published, the writing group makes the following suggestions: . for patients with a cha ds -vasc score of or treated with pci, it is reasonable to omit anticoagu- lant therapy and treat with dapt. . for patients who require dapt or triple therapy, use low-dose aspirin, mg daily. . for stable ischemic heart disease patients who require anticoagulant therapy and treatment with pci, discontinuation of p y inhibitor therapy after months may be reasonable. . for acs patients requiring anticoagulant therapy and treatment with pci (bare metal stent or drug- eluting stent), continuation of aspirin mg daily for year and discontinuation of p y therapy after months may be reasonable. . for patients with a moderate to high risk of bleeding, as assessed by the has-bled score, a shortened duration of triple therapy or warfarin plus clopidogrel may be considered based on the exploratory woest (what is the optimal antiplatelet d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e and anticoagulant therapy in patients with oral anticoagulation and coronary stenting) trial. . prasugrel and ticagrelor should not be used in conjunction with noacs, as a result of excessive bleeding risk. . at present, there are limited data to recommend the routine use of noacs, coupled with clopidogrel alone or dapt after pci. of note, in clinical prac- tice, it can be challenging to reach and maintain therapeutic warfarin levels in certain patients. in these patients, it may be reasonable to combine a noac and clopidogrel after pci. cardioversion of af post hoc analyses from pivotal noac clinical trials have not shown significant differences in outcomes after car- dioversion in those treated with noacs compared with warfarin. – meta-analysis of events across random- ized trials appears to confirm these results, finding no significant difference in stroke/systemic embolism (odds ratio, . ; % ci, . – . ) or major/non- major clinical relevant international society on throm- bosis and haemostasis bleeding events (odds ratio, . ; % ci, . – . ) after cardioversion. , moreover, there is randomized clinical trial of car- dioversion in patients treated with a factor xa inhibitor versus warfarin. more than patients undergoing early (target period of to days after randomiza- tion with transesophageal echocardiography [tee]) or delayed ( to weeks) cardioversion were random- ized in a : fashion to rivaroxaban or warfarin. the primary efficacy end point (composite of stroke, tran- sient ischemic attack, peripheral embolism, myocardial infarction, and cardiovascular death) occurred in . % of the rivaroxaban patients versus . % of the vka- treated patients (rr, . ; % ci, . – . ) with no significant difference in bleeding observed. observational data from clinical practice demonstrate similar findings. data from a large nationwide cohort study demonstrated no difference between outcomes in those treated with dabigatran versus warfarin. in patients un- dergoing cardioversion, the cumulative incidence of stroke, bleeding or death at weeks was . % in those treated with warfarin and . % in those treated with dabigatran (ad- justed hr, . ; % ci, . – . ). high-volume single- center data (> cardioversions) have also failed to iden- tify any difference in postcardioversion thromboembolic or bleeding events across warfarin and noac agents. the ensure-af trial (edoxaban versus enoxaparin- warfarin in patients undergoing cardioversion of atrial fibrillation) randomized patients to either edoxaban or enoxaparin/warfarin during tee or non-tee guided electrical cardioversion. for tee-guided cardioversion, randomization occurred < days from cardioversion and study treatment was continued for at least days. for non–tee-guided cardioversion, study treatment was initiat- ed at least days before cardioversion and extended for at least days. the primary efficacy end point (compos- ite of stroke, systemic embolic event, myocardial infarc- tion, and cardiovascular mortality) and the primary safety end point (major and clinically relevant nonmajor bleeding) occurred at a statistically similar frequency. edoxaban may be an effective and safe alternative to enoxaparin/warfarin for patients with nvaf requiring cardioversion. several practical considerations must be weighed when cardioverting patients on noac therapy with af duration > hours. similar to recommendations with warfarin, pa- tients should be anticoagulated for a minimum of weeks before elective cardioversion. if not, then a tee should be performed to exclude the presence of left atrial append- age or left atrial thrombus. similarly, if a given patient’s adherence to therapy is suboptimal (≥ missed doses) or in question, then a tee should be considered. if a pa- tient has been on a properly dosed noac with weeks of therapy and is found to have left atrial appendage or left atrial thrombus, then consideration should be given to switching to an alternate anticoagulant with special atten- tion to consistent anticoagulant use during the transition. catheter ablation of af catheter ablation is an increasingly used treatment option for rhythm control in nvaf. because of the risks of peri- procedural thromboembolism, anticoagulation is required during the procedure. however, the presence of anticoag- ulation can make the management of bleeding complica- tions more difficult. before the advent of noac therapy, observational , and randomized studies suggested that uninterrupted vka therapy was associated with su- perior outcomes compared with vka interruption with intraprocedural heparin. in particular, the compare (role of coumadin in preventing thromboembolism in atrial fi- brillation [af] patients undergoing catheter ablation) clini- cal trial randomized patients to interrupted warfarin with bridging anticoagulation (n= ) versus continuous warfarin (n= ). bleeding events were less common in the continuous warfarin arm with no significant difference in stroke or transient ischemic attack ( . % versus . % major bleeding, . % versus . % pericardial effusion, and % versus % minor bleeding). how interrupted/continuous noac therapy compares to continuous warfarin has been the subject of intense study over the past years. multiple systematic assess- ments and meta-analyses have demonstrated similar outcomes in patients treated with noacs (interrupted or continuous) versus continuous warfarin. – one randomized study compared uninterrupted rivaroxaban and vka in patients. the occurrence of any throm- boembolic events ( versus ) and bleeding events ( versus ) was similar in the uninterrupted rivaroxa- ban and vka arms. although the study was relatively d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines small with limited power, the results were largely in line with previous observational data that have suggested similar outcomes with noac and vka therapy. another randomized study compared uninterrupted apixaban versus continuous warfarin in subjects with drug- refractory af undergoing ablation and found no differ- ence in thromboembolic or bleeding outcomes. there are several ongoing larger randomized clinical trials of interrupted versus uninterrupted noac therapy and con- tinuous warfarin versus continuous noac therapy. the recommendation to use tee to exclude the pres- ence of left atrial appendage/left atrial thrombus should be similar regardless of whether vka or noac therapy is used. if the patient has not had to weeks of pre- procedural anticoagulation or if the patient is considered at increased risk for stroke, the use of tee is mandatory. however, many laboratories conduct a tee in all patients before ablation since thrombus can be observed even in low-risk patients with paroxysmal af. regardless of whether continuous or interrupted noac therapy is used, on the basis of current consensus recom- mendations, patients should be heparinized with -u/ kg bolus followed by an infusion of u/kg/hour before or immediately after puncture. the activated clotting time should be checked every to minutes until target and then every minutes thereafter. the activated clot- ting time target should be at least to seconds or to seconds in the case of spontaneous echocar- diographic contrast (“smoke”) or severe left atrial enlarge- ment. heparinization before transeptal access may be associated with a lower risk of asymptomatic microembol- ic events as detected by brain magnetic resonance imag- ing. it is important to note that the use of noac therapy before and during the procedure results in the need for an increased dose of heparin to achieve target activated clotting times during the ablation procedure. after the procedure, noac therapy is generally reintroduced within to hours after sheath removal if access site hemo- stasis has been achieved. consistent with consensus rec- ommendations, noac therapy should be continued for a minimum of to months after ablation. thereafter, oral anticoagulation should be based on the patient’s underly- ing risk for stroke (cha ds -vasc score) rather than the current rhythm. electronic device implantation management of oral anticoagulation surrounding cardiac implantable electronic device insertion presents several challenges. oral anticoagulation increases the risk of bleeding and hematoma formation after device implan- tation. furthermore, hematoma formation increases the risk of postoperative infection. based upon the results from randomized clinical trials, uninterrupted warfarin has been shown to lead to less bleeding and superior outcomes compared with interrupted warfarin and par- enteral bridging therapy. , these findings are also consistent with the bridge (perioperative bridging anti- coagulation in patients with atrial fibrillation) trial, which found no significant benefit to bridging for general inter- ruption of oral anticoagulation for invasive procedures in patients with nvaf. however, the optimal manage- ment of noac therapy surrounding cardiac implantable electronic device implantation remains unknown. in general, discontinuation of noac therapy before cardiac implantable electronic device implantation in a manner consistent with the elimination half-life is the most common practice pattern. for apixaban, edoxa- ban, and rivaroxaban, this would include discontinuation hours in advance of the procedure. in the case of dabigatran, discontinuation is recommended hours before in patients with a crcl ≥ ml/min, hours be- fore in those with crcl to ml/min, and hours before in those with a crcl < ml/min. survey data from implanting physicians suggest wide variation in practice patterns reflecting the uncertainly over optimal management. however, the majority of physicians discontinue noac therapy at the time of implantation ( %). although uninterrupted warfarin has the best evidence base (> randomized trial), an increasing num- ber of cardiac implantable electronic device patients are taking noac therapy. whether noac therapy can be continued through cardiac implantable electronic de- vice implantation remains debated and is the subject of a large clinical trial (bruise control- [strategy of continued vs interrupted novel oral anticoagulant at time of device surgery in patients with moderate to high risk of arterial thromboembolic events] study) in which perioperative management will be randomized to a strategy of continued versus interrupted noac thera- py. the few available observational data are limited by their small cohort size but have not identified significant risks of bleeding with uninterrupted noac therapy. , when a decision is made to interrupt noac therapy for cardiac implantable electronic device implantation, the implanting physician must decide when the noac therapy should be restarted. this decision is often influ- enced by patient characteristics, including risk factors for bleeding and the postimplantation physical examina- tion (eg, hematoma). similar to discontinuation, prac- tice patterns regarding resumption of noac therapy after implantation are highly variable. typically, noac therapy was restarted to hours after surgical pro- cedures in the pivotal noac trials. patients with multiple risk factors for bleeding, concomitant antiplatelet ther- apy, or evidence of hematoma on their postoperative examination may benefit from a greater delay to noac resumption ( to days). however, given the lack of evidence to guide these decisions, management should be approached on a patient-by-patient basis, weighing the risks and benefits of earlier versus later resumption of noac therapy. d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e cardiovascular surgery there is limited information regarding the use of noacs in coronary artery bypass grafting or valve replacement surgery. at present, information related to perioperative noac use in cardiac surgery is anecdotal or based on limited subset analyses. , , no significant bleeding event differences were observed between rivaroxaban and warfarin treated patients who underwent cardiac surgery in rocket af. a the atlas acs- -timi- trial tested rivaroxaban to lower cardiovascular events in patients with acs and reported patients undergoing coronary artery bypass grafting after st-segment eleva- tion myocardial infarction. , per the trial protocol, the drug was stopped hours before the procedure and resumed hours after the postprocedural drains were removed or after the last dose of parenteral anticoagu- lant therapy had been administered. the results specific to this group were not reported; therefore, no conclu- sions regarding coronary artery bypass grafting–related care can be made. established indications for noacs in the pericardiac surgery setting include stroke prevention in preoperative af, prolonged or frequent postoperative af, and vte treatment. noac use is contraindicated in patients with mechanical valves; as the re-align (the randomized, phase ii study to evaluate the safety and pharmacoki- netics of oral dabigatran etexilate in patients after heart valve replacement) trial with dabigatran demonstrated, there is an increased rate of thromboembolic and bleed- ing complications compared with warfarin. there are case reports of dabigatran use after left ventricular assist device placement and of rivaroxaban use for heparin induced thrombocytopenia after coronary artery bypass grafting. however, these off-label uses are not supported by available clinical trial evidence. for cardiac surgery, noacs should be stopped in the perioperative setting and restarted after clinical hemostasis has been established. as cardiac surgery is considered a high-bleeding-risk procedure, surgery should be postponed if at all possible until after the appropriate interruption period. bleeding after car- diac surgery should be monitored via standard post- procedure drains. life-threatening bleeding should be treated with supportive therapy, including transfusion of blood products and administration of antifibrinolyt- ics as indicated for hemorrhage resuscitation, and return to the operating room. if contributing to an on- going coagulopathy, administration of noac antidotes as previously described (laboratory measurement of noac effect) could be considered. mild bleeding may be monitored, but noacs should not be reinitiated until there is bleeding control. similarly, the published experience of noac manage- ment in patients undergoing vascular surgery is limited to case reports and very small trial subsets. , , in a subgroup analysis of rocket af, patients with periph- eral artery disease on rivaroxaban had a higher risk of major bleeding and nonmajor clinically relevant bleeding compared with warfarin. noncardiovascular surgery studies examining outcomes among noac users af- ter noncardiovascular surgery largely grouped patients into cohorts spanning minor to major high-risk sur- gery. , , , noacs do not increase the rate of post- operative bleeding events when compared with warfarin. a pooled analysis of dabigatran phase iii trial bleeding data demonstrated no difference in postoperative bleed- ing events between patients on dabigatran and warfa- rin. in the aristotle trial, there was no difference in stroke, myocardial infarction, mortality, or bleeding for patients on apixaban versus warfarin for nvaf. how- ever, small differences may not have been detected as only . % of procedures in this trial were considered emergent and only . % of procedures were consid- ered major. bridging therapy is not recommended during noac therapy interruption for patients undergoing surgery. the dabigatran re-ly study demonstrated an increased risk for major bleeding with bridging therapy. non- bridged patients had a thromboembolic event risk of . %. analysis of periprocedural dabigatran use in the re-ly trial demonstrated no difference in major bleed- ing events between urgent versus elective surgery and major versus minor surgery. there was also no difference in fatal bleeding, reoperation as a result of bleeding, or transfusion requirements. there were few- er bleeding events in patients with shorter interruption periods, though this may not be a causal relationship given that shorter interruptions may indicate patients with characteristics of faster drug clearance. in con- trast, analysis of the dresden noac registry demon- strated increased risk of bleeding in patients with major procedures. heparin bridging still did not reduce car- diovascular events and did not statistically affect bleed- ing risk once the data were adjusted for major versus minor procedures. in the canadian dabigatran cohort study, none of the patients received preoperative bridging, and only . % of patients received postopera- tive heparin or low-molecular-weight heparin. despite this, there was only transient ischemic attack event ( . %) and no major arterial thromboembolic events. in the aristotle study, . % of procedures did not re- quire noac interruption and . % of patients received bridging anticoagulation. in phase iii trials of noac use for vte prevention in high-bleeding-risk orthopedic surgery, the first pro- phylactic dose was administered to hours post- operatively. real-world registries of noac use after orthopedic surgery suggest higher rates of bleeding d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines compared with those observed in the trials. in the dres- den noac registry, out of patients undergoing major orthopedic surgery developed major cardiovas- cular (n= ) or bleeding events (n= ). in the canadian dabigatran cohort, out of patients undergoing major orthopedic surgery developed major bleeding complications. caution should be exercised in man- aging patients on noacs who require major orthopedic interventions. neuraxial anesthesia spinal or epidural hematoma can be a devastating com- plication of neuraxial anesthesia. there are limited data pertaining to the interval between the discontinuation of noacs, the neuraxial anesthesia procedure itself, and subsequent resumption of the noac. rivaroxaban to prevent vte after total knee joint replacement or total hip arthroplasty with neuraxial anesthesia has also been examined. in an analysis of trials, neuraxial hematoma occurred in only of patients in the rivaroxaban group and this occurred before drug administration. of the patients who underwent neuraxial anesthe- sia in the rivaroxaban group in a phase iv cohort study, patient developed intraspinal/hemorrhagic puncture. these data suggest that the incidence of neuraxial he- matoma is low despite concurrent administration of therapeutic doses of a noac. there are no robust clinical outcomes data to address the timing and safety of noac discontinuation and rein- stitution. the american society of regional anesthesia and european society of regional anesthesia and pain therapy recommend stopping dabigatran to days before neuraxial block. for patients with end-stage renal disease, days off dabigatran is recommended. for patients with high risk of vte, dabigatran may be administered hours after the pain intervention. this group recommends stopping apixaban and rivaroxaban to days before neuroaxial block, and resuming either drug hours after the pain intervention if the risk of vte is considered high. no guidance on edoxaban was considered in this document. these recommendations are controversial because discontinuation periods of ≥ days are inconsistent with the return to hemostasis time of these agents, which may expose patients to excess thromboembolic risk. conclusion noacs are no longer novel and are now commonly used in day-to-day medical practice. healthcare providers are encouraged to use well-defined protocols established in collaborations with multiple professional disciplines to address noac dose and continuation or cessation when invasive procedures are required. such protocols should also be encouraged to assist acute care providers who manage bleeding while patients take noacs. simple to administer antidotes are either approved for use, such as idarucizumab for dabigatran, or are currently under investigation. further studies that measure clinical out- comes after noac reversal are needed to optimize pro- tocols for noac-associated bleeding and periprocedural noac management. acknowledgments the aha writing group thanks taylor klein for assistance in reference formatting and anne leonard and connie land for administrative assistance. footnotes the american heart association makes every effort to avoid any actual or potential conflicts of interest that may arise as a result of an outside relationship or a personal, professional, or business interest of a member of the writing panel. specifically, all members of the writing group are required to complete and submit a disclosure questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest. this statement was approved by the american heart asso- ciation science advisory and coordinating committee on octo- ber , , and the american heart association executive committee on october , . a copy of the document is available at http://professional.heart.org/statements by using either “search for guidelines & statements” or the “browse by topic” area. to purchase additional reprints, call - - or e-mail kelle.ramsay@wolterskluwer.com. the american heart association requests that this docu- ment be cited as follows: raval an, cigarroa je, chung mk, diaz-sandoval lj, diercks d, piccini jp, jung hs, washam jb, welch bg, zazulia ar, collins sp; on behalf of the american heart association clinical pharmacology subcommittee of the acute cardiac care and general cardiology committee of the council on clinical cardiology; council on cardiovascu- lar disease in the young; and council on quality of care and outcomes research. management of patients on non–vitamin k antagonist oral anticoagulants in the acute care and peri- procedural setting: a scientific statement from the american heart association. circulation. ; :e –e . doi: . /cir. . expert peer review of aha scientific statements is conducted by the aha office of science operations. for more on aha state- ments and guidelines development, visit http://professional. heart.org/statements. select the “guidelines & statements” drop-down menu, then click “publication development.” permissions: multiple copies, modification, alteration, en- hancement, and/or distribution of this document are not permit- ted without the express permission of the american heart asso- ciation. instructions for obtaining permission are located at http:// www.heart.org/heartorg/general/copyright-permission- guidelines_ucm_ _article.jsp. a link to the “copyright per- missions request form” appears on the right side of the page. circulation is available at http://circ.ahajournals.org. d ow nloaded from http://ahajournals.org by on a pril , mailto:kelle.ramsay@wolterskluwer.com raval et al march , circulation. ; :e –e . doi: . /cir. e writing group disclosures writing group member employment research grant other research support speakers’ bureau/ honoraria expert witness ownership interest consultant/ advisory board other amish n. raval university of wisconsin none none none none none none none sean p. collins vanderbilt university college of medicine none none none none none none none mina k. chung cleveland clinic none none none none none none none joaquin e. cigarroa oregon health and science university none none none none none none none larry j. diaz- sandoval michigan state university none none none none none none none deborah diercks ut southwestern emergency medicine none johnson & johnson* (steering committee member on a study about the treatment of pe using noacs) none none none none none hee soo jung university of wisconsin nih(national center for advancing translational sciences)*; national board of medical examiners* none none none none none none jonathan p. piccini duke university janssen pharmaceuticals† none none none none janssen pharmaceuticals*; medtronic*; bms pfizer* none jeffrey b. washam duke university heart center none none none none none none none babu g. welch ut southwestern medical center st. paul medical foundation† none none none none stryker neurovascular*; covidien* none allyson r. zazulia washington university none none none none none none none this table represents the relationships of writing group members that may be perceived as actual or reasonably perceived conflicts of interest as reported on the disclosure questionnaire, which all members of the writing group are required to complete and submit. a relationship is considered to be “significant” if (a) the person receives $ or more during any -month period, or % or more of the person’s gross income; or (b) the person owns % or more of the voting stock or share of the entity, or owns $ or more of the fair market value of the entity. a relationship is considered to be “modest” if it is less than “significant” under the preceding definition. *modest. †significant. disclosures d ow nloaded from http://ahajournals.org by on a pril , management of patients on noacs in the acute care and periprocedural setting circulation. ; :e –e . doi: . /cir. march , e clinical statem ents and guidelines references . barnes gd, ageno w, ansell j, kaatz s; subcommittee on the control of anticoagulation of the international society on throm- bosis and haemostasis. recommendation on the nomenclature for oral anticoagulants: communication from the ssc of the isth. j thromb haemost. ; : – . doi: . / jth. . . connolly sj, ezekowitz md, yusuf s, eikelboom j, oldgren j, parekh a, pogue j, reilly pa, themeles e, varrone j, wang s, alings m, xavier d, zhu j, diaz r, lewis bs, darius h, diener h-c, joyner cd, wallentin l; re-ly steering committee and investigators. dabi- gatran versus warfarin in patients with atrial fibrillation. n engl j med. ; : – . doi: . /nejmoa . . patel mr, mahaffey kw, garg j, pan g, singer de, hacke w, breithardt g, halperin jl, hankey gj, piccini jp, becker reviewer disclosures reviewer employment research grant other research support speakers’ bureau/ honoraria expert witness ownership interest consultant/ advisory board other tyler w. barrett vanderbilt university nih nhlbi (pi on a k studying a fib that ended in sept )†; janssen (site pi for a multicenter international orbit- af ii registry)†; boehringer ingelheim (pi for a multicenter retrospective cohort that completed in early )* none none none none boehringer ingelheim† none john w. eikelboom mcmaster university (canada) bms†; boehringer ingelheim†; pfizer†; bayer† none bms†; boehringer ingelheim†; pfizer†; bayer†; janssen*; daiichi sankyo* none none none none david a. garcia university of washington daiichi sankyo (local pi and st committee member for clinical trial involving edoxaban)*; janssen (local pi for clinical trial)* none none pfizer*; bms* none pfizer*; bms*; boehringer ingelheim* none gregory y.h. lip city hospital (united kingdom) none none bayer†; bms/ pfizer†; medtronic†; boehringer ingelheim†; microlife†; roche†; daiichi-sankyo† none none bayer/janssen†; astellas†; merck†; sanofi†; bms/ pfizer†; biotronik†; medtronic†; portola†; boehringer ingelheim†; microlife†; daiichi- sankyo† none jeffrey i. weitz thrombosis & atherosclerosis research institute (canada) none none none none none none none this table represents the relationships of reviewers that may be perceived as actual or reasonably perceived conflicts of interest as reported on the disclosure questionnaire, which all reviewers are required to complete and submit. a relationship is considered to be “significant” if (a) the person receives $ or more during any -month period, or % or more of the person’s gross income; or (b) the person owns % or more of the voting stock or share of the entity, or owns $ or more of the fair market value of the entity. a relationship is considered to be “modest” if it is less than “significant” under the preceding definition. *modest. †significant. d ow nloaded from http://ahajournals.org by on a pril , raval et al march , circulation. ; :e –e . doi: . /cir. e rc, nessel cc, paolini jf, berkowitz sd, fox ka, califf rm; rocket af steering committee, for the rocket af inves- tigators. rivaroxaban versus warfarin in nonvalvular atrial fi- brillation. n engl j med. ; : – . doi: . / nejmoa . . granger cb, alexander jh, mcmurray jj, lopes rd, hylek em, hanna m, al-khalidi hr, ansell j, atar d, avezum a, bahit mc, diaz r, easton jd, ezekowitz ja, flaker g, garcia d, geraldes m, gersh bj, golitsyn s, goto s, hermosillo ag, hohnloser sh, horowitz j, mohan p, jansky p, lewis bs, lopez-sendon jl, pais p, parkhomenko a, verheugt fw, zhu j, wallentin l; 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af-timi trial. lancet. ; : – . doi: . / s - ( ) - . d ow nloaded from http://ahajournals.org by on a pril , preliminary screening of hepatitis c virus in pregnant females and their neonates khamis my, rizk ea, kholeif ae, al marsafawi ah, amish sa. introduction hepatitis c virus is a spherical, enveloped, single stranded rna virus.( i ) six major hcv genotypes and more than subtypes have been identified based on molecular relatedness.( ) genotype , & have a worldwide distribution, while genotype , & are localized to specific geographic location.( ) in egypt, hcv is a major cause of liver disease & leading cause of death, with an estimated hcv infection rate of % to %. egypt has a higher incidence of hcv infection than any other country in the world.( ) most egyptian cases of hcv are due to genotype s, which is uncommon in the west. ( ) diagnosis of hcv infection involves the presence of the virus and assessment of the severity of liver infection. the diagnosis workup should include investigations that may help to predict prognosis and response to treatment. diagnosis of chronic hcv infection based upon the presence of elevated serum aminotransferase and anti-hcv antibodies or the presence of hcv rna in the serum. the disease is said to be chronic if amniotransferases were elevated for more than months or the liver biopsy shows chronic pathological changes of hepatitis.( ) aim of the work the aim of this work was to detect the prevalence of hepatitis c virus infection among pregnant women and their neonates at el-shatby maternity university hospital. patients this work is a random cross-sectional study performed at el-shatby maternity university hospital in alexandria, egypt. the study was conducted over one thousand pregnant females in the third trimester attending for labor. counseling and explanation of all aspects of the study to the participants and their consent was taken prior to their participation. withdrawal of the samples started in may and ended in december with the completion of the one thousand cases included in the study. there were no exclusion criteria in this study. methods all patients were subjected to the following: . full history taking including: • personal history: age, occupation, residence and special habits. • history of medical illness: diabetes, liver disease & or treatment • surgical history: previous surgery & or blood transfusion. dr. amani el marsafawi. e-mail: oashkar ©hotmail.com mobile number: 	 egypt.j.fertil.steril. volume , number , june amani el marsafawi • history of dental manipulation & tattooing. • positive family history of liver diseases and any relevant history. • obstetrical & gynecological history. . complete general examination. . complete obstetrical examination. blood sampling and laboratory analysis: blood samples ( m each) were withdrawn from the pregnant females & umbilical cord of their neonates. sera were separated by centrifugation and stored at (- °c) till testing time. at that time the sera were taken out of the freezer, left to thaw, remixed and centrifuged. the supernatant of the maternal samples were tested for hepatitis c antibodies using enzyme linked immunosorbent assay (elisa). the supernatant of the neonates of hcv-antibodies positive mothers were tested for hcv-antibodies by enzyme linked immunosorbent assay (elisa). hcv-antibodies positive samples were examined for liver function tests namely: (a) alt (serum alanine aminotransferase). (b) ast (serum aspartate aminotransferase). fifty hcv-antibodies negative cases were examined for liver function tests as a control group. results in this study a total number of one thousand pregnant females in the third trimester were randomly selected from attendance of shatby maternity university hospital for delivery, they were screened for hepatitis c virus infection using the third generation elisa. blood samples were taken from the selected cases and the umbilical cord of their neonates immediately after birth, the umbilical cord blood samples of the hcv-ab's positive mothers were investigated for the presence of hcv-ab's. forty five pregnant females tested positive for hcv- ab's representing . % of the studied cases and all their neonates (including two sets of twins) tested positive immediately after birth. liver function tests (ast & alt) were done to the hcv-ab's positive cases and compared with (ast & alt) of fifty hcv-ab's negative cases which was used as a control group. table : distribution of hcv-positive pregnant females among the studied sample age group in years hcv-negative (n= ) hcv-positive (n= ) total (n= ) no % no % no < . . . -< . . -< . . . -< . . -< . . . total range mean ± s.d - . ± . - . ± . - . ± . significance x = . p= . =ns x =chi square, p=probability, ns=non significant according to age group the mean age of the studied sample was . years ranging from to years with the highest frequen- cy seen in the to less than years segment amounting to . % of the whole sample. forty five pregnant females were tested positive for hcv- ab's. the mean age of the hcv-ab's positive mothers were . years ranging from to with the highest frequency seen in the to less than years segment representing . % of the hcv-ab's positive group. egypt.j.fertil.steril. volume , number , june 	 amani el marsafawi table : distribution of hcv-positive pregnant females among the studied samples according to their residence residence hcv-negatve (n= ) hcv-positive (n= ) total (n= ) no % no % no % rural . . . urban . . . total significance x = . p= . =ns or= . ( % ci= . - . ) x =chi square, p=probability, ns=non significant, or=odds ratio, cl=confidence interval most of the cases enrolled in this study were from the urban areas ( . %) and of them tested positive for hcv=ab's forming . % of the hcv-ab's positive group. on the other hand, . % of the cases were from the rural areas and of them tested positive making . of the hcv-ab's positive group. table : distribution of hcv-positive pregnant females among the studied samples according to gravidity gravidity hcv-negative (n= ) hcv-positive (n= ) total (n= ) no % no % no % primigravidae . . . multigravidae - > . . . . . . total range mean s.d. - . . - . . - . . the number of the primigravidae was of the studied sample . % and of them tested positive for hcv representing . % of the hcv-positive cases. on the other hand, the number of multigravidae was and of them tested positive for hcv with the highest frequency seen in multigravidae to making . % of the hcv-positive cases. the mean vidity among the studied sample was . ranging from i to . on the other hand, the mean gravidity among the hcv-ab's positive cases was . as compared to . among the non in- fected cases. table : distribution of hcv-positive pregnant females among the studied samples according to the mode of delivery mode of delivery hcv-negative (n= ) hcv-positive (n= ) total (n= ) no % no % no % vaginal . . . cesarean . . . total significance x = . p= . =ns or= . ( % ci= . - . )=ns x =chi square, p=probability, ns=non significant, or=odds ratio, cl=confidence interval total number cases of the studied sample . % were delivered vaginally and of them tested positive for hcv-ab's representing . % of the hcv-positive cases as compared to . % of the hcv-negative cases. on the other hand, cases . % delivered by cesarean section and of them tested positive for hcv-ab's rep- resenting . % of the hcv-positive cases as compared to . % of the hcv-negative cases. 	 egypt.j.fertil.steril. volume , number , june amani el marsafawi table : distribution of hcv- ositive pregnant females among the studied samples according to fetal outcomes fetal outcome hcv-negative (n= ) hcv-positive (n= ) total (n= ) no % no % no % normal . still birth or iufd . - - . congenital malformations . - - . total significance x = . p= . =ns x =chi square, p=probability, ns=non significant, or=odds ratio, ci=confidence interval a total number of pregnant females of the studied sample delivered normal living babies with no congenital fetal malformations and of them tested positive for hcv-ab's. table : distribution of hcv-positive pregnant females among the studied samples according to number of delivered neonates number of babies hcv-negative (n= ) hcv-positive (n= ) total (n= ) no % no % no % single . . . twin . . . triplet . - - . total significance x = . p= . =ns x =chi square, p=probability, ns=non significant a total number of pregnant females in this study delivered single neonates representing . % of the studied sample and of them tested positive for hcv-ab's representing . % of the hcv-positive cases as compared to . % of the hcv-negative group. on the other hand, delivered twins representing . % of the studied sample and of them tested positive representing . % of the hcv-positive cases as compared to hcv-negative group. only one female delivered triplet representing . % of the studied sample. all the neonates of the hcv-positive mothers (including two sets of twins) tested positive for hcv-ab's immediately after birth. table : distribution of hcv-positive pregnant females among the studied samples according to history of blood transfusion history of blood transfusion hcv-negative (n= ) hcv-positive (n= ) total (n= ) no % no % no % . . . significance x = . , p= . *, or= , % ci= . - . x =chi square, p=probability, *=significant difference, or=odds ratio, ci—confidence interval pregnant females of the studied sample . % gave history of at least one episode of transfusion of blood or blood products and of them tested positive for hcv-ab's representing . % of the hcv-positive cases as compared to . % of the hcv-negative group, i.e. nearly about one third of the infected individuals received at least a single transfusion of blood or blood products. egypt.j.fertil.steril. volume , number , june 	 amani el marsafawi table : distribution of hcv-positive pregnant females among the studied samples according to history of operative intervention risk factor hcv-negative (n= ) hcv-positive (n= ) total (n= ) significance no % no % no % previous obstetric operations . . . x = . (c.s, forceps, ventose) p= . , ns or= . ,ns previous gynecologic operations x = . (d&c, laparoscope, myomectomy, repair . . . p= . , ns oper.) or= . ,ns previous surgical operations x = . (tonsillectomy, appendectomy, thyroidectomy, hernia repair) . . p= . * or= . * %c = . - . total . . v.—chi square, p=probability, ns=non significant, *=significant difference, or=odds ratio, ci=confidence interval *some cases had more than one operative intervention. a total number of pregnant females had a positive history of previous operative intervention regardless the indications, representing . % of the studied sample. around of the studied population . % gave a histo- ry of either cesarean section or assisted delivery using forceps or ventose and of them tested positive for hcv representing . % of the hcv-positive cases. on the other hand, . % gave a history of at least one episode of invasive gynecologic procedure including laparoscopy, dilatation and curettage and a variety of major gynecolog- ic surgeries including myomectomies and repair procedures and of them tested positive for hcv representing . % of the hcv-ab's positive cases. finally, of the tested females . % had other surgical (non-obstetric, non-gynecologic) operations ranging from tonsillectomy, appendectomy to hernia repair and thyroidectomy and of them tested positive for hcv representing % of the hcv-ab's positive cases. table : distribution of hcv-positive pregnant females among the studied samples according to history of den- tal procedures and unsafe injections risk factor hcv-negative (n= ) hcv-positive (n= ) total (n= ) significance no % no % no % previous dental procedures x = . . . . p= . * %c = . - . unsafe injections x = . (using non-disposable syringe) . . . p= . , ns %ci= . - . x =chi square, p=probability, ns=non significant, *—significant difference, or=odds ratio, ci=confidence in- terval certain factors were shown to be associated with a high prevalence of hcv-ab's. history of previous dental procedures were positively correlated with a high prevalence of hcv-ab's; around . % of the studied females gave a history of a t least one dental visit and of them tested positive for hcv-ab's representing . % of the hcv-positive cases as compared to . % of the hcv-negative group; this difference is statistically significant. on the other hand, the difference between infected and non-infected individuals as regards history of unsafe in- jection using non-disposable syringe was not statistically significant. 	 egypt.j.fertil.steril. volume , number , june amani el marsafawi table : distribution of hcv-positive pregnant females among the studied samples according to presence of some other risk factors risk factors hcv-negative (n= ) hcv-positive (n= ) total (n= ) significance no % no % no % history of bilharziasis and treat- ment • oral treatment . . - - . . . x = . , p= . ,ns or= . ,ns %c = . - • parenteral treatment . . . x = . , p= . * or= . * %ci= . - . high risk occupation . . . x = . , p= . * or= . * %ci= . - . x =chi square, p=probability, ns=non significant, *=significant difference, or=odds ratio, ci=confidence in- terval fifty four pregnant females of the studied sample gave a history of bilharziasis and treatment; of them were treated with oral therapy and non-of them tested positive for hcv. on the other hand, pregnant females were treated with parenteral therapy and of them tested positive for hcv representing . % of the hcv-ab's pos- itive cases as compared to . % of the hcv-negative cases; this difference was statistically significant. only of the whole studied population gave a history of a high risk occupation including nurses, healthcare workers, employees in laboratories & dialysis units and of them tested positive for tested positive for hcv representing . % of the hcv-positive cases as compared to . % of the hcv-negative cases; this difference was also statis- tically significant. table : multiple regression analysis (linear model) for some predictor risk factors for hcv-positive cases predictor unstandardized coefficients standardized coefficients t significant b std.error beta constant . . . . history of blood transfusion . . . . . * previous obstetric operations - . . - . - . . * previous gynecologic operations . . - . - . . * previous surgical operations . . . . . * previous dental procedures . . . . . * unsafe injections - . . - . - . . * high risk occupation . . . . . * parentral treatment of bilharziasis . . . . . * for regression model: f= , p= . * on doing multiple regression analysis (=multivariate analysis) using linear model it was found that history of blood transfusion, previous obstetric operations, previous gynecologic operations, previous surgical operations, dental procedures, unsafe injections using un-disposable syringes, high risk occupations as well as parenteral therapy for bilharziasis were all statistically significant risk factors for hcv. egypt.j.fertil.steril. volume , number , june 	 amani el marsafawi table : comparison of liver function tests (ast & alt) in hcv-positive and hcv-negative cases (control sgot (ast) (n= ) hcv cv-positive (n= ) total (n= ) no % no % no % normal . . elevated . . total range mean s.d. - . . - . . - significant x = . p= . , ns sgpt (alt) (n= ) hcv cv-positive (n= ) total (n= ) no % no % no % normal . . elevated . . total range mean s.d. - . . - . . - significant x = . p= . , ns x =-chi square, p=probability, ns=non significant ast was elevated in females . % of the hcv-positive cases with the serum level ranging from to and the mean level was . . on the other hand, it was elevated in cases % of the hcv-negative control group with the serum level ranging from to and the mean level was . . alt was elevated in cases . % of the hcv-positive group with the serum level ranging from to and the mean level was . . on the other hand, it was elevated in cases, % of the hcv-negative control group with the serum level ranging from to and the mean level was . . discussion hepatitis c virus is a major health problem throughout the world. it is estimated that % or nearly about one hundred and seventy million people worldwide are hcv antibodies positive; of those, % are women in childbearing period. ( ) our work is cross sectional study depending on the screening of blood samples withdrawn from one thousand pregnant women and their neonates immediately after birth. the withdrawn samples were screened for the presence of hxc antibodies using the rd generation elisa technique. the results of this study revealed that the seroprevalence rate of hcv antibodies among all the tested pregnant females were . %. this goes with the most of the united states and international studies that reported that the seroprevalence rate of hcv antibodies among the pregnant women to be between . and . %. ( , , ) umbilical cord samples of the hcv antibodies positive mothers were all hcv antibodies positive at birth. further confirmation of these results is by detection of hcv-rna using the pcr. ( ) in , a prospective study was performed in blood transfusion service, fukushima medical college, japan, to assess the vertical transmission of hcv. it included parturient women. they were tested for anti hcv antibodies; were positive and of them only were hcv-rna positive. the infants of the hcv antibodies positive mothers (including one st of twins) were hcv antibodies positive at birth and were followed for months. they were tested for hcv- rna. three of the babies ( . %) became hcv- rna positive during the follow up period. none of the 	 egypt.j.fertil.steril. volume , number , june amani el marsafawi babies of the hcv antibodies positive but hcv- . rna negative mothers became positive for hcv_ rna. ( ) 	 . in may , sophie p. et al in france, published a study to evaluate the risk factors for vertical transmission using a case control design. this study demonstrated an increase hcv vertical transmission risk during vaginal delivery and with the use of forceps compared with caesarean section. episiotomy did not appear as a risk factor during vaginal delivery. maternal breast feeding was not found to be a risk factor. furthermore; amniocentesis did not increase the risk of vertical transmission. ( ) in , two separate studies were done to estimate the vertical transmission of hcv and investigate the effect of mode of delivery and infant feed in on the risk of hcv transmission. in the st study, the overall rate of vertical transmission from mothers who wee hcv rna positive at delivery was . %. in the nd study, the overall rate of hcv vertical transmission was . %. membrane rupture for more than hours and internal fetal monitoring were associated with transmission of hcv to infants in both studies. ( ) in our study, the hcv antibodies positive cases were increased with age and gravidity. this may be attributed to increased exposure to other risk factors as increased operative interference and transfusion of blood and blood products. nearly one third of the hcv antibodies positive mothers ( . %) received at least a single transfusion of blood or blood products. this is in agree with arthur et al ( ) who reported a rate of . % among blood donors from different egyptian governorates. ( ) as regards the fetal outcome in our study, all the neonates of the hcv positive mothers were normal, with no congenital anomalies. this is in agreement with kudo t ( ) who reported no association between maternal hcv infection with abortion, still birth, premature birth or congenital anomalies. ( ) in the present study, there were no significant difference between hcv antibodies positive cases and the control group as regards liver enzymes. this goes with gervais et al ( ). ( ). references . houghton m, weiner a, han j, kuo g, choo ql. molecular biology of hepatitis c virus: implication for diagnosis. development and control of virus disease. hepatology ; : - . . sherlock s, duscheiko g. hepatitis c virus update. gut ; : . simmonds p. variability of hcv. hepatology ; : - . arthur rr, hassn nf, abdalla my. hepatitis c antibody prevalence in blood donor in different governorates in egypt. trans r soc trop med hyg ; : - . . kamal sm, madwar a, petres t, fawzy r, rasenack j. interferone therapy in patients with chronic hepatitis c and schistosomiasis. j hepatol ; : - . . gretch t. diagnostic tests for hepatitis c. hepatology ; : s- s. . who consultation organized in collaboration with the viral hepatitis prevention board global surveillance and control of hepatitis c. j viral hepatitis; : - . . marcellin p, bernuau j, martinot-peignoux m. prevalence of hepatitis c virus infection in asymptomatic anti hiv negative pregnant women and their children. dig dissci ; : - - . . marranconi f, fabris p, stecca c. prevalence of anti hcv and risk factors of hepatitis c virus infection in healthy pregnant women. infection ; : - . . . leikin el, reinus jf,schmell e. epidemiology predictors of hepatitis c virus infectionin pregnant women. obstet gynecol ; : - . . lau jy, davis gl, kniffenj. significance of serum hepatitis c virus rna levels in chronic hepatitis c. lancet ; : - . . ohto h, terazawa s, sasaki n, hino k, ishiwata c, kako m, endo c, matsui a. the vertical transmission of hepatitis c virus collaborative study group. n eng j med ; ( ): - . . sophie poiraud. mother to child transmission of hcv: a case control studyof risk factors. ddw may . . european pediatric hepatitis c network. effects of mode of delivery and infant feeding on the risk of mother to child transmissionof hepatitis c virus. j inf dis ; ( ): - . . kudo t. analysis of mother to infant transmission of hcv. j med virol ; : - . . gervais a erlinger s, valla d, marcellin p. decrease in serum alt and increase in serum hcv rna during pregnancy in womenwith chronic hepatitis c. j hepatol ; ( ): - . this study was done in before the era of sofosbuvir for treatment of hepatitis c. our recommendation is to do the study again to find if there is any change in the prevalence of hepatitis c in egypt after introduction of this line of treatment. egypt.j.fertil.steril. volume , number , june 	 	page 	page 	page 	page 	page 	page 	page 	page [pdf] can thrift bring well‐being? a review of the research and a tentative theory | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /j. - . . .x corpus id: can thrift bring well‐being? a review of the research and a tentative theory @article{kasser cantb, title={can thrift bring well‐being? a review of the research and a tentative theory}, author={t. kasser}, journal={social and personality psychology compass}, year={ }, volume={ }, pages={ - } } 	t. kasser 	published 	sociology 	social and personality psychology compass contemporary forms of consumer capitalism encourage people to prioritize materialistic values, an orientation associated with lower personal well-being. such materialistic values stand in contrast to the economic attitude of thrift, which encourages saving, self-sufficiency, reuse of goods, and avoiding debt. this article reviews the existing empirical literature on thrift and well-being, finding it to be very contradictory, with studies showing positive, negative, and null associations between… expand view via publisher selfdeterminationtheory.org save to library create alert cite launch research feed share this paper citationsbackground citations methods citations view all paper mentions news article climate change and sustainable lifestyles phys.org january citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency living both well and sustainably: a review of the literature, with some reflections on future research, interventions and policy 	t. kasser 	business, medicine 	philosophical transactions of the royal society a: mathematical, physical and engineering sciences 	 	 	 	view excerpts, cites methods save alert research feed towards a theory of minimalism and wellbeing 	kasey lloyd, w. pennington 	psychology 	 	pdf save alert research feed tackling materialism and overconsumption on a finite planet: the development, implementation and evaluation of an educational intervention to decrease materialistic values and excessive consumption behaviour 	jane christine genovese 	psychology 	 	pdf 	view excerpts, cites background save alert research feed simply satisfied: the role of psychological need satisfaction in the life satisfaction of voluntary simplifiers 	s. a. rich, s. hanna, bradley j wright 	psychology 	 	 save alert research feed money for happiness: the hedonic benefits of thrift 	j. chancellor, s. lyubomirsky 	psychology 	 	 	pdf 	view excerpts, cites background save alert research feed consumed by the carbuncle of capitalism: bagley’s boil as social sanctioning personified 	kathryn adams-sloan 	economics 	 save alert research feed the psychological science of spending money 	t. carter 	economics 	 	 	pdf 	view excerpt, cites background save alert research feed challenges for job design 	c. korunka 	business 	 	 save alert research feed saving can save from death anxiety: mortality salience and financial decision-making 	t. zaleskiewicz, a. gasiorowska, pelin kesebir 	medicine 	plos one 	 	 	pdf 	view excerpt, cites background save alert research feed teoría de la autodeterminación: una revisión teórica 	j. b. stover, flavia paola bruno, fabiana uriel, m. liporace 	philosophy 	 	 	view excerpt, cites background save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency self-determination theory and the facilitation of intrinsic motivation, social development, and well-being. 	r. ryan, e. deci 	psychology, medicine 	the american psychologist 	 	 , 	highly influential 	pdf 	view excerpts, references background save alert research feed are psychological and ecological well-being compatible? the role of values, mindfulness, and lifestyle 	k. brown, t. kasser 	psychology 	 	 	pdf save alert research feed the high price of materialism 	t. kasser 	sociology 	 	 	view excerpts, references background save alert research feed the art of buying: coming to terms with money and materialism 	miriam tatzel 	economics 	 	 	highly influential 	pdf 	view excerpts, references background save alert research feed self-efficacy: toward a unifying theory of behavioral change☆☆☆ 	a. bandura 	psychology 	 	 , 	view excerpts, references results and background save alert research feed some costs of american corporate capitalism: a psychological exploration of value and goal conflicts 	t. kasser, s. cohn, a. kanner, r. ryan 	sociology 	 	 	pdf save alert research feed the need to belong: desire for interpersonal attachments as a fundamental human motivation. 	r. baumeister, m. leary 	psychology, medicine 	psychological bulletin 	 	 , 	pdf 	view excerpts, references background save alert research feed the use and abuse of consumer credit: application of psychological theory and research 	h. tokunaga 	psychology 	 	 	view excerpt, references background save alert research feed a consumer values orientation for materialism and its measurement: scale development and validation 	marsha l. richins, s. dawson 	psychology 	 	 , 	view excerpt, references background save alert research feed further examining the american dream: differential correlates of intrinsic and extrinsic goals 	t. kasser, r. ryan 	psychology 	 	 , 	pdf 	view excerpt, references background save alert research feed ... ... related papers 	abstract 	paper mentions 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue in this issue of archives of internal medicine in this issue of archives of internal medicine physical activity and the association of common fto gene variants with body mass index and obesity c ommon fto (fat mass andobesity associated) gene vari- ants have recently been associated with increased body mass index and obesity in several large studies. in this study of old order amish adults who were enrolled in the heredity and phenotype intervention heart study, the authors not only repli- cated this finding but also showed that the association between fto variants and body mass index is abol- ished in those who are the most physically active as determined by ob- jective movement monitoring with accelerometers. these findings em- phasize the important role of physi- cal activity in public health efforts to combat obesity, particularly in ge- netically susceptible individuals. see page postmenopausal hormone use and symptoms of gastroesophageal reflux t his study examined the asso-ciation between postmeno- pausal hormone use and symptoms of gastroesophageal reflux disease (gerd) among participants in the nurses’ health study. investi- gators found that use of estrogens, but not necessarily progesterone, is sig- nificantly associated with an in- creased risk for gerd symptoms. the authors observed significant trends in risk for both increasing doses and du- rations of estrogen use in a prospec- tive fashion. in addition, use of selec- tive estrogen receptor modulators, such as tamoxifen and raloxifene, and use of over-the-counter hormone preparations also carries an in- creased risk for gerd symptoms. these findings suggest that there may be a hormonal component to the pathophysiologic characteristics of gastroesophageal reflux. see page association between blood pressure responses to the cold pressor test and dietary sodium intervention in a chinese population c hen et al examined the association between blood pressure (bp) re-sponses to the cold pressor test and to dietary sodium and potassium interventions among study participants in rural china. the dietary in- tervention included three -day periods of low sodium intake ( . mmol/d), high sodium intake ( . mmol/d), and high sodium intake plus potassium supplementation ( mmol/d). compared with the lowest quartile of bp re- sponse to the cold pressor test, systolic bp changes for the top quartiles were – . , – . , and – . mm hg during the low-sodium intervention; . , . , and . mm hg during the high-sodium intervention; and – . , – . , and – . mm hg during the high-sodium intervention plus potassium supple- mentation (all p � . ). these results indicate that bp response to the cold pressor test was associated with salt sensitivity and potassium sensitivity. see page practice-linked online personal health records for type diabetes mellitus w eb-based personal health records (phrs) have been advocated as ameans to improve diabetes care. however, few web-based systems are linked directly to the electronic medical record used by physicians. grant et al randomized primary care practices to receive access to either ( ) a diabetes-specific phr that imported clinical and medications data and en- abled the patient to author a “diabetes care plan” before upcoming 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with more fragmented sleep had a significantly increased risk of falls. see page . . . . . any use short acting long acting use of benzodiazepines o dd s r at io ( % c on fid en ce in te rv al ) association of benzodiazepine use and risk of or more falls during year after the eighth examination. (reprinted) arch intern med/ vol (no. ), sep , www.archinternmed.com © american medical association. all rights reserved. downloaded from: https://jamanetwork.com/ by a carnegie mellon university user on / / expanding the use of real&#x ;time electromagnetic tracking in radiation oncology expanding the use of real-time electromagnetic tracking in radiation oncology amish p. shah,a patrick a. kupelian, twyla r. willoughby, sanford l. meeks department of radiation oncology, md anderson cancer center orlando, orlando, florida amish.shah@orlandohealth.com received february, ; accepted june, in the past years, techniques to improve radiotherapy delivery, such as intensity- modulated radiation therapy (imrt), image-guided radiation therapy (igrt) for both inter- and intrafraction tumor localization, and hypofractionated delivery techniques such as stereotactic body radiation therapy (sbrt), have evolved tremendously. this review article focuses on only one part of that evolution, electromagnetic tracking in radiation therapy. electromagnetic tracking is still a growing technology in radiation oncology and, as such, the clinical applications are limited, the expense is high, and the reimbursement is insufficient to cover these costs. at the same time, current experience with electromagnetic tracking applied to various clinical tumor sites indicates that the potential benefits of electromagnetic tracking could be significant for patients receiving radiation therapy. daily use of these tracking systems is minimally invasive and delivers no additional ionizing radiation to the patient, and these systems can provide explicit tumor motion data. although there are a number of technical and fiscal issues that need to be addressed, electromagnetic tracking systems are expected to play a continued role in improv- ing the precision of radiation delivery. pacs number: . .-d key words: electromagnetic tracking, calypso, localization i. introduction electromagnetic tracking has been in use for a number of medical applications including image-guided interventional therapy and surgery, endoscopic navigation and more recently, localization and tracking systems for prostate radiotherapy. for the past years, development of electromagnetic tracking systems has grown from an early need in surgical navigation to a more recent need for precision radiotherapy. early image-guided surgery and radiation therapy systems relied on optical tracking. optical tracking systems have limitations, however, that have led to increased interest in electromagnetic tracking systems for medical use. optical tracking systems require a line-of-sight between light-emitting diodes and tracking system cameras, whereas electromagnetic systems do not. electromagnetic systems can be used in computer- aided medical procedures by defining position and orientation for guidewires in interventional radiology or catheter placement for bronchoscopic procedures. several electromagnetic tracking systems have been developed for image-guided surgery using wired transponders such as the aurora (northern digital inc., waterloo, ontario) and microbird systems (ascension technology corporation, burlington, vt). another similar system has been developed by superdimension inc. (minneapolis, minnesota) in order to a corresponding author: amish p. shah, department of radiation oncology, md anderson cancer center orlando, s. orange avenue, mp# , orlando, florida ; phone: - - ; fax: - - ; email: amish.shah@orlandohealth.com journal of applied clinical medical physics, volume , number , fall shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall guide endoscopic tools and catheters down the pulmonary tract so as to provide clinicians the ability to navigate to lesions and biopsy for later diagnosis. this electromagnetic technology has also been used in radiation oncology to guide the implantation of radiosurgical markers or fiducials in and around peripheral lung lesions.( ) at the same time, the need for accurate daily targeting during a course of external beam radiotherapy in the treatment of localized malignancies has also led to advancements in elec- tromagnetic tracking systems. these electromagnetic tracking systems provide the clinician with previously unavailable real-time motion information for targets that may have substantial motion. currently, the most prevalent use of electromagnetic tracking technology in radiation therapy is with the localization and tracking system offered through calypso medical technolo- gies (calypso medical technologies, inc. seattle, wa). this system is currently approved by the food and drug administration (fda) for use in prostate and post-prostatectomy prostate bed radiation therapy. other similar electromagnetic tracking systems for linear accelerator radiotherapy have been developed by micropos medical (sweden) and northern digital inc. (waterloo, canada), but these are investigational or prototype devices and are not cleared for sale in the united states for use as tumor tracking devices in radiation oncology. we will discuss their theory of operation, as well as the forecast for electromagnetic tracking including innovations, clinical indications, and challenges within radiation oncology. ii. current state of electromagnetic tracking literature on the use of electromagnetic tracking for radiation therapy was first published in by houdek et al.( ) for stereotactic radiotherapy localization using a wired transponder sensor. this system had a sensor attached to the stereotactic halo and a source to generate the electromagnetic field, which was attached to an accessory mount. more recently in , sieler et al.( ) reported on the development of a magnetic tracking technique, tuloc, for the improvement of the precision of proton radiotherapy. their paper discussed that implantable sensors would be used to continuously monitor patient position during treatment. these sensors were miniaturized induction coils made of insulated copper wire (diameter μm) wound on a piece of soft iron. the sensors had outer dimensions of mm × . mm diameter. currently, information on two electromagnetic tracking systems can be found — a wired and a wireless system — both used for prostate radiotherapy. a. wired system – micropos raypilot the micropos system is an active nonionizing system intended as a stand-alone unit and needs no additional modalities such as x-rays for determining position of the implant and target.( ) with this system, a sterile dilation catheter is inserted into the penis. the internal wired antenna (implant) is located at the tip of the catheter and connected to the control unit, which converts the signal for computer evaluation (standard pc computer). the external antenna is the signal- receiving component and consists of an integrated antennae array; it is also connected to the control unit. this external antenna is located between the patient and the couch. kindblom et al.( ) report the resolution of the tested system in the laboratory was . ± . mm (mean ± sd). in addition to position tracking with the internal antennae, the micropos device houses a radiation detector in order to provide an in vivo dose measuring using a device such as a mosfet. figure shows a representation (fig. (a)) and a schematic drawing (fig. (b)) of the micropos raypilot system. this system has been developed only for investigational use and is not yet commercially available. prior to its clinical use, several issues need to be addressed. first, a reliable and safe technique for transperineal implantation of the wired transponder in the prostate must be developed. secondly, transponder stability within implanted tissue throughout the treatment course must be demonstrated. upon completion of the radiotherapy treatment shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall course, the transponder implant is removed and thus will not interfere with any future imaging needs, such as magnetic resonance imaging. b. wireless system – calypso medical the calypso medical d localization system (calypso system) utilizes radiofrequency (rf) for wireless tracking during radiation therapy. three small ( mm length × mm diameter) beacon transponders are implanted in or near the target. each electronic transponder consists of an ac electromagnetic resonance circuit encapsulated in glass. localization of the transponders is achieved using an electromagnetic array consisting of four radiofrequency signaling coils and receiving coils. rf signals are emitted from the array at selected pulse rates and are used to excite the transponders at their individually unique resonant frequencies. this array is located above the patient. the transponders absorb some of the radiofrequency energy and re- emit that energy in the form of a decaying signal that is detected by the electromagnetic array. the transponder position is then detected relative to the array which, in turn, is calibrated to the room coordinate reference system by three rigidly-mounted infrared cameras. a misalign- ment of the target is detected by a proprietary algorithm that identifies shifts of the target from its prescribed location anytime throughout the treatment. the accuracy of the detection of the target in phantom is less than mm.( - ) figure (a) shows a picture of the calypso system and fig. (b) shows an example of a single beacon transponder. the calypso system displays real-time graphs instantaneously highlighting shifts in position that exceed a user-specified threshold. in order to localize and track with the calypso system for prostate, a minimum of two transponders must be implanted, and the distance between the target centroid and the anterior surface must be < cm. however, centroid localization can occur at distances up to cm. in cases where the prostate isocenter is beyond cm, prone positioning has been found to be an acceptable alternative with the calypso system, provided that the respiration-induced motion is accounted for in treatment planning. although summarized in this manuscript, several publications have discussed, in greater detail, the clinical use, stability, and accuracy of the calypso system.( - ) comparisons of patient localization based on transponder locations versus stereoscopic radiographic images showed an average d difference of . ± . mm.( ) submillimeter accuracy has been reported when tracking calypso transponders moving at cm/s in a volume cm × cm in width and less fig. . the micropos raypilot system: works as an add-on (a) to existing linear accelerators and enables control of a region of interest (roi) position throughout every radiotherapy session; consists of (b) a receiving system which is placed on the existing treatment table, the transmitter that is placed in the roi, and the user-interface software/computer. figure (a) courtesy of micropos medical. figure (b) reprinted from kindblom j, et al.,( ) (radiot oncol.) with permis- sion from elsevier inc. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall than or equal to cm away from the array.( , ) additionally, this submillimeter localization accuracy has also been reported with studies that compared on-board kilovoltage imaging to transponder location from isocenter.( ) significant stability of the geometry of implanted transponders throughout an entire course of external-beam radiation therapy has been reported. the reported mean standard deviation of the intertransponder distances, calculated using tran- sponder coordinates obtained from the ct scans and the calypso system daily localizations, was reported to be . mm.( ) additionally, several added options are available with the calypso system. the system has the capability of real-time translational treatment couch adjustments that are performed as the calypso system calculates the deviation of the transponders from the treatment isocenter. another added option is a radiation monitoring device inside the room that makes it possible to generate position deviation reports synchronized to the radiation delivery. iii. technological advances for electromagnetic tracking increases in technological growth are reflected in improved methods to modulate and direct radiation beams for radiation therapy. the role of position localization and motion tracking in these methods is definitely understated. with electromagnetic tracking, explicit motion data can be recorded and harvested with a frequency of hz. the value of uninterrupted motion tracking for cancer targets is high when trying to provide precise radiotherapy to organs that move within the body independently of the surrounding tissues. this value only increases when clinicians choose to treat these organs by gating the radiation beam produced by the linear accelerator or choosing to dynamically deliver intensity-modulated radiation therapy to these organs in order to decrease dose to normal tissues through a reduction in treatment margins. the improvements in accuracy that positional tracking can provide to radiation oncologists and physicists should help manufacturers of electromagnetic tracking systems push for advance- ments in their technology, as well as integration of their technologies with other major delivery systems over the next several years. fig. . calypso system (a) with sample beacon transponder (b). the calypso system is positioned in the treatment room. the transponders are implanted into the patient and the array panel is positioned over the patient where it will excite and receive a signal of the beacon transponder’s location in the patient. the array is positioned in the room with the use of infrared cameras mounted to the ceiling of the treatment room. images courtesy calypso medical technologies, inc. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall a. technological applications a. linear accelerator gating electromagnetic tracking in partnership with a treatment option, such as linear accelerator gating, potentially is the next major option that may arise with industrial collaborations between device manufacturers within the next ten years. techniques currently used to gate the treatment beam from a linear accelerator utilize a variety of external surrogates to account for respiratory motion. such devices include, but are not limited to, external marker blocks,( ) infrared reflector body markers,( ) as well as strain gauge belts.( ) studies have shown that the correlation between internal anatomy markers and external markers are valid, but in extremely limited cases.( - ) this correlation was reported to be poor or nonexistent unless the external surrogate measuring the skin surface was near the target volume.( ) furthermore, there is no guarantee that these correlations are constant throughout the course of radiotherapy,( ) and these correlations provide the clinician little quantitative information as to the position or location of the radiation target in question. alternative methods for tracking internal markers have focused on kilovoltage imaging. several studies have reported on fluoroscopic imaging systems to provide accurate information of tumor motion.( , ) real-time tracking with systems, such as cyberknife, allow for linear accelerator tracking of lung fiducials at a high frequency with kilovoltage imaging. however, with additional imaging there is an added expense and increased imaging dose to the patient. this is where electromagnetic tracking is most beneficial. direct monitoring of target position may provide an avenue to gate the treatment beam without additional dose to the pa- tient and is conducted in real time, while many imaging techniques cannot be done in real time or can be done for only a fraction of the overall treatment time. published work has reported on the feasibility of gating based on internal position with electromagnetic tracking.( ) this initial study used an electromagnetic tracking system to provide signals to a varian clinac via a “beam-hold” interface of the linear accelerator in order to trigger beam on/off and gate the treatment beam. the signals for beam on/off were determined in a “gating decision box”, which compared real-time position information with a predetermined three-dimensional ( d) volume. ( ) by gating the treatment beam this way, the clinician is able to deliver a therapeutic dose to the target volume based on absolute d position rather than based on phase or amplitude of a respiratory signal produced by the external surrogates for respiration. a. multileaf collimator tracking as part of a multi-institutional and industrial collaboration, a tracking system with the ability of repositioning the multileaf collimator (mlc) dynamically to follow d target motion in real time has been developed.( , ) the goal of mlc tracking is to dynamically find the target location and reposition the treatment beam to compensate for the target motion. this will give clinicians another technique that accounts for respiratory motion but with higher delivery efficiency than a gated treatment.( ) several investigators have published on dynamic mlc tracking of vary- ing target motion.( - ) electromagnetic tracking can be a key component in this effort. with electromagnetic tracking, direct measurement of the target position can be achieved (with an increased frequency of hz), while avoiding issues with fluoroscopic imaging or other imaging techniques to determine tumor position (fig. ).( ) with the industrial collaborations between linac suppliers and electromagnetic tracking system developers, real-time tracking with dynamic mlcs may be a viable option in the near future. electromagnetic tracking systems provide translational position data, as well as reporting rotational information during the localization process. the rotations about the lateral, longitudinal, and vertical axes correspond to the pitch, roll, and yaw, respectively. this type of information can theoretically be implemented with dynamic mlc tracking to account for more complex motion such as in-plane rigid rotation and target deformation.( ) the combination of the mlc tracking and couch robotics integrated with an electromagnetic tracking system could potentially lead to increased conformal dose distributions and reduced dosimetric uncertainties, all with the possibility to improve treatment outcome by reducing the effects of intrafraction motion. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall a. integration with new treatment technologies another opportunity for electromagnetic tracking systems is integration with other radiotherapy technologies such as tomotherapy, cyberknife, new dynamic linear accelerators such as var- ian truebeam, and proton facilities. currently, electromagnetic tracking systems have been installed in standard linear accelerator rooms only, and calypso has established master agree- ments with varian and siemens. however, integration with the treatment control systems for gating or tumor tracking, even with the mainstream linear accelerator manufacturers, has been slow in its development and clinical implementation. high precision delivery technologies could benefit from motion management systems. for example, the opportunity for cyberknife to incorporate em tracking with the motion of the robotic arm and linear accelerator would elevate their real-time tumor tracking for stereotactic lung radiotherapy that currently focuses on fiducial tracking with a ceiling-mounted kilovoltage imaging system. as another example, the effects of target motion are magnified with proton and other particle beam radiotherapy, especially if this motion is along the beam central axis. electromagnetic tracking systems could play a large role in these facilities. a. d dose calculations several institutions have evaluated the dosimetric consequence of intrafraction prostate motion in radiation therapy, either retrospectively or prospectively. with the capability for electromag- netic tracking of prostate motion, motion can be used to calculate four-dimensional ( d) dose distributions. at washington university in st. louis, investigators have developed techniques to utilize this continuous localization data provided by electromagnetic tracking to evaluate dosimetric coverage for prostate cancer patients.( ) investigators have developed a computer- based tool to prospectively determine appropriate rotational and translational motion limits, as well as retrospectively analyze dosimetric target coverage using tracked positions of individual patient data. the investigators believe this application, referred to as swifter (semi-automatic workflow using intrafraction fiducial-based tracking for evaluation of radiotherapy), will allow for evaluation of potentially more effective treatment techniques such as dose escalation, subprostatic boosts, and reduced-margin treatment planning. fig. . the data flow of the experimental system for multileaf collimator tracking with integrated electromagnetic tracking. reprinted from sawant a, et al.,( ) (int j radiat oncol biol phys.) with permission from elsevier inc. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall at md anderson cancer center orlando, two studies have incorporated electromagnetic tracking with dose recalculation, either retrospectively or prospectively. langen et al.( , ) studied the dosimetric impact of intrafraction prostate motion investigated for helical tomother- apy treatments. measured electromagnetic motion tracks were used to calculate the dosimetric impact on delivered target dose distributions in prostate cancer patients. the investigators de- veloped a retrospective dose recalculation method that allowed for the input of motion tracks from an electromagnetic tracking system and dynamically recomputed the treatment delivery for helical tomotherapy treatments. they found that for the observed patient prostate motion, the resulting dosimetric effect on respective tomotherapy plans was small.( ) a second study investigated the feasibility for prospective simulation and visualization of the radiation therapy dose delivery for d lung tumors.( , ) these investigators propose a method of simulating and modeling lung tumors in real time and visualizing dose accumulated to them. santhanam et al.( ) further proposes that the motion tracking data will be recorded via implantable rf devices such as ones offered with electromagnetic tracking systems. they state that the pos- sibility of real-time dose visualization can only be realized through real-time monitoring of patient motion with electromagnetic tracking and utilizing actual patient anatomy through d computed tomography.( , ) b. potential clinical indications the potential clinical benefits from applications of electromagnetic tracking to clinical sites other than the prostate and prostate bed could be significant, especially in regions of large target motion, such as lung and liver. direct measures of motion in target volumes in the thorax and abdomen will improve treatment accuracy and allow clinicians to maintain high dose gradients outside of the treatment volume to prevent excessive dose to the surrounding tissues. several other anatomical sites also fall into this category, where organ motion may be substantial or even minimal motion may be critical. currently, work is being done to incorporate electromagnetic tracking in several sites outside of the prostate. hopefully, while these preliminary investigations are not yet fda-approved, viable options to treat these sites will become clinically available within the next several years based on this early work. b. lung with the increasing use of high-dose radiotherapy along with smaller treatment margins and the trend to use increasingly hypofractionated treatments, accurate placement of radiation fields is critical. one of the main limitations in dose escalation is the additional margin necessary to account for inter- and intrafraction setup errors. respiratory motion of thoracic structures can reach up to . cm,( ) and the interfraction setup error using skin marks or bony landmarks can reach up to cm.( ) this leads to an increase in radiation therapy volume to account for these uncertainties, and limits dose to normal tissues. being able to directly measure the motion of tumors during breathing with electromagnetic tracking would allow clinicians to effectively reduce treatment volumes and decrease normal tissue doses without compromising tumor cover- age. observing and accounting for tumor motion from respiration during radiation treatment of non-small cell lung carcinoma (nsclc) has been the aim of the research proposed by several investigators, most of which are detailed in this section. at md anderson cancer center orlando, preliminary work has been done to directly measure tumor motion using an electromagnetic tracking system under an institutional review board (irb)-approved off-label study for nsclc.( ) additionally, this study requires investigational device exemption (ide) from the fda. to date, a total of twelve rf transmitters have been implanted into five patients with varied success. bronchoscopic implantation of the rf devices was performed along with implantation of gold fiducial markers to improve stability and fixation (for every rf transmitter device, one fiducial marker). the lung implantation was performed using the superdimension system (superdimension inc., minneapolis, mn) that is designed to allow ct-guided bronchial navigation for biopsy, but is used at our clinic for implanting shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall markers( ) as well as rf transmitters in and around lung lesions (fig. ). of the twelve implanted rf devices, three did not remain fixated in the tumor location. motion data were collected for all enrolled patients. the authors reported that this study was meant to collect real-time tumor motion tracks before and after treatment and not to interfere with the standard of care for radio- therapy treatment for the patients enrolled in the investigation at their clinic. figure displays an example of the target tracks obtained for one patient during one session. in a move toward clinical availability for electromagnetic tracking within the lung, an elec- tromagnetic tracking device with a stabilization feature has been developed by calypso medical and investigated by a group at washington university using a canine model.( , ) mayse et al.( ) reported successful bronchoscopic implantation for all of the stabilized beacon transponders. at days follow-up, all of the transponders with working electromagnetic cores had stable intertransponder distances as measured by the calypso system. the stabilization feature incor- porating five nitinol legs was created to house the electromagnetic beacon transponder (fig. ). the nitinol legs are designed to deploy into the bronchus after bronchoscopic implantation. the authors reported that successful implantation and fixation were very sensitive to the implanta- tion technique, in that too much force on the implantation catheter may lead to pneumothorax and not enough force may lead to a low rate of fixation. fig. . images from lung cancer patient with left lobe, posterior lesion approximately . cm in diameter with three rf transmitters and two gold fiducial markers implanted. the fiducial markers were placed to ensure device stability. rf transmitter devices and fiducial markers imaged on (a) computed tomography and (b) a c-arm fluoroscopic system. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall fig. . examples of lung tumor tracks acquired through electromagnetic tracking with the calypso system under an irb-approved off-label study for nsclc. target motion in the same timescale is shown in the left–right (lr) (top), anterior–posterior (ap) (middle), and superior–inferior (si) (bottom) directions for (a) the total time tracked for the single session, and (b) a detailed view of target motion data for seconds. figure (b) illustrates the changes in position from drift and irregular motion, which may not be realized in looking at the tracks given in fig. (a), especially in the anterior– posterior direction where target motion was the largest. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall b. liver/pancreas implanted fiducials are commonly used for image-guided radiation therapy (igrt) of ab- dominal tumors, especially in stereotactic body radiation therapy (sbrt) cases.( , ) fiducial implantation is often performed as an out-patient procedure, with little migration( ) and clear compatibility with kilovoltage imaging; thus, its value for treating moving targets such as liver tumors far outweighs its cost. furthermore, the introduction of electromagnetic tracking for these targets could potentially be quite valuable. there are few studies that have investigated the use of electromagnetic tracking in abdominal targets with the radiofrequency devices. this may be due to a limitation in implantation techniques for the relatively large diameter devices that are currently available on the market, as well as the corresponding concerns with rf device migration after implantation. another concern with implantation of the rf transmitter devices with targets such as the liver is the need for post-treatment magnetic resonance imaging (mri) studies for patient follow-up. the potential for increased image artifact created by the implanted rf transmitters within an mri study would prove too costly for clinicians. this issue is further explained below with regard to rf transmitter size and the need for removable rf devices. at the university of pennsylvania, investigators have implanted rf transmitters into the pancreas of three patients under laparoscopic guidance.( , ) the investigators have reported stability with the rf device implantation and all patients were without complications. b. breast reduction of margins, setup error, and the possibility of intrafraction motion during partial breast irradiation are all factors that may be improved with the introduction of an electromag- netic tracking system. preliminary work has been done to test the feasibility of electromagnetic localization for external beam partial breast irradiation.( ) investigators at swedish medical center in seattle have used an electromagnetic tracking system under an irb-approved pro- tocol to implant rf devices into the lumpectomy cavity of fifteen patients.( ) implantation of electromagnetic transmitters may not be ideal for breast cancer patients due to the need for post-treatment magnetic resonance imaging studies for clinical follow-up. therefore, the investigators took precautions to implant the rf devices temporarily via interstitial catheters and removed them following course of radiotherapy treatment. b. cervix investigators at emory university have recently started a clinical trial to evaluate intrafrac- tional cervical motion utilizing an electromagnetic tracking system in cervical cancer patients. fig. . anchored lung transponder from calypso medical technologies inc. the lung transponder is intended for bronchoscopic implantation in small diameter airways. the anchored transponder consists of the prostate transponder with a -legged nitinol stability feature. images courtesy calypso medical technologies. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall the trial states that two rf devices will be temporarily placed within the cervical os via su- tures and tracked during the course of radiation therapy treatment. the patients will continue with the clinic’s standard of care, but the rf transmitter positions will be continuously tracked and recorded. the end goals for the investigators are to improve the current knowledge of cervical motion and tumor regression during radiation therapy with the use of electromag- netic tracking.( ) b. central nervous system although sbrt is not restricted to the radiotherapy for the central nervous system, it has be- come more common in radiation oncology, and igrt has become a necessary component of this type of treatment. electromagnetic tracking could easily play a critical role in the delivery of sbrt for cns-related systems. the continuous monitoring of target displacements could provide an improved means to deliver accurate dose to the target while reducing dose to normal tissues, especially in cases where the target could move during the treatment delivery. in one such example, willoughby et al.( ) reported on the use of electromagnetic tracking during spinal radiosurgery. these investigators, under fluoroscopic guidance, transcutaneously im- planted rf transmitters into the paraspinal muscles at the level of the targeted vertebra in six patients (fig. ). the implantation was successful in all six of the spine patients. there were no complications related to the implantation processes. all patients had at least two ct scans prior to treatment delivery to confirm stability and geometry of the rf devices. at the time of treatment, all patients also had kv x-rays to confirm stability and geometry in the treat- ment position prior to each delivery. similar to the liver, issues with post-treatment magnetic resonance imaging studies for the spine need to be mentioned and will be addressed in more detail below. tracking data on the available electromagnetic tracking system was obtained on all four patients. an example of this tracking data from one patient can be seen in fig. . a second scenario could also be envisioned for stereotactic radiosurgery patients. currently, many institutions have gone to frameless radiosurgery using fiducials and optical tracking tech- niques. accordingly, an interesting question arises if this could be done using electromagnetic tracking, as proposed in by houdek et al.,( ) for intracranial lesions. for example, rf transmitters could be implanted into a bite-block or similar device, and cranial displacements could be tracked and recorded with tolerances set to gate the radiation beam off as increases to translation or rotational motion occurs.( ) fig. . images from patient who presented with painful bony metastases to the fourth lumbar vertebral body with three rf transmitter devices surgically implanted into the paraspinal muscle for single-fraction stereotactic body radiotherapy for pain relief and quality of life enhancement. rf transmitters imaged on (a) computed tomography and (b) a c-arm fluoroscopic system. (a) (b) shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall fig. . examples of target tracks acquired through electromagnetic tracking with the calypso system under an irb-approved off-label study for spine stereotactic body radiotherapy. target motion in the same timescale is shown in the left–right (lr) (top), anterior–posterior (ap) (middle), and superior–inferior (si) (bottom) directions for the total time tracked before the single fraction treatment. in this example, the target motion in each direction was less than mm. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall iv. challenges to electromagnetic tracking electromagnetic tracking is still an immature technology in radiation oncology, and as such, the clinical applications and clinical experience are both very limited. some of the major chal- lenges with incorporating electromagnetic tracking into daily use for radiation therapy are due to the limited number of available technologies. with the calypso system being the sole fda- approved electromagnetic tracking system used in radiation oncology, development resources are limited. hence, there are many opportunities for improvement over the next decade. first and foremost among limitations, electromagnetic tracking has only been approved for use in prostate cancer treatment. any use at other disease sites must be approved and monitored by the fda and the local irb. while there is utility in tracking the prostate, there is more potential benefit at other disease sites mentioned previously. the development of techniques to treat these clinical sites, along with the regulatory approval to do so, needs to be addressed in the near term. some technical challenges that are inherent to electromagnetic tracking systems are related to the electromagnetic array. in the currently available electromagnetic tracking system, the array rests directly above the prostate cancer patient with a need for direct line of site to the infrared cameras that track the array’s motion and position relative to isocenter. the combina- tion of the array’s position and rf transponder’s position relative to the array, provides the electromagnetic tracking system with the actual room coordinate system and, further, the transponder’s position relative to isocenter. however, several issues arise due to the presence of the electromagnetic array, such as issues with couch angles, distance issues from the array to the implanted rf devices, and collision issues with the gantry due to the proximity of the array to the gantry head. if the need for the array (anterior to the patient) was not there or if the array could be in another location (such as embedded in the treatment couch), many of these issues could be resolved and the infrared cameras in the room could be removed. although for this to happen, treatment couches would be required to maintain millimeter-scale positioning accuracy, or maintain the infrared tracking system but tied directly to the treatment couch. another issue with the electromagnetic tracking systems is in the limited field of view (fov) with the array. the current fov limitation of the available system is not a major problem when treating the prostate (the system’s intended purpose); however, if new clinical indications are to be implemented, this limitation will need to be addressed. even more important, if the flat-field generator were placed in a location adjacent to the patient with no interference from electrical and ferrous objects in the accelerator room, the benefit of tracking implanted needles or devices would be infinite. another challenge to electromagnetic tracking systems is regarding the rf device size. with the currently available rf transmitter device, its large size allows for high signal to noise. however, the mm length × mm diameter device size requires a -gauge needle for transperineal implantation, which is considered large for transcutaneous needle insertion for areas such as the liver or bronchoscopic insertion into the lung. the risk of pneumothorax with transcutaneous approaches is estimated to be in the %– % range, and even higher ( %– %) in patients with obstructive airway disease.( ) the risk of pneumothorax is greatly reduced with transbronchial fiducial placement in the lung, even in peripheral lesions under fluoroscopic guidance.( ) also, when taking into consideration follow-up imaging studies of patients, the rf transmitter’s relatively large ferrite core creates significant artifact on mri or even kilovoltage cone beam ct.( ) it was reported that implanted transponder displacements due to the mr field were minimal; however, the null signal reported due to image artifacts were up to . cm in radius and cm in length, thus creating significant issues with post-treatment imaging with patient follow-up.( ) a reduction in the sizes of rf devices would be beneficial, but would not prevent these image artifacts from occurring. development of removable rf transmitters would be the next expected course of action for treatment areas that benefit from post-treatment mr imaging. shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall another challenge is to provide a system only for motion tracking, not localization and tracking. in the current configuration of electromagnetic tracking systems, there is great care to ensure that the rf transmitter devices are in the correct location relative to the isocenter. in order to perform this calculation accurately and precisely there are limitations to the additional noise from metal or other devices in the room, restrictions on the implantation geometry, as well as procedures to limit the amount of motion the rf devices are experiencing in the implantation location. if tracking-only were allowed, the user would be responsible to localize the patient via some other imaging modality, and the electromagnetic tracking system would gain flexibility by providing only relative coordinates of the implanted rf transmitters. this could be used in computed tomography for d ct, or used during treatment in other sites in the body where localization issues based on single implanted markers may arise due to intrafraction motion and deformation, such as lung and liver. also, if relative displacement numbers were reported, the need for multiple implanted rf devices may not be necessary. multiple transmitters, if implanted correctly, provide the clinician with rotational information and possible organ defor- mation information; however, without imaging, it cannot be known if the reported angles are due to marker migration or due to organ deformation. if rotational information is not desired, then multiple rf devices may not be needed. another challenge to electromagnetic tracking is with competing technologies that may pro- vide tracking via a different delivery system. although radiofrequency-based electromagnetic tracking systems can be used in real time to track tumor location due to its fast update rate, new emerging technologies are constantly being developed that may provide a similar track- ing mechanism. one such technology being developed by navotek inc. (navotek inc., israel) provides the ability to track targets within the body. navotek is developing a gantry-mounted radioactive fiducial tracking system that is reported to provide submillimeter accuracy for pa- tient localization and monitoring.( ) radioactive fiducial tracking, though not fda-approved, challenges technologies such as electromagnetic tracking with technical advancements in im- plantable radioactive materials and localization of these sources. however, this technology may have insufficiencies of its own such as the lack of ability to provide rotational information and possible organ deformation information. in addition, if any migration of the radioactive source does occur, it will be quite difficult to determine by how much and to where it is migrating. the last challenge to electromagnetic tracking is with reimbursement. while not a techno- logical limitation, the up-front capital expense of electromagnetic tracking systems and the ongoing expense of the implantable markers remain barriers to widespread implementation of these technologies. because of the high cost of these rf transmitters and the varying levels of reimbursement in different geographical regions, it is cost prohibitive for many centers to consider this technology for routine clinical use. unless these cost and reimbursement issues are addressed, electromagnetic tracking technology may not experience further growth in radiation oncology. continued research collaborations need to show that (a) electromagnetic tracking is a growing technology with increased development of treatment options, (b) proper and unbiased efficacy tests show an improved therapeutic effect, and (c) effectiveness tests show electromagnetic tracking does help in localization and targeting of the tumor region. for example, the latter two points could be tackled through dosimetric studies that show intrafrac- tion motion during radiotherapy does result in differences in the delivered dose versus the planned dose — similar to some of the studies mentioned above related to retrospective dose recalculations.( , ) v. summary electromagnetic tracking systems enable accurate patient set-up, respiratory correlated radio- therapy, collision avoidance, and adaptive radiation therapy. furthermore, daily use of these systems is minimally invasive and delivers no additional ionizing radiation to the patient. due shah et al.: electromagnetic tracking journal of applied clinical medical physics, vol. , no. , fall to these advantages, electromagnetic tracking systems are expected to play a continued role in improving the precision of radiation delivery. there are a number of technical and fiscal issues that need to be addressed in the near term, however, to ensure the success of these technologies in improving patient care over the next ten years and beyond. acknowledgments the authors would like to thank calypso medical technologies and micropos medical for their assistance in writing this manuscript. 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time in the middle of nowhere. new stories from the midwest, then, is not, by design, a “best of ” collec- tion, though there was this winnowing process to get here. it is more a quilt, not crazy of course, but more a somber monotonic amish quilt. with their subtle fl atness and minute variations these stories are as indicative of the midwest as the township grid is seen from the air as one fl ies over. michael martone university of alabama tuscaloosa, alabama john r. van atta, securing the west: politics, public lands, and the fate of the old republic, – . baltimore: johns hopkins university press, . pp. $ . . in securing the west, john r. van atta seeks to demonstrate just how cru- cial public land policy was in the fi rst decades of the republic. land had, of course, been the great project of british colonialism, and control of the ohio valley the key spark that started the french and indian war. settling this land was not simply an economic issue, but a political and social one as well. after , and especially after , the primary concern was how soon, and by whom, should that land be settled. if the west were settled too quickly, and by the wrong sort, it might well become a breakaway region of its own. instead of providing desperately needed capital to a new anglo american nation, it might prove a crippling albatross, populated by lazy, immoral freebooters with no respect for proper authority. many in congress felt that hardworking, thrifty new englanders would be the best model for settlers. others, concerned about indian resistance and the possibility of foreign invasion, felt that the revolutionary war practice of states awarding bounties to military veterans should be expand- ed. young thomas jefferson, in his more radical pre- presidential years, sought to distribute, rather than sell, the land to yeoman farmers. by , congress settled upon something of a hybrid— selling the land at public auction, but in sections of one- mile square ( acres) at a minimum price of $ per acre- intentionally out of reach for many americans. of course, this and subsequent laws regarding the west tended to breeze over indian land rights, or blissfully assume the lands would be cheerfully sold to the book reviews federal government. further, they set no upper limit on the amount of land one might purchase, which would certainly encourage speculators. con- cern over the vast tracts of land left unpurchased and unimproved led con- gress to repeatedly ease the terms of the land ordinance of . in , they cut the minimum purchase to acres, with four years’ credit to pay, though the price had risen to a minimum of $ per acre. by , jeffer- sonian republicans further cut these amounts to acres and $ . , but kept the credit provision. the efforts to extend credit and ease the purchasing of public lands did increase land sales, though they also contributed to the wild speculation that helped bring on the panic of . the panic, in turn, led some states to reconsider just how the money generated from those sales should be dis- tributed. while the land ordinance had progressively promised one thirty- sixth of the sales— that is, one square mile— from each township to fund public education in the new lands, maryland and other older states began to squawk for a piece of that revenue, arguing that it was benefi tting the new states at the expense of the old. what if the tariffs collected in atlantic ports were used only for projects in those states, they argued. “unsurpris- ingly,” van atta notes, “no new state would ever favor the plan” ( ). as sectional tensions over slavery grew, old east vs. west tensions for a time were coupled with north vs. south frictions. western senators, like democrat thomas hart benton of missouri, continued to champion quick and cheap sales of public lands, while many new englanders, like new hampshire senator samuel foot, tried to apply the brakes. benton became a champion of squatters’ rights to preemption, or claims to land that had been illegally occupied but “improved.” henry clay, in many ways a cham- pion of westerners, felt granting such preemption rights was foolhardy and morally wrong, and it repeatedly cost him votes from men who should have stayed part of his political base. a highlight of this confl uence of sectional tensions came in the famous webster- hayne debate in january . though primarily remembered for the rhetorical genius of massachusetts’ daniel webster and the staunch antitariff position of south carolina’s robert hayne, the debate had really begun with senator foot’s earlier resolution to stop surveying new public lands until those already surveyed had been sold off. given how there were seventy- two million already surveyed acres unsold, the resolution would have effectively stopped surveying for the foreseeable future. hayne thundered for states’ rights and against what later generations would call big government. middle west review • vol. no. webster countered that he rose “to defend the east,” and orderly, rational settlement ( ). though webster won more style points, van atta concludes that, at least temporarily, the preemptionists won the day. as with states’ rights, the slavery controversy remained interwoven with the question of the public lands, and slavery as a political issue became especially prominent in the years after the war with mexico and the mexi- can cession. in the s, the doctrine of popular sovereignty, sometimes called “squatter sovereignty,” helped bring on vicious armed confl icts, especially in the territory of kansas ( ). once the civil war broke out, leaving lincoln and congress without signifi cant southern opposition, they could pass the homestead act of , granting acres to heads of household, and the morrill land grant college act to promote higher edu- cation throughout the nation. securing the west features a number of helpful population maps, as well as tables illustrating the sectional distribution of key votes in congress. it is solidly researched, well written, and cogently argued. in all likelihood it will not garner much of an audience outside of specialists, which is really a shame. the history of public land policy lacks the excitement of studies of the wars against native americans and cannot hope to match the inherent drama of works on american slavery. yet what van atta has ably demon- strated is that one cannot really understand either of those topics without grasping just how inextricable land policy was from both of them. robert m. owens wichita state university wichita, kansas dana elizabeth weiner, race and rights: fighting slavery in the old northwest. dekalb: northern illinois university press, . pp. $ . . in race and rights dana elizabeth weiner examines how americans in the midwest responded to slavery and racial discrimination from to . her monograph focuses on four states: ohio, indiana, illinois, and michi- gan. her central argument is that blacks and whites launched an organized movement to advance civil rights and do away with the remnants of slavery that lingered in defi ance of the ordinance of . they also wanted slav- ery abolished in the country at large. while this book reminds readers of many well known topics from the old northwest territory, weiner makes poster abstracts: linkage mapping/marker polymorphisms section linkage mappinglmarker polymorphisms . linkage analysis and clinical studies in british families with dominant optic atrophy seller, mary'; behnam, j ; spalton, d ; johnston, r ; burdon, m ' dept. of medical and molecular genetics, guy's hospital, london sei rt; dept. of ophthalmology, st thomas's hospital, london sei eh the gene for dominant optic atrophy (kjer type) has been mapped to q -qter, and linkage studies have been reported in cuban, danish and french families. we aimed to perform linkage analysis in british families with this disease, including an extensive five-generation pedigree, and to validate the efficiency of domiciliary screening for affected individuals. ten microsatellite markers in the region of q -q were used. positive lod scores were obtained with several markers and haplotypes segregating with the disease were identified in affected individuals. informative meioses defined a disease interval of cm. domiciliary screening was successful in identifying affected individuals. the location of the opal gene in the british families appears to be in the region of q -q and the same as that reported in families elsewhere in the world, suggesting there is no genetic heterogeneity in this disease. . molecular studies in rett syndrome: a update. thomas, nick ; webb, t ; davies, k ; williams, n ; pereira, j ; kerr, a ; anvet, m ; hanefeld, f ; clarke, a i ' institute of medical genetics, university of wales college of medicine, heath park, cardiff, cf xwu; clinical genetics unit, birmingham maternity hospital, edgebaston, birmingham b tg; curituba, brazil; renfrewshire, uk( stockholm, sweden; t gottingen, germany. one accepted model for rett syndrome [rs] is that of an x-linked dominant disorder observed in females, but lethal to males in utero. all cases should result from the presence of a new mutation. the very infrequent occurrences of affected sister, or other familial cases, has been explained by germinal mosaicism; by differences in x-inactivation patterns between individuals or their tissues; or by the presence of a premutation - a type of anticipation. analysis of the segregation of x chromosomal markers within rs families containing more than one affected individual should, in principle, permit the exclusion of regions of the x chromosome, thus refining the regions to be searched for potential rs candidate genes. we have gathered a panel of familial cases of rs, including families with affected sister-sister, or half-sister, pairs, as well as rs families with affected aunt-niece pairs. these rs families have now been screened with an extensive panel of microsatellite markers from across the x chromosome, searching for evidence of discordancy or concordancy at each of the marker loci. to date, discordant segregation of markers in these families has been observed for many of the loci and no consistent region of concordancy, and hence a potential rs candidate gene region, can yet be defined. . informativity of wiskott-aldrich syndrome (was) gene linked marker at locus dxs in different ethnic groups thompson, lisa j; lalloz, mra; layton dm department of haematological medicine, king's college school of medicine & dentistry, london se pj since cloning the gene over mutations associated with was and its allelic disorder x-linked thrombocytopenia (xlt) have been identified. a short tandem repeat (str) polymorphism (ca)n has recently been identified at locus dxs within kb of the was gene. genotyping errors are less likely than with more distant markers. str allele frequency was studied in individuals ( x-chromosomes) from four ethnic groups by polymerase chain reaction and ca repeat number determined by dideoxy dna sequencing. two novel alleles, (ca) in asian indian and afro-caribbean populations and (ca) in middle eastern subjects were seen. caucasian and oriental populations exhibited five and two alleles respectively, of which the (ca) allele was most frequent ( % and %, respectively). in contrast, (ca) is most common in africans ( %) and afro-caribbeans ( %). the observed heterozygosity (> %) for all groups except orientals ( %) was in accord with that expected. inheritance of the dxs marker was studied in six was families in which % of females were informative. in one kindred a g a mutation was shown to have arisen de novo during grandpatemal gametogenesis. the dxs str offers a rapid approach to the molecular diagnosis of was and xlt. . progressive familial intrahepatic cholestasis (byler disease): evidence that the disease haplotype in the old order amish is also found in the irish "traveller" population. barton, david e ; mcquaid, s '; bourke, b ; carlton, ve ; bull, l ; freimer, nb ; walsh, d , knisley, as ; drumm, b ' national centre for medical genetics, our lady's hospital for sick children, crumlin, dublin , ireland; department of paediatrics, university college dublin, our lady's hospital for sick children, crumlin, dublin , ireland; neurogenetics laboratory and center for neurobiology and psychiatry, department of psychiatry, university of california, san francisco, ca , usa; department of pathology, children's hospital, pittsburgh, pennsylvania, usa. proressive familial intrahepatic cholestasis (pfic, byler disease) is a recessive disorder characterised by impairment of bile flow leading to attacks of jaundice, hepatosplenomegaly and early death. pfic was originally described in an old order amish kindred, but has since been reported in different ethnic groups, often associated with consanguinity. a locus for pfic was mapped to q - by searching for shared segments in two distantly-related amish patients (carlton et al. hum. mol. genet. : - , ). we have studied a irish pfic kindred from the 'traveller" population, a community of closely-related families with a nomadic lifestyle. these patients were investigated for homozygosity for four microsatellite markers in the q -q region. four affected siblings and an affected first cousin were homozygous for all four markers. to our surprise, the alleles observed in our irish traveller family appeared similar to those reported in the amish kindred. inclusion of an amish patient on the same gel as the traveller samples demonstrated that the travellers have an identical haplotype at the pfic locus to that seen in the old order amish. these results strongly suggest that the old order amish and irish travellers share the same pfic mutation, most likely inherited from a common ancestor. r--s- l ahua % enetics o jmedgenet ; s s british human genetics conference j med genet ; : supplement o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . .s u p p l_ .s o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ s poster abstracts: linkage mapping/marker polymorphisms . paget's disease of bone: evidence for linkage to chromosome q and for genetic heterogeneity haslam, sonya; haites, n; thompson, j; ralston, s department of medicine & therapeutics, university of aberdeen medical school, foresterhill, aberdeen ab zd paget's disease is characterised by foci of abnormal bone remodelling. we have studied pedigrees each containing or more affected individuals. in pedigrees where sufficient details were available, the pattern of inheritance was consistent with that of an autosomal dominant trait. a series of affected sibling pairs from this cohort have been identified and genotyping performed using microsatellite markers from q - . using the splink program, preliminary evidence of linkage was found to d s (lod= . ) and d s (lod= . ) in the sib pairs analysed so far. in large sibships it has been possible to construct multimarker haplotypes, infer parental hapotypes and thereby make the genotype data fully informative. in these pedigrees, the haplotypes are fully consistent with the inheritance of an autosomal dominant gene on q ( & informative meioses). haplotype analysis in other pedigrees appear to rule out the possibility of a paget's disease locus in this area thereby raising the possibility of genetic heterogeneity. familial expansile osteolysis (feo) is inherited as an autosomal dominant trait, presents with a severe pagetic phenotype, has been mapped to q - and raises the possibility that paget's disease and feo are allelic disorders.. . corneal stem cell graft mccall, sam ; henderson, trm ; noble ba ; taylor gr i ' regional dna laboratory, ashley wing, st. james's university hospital; beckett street; leeds; ls tf corneal donor stem cell graffing was used to treat symptomatic primary stem cell deficiency in a case of aniridia caused by a pax mutation. with ethical committee approval and informed consent, comeal epithelial samples were taken from the year old aniridic lady % years after limbal stem cell grafts to her right eye. the donor was her non-aniridic but histocompatibly well matched year old son, thus it was possible to look for male (donor) cells against a background of female (recipient) cells. we evaluated methods for the detection of y-specific material in corneal tissue based on the pcr amplification of amelogenin, dyzi and sry respectively. pcr was carried out using fluorescently labelled primers and the products run on a genescanner (abi). the data was analysed using software. all test samples were found to contain less than % male (donor) cells. however lower levels of y material were detected using the sry probe; the significance of this is currently being evaluated. . mapping susceptibility genes for inflammatory bowel disease mirza, mudassarl; lee, john ; naom, lsam'; ford, deborah ; hugot, jean-pierre ; laurent-puig, pierre ; hodgson, shirley'; thomas, gilles ; easton, douglas ; lennard-jones, john ; mathew, christopher' 'umds division of medical and molecular genetics, th floor guy's tower, guy's hospital, london sei rt, st. mark's hospital, london eciv ps; nstitute of cancer research. sutton, surrey sm ng; 'institut curie, paris, france epidemiological studies have shown that first degree relatives of patients with ulcerative colitis (uc) or crohn's disease (cd) have a -fold increased risk of inflammatory bowel disease (ibd). we have collected blood samples from families with multiple members affected with ibd and have been searching for susceptibility genes by linkage analysis with microsatellite markers, using both parametric and non-parametric methods of statistical analysis. there was no evidence for linkage to the hla region (naom et al, am j hum genet, in press). we have therefore analysed markers within or flanking additional candidate genes for linkage, including several t-cell receptor and mucin genes. we have also investigated the loci on chromosome and on chromosome , which showed evidence of linkage to cd (hugot et al, nature : , ) for linkage to ibd. families affected with only uc (n= ) or with both cd and uc (n= ) were typed with markers from chromosome and from chromosome in a total of individuals. an excess of haplotype sharing in affected sib pairs was observed at d s and d s . a detailed statistical analysis of the linkage data will be presented. british human genetics conference j med genet ; : supplement o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . .s u p p l_ .s o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script 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| : | doi: . /s - - - www.nature.com/scientificreports a population-specific reference panel empowers genetic studies of anabaptist populations liping hou , rachel l. kember , jared c. roach , jeffrey r. o’connell , david w. craig , maja bucan , william k. scott , margaret pericak-vance , jonathan l. haines , michael h. crawford , alan r. shuldiner & francis j. mcmahon genotype imputation is a powerful strategy for achieving the large sample sizes required for identification of variants underlying complex phenotypes, but imputation of rare variants remains problematic. genetically isolated populations offer one solution, however population-specific reference panels are needed to assure optimal imputation accuracy and allele frequency estimation. here we report the anabaptist genome reference panel (agrp), the first whole-genome catalogue of variants and phased haplotypes in people of amish and mennonite ancestry. based on high-depth whole- genome sequence (wgs) from individuals, the agrp contains > m high-confidence single nucleotide variants and short indels, of which ~ . % are novel. these anabaptist-specific variants were more deleterious than variants with comparable frequencies observed in the genomes panel. about , variants showed enriched allele frequencies in agrp, consistent with drift. when combined with the genomes project reference panel, the agrp substantially improved imputation, especially for rarer variants. the agrp is freely available to researchers through an imputation server. genetically isolated populations are subgroups that have remained reproductively separated from the surround- ing population over many generations. genetic isolates are often descended from a limited number of founding individuals and may suffer an increased burden of inherited disease. however, genetic isolates also offer many advantages for genetic studies , so have long been of interest to geneticists – . compared with outbred popula- tions, population isolates generally have reduced genetic diversity due to founder effects , . individuals from isolated populations may also share more uniform environments, decreasing the non-genetic sources of variance contributing to complex traits. some population isolates also have well-maintained genealogical records, which can facilitate family ascertainment and estimation of population parameters. previous studies in population iso- lates have successfully identified genes and variants associated with mendelian and complex traits , . anabaptists represent a genetic isolate comprising several groups, including amish and mennonites, whose european ancestors settled in the americas starting in the th century . owing to religious conviction, cul- tural differences, and rural living, most anabaptists have remained genetically distinct, despite rapid popu- lation expansion in recent decades , . a number of otherwise rare mendelian disorders have been identified among anabaptists, with distinctive spectra of observed mutations characteristic of strong founder effects , . high-throughput sequencing has renewed interest in the study of anabaptist groups as a means of identifying genes underlying common, complex traits , . since whole genome sequencing (wgs) is still expensive in large samples, many study designs rely on geno- type imputation of unsequenced individuals. several existing methods can use a framework of common variants human genetics branch, national institute of mental health intramural research program, bethesda, md, , usa. department of genetics, perelman school of medicine, university of pennsylvania, philadelphia, pa, , usa. institute for systems biology, seattle, wa, , usa. division of endocrinology, diabetes and nutrition, department of medicine, university of maryland school of medicine, baltimore, md, , usa. neurogenomics division, translational genomics research institute, phoenix, az, , usa. john p. hussman institute for human genomics, miller school of medicine, university of miami, miami, fl, , usa. institute for computational biology, case western reserve university, cleveland, oh, , usa. department of anthropology, university of kansas, lawrence, ks, , usa. correspondence and requests for materials should be addressed to l.h. (email: liping.hou@nih.gov) or f.j.m. (email: mcmahonf@mail.nih.gov) received: january accepted: may published online: july open correction: author correction http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - x mailto:liping.hou@nih.gov mailto:mcmahonf@mail.nih.gov https://doi.org/ . /s - - - www.nature.com/scientificreports/ scientific reports | : | doi: . /s - - - (obtained, e.g., from a snp array) to infer genotypes from local haplotypes represented within a reference panel of sequenced individuals . common alleles can be imputed with high accuracy, but imputation of rarer alleles can be challenging . in isolated populations, imputation of rarer alleles can be improved by population specific reference panels that better represent founder alleles and haplotypes , . here we present phase of the anabaptist genome reference panel (agrp), the first whole-genome, population-specific imputation reference panel drawn from people of anabaptist ancestry. the agrp is built upon high-depth wgs data obtained from individuals of amish or mennonite ancestry. we describe the population structure and allele frequency spectra represented by the agrp and estimate the impact of this refer- ence panel on the imputation accuracy of variants across the full range of allele frequencies. results sequencing and variant yield. within each sequenced individual, we identified an average of . million autosomal variants, including , non-synonymous snvs, indels, and putative loss of function (lof) variants. in total, we identified ~ . million variants across all anabaptist individuals, including , indels and , putative lof variants (table  ). about . % of the identified variants were novel (including ~ . % of snvs and ~ . % of indels), defined here as variants not present in kaviar , dbsnp (v ) or exac (v . ) . of all identified variants, , ( . %) overlapped with protein-coding sequences; , ( . %) of these were non-synonymous variants. overall, ~ . % of the identified variants were rare (maf < . %). however, a higher proportion of the non-synonymous variants (~ . %), and an even higher proportion of the lof variants (~ . %) were also rare. this is consistent with negative selection. genotype calls were highly consistent between duplicate samples and across genotype platforms. genotype concordance rates calculated based on two pairs of duplicate samples were . % for snvs and . % for indels and substitutions. snv concordance rate was . % within a subset of individuals whose dna was also geno- typed with the illumina omniexpress exome snp array (no indels or substitutions are represented on this array). using the same snp array data, we also estimated the sensitivity of the wgs platform to detect snvs. of the , high quality snv sites identified by the array, , ( . %) sites were also identified within our wgs data. the snv detection sensitivity was similar across a range of maf values (figure s ). many anabaptist populations suffer from high rates of otherwise rare mendelian diseases . thus we investi- gated whether variants seen only in the agrp were more deleterious, as estimated by the combined annotation dependent depletion (cadd) score , than variants of comparable frequencies that were also identified in the genomes project sample. indeed, both coding and non-coding variants seen only in the agrp were signif- icantly more deleterious (p < . ; fig.  ). the agrp contains some individuals with psychiatric or metabolic disorders, so we repeated this analysis in only the healthy individuals. the results were similar (data not shown). imputation. one of the main goals of this project was to provide a population-specific reference panel for genotype imputation in individuals of anabaptist ancestry. here we evaluated the accuracy with which variants of various maf could be imputed. in order to illustrate the specific contribution of the agrp to imputation accuracy, we reserved individuals from the agrp as a test subset and evaluated the results in four different reference panel configurations: ( ) the agrp alone; ( ) the g panel alone; ( ) a merged panel comprising both and ; and ( ) the haplotype reference consortium (hrc) reference panel. the merged agrp and g reference panel provided the highest imputation accuracy across all allele frequency bins (fig.  a). the greatest advantage was observed for rarer alleles. for example, alleles with a g maf < . % were imputed at an aggregate r of . with the merged agrp/ g panel, compared to . with the hrc panel, . with the agrp alone, and . with the g panel alone. the agrp also enabled imputation of a large number of variants apparently unique to anabaptists. overall, ~ . % of variants identified in the agrp were not present in the g and ~ . % were not present in the genome snv indels lofsynonymous non- synonymous total variants number of variants . million , , , , novelty ratea . % . % . % . % . % total variation by frequencyb common (maf > %) . % . % . % . % . % low frequency (maf . – %) . % . % . % . % . % rare (maf < . %) . % . % . % . % . % variants per individual th percentile , , , , , average , , , , , th percentile , , , , , table . discovered autosomal variants. aversus kaviar, dbsnp , and exac. ballele frequencies estimated in agrp. http://s www.nature.com/scientificreports/ scientific reports | : | doi: . /s - - - hrc panel. although some ( . %) of these anabaptist-specific variants were very rare and thus un-imputable, the remaining variants could be imputed well, with aggregate r values in the . – . range (fig.  b). genetic differentiation. principal components analysis (pca) showed that individuals in the agrp clustered with ceu participants from the hapmap project (fig.  ), as expected. in agreement with the pca results, the overall correlation of maf values in the agrp and the european-ancestry subset of g was high (r = . , figure s ). for uncommon variants, however, the correlation was much lower. variants with g maf < % were only weakly correlated with maf estimated in agrp (r = . ). consistent with this result, , variants were rare or absent in g (maf < . %) but reached frequencies > % in agrp (table s ). it is widely believed that founder effects (i.e., population bottlenecks and genetic drift) influence the structure of anabaptist populations , , but the magnitude of these influences is largely unknown. since founder effects lead to depletion of variants that are rare in the ancestral population , we sought to estimate the footprint of founder effects in today’s anabaptists by assessing depletion of rare variants in agrp. the numbers of variants detected figure . variants seen only in agrp are more deleterious. mean combined annotation dependent depletion (cadd) scores for agrp-specific variants (red) and those shared with the g sample (blue), annotated as non-coding (solid lines) or coding (dotted lines), and binned by minor allele frequency. figure . imputation of shared and agrp-specific variants. (a) imputation accuracy (aggregate squared pearson correlation coefficient, r ), across various reference panel configurations. the x-axis (log scale) shows the g eur (non-refernce) allele frequency of the variants being imputed. (b) the stacked bars show proportions of variants found in agrp (left y-axis) that were also shared by various reference panels across a range of (non-reference) allele frequency bins in the agrp. the embedded black line shows the imputation accuracy of agrp-specific variants (right y-axis) when the merged agrp/ g reference panel was used. http://s http://s www.nature.com/scientificreports/ scientific reports | : | doi: . /s - - - in unrelated individuals in agrp (defined as less than third-degree relatives by king ) were compared to those detected in european-ancestry subsets of the g panel that were matched to agrp on sample size. a total of ~ . m rare variants (maf ≤ . %) are reported among the unrelated european-ancestry samples in the g panel. the subsets of unrelated europeans sampled from g carried a mean of , k ± k of these rare variants. among the unrelated anabaptists in agrp, however, only k rare variants were identified (fig.  ). this suggests a substantial depletion of rare variants in anabaptists compared to outbred europeans. the agrp also contained significantly fewer variants with maf between . % and %, but the depletion compared to g was modest. the number of common variants (maf > %) was similar in both groups (fig.  ). the improved imputation accuracy conferred by the agrp could be attributed, at least in part, to increased distant relatedness and reduced genetic diversity, both thought to characterize people of anabaptist ancestry , . to investigate distant relatedness, we compared the proportion of the genome shared identical by descent (ibd) among individuals within the agrp to that shared between agrp and ancestral europeans in the g. ibd sharing was substantially greater within the agrp than within g (fig.  ): % of agrp pairs shared at least % of the genome ibd, while > % of g pairs shared almost none of the genome ibd (< . %). furthermore, very little ibd sharing (< . %) was detected between individuals in agrp and individuals in g, demonstrating that the agrp provides a sample of the ancestral european genome that is largely dis- tinct from that represented by the g. to investigate one measure of genetic diversity, we compared the total length of the genome shared homozy- gous by descent (hbd) among individuals within the agrp to that shared between agrp and europeans in the g. total hbd length was significantly greater in agrp than in g (p < . ; fig.  ). total hbd figure . plots of the first two principal components derived from the agrp samples. (a) agrp samples (black dots) are plotted along with european (ceu, red dots), asian (chb + jpt, violet dots), and african (yri, green dots) samples taken from hapmap phase . (b) mennonite (grey dots), midwestern amish (blue dots), and lancaster county amish (orange dots) within the agrp, and ceu (red dots) from hapmap phase . figure . variant counts by maf group. the error bars for eur (europeans) were estimated based on sub- samples of randomly-selected europeans in the g sample. www.nature.com/scientificreports/ scientific reports | : | doi: . /s - - - length varied among the anabaptist groups represented in the agrp. the longest hbd regions were observed among amish living in lancaster county, pennsylvania, followed by amish living in the midwestern united states. mennonites in agrp carried hbd regions that were only slightly longer on average than those observed in g. discussion imputation of snp-array genotypes against a suitable reference panel represents an efficient and cost-effective strategy for investigating genetic variants not directly represented on inexpensive snp arrays. however, cur- rent reference panels do not always perform well for rare variants and do not capture all of the rare variation present in human populations . our findings demonstrate the value of a population-specific reference panel for studies of anabaptist founder populations. the agrp effectively boosted imputation accuracy, especially for variants that are relatively rare. the increased imputability of rare variants seems to be explained by several key factors: ) increased frequency of some otherwise rare alleles, presumably due to drift; ) distant relatedness and decreased genetic diversity among anabaptists, so that each rare allele is more likely to be represented on a figure . increased distant relatedness among anabaptists. the distribution, over all pairs of individuals, of the fraction of the genome shared ibd (segment lengths ≥ cm) either within agrp (anabaptist, blue), within europeans (eur, grey), or between anabaptists and eur (orange). figure . reduced genetic diversity within anabaptists. total length of the genome homozygous by descent (hbd) within europeans from the g project (eur) and within various anabaptist groups represented in the phase i agrp. www.nature.com/scientificreports/ scientific reports | : | doi: . /s - - - single founder haplotype; ) increased phasing accuracy due to family-relationship aware phasing methods and increased homozygosity by descent, which also increases the length of imputable haplotypes. the agrp performed best when merged with an existing large reference panel from the g project. similar results have been observed in studies of other population-specific reference panels . increasing sample size will likely continue to improve imputation performance, consistent with what has been found by the g project and the haplotype reference consortium . most important for studies of anabaptist populations, the agrp enables imputation of a large number of variants apparently unique to anabaptists. we have shown that such variants tend to be more deleterious than variants of similar maf that are also seen outside anabaptist populations. footprints of the unique histories of anabaptist populations are evident within the whole genome sequences represented by the agrp. common variants showed comparable allele frequencies in the agrp and g european panel, consistent with previous findings . for rare variants, however, the allele frequencies were often quite different. many variants rare or absent in the g panel reached appreciable frequencies in the agrp. on the other hand, many rare variants found in the g panel were not detected in the agrp, suggesting that these variants did not survive the population bottlenecks that occurred early in the anabaptist population history or were lost due to random drift. the substantial reduction of rare variants we observed in the agrp is consistent with previous studies of other founder populations , , but has not, to our knowledge, been previously demonstrated within anabaptists. since we did not sequence a comparable group of outbred europeans in the present study, some of the differences we observed in the abundance of rare variants may be attributable to the sequencing platform and data analysis pipeline we used. however, it is unlikely that methodological differences alone can account for the substantial depletion of rare variants observed within the agrp, since we detected no substantial difference in the sensitivity of variant calls across a broad range of minor allele frequencies (figure s ). principal components analysis (pca) analysis showed that the anabaptist sample we studied was closely clustered with the northern and western european ancestry samples from hapmap. this is not surprising in light of the known recent european ancestry of anabaptist groups. however, a finer scale pca analysis was able to dis- tinguish several different sub-populations within the agrp, which corresponded roughly to known geographic and religious distinctions that have influenced anabaptist mating patterns for most of the last centuries . similar differences were also seen in the hbd analysis. these results suggest that the agrp may perform better when used with studies of the amish, but should still have value in other anabaptist populations. the agrp thus com- plements ongoing projects, such as topmed (g.r. abecasis et al., ashg, abstract) that include individuals drawn from the amish communities in lancaster, pa and surrounding areas. this study has several limitations. first, the sample size is relatively small. this reduces representation of rare alleles and haplotypes. however, this only leads to underestimation of the value of the agrp for imputation of rare alleles. the sample size also limits precision of allele frequency estimates, especially for alleles that are rare in anabaptists. while important, this does not vitiate the agrp for filtering variants that have drifted to high frequencies in anabaptist populations and are thus unlikely to be highly deleterious. second, allele frequen- cies should ideally be estimated separately for each subpopulation identified by the pca analysis, but this will require a much larger sample size, especially for rare variants. third, this phase i of the agrp mainly represents individuals of amish ancestry. mennonites, brethren, hutterites, and other anabaptist groups are not well rep- resented. phase ii of the agrp is underway and will include a better representation of mennonites, including those of dutch-german ancestry (so-called “russian mennonites”.) fourth, the agrp is not a randomly selected sample of anabaptists, but was based on sequences collected in the course of various common disease studies. while an epidemiological sample ascertained without regard to phenotype has advantages, inclusion of cases may actually increase the value of the agrp for future studies focused on the same (or related) diseases, since any disease-associated alleles will presumably be better represented. intuitively, inclusion of people with diseases caused by alleles of large effect could affect imputation of nearby alleles, but this is unlikely to be an important factor for the common, complex diseases represented within the agrp. indeed, a previous study concluded “no loss of imputation quality was observed using a panel built from phenotypic extremes” . we hope that the agrp will facilitate a broad range of genomic studies in anabaptist communities. the agrp is available through an imputation server at https://www.nimh.nih.gov/labs-at-nimh/research-areas/ clinics-and-labs/hgb/data-downloads.shtml. methods sample selection. wgs data were submitted by each of the collaborating groups, including: individuals of amish or mennonite ancestry collected by the human genetics branch, nimh irp and submitted by fjm, lh, and isb ( of whom suffered from a mood disorder), individuals of amish or mennonite ancestry col- lected through the university of maryland’s amish research clinic in lancaster, pa and submitted by ars & jro ( of whom suffered from hypertension or a metabolic disorder); and individuals of amish ancestry collected by janice egeland and colleagues and submitted by mb & rlk ( of whom suffered from bipolar disorder). all participants provided informed consent that explicitly allowed their coded genome sequencing data to be shared with other investigators. the final data set comprised wgs data from individuals, including two pairs of duplicate samples that were used to evaluate sequencing and genotype accuracy. sequencing and data generation. dna derived from whole blood or from lymphoblastoid cell lines was obtained from the rutgers university cell and dna repository (rutgers, nj), the corriell institute (camden, nj), or the university of maryland amish research program. wgs was performed to > × coverage by complete genomics, inc. (cgi) using cgi’s combinatorial probe–anchor ligation (cpal) method . snvs, http://s https://www.nimh.nih.gov/labs-at-nimh/research-areas/clinics-and-labs/hgb/data-downloads.shtml https://www.nimh.nih.gov/labs-at-nimh/research-areas/clinics-and-labs/hgb/data-downloads.shtml www.nature.com/scientificreports/ scientific reports | : | doi: . /s - - - insertions, and deletions were called with the cgi analysis pipeline version . , . or . (described in refs , ), relative to the human reference genome, grch . the cga tools mkvcf command was used by each group to generate vcf files, which include all sites where at least one subject carried a non-reference allele. to confirm that the vcf files included the same types of variants and the genotype data had the same format, the same parameters (–source-names mastervar–field-names gt, ft, gq, gl, dp, ad) were used by each group. the “–source-names mastervar” parameter asks the cga tools to extract variants only from the mastervar files, so that only snvs and short indels are included in the vcf files, while cnvs, svs, and mobile element insertions (meis) are excluded. the “–field-names gt, ft, gq, gl, dp, ad” parameter asks cga tools to extract more information for each genotype call. particularly, for non-reference genotype calls, cga tools extracts gt (genotype), ft (genotype filters), gq (genotype quality), gl (genotype likelihood), dp (total read depth), and ad (allelic depths) from the raw data. previous studies have shown that false positive rates are higher for cnvs, svs and meis than for snvs in cgi data , so these classes of variation were excluded from the reference panel. quality control and variant annotation. all multi-allelic variants and variants located on non-autosomal chromosomes were removed from all three vcf files. all low-quality (marked by mkvcf as low quality; genotype quality < ; allele balance ratio < . or > . (applies to heterozygotes only); read depth < ) and half-called genotypes were set to ‘missing’. to make sure all variants were consistently represented in each vcf file, we normalized all vcf files using the vt variant tool set . we then merged all vcf files and back-filled missing genotypes based on individual mastervar files. all mendelian-inconsistent genotypes were set to missing. after qc, some variant sites no longer showed non-reference alleles; these were excluded. as the final qc step, vari- ants with a missing rate > %, mendelian error rate > % (based on trios), or hardy-weinberg equilibrium p < . × − were also excluded. we assessed the accuracy of the sequencing data by: ) comparing the genotype concordance rate within two pairs of duplicate samples; and ) comparing the genotype concordance rate across sequenced samples that were also genotyped with the illumina omniexpress exome snp array. we also used the same snp array data to estimate the power of the sequencing pipeline to detect snvs. particularly, we calculated the fraction of high-quality variant calls (minor allele count > , missing rate < %, hwe p > . ) detected by snp array that were also identified in the cgi data. all variants called in the agrp were annotated with combined annotation dependent depletion (cadd) raw scores , ensembl gene annotation, and g (aug ) allele frequencies, using annovar . variants were split into either ‘coding’ or ‘non-coding’ according to the ensembl gene model, and categorized as “shared” if they were also identified in the g. otherwise variants were categorized as “agrp-specific.” the wilcoxon rank-sum test was used to test variants for association between sharing status and raw cadd scores. since cadd scores are highly correlated with functional annotation and allele frequency , separate comparisons were carried out for coding and non-coding variants within each of agrp allele frequency bins. reference panel construction. to build the agrp, we first phased the genomes with shapeit . all known relationships were provided for phasing and the–duohmm option was enabled to make shapeit incor- porate pedigree information into the haplotype estimates, which has been shown to greatly improve the phasing accuracy . in order to keep as many as haplotypes as possible but remove duplicate haplotypes for the final reference panel, we used king to extract individuals who were unrelated at the first-degree level. thus phased genomes were extracted to build the reference panel. imputation. to assess imputation accuracy, we randomly selected a “test set” of individuals from the agrp. in the test set, autosomal genotypes were masked at all variable sites not represented on the illumina human omniexpress snp array. we chose this array since it represents an inexpensive and widely used array with over k snps common among people of european ancestry (http://www.illumina.com/products/human_ omni_express_beadchip_kits.html). masked sites were then imputed by impute against: ) the remaining agrp, ) the latest (phase ) genomes project ( g) reference panel alone, and ) a merged panel comprising both the agrp and the g reference panel. we also uploaded the phased vcf file of the test set to the sanger imputation service (https://imputa- tion.sanger.ac.uk/) and imputed the masked sites with the pbwt pipeline against the haplotype reference consortium (r . ) reference panel . variant sites were divided into bins, based on maf in the g panel. imputation accuracy was measured within each maf bin as the aggregate squared pearson correlation coefficient (r ) between imputed and actual allele dosages at the masked sites. to ensure comparability across the various reference panel configurations, only variants present in all panels were included in this comparison. allele frequencies estimation and population structure. the best linear unbiased estimator (blue) , designed to estimate allele frequencies for complex pedigrees, was used to estimate the allele frequencies for all variants identified in the anabaptists. to infer population structure, principal-component analysis (pca) was performed using ~ k ld-pruned snps. since the sample included related individuals, we used a method called principal components analysis in related samples (pc-air) , which identifies principal components that accurately capture population structure, rather than family structure. we ran pc-air twice: once with hapmap phase data (from yri, ceu, and chb + jpt) and once with the ceu subset only. we did this to illustrate how agrp individuals compare to the hapmap reference groups and to explore population structure within agrp. founder effects may lead to depletion of rare variants . to explore this phenomenon in the agrp, we first extracted a list of unrelated individuals from the full set of anabaptists using king . we kept only one http://www.illumina.com/products/human_omni_express_beadchip_kits.html http://www.illumina.com/products/human_omni_express_beadchip_kits.html https://imputation.sanger.ac.uk/ https://imputation.sanger.ac.uk/ www.nature.com/scientificreports/ scientific reports | : | doi: . /s - - - individual from each cluster of those with inferred relationships up to the rd degree, leaving unrelated anabaptists for this analysis. using the same method, we also extracted all unrelated individuals from the european genomes in the final release of the g project, then randomly selected of these for compari- son. we compared the counts of variants in these g individuals with counts in the anabaptists, across various allele-frequency bins. we performed this comparison times by randomly selecting (with replacement) individuals from the unrelated europeans in g. by this procedure, we hoped to distinguish a true depletion of rare variants – which should be confined to variants with lower allele frequencies – from differences attributable to sequencing platform or variant calling pipeline, which should affect a broad range of allele frequen- cies, or differences due simply to sample size. we used only snvs in this analysis, since other variant classes are called less accurately and are more sensitive to differences in sequencing platforms and pipelines. identity-by-descent (ibd) and homozygosity-by-descent (hbd). population isolates such as the anabaptists often show increased ibd and hbd, owing to founder effects, inbreeding, and other population forces . such increases may increase imputation accuracy by improved phasing . to assess these phenomena in the agrp, we identified ibd and hbd regions with ibdseq , a method designed specifically to identify such regions in wgs data. to compare the ibd sharing among anabaptists, among other europeans, and between anabaptists and other europeans, we merged wgs data from the agrp with wgs data from the other europeans in the g project and used only the overlapping variants. we also compared the total length of hbd regions in both groups. references . peltonen, l., palotie, a. & lange, k. use of population isolates for mapping complex traits. nature reviews genetics , – ( ). . arcos-burgos, m. & muenke, m. genetics of population isolates. clinical genetics , – ( ). . hatzikotoulas, k., gilly, a. & zeggini, e. using population isolates in genetic association studies. briefings in functional genomics , – ( ). . kristiansson, k., naukkarinen, j. & peltonen, l. isolated populations and complex disease gene identification. genome biology , ( ). . peltonen, l., jalanko, a. & varilo, t. molecular genetics of the finnish disease heritage. human molecular genetics , – ( ). . sidore, c. et al. genome sequencing elucidates sardinian genetic architecture and augments association analyses for lipid and blood inflammatory markers. nature genetics , – ( ). . nolt,, s. m. a history of the amish: third edition good books ( ). . strauss, k. a. & puffenberger, e. g. genetics, medicine, and the plain people. annual review of genomics and human genetics , – ( ). . hou, l. et al. amish revisited: next-generation sequencing studies of psychiatric disorders among the plain people. trends in genetics: tig , – ( ). . mckusick, v. a., egeland, j. a., eldridge, r. & krusen, d. e. dwarfism in the amish i. the ellis-van creveld syndrome. bulletin of the johns hopkins hospital , – ( ). . puffenberger, e. g. genetic heritage of the old order mennonites of southeastern pennsylvania. american journal of medical genetics part c, seminars in medical genetics c, – ( ). . georgi, b. et al. genomic view of bipolar disorder revealed by whole genome sequencing in a genetic isolate. plos genetics , e ( ). . marchini, j. & howie, b. genotype imputation for genome-wide association studies. nature reviews genetics , – ( ). . carmi, s. et al. sequencing an ashkenazi reference panel supports population-targeted personal genomics and illuminates jewish and european origins. nature communications , ( ). . glusman, g., caballero, j., mauldin, d. e., hood, l. & roach, j. c. kaviar: an accessible system for testing snv novelty. bioinformatics , – ( ). . lek, m. et al. analysis of protein-coding genetic variation in , humans. nature , – ( ). . kircher, m. et al. a general framework for estimating the relative pathogenicity of human genetic variants. nature genetics , – ( ). . crawford, m. h., dykes, d. d. & polesky, h. f. genetic structure of mennonite populations of kansas and nebraska. human biology , – ( ). . chheda, h. et al. whole genome view of the consequences of a population bottleneck using genome sequences from finland and united kingdom. biorxiv ( ). . manichaikul, a. et al. robust relationship inference in genome-wide association studies. bioinformatics , – ( ). . khoury, m. j., cohen, b. h., diamond, e. l., chase, g. a. & mckusick, v. a. inbreeding and prereproductive mortality in the old order amish. i. genealogic epidemiology of inbreeding. american journal of epidemiology , – ( ). . pistis, g. et al. rare variant genotype imputation with thousands of study-specific whole-genome sequences: implications for cost- effective study designs. european journal of human genetics: ejhg , – ( ). . genome of the netherlands c. whole-genome sequence variation, population structure and demographic history of the dutch population. nature genetics , – ( ). . genomes project, c. et al. a global reference for human genetic variation. nature , – ( ). . mccarthy, s. et al. a reference panel of , haplotypes for genotype imputation. biorxiv ( ). . van hout, c. v. et al. extent and distribution of linkage disequilibrium in the old order amish. genetic epidemiology , – ( ). . lim, e. t. et al. distribution and medical impact of loss-of-function variants in the finnish founder population. plos genetics , e ( ). . duan, q. et al. imputation of coding variants in african americans: better performance using data from the exome sequencing project. bioinformatics , – ( ). . drmanac, r. et al. human genome sequencing using unchained base reads on self-assembling dna nanoarrays. science , – ( ). . carnevali, p. et al. computational techniques for human genome resequencing using mated gapped reads. journal of computational biology: a journal of computational molecular cell biology , – ( ). . tan, a., abecasis, g. r. & kang, h. m. unified representation of genetic variants. bioinformatics , – ( ). . wang, k., li, m. & hakonarson, h. annovar: functional annotation of genetic variants from high-throughput sequencing data. nucleic acids research , e ( ). www.nature.com/scientificreports/ scientific reports | : | doi: . /s - - - . delaneau, o., zagury, j. f. & marchini, j. improved whole-chromosome phasing for disease and population genetic studies. nature methods , – ( ). . o’connell, j. et al. a general approach for haplotype phasing across the full spectrum of relatedness. plos genetics , e ( ). . durbin, r. efficient haplotype matching and storage using the positional burrows-wheeler transform (pbwt). bioinformatics , – ( ). . mccarthy, s. et al. a reference panel of , haplotypes for genotype imputation. nature genetics , – ( ). . mcpeek, m. s., wu, x. & ober, c. best linear unbiased allele-frequency estimation in complex pedigrees. biometrics , – ( ). . conomos, m. p., miller, m. b. & thornton, t. a. robust inference of population structure for ancestry prediction and correction of stratification in the presence of relatedness. genetic epidemiology , – ( ). . kong, a. et al. detection of sharing by descent, long-range phasing and haplotype imputation. nature genetics , – ( ). . browning, b. l. & browning, s. r. detecting identity by descent and estimating genotype error rates in sequence data. american journal of human genetics , – ( ). acknowledgements this research was funded by the national institute of mental health (nimh) intramural research program (zia-mh ; nct ). nih: miami and cwru samples in phase ii were collected with support from r ag (jlh), r ag (wks) and r ey (mpv). this work utilized the computational resources of the nih hpc biowulf cluster (http://hpc.nih.gov). author contributions f.j.m. supervised and coordinated the anabaptist genome reference panel project. r.l.k., j.c.r., j.r.o., m.b., w.k.s., m.p.-v., j.l.h., m.h.c., a.r.s., and f.j.m. provided whole genome sequence data. l.h. and r.l.k. performed in-depth data analyses. l.h. and f.j.m. wrote and revised the manuscript. r.l.k., j.c.r., j.r.o., d.w.c., m.b., w.k.s., m.p.-v., j.l.h., m.h.c., a.r.s. provided critical amendments and edited the manuscript. additional information supplementary information accompanies this paper at doi: . /s - - - competing interests: alan r. shuldiner is an employee of regeneron genetics center, regeneron pharmaceuticals, inc. the other authors declare no competing financial interests. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the cre- ative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article’s creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article’s creative commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. © the author(s) http://hpc.nih.gov http://dx.doi.org/ . /s - - - http://creativecommons.org/licenses/by/ . / 	a population-specific reference panel empowers genetic studies of anabaptist populations
 	results
 	sequencing and variant yield. 	imputation. 	genetic differentiation. 	discussion
 	methods
 	sample selection. 	sequencing and data generation. 	quality control and variant annotation. 	reference panel construction. 	imputation. 	allele frequencies estimation and population structure. 	identity-by-descent (ibd) and homozygosity-by-descent (hbd). 	acknowledgements
 	figure variants seen only in agrp are more deleterious. 	figure imputation of shared and agrp-specific variants. 	figure plots of the first two principal components derived from the agrp samples. 	figure variant counts by maf group. 	figure increased distant relatedness among anabaptists. 	figure reduced genetic diversity within anabaptists. 	table discovered autosomal variants. [pdf] mapping biochemistry to metabolism: fdg-pet and amyloid burden in alzheimer's disease. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / - - corpus id: mapping biochemistry to metabolism: fdg-pet and amyloid burden in alzheimer's disease. @article{mega mappingbt, title={mapping biochemistry to metabolism: fdg-pet and amyloid burden in alzheimer's disease.}, author={m. mega and t. chu and j. mazziotta and k. h. trivedi and p. thompson and a. shah and g. cole and s. frautschy and a. toga}, journal={neuroreport}, year={ }, volume={ }, pages={ - } } 	m. mega, t. chu, + authors a. toga 	published 	biology, medicine 	neuroreport we evaluated the relationship between amyloid-beta protein (a beta) concentration and the metabolic abnormality in an alzheimer's disease (ad) patient as measured by [ f]fluorodeoxyglucose positron emission tomography (fdg-pet). across most regions there were significant inverse correlations among fdg-pet intensity values and both insoluble. the temporal lobe samples showed no significant correlation between fdg-pet values and a beta deposition. findings support a beta as contributing to the… expand view on pubmed alzheimer.neurology.ucla.edu save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations view all topics from this paper 	fluorodeoxyglucose f 	alzheimer's disease 	positron-emission tomography 	temporal lobe 	positrons 	brain diseases, metabolic 	biochemistry 	contribution citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency β-amyloid is associated with aberrant metabolic connectivity in subjects with mild cognitive impairment 	f. carbonell, arnaud charil, a. zijdenbos, alan c. evans, b. bedell 	psychology, medicine 	journal of cerebral blood flow and metabolism : official journal of the international society of cerebral blood flow and metabolism 	 	 	pdf save alert research feed spatially distributed amyloid-β reduces glucose metabolism in mild cognitive impairment 	f. carbonell, a. zijdenbos, b. bedell 	medicine 	journal of alzheimer's disease : jad 	 	 	pdf save alert research feed assessment of change in glucose metabolism in white matter of amyloid-positive patients with alzheimer disease using f- fdg pet 	y. jeong, h. j. yoon, d. kang 	medicine 	medicine 	 	 	 save alert research feed basal cerebral metabolism may modulate the cognitive effects of aβ in mild cognitive impairment: an example of brain reserve 	a. cohen, j. price, + authors w. klunk 	psychology, medicine 	the journal of neuroscience 	 	 	highly influenced 	pdf 	view excerpt save alert research feed the art of loss: aβ imaging in the evaluation of alzheimer’s disease and other dementias 	v. villemagne, m. fodero-tavoletti, k. pike, r. cappai, c. masters, c. rowe 	psychology, biology 	molecular neurobiology 	 	 	view excerpt, cites background save alert research feed relations between hypometabolism in the posterior association neocortex and hippocampal atrophy in alzheimer's disease: a pet/mri correlative study 	k. meguro, c. lemestric, b. landeau, b. desgranges, f. eustache, j. baron 	psychology, medicine 	journal of neurology, neurosurgery, and psychiatry 	 	 	pdf save alert research feed tracking the decline in cerebral glucose metabolism in persons and laboratory animals at genetic risk for alzheimer's disease 	e. reiman, r. caselli, g. alexander, k. chen 	psychology 	clinical neuroscience research 	 	 save alert research feed pathologic lesions in neurodegenerative diseases. 	p. thompson, h. vinters 	medicine 	progress in molecular biology and translational science 	 	 save alert research feed neuroimaging alzheimer ’ s disease 	a. toga, m. mega, p. thompson, j. morris 	 	pdf save alert research feed delayed rather than decreased bold response as a marker for early alzheimer's disease 	s. rombouts, r. goekoop, c. stam, f. barkhof, p. scheltens 	psychology, computer science 	neuroimage 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency β-amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage 	j. koh, l. yang, c. cotman 	biology, medicine 	brain research 	 	 save alert research feed selective increase in lipid peroxidation in the inferior temporal cortex in alzheimer's disease 	a. palmer, m. burns 	biology, medicine 	brain research 	 	 save alert research feed neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. 	b. yankner, l. duffy, d. kirschner 	biology, medicine 	science 	 	 , save alert research feed oxidative damage to mitochondrial dna is increased in alzheimer's disease 	p. mecocci, u. macgarvey, m. beal 	biology, medicine 	annals of neurology 	 	 save alert research feed implementation and evaluation of a calculated attenuation correction for pet 	s. siegel, m. dahlbom 	physics 	conference record of the ieee nuclear science symposium and medical imaging conference 	 	 save alert research feed mri‐pet registration with automated algorithm 	r. woods, j. mazziotta, and simon r. cherry 	medicine 	journal of computer assisted tomography 	 	 , 	pdf save alert research feed differential effects of electrical stimulation of sciatic nerve on metabolic activity in spinal cord and dorsal root ganglion in the rat. 	m. kadekaro, a. crane, l. sokoloff 	chemistry, medicine 	proceedings of the national academy of sciences of the united states of america 	 	 	pdf save alert research feed statistical data analysis in the computer age 	b. efron, r. tibshirani 	computer science, medicine 	science 	 	 , 	pdf save alert research feed ieee trans med imaging 	ieee trans med imaging 	 ann neurol 	ann neurol 	 ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue e d i t o r i a l ellis-van creveld syndrome and congenital heart defects: presentation of an additional cases matthew j. o’connor • r. thomas collins ii received: september / accepted: december / published online: january � springer science+business media, llc we read with interest the recent article in pediatric car- diology by hills et al. [ ]: ellis-van creveld syndrome and congenital heart defects: presentation of an additional cases. the authors are to be commended for their work, which sheds further light on the breadth and complexity of congenital heart disease (chd) seen in patients with ellis-van creveld syndrome (evc). we agree with the authors that the diagnostic precision afforded by the pediatric cardiac care consortium (pccc) database is of great value in its application to evc because such anatomic detail has not been provided in prior clinical reports. for a syndrome with a – % incidence of chd, the detailed focus on chd in this paper is of value to pediatric cardiologists, cardiovascular surgeons, and other providers involved in the care of children with evc. with respect to outcomes after chd surgery for patients with evc, however, some of the results described by hills et al. [ ] were unclear to us. we recently reported outcomes of chd surgery for children with evc during the recent era in a smaller, mostly amish, population [ ]. similar to the findings by hills et al. [ ], we noted a relatively high incidence of systemic venous abnormalities ( %) in our study. how- ever, and in seeming contrast to the data reported by hills et al. [ ], we found that chd surgery for patients with evc was associated with a postoperative mortality rate of %, with the deaths occurring at a median of days (range, – days) postoperatively. this incidence is substan- tially higher than that reported in the study by hills et al. [ ], in which no postoperative deaths were reported. in our study, the majority of deaths were related to respiratory complications, presumably on account of the thoracic dystrophy that is a hallmark of evc. it is unclear to us how hills et al. [ ] evaluated follow- up and postoperative mortality in their series of patients. a major limitation of the pccc database is incomplete or missing follow-up data [ ]. in the study by hills et al. [ ], therefore, what was the duration of the follow-up period? a shorter follow-up period (i.e., days), used frequently to determine postoperative mortality, may not be sufficient to capture the true effect of chd surgery on the respiratory system in evc. although we certainly support the concept of repair or palliation of chd in patients with evc, we stress that surgery for chd in patients with evc is a high- risk undertaking. in our study, substantial mortality was not the only notable finding. in addition, all those surviv- ing surgery experienced postoperative respiratory morbid- ity (prolonged need for supplemental oxygen, prolonged mechanical ventilation, and tracheostomy). those involved in the care of children with evc must consider such data when making treatment decisions and counseling families. author responses i appreciate the opportunity to expand on the outcomes for the patients presented in our recent article, ellis-van creveld syndrome and congenital heart defects: presentation of an additional cases. drs. o’connor and collins have reported on an additional cohort of patients who underwent surgery for ellis-van creveld syndrome and demonstrated a higher incidence of postoperative mortality, usually sec- ondary to respiratory compromise due to thoracic dystrophy. the pediatric cardiac care consortium is indeed lim- ited in terms of the long-term follow-up data available. m. j. o’connor (&) � r. thomas collins ii university of arkansas for medical sciences, arkansas children’s hospital, little rock, ar , usa e-mail: mjoconnor@uams.edu pediatr cardiol ( ) : – doi . /s - - - however, the minimum duration of the follow-up period is to the time of discharge from the hospital even if this exceeds the traditional postoperative mortality limit of days. for some patients, additional follow-up data are available, with the reporting of subsequent cardiac cathe- terizations or surgeries. certainly, the patients in our study may represent a somewhat ‘‘healthier’’ subset of patients with ellis-van creveld syndrome in that they were considered appropriate candidates for cardiac catheterization or surgery. this is reflected in the fact that none of the patients admitted outside the newborn period in our study were ventilator dependent at the time of catheterization or surgery. morbidity also may be indirectly evaluated using hospital length of stay data. a significant proportion of admissions for cardiac catheterization alone (n = ) were for days or less ( %), with longer stays related to admissions on the day of birth (length of stay range, – days). as stated in our report, three patients died after cardiac catheterization without additional operative intervention to days sub- sequent to their initial procedure. two of these deaths were attributed to nonrespiratory causes (sepsis and new onset of overwhelming aortic insufficiency), whereas one patient died of cardiac arrest and restrictive chest wall disease. the hospital length of stay for surgery-only admissions (n = ) ranged from to days (overall average, . days). a trend toward shorter hospitalizations has been seen in recent years. in our study, only five admissions were longer than days, and patients were discharged within days after admission. although discharge sum- maries are not available in all cases for assessment of the need for home oxygen therapy, it may be surmised that a short hospital stay suggests a lack of need for prolonged mechanical ventilation. an additional four hospitalizations that included multiple catheterizations or surgeries had lengths of stay ranging from to days, with the longest hospitalization related to prematurity, twin gesta- tion, chronic lung disease, and ultimate need for tracheostomy. the age of the patient at the time of admission also proved to be an important factor in mortality. all three nonsurvivors in our study were younger than months at the time of death. the average age at the time of surgery for all the patients in our study was . years (range, days to years). it is certainly agreed that careful consideration of each patient’s clinical status must be included in the provision of appropriate presurgical counseling for parents of children with ellis-van creveld syndrome. i appreciate the addi- tional cases presented by drs. o’connor and collins and their reminder of the significant morbidities associated with thoracic dystrophy present in this population. christine b. hills department of pediatric cardiology, university of min- nesota, mmc delaware st. se, minneapolis, mn , usa references . hills cb, kochilas l, schimmenti la, moller jh ( ) ellis-van creveld syndrome and congenital heart defects: presentation of an additional cases. pediatr cardiol : – . o’connor mj, rider nl, collins rt, hanna bd, morton dh, strauss ka ( ) contemporary management of congenital malformations of the heart in infants with ellis-van creveld syndrome: a report of nine cases. cardiol young : – . pham pp, moller jh, hills c, larson v, pyles l ( ) cardiac catheterization and operative outcomes from a multicenter con- sortium for children with williams syndrome. pediatr cardiol : – pediatr cardiol ( ) : – 	ellis-van creveld syndrome and congenital heart defects: presentation of an additional cases 	author responses 	references hhe .indd fax + e-mail karger@karger.ch www.karger.com original paper hum hered ; : – doi: . / investigations of the y chromosome, male founder structure and ystr mutation rates in the old order amish toni i. pollin a daniel j. mcbride a richa agarwala b alejandro a. schäffer b alan r. shuldiner a, c braxton d. mitchell a jeffrey r. o’connell a a department of medicine, university of maryland school of medicine, baltimore, md. , b national center for biotechnology information, national institutes of health, department of health and human services, bethesda, md. , c geriatrics research and education clinical center, baltimore veterans administration medical center, baltimore, md. , usa only and . % using up to , observed and inferred meioses combined). conclusions: these data confirm the accuracy and completeness of the male lineage portion of the anabaptist genealogy database and contribute muta- tion rate estimates for several commonly used y chromo- some str markers. copyright © s. karger ag, basel the old order amish (ooa) of lancaster county, pennsylvania, are a closed founder population number- ing approximately , – , , nearly all of whom can trace their ancestors back to a small number of individu- als who immigrated to the united states in the mid- to late s [ , ] . an additional group of ooa immigrat- ed during this period to ohio and indiana, and later some lancaster ooa migrated westward as well. the ooa have a strong interest in their ancestry, and their genea- logical relationships are well-documented [ , ] . these attributes make the ooa an attractive population for ge- netic studies [ ] , and indeed they have been subjects of study of the genetics of both single gene disorders for over years [reviewed in ] and of complex traits for almost as long [ ] , but particularly in the last years by our group [ – ] and others [ , ] . in recent years, the ana- baptist genealogy database [ , , ] has been developed key words amish � str mutation rates � y chromosomes strs � genealogy � founder population abstract objectives: using y chromosome short tandem repeat (ystr) genotypes, ( ) evaluate the accuracy and complete- ness of the lancaster county old order amish (ooa) genea- logical records and ( ) estimate ystr mutation rates. meth- ods: nine ystr markers were genotyped in old order amish males who participated in several ongoing genetic studies of complex traits and could be connected into one of all-male lineage pedigrees constructed using the ana- baptist genealogy database and the query software ped- hunter. a putative founder ystr haplotype was constructed for each pedigree, and observed and inferred father-son transmissions were used to estimate ystr mutation rates. results: we inferred distinct founder y chromosome haplotypes in the male lineages, which encompassed surnames accounting for % of lancaster ooa house- holds. nearly all deviations from founder haplotypes were consistent with mutation events rather than errors. the es- timated marker-specific mutation rates ranged from to . % (average . % using up to observed meioses received: march , accepted: june , published online: september , toni i. pollin, ms, phd west redwood street room c baltimore, md (usa) tel. + , fax + , e-mail tpollin@medicine.umaryland.edu © s. karger ag, basel – / / – $ . / accessible online at: www.karger.com/hhe http://dx.doi.org/ . % f pollin /mcbride /agarwala /schäffer / shuldiner /mitchell /o’connell hum hered ; : – as a computerized resource searchable using the ped- hunter query software by groups with institutional re- view board approved protocols [ ] , which enhances the ability to construct pedigrees defining the relationships between amish subjects of genetic studies. these tools have been particularly useful for projects involving large study samples such as that of the university of maryland, which has recruited well over , lancaster old order amish individuals for several different studies, primarily of complex adult onset conditions including diabetes, os- teoporosis and cardiovascular disease. despite the mul- titude of genetic studies of the ooa, a rigorous analysis of the founder structure has yet to be reported. the work reported here focuses on the lancaster, pennsylvania ooa only. for convenience, throughout the rest of this report, ‘amish’ and ‘old order amish’ will be used to refer to the lancaster, pa old order amish population, which is a subject of study at the university of mary- land. the recent availability of numerous short tandem re- peat (str) markers on the non-recombining region of the y chromosome [ – ] has been applied to population genetics [ – ] , genealogy [ – ] and forensics [ – ] . the rate and mechanism of mutation of an str (whether autosomal or sex-linked) determines not only its degree of polymorphism but also its usefulness for a particular application. high mutation rates with both gain and loss of repeat elements are potential confound- ers in analyses requiring inferences in the presence of substantial missing data. for example, in linkage analysis hypermutable autosomal str mutations that lead to mendelian inconsistencies are often classified as geno- typing errors. while the high mutation rate of str markers also makes using them to track long-term evolutionary pat- terns difficult [ ] , their polymorphic nature makes them useful for distinguishing lineages, helping to understand relationships between lineages [ ] and clarifying recent demographic history [ , ] . by comparing the y chro- mosome haplotypes between male lineages, we would be able to confirm the accuracy of the genealogical records and also determine whether individuals with similar sur- names came from common founders. in addition to allowing us to confirm genealogical re- cords and estimate the number of male founders, such a large number of observed meioses in large families, as are present in the amish, allowed us to estimate mutation rates in the str markers. two approaches have been used to calculate mutation rates in y chromosome str mark- ers: large pedigrees with males connected through com- mon male lineages with observed/inferred meioses and father-son pairs. heyer et al. [ ] typed males from ‘deep rooting’ canadian pedigrees for strs and esti- mated individual marker mutation rates ranging from to . %, with an average of . %. however, since almost all of the meiotic events leading to the apparent mutation events were unobserved, mutations could not be confi- dently distinguished from nonpaternity (although jobling et al. [ ] partially addressed this concern using the mini- satellite marker msy ), and there was insufficient data to infer the direction of the mutations. subsequently, kayser et al. evaluated , meioses in loci in typed father/ son pairs that had undergone paternity testing and ob- served mutation events, for an overall mutation rate of . % [ ] , in contrast to bianchi et al., who found no mutations in , meioses in seven loci [ ] . dupuy et al. studied , confirmed father/son pairs and found an overall mutation rate of . % [ ] . several other investi- gators studying father/son pairs [ , , – ] obtained similar estimates. more recently, bonné-tamir et al. re- vived heyer’s method in male samples from the high- ly isolated israeli samaritan population, which has simi- larly detailed genealogical records as the amish, to arrive at an estimated mutation rate of . % [ ] . additionally similar estimates have been made using sperm samples [ ] . lower ‘evolutionary’ estimates have also been made using cross-population samples [ , , ] . the large number of amish males genotyped in our studies ( males at up to nine different str markers) coupled with extensive pedigrees enabled us to use a large number of genotyped father/son pairs as well as a larger number of transmissions inferred from the pedigree structure to evaluate mutation frequency. subjects and methods subjects as of april , a total of , subjects, including , males, had been recruited for several university of maryland studies. subjects consented to these studies via protocols ap- proved by the university of maryland institutional review board. the construction and usage of the anabaptist genealogy data- base is covered by a human subjects protocol overseen by an in- stitutional review board at nih. of the total , subjects, , subjects ( male) from the amish family diabetes study (afds) [ , ] , amish family osteoporosis/calcification study [ ] or amish osteogenesis imperfecta study were genotyped us- ing dna extracted from leukocytes by the nhlbi mammalian genotyping service for str markers ( cm scan) from sets and (nhlbi mammalian genotyping service), including nine markers on the y chromosome (see below). an additional ( male) subjects from the afds were genotyped in an earlier cm y chromosome studies in the amish hum hered ; : – scan using markers from set only, which included seven of the nine y chromosome markers. y marker data were thus avail- able on a total of males. marker genotyping markers typed by the nhlbi mammalian genotyping ser- vice in all subjects included dys / , dys , dys -i, dys -ii, dys , dys , and dys . the additional mark- ers dys and ggaat b were typed only in the males in the cm scan. because two lineages with no genealogical, his- torical or surname evidence of relatedness shared the same appar- ent nine marker founder haplotype, we sequenced in a subset of individuals in a subset of lineages three additional single copy markers which were chosen based on high diversity statistics cal- culated previously [ ] : dys , dys and dys . because we evaluated these markers in only a small number of individuals, we elected to sequence rather than genotype these markers to as- sure accurate allele calls. genealogy analysis the entire set of , male individuals enrolled in our studies was used in a query of the anabaptist genealogy database version (agdb ), a large searchable database including content from three amish genealogy sources [ , , , , ] . the query utilized the pedhunter software to connect all phenotyped males as far back as possible through male lineages only. statistical analysis founder allele/haplotype designation we used the genotypes of the typed individuals to designate a founder allele for each marker in each lineage. the founder was defined as the most recent common male ancestor (mrcma) for all genotyped individuals within a lineage. all but two lineages for one locus (dys in both cases) had unique putative founder alleles at each locus. for these two lineages at dys we desig- nated the founder allele as the one of two possible alleles that max- imized the number of distinct gene f lows with the fewest number of mutations that fit the data (see results; y chromosome haplo- types, for details). the putative founder haplotype was the set of founder alleles inferred in this manner. we note that for the two lineages above, the founder haplotypes are distinct from all other founder haplotypes regardless of which dys allele is chosen, implying that comparisons made below with regard to similarity of lineages are actually independent of the choice of founder al- lele. y chromosome haplotype reference data we used two publicly available databases for reference data on european y chromosome genotypes and haplotypes. the y-str haplotype reference database (yhrd), and its sister site, the y- str haplotype reference database for u.s. populations (ystr- us) [ ] (now combined into a single yhrd database [ ] ) are freely searchable but restrict submission of genotype data to fo- rensic laboratories that have passed a quality control exercise and are limited to a set of ten strs (dys , dys -i, dys -ii, dys , dys , dys , dys , dys ab, dys and dys ), which includes seven of the nine typed in our study. all samples submitted to str/str-us must minimally be typed for the first eight of the ten markers. in yhrd, any subset of the nine strs can be searched for matching haplotypes, and such search- es yield the worldwide prevalence of a given haplotype along with region- and ethnicity-specific prevalences. the yhrd (release ) currently includes , haplotypes in populations. to facilitate systematic comparison of our data to yhrd data, we downloaded the subset of , haplotypes in european pop- ulations available at the yhrd web site and used in a recent pub- lication describing the use of ystrs to describe european popu- lation history [ ] . another database, ybase: genealogy by numbers, allows un- restricted submission and searching for individual strs, in- cluding all nine typed in our study. however, haplotype searching in ybase requires at least eight markers and is of limited utility for estimating population prevalence since there is no particular minimal set of markers required for inclusion in the database, and denominators are not given for haplotype search results. ybase primarily provides surnames of matching haplotypes with lim- ited geographic information. the distribution maps show that most of the samples are sent by individuals and families residing in the eastern united states, the united kingdom, germany and switzerland. ybase provides periodically updated tables of allele frequencies for individual strs. pedigree errors and mutation analysis we initially reasoned that individuals with apparent non- founder alleles at multiple loci were most likely to represent ped- igree errors and investigated these cases further, including auto- somal loci, to confirm or refute this suspicion. after exclusion of pedigree errors, for a given locus within a lineage, if three or more individuals shared the same non-founder allele and had a mrcma who was not the root and furthermore all of this mrcma’s descendants possessed this same allele, this was con- sidered confirmatory evidence of a mutation. if the same were true in a set of two individuals, this was considered preliminary evidence of a mutation; sequencing of both the individual(s) pos- sessing the putative mutation and additional relatives if available was used to confirm the mutation. similarly, sequencing was used to confirm an apparent mutation appearing in a single individual. sequencing was also used to localize historical mutation events, even those appearing in clusters of three or more individuals, if dna was available from the relevant individuals. pcr product sizes were used with available sequence informa- tion (see below) to convert allele names to repeat lengths named according to standard nomenclature [ ] . a special case is dys -ii, which has the structure [tctg] n [tcta] m [ bp] [tctg] [tcta] q , of which the last portion, [tctg] [tcta] q , defines dys -i [ ] . we provide the repeat length for dys -i as +q (as is standard), and for dys -ii, we provide in table , which lists founder haplotypes, repeat length in the format n+m+ +q n+m , in order to preserve the yhrd nomenclature (n+m+ +q) while simultaneously providing the repeat length of the dys -ii specific segment (n+m) of the marker, which is used in some population and evolutionary studies. this repre- sentation enables our data to be readily compared with other publications and databases, which vary in their formatting of this marker. for example, a founder with the genotype [tctg] [tcta] [ bp][tctg] [tcta] would be denoted as dys -i = and dys -ii = . in discussing specific alleles and mutations in the dys -ii-specific segment in the text, we use the dys -ii-specific format n+m (dys -ii = in the preceding example). pollin /mcbride /agarwala /schäffer / shuldiner /mitchell /o’connell hum hered ; : – two methods were used to estimate mutation rates. first, for each marker, the number of discordant typed father-son pairs was divided by the total number of typed father-son pairs (observed meioses). in the second method, which increased the sample size but also the number of assumptions made, we inferred as many genotypes as possible in each lineage using available genotypes along with our inferred founder genotypes. we then divided the total number of mutation events by the number of inferred/ob- served father-son transmissions for each marker. binomial confi- dence intervals were calculated using the exact method as imple- mented in sas version . (cary, nc). str sequencing sequencing of str markers, including previously typed loci to confirm and/or localize mutations and three additional loci to distinguish lineages, was performed on an abi dna se- quencer. in some cases primers were designed to amplify a pcr product larger than that originally detected by the nhlbi mam- malian genotyping service to guarantee readable sequence with- in the repeat region. for dys , the primer set used by the nhlbi mammalian genotyping service and others, as a result of two binding sites of the forward primer, amplifies two products: the entire dys region, classically denoted as dys -ii, and the dys -i region contained within it. the dys forward pcr primer was redesigned to bind to a unique site upstream of the original upstream binding site so that the entire sequence was only amplified once, allowing us to view within our sequencing result distinct sequences for dys -i and the dys -ii-spe- cific portion. table . putative founder y str haplotypes: lineages are rank ordered by number of male individuals genotyped lineage dys dys dys dys -i dys -iia dys dys dys ggaat b dys nb mrcmac � d a subscript indicates length of the dys -ii specific segment. b number of individuals in lineage genotyped in initial genome scan; includes those with mutations but excludes apparent pedigree errors. c birth year of most recent common male ancestor of putative founder haplotype. d birth year estimated based on birth of first child. y chromosome studies in the amish hum hered ; : – results genealogy analysis querying agdb with pedhunter for all , male subjects resulted in male lineages. two lineages com- prised a total of three individuals with phenotype data, but no genotype data. within each of the genotyped lineages there was a unique surname after accounting for multiple spellings (e.g., stoltzfus/stoltzfoos, the most common amish surname). however, the converse, that each surname corresponded to a unique lineage, was not true. two surnames were each found in two separate lin- eages. the males with y chromosome str markers genotyped could be traced to of the founders, with each of the founders having from one to pheno- typed descendants and from one to descendants gen- otyped for all or some of the str markers. the distri- bution of founder descent of the genotyped individuals is shown in figure , along with the distribution of the cor- responding surnames in the address book of the lancaster county amish. seven founders accounted for % of these males, for % and for %. representativeness of our population sample to assess the representativeness of the general old or- der amish population by our sample, we compared the number of individuals genotyped from each male lineage with the number of families with each corresponding surname as indicated in the address book of the lancaster county amish. results are presented in fig- ure . our sampling of lineages appeared virtually com- plete; the surnames found in our collection of amish males accounted for % of all lancaster county old order amish households in the directory. the same eight surnames accounted for the majority of indi- viduals in our sample ( %) and the majority of house- holds ( %). y chromosome haplotypes after exclusion of pedigree/genotyping errors (see be- low), putative founder y haplotypes for the lineages were inferred and assigned as described in methods and are listed in table , rank ordered by the number of geno- typed males. some lineages had more than one allele at some markers. to assign a putative founder haplotype, for each marker we selected the configuration of the un- typed individuals which minimized the number of muta- tion events and assigned the founder the allele designated in that configuration. the set of founder alleles desig- nated in this manner was then assigned as the putative founder haplotype. in this manner the designation was made unambiguously for all markers in all lineages ex- cept in the cases of lineages # and for one marker, dys . in these two lineages, there were three (equally likely) configurations minimizing the number of muta- tion events for dys ; in each of those cases, the found- er allele associated with the greatest number of these con- figurations ( of in both cases) was assigned as such. in these two lineages, the chosen allele was only marginally more likely than the alternative one; however, the unique- ness of the haplotype was independent of which of the two alleles was chosen. it should be noted that in lineage distribution of surnames/ lineages in genotyped amish malesa b distribution of surnames in , households in the address book of the lancaster county amish ( ) % % % % % % % % % % % % % % % % fig. . distribution of surnames and lin- eages in ( a ) our sample of genotyped males and ( b ) the , households in the address book of the lancaster county amish. surnames are listed in the same or- der in each figure; the most common sur- names in our studies are emphasized with distinct patterns. pollin /mcbride /agarwala /schäffer / shuldiner /mitchell /o’connell hum hered ; : – # , sequencing of a previously unstudied individual eliminated one possible configuration, leading to an equal likelihood of two different founder alleles. how- ever, again, both possible founder haplotypes were unique among the amish. for of the lineages it was possible based on avail- able data to assign a putative full -str founder haplo- type. of the lineages with data from all nine markers available, only two (# and # ) shared the same haplo- type, although they have very distinct surnames and dates of entry into the population. interestingly, one of these lineages entered the population in the mid- s, a rare event for the old order amish. of the five ‘incom- plete’ putative founder haplotypes (# and # – ), three (# – ) could be distinguished from all others on the basis of available markers. one of these three (# ), whose haplotype was later completed by sequencing miss- ing markers, had the same surname as # , from which it differed at four loci, suggesting separate origins of that surname. partial haplotype # matched # and # , and partial haplotype # matched # . sequencing three additional markers (dys , dys and dys ) in selected individuals enabled us to distinguish between haplotypes # and # and be- tween # and # . interestingly, though, haplotypes # and # only differed at one of the three additional mark- ers (for a total of one marker out of genotyped), and only by one repeat unit, suggesting that the founders shared a relatively recent common male ancestor. addi- tional dna was not available from the individual in lin- eage # , so his partial haplotype remained indistin- guishable from haplotypes # and # . six of the seven alleles available for the # individual are the most com- mon alleles (and the seventh is the second most common) for their respective markers according to a large online y chromosome genealogy database which allows unre- stricted submission (ybase). also, in yhrd, the compa- rable -marker haplotype is present on three (including two which match haplotypes # and # ) of the most common marker haplotypes in european americans. thus it is not surprising that at such a low resolution we were unable to distinguish lineage # from the others by haplotype. in any case, we were able to establish that at least of the male founders of our study population had distinct y chromosome haplotypes. as noted previously, in addition to the lineages comprising individuals included in the genome-wide scans, there were two additional male lineages. lineage # was not pursued because the family was not practic- ing amish and the males in the family were not of amish descent. lineage # comprised one individual with the same unusual surname as lineage # who could not be connected to this lineage via the anabaptist genealogy database (agdb). however, successful sequencing of three previously typed str markers (dys , dys and dys ) in the limited dna available on this subject re- vealed that he matched the family on all three alleles. only one other family, # , which had a different sur- name, possessed this same three marker haplotype, which was rare in the both the european ( . % of , hap- lotypes) and worldwide ( . % of , haplotypes) samples as well as in the pennsylvania european-ameri- can ( . % of haplotypes) and national european- american ( . % of haplotypes) samples in the yhrd (release ) [ ] . it is therefore highly likely that the additional individual was either ( ) descended from the founder of lineage # via a path omitted from the genealogy or ( ) shared with founder # a recent com- mon male ancestor who lived prior to the immigration. relationship of the old order amish to other populations the lancaster old order amish reportedly immigrat- ed from western europe, specifically from south ger- many and alsace, where they had originally f led from bern, switzerland, to escape religious persecution [ , ] . we used yhrd data to attempt to corroborate this his- torical account. for the seven loci genotyped in both our data and the yhrd, our amish sample contains com- plete haplotypes, with of these haplotypes being unique. twenty-two of these amish founders’ haplotypes ( unique haplotypes) are found among the , indi- viduals in the pan-european yhrd subset of popula- tions used by roewer et al. [ ] in an analysis of recent european historical events. nineteen founders’ haplo- types ( unique haplotypes) are found in a subset of three german (freiburg, münster and leipzig) popula- tions and the bern, switzerland population. fifteen of the ( of unique) haplotypes are over-represented in this subset of four populations, that is, found at a greater frequency in this subset of , german/swiss individu- als than in the entire set of , , with frequency ratios ranging from . to . . to include the remaining three unique haplotypes, it is necessary to add one polish (byd- goszcz) and one austrian (tyrol) population. if the set of amish founder haplotypes is expanded to include haplotypes differing by one repeat at one locus (‘one step neighbors’) to allow for frequent mutation, then all amish haplotypes except one can be located in the roewer subset. using this same expanded definition, y chromosome studies in the amish hum hered ; : – the set of four german/swiss populations described above includes amish founders’ haplotypes ( unique haplotypes), and including the polish and austrian pop- ulations brings the total founders’ haplotypes found to of ( of unique haplotypes), the most that can be found in the roewer data. thus even this expanded hap- lotype definition fails to locate one of the founder hap- lotypes in one of the european populations analyzed by roewer. interestingly, the founder of this excluded lin- eage (also not found in the entire yhrd database, with one one step neighbor found in a non-european popula- tion), was reported to be kidnapped and brought to the united states as a young boy to work as an indentured servant; thus his family origins are obscure. the single marker genotypes among the founders ex- hibited similar frequencies to those in both the yhrd and ybase online databases (data not shown). distinguishing mutations and pedigree errors in tabulating differences between individual marker genotypes and putative founder alleles to assess marker mutation rates, we first needed to rule out cases of pedi- gree and/or sample errors. we reasoned that multiple al- leles discrepant from respective founder alleles in a single : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : founder haplotype ( / / ) dys -ii = (putative founder = : , : or : ) dys -i = (putative founder = : ) dys = (de novo mutation) marker haplotype not available : : fig. . portion of a pedigree providing a rational explanation for the existence of two non-founder alleles in a single indi- vidual. individuals shown in black have been genotyped and possess the putative founder allele for all three of dys -ii, dys -i and dys . a cluster of indi- viduals with dys -ii = (vs. in founder haplotype) is consistent with a ] mutation in individual : , : or : . a cluster of individuals with a dys -i = allele is consistent with an historic dys -i ] mutation in in- dividual : , a descendant of the putative dys -ii ] founder. another such descendant, : , has a de novo dys ] mutation. pollin /mcbride /agarwala /schäffer / shuldiner /mitchell /o’connell hum hered ; : – individual, while possibly resulting from multiple muta- tions, would have the greatest likelihood of resulting from errors. three samples with multiple mismatches (two in- dividuals each with a one-step mismatch at one locus and a two-step mismatch at another locus, and one individu- al with a one-step mismatch at each of two loci and a two- step mismatch at a third locus) were confirmed to be ped- igree errors based on mendelian inconsistent autosomal data. a fourth individual had only one one-step mis- match with his putative founder haplotype, but was inves- tigated because he was successfully genotyped for only four of the nine markers. he was subsequently found to be discrepant with his founder at two of the three addi- tional markers sequenced (dys and dys , mis- matched by one step each). he was mendelian consistent at all autosomal loci with his two sisters and his mother, but a comparison of his and his two sisters’ chromosome haplotypes (inferred using a markov chain monte car- lo algorithm as implemented in simwalk [ ] ) with oth- er lineage members revealed that he did not appear to share any haplotype segment with these distant cousins, consistent with a paternity error in a recent ancestor. no- tably, this individual and his immediate family were not registered members of the amish church. there were four additional individuals who each had two mismatches with their founder haplotype; however, these cases were consistent with true mutations occur- ring in multiple generations. in lineage # (see table ), individuals possessed a non-founder allele in dys -ii consistent with an historic ] mutation in the dys -ii-specific segment ( fig. ). a cluster of three of these individuals revealed an apparent dys -i ] mutation in a descendant ( : ) of the putative dys -ii ] founder, with the end result being that these three individuals ( : , : and : in the figure) each had two non-founder alleles. in addition, in this same lineage (apparently by coincidence), one individual with the allele ( : in fig. ) had a non-founder allele at dys , consistent with a new ] mutation. of course, even finding evidence of two mutations occurring in the same individual would also not have been surprising sta- tistically, as observed in a study of strs in father- son pairs [ ] . confirmation of apparent mutations those non-founder alleles found in three or more fam- ily members in a pattern consistent with a single histori- cal mutation event were considered to be confirmed evi- dence of a mutation event not requiring further molecu- lar investigation. to increase confidence that apparent mutations manifesting in only one or two family mem- bers were real and not results of genotype errors, we se- quenced relevant markers in putative mutation carriers and discordant ‘normal’ fathers and/or brothers (or the closest relative(s) available) along with previously ungeno- typed sons and/or other relatives expected to share the mutation to confirm those mutations. of the initially observed or inferred mutations, five were confirmed by their presence in clusters of three to individuals, were confirmed by sequencing of individuals possessing the mutation and/or close relatives, and three were re- futed by sequencing. in lineage # , sequencing an addi- tional individual to confirm and localize a dys -ii ] mutation in addition revealed a new or recently inherited dys -i ] mutation. mutation rate analysis prior to estimating mutation rates, we excluded the individuals representing pedigree errors, the three muta- tions refuted by sequencing, and the newly identified dys -i mutation in lineage # . all markers except dys / and ggaat b had at least one apparent mutation event. a summary of these mutation events is shown in table . of , genotypes, differed from the expected family genotype. pedigree analysis revealed that several of these differences could be attributed to a total putative historical and de novo mutation events. table . deviations from familial single marker genotypes ob- served in genome scan data and confirmed by sequencing or clus- tering in or more individuals marker # geno- typed # with non-founder allele # apparent mutation events gains losses dys / dys dys a ( ) ( ) dys -i dys -iib dys dys dys ggaat b totala , ( ) ( ) a numbers in parentheses indicate number of gains or losses if the two cases of ambiguous dys founder alleles (lineages # and # , see text and fig. ) are excluded. b excludes those resulting from dys -i mutations. y chromosome studies in the amish hum hered ; : – we used two methods to estimate mutation rates in our y chromosome markers. we first restricted our anal- ysis to typed father-son pairs ( table ) to generate results that could be compared with a previous study by kayser et al. [ ] and other studies of father-son pairs [ , , , , ] . for the nine markers, the number of such pairs available ranged from to . mutation rates, calcu- lated as the proportion of discordant father-son pairs over the total number typed for each marker, ranged from to . %, with an overall mutation rate of . % ( . % for tetranucleotide repeats only). these rates are similar to those calculated previously [ , , , , – ] . the second method traces haplotypes and mutation events back to putative founders and considers all meio- ses, observed or inferred, as a denominator. this method was used previously [ ] with a much smaller sample size ( individuals from pedigrees) than ours ( indi- viduals from pedigrees). mutation rates ( table ) were similar to those we calculated using father-son pairs: an overall mutation rate of . % ( . % for tetranucleotides only). this method also resulted in a sample size suffi- cient to detect significant departures from the overall and/or tetranucleotide marker mutation rate in three markers, dys (mutation rate = %, fisher’s exact p value = . versus all markers and p = . versus tet- ranucleotide markers), dys (same mutation rate and p values as dys ) and dys -ii (mutation rate = . %, fisher’s exact p value ! . versus all markers and p = . versus all tetranucleotide markers). by fisher’s exact test, no significant differences between mu- tation rates calculated by the two methods were observed for any of the nine markers. in two families, the marker with the highest mutation rate, dys -ii, showed evidence of multiple indepen- dent mutation events. lineage # showed evidence of five independent occurrences of the same mutation in dys -ii ( ] ). furthermore, in this same lineage, there were two additional alleles for dys -ii, both re- sulting from de novo mutations as evidenced by fathers possessing the founder allele: ] (the only two step mutation observed) and ] . there was also evidence of three independent occurrences of a ] mutation in this same marker in lineage # . discussion by examining the genotypes at several str markers on the y chromosome in several hundred amish study volunteers, we have confirmed the historical accuracy of the genealogical records of the ancestors that connect in- dividuals in our current pedigrees recruited for the study of complex phenotypes. the combination of genealogical records and y chromosome genotypes indicates that vir- tually every surname in the amish represents a unique founder. comparison of putative amish founder y chro- table . apparent y str mutation events marker observed meiotic mutation events (typed father-son pairs) all meiotic/mutation events (entire pedigrees) meioses mutations mutation rate, % % exact ci, % meioses mutations mutation rate, % % exact ci, % dys / . . – . , . b . – . dys . . – . . . – . dys . . – . , . . – . dys -i . . – . , . . – . dys -iia . . – . , . c . – . dys . . – . , . b . – . dys . . – . , . . – . dys . . – . , . . – . ggaat b . . – . . . – . total , . . – . , . . – . tetranucleotide only , . . – . , . . – . a excludes those resulting from dys -i mutations. b fisher’s exact p = . vs. all markers and p = . vs. tetranucleotide markers. c p < . vs. all markers and p = . vs. tetranucleotide markers. pollin /mcbride /agarwala /schäffer / shuldiner /mitchell /o’connell hum hered ; : – mosome haplotypes with online european y chromo- some haplotype data support the reported western/cen- tral european origin of the amish. our amish data afforded us the opportunity to use two different but complementary approaches for estimat- ing y str mutation rates. a number of studies have eval- uated mutation rates in y str markers, which are impor- tant for forensic applications [ ] . observation of fre- quent mutations in ystrs is also a reminder of the general mutability of strs, which in autosomes can lead to mendelian discrepancies that may be mistaken for genotype errors when using the markers for linkage anal- ysis. heyer et al. [ ] and jobling et al. [ ] used informa- tion from individuals in ‘deep rooting’ pedigrees table . comparison of single locus mutation rates observed in this study to previously published and online mutation rates study method* dys dys -i dys -ii dys dys dys dys / kayser et al. ( ) [ ] pairs . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) bianchi et al. ( ) [ ] pairs . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) heyer et al. ( ) [ ] pedigrees . ( / ) – – . ( / ) . ( / ) . ( / ) . ( / ) kurihara et al. ( ) [ ] pairs . ( / ) . ( / ) . ( / ) . ( . ) . ( / ) . ( / ) . ( / ) dupuy et al. ( ) [ ] pairs . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) ballard et al. ( ) [ ] pairs . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) budowle et al. ( ) [ ] pairs . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) gusmão et al. ( ) [ ] pairs . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) hohoff et al. ( ) [ ] pairs . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) lee et al. ( ) [ ] pairs . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) domingues et al. ( ) [ ] pairs . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) yhrd pooled (includes all of the above plus un-published studies) mixed . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) bonné-tamir et al. ( ) [ ] pedigrees . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) present study pairs . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) . ( / ) present study pedigrees . ( / ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) . ( / , ) rates given as percents with number of mutations over number of meioses shown in parentheses. * methods: ‘pairs’ refers to studies using only typed, confirmed father/son pairs. ‘pedigrees’ refers to studies using pedigrees that included untyped but inferred transmissions in the calcuations. y chromosome studies in the amish hum hered ; : – and were able to use as a denominator to primar- ily unobserved transmissions [ ] . the disadvantage of this approach, as pointed out by others [ , ] , is that paternity cannot be completely resolved. in fact, heyer et al. used three different scenarios to estimate y chromo- some mutation frequencies because they could not distin- guish multiple apparent mutations in one individual from nonparternity. the minisatellite msy genotyping ap- plied by jobling et al. [ ] to the same pedigrees provided evidence but did not prove definitively that the single marker differences were true mutations and the multiple marker differences represented instances of nonpaterni- ty. to remove nonpaternity concerns from mutation rate estimation, kayser et al. [ ] and later others [ , , , , ] studied father-son pairs in conjunction with auto- somal genotyping. in our amish data, we were able to use both approaches, which yielded similar results and com- plemented each other well. the father-son pairs, which included , meiotic events, led to a . % estimate of the mutation rate, very similar to that calculated by kay- ser et al. since these individuals were genotyped for ap- proximately (in the case of the afds) or (in the case of the afos) autosomal and x-linked strs, we were able to rule out nonpaternity in these pairs to an even greater certainty than kayser et al., who used only – autosomal markers to confirm paternity. the estimated overall mutation rate in the amish was similar using both methods ( . % with father-son pairs and . % with the whole pedigrees). a pedigree based study in the samaritan population similarly estimated a mutation rate of . % [ ] , also consistent with our find- ings. since the landmark study by kayser et al. [ ] , sim- ilar mutation rates have been estimated using father-son pairs in several other populations, including . % in japanese pairs typed for ystrs [ ] , . % in , spanish and portuguese pairs typed for ystrs [ ] , . % in up to mixed uk pairs typed for ystrs [ ] , . % in taiwanese pairs typed for nine ystrs [ ] , . % in north american pairs typed for ystrs [ ] , . % in , german pairs typed for ystrs [ ] , . % in korean pairs typed for ystrs [ ] and . % in ‘afro-brazilian’ pairs typed for ystrs [ ] . similarly, an estimated mutation rate of . – % for repeat gains (losses could not be evalu- ated due to the methodology used) was calculated using strs in sperm samples from three donors; overall mu- tation rate was estimated at . % based on the assump- tion of equilibrium between gains and losses [ ] . these latter results should be interpreted with caution due to the technical limitations of the small-pool pcr and f luo- rescence-based fragment-length analysis methods used, including the inability to detect repeat losses. previously published mutation rates for the seven commonly typed loci are shown in table (study using sperm sample ex- cluded because of its technical limitations) in conjunc- tion with the two sets of mutation rates estimated in the present study. in addition, ‘evolutionary’ mutation rate estimates of . % per years [ ] , . % per years [ ] and . % per generation [ ] have been reported. the dis- crepancy between these estimates and those based on fa- ther-son pairs and pedigree analysis appears to be attrib- utable to several factors, including assumptions about the age of the population [ , , ] , the specific character- istics of the markers and alleles evaluated [ ] , and pos- sibly haplogroup-based selection effects [ ] . the consis- tency of the mutation rates estimated using the pedigree and father-son pair methods in the present study and pre- vious studies suggests that for the purpose of forensic and genetic epidemiology quality control applications, the mutation rate for ystrs is between and % of meioses per marker, varying by specific marker. one marker, dys -ii, showed a significant depar- ture from the overall mutation rate when evaluated using the whole pedigree method. this marker along with two others (tetranucleotide dys / and trinucleotide dys ) showed significant departure from the overall tetranucleotide marker mutation rate. the marker with the highest mutation rate, dys -ii, showed a virtually identical rate between the two methods ( . % in father- son pairs and . % in the whole pedigrees). this marker also had the greatest number of alleles in our study, ex- emplifying the advantages and disadvantages of strs: (autosomal) markers with high diversity are useful for linkage analysis but increase the possibility of mutations to contribute to the overall ‘error’ rate. not surprisingly marker dys / , which has no observed mutations in either our data or the data of kayser et al. and heyer et al., and few mutations in other studies, has very low di- versity, with a single allele accounting for over % of amish founder alleles as well as alleles in yhrd and ybase. summary in summary, our genotype analysis of y chromosome str markers in our amish study subjects has ( ) con- firmed the accuracy of the male lineage portion of the genealogy and completeness of the anabaptist genealogy pollin /mcbride /agarwala /schäffer / shuldiner /mitchell /o’connell hum hered ; : – database; ( ) showed that lancaster amish founder y chromosomes exhibit diversity similar to the general caucasian population, reinforcing that the surnames de- lineate fairly distinct founders, and ( ) added to existing data on mutation rate estimates for several commonly used y chromosome str markers. acknowledgements the authors wish to acknowledge drs. elizabeth streeten and jay shapiro, the amish research clinic staff and amish liaisons for their energetic recruitment of subjects into the amish fam- ily studies, jack shelton for str marker sequencing and the nhlbi mammalian genotyping service for str genotyping. this work would not have been possible without the outstanding cooperation of the amish community. this research was sup- ported in part by the intramural research program of the nih, national library of medicine the american diabetes associa- tion, and nih grants r -dk , r -ar and r - hl . web resources the urls for data presented herein are as follows: nhlbi mammalian genotyping service, http://www.marsh- fieldclinic.org/research/genetics pedhunter, http://www.ncbi.nlm.nih.gov/cbbresearch/schaf- fer/pedhunter.html ybase: genealogy by numbers, http://www.ybase.org the y-str haplotype reference database (yhrd), http:// www.yhrd.org references agarwala r, biesecker lg, hopkins ka, francomano ca, schäffer aa: software for constructing and verifying pedigrees within large genealogies and an application to the old order amish of lancaster county. ge- nome res ; 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( ). hum genet ; : – . carvalho-silva dr, santos fr, hutz mh, salzano fm, pena sdj: divergent human y- chromosome microsatellite evolution rates. j mol evol ; : – . zhivotovsky la, underhill pa, feldman mw: difference between evolutionarily ef- fective and germ line mutation rate due to stochastically varying haplogroup size. mol biol evol ; : – . president’s page: the epidemic of type diabetes and obesity in the u.s.: cause for alarm acc news president’s page: the epidemic of type diabetes and obesity in the u.s.: cause for alarm george a. beller, md, facc president, american college of cardiology diabetes mellitus affects an estimated million people worldwide, and its prevalence is increasing ( ). the preva- lence of type diabetes was . % among u.s. adults from to ( ). according to a centers for disease control and prevention (cdc) study ( ), the number of americans diagnosed with type diabetes jumped an astounding % overall between and , from . % of the population to . %. since many cases go undiagnosed, the actual number of cases was probably higher. this increased incidence of diabetes will surely slow the age-adjusted decline in coronary artery disease incidence in the u.s. even more startling is the fact that the disease is striking at ever-earlier ages. indeed, the cdc study found that americans in their s had an almost % increase in their diabetes rates—the largest increase of any age group ( ). today, children as young as eight are being diagnosed with type diabetes ( ). especially disturbing, noted dr. richard kahn, is that type diabetes was virtually unheard of in children just a decade ago, and now as many as , children have it. dr. kahn was part of a consensus panel of the american academy of pediatrics and american diabe- tes association, which early this year issued recommenda- tions concerning the prevalence of diabetes among children. the panel noted that type diabetes is an “emerging epidemic” among children ( ). the changing demographics of the disease are also reflected in its name. once called “adult-onset diabetes” because of its tendency to strike in middle age or later, the disease is now known simply as “type diabetes” to distinguish it from the entirely different disease once called “juvenile diabetes” and now known as “type diabetes.” as endocrinologist dr. arthur rubenstein of mount sinai school of medicine recently told newsweek, diabetes “is becoming a disease of the young” ( ). clearly dr. rubenstein and other endocrinologists will have their work cut out for them in coming years, but so will cardiovascular specialists. diabetes doubles men’s chances of developing heart disease and quadruples women’s risk ( ). diabetics have a two to three times greater risk of death after myocardial infarction ( ), and cardiovascular disease is the number-one cause of death among people with diabetes. of course, people with diabetes are more likely than nondiabetics to suffer from obesity, hypertension, and other factors that would put anyone at risk of developing coronary heart disease ( ). however, those risk factors account for less than half of the excess mortality associated with diabetes. a consensus document published by the american college of cardiology and the american diabetes associ- ation recently confirmed what physicians have long known: diabetes is itself an independent and potent risk factor for cardiovascular disease ( ). lifestyle and obesity what is behind the soaring diabetes rates? although genetic factors play a role, experts suspect that the main culprits are sedentary lifestyles and poor diets. according to the sur- geon general’s report on physical activity and health, % of american adults are not physically active on a regular basis ( ). even worse, a full quarter of adults aren’t active at all. morbidity and mortality weekly report noted in that, in addition to diabetes, physical inactivity is linked to an increased risk for heart disease, colon cancer, hypertension, obesity, osteoporosis, muscle and joint disor- ders, and symptoms of anxiety and depression ( ). those sedentary habits are being passed on to the next generation of americans. almost half of young people ages to fail to engage in vigorous activity on a regular basis, the surgeon general’s report ( ) revealed, and activity levels decline dramatically during adolescence. schools aren’t do- ing much better than parents when it comes to instilling healthy exercise habits. the new york times reported that one in four children gets no physical education in school, and illinois is the only state that requires daily physical education for all children ( ). high school students’ enrollment in daily physical education classes plummeted from % in to % by ( ). in addition to sedentary lifestyles, americans have also become accustomed to bigger, less healthy meals. although americans have managed to reduce their fat consumption— from % of their diet years ago to % today—they still eat far too much sugar and processed foods ( ). diabetes specialist dr. sanford garfield of the national institutes of health calls it the “mcdonald’s syndrome” ( ). these sedentary lifestyles and poor diets have resulted in an epidemic of obesity. only % of americans were considered obese in ; by , that number had climbed to % ( ). similarly, the percentage of americans considered overweight increased from % to %. the journal of the american college of cardiology vol. , no. , © by the american college of cardiology issn - / /$ . published by elsevier science inc. pii s - ( ) - cdc has reported that, together with physical inactivity, overweight accounts for more than , premature deaths every year in the u.s. ( ). of great concern is the fact that in american children is now obese ( ). reporter jane e. brody ( ) of the new york times, has written that today’s u.s. children are “fatter than ever and less fit than they were in the s.” in another times article ( ), dr. richard s. strauss, of the robert wood johnson school of medicine, attributes the epidemic of obesity largely to inactivity. “we found that for about hours of the day they’re basically doing nothing,” dr. strauss said. “and what we call vigorous activity— moving faster than three and a half miles an hour—that was on average minutes a day.” other researchers have noted that the average child in the six- to eleven-year age range watches h of television per week ( ). meanwhile, a study of elementary school students in san jose, california, found that children who reduced time watching television and playing video games lost a significant amount of body fat in just six months ( ). another important point of concern is that fat children may grow up to be fat adults, therefore more prone to the conditions that plague obese adults— diabetes, hyperten- sion, and arthritis, for example ( ). a study by dr. robert whitaker ( , ), a pediatric obesity expert at children’s hospital medical center in cincinnati, found that children who were obese at six to nine years old had a % chance of becoming obese adults; the percentage climbed to % for obese - to -year-olds. affecting both children and adults is that fact that, as people gain weight, they are more likely to develop diabetes. in the cdc study, . % of the obese patients had diabetes, compared with only . % of those with normal weight ( ). consider the work of dr. alan shuldiner of the univer- sity of maryland, who has compared amish families in pennsylvania with other white americans ( ). although amish adults are just as likely to be fat and eat unhealthy food as other americans, they have about half the rate of diabetes found among other whites. the difference may be that they forgo cars, televisions, computers, and other devices that encourage a sedentary lifestyle. it is especially worrisome that overweight and obesity are becoming so prevalent that the public, and even health care professionals, are becoming inured to these conditions, as noted in the new york times, where writer gina kolata ( ) quoted an assistant professor of pediatrics and medicine at stanford university: “ ‘ten, years ago, the people who were showing up for treatment were percent overweight,’ dr. [thomas] robinson said. now, he said, the children he treats are averaging percent overweight . . . .” the role of cardiovascular specialists as cardiovascular specialists know, diabetes can wreak havoc on the cardiovascular system. high blood-sugar levels dam- age the arteries, for example, weakening capillary walls and causing microvascular damage ( ). fully half of all diabetics have already suffered serious damage to the heart and other organs by the time they finally receive a diagnosis ( ). insulin resistance and compensatory hyperinsulinemia are major pathogenetic factors for atherosclerotic cardiovascular disease. all physicians—whether they’re cardiologists, pediatri- cians, or endocrinologists— have a responsibility to keep cardiovascular complications from developing. although various drugs can help control type diabetes, convincing patients to get regular exercise and eat a healthy diet is crucial. prevention of obesity by diet and regular physical activity is particularly important for children and adoles- cents. the surgeon general recommends that all americans engage in moderate physical activity, such as a half hour of brisk walking or leaf raking, on most days. exercising longer or more vigorously further reduces the chances of diabetes, coronary heart disease, and other causes of early death. physicians should also urge their patients to follow the dietary guidelines issued by the department of agriculture and department of health and human services. sensible portions and reduced fat, salt, and sugar intake are key ( ). with the nurses’ health study, stampfer et al. ( ) have shown that middle-aged women who maintained an appro- priate weight, ate a healthy diet, exercised a half hour a day, and avoided cigarettes and excess alcohol consumption enjoyed % fewer coronary events than other women. but even small changes can make a big difference. researchers in finland have found ( ) that losing as little as pounds can be enough to ward off diabetes. in the study, tuom- ilehto et al. found that beginning a moderate exercise program, eating healthier, and losing only pounds, or less, over a year cut high-blood-sugar patients’ chances of developing full-blown diabetes by %. reductions in weight of even % to % can substantially improve blood pressure, lipid levels, and glucose tolerance and decrease the incidence of both diabetes and hypertension ( ). even more important, physicians need to start prevention efforts early. by encouraging active lifestyles and healthy diets, physicians can help patients avoid obesity and diabetes and, ultimately, cardiovascular disease. physicians must take the time to counsel patients about increasing their physical activity and to communicate persuasively the benefits of regular exercise, particularly to women, racial and ethnic minorities, and members of lower socioeconomic groups, who are at increased risk for obesity ( ). reprint requests and correspondence: george a. beller, md, facc, cardiovascular division, department of internal medi- cine, health system, university of virginia, p.o. box , charlottesville, virginia - . jacc vol. , no. , beller december : – president’s page references . amos af, mccarty dj, zimmet p. the rising global burden of diabetes and its complications: estimates and projections to the year . diabet med ; suppl :s – . . harris mi, flegal km, cowie cc, et al. prevalence of diabetes, impaired fasting glucose, and impaired glucose tolerance in u.s. adults. the third national health and nutrition examination survey, – . diabetes care ; : – . . kluger j. the diabetes explosion. time, september , . . schrof j. the silent killer. u.s. news and world report. april , . . elias m. fighting diabetes’ surge in obese kids. usa today. february , . . adler j, kalb c. an american epidemic: diabetes. newsweek. sep- tember , . . american college of cardiology and american diabetics association. summary report from the consensus development conference on diagnosis of coronary artery disease in people with diabetes. . u.s. department of health and human services. physical activity and health: a report of the surgeon general. executive summary. . monthly estimates of leisure-time physical inactivity—united states, . morbidity & mortality weekly report. may , ; : – . . brody je. fitness gap is america’s recipe for fat youth. new york times, september , . . schultz s. why we’re fat. u.s. news and world report. november , . . cdc report: “epidemic of obesity on rise in u.s.” wall street journal interactive edition. october , . available at http:// interactive.wsj.com. . marquis c. americans need better diet, new health study reports. new york times. may , . . kolata g. while children grow fatter, experts search for solutions. new york times. october , , a , a . . brody je. personal health: planning healthier suburbs, where cars sit idle and people get moving. new york times. oct. , . . whitaker rc, wright ja, pepe ms, et al. predicting obesity in young adulthood from childhood and parental obesity. n engl j med ; : – . . stampfer m, hu fb, manson je, rimm eb, willett wc. primary prevention of coronary heart disease in women through diet and lifestyle. n engl j med ; : – . . manning a. exercise, diet block diabetes at a discount. usa today. june , . . willett nc, dietz wh, colditz ga. guidelines for healthy weight. n engl j med ; : – . . wee cc, mccarthy ep, davis rb, phillips, rs. physician counseling about exercise. jama ; : – . beller jacc vol. , no. , president’s page december : – delineating the expanding phenotype associated with scaper gene mutation r e s e a r c h l e t t e r delineating the expanding phenotype associated with scaper gene mutation james fasham , | gavin arno , | siying lin | mingchu xu , | keren j. carss , | sarah hull , | amelia lane | anthony g. robson , | olivia wenger | jay e. self | gaurav v. harlalka | claire g. salter | lynn schema | timothy j. moss | michael e. cheetham | anthony t. moore , , | f. lucy raymond , | rui chen , | emma l. baple , | andrew r. webster , | andrew h. crosby | nihr bioresource rare diseases consortium medical research, rild wellcome wolfson centre, university of exeter medical school, royal devon and exeter nhs foundation trust, exeter, united kingdom peninsula clinical genetics service, royal devon and exeter hospital (heavitree), exeter, united kingdom ucl institute of ophthalmology, university college london, london, united kingdom moorfields eye hospital, london, united kingdom department of molecular and human genetics, baylor college of medicine, houston, texas human genome sequencing center, baylor college of medicine, houston, texas department of haematology, nhs blood and transplant centre, university of cambridge, cambridge, united kingdom nihr bioresource – rare diseases, cambridge university hospitals nhs foundation trust, cambridge, united kingdom new leaf center, clinic for special children, mount eaton, ohio clinical and experimental sciences, faculty of medicine, university of southampton, southampton, united kingdom division of genetics and metabolism, university of minnesota medical center – fairview, minneapolis, minnesota division of genetics and metabolism, department of pediatrics, university of minnesota, minneapolis, minnesota ophthalmology department, ucsf school of medicine, koret vision centre, san francisco, california department of medical genetics, cambridge institute for medical research, university of cambridge, cambridge, united kingdom correspondence andrew crosby and emma baple, medical research, rild wellcome wolfson centre, university of exeter medical school, royal devon and exeter nhs foundation trust, barrack road, exeter ex dw, united kingdom. emails: a.h.crosby@exeter.ac.uk, e.baple@exeter.ac.uk andrew r. webster, ucl institute of ophthalmology, university college london, london ec v el, united kingdom. email: andrew.webster@ucl.ac.uk funding information fight for sight uk, grant/award numbers: / to al, to elb ahc, early career investigator award to ga; foundation fighting blindness; medical research council, grant/award numbers: g to elb, g to ahc; moorfields eye charity; national centre for the replacement, refinement and reduction of animals in research, grant/award number: to al; national institute for health research, grant/award numbers: academic clinical fellowship to jf, nihr bioresource - rare diseases project rg ; newlife foundation for disabled children, grant/award number: to ac eb; retina uk; university of exeter, grant/award number: vice chancellor scholarship to sl k e y w o r d s : brachydactyly, ccna –cdk , intellectual disability, retinitis pigmentosa, scaper received: march revised: may accepted: may doi: . /ajmg.a. this is an open access article under the terms of the creative commons attribution license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © the authors. american journal of medical genetics part a published by wiley periodicals, inc. am j med genet. ; – . wileyonlinelibrary.com/journal/ajmga https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - mailto:a.h.crosby@exeter.ac.uk mailto:e.baple@exeter.ac.uk mailto:andrew.webster@ucl.ac.uk http://creativecommons.org/licenses/by/ . / http://wileyonlinelibrary.com/journal/ajmga a potential role for the cyclin a –cyclin-dependent kinase complex regulator s-phase cyclin a-associated protein residing in the endoplas- mic reticulum (scaper) in human disease was first suggested by najmabadi et al. ( ), who identified a candidate homozygous frameshift scaper variant as the cause of nonsyndromic intellectual disability (id) in a small iranian family. we subsequently reported a single patient with biallelic loss of function (lof) scaper variants associated with retinal disease (carss et al., ). biallelic lof vari- ants have since been associated with id with or without retinitis pigmentosa (rp) in seven individuals from five families from spain, israel, and iran (hu et al., ; tatour et al., ); in one individual from a jordanian arab family, a homozygous scaper gene variant was identified as the cause of nonsyndromic rp (jauregui et al., ). more recently, wormser et al. ( ) described a scaper gene variant associated with a bardet–biedl syndrome (bbs)-like presenta- tion comprising of id, rp, short stature, obesity, and brachydactyly in eight individuals from two consanguineous bedouin families belonging to the same tribe in southern israel, alongside preliminary functional studies suggesting a possible role for scaper in ciliary dynamics and disassembly. in the current study, we describe clinical and genetic findings, including seven novel scaper variants, in six individuals of amish, caucasian, and south asian descent. together with our molec- ular data, our comprehensive phenotypic assessments enable a more detailed clinical comparison to be drawn between the patient cohort described here (including previously published individual g ; patient in this study, (carss et al., ) with the individuals in whom scaper variants were recently defined (hu et al., ; jauregui et al., ; najmabadi et al., ; tatour et al., ; wormser et al., ), permitting a more precise definition of the clin- ical phenotype arising from pathogenic scaper variation. samples were taken with informed consent (study approved by the ethics committee of akron children's hospital, moorfields eye hospital and baylor college of medicine, in compliance with the declaration of helsinki) for deoxyribonucleic acid (dna) extraction. single nucleotide polymorphism (snp) genotyping was performed (patients and ) using the humancytosnp- v . beadchip array (illumina, cambridge, uk). in patients and – , whole exome or whole genome sequencing (wes or wgs), variant alignment, calling, filtering, and prioritization was performed as previously described (carss et al., ; rawlins et al., ; xu et al., ). allele-specific primers were designed using primer web software to evaluate segre- gation of the candidate scaper gene variants identified via dideoxy sequencing. patient underwent wes at genedx and was identified via genematcher (sobreira, schiettecatte, valle, & hamosh, ) as part of the matchmaker exchange repositories (philippakis et al., ). all variants identified in the study have been submitted to clin- var (https://www.ncbi.nlm.nih.gov/clinvar/). patients and are ohio amish siblings. candidate variants identi- fied through wes of dna from patient were cross-referenced with regions of autozygosity common to both affected siblings, identified through whole genome snp genotyping. this identified only a single plausible candidate variant, located within the largest ( mb) shared region of autozygosity on chromosome (rs –rs ; chr (grch ):g. - ), a novel homozygous duplica- tion in exon of the scaper gene, predicted to result in a frame- shift (nm_ . : c. dupa, p.(ile asnfs* ) chr (grch ): g. dupt; figure ). dideoxy sequencing confirmed the pres- ence and co-segregation of this variant in both siblings. this variant was detected in heterozygous form in five unrelated individuals in a data- base of regional amish controls, corresponding to an estimated allele frequency of ~ . , not uncommon for founder mutations within this population. wes/wgs performed in patients – , identified com- pound heterozygous scaper variants; c. delt, p.(val serfs* ) (chr (grch ):g. dela) and c. c>t, p.(arg *) (chr (grch ):g. g>a) in patient , c. + g>a (chr (grch ): g. c>t) and c. delc, p.(pro glnfs* ) (chr (grch ): g. delg) in patient , c. c>t, p.(arg *) (chr (grch ):g. g>a) and c. _ delct, p.(ser tyrfs* ) (chr (grch ):g. _ delag) in patient , and c. c>t, p. (gln *) (chr (grch ):g. g>a) and c. - c>g (chr (grch ):g. g>c) in patient . the scaper variants in each of these patients were confirmed to be biallelic by familial segregation analy- sis using dideoxy sequencing. none of these variants are present in the genome aggregation (gnomad) or , genomes databases and those in patients , , and – are novel. table summarizes the core phenotypical features of individuals not previously reported, aged between months and years (patients , , and – ), provides additional clinical details for patient (carss et al., ), and compares these to the clinical features of all scaper syndrome patients described to date. id and developmental delay was present in all six affected individuals, and four patients also exhibited hyperactivity and attention deficit hyperactivity disorder (adhd). autism and dyspraxia were each noted in one individual. neuroimaging performed in patients , , , and revealed no abnor- malities. additional dysmorphic features noted in both amish siblings (patients and ) included inverted nipples, brachydactyly, camptodactyly, proximally placed thumbs (figure ), and a characteris- tic facial appearance with frontal bossing and almond-shaped eyes; growth parameters were all normal. patients , , and – all pres- ented between the ages of – with a reduction in night vision and visual field deficits; patient ( months) described no visual symp- toms at the time of presentation. fundus examination in patients – revealed findings typical of rp including optic disc pallor, attenuated retinal vessels and intraretinal mid-peripheral bone-spicule pigmenta- tion, and loss of photoreceptor outer segments with retained central macular structure on optical coherence tomography imaging (figure ; table s ). additional variable ocular features described in some patients with scaper syndrome include cataracts (in two individuals) and myopia and keratoconus in one individual each. our clinical and genetic studies in six affected individuals, includ- ing additional new clinical details for patient , (carss et al., ) take the total number of scaper syndrome patients described to date to . although the extent for which clinical data is available for the pre- viously reported patients is variable, our detailed clinical phenotyping allows a more comprehensive clinical comparison to be made with the previously reported cases, confirming the presence of a variable fasham et al. https://www.ncbi.nlm.nih.gov/clinvar/ pattern of dysmorphic features associated with scaper syndrome. it is now clear that the cardinal clinical features of the disorder include mild/moderate id and developmental delay particularly affecting speech and language and motor milestones. hyperactivity appears to be a common feature, with some affected individuals receiving a for- mal diagnosis of adhd. early adult onset rp is also a key clinical find- ing, and the retinal phenotype appears remarkably consistent. in all individuals for whom we have data, progressive loss of night vision begins in first or second decade of life. together with studies in mice demonstrating expression of scaper in multiple retinal layers, partic- ularly in the retinal pigment epithelium and photoreceptor inner and outer segments, this supports a role for scaper in photoreceptor function and/or maintenance (tatour et al., ). tapering fingers, brachydactyly and proximally placed thumbs, described in eight individuals from two consanguineous bedouin fami- lies of the same tribe in southern israel, were also identified as a con- sistent feature in the two amish siblings, confirming the association of this feature with the scaper syndrome. short stature and obesity were also a common feature amongst the affected bedouin patients, and this constellation of clinical features including rp, obesity, short stature, id, developmental delay, and brachydactyly has consequently led to a suggested diagnosis of bbs in these individuals. although there is some overlap between the clinical features characteristic of ciliopathies and those seen in scaper syndrome, the amish siblings (who are of normal height and weight for age) demonstrate that the digital, retinal, and cognitive abnormalities may occur independently of short stature and obesity. the other common primary features of bbs, including renal anomalies, postaxial polydactyly, hypogonadism (males), and genital abnormalities (females) have not been reported in association with scaper mutation (forsythe & beales, ). the dysmorphic facial features and inverted nipples, noted on examination of both amish siblings, have not been previously noted in other indi- viduals with scaper variants. recently, a single individual homozygous for a c. - a>g scaper variant presenting with nonsyndromic rp and no evidence of id was reported in this journal (jauregui et al., ). the same c. - a>g scaper gene variant has also been reported previ- ously in association with rp, adhd, and mild id (tatour et al., ) indicating the variability in the presence and severity of the extraocular features associated with the scaper syndrome figure (a) simplified pedigree of the amish family investigated, with electropherograms showing the scaper c. dupt sequence variant in all affected and unaffected individuals in generations vi and vii (black arrow identifies the duplicated nucleotide). (b) pictorial representation of the single nucleotide polymorphism (snp) genotypes across the ~ . mb chromosome q - region identified in this family. (c–j) clinical features of scaper syndrome patients. (c, d) brachydactyly, camptodactyly, and proximally placed thumbs identified on examination of patient . (e, f) ocular imaging and investigations from patient illustrating features of rp (e: right eye, f: left eye) fundus photograph (optos plc, dunfermline, uk) showing optic disc pallor, attenuated retinal vessels and mid-peripheral bone spicule pigmentation (g: right eye, h: left eye) faf imaging showing mid-peripheral hypoautofluorescence with a central ring of hyperautofluorescence demarcating the surviving outer retinal structures. (i: right eye, j: left eye) optical coherence tomography (spectralis-oct, heidelberg engineering, heidelberg, germany) of the central retina demonstrating loss of photoreceptor outer segments with retained central macular structure corresponding to faf findings. faf, fundus autofluorescence; oct, optical coherence tomography; scaper, s-phase cyclin a-associated protein residing in the endoplasmic reticulum fasham et al. t a b l e a co m p ar is o n o f cl in ic al fi n d in g s o f al la ff e ct e d in d iv id u al s w it h b ia lle lic p at h o g e n ic s c a p e r v ar ia n ts g e n o ty p e e th n ic it y s e x a g e (y e ar s) a w e ig h t (k g ,s d s ) h e ig h t (c m ,s d s ) o f c (c m ,s d s ) b m i (s d s ) w al k e d (m o n th s) s p e e ch d e la y id b e h av io r is su e s a b n o rm al n e u ro im ag in g r p b ra ch y d ac ty ly o th e r cl in ic al fi n d in g s n aj m ab ad i p .(t y r fs *) /p . (t y r fs *) ir an n a n a n a n a n a n a n a n a ✓ n a n a n a n a n a t at o u r (a :i i: ) c. - a > g / c. - a > g a ra b f n a n a n a n a n o rm al n a m ild (i q ) a d h d m r i: n o rm al ✓ n a n il t at o u r (a :i i: ) c. - a > g / c. - a > g a ra b f n a n a n a n a n o rm al n a m ild (i q ) a d h d n a ✓ n a n il t at o u r (b :i i: ) p .(i le fs *) / p .(i le fs *) s p an is h f n a n a n a n a n a m o d n o n e re p o rt e d c t : n o rm al ✓ n a a lo p e ci a ar e at a t at o u r (c :i i: ) p .(g lu d e l)/ p .(s e r a sn ) s p an is h m n a n a n a n a d e la y e d n a ✓ n o n e re p o rt e d n a ✓ n a n a h u (f am ily ; in d iv id u al s) p .(a rg *) / p .(a rg *) b al o ch n a n a n a n a n o rm al n a n a n a ✓ n a n a n a n a n a ja u re g u i c. - a > g / c. - a > g a ra b m n a n a n a n a n a n a n o n o n p ✓ n a n a w o rm se r (p :v ) p .(l e u *) / p .(l e u *) b e d o u in f (+ . ) (− . ) n o t re d u ce d . (+ . ) n a ✓ m o d n a n p ✓ ✓ g e n u v al g u m /g e n u v ar u m w o rm se r (p :v ) p .(l e u *) / p .(l e u *) b e d o u in m (+ . ) (− . ) n o t re d u ce d . (+ . ) n a ✓ m o d n a n p ✓ ✓ g e n u v al g u m /g e n u v ar u m w o rm se r (p :v ) p .(l e u *) / p .(l e u *) b e d o u in m (+ . ) (− . ) n o t re d u ce d . (+ . ) n a ✓ m o d n a n p ✓ ✓ g e n u v al g u m /g e n u v ar u m w o rm se r (p :v ) p .(l e u *) / p .(l e u *) b e d o u in m (+ . ) (− . ) n o t re d u ce d . (+ . ) n a ✓ m o d n a n p ✓ ✓ g e n u v al g u m /g e n u v ar u m w o rm se r (p :i ii ) p .(l e u *) / p .(l e u *) b e d o u in f . (+ . ) (− . ) n o t re d u ce d . (+ . ) n a ✓ s e v n a n p ✓ ✓ n il w o rm se r (p :i ii ) p .(l e u *) / p .(l e u *) b e d o u in f (+ . ) (− . ) n o t re d u ce d . (+ . ) n a ✓ s e v n a n p ✓ ✓ g e n u v al g u m /g e n u v ar u m w o rm se r (p :i ii ) p .(l e u *) / p .(l e u *) b e d o u in f . (+ . ) (− . ) n o t re d u ce d . (+ . ) n a ✓ s e v n a n p ✓ ✓ n il w o rm se r (p :i v ) p .(l e u *) / p .(l e u *) b e d o u in m . (− . ) (− . ) n o t re d u ce d . (+ . ) n a ✓ m o d a d h d m r i: ab n o rm al b s u sp e ct e d ✓ g e n u v al g u m /g e n u v ar u m p at ie n t p .(i le fs *) / p .(i le fs *) a m is h m . . (+ . ) . (+ . ) . (+ . ) . (+ . ) ✓ m o d h y p e ra ct iv it y m r i: n o rm al n o ✓ p ro xi m al ly p la ce d th u m b s. s h o rt fi ft h fi n g e rs ,p e s p la n u s, fr o n ta lb o ss in g , al m o n d -s h ap e d e y e s, an d in v e rt e d n ip p le s p at ie n t p .(i le fs *) / p .(i le fs *) a m is h f . . (− . ) . (− . ) (− . ) (− . ) ✓ m ild h y p e ra ct iv it y n p n a (a g e ) ✓ p ro xi m al ly p la ce d th u m b s, sh o rt fi ft h fi n g e rs ,p e s p la n u s, fr o n ta lb o ss in g , al m o n d -s h ap e d e y e s, an d in v e rt e d n ip p le s (c o n ti n u e s) fasham et al. t a b l e (c o n ti n u e d ) g e n o ty p e e th n ic it y s e x a g e (y e ar s) a w e ig h t (k g ,s d s ) h e ig h t (c m ,s d s ) o f c (c m ,s d s ) b m i (s d s ) w al k e d (m o n th s) s p e e ch d e la y id b e h av io r is su e s a b n o rm al n e u ro im ag in g r p b ra ch y d ac ty ly o th e r cl in ic al fi n d in g s p at ie n t c (g c ) p .(a rg *) / p .(v al fs *) s o u th a si an f th ce n ti le rd ce n ti le n a n a ✓ m o d a d h d , au ti sm , an d se lf - h ar m m r i: n o rm al ✓ n a n il p at ie n t (g c ) c. + g > a / p .(p ro fs *) c au ca si an f n a n a ( th ce n ti le ) n a ✓ m ild d y sp ra xi a n p ✓ n a n il p at ie n t p .(a rg *) /p . (s e r fs *) n a (u n it e d s ta te s) f n a n a n a o b e se n a n a ✓ n a m r i: n o rm al ✓ n a n il p at ie n t p .(g ln *) / c. - c > g n a (u n it e d s ta te s) f . (+ . ) . (− . ) n a (+ . ) – y e s m ild (i q – s) a d h d m r i: n o rm al ✓ n a m o d e ra te e cz e m a w it h se v e re sk in -p ic k in g b e h av io r s u m m ar y / o b e se / / / a ll n o rm al / / a b b re v ia ti o n s: a d h d ,a tt e n ti o n -d e fi ci t h y p e ra ct iv it y d is o rd e r; b m i, b o d y m as s in d e x; c t ,c o m p u te ri ze d to m o g ra p h y ; f ,f e m al e ; id ,i n te lle ct u al d is ab ili ty ; iq ,i n te lli g e n ce q u o ti e n t (w e ch sl e r a d u lt in te lli g e n ce s ca le ); m ,m al e ; m o d ,m o d e ra te ; m r i, m ag n e ti c re so n an ce im ag in g ; n a ,n o t av ai la b le ; n p ,n o t p e rf o rm e d ; o f c ,o cc ip it o fr o n ta l ci rc u m fe re n ce ; r p ,r e ti n it is p ig m e n to sa ; s d s ,s ta n d ar d d e v ia ti o n sc o re s; s e v , se v e re . n o te .a d u lt s w it h a b m i > ar e cl as si fi e d as o v e rw e ig h t, th o se > ar e cl as si fi e d as o b e se ; th e ✓ sy m b o li n d ic at e s th e p re se n ce o f a fe at u re in an af fe ct e d su b je ct . h e ig h t, w e ig h t, b m i an d o f c z -s co re s w e re ca lc u la te d u si n g a m ic ro so ft e xc e l ad d -i n to ac ce ss g ro w th re fe re n ce s b as e d o n th e l m s m e th o d (p an & c o le , ) u si n g a re fe re n ce e u ro p e an p o p u la ti o n (c o le , f re e m an ,& p re e ce , ). a r e fe rs to ag e o f e xa m in at io n . b a b n o rm al m r i fi n d in g s in cl u d e m ild ly e n la rg e d la te ra l v e n tr ic le s an d se v e ra ll o ci o f ir re g u la r si g n al in th e b ra in p ar e n ch y m a ab o v e th e te n to ri u m ,i n th e p o st e ri o r w h it e m at te r an d al o n g th e e p e n d y m a. c a ls o p at ie n t g (c ar ss e t al ., ). fasham et al. (table ). however, this may also be accounted for by the difficulties in conclusively defining milder developmental delay in some situa- tions, when more subtle clinical findings may not be identified if not specifically assessed. conversely, associated ocular pathology may remain undetected or unrecognized in individuals with id, as such individuals often have difficulty recognizing or articulating their visual symptoms. this highlights the importance of visual screening and ophthalmological assessment in these patients. other common ocular features include cataracts (in particular posterior subcapsular cataracts, which are commonly associated with rp; (pruett, ) and strabismus, with nystagmus and keratoconus noted in a single patient. the high incidence of cataracts, a potentially treatable cause of sight loss, again supports the case for screening in early childhood. the allele frequency (~ . ) of the ohio amish scaper founder mutation suggests that, despite no previous reports, this disorder represents an underrecognized cause of rp and mild id within this community. this further highlights the importance of careful clinical evaluation in children and adults with id and enables targeted genetic testing for this scaper variant for amish individuals with this clinical presentation. together with our clinical review of all previ- ously published patients, this study expands the molecular spectrum of disease-causing scaper variants and enables a clearer delineation of the core (and variable) phenotypical features of scaper syndrome to be characterized. our findings also highlight the importance of prompt visual screening and ophthalmic assessment in all individuals with scaper-associated disease. despite the increasing numbers of individuals identified with scaper syndrome, the precise functions of scaper in human growth and development are not fully under- stood. further studies to elucidate the precise molecular and devel- opmental roles of this molecule in human growth and skeletal, retinal, and brain development and function, will yield important insights into the clinical heterogeneity increasingly observed in scaper-associated disease. acknowledgments first and foremost, we would like to thank the patients and their fami- lies for their participation in and support of this study. we thank kirsty mcwalter at genedx (genematcher and matchmaker exchange). this work was supported by the medical research council (mrc grants g to elb and g to ahc). this paper presents inde- pendent research funded by the national institute for health research (nihr) through the nihr bioresource – rare diseases project [grant number rg ] and nihr academic clinical fellowship to j.f.) the views expressed are those of the author(s) and not necessarily those of the nhs, the nihr or the department of health and social care. fight for sight (grants / to a. l., to e. l. b. and a. h. c., and early career investigator award to g. a.), newlife foundation for dis- abled children (grant to e. l. b. and a. h. c.). further support was pro- vided by nihr biomedical centre of ucl institute of ophthalmology and moorfields eye hospital, national health service foundation trust, and ucl institute of ophthalmology, moorfields eye hospital special trustees, moorfields eye charity, the foundation fighting blindness (united states), and retina uk. s. l. is supported by the university of exeter vice chancellor scholarship and f. l. r. is supported by cam- bridge nihr biomedical research centre and national centre for the replacement refinement and reduction of animals in research (nc rs). author contributions j. f., g. a., s.l. contributed equally to this work. e. l. b., a .r. w., and a. h. c. contributed equally to this work. orcid james fasham https://orcid.org/ - - - gavin arno https://orcid.org/ - - - siying lin https://orcid.org/ - - - emma l. baple https://orcid.org/ - - - andrew r. webster https://orcid.org/ - - - references carss, k. j., arno, g., erwood, m., stephens, j., sanchis-juan, a., hull, s., … raymond, f. l. 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commentary predicting the range of linkage disequilibrium jurg ott laboratory of statistical genetics, rockefeller university, york avenue, new york, ny - a much pondered question in the mid-dle ages was, ‘‘how many angels can dance on the head of a pin?’’ today, we pose more sensible questions but answers are not necessarily getting easier. a generally accepted strategy in the mapping of a disease gene is to initially apply linkage analysis for an approximate estimate of the location of the trait gene and to subsequently make use of linkage disequilibrium (association) for a more accurate localization. the thinking behind this approach has been that disequilibrium extends over much shorter distances from a disease gene than does linkage. on the other hand, biotechnology companies are gearing up to develop large numbers of single nucleotide polymorphism (snp) markers ( ) to localize disease genes by the disequilibrium mapping approach alone, for example, in case-control studies. it is then of interest to know how many such markers will be required on a ge- nome-wide basis. thus, the question is, how rapidly does disequilibrium decay as one moves away from a disease locus? the answer to this question is obviously of more than academic interest. whether several , or several , snp markers are needed will have a major impact on such association mapping stud- ies. an early prediction was that in large outbred populations, disequilibrium should be detectable within kb of a disease locus ( ). so, it is timely that an extensive study addresses this important point in a recent issue of pnas ( ). collins and colleagues ( ) collected information from the literature on auto- somal haplotypes identified through family studies. they focused on haplo- types for pairs of loci, with the two members of a pair being a disease and a marker locus or two marker loci. mark- ers with multiple alleles were dichoto- mized into two-allele systems for a unique estimate of association desig- nated by r. distances (d in kb) between loci were determined on the scale of the physical map. for a disease gene with uncertain map position, its map location was estimated under the malecot model with the aid of the allass program ( ). this approach prev iously has been shown to provide excellent estimates for the locations of trait genes ( ). all in all, approx imately locus pairs were worked up. different classes of locus pairs were distinguished, in particular, those contain- ing a disease locus and pairs of random snp loci. for closely linked loci, the ma- lecot model allowed for the multiple- pairwise estimation of model parameters. one of these parameters, «, depends on the number of generations since forma- tion of the haplotypes and on the ratio between physical and genetic maps. the so-called swept radius, y«, estimates the distance in kb at which association falls to approximately y of its original level. interestingly, this distance turns out to be very similar for disease and random hap- lotypes. for disease haplotypes, the swept radius is estimated between and kb and for random haplotypes it is some- what smaller than kb. the study ( ) concludes with the suggestion that the number of snps required for a genome scan might be on the order of , or less. on the basis of computer simulations, a recent progress report ( ) predicted an extremely short range of useful disequi- librium, only about kb. the report met with widespread skepticism even though it appeared in a so-called high-visibility journal. these predictions are clearly con- tradicted by observed data ( ). the main reason for the discrepancy appears to be that the simulations were carried out un- der the assumption of a continuously ex- panding human population up to its present size of billion, which seems unrealistic ( ). a more likely scenario is that various bottlenecks and cycles of ex- pansion and contraction have occurred in human history. thus, it is reassuring that the study in a recent issue of pnas ( ) projects a required number of , snps rather than the figure of , resulting from the computer simulation. isolated populations often are consid- ered advantageous for association map- ping ( ) but some examples have been found in which the extent of linkage dis- equilibrium is the same in small isolated and large outbred populations ( ). a pre- vious investigation of this situation con- cluded that isolated populations are to some, relatively small degree favorable for association analysis ( ). on the other hand, there are well-known instances of strong disequilibrium in small populations ( ). it seems to depend very much on the history of populations, be they large or small, whether disequilibrium will be ex- tensive around disease loci. in their figure , collins and colleagues ( ) show a graph of the association, r, versus distance, d, between loci on a hap- lotype. visual inspection of the graph reveals two interesting features. first, at least at small distances, there seem to be two clusters of haplotypes, one corre- sponding to low and one with high asso- ciation. further, in each cluster, there does not seem to be a strong relationship between association and distance for ap- proximate values of d , kb. this finding confirms previous observations that disequilibrium and physical distance see companion article on page in issue of volume . table . sample data on linkage disequilibrium (r) and distance (d, in kb) between disease genes and nearby snp markers disease gene population r d x huntington disease ( ) canada . . . . huntington disease ( ) england . . . . cystic fibrosis ( ) caucasians . . . . limb girdle muscular dystrophy ( ) reunion islands . . amish . . hemochromatosis ( ) utah . . – u pnas u january , u vol. u no. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , do not correlate significantly when d , kb ( ). my immediate reaction to these two clusters was that they correspond to isolated versus general populations. thus, i obtained data for a few examples of each from the quoted web site ( ). table shows the observed association values be- tween disease and closest marker loci at very small distances. at least on the basis of this small sample, it appears quite con- vincing that increasing population isola- tion is more or less correlated with in- creasing association. special applications of disequilibrium mapping previously have furnished some spectacular results. a much-quoted exam- ple is that of the locus for diastrophic dys- plasia, which was predicted to be kb from the best marker ( ) and was localized at a distance of kb from it ( ). on the other hand, the same method has been much less successful in other instances ( ). a very dense map of snps can be expected to yield rather accurate results. of course, if candi- date genes are available for a trait, analysis of snp markers in or very near these genes provides a cost-effective solution. . jorde, l. b., watkins, w. s., kere, 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( ). , . inheritance in epidermolysis bullosa letalis harold e. cross, r. s. wells, and john r. esterly from the division of medical genetics, department of medicine, and the department of pathology, the johns hopkins university school of medicine, baltimore, maryland , u.s.a. in herlitz reported cases of a 'new' type of recessively inherited epidermolysis bullosa. be- cause of the severe involvement and death in infancy he used the term 'epidermolysis bullosa hereditaria letalis' to describe these cases and additional cases which had been previously reported (mautner, ; jenny, ; heinrichsbauer, ; kuse, ). the clinical characteristics as described by herlitz ( ) are as follows: the bullae appear at or shortly after birth; they are frequently haemorr- hagic and affect both skin and mucous membranes; the nails may be absent or later show marked dys- trophy; and death usually occurs before the third month. tendons and blood vessels may be seen through the transparent skin in affected areas. the identifying clinical feature is said to be the absence of scarring, pigmentation, or milia in areas ofhealing lesions. since early death of recessive dystrophic cases could be responsible for the lack of these skin changes, some authors (touraine, ; muggler, ; klunker, ) doubt the existence of a separate lethal category of epidermolysis bullosa. those who do accept the lethal type as a distinct entity consider the inheritance to be autosomal recessive though detailed family studies have not been done. a kindred with affected members this report describes a large kindred in which members were affected with a severe, lethal form of epidermolysis bullosa. clinical features. clinical data on all patients were obtained from interviews with the parents. only patients were admitted to hospital and one ofthese (x. (see fig. )) was examined by one of us (h.e.c.) during l&e; intefliew data on one other hospitalized patient (x. ) were supplemented by medical records and ne- cropsy reports. none of the mothers reported an abnormal pregnancy received october , . with any patient and no drugs were taken during gesta- tion. none of the children was premature by weight and two weighed more than - kg. ( lb.) at birth. the dizygotic twins (x. and x. ) were approximately - kg. ( lb.) and - ( lb.), respectively. in one family (ix. - ) two additional sibs died with spina bifida, but all other sibs were normal. although the mouth was always affected with bullae and consequent eroded areas, no difficulty was experi- enced with feeding or swallowing. most patients had no vomiting or diarrhoea, though one (x. ) required a liquid diet in order to maintain nutrition. an initial weight gain was never continued, however, and all lost weight before they died. blisters frequently became secondarily infected. four children had pyrexia and convulsions immediately be- fore death, but no other illnesses were diagnosed during life. in particular, no respiratory infections were re- ported. none of the patients was given systemic corti- sone, and local treatment seemed to be most effective when it dried the lesions. of the affected children, were male and were female. eleven ofthe infants died by weeks of age, the youngest at the age of weeks. two lived for months and one for months. the latter was continuously in hospital from the age of weeks. skin lesions. bullae were occasionally noted at birth and within hours some erythema and blistering developed at the base of several finger-nails. this was usually followed by shedding of the affected nails and infection of the nail-beds. regrowth of nails was rarely observed. during the neonatal period the bullae were often haemorrhagic and developed on all areas of the body except the palms and soles. some of the areas affected by blisters remained erythematous and in rare cases became covered by transparent, atrophic tissue. dystrophic scars, pigmented areas, and milia were never seen. the epidermis was fragile, and it was often im- possible to handle the infants without traumatizing the skin. the mouth was always involved with bullae, and lesions were also generally present in the anterior nares. no ophthalmological abnormalities were noted. pathological features. necropsy was performed on only one patient (x. ). he was grossly under- o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ cross, wells, and esterly : 'v melr x a ' epidermolysis bullosa 'c'zu deceased b spina bifida * miscarriage fig. . pedigree of amish epidermolysis bullosa cases. all parents can be traced to either couple i.a-i.b or ii.c-ii.d. since i.b and ii.c were related, some ancestor common to both probably introduced the gene to the amish. developed and severely emaciated; and the most striking changes were the widespread skin lesions evident around the mouth, nose, and eyes where there were confluent erosions with haemorrhagic encrusted borders (fig. a). large eroded areas were seen on the right elbow, back (fig. b), and lower legs. haemorrhagic bulae were present at the base of the nails and many finger- and toe-nails were missing. blisters and eroded areas were widely distributed on the trunk and limbs. no milia, pigmented areas, or dystrophic scars were seen, nor was it possible to see blood vessels or tendons in the more severely affected areas (e.g. on the right elbow, fig. a). ulceration was also noted on the pos- terior surface of the tongue and most of the pharynx. histological material showed all stages of the disease. sections of skin from the toe demonstrated areas of dermal-epidernal separation (fig. a). the plane and cleavage followed the irregular contour of the rete ridges and descended part of the distance along the dermal-epidermal junctions of hair follicles and sweat ducts. parakeratosis was not present and vacuoles in the epidermal basal cells were infrequent and unrelated to the margins of the bullae. blister fluid, dermal oedema, and inflammatory cells were inconspicuous in the minimal (earliest) lesions. other sections with more extensive bullae showed degenerative changes in the overlying epidermis. both blister and dermal oedema were seen (fig. b). the inflammatory changes at this stage ranged from small aggregates of plasma cells to a denser mixed infiltrate, including occasional cells in the blister fluid. the edges of the bullae were sharp rather than blunted and an acute angle was formed be- tween the dermal and epidermal layers. no evidence of secondary regeneration was seen in the material from this case. a section of abdominal skin showed the most advanced changes (fig. c). the epidermis was absent, and encrusted necrotic debris formed a partial dermal covering. the superficial dermis_had the appearance of non-specific granulation tissue characte?- ized by proliferating capillaries, chronic inflammatory cells, and relatively dense collagen. the lower dermis was unremarkable and the only abnormality in the ad- nexa was dilatation of eccrine ducts. the section through the posterior tongue (fig. ) o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ inheritance in epidermolysis bullosa letalis fig. . post-mortem photograph of x. . (a) large confluent lesions with haemorrhagic encrusted borders are present around the mouth,nares, and eyes. an extensive lesion involving all layers of the skin is present on the right elbow. the finger-nails are absent, the left earis deformed, and there is moderate hirsutism on the arms. (b) note severe emaciation and multiple lesions of all sizes over the trunk andextremities. showed an ulcer and foci of epithelial separation that were identical to the changes in the skin. plasma cells were present around the ulceration and under the adja- cent papillae. no diagnostic changes were found in the remaining tissues. the epithelium was partially detached in the sections of trachea and gall-bladder, but post-mortem artefacts could not be excluded as the cause and no abnormalities were seen in the underlying tissues. genetic aspects. these patients belong to the old order amish, a highly religious and ultraconservative protestant sect with origins in switzerland nearly years ago. all amish today live in genetic isolates in the united states and ontario. each isolate is a closed, self-defined population to which almost no new genes have been added since immigration ceased in the 's. because of the small number of original settlers, the small size of the isolates, and the subsequent strict endo- gamy, a relatively high mean coefficient of consanguinity exists. in an eadogamous population with few founders, such as the amish, the common ancestor approach can be used to suggest autosomal recessive inheritance in certain disorders. fig. shows that all six parents in the present study share a single ancestral couple, i. a. and i. b. alternate pathways are available for the gene since another couple (ii. c and ii. d) is also common to all parents. moreover, indirect evidence (recorded in a study on the mast syndrome) suggests that i. b and ii. c were sibs or at least closely related (cross and mckusick, ). it is likely that an unidentified heterozygous ancestor contributed the epidermolysis bullosa gene to both i. b and ii. c. the amish originated in the canton of berne in switzerland about but no amish live there today. at least reports have described recessively inherited epidermolysis bullosa in th century swiss families. jenny ( ) reported families with the presumed lethal variety who lived in the canton of aargau. later, schnyder, jung, and salamon ( ) found cases of recessively inherited epidermolysis bullosa in switzer- land and, though no distinction was made between the dystrophic and lethal types, it is possible that at least some of these patients did have the lethal type. discussion clinical considerations. the recognized types of epidermolysis bullosa (table i) can usually be distinguished clinically and genetically. there are, however, two exceptions. first, it is not possible to differentiate between dystrophic auto- somal recessive cases and some sporadic cases of the dystrophic autosomal dominant type. secondly, severely affected patients with the recessive o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ cross, wells, and esterly fig. . sections of skin. (a) section from toe showing nearly complete epidermal-dermal separation in the absence of secondary changes. the line of cleavage follows the irregular contour of the . rete ridges (h. + e. x .) (b) a later lesion characterizedl by degeneration of the overlying epithelium and oedema and a moderate ianunamatory infiltrate in the superficial dermis. neither secondary epidermal regeneration nor scarring are present. (h. + e. x ). (c) the chronic ulcerative lesion from abdominal skin hasthe appearance of non-specific granulation tissue. the only abnor- mality in the deeper dermis is dilatation of the eccrine ducts. ~~~~~ k ~~~ ~ ~ ~ ~ ~ ~ ~ ~~~~(h .x ). (a) (b) (c) fig. . an area of epithelial separation and erosion on the posterior tongue. a plasma cell infiltrate is seen in the area of necrosis (right) and underlying the adjacent papillae (h. + e. x .) o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ inheritance in epidermolysis bullosa letalis table i clinical and genetic characteristics of epidermolysis bullosa mode of inheritance clinical category clinical features onset usually within the first two years; bullae precipi- epidermolysisbullosa simplex tated by heat rather than trauma, and heal withoutepidermolysis bullosa simplex scarring; bullae usually subcorneal or intra-epidermal when sectioned epidermolysis bullosa simplex hands andfeet onset often in first two decades; bullae on feet moreautosomal than hands, and heal without scarring dominant onset at birth or early infancy; bullae precipitated by trauma rather than heat, and heal with scarring; epidermolysis bullosa dystrophica mainly on extremities; mucous membranes usually involved; dystrophic changes in nails; epidermal cysts; bullae are subepidermal when sectioned onset at birth; mucous membranes often involved, conjunctivae may be aff&cted, atrophy of teeth, and strictures of oesophagus and pharynx may occur; epidermolysis bullosa dystrophica pigmented areas may be seen; scarring, epidermal cyst formation, and nail dystrophy invariably present; autosomal may develop syndactyly and dwarfism; bullae are recessive subepidermal and may be haemorrhagic onset at birth; severe involvement of skin and mucous membranes at birth, and nails commonly abnormal; epidermolysis bullosa hereditaria letalis usually die in first three months; builae show little tendency to heal and are subepidermal dystrophic type and cases of the recessive lethal condition described by herlitz ( ) may be clinic- ally indistinguishable. the clinical features of the amish cases are com- patible with those previously published for epi- dermolysis b,ullosa hereditaria letalis. in particular, the lesions shown in the post-mortem photographs (fig. ) resemble those in the reports of herlitz ( ), henderson ( ), and roberts and co- workers ( ). characteristically, lesions were first noticed at the base of the nails, and later on the trunk, face, scalp, and extremities. palms and soles were never involved and no dystrophic changes, pigmentation, or milia were seen. some healing was noted but the skin still appeared ab- normal in these areas. no scarring was present, even in the two patients who lived until and months of age. detailed morphological alterations have been described by roberts and co-workers ( ). it was their opinion that the cleavage resulted from coalescence of intracellular vesicles in the germinal epithelium. they also called attention to the mor- phological similarity between the bullae in their cases and those which follow basal cell degenera- tion (lichen planus, incontinentia pigmenti, and lupus erythematosis). the ultrastructural find- ings in another case (pearson, ) suggest that the defect is in the intermembranous space separating the basal and dermal cells. extra-epidermal lesions have been described in previous reports (roberts et al., ; maddison and barter, ; leland and hirschl, ) of epidermolysis bullosa letalis. these have con- sisted of vesicular changes in the trachea and bronchioles similar to those seen in the skin (maddi- son and barter, ; leland and hirschl, ), suggesting that the lesions in the integument are only part of a more widespread abnormality. lesions have also been reported in the pancreatic and biliary ducts (roberts et al., ; maddison and barter, ). the respiratory tract and the pancreatic and biliary ducts, however, are locations in which epithelial separation may result from poor tissue preservation. the present case demon- strates unequivocal evidence of epithelial lesions since the oral ulcerations were noted before necropsy and the tongue is relatively resistant to autolysis. the classification of epidermolysis bullosa must at present be based on clinical and genetic charac- teristics alone, since biochemical and pathological studies have not yielded definitive diagnostic criteria. histological studies of specimens from patients with both recessive dystrophic and the lethal types suggested that bullae form at the dermo- epidermal junction. however, pearson ( ) described ultrastructural changes in epidermo- lysis bullosa letalis, which suggested that separation occurred in the plane of the intermembrane space, i.e. between the cell membranes of the epidermal and dermal layers. by contrast, study of patients with recessive dystrophic type showed the earliest changes in the basal cell layer. these findings suggest that the lethal type has a pathogenesis different from the other varieties, but this has been challenged by electronmicroscopy studies in another patient (lapiere, castermans-elias, and firket, ). o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ cross, wells, and esterly table ii reported families of epidermolysis bullosa letalis age at age at no. affected number onset death reference unaffected (days) (days) parental consanguinity male ifemale jenny ( ) ; second cousins fifth cousins, once removed ; fourth cousins heinrichsbauer ( ) kuse ( ) herlitz ( ) - ; davidson ( ) brandberg ( ) black et al. ( ) - schiffer ( ) ; first cousins leland and hirschl ( ) , ; frank and kern ( ) ? calnan ( ) ; henderson ( ) - third cousins lucini ( ) mercauandcompaytizianozago( ) , lewis, steven, and farquhar ( ) first cousins rosset ( ) - * silver ( ) t roberts et al. ( ) maddison and barter ( ) cox ( ) bergenholtz et al. ( ) mth. klunker ( ) ; lapiere et al. ( ) schwob ( ) present cases - see pedigree - see pedigree see pedigree subtotals mautner ( ) - schroder and wells ( ) birth lamb and halpert ( ) birth matheson and rosner ( ) birth pearson ( ) ( ?)? totals * one patient was still alive at months of age. t patients were alive at months and months of age. genetic considerations. although inheritance of the lethal type of epidermolysis bullosa is pre- sumed to be autosomal recessive, no genetic analysis has been done on the total number of cases reported. because of the strong evidence of autosomal reces- sive inheritance in the amish cases, it is of interest to determine if the consanguinity rate and the segre- gation ratio of previously reported families are con- sistent with this mode of inheritance. epidermolysis bullosa letalis is a rare disorder; including the present report there are only cases on record (table ii). criteria for inclusion of cases with the herlitz lethal form of epidermolysis bullosa were as follows: onset of disease in the neonatal period; death or absence of remissions in the first three months of life; and absence of milia, pigmentary changes, and scarring. since many reports do not comment on parental consanguinity and others contain inadequate family data, only selected families can be used for genetic analysis. among families (upper portion, table ii) in which family data were reported, the parents were definitely known to be related in families. even if it is assumed that there was no consanguinity in the families about which no statement was made, the consanguinity rate would be / , or %. it is difficult to obtain an 'average' consanguinity rae for the world population from which these cases were reported but it is certainly much less than %, and probably no higher than or %. segregation analysis in reported families is usually complicated by lack of information con- o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ inheritance in epidermolysis bullosa letalis cerning the methods of ascertainment and selectivity of reported cases (morton, ; steinberg, ). for practical purposes the families in table ii may be considered as having been ascertained by single ascertainment, i.e. the probability that a family has been reported is dependent on the number of affected offspring, but only one individual served as the proband in each family. if a significant number of the families in table ii were ascertained because of relatedness of previously ascertained probands (multiple ascertainment), then these assumptions clearly would not be valid. however, with the ex- ception of one of the amish sibships reported here, and possibly two of jenny's ( ) families, none of the sibships in table ii are known to have been ascertained in this manner. the proband method of segregation analysis may thus be used since it becomes fully efficient and identical to maximum likelihood methods as ascertainment approaches single ascertainment (steinberg, ). the pro- portion of affected sibs by this method is / , or , for the families in the upper portion of table ii for which adequate family data were available. the standard deviation of this estimate is and the % confidence limits are - to - . at the present time, evidence for the existence of a separate lethal form of epidermolysis bullosa is only suggestive. it must be remembered, however, that when a category of genetic disease is studied closely with combined clinical, genetic, and bio- chemical approaches, heterogeneity is usually found within what previously appeared to be one entity. illustrative examples among skin diseases are albinism, icthyosis, tylosis, the ectodermal dys- plasias, and alopecia congenita. the classification of epidermolysis bullosa has followed a similar pattern in the past, and new evidence will no doubt produce new categories. no doubt some recessive dystrophic patients die before they develop scarring or pigmentary changes, and certainly some patients with the lethal type live longer than the three-month period characteristic of herlitz' cases. in such instances differentiation on clinical grounds alone may not be possible. it is significant, however, that no sibship has yet been reported in which affected individuals with dystro- phic scarring were closely related to affected indi- viduals who survived longer than a year and who ,acked scarring and pigmentary changes. jenny ( ) reported five families belonging to a single kindred who had a lethal form of the disease. muggler ( ) attempted to connect all of jenny's families with a large pedigree of several families with the presumed recessive dystrophic variety since all came from the same canton of switzerland. the critical genealogical evidence was not available, how- ever, since the common ancestors of jenny's cases were different from those of the dystrophic cases. if a lethal variety of epidermolysis bullosa is accepted by present criteria, the pedigree of the amish cases, the increased parental consanguinity in reported families, and segregation analysis all suggest autosomal recessive inheritance. summary three old order amish families with cases of epidermolysis bullosa are presented. the clinical features of these cases are similar to those found in the herlitz or lethal form of epidermolysis bullosa. pathological material in one patient revealed lesions in all stages of the disease and an unequivocal extra- epithelial lesion on the tongue. all parents can be traced to two related immigrant couples. a review of the literature revealed additional cases with the herlitz type of epidermolysis bullosa. in these cases as well as in the amish families re- ported here, increased parental consanguinity and segregation analysis are consistent with autosomal recessive inheritance. the authors would like to thank dr. samuel spector and dr. eugene perrin, cleveland, and dr. a. donald merritt, indianapolis, for providing information on the amish families. this study was supported in part by atomic energy commission contract number at ( - ) and by research grant no. gm- from the national insti- tutes of health. refences bergenholtz, a., olssen, o., arwill, t., and lundstrom, n-r. 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(paris), , . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ mh _ .. w f bynum, anne hardy, stephen jacyna, christopher lawrence and e m tansey, the western medical tradition to , cambridge university press, , pp. xiii, , illus., £ . , $ . (hardback - - - - ); £ . , $ . (paperback - - - - ). textbooks are not only for students. teachers need them too. i remember, as a novice lecturer in the early eighties, being particularly grateful for the cambridge history of science series (to which professor bynum would later contribute) for supplying my lecture notes with a breadth and authority that would otherwise have been sadly lacking. cambridge university press continued this service to the aspiring pedagogues of our specialty with the publication, in , of the western medical tradition, bc to ad . authored collectively by members of what was then the academic unit of the wellcome institute (and is now the wellcome trust centre for the history of medicine at ucl), that major textbook was a product, and an authoritative expression, of the recent surge in the quality and quantity of research in the history of medicine. i therefore opened my review copy of a companion textbook, also by members of the wellcome centre, bringing the period covered up to the end of the twentieth century, with some pleasant anticipation. the authors have divided up the subject matter neatly between them. there are sections on ‘medicine in transformation, – ’ (stephen jacyna), ‘the rise of science in medicine, – ’ (w f bynum), ‘continuity in crisis: medicine, – ’ (christopher lawrence) and ‘medical enterprise and global response, – ’ (anne hardy and tilli tansey). the volume ends with a series of short but very judicious bibliographical essays and a full bibliography. the first sentence of the bibliographic essay for chapter is telling. jacyna writes that ‘‘indispensable to understanding the period is volume one of k. marx, das kapital’’. the reader is also exhorted not to depend on secondary commentators for his/her understanding of the thought of michel foucault, ‘‘[n]one of these can . . . substitute for a direct reading of his works’’ (p. ). this is not, in other words, a textbook which seeks to pander to the casual, less-than-committed, undergraduate. jacyna draws extensively upon anthropology and sociology, to sustain a sophisticated engagement with the historiography of the early nineteenth century and its medicine. this section will challenge many, if not most, honours students, but will be ultimately very rewarding for them (and their teachers). jacyna skilfully deconstructs the ideological role of professional elites and medical heroes, and is tellingly sensitive to the historical significance of the lower ranks of the profession as the ‘‘shock troops of the bourgeois revolution’’. it is curious, therefore, that he is so lacking in sympathy with the apothecaries, describing them as the ‘‘pariahs of medical practice’’, and ‘‘by far the least interesting’’ of the medical orders. uninteresting to whom, one might ask. the following sections are generally less theoretical but, otherwise, just as broad in conception. those who already possess bynum’s previous textbook need have no concern that his section in the current volume merely duplicates what is already on their shelves. while, inevitably, some earlier themes are revisited, the treatment is not only updated but is also both broadened and extended into the early twentieth century. i was pleased to see that bynum, as befits a former student of ackerknecht, has no truck with those revisionist historians who have sought to question the link between british anticontagionism and the ideology of free trade. lawrence explores the great theme of the interaction between medicine and modernity. his account of the impact of the first world war on medicine is balanced and thoughtful; the discussions of specialization and of nosology are very perceptive; and the whole section is peppered with well chosen scientific examples. lawrence is also instructive on the ideological contexts of medicine between the wars and on the social use of medical metaphors. all the sections effectively summarize the relevant scholarly book reviews https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core literature, but the last (hardy and tansey) is particularly welcome, constituting as it does one of the first serious attempts at a comprehensive historical survey of the development of medicine and medical science after the second world war. the authors have a huge task, for western medicine, in their period, became a global rather than european and american project. they also range very widely in subject matter, exploring scientific, social and economic issues. their account of the rise of the pharmaceutical industry is particularly useful. overall it might, however, be said that this section is the least well-organized thematically. this is not, i hasten to add, the authors’ fault— they have more ground to cover and they lack the benefit of the longer and deeper historiographical perspectives enjoyed by the other contributors. having said that, there is the occasional instance of repetition that a more careful editing might have eliminated. there can be no doubt that the second volume of the western medical tradition will be an essential addition to the reading list of every honours and master’s course in the history of medicine. the book is handsomely produced and also very reasonably priced, at least for the paperback edition, given the word count. the prospective reader may, however, be warned that it is not as entertaining a read as the volumes in the cambridge series nor, indeed, as its older companion textbook. this is partly because historians have, in the meantime, increasingly turned away from the sweeping grand narratives that gave the earlier texts, especially the cambridge ones, such rhetorical force. it is also because the very comprehensiveness of the western medical tradition to —its determination to cover all the major countries of europe, as well as north america—sometimes gets in the way of narrative clarity. overall, however, that is a price worth paying for what is a genuinely impressive scholarly and pedagogic achievement. malcolm nicolson, university of glasgow susan lindee, moments of truth in genetic medicine, baltimore, johns hopkins university press, , pp. xii, , £ . , $ . (hardback - - - ). historians of medical genetics have long been preoccupied with the field’s relationship to eugenics. that focus is certainly understandable given the manifest institutional, personal, and ideological entanglements of ‘‘reform eugenics’’ with medical genetics during the s and s, as well as continuing controversies concerning the eugenic content of such medical-genetic technologies as prenatal and pre-implantation genetic diagnosis. lindee notes that research in the field has resulted in more diagnostic tests than it has effective treatments for disease and indeed claims that selective abortion following prenatal diagnosis remains the ‘‘primary intervention’’ associated with genetic medicine (p. ). thus even she can not entirely escape the eugenics issue. nevertheless, the focus of her welcome new book is on aspects of the history of the field that have received much less attention from scholars, such as the central roles played by patient and parent advocacy groups in setting research agendas, financing studies, and providing critical information. moments of truth is not a systematic history of genetic medicine but an analysis of five key developments occurring between and —two decades during which human genetics was transformed from an institutional and intellectual backwater into a research frontier. each case study explores a different facet of the field. thus the routinization of newborn testing for phenylketonuria (pku) following the development of a blood test suitable for hospital-based mass screening is used to investigate the rise of public health genetics, and victor mckusick’s studies of the old order amish, the construction of human pedigrees and rise of mapping studies more generally. similarly, early research on human chromosomes is used to elucidate how standardization has transformed concepts and practices in genetic medicine, the development of the ‘‘twin method’’, a variety of issues in the book reviews https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core r e s e a r c h a r t i c l e high connectivity among argali sheep from afghanistan and adjacent countries: inferences from neutral and candidate gene microsatellites g. luikart • s. j. amish • j. winnie • a. beja-pereira • r. godinho • f. w. allendorf • r. b. harris received: july / accepted: january / published online: april � springer science+business media b.v. abstract we quantified population connectivity and genetic variation in the marco polo subspecies of argali mountain sheep (ovis ammon polii) by genotyping neutral and candidate gene microsatellite loci in individuals noninvasively sampled across five study areas in afghani- stan, china, and tajikistan. heterozygosity and allelic richness were generally high (mean h = . , mean a = . ), but were significantly lower in the china study area (h = . , p \ . ; a = . , p \ . ). one mar- ker in an immune system gene (tcrg ) showed an excess of rare alleles compared to neutral expectations. another immune system gene (glycam- ) showed excessive dif- ferentiation (high fst) between study areas. estimates of genetic differentiation were similar (fst = . vs. . ) with and without the two loci deviating from neutrality, suggesting that selection is not a primary driver of overall molecular variation, and that candidate gene loci can be used for connectivity monitoring, as long as selection tests are conducted to avoid biased gene flow estimates. adequate protection of argali and maintenance of inter-population connectivity will require monitoring and international cooperation because argali exhibit high gene flow across international borders. keywords bottlenecks � habitat fragmentation � gene flow � ovis ammon � pamir mountains � natural selection � adaptation � infectious disease � noninvasive genetic monitoring � mountain ungulate introduction genetic assessments and monitoring are increasingly cru- cial for delineating population boundaries and movement corridors, and possibly for understanding adaptation to environmental change in extreme environments (shackl- eton ; schwartz et al. ). availability of candidate adaptive gene markers (e.g. kohn et al. ) along with neutral loci could make feasible the assessment of both adaptive and neutral connectivity, i.e. gene flow and adaptation to changing environments (black et al. ). argali (ovis ammon) are an ecologically and economi- cally important species, but are increasingly threatened throughout their range. the marco polo subspecies of argali (ovis ammon polii) is among the largest wild sheep and is perhaps the most charismatic wild animal in the pamir mountains of tajikistan, china, kyrgyzstan, and afghani- stan (fedosenko and blank ). the marco polo sub- species is categorized as near threatened on the iucn red list (iucn ). argali in the pamir mountains are important because of their role as a flagship species for the entire ecosystem (schaller and kang ). however, populations are susceptible to human pressures includ- ing poaching, displacement, competition, and disease g. luikart (&) fish and wildlife genomics group, flathead lake biological station, university of montana, polson, mt , usa e-mail: gordon.luikart@mso.umt.edu g. luikart � s. j. amish � f. w. allendorf fish and wildlife genomics group, division of biological sciences, university of montana, missoula, mt , usa g. luikart � a. beja-pereira � r. godinho cibio, centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, - vairão, portugal j. winnie � r. b. harris department of ecosystem and conservation science, university of montana, missoula, mt , usa conserv genet ( ) : – doi . /s - - -z transmission from livestock, as well as possible habitat fragmentation (shackleton ). due to their innate hab- itat preferences, argali are generally distributed patchily, with areas of inappropriate habitat separating populations. unlike the related mountain sheep (e.g., o. canadensis) of north america, argali are generally believed to be willing to traverse long distances, possibly across seemingly inhospitable terrain. thus the degree to which populations are truly fragmented, either demographically or genetically, is often an open question. unfortunately, argali are among the most difficult of wild ungulates to study due to their wary nature, choice of remote and precipitous habitats, and low population den- sity. argali, unlike north american wild sheep, are cur- sorial and will move long-distances to escape predators or disturbance. little is known about argali movements or migrations because they are difficult to capture, tag or collar, and track. in part because they are intolerant of human disturbance, argali typically live only where human access is difficult or infrequent. marco polo argali in the wakhan corridor of afghani- stan are found only in a small section of the big pamir mountains, in the eastern little pamirs, and in the wakhjir valley; thus, possible isolation among populations is a legitimate concern (fig. ). in contrast, it is likely that populations in tajikistan are more contiguous in nature (weinberg et al. ). the exact status of marco polo argali populations in china is uncertain, although they are known to exist in relatively high numbers in most of the taxkorgan nature reserve in kashi prefecture, xinjiang (schaller et al. ; gong et al. ; schaller and kang ). noninvasive and remote genetic sampling (taberlet et al. ; beja-pereira et al. ) facilitates research on elusive species such as argali. molecular genetic markers and metapopulation models allow assessment of migration rates over the recent past (dozens to hundreds of genera- tions, assuming migration-drift equilibrium), and also current migration rates by identifying actual immigrants, e.g. using individual-based assignment tests (cornuet et al. ; paetkau et al. ) or by quantifying immigrant ancestry (wilson and rannala ). molecular markers also allow detection of recent population size reductions or bottlenecks (e.g., cornuet and luikart ). detecting loci under selection is important because selection can bias estimates of population genetic parame- ters, e.g. fst (luikart et al. ). detecting selection signatures also can help infer if a population has experi- enced a recent selection or stress event such as environ- mental change or disease die-off, which could help infer the cause of population declines. for example, simões et al. ( ) studied the genetic response to selection and detec- ted both a reduced effective population size (increased drift at multiple microsatellite loci) and directional selection (fst-outlier effects at a single microsatellite locus) during the adaptation to a new environment in populations of drosophila. the authors suggest that selection at a single locus was associated with adaptive challenges that increased mortality, contributing to genome-wide drift and reduced effective population size. selection can be detected as extremely high (or low) genetic differentiation (fst) between populations at a sin- gle locus compared to neutral loci. researchers have developed ‘‘fst outlier’’ tests (beaumont and nichols ; antão et al. ) and shown that they have rea- sonable power (beaumont and balding ) to detect directional selection between populations. selection can also be detected using neutrality tests within populations (watterson ). for example, paterson ( ) detected even allele frequencies at an mhc locus (major histo- compatibility complex includes more than genes) in a population of soay sheep (ovis aries). microsatellites in genes affected by selection will undergo genetic hitch- hiking and show the selection signature of the gene under fig. map of approximate distribution of the marco polo argali subspecies, o. a. polli (modified from schaller and kang ) showing national boundaries and the five study areas in the pamir mountains. the large grey area is only a coarse range depiction in that argali populations are not distributed continuously throughout the entire shaded area. black filled circles show approximate locations of our murghab (m) study area in southern tajikistan, the taxkorgan (t) study area in xinjiang, china, three study areas in afghanistan: the big pamir (bp), little pamir (lp), wakhjir (waghjir) valley (w) conserv genet ( ) : – selection. this approach has been implemented in large- scale genome scans of thousands of loci to identify genes or genome regions under selection (payseur et al. ; vasemägi et al. ). we genotyped neutral and candidate adaptive gene microsatellites from fecal dna sampled from five study areas within three countries with resident marco polo argali populations (afghanistan, china, and tajikistan) to assess the genetic and demographic status of argali across the region. our specific objectives were to ( ) quantify connectivity of argali among countries and among the three remaining areas with argali in afghanistan, ( ) test for reduced variation and bottleneck signatures within study areas, and ( ) test for signatures of selection at immune system genes that might result from adaptive differentia- tion or stress events such as disease die-offs. methods study area although geographers have not agreed on precise bound- aries, the pamir mountains are generally viewed as con- stituting the eastern portion of tajikistan, the northern half of the wakhan corridor in afghanistan, and the southwest corner of xinjiang, china. this arid (annual precipitation typically * mm/year) and high elevation (highest peaks [ , m) range is typified by broad valleys and relatively gentle slopes (‘‘pamir’’ refers to broad, grassy plateau-like topography). we sampled from argali in five different locations within the pamir mountains; three within wakhan district, badakhshan province, islamic republic of afghanistan, one within gorno-bakakhshan, tajikistan, and one within taxkorgan county, kashi prefecture, xinjiang autono- mous region, people’s republic of china. we also col- lected samples from the karichinai valley in khunjerab national park, pakistan, but because none yielded dna these are not discussed further. we termed our five study areas (fig. ) the big pamir mountains, the little pamir mountains, and the wakhjir valley (all within afghani- stan), the murghab study area (in southern gorno-ba- dakhshan province, tajikistan), and the taxkorgan study area (in xinjiang, china). all five study areas were typified by rolling hills and rugged mountains at elevations of , – , m, vegetated by arid, steppe vegetation com- munities of grasses and low-lying shrubs. the big pamir mountains extend from approximately n to n latitude and � e to � e longi- tude, and are bordered on the south by the wakhan river and on the north by the panj (amu darya) river, which also forms the border between afghanistan and tajikistan. within the big pamirs, we sampled only from approxi- mately km centered on �n latitude and �e longitude. the little pamir mountains, located approxi- mately km east of the big pamir mountains, are cen- tered on � n latitude and � e longitude. the wakhjir valley, located approximately � n latitude and � e longitude, forms a small spur of afghanistan that separates xinjiang, china from gilgit-baltistan of pakistan. our murghab study area in south-eastern taji- kistan extended along an east–west line of approximately km located about – km north of the afghan border. our taxkorgan study area was located within the taxk- organ nature reserve in taxkorgan county, kashi pre- fecture in xinjiang, and was centered on approximately � n latitude and � e longitude. geographic distance between study areas ranged from a minimum of * km between little pamirs and murghab to a maximum of km between taxkorgan and the big pamirs (fig. ). distance within afghanistan ranged from km between the wakhjir valley and the little pamirs to km between the big pamirs and the little pamirs. sampling all field work was conducted either on foot, horseback, or ‘‘yak-back’’. because argali move frequently through dif- ficult terrain and our own movements were circumscribed by the valley systems separated by steep ridges, we made no attempt to impose a standardized geographic sampling regime. instead, we attempted to survey for argali by walking to high vantage points to search for animals during early morning and late afternoon time periods. whenever we encountered fecal pellets we were certain had been freshly deposited by argali, we collected three fecal pellets from each pellet group (i.e. pellet pile). we only collected pellets adjacent to each other within the group, reducing to inconsequential the probability of [ individual argali being represented within each individual sample. we avoided collecting from pellet groups that were scattered over more than approximately a . m area, or that appeared to have been deposited while the animal was moving. we took gps locations for each sample (unless samples were within a few paces of an existing gps fix, in which case we recorded the same location), and noted the date, time, and name of the collector. fecal pellets were stored in sterile cm centrifuge tubes with securely fitting screw-tops to which internal ‘‘sporks’’ were attached (which allowed individual han- dling of each sample without risk of contamination; evergreen scientific, los angeles, ca, usa). we placed three fecal pellets into approximately six parts of % ethyl alcohol (etoh) for each part fecal material, and stored them at room temperature for – months before conserv genet ( ) : – extraction. we collected and extracted dna from one pellet from each of pellet groups. dna extraction, genotyping, and sexing genetic analyses were conducted in two laboratories. ini- tial work was undertaken at ctm/cibio (centro de tes- tagem molecular), portugal, where fecal samples were extracted and eight microsatellite markers were co-ampli- fied in three multiplex pcr reactions as described in harris et al. ( ). all individual fecal samples were initially genotyped twice to quantify the quality of the nuclear dna for producing genotype data. samples with reliable amplifications (electropherogram peak height [ units and identical genotypes from the two replicate genotypings at loci) in this first step were selected to continue the genotyping process. the samples with reliable amplifica- tions were independently re-genotyped three to six times total for each of the loci. the remaining genetic analyses were conducted at the university of montana conservation genetics laboratory (mcgl), missoula, montana, usa. six microsatellite loci (maf , fcb , fcb , adc, maf , krt ) were genotyped in both labs on a large subset of samples as a data quality check. eleven additional loci were genotyped the mgcl for a total of loci. yet another locus (maf ) was genotyped at mcgl in all five populations but was excluded due to allelic dropout and a strong deviation from hardy–weinberg proportions (mean fis = . ). all loci genotyped at mcgl were re-geno- typed three to six times, as in the portugal laboratory. individuals with less than (of ) loci with a consensus genotype (from at least three successful genotypings per locus) were excluded from all analyses. among the microsatellites, were putatively neutral loci, and were located in candidate (functional) genes, including located in introns of genes (krt , mhc (i.e., oladrbps), tcrg , ifng, mmp , glycam- , lif), and one located a few hundred base pairs upstream from the candidate gene (adcyap- ). all candidate genes have some immune system function, except for krt which codes for keratin, a molecule in horn and hair. all loci are described in luikart et al. a, b. multiplex and a single-locus pcrs were optimized and ul reactions were performed on mjr ptc thermocy- clers using touch-down profiles (table ). each reaction contained: . ll of template dna, . ll of qia multiplex mix (qiagen), and either ll of primer mix, or ll of pm forward and reverse primers. two different touch- down profiles with cycles were used, one with an initial annealing temperature of �c stepping down to �c, and another starting at �c and stepping down to �c. fluo- rescently labeled dna fragments were visualized on an abi xl automated capillary sequencer (applied biosystems) in the murdock dna sequencing facility at the university of montana. allele sizes were determined using the abi gs liz ladder (applied biosystems). chromatograms were viewed and analyzed using genemapper software v . (applied biosystems). consensus (i.e. most probable genotypes) genotypes were identified as in previous work (luikart et al. a; harris et al. ). consensus genotypes for microsatellite loci were based on three to six independent sample runs. rules for determining genotypes were as follows: for a sample to be heterozygous at a locus, both alleles had to be observed twice; for a sample to be homozygous the single allele had to be observed in three independent (replicate) genotypings. in addition, ten percent of samples were randomly chosen, re-extracted, and repeat genotyped to monitor for errors. no genotype differences or errors were detected. sex was determined in the mcgl laboratory by pcr amplification of the amelogenin gene as in pidancier et al. ( ). two pcr products (* and bp) were obtained for males but only the longer product for females. due to the large size of the fragments at the amelogenin locus, consensus genotypes were determined as follows: heterozygotes (males) were only accepted only if a male table characteristics of the loci genotyped locus name n a se he se fis maf . . . . . - . maf . . . . . . maf . . . . . . fcb . . . . . - . fcb . . . . . . hh . . . . . . maf . . . . . . maf . . . . . . ilst . . . . . . adcyap- . . . . . - . krt . . . . . - . mhc . . . . . . tcrg . . . . . . ifng . . . . . - . mmp . . . . . . glycam- . . . . . - . lif . . . . . . the top nine loci are presumed to be selectively neutral and are not near coding genes n is the mean number of individuals genotyped among the five study areas, for each locus. a is the mean allelic richness among the five study areas conserv genet ( ) : – band was only observed twice in a heterozygous genotype or if the male band was observed three or more times; homozygotes where only the female band was observed less than three times (e.g. of three independent pcrs) were classified as of unknown gender. sex was determined for of the samples. data analysis the probability that two unrelated individuals (or two random siblings) would have identical genotypes (pid) was computed using dropout (mckelvey and schwartz ) and api-calc (ayres and overall ). principal correspondent analysis (pca) and multilocus genotype matching were conducted in genalex (peakall and smouse ) to identify outliers due to potential genotyping errors or non-argali samples, and to identify identical genotypes. loci contributing significantly more unique individuals than expected were found with dropout (mckelvey and schwartz ). we estimated expected heterozygosity, tested for gametic (linkage) dis- equilibrium, and assessed departures from hardy–wein- berg proportions using exact tests and a markov chain as implemented in genepop . (raymond and rousset ). allelic richness estimates were corrected for sample sizes using rarefaction (kalinowski ). we quantified genetic differentiation among study areas using exact tests for allele frequency differences and using genepop . . we tested for reduced allelic richness and reduced het- erozygosity (e.g. in study areas with low variation) using wilcoxon’s signed-ranks test. this is a nonparametric test for paired comparisons that is appropriate and powerful when homologous loci are examined in related populations. we tested for genetic signatures of recent population bot- tlenecks using heterozygosity excess (i.e., deficit of rare alleles) across multiple neutral loci (cornuet and luikart , luikart and cornuet ). we used two mutation models (the stepwise model smm; and two-phase model tpm with % smm and % multi-step mutations with variance of ) to cover the range of likely mutations models for microsatellite loci (piry et al. ). connectivity among populations was assessed using indices of genetic differentiation (fst) as well as the number of migrants (nm) estimated from both equilibrium models and assignment test approaches that do not assume equilibrium. equilibrium migration rate models included the private alleles method (in genepop . ), and the fst method assuming an island model of migration. two nonequilibrium methods included a bayesian assignment test approach (bayesass; wilson and rannala ) and an individual-based the assignment test of rannala and mountain ( ) coded in geneclass . (piry et al. ). we tested for locus-specific signatures of selection in two ways. first we tested for evenness of allele frequencies at an individual locus within populations using bottle- neck, which gives a probability for each locus being at mutation-drift equilibrium (cornuet and luikart ). we also plotted the probability values of each locus to help assess genome-wide patterns caused by demographic events (e.g. bottlenecks) that affect all loci similarly. selection on individual loci can cause an excess of deficit of heterozygosity (i.e. rare alleles) compared to mutation- drift equilibrium expectations. second, we tested each locus for an excessively high or low fst compared to the mean observed fst by using fst- outlier tests (beaumont and balding , implemented in antão et al. ). an excessively high fst at a locus compared to thousands of simulated neutral loci indicates possible divergent selection; an excessively low fst sug- gests possible balancing selection. results we identified individuals from the five study areas. among the loci, eleven pairs of loci deviated signifi- cantly (p \ . ) from gametic disequilibrium. however among our , tests ( pairs of loci in each of populations), approximately . deviations were expected by chance alone (a = . ). no pair of loci deviated strongly from gametic disequilibrium in more than one population. the pid for identifying sibling pairs was esti- mated to be less than one in one hundred thousand ( - ) in all study areas for the loci, making power high for resolving between random individuals and sibs (table ). genetic variation heterozygosity ranged from a low of . in taxkorgan (china) to a high of . in murghab (table ). allelic table genetic variation in each of the five study areas study area n he (se) a fis pidsibs big pamir . ( . ) . . . e- taxkorgan . ( . ) . - . . e- little pamir . ( . ) . . * . e- murghab (tajik.) . ( . ) . . . e- wakhjir . ( . ) . . . e- mean . . . . . e- n is the number of individuals sampled. he is mean expected heter- ozygosity. a is allelic richness (mean number of alleles per locus corrected for sample size n). pidsibs is the probability of identity among sibs (waits et al. ) * p \ . conserv genet ( ) : – richness ranged from . in taxkorgan to . in murghab. taxkorgan had significantly lower heterozygosity and allelic richness than each other study areas (p \ . ; wilcoxon signed-ranks test). little pamir and wakhjir had the second lowest heterozygosity and allelic richness, respectively. genetic sex identification yielded a sex ratio of % males ( males to females) over all study areas. within study areas, sex ratios ranged from % males in wakhjir, % males in taxkorgan, to % males in big pamir, % males in murghab, to only % (one male) in little pamir. genetic structure and connectivity mean fst for the loci was . among the five study areas. mean fst for the nine putatively neutral loci ( . ) was similar to fst for the candidate loci ( . ) (table ), so most results below are reported for all loci, unless stated otherwise. pairwise mean fst’s ranged from . (between murghab and big pamir) up to . (between taxkorgan and wakhjir). taxkorgan had the highest pair- wise fst’s ranging from a low of . (with murghab) to . (with wakhjir). murghab had the lowest fst’s ranging from only . with big pamir, to the . with taxkorgan. we obtained estimates of . and . migrants per generation using the private alleles method and fst-based method (assuming an island model), respectively. the mean frequency of private alleles p( ) was . . the bayesian approach (bayesass) for estimating the cur- rent number of migrants did not yield informative results because there was not enough information in the data given the relatively low fst (faubet et al. ), despite our fairly large number of loci with high heterozygosity. six highly probable immigrant individuals (p [ . ) were identified in four of the five populations using the individual-based assignment test of rannala and mountain ( ). the probable migrants included the following: two into murghab (females from big pamir), one in little pa- mir (a male from wakhjir), one in big pamir (a male from murghab), and two in wakhjir (a male from big pamir and a male from little pamir). the estimated probability of each of the six putative immigrants actually being an immigrant ranged from . % for the immigrant in little pamir to . % for the immigrant into big pamir. when we lowered the stringency criterion for identification of a migrant (from p [ . to p [ . ), five additional migrants were identified, including two into murghab, two in wakhjir, and one in big pamir. no immigrants were identified in the china study area of taxkorgan. in fact, only one individual of from taxkorgan could potentially be an immigrant, but the probability of that individual being a resident from taxk- organ was still % (fig. ). when we lowered the crite- rion of certainty for the identification of an immigrant (from p [ . to p [ . ), taxkorgan, unlike all other study areas, still showed no evidence of immigrants (e.g., fig. ). selection tests and fst the tcrg gene microsatellite had a significant excess (p \ . ) of rare alleles (i.e., uneven allele frequency distribution compared to neutral expectations) in both the murghab and the little pamir study areas. none of the nine neutral loci or the other seven candidate gene loci deviated from expected allele frequencies under mutation-drift equilibrium (fig. ). two candidate gene microsatellite loci had an fst value significantly different from neutral expectations. gly- cam- had a significantly higher fst (fst = . ; p = . ) and adcyap- had a significantly lower fst (fst = . , p = . ) than expected under neutrality. neither fst deviation was significant at the . level. no neutral loci gave evidence of selection or deviated from mutation-drift equilibrium (fig. ). estimates of genetic differentiation were similar with and without the three outlier loci (glycam- , adcyap- , and tcrg ): the mean fst decreased slightly from fst = . for all loci to fst = . for the loci with no selec- tion signature. pairwise fst, computed after removing out- liers, changed most for the taxkorgan area in china. for example, fst declined from . to . when removing the three outlier microsatellites in candidate genes. discussion our study of neutral and candidate adaptive genes in argali populations suggests relatively high variation within, and low differentiation among populations compared to other mountain sheep (e.g., gutierrez-espeleta et al. ; worley et al. ; epps et al. ; hogg et al. ; luikart et al. a). this is similar to results obtained in table fst between all pairs of sampling areas big pamir taxkorgan little pamir murghab wakhjir big pamir – . . . . taxkorgan . – . . . little pamir . . – . . murghab . . . – . wakhjir . . . . – the putatively neutral loci are included below the diagonal. the eight candidate gene loci are included above the diagonal conserv genet ( ) : – mongolia using mtdna of argali populations previously assumed to represent different subspecies (tserenbataa et al. ). genetic variation and bottlenecks heterozygosity and allelic richness were high compared to many of the same loci genotyped in other wild sheep, in which mean heterozygosity is approximately h = . or a taxkorgan individual number p ro b a b il it y o f o ri g in i n t a x k o rg a n b big pamir individual number p ro b a b il it y o f o ri g in i n b ig p a m ir c murghab individual number p ro b a b il it y o f o ri g in i n m u rg h a b probability threshold for being a resident p< . , p< . , p< . mfmmmf ff fm m m fig. assignment test estimates of the probability of local origin of each individual in the study area from which it was sampled. in a, the individual (# ) least likely to originate from taxkorgan (china) still had an estimated % probability of originating in taxkorgan. in the big pamir b, one individual (# ) had a probability of only . % of originating locally, and was therefore considered an immigrant. in c the murghab (tajikistan) study area, two individuals (# and # ) had a very low probability of local origins (\ . %). the letters m and f designate male and female individuals with a reasonably low probability of being local residents fig. fst-outlier test results showing: a the glycam- locus with excessively high fst among a all five study areas, b between taxkorgan (china) versus big pamir. dots represent loci. white area with most dots represents the expected area for neutral loci ( % confidence area) a - - locus h e te ro zy g o s it y e x c e s s b - - locus h e te ro zy g o s it y e x c e s s tcrg tcrg fig. one locus (tcrg , see arrow) had a significant deviation from mutation-drift equilibrium, i.e., a deficit of heterozygosity (also called an excess of rare alleles), in two populations: a little pamir, and b murghab (tajikistan). loci (dots) at mutation-drift equilibrium will have zero heterozygosity excess (y-axis). loci are in the same order as listed in table . vertical dashed line separates the neutral ( – ) and candidate adaptive gene loci ( – ) conserv genet ( ) : – lower (ozut ; epps et al. ; hogg et al. ; luikart et al. a, b), and even lower in other wild ungulate species (e.g., gebremedhin et al. and papers cited therein). this suggests that marco polo argali popu- lations have relatively high effective population sizes and that our study areas are not yet isolated or inbred, as has been feared (shackleton ; harris et al. ). nonetheless, the significantly lower heterozygosity and allelic richness in taxkorgan (china) compared to our other study areas suggests this population is smaller (schaller et al. , ), and perhaps relatively more isolated than the other populations. the taxkorgan popu- lation is near the southeastern edge of the range of argali and there is a long fence ( km) near the tajik-chinese border (schaller et al. ) that could potentially reduce connectivity of the chinese argali with other populations in murghab and the afghan pamirs; however it is uncertain if the fence is a barrier because, for example, it is not con- tinuous (e.g. open on some high slopes) and argali might jump over it in some locations. the absence of strong bottleneck signatures, even in taxkorgan, and the reasonably high allelic richness suggest no evidence of recent or severe reductions in population size. power for detecting severe reductions is reasonably high when using seventeen highly variable microsatellite loci and individuals (cornuet and luikart ; luikart and cornuet ), as we have from taxkorgan. thus if the taxkorgan population has become genetically bottle- necked or increasingly isolated, which seems likely, the signal might not detectable if the isolation was recent (e.g. \ – argali generations ago). bottleneck signatures can take several generations to become detectable if the bot- tleneck effective size (ne) remains fairly large (e.g. [ ; fig. in cornuet and luikart ). genetic bottleneck signatures also might be obscured by recent immigration. differentiation and connectivity the genetic differentiation (fst) in argali is similar to or lower than other mountain sheep sampled at similar spatial scales in north america. for example, in desert bighorn sheep (o. canadensis nelsoni) from arizona, fst’s ranged from . to . (gutierrez-espeleta et al. ). fur- thermore, over a geographic distance of only km, fst ranged from . to . in desert bighorn sheep pop- ulations without and with a barrier (e.g. road), respectively (epps et al. ). worly et al. ( ) found that genetic differentiation in thinhorn sheep populations (ovis dalli) from western canada was similar to that reported in desert bighorn sheep (gutierrez-espeleta et al. ). rocky mountain bighorn sheep (o. canadensis canadensis) also show fst of * . at this spatial scale (e.g. km across glacier national park, luikart et al. a). the lower differentiation among argali is consistent with their more cursorial nature compared to north american sheep. argali tend to migrate and run for long distances following threat rather short sprints into steep escape ter- rain as bighorn sheep do. argali are built for running, having longer legs than north american sheep. they also will move across large valleys, a behaviour which is less common in north american sheep. the highest differentiation (fst) for taxkorgan among our study areas is consistent with the significantly reduced genetic variation there (compared with other study areas), and suggests increased isolation or a smaller population size. we recommend additional studies of taxkorgan and other argali on the chinese side of international borders, along with monitoring of genetic variation to ensure early detection of population declines, isolation, or recruitment problems, which could potentially be developing. the lowest genetic differentiation in murghab (fst’s . – . ) is consistent with it having the highest genetic variation and being centrally located in the heart of the distribution range of marco polo argali. to estimate gene flow, mean fst values can give only very rough estimates of average number of migrants per generation, and only if populations are near mutation-drift equilibrium (whitlock and mccauley ). because most natural populations are seldom near equilibrium, and vio- late other assumptions, our estimates of * or migrants per generation must be interpreted with great caution; the actual number of migrants could be far higher, for example. current (contemporary) gene flow can be detected from the identification of actual migrants by using individual- based assignment tests (paetkau et al. ). for example, taxkorgan had no detectable immigrants (out of indi- viduals sampled) suggesting relatively low connectivity. the identification of putative immigrants in all other populations suggests they currently are not isolated. the threshold of % certainty for identification of a migrant could be viewed as overly stringent. with our sample size of individuals (and a \ . ), we expect . migrants to be identified by chance alone (as false positives); whereas we identified migrants (p [ . ). because we detected probable migrants, it seems likely that several true migrants exist and that most populations, except per- haps taxkorgan in china, have current migration rates greater than zero. the use of % certainty (a \ . ) for each individual assignment resulted in identification of probable migrants when * were expected by chance alone. selection and adaptation we detected evidence for selection only at candidate gene loci, not at neutral loci, suggesting candidate gene conserv genet ( ) : – approaches can potentially identify loci under selection when using noninvasive sampling in wild sheep. the glycam- microsatellite showed higher fst than neutral expectations (fig. ). this could potentially result from selection at this locus or at other genes nearby such as ifng which is less than centimorgans away from glycam- in domestic sheep, and which has been associated with parasite load in sheep (coltman et al. ) and other ungulates (ezenwa et al. ). glycam- function involves mediating the trafficking of blood-born lympho- cytes into secondary lymph nodes, and also is expressed in the mammary gland of lactating mammals (hou et al. ; rasmussen et al. ). further studies and collec- tion of parasite data are needed to assess if glycam- genotypes are associated with disease resistance in sheep. the lower fst than neutral expectations at adcyap- could reveal balancing selection for even and similar allele frequencies in multiple study areas (e.g., paterson ). the adcyap- gene (adenylate cyclase-activating poly- peptide) is involved in regulating production of interleukin that activates the production of t-helper cell (th ) cytokines involved in defense against helminths and other extracellular parasites (mosmann and sad ). adc- yap- was recently found to be associated with nematode parasite infection in domestic sheep (crawford et al. ), and heterozygotes had lower parasite loads in wild bighorn sheep (luikart et al. b). if argali suffer significant mortalities from disease it is possible that parasites and disease in the pamirs have lead to selection at adcyap- . future research is needed to assess potential effects of disease in argali and on adcyap- . removal of the two fst outlier loci caused little change in mean fst among the five study areas (from . to . , without adcyap- and glycam- ). similarly, removal of the locus (tcrg ) with a heterozygosity- excess had little effect on our multilocus fst estimates among study areas. interestingly, removal of glycam- decreased fst between taxkorgan (china) and other study areas. for example, fst changed from * . to . when we removed glycam- when comparing taxkorgan with big pamir or taxkorgan to wakhjir (fig. b). removal of glycam- did not substantially reduce fst between other study areas, suggesting this glycam- gene contributes substantially to the relatively high multi-locus fst observed between taxkorgan and other study areas. the mhc locus also had the second highest fst between taxkorgan and other study areas. these observations raise the speculative hypothesis that some disease-related selection differential exists between taxkorgan and other study areas. how could selection tests and genotyping of both neu- tral and candidate adaptive loci help advance conservation genetics studies? many candidate adaptive loci will behave as neutral loci, and thus can be used to assess genetic variation, differentiation (fst), and demography (nm and change in ne). however, if a locus reliably shows a response to selection, it could be used to monitor or detect adaptive challenges (e.g. disease die-offs or environmental change) or to identify adaptively-differentiated populations that have exceptionally high fst only at candidate genes associated with selection gradients (e.g. disease or tem- perature). future developments in genomics will allow noninvasive analyses of hundreds of neutral and candidate adaptive genes, which will not only help detect population declines but perhaps infer their cause; for example, if disease candidate genes show high fst then a disease- related die-off could be inferred as the cause of a popula- tion bottleneck (simões et al. ). conclusions our study illustrates the potential usefulness of genotyping both neutral and candidate adaptive loci, which can allow inferences about both demographic status (migration and bottlenecks) and selection events such a disease epizootics and environmental change. our study suggests that candi- date gene loci can be used for connectivity monitoring as long as ‘‘outlier tests’’ are conducted to avoid using non- neutral loci when estimating parameters (e.g. fst) that can be biased by natural selection. future noninvasive studies will include s of loci (e.g., snps) in candidate genes thanks to advances in genotyping technologies for par- tially-degraded dna, such as rt-qpcr assays, which we are developing for use in fluidigm snp-chip dynamic arrays (allendorf et al. ). argali populations appear to have high genetic variation and connectivity in the pamirs within wakhan district of afghanistan, and murghab (tajikistan), but potentially are becoming isolated in taxkorgan, china. we recommend additional studies, including genetic and demographic monitoring of connectivity, along with disease status, to help maintain connectivity and ensure persistence of argali populations. the establishment of international coordina- tion involving afghanistan, china, tajikistan (as well as pakistan, where a few argali remain), would help monitor connectivity and facilitate conservation of argali, their habitat, and other species in the region (schaller ). acknowledgments our study was part of the afghanistan biodi- versity conservation program of the wildlife conservation society (wcs), supported by the united states agency for international development (usaid). for field assistance we thank b. habib, z. moheb, sabir, and a. khairzad. we also thank s. kondratov, k.j. zhang, s. ostrowski, d. bedunah, s. nikzad, z. ejlasi, i. farahmand, q. sahar, k. sediqi, k. sidiqi, g. sediq, a. ahamad, r. king, and l. conserv genet ( ) : – yook. we thank a. dehgan, p. zahler, p. smallwood, p. bowles, and a. simms for advice and administrative assistance. ab-p was sup- ported by sfrh/bpd/ / rg by sfrh/bpd/ / , and this work was supported by poci/cvt/ / all from fundacao para a ciencia e tecnologia (fct), portugal. g.l. and f.w.a. were supported in part by a grant from u.s. national science foundation (grant deb ). g.l. also was supported by grants from nsf (deb ), the walton family foundation, cibio, the university of porto, portugal, and a research grant ptdc/bia-bde/ / from the portuguese science foundation. references allendorf fw, hohenlohe pa, luikart g ( ) genomics and the future of conservation. nature rev genet : – antão ta, lopes rj, beja-pereira a, luikart g ( ) lositan: a workbench to detect molecular adaptation based on an fst- outlier method. bmc bioinformatics : ayres kl, overall adj ( ) api-calc . : a computer program for calculating the average probability of identity allowing for substructure, inbreeding and the presence of close relatives. mol ecol notes : – beaumont ma, balding dj ( ) identifying adaptive genetic divergence among populations from genome scans. mol ecol : – beaumont ma, nichols ra ( ) evaluating loci for use in the genetic analysis 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microsatellites as a source of gene-associated polymor- phisms for detecting signatures of divergent selection in atlantic salmon (salmo salar l.). mol biol evol : – waits l, luikart g, taberlet p ( ) estimating the probability of identity among genotypes in natural populations: cautions and guidelines. mol ecol : – watterson ga ( ) the homozygosity test of neutrality. genetics : – weinberg pi, fedosenko ak, arabuli ab, myslenkov a, romashin av, voloshina i, zheleznov n ( ) the commonwealth of independent states (former ussr) pages – . in: shackl- eton ds (ed) wild sheep and goats and their relatives: status survey and conservation action plan for caprinae. iucn/ssc caprinae specialist group, iucn, gland, switzerland and cambridge, p whitlock mc, mccauley de ( ) indirect measures of gene flow and migration: fst doesn’t equal /( nm ? ). heredity : – wilson ga, rannala b ( ) bayesian inference of recent migration rates using multilocus genotypes. genetics : – worley k, carey j, veitch a, coltman dw ( ) detecting the signature of selection on immune genes in highly structured populations of wild sheep (ovis dalli). mol ecol : – conserv genet ( ) : – 	high connectivity among argali sheep from afghanistan and adjacent countries: inferences from neutral and candidate gene microsatellites 	abstract 	introduction 	methods 	study area 	sampling 	dna extraction, genotyping, and sexing 	data analysis 	results 	genetic variation 	genetic structure and connectivity 	selection tests and fst 	discussion 	genetic variation and bottlenecks 	differentiation and connectivity 	selection and adaptation 	conclusions 	acknowledgments 	references << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma . ) /calrgbprofile (srgb iec - . ) /calcmykprofile (iso coated v % \ eci\ ) /srgbprofile (srgb iec - . ) /cannotembedfontpolicy /error /compatibilitylevel . /compressobjects /off /compresspages true 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/omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) ( . ) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [ ] /pagesize [ . . ] >> setpagedevice rare variants and loci for age-related macular degeneration in the ohio and indiana amish vol.:( ) human genetics ( ) : – https://doi.org/ . /s - - - o r i g i n a l i n v e s t i g at i o n rare variants and loci for age‑related macular degeneration in the ohio and indiana amish andrea r. waksmunski , ,  · robert p. igo jr.  · yeunjoo e. song  · jessica n. cooke bailey ,  · renee laux  · denise fuzzell  · sarada fuzzell  · larry d. adams  · laura caywood  · michael prough  · dwight stambolian  · william k. scott  · margaret a. pericak‑vance  · jonathan l. haines , , received: april / accepted: july / published online: july © the author(s) abstract age-related macular degeneration (amd) is a leading cause of blindness in the world. while dozens of independent genomic variants are associated with amd, about one-third of amd heritability is still unexplained. to identify novel variants and loci for amd, we analyzed illumina humanexome chip data from amish individuals with early or late amd, unaffected amish individuals, and related amish individuals with unknown amd affection status. we retained , polymorphic autosomal variants across samples for association and linkage analyses. after correcting for multiple testing (n = , ), we identified four variants significantly associated with amd: rs (lcn , p = . × − ), rs (rtel , p = . × − ), rs (dlgap , p = . × − ), and rs (cgrrf , p = . × − ). these variants have not been previously associated with amd and are not in linkage disequilibrium with the known amd-associated variants reported by the international amd genomics consortium based on physical distance. genome-wide significant linkage peaks were observed on chromosomes q . –q . (maximum recessive hlod = . ) and q . – . (maximum dominant hlod = . ; maximum recessive hlod = . ). these loci do not overlap with loci previously linked to amd. through gene ontology enrichment analysis with cluego in cytoscape, we determined that several genes in the -hlod support interval of the chromosome locus are involved in fatty acid binding and triglyceride catabolic processes, and the -hlod support interval of the linkage region on chromosome is enriched in genes that participate in serine-type endo- peptidase inhibitor activity and the positive regulation of epithelial to mesenchymal transition. these results nominate novel variants and loci for amd that require further investigation. introduction age-related macular degeneration (amd) is the third lead- ing cause of vision loss in the world (ayoub and patel ). it is characterized by the deterioration of the central field of vision due to the accumulation of lipid deposits (drusen), inflammation, and neurodegeneration in the macula (fritsche et al. ). amd is a multifactorial disease with numer- ous influential genetic and environmental risk factors. envi- ronmental factors constitute about – % of total amd variance and include age, smoking, hypertension, and diet (fritsche et  al. ; seddon ). more than half of amd heritability is explained by independent common and rare genetic variants across genomic loci (fritsche et al. ). therefore, about half of amd heritability is unaccounted for by known genetic polymorphisms and may be partially resolved with the identification of rare variants (manolio et al. ). electronic supplementary material the online version of this article (https ://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. * jonathan l. haines jonathan.haines@case.edu department of genetics and genome sciences, case western reserve university, cleveland, oh, usa cleveland institute for computational biology, case western reserve university, cleveland, oh, usa department of population and quantitative health sciences, case western reserve university, cleveland, oh, usa john p. hussman institute for human genomics, university of miami miller school of medicine, miami, fl, usa department of ophthalmology, university of pennsylvania, philadelphia, pa, usa http://crossmark.crossref.org/dialog/?doi= . /s - - - &domain=pdf https://doi.org/ . /s - - - human genetics ( ) : – as the minor allele frequency (maf) for a variant decreases, the sample size required to detect an associa- tion increases linearly with /maf (manolio et al. ). consequently, traditional genome-wide-association stud- ies (gwas) have been largely unable to detect associa- tions with rarer variation (typically maf < . ) unless the sample size in is the tens of thousands. in contrast, gwas on a sample of large families can increase the power to detect such variants through enrichment of rare variants segregating in families with many affected members (auer and lettre ). isolated founder popu- lations, like the amish, can amplify the power of family- based studies through sharing of founder variants. the amish are descendants of swiss anabaptists who settled in north america –  years ago and maintained their culturally segregated community for generations by mar- rying within their faith group and adhering to a uniform, conservative lifestyle (strauss and puffenberger ). therefore, they have lower genetic and environmental het- erogeneity compared to the general population of euro- pean descent. in addition, amish communities comprise large families with an average of seven to nine children in each nuclear family (mckusick et al. ). extensive genealogical data are available for this community in the anabaptist genealogy database (agdb) making this a valuable population to identify rare genetic variation by combining the advantages of isolated populations and large pedigrees (agarwala et al. ). to identify novel variants underlying the pathophysiol- ogy of amd, we performed association and linkage anal- yses on illumina humanexome chip data for related amish individuals from ohio and indiana. we also exam- ined in silico functional annotations of the genes from our linkage regions to determine whether these genes participate in biological processes that may be relevant for amd pathology. materials and methods study participants the amish individuals were identified from published amish community directories, referral from other amish community members, or due to close relationships to other research subjects. all study participants were at least  years old. the amd affection status was defined by a self-reported diagnosis on a health questionnaire, which asked whether the individual had ever received an amd diagnosis from a physician. we previously demonstrated that self-reported amd statuses have positive and negative predictive values of around % in this population (hoffman et al. ). some ohio amish participants also received a clinical exam at the wooster eye clinic in wooster, ohio where ocular coherence tomography images were obtained for both eyes. participants’ eyes were graded on a scale from to using the modified clinical age-related maculopa- thy staging (carms) system (seddon et al. b; sardell et al. ). individuals with grades , , and in both eyes were designated as unaffected in this study, and individu- als with grades – in at least one eye were designated as affected. grades of were indicative of early/intermedi- ate amd, and grades of or were classified as advanced amd. for this study, amish samples included indi- viduals with confirmed or self-reported amd, unaffected individuals, and related amish individuals with unknown amd phenotype. using the anabaptist genealogy data- base (agarwala et al. ), we constructed a person all-connecting path pedigree for the amish individuals that passed quality control and were closely related (fig.  ). this included affected with amd, unaffected indi- viduals, and relatives with an unknown amd affection status. informed consent was obtained from all study partici- pants, and the study was approved by the institutional review boards at vanderbilt university and case western reserve fig. all-connecting path pedi- gree of the amish individu- als in this study. the pedigree was drawn using information from the anabaptist genealogy database (agdb). circles: females. squares: males. the genotyped individuals are within the shaded blue box human genetics ( ) : – university. study procedures were performed in accordance with the tenets of the declaration of helsinki. genotyping and quality control a total of samples, which included unique amish samples, amish duplicates, and coriell hapmap trio controls of european descent, were genotyped using the illumina humanexome beadchip v . at the vanderbilt technologies for advanced genomics (vantage) dna resources core. genotype data were called and clustered with illumina’s genomestudio (illumina). the accuracy of genotype calling using genomestudio is improved by increasing the sample size (grove et al. ). therefore, an additional non-amish samples that were genotyped by vantage with the same protocols and at the same time as the amish samples were imported into genomestudio and clustered with the amish samples. the data from these non- amish samples were not used in the subsequent association and linkage analyses. all samples demonstrated call rates greater than % and were used for extensive quality con- trol in genomestudio as summarized in supplemental fig.  and supplemental table  (guo et al. ). briefly, we excluded variants for low call rate, gentrain score for cluster quality, cluster separation, and intensity values (supplemen- tal table  ). hemizygous variants were evaluated as well as variants with mendelian and replication errors (supplemen- tal table  ). rare variant calling was performed manually with genomestudio’s gencall algorithm and with zcall, a separate rare variant calling algorithm (goldstein et al. ). zcall is utilized as a post-processing step for call- ing variants with no genotypes assigned after initial variant calling with genomestudio’s gencall algorithm. it uses the intensity profile of the homozygous cluster of the common allele to determine the location of the other homozygous and heterozygous genotype clusters (goldstein et al. ). all variants with at least four new heterozygous calls in zcall were manually reviewed in genomestudio. a total of , variants and samples, includ- ing amish individuals, passed quality control in genomestudio and were evaluated for sex mismatch, race mismatch, and heterozygosity outliers using plink v . (purcell et al. ). sex mismatched samples were evaluated using heterozygosity rates for the x chromo- some and call rates for the y chromosome (supplemen- tal fig.  ). one unresolvable sex mismatch in our amish samples was identified and removed from the study. an xxy amish male and an xo amish female were identi- fied and retained. ancestry mismatch was assessed using the eigenstrat software package and about ancestry-informative markers (aims) from the exome chip (price et al. ). principal component analysis (pca) demonstrated that the combined sample set is multi-ethnic and that the amish samples clustered together with other europeans as expected (supplemental fig.  ). none of our amish samples were in the extremes of the heterozygo- sity distribution calculated by plink v . (purcell et al. ), so none of them were excluded as outliers (sup- plemental fig.  ). the heterozygosity rate can indicate problematic, potentially contaminated dna samples (guo et al. ). of the , variants genotyped, , were polymorphic in the amish sample, passed extensive quality control, and served as input for our association and linkage analyses. variants were considered polymorphic if they had a non-zero minor allele frequency calculated by the robust association-detection test for related individuals with population substructure (roadtrips) software v . . we omitted five amish individuals from our association and linkage analyses due to excessively distant relatedness in the pedigree (supplemental fig.  ). of the remaining amish individuals, are affected, are unaffected, and have an unknown amd status. association analysis the samples in this study are highly interrelated (fig.  ). to account for these relationships and population struc- ture, we performed association testing using the road- trips software v . (thornton and mcpeek ). this program determines associations between genotypes and binary traits while adjusting for the relatedness and population structure of the samples. it calculates and uses an empirical covariance matrix based on the genotypes from the genotype data to account for population struc- ture. roadtrips determines three different test statis- tics for association: the rm, rχ, and rw tests (thornton and mcpeek ). for each of these tests, p values are calculated based on a chi squared distribution with df asymptotic null distribution (thornton and mcpeek ). we used the rm test statistic because it allows inclusion of both unaffected individuals and individuals with unknown phenotype in the analysis and is more powerful than the other two when samples are related and pedigree structure is known (thornton and mcpeek ). we used kinship coefficients for the amish individuals calculated by kininbcoef to account for the pedigree structure in our data (bourgain ). the software program determines the weight of individuals with unknown phenotype in the analysis by a user-defined population prevalence for the trait and the phenotypes of their genotyped relatives (thornton and mcpeek ). we set the population prev- alence of amd to . based on published estimates of its prevalence in individuals of european descent of an age typical of this pedigree (shalev et  al. ; smith et al. ). human genetics ( ) : – linkage analysis we performed multipoint linkage analyses on a panel of autosomal variants chosen to be informative for stretches of identity by descent on the exome chip (abeca- sis lab ). the amish individuals in this study are connected in a multigenerational pedigree of amish individuals (fig.  ). since analyzing very large, complex pedigrees is computationally intractable, we used pedcut to partition the all-connecting path pedigree into densely affected sub-pedigrees with a maximum bit size of (liu et al. ). parametric heterogeneity lod (hlod) scores were determined with the multi-point engine for rapid likelihood inference (merlin) software tool (abecasis et  al. ) under affected-only dominant and recessive models. the penetrance values for the dominant model were , . , or . for , , or copies of the disease allele, respectively. under the recessive model, penetrance values were defined as , , or . for , , or cop- ies of the disease allele, respectively. we set disease allele frequencies to . and . for each of these models. we performed multipoint linkage analyses across all autosomes with hlod scores evaluated at each marker and at every centimorgan (cm; haldane) with a bit size threshold of . for chromosomes with genome-wide significant multi- point hlod scores, we repartitioned the all-connecting path pedigree into sub-pedigrees with maximum bit sizes of and to test the robustness of our findings to changes in sub-pedigree construction. the model parameters for these analyses were consistent with those used in the initial mul- tipoint analyses. for chromosome , we performed a multipoint link- age analysis with liability classes for carriers of the cfh y h (rs ) (edwards et al. ; hageman et al. ; haines et  al. ; klein et  al. ) and p a (rs ) variants (hoffman et al. ). cfh y h failed quality control in our dataset. therefore, we identified a surrogate variant (rs , r = . ) for cfh y h using the ldmatrix module of ldlink to compare link- age disequilibrium statistics among cfh variants that had passed quality control in our study and the y h vari- ant in the ceu population from phase (version ) of the genome project (machiela and chanock ). we identified cfh p a carriers by performing customized taqman genotyping assays of the variant in our amish cohort (unpublished data). briefly, p a genotyping was carried out using a custom taqman snp genotyping assay (thermo fisher) to interrogate the presence of a c (p ) or g (a ) at position in the cfh transcript. assays were carried out per the manufacturer’s instructions using taqman genotyping master mix (thermo fisher) and  ng of genomic dna per reaction. reactions were run on a quantstudio pcr machine, and genotypes were called by the quantstudio analysis software. in our conditional link- age analysis, penetrance values were . , . , and . for , , and copies of the disease allele, respectively, for carriers of only the surrogate for y h. we chose these values based on the population prevalence of amd in the caucasian population ( %) (shalev et al. ; smith et al. ) and the expected prevalence based on the published odds ratio (or) for y h, which is . per copy of the risk allele in individuals of european descent (haines et al. ; sofat et al. ). for carriers of only the p a variant or both variants, penetrance values were . , . , and . for , , and copies of the disease allele, respectively. the penetrance value for the p a risk allele was derived from the number of affected p a carriers we identified in a pre- vious study (hoffman et al. ). for non-carriers of both variants, penetrance values were consistent with those used for the multipoint linkage analyses of the other autosomes. in silico functional analysis of genes from  ‑hlod support intervals of linkage regions to determine whether genes present in the -hlod sup- port intervals (supplemental tables  – ) of our linkage regions on chromosomes and are functionally related to one another, we extracted gene boundaries from ensembl (human genome build ) and performed gene ontology (go) enrichment analyses with the cluego v . . plug-in (bindea et al. ) of cytoscape v . . (https ://cytos cape. org/). specifically, terms from the following classifications in go were considered: immune system process, biological process, molecular function, and cellular component. we included all evidence codes (experimental, non-experimen- tal, author statements from publication, and curator state- ments) in the analysis. we calculated p values using right- sided hypergeometric tests and the benjamini–hochberg correction for multiple testing. we chose medium network specificity to identify representative pathways for our genes of interest. this level of specificity examines go levels – , requires at least three genes per go term, and ensures that mapped genes represent at least % of the total associated genes. we also used a kappa score of . for our go term network connectivity threshold. go terms were iteratively compared, merged into functional groups, and visualized in cytoscape. results association analysis of the , polymorphic autosomal variants that passed quality control, four variants met the bonferroni correction threshold (p < . × − ): rs (chromosome , https://cytoscape.org/ https://cytoscape.org/ human genetics ( ) : – lcn , p = . × − ), rs (chromosome , rtel , p = . × − ), rs (chromosome , dlgap , p = . × − ), and rs (chromosome , cgrrf , p = . × − ) (fig.  ; table  ). p values were not corrected for inflation because the genomic control factor was . (supplemental fig.  ). these variants dem- onstrate novel associations with amd and do not map to the amd susceptibility loci identified by the international amd genomics consortium (iamdgc) based on physical distance (fritsche et al. ). while these variants were identified in the amish, we found that they are rare to low- frequency (maf < . ) in outbred populations of european descent including the iamdgc dataset and the gnomad database (table  ). linkage analysis multipoint linkage analyses under autosomal dominant and recessive models were performed to identify genomic loci linked to amd in amish sub-pedigrees (supplemental figs.  , ; supplemental tables  , ). under the recessive model with a disease allele frequency of . , we identified genome-wide significant signals (hlod > . ) on chro- mosome (table  ; fig.  ). we also identified significant signals on chromosome under the dominant and reces- sive models (table  ; fig.  ). these signals were robust to varying sub-pedigree structure (supplemental figs.  , ). although we initially observed genome-wide significant fig. manhattan plot of p values obtained from association testing using roadtrips. the red line denotes the bonferroni correction threshold for multiple testing (p < . × − ). four variants passed this threshold and are summarized in table  table amd-associated variants identified with roadtrips testing of amish families p values were obtained from the rm test variant positions are given for build (hg ) of the human genome amd, age-related macular degeneration; roadtrips, robust association-detection test for related individuals with population substruc- ture; chr., chromosome; lcn , lipocalin ; rtel , regulator of telomere elongation helicase ; rtel -tnfrsf b, rtel -tnfrsf b readthrough (nmd candidate); dlgap , dlg-associated protein ; cgrrf , cell growth regulator with ring finger domain rsid chr. position alleles gene(s) consequence allele count p affected unaffected unknowns rs , , a/g lcn missense . × − rs , , g/a rtel rtel -tnfrsf b missense non-coding transcript . × − rs , , a/g dlgap synonymous . × − rs , , g/a cgrrf missense . × − table allele frequencies for the amd-associated variants identified with roadtrips testing of amish families in outbred populations (iamdgc and gnomad release . ) amd, age-related macular degeneration; roadtrips, robust association-detection test for related individuals with population substructure; iamdgc, international age-related macular degeneration genomics consortium; gnomad, genome aggregation database variant allele frequency iamdgc gnomad rsid chromosome advanced amd cases controls non-finnish europeans rs . . . rs . . . rs . . . rs . . . human genetics ( ) : – signals (hlod > . ) on chromosome under the dominant and recessive models (supplemental table  ; supplemental fig.  ) and on chromosome under the recessive model (supplemental table  ; supplemental fig.  ), these signals greatly reduced when we re-performed our analyses with dif- ferent sub-pedigree structures (supplemental figs.  , ). in our multipoint linkage analysis under the dominant model with a disease allele frequency of . , we observed a region on chromosome that was nearly genome-wide significant (maximum hlod = . at .  cm; supple- mental table  ). the peak of this region is about  mb from the complement factor h (cfh) gene, which has been repeatedly associated with amd risk in the general popu- lation (fritsche et al. ). we performed a conditional linkage analysis on the markers on chromosome with lia- bility classes for carriers of two amd risk variants in cfh (y h and p a) to determine whether these markers were driving the peak linkage signal on chromosome . of the affected individuals in the linkage analysis, carry at least one copy of the p a variant, and have at least copy of the surrogate for y h. three of the p a carriers are also heterozygous for the surrogate of y h. our conditional linkage analysis on chromosome using distinct classes for carriers of the surrogate for cfh y h and/or the cfh p a variant demonstrated that the cfh locus was contributing to the peak signal we observed in the unconditioned analysis (fig.  ). in silico functional analysis of genes from  ‑hlod support intervals of linkage regions we performed go enrichment analyses using right-sided hypergeometric tests on the genes from the -hlod sup- port intervals our linkage regions to identify any func- tional relationships through go terms. p values were cor- rected for multiple testing using the benjamini–hochberg method. the region we identified with significant evidence of linkage on chromosome q . –q . includes genes (supplemental table  ). the -hlod support inter- val of this peak contains unique genes (supplemental table  ), including those that are involved in fatty acid binding and triglyceride catabolic processes (fig.  a; sup- plemental table  ). the region we found with significant evidence of linkage on chromosome q . – . under the dominant model includes genes (supplemental table  ). the -hlod support interval of this peak con- tains unique genes (supplemental table  ), includ- ing those that are implicated in serine-type endopeptidase inhibitor activity and positive regulation of epithelial to mesenchymal transition (fig.  b; supplemental table  ). table significant linkage loci identified from model-based mul- tipoint linkage analyses of amish families with disease allele fre- quency of . the peak hlod score is the maximum hlod score obtained for the designated chromosome hlod, heterogeneity lod; cm, centimorgans model chromosome peak hlod score region with hlod > . (cm) -hlod sup- port interval (cm) recessive . . – . . – . dominant . . – . . – . recessive . . – . . – . . – . . – . fig. hlod scores obtained from multipoint linkage analysis in merlin under dominant and recessive models on chromo- some . the black line denotes genome-wide significance (hlod score > . ). a genome-wide significant hlod score of . was observed under the recessive model. tick marks along the upper x-axis correspond to the marker positions fig. hlod scores obtained from multipoint linkage analysis in merlin under dominant and recessive models on chromo- some . the black line denotes genome-wide significance (hlod score > . ). the maximum dominant hlod score was . . the maximum recessive hlod score was . . tick marks along the upper x-axis correspond to the marker positions human genetics ( ) : – the significant linkage region we observed on chromo- some q . – . under the recessive model includes genes (supplemental table  ). the -hlod support interval of this peak contains unique genes (supple- mental table  ), which were not overrepresented in any particular go terms in our enrichment analysis. when we analyzed all the genes from our -hlod support intervals on chromosome and together in cluego, the same go terms from our chromosome-specific analyses were identified. discussion we performed association and linkage analyses on amish families from ohio and indiana to uncover novel variants and loci for amd. using roadtrips and kinship coef- ficients derived from the relationships detailed in the all- connecting path pedigree, we identified four novel variants associated with amd in the amish. since we analyzed exome chip data, we recognize that these variants may not be the functional variants underlying these loci and that we might be observing their association signals because they are in linkage disequilibrium with the true functional variants for amd. these four variants are independent and physi- cally distant from the amd-associated variants identified by the iamdgc (fritsche et al. ). association signals were not detected in the directly genotyped variants in the lcn gene on chromosome or the directly genotyped variants in the rtel /rtel -tnfrsf b locus on chromo- some from the iamdgc gwas for advanced amd. however, a few nominal signals (p <  . ) were found in the iamdgc loci for dlgap (n = out of ) on chro- mosome and cgrrf (n = out of ) on chromosome (fritsche et  al. ). these three dlgap variants are not in linkage disequilibrium (ld) with one another (r < . ) but are within  kb of the variant we identi- fied in the amish. the cgrrf variant that achieved a p value less than . in the iamdgc gwas and the variant we identified with roadtrips in the amish are  bp apart but are not in ld (r = . in ceu population). the variant we found on chromosome (rs ) is about .  mb, .  mb, .  mb, and .  mb away from the lead variants identified by the iamdgc on this chromo- some (rs , rs , rs , and rs , fig. conditional linkage analysis on chromosome taking into account the y h and p a carrier statuses. the black line denotes the hlod scores from the multipoint linkage analysis per- formed without liability classes for the cfh variants. the orange line represents the distribution of hlod scores from the condi- tional linkage analysis. the peak hlod score in the uncondi- tioned analysis was . at .   cm ( , ,   bp, build ). the peak hlod score in the conditioned analysis was . at .  cm ( , ,  bp, build ). the cfh gene boundaries are , , – , ,  bp (ensembl, build ) fig. gene ontology (go) enrichment networks for genes from -hlod support intervals from a chromosome and b chromosome . the size of the nodes in each network illustrates the number of mapped genes to the depicted go term, and the color of the node shows the significance of the term in each enrichment analysis. the leading term of each go group is depicted in bold and represents the most significant ontology from the group. full descriptions of these terms are available in supplemental tables  and human genetics ( ) : – respectively) (fritsche et al. ). the variant we iden- tified on chromosome (rs ) is about . and .  mb away from the lead variants from the iamdgc gwas on chromosome (rs and rs , respectively) (fritsche et al. ). the iamdgc did not identify any amd-associated variants on chromosome (fritsche et al. ). the lead variant in the amd locus identified by the iamdgc on chromosome (rs ) (fritsche et al. ) is almost  mb away from the variant we identified in the amish (rs ). the four vari- ants we identified in the amish are rare to low-frequency in the iamdgc data and the gnomad database. while allele frequencies for these variants are fairly similar in advanced amd cases and controls from the iamdgc, these vari- ants were significantly associated with amd in our amish cohort. it was previously demonstrated that the amish popu- lation has a lower genetic burden of known amd variants (hoffman et al. ); therefore, this may suggest that the amish have different etiology for amd than the general population of european descent. of the amd-associated variants that we identified in this study, only the rtel variant (rs ) is cataloged in the clinvar database, but it is considered a variant of unknown significance (https ://www.ncbi.nlm.nih.gov/clinv ar/rcv /). this variant also maps to the natu- rally occurring rtel -tnfrsf b read-through transcript of this locus, which is a candidate for nonsense-mediated mrna decay and unlikely to yield a protein product. rtel encodes a regulator of a telomere elongation helicase, which maintains telomere length and genomic stability (barber et al. ; codd et al. ; ding et al. ). telomere length has been hypothesized as a marker of aging because telomeres shorten with age and the presence of short telom- eres directs the cell to enter senescence (von zglinicki and martin-ruiz ). oxidative stress can also contribute to the reduction of telomere length in cells and has been char- acterized as a contributing factor to amd pathophysiology (shaw et al. ; von zglinicki and martin-ruiz ). lipocalins constitute a family of extracellular proteins that are responsible for transporting small lipids such as fatty acids, retinoids, and steroids (suzuki et al. ). there are no lcn variants documented in the gwas catalog (https ://www.ebi.ac.uk/gwas/). however, another lipocalin family member (lipocalin- , lcn ) may modulate inflam- mation in retinal degeneration by promoting cell survival responses and regulating the production of inflammatory proteins (parmar et al. ). additionally, tear lipocalins constitute a large group of lipid-binding proteins in tears and may serve as potential biomarkers for diabetic retin- opathy and alzheimer’s disease (kallo et al. ; wang et al. ). dlgap encodes a guanylate kinase-associ- ated protein involved in protein–protein interactions with scaffold proteins in the post-synaptic density of excitatory synapses (feng and zhang ; li et al. ). dlgap also is one of nine genes located in a chromosomal region (myopia- , myp ) that demonstrated significant genetic linkage with autosomal dominant high myopia (scavello et al. ; young et al. ). the proteins encoded by dlgap and lipocalin genes (lcn and lcn ) were also found to be expressed in the human choroid-retinal pigment epithelial complex (skeie and mahajan ). the variant we identified in dlgap is about  mb away from an amd variant (rs ) identified in a genome-wide association study accounting for age-stratified effects in the iamdgc data (winkler et al. ). cgrrf encodes a cell growth regulator that has been associated with eye morphology in humans (lee et al. ). while the protein product of this gene is not well-characterized, it has proposed as a modula- tor of evi, which is a transmembrane protein involved in wnt protein secretion (glaeser et al. ). canonical wnt signaling has been implicated in retinal inflammation and may have a role in amd pathology (zhou et al. ). addi- tional studies will be required to elucidate the roles these genes might have in amd etiology. in our linkage screens in the amish, we identified a novel susceptibility locus for amd on chromosome q . –q . under our recessive model. this region does not overlap with amd loci previously identified with genome-wide linkage screens, which occur on chromosomes p and q . (seddon et al. ). in their recent gwas, the iamdgc identified a susceptibility locus for amd on chromosome p . with the most significant signals com- ing from rs and rs in the tnfrsf a/ loc and tnfrsf a genes (fritsche , ). in our linkage analyses of chromosome , we observed a maximum hlod score of . in this gene region under the recessive model with disease allele frequency of . . the strongest single variant p value observed by the iamdgc in our linkage region was . × − , which does not reach classical gwas significance (p < × − ). common vari- ants associated with optic nerve degeneration in glaucoma have also been identified in q , which is near our linkage region (wiggs et al. ). based on our go enrichment analysis with cluego, the genes from the -hlod support interval of our significant linkage region on chromosome q . –q . have func- tional annotations related to triglyceride catabolic processes and fatty acid binding. components of lipid metabolism have been previously implicated in the genetic etiology and pathophysiology of amd. lipids comprise about % of the composition of drusen, which are a hallmark of amd (wang et  al. ). in their most recent gwas, the iamdgc determined that several lipid pathways from the reactome and go pathway databases were enriched for genes from the amd susceptibility loci they identified (fritsche et al. ). known amd-associated genes (apoe and lipc) https://www.ncbi.nlm.nih.gov/clinvar/rcv / https://www.ncbi.nlm.nih.gov/clinvar/rcv / https://www.ebi.ac.uk/gwas/ human genetics ( ) : – (fritsche et al. ) are also described as members of the triglyceride, neutral lipid, acylglycerol, and glycerolipid cat- abolic processes (go: , go: , go: , and go: , respectively), which were enriched in genes from our linkage region. additionally, although con- suming high amounts of saturated and monounsaturated fats has been associated with amd risk (delcourt et al. ), dietary intake of omega- -fatty acids has been attributed to reducing the risk of amd (delcourt et al. ; san- giovanni and chew ; sangiovanni et al. ; seddon et al. a). there is not a consistent association between triglyceride levels and amd risk. some studies observed lower triglyceride levels in patients with early amd (klein et al. ), the choroidal neovascularization subtype of advanced amd (merle et al. ), or any type of amd (paun et al. ; roh et al. ; semba et al. ). how- ever, other studies found higher triglyceride levels were associated with amd status (munch et al. ; nowak et al. ). recently, levels of triglycerides were associ- ated with a decreased risk for amd and smaller drusen area (colijn et al. ). further functional studies would need to be performed to definitively implicate the role of fatty acid binding and triglyceride levels in amd pathology. from our multipoint linkage analyses with dominant and recessive models of inheritance, we identified novel amd loci on chromosome q . – . and q . – . , respectively. these regions do not overlap with the amd- associated variant (rs ) we identified in our asso- ciation test. although both analyses interrogate the genetic etiology of the trait, linkage analyses identify chromosomal segments co-segregating with traits in families (morton ); whereas, association analyses compare allele fre- quencies between individuals with and without the trait of interest (hirschhorn and daly ). therefore, their results can be independent of one another. the iamdgc did not identify any amd susceptibility loci on chromosome in either of their recent gwas, and another study using iamdgc data identified an amd gene × age interaction on chromosome p, which is independent of our linkage peak (fritsche , ; winkler et al. ). the most notable single-marker p value observed in the iamdgc gwas was . × − , which occurred for a variant that falls within our linkage regions under both dominant and recessive models. previous gwas have found suggestive amd variants on chromosomes q . (naj et al. ) and q (sobrin et al. ), which are located outside of the -hlod support interval of our linkage region. the c allele of the q . variant (rs ) has a strong pro- tective effect in never smokers but increases the risk of amd in smokers (naj et al. ). the q variant (rs ), which occurs near the myelin basic protein (mbp) gene, demonstrated suggestive evidence of association with geo- graphic atrophy (sobrin et al. ). the -hlod support interval of our significant linkage region on chromosome q . – . contains genes that have functional annotations in go processes such as positive regulation of epithelial to mesenchymal transition (emt) and serine-type endopeptidase inhibitor activity. another known amd risk gene, tgfbr , (fritsche , ) has been previously described as a part of the positive regula- tion of emt (go: ). the emt of retinal pigment epithelial (rpe) cells is considered one of several biological processes that are responsible for the formation of subretinal fibrosis in the macula following choroidal neovasculariza- tion in advanced amd (ishikawa et al. a). emt has also been proposed as a mechanism employed by rpe cells to survive in the stressful macular microenvironment dur- ing dry amd progression (ghosh et al. ). therefore, it has been suggested that therapeutically targeting rpe cells in emt may help treat patients with advanced amd and subretinal fibrosis (ghosh et al. ; ishikawa et al. b; kobayashi et  al. ). several of the serine proteinase inhibitor (serpin) family members from the -hlod support interval have also been described in the context of amd- related pathologies. the proteins encoded by serpinb (maspin) and serpinb (headpin) are known for their anti-angiogenic properties, which may have implications for the choroidal neovascularization subtype of advanced amd (pescosolido et al. ; shellenberger et al. ; zhang et al. ). a genetic variant near serpinb was identified as a risk factor for amd in smokers and a protec- tive factor in nonsmokers in gene-environment interaction analyses (naj et al. ). another gene from our -hlod support interval, tcf , has repeatedly been association with fuchs endothelial dystrophy, which is characterized by the deterioration of the corneal epithelium (baratz et al. ; kuot et al. ; li et al. ; riazuddin et al. ; thala- muthu et al. ). c and timp are known amd-associ- ated genes identified by the iamdgc that are also described in the following go terms from our analyses: endopepti- dase regulator activity (go: ), peptidase inhibitor activity (go: ), and endopeptidase inhibitor activity (go: ). additionally, the only subtype-specific vari- ant identified for the cnv subtype of advanced amd was located near the mmp gene on chromosome , which is a member of the endopeptidase family that participates in extracellular matrix degradation (fritsche et al. ). although the major peak in our initial linkage analysis on chromosome was diminished in our conditional link- age analysis, a suggestive linkage peak (hlod ~ ) remained around –  cm. this genomic region corresponds to a locus of approximately  mb that contains multiple genes including cfh and cfhr - . these genes are located within the regulation of complement activation (rca) gene clus- ter on chromosome and encode members of the factor h/ cfhr family of proteins (male et al. ). polymorphisms human genetics ( ) : – and deletions in cfh and cfhrs have been previously asso- ciated with amd (edwards et al. ; fritsche et al. ; hageman et al. ; haines et al. ; klein et al. ; lores-motta et al. ; martinez-barricarte et al. ), including rare variants in cfh (hoffman et al. ; ray- chaudhuri et al. ) and a protective deletion of cfhr and cfhr (fritsche et  al. ; hageman et  al. ; hughes et al. ). more detailed mapping of this region in our cohort will be necessary to determine which polymor- phisms in these gene(s) are contributing to our linkage signal. in this study, we performed association and linkage screens in amish families from ohio and indiana. we identified four novel rare variants associated with increased amd risk in the amish and novel susceptibility loci on chromosomes q . –q . (maximum recessive hlod = . ) and q (maximum dominant hlod = . ; maximum reces- sive hlod = . ). these findings suggest novel genetic factors for amd in the amish and demonstrate the benefits of analyzing the genetic architecture of a complex trait, like amd, in closely related individuals from an isolated popula- tion. while family-based studies do not require sample sizes as large as traditional gwas, the sample size for this study (n = ) limited its power to detect novel amd variants and loci. this could be remedied with the ascertainment of additional members of many amish communities. addition- ally, more genetic data could be obtained from whole exome sequencing or dense genotyping for a panel such as the multi- ethnic genotyping array (mega) of about million exonic markers for gwas and ancestry-specific variants. additional studies are needed to replicate their associations and validate their roles in the development or progression of amd in out- bred populations. analyses could also be performed for the subtypes or endophenotypes of amd to identify subtype-spe- cific trends or associations with other binary and quantitative traits that may be pertinent for amd etiology. such studies may help resolve the missing heritability of amd and provide novel insights to amd pathophysiology. acknowledgements we thank the amish families for allowing us into their communities and for participating in our study. we also acknowl- edge the contributions of the anabaptist genealogy database and swiss anabaptist genealogy association. the authors acknowledge the contri- butions of the vanderbilt technologies for advanced genomics (van- tage) dna resources core. this work was supported by the national institutes of health [ey ]. j.n.c.b. was supported in part by a phrma informatics postdoctoral fellowship. this work made use of the high performance computing resource in the core facility for advanced research computing at case western reserve university. compliance with ethical standards conflict of interest the authors have no conflicts of interest to declare. open access this article is distributed under the terms of the crea- tive commons attribution . international license (http://creat iveco mmons .org/licen ses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. references abecasis lab ( ) exome chip design wiki site. https ://genom e.sph. umich .edu/wiki/exome _chip_desig n. accessed may abecasis gr, cherny ss, cookson wo, cardon lr ( ) merlin– rapid analysis of dense genetic maps using sparse gene flow trees. nat genet : – . https ://doi.org/ . /ng agarwala r, biesecker lg, schaffer aa ( ) anabaptist genealogy database. am j med genet c semin med genet c: – . https ://doi.org/ . /ajmg.c. auer pl, lettre g ( ) rare variant association studies: considera- tions, challenges and opportunities. genome med : . https :// doi.org/ . /s - 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corpus id: a case of disseminated sepsis caused by an unusual microorganism in a patient with diabetes @article{ghermanciolac aco, title={a case of disseminated sepsis caused by an unusual microorganism in a patient with diabetes}, author={c. gherman-ciolac and a. patel and s. budha and joanna macve and h. buch}, journal={clinical diabetes : a publication of the american diabetes association}, year={ }, volume={ }, pages={ - } } 	c. gherman-ciolac, a. patel, + authors h. buch 	published 	medicine 	clinical diabetes : a publication of the american diabetes association patients with suboptimally controlled diabetes are susceptible to a higher frequency and severity of infections from common microorganisms ( ). they are also at a higher risk of acquiring sepsis at unusual sites and from uncommon microbes ( ). we report a patient with a background of longstanding type diabetes, multiple comorbidities, and an intra-cardiac device, who had life-threatening disseminated sepsis caused by an unusual bacterium. a -year-old caucasian man with a bmi of . kg/m … expand view on ada clinical.diabetesjournals.org save to library create alert cite launch research feed share this paper citations view all tables and topics from this paper 	 table 	diabetes mellitus 	sepsis 	septicemia 	infection 	diabetes mellitus, non-insulin-dependent 	patients one citation citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency mycobacterium chelonae cutaneous infection: an opportunistic disease in an immunosuppressed patient with myasthenia gravis 	joana granado, a. miranda, marco fernandes, l. santos, k. mansinho 	medicine 	idcases 	 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency disseminated mycobacterium chelonae infection resulting in endocarditis. 	k. galil, r. thurer, k. glatter, t. barlam 	medicine 	clinical infectious diseases : an official publication of the infectious diseases society of america 	 	 	pdf save alert research feed a fatal mycobacterium chelonae infection in an immunosuppressed patient with systemic lupus erythematosus and concomitant fahr’s syndrome 	m. jankovic, l. Žmak, + authors v. katalinic jankovic 	medicine 	journal of infection and chemotherapy : official journal of the japan society of chemotherapy 	 	 save alert research feed fatal disseminated mycobacterium chelonae infection in an immunocompromised host--a unique presentation. 	s. mankad, r. karthik, p. rupali, j. michael 	medicine 	the journal of the association of physicians of india 	 	 	pdf save alert research feed mycobacterium chelonae hand infection following ferret bite 	k. iyengar, j. nadkarni, r. gupta, n. beeching, i. ullah, w. loh 	medicine 	infection 	 	 	view excerpt, references background save alert research feed disseminated mycobacterium chelonae infection: complicating a case of hidradenitis suppurativa 	s. patnaik, i. mohanty, p. panda, s. sahu, muktikesh dash 	medicine 	indian dermatology online journal 	 	 	 	view excerpt, references background save alert research feed injection abscesses in a diabetic due to mycobacterium chelonei var abscessus. 	p. jackson, h. keen, c. j. noble, n. a. simmons 	medicine 	british medical journal 	 	 	pdf save alert research feed disseminated cutaneous infection with mycobacterium chelonae in a patient with steroid‐dependent rheumatoid arthritis 	s. r. lamb, g. i. stables, w. merchant 	medicine 	clinical and experimental dermatology 	 	 save alert research feed insulin injection abscesses caused by mycobacterium chelonae. 	k. finucane, phil ambrey, shalini narayan, c. archer, c. dayan 	medicine 	diabetes care 	 	 	pdf save alert research feed catheter sepsis due to mycobacterium chelonae 	r. wallace, b. a. brown 	medicine 	journal of clinical microbiology 	 	 	pdf save alert research feed catheter related line sepsis resulting from mycobacterium chelonae infection in an immunocompromised host. 	s. antony 	medicine 	infectious disorders drug targets 	 	 save alert research feed ... ... related papers 	abstract 	tables and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue microbiome in mechanisms of asthma tara f carr md, rhonda alkatib md and monica kraft md department of medicine, university of arizona, tucson az corresponding author: tara f carr md assistant professor of medicine and otolaryngology university of arizona n campbell ave tucson, az - tcarr@deptofmed.arizona.edu rhonda alkatib md fellow, allergy and immunology university of arizona n campbell ave tucson, az - ralkatib@deptofmed.arizona.edu monica kraft md professor of medicine university of arizona n campbell ave tucson, az - mkraft@deptofmed.arizona.edu disclosure statement: dr carr reports royalties from wolters-kluwer and has provided consulting services to sanofi-regeneron, astrazeneca, and boehringer ingelheim. dr alkatib has no disclosures to report. dr kraft reports royalties from elsevier, has received research funds paid to the. university of arizona by national institutes of health, american lung association, sanofi-regeneron and chiesi. she has provided consulting services to astra-zeneca, sanofi-regeneron and teva pharmaceuticals . mailto:ralkatib@deptofmed.arizona.edu words including key points, text, references, and figure legends (do not embed figures, provide legend for figure) key words ( – ) microbiome asthma lung gut short chain fatty acids type inflammation abstract/summary [this will be used for indexing services and does not appear with article] (< words) the lung and gut microbiome are factors in asthma risk or protection. relevant elements of the microbiome within both niches include the importance of the early life window for microbiome establishment, the diversity of bacteria, richness of bacteria, and effect of those bacteria on the local epithelium and immune system. mechanisms of protection include direct anti-inflammatory action or induction of non-type- inflammation by certain bacterial colonies. the gut microbiome further impacts asthma risk through the contribution of metabolic products. this chapter will review the mechanisms that connect the lung and gut microbiota to asthma development and severity. key points ( – ) • the human microbiome impacts the development of asthma through direct and indirect mechanisms. • there are independent relationships between the different microbiome niches, particularly lung and gut, and the development of asthma. • the mechanisms through which the microbiome relate to asthma involve immune development, immune tolerance, functional metabolites, and relationship to infections. • further work is necessary to determine how and when manipulation of the microbiome may be implemented toward the prevention or treatment of asthma. introduction the development and implementation of non-culture-based methods to identify bacteria, commonly s ribosomal rna sequencing, have identified an enormous diversity and quantity of bacterial species living within and on the human host. it is estimated that more than , different microbial species live in and on humans, representing trillion organisms. importantly, this so-called human microbiome is in a commensal, symbiotic relationship with the human host, which is thought to be necessary for many host functions, including immune regulation. the microbiome may indeed impact the balance between heath and disease of many organ systems, including the respiratory, cardiovascular, gastrointestinal, and immune systems - . specifically, in asthma, the human microbiome may contribute to both the development and severity of asthma. this chapter will describe the current knowledge of the relationship of human microbiome niches to asthma development and severity, as well as the mechanisms thought to contribute to this relationship. early life exposures the often-discussed ‘hygiene hypothesis’ harbors the idea that exposure to various bacteria and other microbiota in early infancy and childhood can diminish the risk of subsequent allergy and asthma . this relationship has been studied in robust population cohorts, some of which leverage geopolitics and population bottlenecking to describe differences in exposures, microbiome development, and health outcomes. von mutius and colleagues recognized that growing up in a traditional european farm environment seemed to protect children from developing asthma and other atopic disorders, when compared to children growing up in urbanized areas. this group has subsequently described a variety of environmental exposures to be protective against asthma and allergic sensitization, including exposure to pet dogs and farm animals , and measurable differences of endotoxin load in bedding suggested increased diversity of environmental microorganisms in those exposed populations . further, ingestion of raw cow’s milk is inversely correlated with asthma in this population . murine models of allergic asthma have shown that ingestion of raw cow’s milk can prevent house dust mite-induced airways hyperresponsiveness (ahr) and lung eosinophilia by suppressing airway production of chemokine ligand (ccl) and type- inflammatory cytokines interleukin(il)- and il- , suggesting altered epithelial or innate lymphoid cell responses to the raw milk . individuals living across the russia-finland border in the karelia region, while of similar ancestry, have distinctly different rates of disease and different lifestyles, the finnish side having higher socioeconomic status and substantial urbanization. those living on the finnish side have substantially higher rates of asthma, allergen sensitization, and eczema compared with those living on the russian side of the border, and the skin and nasal microbial communities differ between populations . one possible explanation or contributing factor to these epidemiological findings is that those living in russian karelia have significantly higher microbial burden in their drinking water compared with those in finnish karelia, an exposure which seems to be protective against atopy . researchers have determined that health on both sides of that international border relates more to the diversity of overall bacterial environmental exposures than any one exposure. the underlying mechanism may be attributable to immune skewing by less diverse dust to eosinophilic responses, compared with type helper-t cell (th )-transformation of naïve t lymphocytes or induction of il- responses by more diverse bacterial exposure . the amish and hutterite communities of the united states are rural farming populations with closely related european ancestry, who share similarities in diet and family size. however, while the hutterites have embraced industrialized farming, the amish use traditional farming practices that exclude mechanization. the prevalence of asthma and allergy in the amish population is strikingly lower than that of the hutterites . stein et al. made extracts from indoor dust samples collected from representative homes of each of these populations and described marked differences in allergen, endotoxin, and bacterial abundance across the sites with much higher burden in the amish samples. using a mouse model of allergic asthma, the authors also showed that the amish dust extracts were protective against allergic airway inflammation in a manner dependent upon innate immune signaling . these data suggest that one or many components of the dust, particularly the bacterial colonies, function through innate immune mechanisms to protect against the development of allergic inflammation and asthma. these and other population studies strongly support that certain environmental exposures, particularly to bacterial organisms, can be protective against the development of asthma and atopic diseases. as the environment can particularly impact the composition of the lung and gut microbiota, the associations between each of these bacterial niches and development of asthma warrant close evaluation for causal relationships, mechanisms, and therapeutic opportunities. lung microbiome and asthma the role of the lung microbiome in the pathogenesis of asthma is not yet fully understood. however, well described are variables during the first years of a child’s life, including home environment, viral infections, and antibiotic exposures, which have been correlated with the onset of atopy and asthma . as these variables also theoretically relate to differences in respiratory exposures and to the lung microbiome, the need for more careful examination of the relationship between the lung microbiome and asthma has sparked enthusiasm on an international level. the impact of airway bacteria on development of asthma may indeed relate to early life bacterial colony establishment. bisgaard et al. cultured hypopharyngeal aspirates from -month old infants, finding that colonization with the encapsulated bacteria s. pneumoniae, m. catarrhalis, and/or h. influenzae was significantly associated with persistent wheeze, hospitalization for wheeze, and asthma diagnosis at age . in addition, colonization with any of these bacteria related to increased blood eosinophil counts and elevated total immunoglobulin (ig)e at age . a recent study of children with asthma showed that coincident infection of moraxella catarrhalis or streptococcus pneumoniae and rhinovirus conferred greater severity of respiratory tract illness, including asthma exacerbations, suggesting that these, and possibly other, respiratory bacteria contribute to airway inflammation . further, infection or colonization of the airway with atypical bacteria such as mycoplasma pneumoniae are related to both asthma development and severity in epidemiologic and mechanistic studies, and this type of exposure can be detected with readily available culture-based or serologic techniques . for example, m. pneumoniae infection in childhood causes acute wheezing and is associated with both structural changes of the lung and long term lung function deficits , as well as to the development of asthma . for individuals who already have allergic sensitization, m. pneumoniae may contribute to the development of asthma through inducing ahr , inflammation , mucin expression , , and remodeling through collagen deposition . for individuals with asthma, m. pneumoniae may induce pronounced eosinophilic inflammation which may relate to severity of disease or host innate immune response characteristics . treatment of the infection or related inflammation may indeed provide asthma control for the affected patient and underscore the importance of bacterial exposures to asthma. respiratory pathogens, such as those noted above, are relatively easy to identify using culture- based or serologic laboratory techniques that are widely commercially available. however, with the recognition of the myriad bacterial species that are not easily cultured, more recent work has focused on utilizing non-culture-based techniques to examine and characterize the scope of microbiota in human niches, and to relate differences in these populations to clinical outcomes. researchers and clinicians assumed for many years that the lungs were a sterile environment, however we now recognize a plethora of common bacterial phyla as colonizers of the entire respiratory tract, including in the lungs. such phyla include actinobacteria, bacteroidetes, firmicutes, and proteobacteria, with estimates of almost , bacterial genomes present per square centimeter of a single upper lobe of the lung . when utilizing non-culture-based techniques to compare bacteria present in the lungs of healthy adults to asthmatics, asthmatics have lower proportions of bacteroidetes and increased proteobacteria, particularly haemophilus . marri et al. reported alterations in the microbial composition of induced sputum of asthmatics that were similar among asthmatics suffering from mild or severe disease but distinguished asthmatics from normal controls, and which also noted increased proteobacteria in samples from asthmatic patients . huang et al. performed s sequencing for bacterial rrna on bronchial epithelial brushing from asthmatic and healthy subjects . in this study, samples from asthmatic patients had significantly higher bacterial burden and bacterial diversity. further, ahr in the patients with asthma was related to the relative abundance of members of the comamonadaceae, sphingomonadaceae, oxalobacteraceae phylotypes, and interestingly, increased bacterial diversity was related to improvement in ahr following clarithromycin therapy. lung microbiota may contribute not only to presence of asthma, but to asthma phenotypes and severity. durack et al. compared microbiota from bronchial brushings among healthy, asthmatic and atopic patients, showing that subjects with type- inflammation had lower bacterial burden than other asthmatics . a recent study by taylor et al. showed that asthma with prominent airway neutrophilia was characterized by lower microbial richness with enrichment of airway colonies for members of the class gammaproteobacteria, to which haemophilus and moraxella belong, and that these microbiota characteristics relate to asthma severity . green et al. similarly identified bacterial profiles of induced sputum from severe asthmatics, dominated by moraxella, haemophilus, or streptococcal spp., in the airway to be associated with severe airway obstruction and airway neutrophilia. to relate the bronchial microbiome to phenotypic and epigenetic features of asthma, huang et al. performed bronchial bacterial composition by s sequencing in severe asthmatic and healthy control patients. these investigators found that proteobacteria enrichment related to worsening asthma control, fewer airway eosinophils, and the induction of th -related genes . in contrast, actinobacteria were prominent in patients with high epithelial gene expression for fk binding protein, a marker of steroid responsiveness. these findings suggest that the mechanisms relating the bacterial species enrichment and clinical outcomes relate directly to proinflammatory pathways and treatment responsiveness. goleva et al. compared s sequencing from bronchoalveolar lavage in corticosteroid- resistant and corticosteroid-responsive asthmatics, finding relative increased abundance of proteobacteria (including neisseria, haemophilus spp), fusobacteria, firmicutes, and actinobacteria in the steroid-resistant patients . the authors supported these findings with in vitro experiments, showing haemophilus parainfluenzae to activate toll-like receptor , subsequently activating transforming growth factor-β–associated kinase- (tak ), which induces transcription of proinflammatory factors like il- , while inhibiting glucocorticoid-receptor-mediated mitogen-activated kinase phosphatase production and conferring corticosteroid resistance. as inhaled steroids are a mainstay of asthma therapy, this pathway to steroid resistance may help to explain severe and steroid-unresponsive asthma. mechanistic evaluations of these airway microbiotic influences on asthma susceptibility are largely reliant upon murine models. published literature supports the importance of early life lung microbiome development on risk of asthma. herbst et al. utilized a mouse model of allergic asthma in germ-free and control mice to show that the germ-free mice had elevated airway inflammation, type cytokines, and basophils, and increased ahr upon ovalbumin challenge compared with control mice. however, when the neonatal germ-free mice were recolonized with the diverse microbiota from the control mice prior to allergen sensitization, they exhibited reduced levels of circulating ige and reduced airway hyperresponsiveness similar to the control mice . gollwitzer et al. exposed mice at both neonatal and adult stages to house dust mite antigen to compare the effect of age and microbiome development on reactivity to antigen. upon antigen exposure, the neonatal mice showed a substantial increase in airway eosinophilia, blood eosinophilia, type cytokine (il- , il- , and il- ) production, mucus production, and ahr when compared to mice exposed later in development. the airway microbiome of the neonatal mice was composed of firmicutes and gammaproteobacteria, compared with predominance of bacteroidetes in the adults. the authors concluded that temporal shifts in airway microbiota are associated with decreased responsiveness to aeroallergens, via the induction of t- regulatory cells in a programmed death ligand - mediated manner . some bacterial exposures may indeed be protective against a type- airway response. for example, in a murine model of ovalbumin-induced allergic asthma, pulmonary administration of e.coli to the lung induced a tlr -dependent response with induction of γδ-t cells, decreased dendritic cell (dc) activation in the lung, and diminished il- cytokine production, which collectively protected the mice from allergic airway inflammation . in the aforementioned study by durack et al., the investigators studied predicted bacterial functions among the differentially expressed bronchial bacteria. sputum from asthmatic subjects was enriched for members of the haemophilus, neisseria, fusobacterium, and porphyromonas species and the sphingomonodaceae family, and depleted in members of the mogibacteriaceae family and lactobacillales order, when compared with healthy controls. in silico predicted bacterial functions based on the s rrna sequencing showed enrichment for short-chain fatty acid(scfa) metabolism . scfas are important for epithelial barrier function and have been implicated as important for immune tolerance in the gut, as will be discussed below. clearly, patterns of lung microbial dysbiosis are evident among patients with asthma, and proteobacteria seem to consistently relate to asthma presence, cellular phenotype, and severity. further, mechanistic work suggests multiple levels of interaction of microbiota and the immune system can confer protection or risk for asthma development. indeed, the quantity of bacteria, quality of bacteria, time course of exposure and colony establishment, and immunologic function of those bacteria all play a role in the relationship between lung microbiome and asthma (figure ). with these findings, the mechanisms by which microbiota determine early-life immunity may have great implications when applied to the prevention of both asthma and allergic diseases. gut microbiome and asthma the gut microbiome is vitally important for health. evidence underscoring the fundamental need for a gut microbiome includes studies of gnotobiotic mice, in which the mice born in a germ-free environment fail to develop normal intestinal structure or normal immune maturation . these findings may simply confirm that an individual’s gut microbiome contributes substantially to the proper digestion of foods, releasing minerals, vitamins, and digestive products such as short chain fatty acids. however, fascinatingly, introduction of different microbial communities into gnotobiotic mice can confer a specific phenotype, such as obesity , abnormal behavior , and immune development . these and other data suggest that manipulation of the gut microbiome may be preventative or therapeutic for many chronic diseases and may indeed be leveraged to address allergic diseases and asthma. to do so, however, will require a better understanding of gut microbiome development and function as pertaining to asthma. the gut microbiome develops in early life, with a rapid increase in diversity that seems necessary for health . in fact, the microbiome of the gastrointestinal tract, measured by stool sampling, is present even in the first neonatal meconium stool . the stool microbiome develops with time, and evidence supports critical time periods of development of that microbiome for establishing health- protective bacterial communities. longitudinal cohorts describe a relationship between establishment of the early life gut microbiome and development of allergic diseases. for example, a cohort from canada revealed that reduced stool bacterial diversity measured from samples obtained at months of life was associated with risk of asthma through age . a study from sweden revealed that lower microbial diversity within stool samples at week and month of life was related to risk for asthma development through age . in both of these cohorts, the stool communities among those children at risk or not became more similar with time and by months of age there was no relationship between stool diversity and asthma risk. in contrast, a study of the copenhagen prospective studies on asthma in childhood cohort assessed diversity of the gut and found that relative abundance of species at year of age were related to asthma at age , only among children born to asthmatic mothers . a comparison of cellular responses from neonates born in papua new guinea showed that antigen presenting cells from these infants are less active than those from australia, suggesting that the increased bacterial exposures related to the traditional lifestyle in papua new guinea would confer protection against immune responses . cox et al. utilized a mouse model of low dose penicillin exposure in early life to show that this exposure transiently affects the microbiota, but has lasting impact on body composition and metabolism . these, and other studies support that the window for developing and impacting the gut microbiome may be limited to, or crucially important in, early life but may also relate to multifactorial asthma susceptibility and comorbidities , . there are many potential contributing factors to the development of stool microbiome (figure ). much of the neonatal microbiome conferred during birth differs by mode of delivery. that is, the microbiome of infants delivered vaginally represent that of the mother’s vagina and gut. in contrast, the microbiome of infants delivered by caesarian section resembles skin flora . caesarian section, particularly when performed prior to membrane rupture, may indeed be a risk factor for the development of atopy and allergic diseases however studies of this relationship show conflicting results. further, early life exposure to livestock or domesticated pets, particularly dogs, decreases the risk of asthma development through mechanisms that may involve the gut microbiome - . early life exposure to traditional, non-mechanized farming environments, such as in the amish or european cohorts, is protective against asthma in a way that may strongly relate to microbiome through inhaled and ingested exposures , . fujimura and colleagues used a murine model of allergic asthma to show that dog-associated house dust exposure via oral gavage protected against asthmatic changes. further, this protection specifically related to gut microbiome profile- in particular, enrichment for lactobacillus johnsonii- and supplementation of this species could confer protection against the asthma phenotype . dietary exposure may also contribute to gut microbiome development. sordillo and colleagues reported that among other factors, breast-feeding, when compared with bottle-feeding, substantially influenced the gut microbiome development within the first six years of life . breastfed infants had relatively lower abundance of stool clostridales family species in this study. these are findings of interest, as prior work has shown that clostridium difficile enrichment in early life may actually increase risk of wheeze and asthma in childhood . interestingly, atarashi et al. fed gnotobiotic mice a clostridium mixture, noting a subsequent expansion of regulatory t cells in the colon as well as lower specific ige production in a model of ova sensitization, suggesting that this mixture would protect against allergic diseases . however, karimi et al. showed a similar pattern of regulatory, non-atopic immune development in a model feeding mice lactobacillus reuteri . that multiple bacterial taxa are implicated in immune regulation suggests that the individual taxa of bacterium present in the gut may be less important than the presence of bacteria in general, or perhaps identifies a potential shared metabolic or immunologic function of these bacteria. further, the administration of macrolide antibiotics in childhood has been associated to both persistent changes in gut microbiome and risk of asthma development in childhood, suggesting the possibility that the changes induced in the gut microbiome- presumably reduction in diversity or of specific dominant colonies- by these antibiotics put individuals at increased risk for asthma . digestive products of the gut microbiome are very important in modulating health. the metabolic functions of the gut microbiota include production of short-chain(scfas) and medium-chain fatty acids (mcfas) by fermentation of dietary fiber and carbohydrates (implicated in colitis, arthritis, and asthma), formation of secondary bile acids (important for reduction in liver disease and metabolic syndrome), generation of metabolites from meat-derived choline and l-carnitine (implicated in cardiovascular disease), production of vitamins k, b , and folate, and production of indole derivatives (i.e. gamma-aminobutyric acid, the central nervous system neurotransmitter) . of these metabolites, scfas have been most closely implicated in asthma risk. scfas can signal through g protein-coupled receptors, and can inhibit histone deacetylases, controlling gene transcription. acetate, one scfa produced by fermentation of fructose by bifidobacteria, may support colonic epithelial integrity and function . additional scfas, butyrate and propionate, can induce regulatory t cell development which may play an important role in protection against allergic and inflammatory diseases , . acetate and proprionate can induce regulatory t cells in the colon, resulting in increased expression of il- and associated regulatory functions . consumption of dietary fiber may induce more scfa production and induction of tolerogenic dendritic cells in the mesenteric lymph nodes . evidence supporting scfa as protective against asthma includes a study of the gut microbiota of infants at high risk for asthma by arrieta et al. these authors reported a transient gut microbial dysbiosis in the first three months of life, related to lower fecal acetate levels in these high-risk for asthma infants . fujimura and colleagues have synthesized this complex relationship of gut microbiome development, metabolic products, and immunologic outcomes related to asthma . neonatal stool samples from a diverse, longitudinal birth cohort underwent s ribosomal rna sequencing and the patterns of stool microbiota were divided into three “neonatal gut microbiota composition states(ngm - )”. these ngm were compositionally different and conferred different risk of atopy by age and asthma by age . the highest risk group, ngm , had lower relative abundance of bacteria, particularly bifidobacterium, lactobacillaceae, and clostridiaceae, and higher abundance of certain fungi, suggesting a substantial dysbiosis in this group. the sterile fecal water from these ngm samples induced significantly more il- expression in vitro than the low risk group, and metabolites within that sterile water were enriched for , -dihome, a lipid metabolite which can inhibit regulatory t-cell development. in contrast, the sterile fecal water of the low risk groups was enriched for metabolites including the polyunsaturated fatty acids docosapentaenoate and dihomo-γ-linolenate which have anti- inflammatory properties. these findings support that differential production of immunologically active metabolites in the stool by different bacterial species, even very early in life, may be an important mechanism of protection from or risk for asthma (figure ). therapeutics and the microbiome probiotics are viable microorganisms that potentially confer health benefits on the host, however the safety and efficacy of probiotics as organisms that can prevent or treat disease remains uncertain. ongoing investigations on probiotic supplementation may quantify and qualify host health benefits and the long-term impact they may have on the host microbiome. if various microbial exposures are related to asthma through direct immune effects or metabolic products, could a probiotic serve a protective or therapeutic role in those diseases through influencing the host microbiota toward disrupting or redirecting immune outcomes? elazab and colleagues performed a meta-analysis of studies testing prenatal or early life probiotic administration to determine the effects on atopy and asthma in children . variable factors among these trials included timing of supplementation and delivery method. the meta-analysis concluded that while some probiotic supplementation may reduce ige levels and the risk of allergen sensitization, this did not affect the risk of wheezing or asthma. the decreased ige levels were more evident with longer duration of probiotic administration. the mechanisms underlying this potential benefit against atopy have been addressed in specific trials. one randomized, placebo-controlled clinical trial studying feedings of lactobacillus paracasei f between and months of age to placebo identified more pronounced t-cell ifn-y and il- mrna expression in the treatment group, suggesting th promoting effects of this probiotic . another trial of prenatal and postnatal supplementation demonstrated a combination of bifidobacterium bifidum, bifidobacterium lactis and lactococcus lactis was associated with a reduction in in vitro lymphocyte il- and il- production by months of age . interestingly, a comparison of prenatal and postnatal supplementation with bifidobacterium lactis or lactobacillus rhamnosus showed differential effects of those bacteria, with cord blood ifn-γ higher in the l rhamnosus group. the complex host responses to probiotics will likely therefore vary based on dose, strain, treatment timing and duration. additionally, probiotic supplementation may contribute to protection from atopic disease include through improved epithelial barrier function . finally, while successfully implemented in severe gastrointestinal disorders , the frontier of microbiome transplantation to restore health in asthma has not been well studied but may offer an interesting new approach to microbiome manipulation. conclusion risk for asthma is strongly related to both the lung and gut microbiome. important elements of microbiome development consistent with both niches include the early life window for microbiome establishment, the diversity of bacteria, richness of bacteria, and effect of those bacteria on the local epithelium and immune system. mechanisms through which the lung microbiome may protect against asthma include early establishment of rich, non-pathogenic bacterial colonies that are anti-inflammatory or less disposed to type- inflammation. factors influencing the degree of gut microbiome protection from asthma include those that support gut microbial early development and diversity and incorporate the contribution of metabolic products and dietary exposures. with this in mind, the potential application of these findings to asthma treatment and prevention will likely include measures to broaden diversity early in life, supporting this diversity through health-protective early life exposures, and possible 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reconstitution of the microbiota. hum vaccin immunother. : - . figure . exposures and modifying factors contribute to the effect of microbiome on the risk of allergic asthma. figure . the lung and gut microbiome likely impact risk for asthma through direct immune activation and metabolite effects on the immune system. 	microbiome in mechanisms of asthma 	rhonda alkatib md 	monica kraft md 	key words ( – ) 	microbiome 	key points ( – ) 	introduction 	early life exposures 	lung microbiome and asthma 	gut microbiome and asthma 	therapeutics and the microbiome 	conclusion 	references edinburgh research explorer meta-analysis of genome-wide association studies from the charge consortium identifies common variants associated with carotid intima media thickness and plaque citation for published version: bis, jc, kavousi, m, franceschini, n, isaacs, a, abecasis, gr, schminke, u, post, ws, smith, av, cupples, la, markus, hs, schmidt, r, huffman, je, lehtimäki, t, baumert, j, münzel, t, heckbert, sr, dehghan, a, north, k, oostra, b, bevan, s, stoegerer, e-m, hayward, c, raitakari, o, meisinger, c, schillert, a, sanna, s, völzke, h, cheng, y-c, thorsson, b, fox, cs, rice, k, rivadeneira, f, nambi, v, halperin, e, petrovic, ke, peltonen, l, wichmann, he, schnabel, rb, dörr, m, parsa, a, aspelund, t, demissie, s, kathiresan, s, reilly, mp, taylor, k, uitterlinden, a, couper, dj, sitzer, m, kähönen, m, wilson, jf & cardiogram consortium , 'meta-analysis of genome-wide association studies from the charge consortium identifies common variants associated with carotid intima media thickness and plaque', nature genetics, vol. , no. , pp. - . https://doi.org/ . /ng. digital object identifier (doi): . /ng. link: link to publication record in edinburgh research explorer document version: peer reviewed version published in: nature genetics publisher rights statement: published in final edited form as: nat genet. ; ( ): – . doi: . /ng. . general rights copyright for the 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charge consortium identifies common variants associated with carotid intima media thickness and plaque joshua c. bis, phd , , maryam kavousi, md, msc , , , nora franceschini, md, mph , , aaron isaacs, phd , , , gonçalo r abecasis, phd , , ulf schminke, md , , wendy post, md , , albert v. smith, phd , , l. adrienne cupples, phd , , , hugh s markus, md , reinhold schmidt, md , jennifer e. huffman, msc , terho lehtimäki, md, phd , , jens baumert, phd , thomas münzel, md , susan r. heckbert, md, phd , , abbas dehghan, md, phd , , kari north, phd , ben oostra, phd , , steve bevan, phd , eva-maria stoegerer, md , caroline hayward, phd , olli raitakari, md, phd , , christa meisinger, md, mph , arne schillert, phd , serena sanna, phd , henry völzke, md , yu-ching cheng, phd , bolli thorsson, md , caroline s. fox, md, ms , , , kenneth rice, phd , fernando rivadeneira, md, phd , , vijay nambi, md , , , eran halperin, phd , , katja e. petrovic, msc , leena peltonen, md, phd , , h. erich wichmann, md, phd , renate b. schnabel, md, msc , marcus dörr, md , afshin parsa, md, mph , thor aspelund, phd , , serkalem demissie, phd , sekar kathiresan, md , , , muredach p. reilly, mbbch, msce , the cardiogram consortium kent taylor, phd , andre uitterlinden, phd , , , david j. couper, phd , matthias sitzer, md , mika kähönen, md, phd , , thomas illig, phd , philipp s. wild, md , marco orru, md , jan lüdemann, phd , alan r. shuldiner, md , gudny eiriksdottir, msc , charles c. white, mph , jerome i. rotter, md , albert hofman, md, phd , , jochen seissler, md , tanja zeller, phd , gianluca usala, phd , florian ernst, phd , lenore j. launer, phd , ralph b. d'agostino sr, phd , daniel h. o'leary, md , christie ballantyne, md , joachim thiery, md, mba , , andreas ziegler, dr. rer. nat. habil. , edward g. lakatta, md , ravi kumar chilukoti, msc , tamara b. harris, md, phd , philip a. wolf, md , , bruce m. psaty, md, phd , , joseph f polak, md, mph , xia li, md, mph , wolfgang rathmann, md, msph , manuela uda, phd , eric boerwinkle, phd , norman klopp, phd , helena schmidt, md phd , james f wilson, dphil , jorma viikari, md, phd , , wolfgang koenig, md , stefan correspondence should be addressed to j.c.b. (joshbis@uw.edu) or c.j.o. (odonnellc@nhlbi.nih.gov). these authors contributed equally to this work. contributions: study concept and design: j.c.b., m.k., n.f., g.r.a., l.a.c., t.l., s.r.h., k.n., c.h., o.r., c.s.f., v.n., r.b.s., t.a., m.s., m.k., p.s.w., a.r.s., j.i.r., j.s., d.h.o., e.g.l., b.m.p., m.u., e.b., j.v., w.k., s.b., a.b.n., j.w., c.v.d., a.scu.v.g.., c.j.o. acquisition of the data: j.c.b., a.i., g.r.a., u.s., w.p., h.s.m., r.s., t.l., b.o., s.b., e.s., o.r., c.m., h.v., b.t., f.r., k.e.p., h.e.w., r.b.s., m.d., a.p., t.a., s.k., m.p.r., k.t., m.s., m.k., t.i., p.s.w., m.o., j.l., a.r.s., g.e., j.i.r., a.h., j.s., t.z., g.u., f.e., l.j.l., r.b.d., d.h.o., j.t., t.b.h., p.a.w., b.m.p., j.f.p., w.r., e.b., n.k., h.s., j.f.w., j.v., w.k., s.b., a.b.n., g.h., c.v.d., a.scu.g.h., b.d.m., v.g., c.j.o. statistical analysis and interpretation of the data: j.c.b., m.k., n.f., a.i., g.r.a., a.v.s., l.a.c., j.e.h., t.l., j.b., s.r.h., a.d., k.n., c.h., o.r., a.sch.s.s., y.c., k.r., v.n., e.h., k.e.p., t.a., s.d., s.k., p.s.w., c.c.w., r.b.d., a.z., r.k.c., h.s., c.j.o. drafting of the manuscript: j.c.b., m.k., n.f., a.i., k.n., o.r., m.k., j.f.p., j.v., c.j.o. critical revision of the manuscript: j.c.b., m.k., n.f., a.i., g.r.a., u.s., w.p., l.a.c., h.s.m., r.s., t.l., j.b., t.m., s.r.h., a.d., k.n., o.r., c.m., h.v., b.t., k.r., f.r., v.n., h.e.w., r.b.s., m.d., a.p., s.d., m.p.r., k.t., d.j.c., m.s., m.k., t.i., j.l., g.e., j.i.r., a.h., j.s., f.e., l.j.l., r.b.d., d.h.o., c.b., a.z., e.g.l., r.k.c., t.b.h., b.m.p., j.f.p., x.l., w.r., e.b., j.v., w.k., s.b., j.w., c.v.d., a.scu.g.h., b.d.m., v.g., c.j.o. obtained funding: g.r.a., u.s., h.s.m., r.s., t.l., o.r., h.v., l.p., m.d., s.k., m.s., m.k., p.s.w., a.r.s., j.i.r., a.h., j.s., a.z., p.a.w., b.m.p., j.f.p., w.r., m.u., e.b., h.s., j.f.w., j.v., w.k., a.b.n., g.h., c.v.d., g.h., b.d.m., v.g., c.j.o. disclosures: v.n. indicated that he has a non-financial research collaboration with general electric and medipattern inc. nih public access author manuscript nat genet. author manuscript; available in pmc april . published in final edited form as: nat genet. ; ( ): – . doi: . /ng. . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript blankenberg, prof dr med , anne b. newman, md, mph , , jacqueline witteman, phd , , , gerardo heiss, md, phd , , cornelia van duijn, phd , , , , angelo scuteri, md, phd , , georg homuth, phd , , braxton d. mitchell, phd , , vilmundur gudnason, md, phd , , , and christopher j. o’donnell, md, mph , , , cardiovascular health research unit and department of medicine, university of washington, seattle, wa, usa department of epidemiology, erasmus medical center, rotterdam, the netherlands netherlands genomics initiative (ngi)-sponsored netherlands consortium for healthy aging (ncha), rotterdam, the netherlands department of epidemiology, university of north carolina chapel hill, chapel hill, nc, usa genetic epidemiology unit, dept. of epidemiology, erasmus university medical center, rotterdam, the netherlands centre for medical systems biology, leiden, the netherlands center for statistical genetics, department of biostatistics, university of michigan school of public health, ann arbor, mi, usa department of neurology, ernst moritz arndt university greifswald, germany division of cardiology, department of medicine, johns hopkins university icelandic heart association, kopavogur, iceland department of biostatistics, boston university school of public health, boston, ma, usa national heart, lung, and blood institute's framingham heart study, framingham ma, usa clinical neuroscience, st george’s university of london, london, uk department of neurology, medical university graz, austria mrc human genetics unit, institute of genetics and molecular medicine, western general hospital, crewe road, edinburgh, eh xu, scotland department of clinical chemistry, university of tampere, finland tampere university hospital, tampere, finland institute of epidemiology ii, helmholtz zentrum münchen, german research center for environmental health, neuherberg, germany department of medicine , university medical center mainz, mainz, germany cardiovascular health research unit and department of epidemiology, university of washington, seattle, wa, usa group health research institute, group health, seattle, wa, usa carolina center for genome sciences, university of north carolina, chapel hill, nc, usa department of clinical genetics, erasmus university medical center, rotterdam, the netherlands research centre of applied and preventive cardiovascular medicine, university of turku, finland department of clinical physiology, turku university hospital, finland institut für medizinische biometrie und statistik, universitätzu lübeck, universitätsklinikum schleswig-holstein, campus lübeck, lübeck, germany istituto di neurogenetica e neurofarmacologia, consiglio nazionaledelle ricerche, cittadella universitaria di monserrato, monserrato, cagliari, italy institute for community medicine, ernst moritz arndt university greifswald, germany division of endocrinology, diabetes, and nutrition, university of maryland school of medicine, baltimore, md, usa national heart, lung, and blood institute, bethesda, md, usa division of endocrinology, metabolism, and diabetes, brigham and women's hospital and harvard medical school, boston, ma, usa department of biostatistics, university of washington, seattle, wa, usa department of internal medicine, erasmus medical center, rotterdam, the netherlands baylor college of medicine, houston, tx, usa center for cardiovascular prevention, the methodist debakey heart and vascular center, houston, tx, usa ben taub general hospital, houston, tx, usa the blavatnik school of computer science, tel-aviv university, israel the international computer science institute, berkeley, california, usa department of neurology, general hospital and medical university graz, austria wellcome trust sanger institute, wellcome trust genome campus, hinxton, cambridge, uk institute for molecular medicine finland, biomedicum, university of helsinki and national institute for health and welfare, helsinki, finland institute of epidemiology i, helmholtz zentrum münchen, german research center for environmental health, neuherberg, germany department of internal medicine b, ernst moritz arndt university greifswald, germany departments of medicine and epidemiology & public health, university of maryland school of medicine, baltimore, md, usa university of iceland, reykjavik, iceland cardiovascular research center and cardiology division, massachusetts general hospital, boston, ma, usa center for human genetic research, bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript massachusetts general hospital, boston, ma, usa program in medical and population genetics, broad institute of mit and harvard, cambridge, ma, usa the cardiovascular institute, university of pennsylvania, philadelphia, pennsylvania , usa medical genetics institute, cedars-sinai medical center, los angeles, ca, usa department of biostatistics, university of north carolina at chapel hill, chapel hill, nc, usa department of neurology, klinikum herford, germany department of clinical physiology, university of tampere, finland unit of molecular epidemiology, helmholtz zentrum münchen, german research center for environmental health, neuherberg, germany unita operativa semplice cardiologia, divisione di medicina, presidio ospedaliero santa barbara, iglesias, italy institute of clinical chemistry and laboratory medicine, ernst moritz arndt university greifswald, germany program in genetics and genomic medicine, and division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, md, usa ludwig-maximilians university of munich, medical clinic innenstadt, diabetes center, germany interfaculty institute for genetics and functional genomics, ernst moritz arndt university greifswald, germany intramural research program, laboratory of epidemiology, demography, and biometry, national institute on aging, nih, bethesda md, usa mathematcs and statistics department, boston university, boston, ma, usa st. elizabeth's medical center, tufts university school of medicine, boston, ma, usa institute of laboratory medicine, clinical chemistry and molecular diagnostics, university hospital leipzig, germany leipzig research center of civilization diseases, medical faculty, university of leipzig, germany institut für medizinische biometrie und statistik, universitätzu lübeck, universitätsklinikum schleswig-holstein, campus lübeck, lübeck, germany gerontology research center, national institute on aging, baltimore, md, usa neurology, boston university school of medicine, boston, ma, usa cardiovascular health research unit and departments of medicine, epidemiology, and health services, university of washington, seattle, wa, usa department of radiology, tufts university school of medicine, boston ma institute of biometrics and epidemiology, german diabetes center, leibniz center for diabetes research at heinrich heine university düsseldorf, düsseldorf, germany university of texas, school of public health, human genetics center, houston, tx, usa institute of molecular biology and biochemistry, medical university graz, austria centre for population health sciences, university of edinburgh, teviot place, edinburgh, scotland department of medicine, university of turku, finland turku university hospital, finland department of internal medicine ii - cardiology, university of ulm medical center, germany graduate school of public health, department of epidemiology, and school of medicine, division of geriatric medicine, university of pittsburgh, pittsburg, pa, usa cardiology division, department of medicine, massachusetts general hospital, harvard medical school, boston ma, usa keywords genome-wide association study; genetic epidemiology; genetics; subclinical atherosclerosis; carotid intima media thickness; cardiovascular disease; cohort study; meta-analysis; risk coronary heart disease (chd) and stroke rank among the leading causes of death in the industrialized world and a significant genetic component underlies both outcomes. these clinical events are often preceded by the development of subclinical atherosclerosis, typically a thickening of the artery wall due to deposition of cholesterol rich material in the arteries that supply blood to major organs. generalized atherosclerosis results from endothelial dysfunction, inflammation, abnormalities in lipoprotein metabolism , coagulation and fibrinolysis. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript measures of subclinical atherosclerosis, disease that occurs before symptoms are noted, are predictive of incident clinical events and can be detected non-invasively and with reasonable precision in population samples using high resolution ultrasound techniques. both cimt and plaque, reflecting a thickening of the carotid artery wall or the presence of large irregular arterial wall deposits, respectively, are established measures of subclinical atherosclerotic disease. while there may be variation in carotid ultrasound measurement techniques, multiple independent studies have established consistent association of carotid phenotypes with coronary events and stroke in prospective studies of young, middle-aged, and older adults , and recent consensus prevention guidelines cite cimt as a potentially useful measure for prediction. while there is a correlation between common cimt and carotid plaque, common cimt reflects carotid artery wall thickening that may result from multiple vascular etiologies including hypertension and atherosclerosis, whereas carotid plaque is an indicator of the discrete occurrence of carotid atherosclerosis. several recent studies provide evidence that carotid plaque is a better predictor of future cardiovascular disease risk than common cimt. – numerous family studies established consistent evidence for moderate heritabilities for common cimt, internal cimt and carotid plaque (supplementary table ). however, candidate gene studies have not found consistent associations between single nucleotide polymorphisms (snps) and cimt, and genome-wide linkage scans completed to date have revealed only suggestive regions for common cimt. , we performed a gwas of three measures of subclinical carotid atherosclerosis – common cimt, internal cimt, and plaque– in a sample of up to , participants from nine population-based studies that performed genome-wide genotyping with commercial snp arrays and imputed to the approximately . million autosomal snps in the phase ii hapmap ceu reference panel. in addition, we followed-up our discovery findings in a second stage that included , participants from independent studies. results the cross-sectional discovery genome-wide analysis of carotid artery phenotypes included , participants from nine community-based studies whose mean age ranged from to years. characteristics of the samples are presented in the supplementary note. in the studies in which all three carotid measures were available, the correlations between common cimt and plaque ranged from . to . , and between common cimt and internal cimt, from . to . (supplementary table ). the a priori threshold for genome-wide significance was × − , and a p-value > × − but < × − , corresponding to not more than one expected false positive finding over . million tests, was considered suggestive evidence for association in our analyses. figure a provides a plot of −log (p-values) for the associations of the approximately . million snps with common cimt by chromosome and position for the meta-analysis of the nine discovery studies. p-values from the meta-analysis of plaque (n= , participants) and internal cimt (n= , ) are presented according to their genomic positions in figure b and supplementary figure , respectively. overall, from the discovery meta-analysis of common cimt and plaque, we carried forward genome-wide significant snps and suggestive snps to the second stage. our second stage included , participants from seven community-based studies, six of which provided results for common cimt (total n= , ) and three of which provided results for plaque (n= , ). characteristics of the participants in these studies are shown in the supplementary note. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript table presents the genome-wide significant association results for the discovery, second stage, and combined meta-analyses for common cimt and plaque, respectively. we show the discovery gwas results for the kb region surrounding the signal snps for common cimt and plaque along with the recombination rates and the known genes in that region in figures and . figures and show the study-specific findings from the combined meta- analyses of common cimt and plaque, respectively. results for the suggestive loci in the meta-analyses of common cimt and plaque are shown in the supplementary table and supplementary figures – . common cimt for common cimt, independent loci achieved our genome-wide significance threshold (p< × − ) in the combined meta-analysis. the strongest association was for rs , found on q approximately kb from zhx , where the a allele (allele frequency [af]= . ), was associated with lower common cimt (β=− . , p= . × − ), i.e. a . % lower mean common cimt per copy of the a allele. the second association was for rs , located . kb from apoc on q , a region that also includes apoe, apoc , and apoc . the g allele (af= . ) was associated with lower common cimt (β=- . , p= . × − ). the third association was for rs , located within the pinx gene on q . . each copy of the g allele (af = . ) was associated with higher common cimt (β= . , p= . × − ). we also identified a suggestive locus, marked by rs near the slc a gene on p , where the a allele was associated with higher common cimt (β= . , p= . × − ). while our genome-wide significant and suggestive snps from combined meta-analyses for common cimt explained a small proportion of the trait variance (up to . %), we further constructed an additive genetic risk score ( – alleles) comprised of the number of common cimt risk alleles at the four loci. in the discovery samples, the additive risk score showed graded increasing association with common cimt across all studies with an average increase of . % in common cimt from the lowest ( – ) to the highest ( – ) risk category (supplementary figure ). plaque in analysis of carotid artery plaque, independent loci achieved the genome-wide significance threshold (p< × − ) in the combined meta-analysis. the most significant signal was observed for rs , situated . kb from the pik cg gene on q . per copy of the t allele (af= . ), we observed an % increased odds of presence of plaque (p= . × − ). the second signal was centered at rs , located . kb from ednra on q . each copy of the t allele (af= . ) was associated with a % increased odds of the presence of plaque (p= . × − ). furthermore, two snps showed suggestive evidence for association in our combined meta-analysis. the first suggestive locus was rs on p where each copy of the a allele was associated with decreased odds of the presence of plaque (p= . × − ). our second suggestive locus was rs , near ldlr on p . per copy of the t allele we observed a decreased odds of the presence of plaque (p= . × − ). for both cimt and plaque, secondary discovery genome-wide meta-analyses conditioned on the genome-wide significant and suggestive snps from the combined meta-analyses did not reveal any additional associations. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript internal cimt no snp achieved our significance threshold for follow up in the discovery analyses of internal cimt. results for internal cimt snps with p < . × − are shown in supplementary table . cross-phenotype comparisons supplementary table shows the results for the genome-wide significant and suggestive snps from our combined meta-analyses for common cimt and plaque across the three carotid phenotypes. the directions of association were generally consistent and three snps, rs (apoc ) from the common cimt analysis and rs (pik cg) and rsrs (ednra) from the plaque analysis, were associated with all three phenotypes (p < . / / = . ) in cross-phenotype comparisons. associations with coronary artery disease we investigated the genome-wide significant and suggestive snps from our combined meta-analyses for common cimt and plaque for their potential associations with coronary artery disease (cad) in the cardiogram consortium (table ). two snps from our plaque analysis had a p-value for association with cad less than . ( . / tests). the first, rs , near ldlr, where the g allele was associated with both higher plaque risk in our study and higher cad risk (p= . ); and rs , near ednra where the c allele was associated with lower risk of plaque and lower risk of cad (p= × − ). one snp from common cimt analysis, rs near apoc , showed a suggestive association with cad with the same allele (a) being associated with higher common cimt and higher cad risk (p= . ). another snp identified in the plaque analysis, rs near lrig , showed a suggestive association with cad, with the g allele associated with both lower odds of plaque and lower risk of cad (p= . ). conversely, none of snps reported to be associated with coronary artery disease in the cardiogram consortium had a significant association (i.e., a p-value less than . , a conservative bonferroni correction for tests across three phenotypes) in our discovery meta-analyses of common cimt, internal cimt, or plaque (supplementary table ). discussion in this meta-analysis of g was data from nine studies of common cimt and seven studies of plaque, we identified genome-wide significant associations between regions and common cimt and between regions and the presence of carotid plaque in over , participants of european ancestry. interestingly, ednra one of our genome-wide significant regions in the combined meta-analysis of plaque was related to multiple carotid phenotypes and was also associated with coronary artery diseases in the recent large meta- analysis by the cardiogram consortium. three snps emerged as genome-wide significant from our combined meta-analysis of common cimt. the strongest association, on chromosome (rs ), is an intergenic snp located kb from the zhx gene. members of this gene family are nuclear homodimeric transcriptional repressors that interact with the a subunit of nuclear factor-y (nf-ya) and contain two c h -type zinc fingers and five homeobox dna-binding domains. little information about these proteins exists regarding cardiovascular disease or population studies. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript a second association, on q (rs ), fell upstream of the apoc gene. while this region has been of interest for its role in neurological genetics because of the apoe gene, it is also been frequent candidate gene for cardiovascular disease traits. although some previous studies have found associations of variation at the apoe locus and common cimt, among of our discovery studies that had independently measured the apoe epsilon variants, the correlation between rs and the e allele was less than . . further, models that included both the apoe e and the apoc variant indicated that the apoe gene was not associated with common cimt in these studies (supplementary table ), while the apoc variant still showed a significant association with common cimt. while apoe variants have been implicated in cases of familial dyslipidemia and premature atherosclerosis and in recent genome-wide association studies with variation in multiple lipoprotein measures, our results suggest that apoc is the primary variant of interest for carotid traits. the third association (rs ) was located in an intron of the pin -interacting protein (pinx ) gene. the protein, a telomerase inhibitor that plays a role in chromosomal segregation in mitosis, has been investigated in relation to cancers, but was not considered a candidate gene for cardiovascular phenotypes. the region on chromosome marked by rs , which includes the slc a , slc a , and slc a genes showed suggestive evidence for association with common cimt in our combined meta-analysis. this region may merit further investigation as recent genome-wide association studies have implicated this region with uric acid levels. , although high uric acid levels have been associated with cardiovascular disease and all- cause mortality, the contribution to atherosclerotic vascular disease remains controversial. plaque associations for plaque, two regions were genome-wide significant in our combined meta-analysis. the first region was within kb of the pik cg gene, which encodes one of the pi /pi -kinase family of proteins. these proteins are important modulators of extracellular signals, including those elicited by e-cadherin-mediated cell-cell adhesion, which plays an important role of endothelin in maintenance of the structural and functional integrity of epithelia. the fact that this region was reported as a top hit in a recent gwas of both platelet volume and aggregation suggests pleiotropy and highlights the interconnectedness of multiple cardiometabolic traits. the second genome-wide significant region was near the ednra gene. because of the role of endothelin as a potent vasoconstrictor, the endothelin receptor, type a is a target for pharmacologic treatments to reduce blood pressure. in addition, variation in the gene was associated with blood pressure , atherosclerosis and cardiovascular disease endpoints in candidate gene studies. two more regions showed suggestive evidence for association in our combined meta- analysis for plaque. the first region, near the ldlr gene is a particularly interesting candidate for subclinical atherosclerosis because of its role in familial hypercholesterolemia and its appearance in recent genome-wide association studies for lipid traits – and myocardial infarction. , notably, the ldlr snp recently reported to be associated with mi (rs ) is located kb away and is in modest ld (r = . in hapmap ceu) with the signal snp (rs ) in our analysis that also showed an association with cad in the cardiogram consortium. the second was in the vicinity of lrig , which negatively regulates growth factor signaling and is involved in the regulation of epidermal stem cell quiescence. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript interestingly, we found three loci (apoc , pik cg, and ednra) that were associated with all three carotid phenotypes. among these, the ednra locus was also significantly associated with coronary artery disease in the recent large meta-analysis by the cardiogram consortium. these associations may provide important insights into the pathophysiological mechanisms relating the genes to atherosclerosis and subsequent coronary artery disease. in particular, the concordance of association with snps in ednra with both carotid plaque and chd suggests a common etiology for subclinical and clinically apparent disease that warrants further investigation. the strengths of the current study include the large sample size, the population-based designs, the collaboratively designed pre-specified analysis plan, and the high quality of both genotyping and phenotyping. further, our ability to relate our findings to the outcome of cad in a large independent meta analysis provides important additional context to our results. these associations are unlikely to be due to population stratification since the discovery sample was restricted to whites of european origin and was also investigated for global latent population substructure. the study also has limitations. a single cross-sectional imt assessment was used in all studies and ultrasound protocols varied across participating studies. for example, plaque definition included the presence of any plaque in most studies and stenosis greater than % in others. the heterogeneity of measurement techniques may have compromised our ability to detect small associations. despite this heterogeneity, the ability to detect consistent genetic associations for several carotid measures suggests that additional signals may be discovered in future studies utilizing a larger sample size or a higher resolution technique such as magnetic resonance imaging. further, few studies had internal cimt measures since these are more difficult to obtain than common cimt measurements and thus limited our ability to discover associations with this phenotype. although our sample size was reasonably large, we still had limited power to detect associations with small effect sizes. genome-wide association studies are known for revealing associations with common variants and may miss rare variants not covered by the commercial genotyping arrays. for instance, the sparse coverage of the apoc and ldlr gene regions resulted in varying imputation quality and a lower effective sample size for the analysis of these two regions. because we did not conduct follow-up fine mapping of the results, and because some snps were distant from known genes, it is likely that the identified snps are not causal variants, but, instead, may be in linkage disequilibrium with variants that were not analyzed. because some of our associations attained genome-wide significant p-values only in the combined meta-analysis, confirmation of our findings in other populations and further exploration of these genomic regions with dense genotyping, expression, and translational studies will be required to better understand the role of these genes in subclinical atherosclerotic disease. in summary, our meta-analysis of gwas data from nine community-based studies has revealed new loci for common cimt and plaque. these loci implicate ldl metabolism (apoc ), endothelial dysfunction (ednra), platelet biology (pik cg), and telomere maintenance (pinx ). two of our identified loci are also associated with coronary artery disease in the recent large meta-analysis by the cardiogram consortium. exploring the molecular, cellular and clinical consequences of genetic variation at these loci may yield novel insights into the pathophysiology of clinical and subclinical cardiovascular disease. supplementary material refer to web version on pubmed central for supplementary material. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript genome-wide association studies of carotid intima media thickness and plaque: meta-analysis from the charge consortium carotid intima media thickness (cimt) and plaque determined by ultrasonography are established measures of subclinical atherosclerosis that each predict future cardiovascular disease events. we conducted a meta-analysis of genome-wide association data in , participants of european ancestry from nine large studies in the setting of the cohorts for heart and aging research in genomic epidemiology (charge) consortium. we then sought additional evidence to support our findings among , individuals using data from additional studies. in the combined meta-analysis, we identified three genomic regions associated with common cimt and two different regions associated with the presence of carotid plaque (p < × − ). the associated snps mapped in, or near, genes related to cellular-signaling, lipid metabolism, and blood pressure homeostasis and two of the regions were associated with coronary artery disease (p < . ) in the cardiogram consortium. our findings may provide new insight into pathways leading to subclinical atherosclerosis and subsequent cardiovascular events. acknowledgments ages: the age, gene/environment susceptibility reykjavik study is funded by nih contract n -ag- , the nia intramural research program, hjartavernd (the icelandic heart association), and the althingi (the icelandic parliament). amish: the amish studies are supported by grants and contracts from the nih including r ag (amish longevity study), r hl (amish calcification study), u gm - (papi study), and u hl - (hapi study), the university of maryland general clinical research center, grant m rr , the baltimore veterans administration medical center geriatrics research and education clinical center and the paul beeson physician faculty scholars in aging program. we thank our amish research volunteers for their long- standing partnership in research, and the research staff at the amish research clinic for their hard work and dedication. aric: the atherosclerosis risk in communities study is carried out as a collaborative study supported by national heart, lung, and blood institute contracts n -hc- , n -hc- , n -hc- , n - hc- , n -hc- , n -hc- , n -hc- , r hl , r hl and r hl ; national human genome research institute contract u hg ; and national institutes of health contract hhsn c. the authors thank the staff and participants of the aric study for their important contributions. infrastructure was partly supported by grant number ul rr , a component of the national institutes of health and nih roadmap for medical research. erf: the erf study was supported by grants from the netherlands organisation for scientific research, erasmus mc and the centre for medical systems biology (cmsb). we are grateful to all study participants and their relatives, general practitioners and neurologists for their contributions and to p. veraart for her help in genealogy, j. vergeer for the supervision of the laboratory work and p. snijders for his help in data collection. chs: the chs research reported in this article was supported by nhlbi contracts n -hc- , n - hc- through n -hc- ; n -hc- , n hc- , n hc- , n -hc- , n - hc- and nhlbi grants hl , hl , hl , hl with additional contribution from ninds. additional support was provided through ag- , ag- , ag- , and ag- from the nia. see also http://www.chs-nhlbi.org/pi.htm. dna handling and genotyping was supported in part by national center for research resources grant m rr to the cedars-sinai general clinical research center genotyping core and national institute of diabetes and digestive and kidney diseases grant dk to the southern california diabetes endocrinology research center. fhs: from the framingham heart study of the national heart lung and blood institute of the national institutes of health and boston university school of medicine. this work was supported by the national heart, lung and blood institute's framingham heart study (contract no. n -hc- ) and its contract with affymetrix, inc for genotyping services (contract no. n -hl- - ), and by grants from the national institute of neurological disorders and stroke (ns ; paw) and the national institute of aging (ag , ag ; paw and ag ; ss). a portion of this research utilized the linux cluster for genetic analysis (linga-ii) funded by the robert dawson evans endowment of the department of medicine at boston university school of medicine and bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript http://www.chs-nhlbi.org/pi.htm boston medical center. analyses reflect intellectual input and resource development from the framingham heart study investigators participating in the snp health association resource (share) project. rotterdam study i and ii (rs i and rs ii): the rotterdam gwa study was funded by the netherlands organisation of scientific research nwo investments (nr. . . . , - - ), the research institute for diseases in the elderly ( - - ; ride ), the netherlands genomics initiative (ngi)/netherlands consortium for healthy aging (ncha) project nr. - - . this study is further supported by nwo grant (vici, - - ). the rotterdam study is funded by erasmus medical center and erasmus university, rotterdam, netherlands organization for the health research and development (zonmw), the research institute for diseases in the elderly (ride), the ministry of education, culture and science, the ministry for health, welfare and sports, the european commission (dg xii), and the municipality of rotterdam. the authors are very grateful to the participants and staff from the rotterdam study, the participating general practioners and the pharmacists. we thank pascal arp, mila jhamai, dr michael moorhouse, marijn verkerk, and sander bervoets for their help in creating the gwas database. we would like to thank dr. tobias a. knoch, luc v. de zeeuw, anis abuseiris, and rob de graaf as well as their institutions the erasmus computing grid, rotterdam, the netherlands, and especially the national german medigrid and services@medigrid part of the german d-grid, both funded by the german bundesministeriumfuer forschung und technology under grants # ak a-h and # ig g, for access to their grid resources. sardinia: this work was supported by the intramural research program of the national institute on aging, nih. the sardinia (‘‘progenia’’) team was supported by contract no -ag- – from the national institute on aging. the efforts of jbg and gra, were supported in part by contract -ma- from the national institute on aging to the university of michigan and by research grants hg and hl from the national institutes of health (to gra). we warmly thank monsignore piseddu, bishop of ogliastra; mayor enrico lai and his administration in lanusei for providing and furnishing the clinic site; the mayors of ilbono, arzana, and elini; the head of the local public health unit ar ; and the residents of the towns for their volunteerism and cooperation. we also thank harold spurgeon and paul pullen for invaluable help with equipment and readings, and michele evans and dan longo for helpful discussions. ship: ship is part of the community medicine research net of the university of greifswald, germany, which is funded by the federal ministry of education and research (grants no. zz , zz , and zz ), the ministry of cultural affairs as well as the social ministry of the federal state of mecklenburg-west pomerania. genome-wide data have been supported by the federal ministry of education and research (grant no. zik ) and a joint grant from siemens healthcare, erlangen, germany and the federal state of mecklenburg-west pomerania. the ship authors are grateful to the contribution of alexander teumer, anja hoffmann, and astrid petersmann in generating the snp data. the university of greifswald is a member of the ‘center of knowledge interchange’ program of the siemens ag. asps: the research reported in this article was funded by the austrian science fond (fwf) grant number p - p and p . the medical university of graz supports the databank of the asps. caps: the research leading these results has received funding from the european community's seventh framework programme (fp / - ) under grant agreement no. ; atheroremo. ghs: the gutenberg heart study is funded through the government of rheinland-pfalz (“stiftung rheinland pfalzfür innovation,” contract number az - / ), the research programs “wissenschafft zukunft” and “schwerpunkt vaskuläre prävention” of the johannes gutenberg-university of mainz and its contract with boehringeringelheim and philips medical systems including an unrestricted grant for the gutenberg heart study. specifically, the research reported in this article was supported by the national genome network “ngfnplus” (contract number project a gs ) by the federal ministry of education and research, germany. kora: the monica/kora augsburg studies were financed by the helmholtz zentrum münchen, german research center for environmental health, neuherberg, germany and supported by grants from the german federal ministry of education and research (bmbf). part of this work was financed by the german national genome research network (ngfnplus, project number gs ) and through additional funds from the university of ulm. furthermore, the research was supported within the munich center of health sciences (mc health) as part of lmu innovative. imt measurement of the kora cohort was funded by a grant of the karl- wilder foundation. finally, part of this work was financed by the german diabetes center, which is funded by the german federal ministry of health and the ministry of innovation, science, research and technology of the state of north rhine westphalia. orcades: orcades was supported by the chief scientist office of the scottish government, the royal society and the european union framework program eurospan project (contract no. lshg-ct- - ). dna extractions were performed at the wellcome trust clinical research facility in edinburgh. we would like to bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript acknowledge the invaluable contributions of lorraine anderson and the research nurses in orkney, the administrative team in edinburgh and the people of orkney. yfs: the cardiovascular risk in young finns study (yfs) is supported by the academy of finland (grant no. , and ), the social insurance institution of finland, university hospital medical funds to tampere, and turku university hospitals, the finnish foundation of cardiovascular research. cardiogram: we acknowledge the contributions of all of the authors of the cardiogram report, as listed in their primary analysis publication. references . lloyd-jones d, et al. executive summary: heart disease and stroke statistics-- update: a report from the american heart association. circulation. : – . 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[pubmed: ] bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript figure . a–b: genome-wide manhattan plots for common cimt and plaque plots show the individual p-values (based on discovery meta-analysis) against their genomic position for common carotid imt (figure a), the presence of plaque (figure b). within each chromosome, shown on the x-axis, the results are plotted left to right from the p- terminal end. the dashed line indicates the threshold for follow-up, p< × − and the solid line indicates the threshold for genome-wide significance, p< × − . the nearest genes are indicated above points that surpassed our genome-wide significance threshold; genes that are greater than kb from the signal snp are indicated in parentheses. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript figure . regional plots for common carotid imt snps plots are centered on the most significant snp at locus along with the meta-analysis results for snps in the kb region surrounding it. all snps are plotted with their discovery meta- analysis p-values against their genomic position, with the most significant snp in the region indicated as a diamond and other snps shaded according to their pairwise correlation (r ) with the signal snp. the light blue line represents the estimated recombination rates. gene annotations are shown as dark green lines. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript figure . regional plots for plaque snps plots are centered on the most significant snp at each locus along with the meta-analysis results for snps in the kb region surrounding it. all snps are plotted with their discovery meta-analysis p-values against their genomic position, with the most significant snp in the region indicated as a diamond and other snps shaded according to their pairwise correlation (r ) with the signal snp. the light blue line represents the estimated recombination rates. gene annotations are shown as dark green lines. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript figure . forest plots for common carotid imt snp associations plots show the study-specific association estimates (β) and % confidence intervals for the nine discovery and second stage studies, presented as bars. the scale is ln(cimt). the association estimate and confidence interval for the meta-analysis combining discovery and second stage results is presented as a diamond. blank spaces indicate occasions in which a particular study was not able to provide results for a given snp. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript figure . forest plots for plaque snp associations plots show the study-specific association estimates (or) and % confidence intervals for the nine discovery and second stage studies, presented as bars. the association estimate and confidence interval for the meta-analysis combining discovery and second stage results is presented as a diamond. blank spaces indicate occasions in which a particular study was not able to provide results for a given snp. bis et al. page nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript bis et al. page ta bl e a : d is co ve ry , s ec on d st ag e, a nd c om bi ne d m et a- an al ys is fo r c om m on c im t a nd p la qu e d is co ve ry g w a s (c im t ) se co nd s ta ge m et a- an al ys is (c im t ) c om bi ne d m et a- an al ys is (c im t ) sn p c hr n ea re st ge ne a lle le s a f β se n p- va lu e a f β se n p- va lu e β se p- va lu e rs z h x a /g . − . . , . × − . − . . , . − . . . × − rs a po c a /g . − . . , . × − . − . . , . − . . . × − rs pi n x g /a . . . , . × − . . . , . . . . × − d is co ve ry g w a s (p la qu e) se co nd s ta ge m et a- an al ys is (p la qu e) c om bi ne d m et a- an al ys is (p la qu e) sn p c hr n ea re st ge ne a lle le s a f o r ( % c i) n p- va lu e a f o r ( % c i) n p- va lu e o r ( % c i) p- va lu e rs pi k c g a /g . . ( . – . ) , . × − . . ( . – . ) , . . ( . – . ) . × − rs e d n r a t /c . . ( . – . ) , . × − . . ( . – . ) , . . ( . – . ) . × − a lle le s in di ca te s th e co de d (n am ed fi rs t) & n on -c od ed a lle le ; a f in di ca te s al le le fr eq ue nc y fo r t he c od ed a lle le , a n av er ag e w ei gh te d by s tu dy s iz e; o r in di ca te s od ds ra tio , c i, co nf id en ce in te rv al ; n in di ca te s ef fe ct iv e sa m pl e si ze , c al cu la te d by ta ki ng th e su m o f e ac h st ud y’ s sa m pl e si ze m ul tip lie d by th e sn p’ s im pu ta tio n qu al ity . w he n m or e th an o ne s n p at a lo cu s su rp as se d ou r p -v al ue th re sh ol d, w e pr es en te d th e sn p w ith th e lo w es t p -v al ue . nat genet. author manuscript; available in pmc april . n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript n ih -p a a uthor m anuscript bis et al. page ta bl e a ss oc ia tio n of g en om e- w id e si gn if ic an t a nd s ug ge st iv e co m m on c im t a nd p la qu e sn ps w ith c a d in th e c a r d io g r a m c on so rt iu m so ur ce sn p c hr n ea re st g en e a lle le a f o r n p- va lu e c om m . ci m t rs z h x g . . ( . – . ) , . rs a po c g . . ( . – . ) , . rs pi n x g . . ( . – . ) , . rs sl c a g . . ( . – . ) , . pl aq ue rs pi k c g g . . ( . – . ) , . rs e d n r a c . . ( . – . ) , × - rs l d l r g . . ( . – . ) , . rs l r ig g . . ( . – . ) , . a lle le in di ca te s th e co de d al le le in th e c a r d io g r a m c on so rt iu m m et a- an al ys is ; a f in di ca te s al le le fr eq ue nc y fo r t he c od ed a lle le ; o r in di ca te s od ds ra tio , c i, co nf id en ce in te rv al ; n in di ca te s sa m pl e si ze . nat genet. author manuscript; available in pmc april . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ am. j. hum. genet. : – , report identification of c orf (ttdn ) gene mutations and genetic heterogeneity in nonphotosensitive trichothiodystrophy kazuhiko nakabayashi, daniela amann, yan ren, ulpu saarialho-kere, , nili avidan, simone gentles, jeffrey r. macdonald, erik g. puffenberger, angela m. christiano, amalia martinez-mir, julio c. salas-alanis, renata rizzo, esther vamos, anja raams, clifford les, eric seboun, nicolaas g. j. jaspers, jacques s. beckmann, , charles e. jackson, and stephen w. scherer , program in genetics and genomic biology, the hospital for sick children, and department of molecular and medical genetics, university of toronto, toronto; department of molecular genetics, weizmann institute of science, rehovot, israel; department of dermatology, university of helsinki, helsinki; karolinska institutet at stockholm soder hospital, stockholm; the clinic for special children, strasburg, pa; department of dermatology, columbia university, new york; department of pediatrics, university of catania, catania, italy; department of medical genetics, hôpital universitaire erasme, brussels; department of genetics, medical genetic cluster, erasmus university, rotterdam, the netherlands; henry ford hospital, detroit; division of genetics and microbiology, university pierre et marie curie, paris; service of medical genetics, centre hospitalier universitaire vaudois et l’université de lausanne (chuv-unil), lausanne, switzerland; and department of medicine, scott & white memorial hospital, temple, tx we have identified c orf , which localizes to the nucleus and is expressed in fetal hair follicles, as the first disease gene for nonphotosensitive trichothiodystrophy (ttd). c orf maps to chromosome p , and the disease locus has been designated “ttdn ” (ttd nonphotosensitive ). mutations were found in patients with amish brittle- hair syndrome and in other nonphotosensititive ttd cases with mental retardation and decreased fertility but not in patients with sabinas syndrome or pollitt syndrome. therefore, genetic heterogeneity in nonphotosensitive ttd is a feature similar to that observed in photosensitive ttd, which is caused by mutations in transcription factor ii h (tfiih) subunit genes. comparative immunofluorescence analysis, however, suggests that c orf does not influence tfiih directly. given the absence of cutaneous photosensitivity in the patients with c orf mutations, together with the protein’s nuclear localization, c orf may be involved in transcription but not dna repair. trichothiodystrophy (ttd), or sulfur-deficient brittle hair (price et al. ), can be associated with a spectrum of symptoms affecting organs of ectodermal and neu- roectodermal origin (table ). these include nail dystro- phy, mental and growth retardation, ichthyosis, de- creased fertility, and cutaneous photosensitivity, but not cancer (bergmann and egly ). approximately half of patients with tdd display photosensitivity. these cases are associated with defects in nucleotide excision repair (ner) due to mutations in xpd (botta et al. ), xpb (weeda et al. ), or ttd-a (giglia-mari et al. ) that cause a reduction of cellular concen- received november , ; accepted for publication december , ; electronically published january , . address for correspondence and reprints: dr. stephen w. scherer, university avenue, toronto, on m g x , canada. e-mail: steve@genet.sickkids.on.ca � by the american society of human genetics. all rights reserved. - / / - $ . tration of transcription factor ii h (tfiih) (botta et al. ). however, the causal gene or genes for nonpho- tosensitive ttd are unknown. amish brittle-hair brain syndrome (abhs [mim ]) is an autosomal recessive disorder charac- terized by short stature, intellectual impairment, sulfur- deficient brittle hair, and decreased male fertility but not cutaneous photosensitivity (jackson et al. ; baden et al. ). other forms of nonphotosensitive ttd, such as sabinas brittle-hair syndrome (mim ) (howell et al. ) and pollitt syndrome (mim ) (pollitt et al. ), were hypothesized to be allelic with abhs, because of similar clinical pres- entations. initially, to search for the abhs disease locus, we performed homozygosity mapping on a subset of af- fected members from a consanguineous amish kindred (jackson et al. ) and identified a -mb candidate locus on p (fig. a) (seboun et al., in press). we then analyzed seven genes (fig. a) (scherer et al. ; the figure identification of c orf mutations. to screen the two exons and the ′ upstream region of the c orf gene, we used three sets of primer pairs: c orf - upf/ex r , c orf ex -f /r , and c orf ex -f/r (for primer sequences, see table a [online only]). the cycling conditions were initial denaturation at �c for min followed by cycles of denaturation at �c for s, annealing at �c for s, and extension at �c for s. pcr products were purified using microclean (microsone) and were used as the sequencing template. sequencing reactions were performed using the big dye terminator kit (applied biosystems), and an abi- dna sequencer (applied biosystems) was used to obtain sequences. a, physical map of the -mb region on p showing homozygosity in a consanguineous amish kindred. b, diagram of c orf , consisting of two exons spanning kb. the coding and untranslated regions are shown as blackened and unblackened boxes, respectively. the red and blue asterisks (*) indicate the position of the m v mutation in the amish kindred and the - bp deletion in the moroccan siblings with ttd, respectively. black and red bars (numbered from to ) represent the size and location of the pcr products in the deletion mapping for patient . the fragments that were not amplified from patient (because of homozygous deletion) are indicated by red bars. c, five affected families from the amish kindred. gray and blackened symbols indicated affected members, and unblackened symbols indicate unaffected members. the member of family b represented by the blackened symbol is the proband, who had a more severe phenotype, presumably due to a de novo chromosomal abnormality ( ,xy, q�) (jackson et al. ). the genotype at the m v mutation site (a, wild-type; g, mutated) is shown for each member. d, electropherograms for the m v site. e, representative results (from test primers , , , and ) of the pcr-based deletion mapping of the c orf locus in patient (lane c, control; lane p, patient ). unblackened triangles indicate the -bp fragment amplified by control primer djg /g . the blackened triangles indicate the fragment amplified by a test primer pair. the cycling conditions were initial denaturation at �c for min followed by cycles of denaturation at �c for s, annealing at �c for s, and extension at �c for s. detailed information for the test primers pairs and the control primer pair is available in table a (online only). reports figure a, the human c orf protein. the glycine/proline–rich region is shown in green (the low-complexity regions detected by the blastp program are in light green). there are two highly conserved c terminal regions (cr and cr ) among the candidate orthologues. the evolutionary tree (middle) was drawn by the clustalx program (thompson et al. ). the protein sequences used for phylogenetic analysis are human c orf (ncbi accession number np_ ) and orthologues from chimpanzee (ensembl accession number ensptrp ), mouse (ncbi accession number bab ), rat (ensembl accession number ensrnop ), chicken (ensembl accession number ensgalp ), frog (xenopus tropicalis [ncbi accession number np_ ]), pufferfish (tetraodon nigroviridis [ncbi accession number caf ]), fruit fly (drosophila melanogaster [ncbi accession number np_ ]), and mosquito (anopheles gambiae [ncbi accession number xp_ ]). deduced protein sequences were derived from cdna sequences from zebrafish (danio rerio [genbank accession number bc ]) and pig (genbank accession number bp ). the dog protein sequence was predicted from the genomic dna sequence (see ucsc genome browser web site). for each species, the overall amino acid similarity with the human protein sequence, the percentage ratio of glycine/proline content in the region from the n terminus to cr , and the presence (�) or absence (�) of cr and cr are shown. multiple sequence alignments for cr and cr are at bottom. the position of m v in cr is indicated by a red asterisk (*). identical and similar amino acids are highlighted in dark and light blue, respectively. b, subcellular localization assay. transiently expressed myc-tagged c orf in human cultured cells (transformed human embryonic kidney cells [hek ] and sv -transformed wi human embryonic fibroblasts [va ]) was examined by immunostaining, by use of anti-myc antibody and a secondary antibody conjugated with fluorescein isothiocyanate (fitc). bright-field (bf), ’- -diamidino- -phenylindole (dapi), fitc, and the merged image of dapi and fitc signals are shown for each cell line. the myc-tagged cdna construct for c orf was generated by inserting the coding region of c orf (nucleotides – of mrna) in pcdna . � myc-his a (invitrogen). dna transfection was performed using lipofectamine plus (invitrogen). two days after transfection, cells were washed in pbs and were fixed in % acetone plus % methanol at � �c for min. the samples were blocked with % bsa in pbs (blocking buffer) for h and were incubated in blocking buffer containing anti-myc antibody (santa cruz biotechnology) for min. cells were washed three times with pbs, were incubated with fitc-conjugated anti-mouse igg antibody for min, and were washed three times with pbs. after dapi staining, the samples were analyzed by deconvolution microscopy (zeiss). c, in situ rna hybridization for c orf on human fetal skin tissue. a -bp cdna fragment (corresponding to nucleotides – of mrna) was subcloned in pcr-script (stratagene) and was used as template dna to synthesize [ s]utp-labeled riboprobes. [ s]utp-labeled riboprobes (sense and antisense) were hybridized to the sections. radioactive signals were detected by autoradiography with d of exposure. slides were counterstained with hematoxylin-eosin and were photographed under dark-field (df) and bright-field (bf) illumination. the signal detected in epidermis and hair follicles is specific to the antisense probe. the detail conditions used for hybridization, washing, and signal detection were described elsewhere (saarialho-kere et al. ). d, normal level of tfiih in fibroblast cells from one of the moroccan siblings with the - bp deletion. mixed dapi/phase contrast image (top) showing a normal (with large beads) and ttd (with small beads) cells. immunofluorescence with anti-xpb antibody (bottom). for exact quantitative comparison of fluorescence signals between normal and ttd fibroblasts without slide- to-slide variability, two types of fibroblasts were preloaded with cytoplasmic plastic beads of different sizes (large beads for normal and small beads for ttd) and then were mixed, cultured overnight, and processed for immunofluorescence. chromosome annotation project [see web site]) by dna sequencing and identified three homozygous se- quence variations in the affected members of family e (fig. c): a trc variant in intron of c orf , a gra variant in exon of cdc l causing an arginine-to- lysine substitution (r k), and an arg variant in exon of c orf causing a methionine-to-valine substitution (m v). the first two sequence variations were ex- cluded from further analysis, because the same genotype was found in either normal controls or the unaffected parents in family e. we have shown elsewhere that c orf encodes a -aa protein of unknown function (nakabayashi et al. ) and that it is variably ex- pressed in many tissues, including fibroblasts and brain. by sequencing all available members of the amish kin- dred, we confirmed that all affected cases were ho- mozygous for the arg variant and that unaffected members were either heterozygous carriers ( / ) or homozygous for the normal allele ( / ) (fig. c and d). we genotyped controls ( chromosomes), including unrelated amish individuals, and confirmed cosegregation of the m v variant in only carrier or disease chromosomes. we then examined c orf in additional cases of nonphotosensitive ttd and found two deleterious homozygous deletions. in siblings of moroccan origin with ttd (przedborsk et al. ), we found a -bp homozygous deletion in exon (nucleotides and of c orf mrna [genbank accession number nm_ ]) (data not shown), which predicts a - aa truncated protein. in another case—an italian patient with ttd and severe nervous-system impairment (pa- tient ) (rizzo et al. )—our attempts to amplify the coding regions of c orf failed. by multiplex pcr using a primer pair that amplifies a -bp control frag- ment, we determined that part of exon and the entire exon of c orf were homozygously deleted, whereas the flanking genes (cdc l and c orf ) were not (fig. b and e). these patients with homozygous deletions are likely to be genetically null for c orf , which might explain their more-severe neurological phenotype in comparison with that of the patients with abhs. we did not find any mutations in the two exons and ′ up- stream region of c orf in the other cases of non- photosensitive ttd, including two cases of sabinas syn- drome and one case of pollitt syndrome, which suggests that genetic heterogeneity exists in nonphotosensitive ttd. the fibroblasts derived from all but two patients with sabinas syndrome were tested for uv sensitivity by use of various ner parameters, including unscheduled table clinical features and c orf mutations in patients with nonphotosensitive ttd study findinga for isolated ttdb sabinas syndrome abhs moroccan siblings pollitt syndrome patient (present study) ibidsb clinical features: hair defect � � � � � � � nail dystrophy � � � � � � � mental retardation � (iq ) � (iq – ) �c (iq ) � (iq – ) � � � growth retardation � � �c � � � � ichthyosis � � � � � �d � decreased fertility � �e �f other features ataxia ataxia, dental caries severe nervous- system impairment cataract, collodion skin at birth uv sensitivity of fibroblastsg nd nd nd no uv sensitivity no uv sensitivity no uv sensitivity nd mutations in c orf h nd no mutation arg (m v) _ delgg no mutation deletion: part exon and entire exon nd reference alfandari et al. howell et al. baden et al. ; jackson et al. przedborski et al. pollitt et al. rizzo et al. ; stefanini et al. jorizzo et al. a nd p not determined; � p presence; � p absence. b the isolated ttd cases and the ichthyosis, brittle hair, impaired intelligence, decreased fertility, and short stature (ibids) cases were not analyzed in this study but are included in the table to show the extent of the spectrum of ttd symptoms. c mild. d ichthyosiform areas present only on the lower limbs. e male only. f gonadal dysfunction tested. g uv sensitivity tests were performed using various ner parameters, including unscheduled dna synthesis (global ner), recovery from transcription inhibition (transcription-coupled ner), and overall clonogenic cell survival after uv exposure. h genbank accession number nm_ . reports dna synthesis, recovery from transcription inhibition, and overall clonogenic cell survival after uv exposure. microsatellite analysis also excluded the -mb c orf locus from involvement in sabinas syndrome. therefore, we have designated c orf with the symbol ttdn (ttd nonphotosensitive ). we identified predicted proteins with sequence simi- larity to human c orf in six mammalian species as well as chicken, frog, fish, and insects, but not in lower eukaryotic species (c. elegans and yeast). the first two- thirds of the human c orf protein is remarkably gly- cine/proline–rich ( % in aa), and this feature is more evident in higher eukaryotic species (fig. a). there are two highly conserved regions: cr , conserved from zebrafish to human, and cr , conserved from mosquito to human (fig. a). the mutant m v found in pa- tients with abhs is located in the three amino acid res- idues (sml) in cr that are conserved in all species. to examine the subcellular localization of c orf , we transfected myc-epitope–tagged protein into cultured mammalian cells and found that it was predominantly expressed in the nucleus (fig. b); the pattern of the myc- epitope–mutated protein was indistinguishable from the wild type (data not shown). we performed in situ rna hybridization analysis for c orf in human fetal skin tissue and found that it was expressed in epidermis and hair follicles, consistent with presentation of the phe- notype (fig. c). c orf expression was not clearly detected in dermis by in situ hybridization, but it was found by rt-pcr in fibroblast cells. to examine whether c orf mutations affect tfiih cellular concentration, we performed comparative im- munofluorescence analysis, using antibody against the xeroderma pigmentosum group b protein (xpb) (a core component of tfiih), and found that the tfiih levels in normal controls and in fibroblasts from a patient with ttd (one of the moroccan siblings with the -bp de- letion) are the same (fig. d). this result, combined with the observation that mutations could not be detected in any of tfiih subunits and with the genetic mapping data (not shown), suggests that the defect in nonpho- tosensitive ttd is independent of tfiih action. to our knowledge, c orf is the first gene identified as mutated in patients with nonphotosensitive ttd. since these mutations were found in only a subset of nonphotosensitive ttd cases, it is likely that there is more than one disease-causing gene, as was found for photosensitive ttd. photosensitive ttd is caused by mutations in genes encoding subunits (xpd, xpb, and ttd-a) of the tfiih complex that functions in tran- scription and dna repair. since there is an absence of cutaneous photosensitivity in the patients with c orf mutations and since the protein demonstrates a nuclear localization, it is possible that c orf may have a role in transcriptional processes but have no role in dna repair. moreover, the brittle hair observed in patients with ttd is thought to result from the reduced expres- sion of high-sulfur proteins (intermediate keratin fil- aments and matrix proteins) in the late stage of kera- tinocyte differentiation (bergmann and egly ). genes in these pathways and/or proteins associated with c orf would be the primary candidates for involve- ment in other nonphotosensitive ttd cases. acknowledgments we thank alli tallqvist for skillful technical assistance in rna in situ hybridization experiments. we acknowledge the centre for applied genomics, the genome canada/ontario genome institute, the hospital for sick children foundation, the association française contre les myopathies, and the dykstra foundation in detroit (grant to c.e.j.). s.w.s. is an investigator of the canadian institutes of health research, a scholar of the mclaughlin centre for molecular medicine, and an international scholar of the howard hughes medical institute. electronic-database information accession numbers and urls for data presented herein are as follows: the chromosome annotation project, http://www.chr .org/ (for seven candidate genes on p ) ensembl, http://www.ensembl.org/ (for proteins from chimpanzee [accession number ensptrp ], rat [accession number ensrnop ], and chicken [accession number ensgalp ]) genbank, http://www.ncbi.nlm.nih.gov/genbank/ (for human c orf mrna [accession number nm_ ] and cdna sequences from zebrafish [accession number bc ] and pig [accession number bp ]) ncbi, http://www.ncbi.nlm.nih.gov/ (for human c orf [ac- cession number np_ ] and orthologues from mouse [accession number bab ], frog [accession number np_ ], pufferfish [accession number caf ], fruitfly [accession number np_ ], and mosquito [ac- cession number xp_ ]) online mendelian inheritance in man (omim), http://www .ncbi.nlm.nih.gov/omim/ (for amish brittle-hair syndrome, sabinas syndrome, and pollitt syndrome) ucsc genome browser, http://genome.ucsc.edu/ references alfandari s, delaporte e, van neste d, lucidarme-delespierre e, piette f, bergoend h ( ) a new case of isolated tri- chothiodystrophy. dermatology : – baden hp, jackson ce, weiss l, jimbow k, lee l, kubilus j, gold rj ( ) the physicochemical properties of hair in the bids syndrome. am j hum genet : – bergmann e, egly jm ( ) trichothiodystrophy, a tran- scription syndrome. trends genet : – botta e, nardo t, broughton bc, marinoni s, lehmann ar, am. j. hum. genet. : – , stefanini m ( ) analysis of mutations in the xpd gene in italian patients with trichothiodystrophy: site of mutation correlates with repair deficiency, but gene dosage appears to determine clinical severity. am j hum genet : – botta e, nardo t, lehmann ar, egly jm, pedrini am, ste- fanini m ( ) reduced level of the repair/transcription factor tfiih in trichothiodystrophy. hum mol genet : – giglia-mari g, coin f, ranish ja, hoogstraten d, theil a, wijgers n, jaspers ng, raams a, argentini m, van der spek pj, botta e, stefanini m, egly jm, aebersold r, hoeijmakers jh, vermeulen w ( ) a new, tenth subunit of tfiih is responsible for the dna repair syndrome tri- chothiodystrophy group a. nat genet : – howell rr, arbisser ai, parsons ds, scott ci, fraustadt u, collie wr, marshall rn, ibarra oc ( ) the sabinas syndrome. am j hum genet : – jackson ce, weiss l, watson jh ( ) “brittle” hair with short stature, intellectual impairment and decreased fertility: an autosomal recessive syndrome in an amish kindred. pe- diatrics : – jorizzo jl, atherton dj, crounse rg, wells rs ( ) ich- thyosis, brittle hair, impaired intelligence, decreased fertility and short stature (ibids syndrome). br j dermatol : – nakabayashi k, fernandez ba, teshima i, shuman c, proud vk, curry cj, chitayat d, grebe t, ming j, oshimura m, meguro m, mitsuya k, deb-rinker p, herbrick ja, weks- berg r, scherer sw ( ) molecular genetic studies of hu- man chromosome in russell-silver syndrome. genomics : – pollitt rj, jenner fa, davies m ( ) sibs with mental and physical retardation and trichorrhexis nodosa with abnor- mal amino acid composition of the hair. arch dis child : – price vh, odom rb, ward wh, jones ft ( ) trichothio- dystrophy: sulfur-deficient brittle hair as a marker for a neuroectodermal symptom complex. arch dermatol : – przedborski s, ferster a, goldman s, wolter r, song m, ton- nesen t, pollitt rj, vamos e ( ) trichothiodystrophy, mental retardation, short stature, ataxia, and gonadal dys- function in three moroccan siblings. am j med genet : – rizzo r, pavone l, micali g, calvieri s, di gregorio l ( ) trichothiodystrophy: report of a new case with severe ner- vous system impairment. j child neurol : – saarialho-kere uk, chang es, welgus hg, parks wc ( ) distinct localization of collagenase and tissue inhibitor of metalloproteinases expression in wound healing associated with ulcerative pyogenic granuloma. j clin invest : – scherer sw, cheung j, macdonald jr, osborne lr, naka- bayashi k, herbrick ja, carson ar, et al ( ) human chromosome : dna sequence and biology. science : – seboun e, lemainque a, jackson ce. amish brittle hair syn- drome gene maps to p . . am j med genet (in press) stefanini m, giliani s, nardo t, marinoni s, nazzaro v, rizzo r, trevisan g ( ) dna repair investigations in nine italian patients affected by trichothiodystrophy. mutat res : – thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg ( ) the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids res : – weeda g, eveno e, donker i, vermeulen w, chevallier-lagente o, taieb a, stary a, hoeijmakers jh, mezzina m, sarasin a ( ) a mutation in the xpb/ercc dna repair tran- scription gene, associated with trichothiodystrophy. am j hum genet : – head and neck imaging s role of d ct in the evaluation of the temporal bone learning objectives for test after reading this article and taking the test, the reader will be able to: � describe the nor- mal d anatomy of the temporal bone, including the mor- phologic features and interrelationships of individual structures. � list the microana- tomic structures of the temporal bone. � discuss the role of d ct in the evalua- tion of pathologic conditions of the temporal bone. girish m. fatterpekar, md ● amish h. doshi, md ● mohit dugar, md bradley n. delman, md ● thomas p. naidich, md ● peter m. som, md in recent years, three-dimensional ( d) multiplanar reformatted im- ages from conventional cross-sectional computed tomographic (ct) data have been increasingly used to better demonstrate the anatomy and pathologic conditions of various organ systems. three-dimen- sional volume-rendered (vr) ct images can aid in understanding the temporal bone, a region of complex anatomy containing multiple small structures within a relatively compact area, which makes evaluation of this region difficult. these images can be rotated in space and dis- sected in any plane, allowing assessment of the morphologic features of individual structures, including the small ossicles of the middle ear and the intricate components of the inner ear. the use of submillimeter two-dimensional reconstruction from ct data in addition to d refor- mation allows depiction of microanatomic structures such as the osse- ous spiral lamina and hamulus. furthermore, d vr ct images can be used to evaluate various conditions of the temporal bone, including congenital malformations, vascular anomalies, inflammatory or neo- plastic conditions, and trauma. the additional information provided by d reformatted images allows a better understanding of temporal bone anatomy and improves the ability to evaluate related disease, thereby helping to optimize surgical planning. ©rsna, abbreviations: eac � external auditory canal, iac � internal auditory canal, ica � internal carotid artery, mpr � multiplanar reformatted, d � three-dimensional, d � two-dimensional, vr � volume rendered radiographics ; :s –s ● published online . /rg. si ● content codes: from the department of radiology, mount sinai medical center, one gustave l. levy place, new york, ny . presented as an education ex- hibit at the rsna annual meeting. received january , ; revision requested march and received march ; accepted april . all authors have no financial relationships to disclose. address correspondence to p.m.s. (e-mail: peter.som@mssm.edu). ©rsna, r a d io g r a p h ic s cme feature see accompanying test at http:// www.rsna.org /education /rg_cme.html see last page teaching points note: this copy is for your personal non-commercial use only. to order presentation-ready copies for distribution to your colleagues or clients, contact us at www.rsna.org/rsnarights. introduction the temporal bone houses the middle ear, in- cluding the ossicles; the inner ear, which consists of the cochlea, vestibule, and semicircular canals; the bony canals for the facial and vestibuloco- chlear nerves; and the related vasculature and muscles. conventional two-dimensional ( d) imaging has been used extensively to depict the individual structures of the temporal bone. how- ever, the complex, multispatial orientation of these structures, contained as they are within a compact region, often makes it difficult to appre- ciate their three-dimensional ( d) orientation within the temporal bone and their intricate inter- relationships. recent developments in software technology have made it possible to rapidly gener- ate d volumes from conventional d data ( – ). these volume-rendered (vr) images can be sectioned in any plane and rotated in space, al- lowing d insight into the anatomy of the tempo- ral bone ( ). microanatomic structures that are not well seen with conventional d imaging can be clearly depicted using overlapping reconstruc- tion at smaller intervals. in addition, the refor- matted images provide complementary infor- mation about various conditions, including congenital malformations, vascular anomalies, inflammatory or neoplastic conditions, and trauma involving the temporal bone ( – ). in this article, we discuss our retrospective study in terms of patients and procedures, review the normal anatomy of the temporal bone, and discuss and illustrate the utility of d computed tomography (ct) in demonstrating temporal bone anatomy and evaluating related disease. patients and procedures a retrospective study was performed that in- cluded patients who had undergone high-reso- lution ct of the temporal bone for the evaluation of complaints related to the auditory system. each scan was obtained on a -section spiral ct scanner (somatom sensation ; siemens medi- cal solutions, malvern, pa). the studies were performed with the following parameters: . - mm collimation, . -mm section thickness, kvp, mas, pitch of . , a -cm field of view, and a � matrix. the initial data sets were then reconstructed at . -mm intervals. during initial postprocessing, we observed that any amount of gantry tilt on the ct scanner caused distortion of d ct reformatted images. therefore, d ct scans were obtained with a ° gantry tilt and a scanning plane parallel to the inferior orbitomeatal line. in addition, overlap- ping submillimeter reconstruction of the raw data was performed to obtain the best possible d ct reformatted images. three-dimensional vr ct images were gener- ated from the original d data with terarecon aquarius workstation v . (terarecon, san ma- teo, calif). all reformatted images were obtained by a neuroradiology fellow or a postprocessing technologist. the application of different soft- tissue and bone algorithms to the d reformation permitted multiprojectional display of the various temporal bone structures, including the ossicles and the inner ear structures (eg, cochlea, vesti- bule, semicircular canals). with use of a built-in d cut-plane software technique, individual tem- poral bone structures were “removed” and ana- lyzed, allowing optimal display of microanatomic components such as the delicate osseous spiral lamina of the cochlea. three-dimensional refor- matted images were also obtained from temporal bone scans to more effectively demonstrate dis- ease. normal anatomy external ear the external ear consists of the auricle, or pinna, and the external auditory canal (eac). the pinna collects sound waves, and the eac conducts these vibrations to the tympanic membrane. the eac forms an s-shaped curve as it extends from the auricle to the tympanic membrane. the me- dial two-thirds of the eac is osseous and slightly narrower than the lateral third, which is cartilagi- nous. a somewhat more pronounced constric- tion, the isthmus, is seen at the bone-cartilage junction (fig ). the narrowing of the osseous segment of the eac results in the amplification of sound waves as they progress to the tympanic membrane. the tympanic membrane marks the medial boundary of the eac, separating the ex- ternal ear from the tympanic cavity ( – ). s october rg f volume ● special issue r a d io g r a p h ic s teaching point teaching point
during initial postprocessing, we observed that any amount of gantry tilt on the ct scanner caused distortion of d ct reformatted images. therefore, d ct scans were obtained with a -degree gantry tilt and a scanning plane parallel to the inferior orbitomeatal line. in addition, overlapping submillimeter reconstruction of the raw data was performed to obtain the best possible d ct reformatted images. middle ear the tympanic cavity, or middle ear, can be struc- turally divided into three parts. the tympanic cavity proper (mesotympanum) lies at the level of and directly opposite the tympanic membrane; the epitympanic recess (attic) lies above the level of the tympanic membrane; and the hypotympa- num, as its name suggests, lies inferior to the tym- panic membrane (fig ). the tympanic cavity houses three ossicles: the malleus, the incus, and the stapes. the ossicular chain transmits and am- plifies vibrations incident on the tympanic mem- brane across the middle ear cavity, causing deflec- tion of the oval window, which is attached to the footplate of the stapes ( , , ). the malleus, which is the largest of the os- sicles, lies anterolateral to the incus and stapes. a facet on the posterior surface of the head of the malleus articulates with the body of the incus. the head of the malleus and the body of the incus articulate by means of a thin capsular ligament, forming a diarthrodial joint. a mild constriction, the neck of the malleus, is seen at the inferior as- pect of the club-shaped head and provides attach- ment to the tensor tympani. more inferiorly lies the long process, or manubrium, of the malleus. the tip of the manubrium attaches to the tym- panic membrane at the umbo ( – ). small bony spicules, the anterior and lateral processes, pro- ject from the upper portion of the manubrium and provide attachment for the anterior and lat- eral malleal ligaments, which help support the malleus within the middle ear cavity (fig ) ( , , ). the incus is shaped somewhat like a premolar tooth, with two widely diverging processes that differ in length. a facet on the anterior surface of the body of the incus articulates with the head of the malleus. the short process of the incus is di- rected posterolaterally; the long process is di- rected inferiorly and lies parallel to the manu- brium of the malleus ( , ). the tip of the long process bends medially to end in a rounded pro- jection, the lenticular process, which articulates with the stapes (fig ) ( , ). figure . normal anatomy of the auditory apparatus. (a) oblique coronal d vr ct image (anterior view) shows the pinna (p); the eac, including the cartilaginous (caeac) and osseous (oseac) portions; the isthmus (is); the middle ear cavity (me); and the inner ear (ie). the middle ear is divided into the epitympanum (above dotted green line), mesotympanum (between dotted green and dotted yellow lines), and hypotympanum (below dotted yellow line). (b) three- dimensional vr ct image (view from the dissected medial portion of the temporal bone) shows the inner ear, including the cochlea (co), vestibule (ve), superior semicircular canal (sscc), lat- eral semicircular canal (lscc), and posterior semicircular canal (pscc). the internal auditory canal (iac) and the bony canal for the facial nerve (fn) are also seen. rg f volume ● special issue fatterpekar et al s r a d io g r a p h ic s the stapes is the most medial ossicle. it re- sembles a stirrup and consists of a head, a neck, two crura, and a footplate. the head articulates with the lenticular process of the incus. inferior to the head lies the neck, which, at its posterior as- pect, provides insertion to the tendon of the sta- pedius muscle. two crura, the anterior and poste- rior crura, diverge from the neck and are con- nected at their inferior ends by the footplate. the footplate sits on the oval window, attached to its margins by the annular ligament (fig ) ( , , ). the surface area of the tympanic membrane is up to times greater than that of the oval win- dow. thus, the pressure exerted on the tympanic membrane by a sound wave is concentrated through the ossicles onto the much smaller area of the oval window, resulting in a pressure in- crease and amplification of sound transmission. a lever mechanism that exists between the ossicles further contributes to sound amplification. this amplification that occurs within the tympanic cavity is crucial for overcoming the inertia of the perilymph within the osseous labyrinth in the in- ner ear ( , ). inner ear the inner ear consists of the bony labyrinth, which lies within the petrous portion of the tem- poral bone and is the densest structure in the en- tire human body (fig ). the bony labyrinth con- tains the membranous labyrinth, a complex inter- connected series of membranous sacs and ducts that are primarily responsible for balance and hearing. fluid within the bony labyrinth called the perilymph surrounds the membranous laby- rinth, which contains its own unique fluid, the endolymph ( ). the bony labyrinth consists of three structures: the cochlea, the vestibule, and the semicircular canals (fig ). the cochlea is shaped like a coni- cal snail shell. it consists of a bony canal wound around a conical central core called the modiolus. the canal winds around this central axis slightly more than ⁄ times and gradually decreases in figures , . ( ) normal anatomy of the malleus. (a) posterior oblique d vr ct image shows the head of the malleus (hm) and, appearing as a narrow con- striction inferiorly, the neck of the malleus (nm). the manubrium (m) lies infe- rior to the neck. the head of the malleus demonstrates a facet (f) on its posterior aspect that articulates with the body of the incus. (b) anterior oblique d vr ct image shows two bony spicules arising from the upper portion of the manubrium, the anterior process (ap) and the lateral process (lp). the tip of the manubrium (tm) attaches to the tympanic membrane at the umbo. ( ) normal anatomy of the incus. three-dimensional vr ct image (inferolateral view) shows the body of the incus (bi), which demonstrates a facet (f) on its anterior surface that articulates with the head of the malleus. the short process (sp) is directed posterolaterally, and the long process (lp) is directed inferiorly. the tip of the long process bends medially to end in a rounded projection, the lenticular process (lnp), which articu- lates with the head of the stapes. s october rg f volume ● special issue r a d io g r a p h ic s teaching point teaching point
the surface area of the tympanic membrane is up to times greater than that of the oval window. thus, the pressure exerted on the tympanic membrane by a sound wave is concentrated through the ossicles onto the much smaller area of the oval window, resulting in a pressure increase and amplification of sound transmission. a lever mechanism that exists between the ossicles further contributes to sound amplification. diameter as it spirals toward the apex. a delicate osseous spiral lamina projects from the modiolus and divides the bony canal for the cochlea into an upper passage (scala vestibuli) and a lower pas- sage (scala tympani). between these two scalae lies the cochlear duct, or scala media, which con- tains the organ of corti, the sensory organ of hearing. at the apex of the cochlea, the osseous spiral lamina ends freely in a hook-shaped hamu- lus, which partly bounds the helicotrema, an opening through which the two scalae communi- cate (fig ) ( , , ). figure . normal anatomy of the stapes. (a) three-dimensional vr ct image (inferior view) shows the head of the stapes (hs), which articulates with the lenticular process of the incus. inferior to the head of the stapes is the sta- pes neck (ns), which provides attachment to the stapedius muscle. the anterior crus (ac) and posterior crus (pc) connect the stapes neck to the stapes footplate (fp). the stapes is a cartilaginous structure, unlike the bony malleus and incus. therefore, the color rendering of the stapes on the d shaded-surface-display reformatted images was dif- ferent from that of the malleus and incus (cf figs , ). (b, c) three-dimensional vr ct images (view from the dis- sected tympanic cavity looking into the oval window) obtained with (b) and without (c) the stapes (st) present show that the stapes footplate (dotted blue line) sits on the oval window (dotted yellow line), attached to its margins by the annular ligament (al). figures , . ( ) normal anatomy of the bony labyrinth. three-dimensional vr ct image (anterolat- eral view) shows the normal bony labyrinth, which consists of the cochlea (co), vestibule (ve), and semi- circular canals (scc). fn � facial nerve canal, iac � internal auditory canal, ow � oval window. ( ) normal anatomy of the cochlea. three-dimensional vr ct image (inferolateral view after dissection of a portion of the inferior wall of the cochlea) shows the osseous spiral lamina (sl), which divides the bony canal for the cochlea into upper and lower passages, the scala vestibule (sv) and the scala tympani (st), respectively. dissection of the apical cochlear bony labyrinth shows the terminus of the spiral lamina as a conical projection, or hamulus (ha), allowing free communication between the scala vesti- bule and the scala tympani at the helicotrema (he). ow � oval window, rw � round window. rg f volume ● special issue fatterpekar et al s r a d io g r a p h ic s the vestibule is the central and most capacious portion of the bony labyrinth. the vestibule is continuous anteriorly with the cochlea and poste- riorly with the semicircular canals. it contains the utricle and the saccule, parts of the membranous labyrinth, which are primarily involved with bal- ance. superiorly and posteriorly on the medial wall of the vestibule is the elliptical recess, which houses the utricle; inferiorly and anteriorly lies the spherical recess, which accommodates the sac- cule. between these two recesses is an oblique ridge, the vestibular crest, which divides posteri- orly into two limbs bounding a small depression, the cochlear recess. the cochlear recess in turn houses the blind opening of the cochlear duct (fig ) ( , , ). there are three semicircular canals: superior, posterior, and lateral. the planes of the semicir- cular canals are nearly orthogonal, or at right angles, to each other. each of the canals forms about two-thirds of a circle and is enlarged anteri- orly to form the ampulla ( , , ). the posterior end of the superior semicircular canal joins the upper end of the posterior semicircular canal to form a common limb known as the common crus. the perilymphatic space of each semicircular ca- nal opens into and communicates freely with that of the vestibule ( , ). the osseous semicircular canals contain the corresponding membranous semicircular ducts, which are part of the membra- nous labyrinth and communicate with the utricle. collectively, the semicircular ducts are respon- sible for the detection of angular acceleration ( , , ). sound vibrations traveling through the ossicu- lar chain in the tympanic cavity deflect the oval window through its attachment to the footplate of the stapes. this deflection of the oval window compresses the perilymph in the scala vestibuli. vibrations in the perilymph are transmitted to the endolymph in the cochlear duct, which contains the spiral organ of corti. sound transduction oc- curs within the organ of corti, where mechani- cal energy is converted into electrical impulses. these electrical impulses are in turn transmitted by the cochlear nerve to the brainstem. simulta- neously, the compression waves within the scala vestibuli travel along the coils of the cochlea to the helicotrema. the compression waves are transmitted through this opening and travel back down the coils in the scala tympani to reach the round window. the round window serves as a pressure-relief diaphragm, bulging outward with each pressure wave in the scala tympani (fig ) ( , ). figure . normal anatomy of the vestibule. three- dimensional vr ct image (inferolateral view after dis- section of the lateral wall of the vestibule) demonstrates the elliptical recess (er) posteriorly and the spherical recess (sr) anteriorly, which house the utricle and sac- cule, respectively. a bony ridge, the vestibular crest (vc), lies between the elliptical and spherical recesses. the vestibular crest divides inferiorly into two limbs that form an inverted v and bound a small depression, the cochlear recess (cr). the cochlear recess houses the blind opening of the cochlear duct. figure . sound transmission. three-dimensional vr ct image demonstrates the anatomic structures involved in the transmission of sound. sound vibra- tions transmitted through the tympanic membrane (tm) are amplified multifold across the middle ear cavity by the lever mechanism of the ossicles. in addi- tion, the disproportionately larger size of the tympanic membrane allows sound waves to be concentrated onto the smaller oval window (ow), further contributing to sound augmentation. inward deflection of the oval win- dow by the footplate of the stapes (st) compresses the fluid in the scala vestibuli (wavy yellow line). this com- pression wave travels along the coils of the cochlea in the scala vestibuli to the helicotrema (he). it then spi- rals back through the scala tympani (wavy green line) to the round window (rw), which serves as a pressure- relief diaphragm. the vibrations in the scala vestibuli also stimulate the cochlear duct (lying adjacent to the osseous spiral lamina [sl]), where mechanical energy is converted into electrical impulses and transmitted via the cochlear nerve to the brainstem (not shown). in � incus, m � malleus. s october rg f volume ● special issue r a d io g r a p h ic s medial and posterior walls of the tympanic cavity the inner ear structures produce multiple im- pressions, or ridges, on the medial and posterior walls of the tympanic cavity. with use of semi- opaque windowing and careful dissection of over- lying structures, these impressions can be clearly visualized on d vr images. posteriorly and superiorly along the medial wall of the tympanic cavity is the prominence pro- duced by the anterior limb of the lateral semicir- cular canal. inferior to this prominence and ex- tending more anteriorly is the prominence of the bony canal for the facial nerve. anterior to the prominence of the bony canal for the facial nerve is the curved terminus of the septum canalis mus- culotubari, which serves as a landmark for the geniculate ganglion. immediately inferior to this prominence is the oval window. inferior and slightly anterior to the oval window lies the rounded promontory, a convexity produced by the basal turn of the cochlea (fig ) ( , ). the posterior wall of the tympanic cavity con- tains two important recesses: the sinus tympani and the facial nerve recess. the sinus tympani indicates the position of the ampulla of the poste- rior semicircular canal. lateral to the sinus tym- pani is the facial nerve recess, an important surgi- cal landmark when entrance into the tympanic cavity is gained using the mastoid approach. en- trance is initially gained into the middle ear cavity by enlarging the facial nerve recess using the facial nerve and chorda tympani as landmarks ( , , ). the pyramidal eminence is situated between the sinus tympani and the facial nerve recess. this eminence gives rise to the stapedius muscle (fig ) ( – ). osseous neural canals for the facial and vestibulocochlear nerves the internal auditory canal (iac) traverses the petrous portion of the temporal bone and func- tions as a conduit for the facial and vestibuloco- chlear nerves as they course from the brainstem to the inner ear. the medial opening of the iac is known as the porus acusticus. the lateral end of the iac, known as the fundus, is separated from the inner ear by a vertical plate of bone that is perforated to allow passage of the facial and ves- tibulocochlear nerves. the fundus is divided into upper and lower portions by a horizontal ridge of bone known as the falciform crest. a thin verti- cal crest of bone known as the bill bar further divides the upper portion of the fundus into an anterior opening for the facial nerve and a poste- rior opening for the superior vestibular nerve (fig ). the superior vestibular nerve innervates the utricle and the superior and lateral semicircular figures , . ( ) three-dimensional vr ct image (semiopaque windowing) shows the inner ear impressions produced on the medial wall of the tympanic cavity. the prominence produced by the anterior limb of the lateral semicircular canal (lscc) is seen posteriorly, and the prominence produced by the facial nerve canal (fn) is seen anteriorly. the prominence produced by the curved terminus of the septum canalis musculotubari (scm) is seen superior and anterior to the oval window (ow). the septum canalis musculotubari serves as the landmark for the geniculate ganglion. the prominence produced by the cochlear promontory (cop) lies inferior and slightly anterior to the oval window. ( ) three-dimensional vr ct image (semiopaque windowing) shows two recesses on the posterior wall of the tympanic cavity: the sinus tympani (st) lies me- dial to the facial nerve recess (fnr) and indicates the position of the ampulla of the posterior semicircular canal. the pyramidal eminence (pe) is a small conical projection located between the two recesses that gives rise to the stapedius muscle. co � cochlea. rg f volume ● special issue fatterpekar et al s r a d io g r a p h ic s canals (fig ). the lower compartment of the fundus of the iac has a rounded opening anteri- orly through which the bundles of the cochlear division of the eighth cranial nerve pass to inner- vate the cochlea (fig ). the lower compart- ment has a pinpoint opening posteriorly for the passage of the inferior vestibular nerve, which figure . normal iac. coronal (top left), sagittal (top right), and axial (bottom left) vr ct images show the nor- mal iac (arrow). virtual iac otoscopic image (bottom right) shows the fundus of the iac, which is divided into up- per and lower portions by the falciform crest (fcr). the bill bar (bb) further subdivides the upper portion of the fun- dus into openings for the facial nerve (fn) anteriorly and the superior vestibular nerve (sv) posteriorly. the lower portion of the fundus contains an an- terior opening for bundles of the cochlear nerve (cn) and a pinpoint posterior opening for the inferior vestibular nerve (iv). figures – . ( ) normal anatomy of the osseous neural canals for the facial and superior vestibular nerves. three-dimensional vr ct image (superior view) shows the bony canal for the facial nerve (fn) dissected in its proximal portion at the anterosuperior aspect of the internal auditory canal (iac). the bony canal for the superior vestibular nerve (sv) lies posterior to the facial nerve canal and extends to the vestibule (ve). ( ) normal anatomy of the osseous neural canals for the facial and cochlear nerves. three-dimensional vr ct image (medial view) shows that the cochlear nerve canal (cn) lies inferior to the facial nerve canal (fn) and courses to the cochlea. iac � in- ternal auditory canal. ( ) singular canal. three-dimensional vr ct image (posteroinferior view) shows the singular canal (sc), through which courses a branch of the inferior vestibular nerve. the inferior vestibular nerve innervates the posterior semicircular canal (pscc). s october rg f volume ● special issue r a d io g r a p h ic s innervates the saccule. in addition, the inferior vestibular nerve gives off a branch within the iac that arises approximately mm proximal to the fundus and courses within its own bony canal, the singular canal, to innervate the posterior semicir- cular canal (fig ) ( – , ). as illustrated in figure , of the four neural openings identified at the fundus of the iac, the opening for the co- chlear nerve is the largest and that for the inferior vestibular nerve is the smallest ( , ). facial nerve canal the facial nerve takes a winding path through the temporal bone, and its course is divided into three basic segments: the labyrinthine, the tympanic, and the mastoid segments (fig ). with use of multiprojectional d vr ct images, we were able to demonstrate the course of the facial nerve within the temporal bone. the labyrinthine segment of the facial nerve emerges from the anterosuperior aspect of the iac, coursing anterolaterally within its own bony channel, the fallopian canal. this segment of the facial nerve is relatively short, measuring – mm, and lies superior to the cochlea. it makes a subtle anteromedial turn as it courses anteriorly to reach the geniculate ganglion. at the ganglion, the facial nerve reverses its direction, making a sharp posteroinferior turn to continue as the tym- panic segment. this sharp turn, known as the anterior genu, lies superomedial to the cochlear promontory. the tympanic segment is approxi- mately mm long and extends from the genicu- late ganglion to the posterior genu. this portion of the facial nerve courses posteriorly along the medial wall of the tympanic cavity, just inferior to the lateral semicircular canal and superior to the oval window, to reach the sinus tympani. at the sinus tympani, the facial nerve again changes di- rection, making a gentler posteroinferior turn to form the posterior genu. here, the facial nerve assumes a more vertical position and descends just behind the posterior wall of the tympanic cav- ity as the mastoid segment to exit the temporal bone through the stylomastoid foramen. the mastoid segment is the longest portion of the in- tratemporal facial nerve, measuring approxi- mately – mm. the chorda tympani arises from the lateral aspect of the mastoid segment of the facial nerve approximately mm superior to the stylomastoid foramen and follows a subtle curved course superoanteriorly in the canaliculus chorda tympani (fig ). it then proceeds anteri- orly within the tympanic cavity, exiting the tem- poral bone through a minute canal, the anterior canaliculus, near the petrotympanic fissure. it joins the lingual nerve to supply taste sensation to the anterior two-thirds of the tongue ( – , ). figure . normal anatomy of the facial nerve canal. three-dimensional vr ct images (posterior [a], supe- rior [b], and lateral [c] views) show the course of the facial nerve through its bony canal as it exits the anterosupe- rior aspect of the fundus of the internal auditory canal (iac). the labyrinthine segment of the facial nerve courses through the fallopian canal (fc) to the geniculate ganglion, where the nerve makes a hairpin turn known as the ante- rior genu (ag). the tympanic segment (ts) of the facial nerve canal runs below the lateral semicircular canal (lscc) and above the oval window (ow) along the medial wall of the tympanic cavity. it then makes a gentle curve (the pos- terior genu [pg]) and heads vertically downward as the mastoid segment (ms) to exit the temporal bone at the stylo- mastoid foramen. the chorda tympani arises from the lateral aspect of the mastoid segment approximately mm proximal to the stylomastoid foramen and courses superiorly within its own bony canal, the canaliculus chorda tym- pani (cct). co � cochlea, in � incus, m � malleus. rg f volume ● special issue fatterpekar et al s r a d io g r a p h ic s temporal bone disease congenital malformations large vestibular aqueduct syndrome.—the vestibular aqueduct is a curvilinear tubular struc- ture that extends from the posteroinferior surface of the temporal bone to the medial wall of the vestibule. it contains the endolymphatic sac, which is connected to the utricle and saccule by the endolymphatic duct. the vestibular aqueduct normally measures less than . mm in diameter and approximates the size of the posterior semi- circular canal, which runs anterior and parallel to the aqueduct ( , , ). the exact physiologic role of the vestibular aqueduct is not known. however, a dilated vestibular aqueduct has been increasingly recognized as being directly related to sensorineural hearing loss ( , , ). in a study by arcand et al ( ), large vestibular aqueduct syndrome was present in approximately % of children who presented with congenital sensori- neural hearing loss. a dilated vestibular aqueduct can be easily rec- ognized at conventional cross-sectional imaging by identifying its abnormal size in relation to the adjacent posterior semicircular canal. however, d multiplanar reformatted (mpr) ct images can more clearly demonstrate the classic funnel- shaped deformity of the dilated vestibular aque- duct, which occurs due to an enlarged endolym- phatic sac housed within the dorsal vestibular aq- ueduct (fig ) ( ). thus, d ct allows better display of underlying disease. congenital ossicular malformation with oval window atresia.—conductive hearing loss in the pediatric population is usually due to an acquired condition such as acute or chronic otitis media. congenital causes of conductive hearing loss in children are relatively uncommon. most cases of congenital conductive hearing loss are secondary to atresia or stenosis of the eac or, less frequently, to malformed ossicles ( ). ab- sence of the oval window has been suggested as an even more uncommon cause ( – ). recent advances in imaging techniques, such as submilli- meter reconstruction and d vr multiplanar im- aging, have markedly improved the evaluation of congenital anomalies that cause conductive hear- ing loss. to obtain the best possible surgical out- come, it is vital for the neuro-otologist or otolar- yngologist to appreciate the full extent of congeni- tal anomalies present. it is therefore imperative that the radiologist demonstrate the underlying anomalies in the greatest possible detail, thereby allowing optimal presurgical planning. figure illustrates congenital ossicular mal- formation in a young boy. in this case, the con- genitally malformed ossicles were easily identified at routine cross-sectional imaging; however, the atretic right oval window could be clearly de- picted only with d vr images. this additional information is crucial for the clinician because figure . large vestibular aqueduct syndrome in a -year-old girl with progressive sensorineural hearing loss. (a) ct scan shows a dilated vestibular aqueduct (arrowhead). the normal vestibular aqueduct should be approxi- mately the same size as the posterior semicircular canal (arrow). (b) corresponding d ct reformatted image again demonstrates the dilated vestibular aqueduct (arrowhead). the opening of the vestibular aqueduct (curved arrow) into the vestibule (ve) is also seen. straight arrow indicates the posterior semicircular canal. (c) three-dimensional vr ct image (posterior view) shows the classic funnel-shaped deformity of the dilated vestibular aqueduct (*) re- sulting from an enlarged endolymphatic sac housed within the dorsal portion of the vestibular aqueduct. ve � vestibule. s october rg f volume ● special issue r a d io g r a p h ic s teaching point teaching point teaching point
a dilated vestibular aqueduct can be easily recognized at conventional cross-sectional imaging by identifying its abnormal size in relation to the adjacent posterior semicircular canal. however, d multiplanar reformatted (mpr) ct images can more clearly demonstrate the classic funnel-shaped deformity of the dilated vestibular aqueduct, which occurs due to an enlarged endolymphatic sac housed within the dorsal vestibular aqueduct (fig ) ( ). teaching point
recent advances in imaging techniques, such as submillimeter reconstruction and d vr multiplanar imaging, have markedly improved the evaluation of congenital anomalies that cause conductive hearing loss. to obtain the best possible surgical outcome, it is vital for the neuro-otologist or otolaryngologist to appreciate the full extent of congenital anomalies present. it is therefore imperative that the radiologist demonstrate the underlying anomalies in the greatest possible detail, thereby allowing optimal presurgical planning. simply addressing the problem of malformed os- sicles is not sufficient to correct the hearing loss; surgical repair of the atretic oval window is also necessary ( , ). thus, d mpr ct images allow a more complete and thorough evaluation of congenital anomalies, which aids in presurgical planning. vascular anomalies an aberrant course of the internal carotid artery (ica) inside the temporal bone is a rare occur- rence ( ). however, it is crucial to recognize this entity, which can easily be misdiagnosed as a neo- plastic process (eg, paraganglioma) or an inflam- matory condition (eg, effusive otitis media). sub- sequent tympanotomy or biopsy of this “mass” can result in catastrophic hemorrhage. aberrant ica may manifest as pulsatile tinnitus, hearing loss, vertigo, or a sensation of fullness in the ear ( , – ). conventional ct with complementary d reformation is excellent for the evaluation of aber- rant ica. the axial and coronal d ct scans in figure a and b demonstrate bilateral aber- rant carotid arteries. however, by using d mpr ct images (fig c–f), we were able to show a band of tissue attaching the malleus to the aber- rant left ica, accounting for more severe tinnitus on the left side. unlike conventional d images, d vr ct images can be rotated in space and in any plane, an advantage that allows a more com- plete evaluation of the underlying disease. figure . congen- ital ossicular malfor- mation with oval window atresia in a -year-old boy with right-sided mixed hearing loss. (a, b) ct scan (a) and corre- sponding d ct reformatted image (b) show malformed os- sicles with an associ- ated soft-tissue mass (arrowhead) attached to the lateral wall of the right tympanic cavity. a suspicious platelike covering (arrow) is seen in the expected region of the right oval win- dow. the left-sided ossicles (os) and left oval window (ow) are normal. (c) three- dimensional vr ct image (inferolateral view) shows a normal left bony labyrinth with the oval window (ow) and round win- dow (rw) well de- picted. (d) three- dimensional vr ct image (inferolateral view) shows an abnor- mal right bony laby- rinth with absence of the oval window (ar- rowhead). rw � round window. rg f volume ● special issue fatterpekar et al s r a d io g r a p h ic s figure . aberrant icas in a -year-old woman with bilateral tinnitus, which was more pronounced in the left ear. (a, b) ct scans of the right (a) and left (b) temporal bones show bilateral aberrant icas (*). the manubrium of the left malleus (arrow in b) was identified only retrospectively, after evaluation of the d vr ct images (cf c, d), as being in proximity to the aberrant left ica. (c, d) corresponding d vr ct images also demonstrate the bilateral aberrant icas (*). the manubrium of the left malleus (arrow in d) appears to be attached to the aberrant left ica. on the contralateral side, the right malleus (arrowhead in c) is not as close to the aberrant right ica. (e) three-dimensional vr ct image (lateral view) shows the aberrant right ica (*). there is no attachment of the malleus (m) to the artery. in � incus. (f) three-dimensional vr ct image (lateral view) clearly demonstrates attachment of the abnor- mally long manubrium of the left malleus (m) to the aberrant left ica (*) by a band of soft tissue (ar- row). in � incus. s october rg f volume ● special issue r a d io g r a p h ic s inflammatory or neoplastic conditions cholesteatomas are erosive collections of kerati- nous debris arising from an ingrowth of stratified squamous epithelium. cholesteatomas can be acquired or congenital in origin. acquired middle ear cholesteatoma is the most common type, ac- counting for % of cases ( , ). at ct, cho- lesteatomas classically manifest as a soft-tissue mass causing underlying bone erosion ( – ). there are various types of surgeries for the treat- ment of cholesteatomas, including simple, modi- fied radical, and radical mastoidectomies. the type of surgery performed depends on the extent of disease, which is directly related to the extent of the underlying erosions. therefore, detailed preoperative radiologic assessment of the cho- lesteatoma is important ( , ). figure illustrates cholesteatoma in a woman with right-sided hearing loss. this case demon- strates that, although the diagnosis of cholestea- toma is readily established with conventional ct (fig a), the full extent of the underlying ero- sions can be established only by using d vr ct images (fig c, d). more specifically, exten- sion of the erosive process into the incudomalleo- lar joint could be evaluated only on d reformat- ted images in this case. in addition, the stapes, due to its cartilaginous skeleton, can at times be partially masked by the surrounding cholestea- toma at conventional ct, thus precluding its complete evaluation. as figure demonstrates, the use of d ct reformatted images with vary- ing thicknesses helps better assess the integrity of the stapes. such additional information greatly improves presurgical evaluation and allows more appropriate surgical planning. figure . cholesteatoma in a -year-old woman with right-sided hearing loss. (a, b) ct scan (a) and corresponding d ct reformatted image (b) show erosion of the right malleus and incus (arrow). the stapes (st) is intact and is better appreciated on the d image. the left malleus and incus (arrow- head) are normal. (c) three-dimensional vr ct image (lateral view after dissection of the anteroinferior portion of the eac) shows pressure erosion of the head of the right malleus (me) and the body of the incus (bie), along with nearly complete erosion of the short process of the incus (spe). extension of the erosion into the incudomalleolar articulation (ime) is also noted. (d) three-dimensional vr ct image (lateral view after dissection of the anteroinferior portion of the eac) shows the normal left side for com- parison. im � incudomalleolar articulation, in � incus, m � malleus, sp � short process of the incus. rg f volume ● special issue fatterpekar et al s r a d io g r a p h ic s teaching point teaching point
there are various types of surgeries for the treatment of cholesteatomas, including simple, modified radical, and radical mastoidectomies. the type of surgery performed depends on the extent of disease, which is directly related to the extent of the underlying erosions. therefore, detailed preoperative radiologic assessment of the cholesteatoma is important ( , ).
 trauma fractures of the temporal bone can be categorized into three types—longitudinal, transverse, or mixed— on the basis of their orientation relative to the long axis of the petrous temporal bone. fractures that run parallel to the long axis are classified as longitudinal fractures, whereas those that run perpendicular to the long axis are classi- fied as transverse fractures. longitudinal fractures are more common than transverse fractures. lon- gitudinal fractures cross the middle ear and are often associated with ossicular dislocation. trans- verse fractures traverse the fundus of the iac or the bony labyrinth, resulting in sensorineural hearing loss ( , , , ). although temporal bone fractures can usually be identified on con- ventional d scans, their complete extent is best appreciated on d reformatted images ( ). figure illustrates the role of d mpr ct images in evaluating the extent of temporal bone fractures. in this case, extension of the fracture line into the region of the oval and round win- dows was easily seen on the d ct reformatted figure . temporal bone fracture caused by trauma in a -year-old man. (a) ct scan shows a fracture (arrowhead) through the posterior semicircular canal (pscc) extending to involve the ossicles (arrows). (b) corresponding d ct reformatted image also demonstrates the fracture (arrowheads) through the base of the posterior semicircular canal (pscc) extending to the ossicles. the ossicles appear to have an abnormal configuration, suggesting possible dislocation of one of the ossicles (arrow) into the external auditory canal (eac). (c) three-dimensional vr ct image (posteroinferior view) shows the fractured bony labyrinth, with the fracture line (fx) extending through the base of the posterior semicircular canal (pscc) into the round window (rw) and oval window (ow). (d) three-dimensional vr ct image (anterior view after dissection of the anterior wall of the eac) shows incudomalleolar disarticulation, with the incus (in) located lateral to the malleus (m) and partly within the external auditory canal (eac). s october rg f volume ● special issue r a d io g r a p h ic s images (fig b, c) but only retrospectively on the conventional ct scans (fig a). in addition, the ability to rotate the d reformatted images in space allowed more complete evaluation of the associated ossicular dislocation. the characteris- tic shape of the most laterally positioned ossicle on d reformatted images proved that it was the incus, not the malleus as would normally be ex- pected, that was dislocated. this incudomalleolar dislocation was not appreciated on the d ct scans alone. furthermore, because the fracture line extended into the region of the oval window (which was better seen on the d vr ct image), it is possible that, despite undergoing ossicular reconstruction, the patient may continue to expe- rience a degree of hearing deficit from potential scar formation at the oval window fracture site. thus, d ct reformatted images can also help predict the degree of functional recovery expected after surgery. conclusions in this article, we have discussed and illustrated the role of d ct reformation in evaluating both the normal anatomy and pathologic conditions of the temporal bone. the ability to rapidly reformat these images in multiple projections and manipu- late their spatial orientation allows more detailed evaluation of the temporal bone anatomy, includ- ing its microanatomic structures, and of related disease entities. references . reisser c, schubert o, forsting m, sartor k. anatomy of the temporal bone: detailed three- dimensional display based on image data from high-resolution helical ct—a preliminary report. am j otol ; ( ): – . . calhoun ps, kuszyk bs, heath dg, carley jc, fishman ek. three-dimensional volume render- ing of spiral ct data: 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embryology and anatomy. in: som pm, curtin hd, eds. head and neck imaging. th ed. st louis, mo: mosby, ; – . . williams pl, warwick r, dyson m, bannister lh, eds. gray’s anatomy. th ed. new york, ny: churchill livingstone, ; – . . swartz jd, harnsberger hr, eds. imaging of the temporal bone. nd ed. new york, ny: thieme, . . donaldson ja, duckert lg, lambert pm, rubel ew. surgical anatomy of the temporal bone. th ed. new york, ny: raven, . . lemmerling mm, stambuck he, mancuso aa, antonelli pj, kubilis ps. ct of the normal sus- pensory ligaments of the ossicles in the middle ear. ajnr am j neuroradiol ; ( ): – . . yamada m, tsunoda a, muraoka h, komatsu- zaki a. three-dimensional reconstruction of the incudostapedial joint with helical computed to- mography. j laryngol otol ; ( ): – . . lemmerling m, vanzieleghem b, dhooge i, van cauwenberge p, kunnen m. ct and mri of the semicircular canals in the normal and diseased temporal bone. eur radiol ; ( ): – . . hamamoto m, murakami g, 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gaurano jl, joharjy ia. middle ear cholestea- toma: characteristic ct findings in patients. ann saudi med ; ( ): – . . el-bitar ma, choi ss, emamian sa, vezina lg. congenital middle ear cholesteatoma: need for early recognition—role of computed tomography scan. int j pediatr otorhinolaryngol ; ( ): – . . park k, moon sk, cho mj, won yy, baek mg. d micro-ct images of ossicles destroyed by middle ear cholesteatoma. acta otolaryngol ; ( ): – . . jang ch, wang pc. preoperative evaluation of bone destruction using three-dimensional com- puted tomography in cholesteatoma. j laryngol otol ; ( ): – . . meriot p, veillon f, garcia jf, et al. ct appear- ance of ossicular injuries. radiographics ; ( ): – . . lee d, honrado c, har-el g, goldsmith a. pedi- atric temporal bone fractures. laryngoscope ; ( ): – . s october rg f volume ● special issue r a d io g r a p h ic s this article meets the criteria for . credit hour in category of the ama physician’s recognition award. to obtain credit, see accompanying test at http://www.rsna.org/education/rg_cme.html. rg volume • special issue • october fatterpekar et al role of d ct in the evaluation of the temporal bone girish m. fatterpekar, md, et al page s during initial postprocessing, we observed that any amount of gantry tilt on the ct scanner caused distortion of d ct reformatted images. therefore, d ct scans were obtained with a • gantry tilt and a scanning plane parallel to the inferior orbitomeatal line. in addition, overlapping submillimeter reconstruction of the raw data was performed to obtain the best possible d ct reformatted images. page s the surface area of the tympanic membrane is up to times greater than that of the oval window. thus, the pressure exerted on the tympanic membrane by a sound wave is concentrated through the ossicles onto the much smaller area of the oval window, resulting in a pressure increase and amplification of sound transmission. a lever mechanism that exists between the ossicles further contributes to sound amplification. page s a dilated vestibular aqueduct can be easily recognized at conventional cross-sectional imaging by identifying its abnormal size in relation to the adjacent posterior semicircular canal. however, d multiplanar reformatted (mpr) ct images can more clearly demonstrate the classic funnel-shaped deformity of the dilated vestibular aqueduct, which occurs due to an enlarged endolymphatic sac housed within the dorsal vestibular aqueduct (fig ) ( ). page s recent advances in imaging techniques, such as submillimeter reconstruction and d vr multiplanar imaging, have markedly improved the evaluation of congenital anomalies that cause conductive hearing loss. to obtain the best possible surgical outcome, it is vital for the neuro-otologist or otolaryngologist to appreciate the full extent of congenital anomalies present. it is therefore imperative that the radiologist demonstrate the underlying anomalies in the greatest possible detail, thereby allowing optimal presurgical planning. pages s there are various types of surgeries for the treatment of cholesteatomas, including simple, modified radical, and radical mastoidectomies. the type of surgery performed depends on the extent of disease, which is directly related to the extent of the underlying erosions. therefore, detailed preoperative radiologic assessment of the cholesteatoma is important ( , ). r a d io g r a p h ic s radiographics ; :s –s ● published online . /rg. si ● content codes: wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ [pdf] a mutation in a ganglioside biosynthetic enzyme, st gal , results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /hmg/ddt corpus id: a mutation in a ganglioside biosynthetic enzyme, st gal , results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation. @article{boccuto ami, title={a mutation in a ganglioside biosynthetic enzyme, st gal , results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation.}, author={l. boccuto and k. aoki and h. flanagan-steet and c. chen and xiang fan and f. bartel and marharyta petukh and ayla r pittman and robert saul and alka chaubey and e. alexov and m. tiemeyer and r. steet and c. schwartz}, journal={human molecular genetics}, year={ }, volume={ }, pages={ - } } 	l. boccuto, k. aoki, + authors c. schwartz 	published 	biology, medicine 	human molecular genetics 'salt & pepper' syndrome is an autosomal recessive condition characterized by severe intellectual disability, epilepsy, scoliosis, choreoathetosis, dysmorphic facial features and altered dermal pigmentation. high-density snp array analysis performed on siblings first described with this syndrome detected four shared regions of loss of heterozygosity (loh). whole-exome sequencing narrowed the candidate region to chromosome p . . sanger sequencing confirmed a homozygous c. g>a transition (p… expand view on pubmed academic.oup.com save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all figures, tables, and topics from this paper 	 figure 	 table 	 figure 	 table 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure view all figures & tables 	gangliosides 	neurocutaneous syndromes 	glycosphingolipids 	epilepsy 	n-acetylneuraminic acid 	choreoathetosis 	embryo 	loss of heterozygosity 	cohen syndrome 	anabolism 	zebrafish 	intellectual disability 	transcript 	cell death 	glycolipids 	autosomal recessive inheritance 	pigmentation 	apoptosis 	nerve tissue 	whole exome sequencing 	scoliosis, unspecified 	heterozygote 	morpholinos 	sialyltransferases 	neurogenesis citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency diseases of ganglioside biosynthesis: an expanding group of congenital disorders of glycosylation. 	m. trinchera, r. parini, rossella indellicato, ruben domenighini, f. dall'olio 	medicine 	molecular genetics and metabolism 	 	 save alert research feed st gal -related disorders: a deficiency in ganglioside metabolism and a genetic cause of intellectual disability and choreoathetosis 	e. gordon-lipkin, j. cohen, s. srivastava, b. soares, e. levey, a. fatemi 	medicine 	journal of child neurology 	 	 	highly influenced 	 	view excerpts, cites background and results save alert research feed gm synthase deficiency due to st gal variants in two korean 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behavior marie-therese gast *, anke tönjes *, maria keller , annette horstmann , , nanette steinle , , markus scholz , , ines müller , arno villringer , , michael stumvoll , , peter kovacs & yvonne böttcher department of medicine, university of leipzig, leipzig, germany ifb adiposity diseases, university of leipzig, leipzig, germany department for neurology, max-planck-institute for human cognitive and brain sciences, leipzig, germany division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland diabetes and endocrinology section, veterans administration medical center, baltimore, maryland institute for medical informatics, statistics and epidemiology, university of leipzig, leipzig, germany life research center, university of leipzig, leipzig, germany clinic of cognitive neurology, university of leipzig, leipzig, germany keywords addiction, alcohol intake, food intake, human eating behavior, smoking behavior correspondence yvonne böttcher, medical faculty, ifb adiposity diseases, university of leipzig, liebigstrasse , leipzig, germany. tel: ; fax: ; e-mail: yvonne.boettcher@medizin.uni-leipzig.de funding information this work was supported by grants from the german diabetes association (to y. b., a. t., p. k.) and from the dds foundation to y. b. y. b. and p. k. are funded by the ifb adipositydiseases (adi-k d to y. b., and adi-k e to p. k.); m. k. is funded by adi-k - and i. m. is funded by adi-k - . ifb adipositydiseases is supported by the federal ministry of education and research (bmbf), germany, fkz: eo . m. s. is supported by a grant from the dfg (crc ). m. sch. is funded by the leipzig interdisciplinary research cluster of genetic factors, clinical phenotypes, and environment (life center, university of leipzig). life is funded by means of the european union, by the european regional development fund (erdf), the european social fund (esf), and by means of the free state of saxony within the framework of its excellence initiative. ns is supported by p dk from the nih national institute of diabetes and digestive and kidney diseases. received: september ; revised: may ; accepted: may doi: . /brb . *marie-therese gast and anke t€onjes contributed equally to this work. abstract introduction: the glutamate receptor, metabotropic gene (grm ) encodes a g-protein-coupled glutamate receptor and has been associated with smoking behavior and liability to alcoholism implying a role in addiction vulnerability. data from animal studies suggest that grm may be involved in the regulation of the neuropeptide y and melanocortin pathways and might influence food intake and metabolism. this study aimed to investigate the effects of the genetic variant rs within grm on human eating behavior. methods: the ini- tial analysis included sorbs from germany who have been extensively phe- notyped for metabolic traits and who completed the german version of the three-factor eating questionnaire. in addition, we analyzed two independent sample sets comprising subjects from another german cohort and old order amish individuals. genetic associations with restraint, disinhibition, and hunger were assessed in an additive linear regression model. results: among the sorbs the major g allele of rs was significantly associated with increased restraint scores in eating behavior (p = . � ; b = + . ). the german cohort and the old order amish population revealed a trend in the same direction for restraint (p = . ; b = + . ; p = . ; b = + . ; respectively). a meta-analysis resulted in a combined p = . � (z-score . ). conclusion: our data suggest that rs within grm may influ- ence human eating behavior factors probably via pathways involved in addictive behavior. ª the authors. brain and behavior published by wiley periodicals, inc. this is an open access article under the terms of the creative commons attribution license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. introduction eating behavior has been shown to be a complex trait influ- enced by genetic and psychological factors as well as social and environmental circumstances influencing individual food selection, taste preferences, eating pattern, and eating behavior (steinle et al. ; grimm and steinle ). a genetic contribution to individual eating behavior pheno- types has been demonstrated by heritability estimates ( . , . , and . for restraint, disinhibition, and hunger, respectively) in the old order amish population, a geneti- cally isolated caucasian population of central european dissent (steinle et al. ). numerous candidate gene studies support the role of genetics in eating behavior. for instance, genetic variation in tas r has been signifi- cantly associated with eating behavior disinhibition in old order amish (dotson et al. a) and genetic variation in bitter taste receptors has been reported to influence glucose homeostasis (dotson et al. , b). taste perception is predominantly mediated via g-protein-coupled recep- tors. the glutamate receptor (grm ) is a g-protein- coupled glutamate receptor influencing the inhibition of the cyclic amp cascade as well as regulating the presynaptic glutamate release. genetic variation within grm has been reported to significantly influence risk for diseases affecting the central nervous system including depression (terracci- ano et al. ), autism (li et al. ), schizophrenia (takaki et al. ), and attention deficit hyperactivity syn- drome (elia et al. ). interestingly, electrophysiological studies linked variants within grm to increased risk of vulnerability to alcoholism (rangaswamy and porjesz ; chen et al. ). furthermore, rs within gmr has been identified to be at risk for smoking initiation and suggests that members of the glutamate receptor family may associate with nicotine dependence and vulnerability to addiction (vink et al. ). the neurotransmitter glutamate is involved in substance abuse behavior and may influence food intake (stanley et al. ). a glutamate injection into the lateral hypothalamus has led to a dose- dependent eating response in satiated rats (stanley et al. ). although the hypothesis of “food addiction” is under debate, there are further indications implying that alterations in brain reward pathways are similar to those seen in drug addiction, particularly through effects on the dopaminergic system (johnson and kenny ; pandit et al. ). several studies have shown that mechanisms influencing craving for alcohol and other substances may possibly overlap with processes regulating appetite for food, implying a potential relationship with eating behavior (robinson and berridge ; kelley et al. ; volkow and wise ; volkow et al. , , ). moreover, there are indeed similarities reported for both eating disor- ders and substance abuse (umberg et al. ). in line with this, data from studies in chicks indicate that grm may influence the npy system and melanocortin pathway which may play a role in feeding behavior and metabolism via the hypothalamic pathway (higgins et al. ). taken together, grm might be involved in the control of addic- tion behavior and may play a role in the regulation of eating behavior phenotypes. in the present study we aimed to assess the effects of the genetic variant rs within grm on eating behavior determined by the german version of the three factor eating questionnaire (tfeq) (pudel and wes- tenh€ofer ) in the self-contained population of sorbs (veeramah et al. ), and to replicate the findings in two independent study cohorts. methods subjects sorbs all subjects of the discovery cohort are part of an exten- sively phenotyped self-contained population in eastern germany, the sorbs (b€ottcher et al. ; veeramah et al. ). the phenotyping included a standardized interview for past medical history, family history and eating behavior factors (german version of tfeq, pudel and westenh€ofer ), collection of anthropometric data (weight, height, waist-to-hip ratio, body impedance analy- sis), and a g oral glucose tolerance test (ogtt). moreover, data regarding alcohol intake (glasses per week, . l), smoking behavior (cigarettes per day), and coffee consumption (cups per day) have been recorded. in total, sorbs out of completed the german version of the tfeq. seventy subjects with type diabetes (t d) have been excluded from the study (definition of t d according to ada criteria [ada ]). finally, the study included sorbs ( females; males). mean age was � years and mean body mass index (bmi) . � . kg/m². mean eating behavior scores for the sorbs population are shown in table . the study was approved by the ethics committee of the university of leipzig and all subjects gave written informed consent before taking part in the study. german cohort for replication purposes, we analyzed another sample set from germany comprising healthy volunteers ( female, male). subjects were recruited in leipzig, ª the authors. brain and behavior published by wiley periodicals, inc. genetics of eating behavior m.-t. gast et al. germany, via newspaper announcements, posters in public transportation, and announcements on a local internet- based platform (overweight and obese subjects) or the local participant database of the max planck institute for human and cognitive brain sciences. phenotyping of par- ticipants included the following measurements: anthro- pometric data (bmi, weight, height), age, sex, smoking behavior, eating behavior factors were assessed by the ger- man version of tfeq (pudel and westenh€ofer ). mean age was � years and mean bmi . � . kg/m². mean eating behavior scores for the german cohort are shown in table . the local ethics committee of the uni- versity of leipzig approved the study. old order amish (afds) the amish family diabetes study (afds) is an effort to identify genetic contributors to obesity, diabetes, and cardiovascular diseases. recruitment and phenotyping of afds participants has previously been described (hsueh et al. ). briefly, individuals with a previous diagnosis of t d having an age at diagnosis between and years were recruited, as well as all first and second-degree family members over the age of years of each proband (siblings, parents, offspring, grandparents, grandchildren, aunts, and uncles). phenotyping included anthropometric measures (height, weight, waist circumference, hip circum- ference, and body impedance analysis), a g ogtt (performed only among individuals not known to have a diagnosis of t d), blood pressure, blood chemistry, and lipid profiles. whole blood was collected for dna extrac- tion. to examine the relationship of eating behavior, obes- ity and related traits, and genetics, we administered the tfeq (stunkard and messick ). for the purposes of the current analysis, we analyzed eating behavior scores from individuals ( nondiabetic subjects [ %]) from the afds ( % male). the mean age of men was . � . years and of women . � . years. mean bmi was . � . kg/m for men and . � . kg/m for women. eating behavior scores (restraint . � . ; disinhibition . � . ; hunger . � . ) were positively associated with bmi (steinle et al. ). german version of tfeq we investigated specific factors influencing eating behavior as evaluated by the german version of the tfeq quantify- ing three different eating behavior factors influencing human eating behavior: dietary restraint, disinhibition, and hunger (pudel and westenh€ofer ). we assessed all questions by assigning each item either with a score from to or true–false questions with or . the restraint scale includes questions measuring individual cognitive con-t a b le . m e a n e a ti n g b e h a vi o r sc o re s fo r th e s o rb s, g e rm a n co h o rt , a n d a m is h co h o rt ca te g o ri ze d b y b o d y m a ss in d e x (b m i) . e a ti n g b e h a vi o r fa ct o r s o rb s g e rm a n co h o rt a m is h co h o rt b m i < (n = ) ≤ b m i < (n = ) b m i ≥ (n = ) p - va lu e b m i < (n = ) ≤ b m i < (n = ) b m i ≥ (n = ) p - va lu e b m i < (n = ) ≤ b m i < (n = ) b m i ≥ (n = ) p - va lu e r e st ra in t . � . . � . . � . . . � . . � . . � . . . � . . � . . � . . d is in h ib it io n . � . . � . . � . . . � . . � . . � . . . � . . � . . � . . h u n g e r . � . . � . . � . . . � . . � . . � . . . � . . � . . � . . s o rb s, b m i m is si n g fo r tw o in d iv id u a ls ; h u n g e r sc o re m is si n g fo r o n e in d iv id u a l. p -v a lu e s h a ve b e e n ca lc u la te d b y o n e -w a y a n a ly si s o f va ri a n ce (a n o v a ). ª the authors. brain and behavior published by wiley periodicals, inc. m.-t. gast et al. genetics of eating behavior trol of eating. the eating behavior factor disinhibition rep- resents susceptibility to loose cognitive control by external factors resulting in overeating ( questions). realizing hunger feelings based on physiological signals leading to food intake was covered by questions. genotyping grm variant rs was genotyped using the taqman snp genotyping assay (applied biosystems, inc., foster city, ca). the genotyping reaction was amplified on an abi thermal cycler (applied biosystems inc.; °c for min, and °c for sec, and °c for min, for cycles) and fluorescence was detected on an abi real-time pcr system (applied biosystems inc.). to assess genotyping reproducibility, a random ~ % selection of the samples was re-genotyped in all snps; all genotypes matched initial designated genotypes. for replication of the association signals in the old order amish, we used rs which serves as linkage disequilibrium proxy for rs (r = . based on hapmap release ). the genotypes were extracted from a previously completed gen- ome-wide scan using genechip human mapping k set (affymetrix, santa clara, ca) platform (rampersaud et al. ). there were no deviations from hardy–weinberg equilibrium. statistical analysis prior to statistical analysis, non-normally distributed parameters were logarithmically transformed to approxi- mate normal distribution. genetic associations with restraint, disinhibition and hunger were assessed using lin- ear regression models (data analyzed as continuous vari- ables) using age, gender, bmi, and current smoking as covariates for the sorbs and german cohort. significant effects in the sorbs were adjusted for relatedness structure estimated on the basis of genome-wide snp array data. the amish data were adjusted for age, sex, and relatedness structure. current smoking was defined as follows: current smokers versus ever smokers + never smokers. p-values < . were considered to provide nominal evidence for association. two-sided p-values are reported. statistical analyses were performed using spss statistics version . . (spss, inc., chicago, il). for adjustments regarding relatedness structure in the discovery cohort, we used a mixed-model approach implemented in the genabel package of the statistical software environment r (http:// www.r-project.org, amin et al. ). a meta-analysis was performed using metal (http://www.sph.umich.edu/csg/ abecasis/metal/). study specific p-values and effect direc- tions were converted into a z-statistics and weighted with sample size of each study. results association analysis in sorbs an association analysis for variant rs located in intron of grm was performed in the discovery population. using linear regression analysis and an additive inheritance model the major g allele was significantly associated with higher restraint in eating behavior (adjusted p = . � ) in the sorbs (table ). no significant association could be detected for the eating behavior factors disinhibition and susceptibility to hunger feelings. given a low frequency of the a allele (minor allele frequency [maf . ]) we also included a reces- sive model of inheritance which showed significantly higher restraint values in homozygous g allele carriers (table ). when analyzing data regarding alcohol intake, smoking behavior, and coffee consumption, we detected a higher but nonsignificant intake for each category in homo- zygous g allele carriers (table ). association analysis in german cohort in our second cohort comprising a limited sample set of individuals we observed no significant association (table ). however, we detected the same effect direction between the major g allele as the allele show tendency for higher restraint values (table ). no significant associa- tion was detected for the eating behavior factors disinhi- bition and susceptibility to hunger feelings. association analysis in old order amish despite consistent effect directions with the discovery and the german cohort, no significant association was found between rs and restraint eating behavior in the amish using linear regression models (adjusted p = . , b = + . ; table ). of note, there was a significant asso- ciation of rs with hunger (p = . � , adjusted for age, sex, and family structure, data not shown). meta-analysis including all three study populations a sample size weighted meta-analysis including the results from all three study populations (sorbs, german cohort, and old order amish) resulted in a significant association for restraint (combined p = . � , z-score . , table ). discussion the metabotropic receptor grm has been associated with smoking behavior (vink et al. ) and liability to alcohol- ª the authors. brain and behavior published by wiley periodicals, inc. genetics of eating behavior m.-t. gast et al. t a b le . a ss o ci a ti o n a n a ly si s fo r rs w it h e a ti n g b e h a vi o r fa ct o rs u n d e r lin e a r re g re ss io n a n a ly se s. s o rb s g e rm a n co h o rt a m is h co h o rt g g g a + a a p -v a lu e b s e g g g a + a a p - va lu e b s e t t t c + c c p - va lu e b s e m a le /f e m a le / / / / / / a g e (y e a rs ) � . � . � . � . . � . . � . b m i (k g /m ) . � . . � . . + . . . � . . � . . � . . . � . . � . . � . . w h r . � . . � . . � . . n .a . n .a . n .a . n .a . n .a . . � . . � . . . . r e st ra in t . � . . � . . . + . + . . . . � . . � . . + . . . � . . � . . � . . d is in h ib it io n . � . . � . . + . . . � . . � . . . . . � . . � . . . . h u n g e r . � . . � . . + . . . � . . � . . . . . � . . � . . . . a lc o h o l . � . . � . . + . . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . s m o k in g . � . . � . . + . . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . c o ff e e co n su m p ti o n . � . . � . . + . . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . n .a . d a ta a re p re se n te d a s m e a n � s d . p -v a lu e s fo r e a ti n g b e h a vi o r fa ct o rs w e re ca lc u la te d in a lin e a r re g re ss io n m o d e l w it h a d ju st m e n t fo r a g e , g e n d e r, ln (b m i) (e xc e p t fo r b m i) , cu rr e n t sm o k in g (e xc e p t fo r sm o k in g ), a n d fa m ily st ru ct u re (f o r s o rb s) . p -v a lu e s fo r co n su m e r g o o d s a re a d ju st e d fo r a g e a n d se x. s e , st a n d a rd e rr o r; b m i, b o d y m a ss in d e x; w h r , w a is t- to -h ip ra ti o . a d d it iv e m o d e l o f in h e ri ta n ce . d u e to sm a ll sa m p le si ze o f h o m o zy g o u s m in o r a lle le ca rr ie rs w e a p p lie d re ce ss iv e m o d e l o f in h e ri ta n ce (g /g vs . g /a + a /a a n d t /t vs . c /t + c /c fo r rs a n d rs , re sp e ct iv e ly ). n .a . n o t a va ila b le ; b in d ic a te s e ff e ct si ze a n d d ir e ct io n fo r th e ri sk a lle le (m a jo r a lle le s g o r t ). n u m b e r o f g la ss e s p e r w e e k (g e n e ra l si ze . l) . n u m b e r o f ci g a re tt e s p e r d a y. n u m b e r o f cu p s p e r d a y. ª the authors. brain and behavior published by wiley periodicals, inc. m.-t. gast et al. genetics of eating behavior ism (chen et al. ) implying there may be a role in addic- tion vulnerability. moreover, grm has been identified to be differentially regulated during different nutritional states in chicks (higgins et al. ). it has been shown to be co- regulated in the same gene network with proopiomelanocor- tin gene (pomc), one of the most important genes controlling metabolism, highlighting its potential role in food intake (higgins et al. ). pomc is a central player of the melanocortin system within the arcuate nucleus of the hypothalamus (reviewed in cone ). higgins et al. ( ) described a reduced pomc expression in fasted chicks. the coregulation of grm and pomc suggests that glutamatergic neurotransmission may influence feeding behavior in chicks (higgins et al. ). glutamate itself is involved in addiction and may also influence food intake (stanley et al. ). furthermore, it has been postulated that common hedonic mechanisms may underlie obesity and drug addiction (johnson and kenny ). filbey et al. ( ) reported further indications for a potential overlap of neural mechanisms in addiction and compulsive overeat- ing adding further weight on possibly common regulatory processes. in the present study we therefore hypothesized that rs within grm might influence human eating behavior factors in a similar fashion as seen in smoking behavior. using linear regression models we observed in the sorbs significantly increased restraint scores in individ- uals for the homozygous major g allele for rs . restraint eating is known to be a behavioral trait cogni- tively controlling body weight not only in normal weight individuals but also in obese and overweight subjects. indi- viduals representing restraint eating behavior tend to rigor- ously control, for example, the amount of food intake as well as caloric intake. this may also serve as a counteracting behavior in order to attenuate the effects from frequent dis- inhibited eating. thus, as both restraint and disinhibition are associated with increased body weight the restraint eating periods might fail resulting in disinhibited eating episodes which would ultimately lead to higher bmi (gal- lant et al. ). further, it has to be mentioned that albeit not significant, we detected higher intake of consumer goods such as alcohol, coffee, and cigarette smoking in homozygous g allele carriers in our discovery cohort. our data prompted us to replicate the finding in two other independent cohorts, the german cohort and the old order amish population. we identified in both pop- ulations the same effect direction as in the sorbs but did not reach statistical significance which might most likely be due to low sample size. a weighted meta-analysis of all three cohorts revealed nominal associations of rs with increased restraint scores implying the variant might be involved in restraint eating to some extent. however, our data need to be interpreted with caution and can be viewed as a suggestive indication that rs may play a role in influencing restraint scores, especially in our discovery cohort. further, in the old order amish we found significant associations with susceptibility to recognizing hunger feel- ings. it is noteworthy to point out that the three eating behavior factors restraint, disinhibition, and hunger are not considered to be totally independent from each other and thus rs might be involved in the development of different eating behavior factors influencing individual food intake. moreover, it needs to be mentioned that our study is limited at several aspects. first of all, the sample sizes of our study populations are quite small which may have prevented us from significant replication. second, we can- not rule out that various genetic backgrounds of the stud- ied cohorts, especially the old order amish, may have influenced the heterogeneous outcome of the studies. third, data regarding consumer goods intake are available for the sorbs only. therefore, larger studies are necessary to verify the effects we have detected so far. it is further noteworthy to acknowledge that, especially in the context of potential functionality, rs maps near an additional gene encoding microrna . one might hypothesize that the snp may potentially affect posttranslational modifications of grm via regulating the expression of microrna . however, further studies are warranted to investigate underlying functional mecha- nisms. in conclusion, the present study suggests that rs within grm may influence the regulation of human eating behavior and might potentially be involved in affecting human liability to addiction behavior. table . meta-analysis for association of rs with restraint including sorbs, german cohort, and old order amish. chr effect allele sorbs german cohort old order amish combined p-value b se p-value b se p-value b se p-value z-score direction g . + . . . + . . . + . . . . +++ p-values were calculated based on effect sizes from linear regression model using additive inheritance model. all presented data are adjusted for age, gender, and family structure (for sorbs, amish). se, standard error. meta-analysis is based on effect sizes from linear regression models. ª the authors. brain and behavior published by wiley periodicals, inc. genetics of eating behavior m.-t. gast et al. acknowledgments we thank all those who participated in the studies. this work was supported by grants from the german diabe- tes association (to y. b., a. t., p. k.) and from the dds foundation to y. b. y. b. and p. k. are funded by the ifb adiposity diseases (adi-k d to y. b. and adi-k e to p. k.); m. k. is funded by adi-k - and i. m. is funded by adi-k - . ifb adipositydiseases is supported by the federal ministry of education and research (bmbf), germany, fkz: eo . m. s. is supported by a grant from the dfg (crc ). m. sch. is funded by the leipzig interdisciplinary research cluster of genetic factors, clinical phenotypes, and environment (life center, university of leipzig). life is funded by means of the european union, by the european regional development fund (erdf), the european social fund (esf), and by means of the free state of saxony within the framework of its excellence initiative. ns is supported by p dk from the nih national institute of diabetes and digestive and kidney diseases. conflict of interest none declared. references ada. . ada: standards of medical care in diabetes- . diabetes care :s –s . amin, n., c. m. van duijn, and y. s. aulchenko. . a genomic background based method for association analysis in related individuals plos one :e . b€ottcher, y., h. unbehauen, n. kloting, k. ruschke, a. korner, d. schleinitz, et al. . adipose tissue expression and genetic variants of the bone morphogenetic protein receptor a gene (bmpr a) are associated with human obesity. diabetes : – . chen, a. c., y. tang, m. rangaswamy, j. c. wang, l. almasy, t. foroud, et al. . association of single nucleotide polymorphisms in a glutamate receptor gene (grm ) with theta power 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[pdf] heritability of serum sodium concentration: evidence for sex- and ethnic-specific effects. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /physiolgenomics. . corpus id: heritability of serum sodium concentration: evidence for sex- and ethnic-specific effects. @article{wilmot heritabilityos, title={heritability of serum sodium concentration: evidence for sex- and ethnic-specific effects.}, author={b. wilmot and v. s. voruganti and y. chang and y. fu and z. chen and h. taylor and j. wilson and teresa gipson and v. shah and j. umans and m. flessner and r. hitzemann and a. shuldiner and a. comuzzie and s. mcweeney and p. zager and j. maccluer and s. cole and d. cohen}, journal={physiological genomics}, year={ }, volume={ }, pages={ - } } 	b. wilmot, v. s. voruganti, + authors d. cohen 	published 	biology, medicine 	physiological genomics serum sodium concentration is the clinical index of systemic water balance. although disordered water balance is common and morbid, little is known about genetic effects on serum sodium concentration at the population level. prior studies addressed only participants of european descent and either failed to demonstrate significant heritability or showed only modest effect. we investigated heritability of serum sodium concentration in large cohorts reflecting a range of races/ethnicities… expand view on pubmed europepmc.org save to library create alert cite launch research feed share this paper citationsbackground citations results citations view all tables and topics from this paper 	 table 	 table 	 table 	glucose 	sodium, dietary 	serum sodium measurement 	estimated glomerular filtration rate 	diuretics 	exclusion 	stratification 	legg-calve-perthes disease 	fetal hydantoin syndrome 	sample variance 	heredity aspects citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency heritability and individuality of the plasma sodium concentration: a twin study in the united states veteran population. 	andrew k timmons, a. korpak, + authors d. cohen 	biology, medicine 	american journal of physiology. renal physiology 	 	 save alert research feed a population-based approach to assess the heritability and distribution of renal handling of electrolytes. 	f. moulin, b. ponte, + authors m. bochud 	biology, medicine 	kidney international 	 	 save alert research feed clinical and neurohumoral associates of variation in plasma sodium in the prevend cohort. 	niek r hessels, j. j. o. n. van den bosch, m. van londen, s. bakker, i. j. riphagen, g. navis 	chemistry, medicine 	american journal of physiology. renal physiology 	 	 save alert research feed functional polymorphisms affecting the clinically important arginine- residue of avpr do not influence serum sodium concentration at the population level. 	y. fu, t. cheetham, d. bourn, e. orwoll, d. cohen 	biology, medicine 	physiological genomics 	 	 	pdf save alert research feed individuality of the plasma sodium concentration. 	zheng zhang, j. duckart, + authors d. cohen 	chemistry, medicine 	american journal of physiology. renal physiology 	 	 	pdf 	view excerpts, cites background and results save alert research feed nfat and slc a loci associate with plasma osmolality 	c. böger, m. gorski, + authors b. kestenbaum 	 	pdf save alert research feed nfat and slc a loci associate with plasma osmolality. 	c. böger, m. gorski, + authors david m. cohen 	biology, medicine 	journal of the american society of nephrology : jasn 	 	 	pdf save alert research feed new insights into the determinants of serum na+ and the risk for dysnatremias. 	m. rosner 	medicine 	american journal of physiology. renal physiology 	 	 	pdf 	view excerpt, cites background save alert research feed genetics of cardiovascular disease in minority populations 	j. maccluer, j. blangero, a. comuzzie, s. ebbesson, b. howard, s. cole 	biology 	 save alert research feed potassium and its discontents: new insight, new treatments. 	d. ellison, a. terker, g. gamba 	chemistry, medicine 	journal of the american society of nephrology : jasn 	 	 	pdf save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency linkage analysis of albuminuria. 	a. mottl, s. vupputuri, + authors k. north 	medicine 	journal of the american society of nephrology : jasn 	 	 	pdf save alert research feed common genetic variants associate with serum phosphorus concentration. 	b. kestenbaum, n. glazer, + authors c. fox 	biology, medicine 	journal of the american society of nephrology : jasn 	 	 	pdf save alert research feed heritabilities for fifteen routine biochemical values: findings in swedish twin pairs years of age or older 	s. nilsson, s. read, s. berg, b. johansson 	chemistry, medicine 	scandinavian journal of clinical and laboratory investigation 	 	 save alert research feed genome-wide association studies of serum magnesium, potassium, and sodium concentrations identify six loci influencing serum magnesium levels 	t. meyer, g. c. verwoert, + authors a. köttgen 	biology, medicine 	plos genetics 	 	 	pdf 	view excerpts, references results save alert research feed genetic and environmental contributions to cardiovascular disease risk in american indians: the strong heart family study. 	k. north, b. howard, + authors j. maccluer 	medicine 	american journal of epidemiology 	 	 	pdf save alert research feed the effects of inheritance on constituents of plasma : a twin study on some biochemical variables 	j. fie, n. ti 	 	pdf save alert research feed heritability of measures of kidney disease among zuni indians: the zuni kidney project. 	j. maccluer, m. scavini, + authors p. zager 	medicine 	american journal of kidney diseases : the official journal of the national kidney foundation 	 	 	 	view excerpts, references background save alert research feed heritability of biochemical kidney markers and relation to survival in the elderly--results from a danish population-based twin study. 	l. bathum, c. fagnani, l. christiansen, k. christensen 	biology, medicine 	clinica chimica acta; international journal of clinical chemistry 	 	 	view excerpts, references results save alert research feed biological, clinical, and population relevance of loci for blood lipids 	tanya m. teslovich, k. musunuru, + authors s. kathiresan 	biology, medicine 	nature 	 	 , 	pdf save alert research feed toward resolution of cardiovascular health disparities in african americans: design and methods of the jackson heart study. 	h. taylor, j. wilson, + authors s. wyatt 	medicine 	ethnicity & disease 	 	 	pdf save alert research feed ... ... related papers 	abstract 	tables and topics 	 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and type diabetes in pima indians lijun ma, robert l. hanson, lorem n. que, anna m.g. cali, mao fu, janel l. mack, aniello m. infante, sayuko kobes, the international type diabetes q consortium, clifton bogardus, alan r. shuldiner, and leslie j. baier a prior genome-wide linkage scan in pima indians indi- cated a young-onset (aged < years) type diabetes susceptibility locus on chromosome q -q . arhgef , which encodes the rho guanine nucleotide exchange factor , was analyzed as a positional candidate gene for this linkage because this protein may stimulate rho-dependent signals, such as the insulin signaling cascade. the arh- gef gene, and two adjacent genes ntrk and insrr, were sequenced in pima indians who were not first- degree relatives. sequencing of the coding regions, � and � untranslated regions and putative promoter regions of these genes, identified variants in arhgef , vari- ants in ntrk , and variants in inssr. these variants, as well as additional public database variants within/ between these genes, were genotyped for association anal- ysis in the same group of pima indians who had participated in the linkage study (n � , ). an r h in arhgef , and several additional noncoding variants that were in high linkage disequilibrium with this variant, were nominally associated with young-onset type diabe- tes (p � . ; odds ratio . ) after adjusting for sex, family membership, and pima heritage. the risk allele h had a frequency of . . in a subgroup of nondiabetic, full-heritage pima indians who had undergone detailed metabolic testing, the risk allele h also was associated with a lower mean insulin-mediated glucose disposal rate and a lower mean nonoxidative glucose storage rate after adjust- ing for age, sex, nuclear family membership, and percent- age of body fat (p < . ). these findings suggest that variation within arhgef nominally increases risk of type diabetes, possibly as a result of increased insulin resistance. diabetes : – , t he pima indians of arizona have an extremely high prevalence of type diabetes ( ). their diabetes is characterized by obesity, dysfunction of insulin secretion, insulin resistance (de- creased insulin-mediated glucose disposal), and increased rates of postabsorptive endogenous glucose output ( ). studies ( – ) have shown that type diabetes, insulin resistance, the acute insulin response, and obesity are highly heritable in this population. a prior genome-wide linkage scan in pima indians indicated a susceptibility locus for young-onset (onset age � years) type diabetes on chromosome q -q at d s ( ). linkage to type diabetes on chromosome q – has subsequently been observed in seven other populations who have formed the international type diabetes q consortium ( – ). within this region of linkage, there is a cluster of three genes (arhgef , ntrk , and insrr) located between and mb, which encode proteins that have putative roles in the insulin signaling system. arhgef encodes the rho guanine nucleotide exchange factor arhgef (also called pdz-rhogef and kiaa ). arhgef interacts with small gtpases (g-protein, guanine nucleotide-bind- ing proteins), such as rho, that function as molecular switches in signaling pathways that include the insulin signaling cascade ( , ). ntrk encodes the neurotro- phic tyrosine kinase receptor type , which recruits insulin receptor substrate (irs)- and irs- ( ), and insrr encodes the insulin receptor–related receptor, which po- tentially phosphorylates irs- and irs- (available at http://genecards.bcgsc.bc.ca/cgi-bin/carddisp?insrr). in this study, arhgef and the two adjacent genes, ntrk and insrr, were analyzed as positional candidate genes for type diabetes in the pima indians. research design and methods the subjects who were sequenced (n � ) and genotyped (n � , ) were participants in our prior genome-wide linkage study for diabetes susceptibility loci in pima indians ( ) and are part of our ongoing longitudinal study of the etiology of type diabetes among the gila river indian community in central arizona ( ). all individuals in the longitudinal study are invited to participate in a standardized health examination biannually. to determine diabetes from the diabetes molecular genetics section, phoenix epidemiology and clinical research branch, national institute of diabetes and digestive and kidney diseases, national institutes of health, department of health and human services, phoenix, arizona; and the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland. address correspondence and reprint requests to leslie j. baier, phd, diabetes molecular genetics section, phoenix epidemiology and clinical research branch, national institute of diabetes and digestive and kidney diseases, national institutes of health, n. fifth st., suite , phoenix, az . e-mail: lbaier@phx.niddk.nih.gov. received for publication may and accepted in revised form january . published ahead of print at http://diabetes.diabetesjournals.org on jan- uary . doi: . /db - . additional information for this article can be found in an online appendix at http://dx.doi.org/ . /db - . irs, insulin receptor substrate; ld, linkage disequilibrium; snp, single nucleotide polymorphism. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , may status, a -g orally administered glucose tolerance test is given and the results are interpreted according to the criteria of the world health organi- zation ( ). all studies were approved by the tribal council of the gila river indian council and the institutional review board of the national institutes of diabetes and digestive and kidney diseases. among , subjects that were genotyped, of the nondiabetic subjects had additionally been studied for measures of pre-diabetic phenotypes in our clinical research center. only individuals (aged – years) who are con- firmed to be healthy by medical history, physical examination, and routine laboratory tests and are not taking medications are studied. oral glucose tolerance is measured after – days on a weight-maintaining diet of mixed composition. blood for plasma glucose and insulin measurements is drawn before ingesting g of glucose and at , , , and min thereafter. subjects also receive a -g intravenous injection of glucose over min to measure the acute insulin response. blood samples were collected before infusion and at , , , , , and min after infusion for determination of plasma glucose and insulin concentrations. the acute insulin response was calculated as the mean increment in plasma insulin concentrations from to min ( ). the hyperinsulinemic-euglycemic clamp technique was used to determine basal glucose appearance and insulin-stimulated glucose disappear- ance (uptake) rates ( ). briefly, insulin was infused to achieve physiologic plasma insulin concentrations ( � �u/ml) for min. plasma glucose concentrations were held constant at mg/dl by a variable % glucose infusion. tritiated glucose was infused for h before the insulin infusion to calculate glucose disappearance rates during the insulin infusion. during the last min of the insulin infusion, the rate of insulin-stimulated glucose disposal was calculated, adjusted for steady-state plasma glucose and insulin concentration, and normalized to estimated metabolic body size (fat-free mass plus . kg) as described ( , ). ventilated-hood indirect calorimetry was used to estimate rates of glucose and lipid oxidation before and during the insulin infusions ( ). glucose and lipid oxidation rates were normalized to estimated metabolic body size (fat-free mass plus . ) as described ( , ). body composition was estimated by underwater weighing until january and currently is measured by dual-energy x-ray absorptiometry (dpx- ; lunar radiation, madison, wi). a conversion equation derived from comparative analyses is used to make estimates of body composition comparable between methods ( ). single nucleotide polymorphism identification and genotyping. to iden- tify sequence variants, all exons, exon-intron boundaries, � and � untrans- lated regions, and kb of the putative promoter regions of arhgef , ntrk , and insrr were pcr amplified and sequenced in dna samples from non–first-degree–related pima indians. among subjects, developed diabetes before the age of years and were nondiabetic and were at least years of age. sequencing was performed using the big dye terminator (applied biosystems) on an automated dna capillary sequencer (model xl; applied biosystems). single nucleotide polymorphisms (snps) iden- tified by sequencing were genotyped in dna from , subjects using the taqman allelic discrimination assay (applied biosystems), direct sequencing (as described above; applied biosystems), or snpplex (applied biosystems) following the manufacture’s protocol. sequence information for all oligonu- cleotide primers and probes is available from the authors upon request. statistical analysis. for the association analysis with young-onset type diabetes, only siblings who were confirmed to have developed type diabetes before the age of years (n � ) or confirmed to be nondiabetic and at least years of age (n � ) were analyzed. statistical analyses were performed using the software of the sas institute (cary, nc). numeric variables are expressed as means � se. the association of genotypes with diabetes was assessed by logistic regression analysis. for continuous vari- ables, linear regression analysis was used. both linear and logistic models were fit with the general estimating equation procedure because some subjects were siblings. this procedure accounts for family membership by modeling the phenotypic variance/covariance matrix among siblings. for the present analyses, an exchangeable correlation matrix was assumed and the “empirical” se, which is robust to the assumed correlation structure, was used. plasma insulin concentrations and glucose disposal rate during the physi- ological plasma concentration were log transformed before analyses to approximate a normal distribution. for analysis under an additive model, homozygotes for the major allele ( / ) and heterozygotes ( / ) and homozy- gotes for the minor allele ( / ) were coded to a continuous numeric variable for genotype (as , , and ). the recessive model was defined as contrasting genotypic groups / vs. / � / , where allele is defined as the major allele. although the general estimating equation approach accounts for the correla- tion among family members, it does not provide a specific within-family test of association and, thus, still is potentially confounded by population strati- fication. therefore, data also were analyzed using a modification of the method of abecasis et al. ( ) to test for within-family association. in brief, this method partitions the association into between- and within-family com- ponents, represented, respectively, by the sibship mean of the continuous numeric variable for genotype and each individual’s deviation from this mean. the test of the significance of the within-family components is a test of cotransmission among siblings, which is robust to population stratification (although it is less powerful than the more general test). analyses of all traits were adjusted for potentially confounding variables (e.g., age, sex, percentage of body fat); all adjustments were specified a priori based on previous studies of the determinants of these traits. to examine pairwise linkage disequilib- rium (ld), haplotype frequencies were estimated with the estimating haplo- type (eh) program (xie and ott) (available at http://linkage.rockefeller.edu/ ott/), and these haplotype frequencies were used to calculate d� and � ( ). p values of � . were considered to be of nominal statistical significance. results sequencing and genotyping of arhgef . sequenc- ing of the exons, exon-intron boundary regions, and kb of the � (putative promoter) region of arhgef in non–first-degree–related pima indians identified variants, of which were nonsynonymous amino acid substitutions (r q, s g, and r h). these variants, and additional database snps positioned outside of the regions that were sequenced, were geno- typed for association analysis in the same pima indian subjects who were used for our prior type diabetes linkage study (online appendix table [available at http:// dx.doi.org/ . /db - ]). three coding variants in arhgef were nominally associated with young-onset type diabetes. the h allele (frequency � . ) of r h was associated with young-onset type diabetes (p � . ; odds ratio . [ % ci . – . ], recessive model) after adjusting for sex, family membership, and pima heritage (fig. ; table ). the g allele (frequency � . ) of s g also was associated with young-onset type diabetes (p � . , . [ . – . ], additive model; p � . , . [ . – . ], recessive model) after adjusting for sex, fam- ily membership, and pima heritage (fig. ; table ). the q allele of r q was extremely rare (frequency � . ), and although the association data for this variant are included in table , these data may not be reliable due to the limited sample power. thirteen noncoding snps also were nominally associated with young-onset type diabe- tes (listed in table legend). all of these noncoding snps, with the exception of rs (shown in table ), were in high ld (d� � . , r � . ) with either r h or s g (online appendix fig. ). we further assessed whether the r h and s g were associated with metabolic traits predictive of type diabetes in pima indians who had not yet developed diabetes. in a subgroup of nondiabetic, full-heritage pima indians who have undergone detailed metabolic testing, including measurements of body composition, oral glucose tolerance, insulin secretory function, insulin-stim- ulated glucose uptake, and indirect calorimetry, the risk allele (h) for r h was nominally associated with a lower mean glucose disposal rate (p � . ) and a lower mean nonoxidative glucose storage rate (p � . ) during a euglycemic-hyperinsulinemic clamp under physiologic concentrations of plasma insulin infusion, after adjusting for age, sex, nuclear family membership, and percentage of body fat (table ; fig. ). the r h was not associ- ated with insulin secretion as assessed by the acute insulin response to a -g intravenous bolus injection of glucose or the insulin level at min during an oral glucose tolerance test (table ). no association was found be- tween s g and metabolic predictors of type diabetes l. ma and associates diabetes, vol. , may in nondiabetic, full-heritage pima subjects (data not shown). sequencing and genotyping of ntrk and insrr. direct sequencing of all exons, exon-intron boundary regions of ntrk and insrr, and . kb of the common � flanking regulatory region between these two genes in non–first-degree–related pima indians identified variants ( in ntrk and in insrr; online appendix table ). four of these variants predicted nonsynonymous amino acid changes (phe leu, arg leu, pro leu, and arg stop) located in insrr. none of these coding variants were detected when insrr previously was screened in pima indians using the technique of denatur- ing high-performance liquid chromatography on pooled dna samples ( ). the variants, and additional database snps positioned outside of the regions that were sequenced, were genotyped for association analysis in the same pima indian subjects who were used for the type diabetes linkage study. none of the amino acid changes were associated with young-onset type diabetes or metabolic predictors of type diabetes in pima indians. only snp rs , located in the shared common � flanking regulatory region ( . kb) between insrr and ntrk , showed a nominal association with young-onset fig. . association plot of the �log p values (additive model) for snps with young-onset type diabetes versus position along a -kb region of chromosome . the relative positions of exons for insrr, ntrk , and arhgef within this region are shown beneath the plot. table representative* snps associated with young-onset type diabetes in pima indians snp snp ( / ) gene locus minor allele frequency type diabetes by genotype (n)† nondiabetic by genotype (n)‡ association in general (upper) and within-family (lower, italics) models � � p value odds ratio rs c/t insrr/ntrk . . . ( . – . ) intergenic . . ( . – . ) r h g/a arhgef . . § . ( . – . ) exon . § . ( . – . ) s g a/g arhgef . . . ( . – . ) exon . . ( . – . ) r q g/a arhgef . . . ( . – . ) exon na na snp alleles are given as � major and � minor. the risk allele is given in bold. due to the low frequencies of these snps, only the recessive analytical model is given, which is defined as comparing genotype groups / vs. / � / . p values/odds ratios were adjusted for sex, family membership, and pima heritage. odds ratios were calculated by defining allele as the defaulted risk factor. *r h is in nearly perfect ld (d� � . , r � . ) with rs , rs , rs , rs , rs , rs , rs , rs , hcv , and rs . s g is in nearly perfect ld with rs and rs . †subjects with type diabetes diagnosed before the age of years. ‡subjects determined to be nondiabetic by oral glucose tolerance test after the age of years. §the empirical p values obtained with , permutations were . for the general association test and p � . for the within-family test, respectively, which were very similar to the nominal values. arhgef , type diabetes, and pima indians diabetes, vol. , may type diabetes (p � . ; table ; fig. ), but this variant was not associated with any metabolic predictors of type diabetes in nondiabetic, full-heritage pima indians (data not shown). the variants detected in insrr and ntrk are in a distinct ld block compared with variants in arhgef in pima indians (online appendix fig. ). haplotype analyses for arhgef and insrr. to detect an effect of multiple, potentially functional snps on the development of type diabetes, haplotype analyses were done by combining the arg his and ser gly in arhgef and the arg stop and pro leu in insrr. however, neither haplotype analysis provided additional information to the single snp analyses (table ). the arg gln in argef and the phe leu and arg leu in insrr were not included in the haplotypes due to their extremely rare allele frequencies. discussion our data suggest that the h allele at r h in arhgef (or a variant that is carried on the h allele in pima indians) increases risk of type diabetes by reducing insulin- stimulated glucose uptake. among the nondiabetic, full- heritage pima indians, subjects with an h allele (homozygotes and heterozygotes) had a lower mean glu- cose disposal rate during a hyperinsulinemic-euglycemic clamp after adjusting for age, sex, nuclear family member- ship, and percentage of body fat. this lower glucose disposal rate mainly was attributed to reduced nonoxida- tive glucose storage because the other component of glucose disposal rate, glucose oxidation, was similar be- tween subjects with an h allele and subjects who were homozygous for the r allele. these findings indicate that arhgef may alter insulin-mediated glucose storage pathways, such as glycogen synthesis in the muscle and liver. arhgef is ubiquitously expressed and is present in both liver and muscle, which are important organs for fig. . comparison of glucose disposal rates in nondiabetic, full- heritage pima indians grouped by r h genotypes. insulin-mediated glucose disposal rate is measured during a euglycemic-hyperinsuline- mic clamp during infusion of physiological concentrations of plasma insulin and is composed of nonoxidative glucose storage and glucose oxidation. f, glucose oxidation; u, nonoxidative glucose storage. table mean values for insulin action and insulin secretion among nondiabetic, full-heritage pima indians grouped by arhgef r h genotypes r r r h p value* n (male/female) ( / ) ( / ) . age (years) � . � . — percentage of body fat � . � . . glucose disposal rate (mg � min� � kg embs� ) . � . . � . . nonoxidative glucose storage (mg � min� � kg embs� ) . � . . � . . glucose oxidation (mg � min� � kg embs� ) . � . . � . . oral glucose tolerance test fasting glucose (mg/dl) � . � . . -h glucose (mg/dl) � . � . . log 	fasting insulin (miu/ml) . � . . � . . log 	 -h insulin (miu/ml) . � . . � . . log 	acute insulin response (miu/ml) (n � normal glucose tolerance) . � . . � . . log 	insulin � during oral glucose tolerance test (miu/ml) (n � normal glucose tolerance) . � . . � . . data are means � se. among this subgroup there were no subjects homozygous for h h. glucose disposal rate is measured as insulin-stimulated glucose uptake during a hyperinsulinemic-euglycemic clamp at the physiologic plasma insulin concentrations. estimated metabolic body size (embs) equals fat-free mass plus . kg. *p value for sex distribution between two groups was calculated by � . p value for percentage of body fat was assessed using the general estimating equation procedure after adjusting for age, sex, and family membership. p values for glucose disposal rate, oral glucose tolerance glucose, and insulin levels were obtained using the general estimating equation procedure after adjusting for age, sex, family membership, and percentage of body fat. p values for log-transformed acute insulin response was assessed in normal glucose-tolerant full-heritage pima subjects after adjusting for age, sex, percentage of body fat, family membership, and insulin action. p values for log-transformed insulin level at min during an oral glucose tolerance test were obtained after adjusting for age, sex, percentage of body fat, family membership, insulin action, and glucose level at min during oral glucose tolerance tests. l. ma and associates diabetes, vol. , may insulin-mediated glucose metabolism ( ). arhgef is a member of a subfamily of rhogefs that contain regulator of g-protein signaling domains. larg (leukemia-associ- ated rho guanine nucleotide exchange factor; also called arhgef ) also is a member of this regulator of g- protein signaling subfamily. we previously studied larg as a candidate gene for type diabetes and identified a functional tyr cys variant that also was associated with reduced insulin-stimulated glucose uptake among nondiabetic pima indians ( ). although nominally significant associations were ob- served in our analysis of arhgef , it should be cau- tioned that multiple comparisons inherent to association studies can lead to the discovery of false-positives, and for any given variant the prior probability of a true association is probably low. for these two reasons, particularly strin- gent p value thresholds for declaring a significant associ- ation have been proposed ( , ). the p values given in this study are unadjusted, and, if corrected for the variants studied (which could be argued to be an overcor- rection due to high ld among many of the variants), the p values of � . certainly would not be significant (p � � . ). yet, the performance of a correction for multiple comparison proceeds on the assumption that the “global” null hypothesis (no association with any of the variants) is of interest, and it is not clear that this is appropriate for a physiologic candidate gene in a region with replicated evidence for linkage. in this context, its rank relative to other snps genotyped as part of our fine-mapping efforts across this region of linkage, along with potential func- tional consequences of the variant, may provide more relevant information about the importance of this gene. the p value of . for r h puts this variant within the top . % of the 
 , variants genotyped to date in pima indians across a -mb region on chromosome q. the r h also is located in the cooh-terminal region of arhgef protein, which has been shown to regulate arhgef protein activity ( ). replication of associations in other populations also can provide evidence that associations are not spurious. the region spanning arhgef , ntrk , and insrr also has been sequenced and densely genotyped in the amish population, where variants within arhgef also were found to be associated with type diabetes and oral glucose levels in response to an oral glucose tolerance test. however, the specific variants in arhgef demon- strating the best associations differed between the amish and the pima populations ( ). the chromosomal region spanning arhgef also has been genotyped at a � -kb density as part of the fine-mapping efforts of the interna- tional type diabetes q consortium ( ), but among the database variants (which did not include r h) genotyped in the consortium’s multiethnic subjects (n � , ) there was only weak evidence for an association with type diabetes (lowest p value among snps � . ). therefore, we do not propose that the r h in arhgef explains the linkage to type diabetes ob- served in multiple populations on chromosome q – . however, the r h, or another variant carried on the h allele of arhgef , may have played a subtle role in our ability to detect linkage in this region in pima indians. adjustment of the original linkage peak, centered in d s , for the effect of r h results in a % reduc- tion in the evidence for linkage (logarithm of odds drops from . to . ), which is among the top % of the linkage reductions observed among 
 , snps that have been analyzed, to date, within this region in pima indians. in summary, we propose that arhgef may have a minor, or modifying, role in influencing susceptibility to type diabetes via its effect upon insulin action in pima indians. however, due to the relatively low frequency of risk allele h versus the high prevalence of type diabetes in pima indians and the fact that this gene shows little evidence for association in other populations with linkage to type diabetes on chromosome q – , except for the amish, we speculate that there are additional type diabetes susceptibility genes, with larger and more univer- sal effects, on chromosome q -q . acknowledgments this study was supported by the intramural research program of the national institute of diabetes and diges- tive and kidney diseaes, national institutes of health (nih). funding was also provided by nih grant r dk . l.m. was supported by a mentor grant from the american diabetes association. references . knowler wc, bennett ph, hamman rf, miller m: diabetes incidence and prevalence in pima indians: a -fold greater incidence than in rochester, minnesota. am j epidemiol : – , . bogardus c: metabolic abnormalities in the development of non-insulin dependent diabetes mellitus. in diabetes mellitus: a fundamental and clinical text. leroith d, taylor si, olefsky jm, eds. philadelphia, lippincott-raven, , p. – . baier lj, hanson rl: genetic studies of the etiology of type diabetes in pima indians: hunting for pieces to a complicated puzzle. diabetes : – , . ravussin e, bogardus c: energy expenditure in the obese: is there a thrifty gene? infusionstherapie : – , . sakul h, pratley r, cardon l, ravussin e, mott d, bogardus c: familiality of physical and metabolic characteristics that predict the development of table haplotype analyses for ahrgef (arg his and ser gly) and insrr (arg stop and pro leu) haplotype frequency (type diabetes)* frequency (non–type diabetes)† odds ratio ( % ci) (additive model) p value (additive model) corrected p value r- s . . . 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report of a who study group. geneva, world health org., (tech. rep. ser. no. ). . lillioja s, mott dm, spraul m, ferraro r, foley je, ravussin e, knowler wc, bennett ph, bogardus c: insulin resistance and insulin secretory dysfunction as precursors of non-insulin-dependent diabetes mellitus: prospective studies of pima indians. n engl j med : – , . lillioja s, mott dm, howard bv, bennett ph, yki-jarvinen h, freymond d, nyomba bl, zurlo f, swinburn b, bogardus c: impaired glucose tolerance as a disorder of insulin action: longitudinal and cross-sectional studies in pima indians. n engl j med : – , . norman ra, tataranni pa, pratley r, thompson db, hanson rl, prochazka m, baier l, ehm mg, sakul h, foroud t, garvey wt, burns d, knowler wc, bennett ph, bogardus c, ravussin e: autosomal genomic scan for loci linked to obesity and energy metabolism in pima indians. am j hum genet : – , . abecasis gr, cardon lr, cookson woc: a general test of association for quantitative traits in nuclear families. am j hum genet : – , . wolford jk, thameem f, bogardus c, prochazka m: polymorphism screening of the insulin receptor-related receptor gene (insrr) on q in pima indians. mol cell probes : – , . hirotani m, ohoka y, yamamoto t, nirasawa h, furuyama t, kogo m, matsuya t, inagaki s: interaction of plexin-b with pdz domain-containing rho guanine nucleotide exchange factors. biochem biophys res commun : – , . colhoun hm, mckeigue pm, smith gd: problems of reporting genetic associations with complex outcomes. lancet : – , . risch n, merikangas k: the future of genetic studies of complex human diseases. science : – , . chikumi h, barac a, behbahani b, gao y, teramoto h, zheng y, gutkind js: homo- and hetero-oligomerization of pdz-rhogef, larg and p rhogef by their c-terminal region regulates their in vivo rho gef activity and transforming potential. oncogene : – , . fu m, sabra mm, damcott c, pollin ti, ma l, ott s, shelton jc, shi x, reinhart l, o’connell j, mitchell bd, baier lj, shuldiner ar: evidence that rho guanine nucleotide exchange factor (arhgef ) on q is a type diabetes susceptibility gene in the old order amish. diabetes : – , . zeggini e, damcott cm, hansonrl, karim ma, rayner nw, groves cj, baier lj, hale tc, hattersley at, hitman ga, hunt se, knowler wc, mitchell bd, ng mcy, o’connell jr, pollin ti, vaxillaire m, walker m, wang x, whittaker p, xiang k, jia w, chan jcn, froguel p, deloukas p, shuldiner ar, elbein sc, mccarthy mi, the international type diabetes q consortium: variation within the gene encoding the upstream stimula- tory factor does not influence susceptibility to type diabetes in samples from populations with replicated evidence of linkage to chromosome q. diabetes : – , l. ma and associates diabetes, vol. , may doi: . /j.bbabio. . . p/ import and assembly of mitochondrial proteins nikolaus pfanner institute for biochemistry and molecular biology, university of freiburg, germany e-mail: nikolaus.pfanner@biochemie.uni-freiburg.de mitochondria contain about different proteins. % of the proteins are synthesized as precursors on cytosolic ribosomes. the precursors are imported via the translocase of the outer mitochon- drial membrane (tom complex) and are subsequently sorted into the four mitochondrial subcompartments, outer membrane, inter- membrane space, inner membrane and matrix. (i) cleavable preproteins are transported from the tom complex to the presequence translocase of the inner membrane (tim complex). the presequence translocase-associated motor (pam) drives trans- location into the matrix. (ii) hydrophobic inner membrane proteins are transferred through the intermembrane space by a chaperone complex (small tim proteins) and inserted into the inner membrane by the tim complex. (iii) the mitochondrial import and assembly machinery (mia) directs small proteins into the intermembrane space and promotes the formation of disulfide bonds. (iv) beta- barrel proteins are transported from the tom complex to the sorting and assembly machinery of the outer membrane (sam complex). doi: . /j.bbabio. . . p/ new functions for novel mitochondrial transporters ferdinando palmieri university of bari, italy e-mail: fpalm@farmbiol.uniba.it a strikingly large number of mitochondrial dna (mtdna) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. these mitochondrial dis- orders were the first to be investigated after the small mtdna had been sequenced in the 's. only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, hhh syndrome, aspartate/glutamate isoform deficiency, amish microce- phaly and neonatal myoclonic epilepsy; these disorders are characterised by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabo- lism. defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phos- phate and adp, are responsible for diseases characterised by defective energy production. herein, all the mitochondrial carrier- associated diseases known to date are reviewed for the first time. particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders. doi: . /j.bbabio. . . p/ the water oxidizing enzyme a. william rutherford ibitec-s, ura cnrs, cea saclay, gif-sur-yvette, france e-mail: alfred.rutherford@cea.fr photosystem ii, the water oxidising enzyme of photosynthesis, put the energy (or at least a major fraction of it) into the biosphere and the oxygen into the atmosphere. it is certainly one the most influential and important enzymes on the planet. the aim of our research is to understand how this enzyme works as ) a solar energy converter and ) the only known thermodynamically efficient catalyst for oxidizing water. the information obtained is used in the design of artificial catalysts and photocatalysts. a chemical catalyst that has the thermodynamic efficiency of the enzyme could greatly improve the efficiency of ) water electrolysis and photolysis for fuel (e.g. h ) production and ) the reverse reaction, oxygen reduction, in fuel cells. there is therefore a great interest in understanding the mechanism of this enzyme and in reproducing aspects of its function in artificial systems. i will describe our current knowledge of photosystem ii, including some recent experimental studies, as well as recent efforts in our joint saclay/orsay program aimed at producing bio-inspired water oxidizing catalysts. doi: . /j.bbabio. . . p/ the structure of purple bacterial antenna complexes: from single molecules to native membranes richard j. cogdella, alastair t. gardinera, mads gabrielsena, aleks w. roszaka, june southalla, tatas brotosudarmoa, neil w. isaccsa, hideki hashimotob, juergen baierc, silke oellerichc, martin richterc, juergen koehlerc, francesco franciad, giovanni venturolid, dieter oesterhelte adivision of biochemistry and molecular biology, ibls and department of chemistry, university of glasgow, glasgow g qq, uk bcrest-jst and department of physics, graduate school of science, osaka city university, - - sugimoto, sumiyoshi-ku, osaka - , japan cexperimental physics iv, university of bayreuth, d- bayreuth, germany ddepartment of biology, university of bologna, bologna, italy edepartment of membrane biochemistry, max-planck institute for biochemistry, martinsried, germany e-mail: r.cogdell@bio.gla.ac.uk the photosynthetic unit of purple photosynthetic bacteria typically contains two types of light-harvesting complexes, called lh and lh . these antenna complexes are constructed on a modular principle. they are circular or elliptical oligomers of dimers of two low-molecular weight, hydrophobic apoproteins, called a and b, that bind bacteriochlorophylls and carotenoids non-covalently. the lh complex surrounds the reaction centre and, depending on the species, is either a monomer or a dimer. the lh complexes are arranged around the lh -rc complexes. this plenary lecture will present the current status of structural studies on these pigment- protein complexes, based upon a combination of x-ray crystal- lography and single molecule spectroscopy. then an overall view of how they are arranged in their native photosynthetic membranes will be presented. doi: . /j.bbabio. . . p/ catalysis of substrate conversion and electron transfer by mitochondrial complex i judy hirst medical research council dunn human nutrition unit, cambridge, cb xy, uk e-mail: jh@mrc-dunn.cam.ac.uk s abstracts / biochimica et biophysica acta ( ) s –s http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . mailto:nikolaus.pfanner@biochemie.uni-freiburg.de mailto:fpalm@farmbiol.uniba.it mailto:alfred.rutherford@cea.fr mailto:r.cogdell@bio.gla.ac.uk mailto:jh@mrc-dunn.cam.ac.uk ggg .. investigation effect of two lipoprotein (a)-associated genetic variants on plasminogen levels and fibrinolysis hong wang,*, chan e. hong,*, joshua p. lewis,* yanbei zhu,* xing wang,*,† xin chu,‡ joshua backman,* ziying hu,* peixin yang,§ christopher d. still,‡ glenn s. gerhard,‡,** and mao fu*, *division of endocrinology, diabetes, and nutrition and §department of obstetrics, gynecology, & reproductive sciences, university of maryland school of medicine, baltimore, maryland , †department of orthopedic surgery, second affiliated hospital of chongqing medical university, , china, ‡geisinger obesity institute, geisinger clinic, danville, pennsylvania , and **penn state institute for personalized medicine, penn state college of medicine, hershey, pennsylvania orcid id: - - - (m.f.) abstract two genetic variants (rs and rs ) in the apolipoprotein (a) gene (lpa) have been implicated in cardiovascular disease (cvd), presumably through their association with lipoprotein (a) [lp(a)] levels. while lp(a) is recognized as a lipoprotein with atherogenic and thrombogenic characteristics, it is unclear whether or not the two lp(a)-associated genetic variants are also associated with markers of thrombosis (i.e., plasminogen levels and fibrinolysis). in the present study, we genotyped the two genetic variants in subjects of the old order amish (ooa) and recruited subjects according to the carrier and noncarrier status for rs and rs , and also matched for gender and age. we measured plasma lp(a) and plasminogen levels in these subjects, and found that the concentrations of plasma lp(a) were . - and . -fold higher in minor allele carriers of rs and rs , respectively, com- pared with noncarriers (p = . · and p = . · , respectively). by contrast, there was no difference in plasminogen concentrations between carriers and noncarriers of rs and rs . furthermore, we observed no association between carrier status of rs or rs with clot lysis time. finally, plasminogen mrna expression in liver samples derived from caucasian subjects was not significantly different between carriers and noncarriers of these two genetic variants. our results provide further insight into the mechanism of action behind two genetic variants previously implicated in cvd risk and show that these polymorphisms are not major modulating factors for plasma plasminogen levels and fibrinolysis. keywords lipoprotein (a) plasminogen fibrinolysis genetics thrombogenicity cardiovascular disease is one of the leading causes of morbidity and mortality in the world. while progression of cvd is multifactorial, substantial evidence has shown that lipoprotein (a) [lp(a)] is a signif- icant and independent risk factor in the development of cardiovascular diseases (berglund and ramakrishnan ; danesh et al. ; kamstrup et al. ). plasma lp(a) concentration varies up to - fold among individuals, is highly heritable, and is influenced minimally by environmental factors (hobbs and white ; mccormick ). levels of plasma lp(a) are regulated, in part, by the lpa gene located on chromosome q – , which encodes for apo(a) (clarke et al. ). the number of kringle type (kiv- ) repeats can vary from to resulting in apo(a) isoforms with different sizes, and the size of apo(a) is inversely related to the plasma lp(a) concentration (gavish et al. ; lackner et al. ). in addition, single nucleotide poly- morphisms (snps) in the lpa gene are associated with lp(a) levels. among them, a nonsynonymous snp that results in an isoleucine to methionine substitution at position (rs ) and an intronic copyright © wang et al. doi: . /g . . manuscript received june , ; accepted for publication august , ; published early online september , . this is an open-access article distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/ licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. supplemental material is available online at www.g journal.org/lookup/suppl/ doi: . /g . . /-/dc . these authors contributed equally to this work. corresponding author: university of maryland school of medicine, west redwood street, hh , baltimore, md . e-mail: mfu@medicine.umaryland. edu volume | november | http://orcid.org/ - - - http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / http://www.g journal.org/lookup/suppl/doi: . /g . . /-/dc http://www.g journal.org/lookup/suppl/doi: . /g . . /-/dc mailto:mfu@medicine.umaryland.edu mailto:mfu@medicine.umaryland.edu variant (rs ) have been confirmed to be strongly associated with increased levels of plasma lp(a) and the risk of cardiovascular disease (clarke et al. ; li et al. ; thanassoulis et al. ). indeed, a recently published study by our group has provided addi- tional evidence that these variants are significantly associated with lp(a)-cholesterol levels independently of each other and kiv- repeat number (lu et al. ). the elucidation of a potential mechanism of action behind these lp(a)-associated variants for cvd could lead to novel targets for treatment and/or prevention of cvd. lp(a) is recognized as a lipoprotein with atherogenic and thrombo- genic characteristics (caplice et al. ; grainger et al. ; hajjar et al. ; marcovina and koschinsky ). structurally, lp(a) is a lipoprotein particle consisting of apolipoprotein (a) [apo(a)] covalently bound to the apolipoprotein (b) (apob) of an ldl-like particle (koschinsky et al. ). previous studies have shown that apo(a) shares structural homology with plasminogen, including a kringle do- main, a kringle domain, and an inactive protease domain (hancock et al. ; mclean et al. ). plasminogen, a critical protein in fibrinolysis, binds to lysine residues present on fibrin via its kringle domains and gets activated to plasmin by tissue plasminogen activator (tpa) or urokinase (plow and hoover-plow ). due to the struc- tural similarity to plasminogen and the lack of proteolytic activity, it has been suggested that lp(a) competes with plasminogen for fibrin bind- ing, ultimately resulting in impaired fibrinolysis (atsumi et al. ; hervio et al. ). furthermore, plasma plasminogen concentrations vary by about twofold among healthy individuals. the heritability of plasminogen is estimated to range from . to . (ma et al. ). the plasminogen gene (plg) and lpa gene are located on chromo- some q within � kb of each other (crawford et al. ). a recent genome-wide association study (gwas) has identified nine snps within the lpa and plg gene region on chr q to be significantly associated with plasminogen levels (ma et al. ). moreover, lp(a) and plasminogen are primarily produced in the liver and transported into the circulation (koschinsky et al. ; rainwater and lanford ; saito et al. ). we speculate that lpa and plg mrna ex- pressions are likely to be coregulated in the liver. the genetic variants within the lpa and plg gene region on chr q may regulate lp(a) and plasminogen levels to contribute to thrombogenicity. this pro- motes persistence of the clot and the thrombotic process that may contribute to thrombo-atherogenic diseases. lpa snps rs and rs are consistently associated with lp(a) levels and result in increased risk for cardiovascular diseases (arsenault et al. ; clarke et al. ; kutikhin et al. ; thanassoulis et al. ). however, the mechanism of action behind these two genetic variants previously implicated in cvd risk is not known. in an attempt to better understand the relationship between lp(a)-associated genetic variants and thrombogenesis we genotyped the two variants in amish subjects and recruited age- and sex-matched subjects by rs and rs genotype. we mea- sured the levels of plasma lp(a) and plasminogen and compared the levels of lp(a) and plasminogen based on rs and rs genotypes. in addition, we also assessed genotype-specific differences in fibrinolysis using a euglobulin clot lysis assay (ecla). finally, geno- type-specific differences in plg mrna expression were evaluated in liver samples derived from caucasian subjects. materials and methods subjects and genotyping the subjects were from the old order amish community (ooa) of lancaster, pa and were drawn from participants of our previous lp(a) study (lu et al. ), the amish family diabetes study (afds) (fu et al. ), and pharmacogenomics of antiplatelet intervention (papi) (lewis et al. ). details of study design, recruitment, and phenotyping have been previously described (fu et al. ; lewis et al. ; lu et al. ). snps (rs and rs ) were geno- typed in amish subjects using taqman allelic discrimination assay (applied biosystems) and subjects were recruited into the present study (figure ). since carriers of minor alleles for both rs and rs are infrequent (maf: . and . , re- spectively), we recruited carriers of rs and carriers of rs . noncarrier control subjects were matched, as closely as possible, to the carriers according to gender and age ( yr). to ex- clude double mutation of rs and rs in recruited sub- jects, all of the carriers and noncarriers of rs had the same genotype (aa) for rs . likewise, all of the rs individ- uals had the same genotype (tt) for rs . the study was ap- proved by the institutional review board of the university of maryland, baltimore, and all participants provided written informed consent. the methods were carried out in accordance with the approved guidelines. plasma samples venousbloodwasdrawnafteranovernightfast.fourmillilitersofblood wascollectedfromeachindividualinvacutainertubescontainingedta [for lp(a) and plasminogen measurement] or . % sodium citrate (for ecla), respectively. plasma samples were subsequently separated by centrifugation at · g for min at �. the plasma supernatant for ecla measurement was recentrifuged at · g for min at � to remove any residual platelets. multiple aliquots of plasma were stored at � until assays were performed. elisa quantitative determination of lp(a) and plasminogen antigen levels in human plasma samples was performed by an enzyme-linked immuno- sorbent assay (elisa) according to the manufacturer’s instruction (assaypro, st. charles, mo). fifty microliters of standard or plasma using edta as an anticoagulant, diluted with dilution buffer ( : dilution for lp(a); : , dilution for plasminogen), were added into each well of -well plates precoated with a polyclonal antibody specific for human lp(a)/plasminogen. wells were then incubated with the biotinylated polyclonal antibody specific for human figure a flowchart of study design. | h. wang et al. lp(a)/plasminogen, and was recognized by a streptavidin-peroxidase conjugate, and then a peroxidase enzyme substrate. after the reaction was stopped, the absorbance at nm was read on a victor x multilabel plate reader (perkinelmer, waltham, ma). the standard curves were generated by polynomial regression analysis. lp(a) and plasminogen levels were expressed in micrograms per milliliter. euglobulin clot lysis assay eclawasmodifiedfromtheassaydescribedbymanco-johnson(smith et al. ). briefly, the euglobulin fraction was prepared by adding ml of plasma into . ml of . % acetic acid solution, and then samples were incubated in an ice bath for min. the precipitated euglobulin fraction was then resuspended with ml of borate buffer ( mm sodium chloride, . mm sodium borate, ph . ). one hun- dred microliters of sample was pipetted in triplicate (two wells for measurement and one well for blank) into wells of prewarmed � untreated -well plates, followed by addition of ml of . mm cacl to each well ( ml of ddh o added to the blank wells). the plate was read on a victor x multilabel plate reader (perkinelmer) at nm at -min intervals for hr and the temperature was main- tained at �. liver samples seventy-six wedge biopsy liver samples were obtained from caucasian patients undergoing open or laparoscopic roux-en-y gastric bypass operations or laparoscopic adjustable gastric banding procedures for extreme obesity or its comorbid medical problems at geisinger medical center, danville, pa (lu et al. ; still et al. ). simply, we genotyped caucasian patients for rs and rs us- ing taqman allelic discrimination assay (applied biosystems). these liver samples were selected according to the carrier and noncarrier status for rs (n = : for genotypes tt and ct) and rs (n = : : for aa: ag: gg), and also matched for gender, age, and bmi (figure ) (lu et al. ). qrt-pcr total liverrnawas extractedbytrizol(invitrogen,grandisland,ny) according to the manufacturer’s instructions. the resulting rna was subjected to dnase digestion using rnase-free dnase set (qiagen, valencia, ca) and purification using rneasy minelute cleanup kit (qiagen). the rna quantity and quality were determined using a nd nanodrop spectrophotometer, and mg of total rna was reverse-transcribed using a transcriptor first strand cdna synthesis kit (roche, indianapolis, in). real-time pcr was performed using taqman gene expression assay primers and probes [assay id: hs _m for human plg; hs _m for human b-actin (actb)]. steady-state mrna levels were determined by two-step quantitative real-time pcr (qrt-pcr) using the lightcycler (roche) and taqman probe/primer sets (applied biosystems, grand island, ny). relative expression of mrnas was determined after normalization with the reference gene actb levels using lightcycler software . . statistical analysis data were presented as mean se for the clinical profile, qrt-pcr, elisa, and ecla. a two-tailed student’s t-test was used to evaluate the statistical significance for all assays except for the plg mrna expres- sion of rs in which anova was applied (graphpad software, la jolla, ca). a p value , . was considered statistically significant. pairwise linkage disequilibrium (ld) statistics (d and r ) were calculated with haploview version . (https://www.broadinstitute.org/scientific- community/science/programs/medical-and-population-genetics/haploview/ haploview) (barrett et al. ). all statistical tests were two-sided. power estimates in (rs ) and (rs ) pairs of subjects were calculated using the power and sample size calculation software (version . . ) (dupont and plummer ). prior data indicate that the difference in the response of matched pairs is normally distributed with stan- dard deviation. we will be able to detect a true difference in the mean response of matched pairs of . or . and . or . , respectively, with probability (power) . . the type i error probability associated with this test of the null hypothesis that this response difference is zero is . . data availability both the genotypic and phenotypic information for rs and rs used in this study are listed in supplemental material, table s and table s , respectively. results clinical characteristics of the ooa subjects to evaluate whether lpa genetic variants affect plasma plasminogen levels and fibrinolysis, we successfully genotyped rs and n table clinical profile for the ooa subjects by genotype rs rs tt (n = ) ct (n = ) p value aa (n = ) ag (n = ) p value gender male: male: male: male: female: female: female: female: age (years) . . . . . . . . . . bmi (kg/m ) . . . . . . . . . . sbp (mmhg) . . . . . . . . . . dbp (mmhg) . . . . . . . . . hdl (mg/dl) . . . . . . . . . . ldl (mg/dl) . . . . . . . . . . tg (mg/dl) . . . . . . . . . . hct (%) . . . . . . . . . . rbc count (n · , ) . . . . . . . . . . wbc count (n · ) . . . . . . . . . . platelet count (n · ) . . . . . . . . . . data are presented as mean se. volume november | lp(a)-associated snp and thrombosis | https://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview https://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview https://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview http://www.g journal.org/lookup/suppl/doi: . /g . . /-/dc /tables .xlsx http://www.g journal.org/lookup/suppl/doi: . /g . . /-/dc /tables .xlsx http://www.g journal.org/lookup/suppl/doi: . /g . . /-/dc /tables .xlsx rs in amish subjects and identified carriers of rs and carriers of rs . we recruited age- and sex-matched subjects according to their genotype status. the mean age was . yr old, and . % of subjects were female. since carriers of minor alleles for both rs and rs were infrequent (maf: . and . , respectively) in ooa, no minor allele homozygotes were recruited. the clinical characteristics of the ooa subjects are summarized in table . briefly, study subjects were relatively healthy adults, and the mean levels of age, bmi, blood pressure, hdl, ldl, tg, hematocrit, red blood cell count, white blood cell count, and platelet count were not significantly different between the carriers and noncarriers of the two lpa genetic variants rs and rs . association between lpa genetic variants and plasma lp(a) levels to confirm whether lpa genetic variants rs and rs significantly influence plasma lp(a) levels, we measured plasma lp(a) levels by an elisa (assaypro) in our amish participants. we observed that plasma lp(a) levels were significantly higher for the carriers of both rs and rs compared to the noncar- riers. specifically, the lp(a) levels in carriers of rs were . times higher than the lp(a) levels in noncarriers (carriers: . . mg/ml vs. noncarriers: . . mg/ml, p = . · ) (figure a). similarly, the lp(a) levels in carriers of rs were . times higher than the lp(a) levels in noncarriers (carriers: . . mg/ml vs. noncarriers: . . mg/ml ; p = . · ) (figure b). relationship between lpa genetic variants and plasma plasminogen levels to investigate whether lpa genetic variants rs and rs significantly affect plasma plasminogen levels, we measured plasma plasminogen levels by elisa (assaypro) in the amish cohort. we observed that the plasminogen levels in the carriers of rs and rs were very close to the levels in the noncarriers (carriers: . . mg/ml vs. noncarriers: . . mg/ml, and carriers: . . mg/ml vs. noncarriers: . . mg/ml, respectively). the plasminogen levels in the carriers were not significantly different from the levels observed in the noncarriers of rs (p = . ) (figure a) and rs (p = . ) (figure b). figure plasma lp(a) levels among the ooa subjects for the rs genotype (a) and the rs genotype (b). a scatter- gram is shown for individuals of each genotype with the median represented by a black line respectively. figure plasma plasminogen levels among the ooa subjects for the rs genotype (a) and the rs genotype (b). a scatter- gram is shown for individuals of each genotype with the median represented by a black line respectively. | h. wang et al. relationship between lpa genetic variants and fibrinolysis in addition to assessing genotype-specific differences in lp(a) and plasminogen levels, we investigated whether lpa snps rs and rs were associated with fibrin clot lysis. fibrinolysis was assessed using a euglobulin clot lysis time (eclt) and maximum ab- sorbance (od at nm) by the ecla. we observed no difference in eclt between carriersand noncarriersof rs (ct vs. tt, . . min vs. . . min; p = . , figure a) or rs (ag vs. aa, . . min vs. . . min; p = . , figure b), respectively. in addition, the maximum absorbance in carriers of rs and rs were not significantly different from the maximum absorbance in the noncarriers [rs ct vs. tt, . . vs. . . ; p = . (figure c) and rs ag vs. aa, . . vs. . . ; p = . (figure d)]. plg mrna expression in the liver given the close proximately of rs and rs with the plg gene, we tested whether these variants had any influence on plg mrna expression in the liver. total rna was extracted from liver samples derived from caucasian patients undergoing bariatric weight loss procedures for extreme obesity or related comorbid medical con- ditions (lu et al. ). patients were selected according to the genotype for rs (n = and for genotypes tt and ct, respectively) and rs (n = , , and for genotypes aa, ag, and gg) and matched for age and gender. we measured the plg mrna expression by qrt-pcr and found that there was no statistically significant dif- ference in expression between carriers of rs and noncarriers (tt, . . ; ct, . . ; p = . ) (figure a). similarly, no difference in plasminogen rna expression was observed between rs genotype groups (aa: . . , ag: . . , gg: . . , p = . ; ag + gg vs. aa, p = . ) (figure b). linkage disequilibrium analysis weperformedldanalysisforrs ,rs ,andthetopsnps associated with plasminogen on chr. q in previous gwas (ma et al. ). the result of ld analysis showed these two snps did not have high ld with any of the top snps for plasminogen (r from to . , figure ). discussion lp(a) has a causal role in the development of multiple cardiovascular disorders (arsenault et al. ; clarke et al. ; kamstrup et al. ) and is recognized as having both atherogenic and thrombogenic characteristics (caplice et al. ; enas et al. ; gavish et al. ; marcovina and koschinsky ). lpa snps rs and rs are consistently associated with lp(a) levels and result in increased risk for cardiovascular diseases (arsenault et al. ; clarke et al. ; kutikhin et al. ; thanassoulis et al. ). in the present study, we investigated the effects of these variants not only on their impact on lp(a) levels but also on their potential effect on thrombo- genicity in order to provide novel insights regarding the mechanism(s) by which lpa variants confer cvd susceptibility. to our knowledge this study represents the first investigation to simultaneously assess the impact of these variants on both lp(a) levels and markers of thrombosis (i.e., plasminogen levels and fibrinolysis). consistent with previous results, we observed that both of these snps significantly impact lp(a) levels; however, we observed no evidence to suggest that these variants influence plasminogen levels. in addition, we extend these finding to show that neither rs nor rs impact fibri- nolytic activity or plg mrna expression in the liver. figure results of ecla for the ooa subjects with differ- ent genotypes. clot lysis times measured in minutes for the rs genotype (a) and for the rs genotype (b). maximum absorbance (od at nm) measured for the rs genotype (c) and the rs genotype (d). a scattergram is shown for individ- uals of each genotype with the median represented by a black line respectively. volume november | lp(a)-associated snp and thrombosis | the ooa community is a genetically well-defined caucasian foun- der population. more than % of the current lancaster amish pop- ulation can trace their ancestry to one of seven founder couples (agarwala et al. , ). the amish today are a rural, mostly farming, community, and strong religious beliefs help them to maintain the sect as a distinct and closed entity (cross ). they are relatively homogenous in terms of genetic ancestry, environment, and lifestyle characteristics, which minimizes the risk of potentially confounding variables and makes the ooa a particularly advantageous group for genetic studies. in this investigation, we have genotyped the two genetic variants of rs and rs in amish subjects. to maximally limit confounding factors, we recruited subjects according to carrier and noncarrier status for rs and rs , and matched for gender and age. the minor allele frequencies of rs and rs in the ooa are . % and . %, respectively (lu et al. ), which is relatively lower than the minor allele frequencies ( and %, respectively) in outbred european populations (clarke et al. ). we did not recruit subjects who were homozygous for the minor allele of these variants in ooa. fortunately, these two genetic variants are dom- inant genetic disorders meaning that a single allele can control whether the disease develops. we can recruit heterozygotes or/and minor allele homozygotes to study biological function. consistent with previous stud- ies, the plasma lp(a) levels were significantly higher for carriers of rs or rs compared to noncarriers in the ooa subjects (clarke et al. ; laschkolnig et al. ; lu et al. ). plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin, which has an important role in tissue remodeling and blood clot removal after injury (chapin and hajjar ). genetic variants in the lpa gene were identified as significant contributors to plasminogen levels by gwas (ma et al. ). our data showed that rs and rs are not significantly associated with the plasma plasminogen levels. this finding is consistent with a previous report that rs was the most significantly associated snp with lp(a) levels but was not significantly associated with plasminogen levels in healthy subjects (ma et al. ). furthermore, our present study clarified for the first time that another lp(a)-associated snp, rs , also was not significantly associated with plasminogen lev- els. linkage disequilibrium analysis for rs and rs and the top snps associated with plasminogen on chr. q in previous gwas showed that the investigated snps did not have high ld with any plg-associated snps. our data suggest that the lp(a)-associated variants rs and rs in the lpa gene are not the major genetic determinants of plasma plasminogen levels. since apo(a) can bind to fibrin but has no proteolytic activity, it has been hypothesized that apo(a) competes with plasminogen in circula- tion and may attenuate fibrinolytic function (angles-cano et al. ; loscalzo et al. ). we investigated whether genetic variation in lpa (i.e., rs and rs ) influences fibrinolysis and found no difference in clot lysis time and maximum absorbance between carriers and noncarriers of both rs and rs , indicating that clot formation and clot lysis are not affected by these two snps. of note, however, undas et al. reported decreased clot permeability and longer clot lysis time in both healthy subjects and patients with myocardial infarction that have increased lp(a) levels [a cutoff value of lp(a) mg/ml] as a result of small apo(a) size isoforms (undas et al. ). recently, rowland et al. found that the lpa genetic variant rs was associated with decreased clot permeability and longer clot lysis time among caucasians, but was associated with increased clot permeability and shorter clot lysis among non-caucasians (rowland et al. ). mansson et al. however, reported that lp(a) plasma levels had no effect on clot lysis time in diabetic subjects and normal controls (mansson et al. ). these discordant results may be caused by: ( ) differing study designs; ( ) different measure figure plasminogen mrna expression in the liver. (a) plg mrna expression in the subjects with the rs genotype; (b) plg mrna expression in the subjects with the rs genotype. data are presented as mean se. figure linkage disequilibrium pattern of rs , rs , and top plg association snps in chromosome q – . | h. wang et al. methods; ( ) small sample size; and/or ( ) differences in population characteristics. in the present study, we chose relatively homogenous healthy ooa subjects that were matched with regards to sex, age, and the other lipid traits. we had a relatively large sample size and mea- sured fibrinolysis using an ecla method without addition of human thrombin and recombinant tissue plasminogen activator (rtpa). we identified that rs and rs were not major genetic determinants for fibrinolysis. the lpa and plg genes are adjacently located on chromosome and have a high degree of structural homology, with the lpa gene believed to be generated from the duplication of the plg gene (mclean et al. ). since the liver is the major site of both lpa and plg mrna synthesis, it is possible that there is coregulation between he- patic lpa and plg mrna expression. we determined plg mrna levels using total rna extracted from liver samples as described in our previous study (lu et al. ). previously, we determined that levels of lpa mrna were higher in carriers of rs as compared to noncarriers, and were not different between the carriers and noncar- riers of rs (lu et al. ). in the present study, no significant differences in plg mrna levels were observed between the carriers and noncarriers of rs and rs . in addition, a previous animal study reported that there was no correlation between hepatic lpa and plg mrna levels, suggesting an independent regulation for lpa and plg mrna expression (ramharack et al. ). there are some limitations of this study that we acknowledge. according to power estimation, we have nearly % power to detect alargeeffectsizeand %powertodetectamediumeffectsize,butwedo not have power to detect small effect of rs and rs on variation of plasma plasminogen, plasminogen mrna expression, and changeoffibrinolysis,whichmayleadtothenegativefindings.however, our data show these two variants are not the major genetic determinants of plasminogen levels and fibrinolysis although it is known that they contribute to the risk of cardiovascular diseases. in conclusion, our data indicate that two genetic variants in the lpa gene, rs and rs , are significantly associated with ele- vated lp(a) concentration, but not significantly associated with varia- tion in plasma plasminogen concentration. the increased levels of plasma lp(a) with these genetic variants do not affect fibrinolysis in healthy subjects. moreover, these two lp(a)-associated genetic variants were not associated with plg mrna expression in the liver. these two lp(a)-associated variants of rs and rs in the lpa gene are not the major genetic determinants of fibrinolysis, plg mrna expression, and plasminogen levels. acknowledgments the authors thank alan r. shuldiner for his contribution to amish study and the amish research clinic staff for their great efforts in study subject recruitment and characterization as well as all the volunteers in the amish community for their participation in these studies. this work was supported in part by an american heart association grant ( sdg ), an american diabetes associa- tion grant ( - -ct- ), the national institutes of health (u hl , u hl , r ag , r dk ), and the mid-atlantic nutrition obesity research center (grant p dk ). institutional review board: hp- . literature cited agarwala, r., l. g. biesecker, j. f. tomlin, and a. a. schaffer, towards a complete north american anabaptist genealogy: a systematic approach to merging partially overlapping genealogy resources. am. j. med. genet. 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( ): – . communicating editor: r. cantor | h. wang et al. genetic structure of mennonite populations of kansas and nebraska m.h. crawford, d.d. dykes, and h.f. polesky abstract we describe the gene frequency distributions for different blood group, serum, a n d erythrocytic proteins for three m e n n o n i t e c o m m u n i t i e s f r o m kansas a n d nebraska and c o m p a r e their gene frequencies with those of amish, hutterite, a n d m e n n o n i t e p o p u l a t i o n s using the topological m e t h o d of h a r p e n d i n g a n d jenkins ( ). subdivision of these c o m m u n i t i e s into congregations reveals that the "fission-fusion" m o d e l best characterizes the relationship between the genetic patterns a n d historical events. these m e n n o n i t e populations, although reproductively isolated at the t u r n of this century, are presently entering the m a i n s t r e a m of us rural culture. d u r i n g the s a n d s geneticists, anthropologists, a n d physicians o f t e n focused o n the genetics of h u m a n isolates. small, geographically or socially defined p o p u l a t i o n s t h a t were reproductively isolated a n d highly inbred were being studied t h r o u g h o u t the world ( g o l d s c h m i d t ). t h e p r i m a r y p u r p o s e s of those investigations were to d o c u m e n t t h e ef- fect of genetic d r i f t a n d to u n d e r s t a n d t h e m o d e of t r a n s m i s s i o n of r a r e genetic diseases. t h i s interest gave i m p e t u s to the study of such h u m a n p o p u l a t i o n s as t h e h a b b a n i t e jews, t h e samaritans, tristan da c u n h a , p a r m a valley, a n d st. bartholemew. d u r i n g this t i m e the genetics of various a n a b a p t i s t groups, such as t h e amish, dunkers, a n d hutterites, was investigated. t h e s e early genetic isolate studies yielded a b o u n t y of i n f o r m a t i o n o n t h e genetics of rare m u t a n t genes, for example, t h e ellis-van creveld s y n d r o m e , limb-girdle m u s c u l a r dystrophy, h e m o p h i l i a b, a n d congenital d e a f n e s s (mckusick et al. ). t h e d o c u m e n t a t i o n of genetic drift was less d r a m a t i c , even t h o u g h d . f . r o b e r t s ' s study of tristan da c u n h a succeeded in d e m o n s t r a t i n g t h e effects of u n i q u e department of anthropology, university of kansas, lawrence, kansas . war memorial blood bank, minneapolis, minnesota . human biology, august , vol. , no. , pp. - © wayne state university press, key words: mennonites, population genetics, blood groups / crawford et al. historical events and stochastic processes on small p o p u l a t i o n s (roberts ). although the amish and hutterites were intensively studied, the m e n n o n i t e s were by a n d large ignored by the early geneticists and anthro- pologists. w i t h the exception of allen and r e d e k o p ' s ( ) prospectus for f u t u r e research a m o n g the o l d colony m e n n o n i t e s of mexico and a publication of gene frequencies for m e n n o n i t e s encountered by brown et al. ( ) in the g r a n chaco region of paraguay, little is known about the genetics of the mennonites. t h e genetic structure of the m e n n o n i t e p o p u l a t i o n s of kansas a n d nebraska is one p o r t i o n of a long-term research program initiated by the university of kansas during the mid- s on the genetics of differential biological aging. to date, these p o p u l a t i o n s have been described demographically (stevenson et al. ; lin a n d crawford ), anthropometrically (devor et al. a,b; sirijaraya a n d crawford ), physiologically (devor a n d crawford a,b; koertvelyessy et al. ), and genetically (crawford a n d rogers ; rogers ). t h e primary purposes of this article are ( ) t o describe the gene frequency distributions for various blood m a r k e r s in t h e mennonite populations of kansas and nebraska, ( ) t o e x a m i n e the genetic structure of these populations and its relationship to the e t h n o h i s t o r y of the anabaptists, and ( ) to d e t e r m i n e how systematic pressures, such as migration, affect the genetic structure of the m e n n o n i t e s . populations t h e m e n n o n i t e c o m m u n i t i e s of k a n s a s a n d nebraska trace their origins to sixteenth-century europe. these diverse groups, constituting the anabaptist or left wing of the protestant r e f o r m a t i o n , hold in c o m m o n beliefs in the separation of church a n d state, adult baptism, and pacifism. different anabaptist d e n o m i n a t i o n s c a m e to b e identified in europe by their p r o m i n e n t local leaders. for example, t h e followers of jacob hutter are known as hutterites, the followers of jacob a m a n n became the amish, a n d the followers of m e n n o n s i m o n s call themselves the mennonites. persecution of the anabaptists in western europe, particularly in the netherlands and austria, forced t h e m to settle in t h e underdeveloped agricultural regions of eastern europe. in fifteen m e n n o n i t e refugee couples settled in the swampy lowlands south of danzig in west prus- sia and thus founded the przechowka church. t h e congregation increased genetic structure of mennonites / in n u m b e r a n d m a i n t a i n e d m e t i c u l o u s records. in all but seven families of this congregation emigrated to russia a n d settled in the u k r a i n e near t h e molotschna river. t h i s congregation a d o p t e d the n a m e alexanderwohl, in h o n o r of the r u s s i a n czar. e c o n o m i c conditions, changes in russian governmental policies, a n d possibly internal strife i n d u c e d the alexanderwohl m e n n o n i t e s t o emigrate to the u n i t e d states in . virtually the entire congregation emigrated, followed by m a n y of their relatives and descendants, w h o continued the e x o d u s until the soviet revolution e n d e d m o s t of this flow. o n their arrival in the u n i t e d states, c o m p e t i t i o n between railroad agents for l a n d sales a n d additional strife within the group resulted in t h e original alexanderwohl c o m m u n i t y splitting into three m a j o r divisions. o n e group settled west of lincoln, nebraska, in t o d a y ' s h e n d e r s o n . t h e other two groups settled in kansas, miles n o r t h of wichita. as these congregations prospered a n d grew in size, offshoots or subdivisions f o r m e d . today there are ten congregations derived f r o m or related to the original przechowka church. in the k a n s a s g r o u p was subdivided into two c o m m u n i t i e s , hoffnungsau a n d alexander- wohl. shortly after the t u r n of this century ( ), t a b o r split off the alexanderwohl congregation. in the goessel church separated f r o m the alexanderwohl congregation. in a d d i t i o n , t h e nebraska m e n n o n - ite bethesda congregation also underwent a fission f r o m t h e evangelical m e n n o n i t e s . for p u r p o s e s of c o m p a r i s o n , we include a m o r e conservative m e n - nonite group f r o m m e r i d i a n , kansas, in this study. t h i s c o m m u n i t y , k n o w n locally as the h o l d e m a n s of the church of g o d in christ m e n n o , is a heterogeneous p o p u l a t i o n f o u n d e d by j o h n h o l d e m a n in in ohio. t h e m e m b e r s h i p is largely pennsylvania g e r m a n a n d d u t c h (ostroger m e n n o n i t e s w h o migrated in f r o m volhynia in central poland) mixed with the kleine g e m e i n d e m e n n o n i t e s , w h o migrated to m a n i t o b a f r o m southern russia in . methods we collected b l o o d specimens f r o m three c o m m u n i t i e s of k a n s a s a n d nebraska (table ), packed them in ice, a n d shipped t h e m by air to the m i n n e a p o l i s war m e m o r i a l blood bank for analysis. although the sample size for m e r i d i a n appears to be small, it represents % of the total c o m m u n i t y a n d almost the entire adult population. t h e goessel sample represents % of the entire alexanderwohl church m e m b e r s h i p . t h e h e n d e r s o n sample includes m o r e than % of the town residents. / crawford et al. table . size of sample used for blood analyses population sample size (n) goessel meridian henderson total we performed red cell typings for the following systems: abo (anti- a, ai, b), rhesus (anti-c, c, d, e, e), m n s (anti-m, n , s, s), kell (anti- k, k), ( k p a , k p b ) , kidd (anti-jk a , j k b ) , duffy ( a n t i - f y a , f y b ) , p (anti- pi), vel (anti-v+), lutheran (anti-lu a , l u b ) , a n d several low-frequency antigens, including m t a , f r , and b u a . we p e r f o r m e d the red cell typings on microtiter (u) plates using % suspensions of washed erythrocytes and a modification of the m e t h o d of crawford et al. ( ). we diluted antisera to obtain optimal reactivity a n d incubated typings for hr at r o o m temperature ( ° c). before reading, we spun the typing plates for min at rpm. we also carried out tests t h a t required an antiglobulin phase. for these tests we used microtiter plates, washing t h e m three times with saline and rinsing in a c o o m b s rinser; we spun the plates before recording the results. we phenotyped serum proteins either electrophoretically or through isoelectric focusing (ief). transferrin (tf), haptoglobin (hp), a n d ceru- loplasmin (cp) phenotypes were identified f r o m acrylamide slabs with a m i d o black stain (polesky et al. ). t h e group-specific component (gc) and the properdin factor (bf) systems were simultaneously pheno- typed using the agarose electrophoretic m e t h o d described by dykes and polesky ( ). we employed the isoelectric focusing m e t h o d of dykes et al. ( ) for phenotyping gel. we obtained stroma-free hemolysates for d e t e r m i n i n g erythrocytic enzyme phenotypes by washing cells three times in saline, diluting : in distilled water, and centrifuging at high speed. specimens were stored at - ° c until tested. we phenotyped eight red cell protein markers. adenylate ki- nase (ak), -phosphogluconate dehydrogenase ( -pgd), a n d acid phos- phatase (acp) were simultaneously electrophoresed on a single horizontal starch gel (dykes a n d polesky ). t h e esterase d (esd), isocitrate de- hydrogenase (icd), and malate dehydrogenase ( m d h ) phenotypes were identified on a starch gel using a citrate-phosphate buffer system, p h = . , of karp and sutton ( ). locus of the enzyme phosphogluco- mutase (pgm) was phenotyped using the original technique of spencer genetic structure of mennonites / et al. ( ). in a d d i t i o n , glyoxalase (glo) was phenotyped using the m e t h o d s of h a r r i s a n d h o p k i n s o n ( ). allelic frequencies for the b l o o d group systems are m a x i m u m likelihood estimates c o m p u t e d by a modified maxlik program of reed a n d schull ( ). t h e s e gene frequencies are not corrected for the biological relationships t h a t are observed a m o n g the individuals included in t h e sample. analytical methods p o p u l a t i o n structure of the anabaptist groups is represented by the m e t h o d described by h a r p e n d i n g a n d jenkins ( ). sample allelic frequencies are converted to a relationship matrix r of dimension (l x l), where l is the n u m b e r of sample groups. t h e ijlh element of r is where k is t h e n u m b e r of alleles. t h e diagonal elements of /?, rir describe the overall d e v i a t i o n of the allelic frequencies of the array ( h a r p e n d i n g a n d j e n k i n s ; w o r k m a n et al. ). t h e weighted mean of the diagonal of t h e relationship matrix (rst), that is, the mean genetic heterogeneity of all p o p u l a t i o n s , is equivalent to wright's fst- relative genetic relationships a m o n g the anabaptist groups are graphically represented by a least-squares approximation of the r matrix. reduced-space eigenvectorial representations provide two-dimensional "genetic m a p s " of allelic frequency distributions. t h e eigenvector axes of the m a p s are scaled by the root of the corresponding eigenvalues and thus equalize t h e scale of projection (lalouel ). t h e relative c o n t r i b u t i o n s of systematic versus nonsystematic pres- sures on the microdifferentiation of subdivided populations are explored through the m e t h o d of h a r p e n d i n g and ward ( ). t h i s m e t h o d is based on the a s s u m p t i o n that u n d e r u n i f o r m systematic pressure het- erozygosity should decrease in p r o p o r t i o n to the increasing genetic dis- tance f r o m t h e centroid of the array. thus a u n i f o r m negative slope should result f r o m the regression of heterozygosity on a relative ge- netic distance. t h i s relationship is represented by a two-dimensional plot whose o r d i n a t e is genetic distance ( r / ) and whose abscissa is the mean per locus heterozygosity (ho)' (pl - pl)(pj - p l ) pl{\-pl) ( ) ( ) / crawford et al. where pt is the frequency of the zth allele and / is the n u m b e r of loci. to c o m p a r e the genetic relationship of the various mennonite congregations with a historically derived d e n d o g r a m , we p e r f o r m e d a cluster analysis on the gene frequency data. we used a b m d p m c o m p u t e r program (dixon ), which is based on the euclidean distance between two p o p u l a t i o n s (j and k) a n d is defined as / where xtj is the value of the ith variable in the jxh p o p u l a t i o n . t h e algorithm employed by this cluster program uses the distance between centroid clusters as a criterion for a m a l g a m a t i n g clusters. eight blood group systems and alleles were utilized in this cluster analysis. results tables and s u m m a r i z e the phenotypic counts a n d gene frequen- cies for blood groups, s e r u m proteins, and erythrocytic proteins in three m e n n o n i t e communities. of the b l o o d systems tested by the m i n n e a p o l i s blood bank ( systems reported in this publication), loci exhibit variant f o r m s at an incidence of % or m o r e . we call these " p o l y m o r p h i c " (table ). we tested a n u m b e r of rare familial variants, such as froese (fr ), scianna (bu a ), lw, gregory, wright, and miltenberger, for variation in these m e n n o n i t e populations. although these antigens are rare in the general u s population, the presence of some of t h e m , namely f r a n d b u a , has been reported in m e n n o n i t e groups (lewis et al. ). we did not detect any f r * individuals in this study, although out of individuals of o b l o o d group were b u a positive. although the l u t h e r a n b l o o d group system is usually p o l y m o r p h i c in northern europe, l u a ranges f r o m % to %; it is m o n o m o r p h i c in the three m e n n o n i t e c o m m u n i t i e s . o n the basis of the b l o o d group frequencies, the goessel a n d hen- derson populations show genetic similarity a n d b o t h groups differ slightly f r o m the meridian population. these gene frequency patterns reflect the ethnohistory of these communities, with goessel and h e n d e r s o n hav- ing separated reproductively in . the meridian m e n n o n i t e s are a recent mixture of pennsylvania g e r m a n s , swiss, and dutch. because of the small size of the founding population, the gene frequencies consis- tently fall outside the ranges observed in germany, switzerland, and the netherlands. for example, in the m n s system b o t h goessel a n d merid- ian mennonites differ f r o m the gene frequencies observed in western genetic structure of mennonites / table . phenotypes and gene frequencies for blood group antigens system and goessel meridian henderson phenotypes counts gene frequency counts gene frequency counts gene frequency abo a, a, = . a! = . a, = . a a = . a = . a = . b b = . b = . b = . a , b o = . o = . o = . a b mnss ms mss mss ms = . ms = . ms = . mns ms = . ms = . ms = . mnss n s = . ns = . ns = . mns n s = . ns = . ns = . ns nss ns rhesus cde ccdee cde = . cde = . cde = . ccde cde = . cde = . cde = . cde cde = . cde = . cde = . cdee cde = . cde = . cde = . cde cde = . cde = . cde = . cede cde = . cde = . cde = . cdee cde duffy fy a + b - fy a = . fy a = . fy a = . a + b + f y b = . f y b = . f y b = . a - b + kidd j k a + b - jk a = . jk a = . jka = . a + b + j k b = . j k b = . jk b = . a - b + p i + p, = . pj = . p! = . - p = . p = . p = . kell kk k = . k = . k = . kk k = . k = . k = . kk k p a + b - k p a = . k p a = . k p a = . a + b + k p b = . k p b = . k p b = . a - b + / crawford et al. (table cont.) system and goessel meridian henderson phenotypes counts gene frequency counts gene frequency counts gene frequency lewis le a + b - l e ( a + b - ) = . l e ( a + b - ) = . l e ( a + b - ) = . a + b + l e ( a - b + ) = . l e ( a - b + ) = . l e ( a - b + ) = . a - b + l e ( a - b - ) = . l e ( a - b - ) = . l e ( a - b - ) = . a - b - europe, whereas henderson m e n n o n i t e s c o n f o r m m o r e closely to the ob- served european pattern ( m o u r a n t et al. ). these d a t a suggest that the founding mennonites do not represent a r a n d o m sample of either g e r m a n y or the netherlands but constitute a u n i q u e genetic amalgam. there is little comparative i n f o r m a t i o n on the b l o o d genetics of mennonite populations. brown et al. ( ) described t h e blood group frequencies of m e n n o n i t e settlers w h o were encountered by the researchers in the g r a n chaco region of paraguay. t h i s c h a c o colony emigrated f r o m canada in . t h e r e is considerable genetic difference between this small paraguayan sample a n d the m e n n o n i t e s of kansas and nebraska. specifically, the chaco m e n n o n i t e s exhibit high frequencies of o blood group ( %), m s ( %), and c d e ( %) a n d low frequencies of b ( ), ms ( %), and cde ( %). t h e differences between these m e n n o n i t e groups are probably a result of a c o m b i n a t i o n of factors, namely, the genetic composition of t h e f o u n d e r s a n d the small size of the sample f r o m paraguay. most of the serum protein gene frequencies observed in m e n n o n i t e populations fall within or j u s t outside t h e published e u r o p e a n gene frequency ranges. for example, in europe a n p o p u l a t i o n s the group- specific c o m p o n e n t (also t e r m e d the v i t a m i n d b i n d i n g c o m p o n e n t or dbp) varies for the g c allele between % a n d % (constans et al. ). t h e frequency of this allele in the three m e n n o n i t e c o m m u n i t i e s is slightly elevated and beyond the published e u r o p e a n ranges. although the g c i s allele has a frequency of - % in europe, t h e m e n n o n i t e populations range between % a n d %. a sample of m e n n o n i t e s (combined goessel a n d meridian c o m m u n i t i e s ) has been described for gc a n d p g m isoelectric focusing subtypes (dykes et al. ). as expected, these reported frequencies are intermediate between values for each of the two communities. one interesting finding in one of the m e n n o n i t e c o m m u n i t i e s is the presence of the g c a gene. t h i s gene is rarely observed in european populations unless there is a history of african or australian aborigine admixture. although g c a was first detected in australian aborigines and was initially n a m e d g c a b , its highest incidence is in central africa genetic structure of mennonites / table . phenotypes and gene frequencies for serum and red blood cell proteins systems and goessel meridian henderson phenotypes counts gene frequency counts gene frequency counts gene frequency haptoglobin hp - hp = . hp = . hp = . - h p = . h p = . h p = . - h p c a r l = . h p c a r , = . h p c a r l = . -carl ceruloplasmin cpab c p a = . c p a = c p a = . bb c p b = . c p b = . c p b = . bc c p c = . c p c = . c p c = . bf f fs f = . f = . f = . s s = . s = . s = . ff so. = . so. = . so. = . fso. f, = . f, = . fj = . f,s sso. so. s . f gc - g c = . g c = . gc = . - g c = . g c = . g c = . - g c a b = . g c a b = . g c a b = . - a b gc: (isoelectric focus) is is f is = . is = . is = . s if = . if = . if = . if = . = . = . if a = . a = . a = . l a l ( a b ) ada - a d a = . a d a = . a d a = . - a d a = . a d a = . a d a . - adenylate kinase a k = . ak - a k = . a k = . a k = . - a k = . a k = . a k = . / crawford et al. (table cont.) systems and phenotypes goessel meridian henderson counts gene frequency counts gene frequency counts gene frequency -pgd aa p d a = . p d a = . p d a = . ac p d c = . p d c = . p d b = . ab p d c = . acp a ab a c p a = . a c p a = . a = . b a c p b = . a c p b = . b = . bc a c p = . a c p = . c = . ac c esterase d - esd = . e s d = . esd = . - e s d = . e s d = . e s d = . - pgm j - pgm j = . p g m , = . p g m = . - p g m , = . p g m , = . p g m = . - p g m r = . p g m r = . p g m r = . -r pgmj + - + = . l + = . + = . - - = . " = . ~ = . + + + = . + = . + = . + - ~ = . " = . " = . - + it = . lr = . - - + + - lr glo - glo = . n t g l o = . - g l o = . g l o = . - icd - icd = . n t i c d = . - i c d = . i c d = . m d h - m d h = . n t m d h = . genetic structure of mennonites / table . summary of polymorphic and monomorphic loci among at least one of three populations reported in this study polymorphic loci monomorphic loci abo lutheran rhesus ceruloplasmin mns -phosphogluconate dehydrogenase kidd isocitrate dehydrogenase duffy malate dehydrogenase p gregory kell froese (fr ) lewis scianna (bu a ) gm* lw glyoxalase i vel km* wright haptoglobin miltenberger properdin factor group specific component adenosine deaminase adenylate kinase acid phosphatase esterase d phosphoglucomutase * reported in another publication. ( c o n s t a n s et al. ). considering t h e presence of an african marker pgm'j gene in t h e s a m e person with the g c a , it is highly likely that b o t h these genes were introduced through american black gene flow into the p o p u l a t i o n . t h e f r e q u e n c y of the h p allele in the three m e n n o n i t e c o m m u n i - ties is lower t h a n its incidence in germany, switzerland, or the nether- lands. in particular, h p occurs in meridian at . % versus the - % in those regions of e u r o p e f r o m which the mennonites originated. a rare haptoglobin variant, carlberg, was detected in two of the m e n n o n i t e c o m m u n i t i e s . h p carlberg ( h p ca) was originally described by galatius-jensen ( ). t h i s phenotype resembles a mixture of h p - a n d h p - in variable proportions, p r o m p t i n g sutton ( ) to suggest that this variant may be the result of genetic mosaicism. more recently, the h p ca p h e n o t y p e has been explained on the basis of a possible m u t a t i o n of t h e h p a chain followed by a decrease in the synthesis of the p o l y p e p t i d e (bowman a n d kurosky ). / crawford et al. s - l e u t n ( ) l - l e u t i n d i a n a p a r a g u a y ^ ^ ^ © ® ( z ) m e r i d i a n west e u r o p e ( ) ® © g o e s s e l © h e n d e r s o n figure . least-squares reduction genetic map of the eight anabaptist populations based on allelic frequencies for ten alleles and three loci. three kansas and nebraska mennonite communities are represented by meridian, goessel, and hender- son. gene frequencies for the other anabaptist groups were obtained from juberg et al. ( ), brown et al. ( ), and steinberg et al. ( ). population structure figure shows the population structure in the reduced-space ge- netic m a p of the three kansas and nebraska m e n n o n i t e c o m m u n i t i e s compared to other anabaptist groups a n d western europe (represented by mean gene frequencies f r o m the regions of origin of t h e m e n n o n - ites). this plot is based on ten alleles and three loci. t h e n u m b e r of loci used in this analysis was necessitated by the availability of data on other anabaptist groups. samples are arrayed along the first two scaled eigenvectors, which together account for % of the total variation in the sample (eigenvector , %; eigenvector , %). t h e addition of the third eigenvector in a pseudo-three-dimensional representation accounts for % of the total variation. t h e genetic m a p in figure indicates little genetic divergence a m o n g the various anabaptist groups, with the cluster populations in close proximity to the centroid of the distribution. of the three mennonite c o m m u n i t i e s studied in kansas a n d nebraska, the meridian population differs f r o m the other two groups. t h i s genetic difference is a result of their ethnohistory and genetic f o u n d a t i o n , with goessel and henderson being a single c o m m u n i t y until . meridian had few founders. goessel and h e n d e r s o n individuals, w h o are general conference mennonites (the most m a i n s t r e a m of the mennonites), have the closest genetic affinity to the composite western e u r o p e popul a t i on. the paraguay mennonites differ f r o m the other groups probably because of their small sample size (n = ) and may represent a fission of the larger canadian gene pool along familial lines. genetic structure of mennonites / figure . plot of mean per locus heterozygosity h ( / / is used interchangeably) against distance from the centroid rh of the relationship matrix for eight anabaptist communities. the r matrix is based on ten blood group alleles and three loci. figure indicates t h e relationship between mean per locus het- erozygosity ( / / ) a n d the relative distance f r o m the centroid of the allelic d i s t r i b u t i o n ( r / z ) for the seven anabaptist groups and a composite west- ern e u r o p e g r o u p . t h i s c o m p a r i s o n reflects the ethnohistory and origins of the a n a b a p t i s t groups. t h e paraguayan mennonites exhibit low het- erozygosity a n d high rih suggesting that this population is experiencing the action of n o n s y s t e m a t i c pressures because of its geographic repro- ductive isolation a n d small size. by contrast, the indiana amish have high heterozygosity a n d high ru and, like the paraguay mennonites, are located far f r o m expectation, as d e m o n s t r a t e d by their distance f r o m the theoretical regression line. t h i s unusually high level of ru and h a m o n g the a m i s h is probably t h e result of a highly heterogeneous f o u n d i n g pop- ulation acted on by n o n s y s t e m a t i c pressures. however, the goessel and h e n d e r s o n p o p u l a t i o n s exhibit relatively high levels of heterozygosity and low rih suggesting elevated migration rates and exogamy. / crawford et al. wright's fst statistics have been widely used to measure the ge- netic microdifferentiation of subdivided populations. in c o m p a r i s o n with other studies of fst, the anabaptist p o p u l a t i o n s exhibit a low fst level of . . t h i s value indicates t h a t these anabaptist groups can be consid- ered genetically homogeneous (based on alleles) and have experienced little genetic differentiation. in contrast, subdivided c i r c u m p o l a r popu- lations attain fst values of . , a n d the m a j o r geographic entities of homo sapiens exhibit values u p to . (crawford and enciso ). figure contains a least-squares reduction genetic m a p of the three m e n n o n i t e c o m m u n i t i e s subdivided by congregations. we reduced alleles a n d genetic loci into eigenvectors. t h e first two scaled eigenvectors account for almost % of the gene frequency variance in the array. t h e first eigenvector (e\) has almost twice the discriminating power and accounts for . % of the variance, versus . % for e . the first scaled eigenvector separates k a n s a n f r o m nebraskan mennonites. t h e goessel c o m m u n i t y is subdivided into three congregations, namely, the alexanderwohl, tabor, and goessel churches. goessel a n d tabor split f r o m the alexanderwohl church in a n d , respectively. in figure the alexanderwohl church is the m o s t proximal to t h e centroid of the array, whereas the goessel and tabor churches have b e e n separated f r o m their parental groups by the frequencies of the p~ and esterase d alleles. t h e bases for the distribution of the groups along the second axis ( e ^ ) shown in figure is less obvious. t h e reduced-space representation of the alleles used to c o m p u t e the genetic m a p shows that g c f and cde separate the populations along the second axis. figure is a plot of the m e a n per locus heterozygosity and ru for the various congregations. t h i s m e a n of h a n d ru is based on alleles instead of the shown in figure . alexanderwohl, meridian, a n d , to a lesser extent, goessel a n d tabor r e m a i n closest to the theoretical regression line (the predicted relationship between the two variables). t h e henderson mennonite congregation exhibits t h e highest heterozygosity level, whereas the bethesda a n d the evangelical congregations have the lowest h . in this plot the heterozygosity levels of the congregations are reduced to some extent because of the a d d i t i o n of a large n u m b e r of diallelic loci. m a j o r differences are observed in a c o m p a r i s o n of two m e n n o n i t e congregation dendograms, one a historical reconstruction of p o p u l a t i o n fission and fusion (figure ) and the other based on the cluster analysis of gene frequencies (figure ). genetically, the tabor p o p u l a t i o n is sep- arate from the other m e n n o n i t e c o m m u n i t i e s , even t h o u g h historically it split off only two generations ago. t h e evangelical church in h e n d e r - genetic structure of mennonites / bethesda evangelical e x / goessel tabor meridan alexanderwohl henderson m.b. figure . least-squares reduction genetic map ot the three mennonite communities subdivided into seven congregations. frequencies from alleles and genetic loci were used to construct the r matrix. ) o < o a , a c p b k cde b f f b m cde f y b a c p c gc g c , s s j k a cde ada b i f ' ak b f hp b f s acp* cde \ i *= o) _q j- ~ c ® > ° ( © ) <j) co uj o i - < i a c p b s m \ e > l / cde hp bff« / b f b f f b k gc i cde e s d ' a] ada acpa j k a a c p c g c ^ j figure . dispersion of the seven mennonite congregations and alleles along eigenvectors associated with the two largest eigenvectors of r and a matrices. / crawford et al. . . henderson meridan h . . . evangelical . . . . . . . . . . figure . plot of mean per locus heterozygosity ( h ) against distance from the centroid r / of the r matrix for seven mennonite congregations. frequencies from alleles and loci were used. son, nebraska, and the goessel congregation c a m e off the genetic tree next, even though historically their origins m u s t be traced to russia. these findings support the hypothesis t h a t the fission of the m e n n o n i t e gene pools occurred along familial lines a n d does n o t represent a ran- d o m subset of the p o p u l a t i o n . t h e bethesda a n d alexanderwohl com- munities, both r e m n a n t s of the original division of the alexanderwohl congregation in , cluster together genetically. yet, despite their het- erogeneous european origins, b o t h meridian a n d the h e n d e r s o n men- nonite congregations show closer genetic affinities t h a n d o the offshoot congregations. discussion t h e population history of the m e n n o n i t e s can be characterized in t e r m s of a fission-fusion model, which is schematically represented in figure . the founders of the various m e n n o n i t e groups c a m e f r o m the netherlands, switzerland, n o r t h e r n germany, moravia, alsace, and tirol. t h e earliest groups were f o u n d e d by a small n u m b e r of individuals coming from different regions of europe. t h e przechowka church in west prussia, which eventually gave rise to the alexanderwohl congregation, was f o u n d e d by refugee couples f r o m the netherlands a n d switzerland. t h i s congregation, through high m genetic structure of mennonites / w e s t e r n e u r o p e r u s s i a u n i t e d s t a t e s tabor alexanderwohl a l e x a n d e r w o h l a l e x a n d e r w o h l (kansas) g o e s s e l a l e x a n d e r w o h l n e t h e r l a n d s evangelical b e t h e s d a g a l l i c i a n s (nebraska) bethesda w e s t p r u s s i a n s church of g o d in christ church of g o d in christ meridian o s t o r o g e r s mennonites mennonite brethern henderson m b figure . the ethnohistory of the seven mennonite congregations summarized by a dendogram. the dotted lines indicate that various combinations of dutch and german populations contributed to the formation of the nebraskan and kansan mennonites. this dendogram is based on an unpublished flow diagram constructed by john janzen. fertility a n d t h e a d d i t i o n of other migrants f r o m prussia a n d moravia, in- creased in n u m b e r a n d in relocated in the ukraine. the community was r e n a m e d alexanderwohl, a n d when the russian government revoked the military e x e m p t i o n s for mennonites, the entire c o m m u n i t y emigrated t o the u n i t e d states in . o n arrival, alexanderwohl underwent the first fission, with one g r o u p settling in nebraska a n d founding the town of h e n d e r s o n a n d the second group settling in kansas. t h i s kansas sub- division of the alexanderwohl c o m m u n i t y settled in two communities, h o f f n u n g s a u a n d goessel. shortly after the t u r n of the twentieth century, in , t a b o r split f r o m the alexanderwohl congregation (located in the town of goessel) a n d established the goessel church in . in addi- tion, the nebraska m e n n o n i t e bethesda congregation also underwent a fission, with t h e separation of the evangelical mennonites. a similar fission a n d fusion of p o p u l a t i o n s has been described by olsen ( ) for t h e h u t t e r i t e s of n o r t h america. olsen characterized the f o r m a t i o n of h u t t e r i t e colonies on the basis of historic a n d demographic / crawford et al. cd cd amalg. s r- distance ct> co c d co ^t r - ct) c d cd f ^ nt w c\l co co co co hv! meridian h e n d e r s o n mb b e t h e s d a alexanderwohl goessel evangelical t a b o r figure . a dendogram based on the cluster analysis of the frequencies of blood group systems and alleles. this clustering method is based on the distance between centroids as a criterion for amalgamation. evidence for - and likened their structure in m a n y respects to the y a n o m a m o of venezuela. although the m e n n o n i t e s d o not explicitly follow a policy of splitting off daughter colonies because of high fertility, this population subdivision does occur de facto. most of the general conference m e n n o n i t e splits appear to be d u e t o personality conflicts, doctrinal disagreements, or both. although e c o n o m i c considerations are not voiced, they should n o t be ignored as one of the mot i va t i n g factors for population fission. this fission-fusion process is reflected in the genetic structure of the mennonites. for example, the c o n t e m p o r a r y alexanderwohl congre- gation remains proximal to the center of the allelic array, whereas its offshoots, tabor and goessel, show marked genetic difference. given the short time since the fission of these p o p u l a t i o n s ( - generations), it is unlikely that the tabor a n d goessel m e n n o n i t e s would experience this magnitude of genetic microdifferentiation. t h e two m o r e likely explana- tions for this genetic uniqueness are p o p u l a t i o n sampling a n d subdivi- sion by families. sampling may play a m a j o r role, because the t a b o r and genetic structure of mennonites / goessel s a m p l e s are the smallest of all the congregations studied. it is also likely t h a t the fission process involving tabor, goessel, a n d alexan- derwohl t o o k place along familial lines, producing a n o n r a n d o m division of the gene pool. in s u p p o r t of this, rogers ( ) d e m o n s t r a t e d through the use of s u r n a m e analysis t h a t the initial fissioning of the m e n n o n i t e i m m i g r a n t g r o u p was n o n r a n d o m . t h e r e were surnames a m o n g the families t h a t i m m i g r a t e d ; however, only of these s u r n a m e s ( %) are f o u n d in all three settlements. t h e m e r i d i a n c o m m u n i t y (church of g o d in christ m e n n o ) is a heterogeneous p o p u l a t i o n f o u n d e d in . although this group is small a n d highly conservative with regard to endogamy, it has experienced a low level of inbreeding (sirijaraya ). as a result of the genetic heterogeneity of its f o u n d e r s , the m e r i d i a n congregation r e m a i n s close to the regression line in the ru versus h c o m p a r i s o n . meridian displays a relatively high heterozygosity b u t a low distance f r o m the centroid of the allelic f r e q u e n c y d i s t r i b u t i o n (see figure ). t h e k a n s a s m e n n o n i t e p o p u l a t i o n s also experienced various de- grees of r e p r o d u c t i v e isolation a n d inbreeding t h ro u g h o u t their history. according to rogers ( ), their inbreeding coefficient ( f ) varied f r o m % in - to . % in - . at present, the general confer- ence m e n n o n i t e c o m m u n i t i e s of k a n s a s a n d nebraska cannot be char- acterized as genetic isolates because a high p r o p o r t i o n of marriages are with n o n - m e n n o n i t e s . d u r i n g the last - years, approximately % of all the marraiges in hoffnungsau, kansas, were contracted with non- m e n n o n i t e s (kay ). however, f r o m - the c o m m u n i t y was % e n d o g a m o u s . since world war i the reproductive isolation has rapidly b r o k e n d o w n with the congregation m e m b e r s becoming m o r e integrated into m a i n s t r e a m american life. before m o r e than % of the marriages were m e n n o n i t e e n d o g a m o u s . marital partners were m e n n o n i t e b u t f r o m a different village or town. by m e n n o n i t e en- d o g a m y o c c u r r e d in . % of the marriages a n d . % were exogamous (kay ). t h e b l o o d genetics s u p p o r t the conclusion that the general con- ference m e n n o n i t e s described in this study are n o longer m e m b e r s of genetic isolates a n d are culturally approaching the m a i n s t r e a m farming c o m m u n i t i e s . t h e high levels of heterozygosity reflect considerable sys- t e m a t i c pressure in the f o r m of migration. t h e m e a n per locus heterozy- gosity varies f r o m % to %, based on b l o o d systems. in addition, the presence of several african m a r k e r genes suggests gene flow f r o m the american black gene p o o l into the m e n n o n i t e groups. c o n t e m p o r a r y k a n s a s a n d nebraska general conference m e n n o n - ite genetic s t r u c t u r e can best be explained on the basis of episodes of / crawford et al. fission and fusion, followed by a rapid breakdown of reproductive isola- tion. thus these mennonite populations can n o longer be characterized as highly inbred genetic isolates. acknowledgments this research was supported in part by the national institute of aging under grant ago and the university of kansas under faculty research award - . crawford received support through a public health service research career development award (k de - ). we would like to thank the mennonite communities of goessel, henderson, and meridian for their patience and good will in this project. special words of thanks go to john janzen, whose reconstruction of the ethnohistory was used in this publication, and to laurine rogers, who helped organize and administer this research program. revision received june . literature cited allen, g. random genetic drift inferred from surnames in old colony mennonites. hum. biol. 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apo cii, the necessary activator of lpl. the role of apo ciii in lipoprotein metabolism was later expanded by studies in per- fused rat livers demonstrating that apo ciii inhibits uptake of triglyceride (tg) rich lipoproteins and remnants. , the identi- fication of sisters with complete absence of apo ciii because of a homozygous chromosomal deletion that also included apo ai, apo aiv, and apo av, allowed us to demonstrate, in vivo, that absence of apo ciii resulted in a dramatic increase in li- polysis of vldl (very-low-density lipoprotein)-tg. studies in mice overexpressing apo ciii or with targeted deletions of the apoc gene confirmed the human findings and also sup- ported the role of apo ciii as an inhibitor of hepatic uptake of apob -containing lipoproteins. – in addition, mouse- and hepatoma-based studies suggested that apo ciii may increase the incorporation of tg into nascent vldl particles. , although interest in apo ciii’s role in lipid metabolism has continued, enthusiasm for apo ciii as a therapeutic target was limited by uncertainty about the relationship between hyper- triglyceridemia and risk for cardiovascular disease (cvd). , recent genetic studies using both genome-wide association and mendelian randomization approaches have, however, es- tablished the relationship between loss of function of apo ciii, received on: june , ; final version accepted on: october , . from the columbia university vagelos college of physicians and surgeons, new york (g.r.-s., s.h., a.m., t.t., r.n., c.n., w.k., h.n.g., r.r.); maryland school of medicine, university of maryland, baltimore (c.s., r.b.h., t.i.p.); and baltimore va medical center, va research service, geriatric research, education and clinical center and va maryland health care system (c.s., t.i.p.). the online-only data supplement is available with this article at https://www.ahajournals.org/doi/suppl/ . /atvbaha. . . correspondence to gissette reyes-soffer, md, department of medicine, columbia university vagelos college of physicians and surgeons, w th st, ph – , new york city, ny , email gr @cumc.columbia.edu; or toni i. pollin, phd, division of endocrinology, diabetes and nutrition, program for personalized and genomic medicine, departments of medicine and epidemiology and public health, university of maryland school of medicine, health sciences facility iii, room , w baltimore st, baltimore, md , email tpollin@som.umaryland.edu © american heart association, inc. objective—apo (apolipoprotein) ciii inhibits lipoprotein lipase (lpl)-mediated lipolysis of vldl (very-low-density lipoprotein) triglyceride (tg) and decreases hepatic uptake of vldl remnants. the discovery that % of lancaster old order amish are heterozygous for the apoc r x null mutation provided the opportunity to determine the effects of a naturally occurring reduction in apo ciii levels on the metabolism of atherogenic containing lipoproteins. approach and results—we conducted stable isotope studies of vldl-tg and apob in individuals heterozygous for the null mutation apoc r x (ct) and their unaffected (cc) siblings. fractional clearance rates and production rates of vldl-tg and apob in vldl, idl (intermediate-density lipoprotein), ldl, apo ciii, and apo cii were determined. affected (ct) individuals had % reduction in plasma apo ciii levels compared with ccs (p< . ) and reduced plasma levels of tg ( %, p< . ), vldl-tg ( %, p< . ), and vldl-apob ( %, p< . ). these changes were because of higher fractional clearance rates of vldl-tg and vldl-apob with no differences in production rates. cts had higher rates of the conversion of vldl remnants to ldl compared with ccs. in contrast, rates of direct removal of vldl remnants did not differ between the groups. as a result, the flux of apob from vldl to ldl was not reduced, and the plasma levels of ldl-cholesterol and ldl-apob were not lower in the ct group. apo ciii production rate was lower in cts compared with ccs, whereas apo cii production rate was not different between the groups. the fractional clearance rates of both apo ciii and apo cii were higher in cts than ccs. conclusions—these studies demonstrate that % reductions in plasma apo ciii, in otherwise healthy subjects, results in a significantly higher rate of conversion of vldl to ldl, with little effect on direct hepatic uptake of vldl. when put in the context of studies demonstrating significant protection from cardiovascular events in individuals with loss of function variants in the apoc gene, our results provide strong evidence that therapies which increase the efficiency of conversion of vldl to ldl, thereby reducing remnant concentrations, should reduce the risk of cardiovascular disease. visual overview—an online visual overview is available for this article. (arterioscler thromb vasc biol. ; : - . doi: . /atvbaha. . .) key words: apolipoprotein c-iii ◼ cardiovascular diseases ◼ isotopes ◼ lipolysis ◼ lipoprotein lipase effects of apoc heterozygous deficiency on plasma lipid and lipoprotein metabolism gissette reyes-soffer, carol sztalryd, richard b. horenstein, stephen holleran, anastasiya matveyenko, tiffany thomas, renu nandakumar, colleen ngai, wahida karmally, henry n. ginsberg, rajasekhar ramakrishnan, toni i. pollin arterioscler thromb vasc biol is available at https://www.ahajournals.org/journal/atvb doi: . /atvbaha. . translational sciences d ow nloaded from http://ahajournals.org by on f ebruary , mailto:gr @cumc.columbia.edu mailto:tpollin@som.umaryland.edu arterioscler thromb vasc biol january which results in lower plasma tg concentration, and reduced cvd risk. , we found that % of the lancaster old order amish are heterozygous carriers of a null mutation, r x (hgvs nm . c. c>t p.arg ter rs ) in the apoc gene, which converts an arginine to a termination codon, resulting in a % reduction of plasma apo ciii lev- els. in addition to having lower fasting and postprandial tg, higher levels of hdl (high-density lipoprotein)-cholesterol, and lower levels of ldl (low-density lipoprotein)-cholesterol, heterozygous deficient individuals have less subclinical ath- erosclerosis, as determined by coronary artery calcification. our report was followed by large cohort studies of apoc loss of function mutations, including r x, which demonstrated a % reduction in myocardial infarction in carriers. , based on the nonhuman mechanistic studies described above, heterozygous loss of function of apoc might reduce risk for cvd by any or all of the following: ( ) reducing se- cretion of vldl from the liver, ( ) increasing hepatic uptake of vldl and chylomicron remnants by the liver, and ( ) increasing lipolytic conversion of vldl to ldl. we used stable isotope tracers to determine which of these possible results of heterozygous loss of function of apoc would be most important in determining the differences in plasma lipids between r x affected amish and their unaffected siblings. our findings support the development of therapies that lower plasma apo ciii levels as a means of treating mod- erate to severe hypertriglyceridemia , which may reduce risk for cvd. materials methods the data that support the findings of this study are available within the article and its online-only data supplement. study subjects we recruited participants heterozygous for the apoc r x muta- tion, hereafter denoted by ct, and sex-matched, unaffected siblings, hereafter denoted by cc, ages to years, from the lancaster old order amish population. participants were not receiving lipid- altering medications. all study participants provided written in- formed consent and the studies were approved by the institutional review boards of the university of maryland school of medicine and columbia university medical center. stable isotope kinetic studies the sibs, in each pair, were studied on the same day at the amish research clinic in lancaster, pa. the protocol for these studies was one that we have used previously, with minor modifications to allow subjects to complete some visits in their homes. on day , partici- pants fasted for hours, after which a nurse visited their homes and drew baseline bloods for safety and fasting lipid and lipoprotein mea- surements. after pm on day , they were npo (nothing per mouth [oral]) until pm when they started a liquid, isocaloric, % fat diet that was provided every hours for the next hours. at : am (day ), they arrived at the amish research clinic, where intrave- nous were placed in antecubital veins of each arm and baseline bloods were drawn (time hour). immediately after, boluses of h₃-l- leucine ( µmol/kg bw), ring- c₆-l-phenylalanine ( . µmol/kg bw), and h₅-glycerol ( µmol/kg bw) were administered over a -minute period, followed by a constant infusion of h₃-l-leucine ( µmol/kg bw per hour) over hours. additional blood samples were collected at and minutes, and at , , , , , , , , , . , . , hours after the administration of tracers and processed to isolate plasma and serum. after the hours blood sample, the subjects returned to their homes where they continued to consume the liquid meal protocol. eight hours later a nurse drew the final hours blood sample in their homes. vldl, idl (intermediate-density lipo- protein), ldl, and hdl were obtained from the plasma samples via sequential density ultracentrifugation. determination of stable isotope enrichment of apob and tg the isolated lipoprotein fractions were used to determine stable iso- topic enrichments of h₃-l-leucine and ring- c₆-l-phenylalanine in apob in vldl, idl, and ldl. apob was isolated from vldl, idl, and ldl by sds-polyacrylamide gel electrophoresis. the isolated apob bands were excised from the gels, hydrolyzed, and the amino acids derivatized. plasma free amino acids were recov- ered from . ml plasma after precipitation of proteins with ace- tone and extraction of the aqueous phase with hexane. the aqueous phase was dried under vacuum, amino acids were derivatized. enrichments of [ , , - h ]-leucine and [ c ]-phenylalanine tracers in apob -lipoproteins and plasma free leucine and phenylalanine were measured by gas chromatography-mass spectrometer using an agilent gas chromatography and a mass spectrometer with negative chemical ionization. additionally, kinetic analysis of tg in vldl was performed with h₅-glycerol. tg was separated from phospholipid by zeolite binding. the tg was resolubilize with chloroform. we performed transesterfication with methanolic hcl. glycerol was isolated by liquid/liquid extraction (hexane and water added) and derivatized to triacetin (glycerol triacetate) through in- cubation with acetic anhydride. we performed gas chromatography- mass spectrometer positive chemical ionization with selective ion monitoring of m/z and . compartmental modeling of apob and tg metabolism fractional clearance rates (fcrs) and production rates (pr) of tg and apob in vldl, and of apob in idl and ldl were de- termined using a compartmental model to fit stable isotope enrich- ment data. – in our general model, apob and tg are required to have the same pool structure and the same rate constants for each vldl pool, but with different mass distributions. with > pool in the vldl fraction, the different mass distributions lead to different vldl-fcrs for tg and apob, since vldl-fcr is obtained as a weighted average of the individual fcrs (the weights given by the mass distribution). however, if there is only one pool in the vldl fraction, tg and apob necessarily have the same fcr. for each study, the minimum number of pools needed to simultaneously fit the sets of data ( tracers, leucine and phenylalanine, in vldl-, idl-, and ldl-apob and plasma amino acids, and tracer, glyc- erol, in vldl-tg) is chosen for the final model. in the present study, the final model had pool each for vldl, idl, and ldl. the data were fitted by least squares, giving equal weight to all data points (ie, assuming a constant error variance for all measurements) using a computer program, pool fit, which solves the differential equations nonstandard abbreviations and acronyms apo apolipoprotein cvd cardiovascular disease fcr fractional clearance rates hdl high-density lipoprotein idl intermediate-density lipoprotein ldl low-density lipoprotein lpl lipoprotein lipase pr production rates tg triglyceride vldl very-low-density lipoprotein d ow nloaded from http://ahajournals.org by on f ebruary , reyes-soffer et al effects of apo c mutation on lipid metabolism in closed form and computes the fits and parameter sensitivities as sums of exponentials. the fits yielded fractional clearance rates (fcrs) of apob in vldl, idl, and ldl, and tg in vldl. the model also estimated rates of conversion of apob between vldl, idl, and ldl. prs (mg/kg per day) were calculated by multiplying fcrs (in pools/day) by the appropriate lipoprotein pool sizes of apob , which were calculated as each lipoprotein’s con- centration of apob in mg/dl multiplied by an estimate of each individual’s plasma volume ( ml/kg). schematics of the models used to analyze apob , tg-glycerol, and apo ciii in the present study are included in the supplement (figure i in the online-only data supplement). based on the best fit of the present data, we have only one vldl pool for apob and tg, direct conversion of vldl- apob to ldl, as well as conversion from vldl to ldl via idl, no direct out pathway from idl, and a small component of direct secretion of ldl from the liver. determination of stable isotope enrichment of apo cii and apo ciii apo ciii enrichment with h₃-l-leucine was measured using ultraperformance liquid chromatography-mass spectrometry. vldl and hdl fractions were digested with trypsin as previously described. a multiple reaction monitoring method was used to de- termine the following precursor-product ion transitions for a pep- tide specific to apo ciii (gwvtdgfsslk; m : . > . ; m : . > . ) and apo cii (tylpavdek; m : . > . ; m : . > . ). compartmental modeling of apo cii and apo ciii metabolism the h -leucine enrichment data for apo cii and apo ciii in hdl or vldl was fitted by a single pool with the vldl-apob enrich- ment plateau used as the best available estimate of the liver leucine pool enrichment. separate fcrs were estimated for hdl and vldl. while the enrichment of apo ciii was different between hdl and vldl, there was a constant ratio across time-points in any single study. this means that the fcr of apo ciii is estimated to be the same in vldl and hdl; any difference is solely because of random measurement error. we ascribe this to apo ciii moving freely among lipoproteins. the situation was similar with apo cii. therefore, we a calculated single plasma fcr for apo ciii and apo cii. apo ciii and apo cii prs were calculated using the fcr for each apolipoprotein and its plasma pool size, as described above for apob. quantitation of apo ciii in vldl and hdl fractions apo ciii was quantitated by ultraperformance liquid chromatogra- phy-mass spectrometry, using a waters xevo tqs triple quad mass spectrometer coupled with an acquity uplc (waters, milford, ma). vldl and hdl fractions were digested with trypsin as described previously. in brief, μl of ultracentrifuged vldl or hdl from each time-point was desalted, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin overnight. a multiple reaction monitoring method was used to determine the following precursor-product ion transitions for a peptide specific to apo ciii (gwvtdgfsslk; m : . > . ) and a deuterated internal standard (m : . > . ). lower limit of quantitation, defined as the level at which the residual of the calibration line is < % of the expected concentration was determined to be nm. the intra-assay precision for the assay was . %. apo cii was similarly quantitated using the following precursor-product ion transitions for a peptide specific to apo cii (tylpavdek; m : . > . ; m : . > . ). the proportions of plasma apo ciii and apo cii resid- ing in vldl and hdl were then calculated. biochemical and immunologic assays day blood was collected after a -hour overnight fasting period. additional timed blood samples were collected while the subjects were consuming the liquid diet, both before ( hour) and at various time-points after the stable isotope infusion was started ( and minutes, , , , , , , , , , . , . , , and hours.). plasma cholesterol, tg, and hdl cholesterol were measured by integra plus (roche). plasma ldl-cholesterol levels were esti- mated using the friedewald formula. cholesterol and tg were also measured enzymatically in vldl, idl, ldl, and hdl isolated by ultracentrifugation. plasma apo cii, apo ciii, and apoe were meas- ured by human elisa kits (ab [apo cii]; ab [apo ciii]; ab [apoe], abcam, cambridge, ma). apob in plasma and in vldl, idl, and ldl was measured using an apob elisa kit (a alercheck, inc). statistical analysis the data are presented as means and sd. the mutation effects were assessed by analyzing within-pair differences for statis- tical significance using paired t tests. the primary end point was the percent difference in fcr of vldl-tg and vldl- apob between affected and unaffected sib-pairs, and p= . was considered significant. a key secondary end point was the partition of vldl between conversion to ldl and hepatic uptake, and p= . was considered significant. all other comparisons were exploratory. results study population we enrolled participants ( ct and cc), including affected males, affected females, and sex- and age- (within years) matched unaffected siblings. mean ages (ct ± . , cc . ± . ) and body mass index (ct . ± . , cc . ± . ) were similar between the groups (table ). plasma lipid and apolipoprotein levels plasma tg levels were lower in the ct subjects ( ± mg/ dl) versus their cc siblings ( ± mg/dl; table ). the mean (±sd) percent difference for tg between the pairs was ± (p< . ). total plasma cholesterol levels did not dif- fer between sib-pairs. although we have previously shown that apoc r x carriers have higher hdl and lower ldl- cholesterol levels, in this much smaller sample, the levels of cholesterol in these lipoproteins were not significantly dif- ferent between the pairs. plasma levels of apob and apoe were not different between the groups, but, as expected, apo ciii levels were significantly reduced in the ct group (ct ± µg/ml versus cc ± µg/ml) with a mean per- cent difference between pairs of ± (p< . ). of interest, apo cii levels were also lower in the affected ( ± µg/ml) compared with the unaffected siblings ( ± µg/ml), with a mean percent difference of ± (p< . ). the percent of plasma apo ciii in vldl was similar in the ct ( ± %) and cc ( ± %) groups. the percent of plasma apo cii in vldl was also similar in the ct ( ± %) and cc ( ± %) groups. the cholesterol and tg levels in the isolated vldl frac- tions (table ) were lower in the ct ( ± . mg/dl and ± mg/dl, respectively) than in their cc siblings( ± mg/dl and ± mg/dl, respectively; p< . for cholesterol and p< . for tg). the cholesterol and tg levels in the idl d ow nloaded from http://ahajournals.org by on f ebruary , arterioscler thromb vasc biol january and ldl fractions were not different between the sib-pairs. despite similar plasma apob levels, vldl-apob lev- els were lower in the ct ( ± . mg/dl) compared with the cc ( ± mg/dl), with a mean percent difference between pairs of ± (p< . ). there were no differences in idl- or ldl-apob levels between the sib-pairs. apob and tg metabolism as noted in methods, we modeled vldl-tg and vldl- apob metabolism jointly, and the best fits of the stable isotope enrichment data required only a single vldl pool. thus, the vldl-apob fcr and vldl-tg fcr in each subject were the same. the results demonstrate that the lower levels of vldl-tg and vldl-apob in the ct group were because of a significantly greater fcr of vldl-tg and vldl-apob (% increase ± , p< . ; table ). the vldl-apob prs were similar in the ct and cc siblings ( ± and ± mg/kg per day, respectively), as were the vldl-tg prs ( ± and ± mg/kg per day). the ratio of vldl-tg pr to vldl-apob pr, an indicator of the size of newly secreted vldl particles, did not differ between the groups (ct ± versus cc ± ). we also found that the fcr of idl-apob was greater in the ct individuals compared with their cc siblings (% in- crease ± ; p< . ). we did not observe differences be- tween sib-pairs in the fcr of ldl-apob or in the prs of idl- or ldl-apob . there were sources of ldl-apob: conversion of vldl to ldl via idl, direct conversion of vldl to ldl, and direct secretion of ldl from the liver. these made up, respectively on the average, %, %, and % of ldl-pr in the ct siblings and %, %, and % in the cc siblings; none of the sources differed significantly be- tween the groups (table ). a key secondary goal of the study was to determine the effects of reduced apo ciii levels on the partitioning of vldl- apob flux between conversion of vldl to ldl and direct table . effects of apoc r x on mean levels of lipoprotein lipids and apob concentrations cc (mean±sd), mg/dl ct (mean±sd), mg/dl percent differences, (mean±sd) vldl-chol . ± . . ± . − . ± . * idl-chol . ± . . ± . − . ± . ldl-chol . ± . . ± . − . ± . vldl-tg . ± . . ± . − . ± . * idl-tg . ± . . ± . − . ± . ldl-tg . ± . . ± . − . ± . vldl-apob . ± . . ± . − . ± . * idl-apob . ± . . ± . − . ± . ldl-apob . ± . . ± . − . ± . participants are defined as cc (unaffected siblings) and ct (affected siblings). data were obtained from time-points during the -h period of the stable isotope studies. data are presented as means and sd of absolute concentrations as well as the means and sds of the percent differences between cc and ct groups. statistical significance of the percent differences was assessed using paired t tests. apob , apolipoprotein b ; chol, cholesterol; tg, triglycerides; vldl, very-low-density lipoprotein; idl, intermediate-density lipoprotein; and ldl, low-density lipoprotein. *significant at p< . . table . baseline characteristics and plasma lipid and apolipoprotein levels of apoc r x carriers and unaffected sib-pairs id no. sex age, y bmi chol, mg/dl tg, mg/dl ldl-c, mg/dl hdl-c, mg/dl apob , mg/dl apo ciii, μg/ ml apo cii, μg/ml apoe, μg/ml cc f cc f cc m cc m . cc m mean±sd ± ± ± ± ± ± ± ± ± ± ct f ct f ct m ct m ct m mean±sd ± ± ± ± ± ± ± ± ± ± %difference (sd) − . ( . ) . ( . ) − . ( . ) − . ( . )* − . (± . ) . ( ) − . ( . ) − . ( . )† − . ( . )† . ( . ) participants are defined as cc (unaffected siblings) and ct (affected siblings), in order of sib-pairs. all lipid and lipoprotein levels were obtained from time-points during the -h period of the stable isotope studies. data are presented as means and sd of absolute concentrations as well as means and sd of the percent differences between the groups. statistical significance of the percent differences was assessed using paired t tests. apo indicates plasma apolipoprotein; bmi, body mass index; chol, plasma cholesterol; tg, plasma triglycerides; ldl-c, plasma low-density lipoprotein cholesterol; hdl-c, plasma high-density lipoprotein cholesterol. *significance at p< . . †significant at p< . . d ow nloaded from http://ahajournals.org by on f ebruary , reyes-soffer et al effects of apo c mutation on lipid metabolism removal of vldl by the liver. the figure depicts a model of apob -lipoprotein transport from the liver to circulating vldl, and then to either ldl or back to the liver. inherent to this model is the concept that the initial lpl-mediated li- polysis of vldl-tg generates a smaller particle with less tg than present at the time of secretion into the circulation, and at some point in this process, the particle has lost enough tg that it has a density > . and, therefore, leaves the vldl pool. this particle can be found in the idl and ldl pools or be irreversibly taken up by the liver. it is important to un- derstand that although we show a single vldl pool in the figure, it comprises a range of vldl of differing size and number of tg molecules: the largest and most tg-rich vldl are those that have just entered the circulating vldl pool and the smallest and most tg-poor are those that have undergone lipolysis and are about to either leave the circulation directly or become idl or ldl. we show a single pool, despite the heterogeneous nature of the population of vldl within this pool, because when we used a model with > vldl pool, it did not improve the fit of our kinetic data. as noted above, the rates of secretion of newly synthesized vldl from the liver (equal to vldl-pr) were similar for ct and cc siblings and, therefore, the blue arrows from the liver to vldl in each group are the same. however, because lipolysis of vldl-tg was much faster in the cts (depicted by the much thicker black arrow coming out from vldl in that group), the size of the vldl pool in cts was about % smaller than the vldl pool of ccs (table ). the green and red arrows represent the number of vldl plasma pools each day that, after lipol- ysis of tg, move either to ldl or to the liver, respectively. our compartmental analysis indicated that there was a sig- nificant . ± . pools/day difference in the fcrs between ct and cc sib-pairs for the conversion of vldl-apob to idl and ldl (p= . ), but only a . ± . pools/d differ- ence in fcrs between the groups for direct hepatic removal of those particles (p= . ). thus, only the conversion rate of vldl to ldl, for example, the lipolytic pathway, was sig- nificantly greater in the group with partial loss of apo ciii. this is depicted by the thicker green arrow in ct compared with cc, whereas the red arrows are similar in the groups (figure). the individual and mean data for these parameters are presented in table . this greater rate of conversion of vldl to idl and ldl versus uptake by the liver was re- flected by a nonsignificant increase in ldl-apob pr in the ct compared with the cc group (table ). as noted above, the generation of ldl from vldl occurred via direct conversion of vldl to ldl or conversion of vldl to idl followed by conversion of the latter to ldl, with direct conversion accounting for the largest proportion of ldl generated. we were able, using our model, to deter- mine the fcrs of each of these pathways in the ct and cc groups, and these data are presented in table . the fcr for direct conversion of vldl to ldl was significantly greater ( . ± . pools/d) in the ct siblings than in the cc group ( . ± . pools/d; p= . ). there was no difference in the fcrs for conversion of vldl to ldl via idl between the groups (ct: . ± . ; cc: . ± . pools/d; p= . ). these results, together with those for the fcrs of overall conver- sion to ldl versus direct uptake of vldl, support much greater lipolytic activity in the affected versus the nonaffected sib-pairs. apo ciii and apo cii metabolism previous studies by several groups have demonstrated that the kinetics of rapidly exchangeable apolipoproteins such as apo ciii and apo cii can only be characterized by plasma fcrs and prs. – however, because of a lack of unanimity on this issue, we determined enrichments of each apolipoprotein in both vldl and hdl, which together transport nearly all of these proteins in plasma. we found essentially identical fcrs for both apo ciii and apo cii in vldl and hdl (table i in the online-only data supplement) and, therefore, have presented only a single fcr and pr for each apolipoprotein in table . in the cc group, the fcrs of apo ciii ( . ± . pools/d) and apo cii ( . ± . pools/d) were similar, consistent with a com- mon clearance pathway of each of these apolipoproteins. on the other hand, the pr of apo ciii ( . ± mg/kg per day) was almost double that of apo cii ( . ± mg/kg per day), indicative of unique regulation of the synthesis of these apolipopro- teins. similar differences in the prs of apo ciii and apo cii have been reported previously. in the ct group, the fcrs of table . effects of apoc r x mutation on kinetic parameters for tg and apob metabolism cc±sd ct±sd % difference±sd vldl-tg and vldl-apob fcr, pools/d . ± . . ± . ± * idl-apob fcr, pools/d . ± . . ± . ± † ldl-apob fcr, pools/d . ± . . ± . ± vldl-tg pr, mg/kg per d ± ± . ± . vldl-apob pr, mg/ kg per d . ± . . ± . ± idl-apob pr, mg/kg per d . ± . . ± . ± ldl-apob pr, mg/kg per day . ± . . ± . ± ldl-apob pr from vldl via idl, mg/kg per d . ± . . ± . ldl-apob pr directly from vldl, mg/kg per d . ± . . ± . ldl-apob pr from the liver, mg/kg per d . ± . . ± . vldl-tg pr/vldl-apob pr . ± . . ± . . ± . participants are defined as cc (unaffected siblings) and ct (affected siblings). data derived from compartmental modeling of stable isotope enrichment of samples obtained over a h. period. data are presented as means and sd of the absolute fcrs and prs as well as the means and sd of the percent difference in each parameter between the ct and cc groups. statistical significance of the percent differences was assessed using paired t tests. apob indicates apolipoprotein b ; fcr, fractional clearance rate; idl, intermediate-density lipoprotein; ldl, low-density lipoprotein; pr: production rate; tg, triglycerides; and vldl, very low-density lipoprotein. †significant at p< . . *p< . . d ow nloaded from http://ahajournals.org by on f ebruary , arterioscler thromb vasc biol january apo ciii ( . ± . pools/d) and apo cii ( . ± . pool/d) were also similar but, in contrast to the cc group, the prs of apo ciii ( . ± . mg/kg per day) and apo cii ( . ± . mg/kg per day) in the ct group were also similar, reflecting the reduced rate of synthesis of apo ciii in the ct versus the cc subjects, resulting from the apoc r x null mutation. for both apo ciii and apo cii, neither the fcr nor the pr was significantly different between the ct and cc subjects. discussion we used stable isotopes to investigate, in vivo, the effects of heterozygosity for the apoc r x null mutation and the associated % lower levels of plasma apo ciii on apob and tg metabolism. our purpose was to determine the rela- tive importance of haplodeficiency for proposed physiolog- ical roles of apo ciii in vldl metabolism: ( ) inhibition of lpl-mediated lipolysis of vldl-tg, ( ) inhibition of hepatic uptake of vldl or tg-rich remnant particles, and ( ) stimula- tion of the incorporation of tg into vldl in the liver. each of these roles, which have been demonstrated by in vitro assays or by studies in cells and mouse models, could have a significant impact on the efficacy of therapeutic agents that partially in- hibit apo ciii synthesis for treatment of hypertriglyceridemia and for prevention of cvd. the most significant differences we observed between affected ct and unaffected cc sib-pairs were the higher in fcrs for vldl-tg and vldl-apob in the ct. the % lower circulating apo ciii levels in the r x carriers resulted from the doubling of the rate of clear- ance of tg-rich apob -lipoproteins from the bloodstream. the clearance of vldl occurs as a -stage process. first, lpl hydrolyzes the tg in nascent vldl as these particles circu- late through the vascular beds of adipose tissue and muscle, generating what is typically designated as a remnant that still contains less tg, but enough to keep its density < . (within the vldl range). these particles circulate back to the liver, which is believed to be the site of the second stage. the latter actually has components; comprises additional lipolysis of remnant tg by lpl, and probably more importantly hepatic lipase, resulting in conversion of vldl to idl and ldl. the second component involves interaction of apob with one or more receptors and proteoglycans on the cell surface of the liver, resulting in internalization of the remnant particle. – importantly, it is not clear whether these are completely in- dependent parallel pathways or if some additional lipolysis is necessary before remnant uptake by the liver occurs. our kinetic data do not allow us to differentiate between these possibilities and, therefore, we have made them independent, parallel pathways. our compartmental modeling of stable isotope enrich- ments of tg and apob in vldl, and of apob in idl and ldl, enabled us to determine the effect of partial loss of apo ciii on each of the second stage pathways. indeed, our results indicate that the rates of conversion of vldl particles to ldl were significantly greater in the carriers of r x. the findings that the fcr of idl-apob (which was not removed directly) was also significantly greater in the ct group supports higher rates of conversion of vldl to ldl, as does a nonsignificant, numerical increase in the pr of ldl-apob . all of the present results are in accord with those from our previous study in sisters with complete loss of apo ciii because of a large homozygous chromo- somal deletion, where we demonstrated marked increases in fcrs of vldl-apob and vldl-tg as well as normal or increased rates of conversion of vldl-apob to ldl. in figure. the figures depict the effects of partial loss of apo ciii (apolipoprotein ciii) synthesis of the metabolism of vldl (very-low-density lipoprotein). although the rates of secretion of vldl-apob and vldl triglyceride (tg; blue arrows) are similar in cc (unaffected siblings) and ct (affected siblings), the size of the vldl plasma pool (blue circles) is reduced by % in ct because an increase in lpl (lipoprotein lipase) mediated lipolysis of vldl-tg (black arrows) leads to a doubling of the fraction clearance rate of apob and tg from the vldl pool. the lipolysis of vldl-tg generates particles that either undergo conver- sion to idl (intermediate-density lipoprotein) and ldl (green arrows) after additional lipolysis of tg by lpl or hepatic lipase or direct removal by the liver (red arrows). the rate of conversion of vldl to ldl was significantly greater in ct compared with cc whereas the rates of direct removal were similar in the groups (table ). these data indicate that, in individuals heterozygous for loss of function of apoc , who have normal levels of lipoprotein lipase, reduced lev- els of apo ciii in plasma significantly affect the lipolytic but not the hepatic uptake pathways for metabolism of vldl. ffa indicates free fatty acids. d ow nloaded from http://ahajournals.org by on f ebruary , reyes-soffer et al effects of apo c mutation on lipid metabolism the present study, we did not find a significant effect of partial loss of apo ciii synthesis on the uptake of vldl remnants by the liver. this result might seem to conflict with results of re- cent studies of the efficacy of an apo ciii antisense in patients with no lpl, where the significant reductions in plasma tg levels observed would have required increased direct removal of tg-rich lipoproteins, presumably by the liver. however, whereas hepatic removal of tg-rich lipoproteins was the only pathway that would be susceptible for inhibition by apo ciii in patients lacking lpl, our participants would have both of the apo ciii-susceptible pathways available for participation in the metabolism of vldl. a simple explanation for our results might be that more efficient lpl-mediated lipolysis of nascent vldl resulted in smaller vldl that were more depleted of tg than post-lipolysis vldl in nonaffected indi- viduals and, therefore, more likely to be converted to idl and ldl by additional lipolytic activities during the second stage of vldl metabolism than to be internalized by ldl family receptor-mediated pathways present in the liver. , our results also contrast with recent studies of the effects of apo ciii antisense in mice with targeted deletion of lpl, ldl receptors, the ldl receptor-related protein- , and heparin sulfate proteoglycan receptors. their results led the authors to conclude that apo ciii inhibited hepatic uptake of remnant lipoproteins, but had little or no effect on lpl-mediated li- polysis of tg. although those studies were convincing, they were in mice, where apob -apoe enriched lipoproteins are predominant, and where hepatic removal of remnants is much greater than in humans. direct comparisons between the studies are, therefore, very limited. in contrast to the significant effect of reduced apo ciii on clearance of vldl-tg and vldl-apob , we did not find any evidence supporting a role for apo ciii in the incorporation of tg into nascent vldl within the liver. , our result agrees with data showing that treatment of apo ciii transgenic mice with an apo ciii antisense had no effect on rates of vldl secre- tion, and with our previous finding of normal vldl-apob and vldl-tg prs in the patients with complete absence of apo ciii. our results, indicating that partial loss of apo ciii has a significant impact on the lipolytic pathway but not on direct re- moval of vldl remnants, raise obvious questions about the po- tential of therapies that lower apo ciii levels. on the one hand, it is clear that the marked increase in vldl-tg fcr is consistent table . effects of apoc r x mutation on individual and mean fractional rates of vldl-apob clearance, conversion of vldl-apob to ldl, and direct removal of vldl-apob participant vldl-apob fcr, pools/d conversion of vldl- apob to ldl fcr, pools/d conversion of vldl- apob to ldl directly fcr, pools/d conversion of vldl- apob to ldl via idl fcr, pools/d direct removal of vldl-apob fcr, pools/d cc . . . . . cc . . . . . cc . . . . . cc . . . . . cc . . . . . mean±sd . ± . . ± . . ± . . ± . . ± . ct . . . . . ct . . . . . ct . . . . . ct . . . . . ct . . . . . mean±sd . ± . . ± . . ± . . ± . . ± . difference±sd . ± . † . ± . * . ± . * . ± . . ± . participants are defined as cc (unaffected siblings), ct (affected siblings), in order of sib-pairs. vldl: very-low-density lipoprotein, data derived from compartmental modeling of stable isotope enrichments obtained over a h. period. data are presented as means and sd of the absolute fcrs of vldl-apob , the conversion of vldl-apob , and the direct removal of vldl-apob , as well as the means and sd of the absolute differences in each parameter between the ct and cc groups. conversion of vldl-apob to ldl includes both conversion of vldl directly to idl as well as vldl to idl and idl to ldl. there was no direct conversion of vldl to ldl required to fit the kinetic data; all the conversion occurred through idl. statistical significance assessed using paired t tests. apob indicates apolipoprotein b ; fcr, fractional clearance rate; idl, intermediate-density lipoprotein; ldl, low-density lipoprotein; pr: production rate; tg, triglycerides; and vldl, very low-density lipoprotein. *significant p< . . †significant p< . . table . effects of apoc r x mutation on kinetic parameters for apo ciii and apo cii metabolism cc (±sd) ct (±sd) difference apo ciii fcr . ± . . ± . . ± . pr . ± . ± − . ± . apo cii fcr . ± . . ± . ± . pr . ± . ± . ± . participants are defined as cc (unaffected siblings) and ct (affected siblings). data are presented as means and sd. there were no significant differences for any of the variables between cc and ct groups or apo ciii and apo cii. fcr indicates fractional clearance rate; and pr, production rate. d ow nloaded from http://ahajournals.org by on f ebruary , arterioscler thromb vasc biol january with the dramatic reductions in tg levels observed in a study where patients with marked hypertriglyceridemia without muta- tions in lpl were treated with an antisense to apo ciii. those results indicate the utility of apo ciii-lowering therapy to pre- vent pancreatitis in such patients. in contrast, if therapies that reduce apo ciii synthesis result in higher rates of conversion of vldl remnants to ldl with no change or modest increases in ldl-cholesterol or apob levels, , how could that trans- late to risk for cvd? specifically, how do our findings relate to the decreased levels of coronary calcification in r x car- riers and very significant reductions in cvd risk observed in cohorts with loss of function variants in the apoc gene. , a simple answer is that reducing the number of vldl remnants, which carry more cholesterol per particle than ldl, is beneficial regardless of the mechanism involved. individuals with dyslip- idemia and the apo e / genotype accumulate both apob and apob remnants and have significantly increased risk for cvd. , amish individuals heterozygous for the apoc r x mutation and individuals with the combined deletion of apoa /apoc /apoa /apoa had decreased postprandial tg levels, , which have been shown to be atherogenic. , more dramatic reductions in postprandial lipid levels were reported recently in a small number of individuals homozygous for the r x mutation in apoc . of note, individuals in that study who were either heterozygotes or homozygoutes for r x had ldl-cholesterol levels that were similar to noncarriers. a re- cently published study comparing the roles of remnant choles- terol versus ldl-cholesterol in reduced cvd risk in individuals with loss of function of apoc found that lower concentrations of remnant cholesterol accounted for nearly all of the benefit of lower apo ciii levels. importantly, genetic stud- ies have demonstrated that gain of function variants in the lpl gene are associated with reduced risk for cvd, whereas loss of function variants in this gene are associated with increased risk. , our stable isotope kinetic studies of apo ciii and apo cii showed the expected lower apo ciii pr in the ct compared with the cc group, but no difference in apo cii pr between affected and unaffected siblings. these results are in accord with the isolated r x mutation in the ct group and indica- tive of independent regulation of the expression of the apoc and apoc genes which are on different chromosomes. in con- trast, the fcrs of both apo ciii and apo cii were greater in the affected compared with the unaffected siblings, suggestive of a common pathway for the clearance of these apolipopro- teins from the plasma, possibly along with vldl remnants. the fcrs of apo ciii and apo cii are greater than those of both ldl-apob and hdl apo ai, and slower than the fcrs of vldl- and idl-apob . however, as both apo ciii and apo cii are associated with all the major lipoproteins, a model that has reservoirs of apo ciii and apo cii on hdl that would feed a catabolic pathway through vldl remnants seems reasonable to explain our results. a recent article describing the effects of a rare mutation in apoc that affected the binding of the apo- lipoprotein to lipoproteins resulted in clearance of apo ciii via the kidney. we previously demonstrated a role of the kidney in the catabolism of apo ai in hypertriglyceridemic individu- als. while we cannot rule out involvement of the kidney in the increased catabolism of apo ciii and apo cii in the ct group, evidence for a significant pool of free apo ciii or free apo cii in plasma is lacking, , and the mutation in apoc in our ct subjects results in decreased secretion with no evidence of al- tered lipoprotein distribution of apo ciii synthesized by the normal allele. of note, the lower levels of apo cii in the ct group did not seem to restrict or limit the effect of low apo ciii on vldl-tg clearance. this is consistent with the lack of any alteration in plasma lipid levels in heterozygotes for apo cii de- ficiency. despite uncertainties about the effects on cvd risk, which will require large randomized, placebo-controlled cvd outcome trials to resolve, the very large reductions of plasma tg levels observed with apo ciii antisense indicate that thera- pies to significantly lower apo ciii concentrations in the circu- lation could add a new, potent approach for the prevention of pancreatitis in patients with severe hypertriglyceridemia. we also realize that other proatherogenic or proinflammatory effects of apo ciii have been reported, and these may also be reversed by therapies that reduce apo ciii levels. , study limitations this study included a small number of participants, a short- coming that we attempted to alleviate by using sib-pairs. we also did not address the heterogeneous nature of apob - lipoproteins, particularly related to the presence of apo ciii on only a portion of vldl, idl, and ldl, demonstrated first by alaupovic et al and then in a series of stable isotope kinetic studies by zheng et al, mendivil et al, and sacks et al. the finding that there are apob -lipoproteins with and without apo ciii could have an important implication for the present study. we would note, however, that sacks et al have reported that % to % of vldl from individuals with normal tg levels contain to apo ciii molecules per particle. thus, it seems likely that a % reduction of apo ciii in r x carriers would not significantly alter the propor- tion of vldl that containing significant numbers of apo ciii molecules, and that the affected and unaffected siblings would have relatively similar proportions of vldl with and without apo ciii. our finding that the proportions of both apo ciii and apo cii in vldl were similar in affected and unaffected siblings suggests similar proportions of apo ciii-containing vldl in both groups. importantly, we acknowledge that the metabolic differences we are reporting between affected and unaffected siblings may not completely mimic the effects of a therapeutic agent that reduces levels of plasma apo ciii. conclusions the present studies demonstrate the physiological effects of % reductions in plasma apo ciii resulting from heterozy- gosity for a naturally occurring loss of function mutation in the apoc gene. lower apo ciii levels in the circulation resulted in higher rates of lipolysis of vldl-tg and higher rates of conversion of vldl to ldl in the affected siblings. we did not observe changes in the rate of direct remnant re- moval of vldl remnants by the liver or in rates of secretion of vldl. our results, together with those from cohorts with loss of function variants in the apociii gene, provide evi- dence for the increased atherogenicity of vldl (and chylo- micron) remnants, as well as support for therapies that would reduce remnant concentrations regardless of the mechanism d ow nloaded from http://ahajournals.org by on f ebruary , reyes-soffer et al effects of apo c mutation on lipid metabolism involved. the impact of loss of function variants in apo ciii might be of particular importance in the postprandial period. acknowledgments we are grateful for the efforts and support of the amish research clinic nurses, technicians, and staff in lancaster, pa. this study would not have been possible without the outstanding support of the amish research participants. we also thank drs maryam khavandi and marie maraninchi for their assistance in the laboratory processing of the samples and the irving institute for clinical and translational research bionutrition unit for developing and providing all study meals. sources of funding this study was funded by the national institutes of health: r -hl (pollin), r hl (ginsberg), and kl tr (reyes-soffer). additional support was provided by national institutes of health/national center for advancing translational science: ul tr , the mid-atlantic nutrition obesity research center (p dk ), and the geriatric research, 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; : – . doi: . /j.jacl. . . . jensen mk, rimm eb, furtado jd, sacks fm. apolipoprotein c-iii as a potential modulator of the association between hdl-cholesterol and inci- dent coronary heart disease. j am heart assoc. ; :jah -e . . mendivil co, rimm eb, furtado j, chiuve se, sacks fm. low- density lipoproteins containing apolipoprotein c-iii and the risk of coronary heart disease. circulation. ; : – . doi: . /circulationaha. . . alaupovic p, mcconathy wj, fesmire j, tavella m, bard jm. profiles of apolipoproteins and apolipoprotein b-containing lipoprotein particles in dyslipoproteinemias. clin chem. ; ( b):b –b . . zheng c, khoo c, ikewaki k, sacks fm. rapid turnover of apolipopro- tein c-iii-containing triglyceride-rich lipoproteins contributing to the formation of ldl subfractions. j lipid res. ; : – . doi: . /jlr.p -jlr . mendivil co, zheng c, furtado j, lel j, sacks fm. metabolism of very- low-density lipoprotein and low-density lipoprotein containing apolipo- protein c-iii and not other small apolipoproteins. arterioscler thromb vasc biol. ; : – . doi: . /atvbaha. . . sacks fm. the crucial roles of apolipoproteins e and c-iii in apob li- poprotein metabolism in normolipidemia and hypertriglyceridemia. curr opin lipidol. ; : – . highlights • apoc r x null mutation causes % lower levels of plasma apo ciii. • the changes in apo ciii levels were because of a doubling of the rate of clearance of tg-rich apob -lipoproteins from the bloodstream. • these results provide strong evidence that therapies which increase the efficiency of conversion of vldl to ldl, thereby reducing remnant concentrations, should reduce the risk of cardiovascular disease. d ow nloaded from http://ahajournals.org by on f ebruary , multiomics and systems biology are needed to unravel the complex origins of chronic disease challenges commentary multiomics and systems biology are needed to unravel the complex origins of chronic disease david martino ,* , rym ben-othman , danny harbeson and anthony bosco ,* telethon kids institute; university of western australia, nedlands, wa , australia department of pediatrics, division of infectious diseases, university of british columbia, vancouver, bc v z m , canada; rbenothman@bcchr.ubc.ca (r.b.-o.); dannyharbeson@gmail.com (d.h.) * correspondence: david.martino@telethonkids.org.au (d.m.); anthony.bosco@telethonkids.org.au (a.b.) received: december ; accepted: march ; published: march ���������� ������� abstract: modernization has now been linked to poor developmental experience, the onset of immune dysregulation and rising rates of chronic diseases in many parts of the world. research across the epidemiological, clinical, and basic science domains supports the concept that poor developmental experience, particularly during prenatal life, can increase the risk of chronic disease, with enduring effects on long-term health. single ‘omics’ approaches are ill-suited to dealing with the level of complexity that underpins immune dysregulation in early life. a more comprehensive systems-level view is afforded by combining multiple ‘omics’ datasets in order to delineate correlations across multiple resolutions of the genome, and of the genomes of the microorganisms that inhabit us. in this concept paper, we discuss multiomic approaches to studying immune dysregulation and highlight some of the challenges and opportunities afforded by this new domain of medical science. keywords: multiomics; dysregulation; immune system; development; modernity . introduction over the past two centuries, environmental changes associated with the transition to modernity have brought about major demographic, epidemiological, and ecological changes which have had profound effects on human health [ ]. improvements in public health, hygiene, food security, transport, and communication, plus increasing population growth, declining global fertility rates, global migration, and increased exposure to industrial pollutants have had major effects on human immune development and the microbiota. on the scale of human evolution, spanning hundreds of thousands of years, these changes virtually happened yesterday morning, and are now associated with rising rates of chronic non-communicable diseases (ncds) across the globe, due to mismatches between our evolved capacities and modern environments [ ]. ncds now account for the major causes of human mortality across the globe, whereas previously infectious disease constituted the major burden of human mortality [ ]. immune development is a target for the adverse effects of modernization since chronic inflammation and immune dysregulation are common risk factors for a range of ncds, including allergy and autoimmune disease, atherosclerotic disease, diabetes, and cancer, and emerging evidence now suggests links to neurological disease (reviewed in [ ]). efforts to understand the mechanistic basis for immune dysregulation and ncds suggest complex multifactorial pathways of disease induction. given the multiple pathways through which immune trajectories can be manipulated by the sociodemographic changes associated with modernity, there is a need to embrace data-driven approaches of increasing complexity to better enable precision medicine. traditional reductionist approaches are ill-suited to dealing with the emergent properties of complex biological systems, and even single ‘omics’ medicine is proving ill-equipped to meet these challenges [ ]. in this article, challenges , , ; doi: . /challe www.mdpi.com/journal/challenges http://www.mdpi.com/journal/challenges http://www.mdpi.com https://orcid.org/ - - - http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /challe http://www.mdpi.com/journal/challenges challenges , , of we discuss the potential opportunities afforded by integrated multiomic approaches powered by pattern-finding algorithms to better understand the basis for immune dysregulation over a life course. . perinatal influences on immune development and chronic disease it is now recognized that the first thousand days of life between conception and age two are a crucial period of heightened plasticity, in which the developing brain and the immune system are highly sensitive to influences from the environment [ ]. poor developmental experience brought about by adverse exposures (e.g., maternal stress, poor nutrition, infections, toxins and pollutants, alcohol, tobacco smoke, and antibiotics) during the perinatal period have a greater capacity to alter developmental trajectories, potentially increasing the risk for chronic diseases [ ]. preterm birth is a prototypical example of a suboptimal developmental experience with well-described heath consequences in later life. recent findings from a multiomic study of preterm neonates supports the notion of altered developmental trajectories. in this study, cellular immune profiles, blood transcriptomes, plasma proteins, and microbiome profiles were assessed at birth in cord blood, and at , , and weeks of age in a cohort of infants, delivered preterm or at term. these data, in combination with systems biology methods, demonstrated that preterm and term infants followed largely distinct developmental trajectories that converged by around three months of age. this convergence toward a stereotypical immune trajectory was ostensibly linked to microbial interactions in early life [ ]. microbial interactions have long been recognized as potent signals that promote immune development, and disruption of the microbial ecology brought by modernization is increasingly recognized as a key risk factor in chronic disease research [ , ]. given immune development is intimately coupled to microbial signaling, dysbiosis of the microbiome in early childhood has been linked to virtually every chronic disease of the modern era [ ]. nowhere has this been more clearly demonstrated than in the amish and hutterite story. the amish and hutterite populations living in the united states have a similar genetic background, diet, and lifestyle, with the exception that the amish use traditional farming methods and live in close proximity to their animals. by contrast, the hutterites employ highly industrialized farming methods, and the animals are housed in large facilities away from their homes. airborne dust samples collected from amish homes were found to have elevated levels of microbial endotoxin, which was associated with markers of immune maturation from sampled blood, and a four-fold reduction in the rates of childhood asthma and allergic disorders [ ]. accordingly, other studies have also reported that disruptions to the neonatal microbiome is linked to diabetes, asthma, necrotizing enterocolitis, inflammatory bowel disease, obesity, and various other inflammatory diseases, as recently reviewed by amenyogbe et al. [ ]. in addition to the microbiome, other exposures that also constitute poor developmental experience operative during a sensitive window of early development are likely to affect developmental plasticity, causing the induction of disease risk with long-term consequences for health [ , ]. changes in nutrition and lifestyles related to modernization have dramatically altered parental factors like age at conception, body mass at conception, metabolism, and stress with support from animal models and human studies, indicating these factors induce developmental programming of disease risk [ ]. epigenetic mechanisms have been proposed as key mediators of the effects of adverse developmental experience (reviewed in [ ]). critically, at the earliest stages of reproduction, the few cells forming the conceptus are fully exposed to conditions that disturb epigenetic mechanisms, leading to persistent alterations in embryonic gene expression that are heritable across cell divisions and alter developmental trajectories [ ]. these mechanisms have been proposed as exponents of rapidly rising community rates of ncds independently of genetic effects. . moving beyond reductionist biology: systems-level understanding of immune dysregulation elucidation of the mechanisms underlying perinatal induction of immune dysregulation will be extremely challenging because biological phenotypes are emergent properties of highly complex challenges , , of and dynamic interactions between a large number of molecular components [ ]. the advent of high-throughput sequencing and mass spectrometry now enables the profiling of these molecular components across multiple layers of regulation (genome, epigenome, transcriptome, proteome, metabolome, and microbiome). these omic technologies have proven invaluable for enhancing our understanding of the individual layers of regulation, however, efforts are underway to combine these individual layers into a more comprehensive multiomic view of the entire system. parallel initiatives are underway to develop the tools to more comprehensively measure the exposome, which encompasses the totality of the environmental exposures encountered by an individual over their life course [ , ]. we anticipate that incorporation of individual omic data into multiomic space in combination with the exposome will enable a greater understanding of complex biological systems. these systems biology approaches will be necessary in order to make sense of the complexity of interactions that govern immune development and dysregulation. systems biology begins with the understanding that cellular behavior and function are emergent properties of complex and dynamic interactions between genes and biomolecules. emergent properties are characteristics of the whole system that are not present in the individual molecular components and, hence, complex systems are more than the sum of their parts. the application of network graph theory to the study of complex systems has revealed that biological systems are governed by a set of universal organizing principles, or common design elements that can also be observed in man-made systems, such as power grids and the world wide web [ ]. complex biological systems have a scale-free architecture (figure ), where most genes are interlinked with a few genes, and a few genes interact with many, giving rise to hubs [ – ]. challenges , elucidation of the mechanisms underlying perinatal induction of immune dysregulation will be extremely challenging because biological phenotypes are emergent properties of highly complex and dynamic interactions between a large number of molecular components [ ]. the advent of high- throughput sequencing and mass spectrometry now enables the profiling of these molecular components across multiple layers of regulation (genome, epigenome, transcriptome, proteome, metabolome, and microbiome). these omic technologies have proven invaluable for enhancing our understanding of the individual layers of regulation, however, efforts are underway to combine these individual layers into a more comprehensive multiomic view of the entire system. parallel initiatives are underway to develop the tools to more comprehensively measure the exposome, which encompasses the totality of the environmental exposures encountered by an individual over their life course [ , ]. we anticipate that incorporation of individual omic data into multiomic space in combination with the exposome will enable a greater understanding of complex biological systems. these systems biology approaches will be necessary in order to make sense of the complexity of interactions that govern immune development and dysregulation. systems biology begins with the understanding that cellular behavior and function are emergent properties of complex and dynamic interactions between genes and biomolecules. emergent properties are characteristics of the whole system that are not present in the individual molecular components and, hence, complex systems are more than the sum of their parts. the application of network graph theory to the study of complex systems has revealed that biological systems are governed by a set of universal organizing principles, or common design elements that can also be observed in man-made systems, such as power grids and the world wide web [ ]. complex biological systems have a scale-free architecture (figure ), where most genes are interlinked with a few genes, and a few genes interact with many, giving rise to hubs [ – ]. figure . emergent properties of biological networks. scale-free networks follow a power-law degree distribution, meaning that most nodes have few links, and a few nodes have many links. modules are subnets comprised of proteins that function in the same pathway. the innate immune system employs a bow-tie architecture to integrate diverse signals from a fluctuating environment. as biological networks evolve and add new components, the probability of adding new links to an existing component is proportional to the number of links it already has, and this generates hubs through a mechanism known as preferential attachment or rich-get-richer [ ]. biological networks are also modular, meaning that genes which function in the same biological process form communities in the network structure (figure ). smaller modules are embedded within larger modules, giving rise to hierarchical organization. biological networks are small world, meaning that you can “hop” through the network from one gene to any other gene in a few steps. finally, a key figure . emergent properties of biological networks. scale-free networks follow a power-law degree distribution, meaning that most nodes have few links, and a few nodes have many links. modules are subnets comprised of proteins that function in the same pathway. the innate immune system employs a bow-tie architecture to integrate diverse signals from a fluctuating environment. as biological networks evolve and add new components, the probability of adding new links to an existing component is proportional to the number of links it already has, and this generates hubs through a mechanism known as preferential attachment or rich-get-richer [ ]. biological networks are also modular, meaning that genes which function in the same biological process form communities in the network structure (figure ). smaller modules are embedded within larger modules, giving rise to hierarchical organization. biological networks are small world, meaning that you can “hop” through the network from one gene to any other gene in a few steps. finally, a key challenge for the immune system is to interpret and respond to diverse and fluctuating signals in order to maintain homeostasis. challenges , , of this is achieved through a bow-tie signal processing control system [ ]. the bow-tie structure receives diverse input signals and processes them through a core, and this generates complex output signals (figure ). the bow-tie architecture is an important conceptual advance in our understanding of immune dysregulation because genetic or environmental perturbation of the input and output layers will generate lots of noise and complexity but, ultimately, a limited number of core pathways control everything [ , ]. . utilizing systems biology approaches for very early prediction and intervention for immune-mediated diseases very early identification of high-risk children before they develop chronic diseases is extremely challenging because of the multitude of contributory genetic, environmental, and lifestyle factors. recent foundational multiomic studies have begun to pioneer the approaches to identify disease and transitions and intervene early. in a landmark study, price et al. followed healthy adults for nine months, and collected biological samples (saliva, blood, urine, stool) every three months [ ]. multiomic profiles were generated, which included whole genome sequencing, s rrna gut microbiome sequencing, clinical diagnostic tests, proteins, and metabolites. the multiomic assays were designed to assess five health domains (cardiovascular, diabetes, inflammation, nutrition and toxins, stress). the genome sequencing data was summarized into polygenic risk scores for disease traits. these risk scores are a single variable that estimates the genome-wide risk for a given trait by summing the number of risk alleles for each individual, weighted by effect size estimates from large genome-wide association studies (gwas) [ ]. the data were integrated by constructing an interomic correlation network, which captures pairwise interrelationships between the five omic layers. the α-diversity (species richness) of the gut microbiome was positively correlated with height and β-nerve growth factor levels, and negatively correlated with levels of csf- , il- , and flt ligand. clinical diagnostic tests identified deviations from wellness or test results outside of normal reference ranges [ ]. these insights were then leveraged to suggest evidence-based changes to diet (including supplements) and lifestyle (exercise, stress management) that resulted in significant improvements to biomarker levels across multiple health domains—type diabetes (fasting glucose, hba c levels, insulin), cardiovascular disease (total cholesterol, ldl cholesterol), inflammation (il- , tnf), and toxins (mercury). chronic obstructive pulmonary disorder (copd) is a highly heterogeneous, chronic, inflammatory lung disease which has early life origins in a subset of patients [ ]. in a proof-of-concept study, li et al. evaluated the utility of multiomic data to differentiate between copd patients, healthy non-smokers, and smokers with normal lung function [ ]. they found that the mean accuracy of subgroup prediction (healthy, smoker, copd) was extremely poor when each of the omic data blocks were analyzed in isolation. however, combining data from multiple omic platforms increased the mean prediction accuracy to %, even when group sizes were limited to small numbers. these analyses highlight the potential for multiomic approaches to dramatically improve our understanding of highly complex and heterogeneous inflammatory diseases. very early identification of at-risk individuals from birth is now theoretically possible with polygenic risk scores [ ]. these risk scores combine information derived from variants across the entire genome [ ], and are able to identify segments of the population which are at heightened risk (more than three-fold) for a range of complex traits including inflammatory diseases [ ]. one caveat of polygenic risk scores is that information is collapsed across the genome without taking into account the cellular or biological context. to address this issue, cluster analysis of deep immunological and clinical data can be utilized to stratify subjects into distinct developmental trajectories [ ], and the polygenic risk scores can be leveraged to find those clusters enriched for subjects at heightened genetic risk. extending this approach to multiomic layers will undoubtedly increase the resolution of these analyses and further refine the critical windows of opportunity for early intervention. challenges , , of . multiomic studies: challenges and opportunities . . sample collection multiomic studies have been successfully applied to adults to identify molecular correlates of disease risk [ , ]. the application of these tools to early infancy is itself challenging because only limited volumes of blood can be collected, and collection of fasting blood is not feasible. despite this, protocols for small volume collections yielding high-quality data have been developed and published. a recent study of immune cell populations and plasma protein immunoassays was performed on µl of whole blood [ ]. whole blood represents a convenient and attractive tissue for multiomic studies that is amenable to fractionation into different aliquots for individual omic platforms that can be integrated following data generation. the caveat of whole blood is that it is a complex tissue, and omic profiles therefore represent an average profile across all cells in the blood sample, which can limit analyses of cell specific effects. despite this, whole blood is likely to be the tissue of choice for multiomic studies with protocols in development that allow immune profiling, genetics, transcriptomics, epigenetics, metabolomics, and exposome profiling from less than ml of blood. in order to ensure data veracity, it is crucial to stringently standardize the processes of sample preparation even before considering the different omic assays. this strategy starts with sample processing, protocol testing, optimization, and establishment of gold standard procedures and, importantly, technological and biological controls. this focus on quality control includes assurance of lot-consistent performance of supplies and reagents allowing minimal deviations to ensure pristine data collection, compilation, assessment and, finally, analysis (figure ). small differences in materials and supplies or deviations in procedure across sites can entirely derail a multiomic study. challenges , . . sample collection multiomic studies have been successfully applied to adults to identify molecular correlates of disease risk [ , ]. the application of these tools to early infancy is itself challenging because only limited volumes of blood can be collected, and collection of fasting blood is not feasible. despite this, protocols for small volume collections yielding high-quality data have been developed and published. a recent study of immune cell populations and plasma protein immunoassays was performed on μl of whole blood [ ]. whole blood represents a convenient and attractive tissue for multiomic studies that is amenable to fractionation into different aliquots for individual omic platforms that can be integrated following data generation. the caveat of whole blood is that it is a complex tissue, and omic profiles therefore represent an average profile across all cells in the blood sample, which can limit analyses of cell specific effects. despite this, whole blood is likely to be the tissue of choice for multiomic studies with protocols in development that allow immune profiling, genetics, transcriptomics, epigenetics, metabolomics, and exposome profiling from less than ml of blood. in order to ensure data veracity, it is crucial to stringently standardize the processes of sample preparation even before considering the different omic assays. this strategy starts with sample processing, protocol testing, optimization, and establishment of gold standard procedures and, importantly, technological and biological controls. this focus on quality control includes assurance of lot-consistent performance of supplies and reagents allowing minimal deviations to ensure pristine data collection, compilation, assessment and, finally, analysis (figure ). small differences in materials and supplies or deviations in procedure across sites can entirely derail a multiomic study. figure . production workflow for sample collection in multiomic studies. standardization of sample collection can be systematically undertaken using a production workflow. for each sample type, protocols are developed and tested according to pre-determined key performance indicators (kpis) before moving into production. kpis are determined by investigators but could include standard quality control metrics published in the medical literature. the process is iterative, so the methodology is optimized if kpis are not achieved, and proceed through re-testing until targets are met. . . data collection researchers should identify data which may be important to downstream quality control. for instance, tracking the information about storage conditions of a sample, or the time it takes for the sample collection, not only informs about its quality but also can help discriminate outliers from subjects showing meaningful biological differences [ ]. it is also very important to record the right metadata about the study subjects as this will be one of the major components of the multiomic data analysis. correlations between multiomic data and clinical metadata often make up a major portion of the analysis, which is why it is extremely important to put careful consideration into what information will be collected prior to enrollment of the participants [ ]. these clinical metadata are study-specific and depend on the nature of the disease or the biological question asked. the figure . production workflow for sample collection in multiomic studies. standardization of sample collection can be systematically undertaken using a production workflow. for each sample type, protocols are developed and tested according to pre-determined key performance indicators (kpis) before moving into production. kpis are determined by investigators but could include standard quality control metrics published in the medical literature. the process is iterative, so the methodology is optimized if kpis are not achieved, and proceed through re-testing until targets are met. . . data collection researchers should identify data which may be important to downstream quality control. for instance, tracking the information about storage conditions of a sample, or the time it takes for the sample collection, not only informs about its quality but also can help discriminate outliers from subjects showing meaningful biological differences [ ]. it is also very important to record the right metadata about the study subjects as this will be one of the major components of the multiomic data analysis. correlations between multiomic data and clinical metadata often make up a major portion of the analysis, which is why it is extremely important to put careful consideration into what information will be collected prior to enrollment of the participants [ ]. these clinical metadata are study-specific challenges , , of and depend on the nature of the disease or the biological question asked. the identification of the metadata types can be challenging when looking at the impact of sociodemographic changes and modernity on immune development dysregulation. for instance, a study of immune development of neonates should consider prenatal and parental variations in environments, nutrition, lifestyle, age at conception, delivery mode, and many other factors. all of these could be a driving force behind different immune signatures revealed by multiomic analysis. by combining data types, we should be able to see the full spectrum of the global impact of the environment changes on immune development in an unprecedented manner, by leveraging the power of data integration [ ]. . . data management appropriately managing the generated information, collecting the appropriate metrics, centralizing the analysis pipelines, and standardizing the entire process are key fundamental steps that are not easy to implement in large multiomic studies. a first line challenge in any multiomic study involves devising an appropriate strategy to store, index, organize, audit, distribute, and archive vast amounts of big data. initiatives such as the open-source integrated rule orientated data systems (irods) provide an overall framework for data management tasks and allow complete management of primary and derived data with rules and policies to ensure reproducibility of the research [ ]. this includes attaching clinical metadata and metrics, covering the entire range of steps from the bio-sample collection to processing of omic data files. without a pre-defined strategy to build a common infrastructure for data storage, accessibility, and analysis (e.g., code reviewing), the data integrity backing the biological insight can quickly be jeopardized. in fact, the massive amount of data generated needs to be first verified for quality control purposes, and cleaned prior to being analyzed in a transparent and reproducible manner amongst the multiomic study experts. a second challenge involves the integration of clinical patient data with high-dimensional omic datasets. software that enables integration of curated phenotypic data from clinical observations with biomarker data from gene expression and genotyping studies are emerging [ , ]. in order for multiomic studies to be reproducible, these computing and software infrastructures will be mission-critical over the long term. . . data analysis bioinformatic approaches to extract meaning from multiomic data is an obvious challenge. most of the unsupervised data integration methods require cutting edge tools and models using extensive computing capabilities to develop new algorithms and theoretical methods that fit the different layers of omic datasets [ , ]. the challenge for data integration is to apply the appropriate bioinformatic approaches to the study type and data collected. moreover, the different omic datasets have a distinct format, size, and dimensionality, which represent one of the major computational challenges when it comes to data integration [ ]. advances in the field of data integration have made it possible to start generating guidelines on data integration approaches depending on the size of the dataset and its heterogeneity [ ]. these methods and approaches are still in their infancy and, at present, highly specialized, but are anticipated to become more established as more researchers enter the multiomic space. . concluding remarks post-industrial changes to the environment are associated with rising rates of chronic non-communicable diseases in many parts of the world. changes in the microbiome and the effect on immune development are firmly implicated, given the development of the microbiome in early life is particularly sensitive to even minor disturbances. given the complexity and multifactorial nature of immune development, systems-level approaches are now needed to delineate trajectories of immune ontogeny. we posit, therefore, that integrative omics is expected to become increasingly influential for disease prediction, diagnosis, prevention, and prognosis. while these approaches are still in their infancy, the future development of multiomic workflows and community standards will challenges , , of be necessary milestones toward capitalizing on the potential benefits of multilevel data integration. at present, the only cases of omic technologies that have translated to the clinic include genome sequencing and, to a lesser extent, rna sequencing. substantial regulatory and technical hurdles exist before other omic techniques will be approved for clinical use. therefore, in the short-to-medium term, we anticipate integrative omics and associated models of disease risk will enhance the research enterprise and enable a clearer picture of health and disease. the initial stages toward uptake will need to initially demonstrate novel actionable insights and then prove through rigorous trial-based testing that early interventions designed from multiomic data do indeed provide tangible clinical benefits. in parallel, the data standards and precision around individual omic platforms will need to comply with levels acceptable for clinical tests in order to transition into the healthcare setting. although the challenges are substantial, the potential benefits of multiomic studies necessitate research and development. author contributions: writing—original draft preparation, d.m., r.b.o., d.h., a.b. writing—review & editing, d.m., r.b.o., d.h., a.b. funding: this research received no external funding. conflicts of interest: the authors declare no conflict of interest. references . renz, h.; holt, p.g.; inouye, m.; logan, a.c.; prescott, s.l.; sly, p.d. an exposome perspective: early-life events and immune development in a changing world. j. allergy 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[crossref] [pubmed] © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . /omi. . http://dx.doi.org/ . / - - -s -i http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . / - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.atg. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /nar/gky http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /journal.pcbi. http://dx.doi.org/ . /bioinformatics/bty http://dx.doi.org/ . /nmeth. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /rsif. . http://www.ncbi.nlm.nih.gov/pubmed/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	perinatal influences on immune development and chronic disease 	moving beyond reductionist biology: systems-level understanding of immune dysregulation 	utilizing systems biology approaches for very early prediction and intervention for immune-mediated diseases 	multiomic studies: challenges and opportunities 	sample collection 	data collection 	data management 	data analysis 	concluding remarks 	references book reviews tion to technological detail. t good reference material but fox ing. the index is appalling and; to list dyskinesia, dystonia, on- receptors. some can be found ings if you search for them. it such blemishes in an otherwist tion, but i think uk readers prefer gerald stem's recent though not confined to therapy its practical issues, and is a mo: work of reference. his makes for clarity-that is all except th r boring read- author himself points out, th inter alia, fails may be destroyed while th -off effects, or remains intact and the term "t as sub-head- "brain stem death" seems moi is sad to find the best way to practise e useful colla- anticipate problems before the at least, will way, complications are preve text, which effects if they occur are mi embraces all book approaches the subject re satisfactory manner. i am sure neurosurgei and in particular trainees, jms pearce valuable. it should sell well d relatively expensive for its another edition along the sam vascular topics should be cor future. some pitfalls and problems in neurosurgery. series: progress in neurological surgery, vol. . editor: j c de villiers. (pp ; price: £ . .) basel, karger, . this book is the thirteenth in the series "progress in neurological surgery." unlucky for some, perhaps, but the thir- teenth edition which covers transphenoidal operations, frontal and temporal basal tumours, csf shunts, spinal intramedullary lesions, lumbar discs, and brain death, should not prove a disappointment to the editor, dr de villiers. "pitfalls" are interpreted as a complication which may result from incorrect interpretation of clinical signs and investiga- tions, or occur as a result of the operative procedure. the first chapter is an excellent review of the problems encountered with the radiology of pituitary tumours and transphenoidal sur- gery. the text accurately describes the nasal complications following operation (usually omitted from other descriptions) and advises on the ways of minimising these. surpris- ingly, the author fails to mention the need for steroid cover during an anaesthetic or operative procedure in these patients with potential pituitary insufficiency. there follows a rather disappointing chapter on frontal and basal tumours. the radiological examples are all based on angiography, air encephalography and early generation ct scanning. much information with useful advice is buried within a very detailed text which makes heavy reading. again there is no emphasis on the need for steroid cover during operation on suprasellar tumours. shunt complications bedevil us all. a chapter on the ways of minimising these provides practical advice. perhaps the text should have included some mention of the advantages and dis- advantages of the newer variable pressure valves. the next chapter provides helpful infor- mation on spinal intramedullary tumours, incorporating a detailed description of sur- gical technique. a subsequent chapter on lumbar disc prolapse is full of gems often omitted from more standard texts. interest- ing text, however, inevitably contains con- troversy. i was surprised by an introductory sentence stating that "confirmatory tests" (i.e. confirming the clinical findings) "will be required by the majority". surely in this day and age, all patients should undergo at least a ct scan if not myelography? classic root signs do not necessarily result from disc protrusion at the expected level. the "out- patient" operative procedure for disc removal will also raise some eyebrows. the final chapter on the diagnosis of cerebral death by the editor is a model of e title. as the ie "cerebrum" ie brain stem brain death" or re appropriate. surgery is to ty arise. in this nted and their inimised. this in a pragmatic ons at all levels, , will find it lespite seeming size. perhaps e lines covering nsidered in the kw lindsay pain syndromes in neurology. edited by howard l fields. (pp ; price: £ . .) guildford, butterworth scientific ltd. isbn - - - . . much of the most severe chronic intractable pain seen in pain clinics is neuropathic rather than nociceptive pain and whereas mechan- isms of pain with an intact nervous system have been extensively investigated, mechan- isms of neuropathic pain are poorly under- stood, and have received much less attention. this book is particularly concerned with these mechanisms of neuropathic pain and with treatment, but also considers peripheral nociceptive mechanisms and cancer pain, much of which has a nociceptive basis. the editor, fields, opens with a succinct over- view of pain transmission in the normal and damaged nervous system. this is an excellent introduction for the newcomer to this subject. raja and colleagues next consider hyper- algesia and receptor sensitisation, covering both neurophysiological and chemical aspects. devor and rappaport review experimental peripheral nerve injury. perhaps the single most important fact to emerge from all the work on peripheral nerve injury is that damage to sensory axons frequently leads to ectopic impulse generation, and this is a likely basis for at least some of the pain experienced with such lesions. burchiel considers the effects of deafferentation and this is linked with a discussion of the place of the dorsal root entry zone lesion operation described by nasholt. a prominent effect of deafferenta- tion is disinhibition of central neurones with the development of chronic abnormal repetitive neuronal discharges. this explains the common failure ofablative surgery. more damage may lead to more pain. a separate chapter by watson is devoted to post herpetic neuralgia, dealing with clinical features and treatment rather than possible underlying mechanisms of pain. the involvement of the sympathetic nervous system in peripheral nerve injury and in the obscure reflex sympathetic dystrophy syndromes is discussed in two chapters. roberts and kramis consider mechanisms and offer several interesting ideas but we are far from understanding these abnormal states. payne provides a clinical description of reflex sympathetic dystrophy and considers treatment options. the frequent total failure of treatment is under-emphasized here. asbury surveys pain in peripheral neuropathies, drawing attention to the different mechanisms. in a masterly review, tasker considers the place of surgery for pain, both of nociceptive and neuropathic type. stimulation procedures in spinal cord and brain are also discussed and there is brief mention of spinal and intraventricular opiate installation. the literature is comprehensively reviewed and the author admits, with honesty, the influence of personal bias. the unpredictability of results and variability of success of the same procedure in different surgical hands are the two main messages which emerge here. por- tenoy considers cancer pain, in which the multiplicity of possible causes and mechan- isms is stressed and thus the need for careful clinical assessment. the final chapter, again by portenoy deals with the drug treatment of chronic pain. neurologists and neurosurgeons will find much of interest and relevance to their clin- ical practice here. the authors have been well chosen and present difficult subjects clearly, and the book has the advantage ofbeing fairly short. it should find a place in all departmen- tal libraries. the book is not complete in its coverage; those wanting to find full accounts of pain of myelopathic, brainstem, or thalamic origin will be disappointed. these topics are only briefly considered in the chapter by tasker. perhaps correctly, in a book of this length, the emphasis is on the more common painful neurological condi- tions and this is a small criticism. overall, the book can be highly recommended. jw scadding plasticity and morphology of the cen- tral nervous system. a challenge for psychiatry of the nineties. edited by c l cazzullo, e sacchetti, g conte, g inver- nizzi and a vita. (pp ; price: dfi . ; us$ . ; uk£ . ). dordrecht: kluwer academic publishers group, . isbn - - - the title of this book is misleading. it is not about neuroanatomy at all, which is what one might expect. it is a heterogeneous collection ofpapers presented at a conference on schizo- phrenia in milan in . the majority ofthe papers are not even about schizophrenia. there is one on phenylketonuria, one on eating disorders and nine (out of ) on affective disorders. the topics covered include the genetics of depression among the amish population in pennsylvania, ct scan findings in schizophrenics, the description of a new battery of psychometric tests and glucose- -phosphate dehydrogenase deficiency in psychosis. altogether it is a curious mixture. equally puzzling is why it has taken three years for the book to be published, because it is not printed but an amalgam of different type-written manuscripts, a process which i had always assumed speeded up publication. more serious, though, is the fact that some of these contributions would not have passed the refereeing process of even a moderately pres- tigious journal. with five editors available one might have expected a higher standard. in fact, bypassing the refereeing process is not always a bad thing. i have long believed that the reason why we rarely seen papers of the quality of those written by german, french and british neurologists in the first o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn n p .b m j.co m / j n e u ro l n e u ro su rg p sych ia try: first p u b lish e d a s . /jn n p . . . -a o n d e ce m b e r . d o w n lo a d e d fro m http://jnnp.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ www.nmletters.org effective doping of rare-earth ions in silica gel: a novel approach to design active electronic devices d. haranath∗, savvi mishra, amish g. joshi, sonal sahai, virendra shanker (received june ; accepted august ; published online august .) abstract: eu + luminescence spectroscopy has been used to investigate the effective doping of alkoxide-based silica (sio ) gels using a novel pressure-assisted sol-gel method. our results pertaining to intense photolumi- nescence (pl) from gel nanospheres can be directly attributed to the high specific surface area and remarkable decrease in unsaturated dangling bonds of the gel nanospheres under pressure. an increased dehydroxylation in an autoclave resulted in enhanced red (∼ nm) pl emission from europium and is almost ten times brighter than the sio gel made at atmospheric pressure and ∼ ℃ using conventional stöber-fink-bohn process. the presented results are entirely different from those reported earlier for sio :eu + gel nanospheres and the origin of the enhanced pl have been discussed thoroughly. keywords: luminescence; nanophosphor; short-range order; electron microscopy citation: d. haranath, savvi mishra, amish g. joshi, sonal sahai and virendra shanker, “effective dop- ing of rare-earth ions in silica gel: a novel approach to design active electronic devices”, nano-micro lett. ( ), - ( ). http://dx.doi.org/ . /nml.v i .p - introduction luminescent materials have been utilized widely in applications involving lighting to sensing [ ]. the pho- toluminescence (pl) properties of silica have also been an important topic of research for a long time, but the difficulty in the incorporation of rare-earth (re) ions attached covalently to the silica (sio ) network is still considered a great challenge. the weak pl bands with peak energies ∼ . - . ev for both bulk and thin films of sio have been reported [ ]. the sol-gel method has been used to prepare nanomaterials in the form of powders, films, fibers and monoliths that are based on various metal alkoxides [ ]. it has been observed that monodisperse silica nanospheres formed by hydrolysis and condensation of alkoxides using stöber-fink-bohn (sfb) process gives negligible luminescence. incorpora- tion of inorganic luminescent centres into sfb spheres has been demonstrated by some research groups [ ] us- ing re ions, quantum dots and organic flurophores, but the procedure requires multiple processing steps and use of expensive and toxic ligands. during the last few years, there have been few reports [ - ] on intense pl emission in the visible region of the electro- magnetic spectrum, by several different nanostructured materials that are highly disordered such as nanowires, porous silicon, silica-based mesoporous fractals, ferro- electrics with abo type and awo -type perovskite structures (a=ca, ba, sr); and many more. the ori- gin of this type of luminescence is always attributed to unsaturated chemical bonds in these nanostructures. in case of ferroelectrics, as their energy band gaps are lo- cated ∼ - ev, the pl emission in the visible region was correlated to their highly disordered states and many localized electronic levels present within the op- tical gap, causing luminescence [ ]. since sio gel is a well-studied system [ ] having visible transparency and significant absorption peak lying in the ultra-violet national physical laboratory, council of scientific and industrial research, dr k s krishnan road, new delhi, , india *corresponding author. e-mail: haranath@nplindia.org nano-micro lett. ( ), - ( )/ http://dx.doi.org/ . /nml.v i .p - nano-micro lett. ( ), - ( )/ http://dx.doi.org/ . /nml.v i .p - region, the introduction of dopant ions act as a pertur- bation to the well-studied system leading to interesting optical properties. the re ions are used as probes in the sol-gel method due to their sensibility to change with the surrounding matrix probing the local struc- ture [ , ]. thus derived sio and organically modi- fied matrix composites could be the main precursor to prepare many re based smart optical materials [ ]. in this paper, we propose a novel methodology to pre- pare alkoxide-based silica gel nanospheres doped with eu + ions that show enhanced pl brightness, uniform size distribution and improved quantum efficiencies. this is a process by which highly disordered but doped silica gels could be effectively made useful for practi- cal applications involving luminescence. it has already been evidenced that high pressure and temperature leads to more closely packed structures [ ]. the pre- sented analogy is unique and could be extended to many crystalline and non-crystalline phosphor based systems to design new family of sol-gel based nanocomposites, having a wide variety of applications in lasers, chemi- cal sensing, waveguides, bioanalytical assays, blood flow monitoring and effectively harvesting the solar energy for improving the solar cell efficiency. experimental silica nanospheres were prepared with its surface modified by sol-gel method and studied through the incorporation of europium (iii) ions in two differ- ent ways. the europium nitrate (euno ) was mixed with sufficient ethanol (etoh) and this was added to tetraethylorthosilicate (teos). the stoichiometric amount of water, which is essential to carryout hydrol- ysis reaction, was added drop wise under continuous stirring. prior to gelling the low and high resolution transmission electron microscopy (tem) images of col- loidal silica solution (also called silica sol) were taken at a magnification of and kx, respectively as shown in fig. . the silica particles observed are al- most spherical with an average cluster size of ∼ nm. the schematic of the sio gel network [ ] and the probable description of pore and particle sizes were il- lustrated in fig. (c). the silica sol was then divided in two halves for a systematic experimentation. for the first experiment, one of the solution containing vials was allowed to gel at atmospheric pressure ( bar) and a controlled oven temperature of ∼ ℃ (± . ℃). in the second case the solution was kept in an auto- clave, subjected to high temperature and pressure at ∼ ℃ and bars, respectively for about hours. it was intentional to keep the processing time same for both the cases. once the wet silica gels were obtained, sio :eu + nanospheric powders were obtained by dry- ing the gels overnight in vacuum oven at ∼ ℃. all the samples were studied using morphology, composi- tion evaluation, uv-vis absorption and luminescence spectroscopy techniques. fig. (a) tem and (b) hrtem images of silica sol particles aged for hours at room temperature, ℃; (c) schematic illustration of gel network structure showing the pore and particle sizes (courtesy from ref. [ ]). for tem observations, the samples were redispersed in methanol by ultrasonic treatment and dropped on carbon-copper grids. tem images were collected using a tecnai g f s-twin (fei; super twin lens with cs= . mm) instrument operating at an accelerating voltage at kv, having a point resolution of . nm and lattice resolution of . nm) with an edax at- tachment. program digital micrograph (gatan) was used for image processing. scanning electron mi- croscopy was performed using zeiss evo ma . x-ray diffraction (xrd) of the powder sample was performed using bruker d- advance powder x-ray diffractometer with cukα radiation operated at kv and ma. all the samples showed the amorphous na- ture. x-ray photoelectron spectroscopy (xps) studies have been carried out using a perkin elmer model, at k with a non-monochromatic alkα line at . ev. during photoemission studies, small spec- imen charging was observed which was later calibrated by assigning the c s signal at ev. the room temperature photoluminescence (pl) spectra were recorded using an edinburgh lumines- cence spectrometer (model f ) equipped with a xenon lamp. the excitation and emission spectra were recorded in the fluorescence mode over the range of - nm. results and discussion it is important to highlight the pressure-assisted hy- drothermal/solvothermal process for the preparation of variety of nanoparticles of oxides and chalcogenide ma- terials. the solution based nanomaterial synthesis of- ten involves reactions carried out near the boiling point nano-micro lett. ( ), - ( )/ http://dx.doi.org/ . /nml.v i .p - of the solvent. this may lead to poor quality of the nanomaterial and less yield. in order to obtain crys- talline, monodisperse nanoparticles, it is always neces- sary to work at relatively high temperatures and pres- sures. use of acid digestion bomb (commonly called autoclave) is the best alternative to work with. de- tails of the autoclave synthesis of intrinsic silica gels have been reported extensively by haranath et al. since . in the current case, rare-earth (re) doped sil- ica gels were made under pressure at elevated temper- atures as described in latter sections. this method of preparation, which is based on pressure-assisted sol-gel method, has compatibility in modifying the coordinat- ing environment of re (dopant) ions so that the loss in energy of the excited states via. non-radiative mech- anism is minimum. the x-ray diffraction pattern of eu + doped sio gel powder depicted in fig. clearly shows a broad hump at ∼ ◦ indicating the complete amorphous nature of the sio nanospheres. sem im- ages shown in inset of fig. illustrate the quality of the nanospheres with respect to their size and shapes. in other words, the conventional sol-gel method leads to a broad distribution of particle sizes in the range - nm (fig. (a)) whereas the pressure assisted sol- gel method has resulted in almost uniform particles (∼ nm) with spherical shape (fig. (b)). this es- tablishes the fact that high pressure and temperature leads to more closely packed structures and increased charge transfer energies that are efficiently transferred to the eu + ions. the tem and scanning electron mi- crographs (sem) reveal that the optimization of exper- imental and processing parameters allow microscopic preparation of uniform silica nanospheres. fig. x-ray diffraction (xrd) pattern of sio gel pow- der at room temperature (∼ ℃) indicating the amorphous nature of the gel under study. the insets shows the sem micrographs of sio :eu gels prepared at (a) atmosphere pressure ( bar) and ℃; and (b) bars and ℃, re- spectively. moreover, the chemical composition and the relevant surface chemistry of sio :eu + nanospheres were an- alyzed using x-ray photoelectron spectroscopy (xps). the xps observations revealed three peaks correspond- ing to the elements si, o and eu, respectively. pass energy for general survey scan and core level spectra was kept at . and . ev respectively. sur- face contamination was removed by ar ions with kev beam energy. sputtering performed in raster mode with emission current of ma for min at base pres- sure . × − torr. figure shows the survey spectra of sio :eu + nanospheres acquired in the range of - ev. during photoemission studies, small specimen charging was observed, which was later calibrated by assigning the c s signal at ev. survey spectra af- ter sputtering show sharp peaks of c s ( ev), o s ( ev). two clear distinct state of eu were observed at and ev for eu d / and d / respec- tively. the separation between two states is because of spin-orbit splitting. inset of fig. shows the si( p) core level spectra. the value of elemental si( p) is . ev, since, appearance of si( p) at ev confirms the si exist in sio state. the binding energies of various elements match very well with the peaks observed for standard sio :eu +. the presence of c is due to the air atmosphere and the organics used for the preparation of eu + doped in silica matrix. fig. xps survey scan spectra of sputtered and nanopar- ticles of sio :eu + recorded with photon energy of alkα (hν= . ev), inset shows the si( p) core level. figure shows the uv-vis absorbance spectra of eu + doped sio gel samples prepared at ℃, bar; and ℃, bars, respectively. the spectra are the strong indicative two findings. one is being the reduc- tion of surface states of the gel nanospheres and other being the maintenance of almost the same size for the gel particles even after subjecting to high pressure and temperature cycles. the same has been evidenced by the absorption peaks indicated in the inset of fig. . nano-micro lett. ( ), - ( )/ http://dx.doi.org/ . /nml.v i .p - . . . . nm a b s. nm nm wavelength (nm) a b so rb a n ce ( % ) °c, bar °c, bar fig. absorbance spectra of eu doped-sio samples pre- pared at atmosphere pressure ( bar) and bars and ℃, respectively. inset shows the expanded version of the absorption peaks. the observation of weak photoluminescence (pl) at room temperature in various amorphous silica nanos- tructures has been reported in the literature [ - ] but not sufficient to use for any fundamental or potential application. figure shows the pl excitation (ple) and pl spectra from eu + doped and baked sio gel in an autoclave respectively. it is known that the sur- face states and unsaturated dangling bonds play a crit- ical role in determining the overall pl characteristics of nanostructures. if the silica gel is prepared with a rare-earth dopant (eu + in the present case), the pl is dominated by the radiative transitions ( d → fj, j= - ) from the levels of eu + ions [ ] as shown in the inset of fig. . the emission spectra of eu + doped sio gels prepared at ℃, bar; and ℃, bars, were shown in fig. . for recording the pl spectra the excitation energy has been fixed as nm which is the l level of eu + ligand band. the pl from the eu + doped sio gel prepared at atmospheric pressure ( bar) and ℃ was found to be weak and inefficient for any practical application, whereas the pl intensity from the gel turn out to be much stronger (> times) when the sol was gelled under high temperature ( ℃) and pressures ( bars) inside an autoclave. moreover, the ple spectrum also became narrower under auto- clave treated gel sample, which indicate that there is a remarkable decrease in unsatisfied chemical bonds in the final product. the most intense line at ∼ nm corresponds to the hypersensitive transition between the d and f level of the eu + ions and will be rel- atively strong if the surrounding symmetry is low. in the sense, it is generally admitted that the ratio of the emission intensities r=i( d → f )/i( d → f ) is an asymmetry parameter for the eu + sites and a mea- sure of the extent of its interaction with the surround- ing ligands [ ]. this indicates that the environment of the eu + is dictated by the nano-sio host under high pressure and temperature conditions. in addition, broad excitation peaks observed between - nm became distinct and sharp for the sample made under high pressures. the sharp peaks in the pl and ple spectra of sio :eu nanospheres may be due to quantum confinement effects related to size restrictions. a com- bination of unique features of high surface-to-volume ratios, monodispersion and strong photoluminescence suggest that these silica nanospheres will find many in- teresting applications in semiconductor photophysics, inorganic light emitting diodes, solar cells, environmen- tal remediations and optoelectronic devices. °c, bar °c, bar e n er g y , c m _ energy levels of eu + dj d _ f d _ f d _ f d _ f λex= nmλem= nm x ple wavelength (nm) x fj j= j= j= j= pl in te n si ty ( a rb .u n it s) fig. ple and pl spectra of sio :eu gels made at (a) ℃, bar and (b) bars and ℃, respectively. inset shows the mechanism of eu + transitions. conclusions in conclusion, we have demonstrated a mechanism by which photoluminescence enhancement in sio :eu + phosphor nanospheres could be successfully achieved using pressure-assisted sol-gel method. the observed red emission is from typical d − f transition of eu + and is found to be almost ten times brighter than the gel made at atmospheric pressure ( bar) and ∼ ℃ using stöber-fink-bohn process. this kind of process is highly desirable for many crystalline and non- crystalline materials system wherein the doping is inap- propriate. this may lead to design many fundamental and novel optoelectronic applications. acknowledgments the authors (dh, sm and ss) gratefully acknowl- edge the department of science and technology (dst), ministry of science and technology, government of in- dia for their respective fellowships under various spon- sored projects to carryout the work. nano-micro lett. 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[ ] w. j. zhang, x. l. wu, j. y. fan, g. s. huang, t. qiu and p. k. chu, j. phys. condens. matter , ( ). http://dx.doi.org/ . / - / / / 	introduction 	experimental 	results and discussion 	conclusions 	acknowledgments 	references brief communications nature genetics • volume • september bardet-biedl syndrome (bbs) is anautosomal recessive disorder with locus heterogeneity – . none of the ‘responsible’ genes have previously been identified. some bbs cases (approximately %) remain unassigned to the five previ- ously mapped loci . mckusick-kaufma syndrome (mks) includes hydrometrocol- pos, postaxial polydactyly and congenital heart disease, and is also inherited in an autosomal recessive manner , . we ascer- tained unrelated probands with classic features of bbs including retinitis pigmen- tosa (rp), obesity and polydactyly. the probands were from families unsuitable for linkage because of family size. we found mkks mutations in four typical bbs probands (table ). the first is a -year- old hispanic girl with severe rp, pap, men- tal retardation and obesity (bmi > ). she was a compound heterozygote for a mis- sense ( g→a, g d) and a nonsense ( t→a, y stop) mutation in exon . cloning and sequencing of the separate alleles confirmed that the mutations were present in trans. a second bbs proband (from newfoundland), born to consan- guineous parents, was homozygous for two deletions ( delc and - del- gta) in exon , predicting a frameshift. an affected brother was also homozygous for the deletions, whereas an unaffected sibling had two normal copies of mkks. both the proband and her affected brother had rp, pap, mild mental retardation, morbid obe- sity (bmi > and , respectively), lobu- lated kidneys with prominent calyces and diabetes mellitus (diagnosed at ages and , respectively). a deceased sister (dna unavailable) had similar phenotypic fea- tures (rp with blindness by age , bmi > , abnormal glucose tolerance test and iq= , vaginal atresia and syndactyly of both feet). both parents and the maternal grandfather were heterozygous for the deletions. genotyping with markers from the mkks region confirmed homozygos- ity at p in both affected individuals. a third family included a -year-old male proband (the offspring of consan- guineous parents) with reduced visual acu- ity, pap, obesity and cystic kidneys, and a sibling with hypospadias, pap, obesity and lobular cystic kidneys who died at age months (eye examination and dna were unavailable). a fourth bbs family consisted of a female proband diagnosed at age five years because of severe rp, pap, morbid obesity and diabetes mellitus, and a male sibling with rp, pap, obesity, lobulated cys- tic kidneys and diabetes mellitus. although not known to be related, both families are from the same region of newfoundland. sequencing revealed that the three affected individuals in these two families from whom dna is available are homozygous for a frameshift mutation ( delt). both parents of family and the mother of family were heterozygous for this muta- tion. no dna was available from the father of family . families and share a haplo- type of snps within mkks and strps flanking the gene, indicating that they probably inherited the delt mutation from a common ancestor. the mutations reported here were not found in northern european control chromo- somes or, in the case of the mutations in family , in chromosomes from his- panic controls. mkks is expressed in tis- sues affected by bbs, including retina, pancreas, brain and kidney (fig. ). mkks represents a sixth locus for bbs and is the first gene shown to be mutated in patients with bbs. mutations in mkks cause mks in the amish , in whom retinal degeneration, obesity and learning disabil- ity have not been reported , . we hypoth- esize that mks is a distinct phenotype caused by hypomorphic alleles of mkks, whereas bbs is caused by null alleles. although the numbers are small, it is of interest that all known mks patients have at least one missense mutation, whereas homozygous frameshift mutations are pre- sent in three bbs families. alternatively, amino-acid polymorphisms in mkks (ref. ) or variations in the promoter region may influence the phenotype. the frequency of mkks mutations in bbs patients ( / = . %; % ci – %) is consistent with the percentage of cases unassigned to the five previously known loci. identification of mkks as a bbs gene may assist in the identification of other bbs genes. we hypothesize that the clini- cal features of bbs may be caused by the inability of the mkks putative chaper- onin to maintain protein integrity in the retina, brain, pancreas and other organs. our results suggest that genes encoding chaperonins and their substrates are can- didates for other bbs loci, rp, diabetes, obesity and mental retardation. acknowledgements we thank a. mcclain, s. naylor, t. young and d. hefferton for patient ascertainment and collection mutations in mkks cause bardet- biedl syndrome fig. human multiple tissue northern blots analysed with a probe from exons - of the full- length mkks cdna showing a . -kb transcript in tissues affected by bbs. the numbering of muta- tions is based on the ´ end of the mkks cdna with exon b (genbank af ). table • diagnosis of bbs and mks primary (p) or secondary (s) diagnostic major diagnostic clinical feature criteria for bbs criteria for mks family family family family pigmentary retinopathy p + + + + polydactyly p + + + + + obesity p + + + + learning disability p + + + – hypogonadism p – – – – renal anomalies p – + + + diabetes mellitus s – + – + hydrometrocolpos + – – – – congenital heart disease s + – – – – a diagnosis of bbs requires four primary features or three primary features plus two secondary features . a diagnosis of mks requires all three major criteria . © nature america inc. • http://genetics.nature.com © n a tu re a m e ri c a i n c . • h tt p :/ /g e n e ti c s .n a tu re .c o m brief communications nature genetics • volume • september of dna samples; c. searby and h. naik for technical assistance; and r. swiderski for collection of rna and assistance with northern- blot analysis. this work was supported in part by nih grant ey and the foundation fighting blindness (v.c.s. and e.m.s.). v.c.s. is an associate investigator of the howard hughes medical institute. anne m. slavotinek , edwin m. stone , kirk mykytyn , john r. heckenlively , jane s. green , elise heon , maria a. musarella , patrick s. parfrey , val c. sheffield & leslie g. biesecker genetic diseases research branch, national human genome research institute, nih, bethesda, maryland, usa. department of ophthalmology, and howard hughes medical institute and department of pediatrics, university of iowa, iowa city, iowa, usa. department of ophthalmology, harbor-ucla medical center, torrance, california, usa. faculty of medicine, memorial university, st. johns, newfoundland, canada. department of ophthalmology and vision science research program, university of toronto, toronto, canada. long island college hospital, brooklyn, new york, usa. correspondence should be addressed to v.c.s. (e-mail: val-sheffield@uiowa.edu). . green, j. et al. n. engl. j. med. , – ( ). . beales, p., elioglu, n., woolf, a., parker, d. & flinter, f. j. med. genet. , – ( ). . david, a. et al. j. med. genet. , – ( ). . stone, d. et al. nature genet. , – ( ). . kwitek-black, a.e. et al. nature genet. , – ( ). . leppert, m. et al. nature genet. , – ( ). . sheffield, v.c. et al. hum. mol. genet. , – ( ). . carmi, r. et al. hum. mol. genet. , – ( ). . young, t.l. et al. am. j. hum. genet. , – ( ). . bruford, e.a. et al. genomics , – ( ). . mckusick, v.a., bauer, r.l., koop, c.e. & scott, r.b. jama , – ( ). . stone, d. et al. hum. mol. genet. , – ( ). . rosenberg, m.j. et al. am. j. hum. genet. , – ( ). . mckusick, v.a. am. j. hum. genet. , – ( ). fig. cdh inactivation in hdgc tumours. a, pedigrees of families and chg . ‘y’, ‘n’ and ‘u’ indicate cdh germline mutations as present, absent or unde- termined. b, methylation-specific pcr (ref. ) of the cdh promoter , . following sodium bisulphite treatment of genomic dna, the cdh promoter was amplified using pcr primers specific for methylated (m) or unmethylated (u) promoters. cdh methylation is demonstrated in hdgc tumours - , chg -ii- and chg - ii- . ‘mcf methylated’ and ‘mcf ’ are positive and negative controls. c, hdgc tumour summary showing cdh -loh, e-cadherin expression, cdh promoter methy- lation (filled circle, methylated; open circle, unmethylated) and cdh sequence (open box, wild type; filled box, somatic mutation; diagonal hashed box, unknown). ‘x’ designates presumptive silencing of methylated promoters. d, map of the methylation status of the cpg dinucleotides (circles) located in the cdh promoter. a d c b aberrant promoter methylation andthe associated loss of gene expression is a common accompaniment of human cancers . nonetheless, it has been challeng- ing to demonstrate in any given tumour that methylation of a specific gene was causal and not consequent to malignant transformation. in this regard, our atten- tion was drawn to the genesis of gastric can- cers in individuals with hereditary diffuse gastric cancer (hdgc). these individuals harbour germline mutations in the gene encoding e-cadherin, cdh (refs – ), but their cancers have consistently demon- strated absence of loss of heterozygosity at the cdh locus – . these findings sug- gested the hypothesis that cdh promoter methylation might function as the ‘second genetic hit’ in the genesis of these cancers. our study was carried out in two previ- ously identified hdgc kindreds bearing known germline cdh mutations (fig. a). immunohistochemistry showed absence of e-cadherin protein expression from five of six gastric cancers from these families (fig. d–f), suggesting these methylation of the cdh promoter as the second genetic hit in hereditary diffuse gastric cancer © nature america inc. • http://genetics.nature.com © n a tu re a m e ri c a i n c . • h tt p :/ /g e n e ti c s .n a tu re .c o m 	mutations in mkks cause bardet-biedl syndrome 	acknowledgements 	references daily and seasonal variation in light exposure among the old order amish international journal of environmental research and public health article daily and seasonal variation in light exposure among the old order amish ellen e. lee , , , , ameya amritwar , l. elliot hong , iqra mohyuddin , timothy brown ,† and teodor t. postolache , , , ,*,† mood and anxiety program, department of psychiatry, university of maryland school of medicine, baltimore, md , usa; eel @health.ucsd.edu (e.e.l.); iqramohyuddin@gmail.com (i.m.) department of psychiatry, university of california san diego, la jolla, ca , usa sam and rose stein institute for research on aging, university of california san diego, la jolla, ca , usa veterans affairs san diego healthcare system, san diego, ca , usa co-occurring/addiction unit, sheppard pratt hospital, ellicott city, md , usa; aamritwar @gmail.com maryland psychiatric research center, department of psychiatry, university of maryland baltimore, catonsville, md , usa; ehong@som.umaryland.edu centre for biological timing, faculty of medicine, biology and health, university of manchester, manchester m pl, uk; timothy.brown@manchester.ac.uk rocky mountain mental illness research education and clinical center (mirecc), veterans integrated service network (visn) , aurora, co , usa military and veteran microbiome: consortium for research and education (mvm-core), aurora, co , usa mental illness research, education and clinical center (mirecc), veterans integrated service network (visn) , va capitol health care network, baltimore, md , usa * correspondence: tpostola@som.umaryland.edu; tel.: + - - - † timothy brown and teodor t. postolache contributed equally and share senior authorship. received: april ; accepted: june ; published: june ���������� ������� abstract: exposure to artificial bright light in the late evening and early night, common in modern society, triggers phase delay of circadian rhythms, contributing to delayed sleep phase syndrome and seasonal affective disorder. studying a unique population like the old order amish (ooa), whose lifestyles resemble pre-industrial societies, may increase understanding of light’s relationship with health. thirty-three participants (aged – , mean age . ; without physical or psychiatric illnesses) from an ooa community in lancaster, pa, were assessed with wrist-worn actimeters/light loggers for at least consecutive days during winter/spring ( january– april) and spring/summer ( may– september). daily activity, sleep–wake cycles, and their relationship with light exposure were analyzed. overall activity levels and light exposure increased with longer photoperiod length. while seasonal variations in the amount and spectral content of light exposure were equivalent to those reported previously for non-amish groups, the ooa experienced a substantially (~ -fold) higher amplitude of diurnal variation in light exposure (darker nights and brighter days) throughout the year than reported for the general population. this pattern may be contributing to lower rates of sad, short sleep, delayed sleep phase, eveningness, and metabolic dysregulation, previously reported among the ooa population. keywords: seasonal affective disorder; amish; photoperiod; circadian; diurnal; sleep–wake cycles; actigraphy; melanopic illuminance; photopic illuminance int. j. environ. res. public health , , ; doi: . /ijerph www.mdpi.com/journal/ijerph http://www.mdpi.com/journal/ijerph http://www.mdpi.com http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /ijerph http://www.mdpi.com/journal/ijerph int. j. environ. res. public health , , of . introduction human physiology and behavior exhibit seasonal changes during the fall–winter time interval, including lower mood, reduced energy, sleepiness, decreased interest in social interactions, and increased preference for energy-rich starchy foods, leading to possible weight gain, with spontaneous resolution in spring or summer [ , ]. in certain individuals, seasonal changes in mood include episodes of depression during winter that can be improved with bright light exposure [ , – ]. seasonal and daily rhythms govern metabolic processes; therefore, alterations in these processes can have an impact on manifestations of metabolic conditions, such as diabetes, and cardiovascular risk [ , ]. the underlying mechanisms of these health outcomes and of physiological and behavioral seasonal changes could be the consequence of seasonal elongation of the duration of the nocturnal melatonin secretion in response to the shortened photoperiod [ ], or delayed circadian rhythms due to progressively reduced exposure to morning light during fall and winter [ , ]. a large body of literature now indicates that the effects of light on physiology and behavior such as those described above (so-called non-image forming (nif) responses) originate via a specific class of intrinsically photosensitive retinal ganglion cell (iprgcs) [ , ]. the iprgcs integrate phototransduction via their own photopigment, melanopsin, with synaptic input from rods and cones, and relay signals to the master circadian clock in the hypothalamic suprachiasmatic nuclei as well as other subcortical brain sites [ – ]. this arrangement enables iprgcs to detect the pronounced changes in ambient illumination that occur across the solar day and coordinate downstream physiological responses accordingly. mood-modulating effects of light are therefore believed to be mediated by iprgcs, acting via effects on the biological clock, neuroendocrine function, serotonergic tone, and/or more direct impacts on the arousal state [ – ]. given the growing awareness of the nif system’s wide-ranging influence, there is now significant interest in understanding the extent to which ‘unnatural’ patterns of light exposure, associated with modern lifestyles, negatively impact health and well-being. currently, there is an abundance of literature linking shift work as well as night-time artificial light exposure with a variety of negative health outcomes, including increased risk of developing metabolic disease [ ], various forms of cancer [ – ], and mood disorders [ ]. a key unresolved question, however, is the extent to which alterations in the daily and seasonal patterns of light exposure, associated with modern life, have led to impaired physical and psychological health in the general population [ ]. hence, increased night-time light and reduced daytime exposures to natural sunlight, resulting from increasingly dominant indoor occupational patterns and generalized access to modern electric lighting, are expected to alter the diurnal variations in iprgc output. this output is required to appropriately coordinate daily physiological rhythms [ ]. similarly, increased evening exposure to short-wavelength light from led lights, televisions, tablets, tablets, and smartphones [ ] is believed to be particularly disruptive to circadian function [ , ]. indeed, studies in community settings have reported how night-time artificial light exposure is associated with obesity, elevated blood pressure, and depression [ – ]. the old order amish (ooa) are a unique population that can help assess the relationship between modern patterns of light exposure and health. the ooa are a predominantly agrarian society using non-grid-fed lights, and do not use artificial time cues with watches or alarm clocks [ ]. thus, their daily patterns of light exposure are expected to differ qualitatively and quantitatively from the general population. the ooa have lower incidence of seasonal affective disorder (depressive disorder triggered by seasonal changes) (< %) [ , ], lower rates of diabetes [ ], and different seasonal-adjusted sleep patterns (with lesser proportion of short sleepers, and earlier wake and sleep onset time) [ ] relative to non-amish. it is tempting to speculate that such differences between the ooa versus the general north american population may be mediated, at least in part, by differences in daily and/or seasonal patterns of light exposure between them. comparisons of quantitative and qualitative ambulatory light exposure, as well as comparisons of activity patterns in ooa with modern counterparts, may provide clues to understanding the low int. j. environ. res. public health , , of prevalence of metabolic and seasonal mood problems in the ooa community. here, we address this question through a preliminary study that measures seasonal variations in daily activity and ambulatory light exposure (intensity, wavelength, and timing) among the ooa, via wrist-worn monitors. this study primarily aimed to assess seasonal changes in daily light exposure among the ooa and their association with physical activity. we hypothesized that physical activity and the amount and spectral content of light exposure would vary according to photoperiod. . materials and methods we investigated daily patterns of activity and ambulatory light exposure across seasons in subjects from an ooa community in lancaster, pa ( ◦ ′ ′′ n, ◦ ′ ′′ w). lancaster is a rural community which experiences wide seasonal variation in day length ( . h on december to . h on june). in this county, the ooa engage mainly in agricultural activities or traditional businesses, such as woodworking. as their religion prohibits the use of grid-fed electric lighting in their home, the ooa use light sources that are not encountered in more modern homes (work spaces are exempt and can use grid-fed lighting). most houses have portable kerosene or naphtha gas lamps, which can also be portable to provide sources of illumination for the family outside of daylight hours as needed. in addition, many ooa increasingly use battery powered led lamps for individual activities, such as knitting or reading (information gathered during home visits and interactions with ooa liaisons at the amish research center, lancaster). . . study design forty physically and psychiatrically healthy subjects (age range – years, % females, mean age . ) provided informed consent to take part in a study of individual ambulatory measurement of light exposure. this study was approved by the institutional review board of the university of maryland school of medicine (project id code hp- , approval date july ). subjects wore a wrist-worn actimeter/light logger (actiwatch spectrum; philips respironics, bend, or, usa) for consecutive days during two different periods during the year: winter/spring ( january– april) and spring/summer ( may– august). participants were instructed to wear the watch on their non-dominant wrist and to avoid covering the actiwatch spectrum with clothing. overall, study adherence was high with subjects providing > days of continuous light/activity data for both winter/spring and spring/summer conditions. data from the remaining subjects were excluded from the present analysis for not completing both phases of this study (n = ) or providing incomplete data for at least one phase (n = ; due to removing the actiwatch spectrum or covering the device with clothing). . . light and activity measures the actiwatch spectrum device (phillips respironics, bend, or, usa) combined accelerometer- based activity detection with three independent photodiode-based light sensors, with peak response in blue, green or red regions of the spectrum (full width at half maximal response, respectively: – , – and – nm; [ ]). we recorded activity (arbitrary counts) and rgb light exposure (µw/cm ) in min bins across the tested periods. photopic light exposure (lux) was estimated by the actiwatch software as a weighted function of the rgb signal. the actiwatch software also provided automatic detection of sleep onsets/offsets based on periods of inactivity > min. after careful inspection of the data from all subjects, we were able to perceive that the automated scoring was reliable in identifying a main ‘sleep’ bout during the night, with only a few occurrences of significant nocturnal activity or apparent daytime napping. . . data analysis our basic analysis approaches for light exposure and activity data were similar (performed using matlab, the mathworks inc., natick, ma, usa). twenty-four hour daily profiles ( min intervals int. j. environ. res. public health , , of relative to eastern standard time) were calculated for each individual and photoperiod condition by averaging all the measured values obtained for that timepoint. sensors on the actiwatches indicated how long the devices were worn, and rare measurements where the device was not worn were omitted from the resulting average ( , data points from a total of , measurements; ~ % of the data across the two measurement blocks and subjects). equivalent approaches were used to calculate activity/light exposure relative to wake time or onset of sleep, by referencing the actimetry-defined start and end points of a main nocturnal sleep bout (see above). similarly, we also calculated daily light/activity profiles as a function of sun position around dawn and dusk ( . ◦ bins covering solar zenith angles ◦ above and below horizon), with sun position calculated as described previously [ ]. we also calculated each individual’s daily activity/light exposure totals and average values across the following defined epochs: ( ) full h, ( ) pre-dawn (measurements obtained before civil sunrise: solar zenith ◦ below horizon), ( ) dawn (between civil and actual sunrise), ( ) day (between actual sunrise and actual sunset), ( ) dusk (between actual and civil sunset), ( ) post-dusk (after civil sunset), ( ) h post-wake onset, and ( ) h pre-sleep onset. as the precise timing of our winter/spring vs. spring/summer measurements varied across individual subjects, we compared, for each subject, the difference in these parameters measured under long vs. short photoperiod. this was conducted with respect to the difference in civil photoperiod duration (pearson’s correlation) to assess for seasonal changes. for assessment of light exposure, values were first log-transformed. for clarity and ease of comparison with previous work, absolute values for light exposure were included in the analysis to reflect estimated lux values (as described above). similar analyses were performed on raw rgb sensor readings and they provided qualitatively similar results (supplemental table s ). to assess for daily/seasonal variation in the spectral composition of ambulatory light exposure, we then calculated the fraction of total detected light energy across each channel of the rgb sensor array and analyzed as described above. we also specifically investigated the significance of the detected spectral changes for melanopsin photoreception, whose peak spectral sensitivity ( nm) is substantially short-wavelength-shifted relative to the photopic visual system ( nm), by calculating apparent ‘melanopic lux’ [ , , ]. this was achieved using a recently described method for converting actiwatch rgb sensor data [ ]. the analysis was based on the daylight illuminant model provided in the above study, as this approach is predicted to best account for the majority of light sources to which the ooa are exposed (i.e., either daylight or artificial sources with relatively flat spectral power distributions). the calculated values were then converted to the new, si-compliant, metric: melanopic equivalent daylight illuminance (units lux) by multiplying by . as specified in cie s /e: [ ]. this correction ensures that for natural daylight (as formalized by the cie d daylight illuminant) melanopic and photopic illuminance are identical. we then compared the derived melanopic illuminance values with photopic illuminance determined by the actiwatch software. . results the study sample of participants had a mean age of . years (age range – years). twenty-six ( %) of the participants were females. . . daily activity patterns among the ooa we first examined daily activity patterns among the ooa. overall, the average daily activity profiles were broadly similar among the participants, with activity starting early in the morning (before : am in all but one individual) and persisting for . – . h, with a small dip around midday (figure a; see also figure s for data subdivided for subjects tested at timepoints presenting very large or more modest differences in photoperiod duration). closer inspection indicated that wake onset time almost always occurred before civil sunrise (figure b; n = / subjects during winter/spring and / subjects during spring/summer) and sleep always occurred after civil sunset. under both conditions, we also found that activity was reliably higher immediately following wake-onset than int. j. environ. res. public health , , of preceding sleep onset (figure c; % increase in h totals = ± and ± for short and long photoperiods, respectively; paired t-tests both p < . ). int. j. environ. res. public health , , x of within-subject comparisons of actimetry-defined mid-sleep (a surrogate measure approximating circadian timing: ‘chronotype’; [ ]) revealed a strong correlation between values observed under short and long photoperiods (r = . , p = . ). these data instill confidence that the sleep–wake figure . photoperiod-related differences in activity timing in the old order amish (ooa). (a) mean ± sem daily activity patterns for ooa subjects during winter/spring or spring/summer; lower panel indicates wake and sleep onset times for each subject (n = ). (b) mean ± sem activity patterns (upper panels) and wake/sleep onset times (lower panels) for subjects above as a function of sun position around dawn or dusk. (c) mean ± sem activity patterns for subjects above relative to actimetry- defined wake and sleep onset timing. (d) relationship between seasonal change in wake onset time and photoperiod duration for these ooa subjects. (e) relationship between midsleep timing in these subjects under short and long photoperiods. (f) relationship between seasonal change in total activity during the day (top) or post-dusk (bottom) and photoperiod duration. in line with the photoperiod-related difference in wake onset timing (but not sleep onset timing), we also found that overall activity levels across the h day significantly increased with photoperiod duration (table ). as expected based on the activity profiles described above, we also observed a redistribution of activity relative to the solar day, with increases in total daytime and decreases in figure . photoperiod-related differences in activity timing in the old order amish (ooa). (a) mean ± sem daily activity patterns for ooa subjects during winter/spring or spring/summer; lower panel indicates wake and sleep onset times for each subject (n = ). (b) mean ± sem activity patterns (upper panels) and wake/sleep onset times (lower panels) for subjects above as a function of sun position around dawn or dusk. (c) mean ± sem activity patterns for subjects above relative to actimetry-defined wake and sleep onset timing. (d) relationship between seasonal change in wake onset time and photoperiod duration for these ooa subjects. (e) relationship between midsleep timing in these subjects under short and long photoperiods. (f) relationship between seasonal change in total activity during the day (top) or post-dusk (bottom) and photoperiod duration. of particular note, we observed a clear photoperiod-related difference in the timing of wake onset (figure d) but not of sleep onset (not shown; r = − . ; p = . ), with wake onset timing occurring approximately h earlier under the longest vs. shortest photoperiods tested. importantly, within-subject comparisons of actimetry-defined mid-sleep (a surrogate measure approximating int. j. environ. res. public health , , of circadian timing: ‘chronotype’; [ ]) revealed a strong correlation between values observed under short and long photoperiods (r = . , p = . ). these data instill confidence that the sleep–wake onsets defined under our experimental conditions strongly reflect individual sleep onset timing preferences. in line with the photoperiod-related difference in wake onset timing (but not sleep onset timing), we also found that overall activity levels across the h day significantly increased with photoperiod duration (table ). as expected based on the activity profiles described above, we also observed a redistribution of activity relative to the solar day, with increases in total daytime and decreases in total post-dusk activity, in line with increasing photoperiod duration (figure f). total pre-dawn activity also reduced, albeit to a lesser extent, as photoperiod lengthened (table ). by contrast, average levels (counts/min) across all tested epochs were broadly similar, although we did observe a modest trend towards increased daytime and ‘morning’ ( h post wake onset) activity under long photoperiods (table ). table . activity measures under short and long photoperiods. shows total and average activity counts (mean ± sd) for each analysis epoch tested under winter/spring (short) and spring/summer (long) photoperiods. correlation r and p values reflect results of pearson’s correlation for relationship between difference in photoperiod duration (long/short) and activity measures. analysis epochs are: h total (total daily counts for each individual), pre-dawn (total daily counts from wake onset to civil sunrise), dawn (daily counts from civil to actual sunrise), day (daily counts from actual sunrise to actual sunset), post-dusk (daily counts from civil sunset to sleep onset), h post-wake onset and h pre-sleep onset (daily counts for first hours post-wake onset and last hours pre-sleep onset). data for average counts are shown only for those epochs whose duration varies depending on individual and/or photoperiod. total activity (a.u.) average activity (a.u.) winter–spring spring–summer correlation with photoperiod winter– spring spring– summer correlation with photoperiod epoch mean ± sd mean ± sd r p mean ± sd mean ± sd r p h total , ± , , ± , . . pre-dawn , ± , , ± , − . . ± ± − . . dawn , ± ± − . . ± ± − . . day , ± , , ± , . < . ± ± . . dusk , ± , ± − . . ± ± − . . post-dusk , ± , , ± , − . < . ± ± − . . h post-wake onset , ± , , ± , . . h pre-sleep onset , ± , ± , . . . . seasonal changes in light exposure next, we evaluated daily/seasonal variation in photopic light exposure (‘lux’). under both short and long photoperiods, average daytime light exposure was high (> lux), indicating substantial exposure to natural daylight (figure a; table ; see also figure s for data subdivided for subjects tested at timepoints presenting very large or more modest differences in photoperiod duration). by contrast, during waking hours pre-civil sunrise or post-civil sunset, light exposure was very low (figure b; on average < lux), indicative of the relatively dim overall illumination available in amish homes. light exposure fell to well below lux during actimetry-defined sleep (figure c). as a population, the ooa experience a very high amplitude of light–dark cycle (with very bright days and dark nights) regardless of photoperiod. int. j. environ. res. public health , , of int. j. environ. res. public health , , x of figure . photoperiod-related differences in photopic light exposure in the old order amish. (a) mean ± sem daily pattern of photopic light exposure in ooa subjects during winter/spring or spring/summer. (b) mean ± sem pattern of photopic light exposure for subjects above as a function of sun position around dawn or dusk. (c) mean ± sem pattern of photopic light exposure for subjects above relative to actimetry-defined wake onset and sleep onset timing. (d–f) seasonal changes in pre-dawn (d), daytime (e) and post-dusk (f) total light exposure (lux*min) as a function of difference in photoperiod length for the ooa subjects. as one would expect, based on the stark differences in the average amount of light exposure detected in daytime relative to that available outside sunlight hours, we also observed clear seasonal differences. thus, total h and daytime light exposure increased with increasing photoperiod duration, while pre-dawn and post-dusk light exposure decreased (figure d–f; table ). we also found a similar relationship with average light exposure across these epochs (figure b, table ), i.e., higher average daytime light exposure and somewhat lower average light exposure outside of the civil sunlight hours. we interpret that these data reflect greater time spent outdoors during the spring/summer (both during day and ‘night’), coupled with greater availability of direct sunlight (i.e., less overall cloud cover; see figure c,d below). nonetheless, insofar as wake onset times in longer photoperiods were closer to sunrise than in shorter photoperiods (figure b), we observed a positive relationship between total light exposure during the h epoch following wake onset and photoperiod duration (figure c; table ). despite the higher average spring/summer light exposure in the h period immediately preceding sleep onset, a direct correlation with photoperiod duration was not detectable across this epoch (table ). figure . photoperiod-related differences in photopic light exposure in the old order amish. (a) mean± sem daily pattern of photopic light exposure in ooa subjects during winter/spring or spring/summer. (b) mean ± sem pattern of photopic light exposure for subjects above as a function of sun position around dawn or dusk. (c) mean ± sem pattern of photopic light exposure for subjects above relative to actimetry-defined wake onset and sleep onset timing. (d–f) seasonal changes in pre-dawn (d), daytime (e) and post-dusk (f) total light exposure (lux*min) as a function of difference in photoperiod length for the ooa subjects. table . light exposure under short and long photoperiods. shows total (lux values summed over each min of exposure) and average illuminance (mean ± sd) for each analysis epoch tested under winter/spring (short) and spring/summer (long) photoperiods. conventions as in table . total exposure (log lux*min) average exposure (log lux) winter– spring spring– summer correlation with photoperiod winter– spring spring– summer correlation with photoperiod epoch mean ± sd mean ± sd r p mean ± sd mean ± sd r p h total . ± . . ± . . < . pre-dawn . ± . . ± . − . . . ± . . ± . − . . dawn . ± . . ± . − . . . ± . . ± . − . . day . ± . . ± . . < . . ± . . ± . . < . dusk . ± . . ± . . . . ± . . ± . . . post-dusk . ± . . ± . − . < . . ± . . ± . − . . h post-wake onset . ± . . ± . . . h pre-sleep onset . ± . . ± . . . int. j. environ. res. public health , , of as one would expect, based on the stark differences in the average amount of light exposure detected in daytime relative to that available outside sunlight hours, we also observed clear seasonal differences. thus, total h and daytime light exposure increased with increasing photoperiod duration, while pre-dawn and post-dusk light exposure decreased (figure d–f; table ). we also found a similar relationship with average light exposure across these epochs (figure b, table ), i.e., higher average daytime light exposure and somewhat lower average light exposure outside of the civil sunlight hours. we interpret that these data reflect greater time spent outdoors during the spring/summer (both during day and ‘night’), coupled with greater availability of direct sunlight (i.e., less overall cloud cover; see figure c,d below). nonetheless, insofar as wake onset times in longer photoperiods were closer to sunrise than in shorter photoperiods (figure b), we observed a positive relationship between total light exposure during the h epoch following wake onset and photoperiod duration (figure c; table ). despite the higher average spring/summer light exposure in the h period immediately preceding sleep onset, a direct correlation with photoperiod duration was not detectable across this epoch (table ).int. j. environ. res. public health , , x of figure . daily differences in light quality experienced by the old order amish. (a,b) mean ± sem fractional exposure to long (red), medium (green) and short-wavelength light (blue) around dawn and dusk in ooa subjects during winter/spring (a) and spring/summer (b) months. photoperiod- related differences were observed only around dusk (actual-civil sunset; indicated by black line). (c,d) proportional distribution of apparent melanopic and photopic equivalent daylight illuminance experienced by the ooa subjects outside of daylight hours (‘night’; left) and during the day (right) during winter/spring (c) and spring/summer (d) months. outside of daylight hours, there was a clear difference in overall melanopic vs. photopic edi under short and long photoperiods, with apparent brightness, as detected by melanopsin being substantially lower than that for the photopic visual system (mean ± sd: − . ± . log lux; p < . ). thus, estimated melanopsin activation provided by artificial light sources used by the ooa was very low (on average < melanopic edi). however, when comparing the distribution of photopic vs. melanopic light exposure outside of daylight hours, we noted evidence of two distinct classes of light source. while, as indicated above, the majority of light exposure was low intensity (~ – photopic lux) and with very low melanopsin activity, we also detected a secondary higher intensity peak (> lux), where melanopic and photopic lux values were more similar. based on direct actiwatch spectrum sensor readings collected from ooa light sources (not shown) and typical spectral power distributions from such sources [ ], these corresponded, respectively, to naphtha/propane lamps vs. battery powered led lamps sometimes used for reading. in contrast with the majority of nocturnal light exposure, apparent melanopic and photopic light exposure was generally more similar during the day (albeit still moderately lower for melanopsin: − . ± . log lux). however, the distribution of encountered light exposure values during the daytime was remarkably broad, with several distinct peaks (including some periods with little access to natural light). most notably, there was a clear peak in both melanopic and photopic light exposure at very high light levels (~ , lux), presumably indicating exposure to direct sunlight (figure d), which is essentially absent in winter/spring (figure c). however, these peaks did not appear to be associated with any pronounced difference in the ratio of melanopic to photopic light exposure relative to other daytime conditions. . discussion we have provided the first comprehensive description of daily and seasonal patterns of light exposure among the ooa. to our knowledge, only one previous study has undertaken a detailed figure . daily differences in light quality experienced by the old order amish. (a,b) mean ± sem fractional exposure to long (red), medium (green) and short-wavelength light (blue) around dawn and dusk in ooa subjects during winter/spring (a) and spring/summer (b) months. photoperiod-related differences were observed only around dusk (actual-civil sunset; indicated by black line). (c,d) proportional distribution of apparent melanopic and photopic equivalent daylight illuminance experienced by the ooa subjects outside of daylight hours (‘night’; left) and during the day (right) during winter/spring (c) and spring/summer (d) months. . . spectral composition of light we also analyzed the rgb sensor data provided by our light loggers to investigate daily/seasonal changes in the spectral composition of light exposure. under both winter/spring and spring/summer conditions, the proportion of total light energy detected by green and blue sensors decreased and red sensor readings increased in parallel with solar angle around dawn/dusk (figure a,b). thus, there was a pronounced day–night difference in the spectral composition of light exposure. we did not, however, detect any photoperiod-related differences to the spectral composition of light exposure during total daylight hours (pearson’s correlation for fractional rgb, all p > . ; supplemental table s ). therefore, there was no apparent difference in the spectral composition of the artificial light sources used by int. j. environ. res. public health , , of the ooa between winter/spring and spring/summer, nor was there evidence for differences in the proportion of time exposed to natural vs. artificial sources during the day. despite the above, as the proportion of the waking hours spent post-sunset increases as photoperiod shortens (figure ), the typical spectral composition of the lighting to which ooa are exposed at the end of each day should become relatively long-wavelength-shifted during the winter months. as such, we detected a significant negative relationship between photoperiod length and fractional red light exposure during the h preceding sleep onset (r = − . , p = . ). we also observed a similar relationship when we restricted our analysis to fractional light exposure detected between actual and civil sunset (dusk; r = − . , p = . ; figure a,b). altogether, these data are consistent with the idea that, during spring/summer months, a greater proportion of evening light exposure comes from natural sources. equivalent relationships did not attain statistical significance for light exposure in the h period following wake onset or during dawn (r = − . and − . , respectively, both p > . ), suggesting that seasonal differences in artificial vs. natural light exposure are less marked in the morning. given the day–night and seasonal differences in the spectral content of evening light, we aimed to understand the impact of these differences on melanopsin photoreception, an important driver of nif responses [ ]. the distribution of melanopic vs. photopic equivalent daylight illuminance (edi) exposure from all subjects, within and outside of daylight hours as well as under short and long photoperiods, are presented in figure c,d. outside of daylight hours, there was a clear difference in overall melanopic vs. photopic edi under short and long photoperiods, with apparent brightness, as detected by melanopsin being substantially lower than that for the photopic visual system (mean ± sd: − . ± . log lux; p < . ). thus, estimated melanopsin activation provided by artificial light sources used by the ooa was very low (on average < melanopic edi). however, when comparing the distribution of photopic vs. melanopic light exposure outside of daylight hours, we noted evidence of two distinct classes of light source. while, as indicated above, the majority of light exposure was low intensity (~ – photopic lux) and with very low melanopsin activity, we also detected a secondary higher intensity peak (> lux), where melanopic and photopic lux values were more similar. based on direct actiwatch spectrum sensor readings collected from ooa light sources (not shown) and typical spectral power distributions from such sources [ ], these corresponded, respectively, to naphtha/propane lamps vs. battery powered led lamps sometimes used for reading. in contrast with the majority of nocturnal light exposure, apparent melanopic and photopic light exposure was generally more similar during the day (albeit still moderately lower for melanopsin: − . ± . log lux). however, the distribution of encountered light exposure values during the daytime was remarkably broad, with several distinct peaks (including some periods with little access to natural light). most notably, there was a clear peak in both melanopic and photopic light exposure at very high light levels (~ , lux), presumably indicating exposure to direct sunlight (figure d), which is essentially absent in winter/spring (figure c). however, these peaks did not appear to be associated with any pronounced difference in the ratio of melanopic to photopic light exposure relative to other daytime conditions. . discussion we have provided the first comprehensive description of daily and seasonal patterns of light exposure among the ooa. to our knowledge, only one previous study has undertaken a detailed investigation of seasonal patterns of light exposure in human subjects [ ]. moreover, as far as we are aware, our study was the first to investigate spectral changes in light exposure within a community without access to grid-fed electricity. alongside a growing body of literature documenting daily patterns of light exposure in other populations [ – ], our findings help to understand how changes in living and working habits across human history have impacted light exposure and sleep habits. in particular, by providing the first report of daily/seasonal light exposure patterns in an essentially int. j. environ. res. public health , , of pre-industrial-era population for which epidemiological data and documented genetic lineages were readily accessible [ , – ], our data provide an important foundation for future studies geared towards deepening our understanding of the relationship between light exposure and human health. a striking aspect of our findings was that daily patterns of activity and light exposure in the ooa more closely resemble those reported for hunter–gatherer communities in the southern tropics [ ] than those living in temperate-zone climates with access to grid-fed electricity. hence, in accordance with an earlier study [ ], we found that the ooa wake up very early, typically before dawn, with actimetry-defined mid-sleep typically occurring between and am (~ h earlier than for equivalent populations with free access to electricity; [ ]). moreover, daytime photopic light exposure among the ooa was very high (~ lux in spring/summer and lux in winter/spring). these values were similar to those reported for hunter–gatherer communities and north americans camping without access to electric light [ , ], but they were approximately – -fold greater than that reported for subjects living in more urban environments [ , , ]. similarly, nocturnal light exposure for the ooa (~ lux) was one-third of that reported for modern indoor electric lighting [ , , ], which may result in better sleep quality and health. in summary, the ooa experience a substantially more robust day–night difference in light intensity, analogous to pre-industrial era societies [ ], than is the norm in modern society, even for outdoor workers [ ]. our data also allowed us to draw inferences about the primary sources of light and their influence on photoreceptive systems. based on rgb sensory data, we demonstrated that light provided by artificial sources outside of daylight hours was substantially long-wavelength-shifted relative to that experienced during the day. as the photoreceptive systems contributing to human nif responses (melanopsin and perhaps also s-cones) are maximally sensitive to shorter wavelengths, this nocturnal shift would be expected to further reduce effective light intensity outside of daylight hours. indeed, we found effective melanopic excitation was ~ . -fold lower than the corresponding photopic lux values observed across these epochs. given reported interdevice variability in actiwatch rgb sensors [ ], and error associated with the algorithm used to infer melanopsin excitation [ ], these values should only be considered approximate. nevertheless, the resulting nocturnal values (< melanopic edi), were at the lower end of those required to modulate melatonin secretion and/or reset the circadian clock [ – ], and thus less likely to disrupt timing and quality of sleep and wake. it should be noted here that, despite the generally lower nocturnal light exposure experienced by the ooa, our basic finding of a long-wavelength shift was qualitatively similar to other recent studies that have measured ambulatory rgb light exposure in homes with electric lighting [ , ]. thus, we did not identify any direct evidence that the relative amount of short vs. long-wavelength nocturnal light exposure for the ooa differed substantially from those whose homes are lit by commonly used electric sources. however, the light loggers used for these studies might not fully report all relevant light exposure. in particular, televisions and visual displays (laptops, tablets, etc.) are likely to provide a significant source of ocular short-wavelength light exposure for the non-amish [ ], which may not be reliably detected by these wrist-worn devices. by comparison, as ooa do not use such devices, it was less likely that our measurements substantially underestimated nocturnal short-wavelength light exposure in the ooa. thus, the overall lower light levels the ooa were exposed to outside of daylight hours were likely sufficient to result in a measurably reduced impact on nif responses [ , – ]. as expected based on the agrarian lifestyle of the ooa [ ], and our own data indicating that substantial proportions of their light exposure is from natural sources, we also observed pronounced seasonal variation in light exposure. despite the overall higher daytime light intensities experienced by the ooa, the magnitude of this change (~ -fold difference in average photopic exposure during winter/spring vs. spring/summer) was broadly similar to that observed in northern europeans with free access to grid-fed electric lighting [ ]. the decreased long-wavelength and increased short-wavelength light exposure during spring/summer evenings were also similar to previous studies. finally, despite much earlier overall wake up times among the ooa, seasonal variation of wake onset times was similar to that reported for europeans at a similar latitude [ ]. thus, while the ooa experienced int. j. environ. res. public health , , of a higher amplitude of day–night differences in light intensity than is typical for modern society, their relative seasonal variation in light exposure did not exhibit overt differences relative to other modern populations. given these data, it is worth considering the extent to which specific features of light exposure among the ooa could explain the apparently low prevalence of sad in this population [ ]. we see little evidence supporting the notion that this reflects differences in the relative seasonal variation in light exposure. by contrast, the key differences here are likely due to the markedly greater degree of daytime light exposure during the winter, lower levels of nocturnal illumination, and/or the overall higher amplitude of day–night difference in light intensity experienced by the ooa. the decreased nocturnal light exposure may have significant health implications. for example, light exposure at night, even independent of sleep timing, duration and quality, has been implicated in metabolic (obesity and dyslipidemia) and blood pressure abnormalities [ , ]. similarly, aberrant light exposure has been implicated in depression-like behaviors in animals [ ]. nocturnal light exposure increases the risk of subsequent depression in human studies [ ], even when accounting for the duration and timing of sleep. a common component of several current hypotheses regarding the etiology of seasonally-mediated mood and metabolic problems is a reduction in the retinal signal driving nif responses [ ], leading to an impaired circadian alignment relative to the external world [ ] and/or a reduction in direct arousal-promoting and mood-enhancing actions of light (reviewed in [ ]). in either case, the above features noted for winter/spring light exposure among the ooa would be expected to counteract these deficiencies. specifically, the reduction in light exposure that triggers mood and metabolic issues may be corrected by high daytime light exposure in the ooa, even in the winter. additionally, reduced exposure to nocturnal light would be expected to ensure robust alignment of the circadian system to the solar day [ ]. of particular note, the relatively strong day–night signal would undoubtedly be expected to oppose the delayed circadian timing that is relatively common among patients with seasonal affective disorder [ , ]. indeed, in common with previous investigations [ , ], our data certainly indicate a strong morning preference among the ooa. however, the extent to which an association between evening preference and seasonal changes in mood [ , – ] is a contributing factor to developing seasonally-mediated health problems or simply a consequence of the underlying biological mechanisms remains to be further investigated. limitations of this study include the absence of sleep and activity diaries as well as mood ratings in our participants. there is a possibility that exposure to light, as well as rest-activity patterns could have been impacted by the mood states of the participants. these analyses did not account for individual-level characteristics such as health status and bmi, which could also influence chronotype and activity levels. environmental temperature and circadian rhythm-related biomarkers (i.e., melatonin, cortisol, and body temperature) were not assessed during this study, which could potentially impact the behaviors of participants and elucidate the biological mechanisms driving seasonal changes. finally, the actiwatch spectrum was worn on the wrist, and as such, could not precisely assess light exposure at eye level. . conclusions future studies should include mood ratings to account for bidirectional influences between mood and light exposure. biomarkers of circadian rhythm changes (e.g., levels of melatonin and cortisol) may also offer unique insight into the mechanisms underlying these health differences in ooa. recently, a lower frequency of short sleep and a relatively earlier phase of sleep have been reported in the ooa in comparison with non-amish [ ]. these could be, in part, important consequences of light exposure differences, in particular the higher diurnal variation of light exposure, with brighter days and darker nights, potentially contributing to better affective, metabolic and cardiovascular health in ooa relative to non-amish [ , , ]. int. j. environ. res. public health , , of we quantified daily variation in activity and ambulatory light exposure across the seasons within a population without access to grid-fed electricity. collectively, our data support the notion that light exposure patterns typical of the ooa could be protective, at least in part, against conditions characterized by circadian, sleep, affective, and metabolic dysregulation. the well-documented genetic lineages of the ooa, as well as the availability of genetic data and material, and close physical proximity of comparator populations at identical latitudes, present an unparalleled opportunity for future work on genetic, epigenetic, and environmental chronobiological factors and their interactions, with a long-term aim to better understand and target the dysregulation of biological rhythms predictively associated with metabolic, cardiovascular and mental illness. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : activity timing in old order amish subjects compared between epochs with large and smaller differences in photoperiod. figure s : photoperiod related differences in photopic light exposure in old order amish subjects compared between epochs with large and smaller differences in photoperiod table s : relationship between spectral light exposure and photoperiod. author contributions: conceptualization, t.t.p., t.b., and e.h.; methodology, t.t.p., a.a., and t.b.; data collection, a.a.; formal analysis, t.b. and a.a.; results, interpretation and integration: t.b., t.t.p., e.e.l., and a.a.; writing—original draft preparation, t.b., t.t.p., and a.a.; writing—review and editing, e.e.l., t.b., t.t.p., a.a., l.e.h., and i.m.; supervision, t.t.p. and t.b., project administration, t.t.p. and a.a.; funding acquisition, t.t.p. all authors have read and agreed to the published version of the manuscript. funding: this research was funded by several sources. t.t.p. was funded by a senior investigator k grant from the national institute of mental health ( k mh -pi postolache), and in part by the mid-atlantic nutrition obesity research center (norc) pilot & feasibility project (pi, postolache), a sub-award of the parent grant p dk (simeon i. taylor, program director), and the dc department of mental health, also funding a.a. t.m.b. was funded by a biotechnology and biological sciences research council (uk) david philips fellowship (bb/i / ). e.e.l. was funded in part by a k award from the national institute of mental health [nimh k mh - (pi lee)] and a narsad young investigator grant from the brain and behavior research foundation (pi lee). no funding agency or source had any input in the design, analysis, data management interpretation, decision to publish, or have any impact on the manuscript content. the manuscript expresses the opinions of the authors, and not of the funding agencies. specifically, the opinions presented here should not be construed as expressing the positions of the national institutes of health, bethesda, maryland. acknowledgments: the authors thank the entire amish community for supporting our studies on light and seasonality, and the staff of the amish research clinic of the university of maryland in lancaster, pa, for their essential help, and in particular theresa roomet, rn, who went way out of their way for completing the data collection for our project. we also are grateful to alan shuldiner, braxton mitchell, aamar sleemi, uttam raheja and dipika vaswani, as well as mary pavlovitch, sonia y. postolache, patrick donnelly, tam and truffle stubborn, and aline dagdag, for their considerable logistical contributions for this study. conflicts of interest: phillips respironics (bend, or, usa) provided the spectrum actiwatches, the interface and the software freely, and only for the duration of this study. the funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish 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[crossref] [pubmed] © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . /jphysiol. . http://dx.doi.org/ . /rsif. . http://dx.doi.org/ . /s - ( ) - http://dx.doi.org/ . /j.psychres. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j. - . . .x http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /journal.pone. http://www.ncbi.nlm.nih.gov/pubmed/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	materials and methods 	study design 	light and activity measures 	data analysis 	results 	daily activity patterns among the ooa 	seasonal changes in light exposure 	spectral composition of light 	discussion 	conclusions 	references genetics reproductive cloning, genetic engineering and the autonomy of the child: the moral agent and the open future m mameli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . j med ethics ; : – . doi: . /jme. . some authors have argued that the human use of reproductive cloning and genetic engineering should be prohibited because these biotechnologies would undermine the autonomy of the resulting child. in this paper, two versions of this view are discussed. according to the first version, the autonomy of cloned and genetically engineered people would be undermined because knowledge of the method by which these people have been conceived would make them unable to assume full responsibility for their actions. according to the second version, these biotechnologies would undermine autonomy by violating these people’s right to an open future. there is no evidence to show that people conceived through cloning and genetic engineering would inevitably or even in general be unable to assume responsibility for their actions; there is also no evidence for the claim that cloning and genetic engineering would inevitably or even in general rob the child of the possibility to choose from a sufficiently large array of life plans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . correspondence to: m mameli, king’s college, cambridge, cb st, uk; gmm @cam.ac.uk received march accepted march . . . . . . . . . . . . . . . . . . . . . . . . i n many countries there are laws prohibiting the use of cloning and genetic engineering as methods of human reproduction. these laws are controversial. commentators agree that coer- cive and state-directed uses of these reproductive technologies should be avoided. the controversy is about whether would-be parents should be allowed to use such technologies as tools (among others) for satisfying their reproductive desires. whether, when and how to reproduce are among people’s most deeply held desires. they often have a central role in a person’s self- conception, and the satisfaction or frustration of these desires has an important effect on our views about the quality and meaning of our life. some authors have argued that we have a right to choose not only whether to have children or not but also when and by which method to have them, and that we should be allowed to use cloning and genetic engineering as reproductive methods if we wish to do so. – other authors claim that the extension of reproductive freedom to the use of these technologies is problematic, as it may interfere with the rights of those conceived through such methods. one popular argument against the human use of cloning and genetic engineering for reproduction is that these procedures are not ‘‘safe’’ and may result in the birth of children with severe devel- opmental abnormalities. this argument shows (at most) that these biotechnologies should not be used for reproductive purposes until (through, say, experimentation on non-human animals and other experimental procedures) they become as reliable as other accepted reproductive methods. but some commentators maintain that would-be parents should not be allowed to use cloning and genetic engineering even when these procedures become developmentally safe. in their view, these technol- ogies undermine the autonomy of the resulting child. many versions of this objection exist and i shall discuss two different versions. according to the first version, the autonomy of cloned and genetically engineered people is undermined because knowledge of the method by which these people have been conceived make them unable to assume full responsibility for their actions. according to the second version, these biotechnol- ogies undermine the autonomy of those conceived through such methods because they violate what feinberg calls their right to an open future. i shall offer reasons to believe that neither of these versions of the autonomy-of-offspring objection succeeds in showing that the human use of cloning and genetic engineering for reproduction should be banned or heavily restricted. before we start, some terminological and con- ceptual clarifications are needed. if would-be parents were allowed to use cloning and genetic engineering as reproductive methods, they would be given a tool for choosing the genes of their future children. cloning allows would-be parents to give their children the same genes as a pre- existing person, whereas genetic engineering allows them to give their children genes that have been intentionally designed, modified or selected in the laboratory for some particular purpose. i shall call those people conceived through cloning or genetic engineering g-people (and i shall only talk about people conceived through cloning or genetic engineering as a result of parental choice). i shall call genetic choices the choices by which parents can affect their children’s genetic endow- ment.i i shall call environmental choices those choices by which parents can affect their children’s developmental environment. parents make envir- onmental choices whenever they decide to educate their children according to a certain method, to icloning and genetic engineering are not the only ways by which would-be parents can make genetic choices. parents can affect the genetic endowment of their future children simply by choosing a particular reproductive partner rather than another. but obviously cloning and genetic engineering provide a much more direct and precise tool for affecting the genetic endowment of our children. www.jmedethics.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm e .b m j.co m / j m e d e th ics: first p u b lish e d a s . /jm e . . o n ja n u a ry . d o w n lo a d e d fro m http://jme.bmj.com/ make them do certain things and not others, to punish or reward them in certain ways, and so on. both genetic and environmental choices affect the phenotype of children, including the psychological phenotype. every phenotypic trait (henceforth only trait) is the developmental outcome of the interaction between genetic and environmental factors. no trait is entirely genetically determined or entirely environmentally determined. thus, both interventions affecting a child’s genes and interventions affecting a child’s environ- ment can make a difference with respect to the child’s phenotype. by ensuring that their children experience certain environments, parents can raise the probability that their children acquire certain desired traits. similarly, by ensuring that their children have certain genes, parents can—at least in principle, assuming that genetic science becomes sufficiently advanced—increase their children’s chances of developing certain desired traits. but different developmental factors, whether genetic or environmental, affect development in different ways. this means that in some cases the most reliable and practical way to increase the chances of a trait developing may consist in a specific genetic intervention, whereas in other cases it may consist in a specific environmental intervention, and in still other cases both kinds of interventions may be required. the moral agent one influential version of the autonomy-of-offspring objection to the use of cloning and genetic engineering for human reproductive purposes has been elaborated by habermas. he argues that g-people would be unable to conceive of themselves as the ‘‘undivided author’’ (pp – ) of their lives and, as a consequence, would be unable to assume full responsibility for their actions. once informed about the way they were conceived, g-people would inevitably come to believe that the responsibility for their actions falls not on them but on their parents. this belief would undermine the autonomy of the person and his or her status as an ‘‘equal member of the moral community’’ (pp , ). in habermas’s view, in order to be a full member of the moral community, one must be able to conceive of oneself as full members of the moral community. for this to happen, one must be able to assume full responsibility for one‘s actions in the same way and to the same extent as other full members of the moral community do. but, according to habermas, g- people would inevitably believe that they are not responsible for their actions, at least not in the same way and to the same extent as are those conceived in standard ways. as a consequence, g-people would fail to interact with people conceived through standard reproductive methods ‘‘on an egalitarian basis’’ and would be condemned to ‘‘fatalism and resentment’’ (p ). ii many passages in habermas’s book suggest that g-people’s belief that they are not fully responsible for their actions would be true: g-people really are less responsible for their actions than are people conceived through standard methods. the actions of any agent depend partly on the basic psychological makeup of the agent, which includes personality traits. our genome has an important role in the development of our basic psychological makeup. by actually choosing to have a child with certain genes, the parents of a g-person can exercise an influence on the development of the basic psychological makeup of their child. does this mean that their child is going to be less responsible for his or her actions than are other people? no one is fully responsible for his or her psychological makeup. humans can, when they reach the age at which they are capable of conscious reflection, try to modify their psychological makeup, and sometimes these attempts are successful. at least sometimes, we can change our desires, emotional dispositions, habits, beliefs and even personality traits through conscious effort. this explains why we can in some circumstances be held responsible for cultivating or failing to cultivate our minds in one particular way or another. but people are not responsible for the psychological makeup they find themselves with at the age at which they become capable of conscious reflection. if full responsibility for our action requires that we be fully responsible for our psycholo- gical makeup, then obviously no one can be fully responsible for his or her actions. thus, the fact that g-people are not fully responsible for their psychological makeup cannot be used to argue that they are less responsible for their actions than are people conceived through coital means. habermas does not endorse the view that people can be fully responsible for their actions only if they are fully responsible for their psychological makeup. instead, his view seems to be that we can be fully responsible for our actions only if our basic psychological makeup is not the desired outcome of someone else’s choice. if some features of a person’s psychological makeup result from the random mixing of parental chromo- somes—as is the case in humans conceived through coital means—the person’s capacity to be morally responsible for his or her actions is not threatened. it is only when some of the fundamental psychological features of a person are the intended result of another person’s choice that the threat exists. thus, if a person behaves in a certain way (partly) because he or she has certain psychological dispositions, and if those dispositions (partly) due to a parental choice of genes designed to bring about those dispositions, then this person is not fully responsible for his or her behaviour. part of the responsibility for the actions of a person falls on the parents and it does in a way make this person less responsible for his or her actions than are people conceived through standard methods. the problem with this view is that many of the fundamental psychological features of virtually any person—including those conceived through coital means—are the desired outcome of someone else’s choice. in particular, many of the fundamental psychological features of almost everyone are the desired outcome of parental environmental choices. parents often make environmental choices aimed at influencing and directing their children’s psychological development and at raising the probability that their children acquire certain emotional dispositions, desires, values, cognitive skills, personality traits and so on. such parental efforts are often successful, even though not nearly as often as parents would like. children often do acquire at least some of the psychological traits that their parents desire to see in them and they acquire these psychological traits partly as a result of their parents wanting their children to develop these traits. for example, a child may acquire an altruistic disposition partly as a result of his or her parents’ wanting their child to be an altruistic person. the fact that the psychological makeup of g-people is partly the desired outcome of parental choices cannot possibly entail that g-people are less morally responsible for their actions than are people conceived through standard methods, as the psychological makeup of standard people is also (usually) partly the desired outcome of parental choices. the only difference is that in the case of g-people the relevant parental choices include genetic choices, whereas in the case of people conceived through standard means they do not. why should iihabermas talks mainly about genetically engineered people, but he also says that human clones would be in the same situation as genetically engineered people (pp – ). mameli www.jmedethics.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm e .b m j.co m / j m e d e th ics: first p u b lish e d a s . /jm e . . o n ja n u a ry . d o w n lo a d e d fro m http://jme.bmj.com/ this difference matter? habermas argues that the difference is morally important in relation to the violation of autonomy. according to him, environmental choices are ‘‘revisable’’ or ‘‘reversible’’, whereas genetic choices are not (pp , – ). adults or adolescents can—through what habermas calls a revisionary learning process (p )—rid themselves of the effects that their parents’ environmental decisions have produced on their minds, or alternatively, they can endorse their parents’ environmental choices. in contrast, g-people cannot rid themselves of the effects that their parents’ genetic choices have had on them: they have no option but to accept such effects and thereby they cannot but see their parents’ genetic choices as an alienating imposition. such a picture of psychological development is flawed. biologists and psychologists have shown that many environ- mental effects on psychological development are irreversible— and this applies in particular, but not exclusively, to environ- mental effects that occur in early childhood—and they have shown that many genetic effects on psychological development are reversible. with respect to the reversibility of effects, there is no general asymmetry between genetic and environ- mental choices. thus, we can appeal to no such asymmetry to argue that people whose genome has been chosen are less responsible for their actions than those conceived through standard means. sometimes habermas suggests that what really matters in relation to the autonomy of g-people is the moral self- understanding of these people and not whether such self- understanding is accurate: what matters is that g-people would believe that they are not responsible for their actions (p ). even if unjustified, this disavowal of responsibility would undermine their autonomy. it would lead them to see themselves as defective moral agents and would constitute a ‘‘pre-natally induced self-devaluation’’ (p ), a self-devalua- tion induced by the parents’ decision to actively choose their children’s genes. such self-devaluation would make decision- making difficult for g-people and encourage other members of society to treat g-people as less than full moral agents. g-people would be denied (implicitly or explicitly) full membership of the ‘‘moral community of equals’’ and their participation in society would be unjustly restricted. this, says habermas, is sufficient to show that would-be parents should not be allowed to use genetic engineering and cloning as reproductive methods. various replies to arguments of this kind have been elaborated. some authors have claimed that a person’s false beliefs cannot provide moral grounds for restricting another person’s freedom. others have appealed to some form of parfit’s non-identity principle. there is no room to discuss these proposals here. i want instead to explore what i believe is a better (even though not incompatible) reply. the claim that g- people would inevitably come to believe that they are defective moral agents is wrong, or at least (at this stage) unsupported. the costs of refusing to take responsibility are very high. as suggested by habermas himself, the price to pay for the disavowal of responsibility for our actions is the exclusion from full participation in society. given this, a very strong incentive would exist for g-people to assume full responsibility for their actions and to present themselves to other members of society as full moral agents deserving all the privileges (but also all the duties) of full members of society. in a different (but related) context, dennett says something relevant: aren’t we headed toward a percent ‘medicalized’ society in which nobody is responsible, and everybody is a victim of one unfortunate feature of their background or another (nature or nurture)? no, we are not […] people want to be held accountable. the benefits that accrue to one who is a citizen in good standing in a free society are so widely appreciated that there is always a presumption in favor of inclusion. blame is the price we pay for credit, and we pay it gladly under most circumstances. we pay dearly, accepting punishment and public humiliation for a chance to get back in the game after we have been caught out in some transgression. (p ) the benefits resulting from the assumption of full responsi- bility for our actions are likely to be sufficient to hold the line against what dennett calls ‘‘the spectre of creeping exculpa- tion’’. dennett was not thinking about g-people when he wrote this passage, but it is not difficult to see that what he says applies to g-people too. on this view, g-people would not in general believe that they are defective moral agents. they would not believe this because they would in general be aware of (or they would learn very quickly about) the disadvantages that such belief brings with it. it is undoubtedly true that, if the human use of cloning and genetic engineering as reproductive methods were to become legal, some g-people would sometimes be tempted to argue that some of their actions—the socially undesirable ones—are not their fault but their parents’ fault. this by itself does not show that would-be parents should not be allowed to choose the genes of their future children. after all, people conceived through standard means are sometimes tempted to disavow responsibility for some of their actions (again, the socially undesirable ones) and to claim that the responsibility for such actions falls on their parents and on their parents’ educational choices. yet, we do not normally take this as a reason for prohibiting parents in general from choosing how to educate their children. if the benefits associated with the assumption of full responsibility turned out to be insufficient to hold the line against the spectre of creeping exculpation (or perhaps in this case we should call it the spectre of creeping self-devaluation), we could do something about it. we could start teaching people from an early age that some of a person’s traits are the result of his or her parents trying—through genetic or environmental means—to raise the chances of those traits developing and that this does not (at least not by itself) in any way affect the moral status of this person. we could also make sure that people have a good understanding of what they give up—that is, full participation in society—if they disavow responsibility for their actions through the kind of moral self-devaluation described by habermas. and so on. we could perhaps even argue that if, despite all this, some g-people still chose to self-devalue their moral status, the blame for such self-devaluation would have to be assigned to these people themselves and not to their parents or to the fact that society allows parents to determine the genes of their children. but what if the kind of moral self-devaluation that habermas has in mind is similar to certain forms of depression? some forms of depression cannot be cured or avoided by making people aware of the fact that their being depressed is unjustified and irrational and that there are high costs attached to being depressed. could not the same be true of habermasian self-devaluation? in some cases, the process leading to the state of depression is (or involves) a reasoning process. in other cases, the depression is due (merely) to organic causes. in the latter cases, it is not possible to avoid the emergence of the depression by making the person realise that it would be irrational or disadvantageous for him or her to become depressed. but habermasian self-devaluation is not due (merely) to organic causes. it is the outcome of a reasoning process: it emerges when a g-person applies an incorrect view of reproductive cloning, genetic engineering and the autonomy of the child www.jmedethics.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm e .b m j.co m / j m e d e th ics: first p u b lish e d a s . /jm e . . o n ja n u a ry . d o w n lo a d e d fro m http://jme.bmj.com/ genes, parental choice and moral agency to himself or herself. given this, to see whether the analogy is correct, we need to focus only on those cases in which the depression results from a reasoning process. consider the following situation: a person starts thinking about her life and after careful consideration she concludes (perhaps correctly, perhaps incorrectly) that she has reasons to believe that her life is going very badly. she adopts the belief that her life is going very badly, this belief makes her depressed and the depression makes her very unlikely to revise the bleak view she has of her life. once the depression kicks in, telling this person (or even making her realise) that her being depressed is unjustified and irrational has basically no effect on her depression. but as the depression is (partly) the result of a reasoning process, the depression could have been avoided by giving the person reasons to reject the adoption of the belief that her life was going very badly. after all, many people refuse to believe that their life is going very badly (even when they have good evidence for the truth of this belief) and probably (conscious or unconscious) knowledge about the costs attached to being depressed has a role in this. let us apply this to habermasian self-devaluation. perhaps, when people start self-devaluing their moral agency in the way suggested by habermas, it becomes very difficult for them to stop self-devaluing themselves, and showing them that their self-devaluation is unjustified and irrational has basically no effect on them. this may be the case, even though, at the moment, we have no evidence for it. but let us suppose, for the sake of argument, that habermasian self-devaluation has the effects just described. even on this supposition, we can still hold the line against the spectre of creeping self-devaluation. we can do this by making sure that g-people in general do not acquire the belief that they are defective moral agents. we can teach them (from an early age) the correct way of thinking about genes, parental choice and moral responsibility, and we can explain to them (from an early age) the disadvantages and the irrationality of moral self-devaluation. all i have said so far is of course compatible with the claim that would-be parents can in principle use cloning and genetic engineering to make their children unable (when they grow up) to take responsibility for their actions: they could use these biotechnologies in ways that cause their children to actually (and inevitably) develop into defective moral agents. they could choose for their children genes that interfere with the development of the mental abilities required for full-blown intentional action and for moral reasoning. for example, they could choose genes that make their children severely mentally handicapped. such parents would intentionally make their children disabled. arguably, their action would constitute an abuse and would have to be punished accordingly. but the (remote) possibility of such misuses does not (by itself) provide support for general restrictions on the reproductive use of genetic engineering and cloning. this is shown by the fact that parents can of course interfere with the cognitive and emotional development of their children through perverse and abusive environmental interventions, interventions that can result in their children becoming defective moral agents. this can happen, for example, in the case of parents who interfere with the development of their children’s brain by giving them powerful addictive drugs, by beating them violently or by keeping them as recluses for many years. we do not normally take the relatively rare occurrences of abuses of this sort as a reason to ban parents in general from choosing how to bring up their children. similarly, we should not take the remote possibility that some parents may misuse cloning and genetic engineering in the ways described as a reason to ban the reproductive use of these technologies. the open future in this section, i want to discuss another version of the autonomy-of-offspring objection. this version appeals to what feinberg calls ‘‘the right to an open future’’ and has been elaborated by various authors. for example, davis claims that reproductive cloning violates the child’s right to an open future, especially in cases where the child is brought to life with the explicit intention of creating someone who resembles as much as possible a pre-existing person. buchanan et al argue that reproductive genetic engineering can, in some circumstances, infringe a child’s right to an open future and that this fact should seriously be taken into account when deciding whether to ban this reproductive technology. what is the right to an open future? it is not easy to extract a single definition from feinberg’s original article. buchanan et al suggest that the best way to make sense of feinberg’s notion is as follows: the idea is that parents have a responsibility to help their children during their growth to adulthood to develop capacities for practical judgement and autonomous choice, and to develop as well at least a reasonable range of the skills and capacities necessary to provide them the choice of a reasonable array of different life plans available to members of their society. […] in this view, it would be wrong for parents to close off most opportunities that would otherwise be available to their children in order to impose their own particular conception of the good life. (p ) feinberg’s original article is about parents’ environmental rather than genetic choices. the case discussed is the one of wisconsin v yoder. in the early s, parents belonging to the amish community asked to be allowed to withdraw their children from school at the age of years, years before the age at which any child can withdraw from school on his or her own. they argued that schooling beyond age is not necessary for the amish way of life and interferes with amish children acquiring the skills and motivation needed to become an integrated member of the amish community. the wisconsin supreme court accepted their request. feinberg argues that, had the amish asked to withdraw their children from school or years before the age at which other children can withdraw, their request would have been illegitimate. an amish child who undergoes or fewer years of schooling than is required by non-amish children would almost certainly end up being unfit for any way of life other than the amish one. the child would not develop the skills necessary to pursue life projects different from those that can be pursued as a member of the amish community. because of competition with other members of society, this person would not have any real chance of pursuing non-amish life projects with some probability of success. thus, this educational choice would rob this person of the possibility of choosing from a ‘‘reasonable array of life plans’’. according to buchanan et al, the principle that parents should not be allowed to make choices resulting in their children not having a reasonable array of life plans from which to choose should be applied to both environmental and genetic choices. thus, a genetic intervention that makes a child particularly fit to pursue a career as, say, a pianist but unfit to pursue any other (available) career, would be illegitimate, especially in contemporary western societies where a relatively large range of choices is usually available to most people. genetic interventions that make children fit for only a restricted range of ways of life violate the right to an open future and should thereby be banned. mameli www.jmedethics.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm e .b m j.co m / j m e d e th ics: first p u b lish e d a s . /jm e . . o n ja n u a ry . d o w n lo a d e d fro m http://jme.bmj.com/ the ability to choose our own life plan is arguably one of the essential conditions of the good life. what does this ability require? people must have cognitive and emotional skills that make them able to (a) compare (consciously or unconsciously) different life plans, (b) select one among those life plans they are able to consider, (c) transform this choice into the intention to behave in accordance with the chosen plan and (d) transform this intention into behaviour that actually conforms to the chosen option. moreover, people must have skills that allow them to pursue different life plans with some definite chance of success, and they must be in a social context where these different life plans can actually be pursued. if people have skills that make them fit for one and only one particular and very specific life plan, they cannot be said to actually be able to choose their own life plan. despite their decision-making abilities being normal, they have (in some sense) no choice. similarly, if people live in a despotic society where they are allowed to pursue only one kind of career, they are obviously not free to choose their life plan. parents inevitably exert an important influence on the array of life plans available to their children. in many cases, this influence results in children having a larger array of life plans from which to choose than they would otherwise have had. in other cases, the influence results in children having a smaller array of life plans from which to choose than they would otherwise have had. making someone fit for a particular life plan often (but not always) results in making the same person less fit for other life plans. many parents make environmental choices aimed at increasing their children’s chances of succeeding in the pursuit of more or less specific life plans. by doing so, they often make their children less likely to succeed in the pursuit of other (alternative) life plans and, in this sense, they may reduce the range of life plans available to their children. yet this sort of parental behaviour is in most cases considered legitimate, and some see it as an inevitable ingredient of being a good parent. if the practice is legitimate in the case of (most) parental environmental choices, why should it not be legitimate in the case of (most) parental genetic choices? parents are currently allowed to adopt relatively severe educational methods aimed at, for example, transforming their children into successful tennis players or into successful law school graduates. given this, why should they not be allowed to use genetic methods to achieve similar results? the discussion about the legitimacy of the amish parents’ request suggests that there is a moral limit to the extent to which parents can be allowed to reduce the array of life plans available to their children. if some environmental choices (eg, not sending a child to school) reduce the range of life plans available to a child below a certain threshold, then those choices can be said to violate the child’s autonomy and are thereby illegitimate. exactly the same applies to genetic choices. but what is the minimum size of a ‘‘reasonable array of life plans’’? what is the threshold that parental choices—be they genetic or environmental—should never trespass? this is a difficult question and there is no room here for dealing with it properly. the correct answer to this question depends on many different factors. buchanan et al, for example, argue that the answer depends, among other things, on what the correct theory of justice is. despite the question being a difficult one, we can examine current practice for some suggestions about how to think about this issue. for example, we may notice that there exist relatively large disparities in the range of life plans available to different members of society. some of these disparities are due to unjust social arrangements, but arguably some of them are not. rich people usually have more opportunities than poor people, and at least some of these differences in opportunity are usually not considered to be the result of unjust social arrangements. yet it seems wrong to claim that, except perhaps in cases of extreme poverty, poor members of society violate their children’s right to an open future when they decide to give birth to a child and not to give up their child for adoption to rich members of society. this suggests that if our current practices in this domain are correct, the morally permissible minimum size of a ‘‘reasonable array of life plans’’—the size below which the child’s right to an open future is violated—is relatively small and poor parents do not in general trespass the critical threshold. another consideration is that cloning and genetic engineer- ing would probably not be used to reduce the array of life plans available to a child below the morally permissible threshold. let us consider cloning first. in so far as the life plans available to children depend on their physical and mental abilities (rather than on factors extrinsic to the child, eg, the wealth of the parents or the structure of society) and in so far as such physical and mental abilities depend on the children’s genome (rather than on their developmental environment), the array of life plans available to a cloned child is similar to the array of life plans available to the person from whom the child’s genome is derived. thus, unless the parents select for their child the genome of someone who, because of his or her genes, did not have a decent minimum number of different life plans from which to choose, the parental decision to conceive a child by cloning cannot (in general) interfere with the child’s chances to have a reasonable array of life plans at his or her disposal. in fact, if the parents decide to create a child by cloning someone who had many options and opportunities in life, their choice will (in general) positively contribute to the range of life plans available to the child. what about genetic engineering? many would-be parents are likely to want to use genetic engineering to increase the probability that their children develop traits—such as high intelligence—which would make the children more likely to succeed in a whole range of different life plans. such genetic choices would in general enlarge rather than reduce the array of life plans available to the future child. but some parents may want to use genetic engineering to increase the chances of their child becoming fit for a very specific life plan. in many cases, such genetic choices would reduce the range of life plans available to a child to the same extent as currently accepted environmental choices (such as the decision to make a child play a lot of tennis). in other cases, the genetic choices would reduce the range to the same extent as environmental choices that are currently considered illegitimate (such as the decision not to send a child to school at all). in so far as we can make sure that cases of the second kind are relatively rare, the possible occurrence of these cases cannot be used as a reason to ban the reproductive use of genetic engineering. parents can in principle use cloning and genetic engineering to make their children unable to choose their life plan. they could choose for their children genes that interfere with the normal development of the cognitive and emotional abilities required to compare and select life plans or they could choose for their children genes that are likely to make them fit only for a very specific life plan. it is for these reasons that, as in the case of parental environmental choices, parental genetic choices should be regulated and constrained. but they should be regulated and constrained in ways that are similar to the way in which environmental choices are regulated. another interpretation of the open-future objection needs to be discussed. some commentators think that the array of life plans available to people depends not only on their skills and sociophysical context but also on the way with conceive of themselves and of the options available to them. according to these authors, cloned people who know what kind of life was reproductive cloning, genetic engineering and the autonomy of the child www.jmedethics.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm e .b m j.co m / j m e d e th ics: first p u b lish e d a s . /jm e . . o n ja n u a ry . d o w n lo a d e d fro m http://jme.bmj.com/ lived by a pre-existing genetic twin (and what kind of choices the twin made) would feel that their destiny has already been written, that they are condemned to make choices that are similar to those of the twin, and that they are not really free to choose their own life plan. this feeling of pre-determination would be amplified by the interactions between the cloned person and other people. the parents in particular would have expectations about their child, expectations based on what they know about the pre-existing twin. such expectations would exert a powerful psychological pressure on the cloned person and would heavily restrict the array of life plans effectively available to him or her. the cloned person would be condemned to live ‘‘a life in the shadow’’ of the pre-existing twin. – some commentators have extended this argument to the case of genetically engineered people. in their view, genetically engineered people who know that their parents chose for them genes designed to bring about, say, a preference for a particular kind of career would feel heavily constrained in the kind of choices they can make, and parental expectations would amplify this feeling. as in one version of habermas’s objection to the human use of cloning and genetic engineering for reproduction, on this version of the open-future objection the threat to the autonomy of g-people is mainly due to the way g-people are supposed to conceive of themselves. my reply in this case is similar to my reply to habermas. there is no good evidence for the claim that cloned people would inevitably or even in general see themselves as doomed to repeat the choices of their pre- existing twins. because of the disadvantages associated with the feeling of having one‘s destiny already written, it seems likely that they would often rebel against the thought that they have to approach life in the same way as their pre-existing twins. perhaps they would try to differentiate themselves as much as possible from their pre-existing twins. moreover, they would probably learn very quickly—by experimenting with their lives, by observing the behaviour of other clones or by reading about standard monozygotic twins—that they can make choices that differ considerably from those of their pre- existing twins. we could also start teaching people from an early age that the fact that two people have the same genes does not imply that they are destined to live similar lives. we could in this way hold the line against the feeling of pre-determina- tion and all the bad consequences that such feeling produces on one‘s options in life. similar considerations apply, mutatis mutandis, to the case of genetically engineered people. what about parental expectations? would g-peoples’ right to an open future be infringed by such expectations? no good evidence supports this claim either. parental expectations often have an important and positive role in child development, even though they can occasionally harm a child and unjustly constrain his or her (future) freedom of choice. arguably, this would apply to g-people in the same way that it applies to people conceived through standard methods. g-people would, when they reach maturity, be able to rebel against the expectations of their parents, or to endorse them if they wish to do so, and arguably they would be able to do this to the same extent as other people. it should also be noticed that we currently accept very strong and pressing parental expectations as legitimate. consider the example of the firstborn royal child, who has to cope not only with the expectations of the parents but also with the expectations of a whole nation and beyond: everybody expects the child to take the throne when the right moment comes. we do not usually see these expectations as a reason to prohibit kings and queens from having children. this suggests that we do not usually see such expectations as infringing on the royal child’s right to an open future. if we do not want to revise our judgement in this case, we should not see the expectations of the parents of g-people—which in general would probably be less pressing than those concerning the royal child—as infringing on g-people’s right to an open future either. conclusions some authors have argued that the human use of reproductive cloning and genetic engineering should be prohibited because these biotechnologies undermine the autonomy of the resulting child. in this paper, i have considered two versions of this objection. i have argued that there is no evidence that people conceived through cloning and genetic engineering would inevitably or even in general be unable to assume responsibility for their actions. and i have argued that there is no evidence that cloning and genetic engineering would inevitably or even in general rob the child of the possibility to choose from a sufficiently large array of life plans. some would-be parents may use cloning and genetic engineering in ways that violate the autonomy of their future child and, more generally, in ways that constitute abuse. but there seem to be no asymmetry between parental genetic choices and parental environmental choices with respect to the ways, the circumstances and, arguably, the frequency with which these choices would be used to perpetrate abuse. thus, no such asymmetry can be appealed to in arguing that the restrictions on parental genetic choices (and on the biotechnol- ogies that make such choices possible) should be much more severe than current restrictions on parental environmental choices. from a legislative viewpoint, genetic choices and environmental choices should be treated in similar ways. many issues remain unresolved. one issue concerns the best ways to avoid parents from using cloning and genetic engineering to perpetrate abuse. we also need to establish how to distinguish in a principled way between genetic interventions that constitute abuse and genetic interventions that do not. these issues are important, but they should be seen as special cases of general questions about the permissibility of producing certain kinds of effects on children, whether through genetic interventions or through environmental interventions. the fact that a given intervention affects a child’s genome rather than his or her environment does not make that intervention more likely to constitute abuse. and the fact that an abuse has been perpetrated by a choice of genes does not make the abuse worse or its effects more irreversible than if it had been perpetrated through an intervention on the child’s developmental environment. we need to promote healthy and well-balanced relationships between parents and children and to reduce the risk of parental abuse, whether it is perpetrated by old means or by new technologies. a general ban on reproductive cloning and genetic engineering reduces the risk of these biotechnologies being used to perpetrate abuse, but it does so in a far-from-optimal and unjust way, by denying the use of these biotechnologies to many people who would use them in non-abusive ways and to satisfy some of their most fundamental desires. acknowledgements i thank lisa bortolotti, john harris and tim lewens for comments on previous versions of this article. competing interests: none. references harris j. clones, genes and immortality. oxford: oxford university press, . harris j. on cloning. london: routledge, . agar n. liberal eugenics. oxford, uk: blackwell, . dworkin r. sovereign virtue. cambridge: harvard university press, . o’neill o. autonomy and trust in bioethics. cambridge: cambridge university press, . mameli www.jmedethics.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm e .b m j.co m / j m e d e th ics: first p u b lish e d a s . /jm e . . o n ja n u a ry . d o w n lo a d e d fro m http://jme.bmj.com/ habermas j. the future of human nature. cambridge, uk: polity, . feinberg j. the child’s right to an open future. in: aiken w, la fallette h, eds. whose child? children’s rights, parental authority and state power. totowa, nj: littlefield, . davis ds. genetic dilemmas. new york: routledge, . buchanan a, brock d, daniels n, et al. from chance to choice. cambridge: cambridge university press, . mameli m. the inheritance of features. biol philos ; : – . bateson p, martin p. design for a life. london: jonathan cape, . marcus g. the birth of the mind. new york: basic books, . brock d. cloning human beings: an assessment of the ethical issues pro and con. in: nussbaum mc, sunstein cr, eds. clones and clones. new york: norton, . dennett d. freedom evolves. new york: viking, . holm s. a life in the shadow: one reason why we should not clone humans. camb q healthc ethics ; : – . jonas h. biological engineering–a preview. in: jonas h, eds. philosophical essays: from ancient creed to technological man. englewood cliffs, nj: prentice hall, . report of the president’s council of bioethics. human cloning and human dignity, .http://www.bioethics.gov/reports/cloningreport/index.html (accessed jun ). report of the president’s council of bioethics. beyond therapy: biotechnology and the pursuit of happiness, .http://www.bioethics.gov/reports/ beyondtherapy/index.html (accessed jun ). c o r r e c t i o n doi: . /jme. . corr several errors occurred in the paper titled, potential of embryonic stem cells: an ethical problem even with alternative stem cell sources (j med ethics; : – ). a fully corrected pdf is available online at http://jme.bmj.com/. the journal apologises for these errors. reproductive cloning, genetic engineering and the autonomy of the child www.jmedethics.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm e .b m j.co m / j m e d e th ics: first p u b lish e d a s . /jm e . . o n ja n u a ry . d o w n lo a d e d fro m http://jme.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script 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radiofrequency ablation (rfa) is the standard of care for ablation of drug-refractory ventricular tachycardia (vt), particularly in the setting of ischemic heart disease. – ablation success is far from uniform, in part because of techni- cal challenges reaching the vt substrate with currently avail- able technologies because all require some degree of tissue contact with the targeted tissue. epicardial instrumentation may allow reaching vt substrates refractory to endovascular approaches , but remains difficult in patients who have under- gone previous cardiac surgery, and it is not always helpful because many ventricular arrhythmias derive from deep intra- mural origins or in proximity to coronary vessels. – this is particularly true for vt originating from the left ventricular (lv) summit, where an intramural origin, proximity to coro- nary vessels, and inaccessibility to the epicardial approach limit rfa success. transarterial coronary ethanol ablation (tcea) as an alternative treatment modality has been reported exten- sively in the literature. – tcea has become a modality of last resort in the treatment of vt not amenable to alternate contact-based ablation. , tcea is limited by the risks of arterial instrumentation; the dependence on a feasible arterial anatomy, commonly affected by the ischemic disease that led to the vt substrate; the risk of unintended collateral damage; and the logistic challenges of requiring interventional cardiol- ogy support. to overcome these limitations, retrograde coro- nary venous ethanol ablation (rcvea) has been described as an alternative to the arterial approach. its initial application in canines showed feasibility and effective myocardial abla- tion. we have reported feasibility in humans and acute pro- cedural success of rcvea in cases. the venous approach is uniquely suited for detailed activation mapping using an angioplasty wire. here, we report its combination with venous wire mapping in a variety of vt substrates, particularly the lv summit, and report its chronic outcomes. methods data collection a total of patients were included. all were counseled about the unconventional nature of venous ethanol ablation and gave informed consent to the intervention. clinical data collection was performed under an institutional review board–approved protocol. initial data © american heart association, inc. circ arrhythm electrophysiol is available at http://circep.ahajournals.org doi: . /circep. . original article background—radiofrequency ablation (rfa) of ventricular tachycardia (vt) can fail because of inaccessibility to the vt substrate. transarterial coronary ethanol ablation can be effective but entails arterial instrumentation risk. we hypothesized that retrograde coronary venous ethanol ablation can be an alternative bail-out approach to failed vt rfa. methods and results—out of consecutive patients undergoing vt/premature ventricular contraction ablation, patients underwent retrograde coronary venous ethanol ablation. six out of patients had failed rfa attempts (including epicardial in ). coronary venogram-guided venous mapping was performed using a f quadripolar catheter or an alligator-clip–connected angioplasty wire. targeted veins included those with early presystolic potentials and pace-maps matching vt/premature ventricular contraction. an angioplasty balloon ( . – × mm) was used to deliver to cc of % ethanol into a septal branch of the anterior interventricular vein in patients with left ventricular summit vt, a septal branch of the middle cardiac vein, and a posterolateral coronary vein (n= each). the clinical vt was successfully ablated acutely in all patients. there were no complications of retrograde coronary venous ethanol ablation, but patient developed pericardial and pleural effusion attributed to pericardial instrumentation. on follow-up of ± days, vt recurred in out of patients, of whom were successfully reablated with rfa. conclusions—retrograde coronary venous ethanol ablation is safe and feasible as a bail-out approach to failed vt rfa, particularly those originating from the left ventricular summit. (circ arrhythm electrophysiol. ; :e . doi: . /circep. . .) key words: catheter ablation ◼ ethanol ◼ lv summit ◼ premature ventricular contractions ◼ tachycardia, ventricular received may , ; accepted june , . from the division of cardiac electrophysiology, methodist debakey heart and vascular center, houston methodist hospital, tx. *drs kreidieh and rodríguez-mañero contributed equally to this work. correspondence to miguel valderrábano, md, division of cardiac electrophysiology, department of cardiology, houston methodist hospital, fannin st, suite , houston, tx . e-mail mvalderrabano@houstonmethodist.org retrograde coronary venous ethanol infusion for ablation of refractory ventricular tachycardia bahij kreidieh, md*; moisés rodríguez-mañero, md, phd*; paul a. schurmann, md; sergio hugo ibarra-cortez, md; amish s. dave, md, phd; miguel valderrábano, md d ow nloaded from http://ahajournals.org by on a pril , mailto:mvalderrabano@houstonmethodist.org kreidieh et al venous ethanol ablation of ventricular tachycardia relating procedures performed in (n= ) were collected ret- rospectively from patient charts. acute outcomes pertaining to the latter have previously been reported. the subsequent patients were included from a cohort of consecutive patients undergoing vt/pre- mature ventricular contraction (pvc) ablation studied prospectively from january through february . this data included medi- cal history, procedural reports, and major postprocedural and follow- up events. procedural approach: vein mapping in all procedures, efforts were initially made to localize vt sub- strate within an area amenable to rfa. electroanatomical maps were constructed using -dimensional mapping systems (navx; st jude medical, st paul, mn; n= ) or carto (biosense-webster, diamond bar, ca; n= ). mapping strategies included substrate maps to local- ize low-bipolar voltage areas in the presence of structural heart dis- ease, activation maps, and pace-maps. access to the epicardial space via a subxiphoid anterior puncture was undertaken when suitable for epicardial mapping and ablation (n= ). coronary vein mapping was performed by advancing a sheath in the coronary sinus via the right femoral vein (preface; biosense- webster) or via the right internal jugular vein (cps sheath; st jude medical, sylmar, ca). coronary venograms were performed. a multipolar catheter was inserted in the coronary sinus and selected ventricular branches ( f quadripolar ibi; st jude, or deca-nav; biosense-webster). mapping and pacing from small coronary veins was performed by advancing an angioplasty wire (bmw . ”; abbott) connected to an alligator clip in a unipolar configuration with reference electrode as a needle inserted in the thigh skin. this approach led to significantly reduced noise, compared with that produced when using wilson’s central terminal or an indifferent electrode in the inferior vena cava, and provided exclusively local signals, compared with those provided when using a neighboring electrode. selective wire cannulation of different targeted veins was achieved by introducing a left internal mammary artery angioplasty guide catheter and torquing it in the desired direction, or simply by guiding the angioplasty wire with the help of a torquing device. to obtain unipolar signals from selective portions of the targeted vein, the angioplasty balloon was advanced over the wire to cover it except for the most distal to mm, which acted as the active electrode. rcvea was considered in the following situations: ( ) when rfa failed at the best endocardial sites as guided by the earliest activation or best pace-mapping; ( ) when feasible, epicardial rfa failed or was deemed not indicated because of proximity to coronary arteries or because of presence of the earliest activation site at a broad area; and ( ) when optimal pace-mapping and earliest activation was ob- tained from within a coronary vein. the bulk of our experience targeted lv summit vt, which we targeted by mapping septal branches of the anterior interventricular vein (aiv; n= ). other targets included a posterolateral coronary vein (n= ) or the middle cardiac vein (n= ). before ethanol infusion, the presence of vein-to-vein collaterals was assessed by inflating the balloon ( . – mm by mm, depending on vein caliber) to achieve venous occlusion and injecting contrast. what is known •	 rfa of vt fails when the vt substrate is in- accessible as is common for those originating from the lv summit. •	 rcvea has been shown to be feasible, but hu- man experience is limited. what the study adds •	 rcvea was acutely successful in patients with difficult ventricular arrhythmias, of which arose from the lv summit. although vt recurred in , it was then controlled with rfa. •	 there were no major complications attribut- able to ethanol injection. •	 rcvea is reasonable to consider when rfa fails and an appropriate coronary venous target can be identified. table . patient demographics and clinical outcomes patient age, y comorbidities lvef, % previous ablations aicd acute success follow-up interval, d procedure time, h fluoroscopic exposure time, min recurrence interval, d complications htn, dl, af, chf, mechanical aortic valve – yes yes : . none htn, dl, dm ii yes yes : . pericarial effusion; ethanol-induced myocardial injury htn, dm ii, chf s/p lvad < yes yes : . n/a none htn, dl, af, dmii, hypothyroid, chf s/p lvad yes yes : . n/a none htn, dl, dm ii, chf no yes : . none htn, dl, gerd, copd, osa, cad – no yes : . n/a none htn, cad – no yes : . none af indicates atrial fibrillation; aicd, automatic implantable cardioverter defibrillator; cad, coronary artery disease; chf, congestive heart failure; copd, chronic obstructive pulmonary disease; dl, dyslipidemia; dm ii, diabetes mellitus ii, gerd, gastroesophageal reflux disease; htn, hypertension; lvad, left ventricular assist device; and osa, obstructive sleep apnea. d ow nloaded from http://ahajournals.org by on a pril , kreidieh et al venous ethanol ablation of ventricular tachycardia ethanol infusion was injected only in the absence of vein-to-vein col- laterals to deliver ethanol to the capillaries and the myocardium and avoid vein-to-vein shunting. once a vein was selected, the balloon was inflated to to atmospheres (aiming to occlude the selected vein). in the first cases, cc of % ethanol were infused for min- utes. in the subsequent cases, to injections were administered. flushing with normal saline was performed after ethanol. balloon occlusion time ranged from to minutes. an infusion of cold saline was attempted in case, but it failed to terminate the vt, despite an eventual ethanol success, and cold saline was abandoned for subse- quent cases. procedural success in pvc ablation (n= ), ablation success was defined as the cessation of spontaneous ectopy after seconds of ethanol infusion. in vt ablation (n= ), attempts were made to induce the clinical tachycardia before and after ablation, and the procedure was considered success- ful if induction failed post ablation. the electrophysiology testing protocol used - and -ms drive trains followed by to ven- tricular extrastimuli that were ms in duration at twice the diastolic threshold. extrastimuli were decremented down to a coupling interval no shorter than ms. if this protocol proved ineffective, rapid burst pacing up to a cycle length of ms was used instead. clinical follow-up all patients were hospitalized after the ablation procedure. they underwent continuous telemetry monitoring, -lead electrocardi- ography, and -hour ambulatory electrocardiography to document absence of vt/pvc recurrence before discharge. echocardiograms were obtained if clinically indicated. patients were later seen in the clinic at week and month post procedure. they were followed up on a biannual basis thereafter. four patients had an implanted im- plantable cardioverter–defibrillator, and so interrogation and shock history were retrieved during follow-up. for the remaining patients, -lead electrocardiography and -hour ambulatory electrocardio- graphic monitoring were performed on follow-up. statistical analysis gaussian continuous variables are reported as mean±sd and non- gaussian variables as median (minimum–maximum). qualitative findings were described as numbers and percentages. analyses were performed using sigmastat (version . ) and stata software (version figure . mapping of septal branches of the anterior interventricular vein (aiv) in a patient with left ventricular (lv) summit premature ventricular contractions (pvcs) and successful venous ethanol ablation. a, three-dimensional activation map (carto; biosense-webster, sylmar, ca) of the right ventricle (rv), coronary sinus (cs, using a decapolar [deca] catheter), and aiv. the aortic cusps and lv outflow activation map are included in b. c, venogram of the distal cs demonstrating septal branches denoted s , s , and s , at the origin of the aiv. d, surface ecg of the pvc, including signals recorded from the decapolar catheter, and pace-maps (% matching with pvc) obtained with unipolar pacing via an angioplasty wire in each of the septal branches. s has a perfect pace-map match. e–g, angio- plasty wire position and unipolar signals obtained from septal veins s through s , respectively. signal from s precedes qrs by ms. h, balloon occlusion of s and selective s venogram showing myocardial staining (white arrows). i and j, intracardiac echocardiogram images of the lv outflow tract before and after ethanol, showing increased echogenicity of the ethanol-injected myocardium (white arrows). k, ecg before and after ethanol, showing elimination of pvcs. v v v v v v wire h a d % % % % i ii iii avr avl avf v v v v v v pvc deca s s s deca s s s aiv cs s s s e f g v v v v v v wire v v v v v v wire c balloon i j k pre-ethanol post-ethanol pre post s cs cs rv ao aiv aiv rv ao lv b balloon balloon balloon d ow nloaded from http://ahajournals.org by on a pril , kreidieh et al venous ethanol ablation of ventricular tachycardia ). statistical significance could not be attributed because of the in- herent limitation imposed by the small sample size. results baseline characteristics a total of consecutive patients underwent vt/pvc abla- tion in a single tertiary center between january and february . of these, cases were considered suitable for rcvea and underwent the intervention accordingly (table ). three patients underwent ablation for pvc, and for vt. six patients were men, and mean age at procedure time was . ± . years. patient clinical characteristics and comorbidities are detailed in table . of particular relevance, out of the patients had undergone previous vt ablation with recurrence, including epicardial ablation in cases ( . ± . previous ablations). in patient with lv summit vt, an attempt to advance an ablation catheter into the aiv failed because of the small vein caliber, which made it impossible to reach the targeted site. two patients had previously been implanted with a lv assist device (lvad) as well (figures and ), which precluded epicardial access. one lvad patient had recurrent vt in the setting of ischemic cardiomyopathy. the second lvad patient had incessant vt in the setting of a nonischemic cardiomyopathy, which recurred after lvad implant despite maximum tolerated doses of pharmacological therapy including amiodarone and mexiletine. four patients were diagnosed with nonischemic cardiomyopathy and had an implantable cardioverter–defibrillator implanted, whereas was diagnosed with tachycardia-induced cardiomyopathy. mean lv ejection fraction was . ± . %. lv summit vt: ethanol infusion in septal veins six cases had incessant pvc/vt ablated via ethanol infusion in septal veins. five had pvcs arising from the lv summit (figures through )— has been previously reported. in all cases, extensive endocardial mapping in both sides of the septum was initially performed, as well as mapping from the great cardiac vein and aiv with a multipolar catheter. in out of cases, rfa had been previously delivered via the right ventricular endocardium and lv endocardium without suc- cess. epicardial mapping, performed in cases with lv sum- mit pvc/vt ( each), revealed earliest activation over a broad area of the lv anterior base. in case, no suitable ablation targets were found in either right ventricular or lv, and the ablation was performed exclusively with ethanol (figure ). after mapping the great cardiac vein and aiv with a multipolar catheter showed a promising signal from the aiv, selective aiv venograms were performed to identify septal branches. then, detailed unipolar septal vein mapping was performed with the angioplasty wire, which could be used to selectively obtain unipolar signals and pace-maps from the dif- ferent septal branches (figures through ). the septal branch with perfect pace-map match and earliest unipolar signals was selected for ethanol infusion. figure shows septal vein map- ping in a patient with lv summit pvcs in whom the first septal branch was chosen to deliver ethanol after mapping and pacing with the angioplasty wire in other veins. in figure , septal figure . septal vein mapping and ethanol ablation of lv summit premature ventricular contractions (pvcs). a, activation maps (carto) of the right ventricle (rv), left ventricle (lv), coronary sinus (cs), and anterior interventricular vein (aiv), showing earliest site in the aiv. b, ecg and pace-maps of the pvcs (including aiv signals, recorded from the distal cs, csd), obtained from different septal branches: s , s , and s . a near-perfect pace-map is obtained from s . c–e, angioplasty wire cannulation of s , s , and s and their respective unipolar signals, which were earliest in s . f and g, myocardial staining during selective contrast injection in s , shown in right (f) and left (g) anterior oblique (ao) projections. h, increased echo density in the basal lv septum after ethanol injection. i, ecg before and after ethanol injection, with elimination of extrasystoles. this case had not received any previous ablation and radiofrequency ablation of the aiv was not feasible because of its small size. rv lv cs aiv lv aiv i ii iii avr avl avf v v v v v v csp csd i ii iii avr avl avf v v v v v v i ii iii avr avl avf v v v v v v balloon balloon balloon balloon balloon! ii avf v wire ii avf v wire ii avf v wire a b c d e f g pvc deca s s s deca s s s % % % % h pre post i s d ow nloaded from http://ahajournals.org by on a pril , kreidieh et al venous ethanol ablation of ventricular tachycardia branches were mapped as well, and the second one was shown to have earliest unipolar signals with perfect pace-maps. in both figures and , the septal branches were relatively short, and the angioplasty wire only advanced ≈ cm deep into the septum. figure illustrates a case in which a large septal vein was identified and cannulated (figure c and d), and after figure . septal venous mapping and ethanol ablation of lv summit vt. a, navx (st jude, st paul, mn) map of the activation time in the right ventricle (rv) and coronary sinus (cs). the wire tip was localized by navx using an alligator clip. the left ventricle (lv) and epicardium were also mapped (not shown) but showed later activation. the unipolar signal from the septal aiv branch precedes all other signals. b, ecg and unipolar wire signal in sinus rhythm and during a premature ventricular contraction. wire signal obtained from the proximal septal branch (g) preceded qrs by ms. e–g, angioplasty wire mapping of different branches of the septal vein (rao). h–j, lao views of the wire loca- tion relative to earliest sites in the rv outflow tract (rvot, h), lvot (i), and septal vein (j). k and l, intracardiac echocardiography images before and after ethanol infusion, showing echogenicity of the lv basal septal septum (arrows). m, ecg before and after ethanol infusion. s c d e g h i j balloon balloon balloon rvot lvot balloon m pre post wire a k l i ii iii avr avl avf v v v v v v b csp csd wire ms f figure . mapping and ablation of left ventricular (lv) summit ventricular tachycardia (vt) in the context of nonischemic cardiomyopathy. a and b, activation maps of vt, obtained with a pentaray catheter (pr; biosense-webster) in the lv and a decapolar catheter in the coronary sinus (cs). the distal cs signals (csd in d) are earliest. c, absence of endocardial low-voltage areas in either lv or rv. e, cs cannulation with a decapolar. f, cs venogram. g, septal vein wire cannulation and unipolar wire signals during paced rhythm, showing late, post-qrs activations. h, pace-map from the angioplasty wire from a septal vein branch. i and j, septal vein balloon cannulation for ethanol infusion showing perfect qrs match. i ii iii avr avf v v v v wire cs cs lv lv lv rv i ii iii avr avl avf v v v v v v ii v v csd csp pr - pr - a b c e f g i j d h balloon septal lvad lvad pr d ow nloaded from http://ahajournals.org by on a pril , kreidieh et al venous ethanol ablation of ventricular tachycardia mapping with the angioplasty in different locations within the vein (figure e through j), the most proximal region of the septal vein (figure g) offered the best signal and was used for ethanol infusion with successful elimination of pvc. in patient with nonischemic cardiomyopathy and previ- ous lvad implant, endocardial activation mapping of inces- sant vt revealed a large component of the vt cycle length contained in the area of the lv summit (figure a and b), and aiv mapping with a decapolar catheter showed earliest activations (figure a through d). venograms of the aiv were used to navigate an angioplasty wire into the veins and obtain optimal pace-maps (figure g and h). a septal vein was then selected for ethanol infusion (figure i and j). in patient, the third septal branch of the middle cardiac vein was cannulated and subjected to ethanol infusion with elimination of vt originating from an inferoseptal myocardial infarction. lateral lv vein ethanol in a patient with ischemic cardiomyopathy, severe heart failure, and previous lvad implant, endocardial mapping revealed a patchy basal inferolateral scar, from which the exit site of the vt was mapped (figure a through c). mapping of a lv vein with a decapolar catheter revealed earlier sig- nals on the epicardial aspect of the scar (figure d), which prompted more detailed mapping with the angioplasty wire. venograms demonstrated ample postcapillary collaterals (fig- ure f). to deliver ethanol to the myocardium, small venules without such collaterals were selected. recording from the angioplasty wire revealed mid-diastolic signals and optimal pace-maps (figure e through i). ethanol was infused in such venules (figure f through k). capillary and vein obliteration by ethanol targeted veins were selected based on the electrogram timing and pace-mapping. once a targeted vein was cannulated with the angioplasty balloon, selective contrast injection through the inflated balloon was performed. in out of targeted veins, myocardial staining was obtained. in case, the optimal signal was obtained from the proximal aspect of a septal vein (fig- ure ), whereas mapping from more distal in the vein yielded suboptimal signals and pace-maps. ethanol was delivered in the proximal aspect of the vein, which led to pvc elimination and septal vein obliteration (figure a and b). in case, selective septal vein contrast injection opacified a collateral vein return- ing to the coronary sinus, and on ethanol injection, all contrast was directed to the myocardium (figure b and c). in cases, initial pre-ethanol selective venograms led to myocardial staining, whereas postethanol contrast injection demonstrated enhanced collateral flow as well (figure d through i). procedural parameters and outcomes overall, mean procedure time was ± minutes, ± minutes of which were spent under fluoroscopic exposure. three out of procedures included some degree of intraproce- dural failed rfa before rcvea, averaging ± applications figure . posterolateral vein mapping and ethanol infusion in ischemic cardiomyopathy-related ventricular tachycardia (vt) in a patient with an lv assist device (lvad). a, activation map showing earliest site arising from the lv posterolateral base, adjacent to a scar shown in b. radiofrequency ablation failed to terminate the vt at those sites (pink dots). d, a posterolateral vein on the epicardial side of the scar was mapped with a decapolar catheter and showed signals earlier than the earliest endocardial site. e, angioplasty wire mapping the lv vein shows pace-map match (left) and mid-diastolic signals (right), obtained from the wire position shown in f, which also shows the vein anatomy (with an lv pacing lead in situ) and vein-to-cs collaterals. g, selective contrast injection in a vein branch without collaterals (rao view). myocardial staining without collateral flow shunting is seen (white arrows). inset shows signals from the wire that vein. h, lao view. this vein was injected with ethanol. i, additional targeted vein included a small branch without collateral shunting (red arrow). j and k, selective contrast injection in that same small branch showing no collateral flow and myocardial staining (white arrows, rao and lao, respectively). this vein was also targeted with ethanol. c, postethanol endocardial scar map. balloon balloon balloon v v ablendo vein - vein - % a b c d e f g h balloon balloon balloon i j k collaterals i ii iii avr avl avf v v v v v v wire v v v v v v wire vein pre-ethanol post-ethanol d ow nloaded from http://ahajournals.org by on a pril , kreidieh et al venous ethanol ablation of ventricular tachycardia during a span of ± seconds. (an additional patients had previous failed rfa procedures and had no further rfa attempts during rcvea, and patient only had rcvea as the sole ablative technique; figure ). acute procedural success was documented in all patients immediately after venous ethanol ablation. in the cases of lv summit pvc ablation, ectopy was completely abolished after the first seconds of ethanol infusion (figures through ). in one of them, pvcs recurred during the patient’s hos- pitalization. in lv summit vt ablations (figure ), there was acute success because attempts to induce the arrhythmia failed after ethanol ablation. in the lv summit vt patient with an implanted lvad, after ethanol infusion, a separate vt was inducible but not the clinical vt. no major complications occurred during the course of intervention in any patient. one patient developed a small pericardial effusion—attributed to concomitant pericardial access—and ml of bloody fluid was drained from the pericardium at the end of the procedure from the preexistent pericardial access. the patient underwent cardiac magnetic resonance imaging hours post procedure. it revealed significant expansion of basal anteroseptal hyper- enhancement, indicative of ethanol-induced injury. cardiac magnetic resonance images demonstrating this outcome are previously published. one patient developed a coronary sinus dissection that resolved spontaneously and did not prevent aiv cannulation. there were no other adverse events attributable to the venous ethanol infusion protocol. chronic outcomes overall, follow-up interval was ± days. the first patients to undergo the intervention ± days ago have all suffered recurrence of vt/pvc ± days after the ethanol ablation, albeit with a subtly different qrs mor- phology in all . all underwent repeat ablation, this time using rfa: lv summit pvcs were successfully treated with rfa delivered at the left pulmonary artery, and had inferoseptal rfa. after rfa, all patients remain free of vt recurrence to date. one patient had recurrence of his pvcs day after the procedure. all recurrences occurred in cases in which only cc of ethanol was infused: none of the patients in which more than cc ethanol was delivered recurred. no patients suffered any adverse events related to the procedure during the follow-up interval. discussion the salient results of our experience are as follows: ( ) coronary venous ethanol is useful as a bail-out strategy in refractory pvc/vt, particularly when originating from the lv summit; ( ) detailed coronary venous mapping is help- ful in delineating the origin of rfa refractory pvc/vt and can be achieved by unipolar mapping with an angioplasty wire; ( ) capillary obliteration occurs after venous etha- nol infusion; and ( ) despite uniform acute success, recur- rence can occur that seems to decrease with repeated (≤ ) injections. figure . coronary venograms before and after ethanol injection. a and b, septal vein obliteration after ethanol. a large septal vein is shown in a. wire mapping showed best signals from its proximal portion (figure ). ethanol was delivered proximally at a slow rate to allow retrograde flow to keep ethanol close to the injection site, achieving premature ventricular contraction elimination. post ethanol ablation (b), the septal vein was obliterated and contrast injection showed myocardial staining (white arrows). c and d, elimination of postcapillary collaterals with ethanol. a septal vein was selectively cannulated (s in figure ). selective contrast injection opacified a collateral vein shunting blood back to the coronary sinus (cs; arrowheads in c). after ethanol infusion, contrast injection lead to myocardial staining (arrows in d) and no opacification of the collateral. d–f, new collateral shown after ethanol infusion in a septal branch of the middle cardiac vein (mcv). mcv venogram showed multiple septal branches (d), one of which was selectively cannulated (e) and contrast injection led to myocardial staining (white arrows). after ethanol infusion, contrast injection showed new collateral flow aside from myocardial staining. g–i, new collateral flow after anterior interventricular vein (aiv) septal ethanol injection. g, nonselective aiv venogram. a first septal branch was targeted (white arrow). a collateral vein is shown (black arrowheads). h, selective septal vein venogram shows myocardial staining (arrows). i, after ethanol injection, repeated venogram shows enhanced flow to the collateral (black arrowheads). d e f balloon balloon pre-ethanol post-ethanol g h i post-ethanol balloon c d pre-ethanol post-ethanol balloon balloon a b pre-ethanol post-ethanol balloon balloon d ow nloaded from http://ahajournals.org by on a pril , kreidieh et al venous ethanol ablation of ventricular tachycardia current pitfalls in standard treatment the most commonly used modality for vt ablation remains rfa. it has been proven to be % effective in the acute abo- lition of vt, but only ≈ % of patients will remain free of disease during medium-term follow-up. other modalities that have been gaining popularity in this regard include cryo- ablation, laser, and high-intensity focused ultrasound. – all of these interventions, as is the case of rfa, are limited by a need for adequate tissue contact. vt substrate often pres- ents in the epicardium. although pericardial access through a subxiphoid technique is useful, this approach remains dif- ficult in patients who have undergone previous cardiac sur- gery, particularly in post-lvad patients. furthermore, many ventricular arrhythmias derive from deep intramural origins that are difficult to target with contact-based instrumenta- tion. , , pvc/vt originating from the lv summit remain particularly challenging because they combine both an intra- mural origin and inaccessibility to epicardial approaches. proximity to coronary vessels is another factor that limits local application of radiofrequency. , surgical cryoablation or tcea has been reported. strategies such as ablation in the aiv and bipolar ablation have been proposed, but cath- eter size, proximity to coronary arteries, or impedance rises limit the former, and the latter is not universally available. other approaches have been described, such as hot saline or direct ethanol injections. , novel approaches, such as needle radiofrequency irrigation, facilitated radiofrequency with gadolinium, or selected irrigants, , are not available or fully developed. septal coronary venous mapping and etha- nol ablation seem uniquely suited for lv summit vts. coronary arterial ethanol ablation the use of alcohol as an ablative solution preceded the development of rfa and has been reported extensively in the literature. – intra-arterial injection of ethanol clas- sically delivered cytotoxic injury through the vasculature supplying target myocardial tissue. in , brugada et al performed the intervention on patients with incessant vt postmyocardial infarction. they described acute procedural success in all , with recurrence in patient after collateral flow developed and temporary complete atrioventricular block requiring pacemaker implantation in . similarly, kay et al reported tcea in patients, with % acute success and % recurrence. adverse events were common, however, including complete atrioventricular block in % and pericar- ditis in % of patients. more recently, intracoronary ethanol injection has become limited to arrhythmogenic foci that are not amenable to contact-based ablation. , noninducibility of vt after intracoronary ethanol ablation is reported between % and %, with a recurrence rate of ≈ % to %. , , , unfortunately, adverse events are not uncommon, most nota- bly coronary arterial dissection, thrombosis, and myocardial infarction. – furthermore, ethanol infusion may spill over to nontargeted myocardium, resulting in infarction or conduction block. additionally, in patients with coronary artery disease, the technique is limited by difficulty in localizing a terminal arrhythmia-related vessel, especially in the presence of coro- nary stenosis. another limitation stems from the variability of vessel size and flow rate, which influence the cytotoxicity of ethanol and make its outcomes difficult to predict. retrograde venous ethanol ablation to overcome some of these limitations, retrograde venous infusion of ethanol has been described as an alternative to the arterial approach. its initial application in canines showed promise in circumventing arterial damage, thereby avoiding the risk of coronary arterial dissection and myocardial infarc- tion. furthermore, off-target myocardial injury created by ethanol leak is not a concern of rcvea because retrograde flow allows dilution of the ethanol into the coronary sinus rendering it harmless. still, an occlusive balloon is necessary because given the retrograde direction of ethanol infusion, the normal blood flow direction naturally tends to wash out ethanol from the targeted tissue. in our experience, rcvea has proven to be acutely effective in suppressing ventricular arrhythmias, including case in which long-term elimination figure . schematic of coronary venous flow and proposed effects of ethanol. a, redundant epicardial coronary veins show vein branches followed by venules leading to capillaries. precapillary collaterals and postcapillary collaterals exist. not shown are thebesian veins draining into left ventricle (lv) and right ventricle (rv) cavity. selective retrograde contrast injection shows flow into the capillar- ies (myocardial staining) and through postcapillary collaterals (as in figure c). b, after ethanol infusion, capillaries are obliterated, and repeated retrograde contrast cannot reach postcapillary collaterals (as in figure d). if precapillary collaterals exist, flow through them is enhanced after ethanol (as in figure d– i). post-capillary collateral pre-capillary collateral post-capillary collateral pre-capillary collateral ethanol a b d ow nloaded from http://ahajournals.org by on a pril , kreidieh et al venous ethanol ablation of ventricular tachycardia of vt was achieved by rcvea alone without the use of any other ablative technology (figure ). the coronary venous approach offers logistical advan- tages to the operator as well. particularly, when combined with venous mapping, which entails coronary sinus access and selective vein branch cannulation, adding an angioplasty balloon does not contribute increased logistical complexities to the case. most centers using intra-arterial ethanol for vt recruit an interventional cardiologist for this procedure, which adds such logistical complexities. most electrophysiologists are thoroughly familiar with the coronary venous anatomy, and its instrumentation is aided by tools developed for lv lead placement in coronary veins. the coronary venous anatomy is extremely redundant. aside from venous return to the coronary sinus, thebesian veins can drain directly into the lv cavity. within the epi- cardial venous system, collateral veins abound, communicat- ing epicardial veins with one another and the coronary sinus (figure ). when targeting the myocardium with ethanol, it is important to use a vein with direct connection to capillaries to avoid ethanol shunting. collateral veins may be present at baseline injection. in some cases, collateral veins disappeared after ethanol, whereas in others, they became more prominent. we hypothesize that ethanol obliterates capillaries. collateral veins arising after the capillary territory obliterated by ethanol would disappear after ethanol (figure b and c), whereas those collateral veins arising before the capillaries may become more prominent (figure d through i). figure shows a schematic illustrating this concept. long-term outcomes remain challenging because we observed recurrence in out of cases. the fact that the non- recurrent cases underwent repeated ethanol injections (≤ cc versus only cc in the recurrent cases) suggests that successful myocardial ablation may require higher ethanol doses. other potential reasons for failure include inaccurate localization, inadequate tissue destruction, or perhaps other mechanisms. limitations this is a small, observational case series. as long as the inter- vention is considered a bail-out strategy of last resort, and in the presence of alternative bail-out modalities, recruitment of large numbers of clinically indicated patients will be difficult. patients enrolled in this series host exceptionally drug- and ablation-resilient arrhythmias, so recurrence outcomes cannot be generalized to all treatment-indicated lv summit cases. we cannot compare outcomes with other bail-out techniques, but the advantages are appealing. furthermore, all procedures in this series were performed by a single operator who is experi- enced in ethanol ablation (m.v.). hence, additional experience with a larger cohort of patients and by multiple operators will be necessary to assess the global clinical safety, efficacy, and practicality of rcvea. moreover, the specific mechanism of capillary obliteration enforced by this technique remains ill defined and should ideally be explored through histological data derived from animal models. although the technique is promising, it is not without limitations, including the need for a suitable vein without collateral shunting, the technical chal- lenges of vein cannulation, the uncertain mechanisms, and dosing of ethanol. conclusions rcvea seems to be safe and feasible as a bail-out approach in the treatment of failed vt rfa, particularly for lv summit vt. sources of funding m.v. is supported by nih/nhlbi r hl and the charles burnett endowment. m.r.m. was supported by the antonio pacifico, md fellowship grant. disclosures none. references . stevenson wg, wilber dj, natale a, jackman wm, marchlinski fe, talbert t, gonzalez md, worley sj, daoud eg, hwang c, schuger c, bump te, jazayeri m, tomassoni gf, 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transcoronary ethanol ablation for recurrent ventricular tachycardia after failed catheter ablation: an update. circ arrhythm electrophysiol. ; : – . doi: . /circep. . . d ow nloaded from http://ahajournals.org by on a pril , jch_ .. o r i g i n a l p a p e r determinants of blood pressure response to low-salt intake in a healthy adult population may e. montasser, phd; julie a. douglas, phd; marie-hélène roy-gagnon, phd; cristopher v. van hout, bs; matthew r. weir, md; robert vogel, md; afshin parsa, md, mph; nanette i. steinle, md; soren snitker, md, phd; nga h. brereton, rd; * yen-pei c. chang, phd; alan r. shuldiner, md; , braxton d. mitchell, phd from the department of medicine, division of endocrinology, diabetes, and nutrition, university of maryland, school of medicine, baltimore, md; the department of human genetics, university of michigan, school of medicine, ann arbor, mi; the department of medicine, division of nephrology, university of maryland, school of medicine, baltimore, md; the department of medicine, division of cardiology, university of maryland, school of medicine, baltimore, md; and the geriatric research and education clinical center, veterans administration medical center, baltimore, md although the beneficial effects of lowering salt intake in hypertensive patients are widely appreciated, the impact of promoting dietary salt restriction for blood pressure (bp) reduction at the population level remains controversial. the authors used -hour ambulatory bp monitoring to charac- terize the determinants of systolic bp (sbp) response to low-salt intake in a large, relatively healthy amish popula- tion. patients received a high- and low-sodium diet for days each, separated by a - to -day washout period. variance component analysis was used to assess the association of several variables with sbp response to low- salt diet. mean sbp was . � . mm hg and . � . mm hg lower on the low-salt compared with the high-salt diet during daytime (p=. ) and nighttime (p<. ), respectively. sbp response to a low-salt diet was signifi- cantly associated with increasing age and pre-intervention sbp, in both daytime and nighttime, while the association with female sex and sbp response to cold pressor test (cpt) was significant only during nighttime. our results suggest that salt reduction may have greater bp-lowering effects on women, older individuals, individuals with higher sbp, and individuals with higher sbp response to cpt. j clin hypertens (greenwich). ; : – . � wiley periodicals, inc. hypertension (htn) affects % of all americans and is responsible for % of ischemic heart disease, % of stroke, and . % of premature death world- wide. htn is a complex disease influenced by both genes and environmental risk factors, including physi- cal inactivity, obesity, smoking, alcohol consumption, and diets high in fat and salt. the term salt sensitivity (ss) has been in use for more than decades and is broadly used to denote a substantial increase in blood pressure (bp) in response to higher-salt intake. results from epidemiologic studies and clinical trials support a role for ss as an independent risk factor for cardiovascular diseases, and at least one study reported ss to be associated with reduced survival in both hypertensive and normo- tensive individuals. ss has been associated with older age, african american ancestry, female sex, obesity, htn, diabetes, renal disease, , and decreased birth weight. there may also be a genetic predisposition to ss. , finally, the genetic epidemiology network of salt sensitivity (gensalt) study recently reported an association of ss with the bp response to the cold pressor test (cpt), suggesting a role for sympathetic nervous function in the development of ss. the current level of sodium intake among ameri- cans is much higher than the recommended level of mg ⁄ d. although much evidence supports the beneficial effect of lowering sodium intake (down to mg ⁄ d according to the guidelines ) in reducing bp in hypertensive patients, the impact of promoting dietary sodium restriction for bp reduction at the population level remains controversial. , extrapolating to the population level is difficult because many of the published studies have focused on high-risk populations, used arbitrarily defined thresh- olds for ss, had limited sample sizes, and ⁄ or were based on one-time measures of bp, which have notori- ously high variability. rarely has bp response to low salt been studied in young, relatively healthy popula- tions of fit individuals, nor have many studies used detailed measures of bp in their assessment of the response. to address some of these gaps, we examined systolic bp (sbp) response to low-salt intake in relatively healthy adults using -hour ambulatory bp monitoring (abpm) and standardized dietary condi- tions. in this report we describe the distribution and determinants of sbp response to a low-salt diet in this relatively healthy population, using assessments of abpm during daytime and nighttime, defined on the basis of participants’ sleep logs. among the factors we examined were age, sex, pre-intervention sbp, the sbp response to the cpt, and physical activity (pa) levels. a better understanding of the predictors of sbp address for correspondence: braxton d. mitchell, phd, west redwood street, room , baltimore, md e-mail: bmitchel@medicine.umaryland.edu * present address: institute for clinical and translational research, johns hopkins university, baltimore, md manuscript received: february , ; revised: june , ; accepted: june , doi: . /j. - . . .x official journal of the american society of hypertension, inc. the journal of clinical hypertension vol | no | november response to low-salt intake can help to assess its impact on the general population and identify popula- tion subgroups who can most benefit from lowering salt intake. methods study population study patients were participants of the heredity and phenotype intervention (hapi) heart study. hapi participants were members of the old order amish community of lancaster county, pa. individuals and their family members were invited to participate if they were years or older and relatively healthy. exclusions for the study included high bp (> ⁄ mm hg), active cancer, liver, kidney (serum creatinine [cr] > mg ⁄ dl) disease, or untreated thyroid diseases, and being unable or unwilling to discontinue use of nutritional supplements, vitamins, and medications for the period of the study interventions. more informa- tion about patient recruitment, interventions, and mea- surements are available elsewhere. institutional review board approvals were obtained from the uni- versity of maryland and all patients provided written informed consent. study procedures and variables pre-intervention phenotypes were obtained during an initial clinic visit at the amish research clinic in strasburg, pa. all medications, vitamins, and supple- ments were discontinued for at least week prior to the initial clinic visit. pre-intervention bp was mea- sured in triplicate using a standard sphygmomanome- ter in the sitting position after minutes rest, and the average of the measures was used for analysis. pa was assessed during the dietary intervention by actical activity monitors (versions . and . ; mini mitter co inc, bend, or) worn by the patient for consecutive days. these devices incorporate an accel- erometer, sensitive to . times gravity in multiple directions, electronic circuitry, and a memory. acceler- ation of the device is integrated and expressed in units of activity counts within each -second recording interval. the daily mean of these counts was used for this analysis. dietary salt intervention. the dietary salt intervention study began following the initial clinic visit. study patients consumed a standardized high-sodium (na) diet ( meq ⁄ d= mg ⁄ d) for days, and then after a - to -day washout period, consumed a standardized low-na diet ( meq ⁄ d= mg ⁄ d) for days. the potassium (k) level was held constant at meq ⁄ d during both diets. all meals were prepared in a specially outfitted kitchen by an amish cook according to instructions carefully designed by the study nutritionist (nhb), and delivered to the patients’ home by an amish caterer. to test for dietary compli- ance, na, k, and cr levels were measured from the first morning urine sample obtained at the last day of each dietary intervention, and the na ⁄ cr and k ⁄ cr ratios were calculated from this single spot urine speci- men. the hapi heart study includes patients in total, of whom completed both arms of the die- tary intervention. measurement of -hour abpm. twenty four–hour bp and heart rate (hr) measurements were recorded at -minute intervals by an ambulatory bp monitor worn by study patients on the last day of each diet intervention. based on logs maintained by the study participants asking about the times they went to bed and woke up, we divided the -hour period into day- time (after rising in the morning) and nighttime (after bed time) and estimated the mean of the sbp recorded within each period. in daytime and nighttime sepa- rately, we defined the sbp response to low-salt diet as the difference in mean sbp between the low and high standardized salt diets. we excluded from analysis patients with < measurements in any one period (n= ), leaving patients with complete data. cold pressor test. the cpt was performed in a tem- perature-controlled room at the amish research clinic. bp was first measured every minutes for at least minutes or until a stable baseline bp was obtained, and then the patients were asked to immerse their right hand and wrist in ice water ( – �c) for . minutes. bp was measured times during and after the cpt at , , , , , . , , , and min- utes. the calculations of sbp response to cpt were previously detailed. briefly, the sbp response to cpt was calculated as the incremental area under the curve (iauc), defined as the difference between the area under the response curve and the area below baseline (defined as the average of the last measures before the cpt). we further subdivided the iauc into a com- ponent representing the reactivity phase (bp response during the first minutes of the cpt) and a recovery phase (bp response during minutes – of the cpt). statistical analysis we evaluated compliance to the high- and low-salt diets by comparing mean estimated levels of -hour urinary na and k excretion between groups via paired t test. the statistical analysis system programming language (sas . , cary, nc) was used for descriptive analysis. association analysis was carried out using the variance component method implemented in solar. we analyzed measurements of sbp response to low salt obtained from daytime and nighttime separately to allow for the possibility that associations might be stronger during nighttime when patients were supine and during which there might be less variation in bp. results basic characteristics of the study participants for whom sbp response to low-salt diet was computed are the journal of clinical hypertension vol | no | november official journal of the american society of hypertension, inc. bp response to low salt in the amish | montasser et al. presented in table i. the sample included men and women, whose mean (�sd) ages were . � . years and . � . years, respectively. after adjusting for age, men had significantly higher levels of pa counts and lower body mass index compared with women (p<. ). there was no significant difference between men and women in the level of pre-intervention sbp. while . % of men were smokers (mostly light pipe- smoking), none of the women reported tobacco use. mean levels of urinary na and k excretion adjusted for cr level are shown in table ii for the pre-interven- tion period and during the high- and low-sodium diets. mean urinary na ⁄ cr ratios differed little between pre-intervention and the high-salt diet (p=. ) consis- tent with our prior observation that the typical amish diet is high in salt content. in contrast, mean levels of urinary na ⁄ cr ratio decreased by almost -fold during the low-salt diet (p<. ) vs the high-salt diet, which is very close to what one would expect based on the meq ⁄ meq na content of the provided diets, suggesting a near-perfect compliance. mean urinary k ⁄ cr ratios were very similar among the pre-interven- tion and the high-salt diets (p=. ), but slightly higher during the low-salt diet. the distribution of sbp response to low-salt diet in both periods was normally distributed with no indica- tion of bimodality. the mean (�standard deviation) reductions in sbp response to low-salt diet from the high- to the low-salt diet were . � . mm hg (p=. ) and . � . mm hg (p<. ) during day- time and nighttime, respectively, with considerable variability around both means. the correlation in sbp response to low-salt diet measures computed from daytime and nighttime was . . a decrease of at least mm hg in sbp levels under the low- compared with the high-salt diet was observed in . % and . % of patients during daytime and nighttime, respectively. in contrast, . % and . % of patients experienced an increase of at least mm hg in sbp during daytime and nighttime, respectively, on the low-sodium diet. table iii shows the association of baseline character- istics with sbp response to low-salt diet during daytime and nighttime. older age and higher pre-intervention sbp were both significantly associated with higher sbp response to low-salt diet during daytime and nighttime, table i. basic characteristics for the hapi population variable men (n= ) women (n= ) all (n= ) p valuea age, y . � . . � . . � . . pisbp, mm hg . � . . � . . � . . bmi, kg ⁄ m . � . . � . . � . <. activity (counts), � . � . . � . . � . <. values are expressed as mean � standard deviation. abbreviations: bmi, body mass index; hapi, heredity and phenotype intervention heart study, pisbp, pre-intervention systolic blood pressure. ap value for sex differences adjusted for age (except for age). table ii. urinary na ⁄ cr and k ⁄ cr excretion before and during dietary intervention pre-intervention high salt low salt men na ⁄ cr . ( . ) . ( . ) . ( . ) k ⁄ cr . ( . ) . ( . ) . ( . ) women na ⁄ cr . ( . ) . ( . ) . ( . ) k ⁄ cr . ( . ) . ( . ) . ( . ) values are expressed as mean � standard deviation. abbreviations: cr, creatinine, k, potassium; na, sodium. table iii. adjusted sbp response to a low-salt diet during daytime and nighttime by age, sex, pre-intervention sbp, sbp response to cpt, and physical activity sbp response to low-salt dieta daytime nighttime age, y < . � . ) . � . – ) . � . ) . � . > ) . � . ) . � . p for difference by age . . e- sex men ) . � . ) . � . women ) . � . ) . � . p for difference by sex . . pre-intervention sbp, mm hg < ) . � . ) . � . � –< ) . � . ) . � . � ) . � . ) . � . p for difference by pre-intervention sbp . e- . e- sbp response to cptb low ) . � . ) . � . medium ) . � . ) . � . high ) . � . ) . � . p for difference by sbp response to cpt . . physical activity low ) . � . ) . � . medium ) . � . ) . � . high ) . � . ) . � . p for difference by physical activity . . values are expressed as mean � standard deviation. ap values age adjusted for sex and pre-intervention systolic blood pressure (sbp); p values sex-adjusted for age and pre-intervention sbp; p values for pre-intervention sbp adjusted for age and sex; p values for sbp response to cold pressor test (cpt), and physical activity adjusted for age, sex, and pre-intervention sbp. blow: first quartile; medium: second quartile; high: upper quartile. official journal of the american society of hypertension, inc. the journal of clinical hypertension vol | no | november bp response to low salt in the amish | montasser et al. while female sex and higher sbp response to cpt were associated with higher sbp response to low-salt diet only during nighttime. subsequent analyses were carried out to examine the association of sbp response to low- salt diet with the reactivity and recovery phases of the cpt, and the same pattern of association was observed with each phase separately as with the combined cpt response (data not shown). pa levels were not signifi- cantly associated with sbp response to low-salt diet measured during either daytime or nighttime. because hr changes in response to salt intake, we examined whether any of the observed correlations of sbp response to low salt were altered when adjusted for hr changes. we found that lower sbp response to low salt was correlated with increasing hr during daytime (p=. ) and nighttime (p=. ). however, the correlations of age, sex, pre-intervention sbp, and sbp response to cpt with sbp response to low salt remained after adjustment for hr, the only exception was that the association with sex was attenuated from p=. to p=. during nighttime. hypothesizing that sbp response to a low-salt diet might be correlated with other cardiovascular risk factors, we examined its association with body mass index, brachial artery flow–mediated dilation (measured as percent change in brachial artery diameter at peak hyperemia [diameter postprandial ) diameter at baseline] ⁄ diameter at baseline), lipid parameters (low- density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides), inflammation measures (serum levels of c-reactive protein, interleukin [il] , il- b, matrix metalloproteinase [mmp] , mmp- , and white blood cell count), and two measures of kidney function (estimated glomerular filtration rate and microalbumin to cr ratio). none showed any asso- ciation with sbp response to low-salt diet independently of age. we additionally considered whether the sbp reduction in response to low salt was more pronounced in the summer than in the winter because of the possibil- ity of increased salt loss due to perspiration and no significant difference in the sbp response was found. discussion this study used a well-controlled standardized dietary intervention design to investigate the characteristics and correlates of sbp response to low-salt intake in healthy drug-naive amish patients. our study has sev- eral distinguishing features that set it apart from other previously published similar studies. first, our study population was relatively young and healthy, enabling us to evaluate the distribution and predictors of sbp response to low-salt diet in a population without com- orbidities and to make inferences about the impact of low-salt diet at the population level. second, bp in our study was measured using -hour abpm, extracting data during daytime and nighttime based on the partici- pants’ sleep log, to provide as representative a measure of this trait as possible in a population setting. third, the high- and low-salt diets were carefully standardized, and urine collections were obtained to monitor and ver- ify dietary compliance, which was found to be excellent. a salt-sensitivity study, gensalt, was recently carried out in china. both the hapi heart study and gensalt participated in the program for genetic interaction (progeni) network, and the dietary interventions in the gensalt ( mmol and mmol of na ⁄ d for days for the low- and high-salt diets, respectively) and hapi ( mmol and mmol of na ⁄ d for days for the low- and high-salt diets, respectively) studies were coordinated to be comparable. similar to our amish population, the gensalt population was relatively young (mean age= . � . years.). however, in contrast to our study, gensalt was designed to include families at high risk for developing htn by restricting the study to families having a proband and at least one sibling with pre-htn or stage htn (sbp – mm hg and ⁄ or diastolic bp – mm hg). in fact, average sbp decreases in response to low-salt diet in the gensalt study were mm hg and mm hg in men and women, respectively. the benefit of salt restriction for bp reduction in the general population has been debated for a long time. there is now ample evidence from numerous clinical trials showing that salt reduction is associated with a -mm hg to . -mm hg reduction in sbp, with smal- ler reductions ( – mm hg) observed in normotensive individuals and larger reductions ( . – mm hg) observed in hypertensive individuals. similar results were also reported recently from a european longitudi- nal observational study, for which only a . -mm hg increase in sbp was detected with a -mmol increase in sodium excretion in young healthy individ- uals. the magnitudes of changes reported in normo- tensive individuals in these studies are in line with what we have observed in our relatively healthy amish population (ie, . - to . -mm hg reduction). the associations of age and pre-intervention sbp with sbp response to low-salt diet observed in the amish are well established. , – these associations are possibly explained by the age-related decline in both kidney function and the activity of membrane sodium ⁄ potassium-adenosine triphosphate, as well as reduced production of natriuretic factors such as dopamine. , the larger sbp reduction in response to low salt observed in women compared with men in our amish study has been observed in several previous studies, including the dietary approaches to stop hyperten- sion (dash)-sodium trial and gensalt. , this sex effect might be attributable in part to the effect of reproductive hormones on body fluids and sodium reg- ulation ; however, that effect was attenuated after considering the change in hr. similarly, our observation in the amish that patients experiencing a larger reduction in sbp in response to low salt also experienced a larger reduction in their sbp response to the cpt is consistent with previous observa- tions reported in chinese adults and children. the the journal of clinical hypertension vol | no | november official journal of the american society of hypertension, inc. bp response to low salt in the amish | montasser et al. cpt activates a global sympathetic response mediated by catecholamine release that produces vasoconstriction and increased bp. in general, increased na intake requires a corresponding increased renal handling and excretion to avoid retention of blood volume and subse- quent increase in bp. possibly, those with a proclivity for sympathetic overactivity are less able to increase renal na excretion in the face of a higher salt load. , the larger sbp response to low salt observed during nighttime, as well as the associations of female sex and higher sbp response to cpt only during nighttime, may reflect the relative stability of bp measured during night- time, as a result of lower sympathetic activity in the supine position vs the upright position. we looked at the association between sbp response to low salt and habitual levels of pa because exercise is known to improve insulin sensitivity, which may be linked to bp through the sympathetic nervous system. pa was not associated with sbp response to low salt in this population. however, the amish tend to have rela- tively high levels of pa relative to non-amish by virtue of their rural lifestyle and non-adoption of modern tech- nologies in the home, and it is possible that a more sedentary lifestyle than found in the amish may be associated with a larger sbp response to low-salt diet. although the distribution of sbp response to a low- salt diet in this healthy population is relatively similar to the changes in sbp observed with previously published dietary salt restriction trials, it is important to bear in mind that our intervention was of short duration. at present there are limited available data from well-con- trolled feeding studies that address the effects of long- term low-salt intake on bp or other health parameters in normotensive salt-resistant individuals. as such, it remains difficult to ascertain whether the increase in bp in response to low-salt intake represents a regression toward the mean (ie, random variation in salt-resistant individuals) vs an acute or long-term physiological response to salt restriction (eg, activation of the renin- angiotensin system) or a combination of these scenarios. interestingly, the previously mentioned recent longitudi- nal study showed an association between low salt and increased cardiovascular disease outcomes. this observation, as well as other arguments raise the intriguing speculation that there may be possible coun- ter-responses to low-na intake in susceptible individ- uals. – further experimental and longitudinal outcomes studies related to both cardiovascular and renal response to short- and long-term dietary sodium intake, notably in normotensive individuals, are war- ranted to explore this possibility. conclusions in summary, our study confirms the previous observa- tions that bp response to salt intake is a normally distributed trait with no evidence of bimodality and extends the epidemiology of sbp response to low-salt diet to a relatively healthy population in whom the response was measured using rigorous and standardized methods. a sizable proportion of this population experienced an increase in sbp during the low-salt diet. thus, overall, our data suggest that salt restriction (at least in the short-term) might not have 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hypertension. blood press. ; : – . . alderman mh. reducing dietary sodium: the case for caution. jama. ; : – . . jurgens g, graudal na. effects of low sodium diet versus high sodium diet on blood pressure, renin, aldosterone, catecholamines, cholesterols, and triglyceride. cochrane database syst rev. ; :cd . . overlack a, ruppert m, kolloch r, et al. divergent hemodynamic and hormonal responses to varying salt intake in normotensive sub- jects. hypertension. ; : – . the journal of clinical hypertension vol | no | november official journal of the american society of hypertension, inc. bp response to low salt in the amish | montasser et al. discussions in a socrates café: implications for critical thinking in teacher education western connecticut state university from the selectedworks of jody piro august , discussions in a socrates café: implications for critical thinking in teacher education jody piro gina anderson available at: https://works.bepress.com/jody_piro/ / http://www.wcsu.edu https://works.bepress.com/jody_piro/ https://works.bepress.com/jody_piro/ / this article was downloaded by: [dr jody piro] on: august , at: : publisher: routledge informa ltd registered in england and wales registered number: registered office: howick place, london, sw p wg click for updates action in teacher education publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uate discussions in a socrates café: implications for critical thinking in teacher education jody piroa & gina andersonb a western connecticut state university b texas woman’s university published online: aug . to cite this article: jody piro & gina anderson ( ) discussions in a socrates café: implications for critical thinking in teacher education, action in teacher education, : , - to link to this article: http://dx.doi.org/ . / . . please scroll down for article taylor & francis makes every effort to ensure the accuracy of all the 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questioning. results suggested that the nine universal intellectual standards provided an exceptional deductive frame- work for understanding the types and frequencies of socratic questioning occurring in the socrates café. the benefits of using a socrates cafe discussion for instruction to scaffold critical thinking are discussed and implications for teacher education are considered. introduction oliver ( ) suggested that critical thinking is a core learning goal for universities, and it is also central to the values of teacher education in that it promotes civil discourse and democracy in action for individuals and for the larger community. valuing the democratic purposes of educa- tion may be considered a central tenant of teacher education and the ability to critically analyze positions in discussion is a necessary component of those values. these goals are consistent with john dewey’s ( ) educational aim, the cultivation of critical thinkers and socially responsible citizens. teacher education pedagogy can do much to facilitate spaces where students engage with difficult and contrasting ideas within civil discourse. to prepare students who are literate in multiculturalism and an inclusive society (banks, ), the opportunity to participate in demo- cratic learning opportunities must be created. those educators who hope to create learning spaces that foster democratic values may do so by exploring difficult ideas through contem- porary educational dialogue and engaging in deliberative discussions that bring students with diverse viewpoints together. instruction that challenges stereotypes and assumptions is indispens- able in student exchanges, yet without these facilitated instructional spaces, students may remain correspondence should be addressed to jody piro, department of education and educational psychology, western connecticut state university, white street, danbury, ct . e-mail: piroj@wcsu.edu d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t mailto:piroj@wcsu.edu jody piro and gina anderson insulated in comfort zones that perpetuate stereotypes, shade interpretations of social interac- tions, and exaggerate differences between groups of people (naudé, , ). purposeful and deliberative pedagogies may accomplish communication across these differences. linda darling-hammond ( ) stated: america’s capacity to survive as a democracy . . . rests on the kind of education that arms people with an intelligence capable of free and independent thought . . . that helps people to build common ground across diverse experiences and ideas . . . that enables all people to find and act on who they are, what their passions, gifts, and talents may be, what they care about, and how they want to make a contribution to each other and the world. (p. ) recent research indicates critical thinking to be a significant area of improvement needed in preparing preservice and in-service teachers for increasingly diverse classrooms (gorski, ). critical thinking as a common instructional aim in teacher education has broadened to pedagogies supported by asynchronous online discussion boards (yang, newby, & bill, , ). instructors hold aspirations that their students will apply critical analysis to their own thinking and that of their peers in discussion forums. e-learning environments show promise for encouraging critical thinking (thomas, ), and collaborative student learning is supported by social constructivists who maintain that learning does not occur in isolation (vygotsky, ). yet students often use fallacious logic within their arguments and discussion forums can become repositories of distorted thought. as a proponent of critical thinking and civil discourse, christopher phillips ( ) originally established socrates cafés by creating open-invitation discussion groups in coffee shops and libraries and by investigating open ended questions with participants, such as “what is justice? what is good? what is courage?” (phillips, ). the same process can be implemented in online environments as well. as part of a socrates café, open-ended socratic questioning by instructors and students may scaffold the critical thinking of complex educational issues (elder & paul, ; golding, ; knezic, wubbels, elbers, & hajer, ; paul & elder, ). the purpose of this study was to benchmark critical thinking that was occurring in a socrates cafe by way of socratic questioning in an online discussion forum and to explore the implications for teacher education. approaches to critical thinking for the purposes of this article, we explore the connection between diversity curricula and critical thinking. we examined the vast literature on critical thinking within the context of the study. lai ( ) found several approaches to critical thinking bound in the philosophical, educational, and psychological disciplines (lewis & smith, ). all three approaches to critical thinking are addressed as they intersected with the study, along with measures of critical thinking and approaches to critical thinking in online or hybrid class formats. philosophical approach the philosophical approach to critical thinking, a system that emphasizes “reflective and rea- sonable thinking that is focused on deciding what to believe or do” (ennis, , p. ) and d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t discussions in socrates cafÉ “disciplined, self-directed thinking that exemplifies the perfections of thinking appropriate to a particular mode or domain of thought” (paul, , p. ) is an outcome of this approach. the philosophical approach to critical thinking may be found in the works of socrates, plato, aristotle, and, more recently, richard paul and linda elder (lai, ). the philosophical approach to critical thinking promotes the notion that broad critical analysis skills may be taught that are inde- pendent of content and cross subject domains. it is representative of scriven and paul’s ( ) definition of critical thinking as: intellectually disciplined process of actively and skillfully conceptualizing, applying, analyzing, syn- thesizing, and/or evaluating information from, or generated by, observation, experience, reflection, reasoning, or communication, as a guide to belief and action. in its exemplary form, it is based on universal intellectual values that transcend subject matter divisions. (para ) paul and elder ( ) suggested that humans regularly perceive truths and untruths in ways that do not reflect reality. humans do not naturally grasp the truth but see things as they want to see them. it is this distortion in thinking that led elder and paul ( ) to create standards for thought, “standards we can count on to keep our thinking on track, to help us mirror in our minds what is happening in reality, to reveal the truth in situations, to enable us to determine how best to live our lives” (p. ). when students sharpen their critical thinking skills, they create meaning by using sound logic rather than fallacious assumptions (paul & elder, ). universal intellectual standards (elder & paul, ) offer a structure for this process. universal intellectual standards (elder & paul, ; paul & elder, ; paul, ) advance a framework for structuring critical thinking within the philosophical approach to critical think- ing. elder and paul’s ( ) nine universal intellectual standards are clarity, accuracy, precision, relevance, depth, breadth, logic, significance, and fairness. these standards may be applied for assessing the quality of reasoning about a problem (elder & paul, ). in this approach, crit- ical thinking skills may be applied regardless of the content and are viewed as interdisciplinary aptitudes. the philosophical approach is a style of critical thinking that idealizes a person who is inquis- itive in nature, open minded, flexible, fair minded, has a desire to be well informed, understands diverse viewpoints, and is willing to suspend judgment to consider other perspectives may be considered philosophical in approach (facione, ). this style of critical inquiry advances thinking that meets “standards of adequacy and accuracy” and “thinking that is goal-directed and purposive . . . thinking aimed at forming a judgment where the thinking itself meets standards of adequacy and accuracy” (bailin, case, coombs, & daniels, , p. ). the philosophical approach to critical thinking focuses on the student’s ability to logically analyze arguments rather than solely on the teacher’s content expertise (plato, hamilton, & huntington, ). for example, socrates assisted students by employing questions, dialogue, and rebuttal for critically analyzing their understandings and misunderstandings of issues. this form of pedagogy begins with an open-ended question that proceeds to student response. it may be the instructor or other students who pose additional questions, furthering the analysis and discussion outcomes. through the questioning process, students learn to recognize their own limitations in content and in analysis, thus leading to an expanded critical thinking. the profes- sor’s role is facilitative in nature. the professor gently probes students’ responses and guides students through controversial issues (mcavoy & hess, ). the ultimate purpose is enlarged personal understandings of difficult educational issues through social and situated learning. d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t jody piro and gina anderson educational approach educational approaches to critical thinking encompass varying lenses, psychological and philo- sophical. a hierarchical taxonomic approach to information processing skills (bloom, engelhart, furst, hill, & krathwohl, ) is widely used in educational circles to encourage the higher order thinking skills of synthesis and evaluation. some have faulted bloom’s taxonomy for its vagueness (ennis, ) and others for lack of reliability (sternberg, ). still, the notion of applying instructional interventions to scaffold critical thinking undergirds the educational approach. although higher education instructors have pedagogical hopes for critical thinking in their courses, some researchers have suggested that increased critical analysis in classrooms rarely occurs without instructional assistance (abrami et al., ; case, ; facione, ; halpern, ; hew & cheung, ; landsman & gorski, ; paul, ). in teacher education, criti- cal thinking may be viewed as an interdisciplinary form of expression for multiple content areas. further, it may be asserted that using a framework for scaffolding critical thinking in teacher education will increase the probability of a desirable outcome (halpern, ). knezic et al. ( ) examined empirical research (i.e., griessler et al., ; parkinson & ekachai, ; pihlgren, ; yang et al., ) to explore the use of the socratic method in teacher educa- tion. additionally, use of socratic questioning (elder & paul, ; golding, ; paul & elder, ) has been suggested for enhancing critical thinking in classrooms. assessing critical thinking in educational contexts poses some challenges. the debate over whether critical thinking skills may be taught in general or whether they are content specific (case, ; ennis, ; lipman, ; silva, ) is related to instruments that measure critical thinking in general versus specific, domain-oriented measures. there are multiple assess- ments for measuring students’ critical thinking, including: the california critical thinking skills test (facione, ), the cornell critical thinking tests (ennis, millman, & tomoko, ), the ennis-weir critical thinking test (ennis & weir, ), and the watson-glaser critical thinking appraisal (wilson & wagner, ). a criticism of these measurements is that they are general critical thinking tests and are not situated and contextual or subject specific (kennedy, fisher, & ennis, ). furthermore, they are not necessarily designed for online environments. as educational arenas embrace technology for pedagogy, online discussion forums have been touted as a means for engaging students in critical thinking at various levels of education (macknight, ; perkins & murphy, ; trufant, ; yang et al., ). at the k- level, using the internet has been suggested for increasing critical thinking (kurubacak & gonzales, ; salpeter, ), but critical thinking in these contexts has focused mainly on information validity and literacy, whether online sources have valid information. cognitive-psychological approach the ability to problematize issues and understand the perspectives of others is a particularly sig- nificant outcome for critical analysis. the cognitive-psychological practice for increasing critical thinking emphasizes “seeing both sides of an issue, being open to new evidence that disconfirms your ideas, reasoning dispassionately, demanding that claims be backed by evidence, deducing and inferring conclusions from available facts, solving problems, and so forth” (willingham, d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t discussions in socrates cafÉ , p. ). the cognitive-psychological approach to critical thinking emphasizes people’s actual actions and outputs, rather than the merely idealized expressions of critical thinking which typ- ify the philosophical approach. this perspective for increasing critical analysis may be found in the frameworks and taxonomies that this approach promotes for assessing the production of critical thinking. the cognitive psychological approach to critical thinking suggests that behav- iors or skills that imply critical thought may be determined (lewis & smith, ) and that the use of “skills or strategies that increase the probability of a desirable outcome” (halpern, , p. ) may be measured by frameworks intent on increasing critical thinking. the analysis of the data through a framework is suggestive of a cognitive psychological approach (lai, ). reflecting the cognitive-psychological approach to measuring critical thinking, data in this study were analyzed through a taxonomic structure, the universal intellectual standards. philosophical, educational, and cognitive science approaches to critical thinking literature each influenced the development of this study. socratic dialogue and questioning has shown promise in cultivating critical thinking. however, research that measures critical thinking via socratic questioning or that provides a framework for their enhancement in contextual and domain- specific online environments, such as teacher education, warrants further investigation. the lack of research in the assessment of online, subject-specific measures of critical thinking provided the impetus for the inquiry into socrates café online discussion as an instructional tool in teacher education. the socrates cafÉ the socratic method has found novel form in a new expression, the socrates café. a socrates café “reveals people to themselves” and “makes them see what their opinions really amount to.” in that the true philosopher “helps to inculcate men and women against the seductive half-truths of sophists,” the socrates café “is not so much a search for absolute truth and certainty as it is a quest for honesty” (phillips, , p. ). a socrates café promotes reflection, reconceptualization of taken-for-granted notions, and perspective building. a full discussion of our socrates café follows. an online discussion forum, the socrates café, served as the construct for the expression of critical thinking in this study. this construct was chosen to facilitate critical analysis of the issues so that students might increase their awareness of socratic questioning by examining their own and other students’ written expression. the instructors’ role in the online socrates café was to develop opportunities to interact and pose socratic questions in a process-oriented search for students’ own reflective conceptions of taken-for-granted assumptions about current issues and to develop their understanding of those issues. our socrates café parallels a pragmatic philosophical forum in that discussion topics are related to the participants’ own lives as educators (pamerleau, ).we situate our socrates café in parker and hess’s ( ) typology of discussion where the purpose of a socratic semi- nar is to reveal the world with greater clarity and nuance to the participants. in that we require a textual grounding for analysis, our socrates café resembles a socratic seminar, whose pur- pose is to increase students’ powers of understanding, or what adler ( ) termed “enlarged understandings,” through the application of content and reading to open-ended questions. this requirement focuses on a disposition of scholarliness and use of text through readings to inform d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t jody piro and gina anderson the participant’s dialogue in text. like phillips’ ( ) socrates café, we explore big questions in an open-forum style discussion. we also differentiate our use of socrates café from traditional discussion and public socrates cafés. the socrates café in this study is not face-to-face or synchronous whereas the forum of traditional socratic seminars and socrates cafés tend to be face-to-face in organization. our socrates café diverges from socratic seminar in that the purpose is more political and agency oriented than the socratic seminar, which has no specific outcome rather than increased under- standing (parker & hess, ). the socrates café results in a clearer understanding of issues that emerged from the socratic seminar and then culminates in a course of action. in the case of the participants, this action may result in integration of the principles of diversity and democracy into classrooms. unlike a public philosophy forum where participants come of their own voli- tion and shape the flow of discussion in entirety (pamerleau, ), in the study socrates café, “attendance” in the online forum is required. although phillips’ socrates café ( ) and other public philosophical discussions are not evaluated by the facilitator or participants, and in fact, all participant responses may be given equal measure, our socrates café was evaluated by using the intellectual standards (elder & paul, ) as a framework for taxonomic analysis of socratic questions. the nine universal intellectual standards (elder & paul, ) known as clarity, accu- racy, precision, relevance, depth, breadth, logic, significance, and fairness were used to scaffold critical thinking and to develop socratic questions during the discussion, as well as to assess their use at the conclusion of the activity. guidance by and assessment with the universal intellectual standards differentiates the socrates café used in this study from forums of discussion grounded in public philosophy (pamerleau, ). method this inquiry was located within teacher-researcher methodology (cochran-smith & lytle, ). the research context was the online discussion posting from the teacher-researchers’ course and critical thinking via socratic questioning in the online discussion was identified as the as the area of inquiry (mills, ). the purpose of this study was to benchmark the types of socratic questioning in an online socrates café. the research questions were “when socratic questioning occurred in the socrates cafe, what were the types and frequencies of questioning that were employed? what were the implications for scaffolding critical thinking instruction in online and hybrid environments in teacher education?” background and theoretical framework of the context as professors who coteach a graduate-level teacher education course on diversity, the researchers regularly engage in critical conversations about pedagogical practices. the conversations support and challenge the continued practice of fostering students’ critical analysis. we vacillate between the acts of constructing, affirming, deconstructing, and rejecting our practices in iterative cycles; a constant situating and repositioning of ourselves and others within the context of the work of teaching a diversity curriculum. this ongoing process led to the creation of the socrates café in an online discussion forum that asked students to use socratic questioning to investigate their own and peers’ lines of analysis with the goal of increasing critical thinking. this study served to benchmark that attempt. d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t discussions in socrates cafÉ sociocultural and situated learning theories framed this graduate, diversity course in teacher education, revealing the reciprocal nature of teaching and learning. socrates café discussions require students to apply critical thinking and socratic questioning, which are considered higher ordered skills, within an online, social setting. the application of these skills does not occur in a vacuum. rather, sociocultural theory conveys that learning occurs through social interaction with self, others, and the community within specific contexts (vygotsky, ). as students engage with the course content and readings, reflect upon their own diversities and experiences, and discuss the connections and disconnections of these topics and experiences with others within a taxonomic form of scaffolded instruction, new meanings and understandings may emerge. situated learning theorists similarly posit that learning occurs as a function of the activity, context, and culture (korthagen, ). sociocultural and situated learning are social, cultural, and contextual that suggests human development and social interaction are interconnected and cannot be divided. socrates café discussions provide the opportunity for authentic investigations of diversity topics with student colleagues, rather than in isolation by individuals, which is often required for traditional types of assignments. in essence, the real-world context of the diversity topics, the social interactions created in the socrates café discussion boards, and the framework used to help scaffold the critical thinking instruction create opportunities for learning. socio- cultural and situated learning structures complemented the context that situated this inquiry—an online discussion forum titled socrates cafe within a diversity course in teacher education. context of the study the context of this study was in one diversity course within a required graduate teacher educa- tion program with students in preservice and in-service teacher education programs. the goals of the course included understanding the conceptual and philosophical issues of diversity in schools; issues of discrimination, bias and equity surrounding students with diverse needs; issues concern- ing multicultural, pluralistic, and global education; issues of nontraditional families; the impact of specific types of diversity on teaching and learning including race, ethnicity, culture, socioeco- nomic status, language, religion, gender and sexual orientation, cognitive differences, ableness; multicultural curriculum and culturally responsive teaching; and assessment issues related to students from diverse backgrounds. diversity pedagogies construct visions of possibility for students and society. these pedagogies focus on the intersection of power and knowledge and oppose social, economic, and political formations that limit or reduce individual or collective visions of the future (cushner, mcclelland, & safford, ; lusted, ). social justice pedagogies were embedded in the diversity course, constituting a focus on power relations as essential to pedagogy (mclaren, ). the application of social justice pedagogies and the facilitation of our students’ critical analysis of intersections of power, privilege, and the social constructions of knowledge prompted an instructional decision to scaffold critical thought within the course. the socrates café the diversity course utilized an online socrates café discussion with socratic questioning as scaf- folding toward the goal of critical inquiry. through online resources, use of a socrates café and d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t jody piro and gina anderson socratic questioning were introduced and facilitated in the online forum. an ungraded practice socrates café discussion was modeled initially. a model prompt is demonstrated below: read peggy mcintosh’s white privilege located in the corresponding thematic unit. discuss the connections/disconnections you made to the article and any impressions it made upon you. regardless of your race or ethnicity, describe any actions or events you may have encountered or from which you have benefitted based on mcintosh’s reference to this “invisible package of unearned assets. following the instructor prompt, students engaged in the online discussion and the professors gave written feedback to the students. after that, the professor-researchers provided initial topics or questions related to course readings, which then provided a springboard for students to organ- ically write original and reply postings. these resources were the only interventions provided to students. the professor-researchers did not offer any subsequent subject for discussion, and all postings were made by students in the course. students wrote original and response postings each week within a -week semester to issues within the class based upon readings, inter- ests, or current events. sample student postings, which occurred through semester-long diversity courses, included content related to globalization, prejudice, discrimination, immigration laws, and religious diversity. data collection and analysis this study involved a data-collection system consisting of postings within a required online dis- cussion forum titled, the socrates café, through blackboard, a learning management system. the postings from approximately students in two sections of the course were analyzed for two subsequent semesters. data were collected throughout the semester and were selected based upon the following criteria: an original posting or reply that demonstrated at least one socratic question; and postings were selected at multiple points in time throughout the semester. although the coding structure for the reading of the online forum postings was taxonomic, the analysis of the qualitative data was grounded in a constructivist approach (denzin & lincoln, ), as was the researcher reflexivity of the data collection and analysis (kleinsasser, ). both professor- researchers engaged in the collection and analysis of the data. the professor-researchers analyzed a selection of original postings and student replies for a total of socratic questions. none of the data were retouched for grammar or syntactical purposes. the data were analyzed using a directed content analysis approach (gall, gall, & borg, ; hsieh & shannon, ) with a structured deductive reading (kyngas & vanhanen, ) through an already identified situated framework. socratic questioning within a socrates café forum was coded for level of universal intellectual standard. we used elder and paul’s ( ) nine universal intellectual standards: clarity, accuracy, precision, relevance, depth, breadth, logic, significance, and fairness. the taxonomy was used in much the way it was created, to structure the partici- pants’ thought processes away from complete egocentric thinking—“it is true because i think it is true. it is true because i have always believed it. it is true because it is in my selfish interest to be true” (paul & elder, , p. ) with the purpose being “to become infused in the thinking of students, forming part of their inner voice, which then guides them to better and better rea- soning” (paul & elder, , p. ). the use of the taxonomy for the purposes of benchmarking socratic questions in teacher education is a novel use of the standards. table demonstrates the d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t discussions in socrates cafÉ table research method research questions when socratic questioning occurred in a socrates cafe, what were the types and frequencies of questioning that were most often employed? what were the implications for scaffolding critical thinking instruction in online and hybrid environments in teacher education? data collection archival online socrates café postings from one year of a diversity class in a teacher education curriculum. data analysis deductive directed content analysis: taxonomic using the intellectual standards (elder & paul, ). trustworthiness peer debriefing; inter-rater agreement. limitations nongeneralizable; narrowing of data through construct. universal intellectual standard, the type of socratic question that was coded for a standard, and the frequency of that type of socratic question. the coding decisions were based upon the situatedness and contextual nature of the content of the postings. the coding decisions reflected a more holistic analysis of the entire flow of stu- dent contemplation within the discussion board rather than the single, atomistic posting, itself. trustworthiness was addressed by using peer debriefing (marshall & rossman, ) through ongoing data chats. inter-rater agreement of coding was enhanced through the data chats between the researchers. practice in the use of the taxonomy prior to and throughout the analysis fur- ther strengthened trustworthiness. institutional review board protocols were observed. table summarizes the research methods. results when students sharpen their critical thinking skills, they create meaning by using sound logic rather than fallacious assumptions (paul & elder, ). universal intellectual standards offered a structure for this process. the nine standards were used to code socratic questioning events within the socrates café. in general, varying types of socratic questioning were found within the online socrates café. the highest frequencies of postings (n = ) were coded at intellectual standard five-depth and intellectual standard nine-fairness (n = ). the universal intellectual standards with the least amount of postings within the online socrates café forum were intellectual standard seven-logic (n = ) and intellectual standard six-breadth (n = ). the following table, modified from elder and paul ( ), lists the intellectual standard the types of questions that would identify the socratic question at that level, and the frequency of socratic questioning coded for each intellectual standard. analysis of socratic questioning through the universal intellectual standards in this section of the results, examples of socratic questioning types as they were analyzed from the data are provided. adhering to the organization shown in table , we demonstrate repre- sentative postings that were coded at a universal intellectual standard and the types of socratic questions that were created within that posting. d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t jody piro and gina anderson table standards, types, and frequencies of socratic questions level of intellectual standard types of socratic questions asked within the posting frequency of socratic questioning standard one- clarity could you elaborate further? could you give me an example? could you illustrate what you mean? standard two- accuracy how could we check on that? how could we find out if that is true? how could we verify or test that? standard three- precision could you be more specific? could you give me more details? could you be more exact? standard four- relevance how does that relate to the problem? how does that bear on the question? how does that help us with the issue? standard five-depth what factors make this a difficult problem? what are some of the complexities of this question? what are some of the difficulties we need to deal with? standard six-breadth do we need to look at this from another perspective? do we need to consider another point of view? do we need to look at this in other ways? standard seven- logic does all this make sense together? does your first paragraph fit in with your last? does what you say follow from the evidence? standard eight-significance is this the most important problem to consider? is this the central idea to focus on? which of these facts are most important? standard nine-fairness do i have any vested interest in this issue? am i sympathetically representing the viewpoints of others? total socratic questions n = intellectual standard one-clarity within a discussion on religious diversity a student wrote about deciding how to best address diverse celebrations, such as kwanza, christmas, and eid. she asked, “how do i, as the teacher, address varying celebrations in my classroom?” in a follow-up reply, a fellow student continued her line of inquiry. she suggested that celebrations should be a learning activ- ity not a worship experience and followed up with another clarity question, “do you think that is a clear enough distinction between the two instructional activities?” a third student asked, “can you clarify what you mean by teaching celebrations?” these questions in this context were coded as intellectual standard -clarity, in that each sought elaboration from peers. d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t discussions in socrates cafÉ intellectual standard two-accuracy within a discussion on alabama’s immigration law, two students questioned the sovereignty of state law that they felt was unfair to the immigrants in that state. one student asked, “can citizens from other states influence a state decision such as this one?” another student posed the question, “as teachers, would we be legally bound to provide student names of those we suspected were residing illegally?” in the context of quality of education and level of crime, a student questioned other students by stating, “crime is not something that we inherently value,” then went on to question, “if this is indeed true, how does education encourage us against crime? is it because more education reduces our desire for crime because we earn more money?” each of these socratic questions was coded at the level of intellectual standard -accuracy, in that each asked other students to consider how one can determine the veracity of contentions. intellectual standard three-precision in the context of a discussion of the responsibility that teachers have in the affective and cognitive domains, a student maintained that the philosophy of progressivism combines the affec- tive and cognitive domains of education and teaches real-world skills. a fellow student posted a socratic question, asking for more precision for this contention, by using intellectual standard -precision: “can you be more specific? can you give examples of skills that students would be taught by progressives?” in the context of a discussion of a news release that a high school student was not allowed to attend her prom because her date was female a student asked another student for clarification on why she agreed with the school board cancelling the event. the stu- dent asked, “can you offer some clarification or more details on why you argue that cancelling the prom was the wisest decision? isn’t the way to solve the issue to improve tolerance and understanding?” intellectual standard four-relevance in a discussion on setting expectations for students, an original posting discussed her parent’s disciplinary choices for her siblings by questioning the relevance, intellectual standard four, of expectations on the outcomes of each sister and how the different expectations had impacted their lives: “if the same expectations had been set for my sister than for me, would the outcome have been any different for them?” the relevance of globalization on education was discussed, with the context specifically addressing the outsourcing of goods and services to other countries for cost savings. the student making the original post asked, “do you think that the same factor of globalization influences the institutions of education?” intellectual standard five-depth in an original post addressing the a priori contentions of the textbook (i.e., studying diversity is good; it will create a better educational system), a student making an original post examined the inherent difficulty and complexity of the notion of diversity curricula by using intellectual d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t jody piro and gina anderson standard five-depth. she asked peers to question, “what is a ‘better’ educational system? what makes a ‘better’ person? what is this ‘good’ end toward which we are striving by studying diver- sity?” another student, addressing the inherent complexity of this issue asked, “where would we start as classroom teachers?” this last question addressing the depth and complexity of the issue was coded at intellectual standard , rather than just looking for clarification, which would suggest intellectual standard one. intellectual standard six-breadth examples of breadth were found in two different discussions; one regarding a pbs frontline show focusing on a teacher’s hands-on lesson on racial discrimination, and one on the benefits of collaborative teaching and learning. for the former discussion, a student posed the original ques- tion, “could schools include other issues of discrimination besides race, such as gender, sexual orientation, classism, ableism, etc. in similar types of lessons?” for the latter discussion, a student posed the original question, “is collaborative work always the best idea or could competition or individual teaching and learning also yield positive results?” both socratic questions were coded as the intellectual standard six—breadth, because of their comprehensive nature and attention to multiple viewpoints. intellectual standard seven-logic the intellectual standard of logic was noted in a discussion on recent immigration laws viewed as punitive based on several students’ previous posts. a student claimed she was playing devil’s advocate when she posted the reply questions, “are we all suggesting that it’s okay to turn our heads on one thing that is illegal and not another? should all law breaking situations be treated differently, then? how do we discern what is okay to turn our heads at and what is not?” her line of questioning indicated she was concerned with consistency and the elimination of contradiction. intellectual standard eight-significance one discussion investigated the context of assumptions such as the amish not valuing edu- cation or diversity. according to a student’s viewpoint, many amish people assume this stance because, according to her statements, most amish never go to school beyond the eighth grade, marry within their own faith, and often shun the social and economic influences of the dominant culture. follow-up socratic questions that were posted from a second student included, “does their society suffer because it does not share these same ideals? or, would the acceptance of these ideas cause their society to suffer? do the actions (or inactions) of a self-contained society affect those who aren’t a part of it?” a third student responded with additional questions of her own, i wonder, is a public-schooled american with a phd held in higher esteem by his/her community than a respected amish elder within his community? perhaps the ‘education’ valued by the amish is a different type of education than a structured public institution’s curriculum? these response questions were coded as intellectual standard eight due to awareness of what constitutes significance or substantial meaning to the issue at hand; whether the amish value education and diversity. d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t discussions in socrates cafÉ intellectual standard nine-fairness an ongoing discussion of deconstructing arguments from the textbook led to the questions, “is there common knowledge or a majority moral principal we all share as humans? what is the definition of a ‘better’ way of living that education ensures?” a student replied with the following additional questions, coded as intellectual standard nine-fairness, which indicated she was concerned with the viewpoints of others rather than privileging her own position, “what is considered common knowledge? who gets to decide this? what is morality, and who draws the lines? are we talking about religious morality or human instinct morality? is there a ‘better’ education that exists? if there is, what makes it ‘better’?” discussion of results in this study, an online socrates café structured our inquiry into critical thinking in an online dis- cussion. the nine universal intellectual standards provided an exceptional deductive framework for understanding the types and frequencies of socratic questioning occurring in the socrates café. the standards relating to depth, relevance, and fairness represented the most utilized standards. the standard relating to logic was used least often in the socrates café. we found it interesting to discover which levels of universal intellectual standards were applied more often than others. for example, the intellectual standard seven-logic was coded less frequently than other standards. the nature of the course content or teacher education disci- pline may have played a part in in the lower frequency level of this standard. diversity studies, especially in the field of teacher education, often focus on equity and social justice issues. ethical dilemmas that challenge the definitions of what is legal and what is ethical are common. the difference between what is equal, equitable, and fair is often debated, as well. more often than not, issues are problematized rather than simplified, as multiple perspectives are valued over one ideology. thus, it may be that certain intellectual standards are more meaningful to this field than in others (elder & paul, ). the content of diversity curricula often requires a complexity of thought that exceeds a binary analysis of the issues. the questions that demonstrated the intellec- tual standards of depth and fairness reaffirm the contextualized nature of the discipline. critical thinking via socratic questioning may be contextual, content driven, and domain specific (bailin, ; willingham, ). the universal intellectual standards for creating defined categories of critical thinking based upon the production of socratic questioning were found to be beneficial for understanding the types and frequencies of questioning that were being produced in an online discussion forum called the socrates cafe. socratic questioning is assessed by the universal intellectual standards within a socrates cafe for the pedagogical goal of enhancing critical thinking holds promise for practice. the main limitation to the study is that it is contextual and non-generalizable to a wider population. implications if, as the results suggest from this contextual study, a socrates café that employs socratic questioning structured through the universal intellectual standards scaffolds critical thinking d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t jody piro and gina anderson instruction, there are theoretical and practical implications for teacher education. scaffolded crit- ical thinking instruction in online teacher education courses has far-reaching implications toward a much broader, pedagogical goal. socrates café online discussions in this study invited partici- pants to engage with issues that surpassed the self and connected with larger societal problems, which therefore has the potential to enhance civil discourse necessary in a democratic society. critical thinking as a pedagogical goal may be perceived within the larger objective that teacher educators often have for their students, creating a reflective practice (schön, ). establishing a community of critical discourse surrounding educational issues by engaging with contrasting ideas, assisting others to view perspectives outside of their own frameworks of thought, and insist- ing on using evidence support one’s contentions enhances democracy in action (dewey, ). critical thinking by way of socratic questioning within the socrates café supports the combined accumulation of individual and community growth. civil discourse as a value in a democracy is an outcome when individuals and communities use critical thinking to examine difficult issues. the interactions with others as a result of the socratic questions and dialogue stimulates a tran- scendence of the self and further improves the “habits of mind” (meier, ) necessary for the interpretive element of thinking. discussions in socrates café are contextually situated and spring from the participants’ own social, cultural, ethnic, and gendered experiences. this approach is consistent with the sociocultural and situated theoretical foundations of the socrates café discus- sion and the research, itself, and teacher educators interested in grounding their practice in these foundational values may find benefit in socrates café discussions. if critical thinking skills may be taught then there are considerations for practice as well (halpern, ; kennedy et al., ). there are several practical instructional interventions that teacher educators may consider to promote the use of socratic questioning for critical think- ing within online or hybrid courses. a first level of intervention might include an explicit lesson (ennis, ) using the framework of universal intellectual standards. supporting visual and reading materials within the online electronic forum may be provided and an open-ended, domain specific question(s) may guide the socrates café. the questions should be grounded in the con- tent and reflect actual problems in educational settings (halpern, ). this intervention stems from the cognitive science position that students require “deliberate practice” (van gelder, ) in the actual experience of critical thinking and questioning. as some online formats require one or more traditional instructional meetings, a second-level intervention to support socratic questioning might include a face-to-face socrates café lesson that would be fashioned upon the online forum. in the lesson, students would address content related to the class in a collaborative grouping environment and analyze the types of universal intellectual standards they discov- ered within their dialogue (anderson & piro, ). this intervention addresses a collaborative approach to teaching critical thinking (abrami et al., ; heyman, ; paul, ) and a domain-specific approach to critical thinking (bailin et al., ; lipman, ; silva, ). the course content serves as the lens for the collaborative and critical thinking process. using a general approach to critical thinking instruction through a framework, such as the universal intellectual standards, and socratic questioning within a course-specific curriculum, supports the mixed method of teaching critical thinking that suggests that instruction integrating critical think- ing into domain specific academic content and teaching general critical thinking skills (lai, , p. ) has the largest effect-size for increasing critical thinking through instruction (abrami et al., ). d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t discussions in socrates cafÉ an instructional approach for increasing critical thinking through collaborative discussion is established in piagetian and vygotskian traditions (lai, ). piaget’s notion of cognitive conflict in learning and vygotsky’s zone of proximal development suggest that working with more capable peers or adults has instructional value (dillenbourg, baker, blaye, & o’malley, ). collaboration works best when it is scaffolded (nelson, ), such as in a socrates café that uses the universal intellectual standards for analyzing socratic questions. further, it is entrenched in the notion of a community of critical learners. dewey ( ) explained the concept of community: there is more than a verbal tie between the words common, community, and communication. men live in a community in virtue of the things which they have in common; a communication in which they come to possess things in common. what they must have in common in order to form community or society are aims, beliefs, aspirations, knowledge—a common understanding—like mindedness as the sociologists say. (dewey, , p. ). a collaborative intervention would require students to analyze themselves with the assistance of peers within their group, leading to the following questions: what types of socratic questioning am i producing? what types do i tend to omit in my inquiry? in a third level of instructional support, teacher educators might provide structured prompts and replies within the online socrates café developed from the content of the course and paral- leling the assigned readings. instructors might model (hemming, ) varying types of socratic questioning within the forum, and practice coding those socratic questions within the universal intellectual standards. each of these instructional interventions provides fodder for inquiry into the practice of promoting critical thinking in online coursework. conclusion embedded in a socrates café is a search for honesty (phillips, ); and when it is scaffolded with the universal intellectual standards, it is a quest that may sharpen participants’ critical thinking skills by using sound logic rather than fallacious assumptions (paul & elder, ). this contextual study revealed that benchmarking socratic questioning types was beneficial for scaf- folding critical thinking instruction. the use of the universal intellectual standards for creating defined categories of critical thinking based upon the production of socratic questioning was an effective tool for identifying the types and frequencies of questioning that were being produced. the types and frequencies of socratic questioning applied in the socrates café online discus- sions were contextual and situational. diversity issues are complex, and multiple perspectives are often valued over one ideology. the results of this study support the idea that certain intellectual standards are more meaningful in certain fields when compared to others (elder & paul, ). the socratic questions that emerged in the online discussions that demonstrated the intellectual standards of depth and fairness reaffirmed the contextualized nature of the teacher education discipline. the use of the universal intellectual standards to benchmark, typify, and count the socratic questioning occurring within a socrates café for the pedagogical goal of enhancing critical thinking instruction holds promise for practice, especially in online settings where little precedence has been established. d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t jody piro and gina anderson discussion—and a socrates café discussion in particular—is part of a larger curricular goal (parker & hess, ) that intersects the two aspirations of diversity and democracy in an ongoing inquiry. the socrates café makes explicit pedagogical intentions to assist students “to set along- side one perception of the matter under discussion the several perceptions of other participants, challenging our own view of things with those of others” (bridges, , p. ), creating a new dialectic in the process. the socrates café online discussions in this study revealed a symbiotic relationship between the value of critical analysis and values of diversity for teacher educators in that its format has the potential to facilitate civil discourse and democratic engagement for both individuals and for the larger community. references abrami, p. c., bernard, r., borokhovski, e., wade, a., surkes, m., tamim, r., & zhang, d. 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( ). using socratic questioning to promote critical thinking skills through asyn- chronous discussion forums in distance learning environments. american journal of distance education, ( ), – . doi: . /s ajde _ yang, y., newby, t., & bill, r. ( ). facilitating interactions through structured web-based bulletin boards: a quasi- experimental study on promoting learners’ critical thinking skills. computers & education, ( ), – . doi: . /j.compedu. . . d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t http://dx.doi.org/ . /\gdef yes{no}\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}}(issn) - http://dx.doi.org/ . /\gdef yes{no}\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}} - http://dx.doi.org/ . /\gdef yes{no}\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}}s - x( ) - http://dx.doi.org/ . /\gdef yes{no}\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}}s - ( ) - http://dx.doi.org/ . /\gdef yes{no}\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}}(issn) - http://dx.doi.org/ . /\gdef yes{no}\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}}ctch. . . - http://dx.doi.org/ . /\gdef yes{no}\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}}s ajde \gdef yes{no}_\gdef \ {_}\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{_\gdef \ {_}\gdef no{no}\gdef yes{yes}} http://dx.doi.org/ . /\gdef yes{no}\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}\gdef \ \gdef \ {\ }\gdef no{no}\gdef yes{yes}{\penalty \z@ \gdef \ {\penalty \z@ }\gdef no{no}\gdef yes{yes}}j.compedu. . . http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva- www.criticalthinking.org/aboutct/define_critical_thinking.cfm http://files.eric.ed.gov/fulltext/ed .pdf http://files.eric.ed.gov/fulltext/ed .pdf http://net.educause.edu/ir/library/pdf/ncp .pdf http://dx.doi.org/ . /(issn) - http://dx.doi.org/ . / - http://dx.doi.org/ . /s - x( ) - http://dx.doi.org/ . /s - ( ) - http://dx.doi.org/ . /(issn) - http://dx.doi.org/ . /ctch. . . - http://dx.doi.org/ . /s ajde _ http://dx.doi.org/ . /j.compedu. . . discussions in socrates cafÉ jody piro is an associate professor in the doctor of education in instructional leadership program at western connecticut state university. she has been involved in education for over twenty-five years in k- as a social studies teacher and as a dean and principal, and in higher education as a professor and dissertation director. dr. piro’s current scholarly interests focus on accountability outcomes for educators and problematizing discussion for critical analysis and civil discourse. gina anderson is an associate professor of teacher education and the program coordinator of the curriculum & instruction program at texas woman’s university in denton, texas. prior to her work at twu, dr. anderson taught several years of elementary and middle school in oklahoma city, oklahoma and served as a student teacher supervisor and teaching and research assistant at oklahoma state university. culturally-responsive teaching strategies and the scholarship of teaching and learning guide her research and scholarly interests. d ow nl oa de d by [ d r jo dy p ir o] a t : a ug us t 	western connecticut state university 	from the selectedworks of jody piro 	august , 	discussions in a socrates café: implications for critical thinking in teacher education 	abstract 	introduction 	approaches to critical thinking 	philosophical approach 	educational approach 	cognitive-psychological approach 	the socrates cafÉ 	method 	background and theoretical framework of the context 	context of the study 	the socrates café 	data collection and analysis 	results 	analysis of socratic questioning through the universal intellectual standards 	intellectual standard one-clarity 	intellectual standard two-accuracy 	intellectual standard three-precision 	intellectual standard four-relevance 	intellectual standard five-depth 	intellectual standard six-breadth 	intellectual standard seven-logic 	intellectual standard eight-significance 	intellectual standard nine-fairness 	discussion of results 	implications 	conclusion 	references concrete scalp in trauma sir, the scalp as a donor site for the skin graft was first described by crawford in .[ ] the scalp as a donor site allows multiple skin graft harvests. however, multiple complications, including bleeding, alopecia, exudative crusting/“concrete scalp deformity,” staphylococcal folliculitis have been described.[ , ] concrete scalp deformity is a condition seen when thickness of harvested skin graft is more than . mm.[ , ] it is characterised by a boggy granulating mass with enmeshed growing hair. however, this condition is not reported after trauma. deep abrasions in the scalp occurring due to trauma if not treated properly or neglected can lead to infection and proliferation of granulation tissue leading to a condition similar to “concrete scalp deformity” seen after medium thickness skin graft harvested from the scalp. we report a case of neglected abrasion over the scalp following trauma that presented to us after weeks. this lead to the proliferation of the granulation tissue leading to a mulberry — like swelling over the abrasion site with embedded hair that bled on touch [clinical: figure a]. a working diagnosis of foreign body granuloma or concrete scalp deformity was made. a wedge biopsy of the swelling was sent for histopathology. the proliferative granulation tissue was scraped. the histopathology showed inflammatory granulation tissue [histology: figures a and b]. the culture from wound grew staphylococcus aureus. the wound was covered with a split thickness skin graft [clinical: figure b]. deep abrasion of the scalp may sound inconspicuous. if neglected, it can delay wound healing with concrete scalp deformity like picture. the wound should be cleaned well to avoid formation of scabs and dressed with occlusive dressings. gauze should be avoided as a dressing. although described only for scalp as a donor site, concrete scalp can happen after head trauma. the clinician must be aware of this condition and treat early to prevent alopecia. soumya gupta, amish gohil, ashish kumar gupta department of plastic surgery, christian medical collage and hospital, vellore, tamil nadu, india figure : (a) clinical preoperative image showing a cm x cm mulberry like swelling over the forehead abrasion site. (b) clinical postoperative image showing areas of spontaneous epithelisation and growth of hair a b figure : (a) histopathological image showing dermal inflammation (× magnification). (b) histopathological image showing ulceration with fibrin (× magnification) a b address for correspondence: dr. soumya gupta, department of plastic surgery, christian medical collage and hospital, vellore - , tamil nadu, india. e-mail: minisom @yahoo.co.in references . crawford bs. an unusual skin donor site. br j plast surg ; : - . . engrav lh, grube bj, bubak pj. treatment of the concrete scalp donor site. ann plast surg ; : - . . lesesne cb, rosenthal r. a review of scalp split-thickness skin grafts and potential complications. plast reconstr surg ; : - . . carter ym, summer gj, engrav lh, hansen fl, costa ba, matsumura h. incidence of the concrete scalp deformity associated with deep scalp donor sites and management with the unna cap. j burn care rehabil ; : - . access this article online quick response code: website: www.ijps.org doi: . / - . indian journal of plastic surgery september-december vol issue published online: - - article worldwide mutation spectrum in cartilage-hair hypoplasia: ancient founder origin of the major a?g mutation of the untranslated rmrp maaret ridanpää , , pertti sistonen , susanna rockas , , david l rimoin , outi mäkitie and ilkka kaitila*, , folkhälsan institute of genetics, biomedicum helsinki, fi- university of helsinki, finland; department of medical genetics, biomedicum helsinki, fi- university of helsinki, finland; finnish red cross blood transfusion service, fi- helsinki, finland; cedar-sinai medical center, los angeles, california, ca , usa; hospital for children and adolescents, fi- helsinki university central hospital, finland; department of clinical genetics, fi- helsinki university central hospital, finland pleiotropic, recessively inherited cartilage-hair hypoplasia (chh) is due to mutations in the untranslated rmrp gene on chromosome p -p encoding the rna component of rnase mrp endoribonuclease. we describe different mutations in this gene in finnish and non-finnish chh families. based on their nature and localisation, these mutations can be classified into three categories: mutations affecting the promoter region, small changes of conserved nucleotides in the transcript, and insertions and duplications in the ’ end of the transcript. the only known functional region that seemed to avoid mutations was a nucleolar localisation signal region between nucleotides – . the most common mutation in chh patients was a base substitution g for a at nucleotide . this mutation contributed % of the mutations in the finnish chh patients. our results using linkage disequilibrium based maximum likelihood estimates with close markers, genealogical studies, and haplotype data suggested that the mutation was introduced to finland some – years ago, and before the expansion of the population. the same major mutation accounted for % of the mutations among chh patients from other parts of europe, north and south america, the near east, and australia. in the non-finnish chh families, the a g mutation segregated with the same major haplotype, although shorter, as in most of the finnish families. in out of these chromosomes, the common region extended over kb, and, therefore, all the chromosomes most likely arose from a solitary event many thousands of years ago. european journal of human genetics ( ) , – . doi: . /sj.ejhg. keywords: rmrp; mutation; ancestral haplotype; age estimation; untranslated gene; cartilage-hair hypoplasia introduction cartilage-hair hypoplasia (chh) or mckusick-type meta- physeal chondrodysplasia (mim ) is a pleiotropic disease, characterised by disproportionate short stature. other common features include hypoplastic hair, defective immunity and risk for malignancies, hirschsprung disease, hypoplastic anaemia, impaired spermatogenesis, and increased mortality. – the variation in the clinical severity is remarkable, even within affected siblings. chh is espe- cially common among the old order amish in north america and finns. , the carrier frequencies among the amish and finns were calculated to be as high as : received february ; revised march ; accepted april *correspondence: i kaitila; department of clinical genetics, helsinki university central hospital, f - hus, finland; tel: + ( ) ; fax: + ( ) ; e-mail: ilkka.kaitila@hus.fi european journal of human genetics ( ) , – ª nature publishing group all rights reserved – / $ . www.nature.com/ejhg and : , respectively. , there is no precise measure of worldwide incidence, although cases have been observed in numerous populations (international skeletal dysplasia registry; personal observations). after initial mapping of the chh gene to p , linkage disequilibrium was detected among amish and finns. , based on the amish population history and haplotype data, only one mutation was suggested to account for the chh cases, , but recently, multiple allelic mutations have been accepted as being possible. among finns, – % of the chh chromosomes were estimated to originate from a common ancestor and contain an identical mutation. as a result of several years of genetic and physical mapping, the first disease-causing mutations in rmrp were described. the untranslated rmrp gene encodes the rna component of the rnase mrp complex. normally, the rnase mrp complex is involved in multiple cellular and mitochondrial functions though the disease-causing func- tional impairment of the rmrp gene product remains to be characterised. , cartilage-hair hypoplasia is counted as being part of the so-called finnish disease heritage, which consists of some thirty genetic diseases enriched in finland and has its origin in the special population history of finland. the current population is thought to have originated from a relatively small founding population and very little immi- gration has occurred during the last – generations of expansion. – the early settlement up to the s covered only the southwest and southeast regions and the coastal areas of the country. the northern and eastern area, or the late settlement region, was inhabited only – generations ago. the archaeological findings confirm that there has been sparse, although continuous, inhabitation in the southern coastal areas for about years (http://virtual.finland.fi/finfo/english/prehistory.html). in this study, we describe the results of genetic and muta- tion analyses of rmrp in finnish cartilage-hair hypoplasia patients and chh families of other nationalities. the major mutation was introduced to the finnish population – years ago and obviously well before the expan- sion of the population. currently, this mutation contributes % of the rmrp mutations in finland and % of the mutations in a collection of chh patients from several other populations. we have demonstrated that the same haplotype is segregating with this mutation in finland and abroad, thus, suggesting a single ancient occurrence and worldwide distribution. in total, eight different muta- tions in the promoter region and mutations in the rna coding region of nucleotides have now been reported (this study). materials and methods subjects and families the molecular genetic study on chh was approved by the ethical committee of the department of medical genetics at the university of helsinki. the finnish chh patients were referred for diagnostic workup and genetic counselling from all over the country to the helsinki university central hospital, where the chh registry has been maintained from two epidemiological surveys in and in . since the surveys, additional patients have been studied and evaluated, the current number of patients being from families. the clinical diagnostic features include disproportionate short-limbed, short stature, increased lumbar lordosis, joint laxity, extension limitation at the elbows, and bowed legs. usually the patients presented with hair hypoplasia, but for inclusion, hair hypoplasia was accepted as a confirmatory feature only. radiological find- ings included generalised metaphyseal dysplasia, and normal skull and spine in childhood films. a total of patients from families agreed to participate in this study, and the patients or their custodians signed the informed consent. the birthplaces of the finnish patients’ great- grandparents were inspected from the church parish regis- ters. the non-finnish patients from a number of countries were collected through diagnostic consultations with clini- cians and genetic counsellors named in the acknowledgments. the essential diagnostic and demo- graphic data were obtained through clinical descriptions, patients’ photographs, laboratory results, and copies of the skeletal radiographs of most patients. in addition, the consultants filled in a chh data form. the ethnic back- ground of the patients from america and australia was often european. data and samples were obtained from patients from families. seven of these samples were obtained through the international skeletal dysplasia regis- try maintained at the cedars-sinai medical center, los angeles, usa. dna samples from regular blood donors of the finnish red cross blood transfusion service were studied for the finnish major mutation (a g). smaller sets of control samples were studied for the other mutations. the control sample registry consists of a total of samples from – year-old male donors, all of whose grandparents were born in the same geographic area in finland, who were not aware of any of their relatives participating in the study, and had no knowledge that any of their more distant ancestors were from abroad. a sample was accepted after obtaining a written informed consent and fulfilling the criteria above. the samples were collected in eight regional centres, and the sampling density is representative of the finnish population at the start of . a sample consisted of the buffy-coat of the whole donation (about ml) generated as a side product in the routine preparation of a leukocyte depleted red cell unit. dna was extracted using a modified salting out protocol. the ethical committee of the finnish red cross blood transfusion service approved the sample registry. rmrp mutations in cartilage-hair hypoplasia m ridanpää et al european journal of human genetics mutation detection in rmrp dna samples were screened for insertions and deletions in the promoter region using pcr primers rm if ’-ctta- gaaagttatgcccgaaaac- ’ and rm ir ’-gaaagg- ggaggaacagagtc- ’ for amplification and % sequagel (national diagnostics) gels for separation. in case of changes in the length, this region was amplified using rm f ’-ggccagaccatatttgcataag- ’ and rm r ’- cggactttggagtgggaag- ’ primers and sequenced. the finnish major mutation (a g) was amplified using pcr primers rm f ’-gtgctgaaggcctgtatcct- ’ and rm r ’-ctaggggagctgacggatg- ’. sscp analyses were performed in . xmde (fmc bioproducts) gels at room temperature using w overnight. for sequen- cing of the whole rna coding region of rmrp, pcr primers rmf ’-ccaactttctcaccctaacca- ’ and rmr ’-aaggccaagaacagcgtaaa- ’ were used. in some samples, the ’ region was separately amplified for sequen- cing using rm f ’-agagagtgccacgtgcatac- ’ and rm r ’-cttcatagcaaggccaagaac- ’. all page and sscp gels were detected using silver staining. analysis of polymorphic markers and reconstruction of haplotypes primers and separation conditions for d s , ac, tesk a/g, mn/ca c/a, d s , ac, k t sscp, p , ab gs, del , sit s, tp a, ta s, d s, d s, ac , xl s, ccs, and tg s have been described previously. marker delg was amplified using primers ’-tgcatgctg- cttactggttc- ’ and ’-ccaatatcatgggggatgac- ’ followed by separation in . xmde (fmc bioproducts) sscp gels at w overnight. primers for d s and d s were as described (http://www.cephb.fr) and frag- ments were separated in % sequagel (national diagnostics) gels at c. the four frequently polymorphic snps at nucleotides t/a, t/c, c/a, and at t/c were found during sequencing of the promoter and the rna coding region of the rmrp gene (fragments rm , rm and rm as described above). the distance between the transcription initiation site of rmrp and each marker was determined using our genomic contigs and sequences al and al from the ncbi data- base at http://www.ncbi.nlm.nih.gov/entrez. in the case of ab gs and p , the length of bac clone j ( kb) determined by the pulsed-field gel electrophoresis was also needed. haplotypes of the finnish chh families were recon- structed assuming a minimum number of recombinations in each family and grouped according to the mutation in rmrp. haplotypes segregating with the finnish major muta- tion and the healthy haplotypes were used to calculate the age of this mutation in the finnish population. the two patients showing upd were excluded from this analysis. in order to find out if the a g mutation had arisen once or several times in the non-finnish chh patients homozygous or heterozygous for the a g mutation, chh families and two amish ceph (centre d’etude poly- morphisme humain) controls were genotyped. these controls were previously found to be nucleotide g carriers. haplotypes were reconstructed using markers and assuming a minimum number of recombinations in each family. methods of computing the age of the major mutation in the finnish population to estimate the age of the major a g mutation, we used the dmle programme which, in its original form was designed to estimate the recombination distance between a biallelic marker and a disease gene based on observed (or supposed) historical recombinations in the data rather than the age estimation. the possibility of estimating the age of a mutation was implemented in a more recent version (beta . , released april, ) of the programme given the parameters of ‘known’ recombination rate, popu- lation growth rate, frequencies of the disease associated marker alleles, (ie identical by state, ibs) in sampled disease chromosomes and in the general population, and the proportion of the disease chromosomes sampled from the population under study. we chose the following para- meters for the estimate; the recombination rate was based on the estimated physical distance of kb between d s and rmrp, as calculated using the genomic sequence and the bac contig and the genetic distance of . cm in the same interval. these numbers led us to assume that . mb correspond to cm in this chromosomal region. the general approximation mb corresponding to cm was used as well. population growth rate (exponential) was estimated to be . by actual later historical census data from the th century and assuming that the age reaches far beyond the start of the common era. the disease chromosome associated allele frequencies were directly calculated from the data, and their population frequencies from the healthy parental chromosomes. the proportion of sampled disease chromosomes was calculated from the frequency study in the blood donors’ samples ( / ), taking the finnish population size as million. two million monte carlo replicates using different seeds were computed for the . mb correspondence, and the average likelihoods taken to represent the final values because of the poorer convergence to a definite maximum as compared to the mb correspondence to genetic length of cm, where only one million replicates were used. we also computed the means-of-moments estimator for the most recent ancestor (tmrca) to evaluate the agreement with dmle age estimates. we did not apply the correction as suggested by labuda et al. markers tesk a/g sscp, delg, d s, ac , and xl s were selected for the estimation, since they had more than one instance of presumably non- ancestral allele presented in the haplotype in disease chro- mosomes. however, the two most centromeric markers rmrp mutations in cartilage-hair hypoplasia m ridanpää et al european journal of human genetics (ccs and tg s) were not included in any of the calcula- tions because of an obvious recombination hotspot separating them from the rest of the haplotype. results rmrp mutations segregate with specific haplotypes in the finnish population approximately patients with cartilage-hair hypoplasia have been diagnosed in finland (the finnish chh patient registry). after finding the first chh-causing mutations in rmrp, we continued the mutation search in the rest of the finnish patient samples from multiplex and uniplex families. we found the finnish major mutation g at nucleotide (a g) to be homozygous in families (table ). all patients in the remaining families were compound heterozygotes for this mutation and represented one of the four minor mutations for the other allele. the most common minor mutation was also a base substitution (g t) and was found in families. the other minor mutations were detected only in one or two finnish families, as shown in table . all mutations found in the finnish chh patients resided either in the evolutionally conserved nucleotides of the rmrp transcript , or disrupted the function of the promoter region. the g t mutation was not found among anonymous finnish blood donors, nor were the other minor mutations found among of these controls and non-finnish controls. ten heterozygous carriers for the mutation g at nucleotide were found among anonymous blood donors of the finnish red cross blood transfusion service, representing the geographic population distribution in the beginning of the th century. when the dna samples of the parents were available, we genotyped them and reconstructed haplotypes, segregating table rmrp mutations in finnish and foreign chh patients number of families mutations in rmrp nationality uniplex multiplex g/ga finnish d g/ tb finnish g/ g finnish g/ t finnish g/duptactctgtga at c finnish g/g us, canadian, dutch, turkish, english, us amish g/ t australian g/ a us g/ a mexican g/ c dutch, english g/ g us g/dup tctgtga at us g/dup aagctgag at mexican g/dup tgaagctgag at french g/ins cctgag at c german g/dup aagctgaggacgtg at us g/dup ggacgtggtt at brazilian g/ins ttccgcct at french g/dup tg at c canadian g/ t german t/t arabian a/a chinese g/ t canadian a/ g canadian t/ t israelis t/ g brazilian g/ t polish g/ t us t/t israelis a/a austrian t/del ag at english a/dup english tctgtgaagctgaggac at c t/ins ggacgtggtt at us dup tg at /dup tg at turkish dup tg at / times dup swiss actactctgtgaagc at c many of the us patients represent evidence of caucasian family history. aincluding previously reported families ( uniplex and multiplex); bincluding six previously reported families (five uniplex and one multiplex); cpreviously reported family/families; dincluding two patients with uniparental disomy for chromosome (patients a and b in ). rmrp mutations in cartilage-hair hypoplasia m ridanpää et al european journal of human genetics with the different rmrp mutations. the major mutation always segregated with the finnish major haplotype (in this study), the shortest haplotype being less than kb in length between markers d and a base substitution poly- morphism in the first exon of mn/ca (data not shown). the minor mutation g t and the bp duplication at segregated with their own haplotypes as depicted in figure a. all minor haplotypes were more than kb figure haplotype analyses in the finnish and non-finnish chh families. (a) the haplotypes segregating with the finnish minor mutations are different from that segregating with the finnish major mutation g for a at nucleotide of rmrp. the major haplotype determined in finnish chh families is depicted in blue. (b) the non-finnish chromosomes segregating with the major mutation show the finnish major haplotype. the four amish chh haplotypes are reconstructed from an amish chh family and from two amish ceph controls. on top are shown the distances between the markers and the nucleotide of rmrp. the telomere is to the left. n, an undetermined allele. rmrp mutations in cartilage-hair hypoplasia m ridanpää et al european journal of human genetics in length and longer than a large proportion of the major haplotypes suggesting a more recent occurrence of these mutations. the birthplaces of the great-grandparents of chh patients were reported to reside relatively evenly both in the early and late settlement regions of finland (figure a). we separately collected the information about the great-grandparents of the patients with the minor muta- tions and plotted their birthplaces on the map of finland. grandparents of the minor mutation carriers were found more often in the late settlement region (figure b), whereas the birthplaces of grandparents of the major muta- tion carriers were distributed more evenly in both the early and late settlement regions. the distribution of the birth- places of the grandparents of the blood donors carrying the a g mutation in the early and late settlement regions is shown in figure c. the a g substitution is the most common mutation among chh patients worldwide we studied patients from different families, representing nationalities from europe, north and south america, the near east, australia, and china (table ). patients from families were homozygous for the g mutation at nucleotide , whereas patients from families were heterozygous for this mutation and represented one of the numerous rare mutations in the pairing allele (table ). patients from families had only rare mutations. patients from five of these families were homozygous for their private mutation, suggesting parental consanguinity (table ). in addition to the major mutation, the base substitution g at nucleotide was the only mutation found both among the finnish (one family) and non-finnish patients (two families). like the finnish mutations, most of the rare mutations in the foreign samples were base substitutions in the transcribed region. in the ’ end of the coding region, duplications or insertion of – nucleotides represent a new type of mutation in the chh patients. all sites for the different types of mutation in the transcript are evolutionally conserved , and were not found among finnish and non-finnish controls. we did not find any rmrp mutations in nine patients who probably do not have chh but another type of chondrodysplasia. since the mutation g at nucleotide was common in several countries, we asked the question whether this muta- tion had a single origin or if this nucleotide was a hot spot for mutations. eight homozygous and nine heterozygous patients and their family members, and two amish ceph (centre d’etude polymorphisme humain) controls were genotyped and haplotypes were reconstructed. chromo- somes from different nationalities and the old order amish carrying g at the nucleotide shared the same figure geographical distribution of the rmrp mutations in finland. (a) birthplaces of the great-grandparents of chh patients ( chh families). (b) birthplaces of grandparents of the carriers (or chh patients’ parents) of the minor mutations in rmrp. *, g t; &, the finnish duplication; ~, g t; !, c g (c) birthplaces of the grandparents of the blood donors carrying substitution g at nucleotide . in each map, one symbol depicts one great-grandparent/grandparent. the line denotes the approximate border between the old and new settlement in finland. the area depicted by the dotted line belonged to finland before the second world war. rmrp mutations in cartilage-hair hypoplasia m ridanpää et al european journal of human genetics alleles with the finnish major haplotype (figure b). twenty-three of the haplotypes were similar, at least in a region of kb around the rmrp mutation, clearly point- ing to a common origin of this base substitution rather than due to frequent occurrence. the finnish major mutation is ancient according to our maximum likelihood estimates with the dmle program the major rmrp mutation arrived in finland some – generations ago corresponding to an age (given a generation time of years) of approxi- mately – years (figure ). the estimates were computed using five of the markers (tesk a/g sscp, delg, d s, ac and xl s) and averaging the likelihood points, each consisting of either two or one million monte carlo replicates (figure ). using the same five markers and esti- mates for the genetic and physical correspondence, the method of risch et al also resulted in high age estimates, although with much wider limits of – generations. these high age estimates are in keeping with the distribu- tion of the mutation, which also suggests an ancient introduction of this mutation into the finnish population. discussion recently, we have reported that two types of mutation in rmrp can lead to cartilage-hair hypoplasia. duplications and insertions between the tata box and the transcription initiation site silence transcription from that allele, and changes involving at most two highly conserved nucleo- tides of the transcript lead to a homozygous or compound heterozygous mutant transcript. altogether, in a set of finnish and chh families from other countries, we have found different mutations in the rmrp gene; of them are reported for the first time in this paper. these can be classified into three groups. the first group of muta- tions consists of eight insertions and duplications that affect the promoter region. the second group of mutations consists of base substitutions and two other changes in conserved nucleotides of the transcript. in addition, we have characterised herein three insertions and duplications in the ’ end of the transcript, which form a third group of mutations in rmrp. these mutations are gathered around the nucleolar localisation signal region and the regions reported to be important for binding the different proteins of the rnase mrp complex. only heterozygosity of inser- tion and further duplication mutations in the promoter region or ‘null’ alleles of rmrp were detected, further suggesting that expression of the rnase mrp rna is indeed essential for life. , mutations in rmrp seem to avoid nucleotides – which have been reported to be a nucleolar localisation signal region. we found a compound heterozygous muta- tion at nucleotide in one patient, but due to the sequence content of this duplication it does not change the sequence of the nucleolar localisation signal. on the other hand, the mitochondrial localisation signal region has been reported to reside between nucleotides – in the mouse which corresponds to nucleotides – in the human rmrp. this stretch harbors four different substitution mutations; two patients were even homozy- gous for this kind of mutation. functional studies are needed to further clarify the viability of mutations in the nucleolar localisation signal region and the significance of the mutations in the mitochondrial localisation signal region. cartilage-hair hypoplasia and five other diseases of the finnish disease heritage, namely aspartylglucosaminuria (agu), progressive myoclonus epilepsy (pme), infantile neuronal ceroid lipofuscinosis (incl), juvenile neuronal ceroid lipofuscinosis (jncl), and congenital nephrotic syndrome of the finnish type (cnf), show similar distribu- tion in finland, and in each disease, the major mutation has been found in – % of the finnish disease chromo- somes. – the age of these major mutations in finland has not been calculated, but the distribution of the birth- places of the great-grandparents supports a hypothesis that these mutations were present in the founding popula- tion. the most common mutation among the finnish chh patients is the base substitution g for a at nucleotide of rmrp representing % ( / ) of the disease mutations (this study). our computation, using monte carlo sampling, results in an estimate that the major mutation is ancient in finland and was most likely introduced before the hypothesised time of the population expansion. the most conspicuous single parameter in these estimates is the correspondence of the physical map and recombination probabilities at very short map lengths. we are able to esti- mate that . mb corresponds to cm on the telomeric side figure age estimate average plots by the disequilibrium maximum likelihood estimate (dmle) programme using five markers, tesk a/g sscp, delg, d s, ac , and xl s, around the a g mutation in rmrp in finns. a continuous line is for assuming a correspondence of cm to . mb and a broken line for a correspondence of cm to mb. the maximum likeli- hoods are t = generations and t = generations, respec- tively. the lower ci boundary is at around generations for each age estimate, whereas the upper may not be plotted at all. rmrp mutations in cartilage-hair hypoplasia m ridanpää et al european journal of human genetics of the rmrp locus where only one out of the five markers suitable for the age calculations resides. due to the vicinity of the centromere it is possible that cm corresponds to a longer physical distance, while at the same time, we have observed a possible recombination hotspot on the centro- meric side. it was also obvious that the algorithm implemented in the dmle programme does not converge very well when the age of the mutation increased. our assumption that the introduction of the major mutation to the finnish population has occurred only once may be wrong since the major mutation appears to be common worldwide (see below). on the other hand, solitary intro- duction of the mutation is strongly favoured by the length of the shared haplotype segregating among finns. the means-of-moments estimator calculations suffer the same reservations and, furthermore, do not account for the population parameters and are even more sensitive to assumed recombination rate. as an independent observa- tion, the birthplaces of the great-grandparents of chh patients are distributed in the early and late settlement regions relatively evenly, and strengthen the assumption that the major mutation was present at the time of the beginning of the population expansion some – generations ago. , similarly, our haplotype data, and the distribution of different mutations in finland, allow us to assume that the four finnish minor mutations are younger than the major mutation and that the local enrichment of chh in the western finland is, at least in part, due to the enrichment of the rare mutations in that region. the finnish major mutation is also the most common mutation ( %) among the chh patients in families from other countries. patients from families are homozy- gous, whereas patients from families are heterozygous for the a g substitution representing nationalities from europe, north and south america, the near east, and australia. the ethnic background of the patients from america and australia, in particular, was often european and mixed. in all non-finnish chh families, the major mutation segregates with the same major haplotype as in the finnish families. in out of the chromosomes, the common region expands over kb. interestingly, one of the homozygous us patients is amish. in this amish patient and two amish ceph control chromosomes, which were available for genotyping, the haplotypes are the same as the finnish major haplotype extending over kb region around the rmrp locus. a more extensive collection of amish samples is needed in order to find out if the a g substitution accounts for all the frequent chh cases among amish or whether several different mutations are also involved in that population. in conclusion, different insertions and duplications in the promoter region and a large number of mutations in the rmrp transcript can cause chh. our findings clearly support the hypothesis that a single ancient nucleotide g mutation in rmrp causes most of the finnish chh cases and contributes to the majority of the non-finnish cases, possibly also including the amish enrichment. it is very likely that all of the nucleotide g mutations reported in the study represent a single occurrence of this base substitution. the ancient a g mutation was introduced to finland by the founder individuals and is highly enriched in the current population. acknowledgements we acknowledge the following clinicians and genetic counselors for sending clinical data and patient samples for this study: l adès, australia, j bonaventure, france, z borozowitz, israel, j boudames, usa, j campbell, usa, a castriota-scanderbeg, italy, d chitayat, canada, dh cohn, usa, m conley, usa, t costa, canada, a de oliveira, usa, cr dolan, uk, f elmslie, uk, p freisinger, germany, l jobim, brazil, m johnson, usa, h. kayserili, turkey, s langlois, canada, j mattheson, usa, w newman, usa, i pellier, france, r saviriayan, australia, c scott, usa, t simon, germany, s smithson, uk, m splitt, uk, e steichen, austria, a superti-furga, switzerland, i van der burgt, the netherlands, l van maldergem, belgium, w wilcox, usa, l wilson, uk, r winter, uk, b zabel, germany, p zack, uk. professor albert de la chapelle is thanked for useful discussions and critical reading of the manuscript. ahmed mohamed, hanna mäkelä, katarina pelin and riika salmela are acknowledged for laboratory assistance and collaborative help and sinikka lindh for help in the genealogical study and drawing figures b and c. this work was financially supported by the march of dimes birth defects foundation ( -fy - and -fy - ), the academy of finland (grant ), helsinki university’s research funds, the helsinki university central hospital fund, the ulla hjelt fund, finland, and an nih program project grant ( po hd ). references mckusick va, eldridge r, hostetler ja, ruangwit u, egeland ja: dwarfism in the amish. ii. cartilage-hair hypoplasia. bull johns hopkins hosp ; : – . mäkitie o: cartilage-hair hypoplasia in finland: epidemiological and genetic aspects of patients. j med genet ; : – . mäkitie o, kaitila i: cartilage-hair hypoplasia – clinical manifesta- tions in finnish patients. eur j pediatr ; : – . mäkitie o, pukkala e, teppo l, kaitila i: increased incidence of cancer patients with cartilage-hair hypoplasia. j pediatr ; : – . mäkitie o, pukkala e, kaitila i: increased mortality in cartilage-hair hypoplasia. arch dis child ; : – . mäkitie om, tapanainen oj, dunkel l, siimes ma: impaired sper- matogenesis: an unrecognized feature of cartilage-hair hypoplasia. ann med ; : – . sulisalo t, francomano ca, sistonen p et al: high-resolution genet- ic mapping of the cartilage-hair hypoplasia (chh) gene in amish and finnish families. genomics ; : – . sulisalo t, sistonen p, hästbacka j et al: cartilage-hair hypoplasia gene assigned to chromosome by linkage analysis. nat genet ; : – . sulisalo t, klockars j, mäkitie o et al: high-resolution linkage-dise- quilibrium mapping of the cartilage-hair hypoplasia gene. am j hum genet ; : – . mckusick va: ellis-van creveld syndrome and the amish. nat genet ; : – . rmrp mutations in cartilage-hair hypoplasia m ridanpää et al european journal of human genetics vakkilainen t, kivipensas p, kaitila i, de la chapelle a, ridanpää m: integrated high-resolution bac, p , and transcript map of the chh region in chromosome p . genomics ; : – . ridanpää m, van eenennaam h, pelin k et al: mutations in the rna component of rnase mrp cause a pleiotropic human disease, cartilage-hair hypoplasia. cell ; : – . van eenennaam h, jarrous n, van venrooij wj, pruijn gj: archi- tecture and function of the human endonucleases rnase p and rnase mrp. iubmb life ; : – . norio r, nevanlinna hr, perheentupa j: hereditary diseases in finland; rare flora in rare soil. ann clin res ; : – . nevanlinna hr: the finnish population structure. a genetic and genealogical study. hereditas ; : – . de la chapelle a: disease gene mapping in isolated human popu- lations: the example of finland. j med genet ; : – . de la chapelle a, wright f: linkage disequilibrium mapping in isolated populations: the example of finland revisited. proc natl acad sci ; : – . lahiri dk, bye s, nürnberger jr ji, hodes me, crisp m: a non- organic and non-enzymatic extraction method gives higher yields of genomic dna from whole-blood samples than do nine other methods tested. j biochem biophys methods ; : – . pelin k, hilpelä p, donner k et al: mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. proc natl acad sci usa ; : – . sulisalo t, mäkitie o, sistonen p et al: uniparental disomy in cartilage-hair hypoplasia. eur j hum genet ; : – . rannala b, slatkin m: likelihood analysis of disequilibrium mapping, and related problems. am j hum genet ; : – . risch n, teng j: the relative power of family-based and case- control designs for linkage disequilibrium studies of complex human diseases i. dna pooling. genome res ; : – . labuda m, labuda d, korab-laskowska m et al: linkage disequili- brium analysis in young populations: pseudo-vitamin d- deficiency rickets and the founder effect in french canadians. am j hum genet ; : – . schmitt me, bennett jl, dairaghi dj, clayton da: secondary structure of rnase mrp rna as predicted by phylogenetic comparison. faseb j ; : – . sbisà e, pesole g, tullo a, saccone c: the evolution of the rnase p- and rnase mrp-associated rnas: phylogenetic analysis and nucleotide substitution rate. j mol evol ; : – . tremblay m, vézina h: new estimates of intergenerational time intervals for the calculation of age and origins of mutations. am j hum genet ; : – . jacobsen mr, cao l-g, wang y-l, pedersen t: dynamic localiza- tion of rnase mrp rna in the nucleolus observed by fluorescent rna cytochemistry in living cells. j cell biol ; : – . schmitt me, clayton da: yeast site-specific ribonucleoprotein endoribonuclease mrp contains an rna component homolo- gous to mammalian rnase mrp rna and essential for cell viability. genes dev ; : – . li k, smagula cs, parsons wj et al: subcellular partitioning of mrp rna assessed by ultrastructural and biochemical analysis. j cell biol ; : – . ikonen e, aula p, grön k et al: spectrum of mutations in aspartyl- glucosaminuria. proc nat acad sci usa ; : – . vesa j, hellsten e, verkruyse la et al: mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipo- fuscinosis. nature ; : – . järvelä i, mitchison hm, munroe pb, o’rawe am, mole se, syvä- nen ac: rapid diagnostic test for the major mutation underlying batten disease. j med genet ; : – . kestilä m, lenkkeri u, männikkö m et al: positionally cloned gene for a novel glomerular protein nephrin is mutated in congenital nephrotic syndrome. mol cell ; : – . virtaneva k, d’amato e, miao j et al: unstable minisatellite expansion causing recessively inherited myoclonus epilepsy, epm . nat genet ; : – . rmrp mutations in cartilage-hair hypoplasia m ridanpää et al european journal of human genetics 	worldwide mutation spectrum in cartilage-hair hypoplasia: ancient founder origin of the major a→g mutation of the untranslated rmrp 	introduction 	materials and methods 	subjects and families 	mutation detection in rmrp 	analysis of polymorphic markers and reconstruction of haplotypes 	methods of computing the age of the major mutation in the finnish population 	results 	rmrp mutations segregate with specific haplotypes in the finnish population 	the a g substitution is the most common mutation among chh patients worldwide 	the finnish major mutation is ancient 	discussion 	acknowledgements 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: 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to richard l. greaves, chair, committee on research, american society of church history, c / o department of history, florida state university, tallahassee, fl , by july each year. the prize will be announced at the annual meeting of the society in december. cambridge core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core american journal of medical genetics part c (semin. med. genet.) c: – ( ) a r t i c l e pediatric medicine and the genetic disorders of the amish and mennonite people of pennsylvania d. holmes morton,* caroline s. morton, kevin a. strauss, donna l. robinson, erik g. puffenberger, christine hendrickson, and richard i. kelley the clinic for special children in lancaster county, pennsylvania, is a community-supported, nonprofit pediatric medical practice for amish and mennonite children who have genetic disorders. over a -year period, – , we have encountered heritable disorders among the amish and among the mennonites. we emphasize early recognition and long-term medical care of children with genetic conditions. in the clinic laboratory we perform amino acid analyses by high-performance liquid chromatography (hplc), organic acid analyses by gas chromatography/mass spectrometry (gc/ms), and molecular diagnoses and carrier tests by polymerase chain reaction (pcr) amplification and sequencing or restriction digestion. regional hospitals and midwives routinely send whole-blood filter paper neonatal screens for tandem mass spectrometry and other modern analytical methods to detect of the metabolic disorders found in these populations as part of the neogen inc. supplemental newborn screening program (pittsburgh, pa). medical care based on disease pathophysiology reduces morbidity, mortality, and costs for the majority of disorders. among our patients who are homozygous for the same mutation, differences in disease severity are not unusual. clinical problems typically arise from the interaction of the underlying genetic disorder with common infections, malnutrition, injuries, and immune dysfunction that act through classical pathophysiological disease mechanisms to influence the natural history of disease. � wiley-liss, inc. key words: genetic diseases; general pediatric medical care; metabolic diseases; genotype-phenotype correlation introduction features of the population the manuscript of john a. hostetler’s book amish society was reviewed in the early ’s for johns hopkins university press by victor mckusick [hostetler, ]. mckusick recognized the importance of the book as a socio- logical study and realized the potential of amish populations for the study of human genetics. mckusick’s review, and the subsequent publication of amish society in , marked the beginning of a collaboration between hostetler and mckusick that spanned several decades. in mckusick edited and johns hopkins press published medical gene- tic studies of the amish. [mckusick, ] table i lists genetic disorders that were described in this publication, which came from many different amish demes throughout pennsylvania and the midwest. puffenberger and francomano discuss the origins of genetic studies in the mennonite and amish populations in accompaning articles [francomano, ; puffenberger, ]. although these populations are unique in many ways, when compared to the general population of pennsylva- nia, the amish and mennonites are less distinct than is generally appreciated. in each of the or more generations since the early s, – % of the children have left the old order communities [hostetler, ]. lancaster county church cemeteries, public school rosters, and regional phone books include many amish and mennonite family names. therefore, many so-called amish alleles d. holmes morton, m.d., sc.d. (hon.), is co-founder and director of the clinic for special children. he is a pediatrician with an interest in the influence of early diagnosis and treatment upon the natural history and neurobiology of inherited metabolic disorders. caroline s. morton, ed. m., is co-founder and manager of the non-profit clinic and educational organization called the clinic for special children. kevin a. strauss, m.d., is a pediatrician doing post-graduate studies at the clinic focused upon the neurobiology of genetic disorders and the pathophysiology of metabolic injuries of the brain. donna l. robinson, r.n., c.n.p., is a pediatric intensive care nurse practitioner with special interest in developing nursing protocols for management of hospitalized patients with metabolic disorders. erik g. puffenberger, ph.d., is laboratory director at the clinic for special children, with special interest in molecular biology and population genetics. christine hendrickson, rn, is a pediatric nurse who recently joined the clinic staff to work with children with genetic disorders in the outpatient setting. richard i. kelley, m.d., ph.d., is associate professor, department of pediatrics, johns hopkins university, and member of the division of metabolism, kennedy-krieger institute, baltimore md. dr. kelley is consulting pediatrician and geneticist, and a founding board member, of the clinic for special children. *correspondence to: d. holmes morton, clinic for special children, bunker hill rd., strasburg, pa . doi . /ajmg.c. � wiley-liss, inc. or mennonite alleles have diffused out into the general population. the old order amish and mennonite popula- tions of pennsylvania are new compared to the time of origin of these gene muta- tions. all three of the mutations causing phenylketonuria (pku) in the old order amish and mennonites are found throughout europe. the so-called amish mutation for glutaric aciduria type (ga ) is one of the more common mutations found throughout europe and the united states [biery et al., ]. therefore, for most disorders found within the amish and menno- nites, the alleles are not unique, but these alleles are at different frequencies than in the general population. the gene muta- tions carried by the founders of the demes of pennsylvania are not distribut- ed evenly, either within or among the many subpopulations of the state. for of the disorders seen regularly at the clinic, the incidences are high, approxi- mately / to / births. although there is a wide variety of heritable disorders among the plain people (table i), the majority of founder dis- orders are clustered in a few families, and the overall disease prevalence among plain people is relatively low. examples table i. metabolic disorders and syndromes found in medical genetic studies of the amish [mckusick, ] inheritance omim deme b metabolic disorders a phenylalanine hydroxylase deficiency ar in/pa propionyl-coa carboxylase deficiency ar oh/pa pyruvate kinase deficiency ar pa(b) syndromes albinism, oculocutaneous, tyrosinase deficient, type b ar in spastic paraplegia ad pa(l) arthrogryposis, dysmelination, craniosynostosis, and cleft palate ar ataxia-telangiectasia ar in brittle hair, intellectual impairment, and decreased fertility ar byler disease, progressive familiar intrahepatic cholestasis ar pa(lw) cardiomyopathy, asymmetric sepal hypertrophy ad in cardiomyopathy, nonobstructive ad oh cartilage-hair hypoplasia ar pa,oh,in charcot-marie-tooth syndrome ar coclear deafness, myopia, and intellectual impairment ar pa(l) jackson weiss syndrome ad in deafness, congenital ar ellis-van creveld dwarfism ar pa(l) epidermolysis bullosa letalis ar oh familial manic-depressive illness ad pa(l) hypothyroidism and muscular hypertrophy limb girdle dystrophy, type e in/pa mast syndrome ar oh(h) mckusick-kaufman syndrome ar oh/pa muscular dystrophy, limb-girdle type ar in/pa nanophthalmos, familial ar nr oculocerebral syndrome with hypopigmentation ar renal-retinal dysplasia ar troyer syndrome ar oh(h) weill-marchesani syndrome ar pa(l) a disorders in italics are also entered in tables ii and iii. b pa(b) is belleville, pa; pa(l) is lancaster county, pa; pa(lw) is lawrence county, pa, oh(h) is holmes county, oh. the gene mutations carried by the founders of the demes of pennsylvania are not distributed evenly, either within or among the many subpopulations of the state. american journal of medical genetics (semin. med. genet.) article of lower-frequency founder disorders include pku and medium-chain acyl dehydrogenase deficiency (mcadd), which are probably less common in the plain populations of lancaster county than in the general caucasian popula- tion. some common genetic diseases were apparently excluded by founder sampling and/or genetic drift. for example, cystic fibrosis is not seen in the plain people of lancaster county but is found in some other western pennsylvanian demes. this uneven dis- tribution reflects different founders and a -year separation of the various subgroups prior to, and after, settlement in north america. in the plain people of pennsylvania, maple syrup urine disease (msud) is found only in those of mennonite descent and ga is found only in the amish populations. although eight disorders are found in both populations, only crigler-najjar disease and propionic acidemia are currently known to arise from the same mutations in both populations. mcadd, -methylcrotonylglycinuria [gibson et al., ], and at least two forms of osteogenesis imperfecta are found in both populations but are caused by different mutations in the amish and mennonites [puffenberger, ]. medical care and the clinic for special children the early genetic studies of the plain people indicated unique needs for the medical care of people who were geographically and culturally isolated. the clinic for special children was organized to integrate general medical care and modern genetics [hostetler, ; morton, ]. an initial goal of the clinic founders was to diagnose msud and ga in asymptomatic neo- nates and prevent the metabolic crises in order to prevent brain degeneration that characterized these conditions. because common childhood infections often provoke metabolic crisis in children with these disorders, we reasoned that med- ical care for affected children should be directly provided by individuals who were knowledgeable about the disor- ders, who had laboratory facilities to diagnose and monitor biochemical con- trol during infectious illnesses, and who were interested in general pediatric practice [morton et al., ; morton, ; morton et al., b]. because the early symptoms of these serious disor- ders are difficult to distinguish from common diseases, we often evaluate patients who turn out not to have a genetic disorder common to the plain people. infants who present with failure to thrive and irritability or who become unusually ill during otherwise common infections most often do not have under- lying genetic disorders. after the clinic opened in , it was apparent the practice would not be limited to patients with msud and ga . the first ill neonate sent to the clinic to rule out msud did have a family history of msud, but instead had hirschsprung disease with bowel obstruction and sepsis. one of the more recently identi- fied infants with msud also had a second rare recessive disorder, severe combined immune deficiency. two of our patients with msud have first cousins with mcadd. before , all amish children with ga were said to have cerebral palsy. many children with so-called amish cerebral palsy did have ga , but others did not, and their evaluations led to the descriptions of troponin t myopathy [johnston et al., ], amish lethal microcephaly [kelley et al., ; rosenberg et al., ; strauss et al., ], and an auto- somal recessive pelizaeus-merzbacher- like central dysmyelination syndrome [morton et al., unpublished results]. we recently evaluated a neonate with a sibling with ga who fortunately did not have ga , but unfortunately, as we learned from a neonatal screening test, his hypospadias was a sign of another recessive syndrome, -b hydroxyster- oid dehydrogenase deficiency. another family has three children with ga and a fourth with pku. over the years, failure to thrive workups have led to the diagnoses of byler disease, three new disorders of bile salt transport [morton et al., ], bartter syndrome, aldoster- one deficiency [mitsuuchi et al., ], and primary growth hormone defi- ciency [morton et al., a]. clearly, the burden of genetic disease is high and the need for specialized and compre- hensive care is essential to the health of the community. in the last years we clearly, the burden of genetic disease is high and the need for specialized and comprehensive care is essential to the health of the community. have cared for patients from the amish and mennonite populations with more than different single-gene disorders, as shown in tables ii and iii. these tables summarize a clinical experience with – patients since . tables ii and iii are not complete lists of genetic disorders found in the amish and mennonite populations of pennsylvania. however, we have seen patients from other settlements in central and north- western pennsylvania where different founder genes presumably explain the prevalence of different sets of genetic disorders. cultural and economic background of the clinic the clinic for special children was registered in pennsylvania as an inde- pendent nonprofit organization in . the original board of directors included three of the authors of this paper (d.h.m., c.s.m., r.i.k.) and the late dr. john a. hostetler, a descendant of amish parents and a sociologist of rural culture. in , several parents of children with genetic disorders from the amish and mennonite communities joined the board. both communities were aware of significant numbers of children with msud, ga , and other genetic disorders. the goal of these parents was for the clinic to provide comprehensive medical care for child- ren with complex and unstable disor- ders. the clinic was viewed as a way to article american journal of medical genetics (semin. med. genet.) t a b l e ii . g e n e ti c d is o rd e rs o f p e n n sy lv a n ia a m is h il ln e ss d u ri n g in fa n c y h is to ry an d e x am n e o g e n o r p a sc re e n s e ru m am in o ac id an al y si s u ri n e o r (s e ru m ) o rg an ic ac id s b y g c / m s a m in o ac id s an d ac y l c ar n it in e s b y m s / m s d ia g n o se d b y m u ta ti o n d e te c ti o n a c li n ic al n o te s m e ta b o li c d is o rd e rs a d e n o si n e d e am in as e d e fi c ie n c y þ m il ro y a m is h . s e v e re c o m b in e d im m u n e d e fi c ie n c y. l e th al w / t b o n e m ar ro w tr an sp la n t a d re n al h y p e rp la si a ii ß -h y d ro x y st e ro id d e h y d ro g e n as e d e f. þ þ þ þ n e o n at al h y p o n at re m ia / h y p e rk al e m ia . m al e s h av e am b ig u o u s g e n it al ia b u t se v e ri ty v ar ia b le c o n g e n it al g ro w th h o rm o n e d e fi c ie n c y þ p o o r g ro w th in in fa n c y w it h an o re x ia an d lo w ig f - an d g h c o rt ic o st e ro n e m e th y l o x id as e ii d e f. a ld o st e ro n e d e f. þ þ þ p o o r g ro w th , h y p o n at re m ia / h y p e rk al e m ia , v ar ia b le p h e n o ty p e c ri g le r- n aj ja r sy n d ro m e ty p e þ þ k e rn ic te ru s w / t n e o n at al d ia g n o si s an d p h o to th e ra p y. l e th al w / t li v e r tr an sp la n t g al ac to se m ia , d u ar te an d d u ar te / c la ss ic al v ar ia n ts þ þ þ þ c ar ri e rs o f c la ss ic al g e n e id e n ti fi e d b y n e o g e n . c o m m o n in m ar y la n d a m is h g lu ta ry l c o a d e h y d ro g e n as e d e fi c ie n c y þ þ þ þ þ b as al g an g li al d e g e n e ra ti o n w it h c e re b ra l p al sy -l ik e d y st o n ia g m g an g li o si d o si s, b e ta -g al ac to si d as e d e fi c ie n c y þ þ l e th al . f o u n d in m il ro y a m is h d e m e b u t n o t in l an c as te r c o u n ty m e d iu m c h ai n ac y l- c o a d e h y d ro g e n as e d e fi c ie n c y þ þ þ þ þ r ar e . h ig h m o rb id it y an d m o rt al it y w / t n e o n at al sc re e n in g s. in fe c ti o n in d u c e d c ri si s -m e th y lc ro to n y l- c o a c ar b o x y la se d e fi c ie n c y þ þ þ þ þ m il d p h e n o ty p e, se v e ra l as y m p to m at ic ad u lt s an d c h il d re n p h e n y la la n in e h y d ro x y la se d e fi c ie n c y þ þ þ þ þ þ v ar ia b le p h e n o ty p e, se v e re to m il d h y p e rp h e p ro p io n y l- c o a c ar b o x y la se d e fi c ie n c y þ þ þ þ þ v ar ia b le p h e n o ty p e . b as al g an g li al in ju ry , c ar d io m y o p at h y p y ru v at e k in as e d e fi c ie n c y þ þ þ c o m m o n in b e ll e v il le d e m e . e rt h ro b la st o si s, h e p at o p at h y an d h e m o ly si s s h o rt c h ai n ac y l d e h y d ro g e n as e d e fi c ie n c y þ þ þ þ in fa n ts as y m p to m at ic . in c re as e d b u ty ry lc ar n it in e an d u ri n e e th y lm al o n at e s it o st e ro le m ia þ þ c o ro n ar y ar te ry d is e as e in c h il d h o o d . h ig h c h o le rs te ro l an d si to st e ro l s y n d ro m e s a rt e ri al v e n o u s m al fo rm at io n sy n d ro m e þ c o m m o n in l an c as te r a m is h . in tr ac e re b ra l h e m o rr h ag e s in al l ag e g ro u p s. d x b y m r a b la n k sy n d ro m e , fa m il ia r h y p e rc h o la n e m ia þ þ p o o r g ro w th , p ru ri ti s, v it am in k , an d d d e fi c ie n c ie s in in fa n c y b y le r d is e as e , fa m il ia r in tr ah e p at ic c h o le st as is þ þ p o o r g ro w th , p ru ri ti s, v it am in k , an d d d e fi c ie n c ie s in in fa n c y. l e th al w / t li v e r tr an sp la n t american journal of medical genetics (semin. med. genet.) article c ar ti la g e -h ai r h y p o p la si a d w ar fi sm þ þ þ v ar ia b le p h e n o ty p e fo r g ro w th , h ai r, im m u n e d e fi c ie n c y, an d h ir sc h sp ru n g s d is e as e c e re b e ll ar at ax ia w it h p re se rv e d re fl e x e s þ þ g ro ss m o to r an d la n g u ag e d e la y s. a ta x ia an d ab n o rm al g ai t v ar ia b le c o c k ay n e sy n d ro m e þ l e th al . t y p ic al p h e n o ty p e, in fa n ti le fo rm . s e e n in a m is h in b e ll e v il le ar e a l e th al m ic ro c e p h al ly w it h -k e to g lu ta ri c ac id u ri a þ þ þ c o m m o n in l an c as te r. l e th al w it h b ra in m al fo rm at io n s an d p ro g re ss iv e e n c e p h al o p at h y c o n g e n it al su p ra v e n tr ic u la r ar rh y th m ia s þ s e v e re ar rh y th m ia s. l if e -t h re at e n in g c ar d ia c e v e n ts in th e n e o n at e . d x b y e k g e c tr o d ac ty ly , o m p h al o c e le an d c ar d ia c m al fo rm at io n s þ þ r ar e , le th al sy n d ro m e. s o m e in fa n ts h av e e c to d ac ty ly o n ly e ll is -v an c re v e ld d w ar fi sm þ þ þ c o m m o n in l an c as te r. p u lm o n ar y h y p o p la si a, c ar d ia c d is e as e in – % f u c h s e n d o th e li al d y st ro p h y o f th e c o rn e a þ p re se n ts in y o u n g ad u lt s as e ar ly g la u c o m a an d c at ar ac ts m c k u si c k -k au ff m an sy n d ro m e þ þ þ f e m al e in fe rt il it y w it h o u t e ar ly d e te c ti o n an d re p ai r m o y am o y a d is e as e þ o n e c as e in b e ll e v il le a m is h . t h e c h il d al so h ad p y ru v at e k in as e d e fi c ie n c y m u sc u la r d y st ro p h y, li m b -g ir d le ty p e þ b e ta -s ar c o g ly c an g ly c o p ro te in d e fe c t n e u ro fi b ro m at o si s u n c o m m o n . t w o c as e s w it h : p e ri o b it al an d fa c ia l tu m o rs ; o b st ru c ti v e h y d ro c e p h al is o st e o g e n e si s im p e rf e c ta w it h o p al e sc e n t te e th , fr ac tu re s þ þ c o m m o n . d o m in an t v ar ia n t. f ra c tu re s la te r o n se t an d o st e o p o ro si s p o ly c y st ic k in d e y d is e as e , ad u lt ty p e o n se t re n al fa il u re ag e – y e ar s. n o li v e r d is e as e . r e n al tr an sp la n t r e tt sy n d ro m e þ þ t y p ic al p ro g re ss iv e m ic ro c e p h al y in fe m al e s. s p o ra ti c, fu ll d is ab il it y r ie h l sy n d ro m e, p e li za e u s- m e rz b ac h e r- li k e d y sm y li n at io n þ þ a u to so m al re c e ss iv e v ar ia n t. c o n g e n it al n y st ag m u s, m o to r d e la y s. d x b y m r i s w ar e y sy n d ro m e , fe ta l g o n ad al fa il u re , su d d e n in fa n t d e at h þ þ c o m m o n in b e ll e v il le ar e a. l e th al . n o t e n d o c ri n o p at h y o r k e n n e d y sy n d ro m e t ro p o n in ty p e m y o p at h y, in fa n ti le n e m al in m y o p at h y þ þ þ c o m m o n , le th al . t re m o rs in u te ro an d at b ir th . ‘‘ c h ic k e n b re as t d is e as e ’’ a g e n e an d m u ta ti o n id e n ti fi c at io n p u ff e n b e rg e r, . article american journal of medical genetics (semin. med. genet.) t a b l e ii i. g e n e ti c d is o rd e r o f p e n n sy lv a n ia m e n n o n it e s il ln e ss d u ri n g in fa n c y h is to ry an d e x am n e o g e n o r p a sc re e n s e ru m o r (u ri n e ) am in o ac id an al y si s u ri n e o r (s e ru m ) o rg an ic ac id s b y g c / m s a m in o ac id s an d ac y l c ar n it in e s b y m s / m s d ia g n o se d b y m u ta ti o n d e te c ti o n a c li n ic al n o te s m e ta b o li c d is o rd e rs c ri g le r- n aj ja r sy n d ro m e ty p e þ þ þ k e rn ic te ru s w / t n e o n at al d ia g n o si s an d p h o to th e ra p y l e th al w / t li v e r tr an sp la n t c y st in u ri a þ (þ ) þ r e n al st o n e s in e ar ly c h il d h o o d w it h ri sk o f re n al d am ag e. c o m m o n . f o u r m u ta ti o n s g il b e rt sy n d ro m e þ c o m m o n in fa m il ie s w it h c ri g le r- n aj ja r d is e as e h o m o zy g o te s fo r t a t a b o x p o ly m o rp h is m s g ly c o g e n st o ra g e d is e as e , ty p e þ a b n o rm al li v e r e n zy m e s an d h ig h c h o le st e ro l ir ri ta b il it y an d h e p at o m e g al y af te r w e an in g im m u n e d e fi c ie n c y, s c id v ar ia n t þ þ a l c < / m m , n o th y m ic sh ad o w o r t -l y m p h . b o n e m ar ro w tr an sp la n t. ?i l re c e p t d e fe c t b ra n c h e d c h ai n -k e to ac id d e h y d ro g e n as e d e fi c ie n c y þ þ þ þ þ þ c la ss ic al m s d . l iv e r tr an sp la n t w as e ff e c ti v e th e ra p y, b o n e m ar ro w tr an sp la n t w as n o t m e d iu m c h ai n ac y l- c o a d e h y d ro g e n as e d e fi c ie n c y þ þ þ þ þ h ig h m o rb id it y an d m o rt al it y w it h o u t n e o n at al sc re e n in g s. in fe c ti o n in d u c e d c ri si s -m e th y lc ro to n y l- c o a c ar b o x y la se d e fi c ie n c y þ þ þ þ þ m il d p h e n o ty p e, se v e ra l as y m p to m at ic ad u lt s an d c h il d re n m e v al o n at e k in as e d e fi c ie n c y þ þ þ n e o n at al ja u n d ic e , e ry th o b la st o si s. c o m p o u n d h e te ro zy g o te p h e n y la la n in e h y d ro x y la se d e fi c ie n c y þ þ þ þ þ þ v ar ia b le p h e n o ty p e, m il d h y p e rp h e to se v e re p ro p io n y l- c o a c ar b o x y la se d e fi c ie n c y þ þ þ þ þ s am e m u ta ti o n as a m is h . v ar ia b le p h e n o ty p e t y ro si n e m ia ty p e þ þ þ þ þ o n e c as e , n o c li n ic al d is e as e s y n d ro m e s d y st o n ia s d y t - þ þ c h il d h o o d o n se t, is ra e li m u ta ti o n , o n e c as e . d y t re p o rt e d in m id w e st e rn m e n n . g ro u p f am il ia l p e ri o d ic fe v e r (t r a p s ) þ þ þ / � t u m o r n e c ro si s fa c to r re c e p to r m u ta ti o n in fa m il y. t r a p s an d f m f g e n e s n l in an o th e r fa m il y f ra g il e x sy n d ro m e þ þ c o m m o n d is o rd e r. v ar ia b le p h e n o ty p e m ak e s c li n ic al d ia g n o si s d if fi c u lt h e re d it ar y sp h e ro c y to si s þ h e m o ly si s, h y p e rb il ir u b in e m ia , ab n o rm al b lo o d sm e ar . r b c fr ag il it y te st d ia g n o st ic h ir sc h sp ru n g d is e as e þ þ þ / � n e o n at al b o w e l o b st ru c ti o n an d se p si s. p o ly g e n ic d is e as e . h ig h ly v ar ia b le p h e n o ty p e n e p h ro ti c sy n d ro m e, c o n g e n it al þ þ e d e m a an d in fe c ti o n s. l e th al w / t re n al tr an sp la n t o st e o g e n e si s im p e rf e c ta , d e ta c h e d re ti n ae s þ þ c o n g e n it al d e ta c h m e n t o f re ti n ae s. f ra c tu re s, la te o n se t. u n k n o w n m u ta ti o n american journal of medical genetics (semin. med. genet.) article allow the communities to care for their own. this desire was in response to the the goal of these parents was for the clinic to provide comprehensive medical care for children with complex and unstable disorders, as a way to allow the communities to care for their own. many difficulties encountered by the plain people in getting access to, and paying for, specialized medical care. the origins of community support and the economic and medical impacts of the clinic can be understood in relation to the history of msud in the mennonite people. the collection of essays and let- ters what is wrong with our baby?, com- piled and published by john and verna martin, parents of two msud children, is an interesting and sometimes sobering look at modern health care from a parent’s perspective [martin and martin, ]. the oldest surviving mennonite patient with msud was born in , but many undiagnosed or untreated in- fants died before she was born. she was in a hospital in philadelphia for months, did not regain her birth weight for months, and today has severe disability and retardation. her sister, in whom msud was recognized by days of age, became severely intoxicated, remained hospitalized for many weeks, and also has profound retardation. financial difficulties for plain people in traditional medical settings between and , mennonite children were born with msud. over these years, the management of the neonates improved and hospitalization times shortened. however, as funding for research in biochemical causes of mental retarda- tion waned, the direct costs of care paid by the mennonite community in- creased sharply. by , the initial care for a newly diagnosed neonate with msud in regional pediatric hos- pitals was $ , – $ , or more [morton et al., b]. amino acid analyses cost $ , and when coupled with physician charges and other testing charges, the total cost of an outpa- tient evaluation was typically $ , – $ , . these expenses were a major difficulty for the plain people, as they historically have not participated in medical insurance programs. approxi- mately half of the families contribute to a form of major medical insurance called amish aid or mennonite aid, which is administered by businessmen within the churches. prior to the open- ing of the clinic, benefit auctions were held to help pay hospital bills of $ , or higher. other church districts collect alms to help families with large medical bills. members of the old orders are not eligible for medicare or medicaid benefits because of an exemption from social security taxes. when young adults join the old order amish or mennonite churches, they sign an irs form , which indicates that they are ‘‘a member of a religious body that is conscientiously opposed to social secur- ity benefits but that makes reasonable provision for its own dependent mem- bers’’ [hostetler, ]. the exemption from the tax also means that those who sign irs form agree not to accept money from state or federal programs that are paid for by social security funds. this cultural practice is one of many issues community members face when obtaining care for their children. challenges in the medical management of these disorders earlier diagnosis and improved man- agement did, in many cases, allow normal growth and development, yet several otherwise healthy children with msud died unexpectedly from acute cerebral edema after the newborn period. of the children with msud o st e o g e n e si s im p e rf e c ta , a m is h v ar ia n t þ þ f ra c tu re s, la te r o n se t r e tt sy n d ro m e þ t y p ic al p ro g re ss iv e m ic ro c e p h al y in fe m al e s s p o ra ti c, fu ll d is ab il it y r e ti n it is p ig m e n to sa , ar e fl e x ia , se n so ry at ax ia þ þ c o m m o n sy n d ro m e. a x p c , m ap p e d to q - . n o m u ta ti o n in p le x in a . u n k n o w n m u ta ti o n w e rd n ig -h o ff m an d is e as e , in fa n ti le þ þ þ c o m m o n , le th al s m n e x o n d e le ti o n . a m il d e r c li n ic al v ar ia n t h as b e e n se e n in o n e fa m il y f am il ia l m an ic -d e p re ss iv e il ln e ss þ v ar ia b le p h e n o ty p e , c o m m o n . c h il d h o o d o n se t d o e s o c c u r. m u ta ti o n u n k n o w n a g e n e an d m u ta ti o n id e n ti fi c at io n p u ff e n b e rg e r, . article american journal of medical genetics (semin. med. genet.) earlier diagnosis and improved management did, in many cases, allow normal growth and development, yet several otherwise healthy children with msud died unexpectedly from acute cerebral edema after the newborn period. born between and , ( %) died of cerebral edema before the age of years. the typical course of the children was that they became ill with a common infection, had poor appetite, vomiting, and irritability, which pro- gressed to lethargy, and then to coma. by the time arrangements were made to travel from lancaster to the regional hospital, the biochemical intoxication was severe. emergency efforts to reverse the intoxication by intravenous fluids, glucose, and dialysis failed, the cerebral edema worsened, and within hr after entering the hospital, the child died of brain stem herniation. physicians and parents knew that common infectious illnesses provoked serious metabolic in- toxication, but little was known about how to control the metabolic response to such illnesses in the outpatient set- ting. local services for children were limited. specialists and equipment that were needed to study the early stages of cerebral edema were in research labora- tories far from the patients. the available biochemical testing was of no utility for the rapid treatment necessary to avert cerebral edema because results took as long as weeks. routine outpatient visits to metabolic clinics in philadelphia could only be made on one afternoon each week, and outpatient services to assess acutely ill children were not avail- able. instead, ill children were referred to emergency rooms, and the medical staff of the er usually knew less about the management of msud than did the child’s parents. newborn screening for msud was started by pennsylvania in , but the state-administered pro- gram did not substantially improve mat- ters for these patients. until the guthrie method was replaced by the tandem mass spectrometry program in , false positive rates were unacceptably high [morton et al., b]. the state program made no provisions to test and provide immediate results for high-risk infants. the three mennonite infants who were found by the state program were and days old and already ill. more important, the state follow-up program made no provisions to pay for the care of ill neonates, had no plans for the assessment and care of acutely ill older children, did not address the problem of cerebral edema, and did not support care for patients over the age of years. the state program did offer to pay for several amino acid levels per year and offered formula to those who qualified for, and would accept, medicaid, but the program required that mennonite families obtain these services at the hospitals in distant re- gional centers. in , an amino acid analyzer was given to the clinic for special children by two mennonite churches. to date, the clinic has performed , amino acid analyses on this instrument. because of our nonprofit status and community support, we were able to reduce the patient test charge from $ to $ . we also decreased the analysis and reporting time for plasma branched-chain amino acids to min, and we routinely quantify plasma amino acids during an outpatient clinic visit. between and , we managed neonates with msud. twenty-one of these neonates were from high-risk couples recognized by family histories or carrier tests, and the infants were tested for msud and diagnosed before hr of age. with timely diagnosis, the neonates were never ill and could be managed without hospitalization. the diagnosis of msud was made in additional neonates diagnosed because of clinical illnesses; of these infants were found by the pennsylvania screening pro- gram. these ill infants were managed at lancaster general hospital by the clinic staff and had a typical stay of – days with a hospital cost of $ , – , per patient per day. over a - year period, developmental outcomes have been good [morton et al., ]. we currently follow more than patients with msud, most of whom are of mennonite descent. between and we had admissions to our hospital for acute msud in- toxication; however, the average an- nual rate of hospitalization for our msud patients is less than day per patient-year of follow-up. between and , there were, however, no deaths from cerebral edema. the current total annual formula cost for our msud patients is about $ , . again, most mennonite families do not accept formula or reimbursement for costs from the pennsylvania msud follow-up program. the clinic pays half of these costs for families who have limited resources, and the families pay the balance. the charge for an out- patient visit is $ , and medical care and laboratory testing are provided at the clinic regardless of a family’s ability to pay. in summary, a com- prehensive health care system was designed to provide sophisticated medical care of patients with msud within their community. by combining community support and other dona- tions with knowledgeable and sophisti- cated laboratory and clinical care, the morbidity, mortality, and costs have been reduced. care for patients with disorders other than msud we believe that the cost of operat- ing the clinic for special children could be fully justified if we cared for no other patients than mennonite children with msud. however, the medical and economic impacts of care for patients with ga , mcadd, crigler-najjar disease, the bile salt disorders, and many other disorders that affect amish and mennonite children provide equally compelling justification for the clinic. more than % of the disorders we see cause clinical problems during infancy. ten of the syndromes american journal of medical genetics (semin. med. genet.) article can be recognized by history and physical examination, including the common disorders, amish lethal micro- cephaly [kelley et al., ; strauss et al., ], swarey syndrome [morton et al., unpublished results], a variant of cere- bellar ataxia [morton et al., unpublished results], and a newly described syndrome with infantile retinitis pigmentosa and sensory ataxia [higgins et al., ]. five disorders are primarily diagnosed by amino acid analysis, and eight disorders by organic acid analysis with gas chro- matography/mass spectrometry (gc/ ms). genomic dna mutation analysis is performed in the clinic [puffenberger and morton, ; puffenberger, ] and is now used to diagnose disorders, including byler disease and two other disorders that disrupt bile salt transport, crigler-najjar disease, fragile-x syn- drome, glycogen storage disease type vi [chang et al., ], amish osteogenesis imperfecta, infantile spinal muscular atrophy, and methylenetetrahydrofolate reductase deficiency [puffenberger and morton, ]. two common lethal dis- orders, troponin t myopathy [johnston et al., ] and infantile spinal muscular atrophy, can be difficult to diagnose in a newborn by examination, and we now routinely confirm these diagnoses by mutation analyses. neonatal metabolic screenings done by neo gen inc. identify using tandem mass spectrometry routinely identify of the disorders that we see. the preliminary biochemical diagnoses for most of these disorders is confirmed in our laboratory on the original specimen through polymerase chain reaction (pcr)-based mutation detection. the molecular basis of disorders seen at the clinic remains to be identified. seven of these disorders should yield to candidate gene sequenc- ing, including gm gangliosidosis, bartter disease, and polycystic kidney disease, whereas syndromes without candidate genes will require genome- wide studies, including the cerebral arteriovenous malformation syndrome, manic depressive illness, several conge- nital deafness syndromes, and a common seizure/mental retardation syndrome. the importance of presymptomatic care in our clinic, we have emphasized the importance of diagnosis of asympto- matic infants. for example, the diagnosis of ga after degeneration of the basal ganglia allows no opportunity for effec- tive treatment [morton et al., ; strauss and morton, a; strauss et al., b]. the prospective management of ga prevents basal ganglial degenera- tion in at least % of cases. the clinical importance of recognizing asympto- matic infants with mcadd, propionic acidemia, crigler-najjar disease, adrenal insufficiencies, bartter disease, the bile salt transport disorders, and galactosemia is readily apparent to a pediatrician who has cared for infants with these disorders who presented undiagnosed and severely ill. we have also found it helpful to be able to immediately diagnose lethal disorders in neonates. gm gangliosi- dosis can be diagnosed by physical exam and a single inexpensive test of urine for increased urine oligosaccharide excretion. infantile spinal muscular atro- phy and troponin t deficiency are both routinely diagnosed in neonates by molecular methods at a charge of less than $ [puffenberger and morton, ]. because of easy access to the clinic, prolonged hospitalizations invol- ving multiple invasive and expensive diagnostic tests for lethal disorders can usually be avoided. in the primary care setting, undiag- nosed genetic syndromes present differ- ently than in genetic clinics at referral centers. most often, the infants who are seen at the clinic are irritable, feed poorly, gain weight slowly, and in other less well defined ways seem to the parents to be ‘‘just not right.’’ many teenage patients and adults in these populations with underlying genetic syndromes had not been evaluated by modern diagnos- tic methods. the first mennonite girl found to have mcadd at the clinic was years old and had been hospitaliz- ed several times because of vomiting and ketotic hypoglycemia. her sibling died at age months with hypoglycemia and a fatty liver, which was diagnosed as reye syndrome in . here and elsewhere, some genetic disorders have been misdiagnosed as child abuse. approximately % of patients with ga whom we have seen were originally thought to be victims of child abuse. infants with ga may present with subdural hemorrhages after minor head trauma, because % of the infants have open subdural and subar- achnoid spaces that are crossed by bridg- ing veins [strauss and morton, a]. these intracranial hemorrhages are usually large, and retinal hemorrhages have developed spontaneously in asso- ciation with large intracranial hemor- rhages [hymel et al., ]. in the amish population of lancaster county, we follow children with three dif- ferent hepatic bile salt transport disor- ders. nine of these infants ( %) presented before months of age with bruising and bleeding secondary to vitamin k deficiency. one such infant presented in a coma with a full fontanel, retinal hemorrhages, and bruising on bony prominences. shaken baby syn- drome was the suspected diagnosis. the infant was subsequently found to have a vitamin k-responsive coagulopathy, high serum bile salts, a deep, intracer- ebral hemorrhage, and unilateral retinal hemorrhages, which were atypical of shaken baby syndrome. this infant and her sibling are now known to be homozygotes for a mutation in the tight junction protein gene [carlton et al., ]. fractures caused by relatively mild types of osteogenesis imperfecta found in the plain populations have also been mistaken for child abuse. the utility of a detailed understanding of the natural history of disease we have had a unique opportunity to observe the natural history of these more than % of the disorders we see cause clinical problems during infancy. article american journal of medical genetics (semin. med. genet.) disorders in many patients. the men- nonite variant of msud arises from a stop codon in exon of the bckdha gene. thus far, all mennonite patients have been homozygous for this mutation and all have the classical form of the disorder. in spite of the mutational homogeneity, the phenotype ranges from patients who are profoundly dis- abled to those who are apparently normal. poor outcomes in patients with msud can usually be understood in terms of one or more of the following problems: delays in neonatal diagnosis and treatment, chronic essential amino acid deficiencies, ineffective manage- ment of metabolic intoxication during infectious illnesses, and acute vascular brain injuries caused by cerebral edema. neonates with msud who are diag- nosed before – hr of age are not ill, and progressive metabolic intoxication can be prevented by rational manage- ment [morton et al., b]. plasma amino acid quantification allows diag- nosis of the disorder as early as hr of age if high-performance liquid chroma- tography (hplc) or tandem mass spec- trometry is used. it is important to note that the guthrie bacterial inhibition assay is not sufficiently sensitive to be used for specimens collected before hr of age [morton et al., b]. all infants with classical msud identified by newborn screening programs will be very ill if screening results are not available before days of age. infants identified and treated between and days of age can have good neurological outcomes, but the cost of initial treat- ment is higher than those diagnosed within hr. neonatal screening pro- grams for msud wherein specimens are not collected until hr of age and results are not reported before days of age can expect high initial costs of treatment and high morbidity and mor- tality. after msud is diagnosed, the prognosis for the patients depends on several variables, including the time re- quired to lower the plasma and tissue leucine levels to normal, prevention of the vascular compression and ischemic injuries caused by critical brain edema, and prevention of prolonged or severe brain and systemic essential amino acid deficiencies. our impression is that prolonged deficiencies of valine in the brain during the critical period of rapid brain growth between birth and years of age may be a major factor in mental retardation seen in children with poorly treated msud [morton et al., b]. the mainstay of therapy for msud has been the dietary restriction of the branched-chain amino acids. unfortu- nately, in many follow-up programs, leucine levels are measured infrequently and isoleucine, valine, and the other essential amino acids are not monitored at all. it is not possible to understand the relationship of day-to-day metabolic control and neurological outcome when only a few plasma leucine levels are measured per year. intermittent changes in tolerance related to normal variation in growth velocity or illnesses will not be appreciated. deficiencies of branched- chain amino acids and the unbalanced transport of the other neutral amino acids that compete with leucine for entry into the brain cannot be assessed by isolated measurements of leucine. the effects of dietary therapy on intel- lectual outcome can only be understood by reference to the cumulative effects of amino acid metabolism throughout the critical period of brain growth and development. in recent years, we have monitored blood amino acid levels in rapidly growing infants twice weekly and gained an appreciation of how quickly amino acid concentrat- ions change, especially during otherwise minor infectious illnesses. over a -year period, we have admitted patients with msud to the hospital for management of metabolic crisis times. more than of these admissions were necessary because of catabolic intoxication caused by infec- tious illnesses such as gastroenteritis, otitis media, streptococcal pharyngitis, sinusitis, and pulmonary infections. appendicitis, cholecystitis, torsion and infarction of the ovary, and fractures also cause metabolic crises. in addition, we have – msud outpatient visits per year to manage infectious illnesses and other stressors. these illnesses cause significant metabolic changes in patients with msud and require one or more measurements of plasma amino acids and changes to a different treatment regimen to prevent prolonged or progressive metabolic intoxication. the ultimate impact of these illnesses on clinical out- come is influenced by the rapid avail- ability of outpatient and inpatient services, including amino acid analysis, msud hyperalimentation solution, and medical care by physicians and nurses who have experience with the manage- ment of the metabolic crisis and cerebral edema. medical care and the outcome of other genetic disorders eleven of the disorders that affect the amish and mennonites are lethal or invariably cause full disability. although these disorders are lethal, affected children may live months or years and require many human and medical ser- vices. the clinic routinely provides medical care for children who have lethal diseases. most of these children are cared for, and die, at home. much of the community support for the clinic comes from families who need and appreciate this care. the other meta- bolic disorders and syndromes present with a range of clinical problems that require timely diagnosis and ongoing medical care. our daily practice in- cludes management of episodic crises the ultimate impact of these illnesses on clinical outcome is influenced by the rapid availability of outpatient and inpatient services, including amino acid analysis, msud hyperalimentation solution, and medical care by physicians and nurses who have experience with the management of the metabolic crisis and cerebral edema. american journal of medical genetics (semin. med. genet.) article in patients with the adrenal insufficiency syndromes, crigler-najjar syndrome, and various organic acidemias; and the chronic malnutrition and fat-soluble vitamin deficiencies of the bile salt disorders, as well as the cardiac and pulmonary problems of the patients with ellis-van creveld syndrome and the immune and gastrointestinal disorders in patients with cartilage-hair hypopla- sia. the early diagnosis of genetic syndromes and anticipation of predict- able problems is medically important and often limits the adverse effects of the disorder on the life of the patient, especially for metabolic disorders, but also for many other nonmetabolic syndromes. immune deficiency states are known to be associated with several of the disorders that affect the plain people. in infants with cartilage-hair hypoplasia, we have encountered severe varicella infections, pneumoncystis carinii pneumo- nia, hemophilus influenza meningitis in an immunized -month-old, and persis- tent parvovirus infection with a chronic inflammatory disease similar to juve- nile rheumatoid arthritis. our young patients with ellis-van creveld dwarfism who have cardiac lesions and pulmonary hypoplasia are particularly vulnerable to infections with respiratory syncytial virus. our first encounter with the amish-mennonite variant of pro- pionic acidemia was an undiagnosed -month-old who presented with a viral respiratory tract infection and developed acute striatal necrosis and a dilated cardiomyopathy with endo- cardial fibroelastosis. infants with the mennonite variant of nephrotic syn- drome [bolk et al., ] typically die from the immune deficiency state caused by the loss of immune globulins and complement rather than from the loss of albumin or renal failure. gram-negative sepsis must be anticipated in the ill neonate with galactosemia or hirsch- sprung disease. based on our experience in lancaster county, the practice of medical genetics cannot be separated from general pediatrics, and much of our work in general pediatrics is focused on the prevention and treatment of in- fectious illnesses. the disease course of many genetic disorders is directly deter- mined by how effectively the interaction of infectious disease and underlying genetic disorder is understood and managed. biology of genetic disorders our daily clinical work within a population with high incidences of genetic diseases provides a unique opportunity to study the biology of genetic disorders. because of the allelic homogeneity of many disorders among the plain people, we observe a consis- tent, or at least much more limited, spectrum of variation at the primary disease-causing locus than do most physicians. these observations have given us a different perspective on geno- type-phenotype correlation. msud exemplifies the complex relationship of phenotype and genotype and, in addi- tion, the manner in which health care delivery affects clinical outcome. the description of gene mutations is useful for the diagnosis of many disorders, but mutation analysis alone has seldom provided us with insight into the phe- notypic diversity that we encounter in our patients who share the same disease genotypes. our understanding of varia- tions in the natural history of genetic disorders is more often based on the concepts and language of cell biology, pathophysiology, and critical periods of brain development than on specific molecular lesions. msud provides a useful example of the complex relation- ship between gene and disease. descrip- tions of mutations in branched-chain keto-acid dehydrogenase as an under- lying cause of msud do not help us understand the multisystem regulators of protein anabolism or catabolism that cause marked changes in leucine toler- ance. these perturbations of anabolism and catabolism are more important influences on day-to-day metabolic control and illness than are the under- lying mutations. nor do the mutations explain leucine-induced encephalopa- thy, acute cerebral edema, or mental retardation. the biology of msud can only be understood in terms of the acute and chronic effects of high blood and tissue concentrations of leucine on protein turnover in muscle and liver, cell volume regulation, and brain amino acid homeostasis as it relates to neuro- transmitter function, and the devel- opment and maintenance of brain structure. msud arises from a complex derangement of interorgan amino acid transport. our impression is that scientists who work as physicians and care for several patients with the same genetic disorder over long periods of time will develop a different understanding of genetic disease than scientists who study disease mechanisms only in the labora- tory. laboratory scientists implicitly assume that a genetic disease can be adequately characterized by molecular, enzymatic, biochemical, and cellular experiments that are done at one point in time and are independent of experi- ence with an individual patient. our experience suggests that descriptions of disease mechanisms based on in vitro studies are necessarily incomplete and may lead to an oversimplification of models of genetic disorders. msud, ga , and the majority of the other disorders managed at the clinic can only be understood in terms of the patho- physiological mechanisms that unfold in individuals over significant periods of time and in relationship to many dif- ferent disease-causing factors, many of which are not genetic in origin. for ga , msud, and pku, the neurologi- cal syndromes caused by single-gene mutations arise from mechanisms of injury and dysfunction that are linked to critical periods of brain development. in patients with ga , episodes of metabolic illness that cause acute striatal necrosis in an infant are well tolerated in the -year-old or adult. unbalanced amino transport that leads to mental retardation in the infant with pku and msud causes attention deficit disorder, mood disorders in older patients, and presenile dementia in adults. the single- gene mutations of msud and pku give rise to diseases by disruptions of complex biological processes such as the meta- bolic adaptations to fasts and illnesses, cell volume regulation, brain amino acid article american journal of medical genetics (semin. med. genet.) homeostasis, and postnatal organ growth and development. description of a disease process without reference to such biological processes and only by refer- ence to point mutations in genes or changes in enzyme activities seems to us, as clinicians, to be inadequate. assump- tions about the nature of genetic disease and the terms that are used to character- ize these disorders influence how phy- sician-scientists are trained and how, throughout their careers, these scientists ask and answer questions. a recent editorial in the journal of clinical investi- gation indicated that research support in the past years has overwhelmingly directed physician-scientists to the lab- oratory and away from patients, showing that % of physician-scientists who received the howard hughes clinical research awardees elected to do research that did not involve patients [goldstein and brown, ]. our experiences at the clinic over the past years suggest many important aspects of genetic dis- eases will only become apparent to physician-scientists who care for indivi- dual patients over long periods and who are interested in patients, pathophysiol- ogy, and biology in the most general sense. conclusions the clinic for special children was established to provide care for menno- nite and amish children in lancaster county who have msud and ga . however, the need for a similar ap- proach to care for patients with other disorders, in other places and popula- tions, is increasingly apparent. our mennonite patients with msud come from every region of pennsylvania, maryland, new york state, kentucky, michigan, wisconsin, indiana, and iowa. ga is found in all amish populations of pennsylvania and in northern new york state. the most recent cases of ga we have seen were recognized through the supplemental screening program but were not of amish descent. in the general population of the united states, ga is now known to be one of the more common inherited metabolic dis- orders [strauss and morton, ]. around the world, clusters of cases of ga caused by founder genes have been identified in ireland, spain, israel, saudi arabia, chile, and two native american populations. regardless of the under- lying mutation, diagnosis and treatment of msud and ga before the onset of cerebral edema are essential. the med- ical care of a patient with a genetic disorder is no different than the care of a patient with other problems that are medically complex, such as diabetes mellitus, manic depressive illness, leu- kemia, congenital heart disease, renal failure, rheumatoid arthritis, seizures, congenital and acquired immune defi- ciency syndromes, drug dependency, and many other chronic medical trou- bles. rare genetic disorders are like the majority of diseases treated by physi- cians: few curable diseases exist. none- theless, accessible and effective medical care can limit suffering, improve func- tion, reduce dependency, and otherwise limit the effect of disease, genetic and acquired, on the life of a patient. william osler said, ‘‘such is our work.’’ references baric i, zschocke j, christensen e, duran m, goodman si, leonard jv, muller e, morton dh, superti-furga a, hoffmann gf. . diagnosis and management of glutaric aciduria type i. j inherit metab dis : – . biery bj, stein de, morton dh, goodman si. . gene structure and mutations of glutaryl-coenzyme a dehydrogenase: im- paired association of enzyme subunits that is due to an a v substitution causes glutaric acidemia type i in the amish. am j hum genet : – . bolk s, puffenberger eg, hudson j, morton dh, chakravarti a. . elevated frequency and allelic heterogeneity of congenital nephrotic syndrome, finnish type, in the old order mennonites. am j hum genet : – . carlton veh, harris bz, puffenberger eg, batta ak, knisely as, robinson dl, strauss ka, shneider bl, lim wa, salen g, morton dh, bull ln. . complex inheritance of familiar hypercholanemia with associated mutations in tjp and baat. nature genet : – . chang s, rosenberg mj, morton dh, francomano ca, biesecker lg. . identification of a mutation in liver glyco- gen phosphorylase in glycogen storage disease type vi. hum mol genet : – . francomano ca, mckusick va, biesecker l. . medical genetic studies in the amish: historical perspective. am j med genet : – . gibson km, bennett mj, naylor ew, morton dh. . -methylcrotonyl- coenzyme a carboxylase deficiency in amish/mennonite adults identified by de- tection of increased acylcarnitines in blood spots of their children. j pediatr : – . goldstein jl, brown ms. . the clinical investigator: bewitched, bothered, and bewildered—but still beloved. j clin invest : – . higgins jj, morton dh, loveless jm. . posterior column ataxia with retinitis pig- mentosa (axpc ) maps to chromosome q -q . neurology : – . hostetler ja. . amish society. baltimore: johns hopkins press. p – . hymel kp, jenny c, block rw. . intracranial hemorrhage and rebleeding in suspected victims of abusive head trauma: addressing the forensic controversies. child maltreat : – . johnston jj, kelley ri, crawford to, morton dh, agarwala r, koch t, schäffer aa, francomano ca, biesecker lg. . a novel nemaline myopathy in the amish caused by a mutation in troponin t . am j hum genet : – . kelley ri, robinson d, puffenberger eg, strauss ka, morton dh. . amish lethal microcephaly: a new metabolic disorder with severe congenital microcephaly and -ketoglutaric aciduria. am j med genet : – . martin j, martin vm. . what is wrong with our baby? ephrata, pa: grace publishing. p – . mckusick va. . medical genetic studies of the amish. selected papers. baltimore: johns hopkins university press. p – . mitsuuchi y, kawamoto t, miyahara k, morton dh, naiki y, kuribayashi i, toda k, hara t, orii t, yasuda k, miura k, nakao k, imura h, ulick s, shizuta y. . congenitally defective aldosterone biosynthesis in humans: inactivation of the p- c gene (cyp b ) due to nucleo- tide deletion in cmo i deficient patients. biochem biophys res commun : – . morton dh. . through my window— remarks at the th year celebration of children’s hospital of boston. pediatrics : – . morton dh, bennett mj, seargeant le, nichter ca, kelley ri. . glutaric aciduria type i: a common cause of episodic encephalo- pathy and spastic paralysis in the amish of 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rosenberg mj, agarwala r, bouffard g, davis j, fiermonte g, hilliard ms, koch t, kalikin lm, makalowska i, morton dh, petty em, weber jl, palmieri f, kelley ri, schäffer aa, biesecker lg. . mutation in the deoxynucleotide carrier dnc causes con- genital microcephaly. nature genet : – . strauss ka, pfannl r, morton dh. . the neuropathology of amish lethal microce- phaly. am j hum genet :a . strauss ka, morton dh. a. type glutaric aciduria, part : a model of acute striatal necrosis. am j med genet (semin med genet) c: – . strauss ka, puffenberger eg, robinson dl, morton dh. b. type i glutaric aciduria, part : natural history of patients. am j med genet (semin med genet) c: – . article american journal of medical genetics (semin. med. genet.) erratum kir haplotypes defined by segregation analysis in centre d’etude polymorphisme humain (ceph) families m. p. martin & r. m. single & m. j. wilson & j. trowsdale & m. carrington published online: december # springer-verlag erratum to: immunogenetics doi . /s - - -y in the original version of the manuscript, we failed to discuss and cite a key study closely related to ours (norman et al. ). this study described and analyzed data from of the families used in our study. thus, our study extends that of norman et al. by including additional families. we view our manuscript as a source of reference material as opposed to the analytical synthesis provided by the norman et al. study. we sincerely apologize for our omission and wish to correct the manuscript as follows: . norman et al. should be included as one of the citations for the sentence below: human populations can vary remarkably in the frequencies of kir genes and haplotypes, possibly a reflection of selection pressures conferred by population-specific diseases (cook et al. ; crum et al. ; denis et al. ; frassati et al. ; gendzekhadze et al. ; jiang et al. ; kulkarni et al. b; middleton et al. ; norman et al. ; norman et al. ; rajalingam et al. ; toneva et al. ; velickovic et al. ; whang et al. ; witt et al. ; yawata et al. ). . a statement referring to the families used in the study should have read as follows: in order to more fully characterize the kir locus in caucasians, we typed for the presence/absence status of kir genes (martin and carrington ) in members of ceph families of european descent and determined haplo- types based on segregation analysis (figure ). these families included one amish family, ten french families, and forty six families from utah. kir data from of the utah families used in the present study were previously reported by norman et al. . our study includes an additional families and thus is an extension of this previous work. norman pj, cook ma, carey bs, carrington cvf, verity dh, hameed k, ramdath dd, chandanayingyong d, leppert m, stephens haf, vaughan rw ( ) snp haplotypes and allele frequencies show evidence for disrup- tive and balancing selection in the human leukocyte receptor complex. immunogenetics : – doi: . /s - - - immunogenetics ( ) : doi . /s - - - the online version of the original article can be found at http://dx.doi. org/ . /s - - -y m. p. martin : m. carrington (*) cancer and inflammation program, laboratory of experimental immunology, saic-frederick, inc, nci-frederick, frederick, md , usa e-mail: carringt@ncifcrf.gov r. m. single department of mathematics and statistics, university of vermont, burlington, vt , usa m. j. wilson csl limited, bio institute, parkville, victoria , australia j. trowsdale immunology division, department of pathology, university of cambridge, tennis court road, cambridge cb qp, uk http://dx.doi.org/ . /s - - -y http://dx.doi.org/ . /s - - -y 	kir haplotypes defined by segregation analysis in centre d’etude polymorphisme humain (ceph) families << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (none) /calrgbprofile (srgb iec - . ) /calcmykprofile (iso coated v % \ 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http://europepmc.org/abstract/med/ an exemption for sincere believers: the challenge of alberta v. hutterian brethren of wilson colony copyright © sara weinrib, ce document est protégé par la loi sur le droit d’auteur. l’utilisation des services d’Érudit (y compris la reproduction) est assujettie à sa politique d’utilisation que vous pouvez consulter en ligne. https://apropos.erudit.org/fr/usagers/politique-dutilisation/ cet article est diffusé et préservé par Érudit. 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( ). an exemption for sincere believers: the challenge of alberta v. hutterian brethren of wilson colony. mcgill law journal / revue de droit de mcgill, ( ), – . https://doi.org/ . / ar résumé de l'article dans l’affaire alberta c. hutterian brethren of wilson colony, la cour suprême du canada a reconfiguré son approche quant à l’article de la charte canadienne des droits et libertés en statuant que la dernière étape du critère établi dans r. c. oakes (soit la condition de proportionnalité entre les effets salutaires et délétères d’une mesure) formait le cadre essentiel de son analyse. l'auteure suggère que l’accent mis par la cour sur cette dernière étape du critère oakes ne représentait pas la meilleure réponse aux arguments spécifiques avancés par l’alberta en matière d’atteinte minimale. l’alberta soutenait que la province ne pouvait exempter les huttérites de l'exigence de prise de photo de permis, même s’ils s’y opposaient pour des motifs religieux. la province justifiait cette position à la lumière de son interprétation de la décision syndicat northcrest c. amselem, selon laquelle les gouvernements ne pouvaient enquêter sur la sincérité des croyances religieuses. l’ontario, en tant qu’intervenant, a appuyé les arguments de l’alberta. bien que la cour n’ait pas abordé l’analyse de l’atteinte minimale, l’auteure suggère que les provinces ont interprété amselem de façon inutilement stricte. l’auteure propose ainsi une exemption qui adhère aux critères d’amselem tout en remplissant les objectifs de l’alberta en matière de sécurité. de façon plus générale, l’auteure stipule que la préoccupation des provinces dans hutterian brethren démontre le rôle critique que joue le critère de l’atteinte minimale dans oakes pour générer des solutions aux conflits entre les lois d’application générale et les pratiques religieuses minoritaires. par contraste, l’accent mis par la cour sur le critère des effets proportionnels pourrait malheureusement décourager les parties de formuler des alternatives potentiellement novatrices. https://apropos.erudit.org/fr/usagers/politique-dutilisation/ https://www.erudit.org/fr/ https://www.erudit.org/fr/ https://www.erudit.org/fr/revues/mlj/ https://id.erudit.org/iderudit/ ar https://doi.org/ . / ar https://www.erudit.org/fr/revues/mlj/ -v -n -mlj / https://www.erudit.org/fr/revues/mlj/ mcgill law journal ~ revue de droit de mcgill an exemption for sincere believers: the challenge of alberta v. hutterian brethren of wilson colony ————case comment———— sara weinrib* * student-at-law, constitutional law branch, ministry of the attorney general of on- tario. the opinions expressed in this paper are mine alone and do not represent the views of the government of ontario. this paper was written as an independent research project at the university of toronto faculty of law under the supervision of professor lisa austin, whom i wish to thank for her guidance, insightful comments, and encour- agement. i also wish to thank robert e charney, carol rogerson, and the reviewers of the mcgill lj for their feedback on prior drafts. an earlier version of this paper was awarded the patricia julia myhal scholarship in legal writing. © sara weinrib citation: ( ) : mcgill lj ~ référence : ( ) : rd mcgill in alberta v. hutterian brethren of wilson colony, the supreme court of canada reconfigured its approach to sec- tion of the canadian charter of human rights and free- doms by holding that the final step of the r. v. oakes test— the requirement of proportionality between a measure’s salutary and deleterious effects—provided the critical framework for its analysis. the author suggests that the court’s emphasis on the last step of the oakes test was not the most appropriate response to the specific minimal im- pairment argument alberta presented. alberta argued that the reason it could not safely offer an exemption from its li- cence photo requirement to hutterites who objected to pho- tos on religious grounds was because syndicat northcrest v. amselem restricted government inquiries into the sincerity of religious beliefs. ontario intervened in support of al- berta’s concerns. although the court did not address this minimal impairment argument, the author argues that it reflects an unnecessarily strict reading of how amselem’s guidelines would apply in this context. in support, the au- thor presents an exemption that would have cohered with amselem and achieved alberta’s safety objectives. the au- thor then argues more broadly that the provinces’ concerns in hutterian brethren demonstrate the critical role the minimal impairment step of the oakes test plays in generat- ing solutions to clashes between laws of general application and minority religious practices. the court’s new emphasis on the proportionate effects test, in contrast, may unfortu- nately discourage both parties from formulating potentially innovative alternatives. dans l’affaire alberta c. hutterian brethren of wilson colony, la cour suprême du canada a reconfiguré son approche quant à l’article de la charte canadienne des droits et libertés en statuant que la dernière étape du critère établi dans r. c. oakes (soit la condition de proportionnalité entre les effets salutaires et délétères d’une mesure) formait le cadre essentiel de son analyse. l'auteure suggère que l’accent mis par la cour sur cette dernière étape du critère oakes ne représentait pas la meilleure réponse aux arguments spécifiques avancés par l’alberta en matière d’atteinte minimale. l’alberta soutenait que la province ne pouvait exempter les huttérites de l'exigence de prise de photo de permis, même s’ils s’y opposaient pour des motifs religieux. la province justifiait cette position à la lumière de son interprétation de la décision syndicat northcrest c. amselem, selon laquelle les gouvernements ne pouvaient enquêter sur la sincérité des croyances religieuses. l’ontario, en tant qu’intervenant, a appuyé les arguments de l’alberta. bien que la cour n’ait pas abordé l’analyse de l’atteinte minimale, l’auteure suggère que les provinces ont interprété amselem de façon inutilement stricte. l’auteure propose ainsi une exemption qui adhère aux critères d’amselem tout en remplissant les objectifs de l’alberta en matière de sécurité. de façon plus générale, l’auteure stipule que la préoccupation des provinces dans hutterian brethren démontre le rôle critique que joue le critère de l’atteinte minimale dans oakes pour générer des solutions aux conflits entre les lois d’application générale et les pratiques religieuses minoritaires. par contraste, l’accent mis par la cour sur le critère des effets proportionnels pourrait malheureusement décourager les parties de formuler des alternatives potentiellement novatrices. ( ) : mcgill law journal ~ revue de droit de mcgill introduction i. the amselem test for sincere religious belief ii. the shadow amselem cast over hutterian brethren a. alberta’s and ontario’s submissions b. the supreme court of canada’s response iii. an exemption for sincere believers a. drafting an exemption b. does this exemption cohere with amselem? c. does this exemption undermine alberta’s objectives? d. would the supreme court of canada have accepted this exemption? conclusion: future subsection (a) challenges and the role of the minimal impairment test comment: alberta v. hutterian brethren of wilson colony introduction in , alberta terminated the exemptions it previously offered to drivers who objected to licence photos on religious grounds. the hutter- ites interpret the second commandment to forbid them from having their photos taken and therefore objected to the change of policy as a violation of their right to freedom of religion under subsection (a) of the canadian charter of rights and freedoms. alberta conceded this point, but argued that this infringement was justified according to the three-step test in r. v. oakes for establishing the justification of a legislative measure under section of the charter. although the hutterites were successful at trial and at the alberta court of appeal, the supreme court of canada nar- rowly upheld alberta’s amendment as a reasonable limit on the hutter- ites’ rights. hutterian brethren is noteworthy for its lengthy discussion of the last step of the oakes test, which requires proportionality between a meas- ure’s salutary and deleterious effects. this step had not previously played a significant role. nevertheless, chief justice mclachlin affirmed that the statutory basis for these exemptions was alberta’s operator licensing and vehicle control regulation (alta reg / , s ( )(b)), which gave the registrar discretion to determine in what circumstances a photo was required: “before issuing or renewing an operator’s licence ... the register ... may require an image of the applicant’s face, for incorporation in the licence.” the amendment eliminated this discretion by replacing “may” with “must”: operator licensing and vehicle control amendment regulation, alta reg / , s , amending operator licensing and vehicle control regulation alta reg / , s ( )(b). see also alberta v hutterian brethren of wilson colony, abca , ar at para [hutterian brethren (ca)]. part i of the constitution act, , being schedule b to the canada act (uk), , c [charter]. the second commandment prohibits making “an idol, or any like- ness of what is in heaven above or on the earth beneath”: exodus : ; deuteronomy : . see also alberta v hutterian brethren of wilson colony, scc (factum of the appellant at para [foa]). [ ] scr , dlr ( th) [oakes]. alberta v hutterian bretheren of wilson colony, scc , [ ] scr [hutte- rian brethren]. at the alberta court of queen’s bench, lovecchio j held that the mini- mal impairment test was not met and restored the pre- regime (hutterian brethren of wilson colony v alberta, abqb , alta lr ( th) at para ). at the alberta court of appeal, conrad and o’brien jja also held that the amendment did not minimally impair the hutterites’ rights (hutterian brethren (ca), supra note at para , slatter ja dissenting). for a detailed analysis of the court’s application of this step, see sara weinrib, “the emergence of the third step of the oakes test in alberta v hutterian brethren of wil- son colony” : ut fac l rev [forthcoming in ]. frank iacobucci described this step to require only a “resume of previous analysis” (“judicial review by the supreme court of canada under the canadian charter of rights and freedoms: the first ten years” in david m beatty, ed, human rights and ( ) : mcgill law journal ~ revue de droit de mcgill the third step of the oakes test provided the critical methodological framework to assess alberta’s regulation. on this basis, she then con- cluded that the salutary effects of alberta’s amendment outweighed its detrimental effects on the hutterite claimants’ rights. justice abella, who wrote the principal dissent, also affirmed the critical significance of the third step but concluded that the regulation’s deleterious effects out- weighed its marginal security benefits. justice lebel in a separate dis- sent, supported by justice fish, called for a return to the centrality of the court’s minimal impairment test, but did not elaborate on the conclusions the minimal impairment test would have called for in this instance. in this article, i consider whether the court’s emphasis on the last step of the oakes test was an appropriate response to the specific minimal impairment argument alberta presented. the minimal impairment test requires the government to demonstrate that its impugned law impairs the right in question no more than is necessary to accomplish its desired objective. the court accepted that alberta’s objective in terminating its exemptions was to “[maintain] the integrity of the driver’s licensing sys- tem in a way that minimize[d] the risk of identity theft.” however, in its minimal impairment submissions, alberta clearly stated that continuing to offer the exemption to hutterites and other sincere religious believers would not undermine this objective. alberta’s position was, rather, that it could not safely offer this exemption after syndicat northcrest v. amse- lem, a supreme court of canada decision restricting judicial—and pre- sumably also governmental—inquiries into the sincerity of religious be- liefs. alberta thus concluded that an amendment terminating exemptions constituted a minimal impairment of the hutterites’ rights. ontario in- tervened in support of alberta’s concerns; it then addressed them by pro- posing an alternative exemption model that it acknowledged potentially contravened amselem. this article argues that ontario’s and alberta’s reading of amselem was unnecessarily strict, and that the court, by failing to analyze this reading, missed a critical opportunity to assess how amselem’s guidelines judicial review (dordrecht: martinus nijhoff, ) at ). peter hogg similarly concluded that this “[proportionate effect] step has no work to do, and can safely be ig- nored” (constitutional law of canada, th ed (scarborough, ont: thomson carswell, ), vol at . see also pierre blanche, “the criteria of justification under oakes: too much severity generated through formalism” ( ) : man lj at : “[t]he third step has no real weakening impact on the first two steps. it comes too late in the process ... it seems that it is a step that should almost never be reached.” for a discussion of the transformations the minimal impairment test has undergone since oakes, see infra note . hutterian brethren, supra note at para . scc , [ ] scr [amselem]. comment: alberta v. hutterian brethren of wilson colony would apply in this context. this argument proceeds in three sections. section i discusses the amselem test for the demonstration of sincere be- lief, reviewing the framework justice iacobucci put in place as well as the questions he left open for future litigation. section ii describes the shadow amselem cast over the hutterian brethren litigation by recounting al- berta’s and ontario’s concerns with amselem and the court’s response to these submissions. section iii argues that alberta and ontario construed amselem unnecessarily strictly, and proposes an exemption that would, it argues, cohere with amselem while protecting alberta’s objectives. such an exemption would have met the minimal impairment test and made it unnecessary for the court to turn to the proportionate effects test in this instance. what is at stake in this extended treatment of a line of argument to which the court did not respond? clashes between laws of general appli- cation and minority religious practices are likely to escalate in the future as a result of broad factors such as the growth of the administrative state, the development of technologies that may aid the state in address- ing security concerns, the rise of immigration from communities with different conceptions of the significance of seemingly neutral require- ments, and the greater secularization of canadian society as a whole. in hutterian brethren, the court chose to consider such clashes in the third step of the oakes test rather than the second. but the provinces’ concern as to the proper application of the amselem ruling demonstrates paul horwitz asserts that “as the reach of the administrative state extends even further into every aspect of life, law is bound to disturb an increasing number of religious prac- tices.” (“the sources and limits of freedom of religion in a liberal democracy: section (a) and beyond” ( ) : ut fac l rev at ). more particularly, moin yahya re- flects on the significant role driver’s licences have played in the growth of the adminis- trative state: “when the automobile was invented over years ago, few could have imagined that the piece of paper certifying the competence of its drivers would become a major instrument of state control in the st century.” “driver’s licence photos: secu- rity concerns shouldn’t trump religious freedom” the lawyer’s weekly ( august ), online: <http://www.lawyersweekly.ca>. bruce ryder, “the canadian conception of equal religious citizenship” in richard j moon, ed, law and religious pluralism in canada (vancouver: ubc press, ) at . in this respect, alberta argued that two recent developments justified its manda- tory photo requirement: “there are, first, threats to the public that did not exist in , which, second, we may now reduce with facial recognition technology and digital photos of licensees.” hutterian brethren, foa, supra note at para . dieter grimm notes that “new lines of conflict” have appeared as a consequence of im- migration: “general laws that do not have religious implications in western countries or reflect the christian tradition of the western world enter into conflict with the reli- gious norms of the immigrants” (“conflicts between general laws and religious norms” ( ) : cardozo l rev at - ). horwitz, supra note at . ( ) : mcgill law journal ~ revue de droit de mcgill the critical role the minimal impairment test plays in responding to these escalating tensions; it is at this stage in the analysis that the court can scrutinize the legislature’s reasons for not providing an alternative and remove any barriers that may have arisen due to uncertainty as to the na- ture or scope of the charter guarantee. if the court does not respond to these questions at this stage, it may restrict the legislature’s ability to protect all of its citizens and the rights they bear. i. the amselem test for sincere religious belief this section reviews the framework for freedom of religion that jus- tice iacobucci set out in amselem, considering in particular how this framework applied to the particular facts of this case as well as the guide- lines it provided for future litigation. it concludes by noting some particu- lar issues amselem left unresolved—issues squarely raised by alberta and ontario in hutterian brethren. it is important to lay out the facts and reasoning in amselem in some detail in this section, as the sections that follow will return to these passages to assess alberta’s and ontario’s read- ings of amselem, as well as the constitutionality of my proposed exemp- tion. in amselem, orthodox jews, who co-owned condominium units in montreal, asserted a right to build a succah on their balconies to celebrate a nine-day festival. the syndicate of co-ownership claimed that this ac- tion violated a bylaw prohibiting alterations of its balconies, and sought a permanent injunction prohibiting erection of these structures. it proposed that jewish residents set up a communal succah in the garden instead. the parties called experts at trial who differed as to whether jewish law required jews to erect individual succahs: the applicants’ expert, rabbi ohana, testified that the biblical obligation to dwell joyously in a succah required jews to erect their own succahs when factors such as the trans- port of children meant that a communal succah would cause distress; the respondents’ expert, rabbi levy, denied that such factors obligated jews to erect their own succahs. iacobucci j defined a succah as a “small enclosed temporary hut or booth, traditionally made of wood or other materials such as fastened canvas” in which jews are com- manded to dwell temporarily during the festival of succot (amselem, supra note at para ). although the word condominium does not appear in the case, “it is clear from discussion of the parties’ relationship under quebec law that it may be usefully sum- marized by use of this term, which is familiar in the common law provinces”: hutterian brethren, foa, supra note at para , n , amselem, supra note at para . ibid at paras , . comment: alberta v. hutterian brethren of wilson colony justice iacobucci held that the claimants’ rights to religious freedom had been violated. in doing so, he rejected the notion that the court could determine a subsection (a) claim by assessing the doctrinal interpreta- tions of competing religious authorities. instead, he stressed the primacy of subjective articulations of belief over the objective endorsement of these beliefs: [f]reedom of religion consists of the freedom to undertake practices and harbour beliefs, having a nexus with religion, in which an indi- vidual demonstrates he or she sincerely believes or is sincerely un- dertaking in order to connect with the divine or as a function of his or her spiritual faith, irrespective of whether a particular practice or belief is required by official religious dogma or is in conformity with the position of religious officials. justice iacobucci thus affirmed that subsection (a) protects sincere indi- vidual beliefs or practices even if a religious leader denies their signifi- cance. justice iacobucci then demarcated a complex spectrum of religious ex- periences that subsection (a) protects: [p]rovided that an individual demonstrates that he or she sincerely believes that a certain practice or belief is experientially religious in nature in that it is either objectively required by the religion, or that he or she subjectively believes that it is required by the religion, or that he or she sincerely believes that the practice engenders a per- sonal, subjective connection to the divine or to the subject or object of his or her spiritual faith, and as long as that practice has a nexus with religion, it should trigger the protection ... of s. (a) of the cana- dian charter. this critical passage identified three potential sources of sincere reli- gious belief. first, belief may arise from the objective requirements of a religion. second, it may be based on a claimant’s subjective beliefs as to the objective requirements of a religion, affirming that practices whose obligatory nature is a matter of controversy within the religious commu- nity are also protected. finally, beliefs are protected even if they have no basis in a religion’s objective requirements, as long as they have a “nexus with religion” and engender a subjective connection to the divine. ibid at para . although the dispute in amselem arose under the quebec charter of human rights and freedoms’ protection of “freedom of religion” (rsq c c- , s ), iacobucci j repeat- edly affirmed that the principles he established also applied to the guarantee of freedom of religion that is set out in ss (a) of the charter: amselem, supra note at paras , , , . ibid at para [emphasis added, original emphasis omitted]. ( ) : mcgill law journal ~ revue de droit de mcgill justice iacobucci’s first two categories seem relatively straightfor- ward. with respect to the first category, most religions have a core group of uncontested objective requirements, such as attending worship ser- vices. the claimants in amselem, on the other hand, seem to fall into the second category, since they believed that their religion mandated them to set up individual succahs in this instance, despite rabbi levy’s denial of this obligation. justice iacobucci’s third category—beliefs that “engende[r] a personal, subjective connection to the divine” and have a “nexus with re- ligion”—is the most difficult to understand. this difficulty arises partly because the claimants in amselem do not provide an example of this kind of belief. however, as i elaborate below, it is also because this category brings to light some of the deepest tensions involved in the liberal state’s protection of religion. justice iacobucci provided two examples of believers who seem to fall into this third category: jewish women, for example, strictly speaking, do not have a bibli- cally mandated “obligation” to dwell in a succah during the succot holiday. if a woman, however, nonetheless sincerely believes that sitting and eating in a succah brings her closer to her maker, is that somehow less deserving of recognition simply because she has no strict “obligation” to do so? ... should an individual jew, who may personally deny the modern relevance of the literal biblical “obliga- tion” or “commandment”, be precluded from making a freedom of re- ligion argument despite the fact that for some reason he or she sin- cerely derives a closeness to his or her god by sitting in a succah? surely not. justice iacobucci’s two figures—the “observant jewish woman” and the “liberal jewish man”—represent two various responses of those who identify with a traditional religion while accepting certain modern liberal precepts. the “observant jewish woman” may not sincerely believe that she is under an obligation to dwell in a succah, given traditional juda- ism’s gender-based allocation of obligations. yet she claims that subsec- tion (a) should protect her adoption of practices traditional leaders re- quire only of men. the “liberal jewish man”, on the other hand, seems to be a man who is under traditional obligation to dwell in a succah (at least according to some rabbis), but has accepted secular precepts and, as a re- sult, no longer believes he is under this obligation. he calls for subsection (a) to protect a practice that is fulfilling for him even though his inter- pretation of this practice no longer accords with orthodox precepts. these figures illustrate the many complex ways religious identifica- tion may develop in a modern liberal state: the “observant jewish woman” ibid at para . comment: alberta v. hutterian brethren of wilson colony expresses a sincere belief in a practice, but is not recognized by the relig- ion as the appropriate person to hold these beliefs; the “liberal jewish man”, in contrast, meets the formal criteria for recognition, yet does not sincerely believe in the literal biblical obligation. for justice iacobucci, the liberal commitment to state neutrality clearly calls the state to protect a broader spectrum of religious experience than orthodox leaders would sanction. as grimm similarly reflects, in a modern constitutional democ- racy, “religious freedom must not be turned into a protection of ortho- doxy.” on a theoretical level, justice iacobucci’s judgement admirably at- tempts to protect religious practices in both their traditional and modern iterations. however, it also generates a complex practical problem: under amselem, the court and the government cannot simply echo a religious leader’s identification of the beliefs and practices that merit protection. rather, they must conduct their own assessment of whether particular practices merit protection under subsection (a). how is the court or gov- ernment to assess the sincerity of these claims? justice iacobucci set out some broad guidelines. first, he affirmed that the religious claimant had the burden of demonstrating sincere religious belief, and that the court was “qualified to inquire into sincerity of a claimant’s belief, where sincerity [was] in fact at issue.” however, he cautioned that inquiries should be “as limited as possible” and were in- tended “only to ensure that a presently asserted religious belief is in good faith, neither fictitious nor capricious, and that it is not an artifice.” justice iacobucci then provided the following specific directions on a claimant’s use of objective evidence to demonstrate sincere belief: a claimant may choose to adduce expert evidence to demonstrate that his or her belief is consistent with the practices and beliefs of other adherents of the faith. while such evidence may be relevant to a demonstration of sincerity, it is not necessary. since the focus of the inquiry is not on what others view the claimant’s religious obli- gations as being, but rather what the claimant views these personal religious “obligations” to be, it is inappropriate to require expert opin- ions to show sincerity of religious belief. an “expert” or an authority on religious law is not the surrogate for an individual’s affirmation of what his or her religious beliefs are. supra note at . amselem, supra note at para . ibid at para . ibid at para [emphasis added]. ( ) : mcgill law journal ~ revue de droit de mcgill justice iacobucci’s assertion that the court cannot require expert opin- ions as to the sincerity of religious belief seems consistent with his recog- nition of a complex spectrum of religious experiences. requiring a claim- ant to provide evidence of a religious leader’s approval of the practice at issue turns freedom of religion into “a protection of orthodoxy.” the facts of amselem enabled justice iacobucci to provide these guide- lines without directly addressing two difficult and interrelated questions. the first question is what sort of evidence the court or state can request in order to assess sincerity of religious practice when sincerity is in fact at issue (sincerity of belief was hardly at issue in amselem; as ontario later submitted to the supreme court in hutterian brethren, it was highly unlikely anyone would want to build a succah for any reason other than sincere religious belief ). the second question is how broadly to construe justice iacobucci’s third category, the protection of practices that “en- gende[r] a personal, subjective connection to the divine” and have a “nexus with religion” (the jewish claimants in amselem did not provide examples of this category; instead, they claimed the right to a practice with a scriptural basis whose interpretation was contested within their religious community—justice iacobucci’s second category.) this third category could be interpreted broadly to protect idiosyncratic spiritual be- liefs that are not associated with an identified religious community. if it is interpreted in this manner, to bring these two questions together, what evidence can the state require to assess a claim that is not associated with an identified religion, in a situation where sincerity is at issue, and the harm posed by an insincere claim is significant? this question formed the basis of alberta’s and ontario’s critiques of amselem, to which we now turn. ii. the shadow amselem cast over hutterian brethren this section considers the court’s minimal impairment analysis in hutterian brethren from the perspective of both the submissions and the ultimate ruling. part a reviews alberta’s and ontario’s submissions to the court. alberta declared it was not possible for it to provide an exemption after amselem. ontario, after echoing alberta’s concerns, proposed an al- ternative exemption model that set aside some of the central tenets of amselem. part b considers the supreme court of canada’s response to these submissions, and suggests that, since the court did not address al- berta’s and ontario’s readings of amselem, the debate between chief jus- grimm, supra note at . alberta v hutterian brethren of wilson colony, scc (factum of the intervener, attorney general of ontario at para [foi]). comment: alberta v. hutterian brethren of wilson colony tice mclachlin and justice abella as to the extent to which the court, in its minimal impairment analysis, should defer to alberta was premature. a. alberta’s and ontario’s submissions in its minimal impairment submissions, alberta repeatedly admitted to the court that it was able to offer an exemption to the hutterites and other sincere religious believers without compromising its objectives: we note that our concern is not the granting of an exception to peo- ple who assert a religious objection to the photograph requirement as such. our concern is instead the opportunities that an exemption affords wrongdoers. similarly: [o]ur concern is not about numbers as such, or with the numbers of claims for exemption that might be made in good faith. alberta in- creased the security of the operator’s license because wrongdoers lie to take advantage of its currency as identification. in these passages, alberta made clear that offering exemptions to sincere believers would not undermine the integrity of its licensing system. al- berta straightforwardly acknowledged that it could provide an exemption to the claimants at hand without impairing its objectives, as long as this exemption did not enable insincere claimants—“wrongdoers”—to apply successfully. the province then argued that the reason it could not offer this ex- emption to the claimants before it was because amselem restricted its ability to distinguish between sincere and insincere claimants: amselem implies structural constraints on any religious exemption from the photo requirement. the issue in this case is not whether the respondents, as specific claimants, may safely be granted an ex- emption from the photo requirement. rather, the issue is whether the respondents and everyone else who is able to claim the benefit of religious freedom as described in amselem may safely be granted an exemption from the photo requirement. ... [t]he foundational characteristic of freedom of religion—its subjec- tivity—competes directly with the essential purpose of the photo re- quirement, which impedes those who would falsely obtain a driver’s licence. hutterian brethren, foa, supra note at para [emphasis in original]. ibid at para [emphasis in original]. ibid at paras , [emphasis in original]. ( ) : mcgill law journal ~ revue de droit de mcgill alberta’s claim in these passages was that amselem’s conception of relig- ion as subjective situated fraudulent and sincere professions of religious belief within an identical structure: both articulated an idiosyncratic pri- vate sphere that was not objectively verifiable. it was thus not possible for the province to provide an exemption after amselem. alberta’s reading of amselem is troubling. how can the charter prevent the government from distinguishing between fraudulent and sincere religious beliefs, given their dramatically different normative values to society? the presence of religious minorities is a value to be preserved, while those who make fraudulent claims to obtain false documentation undermine the state’s ability to protect its citizens and should be prevented. alberta thus problematically intimated in these passages that the court’s construal of subsection (a) in amselem prevented the province from recognizing the religious minorities the charter obligates it to protect. ontario supported alberta’s contentions, but proposed a different solu- tion. it accepted that amselem made it difficult for provinces to draft an exemption for the hutterites without rendering themselves vulnerable to many false claims. however, whereas alberta declared it was categori- cally not possible for the province to provide this exemption after amse- lem, ontario suggested that amselem should be distinguished to allow the province to craft a narrow and highly structured exemption in situations where sincerity of belief was at issue and the potential for harm from an insincere profession of belief was great. ontario presented its own exemption to the photo-licence requirement as an example of such an exemption. ontario currently provides exemp- s of the charter (supra note ) affirms the value of religious minorities to canada by stating, “this charter shall be interpreted in a manner consistent with the preserva- tion and enhancement of the multicultural heritage of canadians.” for a discussion of the role s could have played in the court’s analysis in hutterian brethren, see robert e charney, “how can there be any sin in sincere? state inquiries into sincerity of religious belief” ( ) sup ct l rev ( d) at - . ontario stated that in light of the court’s analysis in amselem, provincial governments face a dif- ficult policy conundrum. is it possible to draft an exemption from the manda- tory photo requirement that will exempt the respondents’ members (who are a discrete community with a long established and easily verified sincere reli- gious objection to being photographed) but will not exempt false claims of re- ligious belief[?] (hutterian brethren, foi, supra note at para ). this argument is elaborated by charney (supra note at ), who represented ontario in hutterian brethren. hutterian brethren, foi, supra note at para . comment: alberta v. hutterian brethren of wilson colony tions to applicants who can establish the following three indicia of sincere religious belief: . membership in a registered religious organization that prohibits personal photographs. . a letter of support from a religious leader of this organization. . scriptural passages that substantiate the religious prohibition. although ontario’s courts have yet to rule on the constitutionality of ontario’s exemption, each prong seems to challenge amselem: . the requirement of membership in a community that prohibits personal photographs undermines justice iacobucci’s affirmation that subsection (a) protects practices the obligatory nature of which is a matter of controversy. . the requirement of a religious leader’s letter contradicts justice iacobucci’s exhortation that it was “inappropriate to require expert opinions to show sincerity of religious belief.” . the requirement that the claimant’s belief be substantiated by a scriptural passage seems to create a narrower basis for protection than amselem’s broad protection of practices that have a “nexus with religion”. ontario admitted in its factum to the court that this model “ap- pear[ed], on its face, to be inconsistent with this court’s reasons in amse- these criteria were developed under a policy entitled permanent valid without photo (pvwp). the statutory basis for this policy is ss ( ) of the highway traffic act (rso , c h ), which grants the minister discretion to require a digital photo for driver’s licences. see also hutterian brethren, foi, supra note at paras - . the constitutionality of these requirements was challenged in bothwell v ontario (min- ister of transportation) ( ), admin lr ( th) , oac (div ct) [bothwell cited to admin lr], but the court concluded that since the applicant lacked sincere re- ligious belief, it was not necessary to consider this challenge. iacobucci j made this distinction explicit in a passage not discussed in section , above, in which he declared that “any incorporation of distinctions between ‘obligation’ and ‘custom’ or, as made by the respondent and the courts below, between ‘objective obliga- tion’ and ‘subjective obligation or belief’ within the framework of a religious freedom analysis is dubious, unwarranted and unduly restrictive” (amselem, supra note at para ). this distinction is also apparent in iacobucci j’s discussion of the “observant jewish women” and the “liberal jewish man”: see text accompanying note . the question of whether ontario’s requirement of membership in a religious com- munity coheres with amselem is discussed in detail in section iii.b, below (i conclude that this requirement is consistent with amselem). amselem, supra note at para . ibid at para . ( ) : mcgill law journal ~ revue de droit de mcgill lem.” however, it argued that the court should distinguish the guide- lines in amselem to recognize ontario’s model in this circumstance since—unlike in amselem—the potential harm posed by an insincere claim was significant. ontario concluded by stating that, “ontario agrees with alberta that, if amselem precludes the limited approach used in on- tario’s pvwp, then no exemption would be the only alternative that would meet the province’s policy objectives.” ontario’s proposal reflects an attempt to escape the constitutional irony of alberta’s position, discussed above, in which the court’s definition of freedom of religion prevented the province from protecting the claim- ants before it. nevertheless, it still accepts as its starting point both am- selem’s subjective definition of freedom of religion and alberta’s under- standing of amselem’s restrictions on state inquiries into sincerity of be- lief. as a result, ontario’s alternative is underinclusive, since not all the believers whom justice iacobucci stated merited protection would be able to meet its criteria. this underinclusiveness can be demonstrated through a story that has circulated about an application ontario reportedly denied. an old order amish man applied for ontario’s exemption, and included in his applica- tion a letter from a church elder who confirmed the religious prohibition against photographs, but stated that the amish religion also prohibited driving a car. the church elder then asserted that if the applicant was prepared to violate religious precepts by driving a car, he should have no objection to having his photo taken. ontario reportedly denied the amish man’s application on the basis of this letter. however, one could specu- late that the amish man in this story sincerely believed in the prohibition against taking photographs, and yet did not sincerely believe in the pro- hibition against driving. perhaps both prohibitions are contested, or per- haps the applicant had a need to drive that he reconciled with his faith in a manner not accepted by the church elder. in such cases, ontario’s ex- hutterian brethren, foi, supra note at para . see ibid at para . ibid at para . it is unclear how this underinclusiveness plays out in practice in ontario. ontario stated that since its policy had been introduced, it had received eighty applications for exemptions and had not yet granted one, since all applicants “either have not met the ministry’s criteria or have not completed the application process,” but it did not elabo- rate on the basis for these rejections (ibid at para ). charney relates this story but notes that it may be apocryphal: supra note at , n . i rely on it only as an example of a sincere believer who would be excluded from on- tario’s exemption. ibid. comment: alberta v. hutterian brethren of wilson colony emption may turn into a “protection of orthodoxy”, in that it enables the positions of orthodox leaders to hold sway even if the individual claimants can demonstrate the sincerity of their beliefs. of course, since the claimants in hutterian brethren easily met on- tario’s criteria, it was open for the court to accept ontario’s exemption and leave the question of the claimants potentially excluded from it for another day. nevertheless, in section iii, i consider whether ontario’s proposal could be modified to better protect the full spectrum of religious practices justice iacobucci identified in amselem. b. the supreme court of canada’s response the supreme court of canada did not address alberta’s and ontario’s concerns with amselem in hutterian brethren. instead, chief justice mclachlin’s majority judgment and justice abella’s principal dissent each accepted alberta’s proposition that providing an exemption would under- mine its objective of ensuring the integrity of its licensing system, while diverging on the extent of the risk that providing an exemption would pose to this objective. chief jusice mclachlin began her minimal impairment analysis by claiming that this case called for her to accord the legislature a measure of judicial deference, since this was a “complex social issu[e] where the legislature may be better positioned than the courts to choose among a range of alternatives.” the chief justice accorded alberta this deference by refusing to calculate the precise extent to which providing an exemp- tion undermined alberta’s objectives: the claimants’ argument that the reduction in risk would be low, since few people are likely to request exemption from the photo re- quirement, assumes that some increase in risk and impairment of the government goal may occur, and hence does not assist at the stage of minimal impairment. chief justice mclachlin then concluded that even though it was “diffi- cult to quantify in exact terms how much risk of fraud would result from permitted exemptions, it [was] clear that the internal integrity of the sys- tem would be compromised” by an exemption. chief justice mclachlin’s commitment to supporting the legislative process led her to conclude that the minimal impairment analysis should hutterian brethren, supra note at para . ibid at para . ibid at para . ( ) : mcgill law journal ~ revue de droit de mcgill no longer be considered the critical framework for assessing whether in- fringements of freedom of religion were justified: freedom of religion cases may often present this “all or nothing” di- lemma. compromising religious beliefs is something adherents may understandably be unwilling to do. and governments may find it dif- ficult to tailor laws to the myriad ways in which they may trench on different people’s religious beliefs and practices. the result may be that the justification of a limit on the right falls to be decided not at the point of minimal impairment, which proceeds on the assumption that the state goal is valid, but at the stage of proportionality of ef- fects. in this critical passage, the chief justice charts a new course for sub- section (a) litigation, based on the difficulty of governments “tailoring” laws so as not to infringe the myriad of beliefs found in canada’s multi- cultural society. yet in charting this new course, chief justice mclachlin did not address the particular reason alberta found it difficult to tailor this exemption. justice abella criticized chief justice mclachlin’s approach as overly deferential, but she also did not consider alberta’s specific concern with amselem. instead, justice abella stressed that the government had the onus of proving that the risk posed by an exemption was significant in practice, and determined that in this case, alberta had not met this bur- den: there was “no evidence from the government to suggest ... why the exemption [was] no longer feasible, or so dramatically obstructs the gov- ernment’s objective that it cannot be re-instated.” in coming to this con- clusion, justice abella did not reference alberta’s argument that an ex- emption was no longer feasible given the strictures of amselem. likewise, justice abella would have declared alberta’s mandatory photo require- ment unconstitutional, “in the absence of the availability of an exemption on religious grounds,” but she did not offer guidance as to how alberta could draft such an exemption in a manner that cohered with amselem. it is tempting to attribute chief justice mclachlin and justice abella’s contrasting approaches to the minimal impairment test to their different po- sitions in the court’s well-known debate as to the appropriate level of defer- ence to the legislature: chief justice mclachlin called for strong deference while justice abella affirmed the state’s burden to substantiate its projection of risks. however, it seems to me that alberta’s and ontario’s particular ibid at para [emphasis added]. ibid at para . ibid at para . as is well known, the minimal impairment test has undergone a number of transforma- tions. in oakes, dickson cjc articulated this test to require “cogent and persuasive” comment: alberta v. hutterian brethren of wilson colony concerns should have attracted more attention. alberta did not argue that judicial crafting of an exemption would burden its legislative role. rather, alberta claimed that the court’s past jurisprudence restricted its ability to continue to provide an exemption on religious grounds, even though the reli- gious minority requesting the exemption posed no risk. it sought guidance from the court on an issue squarely within the court’s expertise and institu- tional competence. such guidance would have supported alberta’s legislative function, rather than curtailed it. iii. an exemption for sincere believers in this section, i consider how the court could have responded to alberta’s and ontario’s concerns. part a provides an alternative interpretation of amselem, different from that of alberta and ontario, and proposes a three-pronged exemption that modifies ontario’s proposal according to this reading. part b elaborates on how each prong of this proposal coheres with amselem’s restrictions on state inquiries into religious practice as i understand them. part c argues that alberta and ontario could have provided this exemption without impairing their objective of protecting identity fraud, considering their articulations of this objective as well as canadian and american case law on this issue. finally, part d speculates as to whether, had the court been provided with this alternative, chief justice mclachlin would have nevertheless maintained that alberta met the minimal impairment test by refusing to provide an exemption, given the emphasis she placed on deference in her minimal impairment analysis. a. drafting an exemption before setting out my proposed exemption, it is necessary to return to alberta’s and ontario’s interpretations of amselem and consider them in more detail. alberta stated in its submissions to the court that “[t]he subjec- tive character of [the conception of religious freedom in amselem] implies that a person who claims a religious objection to the photo requirement with evidence that the measure impaired rights “as little as possible” (supra note at - ). yet almost immediately afterward, in r v edwards books and art ltd ([ ] scr , dlr ( th) [edwards books]) dickson cjc expressed concern that such a rigorous standard would impede the formation of laws intended to protect vulnerable groups. subsequently, the court articulated other considerations that called for defer- ence, such as laws premised on complex social science evidence, laws that reconcile the interests of competing groups, and laws that allocate scarce resources. see hogg, supra note at - ; timothy macklem & john terry, “making the justification fit the breach” ( ) sup ct l rev ( d) at - . ( ) : mcgill law journal ~ revue de droit de mcgill apparent conviction is presumptively entitled to an exemption.” ontario similarly argued that “[u]nless the province can rely on the kind of objective criteria apparently rejected by this court in amselem, it cannot offer a reli- gious exemption.” alberta and ontario thus read amselem to prohibit the use of evidence to determine the subjective sincerity of belief. this interpretation does not seem to me to follow necessarily from jus- tice iacobucci’s reasoning in amselem. as discussed in section i, justice iacobucci contemplated that a claimant’s beliefs would be objectively verified in several passages. first, he asserted that freedom of religion should not be protected beyond the ability of claimants to demonstrate sincere belief, defin- ing freedom of religion as the freedom “to undertake practices and harbour beliefs, having a nexus with religion, in which an individual demonstrates that he or she sincerely believes.” although he cautioned that these inquiries should be “as limited as possible”, he stressed that the court was “qualified to inquire into the sincerity of a claimant’s belief, where sincerity [was] in fact at issue.” applying these guidelines, justice iacobucci declined to as- sess the claimants’ beliefs rigorously as to their obligation to erect individual succahs. however, as ontario noted, sincerity was not at issue in this case since it was unlikely anyone would insincerely claim a right to build a suc- cah. since the sincerity of applicants for an exemption from a photo-licence requirement is at issue, it is difficult to see how amselem prohibits a more comprehensive inquiry. how could a more comprehensive inquiry be structured? further in his judgment, justice iacobucci made clear that a claimant could choose to adduce any supporting material that would prove sincerity of belief, such as letters from religious leaders, expert opinions, evidence of established practices, evidence that the alleged belief was consistent with other cur- rent religious practices, or general evidence supporting credibility. he imposed only one restriction: the assessor could not require expert opin- ion—or, presumably, any other particular indicia of religious belief. hutterian brethren, foa, supra note at para . hutterian brethren, foi, supra note at para . in ontario’s oral submissions it also requested that the court distinguish amselem in order to permit the province to “re- quire objective verification of a shared religious belief as a condition to qualify for a reli- gious exemption”: hutterian brethren of wilson colony, scc (oral argument, intervener [oai]). amselem, supra note at para [emphasis added]. ibid at para . ibid at para . hutterian brethren, foi, supra note . amselem, supra note at paras - . ibid at para . comment: alberta v. hutterian brethren of wilson colony thus, amselem did not limit the use of evidence to determine the subjec- tive sincerity of belief; rather, it prohibited the province from requiring that claimants prove that their leaders unanimously approved of their particular manifestations of the belief at issue. the following exemption modifies ontario’s proposal in order to bring it in line with the guidelines in amselem: for an applicant to be granted an exemption to the photo-licence re- quirement, he or she must demonstrate sincere religious belief by estab- lishing: a. membership in a religious community: this may be demonstrated by a letter of support from an established member of this community or by other in- dicia of community membership. b. that the objection to photographs arises from membership in this religious community: this may be demonstrated by evidence as to the origins or basis of these practices, for example in religious texts. c. individual commitment to the practice of abstaining from taking photographs: this may be demonstrated by evidence of past or cur- rent practice. the exemption stipulates that the applicant has the onus of demonstrat- ing sincere belief, which it divides into three distinct elements. it then provides examples of what kinds of evidence the applicant could adduce to demonstrate each of these elements. however, instead of singling out any piece of evidence as determinative, it enables the applicant to choose what evidence at the applicant’s disposal would best satisfy these criteria. the province would then assess whether this evidence, in its totality, met the burden of demonstrating sincere belief. b. does this exemption cohere with amselem? the first question to consider, in assessing the feasibility of my pro- posed exemption, is whether it is consistent with amselem. in this section, i evaluate each prong of my exemption according to this standard. the alberta court of appeal interpreted amselem similarly: “[a]lthough amselem may limit the ability of the province to require proof about the objective validity of an appli- cant’s belief, it does not in any way limit the use of evidence to determine the subjective sincerity of the belief”(hutterian brethren (ca), supra note at para ). ( ) : mcgill law journal ~ revue de droit de mcgill prong a: membership in a religious community prong a requires the claimant to prove membership in a particular re- ligious community. this raises the question of whether such a require- ment is consistent with amselem’s broad protection of beliefs that “engen- der[s] a personal, subjective connection to the divine” (justice iacobucci’s third category). as discussed in the conclusion to section i, justice iacobucci did not make clear how broadly he was willing to construe this nexus. in other passages, he described religion in individualistic terms, noting, for example, that freedom of religion “revolves around the notion of personal choice and individual autonomy and freedom.” alberta and ontario both expressed concern that this third category enabled justice iacobucci to protect idiosyncratic spiritual beliefs, making it easier for fraudulent claimants to claim an exemption. however, in this section, i argue that, despite some of the individualistic language used in amselem, this decision did not protect beliefs that emerge in isolation for three rea- sons. first, amselem did not provide the appropriate factual basis for the court to consider protecting idiosyncratic religious beliefs, since the claimants belonged to a discrete religious community. rather, at issue in amselem was whether the claimants had the right to observe individually the precept of building a succah, or whether collective observance of this practice (in a communal succah) would suffice. responding to these facts, justice iacobucci protected a range of doctrinal interpretations regarding the obligations a jewish holiday imposed on individual adherents. how- ever, the facts did not enable him to consider whether religious beliefs that emerge in isolation are protected. supra note at para . ibid at para . ontario argued to the court that after amselem it may no longer be possible to draft an exemption that would not exempt “claims based on personal, political or philosophical concerns like privacy (which, as a conscientious objection may be easily confused with or disguised as religious)”: hutterian brethren, foa, supra note at para . ontario also submitted in oral argument that the court should distinguish amselem as requir- ing “objective verification of a shared religious belief”: hutterian brethren, oai, supra note [emphasis added]. for alberta’s submissions expressing this concern, see hut- terian brethren, foa, supra note at para . iacobucci j asserted that religious beliefs that were not “in conformity with the position of religious officials” were protected (amselem, supra note ). the supreme court of canada has emphasized the critical role facts play in decisions that delineate charter guarantees. in mackay v manitoba ([ ] scr at , dlr ( th) ), cory j asserted that “charter decisions should not and must not be made in a factual vacuum. to attempt to do so would trivialize the charter and inevita- comment: alberta v. hutterian brethren of wilson colony second, the critical passage in which justice iacobucci stressed the significance of preserving individual spiritual beliefs is heavily qualified. recall this passage, discussed above, in which justice iacobucci demar- cated three potential sources of religious belief: [p]rovided that an individual demonstrates that he or she sincerely believes that a certain practice or belief is experientially religious in nature in that it is either objectively required by the religion, or that he or she subjectively believes that it is required by the religion, or that he or she sincerely believes that the practice engenders a per- sonal, subjective connection to the divine or to the subject or object of his or her spiritual faith, and as long as the practice has a nexus with religion, it should trigger the protection ... of s. (a) of the ca- nadian charter. justice iacobucci stipulated here that subsection (a) protects beliefs where “the practice engenders a personal, subjective connection to the di- vine”—a quotation which admittedly raises the spectre of “religions of one.” however, justice iacobucci immediately qualified this broad protec- tion with the clause, “[a]s long as the practice has a nexus with religion.” thus, justice iacobucci did not demarcate a protection for “spiritual” be- liefs that emerge in isolation from “religion”. since justice iacobucci did not define these terms, the question is whether “religion” includes those with only one adherent. this interpretation seems doubtful to me because it would rob justice iacobucci’s qualification of any content; his qualifica- tion would now read as an assertion that subsection (a) protects beliefs that engender a subjective connection to the divine as long as they have a nexus with beliefs that are idiosyncratic. furthermore, justice iacobucci used the word “religion” in a manner synonymous with religious commu- nities elsewhere in this passage. for example, his assertion that subsec- tion (a) protects practices that are subjectively believed to be “required by the religion” would not make sense if justice iacobucci imagined that it was possible for “religion” to consist of one person’s spiritual beliefs, be- cause in this case there would be no conflict between this person’s subjec- tive beliefs and the religion’s objective requirements. a reading of this passage as protecting idiosyncratic religious beliefs is, therefore, difficult to sustain in context. finally, amselem did not engage with judicial and academic treat- ments of this issue. on the judicial side, courts have struggled with how to interpret subsection (a)’s dual protection of “freedom of conscience and religion”. put simply, is religious thought one form of individual conscien- bly result in ill-considered opinions.” see also danson v ontario (ag), [ ] scr - , dlr ( th) . amselem, supra note at para [emphasis added]. see also text accompanying supra note . ( ) : mcgill law journal ~ revue de droit de mcgill tious thought, in which case idiosyncratic spiritual beliefs should be pro- tected? or can religious thought be distinguished from conscientious thought on the ground that it emerges collectively? in the supreme court of canada’s foundational cases on subsection (a), it stressed the similar- ity between religious and conscientious thought: chief justice dickson af- firmed in r. v. big m drug mart ltd. that the purpose of protecting free- dom of religion is “the notion of the centrality of individual conscience”; in edwards books he similarly described freedom of religion as containing “both individual and collective aspects.” however, since these early cases, the court has rarely engaged with freedom of conscience as distinct from freedom of religion, and there is no majority judgment on the issue. the critical literature is likewise conflicted. whereas some assert that “a belief that is spiritual does not need to be shared by anyone else, as long as it is sincerely held,” others stress that the protection of religion rests on the view that religious beliefs “are an integral part of the individual’s cultural identity or membership.” this tension reflects two competing rationales for protecting freedom of religion: the first is the liberal com- mitment to freeing autonomous individual beliefs from state interfer- ence, and the second involves protecting minority groups. the conun- [ ] scr at , ar . supra note at . wilson j’s famous concurring opinion in r v morgentaler held that the decision to ter- minate a pregnancy should be protected as a matter of conscience under ss (a), which she defined as protecting “personal morality which is not founded in religion” ([ ] scr at , dlr ( th) ). lamer cjc’s dissent to rodriguez v british colum- bia (ag) echoed these reflections, maintaining that the “charter has established the es- sentially secular nature of canadian society and the central place of freedom of con- science in the operation of our institutions” ([ ] scr at , dlr ( th) ). horwitz, supra note at . richard moon, “introduction: law and religious pluralism in canada” in richard moon, ed, law and legal pluralism in canada (vancouver: ubc press, ) at . grimm also argues that “[r]eligion presupposes a community that is united in its belief in some truths of a transcendental nature and that develops common forms of worship and interaction. one would [not] call ... the transcendental assumptions of a single per- son a religion” (supra note at ). as stephen l carter notes, “liberals cherish religion, as they cherish all matters of pri- vate conscience, and liberal theory holds that the state should do nothing to discourage free religious choice”: the culture of disbelief: how american law and politics trivial- ize religious devotion (new york, anchor books, ) at . this rationale is buttressed by s of the charter, which requires the charter to “be in- terpreted in a manner consistent with the preservation and enhancement of the multi- cultural heritage of canadians” (supra note , s ). in wilson j’s dissenting opinion in edwards books, she argued that an interpretation of ss (a) that protected the religious comment: alberta v. hutterian brethren of wilson colony drum is this: if religious choice is viewed simply as an expression of pri- vate conscience, what distinguishes religious and secular beliefs? as jer- emy webber reflects, “a definition that treats religious freedom as merely a special case of the freedom to choose one’s conception of the good fails to account sufficiently for the singling out of religion.” returning to the particular context of hutterian brethren, the ques- tion at hand is whether amselem closed the door to an exemption that would require applicants to demonstrate identification with a particular religious community. ontario and alberta seemed to believe it had, but given the facts amselem responded to, justice iacobucci’s carefully quali- fied statements on this point, and the decision’s failure to engage with the line of judicial and critical scholarship outlined above, i would disagree. the question of whether beliefs that are not shared by a community are protected by subsection (a) remains to be litigated on the proper factual foundation. prong b: that the practice arises from membership in this religious community the second prong in my model requires the claimant to establish that the practice at issue is identified with the claimant’s religious community. this prong is reminiscent of ontario’s requirement that the religious or- ganization “prohibit” personal photographs. as discussed in section ii, however, the requirement of a prohibition did not seem consistent with justice iacobucci’s assertion in amselem that subsection (a) should not “protec[t] only those aspects of religious belief or conduct that are objec- tively recognized by religious experts as being obligatory tenets or pre- cepts of a particular religion.” in contrast, my proposal affirms amse- lem’s protection of religious practices that are not obligatory or whose obligatory nature is contested within the religious community. prong c: individual commitment to the practice this prong requires the claimant to demonstrate commitment to the practice at issue by providing evidence of past or current practice. alberta seemed to conclude that such a requirement was inconsistent with amse- lem. alberta claimed, for example, that after amselem, “a person who freedom of individuals but not the groups they belonged to was precluded by s (supra note at para ). “the irreducibly religious content of freedom of religion” in avigail eisenberg, ed, di- versity and equality: the changing framework of freedom in canada (vancouver: ubc press, ) at . see text accompanying note . supra note at para . see also supra note . ( ) : mcgill law journal ~ revue de droit de mcgill claims a religious objection to the photo requirement with apparent con- viction [was] presumptively entitled to an exemption.” alberta likely drew this conclusion from a passage in amselem in which justice iacobucci stressed that a claim should not fail on the grounds that the claimant’s past commitment to this practice was not en- tirely consistent. the issue arose in amselem because some of the claim- ants had not erected individual succahs in previous years, choosing in- stead to celebrate the holiday at the households of family members. in response, justice iacobucci made clear that this variation in practice did not render the claimant’s claim specious. he explained that it was [i]nappropriate ... [to] rigorously study and focus on the past prac- tices of claimants in order to determine whether their current beliefs are sincerely held. over the course of a lifetime, individuals change and so can their beliefs. alberta seemed to extrapolate from this passage that because amse- lem prohibited scrutiny of a claimant’s past practice, a claimant who pro- fessed a certain belief but could not provide evidence of past practice sup- porting this belief would succeed. however, this passage in amselem does not render evidence of past practice irrelevant to the determination of a claim’s sincerity. justice iacobucci made clear that religious practices evolve slowly—over a life- time—and thus it should be rare for a claimant who is not able to offer any evidence of past practice to succeed. furthermore, considering the facts of amselem from which justice iacobucci’s statement arose, the claimants had demonstrated a past commitment to this holiday by observ- ing it with family members. the issue in amselem was the interpretation of the biblical commandment to dwell joyously in a succah, and the par- ticular question before the court was whether the claimants sincerely be- lieved that erecting a communal succah in the garden of the condominium would not satisfy this obligation. the fact that claimants had chosen to celebrate the holiday in the past in the individual succahs of their family members did not undermine the sincerity of their claim that sharing a succah with all condominium members would infringe their beliefs. prong c of my proposed exemption is therefore in line with justice iacobucci’s exhortation in amselem that a believer’s evolving manifesta- tions of religious belief over time should not be subjected to mechanical state scrutiny. in practice, it would mean that that the old order amish hutterian brethren, foa, supra note at para . supra note at para . ibid at para . comment: alberta v. hutterian brethren of wilson colony man’s application, discussed above, would not fail on the basis that his past practices with respect to driving were inconsistent with his past practices with respect to photographs, as long as the claimant could also provide sufficient evidence demonstrating his membership in an amish community (prong a) that objects to the practice of taking photographs (prong b). to conclude, although my proposed exemption develops amselem’s guidelines to suit a more complex context, it does not contradict justice iacobucci’s reasoning in this case or the conclusions he drew based on the facts before him. c. does this exemption undermine alberta’s objectives? the next issue to consider is whether my proposed exemption would enable alberta and ontario to meet their objectives of protecting against identity fraud. after all, even if this exemption cohered with amselem, if it did not provide the province with sufficient criteria to distinguish sin- cere from insincere claims, it would have no potential to alter the court’s minimal impairment analysis in hutterian brethren. in this part, i ad- dress this issue by considering how my exemption would enable the prov- inces to distinguish between insincere claimants and the three kinds of sincere claimants justice iacobucci identified in amselem. recall that justice iacobucci first asserted that subsection (a) pro- tects those whose practices or beliefs were “objectively required by the re- ligion.” this category protects members of an organized religion with a recognized record of objecting to the photograph requirement, such as the amish, molokans, and hutterites. it would be very difficult for an insin- cere believer to provide evidence of membership in such a group (prong a), since these communities isolate themselves from modern secular soci- ety in many other respects and have a well-documented commitment to the practice of not taking photographs. as a result, this model would eas- ily enable alberta and ontario to identify those who insincerely claim to be members of such religions. incidentally, the letter the church elder reportedly wrote on behalf of this applicant seems to me to confirm prongs a and b of my proposed exemption, as the elder recog- nized the claimant as a member of his community and affirmed that old order amish are prohibited from taking photographs. see text accompanying note . amselem, supra note at para . for a discussion of the photo requirement exemption with respect to molokans, see valov v california (department of motor vehicles), cal rptr ( d) , cal app ( th) ( ). ( ) : mcgill law journal ~ revue de droit de mcgill second, justice iacobucci declared that subsection (a) protects claim- ants who sincerely believe a practice is required by their religion, even if this belief is contested by some religious leaders. the american case of quaring v. nebraska (department of motor vehicles) demonstrates how the provinces might determine the sincerity of such a claimant. quaring was a pentecostal christian woman who had long believed that the bible prohibited the taking of photographs; consequently, she possessed no fam- ily photos, did not take pictures of her own wedding, refused to allow any photography in her home, and removed labels displaying pictures on food- stuffs. although some branches of the pentecostal church subscribe to such a belief, it was not shared by quaring’s local church community. as a result, it is unlikely quaring would have been able to satisfy ontario’s current requirements for an exemption, since she would not have been able to offer a letter of support from a religious leader in her community. however, under my proposed exemption, quaring could have demon- strated that she belonged to a local pentecostal church (prong a), that her beliefs were rooted in traditional pentecostal teachings (prong b), and that she had a long history of abstaining from taking photographs (prong c). given the amount of evidence a sincere believer such as quaring would have been required to proffer under this model, it is unlikely that an insincere claimant could similarly demonstrate adherence to a verita- ble religious practice. third, justice iacobucci protected under subsection (a) “a person who sincerely believes that [a] practice engenders a personal, subjective con- nection to the divine,” as long as these beliefs have a “nexus with relig- ion.” as discussed in the conclusion of section i, this category raised par- ticular concerns for ontario and alberta, since an insincere claimant would likely claim that his beliefs were also protected under this category. f ( d) , us app lexis ( th cir, ), aff’d us ( ) [quaring, cited to f ( d)]. interestingly, the state’s argument in this case closely paralleled alberta’s in hutterian brethren. it argued that quaring’s application should not be accepted since she was not able to provide objective criteria of religious faith, and without such criteria, it would become too easy for any applicant to claim this objection, and exemptions would “be available virtually on demand.” (ibid at ). the us court of appeals for the eighth circuit rejected this argument. it stressed quaring’s identification with a historical and scattered community that shared these concerns, and concluded that “by showing that they possess no photos or pictures ,” persons, like quaring “requesting an exemption for religious beliefs based on the second commandment can easily demonstrate the sincer- ity and valid nature of their belief” (ibid). amselem, supra note . comment: alberta v. hutterian brethren of wilson colony in ontario’s submissions to the court, it stated that the bothwell case illustrated this concern. bothwell expressed uneasiness with ontario’s policy of taking all licence photos with a digital camera and storing them in a database. after being informed that ontario only provided exemp- tions for religious objections, he claimed that his “personal relationship with jesus christ” led him to believe that the second commandment pro- hibited photos. he then held a press conference to discuss his case and invited the media to bring cameras. the court dismissed bothwell’s pvwp application for failing to demonstrate sincere religious belief. not- ing bothwell’s press conference, the court held that at the root of his ob- jections were secular concerns about privacy related to government data- bases, rather than a claim with a “nexus to religion.” ontario argued in hutterian brethren that, if it were not allowed to continue requiring its three objective criteria (i.e., membership in a religious organization, a let- ter of support from a religious leader, and scriptural passages substantiat- ing a religious prohibition), it would only be able to catch wrongdoers like bothwell on rare occasions where they, like bothwell, “slipped up” and held a press conference. however, ontario would also be able to distinguish insincere claim- ants like bothwell from sincere claimants under my model. after all, even if bothwell had not called a press conference, he would not have been able to satisfactorily demonstrate any of the three elements of sincere religious belief my proposed exemption requires: he could not have proved mem- bership in a religious community that objected to photographs (prongs a and b) or provided evidence of past or current practice (prong c). the facts of bothwell, therefore, suggest that my proposed modification to on- tario’s criteria is rigorous enough to enable the province to identify insin- cere claimants. since justice iacobucci affirmed in amselem that the claimant held the burden to demonstrate his sincere belief, an applicant who professes sincere belief but who cannot provide objective evidence supporting this belief should not succeed. hutterian brethren, foi, supra note at para . see also bothwell, supra note . ibid at paras , . ibid at para , . hutterian brethren, oai, supra note . the ontario divisional court noted that when bothwell found out about the govern- ment’s exemption policy “he began to search for a congregation to join that had a reli- gious objection to photographs, without success”: bothwell, supra note at para . the ontario divisional court also noted that bothwell had posted photos of himself on his website, and did not object to members of the media taking his photograph: ibid at paras - . supra note at para . ( ) : mcgill law journal ~ revue de droit de mcgill the case law thus far indicates that the objective criteria in my pro- posed exemption—although less rigorous than those in ontario’s current exemption—would still enable the state to identify insincere claimants. d. would the supreme court of canada have accepted this exemption? the final question to consider in this section is whether, if the hutter- ites or an intervener had proposed the exemption i drafted above, chief justice mclachlin would have accepted this alternative and held that al- berta had not met the minimal impairment test, despite her emphasis on the deference she owed alberta in this context. recall that chief justice mclachlin declined to quantify the precise risk exemptions posed within the minimal impairment calculation. as discussed in section ii, she rejected the hutterites’ argument that the risk of an exemption would be low, saying “the claimants argument ... as- sumes some increase in risk and impairment of the government goal may occur, and hence does not assist at the stage of minimal impairment.” my proposed exemption does not seem to alter this minimal impairment reasoning, since any exemption creates more of a risk than a blanket pol- icy that does not allow for an exemption. nevertheless, it is worth considering the nature of the risk this ex- emption would create. elsewhere in her judgment, chief justice mclach- lin elaborated on the manner in which an exemption might undermine alberta’s objectives. for example, she repeatedly noted that even if only sincere religious claimants were granted non-photo licences, this “would not prevent a person from assuming the identity of the licence holder and producing a fake document, which could not be checked in the absence of a photo in the data bank.” this is the sole risk that even a perfectly tai- lored exemption, perfectly applied, could not guard against. let us consider this scenario in more detail. what would happen if a wrongdoer tried to appropriate the identity of a sincere religious claimant in order to obtain a non-photo licence? there are several possible eventu- alities to consider. first, the wrongdoer could attempt to appropriate the identity of a claimant who did not have a licence. however, this situation provides no novel risk given that there are , albertans who do not i am grateful to professor lisa austin for suggesting that i consider this question and for outlining the conclusions that could be drawn in this part. hutterian brethren, supra note at para . ibid. see also ibid at para : “to the extent that licences exist without holder photos in the central photo bank, others can appropriate the identity of the licence holder without detection by the facial recognition software.” comment: alberta v. hutterian brethren of wilson colony have a licence, and thus cannot pose a cause for concern. second, the wrongdoer could attempt to appropriate the identity of a claimant who had been issued a non-photo licence. in this case, the databank would no- tice that an applicant was seeking a licence using the same name, birth date, and other identifying information as a religious claimant who was already in the system, and would recognize that either the licence holder or the applicant was a wrongdoer. the problem here, as chief justice mclachlin noted, is that in the “absence of a photo in the data bank,” it would not be clear which party was the wrongdoer. thus, the central risk posed by my exemption is the risk that an official could not correctly de- termine the wrongdoer in this instance. if we accept the exemption model i proposed above, it is hard to see how this risk would be realized. the objective evidence my exemption re- quires would also provide a basis on which to distinguish wrongdoers from sincere religious believers in this instance. for example, a religious offi- cial who had standing in the community could vouch for the applicant, in the same way that professionals affirm that a passport application cor- rectly identifies an applicant. alternatively, the applicant could provide evidence of long-standing identification with this community, or evidence of commitment to this practice. thus, if the supreme court of canada had addressed alberta’s interpretation of amselem, and interpreted amselem in the manner i outlined above, it is difficult to see on what basis it could have subsequently deferred to alberta’s minimal impairment concerns. conclusion: future subsection (a) challenges and the role of the minimal impairment test although alberta’s concern with how to structure a religious exemp- tion was not recorded in the pages of the supreme court of canada’s deci- sion in hutterian brethren, it provides an important starting point to as- sess the implications of the court’s new approach to subsection (a) claims. as discussed in section ii, above, hutterian brethren marks a sig- nificant methodological departure for the court, because chief justice mclachlin affirmed that the proportionate effects test may often provide the critical framework for assessing freedom of religion claims. this criti- cal passage bears repeating in full: freedom of religion cases may often present this “all or nothing” di- lemma. compromising religious beliefs is something adherents may understandably be unwilling to do. and governments may find it dif- ficult to tailor laws to the myriad ways in which they may trench on different people’s religious beliefs and practices. the result may be ibid at para . ( ) : mcgill law journal ~ revue de droit de mcgill that the justification of a limit on the right falls to be decided not at the point of minimal impairment, which proceeds on the assumption that the state goal is valid, but at the stage of proportionality of ef- fects. what are the potential implications of this methodology for future subsection (a) claims? let us begin by noting that the minimal impair- ment and proportionate effects tests generate distinct products. the minimal impairment test considers whether there are alternative meth- ods of achieving the state’s objective. it thus considers whether there is another way to address each party’s needs, to protect both the integrity of the society as a whole and the functioning of minority groups within it. in contrast, the proportionate effects analysis determines, in the event that no alternative route can be found, whose interests should prevail. grimm’s formulation of the third step of oakes starkly stresses this as- pect: [b]alancing does not save ... the judge from deciding which right or interest shall ultimately prevail in which situation. this means that religious freedom may be on the losing side regardless of the impor- tance of a religious requirement for the believer. there are situa- tions in which the only alternative is adaptation to the secular norm or emigration. the proportionate effects test thus potentially reduces the many al- ternatives presented in the minimal impairment analysis to two stark choices: adaptation or emigration. as grimm acknowledges, this may be appropriate in some very difficult circumstances. however, what are the implications of declaring, as chief justice mclachlin and justice abella did in hutterian brethren, that the proportionate effects test provides the critical framework to assess many of these claims? turning to the proportionate effects test before exhausting the mini- mal impairment inquiry may have the unfortunate consequence of dis- couraging legislative innovation at a time when it is sorely needed. in hutterian brethren the question of whether an exemption to the licence photo requirement was feasible—the classic question of a minimal im- pairment analysis—raised difficult questions as to the scope of the subsec- tion (a) guarantee, such as whether religious beliefs can emerge in isola- tion. the court’s proportionate effects analysis, in contrast, did not re- quire these questions to be considered. thus, if the court turns to the proportionate effects analysis prematurely, it may restrict the legisla- hutterian brethren, supra note at para . supra note at [emphasis added]. for an analysis of why the last step of the oakes test did not provide a suitable frame- work to consider these questions in hutterian brethren, see weinrib, supra note . comment: alberta v. hutterian brethren of wilson colony ture’s ability to craft constitutional alternatives that further the state’s policy objectives. the court’s new methodology may likewise discourage rights claim- ants from considering potential alternatives. as an example, consider the alternative of fingerprinting. in the course of litigation, the hutterites brought up this alternative in fits and starts: they suggested it in oral ar- gument at the alberta court of appeal, and then elected not to pursue it at the supreme court of canada, seemingly after being informed that the act of fingerprinting also involved the taking of a photograph. yet in their application to the court for a rehearing, the hutterites requested that the court consider whether fingerprinting would provide a viable al- ternative to alberta’s photo requirement. although any explanation of the shift in the hutterites’ position remains speculative, their inconsistent positions regarding this alternative brings to mind a potential difficulty in litigating freedom of religion claims. the minimal impairment analysis assumes the religious adherents are able to evaluate precisely how differ- ent alternatives impact their faith. however, since litigation often takes place in response to novel technological or social incursions into the reli- gious sphere, this may not always be the case. the search for alternatives may require religious leaders to learn about the technology at issue as well as to reinterpret foundational guiding texts with these new develop- ments in mind. it is possible that in the course of litigation a communal shift may occur, opening up new options that were not immediately ap- parent. turning to the proportionate effects analysis prematurely may discourage religious claimants, as well as the legislature, from embarking on this difficult process. chief justice mclachlin anchored her assertion of the importance of the proportionate effects test in the difficulty that legislatures have in tai- loring laws to protect the diversity of beliefs found in canada’s multicul- tural society. although it is surely difficult for religious adherents to “compromise” their beliefs, and for governments to “tailor” their laws, the minimal impairment analysis nevertheless calls on all parties to think through the unique challenges of protecting freedom of religion in a lib- eral democracy. if the court had responded to alberta’s and ontario’s concerns pertaining to the nature of a religious-based exemption after amselem, its guidance may have enabled alberta to develop an alterna- alberta v hutterian brethren of wilson colony, [ ] scca no (responding mo- tion record of the appellant at paras - ). alberta v hutterian brethren of wilson colony, [ ] scca no (motion record for order and re-hearing of the hutterian brethren of wilson colony at para ). the court dismissed this application without giving reasons in alberta v hutterian brethren of wilson colony, [ ] scca no at para . ( ) : mcgill law journal ~ revue de droit de mcgill tive in this instance. more critically, the court’s remarks would have en- couraged future parties to these clashes to scrutinize whether the rights of religious claimants truly conflict with the greater good. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ ijdvl_jan_ .pmd indian j dermatol venereol leprol januar y-februar y vol issue therapy in progressive systemic sclerosis. int j dermatol ; : - . generalized hypopigmentationgeneralized hypopigmentationgeneralized hypopigmentationgeneralized hypopigmentationgeneralized hypopigmentation due to imatinib: a fairness boon?due to imatinib: a fairness boon?due to imatinib: a fairness boon?due to imatinib: a fairness boon?due to imatinib: a fairness boon? sir, imatinib (sti , glivec©) is a new selective tyrosine kinase inhibitor that is very useful in the treatment of chronic myelogenous leukemia (cml) and gastrointestinal stromal tumors (gist). more side effects are being reported with its increasing use. we report generalized hypopigmentation with the use of this drug. the majority of cml and gist patients on imatinib mesylate experience some undesirable side effects that involve many organ systems in the body, including the skin. the most common dermatological side effects are periorbital edema and dermatitis. facial edema, pruritus, erythema, dry skin, alopecia, night sweats and photosensitivity reaction are infrequently reported.[ ] over patients of cml or gist are being treated with imatinib at our institute since . for the last one year, during routine outdoor visits, many patients have been reporting that their skin was becoming fairer while on imatinib. we inter viewed patients regarding this side effect; of the patients and their relatives confirmed this experience at varying durations of treatment (median months; range to months). three patients in whom the drug had to be discontinued because of different reasons reported that their skin complexion had reverted to the original one. skin biopsies were performed on seven of these ‘fair’ patients. on h and e staining, even though melanin pigment was seen in all of these biopsy specimens, low melanin content was observed in two of them. in the absence of a pretreatment biopsy, quantification of the melanin pigmentation was not possible. currently, we are doing electron microscopy and tyramine based tyrosinase assay on the skin biopsies performed pre- and post-imatinib treatment for quantification of the melanin deposition in melanosomes. the exact mechanism for this generalized hypopigmentation with imatinib is not known. apart from inhibiting a tyrosine kinase, the bcr-abl oncoprotein, imatinib also inhibits platelet-derived growth factor receptors (pdgfrs) and c-kit receptor tyrosine kinases.[ ] the latter two kinases have important roles in normal pigmentation.[ ] certain hypopigmentary disorders like piebaldism and vitiligo are associated with mutations in the c-kit gene causing alteration in the respective tyrosine kinases.[ ] tsao et al recently reported a similar finding.[ ] however, we found hypopigmentation to be much more common than they did, possibly because it is more apparent in blacks or indians because of their otherwise dark skin complexion. based on our observation, we hypothesize that inhibition of melanocyte c-kit receptor tyrosine kinase by imatinib leads to generalized hypopigmentation. the long-term implications of this observation need to be studied. meanwhile, it will be interesting to see whether topical use of imatinib is possible in the future. atul sharma, amish vora, manisha bhutani department of medical oncology, institute rotary cancer hospital, all india institute of medical sciences, new delhi, india. address for correspondence: dr. atul sharma, assistant professor of medical oncology, department of medical oncology, institute rotary cancer hospital, all india institute of medical sciences, new delhi - , india. e-mail: atul @hotmail.com references . glivec* (imatinib) summary of product characteristics. basel, switzerland: novartis pharma ag; . . buchdunger e, cioffi cl, law n, stover d, ohno jones s, druker bj, et al. abl protein-tyrosine kinase inhibitor sti inhibits in vitro signal transduction mediated by c-kit and platelet- derived growth factor receptors. j pharmacol exp ther ; : - . . luo d, chen h, searles g, jimbow k. coordinated mrna expression of c-kit with tyrosinase and trp- in melanin pigmentation of normal and malignant human melanocytes and transient activation of tyrosinase by kit/ scf-r. melanoma res ; : - . . dippel e, haas n, grabbe j, schadendorf d, hamann k, czarnetzki bm. expression of the c-kit receptor in hypomelanosis: a comparative study between piebaldism, nevus depigmentosus and vitiligo. br j dermatol ; : - . . tsao as, kantarjian h, cortes j, o’brien s, talpaz m. imatinib mesylate causes hypopigmentation in the skin. cancer letters to editor indian j dermatol venereol leprol januar y-februar y vol issue ; : - . amiodarone-induced angioedema:amiodarone-induced angioedema:amiodarone-induced angioedema:amiodarone-induced angioedema:amiodarone-induced angioedema: report of two casesreport of two casesreport of two casesreport of two casesreport of two cases sir, amiodarone, a class ii long-acting anti-arrhythmic, capable of blocking both α and β -adrenoceptors is an iodine-containing highly lipophilic benzofuran derivative.[ , ] adverse reactions are common and are duration-dependent. frequent reactions include nausea and other gi symptoms. ten percent of patients may develop photosensitization (possibly due to phototoxicity) and bluish skin pigmentation, but allergic skin reactions are rare.[ - ] the most serious side- effect related to amiodarone is pulmonary alveolitis and fibrosis.[ ] only a single case of amiodarone-induced angioedema has been reported in the literature.[ ] we came across two unrelated cases of angioedema triggered by the use of amiodarone in the last couple of years. a -year-old lady was admitted with pericardial effusion and af ter a positive pericardial tap antitubercular drugs (atd) were started (isoniazid mg, rifampin mg, ethambutol mg and pyrazinamide mg). she developed atrial fibrillation and an alteration in the ventricular rate. she was put on amiodarone mg twice daily for days and then the dose was tapered to once daily. facial angioedema appeared within a couple of weeks and she complained of anorexia. chest roentgenogram was done and was non-contributory. laboratory evaluation was normal (blood analysis and serum chemistry, stool examination for parasites, ige, thyroid tests (t , t and tsh), and urinalysis). liver function tests yielded a mild rise in liver enzymes like alt, ast and serum alkaline phosphatase; which were , and respectively. she was anicteric. fundoscopy was normal. symptoms of angioedema were unrelated to any physical activity, stress or ingestion of any particular food. she was not an atopic and had no clinical history of nasal polyps, chronic rhinitis, sinusitis, asthma or chronic and chronically relapsing dermatitis. after another four weeks atd were stopped. then they were again started one by one. no improvement was noticed. angioedema persisted. all medicines were stopped and she was put on prednisolone in a reducing dosage. within another four weeks that was also completely tapered off. she improved dramatically and was completely symptom- free within days. atd were started again. no angioedema was noticed then but it appeared within a day when she was given a single dose ( mg) of oral amiodarone. again this symptom improved when amiodarone was taken off. now she is without any symptoms since the last two years. the second patient was a -year-old woman with a history of recurrent episodic facial swelling since one year. this was treated with oral steroids, resulting in clinical improvement. however, facial swelling reappeared after discontinuation of the steroids for which steroids were continued for a year. she presented to our clinic with typical cushingoid habitus. a complete clinical histor y was taken, and she was carefully questioned about past history and recent symptoms. symptoms of angioedema were found to be unrelated to any food ingestion, activity, or stress. she had no clinical history of nasal polyps, chronic rhinitis, sinusitis, or asthma. it was found that she was taking oral corticosteroids and amiodarone mg/day. this last drug had been started by a general practitioner years back for cardiac rhythm abnormalities and had never been discontinued. her chest radiograph was normal and all the laboratory tests (including blood analysis and serum chemistry, stool examination for parasites, ige, thyroid profile and urinalysis) were within range except mild hyperglycemia and low acth level. suprarenal glands were normal. a diagnosis of iatrogenic cushing’s syndrome was made. complete physical examination, ecg and echocardiography were non-contributory. skin patch tests with common allergens were done and found to be negative. in view of the patient’s previous history, the diagnosis of amiodarone-induced angioedema was considered. amiodarone was discontinued, and the symptoms disappeared with the reduction of steroids, which were finally discontinued. in order to confirm that amiodarone was the cause of the patient’s reaction, a double-blind oral challenge with amiodarone was undertaken. within min of receiving the dose ( mg), she began to experience facial flush and facial letters to editor cerebral venous sinus thrombosis, a nonenhanced ct diagnosis? research article cerebral venous sinus thrombosis, a nonenhanced ct diagnosis? ali alsafi, amish lakhani, lalani carlton jones, and kyriakos lobotesis department of neuroradiology, charing cross hospital, imperial college healthcare nhs, london w rf, uk correspondence should be addressed to ali alsafi; ali.alsafi @alumni.imperial.ac.uk received december ; revised april ; accepted april academic editor: andreas h. mahnken copyright © ali alsafi et al. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. purpose. retrospectively evaluate the density of cerebral venous sinuses in nonenhanced head cts (ncts) and correlate these with the presence or absence of a cerebral venous sinus thrombus (cvst). materials and methods. institutional review board approval was obtained and informed consent waived prior to commencing this retrospective study. over a two-year period, all ct venograms (ctvs) performed at our institution were retrieved and the preceding/subsequent ncts evaluated. hounsfield units (hus) of thrombus when present as well as that of normal superior sagittal and sigmoid sinuses were measured. hu of thrombus was compared to that of normal vessels with and without standardisation to the average hu of the internal carotid arteries. results. ctvs were retrieved, with a thrombus. both raw and standardised hu measurements were significantly higher in cvst (𝑝 < . ) compared to normal vessels. both raw and standardised hus are good predictors of cvst. a hu of ≥ and a standardised measurement of≥ . are associated with high probability of cvst on nct. conclusion. cerebral venous sinus hu measurements may help improve sensitivity and specificity of nct for venous sinus thrombosis and avoid potentially unnecessary follow-up examinations. . introduction cerebral venous sinus thrombosis (cvst) is an uncommon cerebrovascular event, accounting for . – % of cases of stroke and affecting in , people [ ]. cvst is a disease of young adults (< years old) predominantly [ ] and is diagnosed based on clinical suspicion with confirmatory neuroimaging [ ]. patients with cvst exhibit a wide range of nonspecific signs and symptoms creating a diagnostic challenge for the clinician and radiologist alike [ ]. headache is the commonest reported symptom in patients with cvst. it is present in ninety percent of cases and reflects raised intracranial pressure [ ]. mri is the noninvasive imaging technique of choice for diagnosing cvst, it is however not universally available in the acute setting [ – ]. ct venography (ctv) has now emerged as an alternative diagnostic test, which is at least as good as mri and in some cases better, with the added advantage of being more readily available [ ]. previously, the gold standard for imaging and diagnosing cvst was digital subtraction cerebral angiography. this is not routinely used and has been superseded by ctv and mrv [ , – ]. as most patients with cvst present with nonspecific symptoms and often cvst is not immediately suspected, patients are likely to have nonenhanced head ct (nct) at presentation. nct is the examination of choice for screening patients with nonspecific neurological presentations in the context of low suspicion of cvst [ ]. nct may be reported as normal in up to two-thirds of patients with venous sinus thrombosis. when abnormal, the findings on nct are often subtle and nonspecific in the early stages and include “hyper-dense” venous sinuses and cerebral swelling. small recent studies have shown that venous sinus hyperdensity is a sensitive sign for cvst [ , ]. venous infarcts and fragmented haemorrhage are late signs [ ]. making a timely diagnosis of cvst is of utmost impor- tance as prompt anticoagulation is thought to prevent thrombus propagation. this in turn prevents ensuing venous hindawi publishing corporation radiology research and practice volume , article id , pages http://dx.doi.org/ . / / http://dx.doi.org/ . / / radiology research and practice table : patients’ characteristics. cvst (𝑛 = ) no cvst (𝑛 = ) median age . [ . – . ] . [ . – . ] sex m = , f = m = , f = haemorrhage same day nct and ctv table : distribution of cvst. vessel number of vessels with thrombus internal jugular vein transverse sinus sigmoid sinus superior sagittal sinus torcula herophili total vessels with cvst ct: computed tomogaphy, nct: non-enhanced ct, ctv: ct venogram, cvst: cerebral venous sinus thrombosis. infarcts and haemorrhage thereby reducing mortality and long-term neurological sequelae [ ]. there remains, how- ever, much debate surrounding optimal management of patients with cvst. . aim in this study we sought to retrospectively evaluate the density (hounsfield units) of cerebral venous sinuses in nonenhanced head ct examinations in patients with and without a cerebral venous sinus thrombus as confirmed on ct venography. we also sought to evaluate whether standardising venous sinus hu measurements to those of the corresponding internal carotid arteries would improve diagnostic accuracy. . materials and methods institutional review board approval was obtained and informed consent waived prior to commencing this retro- spective study. all ctvs performed on adult patients in three teaching hospitals between september and november were retrospectively retrieved. these were reviewed and divided into positive ctvs and negative ones. the preceding or subsequent ncts, within seven days of the ctv, were evaluated. only unique ctvs were included. patients who under- went a neurosurgical procedure in the preceding days were excluded as extra-axial blood products may affect our measurements. follow-up ctvs, where the patient is known to have an old thrombus, were also excluded. we also excluded hypoplastic venous sinuses and heavily calcified icas from the analysis. the site of thrombus was identified from the ctv, where applicable, and the hounsfield units (hus) of the corre- sponding cerebral venous sinus in the nct were measured. in negative studies, hu of the superior sagittal sinus (sss) and both sigmoid sinuses were recorded (figure ). in both groups, hus of both intracranial portions of the internal carotid arteries (icas) as they exit the carotid canal were measured (figure ). the hus of the cerebral venous sinuses with and without thrombus were compared before and after normalisation to the average hu of the corresponding icas (see the following equation): standardised measurment = hu of venous sinus ((hu of right ica+hu of left ica)/ ) . ( ) in our institution patients with cvst are anticoagulated with unfractionated heparin acutely in the absence of contraindi- cations then changed to warfarin once stable and ready for discharge. graphpad prism . for mac os x (graphpad software inc., san diego, ca, usa) was used for statistical analysis. student’s t-test was used to compare hus. fischer’s exact test was used to compare categorical data. receiver operating characteristic (roc) curves were derived and the area under the roc curves (auc) was calculated. % confidence intervals were used to test the hypothesis that the auc is . . microsoft excel for mac v . . (microsoft cor- poration, one microsoft way, redmond, wa) was used to compare the aucs of unpaired roc curves (raw hu versus per patient hu and standardized hu versus per patient hu) using the method described by hanley and mcneil [ ]. medcalc version . . (medcalc software, acacialaan , ostend, belgium), was used to compare the auc of paired roc curves, that is, raw versus standardized hu [ ]. p values< . were considered significant. . . nct and ctv protocols . . . ctv protocol. axial ncts were performed on somatom definition as ct scanners (siemens medical solutions, forchheim, germany) with the following parameters: – mas, kv, and slice collimation of × . mm for the posterior fossa and × . mm for the rest of the brain. ctvs were performed s after administration of intra- venous iodinated contrast at / ml/s. the detector configu- ration was × . mm. all scans were performed from the top of the c lamina to the top of the calvarium, parallel to the floor of the anterior fossa avoiding the eyes. . results ctvs were retrieved. postoperative (postneurosur- gical procedure) studies were excluded. studies were excluded, as there was no preceding or follow-up nct within seven days of the index ctv. five hypoplastic venous sinuses and six heavily calcified icas were excluded from analysis. radiology research and practice (a) (b) (c) figure : images from nct demonstrating sites of hu measurements from (a) superior sagittal sinus at the vertex, (b) sigmoid sinus, and (c) intracranial ica just distal to the carotid canals. h u o f v en ou s s in us thrombus normal sinous thrombus normal sinous r at io o f h u s o f v en ou s s in us /i c a raw cerebral venous sinus hu cerebral venous sinus hu normalised to ica ∗∗∗∗ ∗∗∗∗ figure : comparison of cerebral venous sinus hu before and after standardisation to the icas in patients with and without cvst.∗∗ ∗∗=𝑝< . two-tailed student’s t-test. the remaining unique patients with a median age of had a ctv and nct within seven days of each other with a median of days and a range of [− , . ]. patients had a ctv immediately following their nct. two patients had an nct hours following ctv. no patients underwent an nct less than hours following their ctv. patients had a ctv proven cvst with a median age of . while patients had normal ctvs; they had a median age of . . % ( / ) of those with a cvst an acute intracranial haemorrhage evidence on nct, compared se ns iti vi ty (% ) − specificity (%) roc curve of raw hu. auc = . roc curve of standardised hu. auc = . figure : roc curves comparing the difference between raw hu and standardized measurements. with % ( / ) from the normal ctv group. only . % of patients with a suspected cvst had a proven thrombus on ct venography. there was no significant difference in age, sex, or incidence of intracranial haemorrhage between the two groups with𝑝 values of . (two-tailed student’s t-test), . , and . (fisher’s exact test), respectively. demographic data are shown in table and distribution of cvst is shown in table . radiology research and practice (% ) hu of cerebral venous sinus specificity (%) sensitivity (%) figure : graph demonstrating changes in sensitivity and speci- ficity at different hu cutoffs for standardised hu measurement. the dotted lines represent our proposed hu cutoffs with high negative and positive predictive values for venous sinus thrombosis. . . vessel hu analysis. venous sinuses were analysed, with a cvst and without. the average hu of vessels containing a thrombus was ± . (𝑛 = ) which was significantly higher than that of normal vessels ± . (𝑛 = ) (𝑝< . , two-tailed t-test) (figure ). in an attempt to eliminate hu fluctuations caused by low haemoglobin, haemodilution, dehydration, and so forth, we standardised the venous sinus hus to the average hu of the corresponding icas. the ratio of sinuses containing thrombus was . ± . (𝑛 = ). the ratio in sinuses without thrombus was . ± . (𝑛 = ). this was significantly different (𝑝< . by the two-tailed t-test). . . diagnostic performance of raw versus standardized measurements. roc curves were derived from the raw and standardized hus (figure ). the diagnostic performance of raw venous sinus hus and standardized hus expressed as the area under the curve (auc) were . ( % ci [ . – . ]) and . ( % ci [ . – . ]), respectively. the aucs show that both measurements were good predictors of cvst (𝑝 < . ). standardized hu measurements perform slightly better compared with raw hu albeit not reaching statistical significance (𝑝= . ). . discussion although nonenhanced head ct is thought to have a low sensitivity and specificity for cvst, roland et al. estimate the sensitivity at % [ ]. this may be even lower in normal clinical practice, when cvst is not suspected nor mentioned in the clinical detail. in a recent small study, cerebral venous sinus measure- ments in nct were shown to be of value in detecting cvst [ ]. the study included a small number of controls without venous sinus thrombus and their suggested cutoff hu of is likely to yield a significant number of false positive studies resulting in unnecessary follow-up examinations [ ]. our study has a larger control population showing wide variation in normal venous sinus hu and is likely to be more reflective of general clinical practice. black et al. have previously demonstrated a significant difference in venous sinus densities between patients with and without cvst, although they assumed that patients with a normal nct have no cvst without a confirmatory ctv or mrv [ ]. our results more robustly confirm these findings as all our patients have had a ctv. our results show that when assessing cerebral venous sinuses on nct, hu measurements are a useful objective adjunct to subjective assessment in detecting cvst. black et al. have also demonstrated a correlation between the patient’s hematocrit and venous sinus density [ ]. with that in mind, we standardised venous sinus hu measure- ments to those of the average icas. we postulated that standardization may eliminate some of the physiological variations encountered such as haemodilution, dehydration, and anemia. the area under the roc curves for the standardized hus was . ( % ci [ . – . ]) compared with . ( % ci [ . – . ]) for raw hu measurements although the difference did not reach statistical significance (𝑝 = . ) using delong’s paired measurements roc analysis [ ]. both methods have a strong correlation with the presence or absence of cvst with𝑝< . . black et al. also noted that patients with a cvst often had a hu> , whereas most patients without a cvst had hu < and also showed that a hu/hematocrit ratio of> has a good correlation with cvst. our results, on the other hand, demonstrate that using as a cutoff is likely to result in a significant number of false-negative studies. a recent study demonstrated that the sensitivity of venous sinus hyperdensity on nct may be as high as % with a specificity reaching %. this is also likely to be an overestimate as the reading radiologists had only one task of visually scoring the venous sinuses on ncts [ ]. cerebral venous sinuses with a hu of< are associated with a low probability of cvst (likelihood ration (lr) of . ). a venous sinus with hu of ≥ on the other hand is associated with a high probability of cvst (lr . ). a cerebral venous sinus with a cvs/ica ratio of< is associated with a low probability of cvst (lr . ), while a cvs/ica ratio of ≥ . yields a lr of . and a high probability of cvst (figure ). old thrombus however may also have a low hu and may be missed despite hu measurements. if the patient’s symptoms are long standing, a ctv or mrv may still be warranted. cvst in anaemic patients and those with low haematocrit may also be of low density. low hu measure- ments cannot exclude a thrombus in such cases. the aim of this study is not to provide an alternative to ct or mr venography but to provide the reader with more confidence in their observations on nct. it remains imperative for the reader to analyse the nct images as usual, but with the added benefit of being able to objectively assess radiology research and practice the density of a suspicious venous sinus. although venous sinus hu measurements may be helpful when interpreting a ct, they rely on the reader’s judgment initially as one will not routinely sample every cerebral venous sinus to assess hu. instead, only when there is concern regarding a venous sinus will the reader proceed to hu measurements. the number of positive cases in this study is relatively small potentially affecting the generalizability of our data. this is a function of cvst being a rare diagnosis. a larger study with multicentre collaboration may be more useful to assess the generalizability of our data. the fact that we did not use the raw ct data may intro- duce some errors into our hu measurements. we decided to use this for practical reasons, as the raw data was not readily available, which is the case in usual clinical practice. despite this hu we showed a good correlation between hu measurement with and without standardisation and ctv results making it a clinically useful, practical tool. although, hu measurements correlate well with ctv findings; one cannot extrapolate from our results that this can translate into better pick-up rates of cvst from nct. to assess this, a further study is needed to assess if using hu measurements improves the radiologist’s pick-up rate of cvst. objective hu measurements of suspicious cerebral venous sinus appearances on nct may provide an additional tool in the radiologist’s arsenal to increase reporting confi- dence of the relatively rare clinical entity of cerebral venous sins thrombosis guiding further imaging and management. abbreviations ct: computed tomography nct: nonenhanced ct hu: hounsfield units ica: internal carotid artery cvst: cerebral venous sinus thrombus auc: area under the curve roc: receiver operating characteristic. disclosure partial results were presented at the royal society of medicine’s radiology section: emergency radiology event (november ). conflict of interests the authors declare no conflict of interests. references [ ] m.-g. bousser and j. m. ferro, “cerebral venous thrombosis: an update,” the lancet neurology, vol. , no. , pp. – , . [ ] p. canhão, j. m. ferro, a. g. lindgren, m.-g. bousser, j. stam, and f. barinagarrementeria, “causes and predictors of death in cerebral venous thrombosis,” stroke, vol. , no. , pp. – , . [ ] g. saposnik, f. barinagarrementeria, r. d. brown et al., “diagnosis and management of cerebral venous thrombosis: a statement for healthcare professionals from the american heart association/american stroke association,” stroke, vol. , no. , pp. – , . [ ] c. s. poon, j.-k. chang, a. swarnkar, m. h. johnson, and j. wasenko, “radiologic diagnosis of cerebral venous thrombosis: pictorial review,” the american journal of roentgenology, vol. , no. , supplement, pp. s –s , . [ ] c. s. poon, j.-k. chang, a. swarnkar, m. h. johnson, and j. wasenko, “radiologic diagnosis of cerebral venous thrombosis: pictorial review,” the american journal of roentgenology, vol. , no. , supplement, pp. s –s , . [ ] h. qu and m. yang, “early imaging characteristics of cases of cerebral venous sinus thrombosis,” experimental and therapeutic medicine, vol. , no. , pp. – , . [ ] r. r. ozsvath, s. o. casey, e. s. lustrin, r. a. alberico, a. hassankhani, and m. patel, “cerebral venography: comparison of ct and mr projection venography,” american journal of roentgenology, vol. , no. , pp. – , . [ ] n. janjua, “cerebral angiography and venography for evalu- ation of cerebral venous thrombosis,” journal of the pakistan medical association, vol. , no. , pp. – , . [ ] l. rizzo, s. g. crasto, r. rudà et al., “cerebral venous throm- bosis: role of ct, mri and mra in the emergency setting,” radiologia medica, vol. , no. , pp. – , . [ ] m. wasay and m. azeemuddin, “neuroimaging of cerebral venous thrombosis,” journal of neuroimaging, vol. , no. , pp. – , . [ ] d. f. black, a. e. rad, l. a. gray, n. g. campeau, and d. f. kallmes, “cerebral venous sinus density on noncontrast ct correlates with hematocrit,” the american journal of neurora- diology, vol. , no. , pp. – , . [ ] p.-j. buyck, f. de keyzer, d. vanneste, g. wilms, v. thijs, and p. demaerel, “ct density measurement and h:h ratio are useful in diagnosing acute cerebral venous sinus thrombosis,” american journal of neuroradiology, vol. , no. , pp. – , . [ ] j. coutinho, s. f. de bruijn, g. deveber, and j. stam, “anticoagu- lation for cerebral venous sinus thrombosis,” cochrane database of systematic reviews, no. , article id cd , . [ ] j. a. hanley and b. j. mcneil, “the meaning and use of the area under a receiver operating characteristic (roc) curve,” radiology, vol. , no. , pp. – , . [ ] e. r. delong, d. m. delong, and d. l. clarke-pearson, “comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach,” biometrics, vol. , no. , pp. – , . [ ] t. roland, j. jacobs, a. rappaport, r. vanheste, g. wilms, and p. demaerel, “unenhanced brain ct is useful to decide on further imaging in suspected venous sinus thrombosis,” clinical radiology, vol. , no. , pp. – , . [ ] m. garetier, j. rousset, e. pearson et al., “value of spontaneous hyperdensity of cerebral venous thrombosis on helical ct,” acta radiologica, vol. , no. , pp. – , . infection and immunity, jan. , p. – vol. , no. - / /$ . � doi: . /iai. . . – . copyright © , american society for microbiology. all rights reserved. age-related disparity in functional activities of human group c serum anticapsular antibodies elicited by meningococcal polysaccharide vaccine shannon l. harris, w. james king, wendy ferris, and dan m. granoff * children’s hospital oakland research institute, oakland, california, and department of pediatrics, children’s hospital eastern ontario, university of ottawa, ottawa, canada received july /returned for modification august /accepted september serum bactericidal activity confers protection against meningococcal disease, but it is not known whether vaccine-induced anticapsular antibodies that lack bactericidal activity are protective. we developed an infant rat challenge model using a naturally occurring o-acetylated strain of neisseria meningitidis group c and a strain that was negative for o acetylation (oac). rats to days of age inoculated intraperitoneally (i.p.) with � cfu of either strain developed > � cfu/ml of blood obtained h later. dilutions of preimmu- nization sera given i.p. h before the bacterial challenge had no effect on bacteremia, whereas group c anticapsular antibody in sera from adults immunized with meningococcal polysaccharide vaccine conferred complete or partial (> % decrease in cfu per milliliter of blood) protection against the oac-positive or oac-negative strain, respectively, at antibody doses as low as . �g/rat. anticapsular antibody at doses fivefold higher ( . to . �g/rat) in pooled sera from children immunized at a mean age of . years failed to protect rats, but antibody at the same or fivefold-lower dose in a serum pool from a group of children immunized at years of age gave complete or partial protection. protective activity was observed with some serum pools that lacked detectable complement-mediated bactericidal activity (titers < : ) and correlated with increasing antibody avidity. thus, not only does the magnitude of the group c antibody response to meningococcal polysaccharide vaccine increase with increasing age but there are also age-related affects on antibody functional activity such that higher serum concentrations of vaccine-induced antibody are required for protection of immunized children than for immunized adults. neisseria meningitidis causes serious disease worldwide. in north america and europe, the organism remains the most common cause of bacterial meningitis in children and young adults. meningococci can be subdivided based on distinctive capsular polysaccharides. isolates from five capsular groups— designated a, b, c, y, and w —are responsible for most cases of invasive disease. thirty to forty percent of cases in north america and europe are caused by capsular group c isolates. the group c capsular polysaccharide consists of a ho- mopolymer of �( )-linked sialic acid ( , ). in % of group c strains, the capsular polysaccharide is o acetylation (oac) positive at positions or , while in the remaining %, the capsular polysaccharide is oac negative ( , ). in oac- positive strains, there is nonstoichiometric oac of the capsular polysaccharide (thus, expression of both oac-positive and oac-negative epitopes) ( ). meningococcal polysaccharide vaccines containing oac- positive group c polysaccharide have been available for more than years. these vaccines elicit serum antibodies largely in the absence of t-cell help (so-called thymic-independent anti- gens). in adults, these vaccines elicit high titers of group c complement-mediated serum bactericidal antibodies, and vac- cination has been demonstrated to be highly effective in pre- venting disease ( , , ). however, in infants and young children, the age groups at greatest risk of acquiring meningo- coccal disease, group c polysaccharide is poorly immunogenic ( , , ) and is poorly efficacious ( , ). the presence of serum bactericidal activity remains the se- rologic hallmark of protective immunity against developing invasive meningococcal disease ( , ). although other im- mune mechanisms such as opsonic activity may also contribute to protection against meningococcal disease ( ), it is not known whether sera that contain vaccine-induced anticapsular antibodies but lack complement-mediated bactericidal activity are protective against developing disease. to address this ques- tion, we developed an infant rat model of meningococcal bac- teremia using either an oac-positive or oac-negative group c strain in order to correlate serum anticapsular and bactericidal antibody responses of children and adults vaccinated with me- ningococcal polysaccharide vaccine with the ability of the se- rum antibody to confer passive protection. materials and methods serum samples. we utilized serum samples that had been collected during two previous immunogenicity studies in children (see below), or from laboratory workers with occupational exposure to meningococci and who had been immu- nized with menomune ( �g of a, c, y and w polysaccharide per . -ml dose; aventis pasteur, swiftwater, pa.) by employee health at children’s hos- pital and research center at oakland. the samples were stored frozen at � °c. the adults provided written informed consent, and use of these sera for the present study was approved by the institutional review board (irb) of children’s hospital oakland and research center at oakland. one collection of stored sera from immunized children came from a study performed in in an amish community of genetic factors affecting immune responses to polysaccharide antigens ( ). written informed consent was ob- * corresponding author. mailing address: martin luther king jr. way, oakland, ca . phone: ( ) - . fax: ( ) - . e-mail: dgranoff@chori.org. o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ tained from the parents of all children, and the protocol was approved by the irb of washington university school medicine, st. louis, mo. subjects in this study were given separate intramuscular injections of haemophilus influenzae type b (hib) polysaccharide vaccine ( �g; vaccine prepared at the university of rochester, rochester, n.y.) and meningococcal polysaccharide vaccine ( �g of a and c polysaccharides in . ml; vaccine prepared by connaught labora- tories, swiftwater, pa. [now aventis pasteur]). although the doses used were one-fifth of the usual doses of these vaccines, the lower doses were in the immunogenic range ( , ). blood samples were obtained immediately before vaccination and and months later. the hib anticapsular antibody responses measured in this study have been previously reported ( , ). since only small volumes of sera remained from this study, the sera used in the present study were selected from children, ages . to . years, based on the largest quantities of sera available. the second collection of stored sera were from subjects, ages months to . years, immunized with meningococcal polysaccharide vaccine (menomune; con- naught laboratories) in eastern ontario, canada, as part of control measures instituted by the regional health departments during an outbreak of group c disease in canada. the serum samples were obtained under a protocol approved by the irb of the children’s hospital eastern ontario, ottawa, ontario, can- ada, and written informed consent was obtained from all participants or parents. the group c immunogenicity results in a random sample of participants have been reported ( ). for the present study, pre- and postimmunization sera from children, ages . to . years, were selected randomly. serology. (i) raba. the concentrations of group c anticapsular antibody were measured in the sera by a radioantigen binding assay (raba) modified from that previously described ( ). the group c meningococcal polysaccharide (pro- vided by aventis pasteur) was derivatized with tyramine by a modification of the method of lees et al. ( ) and stored frozen in small portions at � °c. on the week of the assay, an aliquot of antigen was thawed and the tyramine-polysac- charide was radiolabeled with i by the chloramine t method ( ). the average specific activity of the iodinated derivatives was approximately . � cpm/�g of polysaccharide. the raba was performed in . -ml microcentrifuge tubes containing �l of test sera which had been diluted with phosphate-buffered saline containing % (vol/vol) fetal calf serum (pbs-fcs) and an equal volume of radiolabeled polysaccharide that had been diluted in pbs-fcs to contain antigen at � ng/ml (� , cpm/ �l). after incubation at °c for h, �l of % polyethylene glycol in pbs-fcs was added to the reaction mixture, mixed briefly with a vortex, and incubated overnight at °c. the fol- lowing day, the precipitated immunoglobulin and immune complexes containing bound i-labeled group c polysaccharide were harvested by centrifugation performed at °c for min at , � g. the supernatant was removed, and the pellets were washed once in . ml of . % polyethylene glycol , and radioactivity in the precipitate were measured in a gamma counter. antibody concentrations of the test sera were determined by comparison of the percentage of binding of the radiolabeled antigen by different dilutions of the test sample to that of a standard curve of binding at different dilutions of an anti-meningococcal serogroup a/c human reference serum (cdc , obtained from the national institute for biological standards and controls, potters bar, hertfordshire, united kingdom). this reference serum has been assigned a group c anticap- sular antibody concentration of �g/ml ( ). as controls, each assay included a negative serum from an unimmunized adult with no detectable group c anti- capsular antibody and three serum pools prepared from sera of immunized adults with low ( . �g/ml), medium ( . �g/ml), or high ( . �g/ml) anticap- sular antibody concentrations. all test serum samples were assayed in duplicate, and the respective pre- and postimmunization sera were measured in the same assay. for � % of samples, bound polysaccharide in replicate dilutions of individual sera differed by less than %. to calculate interassay variation, the three serum pools described above were assayed on occasions. the respective coefficients of variation for the results from these determinations ranged from . to %. anticapsular antibody avidity was measured in selected sera using a modifi- cation of the raba originally described by griswold et al. for measurement of hib anticapsular antibody avidity ( ). in brief, the total meningococcal group c antibody concentration was measured in each test serum by the raba using radiolabeled group c polysaccharide at � ng/ml of instead of the usual � ng/ml in the final reaction vial. also, incubation of serum and radiolabeled antigen was performed at °c for h, instead of °c for h as done in the usual raba, described above. these changes in the protocol favored binding of both high- and low-avidity antibodies and provided a measurement of total antibody concentration, which in turn was used for calculation of the antibody avidity constant (ka). the raba was then repeated using a -fold-lower dose of radiolabeled antigen ( ng/ml) to assure that the antibody concentration in the reaction vial was in excess, conditions that favor detection of primarily high- avidity antibodies. the fraction of bound antigen was determined at different antibody dilutions. the ka was calculated by the following formula: ka � fraction bound/{( � fraction bound) � [ab]}, where fraction bound is the fraction of antigen bound at a particular antibody dilution, and [ab] is the concentration of group c anticapsular antibody (molecular weight � , ) of the sample dilution tested, determined from the raba using the high concen- tration of radiolabeled antigen ( ). the avidity constant (nanomolar� ) for each sample was calculated from the average ka determined at antibody dilutions giving binding between and %. for each test sample, the ka was measured on at least two to four separate occasions. the assigned ka represented the average ka, omitting outlier results in the calculation. (ii) elisa. immunoglobulin a (iga), igm, and igg anticapsular antibody concentrations in serum were measured by an enzyme-linked immunosorbent assay (elisa). the solid-phase antigen was adipic acid-derivatized polysaccha- ride, prepared from oac-positive meningococcal c polysaccharide as previously described ( ). the blocking buffer in the elisa consisted of pbs-bsa (pbs with % [wt/vol] bovine serum albumin [bsa] [radioimmunoassay reagent grade; sigma, st. louis, mo.]). alkaline phosphatase-conjugated goat antibody specific for human iga, igm (caltag laboratories, burlingame, calif.), or igg (southern biotechnology associates, inc., birmingham, ala.) antibodies were used as the secondary antibody detecting reagents. the elisa employed replicate microti- ter plates in which binding was measured using sera diluted with buffer alone or sera diluted with buffer containing soluble meningococcal group c polysaccha- ride ( �g/ml) as an inhibitor. the antibody concentrations for the test sera were obtained by subtracting the respective absorbance values obtained with the sera diluted with the polysaccharide inhibitor from the corresponding values obtained with the samples diluted with buffer alone. the isotype-specific anti- body concentrations in micrograms per milliliter were assigned to the test sera based on comparison to binding curves obtained with different dilutions of the cdc reference serum, which has been assigned igg, igm, and iga group c anticapsular antibody concentrations of . , . , and . �g/ml, respectively ( ). (iii) inhibition of antibody binding by oac-negative group c polysaccharide. for determination of the proportion of group c anticapsular antibody that was specific for epitopes expressed by oac-negative polysaccharide, the elisa de- scribed above was modified such that dilutions of sera were added to four replicate microtiter plates. the solid-phase antigen was an adipic dihydrazide derivative of the oac-positive polysaccharide ( ) that, because of nonstoichio- metric oac of the capsular polysaccharide, expressed both oac-positive and oac-negative epitopes. the presence of both structures was confirmed by nu- clear magnetic resonance spectroscopy performed by neil ravenscroft, univer- sity of cape town, rondebosch, south africa. in one plate, the serum samples were diluted with buffer alone. in the remaining three plates, the sera were diluted with an excess of soluble oac-positive meningococcal group c or oac- negative meningococcal group c or, as a negative control, meningococcal group y polysaccharides, respectively. the concentrations of inhibitor used in different experiments ranged from to �g/ml ( - to -fold higher than that needed for � % inhibition of specific binding). the results of a typical inhibition experiment are shown in fig. . positive controls in the assay include two murine monoclonal antibodies (mabs), mabs and , with known preferential binding to oac-negative or oac-positive polysaccharide, respectively ( ). (an- tibodies were kindly provided by k. stein, u.s. food and drug administration, bethesda, md. [now at macrogenics, inc., rockville, md.]). these mabs were assayed in parallel with the human sera except that the secondary antibody used to detect binding of the mabs was specific for mouse ig (heavy and light chain; southern biotech) instead of human igg. binding of mab was typically inhibited by � % in the presence of soluble oac-negative or oac-positive polysaccharide, with the inhibition by the oac-positive polysaccharide reflecting nonstoichiometric oac. binding of mab showed significant inhibition only in the presence of oac-positive polysaccharide. as a second positive control, we measured inhibition of binding of a postimmunization serum (serum c), which was obtained from an adult immunized with a group c polysaccharide-tetanus toxoid conjugate prepared from de-o-acylated group c polysaccharide by baxter health care (columbia, md.) ( ) (serum kindly provided by ray borrow, meningococcal reference laboratory, manchester, united kingdom). with this serum, we typically observed � % inhibition with oac-negative or oac-posi- tive polysaccharide (fig. ), a result consistent with nearly all of the group c antibodies in this serum being specific for oac-negative epitopes. for calculation of percent inhibition in test samples, absorbance values in the plate containing serum samples diluted with the oac-positive meningococcal group c polysac- charide inhibitor were considered to represent nonspecific background binding and, therefore, were subtracted as background binding from the absorbance harris et al. infect. immun. o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ values of the respective serum dilutions incubated in the plate with no inhibitor. the resulting specific total absorbance reflected antibody populations specific for both oac-positive and -negative group c polysaccharide. the percent inhibition by the oac-negative meningococcal group c polysaccharide or the negative control group y polysaccharide inhibitor was calculated from the equation {[(to- tal specific absorbance) � (inhibited absorbance)]/(total specific absorbance)} � , using serum dilutions where the total absorbance (optical density) was approximately . to . . the percent inhibition was calculated from the mean results from a minimum of two to three experiments performed on separate days. the negative control meningococcal group y polysaccharide gave no significant inhibition of antibody binding to group c polysaccharide (fig. ). (iv) complement-dependent bactericidal antibody activity. bactericidal activ- ity was measured as previously described ( ) using log-phase test bacteria grown for approximately to . h in mueller-hinton broth that had been supple- mented with . % glucose (wt/vol). two group c meningococcal strains were tested— , which is naturally o-acetylated (oac positive), and , which is naturally not o-acetylated (oac negative). these clinical isolates were provided by trudy v. murphy (now at centers for disease control and prevention [cdc], atlanta, ga.) and were collected during prospective surveillance of invasive meningococcal disease in dallas county, tex. ( ). the two isolates were selected based on resistance to bactericidal activity of normal human serum and for their ability to cause bacteremia in infant rats (see below). the oac status of these strains was determined by relative antibody binding as measured by a whole-bacterial-cell elisa ( ) using the oac-positive specific murine mab . , the oac-negative specific mab . , and the backbone-specific mab c / . ( ). the lack of oac of the capsular polysaccharide purified from strain also was confirmed by nuclear magnetic resonance spectroscopy (kindly performed by neil ravenscroft). for measurement of bactericidal activ- ity, all test sera were heated at °c for min to inactivate intrinsic complement activity. the extrinsic complement source was serum from a healthy adult with no detectable anticapsular antibody to group c polysaccharide as tested by elisa and no detectable intrinsic bactericidal activity against the target strains when tested at a final serum concentration of or % (twofold higher than the serum concentration used for complement to test bactericidal activity in the test sera). passive protection against bacteremia in animal challenge model. the ability of group c anticapsular antibodies to confer passive protection against n. men- ingitidis group c bacteremia was tested in infant rats challenged intraperitoneally (i.p.). in development of the animal challenge model, we tested group c clinical isolates for their ability to cause bacteremia in infant rats; these strains had been passaged in the laboratory fewer than three times. of these, strains and gave the most consistent bacteremia with challenge doses as low as cfu/rat. subsequently, each of these strains was serially passaged three times in infant rats and stored frozen at � °c in skim milk. bacterial challenge of the rats was performed as previously described for group b strains ( ). in brief, - to -day-old pups from litters of outbred wistar rats (charles river, raleigh, n.c.) were randomly redistributed to the nursing mothers. on the day before challenge, freshly thawed bacteria were inoculated onto chocolate agar and grown overnight at °c in % co . on the morning of the challenge, several colonies were inoculated into mueller-hinton broth supplemented with . % glucose (wt/vol). after inoculation of the bacteria to a starting a of � . , the test organism was grown for approximately h with shaking at °c in % co to an a of � . . after washing the bacteria twice in pbs-bsa, the bacterial suspension was diluted in pbs-bsa to contain approximately , cfu/ml. at time zero, groups of animals were treated i.p. with �l of different dilutions of test or control sera. two hours later the animals were challenged i.p. with �l of bacteria ( to , cfu/rat). heparinized blood samples were obtained by cardiac puncture h later, and quantitation of bacteremia was performed by directly plating aliquots of , , and �l of a : dilution (i.e., �l) of blood onto chocolate agar. the number of cfu per milliliter of blood was determined after overnight incubation of the plates at °c in % co . for calculation of geometric mean number of cfu per milliliter of blood, animals with sterile cultures were assigned a value of cfu/ml, and animals whose samples produced a lawn of bacteria on culture plates streaked with �l of blood were assumed to have more than , cfu/ml of blood and were assigned a value of cfu/ml. statistical analysis. for calculation of geometric means, the bactericidal titers and anticapsular antibody concentrations of each subject were logarithmically transformed (base ). titers or concentrations below the lower limit of detec- tion in the assay were assigned as half of the lower limit. for each age group, the mean of the log values of the antibody titers or concentrations, and the associ- ated standard error (se) of the means are reported along with the respective geometric means. in the passive-protection experiments, the proportion of ani- mals with bacteremia and the respective geometric means of the numbers of cfu per milliliter of blood were calculated for each group of animals. the fig. . binding to solid-phase adipic dihydrazide derivative of oac-positive polysaccharide, which is known to express both oac-positive and oac-negative epitopes. sera were tested in the presence of a -�g/ml concentration of soluble oac-positive (gray bars) or oac-negative (striped bars) meningococcal group c polysaccharide or, as a negative control, group y polysaccharide (open bars) (solid bars, no polysaccharide). the control mab is known to recognize an epitope on oac-negative polysaccharide, but its binding is inhibited by both oac-negative and -positive group c polysaccharides because of nonstoichiometric oac. mab preferentially binds oac-positive polysaccharide. the inhibition observed in the elisa is consistent with this binding specificity. human serum c from an adult immunized with the baxter group c conjugate vaccine prepared from oac-negative polysaccharide is completely inhibited by oac-negative polysaccharide. vol. , meningococcal passive protection o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ significance of differences in the respective geometric means of the number of cfu per milliliter of blood from animals treated with different concentrations of group c anticapsular antibody were calculated using a two-tailed student t test (excel software). for the purpose of determining the relationship between an- tibody avidity and passive protective activity, for each serum sample or serum pool, we determined the dose of antibody per infant rat required for a -log decrease in the number of cfu per milliliter of blood (i.e., % decrease), compared to that of control animals pretreated with preimmune serum or pbs. for the regression analyses, protective doses less than . �g/rat were assigned a value of . �g/rat and protective doses greater than . or . �g/rat (the highest doses tested for passive protection against the oac-negative and -posi- tive strains, respectively) were assigned a value of . �g/rat. the correlation coefficient, r, and the r values between the ka of each serum or serum pool and the respective log of the protective antibody dose were calculated by analysis of variance regression (polynomial degree � ) (gb-stat, version . ; ppc, dynamic microsystem, silver spring, md.). results age-related anticapsular antibody response. table sum- marizes the group c anticapsular antibody concentrations measured in serum samples obtained immediately before im- munization and to months later. the results are stratified by the age of the subject at the time of immunization and the study in which the samples were obtained (groups and were from the canadian study, and groups and were from the study done in an amish community in missouri). as expected ( , ), the magnitude of the geometric mean serum anticap- sular antibody concentrations increased with increasing age, being lowest in the -year-olds, intermediate in the -year-olds, and highest in - to -year-olds and adults. for all groups tested, igg anticapsular antibody responses predominated with lesser concentrations of igm anticapsular antibody. table summarizes the serum bactericidal antibody re- sponses measured against two group c test strains: , which expresses o-acetylated capsular polysaccharide (oac posi- tive), and , a strain that expresses capsular polysaccharide which is not o acetylated (oac negative). the adults and the - to -year-old children showed significant increases in the geometric mean bactericidal titers after immunization. the respective postimmunization geometric mean titers of antibod- ies against the oac-negative strain were approximately two- fold higher than those measured with the oac-positive strain. three-quarters or more of the immunized adults or - to -year-old children had postimmunization bactericidal anti- body titers of : or greater when tested against either group c strain. when measured with human complement, this thresh- old correlates with protection against developing meningococ- cal disease ( , , ). table . group c serum antibody responses of adults and children immunized with meningococcal polysaccharide vaccine groupa no. of subjects tested mean age (yr) (range) mean log antibody concn (�g/ml) 	 se (geometric mean) as determined by: raba with: elisa with postimmunization sera and: preimmunization sera postimmunization sera igm igg adults ( – ) � . 	 . ( . ) . 	 . b ( . ) . 	 . ( . ) . 	 . ( . ) children group . ( . – . ) � . 	 . ( . ) . 	 . b,c ( . ) � . 	 . ( . ) . 	 . ( . ) group . ( . – . ) � . 	 . ( . ) . 	 . c,d ( . ) � . 	 . ( . ) . 	 . ( . ) group . ( . – . ) � . 	 . ( . ) . 	 . e ( . ) ndf nd group . ( . – . ) � . 	 . ( . ) . 	 . d,e ( . ) nd nd a groups and were children immunized in a study in canada ( ). groups and were amish children immunized in a study in the united states ( ). preimmunization sera were obtained immediately before vaccination. post-immunization sera were obtained to months later. igm, igg and bactericidal antibody responses were not performed on individual sera from groups and because of insufficient quantities of sera (see methods). b the geometric mean antibody concentrations of the adults and children in group are not significantly different. c the geometric mean antibody concentrations of groups and are significantly different (p . ). d the geometric mean antibody concentrations groups and are significantly different (p � ). e the geometric mean antibody concentrations of groups and are significantly different (p � ). f nd, not done because of insufficient quantities of sera. table . group c serum bactericidal titers of adults and children immunized with meningococcal polysaccharide vaccine groupa no. of subjects tested mean age (yr) (range) reciprocal titer of bactericidal antibody in serum strain (oac positive) strain (oac negative) mean log 	 se (geometric mean) post % � c mean log 	 se (geometric mean) post % � preimmunization postimmunization preimmunization postimmunization adults ( – ) . 	 . ( . ) . 	 . ( . ) . 	 . ( . ) . 	 . ( . ) children group . ( . – . ) . 	 . ( . ) . 	 . ( . ) . 	 . ( . ) . 	 . ( . ) group b . ( . – . ) . 	 . ( . ) . 	 . ( . ) . 	 . ( . ) . 	 . ( . ) a groups and were children immunized in a study in canada ( ). bactericidal titers were not obtained for individual sera from children in groups and because of insufficient quantities. b children in group had lower geometric mean bactericidal antibody responses than did the adults (p . ) or children in group (p . ) for both the oac-positive and oac-negative strains. c percentage of individuals with postimmune bactericidal titers of � : . harris et al. infect. immun. o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ the bactericidal antibody responses of the children immu- nized at years of age were much lower than those of the older children or adults. only half of -year-olds developed titers of : or more when measured against the oac-negative test strain, and only % developed titers of : or more against the oac-positive strain. the low bactericidal antibody re- sponses of the immunized -year-olds were unexpected since most of the children showed high anticapsular antibody re- sponses to vaccination as measured by the raba and/or igg elisa (table ), which used oac-positive group c polysac- charide as the target antigen. the low or absent bactericidal responses despite the presence of relatively high serum anti- capsular antibody concentrations imply that the anticapsular antibodies elicited by vaccination in these young children have poorer functional activity than those elicited by vaccination of older children or adults. to investigate the protective activity of these antibodies in an animal challenge model, we pooled the respective pre- and postimmunization sera of children immunized at different ages (pre- and postimmunization pools , , , and from groups , , , and , respectively, shown in table ). table summarizes the antibody concentrations measured in these serum pools and the results of characterization of the group c anticapsular antibody concentrations, isotype distributions, avidity, and the percentage of the anticapsular antibody response directed against the oac-negative epitopes. for comparison, the cor- responding results are shown for pre- and postimmunization serum samples from three representative control adults given meningococcal polysaccharide vaccine: subject , a low group c responder to vaccination, and subjects and , who were chosen as representative of high responders to vaccination. with one exception, the antibody concentrations measured in the serum pools were similar to the respective geometric mean antibody concentrations of the individual sera that made up the pools. the exception was the postvaccination serum pool from children immunized at to years of age (postim- munization pool ), which had total and igg anticapsular an- tibody concentrations of and �g/ml, respectively. the corresponding geometric means of the antibody concentra- tions of this group were lower, . and . �g/ml. these discrepant results were reproducible upon repeated assays. each of the serum pools was prepared from equal volumes of sera from children in groups to , respectively (table ). sera from two individuals in group had antibody concentrations more than -fold higher than the geometric mean antibody concentration of the group. inclusion of these two outlier sera in the pool may have accounted for the discrepancy between the antibody concentration measured in the pool and the re- spective geometric mean antibody concentration of the group. all of the children and adults in this study were immunized with vaccine prepared from oac-positive group c polysaccha- ride. nevertheless, with the exception of subject , % or more of the igg group c anticapsular antibody binding in the elisa was inhibited by soluble oac-negative polysaccharide (fig. and table ). previous work by arakere and frasch also demonstrated that a substantial amount of the serum anticap- sular antibody in individuals immunized with a meningococcal polysaccharide vaccine was directed towards epitopes on the oac-negative group c polysaccharide ( ). table summarizes the bactericidal antibody titers of the pre- and postimmunization serum pools and the corresponding titers of the three representative pre- and postimmunization sera from the adults. when tested against either group c strain (oac-positive) or (oac-negative), all preimmuni- zation sera had bactericidal titers of : the postimmuniza- tion serum from subject , an adult known to be a low anti- capsular antibody responder (table ), also lacked bactericidal activity. the postimmunization sera from the two high-re- sponder adults (subjects and ), and the postimmunization serum pool from the children immunized at to years of age, had high titers of bactericidal activity against both group c test strains (table ). the postimmunization serum antibody con- centrations required for % killing of the oac-positive strain (bc ) ranged from . to . �g/ml, which were approx- table . group c anticapsular antibody concentrations of selected pre- and postimmunization sera or serum pools subject or pool no. (mean age [yr]) total anticapsular antibody by raba anticapsular antibody by elisa (postimmunization serum) concn (�g/ml) in serum mean ka 	 se of postimmunization serum (nm� ) concn (�g/ml) of: % igg inhibited by oac-negative polysaccharidebpreimmunization postimmunization igm iga igg subjects (adult) . . . ( . , . )c . . . ndd (adult) . . . 	 . . . . (adult) . . . 	 . . . . poolsa ( . ) . . ( . , . )c . . ( . ) . . . 	 . c . . . ( . ) nd . . 	 . e,f . . . ( . ) . . . 	 . f . . . a equal volumes of sera from each individual in groups to described in table were pooled to make pools to , respectively. b in an elisa in the presence of soluble oac-negative group c polysacharide ( to �g/ml). the solid-phase antigen was adipic-dihydrazide-derivatized oac-positive group c polysaccharide, which is known to express both oac-positive and oac-negative epitopes (see text). c se not calculated since only two independent measurements were performed. the values from each determination are given in parentheses. d nd, not done because of insufficient concentrations of anticapsular antibody. e p . (for comparison of mean avidities of pools and ). f p . (for comparison of mean avidities of pools and ). vol. , meningococcal passive protection o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ imately - to -fold higher than the respective concentrations required for killing of the oac-negative strain (table ). note, for calculating the bc concentrations against the oac- negative strains, we only considered the fraction of the serum anticapsular antibody concentrations that was specific for oac-negative epitopes (i.e., inhibited in the elisa by soluble oac-negative polysaccharide). the three postimmunization serum pools from children im- munized before years of age had no detectable bactericidal activity when tested against the oac-positive strain (ta- ble ), and only pool , from the -year-olds immunized in canada, was bactericidal for oac-negative strain (titer � : ). the respective concentrations of anticapsular antibody required for % bacteriolysis in the postimmunization serum pools from children immunized before years of age were two- to sevenfold higher than those for the serum pool from - to -year-olds, or for the individual sera from the two control adult responders. antibody avidity. one possible explanation for low bacteri- cidal activity of the serum pools from the children is low anti- body avidity. figure shows the ability of different concentra- tions of anticapsular antibodies in postimmunization serum pools (mean age of children, . years) and (mean age of children, . years) to bind to radiolabeled group c polysac- charide. for comparison is shown the corresponding binding of group c antibodies in the reference cdc pool prepared fig. . binding of anticapsular antibodies (ab) to radiolabeled group c polysaccharide. (a) when a high antigen dose is used both high- and low-avidity antibodies are detected. (b) when a low antigen dose is used, detection of higher-avidity antibodies is favored. it takes four- to fivefold-higher concentrations of anticapsular antibody from pool , a low-avidity pool, to give equivalent binding to that of antibodies in the cdc reference pool. the dose-response binding of pool in the low-antigen assay is between that of pool and that of the cdc reference pool, and pool is calculated to have an intermediate avidity ka between that of pool and that of the cdc pool (table ). table . serum bactericidal antibody responses of selected adults and children to meningococcal vaccinationa subject or pool no. (mean age [yr]) strain (oac positive) strain (oac negative) reciprocal titer in preimmunization sera postimmunization sera reciprocal titer of preimmunization serab postimmunization sera /titerb bc c /titer bc c subjects (adult) indetd indet (adult) . . (adult) . . pools ( . ) . . ( . ) � . . ( . ) nde � . nd � . ( . ) indet indet a for description of pools, see footnote a of table . b dilution of sera giving % killing in the presence of human complement. note that all sera with titers of : were also negative (titer, : ) when retested with rat complement. c bc for strain was calculated from the total antibody concentration measured by the raba divided by the reciprocal bactericidal titer. a similar calculation was done to calculate the bc of strain except that the total anticapsular antibody concentration was adjusted to take into consideration the percentage inhibition by soluble oac-negative polysaccharide (i.e., the portion specific for oac-negative polysaccharide equals the total antibody concentration measured by raba multiplied by the percent inhibited by oac-negative polysaccharide in the elisa (see fig. ). d indet, indeterminate because of low concentrations of anticapsular antibody. e nd, not done. harris et al. infect. immun. o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ from sera of adults given meningococcal polysaccharide vac- cine ( ). when a high radioantigen concentration is used in the assay (fig. a), which detects both low- and high-avidity antibodies, the respective binding curves of the three pools overlap each other. in a separate experiment not shown, there was similar overlap of the respective binding curves of the cdc reference pool and pools and from the immunized children from canada (mean ages of . and . years, respec- tively). in contrast, when a -fold-lower concentration of ra- dioantigen was used in the raba (fig. b), conditions which favor detection of high-avidity antibodies ( ), it took approx- imately four- to fivefold-higher concentrations of anticapsular antibody from pool to give equivalent binding to that of antibodies in the reference cdc pool. with the low-antigen assay, binding of pool was intermediate between that of pool and that of the cdc pool, and binding of pool was indistinguishable from that of the cdc pool (fig. b). the ka 	 se calculated from these and other raba data (not shown) were 	 . nm� , 	 . nm� , 	 nm� , nm� (se not calculated because the sample was assayed only twice, with values of and nm� ), and 	 nm� , for pools , , , and (table ) and the reference cdc serum pool, respectively. passive protective activity in infant rats. although the pres- ence of serum bactericidal antibody correlates with protection against developing meningococcal disease, absence of bacteri- cidal activity does not necessarily imply susceptibility to dis- ease. to investigate this question in the pools of serum from the children immunized in this study, we adapted the infant-rat challenge model previously used for group b meningococci ( ) to investigate passive protection against group c bactere- mia. as negative controls in these experiments, a total of infant rats were pretreated i.p. at time zero with �l of : or : dilutions of sera obtained before immunization from four adults. of the animals, were challenged i.p. with strain (oac positive), and were challenged i.p. with strain (oac negative). for both strains the challenge bacterial dose used was between and , cfu/rat. all animals pretreated with preimmunization sera had bacteremia in blood obtained h after the bacterial challenge (typically, , to � , cfu per ml of blood). table summarizes the results of pretreatment of rats with pre- or postimmunization sera from the three representative adults on the development of bacteremia h after challenge with group c strain (experiment a) or (experiment b). none of the rats treated with . �g of antibody in postimmunization serum from subject , the control low re- sponder adult with a bactericidal titer of : , was protected against challenge by either strain (table ). however, the same dose of antibody in postimmunization sera from adult subjects or was highly protective against strain , and a dose of . �g per rat gave partial protection against this strain. these data imply that there are important qualitative differ- ences in anticapsular antibodies that may influence protective activity. table shows the result of passive-protection experiments performed with serum pools from immunized children. pool , which was prepared from serum samples of children immu- nized at a mean age of . years and which was highly bacte- ricidal in vitro, was not tested for protection in the animal model since the purpose of these experiments was to deter- mine whether sera from immunized younger children that lacked bactericidal activity could confer protection. experi- ment a compared protection conferred by postimmunization table . passive protection of infant rats challenged with group c n. meningitidis with adult sera subject no. and serumb dilution strain (oac positive) (expt a)a strain (oac negative) (expt b)a antibody dose (�g/rat)c no. of animals positive for bacteremia/total no. tested cfu/ml (geometric mean [ ]) antibody dose (�g/rat) no. of animals positive for bacteremia/total no. tested cfu/ml (geometric mean [ ]) pre : . / � � . / post : . / � � . / � pre : . / � � . / � postd : . / . . / . post : . / . . / post : . / . . / pre : . / � � . / post : . / . . / . post : . / . . / post : . / . . / � a experiments a and b were done on separate days. results of experiments a, b, a, and b are shown in table . b pre, preimmunization; post, postimmunization. c the same respective dilutions of sera from each subject were tested against both strains. the antibody dose per rat for strain was calculated from the total antibody concentration measured by the raba divided by the dilution. the antibody dose per rat for the oac-negative strain was adjusted to take into consideration the percentage of antibody inhibited by soluble oac-negative polysaccharide (see fig. and table , footnote c). d for subject , serum from month postimmunization ( . �g/ml) was used for the passive-protection experiment with strain . for the passive protection experiment with strain , the serum from months postimmunization was used because of insufficient quantity of the sample from month postimmunization. because the -month sample had a slightly higher antibody concentration than the -month sample ( . �g/ml), the -month serum was diluted : , : , and : to achieve doses of . , . , and . �g of total anticapsular antibody/rat, respectively. vol. , meningococcal passive protection o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ serum pools obtained from amish children immunized at mean ages of . (pool ) and . (pool ) years against chal- lenge with strain . experiment b tested the same serum pools in rats challenged with strain . experiment a com- pared protection conferred by postimmunization pools ob- tained from two groups of children immunized at mean ages of . years (pool , u.s. amish children) and . years (pool , canadian children) against challenge with strain . exper- iment b tested the same serum pools in rats challenged with strain . in experiments a and a, all rats pretreated with preimmunization sera from the control adult or pools made from preimmunization sera of children or with pbs had bacteremia following challenge with strain (� , cfu/ml h after challenge). similarly, all animals given these preimmunization sera and challenged with strain in experiments b and b developed bacteremia (geometric mean, � , cfu/ml at h after challenge). in both experiments a and a, a dose per rat of . �g of postim- munization serum from the adult positive control completely protected of rats challenged with strain , and in experiments b and b, a dose per rat of . �g of the positive adult control serum completely protected of rats chal- lenged with strain . the respective results with the positive and negative control sera in these experiments were similar to those of experiment a and b (compare table and table ). in experiment a, doses per rat of . and . �g of anti- capsular antibody in postimmunization serum pool , obtained from amish children immunized at an average age of . years, were completely or partially protective (� % decrease in the number of cfu per milliliter of blood) against strain . in contrast, comparable doses of antibody in pool from amish children immunized at a mean age of . years gave no pro- table . passive protection of infant rats with pediatric serum poolsa expt no. subject or pool no. (age [yr]) and serum sampleb dilution strain (oac positive) (expt a) strain (oac negative) (expt b) antibody dose (�g/rat)c no. of animals positive for bacteremia/ total no. tested cfu/ml (geometric mean [ ]) ab antibody dose (�g/rat) no. of animals positive for bacteremia/ total no. tested cfu/ml (geometric mean [ ]) subject (adult) pre : . / � . / d � post : . / . . / . post : . / . . / d . post : . / . . / � pool ( . ) pre : . / � � . / � f post : . . / � . / f post : . / d � . / � pool ( . ) post : . / . nde nd nd post : . / . . / c . post : . / . . / negative control pbs / � / � g subject (adult) post : . / . . / . post : . / . . / pool ( . ) post : . / . h . / g post : . / . / � pool ( . ) pre : . / � � . / � g post : . / . h . / . post : . / . / � a experiments a and b and experiments a and b were done on separate days. for description of pools, see footnote a of table . b pre, preimmunization; post, postimmunization. c antibody doses were calculated as described in footnotes b and c to table . d one rat in each of these groups was dead h after bacterial challenge. these animals were presumed to be positive for bacteremia. for calculation of geometric mean cfu per milliliter, the dead animals were assigned values of , , cfu/ml of blood. e nd, not done. f p � . (for comparison of geometric mean cfu per milliliter after treatment with postimmunization serum pool to that of animals treated with preimmunization serum). g p � . (for comparison of geometric mean cfu per milliliter after treatment with postimmunization serum pool to that of the combined group of animals treated with either preimmunization serum or pbs). h p . (for comparison of geometric mean cfu per milliliter after treatment with antibody ( . �g/rat) from postimmunization serum pool to that of animals treated with antibody ( . �g/rat) from postimmunization serum pool ). harris et al. infect. immun. o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ tection against this strain (table ). a similar trend was ob- served in animals pretreated with antibody in experiment b and challenged with strain (partial protection with a dose per rat of . �g of serum anticapsular antibody from the . -year-olds but no significant protection at a dose per rat of . �g of serum antibody from the . -year-olds. the postim- munization control serum antibody from adult completely or partially protected against bacteremia caused by either strain at a dose of . to . �g per rat. thus, there was an inverse correlation between age of immunization and dose of serum anticapsular antibody required to confer protection in this model. also, some sera that lack bactericidal activity can be highly protective in vivo. for example, postimmunization serum pools and had no detectable bactericidal activity against strain (table ) but conferred protection against this strain in the animal model (table ). postimmunization serum pools and also had no detectable bactericidal activity when tested with infant or adult rat serum as sources of com- plement; thus, passive protection by these serum pools may have resulted from a mechanism other than bactericidal activ- ity such as opsonization. in experiments a and b (table ), doses per rat of . to . �g of anticapsular antibody in postimmunization serum pool from canadian children immunized at an average age of . years were completely protective against strain and partially protective against strain . this serum pool lacked bactericidal activity against strain and had a titer of an- tibody against strain of : (table ). similar doses of antibody from pool , obtained from amish children with a mean age of . years, were partially protective against strain but in this experiment gave minimal protection against strain . similar doses per rat of antibody from serum of the control immunized adult completely protected against strain and partially protected against strain . relationship between antibody avidity and passive protec- tive activity in infant rats. fig. shows the relationship be- tween the ka calculated for each postimmunization control serum or serum pool tested for passive protection and the protective dose of anticapsular antibody determined for each of the passive-protection experiments. the protective antibody dose was defined as the dose per rat that gave a -log de- crease in the number of cfu per milliliter, compared to that of control animals pretreated with preimmunization serum or pbs. data from one sample (adult with a ka of . nm� ) were excluded from the analyses since the highest antibody dose tested for protection of that serum was . �g/rat (table ) and, thus, the protective dose could not be defined. for both challenge strains, the higher the avidity of the serum anticap- sular antibody, the lower the antibody dose required for con- ferring protective activity. the r value calculated for the oac- positive test strain (fig. a) was . (p . ), and that for the oac-negative test strain (panel b) was . (p . ). excluding the one outlier (see legend to fig. a), the r value calculated for the oac-positive test strain was . (p . ). discussion the relationship between serum group c anticapsular anti- body concentrations and protection from developing meningo- coccal disease is unknown. young children immunized with meningococcal polysaccharide vaccine are reported to develop high serum group c anticapsular antibody responses as mea- sured by elisa ( , ) or raba ( , ). typically, how- ever, there is little or no detectable bactericidal activity in this age group ( , , ), particularly if measured with human complement ( ). our serologic results are consistent with these earlier observations. children immunized at to years of age showed - to -fold increases in anticapsular antibody concentrations as measured by the raba, comparing the re- spective geometric mean concentrations measured in sera ob- tained to months after vaccination to those in preimmuni- fig. . relationship between antibody (ab) avidity and dose of antibody per rat required for passive protective activity (protective activity was defined as a -log decrease in the number of cfu per milliliter, compared to that of control animals pretreated with preimmunization serum or pbs). (a) challenge by group c oac-positive strain . the point shown as an open square was considered an outlier and was excluded for purposes of the curve fit. (b) challenge by group c oac-negative strain . the respective r values calculated by analysis of variance nonlinear regression were . for strain (p . ) and . for strain (p . ). excluding the outlier for strain , the r value was . (p . ). vol. , meningococcal passive protection o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ zation sera (table ). however, the complement-mediated bactericidal antibody responses were poor (tables and ), particularly when measured against the oac-positive strain ; only % of the -year-olds developed titers of antibody to this strain of : or more after vaccination, a threshold predictive of protection against developing disease ( , ). in experiments not shown, we also measured bactericidal anti- body titers of sera from immunized -year-old canadians, including the children in group , using two other oac- positive strains, c (also called e) ( ) and rm , and two other oac-negative strains, i and , as the target organisms. we obtained similar respective results as those shown for strains and . strain c is a group c strain used by many laboratories for measurement of group c serum bactericidal activity ( , , ). thus, the low bactericidal titers measured with the two group c test strains used in the present study are likely to be relevant to titers measured against other group c target strains expressing the oac-posi- tive or oac-negative capsular polysaccharide. considerable data support a relationship between the pres- ence of serum bactericidal antibody and protection from de- veloping invasive meningococcal disease (reviewed by frasch [ ]). however, not all persons whose sera lack bactericidal activity are necessarily susceptible to developing disease ( ). to date, this inference has not been tested experimentally because of lack of a suitable animal model of group c menin- gococcal disease. the infant-rat challenge model described herein for group c bacteremia is similar to that used previously by us for investi- gation of the protective activity of antibodies to group b or- ganisms ( ). for the group c model, we use a relatively low challenge dose of to , cfu of either an oac-positive or oac-negative strain. the challenge dose is prepared from organisms grown to mid-log phase in broth culture, and the bacteria are separated by centrifugation, washed, resuspended in buffer, and given i.p. in unprotected animals, the density of bacteria in the bloodstream increases within h to � , cfu/ml. pretreatment of the animals with antibody can com- pletely prevent bacteremia. thus, the model permits investiga- tion of the protective activity of group c anticapsular antibod- ies with different fine antigenic specificities related to oac in an in vivo setting where, in the absence of protective antibod- ies, the organism is rapidly replicating. our most important findings from this model are as follows. (i) serum anticapsular antibodies that are elicited by menin- gococcal polysaccharide vaccination of children, and which lack complement-mediated bactericidal activity, can confer protection. (ii) in the absence of bactericidal activity, a high antibody avidity correlates with in vivo protective activity. (iii) age of immunization affects antibody functional activity. the serum samples used in our studies were obtained in previous studies of different populations (i.e., canadian children and u.s. amish children) using different vaccines (tetravalent or bivalent) and different vaccine doses. as described below, we do not believe these potential confounders affected the prin- cipal results or conclusions of this study. in the canadian study, all subjects received the same dose of tetravalent vaccine, and their sera were stored under identical conditions (� °c). within each age group, selection of the subset of sera used in our study was random since only a small subset of the samples had been assayed previously. although immunization of the children was performed in response to an outbreak of group c disease, rates of disease in the population were low (� / , population [ , ]). also, colonization by group c strains is infrequent in the general population, even during outbreaks. finally, the group c anticapsular antibody concentrations and bactericidal titers of the prevaccination sera of children in groups and were for the most part below detection (tables and ), results consistent with lack of recent exposure to group c organisms. thus, it is unlikely the antibody responses to vaccination of these subjects were af- fected by prior colonization by group c strains. the amish children in groups and received a bivalent meningococcal polysaccharide vaccine instead of the tetrava- lent vaccine, and one-fifth of the usual dose was given as part of a study of genetic factors affecting immune responses ( ). however, both groups of amish children were immunized in the same study, and the only difference between the two groups was the age of vaccination (mean age of . years for group and mean age of . years for group ). the serum pools from the two groups were compared directly in the same passive- protection experiments in infant rats (table , experiment a, challenge by an oac-positive strain, and experiment b, chal- lenge by an oac-negative strain). the results were unambig- uous. both serum pools lacked detectable complement-medi- ated bactericidal activity against both group c strains (table ). nonetheless, the pool from the older children with higher antibody avidity ( nm� ) protected the rats from bacteremia at doses of . or . �g/rat, whereas the serum pool with lower avidity ( . nm� ) did not confer protection at either dose. in the same experiment, the positive control serum from an immunized adult conferred protection at antibody doses of . , . , and . �g/rat. the superior protection of the adult serum correlated with the presence of complement-mediated bactericidal activity and also had higher-avidity antibody ( . nm� ) than that of the serum pools from the two groups of children. in experiments a and b (table ), we compared protec- tive activity of serum pools prepared from two groups of chil- dren immunized at mean ages of . years (group , canadian) and . years (group , amish). the respective geometric mean pre- and postimmunization anticapsular antibody con- centrations of the two groups were similar (table ), as were the respective total and igg anticapsular concentrations of the pools prepared from the sera from the two groups. although in this experiment there are many potential confounders (i.e., different vaccine, dose, and genetic backgrounds), the data confirm that serum pools without bactericidal activity against strain (pools and ) can be highly protective in the model. also, the adult positive control serum with the highest antibody avidity (ka � . nm� ) gave the best protection, followed by the serum pool from group , which had a some- what lower avidity ( . nm� [canadian children]), with the lowest protection observed in animals pretreated with antibody from serum pool from the amish children, which had the lowest avidity ( nm� ). thus, the results of the different passive-protection experiments with the different serum pools were consistent with respect to our findings that pools that lack bactericidal activity can confer protection, the finding of a correlation between increasing antibody avidity and increasing harris et al. infect. immun. o n a p ril , a t c a r n e g ie m e l l o n u n iv l ib r h ttp ://ia i.a sm .o rg / d o w n lo a d e d fro m http://iai.asm.org/ protective activity, and the effect of older age of immunization and increased protective activity. the raba method used for measurement of antibody avid- ity in this study was adopted from the procedure originally described by griswold et al. ( ) in to measure the avidity of hib anticapsular antibodies. in contrast to subsequently described elisa methods that use chaotropic agents to dis- sociate antigen-antibody complexes ( , , ) and give esti- mates of avidity (avidity index), the raba method (fig. ) allows direct calculation of a ka. using this method, there was a good correlation between antibody avidity and the dose of serum anticapsular antibody required for protective activity (fig. ). similar correlations between antibody avidity and anticapsular antibody functional activity have been observed for hib ( , , ) and streptococcus pneumoniae ( , ). average affinity of the mabs to phosphorylcholine also corre- lated with passive protective activity against experimental pneumococcal infection ( ). however, in the absence of se- rum bactericidal activity against n. meningitidis, predicting pro- tective antibody activity is still a complex undertaking. in ad- dition to antibody concentration and avidity, the quality of the antibody can be affected by the isotype and specific epitopes recognized. our experimental data from the passive protection model are consistent with epidemiologic observations supporting pro- tective efficacy of meningococcal vaccine when given to -year- old children ( , , ), albeit less protective than in adults ( , , ). the efficacy of group c polysaccharide vaccine in -year-old children is controversial, with some studies showing limited or no efficacy ( , ). although the experimental pas- sive protection data reported herein for this age group are limited to sera obtained from only one group of amish chil- dren immunized at to years of age, our results are consis- tent with the low vaccine efficacy in this age group (i.e., absence of both bactericidal and protective activity in pool ). recently, meningococcal group c polysaccharide-protein conjugate vaccines were licensed in europe and canada (two prepared from oac-positive polysaccharide and one prepared from oac-negative polysaccharide). following their introduc- tion into routine and mass catch-up immunization in the united kingdom, the number of cases of group c meningo- coccal disease has declined dramatically ( , , ). the con- jugated polysaccharide has t-cell-dependent antigenic proper- ties. as such, these vaccines are more immunogenic in infants and young children than unconjugated group c polysaccha- ride, and the conjugate vaccines also elicit serum antibodies with greater avidity and bactericidal activity ( , , , , , , ) and prime for memory antibody responses ( , , ). in the future it will be of interest to investigate the protective activity of conjugate-vaccine-induced antibodies in the infant rat model and to dissect the relative protective role of antibodies elicited by oac-positive or -negative polysaccharide. acknowledgments this work was supported, in part, by grants ro ai and ai from the national institutes of allergy and infectious dis- ease, nih, and a grant from aventis pasteur. leslie m. cayco, christian c. dacosta, and quandra t. mcgrue, children’s hospital oakland research institute, provided expert tech- nical assistance. we are grateful to alexander lucas for helpful dis- cussions and to noni macdonald (dalhousie university, halifax, can- ada), a co-investigator of the canadian study, for providing the serum samples from groups and . references . amir, j., x. liang, and d. m. granoff. . variability in the functional activity of vaccine-induced antibody to haemophilus influenzae type b. pedi- atr. res. : – . . anttila, m., j. eskola, h. ahman, and h. kayhty. . differences in the avidity of antibodies evoked by four different pneumococcal conjugate vac- cines in early childhood. vaccine : – . . arakere, g., and c. e. frasch. . specificity of antibodies to o-acetyl- positive and o-acetyl-negative group c meningococcal polysaccharides in sera from vaccinees and carriers. infect. immun. : – . . artenstein, m. s., r. gold, j. g. zimmerly, f. a. wyle, h. 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(lexington: university press of kentucky, . xviii, pp. cloth $ . , isbn - - - - .) kentucky’s first tourist attraction, big bone lick has long deserved a mod- ern account such as stanley hedeen provides. professor emeritus of biology at xavier university, hedeen does a fine job of interpreting the history and significance of the prehistoric and historic salt lick aptly termed “the cradle of american paleontology.” well-grounded in history, geology, and vertebrate paleontology, the au- thor for the most part ably deals with a historical problem compounded by a plethora of obscure and widely scattered references; but in some respects his study supplements rather than supplants the work of the same title by the indefatigable willard rouse jillson (published by the original big bone lick association). hedeen does clarify jillson’s mistaken date for charles de longueuil’s expedition and provides previously unknown nineteenth-century newspaper references. his account is more readable and benefits from more recent advances in our understanding of local geomorphology, paleoecology, and vertebrate taxonomy. he incorporates much unpublished or “underpublished” recent research. hedeen’s account is comprehensive but not quite complete, for both he and jillson omit two significant references to early visitors. in capt. william harrod, hastening to join col. john bowman’s expedition against the shawnee indians of ohio, encountered a group of men transporting a canoe-load of specimens from big bone lick to redstone old fort. some . “bowman’s expedition against chillicothe, may‒june, ,” ohio archaeological and historical publications ( ): ‒ . bo ok reviews of these men joined harrod and bowman on the unsuccessful ohio foray, and subsequently “capt. wm. harrod, with others, went up the ohio to red stone in the two keel boats &c. and took along several bones & tusks got at the big bone lick.” what became of these specimens remains unknown, but this was an important early effort at “mining” the deposits at big bone. similarly, neither jillson nor hedeen mentions thomas rodney’s well-doc- umented stop there on october , , less than a week after meriwether lewis’s collecting visit for thomas jefferson. rodney not only took measure- ments of several of the remaining fossil bones but speculated (incorrectly) that no animals larger than bison ever existed there, larger bones being “only fossil concretions.” rodney himself collected a mastodon tooth and a tusk fragment, but these were lost when his boat sank at natchez. curiously, hedeen merely reproduces without explanation a photograph of three paleo-indian points “collected” at big bone lick. elsewhere, he has written that these were discovered in the lowest level of the big bone de- posits during william clark’s excavations for thomas jefferson and are “now likely” housed at the cincinnati museum center. the significance of associated paleo-indian artifacts and megafauna at big bone lick should certainly be acknowledged but so too should the considerable debate exist- ing about the precise provenance and pedigree of these artifacts. such strictures aside, this book provides ample context for the present big bone lick state park museum and should remain a standard resource for many years. ja m e s l . m u r p h y the ohio state university mennonites, amish, and the american civil war. by james o. lehman and steven m. nolt. (baltimore: johns hopkins university press, . xiv, pp. cloth $ . , isbn - - - .) when war engulfed virginia in the summer of , mennonite christian good was conscripted into the state militia. after good experienced battle and failed to discharge his rifle, his perturbed captain asked him why he . henry hill, “bowman’s campaign— ,” ohio archaeological and historical publica- tions ( ): . . simon gratz, “thomas rodney,” pennsylvania magazine of history and biography . ( ): ‒ . . stanley hedeen, natural history of the cincinnati region. cincinnati museum center scientific contributions no. (cincinnati: the center, ), . in the same publication, these artifacts are captioned only as “most likely [discovered] during william clark’s expedition” (plate ). identification of novel candidate genes for type diabetes from a genome-wide association scan in the old order amish evidence for replication from diabetes-related quantitative traits and from independent populations evadnie rampersaud, coleen m. damcott, mao fu, haiqing shen, patrick mcardle, xiaolian shi, john shelton, jing yin, yen-pei c. chang, sandra h. ott, li zhang, yiju zhao, braxton d. mitchell, jeffery o’connell, and alan r. shuldiner , objective—we sought to identify type diabetes suscepti- bility genes through a genome-wide association scan (gwas) in the amish. research design and methods—dna from type diabetic case subjects and control subjects with normal glucose tolerance were genotyped on the affymetrix k single nucleotide polymorphism (snp) array. a total of , snps were tested for association with type diabetes. type diabetes– associated snps were further prioritized by the following: ) associations with oral glucose tolerance test (ogtt) traits in nondiabetic amish subjects, and ) in silico replication from three independent l snp gwass (framingham heart study caucasians, pima indians, and mexican americans) and a k gwas in scandinavians. results— the strongest association (p � . � � ) was for rs , which is located in growth factor receptor– bound protein (grb ), an adaptor protein that regulate insulin receptor signaling. rs was also strongly associated with ogtt glucose area under the curve in nondiabetic subjects (p � . ). of the , snps associated with type diabetes at p � . , snps demonstrated associations with at least one ogtt trait in nondiabetic individuals; snps were nominally associ- ated with type diabetes in one of the three independent k gwass, snps (rs in mfsd , rs on chromo- some , and rs in bcat were associated with type diabetes in more than one population), and snps were nominally associated with type diabetes in scandinavians. one type diabetes–associated snp (rs , located in fhit) showed replication with ogtt traits and also in another population. conclusions—our gwas of type diabetes identified sev- eral gene variants associated with type diabetes, some of which are worthy of further study. diabetes : – , t ype diabetes, a complex disease that is char- acterized by insulin resistance and impaired �-cell function, represents a serious global pub- lic health problem, with more than million people affected worldwide. while the primary molecular defects in type diabetes remain largely unknown, it is clear that both genetic and environmental risk factors (including diet and physical inactivity) play critical roles. more than genome-wide linkage scans of type diabe- tes have been published, with evidence for linkage re- ported to a number of loci, including regions on chromosomes , , , , , , and ( – ). of the numerous candidate genes studied for their functional role in pancreatic �-cell function, insulin action, or energy metabolism, as well as positional candidate genes identi- fied under linkage peaks, very few have variants that are consistently associated with type diabetes. indeed, com- mon variants in only a few genes (ppar�, kcnj , calpn , tcf l , and hnf a) have been replicated in multiple populations ( ). the old order amish are a closed founder population who emigrated from switzerland in the early s. they are a well-suited population for carrying out genetic studies since they live a relatively homogeneous lifestyle and maintain extensive family history records. the amish family diabetes study (afds) was initiated in with the goal of identifying the genetic determinants of type diabetes ( ). the sibling relative risk (�s) of type diabetes in the amish is . ( % ci . – . ), similar to that observed in other caucasian populations. genome- wide linkage analysis of type diabetes and impaired glucose tolerance conducted in afds pedigrees ( ) re- vealed regions on chromosomes q and q, both of which have been implicated in linkage scans from other popula- tions ( – , ). specific variants in several well-replicated type diabetes susceptibility genes are associated with type diabetes in the amish, including tcf l rs from the division of endocrinology, diabetes and nutrition, university of maryland, baltimore, maryland; and the geriatric research and education clinical center, baltimore veterans administration medical center, balti- more, maryland. address correspondence and reprint requests to alan r. shuldiner, md, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood st., room , baltimore, md . e-mail: ashuldin@medicine.umaryland.edu. received for publication april and accepted in revised form september . published ahead of print at http://diabetes.diabetesjournals.org on sep- tember . doi: . /db - . additional information for this article can be found in an online appendix at http://dx.doi.org/ . /db - . afds, amish family diabetes study; dgi, broad-lund-novartis diabetes genome initiative; gauc, glucose area under the curve; gwas, genome-wide association scan; homa-ir, homeostasis model assessment of insulin resis- tance; iauc, insulin area under the curve; isi, insulinogenic index; ld, linkage disequilibrium; ngt, normal glucose tolerant; ogtt, oral glucose tolerance test; snp, single nucleotide polymorphism. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. original article diabetes, vol. , december (odds ratio . , p � . ) ( ) and hnf a rs ( . , p � . ) ( ). these findings suggest that the common type diabetes gene variants in the amish will likely be relevant to more outbred caucasian populations. increased knowledge of common variation in the human genome learned as part of the hapmap initiative, coupled with advances in technologies, make possible the geno- typing of thousands of single nucleotide polymorphisms (snps) in genome-wide association scans (gwas). this is a powerful approach for identifying novel susceptibility genes for complex diseases ( , ). recently, four gwas studies of type diabetes have identified variants at several novel loci, including slc a , igf bp , ck- dal , cdkn a/ckdn b, and hhex/ide, that show strong replicated association with type diabetes ( – ). in this article, we report results from a gwas of type diabetes in the amish using the affymetrix k snp genotyping platform. we further characterize our findings using diabetes-related quantitative traits measured in non- diabetic amish individuals. lastly, we interpret the results of this scan in the context of three recently completed k gwas studies for type diabetes, as part of the type diabetes k gwas consortium, along with a publicly available k gwas of type diabetes recently per- formed in a scandinavian population. research design and methods study population and phenotype assessment. individuals with type diabetes were identified from the afds. details of the afds have been previously described ( ). phenotypic characterization of participants in- cluded medical and family history, anthropometry, and a -h -g oral glucose tolerance test (ogtt) with insulin levels. we based our primary analyses on type diabetic case and normal glucose tolerant (ngt) control subjects. type diabetes was defined by fasting plasma glucose level (� mmol/l), -h ogtt plasma glucose level (� . mmol/l), random plasma glucose level (� . mmol/l), the use of insulin or prescription oral glucose- lowering agents, or a diagnosis of diabetes documented by a physician. to minimize potentially misclassifying subjects with type diabetes as having type diabetes, case subjects with age of diagnosis � years were excluded. ngt control subjects were aged � years at the time of study and were selected based on fasting plasma glucose level (� . mmol/l) and -h ogtt plasma glucose level (� . mmol/l). we performed secondary quantitative analyses of our mostly highly asso- ciated signals (p � . ) in a set of nondiabetic amish study participants, of whom had impaired glucose tolerance and of whom were part of the ngt control group used in our primary analysis. we estimated the mean levels of two ogtt-derived quantitative glucose traits (fasting glucose and glucose area under the curve [gauc]) and three insulin traits (insulinogenic index [isi], insulin area under the curve [iauc], and homeostasis model assessment of insulin resistance [homa-ir]) according to the snp genotypes in these individuals. total gauc and iauc were calculated based on measurements at , , , , , , and min using the trapezoidal method. the isi was calculated as (insulin at min � fasting insulin)/ (glucose at min � fasting glucose). homa-ir was calculated as fasting insulin (mu/l) � fasting glucose (mmol/l)/ . . table describes the charac- teristics of this sample. the study protocol was approved by the institutional review board at the university of maryland school of medicine, and informed consent was obtained from each study participant. genotyping. genomic dna from leukocytes were genotyped using the affymetrix genechip mapping k array set, which consists of two microar- ray chips (xbai and hindiii) (affymetrix, santa clara, ca). total genomic dna ( ng) was digested with xbai or hindiii restriction enzymes and processed according to the affymetrix protocol. the genechip genotyping analysis software (gtype . ) was used to generate dynamic modeling algorithm– derived genotypes that were reanalyzed with the brlmm (bayes- ian rlmm) genotype calling algorithm (confidence threshold of . ) to improve the proportion of heterozygote calls ( ). as an initial quality-control measure, brlmm-generated chip files with call rates � % for both enzymes across all snps were excluded. the resulting median call rate across all of the remaining case-control samples was . % ( . % for xbai and . % for hindiii). we further removed individual snps with genotype call rates � %, monomorphic snps and snps with minor allele frequency � %, and those deviating from hardy-weinberg equilibrium in control subjects (p � . ). the number of monomorphic and low-frequency snps (n � , ) in the amish was not appreciably different from that observed in more outbred caucasians of the hapmap ceu sample. for this report, we focused our analysis on the , autosomal snps that passed our quality-control standards. the concordance rate for quality-control samples that were run twice on the affymetrix genechip mapping panel was . %. we also calculated a cross-platform concordance rate of % for samples in which snps were genotyped using the affymetrix genechip mapping k panel and an independent illumina -plex goldengate assay. supplementary table (available in an online appendix at http://dx.doi.org/ . /db - ) sum- marizes the quality checks and informativeness of the data. association testing and snp prioritization scheme. our gwas analysis and snp prioritization scheme is shown in fig. . we selected the snps most highly associated with type diabetes in our amish case-control dataset based on p value rankings (p value cutoff � . ) and then used two complementary approaches to further prioritize them. in one approach, we evaluated the most highly type diabetes–associated snps for association with diabetes-related quantitative traits in an expanded set of nondiabetic amish subjects, of whom were ngt control subjects from the primary type diabetes association analysis (internal consistency). in a parallel approach, we as- table description of sample characteristics for type diabetes gwas in the amish characteristics type diabetic case and ngt control subject dataset type diabetic case subjects ngt control subjects n male subjects (%) age (years) . � . . � . bmi (kg/m ) . � . . � . characteristics ogtt-derived quantitative trait dataset all men women n age (years) . � . . � . . � . bmi (kg/m ) . � . . � . . � . fasting glucose (mmol/l) . � . . � . . � . gauc (mmol � l� � h� ) . � . . � . . � . iauc (mu� l� � h� ) . � . . � . . � . homa-ir (mu per mmol/l ) . � . . � . . � . isi (units/g) . � . . � . . � . data are means � sd. all were nondiabetic subjects. a type diabetes gwas in the amish diabetes, vol. , december sessed replication of the most highly associated type diabetes–associated snps in the amish in four independent gwass from other populations (external replication). type diabetes association analysis. we performed case-control associa- tion analysis using a variance component approach as implemented in solar software ( ). using a liability threshold model, we modeled the probability that the individual was a case or control subject as a function of the individual’s age, sex, and genotype, conditional on the correlations in pheno- type among relative pairs. statistical testing was performed using a likelihood ratio test, in which we compared the likelihood of the data under a model in which the genotype effect was estimated with the likelihood of a nested model in which the genotype effect was constrained to be zero. odds ratios (ors) were computed from variance components models. we chose to report the additive model as our primary analysis. supplementary analyses using a dominant or recessive model did not yield any snp showing genome-wide significance. of , snps, had p � . under a dominant model and had p � . under the recessive model. our complete dataset with results from all models is available online (available at http://www.medschool.umary land.edu/amishstudies/index.asp). pairwise linkage disequilibrium (ld) corre- lation statistics (r ) were computed using the helixtree software, version . . (goldenhelix, bozeman, mt). quantitative trait analysis. for quantitative trait analyses performed in nondiabetic amish subjects, we used the measured genotype approach, in which we estimated the likelihood of an additive genetic model given the pedigree structure ( ). before analysis, all insulin traits (iauc, isi, and homa-ir) were transformed by their natural logarithm to reduce skewness. parameter estimates were obtained by maximum likelihood methods, and the significance of association was tested by the likelihood ratio test. within each model, we simultaneously estimated the effects of age and sex. these analyses were performed using the solar program ( ). power calculations. power calculations, based on the genetic power calcu- lator of purcell et al. ( ), indicated that our sample would provide % power to detect a diabetes susceptibility allele having a genotype relative risk of . (for allele frequency of %, case and control subjects, % population prevalence of diabetes, assuming a multiplicative model) and % power to detect a quantitative trait loci accounting for % or higher of the trait variance for a continuously distributed phenotype ( subjects). in silico replication samples. we considered whether our best type diabetes association signals (p value cutoff � . ) replicated in at least one of three distinct populations (framingham caucasians, mexican americans, and pima indians), each with different study designs but performed using the same affymetrix k genotyping platform. descriptions of each of the type diabetes k gwas consortium study populations are provided in accom- panying articles ( – ) and in supplementary table . we directly checked whether any of the , snps with the best type diabetes association signals (p � . ) in the amish were also associated with type diabetes based on generalized estimating equations and family-based association tests in the framingham heart study, fisher’s exact allelic association test in the mexican-american study, and case-control and sib-based association tests in the pima indian study. we also utilized publicly available prereleased data (march ) from a type diabetes gwas carried out in a scandinavian cohort of , type diabetic case and , matched control subjects and genotyped using the affymetrix k platform by the broad-lund-novartis diabetes genetics initiative (dgi) (available at http://www.broad.mit.edu/ diabetes/) ( ). we specifically checked replication of of , of our most highly type diabetes–associated snps that were present on both k and k affymetrix genotyping arrays. since ld structure may differ across populations, and to limit multiple comparisons, we defined replication only if the same snp was associated with type diabetes at p � . with an or in the same direction (i.e., reflective of the same allelic risk). results following quality-control and hardy-weinberg equilibrium checks, , informative snps were included in our analyses. the median physical inter-snp distance was . kb, and the average distance between snps was kb. under the additive model, a total of , snps, some of which were in ld, were associated (p � . ) with type diabetes (fig. ). the most strongly type diabetes– associated snps (i.e., lowest p values) are shown in table . the complete dataset is available online (available at fig. . schematic diagram of analysis and snp prioritization approach for a k type diabetes gwas in the amish. fasg, fasting glucose during an ogtt; fhs, framingham heart study. e. rampersaud and associates diabetes, vol. , december http://www.medschool.umaryland.edu/amishstudies/ afds.asp). no snp was associated with type diabetes at a conservative bonferroni-corrected level. the strongest association (p � . � � ) was for rs on chromosome , which is located in intron of growth factor receptor– bound protein (grb ), an adaptor protein known to regulate signaling of insulin and igf receptors ( – ). in addition to grb , snps were associated with type diabetes at p � � � (fig. and table ). these snps are located in or near msh (chromosome ), prkg (chromosome ), col a (chromosome ), mthfsd (chromosome ), and specc (chromosome ), none of which are obvious candidate genes for type diabetes. adjustment for bmi did not have a large impact on the strength of the associations of these snps with type diabetes (table ). as a measure of internal consistency, we tested whether the , snps associated with type diabetes (p � . ) were also associated with ogtt-derived quantitative traits in nondiabetic individuals. in these analyses, we consid- ered two ogtt glucose traits (fasting glucose and gauc) and three ogtt insulin traits (iauc, homa-ir, and isi), with p � . as our threshold for significance. thirty- eight nonredundant (r � . ) type diabetes–associated snps were also associated with at least one glucose trait and showed the same allelic association as that for diabe- tes (i.e., the diabetes risk allele was also associated with higher glucose levels), while nonredundant type diabetes–associated snps were also associated with at least one insulin-related trait (fig. ; table ). of the top snps associated with type diabetes at p � � � , rs in grb was the only one also associated with an ogtt trait (p � . for gauc). two perfectly correlated (r � ) type diabetes–associated snps in adamts (chromosome ) (p � . – . ) were asso- ciated with one glucose trait (p � . for gauc) and one insulin trait (p � . for iauc). we next sought to determine which of our , most highly type diabetes–associated snps were also asso- ciated with type diabetes in any of three independent populations for which the same k affymetrix plat- form was used or in the dgi scandinavian population for which the k affymetrix platform was used. we identified nonredundant snps for which the same risk allele was also associated with type diabetes in one of the three studies from the type diabetes k gwas consortium (p � . ) and nonredundant snps that showed consistent association in the dgi sample (p � . ) (fig. ; supplementary table ). in total, three snps demonstrated associations in the amish as well as in two independent populations. the t-allele for rs in mfsd on chromosome was associated with decreased risk of type diabetes in the amish (or . , p � . ) and showed nominal asso- ciation in the pima indian dataset (case-control or . , p � . ; sib-based or . , p � . ; and summary or . , p � . ) and also in the mexican-american sample (case-control or . , p � . ). the g-allele in rs on chromosome was associated with de- creased risk of type diabetes in the amish (or . , p � . ) and also with decreased type diabetes risk in mexican-american ( . , p � . ) and dgi ( . , p � . ) samples. the t-allele in rs in bcat on chromosome was associated with decreased risk of type diabetes in the amish ( . , p � . ) and also in the pima indian dataset (sib-based or . , p � . ; summary or . , p � . ) and the mexican-american dataset (or . , p � . ); borderline association was also seen in the dgi sample ( . , p � . ). the direction of effect was the same for all studies. fig. . snp association p values (< . ) across all autosomal chromosomes. a type diabetes gwas in the amish diabetes, vol. , december table highlights our most consistent overall findings. we present type diabetes–associated snps in the amish (p � . ) that also demonstrated either ) association with a diabetes-related quantitative trait (p � . ) in the amish or ) in silico replication of type diabetes association in one independent population (p � . ). of interest, the t-allele in rs in fhit was associated with increased risk of type diabetes in the amish (or . , p � . ) and also in mexican americans ( . , p � . ) and with increased gauc (p � . � � ) in nondiabetic amish subjects. discussion in this article, we described the results of a gwas of type diabetes of , snps in the old order amish, a genetically closed founder population with a homoge- neous lifestyle. we reasoned that this population is likely table fifty snps most highly associated with type diabetes from amish gwas snp chromo- some position* gene† alleles / strand case subjects (n) control subjects (n) allele case subjects allele control subjects type diabetes p‡ or§ type diabetes p (bmi)� rs grb a/g � . . . � � . . � � rs prkg a/g � . . . � � . . � � rs g/t � . . . � � . . � � rs specc a/t � . . . � � . . � � rs col a c/g � . . . � � . . � � rs a/c � . . . � � . . � � rs a/g 	 . . . � � . . � � rs msh g/t � . . . � � . . � � rs g/t � . . . � � . . rs c/g 	 . . . � � . . rs specc a/g � . . . � � . . � � rs c/t � . . . � � . . rs c/g � . . . � � . . rs mthfsd a/t � . . . � � . . rs c/t 	 . . . � � . . rs a/g � . . . � � . . rs specc c/t 	 . . . . . � � rs g/t 	 . . . . . rs a/g � . . . . . rs tceb a/g � . . . . . rs flj c/t 	 . . . . . rs a/g 	 . . . . . rs a/g 	 . . . . . rs flj a/g � . . . . . rs trps a/c � . . . . . rs sidt a/g 	 . . . . . rs samd c/t � . . . . . rs a/g 	 . . . . . rs a/g � . . . . . rs a/t � . . . . . rs a/t � . . . . . rs c/t � . . . . . rs a/g � . . . . . rs gfm a/g 	 . . . . . rs a/g 	 . . . . . rs g/t � . . . . . rs rims c/g � . . . . . rs c/t � . . . . . rs a/g 	 . . . . . rs fbxl c/t 	 . . . . . rs a/g � . . . . . rs g/t � . . . . . rs kiaa a/g � . . . . . rs c/t � . . . . . rs gfm c/t � . . . . . rs kiaa a/c 	 . . . . . rs a/g � . . . . . rs cd a/g 	 . . . . . rs a/g � . . . . . rs kiaa a/g 	 . . . . . *genome build . . †genic region that contains associated snps. ‡p values derived using variance components analysis under an additive genetic model, adjusted for age, sex, and family structure. §or calculated from a liability threshold model in solar and estimated as allele versus allele . �p values derived using variance components analysis under an additive genetic model, adjusted for age, sex, bmi, and family structure. our complete dataset with results from all models are available online (available at http://www.medschool.umaryland.edu/ amishstudies/index.asp). e. rampersaud and associates diabetes, vol. , december table snps associated with type diabetes (p � . ) and at least one ogtt-derived trait (p � . ) in nondiabetic amish subjects snp chromo- some position* gene† strand alleles / frequency allele type diabetes ogtt trait analysis p‡ or§ trait mean � mean � mean � p‡ rs dhcr � a/g . . . gauc . . . . rs pde b � a/g . . . gauc . . . . rs ptger 	 a/t . . . iauc . . . . rs ptger 	 c/t . . . iauc . . . . rs slc a � a/g . . . fasting glucose . . . . rs loc 	 c/g . . . gauc . . . . rs � c/t . . . gauc . . . . rs � c/t . . . gauc . . . . rs 	 a/g . . . gauc . . . . rs esrrg 	 a/g . . . gauc . . . . rs esrrg 	 c/t . . . gauc . . . . � � rs esrrg � a/g . . . gauc . . . . rs 	 a/c . . . homa-ir . . . . iauc . . . . rs � c/t . . . iauc . . . . rs slc a � a/c . . . fasting glucose . . . . gauc . . . . rs 	 a/g . . . homa-ir . . . . rs mfsd � c/t . . . gauc . . . . � � rs � a/t . . . fasting glucose . . . . rs � c/t . . . iauc . . . . rs 	 a/t . . . fasting glucose . . . . rs fhit 	 c/t . . . gauc . . . . � � rs � a/c . . . homa-ir . . . . iauc . . . . rs plscr � c/t . . . iauc . . . . rs plscr 	 a/g . . . iauc . . . . rs plscr � g/t . . . iauc . . . . � � rs leprel � c/t . . . fasting glucose . . . . rs � a/g . . . isi . . . . rs � a/g . . . isi . . . . rs adamts � c/t . . . iauc . . . . rs adamts 	 a/g . . . gauc . . . . iauc . . . . rs adamts 	 a/g . . . gauc . . . . iauc . . . . rs � c/t . . . gauc . . . . rs 	 c/t . . . gauc . . . . � � rs 	 c/t . . . gauc . . . . rs 	 c/t . . . homa-ir . . . . rs � a/t . . . homa-ir . . . . rs spink � a/g . . . homa-ir . . . . rs spink 	 a/c . . . homa-ir . . . . rs odz 	 c/t . . . homa-ir . . . . rs ly � c/t . . . iauc . . . . rs 	 c/g . . . gauc . . . . rs 	 c/t . . . isi . . . . rs � c/t . . . isi . . . . � � rs grb � a/g . . � � . gauc . . . . rs loc � a/g . . . gauc . . . . rs akap � c/t . . . homa-ir . . . . rs akap 	 g/t . . . homa-ir . . . . rs a/g . . . iauc . . . . � � isi . . . . rs kcnd � a/g . . . homa-ir . . . . rs 	 a/c . . . iauc . . . . rs � c/t . . . iauc . . . . rs 	 c/t . . . iauc . . . . rs rims 	 c/g . . . isi . . . . rs � a/g . . . gauc . . . . rs 	 c/t . . . gauc . . . . continued on facing page a type diabetes gwas in the amish diabetes, vol. , december to carry a subset of the same common type diabetes susceptibility variants as those found in the general pop- ulation and that these variants might be easier to identify. gwas studies are prone to false-positives due to the very large number of statistical tests that must be per- formed. we were restricted by our relatively modest sample size and also computationally in our attempts to define a genome-wide significance level for which fol- low-up was justified (i.e., variance components tests were not feasible for the many replications needed for case- control permuted family datasets in the amish). thus, we relied heavily on a prioritization of snps worthy of fol- low-up by testing for ) internal consistency of type diabetes–associated snps with ogtt-derived quantitative traits in nondiabetic amish individuals, ) external repli- cation of type diabetes associations in three independent non-amish k snp gwas studies, and ) external replication in a k snp gwas of type diabetes in a large population of scandinavians. we found that no single snp replicated consistently and in the same direction across all gwas studies, nor were all snps associated with type diabetes also associated with quantitative traits in nondiabetic indi- viduals (supplementary table ). this is not particularly surprising since we expect that an appreciable number of type diabetes–associated snps will be false-posi- tives. furthermore, a true susceptibility gene in one population might not be readily discernible in other populations due to inadequate sample sizes as well as differences in genetic background, ld, and environmen- tal exposures. similarly, a true susceptibility gene for type diabetes might not show association with diabe- tes-related quantitative traits in nondiabetic individuals, especially since our ogtt-derived traits are only surro- gates for gold-standard measures of insulin sensitivity and insulin secretion. nevertheless, we were able to identify a number of candidate genes and loci that showed evidence for association with type diabetes in table continued snp chromo- some position* gene† strand alleles / frequency allele type diabetes ogtt trait analysis p‡ or§ trait mean � mean � mean � p‡ rs 	 a/c . . . gauc . . . . rs � a/g . . . fasting glucose . . . . rs 	 a/c . . . gauc . . . . rs nrp � a/g . . . fasting glucose . . . . rs 	 a/t . . . fasting glucose . . . . � � rs 	 c/t . . . gauc . . . . rs c orf 	 a/c . . . isi . . . . rs � a/c . . . homa-ir . . . . rs 	 a/t . . . gauc . . . . rs 	 g/t . . . homa-ir . . . . rs exph 	 c/t . . . isi . . . . rs cntn 	 a/g . . . fasting glucose . . . . rs cntn 	 c/t . . . fasting glucose . . . . rs cntn � c/t . . . fasting glucose . . . . rs cntn 	 c/t . . . fasting glucose . . . . rs cntn 	 a/c . . . fasting glucose . . . . rs 	 c/t . . . gauc . . . . rs 	 a/g . . . fasting glucose . . . . rs 	 c/t . . . iauc . . . . rs � a/t . . . gauc . . . . rs dcc 	 a/g . . . gauc . . . . rs mbd 	 c/t . . . fasting glucose . . . . rs 	 a/g . . . iauc . . . . rs card � a/g . . . gauc . . . . rs 	 a/g . . � � . fasting glucose . . . . gauc . . . . rs sfrs 	 c/t . . . isi . . . . snps with p � . for type diabetes associations were tested for consistency in a sample of nondiabetic individuals ( of whom overlapped with the type diabetes association dataset). direction of association for glucose traits was required to be higher for diabetes risk allele. neighboring snps in bold are in high ld (r � . ). *genic region that contains associated snps. ‡p values derived using the additive genetic model, adjusted for age, sex, and family structure. the complete dataset including results for dominant and recessive models are available online (available at http://www.medschool.umaryland.edu/amishstudies/index.asp). §or calculated from a liability threshold model for allele versus allele . �mean values for traits are presented by genotype, with alleles shown in alphabetical order as “ / .” all insulin traits (iauc, homa-ir, and isi) were natural log transformed prior to analysis. e. rampersaud and associates diabetes, vol. , december t a b l e s n p s as so ci at ed w it h ty p e d ia b et es in th e a m is h (p � . ) an d p ro vi d in g ev id en ce fo r ei th er in te rn al co n si st en cy (a ss o ci at io n w it h an o g t t -d er iv ed qu an ti ta ti ve tr ai t in th e a m is h ) o r ex te rn al re p li ca ti o n (a ss o ci at ed w it h ty p e d ia b et es in an in d ep en d en t p o p u la ti o n ) s n p c h ro m o - so m e p o si ti o n * g en e† s tr an d a ss o ci at ed w it h ty p e d ia b et es in a m is h (p � . ) in te rn al co n si st en cy w it h o g t t -d er iv ed qu an ti ta ti ve tr ai ts in a m is h (p � . ) e x te rn al re p li ca ti o n w it h ty p e d ia b et es in in d ep en d en t p o p u la ti o n s (p � . ) a ll el e / a ll el e ca se su b je ct s a ll el e co n tr o l su b je ct s p o r ‡ t ra it p p o p u la ti o n p § o r ‡ rs c o r f 	 a /t . . . . p im a in d ia n s . . rs e s r r g 	 c /t . . . . g a u c . rs — 	 a /c . . . . h o m a -i r . ia u c . rs s l c a � a /c . . . . f as ti n g gl u co se . g a u c . rs f h i t 	 c /t . . . . g a u c . � � m ex ic an a m er ic an s . . rs — 	 c /g . . . . f h s . , . . rs c s n � a /c . . . . m ex ic an a m er ic an s . . rs o d z 	 c /t . . . . h o m a -i r . rs — � a /c . . . . f h s . , . . rs — � c /t . . . . is i . rs g r b � a /g . . . � � . g a u c . rs k c n d � a /g . . . . h o m a -i r . rs — 	 c /t . . . . ia u c . rs — 	 c /t . . . . g a u c . rs n r p � a /g . . . . f as ti n g gl u co se . rs — 	 a /t . . . . f as ti n g gl u co se . rs c o r f 	 a /c . . . . is i . rs c n t n 	 c /t . . . . f as ti n g gl u co se . rs g p c 	 a /g . . . . f h s . , . . rs — � a /t . . . . g a u c . rs — 	 a /g . . . � � . f as ti n g gl u co se . * g en o m e b u il d . .† g en ic re gi o n th at co n ta in s as so ci at ed s n p s. ‡o r ca lc u la te d fr o m a li ab il it y th re sh o ld m o d el in s o l a r an d es ti m at ed as al le le ve rs u s al le le . §c as e- co n tr o l ge n er al es ti m at in g eq u at io n an d fa m il y- b as ed as so ci at io n te st p va lu es gi ve n fo r f ra m in gh am h ea rt s tu d y (f h s ) d at a; su m m ar y p va lu e gi ve n fo r p im a in d ia n s. a ll in su li n tr ai ts (h o m a -i r ,i a u c , an d is i) w er e n at u ra l lo g tr an sf o rm ed p ri o r to an al ys is . a type diabetes gwas in the amish diabetes, vol. , december more than one population and/or were also associated with ogtt-derived quantitative traits. these results are intriguing but must be interpreted with caution. none of these loci fall within previously identified linkage re- gions for type diabetes (chromosomes and ) in the amish. our strongest type diabetes association signal in the amish was observed on chromosome in a functionally relevant type diabetes candidate gene, grb . grb encodes growth factor– binding protein and has been shown to bind to activated insulin receptor and act as a negative regulator of insulin action and glucose uptake ( – ). overexpression of grb in mice causes postna- tal growth retardation and insulin resistance ( ). our k gwas contained a total of snps in grb , of which were associated with type diabetes (p � . ) and were in partial ld with each other (r � . – . ). rs , located in intron , provided the lowest p value for association (or . for the g- vs. a-allele, p � . � � ). this snp was also strongly associated with ogtt gauc in nondiabetic amish individuals (p � . ). rs was not associated with type diabetes in the other three populations in which this snp was genotyped or in the k snp scandinavian type diabetes gwas; however, three snps (rs , rs , and rs ) in grb that were genotyped in the scandina- vian cohort were associated with type diabetes (p � . , p � . , and p � . , respectively) and are in partial ld with rs (r � . – . in hapmap ceu). lack of replication could suggest a false-positive or that variation in grb is a true positive specific to the amish due to a founder effect or context-dependent phe- notypic expression of the variant due to genetic back- ground or environmental influences. alternatively, this variant could be in ld with a functional variant, and extended ld in the amish enabled a type diabetes association to be detected in this population and not the others. in a recent report by di paola et al. ( ), the a-allele of rs , a synonymous coding snp in grb , was associated with decreased risk of type diabetes in a relatively homogeneous population of italian caucasians (p � . ). this snp was not part of the k snp panel nor was our most highly type diabetes–associated snp (rs ) genotyped in the italian sample. we found that rs and rs are not in ld (r � ) in hapmap ceu samples. however, rs , another grb snp associated with type diabetes in the amish (p � . ), is in ld with rs (r � . in hapmap ceu). further investigation of grb is currently underway. our gwas and replication strategy have several limita- tions. first, the relatively small sample size limits our ability to detect gene variants of modest effect size. second, we recognize that the definition of external repli- cation of our top snps across three independent k studies of type diabetes might represent a skewed distribution of the overall results since replication was limited to our , most highly type diabetes–associ- ated snps. this approach was used to facilitate compari- sons across populations and also to limit the number of false-positive replications due to multiple comparisons. to the extent that we attempted to pursue signals that repre- sent the “lowest hanging fruit,” we believe that the ap- proach we have taken is reasonable. a formal meta- analysis of the entire set of data from all four k studies is currently underway. third, our replication approach was focused at the level of the snp in order to avoid additional multiple comparisons. however, it is possible that we did not identify significantly associated snps in other populations that were in ld with our top snps. this is particularly relevant for our comparisons with the scandinavian k gwas, for which only % of the snps identified in the amish with p � . were identified in the k shp panel. the likelihood that we missed common variants impor- tant to type diabetes is high due to the relatively sparse density of the k snp panel (mean intermarker dis- tance � kb) compared with other denser gwas snp panels. for example, snps in well-replicated genes (slc a , igf bp , ckdal , cdkn a/ckdn b, and hhex/ide) found in four recently published type dia- betes gwas studies ( – ), as well as previously known type diabetes–associated variants in tcf l , kcnj , hnf a, or capn ( ), were not adequately covered on the k genotyping panel (i.e., r � . between the snp of interest and snps on the k panel). as a positive control, we previously demonstrated that tcf l snp rs and the hnf a promoter snp rs , nei- ther of which is present on the k panel, were associ- ated with type diabetes and impaired glucose tolerance in the amish family diabetes study (or . , p � . ; . , p � . , respectively) ( , ). interestingly, rs in hhex on the k panel (r � . with rs found previously to be strongly associated with type diabetes in other gwas studies) was significantly associated with type diabetes in the amish (or . for the g-allele; p � . ). rs on chromosome , shown to be associated with type diabetes in the other gwas studies ( ), was present on the k panel but was not significantly associated with type diabetes in the amish (or . for the c-allele; p � . ). in summary, we presented results from our initial ex- amination of a gwas of type diabetes in the amish. although we did not identify any genes associated with type diabetes that reached genome-wide significance, we report a number of genes and loci that are worthy of further study based on replication in other studies or on quantitative trait loci consistency. this report (and the three companion articles) provides a valuable resource for other investigators to utilize in the search for the patho- genic variants for type diabetes. acknowledgments this work was supported by the following national insti- tutes of health research grants: training grant in cardiac and vascular cell biology (t hl ), r dk , the university of maryland general clinical research center (m rr ), hopkins bayview general clinical research center (m rr ), the maryland clinical nutrition research unit (p dk ), and the balti- more veterans administration geriatric research and education clinical center. we extend our thanks to our collaborators in the type diabetes k gwas consortium for sharing prepublica- tion results from the starr county health studies, fra- mingham heart study, and the pima indians as well as to the broad-lund-novartis diabetes genomic initiative for access to results from their gwas. we also thank soren snitker for helpful comments and adam naj for help with formatting figures for this manu- script. lastly, we gratefully acknowledge our amish liai- e. rampersaud and associates diabetes, vol. , december sons and research staff and the extraordinary cooperation and support of the amish community, without whom these studies would not be possible. references . elbein sc, hoffman md, teng k, leppert mf, hasstedt sj: a genome-wide search for type diabetes 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suzuki r, muguruma k, kohda t, wakana s, yokoyama m, ishino f, kaneko-ishino t: meg /grb overexpression causes postnatal growth retardation and insulin resistance via negative modulation of the igf r and ir cascades. biochem biophys res commun : – , . di paola r, ciociola e, boonyasrisawat w, nolan d, duffy j, miscio g, cisternino c, fini g, tassi v, doria a, trischitta v: association of hgrb genetic variations with type diabetes in caucasian subjects. diabetes care : – , a type diabetes gwas in the amish diabetes, vol. , december what the amish can tell us about… cholestasis hepatology elsewhere jacquelyn maher, editor san francisco general hospital building , room potrero avenue san francisco, ca advisory committee laurie de leve, los angeles, ca david crabb, indianapolis, in adrian dibisceglie, st. louis, mo emmet keeffe, palo alto, ca joel lavine, san diego, ca michael nathanson, new haven, ct don rockey, durham, nc dwain thiele, dallas, tx what the amish can tell us about. . . cholestasis bull ln, van eijk mjt, pawlikowska l, deyoung ja, juijn ja, liao m, klomp lwj, lomri n, berger r, scharschmidt bf, knisely as, houwen rhj, freimer nb. a gene encoding a p-type atpase is mutated in two forms of hereditary cholesta- sis. nat genet ; : - . (reprinted with permission.) abstract cholestasis, or impaired bile flow, is an important but poorly understood manifestation of liver disease. two clinically distinct forms of inherited cholestasis, benign recurrent intrahepatic cholestasis (bric) and progressive familial intrahepatic cholestasis type (pfic ), were previ- ously mapped to q . haplotype analysis narrowed the candidate region for both diseases to the same interval of less than cm, in which we identified a gene mutated in bric and pfic patients. this gene (called fic ) is the first identified human member of a recently described subfamily of p-type atpases; atp-dependent aminophospho- lipid transport is the previously described function of members of this subfamily. fic is expressed in several epithelial tissues and, surprisingly, more strongly in small intestine than in liver. its protein product is likely to play an essential role in enterohepatic circulation of bile acids; further characterization of fic will facilitate understand- ing of normal bile formation and cholestasis. de vree jml, jacquemin e, sturm e, cresteil d, bosma pj, aten j, deleuze j-f, desrochers m, burdelski m, bernard o, oude elferink rpj, hadchouel m. mutations in the mdr gene cause progressive familial intrahepatic cholestasis. proc natl acad sci u s a ; : - . (reprinted with permission.) abstract class iii multidrug resistance (mdr) p-glycoproteins (p-gp), mdr in mice and mdr in man, mediate the translocation of phosphatidylcholine across the canalicular membrane of the hepatocyte. mice with a disrupted mdr gene completely lack biliary phospholipid excretion and develop progressive liver disease, characterized histologi- cally by portal inflammation, proliferation of the bile duct epithelium, and fibrosis. this disease phenotype is very similar to a subtype of progressive familial intrahepatic cholestasis, hallmarked by a high serum g-glutamyltransfer- ase (g-gt) activity. we report immunohistochemistry for mdr p-gp, reverse transcription-coupled pcr sequence analysis, and genomic dna analysis of mdr from two progressive familial intrahepatic cholestasis patients with high serum g-gt. canalicular staining for mdr p-gp was negative in liver tissue of both patients. reverse transcrip- tion-coupled pcr sequencing of the first patient’s sequence demonstrated a homozygous -bp deletion, starting at codon , which results in a frameshift and introduces a stop codon codons downstream. the second patient is homozygous for a nonsense mutation in codon (c - t) that introduces a stop codon (tga). our results demon- strate that mutations in the human mdr gene lead to progressive familial intrahepatic cholestasis with high se- rum g-gt. the histopathological picture in these patients is very similar to that in the corresponding mdr ( / ) mouse, in which mdr p-gp deficiency induces complete absence of phospholipid in bile. comments progressive familial intrahepatic cholestasis (pfic) is the name given to a group of autosomal recessive disorders in which cholestasis develops in early infancy and progresses to end-stage liver disease before adulthood. , patients with pfic type (pfic ), or byler disease, named for the extended amish kindred in which it was first described, exhibit disproportionately low serum g-glutamyl transpepti- dase (ggtp) and cholesterol levels for the degree of cholesta- sis, along with a bland intracanalicular cholestasis by light microscopy and coarsely granular bile on transmission elec- tron microscopy. by using material from the original byler kindred, the gene responsible for pfic was mapped to chromosome q - . this same region had been linked earlier to benign recurrent intrahepatic cholestasis (bric), a disease characterized by episodic attacks of jaundice and pruritus but without progressive liver disease ; this suggested that the two phenotypes are allelic variants of the same disease. in contrast, there is a second group of patients with pfic who exhibit high serum ggtp levels and bile duct proliferation along with portal inflammatory infiltrates on liver histology. these changes resemble those that have been observed following disruption of the mdr gene, the mouse homolog of human mdr , which encodes a canalicular p-glycoprotein required for biliary phospholipid secretion. in one patient with this second type of pfic, mdr messenger rna was undetectable and in another phospho- lipid in bile was markedly reduced. the biochemical features of pfic had been previously linked to defects in either bile salt synthesis or bile salt secretion. total biliary bile acids were markedly reduced ( . . mmol/l) in seven patients with pfic compared with values in eight control patients with other cholestatic diseases ( . . mmol/l). strikingly low percentages of chenodeoxycholic acid were observed in gallbladder bile from the pfic patients. in this context, two independent groups have now defined the molecular defects underlying various forms of pfic. the gene mutated in pfic and bric patients, termed fic (for familial intrahepatic cholestasis ), exhibits significant simi- larity to a subfamily of p-type atpase genes that encode putative aminophospholipid transporters. five mutations in fic were identified in patients with pfic , including two missense mutations that replace amino acids that are highly conserved in p-type atpases, suggesting that these are critical to normal protein structure and/or function. in patients with bric, two different mutations were identified, including a missense mutation and a small deletion, that are in less highly conserved regions of the gene. this finding provides a potential explanation for the differences among the clinical manifestations of bric and pfic , because the bric muta- tions are expected to have less of an effect on fic structure and function. a surprising finding was preferential expres- sion of fic in the small intestine, consistent with a role for this gene product in the intestinal handling of bile acids. although malabsorption and diarrhea have been described in patients with pfic , , the clinical manifestations of pfic and bric led many to suspect that the defect underlying these disorders would be localized to the liver. furthermore, fic was identified in a wide variety of tissues, including sites not associated with bile acid handling, such as pancreas and stomach. this finding suggests that fic may have a general role in absorptive and secretory processes. other members of the subfamily to which fic belongs are believed to maintain an asymmetric distribution of phospholipids in membranes by the transport of aminophospholipids from outer to inner leaflets. clearly, additional studies are re- quired to determine how fic functions and how mutations cause disease. nevertheless, the identification of fic as the defect in pfic and bric represents a significant shift in our understanding of the pathophysiology of cholestatic liver disease. in the second study, two children with pfic and high serum ggtp levels were found to have mutations in mdr . canalicular staining for mdr p-glycoprotein was absent in liver biopsy specimens in both children. as a control, staining of the canalicular membranes with antibodies directed against mdr p-glycoprotein and mrp /cmoat, the canalicular trans- porter involved in the secretion of nonbile acid organic anions, was normal. two mutations were identified using reverse transcription-polymerase chain reaction; a seven- nucleotide deletion and a single nonsense mutation, both resulting in the introduction of stop codons and truncated messenger rna. this finding is another milestone in our understanding of cholestatic liver disease. by starting with a gene, in this case mdr in the mouse, and determining its function to define a phenotype, it represents a fundamentally different but equally successful approach to that pursued in the first study in which a defined phenotype was used to identify a defective gene. another locus for pfic has been recently identified on chromosome q and designated pfic . this locus corre- sponds to that of the human sister p-glycoprotein (spgp) gene. the protein product of this liver-specific gene is abundantly expressed on the canalicular membrane. rat spgp expressed in xenopus oocytes and in membrane vesicles isolated from transfected sf insect cells was recently found to mediate adenosine triphosphate–dependent bile acid trans- port. furthermore, taurochenodeoxycholate, the concentra- tion of which is strikingly low in gallbladder bile from pfic patients, was the preferred substrate for spgp. these findings suggest that spgp is a canalicular bile acid trans- porter and that mutations of the human liver spgp gene are responsible for pfic type . in three patients with pfic and normal serum ggtp levels, spgp messenger rna was normal but spgp was undetectable on the canalicular mem- brane by immunohistochemistry, consistent with a traffick- ing defect. to date, mutations in spgp have been identified in patients with pfic (richard thompson, depart- ment of pediatrics, university college london medical school, london, uk, personal communication). in summary, the molecular basis for all forms of pfic may now have been identified. however, the implications of these findings may extend far beyond a mere understanding of a group of relatively rare inherited cholestatic disorders. lith , a gene that has been identified as a determinant of cholesterol gallstone susceptibility in inbred mice, co-localizes with spgp. thus, a potential link has been established between canalicular bile salt secretion and cholesterol gallstone forma- tion. further characterization of the function of the gene products of fic , mdr , and spgp holds the promise of even more advances in bridging the gap between the bench and the bedside. richard h. moseley, m.d. va medical center and university of michigan medical center ann arbor, mi references . whitington pf, freese dk, alonso em, schwarzenberg sj, sharp hl. clinical and biochemical findings in progressive familial intrahepatic cholestasis. j pediatr gastroenterol nutr ; : - . . alonso em, snover dc, montag a, freese dk, whitington pf. histologic pathology of the liver in progressive familial intrahepatic cholestasis. j pediatr gastro nutri ; : - . . clayton rj, iber fl, ruebner bh, mckusick va. byler disease: fatal familial intrahepatic cholestasis in an amish kindred. amer j dis child ; : - . . bull ln, carlton veh, stricker nl, baharloo s, deyoung ja, freimer nb, magid ms, et al . genetic and morphological findings in progressive familial intrahepatic cholestasis (byler disease [pfic- ] and byler syndrome): evidence for heterogeneity. hepatology ; : - . . carlton veh, knisely as, freimer nb. mapping of a locus for progres- sive familial intrahepatic cholestasis (byler disease) to q -q the benign recurrent intrahepatic cholestasis region. hum mol genet ; : - . . houwen rhj, baharloo s, blankenship k, raeymaekers p, juyn j, sandkuijl la, freimer nb. genome screening by searching for shared segments: mapping a gene for benign recurrent intrahepatic cholestasis. nat genet ; : - . . maggiore g, bernard o, hadchouel m, lemonnier a, alagille d. diagnostic value of serum g-glutamyl transpeptidase activity in liver diseases in children. j pediatr gastroenterol nutr ; : - . . smit jjm, schinkel ah, oude elferink rpj, groen ak, wagenaar e, van deemter l, mol caam, et al. homozygous disruption of the murine mdr p-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. cell ; : - . . deleuze j-f, jacquemin e, dubuisson c, cresteil d, dumont m, erlinger s, bernard o, hadchouel m. defect of multidrug-resistance gene expression in a subtype of progressive familial intrahepatic cholestasis. hepatology ; : - . . jacquemin e, setchell kdr, o’connell nc, estrada a, maggiore g, schmitz j, hadchouel m, et al. a new cause of progressive intrahepatic cholestasis: b-hydroxy-c -steroid dehydrogenase/isomerase defi- ciency. j pediatr ; : - . . jacquemin e, dumont m, bernard o, erlinger s, hadchouel m. evidence for defective primary bile acid secretion in children with progressive familial intrahepatic cholestasis (byler disease). eur j pediatr ; 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: - . . jansen p, hooiveld g, koning h, van goor h, gouw a, kuipers f, bijleveld c, et al. the spgp gene, encoding the canalicular bile acid transporting protein spgp/cbst, is not expressed in a subgroup of patients with progressive familial cholestasis (pfic- ). j hepatol ; : (suppl ). . lammert f, beier dr, wang doh, carey mc, paigen b, cohen de. genetic mapping of hepatocanalicular transporters establishes sister-p- glycoprotein (spgp) as a candidate for the major gallstone gene (lith ). hepatology ; : a (suppl). hereditary hemochromatosis—sometimes having a real complex can be a good thing feder jn, penny dm, irrinki a, lee vk, lebron ja, watson n, tsuchihashi z, sigal e, bjorkman pj, schatzman rc. the hemochromatosis gene product complexes with the transfer- rin receptor and lowers its affinity for ligand binding. proc natl acad sci u s a ; : - . (reprinted with permission.) abstract we recently reported the positional cloning of a candi- date gene for hereditary hemochromatosis called hfe. the gene product, a member of the major histocompatibility complex class i-like family, was found to have a mutation, cys- tyr (c y), in % of patient chromosomes. this mutation eliminates the ability of hfe to associate with b -microglobulin (b m) and prevents cell-surface expres- sion. a second mutation that has no effect on b m associa- tion, h d, was found in eight out of nine patients heterozygous for the c y mutant. in this report, we demonstrate in cultured cells overexpressing wild-type or mutant hfe proteins that both the wild-type and h d hfe proteins form stable complexes with the transferrin receptor (tfr). the c y mutation nearly completely prevents the association of the mutant hfe protein with the tfr. studies on cell-associated transferrin at c suggest that the overexpressed wild-type hfe protein decreases the affinity of the tfr for transferrin. the overexpressed h d protein does not have this effect, providing the first direct evidence for a functional consequence of the h d muta- tion. addition of soluble wild-type hfe/b m heterodimers to cultured cells also decreased the apparent affinity of the tfr for its ligand under steady-state conditions, both in cells and in hela cells. furthermore, at c, the added soluble complex of hfe/b m inhibited binding of transfer- rin to hela cell tfr in a concentration-dependent manner. scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of tfr for transferrin. these results establish a molecular link be- tween hfe and a key protein involved in iron transport, the tfr, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis. comments in recent years, many disease genes have been successfully cloned by establishing a linkage between the gene and markers from known locations in the genome. one of the virtues of a positional cloning approach to identify a disease- related gene is that information about the function of the gene is not required for success. as long as patients with the disease can be unequivocally identified, the discovery of mutations within a candidate gene in affected individuals but not in healthy individuals should be sufficient to prove that the gene is correct. positional cloning has identified many disease genes; however, there are several situations in which a disease gene bore no resemblance to genes of known function and, thus, provided little insight into pathogenesis. in other instances, a disease gene resembled known genes but the relationship between the gene and the disease process was initially obscure. such was the case for hereditary hemochro- matosis, whose gene named hfe was cloned in . hfe is similar in sequence to major histocompatibility complex class i genes, a family of polymorphic integral membrane proteins that associate noncovalently with a -kd protein b microglobulin (b m). within the endoplasmic reticulum, class i molecules capture and bind peptides derived from proteolytic digestion of intracellular proteins. once peptide is bound to a class i molecule, the complex exits the endoplasmic reticulum and moves to the cell surface where peptides from viral proteins that are expressed in an infected cell can be recognized as foreign and can target the cell for destruction by t-lymphocytes. there is another group of major histocompatibility complex class i proteins that are atypical in that they have maintained the basic structure of class i molecules, but have other biological roles, as in the case of cd and the neonatal fc receptor. although the mechanism by which an abnormality in a class i–like gene would lead to iron overload was initially quite obscure, the observation fit well with previous data that mice homozygous for a targeted disruption in the b m gene develop iron overload (presumably as a consequence of loss of b m and, therefore, loss of the functional hfe-b m complex). subsequently, it has been shown that mice homo- zygous for a targeted disruption of hfe also develop iron overload similar to that in hereditary hemochromatosis. together these findings confirm that functional absence of hfe leads to hemochromatosis. because loss of hfe results in iron overload, it is reason- able to expect that hfe interacts in some fashion with proteins of iron metabolism. to attempt to identify potential hfe-interacting proteins, feder et al. analyzed immunopre- cipitates from cells in which hfe was overexpressed. initially, cell-surface proteins were labeled with n-hydroxysuccin- amide, and hfe was immunoprecipitated with antibodies to the native hfe c-terminus or to an epitope tag inserted in the overexpressed hfe. in these experiments, hfe and b m were coimmunoprecipitated as expected, but in addition, other proteins were identified at approximately and kd. when known proteins of iron metabolism were considered, it became apparent that the - and -kd bands were quite similar to those seen in immunoprecipitations of the transfer- rin receptor (tfr), which can migrate as a dimer as well as in a monomeric form of approximately kd. the identity of hepatology elsewhere hepatology september hydrophobicity of proteins and nanostructured solutes is governed by topographical and chemical context sp ec ia l fe a tu r e c h em is tr y hydrophobicity of proteins and nanostructured solutes is governed by topographical and chemical context erte xia, , vasudevan venkateshwaranb,c, , lijuan lib,c, nicholas regoa, amish j. patela, , and shekhar gardeb,c, a department of chemical & biomolecular engineering, university of pennsylvania, philadelphia, pa ; b howard p. isermann department of chemical and biological engineering, rensselaer polytechnic institute, troy, ny ; and c center for biotechnology and interdisciplinary studies, rensselaer polytechnic institute, troy, ny edited by michael l. klein, temple university, philadelphia, pa, and approved october , (received for review march , ) hydrophobic interactions drive many important biomolecular self- assembly phenomena. however, characterizing hydrophobicity at the nanoscale has remained a challenge due to its nontrivial dependence on the chemistry and topography of biomolecu- lar surfaces. here we use molecular simulations coupled with enhanced sampling methods to systematically displace water molecules from the hydration shells of nanostructured solutes and calculate the free energetics of interfacial water density fluctua- tions, which quantify the extent of solute–water adhesion, and therefore solute hydrophobicity. in particular, we characterize the hydrophobicity of curved graphene sheets, self-assembled mono- layers (sams) with chemical patterns, and mutants of the pro- tein hydrophobin-ii. we find that water density fluctuations are enhanced near concave nonpolar surfaces compared with those near flat or convex ones, suggesting that concave surfaces are more hydrophobic. we also find that patterned sams and pro- tein mutants, having the same number of nonpolar and polar sites but different geometrical arrangements, can display signif- icantly different strengths of adhesion with water. specifically, hydroxyl groups reduce the hydrophobicity of methyl-terminated sams most effectively not when they are clustered together but when they are separated by one methyl group. hydrophobin-ii mutants show that a charged amino acid reduces the hydropho- bicity of a large nonpolar patch when placed at its center, rather than at its edge. our results highlight the power of water den- sity fluctuations-based measures to characterize the hydrophobic- ity of nanoscale surfaces and caution against the use of additive approximations, such as the commonly used surface area models or hydropathy scales for characterizing biomolecular hydropho- bicity and the associated driving forces of assembly. nanotube | curvature | chemical pattern | hydrophilicity | graphene hydrophobic interactions drive many important biolog-ical and colloidal self-assembly processes ( – ). dur- ing such assembly, the hydration shells of the associating solutes are disrupted, replacing hydrophobic–water contacts with hydrophobic–hydrophobic ones. characterizing how strongly water adheres to a given solute is, therefore, directly relevant to the strength of hydrophobic interactions between solutes. macroscopically, surface–water adhesion is quantified by mea- suring the water droplet contact angle on a surface. however, such characterization does not translate usefully to proteins and other nanoscale solutes. indeed, characterizing how strongly or weakly a protein surface or a specific patch on it adheres to water (i.e., its hydrophobicity) is incredibly challenging and has neces- sitated the use of simplifying assumptions. to this end, simple surface area (sa) models have been used to estimate the driving force for assembly, ∆g = γ∆a, where ∆a is the nonpolar sa buried upon assembly and γ is an appro- priate surface tension ( – ). however, the value of γ used in pop- ular models is nearly an order of magnitude lower than the oil– water surface tension ( ), and its temperature dependence is at odds with that of biological assembly ( ), reducing it to a fitting parameter. moreover, not just the magnitude but even the sign of the best-fit γ can depend on the class of solutes used to esti- mate it ( ). as a result, for certain complex solutes the burial of nonpolar sa can even be unfavorable, rendering such mod- els ill-suited for characterizing the driving forces of nanoscale assembly ( ). other approximate approaches that use hydropathy scales (or scoring functions) ( – ) assign an index (or a score) to an amino acid residue based on some measure of its aversion to water (e.g., water to oil transfer free energy) and estimate the hydrophobicity of a protein patch as a sum of the hydrophobic- ities of constituent amino acids ( ). many hydropathy scales exist, and they differ significantly from each other ( , ). importantly, while such methods can efficiently extract informa- tion from large protein databases and classify them into mean- ingful groups, they fail to predict the driving forces in specific situations ( – ). here we trace the failure of such approaches to the assumption that the hydrophobicity of a protein patch can be decomposed into a sum of its constituent parts (i.e., additiv- ity), and that a unique hydrophobicity can be assigned to each residue; our results contribute to the growing consensus that the hydrophobicity of a residue is not unique but depends in a non- trivial manner on its chemical and topographical context in a protein. to study context-dependent hydrophobicity we use molecu- lar dynamics (md) simulations coupled with enhanced sam- pling methods ( , ) to systematically displace water molecules from the solute hydration shell and quantify the corresponding significance numerous biological self-assembly processes, from protein folding to molecular recognition, are driven by hydropho- bic interactions, yet characterizing hydrophobicity at the nanoscale has remained a major challenge, because it requires understanding of the strength of protein–water interactions and the ease with which they can be disrupted. water near a protein responds to its chemistry and topography in a man- ner that is collective and complex and cannot be captured by commonly used surface area models or hydropathy scales. we demonstrate that water density fluctuations near proteins can characterize protein hydrophobicity and reveal its depen- dence on curvature and chemical patterns at the nanoscale. our approach opens new avenues for understanding and effi- cient characterization of biomolecular interactions. author contributions: a.j.p. and s.g. designed research; e.x., v.v., l.l., n.r., and a.j.p. performed research; e.x., v.v., l.l., n.r., and a.j.p. analyzed data; and e.x., v.v., a.j.p., and s.g. wrote the paper. the authors declare no conflict of interest. this article is a pnas direct submission. published under the pnas license. e.x. and v.v. contributed equally to this work. to whom correspondence may be addressed. email: amish.patel@seas.upenn.edu or gardes@rpi.edu. this article contains supporting information online at www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental. www.pnas.org/cgi/doi/ . /pnas. pnas | december , | vol. | no. | – d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/site/aboutpnas/licenses.xhtml mailto:amish.patel@seas.upenn.edu mailto:gardes@rpi.edu http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental http://www.pnas.org/cgi/doi/ . /pnas. http://crossmark.crossref.org/dialog/?doi= . /pnas. &domain=pdf free energetic cost. the free energetics of interfacial water den- sity fluctuations, and especially the rare ones that result in opening of a cavity adjacent to the solute, then serve to quan- tify hydrophobicity. density fluctuations are enhanced and cor- respondingly it is easier to create a cavity near a hydropho- bic surface ( – ), consistent with the weaker surface–water adhesion. theory of inhomogeneous liquids connects water density fluctuations to other quantities, such as water com- pressibility, transverse water density correlations, and the free energy of cavity formation, all of which can also serve as molec- ular measures of context-dependent hydrophobicity ( , – ). such measures have been used previously to study as- pects of context-dependent hydrophobicity of nanoscale surfaces ( – ). here, we build on this work by characterizing the hydropho- bicity of surfaces with systematic variations in curvature and the chemical patterns they display. we show that concave non- polar surfaces are more hydrophobic than convex ones. we also show that hydrophobic patches with variations in chemi- cal pattern and topography, whether on self-assembled mono- layers (sams) or on the surface of a protein, hydrophobin-ii, can display significantly different hydrophobicity. our work high- lights the power of water density fluctuations-based measures to characterize hydrophobicity of nanoscale surfaces and cautions against the use of additive approximations, such as the com- monly used sa models, hydropathy scales, and similar scoring functions, for characterizing biomolecular hydrophobicity and the associated driving forces of assembly. results and discussion effect of nanoscale curvature on surface hydrophobicity. how cur- vature influences the hydration of spherical hydrophobic solutes is known. highly curved solutes smaller than rc ≈ nm are hydrated without significantly perturbing water’s hydrogen bond network, whereas hydrating larger solutes requires the ener- getically unfavorable breaking of hydrogen bonds ( , , ). although typical proteins are larger than rc, their surfaces include bumps and crevices with different curvatures. to under- stand how protein hydrophobicity is influenced by both the mag- nitude and the sign of its local curvature we first study surfaces with homogeneous chemistry and well-defined curvatures, hemi- cylindrical nanotubes (fig. a), which allows us to set one prin- cipal curvature to zero and systematically vary the other, κ. to sample concave (κ< ) and convex (κ> ) curvatures we performed md simulations of hemicylindrical open-ended a b c d fig. . effect of nanoscale curvature on hydrophobicity. (a) simulation snapshot of a ( , ) hcnt in water. only the water molecules in observation volumes, v, near the concave (blue and white) and the convex (red and white) surfaces are shown for clarity. hcnt–water interactions are scaled by λ = . . (b) compressibility of water in the first hydration shell normalized by its value near a flat graphene sheet, χ/χgraphene , as a function of the surface curvature, κ. (c) water density fluctuations, pv (n), in cylindrical-shell volumes on the concave and convex sides of hcnts computed using the indus method ( ). (d) excess chemical potential, βµexv = − ln pv ( ), for creating a cavity of size and shape v near various hcnts. these results show that concave nonpolar surfaces are more hydrophobic than convex ones. surface hydrophobicity is sensitive to and increases substantively with nanoscale curvature of concave surfaces but is rather insensitive to curvature for convex surfaces ( ). (n,n) armchair carbon nanotubes (hcnts) with n = , , and , as well as flat graphene sheets (κ = ) in water. we system- atically varied the strength of surface–water attractions using a scaling factor λ, as described in si appendix. for λ = . , the wettability of the reference graphene surface is similar to that of a ch -terminated sam surface, in that they both have similar water droplet contact angles (si appendix). fig. shows results for systems with λ = . ; results for other λ-values are included in si appendix. fig. b shows the isothermal compressibility of interfacial water near the curved hcnt surfaces, obtained by performing md simulations over a range of pressures and taking the pres- sure derivative of the average number of waters, 〈nv〉, in an interfacial observation volume, v, using χ ≡ −(∂ ln〈nv〉/∂p)t (see si appendix for details). to compare different curvatures on the same footing we deliberately selected subvolumes v, such that they contain about the same number of water molecules, 〈nv〉, on average. the hydration water near the concave sur- face of the ( , ) hcnt is almost twice as compressible as that near a flat graphene sheet. the hydration shell compressibility decreases monotonically with κ as the surface becomes less con- cave, and then increasingly convex, suggesting that concave non- polar surfaces are more hydrophobic than convex surfaces of the same curvature. our results for the convex surfaces are consistent with those of sarupria and garde ( ), who showed that the hydration shell compressibility is the lowest near methane-sized hydrophobic solutes and increases monotonically with increasing solute size (decreasing curvature). for convex surfaces, results in fig. b are also in good agreement with those of jabes et al. ( ), who found that hydration shell compressibility reduces by about % near a hydrocarbon-coated cylinder with κ = . nm− , rela- tive to that near a flat plate. our results highlight an asymmet- ric dependence of hydration shell compressibility on the sign of nanotube curvature. in particular, compressibility is more sensi- tive to concave curvatures (i.e., the absolute value of slope |∂χ ∂κ | is higher for κ< than for κ> ). we note that this asymme- try in slopes depends on the exact definition of curvature. as shown in si appendix, fig. s and discussed in si appendix, sec- tion i-f, defining κ based on the curvature of liquid water inter- face reduces the asymmetry; nevertheless, the observation that compressibility of hydration water is higher near concave sur- faces compared with convex ones holds true. this observation suggests that introduction of both positive and negative curva- tures (of equal magnitude) on an otherwise flat surface ought | www.pnas.org/cgi/doi/ . /pnas. xi et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/cgi/doi/ . /pnas. sp ec ia l fe a tu r e c h em is tr y a c e f d b fig. . how chemical patterns influence surface hydrophobicity. (a) top views of a ch -sam containing n = -oh head groups (red) separated by s = , , , and ch head groups (cyan), respectively. a × × . -nm observation volume, v (white), is placed above the patches. (b) free energies of emptying v near the four patches in a. (c) snapshot of the n = , s = sam surface (space-filled) illustrating the sam–water interface, z(x, y), for a biased simulation that displaces all but a fraction, ρv = . , of waters from v. z is the vertical distance from the bottom of the sam surface. (d) top views of the interfaces for n = and s = , , and for partially wet configurations (ρv = . , . , . from left to right). (e) same as b for n = , and s = , , and . (f) same as d for n = , and s = and . to increase its hydrophobicity. our assertion that a rugged non- polar surface is more hydrophobic than a flat one is supported by mittal and hummer ( ), who find that the interfacial free energy of a sinusoidal hydrophobic surface increases with its amplitude. fig. c shows probability, pv (n ), of observing n water molecules in an interfacial observation volume, v. to compare the various curved surfaces on an equal footing we varied the axial length of v, such that they all contain roughly the same number of waters on average, 〈nv〉≈ . the pv (n ) distribu- tions, for all of the surfaces obtained using the indus method ( ), are gaussian (parabolic) near the mean but display promi- nent non-gaussian low-n tails. patel et al. ( , ) have shown that such low-n fat tails serve as an excellent signature of surface hydrophobicity; the fatter the tail, the more hydrophobic the sur- face. in qualitative agreement with the data for compressibility (fig. b), fig. c shows that while the low-n tails of pv (n ) are rather insensitive to curvature for convex surfaces, curvature sig- nificantly influences the tails, and thereby the hydrophobicity of concave surfaces. these low-n tails in pv (n ) are directly connected to the work required to create a cavity of the size and shape of v near the surface ( , ) through βµexv = − ln pv ( ). fig. d shows the dependence of βµexv on the absolute curvature of the hcnt. it is far easier to create a cavity in the vicinity of concave surfaces compared with convex ones, suggesting that concave nonpolar surfaces are more hydrophobic and ought to bind hydrophobic solutes more strongly than convex surfaces, consistent with the results of setny ( ). these results point to an important short- coming of sa models, which assume that the thermodynamic driving forces for burying concave and convex hydrophobic areas are identical. surfaces with the same chemistry but different patterns can have widely varying hydrophobicities. flat sams provide excellent sys- tems to analyze the effects of chemical patterns on hydrophobic- ity without being encumbered by the effects of surface topogra- phy ( , , ). we study sam surfaces with n hydrophilic sites (–oh head groups) in a background of hydrophobic sites (–ch head groups) (see si appendix for details). we hold n constant but vary the separation, s, the number of ch sites between adja- cent oh sites (fig. a). we studied seven patterns with n = (s = , , , ) and n = (s = , , ) and monitored water den- sity fluctuations in cuboidal volumes, v, of width . nm placed above a square -nm × -nm surface patch containing the chem- ical patterns (fig. a). which of the four patches in fig. a is the most hydropho- bic and which is the most hydrophilic? additive models would predict that all patterns are equally hydrophobic; however, the answer is both nonintuitive and nontrivial. fig. b shows the hydrophilicity of the n = patterns, as quantified by the reversible work, µexv , required to empty the interfacial volume, v; the higher the µexv , the more hydrophilic the patch. although the number of hydrophobic and hydrophilic sites is the same in the four patches, µexv is different and varies nonmonotonically with s. the s = patch with –oh groups separated by one methyl group is the most hydrophilic of the four. thus, embedding a given number of –oh groups in a hydrophobic background decreases xi et al. pnas | december , | vol. | no. | d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf hydrophobicity most effectively not when the –oh groups are adjacent to one another in a cluster (s = ), but when they are separated by a methyl group (s = ). what leads to such a nonmonotonic response to the chemi- cal context presented by the patch? to answer this question, we visualize dewetting of the patches by following the average water density field in indus simulations, where biasing potentials are used to dewet v. for example, consider a biased simulation of the n = , s = sam surface, which displaces all but a fraction, ρv = . , of waters in v on average; in fig. c we show the cor- responding sam–water interface, z (x,y) [si appendix includes details of the interface calculation ( )]. as v is dewetted for the s = pattern (fig. d, top row) the interface detaches from the patch at the borders but remains pinned to the central cluster, forming a doughnut-shaped cav- ity above the patch. the s = pattern (fig. d, middle row) pins the interface over a larger area, even when the observation vol- ume is significantly dewetted, indicating the difficulty of dewet- ting the region in its vicinity. indeed, the work of cavity creation µexv is several kbt larger for the s = pattern than for s = . interestingly, as the –oh groups are further separated (s = ) the interface depins from the space between the hydrophilic sites, making cavity formation easier, as reflected in lower µexv (fig. b). the s = pattern facilitates interface formation between the oh sites as well as pins the interface outside v; thus, the patch defined by v does not bear the full brunt of the pinning by the hydrophilic sites. therefore, v can be emptied more easily, leading to the nonmonotonic dependence of hydrophobicity on s. patterns with seven –oh sites also display similarly context- dependent hydrophobicity that varies nonmonotonically with s (fig. e and f). collectively, these results highlight that even for simple flat surfaces, surface–water adhesion strength depends on chemical patterns in a manner that is complex and not anticipated by addi- tive models. our results are consistent with previous work on the a b c d e f fig. . protein hydrophobicity is nonadditive. (a) wild-type hydrophobin-ii shown in space-fill representation (white, nonpolar; green, polar; red, anionic; blue, cationic). two residues, leu- (gray, at the center) and asp- (red, at the periphery of the patch), were swapped to create the mutant (right). (b) top and front views of hydrophobin-ii are shown along with the observation volume, v, that covers the hydrophobic patch and asp- . (c) the free energetics, βgv (n) = − ln pv (n), of observing n waters in v, is shown for wild type and swap mutant in units of the thermal energy, β− = kb t . (d) the difference in gv (n) for wild-type and mutant proteins is shown; the three lines are fits over the ranges – , – , and – , respectively. (e) interface encompassing dewetted region (orange mesh) near wild-type and mutant proteins for a dewetted state with ≈ (waters hidden for clarity). (f) βgv (n = ) is significantly larger for the mutant than for the wild type, highlighting the diminished hydrophobicity of the patch on the swap mutant. hydration of patterned surfaces. for example, luzar and leung ( ) showed that in confined geometry a regularly spaced distri- bution of hydrophilic sites is much more effective in slowing the formation of vapor tubes that trigger the evaporation process. vanzo et al. ( ) also found that uniformly distributing charges (rather than segregating them) in a hydrophobic surface makes the surface significantly more hydrophilic. protein hydrophobicity is context-dependent and nonadditive. we use water density fluctuations-based measures to characterize the hydrophobicity of a protein, hydrophobin ii (protein data bank id code b ) ( ), which is a small globular (≈ kda) fungal protein secreted in the extracellular environment. it dis- plays a relatively large hydrophobic patch that makes the pro- tein amphiphilic and surface active at the vapor–liquid inter- face of water, enabling the formation of hydrophobic coatings and sheaths that cover fungal spores ( ). we note that when viewed from the atomic-level hydrophobicity scale perspective of kapcha and rossky ( ) the patch appears actually quite heterogeneous (si appendix). acharya et al. ( ) interrogated the hydrophobin-ii surface using binding of small methane-like nonpolar solute probes present in an aqueous solution, which revealed the hydrophobic patch referred to above. we have also used the indus method to characterize the hydrophobic- ity of hydrophobin-ii using larger benzene-shaped probe vol- umes. while the benzene-shaped probes also identified the large hydrophobic patch, interestingly, we found that protein hydrophobicity can also depend on the size and shape of probe itself ( ). here we study how altering the local context affects the hydrophobicity of the patch. to this end, we created a swap mutant by switching the positions of residues asp- and leu- in the wild-type protein to asp- and leu- in the mutant (fig. a). such a swap places a charged residue from the edge to the center of the patch but does not perturb the overall | www.pnas.org/cgi/doi/ . /pnas. xi et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/lookup/suppl/doi: . /pnas. /-/dcsupplemental/pnas. .sapp.pdf http://www.pnas.org/cgi/doi/ . /pnas. sp ec ia l fe a tu r e c h em is tr y structure of the protein; there are four disulfide bonds in the protein, and the backbone root mean square deviation between equilibrium wild-type and mutant structures are within thermal fluctuations (see si appendix for simulation details). to study the collective response of water to the swap we defined an observation volume, v, of width . nm that envelopes a con- tiguous region of nine nonpolar residues and asp- (fig. a and b) and contains roughly waters on average. we calcu- lated the free energetics of water density fluctuations, βgv (n ) = − ln pv (n ), near the wild-type and the swap mutant proteins (fig. c), allowing comparisons between two patches with the same number and type of amino acids but having different geo- metrical arrangement. it is clear that over the entire range of water numbers one expends more work to dewet the patch in the swap mutant compared with that in the wild-type protein, high- lighting the decreased hydrophobicity of the patch in the swap mutant. the additional work, ∆gv (n ), required to dewet the swap mutant relative to the wild type (fig. d) shows three approxi- mately linear regimes. the one at high n arises from the slightly different number of average water molecules in the volumes near the two patches, whereas the differences in the low-n fat tails are visible in the n range from to and reflect the increased hydrophilicity of the mutant patch. further depletion in water numbers encroaches directly on the hydration shell of the charged asp- in the swap mutant, which is costly, leading to the steep linear region for n < . instantaneous interfaces encompassing the dewetted regions near the wild-type and the mutant proteins with roughly water molecules in v (fig. e) show that a large cavity develops near the patch in both cases (see si appendix for details). however, the cavity shape near the wild-type hydrophobin-ii is contiguous, whereas that near the swap mutant is doughnut-shaped in that water remains pinned to the central asp- . importantly, it costs nearly kbt less to displace all but five waters from v and open the cavity near the wild-type patch (fig. f), consistent with the larger hydrophobic- ity of the patch in the wild-type protein. given that the number and types of amino acid residues are identical in the two patches, their disparate hydrophobicities are not be anticipated by addi- tive models. conclusions and outlook characterizing the hydrophobicity of heterogeneous nanoscale surfaces has remained a major challenge. additive and context- independent descriptions of hydrophobicity are frequently used by computational drug design approaches and implicit sol- vent methods to estimate the hydrophobic contribution to association ( ). we showed that molecular measures based on water-density fluctuations effectively capture the collective response of water to complex surfaces and serve as robust nanoscale measures of hydrophobicity. we used these mea- sures to show that the hydrophobicity of proteins and nanos- tructured solutes is strongly influenced by their chemistry and topography in a manner that cannot be captured by additive approaches. in particular, by studying hemicylindrical nonpolar surfaces we showed that concave nonpolar surfaces are more hydrophobic compared with flat or convex surfaces, as reflected in the higher water compressibility, larger water density fluctuations, and eas- ier cavity formation in the vicinity of concave surfaces. the rel- ative ease of displacing water from a concave region suggests an important role for concave features (e.g., clefts or pockets) in binding to hydrophobic solutes. molecular details of water den- sity fluctuations in a concave interfacial region and how they are coupled to an approaching ligand are also known to be important in ligand binding kinetics ( ). the asymmetric dependence of hydrophobicity of nonpolar surfaces on curvature implies that introducing nanoscale topo- graphical features on an otherwise flat nonpolar surface would increase its hydrophobicity. such features may be integral parts of the structure of proteins and other macromolecules, or they may appear fleetingly through conformational changes of a flex- ible molecule or surface, suggesting that flexible nonpolar sur- faces ought to be more hydrophobic than rigid ones. this expec- tation is consistent with the results of andreev et al. ( ), who showed that flexible nanotubes are more hydrophobic, expel water from their interior, and reduce the flow of water through them. enhanced hydrophobicity as a result of flexibility should also influence water phase behavior and evaporation rates under nonpolar confinement. indeed, altabet and debenedetti ( ) and altabet et al. ( ) have shown that water confined between flexible nonpolar surfaces is less stable and evaporates signifi- cantly faster relative to water confined between the correspond- ing rigid surfaces. we also showed that patches with the same chemical composi- tion but different geometrical arrangements, either on sam sur- faces or on proteins, can display significantly different hydropho- bicities. specifically, we showed that hydrophilic sites are most effective in increasing the hydrophilicity of a nonpolar surface not when they are clustered together but when they are sepa- rated from each other by one hydrophobic site. favorable direct (electrostatic) interactions between polar or charged sites and water can pin water molecules not only in direct contact with the site but also in subsequent hydration shells. thus, polar sites separated by ∼ nm can pin water effectively in the region between them, and, similarly, a polar or charged site has a larger impact on hydrophobicity and interactions when placed at the center of a hydrophobic patch instead of at its periphery. such context dependence will play an important role in protein engineering [for example, in engineering of antibodies to opti- mize both affinity and specificity ( )] as well as in materials design. finally, our work suggests that context-dependent hydra- tion of protein surfaces can be characterized effectively using water-density fluctuations and associated quantities. such char- acterization captures many-body effects that are missing in additive models, which presents an important advantage, espe- cially with regard to developing predictive approaches. the suc- cess of bioinformatic approaches in predicting protein struc- ture from sequence has relied on the availability of protein sequence–structure information in the protein data bank ( ). the sequence–structure relationship is nonadditive and com- plex, similar to the relationship between chemistry and topog- raphy, and hydrophobicity. if extensive data on the hydration of diverse proteins were available, we speculate that data ana- lytics approaches could be applied to estimate the hydropho- bicity of protein surfaces. such information about hydration is not available in the protein data bank, which contains information about only the strongly localized crystal waters. however, our approach using water-density fluctuations-based characterization of hydrophobicity combined with advances in high-performance computing provides a route to developing an extensive “protein hydration data bank,” which could not only support development of nonadditive predictive approaches but also help efficient prediction of biomolecular interactions in complex systems. acknowledgments. we thank cuyler bates for preparation of nan- otube coordinates. s.g. thanks the center for computational innova- tions at rensselaer polytechnic institute for high-performance comput- ing resources. this work was supported by national science foundation 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c h m e d i c a l m e d i a original research diabetes publication date: june , sanjay kalra, a panneer selvam, amish v shah, kudugunti neelaveni, navneet agrawal, sambit das, vinay k dhandhania, subodh jain, pankaj kumar jha and neha raval . bharti hospital, karnal, india; . aruna diabetes centre, chennai, india; . advanced diabetes centre pvt. ltd., surat, india; . endocare, hyderabad, india; . diabetes obesity & thyroid centre, gwalior, india; . dr sambit's centre of diabetes and endocrinology, bhubaneswar, india; . diabetes care centre, ranchi, india; . diabetes care centre, allahabad, india; . torrent pharmaceuticals ltd., ahmedabad, india o bjective: despite its beneficial effect on postprandial hyperglycemia, the effect and usage of voglibose for the management of type diabetes mellitus (t dm) in routine clinical practice remains undetermined. the objective of this non-interventional study is to determine the real-world effectiveness of voglibose in terms of efficacy and safety, and the usage pattern as monotherapy or add-on treatment in patients with t dm in india. design: this was a -week, multi-center, prospective, observational study. methods: adults with inadequately controlled t dm (glycated hemoglobin [hba c] . – . % despite diet, exercise and/or antidiabetic agents), treated with voglibose monotherapy or as add-on therapy, were recruited from sites. the primary endpoint was change in hba c. secondary endpoints were change in fasting and postprandial blood glucose (fbg and ppbg) levels and bodyweight, pattern of usage and safety assessments. mean change in hba c, fbg and ppbg levels were analyzed using paired t-test with significance value of %. trial registration: ctri no. ctri/ / / . results: a total of , participants were enrolled and , completed two study visits. mean hba c was . % at baseline, which reduced to . % at week ( . % change, p< . ). at week , mean reductions in fbg and ppbg levels and bodyweight were . mg/dl (p< . ), . mg/dl (p< . ), and . kg (p= . ), respectively. most participants ( %) received voglibose as add-on therapy and the most commonly prescribed concomitant oral antidiabetic drugs were metformin and sulfonylurea ( %). flatulence ( . %) and abdominal discomfort ( . %) were the most commonly reported adverse events in the study. conclusions: voglibose, monotherapy or add-on therapy, significantly reduced hba c, fbg and ppbg and was well tolerated in patients with t dm in a real-world setting. keywords voglibose, hba c, hyperglycemia, diabetes, observational study disclosures: sanjay kalra, a panneer selvam, amish v shah, kudugunti neelaveni, navneet agrawal, sambit das, vinay k dhandhania and subodh jain have received consulting fees from torrent pharmaceuticals ltd. pankaj kumar jha and neha raval are full-time employees of torrent pharmaceuticals ltd. acknowledgments: the authors wish to thank the participating investigators for contributing participants’ data for this study. altaf makwana and ashutosh kakkad were involved in the technical editing of the manuscript. robin nath and saurabh kumar provided administrative support for the conduct of this study. the authors also acknowledge mediception science pvt ltd. for providing writing assistance, and devum research for statistical analysis, which were funded by torrent pharmaceuticals ltd., india. review process: double-blind peer review. compliance with ethics: all ethical approvals required for the study was obtained before the start of the trial and details have been mentioned in trial registration: www.ctri.nic.in (ctri/ / / ). the study was approved by sangini hospital ethics committee, ahmedabad, gujarat. all procedures were followed in accordance with the responsible committee on human experimentation and with the helsinki declaration of and subsequent revisions, and informed consent was received from the patient involved in this study. authorship: all named authors meet the international committee of medical journal editors (icmje) criteria for authorship of this manuscript, take responsibility for the integrity of the work as a whole, and have given final approval for the version to be published. access: this article is freely accessible at touchendocrinology.com ©touch medical media . received: december , accepted: february , published online: march , citation: us endocrinology. ; ( ): – corresponding author: pankaj kumar jha, torrent pharmaceuticals ltd., torrent house, off ashram road, ahmedabad – , india. e: pankajjha@torrentpharma.com support: the study was funded by torrent pharmaceuticals ltd., india. torrent pharmaceuticals ltd. have provided oversight on the conduct of the study, including design, collection, compilation and analysis of data. prospective multicenter observational study of voglibose in type diabetes—victory doi: https://doi.org/ . /use. . . . original research diabetes u s e n d o c r i n o l o g y india, being an epicentre of diabetic population, has faced an increase in disease–prevalence of more than double over the past years, and is projected to have about . million cases by the year . in addition to achieving target glycated hemoglobin (hba c) < %, intensive control of blood glucose levels is important in order to retard or arrest the development and progression of the microvascular complications of diabetes. changes in postprandial blood glucose (ppbg) levels precede those in fasting blood glucose (fbg) and finally in hba c. hence, the management of both fbg and ppbg is critical to achieving full glycemic control; the latter often being more challenging to achieve. voglibose, an alpha-glucosidase inhibitor, reduces ppbg when used alone or in combination with other antidiabetic drugs. its efficacy and safety have been well established in multiple randomized controlled trials (rcts), which conclude it to be an effective oral antidiabetic drug (oad) with a good tolerability profile and cardiovascular benefits. , the design of rcts, often considered the gold standard for evaluating the safety and efficacy of therapeutic agents, necessitates strict eligibility criteria for patient inclusion, which questions the representability of the study population to that encountered in clinical practice. real-world studies, on the other hand, demonstrate the picture of large datasets from diverse patient populations. additionally, studies of an observational nature, wherein data have been collected over a long period, provide evaluation of the long-term safety and effectiveness of the drug in large heterogeneous populations, as well as information on utilization patterns. thus, real-world studies have broader generalizability and are an effective tool to confirm the results of rcts in clinical practice. however, there are scarce data available to demonstrate the effectiveness of voglibose in real-world settings, especially in the indian population. currently available literature covers a few indian studies of voglibose in real-life settings, which include only a small number of patients from one or two centers. , hence, this non-interventional, observational study was planned with an objective to assess the real-world effectiveness of voglibose by determining its efficacy, safety and usage as monotherapy or add-on treatment in patients with type diabetes mellitus (t dm) from multiple sites in india. methods victory (a non-interventional, multicenter, prospective, observational study to understand usage and effect of voglibose as monotherapy or add on treatment in indian type diabetes patients) was a -week, prospective, observational, multicenter study conducted between october and august . participants were recruited by private clinical practitioners (endocrinologists and diabetologists) from both urban and rural sites across india. adult participants, of either sex, aged ≥ years, with inadequately controlled t dm (hba c . – . %) despite diet, exercise and/or antidiabetic agents were included in the study. patients with t dm, those with history of hypoglycemia episodes or ketoacidosis, and other medical conditions that could potentially hinder the safe conduct of the study, were excluded. even though considered safe, use of voglibose is contraindicated in patients with ketoacidosis and is also associated with reports of hypoglycemia. based on these facts, we excluded patients with a history of hypoglycemic or ketoacidosis from this study. patients participating in any other clinical trials, pregnant or lactating women and those of child-bearing potential and not using adequate contraception, were also excluded. data of participants receiving voglibose ( . mg or . mg; either alone or as add-on with other oads based on investigators’ discretion) in routine clinical practice were collected. the study was performed in compliance with the principles of the declaration of helsinki, in accordance with the international conference of harmonization guideline for good clinical practice, and in accordance with applicable regulatory requirements. all participants provided written informed consent. the study was registered at clinical trial registry of india with registration number ctri/ / / . endpoints the primary endpoint was mean change in hba c levels from baseline to week . secondary endpoints were mean change in fbg, ppbg, and body weight from baseline to week ; the usage patterns for voglibose (monotherapy or add-on therapy with other oads); and adverse events (aes) reported during the study period. aes were classified as mild (no interference with usual activity), moderate (significant interference with usual activities), and severe (prevents usual activities). statistical analysis considering the large sample size, it was assumed that data were normally distributed according to the central limit theorem. continuous variables were reported as mean and standard deviation (sd) and categorical variables were presented as absolute frequency and percentage. the data were analyzed, and the mean change in hba c, fbg, ppbg and weight along with sd were subjected to statistical analysis using paired t-test with a significance value of %. the safety review was performed using intent-to-treat dataset (all participants who were recruited in the study irrespective of study completion), while efficacy analysis was performed using per protocol dataset (all participants who completed the study as per the protocol). results a total of , participants were enrolled in the study; of these , participants ( . %) completed the study (figure ). the mean age and body weight of the study participants were . years (sd . ) and . kg (sd . ), respectively. study population included more males (n= ; . %) than females (n= ; . %). the baseline characteristics of study participants are shown in table . efficacy mean hba c was . % (sd . ) at baseline, and decreased to . % (sd . ) at week (p< . ). however, differences between two dose groups ( . mg and . mg) were non-significant for either monotherapy (p= . ) or add-on therapy (p= . ) (figure ). significant (p< . ) reductions in fbg ( . mg/dl; from . to . mg/dl) and ppbg ( . mg/dl; from . to . mg/dl) were reported at week . reduction in fbg was significantly higher among participants who received . mg monotherapy compared to . mg monotherapy of voglibose, while between-dose group differences of fbg reduction for voglibose add-on therapy were non-significant. reduction in ppbg was significantly higher (p< . ) among participants who received . mg dose compared to . mg dose of voglibose with either as monotherapy or add-on therapy (figure ). mean reduction in bodyweight ( . kg; p= . ) was not significant at week (figure ). voglibose efficacy and safety in t dm u s e n d o c r i n o l o g y safety the aes reported during this study were flatulence (n= ; . %), abdominal discomfort (n= ; . %), gastritis (n= ; . %), diarrhea (n= ; . %) and leg pain (n= ; . %) (table ). most of the aes were mild (n= ), and two were moderate. no serious ae or hypoglycemia episode was reported in any participant. aes were managed by the investigators as per their routine clinical practice. usage pattern a total of ( . %) participants received voglibose as monotherapy, while , ( . %) received it as add-on therapy to other antidiabetic drugs. of those who received voglibose as add-on therapy, . % table : baseline characteristics of study participants overall, n= , age, years . ( . ) weight, kg . ( . ) height, cm . ( . ) hba c, % . ( . ) fbg, mg/dl . ( . ) ppbg, mg/dl . ( . ) all data are presented as mean (standard deviation). fbg = fasting blood glucose; hba c = glycated hemoglobin; ppbg = postprandial blood glucose. figure : participant disposition hba c = glycated hemoglobin; pp = per protocol. enrolled at visit (n= , ) visit completed (n= , ) pp dataset analysis (n= , ) lost to follow-up = discontinued voglibose = hba c values missing = figure : changes in glycated hemoglobin levels with monotherapy and add-on therapy with voglibose p value derived using paired t-test for within group differences was p< . for both doses at week . hba c = glycated hemoglobin. overall m e a n h b a c (% ) voglibose monotherapy ( . mg) voglibose monotherapy ( . mg) voglibose as add-on therapy ( . mg) voglibose as add-on therapy ( . mg) . . . . . . (p< . ) (p< . ) (p= . ) (p= . ) (p< . ) (p< . ) (p< . ) . . . . week baseline figure : mean change in fasting blood glucose (a) and postprandial blood glucose (b) from baseline to week p value derived using paired t-test for within group differences was p< . for both doses at week ; p value derived using independent sample t-test for between group differences was p= . and p= . for fbg, and p= . and p= . for ppbg with monotherapy and add-on therapy, respectively. fbg = fasting blood glucose; hba c = glycated hemoglobin; ppbg = postprandial blood glucose. (p= . ) baseline week baseline week (p= . ) (p< . ) (p< . ) (p< . ) (p< . ) (p< . ) overall voglibose monotherapy ( . mg) voglibose monotherapy ( . mg) voglibose as add-on therapy ( . mg) voglibose as add-on therapy ( . mg) m e a n f b g ( m g /d l) (p= . ) (p= . ) (p< . ) (p< . ) (p< . ) (p< . ) (p< . ) overall voglibose monotherapy ( . mg) voglibose monotherapy ( . mg) voglibose as add-on therapy ( . mg) voglibose as add-on therapy ( . mg) m e a n p p b g ( m g /d l) a b original research diabetes u s e n d o c r i n o l o g y received it as part of dual drug therapy, . % as triple-drug therapy, . % as quadruple-drug therapy, . % as five-drug therapy, and . % as other combinations. a total of ( . %) participants received voglibose as add-on with metformin and sulfonylurea; ( . %) with metformin, sulfonylurea and dipeptidyl peptidase inhibitors (ddp -i); ( . %) with only metformin; ( . %) with metformin and ddp -i; and ( . %) with only sulfonylurea. a total of ( . %) participants received voglibose as part of other antidiabetic combinations that included various permutations with sulfonylureas, metformin, ddp -i, and insulin, as well as thiazolidinediones. discussion alpha-glucosidase inhibitors (voglibose and acarbose) were introduced as first-line therapy to attain better ppbg control by the research society for the study of diabetes in india diabetes management guidelines. despite being reported as an efficacious and safe treatment option, the utilization of voglibose remains undetermined for the management of diabetes in real-life settings. the victory study includes analysis of a large population of patients with t dm from varied geographical regions in india. most participants ( . %) in our study were taking multiple antidiabetic agents. voglibose was most commonly used as add-on to metformin and sulfonylureas ( . %)—a finding seen in several clinical trials of patients with t dm treated with voglibose as add-on therapy. – twelve-week treatment with voglibose resulted in significant reduction in hba c ( . %, p< . ). similar reductions in hba c with monotherapy or add-on therapy with voglibose have been reported in randomized studies. in a -month, single-blind, randomized, prospective study that compared voglibose with miglitol and acarbose, voglibose ( . mg three times daily for month followed by . mg three times daily for months) significantly reduced hba c by . %. jindal et al. (n= ; hba c – %) reported reduction in mean hba c of . % with voglibose ( . mg three times daily) when added to glimepiride ( mg twice daily) and metformin ( mg twice daily). in a retrospective study, overweight or obese indian participants with diabetes that was inadequately controlled with metformin and sulfonylureas (body mass index ≥ kg/m ; hba c level > . – . %), received voglibose (n= ; . or . mg three times daily) or acarbose (n= ). voglibose showed significant reduction in hba c level ( . %) at weeks ( . ± . versus . ± . ; p< . ) and at weeks ( . ± . versus . ± . ; p< . ). mean reduction of . mg/dl in fbg was observed in our study. similar reductions of . mg/dl and . mg/dl have been reported by rao et al. and ismail et al., respectively. on the other hand, talaviya et al. and jindal et al. have reported fbg reductions of . mg/dl and . mg/dl, respectively. we observed a mean reduction of . mg/dl in ppbg. this is similar to reductions of . mg/dl and . mg/dl reported by rao et al. and jindal et al., respectively, while it is higher than the reported reduction of . mg/dl in the study by talaviya et al. in the present study, significantly higher reduction of fbg and ppbg was observed among participants on voglibose . mg dose compared to . mg dose, except for fbg reduction in add-on therapy group. the reductions in ppbg with voglibose suggests it may have a major role in the achievement of desired hba c. reductions in ppbg have been shown to significantly reduce hba c levels in participants with t dm. in the present study, there was a non-significant (p= . ) reduction of . kg in bodyweight observed at end of weeks. contrary to our observations, talaviya et al. reported significant reduction in body weight at weeks ( . ± . versus . ± . ; p< . ) and weeks ( . ± . versus . ± . ; p< . ) of treatment with voglibose when compared to baseline. however, jindal et al. have reported no significant change in body weight after months of treatment with voglibose. differences seen between the results of this study and that of the ones by talaviya et al. could be explained by the retrospective nature, obese patient population, and small sample size (n= ) of the latter. in our experience, voglibose monotherapy or add-on therapy was safe for use in participants with t dm, with no serious aes reported. flatulence and abdominal discomfort were the most commonly reported aes and most aes were of mild intensity. this is consistent with the safety and tolerability of voglibose reported in several studies. , , , – however, the under-reporting of safety events, as suggested by the low rates of aes, is a common problem in observational studies. , , in contrast to rcts, the data collection of aes was based on voluntary reporting by the investigators, who are more likely to report serious aes in clinical practice. figure : mean change in bodyweight from baseline to week there was no significant change in bodyweight with voglibose monotherapy. when used as add-on therapy, . mg of voglibose resulted in significant reduction in bodyweight (p< . ). . overall voglibose monotherapy ( . mg) voglibose monotherapy ( . mg) voglibose as add-on therapy ( . mg) voglibose as add-on therapy ( . mg) . . . . . . . . (p= . ) m e a n b o d yw e ig h t (k g ) (p= . ) (p= . ) (p= . ) (p< . ) . . . . . . . . . . . baseline week table : adverse events observed during study adverse event n % flatulence . % abdominal discomfort . % gastritis . % diarrhea . % leg pain . % n= , . voglibose efficacy and safety in t dm u s e n d o c r i n o l o g y . pradeepa r, mohan v. prevalence of type diabetes and its complications in india and economic costs to the nation. eur j clin nutr. ; : – . . schort rj. targeting plasma glucose: preprandial versus postprandial. clin diab. ; : – . . riddle mc. basal glucose can be controlled, but the prandial problem persists-it's the next target! diabetes care. ; : – . . vichayanrat a, ploybutr s, tunlakit m, et al. efficacy and safety of voglibose in comparison with acarbose in type diabetic patients. diabetes res clin pract. ; : – . . lee my, choi ds, lee mk, et al. comparison of acarbose and voglibose in diabetes patients who are inadequately controlled with basal insulin treatment: randomized, parallel, open-label, active-controlled study. j korean med sci. ; : – . . blonde l, khunti k, harris sb, et al. interpretation and impact of real-world clinical data for the practicing clinician. adv ther. ; : – . . riyaz m, imran s, joseph nk, saba m. role of voglibose in prevention of type diabetes in established case of impaired glucose tolerance: an observation study. int j res med sci. ; : – . . lodhi mak, khatoon as. voglibose and its significance in prediabetes: an observational study. int j med health res. ; : – . . torrent pharmaceuticals ltd. prescribing information of voglitor md (voglibose) tablet. . available at: www.torrentian.com/ pisheet/upload/pi_sheet/ .pdf (accessed february , ). . chen x, zheng y, shen y. voglibose (basen, ao- ), one of the most important alpha-glucosidase inhibitors. curr med chem. ; : – . . santra d, totade s. prescribing trends of antidiabetic fixed dose combinations in a rural tertiary care teaching hospital in central india: an observational, cross-sectional study. research journal of pharmacology and pharmacodynamics. ; : . . kumar l, gupta sk, prakash a. assessment of the prescription pattern of anti-diabetic drugs in type- diabetes mellitus participants. the pharma innovation. ; : – . . aparna a, latha s, nagarjun g, et al. a study on drug utilization pattern and effectiveness of oral hypoglycemic agents in diabetes mellitus. pharmatutor. ; : – . . ismail d, deshmukh s. comparative study of effect of alpha glucosidase inhibitors-miglitol, acarbose and voglibose on postprandial hyperglycaemia and glycosylated hemoglobin in type- diabetes mellitus. int j pharm bio sci. ; : – . . jindal a, jindal m, kaur m, et al. efficacy and safety of voglibose as an add-on triple drug in participants of type two diabetes mellitus uncontrolled with glimepiride and metformin in punjabi population. indian journal of basic and applied medical research. ; : – . . talaviya pa, saboo bd, dodiya hg, et al. retrospective comparison of voglibose or acarbose as an add-on therapy to sulfonylureas in western indian participants with uncontrolled overweight/obese type diabetes. diabetes metab syndr. ; : – . . rao c, faruqui aa. efficacy and safety of oral triple drug combination (voglibose, glimepiride and metformin) in the management of type diabetes mellitus. journal of current research and review. ; : . . yamasaki y, katakami n, hayaishi-okano r, et al. alpha-glucosidase inhibitor reduces the progression of carotid intima-media thickness. diabetes res clin pract. ; : – . . seino y, fujita t, hiroi s, et al. alogliptin plus voglibose in japanese participants with type diabetes: a randomized, double-blind, placebo-controlled trial with an open-label, long-term extension. curr med res opin. ; (suppl. ): – . . deb t, chakrabarty a, ghosh a. adverse drug reactions in type diabetes mellitus patients on oral antidiabetic drugs in a diabetes outpatient department of a tertiary care teaching hospital in the eastern india. int j med sci public health. ; : – . . dabhi as, bhatt nr, shah mj. voglibose: an alpha glucosidase inhibitor. j clin diagn res. ; : – . . mathieu c, barnett ah, brath h, et al. effectiveness and tolerability of second-line therapy with vildagliptin vs. other oral agents in type diabetes: a real-life worldwide observational study (edge). int j clin pract. ; : – . the short observation period and the lack of a control group are the main limitations of this study. however, the latter increases the propensity of the results to be more generalizable and replicable in clinical practice, which is necessary to complement the information retrieved from rcts. real-life studies and rcts both have strengths and limitations, and those should be appreciated as complementary to one another. the strength of the victory study is the large sample size from multiple real-life set-ups across india. this clearly supports widespread use of voglibose in patients with t dm in real-world practice. additionally, it also indicates the enthusiasm and interest of both patients and healthcare professionals to be part of such undertakings. having excluded the non-qualifying and incomplete data, we tried to optimize the quality of the data, which were utilized for the final analysis in this study. conclusions the victory study demonstrates the real-life effectiveness of voglibose monotherapy or add-on therapy, which significantly reduced hba c, fbg, and ppbg in patients with t dm. voglibose was most commonly added with metformin and sulfonylureas. it was well-tolerated by the participants with t dm without significant weight change. the efficacy of voglibose in a large population with t dm seen in this study is the real-world reflection of the results obtained in randomized trials. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ brief report evidence that rho guanine nucleotide exchange factor (arhgef ) on q is a type diabetes susceptibility gene in the old order amish mao fu, mona m. sabra, coleen damcott, toni i. pollin, lijun ma, sandra ott, john c. shelton, xiaolian shi, laurie reinhart, jeffrey o’connell, braxton d. mitchell, leslie j. baier, and alan r. shuldiner , rho guanine nucleotide exchange factor (arhgef ), located on chromosome q , is involved in g protein signaling and is a pathway known to play a role in both insulin secretion and action. we genotyped single nucle- otide polymorphims (snps) in arhgef and compared the genotype frequencies of subjects with type diabetes (n � ) or type diabetes/impaired glucose tolerance (igt) (n � ) with those of control subjects with normal glucose tolerance (ngt) (n � ). thirty snps, spanning the entire gene, were significantly associated with type diabetes or type diabetes/igt. the most significantly associated snp was rs (intron ), in which the less common allele was the risk allele (odds ratio [or] . [ % ci . – . ], p � . for type diabetes vs. ngt and . [ . – . ], p � . for type diabetes/igt vs. ngt). in an expanded set of nondiabetic subjects (n � ), most of the type diabetes– and igt-associated snps were significantly associated with glucose levels during an oral glucose tolerance test, with the same snp (rs ) showing the most significant associations (p � . ). all type diabetes– and igt-associated snps were in high linkage disequilibrium and constitute a single -kb hap- lotype block. these results, coupled with similar findings in pima indians, suggest that sequence variation in arhgef may influence risk of type diabetes. diabetes : – , t ype diabetes has a substantial genetic compo- nent, but the polygenic nature of this disease has complicated the identification of susceptibility genes ( ). we previously reported ( ) evidence for linkage of type diabetes and impaired glucose toler- ance (igt) to chromosome q -q (logarithm of odds . , p � . ) in the old order amish that has also been reported in several other populations ( – ). genotyping of multiple single nucleotide polymorphisms (snps) within the region of linkage as part of our linkage disequilibrium (ld) mapping studies in the amish pointed to a region containing rho guanine nucleotide exchange factor (arhgef ). arhgef is of activators of rho gtpases that play a fundamental role in g protein signal- ing and therefore many aspects of cellular regulation ( , ), including �-cell apoptosis, insulin secretion ( – ), insulin signaling ( – ), and lipid metabolism ( , ). we hypothesized that variation in arhgef may affect insulin secretion and/or action and, as a result, type diabetes susceptibility. we sequenced the exons, intron- exon boundaries, and . kb of the putative proximal promoter region of arhgef . a total of variants were identified including nonsynonymous (g s and r h), synonymous (s s and n n), in the � flanking region, in the �-untranslated region, and in the �- untranslated region; all other variants were in introns and did not predict any obvious alterations in rna splicing. fifty-two snps among the snps identified from sequencing and among validated snps in the snp data- base (dbsnp, available at http://www.ncbi.nlm.nih.gov/ projects/snp/) covering the entire . -kb region comprising arhgef and immediately flanking the gene from . kb upstream of the adenine thymine guanine start site to . kb downstream of the polyadenylation signal (average density snp per . kb) were genotyped in type diabetic, igt, and normal glucose tolerant (ngt) individuals. these individuals were also tested for association with type diabetes and type diabetes/igt (fig. ). all snps conformed to hardy- weinberg expectations. using an additive model, of the snps spanning the entire arhgef gene were signif- icantly associated with type diabetes or type diabetes/ igt (p � . – . ) (table and fig. ). the two snps most strongly associated with type diabetes and type diabetes/igt were rs in intron (p � . and from the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland; the diabetes molecular genetics section, phoenix epidemiology and clinical research branch, national institute of diabetes, digestive, and kidney diseases, national institutes of health, department of health and human services, phoenix, arizona; and the geriatric research and education clinical center (grecc), veterans administration medical center, baltimore, maryland. address correspondence and reprint requests to alan r. shuldiner, md, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, w. redwood st., room , baltimore, md . e-mail: ashudin@medicine.umaryland.edu. received for publication october and accepted in revised form february . published ahead of print at http://diabetes.diabetesjournals.org on march . doi: . /db - . additional information for this article can be found in an online appendix at http://dx.doi.org/ . /db - . auc, area under the curve; afds, amish family diabetes study; igt, impaired glucose tolerance; ld, linkage disequilibrium; ngt, normal glucose tolerance; snp, single nucleotide polymorphism. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , may . , respectively) and rs in intron (p � . and . , respectively). the r h variant in exon was more weakly associated with type diabetes (p � . , or . [ % ci . – . ]), with the more common allele (c allele encoding arginine at codon ) being the “risk” allele for type diabetes. the other two coding region variants (d s and s s) did not show signifi- cant association with type diabetes or type diabetes/ igt. the relationship between arhgef snps and quanti- tative traits was assessed in nondiabetic members of the amish family diabetes study (afds). eighteen of the type diabetes– and igt-associated snps located from the � flanking region to intron were significantly asso- ciated with glucose area under the curve (auc) during a -h oral glucose tolerance test (p � . ) (fig. ). these findings in nondiabetic subjects provide additional sup- port for an effect of arhgef polymorphism on glucose homeostasis. the r h was not associated with glucose auc among nondiabetic subjects. there was no evidence fig. . association analysis of snps in arhgef with type diabetes cases (dm; n � ) and combined type diabetes/igt cases (dmigt; n � ) compared with ngt control subjects (n � ). arhgef gene structure is shown above the graph, which plots the p values as a function of physical location on the chromosome. below the graph is an ld plot of the snps in arhgef . d� values are depicted as a color scale with darker shades of orange representing higher values. ld plot was generated using haploview version . . arhgef variation and type diabetes diabetes, vol. , may for association of any snps with bmi, insulin level, insulin secretion index, or homeostasis model assessment of insulin resistance. the snps within the arhgef region were in high linkage disequilibrium (ld) (fig. ). the nine snps that were most significantly associated with type diabetes or type diabetes/igt defined five haplotypes (three with frequencies � . ), accounting for � % of chromosomes in the sample (table ). the ttgttaccg haplotype was associated with a significantly higher risk of type diabe- tes/igt (p � . ), while the agaagctta haplotype was associated with a significantly lower risk of type diabetes/igt (p � . ) (global p value for test � . ). these same haplotypes were also significantly associated with glucose auc in the expanded set of nondiabetic subjects (global p value for test � . ) (table ). to explore whether snps in arhgef were in ld with a potentially functional polymorphism in a neighbor- ing gene, additional snps flanking arhgef in the . - to . -mb region (ncbi build . ) were also genotyped (supplementary table [found in an online appendix at http://dx.doi.org/ . /db - ]). the region is bound by etv and hapln � and � to arhgef , respectively. among these snps that flanked the arhgef gene, snps � of arhgef were also significantly associated with type diabetes and/or type diabetes/igt. seven of these snps were positioned with- in a predicted gene (flj ) immediately � to arhgef , while the remaining five snps (of which four had an allele frequency � . ) were each positioned within different genes (bcan, crabp , insrr, ntrk , and flj ). none of these snps were more strongly associated with type diabetes or type diabetes/igt than rs or rs , and all were in strong ld with the snps in arhgef . no snp centromeric to aghgef within the . - to . -mb region was associated with type diabetes or type diabetes/igt. by quantitative real-time pcr, we determined that arhgef is ubiquitously expressed in various tissues, including that of the liver, muscle, and pancreas (online appendix supplementary fig. ). our region of linkage on chromosome q -q spans � mb and contains at least genes, including many excellent biological candidates. our initial hypothesis-free ld mapping with � snps across the region led us to arhgef , an activator of rho gtpases. although arhgef itself is not known to play a role in glucose homeostasis, previous studies implicate rho subfamily g proteins (e.g., cdc and rac) in physiological insulin secretion ( – ). furthermore, several recent studies implicated rho gtpases in insulin signaling through acti- vation of the jun nh -terminal kinase and p mitogen- activated protein kinase pathways ( , , ). the dynamic actin rearrangement required for insulin-stimulated trans- location of glut is regulated by at least two distinct signals, one leading to the activation of phosphatidylino- table arhgef snps significantly associated with type diabetes and type diabetes/igt in the amish snp id ncbi location snp type minor allele minor allele frequency location (snp type) t d vs. ngt t d/igt vs. ngt p or ( % ci) p or ( % ci) rs a/g g . exon ( � utr) . . ( . – . ) . . ( . – . ) rs c/t t . exon ( � utr) . . ( . – . ) . . ( . – . ) rs a/g a . intron . . ( . – . ) . . ( . – . ) rs t/c t . exon (r h) . . ( . – . ) . . ( . – . ) rs a/t t . intron . . ( . – . ) . . ( . – . ) rs c/t t . exon (n n) . . ( . – . ) . . ( . – . ) rs c/t t . intron . . ( . – . ) . . ( . – . ) rs c/t t . intron . . ( . – . ) . . ( . – . ) rs a/g g . intron . . ( . – . ) . . ( . – . ) rs a/t t . intron . . ( . – . ) . . ( . – . ) rs g/t t . intron . . ( . – . ) . . ( . – . ) rs a/g g . intron . . ( . – . ) . . ( . – . ) rs a/c c . intron . . ( . – . ) . . ( . – . ) rs a/t t . intron . . ( . – . ) . . ( . – . ) rs g/t t . intron . . ( . – . ) . . ( . – . ) rs a/c a . intron . . ( . – . ) . . ( . – . ) rs c/t t . intron . . ( . – . ) . . ( . – . ) rs c/t c . intron . . ( . – . ) . . ( . – . ) rs c/t c . intron . . ( . – . ) . . ( . – . ) rs c/t c . intron . . ( . – . ) . . ( . – . ) rs c/t c . intron . . ( . – . ) . . ( . – . ) rs a/g g . intron . . ( . – . ) . . ( . – . ) rs a/g a . intron . . ( . – . ) . . ( . – . ) rs c/t c . intron . . ( . – ) . . ( . – . ) rs c/t t . intron . . ( . – . ) . . ( . – . ) rs a/g g . intron . . ( . – ) . . ( . – . ) rs a/g g . exon ( � utr) . . ( . – . ) . . ( . – . ) rs a/g g . � flank . . ( . – . ) . . ( . – . ) rs a/t a . � flank . . ( . – . ) . . ( . – . ) rs c/g c . � flank . . ( . – ) . . ( . – . ) ncbi location is based on build . . or � . denotes that the minor allele is the risk allele, while or � . denotes that the major allele is the risk allele. t d, type diabetes; utr, untranslated region. m. fu and associates diabetes, vol. , may sitol -kinase ( ) and the other to the activation of rho family small gtp-binding protein tc ( ). finally, a number of enzymes involved in lipid metabolism are influenced by gtpases including phosphatidylinositol -phosphate -kinase, diacylglycerol kinase, and phospho- lipase d ( , ). arhgef is ubiquitously expressed in various tissues, including liver, muscle, and adipose tissue. together, these data suggest that proteins involved in g protein signaling such as arhgef may play an impor- tant role in glucose homeostasis. our current study provides evidence that variation within arhgef influences risk of type diabetes/igt in the amish. given the strong ld across arhgef , it is difficult to know which snps are the causal genetic variants. in this study, rs in intron and rs in intron were most significantly associated with type diabetes and glucose traits; however, the functional consequences of these variants are unclear. because we sequenced all exons, it is unlikely that we missed common coding variants, although it is possible that other potentially functional variants, perhaps in more distal regulatory regions or within regions of introns not sequenced, may have been missed. r h was the only variant encoding an alteration in amino acid sequence that was significantly associated with type diabetes. how- ever, this variant was not the most significantly associated variant and was not associated with any of the diabetes- related quantitative traits. interesting, the r h substi- tution was significantly associated with type diabetes and insulin resistance among the pima indians who also have linkage to type diabetes on chromosome q -q ( ). however, contrary to our findings in the amish, in fig. . a: examples of two arhgef snps associated with glucose levels during a -h oral glucose tolerance test in nondiabetic subjects. b: eighteen type diabetes–associated snps were significantly associated with oral glucose tolerance test glucose auc (gauc) and post-challenge glucose levels in nondiabetic subjects. *p < . ( , common allele; , minor allele). table association analysis of arhgef haplotypes with type diabetes and type diabetes/igt and glucose auc during an oral glucose tolerance test haplotype frequency t d vs. ngt t d/igt vs. ngt glucose auc haplotype t d igt ngt haploscore p haploscore p haploscore p agaagctta . . . � . . � . . � . . tgaagctta . . . . . . . . . ataagctta . . . . . . . . . atgttaccg . . . . . . . . . ttgttaccg . . . . . . . . . haplotype frequencies were estimated with haploscore for the nine positively associated arhgef snps by case-control analysis and transmissional disequilibrium test (rs , rs , rs , rs , rs , rs , rs , rs , and rs ). global p value for test � . , . , and . for type diabetes vs. ngt, type diabetes/igt vs. ngt, and glucose auc, respectively. bold face data denote statistical significance. t d, type diabetes. arhgef variation and type diabetes diabetes, vol. , may which the r allele was the risk allele for type diabetes, the h allele was the risk allele for insulin resistance and type diabetes in pima indians. associa- tions with opposite risk alleles in the two populations may suggest that r h is not the functional variant but rather is in ld with a true functional variant. less likely, differences in genetic background or environmental expo- sures between these populations could modulate the phe- notypic expression of the r h variant to increase or decrease type diabetes risk in subjects carrying the same allele. finally, it is also possible that one or both associa- tions are false-positives due to multiple comparisons. type diabetes–associated snps in arhgef did not provide evidence for linkage to type diabetes/igt using the same family structures as in our original linkage analysis ( ) (data not shown). these findings are consis- tent with the prevailing idea that there is more than one type diabetes susceptibility gene on chromosome q - q ( ). additional genotyping of snps in neighboring genes and ld analysis revealed that the type diabetes/igt–associ- ated snps were in strong ld and constitute a large haplotype block that include one gene and two predicted genes. indeed, several snps in the predicted gene flj immediately � to arhgef were associated with type diabetes and/or type diabetes/igt. thus we cannot rule out the possibility that the functional variant or haplotype is in this gene or even possibly other nearby genes. in summary, we identified sequence variation in arhgef that may influence risk of type diabetes. further investigation of the role of arhgef in insulin secretion and signaling and functional studies of the type diabetes–associated sequence variants will be necessary to more fully understand the mechanisms underlying these associations. research design and methods the afds was founded in to identify susceptibility genes for type diabetes and related traits. details of the afds design, recruitment, pheno- typing, and pedigree structure have been previously described ( ). our initial case-control analysis included subjects with type diabetes, subjects with igt, and subjects with ngt. ngt subjects included in this analysis were required to be at least years of age at the time of examination to increase the probability of their capacity for diabetes resistance. for analysis of diabetes-related quantitative traits, an expanded set on nondiabetic subjects (with ngt and igt) was used. glucose and insulin auc during the oral glucose tolerance test were calculated using the trapezoidal rule. ho- meostasis model assessment of insulin resistance was calculated as fasting insulin (�u/ml) 	 fasting glucose (mmol/l)/ . , and insulin secretion index was calculated as ln[(insulin at min � insulin at min)/(glucose at min � glucose at min)]. informed consent was obtained from all afds participants, and the study protocol was approved by the institutional review board at the university of maryland school of medicine. sequence analysis and genotyping. direct sequencing was used to screen the exons, exon-intron boundaries, and . kb of the proximal � regulatory region of arhgef for genetic variation in afds participants ( type diabetic subjects from non–first-degree relatives of families providing evi- dence for linkage of type diabetes to chromosome q -q and ngt subjects). this sequencing set ( chromosomes) provides % power to detect at least one copy of the minor alleles for snps with allele frequencies � . . both strands were sequenced on an abi xl dna sequencer (applied biosystems) and analyzed using sequencher software (genecodes). from snps identified by sequencing and from dbsnp, snps in arhgef were genotyped in the full sample. another snps upstream and snps downstream of arhgef spanning from . to . mb were also genotyped (supplementary table ). genotyping was performed using the snpstream ultra high throughput genotyping platform (beckman coulter), snplex (applied biosystems), taqman allelic discrimination assay (applied biosystems), pyrosequencing psq hs a, or illumina golden gate assay (illumina). details regarding assays for specific snps are available from the authors upon request. the error rate, based on blind replicates for the snps examined, was – %. real time pcr. rna was obtained from normal human tissues (ambion). real-time rt-pcr was performed using taqman primers and probe (assay id: hs _m ; applied biosystems) on an abi sequence detection system using standard conditions and cycle times/temperatures. the taqman endogenous control was human cyclophilin a (ppia), and data are shown as relative expression units. statistical analysis. because of the extensive relatedness of study subjects in our amish pedigrees, we tested for associations of snp genotypes while accounting for the pedigree structure. we evaluated the association between snp genotype and disease status (type diabetes vs. ngt and type diabetes/igt vs. ngt) under the additive genetic model using a variance component approach, in which we modeled the probability that the subject was a case or control as a function of the individual’s age, sex, and genotype, conditional on the correlations in phenotype among relative pairs. statistical testing was accomplished using the likelihood ratio test. the or was computed by comparing the odds of disease between subjects carrying one copy of the minor allele and subjects not carrying any copies of the minor allele. the association analyses were carried out using solar as previously described ( ). in an expanded set of nondiabetic subjects (including ngt and igt subjects), we also evaluated the effect of genotype on levels of quantita- tive traits (e.g., bmi, glucose level, and insulin level) by comparing mean trait levels across genotypes as previously described ( ). we compared the likelihood of a model in which the trait values were allowed to vary by genotype (unconstrained model) to that in which the genotype effects were constrained to be zero using the likelihoods ratio test. within each model, we simultaneously estimated the effects of age, sex, and family relationship. pairwise ld and haplotype structure were determined using haploview ( ). haploscore was used to estimate and compare haplotype frequencies between case and control groups ( ). acknowledgments this study was supported in part by the american diabe- tes association and by national institutes of health grants r dk , r dk , and t ag . funding and support was also provided by the university of mary- land general clinical research center (m rr ); the hopkins bayview general clinical research center (m rr ); the national institute of diabetes, diges- tive and kidney diseases clinical nutrition research unit of maryland (national institutes of health p dk ); and the department of veterans affairs and veterans affairs medical center baltimore geriatric research, ed- ucation and clinical center (grecc). we acknowledge the international q type diabetes consortium for performing some of the genotype analysis reported in this study, and we thank the amish research clinic staff for their energetic efforts in study subject recruitment and characterization. this study would not have been possible without the outstanding cooperation of the amish community. references . mccarthy mi, zeggini e: genetics of type diabetes. curr diab rep : – , . hsueh w, jean pls, mitchel bd, pollin pi, knowler wc, ehm mg, bell cj, sakul h, wagner mj, burns dk, shuldiner ar: genome-wide and fine- mapping linkage studies of type diabetes and glucose traits in the old order amish: evidence for a new diabetes locus on chromosome q and confirmation of a locus on chromosome q – q . diabetes : – , . hanson rl, ehm mg, pettitt dj, prochazka m, thompson bd, timberlake d, foroud t, kobes s, baier l, burns dk, almasy l, blangero j, garvey wt, bennett ph, knowler wc: an autosomal genomic scan for loci linked to type ii diabetes mellitus and body-mass index in pima indians. am j hum genet : – , . elbein s, hoffman m, teng k, leppert m, hasstedt s: a genome-wide search for type diabetes susceptibility genes in utah caucasians. diabetes : – , m. fu and associates diabetes, vol. , may . vionnet n, hani eh, dupont s, gallina s, francke s, dotte s, matos fd, durand e, leprêtre f, lecoeur c, gallina p, zekiri l, dina c, froguel p: genomewide search for type diabetes-susceptibility genes in french whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome q -qter and independent replicate of a type -diabetes locus on chromosome q – q . am j hum genet : – , . wiltshire s, hattersley at, hitman ga, walker m, levy jc, sampson m, o’rahilly s, frayling tm, bell ji, lathrop gm, bennett a, dhillon r, fletcher c, groves cj, jones e, prestwich p, simecek n, rao pv, wishart m, bottazzo gf, foxon r, howell s, smedley d, cardon lr, menzel s, mccarthy mi: a genomewide scan for loci predisposing to type diabetes in a uk population (the diabetes uk warren repository): analysis of pedigrees provides independent replication of a suscepitibility locus on chromosome q. am j hum genet : – , . xiang k, wang y, zheng t, jia w, li j, chen l, shen k, wu s, lin x, zhang g, wang c, wang s, lu h, fang q, shi y, zhang r, xu j, wang q: genome-wide search for type diabetes/impaired glucose homeostasis susceptibility genes in the chinese: significant linkage to chromosome q – q and chromosome q – . diabetes : – , . ng mc, so wy, cox nj, lam vk, cockram cs, critchley ja, bell gi, chan jc: genome-wide scan for type diabetes loci in hong kong chinese and confirmation of a susceptibility locus on chromosome q – q . diabetes : – , . etienne-manneville s, hall a: rho gtpases in cell biology. nature : – , . jeffe ab, hall a: rho gtpases: biochemistry and biology. annu rev cell dev biol : – , . hirosumi j, tuncman g, chang l, gorgun cz, uysal kt, maeda k, kain m, hotamisligil gs: a central role for jnk in obesity and insulin resistance. nature : – , . larsen l, storling j, darville m, eizirik dl, bonny c, billestrup n, mandrup-poulsen t: extracellular signal-regulated kinase is essential for interleukin- -induced and nuclear factor kappa b-mediated gene expres- sion in insulin-producing ins- e cells. diabetologia : – , . geiger pc, wright dc, han dh, holloszy jo: activation of p map kinase enhances sensitivity of muscle glucose transport to insulin. am j physiol endocrinol metab :e –e , . kowluru a, velathakal r: rho guanosine diphosphate-dissociation inhibi- tor plays a negative modulatory role in glucose-stimulated insulin secre- tion. diabetes : – , . nevins ak, thurmond dc: a direct interaction between cdc and vesicle-associated membrane protein regulates snare-dependent insu- lin exocytosis. j biol chem : – , . khayat za, tong p, yaworsky k, bloch rj, klip a: insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol -kinase and glut in l myotubes. j cell sci : – , . chiang sh, baumann ca, kanzaki m, thurmond dc, watson rt, neudauer cl, macara ig, pessin je, saltiel ar: insulin-stimulated glut transloca- tion requires the cap-dependent activation of tc . nature : – , . weernink pa, meletiadis k, hommeltenberg s, hinz m, isihara h, schmidt m, jakobs kh: activation of type i phosphatidylinositol -phosphate -kinase isoforms by the rho gtpases, rhoa, rac , and cdc . j biol chem : – , . houssa b, de widt j, kranenburg o, moolenaar wh, van blitterswijk wj: diacylglycerol kinase theta binds to and is negatively regulated by active rhoa. j biol chem : – , . hess ja, ross ah, qiu rg, symons m, exton jh: role of rho family proteins in phospholipase d activation by growth factors. j biol chem : – , . fu m, damcott c, sabra m, pollin ti, ott s, wang j, garant m, o’connell j, mitchell bd, shuldiner ar: polymorphism in the calsequestrin gene on chromosome q is associated with type diabetes in the old order amish. diabetes : – , . ma l, hanson rl, que ln, cali amg, fu m, mack jl, infante am, kobes s, the international type diabetes q consortium, bogardus c, shuldiner ar, baier la: variants in arhgef , a candidate gene for the linkage to type diabetes on chromosome q, are nominally associated with insulin resistance and type diabetes in pima indians. diabetes : – , . hsueh wc, mitchell bd, aburomia r, pollin t, sakul h, gelderehm m, michelsen bk, wagner mj, st. jean pl, knowler wc, burns dk, bell cj, shuldiner ar: diabetes in the old older amish: characterization and heritability analysis of the amish family diabetes study. diabetes care : – , . daly mj, rioux jd, schaffner sf, hudson tj, lander es: high-resolution haplotype structure in the human genome. nat genet : – , . schaid dj, rowland cm, tines de, jacobson rm, poland ga: score tests for association between traits and haplotypes when linkage phase is ambiguous. am j hum genet : – , arhgef variation and type diabetes diabetes, vol. , may wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : 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title). http://europepmc.org/abstract/med/ proc. nail. acad. sci. usa vol. , pp. - , december genetics plasminogen activator inhibitor type gene is located at region q . -q of chromosome and genetically linked with cystic fibrosis (protease inhibitor/serpin/gene mapping) k. w. klinger*t, r. winqvistt§, a. riccio¶, p. a. andreasenii, r. sartorio, l. s. nielsen , n. stuart*, p. stanislovitis*, p. watkins*, r. douglas*, k.-h. grzeschik**, k. alitalo§, f. blasi~tt, and k. dano** *integrated genetics, inc., new york avenue, framingham, ma ; tdepartment of clinical genetics, oulu university central hospital, kajaanintie , sf- , oulu, finland; §department of virology, university of helsinki, haartmaninkatu , sf- helsinki, finland; international institute of genetics and biophysics, consiglio nazionale delle richerche, via marconi, naples, italy; ilfinsen laboratory, rigshospitalet, strandboulevarden, copenhagen o., denmark; **institut fur humangenetik der universitat, vesaliusweg - , d- munster, federal republic of germany; and ttmikrobiologisk institut, university of copenhagen, a oster farimagsgade, copenhagen k., denmark communicated by lester . krampitz, august , (received for review february , ) abstract the regional chromosomal location of the human gene for plasminogen activator inhibitor type (paii) was determined by three independent methods of gene map- ping. paii was localized first to cen-q and then to q . -q by southern blot hybridization analysis of a panel of human and mouse somatic cell hybrids with a pail cdna probe and in situ hybridization, respectively. we identified a frequent hindiii restriction fragment length polymorphism (rflp) of the pail gene with an information content of . . in family studies using this polymorphism, genetic linkage was found between paii and the loci for erythropoietin (epo), paraoxonase (pon), the met protooncogene (met), and cystic fibrosis (cf), all previously assigned to the middle part of the long arm of chromosome . the linkage with epo was closest with an estimated genetic distance of centimorgans, whereas that to cf was centimorgans. a three-point genetic linkage analysis and data from previous studies showed that the most likely order of these loci is epo, pail, pon, (met, cf), with paii being located centromeric to cf. the paii rflp may prove to be valuable in ordering genetic markers in the cf-linkage group and may also be valuable in genetic analysis of plasminogen activation-related diseases, such as certain thromboembolic disorders and cancer. proteolysis caused by plasminogen activation is involved in many diverse biological processes ( - ). the two types of mammalian plasminogen activators, urokinase-type (u-pa) and tissue-type (t-pa), are both serine proteases with similar catalytic specificities but are products of different genes, located on chromosomes and , respectively ( - ). plasminogen activation is regulated at several levels-e.g., by hormonal regulation of the synthesis of the activators and by modulators of their enzyme activity. one such modulator is plasminogen activator inhibitor type (pa l), an - -kda glycoprotein produced by endothelial cells and several neoplastic cell lines and found in thrombocytes and blood plasma ( - ). pail inhibits u-pa and t-pa and belongs to the group of serpins, being an argserpin with an arginine residue at the reactive center ( - ). we have previously described the derivation of monoclo- nal antibodies to pail and their use for purification of the inhibitor ( ). these antibodies and amino acid sequence data have allowed the identification of pail cdna clones from two different sources ( , ). the paii gene was assigned to human chromosome by hybridization analysis of chro- mosomes sorted onto filters ( ). in the present study the results of cell hybrid and in situ hybridization analysis placed paii in the area q -q , close to the chromosomal location predicted for cystic fibrosis (cf) on the basis of genetic linkage between cf and a number of polymorphic loci ( - ). cf is a common autosomal recessive disease with a carrier frequency of : in caucasian populations. the basic biochemical defect in cf is as yet unknown and further genetic linkage studies are needed to identify the responsible genetic locus. we there- fore identified a restriction fragment length polymorphism (rflp) for pail and analyzed genetic linkage between the pail gene and cf as well as other genes located in this region. materials and methods dna probes. the construction and characterization of the pair cdna clone have been described elsewhere ( ). a . -kilobase (kb) bamhi insert of ppai -a plasmid con- taining the entire coding sequence for pail was used for polymorphism screening and linkage studies. p-labeled probes were synthesized by using the procedure of feinberg and vogelstein ( , ). other dna polymorphisms used for linkage analysis have been described already: metd and meth ( , ), epo ( ). the markers at the met locus were analyzed as a haplotype, assuming no recombination between the taq i sites and linkage equilibrium ( ). somatic cell hybrids. the somatic cell hybrids used in this study were produced by the fusion of a mouse cell line (rag or a ) with human cells, including cells containing translo- cation derivatives of chromosome . determination of the chromosomal complement of the hybrids was performed by analysis of g-banded chromosomes combined with testing of known biochemical markers on chromosome ( ), essen- tially as reported ( ). human gm and a and mouse rag cell lines were used as controls. dna analysis. dna from human, mouse, and mouse-hu- man cells was prepared using standard techniques. gene mapping using dna panels of hybrid cell lines and rflp analyses were carried out by using a modified form of abbreviations: u-pa, urokinase-type plasminogen activator; t-pa, tissue-type plasminogen activator; pai , plasminogen activator inhibitor type ; rflp, restriction fragment length polymorphism; cf, cystic fibrosis; pon, paraoxonase; lod, logarithm ofodds; epo, erythropoietin; cm, centimorgans. tto whom reprint requests should be addressed. the publication costs of this article were defrayed in part by page charge payment. this article must therefore be hereby marked "advertisement" in accordance with u.s.c. § solely to indicate this fact. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. natl. acad. sci. usa ( ) southern blotting ( ) and nylon membranes [genetrans (plasco) or hybond-n (amersham)]. the membranes were hybridized to p-labeled ppai -a dna. after hybridiza- tion the filters were washed in successive changes of de- creasing concentrations of nacl/sodium citrate to a final wash in mm naci/ . mm sodium citrate/ . % nadodso at c. autoradiography was at - c. paraoxonase (pon) analysis. six families were tested for the serum arylesterase pon, as described ( ). chromosomal in situ hybridization. hybridization in situ was performed on metaphase chromosomes essentially as described by harper and saunders ( ). pedigrees and families. probands in inbred populations were identified, and the pedigrees were constructed as described ( , ). two large extended kindreds with cf were studied, one amish mennonite family containing affected individuals and a hutterite family containing affected individuals. seventeen two-generation nuclear fam- ilies each with two ( families) or three ( families) affected children were also used in the linkage analysis. the estab- lishment in these families of linkage between the loci and markers colia , epo (erythropoietin), pon, d s , met, and cf has been reported ( , , ). linkage analysis. two-point analyses and multipoint anal- yses were performed using the mlink and linkmap programs, respectively, of the computer package linkage ( ). logarithm of odds (lod) scores z were calculated by assuming no sex difference in recombination. maximal like- lihood estimates of the lod score (i) and recombination fraction ( ) were calculated by using the computer program vaco ( ). the confidence interval for the maximal like- lihood estimate ofthe recombination fraction was determined by graphic interpolation ( ). the large amish mennonite pedigree was divided into three sections to include one inbreeding loop in each section, thus simplifying the analysis and preserving to a large extent the complex interrelation- ships and common ancestry of the members of this pedigree. results chromosomal localization of the paii gene. the chromo- somal localization of the human gene for paii was identified c , l kbp- - +- ho ., ia~~co., cb. fig. . sublocalization of the human pail gene on chromosome by using mouse-human somatic cell hybrids. human (gm , a ), mouse (rag, a ), and hybrid (as indicated in fig. ) cell dna was cleaved with hindili restriction endonuclease, resolved by electrophoresis, and blotted onto membranes hybridized with p-labeled plasmid containing a . -kbp insert of human pati cdna, and specific hybridization was detected by autoradiography. from the autoradiogram it can be seern that the probe detects fragments of kbp and/or kbp in the human gm and a cells and in the hybrids - -cilo, rag ru - , and a it - - . a faint hybridization signal of kbp was also detected for hybrid rag gm - on a longer exposure. this reflects that pail was present in -. % of the cells in this hybrid line. as shown in fig. , the only human chromosomal region these cells have in common is that located between the centromere and band of the long arm of chromosome ( cen-q ). by dna hybridization in mouse-human hybrid cell lines containing different human chromosomes. hybridization with the pail cdna probe to southern blots of hindiii- digested dna revealed two dna fragments of kilobase pairs (kbp) and kbp (see below and fig. ). the presence of one or both of these fragments segregated in a variety of cell lines specifically with the human chromosome , whereas discordance was found with all other human chromosomes (data not shown). to study the sublocalization of the pail gene, cell lines that contained specific regions of human chromosome (fig. ) were investigated. as it appears from fig. , a positive signal was only observed in those cell lines a x- - ( ) it a - - ( x) gm rag - ( x) mh rag - ( x) ru rag - ( x y) ru rag - (x) - -ciio (none) fig. . portion of human chromosome retained in mouse-human hybrid cell lines used for sublocalization of the pail gene by southern blotting analysis. in parentheses are indicated other human chromosomes (or derivatives) present in the respective cell lines. as illustrated in fig. , paii is found in cell lines it a - - , gm rag - , ru rag - , and - -cl . l it i genetics: klinger et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. natl. acad. sci. usa ( ) that had retained the region of the long arm of chromosome located between the centromere and band ( cen-q ). as an alternative approach, the localization of the pail gene was studied by in situ hybridization on metaphase preparations ot human lymphocytes with h-labeled pail cdna. as seen from fig. left, chromosome was the only chromosome that showed a grain number ( %) above background. the majority of the grains on chromosome were associated with bands q l. and q of the long arm ( % of all grains observed, fig. right), verifying and making more precise the localization obtained by the cell hybrid analysis. rflp. dna from four unrelated individuals was digested with a variety of different restriction endonucleases (apa i, bgl ii, msp i, ecorv, pvu ii, xmn i, sac i, bsteii, bcl i, taq i, ecori, bamhi, pst i, hindiii, and nci i) and analyzed by southern blotting with the pail cdna probe. digestion with hindiii revealed allelic fragments of kbp (al) and kbp (a ). a representative autoradiograph of the segregation of the rflp in a family is shown in fig. . the segregation pattern obtained in all of the nuclear families, and both pedigrees, was consistent with mendelian inheritance. the frequencies for al and a were . + . and . ± . , respectively, in a sample ofdna preparations accounting for unrelated chromosomes representative of a general north american population. because of the nearly optimal ratios of the major and minor allele, the polymorphism has a high polymorphism information content ( . ). an additional but much less frequent rflp was found after xmn i digestion. no polymorphisms were detected for the remaining restric- tion enzymes that were tested. linkage analysis. the hindiii polymorphism detected by the pail probe was used to test for linkage between paii and the cf locus and other markers previously mapped to this region of the long arm of chromosome . the segregation pattern of the hindill rflp was analyzed by southern blot analysis using dna prepared from each individual of the families described in materials and methods. lod score [ co z c: l l cc z: z s il i p q jpi q iiiqpjq ip q nsm *i a n r q p q tvl q lp qipjq pqlq x my chromosomes uo * - . i ai * a v . vw epai hindill fig. . inheritance of hindiii rflp. dna samples were di- gested with hindiii and analyzed by southern blotting with a . -kbp fragment of the paiu cdna probe. the family pedigree is shown above. the rflp assignment of each individual is indicated below the pedigree. epai, ppai -a probe. analysis of the cosegregation pattern of the cf disease phenotype and the rflp, or the rflp and additional markers assigned to chromosome q, was performed by using the linkage program (see materials and methods). the results of the two-point linkage analysis are shown in table . evidence was obtained for loose linkage between pail and the cf locus. the maximal likelihood estimate of the recombination distance ( ) between pair and cf was . [ % confidence interval, . - . ; with a maximal lod score (z) of . (odds ratio, : )]. this suggested that the pail locus was located within the established cf linkage group on the long arm of chromosome , albeit somewhat distant from the cf locus. we therefore examined the linkage relationship between paii and other markers within this group. these included the loci for the c-met protooncogene (met) ( ), the erythropoietin gene (epo) ( ), and the gene for the serum protein paraoxonase (pon) ( ). the results of these analyses (table ) demonstrated that paii is most @ * * fig. . localization of human paii gene by in situ hybridization analysis. (left) histogram show- ing the grain distribution in metaphases. abscissa: chromo- somes in their relative size propor- tions. ordinate: number of silver grains. overall, grains were observed, of which ( %) were on chromosome . background grains were found distributed evenly on all chromosomes. (right) sublocalization of pail to q . -q ( grains = % of all grains observed). genetics: klinger et al. i c d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. nati. acad. sci. usa ( ) table . linkage analysis of gene loci for pair vs. cf, epo, the c-met protooncogene (met), and paraoxonase (pon) number of informative lod (z) score at recombinant fraction ( ) loci families . . . . . . . . . o = . ; = . ; % confidence interval = . - . paii vs. cf - . . . . . . . . o = . ; z = . ; % confidence interval = . - . paii vs. epo . . . . . . . . o = . ; t = . ; % confidence interval = . - . pari vs. met - . . . . . . . . o = . ; i = . ; % confidence interval = . - . pail vs. pon - . - . . . . . . . closely linked to epo, with a maximal likelihood estimate for o of . and a maximal lod score of . (odds ratio, . x : ). for linkage ofpail with ponand met, the maximal likelihood estimates for and z were . and . vs. . and . , respectively. a three-locus test was performed to establish the position of pail relative to epo and pon. of the families that were informative for each marker tested for linkage to paii (table ), two families were jointly informative for paii, pon, and epo. results of the three-locus analysis using data obtained from these families are shown in fig. . in this analysis the distance between the epo and pon loci was held constant, and the relative probability of the paii locus was calculated at the indicated intervals. the data are consistent with the order pon, pail, epo with odds of : : over the alter- native orders pon, epo, paii or paii, pon, epo, respectively. discussion we assigned the gene for paii to cen- q based on the pattern obtained when a human pail cdna was hybridized to a panel of dnas isolated from mouse-human somatic cell hybrids, sublocalized to bands q . -q by in situ hybrid- ization. analysis ofthe segregation ofthe hindiii rflp in families previously typed for the loci cf, met, pon, colja , and epo showed that the pahl gene is part of this linkage group on the long arm of chromosome . the paii gene is closely o/o / o °'d i pon epo genetic location (cm) fig. . likelihood of the localization of the paii gene locus in relation to pon and epo. abscissa: genetic distance from pon. ordinate: odds ratio for localization ofpaii at the distance indicated vs. localization of paii at infinite distance from pon (i.e., no linkage). the distance between pon and epo is assumed to be centimorgans (cm). the relative odds for the orders pon, paii, epo: pon, epo, pail: pail, pon, epo are : : . linked to the epo locus (estimated genetic distance, cm), which recently was found to be tightly linked with the collagen locus colja , with no evidence of recombination ( = , z = . ) (p.w., n.s., p.s., and k.w.k., unpublished data). on this basis, the ( - tt)-cm recombination distance of pail from cf found in this study is in good agreement with those of ( - *) and ( - *) cm found for epo and colia , respectively (p.w., n.s., p.s., and k.w.k., unpublished data; ref. ). previous multipoint linkage analysis has established the order (epo, colia ), pon, (met, cf) (p.w., n.s., p.s., and k.w.k., unpub- lished data), with epo, col, and pon centromeric to cf. according to the results of the three-point analysis in the present study, the most probable location of paii is between epo and pon-i.e., (epo, colia ), paii, pon, (met, cf), with paii centromeric to cf (see above) and telomeric to epo. pail may prove to be an important genetic marker. the polymorphism information content of . is high for a diallelic system; thus, the majority of families will be infor- mative for gene mapping by linkage analysis. the paii rlfp may be valuable in genetic analysis of plasminogen activation-related diseases, such as cancer and certain thromboembolic disorders. in preliminary studies, no rearrangements of the pail sequences were found by south- ern blotting either in carcinoma samples or in additional cell lines (r.w. and k.a., unpublished data). these included colon carcinomas, breast carcinomas, and tumor cell lines with previously demonstrated oncogene rearrange- ments and amplifications ( ). however, these results must be interpreted with caution since small changes would have been missed due to the scale of the southern blotting method and since we do not yet know the genomic organization of the pail sequences. in relation to the role of plasminogen activation in the breakdown of matrix proteins in cancer, the close linkage between paii and the collagen locus colia is particularly noteworthy because plasmin is an activator of latent collagenases ( , ), and pail may therefore serve to protect collagen. some thrombotic disorders, including myocardial infarc- tion in young patients and recurrent deep vein thrombosis, are in some cases associated with increased plasma levels of pail ( - ), and familial occurrence of venous thrombosis with high level of pa l has been reported ( , ). the availability of the cloned gene sequence will allow molecular studies of the pathophysiology of these disorders, and the segregation of the rflp in families with thrombosis will allow us to determine whether there is a genetic component involving the pail locus. the excellent technical assistance of helle abildgaard, kirsten lund jakobsen, vivi kielberg, anna margrethe poulsen, aita haataja, and irma kurvinen is gratefully acknowledged. determi- nations of pon serum activity were generously provided by dr. b. # % confidence interval. genetics: klinger et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. natl. acad. sci. usa ( ) ladu (university of michigan). we thank drs. robert schwartz and richard doherty (rochester, ny) for their role in identification and collection of members of the inbred pedigrees. this work was supported financially by the danish canter society, the danish medical research council, pf ingegneria genetica e basi molecolari delle malattie ereditarie and pf oncologia, consiglio nazionale delle ricerche, italy, finnish cancer foundation, and national institutes of health grants hl and am . . astrup, t. 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( ) br. j. haematol. , - . genetics: klinger et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , is there an association between whole-body pain with osteoarthritis-related knee pain, pain catastrophizing, and mental health? clinical research is there an association between whole-body pain with osteoarthritis-related knee pain, pain catastrophizing, and mental health? amish j. dave md, faith selzer phd, elena losina phd, kristina m. klara bs, jamie e. collins phd, ilana usiskin bs, philip band phd, david f. dalury md, richard iorio md, kirk kindsfater md, jeffrey n. katz md, msc received: april / accepted: september / published online: october � the association of bone and joint surgeons abstract background greater levels of self-reported pain, pain catastrophizing, and depression have been shown to be associated with persistent pain and functional limitation after surgeries such as tka. it would be useful for clini- cians to be able to measure these factors efficiently. questions/purposes we asked: ( ) what is the associa- tion of whole-body pain with osteoarthritis (oa)-related knee pain, function, pain catastrophizing, and mental health? ( ) what is the sensitivity and specificity for dif- ferent cutoffs for body pain diagram region categories in relation to pain catastrophizing? methods patients (n = ) with knee oa undergoing elective tka at one academic center and two community orthopaedic centers were enrolled before surgery in a prospective cohort study. questionnaires included the womac pain and function scales, pain catastrophizing scale (pcs), mental health inventory- (mhi- ), and a pain body diagram. the diagram documents pain in anatomic areas. based on the distribution of the anatomic areas, we established six different body regions. our analyses excluded the index (surgically treated) knee. linear regression was used to evaluate the association between the total number of nonindex painful sites on the whole-body pain diagram and measures of oa-related pain and function, mental health, and pain catastrophizing. generalized linear regression was used to evaluate the association between the number of painful nonindex body regions (categorized as ; – ; or – ) with our measures of interest. all models were adjusted for age, sex, and number of comorbid conditions. the cohort included % females and the mean age was years (sd, years). with supported in part by niams grants t ar (ajd) and k ar (el)one of the authors (ajd) was supported by a national institutes of health t grant (ar ) outside the submitted work. one of the authors (dfd) has received personal fees and nonfinancial support from depuy orthopaedics (warsaw, in, usa) outside the submitted work. one of the authors (kk) has received personal fees from depuy orthopaedics outside the submitted work. all icmje conflict of interest forms for authors and clinical orthopaedics and related research editors and board members are on file with the publication and can be viewed on request. each author certifies that his or her institution approved the human protocol for this investigation, that all investigations were conducted in conformity with ethical principles of research, and that informed consent for participation in the study was obtained. this study was done at the orthopaedic and arthritis center for outcomes research, department of orthopedic surgery, brigham and women’s hospital, boston, ma, usa. a. j. dave, f. selzer, e. losina, k. m. klara, j. e. collins, i. usiskin, j. n. katz orthopaedic and arthritis center for outcomes research, department of orthopedic surgery, brigham and women’s hospital, boston, ma, usa a. j. dave, j. n. katz (&) orthopaedic and arthritis center for outcomes research (oracore), division of rheumatology, immunology and allergy, brigham and women’s hospital, francis street, pbb-b , boston, ma , usa e-mail: jnkatz@partners.org a. j. dave, f. selzer, e. losina, j. e. collins, j. n. katz harvard medical school, boston, ma, usa e. losina department of biostatistics, boston university school of public health, boston, ma, usa p. band department of biochemistry and molecular pharmacology, nyu langone orthopaedic surgery, nyu school of medicine, new york, ny, usa clin orthop relat res ( ) : – doi . /s - - - clinical orthopaedics and related research® a publication of the association of bone and joint surgeons® http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - - &amp;domain=pdf removal of the index knee, the median pain diagram score was ( th , th percentiles, , ) with a range of to . the median number of painful body regions was ( th , th percentiles, , ). results after adjusting for age, sex, and number of comorbid conditions, we found modest associations between painful body region categories and mean scores for womac physical function (r = . , p \ . ), womac pain (r = . , p = . ), mhi- (r = � . , p\ . ), and pcs (r = . , p \ . ). a nonindex body pain region score greater than had % ( % ci, %– %) sensitivity for a pain catastrophizing score greater than but a specificity of just % ( % ci, %– %) . a score of or greater had greater specificity ( %; % ci, %– %) but lower sensitivity ( %; % ci, %– %). conclusions we found modest associations between the number of painful sites on a whole-body pain diagram and the number of painful body regions and measures of oa- related pain, function, pain catastrophizing, and mental health. patients with higher self-reported body pain region scores might benefit from further evaluation for depression and pain catastrophizing. level of evidence level iii, therapeutic study. introduction osteoarthritis (oa) of the knee affects more than million adults in the united states [ ]. because there are no effective pharmacologic treatments for stopping or reversing structural progression in patients with oa, management focuses on weight reduction, aerobic exercise, physical therapy, and symptomatic pain relief including corticosteroid injection [ , ]. ultimately, as much as % of patients with knee oa want a tka to improve mobility and quality of life [ ]. although most patients who undergo tka experience substantial pain relief, as much as % with oa have persistent pain and functional limitations months after surgery [ , , ]. studies have shown that pain catas- trophizing, greater medical comorbidity, lower educational attainment, depression, fear avoidance, and other psychologic factors are associated with persistent pain and/ or enhanced functional limitations after tka [ , , , , , ]. pain catastrophizing has been defined as ‘‘an exaggerated negative mental set brought to bear during actual or anticipated painful experience’’ [ ]. prior research on factors associated with persistent oa-related knee pain (lasting more than months) found that pain catastrophizing explained a substantial proportion of vari- ance in measures of pain, psychologic disability, and limitations in walking speed [ , , ]. similarly, worse mental health (including depression) and greater number of pain problems have been associated with worse tha and tka outcomes [ , , ]. given the large number of tkas performed, poor symptomatic outcomes constitute a prevalent and expensive health problem in the united states [ , , ]. as such, risk factors associated with suboptimal responses to treatment are an area of active investigation. body pain diagrams, which have been used for more than years in musculoskeletal pain research, provide a rapid and inexpensive way to measure the extent, distri- bution, and location of pain [ , , , , ]. widespread pain has been recognized as a poor prognostic factor in diseases such as rheumatoid arthritis, cancer, and chronic low back pain, and a poor prognostic factor for outcome of tka [ , ]. we investigated whether the pain diagram, which we used to document widespread pain, is associated with established risk factors for persistent symptoms after tka, including depression, pain severity, and pain catastrophiz- ing. because widespread pain has been associated with poor outcome in various diseases [ , , , , ], and because widespread pain is often clinically understood as a sign of psychologic distress [ , , , , , , ], we posited that widespread pain, as documented on a pain diagram, would be associated with catastrophizing and depression and poor functional status. we therefore asked: ( ) are greater numbers of painful body sites, and painful body regions as measured by a widespread body pain diagram, associated with higher levels of catastrophizing and psychologic distress? ( ) what are the sensitivity and specificity of different cutoffs for body pain diagram regions in relation to pain catastrophizing? patients and methods the study of total knee arthroplasty responses (stars) is a prospective cohort study of patients with the primary diagnosis of osteoarthritis of the knee undergoing elective unilateral tka. stars was designed to evaluate the prevalence and risk factors for suboptimal outcomes after p. band, r. iorio nyu langone orthopaedic surgery, nyu school of medicine, new york, ny, usa d. f. dalury department of orthopaedic surgery, university of maryland st joseph medical center, towson, md, usa k. kindsfater orthopaedic center of the rockies, fort collins, co, usa volume , number , december pain diagram scores before tka tka. institutional review board approval was obtained for all research sites. we recruited patients at one academic center in new york city (nyu langone medical center) and at two community orthopaedic centers (orthopaedic center of the rockies, fort collins, co, usa, and university of maryland st. joseph medical center, tow- son, md, usa) between september and april . subjects included english-speaking community-dwell- ing persons who were at least years old at the time of study entry. at each of the three surgery practices, a local study associate identified all potentially eligible subjects and provided the subject’s contact information to the brigham and women’s hospital research coordinators (kmk, iu). the coordinators contacted all potential sub- jects to confirm eligibility, explain the study, and determine subject interest in participation. baseline data collection occurred before surgery. study participants were reim- bursed (usd ) for returning questionnaires. of patients referred by the clinical sites for enrollment in the study, agreed to participate and were found to be eli- gible on phone screening; of these subjects returned questionnaires before surgery and thus were included in these analyses. the cohort was % female with a mean age of years (sd, years). among the three study enrollment sites, there were no clinically important differences among patients regarding age, sex, or highest level of education. the baseline questionnaire included a body pain dia- gram [ ] based on the widespread pain index [ , ], with a list of checkboxes corresponding to anatomic areas throughout the body that study participants were asked to check if they had any current pain at those sites. we delineated six different body regions using the sites on the body pain diagram. these regions included the left upper extremity (left shoulder girdle, left upper arm, and left lower arm), right upper extremity (right shoulder gir- dle, right upper arm, and right lower arm), left lower extremity (left hip/buttock, left upper leg, and left lower leg), right lower extremity (right hip/buttock, right upper leg, and right lower leg), back or neck (upper back, lower back, and neck), and chest and abdomen. to best capture widespread pain (beyond the index knee), we removed the index knee. the median pain diagram score excluding the index knee was ( th , th percentiles, , ) and the range was between and . when we aggregate pain sites to regions, the median number of painful body regions, excluding the index knee was ( th , th percentiles, , ). the observation that . of the subjects reported pain scores of or less reflects the highly right-skewed distri- butions of the body pain and body region scores. thus, we grouped subjects in categories based on the number painful body region scores. we found no important differences across body region categories in mean age, median bmi, level of education, health insurance type, or site of study enrollment (table ). outcome measures included the womac, a widely used questionnaire validated for patients with hip and knee oa that consists of a five-item pain scale, two items for stiffness, and a -item functional limitation scale. sub- jects responded to each item by checking one of five ordinal likert responses [ ]. we included the womac function and pain scales. in each scale, the responses to questions were summed and scaled from to using a linear transformation with being the worst possible score. we assessed the continuous specification of the womac scales and also categories of scores including to , to , to , and or greater with higher scores indicative of worse oa-related pain or physical function. to assess the degree of pain catastrophizing, including patients’ negative or exaggerated attitudes toward pain, we used the -item pain catastrophizing scale (pcs) [ ]. in addition to the continuous specifica- tion, we used a cutoff of or greater as representing a high degree of pain catastrophizing [ , , ]. the five- item mental health index- (mhi- ) [ , ] was used as a measure of anxiety and depressive feelings. the questions are summed and scaled from to using a linear transformation. we examined the score as a continuous variable and as a dichotomous variable with lower scores (\ ) indicative of worse mental health [ , ]. subjects also answered questions regarding their demo- graphic characteristics, their expectations of having a completely successful surgery, and of having a surgical complication (infection of the knee prosthesis, nerve damage, or dislocation of the knee prosthesis), and medical compli- cation (including pneumonia, blood clot, or heart attack). patients rated the likelihoodofthese eventsoccurringas %to %, % to %, %to %, % to %,or % to %. subjects also completed items documenting use of medica- tions, assistive devices, and appointments with healthcare providers during the prior months. statistical analysis we included only baseline data for this analysis. descrip- tive statistics either were summarized as means (± sd) or medians ( th and th percentiles) for continuous vari- ables, depending on normality, and as percentages for categorical variables. differences between proportions were assessed by the chi-square test or fisher’s exact test and continuous variables were compared by student’s t-tests or wilcoxon nonparametric tests. furthermore, the test for trend was assessed using the jonckheere-terpstra test for continuous variables and the cochran-mantel- haenszel test of trend for categorical variables. linear dave et al. clinical orthopaedics and related research table . baseline demographic, clinical, and resource utilization characteristics by regional body pain groups in stars participants characteristic regional body pain diagram groups p value (n = ) – (n = ) – (n = ) overall number percent, mean (sd) median ( th , th percentiles) number percent, mean (sd) median ( th , th percentiles) number percent, mean (sd) median ( th , th percentiles) age (years) ( ) ( ) ( ) . female . no yes surgical site . maryland colorado nyu langone education . high school or less some college, vocational, or technical education undergraduate or technical school degree white race . no yes current smoker . no yes number of comorbid conditions* . c bmi (kg/m ) ( , ) ( , ) ( , ) . ability to extend knee . completely extend between �– � from full extension between �– � from full extension more than � from full extension ability to bend knee (%) . to � to � to � to � [ � number of pain sites � ( , ) ( , ) ( , ) \ . range, – range, – range, – use of supportive device . no yes volume , number , december pain diagram scores before tka regression was used to evaluate the correlations between body pain scores and continuous measures of oa-related pain and function, mental health, and pain catastrophizing. furthermore, generalized linear models adjusting for age, sex, and number of comorbid conditions were run to assess the association between body region categories and oa- related pain and function and mental health scores. the model output included adjusted mean values (± standard error [se]) and pairwise comparisons for the three body pain regions. pearson correlation analysis was used to examine the associations between the categories of pain- region score and the continuous versions of the womac pain and function scales, mhi- , and pain catastrophizing. we evaluated sensitivity and specificity of the nonindex body region pain score in relation to the pcs using a value of as the threshold for high catastrophizing [ ]. for these calculations, we categorized the number of painful body regions as either to versus or greater or as versus or greater (table ). the second categorization was chosen to investigate any nonindex pain versus none. contingency tables were used to calculate the sensitivity and specificity and positive and negative predictive values of the body pain categories and accepted cutoffs for the pcs. a pcs score of or greater, recognized as a high catastrophizing score [ , ] served as the gold standard in these analyses. we used two different cutoffs ( vs c ; – vs c ) of the body pain category scores to examine tradeoffs between sensitivity and specificity. all statistical analyses were performed using sas software, version . (cary, nc, usa), and a two-sided p value of . or less was considered to indicate statistical significance. table . continued number of different healthcare professional services sought � . or c number of medications taken daily . – – [ current use of medication for knee pain or discomfort . none occasional almost daily rating for expectation of completely successful tka . %– % %– % rating for expectation of complicated surgery . %– % %– % %– % rating for expectation of medical complication . %– % %– % %– % stars = study of total knee arthroplasty responses; * comorbid conditions = diabetes mellitus, depression, stomach ulcers, cancer, chronic kidney disease, liver disease, cardiovascular disease, and hypertension; � visits to orthopaedic surgeons, primary care physicians, rheumatologists, emergency department personnel, physical therapists, chiropractors, acupuncturists, yoga/tai chi instructors, or nurses/physician assistants; � index leg sites have been removed; body regions = left upper extremity = left shoulder girdle, left upper arm, and left lower arm; right upper extremity = right shoulder girdle, right upper arm, and right lower arm; left lower extremity = left hip/buttock, left upper leg, and left lower leg; right lower extremity = right hip/buttock, right upper leg, and right lower leg; back/neck = upper back, lower back, and neck; abdomen/chest = abdomen and chest. dave et al. clinical orthopaedics and related research results overall, we found modest associations between the con- tinuous measure of body region scores and number of painful body sites and worse scores on continuous measures of the mhi- score (regions, r = � . ; sites, r = � . ), pcs score (regions, r = . ; sites, r = . ), womac physical function (regions, r = . ; sites, r = . ), and womac pain (regions, r = . ; sites, r = . ) (p b . for all comparisons). subjects with a greater number of painful body regions were more likely than those were fewer painful body regions to be grouped in the highest pcs score category (c ), indicating more catastrophizing (p = . ). mhi- scores less than also were more common in the subjects with the greatest number of painful body regions (p = . ), as were worse scores on the womac pain and function scales (table ). when adjusted for sex, age, and number of medical comorbidities, we observed associations between increas- ing number of painful body region categories and increasing mean scores for womac physical function, womac pain, and pcs, and decreasing mean scores with the mhi- (table ). for womac physical function, the adjusted mean (se) score for participants reporting no body pain, low ( – regions) and high (c regions) were ( ), ( ), and ( ), respectively. similarly, for womac pain and the three ordered pain-region cate- gories, the adjusted means (se) were ( ), ( ), and ( ), and for pain catastrophizing, the adjusted means (se) were ( ), ( ), and ( ). the adjusted mean mhi- scores decreased with each increase in number of painful body region category. the mean (se) adjusted mhi- scores were ( ) for participants reporting no pain table . sensitivity, specificity, and positive and negative predictive values body regions pain catastrophizing (gold standard) totals c \ – – totals sensitivity ( % ci) % ( %– %) specificity ( % ci) % ( %– %) predictive value positive ( % ci) % ( %– %) negative ( % ci) % ( %, %) – totals sensitivity ( % ci) % ( %– %) specificity ( % ci) % ( %– %) predictive value positive ( % ci) % ( %– %) negative ( % ci) % ( %– %) table . baseline pain and function scores by regional body pain groups in stars participants outcome regional body pain diagram groups p value (n = ) – (n = ) – (n = ) overall number percent number percent number percent womac function categories . – – – c womac pain categories . – – – c mhi- score categories . – – pain catastrophizing categories . \ c stars = study of total knee arthroplasty responses; mhi- = five-item mental health index. volume , number , december pain diagram scores before tka beyond the index joint, ( ) for the low category, and ( ) for participants with three or more painful body regions. the sensitivity and specificity analysis for pain catas- trophizing and nonindex body region pain revealed that a nonindex body region pain score of or more had % ( % ci, %– %) sensitivity for a pain catastrophizing score greater than but a specificity of just % ( % ci, %– %). furthermore, the positive predictive value was % ( % ci, %– %) and negative predictive value was % ( % ci, %– %). a nonindex body pain region score of or more had greater specificity for a catastro- phizing score greater than ( %; % ci, %– %) but lower sensitivity ( %; % ci, %– %), with a positive predictive value of % ( % ci, %– %) and a negative predictive value of % ( % ci, %– %). discussion pain diagrams have been associated with poor outcomes for numerous disorders [ , , , , ]. widespread pain, which can be documented with pain diagrams, has been associated with psychologic distress [ , , , ]. these observations suggest that a body pain diagram might be associated with psychologic distress including catas- trophizing and depression. our objective was to determine whether a body pain diagram was associated with measures of pain catastrophizing, depression, and functional status in patients with advanced oa before tka at three centers. limitations of our study include that the body regions delineated on our pain diagram do not correspond directly to joints, which might result in underreporting of joint pain. in addition, in some studies patients were allowed to record out- of-body or external sites of pain on pain diagrams [ ]. we could not examine this phenomenon because our diagram did not permit subjects to record out-of-body painful sites. in a cohort of preoperative patients with knee oa at three orthopaedic centers, we found that more widespread pain as noted by either a greater number of painful body sites or painful body regions documented on a body pain diagram was associated with greater preoperative levels of pain catastrophizing, worse mental health, greater pain scores, and reduced physical function. these modest associations persisted when adjusted for sex, age, and number of medical comorbidities. several psychologic factors, including anxiety or depression and catastrophiz- ing, are associated with worse outcomes of surgical procedures including tka [ , , , , , ]. thus, the association we documented between painful body regions as determined using a body pain diagram and the mhi- and pcs scores suggests that the pain diagram may be useful in identifying patients at risk for a poor surgical outcome. this hypothesis will be tested in future work. future studies also should examine whether the associa- tions documented here between body pain diagram and measures of catastrophizing, depression, pain, and function are mediated by other musculoskeletal conditions, psy- chologic conditions, or other factors. table . mean scores and ci by regional body pain groups questionnaire regional body pain diagram group pairwise comparisons none low ( – ) high (c ) p value trend none vs low none vs high low vs high womac physical function (n = ) . adjusted mean (se) ( ) ( ) ( ) difference � � � % ci – – – % ci � to � to � � to p value* . . . womac pain (n = ) . mean (se) ( ) ( ) ( ) difference � � � % ci – – – % ci � to � to � � to p value * . . . mhi- (n = ) . mean (se) ( ) ( ) ( ) difference % ci – – – % ci � to – – p value * . . . pain catastrophizing scale (n = ) . mean (se) ( ) ( ) ( ) difference � � � % ci – – – % ci � to � to � � to p value* . . . mhi- = five-item mental health index; * adjusted for multiple comparisons using bonferroni method. dave et al. clinical orthopaedics and related research the % sensitivity of a diagram score of or greater indicates that a normal pain diagram (excluding index joint) will be useful for ruling out catastrophizing. simi- larly, for both body region categorizations, a high negative predictive value ( % and %, respectively) was reported, indicating that subjects with a low number of painful body regions ( – ) may be expected by the clini- cian to have low pain catastrophizing. we found associations between more widespread pain as indicated by a higher number of painful body regions shown on a body pain diagram and measures of pain catastrophizing, mental health, and oa-related pain and function. while patients with to painful body regions had a % negative predictive value for pain catastro- phizing, patients with higher self-reported pain might benefit from additional evaluation for depression and pain catastrophizing. by limiting evaluation for psychologic predictors of tka outcome to a smaller subset of preop- erative candidates, we anticipate fewer burdens for orthopaedic surgeons and patients. these analyses are cross-sectional; future research should investigate whether the degree of generalized pain as assessed from a body 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preliminary diagnostic cri- teria for fibromyalgia and measurement of symptom severity. arthritis care res. ; : – . . wylde v, dieppe p, hewlett s, learmonth id. total knee replacement: is it really an effective procedure for all? knee. ; : – . . wylde v, hewlett s, learmonth id, dieppe p. persistent pain after joint replacement: prevalence, sensory qualities, and post- operative determinants. pain. ; : – . dave et al. clinical orthopaedics and related research 	is there an association between whole-body pain with osteoarthritis-related knee pain, pain catastrophizing, and mental health? 	abstract 	background 	questions/purposes 	methods 	results 	conclusions 	level of evidence 	introduction 	patients and methods 	statistical analysis 	results 	discussion 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" 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imminent demise of liberal education martha nussbaum we may become powerful by knowledge, but we attain fullness by sympathy . . . but we find that this education of sympathy is not only systematically ignored in schools, but it is severely repressed. rabindranath tagore, “my school” achievement comes to denote the sort of thing that a well-planned machine can do better than a human being can, and the main effect of education, the achieving of a life of rich significance, drops by the wayside. meantime mind-wandering and wayward fancy are nothing but the unsup- pressible imagination cut loose from concern with what is done. john dewey, “democracy and education” i begin with three examples, which illustrate, in different ways, a profound crisis in education that faces us today, although we have not yet faced it. all illus- trate the loss of insights contained in the statements by tagore and dewey, two of our greatest educational reformers and thinkers about the role of education in a pluralistic democracy. . it is a hot march in new delhi. i am attending a conference on pluralism and the indian democracy at jawaharlal nehru university, most prestigious of indian universities. much of the conference is concerned with issues of educa- tion. all the papers on education focus on the content of required national text- books, which are to be memorized and regurgitated on examinations. the hindu right wants a content that supports their view of india’s history; the conference presenters, all opponents of the government then in power, inveigh against this aim and propose textbooks with a different, more nehruvian content. but nobody mentions the children: the stultifying atmosphere of rote learning in classrooms, the absence of critical thinking and all cultivation of imagination. tagore once wrote a fable called “the parrot’s training,” in which lots of smart people talk about how to educate a parrot, preparing a fine gilded cage and lots of fancy text- books. nobody notices that along the way the bird itself has died. the education debate at jnu, typical of the larger debate in india, reminds me of that story. . it is a surprisingly warm november in chicago, and i go across the midway to the lab school, the school where john dewey conducted his path- breaking experiments in democratic soul-making. the teachers are having a retreat, and i have been asked to address them on the topic of education for demo- cratic citizenship, something that i undertake with some trepidation because i am journal of social philosophy, vol. no. , summer , – . © blackwell publishing, inc. sure they all know so much more about this topic than i do. as i defend the legacy of dewey and introduce them to the very similar writings of tagore, i discover that i am not where i thought i was, the safe home of dewey’s ideas. i am on a battleground, where teachers who still take pride in stimulating children to ques- tion, criticize, and imagine are an embattled minority, increasingly suppressed by other teachers, and especially by wealthy parents, intent on testable results of a technical nature that will help produce financial success. when i present what i thought of as a very banal version of dewey’s vision, there is deep emotion, as if i have mentioned something precious that is being snatched away. . and finally: one week later i keep a phone appointment to talk with the head of the committee that is searching for a new dean for the school of edu- cation in one of our nation’s, and the world’s, most prestigious universities. here- after i will just refer to the university as x, and its president as y. z. they want my advice, or think that they want it. since, as a result of the first two incidents and many others of a similar nature, i am already alarmed about the future of the humanities and the arts in primary and secondary education, i lay out for this woman my views about education for democratic citizenship, stressing the crucial importance of critical thinking, knowledge about the many cultures and groups that make up one’s nation and one’s world, and the ability to imagine the situa- tion of another person, abilities that i see as crucial for the very survival of demo- cratic self-government in the modern world. to me, it seemed that i was saying the same thing i talk about all the time, pretty familiar stuff. but to this woman, it was utterly new. how surprising, says she, no one else i have talked to has mentioned any of these things at all. we have been talking only about how x university can contribute to scientific and technical progress around the world, and that is the thing that y. z. is really interested in. but what you say is very interesting, and i really want to think about it. she really seems to mean it. well, say i, i will be very happy to tell y. z. exactly what i think, and you can tell him that i’d welcome a call on this topic—although, i add, i am not sure that he would really understand what i am talking about in this case. she agrees that the talk would nonetheless be a good idea. i know y. z., and have a pretty cordial rela- tionship with him, so i actually think this conversation might happen. but i am not going to sit by the phone waiting. and i can just see it now: all over the world, initiatives sponsored by x and the huge money of x, focusing narrowly on sci- entific and technical training, producing many generations of useful engineers who have not a clue about how to criticize the propaganda of their politicians, and who have even less of a clue about how to imagine the pain that a person feels who has been excluded and subordinated. appiah’s book is splendidly argued and also splendidly written. i have little to disagree with concerning its proposal for a “rooted cosmopolitanism.” indeed, although he seems to think that we disagree, it is my belief that we really have very similar views. but i do think that there is a good deal more to be said on the topic of education, which he rightly makes central. i shall begin by setting out the one serious normative disagreement i have with appiah, insisting on the martha nussbaum importance of a distinction between “political liberalism” and “comprehensive liberalism” that does not figure in his argument—although he seems to lean, along with mill, toward the more comprehensive form. i shall then locate the issue of education within a political-liberal conception of political principles, where its role will look subtly different from its role in appiah’s autonomy-focused con- ception. i shall then make some claims about what is truly essential to public edu- cation for democratic citizenship, so construed. finally, i shall make some observations about the crisis that faces us. i. political and comprehensive appiah neglects a distinction that i believe central to good thought about political principles in a pluralistic society: namely, john rawls’s and charles larmore’s distinction between a liberalism that makes comprehensive recom- mendations for how lives should go, using comprehensive epistemological, ethical, and/or metaphysical doctrines, and a liberalism that carefully prescinds from reliance on any such comprehensive doctrines, out of respect for the plu- rality of different reasonable ways citizens may choose to live their lives. (that ethical reason of respect, not skepticism or neutralism for its own sake, is its reason for so prescinding.) appiah does refer to the relevant works of rawls and larmore, but he mischaracterizes them, i believe. he imputes to rawls the view that “governments should be neutral among different reasonable conceptions of the good life” ( ). so far so good, though he does not tell us why this degree of neutrality is recommended. but he then infers from this that “government should not interfere in the ethical dimensions of our lives” ( ). later, similarly, he states that “when antiperfectionists inculcate civic virtues . . . they do so for . . . consequential reasons. the state’s principal concern isn’t with the ethical success of our lives; it’s with the stability and survival of the political order” ( ). since rawls is the only antiperfectionist named in the preceding discus- sion, i take this as a characterization of rawls’s position. (later, in p. , he imputes a similar position to larmore.) the rawlsian state, however, is not ethically neutral. the political sphere is what rawls repeatedly calls a “partial moral conception;” he also characterizes it as a “module” that can be attached to the rest of one’s comprehensive doctrine by people who otherwise have very different views of the meaning and purpose of life. larmore makes similar claims, emphasizing very strongly that the polit- ical conception is not morally neutral. so rawls’s (and larmore’s) “method of avoidance” means only that the political ought to avoid grounding itself in any comprehensive doctrine outside itself: it ought to be both partial in scope and “freestanding,” not invoking comprehensive doctrines in its own justification. so it will not use notions such as those of the soul, or self-evident truth, that are controversial among the major comprehensive doctrines. it will, however, use (partial) ethical notions: a partial ethical conception, indeed, is exactly what it is. the imminent demise of liberal for rawls, as for larmore (and larmore’s own writing about rawls empha- sizes this point) the key ethical notion is that of equal respect, which grounds the political principles (the priority of liberty and the difference principle). it is for an ethical reason, the reason of equal respect, that the rawlsian state will pre- scind from employing comprehensive theological, metaphysical, and epistemo- logical doctrines, and also ethical doctrines about how lives go well in general. rawls adds, however, that the state will have to have some way of talking about issues of objectivity (avoidance of bias and so on), and some way of dealing with issues of motivation to engender support for itself over time; it will therefore use a political notion of objectivity and a “reasonable political psychology,” which try to avoid the most contentious issues that divide the major comprehensive doc- trines. whether he has succeeded in this task the reader must judge; i believe that he does succeed, and i try to develop the psychological doctrine further myself, writing about the political role of compassion, and of the political critique of disgust and stigmatization. all of this can, rawls believes, be endorsed as good, and not just for con- sequential reasons of stability, by people who hold many different comprehen- sive doctrines. that is what the idea of “overlapping consensus” is: the idea that people really affirm the political principles as an ethically rich part, or module, of their own comprehensive doctrine. the idea of an overlapping consensus is carefully distinguished from the idea of a mere modus vivendi focused on stabil- ity. whether such a consensus is possible is a question i will not discuss here. but it is a picture of political pluralism very different from the one that appiah paints. and it is the one i accept, though my specific account of the content of the political is slightly different from rawls’s. what this picture means is that the political is both thicker and narrower than it is in appiah’s sort of pluralism, which seems to me to be a thin sort of com- prehensive liberalism. on appiah’s view, the state can legitimately intervene to promote autonomous lives, so long as it does so with considerable restraint, focus- ing primarily on preventing and correcting obvious obstacles to such lives, such as misinformation, lack of self-control, and ignorance. thus, his view is similar to the views of j. s. mill and joseph raz, both comprehensive liberals, although appiah has a lot more sensitivity to issues of pluralism than mill and probably than raz. on the rawlsian view, by contrast, the state has a sphere that is nar- rower: that of ensuring continued support for the values that form part of the polit- ical conception itself, and of distributing to all citizens some important prerequisites of well-being, but stopping well short of any comprehensive doc- trine of well-being, however thin. it is important to see that the rawlsian state does not limit itself to cultivat- ing sentiments and attitudes required by the political conception and its replica- tion over time, although this will be one of its most important educational tasks. the state also, in rawls’s view and mine, has a further job, namely, giving all citizens some very important prerequisites of general well-being. politics is not martha nussbaum just about politics, it is about and for life. (here lies one of the major disagree- ments between rawls and habermas.) that is why the redistribution of wealth and income is so important: because these are prerequisites for people’s lives in general, not just for the sustenance of the political conception. rawls’s view, then, may overlap considerably with appiah’s in its actual recommendations, but its account of itself will be subtly different. describing the general prerequisites of well-being in a way that does not involve any comprehensive ethical doctrine is a difficult matter, concerning which rawls kept changing his mind until the end of his life. (he began by saying that primary goods such as income and wealth are all-purpose means to the good in all comprehensive doctrines. later, persuaded by thomas nagel and others that it simply was not true that income and wealth play a similar role in all the com- prehensive doctrines, he altered his view of primary goods, saying, instead, that they should be understood in connection with the kantian political conception of the person, which citizens accept for political reasons. he continued to insist, however, that the political must provide for more than itself. and so it remained somewhat unclear why primary goods should be understood primarily in con- nection with the political conception of the person. later still, in making revi- sions for a new edition of political liberalism, which he did not complete due to illness and death, he emphasized that the kantian flavor of the conception of the person troubled him, and he would prefer to recast the whole account in terms of a less divisive conception of reason. all this means that rawls had not fully solved the problem to which i refer, that of getting a conception of the prerequi- sites of well-being that could form part of the overlapping consensus.) my own preferred way of solving the problem is to state the general prerequisites in the form of a list of capabilities, not actual functionings, and to prescind very care- fully from any recommendations as to functioning. thus, the state will make it possible for all citizens to have decent health care, but it will not penalize unhealthy choices. it will give citizens the right to the free exercise of religion, but it will not recommend religious functioning. it will give political rights and opportunities to all, but avoid compulsory voting (which offends some religious doctrines). and so forth. i make exceptions for children in cases where the adult capability requires actual functioning as a child; in this way, once again, there may be considerable convergence between my political liberalism and appiah’s comprehensive liberalism in practice. the theoretical account, however, remains significantly different. the state that i favor should probably not use the notion of autonomy, a notion that has (and had in mill) strong historical links to the rejection of theis- tic sources of meaning. it should not announce, with mill, that non-autonomous lives (such as the lives mill thought calvinism urged one to cultivate, or lives in the military, or lives in a traditional community-based religion like that of the old order amish) are less worthwhile than lives that have thrown off the shackles of authority. but it may reasonably support a general capability for practical the imminent demise of liberal reasoning, understood in a more everyday way, both in order to support the polit- ical conception and in order to give citizens opportunities to think critically about their own way of life, at least enough so that exit options are available to them. supporting human capabilities without showing disrespect for comprehen- sive doctrines that urge people not to use some of these capabilities is a difficult balancing act, as wisconsin v. yoder shows us all. and yet it is plausible to think that the case was rightly decided, supporting sufficient public education to give amish children exit options and the critical thinking relevant to citizenship, without undermining utterly the comprehensive doctrine of the amish. mill would not like the decision, and i am not sure whether appiah would: much depends on how he defines the idea of autonomy, and how comprehensive he makes it. clearly, as rawls says, the state does not need to make it equally easy for all conceptions to accept the overlapping consensus; some will do so only with strain, and some may fail to gain adherents over time. but it will never say to the amish adult, or the soldier, or the roman catholic, “autonomy is a general good in human lives, and your life is therefore ‘pinched’ and ‘hidebound’ [i quote from mill’s discussion of calvinism in on liberty], less rich and satisfactory than the lives of many of your fellow citizens who have dared to throw off the shack- les of authority.” does appiah say this to the amish, or to the career soldier? i am not sure, but i suspect that the answer is yes. so: the political-liberal state narrows its claims. in another way, however, its claims may be considerably richer and deeper than appiah’s thin comprehensive liberalism suggests. anything that lies inside the political module is, ex hypoth- esi, affirmed by all citizens, at least all whose comprehensive doctrines affirm the key idea of equal respect that underlies the constitution, and the principles embed- ded in the constitution itself. so, the state does not have to pussyfoot around with these things: it can teach them flat out, as the best ideas to live with together, using whatever devices of imagery, rhetoric, and emotional suasion seem best suited to the task of imparting them to the young. it has no obligation to be fair to the opposing position. take race, for example. the constitution forbids dis- crimination on the basis of race. so, our schools should not simply teach that we have antidiscrimination laws; it should actively bring up small children as non- racist citizens, in the variety of ways that is familiar to us in daily life, since this is one thing our schools have learned to do pretty well: by not racializing the classroom; by strong opprobrium directed at racist behavior and speech in the classroom; by good teaching about the history of race, teaching that is in no way neutral, but which inculcates anger at racial injustice and hope for a world of racial harmony. small children will, as they so often do, put on plays in which some of them pretend to be people forced to sit in the back of the bus. they will see how they feel when they sit back there, and those emotions will be a topic of classroom discussion—not at all neutrally, since the ethical principle of non- racism is part of the political module that we share. the school will celebrate martin luther king jr.’s birthday, and it will not celebrate strom thurmond’s birthday. it will commemorate the life of rosa parks, and it will not commemo- martha nussbaum rate the life of george wallace—except, perhaps, as an example of regeneration and apology. this non-neutrality is no problem for political liberalism, since it is a partial ethical conception, and neutrality is not what it is about inside the polit- ical module. (about related things that lie outside the boundaries of the politi- cal/ethical conception, however, such as the existence or non-existence of god or gods, and whether god disfavors the racist, the classroom will be normatively silent, although it certainly may and should tell people about the variety of beliefs that is held in their society.) in rawls’s view, and my own, anyone who is willing to accept the basic polit- ical principles can accept all of this. there will be people in any society who do not accept its constitutional principles—who want to reintroduce slavery, for example, or to do away with our constitutional commitment to non-discrimina- tion. those people will not be suppressed, unless they violate the rights of others. they may speak freely, unless their speech poses an imminent threat of public disorder. but they are not going to be treated neutrally. the classroom will not give their views equal weight with the views that support the constitution. it is under no obligation to do so, since, once again, the political conception has a def- inite ethical content, and is not a form of neutralism. even when we are dealing with adults, these racists will be treated asymmetrically with those who do support the constitution: they will not be able to propose legislation that goes in their direction, since the relevant contradictory values are entrenched in the constitu- tion. they will have to amend the constitution itself—or even, in the case of india, call for an entirely new constitutional convention—if they want to change one of their society’s “basic features.” ii. educating citizens in a pluralistic society one thing that a society based upon equal respect needs to do most urgently is to teach young citizens that their society contains many religions and many ethnicities, and that we are all committed to the fair treatment of all of them. indeed, for both rawls and me any such society should teach that this commit- ment to equal respect is the source of our most basic political principles. under- standing this commitment is an essential prerequisite for citizenship, so it is part of the political “module” itself. we do not even need to reach the question of whether it is a consensus-worthy prerequisite of good life in general. this brings me to appiah’s treatment of mozert v. hawkins, the case in which a baptist mother requested an exemption for her children from a series of primary school readers that presented children with pictures of different american ways of life, appealing to them to imagine these different lives. at the outset of his dis- cussion of education, appiah states, and apparently endorses, mill’s view that the liberal state has two tasks, one related to individual autonomous lives, and one related to the good of society (p. ). in the analysis that follows, however, he focuses entirely on the first job, more or less dropping the second. (see “would severely diminish a child’s well-being,” p. ; “to protect the child’s growth to the imminent demise of liberal autonomy,” p. ; “to compromise the possibility of an autonomous adulthood,” p. ; “a child’s prospects for autonomy,” p. . in none of these contexts is the question of society’s good mentioned.) i believe that this neglect of the polit- ical part of the task skews his treatment of the issues involved in mozert. thus, appiah says that the case could “readily” have been resolved by allowing the children to opt out of the reading series: after all, they could have been tested on reading in some other way (p. ). this may be so, if the only issue were personal autonomy. but this is far from being the only issue. these students are going to become citizens in a pluralistic society, so they had better learn about the existence of other ways of life, and they had better be encouraged to imagine those other ways. the mother’s claim that the truths contained in the bible were all her children needed to know is just not reasonable from the point of view of citizenship and the political module. if she does not accept the constitutional principles, then we do not have to give her equal treatment in framing the curriculum, any more than we need give the racist equal treatment. (would appiah say that a racist parent should have an exemption from a reading curriculum that teaches non-racism?) and i think she really cannot be accepting our constitutional principles, when she says that the bible contains everything her children need to know. she is just wrong, from the point of view of the political conception: they need to know the history of race, and religious persecution, and many other facts about america not contained in the bible, if they are to participate in and sustain a political conception based on equal respect and the political principles to which that value gives rise. the school wisely decided that a democracy needs to teach children to think (respectfully and imaginatively) about other ways of life. that is a primary task of the public education system, namely to devise ways of inculcating in the young the values that sustain the political conception. the most significant opinion in the case said exactly this. citing another case in which the court had affirmed that the public schools “serve the purpose of teaching fundamental values ‘essential to a democratic society,’ including ‘tolerance of divergent political and religious views’,” the court then makes a rawlsian distinction between “civil tolerance” and “theological tolerance,” saying that the reading texts do not ask students to accept any particular theo- logical belief (including beliefs about the salvation of others) as true, but they do teach respect and understanding, a legitimate and indeed crucial civil (political) value. correctly, in my view, they hold that such a teaching cannot possibly be an unconstitutional burden on vicky frost’s free exercise of religion. for after all, it is required for the sustenance of the whole set of rights and liberties of which the first amendment is one part. in my view, we can say quite a lot about what education for citizenship in a pluralistic democratic society that is part of an interlocking world should look like. because i have written a book on this topic, i shall simply summarize, in order to turn to the current crisis. i focus, as before, on the humanities and social sciences, leaving the science curriculum for another time. in my book i focused martha nussbaum on liberal education in colleges and universities, but it was always my view that these values need to be cultivated appropriately by primary and secondary edu- cation. colleges will not get very far, unless students have begun much earlier. three values, i argue, are particularly crucial to citizenship in such a nation and for such a world. all, i now add, can be handily accommodated within the political module, as essential underpinnings for its most basic values—although all also have contributions to make to life more generally. the first is the capac- ity for socratic self-criticism and critical thought about one’s own traditions. although some parents may object to this sort of teaching, as they always have since the time of socrates, who lost his life on the charge of “corrupting the young,” we may give them socrates’s own answer: democracy needs citizens who can think for themselves, rather than deferring to authority, who can reason together about their choices rather than simply trading claims and counter-claims. socrates, appropriately, compared himself to a gadfly on the back of a noble but sluggish horse: he was waking democracy up, so it would conduct its business more responsibly. the second ability is the one that was involved in mozert, namely the ability to see oneself as a member of a heterogeneous nation, and world, understanding something of the history and character of the diverse groups that inhabit it. most of my book is taken up with spelling out the curricular dimensions of this one and the pedagogical pitfalls involved, so i shall say no more about it at present. the third ability seems to me possibly the most crucial: it is what i call “the narrative imagination,” the ability to think what it might be like to be in the shoes of a person very different from oneself, indeed a whole range of such persons. for this ability, we need literature and the arts. the great progressive educators of the twentieth century, dewey and tagore, understood this extremely clearly, building the arts into the very core of their curricula for democratic citizenship. (tagore did this through elaborate dance-dramas and other student productions, dewey more abstractly, leaving a lot to the teacher’s own ingenuity, though at the lab school he favored curricular requirements in the arts that are still, though barely, in place. ) both felt that the cultivation of imaginative sympathy was a key prop to good citizenship; that children had this ability to be tapped, if it was not killed off; but that it had to be made more sophisticated and precise through education. iii. democratic education on the ropes education of the type i recommend is doing splendidly in the place where i first studied it, namely the liberal arts portion of college and university curricula. indeed, it is this part of the curriculum, in institutions such as my own, that par- ticularly attracts philanthropic support, as rich people remember with pleasure the time when they read books that they loved, and pursued issues open-endedly. (some of their enthusiasm is related to the personal-life contribution of liberal education, some to its contribution to citizenship.) many nations whose univer- the imminent demise of liberal sity curricula do not include a liberal arts component are now striving to build one, since they acknowledge its importance in crafting a public response to the problems of pluralism their societies face. i have been involved in such discus- sions in the netherlands, in sweden, in india, in germany, in italy, in india and bangladesh. (whether reform in this direction will occur is hard to say: for liberal education has high financial and pedagogical costs. teaching of the sort i rec- ommend needs small classes, or at least sections, where students get copious feed- back on their writing assignments. european professors are not used to this idea, and would at present be horrible at it if they did try to do it; and bureaucrats are unwilling to believe that it is necessary to support the requisite number of faculty positions.) the abilities of citizenship are doing very poorly, however, in the most crucial years of children’s lives, the years known as k through . here the demands of the global market have made everyone focus on scientific and tech- nical proficiency as the key abilities, and the humanities and the arts are increas- ingly perceived as useless frills, which we can prune away to make sure our nation (whether it be india or the united states) remains competitive. to the extent that they are the focus of national discussion, they are recast as technical abilities themselves, to be tested by quantitative multiple-choice examinations, and the imaginative and critical abilities that lie at their core are typically left aside. at one time, dewey’s emphasis on learning by doing and on the arts would have been second nature in any american elementary school. now it is under threat even at the dewey laboratory school. national testing has already made things worse, as national testing usually does: for at least my first and third ability are not testable by quantitative multiple-choice exams, and the second is very poorly tested in such ways. (moreover, nobody bothers to try to test it even in that way.) whether a nation is aspiring to a greater share of the market, like india, or strug- gling to protect jobs, like the united states, the imagination and the critical fac- ulties look like useless paraphernalia, and people even have increasing contempt for them. thus in west bengal, tagore’s pathbreaking santiniketan school, which pro- duced amita sen, amartya sen, satyajit ray, and many more gloriously inde- pendent and imaginative world citizens, is now viewed with disdain as a school for problem children. long ago, jawaharlal nehru sent his daughter indira there, though she spoke little bengali. (and it was the only happy time at school she had, though she attended many famous schools.) today nobody from outside west bengal wants to go there. a parent’s glory is the admission of a child to the indian institute of technology. meanwhile, at the dewey lab school, the arts require- ment is being watered down under pressure of the drive for success on the part of parents and administrators. worse yet, the sheer burden of homework in all disciplines makes it impossible for children to enjoy the use of their critical and imaginative faculties. the united states has some resilience still, thanks to its traditions of local autonomy. thus, indian-american friends of mine wistfully compare the educa- martha nussbaum tion their children receive here (my example of the play about rosa parks comes from one such discussion) with the education they themselves never had in india, where rote learning rules the roost. but the united states is moving toward india, not vice versa. indeed, most outrageously and thoughtlessly, the united states is currently egging on other nations to emulate our worst, not best, traits. the phone call from x university was more upsetting to me than any other confrontation with the recent decline in democratic education: for x is currently investing a lot of money to set up educational outposts all over the world. these narrow ways of thinking about what is worth cultivating will, then, be exported widely, with great money attached. imagine the spectacle of y. z. telling the people of kolkata, who have their own rich and glorious traditions of democratic education, that x can provide wonderful new assistance toward the further destruction of precisely that tradi- tion. and yet exactly this is about to happen. imagine, too, the elites of kolkata lapping all this up, since the prestige of x is superior even to the prestige of the indian institute of technology, so much so that every controversial word of y. z. (and there are so many) makes the front pages of every national indian newspa- per. will y. z. even urge people to think about what makes democracy strong, and to cultivate abilities that strengthen it? not very likely, since the same y. z. has proposed eviscerating the core curriculum at x itself, in such a way that instead of one course in moral reasoning one could take two technical econom- ics courses, and in such a way that the humanities make up a much smaller frac- tion of the entire curriculum than previously. (he may not get his way, but that will not mean that he has broadened his conception of education.) what will we have, if these trends continue? nations of technically trained people who do not know how to criticize authority, useful profit-makers with obtuse imaginations. what could be more frightening than that? indeed, if you look to the indian state of gujarat, which has for a particularly long time gone down this road, with no critical thinking in the public schools and a concerted focus on technical ability, one can see clearly how a band of docile engineers can be welded into a murderous force to enact the most horrendously racist and antidemocratic policies. and yet, how can we possibly avoid going down this road? i believe that outrage is called for, on the part of every person who cares about the future of democracy in the world, and i think philosophers should be leading the expression of outrage. and yet, sad to say, virtually no prominent philosopher writes much about education (one of the great subjects of the pro- fession from plato to locke, rousseau, kant, mill, and sidgwick), and virtually no current philosopher writes on k through in a way that makes a public impact. (here i very much include myself.) appiah’s book is a welcome step back to a philosophical focus on public education. its brilliance and its literary quality suggest that it may reopen a wider debate in the united states, and possibly in other nations as well. the book does not go far enough, however, because it focuses on the personal rather than the the imminent demise of liberal social task, on autonomous lives rather than the prerequisites of citizenship. i call for a second volume, and for all of us to write on the topic in our own ways, with the sense of urgency that our situation demands. notes see my discussion of the indian situation in “education and democratic citizenship: beyond the textbook controversy”, in islam and the modern age ( ), – ; and a slightly different version, “freedom from dead habit,” the little magazine (new delhi) ( ), – . see my reply to his and other essays in for love of country, and the preface to the new edition of that volume. a fuller version of that preface, and the fullest statement of my view on the topic, is in “compassion and terror,” daedalus (winter ): – . a slightly different version, same title, in terrorism and international justice, ed. james sterba (new york: oxford university press, ), – . see my “political objectivity,” new literary history ( ), – . see the discussion of political liberalism in upheavals of thought, chap. and hiding from human- ity, chap. . i give a detailed account of the conception in rawls, justice, and tolerati: an intro- duction to political liberalism, forthcoming from columbia university press. thus, when appiah writes that the doctrine of perfectionism is “controversially” imputed to me and to sen ( n. ), i would rather write—of myself, since sen has not taken a position on this controversy—that it is “falsely” imputed. i have endorsed political liberalism very clearly in arti- cles dating back to and in two books, women and human development ( ) and frontiers of justice ( ). i am not aware of anyone who imputes perfectionism to me, but it may be so. here i am referring to correspondence between rawls and his columbia editor a copy of which is in my possession, and which i have mardy rawls’s permission to cite. see jerome schneewind, the invention of autonomy cambridge: cambridge university press, . putting it this way obscures a problem in rawls’s text that is actually very difficult, since the concept of “reasonable comprehensive doctrines” is actually given more than one definition. in one view, which i take to be the one rawls ought to affirm, a reasonable doctrine is one that accepts and affirms the political “module.” rawls also, however, has an independent characterization of the “reasonable” that involves other ideas such as comprehensiveness, consistency, and ordering of values. i believe that it was mistaken of him to offer this characterization, deep though the reasons for it are, and that it actually sweeps far further than he believes. it is not just new age religion that will be ruled out by these criteria, it is, arguably, christianity itself, since that religion is deliberately grounded on belief in a contradiction: the trinity is standardly understood to be a doctrine belief which humbles reason, because it cannot be given a logically coherent form. i discuss all this in my forthcoming book on pl, columbia university press forthcoming. rawls does not think the brandenburg test speech-protective enough: he recommends protecting all speech unless there is a constitutional crisis and the speech risks upsetting the constitution itself. madison had a similar view, in his debate with george mason. term taken from keshevananda bharati, the indian supreme court case that introduced this doctrine—after indira gandhi had gotten parliament to repeal the whole list of fundamental rights, and voters, thinking this a bad thing, turned her out of office. see mozert v. hawkins county board of education, f. d ( th cir. ): frost, the mother who brought the case, said that the bible “is the totality of my beliefs.” the case was heard by a three-judge panel; there was no majority opinion, though all three reached the same conclusion, because all three wrote separate opinions. i cite from judge lively’s, which was (since he was chief judge) the opinion of the court. the other case, bethel school district no. v. fraser, u.s. ( ), is long on theory but peculiar in content. it concerns a high school student who gave a speech on behalf of a friend who was running for student president; the speech had a mild sexual content that deeply shocked martha nussbaum the justices leading them to wax eloquent about matters of civil toleration—before they proceed to take away the student’s right to be valedictorian of his class! (the majority opinion, written by chief justice burger, refuses even to quote from the speech, saying it is offensive, but justice brennan, in dissent, quotes the entirety of it, and we see just how innocuous and stupid it really is.) this distinction goes back at least to rousseau’s on the social contract, where rousseau argues that societies are entitled to enforce both sorts of toleration, since “it is impossible to live at peace with those whom one believes to be damned.” experience would appear to have proven him wrong. cultivating humanity: a classical defense of reform in liberal education (cambridge, ma: harvard university press, ). we might add leonard elmhirst in england, whose dartington hall school was closely modeled on tagore’s santiniketan, where elmhirst had spent a good deal of time. at the cambridge school, in weston, massachusetts, where my daughter went to high school and where i am a trustee, the dewey values continue in their full force, in part because the boston area contains so many private schools that a dewey-style school can carve out a market niche and avoid the pressure toward conformity that lab, the leading private school in chicago, con- tinually faces. for details, see my the clash within: violence, hope, and india’s future (cambridge, ma: harvard university press, forthcoming ), chap. . one excellent book, philosophical in orientation though not by a professional philosopher, that should have received more attention is leon botstein’s jefferson’s children; but botstein, pres- ident of bard college, has recently been focusing all his attention on post-secondary education (and on his musical career); the book’s recommendations for the public schools have been little discussed. the imminent demise of liberal [pdf] thank you for your continued support | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / . . corpus id: thank you for your continued support @article{keener thankyf, title={thank you for your continued support}, author={t. keener and g. hidy}, journal={journal of the air & waste management association}, year={ }, volume={ }, pages={ - } } 	t. keener, g. hidy 	published 	psychology, medicine 	journal of the air & waste management association view on taylor & francis tandfonline.com save to library create alert cite launch research feed share this paper citations view all citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by most influenced papers sort by citation count sort by recency organizational culture, job satisfaction and turnover intentions: the mediating role of perceived organizational support 	david j. emerson 	political science 	 	 save alert research feed high bandwidth low power operational amplifier design and compensation techniques 	v. kumar 	computer science 	 	 	pdf save alert research feed references thomas (us virgin islands) 	thomas (us virgin islands) related papers 	abstract 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue “prince edward island’s unique ‘brotherly love’ community”: faith and family, communalism and commerce in b. compton limited, – “prince edward island’s unique ‘brotherly love’ community”: faith and family, communalism and commerce in b. compton limited, – ruth compton brouwer acadiensis: journal of the history of the atlantic region / revue d’histoire de la region atlantique, volume , number , winter/spring , pp. - (article) published by university of new brunswick, department of history doi: for additional information about this article [ this content has been declared free to read by the pubisher during the covid- pandemic. ] https://doi.org/ . /aca. . https://muse.jhu.edu/article/ https://doi.org/ . /aca. . https://muse.jhu.edu/article/ “prince edward island’s unique ‘brotherly love’ community”: faith and family, communalism and commerce in b. compton limited, - ruth compton brouwer imprégnée des enseignements millénaristes d’un pasteur insulaire non conformiste du e siècle et animée par le désir de subvenir collectivement aux besoins d’un groupe de parenté tricoté serré qui avait éprouvé des difficultés économiques à l’aube du e siècle, la communauté utopique b.  compton limited, de l’Île-du- prince-Édouard, suscita brièvement l’intérêt général dans le contexte de la grande crise. un sociologue rural de passage remarqua, par exemple, l’excentricité de ses membres tout en constatant leurs liens étroits avec le tissu social insulaire. cet article explore les origines de la communauté, les facteurs qui la gardèrent unie et les changements apportés par la guerre, qui menèrent à sa dissolution officielle en  . imbued with the millenarian teachings of a maverick th-century island clergyman and with a desire to provide communally for a close-knit kin group that had experienced economic hardship at the dawn of the th century, b. compton limited – a utopian community on prince edward island – briefly became an object of widespread interest in the context of the depression. a visiting rural sociologist noted, for instance, its members’ eccentricity while affirming their strong ties within the island’s social fabric. this article explores the community’s background, the factors that held it together, and the wartime changes that culminated in its formal dissolution in . in the producers of “we the people,” a new york-based radio program, wrote to hector d. compton ( - ), the secretary-treasurer of b. compton limited, inviting him to address the program’s national audience. “in a world where there is so much misunderstanding and where disputes are commonplace,” the letter stated, “the story of your community with its model behaviour would serve as a powerful example to the people of america.” the leonard l. bass to hector d. compton, august , hector d. compton collection, private collection, uncatalogued and partially dispersed (hdc collection). the quotation in the title of this article is from flora s. rogers, “brotherly love rules this community,” probably a canadian press story from the guardian (charlottetown): “charlottetown, june [ ] (special to the [halifax] chronicle),” hdc collection. i am grateful to hector’s son george h. compton (d. ) and to members of his extended family for allowing me to borrow papers from this collection, principally copies of his surviving correspondence. the collection provides the ruth compton brouwer, “‘prince edward island’s unique “brotherly love” community’: faith and family, communalism and commerce in b. compton limited, - ,” acadiensis xlv, no. (winter/spring ): - . company about which the radio producers enquired was a small utopian community imbued with the millenarian teachings of a maverick prince edward island clergyman as well as a desire to provide communally for a close-knit kin group that had experienced economic hardship at the dawn of the th century. this article provides some background and a brief sketch of the community before turning to the circumstances that led to the new york invitation. the article outlines the factors that held the community together and the wartime changes that culminated in its formal dissolution. the break-up took place in – little more than a decade after charlottetown journalist flora s. rogers had referred to “prince edward island’s unique ‘brotherly love’ community” as proof that “co-operative community enterprise works.” in a province where local and family history are pursued with zeal and affection, knowledge of this unique communitarian undertaking has largely been lost to the historical record despite occasional attempts by island historians to learn more about it. in canadians and their pasts, margaret conrad and her colleagues report that, overall, canadians believe that family history is the most important history. one prince edward island respondent whose view they cite asked rhetorically why everyone would not view family history as the most important form of history. nevertheless, within and beyond canada, alongside a seemingly insatiable appetite for personal genealogical research and for such popular television programs as pbs’s finding your roots, there are frequent anecdotes by authors of family histories about relatives’ unease with the prospect of having their shared past explored and exposed. nor is the unease confined only to those worried about bringing to view criminal ancestors and moral monsters in the family tree. rather, it extends to matters that, as australian historian marjorie theobald puts it, have now “gone completely from our moral handbook.” it may be that this seeming paradox can be understood only by paying attention to particular cases and contexts, as i try to do in this article. in a brief conclusion i suggest possible reasons why, from the mid- th century, former members and descendants of the compton community so readily let their ancestors’ utopian project slip to the recesses of the family memory bank and politely turned aside outsiders’ attempts to make it known. the article comes out of research for a larger project on the background and life of the compton community. as well as engaging with canadian family and religious history and the history of the atlantic region, the project seeks to contribute to the nascent field of utopian studies in canada. during the late th century in the united states there was a lively interest in such th-century utopian communities as acadiensis single most important primary source for this article, supplemented by information from the few people still living with personal memories of the compton community. i am also grateful to the three anonymous reviewers of the article for wonderfully engaged and engaging comments. rogers, “brotherly love rules this community.” margaret conrad et al., canadians and their pasts (toronto: university of toronto press, ), chap. (p. for pei respondent). accounts of such unease are given in, for instance, carolyn abraham, the juggler’s children: a journey into family, legend and the genes that bind us (toronto: random house, ); mary louise mccarthy, “mixed-race identity black and maliseet: my personal narrative,” acadiensis xliii, no. (winter/spring ): - ; and marjorie theobald, the wealth beneath their feet: a family on the castlemaine goldfields (melbourne: arcadia, ), quotation on . robert owen’s secular social experiment at new harmony, indiana, and john humphrey noyes’s perfectionist commune at oneida, new york, as well as in better-known endeavours such as those undertaken by the shakers, hutterites, and mormons. american scholars’ fascination with utopias arose in the wake of the establishment across north america in the late s and early s of many short- lived hippie or counter-cultural communities. although canada had its share of like-minded movements in this period (they seem to have been particularly salient in the maritimes and british columbia), those movements did not inspire the same degree of scholarly interest in earlier utopian experiments in canada. there have certainly been popular or scholarly accounts of individual communities, such as sointula, a finnish socialist group in early- th-century british columbia, and the religious utopias of the prairie west. in historian colin m. coates provided a brief overview of these and other canadian utopias at a canadian historical association annual meeting. and in an ongoing project that draws on their backgrounds in, respectively, urban planning and human geography, scholars beth moore milroy and brian s. osborne are building on the unfinished work of a deceased colleague to compile a list and briefly describe “built utopias in canada up to .” to date, however, there have been no scholarly book-length studies in english of canadian utopias. like other would-be explorers of utopian communities, i have learned that attempts at definitions of such communities have been fraught with difficulties – excessively broad at one extreme and too narrowly restrictive at the other. in her introduction to the concept of utopia, ruth levitas, for instance, wrote that the constant in all utopias is a “desire for a better way of living and being.” the introduction to a collection of essays to mark the quincentennial of sir thomas more’s birth was similarly expansive, referring to utopianism as “a bright vision of a world where things will be far better than they are now.” writing more recently, the authors of the historical dictionary of utopianism identified what they saw as some defining characteristics: “utopianism, ancient or modern, has almost always been marked by the following: group-based isolation from contemporary worldly corruption, . . . equality of goods and a rejection of luxury; regimentation of the lives of the participants . . . to assure the perceived common good; and direction of the new model society being placed in the hands of leaders . . . endowed with a vision . . . that b. compton limited although one s community is featured in john a. hostetler’s communitarian societies (new york: holt, rinehart and winston, ), the counter-cultural communities of that era are not typically included in the scholarly us literature. see, for instance, sharon ann weaver, “making place on the canadian periphery: back-to-the- land on the gulf islands and cape breton” (phd diss. in history, university of guelph, ); andrew scott, the promise of paradise: utopian communities in british columbia (vancouver: whitecap books, ); and kathleen rodgers, welcome to resisterville: american dissidents in british columbia (vancouver: ubc press, ). scott, promise of paradise; a.w. rasporich, “utopia, sect and millennium in western canada, - ,” prairie forum , no. (fall ): - . colin m. coates, “is there a canadian utopian tradition?” (paper presented to canadian historical association annual meeting, fredericton, may ). see “canadian utopias project/the legacy of professor jeanne m. wolfe,” http://canadianutopiasproject.ca. will bring peace and justice to all adherents within time.” there are, though, some well-known and long-existing communities commonly described as utopias, the amish, for example, in which one or more of these characteristics is weak or absent. given the problematic range of meanings and approaches illustrated in the works just cited, the more limited concept of “communal utopias” seems more helpful for locating the compton community within the broad utopian tradition. in america’s communal utopias, donald pitzer writes: “in this type of close-knit community much or all property is shared communally. members join voluntarily and live in rural settlements or urban housing partly isolated and insulated from the general society. they share an ideology and lifestyle while attempting to implement the group’s ideals.” certainly, communalism seems to have been an essential element in the life of b. compton limited. while it was founded more than half a century after what some scholars of american utopian movements have viewed as their golden age – the s is often highlighted and, in terms of location, the “burned-over district” of new york state – the compton community shared with many communal utopias of that earlier efflorescent period a deep concern with religion and, in particular, a sense of urgency about the coming of the millennium and the importance of awaiting it as a unified body of believers. nevertheless, b. compton limited was also an attempt to respond practically to the earthly needs and social challenges facing a small group of kin in a quite different time and place. sojourners and settlers the ancestors of these prince edward island communitarians were loyalists and planters. william compton, a carpenter like his father (also a william), had come as a boy from new jersey to new brunswick at the end of the american revolution. the family settled first at saint john and then at quaco (later called st. martins). young william’s marriage to mary vaughan linked him to a planter family that had relocated from nova scotia to quaco where, in the early th century, they would become important shipbuilders and ship owners. for a time in the s william acadiensis ruth levitas, the concept of utopia (hemel hempstead, uk: phillip allan, ), ; e.d.s. sullivan, the utopian vision: seven essays on the quincentennial of sir thomas more (san diego: san diego university press, ), ; james l. morris and andrea l. krass, historical dictionary of utopianism (langham, md: scarecrow press, ), . on the amish in canada, see orland gingerich, the amish of canada (waterloo: conrad press, ) and, for a mainly us approach, see donald b. kraybill, ed., the amish and the state, nd ed. (baltimore: johns hopkins university press, ). donald e. pitzer, “introduction,” in america’s communal utopias, ed. donald e. pitzer (chapel hill, nc: university of north carolina press, ), . morris and krass, historical dictionary, xxx; rosabeth moss kanter, commitment and community: communes and utopias in sociological perspective (cambridge, ma: harvard university press, ), ; peyton e. richter, ed., utopias: social ideals and communal experiments (boston: holbrook press, ), ; michail barkunin, crucible of the millennium (syracuse, ny: syracuse university press, ), - . esther clark wright, the loyalists of new brunswick (fredericton: n.p, n.d. [ ]), , and wright, the ships of st. martin’s: shipbuilding and a list of vessels built at st. martin’s new brunswick, - (saint john: new brunswick museum, ). one of several collections of essays on the planters is margaret conrad, ed., intimate relations: family and community in planter nova scotia, - (fredericton: acadiensis press, ), which includes althea and mary lived in cape breton, where william is said to have started a sawmill and a grist mill at malagawatch. by they had settled on prince edward island at belle river (originally called belle creek), in lot , some miles southeast of charlottetown. twenty-five years later, when mary died, pei was home to all but one of their surviving children. william’s death came about a year after his wife’s. in addition to their sons and daughters, the couple left behind grandchildren and great-grandchildren. as residents of belle river and the larger belfast area these comptons were near neighbours and sometimes marriage partners of the successful selkirk settlers and other gaelic-speaking scots presbyterians, many of whom became ardent followers of one of the island’s larger-than-life figures – the rev. donald mcdonald ( - ). born in scotland and trained at the university of st. andrew’s for ministry in the established church of scotland, mcdonald began his career in prince edward island in . as had been the case during his two previous, and reportedly inglorious, years as a missionary preacher in cape breton, he came without a specific call or appointment. and he encountered little in the colony in the way of an overarching and constraining ecclesiastical structure. indeed, the first established church of scotland minister to settle permanently on prince edward island had arrived only three years earlier. given these circumstances, and the charismatic leadership style and powerful revivals that came out of his own transformative conversion experience in , mcdonald was able to move beyond his past reputation for drunkenness and establish what was effectively an indigenous religious movement, eventually winning the support of more than ten per cent of the colony’s protestant population (almost half of which claimed a scottish background). for readers familiar with the history of maritime presbyterianism, b. compton limited douglas’s essay on the vaughans – “connectional history: a gender-related approach to genealogy,” - . pamela hatton compton’s unpublished genealogy on the loyalist comptons, prepared in the s, has been a very helpful departure point for research on compton family links. see “compton: bears; grant; hume; sanders; martin; munn; macdougall,” acc. , public archives and record office, charlottetown, pei. undocumented family memories of their cape breton sojourn are supported by an article by neil macdonald in mac talla ( july ); my thanks to pauline maclean, highland village museum, for e-mail correspondence, september , providing this information the census for prince edward island shows william compton and three sons settled on property in belle creek. see also mary vaughan compton’s obituary, islander, march . j.m. bumsted, “lord selkirk of prince edward island,” island magazine, no. (fall-winter, ): - ; susan hornby, ed., belfast people: an oral history of belfast, prince edward island (charlottetown: tea hill press, ); malcolm a. macqueen, skye pioneers and “the island” ([winnipeg: stovel, ]). jean m. maclennan, from shore to shore: the life and times of the rev. john maclennan of belfast, p.e.i. (edinburgh: knox press, ). david weale, “the ministry of the revd. donald mcdonald on prince edward island, - : a case-study examination of the influence and role of religion in a colonial society” (phd diss. in history, queen’s university, kingston, ); weale, “mcdonald, donald,” dictionary of canadian biography online ix ( - ), www.biographi.ca/en/bio/mcdonald_donald_ _ _ e.html; weale, “‘the minister’: the reverend donald mcdonald,” island magazine, no. (fall-winter ): - . see also rev. m[urdock] lamont, rev. donald mcdonald: glimpses of his life and times (charlottetown: murley & garnhum, ). in this admiring but not wholly uncritical account, lamont dealt with rumours that had followed mcdonald from scotland to cape parallels between mcdonald and another headstrong and idiosyncratic highland- born preacher of the same era, norman mcleod, are likely to come to mind. william and mary compton may have come to know mcdonald during the years when they lived in the same part of cape breton, as some family accounts later claimed. whatever the case, and despite their english ethnicity and their baptist background, they and their numerous offspring became firm disciples of mcdonald and his millenarian message, an important aspect of which was the belief that his followers were part of the so-called ten lost tribes of israel. in the years after mcdonald’s death in , there were divisions among his followers. some former mcdonaldites eventually made their way into the newly formed and proudly national presbyterian church in canada, established in . others, however, remained determinedly apart, even establishing new churches on pei as well as a congregation of expatriate mcdonaldites in cambridge, massachusetts. the comptons remained part of the mcdonaldite fold. but in some of them – the group with whom we are concerned here – were consigned to outsider status as a result of another rupture (discussed below). they became a fragment of the mcdonaldite remnant, and moved on to an even more separatist and anomalous religious existence as communitarians. a second background factor in the establishment of the compton community was rooted in material circumstances. the economic dislocation that afflicted rural areas of the maritimes in the post-confederation decades became part of the compton experience. like thousands of other islanders, and maritimers generally, some of these kin left the province to seek work – a few in western north america, many others in the so-called boston states. leaving behind unviable family farms and underemployment or redundancy, some of the job seekers became permanent exiles. others returned after a period of working abroad. one of the returnees was ben compton, the man who became known as the founder of b. compton limited. when ben came back to the island after a few years acadiensis breton, especially with regard to excessive drinking. without wholly denying the rumours, lamont suggested that they were products of rifts between the few established church of scotland ministers in nova scotia and the more numerous seceders; see, especially, - . r. maclean, “mcleod, norman,” dictionary of canadian biography online ix, www.biographic.ca/en/bio/mcleod_norman_ e.html. lamont, donald mcdonald, - ; weale, “mcdonald, donald.” a belief that they were “latter- day israelites” was common currency among th-century scots presbyterians; see denis mckim, “boundless dominion: providence, politics, and the early canadian presbyterian worldview, - ” (phd diss. in history, university of toronto, ), . british israelism or anglo-israelism is just one variant (albeit an important one) in the long history of the myth of the ten lost tribes of israel. see, for instance, zvi ben-dor benite, the ten lost tribes: a world history (new york: oxford university press, ) and tudor parfitt, the lost tribes of israel: the history of a myth (london: weidenfield & nicolson, ). j.h. bishop, church of scotland in prince edward island (macdonaldite section) (n.p.: n.d. [ ]). alan a. brookes, “islanders in the boston states,” island magazine, no. (spring-summer ): - ; margaret conrad, “chronicles of the exodus: myths and realities of maritime canadians in the united states, - ,” in the northeastern borderlands: four centuries of interaction, ed. stephen j. hornsby, victor a. konrad, and james j. herlan (fredericton: canadian-american center/university of maine and acadiensis press, ), - . working as a carpenter in boston in the s – he may have gone there following the death of his first wife in – he reportedly had a few hundred dollars in his pocket, which he put towards the task of pulling his kin out of poverty. his relatives may not have been worse off than islanders generally, but they included other returnees. among these were one of his brothers and a cousin, hector compton’s father, two family heads who after more than a decade in manitoba and the american west had nothing more to show for their sojourning years than deep impoverishment and numerous offspring. here is hector compton’s account of the start-up years under ben’s leadership as recalled for relatives many years later: he made a start and helped us start lobster fishing. it was hard going at the outset but we kept at it and learned a difficult business. other cousins were invited from abroad, always coming penniless and we added more boats, ran a cannery and besides this began to build up land or soil and buildings. for years we each took our shares and spent it as we pleased. a crisis arose in and benjamin suggested that we all leave our funds in the bank add to it as we could and live from it, thus supporting some dependent relatives jointly etc. we all of us about ten young single men, cheerfully assented. we were standing outside a kitchen door where we chanced to meet. there was never a paper signed or a by law made. hector described ben as “a great leader of men, a leader in spiritual as well as material matters,” and explained that under his guidance the group had gone ahead “financially or in the acquisition of property in miraculous speed the first dozen years.” hector did not elaborate on the nature of the “crisis” that had spurred their communitarian turn; it was perhaps just another influx of impoverished relatives from “abroad.” nor did he refer to the source of ben’s inspiration. but it seems possible, even likely, that ben was familiar with and inspired by the best-known utopian work of the era, edward bellamy’s looking backward: - , first published in boston in . as the editor of a recent edition of bellamy’s novel observes, the book “quickly acquired cult status. almost everyone who was interested in the so- called ‘social question’ debated the book, ‘down to the bootblacks as they s[a]t by the curbstones.’” its influence was certainly felt in later years in western canada on a range of utopian and progressive thinkers, including the leaders of both the social credit and the co-operative commonwealth federation movements. hector may well have inherited his own copy of looking backward from ben. years later he b. compton limited hector d. compton to helen and peter, may , p. , hdc collection. information about benjamin’s role, including the quotation, is from fragment of a letter by hector d. compton, which lacks salutation and date but is marked “p.s” [postscript], hdc collection. in quoting from hector’s writing here and elsewhere i have generally retained his distinctive style of capitalization and punctuation. matthew beaumont, “introduction,” in edward bellamy, looking backward - , oxford world classics ed. (new york: oxford university press, ), vii. rasporich, “utopia, sect, and millennium,” esp. - , - . would lament the fact that a borrower had not returned his copy, and declared to one of his brothers that it had envisioned a system just like theirs. living in community: work, faith, kinship, and fame the community was not incorporated until and then only on the advice of its lawyer and its banker. by that time its net worth was $ , – a figure that included the assets of its satellite farming settlement, some miles across country in bangor near the village of morell. by that time, too, the company was under hector’s management. hector had become secretary-treasurer following founder ben’s death in an accident in the company lumber mill in . hector was no doubt the person best placed to succeed ben by virtue of his ability, his education, and his early and substantial involvement in the community. although his schooling had ended at about age , in , when his family returned to pei following their years in the west, he seems to have received a solid training in the basics during a period when they lived in chicago. as for his initial contribution to the community, writing in the immediate wake of the dissolution process in when there was briefly some tension about allocation of assets, he stated: “at the time we pooled all earnings in , i had a boat & fleet and a share in cannery to throw in, bought ½ acres of land and threw that in, had a trade [carpenter] and kit of tools as against others who had none, had saved $ . [sic; perhaps $ . ?] which later was also thrown into the common pool.” as for the mill that took ben’s life, it employed outside labour as well as community members and was an important source of company income. the company sold off its lobster fishery and cannery in the s in the face of turmoil in the industry. but its assets also included woodlots and farms for its own sustenance and for produce that it carried to markets in eastern nova scotia on a company-owned schooner. workers in a machine shop did custom work as well as maintain and in some cases build company equipment. of most interest to outsiders was the company store. it functioned as a acadiensis hector d. compton to bro. george, november , hdc collection. in her article “brotherly love rules this community,” flora s. rogers, whose husband, the founder of cfcy radio station in charlottetown, also knew hector, quoted from bellamy’s book in her article and claimed that the comptons knew the book well. james m. compton, bangor, and hector d. compton, belle river, liquidators, to provincial secretary and director of income tax, charlottetown, as written april , and redrafted june , hdc collection. the real estate of founder ben compton, who had died intestate in leaving young children, was evidently not part of this total. an online inflation calculator indicates that $ , . canadian dollars in would have purchasing power of $ , , . in ; see http://www.bankofcanada/ca/rates/related/inflation-calculator/. “death of mr. benjamin compton, victim of mill accident,” guardian, march . hector d. compton, untitled statement, april , hdc collection. although ben was the founder of the company and its acknowledged head, hector seems to have functioned as its secretary from the outset; see hector d. compton to george compton, july , hdc collection. hector d. compton, untitled statement, april , hdc collection. regarding the sale of the lobster business, see “agreement” of march signed by m.f. riley and h.d. compton; for the company-owned schooner see, for instance, hector d. compton to dear john and all, november ; regarding the mill and other assets, see, for instance, hector d. compton to commanding officer, royal canadian navy, halifax, october – all in hdc collection. general store to sell merchandise to outsiders while providing community members with goods on a no-cash, needs-based basis. finally, during the s hector evidently invested successfully in the stock market on the company’s behalf. in his defensive statement dealing with the dissolution process, he indicated that between , when ben died, and , prior to the economic downturn that heralded the depression, the company’s net worth had more than doubled, to $ , . . the increase, he claimed, had come mainly from “turnover profits every time we exchanged a security in those years.” even with the losses that followed from “the slump of ,” then, the company was better off than it had been in . when flora rogers wrote about the company in , it was supporting about members in its belle river and bangor settlements on the strength of its varied assets. compton families had been living in bangor at least since the s, along with other families known to be mcdonaldites. not all of them became part of b. compton limited helen jean champion, over on the island (toronto: ryerson press, ; repr. ), - (includes reference to custom-made machinery); “unique pei colony makes progress,” guardian, january . hector d. compton, untitled statement, april , hdc collection. rogers, “brotherly love rules this community.” rogers’s wording does not make it clear that this figure included both the bangor and belle river settlements. plan of lot , illustrated historical atlas of the province of prince edward island (philadelphia: j.h. meacham & co., ; repr. ), . when mcdonaldites built a new church on the bangor road in , the contributors evidently did not include any of the comptons who later established b. compton limited. a record book in the possession of the current secretary of the bangor free church of scotland contains details of the church’s financing. figure : map of prince edward island showing settlements of various sizes, including the two where families belonging to b. compton limited lived. source: will flanagan, cartographer, department of geography, saint mary’s university. b. compton limited, but those who did lived short distances apart on farms along the bangor road adjacent to non-company neighbours. in belle river, the households – five in number in – were closely grouped around the store and other communally owned buildings in a kind of oval formation. what was the glue that held b. compton limited together? two factors bear emphasis: the religious bond among community members and intermarriage among those members. first, however, it is worth noting a factor that was significant for its absence in this community: higher education. like most – though by no means all – of their fellow islanders, compton community members’ schooling was confined to the local one-room school. literacy was evidently a norm among their th- century ancestors. but it appears that, as in the case of the amish, the community’s leaders may have believed that higher education would be a source of potential disruption through its introduction of “alien ideologies.” certainly that was how it appeared to enid charles and sylvia anthony, two scholars who came to know a good deal about the compton community in the course of their research on prince edward island for a article published in the journal rural sociology. with regard to religion, as noted earlier, the group of comptons and related kin who began the community were the smallest and most isolated fragment of the rev. donald mcdonald’s surviving followers. their immediate ancestors had been very much a part of the mcdonaldite fold in the decades following his death in . after hearing of his passing, lydia compton hume, a granddaughter of william and mary, wrote a widely circulated poem that spoke to his followers’ sense of loss. her father, john compton, succeeded his brother-in-law george bears in as “ministering elder” in the belle river and brooklyn congregations, with authority to acadiensis for the bangor settlement in the s, see cummins atlas of province of prince edward island, canada (toronto: cummins map co., n.d. [c. ]), - . hector provided details about both settlements in hector d. compton to commanding officer, royal canadian navy, halifax, october , hdc collection. the bangor members of their community, he wrote, together owned “ acres of best farmland and woodland,” while in belle river they had “over acres of good cultivated land and over acres woodlands.” there were certainly many islanders whose education continued beyond these schools, allowing them to make significant contributions in canada and the united states in such fields as education, religion, medicine, and business. sometimes early support and encouragement had been provided at the local school. see ian ross robertson, sir andrew macphail: the life and legacy of a canadian man of letters (toronto: university of toronto press, ), . macphail’s father, william, a teacher, a farmer, and a strong mcdonaldite, was also a strong advocate of sound education. this and other evidence suggests that the unease about higher education within the compton community did not derive in any direct way from the rev. donald mcdonald’s doctrines. enid charles and sylvia anthony, “the community and the family in prince edward island,” rural sociology , no. ( ): - , esp. for “alien ideologies.” interestingly, however, enid charles that same year commented that within the compton community the “level of culture and of comfort is higher than that of their neighbours”; see “the trend of fertility in prince edward island,” canadian journal of economics and political science , no. (may ): . regarding the amish, see kraybill, amish and the state, , - , and gingerich, amish of canada, . ira mandelker believes that the oneida community was undermined by its founder’s decision to allow some young community members to attend harvard and yale universities, since they returned with disruptive ideas; see mandelker, religion, society, and utopia, . baptize and to preside over communion services. it was as ministering elder rather than farmer that -year-old john was identified under occupation in the census. but in that same year a theological split involving charges of “heresy” separated him and some close relatives from other mcdonaldites. in his account of the dispute, over which he seems to have presided, ewen lamont, the stern gaelic- speaking elder who was one of mcdonald’s earliest and most zealous followers, denounced those “intending communicants” – they went unnamed – whose views of divine judgement were so lax as to hold that “no portion of the human race was to be consigned to endless misery, let them be ever so wicked in this world.” john compton, no longer an elder, suffered a stroke and died in . as a result of the split, the “heretical” comptons had no church building, minister, or elders of their own nor baptism or communion services. their marriages were properly solemnized, but otherwise they did without the services of clergy and conducted home-based, bible-focused worship under the leadership of some of the community men. they seem to have eschewed the flamboyant physical expressions of religious transport that had been noteworthy and much-mocked features of mcdonald’s revivals – known locally as (among other things) “taking the work.” but they remained strongly attached to the lengthy hymns written by mcdonald and his elders, many of which used vivid biblical metaphors to express the mcdonaldites’ millennial expectations; and, still reflecting those expectations, they saw themselves as descendants of the ten lost tribes of israel with, perhaps, a crucial role to play b. compton limited bishop, church of scotland in prince edward island: lydia compton hume’s poem ( - ), and brief references to john compton and other elders and theological disputes among them in the years following mcdonald’s death ( , - ). i am most grateful to dr. david weale for lending me his handwritten copy of a mcdonaldite minute book loaned to him by nathan bears in when weale was writing his doctoral thesis on mcdonald. the minute book provides rich detail about meetings and decisions concerning the brooklyn and belle river congregations between december and june , after which there are no further entries except for a brief account of a meeting in june . ewen lamont, a biographical sketch of the late rev. donald mcdonald ( - ) (charlottetown: john coombs, ), appendix, pp. - . in his later and longer biography of mcdonald (see note ), murdock lamont reprinted part of his late father’s account of divisions following mcdonald’s death but prudently left out details of this controversy ( - ). writing in old age about the theological disputes that had involved his immediate kin – elder john was his grandfather – hector focused on events in the preceding decade rather than this controversy. but the central issue seems to have been the same: a perceived laxity in his people’s views about who could be saved; see hector d. compton to peter and helen, may , and hector d. compton, “p.p.s to emma,” n.d., hdc collection. variations of disputes such as this were playing out in this period among numerous groups of evangelical protestants in other parts of north america; see, for example, benjamin l. hartley, evangelicals at a crossroads: revivalism and social reform in boston, - (durham, nh: university of new hampshire press, ), . denis mckim suggests, however, in “boundless dominion” that presbyterians were particularly noteworthy for their fractiousness over matters of doctrine. see m. lamont, rev. donald mcdonald, chap. x, “the involuntary motions,” for murdock lamont’s attempt to put such phenomena into a broad historical and even global context. for a valuable first-hand account of an mcdonaldite service over which he presided and which was marked by dramatic performative outbursts from “perhaps thirty or forty” of the four hundred communicants present, see the rev. george monro grant’s letter to his wife, as quoted in william lawson grant and frederick hamilton, principal grant (toronto: morang and co., ), - . in the second coming of christ. it bears emphasizing that references to “they” in this discussion of the community’s religious beliefs should be followed by an acknowledgement that for much of this period it was perhaps hector who held to these beliefs most strongly. and notwithstanding the fact that his people had effectively been driven out of the larger mcdonaldite group, hector seems to have regarded his community as the fragment of the movement that most authentically carried forward mcdonald’s millennial hopes and his zeal for religious liberty untrammelled by institutional arrangements. like groups such as the quakers and the amish historically, but to a greater extent, the compton community practised endogamy. cousin marriage was the norm. instances of cousin marriage had certainly taken place among comptons prior to the formation of the community, as it had elsewhere in the th-century anglo- protestant world, and cousin marriage was still for the most part neither rare nor controversial for much of the th century. first cousins charles and emma darwin, for instance, were only following a well-established darwin/wedgwood family tradition when they married in , the year before queen victoria married her first cousin prince albert. closer to home, in the late s, teacher and soon-to-be- famous author lucy maud montgomery was briefly engaged to her cousin ed simpson, whose cavendish people, like families in other rural island settlements, had long intermarried. by the late th century, however, scientific thinking about the potential risks of close conjugal blood ties was changing. as adam kuper writes in incest and influence: the private life of bourgeois england, by the s even cousin marriage was being “routinely condemned” by eugenicists. for the sake of internal unity, then, as well as perhaps for reasons somehow linked to their religious beliefs, the compton community in the early th century was perpetuating a practice that was becoming anomalous. this did not, though, put the group beyond the pale. in contrast to such th- century american utopian groups as the oneida community and the mormons, whose distinctive religious and marital practices put them well outside the societal acadiensis well after the compton community had ceased to exist, some kin, puzzling over their ancestors’ practice of intermarriage, thought perhaps it had been linked to a belief that jesus would be born again into their community. in “dear people/belle river & bangor,” written from boston, december , hector appears to be alluding to some such belief; see hdc collection. because they had much larger faith groups to draw upon for partners, quakers and amish could marry fellow believers without routinely risking forbidden degrees of consanguinity. adam kuper, “introduction,” in kuper, incest and influence: the private life of bourgeois england (cambridge, ma: harvard university press, ). mary henley rubio, lucy maud montgomery: the gift of wings (toronto: doubleday, ), - ; harold h. simpson, cavendish: its history its people (amherst, ns: harold h. simpson and associates, ). kuper, incest and influence, . in the us, kuper writes, from the mid- th century some states began banning first-cousin marriage notwithstanding the “slapdash methodology” behind the studies on which their legislation was based. likewise, there were moves to ban “miscegenation” ( - ). among the issues in the us considered as possible outcomes of consanguineous marriages was not just the matter of potential birth defects but also possible effects on the birth rate and the proportion of the sexes at birth; see george b. louis arner, “consanguineous marriages in the american population” (phd diss. in political science, columbia university, ), . mainstream, the compton community was not subjected to legal pressures or social hostility for its unorthodox way of life – quite the contrary. as enid charles and sylvia anthony observed in in their article in rural sociology: their leader is also regarded as a leader in the belle river neighbourhood and is consulted on all points of communal action. the general attitude of belle river to its eccentric group is one of respect and affection, combined with tolerance of differences in behaviour. a contributory factor to this tolerance is that the group is in no sense alien. its members have relatives in the neighbourhood. they adhere formally to a variety of protestantism which was the religion of some of the founders of the island, and their outstanding characteristics, though expressed in a different way, are those which the ordinary island citizen most respects. charles, the lead author of the article, was a pioneer demographer, socialist, and feminist, a cambridge-educated scholar whose career path briefly included the dominion bureau of statistics. it is possible that charles’s socialist leanings as well as her concern about fertility decline made her unusually sympathetic to the compton community, whose average family size she reported to be “even higher than the large belle river average.” charles and anthony certainly knew about the community’s practice of intermarriage. but it is unlikely that they or perhaps even near neighbours were familiar with intra-community gossip about more troubling sexual irregularities, particularly those involving two brothers born in the mid- th century who were said to have fathered children with their wives’ close kin. goodwill and admiration for the community was a common, even dominant, theme in media coverage of its way of life. such coverage began on the island in the mid- s and was picked up and carried forward in the english-language press in and beyond north america over the next half decade. in addition to the emphasis on “brotherly love” and “model behaviour” in the community (as quoted in the title and the introductory paragraph of this article), the other persistent emphasis in coverage b. compton limited both these groups figure prominently and frequently in studies of american utopias. see, for instance, louis j. kern, an ordered love: sex roles and sexuality in victorian utopias – the shakers, the mormons, and the oneida community (chapel hill, nc: university of north carolina press, ), and pitzer, america’s communal utopias. in “a peculiar people”: anti-mormonism and the making of religion in nineteenth-century america (chapel hill, nc: university of north carolina press, ), j. spencer fluhman “charts how mormonism was defamed and defined as a nineteenth-century american religion.” his book argues that “through condemnation of what mormonism was, protestants defined just what american religion could be” ( ). charles and anthony, “community and the family,” . sylvia wargon, “legacy of enid charles, - ,” canadian studies in population , no. ( ): - . enid charles’s book, the twilight of parenthood (new york: w.w. norton, ), dealt with her concern about fertility decline. charles and anthony, “community and the family,” . speculation about the sexual excesses of these two men and the likelihood that in a later generation their transgressions had resulted in two cases of inadvertent incest persisted among some older community descendants into the early st century. of the community was its prosperity. here, for instance, is travel writer helen jean champion in after visiting the belle river settlement: the houses were large, fine-looking buildings. the barns were barns – par excellence. they were large, with high substantial foundations, and were all made from the same general pattern. we found later that it was quite easy to tell if a family were communistic. just look at their barns! . . . all the houses have running water supplied by underground piping and are equipped with radios. the houses and stables have electric lights from power supplied by the mill. two interrelated factors merit emphasis in connection with this kind of media coverage: it was by no means rigorous investigative journalism, and it reflected the kind of wishful thinking to which the depression gave rise. it is worth recalling that it was also during this period that would-be social reformers from the us and elsewhere descended on the maritimes to investigate the strategies of the antigonish movement, launched by catholic social activists at st. francis xavier university, and the related but autonomous cooperative movement on pei. like the private citizens who wrote to secretary-treasurer hector compton asking how they could join his community, journalists and other enquirers wanted to believe in and share hopeful, feel-good stories about places and ways of life that transcended the failures of the capitalist system. and perhaps the fact that this particular story was set on a tiny island far away from the north american mainstream, and far behind in what pei cooperative movement leader john croteau referred to as the province’s “social evolution,” made it all the more attractive. this is not to say that there was no substance to the positive descriptions of b. compton limited. one piece on the smaller of the company’s two settlements, published in a prince edward island agricultural paper, admittedly contained such wild exaggerations and outright fictions that it was almost certainly written without the author ever having visited the settlement. but other accounts acadiensis champion, over on the island, . j. t. croteau, cradled in the waves: the story of a people’s co-operative achievement in economic betterment on prince edward island, canada (toronto: ryerson press, ), chap. i. i am grateful to john reid for drawing this book to my attention. see also marion bruce and elizabeth cran, working together: two centuries of co-operatives on prince edward island (charlottetown: island studies press, ), esp. chap. . champion, over on the island, ; hector d. compton to helen and peter , may , hdc collection. in the letter hector stated that there had been about mail enquiries and that references to the community had appeared in the english-speaking press as far away as england and new zealand. croteau, cradled in the waves, . as a young economist who came from new england to pei in to teach, and who then worked for years with the island’s mainly catholic-supported cooperative movement, croteau visited numerous impoverished fishing and farming communities and was well placed to observe the changes that took place during that period. but “the constant increase in governmental function” to which he referred evidently did not extend to social services for needy families, as is indicated in note below. combined reasonably accurate details with images filtered through rose-coloured glasses. moreover, demographer charles and her co-author were in substantial agreement with the journalists in terms of substantive matters: the two scholars assured their readers that in the compton community a “communal way of life” had in fact resulted in “an impressive picture of prosperity” and in “ideals of social obligation” that extended beyond the group’s own membership. their picture accords with memories shared by now-elderly descendants of a community whose way of life shielded its members from the worst hardships of pei’s depression years and predisposed them to help their less-fortunate neighbours. the road to dissolution in commitment and community, rosabeth moss kanter’s criterion for classifying a utopian community as a success was that it must have lasted for at least years. by this standard the compton community was successful, lasting almost years. nevertheless, there were vulnerabilities and challenges within the community even before it encountered the stresses of the second world war era. contrary to the images of egalitarianism and equality of condition that prevailed in print media, there were differences in the degree of prosperity existing in the community’s two settlements and even among households within them. none of the homes in bangor, for instance, had electricity, and the homes in either settlement were not all alike in amenities. in regard to leadership, hector certainly provided stable and skilful direction as the company’s secretary-treasurer as well as firm, faith-based spiritual guidance. but j., the man who became leader of the smaller settlement in , and who was, nominally, at least, the spiritual leader of both groups, was something of a loose cannon. the year was unquestionably an annus horribilis for the community: the deaths of three productive members within a few months of one another robbed the small group of valuable male contributors in the prime of life. alcohol use was excessive among some male community members, including j., as it was on the island overall, provincial prohibition notwithstanding. while hector b. compton limited “some interesting sidelights on co-operative experiment in prince edward island,” prince edward island agriculturalist, february ; “unique pei colony makes progress,” guardian, january ; flora s. rogers, “brotherly love rules this community.” charles and anthony, “community and the family,” . as edward macdonald makes clear in if you’re stronghearted (chap. and ), such social and health problems as child malnutrition and tuberculosis were widespread on the island even before the depression, exacerbated by islanders’ reluctance to be taxed for state social services. in this context even modest help from relatively more prosperous neighbours and from religious organizations was important. regarding the latter see heidi macdonald, “doing more with less: the sisters of st. martha (pei) diminish the impact of the great depression,” acadiensis xxxiii, no. (autumn ): - . kanter, commitment and community, . for critical discussion of longevity as a standard for measuring the success or failure of utopian experiments see, for instance, ira l. mandelker, introduction, in religion, society, and utopias in nineteenth-century america (amherst, ma: university of massachusetts press, ), and pitzer, america’s communal utopias, - . hector d. compton to son george compton, july , hdc collection. edward whitcomb, a short history of prince edward island (ottawa: from sea to sea enterprises, ), ; macdonald, if you’re stronghearted, , , . whitcomb writes that for a time pei had the highest rates of alcoholism in canada. personally railed against the harmful effects on his community of what he called “delusive alcohol,” it does not appear that there were ever any formal sanctions against its use as there were in some other communal groups such as the mormons and, in western canada, the doukhobors and the finns at sointula. inevitably, too, there were community members who longed for broader horizons and greater personal freedom than could be found in their island utopia. higher education may have been absent, but there were radios, newspapers and, in time, the reader’s digest to give members a window on a larger world. the restless included some community women. among them was m., a young woman who evidently became the family drudge in the years after her mother’s death. when on one occasion m. dared to attend the local church, her father arrived, threw open its doors, and ordered her to leave. finally, denied the opportunity to take nurses’ training, m. fled from bangor to boston, where she met and married a fellow islander. later, as a hardworking widow, m. took great pride in the fact that both her sons had graduated from elite universities and become successful lawyers. beginning in the early s, when the eldest daughter of the community’s founder married a danish immigrant employed by b. compton limited, there were also marriages out by members who remained in the belle river neighbourhood. finally, even on the matter of religion and even in the s, hector sometimes found it a struggle to bring community members together for sunday evening worship. the war years exacerbated pre-existing problems and tensions by exposing the community to increasing pressures from the outside world. prince edward island experienced a markedly higher level of military enlistment than it had in the first world war. for b. compton limited the wartime manpower drain involved both hired mill labourers and young community members; among them was the only son of founder ben, who was hector’s right-hand man in the store and in the overall management of the company’s business. his absence coincided with the existence of an unprecedented degree of government intervention in business affairs, intervention that even a small communal group could not escape. hence this complaint from hector in to another young kinsman serving overseas: we are this year, since may st under sales tax regulations, accounting each month for % of all taxable sales, then we now have unemp. insurance with a separate book for each hired man and the buying of stamps and accounting for every days work. . . . then we still have to “induce” them to continue contrib. to wars savings and mail a lot of reports to ottawa. . . . we now learn that acadiensis hector d. compton, “compton genealogy,” december , hdc collection. rasporich, “utopia, sect, and millennium.” rogers, “brotherly love rules this community”; charles and anthony, “community and the family,” . as a conservative publication that took a stand against both alcohol and atheistic communism, the reader’s digest, which began in , would have been an acceptable source of reading material in hector’s eyes. in hector wrote to navy officials seeking the young man’s early release from service; see hector d. compton to commanding officer, royal canadian navy, halifax, october , hdc collection. we have got to get out license as food dealers get a sign in our window and account as to quantities and prices charged. as the company’s secretary-treasurer, hector was the person most responsible for carrying these burdens. they came at a time when he was increasingly absorbed by what he took to be clear signs that the millennium was imminent (a perspective shared by many contemporary american prophecy writers). together, these business and religious elements in hector’s wartime life created stress-related health problems and made him a more impatient and judgmental leader. in both settlements, but particularly in belle river, he despaired over what he took to be moral and spiritual declension, especially among the younger generation. thus, in he wrote that he was gravely concerned for the welfare of this small lot of people. we were once a body, united by a living prospect, and now that this prospect is unfolding, we should still be a unit, be watching and waiting the outcome, but it is far from being the case. . . . the greatest world crisis is just ahead of us when the present babylonian systems are due to fall. [but] our children have somehow lost ear for these subjects and do not fear to move out and drink of the world’s cheap pleasures. they are as lambs among wolves, far worse off than worldlings who were always inured to their lot. meanwhile, in bangor, the smaller settlement, there was a growing inclination to dissolve the communal business relationship with the belle river kin. after the death of j. in following a protracted and debilitating illness – he had, as noted, been the nominal spiritual leader of both settlements – there would have been less pressure to continue the relationship. there was also less practical advantage to doing so following the loss by fire in of the lumber mill at belle river that had been a mainstay of b. compton limited’s earning power. calls for the partial, and then total, dissolution of the company evidently came from those operating the four bangor farms. writing in , hector seemed resigned to the separation and independence of this smaller group. but breaking up the communal operation at belle river was a different matter – something he regarded as “mad or impossible,” even shameful, particularly at a time when provincial civil servants were said to be looking at b. compton limited’s communal setup as a possible model for veterans returning from military service to farm but unable to purchase the expensive equipment necessary for successful modern agriculture except on some sort of cooperative basis. b. compton limited hector d. compton to “dear laddie,” september , hdc collection. paul boyer, when time shall be no more: prophecy belief in modern american culture (cambridge, ma: harvard university press, ), , . hector d. compton to “dear cousin belle,” december , hdc collection. hector d. compton to son george compton, july , recalling the “better part” of j. as well as his frailties, illness, and death; see hdc collection. hector d. compton to “dear bro. george” compton, july , hdc collection. hdc to “dear bro. george,” april , and to “dear bro. jas. and all,” april , both in hdc collection. w.r. shaw, provincial deputy minister of agriculture and a future premier, nevertheless, hector came to accept the inevitability of dissolution even for the belle river settlement. several male kinsmen were emulating other young islanders and leaving the province for greener pastures, and even among those who remained there was no reasonable prospect of capable new leadership emerging to take over the load he had carried for more than a quarter century. while he was momentarily hurt by and defensive about what he perceived as criticisms of his decades-long management of the company and his own family’s share in the division of its assets, he ultimately dealt with the complex legal requirements and personal negotiations involved in the dissolution process with what appears to have been remarkable fairness and flexibility. writing to provincial government officials in in regard to legal aspects of that process, he explained that circumstances connected with the community’s incorporation as a limited joint stock company in were now creating difficulties. when asked, unexpectedly, to name their shareholders, “we . . . gave the names of twentytwo ‘active senior members.’ . . . to ourselves this was a mere legal formality and the whole thing belonged to the whole people.” some of those named had since died; other still-living and hard-working contributors had not been listed as members. in these circumstances, adhering to the usual regulations about transferring the assets of a limited joint-stock company only to named shareholders would “work great injustices” and stand in the way of “apportion[ing] things on the basis of merit and of need.” furthermore, he stated that “even without any legal handicap an equitable distribution presents a mass of problems. we have persons of all ages and of various degrees of ability and responsibility. some young men can take a farm and run it. other workers are dependent upon living in and working with some established home. no two of the homes are conditioned quite alike. since our paper securities . . . have been sold and our money about used up, the balancing must be done with real property which is less easily distributed.” by the time hector wrote about these dissolution difficulties to government officials, the “winding up notice” had already been publicly advertised. at the end of , a few weeks after the notice appeared, b. compton limited had ceased to exist. prince acadiensis had been serving on a regional committee for the resettlement of returned service men. he had written to hector, enclosing a article about the company from the toronto star and asking for further information about its structure and operation. hector later met with shaw and gave him further information about the company. accounts of shaw’s interest are provided in both these letters. hector d. compton to “dear bro. george,” september , as well as an undated letter fragment from same period dealing in part with internal discord over company equipment or vehicles among younger male members; see hdc collection. see untitled statement by hector d. compton, april , for hector’s response to perceived criticisms; and, for the account of dissolution difficulties, james m. compton, bangor, and hector d. compton, belle river, liquidators, to provincial secretary and director of income tax, charlottetown, april , and as “rewritten june , .” although these two drafts are very similar, the rewritten draft, from which i have quoted, bears hector’s signature and refers to a august meeting in which the decision was taken to wind up the company and appoint liquidators. “summary of allotments from assets of b. compton limited,” june , indicates that while hector’s family’s share was the largest in belle river, two bangor households were to receive larger amounts. all documents cited are in hdc collection. james m. compton and hector d. compton to provincial secretary & director of income tax, as drafted april and rewritten june , hdc collection. although james m. compton, edward island’s “unique ‘brotherly love’ community” was history. in the end, hector may have felt a sense of relief. freed at last of business responsibilities, he lived on into his st year. in this post-community phase of his life, he regularly contributed money to and corresponded with millenarian groups, particularly in the us, where a new and influential generation of prophetic voices was being heard. if he was unique among former community members in being deeply preoccupied with eschatology, he nonetheless enjoyed the abiding respect of relatives in both settlements and, as long as he lived, provided something of a bond between them. conclusion as much recent scholarship has shown, forms of globalization and transnationalism were transformative personal and societal phenomena long before the terms themselves became common currency. the experience of the people introduced in this article illustrates the extent to which religious currents and diverse ideologies could, like material and human cargoes, travel across the atlantic and across international borders in the centuries preceding our own. as linda colley observes in the ordeal of elizabeth marsh: a woman in world history: “adopting a purely abstract approach to changes and influences that transcend continents means that we understand them only imperfectly . . . there is always a human and individual dimension.” this article, concerned as it is with the “human and individual dimension,” also shows the extent to which transported ideas were subject to adaptation and change through their encounters with local cultures and even particular families. in prince edward island the rev. donald mcdonald created an indigenous religious movement out of evangelical protestant ideas that were making their way across the english-speaking world during the th century. the compton community, in turn, made something distinctively its own out of the mcdonaldite movement by combining it with a utopian vision. such visions had had widespread currency in th-century north america, and then, as the century ended, edward bellamy’s looking backward had provided a uniquely detailed and attractive utopian blueprint. bellamy’s elaborate literary construct was one in which the communitarian comptons found cooperative values and millennial hopes consonant b. compton limited a brother of hector, is shown here as co-liquidator, the details of calculating division of assets, completing income tax, and succession duty reports was the work of hector, who was later paid for his labours by those who had been former shareholders in b. compton limited. see “minutes of meeting,” march , hdc collection. boyer, when time shall be no more; hutchinson and wolffe, short history of global evangelicalism, esp. chap. . for very different examples of such scholarship dealing, respectively, with the th, th, th, and early th centuries, see timothy brooke, vermeer’s hat: the seventeenth century and the dawn of the global world (toronto: viking, ); linda colley, the ordeal of elizabeth marsh: a woman in world history (new york: anchor books, ); “theme section: british north america’s global age,” histoire sociale / social history xlvi, no. (november ): - ; and dana robert, “the first globalization: the internationalization of the protestant missionary movement between the wars,” international bulletin of missionary research , no. (april ): - . colley, elizabeth marsh, . hutchinson and wolffe, short history of global evangelicalism. with their own, notwithstanding the novel’s urban setting and bellamy’s muted religiosity. the compton community was, in effect, an outcome of the coming together of transnational ideas with local circumstances and values. it was, as charles and anthony noted, an “eccentric group” but at the same time “in no sense alien” to the surrounding island culture and not disparaged by it. as early- th-century millenarian utopians, compton community members held to religious views that were no longer a prominent part of the discourse in mainstream congregations. and they worked and married as well as worshipped as a distinct group. nevertheless, their everyday round was necessarily much like that of their neighbours: cows had to be milked, crops harvested, businesses run. and if, beyond the quotidian, their leaders demonstrated abilities and local knowledge that could be put to wider use, they were evidently expected to contribute. charles and anthony perceived that expectation with regard to hector. but decades earlier in the case of founder ben there were also evidently extra-community expectations and involvements, as in when he was named to a provincial committee to recruit men for the navy. indeed, even the much younger second wife who survived ben and lived as a widow to a great old age was proud to recall that she had routinely been called upon by neighbours to help with medical emergencies in advance or in the absence of a doctor. eschatological but not impractical, a discrete community but not unneighbourly, b. compton limited and its leaders lived and interacted effectively if selectively with other island citizens. why, then, did many community descendants from the mid- th century onward seem inclined to wilful forgetting so far as their unusual religious and communitarian past was concerned, more conscious of their ancestors’ eccentricities and foibles than of the fact that their accomplishments had once been admired? writing in the s on “varieties of literary utopias,” northrop frye observed that in the western democracies there was “something of a paralysis of utopian thought and imagination” as a result of the “repudiation of communism” as “the straight utopia” gave way to a taste for such utopian satires as william golding’s lord of the flies. fear and misunderstanding about “communism” may have had some spillover effect even in remote and unlikely corners of north america. one result would have been unease, even about communitarian groups that bore no acadiensis the son of a baptist minister and a baptist minister’s daughter, bellamy became non- denominational in his outlook and was concerned with social rather than personal salvation. in his introduction to the oxford university press edition of looking backward, beaumont speaks of bellamy as having “a profoundly religious sensibility” (xii). see also george e. connor, “the awakening of edward bellamy: looking backward at religious influence,” utopian studies , no. (january ): - , which argues for bellamy’s work as, among other things, an early catalyst for the social gospel movement. “naval recruitment committee named,” guardian, november . see also “attend meeting,” guardian, december , where ben is identified as one of several gentlemen attending a lobster packers’ meeting. hornby, belfast people, . the subject of women in the compton community merits more attention than i have been able to give it in this article. northrop frye, “varieties of literary utopias,” in utopias and utopian thought, ed. frank e. manuel (boston: houghton mifflin, ), quotations on , ; william golding, lord of the flies: a novel (london: faber and faber, ). resemblance to soviet-style socialism, among former members and outsiders alike. two related aspects are also worth considering. in her book family secrets: living with shame from the victorians to the present day, deborah cohen suggests that mid- th-century families may actually have been more inclined than their victorian ancestors to want to hide potentially embarrassing family matters (e.g. mixed-race ancestors, “defective” children, and homosexual uncles). cohen’s suggestion certainly accords with popular stereotypes of s cold war north america as an age of conformity, especially when it came to family norms. at the same time, with regard specifically to prince edward island, the mid-century decade saw the island province drawn increasingly into a larger national and international culture and into the modernizing values and practices that prevailed in those larger worlds. as that process took place, compton community descendants and perhaps especially those of us who were on the threshold of adulthood wanted very much to be regarded not as “other” but rather as conventional, respectable, and “modern.” thus, occasional questions about whether we were part of “those” comptons were perceived as laden with innuendo and condescension. we interpreted them as harking back to our people’s differentness in terms of such matters as “communistic” families, educational deficiencies, intermarriage, and religious identity. with regard to the latter, so far as markers of respectability were concerned, churchgoing within the mainstream denominations remained crucial on prince edward island well into the late th century; interestingly, some adult children of s back-to-the-landers recalled that islanders’ tolerance of their come-from- away parents’ chosen way of life did not extend to their failure to attend church. indeed, across canada until the late s or early s the united church of canada and the other mainstream protestant denominations continued to experience remarkable growth as shown in such measures as church membership and sunday school attendance. speaking personally and as one of “those” comptons, i can attest that as an exceedingly self-conscious teenager i wanted nothing more than to blend into that church-sanctioned culture of respectability and normalcy. it would take many decades of living “away” and a historian’s slowly acquired awareness of social and cultural complexity before i concluded that it would be a worthwhile scholarly initiative and not simply a source of familial discomfort to revisit my ancestors’ religious “otherness” and their attempt to create an island utopia. b. compton limited deborah cohen, family secrets: living with shame from the victorians to the present day (london: viking, ). elaine tyler may, homeward bound: american families in the cold war era (new york: basic books, ; orig. ed. ) and, for a canadian overview, doug owram, born at the right time: a history of the baby-boom generation (toronto: university of toronto press, ), chap. . macdonald, if you’re stronghearted, chap. . the process of modernization, macdonald writes, “would reach its climax in the tumultuous times of the s. . . . but it was in the s that modernization became manifest” ( ). alan maceachern and ryan o’connor, “back to the island: the back-to-the-land movement on pei,” http://niche-canada.org/member-projects/backtotheisland/home.html. stuart macdonald, “death of christian canada? do canadian church statistics support callum brown’s theory of church decline?” canadian society of church history, historical papers ( ): - . see also phyllis d. airhart, a church with the soul of a nation: making and remaking the united church of canada (montreal: mcgill-queen’s university press, ). horticulturae int. j. neonatal screen. , , - ; doi: . /ijns international journal of neonatal screening issn - x www.mdpi.com/journal/neonatalscreening review newborn screening for glutaric aciduria type i: benefits and limitations jana heringer, nikolas boy, peter burgard, jürgen g. okun and stefan kölker * university children’s hospital heidelberg, clinic , division of neuropediatrics and metabolic medicine, heidelberg, germany; e-mail: jana.heringer@med.uni-heidelberg.de (j.h.); nikolas.boy@med.uni-heidelberg.de (n.b.); peter.burgard@med.uni-heidelberg.de (p.b.); juergenguenther.okun@med.uni-heidelberg.de (j.g.o.) * author to whom correspondence should be addressed; e-mail: stefan.koelker@med.uni-heidelberg.de; tel.: + - - - ; fax: + - - - . academic editor: ralph fingerhut received: april / accepted: june / published: july abstract: more than years ago glutaric aciduria type i has been included in newborn screening programmes and pilot studies evaluating the potential benefit of early diagnosis and start of metabolic treatment for patients with this disease have been initiated. at that time many important questions on epidemiology, diagnostic quality, natural history, treatment, and cost effectiveness were not sufficiently answered. in particular, it was rather unknown whether early treatment improves the outcome. after implementation of glutaric aciduria type i in an increasing number of countries, and with careful evaluation of disease course and impact of early treatment, there is now solid evidence that affected individuals do have substantial benefit and that newborn screening for this disease is a cost-effective diagnostic intervention. despite this success, there are still limitations concerning diagnostic sensitivity for patients with a low excreting phenotype and knowledge on long- term disease outcome. in conclusion, it has become evident that tandem mass spectrometry- based newborn screening for glutaric aciduria type i is a powerful and cost-effective tool to prevent the manifestation of prognostically-relevant movement disorders in the majority of early diagnosed patients. keywords: glutaric aciduria type i; newborn screening; tandem mass spectrometry; glutarylcarnitine; guideline; cost-effectiveness open access mailto:jana.heringer@med.uni-heidelberg.de mailto:nikolas.boy@med.uni-heidelberg.de mailto:peter.burgard@med.uni-heidelberg.de mailto:juergenguenther.okun@med.uni-heidelberg.de int. j. neonatal screen. , . introduction glutaric aciduria type i (ga-i) is a rare inherited disorder of l-lysine, l-hydroxylysine and l-tryptophan metabolism first described in [ ], with an overall estimated prevalence of in , newborns [ , ]. high-risk populations with a prevalence of up to in newborns have been identified. this includes the amish community in lancaster county, pennsylvania, usa [ ], the oji-cree first nations in western ontario and manitoba, canada [ ], the lumbee in north carolina, usa [ ], the irish travellers in ireland and the united kingdom [ ], and indigenous south africans [ ]. ga-i is caused by two pathogenic mutations in the gcdh gene which consists of exons ( kb) and is located on p . [ ]. more than disease-causing mutations have been identified so far [ , ]; most of them are private. the most frequent mutation in europe is p.arg trp accounting for %– % of all alleles. other mutations are predominantly or even exclusively found in distinct populations such as p.ala val in the amish community as well as southern parts of germany and switzerland, the original settlement area of the amish, ivs + g > t in the oji-cree first nations [ ], p.pro leu and p.glu lys in turkey [ ], and p.arg pro and p.val met in spain [ ]. inherited deficiency of glutaryl-coa dehydrogenase, a mitochondrial flavoprotein which catalyzes the oxidative decarboxylation of glutaryl-coa to crotonyl-coa in the catabolic pathways of l-lysine, l-tryptophan and l-hydroxylysine, causes cerebral accumulation of glutaric acid (ga), -hydroxyglutaric acid ( -oh-ga) and glutarylcarnitine (c dc). these metabolites can be detected in body fluids by quantitative gas chromatography/mass spectrometry (ga, -oh-ga) or tandem mass spectrometry (c dc). two biochemical subgroups, termed high and low excretors, have been delineated [ ]. high excretors show a (near) loss of gcdh activity and high concentrations of ga (above mmol/mol creatinine), whereas low excreting patients with gcdh residual activity of up to % urinary ga excretion is below mmol/mol creatinine and in some patients is (intermittently) normal. the excretion of -oh-ga in both groups is less variable. about %– % of untreated patients are thought to develop a complex, irreversible movement disorder with predominant dystonia superimposing on truncal hypotonia [ – ] most often following an acute encephalopathic crises precipitated by intercurrent febrile illness, immunization or postsurgical metabolic stress during a defined period in infancy (mostly between age – months). notably, some patients show an insidious onset of movement disorder without preceding encephalopathic crises [ , , ]. in all dystonic patients striatal degeneration is the common neuropathological and neuroradiological finding [ , ]. strikingly, untreated high and low excretors have the same risk of striatal degeneration and, therefore, low excretors should not be mistaken as a “mild” disease variant [ ]. striatal degeneration usually starts in the dorsolateral aspects of the putamen and then spreads over the striatum in a ventromedial direction; three neuroradiologically defined stages–acute, sub-acute and chronic–have been delineated [ ]. morbidity and mortality is significantly increased in dystonic patients [ , ]. in addition to striatal pathology, patients also show a broad spectrum of extrastriatal abnormalities including temporal hypoplasia with concomitantly widening of anterior temporal and sylvian csf spaces, pseudocysts, signal changes of substantia nigra, thalamus, nucleus dentatus, and supratentorial int. j. neonatal screen. , white matter [ ]. extrastriatal abnormalities follow a variable pattern; they could proceed but sometimes regress or even normalize with time. in contrast to striatal degeneration, clinical relevance of extrastriatal changes is still unclear [ ]. since ga-i patients do not present with pathognomonic signs or symptoms in the newborn period or infancy, the majority of them are missed in this age group. therefore, only younger siblings of previously identified index patients have had a realistic change of early diagnosis and treatment before the start of tandem mass spectrometry-based newborn screening programmes [ ]. . to screen or not to screen? in the late s, pilot studies on newborn screening for ga-i were initiated in a few countries (australia, germany, some us states) and in known high-risk populations (usa: amish, republic or ireland: irish travellers). some important epidemiological, diagnostic, clinical, therapeutic, and economic questions could not be adequately addressed at that time. therefore the health technology assessment in the uk in concluded that “ga-i comes quite close to fulfilling the necessary criteria for newborn screening although there is some lack of information on the absolute incidence and natural history” [ ] and that “there was reasonable evidence to support inclusion in extended neonatal screening of (…) glutaric aciduria type ” [ ]. with more than years experience in newborn screening for ga-i, it has become evident that this diagnostic intervention is a powerful tool to prevent the manifestation of movement disorders in the majority of early diagnosed patients [ , , , ]. as a consequence, the american college of clinical genetics has recommended it for screening [ ]. however, despite increasing evidence, not more than one third of european union member states has implemented ga-i in its national disease panel [ ]. in the following, we will evaluate the benefit of existing newborn screening programmes for ga-i based on the original principles of early disease detection as outlined by wilson and jungner [ ]. . . knowledge of the disease as described above, ga-i causes significant health problems. about %– % of untreated ga-i patients present with acute or insidious onset of a complex movement disorder with predominant dystonia during infancy and childhood (age range – months, peak – months) [ , , , ]. symptomatic patients have a significantly reduced life expectancy [ , ]. despite some variability in the clinical presentation among siblings, manifestation of a movement disorder has a high penetrance [ , , ]. importantly, high and low excretors share the same risk of developing a movement disorder. therefore the genotype predicts the biochemical but not the clinical phenotype [ , ]. this intriguing finding is best explained by cerebral entrapment of toxic dicarboxylic acids due to cerebral de novo synthesis and very limited efflux transport of the blood-brain barrier for these metabolites [ , , , ]. affected individuals usually remain asymptomatic or show mild reversible neurological signs, such as truncal hypotonia, during the first three months of life since neonatal onset is a rare finding in this disease [ , , ]. macrocephaly which is found in about % of patients is not specific for ga-i since it has a broad differential diagnoses and is found in % of the normal population (by definition). few patients with late-onset ga-i presenting with white matter abnormalities in mri and a broad spectrum of (progressive) signs and symptoms int. j. neonatal screen. , (macrocephaly, headaches, hand tremor, impaired fine motor function, seizures, ataxia, psychiatric disease) have been described [ , ]. it remains to be elucidated whether this group reflects a specific disease variant or whether white matter changes in adolescent and adult patients are a common finding. the latter notion is supported by the observation that extrastriatal mri changes are also found in early diagnosed and treated ga-i patients and that white matter changes might increase with age [ , , , ]. the major limitation in evaluating the clinical benefit of newborn screening for ga-i patients is the current knowledge on the long-term disease course. this is highlighted by two recent studies. although ga-i was previously thought to exclusively affect the brain, this notion was challenged by recent observation of peripheral neuropathy [ ] and chronic renal failure in adolescent and adult patients [ ]. . . knowledge of the test the diagnostic test is the determination of glutarylcarnitine (c dc) concentration in dried blood spots [ – ]. c dc can be detected in dried blood spot samples by its ion pair m/z → m/z using tandem mass spectrometry. therefore, c dc can be included in tandem mass-spectrometry based newborn screening. ga-i should be considered in newborns with c dc concentrations above the cut-off. neonatal screening centres follow different strategies for defining and updating cut-offs for c dc, and no standardized procedure for the setting of cut-off levels has yet been established worldwide [ , ]. only until recently internal standards for newborn screening have not included a stable isotope of c dc, the c dc concentration has been calculated in relation to the internal standard of octanoylcarnitine (c ). this procedure has resulted in low specificity and low positive predictive value in the early days of c dc screening. these problems can be overcome using multiple reaction monitoring (mrm, m/z → m/z for butylated samples) [ ], a combination of c dc with other acylcarnitines and acylcarnitine ratios using data mining techniques [ ], and two-tier strategies using c dc and -hydroxyglutaric acid [ , , ]. despite improvement of the diagnostic quality of c dc screening, there are reported cases of missed low excretors in the literature, but the exact number, however, is not known since newborn screening programmes often lack a tracking system. in the german newborn screening programme, three patients have been missed in about five million newborns. the majority of these patients have been missed during the start of extended newborn screening, highlighting that screening algorithms and cut-offs have been improved [ , , ]. in high- risk populations with low excretors, mutation-based analysis might be considered as a more reliable strategy [ ]. the overall sensitivity of c dc screening in a mixed population is about % [ ]. a deuterium-labeled analytical standard for c dc is recently available and the implementation in the panel of standards will likely improve quantification of c cd in the future. however, a systematic evaluation of the new standard is still lacking. false positive screening results may be due to glutaric aciduria type ii (multiple acyl-coa dehydrogenase deficiency), pseudoglutarylcarnitinemia (hydroxydecanoylcarnitine) in patients with medium chain acyl-coa dehydrogenase deficiency [ ], newborns with renal failure [ ], and maternal ga-i [ , ]. therefore, these diagnostic pitfalls should be considered for interpretation of positive c dc screening results and for establishment of reasonable diagnostic algorithms [ ]. int. j. neonatal screen. , . . treatment for disease in analogy to other protein-dependent inherited metabolic diseases, metabolic treatment including low lysine diet, carnitine supplementation and intermittent emergency treatment with transient stop of protein intake, energy- and carbohydrate-rich meals or glucose infusions (with or without insulin) during episodes that are likely to induce catabolism (e.g., intercurrent infectious diseases) was introduced for ga-i patients [ , , ]. a meta-analysis on patients, most of them treated after the onset of motor symptoms, however, did not reveal any significant effect on disease course [ ]. a cross-sectional study on patients later on showed that carnitine supplementation slightly reduced the mortality of symptomatic patients [ ]. before ga-i was implemented into national screening programmes, however, there was only very limited evidence that metabolic treatment in asymptomatic newborns or infants could improve the disease course in general and prevent manifestation of movement disorders [ , , ]. in particular, it remained doubtful whether dietary treatment was beneficial [ ]. after the start of newborn screening programmes and prospective follow-up studies in europe, north america, asia and australia there is now increasing evidence that diagnosis and start of treatment during the newborn period has helped to significantly reduce the onset of a complex movement disorder in infancy and childhood and thus to improve the short-term outcome of ga-i patients (table ). table . newborn screening in combination with early start of metabolic treatment improves the neurological outcome of patients with glutaric aciduria type i (selection of studies). ref. country (state) patients n asymptomatic patients * n (%) [ ] australia (new south wales) ( %) [ ] australia (victoria) ( %) [ ] germany total: adherence to guideline: total: ( %) adherence to guideline: ( %) [ ] japan ( %) [ ] usa (pennsylvania) total: cohort – : ; cohort – : total: ( %) cohort – : ( %); cohort – : ( %) [ ] usa (utah) ( %) [ ] taiwan ( %) [ ] spain ( %) * no evidence for the manifestation of acute or insidious onset of dystonia during the follow-up. if successive time points have been evaluated in the same cohort, only the latest published report is listed. the prospective follow-up study in germany showed that the absolute risk reduction for an acute encephalopathic crisis was . in the newborn screening group compared to symptomatic patients [ ] and that the number needed to treat to prevent one acute encephalopathic crisis was . . this huge effect was confirmed in a second evaluation of the same cohort three years later [ ]. the latter study demonstrated for the first time that both metabolic maintenance treatment with low lysine diet and carnitine supplementation and intermittent emergency treatment during infectious diseases and other int. j. neonatal screen. , potentially threatening episodes had a positive impact on the outcome. the best outcome was achieved in the group of neonatally-diagnosed patients who received metabolic maintenance and emergency treatment according to previously-published guidelines [ ]. since the guideline recommendations were developed by an international group of experts and were subsequently tested and approved, the currently available revised version of these recommendations should be regarded as best evidence available for the diagnosis, treatment and follow-up of ga-i patients [ ]. the beneficial effect of newborn screening and combined metabolic treatment has been confirmed independently by other groups in different countries and national healthcare systems [ – , – ]. based on this new therapy, options such as l-arginine supplementation aiming at reducing cerebral l-lysine influx and oxidation are currently discussed [ , – ]. . . cost considerations cost-effectiveness of tandem mass spectrometry-based newborn screening for ga-i was recently assessed in germany based on the results of the prospective follow-up study [ , , ]. it was clearly shown using a markov model that newborn screening for ga-i significantly reduced the number of disability adjusted life years (dalys) by . and that it helped to save about , euro per , screened newborns within a year time horizon [ ]. this study convincingly showed that implementation of ga-i to tandem mass spectrometry-based newborn screening programmes under conditions comparable to the national health care system in germany is highly cost-effective. . conclusions more than years after the start of first newborn screening pilot projects for ga-i, there is now convincing evidence from different countries and health systems that combination of early diagnosis via newborn screening and an early start of metabolic treatment has dramatically improved the neurological outcome and survival of patients with this disease. it is an excellent example how inclusion of a metabolic disease that does fulfill some but not all screening criteria to newborn screening pilot studies can improve the health outcome of affected individuals and how careful evaluation of the screening process, disease course, and economic parameters can help to fill initial gaps of knowledge. despite this success, there are still limitations, including use of optimized combinations of newborn screening parameters to improve the diagnostic quality, continuation of long-term follow-up studies, and establishment of patient registries to identify long-term complications and to optimize therapy and therapy monitoring. acknowledgments we are indebted to all patients and their families who have been willing to contribute to prospective observational follow-up study in germany and to the european registry and network for intoxication type metabolic diseases (e-imd) and to all colleagues who have helped to realize these projects. furthermore, we are grateful to all experts and patient organisations for development, evaluation and review of the international guideline for glutaric aciduria type i. int. j. neonatal screen. , e-imd has received funding from the european commission ( – ), the kindness for kids foundation e.v., munich, germany ( – ), and dietmar hopp foundation e.v., st. leon-rot, germany (since ). guideline development was supported by the european commission, milupa metabolics and nutricia. author contributions manuscript writing: stefan kölker and jana heringer. contributions to the specific topics: nikolas boy (disease course and treatment), peter burgard (diagnostic process), jürgen günther okun (metabolic analysis). proofreading manuscript: all authors. conflicts of interest the authors declare no conflict of interest. references . goodman, s.i.; markey, s.p.; moe, p.g.; miles, b.s.; teng, c.c. glutaric aciduria; a “new” disorder of amino acid metabolism. biochem. med. , , – . . lindner, m.; kölker, s.; schulze, a.; christensen, e.; greenberg, 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lazovic, j.; housman, c.; lanoue, k.; o’callaghan, j.p.; simpson, i.; woontner, m.; goodman, s.i.; connor, j.r.; jacobs, r.e.; et al. mechanism of age-dependent susceptibility and novel treatment strategy in glutaric acidemia type i. j. clin. investig. , , – . . pfeil, j.; listl, s.; hoffmann, g.f.; kölker, s.; lindner, m.; burgard, p. newborn screening by tandem mass spectrometry for glutaric aciduria type : a cost-effectiveness analysis. orphanet j. rare dis. , , . © by the authors; licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution license (http://creativecommons.org/licenses/by/ . /). 	newborn screening for glutaric aciduria type i: benefits and limitations 	 . introduction 	 . to screen or not to screen? 	 . . knowledge of the disease 	 . . knowledge of the test 	 . . treatment for disease 	 . . cost considerations 	 . conclusions 	acknowledgments 	conflicts of interest 	references am. j. hum. genet. : – , long homozygous chromosomal segments in reference families from the centre d’étude du polymorphisme humain karl w. broman and james l. weber marshfield medical research foundation, marshfield, wi summary using genotypes from nearly , short tandem-repeat polymorphisms typed in eight of the reference families from the centre d’étude du polymorphisme humain (ceph), we identified numerous long chromosomal seg- ments of marker homozygosity in many ceph individ- uals. these segments are likely to represent autozygosity, the result of the mating of related individuals. confi- dence that the complete segment is homozygous is gained only with markers of high density. the longest segment in the eight families spanned cm and included homozygous markers. all individuals in family showed at least one segment of homozygosity: the father and mother were homozygous in and segments with an average length of and cm, respectively, and covering a total of and cm, respectively. the progeny in family were homozygous over – seg- ments with average length cm. the progeny in family were homozygous over – segments with average length cm. of the individuals in the other six families, had especially long homozygous segments, and had short but significant homozygous segments. our results indicate that long homozygous segments are common in humans and that these segments could have a substantial impact on gene mapping and health. introduction the reference families from the centre d’étude du poly- morphisme humain (ceph) (white and lalouel ; dausset et al. ) were recruited in the effort to con- struct the first human genetic maps. lymphoblastoid cell lines derived from the ceph individuals have provided received may , ; accepted for publication september , ; electronically published november , . address for correspondence and reprints: dr. karl w. broman, department of biostatistics, school of hygiene and public health, johns hopkins university, north wolfe street, baltimore, md – . e-mail: kbroman@jhsph.edu q by the american society of human genetics. all rights reserved. - / / - $ . a nearly limitless supply of dna, making these families available for genotyping by investigators around the world. many thousands of short tandem-repeat poly- morphisms (strps) have been genotyped within a subset of eight of the ceph families. these data provide a uniquely comprehensive view of the genomes of these individuals, which allows analyses that would not be possible on the basis of data from a more typical genome scan of markers. we recently constructed new genetic maps based on these families (broman et al. ). as part of that work, we screened the data for apparent tight double-recom- bination events indicative of genotyping errors or mu- tations. in the process, we identified several long seg- ments of noninformative markers in family , caused by long stretches of homozygous markers in the parents of that family. these segments likely represent autozygosity. an in- dividual is autozygous at a locus if he or she received two copies of a single ancestral allele at the locus—that is, if the two alleles are identical by descent (hartl and clark ). autozygosity occurs when a couple shares a chromosomal segment identical by descent and both transmit the segment to one of their offspring. autozygosity may be detected at a single locus if ge- netic data are available on many individuals in an inbred pedigree. more generally, one needs to look at sets of linked markers: if an individual is homozygous at a large number of contiguous markers, it is likely that he or she is autozygous for the segment (i.e., that the two chro- mosomal segments have a common origin). we developed a lod score to characterize the sig- nificance of a segment of homozygosity, to distinguish autozygosity from chance homozygosity. we calculated this score for all possible subsets of contiguous auto- somal markers in all individuals from the eight ceph families, in order to discover autozygous seg- ments. all individuals in family showed at least one segment of homozygosity, as did all of the progeny in family . total lengths of homozygous segments in these individuals were – cm. twenty percent of the individuals in the other six families also had signif- icant homozygous segments. these unexpected findings am. j. hum. genet. : – , table probabilities of observed genotype at a marker, given the autozygosity status of the surrounding segment observed genotype probability that segment is ratioautozygous not autozygous aa ( «)p «pa a pa ( «)/p «a ab «pa pb papb « note.—« denotes the combined rate of genotyping errors and mutations. indicate that long homozygous segments are common in humans. material and methods genetic markers and genotype data we used the data described by broman et al. ( ); we summarize them again here. we considered eight of the ceph families ( , , , , , , , and ), excluding individuals - and - , on whom few genotype data were available. these families contain a total of individuals, in- cluding progeny, parents, and grandparents. we considered genotypes for , autosomal strps, including , from généthon (dib et al. ), , from chlc (sheffield et al. ; sunden et al. ), from the utah marker development group ( ), from the center for medical genetics, marshfield medical research foundation, telomeric markers (rosenberg et al. ), and miscellaneous markers. the genotypes for families , , , and were % complete. families , , , and were not typed for the utah markers, but their genotypes were % complete for the other , mark- ers. all the data are available at the center for medical genetics, marshfield medical research foundation web site. the data had previously been cleaned of genotypes causing apparent tight double-recombination events in- dicative of genotyping errors or mutations. the genetic maps and marker order were as determined by broman et al. ( ). the total sex-averaged genetic length of the autosomes was , cm, corresponding to ap- proximately one marker per . cm. in the following, all reported genetic distances are sex averaged. allele frequencies were estimated on the basis of the found- ing individuals in the eight families. the average (sd) of the marker heterozygosities was . (. ). cytogenetic locations of the markers, from collins et al. ( ), were obtained from the genetic location database at the uni- versity of southampton. identification of long homozygous segments our goal was to identify subsets of contiguous mark- ers for which an individual showed an unusual propor- tion of homozygosity. for each individual, we looked at all possible subsets of contiguous markers. for each such subset, we calculated a lod score comparing the hy- potheses that the individual was or was not autozygous (i.e., homozygous by descent) at all markers in the sub- set. large values of the statistic indicate segments in which the individual showed a significant amount of homozygosity. in forming the lod score, we assumed that the mark- ers were in both linkage and hardy-weinberg equilib- rium, and we used a simple model for genotyping errors and mutations, much like that used by broman and we- ber ( ) in the context of the inference of pairwise relationships. the lod score has the following form: k pr(g fautozygous at i)ilod(j,k) = log ,o [ ]pr(g fnot autozygous at i)i=j i where is the individual’s observed genotype at markergi i. the assumed genotype probabilities are displayed in table . in the model underlying the lod score, it is assumed that errors and mutations occur with proba- bility « and that, when such events occur, genotypes are obtained according to their population frequencies. this model serves as a device for obtaining a statistic that facilitates the detection of homozygous segments while both taking account of the varying informativeness of the markers and allowing the presence of a small pro- portion of heterozygous markers within each segment; it was not intended to reflect the details of the error and mutation processes. in calculating the lod score, we assumed that the combined rate of genotyping errors and mutations was . a smaller value of « would give greater weight« = . to heterozygous markers, resulting in shorter identified segments. if « were made larger (smaller) by a factor of , the lod score for a segment would be increased (decreased) by . for each heterozygous marker that it contained; for example, if a segment containing a single heterozygous marker had a lod score of . when , the lod score would be ∼ . and ∼ . when« = . and . , respectively.« = . the above-described lod score was calculated for each possible subset of contiguous markers in each in- dividual. when, for a particular individual, two subsets of markers with large lod scores overlapped, we re- tained information only on that subset with the larger lod score. simulations under linkage equilibrium even under complete linkage equilibrium, individuals may be homozygous for a number of contiguous mark- broman and weber: autozygosity in ceph families figure minimum detectable length (in cm) of an autozygous region for a genome of length , cm, with equally spaced markers of constant heterozygosity. table homozygous segments for individual - chromosome (markers) cytogenetic band(s) length (cm) proportion homozygous lod score (d s –d s ) q -q . / . (d s –d s ) p -p . / . (d s –d s ) q -q . / . (d s –d s ) q -q . / . (dlls –dlls ) p . / . (d s –d s ) q . / . (d s –d s ) q . / . (d s –d s ) q . / . ers somewhere in the genome, completely by chance. in order to estimate the distribution of the above-described lod score under linkage equilibrium, we simulated the genetic data for unrelated individuals # = , having the same pattern of missing data as were seen in the ceph individuals whom we studied. we as- sumed hardy-weinberg and linkage equilibrium and used the allele frequencies estimated on the basis of the founding individuals in the eight families. for each simulated genome, we calculated the maximum lod score, among all possible subsets of contiguous markers, and the length of the subset with the maximum lod score. to determine the effects of marker density and marker informativeness on the minimum detectable length of an autozygous segment, we simulated genomes that were of length , cm and had markers that were equally spaced, had constant heterozygosity, and were in linkage equilibrium. to simplify the simulation, we assumed that the genome consisted of a single long chromosome. for each of various marker densities and heterozygosities, we simulated the genetic data for , genomes and calculated the th percentile of the maximum length of contiguous homozygous markers. this percentile is the minimum detectable length of an autozygous seg- ment when a % genomewide type i–error rate is used. results the minimum detectable length of an autozygous seg- ment, as a function of marker density, in the case of equally spaced markers with constant heterozygosity, is displayed in figure . for markers with . heterozygosity, an autozygous segment as short as cm may be detected when the markers are cm apart, whereas the segment must be > cm long if there are markers every cm. at the density of a more typical genome scan (i.e., cm), only very long autozygous segments can be detected. the simulations under linkage equilibrium that made use of the genetic maps and allele frequencies estimated from the data indicated that, ∼ % of the time, an in- dividual will have a segment of at least nine contiguous homozygous markers, with a lod score . , some- where in the genome. in the following, we present only those segments that had lod scores . . all individuals in family had at least one ho- mozygous segment with a lod score . . the father, individual - , had eight homozygous segments, which are summarized in table . lod scores for these homozygous segments were as high as . . one seg- ment was ∼ cm long; the others were – cm long. overall, these segments covered a total of cm ( . % of the autosomal genome). the mother, individual – , had homozygous segments, which are sum- marized in table . five segments were ! cm long; three were – cm long. overall, these segments cov- ered a total of cm ( . % of the autosomal genome). table contains a summary of the homozygous seg- ments in the other members of family . three of the four grandparents in the family had multiple long ho- mozygous segments; the other grandparent showed just one homozygous segment of markers. the prog- eny in the family showed – homozygous segments with average length . cm and covering, on average, cm ( . % of the autosomal genome). these homo- am. j. hum. genet. : – , table homozygous segments for individual - chromosome (markers) cytogenetic band(s) length (cm) proportion homozygous lod score (d s –d s ) q . / . (gata e –d s ) p -q . / . (d s –d s ) q -q . / . (d s –d s ) q -q . / . (d s –d s ) q -q . / . (d s –d s ) q . / . (d s –d s ) q -q . / . (d s –d s ) q -q . / . (d s –gata e ) q -q . / . (d s –d sl ) q . / . table homozygous segments for progeny and grandparents in family individual no. of segments length (cm) no. of markers total (average) length progeny: . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) grandparents: . – . – . ( . ) . . ( . ) . – . – . ( . ) . – . – . ( . ) zygous segments derived from shared haplotypes in the parents. the grandpaternal chromosome in the father matched the grandmaternal chromosome in the mother in segments covering cm ( . % of the autosomal genome); the grandmaternal chromosome in the father matched the grandpaternal chromosome in the mother in segments covering cm ( . % of the autosomal genome); the grandpaternal chromosomes in the father and mother were identical in chromosomal segments covering cm ( . % of the autosomal genome); and the grandmaternal chromosomes in the father and mother were identical in segments covering cm ( . % of the autosomal genome). the progeny in family showed – homo- zygous segments with average length . cm and cov- ering, on average, cm ( . % of the autosomal ge- nome) (table ). neither parent in family showed any significant homozygous segments; genotype data on the grandparents in this family were not available. a -cm segment of chromosome , containing the markers from d s to d s , is of special interest with regard to family . all progeny were homozygous for at least one portion of this segment. child - was homozygous for markers in this segment, spanning cm; children - and - were homozygous for the entire -cm segment. the other progeny were homozygous for a segment of intermediate size. an inspection of the inferred haplo- types in the two parents shows that they share a common haplotype on one pair of their chromosomes over this entire segment; on their other chromosome pair, they share a common haplotype for an -cm segment con- taining markers. the homozygosity in of the progeny derives from the longer haplotype shared by the two parents; the homozygosity in the other progeny derives from the other shared haplotype. of the individuals in the other six families, had at least one homozygous segment with lod score . . individual - (a grandparent) showed four homozygous segments, including three segments with lod scores , containing markers and spanning – cm. nine individuals were homozygous for seg- ments ! cm long, including one individual homozygous for two short but significant segments. ten other indi- viduals were homozygous for segments – cm long. one of these contained markers; the others contained – markers. a complete list of significant homozygous segments for all individuals in the eight ceph families is available with the electronic version of this article, at the journal’s web site. many of the identified homozygous segments con- tained one or more heterozygous markers. these het- erozygous markers could be due to errors in marker order, genotyping errors, mutations, or gene conver- sions. of the homozygous segments identified, contained one heterozygous marker, and contained two to four heterozygous markers. the other seg- broman and weber: autozygosity in ceph families table homozygous segments for progeny in family individual no. of segments length (cm) no. of markers total (average) length . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) . – . – . ( . ) ments contained no heterozygous markers. among the eight segments with two or more heterozygous markers, there were three cases in which two heterozygous mark- ers were adjacent to each other, separated by cm. (these occurred in a segment on chromosome in progeny from family .) in all other cases, the het- erozygous markers were relatively isolated from each other. the lengths of the homozygous segments that we iden- tified, as well as the proportion of the genome that they cover, are likely to be underestimates. the markers were ordered on the basis of the meioses in these families, and so the order is generally correct only to ∼ – cm. at the ends of a homozygous segment, the presence of a few heterozygous markers, caused by mutation or in- correct marker order, may cause an early truncation of the identified segment. when one assumes that « = , a heterozygous genotype subtracts . from the. lod score, whereas homozygous genotypes for alleles with frequencies . and . add . and . , respectively, to the lod score. thus, for a homozygous segment to be extended past a heterozygous genotype, one must see three to five additional homozygous genotypes. of the homozygous segments described in tables and , contained a single heterozygous genotype, contained two heterozygous genotypes, and contained four het- erozygous genotypes; in all of these cases, there were at least five homozygous genotypes between the hetero- zygous marker and the end of the identified segment. it is likely that a number of autozygous segments were missed, since the segments need to be of a sufficient size, containing a sufficient number of markers, to allow de- tection (see fig. ). although the marker data that we considered were of unusually high density, marker den- sity varied considerably across the genome, with some regions containing markers in ! cm and others con- taining gaps as long as cm with no markers. marker heterozygosity varied considerably as well. although the average heterozygosity was . , % of the markers had heterozygosities <. or >. . discussion autozygosity occurs when two copies of an ancestral haplotype come together in an individual. this may be the result of the mating of close relatives or of linkage disequilibrium in a population, but these are just the two extremes in a continuum of ancestral sharing. it is customary to distinguish between linkage disequilibrium and inbreeding: homozygous segments that are the result of linkage disequilibrium would not ordinarily be called autozygous. however, insofar as linkage disequilibrium is the result of the persistence of ancestral haplotypes in a finite population, homozygosity as the result of linkage disequilibrium is indeed the result of the mating of (very distantly) related individuals. linkage disequilibrium is a local phenomenon and thus would cause only short homozygous segments. long homozygous segments, such as that on chromosome in individual - , which is cm in length and contains markers, can- not be explained by linkage disequilibrium. the length of an autozygous segment reflects its age: haplotypes are broken up by recombination at meiosis, and so a short autozygous region is likely to be of distant origin. (haplotypes can also be brought together by re- combination; if such an event were to occur twice in- dependently, the result could be an autozygous segment composed of portions derived from two distinct ances- tors.) the proportion of an individual’s genome that is autozygous, the expected value of which is the inbreed- ing coefficient (hartl and clark ), is a measure of the degree of relationship between his or her parents. family was of venezuelan origin. the long seg- ments of homozygosity in the progeny of this family, covering on average . % of the autosomal genome, indicates that the parents were relatively closely related. six of the progeny had homozygous segments cov- ering % of their genome. although neither parent showed any inbreeding, the parental haplotypes corre- sponding to the homozygous segments on chromosome imply that the homozygosity in this family is the result of relatedness between at least two pairs of the four grandparents. for example, the grandparents could have been composed of two sets of first cousins, making the parents in the family double–second cousins. if that were true, then one would expect the progeny in the family to be autozygous at an average of . % of the autosomal genome. family is from the old order amish in penn- sylvania. the progeny in this family were autozygous at a smaller proportion of the genome ( . %), and the am. j. hum. genet. : – , autozygous segments were somewhat smaller, than those in family . the parents and grandparents in this fam- ily, however, also showed a large proportion of autozy- gosity. the grandparental origins of the haplotypes shared between the two parents indicates that each of the four grandparents was related to each of the others. the genealogy of this family (egeland ) indicates that the grandparents have no ancestors in common in the three prior generations. for example, if one looks at the five generations preceding father - , he is indicated to be the child of third cousins: one of his father’s great-grandmothers is the sister of one of his mother’s great-grandfathers. the parents of mother - show a similar relationship. the child of third cousins would be expected to be autozygous at, on average, . % of the genome. individuals - and - , however, were autozygous at . % and . % of their genomes, respectively. similarly, this part of the gene- alogy, on its own, would imply an average of . % autozygosity in the progeny, one-fourth the observed av- erage amount of autozygosity in the progeny. thus the homozygosity in family cannot be explained by the inbreeding in four generations preceding the family’s grandparents. prior generations likely showed much more inbreeding, so that the observed autozygosity was the result of drawing from a limited pool of dna five or more generations back in time. the presence of long homozygous segments in the parents of family make this family a less than ideal choice as a reference for genetic maps. future analyses of the ceph data should treat this family with special care. for example, bugge et al. ( ) used the ceph genetic data to calculate crossover counts for each au- tosome. family should have been excluded from those counts for chromosomes for which a parent was homozygous—and, therefore, not informative—for a large portion of the chromosome, since several double crossovers were likely missed. of the individuals in the other six ceph families, all from utah, individual, grandparent - , had one homozygous segment cm long and three segments – cm long, the four segments together covering . % of the autosomal genome, indicating a close re- lationship between her parents; of the other individ- uals in these families had quite small homozygous seg- ments. these six families clearly do not display the sort of inbreeding observed in families and , but the results cannot reasonably be ascribed to chance homo- zygosity; rather, we conclude that the families were sub- ject to a small amount of inbreeding, at least in the past, and that the markers studied show considerable linkage disequilibrium, a modern trace of older relationships in the population from which these families were drawn. the long homozygous segments provide opportunity for study of mutation. heterozygous markers within these homozygous regions are likely to be the result of a mutation event in one of the generations between the last common ancestor and the autozygous individual. an example is marker gata within the long ho- mozygous region on chromosome in - (genotype , ). these heterozygous markers will provide val- uable information about mutation rate—and, when combined with data on the relevant haplotype or allele frequencies, about the nature of the nucleotide change. the presence of long homozygous regions has at least two implications for gene mapping in genetically isolated populations. on the positive side, it may be possible to extend the widely used homozygosity (autozygosity) mapping for rare recessive disorders (smith ; lander and botstein ), to include genetically more complex disorders. the presence of two copies of a rel- atively common allele often increases disease risk com- pared with the presence of a single copy (e.g., see farrer et al. ; rosendaal ). by searching for shared homozygous regions among individuals from an isolated population with extreme phenotypes, it may be possible to map responsible genes. furthermore, a modification of the lod score described here could be used to identify haplotypes shared by distantly related diseased individ- uals. in this case, one would search for segments of many contiguous markers for which the pair shared at least one allele identical by state. on the negative side, if a large sibship contributes substantially to the power of a gene-mapping study and one (or both) of the parents is homozygous at a major gene locus, then evidence in support of linkage may be reduced below the detection threshold. in this respect, it is interesting to note that indications of a gene, on chromosome , predisposing to bipolar affective dis- order have been reported in some studies (berrettini et al. ; stine et al. ; freimer et al. ; mc- mahon et al. )—but not in the old order amish (pauls et al. ; polymeropoulos and schaffer ; ginns et al. , )—and that the mother of amish family (a family with bipolar affective disorder) is homozygous for a long segment of chromosome . although it is impossible to accurately extrapolate from data on only eight families, we suspect that long homozygous segments are common in many human pop- ulations. the old order amish are a clearly recognized genetic isolate, but neither the venezuelan kindred, , nor the remaining six, utah mormon, kindreds derived from any obvious genetic isolates. furthermore, as de- scribed above, the grandparents in amish family were not particularly closely related, and many human populations are probably as isolated genetically as the amish. the total lengths of autozygous segments within individuals from a specific population may provide a useful measure of the isolation of that population. clinical implications of the long homozygous seg- broman and weber: autozygosity in ceph families ments are substantial. first-cousin matings will produce, on average, autozygosity of %. fitness and health of the progeny of first-cousins pairs are well known to be reduced (morton ; jaber et al. ; stoll et al. ; vogel and motulsky ; ober et al. ). yet, our results indicate that many individuals who are not the offspring of obviously consanguineous matings have degrees of autozygosity near or even exceeding that of the offspring of first-cousin matings. autozygosity may play a much larger role in morbidity than previously has been suspected. analogous to a more powerful telescope that makes possible study of new (and often more ancient) astro- nomic phenomena, the high-density whole-genome poly- morphism scan opens to examination a whole new ge- netics realm. the results presented in this report could not have been obtained with low-density scans. current technology does not permit . -cm-density scans to be easily completed today, but many developments are un- derway that should eventually open this new realm to routine exploration (e.g., see wang et al. ). it is interesting to speculate what would be observed, in terms of autozygosity, with an even-higher-densi- ty polymorphism scan; undoubtedly, at least some shorter—and, hence, older—homozygous regions would be detected. the many reported examples of strong link- age disequilibrium in humans virtually guarantee that these short autozygous segments exist (although their number and length are unknown). mutation, especially for strps, will obscure detection of these more ancient segments. for efficient detection, it may become neces- sary to turn to diallelic polymorphisms with lower mu- tation rates. homologous human chromosome pairs may even prove to be a patchwork of alternating short segments that are either largely homozygous or largely heterozygous for low-mutation-rate polymorphisms. acknowledgments mark neff and andrew paterson generously provided com- ments for improvement of the manuscript. janice egeland and colleagues generously provided their time and expertise to trace the genealogy of family . this work was supported in part by a john wasmuth fellowship in genomic analysis, national human genome research institute grant f -hg (to k.w.b.), and by national heart, lung, and blood institute contract n hv for the mammalian genotyping ser- vice (to j.l.w.). electronic-database information urls for data in this article are as follows: center for medical genetics, marshfield medical research foundation, http://www.marshmed.org/genetics genetic location database, http://cedar.genetics.soton.ac.uk/ public_html/ldb.html references berrettini wh, ferraro tn, goldin lr, weeks de, detera- wadleigh s, nurnberger ji, gershon es ( ) chromo- some dna markers and manic-depressive illness: evi- dence for a susceptibility gene. proc natl acad 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sweat electrolytes in an irish trav- eller kindred interests us, as we have observed raised sweat electrolytes in two members of the original byler kindred who have byler’s disease. neither has cystic fibro- sis; both underwent liver transplantation in their second decade and subsequently devel- oped pancreatic disease. one has had recur- rent pancreatitis and the other has a fibrotic pancreas with exocrine insuyciency. in the children without byler’s disease whom we attend, pancreatic disease after liver trans- plantation is not usual.have avected traveller children, particularly older ones, had pan- creatitis? we can provide additional information on the sister and brother with progressive famil- ial intrahepatic cholestasis and raised sweat electrolytes referred to by bourke et al and described by lloyd-still (fig ). neither parent was of irish or amish background; the father (ii. ) came of norwegian and the mother (ii. ) of italian stock. at age . years, the boy (iii. ) had normal serum ã-glutamyltranspeptidase activity ( u/ml; expected, < ) and moderately raised cholesterol concentrations ( . mmol/l; ex- pected . – . ; determination at age year, . ) with marked hyperbilirubinaemia ( µmol/l; expected, . – . ). fasting serum bile acid concentrations were not measured but at age year had been ‘markedly elevated’ ; intense pruritus was present. serum amylase and lipase activities were nor- mal. the (iii. ) girl came to liver transplanta- tion aged years and the boy . years. both died of infection within two months of surgery. on light microscopy, findings in the native livers resembled those in the older traveller children and in the two amish chil- dren who underwent hepatectomy. coarsely granular bile like that seen in byler’s disease (‘byler bile’) was found on transmission electron microscopy (fig ). convergence of phenotypes leads us to believe that these children may have had a lesion at q -q , the byler’s disease locus, to which the disorder in the traveller kindred also has been mapped. we would like to know what was seen if liver tissue from an avected traveller child was examined by transmission electron microscopy. a s knisely denver-aurora pathology associates, east th avenue, denver,co ,usa r m agostini b j zitelli* s a kocoshis** departments of pathology,pediatrics*,and pediatric gastroenterology**, children’s hospital of pittsburgh, pittsburgh,pa,usa j t boyle division of pediatric gastroenterology, rainbow babies and children’s hospital, cleveland,oh,usa dr bourke and professor drumm comment: we thank dr agostini and colleagues for their interest in our paper describing an irish kin- dred with byler syndrome. as yet, we have not examined liver tissue from the traveller kindred using transmission electron micros- copy. as these children likely will need further evaluation and/or transplantation in the coming years we will have the opportunity to undertake further studies including analy- sis of biliary bile acid content and examin- ation of biopsy samples for the presence of ‘byler bile’. we are aware of the report of knisely et al describing pancreatic disease in members of the original amish kindred with byler’s disease. although we have not observed pancreatitis or evidence of pancreatic dys- function in the irish traveller family with byler’s syndrome, one of us (bb) has encountered a child with progressive familial intrahepatic cholestasis and chronic pancrea- titis at another institution (patient of e rob- erts and r superina, hospital for sick chil- dren, toronto). whether this child has a mutation at q -q is currently being evaluated. the presence of raised sweat electrolytes and pancreatitis in a subset of these children with byler’s disease/syndrome is certainly intriguing and raises interesting questions about the function of the mutated allele at q - . ongoing genetic studies of mem- bers of the original kindred and unrelated families such as this irish family should soon provide answers to these questions. bourke b, goggin n, walsh d, kennedy s, setchell kdr, drumm b. byler-like familial cholestasis in an extended kindred. arch dis child ; : - . knisely as, boyle jt, naylor ew, klinger k, freimer nb, kocoshis s. pancreatic dysfunc- tion in byler disease. j pediatr gastroenterol nutr ; : (abstr). lloyd-still jd. familial cholestatic syndrome with elevated sweat electrolytes. in: sturgess jm, ed. proceedings of the th international con- gress on cystic fibrosis. toronto: imperial press, : a (abstr). lloyd-still jd.familial cholestasis with elevated sweat electrolyte concentrations. j pediatr ; : - . bull ln, carlton veh, stricker nl, et al. genetic and morphologic findings in progres- sive familial intrahepatic cholestasis (byler dis- iii ii i figure pedigree of family with byler disease-like progressive familial intrahepatic cholestasis described by lloyd-still. figure transmission electron micrograph of coarsely granular bile, characteristic of bile from children with byler’s disease, within canaliculus of liver obtained at hepatectomy in avected boy (iii. ); % paraformaldehyde/ . % glutaraldehyde in swenson’s phosphate buver,ph . ; oso /uranyl acetate/lead citrate (original magnification × ). archives of disease in childhood ; : – o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ ease and byler syndrome): evidence for hetero- geneity. hepatology ; : - . carlton veh, knisely as, freimer nb. map- ping of a locus for progressive familial intrahe- patic cholestasis (byler disease) to q -q , the benign recurrent intrahepatic cholestasis region. hum mol genet ; : - . barton de, mcquaid s, bourke b, et al. famil- ial progressive intrahepatic cholestasis (byler disease): evidence that the disease haplotype in the old order amish is also found in the irish ‘traveller’ population. am j hum genet ; :a (abstr). intestinal neuronal dysplasia associated with cystic fibrosis editor,—the association between cystic fibrosis and intestinal neuronal dysplasia (ind) has been rarely described. we report a case of full thickness, biopsy proved, ind type b of the ileum and colon associated with cystic fibrosis. the boy was born at full term to non-consanguineous parents. because of obstructive symptoms, several resections were performed: cm of distal ileum after birth; distal ileum and part of ascending colon at the age of days; ileum, part of jejunum, and colon at the age of months. a series of radiographs of the upper gastrointes- tinal tract series showed a normal duodenum at months and no dilatations of the remaining intestinal tract. contrast appeared in the rectum after minutes. by histology, the proximal ileal tract had . neurons/mm of myenteric plexus, according to smith’s method (normal values: – ), the ascending colon . neurons/mm, and the transverse colon neurons/rnm. acetylcholinesterase staining showed an increase of number of submucosal ganglia, neuronal heterotopy, and increase of positive fibres in circular muscular layer and lamina propria. nadph-dh showed an increased number of neurons in myenteric and submu- cosal plexuses. the study with neurofila- ments (nf , nn ) showed a normal maturity of neurons. the results of two sweat tests were abnormal.an homozygosity for the delta f mutation was demonstrated and both parents were carriers of the allele. it is possible that there is an nid-b determining gene linked to the cystic fibrosis transmem- brane conductance regulator locus for cystic fibrosis, localised on chromosome q. we suggest that intestinal neuronal dyspla- sia should be considered as an underesti- mated, underlying cause in patients with cystic fibrosis having functional small bowel dysmotility and obstruction leading to emer- gencies, such as meconium ileus in neonates or meconium ileus equivalent in children and adults. a tozzi g ascione* m l carpentieri a staiano departments of paediatrics and division of paediatric surgery*, university federico ii, via s pansini , naples, italy wildhaber j, seelentag wkf, spiegel r, schoni mh. cystic fibrosis associated with neuronal intestinal dysplasia ype b: a case report. j pedi- atr surg ; : - . smith vv. intestinal neuronal density in childhood: a baseline for objective assessment of hypo- and hyperganglionosis. pediatr pathol ; : - . milla pj, smith vv. intestinal neuronal dyspla- sia. j pediatr gastroenterol nutr ; : - . legislation and drug trials editor,—in their recent leader, walsh and drumm point out important diyculties facing paediatricians wishing to conduct intervention trials where the aim is to prevent disease in children (or anyone incapable of giving fully informed consent) in ireland. it is worth pointing out that the irish legislation that prevents such studies thereby prevents all vaccine studies in children from being conducted in that country. vaccines against haemophilus influenzae type b and more recently acellular vaccines against pertussis have been licensed and introduced in ireland on the basis of immunogenicity and eycacy studies done elsewhere. while it is not neces- sary for each vaccine to be studied in every country, there is a clear need for all countries to be able to contribute clinical studies particularly as the number of new antigens and combinations grows. it is to be hoped that the current stranglehold on research into child health in ireland is loosened in the near future. adam finn department of paediatrics, university of sheyeld, sheyeld children’s hospital, sheyeld s th walsh d, drumm b. legislation and drug trials. arch dis child ; : - . situs inversus and left sided pyloric tumours editor,—the case report by harrington et al reminded me of the procedure to examine for a pyloric tumour taught by dr m j simp- kiss, based on an identical case he had seen decades previously. namely, define the apex beat before examining the abdomen. this combination will occur again, just like the case of ‘the glass eye...’. richard sporik institute of respiratory medicine, university of sydney,nsw , australia harrington b, chambers t, grier d. a diagno- sis obscured:pyloric stenosis with situs inversus [letter]. arch dis child ; : . gordon rm, greene jm, kassirer jp. solution to a ‘medical mystery’[letter]. n engl j med ; : - . head lice in schoolchildren editor,—i am grateful to ibarra and hall for reviewing a common problem in general practice. i recently performed a medline search of the literature regarding the role of hairdressers and head lice. to my concern no references existed and my personal experience compounds my view that some gentlemen’s hairdressers do not wash, let alone sterilise their ‘tools’ or drapes between customers. therapy has an important role in the eradication of head lice, but there is a more important public health issue in relation to prevention. rodger charlton the surgery,fentham hall, marsh lane,hampton-in-arden, solihull,west mdlands b ah ibarra j, hall dmb. head lice in schoolchil- dren. arch dis child ; : - . letters o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://a d c.b m j.co m / a rch d is c h ild : first p u b lish e d a s . /a d c. . . o n s e p te m b e r . d o w n lo a d e d fro m http://adc.bmj.com/ case reports.pmd indian pediatrics volume __july , c artilage-hair hypoplasia (chh) or metaphyseal chondrodysplasia mckusick type (omim # ) is an autosomal recessive disorder [ ]. it is a type of skeletal dysplasia resulting in short-limbed dwarfism with shortening and sometimes bowing of the tubular bones and brachydactyly, with radio- graphs showing disorganized metaphyses as well as cone shaped epiphyses of the fingers [ ]. the rmrp gene was identified as the disease causing gene in chh [ , ]. we report two patients with chh caused by the two novel rmrp mutations c. _ dupagt and c. c>t. case report two children born to a third degree consan- guineously married south indian couple were referred for evaluation of short stature. the elder sibling was a -year-old girl with short fingers at birth. later a delay in all motor milestones and a gradual loss of scalp hair were noticed. she was attending school and was an average performer. when clinically evaluated at the age of -yr, the weight was kg (< th percentile) and height was cm (< rd percentile). the upper and lower segment ratios were infantile with upper segment measuring cm while the lower segment measured cm; the arm span ( cm) was greater than her height. other skeletal abnormalities included pectus carinatum, exaggerated lumbar lordosis, genu valgum and pes planus. the extension of elbows was restricted. brachydactyly with broad finger tips and a single crease in the thumbs were identified. there was no axillary hair and she had not yet attained menarche. there was no history suggestive of immunodeficiency or malabsorption. radiographs revealed cone shaped epiphyses of the hands, metaphyseal irregularities of femoral heads and phalanges, and brachydactyly of the phalanges. the younger sibling was a -year-old boy whose height ( cm) and weight ( kg) were below the th percentile. his phenotype was similar to that of his sister, except for less pronounced scalp hair loss. radiographs revealed similar changes; metaphyseal dysplasia and platyspondyly of the thoracolumbar vertebra was also seen. the patient’s radiographs were submitted to the european skeletal dysplasia network (esdn) clinical-radiographic review panel. as the features were suggestive of chh, analysis of the rmrp gene was proposed. following extraction of dna from blood of the sibs and their parents, the rmrp gene was amplified by polymerase chain reaction (pcr) [ ]. the cartilage-hair hypoplasia caused by novel compound heterozygous rmrp mutations kerstin reicherter, amithkumar iynapillai veeramani* and sujatha jagadeesh* from center for pediatrics and adolescent medicine and faculty of biology, university of freiburg, d- freiburg, germany; and*department of clinical genetics, fetal care research foundation, chennai, india. cartilage-hair hypoplasia is a rare, autosomal recessive skeletal dysplasia, caused by mutations in the rmrp gene. the skeletal abnormalities include irregular metaphyses and cone shaped epiphyses of the hands. molecular diagnosis confirmed two novel rmrp mutations in a compound heterozygous state in two siblings with this condition. key words: india, metaphyseal chondrodysplasia, rmrp mutation, skeletal dysplasia. correspondence to: dr sujatha jagadeesh, consultant and head, department of clinical genetics, fetal care research foundation, chennai, india. fcrfchennai@yahoo.com received: june , ; review completed: january , ; accepted: march , . ccccc aaaaa sssss eeeee r r r r r eeeee ppppp ooooo rrrrr ttttt sssss indian pediatrics volume __july , case reports after having confirmed the diagnosis by mole- cular analysis, the prospective treatment was dis- cussed. unlike most chh patients, these sibs did not have a history of recurring infections or other signs of immunodeficiency. as the clinical outcome of chh patients can only be poorly predicted, they should be carefully followed up because of the possibility of serious infections and malignancies. acknowledgments: we acknowledge the support of dr andreas zankl, dr sheila unger and the esdn clinical-radiographic review panel (www.esdn.org). we thank dr pia hermanns, who was involved in the rmrp mutation analysis and the interpretation of the data. we thank dr suresh s of the fetal care research foundation for his encouragement. contributors: kr: did the molecular analysis, was involved in the interpretation of the data, drafted the manuscript together with aiv and revised the manuscript; aiv: drafted the manuscript initially; sj: managed the case and will act as the guarantor. the final manuscript was approved by all authors. funding: none. competing interests: none stated. references . bonafé l, schmitt k, eich g, giedion a, superti-furga a. complete transcript of the rmrp gene as well as base pairs of the promoter region was sequenced. thereby the novel rmrp mutations c. _ dupagt and c. c>t (fig. ) as well as the previously described polymorphisms g. t>c; g.- c>a and g.+ t>c (all homozygous), were identified in both patients. both siblings were compound heterozygous for the mutations. the correct segregation of the mutations was proven by subcloning and sequencing of the pcr products. analysis of the parents confirmed their carrier status. the father had a c. c>t mutation whereas the mother had the duplication (c. _ dupagt). the father was homozygous for the polymorphisms and the mother was heterozygous. discussion chh is a rare disease with known carrier frequencies of : among the amish and : among the finnish population [ ]. the phenotype of chh is variable. laxity of ligaments, incomplete extension at the elbows, cone shaped epiphyses of the phalanges [ ], rounded distal epiphyses, prominent sternum, chest deformity [ ], mild scoliosis, increased lumbar lordosis and bowing of the lower limbs are described. the affected siblings reported in this study, had most of the clinico-radiographic findings suggestive of chh. the rmrp gene encodes the untranslated rna component of the rnase mrp complex. this ribonucleoprotein complex cleaves rnas and is thereby involved in the replication of mitochondrial dna [ ], processing of the . s ribosomal rna [ ] and control of cell cycle progression at the end of mitosis [ ]. mutations in the rmrp gene lead to a wide spectrum of recessive skeletal dysplasias with different degrees of short stature. the molecular analysis of the rmrp gene in the affected siblings identified two novel mutations, c. _ dupagt and c. c>t. we conclude that these were patho- genic as they were inherited from unaffected parents, both carrying one of the mutations. in addition, the mutations were not identified in controls [ ]. the functional importance could be explained by the localization of both mutations in an evolutionary conserved part of the transcript [ ]. they might alter a very important stem-loop structure [ ]. fig . sequencing of the rmrp gene after subcloning; results of the boy. (a) the paternal allele carries a c. c>t nucleotide substitution within the rmrp gene. (b) the maternal allele carriers a duplication of nucleotides (c. _ dupagt) within the rmrp gene. both patients were compound heterozygous for the novel rmrp mutations c. c>t and c. _ dupagt. indian pediatrics volume __july , case reports rmrp gene sequence analysis confirms a cartilage-hair hypoplasia variant with only skeletal manifestations and reveals a high density of single-nucleotide polymorphisms. clin genet. ; : - . . polmar sh, pierce gf. cartilage hair hypoplasia: immunological aspects and their clinical implications. clin immunol immunopathol. ; : - . . ridanpää m, sistonen p, rockas s, rimoin dl, mäkitie o, kaitila i. worldwide mutation spectrum in cartilage-hair hypoplasia: ancient founder origin of the major a->g mutation of the untranslated rmrp. eur j hum genet. ; : - . . ridanpää, m, van eenennaam h, pelin k, chadwick r, johnson c, yuan b, et al. mutations in the rna component of rnase mrp cause a pleiotropic human disease, cartilage-hair hypoplasia. cell. ; : - . . mckusick va, eldridge r, hostetler ja, egeland ja, ruangwit u. dwarfism in the amish. ii cartilage-hair hypoplasia. bull johns hopkins hosp. ; : - . atypical cogan syndrome mimicking acute rheumatic fever m juneja, r jain and b chakarbarty from department of pediatrics, maulana azad medical college and associated lok nayak hospital, new delhi, india. cogan syndrome is a syndrome of non-syphilitic interstitial keratitis associated with vestibuloauditory deficits. we report a year-old male child who presented with fever, acute polyarthritis, and unilateral red eye and was diagnosed as acute rheumatic fever. subsequently unilateral hearing loss was detected and the child was diagnosed to have atypical cogan syndrome. key words: acute rheumatic fever, arthritis, atypical cogan’s syndrome, polyarthritis. correspondence to: prof monica juneja, c- south extension ii, new delhi , india. drmonicajuneja@gmail.com received: november , ; initial review: february , ; accepted: march , . c ogan syndrome is a syndrome of non- syphilitic interstitial keratitis associated with vestibuloauditory deficits, that progresses to complete deafness within years [ ]. the term ‘atypical cogan’s syndrome’ was suggested later for cases in which sensorineural hearing loss is associated with ocular inflammation distinct from interstitial keratitis, such as uveitis, scleritis, conjunctivitis or retinal vasculitis [ , ]. patients in which the interval between the onset of ocular and audiovestibular manifestations is more than two years are also labeled as atypical cogan’s syndrome [ , ]. majority of patients with this syndrome develop significant systemic symptoms and often present a diagnostic challenge [ - ]. we describe a year old male child who presented to us with acute polyarthritis and was later diagnosed as atypical cogan syndrome. case report a years old male child was referred to our center as a case of acute rheumatic fever. there was history of low grade fever for days, severe pain and swelling in multiple large joints for days and redness of the right eye for days. there was no history of any antecedent upper respiratory tract . chang dd, clayton da. mouse rnaase mrp rna is encoded by a nuclear gene and contains a decamer sequence complementary to a conserved region of mitochondrial rna substrate. cell. ; : - . . schmitt me, clayton da. nuclear rnase mrp is required for correct processing of pre- . s rrna in saccharomyces cerevisiae. mol cell biol. ; : - . . gill t, cai t, aulds j, wierzbicki s, schmitt me. rnase mrp cleaves the clb mrna to promote cell cycle progression: novel method of mrna degradation. mol cell biol. ; : - . . bonafé l, dermitzakis et, unger s, greenberg cr, campos-xavier ba, zankl a, et al. evolutionary comparison provides evidence for pathogenicity of rmrp mutations. plos genet. ; : - . . welting tj, van venrooij wj, pruijn gj. mutual interactions between subunits of the human rnase mrp ribonucleoprotein complex. nucleic acids res. ; : - . a rare functional cardioprotective apoc variant has risen in frequency in distinct population isolates article received jun | accepted nov | published dec a rare functional cardioprotective apoc variant has risen in frequency in distinct population isolates ioanna tachmazidou , george dedoussis , lorraine southam , , aliki-eleni farmaki , graham r.s. ritchie , , dionysia k. xifara , , angela matchan , konstantinos hatzikotoulas , nigel w. rayner , , yuan chen , toni i. pollin , jeffrey r. o’connell , laura m. yerges-armstrong , chrysoula kiagiadaki , kalliope panoutsopoulou , jeremy schwartzentruber , loukas moutsianas , uk k consortium , emmanouil tsafantakis , chris tyler-smith , gil mcvean , yali xue & eleftheria zeggini isolated populations can empower the identification of rare variation associated with complex traits through next generation association studies, but the generalizability of such findings remains unknown. here we genotype , individuals from a greek population isolate on the illumina humanexome beadchip, in search of functional coding variants associated with lipids traits. we find genome-wide significant evidence for association between r x, a functional variant in apoc , with increased high-density lipoprotein and decreased triglycerides levels. approximately . % of individuals are heterozygous for this cardioprotective variant, which was previously thought to be private to the amish founder population. r x is rare (o . % frequency) in outbred european populations. the increased frequency of r x enables discovery of this lipid traits signal at genome-wide significance in a small sample size. this work exemplifies the value of isolated populations in successfully detecting transferable rare variant associations of high medical relevance. doi: . /ncomms open wellcome trust sanger institute, hinxton cb sa, uk. department of dietetics-nutrition, harokopio, university of athens, athens , greece. wellcome trust centre for human genetics, oxford ox bn, uk. european molecular biology laboratory, european bioinformatics institute, hinxton, cb sd, uk. department of statistics, university of oxford, oxford ox tg, uk. division of endocrinology, diabetes, and nutrition, university of maryland school of medicine, baltimore, maryland - , usa. anogia medical centre, anogia , greece. a list of consortium members are presented in supplementary note . correspondence and requests for materials should be addressed to e.z. (email: eleftheria@sanger.ac.uk). nature communications | : | doi: . /ncomms | www.nature.com/naturecommunications & macmillan publishers limited. all rights reserved. mailto:eleftheria@sanger.ac.uk http://www.nature.com/naturecommunications t he escalating burden of cardiovascular and metabolic disease has growing health economics and societal implications. blood lipid levels are measurable indices strongly associated with cardiometabolic disease endpoints. genetic association studies have in the last few years substantially enhanced our understanding of factors underlying traits of high clinical importance, such as body mass index, lipid levels and blood pressure. population isolates have been proposed as useful tools in genetic association studies for complex traits, as they are characterized by reduced phenotypic, environmental and genetic heterogeneity . furthermore, isolated populations can empower next generation association studies focusing on rare variation , as trait-associated alleles may have drifted to higher frequency. however, the generalizability of novel associations identified in population isolates has been the subject of debate. variants private to the isolate may be highly relevant for that particular population but of limited transferability to other populations. for example, a null apoc variant, r x, found to be associated with high-density lipoprotein (hdl) and triglycerides levels in the amish was deemed to be population-specific as it was not detected in non-amish individuals of european descent given available data at the time . there is growing empirical evidence that low-frequency and rare variants have an important role in complex human phenotypes. the illumina humanexome beadchip has been developed as a cost-effective tool to study the role of exonic variants and has been successfully applied to the identification of new loci influencing insulin processing and secretion . to identify potentially functional coding variants associated with cardiometabolic-related traits, we perform exome chip genotyping of , individuals from a greek population isolate stemming from anogia and the surrounding mylopotamos villages on crete (helic (hellenic isolated cohorts) manolis (minoan isolates) study, http://www.helic.org). the mylopotamos villages’ residents anecdotally demonstrate high rates of longevity. although cretans have low mortality rates from cardiovascular disease , their serum lipids are comparable to northern europeans , . we find genome-wide significant evidence for association between r x, a functional variant in apoc , and increased hdl and decreased triglycerides levels. r x is rare (o . % frequency) in outbred european populations, whereas its frequency has increased to . % in manolis, which enables discovery of this lipid traits signal at genome-wide significance in a small sample size. our findings additionally address the generalizability of associations discovered in population isolates, as this cardioprotective variant was previously thought to be private to the amish founder population. results exome chip-wide association analysis. in an analysis of hdl levels using exome chip data in , individuals from the manolis study (supplementary table s ), we find genome- wide significant evidence for association with common-frequency positive control variants in cetp (for example, rs , p ¼ . � � , likelihood ratio test), the well-established hdl-associated locus which has accrued robust evidence for association across multiple populations (fig. ; supplementary fig. s ; table ) – . despite the small sample size, we also find genome-wide significant evidence for association with a functional variant in apoc (rs , r x, p ¼ . � � , likelihood ratio test), which explains . % of the trait variance (table ; fig. ). the strongest r x variant association is with decreased triglyceride levels (p ¼ . � � , likelihood ratio test) and explains . % of trait variance (table ; fig. ). this clear cardioprotective effect is recapitulated in the strong association with high hdl as a dichotomized trait defined as mg dl� (ref. ) (p ¼ . � � ; table , likelihood ratio – lo g p − va lu e chromosome apoc cetp figure | manhattan plot for hdl in manolis. genome-wide statistical association evidence for hdl in manolis. p-values are generated from the likelihood ratio test, as calculated by gemma software. article nature communications | doi: . /ncomms nature communications | : | doi: . /ncomms | www.nature.com/naturecommunications & macmillan publishers limited. all rights reserved. http://www.helic.org http://www.nature.com/naturecommunications test). we observe a trend for the minor allele frequency (maf) to increase with age (supplementary fig. s ). there was no evidence for the association of r x with c-reactive protein (crp) levels, anthropometric, glycaemic and blood pressure traits and no attenuation of the signal was observed when conditioning on smoking status (table ; supplementary table s ). the single nucleotide protein (snp) that led to the original r x discovery in the amish is located in the dscaml gene (rs ) and was highly correlated to r x in that founder population (d ¼ , r ¼ . ). in the manolis cohort, we find that the two variants are poorly correlated (r ¼ . ), whereas only three of the possible four haplotypes are observed (d ¼ , due to low allele frequency). the t allele, which is the derived allele, changes an arginine residue into a stop codon in exon of all three coding transcripts of the apoc gene. the introduction of a premature stop codon at this position means that the mrna transcripts are likely to be subject to non-sense-mediated decay and will not be translated. r x frequency and haplotype analysis. r x has a frequency of . % in the manolis cohort, but is very rare ( . % fre- quency; t: , c: ) in , european-descent exomes from the nhlbi data (exome variant server, nhlbi, http://evs.gs.wa- shington.edu/evs/ (accessed february )) and is not present in the , genome project data . the r x minor allele is table | cetp variants associated with hdl levels in manolis. rs id chromosome position effect allele effect allele frequency beta s.e.* p-valuew rs t . . . . � � rs t . . . . � � rs a . . . . � � rs a . . . . � � rs a . . . . � � *standard error. wp-values are calculated using the likelihood ration test, as calculated by the gemma software. table | distribution of lipid traits by r x genotype. trait cc* ct* effect sizew p-valuez n , — — sex (m:f) : : . ( . , . ) . age (years)y . ( . ) . ( . ) . ( . ) . hdl (mmol l� ) . ( . ) . ( . ) . ( . ) . � � ldl (mmol l� ) . ( . ) . ( . ) � . ( . ) . total cholesterol (mmol l� ) . ( . ) . ( . ) � . ( . ) . triglycerides (mmol l� ) . ( . ) � . ( . ) � . ( . ) . � � high hdl / ( . %) / ( . %) . ( . , . ) . � � low hdl / ( . %) / ( . %) . ( . , . ) . � � *untransformed variables are presented as mean (s.d.) and natural logarithm-transformed variables are presented as median (s.d.). wfor continuous traits beta values (standard error) are reported, whereas for binary traits odds ratios and % confidence intervals are reported. zp-values are calculated using the likelihood ratio test, as calculated by the gemma software. yinverse-normalized for analysis. . . . cc tc a apoc -r x h d l le ve ls ( m m o l l – ) − cc tc b apoc -r x n a tu ra l l o g a ri th m -t ra n sf o rm e d tr ig ly ce ri d e s le ve ls ( m m o l l – ) figure | bean plot of hdl and triglycerides by apoc r x genotype. the y-axis shows the untransformed hdl (a) levels (mmol l� ) and the natural logarithm-transformed triglycerides (b) levels (mmol l� ), respectively. hdl p ¼ . � � and triglycerides p ¼ . � � . p-values are generated from the likelihood ratio test, as calculated by gemma software. nature communications | doi: . /ncomms article nature communications | : | doi: . /ncomms | www.nature.com/naturecommunications & macmillan publishers limited. all rights reserved. http://evs.gs.washington.edu/evs/ http://evs.gs.washington.edu/evs/ http://www.nature.com/naturecommunications carried by out of , whole genome sequenced individuals in the uk k study (http://www.uk k.org) (maf ¼ . %). individuals with the r x variant do not tend to be closely related in manolis, with . % of carrier pairs having r % of alleles identical by descent exome array-wide (supplementary table s ). the age of the r x variant in the general european population is estimated at . kya ( % ci – . kya) , which supports the idea that this variant was present at a low frequency in europe before this cretan isolate was established. to test this, we examined local haplotypes from uk k and find that the four chromosomes with the t (stop) alleles share the same b kb haplotype (b kb-stop-b kb) as we might expect given the frequency of the allele and its age estimated from this frequency. the manolis carriers also all share the same b kb downstream haplotype, which matches b kb of the b kb uk k haplotype, and some share the entire region. we also examined the amish t carrier haplotype data and found that the amish also share almost the same haplotype ( kb- stop- kb) (supplementary fig. s ). these findings suggest that r x has a single recent origin in europe that predates the establishment of the manolis and amish isolates, and that it has risen to a relatively high frequency in the two isolates due to genetic drift. to examine patterns of haplotype sharing around r x more broadly in the manolis cohort, we identified stretches of maximal haplotype identity along chromosome between pairs of samples using an adaptation of the long-range phasing algorithm of kong et al. as expected from an isolated population, haplotype sharing is extensive, such that the median size of maximal haplotype identity around a randomly sampled chromo- somal location is . mb ( . cm). however, around r x, haplotype sharing is no more extensive than elsewhere on the chromosome, nor is it greater among carriers than non-carriers (kolmogorov–smirnov test p ¼ . ; supplementary fig. s ). more- over, there is no evidence for cryptic relatedness, substructure or for any extended correlation between genetic background and carrier status (supplementary fig. s ), as excess relatedness decays exponentially away from the risk locus as expected. around r x, the profile of maximal haplotype identity is well-approximated by a model in which the common ancestor to the genealogical nearest neighbour occurred an average of . generations ago. this provides a lower bound for the age of the mutation at c. years ago assuming a generation time of years. interestingly, around other variants of the same frequency, we find greater levels of cryptic relatedness (supplementary fig. s ), suggesting that these are typically more recent. apoc variant associations with lipid traits. functional coding mutations in the apoc locus have previously been implicated in several traits related to triglycerides and hdl cholesterol levels – . variants in the promoter of this gene have also been associated with hypertriglyceridemia , , changes in response to insulin regulation , , and in the case of rs , with low serum apoc levels and longevity . we find no evidence for association between rs and hdl level in the manolis cohort (p ¼ . , likelihood ratio test). a large-scale meta-analysis of genome-wide association studies across b , individuals has also identified robust associa- tions between a common-frequency variant (rs ) . kb upstream of apoc and hdl and triglycerides levels . we find little evidence for linkage disequilibrium between rs and r x (r ¼ . , d ¼ . ) in the manolis cohort (supplementary table s ), indicating that these are two distinct signals within the same locus. this is further supported by conditional analysis (supplementary table s ). discussion in this study, we examine a newly characterized population isolate from crete in greece. our findings in the greek isolate demonstrate the utility of the exome array in the discovery of low-frequency variant associations in traits of medical relevance. we find r x to be associated with increased hdl and reduced triglycerides levels at genome-wide significance. this work provides insights into the allelic architecture of the apoc locus, in which common variants have also been found to be associated with lipid traits by genome-wide association study (for example, teslovich et al. ) and highlights the dramatic gains in power that can be afforded by studying population isolates. the r x functional rare variant has risen in frequency to b % in this population, enabling discovery of this lipid traits signal at genome-wide significance in a small sample size. the equivalent sample size needed to achieve % power to detect the observed effect size in an outbred european population would be , . our findings additionally address the generalizability of associations discovered in population isolates both at the gene and at the variant level. the r x variant has also risen in frequency and shows association with hdl in the amish founder population . a summary statistic-based meta-analysis across the manolis and amish population isolates for r x leads to a combined p-value of . � � , achieved with a total sample size of b , . apoc r x constitutes to our knowledge the first known example of a clinically important variant that was previously thought to be private to a population but which has in fact drifted in frequency in two independent population isolates and is strongly associated with traits of high clinical relevance. next generation association studies in search of low frequency and rare variants associated with complex disease can be greatly assisted by the study of population isolates. methods anogia population history. anogia is a geographically isolated mountainous village on the island of crete, greece (population size , ). it is located on mount idi and is part of the municipality of mylopotamos, which includes further isolated villages. the name anogia means ‘upper earth’ and refers to the altitude at which the village is located ( m). the residents of anogia demonstrate characteristic cretan resolve, great pride in their land and uphold ancient traditions. in , the settlement numbered citizens. anogia is historically renowned for its residents’ resistance to conquerors. the village has been built three times, as it was burnt down twice by the turks (in and ) and once by the germans (in ). the local dialect is still strongly influenced by the ancient dorian language. helic cohort collection. the helic-manolis collection focuses on anogia and surrounding mylopotamos villages. recruitment of this population-based sample was primarily carried out at the village medical centres. all individuals were older than years and had to have at least one parent from the mylopotamos area. the study includes biological sample collection for dna extraction and lab- based blood measurements, and interview-based questionnaire filling. the phe- notypes collected include anthropometric and biometric measurements, clinical evaluation data, biochemical and haematological profiles, self-reported medical history, demographic, socioeconomic and lifestyle information. biochemical measurements were obtained using enzymatic colorimetric assays and included glucose (hexokinase method), total cholesterol (cholesterol oxidase – phenol aminophenazone method), hdl-cholesterol, triglycerides (glycerol- -phosphate oxidase-phenol aminophenazone) and iron. insulin and ferritin were measured via chemiluminescence and crp through an immunoturbidimetric method. low-density lipoprotein (ldl)-cholesterol levels were calculated according to friedewald equation . the study was approved by the harokopio university bioethics committee, and informed consent was obtained from human subjects. genotyping and qc. samples were genotyped using the infinium humanexome and genotypes were called using illumina genome studio gencall followed by zcall . we undertook a staged quality control (qc) approach; briefly, we performed an initial prefilter on the gencalled data excluding samples and variants that had call rate o %. we carried out a pre-zcall sample qc, excluding samples that had o % call rate or that were heterozygosity outliers based on the distribution at two different maf thresholds (maf o % and maf z %). we also excluded samples that were gender discordant and samples that had genotype discordances when we compared these exome chip genotypes to genome-wide article nature communications | doi: . /ncomms nature communications | : | doi: . /ncomms | www.nature.com/naturecommunications & macmillan publishers limited. all rights reserved. http://www.uk k.org http://www.nature.com/naturecommunications genotypes that were established previously using the illumina omniexpress platform. no variant exclusions were made at this stage because zcall performs variant qc (which includes call rate %, maf %, hardy weinberg equilibrium (hwe) o . ). following zcall, we performed variant qc using the gencalled genotypes (call rate o %, hwe po . , cluster separation score o . ). sample qc was then carried out on the zcall data excluding samples that were ethnicity outliers or duplicates, that had a call rate o %, or that were heterozygosity outliers, performed at two different maf thresholds (maf o % and maf z %). we excluded variants with a zcall call rate o %. we applied a final sample qc step examining again the multidimensional scaling results, maf o % heterozygosity distribution in combination with the number of singletons per sample, and excluded combined visual outliers and any samples that had a minor allele count of for variants. we also applied a final hwe step excluding variants with hwe po . . a total of , samples and , variants that were not monomorphic passed qc (supplementary table s ). trait transformations and analysis. trait values were considered to be outliers if they were five standard deviations away from the average value, and were removed from the analysis. also, ldl values were excluded for individuals with triglycerides level of mg dl� . all phenotypes were analysed directly, apart from trigly- cerides, insulin, crp and ferritin, which were normalized using the natural loga- rithm transformation. age was inverse normalized. an individual was considered to have high hdl if hdl was over or equal to mg dl� , whereas low hdl was defined as hdl o mg dl� . we performed trait association analysis using gemma which incorporates a kinship matrix to account for the relatedness between individuals. p-values were produced using the likelihood ratio test. the kinship matrix was generated using exome array genotype data. for hdl and triglycerides, we performed a three-way meta-analysis using the fisher’s method across the manolis data and the results reported by pollin et al. ( old order amish individuals from the heredity and phenotype intervention heart study, and amish individuals from the amish family calcification study). haplotype analysis to estimate the variant age. four individuals in the uk k data set each have one stop allele at r x. we extracted mb haplotypes each side of the variant from these individuals as well as from other randomly chosen individuals from the uk data set using only the sites which overlapped with the manolis data set. they shared the same b kb haplotype (b kb-stop- b kb) as we might expect given the frequency of the allele and its age estimated from this frequency ( . kya, % ci – . kya). we then constructed haplotypes for the chromosomes with the stop allele in manolis in the same way. one major inferred ancestral haplotype (accounting for / , haplotypes ( . %) in / , individuals ( . %) of the amish cohort) with snps from illumina humanexome beadchip data was compared with the shared haplotype between uk k and manolis. forty-five snps were shared among the three data sets. the amish haplotype shares at least kb ( kb-stop- kb, , , – , , ) with the helic and uk k haplotypes. there are no snps in the amish data between , , and , , where the helic-uk k shared haplotype ends, and the next snp available in the amish data shows that the haplotype is different from the uk k haplotype. the shared haplotype between the amish and helic-uk k may extend some way beyond , , . identification of maximal haplotype sharing. to identify stretches of extended haplotype sharing between individuals, we used a modification of the long-range phasing method of kong et al. briefly, for each target individual, we identify a pair of other individuals from the sample as surrogate parents, such that the target individual’s genotype is compatible with mendelian transmission from the pseudo- parents. we allow for a low level of genotyping error (here %) and recombination (here proportional to the fine-scale hapmap genetic map , such that between any pair of snps, one of the pseudo-parents can change). we find the most likely parent-pair path using the viterbi algorithm and reconstruct shared haplotype lengths. informally, the algorithm can be thought of as attempting to find, for each of an individual’s two chromosomes, those other samples that contain genealogical nearest neighbours. in isolated populations, the method can be used to detect cryptic relatedness and stratification within a sample. the analysis was restricted to those with low levels of relatedness (p̂o . ; individuals of whom are carriers for the risk variant; results are similar with p̂o . ). references . peltonen, l., palotie, a. & lange, k. use of population isolates for mapping complex traits. nat. rev. genet. , – ( ). . zeggini, e. next-generation association studies for complex traits. nat. genet. , – ( ). . pollin, t. i. et al. a null mutation in human apoc confers a favorable plasma lipid profile and apparent cardioprotection. science , – ( ). . huyghe, j. r. et al. exome array analysis identifies new loci and low-frequency variants influencing insulin processing and secretion. nat. genet. , – ( ). . keys, a. coronary heart disease in seven countries. circulation , – ( ). . menotti, a. et al. food intake patterns and -year mortality from coronary heart disease: cross-cultural correlations in the seven countries study. the seven countries study research group. eur. j. epidemiol. , – ( ). . renaud, s. et al. cretan mediterranean diet for prevention of coronary heart disease. am. j. clin. nutr. , s– s ( ). . aulchenko, y. s. et al. loci influencing lipid levels and coronary heart disease risk in european population cohorts. nat. genet. , – ( ). . kathiresan, s. et al. common variants at loci contribute to polygenic dyslipidemia. nat. genet. , – ( ). . ridker, p. m. et al. polymorphism in the cetp gene region, hdl cholesterol, and risk of future myocardial infarction: genomewide analysis among initially healthy women from the women’s genome health study. circ. cardiovasc. genet. , – ( ). . smith, e. n. et al. longitudinal genome-wide association of cardiovascular disease risk factors in the bogalusa heart study. plos genet. 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hashimoto, r. k., ogura, t., hiraga, s. & uzawa, h. molecular cloning of a human apoc-iii variant: thr —ala mutation prevents o-glycosylation. j. lipid. res. , – ( ). . pullinger, c. r. et al. a novel apolipoprotein c-iii variant, apoc-iii(gln — lys), associated with moderate hypertriglyceridemia in a large kindred of mexican origin. j. lipid. res. , – ( ). . von eckardstein, a. et al. apolipoprotein c-iii(lys —glu). identification of an apolipoprotein c-iii variant in a family with hyperalphalipoproteinemia. j. clin. invest. , – ( ). . xu, c. f. et al. association between genetic variation at the apo ai-ciii-aiv gene cluster and familial combined hyperlipidaemia. clin. genet. , – ( ). . dammerman, m., sandkuijl, l. a., halaas, j. l., chung, w. & breslow, j. l. an apolipoprotein ciii haplotype protective against hypertriglyceridemia is specified by promoter and ’ untranslated region polymorphisms. proc. natl acad. sci. usa , – ( ). . talmud, p. j. et al. relative contribution of variation within the apoc /a /a gene cluster in determining plasma triglycerides. hum. mol. genet. , – ( ). . li, w. w. et al. common genetic variation in the promoter of the human apo ciii gene abolishes regulation by insulin and may contribute to hypertriglyceridemia. j. clin. invest. , – ( ). . waterworth, d. m. et al. variants in the apoc promoter insulin responsive element modulate insulin secretion and lipids in middle-aged men. biochim. biophys. acta. , – ( ). . atzmon, g. et al. lipoprotein genotype and conserved pathway for exceptional longevity in humans. plos biol. , e ( ). . friedewald, w. t., levy, r. i. & fredrickson, d. s. estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. clin. chem. , – ( ). . goldstein, j. i. et al. zcall: a rare variant caller for array-based genotyping: genetics and population analysis. bioinformatics , – ( ). . zhou, x. & stephens, m. genome-wide efficient mixed-model analysis for association studies. nat. genet. , – ( ). . altshuler, d. m. et al. integrating common and rare genetic variation in diverse human populations. nature , – ( ). acknowledgements this work was funded by the wellcome trust ( ) and the european research council (erc- -stg -sepi). the amish study was supported by the following grants: r hl , nih u hl , nih u hl , nih p dk and nih k hl . the manolis cohort is named in honour of manolis nature communications | doi: . /ncomms article nature communications | : | doi: . /ncomms | www.nature.com/naturecommunications & macmillan publishers limited. all rights reserved. http://www.nature.com/naturecommunications giannakakis, – . we thank the residents of anogia and surrounding mylopo- tamos villages for taking part. the helic study (www.helic.org) has been supported by many individuals who have contributed to sample collection (including olina balafouti, christina batzaki, georgios daskalakis, eleni emmanouil, chrisoula giannakaki, mar- garita giannakopoulou, anastasia kaparou, vasiliki kariakli, stella koinaki, dimitra kokori, maria konidari, hara koundouraki, dimitris koutoukidis, eirini mamalaki, eirini mpamiaki, maria tsoukana, dimitra tzakou, katerina vosdogianni and niovi xenaki), data entry (thanos antonos, dimitra papagrigoriou and betty spiliopoulou), sample logistics (sarah edkins and emma gray), genotyping (robert andrews, hannah blackburn, doug simpkin and siobhan whitehead), research administration (anja kolb- kokocinski and carol smee) and informatics (martin pollard and josh randall). we would also like to thank klaudia walter and dawn muddyman for their assistance with accessing uk k data. this study makes use of data generated by the uk k con- sortium, derived from samples from uk k_cohorts_twinsuk (the twinsuk cohort) and uk k_cohort_alspac (the avon longitudinal study of parents and children). a full list of the investigators who contributed to the generation of the data is available from www.uk k.org. funding for uk k was provided by the wellcome trust under award wt . author contributions cohort collection: g.d., a.-e.f., c.k., e.t. and e.z. phenotype cleaning: a.-e.f., k.h., a.m., n.w.r., i.t. and e.z. bioinformatics: g.r.s.r., l.s., i.t. and e.z. genotype data processing and cleaning: k.h., a.m., k.p., n.w.r., j.s., l.s., i.t. and e.z. genotype– phenotype association testing: l.s., i.t. and e.z. uk k haplotype analyses: y.c., uk k consortium, chris tyler-smith, yali xue. amish haplotype analysis: t.i.p., j.r.o’c., l.m.y.-a. and y.x. helic haplotype sharing: g.m., l.m. and d.k.x. manu- script drafting: g.r.s.r., l.s., i.t., d.k.x., y.x. and e.z. additional information accession codes: the helic-manolis study genotype data have been deposited to the european genome-phenome archive (ega) under the accession code egas . uk k sequence data have been deposited to the ega under the accession codes egas and egas . supplementary information accompanies this paper at http://www.nature.com/ naturecommunications competing financial interests: the authors declare no competing financial interests. reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ how to cite this article: tachmazidou, i. et al. a rare functional cardioprotective apoc variant has risen in frequency in distinct population isolates. nat. commun. : doi: . /ncomms ( ). this article is licensed under a creative commons attribution . unported licence. to view a copy of this licence visit http:// creativecommons.org/licenses/by/ . /. article nature communications | doi: . /ncomms nature communications | : | doi: . /ncomms | www.nature.com/naturecommunications & macmillan publishers limited. all rights reserved. www.helic.org www.uk k.org http://www.nature.com/naturecommunications http://www.nature.com/naturecommunications http://npg.nature.com/reprintsandpermissions/ http://npg.nature.com/reprintsandpermissions/ http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / http://www.nature.com/naturecommunications 	a rare functional cardioprotective apoc variant has risen in frequency in distinct population isolates 	introduction 	results 	exome chip-wide association analysis 	r x frequency and haplotype analysis 	apoc variant associations with lipid traits 	discussion 	methods 	anogia population history 	helic cohort collection 	genotyping and qc 	trait transformations and analysis 	haplotype analysis to estimate the variant age 	identification of maximal haplotype sharing 	additional information 	acknowledgements 	references doi:** letters to the editor molecular studies in the prader-willi and angelman syn- usually first evident in the pelvic girdle and then spread- drome: an overview. am j med genet : – ing to the upper limbs while sparing facial muscles. on- kuwano a, mutirangura a, dittrich b, buiting k, horsthemke set of symptoms is variable (mean age years), and b, saitoh s, niikawa n, et al ( ) molecular dissection creatine kinase (ck) levels are elevated from early in- of the prader-willi/angelman syndrome region ( q - ) fancy and remain elevated until the individual is well by yac cloning and fish analysis. hum mol genet : – past this age (jackson and strehler ). affected indi- viduals are often wheelchair-bound – years afterlakich d, kazazian hh, antonarakis s, gitschier j ( ) the onset of symptoms. there is variability in the age ofinversions disrupting the factor viii gene are a common death, and most individuals die in middle age.cause of severe haemophilia a. nat genet : – the gene for lgmd a was first linked to chromo-lehrman ma, goldstein jl, russel dw, brown ms ( ) duplication of seven exons in ldl receptor gene caused by some by beckmann et al. ( ). allamand et al. alu-alu recombinatiion in a subject with familial hypercho- ( ) narrowed the region to q . -q . , using lesterolemia. cell : – large kindreds from the isle of la réunion and the marcus s, hellegren d, lambert b, fallstrom sp, wahlstrom northern indiana amish. the muscle-specific calcium- j ( ) duplication in the hypoxanthine phosphoribosyl- activated neutral protease or calpain (canp ) gene, transferase gene caused by alu-alu recombination in a pa- a possible candidate gene in the q . -q . region, tient with lesch nyhan syndrome. hum genet : – was examined by richard et al. ( ). fifteen different nathans j, piantanida tp, eddy rl, shows tb, hogness ds mutations, including missense, splice-site, frameshift,( ) molecular genetics of inherited variation in human and nonsense mutations, were identified in lgmd acolor vision. science : – patients, and many others have subsequently been iden-pentao l, wise ca, chinault ac, patel pi, lupski jr ( ) tified. since the affected patients in la réunion belongcharcot-marie-tooth type a duplication appears to arise from recombination at repeat sequences flanking the . mb to a genetic isolate presumed to derive from a single monomer unit. nat genet : – ancestor who immigrated to the island during the late reiter lt, murakami t, koeuth t, pentao l, muzny d, gibbs s, it was expected that all affected patients from ra, lupski jr ( ) a recombination hotspot responsible la réunion would have the same lgmd a mutation. for two inherited peripheral neuropathies is located near a paradoxically, six different mutations were identified. mariner transposon-like element. nat genet : – this paradox led the investigators to propose digenic robinson wp, bottani a, yagang x, balakrishnan j, binkert inheritance, in which the founder effect is due to an f, mächler m, prader a, et al ( ) molecular, cytogenetic, as-yet-unidentified modulating gene (either nuclear orand clinical investigation of prader-willi syndrome patients. mitochondrial) that permits mutations in canp to ex-am j hum genet : – press lgmd a. this hypothesis does not require therobinson wp, lalande m ( ) sex-specific meiotic recom- presence of multiple mutations, since the genetic princi-bination in the prader-willi/angelman syndrome imprinted region. hum mol genet : – ples of digenic inheritance should apply to all popula- rossiter jp, young m, kimberland ml, hutter p, ketterling tions with lgmd caused by calpain- mutations. rp, gitschier j, horst j, et al ( ) factor viii gene inver- in the amish of northern indiana, richard et al. sions causing severe hemophilia a originate almost exclu- ( ) identified a single mutation in canp sively in male germ cells. hum mol genet : – (cggrcag, r q) in a homozygous state in affected weatherall dj, clegg jb, higgs dr, wood wg ( ) the patients. the authors speculated that the complete pene- hemoglobinopathies. in: scriver cr, beaudet al, sly ws, trance of this disease in the amish and in the other valle d (eds) the metabolic and molecular bases of inherited lgmd a pedigrees might also be under the control of adisease, th ed. mcgraw-hill, new york, pp – second locus. one expectation of the digenic hypothesis address for correspondence and reprints: dr. david h. ledbetter, center for would be that some individuals homozygous for the mu- medical genetics, the university of chicago, east th street, room r , tation would be clinically unaffected (i.e., ck is normalchicago, il - . e-mail: dhl@genetics.uchicago.edu � by the american society of human genetics. all rights reserved. and there are no physical findings suggestive of lgmd). - / / - $ . because of the possible implications in genetic testing and counseling, we analyzed dna samples from amish individuals in one northern indiana county foram. j. hum. genet. : – , the presence of the r q mutation, looking for evi- dna studies of limb-girdle muscular dystrophy dence of phenotypically normal r q homozygotes. type a in the amish exclude a modifying we initiated the countywide screen by first identifying mitochondrial gene and show no evidence for a carrier couples. appropriate informed consent was ob- modifying nuclear gene tained from all individuals. in order to identify r q to the editor: carriers in this population, we specifically approached members of previously studied nuclear lgmd alimb-girdle muscular dystrophy type a (lgmd a) is characterized by slowly progressive muscle weakness, families from this county. we obtained blood samples / a d$$jy - - : : ajhga uc-ajhg letters to the editor table from spouses of already tested siblings of affected indi- viduals and from siblings of known carriers and their results of r q-mutation screening in northern indiana siblings’ spouses, because within this group there was amish couples an increased likelihood of finding couples in which both matingsa no. of couplesmembers were carriers. when carriers were identified outside this group, their siblings and their siblings’ /// and /// bspouses were genotyped. for couples in which both indi- /// and spouse not tested c viduals were heterozygous for r q, blood samples /// and // were requested from their children, for dna studies (fig. // and spouse not tested ( deceased) // and // ) and ck analysis. previous studies (c. e. jackson, obligate // and // dunpublished data) have shown that nonpaternity is rare /// and / in the amish. all children of couples in which both parents were homozygous wild type were assumed to a a plus sign (/) denotes wild type; and a minus sign ( ) denotes be homozygous wild type. the results are summarized r q mutation. b in couples the carrier status of both partners was presumedin table . an exact gene frequency in this particular to be ///, on the basis of the /// status of both parents of eachamish isolate is not known at this time. individual.in addition, we searched for evidence of possible mod- c in individuals the carrier status of one of the partners was ifying mitochondrial effects by studying maternal lin- presumed to be ///, on the basis of the /// status of both parents eages and mtdna haplotypes. genealogical analyses of of that individual. d original couples with known affected children.lgmd patients with the r q mutation embedded in the same haplotype revealed maternal lineages for the northern indiana and the three pennsylvania amish sibships (traceable to ancestors born – years vania lineages and three of the northern indiana lineages covering ( %) of sibships, the mtdna analysesago). this suggests that the mutation came from the same founder. samples from of these lineages were revealed four separate previously known northern euro- pean haplogroups. these four haplogroups are widelyscreened for mtdna restriction-site polymorphisms characteristic of previously identified caucasian groups dispersed over the caucasian phylogenetic tree and are differentiated from each other. hence, any mtdna mu-of related haplotypes (haplogroups). the restriction analyses were conducted on mtdna pcr products, as tation of relevance to the expression of the phenotype would have to have arisen independently in each of thedescribed by torroni et al. ( ). in two of the pennsyl- relevant maternal lineages, an unlikely event. these re- sults strongly suggest that the mitochondrial genetic background does not play a major role in the type of lgmd in this population. the failure to identify any r q-homozygous indi- vidual who was phenotypically normal (i.e., in whom ck is normal) is evidence against a segregating modi- fying gene in this population. since the ratio of affected to unaffected siblings is as expected within nuclear fami- lies, the presence of a modifying mitochondrial gene was considered. since all individuals within a sibship would inherit the same mtdna, the presence of a shared ‘‘per- missive’’ mitochondrial gene would cause that disorder to appear as a simple recessive disorder. our data do not exclude a fixed second nuclear locus in the northern indiana amish population, nor do they exclude the pos- sibility of a second locus for digenic inheritance in the réunion population. a widespread permissive genefigure representative family of five offspring from two carrier could be present in the amish population but not inparents in which r q-mutation analysis by allele-specific oligonu- cleotide assay was performed. the first three children are heterozygous the réunion population. similar studies in the réunion for r q. the fourth child is homozygous for r q, and the fifth population could help to resolve the question of digenic child is homozygous wild type. genomic amplification of exon of inheritance. canp was performed as described by richard et al. ( ). the since digenic inheritance is unlikely in the amish pop-allele-specific oligonucleotide primers for r and q are �-tta- ulation, the réunion paradox still needs to be resolved.ccatgcggtacgcaga- � and �-ttaccatgcagtacg- caga- �, respectively (the variant nucleotide is underlined). zlotogora et al. ( ) observed several different muta- / a d$$jy - - : : ajhga uc-ajhg letters to the editor richard i, broux o, allamand v, fougerousse f, chiannilkul-tions in the genes for metachromatic leukodystrophy chai n, bourg n, brenguier l, et al ( ) mutations inand hurler disease in a small geographic location, lower the proteolytic enzyme calpain cause limb-girdle musculargalilee. they suggested that a high mutation rate and dystrophy type a. cell : – selective advantage of the carriers were responsible for torroni a, lott mt, cabell mf, chen y-s, lavergne l, wal-the multiple mutations, and they proposed that a similar lace dc ( ) mtdna and the origin of caucasians: identi- event may have occurred in the réunion population. fication of ancient caucasian-specific haplogroups, one of perhaps there are multiple founders despite the evidence which is prone to a recurrent somatic duplication in the d- of a single common ancestor in the réunion families, loop region. am j hum genet : – or, less likely, perhaps there is in the canp gene a zlotogora j, gieselmann v, bach g ( ) multiple mutations mutation-rate increase due to exogenous (differential ex- in a specific gene in a small geographic area: a common phenomenon? am j hum genet : – posure to mutagens) or endogenous (unequal distribu- tion of mutator genes) factors. finally, perhaps there are address for correspondence and reprints: dr. gerald l. feldman, medicalunknown environmental or genetic factors that influence genetics and birth defects center, west grand boulevard-cfp , detroit,the manifestations of mutations in the gene in the ré- mi . e-mail: glfeldman@pol.net union population. the search for possible modifying *present affiliation: department of pediatrics, meharry medical college, nashville.genetic or environmental factors should continue, since � by the american society of human genetics. all rights reserved.it could disclose both the mechanism of action of mu- - / / - $ . tated canp in lgmd a and, possibly, factors of therapeutic importance (beckmann ). currently, we are offering this amish population carrier testing and genetic counseling, on the basis of both the r q mutation analysis and monogenic autosomal recessive am. j. hum. genet. : – , inheritance for lgmd a. v. m. pratt, ,* c. e. jackson, d. c. wallace, a c t transition in exon of the atp a gene isd. s. gurley, a. feit, and g. l. feldman associated with exon skipping in an occipital horn henry ford hospital, detroit; and emory university syndrome familyschool of medicine, atlanta to the editor: mutations associated with abnormal splicing accountacknowledgement for % – % of all gene mutations (krawczak et al. the authors wish to thank the amish families for their coop- ). mutations leading to abnormal splicing usually eration. dr. delbert l. gratz, of bluffton, oh, provided valu- are located within the donor or acceptor splice sites. able genealogical data to the project. we thank j. double and however, exonic consensus sequences that are impliedg. taylor for their field studies, and we thank k. dziubek for in the splicing process have been described outside theher technical assistance. this work was supported by a grant splice sites (ligtenberg et al. ; steingrimsdottir etfrom the association française contre les myopathies (to al. ). direct study of the genomic dna may notg.l.f. and c.e.j.) and by nih grants hl and ns easily reveal the splicing mutations. however, reverseand a grant from the muscular dystrophy association (all to d.c.w.). transcription (rt) followed by pcr analysis is likely to detect such mutations. in the atp a gene, which encodes a copper-trans- references porting p-type atpase (chelly et al. ; mercer et al. ; vulpe et al. ), splicing mutations are frequentallamand v, broux o, richard i, fougerousse f, chiannilkul- chai n, bourg n, brenguier l, et al ( ) preferential local- in patients with menkes disease (das et al. ; kaler ization of the limb-girdle muscular dystrophy type a gene et al. , ; tümer et al. ) and also are de- in the proximal part of a -cm q . -q . interval. am scribed in patients with occipital horn syndrome (ohs) j hum genet : – (kaler et al. ; das et al. ). ohs, previously beckmann js ( ) the réunion paradox and the digenic known as ‘‘x-linked cutis laxa’’ (mim ), is a model. am j hum genet : – connective-tissue disorder characterized by skin laxity, beckmann js, richard i, hillaire d, broux o, antignac c, hyperextensible joints, skeletal anomalies, including oc- bois e, cann h, et al ( ) a gene for limb-girdle muscular cipital exostoses, and inconstant mild mental retarda-dystrophy maps to chromosome by linkage. cr acad tion (lazoff et al. ; tsukahara et al. ). here,sci iii : – we report an unusual splice mutation, which wasjackson ce, strehler da ( ) limb-girdle muscular dystro- detected by rt-pcr, in the atp a gene of an ohsphy: clinical manifestations and detection of preclinical dis- ease. pediatrics : – family. / a d$$jy - - : : ajhga uc-ajhg proc. nat. acad. sci. usa vol. , no. , pp. - , december immunology specific human b lymphocyte alloantigens linked to hl-a (lymphocyte subpopulations/non-hl-a alloantigens/genetic control) dean l. mann, leslie abelson, pierre henkart, susan d. harris, and d. bernard amos the immunology branch, national cancer institute, bethesda, maryland; and division of microbiology and immunology, duke university medical center, durham, north carolina communicated by jack l. strominger, october , abstract sera, previously found to react specifically with b lymphoid cultured cells, were tested on isolated t and b peripheral blood lymphocytes in a microcytotoxicity assay. studies were performed on lymphocytes obtained from sever- al large amish families. the sera used in these studies were cytotoxic to peripheral blood, b lymphocytes, but not cytotox- ic to t lymphocytes. the antigens detected followed the in- heritance pattern of hl-a haplotypes. the strong linkage disequilibrium with hl-a antigens suggests that genes con- trolling the expression of b lymphocyte antigens are linked to genes controlling hl-a alloantigens. human histocompatibility antigens (hl-a) are controlled by genes in an apparently large complex that has been desig- nated hl- ( ). the major hl-a antigens are readily iden- tified by serologic techniques. there is ample evidence that the expression of other lymphoid cell surface antigens are controlled by genes in this complex ( - ). such antigens have heretofore been defined by mixed lymphocyte reactions and have been designated ld, or lym- phocyte determined antigens. evidence, therefore, indicates that the lymphocyte determined antigens are expressed only on b lymphocytes, in contrast to the serologically defined antigens (hl-a), which are found on both t and b lympho- cytes. several reports have demonstrated serologic detection of single antigens expressed on b cells, one of which appears to be lymphocyte determined antigen ( , ). winchester et al. ( ) and mann et al. ( ) independently reported the detection of antigens specific for human "b" lymphoid tissue cultured cell lines. our observation was made using nonabsorbed multiparous sera containing no an- tibodies against hl-a. these sera were obtained from women of a religous sect, the amish. (this sect, because of religous and cultural preference, live in relatively isolated communities, intermarry, and have large families.) using these sera, we explored the possibility of serologically identi- fying antigens specific for human "b" lymphoid cells in two relatively large families. a number of specific b lymphoid cell antigens apparently genetically linked to hl-a antigens were found. methods and materials sera were prepared from whole blood obtained from multi- parous women and stored frozen (- ) until use. peripher- al blood lymphocytes were separated from other formed blood elements by ficoll-hypaque (bionetics) sedimenta- tion. human "b" lymphocytes were isolated by a technique that takes advantage of the expression of receptors for the fc portion of complexed immunoglobulin on b lymphocytes ( ). the characteristics of the separated cell populations are described in detail elsewhere (henkart and alexander, sub- mitted for publication). in brief, this technique separates the fc positive, ig positive, and complement receptor cell posi- tive cells (adherent population) from those peripheral blood lymphocytes not bearing the above cell surface markers. in this manuscript we are calling the adherent cells b lympho- cytes and the nonadherent, t lymphocytes. the separation and testing procedures are as follows. polystyrene tissue cul- ture flasks were coated with antigen-antibody complexes by incubating their lower surface with fetal calf serum, wash- ing, modifying the absorbed protein with trinitrobenzene sulfonate, washing, and finally incubating with rabbit anti- body against dinitrophenyl (dnp)-bovine serum albumin. (antibodies to dnp and trinitrobenzene sulfonate are cross- reactive.) after min at the plates were again washed and the prepared lymphocytes were added. the cells were allowed to settle for 'a hr at . nonadherent cells (t lym- phocytes) were removed by pouring the supernatant from the plates and centrifugation. the adherent cells were re- moved by adding a solution of mm dnp-l-lysine in phos- phate-buffered saline containing . m edta and incubat- ing for 'a hr at o. seven to twenty percent of the total pop- ulation of -lymphocytes adhered to the antigen-antibody coated plastic surfaces consistent with reported numbers of human peripheral blood "b" lymphocytes. three thousand t or b lymphocytes were added to ul of serum in microtiter plates and incubated for hr at room temperature. the cells were washed once and gl of whole rabbit serum was added to provide a source of complement. (the rabbit serum was absorbed for hr at with a "b" lymphoid cultured cell line in proportions of ml of sera to x cells.) the complement was removed by "flicking" the microti- ter plate. trypan blue containing . % edta was added. cell death was determined by incorporation of trypan blue dye. serologic reactions were performed in triplicate. a posi- tive control was added to each cell preparation. this consist- ed of a human serum containing antibodies to human pe- ripheral blood lymphocytes which by previous determina- tion was cytotoxic to lymphocytes from approximately % of a normal population. background controls consisted of complement plus heat-inactivated sera from male donor red blood cell type ab. each well was scored for numbers of cells incorporating trypan blue (cell death) in the following manner: = - %; = - %; = - %; = - %; = - %; and = - %. serologic reactions were called positive when numbers were at least greater than the complement controls. these controls were generally or and occasionally . those tests in which the complement controls were greater than were not considered in the com- putation of the data. an example of the triplicate scores given to serologic reaction in a father and mother of one of the families studied is presented in table . sera reacting d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. nat. acad. sci. usa ( ) table . cytotoxic reaction of sera to isolated t and b lymphocytes from wife and husband in le bouf family serum designations and test results* family hl-a cell member antigens type c't lot i , t husband w ,w b ' ,w t l [ wife ,w b * i * numerical scores represent % cells killed in cytotoxicity assays. = - %; = - %; = - %; = - %; = - %; and = - %. t complement control. t positive serum control. specifically with b lymphocytes (shown in boxes) from the husband were , , , , , and . those reacting with the wife were , , , , , , and . all re- actions were scored in a similar fashion and are recorded by plus (+) for a positive reaction and negative (-) for nonreac- tive sera. in some instances in each of the families studied, reactions were not present where expected or cytotoxic reac- tions occurred that did not fit the haplotype association. these were considered to be false negative or false positive reactions. the frequency of these reactions was within the error of the test system ( %), which was determined on re- peated testing of one individual. hl-a phenotyping was performed on the lymphocytes from the individuals studied using the dye exclusion technique as described by kostyu et al. ( ). results two families were studied to determine if the serologic re- actions to b lymphocytes were associated with or linked to an hl-a haplotype. these results are summarized in table . a (+) indicates a positive reaction of the sera to b lym- phocytes using the criteria given above and exemplified in table . due to technical problems, the mother in family was not typed for b cell antigens. however, her hl-a type had previously been determined, allowing assignment of b cell reactivity to maternal hl-a haplotypes. nine sera ( , , , , , , , , and ) reacted with b cells from the father and all of the children expressing the hl-a , haplotype. sera and reacted with b cells from those individuals possessing the paternal hl-a , hap- lotype. another , haplotype was inherited from the moth- er by the offspring in this family. the sera ( , , and ) reacting in association with the maternal , haplotype were different from those reacting with cells from offspring possessing the paternal hl-a , haplotype. in family , the serologic reactions again followed a pat- tern indicating hl-a haplotype association. sera , , and reacted with maternal and offspring cells bearing the hl-a , w haplotype. the other maternal haplotype, hl-a , w , was identified in mother and children by the serologic reactions of and . sera and reacted with b lymphocytes from mother and all of the offspring, thus not differentiating the maternal hl-a haplotypes. sera , , , and reacted in association with the pater- nal hl-a w , w haplotype. sera and reacted with b cells from the father and all of the children and thus did not discriminate between the hl-a , and w , w haplotypes. thus, in both families the inheritance of b cell alloantigens closely follows a pattern of linkage disequili- brium with genes controlling the hl-a antigens. discussion there is ample evidence indicating that the serologic reac- tions seen were not detecting hl-a activity. all lympho- cytes (t and b) express hl-a antigens. the sera used in these assays had been previously screened for hl-a reactivi- ty on preparations of peripheral blood lymphocytes and were found to be nonreactive. these studies were performed by a dye exclusion assay (as described and used in the present study). since b cells represent only - % of the total population of peripheral blood lymphocytes, positive reactions to b cells only could have been easily missed. the separation of the b subpopulation from t cells and the ex- clusive reactivity of these sera to the b cells and not t cells indicates non-hl-a reactivity. studies in mice have demonstrated the presence of non- h- antigens, associated with b lymphocytes and designated ia ( ). genes controlling these antigens map between the h- d and the h- k regions of the h- histocompatibility complex. ia-like antigens may be expressed on the peripher- al blood lymphocytes of rhesus monkeys ( ). human b cell antigens may be analogous to the ia antigens in mice. there are several recent reports of the serologic detection of non-hl-a antigens on human peripheral blood lympho- cytes. legrand and dausett ( ) described sera that caused cytotoxicity to a subpopulation of peripheral blood lympho- cytes. the pattern of reactivity did not associate with the in- heritance of hl-a in family studies. van rood et al. ( ) studied a serum by immunofluorescence, cytotoxicity, and inhibition of mixed lymphocyte reactivity. the antigen de- tected by this serum was inherited with hl-a haplotypes and showed an association with the mlr locus in a family where crossover occurred between mlr and the four series immunology: mann et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. nat. acad. sci. usa ( ) table . summary of results of specific b lymphocyte serologic reactions in two families serum designations and test results hl-a family type ez , + + + + + + + + + + + - _ - (father) , er , + + + + + + + + + - - + + + , id , + + + + + + + - + - - + + + , rb , + + + + + + + + + - - + + + , gr , + + + + + + + + + - - + + + , ar , + + + + + + - + + - - - - - ,x le , - - - - - - - _ - + + + + + , wi , - - - - _ - - _ - + + + + + , rt _ - - - - - - - + + + + + , , + + + + + , hl-a haplotype , , (paternal) , (maternal) association family cell type le , - - - + + - - + + + + - _ (father) w ,w rb ,w + + + - - + + - - - - + + (mother) ,w ma w w - + + + + - + + + + + ,w ve w ,w - - - + + + + + + + + + + ,w le w ,w - - - + + + + + + + - + + ,w ab ,w w ,w + + + + + + - + + + fa , + + + + + - + - - - - + + ,w ed , + + + + + - + + + ,w ) w - + - + + - + - - - - + + wi ,w + + + w ,w + + + + + + + - + + + h -a haplotype association ,w , ,w w ,w ,w w ,w ,w of hl- alloantigens. this antigen appeared to be present only on b lymphocytes. the results of the studies presented here expand the ob- servation of van rood and present evidence for the existence of a number of specific b cell antigens. a minimum of seven different b lymphocyte antigens were detected with the sera studied. however, several sera may be detecting more than one antigen. serum reacts with those individuals in fami- ly where the hl-a , haplotype is expressed, and in family this serum reacts with b cells from those individu- als possessing the w , w hl-a haplotype. other sera re- acting in association with these haplotypes do not share this pattern of reactivity. sera and reacted with b cells from those individuals with the , hl-a haplotype in fam- ily but had different reactivity patterns in family . it ap- pears that there are several antibodies detecting different antigens in these sera and that b lymphocytes may express several specific antigens. the large family studies provide evidence for strong link- age disequilibrium of genes controlling the b lymphocyte antigen expression with genes controlling the expression of hl-a. the b cell antigen reported by van rood also appears to be in linkage disequilibrium with hl-a and is probably an antigen responsible for the stimulation seen in mixed lymphocyte reactions. whether or not the sera used in our studies are detecting the antigen or antigens responsible for immunology: mann et al. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , immunology: mann et al. mixed lymphocyte reactivity remains to be determined. pre- liminary results demonstrate blocking of the mixed lympho- cyte reactions by some but not all of the sera. the relation- ship of the antigens described in this report to the lympho- cyte determined (ld) antigens remains to be determined. more precise definition of the numbers of b cell antigen specificities, their allelic association, and genetic mapping is needed. until this is accomplished, we would propose that these antigens be called l-b antigens. . dausset, j. & colombani, j. (eds.) ( ) "who terminology report," in histocompatibility testing (munksgaard, copenhagen), pp. - . . yunis, e. j., krivit, w., reinsmoen, n. & amos, d. b. ( ) nature , - . . plate, j. d. ( ) j. exp. med. , - . proc. nat. acad. sci. usa ( ) . yunis, e. j. & amos, d. b. ( ) proc. nat. acad. sci. usa , - . . legrand, l. & dausset, j. ( ) transplant. proc. , - . . van rood, j. j., van leeuwen, a., keuning, j. j. & termijtelen ( ) transplant. proc. , - . . winchester, r. j., fu, s. m., wernet, p., kunkel, h. g., du- pont, b. & jersild, c. ( ) j. exp. med. , - . . mann, d. l., abelson, l., harris, s. d. & amos, d. b. ( ) j. exp. med. , - . . dickler, h. b. & kunkel, h. b. ( ) j. exp. med. , - . . kostyu, d., amos, d. b., harris, s., pool, p., johnson, a. h., ward, f. e. & yunis, e. j. ( ) tissue antigens , - . . shreffler, d. c., david, c., gotze, d., klein, j., mcdevitt, h. & sachs, d. ( ) immunogenetics , - . . balner, h. & van vreeswijk, w. ( ) transplant. proc. , - . . van rood, j. j., van leeuwen, a., keuning, j. j., van blusse, x. & van oud alba, a. ( ) tissue antigens , - . d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , untitled genetic epidemiology : – ( ) extent and distribution of linkage disequilibrium in the old order amish cristopher v. van hout, � albert m. levin, � evadnie rampersaud, haiqing shen, jef frey r. o’connell, braxton d. mitchell, alan r. shuldiner, , and julie a. douglas y department of human genetics, university of michigan school of medicine, ann arbor, michigan department of medicine, university of maryland school of medicine, baltimore, maryland geriatric research and education clinical center, veterans administration medical center, baltimore, maryland knowledge of the extent and distribution of linkage disequilibrium (ld) is critical to the design and interpretation of gene mapping studies. because the demographic history of each population varies and is often not accurately known, it is necessary to empirically evaluate ld on a population-specific basis. here we present the first genome-wide survey of ld in the old order amish (ooa) of lancaster county pennsylvania, a closed population derived from a modest number of founders. specifically, we present a comparison of ld between ooa individuals and us utah participants in the international hapmap project (abbreviated ceu) using a high-density single nucleotide polymorphism (snp) map. overall, the allele (and haplotype) frequency distributions and ld profiles were remarkably similar between these two populations. for example, the median absolute allele frequency dif ference for autosomal snps was . , with an inter-quartile range of . – . , and for autosomal snps – kb apart with common alleles (minor allele frequencyz . ), the ld measure r was at least . for and % of snp pairs in the ooa and ceu, respectively. moreover, tag snps selected from the hapmap ceu sample captured a substantial portion of the common variation in the ooa (� %) at r z . . these results suggest that the ooa and ceu may share similar ld profiles for other common but untyped snps. thus, in the context of the common variant-common disease hypothesis, genetic variants discovered in gene mapping studies in the ooa may generalize to other populations. genet. epidemiol. : – , . r wiley-liss, inc. key words: single nucleotide polymorphism; population genetics; human genetics; founder population; linkage disequilibrium; haplotypes contract grant sponsor: nih; contract grant numbers: u hl ; r ca . �the first two authors contributed equally to this work. ycorrespondence to: julie a. douglas, department of human genetics, university of michigan, e. catherine st., buhl building, spc ann arbor, mi - . e-mail: jddoug@umich.edu received january ; revised april ; accepted june published online august in wiley interscience (www.interscience.wiley.com). doi: . /gepi. introduction many genetic studies of complex traits and diseases are being conducted in population isolates, including the old order amish (ooa) of lancaster county pennsylvania [ginns et al., ; hsueh et al., ; mitchell et al., , ; streeten et al., ; post et al., ; douglas et al., ; wang et al., ]. whether results from these studies will generalize to other populations is dependent (in part) on the similarity of allele frequencies and patterns of linkage disequilibrium (ld) between populations. to inform future genetic studies of the ooa and facilitate comparisons of findings with other populations, we conducted the first genome-wide survey of ld in the ooa and compared our findings to the international hapmap project [frazer et al., ]. most of the present-day ooa of lancaster county are the descendants of approximately individuals [cross, ] from central western europe who immigrated to the united states in the early eighteenth century [mckusick et al., ]. although recent data indicate that the dif ferences in ld between isolated and cosmopolitan populations for common alleles are modest [bonnen et al., ; service et al., ], the uncertain but unique demographic history of the ooa necessitates empirical evaluation of ld. subjects and methods ooa study subjects were recruited and genotyped (n ) in the course of the heredity and phenotype intervention (hapi) heart study [mitchell et al., ], which was designed to identify gene-environment inter- actions influencing cardiovascular traits. because many closely related individuals were deliberately ascertained, we used a simulated annealing algorithm [douglas and sandefur, ] to select a set of minimally related individuals ( men and women). the median (range) pair-wise kinship coef ficient was . ( . – . ) for the set of vs. . ( . – . ) for the entire sample of . for comparison with the ooa, we also utilized men and women (or unrelated parents) from a us utah r wiley-liss, inc. population with northern and western european ancestry (abbreviated ceu) in the international hapmap project [frazer et al., ]. genotyping and qc methods dna was extracted from whole blood by standard methods as described previously [mitchell et al., ]. the af fymetrix genechips human mapping k array set was used for the comparison of ld patterns in both the ooa and ceu samples. genotype calls were made using a bayesian robust linear model with mahalanobis (brlmm) distance classifier [af fymetrix, ]. genotype data for the ceu sample and corresponding annotation for the platform, including chromosome and genomic posi- tions for all single nucleotide polymorphisms (snps) on the array, were obtained from the af fymetrix website (www.af fymetrix.com). individuals with % missing genotypes, and/or for men % heterozygous genotypes on the x chromosome, were excluded. a subset of autosomal snps ( , ), which were selected to have high information content (minor allele frequency (maf) z . ), low pair-wise ld (maxi- mum r of . ), and coverage across all autosomes (average intermarker spacing of . cm) in the ooa, were used to infer relationships using the maximum likelihood method implemented in relpair [epstein et al., ]. we excluded individuals who had an inferred relation- ship that dif fered from the pedigree relationship with a likelihood ratio greater than . based on these combined criteria, a total of individuals (out of ) were excluded from further analysis. snps were required to satisfy the following quality control criteria in both samples: ( ) r % uncalled genotypes; ( ) r and r mendelian inconsistencies in ooa and ceu samples, respectively, using pedigree diagnostics as implemented in pedcheck [o’connell and weeks, ]; and ( ) hardy weinberg equilibrium p-valuez � by fisher’s exact test [wigginton et al., ] as implemented in haploview [barrett et al., ]. to assess genotyping accuracy, we used duplicate genotype data for of the ooa subjects for whom data from the af fymetrix genome-wide human snp array . (overlap of , snps with af fymetrix genechips human mapping k array set) were also available. only snps with o duplicate inconsistencies were retained for analysis. of the , genotypes that mapped to a single location in the human genome, , failed at least one qc measure in at least one sample. those snps were removed, leaving a total of , autosomal (table i) and , x chromosome (table ai in the appendix) snps. for the snps that passed our quality control criteria, the genotype consistency rate among duplicate pairs was . %. statistical analyses fisher’s exact test was used to compare allele frequency distributions between the ooa and ceu. for common snps (mafz . ) on the same chromosome and within mb of each other, we used the expectation-maximiza- tion (em) algorithm to obtain maximum likelihood estimates of two-snp haplotype frequencies and mea- sured pair-wise ld by the r and d statistics [lewontin, ]. based on common snps, we also identified haplotype blocks in the ceu using an extension of the four-gamete rule [wang et al., ] and estimated haplotype frequencies in both the ceu and ooa using the em algorithm with a partition-ligation method [qin et al., ] for blocks with snps as implemen- ted in haploview [barrett et al., ]. for each sample, we then calculated and compared the ef fective number of haplotypes in each block, i.e., (spi )� , where pi is the frequency of the ith haplotype in the block. as a measure of redundancy, we identified the number of snps (or proxies) that were in strong ld with each snp at various thresholds of r in each sample. to evaluate the extent to which snps selected to tag variation in the ceu capture common variation in the ooa, we selected common tag snps in the ceu using the greedy algorithm [carlson et al., ] implemented in haploview [barrett et al., ] such that every unselected snp had an r z . with one or more selected snps. we then calculated r between the tag snps and the remaining ‘‘non-tagged’’ but typed snps in the ooa. unless specified otherwise, all analyses were carried out using a combination of in-house r, perl, and c programs. results for the , snps that passed qc, mean hetero- zygosity was . and . for the autosomes in the ooa and ceu, respectively, and . and . for the x chromosome. the slightly lower heterozygosity in the ooa reflects the larger number of monomorphic snps in the ooa relative to the ceu, e.g., , vs. , for the autosomes (table i). among all snps that table i. summary of autosomal snps ooa ceu overlap total genotyped , , , duplicate inconsistencya , na na % missing datab , , , mendelian inconsistenciesb,c , , po � for hwe testd passed qc filtere , , , passed qc in both ooa and ceu monomorphicd , , , polymorphicd mafz . , , , mafz . , , , mafz . , , , ooa, old order amish; ceu, us utah residents from hapmap; maf, minor allele frequency; snps that failed a qc measure in either sample were excluded from further analysis, and snps with mafz . passing qc in both samples (n , ) were used for ld analysis. abased on the ooa individuals who were also genotyped on the af fymetrix . array; snps with more than one duplicated genotype discrepancy were excluded. bbased on ooa and ceu individuals ( trios). csnps with and mendelian inconsistencies in ooa and ceu, respectively. dbased on unrelated individuals ( men and women) from each sample. esnps may fail qc in more than one way, so rows do not sum to the subtotal passing qc. linkage disequilibrium in old order amish genet. epidemiol. were polymorphic in at least one sample, the median absolute allele frequency dif ference was . for the autosomes and . for the x chromosome. at p-valueo � , ooa and ceu allele frequencies were significantly dif ferent for autosomal and x chromosome snps. the percentage of snp pairs within mb of each other and between which strong ld was observed was remarkably similar between the ooa and ceu for the autosomes (table ii) and the x chromosome (table aii in the appendix). for example, for autosomal snps at an inter-marker distance of o kb, no evidence of recombination (d ) was observed for and % of snp pairs, perfect ld (r ) was observed for and % of snp pairs, and useful ld (r z . ) was observed for and % of snp pairs in the ooa and ceu, respectively. based on the ceu sample, we identi- fied , autosomal haplotype blocks, with a median of three snps per block and an inter-quartile range of [ , ]. among all autosomal blocks, the median ef fective number of haplotypes (ne) was . and . in the ooa and ceu, respectively, and the median of the dif ferences in ne (ceu minus ooa) per block was . , with an inter-quartile range of � . to . , suggesting modestly greater haplo- type diversity in the ceu. results based on haplotype blocks defined in the ooa did not qualitatively dif fer from those based on blocks defined in the ceu (data not shown). of common autosomal snps, and % had at least one proxy at r z . and and % had at least one perfect proxy (r ) in the ooa and ceu, respectively, indicating that fewer independent snps are required to represent variation in the ooa relative to the ceu. at r z . , , of , common snps in the ceu were selected as tag snps and captured � % of the ‘‘non- tagged’’ snps in ooa, suggesting that snps selected to tag common variation in the ceu capture much of the same variation in the ooa. snps not captured by the ceu tag snps tended to be of lower maf (data not shown). results for the x chromosome were qualitatively similar. discussion in general, we found a high degree of similarity in allele frequencies and ld patterns in the ooa and ceu samples. allele frequencies were not significantly dif ferent between the ooa and ceu for % of snps. based on common snps, which comprised and % of autosomal snps in the ooa and ceu, respectively, the distribution and extent of ld were remarkably similar between these two samples. these data are consistent with previous theoretical predictions [kruglyak, ; pritchard and przeworski, ] and recent empirical data [bonnen et al., ; service et al., ; navarro et al., ; thompson et al., ], all of which point to modest dif ferences in ld between isolated and cosmopolitan populations for common alleles. the situation for rare alleles, however, is likely to be dif ferent as has been demonstrated in applications of ld mapping for mono- genic diseases and traits. demographic and historical information indicate that the ooa were founded relatively recently (� – generations ago) by a modest number of individuals (several hundred) and then expanded rapidly to a current census population size exceeding , [lancaster county amish, ]. though the precise demographic details are unknown, it is apparent that the number of founders and rate of growth were sufficient and that the subsequent isolation of the ooa was too short for genetic drift and/or recombination to have meaningfully altered the common allele or haplotype frequency spectrum. our recent study of variation on the y chromosome supports these observa- tions in that much of the diversity observed in non-isolated populations of similar ancestry is present in the ooa [pollin et al., ]. it appears that inbreeding due to the finite population size of the ooa was also insufficient to meaningfully alter the allele frequency distribution or extent of ld. based on the ooa individuals included in our analyses, the average inbreeding coef ficient f [wright, ] was . (range of . – . ), which is too weak to generate substantial differences in ld relative to a non- isolated population [hill and robertson, ]. owing to similar allele frequencies and ld patterns in the ooa and ceu, ceu-derived tag snps performed well in capturing common variation in the ooa, consistent with previous studies in other samples of european ancestry, including those from isolated popula- tions [willer et al., ; service et al., ]. these results suggest that the ooa and ceu samples may also share similar ld profiles for other common but untyped snps. thus, findings from gene mapping studies in the ooa may generalize to other populations in the context of the common variant-common disease hypothesis. acknowledgments we gratefully acknowledge the amish research clinic staf f, our amish liaisons, and the amish community, whose extraordinary support and cooperation made this study possible. we also thank drs. alejandro schaf fer and richa agarwala at the nih/ncbi for providing the pedigree information and the center for inherited disease research (cidr), nih for providing duplicate genotypes from the af fymetrix genome-wide human snp array . . table ii. percentage of autosomal snp pairsa showing no evidence of recombination (d ), perfect ld (r ), or where useful ld is observed (r z . ) d r r z . inter-snp distance (kb) ooa ceu ooa ceu ooa ceu r – – – – – – , , – , , – , , – , ooa, old order amish (n ); ceu, us utah residents from hapmap (n ). arestricted to snps with minor allele frequency z . in both samples (n , ). van hout et al. genet. epidemiol. references af fymetrix. . brlmm: an improved genotype calling method for the genechip human mapping k array set. 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selection for finnish individuals based on the ceph utah hapmap database. genet epidemiol : – . wright s. . coef ficients of inbreeding and relationship. am nat : – . appendix summary and percentage of x chromosomes are given in tables ai and aii. table ai. summary of x chromosome snps ooa ceu overlap total genotyped , , , duplicate inconsistencya , na na % missing datab mendelian inconsistenciesb,c po - for hwe testd passed qc filtere , , , passed qc in both ooa and ceu monomorphicd , , , polymorphicd mafz . , , , mafz . , , , mafz . , , , ooa, old order amish; ceu, us utah residents from hapmap; maf, minor allele frequency. snps that failed a qc measure in either sample were excluded from further analysis, and snps with mafz . passing qc in both samples (n , ) were used for ld analysis. abased on the ooa individuals who were also genotyped on the affymetrix . array; snps with more than one duplicated genotype discrepancy were excluded. bbased on ooa and ceu individuals ( trios). csnps with and mendelian inconsistencies in ooa and ceu, respectively. dbased on unrelated individuals ( men and women) from each sample. esnps may fail qc in more than one way, so rows do not sum to the subtotal passing qc. table aii. percentage of x chromosome snp pairsa showing no evidence of recombination (d ), perfect ld (r ), or where useful ld is observed (r z . ) d r r z . inter-snp distance (kb) ooa ceu ooa ceu ooa ceu r – – – – – – , , – , , – , , – , ooa, old order amish (n ); ceu, us utah residents from hapmap (n ). arestricted to snps with minor allele frequency z . in both samples (n , ). van hout et al. genet. epidemiol. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ clinical phenotype and loss of the slow skeletal muscle troponin t in three new patients with recessive tnnt nemaline myopathy géraud j, et al. j med genet ; : – . doi: . /jmedgenet- - original research clinical phenotype and loss of the slow skeletal muscle troponin t in three new patients with recessive tnnt nemaline myopathy justine géraud, klaus dieterich, , john rendu , , emmanuelle uro coste, murielle dobrzynski, pascale marcorelle, christine ioos, norma beatriz romero, , eloise baudou, julie brocard, , anne- cécile coville , julien fauré, , michel koenig, raul juntas morales, emmanuelle lacène, , angéline madelaine, , isabelle marty, , henri pegeot, corinne theze, aurore siegfried, mireille cossee , claude cances diagnostics to cite: géraud j, dieterich k, rendu j, et al. j med genet epub ahead of print: [please include day month year]. doi: . / jmedgenet- - for numbered affiliations see end of article. correspondence to dr claude cances, neuropediatric department, toulouse university hospital, toulouse, france; cances. c@ chu- toulouse. fr jg, kd and jr contributed equally. mc and cc contributed equally. received november revised june accepted july © author(s) (or their employer(s)) . re- use permitted under cc by- nc. no commercial re- use. see rights and permissions. published by bmj. abstract background congenital nemaline myopathies are rare pathologies characterised by muscle weakness and rod- shaped inclusions in the muscle fibres. methods using next- generation sequencing, we identified three patients with pathogenic variants in the troponin t type (tnnt ) gene, coding for the troponin t (tnt) skeletal muscle isoform. results the clinical phenotype was similar in all patients, associating hypotonia, orthopaedic deformities and progressive chronic respiratory failure, leading to early death. the anatomopathological phenotype was characterised by a disproportion in the muscle fibre size, endomysial fibrosis and nemaline rods. molecular analyses of tnnt revealed a homozygous deletion of exons and in patient ; a heterozygous nonsense mutation in exon and retention of part of intron in muscle transcripts in patient ; and a homozygous, very early nonsense mutation in patient . western blot analyses confirmed the absence of the tnt protein resulting from these mutations. discussion the clinical and anatomopathological presentations of our patients reinforce the homogeneous character of the phenotype associated with recessive tnnt mutations. previous studies revealed an impact of recessive variants on the tropomyosin- binding affinity of tnt. we report in our patients a complete loss of tnt protein due to open reading frame disruption or to post- translational degradation of tnt. introduction congenital nemaline myopathies (nms) are rare muscle diseases characterised by abnormal muscle tone and the presence of rod- shaped inclusions in the muscle fibres. these rods are sometimes visible with light microscopy and gomori trichrome staining. the rods are located mainly in the sarco- plasm and more rarely inside the nucleus. they are more easily visualised and authenticated by electron microscopy: they are composed of lattice- like filaments, resembling z- streaks, and are often observed within disorganised myofibrillar networks. the location of the rods in the fibre, their ultrastructural appearance and the fibre type that preferentially contains them are all criteria that provide a genotype orientation. for example, the presence of intranuclear rods indicates that the alpha- actin- (acta ) gene has been affected, whereas a preferential location in the centre of the sarcoplasm more strongly suggests involvement of the troponin t type (tnnt ) gene. at least genes are currently known to be involved in nm. they encode the thin filament proteins of the skel- etal muscle sarcomere (nebulin, actin alpha , beta- tropomyosin- , alpha- tropomyosin- , troponin t type , cofilin- , leiomodin- (lmod ), skeletal troponin- and myopalladin), the kelch domain- associated proteins (kbtbd , klhl and klhl ) and a myosin whose function is unknown (myo b). the ryanodine receptor- gene has also been described in nm. genotyping is now easier with next- generation sequencing (ngs) techniques as variants can be detected, including small deletions and insertions and variations in the number of copies. the involvement of the tnnt gene has been exceptionally reported in various populations. troponin t is one of the three subunits of the troponin complex and has been called the ‘slow skeletal muscle isoform of troponin t (tnt)’ because of its presence in slow skeletal muscle fibres. this protein is responsible for the link between the troponin complex and tropomyosin (tm), which helps to regulate muscle contrac- tion. patients with nms related to the mutations in the tnnt gene show phenotypical variability. initially, this gene was implicated in the so- called amish nm, which affects consanguineous amish families, and linked to a nonsense mutation in exon of tnnt . since then, other patients with nm have been described, implicating the tnnt gene with autosomal recessive inheritance: dutch patients from the same family, hispanic patient and palestinian patients from seven different families. more recently, a new hetero- zygous missense mutation of tnnt c. a>t; p.e v was described, with nm inherited in an autosomal dominant manner. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://crossmark.crossref.org/dialog/?doi= . /jmedgenet- - &domain=pdf&date_stamp= - - http://jmg.bmj.com/ géraud j, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics we describe three new patients with nm due to recessive tnnt mutations identified by ngs and try to better specify the phenotypical spectrum and to identify the consequences of mutations on tnnt mrna and protein levels. materials and methods patients were recruited through the french national network of neuromuscular diseases reference centres. patients or parents gave informed consent for the genetic analysis according to french legislation. sequencing sanger sequencing of tnnt coding exons was performed for patient . ngs was performed on extracted dnas using customised designs of myopathy genes, as previously reported. an in- house bioinformatics spreadsheet was implemented for the copy number variation analyses, as previously reported. this was used to analyse the intersample normalised depth of coverage per exon in a given run. cdnas of the tnnt tran- scripts were analysed on mrna extracted from muscle biopsies; the total transcripts were amplified and sequenced after frag- mentation and library preparation (nebnext new england biolabs). the library was then sequenced on a pgm iontorrent platform. transcript sequences and splice junctions were anal- ysed using rnastar software. western blot the amount of skeletal muscle- specific troponin t (stnt) in the muscle sample biopsies was determined by western blot analysis using antibodies directed against stnt (mab- ct ) with tubulin (mab- tub v. . ) as loading protein sample control. muscle homogenates were prepared from frozen muscle biopsy using minilys homogeniser as described previously. twenty micro- grams of muscle homogenates were separated on a % acryl- amide gel (biorad) and transferred on immobilon as described previously. the patients and an age- matched ( . years old) non- affected control sample were tested. results patient this - month- old patient was born full- term after a normal pregnancy to non- consanguineous parents from the basque region of france. she was eutrophic and showed no difficulty in adapting to extrauterine life. no specific clinical signs were noted at birth. she had one older brother. axial and periph- eral hypotonia was observed at months, associated with facial hypomimia, ‘fishmouth’, non- specific painful manifestations and tremor. respiratory insufficiency appeared gradually, with secondary thoracic dystrophy, requiring ventilatory support by nocturnal non- invasive ventilation at the age of months. she had areflexia of the four limbs. at months, she showed axial weakness and inability to hold her head up or maintain a sitting position, contrasting with better distal mobility. asymmetrical cervical stiffness was observed early on, as well as a limitation in hip abduction and knee retraction. a rapidly progressing kyphoscoliosis required treatment with a rigid brace from months onward. failure to thrive prompted the placement of a gastrostomy tube for enteral nutrition at months. her cognitive abilities were normal. death occurred suddenly at months. creatine phosphokinase (cpk) testing, echocardiog- raphy, electroneuromyography and an mri cerebrospinal flow study showed no abnormalities. there was no phrenic palsy. the muscle biopsy performed at months revealed a clear dispro- portion in fibre size, with type i fibres consistently smaller than type ii fibres, the dissolution of the myofibrillar network in the centre of certain fibres, early endomysial fibrosis, and rare vacuolated degenerative fibres. ultrastructural analysis revealed a large number of rods located in the centre of the fibres in areas of sarcomeric disorganisation (figure ). ngs of the exons and exon–intron junctions of the genes involved in congenital myopathies and muscular dystrophies revealed a homozygous deletion of exons – of the tnnt gene (figure ). the bound- aries of the deletion were determined by genomic sequencing (hg chr : g. _ del and nm_ . : c. + _ - del). this deletion, predicted to be in frame, removed part of the functionally important tm- binding site domain (figure ). the family segregation analysis concluded that the child was homozygous for this variation since she had inherited a mutated allele from each of her parents (table ). figure representative frozen sections (a–e) and electronic microscopy images (f–h). gomori trichrome: patient (a): marked fibre size irregularity. rare atrophic fibres with hyaline core- like intracytoplasmic inclusions (white arrows), slight increased endomysial connective tissue. patient (b): numerous rods and endomysial fibrosis. patient (c): rods are visualised in disorganised fibres. atpase ph . : patient (d) and patient (e): type i fibres are dark; type iia fibres are pale, type iib are intermediate stained; type i fibres are irregular and smaller than type ii. no myopathic grouping. the proportion of type i and type ii is preserved. no fibre- type grouping. ultrastructure: patient (f): the sarcomere structure is disrupted in a large circumscribed central area of a muscle fibre (fibre on the right, white arrows showing the outlines of this area). rods are visualised within this area (white arrow heads showed some rods). detail: a typical rod with homogeneous lattice z disk- like structure in continuity with thin filaments. patient (g): numerous electron dense rods in a disorganised area. patient (h): rods are observed throughout the cytoplasm. detail of a typical rod with lattice structure. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ géraud j, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics analysis of the skeletal muscle transcripts of tnnt showed skipping of exons and (figure ). western blot analysis revealed the total absence of the troponin protein. no smaller band corresponding to the truncated protein was visible (figure ). the set of clinical and anatomopathological data was compatible with an implication of this mutation in the patient’s pathology. patient this - month- old child was born at term to non- consanguineous parents. she was eutrophic, showed a good adaptation to extra- uterine life and presented with a congenital torticollis. axial and peripheral hypotonia was observed only at months, predomi- nant in the pelvic and scapular belts and associated with tongue fasciculations and an abolition of osteotendinous reflexes. at months, a rapid onset of dorsal kyphosis was noted, associated with bilateral limitation of hip and knee amplitudes and pectus carinatum at about months. she developed better motor skills in the forearms and legs. there was no cardiomyopathy. language developments and interactions were normal for her age. death occurred at months. the brain mri was normal, as were the cpk values. elec- troneuromyography revealed a myogenic pattern, but some sequences have neurogenic potentials on the anterior leg. the quadriceps muscle biopsy performed at months was in favour of nm. many of the muscle fibres contained clusters of rods distributed over the entire cytoplasm. numerous annular fibres, irregularly sized fibres and endomysial fibrosis were also observed. no correlation between alterations and fibre type could be made, although most of the small fibres were type . ultra- structural analysis revealed numerous muscle fibres with zones of disorganised structure and rods distributed throughout the fibre. autophagic elements were also observed in several muscle fibres (figure ). molecular ngs analysis detected a heterozy- gous nonsense mutation (c. g>t; p. (glu *)) in exon of the tnnt gene, leading to a stop codon on this allele. in addition, the study of tnnt transcripts from muscle biopsy revealed partial intronic retention of the last bases of intron of tnnt , predicted to interrupt the reading frame (figure ). genomic sequencing of intron then revealed a variant c. – c>a predicted to activate a splice- accepting cryptic site (human splicing finder score of . and maxentscan . ), in agreement with the transcript pattern (figure ). the western blot analysis further revealed the absence of the stnt protein in the presence of those two mutations, confirming tnnt gene involvement in the patient’s pathology (figure ). patient this - month- old child was born at weeks and had no difficulty in adapting to extrauterine life. the parents were of north african figure homozygous deletion of exons – of the tnnt gene. integrative genomics viewer visualisation of the deletion identified by ngs in case (i ). precise breakpoint determination was further obtained by sanger sequencing (hg chr : g. _ del ; nm_ . : c. + _ - del). ngs, next- generation sequencing; tnnt , troponin t type . figure tnnt variants identified in patients. patient revealed a homozygous deletion of exons and of the tnnt gene. these two exons encode part of the tm of slow skeletal muscle troponin t. patient two carried the heterozygous nonsense variant c. g>t in exon leading to a predicted stop codon p.(glu *) in tm . through muscle transcript analysis, a second heterozygous variant was identified in this patient in intron , c. – c>a, activating a cryptic splice- accepting site and predicted to lead to intron retention. patient revealed a very early homozygous stop codon p.(glu *). tm , tropomyosin binding site domain; tnnt , troponin t type . table in- house bioinformatics script results for tnnt cnv familial segregation analysis chr start stop region id labels i i i tnnt nm_ – . . . tnnt nm_ – . . . tnnt nm_ – . . . tnnt nm_ – . . . tnnt nm_ – . . . the in- house bioinformatics script for cnv analysis showed a ratio relative coverage of for tnnt exon and exon in i sample (patient ), in agreement with the homozygous deletion, . and . , respectively, in i sample (father), and . and . , respectively, in i sample (mother), in agreement with a heterozygous deletion of these exons in parents. cnv, copy number variation; tnnt , troponin t type . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ géraud j, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics origin and consanguineous. their older daughter is in good health. this patient presented with neonatal hypotonia, predominantly axial and notably facial, with the progressive onset of respiratory failure and laryngomalacia associated with swallowing disorders and nutritional deficiency. hyporeflexia was also associated. alert- ness and social interactions were normal for this age. the patient died at months from a respiratory infection. the results of cpk testing, brain mri, ocular fundus examination and echocardiog- raphy were normal. right diaphragmatic palsy was observed. the muscle biopsy performed at months revealed an nm, with a disproportion in the fibre size: type i fibres were consistently smaller than type ii fibres. the nemaline rods in the type i fibres were visu- alised with gomori trichrome staining and electron microscopy and showed a homogeneous typical grid pattern. histoenzymological staining revealed no endomysial fibrosis or fibre grouping. the proportion between the fibre types was normal (figure ). sanger sequencing identified a homozygous nonsense mutation in the tnnt gene (c. g>t, p. (glu *), leading to a stop codon on both alleles (figure ). the predicted absence of tnt in the skeletal muscle was confirmed by western blot analysis (figure ). discussion all patients with nm who present with recessive mutations in tnnt have a similar clinical phenotype. the major features are generalised hypotonia with delayed motor development contrasting with conserved fine motor skills and more or less significant joint retraction that may affect the hips, knees and shoulders. thoracic dystrophies like progressive pectus cari- natum indicate progressive respiratory failure that requires inva- sive or non- invasive ventilatory support, as reported in a dutch patient and three palestinian patients. the semiology of our patients was consistent with these reports, summarised in table . the presence of tremor noted in the first month of life of patient has been described in all the patients reported in the literature, with the exception of the dutch patients, who presented either a homozygous mutation at the donor splice site of exon of the tnnt gene causing exon skipping in transcripts, or the same heterozygous mutation associated with a deletion of exon . patient presented tongue fasciculations with a myogenic and discreetly neurogenic electromyography: in the absence of dedi- cated electrophysiological exploration, it is, however, difficult to differentiate between neurogenic damage as already described in patients with tnnt mutations electrophysiologically and anap- athomopathologically by abdulhaq et al or tremor also related to the pathology. kyphoscoliosis was present in two of four patients, as in the dutch patients, in the hispanic patient with a nonsense mutation of exon of the tnnt gene and in the palestinian families. this observation particularly reflects the major axial hypotonia of these patients. in five palestinian patients, foetal hypomobility and spinal stiffness were also reported, which were less observed in the amish patients. an interesting observation in one of the dutch patients was the development of a diaphragmatic hernia at years of life. it was described as secondary to atrophy of the diaphragm. this was the figure transcript analyses from muscle biopsies. visualisation on integrative genomics viewer . in grey are represented the coverage of each sequence. the red and blue crescent represent the splice junction. . . transcript patterns from exons to from a healthy control (a) and patient (b) samples. the complete skip of exons and observed in patient is represented, compared with the normal splicing of exons – in the healthy control . . .transcript patterns from exon to from a healthy control (a) and patient (b) samples. two transcripts were identified in patient : one normal splice pattern between exon and (similar to the healthy control) and another transcript with retention of nucleotides of intron . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ géraud j, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics only patient with this complication. patient presented an eleva- tion of the right diaphragmatic dome, indicating possible diaphrag- matic paralysis. the pathophysiology remains unexplained, and this symptom is suggestive of a spinal muscular atrophy with respiratory distress type . finally, feeding difficulties and slow weight gain were frequently found, requiring the placement of a gastrostomy tube. respiratory failure led to the early death of patients in the absence of ventilatory support: before years of age in the amish population and up to years old in the palestinian population. our three patients died early, despite non- invasive ventilatory support. some of the members of a family of ashkenazi jewish origin were found to carry an autosomal dominant mutation, giving rise to a less severe phenotype that emerged between and years of age. the phenotype included hypomimia and slowly progressing prox- imal muscle fatigability, which in some cases progressed to osteoar- ticular deformities, such as pectus carinatum and/or kyphoscoliosis. intellectual, cardiac and respiratory functions were not impaired. survival was not affected. these cases are less severe because of the heterozygosity of the mutation. nm is characterised by the presence of rod- shaped inclusions in the skeletal muscle fibres. according to the muscle biopsies of our three patients, the rods were more often located in type i fibres. in patient , however, the distribution was more homo- geneous throughout the cytoplasm and associated with areas of sarcomeric disorganisation, located more often in the centre of the fibre instead of its periphery. these features were emphasised by johnston et al. in addition, as frequently observed in the series of abdulhaq et al, our three patients showed a dispro- portion in fibre size, with type i fibres consistently smaller than type ii fibres, and early endomysial fibrosis. the proportion of type i and type ii fibres was preserved, as in previous series. for patient , the rods were not visible with gomori trichrome staining but only with electron microscopy. this should be considered in relation to the findings in the series of abdulhaq et al, who reported that the rods were often rare and difficult to detect, with no rods observed in three of seven muscle biopsies. it should be emphasised that the ultrastructural aspect of the rods that we observed was quite typical, with homogeneous and electron- dense z- disk- like lattice structures in continuity with thin filaments, as in all reported cases of nm associated with alterations of the tnnt gene. in particular, no amorphous material was observed within the rod structure, as was described in nm associated with the lmod gene. the pathophysiology of nm linked to mutations of the tnnt gene is not fully understood, but the absence of slow tnt leads to size defect in both slow and fast fibres, which may be the cornerstone of the disease. functional studies performed in recessive cases due to tnnt truncating mutations at ser and leu and to the rna exon internal deletion revealed a unique impact on the tm- binding affinity of tnt. in patient , deleted for exons and , as the deleted tnnt transcripts were detected, the absence of the protein with western blotting suggests a mechanism of post- translational degradation of tnt, as previously described for the glu nonsense mutation. this degradation could be the consequence of a complete lack of interaction with tm. in patient , the nonsense mutation at glu in exon of the tnnt gene, associated in the other allele with a partial intron retention disrupting the open reading frame, resulted in the total absence of tnt . in patient , the homozygous mutation induced a very early stop codon at glu of the protein and consequently the absence of protein synthesis, as confirmed by the western blot study. in conclusion, the three patients reported in this series reinforce the homogeneous character of the phenotype associated with reces- sive forms of tnnt mutations. in addition, functional studies show a complete loss of protein in all three cases, consistent with the recent results obtained on tnnt knockout mice. author affiliations neuropediatric department, university hospital centre toulouse, toulouse, france inserm u , grenoble alpes university hospital, grenoble, france inserm u , cancer research center of toulouse (crct), department of pathology, toulouse university hospital, toulouse, france inserm u , university of grenoble alpes, grenoble, france maternity department, brest university hospital center, brest, france pathology department, brest university hospital, morvan hospital, brest, france neuropediatric department, garches university hospital center, garches, france umrs , cnrs fre , center for research in myology, inserm, cnrs, sorbonne university, upmc university of paris , paris, france myology institute, assistance publique- hôpitaux de paris, pitié-salpêtrière university hospital, paris, france molecular genetics laboratory, lgmr, montpellier university hospital centre, university of montpellier, montpellier, france contributors jg, kd, jr, mc and cc contributed to medical evidence, data collection, article writing and reading. euc, nbr and as contributed to medical evidence, article writing and reading. pm, a- cc, mk and rjm contributed to article writing and reading. md, eb, ci, jb, jf, el, am, hp, im and ct contributed to data collection and scientific expertise. funding this work was supported by the at c association. we are indebted to pr jian- ping jin for providing the skeletal muscle- specific troponin t antibody. competing interests none declared. patient consent for publication not required. ethics approval the study was approved by the ethical guidelines issued by our institutions for clinical studies in compliance with the helsinki declaration (mr : v ). provenance and peer review not commissioned; externally peer reviewed. figure western blot analysis of stnt. muscular biopsy homogenates were realised for the three patients (patients – ) and an age relative control biopsy (ctrl). an antitubulin antibody was used to control the protein amount loading. an anti stnt was used to evaluate the quantity of stnt. compared with the control, the three patients have nearly null expression of stnt. the very faint bands detected in patients’ biopsy most probably correspond to non- specific reactivity of the antibodies but could also reflect cross- reactivity with other tnnt isoforms. they represent less than . % of the tnnt amount in the control biopsy (tnnt /tubulin amount fixed to % in control). stnt, skeletal muscle- specific troponin t; tnnt , troponin t type . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ géraud j, et al. j med genet ; : – . doi: . /jmedgenet- - diagnostics data availability statement all data relevant to the study are included in the article or uploaded as supplementary information. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by- nc . ) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non- commercial. see: http:// creativecommons. org/ licenses/ by- nc/ . /. orcid ids john rendu http:// orcid. org/ - - - anne- cécile coville http:// orcid. org/ - - - mireille cossee http:// orcid. org/ - - - references koy a, ilkovski b, laing n, north k, weis 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failure to thrive + + – + + + + unk peripheral articular retraction + + – + + + + – rigid spine + + – + – – + unk diaphragm atrophy, diaphragmatic hernia – – + – + – – unk thoracic dystrophy + + – + + + + + kyphoscoliosis + + – – + + + + respiratory failure + + + + + + + – ventilatory support niv – niv niv tracheotomy niv niv/tracheotomy – gastrostomy + – + – + – + – death months months months before years old unk unk between . and years old unk tnnt genetic change homozygous deletion of exons – c. g>t p. 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magdelaine c, giuliano f, gras d, haye d, nizon m, magen m, bieth e, cances c. spinal muscular atrophy with respiratory distress type : a multicenter retrospective study. neuromuscul disord ; : – . piteau sj, rossiter jp, smith rg, mackenzie jj. congenital myopathy with cap- like structures and nemaline rods: case report and literature review. pediatr neurol ; : – . fox md, carson vj, feng h- z, lawlor mw, gray jt, brigatti kw, jin j- p, strauss ka. tnnt nemaline myopathy: natural history and therapeutic frontier. hum mol genet ; : – . amarasinghe c, hossain mm, jin j- p. functional basis of three new recessive mutations of slow skeletal muscle troponin t found in non- amish tnnt nemaline myopathies. biochemistry ; : – . wang x, huang q- q, breckenridge mt, chen a, crawford to, morton dh, jin j- p. cellular fate of truncated slow skeletal muscle troponin t produced by glu nonsense mutation in amish nemaline myopathy. j biol chem ; : – . jin j- p, brotto ma, hossain mm, huang q- q, brotto ls, nosek tm, morton dh, crawford to. truncation by glu nonsense mutation results in complete loss of slow skeletal muscle troponin t in a lethal nemaline myopathy. j biol chem ; : – . oki k, wei b, feng h- z, jin j- p. the loss of slow skeletal muscle isoform of troponin t in spindle intrafusal fibres explains the pathophysiology of amish nemaline myopathy. j physiol ; : – . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm e d g e n e t- - o n s e p te m b e r . d o w n lo a d e d fro m http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /s - - - http://dx.doi.org/ . /j.nmd. . . http://dx.doi.org/ . /j.pediatrneurol. . . http://dx.doi.org/ . /hmg/ddy http://dx.doi.org/ . /acs.biochem. b http://dx.doi.org/ . /jbc.m http://dx.doi.org/ . /jbc.m http://dx.doi.org/ . /jp http://jmg.bmj.com/ 	clinical phenotype and loss of the slow skeletal muscle troponin t in three new patients with recessive tnnt nemaline myopathy 	abstract 	introduction 	materials and methods 	sequencing 	western blot 	results 	patient 	patient 	patient 	discussion 	references rare variant apoc r x is associated with cardio-protective profiles in a diverse population-based survey as part of the epidemiologic architecture for genes linked to environment study | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /circgenetics. . corpus id: rare variant apoc r x is associated with cardio-protective profiles in a diverse population-based survey as part of the epidemiologic architecture for genes linked to environment study @article{crawford rareva, title={rare variant apoc r x is associated with cardio-protective profiles in a diverse population-based survey as part of the epidemiologic architecture for genes linked to environment study}, author={d. crawford and l. dumitrescu and r. goodloe and k. brown-gentry and j. boston and b. mcclellan and cara b. sutcliffe and r. wiseman and paxton baker and m. pericak-vance and w. scott and m. allen and ping mayo and n. schnetz-boutaud and h. dilks and j. haines and t. pollin}, journal={circulation: cardiovascular genetics}, year={ }, volume={ }, pages={ – } } 	d. crawford, l. dumitrescu, + authors t. pollin 	published 	biology, medicine 	circulation: cardiovascular genetics background—a founder mutation was recently discovered and described as conferring favorable lipid profiles and reduced subclinical atherosclerotic disease in a pennsylvania amish population. preliminary data have suggested that this null mutation apoc r x (rs ) is rare in the general population. methods and results—to better describe the frequency and lipid profile in the general population, we as part of the population architecture using genomics and epidemiology i study and the… expand view on wolters kluwer ahajournals.org save to library create alert cite launch research feed share this paper citationsbackground citations results citations view all tables and topics from this paper 	 table 	 table 	 table 	cholesterol 	lipoproteins 	triglycerides 	gene frequency 	angina pectoris, variant citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency frequency and phenotype consequence of apoc rare variants in patients with very low triglyceride levels 	d. crawford, nicole a. restrepo, kirsten e. diggins, e. farber-eger, q. wells 	medicine 	bmc medical genomics 	 	 	pdf 	view excerpts, cites background and results save alert research feed cryptic relatedness in epidemiologic collections accessed for genetic association studies: experiences from the epidemiologic architecture for genes linked to environment (eagle) study and the national health and nutrition examination surveys (nhanes) 	j. malinowski, r. goodloe, k. brown-gentry, d. crawford 	geography, medicine 	front. genet. 	 	 	 save alert research feed finding rare, disease-associated variants in isolated groups: potential advantages of mennonite populations 	f. lopes, l. hou, + authors f. mcmahon 	biology, medicine 	human biology 	 	 	 	view excerpt, cites background save alert research feed population stratification in the context of diverse epidemiologic surveys sans genome-wide data 	m. oetjens, k. brown-gentry, r. goodloe, h. dilks, d. crawford 	biology, medicine 	front. genet. 	 	 	 save alert research feed role of rare and low frequency variants in gene-alcohol interactions on plasma lipid levels 	zhe wang, hai-kui chen, + authors a. morrison 	biology, medicine 	 	 	pdf save alert research feed role of rare and low-frequency variants in gene-alcohol interactions on plasma lipid levels 	zhe wang, h. chen, + authors a. morrison 	biology, medicine 	circulation. genomic and precision medicine 	 	 	 save alert research feed genomics of hypertriglyceridemia. 	hayato tada, m. takamura, m. kawashiri 	medicine 	advances in clinical chemistry 	 save alert research feed apoc loss-of-function mutations, remnant cholesterol, low-density lipoprotein cholesterol, and cardiovascular risk: mediation- and meta-analyses of   individuals 	a. b. wulff, b. nordestgaard, a. tybjærg‐hansen 	medicine, biology 	arteriosclerosis, thrombosis, and vascular biology 	 	 	 save alert research feed emerging evidence that apoc-iii inhibitors provide novel options to reduce the residual cvd 	m. taskinen, c. packard, j. borén 	medicine 	current atherosclerosis reports 	 	 	pdf save alert research feed the role of antisense oligonucleotide therapy against apolipoprotein-ciii in hypertriglyceridemia. 	i. gouni‐berthold 	biology, medicine 	atherosclerosis. supplements 	 	 	view excerpts, cites background save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency a rare functional cardioprotective apoc variant has risen in frequency in distinct population isolates 	i. tachmazidou, g. dedoussis, + authors e. zeggini 	biology, medicine 	nature communications 	 	 	pdf 	view excerpt, references background save alert research feed association of blood lipids with common dna sequence variants at genetic loci in the multiethnic united states national health and nutrition examination survey iii 	mary e. keebler, christopher l sanders, aarti surti, c. guiducci, n. burtt, s. kathiresan 	biology, medicine 	circulation. cardiovascular genetics 	 	 	 save alert research feed the effects of scale: variation in the apoa /c /a /a gene cluster 	s. fullerton, a. buchanan, + authors k. weiss 	biology, medicine 	human genetics 	 	 save alert research feed population-based resequencing of angptl uncovers variations that reduce triglycerides and increase hdl 	s. romeo, l. pennacchio, + authors j. cohen 	biology, medicine 	nature genetics 	 	 	 save alert research feed genetic determinants of lipid traits in diverse populations from the population architecture using genomics and epidemiology (page) study 	l. dumitrescu, c. carty, + authors d. crawford 	biology, medicine 	plos genetics 	 	 	pdf save alert research feed whole-exome sequencing identifies rare and low-frequency coding variants associated with ldl cholesterol. 	l. lange, y. hu, + authors c. willer 	biology, medicine 	american journal of human genetics 	 	 	pdf save alert research feed biological, clinical, and population relevance of loci for blood lipids 	tanya m. teslovich, k. musunuru, + authors s. kathiresan 	biology, medicine 	nature 	 	 , 	pdf 	view excerpt, references background save alert research feed maternal lineages and alzheimer disease risk in the old order amish 	joelle walt, w. scott, + authors m. pericak-vance 	biology, medicine 	human genetics 	 	 	pdf 	view excerpt, references background save alert research feed a spectrum of pcsk alleles contributes to plasma levels of low-density lipoprotein cholesterol. 	ingrid k. kotowski, a. pertsemlidis, + authors h. hobbs 	biology, medicine 	american journal of human genetics 	 	 	 save alert research feed a null mutation in human apoc confers a favorable plasma lipid profile and apparent cardioprotection 	t. pollin, coleen m. damcott, + authors a. shuldiner 	biology, medicine 	science 	 	 	 	view excerpts, references background and results save alert research feed ... ... related papers 	abstract 	tables and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue identification of two novel ercc mutations in old order amish with cockayne syndrome | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / corpus id: identification of two novel ercc mutations in old order amish with cockayne syndrome @article{xin identificationot, title={identification of two novel ercc mutations in old order amish with cockayne syndrome}, author={b. xin and h. wang}, journal={molecular syndromology}, year={ }, volume={ }, pages={ - } } 	b. xin, h. wang 	published 	medicine 	molecular syndromology cockayne syndrome (cs) is a rare autosomal recessive disorder characterized by progressive multisystem degeneration and segmental premature aging. mutations in the dna repair gene ercc are responsible for the majority of cs cases reported. in this study, we identified patients presenting with cs from old order amish families. sequence analysis of the ercc gene revealed novel mutations associated with the disorder in these patients. the patients from family were homozygous for a splice… expand view on pubmed karger.com save to library create alert cite launch research feed share this paper citationsbackground citations results citations view all figures and topics from this paper 	 figure 	 figure 	cockayne syndrome 	sequence analysis 	homozygote 	ercc gene 	introns 	premature aging syndrome 	hepatolenticular degeneration citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency two novel mutations in ercc cause cockayne syndrome b in a chinese family 	c. he, m. sun, g. wang, y. yang, l. yao, y. wu 	biology, medicine 	molecular medicine reports 	 	 	pdf 	view excerpt, cites results save alert research feed abstract. cockayne syndrome (cs) is a rare autosomal recessive disorder characterized principally by progressive growth failure, neurologic abnormality and premature aging. mutations of excision repair cross-complementation group (ercc ) and ercc are predominantly responsible for cs, of which mut 	 save alert research feed identification of two missense mutations of ercc in three chinese sisters with cockayne syndrome by whole exome sequencing 	s. yu, l. chen, + authors jiansheng xie 	biology, medicine 	plos one 	 	 	pdf 	view excerpt, cites background save alert research feed novel frame shift mutation in ercc leads to a severe form of cockayne syndrome with postnatal growth failure and early death 	yao kou, m. shboul, + authors j. tian 	medicine 	medicine 	 	 	 save alert research feed uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing 	n. calmels, géraldine greff, + authors v. laugel 	biology, medicine 	orphanet journal of rare diseases 	 	 	 save alert research feed investigation of the molecular basis of three new disorders of brain growth and development identified amongst the amish 	e. baple 	psychology 	 save alert research feed references showing - of references mutation update for the csb/ercc and csa/ercc genes involved in cockayne syndrome 	v. laugel, c. dalloz, + authors h. dollfus 	biology, medicine 	human mutation 	 	 	view excerpt, references background save alert research feed cockayne syndrome: review of cases. 	m. nance, s. berry 	medicine 	american journal of medical genetics 	 	 	view excerpts, references background save alert research feed structure and expression of the excision repair gene ercc , involved in the human disorder cockayne's syndrome group b. 	c. troelstra, w. hesen, d. bootsma, j. hoeijmakers 	biology, medicine 	nucleic acids research 	 	 	pdf 	view excerpt, references background save alert research feed homozygosity for a novel splice site mutation in the cardiac myosin‐binding protein c gene causes severe neonatal hypertrophic cardiomyopathy 	b. xin, e. puffenberger, john tumbush, j. r. bockoven, heng wang 	medicine 	american journal of medical genetics. part a 	 	 	pdf 	view excerpt, references background save alert research feed ercc , a member of a subfamily of putative helicases, is involved in cockayne's syndrome and preferential repair of active genes 	c. troelstra, a. j. gool, j. wit, w. vermeulen, j. hoeijmakers 	biology, medicine 	cell 	 	 	pdf 	view excerpt, references background save alert research feed the cockayne syndrome group a gene encodes a wd repeat protein that interacts with csb protein and a subunit of rna polymerase ii tfiih 	k. henning, l. li, + authors e. friedberg 	biology, medicine 	cell 	 	 	 	view excerpt, references background save alert research feed the genetic defect in cockayne syndrome is associated with a defect in repair of uv-induced dna damage in transcriptionally active dna. 	j. venema, l. mullenders, a. natarajan, a. v. van zeeland, l. mayne 	biology, medicine 	proceedings of the national academy of sciences of the united states of america 	 	 	pdf 	view excerpt, references background save alert research feed gene-specific dna repair of uv-induced cyclobutane pyrimidine dimers in some cancer-prone and premature-aging human syndromes. 	m. evans, v. bohr 	biology, medicine 	mutation research 	 	 	view excerpt, references background save alert research feed deficient repair of the transcribed strand of active genes in cockayne's syndrome cells. 	a. van hoffen, a. natarajan, l. mayne, a. v. van zeeland, l. mullenders, j. venema 	biology, medicine 	nucleic acids research 	 	 	 save alert research feed related papers 	abstract 	figures and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue parkinson disease loci in the mid-western amish toggle navigation 	 	 	 about 	 login toggle navigation view item  	   institutional repository home 	 electronic theses and dissertations 	 electronic theses and dissertations 	 view item 	   institutional repository home 	 electronic theses and dissertations 	 electronic theses and dissertations 	view item javascript is disabled for your browser. some features of this site may not work without it. search institutional repository this collection browse all of institutional repositorycommunities & collectionsby issue dateauthorstitlessubjectsdepartmentthis collectionby issue dateauthorstitlessubjectsdepartment my account loginregister parkinson disease loci in the mid-western amish davis, mary feller persistent link: https://etd.library.vanderbilt.edu/etd- - http://hdl.handle.net/ / date: - - abstract previous evidence has shown that parkinson disease (pd) has a heritable component, but only a small proportion of the total genetic contribution to pd has been identified. genetic heterogeneity complicates the verification of proposed pd genes and the identification of new pd susceptibility genes. our approach to overcome the problem of heterogeneity is to study a population isolate, the mid-western amish communities of indiana and ohio. we performed genome-wide association and linkage analyses on individuals ( with pd), who are part of a , member pedigree. through these analyses, we identified a region on chromosome q . that shows evidence of association (p-value < x - ) and linkage (multipoint hlod = . ). we also found further evidence of linkage on chromosomes and (multipoint hlod . and . respectively). these data suggest that locus heterogeneity, even within the amish, may be more extensive than previously appreciated. show full item record files in this item 	name: 	mfdavis_thesis.pdf 	size: 	 . kb 	format: 	pdf view/open this item appears in the following collection(s): 	 electronic theses and dissertations connect with vanderbilt libraries 	 	 	 	 	 	 	 your vanderbilt 	 alumni 	 current students 	 faculty & staff 	 international students 	 media 	 parents & family 	 prospective students 	 researchers 	 sports fans 	 visitors & neighbors support the jean and alexander heard libraries gifts to the libraries support the learning and research needs of the entire vanderbilt community. learn more about giving to the libraries. become a friend of the libraries quick links 	 hours 	 about 	 employment 	 staff directory 	 accessibility services 	 contact 	 vanderbilt home 	 privacy policy ggg .. investigation mapping of adaptive traits enabled by a high-density linkage map for lake trout seth r. smith,*,†,‡, stephen j. amish,*,† louis bernatchez,§ jeremy le luyer,§,** chris c. wilson,†† olivia boeberitz,‡‡ gordon luikart,*,† and kim t. scribner‡,‡‡ *conservation genomics group,wildlife biology program, †flathead lake biological station, division of biological sciences, university of montana, polson, mt, ‡department of integrative biology, ‡‡department of fisheries and wildlife, michigan state university, east lansing mi, §institut de biologie intégrative et des systèmes, université laval, québec, canada, **ifremer, ird, institut louis‐malardé, univ polynésie française, eio, f‐ taravao, tahiti, polynésie française, france, and ††ontario ministry of natural resources and forestry, peterborough, ontario, canada orcid ids: - - - (s.r.s.); - - - (l.b.); - - - (j.l.l.); - - - (c.c.w.); - - - (g.l.); - - - (k.t.s.) abstract understanding the genomic basis of adaptative intraspecific phenotypic variation is a central goal in conservation genetics and evolutionary biology. lake trout (salvelinus namaycush) are an excellent species for addressing the genetic basis for adaptive variation because they express a striking degree of ecophenotypic variation across their range; however, necessary genomic resources are lacking. here we utilize recently-developed analytical methods and sequencing technologies to ( ) construct a high-density linkage and centromere map for lake trout, ( ) identify loci underlying variation in traits that differentiate lake trout ecophenotypes and populations, ( ) determine the location of the lake trout sex determination locus, and ( ) identify chromosomal homologies between lake trout and other salmonids of varying divergence. the resulting linkage map contains , single nucleotide polymorphisms (snps) mapped to linkage groups, likely representing the lake trout chromosomes. female and male linkage group lengths ranged from . to . centimorgans, and . to . centimorgans, respectively. we improved the map by determining coordinates for of centromeres, resulting in a map with metacentric chromosomes and acrocentric or telocentric chromosomes. we use the map to localize the sex determination locus and multiple quantitative trait loci (qtl) associated with intraspecific phenotypic divergence including traits related to growth and body condition, patterns of skin pigmentation, and two composite geomorphometric variables quantifying body shape. two qtl for the presence of vermiculations and spots mapped with high certainty to an arm of linkage group sna , growth related traits mapped to two qtl on linkage groups sna and sna , and putative body shape qtl were detected on six separate linkage groups. the sex de- termination locus was mapped to sna with high confidence. synteny analysis revealed that lake trout and congener arctic char (salvelinus alpinus) are likely differentiated by three or four chromosomal fissions, possibly one chromosomal fusion, and or more large inversions. combining centromere mapping information with putative inversion coordinates revealed that the majority of detected inversions differen- tiating lake trout from other salmonids are pericentric and located on acrocentric and telocentric linkage groups. our results suggest that speciation and adaptive divergence within the genus salvelinus may have been associated with multiple pericentric inversions occurring primarily on acrocentric and telocentric chromosomes. the linkage map presented here will be a critical resource for advancing conservation oriented genomic research on lake trout and exploring chromosomal evolution within and between salmonid species. keywords linkage map salvelinus qtl rad genomics lake trout maintaining adaptive phenotypic diversity is a central tenet of conservation biology. in many taxa, diversity is produced through selective pressures that favor reduced intraspecific competition and trophic specialization (skulason and smith ; robinson and schluter ; whiteley ). the evolution of trophically special- ized morphotypes has been observed in multiple fish species volume | june | http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - including arctic char (snorrason et al. ), lake trout (eshenroder ; muir et al. ), multiple coregonid species (lu and ber- natchez ; thomas et al. ), and african cichlids (rüber et al. ), and represents an important pathway by which phenotypic diversity is generated and maintained in nature (pfennig and pfennig ). intraspecific diversity can promote community and ecosystem stability (schindler et al. ); however, the genomic basis for this variation is often poorly understood for non-model species. ad- vancement of our understanding is largely limited by a lack of genomic resources. lake trout (salvelinus namaycush) are a salmonid fish species endemic to north america with substantial cultural, ecological, and economic importance. across their range, lake trout are often the keystone predator of lentic ecosystems (ryder et al. ) and historically supported valuable commercial and subsistence fisheries (waters ; hansen ; brenden et al. ). lake trout express a large degree of sympatric phenotypic variation (muir et al. ) making them a useful species for exploring the genomic basis for phenotypic diversity. multiple morphotypes exist across the species range (muir et al. ; marin et al. ), with diversification largely associated with the ability to exploit resources and habitats at varying depths in large post-glacial lakes (zimmerman et al. ; stafford et al. ; muir et al. ; marin et al. ). in the great lakes, trophic specialization has resulted in the evolution of three widely recognized morphotypes — leans, siscowets, and humpers — that are differentiated by patterns of skin pigmentation, size-at-age, body shape, tissue lipid content, habitat use, and diet (thurston ; eschmeyer and phillips ; burnham-curtis ; harvey et al. ; alfonso ; zimmerman et al. ; zimmerman et al. ; goetz et al. ). similar patterns of divergence exist in other lake trout populations (blackie et al. ; zimmerman et al. ; hansen et al. ; marin et al. ; chavarie et al. ), with some degree of morphological and phenological variation existing among individuals of the same morphotype (bronte ; bronte and moore ). previous studies have evaluated differences in gene expression and signals of adaptive divergence between lake trout morphotypes (goetz et al. ; bernatchez et al. ; perreault-payette et al. ). however, no study has explicitly evaluated which loci control variation in specific traits that underly morphotype divergence. additionally, these studies have relied on de novo assembled markers distributed anonymously across the genome. although these ap- proaches can be powerful (davey et al. ), fully interpreting results requires some knowledge of how loci are ordered along chromosomes. all scans for adaptively significant loci and geno- type-phenotype associations inherently take advantage of linkage disequilibrium between genotyped markers and causal loci. without knowing the relative locations of loci, it can be difficult to determine if genotype-phenotype associations or signals of selection are associated with a single genomic region or multiple regions distributed widely across the genome. information on the order of loci along chromo- somes can be readily attained via linkage mapping or assembly of a reference genome; however, linkage maps are often needed a priori to produce chromosome-scale genome assemblies. linkage maps have been used to map loci associated with disease resistance (houston et al. ; moen et al. ), life history and physiological trait variation (rogers and bernatchez ; miller et al. ; gagnaire et al. a; sutherland et al. ; pearse et al. ), and commercially valuable traits (gonzalez-pena et al. ) in salmonids and have been instrumental in the assembly of salmonid reference genomes (lien et al. , christensen et al. a, b; pearse et al. ; sävilammi et al. ). a linkage map for lake trout would enable the application of cutting-edge genomic tools to questions in lake trout management and evolution and would aid in the identification of loci underlying phenotypic variation and local adaptation. specifically, a linkage map would increase the strength of inference from genome-wide association studies and scans for selec- tion (bradbury et al. ; gagnaire et al. b; mckinney et al. ) and allow for the localization of quantitative trait loci (peichel et al. ; qiu et al. ) and tracts of admixture and homozygosity, and the estimation of historical effective population sizes and ad- mixture dynamics (hollenbeck et al. ; leitwein et al. ). this information would be valuable for selecting stocks for reintroduction and translocation and for estimating the adaptive potential of intact populations under changing climate and abiotic conditions (leitwein et al. ; bay et al. ). comparative analysis of linkage maps and genome assemblies from related species can also shed light on chromosomal evolution and speciation (rastas et al. ; sutherland et al. ; hale et al. ). chromosomal inversions appear to have played an important role in speciation and adaptive divergence within the salmonid lineage (miller et al. ; sutherland et al. , pearse et al. ) and within other taxa (lowry and willis ; küpper et al. ; for review see wellenreuther and bernatchez ). instances of reduced hybrid fitness and hybrid inviability are widespread within the family salmonidae (leary et al., ; fugjiwara et al. ; muhlfeld et al. ). information on the locations of inversions differentiating species and phenotypically divergent populations could shed light on the genetic basis for these phenomena. inversions can contribute to isolation between species and populations because they can suppress recombination over large chromosomal regions, allowing for adaptive differences to accumulate between inverted and non-inverted haplotypes even in the presence of gene flow (berg et al. ; wellenreuther and bernatchez ). inversions can also produce post-zygotic isolation between incipient species if crossing over within heterozygous individuals results in formation of abnor- mal or inviable gametes (wellenreuther and bernatchez ). an improved understanding of the extent to which pericentric (including the centromere) and paracentric (outside the centromere) inversions can accumulate between salmonid species over varied evolutionary time scales, could provide clues about pre- and post-zygotic isolation mechanisms that contributed to adaptive divergence and incipient speciation within salmonids. linkage maps have been constructed for multiple salmonid species including rainbow trout (miller et al. ; palti et al. ; gonzalez-pena et al. ), chinook salmon (brieuc et al. ; mckinney et al. ; mckinney et al. ), coho salmon (kodama et al. ), sockeye salmon (everett, miller and seeb ; larson et al. ; limborg et al. ), chum salmon (waples et al. ); pink salmon (spruell et al. ; lindner et al. ), atlantic copyright © smith et al. doi: https://doi.org/ . /g . . manuscript received february , ; accepted for publication march , ; published early online april , . this is an open-access article distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/ licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. supplemental material available at figshare: https://doi.org/ . / g . . correspnding author: michigan state university, department of integrative biology, wilson road, natural resources building, east lansing, mi . e-mail: smit @msu.edu. | s. r. smith et al. https://doi.org/ . /g . . http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / https://doi.org/ . /g . https://doi.org/ . /g . mailto:smit @msu.edu salmon (moen et al. ; lien et al. ; brenna-hansen et al. ; gonen et al. ), arctic char (nugent et al. ; christensen et al. a), brook trout (sauvage et al. ; sutherland et al. ; hale et al. ), brown trout (leitwein et al. ), european grayling (sävilammi et al. ), lake whitefish (rogers and bernatchez ; gagnaire et al. a), and european whitefish (de-kayne and feulner ). no linkage map has been constructed for lake trout (but see may et al. , johnson et al. , for work on segregation patterns in lake trout x brook trout hybrids), although the lake trout karyotype has been characterized in multiple previous studies (phillips and zajicek ; reed and phillips ) providing a reference for the number of expected chromosomes. here we present a high-density linkage map for lake trout generated using restriction site associated dna (rad) capture (rapture; ali et al. ), a modified rad sequencing protocol that allows variable loci to be preferentially genotyped. the map was used to characterize the lake trout karyotype, estimate recombination rates, determine centromere locations, map the sex determination locus, and identify chromosomal inversions and translocations differenti- ating lake trout from other salmonids. we demonstrate the utility of the linkage map by using available phenotype data to map quanti- tative trait loci (qtl) associated with pigmentation patterns, growth and condition related traits, and variation in body shape — all traits hypothesized to be adaptive in lake trout and other salmonids. materials and methods linkage mapping families two f full-sibling families were created by crossing seneca lake hatchery strain females with parry sound strain males (table , figure ). the seneca lake strain was founded using individuals from seneca lake, new york and this strain has contributed dispropor- tionately to restoring lake trout populations in the great lakes (scribner et al. ). the parry sound strain was founded by wild individuals collected from georgian bay in lake huron. the seneca and parry sound strains are genetically divergent (fst = . ) based on a previous study using microsatellites (scribner et al. ). crosses were produced in using adult lake trout and housed at pendills creek national fish hatchery (u.s. fish and wildlife service, figure ). eggs were fertilized, incubated in heath trays at ambient temperature, and raised until swim-up phase. offspring were then killed using a lethal dose of ms- and preserved in % ethanol. genetic sex was determined for offspring using a sdy presence-absence quantitative pcr (qpcr) assay designed using the approach of anglès d’auriac et al. ( ; see trait mapping methods below). these families were ultimately used for constructing the linkage map and localizing the lake trout sex determination locus. an additional f half-sibling family was created using adult lake trout from the killala lake hatchery strain and wild individuals from kingscote lake, ontario (table , figure ). the killala lake strain was founded by individuals from killala lake, ontario, which is within the lake superior drainage. this hatchery strain is most similar to lean form hatchery strains derived from lake superior based on a previous allozyme genotyping study (marsden et al. ). individuals from the kingscote lake strain also resemble lean lake trout; however, they are small bodied and lack spots and vermicu- lations (wilson and evans ). examination of f offspring at age revealed substantial variation in pigmentation, weight and length at age, and body shape among individuals. these traits are commonly recognized as being adaptively differentiated between lake trout populations and ecophenotypes (eshenroder ; muir et al. ). body shape and early growth rate in particular have been recognized as important traits for differentiating lean, siscowet, and humper ecophenotypes (moore and bronte ; hansen et al. ). the observation that skin pigmentation patterns vary between eco- phenotypes and across depth strata in some lake trout populations also suggests that pigmentation traits might be an important axis of ecophenotypic divergence within lake trout (zimmerman et al. ). the f kingscote x killala family was used for linkage map con- struction, localization of the sex determination locus, and qtl mapping. crosses, culture conditions, and phenotyping procedures are described below. initial kingscote x killala f crosses were produced using adult lake trout using a x factorial mating design. in , mature adults from initial crosses were mated to produce f families. eggs from each family were incubated in heath trays at ambient temperature ( - �). prior to swim-up, hatched sac fry from families were trans- ferred to l laundry tubs ( fry per tub) where they remained until age +. families were manually fed % of tank biomass twice-daily and family sizes were periodically reduced by culling to avoid over- crowding. at age +, families were transferred to l circular tanks with ambient lighting and fed to satiation on an ewos pellet diet. at age , fork length and weight were determined and lateral photo- graphs were collected using the protocol from bernatchez et al. ( ). fish were photographed using a nikon coolpix p digital camera with a focal length of mm mounted on a tripod in fixed position. fish were photographed with the head facing to the left and were cradled in a stretched mesh net as in zimmerman et al. ( ) in order to avoid distorting body shape. fin clips were collected and preserved in % ethanol. photographs were later used for morphom- etric analysis and scoring individuals for presence-absence of spots and vermiculations (see trait mapping methods section below). an additional gynogenetic diploid family was created using a female f resulting from initial kingscote x killala crosses using a protocol similar to that of thorgaard et al. ( ). this family was used for mapping centromeres using half-tetrad analysis (thorgaard et al. ; limborg et al. ). sperm from a male lake trout was diluted : using sperm extender ( . g tris buffer, . g citric acid, . g glycine, . g kcl, g pvp- , and liter of distilled water), mixed thoroughly in a x x inch glass pyrex dish, placed on ice, and irradiated for min using a -watt germicidal uv lamp placed centimeters from the dish. eggs and sperm were then mixed and n■ table family ids, cross type (diploid or gynogenetic diploid), number of genotyped offspring per family, and maternal and paternal origins for the five families used for linkage and qtl mapping. family type no. offspring mother origin father origin s diploid seneca lake parry sound s diploid seneca lake parry sound p diploid killala x kingscote f killala x kingscote f p diploid killala x kingscote f killala x kingscote f g gynogenetic diploid killala x kingscote f none volume june | high-density lake trout linkage map | sperm was activated by adding water. ten minutes after fertilization, eggs were heat shocked at � for min, water hardened, transferred to heath trays for incubation, and raised using the same conditions described for diploid families. all kingscote x killala families were produced at the codrington fisheries research facility (ontario ministry of natural resources and forestry; figure ; codrington, ontario). this facility has a surface water supply which undergoes seasonal and diel temperature variation ranging between - � in winter and - � in summer. sample preparation for all kingscote x killala families, dna from offspring and parents (table ) was extracted using the high-throughput spri bead-based extraction protocol described in ali et al. ( ) with serapure beads (described in rohland and reich ) substituted for ampure xp beads. for the seneca x parry sound crosses, dna was extracted using qiagen dneasy blood and tissue extraction kits ( , qiagen, hilden, germany) using manufacturer recommendations. dna quality was initially assessed using a nanodrop spectro- photometer (thermo fisher scientific, waltham, massachusetts) by evaluating / and / absorbance ratios. samples were diluted to less than ng/ul based on nanodrop readings, then diluted -fold before determining double-stranded dna (dsdna) concentrations using quantit picogreen assays (thermo fisher sci- entific, waltham, massachusetts). sequencing library preparation dsdna concentrations were normalized to ng/ul using an eppen- dorf epmotion tmx liquid handling robot (eppendorf, ham- burg, germany) before proceeding with the bestrad protocol and rad-capture using ng of total input dna (ali et al. ). modifications to the protocol are noted below with a detailed de- scription of methods provided in supplementary material (document s ). first, the enzyme psti was substituted for sbfi and psti was heat- killed at � rather than �. after ligating bestrad adapters and pooling samples, shearing was carried out using a covaris e ultrasonicator (covaris inc., woburn, massachusetts) using the recommended settings for a bp mean fragment length. finished libraries were amplified for cycles, pooled equally in sets of two, and bead cleaned twice using a . : bead-to-dna ratio ampure xp cleanup (a , beckman coulter, brea, california). the two resulting pools were then enriched for , rad loci that were previously found to be variable in lake trout populations in the great lakes, seneca lake, ontario, montana, and alaska using the rad- capture protocol (ali et al. ). target enrichment reactions were carried out using a mybaits custom target enrichment kit using manufacturer recommendations (mycroarray, ann arbor, michi- gan; protocol version ; for more information on capture and bait selection see document s ). finished capture reactions were ampli- fied for an additional cycles, pooled, and sequenced in three lanes of an illumina hiseq x instrument ( x bp paired end reads; illumina, san diego, california) by the novogene corporation (novogene, sacremento california). bioinformatics and genotyping read quality was initially assessed using fastqc v . . (andrews ), and a custom script was used to re-orient paired end reads such that individual specific barcodes and restriction enzyme overhang sequences were always located at the beginning of the first read. reads were demultiplexed using process_radtags v . , duplicate reads were removed using clone_filter v . (catchen et al. ; rochette et al. ), and adapter sequences were clipped from reads using trim- momatic v . (bolger et al. ). at this point, we produced two sets of fastq files: one conservatively filtered dataset used for de novo assembly of rad loci and a slightly less conservatively filtered dataset used for calculating genotype likelihoods that would ultimately be used for linkage mapping and other analyses. for the de novo assembly dataset, reads were trimmed whenever the mean base quality across a sliding window of bp dropped below q , read pairs were removed if one or both reads in a pair were less than bp in length after trimming, and reads were cropped to a length of bp such that all reads were of identical length. for the dataset used to calculate genotype likelihoods, reads were trimmed whenever the mean base quality across a sliding window of bp dropped below q and excluded if one or both reads in the pair were less than bp after trimming. the stringently filtered dataset (read length = bp, trimming threshold of q ) was used to assemble rad loci de novo using modules available in stacks v . (rochette et al. ). rad loci were identified for individuals using ustacks v . , which was run with a minimum depth of coverage of (-m ), a maximum distance between stacks of (-m ), a maximum distance to align secondary reads to primary stacks of (-n ), a minimum of stacks at each de novo locus (–max_locus_stacks ), and disabling calling haplo- types from secondary reads (-h). we then created a catalog of rad loci for the parents of crosses using cstacks v . , allowing for up to two mismatches between sample loci when building the locus catalog (-n ). putative rad loci alleles for all individuals were matched to this catalog using sstacks v . , converted to bam format using tsv bam v . , and then assembled using gstacks v . . consensus sequences for rad loci were obtained by passing the “–fasta-loci” flag to the populations v . module. the fasta file containing rad locus consensus sequences was normalized using picard normalizefasta v . (http://broadinstitute.github.io/picard/), indexed using bwa in- dex v . (li ) and samtools faidx v . (li et al. ), and used as a de novo reference for subsequent analysis. next, the larger set of variable length paired end reads that were trimmed using a q-threshold of were mapped to the de novo figure map displaying the locations of hatchery facilities (dots) and locations of wild progenitor populations (diamonds) used for mapping. locations of hatchery facilities used for conducting crosses are marked with black circles. the locations of the progenitor populations are identified with black diamonds. longitude is displayed on the y-axis and latitude is displayed on the x-axis. | s. r. smith et al. http://broadinstitute.github.io/picard/ assembly using bwa mem v . (li ) with default setting. ge- notype likelihoods were calculated for single nucleotide polymor- phisms (snps) within rad loci using lepmap v . and associated modules (rastas et al. ). sam files produced by bwa-mem were converted to bam format and sorted using samtools v . , then converted to mpileup format using a minimum mapping quality of and a minimum base quality of . the resulting file was filtered using the script pileupparser .awk using a minimum read depth of and a missingness threshold of . . genotype likelihoods were calculated using the pileup posterior.awk script distributed with lepmap v . (rastas et al. , rastas ). we opted to use pileup posterior.awk to calculate genotype likelihoods because the lepmap pipeline was originally validated using likelihoods calcu- lated using this program (rastas ). linkage map construction linkage mapping and additional data filtering were carried out using various programs distributed with lepmap v . (rastas ). first, any missing parental snp genotypes were imputed using parentcall . second, snps showing evidence of segregation distortion were re- moved using filtering with a p-value (–datatolerance) threshold of . . we required that snps be informative for linkage mapping in at least family and removed snps with minor allele frequencies less than . . snps were assigned to linkage groups (lgs) using separate- chromosomes run with logarithm of odds ratios (lod) thresholds ranging from to and a minimum lg size of snps. no single lod threshold produced the expected number of lgs (n = ; phillips and zajicek ; reed and phillips ). beginning with the map produced using a universal lod threshold of , we determined the lod thresholds needed to further split each lg by running separatechromosomes using all lod thresholds be- tween and and specifying the lg targeted for additional splitting using the “lg” and “map” flags (similar to christensen et al. a). we determined that the largest of the initial lgs could be split using lod thresholds ranging from - , with the remaining lgs remaining intact for all lod thresholds between and . the largest lgs were split using the maximum lod threshold that resulted in a new lg containing more than snps, resulting in lgs. unassigned singleton snps were then joined to this map using joinsingles all run iteratively with a lod threshold of and a minimum lod difference of . the order of snps was initially determined by running itera- tions of ordermarkers and selecting the order with the highest likelihood for each lg. lgs were further refined by evaluating lod matrices (output using computelodscores = ). for each snp, the vector of lod scores corresponding to possible map positions was normalized such that values ranged from to . snps were removed if the maximum lod score was less than standard deviation from the mean or if more than one lod ‘peak’ was observed for any given snp, indicating the existence of multiple mapping positions of similar likelihood. lod peaks were identified using the findpeaks function from the r package pracma v . . , a minimum normalized peak height of . and a minimum distance between peaks greater than % of the length of the vector of mapping positions. rad loci were removed from the data set if associated snps mapped to more than one lg. finally, the dataset was thinned to include a single snp for each rad locus, with preference given to the snp closest to the psti restriction cut site. we opted to thin snps after determining which loci could be effectively mapped in order to maximize the number of unique rad loci on the map. maps for each lg were then recon- structed using the evaluateorder and improveorder = options from ordermarkers , with snps that failed the above filtering criteria flagged for removal using the removemarkers option. finally, lgs were inspected for possible mis-ordering using lmplot and any lg marked with possible errors were reordered using ordermarkers for an additional iterations. the linkage map was further improved by trimming snps from the ends of lgs based on manual inspection of lod matrix plots and alignment to rainbow trout, arctic char, and atlantic salmon (salmo salar) genome se- quences (see homology section below). an additional iterations of ordering were conducted after removing potential erroneously placed snps from the ends of lgs. final lgs were sorted based on their number of mapped snps and named as sna -sna . both male and female linkage maps were output by the program. centromere mapping we identified centromeres by estimating the frequency of second division segregation (g) across linkage groups using half-tetrad analysis conducted on gynogenetic diploid offspring from family g (thorgaard et al. ). cells of gynogenetic diploid offspring contain two of the four possible meiosis ii products (a half-tetrad) and the frequency of heterozygous offspring can be used to estimate the frequency of recombination events between the locus in question and the centromere (thorgaard et al. ). reads for these indi- viduals were aligned to de novo assembled rad loci, sorted and indexed using samtools v . (li et al. ), and variable positions within rad loci were genotyped using freebayes v . . (garrison and marth ). genotypes were called without applying population or binomial observation priors, an assumed contamination probability of %, a minimum base quality of , and a minimum mapping quality of . called loci were then converted to their simplest representation using vcfallelicprimatives (https://github.com/vcflib/ vcflib; vcflib v . . ) and loci with more than alleles and indels were removed, such that only snps remained. genotypes were set to missing if there was less than order of magnitude difference in genotype likelihoods between the called genotype and the second most likely genotype using vcftools v . . (gq . ; danecek et al. ). snps were removed from the dataset if more than % of individuals were missing genotypes or if the frequency of the minor allele was less than . . snps were further excluded if they were not placed on the linkage map, not called heterozygous in the mother, or if both possible homozygous genotypes were not observed in off- spring. the mother was removed from the dataset at this point, and observed heterozygosity for the offspring (y) was calculated using the hwe function from seqvartools v . . (gogarten et al. ; https:// github.com/smgogarten/seqvartools). centromeric regions were delineated as the region between the first and last markers with y-values less than . (as in limborg et al. ). results were cross-validated and improved upon using the rfm method (limborg et al. ) applied to the phased genotypes of progeny from families s , s , p , and p . counts of maternal recombination events were reported using ordermarkers with out- putphaseddata = and used to calculate rfm across all maternal haplotypes and identify putative centromeric regions using a cut-off value of . as suggested in limborg et al. ( ). the correct centromeric locations for acrocentric and telocentric chromosomes were identified by selecting the region containing, or neighboring, the lowest y-values from half-tetrad analysis. volume june | high-density lake trout linkage map | https://github.com/vcflib/vcflib https://github.com/vcflib/vcflib https://github.com/smgogarten/seqvartools https://github.com/smgogarten/seqvartools homology rad loci were aligned to the reference genomes for arctic char (refseq accession: gcf_ . ), rainbow trout (onco- rhynchus mykiss; refseq accession: gcf_ . ) and at- lantic salmon (refseq accession: gcf_ . ) using bwa mem v . (li ). rad loci were assigned to their respective linkage map positions, and male and female linkage maps were visualized relative to their order along homologous chromosomes using ggplot v . . (wickham and chang ). chromosomes were considered homologous if or more mapped rad loci aligned to a chromosome with mapping qualities greater than mq . the map was also compared with a linkage map for brook trout (salvelinus fontinalis; sutherland et al. ) using the program mapcomp (sutherland et al. ; https://github.com/ enormandeau/mapcomp) and the arctic char genome as an in- termediate reference in order to detect large structural variants differentiating the two species. putative chromosomal inversions were detected by manually inspecting plots produced by mapping the lake trout linkage map to divergent references. inversion breakpoints were defined by the coordinates with the greatest discrepancy between the divergent physical map and the female linkage map we constructed. inversions were classified as pericentric if putative inversion coordinates over- lapped centromere mapping positions. inversions differentiating lake trout and brook trout were detected by manually inspecting dot plots produced by mapcomp. trait mapping offspring from diploid kingscote x killala crosses were phenotyped for fork length (fl), weight (wt), and condition factor (cf) at age . additionally, photographs collected at age were used to score individuals for presence-absence of spots and vermiculations (vpa) and two composite variables (pca and pca ) summarizing variation in body shape. body shape variables were derived by performing a principal-components analysis (pca) on the coordi- nates of morphometric landmarks that were normalized for slight differences in fish position and rotation using generalized procrustes analysis. using available photographs from families p and p , we placed landmarks using tpsdig v (rohlf ) consistent with those described in muir et al. ( ). landmark coordinates were normal- ized and rotated using generalized procrustes analysis conducted using the function gpagen from the r-package geomorph v . . (adams and otárola‐castillo ). four of landmarks could not be consistently placed using available images ( , , , ) and were therefore excluded from the analysis. synthetic variables pca and pca were calculated by performing pca on the resulting normal- ized coordinates and extracting scores for the first two axes. pca was carried out using the function prcomp from the r-package stats v . . . vpa, pca , and pca phenotypes were available for of individuals. fork length, condition factor, and weight phenotypes were collected for of individuals. phased snp genotypes for offspring were extracted from the final map files reported by ordermarkers using the script map genotypes.awk from lepmap v . . qtl mapping was then carried out for traits of interest using the r-package qtl v . and associated functions (broman et al. ). all traits were mapped to sex-averaged linkage map coordinates. prior to qtl mapping, pseudo-markers were added to the map using insert_pseudomarkers with a step size of cm and genotype probabilities were calculated using calc_genoprob. a kinship matrix was calculated using the calc_kinship function using ge- notype probabilities. a thinned subset of markers obtained using calc_grid (step = ) and probs_to_grid was used as input for the calc_kinship function. qtl scans were carried out using scan and suggestive qtl peaks were identified using find_peaks (drop = , peakdrop = , threshold = ). traits with approximately normal distributions (fl, cf, wt, pca , pca ) were mapped using a mixed linear model with the kinship matrix included as a random effect (model = “normal” and kinship options in qtl ). presence- absence of vermiculations and spots (vpa) was mapped as a binary trait (model = “binary” in qtl ). the kinship matrix was not included as a random effect in the binary trait mapping model because this option was not available in qtl . for each identified lod peak, % credible intervals were calculated using the function find_peaks (prob = . , peakdrop = , threshold = ). finally, p-values were calculated by comparing observed lod scores for each peak with a null distribution obtained from permuting the data times. permutations were carried out using the function scan perm using the same settings as the original tests and p-values were calculated using the ecdf function. the proportion of phenotypic variation explained (pve) by each qtl peak was calculated from lod scores and sample sizes using the equation pve ¼ ð nÞ�lod (broman and sen ). candidate genes for significant lod peaks (p , = . ) were identified by mapping rad loci within % credible intervals to the arctic char genome and determining the three genes closest to each mapping position using the program bedtools closest v . (quinlan and hall ). genes were considered candidates if they were within kb of the mapping position of a rad locus falling within the identified qtl mapping interval. we also mapped the sex determination region using the binary trait model using qtl and assessed significance using the same methodology described above. the sexually dimorphic on the y chromosome gene (sdy) is believed to underly sex determination in lake trout and some other salmonids (yano et al. ). we designed a sdy presence-absence melt curve qpcr assay (similar to anglès d’auriac et al. ) using the lake trout sdy and s primers described in yano et al. ( ). s served as an internal amplification control. each reaction was carried out using a . um concentration of primers sdye s (cccagcactgttttcttgtctca) and sdye as (tgctctctgttgaagagcatcac), and a . um concentration of primers ss (gtycgaagacgatcagatacc- gt) and sas (ccgcataactagttagcatgccg). reaction volumes were ul and contained ul of forget-me-not evagreen qpcr mastermix ( , biotium, fremont, california), . ul of template dna, and . ul of primers eluted in water. s and sdy were amplified in a two-step multiplex reaction using a -minute heat activation step at � followed by cycles of denaturation at c for sec and annealing/extension at � for sec. melt curve analysis was carried out on pcr product for temperatures between � and � using . � temperature shifts and a sec pause between tem- perature shifts. we first tested the assay on a subset of individuals of known sex ( males and females), including the parents used for crosses, in order to verify that males and females could consis- tently be differentiated based on the presence of a male specific sdy peak in the derivative of the melt curve. offspring from all diploid families were subsequently genotyped using the same reaction con- ditions described above. at least one known male and one known female were included on each plate as a control. sex locus mapping was carried out with sdy presence being coded as and sdy absence coded as . | s. r. smith et al. https://github.com/enormandeau/mapcomp https://github.com/enormandeau/mapcomp recombination rate estimation we estimated sex averaged recombination rates for each chromo- some by performing a simple linear regression of pairwise physical distance (base pairs) against genetic distance (cms) and requiring the intercept to pass through . in order to evaluate a pair of rad loci, we required that they map to the same chromosome on the arctic char, rainbow trout, or atlantic salmon genome assemblies and only retained scaffolds and chromosomes with greater than mapped rad loci for which the mapping quality was . for each lg, pairs of rad loci mapping to the same chromosome were randomly sampled from all possible pairs and recombination rate (cm/mb) was estimated using the slope of the resulting regression. this process was repeated times using alignments against the arctic char, rainbow trout, and atlantic salmon genomes. the mean of the distribution of estimates was reported as the chromosome specific recombination rate, and separate values were reported for alignments against the three different divergent reference genomes. regressions were carried out using the r-package lm and recombi- nation rate estimates were visualized using ggplot v . . (wickham and chang ). this process was repeated for male and female maps in order to obtain sex specific chromosomal recombination rates. data availability sequencing data for all individuals used for linkage and qtl map- ping has been made publicly available in a ncbi sequence read archive (prjna ). map information, phenotypes, and geno- types used for qtl and sex locus mapping are available in supple- mentary material (document s ). supplemental material available at figshare: https://doi.org/ . /g . . results bioinformatics and genotyping we obtained a mean of , , demultiplexed paired end (pe) reads for offspring from diploid crosses (range = , – , , , sd = , . ) and a mean of , , reads for parents (range = , , – , , , sd = , . ). on average, . % of reads were removed by clone_filter for these individuals. de novo assembly of rad loci with gstacks produced , rad loci ranging in size from to bp in length. between . % and . % of reads were mapped to de novo assembled rad loci (mean = . %, sd = . %) using bwa mem. the lepmap genotyping pipeline reported genotype probabilities for , snps, , of which were informative for linkage mapping. of those, , snps passed missingness, segregation distortion, and minor allele frequency filters. for gynogenetic diploid offspring, we obtained an average of , , pe reads (range = , , – , , , sd = , , ). we generated , , reads for the mother of this family. on average, . % of reads for these samples were removed by clone_ filter. between . % and . % of those reads were mapped to the de novo assembly using bwa mem (mean = . %, sd= . %). after genotyping with freebayes and filtering data to remove non-informative markers, we identified snps that were informative for half-tetrad analysis. linkage and centromere mapping we were able to assign , rad loci to lgs with between and loci mapped to each lg (figure , table , table s ). the total male map length was . cm and the female map was . cm (overall female:male map ratio = . ). male lg map lengths ranged from . cm - . cm, while female lg map lengths ranged from . cm – . cm (table ). snps were mapped to between and unique positions on linkage groups. as expected, we identified lgs, of which were metacentric and that were acrocentric or telocentric (table s , table s ). these linkage groups likely correspond to the chromosomes identified by previous karyotyping studies (phillips and zajicek ; reed and phillips ). half-tetrad analysis yielded centromere intervals for of metacentric chromosomes and of lgs identified as acrocen- tric or telocentric (figure , figure s , table s ). rfm analysis identified centromeres for of metacentric chromosomes and of acrocentric or telocentric chromosomes (figure , table s ). we were ultimately able to determine the location of centromeres for out of chromosomes using the two methods. we were not able to map the centromere for sna ; however, this chromosome is likely acrocentric or telocentric based on the size of the linkage group relative to others (table ) and karyotyping work suggesting the existence of acrocentric and telocentric chromosomes (phillips and zajicek ; reed and phillips ). homology analysis alignment of the linkage map to divergent salmonid reference genomes revealed that the resulting map was highly congruent with existing assembled salmonid genomes (figures s -s ). large synteny blocks were detected between lake trout linkage groups and the arctic char genome for linkage groups sna -sna (table ). alignments suggested that sna is syntenic with sal ; however, fewer than loci with mq mapped to this chromosome from sna . syntenies were detected between lake trout and all rainbow trout and atlantic salmon chromosomes. mapcomp identified homologies with all brook trout linkage groups identified by sutherland et al. ( ) (figure s ). the lake trout karyotype is differentiated from arctic char by multiple robertsonian translocations including one possible chro- mosomal fusion (sal . and sal . ) and four chromosomal fissions (sal , sal , sal , sal q. . ). sal . and sal . are fused and sal q. . is split into two lgs in lake trout, similar to the arctic char linkage map presented by nugent et al. ( ). the two salvelinus species are also differentiated by at least putative chromosomal inversions (table ), primarily on acrocentric or telocentric chromosomes. arctic char chromosome sal in par- ticular appears to be the result of a fusion between sna and sna . sna also contains multiple chromosomal inversions that differentiate the two karyotypes (figure ). with the exception of inversions detected on sna , all putative inversions differenti- ating the two species were found to be near, or overlapping, the centromere (n = , table ). mapcomp results suggest inversions on sna , sna , sna , and sna are shared with brook trout; however, large inversions differentiating brook trout and lake trout were identified on sna (brook trout bc ), sna (brook trout bc ), and sna (brook trout bc ). trait mapping multiple quantitative trait loci were detected for the evaluated traits (table , figure ). a highly significant qtl for presence of spots and vermiculations mapped to a sex-averaged position of cm on sna (vpa , % ci = - . cm, lod = . , p = . ). we identified candidate genes associated with this peak, including melanore- gulin-like (mreg-l, arctic char scaffold nw_ . : - ; sna , . cm). a second qtl for this trait mapped . volume june | high-density lake trout linkage map | https://doi.org/ . /g . cm on the same linkage group (vpa , % ci = . – . , lod = . , p = . ). a total of candidate genes were identified within this qtl credible interval. the four genes closest the highest lod value were transcription factor -like (tcf -l), retinoic acid induced -like (rai -l), sterol regulatory element binding factor -like (srebf -l), and calcium channel voltage-de- pendent t type alpha i subunit-like (cacna i-l; table s ). these qtl explained . and . % of phenotypic variance, respectively (table ). significant qtl for fork length mapped to two locations on sna (fl , . cm, % ci = . – . cm, lod = . , p = . , and fl , . cm, % ci = . – . cm, lod = . , p = . ) and one location on sna (fl , . cm, % ci = . - . cm, lod = . , p = . ). a significant qtl for condition also mapped to . cm on sna (cf , % ci = . – . , lod = . , p = . ) and a qtl for weight mapped to . cm on sna (w , % ci = . - . cm, lod = . , p = . ). suggestive qtl (lod . , p . . ) were detected on sna ( . cm, % ci = . – . , lod = . , p = . ) and sna ( . cm, % ci = . – . cm, lod = . , p = . ) for weight and condition factor, respectively. we identified candidate genes associated with peak fl , genes associated with fl , and genes associated with fl . we did not search for candidate genes for other growth and body condition related qtl (weight and condition factor) because the locations and credible intervals for these qtl overlapped almost perfectly with those detected for fork length (table ). suggestive qtl were detected for the pca body shape variable on sna ( . cm, % ci = . – . , lod = . , p = . ), sna (pca _ , . cm, % ci = . – . cm, lod = . , p = . ), and sna ( . cm, % ci = – . cm, lod = . , p = . ); however only the peak on sna was statistically signif- icant. suggestive qtl were detected for pca on sna ( . cm, % ci = . – . , lod = . , p = . ), sna ( . cm, % ci = . – . cm, lod = . , p = . ), and sna ( . cm, % ci = – . cm, lod = . , p = . ); however, none of these qtl were found to be statistically significant. candidate genes were identified for the significant qtl interval for pca on sna . our sdy presence-absence assay produced a male specific peak in melt curve derivative plots at approximately �. a melt curve derivative peak existed for s at approximately �. of the known males used to validate the assay, produced sdy peaks and produced s peaks. the known females tested all yielded s peaks; however, sdy peaks were absent in all females as expected. we were ultimately able to determine genotypic sex for offspring produced from diploid crosses. mapping sdy pres- ence-absence as a binary trait using qtl identified strong peaks of association at . cm ( % ci = . – . cm, lod = . , p , . ) and . cm ( % ci = . – . cm, lod = . , p , . ) on sna . recombination rates sex averaged recombination rates estimated by alignment to the arctic char genome ranged from . to . cm/mb with a mean of . cm/mb (table s ; sd = . ) across lgs. in general, recombination rate estimates generated by mapping to the arctic char genome were lower than those obtained from mapping to rainbow trout or atlantic salmon genomes (table s , figure s ). consistent with previous studies on salmonids, male recom- bination rates were considerably lower than those observed for females (figure s ). for example, the mean male recombination rate base on alignment to the rainbow trout genome was . cm/mb (sd = . ), while the mean female recombination rate was . cm/mb (sd = . ). alignment of male and female linkage maps to divergent reference genomes demonstrated that figure map locations of , rad loci along lake trout linkage groups. orange boxes highlight centromeres identified using half tetrad analysis with a y-threshold of . . blue boxes span the intervals of centromeres identified using the rfm method (limborg et al. ) combined with half-tetrad anal- ysis. locations are in centimorgans on the female linkage map. | s. r. smith et al. male recombination is highly suppressed except for near telomeres (figures s -s ). discussion map evaluation multiple lines of evidence suggest this linkage map provides an accurate representation of the lake trout genome. first, we identified a single centromere for each chromosome (except sna ), suggesting that linkage groups were appropriately split. in all cases, centromere mapping locations derived from half-tetrad and rfm analysis either overlapped or were in close proximity (figure , table s ). for acrocentric and telocentric chromosomes, centromeres always mapped to the end of the chromosome with the highest female recombination rate and lowest male recombination rate, which matches results from previous centromere mapping efforts in salmonids suggesting that male recombination occurs almost exclusively near telomeres (moen et al. ; miller et al. ; mckinney et al. ). second, our homology analysis demonstrated a high degree of contiguity between existing genome assemblies and the map presented here (figures s -s ). finally, our sex determination locus mapping results are concordant with cytogenetic studies. previous cytoge- netic analysis of male and female lake trout identified sex-specific quinacrine and c-banding patterns on a large submetacentric chromosome (phillips and ihssen ). mapping sdy presence- absence using the linkage map demonstrated with high certainty that the sex locus exists near one of the telomeres of sna (figure ), which is metacentric or submetacentric based on rfm and half tetrad analysis (figure ). although two significant peaks were detected on this lg, they were in close proximity and credible intervals were adjacent, suggesting that they likely represent a single peak of association. this study and others suggest that salvelinus n■ table summary statistics for each of the constructed linkage groups. no. mapped loci corresponds to the number of unique rad contigs mapped to each linkage group. male and female map lengths are in centimorgans (cm). no. unique positions corresponds to the number of unique linkage map positions to which rad loci were assigned. female:male ratio is the ratio of female length and male length in centimorgans. name no. mapped loci male length (cm) female length (cm) female:male ratio no. unique positions sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . sna . . . volume june | high-density lake trout linkage map | species, and salmonids in general, have highly variable sex chro- mosome configurations. specifically, the brook trout sex determi- nation locus maps to a region that is homologous to the arctic char sex chromosome; however, it localizes to a different arm (sutherland et al. ), while the lake trout sex chromosome identified here lacks homology with those of all species examined (sal p. – nugent et al. ; bc – sutherland et al. ; ssa , ssa , and ssa – kijas et al. ; omy – pearse et al. ). many previous studies have identified variation in sex locus map- ping position both within and between salmonid species (woram et al. ; lubieniecki et al. ; sutherland et al. ; kijas et al. ), even though the same gene ultimately underlies sex de- termination in most cases (yano et al. ). our results add to a growing body of literature suggesting that sdy is a conserved, yet highly mobile, sex determination gene in salmonids. furthermore, the lake trout linkage map presented here is of similar density to those used to scaffold genome assemblies for other salmonids (christensen et al. a, b) and provides valuable information on the order of loci along chromosomes and recombi- nation rates between loci. in general, male:female map length ratios and estimated sex averaged map lengths were highly similar to those observed for other salmonids. for instance, leitwein et al. ( ) found that chromosome specific recombination rates varied from . – . cm/mb (mean = . ) for brown trout, compared with . to . cm/mb (mean = . ) for lake trout based on mapping linkage mapped rad loci to the arctic char reference genome. similar to other salmonids, we observed pronounced het- erochiasmy, with male recombination being almost entirely sup- pressed except for near telomeres (moen et al. ; moen et al. ; mckinney et al. ; leitwein et al. ) n■ table synteny between lake trout linkage groups and arctic char, rainbow trout, atlantic salmon, and brook trout genomes. arctic char, rainbow trout, and atlantic salmon chromosomes were recorded if more than rad contigs from the lake trout linkage group were aligned to a chromosome with a mapping quality of . brook trout linkage groups were recorded if more than aligned markers were detected by mapcomp. graphical depictions of alignment location vs. linkage map position are available in supplementary figures s -s . lake trout arctic char rainbow trout atlantic salmon brook trout sna sal omy ssa , ssa bc sna sal omy ssa bc sna sal omy ssa , ssa bc , bc sna sal omy , omy ssa , ssa bc sna sal . , sal . omy , omy ssa bc sna sal omy ssa bc sna sal omy , omy ssa , ssa bc sna sal omy , omy ssa , ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy , omy ssa , ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal q. : omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal q. omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal q. : omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal p omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal omy ssa bc sna sal a omy ssa bc a sal appears to be homologous with sna , however fewer than rad contigs mapped to this chromosome. | s. r. smith et al. structural variation combining centromere mapping locations with synteny analysis revealed that lake trout are differentiated from arctic char, rainbow trout, and atlantic salmon by multiple pericentric inversions, sug- gesting that centromeric instability (specifically on acrocentric and telocentic chromosomes) is potentially an important component of the salmonid evolutionary legacy (table ). future work should evaluate whether these detected inversions are truly species diagnostic or if they are polymorphic within species. large structural variants have previously been found to be associated with adaptive differen- tiation and life history variation within rainbow trout (miller et al. ; pearse et al. ) and inversions can contribute to pre or post- zygotic isolation between species or ecotypes (kirkpatrick ). future studies should evaluate the extent to which the structural variation detected here contribute to reproductive isolation and adaptive divergence within and between salmonid species. sna presents one of the most striking examples of extensive structural variation in the genus salvelinus, with multiple paracentric and pericentric inversions differentiating the lineages containing arctic char and lake trout (figure ). with the exception of a putative inversion located between and cm, all other inversions on this lg were not observed in other salmonid species examined, suggesting that the other inversions on this chromosome (sna , ssa ; figure , column ) are fixed or segregating within the arctic char lineage or within the salvelinus clade containing arctic char, bull trout (s. confluentus), dolly varden trout (s. malma), and white char (s. albus). this hypothesis is supported by results from mapcomp which suggested that brook trout, the most closely related extant species to lake trout (crête-lafrenière et al. ), and lake trout are not differentiated by any inversions on this linkage group. a large inversion spanning the centromere of sna shows clear evidence of being differentially fixed between lake trout and all other salmonids except brook trout (figure , figures s -s , figure s ). inversions located on sna , sna , and sna also appear to be differentially fixed between the lake trout – brook trout lineage and all other salmonids; however, interpretation is complicated by subsequent translocations and inversions that occurred in other taxa (table ). a large pericentric inversion on sna appears to differentiate lake trout from all other salmonids, including brook trout (figure s ). mapcomp results also suggest that two large inversions on sna (homologous to the brook trout sex chromosome - bc ; sutherland et al. ) and sna (bc ) differentiate lake trout from closely related brook trout (figure s ). it is unclear if these structural variants are truly fixed between species, or if they might be polymorphic within lake trout or brook trout. the inversion polymorphisms identified above could be associated with chromosomal speciation within the genus salvelinus or adaptive divergence within salmonid species and warrant further examination. the majority of detected inversions differentiating lake trout from other salmonids are pericentric, which is not entirely unexpected. repeat-rich eukaryotic centromeres often demonstrate exceptionally high rates of evolution (henikoff et al. ) and are prone to chromosomal breakage and the accumulation of structural variation (kalitsis and choo ; barra and fachinetti ). sutherland et al. ( ) also identified evidence for multiple inversions differentiating salmonid species, including one pericentric inversion differentiating pink, chum, and sockeye salmon from other salmonids. evidence for f inviability and reduced reproductive success between hybrids are widespread (stebbins ), including for pairs of closely related species within the salmonid lineage (renaut and bernatchez ). bull trout and brook trout, for instance, readily produce f offspring but f offspring are rarely observed (leary et al. ). hybrids between westslope cutthroat (oncorhynchus clarkii lewisi) and rainbow trout are viable but have dramatic reductions in reproductive success (muhlfeld et al. ). future work should evaluate if instances of reduced fitness in inter or intraspecific salmonid hybrids might be linked to combined deleterious effects of recombination at multiple pericentrically inverted loci. it would also be interesting to ascertain whether centromeric regions tend to harbor signals of adaptive divergence between salmonid species and morphotypes. for example, ellegren et al. ( ) found elevated levels of divergence between fidecula flycatcher species near centro- meres. given the prevalence of pericentric inversions on acrocentric and telocentric chromosomes, we also might expect these loci to be associated with adaptive ecophenotypic radiations that have oc- curred within salvelinus (eshenroder ) and coregonus (lu and bernatchez ). genomic basis for adaptive traits suggestive qtl for traits that differentiate lake trout morphotypes were detected on multiple linkage groups. this supports the hypoth- esis proposed by perreault-payette et al. ( ) that ecophenotypic divergence in lake trout has a polygenic basis. our results suggest that the presence or absence of spots and vermiculations is controlled by either one or two loci on the same arm of linkage group sna . a search for candidate genes within the qtl mapping intervals iden- tified melanoregulin-like (mreg-l) as a potential causal locus. the homolog of this gene is involved in the transfer of melanosomes from melanocytes to keratinocytes (wu et al. ), and appears to control the distribution of pigments within mice hair (o’sullivan et al. ). pigmentation polymorphisms are common in lake trout (wilson and mandrak ; zimmerman et al. ) and other trout and char (gomez-uchida et al. ), although it is unclear if the genes identified here explain skin pigmentation variation in other species and populations. skin pigmentation variation has been shown to be associated with depth of capture in multiple lake trout populations and is hypothesized to be adaptive in some environments (protas and patel ); however, it is also possible that the trait is simply linked with some other adaptive traits. pigmentation patterns are often linked to variation in behavior, immune response, and energy homeostasis in vertebrates, likely owing to pleiotropic effects of melanocortins (ducrest et al. ). pigmentation traits have also been linked to stress response in rainbow trout, atlantic salmon, and arctic char (hoglund et al. ; kittilsen et al. ). suggestive qtl for the composite body shape variable with the highest explanatory power (pca ) were detected on sna , sna , and sna . interestingly, each of these chromosomes appear to have undergone structural reorganization in relatively recent evolutionary history, based on alignment to the arctic char genome (figure s ). specifically, sna and sna are fused in arctic char and sna is split into two chromosomes. sna in particular appears to have accu- mulated multiple large inversions that differentiate this linkage group from the homologous region of the syntenic arctic char chromo- some. a qtl for condition factor, which is closely related to body shape (froese ), has been previously detected on the brook trout linkage group homologous to sna (linkage group bc , sutherland et al. ). additional mapping crosses, ideally generated using ancestral populations with highly differentiated body shapes (leans vs. siscowet or divergent hatchery strains for example) would be valu- able for further validating the existence of qtl detected here and volume june | high-density lake trout linkage map | improving our understanding of the genetic basis for adaptive di- vergence within lake trout. growth and body condition related traits all have suggestive qtl on linkage groups sna and sna , indicating that genes on these chromosomes likely harbor variation that underlies differences in growth between populations. linkage group sna also appears to harbor an inversion that differentiates lake trout from other salmonid species examined, including brook trout. a previous study identified a putative growth rate qtl on the brook trout linkage group homol- ogous to sna (figure s ; sutherland et al. ; bc ). the same study identified a stress response qtl, measured as change in blood cortisol levels following handling stress, in brook trout on the chromosome homologous to sna (sutherland et al. , bc ). increased cortisol levels have been found to be negatively corelated with growth and condition factor in other salmonids (barton et al. ; reinecke ), suggesting that variation observed in our families could actually be due to variation in stress response. there is evidence for variation in fitness among lake trout hatchery strains used to supplement and restore lake trout populations in the great lakes, with the strain from seneca lake, new york appearing to have a fitness advantage (scribner et al. ). great lakes lake trout populations are heavily impacted by predation by invasive sea lamprey and previous work has shown that larger individuals have a greater probability of surviving lamprey attack (swink ). similarly, size-selective fisheries have also been shown to impose strong natural selection on growth in multiple species (enberg et al. ). future work could examine whether the chromosomal regions identified here are associated with size-at-age or are under selection in n■ table the first column is the lake trout linkage group in question and columns - list the approximate location of any detected inversions that differentiate species. the type of inversion is stated in parenthesis. locations are listed in centimorgans on the female map. whenever multiple inversions were detected on a chromosome, at least one was pericentric. centromeres were not localized for sna , so centricity of inversions could not be determined. linkage group arctic char rainbow trout atlantic salmon sna — — — sna — — — sna — — — sna — — — sna — — — sna — — - (paracentric) sna — — — sna — — — sna — — — sna - (pericentric) �� � sna - (pericentric) - (pericentric) - (pericentric) sna - (pericentric) �� - (pericentric) sna — — — sna — — — sna — — — sna — - (multiple inversions)�� - (multiple inversions) sna — — sna — — sna — - (paracentric), - (pericentric) - (pericentric) sna — - (pericentric) - (pericentric) sna — — — sna — — — sna — — — sna - (multiple inversions) - (pericentric) - (pericentric) sna — — - (pericentric) sna — — — sna — — — sna — — - (pericentric) sna — — — sna � - (pericentric) — sna — — - (pericentric) sna — — — sna — — — sna - (pericentric) � � sna — - (pericentric) — sna — — — sna — — — sna — — — sna — — — sna — — — sna � — — sna — - (unknown) - (unknown) � = suggestive evidence of structure variation but unable to determine if an inversion occurred. �� = inverted region appears to be translocated to a separate chromosome. | s. r. smith et al. populations experiencing lamprey predation or size-selective fisheries. associations between environmental conditions and pheno- type have been observed across the lake trout range and across salmonid species for the afore mentioned traits, suggesting adap- tive significance in some contexts. for example, patterns and intensity of skin pigmentation, along with divergence in other traits, is commonly associated with depth-of-capture in both lake trout (zimmerman et al. ; marin et al. ) and arctic char populations (gomez-uchida et al. ). variation in skin pigmen- tation is potentially involved with predator avoidance and camou- flage, feeding behavior, mate choice (protas and patel ), or protection from ultraviolet radiation (yan et al. ). differences in age specific growth rates are also frequently observed between humper, lean, and siscowet-like lake trout morphotypes (burnham- curtis and bronte ; hansen et al. ) — as well as between arctic char morphotypes (jonsson et al. ; snorrason et al. ; adams et al. ). these differences in growth rate likely reflect figure examples of two linkage groups (sna and sna ) with evidence of inversions differentiat- ing lake trout from other salmonids. female lake trout linkage groups are colored blue (top curves). male lake trout linkage groups are colored red (bot- tom curves). sna (first column) is differentiated from all homologs by a single large pericentric in- version spanning - cm on the female linkage map (left side of each panel). sna is differentiated from omy and ssa by an inversion spanning - cm on the female map. it is unclear if the same inversion exists in arctic char due to extensive structural dif- ferentiation relative to lake trout and other salmonids (sna vs. sal ). n■ table linkage map positions (cm) of qtl peaks detected for the sex determination locus, presence-absence of vermiculations and spots, fork length, shape variable pca , shape variable pca , weight, and condition factor (trait column). ci_low and ci_high are the upper and lower bounds of the % credible interval for map positions for each qtl peak. lg is the linkage group on which the qtl was detected. model lists the model used for qtl mapping in r/qtl . positions are sex averaged map positions. lod scores are the differences in log likelihoods for models assuming presence or absence of a qtl at the locus in question (reported by r/qtl ). the estimates proportion of phenotypic variance explained by each qtl peak is listed in the pve column. estimated additive and dominant effects for the peak in question are also listed. p-values are those obtained via the permutation test described. trait lg position (cm) ci_low ci_high model lod additive effect dominance effect pve p-value sex sna . . . binary . . . . , . ��� sex sna . . . binary . . . . , . ��� vermiculation sna . . . binary . . . . . ��� vermiculation sna . . . binary . . . . . � fork length sna . . . normal . . . . . � fork length sna . . . normal . . . . . � fork length sna . . . normal . . . . . � pca sna . . . normal . . . . . pca sna . . . normal . . . . . � pca sna . . . normal . . . . . pca sna . . . normal . . . . . pca sna . . . normal . . . . . pca sna . . . normal . . . . . weight sna . . . normal . . . . . weight sna . . . normal . . . . . � condition sna . . . normal . . . . . � condition sna . . . normal . . . . . volume june | high-density lake trout linkage map | variation in allocation of resources toward growth and reproduction, adaptation to nutrient stress (arendt ), or plastic responses to environmental variation (hindar and jonsson ). morphotypes can also often be differentiated based on body shape differences, which are hypothesized to be optimized for different feeding behaviors and modes of locomotion (bond ; muir et al. ; perreault-payette et al. ). for example, the streamlined body shape of leans has been hypothesized to be adaptive for swimming and predation in shallower nearshore environments (bond ; muir et al. ), while the more deep-bodied shape of siscowet lake trout is believed to reflect adaptation for vertical migration and foraging in deep-water habitats (webb ; muir et al. ). morphotypes with traits reflecting those observed in the species native range have the potential to emerge rapidly in some introduced invasive populations (stafford et al. ), suggesting a high degree of phenotypic plasticity or exceptionally strong selection favoring divergence. unfortunately, many lake trout metapopulations of conservation concern have experienced reductions in abundance and decreases in ecophenotypic diversity as a result of overexploitation and introduc- tion of invasive species (krueger and ihssen ; hansen ). for example, in the great lakes the siscowet morphotype has been extirpated from all lakes except lake superior (krueger and ihssen ). the results presented here enhance understanding of the genetic architecture of traits that underlie trophic specialization in lake trout and could aid in restoring genetic and phenotypic diversity in lakes where it has been lost. conclusions we identified multiple structural variants potentially involved in speciation and adaptation within the genus salvelinus, mapped the lake trout sex determination locus, and identified qtl for traits believed to be adaptively significant in lake trout populations. future work should use additional qtl mapping crosses and association studies in wild populations to evaluate if the qtl identified here are consistently associated with the phenotypic variation exam- ined, as well as other phenotypes that differentiate lake trout morphotypes. trophically specialized lake trout morphotypes and adaptively diverged populations are differentiated by multiple other traits (i.e., tissue lipid content, fin size, diet; thurston ; eschmeyer and phillips ; zimmerman et al. ; zimmerman et al. ). qtl mapping studies using later gen- eration crosses or genome-wide association studies in wild pop- ulations would be particularly useful for fine-scale localization of genotype-phenotype associations within qtl credible intervals identified here. additionally, qtl mapping efforts can yield different results for different families and the genetic basis for some traits often varies across populations (santure et al. ). the lake trout linkage map will allow further examination of the genetic basis for ecophenotypic variation in lake trout and will enable additional exploration of chromosomal evolution within the genus salvelinus. perhaps most important, this resource will allow for the assembly of a chromosome-anchored reference genome for lake trout, which will greatly facilitate future genomic research on this important species. acknowledgments we would like to thank the staff of pendills creek national fish hatchery and the codrington fisheries research facility for their assistance with producing mapping crosses. we would also like to thank simon bernatchez for his assistance with collecting photo- graphs and two anonymous reviewers for their thoughtful comments and suggestions. ss, sa, ks, and gl were supported by award _ scr_ from the great lakes fisheries commission. gl was also supported in part by the national science foundation grant nsf- dob- . figure panels display lod values on the y-axis vs. sex averaged map position (cm) for qtl scans for (a) the sex determination locus, (b) presence of spots and vermiculations, (c) fork length, (d) weight, (e) condition factor, (f) pca , and (g) pca . the dashed red line corresponds to the p , . significance threshold for lod scores. the solid green line cor- responds to the lod threshold of used to identify peaks putatively associated with each trait. | s. r. smith et al. literature cited adams, c. e., d. fraser, f. a. huntingford, r. b. greer, c. m. askew et al., trophic polymorphism amongst arctic charr from loch rannoch, scotland. j. fish biol. : – . https://doi.org/ . /j. - . .tb .x adams, d. c., and e. otárola‐castillo, geomorph: an r package for the collection and analysis of geometric morphometric shape data. methods ecol. evol. : – . https://doi.org/ 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initial review: january , ; accepted: february , p seudohypoparathyroidism (php) is a group of disorders characterized by resistance to parathyroid hormone, resulting in hypocalcemia. several types of php have been identified: type a- presents with features of albright hereditary osteodystrophy (aho - short stature, obesity, round facies, subcutaneous ossifications, brachydactyly and mental retardation in some patients) and types b and (without features of aho) [ ] . we report a patient with php without features of aho who developed hypothyroidism on follow up. case report an -years-old girl presented with involuntary inward turning of thumb and tingling in hands for months.she had similar complaints one year back, which were diagnosed as hypocalcemia (serum calcium- . mg/dl). she was treated with calcium supplements for months following which she had temporary relief. she weighed kg and measured cm in height (within normal centiles). examination did not reveal skeletal abnormalities or dysmorphism. chvostek’s and trousseau’s sign were positive. laboratory investigation revealed serum calcium . mg/dl, ica++– . mmol/dl (low), serum phosphorous . mg/dl(high) and alkaline phosphatase iu/ml (normal levels). levels of parathyroid hormone were very high (ipth- pg/ml; normal value: . - . pg/ml). complete blood count, oh-vitamin d, and liver and renal function tests were normal. thyroid functions (tft) were normal [free(f)t – . pg/ml; ft – . ng/dl; tsh- iu/ml]. she was treated with calcium supplements and calcitriol. on follow up, serum calcium levels and urinary calcium/ creatinine were monitored to achieve good calcemic control and to prevent calciuria . tft were monitored six monthly. she developed hypothyroidism (tsh: . iu/ ml; ft : . pg/ml; ft : . ng/dl) two years after diagnosis, for which she was treated with thyroxine mcg daily and subsequently increased to mcg. she is now ½-year old, having normal growth and pubertal changes (thelarche). the underlying pathophysiology in php is a defect at the pth-receptor (genetic mutation in the alpha subunit of the receptor g-protein causing altered messenger action), leading to end organ resistance to the action of pth. resistance to other hormones (which function via the alpha subunit of g protein), most commonly thyroid- stimulating hormone and rarely gonadotrophins and growth hormone releasing hormone may be seen in type a [ , ]. php ib typically shows no other endocrine abnormalities, although resistance to thyroid-stimulating hormone (leading to primary hypothyroidism) has been reported rarely [ , ] php type does not present with any other hormone resistance . our patient probably had php type b. php ib is caused by deletions in the differentially methylated region (dmr) of the gnas locus, located on chromosome q . . it is mostly a sporadic disorder, but sex-influenced autosomal dominant inheritance has been reported [ ]. parents of our patient did not have any phenotypic or biochemical evidence of php. php type ib is characterized by renal pth resistance whereas pth responsiveness is preserved in the bone and other tissues resulting in lack of aho features.measurement of camp excretion after pth infusion is a differentiating feature between type b and (decreased in type b and normal in type ) [ ]. however, this test was not affordable. indian pediatrics volume __august , case reports administration of oral calcium and calcitriol remains the mainstay of treatment. the goals of therapy are to maintain serum calcium levels within the reference range so as to avoid hypercalciuria. thyroid function tests should be evaluated periodically even in absence of features of aho, as hypothyroidism develops rarely, as seen in our patient. acknowledgement: we are grateful to dr y k amdekar , medical director b j wadia hospital for children for allowing us to publish this case report. contributors: rj diagnosed and treated the patient. mk was involved in treatment of the patient. both of them wrote the article. funding: none; competing interests: none stated. references . mantovani g, spada a. mutations in the gs alpha gene causing hormone resistance. best prac res clin endocr metab. ; : - . . “online mendelian inheritance in man (omim), a knowledgebase of human genes and genetic disorders”. nucleic acids research (database issue): d –d . available from: http://en.wikipedia.org/wiki/omim. accessed july , . mariot v, maupetit-mehouas s, sinding c, kottler ml, linglart a. a maternal epimutation of gnas leads to albright osteodystrophy and parathyroid hormone resistance. j clin endocr metab. ; : - . . levine ma, downs rw jr , moses am, breslau na, marx sj, lasker rd, et al. resistance to multiple hormones in patients with pseudohypoparathyroidism. association with deficient activity of guanine nucleotide regulatory protein. am j med. ; : - . . heinsimer ja, davies ao, downs rw, levine ma, spiegel am, drezner mk, et al. impaired formation of beta-adrenergic receptor-nucleotide regulatory protein complexes in pseudohypoparathyroidism. j clin invest. ; : - . chronic myeloid leukemia in a child with iga nephropathy amish udani, vijayakumar mahalingam, prahlad nageswaran and *sudha ekambaram from the department of pediatric nephrology and *pediatrics, mehta children’s hospitals, chennai, india. we report an year old boy with iga nephropathy developing chronic myeloid leukemia on follow-up. this association suggests that a b cell defect might be involved in the pathogenesis of these two conditions. key words: chronic myeloid leukemia, iga nephropathy. correspondence to: dr m vijayakumar, consultant pediatric nephrologist, mehta children’s hospitals, no. (e) mc nichols road, rd lane, chetput, chennai , tamilnadu, india. doctormvk@gmail.com, received: september , ; initial review: september , ; accepted: february , . t here is increasing evidence of abnormal glycosylation of immunoglobulin a (iga ) subclass due to b-cell defect in the pathogenesis of immune-complex mediated iga nephropathy [ ]. the occurrence of iga nephropathy and leukemia has been reported rarely in children [ ]. here we report a child with iga nephropathy developing chronic myeloid leukemia (cml) on follow-up. case report an -yr-old boy was diagnosed acute nephritic syndrome at year back in view of hypertension, hematuria, proteinuria (spot urine protein to creatinine ratio . ), mild renal insufficiency (serum creatinine . mg/dl), and normal serum albumin and cholesterol. he had no anemia, leukocytosis or electrolyte disturbances. he was treated with salt and fluid restriction and oral nifedepine for hypertension. serum complement c level was normal, anti nuclear antibody and anti double standard dna was negative. in view of persistent hypertension, renal insufficiency, microscopic hematuria and proteinuria he was referred for evaluation. on examination, the patient was well nourished (weight kg, and height cm) with periorbital edema and blood pressure / mm hg. systemic examination was normal. urinalysis showed + albumin, red blood cells and up/uc ratio of . . blood investigations showed a creatinine of . mg/dl, albumin . g/dl and potassium . meq/l. ultrasonogram of the kidneys showed normal size kidneys. renal biopsy showed seven glomeruli of which two were completely sclerosed and one showed segmental sclerosis and proliferation. remaining [pdf] col a is associated with arterial stiffness by genome-wide association scan | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /circgenetics. . corpus id: col a is associated with arterial stiffness by genome-wide association scan @article{tarasov col a ia, title={col a is associated with arterial stiffness by genome-wide association scan}, author={k. tarasov and s. sanna and a. scuteri and j. strait and m. orru' and a. parsa and ping-i lin and a. maschio and s. lai and m. g. piras and m. masala and t. tanaka and w. post and j. o'connell and d. schlessinger and a. cao and r. nagaraja and b. mitchell and g. abecasis and a. shuldiner and m. uda and e. lakatta and s. najjar}, journal={circulation: cardiovascular genetics}, year={ }, volume={ }, pages={ - } } 	k. tarasov, s. sanna, + authors s. najjar 	published 	biology, medicine 	circulation: cardiovascular genetics background—pulse wave velocity (pwv), a noninvasive index of central arterial stiffness, is a potent predictor of cardiovascular mortality and morbidity. heritability and linkage studies have pointed toward a genetic component affecting pwv. we conducted a genome-wide association study to identify single-nucleotide polymorphisms (snps) associated with pwv. methods and results—the study cohort included participants from the sardinia study for whom pwv measures were available. genotyping was… expand view on wolters kluwer ahajournals.org save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all tables and topics from this paper 	 table 	 table 	nitroprusside 	affymetrix 	basement membrane 	nucleotides 	single nucleotide polymorphism 	chromosomes, human, pair 	cns disorder 	pulse wave velocity 	arterial stiffness 	chromosomes, human, pair 	morbidity - disease rate citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency lack of association between arterial stiffness and genetic variants by genome-wide association scan 	s. park, j. lee, + authors y. jang 	medicine 	blood pressure 	 	 save alert research feed does the p region affect arterial stiffness? results from a cohort of hypertensive individuals 	f. cesana, s. nava, + authors g. mancia 	medicine 	blood pressure 	 	 save alert research feed a genetic risk variant for myocardial infarction on chromosome p is associated with impaired central hemodynamic indexes. 	r. patel, alanna a morris, + authors a. quyyumi 	medicine 	american journal of hypertension 	 	 	pdf save alert research feed a genetic risk score for fasting plasma glucose is independently associated with arterial stiffness: a mendelian randomization study 	m. gottsäter, g. hindy, m. orho-melander, p. nilsson, o. melander 	medicine 	journal of hypertension 	 	 	 	view excerpt, cites background save alert research feed polymorphisms of col a gene are associated with arterial pulse wave velocity in healthy han chinese and uygur subjects. 	dilare adi, x. xie, + authors bang-dang chen 	medicine 	international journal of clinical and experimental medicine 	 	 	pdf 	view excerpts, cites background save alert research feed genome-wide association study identifies loci for arterial stiffness index in , uk biobank participants 	k. fung, j. ramírez, + authors p. munroe 	medicine 	scientific reports 	 	 	pdf save alert research feed genome-wide association analysis of pulse wave velocity traits provide new insights into the causal relationship between arterial stiffness and blood pressure 	m. rode, a. teren, + authors janne pott 	biology, medicine 	plos one 	 	 	pdf 	view excerpts, cites results save alert research feed common genetic variation in the ′-bcl b gene desert is associated with carotid-femoral pulse wave velocity and excess cardiovascular disease risk: the aortagen consortium 	g. mitchell, g. c. verwoert, + authors j. witteman 	biology, medicine 	circulation. cardiovascular genetics 	 	 	 save alert research feed genetic and cellular aspects of arterial stiffness 	a. benetos, a. aviv, p. lacolley, m. safar, v. regnault 	medicine 	 save alert research feed common single nucleotide polymorphisms of the p phox nadph oxidase subunit do not influence aortic stiffness in young, healthy adults. 	c. vlachopoulos, p. xaplanteris, + authors c. stefanadis 	medicine 	hellenic journal of cardiology : hjc = hellenike kardiologike epitheorese 	 	 	pdf 	view excerpt, cites background save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency fibrillin- genotype is associated with aortic stiffness and disease severity in patients with coronary artery disease 	t. medley, t. cole, c. gatzka, w. y. wang, a. dart, b. kingwell 	medicine 	circulation 	 	 	pdf save alert research feed genome-wide association scan shows genetic variants in the fto gene are associated with obesity-related traits 	a. scuteri, s. sanna, + authors g. abecasis 	biology, medicine 	plos genetics 	 	 , 	pdf save alert research feed newly identified loci that influence lipid concentrations and risk of coronary artery disease 	c. willer, s. sanna, + authors g. abecasis 	biology, medicine 	nature genetics 	 	 , 	pdf save alert research feed effect of a col a sp binding site polymorphism on arterial pulse wave velocity: an index of compliance 	d. brull, l. murray, + authors i. young 	medicine 	hypertension 	 	 	 save alert research feed family-based association tests for genomewide association scans. 	wei-min chen, g. abecasis 	biology, medicine 	american journal of human genetics 	 	 	pdf save alert research feed the t-allele of the c t polymorphism is associated with higher arterial stiffness in young healthy males 	j. nürnberger, a. o. saez, + authors r. schaefers 	medicine 	journal of human hypertension 	 	 	pdf save alert research feed genetic risk for myocardial infarction determined by polymorphisms of candidate genes in a japanese population 	y. yamada, k. kato, + authors y. nozawa 	medicine 	journal of medical genetics 	 	 save alert research feed heritability and a genome-wide linkage scan for arterial stiffness, wave reflection, and mean arterial pressure: the framingham heart study 	g. mitchell, a. destefano, + authors d. levy 	medicine 	circulation 	 	 	 save alert research feed heritability of carotid artery structure and function: the strong heart family study 	k. north, j. maccluer, + authors m. roman 	medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 	pdf save alert research feed the glut gene is associated with serum uric acid levels in sardinia and chianti cohorts 	siguang li, s. sanna, + authors r. nagaraja 	biology, medicine 	plos genetics 	 	 	pdf save alert research feed ... ... related papers 	abstract 	tables and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue research article open access association between bilirubin and cardiovascular disease risk factors: using mendelian randomization to assess causal inference patrick f mcardle *, brian w whitcomb , keith tanner , braxton d mitchell , alan r shuldiner , and afshin parsa abstract background: elevated serum bilirubin has been associated with reduced risk of cardiovascular disease (cvd). however, serum bilirubin is also related with several potential confounders related to cvd, such as obesity. mendelian randomization has been proposed as a method to address challenges to validity from confounding and reverse causality. it utilizes genotype to estimate causal relationships between a gene product and physiological outcomes. in this report, we demonstrate its use in assessing direct causal relations between serum bilirubin levels and cvd risk factors, including obesity, cholesterol, measures of vascular function and blood pressure. methods: study subjects included asymptomatic individuals. study subjects were genotyped at the ugt a * locus, which is strongly associated with bilirubin levels. results: serum bilirubin levels were inversely associated with levels of several cardiovascular disease risk factors, including body mass index (p = . ), ldl (p = . ) and total cholesterol (p = . ). in contrast, ugt a * genotype, a known cause of elevated bilirubin levels, was not significantly associated with any of these traditional cvd risk factors. we did observe an association between genotype and brachial artery diameter (p = . ) and cold pressor reactivity (p = . ). conclusions: our findings imply that the observed association of serum bilirubin levels with body mass index and cholesterol are likely due to confounding and suggest that previously established cvd benefits of increased bilirubin may in part be mediated by the early regulation of vascular structure and reactivity. keywords: bilirubin, mendelian randomization, cardiovascular disease background bilirubin is a metabolic byproduct of the breakdown of hemoglobin degradation which itself must be metabo- lized for appropriate excretion. high levels of bilirubin are associated with decreased risk of coronary heart dis- ease (chd) and cardiovascular disease (cvd) [ ]. while the full spectrum by which bilirubin acts to protect against cvd is not fully understood, there has been evi- dence of protecting against oxidative stress by reducing reactive oxygen species and possibly having additional anti-atherogenetic properties [ , ]. previous studies have reported associations of serum bilirubin levels to cardio- vascular disease risk factors, including total cholesterol and blood pressure [ - ]. serum bilirubin levels have also been associated with socioeconomic and behavioral cvd risk factors such as smoking and alcohol intake [ , ]. however, the nature of these associations is unclear, including the potential for residual confounding among serum bilirubin levels and the associated cvd risk factors due to factors measured poorly or not mea- sured at all. notably, the temporal ordering of variation in these variables is hard to determine, raising chal- lenges in separating ‘cause’ from ‘effect’. genotype is uniquely unaffected by most other epide- miologic risk factors. if one accepts the notion that gen- otypes are in a sense “randomly assigned” during gamete formation, then using genetic variation as an * correspondence: pmcardle@medicine.umaryland.edu division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood street, rm. , baltimore, md , usa full list of author information is available at the end of the article mcardle et al. bmc cardiovascular disorders , : http://www.biomedcentral.com/ - / / © mcardle et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:pmcardle@medicine.umaryland.edu http://creativecommons.org/licenses/by/ . independent variable is attractive. relationships between genotype and outcomes have only limited susceptibility to confounding and in particular are generally not sub- ject to temporal misspecification of ‘cause’ and ‘effect’ (i. e., reverse causality). thus, genetic variation can be used to aid in inferring causality between a gene product and some outcome of interest. if a genetic variant is strongly causal of a measured exposure, the variant can be used as a proxy for exposure during modeling. this approach, known as mendelian randomization, can provide evi- dence of causal inference between two variables [ ]. mendelian randomization has particular utility for eva- luation of biomarkers that are highly influenced by a single gene, are of interest in a disease process, and are highly subject to confounding. the gene ugt a codes for a liver-specific glucoro- nosyl transferase that converts bilirubin into a more water-soluble form the body is better able to excrete. homozygosity at the polymorphic promoter repeat locus ugt a * leads to decreased ability of the ugt a enzyme to metabolize bilirubin and subsequent mild hyperbilirubinuria (also known as gilbert’s syn- drome) [ - ]. due to ugt a * ’s strong causal link to bilirubin, use of the ugt a * genotype to more precisely assess bilirubin - cvd risk factor associations is an attractive and informative application of the men- delian randomization approach. in this study, we describe use of the mendelian rando- mization approach to evaluate relations of serum biliru- bin levels with cvd risk factors and subclinical atherosclerosis in a sample of relatively healthy old order amish individuals from lancaster county, pa. we subsequently contrast the observed crude estimates with those obtained utilizing mendelian randomization to illustrate its use. separating causal from non-causal relations provides evidence of bilirubin’s mechanistic role in reducing cvd. methods study sample the hapi heart study began recruitment in with the goal of identifying genes that interact with environ- mental exposure to alter risk of cvd. the study was carried out in an old order amish community in lan- caster county, pa, and the study sample included individuals aged years and older who were relatively healthy. details of the study aims and recruitment pro- cedures have been previously described [ ]. exclusion criteria included severe hypertension (blood pressure > / mmhg), malignancy, and kidney, liver or untreated thyroid disease. the study protocol was approved by the institutional review board at the uni- versity of maryland, school of medicine and informed consent was obtained from each study participant. physical examinations were conducted at the amish research clinic in strasburg, pa. anthropometric vari- ables, including height, weight, and blood pressure, were assessed by study personnel. a fasting blood sample was obtained for measurement of cholesterol and triglyceride levels. biochemical assays were performed by quest diagnostics (baltimore, md). we also measured resting brachial artery diameter. brachial artery diameter was measured using a linear array ultrasound utilizing a pre- viously described technique [ , ]. briefly, the left bra- chial artery was imaged above the antecubital fossa in the longitudinal plane by continuous d gray-scale ima- ging with an mhz ultrasound (phillips hdi cv). using guidelines established by the international bra- chial artery reactivity task force [ ], all subjects were fasting overnight and none were on any vasoactive med- ications. the diameter of each artery was measured in a blinded fashion by a trained technician. high resolution b-mode ultrasound was carried out to image the right and left common carotid arteries. imt was measured between lumen intima and media-adventitia interfaces of the far wall by a single reader using an automated edge detection system. as part of the study protocol, participants underwent a cold pressor test (cpt). while resting in supine state, participants submerged their right hand and wrist to the ulnar styloid in ice water for . min. blood pressure response to the stimulus was measured by calculating the area under the curving using measurements of both systolic and diastolic blood pressure at baseline (pre- emersion), min and . min. genotyping of ugt a * locus the ugt a * locus was genotyped using the method of borlak et. al. [ ] with slight modification to accom- modate the roche lightcycler . pcr was performed on ng of genomic dna with ul of lightcycler genotyping master in a total reaction volume of ul. all other reagents and concentrations were as per bor- lak; pcr conditions were: denaturation ( °c for min), thermocycling for cycles ( °c for s, °c for s, °c for s), single acquisition and ( °c for min, °c for min, °c continuous, acquisition per °c). two alleles were present at this locus in the amish sample, the allele and the allele, with allele frequencies of . and . respectively. the genotype frequencies were slightly out of hardy weinberg equili- brium (p = . ) due to an over representation of heterozygotes. analytic approach mendelian randomization the mendelian randomization approach exploits the fact that genotype precedes life events and is therefore mcardle et al. bmc cardiovascular disorders , : http://www.biomedcentral.com/ - / / page of not affected by lifestyle, socioeconomic or any factors that follow conception. to the extent that alleles are randomly assigned at gamete formation, genotype may be thought of analogously to assigned treatment in ran- domized trials. as previously reviewed [ ], mendelian randomization is an application of instrumental variable analysis, and with certain assumptions the genotype- phenotype relation can be utilized to attain un-con- founded estimates of the relation between the gene pro- duct and outcomes of interest. these assumptions include an adequately strong relation between genotype and phenotype and the absence of alternate pathways from genotype to the outcome of interest (e.g. pleio- tropy, population stratification, linkage disequilibrium). the first assumption–a strong relation between geno- type and phenotype–has been previously demonstrated for ugt a * and bilirubin levels [ - ] and is reflected in our data by the significant differences in bilirubin levels by genotype. as long as these remaining assumptions are met (see below), using genotype as the exposure in analysis will be analogous to modeling assigned treatment in an intention to treat analysis. causal assumptions figure is a causal diagram portraying the assumptions that underlie the relations among ugt a * genotype, serum bilirubin levels and cvd risk factors. the true causal relationship between serum bilirubin levels and cvd risk factors is of interest, but this relationship may be confounded by factors, either measured or unmea- sured, as shown in figure . these factors represent causes of both the exposure and outcome, and failure to appropriately address confounding will distort the rela- tion of interest [ , ]. one can see that the relationship between ugt a * and cvd risk factors are not effected by the confounders. under the assumption that ugt a * ’s affect is primarily mediated by bilirubin, it’s causal effect can be estimated. any potential effect of the ugt a enzyme to glucuronidate other sub- stance affecting our outcome measures, even if limited, cannot be accounted for in this model. statistical methods the principle of mendelian randomization is illustrated by contrasting the correlations observed between serum bilirubin levels and cvd risk factors without considera- tion for ugt a * genotype with the presumed unconfounded results obtained between serum bilirubin levels and cvd risk factors using a two-stage approach that utilizes the genotype ~ outcome and genotype ~ bilirubin regressions [ ]. the simple (potentially con- founded) estimates were obtained by regressing serum bilirubin levels (the independent variable) on each cvd risk factor (the dependant variable) separately. the mendelian randomization estimate of the serum biliru- bin-cvd risk factor correlation was obtained by dividing the regression coefficient representing the genotype- cvd risk factor effect by the regression coefficient representing the genotype-serum bilirubin effect. all of the participants of the hapi heart study are related. to address the correlations potentially existing in phenotype by virtue of the fact that subjects are related, we accounted for residual familial correlations in phenotype using a variance component regression framework. specifically, we modeled variation in the trait as a function of fixed covariates, a polygenic com- ponent and a normally distributed error component. the polygenic component was derived from the kinship coefficient matrix which describes the relationship of each pair of individuals in the sample. the software package solar was used for the analysis (southwest foundation for biomedical research, san antonio, tx). estimates of associations of cvd risk factors with biliru- bin and with ugt a * genotype were evaluated using this variance components approach. bootstrap sampling was used to estimate empirical % confidence intervals for the mendelian randomization estimates. since the existing literature shows that elevated bilirubin was associated with decrease cvd, we a priori tested for measured cvd risk factors within our cohort, as listed in table . these selected cvd risk factors are highly correlated, so a strict bonferroni correction would potentially result in excessive false negatives. therefore we elected to report uncorrected p-values. results sample characteristics the participants of the hapi heart study ranged in age from years to years at the time of the study, and free of any known cvd at that time. table gives the clinical characteristics of the sample. table shows gender, age and bilirubin levels by ugt a * genotype. age was not significantly asso- ciated with genotype although a significantly higher per- centage of individuals with the / genotype were male when compared to carriers of the allele (p = . ). figure a diagram representing assumed relations in the causal system. in this figure, the relation between bilirubin and cvd risk factors is of interest, but is potentially confounded by measured or unmeasured factors. genotype at the ugt a * locus affects cvd risk factors only through its relation with bilirubin and is not subject to confounding. the cvd risk factors listed in table were considered. mcardle et al. bmc cardiovascular disorders , : http://www.biomedcentral.com/ - / / page of there is no evidence for a role of ugt a in gender determination, and the ugt a * locus is not linked to the sex chromosomes. thus, the difference in gender distribution by genotype is most likely not a causal asso- ciation but rather a chance occurrence; however, since gender differences are also a likely cause of variation of many of the traits further studied, statistical control of gender is warranted. as gender is unlikely to have any causes among other factors considered, statistical adjustment will not result in analysis induced bias, also known as collider stratification bias [ ]. association of ugt a * genotype with serum bilirubin as expected, genotype at the ugt a * locus was strongly associated with bilirubin levels, table . among those with / genotype, serum bilirubin level were nearly twice those with / or / (p < . when controlling for gender and age effects and accounting for the expected correlation in the trait due to familial relatedness of the study participants). ugt a * geno- type accounts for % of the variation in bilirubin in this population (f-statistic = . ). this strong asso- ciation between genotype and phenotype supports the first condition for use of mendelian randomization. association of serum bilirubin levels with cvd risk factors simple (i.e., non-mendelian randomization) associations of serum bilirubin levels with selected cvd risk factors, table observational estimates ( % confidence interval) of the phenotype-bilirubin association and the corresponding variance components p-value, mean phenotype value (standard deviation) and significance test of mean difference comparing / homozygotes with carriers of the allele and point estimates and % confidence interval for estimates of effect using mendelian randomization traditional approach mendelian randomization approach trait estimate ( % ci) p / mean (sd) / mean (sd) / mean (sd) / vs carriers p estimate ( % ci) body mass index (kg/m ) - . (- . , - . ) . . ( . ) . ( . ) . ( . ) . . (- . , . ) waist circumference (cm) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . . ( . , . ) sbp (mmhg) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . - . (- . , . ) dbp (mmhg) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . . (- . , . ) heart rate (/s) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . - . (- . , . ) triglycerides (mg/dl) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . . (- . , . ) fasting hdl cholesterol (mg/dl) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . - . (- . , . ) fasting ldl cholesterol (mg/dl) - . (- . , - . ) . . ( . ) . ( . ) . ( . ) . - . (- . , . ) fasting total cholesterol (mg/dl) - . (- . , - . ) . . ( . ) . ( . ) . ( . ) . - . (- . , . ) common carotid imt (mm) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . . (- . , . ) c reactive protein (mg/l) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . - . (- . , . ) pulse wave velocity (m/s) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . . ( . , . ) cpt sbp (mmhg*min) . (- . , . ) . . ( . ) . ( . ) . ( . ) . . ( . , . ) cpt dbp (mmhg*min) . (- . , . ) . . ( . ) . ( . ) . ( . ) . . ( . , . ) flow mediated dilation (%) . (- . , . ) . . ( . ) . ( . ) . ( . ) . . (- . , . ) brachial artery diameter (mm) - . (- . , . ) . . ( . ) . ( . ) . ( . ) . - . (- . , - . ) abbreviations: ci confidence interval, sd standard deviation, mr mendelian randomization, cpt cold pressor test table clinical characteristics (mean (sd)) of the old order amish enrolled in the hapi heart study, lancaster county pa characteristic men (n = ) women (n = ) age (yrs) . ( . ) . ( . ) bmi (kg/m ) . ( . ) . ( . ) total cholesterol (mg/dl) . ( . ) . ( . ) triglycerides (mg/dl) . ( . ) . ( . ) sbp (mmhg) . ( . ) . ( . ) dbp (mmhg) . ( . ) . ( . ) diabetes (%) . . current smokers (%)* . . lipid lowering meds (%)** . . antihypertensive meds (%)** . . * indicates use of cigarettes, cigars and pipes ** medication use assessed at the time of recruitment, before participants were asked to discontinue use table gender, age and bilirubin levels stratified by ugt a * genotype of old order amish enrolled in the hapi heart study, lancaster county, pa trait / (n = ) / (n = ) / (n = ) / vs. / - / gender,% male . % . % . % . age, yrs (sd) . ( . ) . ( . ) . ( . ) . bilirubin, mg/dl (sd) . ( . ) . ( . ) . ( . ) < . mcardle et al. bmc cardiovascular disorders , : http://www.biomedcentral.com/ - / / page of subclinical atherosclerosis, endothelial function and vascular reactivity were evaluated. regression coeffi- cients obtained from these analyses, their % confi- dence intervals, and p-values evaluating whether these estimates differ from zero are shown in table . con- sistent with previous studies showing bilirubin to be inversely associated with cvd and chd, we observed serum bilirubin levels in this study also to be inversely associated with a variety of cvd risk factors. specifi- cally, serum bilirubin levels were negatively associated with bmi (p = . ), ldl cholesterol (p = . ) and total cholesterol (p = . ). higher bilirubin levels correlate weakly with lower imt (p = . ). these associations (or lack thereof) may be causal or non-causal and may be subject to confounding or reverse causality (i.e., measured bilirubin levels affected by the cvd risk factor). association of ugt a * genotype with cvd risk factors we hypothesized that genotype would be related to cvd risk factors primarily through its affect on serum bilirubin levels. mean values (and standard deviation) of the considered cvd risk factors by genotype are pre- sented also in table . genotype was not associated with either bmi (p = . ) or waist circumference (p = . ). similarly there is no significant difference in blood pressure (sbp p = . , dbp p = . ) or choles- terol levels (hdl p = . , ldl p = . ) across geno- type groups. the / genotype, which corresponds to higher serum bilirubin levels, was associated with a sig- nificantly smaller brachial artery diameter (p = . ) and with cold pressor reactivity (p = . ). association of serum bilirubin levels with cvd risk factors: mendelian randomization approach the right-most column of table shows the two-stage estimate (and % confidence interval) of the serum bilirubin-cvd risk factor association derived from the mendelian randomization approach that accounts for ugt a * genotype. to the extent that mendelian randomization assumptions are upheld, these estimates may be considered as unconfounded by all measured and unmeasured factors that fit the role of confounders in figure considered. discordance between the simple and mendelian ran- domization estimates of the serum bilirubin-cvd risk factor relationship suggests the presence of confounding [ , ]. some results indicate that confounding is severe enough to reverse the direction of the point estimates, potentially leading to qualitative differences in inference. for example, point estimates of the crude associations of bmi (- . , p = . ), waist circumference (- . , p = . ) and diastolic blood pressure (- . , p = . ) chan- ged direction in the mendelian randomization analysis (bmi: . , p = . ; waist circumference: . , p = . ; dbp: . , p = . ). discussion associations between serum bilirubin and multiple cvd risk factors have been previously reported, but attribu- tion of causality has been challenged by the potential for confounding and uncertainty regarding temporal order- ing [ ]. importantly, lin et. al. [ ] utilized genotype at the ugt a * locus to suggest a causal role for biliru- bin in cardiovascular and coronary heart diseases. most notably, using longitudinal data over years of follow up, they demonstrated a striking unconfounded associa- tion with cvd events. while that study supports the hypothesis that increased bilirubin levels cause a decreased risk of cvd, it did not provide estimates of the causal relation of bilirubin with markers of cardio- vascular disease burden and hence did not help identify the underlying pathway by which bilirubin might protect against cvd events. in this study, using mendelian ran- domization, we attempt to extend previous studies by exploring the association between bilirubin and sub-clin- ical markers of vascular reactivity and cvd burden in “healthy” subjects, to help delineate potential early mechanistic pathways related to bilirubin. of note, we find an association between bilirubin and brachial artery diameter and cold pressure reactivity, while utilizing genotype as an instrumental variable per mendelian randomization. this association between bilirubin and brachial artery diameter was not present in our data using the traditional regression approach, most likely due to the presence of confounding factors. in this case, it appears that confounding acts to hide or underesti- mate a potentially causal association. if one believes ugt a * locus is a reliable instrument of bilirubin levels, these data provide evidence that bilirubin may act on cvd via mechanism associated with brachial artery diameter. while baseline brachial artery diameter had tradition- ally been measured to derive brachial flow mediated dilation measures as a surrogate of endothelial function, several studies have demonstrated an independent asso- ciation between brachial artery diameter with coronary artery calcification [ ], serum uric acid [ ], blood pressure, serum lipids, smoking and glucose [ , ], car- otid imt [ ], pregnancy [ ], severity of chronic heart failure [ ], angiographic measures of coronary artery disease [ ] and cvd events in a cohort of over subjects [ ]. brachial artery size is also a determinant not only of both fmd and time to peak fmd, but also of non-endothelial related vasodilatory capacity [ ]. these studies clearly demonstrate repeatable and signifi- cant associations between brachial artery diameter and both cvd risk factors, outcomes and measures of mcardle et al. bmc cardiovascular disorders , : http://www.biomedcentral.com/ - / / page of vascular disease, and suggest that in certain populations, it may be more predictive of cvd than fmd measures. as such, our finding of smaller brachial artery diameter in hyperbilirubinemic subjects is consistent with the sig- nificant decrease in cvd disease noted in subjects with gilbert’s syndrome [ , ] and raises a potential new mechanism associated with the cvd protective effects of bilirubin, separate from its described antioxidant and anti-inflammatory properties [ , ]. the cpt, by mea- suring blood pressure change in response to cold stimu- lus, has been used as a measure of global sympathetic activity and/or response. our study shows a modest association between bilirubin and increased cpt reac- tivity. while several studies have shown that increased reactivity in healthy populations may be associated with increased probability for hypertension, the results have not always been consistent, notably in healthy cohorts such as in this study [ , ]. one reason for this could be that increased cpt reactivity may reflect either increased sympathetic activity, which is likely not protective of cvd or may be due to increased reactivity of blood vessels to sympathetic stimulus, which may be a reflection of healthy vasculature and less cvd. indeed, ageing and obesity are associated with decreased vascular response to sympathetic activ- ity and norepinephrine [ - ]. as such, it is plausible that higher bilirubin levels may be associated with increased vascular response capacity as opposed to an increase in sympathetic neural activity. the lack of association between the ugt a * genotype and heart rate also suggests no direct increase in baseline sympathetic activity. mechanistic pathways for the well noted observation of smaller brachial artery dia- meter with decreased measures of cvd have not yet been elucidated. we did not find an association between the ugt a * genotype and markers of more advance vascular pathology such as pwv or imt. this could be secondary to the absence of any such association or also because we studied a rela- tively young and healthy population with a low cvd disease burden. conclusions in conclusion, we have utilized mendelian randomiza- tion techniques to address confounding in the estima- tion of bilirubin’s causal relationship with known cvd risk factors. our data suggest that many of the associa- tions from observational epidemiology studies reported in the literature are subject to un-accounted for con- founding and/or reverse causality. of the tested risk fac- tors, brachial artery diameter and to a lesser degree cpt reactivity were significantly associated with geno- type at the ugt a * locus, providing compelling evi- dence that bilirubin affects cvd through pathways associated with artery size such as vasomotor tone, reac- tivity and possibly arterial wall structure. abbreviations cvd: cardiovascular disease; chd: coronary heart disease; imt: intima-media thickness; fmd: flow mediated dilation; cpt: cold pressor test. acknowledgements we thank the amish research clinic staff for their excellent work conducting the hapi heart study. this study would not have been possible without the outstanding cooperation and support of the amish community. funding this work was supported by the national institute of health [grant number u hl ]; the university of maryland general clinical research center [grant number m rr ]; the clinical nutrition research unit of maryland [grant number p dk ]; and in part by the intramural research program of the eunice kennedy shriver national institute of child health and human development. author details division of endocrinology, diabetes and nutrition, university of maryland school of medicine, west redwood street, rm. , baltimore, md , usa. division of biostatistics and epidemiology, school of public health and health science, university of massachusetts, amherst, ma, usa. geriatric research and education clinical center, veterans administration medical center, baltimore, md, usa. division of nephrology, university of maryland school of medicine, baltimore, md, usa. authors’ contributions pfm participated in the design of the study, performed the statistical analysis and drafted the manuscript. bww participated in the design of the study. kt performed the genotyping. bdm, ars conceived of the study and oversaw data collection. ap interpreted the results and participated in drafting the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. received: september accepted: march published: march references . lin jp, et al: association between the ugt a * allele, bilirubin levels, and coronary heart disease in the framingham heart study. circulation , ( ): - 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/ / /prepub 	abstract 	background 	methods 	results 	conclusions 	background 	methods 	study sample 	genotyping of ugt a * locus 	analytic approach 	mendelian randomization 	causal assumptions 	statistical methods 	results 	sample characteristics 	association of ugt a * genotype with serum bilirubin 	association of serum bilirubin levels with cvd risk factors 	association of ugt a * genotype with cvd risk factors 	association of serum bilirubin levels with cvd risk factors: mendelian randomization approach 	discussion 	conclusions 	acknowledgements 	author details 	authors' contributions 	competing interests 	references 	pre-publication history wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ article section: clinical/epidemiologic research article section: clinical/epidemiologic research systematic investigation of the relationship between high myopia and polymorphisms of the mmp , timp and timp genes by a dna pooling approach kim hung leung, wai chi yiu, maurice k.h. yap, po wah ng, , wai yan fung, pak chung sham, shea ping yip department of health technology and informatics, and centre for myopia research, school of optometry, the hong kong polytechnic university, hong kong sar, china; department of psychiatry, the hong kong of university, hong kong sar, china.g running title: myopia candidate genes by a dna pooling approach corresponding author: shea ping yip, department of health technology and informatics, the hong kong polytechnic university, hung hom, kowloon, hong kong sar, china. e-mail: shea.ping.yip@inet.polyu.edu.hk grant information: the work was supported by grants from research grant council of hong hong (ref. no. polyu / m; account code: b-q a) and from the hong kong polytechnic university (j-bb p, ms and lv). word count: , words (main text) this is the pre-published version. mailto:shea.ping.yip@inet.polyu.edu.hk� abstract purpose. this study examined the relationship between high myopia and three myopia candidate genes, namely matrix metalloproteinase (mmp ) and tissue inhibitor of metalloproteinase and (timp and timp ), involved in scleral remodeling. methods. the study recruited unrelated adult han chinese who were high myopes (spherical equivalent ≤- . d for both eyes; cases) and emmetropes (within ± . d for both eyes; controls). sample set had cases and controls while sample set had cases and controls. forty-nine tag single nucleotide polymorphisms (snps) were selected from these candidate genes. the first stage was an initial screen of case pools and control pools constructed from sample set , each pool consisting of distinct subjects of the same affection status. in the second stage, “positive” snps from the first stage were confirmed by genotyping individual samples forming the dna pools. in the third stage, positive snps from stage were replicated with sample set genotyped individually. results. of the snps screened by dna pooling, passed the lenient threshold of p < . (nested anova) and were followed up by individual genotyping. of the snps genotyped, two timp snps were found significantly associated with high myopia by single-marker or haplotype analysis. however, the initial positive results could not be replicated by sample set . conclusions. mmp , tipm and timp genes were not associated with high myopia in chinese and hence were unlikely to play a major role in the genetic susceptibility to high myopia. ( words) introduction in myopia, the images of distant objects are focused in front of, rather than on, the retina under relaxed accommodation. myopia is the commonest eye anomaly in the world and imposes a huge impact on the public health care system and the economy. in particular, subjects with high myopia, usually defined as ≤- . diopters (d), are more prone to ocular degenerative changes such as glaucoma and retinal detachment. myopia is much more frequent in orientals ( - %) than in caucasians ( - %) although its prevalence varies with time, the age of the subjects, and the ethnic origin of the population concerned. in hong kong, the prevalence is highest ( %) for age - , and then drops after age . both environmental and genetic factors contribute to myopia although the exact cause of myopia remains to be determined. - environmental factors such as lifestyle, schooling, near-work and outdoor activities are known to contribute to differences in the prevalence of myopia. estimates of heritability are high for refractive error and major ocular components, and shared genes between relative pairs could explain the strong correlation between refractive error and axial length. - myopia mainly results from elongated eyeball caused by accelerated postnatal eye growth, rather than changes in corneal or lens power. during myopia development, the sclera undergoes active remodeling, which involves matrix metalloproteinases (mmps) and tissue inhibitors of metalloproteinases (timps) – the enzymes involved in the degradation of extracellular matrix. matrix metalloproteinase (mmp ) is increased in the sclera of the myopic eye induced by form deprivation in chicks when compared to the control eye, and the increased expression has been consistently shown for both the protein - and the mrna transcript. , increased scleral mmp expression in form-deprivation myopia has also been shown in tree shrew at both the protein and the mrna level, , and in guinea pig at the protein level. increased mmp transcript level has also been found in human scleral fibroblasts mechanically stretched in an in vitro system, in lens-induced myopia in tree shrew, but not in lens-induced myopia in chick. on the other hand, there is less extensive study of timp expression in induced myopia. timp expression is found to be reduced in form deprivation myopia in chick and in lens-induced myopia in guinea pig, but at comparable levels as the control eye in lens-induced myopia in both tree shrew and chick. finally, timp transcript level is found to be reduced in lens-induced myopia in tree shrew. these studies did not specifically examine any potential interaction among these three genes. we used a case-control study approach to examine the relationship between high myopia in a han chinese population and the tag single nucleotide polymorphisms (snps) of three candidate genes. these three candidate genes were selected for this study because their involvement in scleral remodeling has been confirmed by extensive studies of animal myopia models, as has been summarized above. we performed the case-control study in three stages: ( ) initial screen of dna pools to identify putatively positive snps, ( ) confirmation of “positive” snps by genotyping of individual dna samples forming the original pools, and ( ) replication of positive snps by an independent sample set (fig. ). the initial dna pooling step served to reduce the cost and time involved in individual genotyping. , dna pools were created by mixing equal amounts of dna from many individuals sharing the same disease status. thus, “case pools” were constructed from subjects with high myopia (cases) and “control pools” from emmetropic subjects (controls) in this study. moreover, we adopted an optimal experimental design in dna pooling step by creating small dna pools each constructed from distinct individuals of the same disease status. methods subjects and dna samples in dna pooling-based initial study, unrelated southern han chinese subjects (sample set ) were recruited: cases of high myopes with spherical equivalent (se) ≤− . d in both eyes, and emmetropic controls with se within ± . d in both eyes. positive snps from the dna pooling- based initial screen were confirmed by individual genotyping of the original sample set and, if confirmed, replicated by testing a second sample set (sample set ). sample set consisted of unrelated han chinese subjects with cases and controls. the same entry criteria were used for subject recruitment of both sample sets. this study was approved by the human subjects ethics subcommittee of the hong kong polytechnic university, and adhered to the tenets of the declaration of helsinki. signed, informed consents were obtained from all participants. all subjects were recruited from the optometry clinic of the hong kong polytechnic university, and collection of blood samples and dna extraction were performed as described previously. construction of dna pools for the dna pooling study, all dna samples were accurately quantified by a picogreen method (invitrogen, carlsbad, ca) according to the manufacturer’s instructions and diluted to a final concentration of ± . ng/μl. equal volumes of dna solutions were mixed to create dna pools. six case pools and six control pools were constructed for sample set , each consisting of distinct individuals of the same disease status. selection of tag snps three candidate genes were selected for study: mmp , timp and timp . tagger implemented in haploview (http://www.broadinstitute.org/haploview/haploview) was used to select tag snps with the following setting: pairwise tagging algorithm, r ≥ . and minor allele frequency (maf) of more than . . the selection was based on the han chinese genotype data from the international hapmap project database (release a, phase ii; http://www.hapmap.org/) for these three loci and their flanking regions ( kb upstream and kb downstream of the genes). in total, tag snps were selected from these genes for analysis by the dna pooling strategy (table ). allele frequency estimation in dna pools the same protocols were used for all snps examined unless stated otherwise. touchdown pcr was performed in a total volume of -μl reaction mixture containing ng of genomic dna template, . µm of each primer (supplementary table s ) and . mm mgcl , . mm of each dntp and . u of hotstartaq plus dna polymerase (qiagen, hilden, germany) in × pcr buffer http://www.broadinstitute.org/haploview/haploview� http://www.hapmap.org/� provided by the manufacturer. there were a few exceptions: . µm of each primer was used for snps (rs , rs and rs ) and . mm of mgcl for snps (rs and rs ). amplification was performed in a geneamp pcr system (applied biosystems, foster city, ca). the touchdown thermocycling program included activation at ºc for min, followed by cycles of ºc for seconds, °c (initial annealing temperature) for seconds and decreased by ºc per cycle, and ºc for seconds, plus additional cycles of ºc for seconds, ºc (final target annealing temperature) for seconds and ºc for seconds; and final extension at ºc for minutes. there were a few exceptions: the initial and final annealing temperatures were ºc and ºc for snps (rs , rs , rs and rs ). pcr products were purified using shrimp alkaline phosphatase and exonuclease i. primer extension (pe) reactions were performed in a -μl reaction volume containing μl purified pcr products, . μm of the specific primer (supplementary table s ), μm of each appropriate ddntp and/or dntp (supplementary table s ) and unit of therminator (new england biolabs, beverly, ma) in a × reaction buffer supplied by the manufacturer. thermocycling was performed with an initial denaturation step at ºc for minute, followed by cycles of ºc for seconds, ºc for seconds and ºc for minute. denaturing high performance liquid chromatography (dhplc) analysis was performed using the wave nucleic acid fragment analysis system (transgenomic inc, omaha, ne). pe products were analyzed with a % linear gradient change of the working elution buffer over a -minute period and a different starting concentration of buffer b, dependent on the snp concerned (supplementary table s ). relative allele frequencies in dna pools were estimated based on the intensity of primer-extended products by dhplc. for each dna pool, the analysis included a single pcr followed by a single pe reaction and a single dhplc analysis. each dna pool was analyzed in triplicates (fig. ). in other words, there were sets of readings for case pools and control pools (sample set ) for each snp. individual genotyping the positive findings ( snps) in the dna pooling-based initial study were confirmed by individual genotyping of the same sample set (set ) with massarray iplex chemistry (sequenom, san diego, ca; supplementary table s ) according to the manufacturer’s instructions (http://www.sequenom.com/). these snps were grouped together with snps of other on-going studies for genotyping using massarray iplex method carried out by a local service provider (http://genome.hku.hk/portal/). the confirmed positive results were tested by a follow-up replication study on sample set . for sample set , two snps of the timp gene (rs and rs ) were genotyped by unlabeled probe melting analysis. this method uses asymmetric pcr to generate single-stranded dna (ssdna) product, and an unlabeled probe that is ’-blocked by a phosphate group to prevent probe extension. after pcr, the unlabeled probe and a saturating dsdna dye are added to ssdna target for high-resolution melting analysis. asymmetric pcr reaction was performed in a -μl reaction mixture containing ng of genomic dna, . mm mgcl , . μm forward primer (excess), . μm reverse primer (limiting) (supplementary table s ), . mm of each dntp, × pcr buffer and . u of hotstartaq plus dna polymerase (qiagen). amplification was performed in -well plates with a geneamp pcr system, including cycle of initial denaturation for minutes at °c, cycles of seconds at °c, seconds at °c for annealing, and seconds at °c, plus cycle of final extension for minutes at °c. after pcr, a -μl reaction mixture containing . μl pcr product, . μm unlabeled probe (international dna technologies, coralville, ia) and . mm syto green fluorescent nucleic acid stain (invitrogen) was prepared in -well white plates. melting was performed in a lightcycler instrument (roche diagnostics, mannheim, germany) with heating of samples to °c for minute and then cooling to °c for minute. the melting data were then collected between °c and °c with a heating rate of . °c/s at acquisitions per °c, using the “melting curves” analysis mode. samples were again cooled to °c for seconds. melting curves were analyzed with lightcycler software (version . , roche). http://www.sequenom.com/� http://genome.hku.hk/portal/� statistical analysis ocular data were analyzed using the stata package (version . ; statacorp, college station, texas). high myopia was examined as a dichotomous trait. subjects were classified as affected (cases) or unaffected (controls). in pe, unequal representation of the two alleles of a snp can result from differential incorporation of ddntps, and was corrected with a correction factor (known as k correction factor) estimated based on the average of three independent replicate readings from a heterozygous sample. relative allele frequencies of a given snp were estimated from the heights (i.e. intensities) of the two peaks representing the two extension products in the dhplc elution profile with correction by the k correction factor. the relative allele frequencies of a snp were compared between the pools of the case group and the pools of the control group by nested analysis of variance (nested anova; see online supplementary materials for explanation) carried out using the stata package. snps with p value ≤ . were followed up by genotyping individual samples forming the dna pools (sample set ). a lenient threshold of p ≤ . was used in order not to exclude any potentially significant snps. genotype data of individual samples (sample set or ) were analyzed by the plink package (ver. . ; http://pngu.mgh.harvard.edu/~purcell/plink/index.shtml). hardy-weinberg equilibrium (hwe) testing was performed with exact test for controls and cases separately. single- marker association analysis was performed with chi-square test or exact test as appropriate. haplotype analysis was also performed with logistic regression based on wald test. multiple comparisons were corrected by permutation tests. permutation tests are based on re-sampling theory, and widely accepted as the gold standard for correction of multiple comparisons. in each permutation, the genotype data structure and the numbers of cases and controls were kept unchanged while the phenotype status of the subjects was randomly swapped (permutated). the statistic was calculated with each permutation and an empirical p value was generated based on , permutations. permutation of the phenotype status among study subjects is valid under the assumption of null http://pngu.mgh.harvard.edu/~purcell/plink/index.shtml� hypothesis. in order to control experiment-wise (instead of marker-wise) type i error rates, each permutation involved all individual snps and all haplotypes for a given sample set genotyped individually (sample set , sample set or combined sets, each separately), and this was repeated , times. as such, the generated empirical p values controlled the experiment-wise (or more correctly, family-wise) type i error rates for a given sample set. results analysis of ocular data this study had two sample sets collected from han chinese in hong kong. sample set consisted of high myopes (cases) and emmetropes (controls). the characteristics of these subjects have been reported previously. cases (n= ) and controls (n= ) of sample set were recruited using the same entry criteria as sample set , and their characteristics are summarized in table with the ocular data being shown for the right eye only, as has been done previously. subjects of sample set were on average older than those of sample set : . years for cases and . years for controls of set (table ); and . years for cases and . years for controls of set . however, cases and controls of set had very similar refractive error and axial length as their counterparts in set . the mean se for the right eye was - . d for cases and . d for controls of set , and - . d for cases and . d for controls of set . the mean axial length of the right eye was . mm for cases and . mm for controls of set , and . mm for cases and . mm for controls of set . analysis of pooled dna results results of pooled dna analysis are summarized in table . the k correction factor ranged from . to . with a mean of . . the estimated frequencies of the first eluted allele ranged from . to . for case pools, and from . to . for control pools. the difference (case pools – control pools) in estimated allele frequencies ranged from - . to . . of the snps tested by the dna pooling approach, only snps showed significant difference in allele frequencies between case pools and control pools: rs (difference= . , nested anova p= . ), rs (difference=- . , p= . ) and rs (difference= . , p= . ). these three snps were then genotyped for individual samples forming the dna pools (sample set ) for confirmation. the remaining snps did not show significant differences in allele frequencies between case pools and control pools, and hence were not tested any further (figure ). confirmation of pooled dna results by individual genotyping the genotypes of the three follow-up snps were in hwe (p> . , exact test) for sample set . the only exception was rs for the case group (p= . ). deviation from hwe in cases can signify marker-disease association. single-marker analysis showed that rs of the timp gene was associated with high myopia (pasym= . , allelic test) while the other two snps (rs and rs ) showed no significant differences between cases and controls (sample set , table ). in addition, haplotypes consisting of rs and rs (both in the timp gene) were also associated with high myopia (pasym= . , omnibus test; sample set , table ). these results remained significant after correction of multiple comparisons across single markers and haplotypes by permutation tests: pemp= . (allelic test, table ) and pemp= . (omnibus test, table ). therefore, both rs and rs were further tested in a replication study using sample set . snp rs was not tested any further (figure ). note that the asymptotic p value is indicated as paysm, and empirical p value as pemp (also see footnotes to tables and ). replication study based on sample set the genotypes of both rs and rs were in hwe (p> . , exact test). single-marker and haplotype analyses did not show any significant differences in allele or haplotype frequencies between cases and controls (sample set , tables and ). we combined the sample sets ( cases and controls in total) and re-analyzed the data with adjustment for age as a covariate because the mean age differed very significantly between sample sets and (difference= . years, p < - for t test). the results remained the same without significant differences in allele or haplotype frequencies between cases and controls (combined, tables and ). in other words, the initial positive results in sample set could not be replicated independently by sample set . discussion we adopted an efficient three-stage approach to investigating the relationship between high myopia and tag snps of three candidate genes (mmp , timp and timp ). there are many experimental studies using animal myopia models which suggest the involvement of these genes in myopia development. in the initial stage, tag snps were screened using a dna pooling approach, and snps passed the lenient threshold of p ≤ . and were followed up. in the second stage, these three “putatively positive” snps were genotyped for individual samples forming the original dna pools. in the third stage, two snps from stage were genotyped for individual samples from a second sample set. however, the initial positive results could not be substantiated in the replication study. it is interesting to note that rs of timp gave an or of . for the combined sample set (pasym= . , table ), but did not survive after correction for multiple comparisons (pemp= . , table ). in view of this borderline significance, we explored the potential functional role of this snp in the literature and using a web-based tool (funcpred; http://manticore.niehs.nih.gov/snpfunc.htm) for prediction of snp functions, but without success. in other words, mmp , timp and timp were not associated with high myopia in the han chinese population under study, and are thus unlikely to play a major role in the genetic susceptibility to high myopia. a recent japanese study examined two functional promoter snps of the mmp gene in a case-control study involving high myopes (se ≤- . d) and population-based controls, and found no association of these two snps with high myopia. these two promoter snps were rs and rs (named as c - t and c - t, respectively, in the report), and were not examined in the present study. the snp rs had an maf of less than . in han chinese and hence did not satisfy the criteria of selecting tag snps in our study while the other snp rs was not documented in the hapmap database. http://manticore.niehs.nih.gov/snpfunc.htm� a us-based group recently examined tag snps from mmp and timp genes for amish families ( individuals, mean se = - . d) and ashkenazi families ( individuals, mean se = - . d). the tag snps were selected from the hapmap caucasian (ceu) database with the criteria of maf ≥ . and r ≥ . . in particular, tag snps from mmp , from timp and from timp were included, which are expectedly less than those examined in our study (table ) because of their less restrictive criteria of snp selection. two snps were found significantly associated with ocular refraction by quantitative trait analysis using family-based association testing in the amish families only, but not the ashkenazi families. both sets of families were sampled from largely endogamous, rapidly expanding, but isolated populations in the usa. the prevalence of refractive errors is high in jewish populations, but relatively low in the old order amish. the behavioral and environmental factors are more conducive to myopia development in the jewish populations than in the amish populations, and could probably explain the discrepancy in the genetic association results, as suggested by the authors. the authors also anticipated that the positive results could not be replicated in south asian chinese and japanese populations with high prevalence of environmentally induced myopia. indeed, our study could not replicate the findings. one of the positive snps in the amish population was rs (p= . ) within the mmp gene. this snp was also screened by the dna pooling approach in the present study: the estimated frequency of the c allele was . in case pools and . in control pools, which were not statistically significant (difference= . , nested anova p value = . , table ). the frequency of the c allele in controls is similar to that in han chinese documented in hapmap database ( . vs . ). it is worth noting that the phenotype definition was different for these two studies: quantitative measures of refractive errors in the american study, but dichotomous trait of high myopia (affected vs unaffected) in our study. all three association studies (japanese, american and our) focus on common polymorphisms in the genes under study and hence assumed the hypothesis of common disease common variants. strong linkage disequilibrium (ld) between common tag snps and common casual variants is critical to the success of this indirect ld mapping approach. sequence variations must have similar allele frequencies in order to be highly correlated and in strong ld. however, rare casual variants may also contribute to myopia development – the other side of the story being the hypothesis of common disease rare variants. this indirect approach is of low power in detecting association with rare variants because of the weak ld between common tag snps and rare casual variants. therefore, direct mapping must be performed to detect association with rare causal variants, which must first be identified. rare variants can be identified for direct association studies by sequencing of good candidate genes or even the whole genome for a very large number of samples. our case subjects had extreme refractive errors (mean se = - . d for set ; and - . d for set , table ). this would enhance the homogeneity of the myopia phenotype, enrich the genetic components of the contributing factors, and hence increase the power of our study (though in a subtle manner). the three candidate genes were chosen for study because they have been shown to be involved in sclera remodeling in myopia development in many studies. - our negative finding might imply that these genes do not carry common sequence variants that are capable of influencing their function and/or regulation in the relevant ocular tissue. however, the contribution of behavioral and environmental effects on high myopia should not be overlooked. our dna pooling-based initial screen adopted a lenient threshold of p ≤ . in order avoid missing potential snps. for rs of timp , the power of the third stage study (sample set ) is % under an allelic model and % under a genotypic model. the power is calculated based on the following assumptions with the online genetic power calculator (http://pngu.mgh.harvard.edu/~purcell/gpc/): or and allele frequencies obtained for rs for sample set (table ), a disease prevalence of . for high myopia in our local chinese population, and a significance level set at α= . because two snps were examined in the third stage. one disadvantage of dna pooling strategy is that it makes haplotype analysis very difficult, if not impossible. algorithms are available for estimating haplotype frequencies in small dna pools constructed from a few (< ) individuals. in other words, our current pooling protocol might miss some potential snps for follow-up in the second stage analysis if high myopia is associated with certain haplotypes, but not individual snps. this is one of the reasons why a lenient http://pngu.mgh.harvard.edu/~purcell/gpc/� threshold of p ≤ . was used to selecting snps for follow-up study by individual genotyping in the second stage. association testing of dna pools has been proven to be an effective initial screen of snps and candidate genes for subsequent detailed follow-up study. , the major advantages are tremendous reduction in dna usage and in the amount of genotyping work when compared to individual genotyping. for example, our study required for each snp pcrs and subsequent analyses for case pools and control pools (fig. ), plus three separate pcrs for heterozygotes to determine the k correction factor. the amount of genotyping work was only about one-fifteenth of that required for genotyping of individual samples. it has also been shown that use of small dna pools of about individuals is superior to use of fewer, larger dna pools for candidate gene studies. in addition to the advantages mentioned above, use of small dna pools allows the use of standard statistical method (nested anova) for data analysis without the need of directly estimating the variance components of the error sources while it properly handles variations arising from sampling of subjects and technical errors, which are due to unequal amounts of dna being mixed together, errors in pcr and primer extension, and in dhplc analysis. the present study used dhplc analysis to estimate the relative allele frequencies of dna pools. dhplc analysis is in fact a rate-limiting step because samples have to be injected and analyzed sequentially. the throughput can be greatly increased if quantitative genotyping is conducted with a mass spectrometer, e.g. using the massarray iplex method (sequenom). however, the local service provider only entertained request of classical genotyping work based iplex method, but not quantitative genotyping. dna pooling strategy may become less attractive as the unit cost of genotyping reduces tremendously with the availability of high-throughput genotyping platforms like whole-genome genotyping arrays. nevertheless, the total cost of whole-genome genotyping for a large number of samples is still prohibitive for many research groups. in fact, genome-wide association studies can be within the reach of even small- to medium-sized research groups if dna pooling strategy is applied. interestingly, errors due to array variations are much greater than those due to pool construction, and hence it is recommended to have multiple arrays per dna pool for a few pools rather than multiple dna pools with less arrays per pool. in conclusion, we used a dna pooling strategy to screen tag snps from three candidate genes (mmp , timp and timp ). three tag snps passed the threshold (p ≤ . ) and were tested by individual genotyping of samples forming the dna pools. two snps from the timp gene were found associated with high myopia by single-marker analysis or haplotype analysis. however, the initial positive results 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: - . . mohlke kl, erdos mr, scott lj, et al. high-throughput screening for evidence of association by using mass spectrometry genotyping on dna pools. proc natl acad sci u s a. ; : - . . macgregor s, zhao zz, henders a, nicholas mg, montgomery gw, visscher pm. highly cost- efficient genome-wide association studies using dna pools and dense snp arrays. nucleic acids res. ; :e . . macgregor s. most pooling variation in array-based dna pooling is attributable to array error rather than pool construction error. eur j hum genet. ; : - . figure legend figure . a three-stage approach to testing genetic association based on an initial screen of dna pools. figure . nested design of the dna pooling study. there are two subject groups (case group, g ;and control group, g ), six dna pools per group (p to p for case group, and p to p for control group), and three technical replicates (rij to rij ) for each dna pool. note that there is no link from any pools of the case group to any pools of the control group. therefore, the level of the case group is not cross-classified with the control group, but is nested with the respective group, i.e. the pools are nested within the group. each dna pool was constructed by mixing equal amounts of dna from distinct individuals of the same subject group. table . summary of tag snps in the mmp , timp and timp genes gene geneid chromosomal location region captioned* no. of tag snps no. of snps captured at mean r =? mmp q -q . kb (r = . ) timp q . kb (r = . ) timp q . . kb (r = . ) * the region captured includes the gene and its flanking region ( kb upstream and kb downstream). table . characteristics of study subjects of sample set (for right eyes only) characteristic cases (n= ) controls (n= ) age, mean (sd), y . ( . ) . ( . ) females, no. (%) ( . ) ( . ) spherical equivalent, mean (sd), d - . ( . ) . ( . ) axial length, mean (sd), mm . ( . ) . ( . ) corneal power, mean (sd), d . ( . ) . ( . ) anterior chamber depth, mean (sd), mm . ( . ) . ( . ) lens thickness, mean (sd), mm . ( . ) . ( . ) table . pooled dna analysis of tag snps in the mmp , timp and timp genes alleles† k correction factor estimated frequencies of st allele nanova snp* ( st/ nd) peak height ratio ( st/ nd) case pools control pools diff (case - control) p value mmp rs t / g . . . . . rs g / a . . . . . rs g / a . . . - . . rs c / t . . . - . . rs c / t . . . - . . rs g / a . . . . . rs g / a . . . . . rs c / t . . . . . rs c / g . . . - . . rs c / t . . . - . . rs g / a . . . . . rs g / a . . . - . . rs g / a . . . - . . rs g / a . . . - . . rs g / a . . . . . rs c / t . . . . . rs g / t . . . - . . timp rs c / t . . . - . . rs c / t . . . - . . rs c / t . . . - . . rs c / a . . . - . . rs g / a . . . - . . rs c / t . . . - . . rs g / a . . . - . . rs c / t . . . . . rs c / t . . . . . rs g / a . . . . . rs c / t . . . . . rs a / t . . . . . rs g / a . . . . . rs g / a . . . - . . rs g / a . . . . . rs g / t . . . - . . rs t / a . . . - . . timp rs g / c . . . - . . rs g / a . . . - . . rs c / t . . . . . rs g / a . . . - . . rs g / a . . . - . . rs c / t . . . - . . rs c / t . . . - . . rs g / a . . . . . rs c / t . . . - . . rs c / a . . . . . rs g / a . . . - . . rs c / t . . . . . rs g / a . . . - . . rs a / g . . . . . rs a / t . . . . . * snps are arranged down the column in the order of ’> ’ along the respective gene. † the st allele has a shorter elution time while the nd allele has a longer elution time. the alleles are named with reference to the sense strand of the respective gene. table . allelic association tests of timp and timp snps genotyped individually alleles* genotype counts ( / / )* minor allele freq allelic test‡ gene, snp cases controls cases controls or ( %ci) † pasym pemp sample set timp , rs a g / / / / . . . ( . - . ) . . timp , rs c t / / / / . . . ( . - . ) . . timp , rs t a / / / / . . . ( . - . ) . . sample set timp , rs c t / / / / . . . ( . - . ) . . timp , rs t a / / / / . . . ( . - . ) . . combined (sets & )§ timp , rs c t / / / / . . . . . timp , rs t a / / / / . . . . . * the major allele is designated as “ ” and minor allele as “ ”; and the genotype counts are indicated as the counts of the genotypes , and , respectively. sample set has cases and controls while sample set has cases and controls. note that the total genotype counts may not add up to these expected numbers because a few samples failed to be genotyped in a random fashion. † the odds ratio (or) is calculated for the minor allele (allele ) with the major allele (allele ) as the reference. the % confidence intervals (ci) are indicated within brackets. since rs is of interest, its genotypic odds ratios and their % ci (in brackets) are given here with cc as the reference genotype: . ( . – . ; set ), . ( . – . ; set ), . ( . – . ; combined set, not adjusted for age here) for genotype ct, and . ( . – . ; set ), . ( . – . ; set ) and . ( . – . ; combined set, not adjusted for age here) for genotype tt. this simply serves to avoid cluttering in the table above. ‡ allele frequencies are compared by chi-square test to give the asymptotic p value (pasym). multiple comparisons are corrected by , permutations across single marker allelic tests (this table) and omnibus tests of haplotypes (table ) for a given sample set (set , set or combined, each separately). the empirical p value is indicated as pemp. § single-marker (this table) and haplotype (table ) analyses are performed for the combined sample set (sets and ) with adjustment for age as a covariate to account for the effect of age. this has to be done using logistic regression, the relevant plink command of which does not give the % ci for the odds ratio (or). table . haplotype analysis of timp snps (rs -rs )* haplotype freq haplotype cases controls or pasym pemp sample set omnibus test - - - . . ta . . . . ca . . . . tt . . . . ct . . . . sample set omnibus test - - - . . ta . . . . ca . . . . tt . . . . ct . . . . combined (sets & ) † omnibus test - - - . . ta . . . . ca . . . . tt . . . . ct . . . . * asymptotic p values (pasym) are obtained from wald test based on logistic regression. multiple comparisons are corrected by , permutations across single marker allelic tests (table ) and omnibus tests of haplotypes (this table) for a given sample set (set , set or combined, each separately). the empirical p value is indicated as pemp. note that plink does not generate confidence intervals for odds ratios (or) in haplotype analysis. † single-marker (table ) and haplotype (this table) analyses are performed for the combined sample set (sets and ) with adjustment for age as a covariate to account for the effect of age. this has to be done using logistic regression, the relevant plink command of which does not give the % ci for the odds ratio (or). figure figure supplementary table s . mmp , timp and timp snps: primers for pcr and primer extension reaction and buffer b concentration ranges for dhplc snp* forward pcr primer ( '> ') reverse pcr primer ( '> ") pe primer ( ’> ’) ddgtp ddctp ddttp ddatp buffer b (%) mmp rs ggtaaattattcatgcttctgcctttt tggcacaagatacaggtcataaa accaaaaccaagatgatgatgag √ √ - % rs tccctcgcaagcccaagt gcactgaaaagtaacccaatgtc tgtgtattgaaactccctaagatg √ √ - % rs catcttagggagtttcaatacaca gctaccaatcatcacgttcctt acagacttgagtttcatacttgct √ √ - % rs ggcttagataggacagtagatg tgcccagtgtgaccaggaaa catctgcccccatgtcaac √ √ - % rs gttggatgatgctggtagaca tcctgtcaatactgccctctta tgtagacactagaggaagggt √ √ - % rs gtaaagagcaggggaaacaatg taccacatacccacattaccac ggatggaggatggcatagac √ √ - % rs cagaatggacagtgcccttg agagagggaaggatgtgaatga gtgtcaatcattttgttgttgggtg √ √ - % rs cagaacagacattgggcatc ctgttggaggaagggaagaa gtctgtatctccaccttttagtc √ √ - % rs cgaccacagccaactacga ggaggtgagtagcagcatct gaccctggacattgccctt √ √ - % rs agacacccacctacccaga gcagtgggaatgtctcttagg gatgctgctactcacctcc √ √ - % rs gttggttcttacagtgaggcta gcagaccttgggctttttcc gagccaggccaaaatagtgaaa √ √ - % rs gcttgcttcttgtcctcctt agcacacctgaacccaaact gtcattctgggaagcatttgg √ √ - % rs agcctccactcacatcttgt tcactcaccacccatacaca gttcccaattcttccctccattt √ √ - % rs tgtgtatgggtggtgagtgac ggatggatgatagaagaatggaag gccctagttccctggagaat √ √ - % rs gtcttcactcttcactactattc ctgatttggatgctggcttc taagtcttcttcatattttgtccac √ √ - % rs cacactgtttggcatgtagtaag cattttcttcttcacctcattgtatc gccatcagctgggtgctc √ √ - % rs cccactcctaatctcctacata cagaaaaaagagatagaaacaatgaca cacctgtagagttcactcctta √ √ - % timp rs cacctcactcagaacaaaatgc ttctgttccctctaccattatctc acggtgtccaggctacag √ √ - % rs ctggagtgctgacctgagt atgtgggagccgaaggact accaccctacacatgttcatc √ √ - % rs † gagtcgagctgaaggggaaa cgctcggaggattttctgct cgggtgggtcgcctggtg √ √ - % rs cttgctcagacttcggcttt gcagccaagagtaaaaggag gcccacaggtgatttaacc √ √ - % rs atccgctcagacactaccac ggaagacaaccacagaacct aaccagctctggggaaagg √ √ - % rs tgggtccgtattagggtttc tcacagacagcagattccag ttcccacataaatcccatcttac √ √ - % rs taatggacgctgtcgaggtat catattggtcagactggttacaaa tgaatctgtgtgtatccatatcc √ √ - % rs cctcctttggcttttcttccta tacgaagtgcctgtgctcta acacctctccctcgttcagatt √ √ - % rs ctgggtagtaagaggtgttcat cacaatggatcatagaccttcat caccaaaagcacaaccaataaac √ √ - % snp* forward pcr primer ( '> ') reverse pcr primer ( '> ") pe primer ( ’> ’) ddgtp ddctp ddttp ddatp buffer b (%) rs ctcagaagcagccacagatg attctgggtcttgggtctgg agactctacaaaaataggtggtg √ √ - % rs gccccatcctgctaaaagaa tctgaaaagggaggtgaagc tctaagctgtctttactgtgatc √ √ - % rs gtgtgatttgagggtgtgga tcatttggctctgggtggaa aggtggactcagctttgttc √ √ - % rs tctcttgattcccattttccttcttt aagaggaggctggtcaaatca tgcccacccccaatttaaag √ √ - % rs agtggtagaatagtgggagcat tcttctttcttcttctgttggag acagacggctttgccctga √ √ - % rs cctcctccttgtctttccag taccctgcctcactgttcct ttctttcctccaacgtccag √ √ - % rs gctctgacacctgactcgtta agtgctgtgctgcgtgtatg ggagtaggagaccagaaatgat √ √ - % rs ggagaaaaggacccgaagataaa tggaaatgtggatggcttgc aacaggaagtgcgagcataag √ √ - % timp rs aagtggaaacaaaaaatcaaaagacct tgcgaaactgatggatggtct tatctttcatccaataaacacagaagt √ √ - % rs gctgagaagtggacaaagaca cttgtgcttgcttttcctacc cctgcccatggctgacag √ √ - % rs agatagatgataaatgataaatagaaatggata aacaaaaacaacaacacgattctcc gccacccaggagtagacta √ √ - % rs caatcacaatggcaggcaag taggtgggagaatcgcttga tgctgcatggtgacctgttt √ √ - % rs atagggaggaggctggtagt tgacctgcctgatgctacct cagcactgaatctgggaac √ √ - % rs acttgggaggctgagggaaa ccagggctcttcttgctatt gagactctgtctcagaaaaagag √ √ - % rs agaaagcaggaggatggtca ctaaaagatgggctaatgacaaca aaaatccacggaaagcatttagc √ √ - % rs ctgaaatgctacctcctcca tcccacagcctcattcattca ctttcctctctttcttccagca √ √ - % rs cagccactcttgatttcttcct gcctcattggacccttagaa gattcctggattcagggcc √ √ - % rs agcaaggcaaacaaggcaca gaagggtctggatttggttga gtttccagaaaagcttactggtg √ √ - % rs tggagaagcacagatgaagtag aataggtggatctggggttg cccaagaattgcctactaagac √ √ - % rs agggcagtattagcatccacat cgtagaaagagaagcacaacct gggaattggaatcaggattgaac √ √ - % rs ttatcccctcagttctcagc gaagagacaccaatgaagagatg gtttataataggaaaatgcctctacttta √ √ - % rs agcaggtactggtacttgttga gatgggcacttaggttgatttc cccatgtgcagtacatcca √ √ - % rs aacattagagactccttacaatttact cagacccctcttccatattc ttaaggccacagagactctc dgtp‡ √ - % rs gagcccacttgaaaccactt ttgcagccctagaaacatcag ggtctgagcagatatagtaagga √ datp‡ - % * snps are arranged down the column in the order of ’> ’ along the respective gene. † dimethylsulfoxide ( %) was added as additive in the pcr. ‡ dntp was used for the primer extension reaction for an optimal discrimination of the extended products. supplementary table s . primers and probes for genotyping individual samples gene, snp primer sequences ( ’> ’)* massarray iplex (sequenom) for sample set timp , rs f: acgttggatgaactatggcacaaagggcag r: acgttggatgaaatgaaagggcgtggccag pe primer: taaggagactctacaaaaataggtggtg timp , rs f: acgttggatgcaatggcaattgggattgag r: acgttggatggatgaacagcacatgtgcaa pe primer: tgaaaatgtgcaatttctggagac timp , rs f: acgttggatggaaaggcattcttccttccc r: acgttggatggcaggtctgagcagatatag pe primer: ctgagcagatatagtaagga unlabeled probe melting analysis for sample set timp , rs f: gaa gaa ggg tct gga ttt ggt t r: agt atg gat cac agt ggc aca a probe: agc tta ctg gtg cgt ctc aag aaa ttg c timp , rs f: ctg act tac agc cct aga aac a r: gac tca aga gcc ctc atg c probe: tct gag cag ata tag taa gga ttg ttg cc * f = forward primer; r = reverse primer; and pe = primer extension supplementary materials for statistical analysis (nested anova) here, we briefly explain the principle of nested analysis of variance (anova) used to analyze the estimated allele frequencies of dna pools. nested anova design is an extension of one-way anova design. all assumptions (e.g. normality of residuals, constant variance, etc) of anova hold true for nested anova. our nested design had one measurement variable (the estimated allele frequency, rijk) and two nominal variables (dna pools and subject groups) with the dna pools (pij) nested within each of the subject groups (gi) (fig. in main text). there were two subject groups (case group and control group, i= , ), six dna pools (j= , , , , , ) within each subject group, and three replicates (k= , , ) per dna pool. nested anova assessed the difference between the case group (high myopia) and the control group (emmetropia) to test for allele frequency difference between these two subject groups – the main objective of the pooling experiments. it could also assess the variability of the dna pools within subject groups, which, however, was not the main objective of the pooling-based screen and was expected to be large because each dna pool was prepared from the dna of distinct subjects within the same subject group. gr jef .. growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging aaron r. jeffries, reza maroofian, claire g. salter, , , barry a. chioza, harold e. cross, michael a. patton, , emma dempster, i. karen temple, , deborah j.g. mackay, faisal i. rezwan, lise aksglaede, diana baralle, , tabib dabir, matthew f. hunter, , arveen kamath, ajith kumar, ruth newbury-ecob, angelo selicorni, amanda springer, , lionel van maldergem, vinod varghese, naomi yachelevich, katrina tatton- brown, , , jonathan mill, andrew h. crosby, and emma l. baple , institute of biomedical and clinical science, university of exeter medical school, rild wellcome wolfson centre, royal devon and exeter nhs foundation trust, exeter, ex dw, united kingdom; genetics research centre, molecular and clinical sciences institute, st. george’s university of london, london sw re, united kingdom; human genetics and genomic medicine, faculty of medicine, university of southampton, southampton, so yd, united kingdom; wessex clinical genetics service, princess anne hospital, southampton, so ya, united kingdom; department of ophthalmology and vision science, university of arizona school of medicine, tucson, arizona , usa; department of clinical genetics, copenhagen university hospital, blegdamsvej b, copenhagen n, denmark; northern ireland regional genetics centre, clinical genetics service, belfast city hospital, belfast, bt ab, united kingdom; monash genetics, monash health, clayton, victoria, vic , australia; institute of medical genetics, university hospital of wales, cardiff, cf xn, united kingdom; north east thames regional genetics service and department of clinical genetics, great ormond street hospital, london, wc n jh, united kingdom; university hospitals bristol, department of clinical genetics, st michael’s hospital, bristol, bs eg, united kingdom; uoc pediatria asst lariana, como, italy; department of paediatrics, monash university, clayton, victoria, vic , australia; centre de génétique humaine and clinical investigation center (inserm), université de franche-comté, , besançon, france; clinical genetics services, new york university hospitals center, new york university, new york, new york , usa; division of genetics and epidemiology, institute of cancer research, london sm ng, united kingdom; south west thames regional genetics service, st. george’s university hospitals nhs foundation trust, london sw qt, united kingdom; peninsula clinical genetics service, royal devon and exeter hospital, exeter, ex ed, united kingdom germline mutations in fundamental epigenetic regulatory molecules including dna methyltransferase alpha (dnmt a) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. we pro- filed genome-wide dna methylation patterns in dnmt a c. g > a; p.(arg gln) carriers in a large amish sibship with tatton-brown–rahman syndrome (tbrs), their mosaic father, and tbrs patients with distinct pathogenic de novo dnmt a variants. this defined widespread dna hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. tbrs patients also displayed highly accelerated dna methylation aging. these findings were most marked in a carrier of the aml-asso- ciated driver mutation p.arg cys. our studies additionally defined phenotype-related accelerated and decelerated epi- genetic aging in two histone methyltransferase disorders: nsd sotos syndrome overgrowth disorder and kmt d kabuki syndrome growth impairment. together, our findings provide fundamental new insights into aberrant epigenetic mecha- nisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders. [supplemental material is available for this article.] dna methylation is an essential epigenetic process involving the addition of a methyl group to cytosine. it is known to play a role in many important genomic regulatory processes, including x-chromosome inactivation, genomic imprinting, and the repres- sion of tumor suppressor genes in cancer, mediating transcription- al regulation as well as genomic stability (jones ). three catalytically active dna methyltransferases (dnmts) are involved corresponding authors: e.baple@exeter.ac.uk, a.h.crosby@exeter.ac.uk, j.mill@exeter.ac.uk article published online before print. article, supplemental material, and publi- cation date are at http://www.genome.org/cgi/doi/ . /gr. . . freely available online through the genome research open access option. © jeffries et al. this article, published in genome research, is available un- der a creative commons license (attribution . international), as described at http://creativecommons.org/licenses/by/ . /. research : – published by cold spring harbor laboratory press; issn - / ; www.genome.org genome research www.genome.org mailto:e.baple@exeter.ac.uk mailto:a.h.crosby@exeter.ac.uk mailto:j.mill@exeter.ac.uk http://www.genome.org/cgi/doi/ . /gr. . http://www.genome.org/cgi/doi/ . /gr. . http://genome.cshlp.org/site/misc/terms.xhtml http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / http://genome.cshlp.org/site/misc/terms.xhtml in the methylation of cytosine: dnmt , which is mainly respon- sible for the maintenance of dna methylation over replication, and dnmt a and dnmt b, which generally perform de novo methylation of either unmethylated or hemimethylated dna. an absence of these enzymes in mice results in embryonic (dnmt and b) or postnatal (dnmt a) lethality (okano et al. ), confirming their essential roles in development. in line with knockout mouse models, pathogenic variants affecting the chromatin binding domains of dnmt have been shown to cause two separate progressive autosomal dominant adult-onset neuro- logic disorders (klein et al. ). biallelic pathogenic variants in dnmt b have been associated with immunodeficiency, centro- mere instability, and facial anomalies (icf) syndrome (jiang et al. ). to date, dnmt a has been linked to a number of physiological functions, including cellular differentiation, malig- nant disease, cardiac disease, learning, and memory formation. somatically acquired pathogenic variants in dnmt a are associat- ed with > % of acute myeloid leukemia (aml) cases, whereas het- erozygous germline pathogenic loss-of-function variants have been found to underlie tatton-brown–rahman syndrome (tbrs; also known as dnmt a-overgrowth syndrome, omim ) (challen et al. ; tatton-brown et al. ). tbrs is character- ized by increased growth, intellectual disability (id), and dysmor- phic facial features. more recently, heterozygous gain-of-function dnmt a missense variants affecting the dnmt a pwwp domain have been shown to cause microcephalic dwarfism and hypermethylation of polycomb-regulated regions (heyn et al. ). there is an emerging group of epigenetic regulatory mole- cule-associated human growth disorders in which the underlying molecular defect is a disruption to the dna methylation and his- tone machinery. there are now over disorders identified within this group, which can be further subgrouped into diseases result- ing from disruption of the “writers,” “readers,” and “erasers” of epigenetic modifications (bjornsson ). example disorders in each group include kabuki, sotos, and weaver syndromes (“writ- ers”); smith-magenis, rett, and bohring–opitz syndromes (“read- ers”); and wilson–turner and cleas–jensen syndromes (“erasers”). the final subgroup occurs because of disruption of chromatin remodelers, with example resulting disorders including charge and floating–harbor syndromes. neurological and cognitive impairment are common features of these conditions, suggesting that precise epigenetic regulation may be critical for neuronal homeostasis. however, a true understanding of the pathogenic mechanism underlying these conditions remains poorly understood. in the current study, we investigated the methylomic conse- quences of a dnmt a pathogenic variant (nc_ . : g. c > t; nm_ . :c. g > a; p.(arg gln)) in a large amish family comprising four individuals affected with tbrs arising as a result of a mosaic pathogenic dnmt a variant in their father (xin et al. ). the occurrence of multiple affected and unaffected individuals in the same sibship, together with the combined genetic and environmental homogeneity of the amish, permitted an in-depth investigation of the genome-wide patterns of dna methylation associated with pathogenic variation in dnmt a. we subsequently extended our analyses to other (non-amish) tbrs patients harboring distinct pathogenic de novo dnmt a variants, as well other methyltransferase-associat- ed overgrowth and growth deficiency syndromes, defining altered epigenetic profiles as common key themes of these growth disorders. results reduced dna methylation at key sites involved in morphogenesis, development, and differentiation in tbrs patients dnmt a encodes a dnmt with both de novo and maintenance activity (okano et al. ; chen et al. ). we first looked for global changes in dna methylation in whole blood obtained from dnmt a c. g > a; p.(arg gln) carriers, using the methylation-sensitive restriction enzyme–based luminometric methylation assay (luma) (karimi et al. ) to quantify dna methylation across gc-rich regions of the genome, finding no ev- idence for altered global dna methylation (luma: mean dnmt a c. g > a carriers = . , wild type = . , t-test p- value = . ). we next quantified dna methylation at , autosomal sites across the genome using the illumina k array. globally, a subtle decrease in mean dna methylation was noted in available age/sex-matched dnmt a heterozygous c. g > a; p.(arg gln) individuals compared with their matched unaffect- ed sibling samples, although this was not statistically significant (wilcoxon rank-sum test p-values for two matched pairs = . and . ) (supplemental fig. s ). in contrast, an analysis of site- specific dna methylation differences in dnmt a c. g > a; p.(arg gln) carriers (including the mosaic father) versus wild- type individuals in the amish pedigree identified differen- tially methylated positions (dmps; benjamini–hochberg false discovery rate [fdr] < . ) (fig. a,b; supplemental table s ), of which dmps were characterized by a > % change in dna methylation. supplemental figure s also highlights dna methyl- ation levels at these dmps across all carriers and control individu- als profiled in this study. technical validation of illumina k array data was performed using bisulfite pyrosequencing for three top-ranking dmps, confirming significant differences in dnmt a c. g > a; p.(arg gln) carriers at each of the tested loci (supplemental fig. s ). the dmps identified were highly enriched for sites character- ized by reduced dna methylation in dnmt a c. g > a; p.(arg gln) heterozygotes (n = dmps, . %, sign-test p-value < . × − ). although there were no statistically signifi- cant differences between dna methylation-based blood cell com- position estimates derived from our data (supplemental table s ), we examined the extent to which the identified dmps were poten- tially influenced by cell-type differences between dnmt a c. g > a; p.(arg gln) carriers and wild-type family mem- bers. there was a highly significant correlation (r = . , p-value < . × − ) (supplemental fig. s ) in effect sizes at the dmps between models, including and excluding cell types as co- variates, indicating that the observed patterns of differential dna methylation are not strongly influenced by cell-type varia- tion. we used dmrcate (peters et al. ) to identify spatially cor- related regions of differential dna methylation significantly associated with the dnmt a c. g > a; p.(arg gln) variant, identifying autosomal differentially methylated regions (dmrs) (for an example dmr, see supplemental fig. s ), all characterized by hypomethylation in dnmt a c. g > a; p.(arg gln) carriers apart from one -bp dmr that showed increased dna methylation (supplemental table s ). the mean size of the identified dmrs was bp (range = – bp), span- ning an average of six probes (supplemental fig. s ). we next investigated whether dnmt a p.(arg gln)-asso- ciated dmps are enriched in specific genic locations (see methods). we found a modest enrichment of dmps in regions ≥ bp jeffries et al. genome research www.genome.org http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc upstream of the transcriptional start site (chi-squared yates-correct- ed p-value = . ) and more prominent enrichment in intergenic regions (chi-squared yates corrected p-value = . × − ) (sup- plementalfig.s ).dmpswerealsosignificantlyenrichedincpgis- land shore regions (chi-squared yates-corrected p-value = × − ) (supplemental fig. s ). we also examined dmp occurrence in ex- perimentally determined cancer and reprogramming-specific dmr locations (doi et al. ), finding a . -fold and b a c d e figure . tbrs dnmt a variants are associated with widespread dna hypomethylation. (a) simplified pedigree indicating the genotyping of individuals in the amish family investigated: (+/−) heterozygous carriers of the dnmt a c. g > a p.(arg gln) variant; (+/− mosaic) the dnmt a c. g > a p.(arg gln) mosaic father; (−/−) wild-type individuals. black shading indicates individuals with a phenotype consistent with tbrs, gray shading, the father with macrocephaly and mild intellectual impairment; and white shading, unaffected individuals. each of these samples was profiled on the illumina k dna methylation array. (b) volcano plot showing site-specific dna methylation differences (x-axis) and −log p-values (y-axis) from an analysis comparing amish dnmt a c. g > a; p.(arg gln) pathogenic variant carriers and wild-type family members using the illumina k array. red values indicate the differentially methylated positions (dmps) detected at a benjamini–hochberg fdr < . . (c) top gene ontology enrich- ment analysis categories associated with the dmps identified in dnmt a c. g > a; p.(arg gln) pathogenic variant carriers versus wild-type family members. (d) comparison of dnmt a c. g > a; p.(arg gln) identified dmps (log fold change) relative to other dnmt a tbrs-associated variants assessed in this study (all variants grouped and measured relative to controls). pearson correlation coefficient = . , p-value < . × − . (e) boxplot illustrating the dna methylation changes observed in association with the dnmt a tbrs variants studied at the dmps identified in the amish dnmt a c. g > a p.(arg gln) carriers. the predicted protein consequence of each dnmt a variant studied is indicated: pink indicates in-frame deletion; yellow, single-nucleotide variant; cyan, duplications predicted to result in a frameshift; green, amish c. g > a; p.(arg gln) variant. epigenetic aging abnormalities in growth disorders genome research www.genome.org http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc . -fold overrepresentation (chi-squared yates-corrected p-value = . × − and chi-squared yates-corrected p-value < . × − , respectively), as well as predicted enhancer elements that showed a . -fold overrepresentation (chi-squared yates-corrected p-value = . × − ). we then undertook gene ontology analysis, ac- countingfor the background distribution of probes on the illumina k array, to functionally annotate the dna methylation differences observed in the dnmt a c. g > a; p.(arg gln) carriers. the dmps identified in this study showed a significant overrepresentation in functional pathways related to morphogenesis, development, and differentiation (top hit go: , multicellular organism development, contains genes associated with dmps; fdr q-value = . × − ) (fig. c; supplemental table s ). we also performed a functional overlap analysis to identify cell- or tissue-specific chromatin signals associ- atedwiththesedmpsusingeforge(breezeetal. ).significant overlap (fdr q-value < . ) was found with dnase i sensitivity hotspots, most apparent with pluripotent cells in encode (the encode project consortium ; davis et al. ) and fetal tis- sues within the nih roadmap epigenomics consortium data set (supplemental data s ; roadmap epigenomics consortium et al. ). chromatin states fromthe nih roadmap epigenomics con- sortium data set show an enrichment of dmps in regions defined as active transcriptional start sites in brain tissue and embryonic stem cells. of particular interest, given the established importance of dnmt a during embryonic development, eforge analysis of blood cell types highlighted an enrichment of dmps in regions characterized by repressed polycomb and enhancer activity. to provide additional evidence to support the notion that dnmt a c. g > a; p.(arg gln) carriers show disruption to developmental pathways, we used the genomic regions enrichment of annotation tool (great) (mclean et al. ) to explore functional pathways enriched in genes annotated to dnmt a c. g > a; p.(arg gln)–associated dmrs. this re- vealed a significant effect on genes implicated in developmental pathways (first ranked go biological process = skeletal system de- velopment, fold enrichment = . , binomial fdr q-value = . × − ), with a specific enrichment for homeobox protein domain encoding genes (interpro; fold enrichment = . , binomial fdr q-value = . × − ), fundamental for normal developmen- tal processes. an enrichment for malignancy terms was also noted (from the molecular signatures database; first ranked term = genes with promoters occupied by pml-rara fusion protein in acute promyelocytic leukemia [apl] cells nb and two apl primary blasts, based on chip-seq data, fold enrichment = . , binomial fdr q-value = . × − ) (see supplemental data s ; liberzon et al. ). to establish whether these dmps are a consistent feature of tbrs, we profiled a further non-amish patients carrying dis- tinct previously published dnmt a pathogenic variants (table ; fig. ) using the illumina epic dna methylation array. examination of the dmps identified in dnmt a c. g > a; p.(arg gln) carriers revealed that the majority of dmps were common to all of the tbrs patients regardless of the underlying causative dnmt a variant (fig. d), with a pearson correlation co- efficient of . (p-value < . × − ) for effect sizes across all dmps. each variant showed some heterogeneity in effect size (fig. e), with dnmt a c. c > t p.(arg cys) associated with the greatest overall changes in dna methylation. this data leads us to conclude that tbrs patients show loss of methylation at sites annotated to key genes involved in development and growth pathways, mirroring the well-characterized overgrowth and neurocognitive features that characterize this disorder. dnmt a mutations are associated with highly accelerated epigenetic aging, particularly the cardinal aml driver mutation p.arg cys dna methylation at a specific set of cpg sites, representing a so-called “epigenetic clock,” has been shown to be strongly table . tbrs dnmt a variants are associated with epigenetic age acceleration id nucleotide change protein change chronological age (yr) epigenetic age (yr) epigenetic age acceleration (fold change) epigenetic age acceleration (percentage increase) single nucleotide variants c. t > a p.(ile asn) . . . % c. g > a p.(gly ser) . . . % c. t > c p.(cys arg) . . % c. t > c p.(leu pro) . . . % c. c > t p.(pro leu) . . . % c. c > t p.(arg cys) . . . % c. g > a p.(arg his) . . . % c. g > a p.(arg gln) – . – . . %a c. a > g p.(asn asp) . . . % c. c > t p.(arg cys) . . . % c. t > c p.(phe ser) . . . % c. c > t p.(pro leu) . . . % in-frame deletions c. _ deltgg p.(trp del) . . . % c. _ deltct p.(phe del) . . . % duplications resulting in a frameshift c. dupa p.(arg fs) . . . % c. _ duptctt p.(ser fs) . . % dnmt a genotype (p.(arg gln), shown in blue; p.(arg cys), shown in red), chronological age, predicted epigenetic age, and percentage of age acceleration calculated for tbrs syndrome cases included in this study. aepigenetic age acceleration taken from the linear regression model applied to four individuals carrying the mutation. jeffries et al. genome research www.genome.org http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc correlated with chronological age (horvath ). deviations from chronological age have been associated with several measures of ac- celerated biological aging and age-related phenotypes (johnson et al. ; levine et al. ; marioni et al. ; chen et al. ). we investigated the dna methylation age of dnmt a c. g > a p.(arg gln) carriers using the dna age calculator (horvath ; http://dnamage.genetics.ucla.edu/), finding that dnmt a c. g > a; p.(arg gln) carriers show evidence for highly accelerated aging—an increase of ∼ % beyond their chro- nological age—compared with wild-type family members (ancova p-value = . ) (fig. a). only one of probes used in the epigenetic clock (horvath ) overlapped with the dmps significantly associated with the dnmt a c. g > a; p.(arg gln) pathogenic variant, leading us to conclude that this finding represented a true acceleration of epigenetic age. furthermore, compared with an extensive number ( ) of wild- type control samples profiled in a previous study from our group (hannon et al. ), dnmt a c. g > a; p.(arg gln), carri- ers were consistent outliers for epigenetic age, suggesting their profiles fall outside the normal distribution of variance observed in the general population (supplemental fig. s ). consistent with this, the mosaic amish father was found to have an interme- diate level of epigenetic age acceleration, with a % increase over his chronological age. this age acceleration was a cumulative pro- cess as indicated by the increased slope of dnmt a c. g > a; p.(arg gln) carriers versus wild-type. epigenetic age could therefore be predicted by the linear regression model as follows: epigenetic age = . + . × chronological age. the cumulative increase of epigenetic age relative to chronological age is also nota- ble compared with a recent meta-analysis of longitudinal cohort data that shows the trajectory of epigenetic age in different popula- tions progresses at a slightly slower rate compared with increasing chronological age (marioni et al. ). we next looked for evidence of elevated epigenetic aging in tbrs patients carrying one of the additional de novo dnmt a pathogenic variants. all tbrs patients showed accelerat- ed epigenetic aging, although the position and type of each variant result in differing degrees of accelerated epigenetic aging (table ). the greatest rate of epigenetic age acceleration (> %) was ob- served in association with the germline p.(arg cys) substitu- tion, somatic mutation of dnmt a arg being the most commonly associated with aml. altered epigenetic aging in methyltransferase-associated human growth disorders to determine whether altered epigenetic aging is a characteristic of other growth disorders associated with disruption of epigenetic regulatory molecules, we extended our study using publicly avail- able illumina k dna methylation data. we first analyzed the data from individuals with sotos syndrome, a congenital over- growth syndrome that results from mutation of the epigenetic modifier nsd (supplemental table s ), a lysine histone methyl- transferase (kurotaki et al. ; qiao et al. ). consistent with dnmt a pathogenic variant carriers, these individuals are characterized by an epigenetic age acceleration of ∼ % (linear re- gression model r = . , p-value = . × − ) (fig. b,d). we then examined data from kabuki syndrome patients carrying path- ogenic variants in the kmt d gene (supplemental table s ), which also encodes a lysine histone methyltransferase (ng et al. ; butcher et al. ). kabuki syndrome is a multisystem dis- order. patients typically present with postnatal growth deficiency (rather than overgrowth), a characteristic facial gestalt, id, and other variable phenotypic features. although there is more hetero- geneity in epigenetic age when compared with the nsd patho- genic variant carriers, there was a significant reduction in epigenetic age of ∼ % seen across these individuals (linear regres- sion model r = . , p-value = . ) (fig. c,d). discussion to date, individuals have been described with the overgrowth condition tbrs. within this group, a wide variety of germline dnmt a pathogenic variants have been reported, including missense, eight stop-gain, seven frameshift and two splice site var- iants, two in-frame and five whole-gene deletions (including a set of identical twins) (tatton-brown et al. ; okamoto et al. ; tlemsani et al. ; hollink et al. ; kosaki et al. ; lemire et al. ; shen et al. ; spencer et al. ; tatton-brown et al. , ; xin et al. ). clinically, the predominant aa prc /eed-ezh complex s-adenosyl-l-methionine - - - aa posi�ons pwwp add mtasedomains interac�ons dnmt and dnmt b aa aa aa aa aa aa aa aascale: missense variant in-frame dele�on frameshi� variant figure . schematic representation of dnmt a. the positions of the disease-associated variants included in this study are indicated relative to the pro- tein domain architecture. pwwp, proline-tryptophan-tryptophan-proline domain; add, atrx-dnmt -dnmt l domain; mtase, methyltransferase domain; aa, amino acid. epigenetic aging abnormalities in growth disorders genome research www.genome.org http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://dnamage.genetics.ucla.edu/ http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc features of tbrs are overgrowth, a characteristic facial gestalt, and neurocognitive impairment. these features show phenotypic overlap with conditions associated with germline pathogenic var- iants in other epigenetic regulatory genes, including sotos and weaver syndromes caused by variants in nsd and ezh histone methyltransferases, respectively (tatton-brown et al. ). these genes encode essential epigenetic regulatory proteins, with a dual somatic/germline role in the pathogenesis of hematological malig- nancies and overgrowth syndromes with variable degrees of intel- lectual impairment (tatton-brown et al. ). the majority of dnmt a pathogenic variants in tbrs have been found to be de novo, with five individuals inheriting the pathogenic variant from two mosaic parents (tlemsani et al. ; xin et al. ) and two individuals inheriting the patho- genic variant from their affected father (lemire et al. ). extensive studies of the role of dnmt a in hematopoietic stem cell (hsc) differentiation are also reported, including the regular occurrence of somatic dnmt a variants in patients with acute myeloid leukemia (aml). the most common somatic pathogenic variant reported in patients with aml affects the amino acid resi- due arg . to date, pathogenic variants predicted to affect this residue have been described in the germline of tbrs patients, five with p.(arg his) and seven with p.(arg cys) (tlemsani et al. ; hollink et al. ; kosaki et al. ; shen et al. ; spencer et al. ; tatton-brown et al. ). despite these studies, the underlying biological mech- anism and outcomes of dnmt a gene mutation in tbrs, as well as the potential risks of hematological malignancy, re- main largely unclear. here we investigated variation in dna methylation associated with a germline heterozygous dnmt a mis- sense pathogenic variant c. g > a; p.(arg gln), affecting the catalytic mtase domain, in a large amish family comprising four children with tbrs, un- affected siblings, and their mosaic father who displayed an intermediate clinical phenotype (xin et al. ). affected in- dividuals were characterized by wide- spread hypomethylation, with dmps enriched in the vicinity of genes/reg- ulatory regions associated with growth and development, tissue morphogene- sis, and differentiation. the magnitude of hypomethylation typically exceeded %, a level often considered to show biological significance (leenen et al. ). the accelerated epigenetic age ob- served did not appear to be driven by overlap of the dnmt a c. g > a; p.(arg gln) variant–associated dmps with the probes that comprise the epige- netic clock as shown by overlap with only one out of the probes used in the epigenetic age estimation (horvath ). although the relevance of blood cells to understanding the etiology of tbrs is not yet known, we hypothesize that our findings will be generalizable across cell types given the ubiquitous developmental expression of dnmt a and given that many age-associated dmps are shared across different cell types (zhu et al. ). nevertheless, it would still be prudent to undertake epigenetic age assessment of other tissues from tbrs patients to determine whether epigenetic agetrulyisaccelerated across all cell typesorafindingthatislimited to blood. although dysregulation of growth control has been linked to numerous developmental disorders and malignancy, the specific molecular basis of this relationship is not fully understood. the as- sessment of dmps associated with the dnmt a c. g >a; p.(arg gln) variant identified an enrichment of pluripotent and fetal dnase i sensitivity hotspots, as well as brain and embry- onic stem cell–associated chromatin sites according to the encode project consortium and epigenomic roadmap consortium data sets (breeze et al. ). similarly, functional an- notation based on gene ontology terms showed an overrepresen- tation of pathways related to morphogenesis, development, and differentiation annotations. dnmt a loss of function has previ- ously been reported to result in up-regulated multipotency genes a b c d w figure . altered epigenetic aging is observed in methyltransferase-associated human growth disor- ders. (a) scatter plot comparing “dna methylation age” derived from the illumina k data (y-axis) and actual chronological age (x-axis) in dnmt a c. g > a p.(arg gln) pathogenic variant carriers (red) versus wild-type family members (blue). green indicates the mosaic individual. the linear regression model is also shown. (b) scatter plot comparing dna methylation age versus chronological age in patients with sotos syndrome. in-frame legend illustrates the different nsd pathogenic variants studied. (c) scatter plot comparing dna methylation age versus chronological age in patients with kabuki syn- drome. in-frame legend illustrates the different kmt d pathogenic variants studied. (d) boxplot compar- ing the epigenetic age acceleration rates found in association with tbrs dnmt a variants, kmt d kabuki syndrome variants, and nsd sotos syndrome variants. each age acceleration observation is plot- ted as a circle. the dotted red line denotes no age acceleration. jeffries et al. genome research www.genome.org and impaired differentiation of neural stem cells and hscs (wu et al. ; challen et al. ; jeong et al. ) compared with a gain-of-function dnmt a variant that may increase cellular dif- ferentiation (heyn et al. ). it is thus conceivable that tbrs-as- sociated dnmt a variants may promote increased proliferation of stem/progenitor cell pool, resulting in increased cell numbers dur- ing organ morphogenesis and clinical overgrowth. our finding of altered epigenetic outcomes in tbrs prompted us to consider similar investigations in other growth disorders as- sociated with epigenetic dysfunction: sotos syndrome, a neurode- velopmental disorder with features overlapping tbrs and with association with overgrowth in childhood owing to histone meth- yltransferase nsd gene alterations, and kabuki syndrome, a dis- tinct neurodevelopmental disorder associated with poor growth and histone methyltransferase kmt d gene alterations. this work defined clear aberrations in epigenetic aging appropriate to the specific nature of each condition. in both overgrowth condi- tions, tbrs and sotos syndrome, we identified accelerated epige- netic aging as measured by the dna methylation age calculator (horvath ). conversely, patients with kabuki syndrome, clin- ically characterized by poor growth, displayed decelerated epige- netic age. epigenetic age has been strongly correlated with chronological age in unaffected individuals in previous studies of a variety of tissue types (hannum et al. ; horvath ). the observation of accelerated epigenetic aging in both tbrs and sotos syndrome potentially results from reduced methyltrans- ferase activity in addition to increased cell turnover associated with the overgrowth seen with these disorders, with the converse being the case for kabuki syndrome. accelerated epigenetic aging has been associated with age-related clinical characteristics and mortality in epidemiological studies. for example, accelerated epi- genetic age in lymphocytes correlates with reduced physical and cognitive function in the elderly and with increased overall mor- tality independent of other variables such as bmi, sex, and smok- ing status (marioni et al. ; chen et al. ). the molecular basis of tbrs has only been determined relatively recently, and as such, most of the affected individuals reported are children and young adults. there is therefore still only very limited data available relating to the progression and prognosis of this disorder, meaning that it is not yet possible to determine whether there might be any clinical evidence of multimorbidity indicative of pre- mature aging or a reduction in average life span in tbrs. further long-term natural history studies of tbrs patients will be extreme- ly helpful for determining the clinical implications of the epige- netic age acceleration observed as a feature of this disorder. accelerated epigenetic age has previously been reported in as- sociation with specific diseases such as huntington’s disease (+ . yr) (horvath et al. ), down syndrome (+ . yr) (horvath et al. ), and werner’s syndrome (+ . yr) (maierhofer et al. ). the accelerated epigenetic aging described in association with these disorders is an average increase in epigenetic age, which is relatively consistent throughout lifespan. a distinguishing feature of carriers of the amish dnmt a c. g > a; p.(arg gln) var- iant is the year-on-year or cumulative increase of accelerated epige- netic aging over life time course, in other words, a true acceleration of epigenetic aging. although it was not possible to undertake these studies for other dnmt a variants, this may be indicative of a similar effect on cumulative epigenetic age acceleration over the life course in tbrs. it is also noted that the gene encoding dnmt l is located on chromosome ; given the previous report of an average dna methylation age acceleration of . yr in blood and brain tissue in individuals with down syndrome (horvath et al. ) and the role of dnmt l in stimulating dnmt a de novo methylation, further investigations are needed to explore the potential relevance of this observation. there are currently only four reported cases of an aml tumor carrying the dnmt a p.arg gln substitution. biochemical measurements of dnmt a show that mutations at both the arg and arg residues result in reduced methyltransferase activity, with a greater degree of reduction resulting from arg variants compared with arg variants ( . -fold difference) (holz-schietinger et al. ). given this reduced methyltransfer- ase activity, we may expect to observe more pronounced changes in dna methylation in patients with germline variants affecting arg compared with variants affecting other amino acid resi- dues such as arg . our data reflected this notion, with alteration arg displaying markedly greater methylation changes com- pared with the other dnmt a mutations investigated in this study. currently, available literature suggests that the risk of hema- tological malignancy in tbrs individuals may vary depending on the specific pathogenic variant underling their condition (hollink et al. ). the significantly advanced epigenetic age that we ob- served in association with p.arg cys may explain why hemato- logical malignancy has to date only been reported in two tbrs patients, one harboring this germline variant and the second the p.tyr ser variant, the latter not being assessed in this study (hollink et al. ; tatton-brown et al. ). in summary, our findings identify widespread dna hypome- thylation in genes involved in morphogenesis, development, dif- ferentiation, and malignancy in tbrs patients. tbrs patients also displayed highly accelerated dna methylation aging. our studies additionally defined phenotype-related altered epigenetic aging in two histone methyltransferase disorders: nsd sotos syn- drome overgrowth disorder and kmt d kabuki syndrome growth impairment. taken together, these findings provide important new insights into the role of dnmt a during development and of relevance to hematological malignancy, and define perturba- tion to epigenetic machinery and biological aging as common themes in overgrowth and growth deficiency syndromes. methods genetic and clinical studies the phenotypic features of the four affected siblings (three females and one male, aged – yr) (fig. a, individuals iii: , iii: , iii: , and iii: ) include macrocephaly, tall stature, hypotonia, mild to moderate id, behavioral problems, and a distinctive facial appear- ance. whole-genome snp genotyping and exome sequencing of dna samples taken with informed consent under regionally ap- proved protocols excluded pathogenic variants in known genes, or candidate new genes, associated with neurodevelopmental dis- orders. subsequent studies defined a heterozygous c. g > a variant in dnmt a, resulting in a p.(arg gln) substitution, as the cause of the condition. full clinical details are previously de- scribed (xin et al. ). further testing revealed mosaicism for the dnmt a c. g > a variant in the father, and xin et al. ( ) showed pathogenic variant load varied in different tissue types. dna methylation profiling genomic dna from blood was sodium bisulfite converted using the ez- dna methylation kit (zymo research) and dna meth- ylation quantified across the genome using the illumina epigenetic aging abnormalities in growth disorders genome research www.genome.org infinium humanmethylation array (illumina k array) (illumina). the additional dnmt a pathogenic variants were profiled using the illumina infinium epic array (illumina). the bioconductor package watermelon (pidsley et al. ) in r . . (r core team ) was used to import idat files, and after check- ing for suitable sodium bisulfite conversion (bisulfite control probe median > %), the dna methylation data were imported and quantile normalized using the dasen function in watermelon and methylation beta values produced (ratio of intensities for methylated versus unmethylated alleles). probes showing a detec- tion p-value > . in at least % of samples or a beadcount < in % of samples were removed across all samples. any samples showing low quality, indicated by a detection p-value > . in ≥ % of probes within a sample, were removed from analysis. probes containing common snps within bp of the cpg site were removed (minor allele frequency > %). nonspecific probes and probes on the sex chromosomes were also removed (chen et al. ; price et al. ). identification of dmps dmps were identified using a limma-based linear model based on pathogenic variant genotype and sex as a covariate (smyth ) and a benjamini–hochberg fdr of % applied (benjamini and hochberg ). when the epigenetic age was used as a covariate, a similar level of dmps were detected ( dmps) with an % overlap to the limma model without age as a covariate. changes in methylation were calculated based on comparison between dnmt a c. g > a; p.(arg gln) carriers versus wild-type in- dividuals in the amish pedigree. the additional dnmt a path- ogenic variants were assessed relative to seven wild-type control samples run on the same epic array run. blood cell counts were unknown and so were estimated using the dna methylation age calculator (horvath ; koestler et al. ) and assessed in the linear model. to identify dmps, the package dmrcate was used with the same limma-based design (peters et al. ). gene ontology and functional enrichment analyses gene ontology enrichment analysis was performed using genes annotated to fdr corrected dmps using the gometh function of the missmethyl package (phipson et al. ), which takes into account potential bias of probe distributions on the beadchip ar- ray. kegg pathway analysis was performed using the gsameth command of missmethyl and kegg annotation files from the bioconductor keggrest package (http://bioconductor.org/ packages/release/bioc/html/keggrest.html). regional enrich- ment analysis based on illumina annotations was performed using a chi-squared test with yates correction in r. dmrs were function- ally annotated using the webtool great (http://great.stanford .edu/public/html/). the top p-value–ranked dmps were also annotated using the eforge tool (https://eforge .altiusinstitute.org/) to perform functional overlap analysis for identifying any cell- or tissue-specific epigenetic signals. quantification of global dna methylation global dna methylation measurements were made using the luminometric methylation assay (luma) (karimi et al. ) based on cleavage by a methylation-sensitive restriction enzyme followed by polymerase extension assay via pyrosequencing on the pyromark q (qiagen). peak heights were obtained using the pyro q cpg . . software and a t-test applied in r . . . global methylation estimates from the illumina k array were assessed through r . . using summary statistics and a wilcoxon rank-sum test on two pairs of samples matched for age and sex. dna methylation age estimation epigenetic age calculations were made using the dna methylation age calculator (https://dnamage.genetics.ucla.edu/) for illumina k data, and illumina epic arrays were assessed using the agep function of the watermelon bioconductor package, the latter based on the original calculator developed by steve horvath ( ). accelerated age was calculated for the amish tbrs dnmt a c. g > a; p.(arg gln) carriers and wild-type family members and sotos syndrome and kabuki syndrome patients and compared with data from control individuals taken from a previous study (hannon et al. ), using linear models of recorded chronolog- ical age and calculated epigenetic age. estimates of age acceleration for the additional tbrs cases were calculated by dividing the calculated epigenetic age with their chronological age. additional nsd sotos syndrome patient illumina dna methylation files were obtained from geo accession gse , with corresponding chronological ages derived from the associated paper (choufani et al. ). kmt d kabuki syndrome dna methylation data and chronological age were obtained from geo accession gse (butcher et al. ). validation of dmps using bisulfite-pyrosequencing bisulfite pyrosequencing was used to validate specific differentially methylated cpg sites originally identified using the illumina k array. primers, designed using pyromark assay design soft- ware (qiagen), and pcr conditions are provided in supplemental table s . bisulfite conversion was performed on ∼ ng of dna using a bisulfite-gold kit (zymo research). pcr was performed with hot firepol dna polymerase (solis biodyne) for min at °c followed by cycles of sec at °c, sec at annealing temperature (shown in supplemental table s ), and sec at °c. a final extension of min at °c was then applied. dna methylation was then assessed using the resulting bisulfite pcr amplicons, together with a pyrosequencing primer on the pyromark q system (qiagen) following the manufacturer’s standard instructions and the pyro q cpg . . software. data access the raw and processed primary data sets generated in this study have been submitted to the ncbi gene expression omnibus (geo; https://www.ncbi.nlm.nih.gov/geo/) under accession num- ber gse . r scripts are provided as supplemental code s and at the following repository: https://github.com/arjeffries/ tbrs . acknowledgments we thank the amish families for participating in this study and the amish community for their continued support of the windows of hope project. the work was supported by the newlife foundation for disabled children (ref: sg/ - / , to c.g.s., a.r.j., a.h.c., and e.l.b.), medical research council (mrc) grant g (to e.l.b.), mrc grant g (to a.h.c.), and mrc grants mr/ m / and mr/k / (to j.m.). author contributions: e.l.b., a.h.c., and j.m. conceived the study. r.m. and b.a.c. performed the genetic analysis. a.r.j. and r.m. performed luma global methylation assay. a.r.j. performed the microarray-based dna methylation analysis and all associated statistical analyses. a.r.j. and e.d. performed the pyrosequencing jeffries et al. genome research www.genome.org http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://bioconductor.org/packages/release/bioc/html/keggrest.html http://great.stanford.edu/public/html/ http://great.stanford.edu/public/html/ http://great.stanford.edu/public/html/ http://great.stanford.edu/public/html/ http://great.stanford.edu/public/html/ https://eforge.altiusinstitute.org/ https://eforge.altiusinstitute.org/ https://eforge.altiusinstitute.org/ https://eforge.altiusinstitute.org/ https://eforge.altiusinstitute.org/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ https://dnamage.genetics.ucla.edu/ http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ https://www.ncbi.nlm.nih.gov/geo/ http://genome.cshlp.org/lookup/suppl/doi: . /gr. . /-/dc https://github.com/arjeffries/tbrs https://github.com/arjeffries/tbrs https://github.com/arjeffries/tbrs 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ye. . dnmt a-dependent nonpromoter dna methylation facilitates transcription of neurogenic genes. science : – . doi: . / science. xin b, cruz marino t, szekely j, leblanc j, cechner k, sency v, wensel c, barabas m, therriault v, wang h. . novel dnmt a germline mu- tations are associated with inherited tatton-brown–rahman syndrome. clin genet : – . doi: . /cge. zhu t, zheng sc, paul ds, horvath s, teschendorff ae. . cell and tissue type independent age-associated dna methylation changes are not rare but common. aging (albany ny) : – . doi: . /aging . received september , ; accepted in revised form may , . jeffries et al. genome research www.genome.org https://www.r-project.org/ https://www.r-project.org/ https://www.r-project.org/ https://www.r-project.org/ https://www.r-project.org/ https://www.r-project.org/ revisão da literatura religiosidade e espiritualidade no transtorno bipolar do humor religiosity and spirituality in bipolar disorder andré stroppa , alexander moreira-almeida professor assistente da faculdade de medicina da universidade federal de juiz de fora (ufjf). professor adjunto da faculdade de medicina da ufjf. recebido: / / – aceito: / / resumo contexto: nos últimos vinte anos, estudos sistematizados têm identificado uma relação positiva entre espirituali- dade/religiosidade (r/e) e saúde, notadamente saúde mental. entretanto, são escassas as informações sobre r/e e transtorno bipolar do humor (tbh). este artigo objetiva revisar as evidências disponíveis sobre estas relações. métodos: foram cruzadas as palavras “bipolar”, “mania” e “manic” com as palavras “religio*” e “spiritu*” nas bases de dados pubmed e psychinfo em novembro de . foram encontrados artigos publicados entre os anos de e . resultado: os estudos apontam que pacientes bipolares tendem a apresentar maior envolvimento religioso/espiritual, maior frequência de relatos de conversão e experiências de salvação e uso mais frequente de coping religioso e espiritual (cre) que pessoas com outros transtornos mentais. indicam ainda, uma relação fre- quente e significativa entre sintomas maníacos e experiências místicas. os estudos mais relevantes encontrados na literatura foram agrupados nesta revisão em cinco tópicos: delírios místicos, religiosidade e espiritualidade, coping religioso-espiritual, recursos comunitários e comunidades tradicionais. conclusão: o tbh e a r/e possuem intensa e complexa inter-relação. estudos sobre práticas religiosas saudáveis, espiritualidade e recursos de coping merecem ser ampliados, bem como sua relação com o cumprimento do tratamento e as recorrências da doença, as inter venções psicoterápicas e a psicoeducação de base espiritual. stroppa a, moreira-almeida a / rev psiq clín. ; ( ): - palavras-chave: transtorno bipolar, espiritualidade, religiosidade. abstract background: over the past twenty years, systematic studies have identified a positive relationship between spirituality/ religiosity (s/r) and health, especially mental health. although there is only scant information about s/r and bipo- lardisorder. methods: the words “bipolar”, “mania” and “manic” were crossed with the words “religio*” and “spiritu*” in the databases pubmed and psychinfo in november . it was found articles published between and . results: the studies indicate that bipolar patients have a greater religious/spiritual concern and involvement, more reports of conversion, experiences of salvation and a more frequent use of spiritual/religious coping, than people with other mental disorders. it also indicates a frequent and significant relationship between manic symptoms and mystical experiences, and changes in the intensity of faith after the onset of the disorder. the most relevant studies in the literature were distributed by subjects: mystical delusions, religiosity and spirituality, spiritual-religious coping, com- munity resources and traditional communities. conclusion: the number of studies about healthy religious practices, spirituality, and coping among bipolar patients should be expanded, as soon as its relation to accession, compliance with treatment and recurrences of the disease. greater attention should be given to investigate the relationships between religiosity, religious coping, psychotherapeutic inter ventions, and based-spiritual psychoeducation. stroppa a, moreira-almeida a / rev psiq clín. ; ( ): - keywords: bipolar disorder, spirituality, religiosity, religiosity and spirituality in bipolar disorder. núcleo de pesquisa em espiritualidade e religiosidade (nupes) da universidade federal de juiz de fora, campus universitário de martelos, juiz de fora, mg. endereço para correspondência: andré stroppa. av. barão do rio branco, / , centro – - – juiz de fora, mg. e-mail: andré.stroppa@ufjf.edu.br stroppa a, moreira-almeida a / rev psiq clín. ; ( ): - introdução a religiosidade e a espiritualidade (r/e) são aspectos importantes na vida da maioria das pessoas e na cultura da maioria dos povos. desde a antiguidade, espirituali- dade e saúde estiveram intimamente relacionadas. no ocidente, religiosos se ocuparam dos cuidados a pessoas enfermas da idade média até bem recentemente. no final do século xix, a ciência se distanciou da religião, diante da necessidade de se firmar como conhecimento autônomo , . nos últimos vinte anos, estudos sistematizados e bem conduzidos passaram a identificar uma relação positiva entre r/e e saúde. estudos sugerem que r/e possam ter efeito protetor sobre a saúde e parecem influenciar a saúde física e mental de várias maneiras: (a) por meio de regras de convivência e do desestímulo a compor- tamentos prejudiciais à saúde, como abuso de álcool e drogas, comportamento violento ou sexual de risco; (b) por meio do uso de crenças religiosas como forma de lidar com situações adversas, como uma doença; (c) por meio da criação de uma rede de suporte social - . a organização mundial da saúde (oms) estima para a próxima década um aumento considerável da participa- ção dos transtornos mentais entre as principais causas de anos de vida perdidos por morte ou incapacidade. o transtorno bipolar do humor (tbh) estará entre as dez principais causas . objetivo revisar a literatura científica a respeito da relação entre tbh e religiosidade/espiritualidade. método as palavras “bipolar”, “mania” e “manic” foram cruza- das com as palavras “religio*” e “spiritu*” nas bases pubmed e psychinfo, no mês de novembro de . também foram consultados os textos e referências citadas em handbook of religion and health (koenig, ) e handbook of religion and mental health (koenig, ). para a presente revisão foram selecionados pri- mariamente os artigos que apresentavam um desenho metodológico adequado e que investigavam diretamente r/e e tbh. resultados foram encontrados artigos publicados entre os anos de e , visto que os estudos realizados até a década de , em sua maioria, vêem na espiritualidade uma evidência de adoecimento mental. predominam os relatos de casos voltados para delírios de conteúdo místico, crenças e conversões religiosas, e os estudos realizados a partir da década de são mais abrangen- tes, envolvem outros aspectos da relação r/e e tb. os estudos mais relevantes foram distribuídos por temas, são eles: delírios místicos, r/e, coping religioso e espiritual (cre), recursos comunitários e inter venções religiosas e comunidades tradicionais. delírios místicos autores clássicos da primeira metade do século xx apontavam esquizofrenia e epilepsia como os transtor- nos mentais mais frequentemente relacionados com sentimentos e sintomas religiosos. emil kraepelin, em , chamou a atenção para a significativa ocorrência de sintomas religiosos entre pacientes bipolares . em estudo transversal de , sedman e hopkin- son estudaram pacientes, sendo sete esquizofrênicos e três bipolares: avaliaram a ocorrência de vivências de influência, alucinações, experiências de conversão e mudanças de humor, acompanhados por vivências místi- cas e religiosas. durante o estudo, mudanças de humor ocorreram nos três pacientes bipolares em vigência de mania e se fizeram acompanhar por experiências místi- cas e religiosas. quatro dos pacientes esquizofrênicos apresentaram períodos de elevação e depressão do humor, visto que em três deles tais fases de mudança de humor foram acompanhadas também por vivências místicas e religiosas. cothran e har vey , em estudo transversal de , compararam pacientes esquizofrênicos e bipolares, porém não identificaram diferenças nas características dos delírios. a incidência de delírios de conteúdo místico nesse estudo foi igual em ambos os transtornos. brewer ton , em estudo transversal de , exa- minou pacientes entre maníacos, esquizofrênicos, esquizofrênicos com alterações eletroencefalográficas, deprimidos em depressão psicótica e pacientes com sintomas psicóticos secundários ao uso de substâncias, sendo cada gr upo representado por dez pacientes. os delírios religiosos foram encontrados em todos os transtornos, com predomínio em pacientes maníacos, seguidos por pacientes com psicoses secundárias ao uso de substâncias psicoativas e esquizofrênicos com alterações eletroencefalográficas. em estudo transversal de , appelbaum et al. ao entrevistarem . pacientes agudamente internados encontraram pacientes delirantes: de pacientes com delírios religiosos, , % tinham diagnóstico de esquizofrenia, , % de transtorno bipolar e , % de transtorno depressivo. getz et al. , em estudo transversal de , estu- daram a influência da atividade religiosa sobre a inten- sidade de delírios místicos em pacientes cristãos com psicose. examinaram católicos, protestantes e pacientes sem afiliação religiosa e encontraram que os pacientes protestantes experimentavam mais delírios que os demais, embora não tenham ocorrido diferenças na intensidade destes. entretanto, quando os grupos foram stroppa a, moreira-almeida a / rev psiq clín. ; ( ): - recombinados, os pacientes mais religiosamente ativos experimentaram delírios místicos mais intensos. hempel et al. , em estudo transversal de , examinaram pacientes de uma instituição penal, deles glossolálicos. segundo os autores, os pacientes glossolálicos eram todos portadores de transtornos do espectro bipolar, com delírios religiosos ou sexuais e hiper-religiosidade. alguns autores atribuem a pacientes bipolares a maior frequência de delírios religiosos e místicos, outros relatam igual prevalência entre bipolares e esquizofrêni- cos. koenig , entretanto, aponta uma diferença qualitati- va entre os delírios de um e de outro: pacientes bipolares apresentam delírios religiosos como intensificação de suas crenças normais e pacientes esquizofrênicos têm delírios místicos autistas e bizarros. religiosidade e espiritualidade a r/e entre pessoas portadoras de transtornos mentais ainda têm suscitado pouco interesse entre pesqui- sadores e clínicos. uma das mais importantes obras dedicadas ao tbh, em sua segunda e recente edição, faz brevíssima referência à relação entre tbh e r/e. os autores ressaltam que o possível efeito protetor de crenças e práticas religiosas não tem sido estudado em indivíduos com tbh . em , nos estados unidos, gallemore et al. rea- lizaram um estudo transversal envolvendo pacientes bipolares e sujeitos-controle de igual idade e sem antecedentes de transtornos mentais. foi utilizada uma entrevista estruturada abrangendo questões relativas à denominação religiosa, educação religiosa, interesse e vida religiosa, além da postura familiar. foram obser va- dos períodos de dúvida e agnosticismo entre os entrevis- tados. os dados para os dois grupos foram comparados evidenciando maior frequência de relatos de conversão e experiências de salvação entre os pacientes bipolares, estivessem bem ou em período crítico da doença. de modo semelhante koenig obser vou entre pa- cientes bipolares uma maior incidência de experiências místicas em suas vidas religiosas, o que poderia estar relacionada ao incremento da afetividade. estudo transversal de , realizado por mitchel e romans na nova zelândia, envolveu pacientes bipolares. a esses pacientes foi enviado um questionário de crenças religiosas, espirituais, filosóficas e práticas religiosas. receberam ( %) respostas completas. a identidade étnica predominante era de origem européia ( , %), seis ( %) eram da etnia maori e cinco ( %) de outros grupos étnicos. a maioria ( %) indicou alguma forma de compreensão religiosa, espiritual ou filosófi- ca a respeito do mundo e relatou uso de suas crenças para lidar com sua doença, e % se disseram cristãos de várias denominações. quanto à prática religiosa, os evangélicos foram mais envolvidos que as denominações tradicionais e liberais. aqueles que não estiveram bem nos últimos cinco anos referiram maior envolvimento religioso, mas suas crenças não os ajudaram a lidar com a doença. não foram obser vadas diferenças entre idades e gêneros. encontrou-se uma relação significativa e inversa entre intensidade da crença e cumprimento das prescrições médicas, aqueles com maiores crenças eram piores cumpridores das prescrições médicas. dezesseis pacientes ( %) referiram redução de sua fé com o surgimento da doença. dantas et al. , em estudo retrospectivo realizado em no brasil, investigaram o papel das crenças e práticas religiosas sobre a doença mental. o trabalho avaliou dados socioculturais e sintomas psicopatológicos associados à presença e à intensidade de sintomas com conteúdos religiosos. para isso, revisaram duzentas internações consecutivas realizadas em hospital univer- sitário. a média da idade dos pacientes foi de , ± , anos, com % de homens (n = ) e as denominações religiosas se distribuíram entre católicos, , % (n = ), protestantes, , % (n = ) e outros, , % (n = ). quanto ao diagnóstico psiquiátrico: esquizofrenia, , % (n = ), depressão, , % (n = ), mania, , % (n = ), neuroses e transtornos da personalidade, , % (n = ) e outros, , % (n = ). não houve diferença estatisticamente significativa entre os grupos religiosos quanto ao diagnóstico e a pontuação média na escala brief psychiatric rating scale (bprs). os sintomas re- ligiosos estiveram presentes em pacientes ( , %), em sua maioria com sintomas do espectro maníaco, mostrando significativa correlação entre sintomas e estados maníacos e experiências místicas. kirov et al. , em estudo transversal de na ingla- terra, investigaram em pacientes ( esquizofrênicos e bipolares) como a doença psicótica influenciou sua fé religiosa e o quanto crenças e práticas religiosas foram usadas como coping. a amostra teve importante participação afro-caribenha e altos níveis de atividade religiosa. dos indivíduos, se declararam religio- sos e não religiosos. a maioria dos que se disseram religiosos relatou ter usado sua religião como coping ( , %) e foi obser vado aumento de crenças religiosas em indivíduos e diminuição em dois após a doen- ça. três variáveis estiveram relacionadas de maneira significativa à maior frequência de coping: ser mulher, não branca e ter tbh. consoante os autores, pacientes com maior frequência de coping apresentaram melhor insight de sua doença, resultando em maior adesão ao tratamento medicamentoso. a experiência do adoeci- mento mental contribuiu para incremento das crenças religiosas nesse estudo. soeiro et al. publicaram em , no brasil, estudo transversal que investigou a relação entre prevalência de transtornos mentais, denominação religiosa e re- ligiosidade. avaliaram pacientes de um hospital geral aplicando um questionário sociodemográfico e um instrumento para o diagnóstico de transtornos mentais (mini-plus): quanto ao grau de religiosidade referido, stroppa a, moreira-almeida a / rev psiq clín. ; ( ): - ( , %) pessoas se disseram muito religiosas, ( , %), religiosas, ( , %), pouco religiosas e três ( , %), não religiosas. a intensidade do envolvimento religioso foi moderadamente associada com maior ocorrência de transtornos mentais, sobretudo com o transtorno bipolar. os pacientes não religiosos e muito religiosos estiveram relacionados a maior adoecimento mental quando comparados a pessoas religiosas. vários autores apontam uma menor ocorrência de sintomas religiosos e místicos nos quadros depressivos, em uma relação inversa entre sintomas religiosos e de- pressivos. frequentemente, crenças e práticas religiosas relacionam-se a menores índices de sintomas depres- sivos. os sintomas psicóticos em quadros depressivos podem ter efeito difuso sobre as crenças religiosas das pessoas. predominam delírios de culpa, menos-valia e ruína, podendo tomar um significado religioso para o paciente. as pessoas deprimidas têm menor frequência de experiências religiosas e menor crença em deus, com maiores sentimentos de culpa pela falta de fé ou perda de sua fé , . os estudos evidenciam a especial importância dos aspectos religiosos e místicos na vida de pacientes bipo- lares, bem como a maior frequência com que utilizam suas crenças religiosas para lidar com situações de estresse e com sua doença. gallemore et al. apontaram diferenças entre certos aspectos da r/e em pacientes bipolares e controles normais, como maior frequência de experiências de conversão e salvação. koenig apon- tou a maior incidência de sintomas místicos e atribuiu às peculiaridades afetivas da doença. dantas et al. encontraram uma relação significativa entre sintomas de mania e sintomas místicos. kirov et al. encontraram entre as mulheres não brancas e portadoras de tbh a maior frequência de uso de coping religioso e espiritual em seu estudo. soeiro et al. apontaram o tbh como o transtorno mental mais frequentemente associado à intensidade do envolvimento religioso. em suma, fica evidenciado pela literatura disponível a significativa relação do tbh com mais frequentes experiências religiosas e espirituais, bem como maior influência de re em suas vidas. coping religioso e espiritual coping pode significar lidar, manejar, adaptar-se ou enfrentar. trata-se de um processo de interação entre o indivíduo e o ambiente, com a função de reduzir ou suportar uma situação estressora que exceda os recur- sos do indivíduo. coping religioso e espiritual (cre) é o modo como pessoas utilizam sua fé em situações de estresse e dificuldade em suas vidas . reger e rogers , em nos estados unidos, es- tudaram cre entre pessoas com transtornos mentais crônicos. o estudo investigou o número de estratégias usadas, o tempo de uso e a percepção de ter sido ajuda- do. participaram do estudo pessoas com transtornos mentais persistentes, sendo ( %) esquizofrênicos, ( %) depressivos, ( %) esquizoafetivos, ( %) bipolares e ( %) com outros transtornos psicóticos, do humor e ansiedade. os resultados apontaram alta prevalência e importância do uso de cre na amostra. para os autores o cre foi mais evidente, duradouro e variado em pacientes esquizofrênicos, esquizoafetivos e bipolares. esses pacientes usaram o cre por mais tempo e perceberam mais benefícios ao lidar com frus- trações e dificuldades diárias. phillips e stein , em nos estados unidos, investigaram o cre e sua relação com as variáveis demográficas e religiosas em um estudo longitudinal, com um grupo-controle de estudantes universitários e duração de um ano. foram estudados adultos jovens esquizofrênicos e bipolares. utilizaram três subescalas de rcope (pargament et al., ): uma de cre posi- tivo e duas de cre negativo. aqueles com transtorno mental grave usaram as mesmas formas de cre que os jovens do grupo-controle. foi, entretanto, estatisti- camente significativo o uso de cre negativo entre os indivíduos com transtorno mental grave no tempo inicial do estudo. os autores chamam a atenção para o fato de que pessoas com transtorno mental grave usam formas de cre positivas e negativas, mas formas negativas merecem atenção por trazerem prejuízo significativo para os pacientes. pollack et al. , em estudo transversal de nos estados unidos, compararam indivíduos brancos e negros bipolares hospitalizados e encontraram entre os pacientes negros melhores recursos internos cogni- tivos, emocionais e espirituais. o enfrentamento mais frequente de situações adversas, uma visão mais positiva dos problemas e uma maior preocupação religiosa du- rante a educação foram apontados pelos autores como aprendizados que persistiram na vida adulta. em pacientes bipolares o cre é frequentemente utilizado, muitas vezes benéfico e variado. são utiliza- das estratégias de coping positivas que resultam em bem-estar, confiança e calma. mas, também, negativas que encerram culpa, medo e autodesvalorização, senti- mentos desvantajosos para a saúde psíquica. atividades psicoeducacionais podem orientar estratégias de cre com importante benefício para pacientes religiosos. recursos comunitários e intervenções religiosas um importante salto de qualidade no tratamento do tbh foi dado com o uso dos sais de lítio e outros esta- bilizadores do humor. entretanto, ainda são frequentes as reagudizações. vários autores têm demonstrado a importância da associação de tratamento farmacológico com abordagens psicossociais . pollack et al. chamam a atenção para a relevância de questões, como adesão ao tratamento e estressores capazes de precipitar novos episódios. vários fatores têm sido relacionados com maior recorrência do tbh: stroppa a, moreira-almeida a / rev psiq clín. ; ( ): - suporte social inadequado, desajuste em atividades so- ciais e de lazer, má qualidade das relações interpessoais, principalmente familiares. chakrabarti et al. , em , na Índia, estudaram pacientes portadores de transtorno do humor e ob- ser varam que a sobrecarga referida por familiares de pacientes bipolares era maior que a referida por familia- res de pacientes depressivos. essa maior sobrecarga era atribuída aos reflexos sociais causados pela ocorrência da mania. o número de episódios de mania, a duração da doença, a severidade e a disfunção das fases foram determinantes da maior sobrecarga. stueve et al. em , nos estados unidos, investi- garam a sobrecarga percebida por cuidadores negros, brancos e latinos de pacientes bipolares. negros tende- ram a referir menor sobrecarga que brancos e latinos. entre as causas apontadas estava o maior envolvimento religioso identificado entre os negros, o que funcionava como importante recurso de coping e rede de apoio social e emocional. phillips et al. , em estudo aberto e não controlado de , nos estados unidos, utilizando recursos religiosos, métodos de coping e técnicas de grupo desenvolveram um programa de tratamento dirigido a pessoas com transtornos mentais graves. foi selecionado um grupo de dez pacientes, todos brancos, a maioria do sexo feminino, por tadores de vários transtornos mentais. o programa de tratamento foi constituído por sete encontros semanais de minutos de duração cada e formato psicoeducacional. foram abordadas questões relacionadas a conflitos espirituais, recursos religiosos e sua influência sobre a doença. os participantes refe- riram maior compreensão de seus sentimentos, de suas preocupações espirituais e relações interpessoais. a preocupação com o desenvolvimento de estratégias psicossociais ganha cada vez maior importância para quem trabalha e quem pesquisa tbh. a qualidade do supor te social, das atividades sociais e das relações interpessoais tem sido relacionada com menor recor- rência da doença. É provável que r/e façam diferença para pacientes bipolares ao proporcionarem, com frequência, suporte social e relações interpessoais de boa qualidade. comunidades tradicionais comunidades tradicionais, com frequência, relacionam transtornos mentais a causas espirituais. suas crenças acerca de saúde e doença não são substituídas por con- ceitos médicos, o que deve ser considerado na condu- ção do tratamento. em estudo na nova zelândia sobre religiosidade e coping pessoas da etnia maori referiram conflitos entre sua compreensão de doença e o modelo médico ocidental, com prejuízos para o cumprimento do tratamento , . raguram et al. em estudo aberto e não controlado, em na Índia, descreveram o tratamento de pes- soas com transtornos psicóticos graves, dentre os quais três pacientes bipolares. os pacientes foram tratados em um templo hindu durante seis semanas, sem o uso de terapêuticas biológicas. os autores observaram redução de % nos escores de sintomas psiquiátricos medidos pela escala bprs. egeland et al. , em estudo aberto e não controlado realizado em nos estados unidos, revisaram alguns dos fatores culturais mais comuns e relevantes para o diagnóstico do tbh entre os old order amish (amish). os amish formam uma comunidade religiosa cristã bas- tante tradicional em seus usos e costumes. um médico não familiarizado com a cultura pode ter dificuldade para identificar uma fase de mania. também a temática religiosa de uma pessoa de outra cultura pode ser facil- mente tomada por vivência psicótica. o diagnóstico de esquizofrenia em pacientes bipolares amish foi recor- rente por longo tempo, em razão de desconhecimento ou desvalorização das diferenças culturais. muitos pacientes bipolares relatam conflito signifi- cativo entre médicos e conselheiros religiosos sobre como entender e lidar com sua doença. É frequente que psiquiatras, psicólogos e outros profissionais de saúde mental ignorem ou critiquem crenças religiosas de seus pacientes. É também frequente que líderes religiosos tenham reser vas em relação aos tratamentos em saúde mental. entretanto, pacientes psiquiátricos dão grande importância às suas crenças e atribuem a elas um papel impor tante no lidar com sua doença. vários autores têm identificado problemas para pacientes que têm um modelo de doença muito diferente de seus médicos, resultando em pior adesão ao tratamento. os resultados sugerem ser essa uma área que merece esforços no sentido de reduzir as incertezas existentes , . comunidades tradicionais chamam a atenção para a diversidade cultural e os paradigmas diferentes de doença. os maori tiveram menor adesão ao tratamento em razão de sua compreensão de doença muito diversa do modelo médico ocidental. os amish foram diagnosti- cados erradamente por décadas em razão das diferenças culturais não consideradas , . discussão mania e depressão são dois momentos de um mesmo transtorno mental, e sintomas psicóticos uma evidência da gravidade desses momentos (fases). nesta revisão a literatura frequentemente tratou o tbh como parte dos transtornos psicóticos, e raramente o relacionou à depressão - . apesar da importante prevalência e gravidade do tbh, sua relação com r/e ainda é pouco conhecida. os estudos obtidos apontam maior ocorrência de relatos de conversão religiosa e experiências de salvação entre pacientes bipolares do que entre pacientes com outros transtornos mentais, além de maior frequência de delí- rios de conteúdo místico na vigência de mania , , , - . stroppa a, moreira-almeida a / rev psiq clín. ; ( ): - os pacientes bipolares estão entre aqueles que com maior frequência utilizam cre, mas não se sabe se pre- dominam cre positivo ou negativo. ainda se conhece pouco sobre estratégias de cre usadas por esses pacien- tes e como lidar com sua r/e em seu benefício , - . não se sabe se r/e e cre poderiam influenciar a adesão ao tratamento e reduzir a ocorrência de fases de adoecimento ou a necessidade de internação hospitalar em uma realidade socioeconômica, cultural e religiosa como a latino-americana. não se encontrou na literatura qualquer estudo comparando r/e e cre em pacientes bipolares quando maníacos, eutímicos ou depressivos, o que deixa em aberto muitas questões relacionadas à doença e à religiosidade no tbh. diante da frequência com que pacientes bipolares se dizem religiosos, estratégias de tratamento psicos- social de conteúdo espiritual podem constituir-se em uma forma de auxiliar e dar qualidade ao tratamento farmacológico - , , . os estudos acerca de comunidades tradicionais e gru- pos étnicos abordam conflitos de paradigmas de doença entre pacientes e profissionais de saúde. estas pesquisas apontam a repercussão negativa desse conflito sobre o tratamento, mas também apontam a possibilidade de conciliação de estratégias de tratamento associadas ao respeito a crenças e cultura locais. os benefícios de uma associação entre os recursos da medicina convencional e outras tradições devem ser considerados em nome do bem-estar de muitas pessoas com crenças religiosas e espirituais , , , . para alguns autores, delírios místicos são mais frequentemente encontrados entre bipolares e esqui- zofrênicos. entretanto, existe na literatura de grande parte do século xx um fator complicador no diagnósti- co diferencial entre esquizofrenia e tbh, com o tbh subdiagnosticado por muitas décadas. na medida em que o tbh vem sendo mais frequentemente identifica- do também sua relação com r/e tem se tornado mais clara - , - , . nesta revisão três estudos indicam igual ocorrência de delírios místicos e religiosos entre pacientes bipola- res e esquizofrênicos. outros cinco estudos apontam a coincidência entre sintomas maníacos e maior ocorrên- cia de sintomas místicos - , , - . o estudo de sedman e hopkinson aponta para uma frequente coincidência entre a ocorrência de delírios místicos e alterações do humor. apesar da pequena amostra, os três pacientes diagnosticados como bipolares e três dos quatro pacien- tes diagnosticados como esquizofrênicos apresentaram delírios místicos durante período de exaltação do humor. essa mesma correlação entre delírios místicos e sinto- mas maníacos foi apontada por dantas et al. . koenig defende, em recente revisão, que delírios religiosos existem em um continuum entre crenças normais e fantásticas. sua maior ocorrência estaria ligada a fatores como diagnóstico, gravidade do trans- torno mental e intensidade do envolvimento religioso do indivíduo. conclusão existem evidências de uma maior preocupação e envol- vimento religioso e espiritual entre pacientes bipolares, bem como mais frequente uso de cre que em outros transtornos mentais. em razão disso, o número de es- tudos sobre práticas religiosas saudáveis e recursos de cre merece ser ampliado, bem como sua relação com a adesão ao tratamento e as recorrências do transtorno. estudos longitudinais com pacientes bipolares exa- minados em suas fases de mania, eutimia e depressão poderão esclarecer aspectos ainda obscuros da r/e no tbh. estratégias psicoeducacionais e psicoterápicas que abordem questões espirituais podem se constituir em importante instrumento no tratamento de pessoas com tbh. 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( ): - . . phillips re rd, lakin r, pargament ki. development and implementation of a spiritual issues psychoeducational group for those with serious mental illness. community ment healt j. ; ( ): - . . raguram r, venkateswaran a, ramakrishna j, weiss gm. traditional community resources for mental health: a report of temple healing from india. bjm. ; ( ): - . . egeland ja, hostetter am, eshleman sk rd. amish study iii: the impact of cultural factors on diagnosis of bipolar illness. am j psychiatry. ; ( ): - . http://jtr.sagepub.com/ journal of travel research http://jtr.sagepub.com/content/ / / the online version of this article can be found at: doi: . / : originally published online june journal of travel research deepak chhabra how they see us: perceived effects of tourist gaze on the old order amish published by: http://www.sagepublications.com on behalf of: travel and tourism research association can be found at:journal of travel researchadditional services and information for http://jtr.sagepub.com/cgi/alertsemail alerts: http://jtr.sagepub.com/subscriptionssubscriptions: http://www.sagepub.com/journalsreprints.navreprints: http://www.sagepub.com/journalspermissions.navpermissions: http://jtr.sagepub.com/content/ / / .refs.htmlcitations: by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ http://jtr.sagepub.com/content/ / / http://www.sagepublications.com http://www.ttra.com http://jtr.sagepub.com/cgi/alerts http://jtr.sagepub.com/subscriptions http://www.sagepub.com/journalsreprints.nav http://www.sagepub.com/journalspermissions.nav http://jtr.sagepub.com/content/ / / .refs.html http://jtr.sagepub.com/ journal of travel research ( ) – © sage publications reprints and permission: http://www. sagepub.com/journalspermissions.nav doi: . / http://jtr.sagepub.com how they see us: perceived effects of tourist gaze on the old order amish deepak chhabra abstract although abundant literature focuses on tourism impact perspectives, folk communities’ perceptions of tourist gaze and its effects on the cultural fabric of host communities as a subject of inquiry has received scant attention. to shed new light on this discourse, this study investigates perceived impacts of tourist gazing within the framework of resistance theory. the old order amish (ooa) are positioned on a resistance scale, with an open-resistance stance at one end and a full-cooperation stance at the opposite end of the spectrum. this study gathers data from ooa families residing in the state of iowa, united states. the results show mixed reactions by the ooa and indicate a middle approach of resistance, termed as “negotiated reciprocity” on the resistance continuum. keywords tourist gaze; folk community; sociocultural impact perceptions; resistance theory; old order amish introduction influence of tourism on local communities has been exten- sively examined within the context of social and cultural impacts using the host community’s perspective. social and cultural impacts pertain to the manner in which tourism plays a role in changing resident value structures, individual conduct, family relations, shared lifestyles, safety zones, moral behavior, innovative expressions, conventional rituals, and community organizations (fox ). ap and crompton ( ) explained the tourism effect on local reserves as “pressure on local resources and facilities, local versus imported labor, local language and cultural effects, and life style changes” ( , p. ), and proceeded to provide a summary of impacts based on their review of the literature. positive social and cultural impacts were found to include enhanced quality of life (pizam ; perdue, long, and allen ), improved understanding and images of other cultures (liu, sheldon, and var ; milman and pizam ), facilitation of cultural exchange (belisle and hoy ), educational experience (liu, sheldon, and var ), preservation of cultural identity of the host population, and increased demand for historical and cultural exhibits (liu and var ). negative social and cultural impacts have included heightened stress, frantic community and personal life (rotham ), and creation of fake folk culture (brougham and butler ; cohen ). similar impacts were reinforced by recent studies. for instance, encouragement of cultural activities was reported by brunt and courtney ( ). according to andereck et al. ( ), residents perceive that tourism enhances their com- munity image and creates awareness of heritage. improved prospects for cultural exchange and revival of local tradi- tions and better quality of life were reported by besculides, lee, and mccormick ( ). with regard to externalities, tosun ( ) suggested social and cultural conflicts arising from sociocultural differences. cultural commercialization was confirmed by chhabra, healy, and sills ( ). gursoy and rutherford ( ) reported negative effects of tourism on the local culture in terms of intrusion. positive effects included cultural activities by local residents, cultural exchange between tourists and residents, and enhanced cul- tural identity. while the aforementioned impacts are useful in providing an overall assessment of benefits and costs (andereck et al. ; ham, brown, and jang ), they have limited value in their ability to unveil root issues or items of concern asso- ciated with host–guest contact at the micro level (nichols, giacopassi, and stitt ). it is no surprise then that the authors of many such studies have recommended a further dissection of identified issues such as impact on the cultural and social fabric of receiving regions within the context of cultural commodification. literature has increasingly recog- nized the significance and sacrifice of authenticity in cultural commodification. this view is heightened by the fact that authenticity is the key message in promotional materials that arizona state university by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ journal of travel research ( ) draw tourist attention. the authenticity notion however has multiple connotations. it has been conceptualized and recon- ceptualized during the past few decades. in fact, previous studies have used different meanings of authenticity from tourist and supplier perspectives to gain an insight of cultural and social impacts on host communities (littrell, anderson, and brown ; waitt ; chhabra, healy, and sills ). in sum, four schools of thought describe the authenticity debate: conventional–essentialist, negotiated, constructed–constructivist, and subjective–existentialist. the conventional concept supports museology and nonin- tervention within the natural phenomena. many heritage attractions, cultural heritage managers, and museums regard authenticity in the chutzpah of frozen or museumfied heri- tage (bruner ; yea ). this view has increasingly gained strength in the face of ongoing threat to cultural changes in the globe today. this view regards commodifica- tion as a threat to objective authenticity. next, the negotiated stance holds that commodification can serve a useful func- tion because it intends to accomplish a middle path between demand and supply (chhabra ). on the other hand, the constructed–constructivist paradigm of authenticity is pre- mised on the notion that all judgments are socially constructed and are affected by the contemporary market forces and environments. last, toward the end of the th century, the constructivist notion of authenticity shifted to a purely sub- jective position. this school of thought argues that subjective negotiation of meanings defines an authentic experience (uriely ). following this trail, another postmodern view argues that authenticity is insignificant and irrelevant. for instance, reisinger and steiner ( ) contended that the whole notion of authenticity is unstable. it is laden with con- flicting concepts because it lies in the eyes of the beholder, be it the tourist or the supplier. nevertheless, authenticity is a most recurrent topic of discussion in tourism impact literature. furthering this line of study, the complex nature of the host–guest or tourist–local contact (kim ; laxon ; moaz ; sutton ) calls for an in-depth and holistic discursive approach from an ethnic perspective to examine the “visualness” or “gazing” phenomena in tourism that has a profound effect on both hosts and guests. previous studies have reported local community perceptions in general while failing to provide an examination of perception dynamism within gazing or nongazing settings. that is, some percep- tions may be caused by noninteraction, visual or physical, with tourists; other perceptions can be the consequence of interactions within the context of gazing or exchange of words or buyer–seller setting. early studies have suggested that tourism creates a dis- tinct type of tourist gaze and locals struggle to deal with it when it lands on their turf. volkman ( , p. ) states that “indigenous peoples are caught up in complex relations with the ethnic tourism industry (and the appetite it stimulates), with tourists, and with major cultural, social, and political transformations at home.” many such communities have tried to reconstruct their culture in a way that it meshes with consumer demands (moaz ), while struggling to stay in harmony with their cultural and religious values. extant lit- erature holds that most indigenous communities commodify their culture or freeze selected remnants of their past as per the market need (beeton ; foster ; light ; mcgregor ). that said, several studies have also reported an active role of other sources such as suppliers (notzke ), the media (beeton ), and the govern- ment (yea ) in the commodification of host community cultures. however, it is also argued that not all local communities walk with tourism; some gaze back (moaz ) to curtail plagued impacts. impressions from the local gaze can cau- tion against unforeseen intrusions or suggest positive discourses if benefits are evident. moaz ( ) introduces the term local gaze when referring to reciprocity gestures of hosts to demonstrate knowingly or unknowingly the power of locals in developing countries. the power of locals over the tourists implies the hosts have the capability to exercise choice and control. against previous assertions that locals in developing countries refrain from gazing and passively hide from the tourist gaze, moaz questions the asymmetrical nature of the gaze power. in the words of this author, “the gaze is not necessarily ocular and is not concerned only with spectacle as some claim, but relies on mental perceptions. it is not ‘how we see them’ but ‘how they see us’” ( , p. ). local residents are often assumed to be mute or submis- sive. that is, it has often been assumed that locals do not gaze but passively hide from the tourist gaze and try to adapt to the expectations and requirements of tourists (haraalam- bopoulos and pizam ; mowforth and munt ; nash ). recent literature, however, has unpacked a more active role of locals. some of it has taken the shape of local gaze to discern and scrutinize tourist behavior so that appro- priate response can be crafted to cope with it. most local gazes are informed and may be based on ste- reotyped notions of the tourists (joseph and kavoori ). alternatively some gazes shape perceptions and consequent behavior toward the tourists. these help craft strategies to shield personal lives or construct staged authenticity by con- structing fake back and front stages. according to chhabra, healy, and sills ( ), front stages are contrived to stage authenticity and these are drawn from predetermined and presold images by those in authority. maccannell ( ) coined the term staged authenticity to explain how barriers are created to block intrusions. in the words of urry, “mac- cannell’s staged authenticity arises from the social relations built around the attempts by visitors visually to consume ‘authentic places’ and people and the resistances to this by mad locals” ( , p. ). in sum, the host community’s response to tourists has taken the form of open resistance, veiled resistance, mediated by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ chhabra resistance, retreatism into traditional norms pertaining to lan- guage and culture, deliberate boundary maintenance, and adaptation to the tourist culture or the revitalization of arts and crafts on a more positive note (dogan ). open resis- tance is used in the form of protest and agitation (westerhausen , p. ). veiled resistance constitutes staging and sell- ing of host images. this resistance has passive connotations in the form of “gossip, obstruction, burlesquing, sulking, and insults” (boissevain , p. ; bramwell , p. ). mediated resistance involves use of effective rhetoric allow- ing an outlet for airing grievances against visitors while deflecting hostile confrontations with tourists. retreatism is cutting oneself off from the outside environment. boundary maintenance, on the other hand, implies retiring of host com- munities behind self-imposed boundaries that afford protection from the tourist invasion (buck ). finally, cooperation approach is used when the host community fully adapts to tourist needs. response or the resistance level used by the host community in the face of the tourist gaze phe- nomena can thus be positioned on a scale punctuated by two extremes: open resistance to full adaptation and cooperation. this study refers to these strategies as cradled within the negotiation framework and argues that resistances are pre- mised on the perspectives and deliberations of locals while gazing back visibly or in a hidden manner. although abundant literature focuses on impact perspec- tives of tourists and tourists in general, a folk community’s perceptions of tourist gaze and its effects on the cultural fabric as a subject of inquiry has received scant attention in tourism literature. to shed new light on this discourse, this study examines perceived impacts of tourist gazing within the framework of resistance theory. the resistance theory holds that negotiation strategy of host communities is guided by singular or multiple resistances to tourist gazing. this argument is premised on the stance that the relationship between guests and hosts is multifaceted and complicated in that “it is mediated while it is being resisted rhetorically” and this mediated resistance functions “to transform an ambiva- lent and disempowered relationship into one that is culturally acceptable to the host community” (joseph and kavoori , p. ). the underpinnings of the negotiation framework while guided by the resistance theory draw inferences from the folk community model. the folk community (fc) model suggests that folk societies change slowly, are immobile, and place emphasis on face-to-face personal relationships. these can however be placed on an urban and nonurban (folk) con- tinuum. “the folk-urban continuum which results from these polar concepts provides a scale along which real societies may be ranged, their position determined by the relative pro- portion of folk or urban characteristics which they display” (redfield cited in foster , p. ). negotiation (adapta- tion toward increasing urbanness) is crafted as one moves toward the urban end of the continuum. the old order amish the folk community under discussion here is the old order amish in iowa, united states. according to cong, the amish have gained a folk “museum type of quality and antique status” ( , p. ). therefore, the ooa community’s per- spective in this study is regarded as the folk community perspective. the old order amish, a diffused group of ana- baptists, are descendents of the swiss radical reformation. they are a branch of the swiss group that originated from suffering and martyrdom in europe by standing firm for their traditions. their extensive migration to north america from europe was encouraged by the relative freedom of move- ment and the opportunities for economic security and ethnoreligious association (reschly ). these led to westward migrations and iowa became one of the preferred locations because of good and cheap land and a chance to create new settlements. even though thirty counties of iowa show records of amish and mennonite inhabitants, bigger settlements are found only in four counties: buchanan, johnson, washington, and wright. the amish–mennonite divisions occurred in the late th century, with mennonites leaning more toward worldliness and mingling with the mainstream culture and the old order amish (often referred to as “the plain people” and the first order amish) clinging to their ancestral traditions. the old order amish (ooa) are usually distinguished from the amish mennonites and the beachy amish by their strict conformity to the use of horses on the farm and as a means of transportation, their refusal to permit electricity or telephones inside their residence, and their more traditional code of dress that includes using hooks-and-eyes fasteners on some forms of clothing. according to reschly ( ), the ooa today are guided by approaches such as biological reproduction, cultural resistance, and a willingness to make compromises with the modern world while keeping the core values of gelassenheit intact. gelassenheit means submis- sion or yielding to the will of god and forms the cornerstone of ooa values. the amish community is religiously rooted in gelassenheit. the amish believe in redemptive living through ordnung and meidung. ordnung is the order and discipline of the community and by spelling out expected behavior, it shapes amish life and identity (kraybill ). meidung is the shunning of erring church members who deviate from the ordnung. it spells out punishment associ- ated with excommunication and social avoidance. the ooa are also referred to as a folk community because they are not in favor of social mobility and are slow to shift their economic and social positions. ooa folkness is thus described by its efforts to stay isolated as well as connected with the mainstream culture in an intellectual, creative, and artistic manner. the folk model lends itself to understanding the traditional character of the amish society (hostetler ). according to redfield, the underlying premise of the by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ journal of travel research ( ) folk society model is nonchange. redfield defines it as “a small, isolated, traditional, simple, homogeneous society in which oral communication and conventional ways are important factors in integrating the whole of life. in such an ideal-type society, shared practical knowledge is more important than science, custom is more valued than critical knowledge, and associations are personal and emotional rather than abstract and categorical” ( , pp. - ). because most tourism images are nucleated on the special character of the ooa reflected in their living styles, and reputation for integrity and quality of agricultural produce and domestic craft products (kraybill ), the impact of tourism and tourist gaze on this community in particular has been considered more profound. it is for this reason that the ooa are the subject of study and are examined using a mixed methods approach. mixed methods research requires both collection and analysis of qualitative and quantitative data (creswell and clark ). it makes use of close-ended as well as open-ended information. by mixing these types of data sets, it is possible to provide an enhanced understanding of the research problem than if either data set were used in isolation. for the purpose of this study, sequential data col- lection was used. the ooa were surveyed in the spring and summer of . information was elicited on amish percep- tions of tourist gaze and its impact on their community. in doing so, the extent and direction of negotiation along the open resistance–full adaptation scale is determined. in sum- mary, this study aims to answer the following research questions: what, according to the ooa, are the reasons of tourists’ gazing at them? what are the benefits and costs of tourist gazing on the ooa community? are certain tourist types preferred by the ooa? where are ooa positioned on the resistance continuum? based on the answers to the afore- mentioned questions, implications for the policy makers and brokers of amish tourism are presented. literature review evidently, many studies have referred to gaze as a singular tourist gazing notion. however, recent literature has also questioned the mundaneness or muteness of gazees. it argues that tourist gaze is reciprocal and not a one-way street because it is countered or returned by the host community (moaz ). gazing is thus not a one-way phenomenon and is a result of both visual spectacle and mental discernment. tourists gaze because they consider hosts inferior and want to exert their superiority or they are impressed with their “authenticness” and culture or are just being inquisitive (bruner ; notzke ). the hosts, on the other hand, gaze out of curiosity, to know the “other” to feel secure in dealing with them (moaz ), or when they consider them to be intruders and modern fools (dogan ). they might respond by resistance, retreating, segregating, and establish- ing demarcations, thereby blending their best to benefit themselves (moaz ). degree of cooperation or resis- tance is often suggested to stem from “social exchange,” which postulates that benefit levels generate positive atti- tudes and perceptions. this section describes local gaze and positions their response on the resistance continuum as pre- sented by previous studies. to begin with, moaz ( ), in his examination of the local gaze on israeli backpackers in the indian himalayas, argued against the asymmetrical nature of gazing in develop- ing countries. the author argued that gazing is mutual although its objective might differ among the guests and hosts. interestingly, he stated that tourist gazing is triggered by the local gaze. moaz’s study identified a range of responses by local indians from cooperation to open resistance to veiled resistance. he pointed out that tourist gaze generates fear as well as a sense of cooperation to ensure tourist demands are met. open resistance happens when “both gazes result in a tendency to didactism and in this case to educate the israelis and teach them to behave” (moaz , p. ). for instance, rules regarding party time and entertainment (not beyond : p.m.) were enforced by local restaurants and guest- houses. examples of veiled resistance included staging and selling of desired images. in this case, a passive role was adopted and hostility revealed in a subtle manner. conclud- ing arguments of the author were that such forms of resistance are often based on stereotyped images and prevent a genuine contact between guests and hosts. local gaze has also been equated with the term reverse gaze, analogous to a second gaze or a questioning gaze that redirects the tourist gaze to the self. the “reverse gaze” term was coined by gillespie ( ) within the context of interac- tion between tourist photo grapher and local photographee. gillespie’s study was conducted in ladakh, india, a popular backpacker destination for tourists. he illustrated how the “reverse gaze” of the ladakhis caused uncomfortable emo- tions and embarrassment among tourists. in line with this argument, based on ladakhi feedback, he concluded that the embarrassment was based on tourists’ self-perceptions, which failed to reflect the actual perspectives of the lada- khis. unpacking the ladakhi view, the author reports that they were generally supportive of tourists in touristic spaces such as the festivals, expected to be photographed, and saw tourists important for the overall economic development of their region. another reason for the ladakhi enthusiasm was related to the assumed significance of ladakhi culture and the impression that their culture was revered across the globe. within the context of resistance theory, gillespie described ladakhi stance as one of boundary maintenance. they acknowledged economic dependence on tourism, and their negotiated response was boundary maintenance. another study conducted by zhihong ( ) discovered local people in an eagerly “contriving act to play different cards in order to attract tourists,” including renovation and inventing traditions. his study focused on bai, china, and by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ chhabra examined how the local culture became touristified and the social landscape altered while responding to tourism devel- opment. he talked about how a local history was made and located. for instance, the culture exhibition center was a showcase that attempted to fix and define, locate, and regu- late fluid and malleable populations and local identities (tap , p. ). the author revealed that the local history, tradi- tions, the place, and the bai people had willingly embraced modifications to create “bainess” that could be sold to the tourists. the aim was to achieve their material ends so they readily exoticized and differentiated themselves. in sum, the author presented a mixed impact. according to the author, the tourist market provided a means for celebrating plural ethnicity with different expectations and interpretations from the guests’ and hosts’ perspectives. hosts were given both the opportunity and the freedom to express who they were and what they chose to represent. this is not to say that they were what they staged and sold but to point out that in this case, tourism served to nurture a sense of being ethnically distinctive and ensured the locals a bigger share of the tourist market. with regard to the resistance theory, the results indi- cated a leaning toward adaptation and full cooperation. evans-pritchard ( ) examined the southwest indian images of the tourist that orchestrate their actual interactions with the tourists when selling arts and crafts. the author speculated that the images held by indians guide their com- munication with tourists while at the same time help with boundary maintenance to ensure privacy and protection. holden ( ) found that most of the references to white visitors were not straightforward. instead they were drawn through “analogies, indirect allusions or traditional narrative figures.” the tourists “dehumanize the indian, patronizingly turn him into something ‘cute’ and see him as a fit object for consumption” (evans-pritchard ). in sum, indians had developed “a mental repertoire of stereotypes” that in turn informed them of multiple ways tourists could be treated when selling handicrafts. this case study projects the coex- istence of boundary maintenance and veiled resistance premised on the author’s argument that despite all negative images of tourists, indians benefit from them. the touristic space does not reduce the level of discomfort between the tourists and the indians but facilitates toleration for eco- nomic benefits. another study by joseph and kavoori ( ) examined the host community’s response to tourism in another asian pilgrimage town (pushkar). although tourism was regarded essential for the economic development of the pushkar town, the local people perceived it as a threat to tradition and reli- gion. the author used the term mediated resistance to explain the local community’s mixed reaction toward tourism. in line with moaz’s argument, the author questioned the asym- metrical relationship between guests and hosts in terms of gazing power. the message was that the locals should not be considered passive recipients. instead, they use many techniques such as reverse gaze (which triggers discomfort) and staged authenticity in contrived settings to protect their back regions. joseph and kavoori ( ) discussed three strategies of mediated resistance that guided the local response. accord- ing to him, three variations, exclusionary, religious, and political, constituted the most significant strategies of medi- ated resistance. exclusionary rhetoric divided the community into insider and outsider (including domestic indian tourists on the periphery) domains, and most problems stemming from tourism were blamed on the domestic outsiders. the religious rhetoric, however, blamed tourists for desacraliza- tion of the town. the political rhetoric on the other hand blamed the government for the negative impacts of tourism. public participation in these different rhetorics acted to serve as channels to vent anger and frustration, thus circumventing the need for further or formal action against the tourism industry. gazing at the amish community in the united states and its repercussions has received a preliminary impetus in the form of a small handful of studies. buck ( ) examined the probable impact of tourist enterprise powered by the tourism organizations in lancaster county, pennsylvania, on the old amish community integration. his study was based on the participant observation technique. according to him, in line with the fact that “the amish are reproduced and cari- catured in tourist promotion materials, area maps, and billboards,” there appears to be a high likelihood of “the amish life being ultimately engulfed and emulsified by the socioeconomic impact of tourist experience” (buck , p. ). however, in the face of the pervasive presence of tourists, the author is intrigued by the apparent success of this community in maintaining and continuing with their unique way of life. in regard to ooa perception of impacts, the main substance of the response was that tourists were considered a “needless nuisance,” who disturbed an other- wise quiet environment and made horse and buggy travel more difficult (buck , p. ). the author found the boundary maintenance approach the most appropriate response. overall, buck’s conclusion was that the ooa did not welcome tourism and viewed tourists as “empty pleasure seeking” people ( , p. ). that said, the author argued that this stance actually strengthened the amish self-identity and contributed to its “culture vigor and personal strength” ( , p. ). this resistance was also an added advantage for the tourism entrepreneurs who used it to discourage the free movement of tourists. in other words, the tourist enterprise sought to confine tourists within selected contact and view zones. this served a dual purpose. first, it prevented direct contact with the amish and made it less intrusive. second, it made the tourists dependent on the tourist enterprise. the entire scene and tour was orchestrated on imagery that “the ooa are out there and all around” even though none of them might actually be seen. by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ journal of travel research ( ) the boundary resistance approach was also noted by loomis and beegle ( ). the authors had found com- pelling arguments to justify this conjecture in the form of self-identity reinforcement of the ooa in lancaster county, pennsylvania. boundary maintenance was described as a form of resistance that “signifies activity to retain identity, value orientation, and interaction pattern of a social system. the process of boundary maintenance requires that the system actively resists forces which tend to destroy the iden- tity and interaction” (loomis and beegle , p. ). the authors also concluded that the boundary maintenance helped to sustain ooa integrity (in a shielded aggressive way) and core values despite being situated in a commercial environment. parallel inferences can be drawn from fagence’s ( ) work focusing on the ooa at st. jacobs in canada. she sug- gested boundary maintenance as a stance taken by the amish in face of intrusion. the author compared st. jacobs with lancaster county in the united states and considered the former as an extreme case of commercialization of amish themes. according to her, the tourism planners at lancaster county have crossed all limits to cater for and retain the interest of visitor groups. although not based on empirical findings, fagence ( ) restated buck’s conjecture and con- sidered “boundary maintenance” a useful shield for ooa. fagence maintained that tourism planning helped facilitate boundary maintenance by checking visitor intrusion. in sum, the aforementioned studies suggest mixed responses. while boundary maintenance is a recurrent response, other responses lie between subtle resistance and adaptation. none of the aforementioned studies reported open resistance. this study endeavors to locate amish com- munity local gaze and the resulting resistance to tourist gazing in a region (iowa state) with characteristics different from those found in canada and lancaster county, pennsyl- vania, in terms of life-cycle stage and visitor numbers. method the aim of this study was to bring to the fore local voices, particularly of those who have been silent receivers of tour- ism (buck ; cong ). attention was on a living culture, a unique set of amish residents: the ooa. this exclusive selection from the amish community was impor- tant because amish tourism heavily draws on the ooa. as reiterated by buck, “in choosing a peculiar way of life, ooa stand out in bold relief against modern society and are tour- istically defined as something worth seeing, a scarce human species” ( , p. ). this paper is not intended to repre- sent all ooa across united states; instead it is intended as an exploratory study that offers an empiricist investigation on a small scale using a mixed methods approach (see figure ). it is hoped that this study will offer a theoretically grounded framework for large-scale studies. the empirical discussion rests on three sets of data gath- ered in the spring and summer of (see figure ). a mixed approach of naturalistic (qualitative) and positivist (quantitative) inquiry was used. the data were collected in three phases to address the research questions. these phases helped to answer the first three research questions inquiring about the ooa perspectives of tourist gaze, its impact in terms of benefits and costs, and preference to certain kinds of tourists. the last research question was discursive and answered by way of inferences drawn from the findings. for the first data set, a total of preliminary interviews were carried out using a purposive sampling technique based on spatial dispersal of ooa families. this form of sampling follows the maximal variation sampling design. the maxi- mal variation design alludes to selection of individuals who can be differentiated according to gender, place of residence, level of schooling etc. (creswell and clark ). the cen- tral argument is that if respondents are “purposefully chosen to be different in the first place, then their views will reflect this difference and provide a good qualitative study (cre- swell and clark , p. ). in this study, county of residence was used as a differentiating point. an adult member of each ooa family was interviewed by two university students the bakery shops (frequented by the ooa) in johnson and buchanan counties. the selection was based on convenience and accessibility. prior to contact, permission was obtained from bakery shop owners. the respondent breakdown was as follows: five ooa families from johnson county and seven from buchanan county (care was taken to ensure an even representation of gender). these initial informant interviews consisted of a series of open-ended standard questions to obtain an understanding of ooa definition of authenticity, examine impressions from local gaze, and determine perceptions of tourist gaze and its impacts. questions included what is your definition of authenticity? why do you think the visitors/tourists seek connections with you or gaze at you? do you benefit from the visitors/tourists who seek to interact with you? while seeking answers, the interviewer was also given autonomy to seek interesting ideas or pursue issues that arose during the intercepts. the students received training prior to the imple- mentation of the first data collection phase. reflexivity (a critical approach for self-assessment) was used by the interviewers. the end result helped reduce personal bias and facilitated objectivity. the answers to open-ended questions were in qualitative format. they were analyzed using the data reduction method. this method helped reduce complex, iterative sets of words (miles and huberman ) and followed four steps: data reduction, data display, and conclusion drawing and verifica- tion. the first step (data reduction) referred to the process of selecting, focusing, simplifying, abstracting, and transforming the data that appeared in field notes or transcriptions derived from recordings. data displayed provided a fresh way of by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ chhabra arrangement and reflection of textual answers. conclusions and verification required retrospection and reflection of the derived meanings and terms from the data. different themes for the open-ended questions emerged from this process. therefore, statements were formulated to represent them. the students accomplished the data reduction task, which resulted in several items for each research question. because multiple opinions resulted from preliminary interviews, they were listed as items to be ranked on a likert-type scale, with = strongly agree and = strongly disagree. this was deemed necessary to determine level of significance attributed by the ooa to each item. the second phase focused on the main questionnaire, which included close-ended questions. close-ended ques- tions included a list of items pertaining to reasons for tourist gazing and its impact in terms of benefits and costs. a sec- tion eliciting information on user profile was added. the ooa were surveyed in public places (such as bakery shops, roadside stands, and quilting shops) using a stratified sam- pling technique. attempt was made to obtain an equal number of responses from each county. although no pres- sure or incentives were placed on the respondents to facilitate participation, refusal rate was % from buchanan county and % from johnson county. during the survey, the objec- tives and the voluntary nature of the study were explained. the ooa were requested to write their answers on the ques- tionnaire. in some cases, the interviewers waited (a total of minutes were taken on an average to answer questions) and in other cases they returned to pick it up. overall, responses were gathered. the third phase of the study focused on posttesting. it consisted of follow-up questionnaires using the member- checking approach to test for credibility and interviewer bias. this process verifies that the meanings gleaned from a questionnaire accurately reflect the perceptions and views of survey participants. alternatively, it also helps to identify doubts and offers the respondents another chance for self- reflection. a sample of six ooa families (three from each county) who had responded to the main survey were selected and requested to answer the questionnaire again. their answers were used to match or test the final results. nonre- sponse bias was also tested by intercepting six additional families (not surveyed earlier) during the posttest stage. the end data resulted in a total of surveys. qualitatively equivalent measures of reliability, validity, and generalizability were applied to determine trustworthi- ness of data. for instance, member checking approach tested for reliability. content validity was confirmed by compari- son with previous studies and a group of experts on the subject, such as two academicians, a visitor center staff, and a member of the historic society located in one of the study counties. potential threat to sequential data was addressed by including same individuals for the second and third phases (creswell and clark ). figure . mixed methods approach (n = ) qualitative data collection & analysis posttest quantitative data collection & analysis quantitative data collection & analysis procedures ) one-on-one semistructured interviews (n = ) ) content analysis: coding & thematic development procedures ) questionnaire with likert scale items and demographic items (n = ) ) univariate analysis: frequencies & averages procedures ) questionnaire with likert scale items and demographic items (n = ) ) testing phase: univariate analysis answers matched product ) field notes ) open-ended respondent answers on paper ) transcripts product ) closed-ended answers: numerical item scores product ) closed-ended answers: numerical item scores phase iiphase i phase iii by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ journal of travel research ( ) findings the first step in analyzing the amish perceptions was to determine the reasons that caused the tourists to gaze at them. table describes amish perceptions of the reasons behind tourist gaze on a likert-type scale. it is evident that the amish perceived that it was their authenticness associ- ated with arts, crafts, woodwork, and food that transpires and inspires the gaze. according to the preliminary interviews, all ooa perceived of authenticity as centered in the primor- dial school of thought and of essentialist character, heavily drawn from past norms. in addition, equal significance was given to curiosity, fol- lowed by desire to learn about other cultures. this view is supported by the general tourist quest to seek the other, where maccannell ( ) argues that the central drive to postmodern tourism is to discover places that seem to exist outside of history. this view is confirmed by hawley ( ) and kreps et al. ( ). another important reason for tourist gaze was perceived to be entertainment. previous research- ers have noted that spectacle often forms the core of constructed or inspired tourist gaze. it is generally transpired by promotional images designed by interest groups (light ). in this case, these are more likely to be tourism orga- nizations such as the convention and visitor bureaus, chamber of commerce, and historic societies. the next significant factor was contemplation or nostal- gia for a simpler life. lowest significance was given to opportunities to be part of their lives and social interactions. it appears that the amish do not consider intimacy and socialization to be the core objective of gazers. it is likely that the gazers have a predetermined image of the amish that they want to seek or confirm without stepping out of their comfort zones (cong ). fleeting experiences and views are sought based on images that are controlled by those inter- ested solely in economic benefits or with a view to distance the ooa. this explanation is supported by buck ( ). it is possible that many tourists who gaze surrender themselves to the parameters of the gaze without questioning or drawing the object of gaze closer to their physical or personal space. similar views were presented by fagence ( ) and buck ( , ). buck noted that the tourists “deliver them- selves into the hands of tourist enterprise induced and reinforced boundaries” facilitated by staged authenticity and pseudo-participation ( , p. ). with regard to benefits, of all the ooa interviewed, % felt that they benefited from tourist gazing. table ranks items that represent positive impacts. with regard to the per- ceived benefits associated with the gazing phenomena, most among the majority felt that their culture and visibility pointed out the negative consequences of the materialistic culture. in other words, their way of life can make the specta- tors question the fast pace of their own lives and thus served an educational purpose of creating awareness of simple life- styles (hawley ). next two significant factors associated with benefits of tourist gaze received parallel ratings (see table ). these were networking and enhancement of amish knowledge to the innovations of the outside world. this view aligns with hostetler’s ( ) contention that amish are vigilant of their surroundings and are often willing to negotiate behind scenes to progressively accommodate contemporary changes in a way that complements their traditions and community. eco- nomic numerations ranked third in order of agreement even though the majority ( %) felt tourist gaze led to higher interest in and increased sale of their merchandize. the amish literature somewhat supports this subtle response. it is with reluctance and careful deliberations that the ooa have embraced microenterprises and deviated from farming as a result of economic hardships. the least beneficial item was “social interactions.” the amish will rather prefer the tourists stay away (buck ). boynton ( ) noted that table . reasons for tourist gazing strongly agree/ disagree/ strongly item disagree (%) neutral (%) agree (%) averagea opportunities to be a part of your life . . . . to have social interactions . . . . to escape from day-to-day complexities of modernity . . . . to explore their roots . . . . nostalgia for a simpler life . . . . to contemplate . . . . entertainment . . . . learn about another culture . . . . curiosity . . . . authentic food . . . . authentic arts, crafts, and woodwork . . . . a. likert-type scale of to , with = strongly agree and = strongly disagree. by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ chhabra the amish prefer physical isolation. preference for zero social interactions is premised on the need to maintain their way of life despite the “seemingly pervasive presence of tourists” (buck , p. ). of those who disagreed with the statement that the tourist gaze results in benefits for the ooa community, the majority felt that tourist gazing was intrusive and facilitated poor understanding of their culture (see table ). many also objected to pictures being taken by tourists during such fleet- ing visits. it appears that enough information of the ooa is not made available to tourists to guide their understanding of amish culture or sensitivity toward it. of the brochures and descriptions of the amish in tour mag- azines presented by the convention and visitor bureaus, none provide information about how to be sensitive and less intrusive to the ooa. instead, the media creates mythological pseudo images of a static culture waiting to be discovered. this image is facilitated by the tour guides and promotional brochures. similar findings were reported by buck ( ). for instance, the tour guide’s tour through the backyards of the amish settle- ment and description of the amish culture reiterated this view. when asked if permission was taken from the amish, the answer was nonaffirmative. throughout the touring experience and the rehearsed narratives, the tour guide gave the impression that an eager ooa community was waiting to unveil itself. on the question if the amish preferred certain tourist types to other, the majority of them stated yes, with prefer- ence for respectful, polite, sensitive, less bold, and not rough-looking tourists. a couple of them stated that they “rather not have them as it is dangerous at the farm and they can hurt themselves.” those in business preferred serious customers who through their purchasing can generate eco- nomic benefits. finally, in response to the question “what do you feel when you see the tourists?” most responses (in their own words) were as follows: we feel like welcoming them; we are comfortable we are also curious; we try to feel if they are happy or not; i do not give them a lot of attention. they are just passers by; uncomfortable; there are occasionally intrusive types wish they were not as many and they minded their own business; hope they will buy our products; we look at their car; we feel their language and sensitivity; i are curi- ous and wonder where they are from? i am interested and concerned about them; wish they stayed away and left us alone; do not know. discussion in regard to the first research question about amish percep- tions of why tourists gaze at them, authentic products and curiosity appear to be the main reason. the authentic demand table . positive impact of tourist gaze strongly agree/ disagree/ strongly item disagree (%) neutral (%) agree (%) averagea social interactions . . . . insight into another culture . . . . alternative income . . . . strengthens outside network . . . . learn new things and increase knowledge . . . . exposure to the negative impacts of modernity . . . a. likert-type scale of to , with = strongly agree and = strongly disagree. table . negative impact of tourist gaze strongly agree/ disagree/ strongly item disagree (%) neutral (%) agree (%) averagea show disrespect . . . . make our community uncomfortable . . . . increase in local crime . . . . stare at us and our children . . . . negative effect on our children . . . . tour buses and cars create crowding and pollution . . . . take pictures . . . . intrusion into our private lives . . . . lack of understanding of our culture . . . . a. likert-type scale of to , with = strongly agree and = strongly disagree. by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ journal of travel research ( ) view finds support in the reasons described for gazing. the ooa thus believe that demand exists for essentialist and pri- mordial products. cohen’s ( ) version of objective authenticity (defining authenticity as an unaltered genuine- ness) and chhabra’s ( ) stance on essentialist authenticity (measured by standards based on unadulterated and real things) resonate with this perspective. arguably, this view is disputed by the contemporary constructivist reflections on authenticity that suggest abandonment of authenticity (reis- inger and steiner ). constructivists hold that authenticity is fluid and is shaped by ongoing social or personal values. thus, despite recent indications by literature of the post- modern disregard and lack of need for object authenticity, rural, handcrafted, and traditional form of authenticity still prevails in special heritage settings. in this study, this need results from the supplier’s prerogative. in argument with maccannell’s ( ) contention that an object ceases to be authentic if labeled so, the findings show that constructed revisions of authentic forms strongly adhere to the museo- logic notion of preservation. this view is also confirmed by joseph and kavoori ( ). buck ( ) points out that the ooa, as custodians in authority, have tightened their grip on heritage in the wake of kaleidoscopic constructivist ideolo- gies associated with the postmodern era. this grip has trickled down and influenced or guided selected assimilation of contemporary economic environments into the daily amish life, thus helping craft an essentialist-driven negoti- ated stance in the form of microenterprises. in these efforts, the authentic ingredients are likely to remain omnipresent and preserved. this view is reiterated by the ooa them- selves and is also supported by fagence ( ). curiosity can be attributed to the notion that “amish products carry a mystique that enhances their marketability. they are viewed as handcrafted, high quality, and unique. public perception is very positive” (kraybill and nolt , p. ), curiosity for the other world or lifestyle is a draw for tourists seeking a break from their daily mundane life. according to fagence ( ), lack of reliance on technology (which is considered indispensable by the modern society) portrays the independent and self-sufficient characteristics of the ooa and this has heightened admiration and inquisi- tiveness of the tourists. moreover, split opinion was noted with regard to the notion that tourists had a genuine desire to be a part of their day-to-day lives. some tourists might have a genuine desire to learn about other cultures. this view is supported by light ( ), who maintains that heritage tourism is an alternative form of tourism that draws a significant number of educated tourists aiming to learn about other cultures. however, paral- lel to this view, another view exists that “tourists are only a nuisance.” this view in its extreme is supported by buck ( ), who notes that the amish have a low opinion of tour- ists in lancaster county because they were out there for cheap entertainment and no inclination to be ontologically a part of the ooa environment. on a positive note, this indi- cates less threat to the amish backstage. alternatively, this can also imply lack of seriousness on the part of tourists, who have no intention to understand deeply the object of their gaze. their act, in their desire to drive through the amish backyards and staring at funeral processions, can be physically intrusive and reflect disrespect for privacy. this view is supported by yoder’s ( ) argument that the amish are often considered unwilling objects of a flourishing tour- ism industry aimed to satisfy the cravings of visitors seeking a glimpse of a simpler and ideal world. the second research question explored benefits of tourist gaze for the ooa. as the findings reveal, the positive bene- fits of tourist gaze were mostly philanthropic. the amish felt that exposure to their way of living transmits a social mes- sage to those entangled in the web of postmodernity, blindly racing toward capitalist-driven materialistic environments. the ooa hoped that visitors, who travel to distance them- selves from their norms, get to reflect on their existing lifestyles from a different perspective. in other words, the agrarian way of ooa life might suggest the need to slow down, build communitas, and promote a simpler life nucle- ated in rural settings. this view is supported by kraybill and nolt ( , p. xii), who hold that “venturing across the fence that separates the two cultures allows us to glance back and see our own society from a different angle.” notzke ( ), in her study of the indigenous communities in the arctic, also demonstrated eagerness on the part of remote communi- ties to educate urbanists of the need to balance life with nature. furthermore, the results also indicate that benefits associ- ated with economic remunerations were considered important. this is evidenced by the number of microenter- prises that have emerged in amish settlements as an alternative to agriculture. the occupational departure from farming to microenterprises suggests that the amish have become directly tied to the economic structure of the main- stream society. economic exigencies have tempered with the isolated ideology of the ooa. that said, this carefully chosen alternative form of income is still viable because it complements togetherness by keeping family home and together, enabling them to retain cultural values. it is proba- ble that this source has made the ooa less isolated and more accessible and visible to the surrounding regions. according to hostetler, “if boundaries are respected, both sides can ben- efit from each other” ( , p. ). such encounters help the amish obtain an insight into human problems in the out- side world, which frequently makes them feel content with their self-imposed isolated path. however, the extent of amish entanglement with the mainstream society for eco- nomic reasons, in their signs “come in, we are open” (kraybill , p. ) also brings with it the risk of a “potential rift in the family structure where the occupational opportunities for all family members had been previously by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ chhabra prescribed by convention and tradition” (fagence , p. ). with regard to negative impacts, intrusion, lack of respect, and picture taking ranked high in terms of agree- ment. the majority of the amish highlighted these issues. moreover, the ooa preference for respectful and less-intru- sive tourist types supports such concerns. similar issues were presented by cong ( ) and buck ( ). last, based on the findings, contrary to previous asser- tions, the amish appear to take a more subtle form of response on the resistance continuum. they can be posi- tioned somewhere between boundary maintenance and full cooperation. this new conjecture is termed as negotiated reciprocity (see figure ). it is highly likely that a more posi- tive response rests on the fact that amish tourism in the study area is still in its development stage. this is in contrast to lancaster county and st. jacobs, where tourism develop- ment has reached a maturity level. hence, in those places negative impacts might have become more visible. alterna- tively, credit can also be given to the environmental factors that have affected both the ooa and the mainstream culture. with the overwhelming awareness on sustainability and the unfolding of postmodern externalities (such as pollution, crowding, financial issues, health problems, etc.), desire to be part of the diminishing rural environment and culture might prove to be real. with regard to the ooa, the financial problems and increasing acculturation of so many ooa fam- ilies into the mainstream culture might have made the remaining ooa more tolerant and eager to craft negotiations with the mainstream culture. in summary, the results indicate that although the ooa share european heritage with their american neighbors, they are not in favor of the progressive underpinnings of the larger culture and are carefully embracing selected elements of it for economic survival. this view is evidenced in hostetler’s ( ) assertion that “the amish people are neither relics of a bygone era nor a people misplaced in time.” even though the amish look old fashioned at first glance, they are up-to- date and selective in their choices of modernity embrace. they are neither frozen or history or a fossilized culture from a bygone era (kraybill ). they are thus not static but moving slowly within the self-defined framework of negoti- ation. over the years, they have negotiated with the ideologies that threaten their existence. contrary to the prediction by sociologists of gradual assimilation and acculturation of the amish into the mainstream culture, this has not happened because the ooa culture is a highly integrated culture and is more resistant to change. they have retained their symbolic and social alienation from the world through their dialect, unique dress, horse and buggy transportation, and lanterns (kraybill ). however, they are constantly changing to survive and their negotiation is driven by gelassenheit principles. over the years, several fragmentations have taken place within the amish communities along the urban and folk spectrum. by rejecting certain types of modernity and accepting others, some amish appear to the outside as con- tradicting themselves. they, in fact, see this strategy as the logic of negotiated selectivity as is indicated by the study results. today, the ooa have created a thriving enterprise of small shops. products made of tools rendered outdated in the larger american society that can no longer be bought in the mass market are still functional and made in amish shops (kraybill & nolt ). even though many outsiders may perceive the old order amish as a people misplaced in time or an ethnic community that will eventually be assimilated into the mainstream of american life, selective negotiation to safeguard cultural capital serves as a protective wall. the primary limitation of this study was the sample size. all ooa families in the study area could not be surveyed because of time constraints. that said, this study is rare in its focus on the perceptions of ooa based 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( ). “residents and transients: community reac- tion to seasonal visitors.” journal of travel research, ( ): - . by hisham gabr on october , jtr.sagepub.comdownloaded from http://jtr.sagepub.com/ the common genetic influence over processing speed and white matter microstructure: evidence from the old order amish and human connectome projects peter kochunov ,*, paul m. thompson , anderson winkler , mary morrissey , mao fu , thomas r. coyle , xiaoming du , florian muellerklein , anya savransky , christopher gaudiot , hemalatha sampath , george eskandar , neda jahanshad , binish patel , laura rowland , thomas e nichols , jeffrey r. o'connell , alan r. shuldiner , braxton d. mitchell , , and l. elliot hong maryland psychiatric research center, department of psychiatry, university of maryland school of medicine, baltimore, md, usa. imaging genetics center, keck school of medicine of usc, marina del rey, ca, usa fmrib centre, oxford university, oxford, uk department of medicine, university of maryland school of medicine, baltimore, md, usa. department of psychology, university of texas at san antonio, san antonio, tx. department of statistics, university of warwick, warwick, uk geriatrics research and education clinical center, baltimore veterans administration medical center, baltimore, md , usa. abstract speed with which brain performs information processing influences overall cognition and is dependent on the white matter fibers. to understand genetic influences on processing speed and white matter fa, we assessed processing speed and diffusion imaging fractional anisotropy (fa) in related individuals from two populations. discovery analyses were performed in individuals from large old order amish (ooa) families and findings were replicated in twins and siblings of the human connectome project (hcp). the heritability of processing speed was h = % and % (both p < . ), while the heritability of whole brain fa was h = % and % (both p < . ), in the ooa and hcp, respectively. whole brain fa was significantly correlated with processing speed in the two cohorts. quantitative genetic analysis demonstrated a significant degree to which common genes influenced joint variation in fa and brain processing speed. these estimates suggested common sets of genes influencing variation in both phenotypes, consistent *please address correspondence to: dr. peter kochunov, maryland psychiatric research center, department of psychiatry, university of maryland, school of medicine, baltimore, md, usa, phone: ( ) - , fax : ( )- - , pkochunov@mprc.umaryland.edu. publisher's disclaimer: this is a pdf file of an unedited manuscript that has been accepted for publication. as a service to our customers we are providing this early version of the manuscript. the manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. hhs public access author manuscript neuroimage. author manuscript; available in pmc january . published in final edited form as: neuroimage. january ; : – . doi: . /j.neuroimage. . . . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t with the idea that common genetic variations contributing to white matter may also support their associated cognitive behavior. introduction information processing is a fundamental cognitive function that supports the higher cognitive and behavior attributes, including working memory, executing function and others (bartzokis et al., ; salthouse, ). speed with which brain processes information follows an inverse u-trend across the lifespan. it rises during maturation, reaches peak in the rd and th decades of life, after which point there is an age-related decline (bartzokis et al., ; salthouse, ). a life-span curve for the cerebral myelination mirrors this trend (bartzokis et al., ; flechsig, ; kochunov et al., ). myelination of the neuronal axons in the cerebral white matter is associated with a ten-fold higher signal transmission speed compared to unmyelinated axons and thirty-fold lower time needed for axonal repolarization to support signal transmission(felts et al., ; waxman and bennett, ). wm supports cognitive and motor functions by facilitating the exchange of information across spatially distributed neural networks. we hypothesize that genetic contributions to white matter fa should have shared genetic control over neurocognitive processing speed performance. toward that end, we use diffusion tensor imaging (dti) methods to provide an in vivo measure of changes in white matter microstructure indexed by fractional anisotropy (fa) of water diffusion. dti-fa describes the directional selectivity of the random diffusion of water molecules (basser, ; conturo et al., ; pierpaoli and basser, ; ulug et al., ). it is not a direct measurement of either myelination or white matter integrity(jones et al., ). instead, fa is sensitive to the anisotropy of the water diffusion created by the barrier of cellular membranes. for example, higher fa values (maximum theoretical value is . ) correspond to heavily myelinated wm tracts. the myelin layer of the axonal cell membranes hinders the diffusion of water molecules in all except the direction along the fiber tract, therefore producing highly anisotropic water diffusion estimates (pierpaoli and basser, ). conversely, fa values are closer to zero for tissue where the water molecule motion is random and isotropic, such csf. thus, the absolute wm fa values are sensitive to many parameters including regional myelination levels, the degree of intra-voxel fiber crossing, axonal density and average axonal diameter (beaulieu, ; jones et al., ). the neuroimaging research suggests a link between white matter fa and processing speed in healthy subjects. among research findings linking neuroimaging measurements to variance in cognition, the strongest association was observed between white matter fa values and other proxy-measurements of white matter microstructure and fiber organization and the neurocognitive processing speed (bartzokis, ; bartzokis et al., ; charlton et al., ; karbasforoushan et al., ; kennedy and raz, ; kochunov et al., a; kochunov et al., b; konrad et al., ; muetzel et al., ; schiavone et al., ; vernooij et al., ). the overall conclusion of these studies was that white matter microstructure as indexed by fa and other neuroimaging indices are associated with the speed of cerebral information processing (bartzokis et al., ). this link between white kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t matter fa and processing speed is consistent and replicable (glahn et al., ; penke et al., ; wright et al., ). moreover a consistent relationship between these measurements has also been observed in healthy subjects(bartzokis et al., ; kochunov et al., ; vernooij et al., ) and in patients with heritable psychiatric disorders(glahn et al., ; karbasforoushan et al., ; penke et al., ; wright et al., ), giving a strong rationale to study the relationship for potential sources of shared genetic contributions. a large proportion ( - %) of the intersubject variance in the dti-fa values(braskie et al., ; chiang et al., ; chiang et al., ; jahanshad et al., ; jahanshad et al., ; kochunov et al., ; shen et al., ) and processing speed measurements(glahn et al., ; glahn et al., ) is explained by additive genetic factors. the high degree of genetic influence of these two traits posits them as important endophenotypes for genetic search for risk factors for heritable psychiatric disorders such as schizophrenia that are associated with both dti-fa and processing speed deficits. this study aims for the first time to determine if shared genetic factors might partly explain the relationship between processing speed and white matter fa (wright et al., ). we set out to (i) establish the degree of additive genetic contribution to processing speed and white matter fa phenotypes using a large pedigree cohort, (ii) test if effects replicate in an independent cohort, and (iii) to determine any regional tract-specificity of the shared genetic associations. evidence that shared genetic factors influence white matter fa and processing speed may inform a more direct analytic approach for discovering influential genes in disorders that affect both phenotypes. toward these goals, we decomposed the phenotypic association between processing speed and fa into its genetic and environmental constituents using a family design with large pedigrees. the family study design offers a structure to test whether phenotypic variances are transmitted by inheritance of additive genetic variance. the old order amish (ooa) are known for their very large family sizes, which include a large number of relatives and thus afford substantial power for genetic correlation analyses, even with a modest sample size. the ooa subjects are european caucasian ancestry. they share similar rural upbringing and lifestyle that includes the same level of basic school education and virtually no illicit substance use. this relative environmental homogeneity in the ooa offers a rarely available setting to test shared heritability across different classes of phenotypes because environmental factors in the general population (for example substance abuse or differences in education levels) might impact each phenotype differently. therefore, the ooa provides an interesting population-level control on heterogeneity and unmeasured sources of individual variation, making it easier to analyze the degree of shared heritability of traits. next we attempted to replicate the findings of shared genetic variance between dti-fa and processing speed using an independent, genetically informative cohort - the publicly available twin-and-sibling based human connectome project (hcp) dataset. in this dataset, both dti-fa and processing speed (though measured differently than ooa) are available. our hypothesis was that the two traits, white matter microstructure and processing speed, would show a consistent pattern of bivariate genetic association in both cohorts, despite the differences in population and methodological variability in data collection. kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t methods ooa sample subjects—brain imaging was conducted in ooa amish individuals ( m/ f, age= . ± . ; - years), in whom processing speed was measured in ( m/ f, age= . ± . ; - years). all individuals were from seventeen nuclear families from lancaster county, pa., who could be combined into a single large pedigree that connected them across eight generations based on genealogical records maintained by the ooa community and incorporated into the nih anabaptist genealogy database (agdb) which traces back to the founders (agarwala et al., ). exclusion criteria included major medical and neurological conditions that might affect gross brain structures such as developmental disability, head trauma, seizure, stroke or transient ischemic attack. subjects with type ii diabetes and hypertension that were controlled with medications were included. psychiatric conditions that were managed with medications were not excluded: the full sample (n= ) included individuals with a lifetime diagnosis of psychiatric disorders, including mood disorders (n= ), anxiety disorders (n= ), psychosis (n= ), and other psychiatric disorders (n= ) based on the structured clinical interview for dsm-iv disorders (scid). among the subjects for whom processing speed measurements were available (n= ), there were individuals with psychiatric disorders. overall, this represented a . % rate of psychiatric illness in the n= sample of the ooa isolate, comparable to the . % prevalence rate of psychiatric illnesses in the general u.s. population (kessler et al., ). all subjects with psychiatric disorders were taking psychotropic medications for their conditions. this included six subjects who were taking antipsychotics, thirteen subjects taking anti-depressants, eleven subjects taking mood- stabilizers and four subjects taking other psychotropic medications, with some subjects taking two medications. heritability and genetic correlation analyses were performed by adding psychotropic medication status as a binary covariate. all subjects provided written informed consent on forms approved by the institutional review board of university of maryland baltimore. processing speed measurements—processing speed was assessed with the digit symbol coding subtest of the wais- , which is considered as a test for speed of information processing and psychomotor response (wechsler, ). the task reports the number of correctly coded symbols within the two-minute interval. besides processing speed, the task requires elements of attention, visuoperceptual processing, and working memory. raw neuropsychological assessment scores were used, and corrections for age and sex were performed as part of the statistical modeling by including age, age , age × sex and age × sex as covariates. imaging protocol—the full protocol is detailed elsewhere(kochunov et al., ). briefly, all data was collected at the maryland psychiatric research center, using a t siemens trio scanner equipped with a -channel phase array coil. the protocol consisted of a single-shot, echo-planar, single refocusing spin-echo, t -weighted sequence with a spatial resolution of . × . × . mm. the sequence parameters were: te/tr= / ms, fov= mm, axial slice orientation with slices and no gaps, isotropically distributed kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t diffusion weighted directions, two diffusion weighting values (b= and s/mm ) and five b= images, calculated with an optimization technique that maximizes the contrast to noise ratio for fa measurements(jones et al., ). the generalized autocalibrating partially parallel acquisition (grappa) acceleration procedure, with an acceleration factor of and a -phase encoding reference line, was used in phase encoding direction. no acceleration along the z-encoding direction was used. the total scan time was about minutes. no cardiac or respiratory gating was used. the hardi data were pre-processed with enigma- dti pipeline. briefly, fmrib software library (fsl) package, was used to perform the eddy-current and motion correction and calculate the fractional anisotropy (fa) images by fitting the diffusion tensor to the raw diffusion data, using default parameters (smith sm, ). the quality of the data was inspected using qa/qc tools implemented in enigma- dti pipeline with all data passing the recommended checks. hcp sample subjects—data were from ( / m/f) participants from the human connectome project (hcp) released in june (humanconnectome.org) after passing the hcp quality control and assurance standards (marcus et al., ). participants were recruited from the missouri family and twin registry (van essen et al., ). all hcp participants were from young adult sibships of average size – that include an mz or dz twin pair and the non-twin siblings. subjects ranged in age from to years ( . ± . years). this age range was chosen as it corresponds to a period after neurodevelopment is largely completed and before the typical age of onset of neurodegenerative changes. this release included twin pairs ( monozygotic and dizygotic pairs), and of their siblings. the full set of inclusion and exclusion criteria is detailed elsewhere (van essen et al., ). in short, the hcp subjects are healthy young adults within a restricted age range and free from major psychiatric or neurological illnesses (edens et al., ; sartor et al., ). all subjects provided written informed consent on forms approved by the institutional review board of washington university in st louis. processing speed measurements—processing speed was assessed using the nih toolbox pattern comparison processing speed (pcps) test (http://www.nihtoolbox.org) (carlozzi et al., ). this test asks participants to discern whether two side-by-side pictures are the same or not, and measures the number of items correct in a -second period. the pcps is appropriate for use across the lifespan (ages, – years) and has high construct validity (carlozzi et al., ). diffusion data collection and preprocessing—diffusion imaging data was collected at washington university, st. louis, using a customized siemens magnetom connectome tesla scanner with a mt/m maximum gradient strength and a channel head coil. details on the scanner, image acquisition and reconstruction reported elsewhere (ugurbil et al., ) and are available online(https://www.humanconnectome.org/documentation/s / hcp_s _release_reference_manual.pdf). diffusion data were collected using a single- shot, single refocusing spin-echo, echo-planar imaging sequence with . mm isotropic spatial resolution (te/tr= . / ms, fov= × mm). three gradient tables of diffusion-weighted directions, and six b= images each, were collected with right-to-left and kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t http://humanconnectome.org http://www.nihtoolbox.org http://https://www.humanconnectome.org/documentation/s /hcp_s _release_reference_manual.pdf http://https://www.humanconnectome.org/documentation/s /hcp_s _release_reference_manual.pdf left-to-right phase encoding polarities for each of the three diffusion weightings (b= , , and s/mm ). the total imaging time for collection of diffusion data was approximately one hour. diffusion data were preprocessed using the hcp diffusion pipeline (glasser et al., ; sotiropoulos et al., ) that included: normalization of b image intensity across runs; correction for epi susceptibility and eddy-current-induced distortions, gradient-nonlinearities, subject motion and application of a brain mask. fa maps were obtained by fitting diffusion tensor model using fsl-fdt toolkit (behrens et al., ). extraction of whole-brain average and regional fa values enigma-dti protocols to extract whole-brain and tract-wise average fa values were used for both datasets. these protocols are detailed elsewhere (jahanshad et al., ) and are available online, at http://enigma.ini.usc.edu/protocols/dti-protocols/. briefly, fa images from hcp subjects were non-linearly registered to the enigma-dti target brain using fsl's fnirt (jahanshad et al., ). this target was created as a “minimal deformation target” based on images from the participating studies as previously described (jahanshad et al., ). the data were then processed using fsl's tract-based spatial statistics (tbss; http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/tbss) analytic method (smith et al., ) modified to project individual fa values on the hand-segmented enigma-dti skeleton mask. after extracting the skeletonized white matter and the projection of individual fa values, enigma tract-wise regions of interest, derived from the johns hopkins university (jhu) white matter parcellation atlas (mori et al., ), were transferred to extract the mean fa across the full skeleton and average fa values for eleven major white matter tracts (table ). the whole brain average fa values were calculated to include all voxels in the enigma-dti skeleton. the protocol, target brain, enigma-dti skeleton mask, source code and executables, are all publicly available (http://enigma.ini.usc.edu/ongoing/dti- working-group/). heritability and genetic correlation analyses we estimated the heritability (h ) of fa values and brain processing speed as the proportion of the total phenotypic variance that can be explained by the additive effects of genes. briefly, we modeled the observed phenotypic co-variance between each pair of individuals as having an expected value given by the product of their coefficient of relatedness (as defined by the pedigree structure) and the observed phenotypic variance of the trait (conditional on covariate effects). parameter estimates including the heritability are assessed by maximum likelihood. the significance of the additive genetic effect (h ) is assessed by the likelihood ratio test that compares the likelihood of a model that includes the family structure versus a model that does not. all analyses were performed using the solar- eclipse software package (http://www.nitrc.org/projects/se_linux). to estimate the extent to which a common set of genes influences variation in brain processing speed and fa values jointly, we used classical quantitative genetic models to partition the phenotypic correlation between two phenotypes into a genetic and environmental components. briefly, the phenotypic correlation can be partitioned into the genetic ρg, and environmental ρe components. for traits a and b this decomposition is kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t http://enigma.ini.usc.edu/protocols/dti-protocols/ http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/tbss http://enigma.ini.usc.edu/ongoing/dti-working-group/ http://enigma.ini.usc.edu/ongoing/dti-working-group/ http://www.nitrc.org/projects/se_linux ( ) where h a and h b denote the heritability for each trait. thus, the shared variance is expressed using shared additive genetic effects and the residual environmental effects. to estimate the additive ρg and the random ρe for pairs of traits, the multivariate phenotype of an individual is modeled as a linear function of kinship coefficients that express relatedness among all pairs of individuals in the pedigree. using standard quantitative genetic theory, the phenotypic variance-covariance matrix and its additive genetic and random environmental components are then obtained. from these matrices, the significance of the ρg and ρe are estimated directly by the likelihood ratio test (almasy et al., ; williams- blangero and blangero, ). this test compares the likelihood of a model in which additive and environmental components, in turn, are constrained to zero against the full model in which all parameters are estimated simultaneously. this provides the estimates for ρg and ρe and their standard error. the significance of these coefficients is determined by a z-test of difference from zero. if the ρg is significantly different from the zero then a significant proportion of the traits’ covariance is considered to be influenced by shared genetic factors (almasy and blangero, ; almasy et al., ). all analyses were conducted with age, age , age × sex and age × sex included as covariates. in ooa sample, psychotropic medication status was included as a binary covariate. inverse gaussian transformation was applied to ensure normality of the measures. the level of statistical significance for regional measurements was set at p= . to correct for multiple (n= ) comparisons. p-values in the interval of . <p≤ . were defined as nominally significant and included into the discussion. results heritability analyses whole-brain fa and processing speed were significantly heritable in both cohorts (h of whole-brain fa = % and % and h of processing speed = % and % in ooa and hcp, respectively) (table ). heritability analysis of regional measurements of fa values in ooa sample demonstrated significant heritability (after correction for n= comparisons) in five tracts: the genu, body, and splenium of the corpus callosum, cingulum and superior longitudinal fasciculus (all p< . ) (figure , table ). nominally significant ( . <p< . ) heritability values were also observed for all other tracts, except for the fornix (fx) (table ). in ooa sample age was the sole significant covariate (p< . ) for whole brain average (p= . ), all regional fa ( . <p< . ) values and processing speed (p= . ). medication status in oaa was included as a covariate and was shown to be non-significant in all heritability analyses (all p> . ). heritability values for regional fa values in hcp data were taken from our previous work (kochunov et al., ) and were significant in all tracts (table ). age was a non- kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t significant covariate for either regional fa values or processing speed in hcp sample. there was a significant and positive correlation (r= . , p= . ) between heritability estimates for regional fa measurements in the ooa and hcp cohorts (figure ). correlations between fa and processing speed significant phenotypic correlation (ρp) was observed between whole-brain average fa and processing speed (ρp= . , p= . ). significant phenotypic correlations were observed for the genu of cc and the sagittal stratum tracts (ρp= . , p= . for both) (table ). nominally significant ρp values were observed for the body and splenium of the corpus callosum, the corona radiata (cr) and superior longitudinal fasciculus (slf) (figure , table ). genetic correlation analyses in oaa sample demonstrated significant shared genetic variance between average fa and processing speed (ρg= . ± . , p= . ). for regional fa values, although none of the coefficients achieved statistical significance at p< . , all genetic correlation coefficients were positive and nominally significant for the genu, splenium and fornix (figure , table ). all environmental correlation coefficients were non-significant (all p> . ). in hcp sample, a positive and significant phenotypic correlation ρp was observed between average fa and processing speed (ρp= . p= . ). the ρp coefficient was numerically lower than that in ooa but there was no significant difference between them (z= . ,p> . ). regionally, the ρp was significant for the slf, cortico-spinal (cst) and ss tracts and nominally significant for the splenium of the corpus callosum and corona radiata (cr) (table ). the phenotypic correlation coefficients were smaller for hcp than for ooa, likely due to a smaller variance in the cognitive performance data (average processing speed ± std dev = . ± . vs. . ± . for ooa and hcp, respectively) and in fa (whole brain fa: = . ± . vs. . ± . ) (table s, see supplement) the ρp coefficients for regional fa measurements and processing speed in hcp were also not significantly different from these in ooa with exception for the genu of corpus callosum (p= . ). the correlation between regional ρp values for ooa and hcp cohorts was positive and significant (r= . , p= . ) (figure , top). likewise, the genetic correlation analyses showed a significant correlation between whole- brain average fa and processing speed (ρg= . ± . , p= . ). there were no significant differences in the genetic correlation coefficients between ooa and hcp samples (p= . ). significant ρg values were observed for the splenium, internal capsule (ic) and ss tracts (p≤ . ). nominal significance was observed for the cr, external capsule (ec), slf and cst tract ( . >p> . ). nominally significant differences between ooa and hcp cohorts were only observed for the splenium of corpus callosum (p= . ). all environmental correlation coefficients were non-significant (all p> . ). the correlation between regional ρg was positive but not significant (r= . , p= . ) (table , figure , bottom) kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t discussion we demonstrated a significant shared additive genetic contribution between intersubject variability in the processing speed and fractional anisotropy (fa) of cerebral wm. this finding was first observed in the old order amish subjects and then replicated in data collected and distributed by the human connectome project. this suggests that the common genetic effects significantly contribute to the phenotypic association between the two traits. this finding was observed despite the differences in the ooa and hcp family structure, sample size, age range, sociocultural background, and the imaging and processing speed assessment tools. both ooa and hcp are robust familial samples, although they also differ in several important aspects. the ooa cohort consisted of subjects ascertained from a large multigenerational pedigree. farm-dwelling ooa subjects have higher environmental homogeneity compared to the urban/suburban hcp sample. ooa are perhaps best known for their old-fashioned dress and resistance to technological change. ooa share rural upbringing and similar diet, income and work environment. all ooa receive uniform th grade level education. illicit drug use was not present in our sample. in comparison, the hcp sample consisted of twin-pairs and siblings with diverse ethnic and economic backgrounds. other distinct features include the age range. the ooa subjects were recruited across the lifespan (age rage= - ) and heritability analyses identified age as a significant covariate for both processing speed and fa values. the hcp recruitment was focused on a narrow age range from to years that corresponds to the plateau in the fa and processing speed aging trends (kochunov et al., ; van essen et al., ). the lack of aging-related trends in fa values were already reported in hcp (kochunov et al., ). likewise, we found no impact of subjects’ age on the processing speed measurements in hcp sample (p> . ). thus, the ooa and hcp samples differ in environmental and likely population genetic contribution, making the similar findings in heritability and genetic correlation more convincing. despite the difference in imaging methods, the heritability estimates for the whole-brain average fa values were similar (h = . vs. . ) for ooa and hcp cohorts. likewise, the tract-wise average fa values in ooa and hcp subjects showed a good agreement (r= . ). heritability is the proportion of the variance that is attributed to the additive genetic variance after correction for covariates. in ooa sample age was the only significant covariate. the hcp sample show no age effect likely because the recruitment strategy was designed to reduce the effects of age on the brain measurements by limiting age-range to that corresponds to a plateau in fa-aging trend ( - years) (kochunov et al., ; van essen et al., ). another source of variability is the difference in methods used to ascertain processing speed in ooa and hcp samples. the digit-symbol coding test used in the ooa sample is a standard neurocognitive assessment tool to assess processing speed, commonly used in psychiatric research(knowles et al., ). the processing speed in the hcp sample was measured using the pattern comparison processing speed test. both tests are accepted measurements of processing speed and share between - % of the intersubject kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t variance(carlozzi et al., ; carlozzi et al., ). despite differences in processing speed tasks in the ooa and hcp cohorts, we found only sporadic differences in phenotypic and genetic correlations across samples; these differences however, were not statistically significant after correction for multiple compassions. the fact that phenotypic and genetic correlation coefficients were positively correlated between the two samples supports the notion that shared variance between fa values and processing speed are not specific to a behavioral task or a particular population. we believe the difference in processing speed measurement tests rather than the imaging protocol may to a greater extent explain why the correlation coefficients in ooa subjects were numerically higher than these in hcp cohort. while, this difference was not significant for the whole-brain average fa values, it was nominally significant for the genu and splenium of corpus callosum for the phenotypic and genetic correlation, respectively. in ooa, the phenotypic correlation between fa values of the genu and processing speed were statistically significant (r= . , p= . ). in comparison, in hcp sample this correlation was only suggestively significant (r= . , p= . ). in the same time, there was no significant difference for the heritability estimate for the fa for this structure (h = . vs. . , for ooa and hcp, respectively, p= . ). the genu contains the primary fibers connecting the left and right frontal lobes and the genetic contribution to processing speed may in part be through genetic influence on the speed of frontal lobe communication (aboitiz, ; aboitiz et al., ; bartzokis et al., ). normal aging and lesion studies suggest that the decision making process in the digit symbol coding task is sensitive to the structural integrity of the frontal white matter, regardless of variance in reaction time(kochunov et al., ; leavitt et al., ; raji et al., ). instead, the pattern of correlation in the hcp had a more posterior/inferior bias with the highest correlations in the corticospinal (cst) and the sagittal stratum (ss) areas with the pcps processing speed task. the pcps test has a heavy visual loading, which may explain the heavy loading on wm structures that serve visual-spatial subsystems including the splenium of corpus callosum (scc) and ss. notably, the phenotypic correlation for these structures were also nominally significant in ooa sample. genetic correlation coefficients were nominally different for the splenium of corpus callosum, with higher ρg values observed in ooa sample (ρg = . vs . in ooa and hcp respectively). nonetheless, both coefficients were significantly different from zero in ooa and hcp cohorts (p= . and . in ooa and hcp, respectively). therefore, the differences in the loading of the digit symbol vs. pcps processing speed tests on specific cognitive domains is the likely explanation of the numerical differences in the phenotypic and genetic correlation coefficients between ooa and hcp cohorts. a potential limitation of this study is the use of psychiatric drugs by n= ooa subjects, with a lifetime diagnosis of mental illness that may have potentially affected brain structure. the use of psychiatric drug was coded as a binary covariate during all analyses. however, as this covariate was robustly non-significant, we believe it to be a minor limitation. another limitation of this study is that we did not collect peripheral pulse oximetry data during the dti acquisition. advanced dti reconstruction techniques allow for use of peripheral data such as pulse and respiration to improve signal-to-noise ratio in tensor fit, especially in the areas of brainstem and cerebellum (mohammadi et al., ). in the absence of cardiac kochunov et al. page neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t gating during the acquisition, it is also possible to vet or edit the input data with publicly available software that allows removal of outliers or volumes corrupted by excessive movement from the tensor calculation, although we did not use such an approach in this study. overall, our findings imply that specific genes influencing variance in fa values may also exert influence over the speed of cognitive information processing. this finding is of importance for neuropsychiatric disorders such as schizophrenia where the reduced speed of information processing and reduced cerebral fa values are highly replicable findings and believed to be interlinked (alba-ferrara and de erausquin, ; ellison-wright and bullmore, ; friedman et al., ; glahn et al., ; kubicki et al., ; nazeri et al., ; penke et al., ; perez-iglesias et al., ; phillips et al., ). together, our results support common sets of genes influencing variation in processing speed and white matter fa phenotypes, consistent with the idea of pleiotropic effects in brain structure and cognitive behavior. our findings may pave a way for multivariate genetic localization analyses that combine processing speed and fa-values to directly identify genes that may have impact in multiple psychiatric disorders. supplementary material refer to web version on pubmed central for supplementary material. acknowledgments we are grateful of the amish families who have supported this research. this research was supported by nih grants u mh , r eb , r da and r mh . this work was supported in part by a consortium grant (u eb ) from the nih institutes contributing to the big data to knowledge (bd k) initiative. this work was also supported by nih grants p mh and t mh . reference aboitiz f. brain connections: interhemispheric fiber systems and anatomical brain asymmetries in humans. biol res. ; 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(p= . ) . ± . (p= × − ) whole-brain average fa . ± . (p= × ) . ± . (p= × − ) regional fa values genu of the corpus callosum . + . (p= . ) . ± . (p= × − ) * body of the corpus callosum . + . (p= . * ) . ± . (p= × − ) * splenium of corpus callosum . + . (p= . ) * . ± . (p= × − ) * fornix (fx) . + . (p= . ) . ± . (p= × − ) * cingulum (cingulate gyrus) - l and r combined (cing) . + . (p= . ) * . ± . (p= × − ) * corona radiata - l and r anterior, superior and posterior sections combined (cr) . + . (p= . ) . ± . (p= × − ) * external capsule - l and r combined (ec) . + . (p= . ) . ± . (p= × − ) * internal capsule - l and r anterior limb, posterior limb, and retrolenticular parts combined (ic) . + . (p= . ) . ± . (p= × − ) * superior longitudinal fasciculus - l and r combined (slf) . + . (p= . ) * . ± . (p= × − ) * sagittal stratum (include inferior longitudinal fasciculus and inferior fronto-occipital fasciculus) - l and r combined (ss) . + . (p= . ) . ± . (p= × − ) * corticospinal tract - l and r combined (cst) . + . (p= . ) . ± . (p= × − ) * analyses in both cohorts were corrected for age, age , age×sex and age ×sex. bolded values are significant at p≤ . . * values that are significant after correction for multiple (n= ) comparisons. neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t kochunov et al. page table phenotypic correlation coefficients (ρp ) between processing speed and fa value (corrected for age, age , age×sex and age ×sex). trait ooa hcp whole-brain average fa . (p= . ) . (p= . ) regional fa values genu of the corpus callosum . (p= . ) * . (p= . ) body of the corpus callosum . (p= . ) . (p= . ) splenium of corpus callosum . (p= . ) . (p= . ) fornix (fx) . (p= . ) . (p= . ) cingulum (cingulate gyrus) - l and r combined (cing) . (p= . ) . (p= . ) corona radiata - l and r anterior, superior and posterior sections combined (cr) . (p= . * ) . (p= . ) external capsule - l and r combined (ec) . (p= . ) . (p= . ) internal capsule - l and r anterior limb, posterior limb, and retrolenticular parts combined (ic) . (p= . ) . (p= . ) superior longitudinal fasciculus - l and r combined (slf) . (p= . ) . (p= . )* sagittal stratum (include inferior longitudinal fasciculus and inferior fronto-occipital fasciculus) - l and r combined (ss) . (p= . ) * . (p= . ) corticospinal tract - l and r combined (cst) . (p= . ) . (p= . ) * bolded values are significant at p≤ . . * values that are significant after correction for multiple (n= ) comparisons. neuroimage. author manuscript; available in pmc january . a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t a u th o r m a n u scrip t kochunov et al. page table genetic correlation coefficients (ρg ) between processing speed and fa value (corrected for age, age , age×sex and age ×sex). trait ooa hcp whole-brain average fa . ± . (p= . ) . ± . (p= . ) regional fa values genu of the corpus callosum . ± . (p= . ) . ± . (p= . ) body of the corpus callosum . ± . (p= . ) . ± . (p= . ) splenium of corpus callosum . ± . (p= . ) . ± . (p= . ) fornix (fx) . ± . (p= . ) . ± . (p= . ) cingulum (cingulate gyrus) - l and r combined (cing) . ± . (p= . ) . ± . (p= . ) corona radiata - l and r anterior, superior and posterior sections combined (cr) . ± . (p= . ) . ± . (p= . ) external capsule - l and r combined (ec) . ± . (p= . ) . ± . (p= . ) internal capsule - l and r anterior limb, posterior limb, and retrolenticular parts combined (ic) . ± . (p= . ) . ± . (p= . ) * superior longitudinal fasciculus - l and r combined (slf) . ± . (p= . ) . ± . (p= . ) sagittal stratum (include inferior longitudinal fasciculus and inferior fronto-occipital fasciculus) - l and r combined (ss) . ± . (p= . ) . ± . (p= . ) * corticospinal tract - l and r combined (cst) . ± . (p= . ) . ± . (p= . ) bolded values are significant at p≤ . . * values that are significant after correction for multiple (n= ) comparisons. neuroimage. author manuscript; available in pmc january . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ [pdf] fdg pet / ct pitfalls in gynecologic and genitourinary oncologic imaging | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. corpus id: fdg pet / ct pitfalls in gynecologic and genitourinary oncologic imaging @inproceedings{bharwani fdgp, title={fdg pet / ct pitfalls in gynecologic and genitourinary oncologic imaging }, author={n. bharwani and a. rockall}, year={ } } 	n. bharwani, a. rockall 	published the role of whole-body positron emission tomography (pet)/ computed tomography (ct) with fluorodeoxyglucose (fdg) is now established in the assessment of many gynecologic and genitourinary malignant tumors. fdg pet/ct has been widely adopted for staging assessments in patients with suspected advanced disease, in cases of suspected disease recurrence, and for determining prognosis in a number of malignancies. a number of pitfalls are commonly encountered when reviewing fdg pet/ct scans in… expand radiology.emory.edu save to library create alert cite launch research feed share this paper figures and tables from this paper 	 table 	 figure 	 table 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure view all figures & tables references showing - of references sort byrelevance most influenced papers recency imaging of pelvic malignancies with in-line fdg pet-ct: case examples and common pitfalls of fdg pet. 	naveen subhas, p. patel, h. pannu, h. jacene, e. fishman, r. wahl 	medicine 	radiographics : a review publication of the radiological society of north america, inc 	 	 	 	view excerpts, references background save alert research feed fdg-pet/ct for diagnosis of primary ovarian cancer 	k. kitajima, kayo suzuki, + authors k. sugimura 	medicine 	nuclear medicine communications 	 	 	view excerpt, references background save alert research feed detection of histologically proven peritoneal carcinomatosis with fused f-fdg-pet/mdct. 	a. dirisamer, w. schima, + authors w. langsteger 	medicine 	european journal of radiology 	 	 	view excerpts, references methods and background save alert research feed the role of fdg-pet/ct in gynaecological cancers 	a. rockall, s. cross, s. flanagan, elizabeth moore, n. avril 	medicine 	cancer imaging : the official publication of the international cancer imaging society 	 	 	pdf 	view excerpt, references background save alert research feed focal fdg uptake in mediastinal brown fat mimicking malignancy: a potential pitfall resolved on pet/ct. 	m. truong, j. erasmus, + authors h. macapinlac 	medicine 	ajr. american journal of roentgenology 	 	 	view excerpt, references background save alert research feed fdg pet evaluation of mucinous neoplasms: correlation of fdg uptake with histopathologic features. 	k. berger, s. a. nicholson, f. dehdashti, b. siegel 	medicine 	ajr. american journal of roentgenology 	 	 	view excerpt, references background save alert research feed etiology and significance of incidentally detected focal colonic uptake on fdg pet/ct 	n. purandare, sachin k gawade, a. puranik, a. agrawal, s. shah, v. rangarajan 	medicine 	the indian journal of radiology & imaging 	 	 	 	view excerpt, references background save alert research feed asymptomatic adnexal masses: correlation of fdg pet and histopathologic findings. 	s. fenchel, d. grab, + authors s. reske 	medicine 	radiology 	 	 	view excerpt, references background save alert research feed diagnostic value of preoperative suvmax on fdg-pet/ct for the detection of ovarian cancer 	yuko tanizaki, a. kobayashi, + authors k. ino 	medicine 	international journal of gynecological cancer : official journal of the international gynecological cancer society 	 	 	view excerpt, references background save alert research feed f-fdg pet/ct delayed images after diuretic for restaging invasive bladder cancer 	dalton a. anjos, e. etchebehere, c. ramos, allan o. santos, c. albertotti, e. camargo 	medicine 	journal of nuclear medicine 	 	 	pdf 	view excerpt, references background save alert research feed ... ... related papers 	abstract 	figures and tables 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking 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using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ delaware faces immunization challenges head-on jim talbott, mpa and paul hess delaware division of public health abstract this article explores the recent history of under immunized vaccine preventable diseases, along with recent outbreaks and how the delaware division of public health has responded to each. delaware’s vaccination efforts for children have achieved high coverage rates for all of the vaccine preventable diseases, leading to low incidences rates in the state. the main reason for these high rates is the law for mandated immunization for children entering kindergarten. as children age out of primary school, vaccination uptake declines. hpv vaccination rates are a prime example, as even though delaware’s rates as compared to the national average are better, they fail to meet healthy people standards of % vaccinated. to get to the heart of an under immunized population, a study to determine where the lower vaccinated rates are, along with the surveillance rates to cross match the results must be undertaken. it is understood that for communities where vaccination rates are lower, risk for disease is greater, with several recent outbreaks corroborating this. a pertussis outbreak in the delaware amish community was managed by the state with education and outreach in the community, with mixed results. hepatitis a is an epidemic in a number of states, and delaware has taken steps to try to protect our vulnerable population, using outreach, education and vaccination clinics to prevent the outbreak from occurring locally. while work continues, resources will need to be continually applied to ensure that vulnerable populations have the means to access the services needed. dph continuously monitors state vaccine coverage level data and disease outbreaks within and outside delaware’s borders and has implemented the above-mentioned public health initiatives to promote immunization awareness and address community health concerns. introduction vaccination efforts save countless lives and billions of dollars each year in avoidable medical expenses. since , the vaccines for children program has enabled the government to vaccinate uninsured and underinsured children. in its first years, this program saved an estimated $ billion in direct health care costs and over $ trillion in societal costs, such as lives and work hours saved. delaware’s vaccination efforts for children have achieved great results. the state of delaware requires that children in licensed daycare centers and entering kindergarten through grade at public, private, and home schools be immunized against certain communicable diseases before enrolling in school. delaware administrative code , “control of communicable and other disease conditions,” requires immunizations against measles, mumps, and rubella (mmr); tetanus, diphtheria, and pertussis/whooping cough (tdap); polio (ipv or opv); hepatitis b; and chickenpox (varicella). these diseases can be fatal or have serious complications that can result in blindness, deafness, and developmental delays. each year, the centers for disease control and prevention’s (cdc) national center for immunization and respiratory diseases (ncird) sponsors the national immunization survey (nis). the nis is a group of telephone surveys used to monitor vaccination coverage among children - months and teens - years, along with flu vaccinations for children months to years. in , delaware’s rate for the series : : : : : : ( +dtap, +polio, +mmr, +hib, +hepb, +varicella, and + pcv) was . %, higher than the national average of . % and close to the healthy people goal of %. due to the successes of vaccination, fewer health care providers and parents have witnessed the serious and sometimes life-threatening consequences of vaccine-preventable diseases. yet small numbers of cases can lead to the re-emergence of vaccine-preventable diseases, especially if there are increasing numbers of unvaccinated people. disease outbreaks sporadically surface nationally and in delaware, and the state’s challenge is to respond quickly to outbreaks to prevent them from spreading rapidly. we describe challenges and opportunities in vaccination and vaccine-preventable disease in delaware by focusing on three case studies: pertussis, human papillomavirus, and hepatitis a. we conclude with discussion of vaccination availability in delaware through the vaccines for children (vfc) program. pertussis challenge occasional outbreaks of whooping cough (pertussis), a highly contagious respiratory disease, occur in delaware. typically, pertussis outbreaks have occurred among kent county’s amish community, a population that is largely unvaccinated. the amish community practices separation from the world through group solidarity and caring for their own. though their religious doctrine does not prohibit vaccination, coverage levels for routine childhood vaccination remain low for various reasons, including misinformation about the safety and/or content of vaccines and a strong belief in naturally acquired immunity. while delaware has seen an improvement in coverage rates in pertussis, identified cases are still being reported every few years. pertussis in delaware has ebbed and flowed, with reported case spikes in and . in , cases of pertussis — the state’s largest caseload since — were reported to the division of public health (dph); most were due to an outbreak among the amish. in may , dph learned of a new pertussis outbreak among the amish. as of december , , the case count for that outbreak was total cases, with confirmed and considered probable. most reported cases were in individuals years of age and younger. for comparison with other years, there were pertussis cases in delaware in , in and in (delaware health and social services [dhss], ). for all years, the actual number of cases was likely higher due to underreporting and misdiagnosis. response to pertussis outbreaks these re-occurring outbreaks demonstrate the challenges of addressing disease in the amish population. dph has served generations of the amish and over the past years has strived to gain their trust. amish leaders and midwives serving the population have educated dph staff about their homeopathic approach to treating disease. some within this community do not consider immunization necessary to prevent diseases from occurring, or in response to outbreaks. instead they view getting some diseases and getting over them naturally without the intervention of a vaccine, a ‘rite of passage’. similarly, a study sought to determine the knowledge, beliefs and attitudes among amish communities in ohio. through it, some respondents shared fears of having too many recommended immunizations and that immunizations would overwhelm a child’s own natural immune system. in response to the pertussis outbreak, dph initiated a multi-pronged effort to control the spread of the disease. one of the early activities was to set up a meeting between the dph director, key staff and the amish leadership (bishops). during this meeting, dph staff learned that the primary drivers behind low vaccination rates were misinformation about the makeup and safety of the vaccine, perceived pain and distress to the children during vaccination, and a belief that if you let the disease run its course that children could not be re-infected. the bishops were happy that dph expressed concern about the community’s well-being and indicated they would be willing to distribute educational materials within the community. these educational materials included flyers highlighting the symptoms of pertussis, the benefits of seeking and completing antibiotic treatment, and the benefits and safety of vaccination. distribution of materials occurred at amish owned businesses, as well as businesses frequented by members of the community, physician’s offices, and schools. generally, the distribution of the flyers was well received, however, dph did encounter resistance to its distribution of information in the schools from some parents. amish schools in delaware are not part of the public school system and so distribution of materials from outsiders must be approved by the community’s leadership. for this reason, dph was asked to stop using the school system to distribute information. an important aspect of dph’s outbreak response was epidemiological surveillance and direct contact with the community. dph spent weeks conducting door-to-door case finding and a contact investigation campaign to maximize active surveillance and control measures. teams of dph epidemiologists and clinic nurses visited homes of community members where reports of pertussis had been confirmed, and asked questions to determine if there were other close contacts at risk. from a treatment perspective, updated standing orders allowed for the distribution of antibiotics for treatment and prophylaxis in households where dph identified symptomatic persons and their contacts. the delaware immunization program also made field visits to offer vaccine and antibiotics at the public health clinics throughout the state. during visits, information gained through administering survey questions informed dph’s response. some of those who had received immunizations in the past, or had their children vaccinated for various reasons, but who subsequently stopped getting vaccinated said it was because dph no longer conducted immunization clinics in their community. in the past, dph conducted immunization clinics in the community but stopped due to lack of attendance. it is apparent that since this community is hesitant to seek and find vaccinations, dph must offer immunizations where the amish are most receptive to receiving them. another way dph addressed this issue was to work with a midwife who sees pregnant amish women to provide pertussis vaccines in accordance with the advisory committee on immunization practices (acip). the midwife is a trusted source of medical guidance for amish women in the community. dph is providing the midwife with tdap vaccine so she can vaccinate her clients during their well visits, if the women agree. dph also contacted, and provided information, to chiropractors, who are another source of medical advice for this population. dph also reached out to holmes county general health district in ohio for public health approaches that were successful with their amish communities. holmes county provides health clinics in the amish community that offer immunizations, health screenings, and other services. they also help organize an annual amish health and safety day, which provides another opportunity to share information. in addition to ohio, dph reached out to the state of pennsylvania, who has a large amish populations to understand how they faced vaccine- preventable disease outbreaks. dph’s educational outreach was not limited to the amish community, because ultimately pertussis spread to the non-amish population in delaware. in the outbreak, pertussis cases occurred among non-amish individuals. dph issued a press release to the public through media contacts, and on august , informed health providers by issuing a health alert (https://healthalertde.org/) through the delaware health alert network (dhan). in addition, dph created and distributed a flyer to all licensed medical providers in kent county and asked them to share it with their patients. the flyer announced the outbreak to help engage patients in conversations about receiving pertussis vaccinations and other ways they could protect themselves and their families. through these multi-faceted measures, dph has re-established open lines of communication with the amish community, and hopes that it will encourage more of these individuals to reach out to the agency in the event of future outbreaks. human papillomavirus according to the cdc, every year , women and men are diagnosed with a cancer caused by human papillomavirus (hpv) infection. hpv vaccination could prevent more than % of these cancers ( , cases ever year) from ever developing. hpv vaccinations coverage levels remain low across the nation. in , only percent of adolescents were up to date on the hpv vaccine, and percent of teens ages - years received the first dose to start the vaccine series. according to the national immunization survey (nis), percent of delaware adolescents were up to date on the hpv vaccine, and percent received the first dose to start the vaccine series. although delaware’s hpv coverage rates are above the national average, hpv vaccination rates are much lower than for other adolescent-recommended vaccines within delaware, such as tdap ( . %) and meningococcal ( . %), both recommended at the same time as the hpv vaccine (delaware hpv vaccination report, december ). the lower vaccination rates are not due to lack of vaccine or vaccine availability. in the fiscal year beginning july , to june , , medical providers in delaware ordered , doses of hpv. in the current fiscal year that began on july , , medical providers in delaware have ordered , doses as of january . the cdc estimates that delaware will have ordered enough vaccine to vaccinate all of the -year-olds in delaware this year. yet while sufficient doses are ordered, some providers are not reporting administered doses to the state immunization information system (iis) suggesting that the true vaccination rate is higher than what is reported. to address the issue of low hpv rates, the delaware immunization program recently collaborated with the delaware cancer prevention program and the delaware cancer consortium to identify and implement activities designed to promote hpv awareness and increase immunization rates. the cancer prevention program provided funding to support three projects in order to increase lagging hpv rates within the state. these activities included initiating an hpv media campaign, continuing state immunization information system (iis) reports training for vaccines for children (vfc) providers, and continuing hpv-education workshops for local providers. the hpv education for vfc provider’s project involved contracting with a vendor to conduct this training for approximately vfc providers. dph established a contract with a vendor to recruit and train these providers from november , thru june , . the vendor developed a hpv training curriculum using the cdc’s “you are the key to hpv cancer prevention” guide. these training sessions range from small groups to one-on-one encounters with providers. topics include the benefits of vaccinating at an early age, role play activities to appropriately address parental safety concerns with the hpv vaccine, and utilizing reports within the iis (i.e. coverage level, not up-to- date, patient roster, and reminder recall reports), to improve vaccination rates. the dph also entered into a contract with another vendor to offer vfc providers training on the delaware’s iis reports module to assist them through the assessment, feedback, incentive and exchange (afix) process and help providers increase their immunization coverage rates. the providers receive training on the following reports: . afix snapshot – report allows providers to run immunization coverage levels for specific age cohorts within their practice. . patient roster reports – report allows the provider to identify all the patients within their practice who are currently active in the iis. . patient inactivation reports — report allows the provider to inactivate any patients in the patient roster report that are no longer active within their practice. . not-up-to- date reports – report allows the provider to identify patients that require additional immunizations to be compliant with immunization recommendations. . reminder recall reports – report allows the provider to generate a list of all their patients that require additional immunizations and print out post-card reminders to mail to these individuals. the primary objective is to attempt to mirror the immunization coverage data from a provider’s medical record with the immunization program’s iis and the nis to get a true picture of the hpv coverage rate in delaware. since implementation of the iis report training, the iis hpv coverage rate rose to . % for the first dose, an increase of . % from june , . the up-to- date coverage for the series completion reflects a % increase from december , . the dph cancer prevention and control program contracted with a vendor to develop and implement a statewide marketing campaign that encourages parents of children ages or to have their children receive the hpv vaccine in the same visit when they are vaccinated for other serious diseases, like meningitis and whooping cough. this campaign consists of print, radio, social media, and digital ads; direct mail; and social influencers targeting parents of - year olds. there is a social media plan for facebook and instagram that includes live chat events with physicians, school nurses, or parents speaking on the advantages of getting children vaccinated for hpv, and polls that engage parents using facts and statistics about hpv and the hpv vaccine. a private school outreach plan encompasses hpv presentations at parent events; hpv messaging in parent newsletters or emails; parent/child hpv vaccination videos; and posters and other printed resources to promote hpv awareness. the immunization program continues to conduct afix provider site visits to all vfc providers within the state as part of the federal requirements for the vfc program. during the initial site visits, a dph staff member reviews the current immunization coverage levels for the provider’s practice and discusses issues or barriers contributing to any lower rates. the staff also works with the provider to develop quality improvement activities that they can incorporate into their existing workflow to increase their immunization coverage rates. special attention is given to their hpv coverage rates at this visit and the staff offer the provider educational materials on hpv and encourage them to offer this vaccine in the same way and on the same day that they offer other vaccines at the and -year-old well visits. providers are also encouraged to take the iis reports training class. dph provides registration information during the site visit. dph conducts a follow-up visit six months later to discuss changes in their coverage rate and the progress made on the quality improvement activities. the immunization program saw increased coverage levels in providers who had received these visits and completed the iis reports training. hepatitis a since march , several state and local health departments have battled hepatitis a outbreaks that spread through person-to-person contact. the outbreaks are occurring primarily among persons who use injection and non-injection drugs, and/or among persons who are homeless and their close direct contacts. hepatitis a outbreaks have occurred in california, utah, arkansas, missouri michigan, ohio, kentucky, west virginia, tennessee, north carolina and the city of philadelphia. those outbreaks compelled the acip on october , to recommend that “all persons at least year old who are experiencing homelessness should be routinely immunized against hepatitis a” . delaware’s hepatitis a response since the beginning of october , dph has contacted homeless shelters, transitional housing organizations, and outpatient facilities to provide hepatitis a vaccinations to persons experiencing homelessness. a questionnaire distributed to homeless and residential shelters asked for the number of residents, their ages, if they are required to leave each morning, and what would be a good time for dph to hold a hepatitis a vaccination clinic there, if interested. if a facility expressed interest in hosting a vaccination clinic, the delaware immunization program shared that information with dph’s northern and southern health services teams, who would reach out to schedule one that would fit everyone’s needs. since homeless individuals are transient, a continuous presence at these sites should occur with further vaccination clinics scheduled. for other agencies with homeless clients, but without the capability to provide the hepatitis a vaccine, dph created an educational flyer to give to individuals at intake to start the vaccination conversation. dph also provides vaccination at state service centers located throughout delaware. it is dph’s hope that the shelters will include hepatitis a immunizations as part of their intake policy. this will protect not only the residents of these shelters, but the community at large as the residence interact in their communities. brandywine counseling and community services (bccs), a leading outpatient care provider in delaware, has sites throughout the state and caters to the population that dph has focused on to prevent the spread of hepatitis a. dph collaborated with bccs to provide hepatitis a vaccine at their facilities. the agreement calls for bccs to screen their clients when they arrive to determine their immunization status for hepatitis a. if clients are not up to date on their hepatitis a coverage, bccs will vaccinate them and schedule a date and time for the final dose. if this initiative is successful, and with appropriate budgets and staff support, dph can visualize an effort to immunize all homeless individuals with all vaccines recommended for adults. section vaccine section of the public health service act authorizes the federal purchase of vaccines to vaccinate children, adolescents, and adults. over its -year history, section -purchased vaccine was directed to priority populations. most recently, this included underinsured children ineligible for vfc and uninsured adults. section discretionary funding also supports immunization program operations at the local, state, and national levels. in delaware, while vfc vaccine covers most, if not all children, section vaccine is used in two areas: pandemic response exercises, and uninsured and underinsured adults and children. each year, every state health department is required to perform a point of dispensing exercise (pod). the pod allows dph staff to practice immunizing a large population in a timely manner. since , dph has operated its largest flu clinics as pods, a maneuver with several benefits. combining efforts allows the state to purchase more influenza vaccine to vaccinate those in the general population, and it enables dph to practice their required pod duties. also in , dph provided section -purchased vaccines to ten local federally qualified health centers (fqhcs) and one non-profit provider clinic to assist with immunizing their under- and uninsured adult populations. these vaccines include: tetanus, diphtheria and pertussis/tetanus diphtheria (tdap/td); hepatitis a and b; measles, mumps, and rubella (mmr); human papillomavirus (hpv), varicella, meningococcal b (men b), meningococcal acwy (mcv ), pneumococcal conjugate (pcv ), pneumococcal polysaccharide (ppsv ), and influenza. dph continues to provide these above-mentioned clinics with all adult vaccines as recommended by the acip with the exception of the zoster vaccine. in fiscal year , the state of delaware received approximately $ , in section -vaccine funding, an amount equal to fiscal year . in , dph was able to supply all needed vaccine to these clinics and utilized all funding by the end of the fiscal year. just two months into fiscal year , dph processed adult vaccine orders and spent % of the budget. as of december , , doses of influenza vaccine were distributed using vaccine funds which was a % increase from the number of influenza doses distributed by december . theses doses were distributed as follows; , doses were used for pod activities and , doses were delivered to section providers. these partnerships allow more underserved at-risk adults to receive the immunizations they need. vaccination data is reported to the state immunization information system (delvax). conclusion the dph diligently monitors state vaccine coverage level data and disease outbreaks within and outside delaware’s borders. as a result, dph has collaborated with stakeholders throughout the state to: • implement hpv provider trainings and a state-wide media campaign designed to promote hpv awareness and increase coverage levels, • establish hepatitis a vaccination clinics in sites that provide services for at-risk populations (i.e. homeless shelters, drug treatment centers), and • rapidly responded to a pertussis outbreak by conducting field case investigations, educational outreach, and medical management (antibiotics and vaccinations) as appropriate within the amish community. in addition; dph issued a public health alert regarding the outbreak, distributed pertussis flyers to medical providers in kent county to share with their patients in order to increase awareness and promote vaccinations, and reached out to other states to identify public health approaches that were successful within their amish communities. resources for readers for more information on immunizations for children, adults, and health care providers, visit dph’s immunizations website, http://www.dhss.delaware.gov/dhss/dph/dpc/immunize.html, or call the immunizations hotline at - - - weekdays between : a.m. and : p.m. children without medical insurance may receive free vaccines through the vfc program. for details, call the hotline or visit http://www.dhss.delaware.gov/dhss/dph/dpc/immunize.html to learn more. the cdc’s website, http://www.cdc.gov/vaccines/, offers additional information such as the recommended immunization schedule. health care providers can view the immunization schedule on tablets or smart phones by downloading the cdc vaccines schedules app at www.cdc.gov. references . jennewein, m. ( , jan).vaccination: more than just your health. sitn.hms.harvard.edu. http://sitn.hms.harvard.edu/flash/ /vaccination-just-health/ . state of delaware. ( ). control of communicable and other disease conditions. delaware.gov. http://regulations.delaware.gov/admincode/title /department% of% health% and% social% services/division% of% public% health/health% promotion% and% disease% prevention/ .shtml . cdc. ( , jan). about the national immunization surveys (nis). cdc.gov. https://www.cdc.gov/vaccines/imz-managers/nis/about.html . kettunen, c., nemecek, j., & wenger, o. ( , june ). evaluation of low immunization coverage among the amish population in rural ohio. american journal of infection control, ( ), – . https://doi.org/ . /j.ajic. . . pubmed . cdc. ( , mar). why is hpv important? cdc.gov. https://www.cdc.gov/hpv/hcp/hpv- important.html . cdc. ( ). adolescent human papillomavirus (hpv) vaccination coverage dashboard. cdc.gov/teenvaxview. https://www.cdc.gov/vaccines/imz- managers/coverage/teenvaxview/data-reports/hpv/dashboard/ .html . cdc. ( , aug). hpv vaccination coverage data. cdc.gov. https://www.cdc.gov/hpv/hcp/vacc-coverage/index.html . cdc. ( , nov). you are the key to hpv cancer prevention – train the trainer. cdc.gov. https://www.cdc.gov/vaccines/ed/hpv/you-are-key.html . walker, m. ( , oct). acip: routinely vaccinate homeless against hepa. medpagetoday.com. https://www.medpagetoday.com/meetingscoverage/acip/ . cdc. ( , feb). questions answered on vaccines purchased with funds. cdc.gov. https://www.cdc.gov/vaccines/imz-managers/guides-pubs/qa- -funds.html https://doi.org/ . /j.ajic. . . https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids= &dopt=abstract 	delaware faces immunization challenges head-on 	abstract 	introduction 	pertussis challenge 	response to pertussis outbreaks 	human papillomavirus 	hepatitis a 	delaware’s hepatitis a response 	section vaccine 	conclusion 	resources for readers 	references 	 . jennewein, m. ( , jan).vaccination: more than just your health. sitn.hms.harvard.edu. http://sitn.hms.harvard.edu/flash/ /vaccination-just-health/ 	 . state of delaware. ( ). control of communicable and other disease conditions. delaware.gov. http://regulations.delaware.gov/admincode/title /department% of% health% and% social% services/division% of% public% health/health% promot... 	 . cdc. ( , jan). about the national immunization surveys (nis). cdc.gov. https://www.cdc.gov/vaccines/imz-managers/nis/about.html 	 . kettunen, c., nemecek, j., & wenger, o. ( , june ). evaluation of low immunization coverage among the amish population in rural ohio. american journal of infection control, ( ), – . https://doi.org/ . /j.ajic. . . pubmed 	 . cdc. ( , mar). why is hpv important? cdc.gov. https://www.cdc.gov/hpv/hcp/hpv-important.html 	 . cdc. ( ). adolescent human papillomavirus (hpv) vaccination coverage dashboard. cdc.gov/teenvaxview. https://www.cdc.gov/vaccines/imz-managers/coverage/teenvaxview/data-reports/hpv/dashboard/ .html 	 . cdc. ( , aug). hpv vaccination coverage data. cdc.gov. https://www.cdc.gov/hpv/hcp/vacc-coverage/index.html 	 . cdc. ( , nov). you are the key to hpv cancer prevention – train the trainer. cdc.gov. https://www.cdc.gov/vaccines/ed/hpv/you-are-key.html 	 . walker, m. ( , oct). acip: routinely vaccinate homeless against hepa. medpagetoday.com. https://www.medpagetoday.com/meetingscoverage/acip/ 	 . cdc. ( , feb). questions answered on vaccines purchased with funds. cdc.gov. https://www.cdc.gov/vaccines/imz-managers/guides-pubs/qa- -funds.html pone. .. fam-mdr: a flexible family-based multifactor dimensionality reduction technique to detect epistasis using related individuals tom cattaert , *, vı́ctor urrea , adam c. naj , lizzy de lobel , vanessa de wit , , mao fu , jestinah m. mahachie john , , haiqing shen , m. luz calle , marylyn d. ritchie , todd l. edwards , kristel van steen , montefiore institute, university of liège, liège, belgium, groupe interdisciplinaire de génoprotéomique appliquée - research, university of liège, liège, belgium, department of systems biology, university of vic, vic, spain, miami institute for human genomics, university of miami, miami, florida, united states of america, department of applied mathematics and computer science, ghent university, ghent, belgium, school of medicine, university of maryland, baltimore, maryland, united states of america, center for human genetics research, vanderbilt university, nashville, tennessee, united states of america abstract we propose a novel multifactor dimensionality reduction method for epistasis detection in small or extended pedigrees, fam-mdr. it combines features of the genome-wide rapid association using mixed model and regression approach (grammar) with model-based mdr (mb-mdr). we focus on continuous traits, although the method is general and can be used for outcomes of any type, including binary and censored traits. when comparing fam-mdr with pedigree-based generalized mdr (pgmdr), which is a generalization of multifactor dimensionality reduction (mdr) to continuous traits and related individuals, fam-mdr was found to outperform pgmdr in terms of power, in most of the considered simulated scenarios. additional simulations revealed that pgmdr does not appropriately deal with multiple testing and consequently gives rise to overly optimistic results. fam-mdr adequately deals with multiple testing in epistasis screens and is in contrast rather conservative, by construction. furthermore, simulations show that correcting for lower order (main) effects is of utmost importance when claiming epistasis. as type diabetes mellitus (t dm) is a complex phenotype likely influenced by gene-gene interactions, we applied fam-mdr to examine data on glucose area-under-the-curve (gauc), an endophenotype of t dm for which multiple independent genetic associations have been observed, in the amish family diabetes study (afds). this application reveals that fam-mdr makes more efficient use of the available data than pgmdr and can deal with multi-generational pedigrees more easily. in conclusion, we have validated fam-mdr and compared it to pgmdr, the current state-of-the-art mdr method for family data, using both simulations and a practical dataset. fam-mdr is found to outperform pgmdr in that it handles the multiple testing issue more correctly, has increased power, and efficiently uses all available information. citation: cattaert t, urrea v, naj ac, de lobel l, de wit v, et al. ( ) fam-mdr: a flexible family-based multifactor dimensionality reduction technique to detect epistasis using related individuals. plos one ( ): e . doi: . /journal.pone. editor: zoltán bochdanovits, vu university medical center and center for neurogenomics and cognitive research, vu university, netherlands received january , ; accepted march , ; published april , copyright: � cattaert et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. funding: the first author is a postdoctoral researcher of the fonds de la recherche scientifique - fnrs. this work is also partially supported by grant mtm - -c - from the ministerio de ciencia y tecnologia, spain, by the belgian network biomagnet (iap p / ), funded by the interuniversity attraction poles programme, initiated by the belgian state, science policy office, and by the ist programme of the european community, under the pascal network of excellence, ist- - . the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. competing interests: the authors have declared that no competing interests exist. * e-mail: tom.cattaert@ulg.ac.be introduction the international hapmap project [ ] was designed to create a genome-wide database of human genetic variation, with the expectation that these data would be useful for genetic association studies of common diseases. this expectation has been fulfilled with just the initial output of genome-wide association analyses, identifying nearly loci for over common diseases and traits [ , ]. despite these successes, it has become clear that usually only a small percentage of total genetic heritability estimates can be explained by the identified loci. for instance, for inflammatory bowel disease (ibd), loci significantly impact disease but they explain only % of disease risk and % of genetic risk [ ]. this may be attributed to the fact that recent findings show many types of genetic associations for various traits, with subtle effects: non- additive genetic effects, non-snp polymorphisms, epigenetic effects, but also gene-environment and gene-gene interactions [ ]. the role of genetic interactions in explaining phenotypic variability has been described in several publications [ , , , , , , , , , ]. interactions may lead to inconsistent results from the masking of associations, they can be suggestive of important pathogenic mechanisms and may elucidate relevant opportunities for intervention [ , ]. epistasis, defined as the deviation from additivity of effects observed at multiple genetic exposures [ , ], may also explain part of the genetic heritability that is left unexplained for most complex disorders [ ]. these reasons have made epistasis an increasingly accepted characteristic of the genetic architecture of common, complex disorders [ , , , ]. one of the potential reasons for the small number of large-scale genetic interaction studies performed in humans so far is that plos one | www.plosone.org april | volume | issue | e although genetic interactions identified from model organisms provide insight into biological processes, these biological processes often lack sufficient overlap with other types of gene/protein associations with traits of interest [ , ]. also, the relatively low success rate of large-scale epistasis searches to date may simply reflect the limited ability to assess the many possible modes of interaction, including pairwise interactions and threshold effects [ ] or inadequate solutions given to a difficult statistical challenge [ ]. in addition, subtle variation in allele frequency can either introduce an interaction effect and likewise remove an interaction effect from a particular dataset; this can make detection of epistasis effects quite challenging [ ]. overviews of methods for epistasis detection were given by cordell [ , ] and by onkamo and toivonen [ ]. one non-parametric approach developed for epistasis analysis is multifactor dimensionality reduction [ , , ] (mdr). since its conception, many methodological and applied papers have emerged that build on or use mdr. to our knowledge, the current state-of- the-art mdr-related method that can accommodate nuclear families of any size and different types of outcome variables is the recently proposed pedigree-based generalized mdr method [ ] (pgmdr) which generalizes the generalized mdr method [ ] (gmdr) to family data. its competitor, the mdr pedigree disequilibrium test [ ] (mdr-pdt), is only suited for case- control data and does not allow for covariate adjustments. similar to mdr, gmdr uses prediction accuracy measures for best model selection. significance assessment is based on random permutations. to easily accommodate continuous traits and variable adjustments, gmdr is based on scores of a (generalized) linear model. in the special case of no covariates and a binary outcome it reduces to the classical mdr. pgmdr first constructs a non-transmitted genotype for every non-founder in the pedigree. when parental genotype information is missing, pgmdr samples one realization of the nontransmitted genotype from the conditional distribution given the minimal sufficient statistic for the null hypothesis through an algorithm that is modified from rabinowitz and laird [ ]. second, the non-founders and the non-transmitted genotypes are analyzed by the gmdr algorithm. significance assessment is again based on permutations. to maintain the correlation structure within the families, families as a whole are used as permuting units and the transmitted and non-transmitted sets in a whole family are randomly shuffled. pgmdr software is available from the url http://www. healthsystem.virginia.edu/internet/addiction-genomics. the need for new statistical methods to overcome some of the remaining statistical hurdles in epistasis detection, has led to the development of fam-mdr. the method combines features of the grammar approach [ ] with features of model-based mdr [ ] (mb-mdr). in the materials and methods section, we introduce the fam- mdr algorithm and describe our extensive simulation study to examine type i error and power of this approach. to examine the application of fam-mdr to determine epistasis in family studies of a complex disease, we examined data on glucose area-under- the-curve (gauc), an endophenotype of type diabetes mellitus (t dm) for which multiple independent genetic associations have been observed, in the extended pedigrees of the amish family diabetes study [ ] (afds). subsequently, we describe the power and type i error performance of fam-mdr in our simulations and application to afds, as well as a comparative study between fam-mdr and pgdmr, in the results section. finally, the discussion elaborates on the significance of our results, and the relevance of its application to finding gene-gene interactions in a complex disease like t dm. materials and methods the fam-mdr algorithm fam-mdr is an acronym for family multifactor dimension- ality reduction and is an adaptation to related individuals of the model-based multifactor dimensionality reduction method [ ] (mb-mdr) for epistasis detection with unrelated individuals. an implementation of the fam-mdr algorithm is available through the url www.statgen.be. the approach consists of two parts. part i: in order to deal with familial correlations between observations, data are first analyzed using a polygenic model yi~mzgizei , ð Þ with i indexing individuals, g distributed mvn( ,ws poly) and e distributed mvn( ,i s env), representing the additive polygenic and environmental effects respectively. the polygenic effect has variance s poly and is correlated within families, with correlation matrix equal to the relationship matrix w. for the calculations we use the polygenic function of r package genabel [ , , , ]. this package can be retrieved from the url http://cran. r-project.org/web/packages/genabel/index.html. the rela- tionship matrix w can be derived either theoretically from the pedigree structure, or can be estimated from the available genomic data, in which case the genomic kinship is computed [ ]. genomic kinship is to be preferred when genome-wide data are available, but of course in a candidate gene study genomic kinship cannot be estimated in a reliable way and one will have to use pedigree kinship [aulchenko, genabel tutorial]. the environ- mental variance s env is assumed to be the same for all individuals. in addition, environmental effects are assumed to be independent between individuals, whether belonging to different families or within the same family, giving rise to an environmental variance- covariance matrix i s env (i : the identiy matrix of rank equal to the number of individuals). the residuals êei~yi{(m̂mzĝgi ), ð Þ that have been derived from ( ), are free from polygenic familial correlation and can serve as new familial correlation-free traits to be used in a genetic association analysis with measured genetic markers. as a remark, m̂m and ĝgi are estimates of mean m and polygenic effect (or breeding value) gi obtained by the expectation-maximisation (em) algorithm using the maximum likelihood (ml) paradigm, hence maximizing the joint likelihood of fixed effects and variance components [ ]. in the grammar approach of aulchenko et al. [ ], such a genetic association analysis targets associations with single markers, one at a time, and is fully parametric in nature. in contrast, in the fam-mdr approach, associations with multiple loci at once are evaluated. part ii: once the data have been prepared in part i, fam- mdr proceeds with investigating the association between the newly defined trait ynew~êei from part i (in particular the aforementioned residuals) and multi-locus measured genotypes, using the potentially fully-parametric mb-mdr method (figure ). it is justified to apply mb-mdr, that was developed for unrelated individuals, to these residuals. indeed, conditional on the observed genotypes all the familial correlation has been accounted for. moreover, mb-mdr flexibly deals with different outcome types, including those measured on a continuous scale. like mdr, mb- mdr reduces a high-dimensional interaction space to a - fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e dimensional space, by assigning genetic effect size labels to multi- locus genotypes, which will be further referred to as risk labels. key steps involved in a fam-mdr part ii analysis are summarized graphically in figure . because some characteristics of mb-mdr have been altered in the fam-mdr algorithm, we summarize the key steps and properties of mb-mdr, and point out changes we made to the initial mb-mdr algorithm. throughout this paper, for ease of exposition, we focus on two-locus models and diallelic markers, although both mb-mdr and fam-mdr are applicable in principle to more general settings as well. in mb-mdr step , for a selection of a pair of snps, each genotype cell is tested against the eight others for association with the trait. the cells that are found to be significantly associated with the outcome, at a liberal threshold of . , are then called high risk (h ) or low risk (l) based on the position of the selected association measure (e.g. t statistic or odds ratio) in the spectrum of all possibilities. those cells that are not significant at the threshold . are labeled as non-evidence cells (o). for a more detailed discussion of the mb-mdr method we refer to calle et al. [ ] and also to their technical report available from the url http:// www.recercat.net/handle/ / . in step , two additional tests are performed for association with the trait: testing h versus fl,og and testing l versus fh ,og. this gives rise to two wald-type statistics of association, wh and wl, that are either derived from a parametric or a non-parametric testing approach. in step , the significance of wh and wl is assessed through permutations. this is different from the classical mb-mdr implementation, but is an elegant way to compensate for the data snooping in mb-mdr steps – and to correct for the otherwise overly optimistic test results. note that in the initial implementa- tion of mb-mdr, depending on the number of combined cells in either the high (low) risk cells pool, a different null distribution for the corresponding wald test statistic wh (wl) was derived. these marginal null distributions were generated by simulating reference data with similar characteristics. the multiple testing issue that arises when considering different snp pair combinations, is tackled by mb-mdr [ ] using bh-fdr (benjamini-hochberg false discovery rate) methodology.[ ] because of the afore- mentioned dependency on number of combined cells, and the complexity of the significance assessment via a simulation-based null distribution derivation in step , we chose to implement a different approach. fam-mdr therefore implements a permuta- tion strategy by simply randomly permuting the familial correlation-free residuals ( ) obtained in fam-mdr part i. this assumes a general null-hypothesis of no association between any of the measured markers and trait (no main effects, no interaction effects). this strategy does not depend on the number of combined cells, nor on reference data.although mb-mdr allows in principle for multiple model selection, we have furthermore restricted the approach to select only the best model, for the purpose of comparison with pgmdr. in particular, we derive the permutation null distribution of the maximum test statistic, i.e. maximized over the two different tests (wh and wl) and all two- snp combinations j: maxh=l,j (wh,j ,wl,j ) and recommend replications for doing so. moreover, we emphasize that corrections for main effects and covariate adjustments are also possible in fam-mdr and we figure . summary of the steps involved in a fam-mdr analysis. the figure shows the three steps of fam-mdr part ii on one of the simulated datasets for model m , p~ : , g ~ : and h ~ : , for the analysis without main effects correction. doi: . /journal.pone. .g fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e choose to carry these out prior to association testing. extending the polygenic model ( ) to a (two-locus) measured genotype (mg) model [ ] with the particular main effect/covariates present in the model, has several advantages over adjusting while identifying risk cells and/or testing for association between new one- dimensional genetic constructs and the trait of interest. first, it leads to more stable parametric estimations when adopting a parametric regression approach in fam-mdr part ii. second, a priori adjustment paves the way for a fully non-parametric epistasis screening and thus faster mb-mdr runs on adjusted residuals. nevertheless, for the purpose of this paper, we will continue to use the parametric linear model paradigm (in fact the one-parameter wald test is equivalent to the student t-test) and not the non-parametric alternative, the wilcoxon rank sum test. third and most importantly, when residuals are free from identified important main effects, the null hypotheses of no association and of no epistasis become equivalent and the permutation procedure is a correct procedure for claiming epistasis, i.e. association above and beyond the main effects corrected for in fam-mdr part i. finally, a note on how fam-mdr handles missing data. for missing genotypes, fam-mdr uses the available cases (ac) paradigm. in other words, if a particular pair is considered, only individuals with complete data for this pair are included in the analysis, while individuals with missing data for one or two snps involved are not considered. individuals with missing trait values are used in fam-mdr part i only when they are useful for deriving the relationship matrix, but never for the polygenic calculations themselves. indeed, if the number of markers is small, the theoretical relationship matrix is used and the complete pedigree is needed for calculating this in a correct way. when the number of markers is large, fam-mdr relies on the genomic kinship matrix and there is no need to keep individuals with missing trait values in the analysis. note that since we are not using the individuals with missing trait values for the polygenic calculations, we essentially are performing a complete case (cc) analysis. also, when correcting for main effects/covariates - since we only use those individuals with complete data on the main effects/covariates that are regressed out in part i, we are again following the cc paradigm. in part ii, fam-mdr obviously does not use missing new trait values, whether these are due to missing trait values or missing main effects/covariates adjusted for. missing new trait values are also not included in the permutation procedure. it is good to recall that both ac and cc are valid under a missing completely at random (mcar) missingness process [ ]. simulation study we simulated data consisting of nuclear families, with the number of children drawn from a multinomial distribution with probabilities / to have one child (hence a trio), / to have two children, and / to have three children. on average, this gave rise to individuals. we assumed that no data were missing. in other words, complete information on genotypes at all loci and complete information on phenotypes were available. to generate genotypes for these individuals, we first generated ten diallelic markers: snpl ,l~ ,:::, , in linkage equilibrium. in addition, we assumed hardy-weinberg equilibrium for every generated marker. in other words, the genotype frequencies can be determined from the allele frequencies as follows: fl ~( {pl ) , fl ~ pl ( {pl ), and fl ~p l . the allele frequencies of a non- functional snp, snpj was fixed at pj ~ : z(j{ ) : , j~ ,:::, , whereas the allele frequencies of the functional snp pair snp ,snp ð Þ were taken to be equal, and varied as (p ,p )~(p,p), p[f : , : , : g. parental genotypes were then drawn according to the population genotype frequencies above. children’s genotypes were assumed to follow mendelian inheritance patterns. assuming the presence of additive polygenes and a residual environmental effect, phenotypes were simulated according to the (two-locus) mg model yi ~mk i ,k i zgizei , ð Þ with the notations of expression ( ) before. note the difference of the fixed effect mean term, m in ( ), which is replaced by mk i ,k i in ( ). here, kli refers to the minor allele count kl[f , , g for individual i and mk k represents the mean trait values according to the functional snp pair. trait values per family were then sampled from a multivariate normal distribution, with as components of the mean vector the trait mean values mk i ,k i corresponding to the two- locus genotypes that the individuals constituting the family belong to, and as variance-covariance matrix the part of ws polyzi s env pertaining to that family. in what follows, we explain in detail the simulation parameter settings of our choice. first, we notice that the population two-locus model variance s loci is computed as a weighted sum of squares, with weights determined by population genotype frequencies (exploiting no ld between the markers) s loci~ x k x k f k f k (mk k {m::) , ð Þ in which the overall mean m:: is computed as m::~ x k x k f k f k mk k : ð Þ in a similar way, the single-locus means and variances are respectively given by mk :~ x k f k mk k , ð Þ m:k ~ x k f k mk k , ð Þ and s ~ x k f k (mk :{m::) , ð Þ s ~ x k f k (m :k {m::) : ð Þ hence, since p ~p , and therefore also s ~s , the epistatic variance s epi , defined as the part of the two-locus model variance that is not explained by the contributing loci separately, is given in our simulation settings by s epi~s loci{ s . different contributions to the two-locus model variance of the epistatic variance s epi or the main effects model variance will be important in interpreting simulation results. fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e the total phenotypic variance, which is given by s tot~s locizs polyzs env, ð Þ was kept fixed at s tot~ for all simulations. the total heritability h of the trait, defined as h ~ s locizs poly s tot , ð Þ was taken to be h [f : , : , : g. the proportion g of the total variance s tot explained by the total two-locus model variance s loci , g ~ s loci s tot , ð Þ varied as g [f , : , : , : , : , : g. because of the afore- mentioned assumptions, and the definitions for h and g , we have s loci~g , s poly~(h {g ), and s env~( {h ). hence, given h and g , the three variance contributions are completely deter- mined. since only snps were considered in all scenarios, the relationship matrix was derived from the nuclear family actual relationships. second, the fixed mean part in ( ) needs to be determined. for this, we consider two two-locus models, m and m , of li and reich [ , ]. the key feature of model m is that phenotypic means increase when for both loci at least one variant allele is present. in contrast, in model m increased phenotypic values occur only when at one (and only one) locus a heterozygous genotype is observed. explicitly, models m and m are respectively determined by mm k i k i ! b@ ca, ð Þ and mm k i k i ! b@ ca: ð Þ it is important to realize that the two-locus variance s loci , can be computed from the genotype-specific means mk k above and the minor allele frequency p. hence, since the total trait variance s tot was set to , and since particular values for the two-locus model heritability g were also pre-specified, the means in ( ) and ( ) are actually proportional to the specifications to the right of the corresponding expression. to enhance the interpretation of future simulation results, it is instructive to more closely look into the decomposition of the two- locus model variances for models m and m into main effects and epistatic variance (table ). we observe that for model m the contribution of main effects becomes increasingly important with increasing p, whereas for model m the reverse is true, leading to a pure epistasis scenario for p~ : . the two different genetic two-locus models (m and m ), three possible minor allele frequencies p of the causal snp pair, different non-zero values for g and three different values for the total heritability h , lead to a total of simulation settings. for each of these settings data sets were simulated. under the general null hypothesis of no association with the trait (no main effects, nor two-way interaction effects), in particular, g (~s loci )~ , the different genetic models m and m are irrelevant. the three possible choices for the minor allele frequency p of the causal snp pair, and the three possible values for h result in nine different general null hypothesis simulation settings. for each of these settings we generated datasets. because we are primarily interested in detecting epistasis effects above and beyond main effects, when these main effects are present, we also generated a different type of null data, under the null hypothesis of no epistasis, but main effects. hence, g w and mmaink k ~mk :zm:k , ð Þ effectively reducing the two-locus variance to s vs loci , while increasing the polygenic variance. for each of the simulation settings, we again generated datasets. to reduce the computational burden of our extensive simulation study, we adopted a sequential approach [ ] during the permutation step of fam-mdr (fam-mdr part ii step ). we carried out the permutation calculations in batches of , and each time evaluated a go or no go to continue or not, setting the maximum number of permutations to . to this end, we determined the binomial ( {c) % confidence interval of the significance level a at the current number of permutations, with a~ : and c~ : . if the current estimate of the permutation p- value fell outside this confidence interval, the permutation procedure terminated and a definite conclusion about significance or non-significance was made. if the current estimate of the permutation p-value fell inside the confidence interval, no decisions were made about significance and an additional batch of permutations were performed. a drawback of this sequential approach is that no accurate estimate of the permutation p-value is output. hence, whereas it serves its purpose for simulation studies, it is less well suited for real data applications. only to enhance an honest comparison of simulations results with pgmdr, we also introduced a liberal version of fam-mdr, referred to as fam-mdr*. in contrast to pgmdr [ ] and gmdr [ ], for which the permutation null distribution is only derived with respect to the actual snp pair identified by the initial epistasis screening, fam-mdr screens all snp pairs in a permuted dataset. by doing so, fam-mdr assesses significance of the best snp-pair, while appropriately correcting for multiple testing. fam-mdr* assesses marginal significance of the best table . relative importance of main effects and epistatic variances for different two-locus models. m m p main epist. main epist. . . . . . . . . . . . . . . . abbreviations: main = ratio s � s loci of each of the main effects variances s to the two-locus variance s loci , epist. = ratio s epi . s loci of the epistatic variance s epi to the two-locus variance s loci . doi: . /journal.pone. .t fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e model by computing the empirical marginal p-value of the pair found. this was done by only maximizing over the two test statistics wh and wl, and by restricting attention to the specific pair jfound identified via the initial epistasis data analysis: maxh=l wh,jfound ,wl,jfound � � . although fam-mdr* is less com- putationally intensive, it leads to overly optimistic results, and increased false positive rates, just like pgmdr. in line with pgmdr [ ], permuations were used to estimate significance, hence no sequential approach is adopted. finally, we analyzed all simulated datasets with both fam- mdr and fam-mdr*. we applied pgmdr to all simulation settings with non-zero epistasis, and to (out of ) generated data sets for each of the simulations settings under the general null hypothesis of no association. every analysis is carried out once with and without main effects correction. the former involves adjusting for snp and snp by assuming a co-dominant mode of inheritance. the analysis with correction for main effects leads to the detection of two-locus effects beyond co-dominant main effects. the analysis without correction for main effects leads to the detection of a two-locus model but without giving a clue about whether this model is genuinely indicative for epistasis or for main effects, or for both. in pgmdr analyses, and when correcting for snp and snp main effects, we created two dummy variables for each of the causal snps and submitted them as covariates to the pgmdr software. amish family diabetes study the amish family diabetes study [ ] (afds) is a study to identify the genetic determinants of type diabetes and related traits in multi-generational extended pedigrees from the old order amish (ooa) community, a genetically-isolated group in lancaster county, pennsylvania. for this analysis, we examined genotype data for snps in five diabetes candidate genes, adipocnectin receptors and (adipor , adipor ), adiponectin (apm , also known as adipoq), calsequestrin (casq ), and hepatocyte nuclear factor a (hnf a), in individuals from a single large multi-generational pedigree subdivided into independent families for analysis and snps. in previous analyses in the afds [ , , , ], rs in hnf a [ ], rs and rs in casq [ ], and rs in adipor [ ] were found to be significantly associated with the continuous trait glucose area under curve (gauc). the latter was estimated using the trapezoid method from glucose levels taken at -minute intervals in a three-hour oral glucose tolerance test (ogtt) administered to all afds subjects without a prior history of diabetes [ ]. we examined log (gauc) as our continuous trait of interest, in non-diabetic individuals with gauc measure- ment available, setting gauc to missing for diabetics and individuals with unknown diabetes status because for diabetics the gauc measurement is expected to be biased. we took the natural logarithm of gauc because the shapiro-wilk test strongly rejected normality of the residuals of the polygenic model in an analysis on gauc itself, whereas it did not so in an analysis on log (gauc). after removing mendelian errors as found by fbat (downloadable from url http://www.biostat.harvard.edu/ ,fbat/default.html), we used the check.marker function of r package genabel on these non-diabetics with gauc measurement available. we put extr.call and extr.perid.call equal to . , so that markers and individuals with call rates below . are removed prior to main analysis. for the main analysis we put callrate and perid.call to . so we discard iteratively markers and individuals with call rates below . . we discarded four snps, rs and rs in adipor , and rs and rs in casq , due to low callrates. all markers had minor allele frequencies above %. we also discarded individuals due to low callrates, by putting their gauc measurement to missing. all analyses are based on the remaining markers and individuals. in order to derive the correct relationships between the individuals of interest, fam-mdr also makes use of the remaining individuals without gauc measurement available and – in order to recognize relationships between offspring – of additional parental information ( parents, without genotype nor phenotype information). hence, ‘‘information’’ of individuals was exploited by fam-mdr. the individuals of interest correspond to independent families. of these, consisted of unrelated single individuals with no genotyped first- degree relatives, were nuclear families (comprising of the individuals or interest), and were multi-generational families. however, these families included or % of the individuals. because the present pgmdr implementation cannot deal with large multi-generational (non-nuclear) pedigrees, and only for reasons of comparison between pgmdr and fam-mdr, we split multi-generational pedigrees into nuclear families. in the resulting set of nuclear pedigrees, individuals were represented twice, most often as offspring in one pedigree and parents in another. the net result of the splitting process was a set of non-independent nuclear families, comprising ( = + ) individuals. of the individuals with gauc measurement available, were represented twice, resulting in a total of individuals of interest to us, in non-independent families. of these families, were part of the extended pedigrees, while the remaining were mutually independent. there is clearly a need to account for the lack of independence between these nuclear families. unfortunately, the pgmdr software is unable to do so. solely for reasons of comparison, fam-mdr was applied to the split-pedigree data, as well as to the original multi- generational family data. analyses were carried out with and without correction for the main effects of rs in hnf a, rs and rs in casq , and rs in adipor . they were regressed out in fam-mdr part i, or entered as covariates in pgmdr, using co- dominant coding. p-values were based on permutations for both pgmdr and fam-mdr, hence no stopping rules were used. the significance level was set at a~ : . without main effects correction, fam-mdr on the original data uses all individuals with gauc value available. when correcting for main effects, missing genotype information contributes to an increased missingness rate for the new trait values. here only or % of the individuals have non- missing new trait values. on the split-pedigree data, individuals are available for fam-mdr without main effects correction, and for fam-mdr with main effects correction. note that this is in fact an artificial increase in sample size of % and % respectively. in contrast to fam-mdr, pgmdr works with the transmitted genotype information. of the individuals with a gauc value available, the unrelated individuals (singletons) obviously did not have parental information available, giving rise to individuals eligible for pgmdr analysis. furthermore, whenever genotypes for any snp are completely missing for a family, pgmdr simply discards the whole family from the entire epistasis screening. as a result, pgmdr uses only individuals in an analysis without correction for main effects, and individuals in an analysis with main effects correction, amounting to reductions of % and % compared to the corresponding fam-mdr analyses. consequently, pgmdr does not use all information available. fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e results simulation study as a quality check on the simulated data, we first assessed the values of the hardy weinberg and linkage disequilibrium correlation coefficients rhw and rld, in the parents only. all correlation coefficients were consistent with the theoretical value of . more specifically, we observed that for all considered simulation settings the % confidence intervals of the correlation coefficients rhw and rld lied within the range ½{ : , : �. we have also fit a full mg model with a covariate coding for the exact dichotomization that is behind our models. in this way we were able to consistently estimate the effect sizes, showing that our data are indeed behaving in the way we intended. we first considered simulation results under the general null hypothesis of no association (no main effects, no two-order interactions). empirical type i error rates were defined as the number of times the selected best -locus model was assessed significant, divided by the number of simulations. from table , we notice that fam-mdr typically gives smaller empirical type i error rates than the nominal type i error rate of a~ : . in other words, fam-mdr has the tendency to be conservative. in contrast, fam-mdr* and pgmdr are much too liberal. the latter is not surprising, since both fam-mdr* and pgmdr completely ignore the multiple testing issue. these results are confirmed by figure , showing the probability-probability (pp) plots of the p-value distributions under the general null hypothesis of no association for p~ : and h ~ : . the pp-plots are based on datasets and permutations, without stopping rule to obtain exact p-values. the plots show the expected ordered p- values (i{ = )= on the horizontal axis and the observed ordered p-values p(i) on the vertical axis. by consequence, a certain theoretical significance level on the vertical axis can be related to a particular empirical type i error rate on the horizontal axis. for fam-mdr (panel a) the curve lies slightly above the diagonal, indicating a rather conservative approach. for fam- mdr* (panel b) and pgmdr (panel c) the curve lies far below the diagonal, pointing to an extremely liberal approach. only when performing the pgmdr on simulated data with two functional (and no non-functional!) snps (panel d), the pp-plot looks acceptable, although rather conservative as well. second, we considered the simulated null data under the null hypothesis of no epistasis, in the presence of main effects. if under this null a two-locus model is identified (that is, a two-locus model is assessed significant), then a type i error has been made with regard to the null hypothesis of no interaction, and the significant result may be largely driven by main effects. type i error rates for a variety of scenarios are given in table . the analyses correcting for main effects have reasonable type i error rates, although also they tend to be rather conservative. this holds for both models m and m . the analyses not correcting for main effects have the tendency to give rise to an increased number of false positives. this increase is sometimes dramatic, going even up to . the estimated type i error rates point towards the importance of using analysis techniques that are able to appropriately account for important lower order effects. the observation that type i error rates increase with increasing values of g , is to be expected. indeed, when g increases, also the main effects variance increases. moreover, as the minor allele frequency p of the functional snps increases, type i error rates increase for model m but decrease for model m . this can be explained by the relative importance of the main effects with varying p for the different models (table ). note that for model m and p~ : no results are stated because this situation corresponds to a pure epistasis scenario. figure s (supporting information) gives a graphical illustration of the liberal type i error rates. panel d shows results for the analysis not correcting for main effects on data generated under the null of no epistasis for model m and the extreme g ~ : . we observe that the probability-probability plot in this setting is perfectly horizontal at height . this extreme case corresponds to the fact that the type i error rate is for this situation. third, we considered simulated data under the alternative hypothesis of epistasis (possibly in the presence of main effects). for every simulation setting, empirical power was defined as the number of times the correct pair snp ,snp ð Þ was selected and declared significant, divided by the number of simulated data sets. for pgmdr, it happened that two or even three models were reported by the software. in this event, the reported models had exactly the same cross-validation consistency, and we selected the model with the lowest permutation p-value. in the rare event that also the reported permutation p-values were tied, we made a random choice, except when the true functional model was among the reported results. in the latter case, we always selected the true functional model among the tied results. table gives an overview of the simulation results under a variety of alternative hypotheses. figure displays our findings graphically for h ~ : . in the supporting information, we give additional power graphs for h ~ : and h ~ : , for model m (figure s ) and model m (figure s ), but these do not fundamentally differ from those for h ~ : . in general, fam-mdr has systematically higher power than pgmdr, even with appropriate correction for multiple testing. note that fam-mdr* may well be the most powerful approach but, like pgmdr, it does not appropriately handle multiple testing problems. there are a few exceptions to these general observations. however, in these exceptional cases, the power gain of pgmdr is always very small, and the apparent better performance of pgmdr can just as well be due to sampling variability. indeed, the standard error of a binomial proportion is about . for simulated datasets. the results of table show that power estimates increase with increasing two-locus heritability g . our simulation data does not show a systematic trend with increasing total heritability h . when correcting for main effects, table . type i error rates under the general null hypothesis of no association. corr. no c. p h f f* pg f f* pg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . results are based on simulated datasets for each setting for fam-mdr and fam-mdr* and datasets for pgmdr. as an aid to interpretation the type i error rates § : are indicated in bold. abbreviations: corr. = with main effects correction, no c. = without main effects correction, f = fam-mdr, f* = fam-mdr*, pg = pgmdr. doi: . /journal.pone. .t fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e the power decreases with minor allele frequency p for model m . the reverse is observed for model m . this can be explained by the proportion s epi . s loci of the epistatic variance to the total two-locus variance, varying with p for the two models in a different way, as given in table . for example, for model m and p~ : , in which case the ratio s epi . s loci is only . , the power is very low. for the analysis without correction for main effects, power is less dependent on p and high power levels are probably indicative for excessive type i error rates due to lower order effects signaling through to the higher order models. correcting for main effects generally gives rise to lower power estimates than not correcting for main effects (table and figure ). there are two reasons for this. first, the epistatic variance s epi (targeted by an analysis with correction for main effects) is generally smaller than the total two-locus variance s loci (which is the focus of an analysis without correction for main effects). consequently, larger sample figure . probability-probability plots based on replicates under the complete null hypothesis of no association. data are generated with no main effects and no two-way interaction effects, with p~ : and h ~ : . analyses are performed without correction for main effects. the first three panels show results of fam-mdr (a), fam-mdr* (b) and pgmdr (c) for the usual situation study considering snps. the final panel (d) shows pgmdr results when only snps are considered. the straight lines indicate the theoretical probability-probability curve (light blue) and the % significance level (dark blue). doi: . /journal.pone. .g fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e sizes are needed to enable epistasis detection. second, there is some efficiency loss because every main effect corrected for contributes two degrees of freedom (as main effects were coded as co-dominant effects). that there is a price to pay for estimating additional parameters can most clearly be deduced from the results for model m and p~ : (pure epistasis), for which we still observe the reduction in power for the analysis with main effects corrections over an uncorrected analysis. another example is that for model m with p~ : the analyses without main effects corrections g ~ : have higher power than the corrected analyses for g ~ : , although in the first case s loci~ : and in the second case s epi~ : . amish family diabetes study table shows the results of epistasis screening with fam- mdr and pgmdr on the split pedigree data, and of fam- mdr on the multi-generational pedigree data, both with and without correction for co-dominant main effects of rs in hnf a, rs and rs in casq and rs in adipor . without correction for main effects, fam-mdr on the cut-pedigree data finds a significant two-locus model involving rs in casq and rs in apm . when performing a main effects correction, the same best interaction model is identified. however, the interaction is significant only at the % significance level, suggesting that the two-locus model is to some extent driven by the main effect of rs . pgmdr without main effects correction does not lead to a significant interaction. with correction for main effects, pgmdr reports a different near-significant two-locus model involving rs in casq and rs in hnf a. fam- mdr on the original data, with or without correction for main effects, does not yield any significant two-locus models. possible explanations for these contradicting results are reviewed in the discussion section. discussion the objectives of this paper were to introduce a new family- based and flexible epistasis detection analysis method, fam- mdr, which is based on multifactor dimensionality reduction of multi-locus genotypes, and to compare it to the current state-of-the art mdr methodology for families, pgmdr. although principles of the initial mdr approach are adopted in fam-mdr, there are some clear differences. these include an alternative way to identify risk categories associated with multi-locus genotypes, the flexibility to use any outcome type, the possibility to correct for lower order effects, covariates or confounding factors, the possibility to assess significance of multiple higher-order interaction models. since model selection is not based on evaluating prediction accuracy but on testing associations, fam-mdr does not involve computa- tionally intensive cross-validation steps. fam-mdr consists of two parts. in part i, residuals are derived from a polygenic model, removing additive polygenic effects and possibly important lower-order effects or confounding factors. these residuals are subsequently considered as new traits for the second part of fam-mdr. in part ii, the familial correlation-free residuals are submitted to the mb-mdr algorithm and either the best model (considered in this manuscript), or multiple epistasis models are checked for their significance. in contrary to first implementations of the mb-mdr algorithm, no simulation-based null distributions are derived to assess significance, but a permutation-based strategy is adopted. under the assumption of familial correlation-free traits in fam-mdr part ii, permutation- based p-values for the best model can easily be derived by randomly permuting the traits. this is in contrast to pgmdr’s implementation of a permutation strategy in that for pgdmr families are considered as the permutation units. the method of removing familial-correlation structure is not new. the grammar approach of aulchenko et al. [ ] and amin et al. table . type i error rates of fam-mdr under the null hypothesis of no epistasis. m m p = . p = . p = . p = . p = . g h corr. no c. corr. no c. corr. no c. corr. no c. corr. no c. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . results are based on simulated datasets for each setting. as an aid to interpretation the type i error rates § : are indicated in bold. abbreviations: corr. = with main effects correction, no c. = without main effects correction. doi: . /journal.pone. .t fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e figure . power of fam-mdr and pgmdr based on replicates, with h ~ : . the different panels show results for m (a) and m (b). abbreviations: corr. = with main effects correction, no corr. = without main effects correction. doi: . /journal.pone. .g fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e table . power of fam-mdr, fam-mdr* and pgmdr. m m corr. no c. corr. no c. p g h f f* pg f f* pg f f* pg f f* pg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . results are based on simulated datasets for each setting. as an aid to interpretation the powers § : are indicated in bold. abbreviations: corr. = with main effects correction, no c. = without main effects correction , f = fam-mdr, f* = fam-mdr*, pg = pgmdr. doi: . /journal.pone. .t fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e [ ] also used this idea in the context of rapid genomewide main effects analysis. for multivariate traits and unrelated individuals, similar principles of first removing trait correlations and then submitting derived residuals to mb-mdr can be adopted. this is work in progress and is particularly useful when measurements over time are available. exploiting the time-relatedness of phenotypic measurements may in part compensate for the large sample sizes needed to detect epistasis in genomic studies. the current implementation of fam-mdr is not scalable to gwas. an efficient c++ implementation and a code version for parallel analyses are on their way. first simulations indicate that these enhancements will make gwas feasible. for now, when large-scale genomic screenings are performed with thousands of markers, since fam-mdr part i is preparatory for subsequent association analysis, fam-mdr can include a pre-selection step of good candidates of markers for epistasis analysis. these candidates may be selected on the basis of information theoretic measures [ , ] using information about the trait under investigation; or on the basis of evidence from other data, i.e. using external and independent information [ ], e.g. omics analyses. depending on the strategy, an additional correction for data snooping in the pre- screening step may be required to control type i error rates. also, a part iii can be added to a fam-mdr analysis to interpret the identified epistasis models. this is an important step of the analysis and may or may not involve deriving good estimates of the significant effects. special care needs to be taken when carrying out this step, in order not to be the victim of the so-called ‘‘winner’s curse’’ [ , ]. currently, mb-mdr and fam-mdr are based on wald statistics, whereas gmdr and pgmdr make use of score statistics. first, the score test is computationally more advantageous because it only needs parameter estimates under the null whereas the wald test needs parameter estimates under the alternative and the likelihood ratio test needs both. second, even though the three tests are asymptotically equivalent, the score test is the most powerful of the three when the true parameter is close to the null value. in line with the first release of an mb-mdr r package for unrelated individuals, wald statistics were implemented in the first version of the fam-mdr software. in the future, score statistics as well as robust non-parametric statistics will be offered as additional options in fam-mdr. one of the major results that our simulation study highlights is that pgmdr is too liberal in identifying epistasis models. this is due to the inadequate correction for multiple testing, implemented in the pgdmr software to date (figure ; panel c). in contrast, fam-mdr correctly deals with multiple testing and consequently leads to appropriate type i error rates (figure ; panel a). in effect, fam-mdr is rather conservative, which is a property inherited from the grammar approach it is built on. indeed, while first removing polygenic effects (fam-mdr part i), an over- correction may take place, resulting in power loss and conservatism to identify remaining genetic association signals. improvements to fam-mdr that can remove this artifact are on the way. a second result is that, generally speaking, fam-mdr has optimal power over pgmdr in virtual all considered simulation scenarios. in addition, we have indicated that occasional better achieved performance of pgmdr in terms of power is probably attributable to sampling variability. also note that when computing the pgmdr power estimates in our simulation study, in case of a tie we gave advantage to the model with the functional snp pair. a third important result is the influence of correcting for lower- order effects when searching for significant epistatic interactions. as was also pointed out by calle et al. [ ], mdr-like analysis that does not account for important marginal effects is prone to report false higher-order interactions, containing the significant lower-order effects not accounted for. although pgmdr accommodates covariate adjustment, more work is needed to enhance flexible implementation. fam-mdr code is currently available as an r-script, in which covariate adjustments are easily incorporated in the model statement of the polygenic function. more work is needed though to develop a genuine screening strategy to search for optimal models, starting from important main effects and ending with higher-order interaction models beyond the two-way interactions considered in this work, with the maximum order pre-defined by the user. this is future research, since our simulations have shown that it is of utmost importance to adjust for previous significant findings when moving to the search space of interactions of the next order. since the way lower-order interactions are accounted for is part of a parametric paradigm, the coding of these effects needs careful reflection. when lower- order effects are important, a correction is warranted. when lower-order effects are not important or not adequately coded, over-adjusting for lower-order effects may lead to there being virtually no variation left with which to identify higher-order interactions. hence, in reality the balance between necessary corrections for important main effects and avoiding over- correction needs to be considered to optimize the performance of any epistasis detection method. the current fam-mdr implementation is only valid under the assumption of no population structure, and our simulations assume a homogeneous population. by using both between- and within-family association – in contrast to pgmdr that uses only within-family association – fam-mdr gains power but this comes at the price of sacrificing the built-in protection against spurious results if population structure is present. a possible solution to this problem lies in the use of genomic control [ ]. although pgmdr is a flexible tool to handle binary or continuous outcome types, and accommodates covariate adjust- ment, our application to real-life data has revealed some important shortcomings that impact the power of a study. these include a rather inefficient use of available information and the inability to table . epistasis analyses of amish family diabetes study data. corr. no c. model p-value model p-value orig. f (f*) rs in casq ; rs in apm . ( . ) rs in casq ; rs in apm . ( . ) split f (f*) rs in casq ; rs in apm . ( . ) rs in casq ; rs in apm . (, . ) split pg rs in casq ; rs in hnf a . rs in adipor ; rs in hnf a . main effects corrections adjust the analyses for rs in hnf a, rs and rs in casq , and rs in adipor . abbreviations: corr. = with main effects correction, no c. = without main effects correction, orig. = original data, split = split pedigree, f = fam-mdr, f* = fam-mdr*, pg = pgmdr. doi: . /journal.pone. .t fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e analyze complex and extended pedigrees with the present pgmdr implementation. regarding missing genotypes, pgmdr discards families entirely when data on one snp are missing. without minimizing the need to also improve fam-mdr’s handling of missing data (whether at the genotype or phenotype level), not being able to account for the full complexity of a pedigree is certainly a drawback of pgmdr. we believe that methods that can accommodate mixed study designs will become more and more important due to the increasing practice of combining data from different groups in consortia collaborations. fam-mdr flexibly deals with both unrelated and related individuals in the same analysis whereas pgmdr excludes unrelated individuals, hereby reducing the power of the association analysis. multi-generational pedigrees may provide more information on inheritance patterns observed on genotype data, which improves the quality of family-based tests of association. due to limitations of the pgmdr software, only fam-mdr analysis was applied to the extended pedigree data as such. with (or without) fam-mdr correction for main effects of rs in hnf a, rs and rs in casq , and rs in adipor , no evidence for a significant interaction (or two-locus model) was found (table ). weak interactions between variations at these loci in contributing to the log (gauc) phenotype may still exist, but if so, fam- mdr was not able to identify them. in this context it is important to note that fam-mdr – like grammar – could be rather conservative, especially for larger extended pedigrees [ ]. on the other hand, the (nearly) significant findings for the simplified pedigree (table ) may potentially be false positive results driven by both the artificial increase in sample size and by not appropriately accounting for family structure. indeed, mcardle et al. [ ] showed that the type i error of detecting snp main effects is elevated when family structure is ignored, and more dramatically with increasing trait heritability, which may naturally extend to (interactive) two-locus models as well. lack of appropriate accounting for multiple testing (pgmdr and fam- mdr*) may also increase the likelihood of observing spurious associations. this is clearly seen for fam-mdr* and to some extent also for pgmdr (table ). improving the ability to detect gene-gene interactions in family studies of complex disease may prove critical in identifying the underlying sources of observed heritability. while it is apparent that type diabetes mellitus (t dm) is polygenic, the genetic sources of the observed heritability have yet to be completely identified [ ], and may include still unobserved gene-gene interactions. detecting interactions between genes associated with t dm has thus far been very challenging, due to a paucity of powerful statistical methods and study datasets with adequate sample sizes [ ]. several studies that have examined interactions between single variants in different genes have shown mixed evidence of two-way interactions in t dm and t dm-related traits like obesity and insulin resistance. using an approach which conditioned on linkage in one region to identify evidence of linkage elsewhere, a linkage study of t dm in mexican americans from starr county, texas, identified the interaction of genes on chromosomes (capn (calpain ), then niddm ) and (near cyp (cytochrome p , family , or aromatase)) in contributiing to t dm susceptibility [ ]. associ- ation studies investigating interactions between variants of the beta- adrenergic receptor (adrb ) and uncoupling protein (ucp ) genes observed in weak [ ] to no [ ] effects on weight gain and insulin resistance in finnish and danish populations, respectively. a study of type ii iodothyronine deiodinase (dio ) and adrb polymorphisms showed a synergistic effect on an increased bmi, suggesting an interaction between these two common gene variants [ ], while a study of intestinal fatty acid binding protein (fabp ) and adrb showed no interaction on levels of fasting plasma glucose or measures of insulin resistance [ ]. in a study of mexican-american families participating in the population-based san antonio family heart study [ ], the combined presence of common variants of peroxisome proliferator-activated receptor gamma (pparc) and adrb are correlated with significantly higher bmi, insulin, and leptin levels than the presence of the pparc variants alone. yet another study [ ] examined two-locus interactions among loci in t dm candidate genes in the risk of t dm, and found a significant interaction between variants in the uncoupling protein (ucp ) and pparc genes. identification of novel interactions and further confirmation of observed interactions may be critical in characterizing the genetic risk factors for t dm and many other complex disease that remain among the unidentified components of the heritability of these diseases, and may have practical application in the identification of individuals who may belong to groups at high risk of disease who can benefit from preventive care. in conclusion, fam-mdr – unlike pgmdr – is able to handle complex and large pedigrees with additional unrelated individuals. in fact, fam-mdr analysis on split pedigree data should not be trusted because it might lead to overly optimistic results. on the other hand, as pedigree size increases, the inherent conservative nature of fam-mdr could become more pronounced. finally, pgmdr results – and in fact also gmdr results – are too liberal as no correction for multiple testing is carried out. supporting information figure s probability-probability plots for fam-mdr analyses under the null hypotheses of no association and no epistasis. the situation considered is p = . and h = . . analyses are performed both with and without correction for main effects. results are based on replicates. panels a and b show results for data generated under the null of no association, whereas panels c and d consider data generated under the null hypothesis of no epistasis, for model m and with g = . . panels a and c show results for analysis with correction for main effects, panels b and d without. found at: doi: . /journal.pone. .s ( . mb tif) figure s additional power results for model m , based on replicates. panels a and b show results for h = . and h = . respectively. abbreviations: corr. = with main effects correction, no corr. = without main effects correction. found at: doi: . /journal.pone. .s ( . mb tif) figure s additional power results for model m , based on replicates. panels a and b show results for h = . and h = . respectively. abbreviations: corr. = with main effects correction, no corr. = without main effects correction. found at: doi: . /journal.pone. .s ( . mb tif) acknowledgments interesting discussions with yurii aulchenko and florence demenais are gratefully acknowledged. author contributions conceived and designed the experiments: mf hs. performed the experiments: mf hs. analyzed the data: tc acn tle kvs. contributed reagents/materials/analysis tools: tc vu vdw mf jmmj hs mlc kvs. wrote the paper: tc acn jmmj mlc mdr tle kvs. developed the method: tc ldl vdw jmmj kvs. designed the simulation study: tc kvs. performed the simulation study: tc kvs. fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e references . frazer ka, ballinger dg, cox dr, hinds da, stuve ll, et al. 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( ) multifactor- dimensionality reduction shows a two-locus interaction associated with type diabetes mellitus. diabetologia : – . fam-mdr epistasis screening plos one | www.plosone.org april | volume | issue | e the “problem” of minority education in an international perspective | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /s - ( ) - corpus id: the “problem” of minority education in an international perspective @article{letendre theo, title={the “problem” of minority education in an international perspective}, author={g. letendre}, journal={international journal of educational research}, year={ }, volume={ }, pages={ - } } 	g. letendre 	published 	sociology 	international journal of educational research the chapters in this issue originated in the comparative and international education seminar given each year at the pennsylvania state university and re#ect nearly two years of ongoing work analyzing minority educational policy from a crossnational perspective. there are three goals of this special issue. the "rst is to provide readers with case studies of the impact of national educational policies on minorities in the us and in other nations. the second is to analyze the degree to which cross… expand view via publisher save to library create alert cite launch research feed share this paper citationshighly influential citations background citations view all citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency factors affecting performance of the ethnic minority students in secondary schools 	m. a. kalam 	sociology 	 	pdf 	view excerpt, cites background save alert research feed managing school behavior: a qualitative 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of political socialization 	kristina brezicha 	sociology 	 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency the world educational revolution, - . 	j. meyer 	economics 	 	 	view excerpt, references methods save alert research feed explaining the origins and expansion of mass education 	j. boli, f. ramírez, j. meyer 	sociology 	comparative education review 	 	 	pdf save alert research feed the political construction of mass schooling: european origins and worldwide institutionalization 	f. ramírez, j. boli 	sociology 	 	 	view excerpt, references background save alert research feed the problem of japan: qualitative studies and international educational comparisons 	g. letendre 	sociology 	 	 	view excerpt, references background save alert research feed exploring and explaining the variability: cross-national perspectives on the school performance of minority students. 	m. a. gibson 	sociology 	 	 	view excerpt, references background save alert research feed world expansion of mass education, - 	j. meyer 	sociology 	 	 	pdf save alert research feed schooling immigrants in france in the s: success or failure of the republican model of integration? 	a. v. zanten 	sociology 	 	 	view excerpt, references background save alert research feed the ideology of childhood and the state: rules distinguishing children in national constitutions, - 	john boli-bennett, j. w. meyer 	sociology 	 	 	view excerpt, references background save alert research feed education in tokugawa japan 	r. dore 	history, sociology 	 	 	view excerpt, references background save alert research feed what do anthropologists have to say about dropouts? : the first centennial conference on children at risk, school of education, stanford university 	h. t. treuba, g. spindler, l. spindler 	history 	 	 	view excerpts, references background save alert research feed ... ... related papers 	abstract 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue use of dental services by immigrant canadians jcda • www.cda-adc.ca/jcda • march , vol. , no. • r epresenting more than . % of the canadian population in and arriving from many countries, foreign- born residents constitute a growing segment of canada’s population. recent literature has firmly established that the health and health- seeking behaviour of canada’s foreign-born residents, including refugees and immigrants, diverge from those of the native-born popula- tion. for example, the “healthy immigrant effect,” whereby the health status of immi- grants is good at the time of arrival but subsequently declines to a level below that of the native-born population, is well docu- mented. – recent immigrants are more likely to rank their health higher than canadian- born people and are less likely to report chronic conditions or disability; these patterns are attributed to the fact that those in good health are more likely to emigrate from their home country and also to the screening process at the time of entry, which may dis- qualify those with serious medical conditions. although it could be argued that the observed decline in health after arrival reflects less con- tact with the health care system than is the case for the native-born population, there is con- flicting evidence about immigrants’ limited use of the health care system. immigrants as a group are typically considered to underuse the health care system, perhaps because of better health status at the time of arrival. however, barriers to care, lack of knowledge of the health care system and recent health care restructuring in canada may discourage health care utilization by immigrants. surprisingly little is known about the dental health of and use of dental services by canada’s foreign-born population. for instance, a recent health canada report on immigrant health was silent on the topic of dental health needs of the immigrant population. yet immigrants dr. newbold email: newbold@mcmaster.ca contact author use of dental services by immigrant canadians k. bruce newbold, phd; amish patel, bsc abstract although the health status and health behaviour of foreign-born residents of canada have been well documented, little is known about their use of dental services. the authors, hypothesizing that foreign-born people would have lower utilization of dental care services than native-born canadians, undertook this study to identify the factors asso- ciated with dental visits by canadians aged years and older and to compare the use of dental services by foreign-born and native-born populations. according to data derived from statistics canada’s – national population health survey, foreign-born people were somewhat more likely than native-born canadians to have visited a dentist within the previous year. higher levels of education, greater income adequacy, and the presence of dental insurance were associated with greater use of dental services, whereas increasing age was associated with lower use. although immigrants reported greater use of dental services than native-born canadians, a variety of barriers to care may be present in this population. mesh key words: canada/epidemiology; dental health services; emigration and immigration/statistics & numerical data; insurance, dental © j can dent assoc ; ( ): this article has been peer reviewed. professional i s s u e s mailto:newbold@mcmaster.ca a jcda • www.cda-adc.ca/jcda • march , vol. , no. • may face greater needs than the native-born population with respect to their dental health because of lack of insur- ance, lower incomes, limited awareness of both facilities and the need for oral care, or other barriers to good dental health such as language. regardless, the general perception is that members of the immigrant canadian population visit the dentist less often, even though they have a greater need for dental services. the objective of this study was to compare the use of dental services by the foreign-born and native-born populations of canada and to determine factors associated with dental visits. methods data for this study were derived from statistic canada’s national population health survey (nphs) cycle ( – ). the target population of the nphs consists of household residents age years and older in all provinces and territories, except those living on reserves, on canadian forces bases and in some remote places. most interviews ( %) were conducted by tele- phone with computer-assisted interviewing techniques; the remainder of the interviews were conducted in person. the analysis compensated for nonresponding households (e.g., because of language barriers) by adjusting the weights assigned to reporting households. the nphs col- lects in-depth information on a number of attributes, including sociodemographic and socioeconomic charac- teristics, lifestyle and health care utilization for each member of the household. it also includes questions about use of dental services. the current paper focuses on use of dental services within the year preceding the survey and reasons for the visit(s). given the complex sampling design of the nphs, the weights developed by statistics canada were used in this analysis. both descriptive and multivariate statistics were used to evaluate patterns of use and differences between the native-born and foreign-born populations. table illustrates differences in the population profiles of the groups. because the age profile of the immigrant population was different from that of the native-born population, it was expected that the groups would have different utilization patterns, all other things being equal. standardized dental use ratios (sdurs), which are similar to standardized mortality ratios, were calculated for the use of dental services according to selected personal attributes, which allowed age and sex standardiza- tion. the sdur was calculated as the ratio of the observed proportion of immigrants reporting use of dental services to the expected proportion of individuals reporting use in the total canadian population, if immigrants experienced the same age- and sex-specific rates of use as the native- born population. a ratio of less than . indicates that the foreign-born population fares better than the native-born population, and vice versa. eight age–sex ( × ) groups were used for standardization. results although previous studies had suggested that foreign- born people would be less likely than native-born canadians to have used dental services, the nphs data analyzed here indicated similar rates of use in the past year ( . % versus . %; p = . ) (table ). relative to the native-born population, a smaller proportion of the foreign-born participants were under years of age and a ––– newbold ––– table characteristics of the foreign-born and native-born populations of canada, years of age and older, – % of population variable foreign-born native-born age (years) – . . – . . – . . ≥ . . sex male . . female . . education some high school . . without graduation high school graduate . . bachelor’s degree or higher . . income adequacy low . . lower middle . . middle . . upper middle . . high . . duration of residence in canada (years) – . na – . na ≥ . na region of origin u.s., europe or australia . na asia . na other . na overall % of all respondents . . n , , , , source: derived from the – nphs. na = not applicable. jcda • www.cda-adc.ca/jcda • march , vol. , no. • b larger proportion were older than years; the slight dif- ference in use of dental services may have been partly due to this difference in age distributions. greater use of dental services by foreign-born participants was observed among those aged and over; among those with middle, upper middle or high income adequacy; and among those with less than a high school education. standardized by age and sex, the sdurs indicated somewhat greater use of dental services among foreign-born respondents as a whole and among foreign-born respondents of male sex, with lower income adequacy and with some high school education. although the type of dental insurance (i.e., private or public) could not be determined, native-born canadians were more likely than immigrants to have dental insurance ( . % versus . %). this difference remained when the data were standardized for age and sex. significant differences in the use of dental services were also noted within the immigrant population. for example, only . % of immigrants who had resided in the country for less than years had used a dentist in the previous year. in contrast, more than % of immigrants who had resided in the country for or more years had visited a dentist. in addition, the region of origin appeared to have a significant impact upon use, with immigrants from asia reporting the lowest use ( . %) (table ). nearly equal proportions of native-born and foreign- born participants reported visiting a dentist because services were covered by dental insurance (about %) (table ). immigrants were more likely than native-born canadians to consult a dentist for treatment reasons, such as care of the teeth, gums or dentures ( . % versus . %). conversely, native-born canadians were more likely to report visits for preventive care, giving such reasons as “to check that everything is okay” ( . % versus . % for native-born and immigrants, respectively), “for good health” ( . % versus . %) and braces ( . % versus . %). logistic regression was used to determine the proba- bility of using dental services with adjustment for other variables such as age, sex and the presence of dental insur- ance (table ). two models were created to explore the determinants of dental use. the first was a pooled model that included both the native-born and foreign-born populations, which enabled determination of whether foreign-born participants were more (or less) likely to have used a dentist than the native-born population, with adjustment for other effects. the second model evaluated use of dental services by immigrants only. the model results are reported as odds ratios (ors), which allow clear interpretation of the effect of a variable. an or greater than . indicates an increased likelihood of use of dental services by participants in that category, and an or less than . indicates the reverse. for example, females in the ––– use of dental services by immigrants ––– table dental use by foreign-born and native-born canadians years of age and older, – % of respondents foreign- native- p variable born born value sdur dental visits % using dental . . . . services n , , age (years) – . . . na – . . . na – . . < . na ≥ . . . na n , , sex male . . . . female . . . . n , , education some high school . . . . without graduation high school graduate . . . . bachelor’s degree . . . . or higher n , , income adequacy low . . . . lower middle . . . . middle . . . . upper middle . . . . high . . . . n , , dental insurance % with insurance . . < . . n , , duration of residence in canada (years) – . na na – . na na ≥ . na na n , na region of origin u.s., europe, australia . na na asia . na na other . na na n , na source: derived from the – nphs. n = unweighted sample size, sdur = standardized dental use ratio, na = not applicable. c jcda • www.cda-adc.ca/jcda • march , vol. , no. • pooled model were . times more likely to have visited the dentist than males, all other things being equal. the pooled model demonstrated that immigrants were significantly more likely than native-born canadians to have visited a dentist (or = . ). in general, individuals with greater income adequacy, those who were better educated, were married or were younger, and those who had dental insurance were more likely to have visited a dentist. among foreign-born participants, recent arrivals (resident in canada for less than years) were less likely to have used a dentist. immigrants from asia were less likely to have used a dentist, whereas those with european origins were more likely to have used a dentist. discussion contrary to expectations, foreign-born residents of canada were significantly more likely than native-born canadians to have visited a dentist in the year preceding the survey, a finding that was supported by multivariate analyses adjusting for age and sex differences in the populations. yet a larger proportion of the native-born population reported having dental insurance. the foreign- born population and the pooled population (foreign- and native-born) were, respectively, . and . times more likely to have visited a dentist if they had insurance than if they did not have insurance. overall, the factors associated with use of dental ser- vices and identified in the logistic model were largely as expected. dental insurance, for example, significantly increased the likelihood of use. in terms of sociodemo- graphic effects, there was a negative correlation between age and use, with greater use among younger people (aged – years) and generally less use with increasing age. among the young, the greater likelihood of use probably reflects social pressure, the presence of parental dental insurance and/or dental care within the school system. conversely, lack of dental insurance coverage probably explains declining use with older age. female respondents and whites were also more likely to have used a dentist. surprisingly, individuals who spoke a language other than english or french were more likely to have used a dentist within the past year, which suggests that language may not be a barrier to dental care. socioeconomic variables also influenced use of dental services. income adequacy, which is defined by statistics canada and is based upon both household size and income level, was positively correlated with use (i.e., increasing likelihood of use with increasing income ade- quacy). education had a similar gradient, and those who had a bachelor’s degree or better were more likely to have used a dentist relative to all other educational levels. in both cases, the results probably reflect greater awareness of the need for care, greater ability to access resources, greater likelihood of dental insurance coverage and greater dis- posable income available for dental care. working status (i.e., working versus not working), which potentially reflects access to dental insurance and care through the employer, was not a significant determinant of use and was therefore not included in the reported model. among foreign-born respondents, other factors were significant predictors of use of dental services: duration of residency within canada and region of origin. with regard to duration of residency, recent arrivals (those who had arrived within the years before the survey) were signifi- cantly less likely to have used a dentist. cross-tabulation of results indicated a difference in use of about percentage points relative to those resident for more than years. social acceptability, adaptation, increasing awareness of ––– newbold ––– table reason for dental visits among foreign- and native-born people years of age and older, – % of respondents reason for visit to dentista foreign-born native-born p value check everything is okay . . . covered by insurance . . . prevention . . . for good health . . . care of teeth, gums, dentures . . < . clean, fluoride, maintenance . . . filling or extraction . . . braces . . < . other . . . n (unweighted) , , source: derived from the – nphs. arespondents were allowed to give more than one reason for dental visits. jcda • www.cda-adc.ca/jcda • march , vol. , no. • d dental resources, income and insurance are possible rea- sons for increasing use with increasing duration of resi- dency. indeed, there was a difference in insurance coverage of about percentage points between the newest immi- grant canadians (about %) and immigrants who had lived in canada for the longest period (about %) (table ). with regard to region of origin, immigrants who had arrived from countries outside north america, ––– use of dental services by immigrants ––– table logistic regression for use of dental services by respondents years of age and older, – a pooled foreign-born variable or % ci or % ci intercept . — . — immigration status (reference: native-born) foreign-born . ( . – . ) — — age (reference: ≥ years) – years . ( . – . ) . ( . – . ) – years . ( . – . ) — — – years . ( . – . ) . ( . – . ) education (reference: less than high school graduation) high school or better . ( . – . ) . ( . – . ) bachelor’s degree or better . ( . – . ) . ( . – . ) ethnic background (reference: nonwhite) white . ( . – . ) . ( . – . ) martial status (reference: not married) married . ( . – . ) . ( . – . ) gender (reference: male) female . ( . – . ) . ( . – . ) income adequacy (reference: low income adequacy) middle . ( . – . ) — — upper middle . ( . – . ) . ( . – . ) high . ( . – . ) . ( . – . ) dental insurance (reference: no insurance) insurance . ( . – . ) . ( . – . ) smoking status (reference: smokes daily) smokes occasionally . ( . – . ) — — language (reference: speaks an official language) other language . ( . – . ) . ( . – . ) origin (reference: other) europe — — . ( . – . ) asia — — . ( . – . ) duration of residence (reference: resident for years or more) arrived within past years — — . ( . – . ) n (unweighted) , , likelihood ratio , . , . rho squared . . % concordant . . source: derived from the – nphs. or = odds ratio, ci = confidence interval asuppressed values (indicated by a dash or not shown) were not statistically significant (working status, lower-middle income, smokers) or were not meaningful (origin, duration of residence) in the context of the model. e jcda • www.cda-adc.ca/jcda • march , vol. , no. • europe and australia were more likely to have used a den- tist in the past year than those of other origins (table ). those from asia had the lowest utilization rate ( . %), about percentage points lower than those from the united states, europe and australia. dental insurance was also one of the factors for which european immigrants had a slight advantage over immigrants from other regions (about percentage points higher than asian immi- grants). unmeasured factors, such as fear, language differ- ence, awareness, and transportation problems, may also have resulted in different utilization patterns among immigrants. although only small proportions of all respondents reported problems with visiting a dentist (data not shown), a somewhat larger proportion of for- eign-born respondents than native-born respondents indicated such problems ( . % vs. . %). about equal proportions of immigrant and native- born respondents identified the presence of insurance as a reason for a visit to the dentist, but these groups may differ in terms of other reasons for dental use. for example, data in table suggest that foreign-born respon- dents obtained dental services to address needs for phys- ical oral care (such as taking care of teeth, gums or dentures, cleaning and maintenance, and getting fillings or extractions) more often than native-born canadians. these results may indicate differences in oral health practices between the groups and may also indicate that immigrants have poorer dental health. the larger propor- tion of immigrants seeking physical oral care could also be due to a lack of services in the region of origin or a lack of prior dental care education. ––– newbold ––– table use of dental services and availability of insurance among foreign-born residents of canada years of age and older, by duration of residence and origin, / % of respondents at least one characteristic visit to dentist insurance duration of residence in canada (years) – . . – . . ≥ . . region of birth u.s., europe, australia . . asia . . other . . source: derived from the – nphs. conversely, the native-born population seemed more likely to have visited a dentist for preventive reasons, potentially reflecting greater dental insurance coverage. two self-reported reasons for use of dental services were to ensure that “everything is okay” and to “maintain good dental health.” although general check-ups and mainte- nance may include some physical corrections (similar to services provided to the immigrant group), it would appear that visiting the dentist was less often associated with a physical ailment and more often to ensure oral health. similarly, dental appointments to check braces, which may be viewed as an expensive health option (com- pared with other conditions), is also a preventive oral health measure. conclusions the lack of attention within the literature to immi- grant oral health is hardly surprising. the recent romanow report on health care in canada did not dis- cuss dental health, and oral health remains a relatively low priority within canada, with public funding of dental care low by international standards. federal funding has decreased over time, and funding for oral health at the provincial level is discretionary, although provisions are made within provincial health plans to cover emergency dental care. other oral health programs, including those for children, expectant mothers, people receiving welfare benefits and elderly people, have been cut back or simply do not exist. even the immigrant health literature, which has grown exponentially over recent years, is noticeably silent on this topic, yet immigrants represent a particu- larly vulnerable population in terms of both overall health status and dental health status. with an average of over , new immigrants per year, this group repre- sents a growing segment of canada’s population, and there is a potential that many new arrivals will have poor dental health or will lack the resources to access care. somewhat unexpectedly, the proportion of foreign- born respondents reporting use of dental services was greater than the proportion of native-born respondents after adjustment for age and sex. in addition, foreign- and native-born individuals reported different reasons for dental visits. there remains much room for improvement in the provision of dental care to the immigrant popula- tion. although dental insurance is an important factor in determining use of dental services, duration of residence since arrival, language, access to appropriate dental insur- ance and other factors may limit use and create barriers to care. in particular, immigrants who speak english as a second language, who speak little or no english at all or who are constrained by social and gender roles may be less likely to use a dentist. increasing the ability to obtain insurance may result in a shift toward prevention among immigrants, similar to the trend observed among native- jcda • www.cda-adc.ca/jcda • march , vol. , no. • f born respondents, with new arrivals benefiting the most. increased education and awareness of the need for dental services may also be appropriate. the question, of course, is who will pay for these enhancements. c references . ali j. mental health of canada’s immigrants. supplement to health reports, statistics canada; . . lillie-blanton m, rushing oe, ruiz s. key facts: race, ethnicity and medical care. the henry j. kaiser family foundation, . . dunn jr, dyck i. social determinants of health in canada’s immigrant population: results from the national population health survey. soc sci med ; ( ): – . . frisbie wp, youngtae y, hummer ra. immigration and the health of asian and pacific islander adults in the united states. am j epidemiol ; ( ): – . . gee em, kobayashi km, prus sg. examining the “healthy immigrant effect” in later life: findings from the canadian community health survey. . newbold kb, danforth j. health status and canada’s immigrant population. soc sci med ; ( ): – . . perez ce. health status and health behavior among immigrants. supplement to health reports, statistics canada; . p. – . . chen j, wilkens r, ng e. life expectancy of canada’s immigrants from to . health reports ; ( ): – (engl); – (fre). . laroche m. health status and health services utilization of canada’s immigrant and non-immigrant populations. can public policy ; ( ): – . . health canada. canadian research on immigration and health. ministry of health: ottawa, . . statistics canada. national population health survey (nphs) cycle ( – ). statistics canada: ottawa; . . romanow r. building on values: the future of health care in canada. final report, commission on the future of health care in canada, november . . birch s, anderson r. financing and delivering oral health care: what can we learn from other jurisdictions? j can dent assoc ; ( ): . available from: url: www.cda-adc.ca/jcda/vol- /issue- / .pdf. . lamotey j. determining family dental health. paper presented at access and care: towards a national oral health strategy. may – , . toronto. available from: url: http://individual.utoronto.ca/accessandcare/ (accessed december , ). ––– use of dental services by immigrants ––– acknowledgement: research for this article was supported by grant from the canadian institutes of heath research. dr. newbold is an associate professor of geography at mcmaster university in hamilton, ontario. mr. patel was an undergraduate student in kinesiology at mcmaster university when the article was written. correspondence to: dr. k. bruce newbold, school of geography and earth sciences, mcmaster university, main st. w., hamilton, on l s k . the authors have no declared financial interests. the authors humoral immunity to stress proteins and periodontal disease j periodontol • october humoral immunity to stress proteins and periodontal disease dennis e. lopatin,* charles e. shelburne,† neal van poperin,* charles j. kowalski,* and robert a. bagramian‡ background: there is evidence that microbial heat shock (stress) proteins (hsp) are immunodominant antigens of many microorgan- isms. immunity to these proteins has been shown in non-oral infections to contribute to protection. this study was undertaken to assess the relationship(s) between immunity to human and microbial heat shock proteins, periodontal disease status, and colonization by periodontal disease-associated microorganisms. methods: subgingival plaque and blood samples obtained from patients during an earlier clinical study were examined for the presence of specific periodontal disease-associated microorganisms and anti- bodies to selected human and microbial heat shock proteins (hsp , hsp , dnak, and groel). particle concentration immunofluorescence assay (pcfia) was used to detect anti-hsp antibodies and slot immunoblot assay (sib) was used to detect subgingival plaque species. regression models were used to examine the contribution of age, gender, gingival index, probing depth, attachment loss, calculus index, plaque index, and microbial colonization to the anti-hsp anti- body concentrations. results: our studies demonstrated that, when evaluated by anova, patients with higher anti-hsp (hsp , dnak, and groel) antibody con- centrations tended to have significantly (p ≤ . ) healthier periodontal tissues. this was most obvious when the relationship between mean probing depths and antibody concentrations were studied. for hsp antibodies, variables (probing depth and p. gingivalis concentration) were found to have significant contributions (r = . , p < . ). the equation derived from the regression model was y = − *pd + *pg. this confirmed the inverse relationship with probing depth and the positive relationship with colonization by p. gingivalis. attempts to model the other stress protein antibodies were not successful. conclusions: we believe that the present observations reflect the presence of protective anti-hsp antibodies, rather than simply the pres- ence of the microorganism in the gingival sulcus. the clinical signifi- cance of these observations lies in the potential of identifying patients who are at risk for developing periodontal disease based on their in- ability to mount an immune response to specific hsp or hsp epitopes, as well as the development of vaccines based on hsp epitopes. j periodontol ; : - . key words periodontal diseases/microbiology; protein, microbial heat-shock; protein, stress; antibody formation; immunity; humoral. a ssociations between host response and severity or progression of periodontal disease have classically been stud- ied in the context of immunity directed against specific micro- organisms. this has been true even though dental plaque repre- sents a highly complex array of microorganisms, each having both unique and common antigens that contribute to its overall antigenicity and immunogenicity. studying the potential role of host immunity in the context of such a complex anti- gen mixture is difficult and one is never certain whether differences in host response represent changes in magnitude of the response to a specific antigen or in qualitative differences in the antigenic array. recently, there has been a greater focus among investigators upon specific purified bacterial antigens such as lipopolysaccharides, lipote- ichoic acids, fimbrial proteins, and other cell membrane-associated antigens. - heat shock proteins (hsp) have been extensively evaluated in numerous medically significant dis- eases such as candidiasis, lyme disease, chlamydia, and tubercu- losis. we questioned whether this family of evolutionarily-conserved proteins had a significant role in periodontal disease. these pro- teins, defined in part by the mol- e c u l a r s i z e o f t h e i r p r o t o t y p e * department of biologic and materials sciences, school of dentistry, university of michigan, ann arbor, mi. † m, biomaterials technology center, st. paul, mn. ‡ department of periodontics, prevention, and geriatrics, university of michigan, ann arbor, mi. members, have significant roles in normal cellular function as molecular chaperones , and defense against environmental stresses. they have been shown to have an important role in inflammatory mechanisms, autoimmune diseases, - and atherosclerosis. in addition, these proteins have been shown to be the immunodominant antigens of many microorganisms including periodontal disease- associated microorganisms such as porphyromonas gingivalis - and actinobacillus actinomycetem- comitans. - because these proteins have been so highly conserved in evolution we felt that they might be used to assess “basal” or “generic” immunity in a variety of disease processes and would not necessar- ily be specifically linked to a single microorganism. in the present study, we have employed a panel of purified stress proteins and measured serum igg antibody levels to them using particle concentration fluorescence immunoassay. these data were then compared to clinical measurements of periodontal disease and colonization by microorganisms com- monly associated with periodontal disease. the spec- imens used in this study were derived from an amish and non-amish population examined in an earlier study. , materials and methods study population this study focused on the amish and non-amish liv- ing in rural southeastern michigan. the clinical data and archived specimens collected from a total of amish (n = ) and non-amish (n = ) specimens were used. this represented all of the subjects recruited during the first year of an earlier study. , the results of that study indicated that the amish did not present as a unique population that could be dis- tinguished from the non-amish. this study was per- f o r m e d u s i n g c l i n i c a l f i n d i n g s a n d s p e c i m e n s archived from that study. participation rate for these subjects was approximately %. ages ranged from to years; % ( ) males and % ( ) females. since the non-significant differences found in clinical measurements between the amish and non-amish were attributable to oral hygiene practice, later analyses treated all subjects as a single popula- tion. subsequent regression modeling confirmed this assumption. the oral disease in this population is consistent with age-associated chronic adult peri- odontitis with no evidence of early-onset periodontal diseases. clinical measurements one dentist conducted examinations of all existing teeth with the exception of third molars using the silness and löe plaque index, löe and silness gin- gival index, and modified ramfjord’s periodontal disease index for probing depth and attachment loss measurements. loss of periodontal attachment and pocket depth were measured to the nearest millime- ter using a periodontal probe. measurements were taken from sites of each tooth: disto-buccal, mid- buccal, mesio-buccal, disto-lingual, mid-lingual, and mesio-lingual. blood collection blood samples were collected in ml vacuum tubes without anticoagulant. after clotting, the tubes were centrifuged and the serum frozen at − °c. plaque specimens plaque samples were collected from the most peri- odontally diseased sites based on probing depth and/or inflammation. these samples were then pooled. this approach allowed us to assess the flora representative of the most diseased sites in each sub- ject. supragingival plaque about the most diseased sites was removed with a sterile curet and discarded. a second sterile curet was introduced into the sulcus or periodontal pocket and extended as far apically as possible. the root surface was then sampled with a curet and the adherent plaque on the scaler tip trans- ferred to a screw-capped cryotube containing . ml phosphate buffered saline (pbs; . m sodium phos- phate, . m nacl, ph . ) with . % formaldehyde, edta ( mm), pmsf ( . mm), pepstatin a ( . mm) and leupeptin ( . mg/l). sample vials were stored at − ° until assayed. slot immunoblot assay (sib) the sib was performed as previously described. the samples were ultrasonicated§ ( to seconds; % power) to disrupt aggregates of plaque particles. nitrocellulose sheets‖ were soaked for minutes in tbs ( . m nacl, . mm tris-hcl, . mm tris- base, ph . ) and inserted into the slot blot manifold.¶ standards (pure cultures) or undiluted plaque sam- ples were applied ( µl) to each well of the slot blot manifold which was then evacuated with gentle appli- cation of vacuum. the nitrocellulose membrane was then removed from the manifold for further process- ing. all subsequent incubations were performed on a rocking table# at room temperature. microbial detection and quantitation was performed after first immersing the nitrocellulose membrane in tbs containing . % non-fat dried milk** for to minutes to block unoccupied binding sites on the nitrocellulose membrane. the appropriate antibacter- ial antibody, diluted in tbs-tween (tbs-t; . % tween ) containing . % milk proteins** was humoral immunity to stress proteins volume • number § kontes instruments. ‖ ba- , schleicher & schuell, keene, nh. ¶ minifold ii, scheicher & schuell. # hoefer scientific co., san francisco, ca. ** blotto, kirkegaard & perry laboratories, gaithersburg, md. j periodontol • october lopatin, shelburne, van poperin, kowalski, bagramian applied and allowed to incubate for hour. following three -minute washes in tbs-t, goat anti-rabbit igg conjugated to alkaline phosphatase†† diluted in tbs- t containing . % milk proteins was applied and incubated for hour. after all washes, the bcip-nbt substrate solution‡‡ was applied and color develop- ment allowed to proceed to its maximum. absorbed polyclonal antisera prepared in rabbits specific for p. gingivalis, a. actinomycetemcomitans, t. denticola, and b. forsythus were used in the sib assay. the specificity of these antisera has been previously described. , particle concentration fluorescence immunoassay (pcfia) pcfia was performed as described by shelburne et al. stress proteins used in these studies were lim- ited to those that were commercially available since periodontal pathogen-specific stress proteins were either not available or were available in amounts insuf- ficient for screening purposes. the initial step in the performance of pcfia was the preparation of antigen- coupled latex particles. briefly, µg of the specific stress protein (human hsp , human hsp , e. coli dnak, and e. coli groel) was diluted to ml in . m sodium phosphate (ph . ) and combined with mg of carbodiimide§§ and ml of carboxyl latex particles‖‖ ( . µm, % w/v,) and incubated for to hours at °c with rotation. the coated particles were washed times in . m sodium phosphate and resuspended in ml of pbs + . % sterile milk proteins. twenty µl of the coated particles were dispensed into each well of well filtration plates.‖‖ sera were diluted : in pbs + . % sterile milk proteins, added to wells ( µl/ well) in triplicate, and incubated minutes at room temperature. the particles were then washed and µl of fitc labeled anti-human igg (various vendors) added. after an additional -minute incubation the particles were washed times and the fluorescence of the labeled anti-human igg captured by the solid phase was measured by epifluorimetry and expressed in rela- tive fluorescence units (rfu). controls included pooled human serum to control for inter-plate variability and fitc-labeled anti-human igg in the absence of : dilution of patient serum to control for non-specific binding. preliminary studies indi- cated that a : serum dilution was optimal, resulting in detection of all sample fluorescence at the same gain setting (gain = ) in the linear range of the machine. the relative amounts of fluorescence in each well was used as a semi- quantitative representation of the amount of anti- body present in the sample. statistical analysis the relationships between specific indices of periodontal disease, antibody level, and colo- nization of subgingival dental plaque were assessed by anova. fisher’s method for multiple comparisons was used for pair-wise comparison of the groups. the contribution of these variables to stress protein anti- body concentration, as well as those of age, gender, and amish/non-amish status, was assessed in multi- ple and stepwise regression models.¶¶ results with p ≤ . were considered significant. results clinical measurements a complete description of the clinical status of this population is available in earlier reports. , an overview of the clinical status of the subjects is pro- vided in table . all analyses described below used full mouth means for all clinical measurement (prob- ing depth, attachment loss, and gingival index). colonization and clinical measurements the relationship between colonization and the clinical measurements was determined by anova. as shown in table , when probing depth was examined, incr easing pr obing depth was associated with increased colonization by all species tested. in all cases but a. actinomycetemcomitans, these increases were significant. we observed no statistically signifi- cant relationships when gingival index or attachment loss were evaluated. anti-stress protein serum antibody concentrations and clinical measurements subjects were stratified into groups ( , , and ) based on their mean whole mouth gingival index ( = to < . ; = . to < . ; = > . ). the anti-stress pro- tein antibody concentrations in these groups were then compared by anova. as shown in figure b, a consistent trend is noted. gingival index strata of and were associated with higher antibody concentrations, while scores greater than (significant amounts of gin- †† bio-rad laboratories, hercules, ca. ‡‡ kirkegaard & perry laboratories. §§ sigma chemical co., st. louis, mo. ‖‖ epicon, idexx, westbrook, me. ¶¶ stat-view, sas institute, cary, nc. table . clinical description of subject population clinical measurement mean ± s.d. range of means by subject (minimum-maximum) plaque index . ± . . - . gingival index . ± . . - . probing depth (mm) . ± . . - . attachment loss (mm) . ± . . - . givitis) were associated with lower levels of antibody. in the case of anti-hsp and anti-groel anti- bodies, the concentrations were significantly (p < . ) lower in the stratum category compared to the category. for the other stress proteins, while trends were apparent, the differences were not within the % confidence level. a similar pattern is seen when examining the relationship between mean probing depth and anti-stress protein antibody con- centration (fig. a). the highest antibody concentrations were found in subjects in the mm and mm strata. subjects who have mean probing depths greater than mm ( mm stratum) have significantly (p < . ) reduced antibody concentrations for all stress proteins, except hsp . figure c shows the same relationships for attachment loss. again, the same trend is obvious and is significant (p < . ) for hsp and dnak. we then examined the relationship between the presence of anti-stress protein antibodies and colo- nization of the subgingival plaque by specific peri- odontal disease-associated microorganisms (fig. ). in this figure, we show the relationship between the antibody concentrations and levels of colonization as measured by the slot immunoblot assay. these levels (low, moderate, and high) correspond to < . × , . to × , and > × microorganisms in the undiluted plaque sample assessed in the assay. as shown in figure a, there is an association between colonization and anti-hsp concentration. in all cases, there is a significantly (p < . ) lower anti- hsp concentration associated with the lowest level of colonization regardless of the bacterial species examined. in all cases, with the exception of a. actino- mycetemcomitans, the highest antibody concentration is associated with the highest level of colonization. there does not appear to be any significant associ- ation in the relationship between the anti-hsp con- centrations and colonization. there is an apparent association with b. forsythus colonization; however, it is not statistically significant. this is also the case when the associations between anti-groel antibodies and colonization are examined (fig. c). in the cases of a. actinomycetemcomitans and b. forsythus while the antibody concentrations appear to be lower at the highest level of colonization, they are not statistically significant. finally, significant (p < . ) relationships between the anti-dnak antibody concentrations and coloniza- tion by p. gingivalis and t. denticola are evident in figure d. though not significant, a. actinomycetem- comitans reveals a trend with anti-dnak antibodies similar to that observed for the other three antibodies. in all cases, the antibody concentration at the highest level of colonization is lower than the preceding con- centration. similarly, b. forsythus reveals this pattern with anti-dnak and anti-groel antibodies. regression modeling of anti-stress protein antibodies in order to begin to understand the variables that con- tribute to the anti-stress protein antibody concentra- tions, we used step-wise and multiple regression models which examined the contribution of age, gen- der, amish/non-amish status, gingival index, probing depth, attachment loss, calculus index, plaque index, and microbial colonization. in the case of the hsp antibodies, variables were found to have significant contributions to the antibody concentration having a r = . and p < . . these variables were prob- ing depth (pd) and p. gingivalis concentration (pg). the equation derived from the regression model was y = − *pd + *pg. this confirmed the inverse relationship with probing depth and the posi- tive relationship with colonization by p. gingivalis. attempts to model the other stress protein antibodies were not successful. discussion there has been a considerable focus on stress proteins in current infectious disease research due to the obser- vations that these protein families are immunodomi- nant antigens of many human pathogens. as a result we felt that it was timely to assess the association between immunity to common stress proteins and one of the most common microbial-mediated diseases in humans, periodontal disease. we selected common, commercially available stress proteins, human and humoral immunity to stress proteins volume • number table . colonization versus mean probing depth mean sib score (mean ± sem) microorganism mm* mm* mm* p† n = n = n = p. gingivalis . ± . . ± . ‡ . ± . < . a. actinomycetemcomitans . ± . . ± . . ± . ns b. forsythus . ± . . ± . ‡ . ± . < . t. denticola . ± . . ± . ‡ . ± . § < . * stratification categories: mm, mean probing depth < . mm; mm, mean probing depth ≥ . and < . ; mm, mean probing depth ≥ . mm. † anova. ‡ significant difference (p < . ) between mm and mm categories. § significant difference (p < . ) between mm and mm categories. j periodontol • october lopatin, shelburne, van poperin, kowalski, bagramian microbial in origin. human hsp and hsp were used because they are amongst the most common of the stress protein families in eukaryotic systems. since there is evidence in studies of candida albicans that conser ved hsp epitopes, shared between humans and candida, were responsible for protection against systemic candidiasis, we felt that evaluating the association between anti-human hsp antibodies and oral disease might be informative. we chose groel and dnak (hsp and hsp ) since they rep- resented the most highly conserved stress proteins of microbial origin. the clinical specimens were derived from a patient population that was from an earlier study of an amish community. , based on an evaluation of the oral health of this population and comparisons with non- amish living in the same geographical location, we felt that, with the exception of a lack of routine oral hygiene, there were no significant differences between the amish and the non-amish in this area. while this is a randomly-selected population with few members pre- senting with advanced periodontitis, there is a clear relationship between disease severity (probing depth) and level of colonization by putative periodontal pathogens. our studies also demonstrated a clear relationship between the serum concentration of of the specific anti-stress protein antibodies and clinical measures of periodontal disease. the serum antibodies (igg) were highest when the subjects have the best oral health. this was most obvious when the mean probing depths are examined. it was interesting that throughout these analyses, while the antibodies reactive with human hsp tended to follow the same trends, they showed the least statistically significant relationships. however, its microbial homologue, dnak, demonstrated consid- figure . relationship between clinical measurements and serum anti-stress protein antibody concentration. antibody concentration for each stress protein is expressed in relative fluorescence units (rfu) as determined by particle concentration fluorescence immunoassay (pcfia). error bars indicate standard error of the mean for the antibody concentration in each clinical category. statistical significance of antibody titers (p ≤ . ): *significant differences between lowest and highest clinical stratum; †significant differences between middle and highest clinical stratum. a. mean gingival index; subjects were stratified into groups ( , , and ; white, gray, and black bars, respectively) based on their mean whole mouth gingival index ( = to < . ; = . to < . ; = > . ). sample sizes for categories , , and were , , and , respectively. b. mean probing depth; subjects were stratified into groups ( mm, mm, and mm; white, gray, black bars, respectively) based on their mean whole mouth probing depths ( mm, < . mm; mm, ≥ . and < . ; mm, ≥ . mm). sample sizes for categories mm, mm, and mm were , , and , respectively. c. mean attachment loss; subjects were stratified into groups ( mm, mm and mm; white, gray, black bars, respectively) based on their mean whole mouth attachment losses ( mm, < . mm; mm, ≥ . and < . mm, ≥ . mm). sample sizes for categories mm, mm and mm were , , and , respectively. humoral immunity to stress proteins volume • number erable disease-associated differences. this would sug- gest that even with the considerable homology within the hsp family, the epitopes that are being recog- nized immunologically are probably those which are not evolutionarily conserved. in contrast, a very signif- icant inverse relationship between disease and human hsp antibody concentration probably indicates that highly conserved epitopes are being recognized. while we did not possess purified microbial hsp protein at the time of this study, investigations in progress in our laboratory demonstrate a high degree of cross-reactivity between human hsp and the hsp homologues of p. gingivalis and a. actinomycetemcomitans based on antibody reactivity (unpublished data). similarly, serum igg antibody concentrations reactive with groel followed similar trends. mean gingival indices and attachment losses also followed these trends. our observations are somewhat similar of a report by schett et al. they examined anti-hsp antibod- ies in sera (igg) and found that the antibody concen- figure . relationship between serum anti-stress protein antibody concentration and colonization. antibody concentration for each stress protein is expressed in relative fluorescence units (rfu) as determined by particle concentration fluorescence immunoassay (pcfia). error bars indicate standard error of the mean for the antibody concentration in each colonization category. colonization by selected microorganisms is expressed as low (sib score = ), moderate (sib score = ) and high (sib score = + ) (white, gray, and black bars, respectively). statistical significance of antibody titers (p ≤ . ): *for significant differences between low and moderate colonization; †for significant differences between low and high colonization; and ‡for significant differences between moderate and high colonization. a) anti-hsp antibodies; b) anti-hsp antibodies; c) anti-groel antibodies; d) anti-dnak antibodies. sample sizes for low, medium, and high colonization categories by microorganism are as follows: p. gingivalis, , , (l,m,h, respectively); a. actinomycetemcomitans, , , ; b. forsythus, , , ; and t. denticola, , , . j periodontol • october lopatin, shelburne, van poperin, kowalski, bagramian trations were highest in periodontitis. this conflicts with our observations that in periodontitis the anti-hsp anti- body levels are lowest. a possible explanation for this discrepancy is not immediately obvious; however, spe- cific differences between our studies exist. there is a considerable difference in sample size between the studies. in their study, a total of subjects were divided into groups. there are also differences between the antigens tested. we tested only e. coli groel and schett et al. reported serum (igg) data only for recombinant mycobacterial hsp . finally, and perhaps the most important difference is that in our random population, we do not have the severe periodontitis that is observed in the schett groups. and as a result, our most diseased group is considerably healthier than theirs and resembles their intermediate (gingivitis) disease. our study also examined the association between colonization by specific microbial species and anti- stress protein antibodies. we found no significant associations between anti-groel or anti-hsp con- centrations and colonization by the specific micro- organisms in our test panel. again, we found this interesting since relationships between antibody con- centration and colonization were observed with anti- dnak antibodies, an hsp homologue, again indicating that, in this case, the non-conserved (anti- bacterial) epitopes are probably more important. however, we observed the most significant associa- tions between the anti-hsp concentrations and microbial colonization. since the antigen used to test for anti-hsp antibodies is derived from human sources, the antibodies detected most likely represent those directed against conserved epitopes, rather than species-specific or anti-bacterial. in all cases, except for a. actinomycetemcomitans, there appeared to be a significant association between increased col- onization and anti-hsp antibody concentration. statistical modeling clearly demonstrated the inverse relationship with probing depth and the positive rela- tionship with colonization. the model very clearly defines the relationship between p. gingivalis and hsp . the most surprising observation is the inverse relationship between the antibody concentrations and oral disease. most prior studies of humoral immunity to periodontal disease-associated microorganisms and oral health have shown a positive correlation. thus, the present observations may actually be identifying the presence of protective antibodies, rather than sim- ply presence of the microorganism in the gingival sul- cus. if these are, in fact, protective antibodies, the next critical step will be to localize the antigens using these antibodies in order to help assess the role of the anti- gen in the virulence of the microorganism. it will be critical to determine if the stress proteins play a role in adherence and colonization, or if they interfere with normal mammalian cell function once the microorgan- ism has established its infection. homologues of specific stress protein families have been demonstrated to be present in oral bacteria, including f. nucleatum, p. intermedia, p. nigrescens, p. melaninogenica, a. actinomycetemcomitans, and p. gingivalis. hsp /groel and hsp /dnak homo- logues have been detected using cross-reactive anti- bodies against prototype members. , , , , homologues of dnak, groel, and groesl have been cloned and sequenced from p. gingivalis and a. actin- omycetemcomitans. , , , , , in addition, the hsp gene homologue of a. actinomycetemcomitans was “accidentally” cloned by winston et al. there have been numerous suggestions that the hsp of pathogens may play a role in their viru- lence , studies by matthews and burnie demon- strate that immunity to candida albicans hsp is important for survival from systemic candidiasis. other studies have shown that the normally non- pathogenic s. cerevisiae can be made pathogenic by causing an over-expression of the hsp gene. mutations in an hsp gene (htra) in salmonella typhimurium result in reduced virulence. finally, reports by kirby et al. have shown that the potent bone-resorbing mediator of a. actinomycetemcomi- tans is homologous to groel. these findings sup- port the importance of understanding the role of stress proteins in microbial virulence and the function of the host response in blocking these effects. our studies employing stress protein homologues reveal that there is considerable conservation of hsp epitopes with periodontal disease-associated microor- ganisms. clearly, since we were able to demonstrate associations using purified human hsp, autoimmunity (as opposed to autoimmune disease) may serve an important function in host defense in periodontal dis- ease. this is not a novel observation and has been proposed by other investigators studying the role of host immunity to stress proteins. the relationship between anti-hsp immunity, oral health, and colonization is reminiscent of the observa- tions by matthews and burnie in their studies of sys- temic candidiasis. in interpreting our findings, it should be clear that we are not using antigens derived from any of the periodontal disease-associated microorgan- isms in our panel. the serum antibodies and the associ- ations that we have described are likely to reflect the contributions of specific antigenic epitopes that have been conserved through evolution due to their impor- tance to the inherent function of the stress protein. we would expect that if we had employed stress proteins derived from our microbial panel, significantly different results would have been obtained. in that case, we would predict that the predominant serum antibody response would be against the species-specific epitopes and would probably reflect infection “history” rather than protective antibodies. our laboratory is currently cloning the hsp homologue of p. gingivalis and once complete we will be able to address issues related to homology between human and p. gingivalis hsp and the epitopes that are involved in this apparent protec- tive response. once sufficient quantities of purified or recombinant hsps of periodontal disease-associated microorganisms are available we will be able to begin to address the importance of immunity to conserved versus microbe-specific epitopes. since matthews and burnie demonstrated that protection could be attrib- uted to the recognition of a specific conserved epitope, it may be possible to establish a genetic basis for resis- tance to p. gingivalis (or other species) colonization based on the ability to recognize a specific epitope. further elucidation of this model is dependent on evalu- ating the immunity to specific hsp epitopes. acknowledgments the authors acknowledge the expert technical assis- tance provided by mrs. christine a. binsfeld in the per- formance of the pcfia analysis. mr. shelburne is a research specialist in the biomaterials technology center of m. this study was supported by u.s. public service grants de , de , and de . references . ebersole 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: - . . matthews r, burnie j. the role of hsp in fungal infection. immunol today ; : - . . schett g, metzler b, kleindienst r, et al. salivary anti- hsp antibodies as a diagnostic marker for gingivitis and a possible link to atherosclerosis. int arch allergy immun ; : - . . koga t, kusuzaki t, asakawa h, senpuku h, nishihara t, noguchi t. the -kilodalton groel-like protein of actinobacillus actinomycetemcomitans. j periodontal res ; : - . . ando t, kato t, ishihara k, ogiuchi h, okuda k. heat shock proteins in the human periodontal disease process. microbiol immunol ; : - . . hotokezaka h, ohara n, hayashida h, et al. trans- criptional analysis of the groesl operon from porphy- romonas gingivalis. oral microbiol immunol ; : - . . yoshida a, nakano y, yamashita y, yu h, ohishi m, koga t. isolation and characterization of the dnakj operon from actinobacillus actinomycetemcomitans. dna sequence ; : - . . winston jl, toth si, roe ba, dyer dw. cloning and characterization of the actinobacillus actinomyce- temcomitans gene encoding a heat-shock protein homologue. gene ; : - . . lathigra rb, butcher pd, garb, tr, young db. heat shock proteins as virulence factors of pathogens. curr top microbiol immunol ; : - . . fer nandez rc, logan sm, lee, sh, hof fman ps. elevated levels of legionella pneumophila stress protein hsp early in infection of human monocytes and l cells correlate with virulence. infect immun ; : - . . hodgetts s, matthews r, morrissey g, mitsutake k, piper p, bur nie j. over-expression of sacchar omyces cerevisiae hsp enhances the virulence of this yeast in mice. fems immunol med microbiol ; : - . . kirby ac, meghji s, nair sp, et al. the potent bone- resorbing mediator of actinobacillus actinomyce- temcomitans is homologous to the molecular chaperone groel. j clin invest ; : - . send reprint requests to: dr. dennis e. lopatin, department of biologic and materials sciences, school of dentistry, university of michigan, north university ave., ann arbor, mi - . fax: / - . accepted for publication february , . short reports journal of medical genetics , , - linkage analysis of manic depression in an irish family using h-ras and ins dna markers manic depression (bipolar affective disorder) is one of the major psychiatric illnesses. it is distinguished from unipolar major depression by the occurrence of manic episodes, symptoms of which show a variable combination of heightened and expansive mood, restlessness, irritability, pressure of speech, disinhibition, increased energy, and grandiose ideas or delusions. it has a lifetime risk of slightly less than % in most populations studied. a large genetic component has been shown using twin and adoption studies. egeland et all have shown linkage between the manic depressive phenotype in one north american pedigree and two polymorphic dna markers on the tip of the short arm of chromosome . other studies ' have failed to show this linkage in icelandic and other north american pedigrees. we have ruled out tight linkage between these dna markers and the manic depressive phenotype, assuming that the disorder is segregating as an autosomal dominant trait in a large irish pedigree. a single, unilateral source of the manic depression is likely but cannot be proven. the diagnoses were based on the research diagnostic criteria of spitzer et al, compiled using a combination of interviews, a review of the case notes, and the schizophrenia and affective disorders schedule-lifetime version (sads-l). all cases were of bipolar affective disorder, apart from one case of unipolar depression. of the unaffected subjects only those over the age of were used to minimise the risk of false negative phenotype assignment. the pedigree is shown in the figure. dna samples from members of the pedigree were received for publication november . revised version accepted for publication january . digested with the restriction enzymes pvuii and sacd. they were separated by electrophoresis on agarose gels and transferred to nylon membranes. hybridisations were carried out with the dna probes h-ras to sacd digests and ins to pvuii digests. both of these loci show high frequency restriction fragment length polymorphisms owing to the insertion or deletion of short, repeated dna sequences (variable tandem repeats). the resulting genotypes are shown in the figure. lod scores were calculated using the computer program liped and the results are presented in the table. these data exclude the possibility of close linkage between manic depression and the chromosome markers h-ras and ins, under the assumption that there is an autosomal dominant gene responsible for the disorder in this family. this would indicate that the gene or genes responsible for manic depression in this family are at other, as yet unknown, loci. further work is needed to identify other manic depressive gene loci in this and other pedigrees. isolation and examination of these genes will lead to a better understanding of the pathogenesis of the disease and its interactions with the environment. michael gill*, patrick mckeont, and peter humphries* *department of genetics, trinity college dublin; and tdepression and manic depression research unit, st patrick's hospital, dublin, ireland. abbcab bc bcab ab bc bc bc bc ac ab bb ab figure pedigree of family showing age, h-ras alleles (a,b,c), and ins alleles ( , , , ). table lod scores calculated between the two marker loci, h-ras i and ins, and the manic depression phenotype, for various values of recombination fraction ( ), using three selected penetrance values for the susceptible genotypes (aa and aa); was the maximum penetrance of the susceptible genotype found in the amish study' and - was the average penetrance found in their sample. penetrance recombination frequency - - - - - - - h-ras - -x - - - - ( - - - - - - - - -x - - ( - - - - - - - - - - - -x - - - - - - - - - - - - ins - -x - - - - - - - - - - - - - -x - - - - - - ( - - - i - - - - - -xc - - - - - - - - - - - - co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ short reports references egeland ja, gerhard ds, pauls dl, et al. bipolar affective disorders linked to dna markers on chromosome . nature ; : - . hodgkinson s, sherrington r, gurling h, et al. molecular genetic evidence for heterogeneity in manic depression. nature ; : - . detera-wadleigh sd, berettini wh, goldin lr, boorman d, anderson s, gershon es. close linkage of c-harvey-ras- and the insulin gene to affective disorder is ruled out in three north american pedigrees. nature ; : - . spitzer rl, endicott j, robins e. research diagnostic criteria: rationale and reliability. arch gen psychiatr ; : - . correspondence and requests for reprints to dr m gill, department of genetics, lincoln place gate, trinity college, dublin , ireland. prenatal diagnosis of mosaicism for del( )(q * q * ) and a normal cell line a year old single hispanic woman had amniocentesis because of maternal age. she had had five pregnancies, resulting in two normal children with a previous partner, one elective abortion, and one tubal ectopic pregnancy with the present year old partner. the medical and family histories were unremarkable. two separate primary amniotic fluid cell cultures at weeks' gestation showed mosaicism for ,xy/ ,xy, del( )(q - q - )(fig ). six of cells ( %) from one primary culture and eight of cells ( %) from the other primary culture showed the deletion. the chromosomes of the parents were normal. the pregnancy was electively terminated by prosta- glandin at weeks' gestation. the fetus weighed g (at about the th centile). noted were the following cranio- facial anomalies: high and narrow forehead, long philtrum, small and thin lips especially the lower (fig ), long and broad thumbs, contractures at the middle interphalangeal joints, bilateral transverse palmar creases, long and broad big toes, and long toes. necropsy showed a large foramen q fig partial gtg banded karyotype from an amniotic fluid cell. the arrows indicate the breakpoints in the normal chromosome (left) and in the idiogrammatic (right). the dark band q - and some ofthe light staining band q - are deleted in the anomalous chromosome . received for publication june . revised version accepted for publication august . fig thefetus at weeks. ovale that was indistinguishable from an atrial septal defect. the deletion was found in % ( / ) of cultured cells from amniotic fluid, in % ( / ) of cells from cord blood, and in % ( / ) of cells from umbilical cord tissue obtained at the time of pregnancy termination. since this pregnancy, the couple has had another pregnancy resulting in a normal son. mosaicism for a structural anomaly at prenatal diagnosis is an uncommon event, occurring in only of about genetic amniocenteses.' since de grouchy et al first described a syndrome associated with partial deletion of the long arm of chromosome , several other physical anomalies associated with deletion of q - *q - have been described. this deletion was found in a proportion of cells from the present fetus, who showed some craniofacial features similar to those of the child previously reported, including a high and narrow forehead and long philtrum. the deletion responsible for this case is more proximal than the deletion associated with de grouchy syndrome, which appears to involve q (probably q - ). we thank mr william herbert who participated in the patient's care. partial support was provided by us public health, maternal and child health services, grant no . miriam g wilson and ming s lin genetics division, department of pediatrics, university of southern california school of medicine, and los angeles county- university of southern california medical center, los angeles, california, usa. references hsu lyf, perlis te. united states survey on chromosome mosaicism and pseudomosaicism in prenatal diagnosis. prenat diagn ; : - . co p yrig h t. o n a p ril , b y g u e st. p ro te cte d b y h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n s e p te m b e r . d o w n lo a d e d fro m http://jmg.bmj.com/ prognostic significance of peritoneal lavage cytology in staging gastric cancer: systematic review and meta-analysis vol:.( ) gastric cancer ( ) : – https://doi.org/ . /s - - -y review article prognostic significance of peritoneal lavage cytology in staging gastric cancer: systematic review and meta‑analysis sara jamel  · sheraz r. markar  · george malietzis  · amish acharya  · thanos athanasiou  · george b. hanna   received: february / accepted: july / published online: august © the author(s) . this article is an open access publication conclusion this study suggests that patients with initial positive cytology may have a good prognosis following neo- adjuvant treatment if the cytology results change to negative after treatment. keywords gastric cancer, stomach neoplasm · laparoscopy · peritoneal cytology · cancer staging · cancer prognosis introduction the main treatment of advanced nonmetastatic gastric can- cer is surgical resection with perioperative chemotherapy or chemoradiotherapy [ , ]. efforts to prolong survival in metastatic gastric cancer have showed little improvement [ , ]. accurate staging of gastric cancer is crucial in selecting the appropriate treatment option, whether curative or pallia- tive. the japanese gastric cancer association included the results of cytological examination of peritoneal lavage fluid as a key prognostic factor in their classification of gastric carcinoma [ , ]. however, recently published guidelines suggested that cytology-positive status in the absence of other noncurative factors, that is, macroscopic disease, can be managed with d gastrectomy and perioperative chemo- therapy [ ]. initial data of those treated with surgery alone showed poor -year survival; however, more recent publica- tions have shown that the use of postoperative chemotherapy improves overall survival rates to %, [ , ]. on the other hand, if the information on cytology status were available before surgery, a chemotherapy-first strategy could be taken whereby patients whose cytology status turned negative could be preferentially treated with curative surgery [ , ]. the incidence of positive peritoneal cytology for patients with gastric cancer varies, in published reports, from % to abstract background peritoneal cytology has been used as a part of the cancer staging of gastric cancer patients. the primary aim of this systematic review was to evaluate the value of peritoneal cytology as part of the staging of gastric cancer and survival prediction. the second aim was to establish if positive cytology may be modified by neoadjuvant therapy, to improve prognosis. methods an electronic literature search was performed using embase, medline, web of science, and cochrane library databases up to january . the logarithm of the hazard ratio (hr) with % confidence intervals (ci) was used as the primary summary statistic. comparative studies were used, and the outcome measure was survival in three groups: ( ) positive versus negative cytology at staging lapa- roscopy immediately preceding surgery; ( ) effect of neo- adjuvant therapy on cytology and survival; and ( ) positive cytology in the absence of macroscopic peritoneal disease was compared with obvious macroscopic peritoneal disease. results pooled analysis demonstrated that positive cytology was associated with significantly reduced over- all survival (hr,  . ; % ci, . – . ; p <  . ). interestingly, negative cytology following neoadjuvant chemotherapy was associated with significantly improved overall survival (hr, . ; % ci, . – . ; p <  . ). the absence of macroscopic peritoneal disease with positive cytology was associated with significantly improved overall survival (hr, . ; % ci, . – . ; p <  . ). * george b. hanna g.hanna@imperial.ac.uk department of surgery and cancer, imperial college london, th floor qeqm building, st mary’s hospital, south wharf road, london w   ny, uk http://crossmark.crossref.org/dialog/?doi= . /s - - -y&domain=pdf prognostic significance of peritoneal lavage cytology in staging gastric cancer: systematic… % [ ]. peritoneal washings positive for cancer cells have been demonstrated to correlate with the extent of cancer (t / t , %; t /t , %; m+, %) [ ] and have been consid- ered as stage iv disease [ ]. the influence of positive cytol- ogy on survival has been shown as a powerful independent predictor of survival when compared to other postoperative pathological variables such as the tumor serosal invasion or lymph node involvement [ , , , ]. positive cytology was shown to be the most powerful predictor of outcome, with a risk ratio of . for patients undergoing curative resection [ ]. furthermore, studies have also shown that the number and arrangement of cytology-positive cells have an effect on survival at the time of gastrectomy [ , ]. the results of the randomized controlled trial by the apan clinical oncology group (jcog ) and korea gastric cancer association (kgca ), comparing gastrectomy plus chemotherapy versus chemotherapy alone in advanced gastric cancer with a single noncurable factor, showed no advantage of resecting the primary gastric cancer in the presence of peritoneal metastasis [ ]. nevertheless, the treatment recommendations for gastric cancer in the event of positive cytology range from palliative chemotherapy to attempts at neoadjuvant therapy followed by surgical resec- tion [ , ]. the aim of this study was to evaluate the value of peri- toneal cytology as part of the staging of gastric cancer and survival prediction. the secondary aim is to establish if positive cytology may be modified by neoadjuvant therapy to improve prognosis. methods literature search strategy an electronic literature search was undertaken using embase, medline, web of science, and cochrane library databases up to january . the search terms ‘gastric can- cer,’ ‘laparoscopy,’ ‘peritoneal cytology,’ ‘cancer staging,’ and ‘prognosis,’ and medical subject headings (mesh) ‘stomach neoplasms,’ ‘neoplasm metastasis,’ ‘laparos- copy,’ and ‘cytology’ were used in combination with the boolean operators and or or (fig.  ). two authors (s.j. and s.r.m.) performed the electronic search independently in january . the electronic search was supplemented by a hand-search of published abstracts from meetings of the society of academic and research surgery, digestive disease week, the association of upper gastro-intestinal surgeons of great britain and ireland, and the american society of clinical oncology for – . the reference lists of articles obtained were also searched to identify fur- ther relevant citations; as was the current controlled trials register (http://www.controlled-trials.com). abstracts of the articles identified by the electronic search were scrutinized by two of the authors (s.j. and s.r.m.) to determine their suitability for inclusion in the pooled analysis. any discord- ances regarding study inclusion between these two authors were settled in discussion with a third independent author (a.a.). the quality of evidence provided by each study was evaluated using the oxford levels of evidence-based medi- cine scoring system (http://www.cebm.net/oxford-centre- evidence-based-medicine-levels-evidence-march- ). publications were included in this review if they meet the following criteria: • studies concerning patients with gastric cancer • comparative studies of patients with positive and nega- tive peritoneal cytology • comparative studies evaluating the effect of neoadjuvant chemotherapy upon patients with positive cytology from gastric cancer • comparative studies of patients with positive peritoneal cytology in the absence of macroscopic peritoneal dis- ease and patients with macroscopic peritoneal disease. publications were excluded if they met any of the follow- ing criteria: • studies published in a language other than english • case reports or cohort studies including fewer than ten patients • noncomparative studies or studies not concerning peri- toneal cytology and gastric cancer. in the situation in which authors from the same institution had published a primary paper and then an updated analysis with a larger patient cohort, the most recent publication was included in the analysis. outcome measures for meta‑analysis of comparative studies the primary outcome measure evaluated was the hazard ratio (hr) for overall survival. three comparisons were made: . positive versus negative cytology at staging laparoscopy immediately preceding surgery. . initially positive cytology that became negative follow- ing neoadjuvant therapy was compared with positive cytology that remained positive despite neoadjuvant therapy. . positive cytology in the absence of macroscopic perito- neal disease was compared with obviously macroscopic peritoneal disease. http://www.controlled-trials.com http://www.cebm.net/oxford-centre-evidence-based-medicine-levels-evidence-march- http://www.cebm.net/oxford-centre-evidence-based-medicine-levels-evidence-march- s. jamel et al. statistical analysis the logarithm of the hazard ratio (hr) with % confi- dence intervals (ci) was used as the primary summary sta- tistic. to estimate hr and its variance, this was extracted from the study directly or required additional calculation depending on the method of data being presented: annual mortality rates, survival curves, number of deaths, or per- centage of freedom from death [ ]. meta-analysis of data was conducted using a random effects model. publication bias was explored graphically with funnel plots to detect asymmetry and any outliers. interstudy heterogeneity was assessed using the chi-square statistic and the i value to measure the degree of variation not attributable to chance alone: this was graded as low (i  <  %), moderate (i  =  – %), or high (i  >  %). the significance level was set at p <  . . calculations were performed by g.m. and verified by t.a. this study was performed in line with cochrane recommendations, following the moose guidelines, using appropriate sta- tistical software (stata/se ) [ ]. results patient demographics the total number of patients included in this meta-analysis was , with a male to female ratio of : . (table  ). the tumor demographics have been described for each group: negative versus positive cytology (table  ). meta‑analysis positive versus negative cytology pooled analysis of studies [ , , , , – ] included patients in total, in the positive and in the negative cytology group at staging laparoscopy. the median follow-up period ranged from to  months. this pooled analysis demonstrated that positive cytology was associated with a significantly reduced overall survival (hr, . ; % ci, . – . ; p <  . ) (fig.  ). there was evidence of significant statistical heterogeneity for this result (i  =  . %). fig. prisma flow diagram of literature search in this meta- analysis prognostic significance of peritoneal lavage cytology in staging gastric cancer: systematic… t ab le d em og ra ph ic s of p at ie nt s w ith c yt ol og y- po si tiv e (c yt + ve ) a nd c yt ol og y- ne ga tiv e (c yt − ve ) r es ul ts st ud y to ta l p at ie nt s f: m ra tio m ed ia n ag e a ge ra ng e n o. p at ie nt s c yt + ve r ec ur re nc e n o. p at ie nt s c yt − ve r ec ur re nc e c om or bi di tie s c om or bi di tie s b ad gw el l / . – b en tr em / . – b ri to / . . . – . c hu w a / . c e ua no ra st er / . fu ka ga w a / . ji an g – k an g . . k at su ra gi / . k od er a l a to rr e / / . – l ee m ak in o / . de m an zo n / . m iy as hi ro / . – n ak ag aw a / . – n od a / . r ib ei ro / . r os en be rg / . w on g to ta l s. jamel et al. table pathological features of gastric cancer in both positive cytology (a) and negative cytology (b) nodal involvement primary tumor (clinical t disease) tumor differentiation histological type angio- lymphatic invasiont –t t –t differentiated poorly dif- ferentiated intestinal diffuse (a) positive cytology  badgwel  bentrem  brito – ( ), – ( )  chuwa  euanoraster  fukagawa  jiang  kang  katsuragi  kodera  la torre  lee  makino  de manzoni  miyashiro  nakagawa  noda  riheiro  rosenberg  wong (b) negative cytology  badgwel  bentrem  brito  chuwa  euanoraster  fukagawa  jiang  kang  katsuragi  kodera  la torre  lee  makino  de manzoni  miyashiro  nakagawa  noda  ribeiro  rosenberg  wong prognostic significance of peritoneal lavage cytology in staging gastric cancer: systematic… following neoadjuvant therapy: positive versus negative cytology pooled analysis of five studies [ , , – ] included patients in total; in the positive cytology and in the negative cytology group. the median follow-up period ranged from to  months. this pooled analysis demon- strated that negative cytology following neoadjuvant chemo- therapy was associated with significantly improved overall survival (hr, . ; % ci, . – . ; p <  . ) (fig.  ). there was evidence of significant statistical heterogeneity for this result (i  =  . %). positive cytology versus macroscopic peritoneal disease pooled analysis of seven studies [ , , , , , ] included patients in total, in the positive cytology and in the macroscopic peritoneal disease group. the median follow-up period ranged from to  months. this pooled analysis demonstrated that positive cytology in the absence of macroscopic peritoneal disease was asso- ciated with a significantly improved overall survival (hr, . ; % ci, . – . ; p <  . ) (fig.  ). there was no evidence of significant statistical heterogeneity for this result (i  =  %). bias exploration/sensitivity analyses funnel plots were created for combined and subgroup analy- sis for the various factors to visually assess the publication bias; these demonstrated symmetry. to determine the influ- ence of each study’s individual dataset, we performed sensi- tivity analyses for the subgroups as described. a single study involved in the pooled meta-analysis was excluded each time, and the corresponding hr was not changed notice- ably (data not shown). discussion this meta-analysis has demonstrated negative peritoneal cytology before treatment improves survival rate when com- pared with positive cytology. cytology should be considered a modifiable factor as it was found in this meta-analysis, that the change in cytology status from a positive to nega- tive result following chemotherapy was shown to carry an improvement in overall survival. furthermore, although pos- itive cytology is considered stage iv disease, the prognosis and overall survival associated with positive cytology versus macroscopic peritoneal metastasis are not equivalent [ ]. fujiwara et al. have shown that the change in cytology status to negative following receiving neo-adjuvant intraperitoneal fig. forrest plot of pooled analysis demonstrated that posi- tive cytology results were asso- ciated with significantly reduced overall survival (hr, . ; % ci, . – . ; p <  . ) s. jamel et al. chemotherapy and systemic chemotherapy improved progno- sis [ ]. it is worth noting that although lorenzen et al. have also shown that the change in cytological status to negative following chemotherapy led to improved prognosis, almost % with negative cytology became positive following chemotherapy, thus worsening their prognosis and outcome [ ]. the prediction of response of patients to neoadjuvant therapy as assessed by cytology remains a challenging area for future research to provide patient- and tumor-targeted therapy. regarding peritoneal disease, this meta-analysis has shown that positive cytology alone carries better sur- vival compared with macroscopic peritoneal dissemina- tion. although survival with no treatment can be similar between these two groups, the use of neo-adjuvant chemo- therapy was shown to improve the -year overall survival from % in gross peritoneal disease to % in positive cytology with no overt peritoneal disease [ ]. therefore, the concept that positive cytology is a potentially modifi- able factor is further supported. interestingly, miyashiro fig. forrest plot for pooled analysis demonstrated that nega- tive cytology results following neoadjuvant chemotherapy were associated with significantly improved overall survival (hr, . ; % ci, . – . ; p <  . ) fig. pooled analysis demon- strated that positive cytology in the absence of macroscopic peritoneal disease was associ- ated with significantly improved overall survival (hr, . ; % ci, . – . ; p <  . ) prognostic significance of peritoneal lavage cytology in staging gastric cancer: systematic… et al. also studied the number of cancer cells detected in cytology studies versus peritoneal metastasis and found that when a higher number of cells was detected, survival was similar to those with peritoneal metastasis [ ]. there was insufficient evidence available to define a threshold of positive cancer cells from cytology that changed sur- vival from this review, which remains an important area for future research. there are a number of limitations to this systematic review and meta-analysis that need to be acknowledged. first, the lack of randomized control trials (rcts) that met the inclusion criteria is therefore reducing the power of the analyses. second, only studies in the english-language lit- erature were included, so it is possible that relevant stud- ies in other languages will be identified in the future. also, patient demographics and co-morbidities were not reported in all the included studies. in conclusion, the results of this meta-analysis support the importance of peritoneal cytology results in the assess- ment of gastric cancer patients. we have shown that nega- tive cytology and the change in cytological status from positive to negative improve survival in gastric cancer. this knowledge justifies the notion to reconsider the presence of positive peritoneal cytology as an absolute indication for palliative intent of treatment without further consideration to changing status following chemotherapy (fig.  ). the change of initial positive cytology to negative subsequent to treatment should be the subject of a prospective large-scale multicenter study that examines long-term survival benefits. compliance with ethical standards conflict of interest no funding sources were used in the prepara- tion of this manuscript, and the authors have no conflicts of interest to declare. ethical approval no ethical approval or informed consent statement was required for this review article. sources of funding sheraz r. markar is supported by the national institute for health research (nihr). the views expressed are those of the authors and not necessarily those of the nhs, the nihr, or the department of health. open access this article is distributed under the terms of the creative commons attribution . international license (http://crea- tivecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. references . japanese classification of gastric carcinoma. japanese gastric can- cer, nd edn. gastric cancer. ; 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: – . . fujiwara y, takiguchi s, nakajima k, et al. neoadjuvant intraperi- toneal and systemic chemotherapy for gastric cancer patients with peritoneal dissemination. ann surg oncol. ; : – . 	prognostic significance of peritoneal lavage cytology in staging gastric cancer: systematic review and meta-analysis 	abstract 	background 	methods 	results 	conclusion 	introduction 	methods 	literature search strategy 	outcome measures for meta-analysis of comparative studies 	statistical analysis 	results 	patient demographics 	meta-analysis 	positive versus negative cytology 	following neoadjuvant therapy: positive versus negative cytology 	positive cytology versus macroscopic peritoneal disease 	bias explorationsensitivity analyses 	discussion 	references lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / open accessr e s e a r c h a r t i c l e research articlepedhunter . and its usage to characterize the founder structure of the old order amish of lancaster county woei-jyh lee , toni i pollin , jeffrey r o'connell , , richa agarwala and alejandro a schäffer* abstract background: because they are a closed founder population, the old order amish (ooa) of lancaster county have been the subject of many medical genetics studies. we constructed four versions of anabaptist genealogy database (agdb) using three sources of genealogies and multiple updates. in addition, we developed pedhunter, a suite of query software that can solve pedigree-related problems automatically and systematically. methods: we report on how we have used new features in pedhunter to quantify the number and expected genetic contribution of founders to the ooa. the queries and utility of pedhunter programs are illustrated by examples using agdb in this paper. for example, we calculated the number of founders expected to be contributing genetic material to the present-day living ooa and estimated the mean relative founder representation for each founder. new features in pedhunter also include pedigree trimming and pedigree renumbering, which should prove useful for studying large pedigrees. results: with pedhunter version . querying agdb version . , we identified , presumed living ooa individuals and connected them into a -generation pedigree descending from founders ( females and males) after trimming. from the analysis of cumulative mean relative founder representation, founders ( females and males) accounted for over % of the mean relative founder contribution among living ooa descendants. discussion/conclusions: the ooa are a closed founder population in which a modest number of founders account for the genetic variation present in the current ooa population. improvements to the pedhunter software will be useful in future studies of both the ooa and other populations with large and computerized genealogies. background the old order amish (ooa) of lancaster county in pennsylvania are a closed founder population, with approximately , - , living individuals that can be connected into a single -generation pedigree. other large ooa populations live in ohio and indiana. there have been numerous medical genetics studies on the ooa. for several decades, the medical genetics studies focused on monogenic diseases such as brittle hair dis- ease [ , ]. more recently, research interests have broadened to include complex traits, such as parkinson disease [ , ], dementia [ ], diabetes [ ], blood pressure [ , ], hip frac- tures/osteoporosis [ ], and vision phenotypes [ ]. inves- tigators at the university of maryland school of medicine (um-som) [ ] have recruited over , ooa from lancaster county for several studies of complex adult- onset diseases beginning in with the initiative of dr. alan r. shuldiner [ , ]. these ooa studies have used genome-wide linkage analysis [ - ], candidate gene association analysis [ ], and most recently genome-wide association studies (gwas) to discover variants influenc- ing traits such as cardiac repolarization [ ], type dia- betes [ ], hypertension [ ], and fasting and post- prandial triglyceride levels [ ] or to validate locus asso- ciations first discovered in other populations, e.g., diabe- tes [ ], human height [ ], fasting glucose levels [ ], * correspondence: schaffer@helix.nih.gov national center for biotechnology information, national library of medicine, national institutes of health, rockville pike, bethesda, maryland , usa full list of author information is available at the end of the article biomed central © lee et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons at- tribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of waist circumference [ ], bilirubin levels [ ] and response to the anti-clotting agent clopidogrel [ ]. the ooa comprise one of several groups in the more general category of anabaptists; other anabaptist groups living in north america include other amish, menno- nites, and hutterites. medical geneticists have been inter- ested in anabaptist populations because they are closed populations and have written genealogies and other fea- tures such as a homogeneous, rural lifestyle and high standard of living [ - ]. their genealogies were mostly published in paper books, which make integration and search challenging and time-consuming tasks. in we set out to construct a computer-searchable genealogy database of the ooa of lancaster county in pennsylvania for use by geneticists [ ]. to support the database, we developed a suite of query software that would solve pedigree-related problems automatically and systematically. the digital genealogy database expanded to include other anabaptist populations and was renamed anabaptist genealogy database (agdb) [ - ], and the query software package was named ped- hunter [ , ]. the initial source to construct agdb was a computer file updating the fisher family history (ffh) book [ ] edited by ms. katie beiler. later versions integrated the amish and amish mennonite genealogies (aamg) book [ ], a large computerized genealogy file from mr. james hostetler and two smaller updates from ms. beiler of recent births, deaths, and marriages in the lancaster area. the queries and utility programs of ped- hunter are illustrated by examples using agdb in this paper. several groups have used agdb in their research. some worked on rare diseases, such as nemaline myopa- thy [ ], congenital microcephaly [ ], and dystonia [ , ]. although agdb contains no explicit phenotype data, lifespan can be inferred when both birth and death dates are available. several studies on lifespan using agdb have established that lifespan is heritable [ ], that lifespan may be associated with other implicit traits [ , ], and that lifespan can be associated with experi- mentally measured traits [ ]. a frequent problem in medical genetics is to recon- struct the pedigree relationships among distant relatives. pedhunter was first designed to solve optimal pedigree connection problems and other problems related to pedi- gree construction, verification, and analysis. pedhunter is not formally tied to agdb; in particular, access to agdb requires ethics approval from an institutional review board (irb), while pedhunter is freely available. ped- hunter has been used to construct genealogies for link- age and haplotype analysis on hutterite families [ - ] and analyses of an icelandic population [ ], a southern italy population [ ], and a northern italy population [ ]. in this paper, we report on new features in pedhunter and on how those features can be used to quantify the impact of the pedigree structure of a founder population on the amount of genetic variation present in the current population. under a model of genetic drift, the number of founder alleles present in the current population will depend on factors such as sibship size and number of new genomes that enter each generation. from agdb we can answer this question: how many ooa founders contrib- uted what expected percent of the genetic material to the present-day living ooa? the answer impacts the genetic architecture, and hence phenotypic distribution of important traits such as low density lipoprotein (ldl) and triglyceride (tg) levels. a recent study characterized the patterns of linkage disequilibrium in the ooa [ ]. in this paper, we quantify the representation of founder genes in the current population that contribute to those patterns of linkage disequilibrium. methods pedhunter versions pedhunter version . [ ] that provided queries was first released in . in versions . , . , . , and the cur- rent . we increased the number of queries to . the full list of query programs and seven utility programs is in the additional file . pedhunter . has been tested on platforms running the linux, sunos, windows, and mac os x operating systems. the queries are imple- mented using transact-sql and c version of open cli- ent db-library. all pedhunter queries and utility programs are available as executable files that can be used from the command line prompt and in unix shell scripts. the current version of pedhunter is freely avail- able and can be downloaded from http:// www.ncbi.nlm.nih.gov/cbbresearch/schaffer/ped- hunter.html. pedhunter queries a genealogy database stored either as a sybase relational database or in structured ascii plaintext files. before version . , the source codes for these two variants were separate with much duplication. the single set of code files in version . can be adapted to either type of database representation with small changes to one code header file. queries in pedhunter . pedhunter . supports four categories of queries as basic operations: ) testing a relationship; ) finding all individuals satisfying a certain relationship; ) printing information; and ) complex queries. we use italics to indicate the names of specific queries. queries that find pedigrees print the pedigrees in linkage format [ ]. in the second category, pedhunter . adds two queries pertinent to our study of ooa founder structure: founder_descendant to find founders of a given individ- http://www.ncbi.nlm.nih.gov/cbbresearch/schaffer/pedhunter.html http://www.ncbi.nlm.nih.gov/cbbresearch/schaffer/pedhunter.html http://www.ncbi.nlm.nih.gov/cbbresearch/schaffer/pedhunter.html lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of ual, and a new query count_descendant to count number of descendants per ancestor from an input file in the for- mat of ancestor-descendant pairs. new complex queries pertinent to our study include: calculate_r to calculate relative founder representation (rfr) for a founder- descendant pair, and average_r to calculate mean rfr per founder in agdb, as explained below. examples of queries useful for analysis of the anabaptist genealogy database to find possible participants in agdb to be recruited into a study, the living query is useful to find living indi- viduals. the founder_descendant query can be followed to find the founders of each living individual. for each founder-descendant pair, the calculate_r tool computes the rfr, defined below. the average_r tool then com- putes the mean rfr for each founder. the person infor- mation for founders can be obtained using the person_info query. prior to running the asp query to find "all shortest paths" pedigree(s) connecting a set of sam- pled individuals, the subset query is required to first find a maximal subset of individuals that shares a common ancestor. pedigree renumbering pedhunter is also useful for genetic evaluations on large pedigrees. for example, at the united states department of agriculture (usda), the number of animals in the hol- stein dairy pedigree used for genomic evaluation of important economic traits has grown from , to , in less than months as new animals are added. the ability of pedhunter to scale to millions of subjects is a feature. other pedigree storage/query packages, such as pedsys [ ], are not able to process such a large pedi- gree (though pedsys adds the capability to connect large amounts of phenotype data with individuals in smaller pedigrees, such as the subset of more than , ooa individuals that have been studied at um-som). to facil- itate handling large pedigrees, we added the renumber_pedigree utility to pedhunter to number and order subject identifiers, and add missing spouses/mates if necessary into the pedigree. the renumbering process ensures the property that identifiers of parents are smaller than the identifiers of their children, which enables more efficient calculation of kinship coefficients [[ ], ch. ]. adding missing parents into the pedigree is necessary for some software packages, such as link- age, which assume that each person has either zero or two parents in the pedigree. the original identifiers are included in the output, so that information can still be connected to these identifiers. anabaptist genealogy database version . agdb was created in sybase sql server release . .x of the sybase relational database management software. each individual in agdb is assigned a unique integer, called the program id. two main tables in the pedhunter pedigree storage system are the person table and the par- ent-child relationship table. there have been several versions of agdb based on the sources mentioned in background and with sizes summa- rized at the beginning of results. the current agdb ver- sion . (agdb ) was created in , after the most recent published description [ ], to combine all the sources and updates. some small batches of corrections and updates have been incorporated based on feedback from users. as indicated by the change in the first word of agdb from the original "amish" to "anabaptist", many of the individuals in agdb are not ooa. as explained in results, we extracted a subpedigree from agdb that should include most living ooa individuals in the lan- caster area and their ancestors. we used the method described in the next subsection to predict which individ- uals are ooa. prediction of old order amish status an adult or near-adult (older adolescent) is of ooa sta- tus if the individual belongs to the ooa church, which means that the individual has chosen to be baptized. for the purpose of our query, we included children and young adults who were not yet baptized but lived with their ooa parents. for individuals in the printed ffh book, determining the ooa status by eye is usually easy, because for most family records, the religious affiliation is included. however, there are some omissions and some cases that are unclear. due to the syntax in ffh, the word "ooa" appeared only once per family record. when agdb was generated, the ooa status was captured only into the record of the person whose name preceded the word "ooa". if the family head was married, the ooa status was usually assigned to the spouse of family head in agdb. for example, the ffh book reads " . christian fisher b april , , d nov. , , farmer, m bar- bara yoder (yost yoder) ooa, ...". christian fisher's per- son record in agdb does not include any ooa status, but barbara yoder's record does. the spouse and children of the person assigned ooa status should also have ooa status in most cases. therefore, to predict if an individual in the genealogy requires some inferences across rela- tionship links. for individuals added into agdb by ms. beiler after the ffh book was printed, the amount of information about religious affiliation rapidly declined to zero. we believe that virtually all individuals added in updates by ms. beiler in and are ooa, due to biased ascertainment. prediction of ooa status is done in three different ways: ) for an individual born in or before , we con- sider person records of self, parents and spouses; ) for an lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of individual born in - , we check person records of self, parents, spouses and spouses' parents; ) for an indi- vidual born in or later, we examine person records of self, parents, grandparents and spouses. for an individual i, if any of the relatives of i considered has ooa status, then we predict that i has ooa status. pedigree trimming on founders working with large pedigrees, trimming redundant or irrelevant individuals (as defined below) from a pedigree will reduce the computational time of some pedigree analysis methods [ ]. in a nuclear family that is at the top of the pedigree and has only one child, we can replace both parent founders with their only child in the analysis because such a child's genomic data represents all genomic data derived from those parents. nuclear fami- lies at the top of pedigree are recursively trimmed, until no more trimming is possible. figure illustrates a mar- ried founder couple hans beuttschi and margaret zum bach with their only son peter beuttschi. after one round of trimming, peter beuttschi replaces his parents as a trimmed founder. peter beuttschi was married to marga- rete oswald, who is a female founder, and gave birth to their only son also named peter beuttschi. after a second round of trimming, peter beuttschi replaces his parents as a new trimmed founder. estimation of birth years for founders many of the older person records in agdb did not have birth dates in the data sources. to report our analysis by birth cohorts, it is necessary to estimate birth years for individuals; the estimated birth years are used for the analysis in this project, but are not recorded within agdb. for simplicity and to allow inclusion of records with known dates missing the month or day, we used only birth years but not actual birth dates. to systematically estimate missing birth years, we performed a frequency test on known parent-child pairs with known birth years for both individuals in agdb . we extracted birth year of each founder record with known birth year, and sub- tracted from birth year of the oldest child with known birth year. there are , known founder-and-oldest- child pairs. the mean birth year difference is " . years", and the median birth year difference is " years". there- fore, we used years per parent-child generation in the estimation of birth years for founders in agdb . if the birth year of a founder was unavailable, we sub- tracted years from the birth year of the (known or esti- mated) oldest child. if birth year of a child is unavailable, we recursively estimated birth years among the children of the child. the following example estimates a founder (unknown name in agdb )'s birth year to be " " by subtracting years (three generations) from the esti- mated oldest child (henry stehly)'s estimated oldest child (magdalena stehly)'s oldest child (catherine sieber) who was born in . mean relative founder representation we created and used the calculate_r tool to calculate the relative representation of each founder in each study par- ticipant. we define the rfr of a given founder in a given descendant as the expected proportion of alleles in the descendant that were inherited identical-by-descent (ibd) from the founder. for example, an offspring inher- its half its genome from each parent, thus the founder representation for each parent is one-half. the expected proportion can be computed as twice the kinship coeffi- cient between the parent and offspring. the kinship coef- ficient is defined as the probability at a given locus that the allele selected from one individual will be identical by descent to a randomly selected allele of a second individ- ual. for example, twice the kinship coefficient between a parent and an offspring, and the rfr of that parent in that offspring, is . . in a three-generation pedigree, the grandparent-grandchild kinship coefficient is . ; thus the relative representation of each grandparent in a grandchild is . . it should be noted that founder repre- sentation represents the average across the genome, whereas at any autosomal locus the two alleles an individ- ual inherits come from at most two founders. from these definitions and the pedigree structure we use average_r to calculate the mean rfr for each founder over all study descendants. by definition the rfrs in a given individual will sum up to . a subtle point is that the founder representation (expected ibd) as described above is calculated assuming no inbreeding in the founders because by definition, their ancestors are not in the genealogy. conceptually, the probability of inheriting a founder allele is a property of mendelian sampling in the pedigree, independent of the figure sample pedigree trimming in agdb . two recursive rounds of pedigree trimming on three founders down to one trimmed founder in agdb . hans beuttschi margaret zum bach peter beuttschi margarete oswald peter beuttschi lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of probability that the two alleles of a founder are inherited ibd from unknown ancestors. thus, the rfr of a parent in an offspring is . only under the assumption of no inbreeding. if the parent were completely inbred (say a mouse strain) then twice the kinship coefficient would be . . the former agrees with what we expect, while the lat- ter would imbalance the founder representation between an inbred father and non-inbred mother. results the size of agdb has grown from agdb , completed in containing , individuals and , marriages, to the current agdb , containing , individuals and , marriages [ - ]. in , we built a special version called fisher that includes , individuals and , marriages to accommodate three updates since ffh was published in . the results here are based on both fisher and agdb . the subjects of interest in the ongoing genetic studies at um-som are in fisher, but some of their ancestors who are not in fisher can be found in agdb . distribution of old order amish the procedure to analyze mean rfr of founders in pre- sumed living ooa individuals is illustrated in figure . by executing the age tool in pedhunter . on the , individuals in fisher, we collected , individuals born in - . using the living tool in pedhunter . , we found , presumed living individuals, with no death date recorded in fisher. by predicting the ooa status among , individuals born in - , we find , to be ooa. among , presumed living individuals, the results showed , presumed living ooa individuals born in - and in fisher. the difference between the , individuals used here and the , - , estimate in the background is due to children born in - and individuals born before who are still alive. we focused attention on the indi- viduals born in - because death records for those born before are rather incomplete, and birth records after are currently incomplete, although we plan to address these deficiencies in agdb. table reports the birth year and gender distribution among above , presumed living ooa individuals in fisher. the results show increasing numbers of births over time. the year with the most newborns is , in which there were , birth records ( females, males, and of unknown gender). the data consistently show more males than females born. we then located these , individuals in agdb by mapping their program ids from fisher to agdb . using the ancestors tool in pedhunter, we constructed a pedigree containing a total of , individuals, includ- ing the above , presumed living individuals, in figure procedure to analyze mean rfr in agdb . flowchart to analyze mean rfr of founders in presumed living ooa descendants. fisher database for each fisher id i: was i born in - without a known death date (as presumed living)? is i an ooa? discard i. no yes no agdb database convert fisher id i to agdb id d.convert fisher id i yes identify all ancestor founders of d. form pairs of married founder couples. for each founder couple (p,m): number of offspring for (p,m)? replace p and m by the offspring c. = form pairs of ma couplep f ma for each founder ouplep number of offspri replace p and m by == form pairs of trimmed founder and presumed living ooa descendant. form pairs of trimme presumed living oo � for each founder- descendant pair (f,d): calculate rfr as r of founder f in presumed living ooa descendant d. calculate mean rfr for each founder f over all presumed living ooa descendants. r sort all trimmed founders by the descending order of mean rfr ’s.r procedure to analyze mean rfr in agdb lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of agdb . the , individuals form a -generation pedigree, in which there are founders. there are living individuals recorded as founders themselves. the ooa study at um-som confirms that many of these are spouses of individuals who left the amish church, although they may have been ooa at some time and may have ooa children. among the founders, are female and are male. there are marriages between female founders and male founders; five male founders had two marriages to female founders, with the second mar- riage following the death of the first spouse in all cases. among marriages, founder couples have exactly one child. by applying the trimming technique described in methods, founders are reduced down to trimmed founders after one round and trimmed founders after two rounds. all subsequent analysis of founders uses this trimmed set of founders. in the trimmed set, there are female founders and male founders, and married founder couples including females and males. table reports the birth years for the founders. the two most ancient founders are a married couple, estimated to have been born in . prior to , birth years of more than % of founders are not recorded in agdb . all but one founder born after have known birth years. old order amish founder structure using the pedigree structure from agdb , one can esti- mate the contribution of the founders to the current ooa gene pool. this is an estimate because, as described in methods, the calculations are based on probabilistic analysis of the pedigree structures rather than observed dna segregation. moreover, there may be additional relationships (not in the genealogy) among the desig- nated founders and some relationships in the genealogy may be inaccurate. table reports on the birth years of founders and numbers of their living ooa descen- dants. the largest coverage number is , living descendants, who are descended from the oldest founder couple. of founders, were born before . however, many of these founders had few descendants. we were interested in calculating the number of founders contributing to the majority of the extant population to evaluate qualitatively and quantitatively our assumptions about the utility of the ooa for gene mapping. we used the calculate_r and average_r tools in ped- hunter . to calculate the relative representation of each founder in an average living ooa individual. by ranking founders in descending order of their founder representa- tion, we were able to count the number of founders repre- sented in the majority of expected alleles in an average individual among living ooa individuals. we computed the average expected genetic contribu- tion of each of the founders ( females and males) to each of the , presumed living ooa descendants as summarized in figure . we found that founders ( females and males) accounted for over % of the average founder contribution, with a sin- gle founder accounting for nearly % of the average con- tribution. half of the total founder representation came from only founders ( females and males). these data quantify the extent to which the ooa are a closed founder population ideal for elucidating the role of genetic variation in complex diseases. to put the coverage analysis in some chronological per- spective, it is useful to consider isaac huyard who was born on february , . he married into the ooa on december , after working as a servant for an amish family, and is anecdotally the last individual who was born and reached adulthood non-ooa, and then became ooa [ ]. of the highest contributing founders (accounting for % of gene pool) were born in table : distribution of birth years and genders for presumed living ooa in fisher. birth year female male unknown gender sum - , - , , , - , , , - , , , - , , , - , , , - , , , total , , , birth year cohorts of ten years are reported in rows, and genders in three categories are reported in columns. the total number of presumed living ooa individuals in fisher is , . lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of or before . the other three high-contributing found- ers were born in , and . on the other hand, founders born after account for only . % of the founder representation. thus, a detailed study of the ped- igree structure of the ooa as catalogued in agdb sup- ports the notion that there has been very little influx of genetic material into the ooa population since the mid- th century. among founders born after isaac huyard, we found that founders are adopted, and another two founders are likely adopted. one of the improvements in going from agdb to agdb was the identification of adopted table : distribution of known and estimated birth years of founders in agdb . birth year female male sum - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) - ( ) ( ) ( ) - ( ) ( ) - - - - - - - - - - ( ) ( ) - total ( ) ( ) ( ) birth year cohorts of ten years are reported in rows, and genders are reported in columns. the numbers represent both known and estimated birth years in the age group; the numbers in parentheses are the number of founders among that cohort whose birth years were estimated, as described in methods. lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of individuals, so that those "non-biological parent to child" links are not in the pedigrees that we construct, if the adoptive relationships are known. relevance of founder structure to published and ongoing studies as of october , the ooa studies at um-som included approximately , participants. % of these participants studied are descended entirely from a subset (numbers of founders per individual ranged between and ) of the founders born before . that is, the entire gene pool of those individuals was derived from founders born before . % of participants studied are descended entirely from a subset (numbers of found- ers ranged between and ) of the founders born before . a subset of founders with more than living descendants comprises the entire gene pool of % of the study participants. a subset of founders with table : distribution of birth years and numbers of descendants among founders in agdb . birth year ≤#(d)< ≤#(d)< ≤#(d)< , , ≤#(d)< , , ≤#(d) sum - - - - - - - total birth year cohorts of fifty years are reported in rows, and numbers of descendants #(d) are reported in columns. founders born earlier have larger numbers of presumed living ooa descendants. figure cumulative mean rfr in agdb . cumulative mean rfr of founders in , presumed living ooa descendants. lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of more than descendants comprises the entire gene pool of % of the study participants. excess of female founders the number of female founders ( ) is larger than the number of male founders ( ) among founders. we evaluated three possible causes for the excess of female founders: ) remarriages of male founders after widow- hood; ) marriages comprising one founder and one non- founder; ) possible overestimate of female founders due to the difficulty of tracking relationships between females with different married surnames. we mentioned previously that there are marriages between female founders and male founders. in addition to the married founder couples, there are female founders married to non-founder males, and there are male founders married to non-founder females. thus, remarriage of widowed male founders is only a minor factor in the excess of female founders. the propensity for female founders to marry non-founders is a more substantial factor, but this may be confounded by difficulty in correctly determining the founder status of females. the lack of knowledge about relationships among female founders is partly due to the male bias in the ffh and aamg books. among female founders, only have partial names given, and only have surnames, making it difficult to track relationships. there are distinct surnames using identical spelling, and after grouping some sets such as {moser, mosser, musser} that are likely to be different spellings of the same surname. if any individuals with the same surname share ancestry, we would be able to reduce the number of female founders. occasionally, the books hint at a hidden relationship among founders, via footnotes, but the footnotes were not used systematically as they are often speculative. we conclude that the excess of female founders is mostly due to cryptic or unknown relationships, so that some of the apparent female founders are not really founders. discussion/conclusions with new queries and utility programs in pedhunter ver- sion . , we identified , presumed living ooa indi- viduals born in - and connected them into a - generation pedigree descended from founders, after trimming. because of the small number of founders, the frequency of consanguineous marriages in ooa steadily increased over time and reached approximately % for individuals born in - . among consanguineous marriages, the median kinship coefficient stayed stable in the th century, but rose in the th century. table reports kinship coefficients of married ooa couples who had at least one offspring in agdb . numbers of couples and kinship coefficients are both increased in temporal statistics. analysis of cumulative mean rfr shows that founders accounted for over % of the average founder contribution among all living ooa descendants. such results confirm that the ooa in lancaster county are truly a closed population. the combination of lack of new genetic material, lack of socio-economic variation, and detailed genealogies make the ooa ideally suited for identifying some rare variants that are associated with complex phenotypes [ ]. the examples of gwas repli- cations cited in background prove that some trait-associ- ated snp alleles seen in other populations can also be found in the ooa. our characterization of the founder structure provides an explanation for why other trait- associated alleles did not enter the ooa population; for example, cystic fibrosis is one of the most common reces- sive diseases in individuals of european ancestry, but hardly seen among the ooa [ ]. that the ooa have a small number of founders, but linkage disequilibrium (ld) patterns similar to outbred european populations [ ] may seem paradoxical. the apparent paradox is explained by noting that ld is a mea- sure of non-random association of alleles at different loci. linkage between the loci maintains this association under random mating and drift, while recombination will decay the association. thus, allele and haplotype frequen- cies drift together. for example, suppose two snps have d' = in the founders, so that only three of the four possi- ble haplotypes are observed, with frequencies . , . and . , respectively, but drift to . , . and . in the current population. both allele and haplotype frequencies have changed, but ld has not changed. the assumption of common alleles is important as random drift over the generations spanned by agdb will not eliminate com- mon variation. for low frequency alleles there is longer ld in the amish due to linkage as shown in [ ] and also in some of our association studies [ , ], where the most significant signal can be more than kbp from the causal allele. one weakness in our characterization of the ooa founder structure is the apparent excess of female found- ers. we tested three hypotheses and concluded that the excess is mostly due to cryptic or unknown relationships, such that some female founders are not really founders. analyses of mitochondrial genomes could provide evi- dence regarding some of these cryptic relationships. the male lineage and y chromosome inheritance have been validated [ ] a unique y chromosome str haplotype bred true within each of the male lineages represented in the um-som study sample. the surnames of the males comprising these lineages captured % of the households in the lancaster county amish address book, which contains distinct surnames. this number lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of is much lower than the total male founders because of genetic drift, conversion of some earlier settlers to other anabaptist sects, and westward migration [ ]. one limitation of our rfr calculations is that the vari- ance of the kinship coefficient due to mendelian sampling variance is not included in the calculation. for example, the kinship coefficient is . for both parent-offspring and full sibs, but the variance is zero for parent-offspring and non-zero for full sibs. analytical expressions of kin- ship variances are not possible for complex pedigrees, but empirical variances can be obtained through simulation by repeated gene-dropping of distinct founder alleles through the pedigree. computationally efficient gene- dropping can be achieved using the ancestor-first pedi- gree renumbering described above. we may implement a function r-empirical in a future version of pedhunter. the notion of "founder" we used is with respect to a trimmed genealogy, not including any explicit conditions on the locations of birth or death. the data sources focus on individuals who lived at least some years in the present united states, but not exclusively. to quantify this, we looked at the source information on founders with known or estimated birth years in and before , and we found that founders ( females and males) probably died in europe or on the sea. therefore, there may be even more female founders who never reached north america. to increase the utility of agdb and other digitized genealogies, we continue to enhance pedhunter with queries and utility programs to assist the discovery pro- cess on pedigrees in large genealogies. for example, agdb has been used to estimate the encatchment popu- lation of specific hospitals to determine the denominator of the hip-fracture incidence [ ]. for a study of osteo- genesis imperfecta, agdb was used prospectively to identify individuals likely to carry a genetic variant of phenotypic interest [ ]. a pedigree constructed from agdb made it possible to trace the origin of a rare apoc null allele conferring a favourable lipid profile and apparent cardioprotective phenotype to a couple born at the turn of the th century [ ]. additionally, pedhunter has been used in other pedigrees, to discover genetic drift and founder effects [ ]. in sum, we quantified the founder structure of the ooa and implemented numerous improvements to the software pedhunter that will be useful in future studies of both the ooa and other populations with large, comput- erized genealogies. additional material abbreviations aamg: amish and amish mennonite genealogies; agdb: anabaptist geneal- ogy database; ffh: fisher family history; gwas: genome-wide association studies; ibd: identical-by-descent; ld: linkage disequilibrium; ooa: old order amish; rfr: relative founder representation; um-som: university of maryland school of medicine. competing interests the authors declare that they have no competing interests. additional file pedhunter . queries and utility programs. table : distribution of birth years and kinship coefficients among married ooa couples with offspring in agdb . birth year #(couples) average % percentile median % percentile - . . . . - . . . . - . . . . - . . . . - . . . . - . . . . - . . . . - . . . . - , . . . . - , . . . . - , . . . . - . . . . union set , . . . . birth year cohorts of ten years are reported in rows. a couple is assigned to the interval containing the average of the two birth years. numbers of married ooa couples #(couples) and kinship coefficients are reported in columns. couples born later tend to have larger kinship coefficients. http://www.biomedcentral.com/content/supplementary/ - - - -s .pdf lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of authors' contributions w-jl implemented the queries added in pedhunter version . , carried out the analysis of founder contributions and agdb shown here, and wrote the paper. tip developed the method for analyzing founder contributions, analyzed the founder contributions using an earlier version of agdb with the pedsys pack- age [ ], suggested additions to pedhunter, and wrote parts of the paper about founder contributions. jro helped develop the method for founder contributions and suggested additions to pedhunter. ra implemented most of the queries added in pedhunter versions . through . and organized data for all versions of agdb including version . and fisher formally announced in this paper. aas conceived agdb and pedhunter, assisted in their implemen- tation, and supervised the project. all authors edited the paper, read and approved all submitted versions including the final manuscript. acknowledgements this research was supported in part by the intramural research program of the nih, nlm. thanks to kathy ryan, braxton mitchell, alan shuldiner and the research team at the university of maryland and amish research clinic for collaborating on pedhunter/agdb usages in studies that stimulated the development of many queries, along with the outstanding cooperation of the amish community, without which the aforementioned studies would not have been possible. thanks to eric grant for suggesting the all_relatives query and for various other suggestions that improved the documentation. author details national center for biotechnology information, national library of medicine, national institutes of health, rockville pike, bethesda, maryland , usa, department of medicine, division of endocrinology, diabetes and nutrition, university of maryland school 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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= lee et al. bmc medical genetics , : http://www.biomedcentral.com/ - / / page of . streeten ea, mcbride dj, lodge al, pollin ti, stinchcomb dg, agarwala r, schäffer aa, shapiro jr, shuldiner ar, mitchell bd: reduced incidence of hip fracture in the old order amish. j bone miner res , : - . . daley e, streeten ea, sorkin jd, kuznetsova n, shapses sa, carleton sm, shuldiner ar, marini jc, phillips cl, goldstein sa, leikin s, mcbride dj jr: variable bone fragility associated with an amish col a variant and a knock-in mouse model. j bone miner res , : - . . pardo lm, mackay i, oostra b, van duijn cm, aulchenko ys: the effect of genetic drift in a young genetically isolated population. ann hum genet , : - . pre-publication history the pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/ - / / /prepub doi: . / - - - cite this article as: lee et al., pedhunter . and its usage to characterize the founder structure of the old order amish of lancaster county bmc medical genetics , : http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ - / / /prepub 	abstract 	background 	methods 	pedhunter versions 	queries in pedhunter . 	examples of queries useful for analysis of the anabaptist genealogy database 	pedigree renumbering 	anabaptist genealogy database version . 	prediction of old order amish status 	pedigree trimming on founders 	estimation of birth years for founders 	mean relative founder representation 	results 	distribution of old order amish 	old order amish founder structure 	relevance of founder structure to published and ongoing studies 	excess of female founders 	discussion/conclusions 	additional material wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ linkage of plasma adiponectin levels to q explained by association with variation in the apm gene toni i. pollin, keith tanner, jeffrey r. o’connell, sandra h. ott, coleen m. damcott, alan r. shuldiner, , john c. mclenithan, and braxton d. mitchell we performed a genome-wide linkage scan of plasma adiponectin levels in nondiabetic participants in the amish family diabetes study. the highest logarithm of odds (lod) score ( . ; p � . ) occurred on chro- mosome q between markers d s and d s , which flank apm /acdc, the adiponectin gene. the apm � a/� insertion/deletion polymorphism in the � untranslated region (single nucleotide polymor- phism [snp] � ; deletion allele frequency . in amish) showed strong association with adiponectin lev- els in a dosage-dependent manner in a direction con- sistent with that reported in previous studies, with deletion heterozygosity increasing adiponectin levels by . � . �g/ml and deletion homozygosity increasing levels by . � . �g/ml (p < . ). two other snps, rs and rs , showed moderate associa- tion. in a subset of subjects genotyped for both snp � and rs , including the apm snp � genotype as a covariate reduced the linkage signal at q by . lod units (from . to . ) and including both snps reduced the signal by . lod units (to . ). these findings, combined with a two-point lod score of . for snp � , provide evidence that variation in apm is responsible for linkage of adipo- nectin levels to q in the old order amish. diabetes : – , a diponectin is a fat-secreted hormone involved in insulin sensitivity, free fatty acid oxidation, and inhibition of inflammation ( , ). circulat- ing adiponectin levels are decreased in obesity ( ), diabetes ( ), hypertension ( ), and dyslipidemia ( ), which are components of the metabolic syndrome, or in- sulin resistance syndrome. adiponectin is one of a number of adipose tissue�secreted proteins collectively called adipocytokines, the discovery of which in the past decade has radically changed the view of adipose tissue from that of a simple energy storage depot to that of an active endo- crine organ. also known as acrp , adipoq, and gbp , adiponectin was discovered in and as the most abundantly expressed sequence tag in an adipose mrna library ( – ). the chromosomal region q , which con- tains apm (also known as acdc), the gene encoding adiponectin, has been linked to several features of the metabolic syndrome ( ) and diabetes ( ). adiponectin levels have been significantly linked to chromosomes ( , ), and ( ), and ( ) and suggestively linked to others ( , ). several investigators have characterized single nucleotide polymorphisms (snps) in the adiponec- tin gene and found one or more of these snps to be associated with diabetes ( ) and other metabolic syn- drome features, as well as with adiponectin levels them- selves ( – ). we measured adiponectin levels in participants in the amish family diabetes study (afds) to ) confirm and further define the relation between adiponectin levels and other metabolic syndrome components ( ) and ) iden- tify loci harboring genetic variants that may influence adiponectin levels (and thus insulin resistance and type diabetes). after finding that our strongest linkage signal for adiponectin levels in our genome scan occurred in the chromosomal region containing apm , the adiponectin structural gene, we performed association and linkage analysis between apm variation and adiponectin levels. our findings suggest that sequence variation in apm influences adiponectin levels. research design and methods recruitment for the afds began in early with the goal of identifying genes influencing the risk of type diabetes and related traits. the study protocol was approved by the institutional review board at the university of maryland school of medicine, and informed consent was obtained from each study participant. with the help of liaisons from the old order amish community, we identified individuals with type diabetes. these probands and their first- and second-degree relatives and spouses age years or older were invited to participate. all subjects could be connected into a single -generation pedigree ( ). among the individuals recruited and included in a -cm genomewide scan, there were subjects with type diabetes ( prevalent cases and incident cases) contained in sibships (including affected subjects in multiplex sibships). stored fasting plasma from subjects (including with diabetes) of the scanned subjects was available for measuring adiponectin levels. because of the potential influence of diabetes and its treatment on adiponectin levels, we excluded type diabetic subjects from the analysis, leaving a total of subjects for the linkage analysis. fasting plasma adiponectin levels were measured in an from the university of maryland school of medicine, baltimore, maryland; and the baltimore veterans affairs medical center, geriatric research, education and clinical center, baltimore, maryland. address correspondence and reprint requests to toni i. pollin, phd, division of endocrinology, diabetes, and nutrition, university of maryland school of medicine, w. redwood st., rm. , baltimore, md e-mail: tpollin@medicine.umaryland.edu. received for publication july and accepted in revised form september . afds, amish family diabetes study; ld, linkage disequilibrium; qtl, quantitative trait locus; snp, single nucleotide polymorphism; solar, se- quential oligogenic linkage analysis routines; utr, untranslated region. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , january additional nondiabetic subjects not included in the original genome-wide scan/linkage analysis; these subjects were included in the association analysis (for a total of subjects). phenotypes. blood samples were obtained from an antecubital vein after an overnight fast at the amish research clinic in strasburg, pennsylvania, or in subjects’ homes. adiponectin levels were measured in fasting plasma samples using a commercial radioimmunoassay kit (linco) according to the manufac- turer’s instructions. the manufacturer reports coefficients of variation of . – . and . – . % for inter- and intra-assay precision, respectively. samples were diluted at : ( �l plasma in , �l milliq water). � counts were measured for min on an automated � counter (packard cobra-ii auto gamma). all samples were assayed in duplicate, and results of samples with � counts differing by � % were discarded and the samples retested. assays in which adiponectin levels for quality control samples did not fall into the expected range were discarded. other phenotyping for the afds has been previously described in detail ( ). short tandem repeat genotyping. genotyping was performed on dna extracted from leukocytes using a screening set of highly polymorphic microsatellite short tandem repeat markers on the autosomes and the x chromosome from the abi prism linkage mapping set (applied biosystems division/perkin-elmer, foster city, ca). the mean marker heterozygosity was . (range . – . ). the average intermarker distance was . cm. the largest gap between markers was . cm, occurring on chromosome . the genotyping error rate based on blind replicates was . % on average. candidate gene snp genotyping. the adiponectin structural gene, apm or acdc, is � kb in length and contains three exons, one noncoding and two coding. from public databases and previous publications ( , ), we selected five snps (rs [promoter]), rs [promoter], rs [intron ], rs [exon ], and rs [intron ]) and one single base pair insertion/deletion polymorphism , bp downstream of the atg start codon in the � untranslated region (hereafter denoted as snp � ). these polymorphisms were chosen because they were distributed throughout the gene, had been previously reported to be common, and in some studies had been found to be associated with diabetes and related phenotypes in cauca- sian populations. the five snps were genotyped using the snpstream ultra high throughput genotyping system (beckman coulter, fullerton, ca) according to the manufacturer’s instructions. briefly, the target genomic sequences containing the snps were amplified as part of -plex pcrs, which were subjected to an extension reaction using �-tagged extension primers and fluorescent dye�labeled terminators. in a thermal cycled extension step, the primers hybridized to the specific amplicons one base adjacent to the snp site and were extended by one base at the � end with a fluorescently labeled nucleotide. these reaction products were transferred to an array plate, where each of the extension products in the multiplex reaction were sorted by hybridization of the unique �-tag sequence to its complementary probe immobilized in a mini-array within each well. the plates were imaged by the snpscope (beckman coulter). snps were called by comparing the two fluorescent signals. the error rates for these snps based on % blind replicates were %, except for snps rs and rs , which had error rates of . and . %, respectively. snp � was genotyped using the psq hs a pyrosequencer according to the manufacturer’s methods (pyrosequencing, uppsala, sweden; www. pyrosequencing.com). briefly, a pcr product was generated from a primer pair that included one primer covalently coupled to biotin, the biotinylated template was bound to streptavidin-coated sepharose hp beads, and this mixture was then annealed to a sequencing primer. stepwise elongation of a sequencing primer strand upon sequential addition of a specified sequence of deoxynucleotide triphosphates and the degradation of nucleotides by apyrase were carried out simultaneously. as the sequencing reaction progressed, the dna strand was extended and the sequence was determined from the measured signal output of light upon nucleotide incorporation. the resulting peaks in the pyrogram were analyzed using pyrosequencing software. the error rate for snp � based on % blind replicates was %. sequencing. to determine if snps found to be associated with variation in adiponectin levels were marking other functional snps, particularly those altering an amino acid sequence, we sequenced the entire coding region of apm , which comprises most of exon and a portion of exon , in a subset of individuals enriched for the presence of associated polymorphisms. sequencing was performed on an abi dna sequencer. statistical analysis. although virtually all subjects could be connected into a single -generation pedigree, we divided the sample into discrete fam- ilies for linkage analysis to reduce computational burden. these families contained – genotyped and phenotyped individuals and provided a large number of relative pairs, including parent-offspring pairs, sibling pairs, avuncular (aunt/uncle�niece/nephew) pairs, and first-cousin pairs. heritability, linkage, and association analyses were carried out using a variance components methodology. for heritability and linkage analyses, we partitioned variation in the adiponectin trait into components attributable to environmental covariates, the additive effects of genes (i.e., residual herita- bility), and a specific quantitative trait locus (qtl, the linkage component). these analyses were conducted using maximum likelihood procedures as im- plemented in the sequential oligogenic linkage analysis routines (solar) program ( ). the residual heritability was modeled as a function of the expected genetic covariances between relatives, and the qtl effect was mod- eled as a function of the identity-by-descent probabilities at the marker locus. allele frequencies were estimated from the data using maximum likelihood methods ( ). the hypothesis of linkage was evaluated by the likelihood ratio test, which determines whether the locus-specific effect is significantly � (ho [null hypothesis]: � qtl 	 vs. ha [alternative hypothesis]: � qtl � ). in each model, we simultaneously adjusted for the effects of sex and sex-specific age and age squared. because of the sensitivity of variance components to violations of normality, the three adiponectin data points whose values were � sd from the mean were truncated and their values set to the value corresponding to sd from the mean (winsorization; ). to estimate the probability of obtaining a false-positive result and to obtain empirical loga- rithm of odds (lod) scores, we used the lodadj routine in solar to generate , fully informative unlinked markers. we evaluated evidence for linkage of adiponectin levels to these simulated markers and defined the probability of obtaining a false-positive result as the proportion of the , replicates for which a lod score greater than or equal to that observed for the original linked locus was obtained. all lod scores in this study were obtained by converting the empirical p value obtained by simulation to its correspond- ing lod score {lod 	 ÷ [ *ln ( )]}. before association analysis, genotypes were checked for mendelian con- sistency using the pedcheck software package ( ) and inconsistencies (� . % of genotypes) were resolved or removed. using variance components, we estimated the effects of the snp genotype on adiponectin levels, adjusting for sex and sex-specific age and age squared). genotype was parameterized according to the number of copies of the minor allele the subject carried (genotype 	 if major allele homozygote, if heterozygote, and if minor allele homozygote). parameter estimates were obtained conditional on the pedigree structure. for each snp, the likelihood ratio test was used to compare a model that included the apm snp as a covariate with a model that did not include the snp. allele frequencies were estimated by the maximum likelihood estimates conditioned on the pedigree structure as implemented in solar. linkage disequilibrium (ld) was estimated using an algorithm implemented in the software package zaplo, which uses both pedigree and ld information to find all possible haplotype configurations of the data under a zero-recombinant assumption ( ). to test whether the apm association explained the linkage to q , variance components linkage analysis was repeated with the associated apm polymorphism genotype as a covariate. the gene-dropping option of mendel . ( ) was used to simulate the segregation , times of an unlinked snp with the same frequency as the apm polymorphism. these simulated snp genotypes were then used as covariates in , repetitions of the linkage analysis to attempt to assess the significance of the lod change that occurred with the inclusion of the apm variant as a covariate. power estimation. we evaluated the power of our sample to detect linkage to qtls accounting for , , and % of the total variation in a model trait using simulation, as previously described ( ). for each of the three effect sizes, qtls were simulated and the power to detect linkage was defined as the proportion of replicates for which we obtained lod scores greater than selected values ( . and . ). results heritability and genome scan. the heritability of the plasma adiponectin trait, adjusted for sex and sex-specific age and age squared, was estimated at . � . . the maximum lod score in the entire genome scan was . (p 	 . ), occurring on chromosome q between markers d s and d s (fig. and table ). we observed four other regions of suggestive linkage (table ): q – , near marker d s (lod 	 . , p 	 . ); q , between markers d s and d s (lod 	 . , p 	 . ); p – , near marker d s (lod 	 . , p 	 . ); and p , near marker d s (lod 	 . , p 	 . ). t.i. pollin and associates diabetes, vol. , january power estimation. the results of the power simulation indicated that the sample provided reasonable power ( %) to detect linkage (at lod scores � . ) for a qtl accounting for � % of the total trait variance. for a qtl accounting for % of the variance, power would be % to detect a lod score � . and % for a lod score � . . the power was much lower for a qtl accounting for only % of the variance ( % for a lod score � . and % for a lod score � . ). candidate gene analysis. the two markers flanking our strongest linkage signal, d s and d s , flank a . -mb region that contains the structural gene (apm ) encoding adiponectin. this observation motivated us to evaluate whether sequence variation in apm might influ- ence adiponectin levels in this population. we genotyped five snps and an insertion/deletion polymorphism (snp � ) distributed throughout the apm gene and known to be polymorphic in caucasian populations ( , ) and then performed single snp association analyses. as shown in table , the strongest association occurred with snp � , the single nucleotide insertion/deletion polymor- phism in the � untranslated region. heterozygosity for the insertion/deletion polymorphism was associated with an increase in the adiponectin level of . � . �g/ml, and deletion homozygosity was associated with an increase of . � . �g/ml (p � . for additive model). the rs and rs snps showed moderate associa- tion in a dosage-dependent manner with adiponectin levels (p 	 . and . , respectively). when individuals with the rs gt or gg genotypes were excluded from the analysis (remaining n 	 ), the association of snp � with adiponectin levels persisted, with 
 	 . � . �g/ml, 
 	 . � . �g/ml, and p 	 . , suggesting that the snp � association with adiponec- tin levels is largely independent of rs . however, when rs was added to a model in the full set already containing snp � , the log likelihood in- creased from � , . to � , . for a nearly signifi- cant p value of . , suggesting an independent effect of rs . the further addition of rs to the two- fig. . genome-wide scan results by chromosome. linkage and association of adiponectin levels diabetes, vol. , january snp model did not alter the association. the snps rs , rs , and rs showed no association with adiponectin levels. including the additive effect of snp � in the vari- ance components model decreased the variance in adi- ponectin levels by . %, and including both snp � and rs decreased the variance by . % from base- line. the combination of all six snps resulted in a de- crease of . % from baseline. as shown in table , the three associated snps are in strong ld with one another, as estimated by pairwise d� coefficients ( . – . ), but weakly predictive of one another (because of differing allele frequencies), as indi- cated by low to moderate pairwise r estimates ( . – . ). the rs g allele, which is much less common than the snp � -dela allele ( . vs. . ), is found almost exclusively in one of the haplotypes containing the snp � -dela allele (table ). the rs -t allele is found on the majority of rs -snp t-dela hap- lotypes and is absent from the g-dela haplotype. taken with the association findings discussed in the preceding paragraph, the association of rs with adiponectin levels may be an independent effect or may be an enhance- ment of the snp � effect. unfortunately, these two possibilities cannot be distinguished by the association data. in contrast, the modest rs association is most likely driven by its ld with its flanking snps. to assess whether the association with apm polymor- phisms explained the linkage at q , we reran the ge- nomewide scan using the snp � and/or rs genotypes as covariates. when snp � was included in the model, the lod score decreased . units from . to . , corresponding to an . -fold (i.e., . -fold) de- crease in the evidence for linkage (lod scores computed on the sample containing subjects genotyped for snp � and rs ) (fig. ). when snp rs was added to the base linkage model, there was a . -unit drop in the lod score, and when both snp � and rs were added simultaneously to the model, there was a . -unit drop in the lod score, for a total . -fold decrease in the evidence for linkage. the two-snp model provided a significantly improved fit over the model in- cluding snp � only (p 	 . ). assessing whether snp � accounted for a “signifi- cant” proportion of the linkage was not straightforward. to gain some insights into this issue, we performed a simu- lation in which we dropped an unlinked snp of the same frequency ( . ) , times through a -generation table multipoint linkage analysis peaks with lod � . (corresponding to p � . ) chromosome location distance (cm) closest marker(s) lod (p) nearby linkages in related traits in old order amish nearby linkages to related traits in other populations q d s /d s . ( . ) — multiple metabolic syndrome qtls (bmi, waist, hip, weight, insulin, insulin:glucose): lod 	 . – . @ – cm ( ) diabetes: mls 	 . and mlb- lod 	 . @ d s ( ) q – d s . ( . ) disinhibition (eating behavior): lod 	 . @ cm ( ) glucose : lod 	 . @ cm ( ) — q d s . ( . ) total serum cholesterol: lod 	 . @ cm ( ) — p -p d s . ( . ) bmi-adjusted leptin: lod 	 . @ cm ( ) adiponectin levels: lod 	 . @ cm ( ) obesity: mls 	 . @ cm ( ) bmi: lodfa 	 . @ cm ( ) p d s . ( . ) bmi: lod 	 . @ cm ( ) leptin: lod 	 . @ cm ( ) fasting glucose to fasting insulin: lod 	 . @ cm ( ) — lodfa, paternal lod; mlb, maximum likelihood binomial; mls, maximum likelihood score. table association of six apm snps with adiponectin levels snp name gene region position (from atg start codon) base change minor allele frequency mean adiponectin level (�g/ml) p (additive model) rs promoter � , c g . . � . ( ) . � . ( ) . � . ( ) . rs promoter � , a g . . � . ( ) . � . ( ) . � . ( ) . rs intron � , a g . . � . ( ) . � . ( ) . � . ( ) . rs exon � t g (gly gly) . . � . ( ) . � . ( ) . � . ( ) . rs intron � g t . . � . ( ) . � . ( ) . � . ( ) . snp � � utr � , insa dela . . � . ( ) . � . ( ) . � . ( ) � . data are means � se (n of individuals). mean adiponectin levels are adjusted for age and age squared; all analyses are adjusted for sex and sex-specific age and age squared. ins, insertion; del, deletion; , homozygous for major allele; , heterozygous; , homozygous for minor allele. t.i. pollin and associates diabetes, vol. , january pedigree connecting virtually all subjects. we then repeat- ed the linkage analysis using the simulated snp as a covariate. none of the replicates resulted in a reduction as great as . lod units, suggesting that association with apm snp � , and possibly rs and/or nearby apm snps, is quite likely the cause of the linkage to q . furthermore, the two-point lod score for apm snp � was . , whereas those for the other poly- morphisms ranged from . (rs ) to . (rs , which was also the next most significantly associated polymorphism). because we did not initially sequence apm in an amish sample, but rather selected polymorphisms for genotyping based on published information, we followed up the pos- itive association results with direct sequencing analysis of the coding region (the � end of exon and the � end of exon ) to determine whether our positive associations might be tracking a potentially functional coding variant. the sample for sequencing consisted of a set of rela- tively unrelated married couples ( individuals, al- leles) in upper generations in which at least one member was heterozygous for the g allele at rs . we did not detect any variants in the coding region that would explain the association. discussion although we were unable to detect chromosomal loci linked to adiponectin levels significant at the lod � . level, five regions ( q , q – , q , p -p , and p ) provided evidence of suggestive linkage. the high- est lod score, . , was observed in the region contain- ing apm , the adiponectin structural gene. although apm might seem an obvious candidate gene for control of adi- ponectin levels, a genome-wide scan for adiponectin levels in pima indians showed no evidence for linkage to this region (although suggestive linkage was observed between markers d s and d s , which map to q ) ( ). in contrast, strong linkage of adiponectin levels to q has been recently reported in the iras (insulin resistance atherosclerosis study) family study (lod 	 . ) ( ). one interesting finding of that study was that evidence for linkage was provided entirely by the hispanic-american sample (lod 	 . ); no evidence for linkage was seen in the african-american families (lod 	 . ) ( ). weak linkage to q (lod 	 . ) was found in another genome- wide scan of adiponectin levels in caucasians, where much higher signals on chromosomes and were seen ( ). linkage in the q – region (lod 	 . ), about cm centromeric to q , has been observed in mexican americans ( ). based on these findings, it is possible that variation in apm is an important determinant of circulat- ing adiponectin levels in some populations, but not others. our linkages for adiponectin on chromosomes q – , q , p – , and p are close to linkages we ob- served in the amish for other obesity-related traits, such as total serum cholesterol, bmi-adjusted leptin, and bmi (table ). although some of these traits are somewhat correlated with adiponectin levels ( ), they provide addi- tional support for the idea that these regions are true linkages rather than false-positives. the p region has also been reported by others to be close to linkages for adiponectin ( ), obesity ( ), and bmi ( ), thus provid- ing additional evidence that a gene influencing obesity and related traits resides in this region. to confirm our suspicion that the linkage signal on q resulted from variation in the adiponectin gene itself, we performed association analysis with six previously report- ed snps (table ). snp � showed strong association with adiponectin levels and appeared to explain most of the linkage signal at q , as accounting for this snp led to a % drop in the lod score, corresponding to an . -fold drop in the evidence for linkage. this insertion/deletion polymorphism has been previously reported to be associ- ated with adiponectin levels ( ). another group has found the corresponding insertion to be in ld with the t/ g haplotype previously associated with type diabetes ( ) and has suggested that snp � may be a causal variant via mechanisms involving regulatory elements in the � region of the apm gene. based on searches in utrsite and utrscan, the polymorphism does not reside within a known functional sequence; the closest known func- tional element identified is a k-box binding region bp downstream. however, functional elements of the � untranslated region (utr) continue to be elucidated, and the possibility that the variant alters a protein or micro- rna binding site and/or rna secondary structure cannot be not ruled out ( ). studies of the effect of the polymor- phism on rna production and stability along with addi- tional characterization of the � utr sequence will be need- ed to confirm the causative nature of this polymorphism. table estimated pairwise ld for apm snps rs rs rs rs rs snp � rs — . . . . . rs . — . . . . rs . . — . . . rs . . . — . . rs . . . . — . snp � . . . . . — upper triangle shows r and lower triangle shows d�. table haplotype frequencies (� %) of associated snps rs rs snp � frequency cumulative frequency t g insa . . t t dela . . g g dela . . t g dela . . ins, insertion; del, deletion. linkage and association of adiponectin levels diabetes, vol. , january in addition to association with snp � , there ap- peared to be a further source of association in rs independent from the snp � association, as evi- denced by ) an apparent additive effect between the two polymorphisms that reached statistical significance in the linkage analysis and ) the persistence of the snp � association when the sample was restricted to those with the rs tt genotype. no common variants were detected in a set of relatively unrelated individuals enriched for the presence of the rs and snp � minor alleles, increasing the likelihood that these variants directly influence adiponectin levels, although the possi- bility remains that variants outside the coding region are influencing the phenotype. as in the amish, the rs -t allele has been asso- ciated with higher adiponectin levels in a japanese popu- lation ( ). the amish data also replicate associations of the rs -g, rs -t, and snp � -del al- leles with increased adiponectin levels in the french population ( ). unlike the french ( ), the amish did not show association of the rs -g allele with decreased adiponectin levels; however, a follow-up analysis of the amish in which subgroups in the lower and upper th bmi percentile were analyzed separately did reveal a recessive association between the rs -g allele and lower adiponectin levels in the high bmi (� . ) group only (p 	 . ). the two-point linkage analysis of the relation of the snps themselves to adiponectin levels provides additional evidence that the sequence variation within apm ex- plains the q linkage, as the highest lod score among the snps, . , which was almost identical to the multi- point peak score, was observed at the polymorphism (snp � ) with the strongest association result. in summary, we have provided evidence for linkage of adiponectin levels to several distinct regions in the ge- nome in the amish. among them, q contains the struc- tural gene for adiponectin itself, apm , in which sequence variation appears to be an important determinant of adi- ponectin levels. because adiponectin is a key regulator of insulin sensitivity and other metabolic syndrome�related traits, we would expect this gene, and likely specific snps therein (e.g., snp � , rs ) to be important con- tributors to these traits. replication of our findings along with further investigation of sequence variation within apm and functional analysis of these variants will be required to further discern the role of apm as a susceptibility gene for obesity, type diabetes, and the metabolic syndrome. acknowledgments this study was supported in part by a research grant from glaxowellcome and axys pharmaceuticals; national insti- tutes of health grants dk- and dk- ; an amer- ican diabetes association research award to a.r.s.; an american heart association mid-atlantic predoctoral fel- lowship to t.i.p.; university of maryland general clinical research center grant m -rr- ; and the general clinical research centers program, national center for research resources, national institutes of health. the authors thank wendy warren, mary ann drolet, denis massey, mary morrissey, janet reedy, and our amish liaisons for their energetic efforts in study subject recruit- ment and characterization; drs. alejandro schäffer and fig. . effect of adjusting for rs and snp � on lod score at q . t.i. pollin and associates diabetes, vol. , january richa agarwala for assistance in pedigree construction; and jack shelton for sequencing. this work would not have been possible without the outstanding cooperation of the amish community. appendix electronic database information for internet-based re- sources used in this study: ) ncbi unists: http://www. ncbi.nih.gov/entrez/query.fcgi?db	unists; ) ucsc hu- man genome browser gateway: http://genome.ucsc.edu/cgi- bin/hggateway; and ) utrscan/utrsite: http://bighost. area.ba.cnr.it/big/utrhome/. references . havel pj: update on adipocyte hormones: regulation of energy balance and carbohydrate/lipid metabolism. diabetes (suppl. ):s –s , . matsuzawa y, funahashi t, kihara s, shimomura i: adiponectin and metabolic syndrome. arterioscler thromb vasc biol : – , . arita y, kihara s, ouchi n, takahashi m, maeda k, miyagawa j, hotta k, shimomura i, nakamura 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genetic factors influencing acquisition of peak bone mass account for a substantial proportion of the variation in bone mineral density (bmd), although the extent to which genes also contribute to variation in bone loss is debatable. few prospective studies of related individuals have been carried out to address this issue. to gain insights into the nature of the genetic factors contributing to variation in bmd, we studied wo- men from large amish families. we evaluated and compared the genetic contributions to bmd in pre- and post-menopausal women, with the rationale that genetic variation in pre-menopausal women is due primarily to genetic determinants of peak bone mass, while genetic variation in post-menopausal women is due to the combined genetic effects of peak bone mass and bone loss. bone mineral density was measured at one point in time at the hip and spine by dual energy x-ray absorp- tiometry (dxa). we used variance decomposition pro- cedures to partition variation in bmd into genetic and environmental effects common to both groups and to pre- and post-menopausal women separately. total variation in bmd was higher in post- compared to pre-menopausal women. genes accounted for – % of the total variation in bmd in pre-menopausal women compared to – % of the total variation in post- menopausal women. in absolute terms, however, the genetic variance was approximately similar between the two groups because the environmental variance was / - to -fold larger in the post-menopausal group. the genetic correlation in total hip bmd was . between pre- and post-menopausal women and differed signifi- cantly from one, consistent with the presence of at least some non-overlapping genetic effects in the two groups for bmd at this site. overall, these analyses suggest that many, but not all, of the genetic factors influencing variation in bmd are common to both pre- and post- menopausal women. keywords bone loss Æ bone mineral density Æ genetics Æ heritability Æ peak bone mass Æ variance introducton osteoporosis is a major public health problem that is associated with significant morbidity and mortality. according to the third national health and nutrition examination survey (nhanes iii), % of u.s. women over age have low bone mass and % of white postmenopausal women have osteoporosis at the femoral neck (hip) [ ]. osteoporotic fractures are one of the most common causes of disability and contributors to medical care costs in many regions of the world [ ]. large prospective studies have shown that almost all types of fracture are increased in adults with low bone mineral density (bmd) [ ]. fractures result in functional impairment, including impaired basic activities of daily living, subsequent nursing home care, the loss of ambulatory ability and loss of the ability to live inde- pendently [ , ]. risk of fracture is particularly acute in women be- cause of the sharp decline in bmd that begins at about osteoporos int ( ) : – doi . /s - - - l.b. brown Æ p.a. peyser department of epidemiology, university of michigan school of public health, ann arbor, mi, usa e.a. streeten Æ d. mcbride Æ a.r. shuldiner Æ b.d. mitchell (&) division of endocrinology, diabetes and nutrition, university of maryland school of medicine, w redwood street, baltimore, md , usa e-mail: bmitchel@medicine.umaryland.edu tel.: + - - fax: + - - j.r. shapiro kennedy krieger institute, baltimore, md, usa j.r. shapiro department of physical medicine and rehabilitation, johns hopkins university, baltimore, md, usa a.r. shuldiner geriatric research and education clinical center (grecc), veterans administration medical center, baltimore, md, usa the time of the menopause. on average, women lose to % of their bmd per year during the first years after menopause, and this rate of bone loss is faster in the spine than other skeletal sites [ , ]. after this period of accelerated bone loss, the rate of bone loss slows down until age , when bone loss begins to accelerate again [ ]. despite this overall trend, there appears to be con- siderable variability among women in the rate of bone loss, with up to % of women losing at least % per year for more than years [ ]. at least one study has shown that with over – years of follow up, the risk of fracture was increased to a similar degree for women starting out with low bone mass (defined as a t score <) sd) and for women starting out with normal bone mass, but experiencing a rapid rate of bone loss, with each group experiencing a doubling of the fracture risk. when baseline bone mass was low and bone loss was rapid, however, the risk of fracture was higher still (odds ratio = . ) [ ]. from studies of young adults, it is well established that peak bone mass is under substantial genetic control [ , , , , , , , , , ]. additionally, women with a maternal history of hip fracture have lower bmd than women without a history of such fracture [ ] and are themselves twice as likely to suffer a hip fracture [ , ]. there are very few published data, however, on the heritability of bone loss. kelly et al. estimated genetic effects on changes in spine and hip bmd measured years apart from a sample of monozygotic (mz) and dizygotic (dz) twin pairs. these investigators observed a significantly higher cor- relation in spine bmd change among mz ( r = . ) compared to dz ( r = . ) twin pairs, with differences consistent with a heritability in spine bmd change as high as % [ ]. correlations in bmd changes at several hip sites were also higher among mz compared to dz twin pairs, although these differences did not achieve statistical significance in this small sample. in contrast, christian et al. reported no difference in -year changes in forearm bmd between and dz twin pairs [ ]. these results may indicate that bmd change is heritable in some sites (e.g., the spine) more than others (e.g., the forearm), or alternatively, that genetic influences on bmd changes at the forearm (and perhaps other sites) are not detectable over a -year period. in a previous study, we used a variance decomposi- tion approach to evaluate the relative contributions of genetic and environmental effects in accounting for variation in male and female differences in bmd [ ]. these analyses did not provide evidence for sex-specific genetic effects, suggesting that genes influencing varia- tion in bmd should be detectable, and in many cases common, in both men and women. in the current study, to begin to dissect genetic components of peak bone mass and bone loss, we evaluated the genetic and envi- ronmental contributions to bmd in women before and after menopause. the rationale for this approach is that genetic variation in pre-menopausal women is due pri- marily to genetic determinants of peak bone mass, while genetic variation in post-menopausal women is due to the combined genetic effects of peak bone mass and bone loss. specifically, we considered the following questions: ( ) is the magnitude of the genetic variation larger in pre-menopausal women than in post-menopausal women?; ( ) does the magnitude of the environmental variation differ between pre-menopausal women and post-menopausal women?; ( ) is there evidence for menopausal-status-specific genetic effects on bmd (i.e., is there a subset of genes that influences variation in bmd in all women and another subset of genes that influences variation in pre- and post-menopausal women separately)? our findings suggest that there are common genetic contributions to bmd in both pre- and post- menopausal women (presumably determinants of peak bone mass), but in addition a separate genetic contri- bution, albeit modest, to bmd in post-menopausal women (presumably determinants of bone loss). materials and methods subjects and measurements the amish family osteoporosis study (afos) began in with the goal of identifying the genetic determi- nants of osteoporosis. details of ascertainment, pheno- typing and clinical characteristics of afos participants were reported previously [ , ]. briefly, individuals believed to be at risk for osteoporosis by virtue of their fracture history or prior bone density measurements were recruited into the study as index cases. these individuals were recruited by word-of-mouth, a com- munity-wide mailing, advertisements in an amish newspaper or by referral from local physicians. the diagnosis of osteoporosis in these individuals was veri- fied by the measurement of bmd using dual energy x- ray absorptiometry (dxa). individuals found to have a t score of – . or less in either the hip or spine were designated as probands. we then invited the probands’ spouses and all first-degree relatives aged years and over to participate in the study. in addition, we recruited into the study the first-degree relatives of any other examined individual (e.g., spouses) having a t score of ) . or lower at the spine or hip on our bone densi- tometry test. between the initiation of recruitment in and february , a total of , individuals ( women and men) were enrolled into the afos, including probands and their relatives. complete information on dxa phenotypes and menopausal status was obtained from of the women enrolled. using the extensive genealogical records maintained by the amish [ , , ], these individuals could be combined into a single -generation pedigree. study participants were evalu- ated by qualified nurses known to the participants at the amish research clinic in strasburg, pa. a medical interview included past medical history, family history of medical problems including fractures and specific details regarding previous fractures, history of medica- tion use and menstrual and reproductive history for women. height was measured using a stadiometer, and weight was recorded with the participant in standard amish clothing, but without shoes. women were con- sidered to be post-menopausal if they reported fewer than two menstrual cycles over the previous months. thirty-three women reported a history of oophorecte- my/hysterectomy and were considered for purposes of these analyses to be post-menopausal. the mineral content at the lumbar spine and hip was measured by dual energy x-ray absorptiometry (dexa) by a registered nurse certified in bone densitometry (hologic w, hologic, inc., bedford, mass.). bmd was determined by dividing the total bone mineral content (g) by the projected area of the region scanned (cm ). for this report, we have restricted analysis of bmd to measures obtained at the spine, femoral neck and total hip. total hip bmd was defined as the sum of the bone mineral content at the femoral neck, trochanter and intertrochanter sites divided by the total area of these three sites. the protocol for the afos was approved by the institutional review board at the university of mary- land. informed consent was obtained from all subjects prior to participation. analytical methods we carried out a series of statistical analyses using a full pedigree-based variance component approach for the purpose of partitioning variation in bmd into selected components [ ]. initially, we modeled variation in bmd as a function of measured environmental covari- ates [e.g., age, age , height and body mass index (bmi)], additive genetic effects (or heritability) and a residual error component. maximum likelihood methods were used to estimate the covariate and genetic effects simultaneously. the covariates selected were included because they were each independently associated with one or more bmd measures in preliminary analyses on pre- and post-menopausal women separately. the sig- nificance of particular components can be assessed by comparing the likelihood of a model with the compo- nent of interest estimated to the likelihood of a model in which the component effect is constrained to a pre- specified value (e.g., zero). the full and restricted models are then compared by likelihood ratio test, which pro- duces a test statistic that is asymptotically distributed as a v distribution. in an initial variance component analysis, we esti- mated the proportion of the total phenotypic variation in bmd (rp ) that could be attributable to the additive genetic effects in pre- and post-menopausal women separately (rg / rp ). this effect corresponds to ‘narrow’ sense heritability since it reflects the degree of additive genetic variance only. we tested whether the heritabilities in bmd differed between pre- and post- menopausal women (i.e., h pre =h post) by comparing the difference between the heritability estimates in the two groups with the estimated variance of the difference. following the approach of blangero, [ , , ], we then expanded the basic variance component model to allow the genetic variances in pre-menopausal and post- menopausal bmd to differ. briefly, we constructed a general model that partitioned variance in bmd into the following terms: an overall mean, a coefficient cor- responding to the effect of menopausal status (bmenostat), coefficients for age and menopausal status*age (bage and bage*menostat), coefficients for age and menopausal sta- tus*age (bage and bage *menostat), coefficients for height (bheight), and bmi (bbmi), pre- and post-menopausal genetic standard deviations (rg-pre and rg-post), pre- and post-menopausal environmental standard devia- tions (re-pre and re-post) and the genetic correlation between pre- and post-menopausal women (qg). men- opausal status was coded as if post-menopausal and if not (i.e., pre- or peri-menopausal). the genetic cor- relation reflects the degree to which the genetic effect on bmd in pre-menopausal women correlates with the genetic effect of bmd in post-menopausal women [ ]. interaction terms of menopausal status with age and age were included because the relationship between age and bmd differs according to menopausal status. a more complete description of the statistical model has been previously published in a study where components of variance could vary by sex [ ]. this expanded model allowed us to test several explicit hypotheses related to menopausal status by gene interactions. first, we considered if the magnitude of the genetic effect was similar between the groups by testing whether the genetic standard deviations were similar between pre- and post-menopausal women (i.e., h : rg-pre=rg-post). rejection of this hypothesis implies that genes account for a larger proportion of the vari- ance in one menopausal status group than in the other. a second hypothesis that we tested is whether the magnitude of the genetic correlation was significantly less than one (i.e., h : qg (pre,post)= ). a genetic cor- relation between pre- and post-menopausal women that is significantly less than one implies that a different gene or suite of genes contributes to variance in bmd in pre- and post-menopausal women. as before, significance testing was conducted using the likelihood ratio test. specifically, we compared likelihoods between models in which values of rg-pre and rg-post were allowed to differ (full model) and in which they were constrained to be the same (restricted model). similarly, we compared the likelihood between a model in which q g(pre,post) was estimated (full model) to that in which its value was constrained to be one (re- stricted model). the degrees of freedom for the likeli- hood ratio test depend on whether the parameter of interest in the nested model is constrained to a boundary value (e.g., h = , where the possible range is to . ) or not [e.g., beta (covariate) = , where the possible range is -¥ to + ¥]. if the parameter constraint is not set to a boundary, then the degrees of freedom are equal to the difference in the number of parameters between the two models. if the parameter constraint is set to a boundary, then the degrees of freedom are based on a / : / mixture distribution with a point mass of zero (in which case the p -value is obtained by dividing the p -value of the one degree of freedom test by two) [ ]. results basic characteristics of the study population are shown in table . pre-menopausal women (n= ) ranged in age from to years, while the age of post-meno- pausal women (n= ) ranged from to . in addition to being older, post-menopausal women had significantly shorter height (p< . ) and higher bmi (p= . ) compared to pre-menopausal women. mean parity in the two groups was . ± . and . ± . births among pre- and post-menopausal women, respectively (p= . ). bmd measurements at the spine (l -l ), total hip and femoral neck were significantly higher in pre-menopausal women compared to post- menopausal women (p< . ). the overall (pheno- typic) variance in bmd was significantly greater in post-menopausal women than in pre-menopausal women at all three bmd sites. the numbers of pre- and post-menopausal relative pairs who were phenotyped and included in the analysis are shown in table . the sample included pre- menopausal pairs ( mother-daughter, sister-sister, aunt-niece and first cousin pairs) and post- menopausal pairs ( mother-daughter, sister-sister, aunt-niece and first cousin pairs). overall, there were , pairs of female relatives in the sample ( mother-daughter, sister-sister, aunt-niece and first cousin pairs). to gain insights into the factors contributing to variation in bmd in pre- and post-menopausal women, we partitioned the total variance in bmd into compo- nents attributable to measured covariates (e.g., age, age , height and bmi), the additive effects of genes and to unmeasured, or residual, environmental factors. re- sults of these analyses are shown in table . in pre- menopausal women, measured covariates accounted for % of the total variation in spine bmd, and from to % of the variation in hip bmd. the additive effects of genes accounted for to % of the total variation in hip bmd and % of the variation in spine bmd. thus, very little of the total variation in bmd in pre- menopausal women could not be accounted for by genes or measured covariates. since relatively little bone loss occurs in healthy adults prior to menopause, the varia- tion in bmd due to genes and environmental factors in this premenopausal group is likely to reflect the varia- tion in peak bone mass, typically achieved in the nd to rd decades of life. in post-menopausal women, the measured covariates accounted for % of the total variation in spine bmd and to % of the total variation in hip bmd. the additive effects of genes ac- counted for an additional % of the variation in spine bmd and to % of the total variation in hip bmd. an additional % of variation in spine bmd and femoral neck bmd and % of total hip bmd could not be accounted for by genes and/or measured covari- ates in the post-menopausal women. the determinants of bmd variation in post-menopausal women are likely to be genes and environmental factors that influence both peak bone mass and bone loss. another way to interpret the genetic effects described in table is in terms of the residual heritability, or the proportion of the unexplained phenotypic variance in bmd after accounting for the effects of the measured covariates age, age , height and bmi. the residual heritability of bmd ranged from . [femoral neck, computed as ( . /( . – . )] to . (total hip) in pre- menopausal women and from . (femoral neck) to . (total hip) in post-menopausal women. at each site, the estimated residual heritability in bmd was signifi- cantly larger in pre-menopausal women than in post- menopausal women (p< . at all sites) (data not shown). we then tested several additional hypotheses, including whether the magnitude of the genetic and environmental variances differed between women before table characteristics (mean ± sd) of female amish family osteoporosis study participants variable pre-menopausal women (n= ) post-menopausal women (n= ) p value equality of variance (p value) age (years) . ± . . ± . < . . height (in) . ± . . ± . < . < . weight (lb) . ± . . ± . . . bmi (kg/m ) . ± . . ± . . . parity . ± . . ± . . . bmd (g/cm ) spine (l -l ) . ± . . ± . < . < . total hip . ± . . ± . < . < . femoral neck . ± . . ± . < . . and after menopause and whether the genetic correlation in bmd differed between pre- and post-menopausal women. to accomplish this goal, we performed a more complete partitioning of bmd into its constituent genetic and environmental components. in these analy- ses, we allowed the genetic and environmental variances in bmd between pre- and post-menopausal women to differ, and also estimated the genetic correlations in bmd between pre- and post-menopausal women. results from the full model, in which all parameters were estimated, are shown in table . following esti- mation of the full set of model parameters, we per- formed a series of nested tests in which we constrained values of selected parameters, which enabled us to test, first, whether the magnitude of the genetic variance in bmd differed between pre- and post-menopausal wo- men; second, whether the magnitude of the environ- mental variance in bmd differed between pre- and post- menopausal women; third, whether the genetic correla- tion in bmd between pre- and post-menopausal relative pairs differed from one. with respect to the first hypothesis, we observed that the genetic sd did not differ significantly between pre- and post-menopausal women (rg-post vs. rg-pre: spine: . vs. . ; femoral neck: . vs. . ; total hip: . vs. . ; p > . for all). in contrast, the environmental sd was greater than three-fold higher at each site in post-menopausal com- pared to pre-menopausal women (spine: . vs. . ; total hip: . vs. ; femoral neck: . vs. . ), al- though in no case did these differences achieve statistical significance, perhaps because of the relatively small magnitude of the environmental variances in this pop- ulation. with respect to the third hypothesis, we ob- served that the genetic correlation between pre- and post-menopausal women did not differ significantly from one for the spine (qg= . , p = . ) and femoral neck (qg= . , p = . ), although the genetic corre- lation did differ significantly between the two groups for total hip (qg= . , p = . ), suggesting the possi- bility that different sets of genes may influence total hip bmd in pre- vs. post-menopausal women. in total, these analyses reveal total variance in bmd to be larger in post- compared to pre-menopausal women, with both measured and unmeasured environ- mental factors contributing to much of the excess vari- ability in the post-menopausal group. in contrast, there was little evidence for meaningful differences in the overall contribution of genetic factors to the variance in bmd between pre- and post-menopausal women, indi- cating that the magnitude of genetic influences on bmd in the two groups was approximately similar. the very high genetic correlations in bmd between pre- and post- menopausal women suggest that common genes, or sets of genes, influence bmd variation in both groups, although at the total hip there was modest evidence for unique or non-overlapping sets of genes that also influ- ence bmd in the two groups. discussion from large family samples, it has been estimated that genes account for – % of the total variation in bmd [ , , , , ]. however, this estimate incorporates genetic effects that occur at different ages. for example, a very large genetic contribution to acquisition of peak bone mass is well established [ ], although in later years table number of relative pairs included in the sample of amish female subjects relative pair class number parent-offspring (mother-daughter) pre-menopausal women post-menopausal women all pairs sibling-sibling (sister-sister) pre-menopausal women post-menopausal women all pairs avuncular (aunt-niece) pre-menopausal women post-menopausal women all pairs cousin-cousin ( st cousin only) pre-menopausal women post-menopausal women all pairs total numbers of female pairs* pre-menopausal pairs post-menopausal pairs total , *does not include grandparent-grandchild, grand avuncular, nd cousins, and more distantly related pairs table components of variance for bmd pre-menopausal women post-menopausal women (n= ) (n= ) bmd site: measured covariates genetic residual environment measured covariates genetic residual environment spine . . . . . . total hip . . . . . . femoral neck . . . . . . measured covariates include age, age , height and bmi another major source of variation, especially in women, is the rate of bone loss, much of which occurs during the peri- to post-menopausal period. the overall contribu- tion of genes to variation in bone loss is much less clear. understanding the factors influencing bone loss is very important from a therapeutic point of view since slowing the rate of bone loss presents a potentially valuable target for prevention of age-related osteoporotic fracture. the optimal approach for understanding the genetics of bone loss would be to follow a cohort of related individuals prospectively. however, only a few such studies have been published, and results have been inconclusive, with some reporting strong genetic effects on bone loss [ ] and others relatively modest effects [ ]. in the absence of more such studies, we have con- sidered an indirect approach using a cross-sectional family sample in which we compared genetic variation in bmd in pre- and post-menopausal women ranging in age from to years. the variance decomposition approach we used enabled us to address whether the same genes control variation in bmd variation in the two groups of women. one would expect there to be some overlap because bmd in older women is influ- enced by genes affecting both peak bone mass and bone loss, while bmd in younger women is influenced by genes affecting peak bone mass only. as expected, we observed significantly greater total variation in bmd in post- compared to pre-menopausal women. we further observed that after accounting for the effects of age, height and bmi on bmd, genes ac- counted for a larger proportion of the residual variation in bmd in pre- compared to post-menopausal women. these results are probably related to the fact that vari- ation in bmd in the post-menopausal group is influ- enced both by factors affecting peak bone mass and factors influencing the rate of bone loss. model parameters: l= mean bdm; b= regression coefficients (for age, age*sex, age , age *sex, height and body mass index); rg-pre= genetic sd in pre-menopausal women; rg-post= genetic sd in post-menopausal women; re-pre= environmental sd in pre-menopausal women; re-post= environmental sd in post-menopausal women; qg= genetic correlation in bmd between pre-menopausal and post- menopausal women. ll log likelihood. a maximum likelihood estimate converged at estimate at lower boundary. *p values for hypothesis based on a / : / mixture of a v and a point mass of zero table model parameters estimated from variance partitioning of bone mineral density parameter spine bmd (· ) total hip bmd (· ) femoral neck bmd (· ) l . . . b(age) ) . ) . ) . b(being post-menopausal) ) . ) . ) . b(age* post-menopausal) ) . . . b(age ) . . . b(age * post-menopausal) ) . ) . ) . b(height) . . . b(bmi) . . . rg-post . . . rg-pre . . . re-post . . . re-pre . [ ] a . qg . . . ll(full model) ) . ) . ) . hypothesis : h : genetic variance equal between pre-menopausal and post-menopausal women parameterization: rg-pre = rg-post ll (nested model): ) . ) . ) . v . . . p . . . conclusion: accept h accept h accept h hypothesis : h : environmental variance equal between pre-menopausal and post-menopausal women parameterization: re-pre = re-post ll (nested model): ) . - . - . v . . . p . . . conclusion: accept h accept h accept h hypothesis : h : genetic correlation similar between pre-menopausal and post-menopausal women parameterization: qg = ll (nested model): ) . ) . ) . v . . . p* . . . conclusion: accept h reject h accept h our partitioning of the variance revealed little dif- ference in the amount of genetic variance between pre- and post-menopausal women, but a / - to -fold higher environmental variance in post-menopausal women. although this difference did not achieve sta- tistical significance, it does nonetheless suggest that environmental factors contribute largely to the greater variability observed in the post-menopausal group. in contrast, there was very little evidence for large differ- ences in the genetic variances between the pre- and post-menopausal groups. furthermore, for spine and femoral neck bmd, the genetic correlation between pre- vs. post-menopausal women did not differ signifi- cantly from one, consistent with the view that the same genes, or sets of genes, act jointly on both groups of women. for total hip bmd, however, the genetic correlation, although high, was significantly less than one, thereby providing modest evidence for incomplete pleiotropy for genes influencing bmd in pre- and post- menopausal women at this site, or for the existence of some distinct genetic effects that do not act jointly on both groups of women. such genes might presumably influence variation in rates of bone loss in the post- menopausal group. the fact that the genetic correla- tions were substantially greater than zero across all three sites indicates that there are at least some sets of genes that influence bmd at these sites in both sets of women jointly. the unique attributes of the old order amish make this population an attractive one for attempting to dissect out the genetic contributions to phenotypic variation. amish families typically tend to be very large, so that there are a large number of sibling rel- ative pairs available for analysis. moreover, the amish have a strong interest in their genealogies, and accu- rate record-keeping dating back many generations al- lows the large amish families to be linked into a single pedigree. finally, the relatively homogenous environment of this population and their reluctance to use prescription medication may allow more clear elucidation of the genetic factors contributing to bmd. in summary, our results support the hypothesis that the same sets of genes influence bmd at the spine and femoral neck in both pre- and post-menopausal wo- men, presumably by influencing acquisition of peak bone mass. however, we also observed some evidence for additional genetic effects acting on one group independently of the other group for total hip bmd. such genes might play a role in bone loss. future studies involving longitudinal follow-up of women as they lose bone will be required to elucidate the nature of these effects. acknowledgements this work was supported by research grants r -ar , r -ag , r -hl and u -hl awarded by the national institutes of health, and the university of maryland general clinical research center, grant m rr , general clinical research centers program, national center for research resources (ncrr), nih. references . looker ac, orwoll es, johnston cc, jr et al ( ) prevalence of low femoral bone density in older u.s. adults from nhanes iii. j bone miner res : – . cummings sr, melton lj ( ) epidemiology and outcomes of osteoporotic fractures. lancet : – . cummings sr, black dm, nevitt mc et al ( ) bone density at various sites for prediction of hip fractures. the study of osteoporotic fractures research group (see comments). lancet : – . gullberg b, johnell o, kanis ja ( ) world-wide projections for hip fracture. osteoporos int : – . lindsay r, cosman f, herrington bs et al ( ) bone mass and body composition in normal women. j bone miner res : – . elders pj, netelenbos jc, lips p et al ( ) accelerated ver- tebral bone loss in relation to the menopause: a cross-sectional study on lumbar bone density in women of to years of age. bone miner : – . hunter dj, sambrook pn ( ) bone loss: epidemiology of bone loss. arthritis res : – . christiansen c, riis bj, rodbro p ( ) prediction of rapid bone loss in postmenopausal women. lancet : – . riis bj ( ) the role of bone loss. am j med : s– s . eisman ja ( ) genetics of osteoporosis. endocr rev : – . christian jc, yu pl, slemenda cw et al ( ) heritability of bone mass: a longitudinal study in aging male twins. am j hum genet : – . pocock na, eisman ja, hopper jl et al ( ) genetic determinants of bone mass in adults. a twin study. j clin in- vest : – . seeman e, hopper jl, bach la et al ( ) reduced bone mass in daughters of women with osteoporosis. n engl j med : – . jouanny p, guillemin f, kuntz c et al ( ) environmental and genetic factors affecting bone mass. similarity of bone density among members of healthy families. arthritis rheum : – . matkovic v, fontana d, tominac c et al ( ) factors that influence peak bone mass formation: a study of calcium balance and the inheritance of bone mass in adolescent females. am j clin nutr : – . ferrari s, rizzoli r, slosman d et al ( ) familial resem- blance for bone mineral mass is expressed before puberty. j clin endocrinol metab : – . lutz j ( ) bone mineral, serum calcium, and dietary intakes of mother/daughter pairs. am j clin nutr : – . krall ea, dawson-hughes b ( ) heritable and life-style determinants of bone mineral density. j bone miner res : – . prentice a ( ) the relative contribution of diet and geno- type to bone development. proc nutr soc : – . seeman e, tsalamandris c, formica c et al ( ) reduced femoral neck bone density in the daughters of women with hip fractures: the role of low peak bone density in the pathogenesis of osteoporosis. j bone miner res : – . zmuda jm, cauley ja, ferrell re ( ) recent progress in understanding the genetic susceptibility to osteoporosis. genet epidemiol : – . kelly pj, nguyen t, hopper j et al ( ) changes in axial bone density with age: a twin study. j bone miner res : – . brown lb, streeten ea, shuldiner ar et al ( ) assessment of sex-specific genetic and environmental effects on bone min- eral density. genet epidemiol : – . streeten ea, mcbride dj, lodge al et al ( ) reduced incidence of hip fracture in the old order amish. j bone miner res : – . beiler k ( ) fisher family history: descendants and history of christian fisher ( – ). ronk’s, eby’s quality print- ing, pa . agarwala r, biesecker lg, hopkins ka et al ( ) software for 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americans: results from the san antonio family osteoporosis study. bone : – 	sec 	sec 	sec 	sec 	sec 	tab 	sec 	tab 	tab 	tab 	bib 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr 	cr position on environmental issues and engagement in proenvironmental behaviors position on environmental issues and engagement in proenvironmental behaviors gene l. theodori department of rural sociology texas a&m university college station, texas, usa a. e. luloff department of agricultural economics and rural sociology pennsylvani a state university university park, pennsylvania , usa using data collected in a general population survey from a random sample of individuals in four communities in pennsylvania, we tested the following two hypotheses: ( ) that differences in sociodemographic characteristics exist among individuals with variant positions on environmental issues; and ( ) that individuals with different positions on environmental issues exhibit dissimilar levels of pro- environmental behaviors. both hypotheses received substantial support. the results indicate that young individuals, the more highly educated, people with higher incomes, and those with liberal political ideologies are more likely than their opposites to maintain proactive positions on environmental issues. the ®ndings also reveal that while both proactive and sympathetic persons engage more frequently in proenvironmental behaviors than do their neutral counterparts, sympathetic indivi- duals partake in these same behaviors less often than do those who expressed proactive positions on environmental issues. keywords environmentalism, environmental movement, position on environ- mental issues, proenvironmental behaviors, social movement the structure or organization of a social movement is often illustrated with con- centric rings or circles (mauss ). the innermost ring, or core, typically contains the principal leaders, along with the most zealous and committed members (mauss ; morrison ). surrounding the core are various rings or layers of the public organized, to a greater or lesser extent, around the issue at hand. each layer is comprised of actors and=or associations with differing levels of interest in and commitment to the success of the movement. received september ; accepted august . support for this research was provided by the pennsylvania department of agriculture (me ) and the pennsylvania agricultural experiment station (regional project ne- and state station project ) . address correspondence to gene l. theodori, department of rural sociology, texas a&m university, tamu, college station, tx ± , usa. e-mail: g-theodori@tamu.edu society and natural resources, : ± , copyright # taylo r & francis - / $ . + . doi: . = as evidenced by its enduring survival, large organizationa l base, and widespread public support, the environmental movement continues to be one of the more successful social movements throughou t the united states and western europe (mertig and dunlap ). despite the vast literature on environmentalism , research on the different layers of individuals involved in the environmental movement has been somewhat limited. surprisingly little empirical research has been conducted on the sociodemographi c characteristic s and=or attitudes , beliefs, and behaviors of these individuals. this article addresses these issues. two hypotheses were tested. first, it was hypothesized that there are differences in sociodemographi c char- acteristics among individuals with variant positions on environmental issues. the second hypothesi s was that individuals with different positions on environmental issues exhibit dissimilar levels of proenvironmental behaviors. background in a review of the trends in public opinion toward environmental quality from the mid s to the late s, dunlap ( ) proposed a ®ve-layer concentric pattern to depict the various segments of the public involved in the environmental move- ment. according to dunlap ( , ± ), activistsÐ``individuals intensely con- cerned about and personally active on behalf of environmental quality’’Ðare at the core of the movement. encompassing the core is a layer referred to as the attentive publicÐ``individuals interested in and informed about environmental issues . . . [who] are likely to provide occasional support for environmental causes, signing petitions, voting for proenvironmenta l issues and candidates and perhaps even contributing time and money to speci®c environmental campaigns.’’ the third and largest layer consists of the sympathetic publicÐ``individuals whoÐalthough not very attentive to environmental issuesÐexpress support for efforts to enhance and protect envir- onmenta l quality.’’ next are the neutralsÐ``persons who have little interest in and typically no opinion concerning environmental issues.’’ last, the outer layer is comprised of opponentsÐ``individuals who are opposed to some degree to the goals of the environmental movement and hold opinions that can be characterized as `anti-environmental.’’’ data from national surveys appear to support dunlap’s ( ) notion of a multilayered public organized around environmental issues. in , an abc news=washingto n post poll asked, ``in recent years, the environmental movement has been very active. do you consider yourself as: an active participant in the environment movement, sympathetic towards the movement, but not active, neutral, or unsympatheti c towards the environmental movement?’ ’ seven percent reported that they were active participants, % said they were sympathetic, but not active, % indicated that they were neutral, and % said they were unsympatheti c (abc news=washingto n post ). more recently, ®ve wirthline worldwide ( , , , , ) national quorum surveys asked respondents a very similar question [do you think of your- self as: ( ) an active environmentalist , ( ) sympathetic towards environmental con- cerns but not active, ( ) neutral, or ( ) generally unsympatheti c to environmental concerns]. the general trends over this -year period re¯ect those reported in the poll. the percentage of respondents who considered themselves active environmentalist s ranged from a low of % in to a high of % in . sympathetic individuals ranged from a low of % in to a high of % in , while neutral individuals ranged from a low of % in to a high of % g. l. theodori and a. e. luloff in . unsympatheti c individuals ranged from a low of % in to a high of % in . despite the consistent pattern of survey ®ndings over nearly two decades, few studies have been conducted on the sociodemographi c characteristics and=or atti- tudes, beliefs, and behavior s of the different layers of individuals involved in the environmental movement. the present study addresses these issues. in this article, we use data collected in a general population survey from a random sample of indivi- duals in four communities to test the following hypotheses: ( ) that differences in sociodemographi c characteristic s exist among individuals with variant positions on environmental issues; and ( ) that individuals with different positions on environ- mental issues exhibit dissimilar levels of proenvironmenta l behaviors. data and measurement the data used in this analysis were drawn from a study that focused on land-use issues at the rural±urban interface in pennsylvania (luloff et al. ; theodori and luloff ). data were collected through a general population survey from a random sample of individuals in four study sites chosen to represent a typology of increasing levels of urban presence and pressure in agricultural areas. the four areas selected were small portions of snyder, bedford, crawford, and lancaster counties. based on major issues identi®ed in key and action informant interviews in each study site, a questionnaire was developed that addressed land use, agri- cultural, development, and natural resource issues, in addition to social issues including community attachment, community participation, stress, and recreation. following a modi®ed total design method (tdm; see dillman ; luloff and ilvento ), data were gathered in the snyder, bedford, and crawford sites using mail survey techniques. data were collected via a questionnaire drop-off=pick-up procedure (melbye et al. ) in the lancaster site due to the presence of a substantial number of old order amish and mennonites. overall, a response rate of % was achieved. this resulted in completed questionnaire s across the sites. position on environmental issues following earlier work (abc news=washington post ; wirthline worldwide , , , , ), position on environmental issues was evaluate d using a single survey item. the question asked: ``which of the following best describes your position on environmental issues?’’ response categories included: ( ) active in environmental issues, ( ) sympathetic to environmental issues, ( ) neither sympathetic nor unsympathetic , ( ) unsympatheti c to environmental issues, and ( ) actively opposed to any action on environmental issues. based on the small number of cases in the latter two categories, respondents who indicated that they were either (a) unsympatheti c to environmental issues or (b) actively opposed to any action on environmental issues were combined with those who reported that they were neither sympathetic nor unsympatheti c on environmental issues. in this article, the three categories describing individuals’ position on environmental issues are referred to as ``proactive,’’ ``sympathetic,’’ and ``neutral.’’ the per- centages of respondents indicating proactive, sympathetic, and neutral positions on environmental issues were . (n = ), . (n = ), and . (n = ), respectively. position on environmental issues engagement in proenvironmental behaviors engagement in proenvironmenta l behaviors was assessed using a list of seven items. respondent s were asked if, during the previous year, they had engaged in any of the following behaviors: ( ) contribute d money or time to an environmental or wildlife conservation group; ( ) stopped buying a product because it caused envir- onmenta l problems; ( ) attended a public hearing or meeting about the environment; ( ) contacted a government agency to get information or complain about an environmental problem; ( ) read a conservation or environmental magazine; ( ) watched a television special on the environment; and ( ) voted for or against a political candidat e because of the candidate’s position on the environment. each proenvironmenta l behavior was dummy coded ( = yes). sociodemographi c variables following earlier research (dunlap and heffernan ; van liere and dunlap ; theodori, luloff, and willits ), age, education, gender, income, and political ideology were included as sociodemographi c factors. age was measured in years. education was coded as follows: ( ) less than high school; ( ) high school equivalent; ( ) some college; ( ) college degree; and ( ) training beyond college. gender was dummy coded ( = male). income was measured by categories, ranging from ( ) less than $ , to ( ) $ , or more. political ideology was measured by the categories: ( ) liberal; ( ) moderate±liberal; ( ) moderate; ( ) moderate±conservative ; and ( ) conservative. analyses differences in sociodemographi c characteristics among the proactive, sympathetic , and neutral individuals were examined by calculating mean scores for each of the ®ve variables for the three groups. the statistical signi®cance of the observed variances were tested by analysis of variance procedures (f-tests). as shown in table , there was considerable support for the proposition that differences in sociodemographi c characteristics exist among individuals with different positions on environmental issues. age, education, income, and political ideology reached statistical signi®cance at the . level and revealed linear-type patterns. of the three groups, proactive persons had the highest incomes and were the youngest, highest educated, and least politically conservative, while neutral individuals had the lowest incomes and were table means and analysis of variance results for sociodemographi c variables by environmental position variable proactive sympathetic neutral f score age . . . . b education . . . . b gender ( = male) . . . . a income . . . . b political ideology . . . . b a signi®cant at p < . . bsigni®cant at p < . . g. l. theodori and a. e. luloff the oldest, least educated, and most politically conservative. gender attained sta- tistical signi®cance at the . level, but did not manifest a linear-type pattern like the other sociodemographi c variables. the results indicated that proactive individuals were most likely to be female, followed by neutral persons. sympathetic individuals were most likely to be male. logistic regression was used to analyze the differences in the levels of engage- ment in proenvironmental behaviors among individuals with different positions on environmental issues. the analysis was conducted in two phases. table reports the bivariate and net odds ratios for the effect of position on environmental issues on engagement in proenvironmenta l behaviors when neutral was treated as the refer- ence category (phase i). phase i bivariate results at the bivariate level, as shown in table , the results indicate that individuals with a proactive orientation on environmental issues were signi®cantly ( p < . ) more likely than neutral individuals to engage in all seven of the proenvironmenta l behaviors. the odds ratios ranged from . to . . this indicated that while proactive persons were almost times more likely than the neutral individuals to stop buying a product because it caused environmental problems, they were approximately times more likely than the neutral individuals to contribute money or time to an environmental or wildlife conservation group. the bivariate results reported in table also indicate that sympathetic individuals were more likely than the neutral individuals to engage in proenvironmenta l behaviors. each of the odds ratios reached statistical signi®cance at the conventional . level, while all but two were signi®cant at the . level. multivariate results as in earlier research, controls for age, education, gender, income, and political ideology were introduced into the model. as noted in table , the results indicate that controlling for these variables had very little effect on the nature or signi®cance levels of the odds ratios for either proactive or sympathetic individuals, although one statistically signi®cant odds ratio did drop to nonsigni®cance. after introducing the control variables, sympathetic individuals did not differ signi®cantly from neutral individuals in terms of attending a public meeting of hearing about the environment. overall, based on the multivariate results reported in table , respondents who expressed either proactive or sympathetic positions on environmental issues were more likely than those who expressed neutral positions on environmental issues to engage in the majority of proenvironmenta l behaviors. the likelihood of proactive individuals who engaged in each of the behaviors was stronger than that for sym- pathetic individuals in both the bivariate and multivariate models. phase ii treating individuals who reported neutral positions on environmental issues as the reference category for the environmental position variable allowed us to test in phase i (table ) whether proactive and sympathetic individuals differed sig- ni®cantly from the neutral individuals in terms of proenvironmenta l behaviors. what we could not test in phase i was whether or not individuals with a proactive position on environmental issues t a b l e o d d s r a ti o s fo r th e e ff e ct o f e n v ir o n m e n ta l p o st io n o n p ro en v ir o n m en ta l b eh a v io r w it h `` n eu tr a l’ ’ a s th e r ef e re n ce c a te g o ry o d d s ra ti o s b iv a ri a te m u lt iv a ri a te a p ro en v ir o n m en ta l b eh a v io rs p ro a ct iv e s y m p a th et ic p ro a ct iv e s y m p a th et ic c o n tr ib u te d m o n e y o r ti m e to a n en v ir o n m en ta l o r w il d li fe co n se rv a ti o n g ro u p (n = )b . e . e . e . e s to p p ed b u y in g a p ro d u ct b e ca u se it ca u se d en v ir o n m en ta l p ro b le m s (n = ) . e . e . e . e a tt en d ed a p u b li c h ea ri n g o r m ee ti n g a b o u t th e en v ir o n m en t (n = ) . e . c . e . c o n ta ct ed a g o v er n m en t a g en c y to g et in fo rm a ti o n o r co m p la in a b o u t a n en v ir o n m en ta l p ro b le m (n = ) . e . d . e . d r ea d a co n se rv a ti o n o r en v ir o n m e n ta l m a g a zi n e (n = ) . e . e . e . e w a tc h e d a te le v is io n sp ec ia l o n th e en v ir o n m en t (n = ) . e . e . e . e v o te d fo r o r a g a in st a p o li ti ca l ca n d id a te b ec a u se o f h is = h er p o si ti o n o n th e en v ir o n m en t (n = ) . e . e . e . e a o d d s ra ti o s co m p u te d co n tr o ll in g fo r a g e , e d u ca ti o n , g en d er , in c o m e, a n d p o li ti c a l id eo lo g y . b n v a lu e s v a ry d u e to m is si n g d a ta . c s ig n i® ca n t a t p < . . d s ig n i® ca n t a t p < . . e s ig n i® ca n t a t p < . . orientation on environmental issues differed from their sympatheti c counterparts in terms of their environmental behaviors. in order to do so, we recoded the envir- onmental position variable. table reports the bivariate and net odds ratios for the effect of environmental position on proenvironmenta l behaviors when the sympathetic-to-environmental-issue s response was treated as the reference category (phase ii). while the odds ratio values for the sympathetic individuals reported in table and those for the neutral persons shown in table are different (due to treating one versus the other as the reference category), it is important to note that the odds ratios for the neutral individuals in table are merely the inverse values of the odds ratios for sympathetic respondents in table . the alpha values (or p values) for the sympathetic individuals in table and those for the neutral respondents in table are identical in both the bivariate and multivariate models. bivariate results as shown in table , the bivariate results indicate that individuals who expressed proactive positions on environmental issues were signi®cantly more likely than their sympathetic counterparts to engage in six of the seven proenvironmenta l behaviors. while proactive individuals were . times more likely than sympathetic individuals to stop buying a product because it caused environmental problems, they were . times more likely than sympathetic respondents to attend a public meeting or hearing about the environment. proactive respondent s did not differ signi®cantly from sympathetic respondents in terms of their likelihood of watching a television special on the environment, despite the fact that they were . times more likely than sympathetic individuals to do so. the results reported in table also indicated that, at the bivariate level, neutral individuals were signi®cantly less likely than sympathetic respondents to engage in each of the proenvironmenta l behaviors (the opposite of the ®ndings reported in table ). multivariate results the multivariate results indicate that controlling for age, education, gender, income, and political ideology had very little effect on the size of the odds ratios for either proactive or neutral individuals (the odds ratios reported for the neutral individuals in table are simply the inverse of the odds ratios for the sympathetic respondents in table ). in brief, the most interesting ®nding in table was that proactive respondents were signi®cantly more likely than sympathetic respondents to engage in six of the seven proenvironmenta l behaviors, net of the other variables in the model. examining the sociodemographi c variables an examination of the sociodeomgraphi c variables indicated that age consistently failed to reach statistical signi®cance (table ). education was positively and sig- ni®cantly related to ®ve of the proenvironmenta l behaviors. more highly educated respondents were signi®cantly more likely than those with lower education to con- tribute money or time to an environmental or wildlife conservation group, to contact a government agency to get information about an environmental problem, to read a conservation or environmental magazine, to watch a television special on the environment, and to vote for or against a political candidate because of his=her position on the environment. while males were signi®cantly more likely than females position on environmental issues t a b l e o d d s r a ti o s fo r th e e ff ec t o f e n v ir o n m e n ta l p o si ti o n o n p ro en v ir o n m en ta l b eh a v io r w it h `` s y m p a th e ti c’ ’ a s th e r ef er e n c e c a te g o ry o d d s ra ti o s b iv a ri a te m u lt iv a ri a te a p ro e n v ir o n m e n ta l b eh a v io rs p ro a c ti v e n eu tr a l p ro a ct iv e n eu tr a l c o n tr ib u te d m o n ey o r ti m e to a n e n v ir o n m en ta l o r w il d li fe co n se rv a ti o n g ro u p (n = )b . e . e . e . e s to p p ed b u y in g a p ro d u ct b ec a u se it ca u se d en v ir o n m en ta l p ro b le m s (n = ) . c . e . c . e a tt en d ed a p u b li c h ea ri n g o r m ee ti n g a b o u t th e en v ir o n m en t (n = ) . e . c . e . c o n ta ct ed a g o v er n m en t a g en cy to g e t in fo rm a ti o n o r co m p la in a b o u t a n e n v ir o n m en ta l p ro b le m (n = ) . e . d . e . d r e a d a co n se rv a ti o n o r en v ir o n m en ta l m a g a zi n e (n = ) . d . e . d . e w a tc h ed a te le v is io n sp ec ia l o n th e en v ir o n m en t (n = ) . . e . . e v o te d fo r o r a g a in st a p o li ti ca l ca n d id a te b ec a u se o f h is = h er p o si ti o n o n th e en v ir o n m en t (n = ) . e . e . e . e a o d d s ra ti o s co m p u te d co n tr o ll in g fo r a g e, ed u ca ti o n , g e n d er , in c o m e, a n d p o li ti c a l id e o lo g y . b n v a lu es v a ry d u e to m is si n g d a ta . c s ig n i® ca n t a t p < . . d s ig n i® c a n t a t p < . . e s ig n i® ca n t a t p < . . t a b l e o d d s r a ti o s fo r th e s o ci o d em o g ra p h ic v a ri a b le s o d d s ra ti o sa fo r so c io d em o g ra p h ic v a ri a b le s p ro en v ir o n m e n ta l b eh a v io rs a g e e d u ca ti o n g en d er in co m e p o li ti ca l id eo lo g y c o n tr ib u te d m o n e y o r ti m e to a n en v ir o n m en ta l o r w il d li fe co n se rv a ti o n g ro u p (n = )b . . d . . d . c s to p p ed b u y in g a p ro d u ct b e ca u se it ca u se d en v ir o n m en ta l p ro b le m s (n = ) . . . c . . a tt e n d ed a p u b li c h ea ri n g o r m ee ti n g a b o u t th e en v ir o n m en t (n = ) . . . d . . c o n ta ct ed a g o v e rn m en t a g e n c y to g et in fo rm a ti o n o r co m p la in a b o u t a n en v ir o n m en ta l p ro b le m (n = ) . . c . . . r ea d a co n se rv a ti o n o r en v ir o n m en ta l m a g a zi n e (n = ) . . e . . . c w a tc h e d a te le v is io n sp ec ia l o n th e en v ir o n m en t (n = ) . . e . . . c v o te d fo r o r a g a in st a p o li ti ca l ca n d id a te b ec a u se o f h is = h er p o si ti o n o n th e en v ir o n m en t (n = ) . . e . . d . a c o m p u te d c o n tr o ll in g fo r p o si ti o n o n e n v ir o n m e n ta l is su es . b n v a lu es v a ry d u e to m is si n g d a ta . c s ig n i® ca n t a t p < . . d s ig n i® ca n t a t p < . . e s ig n i® ca n t a t p < . . to stop buying a product because it caused environmental problems, females were signi®cantly more likely than males to attend a public meeting or hearing about the environment. respondents with higher incomes were signi®cantly more likely than those with lower incomes to contribute money or time to an environmental or wildlife conservation group, and signi®cantly less likely to vote for or against a political candidate because of his or her position on the environment. politically liberal respondents were signi®cantly more likely than their politically conservative counterparts to contribute money or time to an environmental or wildlife con- servation group, read a conservation or environmental magazine, and watch a tel- evision special on the environment. summary and concluding comments these data provided substantial support for our two hypotheses, namely, ( ) that differences in sociodemographi c characteristic s exist among individuals with variant positions on environmental issues, and ( ) that individuals with different positions on environmental issues exhibit dissimilar levels of proenvironmenta l behaviors. the results of the analysis of variance tests revealed that young individuals, the more highly educated, people with higher incomes, and those with liberal political ideol- ogies were more likely than their opposites to maintain proactive positions on environmental issues. while gender reached statistical signi®cance, the results were not as clear-cut. the ®ndings of the logistic regression in phase i indicated that, overall, indivi- duals who maintained either proactive or sympatheti c positions on environmental issues were more likely than those who were classi®ed as neutral to engage in proenvironmenta l behaviors. the likelihood of proactive persons to partake in each of the behaviors was stronger than that for sympatheti c respondents. the odds ratios changed only slightly when controls for a variety of sociodemographi c character- istics were added. taken together, the phase ii logistic regression analyses revealed that proactive individuals were more likely than sympathetic individuals to engage in proenvironmenta l behaviors. again, the odds ratios changed only slightly after the addition of the control variables. although both proactive and sympathetic persons participated more frequently in proenvironmenta l behaviors than did their neutral counterparts , sympathetic individuals engaged in these same behaviors less often than did those who expressed proactive environmental positions. these results have practical implications for environmental protection and natural resource management issues. for example, to the extent that behavior s often change, well-designed environmentally oriented educational program s that motivate and encourage understanding , exploration, participation, and group problem solving might lead to the adoption and facilitation of proenvironmenta l behaviors (kaplan ). moreover, informational campaigns that emphasize environmentally responsible behaviors could foster proactiv e envir- onmenta l positions. despite the statistical signi®cance of our ®ndings, several limitations of these data must be considered. one limitation involved the measurement of position on environmental issues. in this study, position on environmental issues was represented by a global measure. future research examining similar linkages might incorporate measures of domain-speci®c positions. a second limitation of this study dealt with the measurement of proenvironmenta l behaviors. individuals were asked only to indicate whether or not they engaged in any of the listed behaviors. in order to fully g. l. theodori and a. e. luloff understand the links between position on environmental issues and engagement in proenvironmental behaviors, future studies should examine the frequency of parti- cipation in such behaviors. notes . see luloff et al. ( ), theodori, luloff, and willits ( ), and theodori and luloff ( ) for a detailed description of the typology and each study site. . no statistical differences in regard to the available sociodemographic characteristics were found between the lancaster sample and those from snyder, bedford, and crawford. the percentages of old order amish and mennonites from the lancaster site totaled and , respectively. the analysis and reported ®ndings include data on both groups. removal of the amish and mennonites from the sample did not change the nature or pattern of the results reported here. . using mail survey techniques in three study sites, a response rate of about % was obtained, resulting in completed questionnaires from snyder, from bedford, and from crawford. using the drop-off=pick-up technique in lancaster, a response rate of % was achieved, resulting in completed questionnaires. . the percentages of respondents who indicated that they were unsympathetic to environmental issues and actively opposed to any action on environmental issues were . (n = ) and . (n = ), respectively. these respondents were combined with the indi- viduals who reported that they were neither sympathetic nor unsympathetic. the newly cre- ated category contained respondents and represented . % of the sample. . we chose the label ``neutral’’ for this category due to the fact that an overwhelming majority of respondents in this group, roughly % ( of ), indicated that they were neither sympathetic nor unsympathetic to environmental issues. it is important to keep in mind that the small numbers of respondents who reported being unsympathetic to environ- mental issues (n = ) and actively opposed to any action on environmental issues (n = ) are subsumed under this label. . in principal, three of the items could indicate anti- rather than proenvironmental behavior. respondents could have attended a meeting, contacted a government agency, or voted for a candidate to prevent, rather than to promote, environmental protection. however, the correlation of these variables with unambiguously proenvironmental behaviors indicated that such intentions were rare. . an odds ratio (y) is e (natural logarithm) raised to the power of b (the metric logit coef®cient); y refers to the effect of a one-unit change in x on the odds of y. it has a ``times as likely’’ interpretation. y can equal any nonnegative number. when x and y are independent, y equals . a value of generally serves as a baseline for comparison. odds ratios on either side of re¯ect certain types of associations. an odds ratio greater than ( < y < ?) indicates a positive association, while an odds ratio less than ( < y < ) denotes a negative association. values of y farther from in either direction designate stronger levels of association (agresti ; liao ). references abc news=washington post. . abc news washington post april poll. compiled by k. i. wright. interview dates ± , april . survey . abc news polling unit, new york. agresti, a. . an introduction to categorical data analysis. new york: john wiley & sons. dillman, d. a. . mail and telephone surveys: the total design method. new york: john wiley and sons. dunlap, r. e. . public opinion and environmental policy. in environmental politics and policy: theories and evidence, ed. j. p. lester, ± . durham, nc: duke university press. position on environmental issues dunlap, r. e., and r. b. heffernan. . outdoor recreation and environmental concern: an empirical examination. rural sociol. : ± . kaplan, s. . human nature and environmentally responsible behavior. j. social issues : ± . liao, t. f. . interpreting probability models: logit, probit, and other generalized linear models. thousand oaks, ca: sage. luloff, a. e., and t. w. ilvento. . respondents, nonrespondents, and population surveys. j. commun. dev. society : ± . luloff, a. e., l. bourke, s. jacob, and s. seshan. . farm and non-farm interdependencies at the rural±urban interface. final project report for the pennsylvania department of agriculture. university park, pa: pennsylvania state university. mauss, a. l. . social problems as social movements. philadelphia: lippincott. melbye, j., l. bourke, a. e. luloff, p. s. liao, g. l. theodori, and r. s. krannich. . the drop-off=pick-up method for household survey research. working paper ipre-wp- - . university park, pa: institute for policy research and evaluation, pennsylvania state university. mertig, a. g., and r. e. dunlap. . environmentalism, new social movements, and the new class: a cross-national investigation. rural sociol. : ± . morrison, d. e. . how and why environmental consciousness has trickled down. in distributional con¯icts in environmental-resource policy, ed. a. schnaiberg, n. watts, and k. zimmerman, ± . new york: st. martin’s press. theodori, g. l., and a. e. luloff. . urbanization and community attachment in rural areas. society & natural resources : ± . theodori, g. l., a. e. luloff, and f. k. willits. . the association of outdoor recreation and environmental concern: reexamining the dunlap-heffernan thesis. rural sociol. : ± . van liere, k. d., and r. e. dunlap. . the social bases of environmental concern: a review of hypotheses, explanations and empirical evidence. public opin. q. : ± . wirthlin worldwide. . research supplement: the wirthlin report. wirthlin rep. (august): ± . wirthlin worldwide. . research supplement: the wirthlin report. wirthlin rep. (october): ± . wirthlin worldwide. . research supplement: the wirthlin report. wirthlin rep. (august=september): ± . wirthlin worldwide. . research supplement: the wirthlin report. wirthlin rep. (september): ± . wirthlin worldwide. . research supplement: the wirthlin report. wirthlin rep. (november): ± . g. l. theodori and a. e. luloff http://www.greenjournal.org/cgi.pdf outcomes of intended home births in nurse-midwifery practice: a prospective descriptive study patricia aikins murphy, cnm, drph, and judith fullerton, cnm, phd objective: to describe the outcomes of intended home birth in the practices of certified nurse-midwives. methods: twenty-nine us nurse-midwifery practices were recruited for the study in . women presenting for intended home birth in these practices were enrolled in the study from late to late . outcomes for all enrolled women were ascertained. validity and reliability of submit- ted data were established. results: of enrolled women intending home births, % miscarried, terminated the pregnancy or changed plans. another . % became ineligible for home birth prior to the onset of labor at term due to the development of perinatal problems and were referred for planned hospital birth. of those women beginning labor with the intention of deliver- ing at home, ( . %) were transferred to the hospital during labor. ten mothers ( . %) were transferred to the hospital after delivery, and infants ( . %) were trans- ferred after birth. overall intrapartal fetal and neonatal mortality for women beginning labor with the intention of delivering at home was . per . for women actually delivering at home, intrapartal fetal and neonatal mortality was . per . conclusion: home birth can be accomplished with good outcomes under the care of qualified practitioners and within a system that facilitates transfer to hospital care when necessary. intrapartal mortality during intended home birth is concentrated in postdates pregnancies with evidence of meconium passage. (obstet gynecol ; : – . © by the american college of obstetricians and gynecolo- gists.) many accept the premise that because of modern tech- nology, hospitals are the safest place to give birth. since the s, most births in the united states have taken place in hospitals with physicians in attendance. none- theless, there are also more than , out-of-hospital births in the united states annually, most in birth centers, clinics, and the home. of these, nearly , are residential births. over the past years, the frequency of home births has remained stable at approximately . % of all births (table ). – home birth is a controversial issue. debates about the safety of home births focus on the risk of preventable perinatal morbidity and mortality, and on broader issues of appropriate screening and referral. questions also are raised about the ability to predict which women can safely give birth at home and about the risk of unforeseen complications that require emergency trans- port to a hospital. published studies on intended home birth have demonstrated low perinatal mortality and morbidity but have stressed the necessity of planning, risk-assessment and well-qualified attendants. – however, international studies report data from coun- tries in which medical and obstetric care systems may be dissimilar from those in this country and in which the reporting criteria for neonatal and perinatal deaths also may vary, making international comparative data less interpretable. – some us studies of home birth are limited by small size; others reflect limited geo- graphic areas or homogeneous populations. – fi- nally, many studies have inadequate methodology for addressing certain issues. studies using birth certificate data, for example, cannot identify perinatal mortality that may be attributable to planned home birth but occur after transfer to the hospital. certified nurse-midwives have attended approxi- mately home births annually ( – ) in the united states. a retrospective study by one of the authors (pam) reported outcomes of , planned home births. the intrapartum and neonatal mortality among women intending home birth at the onset of labor was two per . although a large sample, the from the department of obstetrics and gynecology, columbia uni- versity college of physicians and surgeons, new york, new york, and the department of family nursing care, university of texas health science center at san antonio, san antonio, texas. this project was supported in part by a columbia university strategic research award and by the acnm foundation. vol. , no. , september - / /$ . pii s - ( ) - findings were limited by the retrospective nature of the report. a prospective evaluation of home birth out- comes in the practices of certified nurse-midwives in the united states was then designed and implemented. this article reports the results of this prospective study. methods a community-based nurse-midwifery practice network was recruited in . nurse-midwifery practices pro- viding home birth services were identified by a mailed survey to the membership of the american college of nurse-midwives, advertisements placed in professional publications, and referral from other midwives. forty- five active nurse-midwifery home birth practices were identified in . of these, twenty-nine practices ( %) providing home birth services agreed to participate in the network. three participating practices later with- drew from full participation because they perceived continued participation to be an excessive work burden but did contribute full data on all patients who were enrolled up to a particular date determined by the investigator. solo and group practices participated, enrolling as few as two to as many as planned home births in the enrollment year; practices were located in rural and urban settings. six were located in california, and five in pennsylvania. new york and virginia were each represented by three—illinois and texas each by two. eight other states were represented by one nurse- midwife home birth practice each. the study was approved by the institutional review board of the principal investigator’s institution. eligible subjects were pregnant women age years or older who requested home birth and were eligible for home birth services according to the practice guidelines of the individual nurse-midwifery practices. the researchers made no attempt to evaluate the equivalency of these practice guidelines, nor to establish practice-related standards. uniform data collection forms were developed and pilot tested in several practices prior to initiation of the study. the forms included demographic and perinatal risk information about individual patients, as well as the outcomes of prenatal, intrapartal, and postpartum care. patients were enrolled between december and december . all new patients seeking home birth in participating practices were invited to enroll; enroll- ment generally occurred at a first or second prenatal visit. initial data were forwarded to the central study office at this time; additional data were collected and forwarded at the time of birth and the postpartum visit. any referral to another provider for care or to a planned hospital birth was recorded. hospital records were requested for all women and newborn infants who were transferred to a hospital during labor or immedi- ately after birth. data were reviewed for completeness and logical consistency by clinically experienced study personnel upon receipt in the study office. when data were called into question, practices were contacted with a request for clarification. data reliability was evaluated by comparing dupli- cate records serendipitously received during the course of the study and in a % random sample of duplicate records specifically requested from each practice. study office personnel abstracted these latter records onto study forms. intrarater and interrater reliability were calculated by simple percent agreement, comparing each item on one form to its counterpart. agreement ranged from % to % for the items compared on both duplicate and random samples. agreement for major outcome variables (such as site of birth, perinatal mortality, and primary birth outcomes) was %. accuracy of data submitted for transfers was as- sessed by examining hospital records requested for all women transferred to the hospital in late pregnancy, during labor, or after birth. the condition of mother and infant on arrival in the hospital and birth outcomes were verified. eighty percent of hospital records re- quested were received and were fully consistent with information provided by the practice. full information was obtained on all intrapartum transfers. in addition, midway through the enrollment year, surveys were sent to women who still were eligible for home birth in late pregnancy. the surveys were mailed months after the estimated date of delivery and included requests for data on the birth, as well as information about patient satisfaction. three quarters of the surveys were re- turned, and again, the data were found to be consistent with data submitted by the practice. data were analyzed primarily as descriptive statis- tics. group comparisons were assessed with � and t test procedures. the probability value for statistical significance was set at . . results one thousand four hundred four eligible women en- rolled in the study. participating practices reported table . home births in the united states, – year total births total home births % of all births , , , . , , , . , , , . , , , . , , , . , , , . murphy and fullerton home birth outcomes obstetrics & gynecology women who refused to enroll, primarily from amish communities. reasons given were cultural or privacy concerns. table describes the sample of women in- tending a home birth. socioeconomic status was de- rived from payment source, occupation of patient and partner, income (if reported by the patient), and an estimate by the nurse-midwife based on family size, reported income, and special circumstances. thirty-two percent of the women were members of amish and mennonite communities, which contributed to the ed- ucational and socioeconomic profile of the sample. there was little evidence of behavioral perinatal risk among the women in this study. few women reported the use of tobacco, alcohol, or other substances prior to or during pregnancy (table ). all but % had at least five prenatal visits. evidence of prior medical or obstet- ric risk in the sample was also minimal. among parous women in the sample, % had had a previous home birth and % a previous birth center birth; overall . % of the parous women had a previous out-of- hospital birth experience. twenty-two percent of the parous women in the sample had a history of one or more intrapartal or neonatal factors that required inter- vention, possibly indicating a higher perinatal risk potential in the current pregnancy. these included a history of pregnancy-induced hypertension ( . %), as- sisted vaginal delivery ( . %), stillborn fetus or neona- tal death ( . %), low birth weight infant ( . %), or postpartum hemorrhage ( . %). a specific question about previous shoulder dystocia was added to the instrument midway through the enrollment year. data on previous shoulder dystocia thus are available for only parous women; . % of this smaller sample had a history of previous shoulder dystocia. of the women who had a previous cesarean delivery ( . % of parous women), % also had had a subsequent suc- cessful vaginal birth following cesarean. one hundred eighty-three women left or were re- ferred out of the home birth practice during the preg- nancy and prior to labor at term. eighty-six ( . %) miscarried, terminated the pregnancy, moved away, or changed their minds about home birth. only seven of these women reported financial problems or lack of insurance coverage as the reason for deciding against home birth. of the remaining women, ( . %) were referred for hospital-based care prior to the onset of term labor. this group includes those women who developed preterm labor or preterm premature rupture of membranes. according to the protocols of the prac- tices, women were referred directly for hospital labor and birth management if labor or membrane rupture occurred prior to a designated gestational age (usually weeks). table presents specific indications for antepartum transfers, and the outcomes of these preg- nancies in known cases. women leaving the home birth service during the antepartal period were more likely to be unmarried ( % compared with %, p � . ), nonwhite ( % compared with %, p � . ), nulliparous ( % compared with %, p � . ) or to have medicaid ( . % compared with . %, p � . ) when compared with women who remained eligible for a home birth. amish and menno- nite women were less likely to be referred out of the home birth practice (p � . ). follow-up data were obtained for . % of the ante- partum referrals that occurred after weeks’ gestation for medical or obstetric risk factors; all of these women had planned hospital births due to the presence of medical or obstetric problems. there were four imme- diate neonatal deaths reported in this group: three due table . characteristics of women intending home birth characteristic initially enrolled and eligible (n � ) eligible for home birth at labor onset (n � ) married/consensual union . % . % white . % . % parity � � . % . % age mean . y (sd . y) . y (sd . y) – y . % . % – y . % . % – y . % . % – y . % . % – y . % . % primary occupation as homemaker . % . % education � y . % . % college graduate or higher . % . % payment self-pay . % . % commercial/military insurance (includes hmos) . % . % medicaid/other government . % . % other payment (barter, etc) . % . % low socioeconomic status . % . % amish/mennonite . % . % drug/substance use . % . % alcohol use: � drinks per wk . % smokes � cigarette per wk . % . % previous cesarean delivery (multiparas only) . % . % prenatal care in st trimester . % . % number of prenatal visits . . sd � standard deviation; hmo � health maintenance organization. vol. , no. , september murphy and fullerton home birth outcomes to congenital anomalies and one due to complications of prematurity. two ( %) of ten antepartum fetal demises were associated with lethal anomalies. among those in this group for whom follow-up data were available, % had cesarean deliveries. one thousand two hundred twenty-one women re- mained eligible for home birth at the time of labor onset. of these, ( . %) were transferred to the hospital during labor, and gave birth at home. ten women and neonates were transferred to the hospital after birth (tables and ). forty women ( . %) deliv- ered before the midwife arrived at the home; all infants and mothers were well after these unattended births. mean birth weight for infants born at home was g (standard deviation ). only four infants ( . %) weighed less than g at birth, infants ( %) weighed more than g at birth, and of these ( . % of all births) weighed more than g. of those low birth weight infants born at home, infants ranged from to weeks’ gestation and weights ranged from to g. no births at home were assisted with vacuum or forceps; four infants were born in breech presentation. forty-six infants ( . %) were reported as not having spontaneous respirations; of these ( . %) were reported to have had some type of resuscitation procedure (ambu bag, cardiac massage, or intubation). thirty-three of these ( . %) had minute apgar scores of less than ; only infants had minute apgars of less than ( . % of attended home births). participating midwives were asked to report on ac- tual or potential obstetric problems occurring during labor and delivery at home. these are listed in table . twenty-seven percent of nulliparous women and . % of parous women were transferred during labor (p � . ). hospital transfer during the intrapartum period was not related to preexisting obstetric risk factors. it was significantly related to a number of actual or potential intrapartum problems (see table ). gesta- tional age of weeks or more and the presence of meconium were factors that greatly increased the risk of transfer to hospital and perinatal mortality. seventeen percent of laboring women with meconium-stained fluid were transferred to the hospital during labor, compared with . % of women with clear fluid. seven- teen percent of women at weeks’ gestation or more who intended to deliver at home were transferred table . antepartal referrals for obstetric or hospital-based management* reason n % of eligible sample† known infant outcomes ptl or preterm prom . : good � wk gestation ( – wk) : hospitalized : nnd (includes also with anomalies) : unknown multiple gestation � . : good : unknown diagnosis of congenital anomalies (excluding above) . : stable : iufd : nnd antepartum fetal demise (excludes those due to anomalies above) . fetal malpresentation . : good : unknown % cesarean delivery rate vaginal bleeding/placenta previa . : good : unknown % cesarean delivery rate suspected macrosomia . : good suspected fetal growth restriction . : good : unknown other suspicion of fetal compromise . : good medical problems . : good : hospitalized : unknown prom at term � . : good pregnancy exceeding wk . : good ptl � preterm labor; prom � premature rupture of membranes; nnd � neonatal death; iufd � intrauterine fetal demise. * these women were no longer eligible for home birth. † n � . excludes from antepartal group spontaneous abortions, voluntary terminations, and those who changed their mind or moved out of the area (n � ). murphy and fullerton home birth outcomes obstetrics & gynecology during labor for hospital birth, compared with . % of women at less than weeks’ gestation. both intrapar- tum fetal deaths occurred in pregnancies of weeks’ or more gestation with meconium-stained amniotic fluid. as previously noted, full birth information was ob- tained from hospital records or managing clinicians for % of these intrapartum transfers; substantial birth information was received for all others. five-minute apgar scores were or higher for % (of those for whom the apgar was reported); all liveborn infants were well at a postpartum evaluation. thirty-two per- cent of these transfers had a cesarean delivery, and % had an assisted vaginal delivery. among those with a previous cesarean delivery who began labor with the intention of delivering at home (n � ), % were transported to the hospital during labor for care (as compared with . % transported among those with no history of cesarean delivery [not statistically significant]). the cesarean delivery rate was . % in women with a previous cesarean; there was one vacuum assisted delivery in this group ( . %). the overall cesarean rate was . %. among the women beginning labor with the intention of delivering at home, there were five perina- tal deaths. thus the total intrapartum and neonatal mortality was . per (see table ). excluding two fetal demises diagnosed at the first labor status evalu- ation by the attending midwife and referred immedi- ately for hospital birth, there were three fetal or infant deaths among women whose labor was managed in whole or in part at home. this results in a total intrapartum and neonatal mortality for planned labor and birth at home of . per . both fetal deaths were in pregnancies of weeks or more gestation with table . intrapartum/postpartum/neonatal transfers to hospital reason n eligible sample (%)* comment intrapartum transfer changed mind regarding home birth . selfreferred to hospital at labor onset midwife unable to attend home birth . transferred to birth center due to other clients in labor or environmental problems in the home no fetal heart tones . fetal demise diagnosed at the first assessment of the patient in labor nonvertex fetal presentation . woman converted to vertex in hospital and returned home for labor and birth fetal distress . one infant stillborn in the hospital after assisted delivery for meconium and fetal distress; others in good condition moderate to thick meconium . excludes the stillborn infant mentioned above; all infants well at mo vaginal bleeding . mothers and infants well prolonged labor or rupture of membranes . all infants well cord prolapse . infant born in good condition postpartum transfer perineal suturing . discharged after suturing retained placenta . discharged after placental delivery postpartum bleeding . discharged after d; all well infant transfer stillborn . resuscitation unsuccessful; meconium and postdates respiratory problems . hospitalized; all well at mo evaluation of anomalies . stable at mo, given condition evaluation for sepsis . well at mo first postpartum month evaluation for possible endometritis discharged after evaluation cholecystitis/cholecystectomy all well other (colitis, hemorrhoidectomy) all well first newborn month neonatal death at h of age; no cause of death established by medical examiner evaluation of possible anomaly all stable for condition at mo respiratory problems all well at mo infection or jaundice all well at mo other† all well at mo * n � . † circumcision, dehydration, failure to thrive, hernia repair. vol. , no. , september murphy and fullerton home birth outcomes evidence of meconium passage. one stillbirth occurred in the hospital; the mother was transferred during labor for slow progress and meconium-stained fluid. the fetal heart rate was reported as normal on arrival in the hospital, and the mother labored for several hours more before delivering the stillborn infant. meconium aspi- ration was reported as the cause of death. the second stillborn was born at home; there was no autopsy, but the reporting midwife indicated the presence of meco- nium as well. a third infant died at hours of age; postmortem studies were unable to establish a cause of death. for labors managed entirely at home, mortality was . per . there were no maternal deaths. discussion this report is one of the few studies of home birth conducted in the united states and is unique in its multisite prospective nature. although the sample has limited ability to reflect stable rates of perinatal prob- lems in home birth settings due to its relatively small size, the antepartum and perinatal transfer rates re- ported are similar to a larger retrospective study that addressed nurse-midwifery home birth practice out- comes. intrapartum and neonatal mortality rates in this study also compare favorably to rates reported in that study (two per ) and in a prospective study of outcomes of planned birth center births ( . per ). the latter study cited comparisons to two studies of uncomplicated hospital births. a study of electronic fetal monitoring published in identified , births as low risk and reported the associated intrapar- tum and neonatal mortality as one per . a report identified , uncomplicated term and post- term pregnancies for an evaluation of perinatal charac- teristics of postdates pregnancies; of these, were uncomplicated term hospital births with an intrapar- tum and neonatal mortality of . per . a number of studies of home birth also report low rates of intrapartum or neonatal mortality, although it is recog- nized that international reporting criteria for perinatal mortality may vary. when possible, raw data available in these published reports were recalculated to reflect a sample similar to that reported here (table ). a recent meta-analysis of six european and us home birth studies revealed no significant difference in perinatal mortality between home and hospital birth. this meta- analysis included data only from developed countries table . summary outcomes of transfers to hospital-based care or management timing of transfer n % of relevant sample adverse outcomes antepartum referral (includes preterm labor or rupture of membranes if no longer eligible for home birth) . (of ) iufd, nnd, information unknown for % intrapartum transfer . (of ) iufd prior to first assessment, iufd in labor (stillborn in hospital) postpartum maternal transfer . (of ) no maternal mortality postpartum infant transfer . (of ) iufd in labor (stillborn at home) total . (of ) later postpartum . (of ) no maternal mortality later newborn . (of ) nnd at home at h of age iufd � intrauterine fetal demise; nnd � neonatal death (within first week of life). table . actual or potential obstetric problems reported by midwives as occurring during labor and birth at home event n (%)* relative risk for transfer to hospital-based care ( % ci) maternal inability to cope with labor/need for analgesia ( . ) . ( . , . ) fetal heart abnormalities in the first stage of labor ( . ) . ( . , . ) fetal heart abnormalities in the second stage of labor ( . ) . ( . , . ) prolonged latent phase of labor ( . ) . ( . , . ) lack of progress in the first stage of labor ( . ) . ( . , . ) lack of progress in the second stage of labor ( . ) ( . , . ) prolonged rupture of membranes ( . ) . ( . , . ) meconium-stained amniotic fluid (any) ( . ) . ( . , . ) moderate to thick meconium ( . ) . ( . , . ) shoulder dystocia or difficulty delivering shoulders ( . ) . ( . , . ) postpartum hemorrhage or excess bleeding in the third stage of labor ( . ) . ( . , . ) ci � confidence interval. * percentages have been corrected for missing data. in some cases transfer to hospital management early in labor created missing data for some variables. murphy and fullerton home birth outcomes obstetrics & gynecology that use similar perinatal reporting criteria and defini- tions. there are several limitations to note in this report. the results reported here reflect a sample of nurse- midwifery practices willing to participate in data col- lection and to permit the resulting scrutiny of their practices. we cannot draw comparisons to nonpartici- pating nurse-midwifery practices or to the home birth practices of physician and other midwife providers. during and , an average of certified nurse-midwives–attended home births per year were reported in vital statistics data. , thus, these data reflect more than % of those births. this study presents only the outcomes of home birth practice; it makes no comment on the process of care. there was no attempt to require adherence to standard- ized guidelines for practice because the intent was to examine home birth practice as it occurs in the commu- nity. however, it may be reasonable to infer that many participating practices developed their individual pro- tocols in accord with the professional practice guide- lines for home birth practice developed by the ameri- can college of nurse-midwives (acnm home birth committee. guidelines for home birth. washington dc: american college of nurse-midwives, ). the equivalency of clinical protocols in the individual prac- tices was not examined, but participating midwives were asked brief questions about their determination of ineligibility for home birth. for example, % of the midwives considered twin pregnancy a contraindica- tion for home birth, and % considered a breech presentation a contraindication. preterm labor was a contraindication as well; for % of practices, labor before weeks’ gestation conferred ineligibility for home birth; two practices held weeks as a minimum gestational age (data on file). seventy-three percent of the midwives accepted women for home birth if they had a previous cesarean delivery; in most cases a previous successful vaginal birth after the cesarean delivery also was required to confer eligibility for home birth. antenatal screening practices were not evaluated per se. however, the observation that referrals to hospital- based care were made upon diagnosis of obstetric complications such as gestational diabetes, multiple gestation, or congenital anomalies suggests that stan- dard obstetric evaluation protocols were followed. variations in skill and experience of participating midwives could affect findings, but these aspects of practice were not evaluated directly. participants aver- aged more than years of experience as midwives and more than in home birth practice, with a range from a few months to years. practices also varied in the number of intended home births enrolled in the study over the course of year. this number ranged from two to . no attempt was made to analyze outcomes according to the experience of the clinician or practice. as noted above, the purpose was to examine home birth outcomes as they occur, given the mix of practice experience and protocol. the absence of a comparison group may be seen as a limitation of this study, creating difficulty in determin- ing whether the intrapartal and neonatal morbidity and mortality seen here are higher than what would be table . perinatal deaths in other studies of home birth*† author site n perinatal or neonatal mortality comment van alten et al wormerveer, – . per those remaining eligible for home birth at labor burnett et al north carolina, – per neonatal mortality only mehl et al us, . per excludes deaths due to prematurity and anomalies schramm et al missouri, – . per neonatal mortality among physician, nurse-midwife, and mma midwife–attended births only campbell et al britain, . per those booked for home birth woodcock et al australia, – home birth . per uncorrected for birth weight and gestational age sullivan and beeman arizona, . per excludes anomalies tyson canada, – per neonatal mortality only hinds et al kentucky, . per neonatal mortality only durand the farm, . per excludes deaths due to anomalies and premature births nrpmsc group britain, . per planned home birth mma � missouri midwife association; nrpmsc � northern region perinatal mortality survey coordinating group. * recalculated from original data where possible to remove twin births, congenital anomalies, and premature births (by standard study participants referred these complications for hospital birth). † only studies with at least births are recorded. vol. , no. , september murphy and fullerton home birth outcomes expected in a similar group of planned hospital births. however the series of risk screening filters through which women interested in home birth passed in these participating practices (at first inquiry, at first visit, and throughout the antepartal and perinatal period) makes it difficult to choose an equivalent comparison popula- tion from other data sources. in addition, bias due to self-selection would remain a threat despite careful matching of risk status. in consideration of these fac- tors, the prospective data are presented without a direct comparison; the low incidence of mortality allows indi- vidual examination of each case. only a randomized clinical trial would remove the issue of selection bias; however, this would be ex- tremely difficult to carry out. researchers in great britain assessed the feasibility of doing a randomized trial comparing home to hospital birth. they found that of women booking for obstetric care, were deemed of low enough risk for a home birth, and of these only ( . % of the original sample) agreed to be randomized. in addition, the authors noted that four of six women randomized to hospital birth were “dis- appointed,” although all randomized to home birth were pleased; those who declined to participate had strong preferences about the place of birth. wiegers et al further suggest that choice in childbirth may have a positive influence on levels of anxiety and apprehen- sion, which in turn could influence outcomes. elimina- tion of choice, as would be necessary with randomiza- tion, could theoretically have a negative impact on the course of childbirth and thus on outcomes. thus the debate over home birth is not likely to be settled with a randomized trial. questions about the relative safety of different birth settings are legitimate but often are answered in terms of ideologies rather than systematic research. the debate usually is framed as one of hospital compared with home. in most situations, and certainly in the practices described here, however, the circumstances are more planned hospital birth compared with in- tended home birth within a system that facilitates transfer to hospital birth when problems arise. thus, issues related to appropriate risk screening and predic- tion of perinatal problems are important when consid- ering out-of-hospital birth settings. the two factors most strongly associated with perinatal mortality and morbidity are congenital anomalies and low birth weight. neither condition is preventable by choice of birth site, although adverse sequelae may be modulated by the availability of emergency medical care. how- ever, in these practices, premature birth and pregnan- cies known to be complicated by fetal anomalies were referred routinely for planned hospital birth. the ability to identify other perinatal problems then becomes more important in out-of-hospital birth. recognizing that risk screening always will be an imperfect science and that some perinatal events cannot be predicted in any event, this report nonetheless underscores the ability of home birth practicing nurse-midwives to select an initial eligible sample at low risk of adverse perinatal out- comes. subsequently developing problems associated with increased perinatal risk, such as multiple gesta- tion, abnormal fetal lie, or concurrent medical illnesses also are referred appropriately for perinatal manage- ment or hospital delivery. although the outcomes of women referred for hospital birth prior to the onset of labor do not reflect on managed home birth, it is of interest to note the concentration of perinatal morbidity and cesarean delivery among women referred for hos- pital birth during the antepartum period. the apparent success of certified nurse-midwives in identifying higher risk pregnancies and appropriately referring these to perinatal care prior to labor is consistent with other reports. , during labor, events associated with increased need for interventions best delivered in hos- pital settings also are identified by nurse-midwives during home birth management, and the mothers or infants are transferred appropriately. one area that might require reassessment is the postdates pregnancy, especially if accompanied by meconium passage. these data and those from another recently published study suggest that eligibility for home birth should be consid- ered carefully in this circumstance; although hospital birth is no guarantee of a good outcome, specialty care is available more readily in that setting for infants with meconium aspiration. when intrapartum, postpartum, and neonatal emer- gencies occur, prompt transport and intervention is critical. perinatal emergencies were rare in this sample. participating midwives were asked at the beginning of this study about routine preparations for emergencies. all carry oxygen, oral suction equipment, intravenous setups, oxytocin, and methylergonovine to the home. all but one midwife carry a doppler device for moni- toring the fetal heart; the remaining midwife uses a fetoscope. all but one carry an ambu bag for resusci- tation and reported having formal certification in neo- natal resuscitation. sixty-eight percent bring a laryngo- scope and endotracheal tubes to home births; twenty percent also bring mechanical suction (data on file). in addition, most home birth practices in this study had eligibility requirements that the birth site be within minutes of a hospital providing obstetric services. ex- amination of hospital transfer records indicated that in the vast majority of cases, the mother arrived in the hospital with ample time prior to delivery to evaluate maternal-fetal status and make perinatal management decisions. in those cases in which birth occurred imme- murphy and fullerton home birth outcomes obstetrics & gynecology diately upon arrival, infants were born in good condi- tion with pediatricians in attendance. obviously, suc- cessful management of such transfers is also contingent on a close collaborating relationship between the mid- wife, the consulting obstetrician, and the hospital staff, especially given that only % of these participating midwives had hospital privileges. this evaluation of planned home birth points to the possibility that home birth can be accomplished, with good outcomes, within a structured system that allows for collaboration with physicians and referral to hospi- tal-based care where necessary. presentation of these data is not intended to posit home birth as a solution for all or most women. demand for home birth is admit- tedly minimal in the contemporary american health care system, but it has been a stable demand, resulting in , births a year. the outcomes, risks, and benefits of home care for childbirth should be assessed to make appropriate decisions regarding the circumstances in which home birth could occur, for those women who are determined to pursue this choice in childbirth. this report is intended to be a step in this direction. references . pearse wh. trends in out of hospital births. obstet gynecol ; : – . . clarke s, martin j, taffel s. trends and characteristics of births attended by midwives. stat bull metrop insur co 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bower c. planned homebirths in western australia – : a descriptive study. med j aust ; : – . . ford c, iliffe s, franklin o. outcome of planned home births in an inner city practice. bmj ; : – . . tyson h. outcomes of midwife-attended home births in toronto, – . birth ; : – . . ackermann-liebrich u, voegeli t, gunter-witt k, kunz i, zullig m, schindler c, et al. home vs. hospital deliveries : follow-up of matched pairs for procedures and outcomes. bmj ; : – . . davies j, reid w, young g, for the home birth study steering group. prospective regional study of planned home births. bmj ; : – . . northern region perinatal mortality survey coordinating group. collaborative survey of perinatal loss in planned and unplanned home births. bmj ; : – . . weigers ta, keirse mjnc, van der zee j, berghs gah. outcome of planned home and planned hospital births in low risk pregnan- cies: prospective study in midwifery practices in the netherlands. bmj ; : – . . mehl le, peterson gh, whitt m, hawes we. 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are they safe? birth ; : – . . anderson re, murphy pa. outcomes of , planned home births attended by certified nurse-midwives: a retrospective de- scriptive study. j nurse midwifery ; : – . . rooks jp, weatherby nl, ernst ekm, stapleton s, rosen d, rosenfield a. outcomes of care in birth centers. n engl j med ; : – . . leveno kj, cunningham fg, nelson s, roark m, williams ml, guzick d, et al. a prospective comparison of selective and univer- sal electronic fetal monitoring in , pregnancies. n engl j med ; : – . . eden rd, seifert ls, winegar a, spellacy wn. perinatal charac- teristics of uncomplicated postdate pregnancies. obstet gynecol ; : – . . olsen o. meta-analysis of the safety of home birth. birth ; : – . vol. , no. , september murphy and fullerton home birth outcomes . dowswell, thornton jg, hewison j, lilfford rjl. should there be a trial of home vs. hospital delivery in the united kingdom? bmj ; : – . . declercq er, paine ll, winter mr. home birth in the united states, – : a longitudinal descriptive report of national birth certificate data. j nurse midwifery ; : – . . campbell r, macfarlane a. place of delivery: a review. br j obstet gynaecol. ; : – . . garite t, snell bj, walker d, darrow v. development and experi- ence of a university-based, freestanding birth center. obstet gy- necol ; : – . . austin d. the process of obstetric triage: management by certified nurse-midwives. j perinat neonatal nurs ; : – . . mehl-madrona l, mehl madrona m. physician- and midwife- attended home births: effects of breech, twin, and post-dates outcome data on mortality rates. j nurse midwifery ; : – . address reprint requests to: patricia aikins murphy, cnm, drph department of obstetrics and gynecology columbia university college of physicians and surgeons west th street new york, ny e-mail: pam @columbia.edu received december , . received in revised form march , . accepted march , . copyright © by the american college of obstetricians and gynecologists. published by elsevier science inc. intrapartum controversies december – , the american college of obstetricians and gynecologists is sponsoring a course offering clinical presentations on a variety of difficult challenges the obstetrician encounters in the delivery room. the meeting is to be held at the sheraton new york hotel and towers, new york, new york. the american college of obstetricians and gynecologists has approved up to credit hours in category (formal learning) for this course. for further information, contact the registrar, the american college of obstetricians and gynecologists, th street sw, po box , washington dc, - , ( ) - . murphy and fullerton home birth outcomes obstetrics & gynecology [pdf] primary prevention of airway allergy | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /s - - - corpus id: primary prevention of airway allergy @article{wikstn primarypo, title={primary prevention of airway allergy}, author={j. wikst{\'e}n and s. toppila-salmi and m. m{\"a}kel{\"a}}, journal={current treatment options in allergy}, year={ }, volume={ }, pages={ - } } 	j. wikstén, s. toppila-salmi, m. mäkelä 	published 	medicine 	current treatment options in allergy purpose of reviewthe aim of this paper is to review and summarize the current knowledge of prevention of airway allergy.recent findingsallergic rhinitis and asthma are allergic airway diseases. due to their increasing incidence and socioeconomic burden, allergic airway diseases have recently gained attention worldwide. the primary prevention of allergic airway diseases focuses on offspring’s gestational and childhood environment, such as maternal smoking and diet during pregnancy and… expand view on springer link.springer.com save to library create alert cite launch research feed share this paper citationsbackground citations view all topics from this paper 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presents with hypertension and proteinuria. women with a history of pre-eclampsia are at increased risk of future cardiovascular complications. this state-of-the-art paper reviews the available evidence that supports the association of pre-eclampsia with future cardiovascular risk and explores potential management options for these high-risk patients. state-of-the-art papers digoxin in worsening heart failure andrew p. ambrosy, javed butler, ali ahmed, muthiah vaduganathan, dirk j. van veldhuisen, wilson s. colucci, mihai gheorghiade the dig (digitalis investigation group) trial revealed that digoxin therapy reduced all-cause and heart failure (hf)–specific hospitalization but had no effect on survival. as a result, the role of digoxin in the management of hf patients remains controversial. this review evaluates the available data on the role of digoxin in the contemporary management of hf and provides a framework for further research. (continued on page a- ) http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - may , (continued) clinical research a- interventional cardiology radial artery catheterization and allen’s test marco valgimigli, gianluca campo, carlo penzo, matteo tebaldi, simone biscaglia, roberto ferrari, for the radar investigators this study investigates the safety and feasibility of transradial catheterization across the whole spectrum of allen’s test (at) results. a total of patients were recruited, of whom , , and presented a normal, intermediate, and abnormal at results, respectively. lactate did not differ among the study groups after the procedure and there were no hand ischemic complications. plethysmography readings and ulnar frame count suggested enhanced ulnar flow in abnormal at patients after transradial access (tra). these results suggest the safety and feasibility of tra across the whole spectrum of at results. editorial comment: olivier f. bertrand, patrick c. carey, ian c. gilchrist, p. interventional cardiology coronary calcification and percutaneous coronary intervention philippe généreux, mahesh v. madhavan, gary s. mintz, akiko maehara, tullio palmerini, laura lasalle, ke xu, tom mcandrew, ajay kirtane, alexandra j. lansky, sorin j. brener, roxana mehran, gregg w. stone the investigators sought to determine the impact of coronary calcification on outcomes after percutaneous coronary intervention (pci) for patients presenting with non–st-segment elevation and st-segment elevation acute coronary syndrome (acs). data from , patients from large randomized trials was pooled. the presence of moderate/severe target lesion calcification was an independent predictor of -year definite stent thrombosis (hazard ratio [hr]: . ) and ischemic target lesion revascularization (hr: . ). the authors conclude that moderate/severe lesion calcification is relatively frequent in patients with non–st-segment elevation acs syndromes and st-segment elevation myocardial infarction, and is strongly predictive of stent thrombosis and ischemic target lesion revascularization at year. editorial comment: david d. waters, rabih r. azar, p. (continued on page a- ) http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) -x http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - may , (continued) a- acute coronary syndromes hypothermia for stemi david erlinge, matthias götberg, irene lang, michael holzer, marko noc, peter clemmensen, ulf jensen, bernhard metzler, stefan james, hans erik bötker, elmir omerovic, henrik engblom, marcus carlsson, håkan arheden, ollie Östlund, lars wallentin, jan harnek, göran k. olivecrona in this investigation, the authors attempted to confirm the cardioprotective effects of hypothermia using a combination of cold saline and endovascular cooling in patients with st-segment elevation myocardial infarction (stemi). a total of patients with stemi (< h) were randomized, and the primary endpoint was infarct size as a percent of myocardium at risk (is/mar). is/mar was not significantly reduced by hypothermia, but the incidence of heart failure was lower. analysis of early anterior infarction ( to h) found a reduction in is/mar of %. the authors conclude that hypothermia did not lower the primary endpoint of is/mar; however, the lower incidence of heart failure and a possible effect among early anterior stemi need confirmation. acute coronary syndromes prognostic value of barc bleeding in stemi wouter j. kikkert, nan van geloven, mariet h. van der laan, marije m. vis, jan baan, jr, karel t. koch, ron j. peters, robbert j. de winter, jan j. piek, jan g. p. tijssen, josé p. s. henriques the aim of this analysis was to compare -year mortality predictions of bleeding academic research consortium (barc)–defined bleeding complications with existing bleeding definitions in patients with st-segment elevation myocardial infarction (stemi) and to investigate the prognostic value of individual data elements of the bleeding classifications. gusto (global utilization of streptokinase and tissue plasminogen activator for occluded arteries) and international society of thrombosis and haemostasis–defined bleeding were not associated with -year mortality, whereas timi (thrombolysis in myocardial infarction) major and barc type b or c bleeding conferred a -fold higher risk of -year mortality. the data elements most strongly associated with mortality were a hemoglobin drop � g/dl, the use of vasoactive agent for bleeding, cardiac tamponade, and intracranial hemorrhage. both the barc and timi bleeding classifications identify stemi patients at risk of -year mortality. editorial comment: harold l. dauerman, p. (continued on page a- ) http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - may , (continued) a- heart rhythm disorders scd risk stratification in nidcm jeffrey j. goldberger, haris suba�cius, taral patel, ryan cunnane, alan h. kadish in this study, the authors evaluated the performance of commonly reported risk stratification tests as predictors of arrhythmic events in patients with nonischemic dilated cardiomyopathy (nidcm). a meta-analysis of studies (n ¼ , ) evaluating the association between arrhythmic events and predictive tests (baroreflex sensitivity, heart rate turbulence, heart rate variability, left ventricular end-diastolic dimension, left ventricular ejection fraction, electrophysiology study, nonsustained ventricular tachycardia, left bundle branch block, signal-averaged electrocardiogram, fragmented qrs, qrs-t angle, and t-wave alternans [twa]) was conducted. the odds ratio (or) was highest for fragmented qrs and twa (or: . and . , respectively) and lowest for qrs duration (or: . ). no autonomic test was a significant predictor of arrhythmic outcomes. the study concludes that the parameters studied provided only modest risk stratification for sudden cardiac death in patients with nidcm. editorial comment: james p. daubert, robert k. lewis, p. heart rhythm disorders ethanol-induced regional left atrial denervation josé l. báez-escudero, takehiko keida, amish s. dave, kaoru okishige, miguel valderrábano the objective of this study was to determine whether ethanol infusion in the vein of marshall (vom) can ablate intrinsic cardiac nerves (icn). patients undergoing catheter atrial fibrillation (af) ablation additionally underwent high-frequency stimulation (hfs) using a multipolar catheter introduced in the vom. parasympathetic activity and af inducibility was assessed before and after vom ethanol infusion. using burst hfs, parasympathetic activity was found in all patients undergoing de novo af ablation and % of patients undergoing repeat ablation. after vom ethanol infusion, the parasympathetic response was abolished in all patients. the results reveal that the vom contains icn that connect with the atrioventricular node and can trigger af. retrograde ethanol infusion in the vom reliably eliminates local icn responses. editorial comment: pradeep s. rajendran, eric buch, kalyanam shivkumar, p. (continued on page a- ) http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - may , (continued) a- cardiac imaging combined ct techniques to assess invasive ffr dennis t. l. wong, brian s. ko, james d. cameron, darryl p. leong, michael c. h. leung, yuvaraj malaiapan, nitesh nerlekar, marcus crossett, john troupis, ian t. meredith, sujith k. seneviratne this study compares the diagnostic accuracy of combined adenosine stress computed tomography myocardial perfusion (ctp) þ computed tomography coronary angiography (cta), transluminal attenuation gradient (tag ) + cta, and ctp + tag + cta (multidetector computed tomography integrated protocol [mdct-ip]) assessment in predicting significant fractional flow reserve (ffr) vessels in patients undergoing coronary angiograms. in vessels, cta predicted ffr-significant stenosis with % sensitivity and % specificity, and mdct-ip showed % sensitivity and % specificity. tag + cta and ctp + cta provided comparable per-vessel diagnostic accuracy, and mdct-ip was superior to both. the authors conclude that mdct-ip may provide the best diagnostic accuracy for functional assessment of coronary artery stenosis. editorial comment: daniel s. berman, richard a. stoebner, damini dey, p. hypertension remodeling after renal denervation independent of hr and bp stephan h. schirmer, marwa m. y. a. sayed, jan-christian reil, christian ukena, dominik linz, michael kindermann, ulrich laufs, felix mahfoud, michael böhm this study investigates the interaction between blood pressure (bp) and heart rate (hr) reduction and changes in left ventricular (lv) structure and function following renal sympathetic denervation (rdn). lv size, mass, and diastolic function were evaluated in patients before and months after rdn. there was a decrease in systolic/diastolic bp ( . � . mm hg/ . � . mm hg to . � . mm hg/ . � . mm hg) and reduction in lv mass index ( . � . g/m . to . � . g/m . ). there was also an improvement of diastolic function as well as an increase in vascular compliance. structural and cardiac changes were not exclusively related to bp or hr changes, suggesting a direct role of the sympathetic nervous system activity on cardiac morphology and function. editorial comment: george bakris, sandeep nathan, p. http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - http://linkinghub.elsevier.com/retrieve/pii/s - ( ) - 	inside this issue 	state-of-the-art papers 	state-of-the-art papers 	pre-eclampsia and heart disease 	state-of-the-art papers 	digoxin in worsening heart failure 	clinical research 	interventional cardiology 	radial artery catheterization and allen's test 	interventional cardiology 	coronary calcification and percutaneous coronary intervention 	acute coronary syndromes 	hypothermia for stemi 	acute coronary syndromes 	prognostic value of barc bleeding in stemi 	heart rhythm disorders 	scd risk stratification in nidcm 	heart rhythm disorders 	ethanol-induced regional left atrial denervation 	cardiac imaging 	combined ct techniques to assess invasive ffr 	hypertension 	remodeling after renal denervation independent of hr and bp men_ .. rad sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic snp assays s t e p h e n j . a m i s h , * p a u l a . h o h e n l o h e , † s a l l y p a i n t e r , * r o b b f . l e a r y , ‡ c l i n t m u h l f e l d , §¶ f r e d w . a l l e n d o r f * a n d g o r d o n l u i k a r t * ¶* * *fish and wildlife genomics group, division of biological sciences, university of montana, missoula, mt , usa, †department of biological sciences, university of idaho, moscow, id - , usa, ‡montana fish, wildlife & parks, university of montana, missoula, mt , usa, §u.s. geological survey, northern rocky mountain science center, glacier national park, west glacier, mt , usa, ¶flathead lake biological station, university of montana, polson, mt , usa, **cibio, centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, - vairão, portugal abstract hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. recently, we identified several thousand candidate single-nucleotide polymor- phism (snp) markers based on rad sequencing of westslope cutthroat trout and rainbow trout individuals. here, we used flanking sequence for of these candidate snp markers to design high-throughput genotyping assays. we vali- dated the assays on a total of individuals from populations and seven hatchery strains. forty-six assays ( %) ampli- fied consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. the snps will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. this technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing snp markers for nonmodel organisms with limited genomic resources. keywords: conservation genomics, hybridization, introgression, invasive species, microfluidic pcr, salmonids, snp, trout species identification received november ; revision received february ; accepted february introduction rainbow trout (rbt; oncorhynchus mykiss), the most widely introduced salmonid in the world (lever ), produce fertile offspring when crossed with cutthroat trout (o. clarkii), and introgression often continues until a hybrid swarm is formed and the native cutthroat genomes are lost (allendorf & leary ). a major consequence of such interspecific hybridization may be outbreeding depression because of the break-up of co-adapted gene complexes and disruption of local adap- tations (allendorf et al. ; muhlfeld et al. ). introgression poses a serious threat to all subspecies of cutthroat trout in western north america owing to wide- spread stocking of rainbow trout and invasion by rainbow trout and hybrids into historical cutthroat trout habitats. currently, range-wide estimates of hybridization in many of the cutthroat trout subspecies and popula- tions are incomplete. westslope cutthroat trout (wct; oncorhynchus clarkii lewisi), the most widely distributed subspecies of cutthroat trout, historically occupied aqua- tic habitats throughout the columbia, fraser, missouri and hudson bay drainages of the united states and can- ada (behnke ). however, nonhybridized populations are estimated to persist in < % of their historical range (shepard et al. ). while over half of the population genetic samples in shepard et al. ( ) found no evi- dence of admixture, only % had enough individuals sampled to detect % admixture at the % level of confi- dence. as a result, only % of the population genetic samples showed no evidence of admixture (< %) with a high degree of confidence. markers detecting low amounts of admixture in popu- lations and individuals will provide an understanding of the mechanisms causing the spread of hybridization, help protect nonhybridized populations from invasion, and aid in identifying nonhybridized populations suitable as sources for hatchery brood stocks or other conservation actions (allendorf et al. ). besides estimates of correspondence: steve amish, fax: - - ; e-mail: stephen.amish@umontana.edu � blackwell publishing ltd molecular ecology resources ( ) , – doi: . /j. - . . .x individual or population levels of admixture, the distri- bution and the frequency of introgressed genotypes within a population or sample can illuminate the dura- tion and extent of hybridization (jiggins & mallet ). for example, a bimodal distribution is thought to result from selection against intermediate genotypes or assorta- tive mating between the species (jiggins & mallet ; weigel et al. ). currently, the number of available species-diagnostic loci for addressing these questions in native cutthroat trout and rainbow trout is limited. the development of additional species-diagnostic genotyping assays and high-throughput snp genotyping systems will provide increased power for detecting hybridization at the individual level and more precisely estimating the structure of hybrid zones. currently, – diagnostic microsatellite markers are often used to detect cutthroat and rainbow trout hybridization at the population level. if we assume that each marker sorts independently, there is no linkage disequilibrium affect- ing the markers, and if genotypes are distributed accord- ing to hardy–weinberg ratios (i.e. the samples are representative of a breeding aggregation or population), then we can calculate the likelihood of failing to detect a single rbt allele in any given fish or in a sample of unre- lated fish using binomial probability (rasmussen et al. ). the probability of detecting % hybridization in a population with % certainty and individual samples requires only eight independent diagnostic markers. in contrast, to detect % admixture in an individual with % certainty will require independent diagnostic markers (table ). similarly, an increase in the number of diagnostic markers will also improve our ability to differ- entiate between parental back-crosses and later genera- tion hybrid crosses. recently, we identified a large set of candidate diag- nostic snps using restriction-site-associated dna (rad) sequencing (hohenlohe et al. ). briefly, we sequenced a single rad library (baird et al. ; etter et al. a) created from fish [ wct, coastal rain- bow trout (o. m. mykiss, crt), and inland or redband rainbow trout (o. m. gairdneri, irt)] and applied strict fil- tering based on observed heterozygosity and deviations from hardy–weinberg equilibrium to remove homeolo- gous loci (paralogs resulting from the ancestral salmonid whole-genome duplication; lie et al. ). that analysis produced a total of rad markers at which there was a single candidate snp fixed between the two spe- cies, and no other polymorphism, within the informative -bp rad tag sequence (hohenlohe et al. ). here, we expand the list of candidate snp markers, and we use microfluidic pcr assays to verify a subset of them for high-throughput estimates of hybridization in trout. we chose to develop a bioinformatics pipeline for this pur- pose because of its cost-effectiveness and because the rainbow trout genome would subsequently be available for the development of additional markers. materials and methods in addition to the single-snp candidate markers from the study by hohenlohe et al. ( ), we identified candidate diagnostic snps in rad tags containing two putative fixed snps in the -bp sequence (an additional markers) and those containing one putative fixed snp and one additional site polymorphic within one of the taxa (an additional markers). we aligned the total set of rad tag sequences against a published database of rainbow trout sequence contigs (sanchez et al. ) using the program bowtie (langmead et al. ). we allowed up to three mismatches between the wct or the crt and the reference sequence. the data set from the study by sanchez et al. ( ) contained contigs ranging in size from to bp, produced by sequencing of a reduced representation genomic library in rainbow trout. a total of ( . %) of our candi- date rad tags aligned against one or more of the contigs from the study by sanchez et al. ( ) with at least bp of flanking sequence on either side of the diagnostic snp. ten of these loci were dropped after preliminary data suggested one of the primers or probes was not amplify- ing. sequences for the remaining candidate markers were submitted to kbioscience for the design of kaspar snp genotyping assays. a total of individuals from populations and seven hatchery strains plus two heterozygous positive controls (f s) were then used to validate the assays on fluidigm . microfluidic pcr chips. the individuals included two cutthroat trout species, wct and yct (yel- lowstone cutthroat trout, o. c. bouvieri), as well as irt and crt (table ). all samples came from putatively nonhybridized populations, based on a current panel of seven diagnostic microsatellite loci and seven indel loci, table the likelihood of detecting a single rbt allele in any given fish or in a population of unrelated fish using binomial probability for a number of independent species-diagnostic loci, samples and percent hybridization number of markers number of samples %hybridization probability of detection . . . . . . . . . . � blackwell publishing ltd s . j . a m i s h e t a l . except for fish from lake koocanusa, which appear to have both a crt and irt genetic component (r. leary, personal communication), and the south fork of the jocko river, which appear to have a crt component. wct samples included two year classes from the washoe park state trout hatchery, anaconda montana, and samples from wild populations, including three populations from the missouri river basin east of the continental divide. yct samples were from the yellow- stone river state trout hatchery, big timber, montana, and a population in slough creek. irt samples were from two populations in the kootenai river drainage in montana. crt were taken from hatchery stock from across the country to account for the multitude of poten- tial sources used currently and historically for stocking. results forty-six of fifty-six assays ( %) were diagnostic for the identification wct, rbt and hybrids (table ). an assay was considered diagnostic if both heterozygous positive controls showed separation from the homozygous genotype clusters and > % of samples had concordant genotypes (i.e. genotype agreed with expectation estab- lished by earlier microsatellite ⁄ indel data). eight of ten assay failures were attributed to the design process, including poor quality or inadequate sequence data being used to design the assay (e.g. using sequence from a paralogous region or sequence containing errors) or errors in the primer or probe design and manufacture process. these failures included three assays where one or both probes did not amplify, and one assay appears to have amplified a homeolog. in fact, this locus had an elevated depth of sequence reads in the original rad sequencing run, in the th percentile among the fixed single-snp candidate markers (hohenlohe et al. ), consistent with the interpretation that it represents two incorrectly assembled homeologs. given a total of assays for detecting rbt and wct hybridization and four samples from a population, we have a . % probability of detecting % introgression in a hybrid swarm. in an individual fish, we have a % probability of detecting > . % introgression with the same number of assays (table ). however, very low table location name, species, whether the fish is of wild or hatchery origin, and the number of samples, basin and subbasin information for the screening panel used to validate species-diagnostic assays. species are labelled as westslope cutthroat trout (wct), yellowstone cutthroat trout (yct), inland or redband rainbow trout (irt), and coastal rainbow trout (crt) location name species wild ⁄ hatchery n basin subbasin anaconda, mt wct h na* na big foot creek wct w columbia upper kootenai copper creek wct w columbia flint-rock cottonwood creek wct w columbia lower flathead davis creek wct w columbia bitterroot flat creek wct w columbia upper kootenai gillispie creek wct w columbia flint-rock hawk creek wct w columbia n. f. flathead humbug creek wct w columbia blackfoot mcginnis creek wct w columbia lower clark fork morrison creek wct w columbia middle flathead ringeye creek wct w columbia blackfoot runt creek wct w columbia yaak s. fork jocko wct w columbia lower flathead six mile creek wct w columbia middle clark fork werner creek wct w columbia n. f. flathead bear creek wct w missouri red rock mcclellan creek wct w missouri upper missouri mcvey creek wct w missouri big hole big timber, mt yct h na na slough creek yct w missouri yellowstone lake koocanusa, bc irt w columbia yaak yahk river, bc irt w columbia yaak abbot creek crt w columbia middle flathead arlee, mt crt h na na eagle lake, ca crt h na na mcconaughy, ne crt h na na fish lake, ut crt h na na erwin ⁄ arlee cross, tn crt h na na *basin and subbasin designations were not made for hatchery stocks. � blackwell publishing ltd t r o u t s p e c i e s - d i a g n o s t i c s n p s f r o m r a d s e q u e n c i n g t a b le s e q u e n ce in fo rm a ti o n fo r th e v a li d a te d a ss a y s. t h e n u m b e r o f a d d it io n a l v a ri a b le si te s in th e r a d ta g a n d th e re fe re n ce se q u e n ce a re li st e d , a lo n g w it h th e ca ll ra te (% o f sa m p le s a ss ig n e d to a g e n o ty p e cl u st e r) a n d th e ca ll co n co rd a n ce (% o f sa m p le s a ss ig n e d to th e co rr e ct cl u st e r) . s e q u e n ce s g iv e n a re fo r th e k b io sc ie n ce s k a s p a ss a y fo rm a t. r b t se q u e n ce n a m e is th e ra in b o w tr o u t re fe re n ce se q u e n ce (s a n ch e z et al . ) fr o m th e r a d lo ci a li g n m e n ts u se d fo r fl a n k in g se q u e n ce in th e s n p a ss a y d e si g n a ss a y n a m e a d d it io n a l v a ri a b le s it e s s n p n % c a ll ra te % c o n co r- d a n ce s e q u e n ce ( ¢- ¢) r a d g e n b a n k a cc e ss io n n o . r b t r e f s e q u e n ce g e n b a n k t ra ce a rc h iv e n o . r a d t a g r e f s e q p ri m e r f a m a ll e le p ri m e r v ic a ll e le c o m m o n p ri m e r r b t w c t o m y _ r a d _ _ a ⁄c . . g t c a g t a g g a g g t g c t a t t g a g a t c a g t a g g a g g t g c t a t t g a g c g c c t g c a g g c t g t c c a g t a g t t jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄a . . t g g g c t g t g t g a g a g a c a g a g a t g g g c t g t g t g a g a g a c a g a g t a c a c c t g c a g g g c c t g t c t g a a jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄c . . t t c c t g t g t a a a g c a g t g g t g g a c t t c c t g t g t a a a g c a g t g g t g a g t g c t c g t a c c a t c c a c c g t c a a jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄g . . g g a g c a a a g c a t t a a a a g t g t g c t g a t g g a g c a a a g c a t t a a a a g t g t g c t t g t t g t t g a t g a g c c a g g g t c t g t t t jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄t . . t t a a t a a t c a c t a c a t t t c a c a t a g a a t t g c t t t t a a t a a t c a c t a c a t t t c a c a t a g a a t t g c t a g c t t a g a t t g t a t a t t c t g c t g c t a g g t t jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄t . . g c t g c t c t g c t g a c g g t t c c t g c t c t g c t g a c g g t t a g g t t g a a c t g g a c g a g c c g g a a jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄g . . c t g t t c a g g g t g a t g a t g c t g t c t g t t c a g g g t g a t g a t g c t g c c a g g a t g a g g g t t g - c c t g g t c a t jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄g . . g a a c t t t g c t g g g c a t g t g g g g a a c t t t g c t g g g c a t g t g g t t g c a c g g a t a a c a t g g t c t t t g a t a a c t t jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . c c c a c g g g a t a c t g g g t g c c c c a c g g g a t a c t g g g t a c c t g c a g g a g c t g g t c a g c t a t jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . t c c t g c a g g g t g t c g g c g c t c c t g c a g g g t g t c g g c a c g c t t t a a a c a g c t g g t g g a c a g t a jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄a . . a t g c a c a c c a c t g c a t c c a g a t a t g c a c a c c a c t g c a t c c a g a c c t a c t g t t a c a a c c t g c a g g a g c t a jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . g g t a g a t t t c c g a c g t a a a t a c g g g g t a g a t t t c c g a c g t a a a t a c g a g a g g c c c t g c a g g a a a t a a c g a t t jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄a . . g a t g t g t g g c t g t t g g t c a a c c a a t g t g t g g c t g t t g g t c a a c c g g c a a g a c c c t c a g a a t c c t c t t c a a jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄t . . g t c g t t c t t c t g g c c c a g g a c t g t c g t t c t t c t g g c c c a g g a a g t c a g g c t c t g a c g g c c t a c t t jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . c c c g c c a g a t g g c c a g g c c c c g c c a g a t g g c c a g a c a t g g a g g a c c t g a g t g c t c t a a a jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄c . . g g t c t g t c c c c c t g t c c g t t c t g t c c c c c t g t c c g g g c a g t g t g a c c c t g c a g g a c a jq jq g n l| ti | _ � blackwell publishing ltd s . j . a m i s h e t a l . t a b le (c o n ti n u e d ) a ss a y n a m e a d d it io n a l v a ri a b le s it e s s n p n % c a ll ra te % c o n co r- d a n ce s e q u e n ce ( ¢- ¢) r a d g e n b a n k a cc e ss io n n o . r b t r e f s e q u e n ce g e n b a n k t ra ce a rc h iv e n o . r a d t a g r e f s e q p ri m e r f a m a ll e le p ri m e r v ic a ll e le c o m m o n p ri m e r r b t w c t o m y _ r a d _ _ c ⁄t . . a t t c t a g a t t c t a g a c a c a t g a c t c c a t t c t a g a t t c t a g a c a c a t g a c t c t t a a t t c a a c t a g c g g t g t g t t g t g t t g t a jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄c . . a t a a t a a g a t c a t g c a a c a g t a a g t g t t t g a t a a t a a g a t c a t g c a a c a g t a a g t g t t t c a t g c c c c t g c a g g c a a g c c a t t jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄c . . g a a c c c a c c c a t t t c a g t g g a c g a a c c c a c c c a t t t c a g t g g a t t t c c t g g g t g a a g t a g g g g a t t g a a jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄c . . a a c c c t c c a t t c g t c a c a t t t a a c c c a a c c c t c c a t t c g t c a c a t t t a a t c t c t t c t a t c t t g t t g a c g t c g a c c t t jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄c . . t g g g t a a t c a c g a g g g t a c a t c t g g t a a t c a c g a g g g t a c a t c g c g t c c a g a g g a g c c a a t g g c a t jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄t . . t c a c a g t a g t c a a c a c t g t g c a g a c t c a c a g t a g t c a a c a c t g t t g t c c t g t t t g t t c a t c t g g t c t c c a a jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄c . . g g t g a a a g t a c a g g t a g c g c t t g a g g t g a a a g t a c a g g t a g c g c t t a a a c a g c t t a c a c c c a g a g c t g c t t jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄g . . a t c t g t t g a c t c c c t c t c c t c t a t c t g t t g a c t c c c t c t c c t c c t t t a c c a g g c g g t g c g g c a g t t jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄c . . t g a a g a a g c c g g a t g t g g a g g t g a a g a a g c c g g a t g t g g a g a c t c a c a a g c g c a g t t c g c a t g t a a jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄a . . g c a g g a t t c a g t c a a g a g c c c t c a g g a t t c a g t c a a g a g c c c c t g t g g a c a a g a t c a g g a c a c g t g t t jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . t g a a g a a g c c g g a t g t g g a g g t g a a g a a g c c g g a t g t g g a g a c t c a c a a g c g c a g t t c g c a t g t a a jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . a g g t c c a t c a a g t c a a a g g c g c c a g g t c c a t c a a g t c a a a g g c a g t g g a t g a c c a c c t g c a g g a c a a jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄g . . a g g g a t g a g a c t c c t c t g a a c c a g g g a t g a g a c t c c t c t g a a g t c t t c c t g c t g c t g a t g t t g c t g t t jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄c . . t c t g c c a g t c t g t c a g g t c g c t c t g c c a g t c t g t c a g g t c a g a t g c t g t g t g g - g a t g c a g g a g a t jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄t . . g t c t t t g t t g g a a t t t a t t g c c a t a t t c t c t t t g t t g g a a t t t a t t g c c a t a t t a a t a t c t c a c c t g c a g g t t t a a g t a c c a a a jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄g . . t a t c g g g t a c c t g c a g g t g a c t a t c g g g t a c c t g c a g g t g a g g c c t t g a c a g t a c a a c a g g c a c t t t jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄t . . a a c t c c a c a a g g t c a g a g g t a a c a a c t c c a c a a g g t c a g a g g t a a a c t a c t c t g c c g a c a t c c t a t c a g a a jq jq g n l| ti | _ � blackwell publishing ltd t r o u t s p e c i e s - d i a g n o s t i c s n p s f r o m r a d s e q u e n c i n g t a b le (c o n ti n u e d ) a ss a y n a m e a d d it io n a l v a ri a b le s it e s s n p n % c a ll ra te % c o n co r- d a n ce s e q u e n ce ( ¢- ¢) r a d g e n b a n k a cc e ss io n n o . r b t r e f s e q u e n ce g e n b a n k t ra ce a rc h iv e n o . r a d t a g r e f s e q p ri m e r f a m a ll e le p ri m e r v ic a ll e le c o m m o n p ri m e r r b t w c t o m y _ r a d _ _ c ⁄t . . g g t g a a t t c g g t g t t g t c t g c c g g t g a a t t c g g t g t t g t c t g t g a g t c t c a t c c c t g c a g g g c t t jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄c . . a a c a c c g a t a t a c a t a a a t g t g c t g t a a c a c c g a t a t a c a t a a a t g t g c t g g g a c t c a g c c t g c a g g g g t c a t a jq jq g n l| ti | _ o m y _ r a d _ _ t ⁄c . . t g g t c a a t g c c a t t a t c a a c a g c c t g g t c a a t g c c a t t a t c a a c a g t c t c a c a g t a c c a g c a c g a c c a a t a jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄t . . t g g a c t c a a a c a g a t c c a a t a a c t a c t g g a c t c a a a c a g a t c c a a t a a c a g g t a c t t c t g t g a a a a c c a t t t g t g t g a a jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄a . . g t a c c t g c a g g g a a a g c t a c t c t t a c c t g c a g g g a a a g c t a c t c g g g a t c c a c c a g t g t g t a t g t g t a g t t jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . g t c a g t t t t c c t t g t c a g g c t c c t t g t c a g t t t t c c t t g t c a g g c t c t g t c g a a g t c t g c c t c a a c c a c a a t a jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄a . . g a g g c c t t a c a g a t t g a t t g c a c a a g g c c t t a c a g a t t g a t t g c a c g g g c a c a g c a g a a g a c c a a t t t c c a t jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄g . . g g a g g a a c c t g c a g g t g g c g a g g a a c c t g c a g g t g g g a a a g t c a g t t a a c t a c a c t a c a g a c c a a t t jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄g . . c c a c a g c g a c c c c a t c g a a c c a c a g c g a c c c c a t c g a g g g t c a a a t g t c a g g g t t a a t c a g a a g t a jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . t c t t a c c a c g a g c t c a g g g a c t c t t a c c a c g a g c t c a g g g a t g c t g g a t c t c a t g g t g g t c c a g a t jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄t . . a g g t g g t g c c a g g a c a g g g c a a g g t g g t g c c a g g a c a g g t c c a g a t c c a g g c c t g c a g g t a a jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . t t g g a g c g g t a c t c t t t c a g g c t t g g a g c g g t a c t c t t t c a g a g g a g t c c c t g c a g g c c a a t g t a jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄a . . t g t g c t g c a g c c c a c a t c a g a a t g c t g c a g c c c a c a t c a g a g t t a a c c t g c a g g a t g a g g a a g g c t t jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄a . . g t c t g t a g c a t a t a c t a t g t t g t c c t t c t g t a g c a t a t a c t a t g t t g t c c c c c t g t c t g g g a a t a a c a g c c g t a t a jq jq g n l| ti | _ o m y _ r a d _ _ c ⁄t . . a c c t c g t c c t g c a g g t c t g c c a c c t c g t c c t g c a g g t c t a c c c t g c t c g a c c c g t g t c t a jq jq g n l| ti | _ o m y _ r a d _ _ a ⁄c . . t g t g t t a c a g c t g c g g g t c c t t t g t t a c a g c t g c g g g t c c t g g g t c a g g c t g c a g t g g a g g a a a jq jq g n l| ti | _ o m y _ r a d _ _ g ⁄t . . g a g c c g t c c t t c a g g a a t c g c a g a g c c g t c c t t c a g g a a t c g a c t g c a g g a g a g g g a g g g g c t t jq jq g n l| ti | _ � blackwell publishing ltd s . j . a m i s h e t a l . levels of introgression will still be difficult to detect at the individual level. our probability of detecting % intro- gression in a fish using markers is only %. because our preliminary screening panel was com- posed of a few individuals from many populations instead of many individuals from a few populations, we have little power to detect alleles at low frequency in these populations. thus, we cannot exclude the possibil- ity that some of the diagnostic markers may share a low-level polymorphism between wct and rbt. in species-diagnostic assays, individuals with genotypes indicative of low-level polymorphisms or rbt hybridiza- tion were detected. the snp from rad_ was not diagnostic in our screening panel. one individual was homozygous for the ‘rbt allele’ at this locus, and others were heterozygotes. these individuals, however, did not possess any other genotypes at the other loci analysed indicative of hybridization. thus, this locus appears to be polymorphic in wct. wct from north- western montana or south-eastern british columbia were heterozygous in five additional assays, and the level of introgression in these populations is uncertain due in part to the potential presence of irt alleles (table s ). fish from lake koocanusa may have low levels of intro- gression from crt or wct. in the fish from yaak, bc and runt creek, these polymorphisms may represent natural levels of introgression between the sympatric wct and irt populations. in five of the remaining six assays, the heterozygous fish came from hatchery popu- lations. because hatchery brood stock samples have been extensively screened for admixture using indels and mi- crosatellites, it is most likely that these alleles represent shared low-level polymorphisms between wct and rbt. testing of additional samples will be required to determine their frequency and the usefulness of the assays for species identification and admixture analysis. discussion our conversion rate of % for diagnostic assays suggests that rad sequencing offers a reliable and relatively quick and inexpensive way to generate large numbers of snp markers that does not require a large screening panel (e.g. seeb et al. ). conversion rates can vary widely and depend on the variability and divergence of the tar- get species, the number of samples sequenced before designing the assays, whether the snp is in a conserved or highly variable region (e.g. diagnostic between species or polymorphic within species) and on the number and extent of samples used to validate the assay. new sample library protocols and next-generation sequencing tech- niques like rads promise to make very low-cost marker development available for most organisms (pennisi ) even when no genomic resources are available. the development of additional species-diagnostic genotyping assays provides increased power for detecting hybridization at the individual level and more precisely estimating the structure of hybrid zones. with the addi- tion of our assays to the previously available snps (finger et al. ; mcglauflin et al. , harwood & phillips ; kalinowski et al. ), the number of cur- rently available diagnostic snps between wct and rbt has increased to . with diagnostic snps, we can detect . % introgression with % certainty at the indi- vidual level. our probability of detecting % introgression in a fish using markers is only %, reaching % with markers (table ). the ability to detect low levels of hybridization at the individual level increases sampling scheme flexibility, removing the requirement that aggre- gations of - samples be considered a population. we developed a bioinformatic pipeline using publicly available reads (sanchez et al. ) for identifying flanking sequence required for assay design that will be eas- ily applied to the rainbow trout genome sequence when it is published (miller et al. ). this reduced our set of candi- date loci from to ( . %). at the time of this experi- ment, using sequencing to produce reads > -nt reads required for snp assay development was beyond our bud- get. the reference genome sequence will allow assay design for most of the snps identified in our rad loci. an alternative approach to using published long read sequence data is to generate longer contiguous sequence reads at each rad tag using over-lapping paired-end sequencing (etter et al. b). this technique holds great promise for allowing assay design on the full set of candi- date snp markers for any species. in addition, this approach should have a higher validation rate, because snp detection and flanking sequence would come from the same individuals and populations. rad sequencing is one of a family of approaches applying high-throughput sequencing to a reduced rep- resentation of a genome to identify and genotype large numbers of snp markers in organisms without substan- tial genetic resources (cosart et al. ; davey et al. ). next-generation sequencing approaches require slightly more bioinformatic effort compared with tradi- tional marker discovery, but a number of publicly avail- able tools are being developed to handle these types of data (catchen et al. ; davey et al. ). one advan- tage of rad over related restriction-enzyme-reduced representation sequencing techniques in taxa with com- plex, repetitive genomes is that the set of markers does not depend on a fragment size selection step, so that it is more consistent across libraries (davey et al. ). this helps reduce variation between runs and allows the com- pilation and re-analysis of large sequence databases across related species, populations and individuals gen- erated using the same rad library technique. we con- � blackwell publishing ltd t r o u t s p e c i e s - d i a g n o s t i c s n p s f r o m r a d s e q u e n c i n g clude that the emerging techniques for the generation and analysis of rad sequencing data provide a relatively quick and cost-effective method for the identification of large numbers of species-diagnostic snps. acknowledgements pah was supported by nih cobre grant p rr (l. forney, pi). funding was provided in part by the great northern landscape conservation cooperative (us department of interior). any use of trade, product or firm names is for descriptive purposes only and does not imply endorsement by the u.s. government. this research was conducted in accordance with the animal welfare act and its subsequent amendments. gl & fwa were supported by nsf deb . references allendorf fw, leary rf ( ) conservation and distribution of genetic variation in a polytypic species, the cutthroat trout. conservation biol- ogy, , 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( ) high-resolution melting analysis for the discovery of novel single-nucleotide polymorphisms in rainbow and cutthroat trout for species identification. transactions of the american fisheries society, , – . miller mr, brunelli jp, wheeler pa et al. ( ) a conserved haplotype controls parallel adaptation in geographically distant salmonid popula- tions. molecular ecology, , – . muhlfeld cc, kalinowski st, mcmahon te et al. ( ) hybridization rapidly reduces reproductive success of a native trout in the wild. biol- ogy letters, , – . pennisi e ( ) using dna to reveal a mosquito’s history. science, , – . rasmussen jb, robinson md, heath dd ( ) ecological consequences of hybridization between native westslope cutthroat (oncorhynchus clarkii lewisi) and introduced rainbow (oncorhynchus mykiss) trout: effects on life history and habitat use. canadian journal of fisheries and aquatic sciences, , – . sanchez cc, smith tpl, wiedmann rt et al. ( ) single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library. bmc genomics, , – . seeb je, carvalho gr, hauser l, naish ka, roberts sb, seeb lw ( ) single-nucleotide polymorphism (snp) discovery and applications of snp genotyping in nonmodel organisms. molecular ecology resources, , – . shepard bb, may be, urie w ( ) status and conservation of westslope cutthroat trout within the western united states. north american journal of fisheries management, , – . weigel de, peterson jt, spruell p ( ) the distribution of introgressive hybridization between westslope cutthroat trout and rainbow trout in the clearwater basin, idaho. ecological applications, , – . sja, pah, rfl, gl conceived and designed the project. rfl, cm supplied the samples. sja, sp, pah analyzed the data. sja, pah, rfl, cm, fwa, gl wrote the paper. data accessibility snp genotypes have been deposited at dryad: doi: . /dryad.b s . supporting information additional supporting information may be found in the online version of this article. table s the sample location and number, rad locus, species and number of heterozygous genotypes (hets) are reported from the validated assays. please note: wiley-blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. � blackwell publishing ltd s . j . a m i s h e t a l . book reviews breeding. however, in a final chapter (chapter ), these aspects of gamete and embryo manipulation are briefly considered in the frame of 'the future of micromanip- ulation and assisted reproduction' and this i consider to be a mistake. it seems unwise to give cursory coverage of such important issues without the proper sensitivity towards the dangers of the manipulation of human eggs and embryos and the social and ethical implications. for example, a page is given to transgenesis in the human and a paragraph to describe the approach to possible injection of genes into human egg nuclei without any consideration of the need or practicality of such genetic interference or the dangers inherent in this approach (inappropriate temporal and spatial expression, insertional mutation, disturbed patterns of inheritance, and the permanence of unforseeable conse- quences). to present gene transfer in the human simply as a technical possibility i think is misleading. it is true that scientists are not trained in ethics (although i consider it is time for ethics to be a compulsory part of scientific training and vice versa). however, i do think that scientists when reporting their work must make every effort to be sensitive to its social and ethical implications and to cover these aspects adequately. overall, i enjoyed reading this book. it is very nicely produced by raven press. i do not think it is of great interest to a general audience but it is a must for the library of every ivf laboratory and assisted concep- tion unit and for all those engaged in micro- manipulation in development with an inter- est in human reproduction. marilyn monk genetic diversity among jews: dis- eases and markers at the dna level. edited b bonne-tamir, a adam. (pp ; ,c .) oxford: oxford university press. . isbn - - - . for social, geographical, or religious reasons population isolates occur, and within these there can be an accumulation of deleterious genes. this has been well documented in the finns, the amish, and the jews. the study of these populations has taught us much about demographic influences on gene frequencies as well as the symptomatology and molecular pathology of a number of rare diseases. this well edited and comprehensive volume addresses such matters in regard to various jewish populations. demographic changes in world jewry are reviewed in a most fascinating opening chapter. from the time of claudius in the th century, when it has been estimated there were between five and eight million jews, the numbers have fluctuated considerably but with a progressive increase over the last years. moreover, the tendency for jews to marry within a community (so called beni- ni's index) has been progressively falling in recent years. furthermore, apart from israel, there has also been an increasing tendency for jews to marry non-jews who do not convert to judaism. the result of all these changes, if continued, will be to have a dilut- ing effect on the gene pool. the present volume is therefore welcome before these changes might possibly have any significant effect. the first part of the volume is concerned with polymorphisms (nuclear and mitochon- drial dna, hla, g pd). the next deals with mendelian disorders particularly preva- lent among ashkenazim (for example, various lipid storage disorders, dysautono- mia, adrenal hyperplasia iii) and non-ash- kenazim (for example, familial mediter- ranean fever). the final part addresses multifactorial diseases which may be particu- larly common in certain jewish communities, including coronary artery disease and idio- pathic inflammatory bowel diseases. this volume is dedicated to richard goodman md ( - ) who was profes- sor of human genetics at tel-aviv univer- sity and who contributed so much in various ways to medical genetics, but particularly to the study of genetic disorders among jews. richard was a personal friend of mine for nearly years and i know he would have been very proud to be associated with such a valuable work of scholarship. alan emery mendelian inheritance in man. th edition, volumes. victor a mckusick. ($ . .) baltimore: johns hopkins univer- sity press. . for a book to go through editions over a year period with the same author must be a rare event. for it to be increasingly valu- able with successive editions is even more unusual. mendelian inheritance in man has become such an essential part of both clinical practice and research in medical genetics that it must seem ungrateful, even disloyal, to criticise it, yet this tenth edition has shown up weaknesses that must be corrected if its future is to remain assured. these were already noted in reviewing the th edition, but are now even more prominent. the edition is produced for the first time in two volumes (a total of around pages compared with rather under for the previous edition). the preliminary section contains, as before, much interesting and valuable material, including citation indices, a table of numbers of genes mapped, a list of molecular defects in mendelian disorders, and detailed tables of mapped genes by chro- mosome, along with the renowned chromo- some maps of 'morbid anatomy' of the human genome. the detailed entries (auto- somal recessive and x linked disorders now occupy volume ) are remarkably up to date, and form a collection of notes and references on rare mendelian disorders that is quite unparalleled in its scope. so what is my criticism? quite simply it lies in what has not been removed in success- ive editions. the entries read increasingly as a series of geological strata, showing accre- tion but very little erosion. this has now reached the stage where it is difficult to find the statements and references that are either new, or old but standing the test of time, among a large mass of material that has become dated and, inevitably in some cases, incorrect. checking a number of entries shows that very little has in fact been removed, and it is this, rather than true growth in knowledge, that accounts for most of the increase in bulk. what can be done to remedy the situation? the answer is simple in principle, but will represent an onerous task for dr mckusick - perhaps even more so for his two assistants. they need to go through the text ruthlessly with a red pencil (or its computer equivalent) and firmly delete all the material that is irrelevant - i would judge about half of the total. if this can be done it will make the book (and its on line version) much more valuable, especially for students who can easily be confused by older material which has since been shown to be wrong. i suspect that a radical pruning may take two years' hard work, but i look forward then to seeing a slimmer and reinvigorated th edition - perhaps again in a single volume! peter s harper o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a p ril . d o w n lo a d e d fro m http://jmg.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ practical consensus recommendations on duration of adjuvant hormonal therapy in breast cancer | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /sajc.sajc_ _ corpus id: practical consensus recommendations on duration of adjuvant hormonal therapy in breast cancer @article{gupta practicalcr, title={practical consensus recommendations on duration of adjuvant hormonal therapy in breast cancer}, author={s. gupta and m. singh and a. vora and g. babu and m. walia and v. nautial and r. saha and b. smruti and j. sharma and r. koul and p. parikh and s. aggarwal}, journal={south asian journal of cancer}, year={ }, volume={ }, pages={ - } } 	s. gupta, m. singh, + authors s. aggarwal 	published 	medicine 	south asian journal of cancer optimization of adjuvant systemic therapy in women with early-stage hormone receptor-positive breast cancer includes the consideration of chemotherapy and duration of hormone therapy. adjuvant hormonal therapy significantly improves long-term survival of breast cancer patients with hormone receptor-positive disease. despite the proven clinical efficacy of tamoxifen and aromatase inhibitors, many breast cancer survivors either fail to take the correct dosage at the prescribed frequency… expand view on wolters kluwer ncbi.nlm.nih.gov save to library create alert cite launch research feed share this paper citations view all tables and topics from this paper 	 table 	 table 	 table 	mammary neoplasms 	tamoxifen 	hormone therapy 	pharmaceutical adjuvants 	systemic therapy 	american society of clinical oncology 	conflict (psychology) 	aromatase inhibitors 	scientific publication one citation citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency breast cancer demographics, types and management pathways: can western algorithms be optimally used in eastern countries? 	s. chakraborty, t. wadasadawala, r. ahmed, c. coles, s. chatterjee 	medicine 	clinical oncology (royal college of radiologists (great britain)) 	 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency duration of adjuvant tamoxifen therapy. 	j. bryant, b. fisher, j. dignam 	medicine 	journal of the national cancer institute. monographs 	 	 	pdf save alert research feed american society of clinical oncology technology assessment on the use of aromatase inhibitors as adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer: status report . 	e. winer, c. hudis, + authors m. somerfield 	medicine 	journal of clinical oncology : official journal of the american society of clinical oncology 	 	 save alert research feed adherence to endocrine therapy for breast cancer 	r. chlebowski, michelle l geller 	medicine 	oncology 	 	 save alert research feed switching to anastrozole versus continued tamoxifen treatment of early breast cancer: preliminary results of the italian tamoxifen anastrozole trial. 	f. boccardo, a. rubagotti, + authors p. sismondi 	medicine 	journal of clinical oncology : official journal of the american society of clinical oncology 	 	 	pdf save alert research feed five versus more than five years of tamoxifen therapy for breast cancer patients with negative lymph nodes and estrogen receptor-positive tumors. 	b. fisher, j. dignam, + authors h. l. lickley 	medicine 	journal of the national cancer institute 	 	 	pdf save alert research feed adherence to adjuvant endocrine therapy in postmenopausal women with breast cancer. 	v. ziller, m. kalder, + authors p. hadji 	medicine 	annals of oncology : official journal of the european society for medical oncology 	 	 	 save alert research feed use of tamoxifen for breast cancer: twenty-eight years later. 	i. jaiyesimi, a. buzdar, d. decker, g. hortobagyi 	medicine 	journal of clinical oncology : official journal of the american society of clinical oncology 	 	 save alert research feed switching of postmenopausal women with endocrine-responsive early breast cancer to anastrozole after years' adjuvant tamoxifen: combined results of abcsg trial and arno trial 	r. jakesz, w. jonat, + authors m. kaufmann 	medicine 	the lancet 	 	 save alert research feed randomized trial of two versus five years of adjuvant tamoxifen for postmenopausal early stage breast cancer. swedish breast cancer cooperative group. 	medicine 	journal of the national cancer institute 	 	 save alert research feed effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and -year survival: an overview of the randomised trials 	early breast cancer trialists' collaborative group 	medicine 	the lancet 	 	 , 	pdf save alert research feed ... ... related papers 	abstract 	tables and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue with program directors in nigms to determine the most appropriate support mechanism for their collaborative work. term/amount: nigms intends to commit up $ - million in total costs in fy to fund both phase i and phase ii awards. nigms anticipates funding up to ten phase i awards; each phase i award will be for $ , in direct costs. two to three phase ii grants will be awarded. because the nature and scope of the proposed research will vary, it is anticipated that the size of the phase ii awards also will vary, and the upper limit for a phase ii award will be $ million per year in annual direct costs. applications of different scales and scope are encouraged. although the financial plans of nigms provide support for this pro- gram, awards pursuant to this rfa are contingent upon the availability of funds and the receipt of a sufficient number of meritorious applications contact: the rfa label available in the phs (rev. / ) application form must be affixed to the bottom of the face page of the application. type the rfa number on the label. failure to use this label could result in delayed processing of the application such that it may not reach the review committee in time for review. in addition, the rfa title and number must be typed on line of the face page of the application form and the yes box must be marked. the sample rfa label available at: http://grants.nih.gov/ grants/funding/phs /label-bk.pdf has been modified to allow for this change. please note this is in pdf format. submit a signed, typewritten original of the application (phase i or phase ii), including the checklist, and three signed, photocopies, in one package to: center for scientific review national institutes of health rockledge drive, room , msc bethesda, md - bethesda, md (for express/courier service). direct inquiries regarding programmatic issues to: dr. michael e. rogers division of pharmacology, physiology and biological chemistry national institute of general medical sciences center drive, msc bethesda, md - telephone: ( ) - fax: ( ) - email: rogersm@nigms.nih.gov deadline: phase i application receipt date: june , . phase ii application receipt date: january , . errata in abstract , “back to basics: utility of peripheral smears in residency training,” on page a of the january issue of jim, the name of the first author, amish desai, was inadvertently left out. the authors apologize for the omission. in the january issue of jim, seven abstracts from the southern society of general internal medicine were inadvertantly left out. these abstracts are printed in this issue of jim on the following pages. the publisher regrets the error. journal of investigative medicine • vol. , no. , may o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jim .b m j.co m / j in ve stig m e d : first p u b lish e d a s . / . . o n d e ce m b e r . d o w n lo a d e d fro m http://jim.bmj.com/ cxcr /cxcl mediate autocrine cell- cycle progression in nf -associated malignant peripheral nerve sheath tumors cxcr /cxcl mediate autocrine cell- cycle progression in nf -associated malignant peripheral nerve sheath tumors wei mo, jian chen, amish patel, liang zhang, vincent chau, , yanjiao li, woosung cho, kyun lim, jing xu, alexander j. lazar, chad j. creighton, svetlana bolshakov, renée m. mckay, dina lev, lu q. le, ,* and luis f. parada ,* department of developmental biology department of dermatology department of cell biology university of texas southwestern medical center, dallas, tx - , usa department of pathology department of cancer biology university of texas md anderson cancer center, houston, tx , usa division of biostatistics, dan l. duncan cancer center, baylor college of medicine, houston, tx , usa *correspondence: lu.le@utsouthwestern.edu (l.q.l.), luis.parada@utsouthwestern.edu (l.f.p.) http://dx.doi.org/ . /j.cell. . . summary malignant peripheral nerve sheath tumors (mpnsts) are soft tissue sarcomas that arise in connective tissue surrounding peripheral nerves. they occur sporadically in a subset of patients with neurofibro- matosis type (nf ). mpnsts are highly aggressive, therapeutically resistant, and typically fatal. using comparative transcriptome analysis, we identified cxcr , a g-protein-coupled receptor, as highly ex- pressed in mouse models of nf -deficient mpnsts, but not in nontransformed precursor cells. the che- mokine receptor cxcr and its ligand, cxcl , promote mpnst growth by stimulating cyclin d expression and cell-cycle progression through pi - kinase (pi k) and b-catenin signaling. suppression of cxcr activity either by shrna or pharmacolog- ical inhibition decreases mpnst cell growth in culture and inhibits tumorigenesis in allografts and in sponta- neous genetic mouse models of mpnst. we further demonstrate conservation of these activated molec- ular pathways in human mpnsts. our findings indi- cate a role for cxcr in nf -associated mpnst development and identify a therapeutic target. introduction the tumor predisposition disorder von recklinghausen’s neuro- fibromatosis type i (nf ) is one of the most common genetic disorders of the nervous system, affecting one in , individ- uals worldwide (zhu and parada, ). a cardinal feature of nf is the growth of benign tumors called neurofibromas, cate- gorized into plexiform and dermal subtypes (le and parada, ). plexiform neurofibromas can undergo malignant transfor- mation into neurofibrosarcomas, known as malignant peripheral nerve sheath tumors (mpnsts), which represent a major source of morbidity for nf patients (ferner, ). despite continued progress in understanding nf biology, mpnst treatment remains limited to surgery, and prognosis remains unchanged (tonsgard, ). the development of murine models has provided an opportu- nity to gain insight into nf -deficient tumor natural history (cichowski et al., ; joseph et al., ; vogel et al., ; zheng et al., ; zhu et al., ). nf and tp , or cdkn a, are the most frequently mutated cancer-related genes in human mpnsts (mantripragada et al., ; rubin and gutmann, ). our genetically engineered mouse models (gemms) reca- pitulate tumors that arise in nf patients, and we and others have shown that genetic ablation of the nf and p tumor suppressors results in spontaneous development of mpnsts (cichowski et al., ; vogel et al., ). benign and malignant nf -deficient tumors can also be induced by subcutaneous implantation of nf - or nf ;p -deficient skin-derived precur- sor (skps), respectively, and are histologically indistinguishable from human counterparts (le et al., ); l.q.l., t. shipman, and l.f.p., unpublished data). here, we examine the chemokine receptor cxcr , which we find enriched in nf -deficient cells and particularly in nf -defi- cient mpnsts. expression of cxcr and its ligand, cxcl , has been reported in solid tumors, (kijima et al., ; koshiba et al., ; laverdiere et al., ; müller et al., ; oh et al., ; righi et al., ; schrader et al., ; sehgal et al., ; sengupta et al., ; taichman et al., ; zeelenberg et al., ; zhou et al., ) as well as non-hodgkin’s lymphoma (bertolini et al., ) and chronic lymphocytic leukemia (burger et al., ). clinical data indicate that high cxcr correlates with poor clinical outcome (bian et al., ; li et al., ; wang et al., ). the proposed paracrine roles for cxcr /cxcl in a variety of tumorigenic processes include cell growth (schrader et al., ; zhou et al., ), cell , – , february , ª elsevier inc. mailto:lu.le@utsouthwestern.edu mailto:luis.parada@utsouthwestern.edu http://dx.doi.org/ . /j.cell. . . http://crossmark.dyndns.org/dialog/?doi= . /j.cell. . . &domain=pdf metastasis (li et al., ), and angiogenesis (sengupta et al., ). a potential role for cxcr in sarcoma pathogenesis has not been examined. here, we provide evidence that the cxcr / cxcl axis is essential for mpnst tumor progression. specif- ically, the autocrine activation of cxcr by cxcl triggers intracellular pi -kinase (pi k) and b-catenin signals to promote g to s phase transition in mpnst cells. the cxcr antagonist, amd , has growth-inhibitory effects on primary cultured mouse and human mpnst cells, tumor allografts, and sponta- neous gemms. moreover, analysis of human primary and cultured mpnst cells, as well as human tissue microarray anal- ysis, reveals conserved pathway activation. thus, cxcr inhibi- tion may represent a therapeutic strategy to treat mpnst. results cxcr in nf -deficient mpnst tumors previously, we demonstrated that nf -deficient skps can give rise to either dermal or plexiform neurofibromas depending on their local microenvironment (dermis versus sciatic nerve) (le et al., ). in addition, dual mutation of the nf and p tumor suppressors in these cells results in mpnsts that exhibit cellular and molecular features of human mpnsts (l.q.l., t. shipman, and l.f.p., unpublished data). these tumors are indistinguish- able from a spontaneous mpnst gemm also based on loss of nf and p (cisnp; cichowski et al., ; vogel et al., ). to gain insight into the molecular and cellular mechanisms underlying development of nf -deficient mpnsts, we used microarray analysis to compare the transcriptomes of mpnsts with those of normal skps (wt), as well as pretumorigenic skps with either nf deletion (nf�/�) or nf and p (np�/�) deletion, as shown in figure a. the comparative array data pre- sented a complex picture of up- and downregulated transcripts for diverse genes (data not shown). however, our attention was drawn to the elevated expression of the chemokine receptor cxcr that exhibited a -fold elevation in both pretumor cell types (nf�/�; np�/�) and a -fold increase in the malignant form of skp mpnst (smpnst). quantitative rt-pcr and western blot analysis (figures b and c) confirmed these array data. we also measured low cxcr levels in schwann cells, which belong to the embryonic lineage of origin of nf -deficient tumors (le et al., ; serra et al., ; zhu et al., ), and high levels in tumor cells from the spontaneous cisnp mouse model of mpnst by western blot (figure s a available online; [vogel et al., ]) and immunohistochemistry (ihc). we further performed ihc on tumor samples from the smpnst-allograft, cisnp, and smpnst-autograft models and a human mpnst sample (figures d and s b). together, these data confirmed elevated cxcr protein in the different sources of nf -deficient malignant tissues compared to controls and, further, provided evidence that cxcr expression is sustained in human nf - associated mpnst. cxcr knockdown impairs cell proliferation and attenuates tumorigenesis to evaluate the functional role of cxcr in mpnst pathogen- esis, we used short hairpin rna (shrna) for knockdown in cell , – , february , ª elsevier inc. smpnst cells. cells were infected with lentivirus containing cxcr shrna or a control scrambled shrna (plko ctrl), and stable cell lines were established using puromycin selection. two different shrna sequences, targeting either the coding region (plko-mcxcr ) or the utr (plko-mcxcr -utr) reduced cxcr messenger rna (mrna) levels by � % (figures s a and s b). to address whether cxcr influences smpnst cell metabolism, we measured atp levels using a luminescence assay and found that tumor cells with depleted cxcr were less metabolically active. introduction of exoge- nous cxcr complementary dna (cdna) lacking the utr sequences (so that it is not targeted by the shrna) re-estab- lished cxcr protein levels and overcame the cell growth inhi- bition (figures a and s b). we turned to a doxycycline (dox)-inducible mir -based shrna system to enable acute knockdown of cxcr . using this system, murine and human cxcr mrna levels were decreased to % and % of the origin level, respectively (figures s c–s f). in contrast, induction with dox had no effect on cxcr expression in mpnst-tripz-scrambled control cells (figures s c–s f). similar to the plko knockdown results, we observed cell growth arrest in cxcr -depleted smpnst and human mpnst (s ) cells upon dox treatment (figure b). together, these results confirm that cxcr plays a role in murine- and human-derived mpnst cell proliferation. we next investigated the growth properties of tumor cells in vivo after cxcr knockdown. or plko-mcxcr or plko-ctrl smpnst cells were injected subcutaneously into nude mice and monitored for tumor growth (smpnst-allografts). the mice were sacrificed and tumors were dissected month after injection (figure s g). quantification of tumor size and weight showed that mpnst cells with cxcr knockdown generated smaller tumors than control cells (figure s h), and additionally, time to tumor appearance was significantly in- creased (figure s i). we also analyzed cell proliferation in excised tumors and found the average percentage of ki - positive, proliferating cells was . % ± . % in cxcr - depleted mpnsts versus . % ± . % in controls (figures s j and s k). similar results were obtained when the inducible shrna tumor cells were implanted and subjected to dox-mediated cxcr knockdown after the tumor cells had successfully seeded in the allograft. this approach eliminated the possibility that cxcr knockdown in culture impeded subsequent tumor cell implantation. or mpnst-tripz-cxcr cells were in- jected subcutaneously into nude mice, and one group received dox ( mg/ml) in the drinking water (figure c). compared to controls, tumor appearance in the dox-treated group was de- layed by week, and tumor progression was impaired (figures c and d). all mice were sacrificed on day , and tumors were excised. western blot analysis showed an � . % deple- tion of cxcr protein in the tumors harvested from dox-treated mice (figure e). when cells were injected, / control mice bore tumors ( ± mm in size and . ± . g in weight), and / dox-treated mice developed tumors that were smaller both in size ( ± mm ) and weight ( . ± . g) (figures f and g). notably, when cells were injected, no dox-treated mice developed tumors, whereas control group a b c d figure . cxcr is overexpressed in mpnsts (a) schema of the microarray analyses. (b) qrt-pcr analysis of cxcr mrna levels in wt, nf�/�, and np�/� (nf�/�;p �/�) skps, as well as mpnst skp model tumors and the cells derived from the tumor. (c) western blot analysis of cxcr protein levels in nf�/� and np�/� skps and smpnst cancer cells. (d) quantification of cxcr -positive cells in tumor tissues from different mouse models of mpnst and an mpnst from an nf patient (smpnst = smpnst autograft; athymic model = smpnst allograft). all statistics represent mean ±sd. see also figure s . cell , – , february , ª elsevier inc. a b c d e f g figure . knockdown of cxcr expression in mpnst cells decreased proliferation in vitro and tumorigenesis in vivo (a and b) atp luminescence assays were used to measure growth of indicated cells. (c and d) representative pictures of the tumors from nude mice injected with smpnst-tripz- mcxcr cells. mice – : without dox treatment; mice – : dox treatment. (e) cxcr protein level is downregulated in dox- treated mpnsts. (f) kinetic curve of tumor growth as measured by tumor volume. (g) quantification of tumor weight. all statistics represent mean ±sd. see also figure s . mice developed tumors (figure d). thus, both chronic and acute suppression of cxcr in vivo substantially decreased the tumorigenic capacity of mpnst cells. cxcr depletion alters the mpnst cell cycle we investigated possible mechanisms of cxcr function in pro- moting mpnst progression. shrna depletion caused growth arrest of smpnsts (figure ) rather than apoptosis (figures cell , – , february , ª elsevier inc. s a and s b) or senescence (figures s c and s d). bromodeoxyuridine (brdu) incorporation and fluorescence- activated cell sorting (facs) analysis showed significant reduction in brdu incorporation in cxcr -depleted cells ( . % ± . % versus . % ± . %; figure a). when cxcr protein level was restored, the percentage of brdu- positive cells was also restored to that of cxcr -wt cells (figure a). addi- tional cell-cycle analysis revealed that the percentage of cells in g phase was � % in cxcr -depleted smpnst cells versus � % in cxcr -wt cells; by contrast, the number of cells in g /m phase was only slightly changed (figures b and s e). these data suggest that cxcr -depleted mpnst cells undergo g /s arrest. we also performed brdu incorporation studies in vivo. tumor- bearing mice were injected with brdu hr before sacrifice. brdu ihc revealed % ± % brdu-positive cells in the tumor samples from mice injected with control smpnst cells. in contrast, the brdu-positive cell number decreased to % ± % in the tumor samples from mice injected with cxcr -depleted smpnst cells (figures c and d). similar to the mpnst cell culture results, there was no obvious apoptosis in either tumor group (figures s f and s g). in summary, knockdown of cxcr expression, in culture and in vivo, led to cell-cycle arrest manifested by reduced s phase and brdu incorporation, rather than cell death or senescence. cyclin d expression is regulated by cxcr we next investigated whether cxcr perturbation impacts the expression of cell-cycle-regulatory genes (johnson and walker, ; lee and yang, ; obaya and sedivy, ). although quantitative rt-pcr (qrt-pcr) analysis indicated that ex- pression of most genes examined was unchanged in cxcr - depleted smpnst cells and tumors, cyclin d mrna levels decreased � % (figures e and s h). a slight reduction in cdk / and cyclin e mrna levels was also observed (figures e and s h). quantification of western blots confirmed the cyclin d protein level decrease in cxcr -depleted mpnst cells and tumors (smpnst allografts) (figures f and s i). both cyclin d mrna and protein levels were fully restored in cxcr -depleted mpnst cells with exogenous cxcr expression (figures e and f). moreover, reintroduction of cyclin d cdna restored protein levels and counteracted the cxcr depletion effect, allowing cells to resume progression through the cell cycle (figures g and h). these data demonstrate that, in smpnst cells, cell-cycle progression mediated by cyclin d is dependent on cxcr function. cxcr activates the akt/gsk- b/b-catenin network signaling pathways known to regulate cyclin d include the nfkb, ras, erk, wnt/b-catenin, and jak/stat pathways (sherr, ). to determine whether any of these candidate pathways mediate cxcr regulation of cyclin d in neurofibro- sarcomas, fractionated nuclear and cytoplasmic extracts of smpnst cells were immunoblotted with the indicated anti- bodies to examine changes following cxcr depletion. we observed that both cyclin d and b-catenin levels decreased . % and . %, respectively, in the nuclear fraction of cxcr -depleted mpnst cells (figure a). no changes were seen in other pathways examined (figure a). transcriptional activation assays using a luciferase reporter linked to the t cell factor (tcf) promoter—a physiological target of b-catenin— showed a % reduction in luciferase activity in cxcr - depleted smpnst cells compared to controls, which is con- sistent with the model that suppression of cxcr attenuated b-catenin activity (figure b). to test whether nuclear b-catenin is required for cyclin d expression, we directly blocked tcf function (hoppler and kavanagh, ). expression of domi- nant-negative tcf (dntcf) dramatically decreased cyclin d mrna and protein levels in cxcr -wt-mpnst cells (figures c and d), whereas overexpression of b-catenin in cxcr - depleted mpnst cells restored cyclin d expression (figure e) and, further, rescued the cell growth arrest caused by cxcr knockdown (figure f). functionally, downregulation of the b-catenin pathway by dntcf resulted in cell growth arrest in mpnst cells (figure s a). we also examined tumors for b-cate- nin expression. ihc on mpnst tumor tissues from smpnsts (smpnst allografts) (figure s b) and one human patient sample (figure s c) showed robust nuclear immunoreactivity consistent with activation of b-catenin signaling in these tumors. the above data demonstrate a role for b-catenin downstream of cxcr and upstream of tcf and cyclin d in stimulating mpnst growth. b-catenin stabilization is required for its role as a transcription factor. in the inactive state, b-catenin is destabi- lized by phosphorylation at ser , ser , and thr by gsk- b or at ser by ck a (liu et al., ). western blot analysis showed that cxcr depletion increased b-catenin serine phos- phorylation at sites / , but not (figure g), suggesting that cxcr suppression resulted in gsk- b kinase activation. we also examined gsk- b serine inactivating phosphorylation and observed a reduction in cxcr -depleted mpnst cells (figure g). thus, in mpnst, cxcr activity maintains gsk- b in the inactive state. gsk- b is in turn a downstream element of the pi k/akt signaling pathway (cross et al., ) whose kinase activity is inhibited by akt-mediated phosphorylation at ser (srivastava and pandey, ). we reasoned that the decreased phosphoser -gsk- b level in cxcr -depleted mpnst cells might result from attenuated akt kinase activity and confirmed this by western blot analysis with pakt (ser ) antibody (figure g). we also found reduced levels of activated b-catenin, pgsk- b (ser ), and pakt (ser ) in cxcr - depleted mpnst tumor samples compared to control tumors (smpnst allografts) (figure h). these data support the model that cxcr promotes mpnst growth by activating the pi k/ akt and the gsk- b/b-catenin pathways. previous in vitro studies using human and murine mpnst cell lines, as well as in vivo studies using the cisnp mouse model, demonstrated that the mtor pathway can regulate mpnst growth by posttranscriptional regulation of cyclin d (johannessen et al., ). we observed modest reduction in phospho-mtor upon cxcr knockdown, but this was not accompanied by obvious change in the levels of phosphorylated-s ribosomal protein (figure s d). collectively, our data indicate that cxcr regu- lates the activity of b-catenin via the akt/gsk- b cascade, which ultimately controls cyclin d transcription and protein levels. cxcl activates cxcr in an autocrine loop we sequenced cxcr cdnas from two independent murine mpnsts, one derived from the smpnst-allograft model and one from the cisnp model, and found no missense or nonsense mutations (data not shown). moreover, cxcr -activating muta- tions have not been identified in mpnst cell lines or primary cells derived from human patients (see below and figure s a). there- fore, in these tumors, receptor activity must rely on the presence of ligand—cxcl . qpcr analysis showed that cxcl mrna was indeed elevated in pretumorigenic skps (np�/�) and was even more highly expressed in smpnst tumor cells (figure s b). to test the role of cxcl in mpnst cell growth, we treated cultured mpnst cells with recombinant cxcl protein and found that it promoted cell growth, generating a . -fold increase in cell number at a concentration of ng/ml by day , when compared to nontreated cells (figure a). in contrast, cell number decreased by % and % at days and , respec- tively, when cells were cultured with anti-cxcl antibody compared to igg or pbs control (figure b). these data suggest that cxcl is critical for mpnst cell proliferation. to verify the autocrine source of cxcl ligand in the mpnst cell culture, we performed an elisa on the medium conditioned by mpnst cells using an anti-cxcl antibody. we observed no detectable cxcl protein in control media (with no cells) compared to the conditioned media, indicating that the in vitro source of cxcl protein is the mpnst cells (figure c). consistent with the elisa showing that conditioned media from cxcl - overexpressing mpnst cells (pbabe-cxcl ) and knockdown cells (plko-cxcl ) contained elevated and reduced cytokine, respectively (figures c and s c), cxcl -overexpressing cell , – , february , ª elsevier inc. a b c d e f g h figure . cxcr alters the cell cycle in smpnst cells by regulating cyclin d expression smpnst cells were incubated with brdu for min and then stained with propidium iodide. (a and b) facs analysis showed that cxcr depletion in smpnst cells decreased the percentage of brdu-positive cells (plko-mcxcr and plko-mcxcr - utr compared to control plko-ctrl) (a) and altered the proportion of cells in g and s phases (b). expression of cxcr in the knockdown cells (plko-mcxcr - utr + ub-cxcr ) restored the cells to the control state. (legend continued on next page) cell , – , february , ª elsevier inc. cells had a significant growth advantage (figure a), whereas cxcl knockdown cells exhibited a significant reduction in growth at days and (figure d). importantly, the cell growth inhibition could be rescued by addition of cxcl protein (figure e). therefore, mpnst cells synthesize and secrete cxcl protein, causing increased cell growth that corre- sponded with cxcl levels. to confirm that the effect of cxcl on mpnst growth is dependent on cxcr , we added cxcl protein to cultured cxcr -depleted mpnst cells. we found no detectable change in cell proliferation after ligand stim- ulation compared to control cxcr -depleted mpnst cells (fig- ure e). additionally, cxcl modulation in the presence of receptor induced the same signaling pathway changes brought about by cxcr modulation (figure f). taken together, these data demonstrate that cxcl , secreted by mpnst cells, is sufficient to sustain autocrine activation of its receptor, cxcr , and triggers specific downstream signaling pathways, leading to mpnst cell-cycle progression and proliferation. given the important roles of cxcr /cxcl in tumor angiogenesis, we also investigated whether cxcr depletion in mpnst cells altered the microvasculature. both control and cxcr -depleted tumors exhibit strong cd staining (fig- ure s d) and similar vessel densities (figure s e), indicating that the tumor growth arrest induced by cxcr depletion is cell autonomous and not secondary to effects on the microvasculature. pharmacological inhibition of cxcr arrests mpnst proliferation and tumor growth we next tested a clinically validated, highly specific cxcr antagonist, amd , in our system. we performed a dose- response analysis of amd on primary murine mpnst cells (smpnsts and cisnp), one primary human mpnst cell line, three established human mpnst cell lines (s ; snf . ; snf . ), and pretumor cells (nf ;p null skps) (figures a and b). four of six cell lines were potently inhibited by low doses of amd (ic values & . um; figure c). one human mpnst cell line, snf . , and the pretumor cells were less sensitive (ic values of – um; figure c). only one human mpnst cell line, snf . , showed no response, which is consis- tent with its low endogenous cxcr levels relative to the other mpnst cells (figure s a). of note, we were unable to seed xeno- graft tumors using snf . cells, suggesting that this cell line has drifted significantly from the original tumor of derivation (data not shown). in contrast, the cell viability of wild-type (wt) schwann cells and skps was unaffected by amd (fig- ure a). inhibition of cxcr by amd also reduced cyclin d levels via the akt/gsk- b/b-catenin pathway (figure d). to assess the therapeutic potential of amd in vivo, we first tested subcutaneous allografts of primary smpnsts. trans- (c) paraffin sections of mpnsts (harvested from smpnst-allograft mice injecte cells) were stained with antibody against brdu and counterstained with hematox (d) the percentage of smpnst cells that were brdu positive was determined fo (e and f) analysis of the expression levels of various cell-cycle genes/proteins in (g) cell growth curves of indicated cells as measured with an atp luminescence (h) cxcr and cyclin d protein levels were examined by western blotting in ind scale bar, mm. all statistics represent mean ±sd (*p < . and **p < . ). s plant recipient animals with palpable tumors were treated with amd and, after weeks of treatment, were sacrificed for analysis (figure e). amd treatment potently suppressed mpnst growth, resulting in reduced tumor size and weight that was % and %, respectively, of the control group (fig- ure s b). this was accompanied by commensurate reduction in brdu incorporation and ki staining (figures s c and s d). consistent with the in vitro data, amd -treated tumors showed decreased levels of cyclin d , activated b-catenin, phospho-akt, and phospho-ser -gsk- b (figure s e). to further validate the role of cxcr signaling in mpnst growth and the potent cytostatic effect of amd on tumor growth, we turned to a spontaneous endogenous gemm. a mouse strain in which null p and nf alleles are configured in cis on the same chromosome spontaneously develop mpnst via loss of heterozygosity (loh) of both tumor suppressor alleles (cisnf/p ; vogel et al., ). although a majority of the sponta- neous tumors are sarcomas, these cisnf/p mice are also prone to other nf -initiated tumors such as lymphomas and gliomas, as well as to spontaneous p -based tumors (cichow- ski et al., ; vogel et al., ). based on kaplan-meier curve data that indicate spontaneous neurofibrosarcoma appearance with % mortality at about weeks of age and percent mortality at weeks (n = ; vogel et al., ), we com- menced the amd drug trial on cisnf/p mice at weeks of age. as shown in figure f, at months posttreatment ( weeks of age), / control mice ( %) had perished, whereas, in the amd group, only / ( %) had perished. all perished mice had large tumors, and histopathological anal- ysis of the tumor tissues revealed that one mouse from the control group and one from the experimental group had clear evidence of a primary tumor of hematopoietic origin (figures s f and s g). therefore, these mice were removed from the study, resulting in a total of nine control mice and amd -treated mice (figure g). six out of nine control mice developed lethal sarcomas. five developed mpnst, as evidenced by histopathological analysis and s b and gap immunoreactivity (figure f, c , c , and c –c ; fig- ure s h). a sixth control mouse died with a malignant triton tumor (mtt), which is an nf -associated sarcomatous tumor having high myoglobin expression (figure f, c ; and fig- ure s i). all six tumors displayed cxcr expression (figure f). in contrast to the high mortality rate in the control group, at months posttreatment, only two out of mice from the amd treatment group had perished with a solid tumor (fig- ure f, amd and amd ; figure g). by histological analysis, we determined that one of the tumors was a rhabdomyosarcoma (rms) with very low levels of cxcr expression (figure f, amd , and figures s j and s k). the second treated mouse developed a leiomyosarcoma (lms) with cxcr expression d with either control plko-ctrl or cxcr -depleted plko-mcxcr smpnst ylin (blue). r each tumor. the indicated cells as measured by qpcr (e) and western blotting (f). assay. icated cells. ee also figure s . cell , – , february , ª elsevier inc. a b c d e f g h figure . cxcr regulates cyclin d expression through the akt/gsk- b/b-catenin network (a) immunoblot of nuclear and cytoplasmic extracts from smpnst cells with indicated antibodies. (legend continued on next page) cell , – , february , ª elsevier inc. a b c d e f figure . an autocrine mechanism involving cxcl and cxcr maintains growth of mpnst cells (a and b) growth curve of smpnst cells treated with recombinant cxcl protein (a) or cxcl /cxcr antibodies (b). (c) elisa to measure cxcl protein level in pbs, % fetal bovine serum (fbs) dulbecco’s modified eagle’s medium (dmem) media, and conditioned media from smpnst cells or smpnst cells expressing pbabe-cxcl (overexpression) or plko-cxcl (knockdown). (d) growth curve of control smpnst cells (plko ctrl) and cxcl -depleted smpnst cells (plko-cxcl ). (e) growth curve of smpnst cells, cxcr -or cxcl -depleted smpnst cells, and cxcr -or cxcl -depleted smpnst cells treated with recombinant cxcl protein. (f) immunoblot of cell lysates from smpnst cells and smpnst cells expressing plko-cxcl or pbabe-cxcl with indicated antibodies. all statistics represent mean ±sd (**p < . ). see also figure s . (figure f, amd , and figures s l and s m), suggesting that this tumor escaped amd treatment. whether this outlier reflects failed drug delivery or, alternatively, an independent mechanism of tumor promotion will require additional studies. in summary, the allograft and genetic tumor model data demonstrate that in vivo pharmacological blockade of cxcr can potently inhibit spontaneous mpnst tumor development in a gemm by blocking the same signaling pathways as ob- served in primary tumor cultures. (b) luciferase reporter assay to measure tcf activation in control smpnst ce mcxcr -utr), cxcr -depleted smpnst cells rescued by cxcr overexp dominant-negative tcf (plko-ctrl+ub-dntcf). (c and d) the mrna and protein levels of cyclin d in smpnst cells (ctrl) or smpn by western blotting (d). (e) immunoblot of cell lysates from smpnst cells, cxcr -depleted smpnst b-catenin with indicated antibodies (abc: antibody against activated b-catenin). (f) atp luminescence assay to measure growth of indicated cells. (g) immunoblot of cell lysates from smpnst cells and cxcr -depleted smpns (h) graph showing fold change versus control of tumor tissue lysates from con indicated antibodies. all statistics represent mean ±sd (**p < . ). see also figure s . conserved signaling pathway activation in human mpnst the above studies reflect analyses of genetic mouse models extended to a single human tumor and primary mpnst line plus three established human mpnst lines. to further assess the relevance of our findings to human mpnst, we examined cxcr in mpnsts from patients with nf . we sequenced coding exons and (see experimental procedures) of cxcr from a series of nf -associated human mpnst lls (plko-ctrl), cxcr -depleted smpnst cells (plko-mcxcr and plko- ression (plko-mcxcr -utr+ub-cxcr ), and smpnst cells expressing st cells expressing dntcf (dntcf) were determined either by qpcr assay (c) or cells, and cxcr -depleted smpnst cells ectopically expressing cxcr or t cells with indicated antibodies. trol smpnsts (ctrl) or smpnsts from cxcr -depleted cells (mcxcr ) with cell , – , february , ª elsevier inc. a b c d e f g figure . amd specifically inhibits the proliferation of smpnst cells and suppresses the growth of mpnsts in vivo (a and b) dose curve showing effect of amd on proliferation of murine (primary cells from mpnst skp model and cisnp model) and human (snf . , snf . , s , and primary cells from a human nf patient) mpnst cells, pretumor skp np�/� cells, and wt murine schwann cells and skps. (c) experimentally derived amd ic values. (d) immunoblot with the indicated antibodies of cell lysates from murine cells in the absence or presence of amd . (e) representative picture of the smpnst-allograft tumors (from nude mice injected with smpnst cells) during the treatment study with tumor histopathology and immunohistochemistry. (f) list of control (c-) and amd -treated (amd-) mice that died during the treatment study, with tumor histopathology and immunohistochemistry. (g) kaplan-meier survival curve of mice in the amd treatment study (control group, n = ; amd -treated group, n = ; p = . by mantel-cox test). other statistics represent mean ±sd. see also figure s . samples and cell lines (figure s a), including s , which requires cxcr function for growth (figure b); all cxcr coding regions analyzed had wt sequence (figure s a). thus, similar to the murine mpnsts, human mpnst cxcr activity must depend on ligand presence. we next examined cxcr protein expression using a human tissue microarray (tma) that included plexiform neurofibromas (from nf patients har- boring mpnsts) and mpnst samples ( from nf patients and sporadic) (ghadimi et al., ; zou et al., ). the data showed that % of neurofibromas and % of nf - deficient mpnsts displayed cxcr immunoreactivity (figures cell , – , february , ª elsevier inc. a and b), whereas only % of sporadic mpnsts tissues showed cxcr immunoreactivity. similarly, % of neuro- fibromas and % of nf -associated mpnsts exhibited cxcr expression in > % of tumor cells, as compared to % of sporadic mpnst tissues (figure a). spearman correla- tion analysis showed a statistically significant difference in cxcr intensity and distribution when comparing nf -associ- ated tumors to sporadic tumors (two-sided p = . and . , respectively) (figure b). we also assessed p-akt, b-catenin, and cyclin d expression and observed a direct con- cordance between their levels and those of cxcr (p = . , a b c d figure . analysis of cxcr expression and downstream pathway signaling reveals conservation in human mpnsts (a) quantification of the intensity and distri- bution of cxcr immunoreactive cells in all mpnst tmas. (b) schema of the cxcr distribution in tmas. spearman correlation identified a statistically sig- nificant difference between nf -associated and sporadic tumors (p = . ). (c) spearman correlation coefficient analyses between cxcr , pakt, b-catenin, and cyclin d in nf -mpnst. (d) quantification of pakt, b-catenin, and cyclin d expression levels in mpnst-nf tma and categorization based on cxcr expression level. staining distribution of positively staining cells: ( ) = < %, ( ) = % – %, ( ) > %, and intensity: ( ) = negative, ( ) = low, and ( ) = intermediate to high. spearman’s correlation coefficient analyses were used to determine the concordance between cxcr expression and nf status and between cxcr and other biomarkers. . , and . � , respectively) (figures c and d). thus, tma data confirm that akt/gsk- b//b-catenin pathway activation is conserved in human nf -associated mpnsts. extension of our functional data in murine and human mpnst cells, coupled with the tma studies, indicate that cxcr and cyclin d are closely associated with clinical nf -associated pathogenesis. discussion we exploited the unique properties of gemms for nf -associ- ated mpnst that permitted isolation and direct comparison with tumor cells of origin and precursor nontumor cells. the progressively elevated expression of the chemokine receptor cxcr along the increasingly tumorigenic series from premalig- cell , – , f nant nf null and nf/p double null tumor progenitors to nf null benign tumors (neurofibromas) and, finally, to nf/p double null mpnsts drew our attention and motivated the present study. cxcr /cxcl : an mpnst autocrine loop we also found increased cxcr expres- sion in human nf patient tumor samples and primary cells. cxcr is commonly found in human and experimental murine cancers. protumorigenic paracrine func- tions attributed to this g-protein-coupled receptor/ligand include cell homing, migration, and induction of neovasculari- zation (li et al., ; sengupta et al., ; singh et al., ). in the present study, we identify an autocrine loop in cells that sustains activated cxcr . mpnst cells with cxcr depletion exhibit proliferative arrest rather than cell death, with fewer cells in s phase. consistent with this, we found that the g /s phase transition protein cyclin d responded to altered cxcr expression. cxcr signaling differs in normal and cancer cells both at the functional and molecular signaling levels. for example, cxcl treatment of wt primary neonatal astrocytes induced apoptosis, whereas it promoted enhanced survival in astrocy- toma cultures (warrington et al., ). in our study, cxcr suppression inhibited mpnst cell progression from g to s phase in the cell cycle and resulted in potent inhibition of mpnst cell growth in vitro and also in vivo tumor growth in several exper- imental paradigms. mpnst cells with either chronic or acute cxcr knockdown were deficient in allograft transplantations. ebruary , ª elsevier inc. administration of the cxcr inhibitor, amd , also impeded efficient tumor allotransplantation of murine mpnsts. in perhaps the most stringent assay, spontaneous mpnst devel- opment was dramatically inhibited in a cisnf/p gemm. this result illustrates the potential power of spontaneous genetically relevant malignant tumor mouse models as experimental tools for cancer treatment discovery. our data point to a cell-autonomous pathway of activation but do not rule out potential ligand contribution from other tumor- associated sources. however, mpnst cells themselves provide sufficient ligand to sustain autocrine cell growth in culture. we make note that, although our cxcr shrna experiments provide similar allograft growth inhibition to that of amd , our studies do not address the possibility that pharmacological inhibition of cxcr may extend to additional targets such as bone-marrow-derived cells that may result in additional tumor inhibitory effects. cxcr promotes mpnst proliferation via pi k and gsk- b the function of the nf protein neurofibromin as a ras-gtpase- activating protein has traditionally focused attention on the two main ras effector pathways: raf/erk and pi k/mtor. inhibi- tion of the erk pathway can impact and even lead to reversal of certain nf mutation-based phenotypes (cui et al., ; wang et al., ). other studies have reported that hyperactiva- tion of the mtor pathway underlies a growth advantage in nf - deficient mpnst cells and that rapamycin-mediated inhibition of mtor blunts mpnst growth in vivo (johannessen et al., ; johansson et al., ). we saw modest downmodulation of p-mtor but saw no effects on p-s signaling in our smpnst system. in our study, nf loss specifically caused upregulation of akt/pi kinase, but not of erk. by recent accounting, more than positive and negative effectors of akt signaling have been described (http://www.proteinlounge.com). how specific pathways are selected for activity in a given cellular context may depend in part on the akt isoform, intracellular localization of the active intermediates, and other factors not yet understood. the association of cxcr with various cancers has implicated activation of several signaling effectors, including plcg/ca +, pi k/akt/nfkb, and erk/p , leading to chemotaxis, sur- vival, angiogenesis, etc. (balkwill, ). in mpnsts, however, cxcr depletion deactivated akt, but not erk or plcg, thus revealing cell context-specific pathway regulation. mtorc and gsk- b are important downstream effectors of phospho- akt, and our results show that, in mpnsts, cxcr depletion more strongly impacts gsk- b rather than mtor through pi k and that gsk- b mediates cyclin d expression. thus, we propose that the pi k/gsk- b branch downstream of cxcr is sufficient to promote mpnst proliferation. these studies, however, do not rule out that additional signaling path- ways and effectors of akt, b-catenin, and tcf may have contributory functions in mpnst progression. soft tissue sarcomas are among the most therapy-resistant and life-threatening forms of cancer. our data indicate that correction of abnormal cxcr signaling may be a promising therapeutic target for nf -associated mpnsts. in addition, a subset of sporadic mpnsts may also co-opt this signaling cell , – , february , ª elsevier inc. mechanism, in which case cxcr attenuation could poten- tially benefit this patient population. clinically-relevant cxcr inhibitors were initially developed for the treatment of hiv (peled et al., ). these include compounds of four different classes: small-molecule inhibitors (e.g., amd ), small modified pep- tides (e.g., btk ), antibodies (e.g., alx- ), and modified cxcl antagonists (e.g., ctce- ). given the accumulating evidence for the important role of this paracrine axis in cancer, such compounds are currently being tested in early-phase clin- ical trials (http://www.clinicaltrials.gov). our preclinical observa- tions supplemented by strong evidence that the cxcr axis activity is sustained in human samples support the inclusion of mpnst patients in such studies. notably, effects of cxcr blockade are cytostatic rather than cytotoxic. however, taking into account that surgery is the mainstay of mpnst treatment, therapeutic approaches such as cxcr inhibition may improve local growth control and enhance resectability. in addition, sustained blockade of tumor development may provide an opportunity for endogenous inflammatory and immune anti- tumor responses to gain a foothold. moreover, the identification of additional pathways that can be targeted in combination with cxcr will hopefully result in synthetic lethality and superior anti-mpnst effects. further studies will resolve this important question. experimental procedures all experimental procedures are described in detail in the extended experi- mental procedures. mpnst mouse models skp mpnst model skps were isolated as previously reported (le et al., ) from nf f/f; p f/f;rosa -lacz mice. np�/� skps were implanted subcutane- ously into the same mouse from which the skps were originally isolated (smpnst autograft). the autologously transplanted skps gave rise to mpnsts within months after implantation % of the time. the tumors ex- hibited all the characteristics of human mpnst, including fascicular patterns of tightly packed spindle cells with hyperchromatic nuclei and frequent mitoses, as well as s b expression (l.q.l., t. shipman, and l.f.p., unpub- lished data). smpnst allograft model smpnst cells were subcutaneously injected into the backs of - to -week- old athymic nude mice. cisnp mpnst model a spontaneous endogenous gemm for mpnsts. we utilized a mouse strain in which null p and nf alleles are configured in cis on the same chromosome and spontaneously develop mpnst via loss of heterozygosity of both tumor suppressor alleles (vogel et al., ). amd and doxycycline treatment in vivo; measurement of tumor volume amd treatment of mpnst allograft model mpnst cells were subcutaneously injected into the backs of - to -week-old female athymic nude mice. the cxcr inhibitor amd was administered subcutaneously daily at mg/kg of body weight, beginning at the time of tumor detection. amd treatment of gemm amd ( mg/d) or saline was administered using subcutaneously im- planted osmotic minipumps (durect corporation). two month treatment began when the mice were weeks old. doxycycline was delivered in % sucrose-containing drinking water at mg/ml. tumor size was calculated by measuring length and width of the http://www.proteinlounge.com http://www.clinicaltrials.gov lesion with the formula (length) (width) ( . ). all statistical analyses were done with student’s t test. supplemental information supplemental information includes extended experimental procedures and six figures and can be found with this article online at http://dx.doi.org/ . /j.cell. . . . acknowledgments we thank karen cichowski for the generous gift of the human mpnst cell lines. this work was partially supported by funding from the national cancer institute (grant r ca to l.q.l.), department of defense (grant w xwh- - - to l.q.l. and postdoctoral fellowship award grant w xwh- - - to w.m.), and the national institutes of health (grant p ns to l.f.p.). l.q.l. is a disease-oriented clinical scholar and holds a career award for medical scientists from the burroughs wellcome fund. l.f.p. is an american cancer society research professor. received: june , revised: november , accepted: january , published: february , references balkwill, f. 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( ). neurofibromin, a tumor suppressor in the nervous system. exp. cell res. , – . zhu, y., ghosh, p., charnay, p., burns, d.k., and parada, l.f. ( ). neuro- fibromas in nf : schwann cell origin and role of tumor environment. science , – . zou, c.y., smith, k.d., zhu, q.s., liu, j., mccutcheon, i.e., slopis, j.m., meric-bernstam, f., peng, z., bornmann, w.g., mills, g.b., et al. ( ). dual targeting of akt and mammalian target of rapamycin: a potential thera- peutic approach for malignant peripheral nerve sheath tumor. mol. cancer ther. , – . 	cxcr /cxcl mediate autocrine cell- cycle progression in nf -associated malignant peripheral nerve sheath tumors 	introduction 	results 	cxcr in nf -deficient mpnst tumors 	cxcr knockdown impairs cell proliferation and attenuates tumorigenesis 	cxcr depletion alters the mpnst cell cycle 	cyclin d expression is regulated by cxcr 	cxcr activates the akt/gsk- β/β-catenin network 	cxcl activates cxcr in an autocrine loop 	pharmacological inhibition of cxcr arrests mpnst proliferation and tumor growth 	conserved signaling pathway activation in human mpnst 	discussion 	cxcr /cxcl : an mpnst autocrine loop 	cxcr promotes mpnst proliferation via pi k and gsk- β 	experimental procedures 	mpnst mouse models 	skp mpnst model 	smpnst allograft model 	cisnp mpnst model 	amd and doxycycline treatment in vivo; measurement of tumor volume 	amd treatment of mpnst allograft model 	amd treatment of gemm 	supplemental information 	acknowledgments 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete 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click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ microsoft word - hhe bf.rtf further section hum. hered. ; : - subject index abo groups, andhra pradesh , --, himachal pradesh abortions and tfc subtypes achromatopsia, congenital acp groups, andhra pradesh --, belgium --, pyreneans --, west bengal ada groups, belgium --, pyreneans affective disorders and blood groups afghanistan, properdin factor b ag groups, belgium - -‚ south african negroes and indians ak groups, affective disorders --, andhra pradesh --, belgium --, pyreneans albumin variants in south india alpha- -antitrypsin levels in pim subtypes types in newborns sweden ‚ see pi types - - thalassaemia and hb new york am groups in siberia amish mennonites, dermatoglyphics andhra pradesh, blood and serum groups , ‚ arabs, ina antigen bantu-speaking negroids, ag groups basques, blood and serum groups belgians, protein and enzyme groups bengal, west, acp groups beta-thalassaemia and gpt -- intermedia --, turkey bf groups, afghanistan - -‚ parentage testing brachydactyly breast cancer and hp groups cameroon, sickle cell trait and g- -pd deficiency -‚ hbs, g- -pd deficiency and malaria canada, pi types , cancer of breast and hp groups c esterase in pyreneans chile, haemophilia a cholinesterase, serum, in affective disorders chromosome no. , regional duplication -- ptrisomy consanguinity and mortality -‚ england c in norwegian lapps c deficiency, familial, and lipodystrophy - groups, uremia --, belgium c groups in graves’ disease denmark, galt groups dental disease and salivary proteins dermatoglyphics, absent d triradius in sibs -‚ amish mennonites -‚ bilateral assymmetry -‚ genetics -‚ hypospadias -‚ maharashtra - of sole, linkage study diabetes, association with glo groups esd groups, andhra pradesh --, belgium --, france --, west bengal fertility and consanguinity finland, glo groups france, esd and glo groups galactose- -phosphate uridyl transferase types in denmark gamma-gct groups gc groups, andhra pradesh --, belgium - -‚ himachal pradesh --, iceland gene frequency estimation genotype probabilities in pedigrees, calculation glo groups, associations with graves’ disease and diabetes --, finland --, france subject index --, iceland --, iraq gm groups, belgium --, siberia - -‚ singapore and sri lanka gpt groups, belgium --, italy --, japan --, pyreneans graves’ disease, c groups --, glo groups greece, serum protein and enzyme groups gsh-px activity in israeli populations g- -pd, affective disorders -‚ afro-americans -.cameroon -‚ pyreneans -‚ south vietnamese -‚ turkey - interactions with sickle cell trait and malaria , : haemophilia a in chile hb new york and alpha-thalassaemia s in cameroon types in turkey heritability estimates himachal pradesh, abo groups hp groups, affective disorders --, andhra pradesh , --, belgium - -‚ breast cancer --‚ himachal pradesh --.turkey - levels, africans -o‚mali hypospadias, dermatoglyphics iceland, gc groups -‚ glo groups immunoglobulins, ige twin study ina antigen in iranians and arabs india, andhra pradesh, albumin, blood and serum groups , , , himachal pradesh, abo groups maharashtra, dermatoglyphics -mem groups punjabis, gm and km groups west bengal, enzyme groups iranians, ina antigen iraq, glo groups israeli populations, gsh-px activity italy, gpt groups japan, gpt groups km groups, belgium --, siberia - -‚ singapore and sri lanka lap in affective disorders lapps, norwegians, c types ldh groups, affective disorders --, andhra pradesh --, west bengal linkage, beta-thalassaemia and gpt locus - dermatoglyphics of soles and blood groups lipodystrophy and c deficiency lung disease and pi types mdh, pyreneans -‚ west bengal mem groups in india mn groups in andhra pradesh monoamine oxidase activity, twin study nadh diaphorase in pyreneans norway, c types in lapps pgd groups, belgium pgmi groups, andhra pradesh --.belgium --.pyreneans --‚ west bengal phi groups in andhra pradesh pi m, subtypes and alpha- -antitrypsin levels -.variant - groups, canada --, lung disease placental alkaline phosphatase variants properdin factor b in afghanistan punjabis, gm and km groups pyreneans, blood, serum and enzyme groups rh groups, andhra pradesh , - -‚ himachal pradesh salivary proteins and dental disease schizophrenia, genetic model subject index serum cholinesterase, pyreneans siberia, am, gm and km groups sickle cell trait and g- -pd deficiency in cameroon , singapore, gm and km groups -pgd, affective disorders -‚ andhra pradesh -‚ pyreneans sjögren-larsson syndrome sod groups in pyreneans somatotype components, family study south africa, ag groups sri lanka, gm and km groups sweden, achromatopsia -‚ pi types jung disease tf c groups, andhra pradesh , --, belgium --, himachal pradesh subtypes, abortions -‚ us black and whites tgp in pyreneans triradius d, see dermatoglyphics trisomy p turkey, beta-thalassaemia, g- -pd, hb variants and hp groups twin studies, ige levels --, mao activity - zygosity diagnosis uremia and c groups vietnamese, south, g- -pd deficiency wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to 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m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ original article linkage analysis of circulating levels of adiponectin in hispanic children me tejero , g cai , hhh göring , v diego , sa cole , ca bacino , nf butte and ag comuzzie department of genetics, southwest foundation for biomedical research, san antonio, tx, usa and department of pediatrics, usda/ars children’s nutrition research center, baylor college of medicine, houston, tx , usa introduction: adiponectin, a hormone produced exclusively by adipose tissue, is inversely associated with insulin resistance and proinflammatory conditions. the aim of this study was to find quantitative trait loci (qtls) that affect circulating levels of adiponectin in hispanic children participating in the viva la familia study by use of a systematic genome scan. methods: the present study included extended families with at least one overweight child between and years old. overweight was defined as body mass index (bmi) th percentile. fasting blood was collected from children from families. adiponectin was assayed by radioimmunoassay (ria) technique in fasting serum. a genome-wide scan on circulating levels of adiponectin as a quantitative phenotype was conducted using the variance decomposition approach. results: the highest logarithm of odds (lod) score ( . ) was found on chromosome q . – q . , and a second significant signal (lod score ¼ . ) was found on chromosome q . – q . . in addition, a signal suggestive of linkage (lod score ¼ . ) was found between q . and q . . after adjustment for bmi-z score, the lod score on chromosome remained unchanged, but the signals on chromosomes and dropped to . and . , respectively. two other signals suggestive of linkage were found on chromosome (lod score ¼ . ) and (lod score ¼ . ). although the region on chromosome has been associated with obesity and diabetes-related traits in adult populations, this is the first observation of linkage in this region for adiponectin levels. our suggestive linkages on chromosomes and replicate results for adiponectin seen in other populations. the influence of loci on chromosomes and on circulating adiponectin seemed to be mediated by bmi in the present study. conclusion: our genome scan in children has identified a novel qtl and replicated qtls in chromosomal regions previously shown to be linked with obesity and type diabetes (t d)-related phenotypes in adults. the genetic contribution of loci to adiponectin levels may vary across different populations and age groups. the strong linkage signal on chromosome is most likely underlain by a gene(s) that may contribute to the high susceptibility of these hispanic children to obesity and t d. international journal of obesity ( ) , – . doi: . /sj.ijo. ; published online august keywords: adiponectin; genetics; linkage; childhood obesity; qtl; genome scan introduction the hormone adiponectin is produced exclusively by adipose tissue and has insulin-sensitizing and anti-inflam- matory properties. as opposed to other adipose tissue products, low circulating levels of this protein are associated with obesity, type diabetes (t d) and the metabolic syndrome. , consistent negative correlations between adi- ponectin, insulin resistance and inflammatory states have been reported. these latter two conditions improve after weight loss with concomitant increase of adiponectin levels. adiponectin structure is similar to tumor necrosis factor-a and seems to have a counter-regulatory activity with respect to this proinflammatory cytokine. as observed in adults, obese children and adolescents have lower adipo- nectin levels than their normal weight counterparts, and serum adiponectin is positively correlated with insulin sensitivity and high-density lipoprotein, and negatively to fasting proinsulin and proinsulin/insulin ratio. a study by reinehr et al. found that weight loss in children is associated with a significant increase of circulating adipo- nectin and a decrease of insulin resistance. a significant heritability for adiponectin levels in hispanic children has been previously reported by our group. heritability (h ) of serum adiponectin was . . received november ; revised may ; accepted june ; published online august correspondence: dr anthony comuzzie, department of genetics, southwest foundation for biomedical research, nw loop , po box , san antonio, tx - , usa. e-mail: agcom@darwin.sfbr.org international journal of obesity ( ) , – & nature publishing group all rights reserved - / $ . www.nature.com/ijo (p ¼ . � � ). adiponectin differed by age (p ¼ . ), sex (p ¼ . ) and weight (p ¼ . ). numerous investigations have analyzed the genetic component of obesity and t d- related phenotypes in humans and other species. kissebah et al. conducted linkage analyses on phenotypes of the metabolic syndrome, including body weight, hip and waist circumference, insulin and leptin levels. the multipoint linkage analyses of these phenotypes identified significant logarithm of odds (lod) scores on chromosome at – cm. vasseur et al. reported association between some single nucleotide polymorphisms (snps) and circulating levels of this adipose tissue protein. four studies have conducted linkage analysis using adiponectin levels as a phenotype. comuzzie et al. identified regions on chromo- somes and , and secondary signals on chromosomes and in caucasian adults. a study by pollin et al. in old order amish found linkage of adiponectin levels to chromo- some p , and an investigation in pima indians, a population characterized by a high prevalence of obesity and diabetes, found significant linkage to chromosome p and suggestive evidence of linkage on chromosomes , and . a fourth study by chuang et al. found suggestive linkage of adiponectin on chromosome at cm for chinese (lod ¼ . ) and on chromosome at cm for japanese adults. some of these regions have been previously reported in association with obesity and diabetes-related phenotypes. the present study is the first linkage analysis on circulating levels of adiponectin in children. the aim of the present investigation was to find quantitative trait loci (qtls) that affect circulating levels of adiponectin in hispanic children participating in the viva la familia study by use of a systematic genome scan. materials and methods study design and subjects genetic and environmental factors influencing fasting serum adiponectin were investigated in a subsample of children from the enrolled in the viva la familia study, which was designed to genetically map childhood obesity in the hispanic population. each family was ascertained on an overweight proband between the ages and years using a bivariate ascertainment scheme (i.e., th percentile for body mass index (bmi) and th percentile for fat mass. once identified, the overweight proband and all siblings, – years of age, and their parents were invited to the children’s nutrition research center for a tour and full explanation of the study. all children and their parents gave written informed consent. the protocol was approved by the institutional review board for human subject research for baylor college of medicine and affiliated hospitals. the overweight proband and all siblings were then characterized for body size, and endophenotypes associated with the development of obesity. here, we report our findings on the linkage analysis using adiponectin levels as a phenotype. phenotyping body weight was measured with a digital balance and registered to the nearest . kg. and height to the nearest mm was measured with a stadiometer. fasting serum adiponectin levels were measured by radioimmunoassay (ria) (linco research inc., st charles, mo, usa). genotyping the participants (children and their parents) were genotyped in the present study. dna was isolated from whole blood using the wizard genomic dna purification kit (promega, madison, wi, usa). to genotype each participant, we used the autosomal markers from the abi prism linkage mapping set-md version . (applied biosystems, foster city, ca, usa). this mapping set consists of fluorescently labeled polymerase chain reaction (pcr) primers that amplify dinucleotide single tandem repeats (strs) selected from the genethon human linkage map. the set is designed to create a map with markers spaced an average of cm apart (range . – . cm). dna from study participants was arrayed on -well pcr plates using the robbins hydra- microdispenser (sunnyvale, ca, usa). each marker was amplified in a separate pcr reaction to avoid the preferential amplification that can occur in combined reactions. pcr reactions used the true allele pcr premix (applied biosystems), and amplification oc- curred in applied biosystems thermocyclers, according to the manufacturer’s specifications. after pcr, the products of separate pcr reactions, for each individual, were pooled using the robbins hydra- microdispenser, and a labeled size standard was added to each pool. the pooled pcr products were loaded into an abi prism genetic analyzer for laser-based automated genotyping. the strs were detected and quantified by fluorescent emissions, their sizes were estimated by comparison with the labeled size standard and genotypes were scored using the genotyper software package (applied biosystems). pedigree errors were detected using prest (pedigree relationship statistical tests), which employs likelihood- based inference statistics for genome-wide identity-by- descent (ibd) allele sharing. pedigree errors were resolved by making changes to the existing pedigree structure that required the fewest assumptions and that were consistent with the genetic data. using simwalk genotypes that resulted in mendelian inconsistencies and spurious double recombinants were blanked if not resolved by the laboratory. we used loki to compute the ibd matrix needed for our linkage analyses. all three programs, prest, simwalk and loki, require information on the relative distances between linkage analysis of circulating levels me tejero et al international journal of obesity markers. we used the sex-averaged chromosomal maps obtained at the marshfield center for medical genetics. analysis using a variance component model, we tested the null hypothesis that the additive genetic variance owing to a qtl (sq ) equals zero or absence of linkage by comparing the likelihood of this restricted model with that of a model in which sq is estimated. the difference between the two log likelihoods produces a lod score that is the equivalent of the classical lod score of linkage analysis. twice the difference in loge likelihoods of these models yields a test statistic that is asymptotically distributed as a mixture of a w variable with df and a point mass at zero. a genome scan was conducted in solar using adiponec- tin levels as a phenotype and sex, age, age and their interactions as covariates. empirical lod score adjustment was conducted by the method described by blangero et al. the effect of bmi-z score was tested by including it as a covariate and repeating the genome scan. the chromosomal region investigated for positional candidate genes was defined as the lod score unit support interval. the selection of positional candidate genes was conducted using the ncbi map viewer database. results the present study included a total of children from families, with approximately % each of boys and girls in the sample. descriptive data of the cohort are shown in table . table includes information on the relative pairs in this study. the average age of the children was . years. the distribution across the tanner stages of development was as follows: for the genital/breast criteria children corresponded to tanner stages i, to ii, to stage iii, to stage iv and to tanner stage v. for the pubic hair criteria, children were classified as tanner stage i, as ii, as iii, as iv and as v. the analysis of the effect of age, sex, tanner stage and body composition on adiponectin levels in these children showed a significant difference between children classified as tanner stage i and ii–v. in the present study, the effect of this variable was not significant after adjustment for sex, age and bmi-z score. mean fasting serum levels of adiponectin was . mg/ml in boys and . mg/ml in girls with a significant heritability of . . (p ¼ . � � ). results of the linkage analysis are shown in figure . the highest lod score was observed on chromosome q – between markers d s and d s , with an lod support interval from to cm (lod ¼ . ). a second signal was identified on table anthropometry of the non-overweight and overweight hispanic children n boys, girls, n ¼ n ¼ age (years) . . . . weight (kg) . . . . height (m) . . . . bmi (kg/m ) . . . . bmi-z score . . . . abbreviations: bmi, body mass index. mean s.d. table relative pairs in the analyzed cohort of hispanic children in the viva la familia study relationship n siblings half-siblings half-avuncular first cousins half-first cousins half-siblings and half-cousins identical sibpair cm cm lod lod lod figure string plot of fasting serum adiponectin in hispanic children using sex, age and age as covariates. lod scores were empirically adjusted using simulations. linkage analysis of circulating levels me tejero et al international journal of obesity chromosome , between markers d s and d s ( – cm, lod ¼ . ), and finally a signal suggestive of linkage was observed on the region d s –d s on chromosome (lod ¼ . ). after adjustment for bmi-z score (figure ), the signal on chromosome remained unchanged; however, the signal dropped on chromosomes and to lod scores of . and . , respectively. two other regions suggestive of linkage were found on chromosomes and . on chromosome , suggestive linkage was found between markers d and d s ( – cm) with an lod score of . , and on chromosome a signal was detected between d s and d s ( . – . cm) with a lod score of . . figure shows the marker distribution and second genome scan on chromosome . other regions showing lod scores . were found on chromosomes q ( . ), q ( . ) and q ( . ). discussion few linkage studies on childhood obesity and related phenotypes have been conducted until now. the present study represents the first genome-wide search for adiponec- tin levels in children. this population is at high risk for the development of obesity-related comorbidities, such as t d. the children in this investigation showed significant meta- bolic abnormalities in association with excessive adiposity, as reported previously. given the high heritability of fasting serum adiponectin, and its inverse associations with insulin resistance, dyslipidemia and blood pressure in the viva la familia study, , and the high prevalence of obesity and t d in the united states hispanic population, we sought to identify genetic loci contributing to circulating adiponectin in hispanic children. previous investigations have found significant linkage for adiponectin in adults (table ). – the present analysis identified three new regions on chromosomes , and linked to adiponectin levels in hispanic children, and replicated two regions on chromosomes and reported in adults. our strongest linkage for fasting serum adiponectin was on chromosome and its strength was not diminished after adjustment for bmi-z score. although this is the first identification of a linkage signal on chromosome for adiponectin, this chromosomal region has been linked with obesity and diabetes-related traits (table ). stein et al. identified moderate evidence for linkage on chromosome near marker d s ) using five components of the metabolic syndrome. their analysis confirmed observations by other investigators who reported significant linkage of body size, diabetes mellitus and insulin resistance to chromosome q. significant linkage to chromosome q was found for obesity in subjects with t d in a study of siblings by van tilburg et al. after fine mapping this cm cm lod lod lod figure string plot of fasting serum adiponectin of hispanic children using sex, age, age and bmi-z score as covariates. lod scores were empirically adjusted using simulations. chromosome chromosome position (cm) l o d d s d s d s d s d s d s d s d s d s d s d s d s d s d s d s d s d s d s figure markers on chromosome . linkage analysis of circulating levels me tejero et al international journal of obesity table linkage studies for circulating adiponectin levels in adults population identified chromosomes map distance (cm) lod score lod score after adjustment for bmi reference white northern . comuzzie european ancestry . . . pima indians . . lindsay . . . . . . hawaiian . . chuang japanese . . . . chinese . . amish . pollin . . . . abbreviations: bmi, body mass index; lod, logarithms of odds. table replications of previously reported qtls linked to obesity and diabetes-related phenotypes chromosomal region markers phenotype population lod score reference q – q . d s –d s bmi white . van tilburg q –q f f f f f q –q d s bmi pima indians . norman q – d s body fat nigerian . adeyemo p . – q d s metabolic syndrome white p ¼ . stein bmi white (amish) p ¼ . platte q . d s body fat % pima indians . norman body fat % pima indians . norman q . d s bmi pima indians . lindsay q . d s h energy expenditure pima indians . norman q . d s bmi pima indians . lindsay bmi whites . stone bmi mexican-american . arya q . d s bmi f . stone p . d s obesity whites and npl ¼ . dong african-american p ¼ . price whites and african– american p . d s obesity white children and adolescents . saar p . d s bmi french population . hager p . d s obesity f . hinney q . d s bmi-adjusted leptin old order amish . hsueh q d s – bmi african . palmer d s apnea–hypopnea index american . p . d s bmi mexican–american . mitchell q . d s leptin mexican–american . comuzzie q . d s bmi mexican–american p ¼ . gorlova q . d s bmi whites . chagnon q d s young onset type diabetes british/irish descents . frayling p . d s bmi, sbp, dbp whites . turner q . d s bmi kg/m whites npl ¼ . li q . d s obesity finns . ohman q . d s bmi and blood pressure whites . north q mc r respiratory quotient whites p ¼ . chagnon q – d s fasting glucose european americans . li abbreviations: bmi, body mass index; dbp, diastolic blood pressure; lod, logarithms of odds; npl, non-parametric linkage; qtl, quantitative trait loci; sbp, systolic blood pressure. linkage analysis of circulating levels me tejero et al international journal of obesity region, they found a lod score of . on chromosome q –q using bmi as a phenotype. the combined phenotype ‘diabesity’ had a highly sig- nificant lod score of . at q – in pima indians. this study reported snps associated with bmi and a second region harboring a cluster of snps related to diabetes in this population. this group sequenced physiological candi- date genes in this region encoding serotonin receptor, dopamine receptor, three apolipoproteins, three zinc-finger proteins, two potassium-channel proteins and glucose- - phosphate transferase. none of these candidates had nucleo- tide variants that account for the linkage signal for bmi and diabetes. one of the candidates in the q – region is the dopamine d receptor gene (drd ). jenkinson et al studied polymorphisms of this gene in pima indians, and determined that heterozygotes at the ser cysdrd poly- morphism had a higher bmi than homozygotes. the list of positional candidates proposed in the present study is included in table . adipocyte-specific adhesion molecule (asam) is specifically expressed in adipose tissue, the transforming growth factor beta regulator regulates the development and homeostasis of tissues, the endothelial cell adhesion molecule is a type i transmembrane protein and is a new member of the immunoglobulin superfamily, similar to asam, and is considered an adhesion mole- cule. cdon (omim ) cell adhesion molecule- related/downregulated oncogenes is a cell surface receptor of the immunoglobulin (ig)/fibronectin type iii repeat family involved in myogenic differentiation. toll-interleu- kin receptor domain/adaptor protein (tirap) (omim ), which is a protein involved in the inflammatory response in mice. potassium inwardly rectifying channel (kcnj ) (omim ) are important regulator of resting membrane potential and cell excitability and interacts with the phosphatidylinositol , -biphosphate (pip ). most of these new candidates seem to be associated to the immune response. our genome scan replicated linkage findings in adults. the qtl on chromosome for adioponectin was previously reported in pima indians and hawaiian japanese adult subjects. in contrast to our study, adjustment for bmi did not alter their lod scores. other phenotypes such as bmi and blood pressure have been linked to this chromosome region. , the region localized on chromosome has been linked to adiponectin levels in pima indians, white americans of northern european ancestry and old order amish. in addition, linkage has been reported for other obesity-related phenotypes on this chromosomal region in numerous studies, including a previous investigation in children and adolescents. the region found on chromosome between markers d s and d s has shown significant linkage with the phenotypes bmi and leptin. a study by frayling et al. identified a locus on chromosome q linked to early-onset t d in a study conducted in the united kingdom. the region on chromosome has been linked to bmi in a previous study in caucasians of northern european back- ground. the investigation by chuang et al. identified significant linkage signals for adiponectin levels in hawaiian japanese population on chromosomes p and p (table ). the signals on chromosome were located at and cm and do not overlap the region found in our study. that study reported that the lod score on chromosome remained unchanged after adjustment for age, sex and bmi, whereas the signal on chromosome decreased from . to . , suggesting that the contribution of this qtl may be mediated by body mass. ohman et al. reported suggestive linkage in this region using obesity as a phenotype. this region has been linked to the respiratory quotient in caucasians and to fasting glucose. the lod score on the chromosome regions found on chromosomes and dropped to nonsignificant values after adjustment for bmi-z table positional candidates on chromosome chromosome markers lod score distance in cm candidates q – d s –d s . – asam – adipocyte-specific adhesion molecule tbrg – transforming growth factor beta regulator esam – endothelial cell adhesion molecule cdon – cell adhesion molecule-related/down regulated oncogenes tirap – toll-interleukin- receptor domain/adaptor protein kcnj – potassium inwardly rectifying channel table linkage findings for fasting serum adiponectin in hispanic children chromosome distance (cm) flanking markers lod score lod score model model d s . . d s d s . . d s d s . . d s d s . . d s d s . . d s model : sex , age, age as covariates. model : sex, age, age and bmi-z score as covariates. linkage analysis of circulating levels me tejero et al international journal of obesity score, interestingly both of them have been linked to this trait in previous studies in adults. in summary, the present investigation identified novel chromosomal regions linked with circulating adiponectin levels on chromosomes , and , and suggestive of linkage on chromosomes and . the last two replicate findings in other populations. all the chromosome regions identified in the present study have been linked to obesity and diabetes-related phenotypes in adults across different ethnicities. further fine mapping of these regions will allow the identification of genetic polymorphisms that influence the circulating levels of adiponectin. acknowledgements we thank the families who participated in this study, and to acknowledge the contributions of mercedes alejandro and marilyn navarrete for study coordination, and sopar seribu- tra for nursing and theresa wilson, tina ziba, maurice puyau, firoz vohra, anne adolph, roman shypailo, joann pratt and maryse laurent for technical assistance, grace meixner and daniel zamarripa for genotyping and jennifer darling for the data analysis. this work is a publication of the us department of agriculture (usda)/agricultural research service 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heritage. j appl physiol ; : – . li w, dong c, li d, zhao h, price r. an obesity-related locus on chromosome region q – . diabetes ; : – . north ke, rose km, borecki ib, oberman a, hunt sc, miller mb et al. evidence for a gene on chromosome influencing postural systolic blood pressure change and body mass index. hypertension ; : – . linkage analysis of circulating levels me tejero et al international journal of obesity 	linkage analysis of circulating levels of adiponectin in hispanic children 	introduction 	materials and methods 	study design and subjects 	phenotyping 	genotyping 	analysis 	results 	discussion 	acknowledgements 	references innate immunity and asthma risk in amish and hutterite farm children the new england journal of medicine n engl j med ; nejm.org august , established in august , vol. no. the authors’ affiliations are listed in the appendix. address reprint requests to dr. vercelli at the arizona respiratory cen- ter, university of arizona, the bio insti- tute, rm. , e. helen st., tucson, az , or at donata@ email . arizona . edu; to dr. ober at the department of human genetics, university of chicago, e. th st., clsc , chicago, il , or at c-ober@ genetics . uchicago . edu; or to dr. sperling at the department of medi- cine, university of chicago, e. th st., jfk r , chicago, il , or at asperlin@ uchicago . edu. ms. stein, dr. hrusch, and ms. gozdz and drs. von mutius, vercelli, ober, and sperling contributed equally to this article. this article was updated on august , , at nejm.org. n engl j med ; : - . doi: . /nejmoa copyright © massachusetts medical society. background the amish and hutterites are u.s. agricultural populations whose lifestyles are remark- ably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. the populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities. methods we studied environmental exposures, genetic ancestry, and immune profiles among amish and hutterite children, measuring levels of allergens and endotoxins and assess- ing the microbiome composition of indoor dust samples. whole blood was collected to measure serum ige levels, cytokine responses, and gene expression, and peripheral- blood leukocytes were phenotyped with flow cytometry. the effects of dust extracts obtained from amish and hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma. results despite the similar genetic ancestries and lifestyles of amish and hutterite children, the prevalence of asthma and allergic sensitization was and times as low in the amish, whereas median endotoxin levels in amish house dust was . times as high. differences in microbial composition were also observed in dust samples from amish and hutterite homes. profound differences in the proportions, phenotypes, and func- tions of innate immune cells were also found between the two groups of children. in a mouse model of experimental allergic asthma, the intranasal instillation of dust ex- tracts from amish but not hutterite homes significantly inhibited airway hyperreactiv- ity and eosinophilia. these protective effects were abrogated in mice that were deficient in myd and trif, molecules that are critical in innate immune signaling. conclusions the results of our studies in humans and mice indicate that the amish environment provides protection against asthma by engaging and shaping the innate immune re- sponse. (funded by the national institutes of health and others.) a b s t r a c t innate immunity and asthma risk in amish and hutterite farm children michelle m. stein, b.s., cara l. hrusch, ph.d., justyna gozdz, b.a., catherine igartua, b.s., vadim pivniouk, ph.d., sean e. murray, b.s., julie g. ledford, ph.d., mauricius marques dos santos, b.s., rebecca l. anderson, m.s., nervana metwali, ph.d., julia w. neilson, ph.d., raina m. maier, ph.d., jack a. gilbert, ph.d., mark holbreich, m.d., peter s. thorne, ph.d., fernando d. martinez, m.d., erika von mutius, m.d., donata vercelli, m.d., carole ober, ph.d., and anne i. sperling, ph.d. the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , t h e n e w e n g l a n d j o u r n a l o f m e d i c i n e many genetic risk factors have been reported to modify susceptibility to asthma and allergy, , but the dra- matic increase in the prevalence of these condi- tions in westernized countries in the past half- century suggests that the environment also plays a critical role. the importance of environmental exposures in the development of asthma is most exquisitely illustrated by epidemiologic studies conducted in central europe that show signifi- cant protection from asthma and allergic disease in children raised on traditional dairy farms. in particular, children’s contact with farm animals and the associated high microbial exposures , have been related to the reduced risk. , however, the effect of these traditional farming environ- ments on immune responses is not well defined. to address this gap in knowledge, we de- signed a study that compares two distinctive u.s. farming populations — the amish of indi- ana and the hutterites of south dakota — that recapitulate the differences in the prevalences of asthma and allergy observed in farmers and nonfarmers in europe. these two particular groups of farmers originated in europe — the amish in switzerland and the hutterites in south tyrol — during the protestant reformation and then emigrated to the united states in the s and s, respectively. both groups have since remained reproductively isolated. , their lifestyles are similar with respect to most of the factors known to influence the risk of asthma, including large sibship size, high rates of child- hood vaccination, diets rich in fat, salt, and raw milk, low rates of childhood obesity, long dura- tions of breast-feeding, minimal exposure to tobacco smoke and air pollution, and taboos against indoor pets. however, whereas the amish practice traditional farming, live on single- family dairy farms, and use horses for fieldwork and transportation, the hutterites live on large, highly industrialized, communal farms. strik- ingly, the prevalence of asthma in amish versus hutterite schoolchildren is . % versus . % and the prevalence of allergic sensitization is . % versus . %, as previously reported. , m e t h o d s overview we characterized the immune profiles of amish and hutterite schoolchildren. furthermore, we used mouse models of asthma to study the effect of the environment on airway responses and to create a mechanistic framework for the interpre- tation of our observations in humans. study participants and study oversight in november , we studied amish children to years of age who lived in indiana, and in december we studied hutterite children who lived in south dakota and were matched with the amish children for sex and for age within year. (for information on the characteristics of the children see table , and the methods section in the supplementary appendix, available with the full text of this article at nejm.org.) written informed consent was obtained from the parents and written assent was obtained from the chil- dren. one parent of each child responded to a questionnaire on asthma symptoms and previous diagnoses. the study was approved by the insti- tutional review boards at the university of chi- cago and at st. vincent hospital in indianapolis. blood-sample collection and analysis whole blood was collected in tubes that con- tained culture medium alone, medium plus . μg per milliliter of lipopolysaccharide, or medium plus . μg per milliliter of anti-cd plus . μg per milliliter of anti-cd monoclonal antibod- ies (truculture blood collection system, myriad rbm). after incubation at °c for hours, a quick take is available at nejm.org characteristic amish (n = ) hutterite (n = ) age (yr) median range – – girls (no.) sibships (no.) children with asthma (no.) positivity for allergen-specific ige (no.) > . kua/liter > . kua/liter serum ige (ku/liter) median interquartile range – – * ua denotes allergen-specific unit. table . demographic and clinical characteristics of the study populations.* the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , immunit y a nd a s thm a in a mish a nd hu t ter ite childr en supernatant and cells were frozen for use in gene-expression and cytokine studies. levels of cytokines were measured with the use of the milliplex map human th magnetic bead panel (emd millipore) or enzyme-linked immuno- absorbent assay (ebioscience) in the supernatant, in accordance with standard protocols. addi- tional blood was collected to obtain peripheral- blood leukocytes for flow cytometry and dna isolation, and serum was collected for ige stud- ies (as described in the methods section in the supplementary appendix). cryopreserved human peripheral-blood leuko- cytes were incubated for minutes with pooled human igg antibodies (fcx, biolegend) to block nonspecific antibody binding before undergoing surface staining with f luorescently conjugated antibodies (see table s in the supplementary appendix) and intracellular staining for foxp (ebioscience). flow-cytometry data were acquired on an lsrfortessa cell analyzer (bd biosciences), and acquisition data were analyzed with flowjo software (tree star). dust collection and extract preparation electrostatic dust collectors were placed in one bedroom and the living room in each of amish and hutterite homes to collect air- borne house dust. all amish homes and of hutterite homes housed children who partici- pated in the study. after month, dust was ana- lyzed for endotoxin and allergen levels, and ex- tracts were prepared for studies in mice. in addition, a vacuum was used to collect dust from the living-room floor in amish homes and from mattresses in amish and hutterite homes for use in microbiome studies (as described in the methods section in the supplementary appendix). aqueous extracts of house dust from amish and hutterite homes were prepared as described in the methods section in the supplementary appendix. genetic studies rna was extracted from thawed cells with the use of allprep dna/rna mini kits (qiagen). rna underwent complementary dna synthesis and was then hybridized to humanht- v expression beadchip arrays (illumina). a com- mon set of , single-nucleotide polymor- phisms (snps) was genotyped or imputed in the children in the study (see fig. s in the sup- plementary appendix). mouse models we instilled μl of house-dust extract intra- nasally every to days (for a total of times) into -week old balb/c mice (harlan laborato- ries), beginning on day . the mice had been sensitized intraperitoneally with μg of oval- bumin (grade v, sigma) plus alum (pierce) on days and and were challenged intranasally with μg of ovalbumin on days and . beginning days before day , we also instilled μl of dust extract from amish homes intra- nasally every to days (for a total of times) in -week old, c bl wild-type, myd -deficient mice and in mice deficient in both myd and trif (jackson laboratories). these mice were sensitized intraperitoneally with μg of oval- bumin plus alum on days and and chal- lenged intranasally with μg of ovalbumin on days , , and . statistical analysis statistical analyses were performed with the use of r or prism software (graphpad). differences in the distributions of median cytokine levels between amish and hutterite children were de- termined with use of a wilcoxon signed-rank test. we used linear regression to identify differ- entially expressed genes in the amish and hut- terite untreated samples of peripheral-blood leukocytes. the methods of benjamini and hoch- berg were used to control the false discovery rate. for flow cytometric studies and the study in mice, differences in cell populations and air- way resistance were assessed with the use of an unpaired student’s t-test. additional details on sample processing, quality control, and statisti- cal analysis for all methods described here are provided in the methods section in the supple- mentary appendix. r e s u l t s asthma and allergic-sensitization rates and genetic ancestry none of the amish children and six ( %) of the hutterite children had asthma, rates similar to those reported in earlier studies. , levels of total serum ige and the number of children whose levels of ige against common allergens were high (defined as more than . kua [allergen- specific unit] per liter) were lower in the amish group than in the hutterite group (table , and the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , t h e n e w e n g l a n d j o u r n a l o f m e d i c i n e table s in the supplementary appendix). no statistical differences were observed in levels of serum ig isotypes other than ige (fig. s in the supplementary appendix). to evaluate whether the differences in the prevalence of asthma and of allergic sensitiza- tion and differences in ige level could be attrib- uted to population history, we assessed ancestry by conducting principal component analysis and compared allele frequencies using genomewide snps. these studies revealed remarkable genetic similarities between the amish children and the hutterite children, as compared with other euro- pean populations (fig. a, and fig. s in the supplementary appendix). exposures to allergens, microbes, and microbial products common allergens (from cats, dogs, house-dust mites, and cockroaches) were detectable in air- borne dust from of amish and of hut- terite homes (table s in the supplementary appendix). in contrast, endotoxin levels were measurable in airborne dust from all homes, and median levels were strikingly higher ( . times as high) in amish homes than in hutterite homes ( endotoxin units [eu] per square meter vs. eu per square meter, p < . ) (fig. b). analysis of a single pooled sample of mattress dust from each population revealed different profiles of the relative abundance of bacteria at the family level (fig. s in the supplementary appendix). composition and phenotype of peripheral- blood leukocytes peripheral-blood leukocytes from amish children had increased proportions of neutrophils, de- creased proportions of eosinophils, and similar proportions of monocytes as compared with samples from hutterite children (fig. a). neu- trophils from amish children expressed lower levels of the chemokine receptor cxcr and the adhesion molecules cd b and cd c than did neutrophils from hutterite children, suggesting that these cells may have recently emigrated from the bone marrow (fig. b). although propor- tions of monocytes were similar in amish and hutterite children, monocytes from amish chil- dren, unlike those from hutterite children, ex- hibited a suppressive phenotype characterized by lower levels of human leukocyte antigen dr (hla-dr) and higher levels of the inhibitory molecule immunoglobulin-like transcript (ilt ) , (fig. c). in contrast with previous studies, , no significant differences in percent- figure . ancestries and environments of amish and hutterite children. panel a shows a principal components plot of the first two principal com- ponents (pc and pc ) of the analysis of , single-nucleotide poly- morphisms (snps). amish and hutterite genotypes were projected onto the sample space created by human genome diversity project (hgdp) for european populations. panel b shows endotoxin levels in airborne dust from amish and hutterite homes. box-and-whisker plots show a hor- izontal line indicating median value, a box representing the interquartile range, and whiskers showing the % confidence interval. the p value was calculated with the use of the wilcoxon rank-sum test. eu denotes endo- toxin units. a snp analysis of genetic association p c − − − − − pc b endotoxin levels in airborne dust e u /m amish homes hutterite homes , , , , p< . amish hutterite basque french north italian russian russian caucasus sardinian scottish tuscan the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , immunit y a nd a s thm a in a mish a nd hu t ter ite childr en figure . proportions of peripheral-blood leukocytes and cell-surface–marker phenotypes in amish and hutterite children. the percentages of total peripheral-blood leukocytes (panel a) were determined with flow cytometry for neutrophils (defined as cd b+cd +), eosinophils (defined as ccr +siglec- +), and monocytes (defined as cd +cd b−). box-and-whisker plots show a line indicating median value, with the box showing the interquartile range and whiskers showing the % confidence interval. neutrophils (panel b) were characterized according to the surface expression of cxcr , cd b, and cd c (shown here), along with cxcr and cxcr , expressed as mean fluorescence intensity (mfi). the expression of the interleukin- coreceptors cxcr and cxcr was not signifi- cantly different between groups (p = . and p = . , respectively). monocytes (panel c) were characterized for the surface expression of hla-dr and immunoglobulin-like transcripts (ilts), including ilt (shown here). there was no significant difference in the mfi of in- hibitory receptors ilt and ilt between amish and hutterite children (p = . and p = . , respectively; data not shown), whereas the surface expression of ilt was increased on amish monocytes (p = . ; data not shown). all p values were calculated with the use of an unpaired student’s t-test. cell proportions and phenotypes after the exclusion of children with asthma or allergic sensitization are shown in table s in the supplementary appendix. c d b + c d + ( % ) amish hutterite b cell-surface markers on neutrophils a cell proportions of peripheral-blood leukocytes neutrophils p= . c c r + s ig le c- + ( % ) amish hutterite eosinophils p< . c d + c d b – ( % ) amish hutterite monocytes p= . m f i amish hutterite cxcr p< . m f i , , , , , amish hutterite cd b p< . m f i amish hutterite cd c p= . c cell-surface markers on monocytes m f i amish hutterite hla-dr p= . m f i amish hutterite ilt p= . the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , t h e n e w e n g l a n d j o u r n a l o f m e d i c i n e ages of t regulatory cells (defined as cd +, cd +, foxp +, and cd −) was observed in amish and hutterite children ( . ± . % vs. . ± . % of peripheral-blood leukocytes, p = . ). cytokine responses to innate and adaptive stimulation cytokine levels were measured in supernatants from peripheral-blood leukocytes that were cul- tured for hours, with or without innate stimuli (lipopolysaccharide) or adaptive stimuli (combined anti-cd and anti-cd antibodies). twenty-three cytokines were detectable in the supernatants from peripheral-blood leukocytes treated with lipopolysaccharide. median levels of each of these cytokines were lower in the amish children than in the hutterite children, and these distributions were significantly differ- ent (p < . by wilcoxon signed-rank test) (see tables s and s in the supplementary appen- dix). results were similar after the exclusion of children who were known to have asthma or allergies (table s in the supplementary appen- dix). in contrast, after adaptive stimulation, the overall distributions of median cytokine levels were not significantly different in peripheral- blood leukocytes from amish and hutterite children (p = . by wilcoxon signed-rank test) (table s in the supplementary appendix). gene-expression profiles the striking differences in the proportions of peripheral-blood leukocytes observed in amish and hutterite children were reflected in the gene- expression profiles of these cells (fig. s in the supplementary appendix). at a false discovery rate of %, genes were up-regulated in the peripheral-blood leukocytes of amish children (blue points in fig. a) as compared with genes up-regulated in the cells of hutterite chil- dren (red points in fig. a). these differentially expressed genes were organized into coexpres- sion modules with the use of the whole genome co-expression network analysis (table s in the supplementary appendix). to better understand the biologic relationships within each set of genes in each module, we used ingenuity pathway analysis (qiagen) to construct unsupervised net- works on the basis of prior knowledge of the physical and functional connections between the molecules encoded by the genes. the most sig- nificant network (p = . × − by fisher’s exact test) was in a module that contained genes. this module was associated with both amish and hutterite status (p = . × − ) and was therefore also associated with the proportions of neutro- phils (p = . × − ) and eosinophils (p = . × − ). eighteen of the genes in this module were over- expressed in amish peripheral-blood leukocytes, and all were clustered in a network that had as hubs tumor necrosis factor (tnf) and interferon regulatory factor (irf ), two key proteins in the innate immune response to microbial stimuli (fig. b). effects of house-dust extracts on experimental asthma to create a framework that would help us to interpret our observations, which were pointing toward a protective role of innate immunity, we used a classic ovalbumin mouse model of aller- gic asthma, comparing the effects of house dust obtained from amish and hutterite homes by administering extracts intranasally to mice over the course of to weeks. eosinophilia was observed in bronchoalveolar-lavage samples, and airway hyperresponsiveness was exacerbated in mice treated with ovalbumin and hutterite dust extracts as compared with mice treated with ovalbumin alone, findings that were consistent with the absence of protection from asthma observed in hutterite children (fig. a). in con- trast, inhalation of amish dust extracts was suf- ficient to significantly inhibit ovalbumin-induced airway hyperresponsiveness, eosinophilia in the bronchoalveolar lavage, and the elevation of se- rum ovalbumin-specific ige levels (fig. a, and table s in the supplementary appendix). levels of lung t regulatory cells (defined as cd +, cd +, and foxp +) were not increased (table s in the supplementary appendix), and all cyto- kines measured in bronchoalveolar-lavage sam- ples, including interleukin- , were suppressed in mice that received amish dust extracts (table s in the supplementary appendix). the inhibi- tory effects of these extracts in wild-type mice probably required innate immunity, because protection was strongly reduced in mice defi- cient in myd (fig. c) and completely abro- gated in mice deficient in both myd and trif (fig. d), two molecules that are critical to the development of multiple innate immune-signal- ing pathways. the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , immunit y a nd a s thm a in a mish a nd hu t ter ite childr en d i s c u s s i o n our studies in amish and hutterite schoolchil- dren revealed marked differences in the preva- lence of asthma despite similar genetic ances- tries and lifestyles. as compared with the hutterites, the amish, who practice traditional farming and are exposed to an environment rich in microbes, showed exceedingly low rates of asthma and distinct immune profiles that sug- gest profound effects on innate immunity. data generated in an experimental model of asthma support this notion by showing that the protec- tive effect of the amish environment requires the activation of innate immune signaling. analyses of the proportions and gene-expres- sion profiles of peripheral-blood immune cells in amish and hutterite children revealed differences in the cells and genes involved in innate immune responses to microbes. indeed, neutrophils, eosino- phils, and monocytes appeared to be major tar- gets of the distinct environments to which amish and hutterite children are exposed be- cause these cell types differed between the two groups in terms of their relative abundance, their phenotypes, or both. moreover, the network most associated with these differences consisted of innate immune genes. notable among the genes that were more highly expressed in the amish children was tnfaip , which encodes a , a ubiquitin-editing enzyme that limits the activity of multiple inflammatory pathways that depend on nuclear factor κb (nf-κb) and that has also been shown to mediate the protective effects of european farm-dust extracts in murine models of allergic asthma. irf , a hub in this network, figure . gene-expression profiles in peripheral-blood leukocytes from amish and hutterite children. in panel a, a volcano plot shows differences in baseline gene expression in peripheral-blood leukocytes from amish and hutterite children.the x axis indicates the log differences in gene-expression level between groups, with larger positive values representing genes with higher expression in the hutterites relative to the amish ( genes, shown in red points) and larger negative values representing genes with higher expression in the amish relative to the hut- terites ( genes, shown in blue points). the y axis shows the –log of the p values for each gene, with larger values indicating greater statistical significance. the solid horizontal line indicates the % false discovery rate. black points represent genes from amish and hutterite cells for which there was no significant difference in gene expression. differences in gene expression remain after the data for children with asthma or allergic sensitization were excluded (figs. s and s in the supplementary appendix). changes in gene expression between the two groups after correct- ing for differences in cell proportion are shown in figure s in the supplementary appendix. in panel b, a network of differentially expressed genes in untreated peripheral-blood leukocytes is shown. genes shown in blue have in- creased expression in amish children, and the gene shown in red has increased expression in hutterite children. the gene shapes indicate the class of each gene’s protein product (spirals denote enzymes, a v-shape denotes cyto- kines, conjoined circles denote a transcription regulator, hollow upside-down triangles denote kinases, cups denote transporters, and circles denote other products). lines represent different biologic relationships (solid lines indicate direct interaction, dashed lines indirect interaction, arrows direction of activation, arrows with a horizontal line direc- tion of activation and inhibition, and lines without arrows binding only). steap zc h a irak trim trim tnfaip parp tap parp samd l psme irf dhx map k stat tnfaip sco rhbdf tnf − − log (difference in gene expression) − lo g (p v al u e) ba the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , t h e n e w e n g l a n d j o u r n a l o f m e d i c i n e f ig u re . e ff ec ts o f a m is h a n d h u tt er it e h o u se -d u st e xt ra ct s o n a ir w ay r es p o n se s in m o u se m o d el s o f a lle rg ic a st h m a. p an el a s h o w s th e ef fe ct s o f th e in tr an as al i n st ill at io n o f μ l o f a m is h o r h u tt er it e d u st e xt ra ct i n -w ee k- o ld m ic e (b a lb /c s tr ai n ) ev er y to d ay s fo r a to ta l o f t im es b eg in - n in g at d ay . t h e m ic e w er e se n si ti ze d w it h o va lb u m in ( o v a ) in tr ap er it o n ea lly o n d ay s an d an d c h al le n g ed w it h o va lb u m in i n tr an as al ly o n d ay s an d . a ir w ay r es is ta n ce (s h o w n a s ce n ti m et er s o f w at er p er m ill ili te r p er s ec o n d a n d s ti m u la te d i n r es p o n se t o i n cr ea si n g d o se s o f ac et yl ch o lin e ad m in is te re d i n tr av en o u sl y) a n d b ro n ch o al ve o la r- la va g e (b a l) c el lu la ri ty w er e m ea su re d o n d ay ( t o m ic e p er g ro u p ). t h e to ta l am o u n t o f a m is h a n d h u tt er it e d u st e xt ra ct a d m in is te re d o ve r th e co u rs e o f th e ex p er im en t re p re se n t- ed t h e to ta l lo ad o f ai rb o rn e d u st d ep o si te d o n e le ct ro st at ic d u st c o lle ct o rs p la ce d i n a m is h o r h u tt er it e h o m es f o r m o n th . s ta ti st ic al d if fe re n ce s in e xp er im en ta l m ea su re s w er e as se ss ed w it h t h e u se o f s tu d en t’ s t- te st . a m is h h o u se -d u st e xt ra ct s ( . m g o f d u st e q u iv al en t in μ l) w er e in st ill ed i n tr an as al ly e ve ry t o d ay s fo r a to ta l o f t im es b eg in n in g d ay s b ef o re d ay i n to -w ee k o ld w ild -t yp e m ic e (p an el b ), m ic e d ef ic ie n t in m yd (p an el c ), a n d m ic e d ef ic ie n t in m yd an d t ri f (p an el d ) (a ll c b l s tr ai n s) . t h es e m ic e w er e se n si ti ze d i n tr ap er it o n ea lly w it h μ g o f o va lb u m in o n d ay s an d an d w er e ch al le n g ed i n tr an as al ly w it h μ g o f o va lb u m in o n d ay s , , an d . a ir w ay r es is ta n ce (s h o w n a s an i n cr ea se f ro m b as el in e in r es p o n se t o i n cr ea si n g d o se s o f n eb u liz ed m et h ac h o lin e) a n d b ro n ch o al ve o la r- la va g e ce llu la ri ty w er e m ea su re d o n d ay ( m ic e p er g ro u p f o r w ild -t yp e m ic e an d m ic e p er g ro u p f o r th o se d ef ic ie n t in m yd o r m yd an d t ri f) . s ta ti st ic al d if fe re n ce s in e xp er im en ta l m ea su re s w er e as se ss ed w it h t h e u se o f s tu - d en t’ s t- te st . i b ar s re p re se n t th e st an d ar d e rr o rs o f th e d at a. n s d en o te s n o t si g n if ic an t an d p b s p h o sp h at e- b u ff er ed s al in e. s al in e o v a o v a –a m is h s al in e o v a o v a –a m is h s al in e o v a o v a –a m is h p b s o v a o v a – a m is h o v a – h u tt er it e airway resistance (cm of water/ml/sec) . . . . . a ce ty lc h o lin e (µ g/ g m o u se ) b w ild t yp e c b l c c b l m yd k n o ck o u t d c b l m yd –t r if k n o ck o u t a b a lb /c p = . p = . p = . p < . p < . p = . airway resistance (cm of water/ml/sec) m et h ac h o lin e (m g/ m l)p = . airway resistance (cm of water/ml/sec) m et h ac h o lin e (m g/ m l)n s airway resistance (cm of water/ml/sec) m et h ac h o lin e (m g/ m l)n s bal cells (%) bal cells (%) bal cells (%) bal cells (%) e os in op hi ls n eu tro ph ils m ac ro ph ag es p < . p = . p = . p < . p = . p < . n s n s n s p = . n s eo sin op hi ls n eu tro ph ils m ac ro ph ag es eo sin op hi ls n eu tro ph ils m ac ro ph ag es eo sin op hi ls n eu tro ph ils m ac ro ph ag es p b s o v a o v a – a m is h o v a – h u tt er it e s al in e o v a o v a –a m is h s al in e o v a o v a –a m is h s al in e o v a o v a –a m is h the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , immunit y a nd a s thm a in a mish a nd hu t ter ite childr en regulates type i interferon transcription and is therefore essential for innate airway responses against viruses that are linked to susceptibility to asthma. , in turn, trim , the one gene in the network that was more highly expressed in the hutterites, acts as a positive regulator of tnf-α– and interleukin- β–induced activation of nf-κb. these findings suggest that in the amish, intense and presumably sustained exposure to microbes activates innate pathways that shape and cali- brate downstream immune responses. sustained microbial exposure was also reflect- ed in the phenotypes of peripheral innate im- mune cells in the amish. repeated microbial stimulation can lead to reduced expression of hla-dr on monocytes , and drive immature neutrophils from the bone marrow. - indeed, amish children had immature neutrophils bear- ing markers suggestive of recent emigration from the bone marrow, and they had monocytes with reduced expression of hla-dr and in- creased expression of ilt , all of which are sug- gestive of antiinflammatory function. proportions of t regulatory cells and levels of interleukin- , which typically mediate immune-balancing effects, were not increased in the amish children. how- ever, qualitative and functional differences in regulatory-cell populations remain to be defined. innate immunity has evolved to sense the environment and transduce signals that cali- brate adaptive responses to exogenous antigens. the proteins myd and trif are located at the convergence of multiple innate signaling path- ways, and deletion of these molecules virtually disables innate immune responses, thereby also dysregulating adaptive immunity. the fact that the loss of protection was more marked in mice deficient in both myd and trif than in mice deficient only in myd points to the involve- ment of multiple innate pathways. the concor- dance between findings from studies in humans and in mice was remarkable: in both studies protection was accompanied by lower levels of eosinophils, higher levels of neutrophils, general- ly suppressed cytokine responses, and no increase in levels of t regulatory cells or interleukin- . thus, the finding that these features were largely dependent on innate immune pathways in mice suggests that innate immune signaling may also be the primary target of protection in the amish children, in whom downstream adaptive immune responses may also be modulated. our study has several limitations. first, we were unable to include children younger than years of age, we collected samples at a single time point, and the numbers of amish and hutterite children in our study were relatively small. as a result, we may have missed important windows of immune development or lacked the ability to detect early, subtle shifts in cell composition, response, or phenotype that are critical for im- mune maturation. second, our microbiome as- sessments were limited, since only pooled dust samples from a limited number of homes were available for the studies in which we assessed bacterial composition. therefore, we cannot fur- ther dissect microbial composition and identify potentially protective microbes to target. how- ever, the striking differences found in endotoxin levels support the notion that the amish indoor environment is much richer in microbial expo- sures than the hutterite environment. third, the strategy used for sampling the hutterite chil- dren enriched selection for those with asthma, although the prevalence of asthma in our sample was similar to that reported in previous popula- tion-based studies. moreover, the exclusion of children with asthma or allergic sensitization from our analyses of gene expression, cell com- position, and immune phenotypes did not affect the outcomes. our study in a small number of children was sufficient to show significant differences in the prevalence of asthma and in immune profiles, suggesting that very strong environmental fac- tors must account for these differences. indeed, we showed that there are remarkable genetic similarities between amish and hutterite chil- dren. although we interrogated only common variants, other variants that occur at very low frequency in these populations are unlikely to account for the observed large differences in the prevalence of asthma. in the end, the novelty of our work lies in the identification of innate im- munity as the primary target of the protective amish environment, a finding supported by re- sults obtained in both humans and mice. con- versely, our work suggests that susceptibility to asthma may be increased when innate immune stimulation is weak. a deeper understanding of the relevant stimuli and the innate immune path- ways they engage may ultimately pave the way for the development of effective strategies for the prevention of asthma. the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , t h e n e w e n g l a n d j o u r n a l o f m e d i c i n e supported by the national institutes of health, st. vincent foundation, and the american academy of allergy, asthma, and immunology foundation. disclosure forms provided by the authors are available with the full text of this article at nejm.org. we thank the hutterite and amish volunteers and their families for participating in this study; gorka alkorta-aranburu, maitane arrubarrena orbegozo, kathleen bailey, christine billstrand, kelly blaine, daniel cook, donna decker, mohammad jaffery, courtney burrows, katherine naughton, raluca nicolae, rob stanaker, meghan sullivan, and emma thompson for assistance on field trips and with sample processing; peace ezeh, amanda herrell, ashley horner, kenneth addison, dominik schenten, and shane snyder for their contributions to the studies in mice; and minal Çalışkan, yoav gilad, jessie nicodemus-johnson, john novembre, and matthew stephens for helpful comments and statistical advice. appendix the authors’ affiliations are as follows: the department of human genetics (m.m. stein, c.i., r.l.a., c.o.), the department of medi- cine, section of pulmonary and critical care medicine, and the committee on immunology (c.l.h., a.i.s.), the department of ecology and evolution (j.a.g.), and the department of surgery (j.a.g.), university of chicago, chicago, and the institute for genomic and systems biology, argonne national laboratory, argonne (j.a.g.) — all in illinois; the niehs training program in environmental toxicology and graduate program in cellular and molecular medicine (j.g.), and the departments of cellular and molecular medicine (v.p., d.v.), medicine (j.g.l.), chemical and environmental engineering (m. marques dos santos), and soil, water, and environmental science (j.w.n., r.m.m.), university of arizona, and the arizona respiratory center and bio institute (j.g., v.p., s.e.m., j.g.l., f.d.m., d.v.) — all in tucson; the department of occupational and environmental health, university of iowa, iowa city (n.m., p.s.t.); allergy and asthma consultants, indianapolis (m.h.); and dr. von hauner children’s hospital, ludwig maximilians university munich, munich, germany (e.m.). references . ober c, yao tc. the genetics of asth- ma and allergic disease: a st century perspective. immunol rev ; : - . . meyers da, bleecker er, holloway jw, holgate st. asthma genetics and per- sonalised medicine. lancet respir med ; : - . . bach jf. the effect of infections on susceptibility to autoimmune and allergic diseases. n engl j med ; : - . . braun-fahrländer c, riedler j, herz u, et al. environmental exposure to endo- toxin and its relation to asthma in school- age children. n engl j med ; : - . . ege mj, mayer m, normand ac, et al. exposure to environmental microorgan- isms and childhood asthma. n engl j med ; : - 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other uses without permission. copyright © massachusetts medical society. all rights reserved. n engl j med ; nejm.org august , immunit y a nd a s thm a in a mish a nd hu t ter ite childr en . orden s, de pablo c, rios-navarro c, et al. efavirenz induces interactions be- tween leucocytes and endothelium through the activation of mac- and gp , . j antimicrob chemother ; : - . . devi s, wang y, chew wk, et al. neu- trophil mobilization via plerixafor-medi- ated cxcr inhibition arises from lung demargination and blockade of neutro- phil homing to the bone marrow. j exp med ; : - . . yamada m, kubo h, kobayashi s, et al. the increase in surface cxcr expres- sion on lung extravascular neutrophils and its effects on neutrophils during endotoxin- induced lung injury. cell mol immunol ; : - . . netea mg, wijmenga c, o’neill laj. genetic variation in toll-like receptors and disease susceptibility. nat immunol ; : - . copyright © massachusetts medical society. tucson, arizona gregory koshkarian, m.d. the new england journal of medicine downloaded from nejm.org at carnegie-mellon univ on april , . for personal use only. no other uses without permission. copyright © massachusetts medical society. all rights reserved. author's personal copy review diseases caused by defects of mitochondrial carriers: a review ferdinando palmieri ⁎ department of pharmaco-biology, laboratory of biochemistry and molecular biology, university of bari, via e. orabona , bari, italy a r t i c l e i n f o a b s t r a c t article history: received january accepted march available online march a strikingly large number of mitochondrial dna (mtdna) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. these mitochondrial disorders were the first to be investigated after the small mtdna had been sequenced in the s. only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, hhh syndrome, aspartate/glutamate isoform deficiency, amish microcephaly, and neonatal myoclonic epilepsy; these disorders are characterized by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabolism. defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phosphate and adp, are responsible for diseases characterized by defective energy production. herein, all the mitochondrial carrier- associated diseases known to date are reviewed for the first time. particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders. © elsevier b.v. all rights reserved. keywords: carrier deficiencies mitochondria mitochondrial carriers mitochondrial diseases mitochondrial carrier-related diseases transporters . introduction mitochondrial diseases are hundreds of clinical phenotypes caused by defects in one or more components of the mitochondrial proteome. this proteome consists of more than proteins that are encoded by either nuclear dna or mitochondrial dna (mtdna). the mitochon- drial genome encodes only proteins which are subunits of the four respiratory chain enzyme complexes (i–iv) and atp synthase complex (v). both the subunits encoded by mtdna or nuclear dna are essential for the activity of these complexes and therefore for the accomplish- ment of oxidative phosphorylation. since , when the first mutations in mtdna were described [ , ], more than mutations in mtdna have been identified as being responsible for respiratory chain and oxidative phosphorylation diseases (http://www.mitomap.org). these mutations concern not only mtdna genes encoding the above-mentioned proteins but also mtdna genes encoding ribosomal rnas and transfer rnas which are part of the protein synthesis machinery present in the mitochondrial matrix. respiratory chain and oxidative phosphoryla- tion diseases can also be caused by mutations of nuclear dna genes that encode proteins required for a) the replication, transcription, translation, repair and maintenance of mtdna; b) the functioning of complexes i–v (the nuclear-coded subunits) and their assembly; c) the import of the substrates of oxidative phosphorylation (see section of this review); and d) the import, modification and insertion of respi- ratory chain cofactors such as heme, metals and the iron–sulphur clusters. mtdna-dependent oxidative phosphorylation diseases are due either to large-scale rearrangements including deletions and duplications, which are usually sporadic and invariably heteroplasmic, or to point mutations which may be heteroplasmic or homoplasmic and are transmitted maternally. nuclear-dependent oxidative phos- phorylation diseases can induce secondary multiple mtdna deletions or loss of mtdna in affected tissues and follow the mendelian pattern of inheritance. although the primary function of mitochondria is the synthesis of atp by oxidative phosphorylation, these organelles play many other important roles. they contain several metabolic pathways, such as the citric acid cycle, or parts thereof, are the main site where reactive oxygen species (ros) are generated, and are fundamental for certain biological processes such as ca + cell signaling, cell proliferation and necrotic or apoptotic cell death. some enzymes involved in ros management, in amino acid catabolism and in the biosynthesis of fatty acids, cholesterol, steroid hormones, farnesyl and geranyl side chains, cardiolipin, ubiquinol, heme and urea are located inside mitochondria. furthermore, mitochondria communicate with other cell compart- ments by means of transport proteins (carriers) present in the mito- chondrial membrane that allow the selective passage of solutes in and out of the mitochondrial matrix. all these functions are accomplished by a large number of proteins which are encoded by the nuclear genome, translated in the cytosol and imported into the mitochondria. in recent years, the completion of human nuclear genome se- quencing and the ongoing identification of nuclear gene function have accelerated the disclosure of several mitochondrial disorders (apart biochimica et biophysica acta ( ) – ⁎ tel.: + x ; fax: + . e-mail address: fpalm@farmbiol.uniba.it. - /$ – see front matter © elsevier b.v. all rights reserved. doi: . /j.bbabio. . . contents lists available at sciencedirect biochimica et biophysica acta journal homepage: www.elsevier.com/locate/bbabio author's personal copy from oxidative phosphorylation diseases) resulting from mutations in nuclear genes encoding mitochondrial proteins. these include disorders caused by mutations of proteins involved in the citric acid cycle [ ], fatty acid β-oxidation [ ], cardiolipin metabolism (barth's syndrome) [ , ], the utilization of iron (friedreich's ataxia, x-linked ataxia and sideroblastic anemia) [ ], antioxidant defense [ ], protein import (x-linked deafness distonia) [ ], network dynamics (fission/ fusion) [ ] and mitochondrial metabolite carriers (this review). in addition, some cancers are caused by mutations in nuclear genes en- coding mitochondrial proteins [ , ]. several reviews on mitochon- drial diseases resulting from defects in the respiratory chain and oxidative phosphorylation have been published [ – ]. the con- tribution of mtdna mutations, ros generation and, in general, mito- chondrial dysfunction to cardiovascular disease, diabetes, cancer, aging and aging-related neurodegenerative disorders, such as parkin- son's and huntington's disease, has also been reviewed [ , – ]. this review highlights for the first time the current molecular, biochemical, clinical and pathophysiological information about all the diseases known thus far to be associated with defects of mitochondrial carriers. as a premise, some features of mitochondrial carriers are summarized. . characteristics and physiological role of mitochondrial carriers because mitochondria and cytosol cooperate in a large number of metabolic cellular processes, a continuous and highly diversified flux of metabolites, nucleotides, and cofactors into and out of the mitochondria is needed. the transport of solutes across the inner mitochondrial membrane is catalyzed by a family of nuclear-coded, membrane-embedded proteins called mitochondrial carriers. the carriers are involved in the krebs cycle, oxidative phosphorylation, modulation of the nucleotide and deoxynucleotide pools in the mito- chondrial matrix, the synthesis and breakdown of mtdna, mtrna and mitochondrial proteins, fatty acid oxidation, gluconeogenesis, lipo- genesis, transfer of reducing equivalents, urea synthesis, amino acid degradation, and other functions shared by cytosol and mitochondria. the central role in cell metabolism of most mitochondrial carriers identified so far (except the pyruvate carrier, pyc) is illustrated in fig. . in man, mitochondrial carriers are encoded by the slc gene family ([ ] for a review). several carriers, such as the adp/atp, atp- mg/pi, glutamate and ornithine carriers, have isoforms encoded by different genes, and only the phosphate carrier (pic) has two alternatively spliced isoforms. the main carriers importing substrates for oxidative phosphorylation are widely distributed in human tissues. in contrast, other carriers are tissue specific and have a limited dis- tribution reflecting their importance in special functions. the majority of mitochondrial carriers catalyze obligatory solute exchange reac- tions. those mediating h+-compensated undirectional substrate flux may also fall into the above category, given that at least pic has been shown to function in a phosphate/oh− antiport mode. the carnitine– acylcarnitine carrier catalyzes both unidirectional transport of carni- tine and the carnitine/acylcarnitine exchange, whereas the uncoupling protein catalyzes uniport as the exclusive transport mode. as their driving force, mitochondrial carriers utilize the concentration gradient fig. . metabolic roles of mitochondrial carriers. the scheme shows carriers catalyzing metabolite transport through the inner mitochondrial membrane. these carriers are involved in: oxidative phosphorylation (aac, pic, ucp); oxidation/reduction pathways (agc, ogc, dic, cic, cac, pyc); homeostasis of the intramitochondrial adenine nucleotide pool (apc); methylation of mtdna, mtrna and some intramitochondrial proteins (samc); import of the essential coenzyme thiamine pyrophosphate (thpp) required for pyruvate– and oxoglutarate–dehydrogenase complex activities (tpc); and, with partial overlap, amino acid metabolism (agc, orc, gc, odc). the scheme does not show all the metabolic pathways in which the already identified carriers are involved. f. palmieri / biochimica et biophysica acta ( ) – author's personal copy of the solutes and/or the h+ electrochemical potential gradient (Δμh+) generated across the inner mitochondrial membrane by the function of the respiratory chain. mitochondrial carriers can be divided into electrogenic (or electrophoretic) and electroneutral transporters depending on whether their transport reactions result in charge imbalance. the intra-/extra-mitochondrial distribution of solutes transported by electophoretic carriers is regulated by the electrical component of Δμh+, whereas the distribution of solutes carried by the proteins transporting anions together with an equivalent amount of h+ (anion/h+ symport) or in exchange for oh− (anion/oh− antiport) is regulated by the transmembrane ph gradient [ ]. all primary structures of the mitochondrial carrier family mem- bers consist of three tandemly repeated homologous domains of about amino acids in length. each repeat contains two hydrophobic stretches, that span the membrane as α-helices, and a characteristic signature motif p-x-[de]-x-x-[rk]-( – residues)-[de]-g-x-x-x-x- [ywlf]-[rk]-g. based on the sequence features described above and on the accessibility of carriers to peptide-specific antibodies, proteo- lytic enzymes and impermeable reagents, each mitochondrial carrier monomer has six helices traversing the membrane connected by hydrophilic loops with both the n- and c-termini on the cytosolic side of the inner mitochondrial membrane. in , the d structure of a member of this protein family, the adp/atp carrier in complex with its inhibitor, carboxyatractyloside, was determined [ ]. this structure, whose pseudo-three-fold symmetry [ , ] is in agreement with the three-fold sequence repeats, consists of six transmembrane α-helices (h –h ) and three short α-helices (h , h and h ) parallel to the membrane plane on the matrix side [ ]. in each repeated domain, the first part of the signature motif p-x-[de]-x-x-[rk] is located at the end of the odd-numbered transmembrane α-helices, whereas the second part [de]-g-x-x-x-x-[ywlf]-[rk]-g is located immediately before the even-numbered transmembrane α-helices. the prolines of the signature motif p-x-[de]-x-x-[rk] sharply kink the odd-numbered transmembrane α-helices, and the charged residues of the three signature motifs form a salt bridge network that closes the water- accessible cavity that is exposed towards the cytosolic side of the mitochondrial membrane and is occupied by the inhibitor. by using comparative models of carriers with known substrate specificity, a common substrate-binding site was proposed [ ]. this site is formed by residues which belong to the three even-numbered transmembrane α-helices and protrude into the cavity at the midpoint of the membrane, one-and-a-half helix turns above the salt bridge network. the significant sequence conservation in the mitochondrial carrier family suggests that the structural fold and transport mechan- ism are similar for all carriers. although the conformational changes involved in substrate translocation are not yet known, it is currently thought that translocation might be triggered by substrate-induced rearrangement of the salt bridge network from inter- to intra-domain interactions [[ ] and references therein]. the human genome encodes about different mitochondrial carriers. in the last decade, a significant advance in the field of mitochondrial transport has been the identification of many mito- chondrial carrier family members by over-expression in escherichia coli and/or saccharomyces cerevisiae, purification and reconstitution into liposomes for functional characterization by transport assays. until now, almost human carriers have been characterized in terms of substrate specificity, inhibitor sensitivity, transport mechanism, tissue distribution and kinetic characteristics [[ ] for a review, [ – ]]. naturally, this extension of the mitochondrial carrier family members has greatly favoured the discovery of their involvement in several diseases. . diseases associated with mitochondrial carriers mitochondrial carrier-related diseases are rare errors of metabo- lism caused by alterations of nuclear genes encoding mitochondrial carriers. the nine known disorders are listed in table . carrier acronyms throughout the text are those used in ref. [ ]; aliases are mentioned in the same reference. these disorders are all transmitted by mendelian inheritance; with the exception of autosomal dominant progressive external ophthalmoplegia (adpeo), the others are inher- ited in an autosomal recessive manner. mitochondrial carrier-related diseases can be divided into two groups. the first concerns defects of mitochondrial carriers directly linked to oxidative phosphorylation, such as the adp/atp carrierand the pic. the symptomatology of these diseases is characterized by insufficient energy production in tissues where these carriers are expressed and play an important role in oxidative phosphorylation. these diseases should also include adpeo, which is caused by hete- rozygous mutations of the adp/atp carrier isoform gene (as well as of other nuclear genes), because these mutations cause mtdna instability. the second group of diseases is due to alterations of the genes encoding mitochondrial carriers that are required for mitochondrial functions other than oxidative phosphorylation and, in particular, intermediary metabolism. their symptomatology depends on the metabolism affected and its significance in specific tissues. . diseases due to defects of mitochondrial carriers directly linked to oxidative phosphorylation given that the catalytic site of the atp synthase complex is exposed towards the mitochondrial matrix, the uptake of inorganic phosphate (pi) and adp into the mitochondria is essential for the oxidative phosphorylation of adp to atp (see fig. ). these uptakes are catalyzed by two distinct carriers, the pic and the adp/atp carrier, respectively; very recently, deficiencies of one isoform of both carriers have been described. . . aac deficiency in , a homozygous mutation (c. c→a) was found in the slc a gene of a -year-old male patient of slovenian origin [ ]. slc a encodes the heart-/muscle-specific adp/atp carrier (aac , also termed ant ). the mutation involves a highly conserved alanine at position that is replaced with an aspartic acid (a d). in the crystallographic structure of bovine aac , a protrudes into the central cavity two helix turns above the salt bridge network (slightly above the level of the proposed binding site) and is localized in the third transmembrane segment within the consensus sequence gxxxg, which is thought to be involved in high-affinity associations between transmembrane α-helices [ ]. the substitution of a d results in a complete loss of the protein's ability to transport adp and atp in reconstituted liposomes [ ]. interestingly, in contrast with the other aac mutations found so far in humans (see . ), a d is the first recessive mutation found in the aac gene. the patient affected with aac deficiency (omim ) mani- fested exercise intolerance, lactic acidosis, hypertrophic cardiomyo- pathy and a mild myopathy with no peo. exercise intolerance with table diseases associated with mitochondrial carriers disorder gene carrier substrates aac deficiency slc a aac adp/atp sengers' syndrome ? aac adp/atp pic deficiency slc a pic phosphate adpeo slc a aac adp/atp cac deficiency slc a cac carnitine/acylcarnitines hhh syndrome slc a orc ornithine/citrulline niccd/ctln slc a agc aspartate/glutamate amish microcephaly slc a tpc thiamine pyrophosphate neonatal myoclonic epilepsy slc a gc glutamate carrier acronyms are taken from ref. [ ]; aliases are mentioned in the same reference. f. palmieri / biochimica et biophysica acta ( ) – author's personal copy easy fatigability and muscle pain was present since early childhood and ventricular hypertrophy was diagnosed during puberty, indicating that the disease is slowly progressive. serum lactate levels in fasting conditions and at rest were higher than normal. in the skeletal muscle of the patient, numerous ragged-red fibers and multiple mtdna large- scale deletions were present (without a reduction of the wild-type mtdna copy number). the activities of the three mtdna-dependent respiratory chain complexes (i, iii and iv) were lower than normal and citrate synthase activity was much higher than normal, probably due to mitochondrial proliferation. interestingly, this symptomatology closely resembles that of the aac knock-out mice [ ]. as in the mouse model, loss of aac function is compatible with adult life, presumably due to compensation by glycolysis and/or transport activities of aac isoforms or other adenine nucleotide mitochondrial carriers, such as the atp-mg/pi carrier. . . sengers' syndrome aac deficiency was previously demonstrated in two unrelated patients with sengers' syndrome (omim ), an inherited autosomal recessive disease characterized by congenital cataracts, hypertrophic cardiomyopathy, mitochondrial myopathy and lactic acidosis but not peo [ ]. in the muscle tissues of both patients, the protein content of aac was drastically reduced. furthermore, ade- nine nucleotide transport was strongly impaired in liposomes reconstituted with mitochondrial extracts of the patients' skeletal or heart muscle. no mutation was found in the slc a gene and involvement of the aac locus was excluded by linkage analysis. the genetic defect underlying sengers' syndrome is still unknown. nevertheless, aac deficiency appears to be causally related to the clinical symptoms and tissue specificity of the disease. it may be that defective transcription, translation or targeting of aac is responsible for this disorder. . . pic deficiency in , a homozygous mutation in the gene encoding the pic (slc a ) (c. g→a) was found in two ill siblings of non- consanguineous turkish parents (the parents were heterozygous for the same mutation) [ ]. the human gene, which is spread over . kb of dna, maps to q . and contains coding exons; of these, exons a and b are closely related and spliced alternatively [ ]. the resulting isoforms, termed pic-a and pic-b, differ in amino acids and kinetic parameters but have the same substrate specificity and inhibitor sensitivity [ ]. both catalyze the uptake of phosphate, either by h+ co-transport or in exchange for oh−, into the mitochondria at the expense of the Δph component of the protonmotive force generated by electron transport. this uptake of pi into mitochondria is essential for oxidative phosphorylation of adp to atp. like isoforms of other mitochondrial proteins involved in oxidative phosphoryla- tion, pic-a is abundantly expressed only in heart and muscle, whereas pic-b is expressed in all tissues [ , ]. the differences between the two pic isoforms in kinetic properties (km of pic-b is -fold lower than that of pic-a, and vmax of pic-b is -fold higher than that of pic-a) and abundance in tissue ( and pmol pic-a per mg protein, and and pmol pic-b per mg protein in heart and liver bovine mito- chondria, respectively ) may account for the variation in reliance of the tissues on oxidative phosphorylation. the ubiquitous pic isoform b might match the basic energy requirement of all tissues, and isoform pic-a might become operative to accommodate the higher energy demands associated with contraction of striated muscle fibers. interestingly, the c. g→a mutation found in the two patients is localized in exon a and therefore affects the heart-/muscle-specific isoform, pic-a. in the pic-a of both patients glycine , that is conserved in all known pic sequences and in most other mitochon- drial carriers and is located in the first transmembrane helix three helix turns from the membrane's cytosolic side, is substituted by a glutamic acid residue; this substitution is deleterious for protein function [ ]. pic deficiency (omim ) is characterized by muscular hypotonia, progressive hypertrophic cardiomyopathy, elevated plasma lactate levels, and lactic acidosis. in one of the two affected siblings, cyanosis and poor weight gain were also observed. the patients died of heart failure at and months. in accordance with the tissue specificity of disease symptomatol- ogy, the synthesis of atp by oxidative phosphorylation was defective only in muscle. indeed, it was found that in heart mitochondria, but not in digitonin-permeabilized fibroblasts, adp-stimulated respira- tion of pyruvate was drastically reduced, whereas uncoupler-stimu- lated respiration was normal [ ]. furthermore, the activities of respiratory chain enzymes, pyruvate dehydrogenase and oligomycin- sensitive atp-ase were normal. no mutation was detected in the genes of atp synthase subunits encoded by mtdna or in slc a encoding the heart-/muscle-specific isoform of the adp/atp carrier. in view of the above-reported results, the disease is due to a defect in the pic-a- mediated import of pi into mitochondria, which prevents atp synthesis by oxidative phosphorylation. . . autosomal dominant progressive external ophthalmoplegia (adpeo) adpeo is a clinically and genetically heterogeneous disorder that is usually inherited as a dominant trait. it is caused by defects in certain nuclear genes and is characterized by multiple deletions of mtdna in post-mitotic tissues [ – ]. therefore, adpeo constitutes an example of nuclear gene defects affecting mtdna. the mtdna deletions result in a lack of respiratory chain proteins that lead to defective energy production. adpeo manifests an adult-onset phenotype (typically at – years of age); however, early onset has also been reported. typical symptoms of the disorder are peo due to weakness of the external eye muscles, ptosis, and mild descending myopathy. addi- tional variable symptoms include bilateral cataract, sensorineural hypocusia, tremor, ataxia, sensorimotor peripheral neuropathy, gen- eralized muscle weakness, exercise intolerance, depression, parkin- sonism and endocrine dysfunction. serum lactate levels are normal or slightly increased. muscle biopsies of affected individuals exhibit ragged-red fibers, cytochrome c oxidase-negative fibers, and dimin- ished activity of respiratory chain complexes. southern blot analysis of muscle dna reveals the presence of heterogeneous mtdna species due to multiple large-scale deletions ranging from . to . kb in length. mtdna lesions are present and accumulate in post-mitotic tissues, mainly brain, muscle and heart. this is consistent with the tissue distribution of aac in man, since aac is the predominant isoform of the adp/atp carrier in skeletal and heart muscle but is also present in brain. peo is one of the most common diseases caused by primary mtdna deletions. however, more recently, mutations of three nuclear genes have also been shown through linkage analysis to be responsible for adpeo by causing secondary multiple deletions of mtdna. one of these nuclear genes is slc a located on chromosome q and encoding the heart-/muscle-specific mitochondrial aac (omim ). the other two genes are twinkle, on chromosome q encoding an mtdna helicase (omim ), and polg on chromosome q encoding the catalytic subunit of mtdna-specific polymerase γ (omim ). furthermore, at least a fourth locus must be implicated in view of cases that could not be linked to any of the three genes reported above. nuclear-dependent peo is usually inherited as an autosomal dominant disorder, but cases with autosomal recessive inheritance it should be mentioned that according to mayr et al. [ ], “the small proportion of isoform b” detectable in all muscle tissues “may originate from either myoblasts or contaminating connective tissue or blood”. f. palmieri / biochimica et biophysica acta ( ) – author's personal copy have been described in families carrying heterozygous mutations in the polg gene. mutations in the polg and twinkle genes are the most frequent cause of peo, whereas those in the slc a gene are rarer. four heterozygous missense mutations of aac (a d, a p, l p, d g) have been found in adpeo families and one (v m) in a sporadic case. in the crystallographic structure of bovine aac , these mutations interface with the membrane except for d g which is located in the cytosolic loop between h and h . all the mutations are near the cytosolic side of the carrier protein, three to four helix turns from the common binding site. aac -related adpeo is relatively benign because the symptoms are usually limited to skeletal and facial muscles. as for all diseases caused by mutations of mtdna, treatment of adpeo is symptomatic and includes eye props and ptosis surgery. the twinkle and polg genes are directly involved in the repair and replication of mtdna, whereas aac catalyzes the exchange of cytosolic adp for intramitochondrial atp. therefore, the mechanism by which aac mutations cause mtdna instability is unclear. several hypotheses have been set forth; the mutations may: modify the transport properties of aac ; alter the intramitochondrial pool of adenine nucleotides causing a shortage of adp, which may be insuf- ficient for datp synthesis through the action of ribonucleotide reductase and nucleoside diphosphokinase and, consequently, yield a high error rate of mtdna polymerase; and cause a defective import of the mutated proteins into mitochondria, leading to misfolded proteins that are more susceptible to ros or to an increased pro- duction of ros within the mitochondria and damage of mtdna. to investigate the functional consequences of the human aac muta- tions found in adpeo patients, the mutations were introduced at equivalent positions in aac , the yeast ortholog of human aac [ ]. interestingly, expression of some of these mutants in aac -defective haploid strains of s. cerevisiae caused a growth defect on nonfermen- table substrates, a decrease of cytochrome content and reduced respiratory activity, but variable inhibition of atp and adp transport as deduced from measurements in liposomes reconstituted with mitochondrial extracts obtained from cells transformed with either each mutant or wild-type aac . in addition, heteroallelic strains, expressing both the aac mutant allele and wild-type aac , behaved as a recessive trait for oxidative growth and as a dominant trait for mtdna instability. therefore, the dominant feature of mtdna alteration induced by pathogenic aac alleles is similar in yeast and adpeo patients. . diseases due to defects of mitochondrial carriers involved in intermediary metabolism the diseases of this group known so far are cac deficiency, hhh syndrome, agc deficiency, amish microcephaly and neonatal myoclonic epilepsy. they will be reviewed in the following sections in the order in which they have been associated for the first time to alterations of a specific mitochondrial carrier gene. . . cac deficiency carnitine–acylcarnitine carrier (cac) deficiency (omim ) is the first disorder to have been associated with a member of the slc gene family, slc a [ , ]. this gene, which spans about kb, contains coding exons, maps to chromosome p . and encodes the cac, a protein of amino acids. the rat cac was purified in and years later its cdna was cloned in rat and man [[ , ] and references therein]. cac catalyzes a : electroneutral exchange between a mo- lecule of acylcarnitine, that enters mitochondria, and a molecule of free carnitine, that exits the organelles (see fig. k of ref. [ ]). the cac function is conserved in all eukaryotes, although mammalian cac has a higher affinity for long-chain rather than short-chain acylcarnitines and the lowest affinity for free carnitine, in contrast with the fungal carriers [[ ] and references therein]. in particular, cac is the key component of thecarnitine cyclewhichconsists of three steps: i)transferof acyl groups from acyl-coa to carnitine through carnitine palmitoyltransferase (cpt i), an outer mitochondrial membrane enzyme; ii) cac-mediated transport of acylcarnitines across the inner mitochondrial membrane in exchange for free carnitine; and iii) transfer of acyl groups from acylcarnitines to coa inside the mitochondria through cpt ii, an inner mitochondrial membrane enzyme (see fig. k of ref. [ ]). by catalyzing the carnitine/acylcarnitine exchange, cac allows the import of fatty acyl moieties into the mitochondria where they are oxidized by the β- oxidation pathway. this pathway is the major source of energy for heart and skeletal muscles during fasting and physical exercise. besides the exchange, cac is also able to perform unidirectional transport of substrates across the inner mitochondrial membrane but at a much lower rate (about one tenth that of the exchange); nevertheless, uniport of carnitine into carnitine-depleted mitochondria is physiologically important to balance the matrix level of carnitine with that present in the cytosol, a prerequisite for optimal carnitine/acylcarnitine activity. given that cac-mediated transport is an essential step in the long-chain fattyacid β-oxidationpathway, cac deficiency is a fatty acid β-oxidation disorder that prevents the body from converting long-chain fatty acids into energy. cac deficiency was first described in by stanley et al. [ ]. it is a severe autosomal recessive, nonpopulation-specific disorder pre- senting an equal male-to-female ratio. the most affected organs are heart, liver, brain, and skeletal muscles. the disorder manifests with life-threatening episodes of coma upon fasting (due to hypoglycemia, since the liver is unable to produce ketone bodies from fat during fasting and muscles use glucose), cardiomyopathy, cardiac arrhythmia, muscle weakness and abnormal liver function (all likely due to the accumulation of long-chain fatty acids and acylcarnitines that cannot be oxidized). other symptoms include vomiting, lethargy, hy- potonia, weakness, hepatomegaly, cardiac insufficiency, respiratory distress, and seizures. in addition to hypoglycemia, metabolic alte- rations in blood include hypoketosis (caused by lack of hepatic fatty acid β-oxidation), acidosis, hyperammonemia (possibly due to a compensatory increase in amino acid oxidation), dicarboxylic acid- uria, increased long-chain acylcarnitines, decreased free carnitine, mildly elevated creatine kinase and liver enzyme levels and occa- sional hypocalcemia. when examined in cultured cells, cac and fatty acid β-oxidation activities are markedly reduced or nearly abo- lished, although the other carnitine cycle enzymes, cpt i and cpt ii, and β-oxidation enzymes exhibit normal activity. cac deficiency presents two infantile phenotypes: the first, and most common, with early onset in the neonatal period, and the second milder form with onset in infancy or, less frequently, in childhood. neonates with the early-onset variety deteriorate rapidly after birth; their mortality rate is high, generally displaying a good correlation between the severe phenotype and null genotype. infants whose cac retains some residual activity usually have no cardiac involvement and can respond to treatment with medium-chain triglycerides that do not require carnitine to enter the mitochondria. when both phenotypes are untreated, patients with cac deficiency experience hypoglycemic crises that may lead to brain damage, coma and death, or may die from cardiac arrest. since the first reported mutation in the slc a gene [ ], others have been identified (fig. ). interestingly, some missense mutations (d n, r w, p r and d h; fig. a) regard residues of the signature motifs present in all mitochondrial carriers, and two others (r q and g r; fig. b) concern residues whose equivalents in yeast cac have been proposed to bind the substrate [ ], underscoring the importance of the motifs and of the proposed “common substrate-binding site” [ ] for the structure and function of these proteins. also known as carnitine/acylcarnitine translocase (cact) deficiency. f. palmieri / biochimica et biophysica acta ( ) – author's personal copy early recognition and treatment is crucial in cac-deficient patients. cac deficiency can be identified by newborn screening programs using tandem mass spectrometry, although in severe infantile cases the results of testing might return after the child is already symp- tomatic. management and long-term treatment of the cac patient consist of fasting prevention with frequent meals, a diet rich in carbohydrates, low in lipids (of which about % should be provided as medium-chain triglycerides), and supplemented with essential poly- unsaturated fatty acids. patients should have an emergency protocol in place to assure administration of intravenous glucose in case of fever, vomiting or other illness preventing oral intake. intake of l-carnitine may be helpful, particularly in individuals with severely deficient car- nitine levels. cac deficiency can be differentiated from other disorders of the carnitine cycle and fatty acid β-oxidation in cultured fibroblasts (e.g., medium-chain acyl-coa dehydrogenase deficiency, omim ) by measurement of urine organic acid and plasma acylcarni- tine profiles. diagnosis should be confirmed in vitro by measurements of fatty acid β-oxidation activity, cac activity (in cultured fibroblasts [ ], in reconstituted liposomes [ ] or by complementation in s. cerevisiae [ ] or aspergillus nidulans [ ]) and dna testing. determi- nation of carrier activity is also important to establish that a newly found mutation is disease-causing. . . hhh syndrome another error of metabolism is hhh syndrome (omim ) that is caused by mutations in the slc a gene [ ]. this gene, which maps to band q . , spans about kb and contains coding exons, encodes isoform of the ornithine carrier, named orc . the ornithine carrier was purified from rat liver mitochondria in and termed ornithine/citrulline carrier for its ability to catalyze a highly active ornithine/citrulline exchange and for its importance in the urea cycle [ ]. it was cloned for the first time in s. cerevisiae in [ ]. later, starting from the yeast ortholog, two human isoforms, orc and orc , were identified [ , ]. the two human isoforms were over- expressed and characterized in reconstituted liposomes. both trans- port l-isoforms of ornithine, lysine, arginine, and citrulline by a : substrate exchange or, to a lesser extent, by an exchange of basic amino acids for h+; both are inactivated by spermine and spermi- dine and stimulated by malate and phosphate. however, these iso- forms differ in some respects. firstly, orc has a broader specificity than orc , since it transports l- and d-histidine, l-homoarginine and d-isoforms of ornithine, lysine and arginine with the same efficiency as l-isoforms. secondly, orc has a higher affinity for lysine and arginine and a lower affinity for ornithine and citrulline than does orc . thirdly, orc is expressed at higher levels than orc in all the tissues investigated, particularly in liver, lung, pancreas and testis [ ]. orc plays a number of important roles in cell metabolism. for example, under conditions of low arginine content in the diet and/or in tissues where argininase activity is negligible, ornithine produced intramitochondrially from glutamate must be exported to the cytosol to accomplish polyamine biosynthesis. conversely, when dietary content of arginine is high, the ornithine formed by arginine hydro- lysis in the cytosol must be imported into the mitochondria where it is converted to glutamate and proline by ornithine aminotransferase, an enzyme that is located in the mitochondrial matrix and abundant in liver and kidney. hepatic ornithine aminotransferase is localized in the pericentral hepatocytes that contain glutamine synthetase and not in the periportal hepatocytes containing the urea cycle enzymes [ ]. in periportal hepatocytes, orc carries out the important function of exchanging cytosolic ornithine for mitochondrial citrulline. by provid- ing this function, orc links the enzyme activities of urea synthesis in the cytosol to those in the mitochondria and is therefore an essential component of the urea cycle, as previously proposed (see fig. e of ref. fig. . mutations in patients with cac deficiency. (a) lateral view of the structural-homology model of human cac (in cartoon representation) showing the positions of the amino acid substitutions found to date in cac deficiency. (b) a view of the proposed substrate-binding site from the cytoplasmic side. the carnitine substrate is shown in stick representation. (c) list of type and number of mutations found to date in patients with cac deficiency. all mutations are from the hgmd database (http://www.hgmd.cf.ac.uk/ac/ index.php). the colour code for the transmembrane α-helices is as follows: h , red; h , orange-green; h , light green; h , dark green; h , light blue; and h , blue. purple surfaces highlight the salt bridge network between residues k and d , k and e , and k and d . f. palmieri / biochimica et biophysica acta ( ) – author's personal copy [ ]) [[ ] and references therein]. under physiological conditions, it is highly probable that the key step of the urea cycle consisting in the ornithine/citrulline exchange is catalyzed, mainly or exclusively, by orc , since orc has a lower affinity for ornithine and citrulline and is expressed in liver at a much lower level. deficiency of orc in patients with hhh syndrome supports this interpretation. as indicated by its name, hhh syndrome is characterized by hyperammonemia, hyperornithinemia and homocitrullinuria. these metabolic alterations result from a defect in orc as do many clinical symptoms of the disease. hyperammonemia is due to impairment of the urea cycle at the level of orc . defective orc does not allow import of ornithine from the cytosol to the mitochondria and, con- sequently, impedes the function of intramitochondrial ornithine transcarbamoylase that condenses carbamoyl phosphate and orni- thine to form citrulline. accumulation of ornithine in the cytosol explains hyperornithinemia and leads to an increased production of polyamines. in the absence of ornithine inside the mitochondria, car- bamoyl phosphate accumulates and either condenses with lysine to form homocitrulline, leading to homocitrullinuria, or enters the cytosolic pyrimidine biosynthetic pathway, leading to increased excretion of orotic acid and uracil. other laboratory findings include increased levels of glutamine, alanine, liver transaminases and alka- line phosphatase. hhh syndrome may present at any age from the neonatal phase to adulthood. it usually manifests in early childhood, with % of patients presenting in the neonatal period. the most common symptoms of the disorder are episodes of confusion, lethargy, and coma due to hyper- ammonemia, and neurologic features such as mental retardation, learning difficulties, spastic paraplegia and seizures. interestingly, most patients present with pyramidal dysfunction, often resulting in early adulthood in frank spastic paraplegia, whose course appears to be unrelated to treatment and whose pathogenetic mechanism(s) is still poorly understood [ ]. patients also often present abnormal liver function leading to hepatitis-like attacks and coagulopathy with factor-specific defects (factors vii, ix and x). some symptoms present in an acute way (e.g., intermittent episodes of vomiting, ataxia, lethargy, confusion, coma, myoclonic jerks, and hepatitis-like attacks), whereas others follow a more chronic course (e.g., aversion for protein-rich foods, coagulation abnormalities, hypotonia, develop- mental delay, progressive encephalopathy with mental regression, pyramidal dysfunction). similarly to other urea cycle defects, neonates may manifest symptoms soon after feeding. children and adults have a tendency to refuse high-protein foods. although variable, the phenotype of hhh patients is somewhat milder than that associated with defects of urea cycle enzymes. it may be that orc partially compensates for orc . gain of function polymorphism of orc and a haplogroup (mtdna lineage) have been suggested [ ] as possible factors that may influence the orc deficiency phenotype. because in mouse isoform of orc is a pseudogene (given the presence of a single nucleotide deletion near the ′-end causing a frame shift), it would be interesting to delete the murine orc and compare the resulting phenotype with that of hhh patients and the phenotype of urea cycle enzyme knock-out mice. hhh syndrome is an autosomal recessive disease and has a pan- ethnic distribution with a higher proportion of cases reported in canada (a founder mutation located in quebec), italy, and japan. the male-to-female ratio is approximately : . since its first description in by shih et al. [ ], additional cases have been reported. in fig. , all the naturally occurring mutations in orc known to date are listed, including from dr. dionisi-vici that are yet unpublished. many of the mutations have been functionally analyzed in recon- stituted liposomes and found to inactivate the carrier [ , ]. inter- estingly, two of the disease-causing mutations, r q and e k, concern residues involved in the proposed substrate-binding site, fig. . mutations in patients with hhh syndrome. (a) lateral view of the structural-homology model of human orc (in cartoon representation) showing the positions of the amino acid substitutions found to date in hhh syndrome. the transmembrane α-helices are coloured as reported in fig. . purple surfaces highlight the salt bridge network between residues k and d , k and e , and k and d . (b) a view of the proposed substrate-binding site from the cytoplasmic side. the ornithine substrate is shown in stick representation. (c) list of type and number of mutations found thus far in patients with hhh syndrome. seventeen mutations were taken from the hgmd database (http://www. hgmd.cf.ac.uk/ac/index.php); the remainder was obtained through the courtesy of dr. dionisi-vici. f. palmieri / biochimica et biophysica acta ( ) – author's personal copy whereas t r and p r residues belonging to the signature motifs of the mitochondrial carrier family (fig. ). furthermore, of the missense mutations affect residues protruding toward the cavity occupied by the entering substrate in the cytosolic conformation of the carrier (fig. ). early diagnosis and treatment of hhh syndrome are important to prevent neurologic and cognitive deterioration. however, diagnosis is often delayed as a result of non-specific symptoms. differential diag- nosis is based mainly on biochemical characteristics: persistently elevated plasma ornithine levels to times higher than normal, which distinguishes the disorder from other urea cycle defects; the presence of homocitrulline and orotic acid in urine; and episodic or postprandial hyperammonemia, differentiating hhh syndrome from gyrate atrophy (omim ) which is caused by ornithine trans- aminase deficiency. measurement of carrier activity in cultured fibro- blasts [ ] and in reconstituted liposomes followed by genetic testing constitutes evidence of the disease. treatment of hhh syndrome is similar to that of other urea cycle defects and is based mainly on a low-protein diet. it is usually accompanied by supplementation of citrulline and ornithine (to reduce ammonemia), although the long-term side effects of hyper- ornithinemia are not known. arginine, sodium benzoate and sodium phenylbutyrate have also been administered to decrease ammonia levels. treated patients exhibit good metabolic control and very infrequent relapses of hyperammonemia. the mortality rate for the disease is low. . . agc deficiency aspartate/glutamate carrier isoform (agc ) deficiency is a highly widespread autosomal recessive disease in the far east and southeast asia with a carrier prevalence of about in individuals and a male- to-female ratio of . : [ ]. agc deficiency was also detected in the middle east, including israel. more recently, some cases have been found in the us and the united kingdom, pointing towards a pan- ethnic distribution. agc deficiency has been extensively studied by dr. saheki in japan who identified slc a as the gene responsible for the disease by means of homozygosity mapping and positional cloning [ ]. the slc a gene, spanning about kb, maps to chromosome q . and consists of exons. subsequently, it was found that the gene product of slc a , previously termed citrin, and its closely related human homolog, aralar, function as ca +- regulated aspartate/glutamate carriers, abbreviated as agc and agc , respectively [ ]. agc (encoded by slc a on chromosome q ) is highly expressed in brain, heart, and skeletal muscle, whereas agc is found in several tissues but most abundantly in liver where agc is absent [ , ]. these proteins are localized in the inner mitochondrial membrane, bind ca +, and consist of two domains: the c-terminal domain, containing all the sequence features of the mito- chondrial carrier family, and the n-terminal domain, a long extension containing four ef-hand ca +-binding motifs and protruding outside the inner mitochondrial membrane. in reconstituted liposomes, agc and agc transport aspartate and glutamate by an obligatory : exchange. the exchange is electrophoretic because aspartate is tran- sported as an anion, whereas glutamate is transported with a proton. consequently, in energized mitochondria with a positive membrane potential outside, exit of aspartate and entry of glutamate are strongly favoured and therefore the agc-catalyzed reaction is essentially unidirectional and irreversible. the km values of both isoforms for external aspartate and glutamate are about . and . mm, re- spectively, do not change with membrane potential in reconstituted liposomes, and are virtually identical to those determined with native agc. the c-terminal domains of agc and agc account for the activities and all transport properties of the entire two proteins and constitute their catalytic portion, whereas the n-terminal domains, containing the ca +-binding sites, make up their regulatory portion. in fact, the activity of agc and agc is stimulated by ca + on the external side of the inner mitochondrial membrane, since this stimulation is not affected by ruthenium red, a specific inhibitor of ca + entry into mitochondria [ ]. furthermore, by using mitochond- rially targeted luciferase to monitor atp formation, a surplus production of atp was observed in the mitochondria of agonist- stimulated cells overexpressing agc or agc but not in stimulated cells overexpressing the c-terminal domains lacking the ca +-binding sites. notably, the intramitochondrial ca + level was the same in cells overexpressing either the entire protein (agc or agc ) or each of the two c-terminal domains alone [ ]. therefore, cytosolic ca + activates mitochondrial oxidative metabolism through its binding to the n- terminal domain of agc. the major function of agc is to supply aspartate to the cytosol. this is particularly important in hepatocytes that have a negligible capacity of taking up aspartate from the blood. in hepatocytes, agc plays a fundamental role in urea synthesis by supplying aspartate to argi- ninosuccinate synthetase (ass), a urea cycle enzyme that is localized in the cytosol and condenses citrulline and aspartate to produce argininosuccinic acid. the reactions of the urea cycle, their subcellular localization and site of aspartate involvement are shown in fig. e of ref. [ ]. besides urea synthesis, cytosolic aspartate is necessary for gluconeogenesis from reduced substrates and for protein, purine and pyrimidine synthesis. cytosolic aspartate is also very important for the oxidation of cytosolic nadh+h+, since agc is a key component of the malate–aspartate shuttle that transfers the reducing equivalents of nadh+h+ from cytosol into mitochondria (see fig. h of ref. [ ]). in this shuttle, agc again provides the cytosol with aspartate; in the cytosol, aspartate is transaminated to oxaloacetate which is reduced by cytosolic malate dehydrogenase to malate, leading to the oxidation of cytosolic nadh+h+. malate enters the mitochondria via the oxoglutarate carrier and in the matrix is reduced to oxaloacetate by mitochondrial malate dehydrogenase, resulting in the reduction of intramitochondrial nad+. subsequently, oxaloacetate is transami- nated to aspartate by mitochondrial aspartate aminotransferase, com- pleting the cycle. it should be emphasized that the malate–aspartate shuttle operates in the direction of transferring reducing equivalents from cytosol to mitochondria because the reaction catalyzed by agc is unidirectional and irreversible. by oxidizing cytosolic nadh, the malate–aspartate cycle allows aerobic glycolysis and alcohol metabo- lism to occur. it is important to note that the glycerol- -phosphate shuttle, which is the main physiological alternative mechanism for the oxidation of cytosolic nadh, exerts low activity in human liver (whereas it is very active in murine liver). because agc is the only isoform, or at least the most prevalent isoform, of this carrier expressed in liver, lack of its function in this tissue explains many symptoms of the disease. agc deficiency causes two age-dependent phenotypes: neonatal intrahepatic cholestasis caused by citrin (agc ) deficiency (niccd, omim ) and adult- onset type ii citrullinemia (ctln , omim ). type ii citrulline- mia usually manifests in adults between the ages of and years. a shortage of aspartate in the cytosol of hepatocytes causes citrulline- mia, hypoproteinemia, and recurrent episodes of hyperammonemia responsible for the observed encephalopathy and neuropsychiatric symptoms (e.g., sudden aberrant behavior, disturbance of conscious- ness, disorientation, delirium, tremor, convulsive seizures and coma). the inhibition of cytosolic nadh oxidation, with the consequent increase in the nadh/nad+ ratio, causes a distaste for carbohydrates, a preference for fat and especially for proteins as well as an incapacity to consume alcohol. in fact, in patient dietary surveys carbohydrates average only ~ % of total caloric intake, whereas proteins average ~ %. in particular, a strong desire for beans and nuts, which are the activity, which is based on measurement of the incorporation of [ c]ornithine, as compared to that of [ h]leucine, into cellular protein, is also markedly decreased or null in patients with gyrate atrophy. f. palmieri / biochimica et biophysica acta ( ) – author's personal copy particularly rich in aspartate and asparagine, has been described. citrullinemia and hyperammonemia are also due to a unique feature of the disease, i.e., a variable liver-specific reduction in ass protein and activity with no alterations in either its gene or hepatic ass mrna levels [ ]. conversely, in type i citrullinemia (ctln , omim ) the gene for ass is mutated, leading to ass deficiency in all tissues [ ]. it is not yet clear why the level of hepatic ass is low in ctln . it may be that critical aspartate levels are required to make ass active or to stabilize the enzyme. patients also manifest fatty liver most likely because they oxidize cytosolic nadh+h+ by yet another pathway, the citrate–malate shuttle (see fig. d of ref. [ ]), whose normal function is to synthesize fatty acids by transferring acetyl-coenzyme a from the mitochondrial matrix to the cytosol. in fact, the key enzyme of the citrate–malate shuttle, the citrate carrier, exports citrate from mitochondria to the cytosol where it is cleaved to acetyl-coenzyme a and oxaloacetate by atp-citrate lyase. furthermore, some patients develop hyperlipidemia which likely results from the functioning of the citrate–malate shuttle and from the accumulation of glycerol- - phosphate due to poor activity of the glycerol- -phosphate cycle in human liver. other laboratory findings are increased arginine and pancreatic secretory trypsin inhibitor levels, an elevated threonine-to- serine ratio, and a decreased ratio between branched-chain amino acids (bcaas) to aromatic amino acids (aaas) in patient sera. finally, pancreatitis and hepatocellular carcinoma occur with a higher incidence in ctln patients. adult-onset type ii citrullinemia is pro- gressive and patients usually die from hyperammonemic encephalo- pathy and complications of brain edema. given the poor prognosis of the adult agc -deficiency phenotype, patients should consider undergoing liver transplantation. the neonatal phenotype, niccd, was recognized more recently by saheki and co-workers [ ]. affected children commonly present transient intrahepatic cholestasis, fatty liver, hepatomegaly, growth retardation, citrullinemia, aminoacidemias (elevated methionine, threonine, tyrosine, phenylalanine, lysine and arginine), an increased threonine-to-serine ratio and galactose concentration, ketotic hypo- glycemia, and hypoproteinemia. some patients present with hepatitis, jaundice, decreased coagulation factors, hemolytic anemia and bleed- ing diathesis as a result of liver dysfunction and hypoproteinemia. high levels of plasma α-fetoprotein have been detected in niccd but not in ctln . the neonatal phenotype of agc deficiency is usually benign. symptoms disappear by the age of months; afterwards, these patients become seemingly healthy (or manifest non-specific symp- toms) but display a preference for protein- and lipid-rich foods and an aversion for foods high in carbohydrate content. one or several decades later, some agc -deficient individuals go on to develop ctln . the clinical spectrum of agc deficiency has recently been expanded with the report of infantile presentation characterized by failure to thrive and bleeding diathesis and no evidence of neonatal intrahepatic cholestasis [ ]. in the compensatory period (which corresponds to the transition from niccd to the onset of ctln ), affected individuals live with metabolic defects that mainly involve liver, i.e., a high cytosolic nadh/ nad+ ratio and impairment of urea synthesis. it is likely that in the agc -deficient liver, partial oxidation of cytosolic nadh is accom- plished by the low activity of the glycerol- -phosphate nadh shuttle and by the citrate–malate cycle, as mentioned above. given that in patient liver the export of aspartate from the mitochondria is nearly null and aspartate is virtually not taken up by the blood, ureagenesis is dependent on aspartate production in the cytosol. aspartate can be produced in the cytosol in two ways: from asparagine by the action of asparaginase and from oxaloacetate by cytosolic aspartate amino- transferase. asparagine can be synthesized by asparagine synthetase or can be supplied from the diet via the portal venous blood. oxa- loacetate can be formed from fumarate, a product of the urea cycle enzyme argininosuccinate lyase, by the successive actions of cytosolic fumarase and malate dehydrogenase. in both cases serious draw- backs emerge, i.e., the production of mol of ammonia per each mol of aspartate provided by asparagine and the generation of mol of nadh+h+ per each mol of aspartate provided by oxaloacetate origi- nating from malate (see fig. b of ref. [ ]). therefore, in the agc - deficient patient, the deficit of ureagenesis is due not only to a decreased availability of aspartate to ass but also to an increased cytosolic nadh/nad+ ratio as a consequence of the defective malate– aspartate nadh shuttle and of aspartate production from fumarate, as mentioned above. slc a is the only gene known to be associated with agc deficiency. the first mutations observed in slc a led to either truncation of the protein or extensive deletions causing agc de- ficiency [ ]. more recently, missense mutations were also detected. until now, no significant correlation has been observed between slc a mutation types and the age of ctln onset. forty-four mutations have been found thus far in patients with agc deficiency, of which have not yet been published (t. saheki, personal communication). all together, missense mutations have been found. the newly identified mutation r q that inhibits transport of glutamate and aspartate in reconstituted liposomes by about % [ ] regards a residue belonging to the proposed common substrate-binding site formed by r , r , e and k — residues equivalent to those proposed to bind the substrate in yeast agc (r , r , e and k ) [ ]. r corresponds to r of the bovine oxoglutarate carrier, to r of theyeast aac , and to r of the murine ucp which also do not tolerate replacement with other amino acids [[ ] and references therein]. r also corresponds to r of the human orc ; its substitution with q causes hhh syndrome (see . ). to examine the consequences of the r q mutation, docking of glutamate was performed using the residues of the common substrate- bindingsite of the human agc [ ]. docking with thehumanwild-type agc shows the substrate directly on top of the salt bridge network interacting with r and the charged residues k and k of the network (fig. a). in the case of the r q mutant, the substrate is positioned far from the salt bridge network at the level of the binding site (fig. b). therefore, lack of r in position impedes the interaction of the substrate with the salt bridges causing loss of activity. it is tempting to speculate that residue r functions as a mobile side-chain that transports the substrate onto the salt bridges, determining their aperture and the substrate's progression through the protein toward the matrix. the pathophysiology of agc deficiency was recently investigated by elegant studies conducted on knock-out mice [ ]. slc a −/− mice exhibited a marked decrease in mitochondrial aspartate tran- sport and malate–aspartate shuttle activities in vitro. in liver perfused with ammonia, the rate of urea production was drastically reduced and the lactate-to-pyruvate ratio, which reflects the cytosolic nadh/ nad+ redox state, was increased. under these conditions, both the deficit in ureagenesis and increase in the nadh/nad+ ratio were partially corrected by the administration of asparagine that enters hepatocytes and is hydrolyzed to aspartate. likewise, pyruvate (but not aspartate or citrate) reduces the deficit in ureagenesis and decreases the nadh/nad+ ratio in the liver-perfused system. further- more, in liver perfused with lactate (but not pyruvate) gluconeogen- esis was diminished. these results demonstrate that hepatic cytosolic aspartate is rate-limiting in slc a −/− mice. however, agc knock- out mice did not manifest the key symptoms of agc deficiency present in man. the mice exhibited neither hyperammonemia and changes in amino acid levels even following administration of protein- rich diets nor hypoglycemia upon fasting. the most likely explanation for the lack of ctln -like phenotype in the agc knock-out mice is the high activity of the glycerol- - phosphate cycle in their liver. consequently, saheki et al. generated mice with a combined disruption of the agc gene and the gene for mitochondrial glycerol- -phosphate dehydrogenase, a key compo- nent of the glycerol phosphate cycle [ ]. double knock-out mice f. palmieri / biochimica et biophysica acta ( ) – author's personal copy investigated after days of age manifested: (i) a reduction in growth rate, which is consistent with the observation that usually ctln patients are thin and niccd patients exhibit growth retardation; (ii) hyperammonemia and increased lactate-to-pyruvate ratio under fed conditions, which were further increased after sucrose administra- tion; (iii) elevated plasma citrulline levels and threonine/serine ratio under fed and fasted conditions (not altered by sucrose administra- tion); (iv) decreased plasma bcaa/aaa ratio and alanine level after sucrose administration (both were normal under fed or fasted conditions); (v) hypoglycemia upon fasting due to the increased cytosolic nadh/nad+ ratio leading to decreased gluconeogenesis from reduced substrates and to the lowered oxaloacetate level re- quired by phosphoenolpyruvate carboxykinase; and (vi) fatty liver, elevated levels of plasma free fatty acids and glycerol; the latter being due to loss of the glycerol- -phosphate shuttle activity in all the tissues and to loss of the malate–aspartate shuttle in liver. finally, the hepatic aspartate content was lower in the double knock-out mice compared to wild-type, glycerol- -phosphate shuttle and agc single knock-out mice [ ]. therefore, the double knock-out mice exhibited most of the features of the human agc -deficient phenotype. agc deficiency should be suspected in children with hypoglyce- mia, hepatomegaly, growth retardation or failure to thrive. in addition, agc deficiency should also be considered in children presenting with elevated blood galactose levels. it is important to confirm the diagnosis of agc deficiency, as treatment of these patients highly contrasts that of urea cycle disorders. individuals with urea cycle enzyme defects are given protein-restricted, high-caloric diets to prevent episodes of hyperammonemia. in contrast, ctln -affected patients manifest severe episodes of hyperammonemia upon consumption of high- carbohydrate diets. likewise, severe episodes of hyperammonemia have been observed after administration of glycerol or fructose for the treatment of hyperammonemia and brain edema [ ]. alcohol intake and the use of anti-inflammatory and analgesic drugs and/or surgery may often provoke ctln symptoms. at present, liver transplantation is the only effective treatment for type ii citrullinemia. to prevent hyperammonemia (and resolve growth retardation in children), a diet high in protein and lipid content and low in carbohydrates is highly recommended. this recommendation is further supported by the observed food preference of these patients. treatment with pyruvate has also been suggested to decrease the cytosolic nadh/nad+ ratio [ ]. moreover, in children with niccd, supplementation with fat- soluble vitamins and use of lactose-free formula or high protein and low carbohydrate diet have also been applied [ ]. in summary, treatment of niccd requires continued dietary control and growth monitoring. continuous monitoring of these patients is necessary to prevent a severe outcome later in life. . . amish microcephaly (mcpha) amish microcephaly (mcpha, omim ) is an inborn error of metabolism presenting severe microcephaly and -oxoglutaric acid- uria [ ]. the disorder is caused bya defect in the slc a gene which encodes a protein of residues [ ]. the protein was first identified as a deoxynucleotide carrier (dnc) [ ] and later as a thiamine pyro- phosphate carrier (tpc) [ ]. in reconstituted liposomes, tpc trans- ports the important coenzyme thiamine pyrophosphate (thpp), thiamine monophosphate (thmp) and deoxynucleotides in descend- ing order of potency, dndpndntpndnmp, by an obligatory exchange mechanism. nucleotides are also transported, though less efficiently than the corresponding deoxynucleotides. furthermore, the dideox- ynucleoside triphosphates that are produced in the cytosol from dideoxynucleosides, including antiviral and anticancer nucleoside analogs, are equally efficient substrates as dndps [ ]. to date, amish microcephaly has only been observed in the old order amish community in pennsylvania, u.s.a. with a high pre- valence of about in individuals. in this community, nuclear families affected with mcpha are connected to a single ancestral couple that lived in the s. the disease is characterized by severe fig. . structural-homology model of human agc illustrating the role of r in substrate translocation through the carrier. docking of glutamate in the r wild-type agc (a) and the q mutated agc (b) viewed from the lateral side. glutamate is shown in the van der waals representation and coloured in yellow. stick representations highlight some residues that are located within Å from the substrate. rectangular boxes contain residues r (a) and q (b). portions of helices iv, v and vi (in a and b) and of the salt bridge network (in a) were rendered transparent to facilitate viewing of the substrate and side-chains of some amino acids. the transmembrane α-helices are coloured as reported in fig. . purple surfaces highlight the salt bridge network between residues k and d , k and e , and k and d . f. palmieri / biochimica et biophysica acta ( ) – author's personal copy congenital microcephaly, elevated levels of α-ketoglutarate in urine, premature death (observed maximum survival, months) and, as a result of microcephaly, nearly absent cranial convexity, frontal sloping and distorted facial features. the only non-cns physical anomaly is moderate micrognathia. patients manifest no orientation to sight or sound and no fine or gross motor development, have metabolic acidosis enhanced by episodic viral illnesses, in some cases mild hepatomegaly, difficulty maintaining normal body temperature and develop increasing irritability. it was first considered that the increased levels of α-ketoglutarate might be the result of a defect in the α-ketoglutarate dehydrogenase complex which comprises three enzymes: -oxoglutarate dehydro- genase (omim ), dihydrolipoamide succinyltransferase (omim ) and dihydrolipoamide dehydrogenase (omim ). the genes encoding these enzymes were excluded on the basis of genetic linkage and haplotype analysis, indicating that this disorder must be associated with another genetic locus. through a whole-genome scan, fine mapping and haplotype ana- lysis, the gene affected in mcpha was localized to a region of mb, on chromosome q [ , ]. in this region, the genes encoding proteins with known mitochondrial functions were analyzed in view of the oxoglutarate abnormality in mcpha, which suggested mitochondrial dysfunction. a homozygous nucleotide change in the coding region of tpc, c. g→c (nm_ ), was identified in affected children. the parents of individuals affected by mcpha are obligate heterozygotes. the mutation, which produces a glycine-to-alanine substitution at position , alters a highly conserved residue in the mitochondrial carrier family that is located in the second part of the signature motif between helices h and h , in proximity to one of the important salt bridges that close the substrate translocation path in the cytosolic conformation of the carrier (fig. ). the substitution co-segregates with the disease in the families investigated [ ] and is deleterious for protein function. indeed, in reconstituted liposomes the transport activity of thpp, thmp or datp mediated by the g a mutant tpc was % to % that mediated by the wild-type tpc [ ]. in addition, substitutions of g with bulkier residues such as valine, cysteine and leucine are not tolerated at all (f. palmieri, unpublished data), as was also found for the equivalent glycine in other members of the mito- chondrial carrier family [[ ] and references therein]. it may be hy- pothesized that g functions as a hinge between helices h and h during the conformational changes that occur in the catalytic cycle of the carrier. slc a is the only gene known to be associated with amish microcephaly. to elucidate the pathogenic mechanism of this disease, a knock-out mouse for the slc a gene was generated, and the phenotype of both the knock-out mice and patients was studied at the cellular level [ ]. all slc a −/− mice died prior to . days' gestation. the homo- zygous embryos at e . exhibited an abnormally developed cns with a neural-tube closure defect and exencephaly that commonly fig. . structural-homology model of human tpc showing the position of the mutation g a responsible for amish microcephaly. cartoon representations of the g wild-type tpc (a) and the a mutated tpc (b) viewed from the cytoplasmic side. the residues g and a are in surf representation and coloured in yellow. the transmembrane α-helices are coloured as reported in fig. . purple surfaces highlight the salt bridge network between residues k and d , r and d , and k and d . cartoon and stick representations showing the proximity of g (c) and a (d) to the salt bridge between k and d . g and a are in surf representation and coloured in yellow. in c and d, the signature motifs (p-x-d/e-x-x-k/r) of h , h and h are coloured blue, green and orange, respectively, with the side-chains in stick representation. the salt bridges are indicated by black dotted lines. f. palmieri / biochimica et biophysica acta ( ) – author's personal copy involves the entire brain. in addition, they manifested erythropoietic failure, being nearly devoid of erythrocytes at e . , and elevated oxoglutarate levels in the amniotic fluid. in the mitochondria of knock-out mice fibroblasts and patient lymphoblasts, no differences in ribonucleoside- and deoxynucleoside- triphosphate levels, aswell as nodepletion or deletions in mitochondrial dna, were found in comparison to controls. these findings demonstrate that although slc a may be able to transport dndps and dntps in vivo, this function is not critical, owing perhaps to a redundancy of deoxynucleotide carriers in mammalian mitochondria. in agreement with the other function of tpc (i.e., to import thpp produced in the cytosol into the mitochondria in exchange for intra- mitochondrially generated thmp), it was found that thpp and thmp were not detectable in the mitochondria of knock-out mice fibroblasts and were markedly decreased in the mitochondria of patient lym- phoblasts as compared to controls. (of note, thpp and thmp were increased in the cytosol.) secondly, in the media of both cell lines lactate was markedly increased, indicating mitochondrial dysfunction. lastly, the mitochondrial thpp-dependent ketoglutarate dehydrogen- ase (kgdh) and pyruvate dehydrogenase (pdh) complex activities were nearly absent in knock-out mice mitochondrial extracts and strongly diminished in patient mitochondrial extracts as compared to controls. interestingly, the addition of thpp to the kgdh and pdh assay mixtures restored full activity, showing that the lower activities of kgdh and pdh in knock-out mice and patients is caused by lack of thpp in mitochondria [ ]. taken together, these data suggest that transport of thpp is the primary physiological function of tpc. table summarizes and compares the phenotypes of the slc a knock-out mice and patients with amish microcephaly. it can be observed that all the metabolic abnormalities of the human disease, starting from the decrease in thpp level in the mitochondria, were also present in the knock-out mice. however, these abnormalities, which are caused by lack of thpp in the mitochondria, were more severe in the knock-out mice. the only exception concerns increased oxoglu- tarate levels in amniotic fluid which were found to be less dramatic than those in the urine of patients. this is due to the fact that mouse embryos died prior to kidney formation and therefore did not concentrate the metabolite. the increased severity of the mouse model is also illustrated by the embryonic lethality of the slc a knock-out animals, by the arrest of brain formation at the level of the neural tube closure, probably caused by decreased fluxes through kgdh and pdh reactions, and by erythropoietic failure perhaps caused by lack of succinyl-coa, a product of kgdh. the most likely explanation for the differences in the mouse and human phenotypes is that human point mutation retains some thpp transport activity whereas the murine null mutation does not. in conclusion, amish microcephaly is due to a defect of thpp transport into mitochondria, which primarily affects brain development because of the brain's great need for oxidative metabolism. metabolic screening and eventual imaging studies are warranted for all children with congenital microcephaly. no intervention or vitamin therapy has proven to be effective for treating and/or im- proving the disorder. only supportive therapy is available for mcpha- table phenotypes of the knock-out mice and patients with amish microcephaly knock-out mice patients tpc activity absent reduced to – % lethality embryonic – months cns defects arrest of brain formation (open neural tube) microcephaly mitochondrial thpp and thmpa not detectable decreased cytosolic thpp and thmpa highly increased increased kgdh and pdh activitiesa highly decreased decreased kga increasedb highly increased lactatea highly increased increased mtdna abnormalitiesa absent absent mitochondrial (d)ntp levelsa normal normal a data obtained from slc a −/− mouse fibroblasts and patient lymphoblasts. b in amniotic fluid. fig. . partial sequence alignment of the glutamate and aspartate/glutamate carriers in man and other species displaying high conservation of p and g of the human glutamate carrier isoform . abbreviations: hs, homo sapiens; mm, mus musculus; bt, bos taurus; rn, rattus norvegicus; xt, xenopus tropicalis; dr, danio rerio; gg, gallus gallus; dm, drosophila melanogaster; ag, anopheles gambiae; nv, nasonia vitripennis; sc, saccharomyces cerevisiae; nf, neosartorya fischeri; af, aspergillus fumigatus; nc, neurospora crassa; dd, dictyostelium discoideum. f. palmieri / biochimica et biophysica acta ( ) – author's personal copy affected patients. phenobarbital has been administered for the treat- ment of seizures, and physical therapy is encouraged for alleviating contractures or other secondary neurologic manifestations. genetic risk of mcpha is best determined in the pre-gestational phase. . . neonatal myoclonic epilepsy mutations in the slc a gene encoding isoform of the gluta- mate carrier (gc ) cause a form of early myoclonic epilepsy (eme), named neonatal myoclonic epilepsy (omim ) [ ]. the slc a gene maps to chromosome p . and contains coding exons. gc and its isoform gc , which is encoded by slc a , were identified upon gene expression in e. coli as glutamate carriers by their transport properties in reconstituted liposomes [ ]. both iso- forms are located in the mitochondrial membrane (m. lasorsa and f. palmieri, unpublished data) and catalyze a glutamate/h+ symport with high specificity. however, gc has a high km for glutamate (∼ mm), whereas the km value of gc is low (∼ . mm) and the vmax value of gc is higher than that of gc . furthermore, isoform is expressed at higher levels than isoform in all the tissues investigated, particularly in liver, pancreas and brain. the differences in expression levels and kinetic parameters of the two isoforms suggest that isoform matches the basic requirement of all tissues, especially with respect to amino acid degradation, and that isoform becomes operative to accom- modate higher demands associated with specific metabolic functions. because glutamate is co-transported with an h+ by the gc, and therefore its distribution across the mitochondrial membrane is dependent on Δph, entry of glutamate is favoured in energized mi- tochondria. however, when glutamate is generated intramitochond- rially (e.g., by proline oxidation), the gc may operate in the reverse direction to limit intramitochondrial accumulation of glutamate. the first gc mutation, a substitution of proline with leucine, was found in a consanguineous arab muslim family with affected children among [ ]. a second mutation (a change from glycine to tryptophan) was identified in a consanguineous family from algeria with affected child among (l. colleaux and f. palmieri, unpublished data). all affected individuals were homozygous for the above- mentioned mutations, whereas the parents were heterozygous. the symptoms of the affected children in both families were similar: very-early-onset intractable myoclonic seizures, hypotonia, progressive microcephaly, abnormal visual nerve conduction, and rapid evolution into encephalopathy and spasticity. concerning labo- ratory features, neonatal myoclonic epilepsy is characterized by a typical eeg pattern with suppression burst and by an abnormal visual- evoked potential with low amplitude signal and slow response. both the p l and g w substitutions, which are located in h and h two and three helix turns from the cytosolic membrane side, respectively, alter highly conserved residues in the glutamate and aspartate/glutamate carriers found in man and other species (fig. ) and abolish glutamate transport in reconstituted liposomes. in digitonin-permeabilized fibroblasts from patients, glutamate oxida- tion was also strongly defective [[ ] and unpublished data]. these results have provided the first evidence that, despite normal oxidative phosphorylation, impaired mitochondrial glutamate import/metabo- lism leads to an alteration of neuronal excitability. although the pathogenesis of neonatal myoclonic epilepsy remains an open question, the following information should be stressed: i) during fetal development, gc is expressed first in brain, specifically within territories that contribute to the genesis and control of myo- clonic seizures [ ]; ii) gc expression is much greater in astrocytes than in neurons [ ]; iii) astrocyte mitochondria do not contain the aspartate/glutamate carrier [ ] and thus take up glutamate only via the glutamate carrier; iv) glutamate is a major excitatory neurotrans- mitter; and v) after its release into the synapse glutamate is taken up by astrocytes. it may be that gc -dependent epilepsy is due to a defect of mitochondrial glutamate transport in astrocytes, which would consequently lead to an increase in intrasynaptic glutamate concentration. there is no effective treatment for neonatal myoclonic epilepsy; affected children die within to years after birth or survive in a vegetative state. some severe cases of the disorder may be confused with febrile seizures, which are of shortened duration and recur less frequently. it is also important to distinguish neonatal myoclonic epilepsy from benign neonatal sleep myoclonus. the latter condition is seen in healthy infants, occurs only in sleep, and eeg is normal. aside from metabolic errors, eme syndromes may be caused by perinatal insults and brain malformations. a lumbar puncture may help to rule out neurodegenerative diseases. . conclusions since , when carnitine–acylcarnitine carrier deficiency was associated to the nuclear slc a gene, the number of nuclear gene defects impairing the function of mitochondrial metabolite transpor- ters has been rapidly increasing. to date, eight mitochondrial carrier- related diseases have been well characterized biochemically and genetically. this new group of diseases contributes to the current expansion of the field of mitochondrial disorders which were first thought to be limited to those related to respiratory chain dysfunction. in the last decade, the number of mitochondrial diseases caused by defects of the nuclear-coded components of the mitochondrial proteome has been growing at a booming rate to justify what is now known as “mitochondrial medicine”. despite the substantial progress that has been made in our understanding of the molecular bases of mitochondrial carrier-asso- ciated diseases, etiologic therapy is not yet available. current therapy is symptomatic and also addresses disease complications. however, some mitochondrial carrier-associated diseases, like other metabolic disor- ders, benefit enormously from appropriate dietary measures. effective disease management should include wider diffusion of newborn screening and close collaboration among physicians, geneticists and metabolic disease experts in specialized centers. preventive therapy through genetic counseling and prenatal diagnosis is essential and already available. as we wait for gene therapy to become available for practical application, further understanding of pathogenetic mechan- isms through a multidisciplinary approach combining in vitro assays with in vivo studies on patients, cellular and animal models will help to develop new therapeutic strategies that may prove useful to this group of metabolic disorders. further studies are likely to reveal additional mitochondrial carrier-related diseases (particularly in view of the fact that the function of about human genes of the slc family has yet to be identified) and provide new insight into this exciting field. acknowledgments this work was supported by grants from ministero dell'università e della ricerca (firb and prin), ministero della salute, apulia region, center of excellence in genomics (cegba) and european community contract lshm-ct- - . references [ ] i.j. holt, a.e. harding, j.a. morgan-hughes, deletions of muscle mitochondrial dna in patients with mitochondrial myopathies, nature ( ) – . 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find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ [pdf] cerebral haemodynamics in patients with glutaryl-coenzyme a dehydrogenase deficiency. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /brain/awp corpus id: cerebral haemodynamics in patients with glutaryl-coenzyme a dehydrogenase deficiency. @article{strauss cerebralhi, title={cerebral haemodynamics in patients with glutaryl-coenzyme a dehydrogenase deficiency.}, author={k. strauss and p. donnelly and m. wintermark}, journal={brain : a journal of neurology}, year={ }, volume={ pt }, pages={ - } } 	k. strauss, p. donnelly, m. wintermark 	published 	medicine 	brain : a journal of neurology in glutaric aciduria type , glutaryl-coenzyme a and its derivatives are produced from intracerebral lysine and entrapped at high concentrations within the brain, where they interfere with energy metabolism. biochemical toxicity is thought to trigger stroke-like striatal degeneration in susceptible children under years of age. here, we explore vascular derangements that might also contribute to brain damage. we studied injured and non-injured amish glutaric aciduria type patients using… expand view on pubmed academic.oup.com save to library create alert cite launch research feed share this paper citationshighly influential citations background citations results citations view all figures, tables, and topics from this paper 	 table 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure view all figures & tables 	coenzymes 	necrosis 	adverse reaction to drug 	glutaric aciduria, type 	cerebral blood volume 	retinal hemorrhage 	cerebrovascular circulation 	brain injuries 	coenzyme a 	x-ray computed tomography 	ultrasonography 	glutaryl-coa dehydrogenase 	cerebrovascular accident 	structure of middle cerebral artery 	white matter 	structure of basilar artery 	gray matter 	homeostasis 	blood capillaries 	structure of cerebral artery 	gastritis, atrophic 	edema 	lysine 	infant, newborn 	acetylsalicylic acid lysinate citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency impairment of astrocytic glutaminolysis in glutaric aciduria type i 	shoko komatsuzaki, raga deepthi ediga, j. okun, s. kölker, s. sauer 	chemistry, medicine 	journal of inherited metabolic disease 	 	 	view excerpts, cites background save alert research feed glutaric aciduria type i: a translational approach to an enigmatic disease 	s. boy, silvana opp, j. heringer, j. okun, s. sauer, s. kölker 	medicine 	 	 	view excerpt, cites background save alert research feed disruption of brain redox homeostasis in glutaryl-coa dehydrogenase deficient mice treated with high dietary lysine supplementation. 	b. seminotti, a. u. amaral, + authors m. wajner 	biology, medicine 	molecular genetics and metabolism 	 	 save alert research feed arachnoid cysts in glutaric aciduria type i (ga-i) 	n. boy, s. kölker 	biology 	 save alert research feed mechanism of metabolic stroke and spontaneous cerebral hemorrhage in glutaric aciduria type i 	w. zinnanti, j. lazovic, + authors l. steinman 	medicine 	acta neuropathologica communications 	 	 	 	view excerpts, cites results and background save alert research feed mouse model of encephalopathy and novel treatment strategies with substrate competition in glutaric aciduria type i. 	w. zinnanti, j. lazovic 	biology, medicine 	molecular genetics and metabolism 	 	 	view excerpt, cites background save alert research feed unravelling the complex mri pattern in glutaric aciduria type i using statistical models—a cohort study in patients 	s. garbade, c. greenberg, + authors s. kölker 	medicine 	journal of inherited metabolic disease 	 	 	view excerpts, cites background save alert research feed glutaric acidemia type : treatment and outcome of patients over three decades. 	k. strauss, katie b williams, + authors d. morton 	medicine 	molecular genetics and metabolism 	 	 	 	view excerpts, cites background save alert research feed electroencephalography and transcranial doppler ultrasonography in neonatal citrullinemia. 	p. su, j. chen, y. chen, dau-ming niu, ju-hui hsu, inn-chi lee 	medicine 	journal of the formosan medical association = taiwan yi zhi 	 	 	 save alert research feed current concepts in organic acidurias: understanding intra- and extracerebral disease manifestation 	s. kölker, p. burgard, s. sauer, j. okun 	biology, medicine 	journal of inherited metabolic disease 	 	 	view excerpts, cites background save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency multimodal imaging of striatal degeneration in amish patients with glutaryl-coa dehydrogenase deficiency. 	k. strauss, j. lazovic, m. wintermark, d. morton 	medicine 	brain : a journal of neurology 	 	 	pdf 	view excerpts, references background and methods save alert research feed excitotoxicity and bioenergetics in glutaryl-coa dehydrogenase deficiency 	s. kölker, d. koeller, + authors j. okun 	biology, medicine 	journal of inherited metabolic disease 	 	 	view excerpts, references background save alert research feed dynamic changes of striatal and extrastriatal abnormalities in glutaric aciduria type i. 	i. harting, e. neumaier-probst, + authors s. kölker 	psychology, medicine 	brain : a journal of neurology 	 	 	pdf 	view excerpts, references background save alert research feed mechanism of age-dependent susceptibility and novel treatment strategy in glutaric acidemia type i. 	w. zinnanti, j. lazovic, + authors k. cheng 	medicine, biology 	the journal of clinical investigation 	 	 	highly influential 	pdf 	view excerpts, references background save alert research feed glutaryl-coa dehydrogenase deficiency: region-specific analysis of organic acids and acylcarnitines in post mortem brain predicts vulnerability of the putamen. 	s. kölker, g. hoffmann, + authors r. chalmers 	biology, medicine 	neuropediatrics 	 	 	view excerpts, references background save alert research feed vascular dysfunction as an additional pathomechanism in glutaric aciduria type i 	c. mühlhausen, s. ergün, + authors t. braulke 	medicine 	journal of inherited metabolic disease 	 	 	view excerpts, references background save alert research feed type i glutaric aciduria, part : a model of acute striatal necrosis 	k. strauss, d. morton 	medicine 	american journal of medical genetics. part c, seminars in medical genetics 	 	 	view excerpt, references background save alert research feed bioenergetics in glutaryl-coenzyme a dehydrogenase deficiency 	s. sauer, j. okun, + authors s. kölker 	biology, medicine 	journal of biological chemistry 	 	 	pdf 	view excerpts, references background save alert research feed transport and distribution of -hydroxyglutaric acid before and during induced encephalopathic crises in a mouse model of glutaric aciduria type . 	b. keyser, m. glatzel, + authors c. mühlhausen 	biology, medicine 	biochimica et biophysica acta 	 	 	pdf 	view excerpts, references background save alert research feed a diet-induced mouse model for glutaric aciduria type i. 	w. zinnanti, j. lazovic, + authors k. cheng 	biology, medicine 	brain : a journal of neurology 	 	 	highly influential 	pdf 	view excerpts, references background save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue hindawi publishing corporation case reports in dentistry volume , article id , pages http://dx.doi.org/ . / / case report modified bluegrass appliance: a nonpunitive therapy for thumb sucking in pediatric patients—a case report with review of the literature amish diwanji, preet jain, jigar doshi, prakash somani, and dhaval mehta department of pedodontics and preventive dentistry, faculty of dental science, nadiad, gujarat, india department of prosthodontics, pacific dental college, udaipur, india department of orthodontics and dentofacial orthopedics, darshan dental college, udaipur, india department of oral medicine and radiology, karnavati school of dentistry, gandhinagar, gujarat, india correspondence should be addressed to amish diwanji; amishdiwanji@yahoo.co.in received march ; accepted may academic editors: p. g. arduino and d. ram copyright © amish diwanji et al. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. oral habits in form of digit/thumb sucking are common phenomenon and part of childhood behavior. they are normally associated with oral pleasure, hunger, anxiety, and sometimes psychological disturbances. chronic practice can cause major orthopedic alterations to the skeletal structures of the oral cavity and lower face. aversive approaches in form of punitive therapy have been moderately effective. modified bluegrass appliance is nonpunitive therapy to treat sucking habits. it acts as a habit reversal technique and installs positive reinforcement in children. modified blue grass appliance proved to be very comfortable to patients and encourages neuromuscular stimulations. . introduction oral habit is a part of normal development in children. habits are learned patterns of muscle contraction with complex nature. oral habits are repetitive act seen commonly from infancy and should finish automatically as age advances. common repetitive behavior seen in infants is hand or figure sucking [ ]. sucking is one of the most common reflexes seen in infants. it manifests when they are in womb around wk of age [ ]. this is the first pattern of behavior observed in infant. infants and young children may use finger, thumb, pacifiers, or other objects to feel secure and learn the outside world. this is commonly seen when the child is anxious, insecure or surrounded by strangers and in families when they are separated from their parents. sucking habit induces sleep and hence makes infant and child calm and relaxed [ ]. hand sucking is naturally developed in % of infants in the second month and increases by first year of life [ ]. it is normal up to – years of age. it becomes a concern when continued for longer time and even seen in mixed dentition phase. this is the first sign for child to manifest future malocclusion or discrepancy during mixed dentition. the prevalence of oral habit has been reported up to % and % in high school girls and boys, respectively [ ]. % of prevalence has been reported in other literature [ ]. it has been documented that parental education, child’s nutrition, and sucking habits are associated with each other [ ]. however, higher prevalence rate has been claimed with high stress level among children in recent time [ ]. when child performs sucking habit in the first year of life, parents can move away his/her thumb smoothly and attract the child to other things. after second year of age, sucking habit should start decreasing and should appear only when child goes to sleep [ ]. when it continues in mixed and permanent dentition, it becomes a parental concern, as it is believed to affect growth of maxilla palate leading to skeletal changes, showing side effects on developing occlusion and resulting in malocclusion and constriction of skeletal structures [ , ]. the effect of prolonged sucking habit in children can affect development of occlusion. it may result in anterior open bite, increased over jet, lingual inclination of lower http://dx.doi.org/ . / / case reports in dentistry incisor and labial inclination of maxillary anterior, posterior crossbite, deep palate, compensatory tongue thrust, and sometimes speech defect [ , ]. the changes in dentition depend upon duration and frequency of habit being per- formed. during active eruption phase of permanent teeth, children who perform sucking habit for longer duration (more than hrs in a day), especially during sleep, tend to develop minor skeletal abnormalities and sever dentoalveolar changes [ ]. historically, correction of habit revolves around direct counseling of child, encouragement to improve self- confidence by rewarding the child, appliance therapy, and, in case of more complex dental changes, orthodontic therapy along with habit breaking appliances [ ]. the use of pacifier may cause severe and harmful effects on dentition if used for more than -year-old child [ ]. tongue, cribs, hay rakes and other sharp points employ an aversive negative stimulus to cease the undesirable oral habits. they are moderately effective and may trigger unexpected behavior sometimes. in , haskell and mink introduced blue grass appliance, also known as habit correction roller which gained universal attention and acceptance [ ]. it is userfriendly, nondestructive, easy to wear appliance replacing the common destructive habits. it is useful in avoiding traditional physical barriers of appliance in form of cribs and helping child with positive reinforcement. later, similar appliance called lingual pearl was used as a habit breaking and for multiple clinical applications [ ]. further, baker modified blue grass appliance with multiple rollers/beads and thus expanding its use from primary to permanent dentition [ ]. . management management of sucking habit depends upon the age. many questions arise in parents’ mind and among pediatricians regarding intervention by specific therapy to break the habit. counseling by pediatric dentist and pediatrician is important. if habit is stopped by - years of age, however, when they persists during eruption of permanent teeth child should be motivated to stop the habit [ , ]. appliance therapy involves use of either fixed or remov- able design in form of palatal crib or spurs. it is reminder therapy for child to make the habit unpleasant and difficult to practice. however, it causes difficulty in speech and eating and can cause iatrogenically inflicted wound and make child emotionally disturbed [ , ]. here, we present a case of thumb sucking habit where habit was corrected using bluegrass appliance as a non punitive therapy. . . case report. we present a case of an -year-old child, gir,l whose parents reported proclination of maxillary teeth and also complained of thumb sucking habit since birth. the patient used to suck her thumb regularly, - hrs/day, uncon- sciously in sleep or when idle from the primary dentition period. callous formation was seen over her digits (figure ). the patient reported with proclination of teeth, increased overjet and overbite with high palatal vault (figures (a) and (b)). management was started by counseling the parent figure : callous formation was seen over digits of patient. and the child regarding the ill effects of digit sucking on the developing dentition during the first visit. on the second visit, the patient was willing to discontinue the habit by treatment. a modified blue grass appliance was planned. molar bands were fabricated and adapted on maxillary molars. alginate impression was taken and casts were poured with dental stone over which molar bands were transferred. stainless steel wire ( . mm) was adapted over the palate extending from either side of molars. acrylic beads were made in laboratory using dental monomer and polymer. later, beads were inserted into stainless steel wire over palatal rugae area. no contact was established by beads with palatal tissues. the wire was soldered to molar bands by protecting the beads. the appliance was cemented using luting cement (figure ). the patient was instructed to roll the bead with tongue whenever she feels like sucking her thumb. the patient was kept on followup every month for checkup. the child was comfortable with the appliance and played by rolling the beads with the tongue. by end of months, callous formation had almost disappeared. patient was asked to wear appliance for almost months after correction to avoid relapse of the habit. appliance was removed after the discontinuation of habit. . discussion digit sucking is common phenomenon in pediatric age group that reflects the earliest form of habitual manipulation of body. many questions arise in the minds of general dentist, pediatricians, pediatric dentists and psychiatrists regarding impact of sucking habits on developing dentition. when should an attempt be made to break the habit? how does it disturb the child psychologically? in case of major orthopedic alterations to the skeletal structures of the oral cavity and lower face, intervention of orthodontist may require correct malocclusion. since years, habit breaking appliances in form of palatal cribs, spurs, palatal bars, hay rakes, and cage type appliances have been given to pediatric age group. however, emotional disturbances, difficulty in speech and eating, and iantrogenically self-inflicted wounds can occur with such appliances. hay rake and cage type appliances tend to get mutilated or destroyed while eating or due to habitual sucking habit. it reminds the child as punitive case reports in dentistry (a) (b) figure : proclination of teeth and increased overjet and overbite with high palatal vault. figure : modified blue grass appliance. therapy to cease the habit [ ]. haskell and mink described that blue grass appliance which is easy to wear, and did not have problems associated with traditional palatal cribs. the design consisted of hexagonal teflon roller on a cross-palatal wire which was effective to ending the sucking habit in several days [ ]. in the present study, modified blue grass appliance was used using mm acrylic beads as per recommended by baker [ ]. it encourages neuromuscular stimulations by using multiple beads as per what was the principles of castillo-morales [ ]. between – -year-old children can be instructed to play with the beads with the tongue immediately after placement. this allows the child to accept the appliance and learn the neuromuscular activity to normalize the tongue position. when a spinning roller is placed in close proximity to the tip of the tongue, “fascinating” response is quickly implemented due to neuromuscular and sensitive nature of tongue. since teflon rollers are not in contact with palatal tissues, children can roll them with their tongues. within few days, the tongue establishes new nonharmful habit of playing with roller. hence, this appliance works through counter conditioning response to the original conditioned stimulus for thumb sucking. psychologically, it is acceptable for parents also as they can encourage the child to play with beads instead of instructing the child to cease the habit all the time and thus making him/her anxious. reduced bulk of bead does not obstruct while eating, presents minimum disturbances with speech, and stimulates tongue movement. it is esthetic and child becomes comfortable quickly. the patient believes to have acquired a new toy in mouth to play with tongue. on the other side, direct relationship between age and time of appliance placement has been observed. the younger the patients are, the more quickly and completely the tongue position becomes normalized and the lesser the time required for cessation of the habit is. limbrock et al. [ ] suggested the appliance design even for toddler group to year old child. cessation of habit was reported on very st day in toddlers, whereas it takes few weeks in case of – -year-old children. hence, in early mixed detention or even in younger group, appliance could be used comfortably. if habit persists for longer time exhibiting posterior cross bite, modified blue grass appliance can be given with quad helix [ ] to expand arch. hence, two stage treatments can be completed with single appliance with correction of habit. . conclusion hence, modified blue grass appliance is a non punitive appliance and esthetic and child can wear it comfortably. it can be given as a supportive therapy as it requires no reminding or bribing, and parents can be freed of anxiety and frustration. it does not interfere with child’s growth and eliminates the habit with limited complications. references [ ] j. a. maguire, “the evaluation and treatment of pediatric oral habits,” dental clinics of north america, vol. , no. , pp. – , . 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[ ] b. s. haskell, “construction of a habit correction roller with arch expansion: chair side application of a new piece bondable “bluegrass” appliance,” journal of southeastern society of pedi- atric dentistry, vol. , no. , pp. – , . variants in scavenger receptor class b type i gene are associated with hdl cholesterol levels in younger women fax + e-mail karger@karger.ch www.karger.com original paper hum hered ; : – doi: . / variants in scavenger receptor class b type i gene are associated with hdl cholesterol levels in younger women caroline g.p. roberts a haiqing shen b braxton d. mitchell b coleen m. damcott b alan r. shuldiner b, c annabelle rodriguez a a department of medicine, the johns hopkins university school of medicine; b department of medicine, the university of maryland school of medicine, and c department of veterans affairs and veterans affairs medical center baltimore geriatric research, education and clinical center (grecc), baltimore, md. , usa sense mutation (ile val), which may be functional. more- over, rs (p = . ) and rs (p = . ) were sig- nificantly associated with higher hdl-c levels in women younger than years but not in women aged years or older ( p for interaction between age and rs = . ). none of the snp effects on hdl-c were modified in the pres- ence of diabetes, in either men or women. conclusions: scarb snps influence hdl-c levels in women, particularly in those less than years old. condensed abstract: we as- sessed associations between scarb snps and lipid traits in amish men and women. two snps, rs and rs , were significantly associated with higher hdl-c levels in women younger than years but not in women aged years or older, supporting an interaction between common sequence variants in scarb and estrogen on hdl-c. copyright © s. karger ag, basel the metabolism of high-density lipoprotein (hdl) is complex, with many factors inf luencing its circulating plasma levels. one such factor is the scavenger receptor, class b, type i (sr-bi), first characterized by acton et al. [ ] , and subsequently shown to be a physiologically rele- vant receptor that exerts a major inf luence on hdl cho- key words atherosclerosis � autosomal snps � snp abstract objective: variants within the scavenger receptor class b type i ( scarb ) receptor gene have been previously associ- ated with lipid levels, especially in women, with some stud- ies reporting the association to be stronger in the presence of diabetes or post-menopausal estrogen use. based on the reported gender-specific association and modification ef- fect of estrogen on lipid levels according to scarb variants, we explored the relationship between scarbi single nucleo- tide polymorphisms (snps) and lipid levels in an amish pop- ulation to assess sex and age differences. methods: eight scarb snps, identified from public databases, were geno- typed in subjects. results: rs and rs were in high linkage disequilibrium (ld), with r > . . none of the snps were significantly associated with lipid levels in men; however in women, rs (p = . ) and rs (p ! . ) were significantly associated with higher hdl-c levels. rs had an allele frequency of % and predicts a mis- received: october , accepted after revision: january , published online: may , dr. annabelle rodriguez the johns hopkins bayview medical center, division of endocrinology mason f. lord building, center tower, room , eastern avenue baltimore, md (usa) tel. + , fax + , e-mail arodrig @jhmi.edu © s. karger ag, basel – / / – $ . / accessible online at: www.karger.com/hhe c.g.p. roberts and h. shen are co-first authors. http://dx.doi.org/ . % f roberts /shen /mitchell /damcott / shuldiner /rodriguez hum hered ; : – lesterol (hdl-c) levels in rodents [ ] . it is highly ex- pressed in the liver and in steroidogenic tissues [ , ] , where its primary function is to mediate the uptake of cholesteryl esters from the hdl particle core. much less is known regarding the role of sr-bi in humans, although recent studies have shown some vari- ants in the sr-bi gene ( scarb ) to be associated with lipid levels, lipoprotein particle size and body mass in- dex (bmi) [ – ] . scarb variants have also been stud- ied in populations characterized with type diabetes mellitus (t dm) [ , ] and, interestingly, a scarb variant in exon was found to associate with differenc- es in insulin sensitivity in healthy people during the consumption of an olive oil-rich diet [ ] . in addition, a scarb variant was studied in older women using hor- mone replacement therapy [ ] . osgood et al. [ ] exam- ined scarb gene variants in the participants of the framingham study, and reported that three common single nucleotide polymorphisms (snps) rs (exon , + g ] a), rs (exon , + c ] t) and an intron (+ c ] t) snp were associated with variation in plasma lipoprotein concentrations and par- ticle size, particularly in subjects with t dm. while there was no association of snps with t dm, the g al- lele in rs was significantly associated with low- er hdl-c levels and smaller hdl particle sizes, al- though only among subjects with t dm. no interaction was observed for the rs snp with t dm, whereas the interaction between the intron snp and t dm on hdl metabolism was seen only in men. gender-specific associations have been reported be- tween some of the scarb variants and lipid levels, an observation that led richard et al. [ ] to examine the effect of exogenous estrogen on hdl-c levels by scarb variants in elderly women in the rancho bernardo study. these investigators found no significant associa- tions between either the intron snp or rs and hdl-c levels after adjusting for age, bmi, alcohol, smoking, triglycerides, fasting plasma glucose and es- trogen use. however, they did report significant estro- gen interaction with the rs snp on hdl-c levels, with hdl-c levels significantly higher in estrogen users than non-users [ ] . motivated by these previous observations, we assessed the relationship of eight different scarb variants, in- cluding the previously studied rs , intron (+ c ] t), and rs variants, with lipid levels in adult men and women participants of the amish family diabetes study (afds) and to determine if associations were mod- ified by the effects of age, gender and/or t dm. methods study population the old order amish (ooa) is a genetically homogenous population of central european ancestry. the original founders of this population immigrated to eastern pennsylvania (now lancaster county, pa., usa) in the mid to late s, with the lancaster county population now consisting of approximately , individuals [ ] . the ooa today are still characterized by their social cohesiveness and rural lifestyle [ ] . in , afds was begun to identify susceptibility genes for t dm and related traits. probands with t dm onset between and years of age were identified and all willing first-degree family members years of age and older were invited to participate. first-degree relatives of any family members subsequently diagnosed with t dm were also invited to participate. a total of subjects with lipid data and dna available for genotyping were included in this study. participating subjects were examined at the amish research clinic in strasburg, pa following a -hour overnight fast. height and weight were measured and body mass index (bmi) was calculated as the weight in kilograms divided by the height in meters squared (kg/m ). fasting lipid profiles [total choles- terol (tc), hdl-c, triglycerides (tg)] were performed by quest diagnostics (baltimore, md). intra-assays cvs of duplicate samples ranged between . and . %, and inter-assay cvs ranged between . and %. low-density lipoprotein cholester- ol (ldl-c) concentrations were estimated using friedewald’s formula [ ] . a g oral glucose tolerance test (ogtt) was administered, and diabetes mellitus was classified based on the american dia- betes association plasma glucose criteria [ ] of a fasting plasma glucose level ( mmol/l) or a -hour ogtt plasma glucose lev- el ( . mmol/l). subjects were also considered to have diabetes if they reported current use of insulin or oral glucose-lowering agents. subjects were excluded from these analyses if they had a diagnosis of diabetes before the age of years and reported cur- rent use of insulin (n = ). impaired glucose tolerance was diag- nosed based on ogtt plasma glucose levels ( -hour ogtt plas- ma glucose level . but ! . mmol/l). normal glucose toler- ance was defined as fasting plasma glucose level ( ! . mmol/l) and -hour ogtt plasma glucose level ( ! . mmol/l). informed consent was obtained from all afds participants, and the study protocol was approved by the institutional review board at the university of maryland school of medicine. details of the study design have been previously described [ ] . genotyping the scarb gene is . kb in length and consists of exons. we selected snps for genotyping within the coding and intronic regions of scarb from public databases (nih dbsnp, wellcome trust sanger institute and applied biosystems) that were vali- dated by frequency (minor allele frequency %) as well as snps reported in published articles [ – , ] . snps were genotyped us- ing the snpstream uht genotyping system (beckman coulter, fullerton, ca) [ ] . eight of the eighteen snps genotyped were polymorphic in this sample: rs , rs , rs , rs , rs , rs , rs , rs and the intron (+ c ] t) snp. error rates based upon blind replicates were – . %. scarbi snps and hdl-c levels in young women hum hered ; : – statistical analysis genotypes were checked for mendelian consistency using the pedcheck software program in the extended amish pedigree [ ] . a small number of mendelian errors were resolved or re- moved prior to analysis. the distribution of all snp genotypes conformed to those expected under hardy-weinberg equilibrium using the � test. association analyses were performed using a variance com- ponents methodology implemented in the sequential oligogenic linkage analysis routines (solar) software program, which allowed us to account for the relatedness of study subjects [ ] . brief ly, we evaluated the effects of snp genotype on hdl levels, while simultaneously adjusting for the effects of age, age , sex, bmi, and diabetes status. we accounted for the non-indepen- dence among family members by modeling the residual correla- tions in hdl levels between relative pairs explicitly as a function of the kinship matrix. our primary analysis was based on an ad- ditive genetic model, which implies a dose-response relationship between number of ‘risk’ alleles and hdl level. genotypes were thus assigned values of , , or , corresponding to the number of risk alleles. subjects currently taking lipid-lowering medica- tions (n = ) were excluded from the analysis. the significance of the genotype effects was assessed using the likelihood ratio test, in which we compared the likelihood of the data under a model in which the genotype effect was estimated against the likelihood of a nested model in which the genotype effect was constrained to zero. based upon our a priori hypothesis regarding an interaction between scarb genotype and age or sex, genotype-phenotype associations were estimated in men and women (and older and younger women) separately. interaction terms (e.g., genotype by sex, genotype by diabetes status, and genotype by age) were in- cluded in the model to assess whether the magnitude of the geno- type effect on hdl levels differed by group. given negligible ld between snps (except rs and rs ), haplotypes could not be reliably assigned, and thus haplotype association analysis was not performed. results the study subjects included t dm and non-t dm individuals from families, ranging in size from to . the characteristics of the study cohort are summarized in table . overall, there were slightly more women than men ( vs. %). women had higher bmi ( . vs. . kg/m , p ! . ), higher prevalence of t dm ( . vs. . %, p ! . ), higher mean hdl-c levels ( . vs. . mg/dl, p ! . ) and lower mean ldl-c levels ( . vs. . mg/dl, p ! . ). the use of prescription medications such as lipid-lowering agents and hormone replacement therapy is low in the amish. the frequency of cigarette smoking is also low in the amish. physical activity levels in the old order amish are typically high, especially compared to non-amish caucasians. the amish diet is generally high in calories and fat. we next examined the associations between scarb snps and lipid levels stratified by sex. in these analyses, we adjusted for age, age , bmi, diabetes status, family struc- ture and excluded those using lipid-lowering medications. none of the scarb snps were associated with tc, hdl- c, ldl-c, or tg levels in men (data not shown). however, in women, rs (p = . ) and rs (p ! . ) were significantly associated with higher hdl-c levels ( fig. ). the genotype ! gender interaction was significant for rs using a dominant model ( p ! . ), whereas no significant interaction was found for rs . as the ran- cho bernardo study [ ] suggested an interaction between estrogen use and rs snp genotypes on hdl-c and because estrogen levels are related to age, we examined as- men women age-adjusted p n age . . . . . bmi, kg/m . . . . < . diabetes . % . % . impaired glucose tolerance . % . % . total cholesterol, mg/dl . . . . . ldl-c, mg/dl . . . . . hdl-c, mg/dl . . . . < . triglycerides, mg/dl . . . . . lipid-lowering medications, % . % . % . estrogen users, % na . % na oral hypoglycemic agents, % . % . % . insulin therapy, % . % . % . na = not applicable. table . characteristics (mean sd, %) of the amish family diabetes study cohort roberts /shen /mitchell /damcott / shuldiner /rodriguez hum hered ; : – sociations between scarb snps and hdl-c levels in women older and younger than age years. as shown in table , women less than years of age who carried the rs (p ! . ) or rs (p ! . ) minor allele had significantly higher hdl-c levels compared with women homozygous for the major allele (hdl-c levels ap- proximately and % higher, respectively). in women greater than years of age, these two snps were not as- sociated with hdl-c levels. the genotype ! age group interaction term was statistically significant for rs genotypes (p = . ). by contrast, rs , which encodes a missense mutation (ile val), was associated with hdl- . kb rs rs rs intron rs rs rs rs rs rs rs in t r o n rs rs rs rs . . . . . r^ . . . . . d fig. . gene structure and pairwise ld among snps in scarb . the upper por- tion of the figure shows the gene structure and location of polymorphisms. the lower portion of the figure shows a schematic pairwise ld, calculated as d � and r among the polymorphic snps in the amish. the lines connect each snp name and the posi- tion with the corresponding cell in the ld matrix. magnitude and significance of the d � and r is illustrated by shading with a red to white or a blue to white gradient re- f lecting higher to lower ld values. table . mean hdl-c levels (se) according to scarb snp genotype in women ≥ and < years of age snp name maf women ≥ years (n = ): genotype women < years (n = ): genotype p* p* rs (c/t) . . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . rs (g/a) . . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . rs (c/t) . . ( . ) . ( . ) . . ( . ) . ( . ) . rs (g/a) . . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . intron (t/c) . . ( . ) . ( . ) . . ( . ) . ( . ) . rs (a/g) . . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . rs (c/t) . . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . rs (t/c) . . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . * p is for additive genetic model, adjusted for age, bmi, family structure, diabetes, and excluded lipid medications. significant p values are printed in bold. scarbi snps and hdl-c levels in young women hum hered ; : – c levels in women from both age groups, although this snp was uncommon ( %) in this population. none of the snps were associated with diabetes, in either men or wom- en, nor were the effects of any snp on hdl-c levels mod- ified in the presence of diabetes. excluding subjects with diabetes did not appreciably change the hdl-c associa- tion results (data not shown). a description of the gene structure showing snp loca- tions and pairwise ld between snps is provided as figure . rs (exon ; silent) and rs (intron ), both of which were associated with hdl-c levels in women younger than years old, are located . kb from each other and are in high ld with each other. by contrast, rs (exon ; ile val), which was also associated with hdl-c levels, is rarer and not linked to the other two hdl-c-associated snps, suggesting that its association with hdl-c levels is independent of the other two snps. this was supported by the fact that the association of rs with hdl-c levels remained statistically signifi- cant even after adjusting for the effects of the other two snps in either total women or women of each age group. the opposite analyses were done on rs or rs while adjusting for rs . significant association between rs or rs and hdl-c remained in total women and women younger than years old (data not shown). because of the relative absence of ld between all pairs of snps (r ̂ . ), with the exception of snps rs and rs , which were in very high ld (r = . ), no hap- lotype association analyses were performed. discussion we have observed several scarb polymorphisms to be associated with hdl-c levels in amish women, but not in men. furthermore, the associations were stronger in younger as compared to older women. because amish women, at least in lancaster county, rarely take estrogen ( . % in our cohort), the stratification of women accord- ing to those younger and older than age years is a proxy for examining the interaction of estrogen on lipid levels according to scarb genotype. our results are consis- tent with richard et al. [ ] in that hdl-c levels were significantly higher in amish women younger than years of age (presumably pre-menopausal and having ad- equate endogenous estrogen levels) who carried the rs variant, suggesting an important interaction be- tween estrogen and sr-bi. in addition, we found signifi- cantly higher hdl-c in younger female carriers of the minor allele of rs . however, unlike the rancho bernardo population, we did not find a difference in ldl-c levels in the old order amish women according to age and scarb genotype. osgood et al. [ ] did not show a sex difference between lipid levels and the rs snp. after multivariate adjust- ments, they showed that men homozygous for the minor allele had significantly higher total hdl-c, higher hdl -c levels and larger hdl particle size compared with men homozygous for the major allele. however, in women, while there were no apparent differences in hdl-c levels between minor allele homozygotes and major allele homozygotes, ldl-c levels were lower and hdl particle size was larger in the minor allele homozy- gotes [ ] . acton et al. [ ] also did not find significant as- h d l -c (m g /d l) gg ga aa p = n.s. rs gg ga aa p < . h d l -c (m g /d l) cc ct tt p = n.s. rs cc ct tt p = . men women fig. . adjusted hdl-c levels in men and women according to rs and rs genotype. mean hdl-c levels for men and women according to rs and rs genotype are shown. the p value for women is statistically significant for trend while the p value for men is not significant. there is a significant gene ! gen- der interaction for rs (p ! . ). roberts /shen /mitchell /damcott / shuldiner /rodriguez hum hered ; : – sociations between lipid levels and the rs snp in men in a population sampled from zaragoza, spain. while they also did not find significant associations between hdl-c levels and rs variant in women, they report- ed that women carrying the minor allele had significant- ly lower ldl-c levels compared to women homozygous for the major allele. other groups examining the rs snp have also reported association of this snp with lipid levels [ , ] . thus, our findings, coupled with reports in the literature, provide evidence for replicated association of rs with lipid levels, although there appear to be inconsistencies with respect to which lipid traits. in our population we did not find associations be- tween scarb snps and triglycerides (p ranges . – . ). only one scarb snp was significantly associated with bmi (rs , p = . ). relationships between bmi, triglycerides, and scarb snps have been found in a spanish population [ ] , and relationships between tri- glycerides and the scarb intron snp have been de- scribed in an obese population undergoing gastric bypass surgery [ ] . these differences may be due to the differ- ent populations studied. the relationship between scarb and estrogen may have biological relevance given that in animals estrogen regulates the expression of sr-bi and its isoform sr-bii [ , ] , and that estrogen response elements are found within the scarb promoter [ , ] . in humans, we have shown that women undergoing oocyte retrieval for in vitro fertilization who have low estradiol levels are also low expressors of sr-bi mrna [ ] . it is possible that rs or rs may be in linkage disequilibrium with a functional variant in estrogen response element in scarb such that carriers of variant alleles for scarb may have altered expression of sr-bi that becomes appar- ent only in the setting of estrogen. interestingly, rs , which encodes a missense mutation (ile val), is associ- ated with hdl-c levels in both age groups. although rare, it is possible that the effect of this potentially func- tional variant is strong enough to be independent of es- trogen status. the limitations of our study merit further discussion. the ooa are a closed founder population with relatively homogeneous environmental factors and fewer con- founders of disease and related phenotypes (e.g., low rates of smoking, alcohol consumption, and medication usage) [ ] . although this homogenous lifestyle might enhance our ability to discern genetic inf luences on disease and related traits, the ability to generalize our findings will require further study in the general population. in addi- tion, the high fat diet of the ooa (approximately % saturated fat (shuldiner, in preparation)) may also con- tribute to an interaction between diet, genotype and lipid levels. perez-martinez explored the effect of dietary fat content (saturated fat diet, carbohydrate-rich diet and a monounsatured olive oil diet) on lipid levels in subjects stratified by the scarb exon snp genotype. subjects consuming a saturated fat diet and expressing the minor allele in exon had significantly higher ldl-c levels compared to subjects homozygous for the major allele [ ] . this suggests the possibility that dietary fat might also have an important interaction with other scarb variants. furthermore, spady et al. [ ] have previously shown that polyunsaturated fatty acids up-regulate sr-bi in the hamster. it should be noted that we reported nominal p values in our statistical analysis instead of p values adjusted for multiple comparisons. however, since a major motiva- tion of our study was to determine if observed associa- tions between scarb snps and hdl-c levels were in- f luenced by age and gender given prior speculations that genetic effects at this locus might be estrogen sensitive, the comparisons we considered were not wholly indepen- dent. nonetheless, we could not exclude the possibility of a false-positive association and we need to be cautious in our interpretation of the results. in summary, we have shown that some scarb vari- ants are significantly associated with hdl-c levels in participants of the afds. one of these variants encodes a relatively rare conservative missense mutation and thus may be functional. interestingly, there was a significant age or estrogen interaction with two more common non- coding scarb variants. the clinical relevance of these associations and the mechanism by which these variants inf luence hdl-c levels, especially in younger women, warrants further study. acknowledgments this work was supported by research grants r -dk , u -dk , and k -ca awarded to the university of maryland and to hl awarded to dr. annabelle rodriguez. other support was received from the university of maryland general clinical research center grant m rr ; 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[pdf] commentary: physical activity and weight control. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /ije/dyt corpus id: commentary: physical activity and weight control. @article{hill commentarypa, title={commentary: physical activity and weight control.}, author={j. hill and j. peters}, journal={international journal of epidemiology}, year={ }, volume={ }, pages={ - } } 	j. hill, j. peters 	published 	political science, medicine 	international journal of epidemiology references luke a, cooper rs. physical activity does not influence obesity risk: time to clarify the public health message. int j epidemiol ; doi: . /ije/dyt . ng sw, popkin bm. time use and physical activity: a shift away from movement across the globe. obes rev ; : – . dugas lr, harders r, merrill s et al. energy expenditure in adults living in developing compared with industrialized countries: a meta-analysis of doubly labeled water studies. am j clin nutr ; : … expand view on pubmed academic.oup.com save to library create alert cite launch research feed share this paper citationsbackground citations view all topics from this paper 	ethanol 	exercise 	tracer 	obesity 	food 	published comment 	disease response domain 	fast foods paper mentions blog post let’s ask marion: can exercise balance out soda drinking? food politics august citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency authors' response to commentaries on 'physical activity does not influence obesity risk'. 	a. luke, r. cooper 	medicine 	international journal of epidemiology 	 	 	pdf save alert research feed obesity and energy balance: what is the role of physical activity? 	r. shook 	medicine 	expert review of endocrinology & metabolism 	 	 save alert research feed accelerometer-measured physical activity is not associated with two-year weight change in african-origin adults from five diverse populations 	l. dugas, stephanie kliethermes, + authors a. luke 	medicine 	peerj 	 	 	pdf save alert research feed the role of physical activity and exercise in obesity and weight management: time for critical appraisal 	p. wiklund 	medicine 	journal of sport and health science 	 	 	pdf save alert research feed global, regional, and national prevalence of overweight and obesity in children and adults during – : a systematic analysis for the global burden of disease study 	marie ng, t. fleming, margaret m. robinson, blake thomson, e. gakidou 	medicine 	the lancet 	 	 , 	pdf save alert research feed long-term effects of physical activity level on changes in healthy body mass index over years in young adult women. 	t. pavey, g. peeters, sjaan r. gomersall, w. brown 	psychology, medicine 	mayo clinic proceedings 	 	 save alert research feed the retail food environment in relation to socio-economic characteristics, weight status and diabetes 	yavgenia jane polsky 	business 	 	pdf save alert research feed extremes of weight gain and weight loss with detailed assessments of energy balance: illustrative case studies and clinical recommendations 	r. falck, r. shook, g. hand, c. lavie, s. blair 	medicine 	postgraduate medicine 	 	 	view excerpt, cites background save alert research feed correlates of adherence to an adolescent weight management program: a secondary data analysis 	meredith walker hanson 	medicine 	 	view excerpt, cites background save alert research feed relaxed natural selection contributes to global obesity increase more in males than in females due to more environmental modifications in female body mass 	wenpeng you, m. henneberg 	biology, medicine 	plos one 	 	 	pdf save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency physical activity and appetite control: can we close the energy gap? 	j. blundell 	medicine 	 	 	highly influential 	view excerpts, references background save alert research feed physical activity in an old order amish community. 	d. bassett, p. schneider, gertrude e. huntington 	medicine 	medicine and science in sports and exercise 	 	 	pdf save alert research feed time use and physical activity: a shift away from movement across the globe 	s. w. ng, b. popkin 	geography, medicine 	obesity reviews : an official journal of the international association for the study of obesity 	 	 	 save alert research feed energy expenditure in adults living in developing compared with industrialized countries: a meta-analysis of doubly labeled water studies. 	l. dugas, r. harders, + authors a. luke 	medicine 	the american journal of clinical nutrition 	 	 	pdf save alert research feed reduction in obesity and related comorbid conditions after diet-induced weight loss or exercise-induced weight loss in men 	r. ross, d. dagnone, + authors i. janssen 	medicine 	annals of internal medicine 	 	 , 	pdf save alert research feed the global obesity pandemic: shaped by global drivers and local environments 	b. swinburn, gary sacks, k. hall, k. mcpherson, s. gortmaker 	medicine 	the lancet 	 	 , 	pdf save alert research feed from instinct to intellect: the challenge of maintaining healthy weight in the modern world 	j. peters, h. wyatt, w. donahoo, j. hill 	psychology, medicine 	obesity reviews : an official journal of the international association for the study of obesity 	 	 save alert research feed effects of mo of verified, supervised aerobic exercise on macronutrient intake in overweight men and women: the midwest exercise trial. 	j. donnelly, e. kirk, d. jacobsen, j. hill, d. sullivan, s. johnson 	medicine 	the american journal of clinical nutrition 	 	 	pdf save alert research feed covert manipulation of the ratio of dietary fat to carbohydrate and energy density: effect on food intake and energy balance in free-living men eating ad libitum. 	r. stubbs, p. ritz, w. a. coward, a. prentice 	medicine 	the american journal of clinical nutrition 	 	 save alert research feed physical inactivity as the culprit of metabolic inflexibility: evidence from bed-rest studies. 	a. bergouignan, floriane rudwill, c. simon, s. blanc 	medicine 	journal of applied physiology 	 	 	pdf save alert research feed ... ... related papers 	abstract 	topics 	paper mentions 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators blog posts, news articles and tweet counts and ids sourced by altmetric.com terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue the journal of academic social science studies international journal of social science doi number:http://dx.doi.org/ . /jasss number: , p. - , autumn ii yayın süreci yayın geliş tarihi / article arrival date - yayınlanma tarihi / the published date . . . . dÎvÂnu lugÂtİ’t tÜrk’te gİyİm kuŞam kÜltÜrÜ İle İlgİlİ kelİmeler words about clothing in dÎvÂnu lugÂti’t turk yrd. doç. dr. recep tek nevşehir hacı bektaş veli Üniversitesi fen-edebiyat fakültesi türk halk bilimi bölümü Öz bireyin bedenini örten, onu dondurucu soğuktan, yakıcı güneşten, çeşitli saldırılardan vb. dış etmenlerden koruyan giyinme, insana has bir özelliktir. Örf ve âdetler, inançlar, coğrafya, yaşam biçimi, estetik anlayış gibi hususlar, folklorik bir öge olan giyim kuşamın şekillenmesinde etkili olan faktörler olarak karşımıza çıkmaktadır. bu faktörler, milletlerin kendi milli giyimlerini yaratmalarında da mühim bir rol oynamıştır. tarihî süreç içerisinde milletleri birbirinden ayıran özelliklerden biri olarak giyim kuşam, milli kültür içerisinde yer alan önemli bir maddi kültür ögesi olmuştur. giyim kuşam, insanoğlu için aynı zamanda bir göstergedir. bu gösterge, insanın sosyal durumunu, statüsünü, dünya görüşünü, değerlerini ifade edebilir; onun cinsiyeti, yaşı, inancı, mensubu olduğu topluluk, medeni hâli ve hatta o andaki ruh hâli hakkında karşısındakilere bir fikir verebilir. türklerde giyim kuşam eşyalarının ana malzemesini deve, koyun, kuzu, sığır, tilki, samur ve ayı gibi hayvanların derisi ve kürkü ile koyun, keçi ve deve yünü oluşturmaktaydı. medeniyetin gelişmesi ve uygarlığın ilerlemesi ile birlikte bunlar yerini kumaşlara ve bezlere bırakmıştır. Özellikle yerleşik hayata geçiş ile birlikte dokumacılığın yaygınlaşması, pamuk, ipek, keten ve yünlü dokuma giyim kuşam eşyalarını ön plana çıkarmıştır. dîvânu lugâti’t türk’te giyim-kuşamla ilgili tespit edilmiş ve tespit edilen bu kelimeler dört alt başlıkta sınıflandırılmıştır. tespit edilen bu kelimelerden yola çıkarak milli kültürün önemli bir ögesi olan o dönem türk giyim kuşam gelenekleri hakkında genel bir bilgi edinilebilmektedir. dîvânu lugâti’t türk’te geçen kelimelerden, türklerin süse ve süslenmeye de büyük önem verdiklerini görmekteyiz. bu kelimeler göstermektedir ki türkler için giyinmek sadece bedeni örtmek ve çeşitli dış etmenlerden korunmak anlamına gelmeyip; kendilerini güzel, zarif ve şık göstermenin bir aracı olarak da karşımıza çıkmaktadır. eserde geçen giyim kuşam ile ilgili kelimelerin bazılarının beyit ve dörtlüklerle kalıplaşmış sözler olan atasözleri içerisinde geçtiği de görülmektedir. bu durum, bu kelimelerin o dönemdeki kullanımı, - nisan tarihlerinde karabük Üniversitesi, mahmud kaşgari barsgani doğu Üniversitesi ve türk dünyası belediyeler İşbirliği ile kırgizistan’ın başkenti bişkek’te düzenlenen “ii. uluslar arası kaşgarlı mahmud sempozyumu: bilim dünyasına katkıları” başlıklı sempozyumda sunulan yayımlanmamış bildiri metninin genişletilmiş şeklidir. recep tek kullanım yaygınlığı ve türklerin yaşamındaki yerini ve ehemmiyetini göstermesi açısın- dan da önemlidir. anahtar kelimeler: türkler, milli kültür, giyim-kuşam ve süslenme, kaşgarlı mahmut, dîvânu lugâti’t türk, giyim-kuşam ve süslenme ile İlgili kelimeler abstract clothing is a speciality belongs to human. it protects him from cold, hot, attacks and other factors. moreover, it covers his body. traditions, beliefs, geography, lifestyle, sense of aesthetics are factors to shape of clothing. these factors have important place in creating their own clothing style. at the same time clothing separates nations from each other and it symbolizes material culture in national culture. clothing is an indicator for human being. this indicator can explain a human social situation, world- view and val- ues, it can give information about his sex, age, belief, society, marital status and mood. in turks, there are some basic clothing materials. some of them are camel, sheep, cattle, lamb, fox, sable and bear. these animal skins and furs had been used for producing clothes. these materials replaced with fabrics and cloth along with rising civilization. especially, along with settled life textile industry has improved and cotton, silk, linen and wool came into prominence at that term. about words about clothing were de- termined in dîvânu lugâti’t türk and these words were classifed under four titles. the- se words can give general information about turkish clothing and traditions at that term. we can understand from dîvânu lugâti’t türk how adornment is important for them. these words show that clothing is not only to cover a body and protect from other factors but also it is a tool to be seen as smart and good. in this study, it is seen that these clothing words are used inside proverbs. this situation is important because it shows us that the use of words, prevalence and place in turkish life at that term. keywords: turks, national culture, clothing and adornment, kaşgarli mahmut, dîvânu lugâti’t turk, words coupled with clothing giriş türk dilinin bilinen ilk sözlüğü olan dîvânu lügati’t türk, xi. yüzyılın ikinci yarı- sında kaşgarlı mahmud olarak bilinen mahmûd bin el-hüseyn bin muhammed el- kâşgarî tarafından yazılmıştır. türkçe’den arapça’ya ansiklopedik bir sözlük mahiyetinde olan dîvânu lügâti’t türk, karahanlılar döneminin en önemli eser- lerinden biridir (ercilasun & akkoyunlu, : xvi). veya yılında muhteme- len bağdat’ta tamamlanan eser, barındırdığı türkçe kelimelerin yanı sıra dönemin türkçe- sinin grameri, türk boyları ve bu boyların ağızları, türk tarihi, türk coğrafyası, türk halk edebiyatı ve folkloru, türk mitolojisi gibi çok çeşitli alanlarda verdiği bilgiler bakımın- dan bir türkiyat ansiklopedisi özelliği gös- termektedir (atalay, : x ; kaçalin, : ; ercilasun & akkoyunlu, : xvi). türk kültürünü bütün yönleriyle der- lemeye çalışan kaşgarlı mahmut, bilgeliği ve akademik kişiliğiyle dönemin türk toplum yaşamını eserinde gözler önüne sermeye ça- lışmıştır (yavuz, : ). kaşgarlı mahmud, türkçenin ilk ta- nıklı sözlüğü olan eserine türkçe olduğunu düşündüğü kelimeleri almış ve bu kelimelerin cümle içindeki örnek kullanımlarını göstere- rek de arapların türkçeyi kolay bir biçimde öğrenmelerini amaçlamıştır (bulut, : ). dîvânu lügâti’t türk’ün günümüze ulaşmış tek nüshası İstanbul fatih’teki millet yazma eserler kütüphanesi ali emirî efendi yazmaları kısmında numarada bulun- maktadır (ercilasun & akkoyunlu, : xxxvi). eser, muhammed bin ebî bekr bin ebi’l feth es-sâvî tarafından müellif nüsha- sından Şam’da istinsah edilmiş ve istinsah ağustos ’da tamamlanmıştır (ercilasun & akkoyunlu, : xxxvi). ali emirî efendi tarafından tesadüfen bulunan eser (atalay, : xviii), ilk olarak - yılları arasında kilisli rıfat tarafın- dîvânu lugâti’t türk’te giyim kuşam kültürü İle İlgili kelimeler dan yayınlanmıştır (kaçalin, : ; ercila- sun & akkoyunlu, : ixx). türklerin sosyal hayatına ilişkin önemli veriler sunan eserde, giyim-kuşamla ilgili bilgiler de yer almaktadır. bireyin bede- nini örten, onu dondurucu soğuktan, yakıcı güneşten, çeşitli saldırılardan vb. dış etmen- lerden koruyan giyinme, insana has bir özel- liktir. Örf ve âdetler, inançlar, coğrafya, ya- şam biçimi, estetik anlayış gibi hususlar folk- lorik bir öge olan giyim kuşamın şekillenme- sinde etkili faktörler olarak karşımıza çıkmak- tadır. bu faktörler, milletlerin kendi milli gi- yimlerini yaratmalarında da mühim bir rol oynamıştır. tarihî süreç içerisinde milletleri birbirinden ayıran özelliklerden biri olarak giyim kuşam, milli kültür içerisinde yer alan önemli bir maddi kültür ögesi olmuştur. dîvânu lugâti’t türk’te geçen oğuz elbisesi giymek anlamına gelen “oguzlan-” (ercilasun & akkoyunlu, : ), yağma gibi giyin- mek anlamına gelen “yagmalan-” (ercilasun & akkoyunlu, : ), türk çarığı giymek anlamına gelen “çarukla, çarıkla” (ercilasun & akkoyunlu, : ), türk ayakkabısı anla- mına gelen “izlik” (ercilasun & akkoyunlu, : ) ve Çiğillerin giydiği börk demek olan “kıymaç börk” (ercilasun & akkoyunlu, : ) gibi sözcükler ile balasagun’da yaşayan ve soğdak diye adlandırılan bu kav- min türkler gibi giyindiklerinin kaşgarlı mahmud tarafından açıklamada belirtilmesi (ercilasun & akkoyunlu, : ) giyim kuşamın bir milletin kültürünün ayrılmaz bir parçası, onun simgesi ve sembolü olduğunu kanıtlar niteliktedir. giyim kuşam, insanoğlu için aynı zamanda bir göstergedir. bu gösterge, insanın sosyal durumunu, statüsünü, dünya görüşü- nü, değerlerini ifade edebilir; onun cinsiyeti, yaşı, inancı, mensubu olduğu topluluk, me- deni hâli ve hatta o andaki ruh hâli hakkında karşısındakilere bir fikir verebilir (ennınger, Örneğin uygur kadınları gençlik döneminde renkli (kırmızı, yeşil) desenli kumaşlardan elbiseler giymeyi severken; yaşlılık dönemlerinde beyaz ve siyah giysiler giymeyi tercih etmişlerdir. gençlik ve ihtiyarlık dönemle- rindeki bu farklılık, elbisenin şekil ve biçiminde de gö- rülmüştür. budist rahipler, müritlerden farklı bir biçimde beyaz kumaştan önü açık gömlek ve pantolon giyip üstüne sarı renkli kumaş örtmüşler veya yere değinceye kadar sarkıtmışlardır. başlarını da sık sık kazıtıp şapkasız dolaşmışlardır. müslüman din adamları ise başlarına sarık sarıp ayaklarına mes ve lastik üstüne de siyah cüp- pe giymişlerdir. toplum içerisinde yüksek rütbeli kimse- ler elbiselerinin önüne ve arkasına derecelerini gösteren altın ve kalotun iplerden kuş veya çeşitli hayvan şekilleri işlemişlerdi. yine elbisenin rengi, içinde bulunulan ruh hâlinin bir göstergesi kabul edilmiş ve elbisenin rengi ruh hâlini yansıtır biçimde seçilmiştir. mesela uygurlarda kırmızı, sevinç ve şadlığın; beyaz, saflık ve kutluluğun; siyah ise kaygı ve sükûnetin sembolü olarak algılanmıştır. ayrıntılı bilgi için bkz. rahman, : - . başka bir kaynakta ise bu konuda şu bilgileri görmekteyiz: “İslam devletlerinde daha halife Ömer zamanında müslüman olmayan tebaanın müslümanlardan ayırt edici bir giysi giymeleri ya da belirli bir işaret taşımaları öngörülmüştü. türk tarihinde de eski dönemler için dinlere göre ayrı ayrı giyimler söz konusu olmasa bile selçuklulardan başlayarak böylesi ayrımlara gidildiği bilinmektedir. osmanlı İmparatorluğu döneminde bu ayrım, daha da belirginleşmiş, zımmi denen, yani devletin güveni altında kabul edilen müslüman olmayan tebaanın, giyim- kuşamda müslümanlar gibi görünmemesine büyük önem verilmiştir. bu amaçla da zaman zaman yayımlanan fermanlar, çıkartılan divan kararları, yani hükümlerle, başa sarılan tülbentten ayağa giyilen pabuca dek, müs- lüman olmayanların giyecekleri şeyler, renk, kalite, biçim hatta uzunluk-kısalık bakımından birbirinden ayrı tutul- muştur. Örneğin daha xiv. yüzyıl sonlarında, müslü- manlar külahlarına beyaz tülbent sararlarken, rumların mor ya da mavi, yahudilerin ise sarı renkte şerit takmala- rı zorunlu kılınmıştı. böylece müslüman olmayan kişiler ayırt edilmiş, cizye tahsildarlarınca da kolaylıkla tanınmış oluyorlardı. xvi. yüzyıla gelindiğinde, atlas kemha ipekli gibi değerli kumaşların, ince tülbentlerin ve pahalı pabuç- ların müslüman olmayanlarca kullanılmasının önlendiği görülmektedir. zımmi sayılanlara bu yasaklar getirilir- ken, müslümanların da şapka giymeleri yasak edilmişti. tarihli bir belgede de vurgulandığı gibi daha fatih mehmet döneminde İstanbul’daki yahudilerin kırmızı, hıristiyanların ise siyah renk şapka giymeleri öngörül- müştü.” (turan, : ). ennınger ise bu konuda şu örneği verir: “Örneğin amish erkekleri tarafından giyilen mavi ya da siyah renkli mutze, bir yabancı için sadece bir cekettir. amish kültüründeki giysi normlarını bilen bir kimse için ise mutze, bu giysiyi giyen kimsenin bir amish olduğunu ve vaftiz edildiğini (bu işlem genellikle yaşında gerçekleşir.) gösteren bir giyim eşyasıdır. mutze mavi renkli ise giyen kimsenin ile yaşlarında; siyah recep tek : - ; uçar, : ; arığ, ). zaman içerisinde çeşitli işlevlere sa- hip olan giyim kuşam geleneklerinde, coğrafî şartlara uyma, korunma ihtiyacı, dinî inanç- lar, siyasal düzenlemeler, ekonomik şartlar ve meslekî nedenler gibi faktörler (turan, : - ) etkili rol oynamıştır. türklerde giyim kuşam eşyalarının ana malzemesini deve, koyun, kuzu, sığır, tilki, samur ve ayı gibi hayvanların derisi ve kürkü ile koyun, keçi ve deve yünü oluştur- maktaydı (Ögel, : - ; tezcan, : - ; arığ, ; Özbay, : ; kafe- soğlu, : ). medeniyetin gelişmesi ve uygarlığın ilerlemesi ile birlikte bunlar yerini kumaşlara ve bezlere bırakmıştır. Özellikle yerleşik haya- ta geçiş ile birlikte dokumacılığın yaygınlaş- ması, pamuk, ipek, keten ve yünlü dokuma giyim kuşam eşyalarını ön plana çıkarmıştır (Özbay, : ; rahman, : - ; turan, renkli ise bu giysiyi giyen kişinin yaşından büyük olduğunu göstermektedir. bir yabancıya göre bir rus kalpağı, kırmızı renkli basit bir şapkadır. bir rus için ise bu kalpak, bu şapkayı giyen kimsenin doktor olduğuna işaret etmektedir.” (ennınger, : ). nezihe araz ise “anadolu-kadın giyimi Üstüne notlar başlıklı yazısında bu konuda önemli bilgiler vermektedir. Örneğin kişinin giydiği çorabın burnundaki mühürden mensup olduğu yörük boyunun yanı sıra çoraptaki motiflerden hareketle giyenin evli mi bekâr mı, ovalı mı dağlı mı, büyük ağa mı küçük ağa mı olduğunun anlaşılabileceğini belirtir (araz, : - ). yine kadının alnındaki altın sayısının kadı- nın çocuk sayısını göstermekle birlikte gelinin başındaki süslemelerin ve işlemelerin her birinin de ayrı bir anlam içerdiğini ifade eder. mesela başta bulunan mezar taşı oyası gelinin kaynanası ile arasının bozuk olduğunu anlatırken Çayır çimen oyası gelinin kaynanası ile mutlu olduğunu, kırmızıbiber oyası gelinin kocasıyla arasının iyi olmadığını, hercai oyası ise kocasının gözünün dışa- rıda olduğunu anlatır (araz, : ). ayrıntılı bilgi için bkz. araz, : - . giyimin işlevleri için bkz. tezcan, : - ; ennınger, : - . türklerde pamuklu, ipekli kumaşlar ve keten dokumalarla ilgili ayrıntılı bilgi için bkz. Ögel, : - . bahaeddin Ögel, türk kültürünün gelişme Çağları adlı eserinde de ayrıca turfan’da dokunan çiçekli uygur kumaşlarının çok meşhur olduğunu ve Çin elçisinin bu kumuşlara hayran kaldığını, beş-balıg’da dokunan kumaşların da arap ve İran kumaşları ile rekabet hâlinde olduğunu belirtir (Ögel, : ). : ; Öger & İnce, : - ). tarihin eski dönemlerinde atlı-göçebe yaşayan türklerde erkekler, deri pantolon , belden süslü kemerlerle sıkılan uzun etekli ceketler, uzun kaftan, kürk, soğuk ve sıcak havalar için ayrı ayrı pelerinler, hırka, gömlek ve börk (Ögel, : - ; esin, : ; turan, : - ; tezcan, : - ; Özel, : - ; tavkul, : - ; lale, : - ; kafesoğlu, : ); kadınlar ise şalvar, gömlek, cepken, dolama ya da kaf- tan ve ayakkabı; başlarına ise çok çeşitli baş- lıklar giymişlerdir (Özel, : - ; Özda- rıcı, : - ; turan, : - ). bildiride, dîvânu lugâti’t türk’te tes- pit edilen giyim-kuşam ile ilgili kelimeler dört alt başlıkta sınıflandırılmıştır. bu alt başlıklar ve başlıklar içerisinde yer alan kelimeler şun- lardır : . giyim kuşamda kullanılan mal- zemeler ile İlgili kelimeler: agı: İpek kumaş (ercilasun & akkoyunlu, : ). Âl: turunç renginde bir kumaş ( ). ay: turuncu renkli ipek ( ). barçın: İpekli kumaş, ipek ( ). böz: bez ( ). buçgak: devenin ayak derisi ( ). Çarukluk: ayakkabı yapmak için hazırlanan deri ( ). Çaydam: yağmurluk yapmaya veya yatak doldurmaya yarayan ince bir keçe ( ). Çınaxsı: nakışlı Çin ipeği ( ). Çit: Üzerine nakışlar basılmış Çin ipeği ( ). Çuz: kırmızı ve siyah altınla işlenmiş Çin kumaşı (atlas) ( ). edēd: kumaş vb. imal edilen her şey ( ). İsmail hami danişmend, paris antropoloji mektebi etnoloji profösörü dr. george montandon’un “traite d’ethnologie culturelle” adlı eserinde Çin’e pantolunun ilk olarak orta asya türkleri tarafından ithal edildiğinin söylendiğini belirtmektedir (danişmend, : ). selçuklu ve osmanlı dönemindeki giyim-kuşam gelenekleri ve kullanılan kumaşlar ile ilgili ayrıntılı bilgi için bkz. Özel, : - . Çalışmada ahmet b. ercilasun ve ziyat akkoyunlu tarafından hazırlanan dîvânu lugâti’t türk, tdk yay., ankara adlı eserden faydalanılmıştır. dîvânu lugâti’t türk’te giyim kuşam kültürü İle İlgili kelimeler eldiri: oğlak derisi ( ). eşgürti: İpek cinsinden işlemeli bir Çin kuma- şı ( ). eteklik böz: etek yapmak için kullanılan ku- maş ( ). xulın: değişik renklerde olan ve Çin’den geti- rilen bir ipek ( ). kaçaç: bir Çin ipeklisi ( ). kāfgar: behremān ipeği ( ). keçe: oğuzcada keçe ( ). kedüklük: yağmurluk yapmak için hazırlan- mış keçe malzeme ( ). kemek: pamuktan yapılan nakışlı ve şeritli bir kumaş ( ). kenzi: değişik renklerde kırmızı, sarı, yeşil Çin kumaşı ( ). kerim: nakışlı kumaş ( ). kez: bir tür Çin kumaşının adı ( ). kimişge: nakışlı kaşgar keçesi ( ). kuyka: deri ( ). loxtay: Üzerinde sarı pullar olan Çin ipeklisi ( ). mındatu: İpek ( ). sagrı: deri ( ). Şalaşu: bir Çin dokuma türü ( ). tatırga: tirşe, tabaklanmış beyaz deri ( ). taxçek: bir tür Çin ipeği ( ). taxtu: eğrilmeden önceki ham ipek ( ). tewren: Şalvar uçkuru yapmak için bir araya getirilip burulan ipler ( ). tıgrak: deri ( ). torku: İpek ( ). uyma: türkmenlerin ayakkabı yapımında kullandıkları keçe ( ). yatuk: İki cins iplikten örülen yün, argacı pamuk olan yün ( ) yūnyun: yün, deve tüyü ( ) yun: pamuk ( ). yuvlıç: tiftik, keçinin ince, yumuşak yünü ( ). züngüm: bir tür Çin ipeği ( ). . kadın-erkek giyim kuşamı ile İl- gili kelimeler: artıg: kadın yeleği ( ). aşuk: demir tolga ( ). bagırdak: kadın göğüslüğü ( ). başmak: oğuzlar ve kıpçaklarda ayakkabı ( ). bertü: Üst kısma giyilen elbise, hırka ( ). beçkem: İpekten veya yaban öküzü kuyru- ğundan yapılan ve savaşta kahramanın kim olduğunu gösteren alamet ( ). börk: başa giyilen şey, börk ( ). büküm etük: oğuzcada kadınların giydiği ayakkabı ( ). bürünçük: burnu ve burundan aşağısını örten yarım peçe, yaşmak ( ). Çekrek: kölelerin giydiği yünden yapılmış aba ( ). Çenşü: küçük hırka ( ). didim: gerdek gecesi gelinin giydiği taç ( ) eliglik: eldiven ( ). eşük: sarınmak için kullanılan örtü, üste giyi- nilen ve bürünülen şey ( ). etek: etek ( ). etik/etük: ayakkabı, edik, çizme ( ). İçmek: kuzu derisinden kürk ( ). İçük: samur, sincap vb. hayvanlardan elde edilen kürk ( ). İzlik: kesilen hayvanın derisinden yapılan türk ayakkabısı ( ). kadış: kesilen hayvanların derisinden diline- rek yapılan kayış ( ). kaftan: kaftan ( ). kars: koyun yününden veya deve tüyünden yapılan elbise ( ). kedgü: giyilen şey, elbise ( ). kedük: kepenek ( ). kedük: kuş tüyünden başlık ( ). kedüt: giyilen her çeşit elbise ( ). kewşek: yumuşak ve gevşek olan şey, ince (elbise vb.) ( ). keyük: “d”yi y yapanların lehçesinde kepe- nek ( ). kıdıklıg börk: dikilmiş kenarı olan başlık ( ). kıymaç börk: Çiğillerin giydiği tiftikten beyaz börk ( ). Çobanların omuzlarına aldıkları dikişsiz, kolsuz, keçeden üstlük, aba (türk dil kurumu, : ). recep tek könlek: gömlek ( ). kŏşik: yüz örtüsü, örtü ( ). kulak ton: kısa yenli elbise ( ). kur: kuşak, kemer ( ). kurşag: kuşak ( ). kuturma börk: Önde ve arkada kanadı olan başlık ( ). kuyka: kürk ( ). mükim (etük)/mükin (etük): kadınların giy- diği ayakkabı ( ). otran: yağmalarda paçalı giyecek ( ). sakalduruk: başlık sıkı dursun ve düşmesin diye çene altından bağlanan ipekten örülmüş ip ( ). samda: Çiğil lehçesinde giyilen sandal ( ). saraguç: kadının yaşmağı ( ). sırdım: oğuzcada deri ip, kayış ( ). suf: yün ipliklerden elde edilen bir kuşak ( ). suwluk: sarık ( ). terinçek: oğuzcada ince kadın çarşafı ( ). tŏnton: elbise ( ). uguk: tozluk, çorap ( ). Ümǖm: Şalvar, tuman, pantolon ( ). yagku/yaku: yağmurluk ( ). yalma: kaftan ( ). yaptaç: Çobanların yağmur ve karda giydiği küçük keçe ( ). yarındak: türk sırımı, kayış. keçi derisinden yapılır ( ). yişim: tozluk, soğukta iki bacağa giyilen bir çift tozluk ( ). . kıyafetin bölümleri ile kıyafe- ti/elbiseyi tamamlayıcı unsurlar ile İlgili kelimeler: bogmak: gömlek düğmesi ( ). burtala-: börk vb. şeylere altın pullar yapış- tırmak ( ). burtalan-: börk vb. şeyler altın varaklarla altınlanmak ( ). cigi: kıpçak vb. lehçelerde sağlam, sık dikiş, dikişi sağlam dikiş ( ). Çırguy: böğrün iki yanından kemerin geçtiği ilik ( ). Çikin: altın ipekle ipekli kumaşı nakışlama; altın ipekle kumaşa işlenen tasvir ( ). esrile: kaplan derisi gibi nakışlamak ( ). İlersük: uçkur ( ). kırgag: elbisenin kenarları ve çevresi ( ). kimsen: başa giyilen başlık vb. şeylerde kul- lanılan ince, altın yapraklar ( ). koy: kaftanın koyun kısmı ( ). könçek: oğuz lehçesinde yaka ( ). kudurgak: kaftanın arkadaki iki kuyruğun- dan biri ( ). Ör: kaftanın koltuk altı ( ). saçu: elbise, mendil vb. şeylerin saçağı ( ). saxt: oğuzlarda tokalar, kemer başları ve eyerdeki altın ve gümüş kakmalar ( ). sıdıg: kaftanın uçlardan göğse kadar uzanan iki eteğinden biri ( ). sıgzıg: mest vb. şeylerde iki dikiş deliği arası- na konan şerit ( ). sidig: kaftanın yanlardaki iki eteğinden biri ( ). tizildürük: ayakkabıların ucuna takılan bakır pullar ( ). tizme: İçine ip geçirildikten sonra ağzı bağla- nan şalvarın uçkurluğu ( ). toku: kemerin tokası ( ). tūş: altından veya gümüşten yapılan ve ke- merin ucuna takılan toka ( ). tügme: düğme ( ). ulag: elbise yaması ( ). uldan: ayakkabının altı ( ). Üstem: toka, kemer başı ( ). yaka: elbise yakası ( ). yamag: yama ( ). yanalduruk: kepeneğin omuzlarına dikilen, tipi ve yağmurdan korunmak için başa örtü- len keçe parçası ( ). giyim kuşamı tamamlayıcı unsurlar- dan birisi de şüphesiz ki süslenme ve süs eşyalarıdır. kaynaklardan hareketle eski türk toplum yaşamında kadınların yanı sıra erkek- lerin de saçlarını uzatıp tokalar taktıklarını, ördüklerini ya da topuz yaptıklarını, kulakla- rına küpeler taktıklarını, kadınların süs eşyası olarak ya da değişik amaçlarla bilezik; erkek- lerin ise kolbağı/kolçak kullandıklarını, par- maklarına yüzükler taktıklarını öğrenmekte- yiz (Ögel, : - ; esin, : - ; Ögel, : ; sevin, : ; Özel, : - ; tavkul, : - ; Ögel, : ; Öz- dîvânu lugâti’t türk’te giyim kuşam kültürü İle İlgili kelimeler darıcı, : - ; turan, : ). bun- lara ilave olarak yukarıda sıralanan kelimeler içerisinde yer alan börk vb. başlıkların altın pullar, yapraklar ve varaklarla süslenmesi, ipekli kumaşın altın ipekle nakışlanması, altından veya gümüşten yapılan ve kemerin ucuna takılan tokalar ile tokalar ve kemer başlarındaki altın ve gümüş kakmalar, ayak- kabıların ucuna takılan bakır pullar bu süs eşyaları içerisine dâhil edilebilir. aynı za- manda dîvânu lugâti’t türk’te geçen “bezen- ”, “enlik”, “bogmak”, “önik” gibi sözcükler- den hareketle kadınların süslenmek, kendile- rini güzel göstermek için makyaj yaptıklarını, yanaklarına kırmızı allıklar sürdüklerini, altın vb. şeylerden yapılmış gerdanlıklar ve kolye- ler, keçi kılından yapılma sahte zülüfler tak- tıklarını da öğrenmekteyiz. dîvânu lugâti’t türk’te geçen süs ve süslenme ile ilgili bu kelimeler şunlardır: bakan: pirinçten yapılan halka ve boyun hal- kası (tavk), genel olarak boyunluk ve halka ( ). bezen: bezenmek, kadın süslenmek, kadın makyaj yapmak ( ). bogmak: İnci ve mücevher kakılmış altın vb. şeylerden yapılan gerdanlık, kolye. gelin bununla gerdeğe sokulur ( ). būt: kıymetli ve büyük firuze ( ). o, bü- yüklerin oğul ve kızlarının perçemlerinin altına takılır ( ). enlik: allık, kadınların yanaklarını boyadıkla- rı kırmızı bir boya ( ). kirşen: kirşan, üstübeç ( ). *yüze sürülen allık, pudra+. Önik: kadınların kullandıkları keçi kılından yapılma sahte zülüf ( ). “burtala-“, “burtalan-“, “kimsen”. “Çikin”. “tūş”. “saxt”. “tizildürük”. küpe ve yüzük taşı gibi süslemede kullanılan, mavi renkli, saydam olmayan hidratlı doğal alüminyum ve fosfattan oluşan değerli bir mineral (türk dil kurumu, : ). Örçüg: oğuzcada saç örgüsü ( ). Örgüç: kadının saç örgüsü ve saç öbeği ( ). Örgüçlen: saç örgüsü olmak ( ). Örme saç: saç örgüsü ( ). Örük saç: Örgülü saç ( ). sulundı: adamın saç örgüsü ( ). tartıg: adamın saç örgüsü ve onun bağı ( ). tod monçuk: parfüm ve miskten yapılmış boncukların adı. bunu cariyeler takınır ( ). tolgag: küpe, kadın küpesi ( ). Ügmek: altından veya gümüşten kadın hal- kası, küpe ( ). yinçü: İnci, büyük inci ( ). yüzük: yüzük ( ). . giyim kuşam ile İlgili diğer ke- limeler: beçkemlen-: savaş günü vb. zamanlarda beç- kem denilen alameti takınmak ( ). bertülen-: hırka giymek ( ). boxtay: elbise bohçası ( ). börk yanı: börk kalıbı ( ). burış: deri ve elbisedeki buruşukluk ( ). burkug: deri vb. şeylerin çekilip büzülmesi ( ). bürük: torba, şalvar gibi şeylerin ağzını büz- meye yarayan ip ( ). bürün: bürünüp yüzünü kapatmak ( ). Çarukla-çarıkla: türk çarığı giymek ( ). Çaruklan-: ayakkabı giymek ve ona sahip olmak ( ). Çaruglug: Çarıklı ( ). Çekreklen-: kepenek giyinmek ( ). Çikne-: altın ipekle ipekli kumaşı işlemek, üzerine tasvir yapmak ( ). Çöpre: eski elbise ( ). eteklen-: elbise eteklenmek ( ). eteglig: eteği olan ( ). etüklen-: ayakkabı sahibi olmak ( ). İçle-: (elbiseye) astar yapmak ( ). İçmeklen-: kuzu kürkü giymek ( ). İçükle-: elbiseye kürk yapıştırmak ( ). kadıg: sağlamlaştırılmış dikiş ( ). kadıl: elbiseyi teyellemek ( ). teyel: seyrek ve eğreti dikiş. teyel yapmak: dikilecek parçaları birbirine teyelle tutturmak, kumaşın üzerinde recep tek kadışla: kayış yapmak ( ). kadu: teyellemek ( ). kātunlan: hatun elbisesiyle bezenmek ( ). kaw kuw bol-: (elbise) kötü dikiş sonucu büzülüp yırtılmak ( ). ked: giymek ( ). kedil-: giyilmek ( ). kedindi: Çok giyilmiş elbise ( ). kedrül-: giydirilmek ( ). kedür-: giydirmek ( ). kirşenlen-: yüzüne düzgün sürmek ( ). kiz: elbise sandığı, elbise bohçası ve saklayıp koruyan her şey ( ). könçüklen-: (elbiseye) yaka yapılmak ( ). könleklen-: gömlek giymek ( ). köpül-: (elbise) kaftan gibi kabartılarak di- kilmek ( ). kurşa-: kemer bağlamak ( ). kurşag: kuşakla kuşanma ( ). kurşan-: kemer kuşanmak ( ). oguzlan-: oğuz elbisesi giymek ( ). opra-: elbise eskimek, yıpranmak ( ). sagrıla-: hayvan derisinden sahtiyan yap- mak ( ). saraguçlan-: baş örtüsüyle baş örtmek ( ). sarın-: sarınmak, sarık sarınmak ( ). sarın-: yaşmaklanmak, örtünmek ( ). sarıt-sarut-: (sarık vb. şeyleri) sarınmak ( ). sarlan-: Örtü vb. bir şeyi sarınıp örtünmek ( ). sedre-: (elbise) havı dökülüp eskimek ( ). sedreş-: (elbise vb.) için kalınlığı gitmek, yo- ğunluğu azalmak, seyrelmek ( ). serdet-: elbiseyi seyreltmek ( ). tıtıl-: (elbise vb.) eskiyip parçalanmak ( ). tik-: (elbise vb. şeyleri) dikmek ( ). tikiglik: dikilmiş olan (elbise vb.) ( ). tokula-: kayışa toka takmak ( ). tonat-: elbise giydirmek ( ). tonlan-: elbise giymek ( ). tŏnluk: elbise için planlanan kumaş ( ). topul-: oğuzcada (elbiseyi) çıkarmak ( ). dikilecek yerleri teyelle belirtmek. teyellemek: teyel yapmak (türk dil kurumu, : ). sahtiyan: tabaklanarak boyanmış ve cilalanmış genellikle keçi derisi (türk dil kurumu, : ). Ügmeklen-: küpe takınmak ( ). Ǖmlüg: Şalvarlı, tümenli ( ). Ütüg: kırışıklıkları gitsin diye kızdırılarak elbisenin dikiş yerlerine bastırılan mala şek- linde demir ütü ( ). yagmalan-: yağma gibi giyinmek, yağma gibi davranmak ( ). yama-: yamamak ( ). yamaglıg: yamalı ( ). yamaglıg böz: yamamak için hazırlanmış kumaş vb. bir şeyin parçası ( ). yamagu: yamamaya değer, yamanacak (elbi- se vb.) ( ). yamal-: (elbise vb.) yamamak ( ). yarat-: oğuzlarda (elbise, ayakkabı vb. şeyler) yapmak. yıgrıl: elbise çekmek ( ). yi: dikiş, dikiş yeri ( ). yişimlen-: tozluk giyinmek ( ). yorış-: (İpek vb.leri üzerinde) yıpranmadan dolayı yollar, çizgiler belirmek ( ). dîvânu lugâti’t türk’te geçen kelime- lerden bazılarının günlük yaşamın yanı sıra beyitler, dörtlükler ve atasözleri içerisinde de kullanıldıkları görülmektedir. giyim kuşamla ilgili kelimelerin geçtiği beyit ve dörtlükler şöyledir: türlüg çeçek yazıldı barçın yadım kerildi uçmak yeri körüldi tumlug yana kelgüsüz. bardı sana yek utru tutup bal barçın kedipen teli yuwga bolup kāl ( ) ulşıp eren bȫrleyü yırtıp yaka orlayu sıkrıp ǖni yurlayu “türlü çiçekler açıldı. sanki ipek halı yayıldı. böylece cennet yeri görüldü. mevsim öyle ılıklaştı ki soğuk hiç dönmeyecek.” (ercilasun & akkoyunlu, : ) “Şeytan sana bal verdi. hatta ona kanıp ondan ipek bile giyindin. madem ki onun entrikasını anlamıyorsun öyle kal ve delilik içinde yaşa.” ( ). dîvânu lugâti’t türk’te giyim kuşam kültürü İle İlgili kelimeler sıgtap közi örtülür. ( ) İçerisinde giyim kuşamla ilgili kelime- lerin yer aldığı atasözleri ise şunlardır: barçın yamagı barçınka kars yamagı karska. ( ) tatsız türk bolmas başsız börk bolmas. ( , ). taz keligi börkçile. ( ). suw körme ginçe etük tartma. ( ). İzlik bolsa er uldımas, içlik bolsa at yagrı- mas. ( ). kılnu bilse kızıl keder yaranu bilse yaşıl ke- der. ( , ). neçe me oprak kedük erse yagmurka yarar. ( ). kin ton opramas keneşlig bilig artamas. ( ). tawgaç xānın torkusu telim tenlemedip bıç- mas. ( ). “afrasiyâb’a üzüldükleri için adamlar kurt gibi ulumaya başladılar. feryat ve figan ederek yakalarını yırtıyorlar.” ( ). “İpeğin yaması ipekle daha uygun olur. yünün de yaması yünle yakışır. bu, her cinsin kendi cinsine daha meyilli olduğunu anlatmak için kullanılır.” ( ). “başsız börk olmadığı gibi farssız türk de olmaz.” ( ); “türklere karışmamış hiçbir fars yoktur; üzerine konacak baş olacak ki börk de olsun.” ( ). “kelin gelişi börkçüyedir.” ( ). “su görülmeden ayakkabı çıkarılmaz. İşlerde temkinli davranmak için söylenir.” ( ) “adam ayakkabılı ise yalınayak kalmaz; atın sırtında örtü varsa (atın) sırtı yaralanmaz. bu söz, işlerde tedbirli olmak için söylenir.” ( ). “kadın kocasıyla iyi geçinmek isterse kırmızı ipek giyer; naz ve cilve yapmak isterse yeşil ipek giyer. bu söz kadınlar için kullanılır; kadın iyi davranırsa iyi karşılık görür.” ( ). ayrıca bkz. s. . “keçe ne kadar yıpranmış olursa olsun yağmurda işe yarar. kötü davranan ve kibirlenen hizmetçiyi çıkarmak isteyen adam için kullanılır. böyle olsa bile belki onu bazı işlerinde kullanırsın ve sen de rahat edersin.” ( ). “geniş elbise çabuk yıpranmaz; meşveretle aşılanmış akıl bozulmaz. bu, işi yaparken başkasına danışılması ve kendi başına yapılmaması için söylenir.” ( ). “Çin hakanının ipeği çoktur. buna rağmen ölçmeden elbiselik kesmez. bu söz israfı önlemek ve bir işi bilerek yapmak için kullanılır.” ( ). itka uwut atsa uldan yimes. ( ). ernenke elig karı bözün ǖm tükemes. ( ). bütün ǖmlüg kança kolsa olturur. ( ). yakadaki yalgagalı eligdeki ıçgınur. ( , ). sonuÇ kaşgarlı mahmut’un dîvânu lugâti’t türk adlı eserinde “giyim kuşamda kullanı- lan malzemeler ile İlgili kelimeler” başlığı altında , “kadın-erkek giyim kuşamı ile İlgili kelimeler” başlığı altında , “kıyafetin bölümleri ile kıyafeti/elbiseyi tamamlayıcı unsurlarla İlgili kelimeler” başlığı altında ve “giyim kuşamla İlgili diğer kelimeler” başlığı altında ise kelime olmak üzere top- lam kelime tespit edilmiştir. bu kelime- lerden yola çıkarak milli kültürün önemli bir ögesi olan o dönem türk giyim kuşam gele- nekleri hakkında genel bir bilgi edinilebilmek- tedir. kaşgarlı mahmud’un eserinde yer ver- diği ve açıklamalarda bulunduğu kelimeler- den hareketle türklerin giyim kuşam eşyala- rını elde etmede çeşitli renklerde düz ve na- kışlı ipekler, pamuklu ve keçi, koyun gibi hayvanların yününden elde edilen dokuma- lar, deve, oğlak, kuzu, samur, sincap, keçi derileri, keçeler , deve ve kuş tüyü kullandık- “köpeğe hayâ atılırsa (utandırılırsa) ayakkabının altını yemez. birsinin hayâ ile iş yapması istendiği zaman söylenir ve bu sözle birisi hayâlı olmaya zorlanırsa, hayâdan dolayı kötü işlerini terk eder.” ( ). “bekâr adamın şalvarı için elli arşın kumaş yetmez. Çünkü ona yabancı öğüt vermez. evlenmesi istenen kişi için söylenir.” ( ). “Şalvarı sağlam olan istediği şekilde oturur. kendisinden emin olup hiçbir töhmeti umursamayan kimse için kullanılır.” ( ) “yakadaki yemek döküntülerini yalayanın elindeki kap vb. kaçar veya kaçırılır. elindeki kendisine yetiyorsa onunla yetinilmesi ve o şeyin muhafaza edilmesi için söylenir.” ( , ). robert lewie adlı amerikalı bir antropoloji profesörünün fransızcaya çevrilen eserinde, keçenin bir türk icadı olduğu ve bu icadın Şark âleminde yunanistan ile garp âleminde roma’ya türklerden geçtiği, ayrıca yunanlılarla romalıların türk yamçısına bürünerek recep tek larını; bunlardan yararlanarak elbise, panto- lon, şalvar, etek, gömlek, yelek, hırka, şal, kürk, kaftan, kepenek/aba, yağmurluk, ayak- kabı, çarık, börk ve çeşitli başlıklar ile kayış ve kuşak gibi giyim kuşam eşyaları ürettikleri görülmektedir. dîvânu lugâti’t türk’te geçen kelime- lerden, türklerin süse ve süslenmeye de bü- yük önem verdiklerini görmekteyiz. burada geçen kelimelerden hareketle kadınların mak- yaj yaptıklarını yanaklarına allıklar sürdükle- rini, keçi kılından yapılma sahte zülüfler, inci ve mücevher kakılmış altın vb. şeylerden ya- pılan gerdanlık ve kolyeler taktıklarını, kadın- lar gibi erkeklerin de saçlarını uzatıp ördükle- rini, altından ve gümüşten küpeler, yüzükler taktıklarını, yine altından veya gümüşten yapılan altın, gümüş kakmalı kemer başları, tokalar kullandıklarını öğrenmekteyiz. bu kelimeler göstermektedir ki türk- ler için giyinmek sadece bedeni örtmek ve çeşitli dış etmenlerden korunmak anlamına gelmeyip; kendilerini güzel, zarif ve şık gös- termenin bir aracı olarak da karşımıza çık- maktadır. “giyim kuşamla İlgili diğer keli- meler” içerisinde yer alan ve “ütü” anlamına gelen “ütüg” kelimesi de bu fikri destekler mahiyettedir. zira bu, türklerin tertipli ve düzenli olmak, hoş ve güzel görünmek için kıyafetlerini kırışıksız ve düzgün bir şekilde giymeyi âdet edindiklerini, buna önem verip bu konuda titiz davrandıklarını bize göster- mektedir dîvânu lugâti’t türk’te geçen giyim kuşam ile ilgili kelimelerin bazılarının beyit ve dörtlüklerle kalıplaşmış sözler olan atasöz- leri içerisinde geçtiği de görülmektedir. bu durum, bu kelimelerin o dönemdeki kullanı- mı, kullanım yaygınlığı ve türklerin yaşa- mındaki yerini ve ehemmiyetini göstermesi açısından da önemlidir. kaynakÇa araz, n. ( ). anadolu kadın giyimi Üstü- ne notlar. türk halk edebiyatı ve folk- yağmurdan korundukları belirtilmektedir (danişmend, : ). lorunda yeni görüşler i (s. - ), an- kara: konya kültür ve turizm derne- ği yayınları. arığ, a. s. ( ). türklerdeki kıyafetin kısa tarihi, atatürk araştırmaları merkezi dergisi, / - - . http://www.atam.gov.tr/dergi/sayi- - - /turklerdeki-kiyafetin-kisa-tarihi. adresinden . . tarihinde eri- şildi. atalay, b. ( ). divanü lûgat-it türk tercü- mesi ( . cilt). ankara: türk tarih ku- rumu basımevi. bulut, s. ( ). divânü lügâti’t türk’ten ahıska türkleri yazılı ve sözlü diline geçen atasözleri, littera turca journal of turkish language and literature, / , - . danişmend, İ. h. ( ). medenî kıyafetin batı’ya türklerden İntikali, türk folk- lor araştırmaları, / , - . ennınger, w. ( ). giyim (Çeviren: n. Öz- demir), (yay. haz.: m. Ö. oğuz vd.) halkbiliminde kuramlar ve yaklaşımlar (s. - ), ankara: geleneksel ya- yıncılık. ercilasun a. b. & akkoyunlu z. ( ). dîvânu lügâti’t türk, ankara: tdk yay. esin, e. ( ). İslamiyet’ten Önce türk kültür tarihi ve İslam’a giriş, İstanbul: edebi- yat fakültesi matbaası. kaçalin, m. s. ( ). dîvânü lugâti’t türk, diyanet vakfı İslam ansiklopedisi (s. - ), İstanbul: türkiye diyanet vakfı yay. kaya, m. ( ). divânü lûgati’t türk’ün halkbilimi açısından Önemi, folk- lor/edebiyat, viii/ , - . kafesoğlu, İ. ( ). türk milli kültürü ( . baskı). İstanbul: Ötüken yayınları. lale, m. ( ). ii. mahmud dönemi kıyafet alanında yapılan yenilikler, yayım- lanmamış yüksek lisans tezi, isparta: süleyman demirel Üniversitesi sosyal bilimler enstitüsü. oğuz, m. Ö & sever m. ( ). dîvânu luga- http://www.atam.gov.tr/dergi/sayi- - - /turklerdeki-kiyafetin-kisa-tarihi http://www.atam.gov.tr/dergi/sayi- - - /turklerdeki-kiyafetin-kisa-tarihi dîvânu lugâti’t türk’te giyim kuşam kültürü İle İlgili kelimeler ti’t türk’ün nesne dünyası, (editör: f. s. barutçu Özöndör) kaşgarlı mah- mud kitabı (s. - ), ankara: kültür ve turizm bakanlığı yayınları. Ögel, b. ( ). türk kültür tarihine giriş ( . cilt). ankara: kültür bakanlığı yay. Ögel, b. ( ). İslamiyet’ten Önce türk kültür tarihi ( . baskı). ankara: türk tarih kurumu basımevi. Ögel, b. ( ). türk kültürünün gelişme Çağ- ları ( . baskı). İstanbul: türk dünyası araştırmaları vakfı yayınları. Öger, a. & İnce, g. ( ). halı dokumacılı- ğının avanos yöresi sosyo-kültürel ve ekonomik yaşamındaki yeri, ava- nos sempozyumu bildiriler kitabı - ekim , (s. - ), ankara: grafiker yay. Özbay, m. ( ). İlk yazılı belgelerimize göre türklerde giyim, milli folklor, / , - . Özdarıcı, Ö. ( ). divânü lugat-it-türk’te kadın ve kadına İlişkin unsurlar, sosyal bilimler, , - . Özel, m. ( ). folklorik türk kıyafetleri, ankara: türkiye güzel sanatları ge- liştirme vakfı yayınları. tavkul, u. ( ). tarihi türk erkek kıyafet- leri, milli folklor, , - türk dil kurumu ( ). türkçe sözlük ( . baskı), ankara: tdk. tezcan, m. ( ). giyim olgusuna sosyo- kültürel bakış ve türklerde giyim, ankara Üniv. eğitim bilimleri fakültesi dergisi, / , - . turan, Ş. ( ). türk kültür tarihi ( . baskı). ankara: bilgi yay. rahman, a. ( ). uygur folkloru (Çev. s. yalçın, e. emet), ankara: kültür bak. yay. sevin, n. ( ). on Üç asırlık türk kıyâfet tarihine bir bakış, ankara: kültür bakanlığı yayınları. uçar, z. ( ). battal gazi ve digentis akri- tas destanında simgeleşen kıyafet kültürü Üzerine, akademik bakış der- gisi, , - . yavuz, s. ( ). dîvânü lügâti’t türk’teki akrabalık adları ve bu adların tür- kiye türkçesi ağızlarındaki karşılık- ları, ekev akademi dergisi, / , - . recep tek journal ofmedical genetics, , , - pitfalls of genetic counselling in pfeiffer's syndrome m baraitser,* mary bowen-bravery, and p saldana-garcia from the kennedv-galton centre for clinical genetics, harperbury hospital, harper lane, shenley, radlett, herts wd hq summary a family with pfeiffer's syndrome is presented in which members of two generations showed only partial but relevant syndactyly before a child was born, in the third generation, with the full acrocephalosynd:ctyfy syndrome. the acrocephalosyndactyly syndromes are a group of hereditary disorders which manifest with anoma- lies of the cranium, hands, and feet. as with other dominantly inherited syndromes the clinical picture varies considerably and this has led to conflicting views about the classification of the acrocephalies. most authors recognise apert's, pfeiffer's and chotzen's syndromes and there is uncertainty about *also at the division of inherited metabolic diseases, clinical research centre, northwick park hospital, watford road, harrow, middlesex hal uj. received for putlication november a fourth entity with features of both apert's and crouzon's syndrome.' the acrocephalic syndrome of carpenter can be distinguished because of the presence ofpolydactyly. others have documented the presence of apert's, pfeiffer's, and transitional syndromes in multiple generations of the same family suggesting that the subdivision is spurious. - the variable expression and improvement of the cranio- facial deformities with age has also led to difficulties in detecting minimal manifestations, thereby in- creasing the possibility of missing gene carriers and counselling incorrectly. the following report of a family (fig ) with fig i pedigree of the family. large big toes, partial syndactyly of toes examined personally e- o fig proband iv.]. photograph and x-ray of skull. ii ill lv o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ pitfalls ofgenetic counselling in pfeiffer's syndrome al /& -t /.' .. ui k., . l/)) fig photographs of hands andfeet of (a) iv.i, (b) iii.s, (c) iii. , (d) ii . ig: :: li .. i o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ m baraitser, mary bowen-bravery, and p saldana-garcia pfeiffer's syndrome illustrates the pitfalls in genetic counselling. case reports case iv. the patient, born in , was referred to the counselling clinic because of an unusually shaped skull (fig ). the pregnancy was complicated at months by loss of blood but was otherwise unevent- ful. examination of the male child showed acro- cephaly, hypertelorism, antimongoloid slant of the eyes, a high arched palate, and a flat nasal bridge. the patient's hands showed no abnormality. there was cutaneous syndactyly of the second and third toes bilaterally (fig ) and the big toes were unusually broad. when seen at the age of months the developmental milestones were normal. case . the patient's mother, born in , had no cranial manifestations of acrocephaly (fig ) but had partial cutaneous syndactyly of the second, third, and fourth toes and large big toes (fig ). her brother is said to be similarly affected. case iii. the patient's maternal aunt, born in , had no cranial manifestations of acrocephaly (fig ) but had partial cutaneous syndactyly of the second and third toes and large big toes (fig ). case . the patient's maternal grandfather, born in , had a high forehead but his facial features were otherwise unremarkable (fig ). he had partial cutaneous syndactyly of the second and third toes and large big toes (fig ). his mother, his brother, and his brother's two children were said to have similar deformities of the feet, but were of normal facial appearance. in all affected family members examined there were no visible joint creases between the middle and terminal phalanges of some of the fingers. radiographic findings x-rays of the hands (fig ) show fusion between the middle and terminal phalanges of fingers and in the patient's mother ( . ) and maternal aunt ( i. ), and of finger in the maternal grandfather ( . ). in the feet the changes include medial deviation and partial fusion of the phalanges of the big toes (fig ). x-rays of the patient's hands are reported to be within normal limits apart from incurved little fingers. a summary of the clinical and radiographic findings appears in the table. dermatoglyphs the fingerprint patterns of the proband and the affected members of his family are of great size and contain a large number of ridges (see appendix). the digital triradii on fingers , , and are in most cases placed near the limit of the ridged skin. unusually high ridge counts of near or over ridges occur on fingers and in the proband's mother and maternal aunt. the proband's finger ridge counts could not be ascertained for technical reasons. his plantar configurations show features in common with those observed in his affected relatives. for example, his pattern intensity ( . ) is above the corresponding normal average of . loops per sole fig (a) mother ofproband, (b) maternal aunt, (c) maternal grandfather. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ pitfalls ofgenetic counselling in pfeiffer's syndrome (a) (d) fig x-rays of hands andfeet of (a) iv.j (hands only, permission withheld for x-ray offeet), (b) iii.s, (c) . , (d) i . . (c) f /) o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ m baraitser, mary bowen-bravery, and p saldana-garcia table summary of clinical and radiological findings . . . iv.] head acrocephaly + high forehead + + hypertelorism + antimongoloid slant of eyes + flat nasal bridge + high arched palate + upper limbs absent joint creases + + + fusion of phalanges + + + lower limbs large big toes + + + + cutaneous syndactyly of toes + + + + medial deviation of phalanges of big toes + + + x-ray not partial fusion of permitted phalanges of big toes + + + and zygodactyly is represented in both feet. his plantar configurations were not found in a sample of english male controls. the dermatoglyphs of the unaffected father differ from those of his son and show nothing remarkable. discussion pfeiffer's syndrome is characterised by acrocephaly, normal intelligence, hypertelorism, antimongoloid slant to the eyes, prominent eyes, flat nasal bridge, irregularly placed teeth, soft tissue syndactyly, broad thumbs and toes, brachymesophalangia, ulnar deviation ofthe proximal phalanx ofthe thumbs, and radially deviated phalanges of the fingers. genetic counselling is usually straightforward as there have been at least three families described with three affected in two generations, and subjects affected in many generations in whom dominant inheritance was the most likely pattern of trans- mission. in the large mid-western amish kindred reported by jackson et a variable expression was found. a total of members and possibly others were affected. all those affected had clinical or radiological abnormalities of the feet, but enlarge- ment of the thumb did not occur. several members looked clinically normal or had features ofcrouzon's disease, but there were radiographic changes in the feet compatible with pfeiffer's syndrome. naveh and friedman reported a family with affected members in three generations in whom the signs of pfeiffer's syndrome were confined to the head and feet, sparing the upper limbs. in the family presented here members were aware of the transmission of webbed toes over three generations, without noticeable acrocephaly. the main deformities were syndactyly of the second and third toes, large big toes, and the inability to flex the terminal phalanges of the fingers. only the proband had, in addition, the facial features of pfeiffer's syndrome. the gene carriers in this family showed in common certain unusual dermatoglyphic features, but there is as yet no evidence that they are specific for pfeiffer's syndrome. it came as a shock to the mother to learn that there was an association between what had been regarded as a mild familial trait and the cosmetically more unacceptable craniofacial manifestations of this disorder. other members of this family, with only the mild limb abnormalities, run a risk of having off- spring with the full syndrome, but the risk is probably small. syndactyly of the second and third toes is a common dominantly inherited malformation so that only the presence of large big toes or thumbs would make pfeiffer's syndrome a possibility. addendum the possibility that separate genes are responsible for the limb and craniofacial abnormalities is made less likely by a recent encounter with a second family showing the same variation in expression as the family described above. the authors thank dr m a c ridler for his help with the preparation of this article and mrs helen butcher for typing the manuscript. references mckusick va. mendelian inheritance in man. th ed. baltimore and london: john hopkins university press, : - . smith dw. recognizable patterns ofhuman malformation. major problems in clinical pediatrics. vol . philadelphia: saunders, : - . robinow m, sorauf tj. acrocephalopolysyndactyly, type noack, in a large kindred. birth defects ; : - . jackson ce, weiss l, reynolds wa, forman tf, petersen ja. craniosynostosis, midfacial hypoplasia and foot abnormalities: an autosomal dominant phenotype in a large amish kindred. j pediatr ; : - . escobar v, bixler d. the acrocephalosyndactyly syn- drome: a metacarpophalangeal pattern profile analysis. clin genet ; : - . martsolf jt, cracco jb, carpenter gg, o'hara ae. pfeiffer syndrome: an unusual type of acrocephalo- syndactyly with broad thumbs and great toes. am j dis child ; : - . saldino rm, steinbach hl, epstein cj. familial acro- cephalosyndactyly (pfeiffer syndrome). ajr ; : - . naveh y, friedman a. pfeiffer syndrome: report of a family and review of the literature. j med genet ; : - . requests for reprints to dr m baraitser, kennedy- galton centre for clinical genetics, harperbury hospital, harper lane, shenley, radlett, herts wd hq. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ pitfalls ofgenetic counselling in pfeifjer's syndrome appendix dermatoglyphs of the family. (a) iv. ; (b) ii . ; (c) iii. ; (d) h . ; (e) . . j. ( 'a i (a) (b) £, ( c) o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ m baraitser, mary bowen-bravery, and p saldana-garcia (d) i~ ~ - (e) o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n a u g u st . d o w n lo a d e d fro m http://jmg.bmj.com/ <bfaac d c b c f d c a e > 서 론 [ ]. . (statistical power) (heterogeneity) . . . (initiation) (progr- ession) . (severe case) (recall bias) [ ]. . (unrelated subjects) (adjustment) [ ]. imprinting (parent-of-origin) effect . . , . (framingham cohort) . (framingham study) . ( ). “ ” (family- based cohort population-representative family) . 가족기반 코호트와 가족연구의 사례 . 프램험 연구 (the framingham heart study , ( , ) , . (systematic sample . , . . , . 가족기반 코호트 연구의 사례와 전망 설재웅 박수경 오희철 지선하 서울대학교 의과대학 예방의학교실 연세대학교 의과대학 예방의학교실 연세대학교 보건대학원 국민건강증진연구소 원 저 . . ( ) . . , - , , , , . , affymetrix k genechip (genotyping) . (genotype) (cleaning) , ( , , ) . , , . (phenotype) , (arterial stiffness), , , , , (subclinical atherosclerosis), , , , . , . , linkage . linkage (candidate gene approach) . esr c. - t>c polymorphism . cc (genotype) ct tt (genotype) (p value= . ), (p value= . )[ ]. whole-genome wide association . , snps (genotype) . (quantitative traits) pbat “conditional mean model” (multiple testing) (two-stage testing) . , screening , (gen- otype) , snp (power) . no. country starting year study name study phenotype no. of subjects study design usa framingham share phenotypes group , community-based, longitudinal, family based cohort eu countries, and usa international multi-center adhd genetics project adhd , parent-offspring trios australia the victorian family heart study (vfhs) cvd , ( families) family based cohort usa the amish family diabetes study diabetes family study usa the nimh alzheimer’s disease study alzheimer’s disease ( families) family based cohort usa, canada, and australia the breast cancer family registry , families cancer family registry :cupples et al.[ ], :kuntsi et al.[ ], :ellis et al.[ ], :pollin et al.[ ], :bertram et al.[ ], :john et al.[ ] table table table table . . . . examples of family-based study snp , snp . , screening , , , snp power snp , . ( , p-value x - ) power over-correction unrel- ated population - . fbat snp rs p-value . . snp . kora , snp linear regression p-value . . maywood african-american pbat snp p-value . [ ]. k genechip [ ]. (phen- otype) . linkage , fbat generalized estimating equation (gee) . . linkage association . monocyte chemoattractant protein- lod score= . or j or j rs rs snp . factor vii rs snp snp . , , snps whole-genome wide association . . international multi-center adhd genetics (image) project image project (attention deficit disorder with hyperactivity adhd) (parent-child trios) . . , [ ]. adhd - %, - % (neurodeve- lopmental) . - adhd , . , single-nucl- phenotype working group trait snp rs id chr gee p-value fbat p-value in/near gene select biomarkers monocyte chemoattractant protein- rs . * - . * - fceria, orioj monocyte chemoattractant protein- rs . * - . * - orioj kidney/endocrine cystatin c rs . * - . cst l/cst diabetes fasting plasma glucose rs . * - . zmat neruology total cerebral brain volume (atcbv) rs . * - . cdh hemostatic factors factor vii rs . * - . * - f source: cupples et al.[ ] table table table table . . . . results of framingham k genechip eotide polymorphisms (snp) [ ]. haplotype . (multiple testing) type error snp haplotype . whap snp sliding window . whap (http://pngu.mgh.harvard.edu/~purcell/whap/). tph haplotype adhd (transmitted) (psedu-control) (tra- nsmitted) haplotype adhd (p-value = . ). , tag snp genome- wide association scan . . the victorian family heart study (vfhs) , vfhs . , ( - ) ( - ) . (caucasian) . . vfhs genome-wide associ- ation , (height) genome-wide linkage (lod-score . )[ ]. microsatellite marker x cm (resolution) genome-wide linkage . . (region) - cm (resolution) fine-mapping linkage cm . . the amish family diabetes study the amish family diabetes . ( ) . . (lipid) (serum lipid level) linkage [ ]. , . linkage microsatellite markers . centimo- rgans (average density) genome- wide linkage . . gene marker window p-value transmitted non- transmitted or haplotype-specific p-value net - - - - . . . tph - - - - . . . per - - - - . . . adrb - - - - . . . htr e - - - - . . . maoa - - - - . . . chrna - - - - . . . source: brookes et al.[ ] table table table table . . . . haplotype analysis using -snp sliding window method and analyses using unphased ldl p , p , p lod-score . whole-genome wide association . . the nimh alzheimers disease study , . . , . . linkage q snp . fbat cond- itional logistic regression . ubqln snp [ ]. . the breast cancer family registry the breast cancer family registry , , , . , population-based case , , population-based control , , clinic-based control , , clinic-based control [ ]. atm . odds ratio= . [ ]. 가족기반 코호트 연구에서 주로 사용되는 통계분석방법 . linkage 분석 linkage . linkage (disease locus) (recombination) . (recombination) (crossing-over) . , link- age (segregate) (no linkage) . , (recombination) (θ) . % (recombination) . (marker) (disease gene) (recombination rate) chromosome location distance (cm) closet marker(s) trait lod (p-value) positional candidate genes p d s /d s ldl-c . ( . ) apob, lpin , abcg , abcg p d s ldl-c . ( . ) pparg q d s lntg . ( . ) apoc , apoa , apoa , apoa p d s ldl-c . ( . ) ldlr d s . ( . ) ldl-c: serum low density lipoprotein cholesterol, lntg: ln-transformed tg candidate genes: abcg : atp-binding cassette, subfamily g, member ; abcg : atp-binding cassette, subfamily g, member ; apoa : apolipoprotein a ; apoa : apolipoprotein a ; apoa : apolipoprotein a ; apob: apolipoprotein b; apoc : apolipoprotein c ; ldlr: low density lipoprotein receptor; pparg: peroxisome proliferators-activated receptor-gamma source: pollin et al.[ ] table table table table . . . . multipoint linkage analysis peaks with lod>= . (p< . ) in the amish family diabetes study . linkage [ ]. marker locus restriction fragment length polymorphisms(rflp), variable number of tandem repeats(vntr), microsatellite ( : ca repeats), single nucleotide polymorphism(snp) microsatellite snps . vfhs micros- atellite cm (resolution) whole genome linkage . , vfhs (resolution) fine-mapping linkage . linkage . (extended families) (nuclear families) (pairs of sibs) linkage . linkage (parametric) linkage (non-parametric) linkage . linkage merlin . merlin web-site (http://www.sph.umich.edu/csg/abecasis/merlin/). . tdt 분석과 fbat (association) case-parents trio trans- mission disequilibrium test (tdt) (extended family-based association study data) family based association test (fbat) package . tdt trios ( , ) . tdt . marker allele (transmitted) % . general model m (allele) (transmitted) m (allele) (transmitted) . , m % . m allele (risk allele) ( ). (example) (dad) m allele (transmitted) , m allele (m allele) (transmitted) . b cell (count) . (mom) m allele(m ) , m allele(m ) . d cell (count) . trios a,b,c,d cell (count) . m % . b cell c cell m m m m m m m m m m m m m m m m m m null hypothesis (ho): probability of transmitting the m allele is . mcnemer’s chi-square test:  =(b-c) /(b+c) degree of freedom (d.f.)= if  ＞ . , then reject hom a b c d m m tra n sm itte d not transmitted m m m m tra n sm itte d not transmitted m m m m tra n sm itte d not transmitted m a) general model b) example c) example fig.fig.fig.fig. . . . . example of tdt analysis . mcnemar chi-square test: Χ =(b-c) /(b+c) [ ]. fbat tdt trios (extended family-based asso- ciation study data) . tdt (binary trait) fbat (quantitative trait) . (missing) fbat . fbat website (http://www.biostat. harvard.edu/~fbat/fbat.htm) [ ]. illumina affymerix gene chip whole-genome association study . tdt plink [ ]. . imprinting (parent-of-origin) effect 분석 imprinting(parent-of-origin) effect screening cordell stata . cordell (maternal transmission) (paternal transmission) tdt [ ]. cordell imprinting effect . bipolar disorder tdt . snp rs (transmission) p-value . , (transmission) p-value . , (transmission) p-value . . snp bipoloar disorder [ ]. , plink snp (gwa ) imprinting effect screening [ ]. imprinting effect weinberg log-linear . imprinting (genotype effect) (genotype effect) (adjustment) imprinting [ ]. 가족기반 코호트 연구의 장점, 단점 및 문제점 . 장점 . , popu- lation stratification . population stratification (allele freq- rs rs t u p-value t u p-value stratified tdt paternal . paternal . maternal . maternal . total . total . source: mulle et al.[ ] t: transmitted, u: untransmitted table table table table . . . . transmission of alleles to bipolar i offspring, stratified by parent-of-origin of alleles uency) - (false positive) . , linkage association . linkage association [ ]. , (heritability) . , familial risk . epigenetic study (unrelated individual) (information) . , , , imprinting . imprinting imprinting [ ] , imprinting . . 단점 및 문제점 - [ ]. , - [ ]. , tdt main effect . , , main effect [ ]. , “ ” (gee ) , genetic correlation environmental correlation . 가족기반 코호트 연구의 방향 및 전망 linkage (candidate gene) , genome-wide association (gwa) . gwa snp (multiple testing) . - multi-stage . multi-stage , - data set screening testing [ ]. gwa . - . , - population substructure . wellcome trust case control conso- rtium genome-wide association population structure [ ]. gwa - [ ]. imprinting . , image project gwa . gwa [ ]. gwa . 요 약 . population structure , imprinting (association) linkage . image project genome-wide association . linkage tdt , imprinting effect . . - genome-wide association . genome-wide association . genome- wide association genome-wide imprinting . . 참고문헌 . collins fs. the case for a us prospective cohort study of genes and environment. nature. may ; ( ): - . . gauderman wj, conti dv. commentary: models for longitudinal family data. int j of epidemiology ; : - . . cupples la, arruda ht, benjamin ej, d’agostino rb sr, demissie s, destefano al, dupuis j, falls km, fox cs, gottlieb dj, et al. the framingham heart study k snp genome-wide association study resource: overview of phenotype working group reports. bmc med genet. ; suppl :s . . kuntsi j, neale bm, chen w, faraone sv, asherson p. the image project: methodological issues for the molecular genetic analysis of adhd. behav brain funct. aug ; : . . ellis ja, scurrah kj, duncan ae, lamantia a, byrnes gb, harrap sb. comprehensive multi-stage linkage analyses identify a locus for adult height on chromosome p in a healthy caucasian popul- ation. hum genet. apr; ( ): - . . pollin ti, hsueh wc, steinle ni, snitker s, shuldiner ar, mitchell bd. a genome-wide scan of serum lipid levels in the old order amish. atherosclerosis. mar; ( ): - . . bertram l, hiltunen m, parkinson m, ingelsson m, lange c, ramasamy k, mullin k, menon r, sampson aj, hsiao my, elliott kj, velicelebi g, moscarillo t, hyman bt, wagner sl, becker kd, blacker d, tanzi re. family-based association between alzheimer’s disease and variants in ubq ln . n engl j med. mar ; ( ): - . . john em, hopper jl, beck jc, knight ja, neuha- usen sl, senie rt, ziogas a, andrulis il, anton- culver h, et al. the breast cancer family registry: an infrastructure for cooperative multinational, interdisciplinary and translational studies of the genetic epidemiology of breast cancer. breast cancer res. ; ( ):r - . . shearman am, cupples la, demissie s, peter i, schmid ch, karas rh, mendelsohn me, hous- man de, levy d. association between estrogen receptor alpha gene variation and cardiovascular disease. jama. nov ; ( ): - . . herbert a, gerrynp, mcqueen mb, heid im, pfeufer a, illig t, wichmann 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oxford universitypress. ; - . . spielman rs, mcginnis re, ewens wj. transm- ission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (iddm). am j hum genet. mar; ( ): - . . laird nm, lange c. family-based designs in the age of large-scale gene-association studies. nat rev genet. may; ( ): - . . purcell s, neale b, todd-brown k, thomas l, ferreira ma, bender d, maller j, sklar p, de bakker pi, daly mj, sham pc. plink: a tool set for whole-genome association and population-based linkage analyses. am j hum genet. sep; ( ): - . . cordell hj, barratt bj, clayton dg. case/pseudoc- ontrol analysis ingenetic association studies: a unified framework for detection of genotype and haplotype associations, gene-gene and gene-envi- ronment interactions, and parent-of-origin effects. genet epidemiol. apr; ( ): - . . mulle jg, fallin md, lasseter vk, mcgrath ja, wolyniec ps, pulver ae. dense snp association study for bipolar i disorder on chromosome p suggests two loci with excess paternal transmi- ssion. mol psychiatry. apr; ( ): - . . weinberg cr. methods for detection of parent- of-origin effects in genetic studies of case-parents triads. am j hum genet. jul; ( ): - . . le stunff c, fallin d, bougnères p. paternal transmission of the very common class i ins vntr alleles predisposes to childhood obesity. nat genet. sep; ( ): - . . hopper jl, bishop dt, easton df. population- based family studies in genetic epidemiology. lancet. oct - ; ( ): - . . weinberg cr, umbach dm. a hybrid design for studying genetic influences on risk of diseases with onset early in life. am j hum genet. oct; ( ): - . . van steen k, mcqueen mb, herbert a, raby b, lyon h, demeo dl, murphy a, su j, datta s, rosenow c, christman m, silverman ek, laird nm, weiss st, lange c. genomic screening and replication using the same data set in family-based association testing. nat genet. jul; ( ): - . . wellcome trust case control consortium. genome- wide association study of , cases of seven common diseases and , shared controls. nature. jun ; ( ): - . . sung j, cho si. strategy considerations in genome cohort construction in korea. korean j prev med. mar; ( ): - . family-based designs are commonly used in genetic association studies to identify and to locate genes that underlie complex diseases. in this paper, we review two examples of genome-wide association studies using family-based cohort studies, including the framingham heart study and international multi-center adhd genetics project. we also review statistical methods of family-based designs, including the transmission disequilibrium test (tdt), linkage analysis, and imprinting effect analysis. in addition, we evaluate the strengths and limitations of the family-based cohort design. despite the costs and difficulties in carrying out this type of study, a family-based cohort study can play avery important role in genome wide studies. first, the design will be free from biases due to population heterogeneity or stratification. moreover, family-based designs provide the opportunity to conduct joint tests of linkage and association. finally, family-based designs also allow access to epigenetic phenomena like imprinting. the family-based cohort design should be given careful consideration in planning new studies for genome-wide strategies. : family-based cohort study, transmission disequilibrium test (tdt), linkage study examples and outlook of family-based cohort study jae woong sull , ) , sue kyung park ) , heechoul ohrr ) , sun ha jee , ) diabetes care, volume , number , january o b s e rvat i o n s diabetes scre e n i n g practices among i n d i v i d u a l s a g e d ye a r s a nd o l d e r i n , the american diabetes associa- tion (ada) adopted new re c o m m e n d a- tions for screening the general popula- tion aged years for diabetes every years with an emphasis on those at high risk for undiagnosed diabetes ( ). few studies have examined the extent to which this screening has been adopted. this re p o rt describes the results of a telephone s u rvey of montana residents aged years to assess the diabetes screening prac- tices in this population. f rom october to december , the montana department of public health and human services conducted a random household telephone survey of montana residents aged years living in counties. respondents indicated whether they ever had been told by a physician that they had diabetes, the number of visits they made to a health care provider during the past year, their family history of dia- betes, whether they had ever been told they had high cholesterol and/or high blood pre s s u re, and their height and weight. respondents were asked the fol- lowing question to identify whether they had ever been screened for diabetes: “glu- cose or sugar is a substance found in your blood. have you ever had your blood glu- cose or sugar checked to see if you have diabetes?” when respondents re s p o n d e d “yes” to this question, they were asked to identify when screening was completed (“when was the last time your blood glu- cose or sugar level was measured by a health care professional?”). the re s p o n s e categories for this question included within the past year, within the past years, years ago, do not know/not sure, and refused to answer. pearson tests were used to assess associations between dia- betes screening and risk factors for dia- betes. logistical re g ression analyses were conducted to identify independent vari- ables associated with screening for diabetes during the past year. odds ratios ( % cis) were calculated. of the , respondents, ( . %) re p o rted that they had diagnosed diabetes. the remaining , respondents re p o rt e d that they did not have diagnosed diabetes and are included in the following analyses. of the respondents, % re p o rted a family h i s t o ry of diabetes, % re p o rted a bmi kg/m , % re p o rted having hyper- tension, and % re p o rted having high c h o l e s t e rol. excluding age, % of re s p o n- dents had one risk factor for diabetes, and % had two or more risk factors. of the , respondents without diagnosed dia- betes, % re p o rted that they had been s c reened for diabetes during the past year, % re p o rted screening from to years ago, and % re p o rted screening years ago or having never been scre e n e d . respondents who re p o rted being s c reened for diabetes during the past year w e re more likely to be age years and to have a family history of diabetes, two or m o re visits to a health care provider dur- ing the past year, hypertension, and high c h o l e s t e rol levels (table ). we found no association between recent screening and sex ( % men vs. % women), ameri- can indian ancestry ( % yes vs. % no), or bmi ( % kg/m vs. % kg/m ). respondents with three or m o re risk factors (e.g., aged years, american indian ancestry, family history of diabetes, hypertension, high choles- t e rol, or bmi kg/m ) were more likely to be screened for diabetes compared with respondents with only one risk factor ( vs. %, respectively). however, % of individuals with two risk factors for dia- betes and % of individuals with more than three risk factors had not been s c reened during the past years. based on logistical re g ression analysis, t h ree factors were associated with scre e n i n g for diabetes during the past year: two or m o re visits to a health care provider during the past year ( . [ % ci . – . ]), high cholesterol level ( . [ . – . ]), and family history of diabetes ( . [ . – . ]). respondents aged – years were less likely to re p o rt re c e n t s c reening than those aged years ( . [ . – . ] ) . a limitation of this assessment is that these data are self-re p o rted. pre v i o u s studies, however, have found that self- re p o rts of conditions such as diabetes and h y p e rtension are reliable ( , ). in addi- tion, the survey was conducted by tele- phone and does not re flect the experience of individuals in montana homes without t e l e p h o n e s . the findings suggest that diabetes s c reening is being adopted by physicians for individuals aged years at risk for diabetes and that the ada re c o m m e n d a- tions are being implemented in the general c o m m u n i t y. however, these data also indi- cate a need to develop strategies to encour- l e t t e r s table —characteristics of respondents aged years reporting screening for diabetes in montana in s c reening for diabetes past year – years years or never n age (years) – ( ) ( ) ( ) – ( ) ( ) ( ) ( )* ( ) ( ) family history of diabetes ye s ( )* ( ) ( ) n o ( ) ( ) ( ) visits to a health care provider during the past year ( )* ( ) ( ) ( ) ( ) ( ) h y p e rt e n s i o n ye s ( )* ( ) ( ) n o ( ) ( ) ( ) high cholestero l ye s ( )* ( ) ( ) n o ( ) ( ) ( ) data are n (%). *p . . diabetes care, volume , number , january letters age screening among all individuals at high risk for diabetes. todd s. harwell, mph jane g. smilie, ba janet m. mcdowall, rn, bsn steven d. helgerson, md, mph dorothy gohdes, md f rom the montana diabetes project, montana d e p a rtment of public health and human serv i c e s , helena, montana. a d d ress correspondence to todd s. harw e l l , mph, montana department of public health and human services, cogswell building, c- , p. o . box , helena, mt - . e-mail: t h a rw e l l @ s t a t e . m t . u s . a c k n o w l e d g m e n t s — this project was sup- p o rted through a cooperative agreement (u- / ccu- - ) with the centers for disease c o n t rol and prevention, division of diabetes translation, atlanta, georg i a . we thank linda priest and the staff mem- bers at northwest resource consultants for their work on the telephone surv e y. the contents of this letter are solely the responsibility of the authors and do not neces- sarily re p resent the official views of the centers for disease control and pre v e n t i o n . r e f e re n c e s . american diabetes association: screening for type diabetes. diabetes care (suppl. ): s –s , . kehoe r, wu sy, leske mc, chylack lt jr: comparing self-re p o rted and physician- re p o rted medical history. am j epidemiol : – , . jackson c, jatulis de, fortmann sp: the behavioral risk factor survey and the stan- f o rd five-city project survey: a comparison of cardiovascular risk behavior estimates. am j public health : – , h b a c is not recommended as a s c reening test for diabetes in cystic fibro s i s i n the june issue of diabetes care, h u n k e rt et al. ( ) recommend the use of h b a c for early detection of cystic fib ro- s i s – related diabetes (cfrd). their re c o m- mendation is based on the finding that mean hba c is slightly higher in cystic fib rosis (cf) patients requiring insulin t h e r a p y, compared with cf patients with i m p a i red or normal glucose tolerance. h o w e v e r, no data on the validity of this a p p roach for the diagnosis of asympto- matic diabetes in patients with cystic fib ro- sis are pre s e n t e d . our group has a long-standing intere s t in the early diagnosis of cfrd, comparing fasting blood glucose levels with oral glu- cose tolerance test results ( ). in our series, we have now cf patients with newly diagnosed diabetes based on a -h venous plasma glucose value mg/dl ( . mmol/l), and simultaneous determ i n a t i o n of hba c ( h i g h - p e rf o rmance liquid chro- matography method [pharmacia, erlan- gen, germany], normal range . – . %). only out of cf patients ( %) diag- nosed as diabetic according to the ameri- can diabetes association and world health o rganization criteria ( ) had an hba c value above the normal range (individual values . , . , . , and . %). in nine diabetic cf patients with normal hba c, values between . and . % were e n c o u n t e red (mean ± sd, . ± . %). the mean -h blood glucose value after inges- tion of oral glucose was not signific a n t l y d i ff e rent between diabetic cf patients with n o rmal hba c ( ± mg/dl, mean ± sd) and diabetic cf patients with elevated h b a c ( ± mg/dl, student’s t t e s t ) . these data clearly demonstrate that the d e t e rmination of hba c is not able to sub- stitute for the oral glucose tolerance test in the early diagnosis of cfrd. our fin d i n g s a re in agreement with several re p o rts in the l i t e r a t u re ( – ) as well as the consen- sus conference on cfrd ( ). in addition to its low sensitivity when used as a diagnos- tic tool for the detection of cfrd, the mea- s u rement of hba c has the disadvantage of considerable interassay variability and lack of standardization. there f o re, we stro n g l y advise against the use of glycosylated hemoglobin as a screening test for the early diagnosis of diabetes in patients with cystic fib ro s i s . reinhard w. holl, md christian buck, md christine babka, md anna wolf, md angelika thon, md f rom the department of pediatrics (r.w.h.), uni- versity of giessen, giessen; the department of pedi- atrics (c.bu., c.ba., a.w.), the university of ulm, ulm; and the medical school hannover (a.t. ) , h a n n o v e r, germ a n y. a d d ress correspondence to pd dr. reinhard w. holl, universitätskinderklinik giessen, feulgenstr. , d- giessen, germ a n y. r e f e re n c e s . h u n k e rt f, lietz t, stach b, kiess w: potential impact of hba c d e t e rm i n a t i o n on clinical decision making in patients with cystic fib ro s i s – related diabetes (let- ter). diabetes care : – , . holl rw, buck c, cario h, wolf a, thon a, heinze e, kohne e, debatin k-m: diag- nosis of diabetes in cystic fib rosis and tha- lassemia major. diabetes care : – , . the expert committee on the diagnosis and classification of diabetes mellitus: r e p o rt of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care : – , . lanng s, hansen a, thorsteinsson b, n e rup j, koch c: glucose tolerance in patients with cystic fib rosis: five year p rospective study. b m j : – , . deluca f, arrigo t, nibali sc, sferlazzas c, gigante a, dicesare e, cucinotta d: insulin secretion, glycosylated haemoglo- bin and islet cell antibodies in cystic fib ro- sis children and adolescents with diff e re n t d e g rees of glucose tolerance. h o rm metab r e s : – , . moran a, doherty l, wang x, thomas w: a b n o rmal glucose metabolism in cystic fib rosis. j pediatr : – , . moran a: highlights of the febru a ry consensus conference on cfrd. bonn, g e rm a n y, cystic fibrosis foundation, p ro gln p e roxisome p ro l i f e r a t o r- a c t i v a t e d r e c e p t o r- and o b e s i t y r istow et al. ( ) re p o rted an activating mutation in the peroxisome pro l i f- e r a t o r-activated re c e p t o r- g e n e ( p ro gln ppa r - ), which was pre s e n t in % ( of ) of obese and % ( of ) of nonobese german caucasians. these findings may have profound implica- tions, particularly if the presence of this variant and its association with obesity are c o n firmed in other populations. we perf o rmed polymerase chain re a c- t i o n – restriction fragment length polymor- phism analysis for the pro gln ppa r - variant as described ( ) on dna samples f rom several independent populations, including lean and obese caucasians fro m the baltimore, maryland, region; african- diabetes care, volume , number , january letters americans from jackson, mississippi, and forsyth county, north carolina; pima indians from arizona; and old ord e r amish from lancaster county, pennsylva- nia (table ). a pcr fragment corre s p o n d- ing to gastric insulinotropic peptide, which has two known restriction sites for h i n dii, was mixed with each sample as a positive control. among a total of sub- jects ( , alleles), the pro gln variant was not detected in a single subject. these findings were unexpected because there is substantial overlap of gene pools of caucasians from central e u rope and the baltimore and amish caucasians studied ( ). the germ a n caucasians studied by ristow et al. were said to be unrelated and re c ruited fro m the nord rh e i n - westfalen region, but they may be a genetic isolate whose gene pool does not re flect that of other caucasian populations. altern a t i v e l y, if the individ- uals carrying the mutation were re l a t e d , the true frequency of the pro g l n p pa r - variant may have been overe s t i- mated. the absence or very low fre- quency of this variant has also been doc- umented in danish ( ) and german ( ) populations. this study is the first, to our knowledge, to examine american popu- lations for this variant. in summary, the study by ristow et al. demonstrating that the pro g l n p pa r - variant is activating and can influence body weight in people is important. however, this variant appears to be absent or very r a re in the american populations studied. additional studies are re q u i red in other regions of europe and the u.s. to furt h e r d e fine the relevance of this intere s t i n g genetic variant to susceptibility to obesity. alan r. shuldiner, md william nguyen, bs w.h. linda kao, phd brock a. beamer, md ross e. andersen, phd richard pratley, md frederick l. brancati, md, phd f rom the department of medicine (a.r.s., w. n . ) , university of maryland school of medicine; the d e p a rtments of medicine (b.a.b., f.l.b.) and epi- demiology (w.h.l.k.), johns hopkins university school of medicine, baltimore, maryland; and the national institute of diabetes and digestive and kidney diseases (r.p.), national institutes of health, phoenix, arizona. a d d ress correspondence to alan r. shuldiner, md, professor and head, division of endocrinol- o g y, diabetes, and nutrition, department of medi- cine, university of maryland school of medicine, w. lombard st., room s- , baltimore, md . e-mail:ashuldin@medicine.umary l a n d . e d u . a c k n o w l e d g m e n t s — this study was sup- p o rted by nih r dk- , the american diabetes association, glaxowellcome, the b a l t i m o re geriatrics research and education clinical center of the baltimore ve t e r a n s administration medical center. r e f e re n c e s . ristow m, muller- wieland d, pfeiffer a, k rone w, kahn cr: obesity associated with a mutation in a genetic regulator of adipocyte diff e rentiation. n engl j med : – , . c a v a l l i - s f o rza ll, menozzi p, piazza a: t h e h i s t o ry of human genes. princeton univer- sity press, princeton, nj, . elk j, urhammer sa, sorensen ti, ander- sen t, auwerx j, pedersen o: homozygosity of the pro ala variant of the pero x i s o m e p roliferation-activated re c e p t o r- g a m m a ( p par-gamma ): divergent modulating e ffects on body mass index in obese and lean caucasian men. diabetologia : – , . mamann a, munzberg h, buttron p, busing b, hinney a, mayer h, siegfried w, hebe- brand j, greten h: missense variants in the human peroxisome pro l i f e r a t o r- a c t i v a t e d re c e p t o r-gamma gene in lean and obese subjects. eur j endocrinol : – , insulin secre t i o n , insulin sensitivity, and glucose e ffectiveness in nonobese individuals with va ry i n g d e g rees of glucose to l e r a n c e a lthough it is well known that insulin s e c retion, insulin sensitivity, and glu- cose effectiveness are impaired in type diabetic patients ( – ), little is known about the role of each of these fac- tors individually on the evolution of type diabetes. in this context, a major issue is that hyperglycemia per se impairs insulin s e c retion and insulin sensitivity and that obesity observed in type diabetic patients per se causes insulin resistance ( , ). to o v e rcome this problem, we studied u n t reated nonobese subjects classified as having normal glucose tolerance (ngt) (n = ; bmi . ± . kg/m [ r a n g e . – . ], mean ± sem), impaired glu- cose tolerance (igt) (n = ; bmi . ± . kg/m [ . – . ]), and type dia- betes (n = ; fetal bovine serum . ± . mmol/l [range . – . ], bmi . ± . k g / m [ . – . ]), based on the criteria of the world health organization ( ). they were normotensive and had norm a l renal, hepatic, and thyroid function. insulin sensitivity and glucose eff e c t i v e n e s s w e re estimated by the minimal model a p p roach ( – ). insulin secretion was e x p ressed as the area under the insulin c u rve between and min after an intra- venous glucose injection ( ). after the data w e re analyzed by one-way analysis of vari- ance, bonferroni correction was used to evaluate the diff e rences between any two of the three groups we studied ( ). no signifi- cant difference was observed in bmi among the three groups. compared with the subjects with ngt, the subjects with table —characteristics of subjects screened for pro gln ppa r - n ( a l l e l e s age ± sd f e m a l e bmi ± sd p o p u l a t i o n t y p e d ) ( y e a r s ) ( % ) ( k g / m ) c a u c a s i a n s b a l t i m o re longitudinal ( ) . ± . . . ± . study on aging johns hopkins we i g h t ( ) . ± . . . ± . management center amish, lancaster, pa ( ) . ± . . . ± . a f r i c a n - a m e r i c a n s a t h e ro s c l e rosis risk in ( ) . ± . . . ± . communities study pima indians a r i z o n a ( ) . ± . — . ± . all non-amish subjects were unrelated; amish subjects were not fir s t - d e g ree relatives of each other. diabetes care, volume , number , january letters igt had significantly lower insulin secre- tion ( , ± vs. , ± pmol l m i n , p = . ) and glucose eff e c- tiveness ( . ± . vs. . ± . m i n , p . ). insulin sensitivity index was lower in subjects with igt ( . ± . m i n pmol l) than in those with ngt ( . ± . min pmol l ) , but was not statistically significant (p = . ). in con- trast, disposition index calculated by the p roduct of insulin secretion and insulin sensitivity was significantly lower in sub- jects with igt ( , ± ) than in those with ngt ( , ± , p = . ). on the other hand, patients with type dia- betes had significantly lower insulin secre- tion ( ± pmol l m i n , p = . ) compared with subjects with igt. although no significant diff e rence was observed in insulin sensitivity index between subjects with type diabetes and igt ( . ± . vs. . ± . min pmol l, p = . ), disposition index was s i g n i ficantly diminished in type diabetic patients as compared with subjects with igt ( ± vs. , ± , p = . ). glucose effectiveness in type diabetic patients ( . ± . min ) was similar to that in subjects with igt ( . ± . m i n , p = . ) but was signific a n t l y lower than that in the subjects with ngt (p . ). from these results, the fol- lowing may be hypothesized: ) impair- ments in insulin secretion and disposition index and decreased glucose eff e c t i v e n e s s , but not insulin resistance, seem to consti- tute the basic characteristics of patients with igt or type diabetes in nonobese japanese populations. ) risk factors wors- ening to type diabetes in subjects with igt are associated with further impair- ments in insulin secretion and disposition index, but not associated with furt h e r derangement in glucose effectiveness in japanese populations. ataru taniguchi, md mitsuo fukushima, md masahiko sakai, md itaru nagata, md kentaro doi, md shoichiro nagasaka, md kumpei tokuyama, md yoshikatsu nakai, md f rom the first department of internal medicine ( a . t., m.s., i.n.), kansai-denryoku hospital, and the department of internal medicine, hoshida- minami hospital (m.f.), osaka; the second depart- ment of internal medicine (k.d.) and the college of medical technology (y.n.), kyoto university, kyoto; the department of internal medicine (s.n.), jichi medical college, tochigi; and the laboratory of biochemistry of exercise and nutrition (k.t. ) , tsukuba university, tsukuba, japan. a d d ress correspondence to ataru ta n i g u c h i , md, first department of internal medicine, kansai- d e n ryoku hospital, - - , fukushima-ku, fuku- shima, osaka city, osaka - , japan. e-mail: k @ k e p c o . c o . j p . r e f e re n c e s . b e rgman rn: to w a rd physiological under- standing of glucose tolerance: minimal- model approach. d i a b e t e s : – , . welch s, gebhart ssp, bergman rn, phillips ls: minimal model analysis of intravenous glucose tolerance test-derived insulin sensitivity in diabetic subjects. j c l i n endocrinol metab : – , . taniguchi a, nakai y, fukushima m, kawamura h, imura h, nagata i, tokuyama k: pathogenic factors re s p o n s i- ble for glucose tolerance in patients with niddm. d i a b e t e s : – , . nagasaka s, tokuyama k, kusaka i, hayashi h, rokkaku k, nakamura t, kawakami a, higashiyama m, ishikawa s, saito t: endogenous glucose pro d u c t i o n and glucose effectiveness in type diabetic subjects derived from stable-labeled mini- mal model approach. d i a b e t e s : – , . best jd, kahn se, ader m, watanabe rm, ni t-c, bergman rn: role of glucose eff e c- tiveness in the determination of glucose tol- erance. diabetes care : – , . rossetti l, giaccari a, defronzo ra: glu- cose toxicity. diabetes care : – , . taniguchi a, nakai y, doi k, fukuzawa h, fukushima m, kawamura h, to k u y a m a k, suzuki m, fujitani j, tanaka h, nagata i: insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis. m e t a b o l i s m : – , . world health organization: diabetes melli - tus: report of a who study gro u p . g e n e v a , world health org., (tech. rep. ser. , no. ) . winer bj: statistical principles in experimen - tal design. nd ed. new york, mcgraw-hill, , p. – late-onset tro g l i t a z o n e - i n d u c e d hepatic dysfunction r e c e n t l y, iwase et al. ( ) re p o rted a case of liver dysfunction occurring after months of troglitazone therapy. because it was thought before this re p o rt that the risk of liver dysfunction with tro g l i- tazone after months was negligible, we wish to re p o rt another patient who took t roglitazone intermittently and developed hepatic dysfunction after months. a -year-old white man with type diabetes, ischemic heart disease (post angioplasty and stent placement), hyper- tension, degenerative joint disease, benign p rostatic hypert ro p h y, dyslipidemia, and g a s t roesophageal re flux disease had tro g l i- tazone mg daily added to his re g i m e n of glimeperide mg daily and metform i n mg b.i.d. because of poor glycemic c o n t rol (hba c . % [normal – %]). the other medicines he used were aspirin and pravastatin. after months of triple oral therapy, his hba c level dropped to . %, and, after months, to . %. after months, the patient’s hba c began to rise: . % at months, . % at year, and . % at months. liver function tests were normal until months, when his aspartate amino- transferase (ast) was found to be (nor- mal – u/l) and alanine aminotrans- ferase (alt) (normal – u/l). the t roglitazone regimen was discontinued, and testing for hepatitis b and c, h e m a c h romatosis, autoimmune liver dis- ease, and gallbladder disease were nega- tive. two months after discontinuing t roglitazone, the patient’s ast and alt had decreased to and u/l, and, after months, had re t u rned to normal at and u/l, re s p e c t i v e l y. his ast and alt have remained normal since then and he has continued to take pravastatin, aspirin, m e t f o rmin, and glimeperide. when the patient was told to discon- tinue troglitazone, he admitted that he had been taking it only interm i t t e n t l y. he esti- mated that he took the drug regularly at first, but after the first months, he took the drug only once or twice weekly on aver- age. he gave the following three reasons for his lack of compliance: a lack of funds, a fear of liver disease, and a tendency to avoid taking drugs whenever possible. this case, like the case described by iwase et al., illustrates that the hepatic dysfunction caused by troglitazone can occur after months and further sup- p o rts the u.s. food and drug administra- t i o n ’s current recommendation that quar- terly liver function tests should be obtained when troglitazone utilization extends beyond year ( ). in this case, could the onset of hepatic dysfunction have been delayed because the diabetes care, volume , number , january letters d rug was being taken only interm i t t e n t l y after the first months, and the estimated total load presented to the liver would be equivalent to the exposure at months in a compliant patient? we doubt this, since t ro g l i t a z o n e ’s hepatic effects are thought to be idiosyncratic and there f o re the total e x p o s u re should be irre l e v a n t . david s.h. bell, mb fernando ovalle, md f rom the division of endocrinology and metabo- lism, department of medicine, school of medicine, university of alabama, birmingham, alabama. a d d ress correspondence to david s.h. bell, mb, th ave. s., birmingham, al . d.s.h.b. and f.o. have served on an advisory panel for sankyo parke-davis and have received con- sulting fees, re s e a rch grant support, and honoraria for speaking engagements from sankyo parke-davis. r e f e re n c e s . iwase m, yamaguchi m, yoshinari m, oka- mura c, hirahashi t, tsuji h, fujishima m: a japanese case of liver dysfunction after months of troglitazone tre a t m e n t . diabetes care : – , . parke-davis, division of wa rn e r- l a m b e rt : rezulin package insert. morris plains, nj, g l y b u r i d e - i n d u c e d hemolysis in m y e l o d y s p l a s t i c s y n d ro m e g lyburide, also known as gliben- clamide, is a widely used sulfonylure a to treat patients with type diabetes. hemolytic anemia is an extremely rare side e ffect of which there have been only a few re p o rts ( – ). we describe a patient with myelodysplastic syndrome who pre s e n t e d with glyburide-induced hemolysis. a -year-old man with a long history of type diabetes presented with left foot cellulitis of week’s duration. this patient was known to have slowly pro g re s s i v e pancytopenia for years, for which no work-ups had been perf o rmed. his med- ications included the following: glyburide, mg per day, which he had taken for m o re than year; buformine, mg per day; and boglibose, . mg per day. he was afebrile, and the physical examination was normal, except for localized cellulitis on his left foot, for which he was start e d on intravenous antibiotics. the laboratory studies revealed a white blood cell count of . / l , hemoglobin . g/dl, platelet count /l, reticulocyte count . %, mean cor- puscle volume fl, moderate anisocyto- sis, fasting plasma glucose mg/dl, h b a c . %, lactate dehydrogenase iu/l, total bilirubin . mg/dl, and hapto- globin mg/dl. red cell glucose- - phosphate dehydrogenase level was ade- quate. cold agglutinin test, ham’s test, and sugar water test were normal. both dire c t and indirect coombs’ tests were negative. red cell resistance to osmolarity was mildly low (parpart ’s method). urinalysis demonstrated no urobilinogen. endo- scopic studies did not reveal gastro i n t e s t i- nal bleeding. ultrasonography of the abdomen showed no splenomegaly. the result of bone marrow aspiration was equivocal. on the basis of pre s u m p t i v e glyburide-induced hemolysis, glyburide was discontinued and the patient was switched to subcutaneous insulin on the seventh day. there a f t e r, his hemoglobin level increased to . g/dl, re t i c u l o c y t e count decreased to . %, and anisocytosis d i s a p p e a red pro m p t l y. he was discharg e d with insulin therapy after month in the hospital, which is when the cellulitis re s o l v e d . t h ree months later, his hemoglobin level was . g/dl and his haptoglobin remained low. repeated bone marro w aspiration confirmed the diagnosis of myelodysplastic syndro m e . we conclude that this patient devel- oped glyburide-induced hemolysis super- imposed on red cell fragility secondary to an underlying bone marrow disord e r. t h e re have been several re p o rts of hemoly- sis caused by sulfonylureas, most of which have been considered immune-mediated ( , , ). our case points to the possibility that glyburide could cause hemolysis by a non–immune-medicated mechanism. it is i m p o rtant to be aware of this potential side e ffect of glyburide in light of this medica- t i o n ’s widespread prescription, even though such a side effect is rare . hiroshi noto, md kazuhisa tsukamoto, md satoshi kimura, md f rom the department of diabetes and metabolism, tokyo university hospital, tokyo, japan. a d d ress correspondence to hiroshi noto, md, d e p a rtment of diabetes and metabolism, tokyo uni- versity hospital, - - hongo, bunkyo-ku, to k y o - , japan. e-mail: noto-tky@umin.ac.jp. r e f e re n c e s . nataas ob, nesthus i: immune haemolytic anaemia induced by glibenclamide in selective iga defic i e n c y. b m j : – , . abbate sl, hoogwerf bj: hemolytic ane- mia associated with sulfonylurea use. d i a - betes care : – , . meloni g, meloni t: glyburide-induced acute haemolysis in a g pd-defic i e n t patient with niddm. br j haematol : – , . kopicky ja, packman ch: the mechanism of sulfonylurea-induced immune hemoly- sis: case re p o rt and review of the literature . am j hematol : – , e ffects of exposure at an altitude of , m on p e rf o rmance of glucose meters s elf-monitoring of blood glucose is m a n d a t o ry for type diabetic p a t i e n t s who participate in sports to adjust insulin dose and carbohydrate ingestion ( ). sports also include activities p e rf o rm e d at moderately high altitudes, such as hiking or skiing. capillary blood glucose monitors (bgms) have been shown to underestimate blood glucose values at an altitude of , m ( ) and at a simulated altitude of , m with t e m p e r a t u re and humidity kept constant ( ). the aim of the present study was to assess the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and p o can result in errors in blood glucose d e t e rmination ( ). two bgms, the lifescan one touch ii (ot) (ortho diagnostics, milpitas, ca) and the glucometer elite ii (ge) (bayer diagnostics, brussels, belgium), were tested during a study on the effects of acute exposure at an altitude of , m and exercise on blood pre s s u re and albu- min excretion rate in six type diabetic p a t i e n t s . all subjects (four men and two women) were free of disease-related com- plications and in good and stable glycemic control (ghb . ± . %). all subjects gave their informed and written consent to participate in the study pro t o- col. all subjects were investigated both at diabetes care, volume , number , january letters sea level and after ascent by car and cable car to the angelo mosso institute at col d’olen ( , m altitude), gressoney la trinité, italy. at sea level and at a moderately high altitude, bgm reliability at diff e rent blood glucose levels was tested, and blood glu- cose was assessed in fasting and re s t i n g conditions at : a.m.; at : a.m. b e f o re an in-field exercise test; and imme- d i a t e l y, min, and min after the exer- cise stopped. capillary glucose was simul- taneously assessed with the ot and the ge. both of these bgms measure capillary blood glucose through the glucose oxi- d a s e - p e roxidase reaction. bgms were cali- brated at the beginning of each test ses- sion. a venous blood sample was simulta- neously drawn from the contralateral antecubital vein in a sodium fluoride tube, centrifuged, and stored at °c. plasma glucose was assayed with the glucose oxi- dase method (go) within days. this last assessment was taken as a re f e re n c e method. statistical analysis compare d bgm capillary glucose values and go plasma glucose values for each blood col- lection time. measurement linearity was tested with pearson’s correlation coeff i- cient. the mean of the diff e rences between the bgm and go results re p resents the mean bias between the methods with accuracy expressed as percent error (pe): pe (%) = bmg – go % g o the level of statistical significance was c o n s i d e red to be p . . the ge and ot measurements had a good correlation with plasma glucose both at moderately high altitude and at sea level. p e a r s o n ’s correlation coefficients were . and . for the ge and . and . for the ot at sea level and at moderately high altitude, re s p e c t i v e l y. biases between plasma glucose and bgm measure m e n t s w e re as follows: for the ge, . ± . at sea level and . ± . at moderately high altitude; for the ot, . ± . at sea level and . ± . at moderately high alti- tude. at sea level, both the ge and the ot tended to underestimate glucose values (ns); at moderately high altitude, the ge tended to overestimate and the ot tended to underestimate glucose values (ns). mean pes between plasma glucose and bgm m e a s u rements were . (ot) and . (ge) at sea level and . (ot) and . (ge) at moderately high altitude. pe tended to be higher for both bgms at moderately high altitude (ns). figures and show the bias between the single measurements with both devices at sea level and at moderately high altitude, re s p e c t i v e l y. at moderately high altitude (fig. ), the tendency of the ge to o v e restimate was more evident for low ( mg/dl) and intermediate ( – mg/dl) blood glucose values, whereas the ot tended to underestimate mainly high blood glucose values (ns). in our study, bgm perf o rmance was similar and good at sea level. at a moder- ately high altitude, a tendency to overe s t i- mate blood glucose for the ge and to u n d e restimate for the ot was observ e d . the overestimation for the ge involved mainly low ( mg/dl) and interm e d i- ate ( – mg/dl) blood glucose val- ues. this could present a problem in the p resence of symptoms suggesting hypo- figure —relationship between plasma and capillary glucose at sea level. figure —relationship between plasma and capillary glucose at moderately high altitude. lifescan one touch ii glucometer elite ii lifescan one touch ii glucometer elite ii diabetes care, volume , number , january letters glycemia and normal blood glucose val- ues. the ot tended to undere s t i m a t e mainly high blood glucose values, although its perf o rmance with low to i n t e rmediate values was good. the pre s- ent study assessed the accuracy of two bgms at a moderately high altitude in which changes in temperature, humidity, and po can result in errors in blood glu- cose determination ( ). our results are consistent with previous studies ( , ). the decrease in po could alter the sec- ond phase of the chromogen reaction and u n d e restimate blood glucose values ( ); on the other hand, an increase in atmos- pheric pre s s u re could overestimate blood glucose values ( ). in our study, minimal o v e restimation by the ge at low interm e- diate blood glucose values at moderately high altitude cannot be explained by the a l t e red po . an increase in hematocrit, which is known to alter blood glucose m e a s u rements with bgms ( ), may also occur after prolonged exposure to high altitude or as a consequence of dehydra- tion. although our study did not deter- mine hematocrit, the exercise test was s h o rt, and the patients were instructed to drink according to their thirst during the , -m exposure; there f o re, dehydration was not likely to have occurred. in con- clusion, bgm perf o rmance is similar and good at sea level. at a moderately high altitude similar to that experienced during winter skiing or summer hiking, a ten- dency to overestimate low to norm a l blood glucose values for the ge and to u n d e restimate high blood glucose values for the ot was observed. the bias is not clinically meaningful for either bgm, both of which can be safely used by dia- betic patients during exposure to moder- ately high altitudes. some care in the eval- uation of low and intermediate blood glu- cose values measured with the ge is n e v e rtheless re c o m m e n d e d . oriana pecchio, md simona maule, md marco migliardi, md marina trento, bsc massimo veglio, md f rom the italian alpine club medical commission ( o . p.); the department of internal medicine (m.t. ) , university of turin; the s. giovanni battista hospital (s.m.); and the department of endocrinology (m.m., m . v.), mauriziano umberto i hospital, turin, italy. a d d ress correspondence to dr. m. veglio, via mancini , torino, italy. e-mail: veglio@ o n w. n e t . r e f e re n c e s . h o rton es: role and management of exer- cise in diabetes mellitus. diabetes care : – , . g i o rdano bp, trash w, hollenbaugh l, dube wp: perf o rmance of seven blood glucose testing systems at high altitude. diabetes educ : – , . gautier jf, bigard ax, douce p, duvallet a, cathelinau g: influence of simulated alti- tude on the perf o rmance of five blood glu- cose meters. diabetes care : – , . b a rnett c, ryan f, ballonoff l: effect of altitude on the self monitoring of blood glucose (smbg) (abstract). diabetes (suppl.): a, . piepmeier eh, hammett-stabler c, price me, kemper gb, davis mg: atmospheric p re s s u re effects on glucose monitoring devices (letter). diabetes care : – , . b a rreau pb, buttery je: effect of hematocrit concentration on blood glucose value d e t e rmined on glucometer ii. diabetes care : – , c o m m e n t s a n d r e s p o n s e s deterioration of glycemic contro l after long-te rm treatment wi t h troglitazone in nonobese type diabetic patients t roglitazone is an oral antidiabetic d rug used to treat type diabetic patients with insulin re s i s t a n c e . troglitazone improves overall insulin sen- sitivity in the liver and skeletal muscles, which are the largest consumers and metabolizers of glucose in the body ( – ). recent re p o rts showed that troglitazone is also effective in nonobese type diabetic patients whose hyperglycemia could not be controlled with sulfonylurea therapy ( , ). however, we aware that in some patients in whom adequate glycemic con- t rol is obtained during the first several months of troglitazone treatment, their glycemic control deteriorates several months later. we assume that two distinct g roups of type diabetic patients exist who respond diff e rently to long-term administration of troglitazone, one gro u p that maintains a steady response and another group that has a decre a s i n g response after certain periods. in this s t u d y, we re t rospectively examined patients with type diabetes who were t reated with troglitazone for months and whose hba c levels had improved by % with troglitazone by month . in of the patients ( %), hba c levels increased by . % after – months despite continuous tro g l i t a z o n e t reatment (group p). in contrast, the rest of the patients experienced steady glycemic c o n t rol with . % of hba c flu c t u a t i o n ( g roup g). during the first months, h b a c levels decreased from means ± sem . ± . to . ± . % in group p and fro m . ± . to . ± . % in group g, re s p e c- t i v e l y. no significant diff e rences were evi- dent between the two groups re g a rding the d e c rease in hba c during the first months (fig. ). from month onward, hba c l e v- els in group p climbed gradually by . % a month up to the baseline level at month , but hba c levels were stable in group g t h roughout the treatment period. a signifi- cant diff e rence in hba c levels was evident during months – (p . ) . among clinical characteristics, gro u p p had a significantly lower bmi ( . ± . vs. . ± . kg/m , p . ) and s i g n i ficantly lower fasting insulin levels ( . ± . vs. . ± . µu/ml, p . ). of the patients with a bmi of kg/m ( %), exhibited deteriora- tion of glycemic contro l . in this study, we re p o rt a group of patients who showed a renewed decline in glycemic control after long-term tre a t m e n t with troglitazone. these results seemed to suggest a secondary failure of tro g l i t a z o n e . our study demonstrates that this drug is indeed useful for a long-standing obese i n s u l i n - resistant diabetes but not for a nonobese type diabetes. yuko murase, md takanobu wakasugi, md kunimasa yagi, md hiroshi mabuchi, md f rom the department of internal medicine (y. m . , t. w.), fukui perfectural hospital; and the second d e p a rtment of internal medicine (k.y., h.m.), kanazawa university, ishikawa, japan. a d d ress correspondence to yuko murase, md, the second department of internal medicine, kanazawa university, - takara-machi, kanazawa, ishikawa - , japan. e-mail: diabe@med. k a n a z a w a - u . a c . j p . diabetes care, volume , number , january letters r e f e re n c e s . suter sl, nolan jj, wallace p, gumbiner b, olefsky jm: metabolic effects of new oral hypoglycemic agent cs- in niddm subjects. diabetes care : – , . o’rourke cm, davis ja, saltiel ar, corn i- celli ja: metabolic effects of troglitazone in the goto-kakizaki rat, a non-obese and n o rmolipidemic rodent model of nonin- sulin-dependent diabetes mellitus. m e t a b - o l i s m : – , . troglitazone study group: the metabolic e ffects of troglitazone in non-insulin dependent diabetes (abstract). d i a b e t e s (suppl. ): a, . mori k: the effect of troglitazone in combi- nation with sulfonylurea in non-insulin dependent diabetes mellitus (abstract). j japan diabetes soc (suppl. ): , . h o rton es, venable tc, whitehouse f, the troglitazone study group, ghazzi mn, whitcomb rw: troglitazone in combina- tion with sulfonylurea re s t o res glycemic c o n t rol in patients with type diabetes. diabetes care : – , figure —change from baseline in hba c. values are means ± sem. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ religious culture and economic performance: agricultural productivity of the amish, - m e t i n m. co§gel the fanning practices of the amish have differed in various ways from those of other farmers. i use the information from federal census schedules to examine the differences during the period to in light of amish culture. amish farmers did not resist technological change during - , but the evidence shows other systematic differences. they adopted distinct patterns in investment to ensure the survival of their religious culture, which in turn affected their relative perfor- mance. do religious beliefs affect economic activities and performance? thisis an old and important question, but also a complicated one to address. for large groups, the role of religion is particularly elusive because it is difficult to identify precisely which religious beliefs have a direct impact on economic behavior and to isolate the effect of this behavior on productivity. the problem is highly simplified, however, for smaller religious groups such as the amish, who are well known for their distinct religious beliefs and lifestyle. historians and sociologists have extensively studied the origins, evolution, and significance of amish customs. the effect of amish beliefs on economic activities and performance, however, has received little systematic analysis. in this article i investigate the agricultural practices and productivity of amish farmers in a comparative frame- work, focusing particularly on amish settlements around kalona, iowa, during the period to . the analysis is based primarily on the information recorded in the agriculture and population schedules of the u.s. censuses. comparisons between the amish and other farmers reveal various persistent differences in farming that reflect some of the distinct features of amish culture. the journal of economic history, vol. , no. (june ). © the economic history association. all rights reserved. issn - . the author is assistant professor of economics at the university of connecticut, storrs, ct - . i am grateful to lester j. miller, the staff at the state historical society of iowa city, and the iowa mennonite museum and archives for information on kalona history. george grantham, david wishart, and the editors of this journal provided helpful comments and suggestions. i also thank brad andrew, jennifer easdon, and linda rudolph for research assistance, and the university of connecticut research foundation for financial support. co§gel the amish in iowa the history of the amish people began with the upheavals of the protestant reformation. in the s they emerged as the most orthodox followers of the swiss anabaptist leader, jacob amman. escaping religious persecution, they migrated to america in two waves, first in the early s, and later between and . the first amish families moved to iowa in and formed the largest amish settlement west of the mississippi river in johnson county, near the present town of kalona. this study focuses on the two townships in johnson county where the amish concentration was heaviest, sharon and washington. it also focuses on the period to , because the community had no major schisms before the s, and the current opposition to techno- logical advances had not yet emerged. the amish in these townships differed from other farmers in their religious culture, but faced the same physical constraints in farming. the two townships thus provide a setting where it is possible to assess the effect of religious culture in a comparative framework. moreover, the agriculture schedules of the u.s. censuses for this period provide detailed information about the operations of each farm, allowing for quantitative comparisons. genealogical studies and oral histories make it possible to distinguish amish from other farmers in census records. agriculture schedules recorded amish farmers in , in , in , and in . during the same time period, there were also , , , and non-amish farmers, whose records are available in complete form. farming and the amish how would we expect the religious beliefs of the amish to affect their farming practices and productivity? the amish regulate their economic practices according to their interpretations of the bible and prefer farming over other occupations for religious reasons. the precise rules and norms are recorded in the ordnung, the amish form of common law. it is well known, for example, that amish farmers have generally rejected the use of tractors and various other machinery in farming during the twentieth century and have deviated from mainstream see, for example, hostetler, amish society; kraybiu, riddle; and yoder, tradition, for the history of the amish. see gingerich, mennonites; schwieder and schwieder, peculiar people; wick, amish mennonites; and yoder, "my amish boyhood," for the history of the amish in iowa. these numbers are slightly less than the actual number of farms, because for and the agriculture schedules enumerated only farms with annual produce worth $ or more. for and they enumerated only farms of three or more acres or with an annual produce worth $ or more. amish productivity, - farming practices in other ways. one might therefore expect amish farmers of today to be less productive than their non-amish neighbors because of such differences in farming technology. the amish resistance to technological change in farming, however, is a relatively recent phenomenon. there is little evidence of opposition to mechanized farming before the twentieth century, particularly during the period studied in this paper. amish farmers were some of the first to adopt, and even invent, new developments in technology. therefore, the causes of any difference in their productivity must be sought elsewhere. commentaries on the farming practices of the amish before the twentieth century have consistently praised them as being among the best farmers. the distinct and superior farming practices of the amish are often attributed to their persecution in europe and the confiscation of their land. they were forced to become tenants on marginal land and are said to have excelled in farming to ensure the survival of their communities. it has also been observed that for the iowan amish, success at farming is a sign of god's blessing and strength of character. such observations represent a common sentiment among students of the amish and suggest a positive effect of amish religious beliefs and history on economic performance, relative to other farmers. the performance itself, however, has never been systematically studied, and the superior performance of amish farmers (during the nineteenth century) has almost become an unquestioned presumption. were the amish farmers more productive? agriculture schedules itemize the inputs and outputs of each farm recorded in the censuses, making it possible to calculate the cost and revenue of operating farms. we can aggregate the information on the basis of religious affiliation and use the ratio of revenue to cost as a see, for example, kraybill, riddle, pp. - ; and hostetler, amish society, pp. - , for current agricultural practices and farm technology among the amish. similarly, the ordnung rejects various conveniences such as electricity, television, and cars as "worldly" and sets standardized patterns in clothing, household appliances, and even grooming. but see, for example, berry, unsettling, pp. - , for the superiority of amish practices in other respects. fisher, farm life, chap. ; and yoder, tradition, pp. - . also see gingerich, menno- nites, pp. - ; and yoder, "my amish boyhood," pp. - , for the amish in iowa. the amish do not oppose new technology per se, but for the danger it might present to their community. see correll, schweizerische tdufermennonitentum; and its review by maurer, "review," for the success of mennonite groups in europe. see also kollmorgen, culture, pp. - . gingerich, mennonites, p. . see, for example, hostetler, amish society, pp. - ; kraybill, riddle, pp. - ; stoltzfus, "amish agriculture," pp. - ; and the references in getz, "economic organization," pp. - . co§gel simple measure of productivity to compare the economic performance of amish and other farmers. the information provided by census schedules on cost and revenue is less detailed before . on the cost side, the schedules for and provide only the values of farms, farming implements and machin- ery, and livestock. the estimate of total cost is thus restricted to the user cost of capital only (calculated by multiplying the total value of these assets by the rate of interest). on the revenue side, the schedules provide only earnings generated from orchard products, market gar- dens, home manufactures, and animals slaughtered (or sold for slaugh- ter). we thus need to estimate the revenue from grain production by using market prices and the reported quantities that each farmer produced. the productivity estimates for and thus need to be interpreted with some caution, because not all inputs and products are accounted for in the calculation. if the omitted items are not reasonably proportional to those included, then the estimates will not reflect relative productivities accurately. moreover, the size of the samples, especially for , may be too small to reach any definitive conclu- sions. the census information for and is more detailed, allowing for a more reliable comparison. for example, in both years farmers reported the "estimated value of all farm productions (sold, consumed, or on hand)." furthermore, information about cost included the wages paid (for both and ), the cost of building and repairing fences (for only), and the cost of fertilizers purchased (for only). in addition to the revenue-cost ratio, we can use the average revenue of land as another proxy for productivity. this measure (calculated by dividing the total revenue of each farmer by the acres of improved land he owned) would indicate the productivity of land. although this measure may fail to reflect the productivity of the overall farming operations accurately in the presence of other variable inputs, the two productivity proxies nevertheless appear consistent in this case. federal government bond yield for and railroad bond yields for - are used to calculate the cost of capital, as reported in homer and sylla, history, pp. - . productivity comparisons, reported in table , are robust to other estimates (for example, local mortgage rates reported by bogue, from prairie, p. ), because the same rate applies to all farmers. there are reliable estimates of agricultural prices for iowa beginning in . see strand, prices. prices for are estimated using the price indices for cincinnati, reported by cole, wholesale commodity prices, p. ; and berry, western prices, pp. - . one might object to using the estimates provided by the farmers, because of possible errors or bias in reporting. reported estimates, however, closely reflect market values, as we can infer from the high correlation between farmers' estimates and those calculated from market prices and the quantities reported by each farmer. the simple correlation coefficients between the two estimates of cost and revenue range between . and . across censuses, with no systematic differences between the amish and other farmers. amish productivity, - table comparison of average productivity, amish and non-amish farmers, - revenue-cost ratio amish other average revenue of land ($/acre) amish other sample size amish other . . ( . ) . . ( . ) . . ( . ) . . ( . ) . . ( . ) . . ( . ) . . ( . ) . . ( . ) notes: see the text for an explanation of the calculation of cost and revenue. the r-statistics are in parentheses and test for the difference between the two means. at the percent level, amish farmers had significantly lower revenue-cost ratios during - and lower average revenue of land during - . source: u.s. bureau of the census, census schedules, - . also see notes and . as table demonstrates, comparison of productivities reveals a consistent differential in favor of non-amish farmers. the revenue-cost ratios for and are lower than those for and , due primarily to the difference in the items included in cost. the decline in the ratios for reflects the additional items included in cost and the fall in agricultural prices during the census year. despite such fluctua- tions in the absolute levels of revenue-cost ratios, the relative difference in productivity between amish and non-amish farmers persisted, challenging the conventional view about the superior productivity of amish farmers. note that the productivity proxies used above may be biased because they omit labor inputs not reported in the schedules, such as the unpaid labor input of family members and friends. the productivity differential may thus simply be the result of a difference in the ratio of unpaid labor to other inputs between the two groups of farmers. in our case, however, the direction of the bias actually reinforces the conclusion of table . on amish farms, the labor input of family members was probably higher, because the average family size was significantly higher. amish farmers might also have had greater access to non- household labor within the religious network because of their well- known cooperation in farming operations. see note . co§gel . * ( . ) - . ( . ) - . ( . ) . ( . ) - . * ( . ) - . ( . ) . * ( . ) - . ( . ) - . * ( . ) . ( . ) . . * ( . ) - . ( . ) - . * ( . ) . ( . ) . ( . ) - . ( . ) . * ( . ) - . ( . ) - . * ( . ) . ( . ) . . * ( . ) - . ( . ) . ( . ) - . ( . ) - . ( - ) - . ( . ) - . ( . ) . ( . ) - . * ( . ) . ( . ) . table determinants of productivity, amish and non-amish, native and foreign-born farmers, - variable constant amisha age amish x age nativity " amish x nativity number of products amish x number of products total value of farming assets amish x value of farming assets degrees of freedom r * = significant at the percent level. a dummy variable assigns to amish and to non-amish farmers. b dummy variable assigns to native-born and to foreign-born farmers. c this represents the sum of the values of farm, implements and machinery, and livestock. notes: the revenue-cost ratio is used as the productivity proxy. figures in parentheses are /-statistics. source: u.s. bureau of the census, census schedules, - . explanations of productivity differential to understand why amish farmers were less productive, we must first identify the determinants of productivity. i tested for the effect on productivity of age, nativity, number of products, and the total value of farming assets, using a dummy variable to test for the difference between amish and non-amish farmers in the way each variable affected their productivity. table reports the results. the degree of standardization of our sample can be established by examining the differences in the composition of the amish and non- amish groups that might have contributed to the differential in produc- tivity. the differential might have resulted, for instance, from a gap in the farming experience between the two groups. although census records do not provide information on longevity in farming, we can use age as a proxy for experience. non-amish farmers were indeed older on average by about three years in and (though the average age was approximately equal in ). but the difference in age does not amish productivity, - seem to explain the productivity differential; table reveals either an insignificant (for and ) or a negative (for ) relationship between age and productivity. it would seem that the nativity of farmers might also have affected their productivity. for example, if new immigrants followed traditional methods in farming, their practices and performance might have differed from those of native-born farmers, and significant disparities in the proportion of new immigrants in the two groups could have contributed to the difference in their average productivities. the evidence from our sample, however, does not support this conclusion. although the coefficient of nativity is statistically significant for , the difference in the proportion of foreign-born farmers between the two groups was too small ( percent for the amish and percent for other farmers) to account for the productivity differential. moreover, although the differ- ence in the proportion of foreign-born farmers was substantial in and ( percent and percent for the amish and percent and percent for other farmers), the coefficient of nativity is insignificant in both years, suggesting that nativity had little or no effect on the relative productivities of the amish and other farmers. population characteristics such as age and nativity appear to be insufficient explanations for the productivity differential. to understand why amish farmers were less productive, the relationship between religious beliefs and farming practices must be examined in more detail. amish farmers might have pursued a distinct objective in selecting farming products, which could have reduced their productivity. for example, they might have been highly averse to the risks their commu- nity would have faced by exposure to outside markets and sought to ensure their own self-sufficiency by producing a diverse selection of products. as john hostetler has observed, rather than specialize in certain products, "amish farmers today, as in earlier periods, prefer general farming or a diversity of crops." we can check the applicability of this observation to the period considered in this article by comparing the number of items produced by the amish with those produced by other farmers. among the products listed in the agriculture schedules, amish farmers produced an average of . different items in , . in , . in , and . in . by contrast, other farmers produced an average of . , . , . , and . items. the comparison thus shows greater diversity in the product choices of the amish farmers, especially after . hostetler, amish society, p. . but see stoltzfus, "amish agriculture," pp. - , for recent changes in practices. between and , each new census required the reporting of additional products, which accounts for part of the increase in the average number of items produced by the farmers. the f-statistics for the test of the difference in the averages are . for , . for , . for , and . for . the average number of items produced by the amish farmers was thus significantly higher for and at conventional levels. co§gel the important question in the present context is whether the amish preference for diversity was costly. although product diversity might have helped to insure controlled interaction with the outside world, the amish might have paid a premium for it in lost income. the regression results of table , however, show either a positive (for and ) or an insignificant (for ) relationship between the number of products and productivity. this finding suggests that the product choices of amish farmers did not cause them to be less productive. systematic differences in the use of farming inputs between the amish and non-amish farmers might have caused the differential in productivity. although the amish did not oppose technological change before the twentieth century, they might have had other restraints on the use of farming inputs based on their religious beliefs. for example, as melvin gingerich reports, amish farmers in iowa "object[ed] to mules because they believefd] it contrary to the plan of nature that two species of animals should be crossed." accordingly, census records show no mules and asses on amish farms during the period to . it is difficult to assess, however, how much of the differential in productivity can be attributed to such variations in the composition of livestock holdings or other inputs. more important is the difference in the level of all inputs. as table shows, the coefficient of the total value of farming assets is consistently significant and negative, demonstrating a negative relationship between farm size and productivity. moreover, as table shows, the amount of land as well as the values of farms, farm implements and machinery, and livestock were all significantly greater on average for amish farms. given the differential in productivities, comparison of inputs therefore suggests that amish farmers overinvested in their farming operations, exceeding the optimal size. the question that remains is why the amish invested so heavily in farms, machinery and implements, and livestock, when additional investment actually reduced the efficiency of their operations. the bequest motive the amish were primarily concerned with preserving their religion and maintaining a stable community. because of their belief that they should separate from the world, they minimized contact with outsiders and did not actively seek to recruit members, except from their own families. therefore, although they did not face persecution in iowa, they recognized that their religious traditions could survive only if their children chose to stay in the community and retain the amish religion. therein lies the clue to understanding the effect of religious culture on gingerich, mennonites, p. . amish productivity, - table average size of land and values of farming assets, amish and non-amish, - asset by group land" (acres) amish other value of farm ($) amish other value of machinery and implements ($) amish other value of livestock ($) amish other total value of assets" ($) amish other ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) ( . ) a this represents the sum of improved and unimproved land. b this represents the sum of the values of farm, implements and machinery, and livestock. notes: the /-statistics are shown in parentheses and test for the significance of the difference between two averages. the average size of land and the total value of the assets of amish farmers was significantly higher in and at conventional levels. source: u.s. bureau of the census, census schedules, - . the investment practices of the amish. because the preservation of their religion depended on children, they encouraged large families and invested in the future of their religious community. the amish have always preferred large families. they interpret the biblical passage "go forth and multiply" literally, and advocate large families by encouraging early marriages and opposing birth control. the amish farms in our sample thus housed consistently greater numbers of people than non-amish farms, by an average of about one person per farm. the average household size for amish farms was . in , . in , . in , and . in . by contrast, non-amish farms housed on average . , . , . , and . people during the same years. large families, however, also presented the amish with a problem',' see ericksen et al., "fertility patterns," for recent trends. household size is calculated from the population schedules, and includes members of extended family such as stepchildren and parents, but excludes those boarding on farm for work-related reasons, such as farm laborers and domestic servants. the r-statistics for the test of the difference between the two averages are . for , . for , . for , and . for . the average size of the amish household was significantly higher in all years at the percent level. co§gel table the bequest motive: wealth" regressed on family size variable constant family size (common slope) dummy " x family size (differential effect for the amish) degrees of freedom r . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . . ( . ) . ( . ) . ( . ) . a this represents the total value of the farm, machinery and implements, and livestock. b dummy variable assigns to amish and to other farmers. notes: figures in parentheses are /-statistics. all coefficients, except for the differential effect for i , are significant at conventional levels. source: u.s. bureau of the census, census schedules, - . because children could stay in the community only if they were able to support their own families after they got married. as eugene ericksen, julia ericksen, and john hostetler have found, "[w]hen families are able to set their children up in farming, the children are likely to remain amish." in the absence of established institutions for credit, the ultimate responsibility rested on the parents to secure farms for their children. as gingerich observes for the amish in iowa, "[m]any a father has had an ambition to acquire enough land in a solid block so that each son could be given a farm." large families thus required amish parents to invest heavily in farming inputs (with the expectation that they would be passed down to future generations), suggesting the presence of a bequest motive as an explanation of their investment patterns. a common procedure to test for the presence of the bequest motive is through the relationship between wealth (total value of farming assets) and family size. if the motive was present, we would expect farmers with larger families to hold more wealth. as table shows, the coefficient of family size is positive and significant for all farmers, confirming the presence of the bequest motive in the overall sample. more important, however, is whether the motive was significantly different for amish farmers, an expectation that can be tested by using a dummy variable that differentiates amish from non-amish farmers. as table shows, the coefficient of the differential effect (dummy x family size) is positive and significant for and , confirming the presence of a higher bequest motive for amish farmers. how successful was the bequest motive? comparison of the agricul- ture and population schedules for two consecutive censuses reveals the e r i c k s e n , e r i c k s e n , and h o s t e t l e r , " c u l t i v a t i o n , " p p . - . gingerich, mennonites, p . . s e e , for e x a m p l e , h u r d , " r e s e a r c h , " p p . - , for a review of tests for the bequest m o t i v e . amish productivity, - table success of the bequest motive (number of fanners) status in census records total recorded in first census recorded in both censuses farm taken over in second census not recorded in second census new farmer in second census (x) child in first census " (y) percentage of children among new fanners (y/x) and censuses amish other and censuses amish other " farmer died or retired between censuses; farm taken over by child or spouse. b farmer was recorded as child of farmer parents in the population schedules of the first census and separately as a new farmer in the agriculture schedules of the second census. notes: in addition to population schedules, genealogical studies were used to determine family connections for the amish in a few uncertain cases. similar information is not available for other farmers; thus, the number of farmers reported as "child in first census" may be slightly lower than actual, if the census schedules are incomplete. sources: u.s. bureau of the census, census schedules, - ; and gingerich, cemetery directory. changes in farm ownership that occurred over the decade, making it possible to test the success of the amish in maintaining their religious community. as table shows, among the new farmers recorded in and , a higher percentage of amish than non-amish had farmer parents living in the area a decade ago. similarly, a lower percentage of amish farmers left the area during the period to . these results testify to the stability of the amish community and to the success of amish parents in affecting the decisions of their children to remain amish and continue farming in the same location. the bequest motive of the amish farmers thus clarifies their invest- ment patterns during the period and . the amish sought and successfully achieved the survival of their religious community by investing heavily in land and other inputs in farming. by this means, they secured farms for their children's future, even though the produc- tivity of their own farms lagged behind those of other settlers. they sacrificed current income in order to raise their bequests and invested heavily in the next generation. conclusion the experience of amish fanners who lived near the present town of kalona, iowa, during the period to demonstrates how religious culture can affect economic practices and performance. the amish were primarily concerned with the survival of their religion and co§gel the stability of their communities. their farming practices accordingly differed from those of their non-amish neighbors, particularly in the selection of inputs: they held larger assets in farming in order to safeguard the future of their religious community. such differences in farming practices resulted in lower levels of productivity, contrary to widely held presumptions about the relative performance of amish farmers during the nineteenth century. it would be presumptuous to generalize about the relationship be- tween religion and economic performance from the experience of this one community. amish practices in farming have evolved over time, and other religious groups assuredly have entirely different beliefs and practices. the case of the amish in iowa does show, however, that the shared beliefs and objectives of a religious community can produce distinct patterns of economic behavior and affect their productivity. references berry, thomas s., western prices before (cambridge, ma, ). berry, wendell, the unsettling of america: culture and agriculture (new york, ). bogue, allan g., from prairie to corn belt (chicago, ). cole, arthur h., wholesale commodity prices in the united states: - (cambridge, ma, ). correll, ernst, das schweizerische tdufermennonitentum (tubingen, germany, ). ericksen, eugene p., julia a. ericksen, and john a. hostetler, "the cultivation of the soil as a moral directive: population growth, family ties, and the maintenance of community among the old order amish," rural sociology, (spring ), pp. - . ericksen, eugene p., et al., "fertility patterns and trends among the old order amish," population studies, (july ), pp. - . fisher, gideon, farm life and its changes (gordonville, pa, ). getz, jane c , "the economic organization and practices of the old order amish of lancaster county, pennsylvania," mennonite quarterly review, (jan. ), pp. - ; (apr. ), pp. - . , gingerich, mary a., cemetery directory of amish and mennonites in iowa, johnson, and washington counties of iowa (kalona, ia, ). gingerich, melvin, the mennonites in iowa (iowa city, ia, ). homer, sidney, and richard sylla, a history of interest rates ( st edn., ; rd edn., new brunswick, ). hostetler, john a., amish society ( st edn., ; rd edn., baltimore, ). hurd, michael, "research on the elderly: economic status, retirement, and con- sumption and saving," journal of economic literature, (june ), pp. - . kollmorgen, walter m., culture of a contemporary rural community. rural life studies no. (washington, dc, ). kraybill, donald b., the riddle of amish culture (baltimore, ). maurer, h. m., "review of correll, das schweizerische tdufermennonitentum," american journal of sociology, (mar. ), pp. - . amish productivity, - schwieder, elmer, and dorothy schwieder, a peculiar people: iowa's old order amish (ames, ia, ). stoltzfus, victor, "amish agriculture: adaptive strategies for economic survival of community life," rural sociology, (summer ), pp. - . strand, norman v., prices of farm products in iowa, iowa state college of agriculture and mechanic arts, research bulletin no. (ames, ia, ). u.s. bureau of the census, federal census schedules, national archives and records service, agriculture and population schedules (microfilm, washington, dc, - ). wick, barthinius l., the amish mennonites (iowa city, ia, ). yoder, paton, tradition and transition (scottdale, pa, ). yoder, sanford c , "my amish boyhood," the palimsest, (mar. ), pp. - . doi: . /j.bbabio. . . p/ import and assembly of mitochondrial proteins nikolaus pfanner institute for biochemistry and molecular biology, university of freiburg, germany e-mail: nikolaus.pfanner@biochemie.uni-freiburg.de mitochondria contain about different proteins. % of the proteins are synthesized as precursors on cytosolic ribosomes. the precursors are imported via the translocase of the outer mitochon- drial membrane (tom complex) and are subsequently sorted into the four mitochondrial subcompartments, outer membrane, inter- membrane space, inner membrane and matrix. (i) cleavable preproteins are transported from the tom complex to the presequence translocase of the inner membrane (tim complex). the presequence translocase-associated motor (pam) drives trans- location into the matrix. (ii) hydrophobic inner membrane proteins are transferred through the intermembrane space by a chaperone complex (small tim proteins) and inserted into the inner membrane by the tim complex. (iii) the mitochondrial import and assembly machinery (mia) directs small proteins into the intermembrane space and promotes the formation of disulfide bonds. (iv) beta- barrel proteins are transported from the tom complex to the sorting and assembly machinery of the outer membrane (sam complex). doi: . /j.bbabio. . . p/ new functions for novel mitochondrial transporters ferdinando palmieri university of bari, italy e-mail: fpalm@farmbiol.uniba.it a strikingly large number of mitochondrial dna (mtdna) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. these mitochondrial dis- orders were the first to be investigated after the small mtdna had been sequenced in the 's. only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, hhh syndrome, aspartate/glutamate isoform deficiency, amish microce- phaly and neonatal myoclonic epilepsy; these disorders are characterised by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabo- lism. defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phos- phate and adp, are responsible for diseases characterised by defective energy production. herein, all the mitochondrial carrier- associated diseases known to date are reviewed for the first time. particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders. doi: . /j.bbabio. . . p/ the water oxidizing enzyme a. william rutherford ibitec-s, ura cnrs, cea saclay, gif-sur-yvette, france e-mail: alfred.rutherford@cea.fr photosystem ii, the water oxidising enzyme of photosynthesis, put the energy (or at least a major fraction of it) into the biosphere and the oxygen into the atmosphere. it is certainly one the most influential and important enzymes on the planet. the aim of our research is to understand how this enzyme works as ) a solar energy converter and ) the only known thermodynamically efficient catalyst for oxidizing water. the information obtained is used in the design of artificial catalysts and photocatalysts. a chemical catalyst that has the thermodynamic efficiency of the enzyme could greatly improve the efficiency of ) water electrolysis and photolysis for fuel (e.g. h ) production and ) the reverse reaction, oxygen reduction, in fuel cells. there is therefore a great interest in understanding the mechanism of this enzyme and in reproducing aspects of its function in artificial systems. i will describe our current knowledge of photosystem ii, including some recent experimental studies, as well as recent efforts in our joint saclay/orsay program aimed at producing bio-inspired water oxidizing catalysts. doi: . /j.bbabio. . . p/ the structure of purple bacterial antenna complexes: from single molecules to native membranes richard j. cogdella, alastair t. gardinera, mads gabrielsena, aleks w. roszaka, june southalla, tatas brotosudarmoa, neil w. isaccsa, hideki hashimotob, juergen baierc, silke oellerichc, martin richterc, juergen koehlerc, francesco franciad, giovanni venturolid, dieter oesterhelte adivision of biochemistry and molecular biology, ibls and department of chemistry, university of glasgow, glasgow g qq, uk bcrest-jst and department of physics, graduate school of science, osaka city university, - - sugimoto, sumiyoshi-ku, osaka - , japan cexperimental physics iv, university of bayreuth, d- bayreuth, germany ddepartment of biology, university of bologna, bologna, italy edepartment of membrane biochemistry, max-planck institute for biochemistry, martinsried, germany e-mail: r.cogdell@bio.gla.ac.uk the photosynthetic unit of purple photosynthetic bacteria typically contains two types of light-harvesting complexes, called lh and lh . these antenna complexes are constructed on a modular principle. they are circular or elliptical oligomers of dimers of two low-molecular weight, hydrophobic apoproteins, called a and b, that bind bacteriochlorophylls and carotenoids non-covalently. the lh complex surrounds the reaction centre and, depending on the species, is either a monomer or a dimer. the lh complexes are arranged around the lh -rc complexes. this plenary lecture will present the current status of structural studies on these pigment- protein complexes, based upon a combination of x-ray crystal- lography and single molecule spectroscopy. then an overall view of how they are arranged in their native photosynthetic membranes will be presented. doi: . /j.bbabio. . . p/ catalysis of substrate conversion and electron transfer by mitochondrial complex i judy hirst medical research council dunn human nutrition unit, cambridge, cb xy, uk e-mail: jh@mrc-dunn.cam.ac.uk s abstracts / biochimica et biophysica acta ( ) s –s http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . http://dx.doi.org/doi: . /j.bbabio. . . mailto:nikolaus.pfanner@biochemie.uni-freiburg.de mailto:fpalm@farmbiol.uniba.it mailto:alfred.rutherford@cea.fr mailto:r.cogdell@bio.gla.ac.uk mailto:jh@mrc-dunn.cam.ac.uk virtual mentor virtual mentor american medical association journal of ethics september , volume , number : - . history of medicine a brief history of environmental bioethics cristina richie as a response to anthropogenic—that is, human-caused—climate change, nearly every sector of public and private life has been scrutinized by ecologists. automobile pollution, greenhouse gas emissions from livestock, aerosol cans, and individual reproduction have all been topics of environmental policy and practice in the united states since the s [ ]. nearly years ago medicine’s attention was first brought to the effects of ecological matters such as pollution and carbon emissions on human health, concerns that have come to be referred to as “environmental bioethics.” efforts to quantify “the environmental impact of health care…to determine the potential value of mitigation efforts and to reduce harm associated with health care delivery” [ ] have come primarily from several scholars and a few notable journals. this article will provide background on the integration of environmental ethics into health care [ ]. i will move chronologically through a brief history of the field. van rensselaer potter van rensselaer potter’s first book bioethics: bridge to the future expanded the concerns of medical ethics—such as responsibility and rational action—to other branches of life like ecology [ ]. potter saw the interconnectedness of human life and nature as self-evident, given that we humans are situated in a natural environment, and sought to connect us not just to health within the hospital, but to holistic life in the world as well. in he published his second and last book global bioethics: building on the leopold legacy [ ]. global bioethics attempted to link the medical industry back to our earthy origins. although medicine was made by and for humans, we had come to dominate “nature” instead of live harmoniously with it. in the opening pages potter laments that, “with the focus on medical options, the fact that bioethics had been proposed to combine human values with ecological facts was forgotten by many” [ ]. potter considered continuation of the species to be of the utmost importance, but he recognized that there was an “ecological need to limit the exponential increase in the human population…[and] no program [of conservation or advancement] can hope to succeed without the acceptance of controlled human fertility as a basic ethical imperative for the human species” [ ]. potter’s work located bioethics in the bios—the life in the world—and drew a connection between medicine and conservation. his foundational writings opened the door for multidirectional progress in environmental bioethics in the years to follow. www.virtualmentor.org virtual mentor, september —vol http://www.virtualmentor.org/ jessica pierce in the late s, about a decade after potter’s second book, dr. jessica pierce appeared as a major advocate for environmentally sustainable advances in medical and hospital practices, taking up potter’s work for a “second generation” of environmental bioethicists. in pierce examined the idea of “greening” health care products [ , ]. connecting what happens within the walls of hospitals to the natural world suggested avenues for change. she discussed reducing the use of hazardous chemicals in facilities and using environmentally friendly cleaning products, now common practices in many hospitals and other businesses. noting that “about percent of health problems are already environmental in origin” [ ], in pierce demanded that the health care industry examine the way in which human health was inextricably linked to our ecosystem and dependent on a healthy planet. she called for bioethics to examine “health care’s shared responsibility for the environmental problems created by the acquisition, processing and transportation of natural resources required to make the supplies and energy used by consumers” since “health care services represent a significant sector of intensive north american economies” [ ]. her vision for sustainable health care combined the conservationist sensibilities of ecology, the call for slowing the rapid development of the global marketplace, and the more specific conclusion that health care, too, must become “smaller.” the canadian medical association journal and the journal of medical humanities the years between and saw growing academic interest in environmental bioethics and issues related to human health and planetary sustainability. the canadian medical association journal and the journal of medical humanities both ran series exploring ecology and medical ethics. the editorial piece introducing the canadian medical association journal’s - series on environmental bioethics credited michael mccally, md, for suggesting the articles on human health, the economy, social justice, and national environmental security [ ]. the nine articles in the series covered population and consumption, climate change, ozone depletion, cancer, war, endocrine disruption, species loss, sustainable health care, and risk assessment [ ]. the diversity of publications attested to the varied concerns of environmental bioethics. dovetailing with this effort, in the journal of medical humanities dedicated an entire issue to the declining environment and health care [ ]. authors wrote on such varied topics as “environmental thinking,” the role of natural light in human health, the amish ethos of placing communal needs above individual rights to prolonged life, the need for simple living and a restructuring of the global economy to aid public health, connections between ecofeminism and feminist bioethics, the allure of biotechnologies and implications of resource diversion on a planet with limited resources, and the role of nature and childlike wonder in our declining years. this virtual mentor, september —vol www.virtualmentor.org expanded the notion of environmental bioethics from a field solely relevant to medicine to one that was also of interest to literature, religion, feminism and global justice, thus solidifying the interdisciplinary character of environmental bioethics. in , jessica pierce co-authored (with andrew jameton) the ethics of environmentally responsible health care [ ]. this full-length treatment of environmental bioethics was one of the first books since potter’s to address the health needs of human beings—both current and future—and the limits of the shared ecosystem that sustains us. focusing specifically on the united states health care system, the book highlights the tensions between health care needs in developed and the developing world, the individual and the community, and the limits of the planet and the demands of a growing human population. recent trends nearly ten years passed before another “generation” of environmental bioethics emerged. in two promising developments brought environmental bioethics back to the forefront of medicine. david resnik’s environmental health ethics revisited the trails that potter and pierce had blazed and expanded on issues of nutrition, natural disasters, and public health [ ]. in addition, the american society for bioethics and humanities (asbh), which has an affinity group for environmental bioethics, sponsored an undergraduate conference entitled “bioethics: intersections of global health and environmental policy” [ ]. the impact of climate change on the human population has continued to receive interest in the medical industry, urging conservation to better the lives of those who currently suffer under the effects of global warming, including conditions of food scarcity, respiratory disease and drought. it seems that at every turn there is a new organization [ ], ethicist [ , ], or initiative [ ], like the healthy hospitals initiatives [ ] and practice greenhealth [ ], ready to take on the challenge of environmental degradation and human health, health care, and personal responsibility. health care professionals and those who teach them must be prepared to examine the implications of carbon dioxide emissions on human well-being and make decisive steps towards sustainability. references . many trace the inception of the environmental movement in the us back to the seminal text by carson r. silent spring. boston: houghton mifflin; . . chung jw, meltzer do. estimate of the carbon footprint of the us health care sector. jama. ; ( ): . . i have integrated ecological ethics in two different settings: in the course interdisciplinary approaches to bioethics at the experimental college of tufts university in the spring of and om the course health care ethics at massachusetts college of pharmacy and health sciences. . potter v. bioethics: bridge to the future. upper saddle river, nj: prentice hall; . . potter v. global bioethics: building on the leopold legacy. east lansing, mi: michigan state university press; . www.virtualmentor.org virtual mentor, september —vol http://www.virtualmentor.org/ . potter, global bioethics, - . . potter, global bioethics, . . pierce j. can you use a “greener” cleaner? hosp mater manage. : - . . product review yields cleaner, greener use of chemicals. healthc facil manag. : - . . pierce j, jameton a. sustainable health care and emerging ethical responsibilities. cmaj. ; ( ): . . pierce, jameton, . . ecosystem evasion and health (editorial). cmaj. ; ( ): , . . a list of the article in the series can be found at bailer j, bailer a. environment and health: . the science of risk assessment. cmaj. ; ( ) - : . . see the environment-themed articles in j med humanit. ; ( ): - . . pierce j, jameton a. the ethics of environmentally responsible health care. new york: oxford university press; . . resnik d. environmental health ethics. new york: cambridge university press; . . american society for bioethics and humanities. national undergraduate bioethics conference at georgetown university. april - , . http://nubc .org/. accessed july , . . catholic health association and practice greenhealth. environmental sustainability: getting started guide. st. louis: the catholic health association of the united states; . http://www.chausa.org/docs/default-source/general-files/gettingstartedguide- pdf.pdf?sfvrsn= . accessed july , . . most recently discussions have turned from environmental bioethics to “green bioethics.” see richie c. building a framework for green bioethics: integrating ecology into the medical industry. health care ethics usa. ; ( ): - . . richie c. what would an environmentally sustainable reproductive technology industry look like? j med ethics. epub ahead of print july , . . roberts i. the nhs carbon reduction strategy. bmj. ; :b . . healthier hospitals initiative. lead your community to a healthier future: - . http://healthierhospitals.org/sites/default/files/imce/public_files/pdfs/hhi-brochure.pdf accessed may , . . practice greenhealth website. https://practicegreenhealth.org/. accessed may , . cristina richie is writing her phd dissertation on green bioethics in the theology department at boston college. she has taught health care ethics at massachusetts college of pharmacy and health sciences (mcphs) in boston and published in more than a dozen journals. related in vm medicine’s role in mitigating the effects of climate change, june greener clinics, better care, september caring for the health of the community means caring for the health of the environment, june the viewpoints expressed on this site are those of the authors and do not necessarily reflect the views and policies of the ama. copyright american medical association. all rights reserved. virtual mentor, september —vol www.virtualmentor.org http://virtualmentor.ama-assn.org/ / /msoc - .html http://virtualmentor.ama-assn.org/ / /stas - .html http://virtualmentor.ama-assn.org/ / /pfor - .html http://virtualmentor.ama-assn.org/ / /pfor - .html influence of demographic characteristics on production practices within the ohio maple syrup industry gary w. graham, p. charles goebel, randall b. heiligmann, and matthew s. bumgardner maple syrup production contributes approximately $ million annually to ohio’s economy and provides supplemental nontimber forest product income for forestland owners. to better understand the factors that influence this important nontimber forest industry in ohio, including producer heritage, producer age, sap collection methods, size of maple operation, and educational programming, we conducted a detailed survey of all known ohio maple syrup producers ( total producers). over % of producers responded to the survey ( respondents), making our analysis one of the most extensive of a maple industry in north america. in general, most maple operations in ohio are part-time, family-based enterprises and over % of ohio’s maple producers are of amish heritage. although we estimate that there are over , taps in the state, the typical sugarbush is relatively small—the average sugarbush is ac in size and over a third of the operations have fewer than taps. chi-square analyses did reveal several significant (� � . ) associations among producer characteristics. although amish producers were significantly younger and had significantly larger operations than their english or non-amish counterparts (p � . ), a higher proportion of english producers reported using tubing collection systems than amish producers (p � . ). additionally, while larger maple operations tended to use tubing systems more frequently (p � . ), we did not detect a significant association between sap collection method (bucket versus tubing) and producer age (p � . ). finally, english producers tend to be older. older producers (� years old), producers using tubing collection systems, and producers with more than taps were significantly more likely to participate in ohio state university (osu) extension educational programming (p � . ). these results suggest significant relationships among producer demographics and the characteristics of maple operations in ohio, and future osu educational programming should be tailored to reflect these important relationships. keywords: extension programming, maple production, maple producer characteristics, programming impacts, amish r ecently, there has been an increased interest in production and management of nontimber forest products. this is par- ticularly true with regard to the commercial production of maple syrup and related products that occurs primarily in the east- ern united states and southeastern canada where in late winter and early spring cold nights (below °c) and warm days (above °c) result in the flow of sweet sap (tiree , chapeskie et al. ). although there are seven native and at least one fairly common exotic maple species in this region, sugar maple (acer saccharum marsh.), black maple (acer nigum michx.), red maple (acer rubrum l.), and, occasionally, silver maple (a. saccharinum l.) are the most often used maple species (heiligmann et al. b). since the usda, national agricultural statistical service (usda-nass – ) has included ohio in its annual statis- tics reporting of maple syrup production in the united states. over the past years vermont and new york have combined, on aver- age, to produce annually, over , gal of maple syrup, which has contributed annually over $ million to vermont and new york economies (usda-nass – ). ohio, which ranks as the fifth largest maple syrup–producing state behind vermont, new york, maine, and wisconsin, is typical of the smaller maple syrup– producing states and canadian provinces, with approximately , taps producing , gal a year at an average retail price $ . /gal (table ; usda-nass – ). usda-nass, with a limited sampling of the states producers, estimates that maple syrup production contributes approximately $ . million annually to ohio’s economy. (usda-nass – ). however, our comprehensive data place its economic value closer to a $ million annual contribution to the state’s economy and income from maple products provides important supplemental income for many ohio families (graham , graham et al. ). to serve this important clientele, many state and provincial agencies, universities, and extension services throughout the maple region have engaged in research, education, and outreach programs designed to improve maple syrup production and marketing practices. however, little is known about the relationships among demographics, production practices, and marketing strat- egies of maple producers and consumers in the united states or received april , ; accepted january , . gary w. graham (graham. @osu.edu), ohio state university extension center at wooster, madison avenue, wooster, oh . p. charles goebel (goebel. @osu.edu), school of environment and natural resources, ohio agricultural research and development center, the ohio state university, madison avenue, wooster, oh . randall b. heiligmann (heiligmann. @osu.edu), school of environment and natural resources, the ohio state university, coffey road, columbus, oh - . matthew s. bumgardner (mbaumgardner@fs.fed.us), us forest service, northeastern research station, main road, delaware, oh . salaries and financial support for this research were provided by osu extension, the ohio agricultural research and development center (oardc), and the us forest service, northeastern research station. the authors also thank the maple producers who responded to our survey and to cheryl fischnich, and lisa troyer associated with osu extension who helped manage the survey database. additionally, we appreciate the editorial assistance of marie semko-duncan. copyright © by the society of american foresters. north. j. appl. for. ( ) a b s t r a c t canada. although usda-nass has surveyed limited aspects of the us maple industry, a complete statewide or provincewide assess- ment of the maple industry has not been attempted despite the important contributions the industry provides to the economies of the region. there are a variety of reasons for this including the lack of available financial resources to complete such assessments and the perception that maple producers often are a somewhat reclusive and independent group concerned with protecting the privacy of their cash-based supplemental income (lawrence et al. , demchik et al. ). additionally, few states or provinces require maple pro- ducers to register their operations or their production records, mak- ing the identification of a producer population challenging. in ohio, ohio state university (osu) extension is the primary source of technical production and marketing information for ma- ple producers (graham et al. ). major educational strategies include workshops, fact sheets, the ohio maple news newsletter, the north american maple producers manual (heiligmann et al. b), work through the ohio maple producers association, and personal consultation. among the osu extension’s workshop efforts, three -day workshops (“ohio maple days”) have been the most at- tended, and based on our analyses, have historically had the greatest impact on maple producers (graham , graham et al. ). these all-day workshops are held each winter at three different locations across the state and include training and outreach on a variety of topics ranging from sugarbush management to marketing of maple products. from surveys of these attendees, graham et al. ( ) have identified the major needs in the areas of production technologies, resource management, and marketing. in this study, our objective was to better understand industry characteristics and demographics and how educational strategies might be better struc- tured to meet producer needs. specifically, we ( ) examined pro- ducer age, sap collection method, cultural heritage, and participa- tion in the ohio maple days conferences and how these character- istics were associated with operation size; ( ) explored the implica- tions these results might have on the maple industry in ohio; and ( ) suggest how these results can guide future extension educational programming and materials designed for the maple industry throughout the maple producing region of the united states and canada. methods maple producer survey an initial survey list was compiled from an osu extension da- tabase and private sources within the ohio maple industry. in may , producers on this master list (n � , ) were mailed a multisectional questionnaire designed following the methods out- lined in dillman ( ). data were collected using an -question questionnaire that addressed producer demographics, sap collection techniques, sugarbush and sugarhouse characteristics, type of equip- ment used, syrup production and grading practices, and marketing methods. a final set of questions assessed educational resources that respondents used and their perspective on important educational program needs for the maple syrup industry. the design of the questionnaire, the questions asked, and the structure of the ques- tions were selected to reduce perceived intrusiveness and increase return rates. all mailings were sent with return postage, instructions, and cover letters to encourage response rates. after the initial survey and two reminder notices had been mailed, all nonrespondents were sent a second identical question- naire (n � ). individuals not responding to the second question- naire received a third, shortened questionnaire (n � , with % [n � ] sent by priority mail). follow-up phone calls were not used, although suggested by dillman ( ), because of a signifi- cant amish producer population that would not have been reached using this method. an overall response rate of % (n � ) was achieved. the initial survey list of , had entries removed due to blank responses or an indication that the respondent no longer (or never) produced maple syrup, resulting in an adjusted list of maple producers. of the questionnaires returned from active maple operations, ( %) were first-questionnaire re- sponders, ( %) were second-questionnaire responders, and the remaining ( %) were third-round responders. data analysis basic summary statistics of the maple syrup industry were con- ducted using minitab release software (minitab, inc., ). on completion of these analyses, we determined there were five categories of information obtained from the questionnaire that were important factors influencing the maple syrup industry: ( ) pro- ducer heritage (english, amish); ( ) sap collection methods (buckets, tubing); ( ) producer age compared with average age of producers (less than years old, years or older); ( ) attendance at the osu extension sponsored “ohio maple days” educational workshops (attend, do not attend); and ( ) sugaring operation size based on number of taps. producers were placed into one of five categories (table ) based on operation size (e.g., number of taps). using the total number of taps reported on the questionnaires, each producer’s annual production potential (gallons of syrup) was estimated using the state average of qt of syrup per tap. chi-square analyses were used to identify significant relation- ships among producer heritage, sap collection method, attendance at the ohio maple days workshops, and age. categorical variables were defined based on osu extension’s experience working with the maple industry. additionally, chi-square analysis was used to evaluate the relationship between each of those four producer char- acteristics and size of operation. pearson’s chi-square test of associ- ation was selected because of its strength in evaluating associations among independent, categorical variables (steel et al. ). anal- yses were conducted using minitab release software (minitab, table . average maple syrup production of the top five maple syrup–producing states from to . average production (gal) average taps ( , ) average yield per tap (gal) average price (per gal) average value of crop to state’s economy vermont , , . $ . $ , , new york , , . $ . $ , , maine , , . $ . $ , , wisconsin , . $ . $ , , ohio , . $ . $ , , source: data are from the usda-nass ( ). north. j. appl. for. ( ) inc., ) and significant relationships were recognized at � � . . results producer age and cultural heritage over % of ohio’s maple operations are family owned, and the average age of ohio maple producers is years old (ranging from to years). on average, these producers have years of expe- rience in the maple industry and have been producing maple syrup for more than one generation. almost one-half ( %) of ohio’s producers indicated they have a new generation that will take over maple syrup production when they retire. almost all producers ( %) indicated that maple production was a part-time occupation, with most producers indicating their full-time occupation as a tech- nical or trade field or agriculturally related. finally, over % of the producers indicated they were of amish heritage. when we analyzed these patterns in more detail, amish producers were significantly younger than their english or non-amish counterparts (x ( ,n� ) � . ; p � . ). only . % of amish respondents were above the average producer age of years, and . % of the english respondents were older than years. sugarbush the typical ohio sugarbush is ac (ranging from . to ac); however, the majority of sugarbushes are below the average size ( % range from to ac; % range from to ac). when maple syrup operations were classified into categories based on the number of taps (table ), we found that . % are best described as “hobby” operations (less than taps) despite comprising only % of the total taps in the state. the remaining operations are distrib- uted among “small retail-wholesale” ( – taps), “medium re- tail-wholesale” ( – taps), “large retail-wholesale” ( – , taps), and “commercial” (more than , taps) operations. despite only representing . % of all operations in the state, commercial operations account for % of all taps. amish producers were more likely to have larger sugaring oper- ations (based on the number of taps) than english producers (x ( ,n� ) � . ; p � . ). eighty percent of the amish oper- ations contained more than taps, and only % of english operations contained more than taps (figure a). however, no significant association was found between operation size and pro- ducer age (x ( ,n� ) � . ; p � . ), with . % of producers less than years of age and . % of producers more than years of age associated with operations with more than taps (figure b). sap collection and processing the survey indicated that most producers have adopted some conservative tapping guidelines that are designed to protect tree health. for example, % of producer’s indicated they are only using taps/tree or the recommended tapping practices in ohio, and % adjusted the number of taps per tree because of the tree health and environmental conditions. additionally, % responded that they have the potential to expand their operations in the future. we estimate there currently are over , taps in the state, most ( %) associated with traditional bucket collection systems. the average bucket collection operation has taps, with most produc- ers using galvanized metal buckets rather than plastic containers. of those operations using a tubing collection system, most used a den- dritic layout ( %) and gravity to transport the sap from the tree to the sugarhouse. there was a significant association between the heritage of maple syrup producers and their sap collection method. a higher propor- tion of english producers reported using tubing collection systems than amish (x ( ,n� ) � . ; p � . ), with . % of english and only . % of amish respondents reporting that tubing was their primary collection method. there was, however, no significant association between sap collection method and producer age (x ( ,n� ) � . ; p � . ). seventy-six percent of producers who were less than years old and . % of producers who were more than years old reported using buckets for sap collection, and . % of producers who were less than years of age and . % of producers who were more than years of age collect sap with tubing systems (figure b). sugaring operations using tubing systems to collect sap were more likely to be in the three larger size classification categories (x ( ,n� ) � . ; p � . ). only . % of bucket collection operations were associated with the three larger size categories (more than taps), and . % of tubing collection operations were associated with the larger size operations (figure c). almost all ( %) of maple producers indicated they boil col- lected sap the same day of collection or within one to two days of collection. additionally, over % indicated that they filter the sap before boiling, most using a cloth filter ( %). most producers also have a sugarhouse located near the sugarbush that houses a wood- fired evaporator. very few producers blend their syrup to adjust color, density, or flavor. additionally, although syrup grading is not required by ohio law, % of producers currently grade their maple syrup, most ( %) using the vermont temporary grading kit (vermont maple sugar maker’s assn. inc., south royalton, vt). educational activities ohio maple days workshops typically provide attendees with programs built around the latest research-based knowledge and ex- perience on efficiently and effectively producing and marketing ma- ple syrup. the role these workshops play and their effectiveness in the ohio maple products community have been reported elsewhere (graham et al. ). the purpose of this analysis was to character- ize those ohio maple producers participating in the workshops us- ing the variables evaluated in this study. table . relative proportion of maple producers and taps associated with maple syrup operations in ohio by size category. category number of taps projected productiona (gal of syrup) relative proportion of producers (%) relative proportion of taps (%) hobby � � . small retail/wholesale – – . medium retail/wholesale – – . large retail/wholesale – , – . commercial � , � . aprojected production, qt/tap. north. j. appl. for. ( ) attendance at the ohio maple days workshops was significantly related to producer heritage, age, sap collection methods, and size of operation. english producers were more likely to attend ohio ma- ple days than amish (x ( ,n� ) � . ; p � . ) with % of english respondents and % of amish respondents reported par- ticipating. producers who were more than years old also were more likely to attend ohio maple days than their younger coun- terparts (x ( ,n� ) � . ; p � . ); only . % of respondents below the average producer age (less than years of age) attended the workshops, and . % of respondents greater than the average producer age (more than years of age) reported attending. in terms of collection systems, producers with tubing collection sys- tems were more likely to attend the workshops than producers using bucket collection systems (x ( ,n� ) � . ; p � . ). finally, producers with larger operations (more than ) were more likely to attend ohio maple days than producers from smaller (less than taps) operations (x ( ,n� ) � . ; p � . ). only . % of producers in the two smaller size categories (hobby and small retail-wholesale) attended ohio maple days workshops, and . % of producers within the three larger size categories (medium retail- wholesale, large retail-wholesale, and commercial) attended the workshops (figure d). discussion maple syrup production is a labor-intensive enterprise. because most maple syrup operations in ohio are family oriented businesses, many members of the extended family, multiple families, friends, and, in some instances, neighbors are involved (whitney and up- meyer ). this fact is reflected in the typical maple producer in ohio who was a -year-old male, second-generation producer. additionally, almost one-half ( %) of the producers indicated that a next generation was involved in their operation. we observed no association between producer age and sap collection method or producer age and operation size (based on the number of taps). this is not surprising because most maple syrup operations are small and, consequently, do not require more technologically advanced equip- ment. despite this lack of association between producer age and sap collection method or producer age and operation size, there were significant associations among other demographic and production characteristics. in particular, we observed significant differences related to cul- tural heritage. over one-quarter of the maple producers in ohio indicated they were amish. our analyses showed that amish pro- ducers tend to be younger than their english counterparts and also figure . association of producer (a) heritage and (b) age and (c) sap collection method with operation size for ohio’s maple industry; (d) operation size with attendance at ohio maple days workshops. north. j. appl. for. ( ) tend to have larger operations (more than taps). not surpris- ingly, amish producers also tended to be less technologically ad- vanced because they tended to use bucket collection systems rather than tubing collection systems and were less likely to attend the ohio maple days workshops. although there may be many reasons for these relationships, there are cultural aspects that can help ex- plain these patterns. ohio’s amish population has increased from communities in the early s to over communities in with a total population of , . thirty-three of ohio’s coun- ties are home to % of all known amish (donnermeyer ). the result is generally large families and close-knit church networks that can help support more traditional, often less efficient maple syrup collection and production practices, such as the use of bucket collection systems. however, this is not to imply that all amish operations are either traditional or inefficient. some of ohio’s most progressive and modern maple operations are amish owned and operated. amish also believe in separation from the world and that hard- ship in life is part of their religious beliefs and a factor that helps maintain community harmony (schreiber , moore et al. ). this belief requires them to separate themselves from most non-amish social, religious, and political activities. this separation also discourages participation with educational and government as- sistance programs (stinner et al. , donnermeyer ). for example, two of the three annual ohio maple days workshops are intentionally held in highly concentrated amish communities to be within buggy travel distance, but a low percentage of the amish producers attend the workshops. these results suggest that to reach effectively the amish community, educational programming by osu extension and others with similar objectives may need to be coordinated within the amish communities or church districts. ad- ditional outreach activities also may be necessary, including sharing information in other formats (e.g., newsletters, newspapers, and amish-based publications) and the distribution of fact sheets to current amish participants for distribution to other producers in their communities and church districts. results suggest that both amish and english producers would benefit from additional educational programming related to the latest technological advances to help improve sap collection effi- ciency. as noted, % of all ohio sugaring operations use bucket systems and only % currently use the more efficient tubing col- lection systems (although english producers are more likely to using tubing systems). a properly constructed and maintained tubing collection system has been shown to significantly increase sap pro- duction (walters , coons et al. , chapeskie et al. ) and benefit sugarbush health (coons et al. , houston et al. , heiligmann et al. a). the economic and ecological value and use of tubing sap collection systems may be an important pro- duction practice that needs to be more strongly emphasized in ohio and similar states where bucket collection systems are the norm. such programming should focus on the benefits of tubing to sugar- bush health, its reduced labor requirements, and the increased pro- duction potential. future osu extension programming should ad- dress these issues and expand efforts on the economic and marketing aspects of the maple industry. implications for management and education our results indicate that the size of an operation and the age of the producer are important determinants of participation in educa- tional programming such as the ohio maple days workshops. pro- ducers with larger (more than taps) tubing collection operations are more likely to attend as are english producers. however, there is still a large segment of ohio’s maple syrup industry (both amish and english) that does not participate in educational programming. analysis shows that extension programming is reaching the larger operations (more than taps), which represent approximately % of the taps in the state (figure ). however, this only represents % of the producer population, suggesting the need to promote more effectively educational programs to smaller producers. in ad- dition, strategies for targeting younger producers also seem war- ranted, especially given that many producers involve a younger gen- eration in their operations. although there may be many reasons we have been less successful in reaching smaller producers (less than taps), three can be suggested. first, maple sugaring in ohio is a part-time business and smaller producers are less likely to take time off from their primary employment to attend educational programs. second, although many small producers will attend a workshop promoted as a “hobby and small producer” workshop, many may perceive that ohio ma- ple days workshops are “geared” to the larger, commercial produc- ers, and therefore, not address their needs. third, as noted earlier, many producers are protective of their privacy and do not want anyone, especially a group associated or perceived to be associated with the government, to know the scope of their business. increasing participation among these producers will be challenging, but most certainly involves targeting them directly in both the advertising of and the content of the programs. three other strategies to meet the needs of maple-producing communities are suggested by this study. as discussed, to effectively serve the amish, educational programming may need to be coordi- nated within the amish communities or church districts. addition- ally, to reach most effectively many of the younger producers of the web-based information generation, workshop information and pub- lications should be made available electronically. finally, although maple producers express and exhibit a strong interest in programs addressing the technical aspects of maple product production, and to a lesser extent sugarbush management, generally, they have far less interest in or enthusiasm for programs devoted to economics or marketing topics. creative, effective ways need to be developed to figure . proportion of taps and producers reached through current ohio maple days workshops organized by the osu extension. north. j. appl. for. ( ) provide attractive programs in those areas that will provide produc- ers with the information necessary to make pricing and processing decisions appropriate to their operations, which will contribute to their achieving their entrepreneurial goals. literature cited coons, c.f., ontario, ministry of natural resources, ontario, and ministry of agriculture and food. . sugarbush management for maple syrup producers. rev. june ed. ontario ministry of natural resources, toronto. p. chapeskie, d., t. wilmot, b. chabot, and t.d. perkins. . maple sap production-tapping, collection, and storage. p. – in north american maple producers manual, nd ed., heiligmann, r.b., m.r. koelling, and t.d. perkins. (eds.). the ohio state univ., columbus, oh. demchik, m.c., j.c. finley, a.l. davenport, and r.d. adams. . assessing the characteristics of the maple syrup industry in pennsylvania to aid in the development of extension programs. north. j. appl. for. ( ): – . dillman, d.a. . mail and internet surveys: the tailored design method, nd ed. j. wiley, new york. p. donnermeyer, j.f. . amish society: an overview. j. multicult. nurs. health ( ): – . donnermeyer, j.f. . amish population estimates for ohio counties. department of human and community resources development, ohio state univ., columbus, oh. p. graham, g .w. . analysis of production practices and demographic characteristics of the ohio maple syrup industry. ph.d. dissertation, the ohio state university, columbus, oh. p. graham, g.w., p.c. goebel., r.b. heiligmann, and m.s. bumgardner. . maple syrup production in ohio and the impact of ohio state university (osu) extension programming. j. for. : – . heiligmann, r.b., p. smallidge, g.w. graham, and b. chabot. a. managing maple trees for sap production. p. – in north american maple producers manual, nd ed. heiligmann, r.b., m.r. koelling, and t.d. perkins. the ohio state univ., columbus, oh. heiligmann, r.b., m.r. koelling, and t.d. perkins. b. north american maple syrup producers manual, nd ed. heiligmann, r.b., m.r. koelling, and t.d. perkins. the ohio state univ., columbus, oh. p. houston, d.r., d.c. allen, and d. lachance. . sugarbush management: a guide to maintaining tree health. us for. serv. gen. tech. rep. ne- , northeastern forest experiment station, radnor, pa. p. lawrence, j., r. martin, and p. boisvert. . sweet maple: life, lore, and recipes from the sugarbush. vermont life, montpelier, vt. p. minitab, inc. . minitab statistical software, release for windows. state college, pa. moore, r.h., d.h. stinner, and d. kline, and e. kline. . honoring creation and tending the garden: amish views of biodiversity. p. – in traditional agriculture and soil management, cultural and spiritual values of biodiversity: a complementary contribution to the global biodiversity assessment of the un environmental program. plenderleith, k. (ed.). intermediate technologies, london, u.k. schreiber, w.i. . our amish neighbors. university of chicago press, chicago, il. p. steel, r.g.d., j.h. torrie, and d.a. dickey. . principles and procedures of statistics: a boimetrical approach, vol. , rd ed. the mcgraw-hill companies, inc., new york. p. stinner, d.h., m.g. paoletti., and b.r. stinner. . in search of traditional farm wisdom for a more sustainable agriculture: a study of amish farming and society. agric. ecosyst. environ. : – . tyree, m.t. . sap uptake, exudation, and pressure changes correlated with freezing exotherm and thawing endotherm. plant physiol. : – . us department of agriculture–national agricultural statistical service (usda-nass). – . maple syrup production, davis, a.r. (ed.). usda, new england agricultural statistics service, concord, nh. walters, r.s. . sugar maple sap collection. p. – in sugar maple research: sap production, processing, and marketing of maple syrup. us for. serv. gen. tech. rep. ne- , northeastern for. exp. stn., burlington, vt. whitney, g.g., and m.m. upmeyer. . sweet trees, sour circumstances: the long search for sustainability in the north american maple products industry. for. ecol. manag. : – . north. j. appl. for. ( ) developmental and degenerative features in a complicated spastic paraplegia developmental and degenerative features in a complicated spastic paraplegia m. chiara manzini, phd, anna rajab, phd, thomas m. maynard, phd, ganeshwaran h. mochida, md, mmsc, , , wen-hann tan, bmbs, ramzi nasir, md, mph, r. sean hill, phd, danielle gleason, ba, muna al saffar, mbchb, ccgc , jennifer n. partlow, ms, cgc, , brenda j. barry, ms, cgc, , mike vernon, msc, anthony-samuel lamantia, phd, and christopher a. walsh, md, phd , objective: we sought to explore the genetic and molecular causes of troyer syndrome, one of several compli- cated hereditary spastic paraplegias (hsps). troyer syndrome had been thought to be restricted to the amish; however, we identified omani families with hsp, short stature, dysarthria and developmental delay— core fea- tures of troyer syndrome—and a novel mutation in the spg gene, which is also mutated in the amish. in addition, we analyzed spg expression throughout development to infer how disruption of this gene might generate the constellation of developmental and degenerative troyer syndrome phenotypes. methods: clinical characterization of non-amish families with troyer syndrome was followed by linkage and sequencing analysis. quantitative polymerase chain reaction and in situ hybridization analysis of spg expression were carried out in embryonic and adult human and mouse tissue. results: two omani families carrying a novel spg mutation displayed clinical features remarkably similar to the amish patients with troyer syndrome. spg mrna is expressed broadly but at low relative levels in the adult brain; however, it is robustly and specifically expressed in the limbs, face, and brain during early morphogenesis. interpretation: null mutations in spg cause troyer syndrome, a specific clinical entity with developmental and degenerative features. maximal expression of spg in the limb buds and forebrain during embryogenesis may explain the developmental origin of the skeletal and cognitive defects observed in this disorder. ann neurol ; : – hereditary spastic paraplegias (hsps) comprise severaldisorders commonly divided into subgroups: “pure” hsps characterized by progressive spasticity in the lower limbs due to pyramidal tract degeneration and “complicated” hsps, where lower limb spasticity is asso- ciated with a variety of other neurological signs and clin- ical features. complicated hsps are clinically heteroge- neous, mainly autosomal recessive syndromes, frequently described and mapped in sporadic families within inbred populations. – because of this heterogeneity, diagnosis published online in wiley interscience (www.interscience.wiley.com). doi: . /ana. received jun , , and in revised form oct . accepted for publication nov , . address correspondence to dr walsh, chief, division of genetics, children’s hospital boston, howard hughes medical institute, beth israel deaconess medical center, harvard medical school, room . , center for life sciences building, longwood ave cls . , boston, ma . e-mail: christopher.walsh@childrens.harvard.edu from the department of neurology and howard hughes medical institute, beth israel deaconess medical center, harvard medical school, boston, ma; genetic unit, directorate general of health affairs, ministry of health, muscat, sultanate of oman; department of cell and molecular physiology and unc neuroscience center, university of north carolina school of medicine, chapel hill, nc; pediatric neurology unit, massachusetts general hospital, boston, ma; division of genetics and manton center for orphan disease research, children’s hospital boston, boston, ma; and division of developmental medicine, children’s hospital boston, boston, ma. additional supporting information can be found in the online version of this article. original article © american neurological association and recommendations for genetic testing in these disor- ders have been daunting tasks. troyer syndrome (online mendelian inheritance in man # ) is a complicated hsp associated with short stature, skeletal abnormalities, dysarthria, and developmen- tal delay, first described in the old order amish. , since the original description in , several troyer-like syn- dromes have been reported, – but they often differed from classical troyer syndrome in their neurological or skeletal features. the amish founder mutation is a single nucleo- tide deletion in the spg gene, leading to the loss of the spartin protein ; however, no additional spg mu- tations were subsequently identified, and it was suspected that troyer syndrome may be restricted to the amish. we identified an omani kindred presenting with clinical features resembling troyer syndrome. all affected omani individuals had a novel homozygous null mutation in spg , and their clinical descriptions matched closely to those of amish troyer syndrome individuals of comparable ages. because troyer syndrome is associated with develop- mental features, such as short stature, skeletal abnormali- ties, and global developmental delay, we investigated the sites of action of spg during development in humans and mice. spg /spg expression in the adult is relatively modest and widespread in the nervous system; however, maximal and focal expression is observed in the embryonic limb buds, face, and forebrain during early morphogenesis, which might explain the developmental phenotypic changes involving the extremities, face, and brain. subjects and methods enrollment and clinical studies the subjects belonged to families residing in a remote region of oman and were originally ascertained by a local clinical geneticist. detailed family and medical histories were obtained by a genetic counselor, who is a native arabic speaker, and a developmental pediatrician, also a native arabic speaker, conducted a develop- mental assessment. a pediatric neurologist performed a standard neurological examination, and a second clinical geneticist obtained anthropometric measurements. the height of all subjects and head circumference of subjects under the age of months were plotted on the centers for disease control and prevention growth charts, and head circumference of subjects above months and all other anthropometric measurements were plotted on standard charts. written, informed consent was obtained from the subjects or their legal guardians. the ministry of health in oman and the institutional review board of children’s hospi- tal boston approved this study. linkage analysis genomic dna was purified from lymphocytes separated from peripheral blood using commercial kits (qiagen, valencia, ca). six hundred nanograms of genomic dna was used to hybridize affymetrix human snp array . at the genomic analysis core of the university of north carolina (unc) neuroscience center of the unc school of medicine, chapel hill, north carolina. after removal of low-quality calls and of mendelian and non-mendelian errors using merlin software, the single nucleotide polymorphism (snp) data were analyzed using alle- gro software under a fully penetrant autosomal recessive model. for microsatellite analysis, highly polymorphic microsat- ellite markers were chosen from the marshfield database in the university of california at santa cruz genome browser. fluo- rescently labeled polymerase chain reaction (pcr) primers (ap- plied biosystems, foster city, ca) were used to amplify dna samples using standard conditions, and pcr products were re- solved on an applied biosystems xl genetic analyzer. spg sequencing analysis sequencing of spg (nm_ ; nm_ - ) cod- ing region was performed by seqwright (houston, tx) on pcr products after amplification of genomic dna. pcr prim- ers were designed for each exon including at least bp of flank- ing intronic sequences. primer sequences are available on re- quest. once a putative change was identified, at least control dnas ( alleles) were tested to exclude the possibility of a benign polymorphic change. patient cell lines and mutated protein/mrna detection transformed lymphoblastoid cell lines were established from pe- ripheral blood of affected and unaffected individuals at the part- ners center for personalized genetic medicine (cambridge, ma). cell lines were maintained in rpmi medium supplemented with % fetal bovine serum, glutamine ( mm) and penicillin/strep- tomycin ( u/ �g) (all from gibco, grand island, ny). pro- tein lysates were obtained by boiling a cell pellet in laemmli sam- ple buffer (bio-rad laboratories, hercules, ca). western blotting was performed using standard protocols on bio-rad equipment. presence of spartin was assessed using a rabbit anti-spg anti- body (proteintech group, chicago, il) and a goat anti-rabbit irdye- cw secondary antibody (li-cor biosciences, lin- coln, ne) detected on an odyssey imager (li-cor biosciences). total rna was purified from patient cell lines using the mirvana rna isolation kit (ambion applied biosystems, foster city, ca). random primed cdna was generated using reverse transcriptase (promega, madison, wi) and analyzed by quantita- tive pcr (qpcr) using sybr green reagents (applied biosys- tems) on an abi qpcr platform as previously described. primer sequences are available in supplementary table. spg /spg expression analysis cdna samples from human fetal and adult postmortem brain tissue were obtained commercially (biochain institute, hayward, ca and clontech, mountain view, ca). cdna from embryonic and postnatal mouse brains was generated as described above. qpcr was performed as described above (see supplementary ta- ble for primer sequences). dna templates for in situ hybridiza- tion probes were cloned using a bp fragment of the spg manzini et al: novel spg mutation april, coding sequence using the following primers: spg -bamhi �- ctacatggatccgatatcaaccggaggagcagccaaagtcagc- � and spg -sali �-gtccttgtcgactgcccctggcttctcttcctccacctg- �. digoxigenin-labeled riboprobes were synthesized and in situ hybridization was per- formed as previously described. video images of the hybridized sections were obtained on a wild leitz (heerbrugg, switzerland) photomicroscope or leica (nussloch, germany) dmr micro- scope under consistent illumination conditions. results identification of a novel spg mutation in an omani kindred we examined related omani families presenting with short stature, dysarthria, motor and cognitive develop- mental delay, and increased muscle tone (families and in fig a). the fathers (individuals - and - ) were brothers, and the mothers ( - and - ) were aunt and niece. family had children: were reportedly unaf- fected females, who were married and lived elsewhere, was a male ( - ), who was unaffected by clinical exami- nation, were affected males ( - , - , and - ), and was an affected female ( - ). family had children: unaffected girls and unaffected boy, of whom were present at the time of our evaluation ( - , - and - ), affected male ( - ), affected female ( - ). the young- est child ( - ) was small for her age and could neither talk nor walk, but did not display overt neurological symptoms; we were not able to obtain dna from her for genotyping or sequencing. figure : affected individuals carry a homozygous null mutation in the spg gene. (a) pedigree of related omani families affected with short stature, spasticity, dysarthria, and developmental delay. affected individuals are in black and unaffected in white. the status of individual - (in gray) could not be determined, because she is too young to properly assess neurological symptoms. all numbered individuals were examined, but genomic dna was only collected from indi- viduals for whom microsatellite analysis is shown. microsatellite analysis revealed common maternal (in yellow) and paternal (in blue) haplotypes in all affected. microsatellite markers in the linkage region identified by the single nucleotide poly- morphism analysis are in bold. one homozygous marker (d s ) is common to all affected individuals (highlighted in orange). (b) the homozygous region contains genes, including spg (in orange). (c) the affected individuals carried a homozygous bp deletion in spg , which was present in heterozygosity in the carriers. (d) western blot analysis of patient cell lines showed that full-length spg protein is missing in the affected individuals (a) compared with a nonaffected noncarrier sibling (na). (e) quantitative polymerase chain reaction analysis of cdna from the patient cell lines indicated that spg mrna is not present in the affected individual. deltarn � signal magnitude expressed by the difference in the normalized reporter (rn) values. annals of neurology volume , no. genomic dna was obtained from most individuals in the pedigree and hybridized to affymetrix human snp array . chips for genome-wide genotyping. we suspected the presence of a homozygous ancestral mutation because the families resided in an isolated mountain village and large stretches of homozygous dna identified in all indi- viduals suggested some degree of consanguinity; however, we could not ascertain any shared ancestry as far as gen- erations removed from the probands by pedigree analysis. thus, the snp data was first analyzed following an auto- somal recessive model assuming no consanguinity. linkage analysis identified region of . mb flanked by snps rs and rs on chromosome q . (log of odds � . ). microsatellite analysis confirmed a shared maternal and paternal haplotype in all affected individuals (see fig a). individual - , who was unaffected, was ho- mozygous for the maternal haplotype throughout most of the linkage region, excluding marker d s , which was homozygous on all affected individuals. at this locus, the snp data indicated an kb region of homozygosity be- tween markers rs and rs , shared only by affected but not unaffected individuals. among the genes in this region (see fig b), spg was the strongest can- didate gene, because individuals carrying an spg muta- tion were affected with a remarkably similar phenotype, a complicated form of hereditary spastic paraplegia associated with short stature, dysarthria, and developmental delay, called troyer syndrome. , sequencing of the spg coding regions revealed a homozygous bp deletion (c. _ delat), which re- sulted in an amino acid substitution followed by a stop codon in the first coding exon (p.m vfsx ; see fig c). this mutation was confirmed in all affected individ- uals, and the inheritance pattern was confirmed in the rest of the families. this mutation was not identified in con- trol individuals, indicating that this is not a common polymorphism. since the spg mutation in the amish (c. dela) was reported to be a null mutation, we generated lympho- blastoid cell lines from affected ( - and - ) and unaf- fected individual ( - ). we found that the full-length spartin protein was absent in the affected individuals (see fig d). we also carried out qpcr for spg on cdna samples from of the lymphoblastoid lines and could not detect any mrna in the affected individual (see fig e), indicating that this mutation is a null allele. clinical characterization of patients carrying a novel spg mutation to ascertain phenotypic similarities or differences among individuals carrying spg mutations, we compared the clinical presentations of the omani individuals with the clinical description of amish patients by proukakis et al (table). all affected individuals in the omani cohort presented with short stature and dysarthria, and were de- layed in reaching motor and cognitive developmental milestones. they all had some difficulties walking, with clumsy, mildly spastic gait, which their parents reported to be worsening over time. the most common physical features were relative hypertelorism and overgrowth of the maxilla leading to overbite, as well as hand and feet anomalies such as brachydactyly ( / patients), hammer toes, and pes cavus (fig ). additional nonspecific skeletal malformations observed in the hands were clinodactyly, camptodactyly, and hypoplastic th middle phalanges (see fig e, f). most affected individuals had persistent cog- nitive deficits and poor performance in school, but no emotional lability was reported. detailed neuropsycholog- ical testing was not available. the neurological examination revealed distal amyot- rophy ( / patients), dysmetria in the upper extremities ( / patients), hyperreflexia, which was more severe in the lower limbs ( / patients), and ankle clonus ( / pa- tients). heel cords were tight in the older affected in- dividuals. brain magnetic resonance imaging (mri) was performed in individuals ( - at years and - at years). both individuals had mild atrophy of the cerebellar vermis, mild white matter volume loss, and revealed periventricular white matter hyperintensity in t - weighted images, consistent with gliosis (see fig i, j). the mean age of our cohort ( . years) was younger than the amish cohort, which might explain the milder and more variable phenotype observed in the omani individuals; therefore, we subdivided the amish cohort into a younger subset (amish i) comparable in age to our cohort (� years; mean age, years) and an older subset (amish ii) (� years; mean age, . years). our cohort closely matched the description of the amish i group, whereas the amish ii group was more severely affected, consistent with the progressive, degener- ative aspects of troyer syndrome (see table). expression of spg in the developing and adult nervous system there appear to be major components in troyer syn- drome: an early developmental aspect and a neurodegen- erative process. the combination of phenotypes, includ- ing limb, craniofacial, and behavioral anomalies, is common in complex genetic disorders affecting early mor- phogenetic events, and the relevant genes are often spe- cifically expressed in the embryonic rudiments of the af- fected adult structures. we first analyzed spg expression in the developing and adult human brain using qpcr. spg was expressed at modest levels in the fetal manzini et al: novel spg mutation april, and adult human brain compared to a highly expressed neurally specific gene, synaptophysin (syp; fig a, b). levels of spg were highest in the amygdala, cortex, and thalamus, and lowest in the hippocampus and cere- bellum. although clearly detectable, spg was expressed at substantially lower levels than syp. expression levels and distribution of the mouse orthologue of spg , spg , in the adult mouse brain parallel those of the hu- man, with some divergence; particularly, relative expres- sion of spg in the hippocampus was slightly elevated compared with humans. in addition, we analyzed spg expression in mouse spinal cord and brainstem and found that it was highest in the spinal cord of all brain regions measured. as in the human, spg was expressed at com- paratively lower levels than syp. spg is not a brain- specific gene, as it is also expressed in several other or- gans. , it is, however, developmentally regulated, as expression was maximal at midgestation and embryonic day (e) , and declined precipitously thereafter (see fig c–f). we then used in situ hybridization to localize spg in adult, fetal, and embryonic mouse brain (fig ). spg was expressed at relatively low levels in neurons and glia throughout the adult brain, including glia in fiber tracts. modestly elevated expression was seen throughout hip- pocampal stratum pyramidale of the ca fields and den- tate gyrus and the transitional parahippocampal/entorhi- nal cortex. low expression was seen in the cerebral cortex with no apparent laminar or cell class specificity. the only additional site of elevated expression in the forebrain was the habenular complex, including the habenular re- cess appended to the corpus callosum. spg is expressed throughout the cerebellum in purkinje cells, granule cells, and scattered cells in the molecular layer. in the brain- table: clinical features of the omani troyer syndrome individuals and comparison with the amish cohort characteristics individuals omani amish i amish ii – – – – – – age (yr) mean . mean mean . sex m f m m m f height (cm) na short short short % � rd � rd � rd � rd � rd ofc (cm) . . . . . . normal normal normal % rd– th � rd th th– th th– th rd– th developmental milestones and cognition talked at years years years late late late late ( %) late ( %) late ( %) walked at years years . years . years years . years late ( %) late ( %) late ( %) poor school performance � � � � � na �( %) �( %) �( %) emotional lability � � � � � na no �( %) �( %) neurological exam dysarthria � � � � � na �( %) �( %) �( %) tongue dyspraxia � � � � � na �( %) often often distal amyotrophy � � � � � � �( %) �( %) �( %) hyperreflexia upper � � � � � � �( %) �( %) �( %) lower � � � � � � �( %) �( %) �( %) ankle clonus � � � � � � �( %) �( %) �( %) clumsy, spastic gait � � � � � � �( %) �( %) �( %) dysmetria � � � � � � �( %) �( %) �( %) skeletal abnormalities overbite � � � � � ? �( %) no no hypertelorism � � � � � � �( %) no no hand/foot abnormalities � � � � � � �( %) �( %) �( %) ofc � occipitofrontal circumference; na � not available. annals of neurology volume , no. stem, there is robust expression in large neurons, probably motor neurons, and in the facial nucleus. spg is also expressed in cells distributed throughout the spinal cord with no noticeable discontinuities. as predicted from the qpcr data, expression in the fetal brain is relatively low, with no apparent regional distinctions. aside from ele- vated expression in the lens placode and pigment epithe- lium, cochlear epithelium, and condensing mesenchyme at sites of myogenic or cartilage formation, there is little focal expression at fetal stages (see fig l–n; e . and e . are shown). in contrast to the later stages, and, as predicted from the qpcr analysis, spg expression in the midgestation embryo is elevated and highly patterned. in the e . embryos, shortly after neural tube closure, spg is spe- cifically expressed in the initial frontonasal mass/forebrain, craniofacial structures, aortic arch/heart primordium, and limb buds during morphogenesis, with lowest expression in the heart (fig o, p). qpcr analysis confirms this dis- tribution; expression levels in limb buds, branchial arches, and frontonasal mass/forebrain are substantially elevated compared with the heart as well as with whole e . em- bryo (fig r). our observations suggest that spg has its most specific and maximal activity in the limbs, face and forebrain during early morphogenesis. this is remarkable, because the sites of phenotypic manifestations in the af- fected members of the omani troyer syndrome kindred described here include anomalies of the limb extremities, face, and brain. discussion our identification of a novel, disease-associated spg mutation in an omani kindred indicates for the first time that troyer syndrome is not restricted to the amish, as previously proposed. complicated autosomal recessive hsps are heterogeneous disorders often lacking clear clin- ical and molecular diagnostic guidelines. of the dis- tinct loci for complicated hsp identified to date, more figure : morphological and radiological features of omani individuals with troyer syndrome. comparison between (a, c) an unaffected and (b, d) an affected individual revealed (a, b) relative hypertelorism and (c, d) pronounced overbite. examples of skeletal anomalies in the extremities: brachydactyly (short digits; e, f), camptodactyly (flexed digit; asterisks in e, f), hammer toes (asterisks in g, h), and clinodactyly (curved digit; arrowheads in g, h). brain magnetic resonance imaging shows atrophy of the cerebellar vermis (arrowhead in i) and white matter hyperintensity in t -weighted images, which is more prominent posteriorly (arrowheads in j). manzini et al: novel spg mutation april, than half have been described in single families or isolated populations. , in the past decades, several hsp syn- dromes with troyer-like features have been reported, but none matched the clinical presentation in the amish cases. some of these disorders were later mapped to dif- ferent loci such as spg , , spg , and arsacs. , even within the isolated region where these omani families reside, we had described a similar extended pedigree with dysarthria, mental retardation, cerebral palsy, and micro- cephaly. linkage analysis ruled out the spg locus in the second kindred, confirming that the disorders are separate clinical and genetic entities (a.r., r.s.h., c.a.w., unpublished data). four individuals in this ex- tended family (n � individuals) also were heterozy- gous carriers for the spg mutation, highlighting its presence in this isolated population. thus, this novel mu- tation in spg is geographically and genetically distinct from that in the amish population; nevertheless, both mutations result in the phenotypic spectrum associated with troyer syndrome. figure : quantitative polymerase chain reaction analysis of spg /spg expression in human and mouse tissues. (a) there is modest variation in spg local expression in the adult human brain when distinct regions are compared to whole brain samples. (b) human spg expression is substantially lower than that of the brain-specific gene synaptophysin (syp) across all brain regions with the exception of the amygdala. (c) a similar profile of regionally variable spg expression, with some modest differences, is seen in the mouse brain. (d) lower spg expression is observed when compared with syp. (e) spg is expressed in several murine tissues beside the brain. (f) spg is developmentally regulated in the mouse embryo with highest expression levels in the whole embryo at e . . �ct � ; bas gang � basal ganglia; deltact � signal magnitude expressed by the difference in ct values. hippoc � hippocampus; mesen � mesenchyme; cerebel � cerebellum; sp cord � spinal cord; sk muscle � skeletal muscle; rel. � relative. annals of neurology volume , no. although a clear troyer syndrome diagnosis is dif- ficult to reach in young subjects due to the variability and mildness of their symptoms, direct comparison of clinical features showed that the omani cohort closely resembled the age-matched amish troyer syndrome group (amish i), suggesting that spg null mutations cause a well- defined phenotype. as in the amish, we observed short stature, skeletal abnormalities in the extremities, develop- mental delay, and dysarthria of possible cerebellar origin from an early age. therefore, a diagnosis of troyer syn- drome must be considered when this constellation of phe- notypes is present in young children. facial dysmorphism figure : in situ analysis of spg expression in mice. (a–j) spg expression in the adult brain is modest and focal. (e) cortical expression is moderate and not layer-specific. relatively higher expression is observed in (b) the hippocampus, (c) glial cells in the corpus callosum, (d) entorhinal cortex, (f, g) habenular complex, (h) cerebellum, (i) facial nucleus (fn), and (j) spinal cord. (k) fetal expression is also modest and distributed in the brain, with higher expression in (l) the lens placode (le), (m) the cochlear epithelium, and (n) condensing mesenchyme at sites of myogenic and cartilage formation (asterisks). (o) early embryonic expression (embryonic day . ) is highly patterned and enhanced in the forebrain (fb), frontonasal mass (fnm), maxilla (mx), branchial arches (ba , ba ), heart (h), and limb buds (flb). (p) the antisense control is completely unlabeled. (q) selective, focally elevated expression in the fnm/fb, flb, and ba /ba is confirmed by qpcr after microdissection compared to the heart (h) and whole embryo (wh). ca � ; ca � ; ca � ; cc � corpus callosum; m � molecular layer; p � purkinje cells layer; g � granule cell layer; dh � dorsal horn; vh � ventral horn; ctx � cortex; lge � lateral ganglionic eminence; mge � medial ganglionic eminence; r � retina. manzini et al: novel spg mutation april, and skeletal features are subtle, and in single cases in non- consanguineous populations this disorder could be ini- tially diagnosed simply as cerebral palsy. spasticity and distal amyotrophy appeared in the teenage years and worsened slowly over time. in the amish, brain mri re- vealed white matter abnormalities, which were less severe in the youngest patient examined (age years), suggest- ing a progressive worsening of the condition. mri anal- ysis indicates that the omani troyer syndrome individu- als of comparable ages also have mild white matter abnormalities, supporting the hypothesis that white mat- ter degeneration accompanies disease progression, al- though direct neuronal degeneration cannot be ruled out. in addition, we identified mild cerebellar atrophy, which is consistent with the cerebellar signs. serial mri scans of the omani individuals might resolve whether progressive neurological symptoms are correlated with increased white or gray matter degeneration. despite many similarities to the amish cohort, a few specific differences were observed in the affected omani individuals, including the presence of hypertelorism and a pronounced overbite, and the ab- sence of inappropriate emotional responses. as new cases are identified it will be interesting to assess whether these traits have variable penetrance or are population specific. functional studies on spartin, the protein encoded by spg , have identified multiple roles in protein ubiq- uitination – and lipid droplet formation, , , and a possible link to endothelial growth factor receptor signal- ing. previous expression analyses by northern blot or qpcr identified widespread spg /spartin localization in the brain and in other tissues , ; however, it was unclear whether regional and cellular dynamism in spg expres- sion could explain the phenotypes observed in the affected individuals. our study shows that whereas spg /spg expression is modest and virtually ubiquitous in the adult and developing brain, early stages of embryonic develop- ment show maximal levels of spg in the limbs, face, and forebrain primordia. this localized expression sug- gests a parallel role for spg /spg in morphogenesis and differentiation at these phenotypic sites; accordingly, a loss of function mutation may contribute to the pheno- typic spectrum of troyer syndrome. acknowledgment this work was supported by the ninds (grant ro ns , c.a.w.), nichd (grant ro hd , a.-s.l.), the dubai harvard foundation for medical re- search (c.a.w.), the manton center for orphan disease research (c.a.w.), the muscular dystrophy association (development grant, m.c.m.), and the university of north carolina (reynolds faculty fellowship, a.-s.l.). c.a.w. is an investigator of the howard hughes medical institute. we thank the families for their cooperation and their hospitality; the local nurses and doctors for their as- sistance; megumi aita at the university of north caro- lina in situ core for help with the in situ analysis; daniel rakiec at children’s hospital, boston for help with the sample preparation and analysis; and the following col- leagues at children’s hospital boston for helpful discus- sion on the clinical features of the patients: hope e. dickinson, a speech pathologist from the department of otolaryngology and communication enhancement, and janice ware, a child psychologist and associate director of the developmental medicine center. authorship m. chiara manzini and anna rajab contributed equally and are co-first authors. potential conflicts of interest nothing to report. references . harding ae. classification of the hereditary ataxias and paraple- gias. lancet ; : – . . depienne c, stevanin g, brice a, durr a. hereditary spastic paraplegias: an update. curr opin neurol ; : – . . salinas s, proukakis c, crosby a, warner tt. hereditary spastic paraplegia: clinical features and pathogenetic mechanisms. lan- cet neurol ; : – . . cross he, mckusick va. the troyer syndrome. a recessive form of spastic paraplegia with distal muscle wasting. arch neurol ; : – . . proukakis c, cross h, patel h, et al. troyer syndrome revisited. a clinical and radiological study of a complicated hereditary spastic paraplegia. j neurol ; : – . . farag ti, el-badramany mh, al-sharkawy s. troyer syndrome: re- port of the first “non-amish” sibship and review. am j med genet ; : – . . farah s, sabry ma, al-shubaili af, et al. hereditary spastic para- paresis with distal muscle wasting, microcephaly, mental retarda- tion, arachnodactyly and tremors: new entity? clin neurol neu- rosurg ; : – . . auer-grumbach m, fazekas f, radner h, et al. troyer syndrome: a combination of central brain abnormality and motor neuron disease? j neurol ; : – . . bertini e, sabatelli m, di capua m, et al. familial spastic para- plegia, axonal sensory-motor polyneuropathy and bulbar amyot- rophy with facial dysmorphia: new cases of troyer-like syndrome. eur j paediatr neurol ; 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: – . . lamantia as. forebrain induction, retinoic acid, and vulnerability to schizophrenia: insights from molecular and genetic analysis in developing mice. biol psychiatry ; : – . . robay d, patel h, simpson ma, et al. endogenous spartin, mu- tated in hereditary spastic paraplegia, has a complex subcellular localization suggesting diverse roles in neurons. exp cell res ; : – . . wilkinson pa, simpson ma, bastaki l, et al. a new locus for autosomal recessive complicated hereditary spastic paraplegia (spg ) maps to chromosome p . - q . j med genet ; : – . . rainier s, bui m, mark e, et al. neuropathy target esterase gene mutations cause motor neuron disease. am j hum genet ; : – . . bouchard jp, barbeau a, bouchard r, bouchard rw. autosomal recessive spastic ataxia of charlevoix-saguenay. can j neurol sci ; : – . . engert jc, berube p, mercier j, et al. arsacs, a spastic ataxia common in northeastern quebec, is caused by mutations in a new gene encoding an . -kb orf. nat genet ; : – . . rajab a, yoo sy, abdulgalil a, et al. an autosomal recessive form of spastic cerebral palsy (cp) with microcephaly and mental retardation. am j med genet a ; : – . . eastman sw, yassaee m, bieniasz pd. a role for ubiquitin ligases and spartin/spg in lipid droplet turnover. j cell biol ; : – . . edwards tl, clowes ve, tsang ht, et al. endogenous spartin (spg ) is recruited to the endosomes and lipid droplets and interacts with the ubiquitin e ligases aip and aip . biochem j ; : – . . milewska m, mcredmond j, byrne pc. identification of novel spartin-interactors shows spartin is a multifunctional protein. j neurochem ; : – . manzini et al: novel spg mutation april, association of nos ap genetic variants with qt interval duration in families from the diabetes heart study allison b. lehtinen, , christopher newton-cheh, , julie t. ziegler, carl d. langefeld, barry i. freedman, kurt r. daniel, david m. herrington, and donald w. bowden , , objectives—prolongation of the electrocardiographic qt interval is a risk factor for sudden cardiac death (scd). diabetic individuals are at increased risk for prolonged qt interval and scd. we sought to replicate the finding that genetic variants in the nitric oxide synthase adaptor protein (nos ap) gene are associated with qt interval duration in a type diabetes– enriched sample of european ancestry. research design and methods—two single nucleo- tide polymorphisms (snps) in nos ap were genotyped in european americans and african americans from pedi- grees enriched for type diabetes. an additive genetic model was tested for each snp in ancestry-specific analyses in the total sample and the diabetic subset (european americans, n � ; african americans, n � ), excluding from the analyses individuals taking qt-altering medications. results—in european americans, rs minor homozy- gotes had a . -ms-longer qt interval compared with major homozygotes (p � . � � ); rs minor homozygotes had a . -ms-longer qt interval compared with major homozy- gotes (p � . � � ). restricting analyses to the diabetic european americans strengthened the effect despite the reduc- tion in sample size ( . -ms difference, p � . � � ; . -ms difference, p � . � � , respectively). no association between the nos ap snps and qt interval duration was observed in the limited number of african americans. conclusions—two nos ap snps are strongly associated with qt interval duration in a predominately diabetic european- american sample. stronger effects of nos ap variants in dia- betic individuals suggest that this patient subset may be particularly susceptible to genetic variants that influence myo- cardial depolarization and repolarization as manifest in the qt interval. diabetes : – , s udden cardiac death (scd) claims � , lives per year in the u.s. ( ). numerous community- based studies have established qt interval as an independent risk factor for scd ( – ). moreover, qt interval prolongation and resultant arrhythmia on exposure to cardiac and noncardiac medications is a major barrier to drug development ( ). genetic factors contribute significantly to both scd risk and qt duration ( – ). however, with the exception of rare monogenic forms of congenital long or short qt syndromes, the precise genetic variants that account for the heritability of these traits in the general population have not been established. data from a recently staged genome-wide association study of german women from the extremes of the qt interval distribution identified an association between common variants in the nitric oxide synthase adaptor protein (nos ap) gene and adjusted qt interval duration ( ). this association was replicated in two other popula- tion-based studies ( ), an elderly population ( ) and the genetically more homogeneous old order amish popula- tion ( ). however, the propensity for false-positive reports in genetic association studies warrants replica- tion in additional cohorts: especially cohorts with high risk for prolonged qt interval and scd due to compet- ing factors, such as diabetes and coronary disease. in addition, because the common variants at the nos ap locus are highly differentiated across continental ances- tral groups, the extension of findings into samples of non-european ancestry is important ( ) (http:// www.hapmap.org/cgi-perl/gbrowse/hapmap_b /). in the current study, we attempted to replicate the association of two previously implicated nos ap single nucleotide polymorphisms (snps), rs and rs , with adjusted qt interval duration in the diabetes heart study, a sample of european-american and african-american pedigrees enriched for type diabetes. the extent to which qt interval is influenced by genetic variants will expand our understanding of myocardial electrical activity and could facilitate the development of new clinical therapies to reduce the risk of scd. research design and methods the study sample consists of european-american individuals ( type diabetes–affected individuals and unaffected individuals from fami- lies) and african-american individuals ( type diabetes–affected individuals and unaffected individuals from families) from the diabetes heart study. ascertainment and recruitment have been described previously ( – ). briefly, siblings concordant for type diabetes and lacking advanced renal insufficiency were recruited. type diabetes was defined clinically as diabetes developing after the age of years treated with insulin and/or oral agents, in the absence of historical evidence of ketoacidosis. all protocols were approved by the institutional review board of wake from the department of biochemistry, wake forest university school of medicine, winston-salem, north carolina; the center for human genomics, wake forest university school of medicine, winston-salem, north carolina; the cardiology division, massachusetts general hospital, boston, massachu- setts; the program in medical and population genetics, broad institute of harvard and mit, cambridge, massachusetts; the department of public health sciences, wake forest university school of medicine, winston-salem, north carolina; and the department of internal medicine, wake forest university school of medicine, winston-salem, north carolina. address correspondence and reprint requests to donald w. bowden, phd, department of biochemistry, wake forest university school of medicine, medical center boulevard, winston-salem, nc . e-mail: dbowden@ wfubmc.edu. received for publication september and accepted in revised form january . published ahead of print at http://diabetes.diabetesjournals.org on jan- uary . doi: . /db - . a.b.l. and c.n.-c. contributed equally to this work. additional information can be found in an online appendix at http:// dx.doi.org/ . /db - . ecg, electrocardiogram; nos ap, nitric oxide synthase adaptor protein; qtc, corrected qt interval; scd, sudden cardiac death; snp, single nucleotide polymorphism. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. original article diabetes, vol. , april forest university school of medicine, and all participants gave informed consent. participant examinations were conducted in the general clinical research center of the wake forest university baptist medical center and included interviews for medical history and health behaviors, anthropometric measures, resting blood pressure, fasting total cholesterol, electrocardiogra- phy, and spot urine collection. not all measurements were available for all participants. excluded from these analyses were european-american individuals ( . %) and african-american individuals ( . %) from the total diabetes heart study population who were identified by the minnesota code ( ) as having had a myocardial infarction. individuals were scored as using qt-altering medications if they were on at least one of the following medications: ) a medication known to prolong qt interval as listed at http://qtdrugs.org (http://www.arizonacert.org/medical-pros/drug-lists/drug-lists.htm); ) digoxin (known to shorten qt interval duration); or ) hormone replacement therapy (personal communication, d.m.h., md, mhs), resulting in the exclusion of european-american individuals ( . %) and african-american individuals ( . %) from analysis. qt interval measurement. a resting -lead electrocardiogram (ecg) was obtained for each study participant using a marquette mac ecg instrument (marquette electronics, milwaukee, wi) using a standardized protocol with special attention to precise placement of the electrodes. all ecgs were performed in the general clinical research center at wake forest university school of medicine and were electronically transmitted to a central reading center (epicare). each ecg was visually analyzed for recording errors and graded for quality. the ecgs were then analyzed using the marquette -sl program, version (ge healthcare, waukesha, wi) ( ). qt interval duration was defined as the duration in milliseconds from the initial deflection of the qrs complex until the return of the t-wave to the electrical baseline. the qt duration was measured in each of the ecg leads, and the longest qt duration was selected as the trait for analysis. the heart rate– corrected qt interval (qtc) was calculated using bazett’s formula: qtc � qt �hr � ( ). the qtc is commonly interpreted as the qt interval adjusted to a standard heart rate of beats per minute. genetic analysis. total genomic dna was purified from whole-blood sam- ples obtained from subjects using the puregene dna isolation kit (gentra, minneapolis, mn). dna concentration was quantified using standardized fluorometric readings on a hoefer dyna quant fluorometer (hoefer pharmacia biotech, san francisco, ca). each sample was diluted to a final concentration of ng/�l. genotypes for rs and rs were determined using a mass- array snp genotyping system (sequenom, san diego, ca) ( ). this genotyping system uses single-base extension reactions to create allele- specific products that are separated and scored in a matrix-assisted laser desorption ionization/time of flight mass spectrometer. primers for pcr amplification and extension reactions were designed using the massarray assay design software (sequenom). statistical analysis. maximum likelihood allele and genotype frequencies for each snp were calculated from unrelated probands and were tested for departures from hardy-weinberg equilibrium using both � and exact tests. estimates of linkage disequilibrium between snps were determined by calculating pairwise d� and r statistics in unrelated individuals. as previously reported, the microsatellite markers from a -cm genome scan were used to examine and correct self-reported familial relationships ( ). association between each snp and the phenotype under an additive genetic model was tested using variance components methods implemented in solar ( ). tests of statistical significance for the additive genetic model were based on the likelihood ratio statistic incorporating the familial correlation structure. despite the strong a priori nature of the hypothesis, we used two-sided tests of significance. all analyses were performed separately in european ameri- cans and african americans. within each ethnic group, analyses were performed in all participants and in diabetic subjects only, excluding subjects using any qt-altering medication. the following procedure was used to estimate the ethnicity-specific magnitude of the effect of each polymorphism while adjusting for the covariates. first, the residuals from the variance components model adjusting for familial relationships and containing the covariates but not the polymor- phisms were computed. second, using the above variance components model without the polymorphisms, the predicted value of a woman, diagnosed with diabetes, with the mean rr interval (european american � . , african american � . , european american with diabetes � . , and african american with diabetes � . ), the mean age (european american � . , african american � . , european american with diabetes � . , and african american with diabetes � . years), and mean bmi (european american � . , african american � . , european american with diabetes � . , and african american with diabetes � . kg/m ) was added to the residuals. third, the genotypic means of the residuals scaled to the predicted value of the above “individual” were computed to give a sense of the observed differences between the three genotypes for the respective polymorphisms. to estimate the ethnicity-specific genetic contribution to qt interval duration, a series of heritability analyses were computed using solar ( ). in these analyses, the overall phenotypic variation was partitioned into individ- ual variance components due to polygenic effects (multiple unmeasured genes under an additive variance), covariates (e.g., rr interval, age, sex, diabetes affection status, and bmi), and random environmental effects. the estimated heritability (h ) is defined as the ratio of the genetic variance component to the residual phenotypic variance and is an estimate of the familiality of the trait. to estimate the heritability and the proportion of variation explained by each polymorphism while adjusting for the above covariates, the heritability and proportion of variation in qt interval duration were computed with and without each individual polymorphism. results the clinical characteristics of the european-american men and women in families and african-american men and women in families are presented in table . as expected, the diabetes heart study participants carry a high burden of coronary artery disease risk factors with elevated bmi, rates of hypertension, and current and past smoking (table ). table clinical characteristics of european-american and african-american diabetes heart study participants after exclusion of individuals with history of myocardial infarction or on qt-altering medications european- american men european- american women african- american men african- american women n age (years) . 	 . . 	 . . 	 . . 	 . bmi (kg/m ) . 	 . . 	 . . 	 . . 	 . total cholesterol (mg/dl) . 	 . . 	 . . 	 . . 	 . smoking current . ( ) . ( ) . ( ) . ( ) past . ( ) . ( ) . ( ) . ( ) never . ( ) . ( ) . ( ) . ( ) diabetes diagnosis . ( ) . ( ) . ( ) . ( ) duration of diabetes (years) . 	 . . 	 . . 	 . . 	 . hypertension . ( ) . ( ) . ( ) . ( ) rr interval . 	 . . 	 . . 	 . . 	 . unadjusted qt interval duration (ms) . 	 . . 	 . . 	 . . 	 . qrs duration (ms) . 	 . . 	 . . 	 . . 	 . data are means 	 sd. a.b. lehtinen and associates diabetes, vol. , april dna from these participants was genotyped for two snps (rs and rs ) selected a priori based on the original report of association with qt interval duration ( ). for both snps, the genotyping success rate was %, and the genotyping consensus rate for dupli- cate dna samples within and across dna plates was %. in the european-american individuals, genotype frequencies were consistent with those expected under hardy-weinberg equilibrium (p . ). the two snps (minor allele frequency for g allele . and g allele . , respectively) were in strong linkage disequilibrium (d� � . ) and were correlated (r � . ). in the african- american individuals, rs genotypes (major allele frequency for g allele . ) were consistent with hardy- weinberg expectations. however, rs (major allele frequency for g allele . ) did not conform to hardy- weinberg expectations because of a deficiency in the observed number of heterozygotes (p � . ), possibly due to admixture of chromosomes of african and euro- pean ancestry, in which substantial frequency differences exist. in african americans, the two snps showed less linkage disequilibrium with each other (d� � . ) and substantially less correlation (r � . ). in european-american individuals not taking any medi- cation known to alter qt interval (n � ), the minor allele g at each locus was strongly associated with qt interval duration in an additive genetic model adjusting for rr interval, age, sex, diabetes affection status, and bmi (table ). there were . - and . -ms differences in adjusted qt interval duration for each additional minor allele for the two snps, respectively (p � . � � , p � . � � ). minor homozygotes of rs had a . -ms difference in qt interval duration, and rs minor homozygotes had a . -ms difference in a qt interval duration compared with major homozygotes. in analyses restricted to the diabetic subsample of euro- pean-american men and women, there was a . - and . -ms difference in adjusted qt interval duration for each additional minor allele for the two snps, respectively (p � . � � , p � . � � ). minor homozygotes for the two snps had longer qt interval duration compared with major homozygotes ( . - and . -ms difference, respec- tively). statistical significance was retained for both snps in the european-american and european-american dia- betic individuals when analysis of the heart rate– corrected qtc was performed (additive model p values of . and . for rs and rs , respectively; data not shown). when individuals with atrial fibrillation or bundle branch blocks were excluded, there were no appreciable differences in the point estimates or statistical significance (additive model p values of . � � and . � � in european-american individuals; additive model p values of . � � and . � � in european-american diabetic subjects). in addition, similar effects were ob- served when analyses were performed on the excluded sample of european-american individuals known to be taking a qt-prolonging medication (p � . ; data not shown). in the entire european-american sample, rs and rs explained . and . % of the variation in qt interval duration, and in the diabetic european-amer- ican sample, these snps explained . and . % of the variation in qt interval duration, respectively (table ). the heritability of qt interval duration adjusted for rr interval, age, sex, diabetes affection status, and bmi was . 	 . in the total sample and . 	 . in the diabetic subsample. a formal test of interaction of diabe- tes affection and snp-qt interval effect in the european- american sample was not significant (p � . for rs ; p � . for rs ) but was underpow- ered because of the small number of individuals free of overt diabetes. in african-american individuals not taking any qt- altering medications, there was no evidence of association between rs and qt interval duration in the total sample (n � ; p � . ) or in the diabetic subsample (n � ; p � . ). however, rs exhibited a trend toward association with qt interval in both the total african-american sample (two-sided p � . ; one-sided p � . ) and the diabetic african-american subsample (two-sided p � . ; one-sided p � . ). the results for rs should be interpreted with caution, however, because this snp did not conform to hardy-weinberg expectations in african-american individuals. in the afri- can-american sample, the g allele for each polymorphism is the major allele, but the observed effect is in the same direction as in the european-american sample with gg homozygotes having a longer qt interval duration than the alternate homozygotes. power in the african-american sample was limited to detect an effect of nos ap snps due to small sample size. specifically, under an additive model, the african-american sample provides � . power at � � . to detect a difference of . ms in qt interval per major allele copy; here, the difference of . ms is obtained by multiplying the approximate within- genotype sd for qt interval in african americans of � ms by the proportion of sd detectable of . (i.e., � . � . ) (supplemental table, available in the online appendix at http://dx.doi.org/ . /db - ). discussion these data strongly replicate the findings from three previous studies that common noncoding variants at the nos ap locus are associated with qt interval duration ( – ). the two snps evaluated—rs , located in the promoter region upstream of nos ap, and rs , located in intron of nos ap—are separated by kb genomic dna, have no known biological func- tion, and are not highly correlated with any known func- tional polymorphisms. taken together, these observations suggest that a causal untyped variant exists that is highly correlated with rs and rs . the data reported in the current study extend the prior findings to a cohort of families aggregating type diabetes, which are known to be at increased risk for disordered myocardial repolarization and increased risk of ventricular arrhythmias ( , ). in diabetic patients, the magnitude of the estimated effect (
) of nos ap snps on qt interval duration was larger than that observed in the total sample of diabetic and nondiabetic subjects or in the prior reports of community-based samples ( – ). although the formal test of interaction of diabetes affection and snp-qt inter- val effect was not significant (p � . for rs , p � . for rs in european-american individuals), the test was underpowered due to the small number of nondiabetic individuals in the study population. it should be noted that the nondiabetic subjects were also relatively obese, with many of these individuals having hypertension and metabolic syndrome (many were likely pre-diabetic). however, when comparing the estimated effect sizes ob- served in our european-american diabetic subjects to nos ap and qt interval in diabetes diabetes, vol. , april t a b l e a sso ciatio n an alysis o f tw o n o s a p s n p s an d q t in terval in e u ro p ean -a m erican an d a frican -a m erican d iab etes h eart s tu d y p articip an ts free o f m yo card ial in farctio n o r q t -alterin g m ed icatio n u se s n p † a lleles (m ajo r/m in o r) f requ en cy o f g allele g en o typ ic m ean s (q t in terval in m s) 
 fo r ad d itive gen etic m o d el p valu e fo r ad d itive gen etic m o d el* a ll e u ro p ean a m erican s rs t /g . t t ( n � ) t g ( n � ) g g ( n � ) . 	 . . � � . 	 . . 	 . . 	 . rs c /g . c c ( n � ) c g ( n � ) g g ( n � ) . 	 . . � � . 	 . . 	 . . 	 . d iab etic e u ro p ean a m erican s o n ly rs t /g . t t ( n � ) t g ( n � ) g g ( n � ) . 	 . . � � . 	 . . 	 . . 	 . rs c /g . c c ( n � ) c g ( n � ) g g ( n � ) . 	 . . � � . 	 . . 	 . . 	 . a ll a frican a m erican s rs g /t . t t ( n � ) t g ( n � ) g g ( n � ) . 	 . . . 	 . . 	 . . 	 . rs g /c . c c ( n � ) c g ( n � ) g g ( n � ) . 	 . . . 	 . . 	 . . 	 . d iab etic a frican a m erican s o n ly rs g /t . t t ( n � ) t g ( n � ) g g ( n � ) . 	 . . . 	 . . 	 . . 	 . rs g /c . c c ( n � ) c g ( n � ) g g ( n � ) . 	 . . . 	 . . 	 . . 	 . * p valu es rep o rted are fo r th e ad d itive gen etic m o d el fo r asso ciatio n w ith q t in terval ad ju sted fo r r r in terval, age, sex , d iab etes affectio n statu s (w h ere ap p ro p riate), an d b m i. p valu es sh o w n in b o ld rep resen t sign ifi can ce at th e p � . level. †c h ro m o so m e p h ysical p o sitio n s: (rs ) an d (rs ) relative to n atio n al c en ter fo r b io tech n o lo gy in fo rm atio n d b s n p b u ild . a.b. lehtinen and associates diabetes, vol. , april those in the subjects of the community-based rotterdam study and the old order amish, our estimates are statis- tically larger (rs european-american diabetic subjects 
 � . , rotterdam study 
 � . , p value � . ; rs european-american diabetic subjects 
 � . , old order amish 
 � . , p value � . ; and rs european-american diabetic subjects 
 � . , rotterdam study 
 � . , p value � . ). in addition, a test comparing diabetic and nondiabetic individuals sug- gests that there is a significant difference in qt interval duration between the two groups (two-sided p values of . and . for rs and rs , respec- tively) in our study population. we observed modest association of one nos ap variant in the african-ameri- can sample, in whom the minor qt-prolonging allele in european americans is the major allele, but power was limited to detect an effect because of the small sample size. notably, the variant modestly associated with qt interval in the african americans did not conform to hardy-weinberg expectations because of a deficiency in the observed number of heterozygotes compared with the expected number. however, because the genotyping suc- cess rate was . % and the genotyping consensus rate for duplicate dna samples within and across dna plates was %, it is unlikely that this deviation from hardy- weinberg expectations is due to genotyping error. for variants such as these nos ap snps with substantial frequency differences between chromosomes of african and european ancestry, deviation from hardy-weinberg expectations due to a deficiency of heterozygotes can be observed simply because of admixture of chromosomes of different ancestry, as is found in the african americans ( ). genetic association studies have been plagued by lack of reproducibility of reported associated variants ( ). in large part, inappropriately permissive thresholds for de- claring statistical significance and samples underpowered to detect modest effects underlie the previously chaotic field of genetic associations. the problem was previously more modest in scope because genotyping platforms lim- ited association tests to a handful of variants in candidate genes that have higher prior probabilities of harboring functional alleles. in the era of genome-wide association studies, the problem is compounded by the availability of hundreds of thousands of tests of variants with generally lower prior probability for association with a trait. use of appropriately rigorous statistical thresholds and replica- tion in multiple independent samples is critical to assure that apparent associations are true-positive results. thus, our finding that the previously reported association of common variants in nos ap with qt interval duration is strongly replicated is an important validation of the origi- nal report. diabetes is a potent risk factor for scd ( , , ). qt interval prolongation is known to be associated with both type and type diabetes ( , , – ). qt interval prolongation is also known to predict increased risk of scd and coronary heart disease death in the general population ( ) and is a particularly potent risk factor in the diabetic population ( ). whether the relationship of dia- betes to qt prolongation is through acquired autonomic dysfunction ( ), which presumably might have a nonge- netic basis or reflects some fundamental interaction with genetic factors of the diabetic host, is unknown. however, the demonstration that genetic variants in nos ap repro- ducibly alter qt interval duration in families aggregating diabetes demonstrates that genetic effects are not swamped out by the strong effect of diabetes on repolar- ization. rather, the opposite appears to be true: the reduced repolarization reserve of diabetes ( , ) shows a synergistic interaction with genetic variants that alter myocardial repolarization. it is thus notable that the strength of the effect of nos ap snps in european- american diabetic subjects in the current report, . - to . -ms differences between alternate homozygotes, is much greater than that observed in the previous reports by arking et al. ( ) ( – ms) aarnoudse et al. ( ), ( . – . ms) and post et al. ( ) ( . – . ms). of additional interest, % (n � ) of all european-american diabetic subjects (n � ) in the diabetes heart study is using a qt-altering medication (data not shown). it is therefore possible that this medication use in the diabetic popula- tion, in concert with the presence of qt-prolonging alleles of nos ap, may increase the risk for arrhythmias and scd in this patient subset. this is an important issue to address in the future. strengths of our study include the well-phenotyped study sample, the precise electrocardiographic measures, the ability to adjust for multiple potential confounders, and the modest heritability of qt interval in the study population. in fact, our heritability results in european americans (h � . ) are notably higher than the esti- mates in the framingham heart study (h � . ) ( ) or in female (h � . ) ( ) or male (h � . ) ( ) twins. in addition, the availability of detailed medication information allowed exclusion of individuals on qt-altering medications. some limitations apply. our study had better power to detect nos ap effects on qt interval duration in individ- uals of european ancestry but was clearly underpowered to demonstrate convincingly whether nos ap variants table proportion of variance in qt interval duration explained by covariates and estimates of heritability in the european-american sample with and without each snp individually under an additive genetic model covariates proportion of variance attributable to covariates proportion of variance attributable to each snp h r all european americans rr interval, age, sex, diabetes, and bmi . . 	 . covariates plus rs . . . 	 . covariates plus rs . . . 	 . diabetic european americans only rr interval, age, sex, and bmi . . 	 . covariates plus rs . . . 	 . covariates plus rs . . . 	 . h r data are means 	 se. h r represents the residual heritibility after adjusting for covariates and each snp individually. nos ap and qt interval in diabetes diabetes, vol. , april influence qt interval duration in individuals of african ancestry. ultimately, testing in large numbers of african americans will be required to establish whether common genetic variants at nos ap are related to qt interval duration. differences in the genetic architecture of qt interval duration or in patterns of linkage disequilibrium between individuals of european and african ancestry could contribute to lack of association in african ameri- cans. the small number of scds and difficulty in its ascertainment precluded an analysis of the influence of nos ap variants on scd risk. in summary, these data provide convincing replication of the association with continuous qt interval duration of nos ap variants in families of european ancestry aggre- gating diabetes. further studies in diabetic individuals will be needed to test the hypothesis raised here that genetic variants influencing qt interval duration are particularly important with regard to the burden of scd among diabetic subjects. genetic studies of qt interval variation in diabetic individuals, who have reduced repolarization reserve, may be particularly fruitful in identifying variants contributing to risk of arrhythmia. acknowledgments a.b.l. has received national heart, lung, and blood institute grant f -hl- . c.n.-c. has received na- tional heart, lung and blood institute grant k -hl- , a doris duke charitable foundation clinical scientist development award, and a burroughs wellcome fund career award for medical scientists. k.r.d. has received national institutes of health grant t -hl- - . d.w.b. has received general clinical research center of the wake forest university school of medicine grant m rr and national heart, lung, and blood institute grant r -hl- . we acknowledge dr. wendy post and her collaborators for kindly providing us with the effect size estimates for nos ap snps in the old order amish population. references . heart disease and stroke statistics: update. dallas, tx, american 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spooner pm, meitinger t, chakravarti a: a common genetic variant in the nos regulator nos ap modulates cardiac repolarization. nat genet : – , . aarnoudse aj, newton-cheh c, de bakker pi, straus sm, kors ja, hofman a, uitterlinden ag, witteman jc, stricker bh: common nos ap variants are associated with a prolonged qtc interval in the rotterdam study. circulation : – , . post w, shen h, damcott c, arking de, kao wh, sack pa, ryan ka, chakravarti a, mitchell bd, shuldiner ar: associations between genetic variants in the nos ap (capon) gene and cardiac repolarization in the old order amish. hum hered : – , . frazer ka, ballinger dg, cox dr, et al.: a second generation human haplotype map of over . million snps. nature : – , . wagenknecht le, bowden dw, carr jj, langefeld cd, freedman bi, rich ss: familial aggregation of coronary artery calcium in families with type diabetes. diabetes : – , . bowden dw, rudock m, ziegler j, lehtinen ab, xu j, wagenknecht le, herrington d, rich ss, freedman 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reserve in mammalian heart. cardiovasc res : – , . carter n, snieder h, jeffery s, saumarez r, varma c, antoniades l, spector td: qt interval in twins. j hum hypertens : – , . russell mw, law i, sholinsky p, fabsitz rr: heritability of ecg measure- ments in adult male twins. j electrocardiol (suppl.): – , nos ap and qt interval in diabetes diabetes, vol. , april wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ fontographer . genetic influences on premature parturition in an australian twin sample susan a treloar , george a macones , laura e mitchell and nicholas g martin queensland institute of medical research and joint genetics program, the university of queensland, brisbane, australia the university of pennsylvania health system the children’s hospital of philadelphia, philadelphia, pa, usa we investigated possible genetic influences on women’s liability to preterm birth, using data from a large sample of australian female twin pairs. in a – questionnaire survey, both members of parous twin pairs ( monozygotic and dizygotic) reported on whether deliveries had been more than two weeks preterm. tetrachoric twin pair correlations for first birth were rmz = . ± . and rdz = – . ± . , and for any birth were rmz = . ± . and rdz = . ± . . best-fitting models to data contained only additive genetic influences and individual environmental effects. heritability was % for preterm delivery in first pregnancy, and % for preterm delivery in any pregnancy. in the former case, however, we could not reject a model without genetic influences. although our data did not allow for differentiation of the varying aetiologies of premature parturition, results from this exploratory analysis suggest that further investigation of genetic influences on specific reasons for preterm birth is warranted. twin research ( ) , – . keywords: preterm birth, pregnancy outcome, twins, genetics the risk of preterm birth has been found to be higher for women who were themselves born prematurely, suggesting a repeating pattern across generations. the relevance of genetic influences on prematurity had been highlighted by an earlier study using old order amish records in lancaster county, pennsyl- vania, which suggested that prematurity was mostly related to the maternal genotype. in order to explore the possibility of genetic influences on a woman’s liability to preterm births we have examined self-reported, retrospective data on premature birth provided by a large number of australian, female twin pairs. these twins are members of the australian national health and medical research council twin registry, and partic- ipated in a health and lifestyle survey conducted between and . – responses to this survey were obtained from both members of female monozygotic (mz) and dizygotic (dz) twin pairs aged and older, for a pairwise response rate of %. the study questionnaire included questions on their menstrual cycles and reproductive events, including specific obstetric factors relating to each delivery. – premature delivery (preterm birth) was defined in the questionnaire as delivery ‘over weeks early’, rather than the standard definition of curtailment of pregnancy before the th week of gestation. we did not ask for number of weeks gestation. we investi- gated parous twins’ independent reports of pre- mature delivery of first baby, and also reports of whether any birth was premature using the > week criterion. premature births may have been sponta- neous or induced, vaginal deliveries or performed by caesarean section. data were also available on these items, but analyses are not presented here. twin pair matrices of tetrachoric correlations, and corresponding asymptotic covariance matrices, were computed separately for mz and dz twin pairs, using the windows version of prelis . . these correlations apply appropriately to dichotomous categorical data where the underlying distribution is assumed to be continuous and normally distributed. genetic models were fitted by the method of asymp- totic least squares to estimate the contributions of additive genetic, shared and non-shared environ- mental effects, using mx. we proceeded by system- atically testing the significance of dropping parame- ters in turn. in addition to the likelihood ratio ø test (lr), the akaike information criterion (aic, meas- ured as ø - df) was used as an additional indicator of fit. on the grounds of parsimony the model with the least number of parameters that offered a fit not significantly worse than the full model was chosen. data analysis methods are described more fully elsewhere. correspondence: susan a treloar, phd, queensland institute of medical research, po royal brisbane hospital, queensland , australia. tel: ; fax: ; e-mail: suet@qimr.edu.au received november ; accepted november twin research ( ) , – y © macmillan publishers ltd all rights reserved – / $ . www.nature.com/tr both members of parous twin pairs ( mz pairs and dz pairs) answered the question concerning prematurity of their first and any sub- sequent birth. ages of parous twins ranged from to years, and mean age was . ± . years. mean ages of mz twins ( . years) and dz ( . ) twins did not differ significantly. the prevalence of premature first birth in the sample was . % of individuals in mz pairs and higher at . % in the dz twins, giving an overall prevalence of . %. premature parturition relating to any birth using the > week criterion was reported by . % of individ- uals in mz pairs and . % of twins in dz pairs. the percentage of nulliparae was the same ( %) for both mz and dz individuals. non-parametric one-way median score tests showed that parous mz and dz twins did not differ significantly on median total pregnancies. however, they did differ on median number of full-term (vs aborted) pregnancies (p < . ) and of offspring (p < . ), the latter median being higher for dz twins ( ) than for mz twins ( ). this suggested a higher prevalence of multiple births in the dz twins. the median number of preterm babies reported by dz twins was significantly higher than for mz twins (p < . ), consistent also with a higher rate of multiple births. the ratio of mz to dz twin pair correlations ( > : ) shown in table suggests that genetic influ- ences may be operating on premature parturition. for both prematurity variables, using the aic as the indicator, best-fitting most parsimonious models contained only additive genetic variation (a) and individual environmental effects (e). for having had a premature delivery for any birth, the best fitting model (aic = – . ) included a heritability of %. a model including non-additive genetic influ- ences resulted in a better fit than a model containing shared environment (c), but was not a significant improvement on the ae model. a ce model contain- ing no additive genetic variation was rejected on significance grounds. for premature delivery, the ae model was again the model of choice (aic = – . ) with a heritability of %; however, in this case a model containing no genetic influences could not be rejected on significance grounds. evidence suggests that in australia the impact of changes in obstetric care that might influence pre- mature labour were minimal up to . we would not expect the impact of obstetric practices to differ for individuals on the basis of their zygosity. increased prevalence of multiple births to dz twins may have influenced pairwise concordance for pre- term birth, but any effect would have been too small to detect in this sample. data were retrospective and collected in – , before the widespread availa- bility of assisted reproductive technologies, which are increasing the prevalence of non-monozygotic multiple births, and hence may influence prevalence of premature delivery. we cannot unequivocally separate out the differ- ent aetiologies for preterm birth in our data. the aetiologies of spontaneous premature membrane rupture or preterm labour may well differ, and those of indicated preterm births relating to foetal or maternal morbidity clearly may vary even further. retrospectivity may have influenced accuracy of recall of preterm delivery. data on length of gestation or date of last menstrual period (lmp) were not available. furthermore, genetic heterogeneity may be impli- cated in the predisposing conditions. for example, the prevalence of pre-eclampsia, which may be associated with indicated premature delivery, is higher for first than subsequent births. if the aetiol- ogy is foetal in origin rather than maternal, which may well be the case, the effect may be to reduce the (maternal) twin pair correlations. this might suggest that the heritability we have observed is occurring in the spontaneous rather than the indi- cated preterm deliveries, but to test this hypothesis would require a very large sample indeed. it would be fascinating to know whether, for example, there is a genetic contribution to factors such as cervical incompetence per se. nevertheless, our data do suggest that genetic influences may be important in influencing preterm birth, and further investigation of this hypothesis may well be warranted. acknowledgements we thank the national health and medical research council of australia for support (no. , no. ) and the twins for their co-operation. references porter t, fraser a, hunter c, ward r, varner m: the risk of preterm birth across generations. obstet gynecol ; : – . khoury m, cohen b: genetic heterogeneity of prematurity and intrauterine growth retardation: clues from the old order amish. am j obstet gynecol ; : – . table tetrachoric twin pair correlations for prematurity of first delivery and of any delivery mz pairs (n= ) dz pairs (n= ) premature by twin pair twin pair > weeks correlation se correlation se first birth . . – . . any birth . . – . . twin research genetic influences on preterm birth in australian twin mothers sa treloar et al y do k-a, treloar sa, pandeya n, purdie d, green a, heath ac, martin ng: predictive factors of age at menopause in a large australian twin study. hum biol ; : – . heath a, cloninger c, martin n: testing a model for the genetic structure of personality: a comparison of the person- ality systems of cloninger and eysenck. j pers soc psychol ; : – . treloar s, martin n, heath a: longitudinal genetic analysis of menstrual flow, pain and limitation in a sample of australian twins. behav genet ; : – . treloar s, martin n, dennerstein l, raphael b, heath a: pathways to hysterectomy: insights from longitudinal twin research. am j obstet gynecol ; : – . jöreskog k, sörbom d: prelis . for windows. scientific software international inc: chicago, . neale m: mx: statistical modeling. department of psychiatry: mcv, richmond, va, . neale m, cardon l: methodology for genetic studies of twins and families nato asi series. kluwer academic publishers: dordrecht, . heath ac, martin ng, eaves lj, loesch d: evidence for polygenic epistatic interactions in man? genet ; : – . stanley f: perinatal outcome in western australia, to . perinatal mortality and birthweight. med j aust ; : – . thornton j, macdonald a: twin mothers, pregnancy hyper- tension and pre-eclampsia. br j obstet gynaecol ; : – . twin research genetic influences on preterm birth in australian twin mothers y sa treloar et al wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ r e v i e w a r t i c l e – g a s t r o i n t e s t i n a l o n c o l o g y use of tumor markers in gastrointestinal cancers: surgeon perceptions and cost-benefit trade-off analysis amish acharya, mrcs, sheraz r. markar, mrcs, michael matar, mbbs, melody ni, phd, and george b. hanna, phd division of surgery, department of surgery and cancer, st mary’s hospital, imperial college london, london, uk abstract background. gastrointestinal cancers constitute the third most common cancers worldwide. tumor markers have long since been used in the postoperative surveillance of these malignancies; however, the true value in clinical practice remains undetermined. objective. this study aimed to evaluate the clinical utility of three tumor markers in colorectal and esophagogastric cancer. methods. a systematic review of the literature was undertaken to elicit the sensitivity, specificity, statistical heterogeneity and ability to predict recurrence and metas- tases for carcinoembryonic antigen (cea), cancer antigen (ca) - and ca . european surgeons were surveyed to assess their current practice and the characteristics of tumor markers they most valued. data from the included studies and survey were combined in a cost-benefit trade- off analysis to assess which tumor markers are of most use in clinical practice. results. diagnostic sensitivity and specificity were ranked the most desirable characteristics of a tumor marker by those surveyed. overall, studies were included to inform the cost-benefit trade-off. the cost-benefit trade-off showed that cea outperformed both ca - and ca , with lower financial cost and a higher sensitivity, and diagnostic accuracy for metastases at presentation (area under the curve [auc] . vs. . vs. . ), as well as similar diagnostic accuracy for recurrence (auc . vs. . ). conclusions. cost-benefit trade-off analysis identified cea to be the best performing tumor marker. further studies should seek to evaluate new tumor markers, with investigation tailored to factors that meet the requirements of practicing clinicians. gastrointestinal cancers are the third most common cancers worldwide, with a prevalence of , , in the us in . survival for gastrointestinal malignancies have been improving worldwide due to advances in mul- timodality treatments, diagnostic strategies and expanding the criteria for treatable disease. while these strategies commonly involve a combination of radiological and endoscopic techniques, some studies have shown serum tumor markers may have a diagnostic, as well as thera- peutic, monitoring role. , a tumor marker is defined as a compound produced by the tumor or the host, in response to a malignancy. tra- ditionally, markers such as carcinoembryonic antigen (cea) and, to a lesser extent, cancer antigen (ca) - , have been used clinically to monitor disease response, whereby the efficacy of treatments can be assessed by noting a reduction in the level of a marker, which was previously high. in colorectal cancer, the use of tumor makers in postoperative surveillance has been recom- mended by the american society of clinical oncology; however, their use in the identification of metastasis and diagnostic accuracy has not been established despite some clinicians incorporating them into regular practice. in contrast, the use of tumor markers in esophagogastric malignancies in any capacity remains controversial. a number of studies have assessed the use of several tumor markers in prognosis and diagnosis, however they are of electronic supplementary material the online version of this article (doi: . /s - - -y) contains supplementary material, which is available to authorized users. � the author(s) . this article is published with open access at springerlink.com first received: april ; published online: december g. b. hanna, phd e-mail: g.hanna@imperial.ac.uk ann surg oncol ( ) : – doi . /s - - -y http://dx.doi.org/ . /s - - -y http://crossmark.crossref.org/dialog/?doi= . /s - - -y&amp;domain=pdf http://crossmark.crossref.org/dialog/?doi= . /s - - -y&amp;domain=pdf limited quality. , as such, there is no clear consensus on the use of tumor markers, and current practice is dependent on the individual clinician’s choice. in both cancer types, there is hence a clear need for an objective evaluation of common tumor markers in several clinical scenarios. this appraisal requires a comparison of clinical utility and costs or negatives with the utilization of the marker; however the relative importance of these benefits and costs, or performance characteristics, as dri- vers to uptake has not yet been quantified by the literature and would be dependent on how clinicians perceive the use of tumor markers. this study aimed to critically assess the cost-benefit trade-off of three common tumor markers in gastrointestinal cancers by means of evaluating the per- ceptions of surgeons to common markers. methods literature search strategy a literature search of the pubmed, ovid medline, embase and google scholar electronic databases was conducted from january up to and including december for studies regarding the use of tumor markers in the diagnosis, postoperative surveillance, or prediction of metastasis in colorectal and esophagogastric cancer (online appendix ). search terms used included ‘colorectal neoplasms’, ‘esophageal cancer’, ‘gastric can- cer’, ‘tumour markers’, ‘neoplasm antigens’, ‘tumour- associated antigens’, ‘prognosis’, ‘recurrence’, ‘metastasis’ and ‘staging’ in various combinations, as well as the name of the specific markers, relevant surgical procedures, and alternative spellings, e.g. tumor. research titles were then screened for suitability, with full-text copies retrieved. all studies that investigated the diagnostic, prognostic, or predictive ability of a single or multiple tumor marker in colorectal or esophagogastric cancers that could be tested in patients were included. exclusion criteria involved studies with no available eng- lish translation, published abstracts only, and those assessing the predictive ability for metastases in which no diagnostic accuracy or recurrence data could be calculated. of those studies meeting the inclusion criteria, the stated specificity and sensitivity were extracted. studies that did not explicitly state the sensitivity and specificity of the marker were independently calculated and verified by two authors (aa and srm), provided sufficient data were available. literature standard the quadas- tool, which involves four domains, i.e. patient selection, index test, reference standard, and flow of subjects through the study, was used to appraise the standard of the literature, and was implemented to assess the quality and risk of bias of the included studies. the reference standard was histological confirmation of malignancy or recurrence. tumor marker survey surgeons affiliated with the european association of endoscopic surgery were invited to complete an anony- mous survey regarding tumor markers. these surgeons were asked to rank attributes of the ‘ideal marker’ in order of their perceived importance to routine clinical practice (online appendix ). no duplication of rank was permit- ted. these characteristics included diagnostic sensitivity, specificity, consistency across demographics, patient acceptability, cost, time for result, and predictive of recurrence and metastases (defined by the auc). a sum- mative rank was then calculated and informed the weighting for the cost-benefit trade-off analysis. statistical methodology for each of the assessments of cancer diagnosis, recur- rence, and metastasis, paired sensitivity and specificity were calculated from each eligible study, as appropriate. a bivariate model for meta-analysis of statistical accuracy provides more accurate results than fixed-effects modeling. following the validated methodology of harbord et al. , bivariate meta-analyses were performed to generate pooled point estimates and % confidence intervals (cis) for the sensitivity and specificity of the tumor marker under investigation, with histopathological confirmation of malignancy, together with hierarchical summary receiver operating characteristic (roc) curves. the software used for this analysis was the custom-designed statistical pack- age michigan interactive data analysis system (midas). areas under the hierarchical summary roc curves, as well as i statistics, were obtained directly from the midas output (see zhou and tu for an in-depth description of the statistical methods used. ) performance characteristics the performance of the three tumor markers, with respect to the eight characteristics surgeons were asked to rank in the survey, was calculated (tables , ). cost and speed of result were taken as the stated laboratory process costs from st mary’s hospital, paddington, uk. sensitiv- ity, specificity, prediction of metastases at primary diagnosis, and recurrence following resection were calcu- lated from the aforementioned pooled analyses, with the a. acharya et al. t a b l e p e rf o rm a n c e o f c e a , c a - a n d c a w it h re sp e c t to th e e ig h t p e rf o rm a n c e c h a ra c te ri st ic s fo r c o lo re c ta l c a n c e r c h a ra c te ri st ic d e si ra b il it y fr o m th e su rv e y c h a ra c te ri st ic c e a c a - c a d ia g n o st ic se n si ti v it y . . . d ia g n o st ic sp e c ifi c it y . . . p re d ic ti v e o f re c u rr e n c e . . . p re d ic ti v e o f m e ta st a si s . . . l o w c o st (£ ) . . . s p e e d o f re su lt (h o u rs ) . . . p a ti e n t a c c e p ta b il it y g o o d g o o d g o o d c o n si st e n c y . . . c e a c a rc in o e m b ry o n ic a n ti g e n , c a c a n c e r a n ti g e n t a b l e p e rf o rm a n c e o f c e a , c a - a n d c a w it h re sp e c t to th e e ig h t p e rf o rm a n c e c h a ra c te ri st ic s fo r e so p h a g o g a st ri c c a n c e r c h a ra c te ri st ic d e si ra b il it y fr o m th e su rv e y c h a ra c te ri st ic c e a c a - c a d ia g n o st ic se n si ti v it y . . . d ia g n o st ic sp e c ifi c it y . . . p re d ic ti v e o f re c u rr e n c e . . . p re d ic ti v e o f m e ta st a si s . . . l o w c o st (£ ) . . . s p e e d o f re su lt (h o u rs ) . . . p a ti e n t a c c e p ta b il it y g o o d g o o d g o o d c o n si st e n c y . . . c e a c a rc in o e m b ry o n ic a n ti g e n , c a c a n c e r a n ti g e n use of tumor markers in gastrointestinal cancers latter two represented by the area under the curve (auc). consistency was calculated from the i heterogeneity statistic from the included studies, representing the per- centage of total variation across the studies, with a higher number meaning lower consistency. cost-benefit trade-off analysis the eight performance characteristics were broadly divided into either costs (time for result and financial cost) or benefits (sensitivity, specificity, predictive ability for recurrence or metastases and consistency). to assess trade- offs between costs and benefits among the tumor markers, we employed multi-criteria decision analysis (mcda) methods. – to achieve this, we rated each tumor marker on all the performance characteristics (criteria ratings), and assessed the relative importance of performance characteristics (criteria weights) based on the average rankings retrieved from the tumor marker survey (see above). using a weighted average model, we then com- bined the ratings to produce an overall benefit score for each tumor marker, and then contrasted the benefit scores against the scores on costs and time criteria, respectively. criteria ratings within mcda, the performance of each tumor marker (so-called a criteria rating), with respect to each charac- teristic, is bounded between , assigned to the worst- performing tumor marker (e.g. most expensive or least sensitive), and for the best-performing marker (e.g. cheapest or most sensitive). we assumed linearity between performance and rating, using linear interpolation to assess criteria ratings of any intermediate performance. for instance, if the costs of one tumor marker were halfway between the most expensive and the least expensive options, then this tumor marker received a criteria rating of with respect to cost. criteria weights trade-offs between the criteria are achieved through ‘criteria weights’, which capture the relative importance of the eight performance characteristics. from the tumor marker survey, respondents provided rankings as to the desirability of each of the characteristics. we converted the average rankings into numerical weights by assigning a criteria weight of to the highest ranked performance characteristic, to the second highest ranked, and so forth. we then normalized all the weightings so that they totaled . for example, if sensitivity received a criteria weight of , whereas specificity received a weight of , this would imply that the surgeons considered a difference of %, with respect to the diagnostic sensitivity between two tumor markers, to be equivocal to a difference of % in their relative diagnostic specificities. overall benefit scores our aim was to assess trade-offs between the costs and benefits of using a tumor marker. we therefore combined the ratings of the characteristics previously designated as benefits into an overall score. under the assumption that these characteristics are independent of each other, we can assess the overall score by a weighted average model: benefit ¼ x k wkrk where rk is the rating on the kth benefit criterion and wk is the weight assigned to that criterion. for instance, suppose the benefit of tumor markers comprises their sensitivity and specificity alone. assuming that a tumor marker receives ratings of and on sensitivity and specificity, respectively, which have nor- malized weights of . and . , it follows that the benefit score of this tumor marker is . (= . ? ? . ). trade-offs using normalized weights, the benefit scores are boun- ded between and ; a hypothetical tumor marker that has the worst performances on any criteria is scored , whereas a tumor marker that has the best performances is scored . the higher the score, the more beneficial the tumor marker is and vice versa. we plotted two-way cost- benefit maps to compare benefit against financial costs, and separately from time to results. the analysis was performed using the decision analytic software hiview, version . . . (educational copy). results literature search a total of full-text articles met the inclusion criteria and were appraised following the literature search (online appendix ), constituting a total of , patients ( , with colorectal malignancy and , with esophagogastric malignancy). overall, articles assessed colorectal tumor markers ( on diag- nostic ability, on prediction of recurrence, and on prediction of metastases), and articles assessed esophagogastric tumor markers ( on diagnostic ability, on prediction of recurrence, and on prediction of metastases). a. acharya et al. quadas- evaluation the results of the quadas- evaluation are shown in fig. . of the studies included, % had a ‘high risk’ of bias with respect to patient selection, while a further % did not provide sufficient detail on exclusion criteria for patients enrolled in the study. with respect to the ref- erence standard, % had a ‘high risk’ of bias due to their retrospective nature. pooled analysis for diagnostic sensitivity and specificity the pooled diagnostic sensitivity and specificity of cea in colorectal cancer was . ( % ci . – . ) and . ( % ci . – . ), respectively (table ). for ca - , sensitivity was . ( % ci . – . ) and specificity was . ( % ci . – . ) and, for ca , pooled diagnostic sensitivity was . ( % ci . – . ) and specificity was . ( % ci . – . ). heterogeneity for diagnosis determined using the i statistic, which repre- sented the consistency, was . for cea and . for ca - and ca . similar results were found for esophagogastric cancer (table ). tumor marker survey the survey was distributed online from august to september , and a total of responses were t a b l e r e su lt s o f th e p o o le d se n si ti v it y , sp e c ifi c it y a n d a u c fr o m th e li te ra tu re se a rc h fo r d ia g n o st ic a b il it y , p re d ic ti v e a b il it y o f re c u rr e n c e a n d m e ta st a se s fo r c e a , c a - a n d c a , w it h re sp e c t to c o lo re c ta l c a n c e r t u m o r m a rk e r d ia g n o si s d ia g n o si s o f re c u rr e n c e d ia g n o si s o f m e ta st a se s s e n si ti v it y s p e c ifi c it y a u c s e n si ti v it y s p e c ifi c it y a u c s e n si ti v it y s p e c ifi c it y a u c c e a . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . c a - . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . . . * . * . * c a . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . . * . * . * v a lu e s in p a re n th e se s re p re se n t % c o n fi d e n c e in te rv a ls a u c a re a u n d e r th e c u rv e , c e a c a rc in o e m b ry o n ic a n ti g e n , c a c a n c e r a n ti g e n * in su ffi c ie n t d a ta a v a il a b le risk of bias applicability flow reference standard reference standard index selection index selection patients selection patients selection percentage of studies percentage of studies high high low low unclear unclear fig. results of the quadas- evaluation of the included studies with respect to the risk of bias and applicability use of tumor markers in gastrointestinal cancers collected, representing a response rate of . %. there were respondents with an interest in colorectal disease ( consultants, registrars, and who were primarily academic), and who had completed a median of more than cancer operations. cea was the most commonly utilized tumor marker in col- orectal cancer (fig. a), with surveillance for recurrence the most common indication (fig. c). with respect to upper gastrointestinal surgery, respondents ( consultants, registrars, and primarily academic) had completed a median of more than cancer operations. cea was the most commonly employed tumor marker (fig. b), with assessment for recurrence being the most common indication (fig. d). of the eight performance characteristics, the ideal tumor marker would have, diagnostic sensitivity ranked the overall highest (most desirable), followed by diagnostic specificity. consistency across demographics was considered the least desirable (tables , ). cost-benefit trade-off all tumor markers had identical performances with regard to patient acceptability and speed for result. as all were derived from serum, with a result returned within h, these characteristics were removed from the analysis. tables and display the performance of each tumor marker with respect to the eight char- acteristics and the associated importance ranking derived from the survey. figure displays the trade-offs between the ben- efits (high diagnostic sensitivity, specificity, consistency, predictive ability for recurrence and metastases) and costs (financial). cea outperformed both ca - and ca with respect to overall utility; it had lower associated financial costs and higher benefits, as weighted by the importance placed on the characteristics. this pattern was seen with both colorectal and esophagogastric cancer. discussion the present study has highlighted the variable performance of common tumor markers in the assessment of gastrointestinal cancers. despite this variability, the majority of surgeons who were sur- veyed utilize tumor markers in their practice. cea was found to have high utility, with a high ability to predict recurrences and metastases, and was also associated with the lowest costs, primarily financial,t a b l e r e su lt s o f th e p o o le d se n si ti v it y , sp e c ifi c it y a n d a u c fr o m th e li te ra tu re se a rc h fo r d ia g n o st ic a b il it y , p re d ic ti v e a b il it y o f re c u rr e n c e a n d m e ta st a se s fo r c e a , c a - a n d c a , w it h re sp e c t to e so p h a g o g a st ri c c a n c e r t u m o r m a rk e r d ia g n o si s p re d ic ti o n o f re c u rr e n c e p re d ic ti o n o f m e ta st a se s s e n si ti v it y s p e c ifi c it y a u c s e n si ti v it y s p e c ifi c it y a u c s e n si ti v it y s p e c ifi c it y a u c c e a . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . c a - . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . c a . ( . – . ) . ( . – . ) . . * . * . * . ( . – . ) . ( . – . ) . v a lu e s in p a re n th e se s re p re se n t % c o n fi d e n c e in te rv a ls a u c a re a u n d e r th e c u rv e , c e a c a rc in o e m b ry o n ic a n ti g e n , c a c a n c e r a n ti g e n * in su ffi c ie n t d a ta a v a il a b le a. acharya et al. as all three tumor markers took the same timeframe to attain the result. therefore, cea outperformed ca - and ca in cost-benefit trade-off with respect to both colorectal and esophagogastric cancer. however, all markers had poor sensitivity, which would suggest their use in diagnosis is significantly limited. , cea is a glycoprotein produced in minimal amounts after fetal development, and which is involved in cell adhesion. the use of cea in postoperative surveillance is well established, with the majority of surgeons surveyed using tumor markers for monitoring recurrence. we have shown that cea could be of use in the assessment of metastasis, having a predictive ability of %, due to its association with the spread of cancer and increasing tumor burden. however, the use of cea in diagnosis is not widely endorsed. its use is confounded by its association with smokers, and the need for repeated measurements to mit- igate limited sensitivity. despite this, our survey showed % of surgeons still utilize tumor markers for diagnosis, whereas we have shown cea performs similarly to chance, with a diagnostic sensitivity of %. moreover, high diagnostic sensitivity and specificity were the two most desired characteristics of the ‘ideal tumor marker’, despite monitoring of recurrence being the primary role for cea. hence, there is a clinical need for an improved diagnostic tumor marker for colorectal cancer. while utilizing a single diagnostic marker is challenging, a diagnostic tumor mar- ker that could be used in conjunction, or even triage further more specific testing, would be of use. tumor markers are most commonly used in clinical practice in combination rather than in isolation; however, it was not possible to test the combination of tumor markers in this current study due to limitations in the data. none of the traditional tumor markers would satisfy the criteria for use alone clinically, and, as such, research should focus on novel markers with diagnostic ability. , in contrast to colorectal cancer, there remains no con- sensus for the use of tumor markers in esophagogastric cancer. despite this, they appear widely used in clinical practice, with cea and ca - the most often employed, and monitoring for recurrence the most common indica- tion, but, in as many as % of cases, surgeons admitted to using tumor markers for diagnostic investigation. while cea again outperformed ca - and ca overall, all three tumor markers were found to have low diagnostic accuracy, as determined by the auc. unlike in colorectal cancer, all three were also found to have a low capacity to predict recurrence, which should preclude their use in widespread clinical practice. (a) colorectal cancer cea monitor recurrence predict metastases assess surgical difficulty other diagnosis monitor recurrence predict metastases assess surgical difficulty other diagnosis ca - ca other none cea ca - ca other none % % % % % % % % % % % % % % % % % % % % (c) colorectal cancer (b) oesophago-gastric cancer (d) oesophago-gastric cancer fig. survey results detailing the most commonly used tumor markers and the most commonly cited indications in (a, c) colorectal cancer and (b, d) esophagogastric cancer. cea carcinoembryonic antigen, ca cancer antigen use of tumor markers in gastrointestinal cancers discrepancy with the use of tumor markers in esopha- gogastric cancer would suggest further prospective evaluation is warranted, with a degree of discrimination with respect to their clinical interpretation. furthermore, given the sensitivity of upper gastrointestinal endoscopy of %, the capacity for histological evaluation, and surveil- lance for barrett’s esophagus, the pragmatic value of traditional tumor markers for esophagogastric cancer is restricted. this study has also highlighted the importance of a holistic evaluation of tumor markers prior to incorporating them into clinical practice. while ‘benefits’ such as diag- nostic sensitivity and specificity are key characteristics, as seen by their relative importance denoted in the survey, there must be an appreciation of costs. a marker that has high sensitivity but has a high financial cost or requires excessive processing time may be practically precluded from widespread use, which may explain the fact that despite poor diagnostic accuracy and only relatively high predictive ability, the tumor markers this study has appraised are still in widespread use. while novel markers may offer greater diagnostic use, they are also likely to require more esoteric, and therefore expensive, assays. the limitations of this study are as a result of the pub- lished studies included to inform the cost-benefit trade-off. quadas evaluation revealed a ‘high’ level of bias in % of the studies due to their retrospective design, limiting the reliability of the results obtained from the pooled analyses. moreover, the potential for bias in some of these studies was high due to their case-control design and restrictive exclusion criteria, as was seen by the ‘high’ risk of bias with respect to patient selection. as only a few studies assessed the use of ca - and ca for the prediction of recurrence or metastases, there were insufficient data to undertake the analyses, and an assumption was therefore required, with the marker scoring zero for that performance characteristic. this would lead to cea spuriously appearing to have higher benefits, simply as a product of it being more extensively investigated. the trade-off analysis is also informed by the survey, which would only represent the views of members of the euro- pean society who responded. this suggests some positive selection bias, especially given the response rate of approximately %. conclusions tumor markers have long been utilized in the moni- toring of gastrointestinal cancers, with variable success. while traditional markers have a use in colorectal cancer surveillance, their use in esophagogastric malignancies is somewhat less defined and requires clarification. in both cases, there appears to be a need for a tumor marker with higher diagnostic accuracy. this would suggest that further areas of research should focus on the search for new novel biomarkers for diagnosis and therapeutic monitoring (see online appendix for references of included papers). funding mr. sheraz markar is funded by the national institute for health research (nihr). in addition, this research was supported by the nihr diagnostic evidence co-operative london at the imperial college healthcare nhs trust. the views expressed are those of the authors and are not necessarily those of the nihr or the department of health. disclosure none. open access this article is distributed under the terms of the creative commons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. summation of benefit scores s u m m at io n o f c os t s co re s ca , ca , cea , summation of benefit scores s u m m at io n o f c os t s co re s ca , ca , cea , (a) (b) fig. cost-benefit trade-off for use of the tumor markers in (a) colorectal cancer and (b) esophagogastric cancer. higher summated cost score represents lower costs, while higher summated benefit score represents higher benefits. cea carcinoembryonic antigen, ca cancer antigen a. acharya et al. http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / references . national cancer institute. seer stat fact 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utility of tumor marker cutoff ratio and a combination scoring system of preop- erative carcinoembryonic antigen, carbohydrate antigen - , carbohydrate antigen - levels in gastric cancer. j korean surg soc. ; ( ): – . . narita m, oussoultzoglou e, chenard mp, fuchshuber p, yamamoto t, addeo p, et al. predicting early intrahepatic recurrence after curative resection of colorectal liver metastases with molecular markers. world j surg. ; ( ): – . . gold p, freedman so. specific carcinoembryonic antigens of the human digestive system. j exp med. ; ( ): – . . choi se, hur c. screening and surveillance for barrett’s esophagus: current issues and future directions. curr opin gas- troenterol. ; ( ): – . use of tumor markers in gastrointestinal cancers http://seer.cancer.gov/statfacts/html/colorect.html http://seer.cancer.gov/statfacts/html/colorect.html https://babel.hathitrust.org/cgi/pt% fid% dmdp. % bview% d up% bseq% d https://babel.hathitrust.org/cgi/pt% fid% dmdp. % bview% d up% bseq% d 	use of tumor markers in gastrointestinal cancers: surgeon perceptions and cost-benefit trade-off analysis 	abstract 	background 	objective 	methods 	results 	conclusions 	methods 	literature search strategy 	literature standard 	tumor marker survey 	statistical methodology 	performance characteristics 	cost-benefit trade-off analysis 	criteria ratings 	criteria weights 	overall benefit scores 	trade-offs 	results 	literature search 	quadas- evaluation 	pooled analysis for diagnostic sensitivity and specificity 	tumor marker survey 	cost-benefit trade-off 	discussion 	conclusions 	references pii: s - ( ) - now that the human genome is sequenced (more or less), what shall we do with it? if we want to benefit from it to gain an improved understanding of the genetic basis of disease and the different responses to drugs, knowing the three billion bases that all of us have in common is only the beginning. the second leg that future medical genetics will need to stand on is the knowledge of the several million bases that we don’t have in common, from which the most widespread types of genetic variation originate. the most important group of variabilities is described as ‘single nucleotide polymorphisms’ or snps. they are defined as base exchanges occurring in at least % of a population, as opposed to a random mutation that is normally found with a frequency of much less than %. snps essentially define the genetic differences between individuals, including — most importantly for medical research — their different susceptibilities to diseases and different responses to drugs. so far, the links between genes and diseases have only been established in relatively few, mostly rare and monocausal diseases, like cystic fibrosis. how can the more common multifactorial diseases (like coronary heart disease or type ii diabetes) be linked to patterns in genetic variability? this is one of the major challenges awaiting science in the postgenomic era, and there are different approaches to it, which can be broadly divided into those which use small, inbred populations and those based on large populations. one large-scale study is being launched in the uk with funding from the medical research council and the wellcome trust. the plan is for a major long-term study of around , middle-aged volunteers as a representative sample of the uk population. alongside genetic analysis, basic measurements such as height, weight and blood pressure will be made and followed up with data on health and lifestyle. the hope is that within a few years meaningful research on a significant number of ill people should be possible. researchers who believe in the benefit of studying small populations are the luckier ones in practical terms, as they get to work in sunnier climates. hotspots of genomics research have emerged in remote italian villages such as perdasdefogu and talana, both situated in the ogliastra region in northwestern sardinia, near the town of alghero. between them, these villages have less than inhabitants, who mostly descend from a small group of families who founded the villages around years ago. in the case of talana, it is precisely known that all villagers descend from eight males and eight females. less than % of marriages have been to outsiders. magazine r news focus european genomics: think big or small? with the first draft of the human genome completed, michael gross looks at some of the plans to study a variety of european populations to exploit the new information. iceland cometh: local people celebrating national day in reykjavik may be among the first to provide insight into the links between human genes and common diseases through the establishment of a national genetic database. the man who pushed these villages to the forefront of genomics research is mario pirastu, a native sardinian who returned to his island after research work in san francisco and is now the director of the istituto de genetica molecolare in alghero, funded by italy’s national research council, cnr. he is a firm believer in the approach that uses small inbred populations which should be less than generations old. outside sardinia, the best- known cases are the amish, some communities in newfoundland, along with some villages in switzerland. pirastu and his colleagues argue that the genetic homogeneity of such small inbred populations makes it easier to track down genetic factors contributing to multifactorial diseases, as differences irrelevant to the questions under study will be producing less noise than they would in a large and diverse population. the main criterion used in such studies is linkage disequilibrium which essentially relies on the fact that genes are more likely to be passed on together, the closer they are on the chromosome. last summer, pirastu obtained large scale support from various sources to set up an international research centre at perdasdefogu and make the most of the rare and valuable gene pool found in this village. for the native population, this comes mainly as a welcome boost to the local job market, so the local politicians are quite supportive of the research. in other remote parts of italy, similar projects are on the way. paolo gasparini and his coworkers from the medical genetics service at rotondo have picked the southern italian village of carlantino, where the rate of marriage within the community was . % over the last century. and in the surroundings of naples, graziella persico is studying a whole cluster of similarly isolated villages. however, this cottage industry also has its limitations. although some disease-related variants will be abnormally frequent in such inbred populations, others will be totally absent and thus cannot be studied until one finds another suitable group that has them. this is where large-scale, nationwide genome projects are hoping to make their mark. the oldest study of this kind is the one in iceland. like the sardinian villages, iceland has remained isolated for centuries, but it started from a much bigger gene pool, gradually expanding to today’s population of , . in december , the icelandic parliament passed a bill enabling the creation of a centralized medical database, after a year-long public debate on ethical and privacy problems arising from this proposal. much of the debate was based on the issue that a private biotech company, namely decode genetics, chaired by the icelandic geneticist kari stefansson, is running and controlling the database. to defuse this issue, the parliament created a governing committee including members of the public, that acts as a kind of consumer watchdog to oversee this project. public debate is remarkably absent in a country that tries to beat iceland’s genome project with larger numbers and a more diverse gene pool: estonia. although the core estonian population has been sedentary on the baltic shore for more than years, some mingling with neighbours must have taken place. among today’s . million citizens of the estonian republic, russians form the strongest ethnic minority with %. thus, the proponents of the estonian genome project argue, estonians are less exceptional than the isolated populations, while still being sufficiently homogenous for meaningful genetic studies. the estonian genome foundation (egf) is currently seeking corporate sponsors and parliamentary approval for what would become the world’s largest database of medical, genetic and genealogical data. more than % of the estonian population are expected to consent to part with ml blood and their medical records.if successful, this project would result in a database holding sensitive information on . million individuals, including people’s health status, diseases, important health and behavioural risks and genealogical information, as the egf cheerfully proclaims on its website. even though the names of the subjects will be replaced by anonymous codes, it is hard to imagine how a database of this size and with such sensitive personal information could be handled without at least a small risk of leakage and misuse of data. using only dna samples from deceased hospital patients (who have consented before dying) may be a way around the privacy problems arising from such large projects. while the village-sized projects certainly have the advantage that the smaller databases are more easily kept safe from misuse, ethical issues are far from absent in these cases. like in anthropological studies of native tribes in africa or south america, the very remoteness of the villages under study throws up ethical concerns. in this case the question has to be asked whether agreement to give a blood sample constitutes informed consent to a full scale characterization of genomic markers. as even the geneticists don’t know yet what these markers will tell them, the subjects of the research cannot possibly know — and probably lack the ability to imagine — what kind of information about themselves they are handing over. if geneticists don’t want to limit themselves to studying the dna of deceased patients, they may have to redefine what informed consent means in the postgenomic era. michael gross is a science writer based in oxford, uk. url: www.michaelgross.co.uk r current biology vol no j. chil. chem. soc., , nº ( ) synthesis, characterization and antimicrobial studies of co-ordination polymers amish i. shaha, hemang m. shuklaa, purvesh j. shahb and dilipsinh s. raja* a chemistry department, m.b. patel science college, anand - (india). b department of chemistry, shri a.n.patel p.g.institute, anand- (india). (received: may , - accepted: august , ) abstract a novel heteronuclear bis-ligand namely -[ -( -carboxyphenyl carbonyl amino)-phenyl- -carbonyl methyl amino)- -hydroxyquinoline was synthesized and characterized. this bis-ligand was designated as cphq. co-ordination polymers of this cphq bis-ligand were prepared with cu+ , co+ , ni+ , mn+ and zn+ metal ions. all of these co-ordination polymers and the cphq ligand were characterized by elemental analysis, ir, nmr spectral studies, thermogravimetry, electronic reflectance spectral studies and magnetic susceptibility measurement. the synthesized novel bis-ligand and their co-ordination polymers were screened for their antimicrobial activity. key words: heteronuclear bis-ligand; co-ordination polymer; spectral studies; magnetic moment; antibacterial and antifungal activities. e-mail:purvesh @gmail.com introduction the research on co-ordination polymers by linking transition metal ions with ligands has been constantly developed in past years. because of their excellent properties, such as semiconducting catalytic properties, waste water treatment for metal recovery, in protective coating, as antifouling paints and anti fungal properties , . recently new coordination supramolecules and polymers bis(oxine), bidentate ligand based on transition metal compounds and multidentate organic ligand has attracted much interest. in this context bis(oxine) ligand with two oxine units link by a bridge of , ’- methylene (-ch - ), , ’-sulfonyl (-so -), , ’-dimethylene sulfide (-ch -s-ch -) and -ch - o-ch - are reported in literature - . most of bichelating ligands are derived from well known chelating agents like -hydroxy quinoline and salicylic acid etc , . -hydroxyquinoline ( -hq) moiety has received constant consideration due to their efficient ionophores, therapeutic and fluorescences properties - . -hydroxyquinoline containing polymers are used in areas such as waste water treatment to recover metals, protective coatings, water disinfectants, ion-exchange resin, antifouling paints, antimicrobial, surgical materials, gels and ointment for medical uses - . -hydroxyquinoline containing polymers and copolymers reveal complexing ability and biological activity - . the study of -quinolinol containing polyester has also been reported . the area in which the co-ordination polymers having bis-azo dye containing ligands has not been developed so far. such ligand may afford the co-ordination polymer with different properties. hence, it was thought interesting to explore the field of co-ordination polymers based on hetronuclearting bis-ligand having -hydroxyquinoline and amic acid. though the amic acid is generally derived by condensation of amines with anhydrides having carboxylic and amide group. the metal complexation study of various amic acids has been reported recently - , so the proposed present work is in connecting with the co-ordination polymers based on hetronuclearting bis-ligand. the synthetic route for the preparation of bis-ligand and its co-ordination polymers are shown in scheme . experimental materials all the chemicals used were of analytical grade. the compound -amino- - hydroxyquinoline hydrochloride (ahq) was prepared by reported methods. , -chloro acetamido phenacyl chloride (apc) was prepared by known method. synthesis of -[ -( -carboxyphenyl carbonyl amino)-phenyl- -carbonyl methyl amino)- -hydroxyquinoline (cphq): the -[ -( -carboxyphenyl carbonyl amino)-phenyl- -carbonyl methyl amino)- - hydroxy quinoline cphq was prepared in two steps. . synthesis of -( -phenyl carbonyl methyl amino)- -hydroxyquinoline (phq): a solution of -chloro acetamido phenacyl chloride (apc) ( . g, . mole) in acetone was treated with -amino- -hydroxyquinoline hydrochloride (ahq) ( . g, . mole) in acetone at room temperature for . hours. the resultant product was filtered and hydrolyzed by : hcl and etoh mixture. the yield of cphq was % ( . g); m.p. - ° c (uncorrected). . synthesis of -[ -( -carboxyphenyl carbonyl amino)-phenyl- - carbonyl methyl amino)- -hydroxyquinoline (cphq): a solution of -( -phenyl carbonyl methyl amino)- -hydroxy quinoline (phq) ( . g, . mole) in acetone was cooled to ° c and then a solution of phthalic anhydride ( . g, . mole) was added with constant stirring. the resulting product filtered and air-dried. the yield of cphq was % ( . g); m.p. - ° c (uncorrected). anal. found for c h n o ( ): c, . ; h, . ; n, . . calcd. c, . ; h, . ; n, . . (scheme- ) scheme j. chil. chem. soc., , nº ( ) preparation of co-ordination polymers all co-ordination polymers were synthesized by using metal acetates in a general method described as follows: a warm and clear solution of cphq ( . g, . mole) in % aqueous formic acid ( ml) was added to a solution of copper acetate ( . g, . mole) in % aqueous formic acid ( ml) with constant stirring. after complete addition of metal salt solution, the ph of reaction mixture was adjusted to about with dilute ammonia solution. polymeric chelates were separated out in the form of suspension and then digested on a water bath for one hour and eventually filtered, washed with hot water followed by acetone and n,n- dimethyl formamide (dmf) and then dried in air at room temperature. the yields of all co-ordination polymers were almost quantitative. the obtained solid metal complexes and their colors are shown in table . the complexes are stable solids, decomposing above ° c without melting and insoluble in diethyl ether, acetone, ethanol, methanol and chloroform; however, they are soluble in dimethyl sulfoxide (dmso) and dmf. measurements elemental analysis of cphq and its co-ordination polymers were carried out on a c, h, n elemental analyzer (italy). ir spectra of the bis-ligand and the polymeric chelates were scanned on a nicolet- d ftir spectrophotometer in kbr. h and c nmr spectra were carried out at room temperature in dmso-d and tetramethylsilane (tms) as an internal reference using brucker spectrophotometers ( mhz and mhz). the metal content analyses of the polymeric chelates were performed by decomposing a weighed amount of each polymeric chelates followed by edta (disodium ethylene diamine tetra acetate) titration as reported in the literature. magnetic susceptibility measurements of all the polymeric chelates were carried out at room temperature by the gouy method. mercury tetrathiocynatocobaltate (ii) was used as a calibrantion agent. molar susceptibilities were corrected for diamagnetism of component atoms using pascal’s constant. the diffuse reflectance spectra of the solid polymeric chelates were recorded on a beckman dk- a spectrophotometer with a solid reflectance attachment. mgo was employed as the reference compound. the number average molecular weight ( mn ) of all the co-ordination polymers were determined by method reported in earlier communications. thermogravimetric analysis of co-ordination polymers were carried on dupont tga analyzer in air at a heating rate of ° c/min. screening of antibacterial and antifungal activities antibacterial activities in vitro antibacterial screening of cphq and its co-ordination polymers were studied against gram-positive bacteria (bacillus subtilis and staphylococcus aureus) and gram-negative bacteria (escherichia coli and salmonella typhi) by cup-plate method using nutrient agar as medium. in a typical procedure, molten nutrient agar kept at ° c was then poured into petri dishes and allowed to solidify. then holes of millimeter diameter were punched carefully using a sterile cork borer and these were completely filed with test solutions ( mg/ml in dmf). the diameter of the zone of inhibition for all the compounds was measured and the results were compared with the standard drug streptomycin of the same concentration as that of the test compound under identical conditions. table . analytical and spectral data of the cphq and its co-ordination polymers. ligand/co- ordination polymers (empirical formula) yield (%) color molecular weight analyses found (calcd.) % meff. (b.m.)a ( mn )+ b dp c m c h n cphq c h n o lightyellow - . ( . ) . ( . ) . ( . ) - - - [cu(cphq)(h o) ]n cu.c h n o . h o lightgreen . . ( . ) . ( . ) . ( . ) . ( . ) . [co(cphq)(h o) ]n co.c h n o . h o brown . . ( . ) . ( . ) . ( . ) . ( . ) . [ni(cphq)(h o) ]n ni.c h n o . h o green . . ( . ) . ( . ) . ( . ) . ( . ) . [mn(cphq)(h o) ]n mn.c h n o . h o lightgreen . . ( . ) . ( . ) . ( . ) . ( . ) . [zn(cphq)(h o) ]n zn.c h n o . h o yellow . . ( . ) . ( . ) . ( . ) . ( . ) diamagnetic d a magnetic susceptibility; b number average molecular weight ; c average degree polymerization ; d diamagnetic behaviour. antifungal activities the antifungicidal activity of all the compounds was evaluated against penicillium expansum, nigrospora sp., trichothesium sp., and rhizopus nigricum by cup-plate method cultured on potato-dextrose agar medium adapting similar procedure describe above. the plates were incubated at ° c for hours. the diameter of the zone of inhibition for all the compounds was measured and the results were compared with standard drug chlotrimazole in the same experimental conditions. results and discussion the synthesis of the bis-ligand, -[ -( -carboxyphenyl carbonyl amino) phenyl carbonyl methyl amino)- -hydroxyquinoline (cphq) has not been reported in the literature. all the metal complexes were sparingly soluble in common organic solvents but soluble in dmf, dmso and acetonitrile. the results of elemental analyses of bis-ligand cphq and its co-ordination polymers are agreed with those predicted on the molecular formula (table ). j. chil. chem. soc., , nº ( ) the analytical data indicates that the metal complexes are agreed well with : metal to ligand (m/l) stoichiometry. the structure of cphq and its co- ordination polymers was confirmed by the elemental analysis, ftir, h-nmr and c-nmr, reflectance spectra, magnetic susceptibility measurements and tga analysis. elemental (chn) analysis, physical properties and ir data provided good evidence that the chelates are polymeric in nature. , possible structure of the co-ordination polymers is shown in scheme . magnetic susceptibility the magnetic susceptibility measurements of the metal complexes were performed at room temperature (table ). the magnetic susceptibility value for cu+ complexes of the ligand cphq is . b.m., it is less than the normal value ( . - . b.m.). the lowered magnetic moment value observed for cu+ complex under the present study is due to distorted octahedral geometry. , on the other hand magnetic moment values of co+ and ni+ complexes indicate an octahedral geometry for these complexes. mn+ complex exhibited magnetic moment at . b.m. it indicates that the complex is high-spin type paramagnetic, it lies within the octahedral range. the obtained result is very close to spin value . b.m. as the ground term is a g and thus supports the octahedral geometry. infrared spectra the ir spectrum of cphq shows a broad band at cm- attributed to the -oh stretching vibrations. the weak bands around and cm- may be due to asymmetric and symmetric stretching vibrations of methylene groups. the bands at cm- and cm- are due to nh (sec) and co group respectively. the bands around , , and cm- are attributed to the -hydroxyquinoline nucleus. the band at cm- is recognized cooh group. the bands around , and cm- are attributed to amide group. comparison of the ir spectrum of the bis-ligand cphq and those of the co-ordination polymers reveals certain characteristic differences. the broad band at cm- for cphq has almost disappeared for the spectra of polymers. however, the weak band around cm- in the spectra of cphq- co +, cphq-ni +, cphq-mn + indicate the presence of water molecules which may have been strongly absorbed by the co-ordination polymers. the c-o stretching in the ligand may be suggested due to the absorptions at and cm- . in the complexes, these bands are shifted to and cm- respectively. these results indicate the formation of c-o-m bond. the peak at cm- in the ligand is assigned to oh-bending of the phenolic moiety. this band is absent in the co-ordination polymers. in addition to these bands, the spectrum of cphq has many characteristic absorption bands, which are identical to those that occur in -( -phenyl carbonyl methyl amino)- -hydroxy quinoline (phq) and phthalic anhydride. the new bands observed in the region - cm- and cm- are probably due to the formation of m-o and m-n bonds respectively. thus, it is possible that the oxygen and nitrogen atoms of -hydroxyquinoline groups are coordinated to the metal. , from the above data the following structure has been proposed for the co-ordination polymers shown in scheme . h nmr and c nmr spectra of the bis-ligand (cphq) h nmr spectrum shows signal at δ . ppm (s, h) which is due to -ch protons. also single at δ . , . ppm (s, h) due to –nh proton and single at δ . ppm (s, h) due to –oh proton. the signal at δ . - . ppm (m, h) is assign to aromatic protons of quinolone and phenyl moieties. a sharp peak at δ . ppm (s, h) is assign to –cooh group. these all features confirm the proposed structure of cphq bis-ligand. in the c nmr spectrum of cphq is observe a signal at . ppm it is assign to methylene carbons. signals at . - . ppm are assign to c=o carbons. the signals observe between . - . ppm are assigned to aromatic carbons of the ligand. electronic spectra the diffusion electronic spectrum of cu+ , co+ , ni+ , mn+ and zn+ complexes were recorded in dmf. the diffusion electronic spectrum of cphq-cu + co-ordination polymers shows two broad bands at , cm- and , cm- . the first bands may be due to t g à eg transition, while the second may be due to charge transfer. the first band assigns distorted octahedral structure for the cphq-co + complex. the higher value of µeff of the cphq- cu + polymer support distorted octahedral structure. , the cphq-ni + and cphq-co + polymers give two absorption bands at , and , cm- and at , and cm- respectively. these bands can be assigned to t gà t g and t gà t g(p) transitions respectively. the absorption bands and the values of µeff indicate an octahedral configuration for the cphq-ni + and cphq-co + polymers. , the spectrum of cphq-mn + showed weak bands at , , , and , cm- assigned to the transitions a g→ t g( g), a g→ t g( g) and a g→ a g, eg respectively, suggesting an octahedral structure for the mn + complex. the spectrum of the zn + complex is not well resolved, so it is not interpreted, but its μeff value shows that it is diamagnetic as expected. thermogravimetric analyses the thermal behavior of co-ordination polymers and parent bis-ligand was investigated by tga analysis. the tga data of all the samples are presented in table . the weight loss of the polymer samples at different temperature indicates that the degradation of the polymers is noticeable beyond ° c. table . thermogravimetric analysis of bis-ligand (cphq) and their co-ordination polymers. ligand/ co-ordination polymers % weight loss at different temperature (° c) activation energy (kcal/mol) cphq . . . . . . . . [cucphq (h o) ]n= . . . . . . . . [cocphq (h o) ] n= . . . . . . . . [nicphq (h o) ] n= . . . . . . . . [mncphq (h o) ] n= . . . . . . . . [zncphq (h o) ] n= . . . . . . . . a very slight decrease in weight loss depicted from the thermogram in the temperature range - ° c for the parent ligand may be attributed to loosely bonded moisture. however, the initial slow weight loss occurring below ° c in all of the co-ordination polymers may be attributed to the removal of water. inspection of the thermograms of all coordinated polymer samples reveals appreciable weight loss in the range to ° c which might be due to metal-co-ordinate water molecules (scheme ). it was reported that the water molecules are coordinated to the metal ions. the rate of degradation becomes maximum at temperature between ° c and ° c. above this temperature, co-ordination polymers are most stable and each co-ordination polymers loses about % of its weight when it is heated at ° c originating metal oxides. the thermodynamic activation parameter of the decomposition process of the co-ordination polymers such as energy of activation (ea) and order of reaction (n), were evaluated graphically by employing the freeman–carroll method using the following relation: [(-ea/ . r)∆( /t)]∆log wr = -n +∆log (dw/dt) ∆log wr where t is the temperature in k, r is gas constant, wr = wc-w; wc is the weight loss at the completion of the reaction and w is the total mass loss up to time t. ea and n are the energy of activation and order of reaction respectively. a typical curve of [∆log(dw/dt)/∆log wr] vs [∆( /t)/ ∆logwr] for the co + co- ordination polymer is shown in figure . the slope of the plot gave the value of ea/ . r and the order of reaction was determined from the intercept. j. chil. chem. soc., , nº ( ) figure . freeman-carroll plot for thermal degradation of [cocphq(h o) ]n complexe. the kinetic parameter, especially activation energy (ea) is helpful in assigning the strength of the co-ordination polymers. the calculated ea values of co-ordination polymers indicate that volatile gas is produce in the range of . - . kcalmol– . the relatively high value of ea (table ) indicates that the ligand is strongly bonded to the metal ion. - based on the activation energy values, the thermal stability of the co-ordination polymers in the decreasing order is: mn> co> ni > zn> cu. antimicrobial activity of the bis-ligand and its co-ordination polymers all the novel synthesized compounds and standard drugs were prepared in freshly distilled dmf. the zone of inhibition evaluated was negligible. these data reveal that the bis-ligand cphq and its co-ordination polymers shows weak to good antimicrobial activity (table ). these data are compared with the standard drug streptomycin, the bis- ligand and its cu + and co + complexes showed weakly active against the both organisms with zones of inhibition - mm and - mm respectively. on the other hand, the ni + and mn + complexes can be classified of moderately active compared to its ligand with zone of inhibition - mm while the zn + complex exhibited good activity with zone of inhibition - mm. table . antibacterial activities of bis-ligand (cphq) and their co-ordination polymers. zone of inhibition (mm) ligand/ co-ordination polymers gram +ve gram –ve bacillus subtilis staphylococcus aureus salmonella typhi escherichia coli cphq [cucphq(h o) ]n [cocphq(h o) ]n [nicphq(h o) ]n [mncphq(h o) ]n [zncphq(h o) ]n streptomycin dmf (control) bore size the antifungal activity results revealed that the ligand cphq and its co-ordination polymers show weak to good activity (table ). bis-ligand and its cu +and co +complexes showed weak active with zone of inhibition - mm. the ni + and zn + complexes show moderate activity with zone of inhibition - mm, while the mn + complex exhibited good activity with zone of inhibition - mm. table . antifungal activity of bis-ligand (cphq) and their co-ordination polymers. zone of inhibition at ppm (%) ligand/ co-ordination polymers penicillium expansum nigrospora sp. trichothesium sp. rhizopus nigricum cphq [cucphq(h o) ]n [cocphq(h o) ]n [nicphq(h o) ]n [mncphq(h o) ]n [zncphq(h o) ]n chlotrimazole j. chil. chem. soc., , nº ( ) conclusion the novel -[ -( -carboxyphenyl carbonyl amino)-phenyl- -carbonyl methyl amino)- -hydroxyquinoline (cphq) and its octahedral metal (ii) complexes ( : metal to ligand ratio) were synthesized and characterized. further, the co-ordination polymers were thermally more stable than the ligand. all the co-ordination polymers showed good antibacterial and antifungal activities compared to cphq ligand due to the insertion of the metal ions. the information regarding geometry of the complexes was obtained from their electronic and magnetic moment values. the magnetic moment values indicate an octahedral geometry. references . h. m. shukla, a. i. shah, p. j. shah and d. s. raj, der pharmacia sinica ( ), , ( ). . h. m. shukla, a. i. shah, p. j. shah and d. s. raj, j. chem. pharm. res. 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( ), , ( ). deb_pone. .. uc san diego uc san diego previously published works title analysis of the gut microbiota in the old order amish and its relation to the metabolic syndrome. permalink https://escholarship.org/uc/item/ g c nw journal plos one, ( ) issn - authors zupancic, margaret l cantarel, brandi l liu, zhenqiu et al. publication date doi . /journal.pone. peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ g c nw https://escholarship.org/uc/item/ g c nw#author https://escholarship.org http://www.cdlib.org/ analysis of the gut microbiota in the old order amish and its relation to the metabolic syndrome margaret l. zupancic ., brandi l. cantarel , ., zhenqiu liu , elliott f. drabek , kathleen a. ryan , shana cirimotich , cheron jones , rob knight , william a. walters , daniel knights , emmanuel f. mongodin , , richard b. horenstein , braxton d. mitchell , nanette steinle , , soren snitker , alan r. shuldiner , , claire m. fraser , * institute for genome sciences, university of maryland school of medicine, baltimore, maryland, united states of america, department of medicine, university of maryland school of medicine, baltimore, maryland, united states of america, department of microbiology and immunology, university of maryland school of medicine, baltimore, maryland, united states of america, department of epidemiology and preventive medicine, university of maryland school of medicine, baltimore, maryland, united states of america, program in personalized and genomic medicine, division of endocrinology, diabetes, and nutrition, department of medicine, university of maryland school of medicine, baltimore, maryland, united states of america, howard hughes medical institute, department of chemistry and biochemistry, university of colorado at boulder, boulder, colorado, united states of america, department of molecular, cellular and developmental biology, university of colorado at boulder, boulder, colorado, united states of america, veterans administration medical center, baltimore, maryland, united states of america abstract obesity has been linked to the human gut microbiota; however, the contribution of gut bacterial species to the obese phenotype remains controversial because of conflicting results from studies in different populations. to explore the possible dysbiosis of gut microbiota in obesity and its metabolic complications, we studied men and women over a range of body mass indices from the old order amish sect, a culturally homogeneous caucasian population of central european ancestry. we characterized the gut microbiota in subjects by deep pyrosequencing of bar-coded pcr amplicons from the v –v region of the s rrna gene. three communities of interacting bacteria were identified in the gut microbiota, analogous to previously identified gut enterotypes. neither bmi nor any metabolic syndrome trait was associated with a particular gut community. network analysis identified twenty-two bacterial species and four otus that were either positively or inversely correlated with metabolic syndrome traits, suggesting that certain members of the gut microbiota may play a role in these metabolic derangements. citation: zupancic ml, cantarel bl, liu z, drabek ef, ryan ka, et al. ( ) analysis of the gut microbiota in the old order amish and its relation to the metabolic syndrome. plos one ( ): e . doi: . /journal.pone. editor: farook thameem, the university of texas health science center (uthscsa), united states of america received november , ; accepted july , ; published august , copyright: � zupancic et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. funding: the work in this study was supported by uh /uh award dk from the national institutes of health to cmf-l and ars, and u gm and p dk (mid-atlantic nutrition and obesity research center) to ars. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript competing interests: the authors have declared that no competing interests exist. * e-mail: cmfraser@som.umaryland.edu . these authors contributed equally to this work. introduction obesity, the accumulation of excess body fat has a negative impact on morbidity, mortality, and quality of life through its complications, which include cardiovascular disease, type diabetes, osteoarthritis, and certain cancers [ ]. globally in step with the increase in industrialization, obesity has reached epidemic proportions such that overweight or obese humans now out- number those suffering from malnutrition [ ]. the etiology of obesity and its metabolic complications, including hyperlipidemia, hypertension, glucose intolerance and diabetes reflect the complex interactions of multiple genetic, behavioral, and environmental factors. great inter-individual variation is apparent in the propensity toward obesity, the location where excess fat is deposited, and the extent to which this results in metabolic derangements and adverse health outcomes. available treatments for obesity include lifestyle modification (diet and exercise), drugs, and bariatric surgery. with the possible exception of surgery, individuals often fail to maintain long term weight loss with these modalities. to offer better treatment and prevention modalities, deeper understanding of the etiology of obesity is needed. novel lines of investigation implicate chronic inflammation [ ] and the gut microbiome [ – ] in the development of obesity and its metabolic complications. a publication by ley et al. [ ] provided evidence for a link between gut microbial ecology and obesity in a genetically homogeneous strain of leptin-deficient mice maintained in a highly controlled laboratory environment. more recent studies have suggested that microbes present in the human gut may also play a role in metabolism and adiposity; however, the results of these studies have been more variable, perhaps reflecting the complexity of human genetics and/or heterogeneity in lifestyle [ ]. the emerging evidence that the microbiota may contribute in important ways to human health and disease has led us and others to hypothesize that both symbiotic and pathological relationships between gut microbes and their host may be key contributors to obesity and the metabolic complications of obesity [ – ]. we hypothesize that the gut microbiota influences host plos one | www.plosone.org august | volume | issue | e energy homeostasis, metabolism, and inflammation, and is an important determinant of obesity and its adverse health con- sequences. to explore the possible dysbiosis of gut microbiota in obesity and its metabolic complications in humans, we studied old order amish subjects from lancaster county, pennsylvania. the amish are a genetically closed homogeneous caucasian population of central european ancestry ideal for such a study because of their high degree of social cohesiveness and common lifestyle [ , ]. there is great uniformity of socioeconomic status and lifestyle among the amish, and prescription medication usage is minimal, reducing the potentially confounding influences of variation in environmental exposures on complex traits. extensive genealogies document a small number of founders and genetic analyses confirm less genetic heterogeneity relative to the general popula- tion. these attributes make the amish a highly desirable population in which to study the composition of the gut microbiota, its heritability, and its relationship to obesity and metabolic complications. the specific questions we set out to address in this report were: ( ) what are the major gut microbial subpopulations in this population and how does their distribution vary in obesity and with metabolic syndrome phenotypes? and ( ) are socio-demographic and other factors associated with the major gut microbial subpopulations? results we enrolled a total of adult subjects, of whom were male and were female. in this cohort, mean age and bmi was higher, and manifestations of the metabolic syndrome [ ], were more common in women than in men (table ). we performed s rrna multiplex pyrosequencing of v –v amplicons using bar-coded primers on the titanium platform [ ] to characterize the fecal microbiota. we obtained , pyrosequencing reads per sample, with an average read length of bp (table s ). reads were binned into individual samples based on the barcode sequence, and complementary phylogenetic and taxon-based analysis methods were used to compare s rrna sequences across the fecal microbial communities (see methods). in total, genera were identified in the gut microbiota in the amish; the most abundant genera accounted for % of the reads (figure s ). seven species were each represented by more than % of the total sequence reads, including three species in the bacteroidetes (prevotella copri, bacteroides vulgatus and bacteroides plebius) and four species in the firmicutes (faecalibacterium prauznitzii, eubacterium rectale, eubacterium biforme, and roseburia faecis) (figure s ). the percent of reads for which a species-level taxonomic assignment could not be made averaged % across all the samples (range of % to %). all but one of the twenty-five most abundant genera were present in at least % of all subjects, and these include members of the firmicutes, bacteroidetes, tenericutes, actinobacteria, and proteobacteria (figure s ). the prevalence of genera in the gut microbiota follows a bi-modal distribution, with a large peak above zero associated with many genera that are present in a small number of subjects and a second peak of genera that are present in % or more of the subjects (figure s ). these genera were deemed to comprise the core microbiota [ ] in the amish (table ) and represent members of the firmicutes, bacteroidetes and tenericutes. despite the substantial overlap in genera present in the gut microbiota of the subjects, a significant amount of inter- individual variation was observed with respect to the relative abundance of both the predominant and rare genera (figure s ). for example, the relative abundance of bacteriodetes across all the samples ranges between and %. phyla-level phylogenetic binning of s rrna data revealed a nominally significant correlation between the bacteroidetes:firmicutes ratio and age- and sex- adjusted bmi (r = . ; p = . ), but no significant correlation with metabolic syndrome traits [fasting glucose (r = . ; p = . ), systolic or diastolic blood pressure (r = . ; p = . and (r = . ; p = . ), respectively), fasting triglycerides (r = . ; p = . ), hdl-cholesterol (r = . ; p = . ); all adjusted for age and sex) results from unweighted unifrac analysis [ ] also did not distinguish among the subjects based on bmi (figure s ). using a random matrix theory-based framework [ ] we identified three networks of interacting bacteria in the human gut, that correlated with the three gut enterotypes recently reported by arumugam et al. [ ] (figure a–c). in our dataset, these groups appear to represent a strong gradient effect between a few dominant taxa, rather than distinct clusters (figure ) because the silhouette width statistic [ ] does not prefer three (mean silhouette width of . ) clusters over two (mean silhouette width of . ). each subject was assigned membership to one of the bacterial groups based on the predominant genus present in his/her microbiota. the most prevalent group (i) in this cohort ( % of subjects: % male and % female) is dominated by prevotella, the most abundant genus in the ooa gut microbiota. the least common group (ii) in this cohort ( % of subjects: % male and % female), is bacteroides-dominated. a firmicutes- dominated group (iii; % of subjects: % male and % female) is characterized by diverse firmicutes genera, oscillospira being the most abundant. group iii displays a statistically significant higher shannon diversity index of both otus and named taxa than either network i or ii (figure d; p = . by wilcoxon rank-sum test). the three networks appear to reflect the main large-scale trends in gut microbial populations and suggest that gut bacterial community structures are driven by high abundance and high variability in populations of prevotella, bacteroides, or firmicutes (with a degree of mutual exclusion between them). prevotella and bacteroides appear to co-exist at lower levels if the community is firmicutes-dominated, but are nearly mutually exclusive when either is abundant in bacteroidetes-dominated communities. using community structure as a proxy, we examined the stability of the dominant taxa in second samples from the subjects in this study – months following initial samples. the dominating taxa in of the subjects ( %) did not change (figure s ). the communities became prevotella-dominated in subjects where a change was observed; in cases first samples were collected in winter and the second samples were collected – months later in the spring. these changes may represent seasonal changes in diet that will need to be investigated further. our cohort of subjects included nuclear families having two or more members phenotyped. these nuclear families contributed a total of spouse pairs, sibling pairs, and parent-offspring pairs. community structure concordance rates tended to be lower for the spouse pairs ( . %) than for the sibling ( . %) and parent-offspring ( . %) pairs (p = . for compar- ison of spouse pairs with the combined set of sibling and parent- offspring pairs). larger sample sizes will be required to get better estimates of the relative contributions of relatedness and household effects to community structure. we tested the association of groups (coded as a class variable with levels) with each available clinical factor, while adjusting for age and sex (a df test). neither bmi nor any metabolic syndrome trait was associated with a particular group (table ). these data obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e suggest that individual traits associated with obesity and its metabolic complications do not correlate with a major shift in the relative abundance of these two predominant phyla in the amish, an observation reported in other cohorts [ , ]. to determine if gut community type was associated with occupation, we classified study subjects into occupational classes (farmers, tradesmen, farmer’s wives, teachers/shopkeepers, and unknown/retired) and tested the association of phenotype with each occupational class. in men, the occupation of farming was over-represented among those with the prevotella-dominated network ( . %) compared to those with either the bacteroidetes- dominated ( . %) or firmicutes-dominated ( . %) networks (p = . ; table ). the distribution of networks did not differ significantly within any other occupational class in men or with any occupational class in women (table ). the availability of extensive clinical data from the ooa cohort allowed us to evaluate the potential role of the microbiota (both species and otus) in obesity and its metabolic derangements using network analysis, independent of enterotype. twenty-two species of bacteria from the phyla bacteroidetes, firmicutes, and actinobacteria, and four otus from the order clostridiales, displayed both positive and inverse correlations with bmi, serum triglycerides, hdl cholesterol, total cholesterol, fasting glucose levels and c-reactive protein (figure a) and with each other (figure b). these bacteria differ in both abundance and prevalence. collectively, this group of species and otus represent between . and % of the total s rrna sequence reads in the subjects. phylogenetic analysis revealed that the majority of these otus, along with the majority of known species associated with clinical phenotype are members of the order clostridiales (figure c). while a majority of the correlations observed were between one metabolic trait and one taxon, lachnobacterium bovis and anaerotruncus colihominis were inversely correlated with both high bmi and elevated serum triglycerides. although these two species of gut bacteria are known to produce short chain fatty acids as end-products of metabolism, a more thorough understanding of the potential impact of these species on host metabolism awaits further studies that will be facilitated by completion of ongoing reference genome sequencing projects for gut-associated bacterial species [ ]. in a subset of older ($ years of age) obese subjects with more severe manifestations of the metabolic syndrome there was an increase in the relative abundance of three gram-negative bacterial genera, fusobacterium, j – , and tenacibaculum, as compared to younger subjects (table s ). it is not possible to determine whether these represent age- or disease-related changes table . characteristics of subjects enrolled in this study. men women n age (yr) . . . . bmi (kg/m ) . . ( . – . ) . . ( . – . ) waist circumference (cm) . . . . systolic bp (mmhg) . . . . diastolic bp (mmhg) . . . . total cholesterol (mg/dl) . . . . hdl-cholesterol (mg/dl) . . . . ldl-cholesterol (mg/dl) . . . . triglycerides (mg/dl) . . . . glucose (mg/dl) . . . . at least metabolic syndrome trait (%)* . . blood pressure (%) . . hdl-c (%) . . triglycerides (%) . . glucose (%) . . *metabolic syndrome traits were defined by nhlbi criteria: ( ) fasting triglycerides . mg/dl (or on triglyceride lowering medication prescribed by a physician); ( ) fasting hdl# mg/dl for women or , mg/dl for men (or on hdl raising medication prescribed by a physician); ( ) either or both systolic or diastolic blood pressure . / mm hg (or on anti-hypertension medication prescribed by a physician); ( ) fasting glucose $ mg/dl (or on anti-diabetes medication prescribed by a physician). waist circumference was not included in our definitions because of the high correlation between waist circumference and bmi. doi: . /journal.pone. .t table . core gut microbiota at the genus level (present in $ % of subjects). firmicutes bacteroidetes tenericutes clostridium bacteroides incertae sedis ruminococcus prevotella faecalibacterium subdoligranulum oscillospira coprococcus blautia roseburia lachnospira incertae sedis lachnobacterium dorea eubacterium streptococcus doi: . /journal.pone. .t obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e in the gut microbiota [ ]; however, the increase in the abundance of fusobacterium is of interest in light of previous findings that periodontal fusobacterium species have been associated with in- flammation, atherosclerosis [ ] and colon cancer [ ]. discussion the prevalence of obesity has dramatically increased around the world over the last twenty years, in large part linked to the western life-style. many lines of evidence point to a complex etiology for obesity that includes both genetic and environmental factors. obesity is associated with a panoply of co-morbidities including hypertension, dyslipidemia, insulin resistance, and diabetes, collectively known as the metabolic syndrome, that increase the risk of cardiovascular disease [ ]. while numerous obesity-related changes in human physiology have been described, the gut microbiota has also been implicated in obesity, perhaps by influencing energy homeostasis, host signaling, insulin resistance, gut permeability, and inflammation and the innate immune response. the present study used sequencing of s rrna amplicons to characterize the gut microbiota in a metabolically well-character- ized cohort of amish subjects over a range of bmi. the amish are an excellent population to study the relationship between gut microbiota and metabolic traits because of their relatively homogeneous lifestyle. furthermore, we believe that knowledge gained from the amish is relevant to the general population because the clinical characteristics of obesity and its related complications in the amish are indistinguishable from that in the general caucasian population [ ], and the amish gene pool originates in central europe and thus host genetic findings in the amish are likely to represent a subset of those present in the general caucasian population. our analyses revealed several novel insights into the structure and role of the gut microbiota in obesity and the metabolic syndrome. first, we identified three groups of bacterial species, each of which include dominant organisms in the three enterotypes previously identified in a small set of european subjects [ ]. these bacterial groups likely define an interactive functional community, where some members play redundant functional roles. approximately % of the subjects enrolled in this study were members of nuclear families allowing us to compare concordance rates of community structure groups between related and unrelated individuals; however, these results did not provide any evidence that these structure groups aggregated within families. interestingly, there was a correlation between occupation and community structure with farmers more likely than other occupations to harbor the prevotella-dominated figure . bacterial networks in the human gut microbiota. bacterial networks were identified based on statistically significant correlations among genera using the louvain algorithm. network i: prevotella-dominated (a), network ii: bacteroides-dominated (b) and network iii: oscillospira- dominated (c) are illustrated, where the dominant bacterial genus is highlighted in yellow and other genera in green. the size of the circles represents the mean relative abundance of each genus in the ooa population. solid lines represent positive correlations and dashed lines represent inverse correlations (all p, . ). numbers connecting microbes are the correlation coefficient. (d) diversity in the three networks as measured by the shannon diversity metric. doi: . /journal.pone. .g obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e microbiota. to the extent that amish famers come in closer contact with livestock than amish in other occupations, this observation is intriguing in that the gut microbiota of various livestock species has been reported to contain a high relative abundance of the xylanolytic bacterial species prevotella [ – ], although this result is also consistent with the interpretation that the ‘over-representation’ of the prevotella-dominated microbiota is really due to an under-representation of the bacteroidetes and firmicutes-dominated phyla. regardless, this leaves open the speculation that the environment may in some situations play an figure . unifrac principle co-ordinates analysis plot, showing each study sample positioned according to its first two principle coordinates. these are determined by the classical multidimensional scaling algorithm so that the euclidean distance between points approximates the unweighted unifrac distance between the otu profiles of the corresponding samples, colored by abundance of (a) prevotella, (b) bacteroides, and (c) firmicutes. doi: . /journal.pone. .g table . regression analyses between bacterial networks and metabolic phenotypes. variable network network network p-value for network effect contrast p-value age (yrs) . . . . . . . sex (% male) . . . . network vs. network network vs. network network vs. network . . . bmi (kg/m ) . . . . . . . waist (cm) . . . . . . . hdl-cholesterol (mg/dl) . . . . . . . triglycerides (mg/dl) . . . . . . . glucose (mg/dl) . . . . . . . systolic bp (mm hg) . . . . . . . diastolic bp (mm hg) . . . . . . . reached menopause (%) . . . . has one or more metabolic syndrome traits (yes/no) (%) . . . . all analyses adjusted for age and sex except analyses of age and sex, which were unadjusted. see table legend for definitions of metabolic syndrome traits. doi: . /journal.pone. .t obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e important role in modulating community composition, and may even suggest that transmission of gut microbes may occur across host species. this possibility is highly speculative, but suggests a potential avenue for future follow-up. we hypothesized that we would identify differences in the gut microbiota that would be associated with body weight and/or features of the metabolic syndrome in this cohort. neither b/f ratio nor community structure was associated with bmi or metabolic syndrome traits. further investigation using network analysis identified bacterial species and otus that represent between . and % of the total sequence reads that were statistically significantly correlated with bmi and several features of the metabolic syndrome. approximately half of these species are members of the core gut microbiota in the amish and members of the bacteroidetes and firmicutes phyla. in summary, we have carried out a survey of the gut microbiota and its relationship to obesity and metabolic syndrome in the ooa, a population of common genetic background and similar lifestyle. our results have identified a subset of bacterial taxa that are linked to metabolic syndrome traits; although the cross- sectional nature of this study makes it difficult to infer cause and effect with these data alone. follow-on longitudinal studies can begin to address whether specific gut bacterial taxa a play a causal role in the predisposition to or development of the metabolic syndrome, as well as the utility of interventions that modulate the composition of the gut microbiota to mitigate the risk of cardiovascular complications associated with metabolic syndrome. materials and methods study population and sample collection our study population consisted of old order amish adults from lancaster county, pennsylvania. the amish sect originated in berne, switzerland in as an ultraconservative wing of the mennonite movement [ , ]. over approximately years, beginning in , a small number of amish immigrated to eastern pennsylvania. the amish in lancaster county have expanded to over , today. our analyses indicate the lancaster amish population descended from a total of founders, of whom ( men and women) account for over % of the average founder contribution [ ]. all recruitment was performed between april and september . eligibility criteria included the following: of amish descent; age between and years. we recruited subjects over a wide range of bmis. exclusion criteria included the following: currently pregnant or have been pregnant in the last months; antibiotic treatment within the prior months; currently taking a medication (e.g., antibiotic, anti-inflammatory agents, glucocorticoids or other immune modulating medications), un- willing to discontinue vitamin or supplements, including probio- tics, potentially affecting the gut microbiome (vitamins/supple- ments and medications that were judged to possibly affect the gut microbiome were discontinued for at least days prior to stool collection); renal insufficiency (serum creatinine . mg/dl); hematocrit , %; uncontrolled thyroid disease (tsh , . or . . miu/; co-existing malignancy; history of intestinal surgery (except appendectomy or cholecystectomy); history of inflamma- tory bowel disease, celiac disease, lactose intolerance, chronic pancreatitis or other malabsorption disorder. recruitment was performed during an initial home visit by a field team consisting of a nurse and an amish liaison. a screening questionnaire, height, weight, and hip measurement, and blood tests (comprehensive metabolic panel, complete blood count, thyroid stimulating hormone (tsh), celiac screen; quest diagnostics, inc, horsham, pa) were obtained to rule out exclusions (see below). eligible and consenting volunteers were provided with a stool sample collection kit and instructions for collection (see below). a follow-up home visit was conducted after an overnight fast to obtain additional information through questionnaires (such as medical and family history, food frequen- cy), blood pressure measurements, and collection of blood, urine, and fecal samples. all procedures were performed by trained personnel using standard operating procedures following the guidelines of the university of maryland and the amish research clinic [ ]. height and weight were measured by trained nurses in subjects without shoes and in light clothing using a stadiometer and calibrated scale. body mass index was calculated as weight in kilograms divided by height in meters squared. waist circumfer- ence was measured to the nearest . cm using an inelastic tape. blood pressure was obtained manually with the subject in the sitting position after he or she had been sitting quietly for minutes and the average of measurements was used for analysis. blood was drawn after a . hour fast and serum glucose, total cholesterol, hdl-cholesterol, and triglycerides, and c-reactive protein were assayed by quest diagnostics (horsham, pa). ldl- cholesterol was calculated using friedewald’s formula. for collection of feces, subjects were instructed to collect a stool sample within day of the scheduled home visit. the sample was collected in a disposable ‘‘nuns’’ hat, and a portion of the sample (approximately – g) was immediately dispersed in rnalater (qiagen) and refrigerated overnight. rnalater-stabilized sam- ples were then frozen at uc and transported on dry ice to the institute for genome sciences for long-term storage. in a subset of table . distribution of subjects in each microbiota network, according to occupational class. men women network prevotella (n = ) network bacteroides (n = ) network firmicutes (n = ) age adj. p-value* network prevotella (n = ) network bacteroides (n = ) network firmicutes (n = ) age adj. p-value farmers ( . ) ( . ) ( . ) . ( . ) ( . ) ( . ) – tradesmen ( . ) ( . ) ( . ) . ( . ) ( . ) ( . ) – farmer’s wives ( . ) ( . ) ( . ) – ( . ) ( . ) ( . ) . teachers/shopkeepers ( . ) ( . ) ( . ) . ( . ) ( . ) ( . ) . unknown/retired ( . ) ( . ) ( . ) . ( . ) ( . ) ( . ) . *p-value for test of association of occupational class with microbiota network. doi: . /journal.pone. .t obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e subjects, a second fecal sample was obtained to months later through a home visit. an interval history and follow-up anthropometry was also obtained at that home visit to assess any changes in health status or medication usage (including anti- biotics). protocols and procedures for this visit were similar to those in which the first fecal sample was obtained. the institutional review board at the university of maryland school of medicine approved the protocol and informed consent was obtained from all subjects. dna extraction for dna extraction, a . g stool aliquot was transferred to a dna/rna-free sterile tube, and ml of phosphate-buffered saline was added to the sample. cell lysis was initiated by adding ml of lyzosyme ( mg/ml) and ml of mutanolysin ( , u/ml; sigma- aldrich). after a hour incubation at uc, each sample was further lysed by addition of ml proteinase k and ml % sds, followed by incubation at uc for minutes. the samples were then disrupted by bead beating, which was performed in a fp fastprep at . m/s for sec using . mm silica spheres (qbiogen lysis matrix b). the resulting crude lysate was processed using the zymo fecal dna kit (zymogen) according to the manufacturer’s recommendations. negative extraction controls, where stool samples were omitted, were performed to ensure the samples were not contaminated by exogenous bacterial dna during the extraction process. the dna concentrations in the samples were measured using the quant-it picogreen dsdna assay kit from molecular probes (invitrogen). pyrosequencing of barcoded s rrna gene amplicons universal primers f and r were used for pcr amplification of the v –v hypervariable regions of s rrna genes. the r primer included a unique sequence tag to barcode each sample. the primers were as follows: f- - gccttgccagcccgctcagtcagagttt- gatcctggctcag- and r- - gcctccctcgcgccatcagnnnnnnnn- catgctgcctcccgtaggagt- , where the underlined sequences are the life sciences flx sequencing primers b and a in f and r, respectively, and the bold letters denote the universal s rrna primers f and r. the -bp barcode within primer r is denoted by ns. using barcoded r primers [ ] the v –v regions of s rrna genes were amplified in -well microtiter plates using amplitaq gold dna polymerase (applied biosystems) and ng of template dna in a total reaction volume of ml. reactions were run in a ptc- thermal controller (mj research) using the following cycling parameters: min of denaturation at uc, followed by cycles of s at uc (denaturing), s at uc (annealing), and s at uc (elongation), with a final extension at uc for min. negative controls without a template were included for each barcoded primer pair. the presence of amplicons was confirmed by gel electrophoresis on a % agarose gel and staining with sybrgreen. pcr products were quantified using a geldoc quantification system (biorad) and the quant-it picogreen dsdna assay. equimolar amounts ( ng) of the pcr amplicons were mixed in a single tube. amplification primers and reaction buffer were removed from each sample using the ampure kit (agencourt). the purified amplicon mixtures were sequenced by flx titanium pyrosequencing using life sciences primer a by the genomics resource center at the institute for genome sciences, university of maryland school of medicine, using protocols recommended by the manufacturer as amended by the center. sequence analysis sequences were binned and trimmed, using the sample-specific barcode sequences, using mothur and the following criteria: (i) sequence length . bases, (ii) sequence length , bases, number of ambiguous bases = , exact barcode matching, nucleotide mismatch in primer matching, and maximum homo- polymer string of bases [ ]. taxonomy assignments were done by kmer-based naive-bayes classification via mothur classify.seqs applied to the greengenes [ ] reference sequences and taxonomy, with a confidence cut-off of . . operational taxonomic units (otus) were determined using mothur by (i) alignment to the silva s rrna database [ ], (ii) clustering by bacterial family [ ], and a distance matrix cutoff of . . jensen- shannon divergence between genus-level frequency distributions was calculated for each pair of samples, and the square-root of this was used to perform hierarchical clustering with complete linkage via the r hclust command. the three top-level clades were then used as the three groups. construction of bacterial networks for each network, we transformed the s rrna sequence reads into relative abundance, computed the spearman rank correlation, and then constructed a genera network and identified a sub-network (module) with those genera that have a direct connection. the network was constructed with the cutoff p value of less than . . network analysis with integrated s rrna sequence and clinical data to visualize the interconnectivities between species in the human gut microbiota and clinical phenotypes, we transformed the number of s rrna sequence reads from each sample into relative percentages, and then computed the cross-correlation matrix between clinical metadata and sequence data with the spearman rank correlation. the network was constructed with correlations that have a p-value of . or less. the sub-networks (modules) are identified with the louvain algorithm [ ]. phylogenetic tree construction representative s rrna sequences assigned to the species and otus with correlations to mst, were aligned to the silva database of reference s rrna genes [ ] using figure . bacterial species and otus correlated with metabolic syndrome phenotype. (a) known species and operational taxonomic units (otus) (green circles) linked to metabolic syndrome traits (yellow diamonds), illustrating statistically significant correlation coefficients using the louvain algorithm. the size of the circles represents the mean relative abundance in the amish cohort studied. numbers connecting microbes are the correlation coefficient (p, . for all). solid lines represent positive correlations and dashed lines represent inverse correlations. (b) the same network as shown in panel a, but also including the statistically significant associations between bacterial taxa. (c) phylogenetic tree of s rrna sequences from the bacterial taxa in this network using the r implementation of dnadist and fastme. otus and known species that are inversely correlated with metabolic syndrome traits are colored in red and that are positively correlated with metabolic syndrome traits are colored in blue (p, . for all). doi: . /journal.pone. .g obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e mothur (align.seq) and trimmed based on reference coordi- nates using screen.seqs/filter.seqs to include sequences that were atleast bp over the v –v region. distances were calculated with the trimmed multiple sequence alignment using dnadist [ ] in the r ape package. a phylogenetic tree was constructed using fastme [ , ]. association of major networks with demographic and other factors we compared the distribution of the three major groups across a variety of factors including age, sex, occupation, season of feces collection, and metabolic syndrome-related phenotypic character- istics of the study subjects. we tested the association of bacterial groups with demographic and metabolic factors by regressing group, coded as a class variable with levels, against each metabolic variable separately, and adjusting for age and sex. this was a df test. continuously distributed variables were compared across groups using analysis of variance. whenever a significant association was detected, we then tested each pairwise contrast separately (e.g., group vs groups and ; group vs groups and ; and group vs groups and ) to determine the relative contributions of each contrast to the difference. these analyses were run in sas using the glm procedure. because our sample of individuals included nuclear families, we were able to estimate the heritability of enterotype by comparing concordance rates for enterotype class between spouse pairs (who are unrelated) and sib-pairs and parent-offspring pairs (who share % of their genes in common). we estimated heritability as twice the difference in concordance rates between the first-degree relative pairs and the spouse pairs. supporting information figure s rank abundance of genera in the gut micro- biota of the amish. relative abundance of the top species identified across all samples in this study. (eps) figure s prevalence of bacterial genera in the amish. the prevalence of a genus was calculated as the percent of subjects in which that genus is present. genera are colored by percent mean relative abundance, – . (blue), . – . (purple), . – . (navy) and . – (red). (eps) figure s distribution of genera in the amish. histogram of prevalences among all genera observed in the amish cohort studied. the peak on the left represents the many genera observed in a small number of samples, while the peak on the right represents the core microbiota, observed in % or more of the samples. (eps) figure s bacteroidetes and firmicutes composition in the gut microbiota of old order amish subjects. bar plots show the fraction of total sequence reads assigned to the bacterial families bacteroidaceae, prevotellaceae, lachnospira- ceae, and ruminococcaceae in lean women (fn), lean men (mn), overweight/obese women (fb) and men (mb) without, and overweight/obese women (fm) and men (mm) with metabolic syndrome traits. (eps) figure s unifrac distribution of ooa subjects based on bmi. unifrac principle co-ordinates analysis plot, showing each study sample positioned according to its first three principle coordinates. these are determined by the classical multidimen- sional scaling algorithm so that the euclidean distance between points approximates the unweighted unifrac distance between the otu profiles of the corresponding samples. for this analysis, subjects were divided into non-overlapping phenotype groups: normal weight (bmi, kg/m )(n = red, n = ); overweight (bmi . – , kg/m ) with no features of the metabolic syndrome (ovb = yellow, n = ); overweight with one or more features of the metabolic syndrome (ovm = green, n = ), obese (bmi . . kg/m ) with no features of the metabolic syndrome (ob = green, n = ), and obese with one or more features of the metabolic syndrome (om = violet, n = ). features of the metabolic syndrome were defined by nhlbi criteria: ( ) fasting triglycerides . mg/dl (or on triglyceride lowering medication prescribed by a physician); ( ) fasting hdl# mg/dl for women or # mg/dl for men (or on hdl raising medication prescribed by a physician); ( ) either or both systolic or diastolic blood pressure . / mm hg (or on anti-hypertension medication prescribed by a physician); ( ) fasting glucose . mg/dl (or on anti-diabetes medication prescribed by a physician). waist circumference was not included in our definitions because of the high correlation between waist circumference and bmi. (eps) figure s community profile comparison in samples from the same subject. principal co-ordinates analysis plot, showing each study sample positioned according to its first two principle coordinates of the square root of the jensen-shannon divergence. samples belonging to the same sample are denoted with the same symbol. phenotype is indicated by specifically normal weight, bmi, kg/m (red); overweight and obese with no features of the metabolic syndrome, bmi . . kg/m (purple) and overweight and obese with features of the metabolic syndrome, bmi . . kg/m (blue). (eps) table s sequencing statistics. (docx) table s regression analyses for phenotype clusters in the ooa. (docx) acknowledgments the authors wish to acknowledge the efforts of luke tallon, lisa sadzewicz, kristine jones, ivette santana-cruz, and cesar arze from the genomics resource center at the institute for genomic research for s rrna amplicon pyrosequencing. the authors would also like to acknowledge patrick d. schloss for advice on the optimization of s rrna sequence analysis using mothur. data generated in this study are available at dbgap under study id phs and at sra under accession srp . author contributions conceived and designed the experiments: cmf ars ns rh ss. performed the experiments: kar sc cj em mlz. analyzed the data: blc zl efd rk waw bm dk. wrote the paper: cmf ars blc efd. obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e references . mokdad ah, marks js, stroup df, gerberding jl ( ) actual causes of death in the united states. jama : – . . power ml, schulkin j ( ) sex differences in fat storage, fat metabolism, and the health risks from obesity: possible evolutionary origins. br. j. nutrition : – . . hotamisligil gs ( ) endoplasmic reticulum stress and the inflammatory basis of metabolic disease. cell : – . . ley re, backhed f, turnbaugh p, lozupone ca, knight rd, et al. 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( ) silva: a comprehensive online resource for quality checked and aligned ribosomal rna sequence data compatible with arb. nucleic acids res. : – . . blondel vd, guillaume j-l, lambiotte r, lefebvre e ( ) fast unfolding of communities in large network. journal of statistical mechanics: theory and experiment , p . . felsenstein j, phylip (phylogeny inference package). . c ed. seattle: distributed by author. . desper r, gascuel o ( ) fast and accurate phylogeny reconstruction algorithms based on the minimum-evolution principle. : – . . desper r, gascuel o ( ) getting a tree fast: neighbor joining, fastme, and distance-based methods. curr. prot. bioinformatics chapter : unit . obesity and the gut microbiota in the amish plos one | www.plosone.org august | volume | issue | e wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ msy .indd fax + e-mail karger@karger.ch www.karger.com mol syndromol ; : – doi: . / severe intellectual disability associated with recessive defects in cntnap and nrxn c. zweier institute of human genetics, friedrich-alexander-university erlangen-nuremberg, erlangen , germany bakkaloglu et al., ; friedman et al., ; kim et al., ; kirov et al., ; marshall et al., ; vrijenhoek et al., ; zahir et al., ; bucan et al., ; rujescu et al., ; awadalla et al., ; bradley et al., ; ching et al., ; magri et al., ; mefford et al., ; wiśniowiecka-kowalnik et al., ]. a homozygous stop mutation in cntnap in old order amish children was reported to cause a distinct disorder, cortical dysplasia-focal epilepsy syndrome (mim ), characterized by cortical dysplasia and early onset, intractable focal epilepsy leading to language regression, and behavioral and mental deterioration [strauss et al., ; jackman et al., ]. recently, homozygous or compound heterozygous defects in cntnap or nrxn were reported to cause a severe in- tellectual disability disorder resembling pitt-hopkins syndrome (mim ) [zweier et al., ]. these patients had an initially negative tcf -testing and showed severe intellectual disability and additional vari- able symptoms such as epilepsy, breathing anomalies and stereotypies. in total, patients with homozygous or compound heterozygous defects in cntnap and patient with a compound heterozygous defect in nrxn are reported to date. key words cntnap � epilepsy � intellectual disability � mental retardation � nrxn abstract while heterozygous variants in cntnap and nrxn are re- ported as susceptibility factors for neuropsychiatric disor- ders, homozygous or compound heterozygous defects in either gene were reported as causative for severe neurode- velopmental disorders. this review provides an overview of the clinical aspects in patients with recessive defects in cntnap and nrxn . copyright © s. karger ag, basel history of the syndrome during recent years, heterozygous copy-number and missense variants in cntnap and nrxn have repeat- edly been reported as susceptibility factors for a wide spectrum of neuropsychiatric disorders such as develop- mental language delay and autism spectrum disorders, epilepsy and schizophrenia [verkerk et al., ; feng et al., ; autism genome project consortium, ; bel- loso et al., ; alarcón et al., ; arking et al., ; published online: september , christiane zweier institute of human genetics, university erlangen-nuremberg schwabachanlage de– erlangen (germany) tel. + , e-mail christiane.zweier   @   uk-erlangen.de © s. karger ag, basel – / / – $ . / accessible online at: www.karger.com/msy zweier mol syndromol ; : – clinical features ( table  ) facial gestalt in contrast to patients with pitt-hopkins syndrome, the facial phenotype in patients with recessive cntnap and nrxn defects is rather unsuspicious. two patients with a homozygous deletion within cntnap [zweier et al., ] had previously been published as possible pitt- hopkins syndrome patients due to a wide mouth and thick lips [orrico et al., ], but as indicated by peippo et al. [ ] and supported by the present knowledge and perspective, they do not have the distinct pitt-hopkins syndrome facial phenotype. the remaining patients do not show specific facial dysmorphisms [zweier et al., ] ( fig.  ), neither are any reported for the amish pa- tients [strauss et al., ; jackman et al., ]. intellectual disability intellectual disability is reported to be severe in all pa- tients. developmental testing of patients with cortical dysplasia-focal epilepsy syndrome at the ages of , and months revealed global mental ages of , and months, respectively [strauss et al., ]. particularly speech impairment is severe with either no or very lim- ited speech development or regression of speech abilities. both receptive and expressive language was reported to be maintained at the -year level in a -year-old patient [jackman et al., ]. in comparison, motor delay is rather mild with a normal or mildly delayed walking age. developmental regression was not noted in patients table . clinical findings in patients with recessive cntnap or nrxn defects cntnap (n = ) nrxn (n = ) age at clinical assessment – years years normal body height / / head circumference <p , others p –p , mean >p p –p severe intellectual disability / / age of walking normal– months years speech none or single words none developmental regression / / seizures with age of onset / , – months / mri anomalies cd / , ch / , pl / / behavioral anomalies / / decreased deep tendon reflexes / / breathing anomalies / / p = centile; cd = cortical dysplasia; ch = cerebellar hypoplasia; pl = periventricular leukomalacia. data for cntnap : jackman et al., ; strauss et al., ; zweier et al., . data for nrxn : zweier et al., . a b fig. . facial appearance of patients with compound heterozygous defects in cntnap ( a ) or nrxn ( b ). note a wide mouth in the patient with nrxn defects but otherwise unsuspicious facial ge- stalts in both patients. reprinted from zweier et al. [ ], with permission from elsevier. recessive defects in cntnap and nrxn mol syndromol ; : – [orrico et al., ], but was reported in the other patients with cntnap defects, starting at the same age as onset of epilepsy between and months [strauss et al., ; zweier et al., ]. later on, the degree of mental impair- ment seems to be stable. the early onset mandatory epi- lepsy with concurrent developmental regression in pa- tients with cntnap defects might be a discriminating aspect regarding other disorders with regression pheno- types such as rett syndrome. furthermore, the discrep- ancy between severe speech impairment and rather mild motor delay seems to be quite specific for recessive cntnap defects. similarly, in the patient with the nrxn defect, only mild motor delay with a walking age of years but no speech development was reported [zwei- er et al., ]. seizures the single patient with biallelic nrxn defects does not have epilepsy [zweier et al., ]. due to the limited number of patients, no conclusion about frequency of seizures in nrxn related intellectual disability can be made. all patients with recessive defects in cntnap do show epilepsy with an early onset between and months of age [strauss et al., ; jackman et al., ; zweier et al., ]. concurrent with the onset of epi- lepsy, language regression and deterioration of social behavior occur in most of the patients [strauss et al., ]. regarding available data reported by strauss et al. [ ], the average peak seizure frequency (number of events per week) is with complex partial, simple par- tial, secondarily generalized, and status epilepticus types of seizures. growth parameters as far as data is available, birth measurements are nor- mal. apart from siblings with short stature [orrico et al., ; zweier et al., ], later body measurements are reported to be normal as well. interestingly, many pa- tients show a tendency to rather large head circumfer- ences in relation to body height [strauss et al., ]. this might be a suitable discriminating factor in differential diagnosis to other severe intellectual disability disorders where microcephaly is common. behavioral observations behavioral anomalies with autistic traits or pervasive developmental delay, stereotypic movements and atten- tion deficit-hyperactivity disorder are common [strauss et al., ; jackman et al., ; zweier et al., ]. magnetic resonance imaging mri examination of the brain showed focal malfor- mations in patients. two of them had unilateral dyspla- sia of the anterior temporal lobe, and had a malforma- tion of the left striatum [strauss et al., ]. in other patients, periventricular leukomalacia [jackman et al., ] and cerebellar hypoplasia were observed, respec- tively [orrico et al., ; zweier et al., ]. other findings breathing anomalies [zweier et al., ] and de- creased deep tendon ref lexes [strauss et al., ; jack- man et al., ] were reported in some patients; hepato- megaly was observed in girl [jackman et al., ]. natural history cntnap early infant development appears to be normal or with only mild motor delay and is followed by onset of sei- zures, language regression or no speech development, with social and behavioral disturbances, and moderate to severe intellectual disability by late childhood [orrico et al., ; strauss et al., ; jackman et al., ; zweier et al., ]. nrxn the development of the patient was reported to be nor- mal for the first year before severe intellectual disability with a walking age of years and no speech development was noted [zweier et al., ]. recommendations for management not much information about treatment is available. in patient, zonisamide was used for treatment of seizures [jackman et al., ]. electrocorticography-guided epi- lepsy surgery for disabling complex partial seizures in patients resulted in a temporarily seizure-free period of – months but with recurrence after this time [strauss et al., ]. genetics information about the genes and protein function cntnap is one of the largest genes in the human genome spanning . mb on chromosome q – . and zweier mol syndromol ; : – consisting of coding exons (nm_ ). nrxn also belongs to the largest known genes in humans with span- ning . mb on chromosome p . . the classical neurex- in genes in mammals have promoters, generating longer � - and shorter � - neurexins, and are subject to addition- al extensive alternative splicing, generating a large num- ber of variants [missler and südhof, ]. the represen- tative nrxn isoform � (nm_ ) consists of coding exons. cntnap encodes for caspr , a protein distantly related to the neurexins and regulating neuron-glia con- tact in vertebrates and glia-glia contact in insects [bellen et al., ]. vertebrate caspr has been shown to colo- calize with shaker-like k+ channels in the juxtaparanod- al areas of ranvier nodes in myelinated axons of both the cns and pns [arroyo et al., ; poliak et al., ]. furthermore, it seems to play a role in human cortical histogenesis as signs of neuronal migration anomalies were observed in brain samples of patients with a homo- zygous cntnap mutation [strauss et al., ]. the presynaptic neurexins like nrxn and their postsynaptic binding partners, the neuroligins, are cru- cial synapse molecules [li et al., ], with � -neurexins playing a role in normal neurotransmitter release and the function of synaptic calcium channels [missler et al., ]. recently, findings in drosophila indicated that not only nrxi (nrxn ), but also nrxiv (cntnap ) might be involved in synaptic organization and that both pro- teins might be linked by a common target, presynaptic protein bruchpilot [zweier et al., ]. mode of inheritance the mutations and deletions in the published patients were inherited in an autosomal recessive manner with homozygous or compound heterozygous defects in the patients and heterozygosity for of the defects in each parent [strauss et al., ; jackman et al., ; zweier et al., ]. the carrier parents were reported to be healthy; therefore, penetrance of clinical symptoms as- sociated with heterozygous defects in either gene might be lower than possibly appreciated from previous reports on neuropsychiatric disorders [verkerk et al., ; feng et al., ; autism genome project consortium, ; belloso et al., ; alarcón et al., ; arking et al., ; bakkaloglu et al., ; friedman et al., ; kim et al., ; kirov et al., ; marshall et al., ; vri- jenhoek et al., ; zahir et al., ; bucan et al., ; rujescu et al., ; awadalla et al., ; bradley et al., ; ching et al., ; magri et al., ; mefford et al., ; wiśniowiecka-kowalnik et al., ]. however, an increased risk for variable neuropsychiatric disorders has to be considered in carrier individuals. frequency of certain mutations/copy number variations in certain patient cohorts the number of patients carrying recessive defects in cntnap or nrxn is too small to give frequencies of certain mutations/copy number variations. in the amish population, specific stop mutation c. delg in the c- terminal region of cntnap is reported [strauss et al., ]. the other published patients either harbor a ho- mozygous deletion of exons – or a compound hetero- zygous intragenic deletion of exons – and a splice site mutation in exon of cntnap [zweier et al., ]. for nrxn , to date only a deletion of exons – in com- pound heterozygosity with a stop mutation in exon is known. genotype-phenotype correlation some phenotypic differences such as lack of speech development versus regression or the presence/absence of cortical dysplasia and episodes of hyperbreathing be- tween the amish patients with the c-terminal stop muta- tion and the other patients with rather n-terminal defects in cntnap can be noted [zweier et al., ]. however, it remains currently elusive if these observations might indicate a real genotype-phenotype correlation or if they are due to clinical bias as the phenotype in the amish pa- tients was classified as an epilepsy syndrome with devel- opmental deterioration, while the other patients were ini- tially classified as having primary intellectual disability with epilepsy. references alarcón m, abrahams bs, stone jl, duvall ja, perederiy jv, et al: linkage, association, and gene-expression analyses identify cntnap as an autism-susceptibility gene. am j hum genet : – ( ). arking de, cutler dj, brune cw, teslovich tm, west k, et al: a common genetic variant in the neurexin superfamily member cntnap increases familial risk of autism. am j hum genet : – ( ). arroyo ej, xu t, poliak s, watson m, peles e, scherer ss: internodal specializations of my- elinated axons in the central nervous system. cell tissue res : – ( ). autism genome project consortium, szatmari p, paterson ad, zwaigenbaum l, roberts w, et al: mapping autism risk loci using genetic linkage and chromosomal rearrangements. nat genet : – ( ). recessive defects in cntnap and nrxn mol syndromol ; : – awadalla p, gauthier j, myers ra, casals f, hamdan ff, et al: direct measure of the de novo mutation rate in autism and schizo- phrenia cohorts. am j hum genet : – ( ). bakkaloglu b, o’roak bj, louvi a, gupta ar, abelson jf, et al: molecular cytogenetic analysis and resequencing of contactin asso- ciated protein-like in autism spectrum dis- orders. am j hum genet : – ( ). bellen hj, lu y, beckstead r, bhat ma: neurex- in iv, caspr and paranodin – novel members of the neurexin family: encounters of axons and glia. trends neurosci : – ( ). belloso jm, bache i, guitart m, caballin mr, halgren c, et al: disruption of the cntnap gene in a t( ; ) translocation family without symptoms of gilles de la to- urette syndrome. eur j hum genet : – ( ). bradley we, raelson jv, dubois dy, godin e, fournier h, et al: hotspots of large rare dele- tions in the human genome. plos one :e ( ). bucan m, abrahams bs, wang k, glessner jt, herman ei, et al: genome-wide analyses of exonic copy number variants in a family- based study point to novel autism suscepti- bility genes. plos genet :e ( ). ching ms, shen y, tan wh, jeste ss, morrow em, et al: deletions of nrxn (neurexin- ) predispose to a wide spectrum of develop- mental disorders. am j med genet b neuro- psychiatr genet b: – ( ). feng j, schroer r, yan j, song w, yang c, et al: high frequency of neurexin beta signal peptide structural variants in patients with autism. neurosci lett : – ( ). friedman ji, vrijenhoek t, markx s, janssen im, van der vliet wa, et al: cntnap gene dos- age variation is associated with schizophre- nia and epilepsy. mol psychiatry : – ( ). jackman c, horn nd, molleston jp, sokol dk: gene associated with seizures, autism, and hepatomegaly in an amish girl. pediatr neu- rol : – ( ). kim hg, kishikawa s, higgins aw, seong is, donovan dj, et al: disruption of neurexin associated with autism spectrum disorder. am j hum genet : – ( ). kirov g, gumus d, chen w, norton n, geor- gieva l, et al: comparative genome hybrid- ization suggests a role for nrxn and apba in schizophrenia. hum mol genet : – ( ). li j, ashley j, budnik v, bhat ma: crucial role of drosophila neurexin in proper active zone apposition to postsynaptic densities, synap- tic growth, and synaptic transmission. neu- ron : – ( ). magri c, sacchetti e, traversa m, valsecchi p, gardella r, et al: new copy number varia- tions in schizophrenia. plos one :e ( ). marshall cr, noor a, vincent jb, lionel ac, feuk l, et al: structural variation of chromo- somes in autism spectrum disorder. am j hum genet : – ( ). mefford hc, muhle h, ostertag p, von spiczak s, buysse k, et al: genome-wide copy num- ber variation in epilepsy: novel susceptibility loci in idiopathic generalized and focal epi- lepsies. plos genet :e ( ). missler m, südhof tc: neurexins: three genes and products. trends genet : – ( ). missler m, zhang w, rohlmann a, kattenstroth g, hammer re, et al: alpha-neurexins cou- ple ca + channels to synaptic vesicle exocy- tosis. nature : – ( ). orrico a, galli l, zappella m, lam cw, bonifa- cio s, et al: possible case of pitt-hopkins syn- drome in sibs. am j med genet : – ( ). peippo mm, simola ko, valanne lk, larsen at, kähkönen m, et al: pitt-hopkins syndrome in two patients and further definition of the phenotype. clin dysmorphol : – ( ). poliak s, salomon d, elhanany h, sabanay h, kiernan b, et al: juxtaparanodal clustering of shaker-like k+ channels in myelinated ax- ons depends on caspr and tag- . j cell biol : – ( ). rujescu d, ingason a, cichon s, pietiläinen op, barnes mr, et al: disruption of the neurexin gene is associated with schizophrenia. hum mol genet : – ( ). strauss ka, puffenberger eg, huentelman mj, gottlieb s, dobrin se, et al: recessive symp- tomatic focal epilepsy and mutant contactin- associated protein-like . n engl j med : – ( ). verkerk aj, mathews ca, joosse m, eussen bh, heutink p, et al: cntnap is disrupted in a family with gilles de la tourette syndrome and obsessive compulsive disorder. geno- mics : – ( ). vrijenhoek t, buizer-voskamp je, van der stelt i, strengman e; genetic risk and outcome in psychosis (group) consortium, et al: recurrent cnvs disrupt three candidate genes in schizophrenia patients. am j hum genet : – ( ). wiśniowiecka-kowalnik b, nesteruk m, peters su, xia z, cooper ml, et al: intragenic rear- rangements in nrxn in three families with autism spectrum disorder, developmental delay, and speech delay. am j med genet b neuropsychiatr genet b: – ( ). zahir fr, baross a, delaney ad, eydoux p, fer- nandes nd, et al: a patient with vertebral, cognitive and behavioural abnormalities and a de novo deletion of nrxn alpha . j med genet : – ( ). zweier c, de jong ek, zweier m, orrico a, ous- ager lb, et al: cntnap and nrxn are mutated in autosomal-recessive pitt-hop- kins-like mental retardation and determine the level of a common synaptic protein in drosophila . am j hum genet : – ( ). conflict resolution among peaceful societies: the culture of peacefulness conflict resolution among peaceful societies: the culture of peacefulness author(s): bruce d. bonta source: journal of peace research, vol. , no. (nov., ), pp. - published by: sage publications, ltd. stable url: http://www.jstor.org/stable/ . accessed: / / : your use of the jstor archive indicates your acceptance of the terms & conditions of use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . jstor is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. we use information technology and tools to increase productivity and facilitate new forms of scholarship. for more information about jstor, please contact support@jstor.org. . sage publications, ltd. is collaborating with jstor to digitize, preserve and extend access to journal of peace research. http://www.jstor.org this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/action/showpublisher?publishercode=sageltd http://www.jstor.org/stable/ ?origin=jstor-pdf http://www.jstor.org/page/info/about/policies/terms.jsp http://www.jstor.org/page/info/about/policies/terms.jsp ? journal of peace research, vol. , no. , , pp. - conflict resolution among peaceful societies: the culture of peacefulness bruce d. bonta general reference section, pennsylvania state university libraries the literature about peaceful peoples was examined to determine if their ways of conflict resolution differ from the approaches to conflict found in other, more violent, societies. while the strategies for managing con- flicts employed by these peoples are comparable to those used in many other small-scale societies, their world- views of peacefulness and the structures they use to reinforce those world-views do distinguish them from other societies. several common notions about conflict and conflict resolution that are asserted by western scholars can be questioned in light of the success of these societies in peacefully resolving conflicts: namely, that violent con- flict is inevitable in all societies; that punishment and armed force prevent internal and external violence; that political structures are necessary to prevent conflicts; and that conflict should be viewed as positive and necess- ary. the contrary evidence is that over half of the peaceful societies have no recorded violence; they rarely punish other adults (except for the threat of ostracism); they handle conflicts with outside societies in the same peaceful ways that they approach internal conflicts; they do not look to outside governments when they have internal dis- putes; and they have a highly negative view of conflict. . introduction and forgive us our trespasses as we forgive those who trespass against us. from 'the lord's prayer' nyam, the articulate son of a former headman, had been accused of planting durian trees on lands that traditionally belonged to others. in recent years the semai, peaceful aboriginal people who live in the rugged mountains of the malay peninsula, have been harvesting durian fruit and packing it out to the road which comes up from the lowlands, so they can sell it and buy the consumer goods that have become essential to them - tobacco, machetes, radios, and so forth. planting trees on other properties threat- ened and angered nyam's neighbors, some of whom belonged to different families. tensions were mounting. tidn, the headman of the village affected by nyam's actions, recognized the potential for conflict so he convened a becharaa', a proceed- ing which the villagers use to try to resolve dis- putes. nyam and his relatives were invited to attend to discuss and settle the matter. since his land also had been invaded by nyam, tidn was a party to the dispute; he invited entoy, head- man from a nearby valley, to preside over the becharaa'. nyam arrived near dusk at the semai village. conversation was casual, as everyone was well acquainted and was generally familiar with the nature of the conflict. nyam, a picture of studied indifference, talked animatedly with various people. after a while, the villagers gathered in a circle and the formal discussions began with preliminary speeches about the im- portance of settling the dispute before it got out of hand. each of the parties to the conflict gave his version of events, justifying his actions in an unemotional manner. nyam denied some of his trespasses and sought to rationalize others. speakers advanced their points of view, but no one acted as witnesses except for the principals in the case; there was no direct confrontation or cross-examination. the speeches went on and on, with people frequently talking past one another and not answering the comments of others. when no one had anything more to say - points had been emphasized and re-emphasized until all were exhausted from the proceedings - the becharaa' was ready to be concluded. it was obvious to everyone that nyam's actions were wrong, but the consensus was that he could keep and use the trees that he had already planted, though he must plant no more. entoy could have levied a small fine on nyam but everyone felt it was more important for the group to keep its harmony than to treat the guilty party too roughly. entoy lectured the assembled people on the importance of their tradition of unity, this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp bruce d. bonta peacefulness, sharing food, and not fighting. he made it clear to all that the matter had been completely settled and that no one was allowed to bring it up again (robarchek, , , ). contrast that scene reported in the anthropol- ogy literature with a comparable one from the legal proceedings of pennsylvania. for two years charles peterman leased acres of farm land in columbia county, pennsylvania, but a crop of winter wheat which he planted the sec- ond fall could not be harvested before the lease expired the following april. late the next spring, peterman tried to harvest his grain any- way, despite being warned away by the agent of the owner; when he continued to enter the land, the agent had him arrested. the matter soon reached the court of quarter sessions in the county seat of bloomsburg, where peterman was found guilty of criminal trespass. he ap- pealed his conviction to the superior court of pennsylvania, which reversed the ruling of the lower court (commonwealth v. peterman, ). the ways that conflicts are handled in pennsylvania courthouses differ significantly from the approaches the semai take with their becharaas', though the latter may have a super- ficial resemblance to american trials: the semai are most concerned with resolving con- flicts peacefully, while americans are primarily focused on fulfilling justice. everyone in the semai village knows the parties to a conflict and are already familiar with the facts; if a case in pennsylvania goes to a formal jury trial, the ju- rors must have no previous knowledge of the parties or the case. the parties to a conflict in the us usually hire attorneys to present their ar- guments aggressively; the semai present their own positions without confrontation or aggres- siveness. near the conclusion of the proceed- ings, the pennsylvania judge explains the legal issues to the jury and tells them that their role is to decide the truth of what happened; the semai headman lectures the whole village on the over- riding importance of the peaceful resolution of the conflict. punishment is the normal conclu- sion of the us trial; it is relatively unimportant to the semai. and finally, at the end of the becharaa', the semai headman prohibits any further consideration of the case, since it has been thoroughly resolved; the pennsylvania cit- izen is free to appeal his conviction, as peterman did. the important point in the us courtroom is winning; the major issue for the semai is resolving the conflict, removing the emotions from the parties to the dispute, and reaffirming correct, peaceful behavior. the lives of their children and grandchildren depend on it, they believe (robarchek, , , ). while these brief, simplified sketches repre- sent only a couple of the many ways that societies resolve disputes, they do illustrate fun- damental differences in perceptions of conflicts, resolution of disputes, and tolerance for viol- ence. the semai are among more than so- cieties that have evolved highly peaceful lifestyles, that rarely if ever resort to violence; us citizens are among the thousands of soci- eties that do use violence, if need be, to settle their differences. the processes of settling dis- putes in the usa, such as the jury trial, are based on assumptions about conflict that differ from those of the peaceful societies. the goal of this article is to explore those differences. the basic issue is to gain an understanding of why dozens of peaceful peoples are able to resolve conflicts nonviolently virtually all the time, while the rest of the world is not so successful. as the examination of conflict resolution in these small-scale societies proceeds, one funda- mental fact emerges: the peacefulness of their conflict resolution is based, primarily, on their world-views of peacefulness - a complete rejec- tion of violence. that argument may appear to be circular, but a careful look at conflict resol- ution in those societies seems to support it. in contrast, the western world-view boils down to an acceptance of the inevitability of conflict and violence. peace and conflict studies, for western scholars, is frequently a process of understanding the reasons for conflict, and the study of conflict resolution often focuses on strategies for preventing and resolving dis- putes. some of the major facets of western be- liefs that will form a framework for this essay include the following concepts: ( ) all societies have violent conflict and warfare (boulding, ; deutsch, ; knauft, ); ( ) pun- ishment deters internal conflicts and violence (greenawalt, ); ( ) the threat of armed force helps prevent external conflicts and viol- ence (brown, ; ceadel, ); ( ) conflict is best managed through reliance on political this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp conflict resolution among peaceful societies structures such as governments (boulding, , p. ); and ( ) conflict has many positive functions, and as long as it is managed properly it should be viewed as normal, reasonable, beneficial and helpful (augsburger, ; deutsch, ), or at least neither desirable nor undesirable (nader, ; ross, b). the purpose of this investigation is therefore to examine conflict and conflict resolution among the peaceful societies and to compare them with the corresponding western beliefs. since much of the literature of conflict resol- ution is based on the experiences of the thou- sands of relatively violent societies, a balance is needed from the perspective of peaceful peo- ples. in this paper i attempt to show that conflict resolution in peaceful societies is founded on overarching world-views that conflicts are the exception, not the norm, and that they are neither reasonable nor desirable. conflicts, to these peoples, must be avoided as much as poss- ible, resolved as quickly as possible, and har- mony restored as soon as possible in order for people to live peacefully with one another and with outsiders. in order to achieve nonviolent conflict resolution in practice, individuals and groups of people should rely on themselves to settle disputes within their groups as well as conflicts with other peoples; furthermore, they should use resolution strategies that dissipate tensions as well as settle the issues. this resol- ution should be achieved as much as possible without the threat of punishment (other than os- tracism). before getting to the information about peaceful societies and the reasons they provide a challenge to western thinking about conflict resolution, it is necessary to pause a moment, define terms, and introduce some basic under- standings. . background and definitions peacefulness is a condition of human society characterized by a relatively high degree of in- terpersonal harmony; little if any physical viol- ence among adults, between children and adults, and between the sexes; workable strategies for resolving conflicts and averting violence; a commitment to avoiding violence (such as warfare) with other peoples; and strategies for raising children to adopt and continue these nonviolent ways. a people or a society (those terms are used in- terchangeably, for variety) is a group of human beings who share a common ancestry for the most part, who share common beliefs and cul- tural value systems, and who primarily live in the same area. the peoples.: evidence demonstrates that a modest number of societies have developed highly, and in some cases totally, nonviolent social systems. several writers have provided different, but overlapping, lists of these peace- ful, peaceable, nonviolent, or low-conflict soci- eties (e.g. bonta, ; fabbro, ; howell & willis, a; montagu, ; ross, a; sponsel & gregor, ). the peoples included in this paper are based on the peace- ful societies included in bonta ( ); these were selected because there is at least some information about their styles of conflict resol- ution in the literature. most of the societies dis- cussed here are far from being utopias: many of them are plagued by the same jealousies, gossip, resentments, and backbiting as the rest of hu- manity (see robarchek, , p. ). some have social and cultural practices that would repel outsiders; they vary greatly from one to the next. the common denominator among all of them is that they are able to resolve their con- flicts peacefully, and that they fit the definition of peacefulness as given above. some are pri- marily hunting and gathering peoples; others rely mostly on shifting cultivation (swidden agriculture); others are settled farmers and are very much a part of the modern, world-wide trading society. these societies are listed in the appendix, with a brief paragraph describing each one. conflict is variously defined by scholars. some think of it in economic terms, such as 'a phenomenon that necessarily implies scarcity' (padilla, , p. ), or as an 'incompatibility between the preferences or goals of two or more parties' (schmidt, , p. ), or as the exist- ence of incompatible activities (deutsch, , p. ). these definitions do not go far enough, so conflict is defined here as: the incompatible needs, differing demands, contradictory wishes, opposing beliefs, or diverging interests which produce interpersonal antagonism and, at times, hostile encounters. conflict situations thus range from antagonist behavior to verbal abuse to physical violence to, ultimately, killing. this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp bruce d. bonta conflict resolution among peaceful peoples is the settlement or avoidance of disputes between individuals or groups of people through sol- utions that refrain from violence and that at- tempt to reunify and re-harmonize the people involved in internal conflicts, or that attempt to preserve amicable relations with external societies. basic understandings: a few basic under- standings must be introduced before proceeding any farther. first of all, while the peaceful societies are quite different from one another, they can be grouped together for this analysis because they do share at least one major charac- teristic: they rarely, if ever, have violent con- flicts. also, comparing conflict management in widely differing cultures is risky, though ross ( a) is confident that the shared features of conflict resolution in different cultures can be analyzed successfully. and while conflict resol- ution is commonly practiced by almost all soci- eties (sponsel, ), the unifying feature of nonviolence among the small group of peaceful peoples makes the study of their strategies and attitudes toward peace particularly worthwhile. another caveat: this work is not based on statis- tical research about cross-cultural peacefulness. social scientists doing research on conflict (e.g., ember & ember, ; ross, b) often use statistically valid samples of cultures, such as from the human relations area files, but this essay does not follow that approach. it is based, instead, on a careful examination of the litera- ture about all of these societies: a sampling would not have served the purposes of the in- vestigation. . strategies of conflict resolution the peaceful societies use a variety of strat- egies to try to prevent, control, manage, and re- solve the conflicts that do come up, such as the semai becharaa' that was mentioned at the opening of this paper. an examination of these various strategies provides an overview of the common processes used by these peoples to re- solve conflicts, and helps set the stage for the discussion that follows. unfortunately, there is no standard listing of conflict-resolution strat- egies, which have been described in many ways (e.g., takie sugiyama lebra, as described by augsburger, , pp. - ; boulding, ; lederach, ; le vine, ; ross, a and scimecca, ). since a commonly- agreed upon list of strategies is not available, it seemed best to look directly at the literature on the peaceful peoples and see what common strategies are suggested there. the following six are suggested by the literature. . self-restraint the literature explicitly describes the ways that the ifaluk (lutz, ), tahitians (levy, ), paliyan (gardner, , , ), and toraja (hollan, ) use variations of self-re- straint as a means of moving away from conflict situations once they arise. (their approach is doubtless followed by other peaceful societies, such as the amish, mennonites, and hutterites, though the literature about those peoples is not as explicit on the subject.) these peoples feel that heightened emotional states lead quickly to further trouble, so they actively try to dissipate their emotions whenever a conflict seems poss- ible. a first-stage approach for a toraja individ- ual experiencing heated emotion is to remind himself or herself that any open expression of the feeling might be dangerous: the expression of such feelings would be ridiculed, might lead to hostile supernatural actions, and would open oneself to serious illness (hollan, ). . negotiation negotiation is often considered in a positive light by western writers (rubin, ), particu- larly when it is broadly defined as the interac- tion between parties to a dispute who work toward an agreement without the intervention of third parties who might make compulsory de- cisions (gulliver, , p. ). but the literature on the peaceful societies, other than the montagnais-naskapi (lips, ), semai (robarchek, ) and amish (cong, ), has little to say about direct negotiations by dis- putants. people in many of these societies do not want to confront one another directly, and they prefer indirection rather than assertion, infer- ence rather than confrontation. the parties to a conflict are encouraged to settle their problems on an internal level, through self-restraint, but not necessarily through the confrontational tac- tics of direct negotiation. other techniques are more effective. this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp conflict resolution among peaceful societies . separation at least ten of the peaceful societies separate in order to avoid conflicts (which is equivalent to resolving them). clearly, walking away from a dispute is one of the most favored ways of re- solving conflicts among these peoples. among the malapandaram (morris, , ), paliyan (gardner, , , ), birhor (sinha, ), buid (gibson, , , ), and !kung (draper, ; lee, ), in- dividuals, including spouses, separate when a quarrel cannot be easily resolved, and whole communities will split apart to avoid conflicts. the literature about these peoples is filled with examples of individuals or whole communities moving away from an area, in some cases quite abruptly, because they faced conflicts. among the toraga (hollan, ) and balinese (howe, ), separations to avoid conflicts appear from the literature to be somewhat less perma- nent than among the other five peoples men- tioned above. the historical literature about some of the western peaceful peoples - the amish, hutterites, and mennonites - makes it clear that they moved away from domination and conflict by stronger societies numerous times. while they would doubtless not abandon their communities and flee on a moment's no- tice because of a minor conflict, there is no question that they might well move again if faced with an unresolvable conflict with the larger society. . intervention western writers on conflict resolution concen- trate heavily on the importance of third-party in- tervention in disputes (keashly et al., ; augsburger, ; fisher & keashly, ). among peaceful peoples, intervention by others is an effective technique for resolving conflicts. in several of these societies, such as the ifaluk (lutz, ), !kung (lee, ), mala- pandaram (morris, , ), nubians (femea, , ), toraja (hollan, ), zapotec (paddock, ), montagnais-naskapi (lips, ), paliyan (gardner, ), and yanadi (raghaviah, ) the ethic of avoiding conflicts is so strong that it is incumbent on by- standers to become involved in virtually any cir- cumstances where controversies threaten to be- come serious or where a conflict situation seems to be developing. among some peoples, certain individuals are noted as being particularly skilled at helping defuse conflicts, but in others the literature indicates that any bystander will step in to mediate. the common thread of these mediators is their desire to get a dialogue going - and keep the potential contestants talking until the tensions are defused. . meetings humor and meetings, such as the semai becharaa' mentioned earlier, are specific tech- niques used by third parties, but they deserve to be mentioned separately because they are fre- quently used by several peaceful societies. as with the other strategies, the purpose of the meeting is to lessen tensions more than it is to confront or decide, though those elements may also be present. these meetings provide forums for the airing of hostilities: frequently the simple discussion of grievances is enough to defuse problems. the meetings also serve to contain conflicts before they can disrupt society, either by minimizing issues as private rather than public concerns, or by restricting involve- ment in order to allow informal mechanisms of social control to operate. meetings are used heavily, as a major part of the strategy for re- solving conflicts, by the birhor (sinha, ), buid (gibson, , ), ladakhis (norberg- hodge, ), zapotec (o'nell, , and nubians (femea, ; callender, ). . humor humor is undoubtedly a useful strategy for re- ducing tensions and resolving conflicts in many societies, but it has been mentioned only a few times in the literature of peaceful peoples. the !kung (marshall, ) try hard to maintain a joking atmosphere in their camps, frequently pointing out one another's faults in a facetious manner to resolve their tensions. when a leader in a paliyan community becomes involved in helping to resolve a conflict, he will often use joking or soothing to defuse the situation (gardner, ). if a tristan islander ever lost his temper in a quarrel he would have that scar on his reputation for life; people who defuse tense situations with jokes gain general respect (munch, ). the inuit joke to avoid and defuse conflicts; joking also allows them to con- front problems with enough ambiguity that this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp bruce d. bonta grievances can be aired without fear of provok- ing others (briggs, ). in the past, the inuit had song duels to resolve conflicts in a humor- ous fashion before they became serious enough to provoke violence, and to laugh off animosi- ties and return to friendship, or at least restraint (eckert & newmark, ). these strategies seem to dissipate tensions and resolve issues effectively when conflicts do arise in the peaceful societies. in some societies authority figures make judgments while in others the people decide by consensus, but the overall effect is the same - healing, continuation of the community, or separation. furthermore, the traditional forms that those strategies take among these peoples appear to be important fac- tors in their success. that is, peoples are con- scious of their own traditional ways of handling problems and seem able to keep the peace in part through the force of their traditions. for these peoples, the ways they resolve disputes are logical and effective - and they seem to work. when the traditional ways are not used, conflicts can result. for instance, the failure of a group of buid to follow their traditional meet- ing-style of conflict resolution (called a tul- tulan) on one occasion resulted in tragedy (gibson, ). . conflict is a normal aspect of all societies some scholars have maintained that conflict and violence is the normal condition of small-scale societies, which typically rely on a superior state authority to prevent warfare (ferguson, , pp. - ). others argue that all societies have to contend with violence (knauft, , ). the literature on the peaceful peoples flatly contradicts these assertions. while viol- ence exists in very modest amounts in some of these societies, in others it appears to be rare or completely absent. there are a few basic differences in strategies for resolving conflicts among these societies. some of the ones that experience occasional vi- olence use moderately aggressive techniques for resolving disputes, such as stylized rhetorical speaking referred to as 'talking' by some an- thropologists. when the !kung are discussing a contentious issue and their emotions begin to rise, they may pour out their thoughts at a very rapid rate - a sudden, spontaneous discussion by the various people involved with the issue (marshall, ; lee, ). when the g/wi have a conflict that is threatening to escalate, one party to the problem will talk out the diffi- culty to a third person within the hearing of the whole band, and the other party may answer to a fourth, again so everyone will hear (silberbauer, ). when the temair become too angry for mediation to work, instead of a face-to-face confrontation the angry people may conduct night time harangues so everyone in the longhouse can hear without specifically naming individuals (roseman, ). these practices allow everyone to be a party to the dispute, to get feelings about an issue into the open without provoking direct confrontations, and to settle the contentious issues. they also save face for all participants, a universal need according to some (augsberger, ). on the other hand, many of the societies that almost never experience any violence tend to be meek and to have world-views that advocate meekness. for instance, the highly peaceful chewong, ifaluk, paliyan, and semai generally describe themselves as fearful people; the batek, chewong, paliyan, and semai flee from violence; and the amish, hutterites, chewong, semai, tristan islanders, and yanadi are no- table for their belief in nonresistance (not resisting aggression by the state or other indi- viduals). but, while the most highly peaceful peoples are strongly characterized by a general fearfulness, passiveness, meekness, flight from conflict, and a belief in nonresistance, the soci- eties which appear to take a more active role in promoting peacefulness do have patterns of oc- casional violence. there are elements of aggres- siveness in these peoples - perhaps it could be described as an aggressive pursuit of non- violence in resolving conflicts. . punishment deters conflict and violence western peoples believe that punishment is necessary to deter crime and violent conflict. they feel it creates fear in potential offenders that they will suffer as a result of their actions, and it is a just retribution for violations of the normal moral order (greenawalt, ). it seemed reasonable to look for evidence of pun- ishment in the literature about these societies to see if it is part of their conflict resolution this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp conflict resolution among peaceful societies practices. as it turns out, except for the punish- ment that parents in a few of these societies use for disciplining their children, these peoples use very little adult punishment. in fact, the absence of punishment appears to be one of the defining characteristics of a peaceful society. these peo- ples seem to rely on the strength of their other mechanisms to prevent and resolve conflicts peacefully and effectively. the threat of punish- ment is not needed, except for the practice of os- tracism, a form of punishment. ostracism is practiced by a range of societies worldwide to enforce social standards, according to gruter & masters ( , p. ), who define it in general terms as 'the general process of social rejection or exclusion'. from the perspective of the peaceful peoples, ostracism may be defined as complete banishment from the society or, per- haps less severely, as rejection by a people of an individual's participation in some or all of the group's activities. the societies that use it at all use it quite infrequently, but the possibility is al- ways there. probably the most dramatic practice of os- tracism in this body of literature is the amish strategy of shunning. if an amish person has a problem accepting one of the rules of their church, and he or she refuses to give in to the will of the group, the individual will be os- tracized by all members of the community, in- cluding the spouse, children, parents, siblings, and friends. no one may speak to the shunned person or hand food or other goods to him or her - food or other articles will be placed on a table for the shunned person to pick up (gruter, ; hostetler, ). the person may continue to live at home and try to carry on a normal life - though that is, of course, nearly impossible. the hutterites have a similar style of excommuni- cating members without expelling them from their colonies (hostetler, ). a comparable example can be found in ladakh, where again ostracism does not necess- arily mean the person is sent away from the community. if someone refuses to stop provoca- tive or offensive behavior, the lamas may cease serving the religious needs of the individual, which would be highly demoralizing to a ladakhi. no one would visit the ostracized per- son; no one would help the offender or his fam- ily in any endeavor; no one would offer food to, or accept food from, the individual; and there would be no possibilities of marriage alliances with other families. a harsh punishment such as that could only be relieved when the offender sought the pardon from the village civil and re- ligious leaders (norberg-hodge, ). ostracism in other societies usually means totally excluding offenders from the group - e.g., the nubians (fernea, ) - though in some cases it is done very gently. when a mem- ber of a g/wi band does not heed the consensus judgment of the group about a conflict, and when he ignores the barbed comments of others and does not mend his ways, the people may have to ease the offender out. this is done not by overt antagonism, but rather by subtly frus- trating the offender, by misunderstanding his wishes on purpose, by not hearing him: by, in effect, rejecting him without causing him to feel rejected or offended. the process prompts the offender to feel disgusted with his life in the band, so that he'll leave of his own accord with- out feeling a need for revenge. sometimes the offender will find another band to be more com- patible and will settle into acceptable behavior patterns. some g/wi, of course, never adapt and move about from band to band, accepted by all as individuals who have to be tolerated for a time (silberbauer, ). . armies are necessary to deter external conflict many western writers maintain that the exist- ence of armies and the threat of military force is the only thing that keeps the peace between nations. states would invade one another con- stantly in their egocentric drives to acquire more territory, goods, trade markets, resources, and security, according to this argument, if it weren't for the certainty that the invaded state would fight back (brown, ; ceadel, ). this kind of argument is also extended to peace- ful societies, which, it is argued, exist only in re- lation to, and through the sufferance of, more aggressive neighboring societies. these peace- ful peoples must have relatively peaceful neigh- bors, live where they are relatively isolated from attack, live where flight from attack is a reason- able option, or be much stronger than potential attackers so that others wouldn't dare try an at- tack (argument and literature summarized by ross, a, pp. - ). some of the peaceful societies under this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp bruce d. bonta consideration here follow this generalization, at least superficially. the problem with the idea is that it views the relationship of the nonviolent society and the aggressive society only from the perspective of the latter: the peaceful society must be isolated from the strong society or it can't exist; it must be able to flee quickly from an attack by the neighboring violent people or it would quickly be destroyed. the literature on these peoples and their relationships with dominating societies provides insights into this issue of peaceful peoples getting along with ag- gressive peoples - and it allows the simple gen- eralizations to be challenged. some of the peaceful societies fit those stereotypes but others do not. clearly, a few of the peoples live in very isolated locations, such as the ifaluk and the tristan islanders, who inhabit, respectively, is- lands in the pacific and atlantic oceans. other societies, such as the paliyan (gardner, , ), semai (dentan, a), batek (endicott, ), malapandaram (morris, ), buid (gibson, , ), and chewong (howell, , ) solve problems with outsiders, par- ticularly with more powerful outsiders, by flee- ing from danger. at the first sign of conflict, these peoples will abandon their villages and melt back into the forest, where they may stay for weeks or even years. but it is a mistake to assume that their relationships with their more powerful neighbors or the nations that they live in can be characterized only by isolation or flight from danger. they take their nonviolence seriously - as a positive approach to human re- lationships and as the basis of their lives - and avoiding conflict is only part of their logic. conflict resolution, such as the semai becharaa', is more complex and ingenious than the simple term 'separation' would imply. the semai are highly committed to their peaceful ways, and they try hard to resolve conflicts with their more powerful neighbors, the malay people, nonviolently. they have been invaded, dominated, and enslaved by the malays for a long time (endicott, ; robarchek, , pp. - ), but they still agonize over the di- lemma of how to continue to maintain their own ethic of peacefulness in the face of this domi- nation (robarchek, , pp. - ); they do not easily accept the ethic of the aggressor (dentan, ). a number of the peaceful peoples do have frequent contacts with outsiders and have been able to maintain their peacefulness despite it. the literature on these peoples suggests that they are able to get along with larger and more aggressive societies. not all conflicts with people and government officials from outside their societies can be avoided, of course, yet they handle conflicts with outsiders in a similar fashion to their handling of internal conflicts - i.e., peacefully. for instance, the anarchistic tristan islanders historically disliked the idea of any outside in- stitutional authority in their midst, but they have always had a knack for resolving conflicts with outsiders in a highly deferential, but still quite effective, fashion. in the late s an english minister on the island tried to run the lives of the people in an imperious, dictatorial manner, which the islanders didn't care for. he estab- lished a storehouse over which he exerted absolute control, in an attempt to bend the is- landers to accepting his will. the tristan islanders never got to the point of openly con- fronting the minister, however, since they could not endure the strain of confrontation with him; they would buckle under to his will with a meek 'yes, father', out of respect for his power and high office. they accepted his orders if they couldn't avoid them, to placate him and ignored everything else that they could get away with. they felt that his antics in trying to run their lives were simply part of the fun he enjoyed being among them. besides, they realized that in a few years he would be replaced by another minister who would have different ideas (munch, ). in the early s the entire population of tristan da cunha was brought to great britain by the british government when a volcano on the island threatened to destroy the settlement. however, when the government decided to make the evacuation permanent, the islanders united for the first time in their history to ex- press their feelings. they affirmed their belief that people should not control the lives of others; they agreed on a dislike for the ag- gression and self-assertion that they witnessed in britain; and they recognized that the violence in british society was too different from their own nonviolent culture for them to tolerate. they decided to return to their island on their this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp conflict resolution among peaceful societies own. faced with this determination, and with a lot of criticism in the media, the colonial office backed down and agreed to return them to tristan da cunha (munch, ). the tristan islanders succeeded in resolving the conflict with the government by confronting it in the most unobtrusive and peaceful manner they could figure out - by trying to find a way to get themselves back without the assistance of the government. the peaceful anabaptist peoples that live in north america - the mennonites, hutterites, and amish - have been persecuted for centuries but, like the tristan islanders, they have tried to settle conflicts with the larger societies in the same spirit of nonviolence that characterizes their internal relationships. for instance, during world war i draftees from these groups were punished, beaten, and tortured in us military prisons (unruh, ; juhnke, ), and civil- ians who refused to contribute money to the war effort through the purchase of war bonds were named in local newspapers, harassed, and physically abused by the local citizens (juhnke, ). as a result of these experiences, the con- troversial civilian public service program (cps) of world war ii (bush, ), and a range of other factors (toews, ), the basic beliefs of most mennonites in nonresistance have been slowly changing. from the s through the s mennonite commitment to 'nonresistance' (taken from matthew . , 'do not resist one who is evil'), has changed into a belief in 'peacemaking', the feeling that they have a responsibility to engage the broader american and canadian societies and work ac- tively for peace, rather than avoid outsiders as nonresistance had previously implied (driedger & kraybill, ; bush, ; nisly, ). the two other anabaptist peoples, the hutterites and the amish, have not developed a spirit of engaging the larger societies of the usa and canada as the mennonites have done; but they still have conflicts or the threats of con- flicts with outsiders to deal with. the hutterite colonies try to prevent conflicts from arising by fostering frequent contacts with their farming neighbors and by generous exchanges of farm produce (bennett, ). the amish have prob- lems resolving individual conflict situations with outsiders since they cannot file lawsuits against others - that would violate their belief in nonresistance. business competitors, buyers, and suppliers, knowing of that prohibition, take advantage of them by cheating and exploiting them (kraybill, ). much as they say they do not deal with the outside society, in fact the amish have devel- oped a pattern of adjustments to external con- flicts. non-amish leaders and supporters of the amish help them informally to resolve their conflicts with outsiders in positions of power and influence, sometimes through helpful advo- cacy, sometimes through finding creative sol- utions to their problems. if an amishman were taken into court, he would never contest charges and hire an attorney because of his belief in non- resistance, but an attorney friend might go along, just to sit there and make sure the courts acted fairly. the lawyer would not be paid, but the amish would give him some garden vegetables or freshly baked bread. when the pennsylvania government passed a new state requirement that all teachers had to be certified and had to meet minimum educational require- ments, which the amish teachers in their one- room schoolhouses couldn't do, the amish got around the regulation by declaring that all their teachers were substitutes, and thus exempt from the regulation. the rural amish people have little concern or interest in these pressures, counterpressures, and maneuverings - they be- lieve in nonresistance and, if necessary, mi- gration to avoid problems. even their leaders do not frame their advocacy in the terms of the out- siders: rather, they see their activity as 'working things out', being helpful in resolving issues, and liberating officials from their constant need to obey rules (kidder & hostetler, ). while their strategies are not precisely the same as those of the tristan islanders, the similarities are striking. conflicts with outsiders are thus resolved by peaceful peoples in a variety of ways, but the conclusion from these examples is that armies, killing, or other forms of violence are never part of their thinking, as they are to the rest of the world. the non-western peaceful peoples like- wise, such as the yanadi (raghaviah, ), try to resolve their conflicts with outsiders in fashions that are consistent with their overall commitments to peacefulness. the cumulative story is thus of peaceful peoples resolving con- this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp bruce d. bonta flicts with larger, more aggressive societies through meekness, active involvement, and at- tempts to resolve difficulties peacefully - in complete accord with their world-views. . conflict is best managed through political structures boulding sees conflict management as extend- ing from the family to the tribe, to the nation, to the superpower, to the evolving world govern- ment. 'conflict control is government', he writes, 'and though government has broader functions than this, conflict control is perhaps its most important single task' (boulding, , p. ). the literature about the peaceful peoples suggests that avoiding governments may also provide a viable model for peacefully resolving conflicts. in many traditional societies, people avoid calling in outside authorities and try to settle their internal conflicts themselves (just, ; nader, ). outside police are to be avoided if at all possible. the peaceful societies likewise try to keep their conflicts to themselves. the idea that an outside government or political structure is an essential part of solving their con- flicts, or would even be helpful in such situ- ations, would be alien to these peoples. they see government agencies as highly threatening and they avoid such outsiders as much as possible during conflicts, though there are some excep- tions (hollan & wellenkamp, ). for instance, a peaceful zapotec town voids having government officials involved in the af- fairs of their community since people feel that they would be treated much as any other mexican town - and they are convinced that their town is different from the rest in its oppo- sition to violence (paddock, ). likewise, nubian communities don't reveal serious prob- lems to outsiders, particularly to authorities such as the egyptian police; they feel that the best chance for their villages to survive is to be ignored by authorities (fernea, ). the amish also settle their conflicts within; an in- stance where an amish man sued his own church officials in court because he was ostra- cized (gruter, ) was exceedingly unusual. in fact, none of the peaceful peoples included in the group of , to judge by the available litera- ture, appear to rely on intervention by outside agencies of any kind, with the possible excep- tion of the mennonites, many of whom today no longer feel the strict need to remain absolutely separate from all government functions, par- ticularly in canada (driedger & kraybill, ). in the peaceful societies, conflicts are handled by the individual parties to the conflict and by the group - rarely by outsiders. individuals are expected to deal with conflict situations by walking away from them, by laughing them off, by displacing their feelings of anger in various ways, by smiling and being pleasant to every- one, by actively socializing with people with whom they may have unpleasant inner feelings, and so on. individuals should try to solve their problems internally if they can. when that doesn't work, the parties to a con- flict should resolve the issues between them- selves, or, more frequently, bring them to larger groups of people or authority figures within the society for discussion and resolution. but even group resolutions of conflicts, such as the semai becharaa', rely on the group to foster the dissi- pation of tensions so that individual, personal controls may keep the peace. none of these so- cieties rely on the power of people as a political body to enforce the peace, with the sole excep- tion of the threat of ostracism. but if the ulti- mate approach to resolving difficult conflicts for western peoples is outward, to the next larger political or governmental body, as boulding as- serts, the ultimate focus for the peaceful peoples (and many other traditional societies) is inward, towards individuals and the group. . world-view of conflict resolution 'conflict is ... inevitable in human life .... eliminating conflict is clearly impossible, and likely undesirable, because of the close link be- tween conflict and creative, constructive change' (augsburger, , pp. , ). two decades earlier, deutsch ( ) expressed simi- lar ideas, and popular writers often reflect this thinking: 'conflict is a necessary part of every marriage.... if there is no conflict... it is a sign that something is wrong with the marriage' (warren, ). other scholars (e.g., ross, b; nader, ), though not necessarily so enthusiastic about conflict as those writers, con- sider it simply a cultural behavior, and as such this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp conflict resolution among peaceful societies not to be judged desirable or undesirable. these ideas reflect the predominant world-views of western societies that proclaim the ideology of love, peace, cooperation, and generosity, but ac- cept conflict, aggression, competition, and viol- ence as inevitable aspects of human nature and human societies. conflict resolution, in this view, is just a process - a strategy or series of strategies for settling disputes. such attitudes toward conflict would not be shared by peaceful peoples. while many of them would recognize that conflict is a problem at times in their societies, none would see it as beneficial. the purpose of this section is to look at the (mostly) positive ways these peaceful so- cieties view their lack of conflict - their world- views of peacefulness - and to compare those views with the thinking of western writers. to start with some western thinking, scholars using cross-cultural data have sought to explain the phenomenon of conflict and conflict resol- ution based on either the structural factors in so- cieties or on psychological/cultural elements (ross a, b). the social structural analysis concentrates on economic, political and social organization as the source of conflict; the psy- chocultural approach focuses on deep-seated 'we-they' conceptions of human opposition. the former argues that stronger ties, such as kinship, will reduce conflicts, while the latter sees ambiguity in social actions, and thus tries to explain why some disputes are far more in- tense than others. based on his own extensive cross-cultural analysis, ross feels that both have validity: psychocultural factors may determine the intensity of a conflict, while structural fac- tors may point out the targets of hostile actions and the ways conflicts are organized. he argues that low-conflict societies are characterized by both a psychocultural atmosphere of warmth and affection and cross-cutting social structures (ross, a). these arguments, and the impressive amount of cross-cultural data assembled, make a lot of sense but are not completely supported by the literature on peaceful societies. ross's descrip- tion ( a, pp. - ) of the strong sense of interpersonal trust that exists in low-conflict so- cieties, with a corresponding lack of fear of iso- lation and abandonment, is contradicted by briggs's ( , a, , ) writings on peaceful inuit groups, lutz's ( , ) descriptions of the ifaluk, wikan's ( ) work on the balinese, and other writings about non- violent peoples. these societies try to eliminate expressions of anger and aggression by devel- oping fears, anxieties, and uncertainties in chil- dren about other people. if others are not to be depended on to love them, if affection and sup- port can never be taken for granted, the children internalize a constant need to live up to the society's peaceful values. aside from that, ross's theory of the culture of conflict is impressive, but his bias is similar to other western thinkers - that conflict is in- evitable, though it can be managed better. his choice of terminology reflects his thinking: he frequently refers to 'the culture of conflict', the title of one of his works ( b); yet nowhere in either volume does he use the phrase, 'the cul- ture of low conflict'. conversely, he refers to a group of five peaceful societies as 'low-conflict societies', but he does not refer to other, more violent, peoples as 'high conflict' or even 'nor- mal conflict' societies. conflict is normative, in this view, while the lack of conflict is the ex- ception. of course, the literature on a wide range of peoples, such as ross has studied, does show that conflict is normative, and 'low-con- flict' societies are the exception. but - and this is the critical point - viewed from within the literature of the peaceful societies, from the per- spective of those peoples, the 'high conflict' so- cieties are the ones that vary from their norm. perhaps this alternative norm should be called 'the culture of peacefulness', or as unesco has designated one of its new programs, the culture of peace (mayor, ). conflict resolution among the peaceful so- cieties, their culture of peacefulness, is based on more than psychocultural and social structures: just as significant are their world-views of peacefulness. gregor ( ) touches on this when he points out that the ideologies and sym- bolic values that societies hold to are also criti- cal elements in providing the basis of a peaceful (or a violent) society. deutsch ( ) makes the same point in his so-called 'crude law of social relations', namely that 'the characteristic pro- cesses and effects elicited by a given type of social relationship (e.g., cooperative or competi- tive) also tend to elicit that type of social relationship'. in other words, cooperation breeds cooperation, competition breeds compe- this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp bruce d. bonta tition. likewise, howell and willis ( b, p. ), introducing the peaceful societies included in their anthology, conclude that these peoples all place an emphasis 'on peaceful interaction among the members of the society, and this emphasis is cosmologically constructed and morally embedded in a cosmological universe of meaning'. the literature on the societies considered for this essay shows that their peace- ful conflict resolution practices are fostered by their beliefs in peacefulness, which are in turn bolstered by the successful practices. to a western analyst, 'the goal of conflict resolution is to shape new political and social arrange- ments . . . ' (kelman, , p. xi). to the members of these peaceful societies, the goal of conflict resolution is to maintain social harmony through traditional means of prompting indi- viduals to remember and act on their shared beliefs. the basic reason for peacefulness in these so- cieties is that the people are strongly opposed to actual physical violence and firmly in favor of nonviolence, in contrast to neighboring, and sometimes very similar, communities that may only pay lip service to the ideals of peace and are, in actual practice, far more violent. the peaceful peoples not only believe fervently in their world-views of nonviolence: in general, they have internalized those beliefs and adhere to them very strictly, using primarily internal controls to prevent and resolve conflicts, as has been discussed earlier. in other societies that claim they have nonviolent values, but have not really internalized them, people rely primarily on external controls for preventing and resolv- ing conflicts. for instance, several scholars have written about a zapotec town in the state of oaxaca, mexico (dubbed 'la paz'), which is much more peaceful, and experiences a lot less violence, than other nearby towns. in la paz, violence is never acceptable: people avoid problems with others, deny that they have interpersonal diffi- culties, and refuse to fight. by way of contrast, in a nearby, more violent community, even though the people talk about themselves as having a peaceful town, they rationalize that sometimes humans get violent and sometimes fighting is understandable, particularly if pro- voked by alcohol or sexual jealousies (fry, ). the semai, as indicated at the beginning of this essay, emphasize and re-emphasize their shared value of peacefulness (robarchek, ). other peoples are highly conscious of, or take active pride in, their peacefulness as the defining characteristic of their societies, e.g., the paliyan (gardner, ), nubians (fernea, ), toraja (hollan, ), mennonites (driedger & kraybill, ), malapandaram (morris, ), tristan islanders (loudon, ), and so on. even in those peaceful so- cieties in which people fear their violent nature (as they conceive it), such as the inuit (briggs, b), strongly held values promote their non- violence. in addition, the point made at the beginning of this essay can't be emphasized too strongly, that the peacefulness of these societies is not based on utopian thinking. people such as the semai do not conceive of nonviolence as an ideal they should strive for; rather, they think of themselves as nonviolent. according to dentan ( b, p. ) the semai would not describe anger as bad in the abstract; instead they would say, 'we do not get angry'. the practice of non- violence of these peoples combines their world- views of peace with a very realistic, pragmatic understanding of the results of violence (thomas, ). for instance, the anabaptist societies and the tristan islanders see a con- stant, practical benefit to themselves in main- taining their meek, non-confrontational, peace- ful relationships with each other and with outsiders. the literature on the peoples who live on the fringes of indian society - the ladakhis, paliyan, malapandaram, birhor, and yanadi - emphasizes the practical ways their economic and social structures are integrated with their peacefulness. to sum up this section, the peaceful peoples are intolerant of internal strife; they do not rationalize conflict and would not accept the possibility that violence is excusable in some circumstances. few individuals in these so- cieties would admit that, while they know they should be peaceful, sometimes they just have to use violence - that's the way humanity is. to them, other peoples are obviously violent, ag- gressive, and filled with conflicts and warfare; but they themselves are peaceful and highly conscious of it. peacefulness is an absolute commitment for them. most of their social, reli- gious, mythical, cultural, psychological, and this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp conflict resolution anmong peaceful societies educational beliefs are derived from this world- view of their own peacefulness. . conclusion attitudes about conflict and approaches to con- flict resolution among of the earth's peaceful peoples differ from those of the other societies on earth. personal development and social life in the peaceful societies is based on striving for - and achieving - an absence of conflicts and violence. people in most of these societies do not view conflict as normal and productive, as westerners often do; they view it as harmful and destructive. they avoid all types of conflicts if they possibly can, and if they can't they almost always resolve them quickly and nonviolently. while these peoples resolve conflicts by using techniques that other societies also use, they emphasize certain strategies in unique ways. for instance, direct negotiation between the parties to a dispute, an important approach in western societies, is not used too often by the peaceful peoples. instead of negotiating, most of them rely on self-restraint to prevent conflicts and to help people settle the disputes that do arise. people in many of the peaceful societies prefer to avoid controversy, to walk away from conflicts, to separate families or communities in order to circumvent hostilities. one-on-one negotiation is too confrontational for many of these peoples. also, bystanders in several of the peaceful societies will intervene enthusiastically to help resolve conflicts - a contrast to modem urban areas where strangers often fear getting involved in confrontations. a number of the peaceful societies depend on community meetings as a technique to help settle disputes, while western societies settle conflicts by relying on formal court trials to de- termine guilt or innocence, right from wrong. the peaceful community meetings exemplify the importance of preventing and resolving con- flicts, while the western belief in trials is founded on abstract conceptions of justice. also, angry individuals in several peaceful so- cieties may talk out problems without specifi- cally addressing other people - a rhetorical discussion of grievances with the community at large which does not directly confront the other parties to the problems. to repeat, in these so- cieties avoiding conflict is more important than confronting it, and resolving disputes by what- ever nonviolent means possible is the highest goal of society. however, conflict resolution in the peaceful societies relies on more than just strategies and techniques. it is based on assumptions about human relations and social patterns that are quite different from those of modern societies. for instance, people in the peaceful societies strongly believe they should avoid, and if they can't avoid then they should quickly resolve, all conflicts. they view nonviolence as absolutely essential to the proper functioning of their so- cieties. in contrast, western social scientists and popular writers believe that conflict is an in- evitable, and to some extent productive, aspect of human societies which we must learn to manage effectively. the peaceful peoples settle conflicts with outsiders by using nonviolent strategies which are quite comparable to the techniques they use for resolving internal dis- putes. western societies, in general, view force and violence as a necessary, and at times justifi- able, aspect of external relations. other ways that the peaceful societies con- trast with the rest of the world are that they do not punish those who violate social norms, ex- cept for the occasional use of ostracism; and they place very little reliance on political struc- tures larger than their own communities for achieving peace. the most peaceful of them have ideologies that encourage meekness and nonresisting behavior. most important of all, the peacefulness in these societies - and their suc- cess in resolving conflicts - is founded on world-views which include nonviolence as one of the defining characteristics of humanity. their world-views are not just ideology: they include, and integrate, psychological, social, re- ligious, and ethical structures that constantly re- inforce their shared beliefs in living peacefully. the natures of these structures, of course, vary widely among all the peaceful societies. how do the conflict-resolution strategies and beliefs of the peaceful peoples relate to the com- plex societies of today's world? on a practical level, professionals in the dispute resolution field might find some of the techniques used by these societies to be applicable at times, such as relying more on humor to defuse tensions, or placing more emphasis on building up individ- ual restraints on hostility in conflict situations. this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp bruce d. bonta but the peaceful societies exemplify a more basic lesson about resolving conflicts without violence. they demonstrate that peaceful con- flict resolution, in order to be an integral part of modern social life, must be based on a fervent commitment to nonviolence. if the examples of the peaceful peoples have any validity, nonviol- ence has to be accepted as one of the highest ideals, one of the most strongly accepted be- liefs, of today's societies. we can gain glimpses of a world which resolves conflicts nonviolently through the vision provided by the peaceful peoples: a vision of individuals who always pre- fer peaceful behavior over aggression, and who always avoid confrontation and conflict; a vision of societies which look to their widely varying ethical, religious, and social traditions to support world-views of peace; and a vision of humanity successfully building and reinforcing peaceful beliefs into nonviolent social lives. unesco has launched a comparable vision, a new culture of peace program, which seeks ways of building nonviolent world-views among nations (mayor, ): the peaceful societies discussed here should provide inspi- ration and support for unesco's work. the example of the peaceful societies cannot be extended too far - they do not provide clear answers to many of the complex issues of con- flict in today's world. the peaceful peoples do, however, provide a basis for understanding successful conflict resolution and they do in- spire a vision of a potentially peaceful world. arguments about the complexity of modem so- cieties (compared to the small-scale peaceful peoples) may try to justify conflict as inevitable, but these are rationalizations which fade under the vision of peacefulness provided by these peoples: that human societies can be peaceful, that people can build virtually fail-safe struc- tures for avoiding and resolving conflict, that punishments and armed conflicts are not es- sential for keeping the peace. the answer is for us to build, in our societies, world-views of peacefulness that are as strong as those of the peaceful peoples. this is the first step. notes . variation of the standard text of matthew . , used in some protestant churches such as the episcopal, methodist, congregational, and others. . i have served twice on criminal court juries, and i base this description of a pennsylvania trial on those experi- ences. . it would be ideal to include in the rest of this essay more descriptions of ethno-concepts such as the semai becharaa'; the analysis to be presented would be con- siderably enriched by looking at peacefulness primarily through the languages of the peoples themselves. unfortunately, that is not possible: there is not enough space to add the additional discussions, and many of the works about these societies are not enriched by that level of detail. . this is not meant to demean the work of researchers within the western tradition on war, conflict, and conflict resolution - much of it is immensely valuable. my point is to argue for a peaceful basis of understanding conflict. . the definitions of 'peacefulness' and 'people' or 'society' are taken from bonta ( ) with some updat- ing and modifications. . the present tense is used throughout this article for peo- ples discussed in the anthropological literature, even though the information may or may not be current; the past tense is used for references that are from the histori- cal literature. . i would define 'world-view' as a system of thoughts and emotions about individual, social, and spiritual life which includes the human actions guided by those thoughts and emotions, while 'ideology' is a system of beliefs which may or may not influence individual acts. . see bonta ( ) for a listing of the literature. . in some of these societies, on rare instances murderers or dangerously insane individuals have been killed by other members of their groups. the people evidently felt they had no other ways to handle these dangerous situations. references augsburger, david. w., . conflict mediation across cultures: pathway's and patterns. louisville, ky: westminster/john knox. bennett, john w., . hutterian brethren: the agricultural economy and social organization of a communal people. stanford, ca: stanford university press. bonta, bruce d., . peaceful peoples: an annotated bibliography. metuchen, nj: scarecrow. boulding, kenneth e., . conflict and defense: a general theory. new york: harper & row. briggs, jean l., . 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'work it out: the triumphant marriage', focus on the family, october, pp. - . wikan, unni, . managing turbulent hearts: a balinese formula for living. chicago, il: university of chicago press. appendix. twenty-four peaceful societies the following list provides a brief description of where and how each of the societies mentioned in this article lives. for a full bibliography of works describing the peacefulness of these societies, as well as the works of detractors who dis- cuss their violence, please consult bonta ( ). amish. over , amish live in canada and the united states, mostly on traditional family farms in the eastern states of pennsylvania, ohio, and indiana, though many are now engaging in small business enterprises. balinese. over two million people, most of whom practice hindu beliefs, live on the indonesian island of bali and work as either farmers or business people. batek. peoples of the mountainous malay peninsula who gather forest products for trade as well as hunt and gather their food. this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp bruce d. bonta birhor. a gathering and hunting people who also trade with other peoples in southern bihar state, east central india. buid. shifting agricultural peoples of the forested highlands of mindoro island in the philippines. chewong. a hunting and gathering people of the mountain- ous interior, peninsular malaysia, who also undertake some swidden agriculture. g/wi. a san people of central botswana, southern africa, who used to live as hunting and gathering nomads in the kalahari desert, but who now mostly work as hired la- borers on ranches. hutterites. an anabaptist people who live in colonies scat- tered across the plains of rural north central united states and central canada. ifaluk. a fishing and agricultural people who live on a pacific atoll in the federated states of micronesia, near the large island of yap. inuit. the anthropologist jean briggs has written many arti- cles and monographs about the strategies that two differ- ent inuit groups, one in the central canadian arctic and the other on baffin island, use to control anger and pre- vent violence from occurring. these peoples traditionally survived on fishing and hunting, though they now are part of the cash economy. !kung. one of the most studied of traditional societies, the !kung, a so-called san people, live in the boundary area of botswana and namibia, in southern africa. traditionally they were nomadic gatherers and hunters. ladakhis. buddhist agricultural and pastoral people who live south of the karakoram range in the northwest cor- ner of india, the state of jammu and kashmir. malapandaram. the malapandaram, or hill pandaram, live in the hills at the southern end of the western ghats in india. mennonites. nearly , mennonites live today in canada and the united states, some as traditional farmers who live without much technology, much as the amish do, and others in quite contemporary businesses, trades, and professional positions. montagnais-naskapi. an indian society of the labrador peninsula, eastern canada, the montagnais-naskapi live on trapping, trading, hunting, gathering and seasonal em- ployment. nubians. before the completion of the aswan high dam in upper egypt in the s, the nubian people lived in tra- ditional farming villages along the nile river, though many of the men had to leave for periods of time to work in cities to the north. paliyan. a gathering people who live in a range of hills at the southern end of the western ghats of india. semai. the semai, people of the mountainous malay peninsula, live (or formerly lived) primarily on their hunt- ing, fishing, gathering, and swidden agriculture. tahitians. residents of the society islands, part of french polynesia in the central pacific, live off their gardening, fishing, trade and business pursuits. temiar. the temiar, primarily agricultural peoples who do some hunting and gathering, live in permanent villages in longhouses built, in the past, for defense from the malay slave raiding. toraja. several hundred thousand toraja, most of whom have converted to christianity, live primarily by farming in the mountains of south sulawesi, in indonesia. tristan islanders. a small population of mixed european and either african or south east asian ancestry who have lived on the isolated island of tristan da cunha, a british dependency in the south atlantic, since the early nine- teenth century. these people have traditionally engaged in fishing, gathering, and agriculture, though in recent decades they have also had cash income from a fishing factory and tourists. yanadi. several hundred thousand yanadi live mostly in the eastern coastal areas of india, where they engage in gath- ering, work for wages, and subsistence hunting. zapotec. while the zapotec, an indigenous agricultural people of oaxaca state, southern mexico, may not be ex- ceptionally peaceful, a few highly nonviolent communi- ties not too far from the city of oaxaca have been studied by a succession of social scientists. bruce d. bonta, b. , mls in library science (university of maine, ); reference librarian at the library of congress, colby college (waterville, me), and the pennsylvania state university (university park, pa), history/area studies librarian, pennsylvania state ( - ); author of peaceful peoples: an annotated bibliography (scarecrow press, ). this content downloaded from . . . on thu, aug : : pm all use subject to jstor terms and conditions http://www.jstor.org/page/info/about/policies/terms.jsp 	article contents 	p.[ ] 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	p. 	issue table of contents 	journal of peace research, vol. , no. (nov., ), pp. - 	volume information [pp. - ] 	front matter [pp. - ] 	focus on 	antinomies of postmodernism in international studies [pp. - ] 	united states military intervention and the promotion of democracy [pp. - ] 	conflict resolution among peaceful societies: the culture of peacefulness [pp. - ] 	when the individual soldier says 'no' to war: a look at selective refusal during the intifada [pp. - ] 	explaining territorial disputes: from power politics to normative reasons [pp. - ] 	the nordic area as a 'zone of peace' [pp. - ] 	when officers need internal enemies: aspects of civil-military relations in scandinavia between the world wars [pp. - ] 	the political economy of defense spending in south korea [pp. - ] 	review essay 	war termination [pp. - ] 	book notes 	untitled [p. ] 	untitled [p. ] 	untitled [pp. - ] 	untitled [p. ] 	untitled [pp. - ] 	untitled [p. ] 	untitled [p. ] 	untitled [pp. - ] 	untitled [p. ] 	untitled [p. ] 	untitled [pp. - ] 	untitled [p. ] 	untitled [p. ] 	untitled [pp. - ] 	untitled [p. ] 	books received [pp. - ] 	back matter [pp. - ] science magazine april vol science www.sciencemag.org five years ago, a team headed by jeffrey gordon of washington university in st. louis (wustl) in missouri made a surpris- ing discovery: the guts of obese mice and people harbor an array of microbes different from that of their lean counterparts. more provocatively, when they gave lean mice certain gut-dwelling microbes, the rodents became fat (science, may , p. ). the fi ndings sparked headlines and fueled popular speculation that manipulating gut bacteria might keep weight down in people. already, martin blaser had been head- ing down a similar track. blaser, a microbi- ologist at new york university in new york city, was struck by how successful farmers are at increasing the growth rates of livestock by adding low doses of antibiotics to their feed. “the earlier in life they start the antibi- otic, the more profound the effect,” he points out. he began to wonder whether antibiotic use, particularly in children, might affect the long-term establishment of a balanced micro- bial community in the human gut, eliminat- ing bacteria there that could help ward off obesity. he started conducting mouse studies to examine the hypothesis. since then, several other groups have joined in. a raft of intriguing obesity- related fi ndings was presented at a meeting last month on the microbiome, the bacteria that live inside the guts and other tissues of animals. yet many in the fi eld caution that it remains diff icult to determine whether changes in gut microbes drive or contribute to obesity or whether the excess weight itself triggers those changes. “the jury is still out [about] what the role of the gut microbiota may be in obesity in humans,” says claire fraser-liggett, a microbiologist at the uni- versity of maryland school of medicine in baltimore who has studied gut bacteria and obesity in the amish. case of the missing microbes the farm animal–antibiotic connection was one clue that led blaser to wonder about microbial causes of the obesity epi- demic. another was the fact that very few people now harbor the ulcer-causing bacte- rium helicobacter pylori in their stomachs. h. pylori, which has also been linked to stomach cancers, is one of up to differ- ent microbes that call the human body home. once ubiquitous in the human microbiome and still so in the guts of people from devel- oping countries, it is now found in just % of u.s. children. that might seem like good news, as there should be fewer ulcers and cancers. but blaser suspects that it is also bad news, as studies suggest that h. pylori’s presence in the gut helps regulate the stom- ach’s production of the hormone ghrelin, which stimulates food intake. this bacterium may not be the only species disappearing from our microbiome. after a person takes antibiotics, “it has always been pre- sumed that the micro- biota will spring back,” blaser says. but the fate of h. pylori suggests otherwise. its vanishing act and other shifts in the microbiome may con- tribute to an increased risk for weight gain, blaser worries. he has started to investigate this theory by giving mice either low doses of antibiotics over long periods, akin to what farm ani- mals receive, or short-term, high doses, more like what a sick infant or adult would get. he then com- pares the physiology and microbi- omes of these treated rodents with those of mice raised under similar conditions but given no antibiot- ics. in one set of studies, the mice fed low doses of antibiotics long- term wound up with % more body fat than the control mice, blaser reported last month at the international human microbiome congress in vancouver, canada. the chubbier, antibiotic-fed mice also had about % more fat in their livers. the treated mice also had a different set of bacterial species inhabiting their guts. and several hundred bacterial genes, includ- ing ones for fatty acid production, exhibited different levels of activity—some increas- ing, others decreasing—in these mice com- pared with the controls. similar changes occur in the rodents given short pulses of antibiotics, he noted. antibiotics “may be driving the gut microbiome to a place where it shouldn’t be,” fraser-liggett says. “we do not know the functional consequences, but with these miracle drugs now years later, we may be seeing effects that change susceptibility to various diseases.” blaser will examine the gut microbiomes of children to see whether his results are applicable to humans. if so, “that would be a remarkable connection that could have a sig- nifi cant impact on medical care,” says genome scientist george weinstock of wustl. but he’s cautious: “in a lot of cases, the micro- biome in mice doesn’t translate into humans.” patterns in genes s. dusko ehrlich has avoided that issue, bypassing mice and instead directly exam- ining whether patterns in the microbiome of people relate to body mass index and obesity. a microbiologist at the inra microbiology and food chain divi- sion in jouy-en-josas, france, ehrlich is part of a group, the meta- newsfocus c r e d it s ( t o p t o b o t t o m ): r a d e l u k o v ic /t h in k s t o c k ; j a s o n h e go with the gut. antibiotic use may adversely affect the long-term makeup of the intestine’s bacterial communities. girth and the gut (bacteria) mouse, human studies begin to clarify gut bacteria’s role in obesitymouse, human studies begin to clarify gut bacteria’s role in obesity m i c r o b i o l o g y published by aaas o n j a n u a ry , w w w .s ci e n ce m a g .o rg d o w n lo a d e d f ro m http://www.sciencemag.org/ www.sciencemag.org science vol april newsfocus c r e d it : l a u r a k y r o / c e n t e r f o r g e n o m e s c ie n c e s & s y s t e m s b io l o g y hit consortium, investigating connections between microbial genes in human intestines and human health. by comparing such genes from obese and nonobese individuals, he and his colleagues have found that certain sets of bacterial genes and bacteria correlate with excess weight and insulin resistance. the researchers fi rst sequenced all the bac- terial genes in stool samples of danes, who were thin and who were either over- weight or obese. although the researchers concluded that most participants in the study had roughly , distinct bacterial genes in their guts, almost one-third of the obese study participants had only about , such genes, % to % fewer. a similar per- centage of obese french people had a com- parable dearth of gut bacteria genes, ehrlich reported at the vancouver meeting. moreover, the obese people “don’t have as great a bac- terial diversity” in their guts, ehrlich reported. one missing microbe in that group was a methane pro- ducer, leading ehrlich to wonder whether “the car- bon that does not get out [of the body] as gas could be incorporated as fat.” when they looked at medical histories of all their study subjects, ehrlich and his colleagues found that the obese peo- ple with fewer gut bacte- ria genes were more likely to be insulin resistant than were the obese people who had a typical tally of intestinal microbial genes. these obese people also tended to have higher than normal white blood cell counts, suggesting that they were in a state of low-level infl am- mation, ehrlich said. some researchers have found evidence of a link between inflam- mation and obesity (science, december , p. ). ehrlich and his colleagues have also tested whether the types of bacteria in a person’s gut can “diagnose” obesity. using just six meta- species, they were able to correctly predict whether a person was lean or obese more than % of the time, he reported. when research- ers try to make the same predictions by con- sidering all of a person’s genetic risk factors for obesity, they are right only % of the time, ehrlich pointed out. at this point, however, it’s unclear whether the differences in intestinal microbes are “the cause, a contribution to, or the consequence” of obesity, notes ehrlich. “if we can provide evidence that they [at least] provide a contri- bution, then we can go and fi nd a treatment.” help from the amish other work presented at the microbiome meeting indicates that sorting out this cause- and-effect puzzle will be tough. frustrated by the inconsistent results others were getting when they looked for connections between the microbiome and obesity, fraser-liggett and her colleagues examined adult amish liv- ing in pennsylvania. amish marry within their group and have very similar lifestyles, envi- ronment, and eating habits—they even cook in communal kitchens. thus, fraser-liggett hoped to eliminate some of the variables that might have confounded other studies. the body mass index of amish ranged from to ( is obese), and some of the obese ones also had metabolic syndrome. fraser-liggett and her colleagues captured a snapshot of the gut microbiome of each amish by obtaining stool samples, sequenc- ing the dna in them, and using the s ribo- somal subunit gene often used to tell bacteria apart, identifying any microbial components. although the scientists did detect some dif- ferences in certain bacteria between obese and lean amish, they didn’t fi nd the dramatic shifts that gordon had documented between lean and obese mice, fraser-liggett reported at the meeting. one problem may be that simply taking a census of the bacteria present in a person’s gut may not be enough. “ s [analysis] is not very informative,” ehrlich says. “we need to go to more precise measures.” increasingly, micro- biome researchers are looking at what bacte- rial genes are active in a person and not just at which bacteria are there. scientists have found that although the species mix of the micro- biome may vary signifi cantly from one person to the next, those individuals often still have equivalent complements of bacterial genes at work inside them. through such gene analy- ses, researchers can begin to better assess what the bacteria in the gut are really doing to, or for, their host, fraser-liggett notes. gordon suggests that more clarity on the obesity-microbiome issue will also come from using the guts of mice as bioreactors for human microbes. his group has pio- neered the study of germ-free mice, which are grown in a sterile environment from birth, and he is now exposing such mice to bacteria from human guts. once those human microbes have established residence in the guts of the mice, gordon then feeds the animals a variety of human diets. using these rodent proxies, he can thus track how different diets affect the “human” microbiomes, assessing the bacteria as often as he needs to to get a dynamic picture. “you can sample many features between the microbial community and the host,” he says. despite his role in ignit- ing the study of obesity and microbiomes, gordon resists the idea that our gut bacteria are the sole expla- nation for the growing number of obese people. “it’s not the dominant part of the problem; excessive energy intake is,” he con- tends. the simplistic notion of only chang- ing one’s gut bacteria to lose weight has been “hyped a lot,” he complains. others, such as microbial ecologist lip- ing zhao of shanghai jiao tong university in china, are more convinced that the micro- biome will prove important when it comes to obesity. “increased abundance of ‘bad genes’ and [a] decrease of ‘good genes’ in our diet-disrupted gut microbiome [may] be the primary driving force for this obesity epidemic,” zhao says. even if zhao’s prediction proves right, the studies to date make clear that the con- nection between the microbiome and excess weight is complex, fraser-liggett says. for those looking to bacteria to stem the obesity epidemic, she concludes, “there’s clearly no magic formula.” –elizabeth pennisi personalized microbiota. germ-free mice grown in sterile environments and given human gut microbes provide a way to test links between bacteria and obesity. published by aaas o n j a n u a ry , w w w .s ci e n ce m a g .o rg d o w n lo a d e d f ro m http://www.sciencemag.org/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ ellis-van creveld syndrome: prenatal diagnosis, molecular analysis and genetic counseling taiwan j obstet gynecol • december • vol • no ■ short communication ■ introduction ellis-van creveld (evc) syndrome (omim ), or chondroectodermal dysplasia, is an autosomal reces- sive ciliary disorder associated with a wide spectrum of developing abnormalities involving the ectoderm, skeleton and heart. evc syndrome is a relatively rare ellis-van creveld syndrome: prenatal diagnosis, molecular analysis and genetic counseling chih-ping chen , , , , , *, yi-ning su , chin-yuan hsu , schu-rern chern , fuu-jen tsai , , pei-chen wu , po-tsang chen , wayseen wang , departments of obstetrics and gynecology and medical research, mackay memorial hospital, institute of clinical and community health nursing, department of obstetrics and gynecology, national yang-ming university, department of medical genetics, national taiwan university hospital, and department of bioengineering, tatung university, taipei; department of biotechnology, asia university, school of chinese medicine, college of chinese medicine, china medical university, and departments of medical genetics and medical research, china medical university hospital, taichung, taiwan. summary objective: to present the perinatal findings and molecular genetic analysis of two siblings with ellis-van creveld (evc) syndrome. materials, methods and results: a -year-old woman, gravida , para , was referred for genetic counseling at gestational weeks because of recurrent fetal skeletal dysplasia. two years previously, she had delivered a , -g dead male baby at gestational weeks with a karyotype of ,xy, postaxial polydactyly of the hands, thoracic narrowness, endocardial cushion defects, transposition of the great arteries, shortening of the long bones, malposition of the toes, and hypoplastic nails. during this pregnancy, prenatal ultrasound at gesta- tional weeks revealed shortening of the long bones (equivalent to weeks), postaxial polydactyly of both hands, thoracic narrowness, and endocardial cushion defects. the pregnancy was subsequently terminated, and a -g female fetus was delivered with a karyotype of ,xx, postaxial polydactyly of the hands, thoracic dysplasia, endocardial cushion defects, shortening of the long bones, and malposition of the toes and hypoplastic nails. the phenotype of each of the two siblings was consistent with evc syndrome. molecular analysis of the evc and evc genes revealed heterozygous mutations in the evc gene. a heterozygous deletion mutation of a -bp deletion of c. - _ del encompassing the junction between intron and exon of the evc gene was found in the mother and two siblings, and a heterozygous nonsense mutation of c. c > t, p.r x in exon of the evc gene was found in the father and two siblings. conclusion: prenatal sonographic identification of endocardial cushion defects in association with shortening of the long bones should alert clinicians to the possibility of evc syndrome and prompt a careful search of hexa- dactyly of the hands. molecular analysis of the evc and evc genes is helpful in genetic counseling in cases with prenatally detected postaxial polydactyly, thoracic narrowness, short limbs and endocardial cushion defects. [taiwan j obstet gynecol ; ( ): – ] key words: ellis-van creveld syndrome, evc, evc , prenatal diagnosis, ultrasound *correspondence to: dr chih-ping chen, department of obstetrics and gynecology, mackay memorial hospital, , section , chung-shan north road, taipei, taiwan. e-mail: cpc_mmh@yahoo.com accepted: september , disorder, but is most prevalent in the amish popula- tion [ , ] and in some arab populations [ ] because of consanguinity. the birth prevalence is estimated to be . per , of live births in the non-amish population [ ], . per , of live births in the united arab emirates [ ], and per , of live births in the amish of lancaster county, pennsylvania, usa [ ]. evc syndrome is characterized by short ribs, short limbs, postaxial polydactyly of the hands, poly- dactyly of the feet (in % of cases), ectodermal dys- plasia such as dysplastic nails and teeth, sparse hair and an absent gingival sulcus, and congenital heart defects (in % of cases) such as a common atrium, atrioventricular septal defects (avsds) and patent ductus arteriosus [ , ]. evc syndrome is caused by mutations in the evc gene (omim ) [ ] or evc gene (omim ) [ ] that encodes cilia- related proteins evc or evc , respectively. mutations in the evc gene or evc gene may also cause weyers acrodental dysostosis (omim ), an autosomal dominant disorder characterized by postaxial poly- dactyly and abnormalities of the lower jaw, dentition taiwan j obstet gynecol • december • vol • no c.p. chen, et al figure . whole body x-ray of proband at gestational weeks. a b figure . postaxial polydactyly of the hands in proband . and oral vestibule. the evc and evc proteins are localized in the basal bodies of primary cilia. evc is a basal body component of hedgehog signaling indis- pensable for normal endochondral growth and normal transcriptional activation of indian hedgehog-regulated genes [ ]. both evc syndrome and weyers acroden- tal dysostosis are caused by hedgehog signaling de- fects in the primary cilia due to mutations in the cilia-related proteins resulting in an aberrant response to the hedgehog ligands [ ]. we previously reported perinatal findings of hexadactyly-associated ciliary dis- orders of meckel syndrome [ ], joubert syndrome [ ], and short rib-polydactyly syndrome (srps) [ – ]. we present the perinatal findings and molecular genetic analysis of two siblings affected by hexadactyly and evc syndrome. materials, methods and results a -year-old woman, gravida , para , was referred for genetic counseling at gestational weeks because of recurrent fetal skeletal dysplasia. she and her hus- band were non-consanguineous. she had experienced one spontaneous abortion and delivered a baby with skeletal dysplasia. two years previously, she had deliv- ered a , -g dead male baby (proband ) at ges- tational weeks with a karyotype of ,xy, postaxial polydactyly of the hands, thoracic narrowness, endo- cardial cushion defects, transposition of the great arteries (tga), shortening of the long bones, malposi- tion of the toes and hypoplastic nails (figures – ). during this pregnancy, prenatal ultrasound at ges- tational weeks revealed shortening of the long bones (equivalent to weeks), postaxial polydactyly of both hands, thoracic narrowness, and endocardial cushion defects (figures – ). the pregnancy was subsequently terminated, and a -g female fetus (proband ) was delivered with a karyotype of ,xx, postaxial poly- dactyly of the hands, thoracic dysplasia, endocardial a b figure . malposition of the toes with hypoplastic nails in proband . cushion defects, shortening of the long bones, malpo- sition of the toes and hypoplastic nails (figures – ). the phenotype of each of the two siblings is consistent with evc syndrome. molecular analysis of the evc and evc genes showed heterozygous mutations in the evc gene. a heterozygous deletion mutation of a - bp deletion of c. - _ del encompassing the junction between intron and exon of the evc gene was found in the mother and two siblings, and a het- erozygous nonsense mutation of c. c > t, p.r x taiwan j obstet gynecol • december • vol • no ellis-van creveld syndrome figure . prenatal ultrasound shows thoracic narrowness in proband . figure . prenatal ultrasound shows hexadactyly of the hands in proband . figure . prenatal ultrasound shows endocardial cushion defects in proband . figure . whole body x-ray of proband at gestational weeks. figure . proband at birth. a b figure . postaxial polydactyly of the hands in proband . in exon of the evc gene was found in the father and two siblings (figure ). discussion the present case prenatally manifested shortening of the long bones, thoracic dysplasia, hexadactyly of the hands, and avsd on the second-trimester ultrasound. prenatal diagnosis of evc syndrome by direct visualiza- tion of the fetus using fetoscopy has previously been reported [ , ]. first-trimester transabdominal embry- ofetoscopy for the detection of limb or facial abnor- malities has recently been applied to detect skeletal dysplasia such as srps [ ]. recurrent evc syndrome can be diagnosed in the first trimester by the ultra- sound findings of avsd, polydactyly and short limbs [ ] as well as increased fetal nuchal translucency thickness [ ]. in the second trimester, the diagnosis of evc syndrome can be made based on a positive family history and the ultrasound findings of shortness of the long bones, hexadactyly of the hands, a narrow thorax and congenital heart defects, especially abnor- malities of atrial septation and avsd [ – ]. our first proband manifested avsd and tga, and our second proband manifested avsd on prenatal ultrasound. congenital heart defects occur in approxi- mately % of patients with evc syndrome with most patients being variants of avsd [ ]. atrioventricular septation of the mammalian heart into four chambers requires sonic hedgehog signaling-dependent cellular contributions from the extracardiac tissues of the dor- sal mesocardium as well as contributions from the muscular and mesenchymal atrial septum and the endocardial cushions [ ]. sund et al [ ] found that the expression of evc and evc mrna and proteins taiwan j obstet gynecol • december • vol • no c.p. chen, et al figure . malposition of the toes with hypoplastic nails in proband . proband proband c. – _ delaggttctgccgcaccacggcctccac c. – _ delaggttctgccgcaccacggcctccac c. – _ delaggttctgccgcaccacggcctccac evc - evc - evc - mother father control wt wt wt e evc - evc - evc - evc - proband proband mother father control c. c > t c. c > t c. c > t e wt wt wt evc - evc - evc - evc - proband proband father mother figure . a heterozygous deletion mutation of the -bp deletion encompassing the junction between intron and exon of the evc gene (c. - _ del ) in proband , proband and the mother, but not in the father, and a heterozygous nonsense mutation in exon of the evc gene (c. c > t, cga > tga, arg stop, r x) in proband , proband and the father, but not in the mother. wt = wild type. was high in the outflow tract and dorsal mesenchymal protrusion and was also present in the mesenchymal structures of the atrial septum and the endocardial cushions. this finding suggested that evc and evc proteins function coordinately in cardiac development and that loss of this coordinate function results in characteristic evc syndrome. in the present case, we identified heterozygous evc mutations in the affected fetuses. the nonsense mutation of c t, r x, has been described pre- viously [ ]. however, the deletion mutation of c. - _ del is novel. sequencing the evc and evc genes has been reported to identify mutations in only two-thirds of evc patients, indicating the possibility of genetic heterogeneity in evc syndrome [ ]. in a study of individuals with evc syndrome, tompson et al [ ] identified evc mutations in cases ( %), all of whom had mutations on each allele, found evc muta- tions in cases ( %), of whom had mutations on each allele, and three had only one mutation, and there was no mutation in either gene in cases ( %). the three patients in their study with an evc mutation on only one allele had a frameshift or a nonsense codon and a more severe phenotype than weyers acrodental dysostosis. there is a risk of % recurrence in subsequent pregnancies in fetal evc syndrome. genetic counseling of fetal evc syndrome should include differential diag- noses of srps, jeune asphyxiating thoracic dystrophy (jatd), and mckusick-kaufman syndrome (mkks). srpss are a heterogeneous group of lethal autosomal recessive skeletal dysplasias. four types of srps have been recognized [ ]. type i srps (saldino-noonan) (omim ) is characterized by flipper-like extrem- ities, polydactyly, polycystic kidneys and pointed meta- physes. type ii srps (majewski) (omim ) is characterized by polydactyly, micromelia, cleft lip/palate, polycystic kidneys, a disproportionately short ovoid tibia and occasionally hypoplastic epiglottis and larynx. type iii srps (verma-naumoff) (omim ) is characterized by polydactyly, micromelia, metaphyseal spurs and occasionally situs inversus totalis. type iv srps (beemer-langer) (omim ) clinically resembles type ii srps other than polydactyly. overlapping clinical and radiological manifestations have led to the hypoth- esis that the different subtypes may be a single genetic disorder with variable expressivity [ – ]. type iii srps is caused by mutations of the dync h gene (omim ) [ , ]. jatd (omim ) is an autoso- mal recessive disorder characterized by thoracic dys- trophy, chondrodysplasia, short ribs, short long bones, inconstant polydactyly and a trident acetabular roof with occasional involvement of the liver, retinal degen- eration, and cystic renal disease. jatd is caused by muta- tions of the ift gene (omim ) and dync h gene [ – ]. jatd and type iii srps have been sug- gested to be variants of a single ciliary disorder [ ]. mkks (omim ) is an autosomal recessive dis- order characterized by polydactyly, congenital heart defects, hydrometrocolpos, and it clinically overlaps with bardet-biedl syndrome (omim ) comprising obesity, retinitis pigmentosa, polydactyly, mental retardation, renal malformation and genital hypoplasia. mkks and bardet-biedl syndrome can be caused by mutations of the mkks gene (omim ) [ , ]. in summary, we have presented prenatal diagnosis, molecular analysis and genetic counseling of recurrent evc syndrome. prenatal sonographic identification of en- docardial cushion defects in association with shortening of the long bones should alert clinicians to the possibil- ity of evc syndrome and prompt a careful search of hexa- dactyly of the hands. molecular analysis of the evc and evc genes is helpful in genetic counseling in cases with prenatally detected postaxial polydactyly, thoracic nar- rowness, short limbs, and endocardial cushion defects. acknowledgments this work was supported by research grants nsc- - -b- - -my and nsc- - -b- - -my from the 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biesecker lg. mutation of a gene encoding a putative chaperonin causes mckusick-kaufman syndrome. nat genet ; : – . taiwan j obstet gynecol • december • vol • no c.p. chen, et al deletion of a genomic segment containing the cardiac troponin i gene knocks down expression of the slow troponin t gene and impairs fatigue tolerance of diaphragm muscle* deletion of a genomic segment containing the cardiac troponin i gene knocks down expression of the slow troponin t gene and impairs fatigue tolerance of diaphragm muscle* received for publication, may , , and in revised form, september , published, jbc papers in press, september , , doi . /jbc.m . han-zhong feng, bin wei, and jian-ping jin from the department of physiology, wayne state university school of medicine, detroit, michigan the loss of slow skeletal muscle troponin t (tnt) results in a recessive nemaline myopathy in the amish featured with lethal respiratory failure. the genes encoding slow tnt and cardiac tro- ponin i (tni) are closely linked. ex vivo promoter analysis sug- gested that the �-enhancer region of the slow tnt gene overlaps with the structure of the upstream cardiac tni gene. using trans- genic expression of exogenous cardiac tni to rescue the postnatal lethality of a mouse line in which the entire cardiac tni gene was deleted, we investigated the effect of enhancer deletion on slow tntgeneexpressioninvivoandfunctionalconsequences.thelev- els of slow tnt mrna and protein were significantly reduced in the diaphragm muscle of adult double transgenic mice. the slow tnt-deficient (sstnt-kd) diaphragm muscle exhibited atrophy and decreased ratios of slow versus fast isoforms of tnt, tni, and myosin. consistent with the changes toward more fast myofila- mentcontents,sstnt-kddiaphragmmusclerequiredstimulation at higher frequency for optimal tetanic force production. the sstnt-kd diaphragm muscle also exhibited significantly reduced fatigue tolerance, showing faster and more declines of force with slower and less recovery from fatigue as compared with the wild type controls. the natural switch to more slow fiber contents dur- ing aging was partially blunted in the sstnt-kd skeletal muscle. the data demonstrated a critical role of slow tnt in diaphragm function and in the pathogenesis and pathophysiology of amish nemaline myopathy. troponin t (tnt) is a subunit of the troponin complex in striated muscles. through interactions with troponin c, tropo- nin i (tni), and tropomyosin, tnt anchors the troponin com- plex to actin thin filament and plays a central role in the ca � regulation of muscle contraction ( ). three tnt genes are pres- ent in the vertebrates: cardiac tnt, fast skeletal muscle tnt, and slow skeletal muscle tnt (sstnt). they encode three mus- cle fiber type-specific tnt isoforms with differentiated expres- sion and function ( ). a nonsense mutation at codon glu in the sstnt gene (tnnt ) causes a postnatal lethal form of inherited nemaline myopathy in the amish ( ). this mutation prematurely termi- nates the sstnt polypeptide, and the c-terminally truncated sstnt-( – ) has a much decreased binding affinity for tro- pomyosin and fails to incorporate into the myofilaments ( ). as a result, the truncated sstnt protein was degraded and unde- tectable in the muscle of amish nemaline myopathy patients, consistent with the recessive phenotype of the disease ( ). accordingly, the pathogenesis and muscle pathophysiology of amish nemaline myopathy is due to the loss of slow tnt. the genes encoding the muscle type-specific tni and tnt isoforms are linked in tandem in three pairs in the vertebrate genome ( ). the cardiac tni (ctni) (tnni ) and sstnt genes are very closely linked. shown in ex vivo promoter analysis, the � upstream enhancer region of the sstnt gene overlaps with the structure of the ctni gene ( ). this struc- tural integration of ctni and sstnt genes suggested that a previously developed mouse line in which the entire tnni gene was deleted through embryonic stem cell gene targeting ( ) would have lost a large portion of the �-enhancer region of the tnnt gene (fig. ). although the tnni -deleted mouse line is a potentially useful model to study the phenotypes of sstnt deficiency, a major hurdle is that the tnni -deleted mice die – days after birth from heart failure when the fetal expression of slow skeletal muscle tni in the heart ceases ( ). this postnatal lethality pre- cluded functional examination on adult skeletal muscle for the phenotypes of sstnt deficiency. however, adult skeletal mus- cle is necessary for investigating the pathophysiology of amish nemaline myopathy because the fetal and neonatal expression of cardiac tnt in skeletal muscles ( ) may functionally com- pensate for the loss of sstnt in ( ). we previously developed two transgenic mouse lines overexpressing an n-terminal truncated ctni (ctni-nd) driven by an �-myosin heavy chain (mhc) promoter ( ). ctni-nd was originally found as a product of restricted pro- teolysis in cardiac adaptation to simulated microgravity, which selectively removes the ctni-specific n-terminal extension and leaves the conserved structure intact ( ). ctni-nd transgenic mice exhibit normal base-line life activ- ity with an increased relaxation of the cardiac muscle ( ). using this postnatally up-regulated ( ) transgene allele encoding a non-destructive exogenous tni, we successfully * this work was supported, in whole or in part, by national institutes of health grants ar- and hl- (to j.-p. j.). to whom correspondence should be addressed: dept. of physiology, wayne state university school of medicine, e. canfield, detroit, mi . tel.: - - ; fax: - - ; e-mail: jjin@med.wayne.edu. the abbreviations used are: tnt, troponin t; tni, troponin i; ctni, cardiac tni; ctni-ko, ctni knockout; ctni-nd, n-terminal truncated ctni; mab, mono- clonal antibody; mhc, myosin heavy chain; pbs, phosphate-buffered saline; sstnt, slow skeletal muscle tnt; sstnt-kd, sstnt knockdown. the journal of biological chemistry vol. , no. , pp. – , november , © by the american society for biochemistry and molecular biology, inc. printed in the u.s.a. journal of biological chemistry volume • number • november , this is an open access article under the cc by license. http://creativecommons.org/licenses/by/ . / rescued the postnatal lethality of the tnni gene-deleted mice ( ) to produce adult skeletal muscle for investigating the functional effects of sstnt deficiency. amish nemaline myopathy is a rapidly progressive disorder, with affected children dying primarily from respiratory insufficiency due to weakness of the respiratory mus- cle, usually in the second year of life ( ). these clinical features suggest a critical role of sstnt in diaphragm function. therefore, we examined in the present study the phenotypes of diaphragm muscle of adult dou- ble transgenic mice. the results revealed significantly decreased expression of sstnt mrna and protein. the sstnt deficiency pro- duced atrophy and a change toward more fast fibers in the diaphragm muscle. the slow tnt-defi- cient diaphragm muscle had significantly decreased tolerance to fatigue, demonstrating a critical role of sstnt in respiratory function. materials and methods tnni -deleted/ctni-nd double transgenic mice—as de- scribed recently ( ), we crossed transgenic mouse line ctni- nd# ( ) with heterozygotes of tnni -deleted mice ( ), pro- vided by dr. xupei huang (florida atlantic university). the genotyping of the double transgenic offspring was performed on genome dna isolated from tail biopsies using pcr, and the compensation for the loss of endogenous ctni by ctni-nd in cardiac muscle was confirmed by western blotting as described ( , ) (fig. ). the mice were maintained on a -h light/ -h dark cycle ( : a.m./ : p.m.) and standard pellet diet. except for aging studies, mice at age – months of both sexes were used for phenotype characterization. immediately after euthanasia with pentobarbital (intraperitoneally, mg/kg), diaphragm mus- cle strips were dissected from the o’clock and o’clock sectors with the dorsal side as and o’clock positions ( ) for use in the experiments to avoid the difference between different por- tions of the diaphragm. all animal procedures were approved by the institutional animal care and use committee. quantitative pcr—total rna was extracted from mouse diaphragm muscle samples using the trizol reagent (invitro- gen). integrity of the isolated rna was verified using agarose gel electrophoresis. one �g of each rna was reverse tran- scribed using an anchored oligo(dt) primer (tv ) at °c for h. quantitative pcr was carried out with power sybr green pcr master mix (applied biosystems) to determine the level of sstnt cdna relative to the level of glyceraldehyde- -phos- phate dehydrogenase, using an applied biosystems real time pcr system (applied biosystems, foster city, ca). the pcr primers (fig. a) were designed to target the common sequence for all three alternatively spliced isoforms of sstnt mrna ( ) without cross-reaction with fast skeletal muscle tnt cdna (fig. b). the quantitative pcr was carried out in a -�l volume with a -min preheating at °c followed by cycles of s at °c and s at °c. melting curve analysis was performed at the end to verify that there was no significant figure . targeted deletion of ctni gene in the mouse genome removes the � portion of the sstnt gene promoter-enhancer region. the ctni gene and sstnt gene are very closely linked in the mouse genome with the � portion of the sstnt gene promoter-enhancer region integrated in the structure of the ctni gene ( ). the targeted deletion of the entire ctni gene in a ctni knock-out mouse line ( ) had concurrently removed a segment containing �-enhancer(s) of the sstnt gene promoter, which would reduce transcriptional activity as indicated by significantly decreased transcriptional activity of the � . kb promoter construct versus the � . kb control in previous ex vivo experiments ( ). figure . rescue of ctni gene deletion mice by transgenic expression of ctni-nd. a, the two agarose gels demonstrated the pcr genotyping of dou- ble transgenic mice using two pairs of specific primers recognizing wild type and tnni deletion alleles, respectively ( ), to identify homozygote of ctni gene deletion (top) and the presence of �-mhc promoter-directed transgene encoding ctni-nd (bottom). b, the sds-gel and western blot using mab tni- showed that the cardiac muscle of adult double transgenic mice expressed ctni-nd in the absence of endogenous ctni, demonstrating a successful res- cue of the postnatal lethality of ctni-ko mice. the mab ct western blot showed normal expression of cardiac tnt (ctnt) in the double transgenic mouse cardiac muscle. slow troponin t and diaphragm fatigue november , • volume • number journal of biological chemistry formation of primer dimmers under the pcr conditions. dia- phragm samples from three wild type and three double trans- genic mice were examined, each with triplicate reactions. the results were analyzed using the ���ct method as per the applied biosystems user’s instructions. sds-page and western blot—endogenous ctni and exoge- nous ctni-nd in the heart and tnt and tni isoforms in the diaphragm muscle were examined by sds-page and western blotting. total protein was extracted from freshly isolated mus- cle tissue by homogenization in sds-page sample buffer con- taining % sds to inactivate tissue proteases. the samples were heated at °c for min, centrifuged at top speed in a micro- centrifuge for min to remove insoluble materials, and stored at � °c until use. the protein samples were resolved on % laemmli gels with an acrylamide/bisacrylamide ratio of : . the resulting gels were stained with coomassie blue r or electrically trans- ferred to nitrocellulose membrane using a semidry transfer apparatus (bio-rad). after blocking in tris-buffered saline containing % bovine serum albumin, the membranes were probed using anti-tni monoclonal antibody (mab) tni- , anti- slow and cardiac tnt mab ct ( ), or anti-fast tnt mab t (a gift from prof. jim lin, university of iowa) ( ). the first antibody reactions were in tris-buffered saline containing . % bovine serum albumin at °c overnight. the subsequent washes, alkaline phosphatase-labeled anti-mouse igg second antibody (santa cruz biotechnology, inc., santa cruz, ca) incubation, and -bromo- -chloro- -indolylphosphate/nitro blue tetrazolium substrate reaction were carried out as described previously ( ). the relative amounts of tnt and tni isoforms were quantified by two-dimensional densitometry of the blots. myosin heavy chain isoforms were examined as described previously ( ). the muscle protein samples were resolved on % sds-polyacrylamide gel containing % glycerol run in an icebox overnight. the gels were stained with coomassie blue r , and the relative amounts of mhc isoforms were quanti- fied by two-dimensional densitometry. hematoxylin-eosin staining of paraffin sections—after euthanasia of the mice, diaphragm tissue was rapidly excised and fixed in . % formaldehyde in phosphate-buffered saline (pbs). the diaphragm samples were embedded in paraffin with precise orientation and thin cross sections were cut and pro- cessed with hematoxylin-eosin staining at a service facility for light microscopic examinations. immunohistochemistry—mouse diaphragm muscle samples were rapidly frozen in a small drop of o.c.t. compound in isopentane at � °c and then embedded in o.c.t. for thin frozen cross sectioning. the sections were fixed in % acetone, % ethanol for min. after blocking in pbs containing . % tween (pbs-t) and % bovine serum albumin for min, the sections were incubated with % h o in pbs for min to inactivate endog- enous peroxidases. the sections were then washed with pbs-t three times and incubated with anti-mhc-i mab fa or sp / myeloma culture supernatant at °c overnight. after washes with pbs-t to remove unbound mab, the sections were incu- bated with horseradish peroxidase-labeled anti-mouse second- ary antibody at room temperature for h, washed again, and developed in , �-diaminobenzidine-h o substrate solution in the dark for – s. the substrate reaction was stopped by washes in mm tris-hcl, ph . , for six changes. after they were counterstained with hematoxylin for min and water washes, the sections were immersed in a drop of % glycerol in pbs and mounted with cytoseal. the slides were examined under a zeiss observer microscope and photo- graphed. fa -positive (type ) fibers were counted per unit of cross-sectional area for comparisons. contractility measurements—mouse diaphragm muscle was isolated immediately after euthanasia as above. immersed in krebs solution ( mm nacl, mm nahco , . mm kcl, . mm kh po , . mm mgso , . mm cacl , and mm d-glucose, ph . ) continuously bubbled with % o plus % co at room temperature, -mm-wide diaphragm muscle strips were dissected with a piece of central tendon at one end and a piece of the rib cage tissue at the other end. the rib cage end of the muscle strip was tied to a triangle-shaped stainless steel hook with a # - suture. the muscle strip was then mounted to the bottom pole of a vertical bath (radnoti) con- taining krebs bubbled with % o plus % co at °c, and the tendon end was tied to another stainless steel hook for con- nection to a motorized force transducer (model b-lr, aurora scientific, inc.). isometric twitch contractions were stimulated by -v field electrical pulses of . -ms duration using an aurora b stimulator. force of the muscle strip was measured, and length was controlled through the b-lr transducer, and data were collected via a digital controller a/d interface (model c, aurora scientific inc.). the mounted muscle strip was adjusted to an optimal length for the production of maximal twitch force during min of equilibration. -ms tetanic contractions were then stimu- lated every min with -hz -v field electrical pulses of . -ms duration for min. the force-frequency relationship was determined from isometric tetanic contractions stimulated with electrical pulses at various frequencies ( – hz). high frequency and intermittent fatigue protocols—the dia- phragm muscle was examined for high frequency fatigability in which tetanic contractions were stimulated with -v field electrical pulses of . -ms duration at the optimal frequency for s, followed by stimulations with electrical pulses of . -ms duration for s to bypass the membrane electrical fatigability ( ). the effect of bypassing membrane electrical fatigability was verified by pulse stimulations of . -ms duration for s followed by . -ms pulses for s. one minute after the high frequency fatigue contractions, -ms tetanic contractions were stimulated every min with -v field electrical pulses of . -ms duration at the optimal frequency for min to monitor the recovery. fatigability of the diaphragm muscle was also examined with intermittent tetanic contractions in which ms of tetanic contraction was evoked every s (a duty cycle of . %), as described previously ( , ) with -v field electrical pulses of . -ms duration at the optimal frequency for min. one minute after the intermittent fatigue contractions, min of recovery was monitored as above. slow troponin t and diaphragm fatigue journal of biological chemistry volume • number • november , all muscle strips used for the functional studies were recov- ered at the end of the experiment and examined by sds-page and western blot to verify sstnt and other myofilament pro- tein contents. data analysis—two-dimensional densitometry was used to quantify the sds-gel and western blots scanned at dots/ inch using image j software. using image j software, the average thickness of the diaphragm was measured in -�m intervals along the tissue sections, and the cross-sectional area of muscle fibers was calculated. the data are shown as mean � s.e., and statistical significance was determined using two-tailed unpaired student’s t test. difference was considered significant when p was � . . results decreased sstnt mrna in diaphragm muscle of adult dou- ble transgenic mice—quantitative pcr with primers recogniz- ing all three alternatively spliced isoforms of sstnt but not fast tnt mrna showed significantly decreased expression of sstnt mrna in diaphragm muscle of adult ctni-ko/ ctni-nd double transgenic mice as compared with that in wild type mouse diaphragm muscles (fig. ). therefore, the deletion of the � portion of the sstnt gene enhancer-promoter region integrated in the ctni gene resulted in a knockdown effect on the transcription of the sstnt gene in vivo. decreased sstnt protein in diaphragm muscle of young adult double transgenic mice—western blots using mab ct detected decreased sstnt protein (down by � %) in dia- phragm muscle of ctni-ko/ctni-nd double transgenic mice as compared with that in wild type control (fig. a). together with the decreased sstnt mrna, the results demonstrated that the double transgenic mice provide a model of sstnt deficiency for investigating the function of sstnt in adult diaphragm muscle. the double transgenic mice are referred as sstnt-kd in the follow- ing phenotype characterizations. corresponding to the decreased sstnt, western blots using mab t showed that fast tnt in the sstnt-kd diaphragm muscle was increased by � % as compared with the level in wild type mouse diaphragm muscle (fig. a). decreases in the slow isoforms of tni and mhc in sstnt-kd diaphragm—in – -month-old young adult sstnt-kd mice, western blots with mab tni- recognizing all tni isoforms showed that slow tni was signifi- cantly decreased (down by � %) and fast tni increased as com- pared with the wild type controls (fig. a). glycerol sds-page showed decreases of mhc-i in the adult sstnt-kd diaphragm muscle (� . % of total mhc versus � . % in wild type control diaphragm) (fig. b). trends of reduction of mhc-iia and potentially compensatory increases in mhc-iix and mhc-iib were also observed (fig. b). decreased number of type slow fibers and atrophy in sstnt-kd diaphragm muscle—type slow fibers in mouse diaphragm muscle were identified by staining frozen thin cross- sections using anti-mhc-i mab fa . the results detected a significantly decreased number of type fibers in – -month- old sstnt-kd mouse diaphragm muscle (� % of the wild type control normalized to unit area) (fig. a). correspond- ingly, fa -negative type fibers were increased by � % in the sstnt-kd diaphragm muscle as compared with that in wild type control. these changes resulted in a significant decrease in the ratio of slow/fast fiber in the young adult sstnt-kd dia- phragm muscle (� . ) versus that in wild type control (� . ) (fig. a). microscopic measurement of hematoxylin-eosin-stained paraffin cross-sections showed that the young adult sstnt-kd mouse diaphragm was thinner than that of wild type control (� . versus � . mm; fig. b), indicating muscle atrophy. comparing the cross-sectional area of type and type fibers, we further observed significant atrophy of type fibers (� % of the wild type control; fig. b). interestingly, the cross-sec- tional area of type fibers was also decreased in sstnt-kd diaphragm muscle although less severely than that of the type fibers (� % of the wild type controls; fig. b). higher stimulating frequency required for the production of maximum tetanic force in sstnt-kd diaphragm muscle—we studied the force-frequency relationships in tetanic contraction of wildtypeandsstnt-kdmousediaphragmmusclestrips.aright- figure . decreased expression of sstnt mrna in diaphragm muscle of double transgenic mice. a, for quantitative pcr, a pair of oligonucleotide primers were designed for a region identical in the three alterna- tively spliced isoforms of sstnt mrna. b, the agarose gel verified the specificity of the primers in pcr on cloned sstnt and fast skeletal muscle tnt (fstnt) cdna templates. c, quantifications using real-time pcr detected a significantly decreased level of sstnt mrna in the diaphragm muscle of ctni-ko/ctni-nd double transgenic mice in comparison with the wild type control. values are shown as mean � s.e., n � mice in each group. ***, p � . in two-tailed student’s t test. slow troponin t and diaphragm fatigue november , • volume • number journal of biological chemistry ward shift of the force-frequency relationship curve was found in young adult sstnt-kd diaphragm muscle as compared with wild type control (fig. ). the optimal frequencies at which the maxi- mal tetanic force was generated in wild type and sstnt-kd dia- phragm muscles were around and hz, respectively. this shift indicates an increased dependence of the sstnt-kd dia- phragm muscle on stimulating frequency in tetanic force produc- tion, consistent with a faster muscle phenotype. whereas most of the twitch contraction parameters did not show significant difference between the sstnt-kd and wild type mouse diaphragm muscle strips, the shorter time of ten- sion development (tpt and tp ) of the sstnt-kd diaphragm muscle was also consistent with a faster muscle phenotype (table ). although sstnt-kd diaphragm muscle produced lower tetanic force at the stimulating frequency optimal for wild typediaphragmmuscle(fig. ),tetaniccontractileforcegenerated at its own optimal frequency of stimulation was not significantly different from that of the wild type control (table ). unchanged electrical fatigability and increased myofila- ment fatigability of sstnt-kd diaphragm muscle—because electrical pulses of short duration induce skeletal muscle con- traction dependent on the propagation of action potential along the plasma membrane, we used a high frequency fatigue protocol to study the membrane electrical fatigability of the mouse diaphragm muscle strips. the results in fig. a showed that wild type and sstnt-kd young adult mouse diaphragm muscles had no difference in membrane fatigability during a -s tetanic contraction stimulated with field electrical pulse of . -ms duration at the optimal frequencies. switching to elec- trical pulse stimulations of -ms duration at the end of the pro- tocol significantly increased the tetanic force production of both wild type and sstnt-kd groups to the same extent (fig. a). the similar increases in force production upon bypassing membrane fatigability using brief wider electrical pulse stimu- lations ( ) indicated similar extents of myofilament fatigability in the wild type and sstnt-kd mouse diaphragm muscle strips when the contractile apparatus was protected by membrane fatigability. consistently, both wild type and sstnt-kd dia- phragm muscle strips completely recovered from the electrical fatigue treatment (data not shown). in a modified high frequency fatigue protocol using pulse stimuli of . -ms duration followed by a switch to . -ms pulse duration, we further investigated the fatigability of mouse dia- phragm muscle bypassing the membrane electrical depend- ence. the results in fig. b revealed a significant difference between sstnt-kd diaphragm muscle strips and the wild type controls. the faster and more extensive decreases in tetanic force of sstnt-kd diaphragm muscle indicated increased myofilament fatigability in comparison with the wild type con- trol. when the duration of electrical pulses was switched from . to . ms, there were small increases in tetanic force in both sstnt-kd and wild type groups, suggesting that the calcium mobilization capacity ( ) was not exhausted in the diaphragm muscle fibers when stimulated by -v field pulses of . -ms duration. increased fatigability and decreased recovery of sstnt-kd diaphragm muscle in intermittent tetanic contractions—the increased myofilament fatigability in sstnt-kd diaphragm muscle was further examined using a -min intermittent fatigue protocol. the results in fig. a showed that cycles of intermittent tetanic contraction at . % duty cycle ( -ms trains) stimulated with . -ms electrical pulses at optimal fre- quency exhibited force decreases to � . % of the prefatigue level in sstnt-kd diaphragm muscle as compared with figure . decreased sstnt and the slow isoforms of tni and mhc in dia- phragm muscle of young adult sstnt-kd mice. a, sds-gel and mab ct western blot densitometry detected significantly lower levels of sstnt pro- tein in diaphragm muscle of the sstnt-kd mice as compared with the wild type controls (down by . � . % normalized to the level of actin in accom- panying gels). correspondingly, mab t western blot and densitometry analysis detected higher levels of fast skeletal muscle tnt (fstnt) in the sstnt-kd diaphragm muscle than the wild type controls (up by . % nor- malized to the level of actin in accompanying gels). densitometry analysis of mab tni- western blot normalized to the amount of total tni found decreased the slow versus fast isoform ratio of tni in the sstnt-kd diaphragm muscle as compared with the wild type controls. b, glycerol-sds-gel and den- sitometry analysis normalized to total mhc showed a decreased level of mhc-i in sstnt-kd diaphragm muscle ( . % of total mhc versus . % in wild type control). *, p � . by two-tailed student’s t test. slow troponin t and diaphragm fatigue journal of biological chemistry volume • number • november , � . % in wild type control muscle strips. the -min recovery from fatigue treatment showed that tetanic force went back to � . % of the prefatigue level in wild type diaphragm muscle but only to � . % in the sstnt-kd group (fig. b). the data indicated that the slow tnt deficiency resulted in significantly increased myofilament fatigability in the young adult dia- phragm muscle of sstnt-kd mice. effects of sstnt-kd on contractility and myofilament pro- tein isoform contents of aging diaphragm—we further exam- ined diaphragm muscle from – -month-old mice for con- tractility and myofilament protein isoform expression to investigate the effect of sstnt-kd. the results in fig. a showed that aging wild type and sstnt-kd diaphragm muscles both switched toward slower fiber type, whereas the sstnt-kd group remained faster than the wild type control, consistent with the previous observation that aging skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower twitching fiber population ( , ). slow tnt and slow tni remained low in aging sstnt-kd diaphragm muscle (fig. b). consistent with the shift toward slower muscle type, the slow/fast isoform ratio of tni increased in aging wild type diaphragm muscle but was insignificant in sstnt-kd diaphragm (fig. b). the level of mhc-iia increased in aging diaphragm muscles of wild type mice with a lesser degree in sstnt-kd group (fig. b). mhc-iix decreased during aging in both groups (fig. b). figure . atrophy and type fiber reduction of sstnt-kd diaphragm muscle. a, immunostaining of frozen sections using anti-mhc-i mab fa showed . � . % decrease in type fibers and . � . % increase of type fibers in sstnt-kd diaphragm muscle as compared with the wild type control. accordingly, the ratio of type /type fibers was much lower in sstnt-kd mouse diaphragm muscle ( . � . %) than that in wild type control muscles ( . � . %). b, hematoxylin-eosin-stained cross-sections showed that the thickness of adult sstnt-kd mouse diaphragm was smaller ( . � . mm) than that of wild type controls ( . � . mm), indicating muscle atrophy. morphological analysis further found that the cross-sectional area (csa) of type fibers was significantly decreased by . � . % in sstnt-kd diaphragm as compared with the wild type control. the cross-sectional area of type fibers in sstnt-kd diaphragm was also decreased, although by a lesser extent ( . � . %), as compared with the wild type control. *, p � . by two-tailed student’s t test. figure . shift of force-frequency relationship in sstnt-kd diaphragm muscle. tetanic force production was examined with electrical pulse stimu- lations of . -ms duration at – hz in -hz increments. the results showed that sstnt-kd diaphragm muscle had optimal frequency for maxi- mum force at hz, increased from hz for the wild type mouse dia- phragm muscle, consistent with increased fast fiber content. *, p � . by two-tailed student’s t test, n � mice in each group. table parameters of twitch and tetanic contractions data are presented as mean � s.e. n � mice for wild type and n � mice for sstnt-kd groups. mn, millinewtons; dt/dt, develop tension; tp , time to reach % peak tension; tpt, time to reach peak tension; tr , time to reach % relaxation. wild type sstnt-kd developed twitch tension (mn/mm ) . � . . � . �dt/dt (mn/s) . � . . � . �dt/dt (mn/s) � . � . � . � . tp (ms) . � . . � . a tpt (ms) . � . . � . b tr (ms) . � . . � . optimal tetanic tension (mn/mm ) . � . . � . a p � . in two-tailed student’s t test. b p � . in two-tailed student’s t test. slow troponin t and diaphragm fatigue november , • volume • number journal of biological chemistry in contrast to the significant decrease in mhc-iib in aging wild type mouse diaphragm muscle, the level of mhc-iib increased in sstnt-kd mouse diaphragm during aging (fig. b). although determination of its functional significance requires follow up studies, this finding indicated that the decrease in slow tnt induced adaptive gene regulations for the expression of other myofilament proteins with differentiated effects in young and aging skeletal muscles. discussion troponin t abnormalities have been found in causing dis- eases in both cardiac and skeletal muscles ( ). we and others have extensively studied the biochemical mechanisms for the structure-function relationship of tnt isoforms ( , ). it has been shown that slow tnt has differentiated binding affinity for tni and tropomyosin and confers higher ca � sensitivity in comparison with that of fast tnt ( , ). the present study demonstrated the importance of slow tnt in the function of skeletal muscle. our findings provide some new insights into the following aspects. the structural linkage of ctni and sstnt genes—cardiac tni and sstnt genes are very closely linked in the vertebrate genome. we previ- ously detected in ex vivo promoter analysis that the kb � . to � . upstream region of the sstnt gene, which overlaps with the � region of the ctni gene, contained major enhancer activity required for high level transcription of the sstnt gene ( ). the present study further demonstrated that deletion of the ctni gene between the two bamhi sites (fig. a) ( ), equivalent to the � . kb deletion of the upstream region of sstnt gene tested ex vivo, had a destructive effect in vivo on the promoter activity of the sstnt gene. because a single copy of a functional sstnt gene was sufficient in sustaining muscle function in heterozygote careers of amish nemaline myopathy ( ), the decreased level of sstnt in the diaphragm muscle of sstnt-kd mice indicated a more than % decrease of sstnt gene promoter activity due to deletion of the � . kb upstream genomic enhancer segment. although the sstnt gene is iden- tified to be the newest member evolved among the three muscle type tnt isoform genes ( ), its function is indispensable, and the loss of sstnt causes a lethal phenotype in amish nemaline myopathy ( ). this functional importance might have been responsible for the sstnt gene remaining closely linked to the upstream ctni gene during evolution, because a destruc- tion of either the �-coding exons of the ctni gene or the �-enhancer region of the sstnt gene would cause infantile lethality ( , ). the effects of sstnt deficiency on myofilament protein iso- forms and atrophy of diaphragm muscle—the primary sstnt deficiency resulted a series of secondary changes in the dia- phragm muscle of young adult sstnt-kd mice. in addition to an increased ratio of fast tnt, sstnt-kd diaphragm muscle showed decreased slow tni and a trend of increase in fast tni. mhc-i was decreased together with trends of increases in mhc-iix and mhc-iib. therefore, the primary deficiency of sstnt resulted in reductions of the slow isoforms of both thin and thick filament proteins. the decreased slow/fast isoform figure . high frequency fatigue analysis. a, a high frequency fatigue protocol was tested on diaphragm muscle strips with field electrical stimuli of . -ms duration for s followed by . -ms stimuli for s. the results showed no difference in electrical fatigability between the sstnt-kd and wild type diaphragm muscles. switch of the electrical pulses from . -ms duration to . -ms duration generated similar partial recovery of the contractile force in both groups, suggesting similar protection of the muscle by electrical fatigue. b, high frequency fatigue analysis was modified to bypass the membrane dependence using electrical stimuli of . -ms duration for s followed by stimuli of . -ms duration for s. the results showed significantly higher myofilament fatigability of sstnt-kd diaphragm muscle as compared with the wild type control. *, p � . by two-tailed student’s t test, n � mice in each group. figure . intermittent fatigability analysis. a, an intermittent fatigue protocol was applied to examine wild type and sstnt-kd mouse diaphragm muscle strips. tetanic contractions stimulated at optimum frequency with -ms/ -s trains for min demonstrated force declines to . � . % in wild type and to . � . % in sstnt-kd diaphragm muscle strips. b, during a -min recovery, the force of wild type diaphragm muscle returned to . � . % of the prefatigue level, whereas that of sstnt-kd diaphragm muscle returned to . � . % of the prefatigue level. *, p � . by two-tailed student’s t test, n � mice in each group. slow troponin t and diaphragm fatigue journal of biological chemistry volume • number • november , ratio of myofilament proteins formed a basis of the fast-ward change of fiber types. because the diaphragm is a mixed fiber muscle ( ), the decreases in the slow isoform myofilament proteins could reflect decreases in the number and/or size of slow fibers as well as increases in the number and/or size of fast fibers as a com- pensation for the decreases in sstnt. our results first showed reductions of both number and size of type slow muscle fibers, which would directly contribute to the atrophy of sstnt-kd mouse diaphragm. although the number of type fibers increased to compensate for the decreased number type fibers, the cross-sectional area of type fibers in sstnt-kd diaphragm was decreased (fig. b). therefore, the atrophy of type fibers that are the major fiber type in mouse diaphragm muscle (fig. a) further contributed to the atrophy of sstnt-kd diaphragm muscle. the mechanism for sstnt defi- ciency to cause atrophy of fast type fibers in diaphragm muscle remains to be investigated. the effects of slow tnt defi- ciency on diaphragm myofilament protein contents exhibited aging related changes. although both wild type and sstnt-kd diaphragm muscles turned toward a slower fiber type in aging (fig. ), an inter- esting adaptation to sstnt-kd was that mhc-iib was significantly decreased in wild type but increased in sstnt-kd diaphragm muscle. this adaptive sustainment of mhc- iib that produces the fastest in vitro motility among mhc isoforms ( ) mayhavecontributedtothelessslow- wardshiftoftheagingsstnt-kddia- phragm muscle (fig. a). the finding that decreased expression of slow tnt resulted in a series of decreases in slow tni and mhc-i indicated a consequence of fiber type switching. the finding that a change in one thin filament protein in skeletal muscle would cause multiple secondary changes in gene regulation suggests an experimental model to investigate the highly plastic nature of skeletal muscle during physiological and pathological adaptations. slow tnt deficiency reduces fatigue tolerance of diaphragm muscle—it has been established that stimulating skeletal muscle contraction with an electrical pulse of short duration ( . – . ms) significantly depends on the propagation of action potential along the plasma membrane ( ). in contrast, electrical pulses with longer duration of . ms can propagate directly to the transverse tubular membranes, in which a majority of the l-type calcium channels are pres- ent, to activate calcium mobilization mechanisms with less dependence on plasma membrane action potential propaga- tion, thus bypassing the fatigue mechanism of plasma mem- brane inexcitability ( ). therefore, we applied a high fre- quency fatigue stimulation protocol to dissect plasma membrane electrical fatigability and myofibril fatigability. the results clearly demonstrated that sstnt deficiency significantly increased myofilament fatigability with un- changed plasma membrane excitability. the increased myofilament fatigability of the sstnt-kd mouse diaphragm muscle was further demonstrated using an intermittent tetanic fatigue protocol mimicking the respiratory contractions. the results confirmed the physiological impor- figure . effects of sstnt-kd in aging diaphragm muscle. a (left), twitch and tetanic force measurements found no significant difference between young and aging diaphragm muscles from wild type and sstnt-kd mice. right, normalized twitch contraction curves revealed that wild type and sstnt-kd mouse diaphragm muscles both shifted toward slower fiber type in aging, whereas the sstnt-kd group remained faster than the wild type control. b (top), densitometry analysis of ct , t , and tni- mab western blots showed that slow tnt remained low in aging diaphragm muscle of sstnt-kd mice along with the trend of higher fast tnt contents and decreased slow versus fast isoform ratio of tni as compared with the wild type controls. bottom, glycerol- sds-gel and densitometry analysis showed that mhc-i went down in aging diaphragm muscle with much more severity in the sstnt-kd group than in the wild type control. mhc-iia was increased during aging to a lesser degree in sstnt-kd than that in wild type diaphragms. there was a trend of decrease in mhc-iix in both wild type and sstnt-kd diaphragm during aging. mhc-iib was significantly decreased in aging wild type mouse diaphragm muscle, whereas aging sstnt-kd diaphragm had an increase in mhc-iib. *, p � . aging versus young groups of the same genotype; #, p � . , sstnt-kd versus wild type in the aging group, analyzed by two-tailed student’s t tests. mn, millinewtons. slow troponin t and diaphragm fatigue november , • volume • number journal of biological chemistry tance of sstnt in diaphragm function. the fatigability of sstnt-kd diaphragm muscle shown by normalized force pro- duction (fig. ) demonstrated that in addition to the overall muscle atrophy, the qualitative changes of decreased number of slow fibers and slow isoforms of myofilament proteins severely impaired the fatigue tolerance of diaphragm muscle. in addi- tion to explaining the terminal respiratory failure seen in virtu- ally all amish nemaline myopathy patients, the ctni-ko/ ctni-nd double transgenic mouse model provides a highly valuable experimental system to study the pathogenesis, patho- physiology, and treatment of this devastating and lethal genetic disease. acknowledgments—we thank hui wang for pcr genotyping of trans- genic mice, dr. xupei huang (florida atlantic university) for provid- ing the ctni-ko mouse line, dr. jim lin (university of iowa) for pro- viding the t mab, and dr. jeffrey robbins (university of cincinnati) for the mouse cardiac �-mhc gene promoter used in the construction of ctni-nd transgenic mouse lines. references . perry, s. v. ( ) j. muscle res. cell motil. , – . jin, j. p., zhang, z., and bautista, j. a. ( ) crit. rev. eukaryot. gene expr. , – . johnston, j. j., kelley, r. i., crawford, t. o., morton, d. h., agarwala, r., koch, t., schäffer, a. a., francomano, c. a., and biesecker, l. g. ( ) am. j. hum. genet. , – . wang, x., huang, q. q., breckenridge, m. t., chen, a., crawford, t. o., morton, d. h., and jin, j. p. ( ) j. biol. chem. , – . jin, j. p., brotto, m. a., hossain, m. m., huang, q. q., brotto, l. s., nosek, t. m., morton, d. h., and crawford, t. o. ( ) j. biol. chem. , – . huang, q. q., and jin, j. p. ( ) j. mol. evol. , – . huang, x., pi, y., lee, k. j., henkel, a. s., gregg, r. g., powers, p. a., and walker, j. w. ( ) circ. res. , – . jin, j. p. ( ) biochem. biophys. res. commun. , – . barbato, j. c., huang, q. q., hossain, m. m., bond, m., and jin, j. p. ( ) j. biol. chem. , – . yu, z. b., zhang, l. f., and jin, j. p. ( ) j. biol. chem. , – . subramaniam, a., gulick, j., neumann, j., knotts, s., and robbins, j. ( ) j. biol. chem. , – . feng, h. z., hossain, m. m., huang, x., and jin, j. p. ( ) arch. biochem. biophys. , – . brotto, m. a., biesiadecki, b. j., brotto, l. s., nosek, t. m., and jin, j. p. ( ) am. j. physiol. cell physiol , c –c . jin, j. p., chen, a., and huang, q. q. ( ) gene , – . lin, j. j. ( ) proc. natl. acad. sci. u.s.a. , – . feng, h. z., biesiadecki, b. j., yu, z. b., hossain, m. m., and jin, j. p. ( ) j. physiol. , – . feng, h. z., chen, m., weinstein, l. s., and jin, j. p. ( ) j. biol. chem. , – . cairns, s. p., chin, e. r., and renaud, j. m. ( ) j. appl. physiol , – . yu, z. b., gao, f., feng, h. z., and jin, j. p. ( ) am. j. physiol. cell physiol. , c –c . van lunteren, e., and moyer, m. ( ) respiration , – . larsson, l., grimby, g., and karlsson, j. ( ) j. appl. physiol. , – . gannon, j., doran, p., kirwan, a., and ohlendieck, k. ( ) eur j. cell biol. , – . gomes, a. v., barnes, j. a., harada, k., and potter, j. d. ( ) mol. cell. biochem. , – . reiser, p. j., westfall, m. v., schiaffino, s., and solaro, r. j. ( ) am. j. physiol. , h –h . chong, s. m., and jin, j. p. ( ) j. mol. evol. , – . degens, h., and larsson, l. ( ) j. musculoskelet. neuronal interact. , – . cairns, s. p., taberner, a. j., and loiselle, d. s. ( ) j. appl. physiol. , – slow troponin t and diaphragm fatigue journal of biological chemistry volume • number • november , 	deletion of a genomic segment containing the cardiac troponin i gene knocks down expression of the slow troponin t gene and impairs fatigue tolerance of diaphragm muscle* 	materials and methods 	results 	discussion 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ research open access modeled nitrate levels in well water supplies and prevalence of abnormal thyroid conditions among the old order amish in pennsylvania briseis aschebrook-kilfoy , , , sonya l heltshe , john r nuckols , mona m sabra , alan r shuldiner , , braxton d mitchell , matt airola , theodore r holford , yawei zhang and mary h ward * abstract background: nitrate is a widespread contaminant of drinking water supplies, especially in agricultural areas. nitrate intake from drinking water and dietary sources can interfere with the uptake of iodide by the thyroid, thus potentially impacting thyroid function. methods: we assessed the relation of estimated nitrate levels in well water supplies with thyroid health in a cohort of , old order amish residing in lancaster, chester, and lebanon counties in pennsylvania for whom thyroid stimulating hormone (tsh) levels were measured during - . nitrate measurement data ( - ) for , wells in the study area were obtained from the u.s. geological survey and we used these data to estimate concentrations at study participants’ residences using a standard linear mixed effects model that included hydrogeological covariates and kriging of the wells’ residuals. nitrate levels estimated by the model ranged from . mg/l to . mg/l n-no -, with a median value of . mg/l, which was used as the cutpoint to define high and low nitrate exposure. in a validation analysis of the model, we calculated that the sensitivity of the model was % and the specificity was %. tsh levels were used to define the following outcomes: clinical hyperthyroidism (n = ), clinical hypothyroidism (n = ), subclinical hyperthyroidism (n = ), and subclinical hypothyroidism (n = ). results: in women, high nitrate exposure was significantly associated with subclinical hypothyroidism (or = . ; % ci: . - . ). nitrate was not associated with subclinical thyroid disease in men or with clinical thyroid disease in men or women. conclusions: although these data do not provide strong support for an association between nitrate in drinking water and thyroid health, our results do suggest that further exploration of this hypothesis is warranted using studies that incorporate individual measures of both dietary and drinking water nitrate intake. keywords: nitrate, thyroid conditions, tsh, old order amish, water pollution, drinking water background nitrate is a widespread contaminant of drinking water supplies, especially in agricultural areas. the thyroid can concentrate univalent anions such as nitrate (no -), which subsequently interferes with the uptake of iodide (i-) by the thyroid and may cause reduced production of thyroid hormones [ - ]. the result of the reduced thyr- oid hormone production is a compensatory increase in thyroid stimulating hormone (tsh), a sensitive indicator of thyroid function. high and low tsh levels reflect hypo- and hyperfunction of the thyroid gland, respec- tively. chronic stimulation of the thyroid gland by excessive tsh has been shown in animals to induce the development of hypertrophy and thyroid disease, as well as hyperplasia, followed by adenoma and carcinoma [ ]. at least two epidemiological studies have shown high nitrate intake to be associated with thyroid dysfunction, * correspondence: wardm@mail.nih.gov occupational and environmental epidemiology branch, division of cancer epidemiology and genetics, national cancer institute, national institutes of health, department of health and human services, rockville, md, usa full list of author information is available at the end of the article aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / © aschebrook-kilfoy et al; biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:wardm@mail.nih.gov http://creativecommons.org/licenses/by/ . including hypertrophy and changes in tsh levels [ , ]; however, the impact of nitrate intake on specific thyroid conditions, including hyperthyroidism and hypothyroid- ism is not clear. elevated concentrations of nitrate in groundwater ori- ginate from a number of sources, including leaking sep- tic tanks, animal waste, and overuse of nitrogen fertilizers [ ]. nitrate is very soluble and it readily migrates to groundwater. nitrate contamination of groundwater is an exposure of interest as groundwater serves as the primary drinking water supply for over % of the rural population and % of the total popu- lation of north america [ ]. although the u.s. environ- mental protection agency (epa) maximum contaminant level (mcl) for nitrate as nitrogen (nitrate-n) is mg/ l in public water sources [ ], the levels in private wells are not regulated and the task of monitoring is left to residential owners, presenting opportunities for high levels of human exposure. the u.s. geological survey (usgs) estimates that nitrate concentrations exceed the epa’s standard in approximately % of agricultural and rural areas, exposing over million people in the united states [ ]. the mcl for nitrate in drinking water was established to protect against methemoglobinemia, or “blue baby syndrome,” to which infants are especially susceptible. however, this health guideline has not been thoroughly evaluated for other health outcomes such as thyroid disease and cancer. the old order amish community is a population characterized by a homogeneous lifestyle, including intensive farming practices and low mobility, and has been relatively unchanged across generations [ ]. in areas where many large dairy and poultry farms are con- centrated, the land area for disposal of animal wastes is limited. this situation often results in overloading the available land with manure, with considerable nitrogen ending up in groundwater or surface water [ , ]. lan- caster county in southeastern pennsylvania is an exam- ple of such an area where extensive dairy enterprises with high stocking rates prevail. high levels of nitrate in the groundwater [ ] suggest that the amish are a poten- tially highly exposed population. given the biological effects of nitrate intake on the thyroid, investigation of whether the amish in this area exhibit an increased pre- valence of thyroid dysfunction and thyroid disease is of interest. the aim of this study is to assess whether nitrate con- centrations in well water are associated with levels of tsh and thyroid disease. our goal was to use survey data on nitrate levels in well-water obtained from the usgs to conduct a cross-sectional analysis of the asso- ciation between nitrate exposure and thyroid health. this study builds upon several ongoing studies of dia- betes, obesity, osteoporosis, hypertension, and cardiovascular disease in the amish, initiated in at the university of maryland [ - ]. methods study population subjects included in this analysis were , old order amish aged years and older from lancaster, chester, and lebanon counties, pennsylvania, for whom thyroid health was assessed through measure- ment of thyroid stimulating hormone (tsh) levels in their prior participation in one or more studies of health by investigators at the university of maryland, baltimore [ - ]. we excluded participants whose residences were located outside of lancaster, chester, or lebanon counties (n = ) due to sparse nitrate measurement data, and persons who reported use of thyroid medication (n = ) leaving a total of , persons ( , females and , males) in the final analysis. nearly all of the enrolled individuals are des- cendants of a small number of amish who settled in lancaster county, pennsylvania, in the mid-eighteenth century [ , , ]. this study was approved by the institutional review boards of the university of mary- land and the national cancer institute. all subjects included in this analysis received a stan- dardized examination at the amish research clinic in strasburg, pennsylvania or in the participant’s home during the time period - . as part of this exam- ination, a fasting blood sample was collected from which tsh levels were measured with the siemens tsh assay (immulite ; deerfield, il) according to the manufacturer’s instructions. the method is a solid- phase, chemiluminescent, competitive analog immu- noassay and has analytical sensitivity of . μiu/ml and upper limit of μiu/ml of tsh. residential street addresses were geocoded using the teleatlas (lebanon, nh) matchmaker sdk profes- sional version . (october ), a spatial database of roads, and a modified version of a microsoft visual basic version . program issued by teleatlas to match input addresses to the spatial database. we assigned residence location using an offset of ft from the street centerline. addresses that were not successfully geocoded were checked for errors using interactive geocoding techniques. where only a street intersection was available for the residential location ( . % of residences), we assigned the geographic loca- tion of the residence to the middle of the intersection. where only a zip code was available for the residential location ( . % of residences), we assigned the geo- graphic location of the residence to the centroid of the zip code. the geocoded location of the residences and the geographic boundary of our study area is shown in figure . aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of historical assessment of nitrate levels a survey of nitrate levels in well water in lancaster, chester, and lebanon counties was carried out from - by the usgs. the usgs collected data from active monitoring wells in the county and from a well- owner monitoring program conducted by the state department of natural resources in collaboration with pennsylvania state university. water samples ( - ul) from all programs were measured for nitrate using ion chromatography with a detection limit of . mg/ l as nitrate-n [ ]. a total of , unique wells were measured in our study area during the survey period. the measurements were not from wells chosen at random but included monitoring data reported by the usgs and samples from individual well owners. a total of , wells had measurement; wells had measurements; wells had measurements; and wells had more than measurements. figure shows the geographic distribution of the wells in our study area in relation to the location of the , participant residences the median distance between a residence and the closest measured well was . m (interquartile range: . - . m). the median nitrate concentration by season ranged from . mg/l as nitrate-nitrogen (hereafter mg/ l) for summer months (interquartile range: - . mg/l) to . mg/l in spring months (interquartile range: - . mg/l). for wells with multiple measures, the median difference between the maximum and minimum value was . mg/l (iqr: . - . ). the mean of the measure- ments was used for wells with multiple measurements when we did the exposure modeling (see below). prediction of nitrate levels in well water of participants’ residences we assumed the drinking water supply for participants to be a well located at their reported residence. to esti- mate nitrate levels at this location, we first determined whether nitrate concentrations in the usgs wells varied across the types of aquifers in the study area (table ). maps of the primary aquifers were obtained from the usgs (created from m pixel satellite imagery) [ ]. there are five principal aquifers in the study area (fig- ure ). the differentiation of aquifer type is important because the transport of contaminants in groundwater is generally confined to within these hydrogeologic figure location of participant residences and wells with nitrate measures in study area. aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of boundaries. using the usgs national land cover data set (nlcd) [ ], we also evaluated nitrate levels in the usgs wells across thirteen types of land use (pas- ture, deciduous forest, row crops, low intensity residen- tial, mixed forest, commercial or industrial, evergreen forest, high intensity residential, water, quarries/gravel pits, transitional, urban grasses, and woody wetlands) (table and figure ). we found limited temporal var- iation by season and by decade within each of the five aquifer types over the well measurement period, as well as across the land use classifications used in our analysis (figure ). we used a standard linear mixed effects statistical model to develop a predictive model including the vari- ables principal aquifer and land use. nitrate levels were log normally distributed so we modeled the natural logarithm of the concentration. spatial correlation existed in the nitrate measurements even after covariate adjustment [ ], so we performed kriging on the wells’ residuals from the predictive nitrate model. if a well had more than one measurement, the mean of the measure- ments and its residual was used in the modeling. we assumed that the residuals in the model have a single, normally distributed mean structure centered at zero, allowing for universal kriging across the study area. the kriging procedure predicts a ‘residual’ for each study participant based on a weighted average of the neigh- boring wells’ residuals (within the respective aquifer and land use category). for comparison, we also applied the kriging procedure based on the weighted average of the five neighboring wells’ residuals. for example, if for a particular region of our study area, the regression model tends to underestimate the true observed log nitrate values (positive residuals) then individuals in this region will be given a representative positive residual prediction that is added to the log nitrate estimate based on the individuals’ covariates and the regression parameters. the antilog gives an unbiased predictor of median nitrate value resulting in estimates that are more robust to outlier observations than a mean estimator. nitrate levels estimated by the model ranged from . mg/l to . mg/l, with a median of . mg/l and a mean of . mg/l (sd = . mg/l). the predicted nitrate level mean was similar to the mean of the measured values used for modeling ( . mg/l; sd = . mg/l) although the standard deviation was smaller. model validation the validity of the predictive model was assessed for validation wells by comparing the predicted nitrate table distribution of nitrate concentration in us geological survey wells by aquifer type and categories of land use in lancaster, lebanon, and chester counties, from - well location nitrate mg/l (no -n) aquifer n median mean std. dev. minimum maximum piedmont and blue ridge crystaline-rock . . . < . . piedmont and blue ridge carbonate rock . . . . . early mesozoic basin . . . < . . valley and ridge carbonate rock . . . < . . valley and ridge . . . . . land use n median mean std. dev. minimum maximum pasture . . . < . . deciduous forest . . . . . row crop . . . . . low intensity residential . . . . . mixed forest . . . < . . commercial or industrial . . . . . evergreen forest . . . . . high intensity residential . . . . . water . . . . . quarry mine gravel pit . . . . . transitional . . . . . urban grasses . . . . . woody wetland . . . . . overall . . . < . . spatial classification is based on the national land cover data set; , usgs [ref ]. aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of concentration to the observed nitrate concentrations. the validation wells were randomly selected from wells with usgs measurements from - . the limited date range was chosen to be consistent with the time frame of the nlcd land use database used in our analyses. we evaluated model sensitivity, specificity, and percent agreement using the median of the predicted nitrate level ( . mg/l as nitrate-n) as a cutpoint for high and low exposure categories. the sensitivity of the model was % and the specificity was %. the spear- man’s rank correlation between the continuous pre- dicted and measured concentrations was . . cross tabulation of predicted and observed nitrate concentra- tions by quartiles of the measured nitrate concentrations demonstrated a percent agreement of % (table ). data analysis we used generalized linear regression to assess the asso- ciation between estimated nitrate levels in well water and continuous tsh measures. tsh levels were also used to define disease status based on clinical guidelines [ ]. a “normal” range for tsh was defined as . - miu/ml. a tsh level of > miu/ml- miu/ml was defined as subclinical hypothyroidism (n = ) and more than miu/ml was defined as clinical hypothyr- oidism (n = ). a tsh value of . miu/ml to . miu/ml was defined as subclinical hyperthyroidism (n = ) and less than . miu/ml was defined as clinical hyperthyroidism (n = ). all of the disease definitions are based on the assumption that tsh was marking pri- mary disease in the thyroid since other causes of tsh abnormalities, e.g., primary pituitary disease, thyroid hormone resistance, are very uncommon by comparison [ ]. estimated nitrate levels in participants’ drinking water were categorized into quartiles and by the median of the predicted well nitrate level ( . mg/l). we evaluated the association of the nitrate levels with each thyroid disease group using unconditional logistic regression to com- pute the odds ratio (or) and % confidence intervals. all models were adjusted for potential confounding fac- tors including age (continuous) and bmi ((normal (< figure principle aquifers in the three study area counties in southeastern pennsylvania. data from principal aquifers of the conterminous united states, hawaii, puerto rico, and the u.s. virgin islands: u.s. geological survey. madison, wi; aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of kg/m ), overweight ( - kg/m ), and obese (> kg/ m )). we conducted analyses stratified by gender as well as for men and women combined. tests of linear trend were performed by modeling the continuous nitrate esti- mates. a p-value < . was considered significant and all data analyses were conducted using sas version . . we conducted two sensitivity analyses. in the first analysis, we excluded participants whose residences were located within boundaries of the u.s. census places (uscb ) and were therefore possibly con- nected to public water supplies with nitrate levels below the mcl ( % of study population). in the second ana- lysis, we excluded those whose residence was greater than m from the nearest well with measurement data ( %) to reduce the probability of measurement error. we recomputed the or for subclinical hypothyr- oidism after correcting for exposure misclassification (i. e. by reclassifying false positives and false negatives) using our estimates of sensitivity and specificity and the prevalence of exposure ( %). results the mean tsh level was . miu/ml ( . miu/ml for women and . miu/ml for men). based on the tsh measures, the prevalence of clinical hyperthyroidism was . % and the prevalence of subclinical hyperthyroidism was . %. the prevalence of clinical hypothyroidism was . % and the prevalence of subclinical hypothyroidism was . %. the mean age of participants was years (range: - ). the mean bmi was . kg/m for men and . figure land use in in the three study area counties in southeastern pennsylvania. data from principal aquifers of the conterminous united states, hawaii, puerto rico, and the u.s. virgin islands: u.s. geological survey. madison, wi; table comparison of quartiles of the predicted nitrate concentration by quartiles of the measured nitrate concentrations, wells used for the model validation quartiles of the predicted nitrate concenytration quartiles of measured nitrate concentration q q q q total q q q q total percent agreement = % aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of kg/m for women (table ). the average bmi of males with clinical hyperthyroidism was lower than that of those in the general study population but females with clinical hyperthyroidism had a slightly higher average bmi than the general study population. the average age of persons with thyroid disease was higher in all cate- gories compared to the group with normal tsh levels. although smoking data was not available for the entire study population, among those for whom these data were collected, less than % of women ( of ) and % of males ( of ) reported ever smoking tobacco. adjusting for age and bmi, and modeling tsh con- centration as the outcome, we observed no significant relationship with nitrate concentration. the b coefficient for men and women combined was - . (p-value = . ), - . for men (p-value = . ), and - . for women (p-value = . ). modeling the dichotomized high/low nitrate predictor, the b coefficient for men and women combined was - . (p-value = . ), - . for men (p-value = . ), and - . for women (p-value = . ). neither clinical or subclinical hyperthyroidism were associated with nitrate concentrations (table ), although the number of cases was low (n = cases of clinical hyperthyroidism and n = cases of subclinical hyperthyroidism). the results for hypothyroidism are presented in table . overall, there was a borderline significant positive association between subclinical hypothyroidism and high nitrate exposure (age- and bmi-adjusted or = . ; % ci: . - . ), with further analyses revealing the association to be present in women (or = . ; % ci: . - . ), but not in men (or = . ; % ci: . - . ). however, the association among women did not increase monotonically with increasing quartiles of esti- mated nitrate concentrations in their water supply expo- sure. the interaction for gender and nitrate was not significant (p-interaction = . ). no significant associations were observed for clinical hypothyroidism. the results were consistent when stratified by age and bmi. the results were unchanged in a sensitivity analysis that excluded participants whose residences were possi- bly connected to public water supplies (data not shown). the exclusion of persons who reside more than m from the nearest well also did not result in a material change in our results (data not shown), although it did decrease the odds ratio for high nitrate intake and sub- clinical hypothyroidism in women from . - . . we also estimated the or for subclinical hypothyroidism among women in the absence of exposure misclassifica- tion as . (versus . observed). discussion our results provide limited support for an association between nitrate levels in private wells and subclinical hypothyroidism among women but not men. with esti- mated exposure to nitrate in drinking water at or above . mg/l, we observed a significantly increased preva- lence of subclinical hypothyroidism in women, although there was not a monotonic increase with increasing quartiles of nitrate. these findings of an increased pre- valence of hypothyroidism among women are consistent with our hypothesis, namely that the competitive inhibi- tion of iodide uptake associated with increased nitrate exposure would result in decreased systemic active thyr- oid hormone (as indicated by increased tsh levels). we did not observe an association for clinical hypothyroid- ism, but the number of cases in this group was much lower. the mean tsh level in our study population was . μiu/ml in women and . μiu/ml in men. these levels are higher than tsh levels in the general us population surveyed by the national health and nutrition examina- tion survey (nhanes) from - [ ], in which the means among women and men were . μiu/ml and . μiu/ml, respectively. the prevalence of table characteristics of the study population by thyroid disease status normal subclinical hyperthyroidism clinical hyperthyroidism subclinical hyperthyroidism clinical hyperthyroidism total overall (n, %) ( . %) ( . %) . %) ( . %) ( . %) , male (%) . . . . . age in years (mean) . . . . . . bmi males kg/m (mean) . . . . . . bmi females kg/m (mean) . . . . . . ever smoker (%)* . . . . . . includes persons who reside in lancaster, chester, or lebanon counties and excludes persons younger than or who report use of thyroid medications. *smoking status is based on . % of the study population aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of hypothyroidism and hyperthyroidism in the us popula- tion is estimated to be . % ( . % clinical and . % sub- clinical) and . % ( . % clinical and . % subclinical), respectively [ ], compared with . % and . % in our study population. however, as the risk of thyroid disease increases with age, the higher prevalence in the amish could be partially due to the older age distribution in this study (mean = . years) population compared to the age distribution of the nhanes study population (mean = . years). when hypothyroidism is compared by sex in this study population and the nhanes popu- lation, the prevalence is . -times more common in amish women than men whereas it is - times more common in women than men in the us population [ ]. in previous epidemiological studies, investigators have identified a relationship between nitrate contamination of water supplies and thyroid dysfunction and thyroid disease. in a cross-sectional study of school children liv- ing in areas of slovakia with high and low nitrate expo- sure via drinking water, children in the high nitrate area had increased thyroid volume and increased frequency of signs of subclinical thyroid disorders (thyroid hypoe- chogenicity by ultrasound, increased tsh level and positive anti-thyroid peroxidase (tpo)) [ ]. the nitrate levels ranged from . to . mg/l (as nitrate-nitro- gen) in the highly polluted area and were < . mg/l nitrate-nitrogen in the low nitrate area. similarly, inves- tigators in the netherlands conducted a cross-sectional table odds ratios (ors) and % confidence intervals (cis) for the prevalence of hyperthyroidism associated with estimated nitrate levels in residential wells overall men women mg/l nitrate-nitrogen cases or %ci cases or %ci cases or %ci clinical hyperthyroidism low nitrate (< . ) . . . high nitrate (= > . ) . ( . - . ) . ( . - . ) . ( . - . ) subclinical hyperthyroidism low nitrate (< . ) . . . high nitrate (= > . ) . ( . - . ) . ( . - . ) . ( . - . ) subclinical hyperthyroidism q [ . - . ] . . . q [ . - . ] . ( . - . ) . ( . - . ) . ( . - . ) q [ . - . ] . ( . - . ) . ( . - . ) . ( . - . ) q [ . - . ] . ( . - . ) . ( . - . ) . ( . - . ) p-trend . . . models adjusted for age and bmi; model with men and women combined is also adjusted for gender table odds ratios (ors) and % confidence intervals (cis) for the prevalence of hypothyroidism associated with nitrate levels in residential wells overall men women mg/l nitrate-nitrogen cases or %ci cases or %ci cases or %ci clinical hypothyroidism low nitrate (< . ) . . . high nitrate (= > . ) . ( . - . ) . ( . - . ) . ( . - . ) subclinical hypothyroidism low nitrate (< . ) . . . high nitrate (= > . ) . ( . - . ) . ( . - . ) . ( . - . ) subclinical hypothyroidism q [ . - . ] . . . q [ . - . ] . ( . - . ) . ( . - . ) . ( . - . ) q [ . - . ] . ( . - . ) . ( . - . ) . ( . - . ) q [ . - . ] . ( . - . ) . ( . - . ) . ( . - . ) p-trend . . . models adjusted for age and bmi; model with men and women combined is also adjusted for gender; nitrate concentrations in mg/l aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of study of women who obtained their drinking water from public supplies and private wells with varying nitrate levels [ ]. they observed a dose-dependent increase in the volume of the thyroid associated with increasing nitrate concentrations in drinking water from a combi- nation of public and private supplies, with nitrate levels ranging from . mg/l to . mg/l (as nitrate-nitro- gen). women with nitrate levels exceeding . mg/l as nitrate-nitrogen had a significant increased prevalence of thyroid gland hypertrophy. our results for women are consistent with the findings in slovakia and indir- ectly support the associations observed in the nether- lands. however, the reason for our finding of in women but not men is unclear particularly since men consume more water than women on average [ ]. it is possible that women may be more sensitive to exposures that perturb the thyroid as indicated by their higher preva- lence of thyroid disease [ ]. a previous epidemiologic investigation of the associa- tion of nitrate intake from public water supplies and diet with the risk of self-reported hypothyroidism and hyperthyroidism was conducted in a cohort of , older women in iowa [ ]. the investigators found no association between the prevalence of hypo- or hyperthyroidism and nitrate concentrations in public water supplies; nor was there an association for those who were using private wells. however, intake of nitrate from the diet can be a primary source of exposure when drinking water nitrate levels are below the mcl of mg/l nitrate-n [ - ]. in the iowa study, increasing intake of nitrate from dietary sources was associated with an increased prevalence of hypothyroidism (or q = . ; % ci = . - . , p for trend = . ) while no association was observed with hyperthyroidism [ ]. in addition to consumption of tap water, people living in areas with high nitrate concentration in their water supplies may be exposed through their use of water for cooking, irrigation of crops used as a food source, and through milk products from local farm animals. nitrate is a natural component of plants and is found at high concentrations in leafy vegetables, such as lettuce and spinach, and some root vegetables, such as beets [ ]. the lack of dietary questionnaire data in our study is a limitation since estimates of well-water nitrate were below the mcl of mg/l for % of participants [ - ]. the lack of dietary information in general likely resulted in exposure misclassification in our study population. a strength of this study is the availability of valid mea- sures of tsh using study participant serum samples. although only one measure was available for each study participant, the use of tsh rather than self-reported thyroid disease is likely to more accurately define thyr- oid disease. although factors such as pregnancy and obesity can affect tsh, the levels are a reliable index of the biological activity of thyroid hormones. anti-tpo was not available, which can also be helpful in the diag- nosis of thyroid disease as an autoimmune disease. in addition to measuring tsh and anti-tpo in blood, future studies would be further strengthened by the use of ultrasound technology to determine thyroid volume, which could provide insight into nitrate exposure levels that may cause hypertrophy of the thyroid. an additional strength of our study was that we vali- dated our exposure metric and characterized the sensi- tivity and specificity based on the median observed versus predicted nitrate level in wells monitored by the usgs in our study area. specificity was high ( %) indi- cating that our model classified those with lower nitrate levels accurately. the lower sensitivity ( %) indicated that the model underestimated nitrate concentrations for those with higher levels. the result of this misclassi- fication, if nondifferential by disease status [ ], would be to attenuate ors as we demonstrated for subclinical thyroid disease. our study was limited by a lack of information about the study population’s complete residential history. however, we know that the majority of this amish cohort reside in rural areas, with low relocation rates, and that it is typically the women who relocate to live in the homes or on the same land as their husband’s family [ ]. most amish men would subsequently have a stable residential history and exposure to nitrate con- tamination of well water over time. it is not clear to what degree a complete residential history would have affected our findings for both men and women. it is possible that the association we observed between sub- clinical hypothyroidism in women was attenuated due to this source of misclassification. the well measurements were also not randomly selected but represent data col- lected by usgs and individuals that potentially reside in areas with higher levels of nitrate than those who did not receive monitoring attention from usgs or who were not aware of a problem in their well. we identified a large standard deviation for the wells with repeat mea- sures and were unable to fully explore the reasoning beyond having a small proportion ( %) of repeat sam- ples. additional data on well depth, other hydrogeologi- cal factors, or why multiple samples were taken could provide more insight into this observed variation, but was not available. our study is also limited by the fact that we did not have data on actual water supply source to the resi- dence, nor personal water consumption. because most residences were located outside of areas served by public water utilities, we assumed the drinking water supply for participants was a well located at their residence. we did not have data on tap water consumption, and thus aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of the approximate daily intake, which can be an important variable in determining exposure. most people in the united states drink about . - l of water per day [ ]. similarly, because of the attention given to water con- tamination in the lancaster area, it is possible that some study participants obtain their water from sources that have been purified via reverse osmosis or from bottled water. these limitations would clearly affect the expo- sure estimates and result in misclassification of the exposure. future work in this area would be enhanced by the assessment of multiple contaminants present in water sources and the general environment that could be simultaneously affecting thyroid health. multiple envir- onmental pollutants from industrial as well as agricul- tural activities may be an important consideration for future investigation. of particular interest is pesticides as there is increasing evidence of their ability to alter thyroid hormone homeostasis, causing thyroid dysfunc- tion and thyroid disease [ , ]. the varied effects of these chemicals on thyroid function could affect study findings. determination of these exposures should be a future study design consideration. furthermore, the effect of contamination from other univalent anions which interfere with the uptake of iodide by the thyroid should be considered in future investigation into the effects of nitrate in drinking water. for example, perchlorate, the oxidizer for solid rocket fuel and a component of some fertilizers, is found in both food and water [ ], and interferes with iodide uptake much like nitrate. similarly, thiocyanate, another univalent anion that causes thyroid dysfunction, is a metabolite from tobacco smoke and is found in certain foods [ - ]. conclusions the present study provides limited evidence that nitrate in residential well water is associated with subclinical hypothyroidism in women but not men. future studies that include validated biomarkers, as well as individual level nitrate exposure estimates of dietary and drinking water intakes, and an assessment of co-contaminants, are needed to provide information about the relevance of nitrate intake and thyroid disease. list of abbreviations tsh: thyroid stimulating hormone; no -: nitrate; epa: u.s. environmental protection agency; mcl: maximum contaminant level; nitrate-n: nitrate as nitrogen; usgs: united states geological survey; nlcd: national land cover data set; nhanes: national health and nutrition examination survey acknowledgements we gratefully acknowledge our amish liaisons and field workers and the extraordinary cooperation and support of the amish community, without whom these studies would not be possible. the study was supported by research grants from the american federation of aging research (f.s.c.), nih r dk (a.r.s.) the american diabetes association (a.r.s.), the baltimore veterans administration geriatric research and education clinical center (grecc), and the university of maryland general clinical research center, grant m rr , the general clinical research centers program, national center for research resources (ncrr), nih. this research was supported in part by the intramural research program of the nih/national cancer institute, including an interagency personnel agreement between nci and colorado state university. partial funding was also provided by the mid-atlantic nutrition and obesity research center (grant p dk ). briseis aschebrook-kilfoy was supported by nih training grant tu ca . author details occupational and environmental epidemiology branch, division of cancer epidemiology and genetics, national cancer institute, national institutes of health, department of health and human services, rockville, md, usa. yale school of public health, yale university, new haven, connecticut, usa. biostatistics branch, division of cancer epidemiology and genetics, national cancer institute, national institutes of health, department of health and human services, rockville, md, usa. department of environmental and radiological health sciences, colorado state university, fort collins, co, usa. endocrine service, department of medicine, memorial sloan-kettering cancer center, new york, ny, usa. division of endocrinology, diabetes and nutrition, department of medicine, university of maryland school of medicine, baltimore, md, usa. geriatrics research and education clinical center, veterans administration medical center, baltimore, md, usa. westat, rockville, md, usa. department of health studies, university of chicago, chicago, il, usa. authors’ contributions ba-k: participated in the conceptualization of the study and the exposure assessment design; conducted data analysis and drafted the manuscript; sh: conducted the nitrate modeling work and gave important intellectual input for the data analysis, and helped draft the manuscript; jn: conceptualized the exposure assessment approach, advised in the creation of the hydrogeological covariates and exposure modeling, and provided important intellectual content; ms: advised on the medical interpretation of the findings, and provided important intellectual content; as: as the pi of the cohort study, as designed the study, oversaw the recruitment of subjects and conducted much of the primary data gathering; bm: as a co-pi, bm participated in the design of the study and ongoing study efforts, the interpretation of the results for the nitrate investigation, and drafting of the manuscript; ma: obtained the hydrogelogical dataset, gis analysis, reviewed the manuscript, and revised it critically for important intellectual content; th: helped conceptualize the study, provided statistical support and modeling guidance, and provided important intellectual content for the manuscript; yz: helped conceptualize the study and provided important intellectual content for the manuscript; mw: mentored in the conceptualization of the study, participated in study design, method development and data analysis, helped in the drafting of the manuscript. all authors assisted in the interpretation of results and contributed towards the final version of the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. received: september accepted: february published: february references . dai g, levy o, carrasco n: cloning and characterization of the thyroid iodide transporter. nature , : - . . eskandari s, loo dd, dai g, levy o, wright em, carrasco n: thyroid na symporter. mechanism, stoichiometry, and specificity. the journal of biological chemistry , : - . . van sande j, massart c, beauwens r, schoutens a, costagliola s, dumont je, wolff j: anion selectivity by the sodium iodide symporter. endocrinology , : - . . tonacchera m, pinchera a, dimida a, ferrarini e, agretti p, vitti p, santini f, crump k, gibbs j: relative potencies and 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in workers exposed to perchlorate long-term. the journal of clinical endocrinology and metabolism , : - . . vogelmann je, howard sm, yang l, larson cr, wylie bk, van driel jn: completion of the ’s national land cover data set for the conterminous united states. photogrammetric engineering and remote sensing , : - . doi: . / - x- - cite this article as: aschebrook-kilfoy et al.: modeled nitrate levels in well water supplies and prevalence of abnormal thyroid conditions among the old order amish in pennsylvania. environmental health : . submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www.biomedcentral.com/submit aschebrook-kilfoy et al. environmental health , : http://www.ehjournal.net/content/ / / page of http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/ ?dopt=abstract 	abstract 	background 	methods 	results 	conclusions 	background 	methods 	study population 	historical assessment of nitrate levels 	prediction of nitrate levels in well water of participants’ residences 	model validation 	data analysis 	results 	discussion 	conclusions 	acknowledgements 	author details 	authors' contributions 	competing interests 	references original paper growth charts for children with ellis–van creveld syndrome sabine verbeek & paul h. c. eilers & kate lawrence & raoul c. m. hennekam & florens g. a. versteegh received: july /accepted: august /published online: september # the author(s) . this article is published with open access at springerlink.com abstract ellis–van creveld (evc) syndrome is a congenital malformation syndrome with marked growth retardation. in this study, specific growth charts for evc patients were derived to allow better follow-up of growth and earlier detection of growth patterns unusual for evc. with the use of observations of evc patients ( males, females), growth charts for males and females from to years of age were derived. longitudinal and cross-sectional data were collected from an earlier review of growth data in evc, a database of evc patients, and from recent literature. to model the growth charts, the gamlss package for the r statistical program was used. height of evc patients was compared to healthy children using dutch growth charts. data are presented both on a scale for age and on a scale for the square root of age. compared to healthy dutch children, mean height standard deviation score values for male and female evc patients were − . and − . , respectively. the present growth charts should be useful in the follow-up of evc patients. most importantly, early detection of growth hor- mone deficiency, known to occur in evc, will be facilitated. keywords growth . body height . ellis–van creveld syndrome . growth charts introduction ellis–van creveld syndrome (evc) (omim # ) is a skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly, and dysplastic nails and teeth. sixty percent of affected individuals have a congenital cardiac defect, most commonly an atrial septum defect [ , ]. the syndrome is named after the physicians ellis and van creveld, who described the first cases in [ ]. evc is uncommon, the incidence in the general population being per , , live births [ ]. in the old amish population, the incidence is much higher, with per , live births and as many as % of the amish people are carrier of the mutation [ ]. the disorder follows an autosomal recessive pattern of inheritance. mutations in two genes (evc and evc ) on chromosome p have been identified to be responsible for the evc phenotype [ ]. after birth, children with evc present with short limbs. the shortening of the limbs increases from proximal to distal, with the most pronounced shortening of the distal interphalangeal bones. a recent study showed that growth hormone deficiency can play a role in the retarded growth in at least some evc patients [ ]. the same study showed variation of the growth retardation from − . to − . standard deviation score (sds) of average height. the s. verbeek (*) : f. g. a. versteegh department of pediatrics, groene hart ziekenhuis, p.o. box , gouda, the netherlands e-mail: sabineverbeek@hotmail.com f. g. a. versteegh e-mail: florens.versteegh@ghz.nl p. h. c. eilers department of medical statistics, erasmus medical centre, rotterdam, the netherlands k. lawrence springburn health centre, glasgow, scotland, uk r. c. m. hennekam institute for child health, great ormond street hospital for children, ucl, london, uk r. c. m. hennekam department of pediatrics, academic medical center, amsterdam, the netherlands eur j pediatr ( ) : – doi . /s - - - authors argued that growth charts specifically for evc patients would be useful, as has been found for other entities that go along marked growth retardation, such as down syndrome, turner syndrome, and achondroplasia [ , , , ]. such dedicated growth charts allow better follow-up of growth and earlier detection of growth patterns unusual for the entity involved. this urged us to create growth charts specifically for children with evc. methods patients growth data of evc patients were available for analysis. data were gathered from different sources. for our previous study [ ], we gathered measurements of patients. from these studies, four patients were known to receive therapy for growth hormone deficiency and were therefore excluded. all cases from literature that were molecularly proven were added; patients of whom molec- ular results were not available were included if the diagnosis was thought to be right by an experienced clinical geneticist. one hundred and three measurements of patients from these studies were used for analysis. secondly, we used the database of an evc website (www.ellisvancreveld.co.uk). this database provided data points of subjects with evc. in all latter patients, the diagnosis was molecularly proven. there was no overlap between subjects of this database and subjects from our previous studies. thirdly, we added data from literature published after our study was concluded [ , , ]. this provided us eight measurements of six patients. in total, usable data were available of patients— data points of males and data points of females. statistical analysis to model the growth charts, the gamlss package for the r statistical program (r development core team, ) was used [ ]. gamlss models the conditional distribu- tion of height for a given age by means of three curves: one for the mean, one for the (logarithm of the) standard deviation, and one for a power transformation to normality. to find the best balance between complexity and fit to the data, transformation to normality was not performed, and linear trends (in the square root of age) were taken for the mean curve and the logarithm of the standard deviation. with this model, percentile curves were computed. to compare height of evc patients with height in healthy children, we presented the data in digital dutch growth curves [ ]. results a total of measurements on subjects were used for the analysis. of subjects, we gathered more than , and of subjects, we gathered more than measurements (table ). of the female subjects, were aged between and years and subjects were above years. we assumed height of these patients would be comparable with -year-old patients and therefore treated these data points as such. indeed, if leaving these data points outside the analysis, the growth curves did not change. of the male subjects, were aged between and years and were above years of age. again, we treated the data of these patients as if they were -year-old patients. as in the females, the growth curve did not change if these data points were left out of the analysis. data from the literature were published between and . ethnicity differed since data were gathered from all over the world. most of the patients (n= ) were of caucasian descent, were hispanic, patients had other ethnic backgrounds (african-american, jewish, turkish, chinese, mongolian, japanese, indian), and of patients from literature, the ethnicity was not known. consanguinity of parents in patients collected from the literature was mostly unknown to us. in the uk database, none of the parents was consanguineous. the growth charts were derived for males from to years of age (figs. , , and ) and females from to (figs. , , and ). transformation of age to a different scale, by taking square roots, led to almost linear curves (figs. and ). figures and show growth charts for boys and girls, respectively, with all the collected data points. figures and show the same growth charts without these data points, to allow use for patient care purposes. curves are made for the specific age group. the statistical program used here does not make it possible to extrapolate data points to adulthood. height sds values for evc patients are shown on dutch growth charts for boys (fig. ) and girls (fig. ) of – table patient characteristics age group (years) male data points (n)/ male patients in age group (n) female data points (n)/ female patients in age group (n) – / / – / / – / / – / / > / / total / / data points of male and female patients per age group eur j pediatr ( ) : – http://dx.doi.org/http://www.ellisvancreveld.co.uk years. sds values vary between + . and − . , with a mean of − . sd for male evc patients and − . sd for female evc patients [ ]. discussion syndrome-specific growth charts have been developed for several syndromes, such as down syndrome, turner syndrome, and williams syndrome [ , , ]. these charts have proven to be a helpful tool in the medical care of these children. growth in evc is known to be impaired, with an estimated deviation of − . to − . from standard growth [ ]. until recently, it was assumed to be explainable merely because of the skeletal dysplasia. however, growth hormone deficiency has been reported in patients with evc and its frequency remains uncertain [ ]. this may aggravate the growth retardation. to detect growth hor- mone deficiency and other disorders that cause additional growth deviation in evc patients, specific growth charts for square root of age (in years) h e ig h t (c m ) males . . age fig. growth chart for boys from to years of age with ellis–van creveld syndrome (n= ). curves presented as height (centimeters) versus square root of age (years) age [years] h e ig h t (c m ) males fig. growth chart for boys from to years of age with ellis–van creveld syndrome (n= ). curves presented as height (centimeters) versus age (years) square root of age (in years) h e ig h t (c m ) females . . age fig. growth chart for girls from to years of age with ellis–van creveld syndrome (n= ). curves presented as height (centimeters) versus square root of age (years) h e ig h t (c m ) females age [years] fig. growth chart for girls from to years of age with ellis–van creveld syndrome (n= ). curves presented as height (centimeters) versus age (years) eur j pediatr ( ) : – children with evc should be of great value. we present here growth charts for males and females with evc. since the data set was small, a simple model was aimed for. transformation of age to a different scale, by taking square roots, led to almost linear curves. furthermore, it spreads out the data for the younger age group, which makes it more clarifying. we treated the data as cross-sectional although a number of measurements come from the same individuals. there is no provision in the statistical program gamlss for grouped data. the alternative would be to select one measurement per individual or leave repeated measure- ments out completely. both choices were unattractive. because our analysis is mainly descriptive, we believe that little harm was done by using all data. to compare growth of evc patients to healthy children, data were shown in dutch growth charts. mean growth deviation was − . sd for male patients and − . sd for female patients, comparable to our previous study [ ]. a drawback of the present study is the possible influence of the secular trend in growth. data were gathered from h e ig h t (c m ) males age [years] fig. growth chart for boys from to years of age with ellis–van creveld syndrome age [years] h e ig h t (c m ) females fig. growth chart for girls from to years of age with ellis–van creveld syndrome age [years] h e ig h t (c m ) males fig. dutch growth charts for boys ( – years, blue line) with height sds of boys with ellis–van creveld syndrome ( – years, dotted line) age [years] h e ig h t (c m ) females fig. dutch growth charts for girls ( – years, blue line) with height sds of girls with ellis–van creveld syndrome ( – years, dotted line) eur j pediatr ( ) : – publications between and . nowadays, boys and girls are taller than in the s [ , ]. although the proportion of data from more than years ago is relatively small ( data points), the influence of the secular trend cannot be ruled out. another drawback is that data were gathered from all over the world. consequently, the here presented growth charts represent different cultures and ethnicities, which can differ greatly [ , , ]. we reasoned, however, that in evc the growth retardation would be that extreme that this would easily outweigh the ethnic growth differences and we therefore used all data. when compared to regular growth charts, the here presented growth charts differ in form. it seems that growth does not slow down to a complete horizontal curve after puberty. the reason for this is the statistical approach used in this study. since the data set was small, the formation of a splined curve was not possible. regular growth charts show an s-shaped or “splined” curve. these curves flatten in adolescence, leading to an end height or target height. the here presented growth charts are developed with the use of another statistical approach, used for smaller databases [ ]. this method does not allow extrapo- lation of the curve. hence, the here presented growth charts can only be used in a specific age group. more research with a larger database would be necessary to develop growth charts with a splined curve. open access this article is distributed under the terms of the creative commons attribution noncommercial license which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. references . abeles ai, tobias jd ( ) anaesthetic implications of ellis– van creveld syndrome. j clin anesth : – . de onis m, onyango aw, borghi e et al ( ) development of a who growth reference for school-aged children and adolescents. bull world health organ : – . digilio mc, marino b, ammirati a et al ( ) cardiac malforma- tions in patients with oral–facial–skeletal syndromes: clinical similarities with heterotaxia. am j med genet : – . ellis rw, van creveld sa ( ) syndrome characterized by ectodermal dysplasia, polydactyly, chondro-dysplasia and con- genital morbius cordis: report of three cases. arch dis child : – . gawlik a, gawlik t, augustyn m et al ( ) validation of growth charts for girls with turner syndrome. int j clin pract : – . gohlke b, woelfle j ( ) growth and puberty in german children: is there still a positive secular trend? dtsch arztebl int : – . growth analyser junior ( ) . ide se, ortiz ri, francomano ca, polymeropoulos mh ( ) exclusion of the msx homeobox gene as the gene for the ellis–van creveld syndrome in the amish. j human genet : – . lejarragaa h, del pinoa m, fanoa v et al ( ) growth references for weight and height for argentinian girls and boys from birth to maturity. incorporation of data from the world health organisation from birth to years and calculation of new percentiles and lms values. arch argent pediatr : – . martin nd, smith wr, cole tj, preece ma ( ) new height, weight and head circumference charts for british children with williams syndrome. arch dis child : – . ogden cl, kuczmarski rj, flegal km et al ( ) centers for disease control and prevention growth charts for the united states: improvements to the national center for health statistics version. pediatrics : – . polymeropoulos mh, ide se, wright m et al ( ) the gene for the ellis van creveld syndrome is located on p . genomics : – . rigby ra, stasinopoulos dm ( ) generalized additive models for location, scale and shape. appl statist : – . roelants m, hauspie r, hoppenbrouwers k ( ) references for growth and pubertal development from birth to years in flanders, belgium. ann hum biol : – . stoll c, dott b, roth mp, alembik y ( ) birth prevalence rates of skeletal dysplasias. clin genet : – . styles m, cole t, dennis j, preece m ( ) new cross sectional stature, weight, and head circumference references for down’s syndrome in the uk and republic of ireland. arch dis child : – . thapa r, mukhopadhyay m, bhattacharya a ( ) discordance for ellis–van creveld syndrome in twins. singapore med j : – . trotter tl, hall jg, committee on genetics ( ) health supervision for children with achondroplasia. pediatrics : – . versteegh fga, buma sa, costin g et al ( ) growth hormone analysis and treatment in ellis–van creveld syndrome. am j med genet a: – growth data of children with proven evc can be sent to evc@crydee.plus.com. eur j pediatr ( ) : – http://evc@crydee.plus.com 	growth charts for children with ellis–van creveld syndrome 	abstract 	introduction 	methods 	patients 	statistical analysis 	results 	discussion 	references << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma . ) /calrgbprofile (srgb iec - . ) /calcmykprofile (iso coated v % \ eci\ ) /srgbprofile (srgb iec - . ) /cannotembedfontpolicy /error /compatibilitylevel . /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false 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/pagesize [ . . ] >> setpagedevice pathogenic variants in kptn gene identified by clinical whole-genome sequencing pathogenic variants in kptn gene identified by clinical whole-genome sequencing isabelle thiffault, , , andrea atherton, bryce a. heese, ahmed t. abdelmoity, kailash pawar, emily farrow, , , lee zellmer, neil miller, sarah soden, , , and carol saunders , , center for pediatric genomic medicine, children’s mercy hospital, kansas city, missouri , usa; department of pathology and laboratory medicine, children’s mercy hospitals, kansas city, missouri , usa; university of missouri–kansas city school of medicine, kansas city, missouri , usa; department of pediatrics, children’s mercy hospitals, kansas city, missouri , usa abstract status epilepticus is not rare in critically ill intensive care unit patients, but its diag- nosis is often delayed or missed. the mortality for convulsive status epilepticus is depen- dent on the underlying aetiologies and the age of the patients and thus varies from study to study. in this context, effective molecular diagnosis in a pediatric patient with a geneti- cally heterogeneous phenotype is essential. homozygous or compound heterozygous var- iants in kptn have been recently associated with a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. we describe a comprehensive investigation of a -yr-old male patient who was admitted to the intensive care unit, with focal epilepsy, stat- ic encephalopathy, autism spectrum disorder, and macrocephaly of unknown etiology, who died of status epilepticus. clinical whole-genome sequencing revealed compound het- erozygous variants in the kptn gene. the first variant is a previously characterized -bp in-frame duplication (c. _ dup) in exon , resulting in the protein change p.met _gln dup. the second variant, c. + g > a, affects the splice junction of exon . these results are consistent with a diagnosis of autosomal recessive kptn-related disease. this is the fourth clinical report for kptn deficiency, providing further evidence of a wider range of severity. [supplemental material is available for this article.] introduction in recent years, next-generation sequencing (ngs) technologies have revolutionized ap- proaches in clinical genetics. whole-exome sequencing (wes) or whole-genome sequenc- ing (wgs) allows diagnoses in many patients with complex phenotypes and unusual clinical presentations. as the cost of ngs falls, it has become feasible to use this powerful technol- ogy in clinical care, simultaneously unraveling variations in about , genes. technological advances have led to the ability to sequence, analyze, and interpret entire ge- nome data in a timely manner, clearly changing the diagnostic paradigm and proving to be cost-effective in many cases (soden et al. ). clinical diagnostic sequencing currently fo- cuses on identifying causal mutations in the exome (∼ % of the genome), where most dis- ease-causing mutations are known to occur. wgs permits analysis of coding regions as well as regulatory elements that control gene expression; however, noncoding variants corresponding author: csaunders@cmh.edu © thiffault et al. this article is distributed under the terms of the creative commons attribution-noncommercial license, which permits reuse and redistribution, except for commercial purposes, provided that the original author and source are credited. ontology terms: complex febrile seizures; delayed fine motor development; delayed gross motor development; epileptic encephalopathy; intellectual disability, severe; macrocephaly at birth; polymorphic focal epileptiform discharges published by cold spring harbor laboratory press doi: . /mcs.a | research reportc o l d s p r i n g h a r b o rmolecular case studies cite this article as thiffault et al. cold spring harb mol case stud : a of cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from mailto:csaunders@cmh.edu http://www.molecularcasestudies.cshlp.org/site/misc/terms.xhtml http://creativecommons.org/licenses/by-nc/ . / http://creativecommons.org/licenses/by-nc/ . / http://creativecommons.org/licenses/by-nc/ . / http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com remained largely unexplored in clinical diagnostics because of the interpretive challenges (warman chardon et al. ). although it lacks the depth of coverage of an exome, wgs can have more reliable and uniform sequence coverage, particularly in regions of the genome with low sequence complexity or high gc-rich content. both wgs and wes ap- proaches have benefits and limitations, but the limitation of ngs gene panels and wes should be considered prior to clinical testing (williams et al. ; soden et al. ; ankala et al. ; warman chardon et al. ). in this context, the present work demon- strates the application of clinical wgs in the pediatric population in which we provided a fast, accurate, and cost-effective molecular diagnosis in a pediatric patient with a genetically het- erogeneous phenotype. results clinical presentation a -yr-old caucasian male was admitted to the children’s mercy hospital pediatric intensive care unit (picu) for status epilepticus and further evaluation of a suspected underlying genet- ic condition because of his history of macrocephaly, intractable epilepsy, autism, severe developmental delays, hypotonia, and hypoglycemia. dysmorphic features include frontal bossing, sunken eye sockets, downslanting palpebral fissures, small ears, thin upper lip, and small nose (fig. ). the prenatal history was unremarkable: delivery was at full term, and neonatal development was normal. he was the third child for his parents together, born to a -yr-old mother and -yr-old father. family history was noncontributory. at birth, he weighed . kilograms and was . cm long. his head circumference was noted to be ba c figure . our patient was a -yr-old male with epilepsy, static encephalopathy, autism spectrum disorder, and (a) macrocephaly of unknown etiology who died of status epilepticus. (b) several facial dysmorphisms were found, including frontal bossing, sunken eye sockets, downslanting palpebral fissures, small ears, thin upper lip, and small nose. (c) at yr old, he was normal height and weight, but his head circumference measured cm (> th percentile). kptn deficiency identified by wgs c o l d s p r i n g h a r b o r molecular case studies thiffault et al. cold spring harb mol case stud : a of cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com greater than the th percentile at birth. at yr old, he was of average height and weight, but his head circumference measured cm (> th percentile, + sd). he had poor tracking and eye contact but a normal ophthalmologic exam. a head ct was completed at mo of age and reported to be normal. at mo of age, he started to have partial seizures but eeg was normal. at mo of age he was noted to have hepatosplenomegaly accompanied by hypo- glycemic episodes. developmentally, he smiled socially at mo, rolled at mo, sat without assistance at – mo, crawled at mo, and walked at . yr. a brain mri was normal at mo of age; however a second, performed at yr of age, was concerning for a possible ar- teriovenous malformation, with abnormal hyperintense t /flair signals in the subcortical white matter of the right anteromedial temporal lobe. an extensive etiologic workup was normal, including high-resolution karyotype, cgh microarray, pten sequencing, fragile x, enzyme analysis for gaucher, mucopolysaccharidosis i and ii, plasma amino acids, pristanic and phytanic acids, very long chain fatty acid (vlcfa), and urine organic acids. mild eleva- tions of c species on an acylcarnitine profile in were detected in conjunction with ketotic hypoglycemia and were likely associated with physiologic response to fasting. previously, the seizures occurred at a frequency of – per hour, consisting of focal tonic- clonic seizures involving his left or right side of the body but occasionally progressed to status epilepticus, and a pentobarbital infusion was started with the goal to acquire burst suppression. unfortunately, this was unsuccessful, and he passed away at age . genetic analysis clinical wgs was performed on the patient and wes was performed on his healthy mother, following informed consent. the patient was compound heterozygous for two pathogenic variants in the kptn gene (table ). this gene was ranked st in the phenomizer gene list. the first variant identified was an -bp in-frame duplication (c. _ dup accgaccacatctgcaga; rs ) in exon , resulting in the protein change p.met _gln dup. this variant has been previously reported in trans with a second trun- cating variant in multiple affected individuals from two amish families (baple et al. ). prior in vitro transfection studies indicated the mutant protein is mislocalized and accumu- lates in neurons of affected individuals, leading to a dominant negative effect (baple et al. ). the p.met _gln dup variant is not found in population databases such as dbsnp, exome variant database, or exac but was found in of amish control chromo- somes (baple et al. ); / in gnomad data set ( . %). the second variant, c. + g > a, affects the splice junction of exon . this variant has not been reported in affected individuals but is predicted to cause aberrant splicing. this variant was observed in ∼ . % individuals of european ancestry in the nhlbi exome sequencing project and exome aggregation consortium (exac); / in gnomad data set ( . %). the c. + g > a variant, but not the c. _ dup variant, was inherited from the mother, suggesting these variants are in trans. this genotype was confirmed by sanger sequencing and is consistent with a diagnosis of autosomal recessive kptn-related disease. in addition, two variants of unknown significance were reported ((chd -nm_ . :c. g > c (p.gln his); ctdp -nm_ . :c. t > c (p.trp arg)). no incidental findings were reported for this case. the turnaround time was d. discussion the use of ngs techniques by clinical laboratories has risen tremendously and has greatly facilitated the elucidation of the etiologic diagnosis in patients suspected of having a genetic disease. the various approaches, including wes, wgs, and targeted panels, all have ben- efits and limitations. targeted panels offer a specific list of genes relevant to the clinical kptn deficiency identified by wgs c o l d s p r i n g h a r b o r molecular case studies thiffault et al. cold spring harb mol case stud : a of cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com t ab le . v ar ia n t ta b le g e n e c h ro m o so m e h g v s d n a re fe re n ce h g v s p ro te in re fe re n ce v ar ia n t ty p e p re d ic te d e ff e ct (s u b st it u ti o n , d e le ti o n , e tc .) d b s n p /d b v ar id g e n o ty p e c lin v ar id p ar e n t o f o ri g in c o m m e n ts k p t n : - c > t n m _ . :c . + g > a p .? lo f n /a rs h e t. m at e rn al ly in h e ri te d g n o m a d s w e d is h , ( / ), . % k p t n : - → a c c g a c c a c a t c t g c a g a n m _ . :c . _ d u p t c t g c a g a t g t g g p .l e u _v al d u p in -f ra m e b lo su m : rs h e t. p at e rn al ly in h e ri te d g n o m a d s w e d is h , ( / ), . % c h d : - g > c n m _ . :c . g > c p .g ln h is m is se n se s if t : to le ra te d _l o w _c o n fid e n ce ( . ) p o ly p h e n - : p o ss ib ly _ d am ag in g ( . ) rs h e t. c lin v ar m at e rn al ly in h e ri te d g n o m a d a sh ke n az i je w is h , ( / ), . % c t d p : - t > c n m _ . :c . t > c p .t rp a rg m is se n se s if t : to le ra te d ( . ) p o ly p h e n - : b e n ig n ( . ) rs h e t. n /a m at e rn al ly in h e ri te d g n o m a d o th e r, ( / ), . % cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com phenotype in question and often boast higher coverage than wes/wgs. however, whether this improves sensitivity, particularly in mendelian disorders, is not clear. in addition, a wide range of interlaboratory variability exists for gene lists offered for the same condition. for in- stance, commercial clinical ngs panels for macrocephaly include anywhere from to genes. although targeted panels may appear to be a more economical approach to wes/ wgs, this is only the case if the gene relevant to the patient being tested is present on the panel, which is often difficult to know a priori, particularly in patients with nonspecific symptoms such as intellectual disability or seizures. in many cases, serial testing of additional genes and panels quickly surpasses the expense of wes/wgs (soden et al. ). in addi- tion, targeted panels may fail to incorporate newly discovered disease-associated genes. for example, new genes associated with both syndromic and nonsyndromic macrocephaly have been identified, further expanding the genetic heterogeneity to more than conditions, including metabolic disorders, associated with macrocephaly (williams et al. ; keppler-noreuil et al. ; koh et al. ; marchese et al. ; sugathan et al. ; nevado et al. ; nguyen et al. ). one such gene, kptn, encoding kaptin, was first reported in january of in patients with a syndrome typified by macrocephaly, neurode- velopmental delay, and seizures (mrt ; omim# ) (baple et al. ; pajusalu et al. ). kptn is not currently offered in any clinical panels for macrocephaly in the united states. of the clinical laboratories listed in the nextgxdx website, only two of offer kptn as part of an autism/intellectual disability panel, and only two of include it as part of an epilepsy panel. the comparison of such gene lists is difficult for clinicians and the curation is onerous for the clinical laboratory to manage. clinical wes/wgs removes the guesswork as far as which gene to include, as all genes relevant to the patient’s phenotype are queried in the analysis process. in the current case, by using clinical wgs in a -yr-old caucasian male admitted in pediatric intensive care unit for status epilepticus, a diagnosis of kptn-related disease was made. a comparison of the individual clinical signs in patients reported with kptn-related dis- ease are shown in table . the most unifying features are macrocephaly ( %), developmen- tal delay ( %), and intellectual disability ( %). recurrent dysmorphic features included frontal bossing, abnormal head shape, and prominent chin. with the exception of the p.met _gln dup, which is predicted to be nonfunctional by producing a misfolded al- tered protein, all patients have truncating variants (p.s ∗; p.s qfs∗ ; p.s ifs∗ ); no patients were homozygous for the p.met _gln dup. rt-pcr or western blot experi- ments were not performed to assess if those variants result in nonsense-mediated mrna de- cay or whether a truncated protein (lacking the carboxy-terminal amino acids) is produced. the second variant uncovered in our patient, c. + g > a, is located upstream of the two previously reported truncating variants. this is the first splicing variant reported in kptn, it is likely to result in nonsense-mediated mrna decay, which may explain the more severe outcome. additional external factors such as modifier genes could also influ- ence the phenotype. of the previous patients described, seven have survived into their second and third decades, with two having expired at ages and to a head injury and pneumonia (table ; baple et al. ; pajusalu et al. ; lucena et al. ). at this time, limited data exist for genotype–phenotype correlations; further phenotyping and natural his- tory of disease study are needed to understand the clinical spectrum and prognosis of kptn- related syndrome. this is the fourth clinical report for kptn-deficiency, the th patient but the first to die in childhood with status epilepticus, providing further evidence of a wider range of severity. several studies have shed new light on the molecular and cellular processes that orchestrate the human neuronal circuitry and is defective in neurological disorders (ropers et al. ; riviere et al. ; baple et al. ; koh et al. ; sugathan et al. ). kptn deficiency identified by wgs c o l d s p r i n g h a r b o r molecular case studies thiffault et al. cold spring harb mol case stud : a of cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com t ab le . a co m p ar is o n o f cl in ic al fin d in g s o f af fe ct e d in d iv id u al s re p o rt e d w it h p at h o g e n ic va ri an ts in th e k p t n g e n e o ri g in a m is h , o h io a m is h , o h io e st o n ia m ix e d , k an sa s∗ g e n o ty p e h o m o zy g o u s p .s ∗ p .s ∗ /p .m q d u p h o m o zy g o u s p .s q fs ∗ c. + g > a /p .m q d u p n = p at ie n ts # % g e n d e r m m m m m f f m f m f m a g e at e va lu at io n (y e ar s) . . . . . . . a g e at d e at h (y e ar s) n .a (h e ad in ju ry ) (p n e u m o n ia ) n .a n .a n .a n .a n .a n .a n .a n .a g ro w th p ar am e te rs b ir th w e ig h t, kg (s d ) . ( . ) . ( ) . (+ . ) . (− . ) . (− . ) . (+ . ) . (− . ) . (+ . ) . (+ . ) ? ? . b ir th o f c , cm (s d ) o r b ir th m ac ro ce p h al y re p o rt e d ? . (+ . ) . (+ . ) ? (+ . ) ? ? m ac ro ce p h al y m ac ro ce p h al y (n ) (n ) m ac ro ce p h al y . h e ig h t, cm (s d ) at e va lu at io n . (− . ) ? . (− . ) (− . ) . (+ . ) . (− . ) . (− . ) ? . (− . ) n n . ( th ) w e ig h t, kg (s d ) . (+ . ) ? . (− . ) . (+ . ) . (+ . ) . (+ . ) (+ . ) ? ? n n . ( th ) o f c , cm (s d ) (+ . ) ? . (+ . ) . (+ . ) (+ . ) (+ . ) (+ . ) . (+ . ) . (+ . ) (+ . ) (+ . ) . ( th ) . p ar e n ta l o f c , cm (s d ) ? m o th e r . (+ . ); f at h e r (+ . ) ? m o th e r . (+ . ); f at h e r (+ . ) ? m o th e r . ( . ) ? m o th e r (+ . ); f at h e r . (+ . ) m o th e r (+ . ); f at h e r . (+ . ) ? ? n d e ve lo p m e n t w al ke d (y e ar s) . . . . > . . n .a n .a . e xp re ss iv e an d re ce p ti ve ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . la n g u ag e d e fic it ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ n o n ve rb al . in te lle ct u al d is ab ili ty ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . h e ar in g n n n n n n n n n n n n . n e u ro lo g y c h ild h o o d h yp o to n ia – – – – – – ▪ ▪ ▪ ▪ ▪ ▪ ? ? ▪ . s e iz u re s (o n se t) a s / g t c s ( m o ) a s /g t c s ( yr ) g t c s ( yr ) g t c s ( m o ) g t c ( m o ) . n e u ro im ag in g n ? v e n tr ic u lo m e g al y ? ? ? ? w id e n in g o f m e to p ic su tu re n n n a b n o rm al ∗∗ . b e h av io ra l ch ar ac te ri st ic s r e p e ti ti ve sp e e ch ▪ – – – – – – ▪ ▪ ▪ – – – – ▪ – – ▪ . s te re o ty p ie s – – – – ▪ ▪ ▪ ▪ ▪ – – – – ▪ – – ▪ . h yp e ra ct iv it y – – – – – – ▪ – – – – – – – – – – ▪ – – ▪ . a u ti st ic fe at u re s ? ? ? ? ? ? ? ? ? ▪ ▪ ▪ . a n xi e ty ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . (c o n ti n u e d o n n e xt p ag e .) cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com t ab le . (c o n ti n u e d ) o ri g in a m is h , o h io a m is h , o h io e st o n ia m ix e d , k an sa s∗ g e n o ty p e h o m o zy g o u s p .s ∗ p .s ∗ /p .m q d u p h o m o zy g o u s p .s q fs ∗ c. + g > a /p .m q d u p n = p at ie n ts # % – – – – p h o b ia – – – – – – – – – – – – – – ▪ – – ? ? – – . p h ys ic al an o m al ie s f ro n ta lb o ss in g ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . h ig h p al at e ? ? ? ? ? ? ? ? ? ▪ ▪ – – . h yp e rt e lo ri sm – – – – – – ▪ – – – – – – ▪ – – – – – – – – . p la g io ce p h al y – – – – – – ▪ ▪ – – ▪ ▪ – – – – – – – – . p ro m in e n t ch in ▪ ▪ ▪ ▪ – – – – – – – – ▪ ▪ ▪ ▪ . h e p at o sp le n o m e g al y – – – – – – – – – – – – ▪ – – – – – – – – ▪ . s p le n o m e g al y – – – – – – – – – – – – – – ▪ – – – – – – – – . li ve r ci rr h o si s – – – – – – – – – – – – ▪ – – – – – – – – – – . a n e m ia – – – – – – – – – – – – – – ▪ – – – – – – – – . r e cu rr e n t in fe ct io n s – – – – ▪ – – – – – – ▪ ▪ – – ? ? ▪ . th fin g e r cl in o d ac ti ly ▪ ▪ ▪ – – – – – – – – – – – – – – – – – – . f e ta lf in g e r p ad – – – – – – – – – – – – – – – – ▪ – – – – – – . r e fe re n ce b ap le e t al . b ap le e t al . b ap le e t al . b ap le e t al . b ap le e t al . b ap le e t al . b ap le e t al . b ap le e t al . b ap le e t al . p aj u sa lu e t al . p aj u sa lu e t al . n .a . (f ) f e m al e , (m ) m al e , (o f c ) o cc ip it o fr o n ta l ci rc u m fe re n ce , (s d ) st an d ar d d e vi at io n sc o re , (n /p ) n o t p e rf o rm e d , (▪ ) in d ic at e s p re se n ce o f th e cl in ic al fe at u re s in an af fe ct e d in d iv id u al , (– ) in d ic at e s ab se n ce o f th e cl in ic al fe at u re s in an af fe ct e d in d iv id u al , (? ) in d ic at e s th e p re se n ce o r ab se n ce o f cl in ic al fe at u re s in an af fe ct e d in d iv id u al is u n kn o w n , (a s ) ab se n ce o f se iz u re s, (g t c ) g e n e ra liz e d to n ic -c lo n ic se iz u re s, (n ) n o rm al , ( n .a .) n o t ap p lic ab le . cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com evidence implicating defects in proteins involved in actin dynamics, microtubule homeosta- sis, and vesicle trafficking in developmental disorders via effects on neuronal development and migration and brain architecture has accumulated over the past decade. the role of microtubule homeostasis (lis and dcx) or tubulin (tuba a, tuba , tubb a, tubb a, tubb b, tubb , and tubb and tbce) genes has been established in several brain disorders. more recently, the actin-encoding genes actb and actg have been shown to be involved in brain malformations causing baraitser–winter syndrome (omim # ) (riviere et al. ). additional intellectual disability genes involved in actin dy- namics and vesicle trafficking include stxbp and syp (barcia et al. ; kato ). patients with kptn deficiency consistently exhibit macrocephaly, intellectual disability, and developmental delay. furthermore, kptn is a novel actin binding protein, enriched in neuronal growth cones and cortical sites of neurons at early developmental stages, likely playing a central role in modulating neuron morphology and growth. although genes af- fecting many of these pathways are associated with hearing loss, to date, no patients with ktpn-related disease, including our patient, have been reported with deafness. as with several other published studies (riviere et al. ; soden et al. ; ankala et al. ; warman chardon et al. ; willig et al. ), wgs was found to be a rapid and cost-efficient approach for molecular diagnostic of a genetically heterogeneous condi- tion (thiffault et al. ). thus, this report confirms the occurrence of kptn-related syn- drome outside of the amish population and demonstrates the variability in the phenotypic spectrum and severity. pathological mechanisms of abnormal neuronal actin cytoskeleton and discrepancy between the underlying phenotypes caused by kptn defi- ciency remain to be elucidated. methods genomic dna was extracted from peripheral blood mononuclear cells using a chemagen msm robot (perkinelmer). wgs was prepared using the kapa hyper library prep omitting pcr. sequencing was completed on an illumina hiseq instrument utilizing paired end × base pair reads with v chemistry (illumina). the proband’s sample was sequenced to a depth of . gb for a mean coverage of ∼ × (supplemental table s ). bidirectional sequence was assembled, aligned to reference gene sequences based on human genome build grch /ucsc hg , and analyzed using custom-developed software, runes, and viking (saunders et al. ; soden et al. ; willig et al. ). variants were filtered to a % minor allele frequency and then prioritized by the american college of medical genetics (acmg) categorization (richards et al. ), omim identity, and phenotypic as- sessment. alignments were viewed using integrative genomic viewer software version . . (igv; broad institute). a candidate gene list was generated by phenomizer using human phenotype ontology (kohler et al. ) terms: macrocephaly (hpo: ), muscular hypotonia (hpo: ), autism (hpo: ), seizures (hpo: ), global developmental delay (hpo: ), and abnormal facial shape (hpo: ), with a cutoff at p-value of . . this gene list contained approximately genes and was im- ported into viking to guide the analysis; however, a separate analysis, not limited by this gene list, was performed in parallel to look for relevant pathogenic genotypes in genes not included in the hpo gene list. pathogenic, likely pathogenic, and variants of unknown significance in hpo genes were reported; likely benign and benign variants are not reported but are made available upon request. incidental findings in the genes recommended by acmg were requested by the family and analyzed for pathogenic and likely pathogenic variants only. kptn deficiency identified by wgs c o l d s p r i n g h a r b o r molecular case studies thiffault et al. cold spring harb mol case stud : a of cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://www.molecularcasestudies.org/lookup/suppl/doi: . /mcs.a /-/dc http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com additional information data deposition and access our patient consent does not permit patient sequence data to be uploaded to a data repos- itory. the variants reported have been deposited in the clinvar database (https://www.ncbi .nlm.nih.gov/clinvar/) and can be found under accession numbers vcv . , vcv . , and vcv . . ethics statement written informed consent was obtained from the patient’s legal guardians for publication of this case report and governed by the institutional review board of children’s mercy hospitals and clinics. a copy of the written consent is available for review by the editor- in-chief of this journal. acknowledgments we are very grateful to this family for allowing this case to be published. we thank our col- leagues in the center for pediatric genomic medicine and children’s mercy hospital in kansas city. this work was not supported by a grant. author contributions c.j.s., e.f., i.t., l.z., and s.s. conceived and designed the experiments; c.j.s., e.f., and i.t. performed the experiments; n.m. and e.f. contributed reagents/materials/analysis tools; i.t. and c.j.s. wrote the paper; e.f., s.s., l.z., b.h., a.a., and n.m. reviewed the manuscript; k.p. reviewed the manuscript and contributed to the clinical investigation of the patient; and a.a., b.h., and a.t.a. contributed to the recruitment and clinical investigations of the patient for the study. references ankala a, da silva c, gualandi f, ferlini a, bean lj, collins c, tanner ak, hegde mr. . a comprehensive genomic approach for neuromuscular diseases gives a high diagnostic yield. ann neurol : – . doi: . /ana. baple el, maroofian r, chioza ba, izadi m, cross he, al-turki s, barwick k, 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studies thiffault et al. cold spring harb mol case stud : a of cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com . /mcs.a access the most recent version at doi: a originally published online may , : , cold spring harb mol case stud isabelle thiffault, andrea atherton, bryce a. heese, et al. whole-genome sequencing gene identified by clinicalkptnpathogenic variants in material supplementary c http://molecularcasestudies.cshlp.org/content/suppl/ / / /mcs.a .d references http://molecularcasestudies.cshlp.org/content/ / /a .full.html#ref-list- this article cites articles, of which can be accessed free at: license for commercial purposes, provided that the original author and source are credited. attribution-noncommercial license, which permits reuse and redistribution, except this article is distributed under the terms of the creative commons service email alerting click here.top right corner of the article or receive free email alerts when new articles cite this article - sign up in the box at the © thiffault et al.; published by cold spring harbor laboratory press cold spring harbor laboratory press on april , - published by molecularcasestudies.cshlp.orgdownloaded from http://molecularcasestudies.cshlp.org/lookup/doi/ . /mcs.a http://molecularcasestudies.cshlp.org/content/suppl/ / / /mcs.a .dc http://molecularcasestudies.cshlp.org/content/suppl/ / / /mcs.a .dc http://molecularcasestudies.cshlp.org/content/ / /a .full.html#ref-list- http://molecularcasestudies.cshlp.org/cgi/alerts/ctalert?alerttype=citedby&addalert=cited_by&savealert=no&cited_by_criteria_resid=protocols; . /mcs.a &return_type=article&return_url=http://molecularcasestudies.cshlp.org/content/ . /mcs.a .full.pdf http://molecularcasestudies.cshlp.org/ http://www.cshlpress.com linkage and association mapping of a chromosome q -q type diabetes susceptibility locus in northern european caucasians swapan kumar das, sandra j. hasstedt, zhengxian zhang, and steven c. elbein , we have identified a region on chromosome q -q that was significantly linked to type diabetes in mul- tiplex families of northern european ancestry and also in pima indians, amish families, and families from france and england. we sought to narrow and map this locus using a combination of linkage and association approaches by typing microsatellite markers at . and . cm densities, respectively, over a region of cm ( . mb). we tested linkage by parametric and non- parametric approaches and association using both case- control and family-based methods. in the multiplex families that provided the previous evidence for link- age, the highest parametric, recessive logarithm of odds (lod) score was . at marker d s ( . cm, . mb) without heterogeneity. nonparametric link- age (npl) statistics (p � . ), simwalk statistic a (p � . ), and sib-pair analyses (maximum likeli- hood score � . ) all mapped to the same location. the one lod ci was narrowed to . – . mb. under recessive, two-point linkage analysis, adjacent markers d s ( . cm, . mb) and d s ( cm, . mb) showed lod scores > . . nonparametric analyses revealed a second linkage peak at cm near marker d s ( . mb, npl score . , p � . ), which was also supported by case-control (marker d s , cm, . mb; p � . ) and family-based (marker ata a , cm, . mb; p � . ) association studies. we propose that the repli- cated linkage findings actually encompass at least two closely spaced regions, with a second susceptibility region located telomeric at . – . mb. diabetes : – , t ype diabetes (mim ) likely encompasses a diverse set of diseases marked by elevated levels of plasma glucose. among caucasian pop- ulations, individuals with type diabetes, indi- viduals with the intermediate phenotype of impaired glucose tolerance, and likely individuals at risk of diabetes are all characterized by variable degrees of both decreased insulin action, particularly resistance to insulin-mediated muscle glucose uptake, and impaired insulin secretion in response to that decreased insulin action ( ). defects of both insulin action and insulin secretion among individu- als with normal glucose tolerance predict later onset of diabetes ( ). despite the diverse phenotypic nature of type diabetes, monozygotic and dizygotic twin studies, family studies, and marked differences in disease prevalence across populations all provide convincing evidence for an important role of genetic susceptibility loci in type diabetes pathogenesis ( ). based on epidemiological data, the total sibling relative risk (�s) has been estimated at – ( ), although the number of loci that contribute to this risk is unclear. based on these data supporting type diabetes suscep- tibility genes, genome scans for both type diabetes and type diabetes–related traits have been undertaken by multiple laboratories in caucasian, pima indian, african- american, and asian populations ( , , ), among others. these scans have identified possible susceptibility loci throughout the genome, but to date only the niddm locus on chromosome q in mexican-american subjects has been mapped to a single gene, the calpain gene ( ). calpain plays a small role in most other populations, however, and has been inconsistently replicated by link- age and association. other regions with evidence for replication include chromosome q ( – ) and chromo- some ( – ). a region on chromosome q -q was identified independently among pima indian sib-pairs dis- cordant for type diabetes or pima indian sib-pairs with onset of diabetes before age years ( ) and in studies from our laboratory of multiplex kindreds of northern european ancestry ascertained in utah ( ). subsequent studies in french families ( ), english sib-pairs ( ), and amish families ( ) and in preliminary studies of chinese sib-pairs ( ) have identified linkage of type diabetes to this same region, very near the original pima and utah linkage peaks. furthermore, this region was linked to hba c in the framingham offspring study ( ), to meta- from the department of medicine, university of arkansas for medical sciences, little rock, arkansas; the department of human genetics, univer- sity of utah health sciences center, salt lake city, utah; and the department of medicine, central arkansas veterans healthcare system, little rock, arkansas. address correspondence and reprint requests to steven c. elbein, md, professor of medicine, university of arkansas for medical sciences, endocri- nology j- /lr, john l. mcclellan memorial veterans hospital, w. th st., little rock, ar . e-mail: elbeinstevenc@uams.edu. received for publication august and accepted in revised form november . additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. hlod, heterogeneity lod; idb, identical by descent; lod, logarithm of odds; mls, maximum likelihood score; npl, nonparametric linkage; pklr, liver- and red cell–type pyruvate kinase; rorc, retinoid-related orphan receptor �; snp, single nucleotide polymorphism; tdt, transmission disequi- librium test. © by the american diabetes association. diabetes, vol. , february bolic syndrome traits in nuclear families from hong kong ( ), and to the possibly related phenotype of familial combined hyperlipidemia ( , ). given the difficulty in replicating linkage in complex diseases, the finding of diabetes and related traits in at least studies from diverse populations is striking. however, the exact map location of the linkage peaks, the specific trait or disease definition for the study, and the subgroup providing the evidence for linkage differs among studies. in previous studies from our laboratory ( ), the most significant linkage peak (logarithm of odds [lod] � . ) was found using pedigrees trimmed to fit into the gene- hunter program ( ) under a partially penetrant recessive parametric model. the linkage peak was quite broad, with a lod ci that extended from between d s and crp to d s , or � cm. a similar location, albeit with lower significance, was identified with both sib-pair analysis (mapmaker/sibs) and nonparametric linkage (npl). stud- ies in pima indians and in french families placed the linkage peak within cm of our data, although initial amish and english studies placed the peak centromeric or telomeric, respectively. in post hoc analyses from our laboratory, the lod score was reduced when full families were used for fewer markers, when unaffected individuals were removed from the analysis, and when individuals with intermediate diagnoses were removed. in contrast, removal of two families that segregated hepatocyte nu- clear factor � variants increased the lod score to . in the remaining families ( , ). finally, we found no linkage to chromosome in either smaller replication families or when all families were analyzed together without heterogeneity ( ). the goal of the present study was to localize the well-replicated type diabetes suscep- tibility gene in this region using a dense microsatellite map across a -cm region for linkage, case-control association studies, and family-based association studies. research design and methods we performed a number of analyses using both family-based and case-control studies to narrow the regions of susceptibility genes on chromosome q. for linkage analyses, we first attempted to replicate the earlier analyses showing linkage under a recessive model ( ), but using a dense marker map. although software is now available that permits multipoint analysis of full families, we included recessive analysis using genehunter v. . and genehunter-sized families to be comparable with our earlier analysis. while our highest linkage peak was under a recessive model, based on the variable location of the linkage peak from other laboratories and unpublished data from our labora- tory suggesting associations in multiple locations, we considered the possi- bility that multiple susceptibility loci might be present and that these loci might have different modes of inheritance. to test this hypothesis, we included two nonparametric (model independent) analyses, one using the statistics implemented in simwalk ( ) and the other using the sib-pair analysis that provided the highest nonparametric score in our previous study ( ). by using multiple analytical methods, we were also able to assess whether the localization of the linkage peaks was robust to model assump- tions. finally, based on the success of microsatellite association studies in mapping other complex disease genes ( , ), we included both case-control and family-based studies of a dense microsatellite map as a framework for mapping genes by association. we included two closely related study populations. both linkage and family-based association studies were conducted in samples from previously described families ( ). briefly, the primary studies were conducted on members of families ( nonfounders). the mean number of individuals tested was . per family, and the mean number of affected individuals per family was . , with a mean age of onset of . years. an additional smaller families (mean number of individuals tested: . ), which included six families that were not previously typed, were used as a replication set and were typed for all markers in the present study. the replication families were ascertained under the same criteria as the initial families, but the families were smaller and had fewer available members for testing. all families were ascertained for at least two siblings with type diabetes diagnosed before the age of years and with no more than one parent known to have type diabetes. all subjects were ascertained in utah for northern european ancestry. all available parents and siblings of the index sib-pair, as well as all available offspring of diabetic siblings, were studied. all nondiabetic individuals underwent a -g oral glucose tolerance test. subjects were classified as affected if they had a previous diagnosis of type diabetes and were on medical therapy. to incorporate young-onset impaired glucose tolerance into the affection status, individuals were considered affected if the fasting glucose exceeded . mmol/l or if the -h postchallenge glucose was � . mmol/l for participants under age years, . mmol/l for participants aged – years, or . mmol/l for those over age years. all other individuals with abnormal glucose tolerance tests were considered to be of unknown affection status. this scheme closely follows the world health organization criteria for impaired glucose tolerance (under age years) and type diabetes (age – years) but raises the postchallenge glucose for elderly subjects based on epidemiological data. all diagnoses were the same as in our previous study ( ). uncertainty was programmed into parametric models for individuals considered affected but who did not meet the criteria for type diabetes. case-control association studies were conducted on unrelated individ- uals with known type diabetes and ethnically matched, unrelated control individuals. of the type diabetic individuals, were selected from the linkage families and additional individuals were selected from the same population for type diabetes and a family history of type diabetes in a first-degree relative. control individuals included spouses from linkage fami- lies who had normal glucose tolerance tests ( subjects) and caucasian individuals ascertained in utah or arkansas ( subjects) who had normal glucose levels or glucose tolerance tests and no family history of diabetes in a sibling, parent, or grandparent. all individuals provided written informed consent under protocols ap- proved by the university of utah institutional review board (diabetic kindreds and case-control population) or the university of arkansas for medical sciences institutional review board (additional case-control sam- ples). marker selection and typing. for linkage studies of chromosome , we added microsatellite markers to the markers previously typed ( ), with new markers in the region between d s and d s , where previous linkage signals were found. marker order and spacing was derived from published maps ( , ) with reference to the physical map to establish the order and distance for closely spaced markers (national center for biotech- nology information [ncbi] build ). the average marker distance between d s and d s was . cm. for the population-based case-control association study, we typed microsatellite markers between markers d s and d s , with an average inter-marker distance of . mb. microsatellite markers were amplified in the presence of universal m forward primers that were labeled with li-cor ir and ir dyes, and the products were separated and detected on li-cor sequencers using standard methods (li-cor, lincoln, ne). genotypes were scored automati- cally using either sagagt software ( ) (li-cor) or semiautomatically using geneimage ir . software (scanalytics, fairfax, va). all readings were reviewed independently, and between and blinded duplicate samples were included for all markers for both linkage and association studies. all gels included at least two additional samples from selected grandparents of ceph (centre d’etude du polymorphisme humain) families as an additional quality control. before linkage analysis, all data were checked for inconsistencies in size, inconsistencies between duplicates, and inconsistencies in mendelian inheritance using the pedcheck program (v. . ) ( ). all blinded duplicates were in agreement with the exception of four samples that were consistently incorrect and appeared to be incorrectly identified duplicate samples. we identified . % genotyping errors ( of , genotypes that were auto- matically read without reference to pedigree data) that resulted in noninher- itance and were changed to unknown before analysis. linkage analysis. the marker map used for all multipoint studies was derived from primary reference to the marshfield map (http://research.marsh- fieldclinic.org/genetics), which included all of the typed markers. to properly space markers that were too close to be resolved on the marshfield linkage map, we set the distance between markers with recombination fractions to . cm, with marker order based on the physical map. consequently, our map over the region from d s to d s was expanded by cm from the marshfield map and by cm from the recently published decode map ( ). thus, exact locations used in the current study differ slightly from those cited in the most recent marshfield map. despite careful quality control and retyping of markers with excess recombination events, recombination between closely linked markers ex- s.k. das and associates diabetes, vol. , february ceeded expectations for many intervals. inspection of genotypes failed to identify errors leading to increased recombination. consequently, before multipoint analysis we used a mistyping analysis implemented in simwalk (v. . ) ( ) to remove all genotypes that had a % or greater posterior probability of error based on excess recombination. these genotypes were considered missing for all multipoint analyses. we removed a total of of , genotypes for all families ( . %). expected and observed recombi- nation rates for each interval are shown in the online supplemental data (table ). we conducted multipoint linkage analysis under a recessive parametric model that provided the maximum lod score in our previous studies using genehunter version . _r beta ( , ) and families trimmed to fit this program. nonparametric analyses were performed using statistics a through e in simwalk (v. . ) ( ). additionally, based on previous results showing the highest lod score under a sib-pair analysis, we performed sib-pair linkage analysis using genehunter (v. . _r ) under models of dominance variance and no dominance variance ( ). the recessive parametric model set the disease allele frequency at . and included a linear, age-dependent pen- etrance function that varied from . below age years to . over age years ( ). the allele frequency of each microsatellite marker used for linkage analysis was estimated from unrelated pedigree members, assuming hardy- weinberg equilibrium. linkage studies were conducted on the full -family set (original families and replication families) and on the families that provided the maximum evidence for linkage in our previous study. these families were selected from the families of the previous study but excluded two families that segregated hepatocyte nuclear factor � variants ( ). to fit the large families into genehunter plus, individuals who were unaffected or of unknown affection status were trimmed before analysis as described previ- ously ( ). the location score was also calculated in simwalk using full families. parametric recessive lod scores were calculated assuming homo- geneity (� � ) and allowing for heterogeneity. the maximum likelihood estimate of alleles shared identical by descent (ibd) among sib-pairs from the kindreds that were primarily responsible for earlier linkage findings was calculated both with and without weighting to correct for multiple sib-pairs and both with and without dominance variance (�s � �o). because of the increased recombination observed in this study despite elimination of clear genotyping errors and to minimize the impact of map errors, particularly between closely spaced markers, we supplemented the multipoint analyses with a two-point linkage analysis of the -family set under the recessive model using the fastlink program ( ). to further minimize the errors in recombination fractions resulting from sex-averaged estimates of recombination, we incorporated sex-specific recombination fractions in these analyses. tests of association. population association tests for microsatellite alleles were conducted for markers using clump v. . software ( ). we report the maximized � test (t statistic), which calculates the maximum � value found by collapsing the contingency tables over each allele in turn to form � contingency tables. the significance was assessed using a monte carlo approach with , simulations. family-based associations with type diabetes were tested in families using a modification of the transmission disequilibrium test (tdt) ( ), as implemented in the pedigree analysis package ( ) and described previously ( ). this analysis tests the probability that a heterozygous parent transmits an allele to an affected offspring more often than expected by chance, similar to the gamete-competition model described by sinsheimer et al. ( ). increased transmission from parents to affected offspring was tested by maximum likelihood analysis against equal transmission of the alleles. all alleles at a marker were tested simultaneously with k- df, where k represents the number of alleles. the pedigree is analyzed as an intact unit, so that trios and nuclear families were not examined separately. because linkage in this region was established, this likelihood test was a test of association. data are presented without correction for the number of markers tested. in a case control study of a two-allele marker, our power for a test of allelic association with individuals in each group exceeds % for differences in allele frequency of % or greater. linkage disequilibrium between microsatellite adjacent markers was calculated from the case-control study of unrelated individuals (both case and control subjects included) as a multilocus d	 value using the expectation maximization algorithm as implemented in the ld program (http://linkage.rockefeller.edu). haplotype estimation and haplotype sharing analysis. haplotypes were inferred in large pedigrees using ordered markers from d s to d s and simwalk (v. . ) following the methods of saarela et al. ( ). sharing of maternal and paternal haplotypes between affected siblings within each family was determined by manual inspection ( ). results we examined a total of microsatellite markers on chromosome , including markers previously reported ( ) and markers newly typed. we typed a total of markers in the region of the previously described linkage peak from marker d s ( cm) to marker d s ( cm), with all locations referenced to the marshfield map (http://research.marshfieldclinic.org/genetics) ( ). in our earlier analysis, we considered two family sets: the families that constituted our primary genome-wide scan, and smaller replication families. for the present study, we considered all available families ( families; the original families and replication families, including families not considered in the earlier study) and the families from the original families for which we had not identified another potential diabetes susceptibility gene. based on our earlier data, we chose the recessive para- metric model that provided the best evidence for linkage previously as the primary tool for narrowing the linkage peak. however, to determine whether that localization was robust to model assumptions, we also analyzed the linkage data under nonparametric models. parametric linkage analysis. as in our previous report of replication families ( ), we found no evidence for linkage in the replication families despite the dense map. using the full available pedigree set ( families), we only found evidence for linkage under models that incor- porated heterogeneity, with a maximum heterogeneity lod (hlod) score of . with % of families linked at position . cm (marker d s ). when the families from the original linkage study that did not segregate hepatocyte nuclear factor � variants were tested, the maximum lod score using families trimmed to fit gene- hunter requirements was . at the same location (posi- tion . cm; marker d s ), which was increased from . in our previous study. in contrast to the full family set, we found little evidence for heterogeneity (hlod � . ; � � . , . cm) using the families that were trimmed of many unaffected individuals. as in our previ- ous analysis ( ), inclusion of all unaffected individuals using the simwalk program dropped the nonheterogene- ity location score to . and the heterogeneity lod score to . (� � . ) without moving the location of the peak (marker d s ; . cm) (fig. ). based on the gene- hunter analysis, the one lod ci was narrowed to . – . cm, corresponding to locations . – . mb on the physical map (ncbi build ). nonparametric analyses. to determine whether local- ization of the chromosome q type diabetes susceptibil- ity locus was robust to model assumptions, we tested linkage also using nonparametric approaches (fig. ). our primary nonparametric analyses used simwalk , which could handle full families (fig. ), and affected sib-pair analysis using the genehunter program (fig. ). although these analysis corroborated the location of the first peak at . cm (marker d s , . mb; genehunter npl score . ; p � . ), they showed a prominent second peak not seen in the parametric analysis � cm telomeric to the first peak at cm, between markers d s ( . mb) and d s ( . mb; npl score . ; p � . ) (fig. ). using no weighting for sibships and assuming dominance variance, the highest maximum like- type diabetes locus on chromosome q -q diabetes, vol. , february lihood score (mls) was . at . cm and . at cm (fig. ). additionally, a third peak was evident on sib-pair analysis (unweighted; mls � . ) centromeric to the larger peaks at . cm and just proximal to marker d s ( . mb) and near candidate genes liver- and red cell–type pyruvate kinase (pklr) ( . mb), retinoid- related orphan receptor � (rorc), and interleukin receptor (il r; . mb). nonparametric statistics exam- ined in simwalk , which did not permit simultaneous consideration of the full map region, nonetheless showed similar trends for location (fig. ). the most significant simwalk results were seen with statistic a, which is strongest under recessive models, at p � . and location . cm at marker d s . the second peak was less obvious with the simwalk statistics but was most significant near marker d s ( cm, . mb; p � . for statistic c, p � . for statistic d) (fig. ), which was � cm or . mb telomeric to the genehunter npl and sib-pair analyses. when the full family set ( families) was examined together, the highest mls scores on sib-pair analysis were . at cm (apoa ; . mb) and . at cm (d s ; . mb). thus, when all families were considered, the proximal peak moved slightly telomeric and the distal peak slightly centromeric but retained approximately the same locations. two-point lod score. we observed an unexpectedly high recombination fractions between closely spaced markers despite retyping several markers and careful scrutiny of recombination events (online supplemental data, table ). to reduce the effect of these potential errors and to incorporate sex-specific recombination frac- tions, we calculated two-point parametric lod scores fig. . multipoint parametric linkage tests. curves are listed from highest to lowest. �, -family recessive lod score in genehunter-sized families; Œ, -family hlod scores in full families; ‚, -family recessive lod score, full families; ——, hlod in families (full families). fig. . multipoint nonparametric link- age tests. scores are shown for fam- ilies using simwalk (statistics a, c, and d) in full families or genehunter npl statistic in families trimmed to fit the genehunter program. all scores are � log of the p value except for the genehunter npl score, which is shown as the npl statistic. curves are listed from highest to lowest. �, gene- hunter npl; f, simwalk statistic a; ‚, simwalk statistic c; Œ, simwalk sta- tistic d. s.k. das and associates diabetes, vol. , february using recessive parametric model described above. as shown in table of the online supplemental data, lod scores exceeded . for markers d s (lod . ) and d s ( . ), which are located at . cm ( . mb) and cm ( mb), respectively, just telomeric to the recessive multipoint linkage peak near markers d s and d s ( . cm or . mb and cm or . mb, respectively). association studies. to further localize the type dia- betes susceptibility locus, we tested association in a case-control population comprising diabetic case subjects and nondiabetic control subjects ascertained in utah or arkansas for microsatellite markers. we also tested the markers used in the linkage studies for excess trans- mission of any allele from parents to affected offspring using maximum likelihood methods. in case control stud- ies, markers d s ( cm, . mb) and d s ( cm, . mb) were nominally significant at p � . and p � . , respectively, based on monte carlo assessment of significance tested using the clump statistic t to examine all alleles simultaneously ( ). marker ata a at cm ( . mb) was most strongly associated by tdt (p � . ). these markers fall under the second linkage peak, with both d s and ata a falling within the lod ci for the sib-pair analysis. the data for all microsatellites is shown in table of the online supple- mental data. multipoint linkage disequilibrium between adjacent pairs of markers ranged from not significantly different from to the highest d	 value of . (table of online supplemental data). haplotype sharing. we followed the methods of saarela et al. ( ) to establish shared haplotypes for the markers that spanned the -cm region between markers d s and d s . haplotypes were inferred in sim- walk and were examined manually for sharing among the sibships that had two or more affected individuals from the families. although no single haplotype was shared by all sibships, a . -cm region centered on the first linkage peak and flanked by markers d s and d s was shared by of sibships (table of online supplemental data). discussion multiple genome-wide scans for type diabetes have implicated a large number of regions for possible suscep- tibility genes. to date, only a single gene has been cloned, niddm or calpain on chromosome q, but the at-risk haplotype at this locus is rare outside of hispanic popula- tions. other regions with evidence for replication include chromosomes q and , but the replication has generally been at some distance from the original description. chromosome has now been identified in pima indians ( ), our studies described here, amish caucasians ( ), british caucasians ( ), french caucasians ( ), and in preliminary reports of both chinese and african ameri- cans ( ). furthermore, a syntenic region was identified in the gk rat ( ). the location of these linkage peaks is remarkably consistent but nonetheless spans the three peaks observed in the present study ( ). thus among amish with both type diabetes and impaired glucose homeostasis, the peak was near cm (marker d s ), with a second peak that was centromeric on the p arm. this first peak falls just centromeric to our primary peak at cm. among pima indians, the highest scores were at cm (sib-pairs discordant for diabetes) and cm (sib-pairs with onset before age years), and thus fall more into our second linkage peak. initial reports from wiltshire et al. ( ) placed their linkage in the region of our second peak at cm (d s ), although additional markers are reported to have moved the highest score more centromeric to the location of our first and largest linkage peak. the results of vionnet et al. ( ) place their chromosome peak in nearly the same location as our first peak, albeit only in lean (bmi kg/m ) individuals. the loci reported in other studies are not precisely localized ( ). these studies thus support the possibility that several fig. . mls of ibd sharing by sib-pair analysis. �, -family analysis with dominance variance, no weighting; f, -family analysis with no dominance variance, no weighting; ‚, -family analysis, dominance variance with weighted sib-pairs; Œ, -family analy- sis, no dominance variance, weighted sib-pairs; ——, -family analysis, dom- inance variance, and no weighting. type diabetes locus on chromosome q -q diabetes, vol. , february susceptibility loci account for the apparent replication across studies, as suggested by our distinct linkage peaks. we focused the current study on the multiplex families that provided the majority of the evidence for linkage in our initial report and that did not segregate other known mutations. unlike the original study, the dense map of approximately one marker every centimor- gan has resolved the broad linkage peak observed initially into at least two narrow peaks. the first of these peaks has moved slightly centromeric from the original peak at apoa ( cm) to the present location of d s ( . cm). with additional markers, the lod score has in- creased to . under the recessive model and using genehunter-sized pedigrees. similarly, using multipoint sib-pair analysis, the mls has increased from . in the original study to . in the present study. despite the large variation in significance levels for the first peak with different analytical methods, the location of this peak was remarkably consistent. both the simwalk statistic a and the parametric analysis continue to support a recessive- like mode of inheritance for the susceptibility gene or genes that accounts for the first peak. based on the present analyses, we have narrowed the lod ci for this peak to a region from . cm ( . mb) to . cm ( . mb). this peak includes at least refseq genes, including a number of strong candidate genes, many of which have been evaluated by our laboratory and others. among the candidate genes previously evaluated in this region are apolipoprotein a (apoa ) at cm ( . mb) ( ); phosphoprotein enriched in astrocytes (pea ), which may be involved in insulin action ( ); c-reactive protein, which may be involved in inflamation ( ); and two inwardly rectifying potassium channel genes, kcnj and kcnj ( , ). none of the reported associations of single nucleotide polymorphisms (snps) in these genes can convincingly account for the strong linkage signal in our families, however. in contrast, we have identified two regions within the lod support interval in which a cluster of snps shows strong associations with type diabetes in case-control studies. these associations thus appear to support the linkage findings. neither region falls close to a strong candidate gene, but work is in progress to identify additional polymorphisms within these regions and to evaluate nearby coding genes. additional support for an association under this peak has come from other groups with linkage in this region ( ). unlike our original report, the present study suggests a second peak at cm, � cm from the first peak at cm. based on the -family sib-pair analysis, the lod support interval is . – . cm, or � . to . mb. unlike the first peak, this second region is much less prominent using the recessive parametric models and is most prominent using multipoint sib-pair analysis, under which this peak nearly equals the first peak with a mls of . . these data suggest that the susceptibility locus accounting for the second peak acts less like a recessive locus. furthermore, this peak has a higher mls score than the first peak when all families are considered, thus suggesting that the susceptibility allele accounting for this peak may be more prevalent than that accounting for the first peak. the most prominent candidate genes for type diabetes in the lod support interval are the rxr� ( ), for which we found an association with lipid abnormalities but a less prominent association with type diabetes, and the overlapping homeobox transcription factor lmx a ( ). the microsatellite associations found in the present study also support one or more susceptibility genes that account for this peak. marker d s , which was associ- ated with type diabetes in the case-control study, lies just telomeric to rxr� ( . mb), whereas marker d s lies nearly mb telomeric to the lod ci ( . mb). however, the only marker identified as overtransmitted in family members in a tdt-like test, marker ata a , also lies within this second peak ( . mb). we cannot ex- clude the possibility that one or more of the associations are spurious, particularly given the modest p values and the span of nearly . mb between associated microsatel- lite markers. additional snp typing in these regions will be needed to confirm these associations and to narrow the genes responsible for these associations. although this study narrowed the most prominent link- age and association signals to the region between and mb, we have previously demonstrated an association of multiple noncoding snps within the pklr gene with type diabetes ( ), which is centromeric to the first linkage peak. this association would fall under the most centromeric linkage peak that was observed only on the unweighted sib-pair analysis (fig. ). the physical dis- tance encompassed by this peak might extend from mb to at least . mb. among possible candidates in this region besides pklr are rorc ( ), �-endosulfine (ensa) ( ), and interleukin- receptor (s.c.e., unpub- lished data). of these candidates, a prominent association in this population was observed only with pklr. because of unusually strong linkage disequilibrium extending for large distances in this centromeric region, the actual genes accounting for the linkage peak and the association may lie at some physical distance from the observed association. were a single variant responsible for our linkage signal on q -q , we would expect to identify one haplotype of the microsatellite markers across the linkage peak that was shared among affected individuals. in contrast, in the region between d s to d s , no single haplotype was shared. this finding is consistent with the existence of at least two and possibly three linkage peaks, suggesting more than one susceptibility gene in this region. the finding of several association peaks in this region offers further support for multiple susceptibility loci. we did identify a . -cm region flanked by markers d s and d s in which affected siblings of % of the sibships from the families shared the same haplotype, but no single haplotype was shared, even in this narrow region. this finding is consistent with other common disease susceptibility genes and suggests that even within this first linkage peak, multiple at-risk haplotypes contrib- ute to the linkage signal. in summary, using combined linkage mapping, haplo- type sharing and association studies with a dense marker map, we were able to confirm and narrow our original peak of linkage to a . -cm region or � . mb. we have resolved a second linkage peak that is � cm telomeric to our largest peak but in a region of both association and linkage in other studies. our analysis strongly suggests s.k. das and associates diabetes, vol. , february that the replication in this region comes in part from the coalescence of several susceptibility loci in a region that could not be resolved on a -cm genome scan. the region harbors many strong candidate genes for type diabetes, as well as a large number of poorly characterized tran- scripts that may also be good candidates. international collaborative efforts are underway to map these loci using positional candidate and linkage disequilibrium ap- proaches in the populations with linkage to this region. acknowledgments this work was supported by grant dk from the national institutes of health/niddk. subject ascertain- ment was supported in part by the research service of the department of veterans affairs, by the american diabetes association, and by national institutes of health/ncrr support of the general clinical research centers of uni- versity of arkansas for medical sciences (m rr ) and the university of utah (m rr ). we thank demond williams and winston chu for technical assis- tance, terri hale and judith cooper for assistance with subject ascertainment, and the gcrc nursing and labora- tory staff for assistance with subject assessment. references . elbein sc, chiu kc, permut ma: type diabetes mellitus. in the genetic basis of common diseases. king ra, rotter ji, motulsky ag, eds. new york, oxford university press, , p. – . weyer c, bogardus c, mott dm, pratley re: the natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type diabetes mellitus. j clin invest : – , 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . j med genet ; : – . doi: . /jmg. . t he term hereditary spastic paraplegia (hsp) is used to describe a group of clinically and genetically hetero- geneous disorders in which the defining clinical feature is progressive spasticity and weakness of the lower limbs. the phenotype is traditionally classified as ‘‘pure’’ when symp- toms and signs are generally confined to those of a progressive spastic paraparesis, or ‘‘complicated’’ when associated with additional neurological or other clinical features. inheritance may be autosomal dominant, auto- somal recessive, or rarely x linked. overall autosomal dominant inheritance is most commonly associated with pure forms of the disease, whereas autosomal recessive hsp shows greater phenotypic variability, including several well defined syndromes. to date nine autosomal recessive hsp loci have been identified and causative mutations found in three genes: spg (paraplegin), spg (spartin), and spg (maspar- din). spg encodes paraplegin, a mitochondrial protein, which is a member of the aaa protein superfamily (atpase associated with diverse cellular activities) and is homologous to a number of yeast mitochondrial metalloproteases. spg mutations may result in either pure or complicated hsp phenotypes. muscle biopsy analysis of patients with spg mutations may show histological evidence of mitochondrial dysfunction and recently biochemical studies have shown specific defects in mitochondrial respiratory chain function. mutations in the spg and spg genes have so far only been identified in the old order amish population in association with well characterised complicated hsp pheno- types. spartin, the protein product of spg mutated in troyer syndrome, contains a mit domain that is found almost exclusively in molecules thought to play a role in subcellular trafficking. only one study has so far been published relating to the function of maspardin—the protein product of the spg gene mutated in mast syndrome. this suggests co-localisation with transportation vesicles and implies a role in protein transportation or sorting. in the current study we have ascertained a large consanguineous family comprising five affected and seven unaffected siblings in which the parents were first cousins of bedouin ancestry. a uniform early onset of disease at between seven and eight years of age was noted. at the time of examination all affected individuals had signs of a progressive spastic paraparesis with dysarthria and distal amyotrophy in both upper and lower limbs. the three eldest affected subjects were also felt to have a degree of intellectual impairment (table ) with reduced iq, although it is unclear whether this represents progressive cognitive decline. neurological examination of the parents and the remaining siblings was unremarkable. routine biochemical studies and neurophysiological testing including nerve conduction velocities and eeg were normal. the appearances on magnetic resonance imaging were normal. linkage to the previously described autosomal recessive hsp loci was excluded using polymorphic microsatellite markers spanning these regions (data not shown). genome- wide linkage analysis, using parents and affected individuals only, was carried out with the abi linkage marker set (version . ) with an abi genetic analyser and genotyper software (version . ). a single region of homozygosity was identified on chromosome flanked by markers d s and d s . marker saturation analysis demonstrated a . cm region of homozygosity co-segregating with the disease in all affected individuals flanked by markers d s and d s (fig ). multipoint lod scores across the region—calculated using genehunter (version . ) under the assumption of equal allele frequencies and equal male and female recombination rates, with the disease modelled as an autosomal recessive trait with complete penetrance (allele frequency )—were significantly positive, with a maximum score of . between markers d s and d s (fig ). a database search of the critical interval identified approximately known or predicted genes including kif a, in which a missense mutation (a g) has previously been reported in a family with an uncomplicated autosomal dominant form of hsp. direct sequencing of all exons and splice junctions of the kif a gene, as previously described, in parents and affected individuals did not reveal any pathogenic mutations. key points n the hereditary spastic paraplegias (hsps) are a genetically and clinically heterogeneous group of neurodegenerative disorders. n a genome-wide screen was carried out in a con- sanguineous kuwaiti family with autosomal recessive hsp complicated by dysarthria, distal amyotrophy, and mild intellectual impairment in some affected individuals. n this defined a single region of homozygosity co- segregating with the disease spanning . cm of chromosome p . – q , flanked by markers d s and d s (multipoint lod score = . ). n this hsp neuropathy represents a novel genetic entity designated spg . abbreviations: hsp, hereditary spastic paraplegia www.jmedgenet.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ however, we cannot exclude the possibility of an intronic mutation or a mutation in a distant regulatory sequence. comment using this consanguineous kuwaiti family we have identified a novel locus for autosomal recessive complicated hsp to a . cm region on chromosome . although this region contains the kif a gene, the fact that no pathogenic mutations were identified in affected individuals in any of the exons and flanking splice junctions in this pedigree is strongly supportive of a further causative gene in this region. the different mode of inheritance and different phenotype compared with that of kif a offers further support of this hypothesis. this study should prompt the investigation of additional autosomal recessive hsp families for linkage to this region on chromosome , which may aid in the refinement of this currently large locus. based upon the proposed functions of the genes so far identified in the various forms of hsp, various different underlying patho- genic mechanisms have been proposed, including the disrupted development of the corticospinal tracts, mitochon- drial dysfunction, and defects in subcellular transportation and sorting processes. – the ultimate identification of the spg causative gene will further elucidate the pathogenesis table clinical features feature affected family member age at examination (years) onset years years years years years dysarthria +++ +++ +++ +++ tongue tremors + + + + + atrophy of small hand muscles +++ + +++ +++ +++ muscle tone spastic ll spastic ll spastic ll spastic ll slightly spastic ll knee reflex +++ +++ +++ +++ clonus +++ + + extensor plantar response +++ +++ +++ gait spastic ataxic spastic spastic spastic spastic emotional lability + +++ + +++ iq total total total total total verbal verbal verbal verbal verbal performance performance performance performance performance the case numbers – correspond to the affected cases from left to right in fig below. findings are graded by relative severity: absent; +mild; ++moderate; +++severe. iq, wechsler adult intelligence scale; ll, lower limb. figure pedigree of the kuwaiti family used to map the spg locus, showing haplotypes across the interval. figure multipoint lod scores for markers situated across the spg locus, flanked by markers d s and d s , producing a peak score of . . the spg locus www.jmedgenet.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ of this form of hsp and improve our understanding of the mechanisms of neurodegeneration in this heterogeneous disease. acknowledgements we are grateful to all the patients and their relatives who participated in this study. this work was supported by grants from action medical research uk, the birth defects foundation (uk), and the wellcome trust. the work was carried out within the network of the london ideas knowledge park. authors’ affiliations . . . . . . . . . . . . . . . . . . . . . p a wilkinson, m a simpson, h patel, j a reed, k kalidas, a h crosby, department of medical genetics, st george’s hospital medical school, london sw , uk p a wilkinson, t t warner, department of clinical neurosciences, royal free and university college medical school, london nw , uk l bastaki, e samilchuk, r khan, kuwait medical genetics centre, maternity hospital, kuwait competing interests: none declared correspondence to: dr andrew h crosby, department of medical genetics, st george’s hospital medical school, cranmer terrace, london sw re, uk; acrosby@sghms.ac.uk references harding ae. the hereditary ataxias, related disorders. edinburgh: churchill livingstone, . coutinho p, barros j, zemmouri r, guimaraes j, alves c, chorao r, lourenco e, ribeiro p, loureiro jl, santos jv, hamri a, paternotte c, hazan j, silva mc, prud’homme jf, grid d. clinical heterogeneity of autosomal recessive spastic paraplegias: analysis of patients in families. arch neurol ; : – . polo jm, calleja j, combarros o, berciano j. hereditary ‘‘pure’’ spastic paraplegia: a study of nine families. j neurol neurosurg psychiatry ; : . casari g, de fusco m, ciarmatori s, zeviani m, mora m, fernandez p, de michele g, filla a, cocozza s, marconi r, durr a, fontaine b, ballabio a. spastic paraplegia and oxphos impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. cell ; : – . wilkinson pa, crosby ah, turner c, bradley lj, ginsberg l, wood nw, schapira ah, warner tt. a clinical, genetic and biochemical study of spg mutations in hereditary spastic paraplegia. brain ; : – . mcdermott cj, dayaratne rk, tomkins j, lusher me, lindsey jc, johnson ma, casari g, turnbull dm, bushby k, shaw pj. paraplegin gene analysis in hereditary spastic paraparesis (hsp) pedigrees in northeast england. neurology ; : – . atorino l, silvestri l, koppen m, cassina l, ballabio a, marconi r, langer t, casari g. loss of m-aaa protease in mitochondria causes complex i deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. j cell biol ; : – . patel h, cross h, proukakis c, hershberger r, bork p, ciccarelli fd, patton ma, mckusick va, crosby ah. spg is mutated in troyer syndrome, an hereditary spastic paraplegia. nat genet ; : – . simpson ma, cross h, proukakis c, pryde a, hershberger r, chatonnet a, patton ma, crosby ah. maspardin is mutated in mast syndrome, a complicated form of hereditary spastic paraplegia associated with dementia. am j hum genet ; : – . ciccarelli fd, proukakis c, patel h, cross h, azam s, patton ma, bork p, crosby ah. the identification of a conserved domain in both spartin and spastin, mutated in hereditary spastic paraplegia. genomics ; : – . zeitlmann l, sirim p, kremmer e, kolanus w. cloning of acp as a novel intracellular ligand of cd . j biol chem ; : – . farag ti, el-badramany mh, al-sharkawy s. toyer syndrome: report of the first ‘‘non-amish’’ sibship and review. am j med genet ; : – . reid e, kloos m, ashley-koch a, hughes l, bevan s, svenson ik, graham fl, gaskell pc, dearlove a, pericak-vance ma, rubinsztein dc, marchuk da. a kinesin heavy chain (kif a) mutation in hereditary spastic paraplegia (spg ). am j hum genet ; : – . crosby ah, proukakis c. is the transportation highway the right road for hereditary spastic paraplegia? am j hum genet ; : – . crosby ah. disruption of cellular transport: a common cause of neurodegeneration? lancet neurol ; : – . reid e. science in motion: common molecular pathological themes emerge in the hereditary spastic paraplegias. j med genet ; : – . wilkinson, simpson, bastaki, et al www.jmedgenet.com o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . o n ja n u a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ © indian journal of plastic surgery | published by wolters kluwer - medknow introduction ear piercing is practiced from ancient times. ear piercing is frequently associated with keloid formation.[ ] various treatment modalities have been described for ear keloid.[ ] pressure therapy following surgical excision and intralesional steroid injection has the least recurrence rate. pressure can be applied on the ear by various methods using spring clips, binder clips and magnet discs but the pressure applied by these devices is access this article online quick response code: website: www.ijps.org doi: . / - . simple device to determine the pressure applied by pressure clips for the treatment of earlobe keloids aashish sasidharan, ann david , amish gohil, ashish kumar gupta departments of plastic and reconstructive surgery and bioengineering, christian medical college and hospital, vellore, tamil nadu, india address for correspondence: prof. ashish kumar gupta, department of plastic surgery, christian medical college and hospital, vellore - , tamil nadu, india. e-mail: ananyashish@gmail.com abstract background: keloids of the ear are common problems. various treatment modalities are available for the treatment of ear keloids. surgical excision with intralesional steroid injection along with compression therapy has the least recurrence rate. various types of devices are available for pressure therapy. pressure applied by these devices is uncontrolled and is associated with the risk of pressure necrosis. we describe here a simple and easy to use device to measure pressure applied by these clips for better outcome. objectives: to devise a simple method to measure the pressure applied by various pressure clips used in ear keloid pressure therapy. materials and methods: by using a force sensitive resistor (fsr), the pressure applied gets converted into voltage using electrical wires, resistors, capacitors, converter, amplifier, diode, nine-volt ( v) cadmium battery and the voltage is measured using a multimeter. the measured voltage is then converted into pressure using pressure voltage graph that depicts the actual pressure applied by the pressure clip. results: the pressure applied by different clips was variable. the spring clips were adjustable by slight variation in the design whereas the pressure applied by binder clips and magnet discs was not adjustable. conclusion: the uncontrolled/suboptimal pressure applied by certain pressure clips can be monitored to provide optimal pressure therapy in ear keloid for better outcome. key words clips; device; earlobe; magnetic discs; measure; pressure therapy; splint this is an open access article distributed under the terms of the creative commons attribution-noncommercial-sharealike . license, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. for reprints contact: reprints@medknow.com how to cite this article: sasidharan a, david a, gohil a, gupta ak. simple device to determine the pressure applied by pressure clips for the treatment of earlobe keloids. indian j plast surg ; : - . ideas and innovations published online: - - sasidharan, et al.: novel method to assess pressure of clips on ear keloids not monitored and can cause suboptimal or uncontrolled excessive pressure leading to skin necrosis. we describe a simple device to measure the pressure applied by these clips for optimal compression therapy. device design and operation we have devised an instrument [figure ] with the help of our bioengineering department using the following components: force sensitive resistor (fsr), electrical wires, resistors, capacitors, converter, amplifier, diode, nine-volt ( v) cadmium battery and a multimeter. for the assessment of pressure, the sensor has to be compatible and non-invasive to the human body as the sensor will be applied on the ear along with the pressure clips. we chose the force sensitive resistor (fsr -probots techno solutions, bangalore, india) for assessing the pressure applied by the pressure clips on the earlobe. the sensors have . mm round sensing area which can be used to detect any force or pressure applied on them. fsr is a thick polymer film device which senses the change in resistance based on the force applied on it. the active area of fsr detects the force applied over it. so, to measure the pressure between the heads of a clip, fsr needs to be placed in between the heads of the clip on earlobe [figure ]. when the clip closes, it applies force over the active area of fsr and this is reflected as a change in resistance across its leads. let resistance of fsr be r fsr , then force α /r fsr pressure = force/area, hence pressure α /r fsr the fsr has two output wires. the signal is transmitted to the circuit of the device in order to detect the force applied. the circuit works on a v battery and using a voltage converter it gives- v output. this voltage is used as supply voltage and a zener diode is used to give constant v output (v ref ). the final section of the circuit converts force applied to output voltage (v out ) which is measured using a multimeter. the voltage developed is directly proportional to the force applied at the fsr. the voltage detected is then converted to pressure in millimetres of mercury (mm of hg) using pressure voltage graph as shown in figure . the pressure clips used in the study were spring clips and magnetic discs. the clips with spring action were customised using g dental wire, acrylic buttons, and binder clips ( mm, mm and mm). the magnetic discs of sizes mm × mm and mm × . mm were also used. we used this device and measured the pressure applied by various clips and magnet discs on the ear and the findings were noted. figure : device and its components figure : ear clip applied on earlobe with fsr to measure pressure figure : pressure voltage graph indian journal of plastic surgery september-december vol issue sasidharan, et al.: novel method to assess pressure of clips on ear keloids results the pressure applied by these ear clips varied and the findings are tabulated in table . the pressure applied by certain clips such as binder clips and magnetic discs was not adjustable, whereas with spring clip , pressure could be adjusted by changing the width of u loop in between the limbs. by narrowing the width, the pressure applied between the clip heads could be increased.[ ] using spring clips and the pressure could be adjusted according to the memory of the coil/turn of wires. the tensile strength of the wire used also affects the pressure applied by the clip. the force distributed on the fsr may not be uniform and, on assessment, it was found to have % deviation from the normal value, mostly the deviation was towards higher end and that has been considered while interpreting the results. discussion pressure therapy is a widely practiced method for the prevention and treatment of hypertrophic scar and keloid. although there is no consensus regarding optimal pressure to be applied, the majority recommend pressure in the range of - mm of hg for good outcome. pressure therapy on ear is difficult due to its peculiar shape. an efficient compression device should provide calibrated uniform compression. many pressure devices have been described for ear, including button compression,[ ] oyster splint,[ ] cloth clip,[ ] silicone mould,[ ] compression using spring clips,[ , , ] binder clips[ ] and magnetic discs.[ , ] the use of binder clips and cloth clips forced us to think whether these clips apply much more pressure than the recommended range. in all splints, excessive pressure is judged by subjective symptoms such as pain, numbness and redness and it depends on patient’s intelligence. this method is an objective method of measurement. this study showed that spring clips are better than binder clips. out of the three spring clips, type i spring clip was the best. the use of binder clips should be discouraged. magnets are safe if they are of correct size and strength. considering that this device has % error towards the higher side, we calibrate spring clip for mm of hg. recently, there has been an increase in the application of magnet discs for pressure therapy on the ear. magnets are available in different power, size and thickness. the force applied by the magnets can also be assessed using gauss meter and pressure can be determined using simple conversion,[ ] although this device is costly and not used frequently. the presently available magnets are neodymium magnets. chang et al.[ ] have described the use of magnet discs of mm ( , gauss) and mm ( , gauss) size with pressure of mm of hg. our device had few disadvantages as mentioned below: measurement of pressure depends upon the area across which the force is applied; it has % error towards the higher side.repeatability of measurement depends on the region on which the force is applied.the response time for the system is high. conclusion the currently available pressure devices for ear keloids can exert uncontrolled pressure over and above the recommended pressure. too much pressure may be indicated by pain sensation but this is subjective and it depends upon the patient’s intelligence. certain pressure clips, such as binder clips and cloth clips, should be avoided as they cause uncontrolled pressure which cannot be adjusted. so, it is always better to assess the pressure applied by these clips before using them. our simple and easy to use device can be used to measure the pressure applied by these clips before their application, and the over-the-counter availability of the device would make things easier for most of the plastic surgeons who do not have the resources to have such an instrument made. financial support and sponsorship nil. table : values of pressure applied by various clips and after deduction of % error types of pressure clips pressure pressure after % error correction spring clips - mm of hg - mm of hg spring clips - mm of hg - mm of hg spring clips - mm of hg - mm of hg binder clips - mm of hg - mm of hg magnetic discs ( mm mm) mm of hg mm of hg mm of hg and mm of hg indian journal of plastic surgery september-december vol issue sasidharan, et al.: novel method to assess pressure of clips on ear keloids conflicts of interest there are no conflicts of interest. references . zuber tj, dewitt de. earlobe keloids. am fam physician ; : - . . nason lh. keloids and their treatment. n engl j med ; : - . . vachiramon a, bamber ma. a u-loop pressure clip for earlobe keloid. j prosthet dent ; : - . . snyder gb. button compression for keloids of the lobule. br j plast surg ; : - . . mercer dm, studd dm. “oyster splints”: a new compression device for the treatment of keloid scars of the ear. br j plast surg ; : - . . kulkarni a, abhyankar s, singh r, bhatia s, desai r. innovative pressure clip for ear keloid. indian j otol ; : . . sand m, sand d, boorboor p, mann b, altmeyer p, hoffmann k, et al. combination of surgical excision and custom designed silicon pressure splint therapy for keloids on the helical rim. head face med ; : . . brent b. the role of pressure therapy in management of earlobe keloids: preliminary report of a controlled study. ann plast surg ; : - . . agrawal k, panda kn, arumugam a. an inexpensive self- fabricated pressure clip for the ear lobe. br j plast surg ; : - . . murina at, ellis dl. binder clips in the treatment of auricular keloids. j am acad dermatol ; :e . . chang ch, song jy, park jh, seo sw. the efficacy of magnetic disks for the treatment of earlobe hypertrophic scar. ann plast surg ; : - . . park th, seo sw, kim jk, chang ch. outcomes of surgical excision with pressure therapy using magnets and identification of risk factors for recurrent keloids. plast reconstr surg ; : - . . magnetic pressure [internet]. wikipedia, the free encyclopedia. availablefrom:http://en.wikipedia.org/w/index. php?title=magnetic_pressure and old id= . [last accessed on may ]. indian journal of plastic surgery september-december vol issue arsenic exposure is associated with diminished insulin sensitivity in non-diabetic amish adults arsenic exposure is associated with diminished insulin sensitivity in non-diabetic amish adults sung kyun park , * qing peng lawrence f. bielak kristi d. silver patricia a. peyser braxton d. mitchell , department of epidemiology, school of public health, university of michigan, ann arbor, mi, usa department of environmental health sciences, school of public health, university of michigan, ann arbor, mi, usa departments of medicine and epidemiology and public health, university of maryland school of medicine, baltimore, md, usa department of veterans affairs and veterans affairs medical center baltimore geriatric research education and clinical center (grecc), baltimore, md, usa *correspondence to: sung kyun park, department of epidemiology, university of michigan school of public health, washington heights, ann arbor, mi, , usa. e-mail: sungkyun@umich.edu abstract background substantial evidence supports an association between diabetes and arsenic at high exposure levels, but results are mixed at low exposure levels. the aetiology of diabetes involves insulin resistance and β-cell dysfunc- tion. however, only a few epidemiologic studies have examined measures of insulin resistance and β-cell function in relation to arsenic exposure, and no studies have tested for associations with the oral glucose tolerance test (ogtt). we examined the association between urinary total arsenic and ogtt-based markers of insulin sensitivity and β-cell function. methods we studied non-diabetic adults (mean age = . years) from the amish family diabetes study. we computed ogtt-based validated measures of insulin sensitivity and β-cell function. generalized estimating equations accounting for sibship were used to estimate associations. results after adjusting for age, sex, waist-to-hip ratio and urinary creatinine, an interquartile range increase in urinary total arsenic ( . μg/l) was significantly, inversely associated with two insulin sensitivity measures (stumvoll metabolic clearance rate= � . mg/(kg min), ( % ci: � . , � . ), p = . ; stumvoll insulin sensitivity index= � . μmol/(kg min pm), ( % ci: � . , � . ), p= . ). urinary total arsenic was also significantly associated with higher fasting glucose levels ( . mg/dl ( % ci: . , . ) per interquartile range increase, p = . ). no significant associ- ations were found between urinary total arsenic and β-cell function measures. conclusions this preliminary study found that urinary total arsenic was associated with insulin sensitivity but not β-cell function measures, suggesting that low-level arsenic exposure may influence diabetes risk through impairing insulin sensitivity. copyright © john wiley & sons, ltd. keywords arsenic; β-cell function; insulin sensitivity; oral glucose tolerance test introduction arsenic is the top hazard that poses the most important potential threat to human health including diabetes on the priority list of the us agency for toxic substances and disease registry [ ]. the main sources of arsenic are contaminated drinking water and food [ , ]. potential biological mechanisms by which arsenic influences diabetes include high affinity of arsenic with sulfhydryl groups in insulin, insulin receptor and glucose transporters; increased research article received: july revised: october accepted: november copyright © john wiley & sons, ltd. diabetes/metabolism research and reviews diabetes metab res rev ; : – . published online january in wiley online library (wileyonlinelibrary.com) doi: . /dmrr. oxidative stress that can lead to formation of amyloid in pan- creatic islet cells, causing β-cell dysfunction; interference with gene expression involving signal transduction and gene transcription related to insulin pathways [nuclear factor-κb (nf-κb), tumor necrosis factor α (tnfα), il- , peroxisome proliferator-activated receptor gamma (pparγ)], leading to insulin resistance [ – ]. substantial evidence supports an association between arsenic and diabetes at high exposure levels, but results are mixed at low exposure levels [ , ]. the aetiology of diabetes involves insulin resistance and β-cell dysfunction [ , ]. however, only a few epidemiologic studies have examined measures of insulin resistance and β-cell function in relation to arsenic exposure, and such studies utilized indices derived from fasting glucose and insulin [ , ]. no studies have tested for associations with the oral glucose tolerance test (ogtt). we examined the association between urinary total arsenic and the ogtt-based markers of insulin sensitivity and β-cell function. materials and methods study population this is a preliminary study of the association between uri- nary arsenic and glucose homeostasis measures, con- ducted in the amish family diabetes study (afds), a genetic epidemiology study of type diabetes in the old order amish living in lancaster, pennsylvania [ ]. in to- tal, the afds included subjects aged ≥ years from multigenerational families recruited between and . detailed participant recruitment procedures of the afds can be found elsewhere [ ]. all participants gave written informed consent and underwent a detailed clinical examination at the amish research clinic. participants were instructed to fast for h before their appointment and to bring a first morning void urine sample. this preliminary study was based on afds partici- pants with normal (n= ) or impaired (n = ) glucose tolerance. subjects were sampled from non-diabetic indi- viduals who had undergone a -h ogtt (n = subjects: with normal; impaired glucose tolerance) and who had sufficient volumes ( ml) of stored urines remaining in our biorepository for the heavy metal assay. the mean (±sd) age of this sample ( . ± . years) was slightly higher than that of the full afds ( . ± . years). outcome assessment after acquisition of a fasting blood sample, a -g ogtt was administered. blood samples were then drawn for de- termination of glucose and insulin values at -min intervals for h. glucose and insulin concentrations were assayed with a beckman glucose analyser (beckman coulter, fullerton, ca) and radioimmunoassay (linco, st. louis, mo), respectively. using ogtt results, we computed three validated mea- sures of insulin sensitivity (stumvoll estimated metabolic clearance rate (stumvoll mcr) [ ], stumvoll insulin sen- sitivity index (stumvoll isi) [ ] and matsuda index [ ]) and three validated measures of β-cell function (stumvoll insulin secretion, phase and phase [ ] and insulin- ogenic index [ ] (refer to detailed formula in table )). as secondary measures, we also computed one fasting state-based index of insulin sensitivity (homeostatic model assessment-insulin resistance (homa-ir)) and one fasting state-based index of β-cell function (homa-%β) [ ]. de- tails of each measure including mathematical formula and clinical significances are provided in table . arsenic assessment urinary total arsenic concentrations were determined using inductively coupled plasma-mass spectrometry by the university of michigan environmental health sciences core center’s trace metals laboratory. all arsenic concen- trations among participants were above the limit of detection ( . μg/l). we conducted quality control proce- dures including analysis of urine-based reference mate- rials before, during and after every analytical run and use of calibration standards, procedural blanks, duplicate samples and spiked samples. the coefficient of variation was %. urinary creatinine was measured in the afds and used to adjust for urine dilution. data analysis to account for correlations among participants in the same sibship, we used generalized estimating equations with an exchangeable correlation structure where pair-wise correlations between participants from the same sibship were equal, to estimate differences for an interquartile range (iqr) increase in urinary arsenic ( . μg/l). all models were adjusted for age and sex (model ) and further adjusted for waist-to-hip ratio and bmi (model ). in model for stumvoll mcr and stumvoll isi, however, bmi was not included because bmi is used in the formula to calculate each of these indices. both models were also adjusted for urinary creatinine to account for urine dilution [ ]. how- ever, adjustment for urinary creatinine may introduce bias if creatinine production is influenced by diabetes and/or arsenic [ ]. we, therefore, report regression results for both models without urinary creatinine adjustment as well. two- sided p < . was considered statistically significant. s. k. park et al. copyright © john wiley & sons, ltd. diabetes metab res rev ; : – . doi: . /dmrr results the mean (sd) age was . ( . ) years, and parti- cipants ( %) were female (table ). the means (sds) of insulin sensitivity and β-cell function measures were . ( . ) mg/(kg min) for stumvoll mcr, . ( . ) μmol/(kg min pm) for stumvoll isi, . ( . ) for matsuda index, . ( . ) for homa-ir, . ( . ) for homa-%β, . ( . ) pm for stumvoll insulin secretion phase , . ( . ) pm for stumvoll insulin secretion phase and . ( . ) for insulinogenic index. the median urinary total arsenic concentration was . μg/l (iqr: . – . ) (table ). the creatinine-adjusted median concen- tration was . μg/g (iqr: . – . ). participants aged and older had higher concentrations (both crude- and creatinine-adjusted) than younger participants. men had higher crude total arsenic concentrations than women ( . vs. . μg/l) but lower creatinine-adjusted total arsenic concentrations ( . vs. . μg/g). participants with im- paired glucose tolerance had higher crude- and creatinine- adjusted urinary total arsenic concentrations than those with normal glucose tolerance. table . the meaning and equation of each fasting state measure and ogtt-derived index index definition/clinical significance equation reference measures of insulin sensitivity stumvoll metabolic clearance rate correlates with the metabolic clearance rate (mcr) derived from the hyperinsulinaemic-euglycaemic clamp that measures the rate of glucose uptake into tissues (primarily muscle and adipose tissue). mcr from clamp studies is calculated as the average glucose infusion rate divided by the average plasma glucose concentration during the last hour of a hyperinsulinaemic-euglycaemic clamp. higher levels indicate greater insulin sensitivity. . � . × bmi � . × i � . × g stumvoll insulin sensitivity index correlates with the insulin sensitivity index (isi) derived from the hyperinsulinaemic-euglycaemic clamp and represents the amount of glucose metabolized per unit of plasma insulin. isi from clamp studies is calculated as the metabolic clearance rate divided by the mean insulin concentration during the same period of the clamp. higher levels indicate greater insulin sensitivity. . � . × bmi � . × i � . × g matsuda index correlates with the rate of whole-body glucose uptake into tissues (primarily muscle and adipose tissue) during the hyperinsulinaemic-euglycaemic clamp. higher levels indicate greater insulin sensitivity. / ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi g �i �gmean�imean p (glucose in ‘mg/dl’ and insulin in ‘μu/ml’) measures of β-cell function stumvoll insulin secretion, phase correlates with first-phase insulin secretion (rise in insulin levels during the min immediately after starting the glucose infusion that rapidly raises glucose levels) during a hyperglycaemic clamp. higher levels indicate greater insulin secretion capacity. + . × i � . × g + . × i stumvoll insulin secretion, phase correlates with second-phase insulin secretion, the steady-state insulin levels during the last hour of the hyperglycaemic clamp. higher levels indicate greater insulin secretion capacity. + . × i � . × g + . × i insulinogenic index measure of early phase insulin secretion. for a given rise in plasma glucose during the first min of an ogtt, a larger index indicates greater insulin secretion. (i � i ) / (g � g ) fasting-state measures homa-ir insulin resistance index calculated from fasting glucose and insulin based on a physiologic model of the glucose and insulin relationship in vivo (homeostasis model). lower levels indicate greater insulin sensitivity. (g × i ) / . (insulin in μu/ml) homa-%β beta-cell function index calculated from the homeostasis model. it is expressed as a percent of normal β-cell function. ( × i ) / (g � . )% body mass index (bmi) in kg/m . insulin in pmol/l and glucose in mmol/l unless stated otherwise. g , fasting glucose; g /g , glucose and min after the administration of g glucose; gmean, mean glucose during oral glucose tolerance test (ogtt); i , fasting insulin; i /i , insulin and min after the administration of g glucose; imean, mean insulin during ogtt. arsenic and insulin sensitivity/β-cell copyright © john wiley & sons, ltd. diabetes metab res rev ; : – . doi: . /dmrr urinary total arsenic was significantly and inversely asso- ciated with all insulin sensitivity indices with adjustment for age, sex and urinary creatinine (model , table ). after further adjusting for adiposity (model ), associations remained significant for two of the three ogtt-based insu- lin sensitivity measures; an iqr increase in urinary total ar- senic ( . μg/l) was significantly, inversely associated with stumvoll mcr (� . mg/(kg min), % confidence interval (ci): � . , � . ; p= . ) and stumvoll isi (� . μmol/(kg min pm), % ci: � . , � . ; p= . ). urinary total arsenic was also significantly as- sociated with higher glucose levels ( . ( % ci: . , . ) mg/dl per iqr increase; p = . ). no significant associations were found between urinary arsenic and measures of β-cell function. the results remained un- changed in the models without urinary creatinine ad- justment (table ). discussion this is the first epidemiologic study to examine arsenic ex- posure and ogtt-based measures of insulin sensitivity and β-cell function. in this preliminary study of non- diabetic amish adults, urinary total arsenic was inversely associated with ogtt-based insulin sensitivity measures. notably, these associations were stronger and remained statistically significant following covariate adjustment compared with the widely used index of insulin resis- tance, homa-ir, which is based on fasting measures of insulin and glucose. possibly, the previous mixed results [ , , – ] may be partly because of low sensitivity of homa-ir. the ogtt-based insulin sensitivity measures, such as stumvoll mcr, showed better correlations with the hyperinsulinaemic-euglycaemic clamp-based insulin sensitivity than the fasting indices, such as homa-ir [ ]. we did not observe significant associations of uri- nary total arsenic with any measures of β-cell function. to our knowledge, only two human studies have ex- amined arsenic exposure and measures of both insulin sensitivity and β-cell function [ , ]. in mexican subjects with mean urinary total arsenic concentrations of . (sd = ) μg/l for non-diabetic (n = ) and . (sd = . ) μg/l for type diabetic subjects (n = ), urinary total arsenic was inversely associated with homa -%β but was not significantly associated with homa -ir [ ]. a national survey conducted in korea (n = ; median urinary total arsenic concentra- tions= . μg/g creatinine) also reported a significant in- verse association of urinary total arsenic with homa -%β but no significant association with homa -ir [ ]. their arsenic exposure levels were much higher than those found in our study ( . μg/l), which is comparable with that found in non-hispanic white, non-fish eating, never- smoker adults from nhanes – data ( . μg/l (iqr: . – . ), unpublished data). a recent national tox- icology program (ntp) workshop review suggested that the arsenic effects on β-cell function are concentration de- pendent [ ]: low concentrations (in the submicromolar range) may lead to impaired glucose-stimulated insulin se- cretion through adaptive cellular responses to arsenic- induced oxidative stress, whereas high concentrations may lead to apoptosis or necrosis via irreversible oxidative dam- age to β-cells. the ntp workshop review also suggested that low concentrations may inhibit insulin signalling and table . population characteristics (n = ) mean ± sd or otherwise specified age (years) . ± . female, n (%) ( ) bmi (kg/m ) . ± . waist-to-hip ratio . ± . fasting glucose (mg/dl) . ± . fasting insulina (μu/ml) . ± . glucose min (mg/dl) . ± . stumvoll mcrb (mg/(kg min)) . ± . stumvoll isib (μmol/(kg min pm)) . ± . matsuda indexa . ± . stumvoll insulin secretion, phase c (pm) . ± . stumvoll insulin secretion, phase c (pm) . ± . insulinogenic indexc . ± . homa-ira . ± . homa-%βa . ± . an = . bn = . cn = . table . distributions (median and interquartile range (q and q )) of urinary total arsenic by covariates n creatinine unadjusted (μg/l) creatinine adjusted (μg/g) all . ( . , . ) . ( . , . ) age (years) – . ( . , . ) . ( . , . ) – . ( . , . ) . ( . , . ) ≥ . ( . , . ) . ( . , . ) sex male . ( . , . ) . ( . , . ) female . ( . , . ) . ( . , . ) bmi (kg/m ) < . ( . , . ) . ( . , . ) – . ( . , . ) . ( . , . ) ≥ . ( . , . ) . ( . , . ) high waist-to-hip ratioa no . ( . , . ) . ( . , . ) yes . ( . , . ) . ( . , . ) ogtt normal . ( . , . ) . ( . , . ) impaired . ( . , . ) . ( . , . ) ahigh waist-to-hip ratio was defined as waist-to-hip ratio ≥ . for men and ≥ . for women [ ]. s. k. park et al. copyright © john wiley & sons, ltd. diabetes metab res rev ; : – . doi: . /dmrr insulin-dependent glucose uptake by adipocytes or skeletal muscle cells. although it is unclear why our findings are in- consistent with the previous ones, low-level arsenic expo- sure found in the present study may result in insulin resistance through inhibition of insulin signalling and insulin-dependent glucose uptake [ , , ]. more epi- demiologic studies with a wide range of exposure levels and measures of insulin sensitivity and β-cell function are warranted to investigate concentration-dependent mechanisms. non-significant associations between urinary total arse- nic and fasting state-based measures seem to be because of confounding by adiposity. the age- and sex-adjusted as- sociation between urinary total arsenic and homa-ir was statistically significant (model , table ), but the effect estimate was substantially attenuated after adjustment for adiposity (bmi and waist-to-hip ratio) (model , table ). in this population, urinary total arsenic concentrations were modestly correlated with bmi and waist-to-hip ratio (pearson correlation coefficient for both measures = . ). adiposity is a well-known risk factor for insulin resistance and diabetes [ ]. given that drinking water and diet are major environmental sources of arsenic exposure [ ] and more food and water consumption is expected in obese in- dividuals, adiposity may play a role as a positive con- founder, and therefore, reduced effect estimates are expected with adiposity adjustment. however, previous lit- erature has reported an inverse association between bmi and arsenic biomarkers [ – ]. this is because obese individuals are more likely to consume more methyl donors, such as methionine, folic acid and vitamin b , that facili- tate arsenic methylation, resulting in faster arsenic ex- cretion [ ]. gruber et al. found an inverse association between toenail arsenic and dietary fat intake, suggesting that dietary fat may inhibit arsenic absorption [ ]. differ- ent population characteristics including dietary habits, life- style and genetic variations may explain the inconsistency observed in our population, but our study is limited to fully understand plausible links between adiposity and arsenic metabolism and excretion because of the lack of arsenic species data. future studies of the role of adiposity in arse- nic metabolism and in the arsenic diabetes association in the amish population are needed. there are several limitations. we did not measure arse- nic species. total urinary arsenic reflects all arsenic spe- cies including inorganic forms of arsenic and their methylated metabolites and the organic forms. a ntp workshop recommended arsenic speciation analysis because it is assumed that the inorganic arsenic and methylated metabolites, but not the organic forms, may be associated with type diabetes [ ]. it is also important to consider the organic forms of arsenic (e.g. arsenobetaine), a less-toxic species of arsenic found in seafood, in data anal- ysis. however, it is unresolved whether the organic species of arsenic should be adjusted as a covariate or subtracted from total arsenic concentrations [ – ]. given that expo- sure to the organic forms of arsenic occurs through fish con- sumption and fish is not a common component of the amish table . differences in ogtt-based insulin measures per interquartile range ( . μg/l) increase in urinary total arsenic (n = ) with creatinine adjustment without creatinine adjustment model a model b model model fasting glucose (mg/dl) . ( . , . )* . ( . , . )** . ( . , . )* . ( . , . )** glucose ( min) (mg/dl) . (� . , . ) . (� . , . ) . ( . , . )** . (� . , . ) measures of insulin sensitivity stumvoll mcr (mg/(kg min)) � . (� . , � . )* � . (� . , � . )* � . (� . , � . )* � . (� . , � . )* stumvoll isi (μmol/(kg min pm)) � . (� . , � . )* � . (� . , � . )* � . (� . , � . )* � . (� . , � . )* matsuda index (percent difference) � . (� . , � . )* � . (� . , . ) � . (� . , � . )* � . (� . , . ) measures of β-cell function stumvoll insulin secretion, phase (pm) . (� . , . ) . (� . , . ) . (� . , . ) . (� . , . ) stumvoll insulin secretion, phase (pm) . (� . , . ) . (� . , . ) . (� . , . ) . (� . , . ) insulinogenic index (percent difference) . (� . , . ) � . (� . , . ) . (� . , . ) � . (� . , . ) fasting-state measures homa-ir (percent difference) . ( . , . )* . (� . , . ) . ( . , . )* . (� . , . ) homa-%β (percent difference) � . (� . , . ) � . (� . , . ) � . (� . , . ) � . (� . , . ) amodel adjusted for age and sex in generalized estimating equations accounting for sibship. bmodel additionally adjusted for waist-to-hip ratio and bmi. bmi was not adjusted when modeling stumvoll mcr and stumvoll isi because bmi is used to calculate those indices. for both models and , results when urine creatinine was included as a covariate and when it was not are presented separately. *p value < . . ** . ≤ p value < . . arsenic and insulin sensitivity/β-cell copyright © john wiley & sons, ltd. diabetes metab res rev ; : – . doi: . /dmrr diet, the contribution of the organic forms to the urinary to- tal arsenic concentrations might be minimal in our study. al- though the study participants are not diabetic subjects, they have a family history of diabetes given the study design of afds; thus, they may be at higher risk of diabetes than those without a family history. our study was conducted in a cross-sectional setting that raises concerns of the validity of causal inferences between urinary arsenic and insulin sensitivity. this preliminary study suggests several future direc- tions. given that arsenic metabolism, such as arsenic methylation efficiency, has been associated with diabetes in several studies including prospective evidence with in- cident diabetes [ ], it will be important to evaluate the associations between arsenic metabolism and ogtt- based measures of insulin sensitivity and β-cell function. future studies will also need to evaluate potential sources of arsenic exposure in this population of the amish. al- though the exposure level found in this preliminary study was low, given that all of the amish use well-water for drinking and they adhere to traditional lifestyle and dietary habits, it will be important to identify main sources of arse- nic (especially inorganic arsenic) in this community. in conclusion, this preliminary study using ogtt-based measures of insulin sensitivity and β-cell function suggests that low-level arsenic exposure may influence diabetes risk through impairing insulin sensitivity rather than insu- lin secretion through pancreatic β-cells. acknowledgements this study was supported by the national institutes of health p es , p dk and p dk and the univer- sity of michigan office of the vice president for research, faculty grants and awards program. conflicts of interest the authors have no conflicts of interest. statements all of the authors have read and approved the article, and it has not been published previously nor is it being consid- ered by any other peer-reviewed journal. all authors have agreed to submit the article to diabetes/metabolism re- search and reviews. references . priority list of hazardous substances [article online], . available from http://www.atsdr.cdc.gov/spl/. accessed june . . heikens a, panaullah gm, meharg aa. arsenic behaviour from groundwater and soil to crops: impacts on agriculture and food safety. rev environ contam toxicol ; 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( ): – . . kuo cc, howard bv, umans jg, et al. arsenic exposure, arsenic metabolism, and incident diabetes in the strong heart study. diabetes care ; ( ): – . . who. waist circumference and waist-to- hip ratio. world health organization: geneva, . arsenic and insulin sensitivity/β-cell copyright © john wiley & sons, ltd. diabetes metab res rev ; : – . doi: . /dmrr biron - birkbeck institutional research online hill, katherine ( ) memories from the margins? anniversaries, anabaptists and rethinking reformations. palgrave communications , p. . issn - . downloaded from: http://eprints.bbk.ac.uk/id/eprint/ / usage guidelines: please refer to usage guidelines at https://eprints.bbk.ac.uk/policies.html or alternatively contact lib-eprints@bbk.ac.uk. http://eprints.bbk.ac.uk/id/eprint/ / https://eprints.bbk.ac.uk/policies.html mailto:lib-eprints@bbk.ac.uk comment memories from the margins? anniversaries, anabaptists and rethinking reformations kat hill abstract with the recent -year jubilee of the lutheran reformation, reformation anniversaries have become big business and the subject of much scholarly debate. this paper considers the question of anniversaries in relation to suppo- sedly marginal religious groups in the era of the reformation. what do they choose to commemorate? how did they fit into our accounts of religious change? and what does memory from the margins tells us? the paper argues that con- sidering memories and anniversaries amongst these communities allows us to reassess our categories of mainstream and marginal in relation to religious change in the early modern world and beyond, and to reconsider some of our narratives about the legacies of religious change. https://doi.org/ . /s - - - open birkbeck college, university of london, london, uk. correspondence and requests for materials should be addressed to k.h. (email: katherine.hill@bbk.ac.uk) palgrave communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/palcomms () :,; mailto:katherine.hill@bbk.ac.uk www.nature.com/palcomms www.nature.com/palcomms introduction was ‘luther year’, the -year anniversary of the posting of the ninety-five theses and the start of the reformation. ten years of preparation from the lutheran church and german authorities culminated in celebrations all over the world. from poetry slams to movies, panorama installations to conferences, heritage tours to a wave of publications, luther sparked intense intellectual and academic analysis about the meaning of religious change in early modern europe. (marshall, ; roth, , pp. – ). anniversaries and moments of memory activation invite us to consider what narratives are being shaped about the past and what narratives we as historians and scholars want to tell. remembrance inevitably sparks renewed debates about origins and outcomes, and we can learn much about a movement, nation or community at times of commemorative celebration. the first big -year reformation jubilee, that of luther’s birth, initiated a battle for ownership of his memory in a fractured germany in the dying days of the gdr (hoffmann, ; scott, ). from – , visitors might have experienced the nuanced differ- ences of nationally inflected exhibitions commemorating the anniversary of world war one in, for example, belgium, france, england, and germany. the -year anniversary of women’s right to vote and the suffragettes has struck chords with the contemporary #metoo movement. the luther anniversary was, it seemed to many, a chance to embrace a more ecumenical luther, though there have also been concerns about the way in which commemorations renewed a hero image of the reformer (evangelical church in germany, ; roth, ). but what if we did not focus on luther’s story? the years and will mark the anniversary of a different part of the reformation. these are the dates that the mennonite world conference (mwc) has picked to commemorate the origins of anabaptism. recalls the moment at which conrad grebel baptized george blaurock in zurich ‘to ignite a brand-new anabaptist movement which countered the movements of luther, zwingli and catholicism’. coincides with years since the appearance of the schleitheim articles, a statement of ana- baptism’s separation from the world, and the martyrs’ synod, a meeting which established a principle of mission. (it earned its name because many of its principle participants died soon after). the significance of these dates is clear, but any such immediate connection is sidestepped on the front page of the mwc’s website for the anniversary which focuses on the message of what has been titled ‘renewal ’. renewal was launched in the same year as the -year anniversary of the lutheran refor- mation and is presented as a chance for ecumenical discussion within and beyond the mennonite church, with ten years of events designed to invigorate anabaptist faith globally. these events are not likely to be on most people’s radar and will not spark the same rash of publishing as luther’s . nor are the events uncontroversial amongst mennonites some of whom question whether these anniversaries are an appropriate or rele- vant celebration of mennonite identity (goossen, b). for mennonite communities and mennonite scholars the question of anniversaries has stoked intense debate about the historical nar- ratives that surround these public rituals of commemoration. (roth, , pp. – ; osborne, ). considering the anni- versary celebrations and controversies of the lesser known part of the reformation story, however, is also a chance for reformation scholars to revisit some of our accounts of religious change and its legacies. what if we switched our perspective to those who were supposed to be on the edges of the story? what do the debates within the mennonite community reveal about these memories and anniversaries more broadly? what new questions or old problems might we consider by examining remembrance from the edges of the reformations? memory on the margins makes us reconsider the centre. the mennonite church, which is coordinating the -year anniversary, has its roots in anabaptism. this reforming move- ment of the sixteenth century rejected infant baptism along with many of the conventional structures of society, such as swearing oaths and serving in the military (stayer, ; goertz, ). anabaptism is not well integrated into reformation histories. histories of the radical or ‘left-wing’ of the reformation remain understudied and marginalized from the mainstream historio- graphical scholarship on the reformation, and despite a spate of histories produced in the s and s on early modern ana- baptism, anabaptist studies have not seen the same energy since. new waves of scholarship have started to redress this problem, recognizing the fundamentally problematic label of the ‘radical reformation’ and a group of new researchers are spearheading moves to bring anabaptist histories into the digital age with the open access website, anabaptist historians. scholarship in the last fifteen years has seen a handful of important monographs on anabaptist and mennonite histories (driedger, ; räisänen- schröder, ; monge, ; hill, ; goossen, a). but the scholarly imbalances and the division between mainstream and radical persist, especially for the pre-modern era. not everyone agrees that anabaptist studies are an endangered enclave (dipple, ; p. ). anabaptism does have a rich historiography, and there are mennonite presses and journals, notably the mennonite quarterly review and journal of menno- nite studies, which are energetic and stimulating fora. however, the specialist focus of this publishing can also reiterate the boundaries separating scholarship. research is often produced in confessional contexts, and though confessional history in itself is not inherently problematic, i would agree with academics who stress that there has been a return to confessionalised scholarship in reformation studies. the anniversary sharpened this in many cases, despite its claims to ecumenism, and has reinforced a sense of separatism. even sources may be divided. a special series exists for documents on the anabaptist reform movement (with the tactical name change from the pejorative ‘wiedertäufer’ to ‘täufer’ in the late s). so, while students of the european reformation may have a week on radical anabaptists in the sixteenth century, few courses touch on much beyond this or examine the longer-term traditions that evolved from non- conformist impulses. the distorting nature of this scholarship has broader impli- cations. it matters that anabaptists have been on the edges of historiography since it is partly a symptom and consequence of on one of the biggest, most problematic debates in reformation historiography: confessionalisation (brady, ). reformation scholars have for some time sought ways to write histories which go beyond the confessionalisation thesis which ties long-term narratives of religious change into accounts of state, discipline, and institutions. confessionalization neglects alternatives to the model of institutional faith, faiths often related to national identity and larger historical narratives of the rise of nationalism, and thus it omits a fundamental and long-lasting element of confessional change (lotz-heumann, ). moments of mem- ory and celebration, therefore, are a chance for academics to address these questions. anniversaries bring to the fore big debates about origins and futures, as well as the place of com- munities in the contemporary world, and so often implicate national and international institutions. though there are ques- tions about its success, a major emphasis of the luther commemoration, for example, was to attempt to move away from national and confessionalised narratives and embrace a more ecumenical vision for lutheranism’s future in the modern comment palgrave communications | https://doi.org/ . /s - - - palgrave communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/palcomms www.nature.com/palcomms world (lwf and pcpcu, ). the run of reformation anniversaries can stimulate conversations which re-invigorate scholarship. anabaptists memories there are over million anabaptists in the world, and the mwc -year celebrations for many are a remembrance of those things that characterize the collective memory of their commu- nities—an emphasis on the separation between church and state, a history of persecution and martyrdom, freedom of religion, liberty of conscience, and elective entry into the church. it is also seen as a chance to assess and reinvigorate mennonite identity in the contemporary world (roth, ). clearly memory matters to mennonites (and their close cousins the amish and hutterites). for numerous mennonites, there exists a deep sense of connec- tion to their early modern past, its martyrs and heroes. but whilst the / centenary will be a celebration coordinated by the mwc, the reality is of course that cultures of memory amongst anabaptist descendants go far beyond the official celebration and central structures. the attitudes which form the global backdrop to the planned commemorations are rooted not in national churches and state institutions, but family, communal and personal memories which have preserved the sense of the past and collective remembrance. mennonites, amish and hutterites all migrated great distances from the sixteenth century onwards. this was not always the result of persecution and exile, as a traditional anabaptist narrative might suggest, but anabaptists undeniably traveled far. communities moved from northern and central europe to russia and ukraine, migrated to the americas, or undertook confessional work in india, south east asia, and africa. as people move, memories travel with them. indeed, for mennonites, amish and hutterites, memory was a critical tool for bridging the gap between communities dispersed across wide areas and separated from society in a variety of ways. memory is central to diasporic identity (agnew, ; baronian, et al., , pp. – ). anabaptist memories existed in the records kept by their own churches, family possessions or per- sonal recollections which bound the community together. men- nonites in contemporary manitoba or kansas, for example, have objects, documents and shared narratives which trace cross- generational diasporic histories in europe, russia and beyond. we can only follow these histories and memories if we look beyond conventional institutional and geographical boundaries, since the very core of mennonite, hutterite and amish identity is in the communities of dispersion which stretched across regions. memory in these diasporic communities could not tell the same story as institutional churches. lutheran memories had an inclusive message in which looked to a global lutheran culture but also reasserted the dominance of wittenberg. calvi- nist memories are focused on geneva. anabaptist memories have been of a more disparate nature, though the mwc’s focus on and the swiss origins of the movement has caused con- troversy by centring anabaptist histories on one location deter- mined by a central committee. but why does it matter to think about this type of memory? is it not just one more anniversary, one more strand of the legacy of the reformation era? first, it reveals issues concerning con- temporary mennonite identity in relation to an early modern past and a diasporic identity. debates arose whether the date for a mennonite anniversary should be or , or indeed whether should have been the date for celebration. the result is ten years of renewal announced in , culminating in but with a celebration of in switzerland along the way. deeper unease amongst some members of the community has focused on the way in which any anniversary of this kind reinforces a monogenetic heritage which excludes the global church and diversity, and even alludes to a form of european ethnic purity (goossen, b). the monogenesis versus poly- genesis debate about anabaptist origins is long-standing but seems not to have died (stayer et al., ). an official celebration which gives one line of interpretation and reinforces one nor- mative view of anabaptist and mennonite heritage, rather than recognize all the alternatives and global perspectives, is proble- matic. it is possibly in dialogue with these concerns that africa is a suggested location for the th mennonite world conference assembly (roth, , p. ). some counterpoint views to the and dates also argue that the anabaptist church did not originate in the sixteenth century since it was a continuation and successor to the apostolic church of christ (roth, , pp. – ; goossen, b). this was the line taken by the two most famous anabaptist historical works, the seventeenth-century martyrs mirror and the hutterite great chronicle. such a view, however, can be equally proble- matic and elide the constant and shifting creative power of memory formation and the way in which recollections have been reinvented across the centuries. there are, too, always political questions at stake in commemorations (goossen, b). men- nonites, not the amish or hutterite, are driving the -year anniversary, but it is presented as celebration for all anabaptists. what does it tell us that this celebration matters most for the better integrated and politically active part of the anabaptist legacy, and that this is intertwined with institutional and con- fessional narratives? anniversaries after all always serve some purpose, and also coincides with the -year anniversary of the foundation of the mennonite central committee, so some would argue that this has more to do with institutional and political positioning than organic commemoration (goossen, b). we should remember that the official celebrations are only one part of memory; we have to look beyond to broader memory cultures, to individuals and local communities. memory is the aggregation of these narratives, as much as central cele- brations. the intense debate amongst different anabaptist tra- ditions is a stark reminder of the power and importance of diasporic memory, but also the need to recover alternatives to centralized commemorations. scholarship should consider how memory cultures themselves are created and recreated, why and when, and in what contexts. centenaries and anniversaries of origins, celebrated centrally, are a relatively modern phenomenon. marked the first major mennonite anniversary when the new mennonite world con- ference convened in basel and there do not seem to have been parallel events in or before. earlier celebrations and asso- ciated controversies existed. in various mennonite com- munities celebrated the death of menno simons and a call was issued in by august heinrich neufeld, pastor of the iber- sheim mennonite congregation in rhine-hesse, for ‘every men- nonite congregation in the old world and the new’ to plan for the date. the proposed celebrations sparked considerable con- troversy which revealed the fault lines in nineteenth-century mennonite society (roth, ; urry, ). thus, historians should be sensitive to the way in which memories are chosen, contested, and narrated, how they vary and shift, and how they exist at different levels. for specific communities, individuals, and families, it might be personal or local commemorations which held the most weight, and memory cultures had local inflections. mennonites in chortitza (zaporizhia oblast, ukraine) erected monuments in the late nineteenth century to figures of their past, to johann bartsch and jakob höppner, the men who had nego- tiated the details of the settlement for mennonite migration from prussia at the end of the eighteenth century (urry, ). research needs to understand the way in which memory has been palgrave communications | https://doi.org/ . /s - - - comment palgrave communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/palcomms www.nature.com/palcomms www.nature.com/palcomms constructed at local, regional and familial levels, as well as focusing on global and international commemorations. second, i would argue that by interrogating anabaptist memories as this anniversary approaches, we can uncover novel trajectories for the long-lasting legacies of the reformation throughout the world and in so doing address some of the fun- damental debates of the reformation era. it opens up new per- spectives on both our histories of non-conformist groups in the early modern world, and the implications for how religious change has shaped global culture. the debates over anniversaries have sharpened questions of the interaction between the refor- mation legacies of anabaptism and lutheranism (and other tra- ditions) and brought to the fore the continued global power of these anabaptists networks of memory. such trends undermine the sense of marginality and separateness that has shaped ana- baptist historiography, but also call into question broader reli- gious narratives which still seem to rest on notions of confessionalisation. the nature of the relationship between these different religious communities and the -year celebrations matters, and any student of the reformation needs to understand those labeled both marginal and mainstream. it is only from the margin though that we can rewrite the centre. until we question the narratives of the marginal, we will be forever locked into the modes of persecuted and persecutor. anniversaries do not always help in this reassessment since they have in some ways reasserted confessional narratives and nor- mative divisions. in , as preparations for were getting under way, the lutheran world fellowship offered an official apology to anabaptists for past persecutions. the divided mem- ory cultures sought rapprochement but also in many ways reit- erated division (roth, , – ). such an event reinforces normative mennonite self-definitions of the confession as a per- secuted minority without asking how this cultural narrative evolved, or what happened when communities or individuals chose not to follow this path. looking from the edges, we can see that anabaptists were not always a persecuted minority, they were not marginal to histories, and they did not always search for the peaceful, quiet way (urry, ). and looking from the centre, we have neglected the impor- tance of supposedly marginal groups in our long histories of the reformation. anabaptist descendants are a visible presence across much of america. nearly every north american, it seems, has a mennonite, hutterite or amish story. they have seen the buggies, been to the farms, or visited the churches. yet these communities still seem a curiosity. amish communities in pennsylvania, for example, are a tourist attraction more than a subject of scholarly discussion. amish, hutterites and more conservative mennonites of the old order tradition do not necessarily sit easily in our narratives of historical development and modernity. for those from outside the tradition, they seem an outdated relic of an older time, but their existence problematizes our notions of reforma- tion legacies. it becomes difficult to argue, for example, that the unintended effect of the reformation was secularization (gregory, ). in a series of six essays, gregory proposed that the protestant refor- mation questioned authority in ways which led to a multiplicity of competing claims to truth and this resulted ultimately in the privatization of faith, the power of the state over church, and secularization. but there are problems with this account. if we are to write narratives which escape the pitfalls of a return to con- fessionalisation but also appreciate reformation legacies on their own terms we have to be able to understand the dynamics of the communities of pennsylvanian amish or old order mennonites in belize. and we also have to be able to contain accounts of mennonites who are integrated into modern canada, hutterites who reject televisions and much modern media but embrace the best farming technologies, or amish communities who live separate lives but actively engage in a form of tourism which plays on their traditionalism. such solutions do not represent secular- ization necessarily but alternative models for recreating faith and adapting. for gregory, division has shattered a more universal sense of faith but for mennonites, for example, the broken body of the church can also be a symbol of the quest for faithfulness and of following the right path which diverts from the main- stream (roth, , p. ). splintering may have led to diversity, but it can also provide energy. furthermore, a sense of secular- izing decline can also be seen as a particularly western-centric narrative, and reformation legacies must be able to appreciate the explosion of christianity in the global south. mennonite memory brings into sharper focus other issues about reformation memory and the need to look beyond the official celebrations. focusing on luther in a way which reinforces the norms of the mainstream and the marginal absents a major and important part of the reformation legacies in eur- opean, eurasian and north american history, and beyond into africa, japan, india, and korea. deeply rooted identity and multifaceted memories have shaped anabaptist communities and cultures across the world: from prussia militarism to tsarist expansion, from the american west to the world wars, from south american colonies of mennonites to the power of contemporary protestant churches in the global south, even to questions of how mennonites should respond to trump. understanding the geo- graphical spread of anabaptist groups and the way in which their communities evolved across the diaspora opens up other archives, resources and memory cultures. new archives, new sources and new regions all offer promising concrete areas for research on reformation legacies. first, scholarship can look to understudied or neglected archives and records which provide alternative histories to accounts of confessional change. whilst mennonite archives in north america, for example, are an important resource for scholars of mennonite history and communities themselves, they remain little studied by reformation historians more generally. yet, there is a wealth of material in the church and family fonds which continues to come into these repositories. there are sur- prising finds in archives across europe and beyond, such as the histories of mennonite communities in regional polish archives or the church books of mennonites, quakers and other groups that have made their way into german state archives. a com- prehensive understanding of the complex archival traces would allow a much richer account of the long legacies of the reformation. second, we must think about the diverse way in which memory was enacted and histories recorded. this might be by paying closer attention to the way in archives themselves and their construction shape memory. the contents, materials and orga- nization of archives reflect power relations (stoler, ) and scholars have started to write the social history of archives (ketelaar, ). unpicking these archival histories, such as the reasons why mennonites have come to create their own archives, will in turn shed light on the history of confessional memory and memory making. furthermore, focusing on the transmission and function of memory could lead us to think about the very dif- ferent memory cultures amongst all those included amongst anabaptists. the memory practices of mennonites who have created extensive records differ from the old order amish who eschew formal archiving but rely on family and oral histories. the implications of different practices of recalling pasts and the power relations between different memory cultures is essential to the construction of more dynamic narratives of religious cultures. third, we might think about the way in which memory and confessional legacies were enacted across different media. comment palgrave communications | https://doi.org/ . /s - - - palgrave communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/palcomms www.nature.com/palcomms discussions of reformation anniversaries have tended to focus on written narratives, documents and recorded history. but memory and connections to the past exist in other ways. how did objects function as embodiments of tangible connections with a past? how did the landscape and environment tell stories of connection? mennonites draw on memories of the great chortitza oak which grew in ukraine where mennonites settled in the late eighteenth century but whose acorns have traveled across the atlantic. considering material and environmental sources, as well as written documents will help us understand legacies in diachronic and global perspective or the ways in which communities negotiate their interaction with the contemporary world. finally, scholarship can think about memory, community and identity in areas which have not been part of more conventional narratives of reformation legacies. this means uncovering new trajectories for communities who are not part of the traditional accounts of survival, such as mennonite who migrated down to the black sea from russia and ukraine rather than crossing the atlantic. more broadly, it means focusing on global histories of mennonites and protestantism in general, particularly in the global south, exploring questions of community, race and place in a post-colonial era. these new avenues for research will help reveal religious communities which existed beyond institutional and national structures and the problematic way in which big anniversaries reinforce grand narratives. paying more attention to the local and the individual has the effect conversely of broadening memories across seas and generations. the contentious / cele- bration not only provokes questions about anabaptist history but demands we find more nuanced ways of thinking about memory, the ways it is embedded in language, landscape, and people, and the traces it leaves. conclusions i would like to finish with one example of this power of memory at the level of the individual and the family which also touches on the way in which it expands our global and cross-period histories of religious change. in the mennonite archives in bethel college in north newton, kansas there are boxes which contain family papers donated to the archive for safe-keeping. papers inside have not been categorized into type and are often an eclectic mix of items preserved and curated by the family over generations. opening these folders, i was given a snapshot into the way in which histories, church, and communities are built on individual and family memories but also the importance of placing these in the context of globally connected communities. these collections are not the usual stuff of institutional or national memory. however, precisely because they are personal and have been kept by groups and families on the move, they embody the connected communities of dispersion which are at the core of mennonite identity. one folder has a tiny notebook, meticulously translating and transcribing the daily record kept by their forebears who traveled from russia, russian passports, a handwritten book of early nineteenth century remedies for colic and much spider bites. the jacob f. and marie banman fond contains beautiful eighteenth century fraktur examples, modern hand-written genealogies and family record and copy books. like their owners, these objects had migrated across land and sea, and the juxtaposition of documents which record lives lived across centuries and borders encapsulates the connections, memories and emotions that sustained mennonite communities. these collections of memories go far beyond the -year anniversary and remind us of the power of the local and the glocal (the term coined to express the interplay between the global and the local) in communities bound together by remembrance (freist, , p. ). whether or not mennonites celebrate the -year centenary in or , their debates reveal the global and diachronic power of anabaptist memories, and these memoryscapes offer not only ways of thinking about mennonite identity but broader memory cultures of early modernity and reformation history. memories of the reformations and their global legacies must be understood in cross-confessional contexts. scholarship can use these comparative histories of memories and legacies as a way out of the debates of marginal versus mainstream and con- fessionalisation to consider questions of global protestantisms, long term legacies, and concepts of diaspora and exile. anniver- saries always offer us a chance to rethink histories. in analyzing the various reformation anniversaries, it is not a question of ‘memory wars’—whose anniversary we should be celebrating, whose reformation was better, or which had more positive or long-lasting effects. rather scholars can take the opportunity of these discussions over commemorations to diversify our concepts of the reformation and its legacies. received: january accepted: july notes for information on the variety of celebrations see the official luther website. https://www.luther .de/en/ /reformation-anniversary/. german plans to spend relatively little on centenary events compared to the uk and france were criticized. see https://www.theguardian.com/world/ /mar/ / germany-plans-first-world-war-centenary. an example of a more subtle and low-key exhibition was that at the neues museum weimar, ‘krieg der geister: weimar als symbolort deutscher kultur vor und nach ’. http://mennoworld.org/ / / /the-world-together/anabaptisms- th- anniversary-is- -not- /. see also https://themennonite.org/daily-news/how-to- celebrate- -years/. https://mwc-cmm.org/renewal . https://anabaptisthistorians.org/. this is the series quellen zur geschichte der täufer. see for example the joint statement by the lutheran world federation and the vatican. https://press.vatican.va/content/salastampa/en/bollettino/pubblico/ / / / a.html. a booklet was produced to recognize this by the lwf and the pontifical council for promoting christian unity, from conflict to communion: lutheran-catholic common commemoration of the reformation in ( ). the latest triennial census conducted by the mwc counted , , members as of november . this includes various groups such as the mennonites, the amish, and hutterites. this was the first known anabaptist confession written by michael sattler. references agnew v ( ) diaspora, memory and identity: a search for home. university of toronto press, toronto baronian m-a, besser s, jansen y ( ) diaspora and memory: figures of dis- placement in contemporary literature, arts and politics. rodopi, amsterdam and new york brady jr. ta ( ) confessionalizaiton—the career of a concept. in: headley jm, hillerband hj (eds) confessionalization in europe, – : essays in honor and memory of bodo nischan. ashgate, aldershot dipple g ( ) the radical reformation will not be televised. sixt century journal ( ): – driedger md ( ) obedient heretics: mennonite identities in lutheran hamburg and altona in the confessional age. ashgate, aldershot evangelical church in germany (ekd) perspectives : writings on the reformation ( ). evangelische kirche in deutschland, hanover freist d ( ) lost in time and space? glocal memoryscapes in the early modern world. in: kuijpers e, pollmann j, müller j, van der steen j (eds) memory before modernity: practices of memory in early modern europe. brill, leiden, p. – goossen b ( a) chosen nation: mennonites and germany in a global era. princeton university press, princeton goossen b ( b) why years?: a critique of anabaptism’s upcoming anni- versary celebration. mennonite life palgrave communications | https://doi.org/ . /s - - - comment palgrave communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/palcomms https://www.luther .de/en/ /reformation-anniversary/ https://www.theguardian.com/world/ /mar/ /germany-plans-first-world-war-centenary https://www.theguardian.com/world/ /mar/ /germany-plans-first-world-war-centenary http://mennoworld.org/ / / /the-world-together/anabaptisms- th-anniversary-is- -not- / http://mennoworld.org/ / / /the-world-together/anabaptisms- th-anniversary-is- -not- / https://themennonite.org/daily-news/how-to-celebrate- -years/ https://themennonite.org/daily-news/how-to-celebrate- -years/ https://mwc-cmm.org/renewal https://anabaptisthistorians.org/ https://press.vatican.va/content/salastampa/en/bollettino/pubblico/ / / / a.html https://press.vatican.va/content/salastampa/en/bollettino/pubblico/ / / / a.html www.nature.com/palcomms www.nature.com/palcomms goertz h-j ( ) the anabaptists, trans. trevor johnson. routledge, london and new york gregory b ( ) the unintended reformation: how a religious revolution secu- larized society. belknap press of harvard university press, cambridge, mass and london hill k ( ) baptism, brotherhood, and belief in reformation germany: ana- baptism and lutheranism, – . oxford university press, oxford hoffmann sp ( ) the gdr, luther and the german question. rev polit ( ): – ketelaar e ( ) prolegomena to a social history of dutch archives. in: blok a, lucassen j, sanders h (eds) a usable collection: essays in honour of jaap kloosterman on collecting social history. amsterdam university press, amsterdam lotz-heumann u ( ) the concept of “confessionalization”: a historiographical paradigm in dispute. mem y civón : – lwf (the lutheran world federation and pcpcu (the pontifical council for promoting christian unity)) ( ) from conflict to communion: lutheran- catholic common commemoration of the reformation in . evangelische verlagsanstalt: leipzig and bonifatius marshall p ( ) : martin luther and the invention of the reformation. oxford university press, oxford monge m ( ) des communauteś mouvantes: les societ́eś des frer̀es chret́iens en rheńanie du nord: juliers, berg, cologne vers – . libraire droz, geneva osborne t ( ) “golden age” or “global age”: commemorating anabaptists past, present and future. mennonite life räisänen-schröder p ( ) ketzer im dorf: visitationsverfahren, täuferbe- kämpfung und lokale handlungsmuster im frühneuzeitlichen württemberg. universitatsverlag konstanz, konstanz roth jd ( ) how to commemorate a division? reflections on the th anniversary of the lutheran reformation and its relevance for the global anabaptist mennonite church today. mennon q rev : – scott t ( ) the luther quincentenary in the gdr. ger hist ( ): – stayer jm ( ) anabaptists and the sword. new edition of edition. wipf and stock, eugene stayer jm, depperman k, packull wo ( ) from monogenesis to polygenesis: the historical discussion of anabaptist origins. mennon q rev : – stoler al ( ) along the archival grain: epistemic anxieties and colonial com- mon sense. princeton university press, princeton and oxford urry j ( ) mennonites, politics, and peoplehood: europe-russia-canada, to . university of manitoba press, winnipeg urry j ( ) memory: monuments and the marking of pasts. conrad grebel rev ( ) acknowledgements thanks to proofreaders in the early modern work in progress group and james urry for references and discussions on mennonites. additional information competing interests: the author declares no competing interests. reprints and permission information is available online at http://www.nature.com/ reprints publisher’s note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article’s creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article’s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/ . /. © the author(s) comment palgrave communications | https://doi.org/ . /s - - - palgrave communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/palcomms http://www.nature.com/reprints http://www.nature.com/reprints http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / www.nature.com/palcomms 	memories from the margins? anniversaries, anabaptists and rethinking reformations 	introduction 	anabaptists memories 	conclusions 	references 	references 	acknowledgements 	competing interests 	acknowledgements will be an adjunctive text in a medical library, i am not convinced that the usefulness of this text outweighs its shortcomings. tillman farley, md fort lupton, colo fundamentals of clinical practice: a textbook on the pa- tient, doctor and society. edited by mark b. mengel and warren l. holleman. pp. new york, plenum publishing, . . . isbn - - - . this book would be an excellent addition to commu- nity and behavioral medicine curricula in both resi- dency and medical school settings. several of the au- thors are well-known teachers and researchers in family medicine. the textbook addresses the patient- physician relationship from a variety of angles. the first section addresses the patient, with chapters on hu- man health and disease and individual and family de- velopment; the second section deals with becoming and being a physician in today's challenging health care environment; and the third section describes the nu- merous contexts of family, community, the workplace, environment, culture, and economics that have an im- pact on the physician-patient relationship. the re- mainder of the book is devoted to special issues of health policy and economics, medical ethics, and the current problems of tobacco, alcohol, and drug abuse; violence; mental illness; sexually transmitted diseases; vulnerable and indigent populations; and maternal and child health. overall, i found the book to be fascinating and well researched. this book successfully keeps the focus on the pa- tient-physician relationship by inserting case presenta- tions at regular intervals. the chapters are designed with cases and questions for small-group discussion at the end of each chapter. "\\'hen i used several of the chapters this past year during our community medicine rotation, the answers to some of the questions accompanying the case pre- sentations did not appear to be straightforward. a fa- cilitator's guide to accompany the cases and questions might be helpful. additionally, the residents found that discussing cases which might not be applicable to their practice community to be less meaningful. neverthe- less, the cases raised lots of good issues for discussion. this attempt at covering a broad range of topics is both successful and comprehensive. the book begins by focusing on the physician-patient relationship, adding layer upon layer until we can see in its entirety how complex and diverse practicing medicine can be, particularly in the context of worrisome public health issues. medical schools and residency programs will find this textbook on the relationship of the patient, physician, and society a welcome resource as they strive to impart the community and public health im- plications of the physician's role. sara cate, md central washington family medicine yakima jabfp jan_-feb. vol. no. manual of skin surgery-a practical guide to dermato- logic procedures. by david] lefjell and marc d. brown. pp., illustrated. new york, wiley-liss, . $ . isbn: - - - . this textbook of excisional cutaneous surgery is de- signed for medical students, residents, and practicing physicians. the text begins with a review of basic prin- ciples of anatomy and skin pathophysiology and then focuses on specific procedural skills training for office- based care and procedures. the book contains chapters on diagnosis, practical anatomy, wound heal- ing, skin biopsy, local anesthesia, surgical instruments, wound closure materials, patient preparation, basic ex- cisional surgery, surgical complications, special topics in dermatologic surgery, and risk management. the chapter on special topics helps the practitioner modify general approaches for special circumstances. one ap- pendix includes action guides for skin biopsy, pig- mented lesions, basal cell cancer, squamous cell cancer, and complications. these action guides are suggested algorithms in the work-up and treatment strategies. another appendix includes vendors for dermatologic surgery products. the book can be understood by learners at any level. the index is detailed and easy to follow. the text uses drawings and color pictures to focus the reader. the drawings are extremely helpful, particu- larly in the challenging area of the face, where special anatomic consideration is given to recommended planes for elliptical excision. the strategy of the au- thors, beginning with basic principles of defining the lesion, reviewing the anatomy, and describing normal wound healing, followed by procedural considerations of anesthesia and biopsy, is very effective. although there is no formal bibliography, there are suggestions for further reading with descriptions of seven other texts. it is important to understand that this text does not relate to skin disease but rather surgical ap- proaches to lesions. i believe that this reference is useful for the family practice clinic, is an excellent learning guide for family medicine residents, and provides a strong foundation for medical students. best of all, its relatively low cost should allow even those on a limited budget to include it in a personal library. . scott a. fields, md oregon health sciences university portland a measure of my days: the journal of a country doctor. by david loxterkamp. pp. hanover, nh, university press of new england, . . (paper), isbn - - - . this is an engaging and important book that should take its place next to the classic photo-essay by berger and mohr, a fortunate man, which describes the life of the english country doctor john sassall (berger j, mohr j. a fortunate man. new york: pantheon books, ). each provides an intimate look inside the prac- o n a p ril b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://w w w .ja b fm .o rg / j a m b o a rd f a m p ra ct: first p u b lish e d a s . / - - - c o n ja n u a ry . d o w n lo a d e d fro m http://www.jabfm.org/ tice of medicine and the life of the practitioner in the community. david loxterkamp is a family physician in a small coastal town in maine. in his th year in practice he chose to record his life with regular journal entries, which he then worked into this book. in doing so, he pulls the reader into his life and provides a very enjoy- able reading experience. in the process of examination he seems to have developed a fuller sense of self, home, family, and community, and we are privileged to share that journey. there is a chapter for each month of the year and for each of the four seasons, but the book is much more than an accounting of those days. by moving from the events of the day back into memory of earlier years and by anticipating the future, the author weaves a captivating narrative. the title indicates that the pages will reveal the daily life of a country doctor, and the book delivers on that promise. it reveals with hon- esty and candor the highs and lows and the growth of its author as physician, husband, father, and person of faith. it is a book about community and a deepening commionent to one community as home. dr. loxterkamp's gift with words ensures that the reader will experience the events and emotions of the days recorded. during the several days i was reading the book, i found myself experiencing elation, depres- sion, anxiety, and contenonent. at first i thought these emotions reflected the undulating circumstances of my own days, but i later concluded that was not so. i be- lieve the writing evokes in the reader the emotions and feelings of the author as he reacts to the circumstances of his life. i suspect also that each reader will react dif- ferently to the events described through memory of personal experience. a physician with community practice background will find this journal stimulates recall of the early years of establishing practice, home, and family. some who felt isolation and frustration in those years might find reassurance that their experiences were not unique. anyone who seeks to understand the practice of medi- cine in community would benefit from reading this work, as would those considering such a career. this book should be read by persons responsible for plan- ning, shaping, financing, or regulating community health care systems and organizations, for in doing so, they would develop an intimate understanding of the workings of such systems and the very personal needs they try to meet. such an understanding cannot be found in the collection of diagnostic classifications, lists of symptoms, reports of encounters and proce- dures, and similar tabulations used to describe or eval- uate a medical practice. dr. loxterkamp reveals his life to be one of inten- sity, commitment, and searching. he also shares his contenonents and satisfactions. one might wish him more sheer joy and happiness than what seems to come through in the narrative. but, he says of himself (page ): "there is always a tension between enjoying life and examining it." theodore j. phillips, md lopez island, wash patients are a virtue: practicing medicine in the pennsyl- vania amish country. by henry s. wentz. pp. morgan/gum, pa, masthofpress, . . . isbn - - - . have you wondered what it would be like to practice medicine from to among the amish in lan- caster county, pennsylvania? this fascinating period in medicine spanned the early years of antibiotics, the scourges of polio and rheumatic fever, the shift from solo to group practice, and the changes in health care financing from the $ fee-for-service office visit to medical insurance to managed care. dr. wentz chronicles these events, covering four decades, through short vignettes. he writes in a matter-of-fact, straightforward, conversational style that is most likely consistent with the way in which he practiced and cared for his patients. as he relates the satisfactions and frustrations of his practice, he inter- sperses reflections on lessons he learned, such as the value of nurses, the power of suggestion, the courage of patients facing adversity, and the importance of a supportive family and community for both patient and physician. perhaps his most interesting insights evolve from experiences with his amish patients that illustrate how their beliefs sustained them as they dealt with the challenges of life and death. he also describes the diffi- culties of getting the amish to accept such preventive measures as prenatal care, well-baby examinations, and immunizations. throughout these episodes dr. wentz conveys ac- ceptance of his patients, himself, and his times without undue moral judgment or philosophical speculation. nevertheless, the reader becomes aware of the unique problems associated with practicing alone out of an of- fice in one's home, the stresses on marriage and chil- dren, the interruptions of family gatherings by patients with emergencies, and the concerns of losing patients to a competing physician in the community. most of these stories are not particularly dramatic. their impact arises from the cumulative descriptions of a type of practice that will not be repeated. a physi- cian emerges who appeared to possess the qualities necessary to succeed during a unique time in medical history within a geographical location enriched by people with distinctive motivations and moral princi- ples. thus, this book might be of interest to those physicians who would like to know more about this way of life, to people living in the vicinity of lancaster county, and to lay persons who wonder about the daily activities of a busy general practitioner. vincent r. hunt, md brown university-memorial hospital of rhode island pav.'tucket book reviews o n a p ril b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://w w w .ja b fm .o rg / j a m b o a rd f a m p ra ct: first p u b lish e d a s . / - - - c o n ja n u a ry . d o w n lo a d e d fro m http://www.jabfm.org/ evaluation and management of right-sided heart failure: a scientific statement from the american heart association may , circulation. ; :e –e . doi: . /cir. e marvin a. konstam, md, chair michael s. kiernan, md, ms, faha, co-chair daniel bernstein, md biykem bozkurt, md, phd, faha miriam jacob, md navin k. kapur, md robb d. kociol, md, ms eldrin f. lewis, md, mph, faha mandeep r. mehra, md francis d. pagani, md, phd, faha amish n. raval, md, faha carey ward, md on behalf of the american heart association coun- cil on clinical cardiology; council on cardiovas- cular disease in the young; and council on cardiovascular surgery and anesthesia © american heart association, inc. circulation http://circ.ahajournals.org background and purpose: the diverse causes of right-sided heart failure (rhf) include, among others, primary cardiomyopathies with right ventricular (rv) involvement, rv ischemia and infarction, volume loading caused by cardiac lesions associated with congenital heart disease and valvular pathologies, and pressure loading resulting from pulmonic stenosis or pulmonary hypertension from a variety of causes, including left-sided heart disease. progressive rv dysfunction in these disease states is associated with increased morbidity and mortality. the purpose of this scientific statement is to provide guidance on the assessment and management of rhf. methods: the writing group used systematic literature reviews, published translational and clinical studies, clinical practice guidelines, and expert opinion/statements to summarize existing evidence and to identify areas of inadequacy requiring future research. the panel reviewed the most relevant adult medical literature excluding routine laboratory tests using medline, embase, and web of science through september . the document is organized and classified according to the american heart association to provide specific suggestions, considerations, or reference to contemporary clinical practice recommendations. results: chronic rhf is associated with decreased exercise tolerance, poor functional capacity, decreased cardiac output and progressive end-organ damage (caused by a combination of end-organ venous congestion and underperfusion), and cachexia resulting from poor absorption of nutrients, as well as a systemic proinflammatory state. it is the principal cause of death in patients with pulmonary arterial hypertension. similarly, acute rhf is associated with hemodynamic instability and is the primary cause of death in patients presenting with massive pulmonary embolism, rv myocardial infarction, and postcardiotomy shock associated with cardiac surgery. functional assessment of the right side of the heart can be hindered by its complex geometry. multiple hemodynamic and biochemical markers are associated with worsening rhf and can serve to guide clinical assessment and therapeutic decision making. pharmacological and mechanical interventions targeting isolated acute and chronic rhf have not been well investigated. specific therapies promoting stabilization and recovery of rv function are lacking. conclusions: rhf is a complex syndrome including diverse causes, pathways, and pathological processes. in this scientific statement, we review the causes and epidemiology of rv dysfunction and the pathophysiology of acute and chronic rhf and provide guidance for the management of the associated conditions leading to and caused by rhf. aha scientific statement evaluation and management of right-sided heart failure a scientific statement from the american heart association endorsed by the heart failure society of america and international society for heart and lung transplantation key words: aha scientific statements ◼ causality ◼ disease management ◼ heart failure ◼ ventricular dysfunction, right d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines o ver the years, there have been intermittent surges in interest in and investigation of the right ven- tricle (rv). one such period was the early s, with the emergence of novel imaging techniques, ad- vanced surgical approaches, enhanced understanding of rv infarction and ischemia, and intense physiological in- vestigation of right-sided hemodynamics and ventricular interdependence. now, once again, we enjoy intense in- terest in this area, owing in large part to advanced phar- macology for managing pulmonary hypertension (ph) and a mushrooming of both diagnostic techniques and durable and temporary mechanical circulatory support (mcs) devices, yielding expanded treatment options and enhanced outcomes in patients with acute and chronic disorders of the right side of the heart. rv dysfunction (rvd), defined here as evidence of ab- normal rv structure or function, is associated with poor clinical outcomes independently of the underlying mecha- nism of disease: across the spectrum of left ventricular (lv) ejection fraction (ef) in patients with acute and chronic heart failure (hf), after cardiac surgery, acute myocardial infarction (mi), congenital heart disease (chd), and ph. to distinguish right-sided hf (rhf) from structural rvd, we define rhf as a clinical syndrome with signs and symp- toms of hf resulting from rvd. rhf is caused by the inability of the rv to support optimal circulation in the presence of adequate preload. this scientific statement provides evidence-based guidance in the following areas: pathophysiology of rvd, assessment and evaluation of rv function, epidemiology and prognosis of rhf, medical and surgical management of acute (arhf) and chronic rhf (crhf), and proposed areas for future investigation. the recommendations and suggestions/considerations listed in this document are, whenever possible, evidence based. an extensive literature review was conducted through may , with references selected as appropri- ate. searches were limited to studies, reviews, and other evidence conducted in human subjects and published in english. in addition, the committee reviewed documents related to the subject matter previously published by clini- cal practice guideline task forces from the american heart association (aha) and american college of cardiology. references selected and published in this document are representative but not all inclusive. to provide clinicians with a representative evidence base, whenever deemed appropriate or when published, it was felt that critical appraisal of the quality of study be maintained and, whenever possible, robust statistical data be provided. organization of the writing committee the committee was composed of physicians with a broad knowledge base in the epidemiology and patho- physiology of hf with expertise in the evaluation, care, and management of patients with rhf. the authors’ expertise included general cardiology, advanced hf and transplantation cardiology, cardiac surgery, interven- tional cardiology, ph, and chd; physicians with meth- odological expertise also were included. the committee included representatives from the aha council on clini- cal cardiology, council on cardiovascular disease in the young, and council on cardiovascular surgery and an- esthesia; the heart failure society of america; and the international society of heart and lung transplantation. document review and approval this document was reviewed by official reviewers, each nominated by the aha. all information on review- ers’ relationships with industry was distributed to the writing committee and is published in this document. this document was approved for publication by the governing bodies of the aha. suggestions/considerations and reference to clinical practice guideline recommendations to make certain that this document is aligned with the appropriate guideline statements but does not preempt those guidelines, the authors have opted to reference evidence-based clinical practice recommendations only and to refer the reader to the most recently published clinical practice guideline statement for more specific alignment with extant guidelines. suggestions/con- siderations are included when the evidence does not warrant recommendations but there is still a desire to provide some guidance to the community. scope of this scientific statement with reference to other relevant guidelines or statements this scientific statement focuses on the evaluation and management of rhf. some topics may have been reviewed in other clinical practice guidelines and scientific statements published by other working groups, including the ameri- can college of cardiology/aha task forces. the writing committee saw no need to reiterate the recommendations contained in those guidelines but chose instead to provide current suggestions or considerations for clinical practice and to clarify previous discrepancies if present. anatomy and embryology of the rv several developmental and anatomic features distin- guish the rv from the lv. , the rv and rv outflow d ow nloaded from http://ahajournals.org by on a pril , may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es konstam et al evaluation and management of right-sided heart failure tracts originate from cells of the secondary (anterior) heart field, whereas the lv and left atria originate from the primary heart field. the identification of the sec- ondary heart field has provided new insight into the development of congenital heart defects and may lead to discoveries identifying distinct intercellular signaling pathways and transcriptional regulation in response to injury by the rv in contrast to the lv. – beginning in the third week of embryonic development, the primi- tive heart tube begins beating and undergoes a series of twisting and folding movements to generate a sin- gle primitive ventricle that receives blood from a single atrium and ejects blood through a common outflow tube known as the truncus arteriosus (figure  ). by the end of the fourth week, a muscular ventricular sep- tum emerges from the floor of the primitive ventricle to form the earliest signs of distinct rvs and lvs. between the fifth and eighth weeks, ridges within the truncus ar- teriosus grow into the aorticopulmonary septum, which fuses with the endocardial cushions and muscular inter- ventricular septum to form the membranous septum. at the end of the eighth week, distinct pulmonary and systemic circulations exist. for the remainder of fetal development, the rv will account for ≈ % of total cardiac output (co), which provides systemic perfusion via the foramen ovale and the ductus arteriosus. at birth, the lv becomes the dominant systemic ventricle while the rv adapts to provide flow through the pul- monary circulation alone, assuming that the foramen ovale and ductus arteriosus close appropriately. anatomically, the rv free wall is thin ( – mm) and compliant and forms a hemi-ellipsoid shape that ad- heres to the lv (figure  ). a large sinus for venous in- flow and a tubular outflow tract provide a funnel-like configuration to the heavily trabeculated rv. unlike the shared annulus of the aortic and mitral valves, the crista supraventricularis is a muscle bridge that is unique to the rv and separates the rv inflow (tricuspid annulus) from the outflow tract (pulmonic annulus). the crista supraventricularis shares muscle fibers with the inter- ventricular septum and the rv free wall and serves to contract the orifice of the tricuspid valve (tv) while pull- ing the rv free wall toward the interventricular septum during systole. , physiology of the rv normal rv function is governed by systemic venous return, pa load (rv afterload), pericardial compliance, and native contractility of the rv free wall and inter- ventricular septum. generating rv output requires one sixth the energy expenditure of the lv because much of rv stroke work maintains forward momentum of blood flow into a highly compliant, low-resistance pul- monic circulation. this difference is exemplified by the rv pressure-volume (pv) loop, which lacks isovolumic figure . cardiac embryogenesis. during embryogenesis, the primary heart field is formed by early cardiac progenitor cells in the anterior mesoderm. the sec- ondary heart field is derived from the pharyngeal mesoderm located medial and anterior to the primary heart field. cells from the primary heart field migrate to the midline to form a linear heart tube, serving as a scaffold for subsequent heart growth. the heart tube is expanded posteriorly and anteriorly with cells migrating from the secondary heart field, giving rise to the arterial and venous poles. the linear heart tube undergoes a rightward looping, leading to the formation of primitive ventricles and atria. as a result, the venous pole moves anteriorly, positioning the future cardiac chambers for proper development. heart maturation involves septation formation in the ventricles and atria, as well as valve formation. the primary heart field contributes to the left ventricle and right and left atria. the secondary heart field contributes to the right ventricle, outflow tract, and right and left atria. cardiac neural crest cells migrating from the dorsal neural tube into the arterial pole participate in separation of the outflow tract. reprinted by permission from macmillan publishers ltd. adapted from xin et al. nature reviews molecular cell biology. copyright © , macmillan publishers ltd. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines phases of contraction and relaxation during systole and diastole, has a lower peak systolic pressure, and exists at a higher steady-state volume compared with the lv. in contrast to the lv, peak rv pressure occurs before the end of systolic ejection, which leads to a more trap- ezoid-appearing rv pv loop (figure  ). – afterload is a primary determinant of normal rv function, and rvef is inversely proportional to pulmo- nary artery (pa) pressure (pap). the rv has a shal- lower end-systolic pv slope than the lv, which results in lesser change in end-systolic pressure, generating greater change in end-systolic volume. , accordingly, rv systolic function is highly sensitive to changes in afterload, with minor increases in afterload causing large decreases in stroke volume (sv) (figure  ). as dictated by the law of laplace, wall stress (afterload) is directly proportional to intracavitary pressure and to internal ventricular diameter and inversely related to ventricular wall thickness. however, regional wall stress may vary widely as a result of the nonspherical rv shape. rv afterload is most appropriately defined as the rv wall stress during systolic ejection estimated by the summation of the resistive and pulsatile com- ponents of blood flow. , commonly used measures of rv afterload, including pa systolic pressure (pasp) and pulmonary vascular resistance (pvr), provide an inadequate description of rv afterload because they do not account for contributions of pulsatile loading. as blood ejects from the rv into the lungs, antegrade flow away from the pulmonic valve (pv) encounters waves of retrograde flow generated by multiple bifur- cations throughout the pulmonary vasculature. this retrograde impedance wave reduces antegrade flow and increases peak pasp. in the setting of left-sided heart (lh) disease, eleva- tions in left atrial pressures lead to lower pa compliance than would be anticipated from elevated pvr alone. , similarly, lowering of pulmonary capillary wedge pres- sure (pcwp) increases pa compliance more than would be anticipated from a fall in pvr alone. thus, elevated lh filling pressures directly increase rv afterload, sec- ondarily reduce pa compliance, and increase pa resis- tance through acute vasoconstriction and chronic vas- cular remodeling. under steady-state conditions, optimal ventricu- lar efficiency is achieved if end-systolic elastance (ees) is matched by vascular load, defined by arterial elas- tance , – (figure  ). the ratio of ees to arterial elas- tance is known as ventriculo-arterial coupling, a frame- work to consider contractility in the context of load. the optimal mechanical coupling of rv function to afterload corresponds to a ratio of ees to arterial elas- tance of . , with uncoupling occurring below a ratio of . to . . , , when pasp increases acutely, rv sv decreases significantly and arterial elastance increases out of proportion to ees. as a result, rv function be- comes inefficient, and more energy is expended to maintain adequate rv output. in contrast, an increase in aortic systolic pressure results in smaller decreases in lv sv, thereby maintaining a near-normal ventriculo- arterial coupling ratio. unlike the predominantly diastolic coronary flow of the lv, normal rv coronary perfusion occurs during both systole and diastole. the pressure-overloaded rv is at increased risk for developing ischemia as a result of decreased perfusion pressure in the setting of in- creased rv intramural pressure and decreased system- ic arterial pressure. , any process that increases rv figure . right ventricular (rv) geometry in health and disease. three-dimensional reconstructions of the rv illustrating its complex shape in a normal subject (a). rv remodeling in diseased hearts can result in profound shape change with rv dilation caused by chronic volume or pressure overload (b). the red mesh surface is the left ventricle (lv), and the solid blue surface is the rv. p indicates pulmonary valve; and t, tricuspid valve. reprinted from sheehan and redington with permission from bmj publishing group, ltd. copyright © , bmj publishing group, ltd. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es end-diastolic pressure leads to decreased rv coronary blood flow and has the potential to induce subendo- cardial ischemia. pathophysiology of rhf acute rhf arhf can occur because of abruptly increased rv af- terload (pulmonary embolus, hypoxia, acidemia) or de- creased rv contractility (rv ischemia, myocarditis, post- cardiotomy shock). each condition represents a unique hemodynamic challenge for the rv. the rv is coupled to the high-compliance, low-resistance pulmonary cir- culation and is suited to adapt to changes in volume rather than pressure. in a healthy individual, pvr is < / th of the systemic vascular resistance. in contrast, the lv is coupled to the lower-compliance, higher-re- sistance systemic arterial circulation and adapts better to changes in pressure than volume. thus, an acute in- crease in rv afterload such as can result from a large pulmonary embolism (pe) may abruptly decrease rv sv, with minimal increase in rv systolic pressure. acute reductions in rv contractility may also be caused by direct myocardial injury from mechanisms such as myocardial inflammation (myocarditis) and ischemia. reduced rv sv results in rv dilation, which promotes tricuspid regurgitation (tr), exacerbates rv dilation, and drives a ventricular-interdependent effect on lv filling. ventricular interdependence is defined as the forces directly transmitted from one ventricle to the other through the myocardium and pericardium. mechanical flattening with a leftward shift of the inter- ventricular septum increases lv end-diastolic pressure, reduces lv transmural filling pressure, and impedes lv diastolic filling, contributing to systemic hypoperfusion (figure  ). diastolic interaction is described as ventricu- lar competition for diastolic distension/filling within an acutely confined pericardial space. systolic interactions also exist because it is estimated that % to % of rv systolic pressure results from lv contraction. , elevated filling pressures of the right side of the heart also cause coronary sinus congestion, which re- duces coronary blood flow and can provoke rv isch- emia. , high right-sided filling pressure with systemic figure . right ventricular (rv) pressure-volume (pv) loops. rv pv loops obtained by a conductance catheter. white solid lines reflect the end-systolic pv relationships (espvr) of a series of loops generated by varying the loading conditions. the slope of espvr line reflects the rv end-systolic elastance (ees). a steeper slope represents higher ees. loop a depicts a normal rv pv loop. a lower proportion of rv stroke work goes to pressure generation, with a higher proportion going to blood momentum. in the normal state, in contrast to the left ventricle (lv), there is a relative absence of rv isovolemic periods. the high momentum of blood ejecting from the rv into the low-pressure pulmonary circulation results in con- tinued rv ejection after lv systolic ejection has ended into rv relaxation. loop b represents a compensated, chronically hypertensive rv. loop c is obtained from a decompensated hypertensive rv. note the decrease in rv ees from the com- pensated rv depicted in loop b to the decompensated rv depicted by loop c. reproduced from friedberg and reding- ton with permission. copyright © , american heart association. figure . relationship of right ventricular (rv) and left ventricular (lv) stroke volumes to increases in after- load. response of the rv and lv to an experimental increase in afterload. note the comparatively steep decline in stroke volume associated with increases in pressure compared with the smaller reductions seen in lv stroke volume associated with similar pressure increments. reprinted with permission of the american thoracic society. copy- right © , american thoracic society. macnee w. pathophysiology of cor pulmonale in chronic obstructive pulmonary disease: part one. am j respir crit care med. ; : – . the american journal of respira- tory and critical care medicine is an official journal of the american thoracic society. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines venous congestion also negatively affects hepatic and renal function, aggravating further fluid retention and worsening rhf. chronic rhf crhf most commonly results from gradual increases in rv afterload caused by ph most frequently from lh failure (lhf), although chronic volume overload from right-sided lesions such as tr can also lead to its development (figure  ). long-standing pressure or volume overload imposed on the rv initially pro- motes compensatory myocyte hypertrophy and fibro- sis analogous to the remodeling that occurs in lhf. if the load persists, then the rv transitions from a compensated to decompensated phenotype charac- terized by myocyte loss and replacement/fibrosis. during the initial compensated phase, the hyper- trophied rv begins to develop isovolumic phases of contraction and relaxation with increased rv systolic pressure and higher end-diastolic volume (figure  ). in the decompensating phase, there is a concomitant rise in pvr and right atrial (ra) pressure (rap). while pvr remains persistently elevated, co subsequently declines, followed by a reduction in pap , (figure  ). declining pap in the setting of high pvr is an ominous clinical finding. in the presence of an intact pericardium, rv dila- tion eventually compresses the lv cavity, impeding lv filling and equalizing biventricular diastolic pressures (figure  ). although it is true that patients with crhf may require higher rv end-diastolic pressure (preload), reduced lh filling is more likely caused by rv dilation and ventricular interdependence than reduced rv for- ward output. that is, increased transmural pressure caused by rv dilation with pericardial constraint impairs lv filling (preload). the combination of rv systolic and biventricular diastolic dysfunction reduces co, impairs coronary blood flow, and exacerbates peripheral and abdominal congestion. epidemiology and pathogenesis of rhf hf with reduced ef progressive rhf was first described as a component of the hf clinical syndrome in . regardless of pathogenesis, rvd increases in prevalence with more advanced lhf. in this setting, rvd may occur second- ary to increased rv afterload from postcapillary ph, vol- ume overload, arrhythmias, or the underlying myocar- dial disease process affecting the lv (table  ). the last factor may contribute to the higher prevalence of rvd observed in nonischemic dilated cardiomyopathy com- pared with ischemic cardiomyopathy, especially given the possible genetic predisposition in many of these patients. the overall prevalence of rvd in hf with re- duced ef (hfref) varies widely with distinct differences in varied populations, but its presence is universally associated with increased mortality. the prevalence of rvd in a meta-analysis of patients with hfref was %. in a small cohort of patients with dilated car- diomyopathy, rvd was seen in ≈ % of patients and was associated with greater mitral regurgitation and tr, more rapid progression of clinical hf, and decreased survival. likewise, in a separate series, patients with nonischemic dilated cardiomyopathy had a higher pro- portion of rvd than those with an ischemic pathogen- esis: % versus %. among patients with hfref who underwent echocardiography during acute hf hospitalization, % had rvd. these patients had a . -fold increased risk of mortality, urgent transplanta- figure . pressure-volume (pv) loop. right ventricular–pulmonary arterial (rv-pa) coupling. the pv loop is a comprehensive description of the relationship between pressure and volume during the cardiac cycle. the area within the loop defines the stroke work of the rv, with the width of the loop representing stroke volume (sv). end-systolic elastance (ees), a load-independent measure of contractility, is determined by a tangent fitted on the end-systolic portions of a family of pv. rv afterload is determined by dividing the end-systolic pressure by the sv, providing the effective arterial elastance (ea). ea is measured as the slope of a straight line drawn from the end-systolic to end-diastolic pv relationship (to end-diastolic volume [edv] at p= ). the relationship of these parameters (ees:ea) provides a ratio defining rv-pa coupling, which reflects contractility in the context of afterload. determinations of ees and ea require instantaneous measurements of rv pres- sure and volume to generate sequential pv loops obtained by a decrease of venous return via stepwise inflation of an inferior vena cava balloon or a valsalva maneuver. reprinted from guazzi and naeije with permission from elsevier. copyright © , elsevier. d ow nloaded from http://ahajournals.org by on a pril , may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es konstam et al evaluation and management of right-sided heart failure tion, or urgent lv assist device (lvad) placement at days compared with those without rvd. rvd is also associated with decreased exercise ca- pacity measured by peak oxygen consumption and worse new york heart association functional class. in a study of patients with hfref, rv exercise con- tractile reserve and rv-pa coupling were assessed with tricuspid annular plane systolic excursion (tapse) ver- sus pasp and the slope of mean pap versus co. pa- tients were grouped according to whether their rest- ing tapse was ≥ mm. those with tapse < mm were further subdivided by whether tapse at peak figure . ventricular interdependence in right-sided heart failure. pathological increases in right ventricular (rv) filling pressures are transmitted to the interventricular septum. as the rv is constrained by the pericardium (arrows), these forces result in leftward shift of the septum, altering left ventricular (lv) ge- ometry. these changes contribute to reduced cardiac output by decreasing lv distensibility, preload, and ventricular elastance, adversely affecting lv diastolic filling. leftward septal shift secondary to pericardial constraint from elevated rv end-diastolic pressure distorts the normal geometric ventricular relationship, also impairing rv contractile function. adapted from haddad et al with permission. copyright © , american heart association. figure . pathophysiology of right-sided heart failure. lv indicates left ventricular; lvedp, left ventricular end-diastolic pressure; rap, right atrial pressure; rv, right ventricle; and rvedd, right ventricular end-diastolic dimension. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines exercise was ≥ . mm. although patients had similar baseline profiles of biventricular function, those with higher tapse in response to exercise demonstrated improved rv contractile reserve and some degree of favorable rv-pa coupling in contrast to those patients with persistently low tapse. thus, many patients with resting rvd can have residual contractile reserve with the ability to improve rv-pa coupling during exercise. data directly linking rvd to reduced performance on structured health-related quality of life (hrqol) ques- tionnaires are sparse. hf with preserved ef rv function is equally important in patients with hf with preserved ef (hfpef). in this population, however, it is difficult to distinguish primary rv pathology from that resulting from secondary ph, given the afterload dependency of rv function. nevertheless, several small cohort studies have evaluated the prevalence of concomitant rv systolic dysfunction in the setting of hfpef. in a mayo clinic cohort, % of patients with hfpef had rvd defined as rv fractional area change (rvfac) < %. in a separate study of patients, depending on the criteria used, rvd was present in % to % of patients with hfpef in contrast to % to % of those with hfref. other groups have reported similar findings. a meta-analysis including patients reported varied prevalence depending partly on the modality used to assess rv function: % by tapse, % by rv s’, and % by rvfac. approximately % of these patients with hfpef with rvd had concomitant ph at rest. more novel indexes of rv function have led to higher prevalence estimates. rv longitudinal systolic strain abnormalities were iden- tified in % of patients with hfpef, whereas rv- fac < % was seen in only %. compared with patients with hfpef without rvd, those with rvd were more likely to be male, to have more renal impair- ment, and to have a higher prevalence of atrial fibrilla- tion and coronary artery disease. , analogous to outcomes in hfref populations, rvd is associated with increased morbidity and mortality in hfpef populations. two-year mortality in study was ≈ % for patients with rvd compared with % figure . hemodynamics in progressive pulmonary vascular disease. a decrease in pulmonary arterial pressure (pap) in patients with pulmonary hypertension may be a sign of low cardiac output (co) and severe right ventricular dysfunction. mpap indicates mean pap; pcwp, pulmonary artery capil- lary wedge pressure; pvr, pulmonary vascular resistance; and rap, right atrial pressure. adapted from haddad et al with permission. copyright © , american heart association. table . causes of rhf decreased rv contractility rv volume overload rv pressure overload acute sepsis acidosis lvad support hypoxia rvmi excessive transfusion pe myocarditis ards perioperative injury/ischemia (postcardiotomy) positive pressure ventilation chronic rv cardiomyopathy lh disease arvc single ventricle ebstein anomaly pericardial disease pr pah tga chronic thromboembolic ph tr ps left-sided valvular heart disease restrictive cardiomyopathy ards indicates acute respiratory distress syndrome; arvc, arrhythmogenic right ventricular cardiomyopathy; lh, left- sided heart disease; lvad, left ventricular assist device; pah, pulmonary arterial hypertension; pe, pulmonary embolism; ph, pulmonary hypertension; pr, pulmonary regurgitation; ps, pulmonary stenosis; rhf, right-sided heart failure; rv, right ventricular; rvmi, right ventricular myocardial infarction; tga, transposition of the great arteries; and tr, tricuspid regurgitation. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es in those without rvd. exercise intolerance is com- mon in people with hfpef, and those with evidence of rvd have lower new york heart association classifica- tion. in an exercise comparison between patients with hfpef and control subjects, those with hf- pef had impaired rv systolic and diastolic functional enhancement measured by invasive cardiopulmonary exercise testing and simultaneous echocardiogra- phy. increased left- and right-sided filling pressures and limitations in co reserve correlated with abnor- mal augmentation in biventricular mechanics during stress, suggesting limited rv reserve with rv-pa un- coupling during. myocarditis inflammatory myocardial disease has varied clinical presentations and outcomes. the focus of myocarditis studies has been predominantly on characterization of lv function. rv involvement, however, may reflect a greater burden of inflammation, preexisting vulnerabil- ity to an acute process, or increased afterload caused by lhf. when treatment options are considered, the presence of rvd, depending on the severity, may dic- tate the need for biventricular support. in patients with active or borderline myocarditis, rvd was pres- ent in % of patients with anti-heart autoantibodies compared with % of those without anti-heart auto- antibodies. the presence of rvd by cardiac magnetic resonance imaging (mri) in a study of patients with myocarditis was associated with a hazard ratio of . for death or heart transplantation and was the stron- gest predictor of death. rv myocardial infarction acute rvmi is prevalent in ≈ % of patients with an acute inferior mi. a functionally-relevant acute rvmi generally requires disruption of blood flow to both the rv free wall and a portion of the interventricu- lar septum. it typically occurs when a dominant right coronary artery is occluded proximally to the major rv branch(es), leading to reduced rv systolic function and acute rv dilation. a smaller proportion of patients have rvmi resulting from circumflex coronary artery occlusion in a left-dominant coronary system and rare- ly in association with left anterior descending coronary artery occlusion, in which this artery supplies collater- als to an otherwise underperfused anterior portion of the rv free wall. rvmi is associated with hemodynamic compromise in % to % of patients presenting with this infarct pattern. early mortality is highest among patients with evidence of hemodynamic compromise. , patients with rvmi have a greater burden of arrhythmias, con- tributing to mortality. most patients recover rv func- tion within days to weeks after the infarct. one-year mortality after rvmi is reported to be % in patients with isolated right coronary artery lesions compared to % in the presence of combined right and left coro- nary artery disease. in long-term follow-up, mortality beyond the first year remains at an additional %/y to %/y through year . in series, mortality among patients with inferior mi with rvmi was % to % compared with % in patients without rvmi. similar- ly, among patients with acute mi undergoing per- cutaneous coronary intervention, excluding those with cardiogenic shock on admission, electrocardiographic (st-segment elevation of . mv in lead v r or v r) and echocardiographic evidence of rvmi (rv free wall motion abnormalities or rv dilatation) was associated with higher in-hospital and - and -month mortality compared with patients with either anterior or inferior mi without evidence of rvmi (although findings at months were not statistically significant). although patients with inferior mi have, in general, a better prognosis than those with anterior mi, the presence of rv involvement increases the risk of death, shock, and arrhythmia. among patients with mi complicated by cardiogenic shock enrolled in the shock trial (should we emergently revascularize occluded coronaries for cardiogenic shock), % had a picture of predominant rhf and the remainder had a shock syndrome charac- terized by lhf. despite some favorable clinical charac- teristics among the patients with rhf, the mortality rate was similar between these groups. postsurgical arhf arhf may occur during or after noncardiac surgery as a result of the development of acute ph or intraopera- tive myocardial ischemia. the prevalence of rhf after noncardiac surgery is difficult to determine. there is the potential for a survival bias whereby patients with more profound rhf die before full cardiac evaluation. furthermore, preexisting hf may make the diagnosis of arhf more challenging to differentiate. during cardiac surgery, arhf can be caused by hy- poxia/myocardial ischemia, microemboli, air emboli leading to mi, arrhythmias, and excessive volume load- ing. , furthermore, there is a disruption in the na- tive rv contractile pattern after cardiothoracic surgery. although overall rv function remains preserved, the combination of cardiopulmonary bypass and pericardi- otomy leads to a reduction in longitudinal contraction and an increase in transverse shortening. in the nor- mal rv, longitudinal shortening accounts for ≈ % of rv function. whether release of pericardial constraint after complete pericardiotomy predisposes the at-risk rv to the development of arhf remains uncertain. rvd is frequently seen within days of cardiac surgery and may persist despite improvements in lv function. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines postoperative arhf is associated with increased mor- tality, prolonged length of stay, and increased resource use. in a cohort of patients undergoing coronary ar- tery bypass graft surgery, most patients did not dem- onstrate a significant postoperative change in rv func- tion, although modest decreases in longitudinal strain were noted. after cardiac transplantation primary graft dysfunction (pgd) affects ≈ % or more of patients after cardiac transplantation and is the lead- ing cause of early mortality. , pgd may be classified as pgd of the lv, which includes biventricular dysfunc- tion, versus pgd of the rv alone. diagnosis of pgd of the rv alone requires both ( ) rap > mm hg, pcwp < mm hg, and cardiac index < . l·min− ·m− and ( ) transpulmonary gradient < mm hg and pasp < mm hg or ( ) the need for an rv assist device (rvad). the pathogenesis of pgd is complex and likely multifac- torial. contributing causes consist of donor, procedur- al, and recipient-level factors, including inflammatory mediators resulting from brain death, elevated pvr, and ischemia/reperfusion injury associated with pres- ervation issues. , although management of arhf is discussed elsewhere, a decision on the need for right- sided mcs should be made before leaving the opera- tive room, pending the initial response to medical inter- ventions. , after lvad twenty percent or more of patients undergoing isolated lvad implantation experience arhf, which is a lead- ing cause of premature morbidity and mortality. – rates of rhf associated with lvad insertion may be partially dependent on the underlying cause of myopa- thy. patients with a history of chemotherapy-associated cardiomyopathy appear to be at higher risk than those with other forms of nonischemic or ischemic disease. the physiology of arhf after lvad implantation is com- plex. from a hemodynamic perspective, activation of an lvad increases venous return, potentially overwhelm- ing a functionally impaired rv, leading to rv dilatation, tr, leftward shift of the interventricular septum, and decline in rv sv. as rv output falls and the septum shifts leftward, lv preload and lvad flows are reduced. the rv is dependent on the lv for a significant portion of its contractile function, and leftward septal shift re- sulting from lv unloading can have a direct, detrimental effect on rv contraction. , furthermore, anchoring of the lvad to the lv apex may alter the normal twisting contractile pattern of the heart. whether the direction of apical deformation (ie, apical pull versus push, de- pending on device configuration and placement) alters the risk of rvd remains uncertain. lv unloading with mechanical support may improve rv contractility via a reduction in pap after the acute decline in pcwp associated with lvad activation. , ph, however, is a risk factor for the development of arhf in lvad recipients, , and residual, fixed ph likely contributes to rv-pa uncoupling when other intraoperative complications are encountered. even if compensated for in the preoperative period, a chroni- cally dysfunctional rv coupled to a fixed and elevated pulmonary afterload may not be able to tolerate intra- operative insults such as ischemia and volume loading, which then precipitate arhf. in addition, it is possible that a reduction in systemic afterload after insertion of rotary blood pumps leads to a decline in lv contractility with a resultant second- ary decline in rv contractility. the anrep effect is the physiological consequence whereby increases in arterial afterload lead to increases in ventricular contractility. although this relationship remains somewhat hypo- thetical in the lvad-supported circulation, the converse of this is also true: reductions in afterload may lead to reduced contractility. late rhf in the lvad recipients, after initial hospital discharge, occurs in ≈ % of patients and is similarly associated with reduced survival and lower hrqol and functional capacity. , the development of ventricular and atrial tachyarrhythmias may be a significant factor contributing to the development of late rhf. pe with arhf acute pe can lead to acute rv strain as a result of pres- sure overload within minutes of occlusion of a major pa segment and is a common cause of arhf. , physi- cal presentation often includes initial syncope or right- sided atrial arrhythmias. the prevalence of arhf in the setting of acute pe ranges from % to %. , predictors of rvd include > % of the pa tree oc- cluded by thrombus. patients with evidence of rvd have a . - to . -fold increase in mortality compared with those without rvd. , given the poor prognosis, guidelines on the management of acute pe recommend early detection of rvd to guide risk stratification and therapeutic decision making. arrhythmogenic rv cardiomyopathy arrhythmogenic rv cardiomyopathy (arvc) is a dis- ease of the cardiac myocytes caused by impaired des- mosome function. desmosomes are intercellular junctions that provide adhesion between cells. muta- tions in desmosomal proteins such as plakophilin and desmoplakin decrease the ability of the cells to toler- ate mechanical stress, resulting in myocyte detachment and cell death. the inflammation that accompanies this process manifests as fibrofatty infiltration, causing ven- d ow nloaded from http://ahajournals.org by on a pril , may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es konstam et al evaluation and management of right-sided heart failure tricular irritability and arrhythmias and eventually ven- tricular dysfunction. in affected patients, this process shows a predilection for the thinnest portions of the rv where mechanical stress is greatest. however, the lv also is often affected in advanced disease. the prevalence of arvc is estimated to be in to , and arvc affects men more frequently than women. a familial component is identified in > % of patients. transmission is autosomal dominant with in- complete penetrance. diagnosis of arvc can be diffi- cult, so standardized criteria have been developed that are based on family history, ventricular dysfunction, tissue characterization, electrocardiographic changes (figure  a), and history of arrhythmias. the sensitivity of electrocardiographic criteria alone (table  ) for the diagnosis of arvc is low. diagnosis requires a specific combination of major and minor criteria from the ecg, rv imaging, and family history that are reviewed in detail elsewhere. genetic screening, although not re- quired, may be helpful when screening family members of a recently diagnosed patient. , recommendations for the role of genetic testing of probands and first- degree relatives have been published. arvc is more often associated with arrhythmia than isolated rvd. in italy, where young athletes undergo detailed screening before participation in sports, arvc was a cause of sudden cardiac death (scd) in > %. in a registry of scds in athletes from the united kingdom, arvc was detected in % of subjects. among us patients diagnosed with arvc, the median age at presentation was years, but age at presentation varied widely, from to years. thirty-one patients experienced scd, and patients progressed to rhf, one of whom died while awaiting cardiac transplantation. figure . ecgs in patients with right-sided heart disease. a, ecg from a patient with arrhythmogenic right ventricular cardiomyopathy. ecg from a patient with t-wave inversion in v through v and prolongation of the terminal activation of a -millisecond duration measured from the nadir of the s wave to the end of the qrs complex in v . reproduced with permission from marcus et al. copyright © , american heart association. b, ecg with right ventricular hypertrophy. ecg demonstrating the changes of right ventricular hypertrophy. long arrow indicates dominant r wave in v ; short arrow, right-axis deviation; black arrowhead, right atrial abnormality; and open arrowhead, secondary st-t changes. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines more than % of patients had a life-threatening ar- rhythmia at some point during follow-up. this statistic is, of course, influenced by selection bias because the presence of ventricular arrhythmia increases the prob- ability of ascertaining the diagnosis of arvc. indica- tions for implantable cardioverter-defibrillator therapy in arvc are available. tricuspid regurgitation tr is a common echocardiographic finding, and mild tr is present in % to % of individuals. although less common, moderate to severe tr affects > mil- lion people in the united states. tr can be related to principal types: primary valvular tr and secondary functional tr. functional changes of the tv related to annular dilation and leaflet tethering in the setting of rv remodeling caused by pressure and volume over- load are the most common causes of significant tr, ac- counting for % of cases. in contrast, primary tr is secondary to lesions of the valve structure itself such as endocardial cushion abnormalities, ebstein anomaly, endocarditis, and carcinoid heart disease. the severity of tr affects prognosis even when con- trolling for lv dysfunction or ph. in a study of pa- tients at veterans affairs medical centers, -year sur- vival rates were %, %, %, and % in patient groups with no, mild, moderate, or severe tr, respec- tively. moderate or greater tr was associated with increased mortality regardless of pasp or lvef. severe tr, older age, lower lvef, inferior vena cava dilation, and moderate or greater rv enlargement were associ- ated with worse survival. pv disease pulmonary stenosis (ps) occurs in ≈ % of children with chd. ps can be encountered as part of tetralogy of fallot (tof), the incidence of which is in live births, , or other complex chds such as trans- position of the great arteries (tga), ventricular septal defect, and ps or an isolated valve abnormality. pv atre- sia can be seen in patients with tof but can also be encountered in those with an intact ventricular septum. ps can also be seen in patients with noonan syndrome, in whom it can be isolated or seen in combination with cardiomyopathy. pv disease can be found in the setting of endocarditis, especially in intravenous drug users, or in carcinoid heart disease. , patients with isolated ps do quite well, usually treated with balloon valvuloplasty alone. when treated, long-term survival of patients with ps is not different from that in individuals without ps. the development of rhf in patients with isolated ps is rare. pulmonary insufficiency is most often a consequence of balloon valvuloplasty or surgical repair of congeni- tal abnormalities and is commonly seen after complete repair of tof. the highest-risk group is made up of patients with a small pv annulus, in whom surgical re- pair involves placement of a transannular patch, leav- ing the patient with an incompetent pv. historically, surgeons have attempted to make this patch as large as possible to relieve ps; however, more contemporary techniques use a smaller transannular patch, recogniz- ing that a small degree of residual ps is preferable for preservation of long-term rv function than wide-open pulmonary insufficiency. mri studies have shown rv fibrosis in % of patients with repaired tof and lv fibrosis in %. in % to % of patients, pulmo- table . revised task force criteria for electrocardiographic diagnosis of arvc repolarization abnormalities major inverted t waves in right precordial leads (v –v ) or beyond in individuals > y of age (in the absence of complete right bundle-branch block qrs ≥ ms) minor inverted t waves in leads v and v in individuals > y of age (in the absence of complete right bundle-branch block) or in v , v , or v inverted t waves in leads v –v in individuals > y of age in the presence of complete right bundle-branch block depolarization/conduction abnormalities major epsilon wave (reproducible low-amplitude signals between end of qrs complex and onset of the t wave) in the right precordial leads (v –v ) minor late potentials by saecg in ≥ of parameters in the absence of a qrs duration ≥ ms on the standard ecg filtered qrs duration ≥ ms duration of terminal qrs < v (low-amplitude signal duration) ms root-mean-square voltage of terminal ms ≤ μv terminal activation duration of qrs ≥ ms measured from the nadir of the s wave to the end of the qrs, including r, in v , v , or v , in the absence of complete right bundle- branch block arvc indicates arrhythmogenic right ventricular cardiomyopathy; and saecg, signal-averaged ecg. adapted from marcus et al with permission. copyright © , american heart association. table . classification of ph ph category characteristics clinical group precapillary mpap ≥ mm hg pcwp ≤ mm hg who class , – postcapillary mpap ≥ mm hg pcwp > mm hg who class , isolated postcapillary ph dpg < mm hg and/ or pvr ≤ wu combined precapillary and postcapillary ph dpg ≥ mm hg and/ or pvr > wu dpg indicates diastolic pulmonary gradient; mpap, mean pulmonary artery pressure; pcwp, pulmonary capillary wedge pressure; ph, pulmonary hypertension; pvr, pulmonary vascular resistance; who, world health organization; and wu, woods units. reproduced from galiè et al with permission. copyright © , oxford university press. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es nary insufficiency leads to progressive rv dilatation and dysfunction and may require pv replacement later in life. , left unrepaired, rv dilatation and dysfunction can lead to ventricular arrhythmias with a rate of scd in this population estimated to be . %/y. pulmonary hypertension the evaluation of a patient with rvd includes an as- sessment for increased rv afterload. ph is characterized by alterations in the pulmonary vasculature leading to increased pvr and ultimately rvd. the increased after- load on the rv leads to rv hypertrophy, dilation, and systolic dysfunction. ph is defined as a mean pap ≥ mm hg and when present is associated with impaired survival. ph is found in isolation and as a consequence of other diseases as detailed below. patients are catego- rized on the basis of the mechanism of disease (table  ). group : pulmonary arterial hypertension pulmonary arterial hypertension (pah) describes a group of disorders characterized hemodynamically by the presence of precapillary ph, defined by a pcwp ≤ mm hg and a pvr > woods units in the absence of other causes of precapillary ph such as chronic lung disease. , initial ph screening is typically performed with echocardiography, but right-sided heart catheter- ization is required for definitive diagnosis. the incidence for all group pah is . cases per million adults with an overall prevalence of . per million adults. group includes those patients with idiopathic pah; hereditary pah; pah caused by drugs and toxins; pah associated with connective tissues disease, portal hy- pertension, hiv, chd with persistent pulmonary-to- systemic shunt, eisenmenger physiology, and schisto- somiasis; persistent pah of the newborn; pulmonary veno-occlusive disease; and pulmonary capillary hem- angiomatosis. patients with pah benefit from care provided in cen- ters with expertise in this condition. despite the intro- duction of new pulmonary vasodilator therapies, group ph continues to be associated with high morbidity and mortality. in a us cohort of patients in the reveal registry (registry to evaluate early and long-term pul- monary arterial hypertension disease management), - and -year survival rates were % and %, respec- tively. pah secondary to connective tissue disease or portopulmonary hypertension is associated with a worse prognosis. the presence of rvd in patients with pah is a strong predictor of adverse outcomes and more closely associated with clinical outcomes than the paps. hemodynamic and echocardiographic mark- ers of rvd associated with increased mortality include ra and rv dilation, elevated rap, rv systolic dysfunc- tion, the presence of a pericardial effusion, decreased pa capacitance, and reduced co. , , assessment of rv strain may add prognostic value to traditional markers of rvd in the assessment of pah. , there is significant variability in the timing of the onset of rhf among patients, including those with similarly elevated pap. , , , other variables associated with reduced survival include lower functional class, lower blood pressure (bp), higher heart rate, increased bnp (b-type natriuretic peptide), reduced diffusion capacity of the lung for carbon monoxide, and reduced -minute walk distance. in the subset of patients with elevated rv pressure secondary to eisenmenger syndrome or con- genital ps, chronic rv pressure overload can be reason- ably well tolerated for decades. – pulmonary veno-occlusive disease represents a small subgroup of group pah associated with a particularly poor prognosis. in contrast to other forms of pah, pulmonary veno-occlusive disease results in postcapil- lary ph. vasodilatory therapy can lead to clinical wors- ening in patients with pulmonary veno-occlusive dis- ease, and these patients should be considered for lung transplantation. group : lh disease chronically elevated left-sided filling pressures from lv systolic and diastolic dysfunction or significant left-sid- ed valvular heart disease can lead to ph. a study of patients with hf found that % of those with hfref and % with hfpef had ph. the definition of group ph requires a mean pap ≥ mm hg with a pcwp > mm hg or lv end-diastolic pressure ≥ mm hg. most patients with hf have postcapillary ph, characterized by low pvr (< woods units) and low transpulmonary gradient (≤ mm hg). others, howev- er, have elevation of pvr and transpulmonary gradient, historically described as out-of-proportion or mixed ph. the commonly used measures of out-of-proportion ph include transpulmonary gradient, pvr, and the diastolic pulmonary gradient (diastolic pulmonary gradient=pa diastolic pressure–pcwp). a diastolic pulmonary gra- dient ≥ mm hg suggests pulmonary vascular disease superimposed on left-sided pressure elevation. clas- sification of postcapillary ph in the european society of cardiology guidelines for the diagnosis and treatment of ph identified patients as having either isolated postcapillary ph, defined by a diastolic pul- monary gradient < mm hg, or combined precapillary or postcapillary ph (cpc-ph) with diastolic pulmonary gradient ≥ mm hg and concomitantly elevated pvr > woods units (table  ). in study, cpc-ph was observed in % of patients with hf with an equal prevalence in hfref and hfpef. predictors of cpc-ph included younger age, coexistent chronic obstructive d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines pulmonary disease, and valvular heart disease. com- pared with isolated postcapillary ph, cpc-ph was asso- ciated with increased mortality. rv–pulmonary vascular coupling is poor in cpc-ph and has been proposed as an explanation for poor outcomes associated with this syndrome. , , group : chronic lung disease and hypoxia ph caused by chronic obstructive pulmonary disease or interstitial lung disease is usually mild to moder- ate in severity. the small number of patients with severe ph in this category may have an additional underlying pathogenesis for an elevated pvr. ph is seen in ≈ % of patients with obstructive sleep ap- nea. , obesity hypoventilation syndrome is similarly associated with a high prevalence of ph and rvd – and a high rate of overlap among patients with these diagnoses. group : chronic thromboembolic disease when ph is identified, exclusion of chronic thrombo- embolic ph is required, given that surgical interven- tion can alter the natural history of this disease. a significant proportion of patients without a history of pe are ultimately diagnosed with chronic thromboem- bolic ph. ventilation/perfusion scan is standard in the evaluation of newly diagnosed ph. , among pa- tients undergoing pulmonary endarterectomy at expe- rienced centers, -year survival is as high as %, and -year survival is %. limited data from obser- vational studies describe a potential role for percuta- neous balloon angioplasty in patients with inoperable disease. – group : miscellaneous group ph is characterized by diseases with multiple or unclear mechanisms contributing to the develop- ment of ph. this group includes systemic illnesses such as sarcoidosis, chronic hemolytic disorders, and chronic kidney disease. myocardial depression during sepsis is a well-known phenomenon, and rhf can occur inde- pendently of ph or respiratory status. rvd can oc- cur in isolation or concomitantly with lv dysfunction. a combination of reduced rv contractility and decreased preload caused by systemic vasodilation can impair circulation. acute respiratory distress syndrome and mechanical ventilation are associated with increased pvr, which can also precipitate rvd. in a small study of septic patients evaluated with radionuclide imaging, patients with worsening respiratory status and ph were less likely to have recovery of rv function. congenital heart disease chd affects nearly % of births per year. , because of improved surgical techniques and advances in the medical care of patients with chd, the prevalence of congenital heart defects in adult patients has grown substantially over the past decade. as of , the esti- mated . million adults in the united states living with chd exceeded the number of children with chd. many patients with chd have significant rv involve- ment because of right-sided valvular lesions, intracar- diac shunting, or the presence of a systemic rv. the prognosis for each lesion of chd is dependent on the severity of the residual hemodynamic abnormalities and other associated defects. atrial septal defect there are types of atrial septal defects (asds): ostium secundum (most common), ostium primum, and sinus venosus. , larger asds pose little or no resistance to intra-atrial flow, yielding equalization or near equaliza- tion of left atrial pressure and rap. in the absence of pulmonary vascular obstructive disease (eisenmenger physiology), the magnitude of the left-to-right shunt in unrestrictive asds relates primarily to the relative com- pliance characteristics of the ventricles. the shunt poses a volume load on the rv, dilating this chamber, as well as the pulmonary vasculature and left atrium, but if not complicated by additional anomalies or ph, it seldom results in rhf in early life. rv volume overload is associated with lv dysfunction secondary to altered chamber geometry and decreased myofiber preload, which is immediately reversible after asd closure and reflective of the ventricular interdependence. across multiple disease types, in the absence of primary rv pathology, rvef tends to correlate with pap. however, because of increased preload associated with an asd, rvef is higher than would otherwise be expected for any degree of rv contractile dysfunction or afterload. asds do not close spontaneously, and in developed countries, they are generally surgically corrected in childhood. left uncorrected, they are associated with pulmonary vascular obstructive disease and eisen- menger physiology in a small percentage of cases. eisenmenger syndrome is characterized by progressive pvr, ph, reversal (or bidirectionality) of the intracardiac shunt, cyanosis, and rv hypertrophy and failure. the frequency of this syndrome is much lower with an asd than a ventricular septal defect because in the latter case increased pulmonary blood flow is accompanied by increased pressure caused (effectively) by direct lv ejection into the pulmonary circulation, impeded only by the resistance posed by the intracardiac defect it- self. when asds are allowed to persist into late adult life, in the absence of eisenmenger physiology, an in- d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es creased rate of morbidity and mortality may be driven by an increased magnitude of the left-to-right shunt, the result of progressive lv stiffness caused by system- ic hypertension or aging. , in these cases, rhf may ensue as a result of the increase in both volume and pressure load, the latter generally driven more by the increased magnitude of pulmonary flow than obstruc- tive pulmonary vascular disease. therefore, if the asd is missed or ignored in childhood, it is not unusual for the patient to present symptomatically for the first time in late life. ebstein anomaly in ebstein anomaly, ≥ leaflets of the tv are adherent to the rv wall, leading to atrialization of a portion of the rv chamber with varying degrees of tr. in neonates, severe ebstein anomaly can lead to profound cyanosis caused by the inability of the rv to eject into the pul- monary circulation. in older patients with milder forms, it can lead to cyanosis with exercise caused by shunting across a foramen ovale or an asd. the degree of long- term rhf depends on the degree of rv hypoplasia or the success of tv reparative surgery. transposition of the great arteries l-tga is commonly referred to as corrected transposi- tion. in this lesion, the morphological rv serves as the systemic ventricle and is coupled to the high-pressure systemic circulation. the majority of patients with l- tga have other significant cardiac defects, most com- monly including ventricular septal defect and ps. the tv is almost always abnormal, frequently with ebstein- like displacement, leading to tr and rv volume over- load. rhf occurs in up to % of patients with l-tga by middle age, with the risk increased by the presence of associated lesions such as tv disease. d-tga results in cyanosis in the newborn period. be- fore the s, surgical repair involved placement of an atrial baffle (atrial switch, mustard or senning pro- cedure) to redirect venous return, leaving the rv as the systemic ventricle. analogous to patients with l-tga, the rv in these patients is at increased risk of dilatation and failure. the arterial switch operation involves tran- section of both great arteries and translocation of the vessels to the opposite root, creating ventriculoarterial concordance, and has become the standard corrective procedure for this lesion. single-ventricle (rv) physiology there are various forms of single-ventricle morphology. in the most severe forms of chd involving hypopla- sia or total absence of the lv (ie, hypoplastic lh syn- drome), surgical palliative procedures result in the rv being used as the systemic ventricle, coupled to the high-resistance systemic circulation. in these patients, pulmonary blood flow is achieved by connecting the vena cavae directly to the pulmonary arteries (glenn and fontan operations). there are a number of long- term complications in patients with a single ventricle, including protein-losing enteropathy, plastic bronchitis, and hepatic fibrosis. however, systemic rv dilation, tr, and rhf become increasingly common as patients en- ter their third and fourth decades. patients with a sys- temic rv are at greater risk of developing hf than pa- tients with a systemic lv. fibrosis is present in > % of patients with a fontan operation. clinical manifestations of rhf acute rhf arhf is generally characterized by acute rv dilation, a ventricular-interdependent effect limiting lv fill- ing, reduced rv forward flow, and elevated systemic venous pressure. patients with arhf typically show signs of hypoperfusion and hypotension, including diaphoresis, listlessness, cyanosis, cool extremities, hypotension, and tachycardia. although chest aus- cultation may point to underlying lung pathology, the finding of pulmonary edema is not consistent with isolated arhf. instead, if pulmonary edema is pres- ent, it suggests arhf combined with or secondary to lhf. in this setting, many of the common clinical findings of crhf (see chronic rhf) such as peripheral table . manifestations of rhf clinical manifestations of rhf increased mortality fatigue/decreased functional capacity cardiorenal abnormalities cardiohepatic abnormalities protein malnutrition coagulopathy cachexia signs and symptoms elevated jugular venous pressure with prominent v wave peripheral edema bloating/early satiety/abdominal discomfort ascites and hepatomegaly pleural effusion prominent s (p ) (ph) right-sided s gallop holosystolic murmur llsb (tr) rv parasternal heave llsb indicates left lower sternal border; ph, pulmonary hypertension; rhf, right-sided heart failure; rv, right ventricular; and tr, tricuspid regurgitation. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines edema may initially be less prominent or absent al- together. besides systemic hypoperfusion, prominent clinical findings in arhf include shortness of breath resulting from diminished peripheral oxygen deliv- ery, as well as atrial and ventricular arrhythmias. on clinical examination, signs of arhf include increased jugular venous pressure with a prominent v wave, prominent midprecordial cardiac impulse, right-sided third heart sound, and holosystolic murmur of tr. hepatomegaly, ascites, and peripheral edema may be present when arhf is superimposed on crhf. right upper quadrant discomfort may be caused by stretch of the hepatic capsule by hepatic congestion. if a pat- ent foramen ovale is present (≈ % of adults), right- to-left shunting at the atrial level can lead to systemic hypoxemia and cyanosis. chronic rhf peripheral edema is often the most prominent clinical feature in patients with crhf (table  ). although pa- tients with early stages of crhf may initially have mild symptoms, as rv function worsens, the reduction in co leads to progressive exercise intolerance and fatigue. atrial tachyarrhythmias are common in the setting of elevated rap and can lead to hemodynamic deteriora- tion in crhf. ventricular tachycardia and heart block are additional electrophysiological complications and common causes of scd in this population. in patients with ph, crhf closely correlates with increased morbid- ity and mortality. , the hemodynamic constellation driving the del- eterious effects of crhf on end-organ function dif- fers between crhf and primary lhf, the former rep- resenting a combination of increased central venous pressure (cvp) and reduced lv filling resulting from ventricular interdependence as a consequence of rv dilation. , recent studies have also suggested a pri- mary role for elevated cvp in the pathophysiology of end-organ dysfunction occurring in the setting of pri- mary lhf. in the later stages of crhf when systemic output is reduced, impaired end-organ function may be caused by both elevated central venous filling pres- sure and reduced co (figure  ). increased systemic venous pressure impedes lung lymphatic drainage, decreasing lung fluid clearance and exacerbating pul- monary edema and the development of pleural effu- sions in the setting of concomitant postcapillary ph. the organs most affected in crhf are the kidneys and liver, with recent studies implicating the gastrointesti- nal tract. , cardiorenal syndrome in patients with rhf, the increase in cvp and conse- quent rise in renal vein pressure worsen renal function, even in the absence of decreased co (figure  ). in- creased cvp has been identified as an independent risk factor for impaired renal function in patients with hf of diverse origins. , clinical features include decreased urine output, worsening fluid retention, and increased diuretic requirements. laboratory abnormalities in- clude increased blood urea nitrogen and creatinine. elevated and worsening levels of serum creatinine and blood urea nitrogen are independent predictors of ad- verse outcomes, unless they are accompanied by signif- icant decongestion in the setting of acute hf. these findings, connoting worsening renal function, may be causally linked to adverse outcomes or may, in part, represent epiphenomena connoting worsening hf and cardiac function. furthermore, these findings may limit the clinician’s aggressiveness in pursuing diuresis and using guideline-directed medical therapy, includ- ing renin-angiotensin-aldosterone system–inhibiting agents, and may promote the use of inotropic agents with their associated deleterious effects. considerable efforts to identify treatments that simultaneously re- solve congestion, improve clinical hf, and preserve or improve renal function have had limited success. – continued investigation into the interdependency of right-sided heart function and the cardiorenal syn- drome is warranted. increasing evidence of acute kidney injury in the set- ting of elevated right-sided filling pressures may moti- vate clinicians to erroneously reduce loop diuretic ther- apy. however, this action may be deleterious because there is a proverbial “hump” one must get over to im- prove hemodynamics sufficiently that diuresis becomes increasingly effective. if the volume status is unclear or there is concern that the clinical presentation represents isolated rhf, then placement of a pa catheter may be informative. cardiohepatic syndrome the term congestive hepatopathy is a misnomer be- cause it generally results from a combination of he- patic congestion and reduced hepatic perfusion. over time, congestive hepatopathy can lead to the devel- opment of cardiac cirrhosis. , the most prominent laboratory abnormalities include markers of cholesta- sis (elevated bilirubin, γ-glutamyl transpeptidase, and alkaline phosphatase) and altered synthetic function (prolonged prothrombin time). these laboratory ab- normalities are more commonly encountered than el- evations in transaminases. , severity of tr has been found to be closely associated with liver function ab- normalities, and markers of cholestasis are indepen- dently associated with mortality among patients with hf. , likewise, hyperbilirubinemia is a risk factor for poor outcomes in patients with ph. in the set- ting of these abnormalities, patients being considered for advanced hf therapies such as cardiac transplan- tation may require a liver biopsy to exclude cirrhosis, d ow nloaded from http://ahajournals.org by on a pril , may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es konstam et al evaluation and management of right-sided heart failure particularly if hepatic imaging is also abnormal. furthermore, patients with end-stage liver disease are at risk for the developing portopulmonary hyperten- sion, which is associated with worse outcomes after liver transplantation. isolated and markedly elevated transaminases are more commonly caused by severely reduced co/cardiogenic shock and respond better to inotropes than diuretic therapy. increased serum am- monia is also associated with adverse prognosis in de- compensated hf. gastrointestinal involvement gastrointestinal tract function can be impaired by rhf as a consequence of increased cvp and reduced co, leading to reduced absorption and malnutrition. , splanchnic venous congestion with deficient abdominal figure . pathophysiology of cardiorenal disease: acute decompensated heart failure leading to kidney injury. acei indicates angiotensin-converting enzyme inhibitor; arb, angiotensin- receptor blocker; co, cardiac output; cvp, central venous pressure; gfr, glomerular filtration rate; lvedp, left ventricular end-diastolic pressure; nsaid, nonsteroidal anti- inflammatory drug; raas, renin-angiotensin-aldosterone system; sns, sympathetic nervous system; and sv, stroke volume. reproduced with permission from kiernan ms, udelson je, sarnak m, konstam m. cardiorenal syndrome: definition, preva- lence, diagnosis, and pathophysiology. in: post tw, ed. uptodate. waltham, ma: uptodate. accessed on january , . copyright © , uptodate, inc. for more information, visit www.uptodate.com. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines lymph flow causes interstitial edema with ensuing in- creases in intra-abdominal pressure that may contribute to rhf-induced renal failure. , visceral edema also impairs the barrier function of the intestine, allowing entrance of toxins produced by microorganisms in the gut lumen into the bloodstream, which can further sup- press cardiac and renal function and contribute to sys- temic inflammation. , protein-losing enteropathy, a complication most commonly associated with single- ventricle physiology, is also less commonly seen with other forms of rhf, including severe tr. , labora- tory abnormalities include reduced serum albumin and increased stool α -antitrypsin. evaluation of rhf physical examination patients with severe rhf may appear emaciated, tachy- pneic, and cyanotic. patients with significant rhf typically have elevated jugular venous pressure with a prominent v wave from tr. atrial fibrillation is common. those in sinus rhythm may have a prominent a wave caused by rv diastolic abnormalities. increased jugular venous pressure with inspiration (kussmaul sign) may also be seen with a noncompliant rv. the abdomino- jugular reflex test, defined as a sustained rise of > cm in the jugular venous pressure for at least seconds with calm spontaneous respiration, may unmask subtle venous hypertension. a precordial rv heave, hepa- tomegaly and ascites, and lower extremity or presacral edema may be observed. for patients with rhf second- ary to ph, a prominent pulmonic component of the sec- ond heart sound (p ) may be heard on auscultation. in contrast, for those with chd, p may be soft or even absent in cases when there is a structural or postsurgi- cal abnormality of the pv. a low-amplitude holosystolic murmur of tr may be present. an increase in the ampli- tude of the systolic murmur during inspiration (carvallo sign) may distinguish tr from mitral regurgitation. for patients with chd such as repaired tof, there may be a to-and-fro murmur of combined ps and insufficiency at the upper left sternal border, with radiation to the lungs. electrocardiographic evaluation crhf is often associated with right-axis deviation, r:s amplitude ratio of > in lead v , r wave > . mv in v , and p-wave amplitude of > . mm in ii, iii, avf or > . mm in v indicative of ra enlargement (figure  b). arhf may be associated with sinus tachycardia and a qr pattern in lead v . , an initial s deflection in i, initial q deflection in iii, and inverted t in iii (si, qiii, tiii) may point to acute rv strain such as in the case of large pe (high specificity, low sensitivity). atrial arrhythmias, especially atrial flutter, are common. serum markers in crhf, the transaminases may be normal or mini- mally elevated; however, in arhf, transaminase levels are commonly high. in advanced crhf, liver synthetic function may be impaired as evidenced by reduced al- bumin and elevated international normalized ratio. in- creased bilirubin can be related to passive congestion or cholestasis or could suggest the onset of fibrosis and cirrhosis. in more severe cases, venous congestion com- bined with systemic hypoperfusion can lead to renal insufficiency characterized by an elevation in the blood urea nitrogen and creatinine. echocardiography guidelines on the echocardiographic assessment of the rv are available elsewhere. there are limitations to the quantification of rv function by -dimensional echocardiography because of the complex geometry and retrosternal position of the rv, significant intrao- bserver variability, and load and angle dependence of standard imaging parameters, including tapse, rvfac, and tricuspid annular systolic velocity by tissue dop- pler. although strain and strain rate are indepen- dent of ventricular morphology and angle independent when obtained by speckle tracking, these measures are intrinsically load dependent. rv strain imaging is be- coming an increasingly popular tool in the evaluation of ph and, when performed in experienced hands, is in- dependently prognostic. , however, given the high variability in addition to a current lack of standardiza- tion and normative data, the american society of echo- cardiography does not yet recommend tissue doppler imaging for this purpose. rv size compared with volumetric mri, -dimensional mea- surements of rv size can be erroneous because of the complex shape of the rv. rv enlargement is suggest- ed if the rv area dimension is larger than the lv area in end diastole in the apical -chamber view. a linear rv basal dimension of > . cm also suggests significant enlargement. rv end-diastolic wall thickness of > mm in the subcostal view indicates hypertrophy. rv function rv motion is restricted mainly to longitudinal (base to apex) shortening and systolic thickening. longitudi- nal shortening of the rv may be measured by tapse (normal reference limit ≥ . cm). tapse reflects systolic motion of a single point of tricuspid annulus, disregard- ing the contribution of mid, apical, and free wall seg- ments. motion of the tv annulus is affected by prior cardiac surgery, which may render tapse less useful in this population. additional measures of rv systolic dys- function include rvfac < % and rv tissue doppler s’ d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es velocity < cm/s at the plane of the tricuspid annulus. three-dimensional echocardiography is emerging as a potential alternative, perhaps more accurate, method of evaluating rv size and contractility. , close corre- lation exists between -dimensional echocardiography and mri for rv size in patients with ph and chd. , rv diastolic dysfunction may be measured with tis- sue doppler early diastolic myocardial velocity at the tricuspid lateral annulus (e) and early diastolic tricuspid inflow (e’) ratio (e/e’) as demonstrated in patients with ph. , an estimate of pasp can be made by doppler inter- rogation of tr, assuming an adequate doppler enve- lope. pasp is calculated as the doppler gradient across the tv plus estimated rap. pasp may be underestimat- ed with poor ultrasound alignment and overestimated if the patient is anemic, as well as in cases when rap is severely elevated. the tapse/pasp ratio has recently been proposed as a potential marker of rv-pa coupling because tapse reflects rv contractile function and pasp serves as a surrogate of afterload. this ratio was lower in sub- jects who died with a similar distribution in the hfref and hfpef populations. the inferior vena cava diameter and collapsibility with respiration can be used to estimate rap as a mark- er of volume status. hepatic vein flow reversal indicates severe tr, whereas leftward interatrial septal bowing also indicates rap or volume overload. although vol- ume overload and pressure overload frequently occur simultaneously, diastolic septal flattening is associated with a volume-overloaded state, whereas systolic flat- tening is more consistent with pressure overload. cardiac mri cardiac mri offers -dimensional, tomographic imaging of the entire heart and has become the gold standard for quantitative noninvasive measurement of rv vol- ume, mass, and ef, including patients with chd. – delayed gadolinium enhancement methods can iden- tify areas of rv fibrosis, although given the thinner wall of the rv, this may be more challenging compared with use for quantification of lv fibrosis. dynamic real-time imaging during deep inspiration and expiration has been developed to detect rv volume changes and in- termittent interventricular septal flattening. velocity- encoded methods also measure co. multidetector computed tomography similar to cardiac mri and -dimensional echocar- diography, multidetector computed tomography provides volumetric information about rv size and function. , the high spatial resolution of contrast- enhanced computed tomography, combined with its rapid scan sequence, makes it an appealing modality for rv assessment. limitations with computed tomog- raphy include the need to bolus potentially nephrotoxic iodinated contrast material and the need for ionizing radiation, which is a problem for radiosensitive patient populations, including children, and for serial surveil- lance studies. although the patient must be able to lie flat and perform a breath hold, the scan process occurs over a shorter period compared with cardiac mri. fur- thermore, implanted devices do not prevent the use of computed tomography, and claustrophobia is rarely an issue given the short bore length. radionuclide imaging radionuclide imaging can be used for the assessment of rv size, function, and infiltration. it is less fre- quently used for these purposes in the contemporary era given its poorer spatial resolution compared with other imaging modalities and the need for ionizing radiotracers. however, radionuclide ventriculography can be more accurate than echocardiography in mea- suring rv volumes given its reliance on count density rather than on geometric assumptions, which may be stymied by complex rv geometry. increased rv uptake of [ f]- -fluoro- -deoxy-d-glucose has been described in cases of ph, including its potential utility in tracking response to pulmonary vasodilator therapy. , tech- netium pyrophosphate imaging is a sensitive means of detecting certain infiltrative processes, particularly car- diac involvement by transthyretin amyloidosis. it is most frequently used via ventilation/perfusion scan to screen for chronic thromboembolic ph in patients with ph and rv enlargement detected by other imaging modalities. chest radiograph in cases of significant rv enlargement, the cardiac sil- houette on a chest radiograph will have a globular ap- pearance. loss of the retrosternal airspace on a lateral projection also indicates rv enlargement. rightward displacement of the cardiac silhouette, well beyond the spine, almost always represents ra enlargement (the alterative being a giant left atrium). with coexisting ph, the main pa will be enlarged and the distal pa branches may have a “pruned” appearance. , elevated cvp may sometimes be recognized by an enlarged azygous vein. pulmonary vascular redistribution, increased inter- stitial markings, and kerley lines are signs of pulmonary venous hypertension, evidence of which often dimin- ishes as pulmonary vascular obstructive disease pro- gresses and signs of rhf dominate those of lhf in the presence of chronic biventricular failure. pleural effu- sions are frequent in the presence of severe hf, particu- larly when pulmonary and systemic venous pressures are both elevated. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines hemodynamic assessment of rv function several hemodynamic variables have been identified as risk factors for the development of rhf, predominantly after lvad surgery (table  ). as with imaging pa- rameters of rv function, hemodynamic correlates are largely inconsistent across studies. elevated rap, par- ticularly when this elevation is disproportionate relative to the rise in pcwp, is a marker of rvd. , a normal ra/pcwp ratio is ≈ . ; higher ratios imply rvd. , a preoperative ra/pcwp ratio > . is associated with rhf after lvad surgery. pa pulsatility is another po- tential correlate of rhf. , pa pulse pressure index, defined as the ratio of pa pulse pressure (pasp minus pa diastolic pressure) to rap, was recently proposed as a sensitive marker of disproportionate rvd and a pre- dictor of post-vad rhf. this metric, however, has also yet to be validated in larger, prospective cohorts. rv stroke work index [(mean pap−rap)×sv index] is an established marker of rv function. sv index is calcu- lated by dividing cardiac index by heart rate. rv stroke work index is influenced by preload, however, and its calculation is dependent on multiple measured param- eters susceptible to acquisition error. , – risk models of rhf durable mechanical circulatory assist devices are increas- ingly offered to patients with advanced hf as a bridge to transplantation or as long-term destination therapy. despite technological advances in the use of continuous- flow lvads, rhf remains a major cause of morbidity and mortality after lvad implantation. , postoperative rhf can occur immediately, before the patient leaves the operating room, whereas in other cases, it may develop over weeks, months, or even years as a result of the pro- gression of underlying myocardial disease, worsening tr, or ph. several clinical prediction scores have been developed to facilitate preoperative identification of pa- tients at risk for post-lvad rhf. , , , , , , age, vital signs, invasive hemodynamic metrics, echocardio- graphic parameters, indexes of end-organ function, and the need for cardiorespiratory support have been used to identify high-risk individuals. although these scores perform well in their derivation cohorts, they perform less well in external validation studies. the complex physiology of rhf complicates accurate prediction of postoperative events, and these scores are unable to in- corporate intraoperative parameters or events that can precipitate arhf. such events include volume loading from transfusions and short-term increases in pvr sec- ondary to hypoxia, acidemia, or increases in airway pres- sure from mechanical ventilation. furthermore, predic- tion models cannot model rv-lvad interactions and the direct impact of lvad hemodynamics on rv function, including volume loading of the right-sided circulation and geometric changes in the contractile pattern of the interventricular septum that affect rv sv. other limita- tions of risk scores include the heterogeneity of popu- lations studied and the variability in definitions of rhf used. the overall clinical profile of the patient tends to be the strongest correlate of the failing rv, with greater acuity of illness being associated with more severe rhf. severe rhf with diminished systemic perfusion is com- monly characterized by a state of vasodilation, inter- stitial fluid leakage, and systemic inflammation, poten- tially with fever in the absence of infection. profound vasodilation can be present and should not be mistaken for alternative causes of distributive shock. refractory cardiogenic shock is generally accompanied by severe biventricular dysfunction. given the known limita- tions of echocardiographic and hemodynamic variables, no parameter in isolation can adequately identify clini- cally significant rhf with a high sensitivity or specificity. the assessment of rv function requires a multimodal- ity approach with careful evaluation of trends across hemodynamic, hematologic, and imaging parameters. confidence in the clinical diagnosis of rhf increases when multiple parameters across modalities collectively suggest a state of rvd. biomarkers to assess rv function there has been much interest in identifying biomarkers such as nt-probnp (n-terminal pro-bnp) to help guide table . hemodynamic assessment of rh function hemodynamic parameters associated with rv function variable calculation thresholds associated with clinical events in specific populations rap rap (or cvp) > mm hg (rhf after lvad) , right-to-left discordance of filling pressures rap:pcwp > . (rhf after lvad) > . (rhf in acute mi) pa pulsatility index (pasp−padp)/rap < . (rhf in acute mi) < . (rhf after lvad) rv stroke work index (mpap−cvp)×svi < . – . mm hg·l/m (rhf after lvad) , pvr (mpap−pcwp)/co > . wu (rhf after lvad) pa compliance sv/(pasp−padp) < . ml/mm hg (rhf in chronic hf, rv-pa coupling in pah) , co indicates cardiac output; cvp, central venous pressure; lvad, left ventricular assist device; mi, myocardial infarction; mpap, mean pulmonary artery pressure; pa, pulmonary artery; padp, pulmonary artery diastolic pressure; pah, pulmonary artery hypertension; pasp, pulmonary artery systolic pressure; pcwp, pulmonary capillary wedge pressure; pvr, pulmonary vascular resistance; rap, right atrial pressure; rh, right-sided heart; rhf, right-sided heart failure; rv, right ventricular; sv, stroke volume; svi, stroke volume index (svi=cardiac index/heart rate); and wu, woods units. adapted with permission from kapur et al. copyright © , american heart association. d ow nloaded from http://ahajournals.org by on a pril , may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es konstam et al evaluation and management of right-sided heart failure the identification and management of patients with hf. the focus, however, has historically been on iden- tifying patients with lv dysfunction. nt-probnp may also be useful in patients with predominantly rhf but is relatively nonspecific. , likewise, elevated bnp is observed in both lh and right-sided heart dysfunction and is not a reliable marker to distinguish the level of dysfunction within each ventricle. , bnp, however, has emerged as a useful marker of prognosis in rhf ac- companying pah. the reliability of other biomarkers such as creatine phosphokinase-mb and troponins is less well established in the setting of rhf. – elevated bnp and troponin have adverse prognostic implications in the setting of acute pe. , several small, predominantly exploratory studies have examined rv-specific gene expression patterns, micrornas, exosomes, and proteins associated with rhf, primarily in patients with chd and ph. – one study used unbiased rna sequencing to identify several differentially expressed genes in patients with rvd. of these, steap , sparcl , and vsig were differentially expressed between the rv and lv, suggesting their role as rv-specific biomarkers. lewis et al identified metabolites that were closely related to hemodynamic indexes of rvd using mass spectrometry–based meth- ods in subjects with ph. similarly, early animal stud- ies have identified unique micrornas associated with rv but not lv failure. , identifying novel rv-specific biomarkers may lead to targeted therapies for rhf. medical management of arhf management of arhf focuses on management of volume and preload, myocardial contractility, and rv afterload with pharmacotherapy and, if needed, mcs (figure  ). , abnormalities in the pulmonary circu- lation and lv filling should be identified as targets for reducing rv afterload and augmenting rv function. volume management volume management is a critical consideration in arhf, and the volume status of the patient should be deter- mined on initial examination. a primary goal should be a reduction in left atrial pressure with an aim of reducing congestion and pulsatile rv loading. in the absence of gross volume overload, evident by findings such as pe- ripheral edema, careful inspection of the jugular venous pulsation should allow the clinician in most cases to de- termine the presence or absence of elevated cvp. this examination may be confounded by the large a waves of atrial contraction against a poorly compliant rv and large v waves caused by a poorly compliant ra or significant tr. hemodynamic monitoring with a central venous cath- eter or pa catheter can be informative if the volume status is uncertain or if a patient has hemodynamic instability or worsening renal function in response to therapy. , the teaching that arhf is a preload-dependent condition requiring volume loading is overly simplistic. figure . management of acute right-sided heart failure. all management must be undertaken with an awareness of the patient’s hemodynamic status. if this status is not clear clini- cally, then invasive assessment/monitoring should be undertaken. hemodynamic targets provide rough guidelines for tai- lored therapy. av indicates atrial-ventricular; ci, cardiac index; cvp, central venous pressure; cvvhf, continuous venovenous hemofiltration; dccv, direct current cardioversion; iv, intravenous; lv, left ventricular; map, mean arterial pressure; ns, normal saline; pah, pulmonary arterial hypertension; pcwp, pulmonary capillary wedge pressure; pm, pacemaker; rap, right atrial pressure; rhc, right-sided heart catheterization; rv, right ventricular; uf, ultrafiltration; and uop, urine output. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines although it may be reasonable to consider a small in- travenous fluid bolus in the setting of arhf compli- cated by hypotension, excess rv preload can lead to further clinical deterioration if it results in increased rv dilation, tr, rv afterload, and myocardial wall ten- sion causing ischemia. , – as the rv dilates, the interventricular septum is pushed leftward in diastole. limits of pericardial compliance result in rv constraint, causing impaired lv filling and a reduction in co. this ventricular-interdependent effect is often more impor- tant than impaired rvef in reducing co. if the cvp exceeds to mm hg, the patient will likely benefit from decongestion to restore more favorable intraven- tricular loading conditions and normalized interventric- ular interaction. invasive hemodynamic monitoring is very useful to help determine the optimal rap needed to maintain appropriate preload. decongestion leads to rv decompression, reduced ventricular interdepen- dence, downward displacement of the effective lv dia- stolic pv curve, improved lv filling, and augmentation of sv, co, and bp. diuretics diuretic resistance can be a significant barrier to ef- fective therapy in the setting of acute decompensa- tion. , patients may not respond to high doses of intravenous loop diuretics because of a combination of factors, including elevated cvp and renal venous congestion, hypotension, low co, and oliguric acute kidney injury. although efficacy has not been clear- ly demonstrated, there is mounting evidence for the safety of an early, aggressive high-dose diuretic strat- egy, with rapid titration in patients failing to respond to initial interventions , (table  ). it is reasonable to consider adding a thiazide diuretic to augment natri- uresis with intravenous loop diuretics. , aldosterone antagonists may help to maintain potassium homeo- stasis from potassium losses, although a trial of high- versus low-dose spironolactone in acute hf did not lead to improved clinical outcomes. likewise, carbonic anhydrase inhibitors can improve the hypochloremic metabolic alkalosis resulting from aggressive loop and thiazide diuresis. diuretics should not be held in a hypotensive patient who is clearly volume-overloaded. hypotension in this setting is a marker of a critically ill patient, and mea- sures should be taken to support the bp with vasoactive therapies while concomitantly attempting to improve clinical congestion with diuretic or renal replacement therapies. hypotension requiring vasoconstrictors and persistent poor hemodynamics should prompt strong consideration for mcs. renal replacement therapies among patients who fail to respond to escalating di- uretics therapy, continuous veno-venous hemofiltration or ultrafiltration may be needed to mechanically re- move intravascular volume. , care should be taken not to remove fluid at a rate that exceeds the ability of the body to shift extravascular fluid into the intravas- cular space (the plasma refill rate) because such an ex- cess is likely to result in new or worsening acute kidney dysfunction or injury. markers of hemoconcentration include increases in hemoglobin, hematocrit, serum al- bumin, and total serum protein levels. , ultrafiltration, which refers to the removal of isoton- ic fluid from the venous compartment via filtration of plasma across a semipermeable membrane, has been evaluated for the treatment of acute hf in multiple tri- als, although not specifically targeting subjects with rhf. the unload trial (ultrafiltration versus iv diuret- ics in patients hospitalized for acute decompensated table . stepped pharmacological care: treatment algorithm from the carress-hf trial stepped pharmacological care treatment algorithm uo goals to be assessed daily from randomization to h uo > l/d→reduce current diuretic regimen if desired uo – l/d→continue current diuretic regimen uo < l/d→see diuretic grid -h assessment uo recommendations as above advance to next step on grid if uo < l/d -h assessment uo recommendations as above advance to next step on grid if uo < l/d consider dopamine or dobutamine at μg·kg− ·h− if sbp < mm hg and ef < % or rv systolic dysfunction consider nitroglycerin or nesiritide if sbp > mm hg (any ef) and severe symptoms - and -h assessments uo recommendations as above advance to next step on grid if uo < l/d consider dopamine or dobutamine at μg·kg− ·h− if sbp < mm hg and ef < % or rv systolic dysfunction consider nitroglycerin or nesiritide if sbp > mm hg (any ef) and severe symptoms consider hemodynamic-guided iv therapy, lvad, dialysis, or ultrafiltration crossover diuretic grid suggested dose current dose daily loop dose thiazide a < mg mg iv bolus mg/h none b – mg mg iv bolus+ mg/h mg metolazone once daily c – mg mg iv bolus+ mg/h mg metolazone twice daily d > mg mg iv bolus+ mg/h mg metolazone twice daily carress-hf indicates cardiorenal rescue study in acute decompensated heart failure; ef, ejection fraction; iv, intravenous; loop, loop diuretic dose in furosemide equivalents; lvad, left ventricular assist device; rv, right ventricular; sbp, systolic blood pressure; and uo, urine output. reproduced from bart et al with permission. copyright © , elsevier. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es congestive heart failure) showed that early ultrafiltra- tion produced greater weight and fluid loss compared with intravenous diuretics. the subsequent carress- hf trial (cardiorenal rescue study in acute decompen- sated heart failure), however, failed to demonstrate a benefit of ultrafiltration compared with a prescribed stepwise escalation of diuretic therapy based on uri- nary response in patients with acute hf and worsen- ing kidney function. the diuretic algorithm (table  ) included high doses of loop diuretics via both bolus and continuous infusion, the addition of thiazide diuret- ics, and selected intravenous inotrope and vasodilatory therapy. the avoid-hf trial (aquapheresis vs intrave- nous diuretics and hospitalizations for heart failure) was designed to clarify the role of ultrafiltration in the management of acute hf but was terminated prema- turely after the sponsor withdrew financial support. whereas ultrafiltration may be helpful for fluid removal, available evidence does not support ultrafiltration as a first-line therapy for acute hf, and its use should be re- served for patients with persistent congestion refrac- tory to escalation of diuretic therapies. vasoactive therapies vasoactive medical therapy plays an important role in the management of arhf. global goals of therapy in- clude reducing rv afterload, enhancing forward flow, and augmenting rv perfusion. there are few clinical trials to guide selection of vasoactive agents for arhf, and most available data are from observational se- ries. , , medication choice relies on clinician experi- ence, expert consensus opinion, and a firm understand- ing of the mechanism of action of chemotherapeutics and cardiovascular physiology. if a patient remains re- sistant to therapy, a pa catheter may be helpful to mea- sure biventricular filling pressures and co. afterload reduction correcting reversible causes of elevated pvr such as hy- poxia and acidosis is a primary consideration. by relax- ing vascular tone, vasodilators decrease systolic stress, enabling more effective systolic contraction. nonselec- tive vasodilators, including intravenous nitroglycerin and sodium nitroprusside, decrease both pvr and sys- temic vascular resistance, augmenting rv and lv sv and facilitating decongestion of the pulmonary and systemic circulations. – conceptually, vasodilator therapy may also relieve renal venous congestion and augment renal perfusion, although this strategy has not had positive results when studied in patients with acute hf and cardiorenal disease. both nitroglycerin and sodium nitroprusside have short half-lives, which is advantageous in the event of hypotension. partially selective pulmonary vasodilators provide cli- nicians another option to decrease pvr in an effort to improve rv sv. for appropriately selected patients, available agents include inhaled and parenteral epo- prostenol and nitric oxide. when long-term use of pulmonary vasodilatory therapy is being considered, it should be recognized that adverse effects have been observed in patients with lh disease. inhaled agents have the advantage of avoiding systemic hypotension and worsening ventilation/perfusion mismatch. in pri- marily observational studies, short-term use of inhaled nitric oxide resulted in lower pvr, increased rv ejection, less rv distension, and improved oxygen consumption in patients with ph, – rvmi, or pe , and after lvad implantation. – in chronic hf, however, aug- mented rv co from inhaled nitric oxide administration can precipitate acute pulmonary edema caused by an abrupt increase in the filling of an already volume-over- loaded or poorly compliant lv. oral agents include the phosphodiesterase- inhibi- tors (pde is). there are anecdotal reports of the ben- eficial impact of this class of medications on persistent ph after lvad implantation complicated by arhf, al- though these findings have been inconsistent. , augment contractility in broad terms, inotropes augment myocardial con- tractility and augment failing rv sv while reducing rv end-diastolic volume and pressure. if the co and bp are inadequate, inotropes should be considered to increase forward flow and possibly renal perfusion, recognizing the potential for inducing ischemia and ar- rhythmia. milrinone and dobutamine have combined inotropic and vasodilator properties. – therefore, they can precipitate or worsen hypotension, although this is less likely if lv preload is adequate, and their use results in improved co. overall, direct comparisons of dobutamine and milrinone have shown similar clinical outcomes, including similar hemodynamic efficacy and arrhythmogenic potential. , compared with dobutamine, milrinone leads to greater reductions in rv and lv end-diastolic pressure because of its potent pulmonary and systemic vasodi- latory properties. , in addition, milrinone less com- monly causes tachycardia and may be considered a more rational choice in the setting of concomitant β-blocker therapy. patients are also less likely to develop drug toler- ance during prolonged infusions of milrinone. however, milrinone is more likely than dobutamine to provoke hypotension, particularly during bolus administration, which is subsequently less easily reversed by discontinua- tion of therapy. given the long half-life, vasopressor sup- port may be necessary to counteract vasoplegia in this setting. in addition, milrinone is partially renally cleared, and the estimated glomerular filtration rate must be con- sidered in the determination of appropriate dosing. dobutamine has the advantage of a short half-life with rapid onset and offset of effect when discontin- d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines ued. it has only a modest direct vasodilator effect, me- diated through β -adrenergic peripheral vasodilation and thus a lower propensity for causing hypotension. accordingly, it also has less effect on rv and lv after- load. in rare cases, dobutamine can cause an eosino- philic myocarditis, which may result in further deterio- ration of cardiac function. although short-term administration of inotropic agents leads to hemodynamic improvement, long-term administration is associated with increased myocardial oxygen consumption and possibly increased mortal- ity. , – clinical guidelines recommend against the routine use of these agents in hospitalized patients with acute hf. maintain perfusion in cases of hypotension, a drug with combined ino- tropic and vasopressor properties may be necessary to maintain adequate perfusion. studies comparing out- comes of vasoactive agents are limited. given the inherent inotropic properties and a dose-dependent vasopressor effect from α agonism, dopamine, nor- epinephrine, and epinephrine are useful adjuncts to augment contractility in the setting of significant hy- potension (systolic bp < – mm hg). , norepi- nephrine, for example, is a potent α -receptor ago- nist with weaker β-receptor activity. its administration leads to vasoconstriction with less prominent inotropic and chronotropic effects. recognizing the downside of increasing ventricular afterload, cases of refractory hypotension caused by peripheral vasodilation not responding to initial inter- ventions may require the use of more pure vasopres- sors such as arginine vasopressin or phenylephrine. arginine vasopressin causes peripheral vasoconstric- tion with less impact on pvr and has beneficial effects supporting glomerular filtration via selective efferent arteriole constriction. , these agents can aug- ment mean arterial pressure to improve coronary ar- tery perfusion and to reduce the risk of rv myocardial ischemia. furthermore, supportive care with arginine vasopressin may be beneficial to limit the arrhythmo- genic properties of catecholamines. attempts should be made to wean off these treatments as rapidly as possible. medical management of crhf diuretics and sodium restriction similar to arhf, diuretics remain the mainstay of ther- apy to treat congestion in crhf. , the intensity of diuretic therapy needed may vary according to the pathogenesis and severity of rhf, in addition to other factors such as coexisting renal disease. because pa- tients with rhf can have normal or low lv filling pres- sures, clinical monitoring is necessary to prevent the development of prerenal azotemia and worsening re- nal function. akin to arhf, the goals of volume man- agement in crhf are to maintain sufficient preload for adequate cardiac filling while providing relief from rv volume overload, ventricular interdependence, and congestion. patients with crhf frequently require large diuretic doses because of neurohormonal activation, includ- ing upregulation of the renin-angiotensin-aldosterone system axis, resulting in fluid and sodium retention. congestion leads to increased volume of distribution, visceral edema causing impaired drug absorption and tubular drug delivery, and rebound sodium absorp- tion in the hypertrophied distal nephron resulting from chronic na-k- cl blockade. , combination therapy including loop diuretics with thiazides may be helpful to augment natriuresis via sequential nephron blockade of sodium reabsorption. compared with furosemide, torsemide has more consistent absorption, especially during decompensation, and may be a preferred loop diuretic in crhf. , according to the aha/american college of cardiol- ogy hf practice guidelines, sodium restriction is consid- ered reasonable for patients with symptomatic hf to re- duce symptoms of congestion. this recommendation is applicable to patients with symptomatic biventricu- lar failure or isolated rhf, but it should be noted that no large-scale studies have demonstrated the safety or efficacy of sodium restriction in these populations. clinicians should consider some degree (ie, < g/d) of sodium restriction in patients with symptomatic crhf, although insufficiency of data and inconsistency of rec- ommendations across guidelines make it difficult to provide precise recommendations. , similarly, fluid restriction ( . – l/d) is considered reasonable in pa- tients with refractory congestion and hyponatremia. renin-angiotensin-aldosterone system inhibitors, β-blockers, and hydralazine patients with biventricular dysfunction should be man- aged according to current practice guidelines for the management of chronic hf. , in contrast to clear guidelines available for the management of hfref, less evidence is available to guide therapy of predominant rhf syndromes. small-scale, single-center studies sup- port the use of β-blockers, – renin-angiotensin-al- dosterone system inhibitors, – and hydralazine, although results are inconsistent and vary across popu- lations, depending on the pathogenesis of rhf. – early studies with systemic vasodilators, including hy- dralazine, produced inconsistent hemodynamic benefits in patients with ph. , , these agents were also fre- quently associated with serious adverse events. – at present, the use of angiotensin-converting enzyme in- hibitors, angiotensin- receptor blockers, and β-blockers d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es is not recommended in patients with ph regardless of rhf, unless associated with hypertension, coronary artery disease, or lhf. prospective trials are also not available on the role of mineralocorticoid antagonists, including spironolactone, in the setting of rhf caused by ph. digoxin few studies have evaluated the use of digoxin use for crhf. in early small studies, digoxin administration was associated with acute increases in co or rvef when ad- ministered to patients with ph and rvd. – in other studies, digoxin in subjects with severe chronic airflow obstruction and rvd did not improve exercise capacity or rvef. likewise, a meta-analysis did not find digox- in to be associated with improvement in rvef, exercise capacity, or new york heart association class. the clinical efficacy of digoxin remains unknown when ad- ministered long term in patients with rhf. pulmonary vasodilators rhf is the final common pathway of ph, and vasodilator therapy relieving rv afterload has led to improved out- comes among group patients with rhf. , medical therapy is also now available for treatment of refractory group disease because riociguat, a soluble guanylate cyclase stimulator, has been shown to improve exercise capacity and pvr in patients with persistent chronic thromboembolic ph after pulmonary endarterectomy or in patients with inoperable disease. although some trials have investigated pulmonary vasodilator therapy in patients with lh disease (group ), these tri- als have not specifically targeted patients with evidence of ph. comprehensive guidelines for the treatment of ph are available elsewhere. the soprano trial (clini- cal study to assess the efficacy and safety of maciten- tan in patients with pulmonary hypertension after left ventricular assist device implantation; nct ) is actively investigating the potential for macitentan to lower pvr after lvad. prostacyclin analogs the role of pulmonary vasodilators with prostacyclin analogs has been extensively studied in patients with group ph, a significant proportion of whom have rvd. among patients with pah, intravenous epopro- stenol has long-term clinical benefits, including im- proved survival and functional capacity. , compared with epoprostenol, other prostacyclin analogs, includ- ing treprostinil and iloprost, are more easily adminis- tered, have longer half-lives, and are able to be admin- istered subcutaneously or by inhalation, with similar pharmacological actions and comparable hemodynam- ic effects. these agents also improve exercise tolerance and hrqol in group ph. – parenteral prostanoids, however, remain the first-line therapy for patients with advanced disease. , in contrast, epoprostenol resulted in increased mor- tality when used to treat patients with lhf. a study of patients with severe hfref was terminated early because of a strong signal of decreased survival in patients treat- ed with epoprostenol, despite increases in cardiac index and decreases in pcwp. similarly, epoprostenol was not associated with improvement in distance walked or hrqol in this population. the use of prostacyclin ana- logs is not recommended to treat group ph. phosphodiesterase- inhibitors oral pde is, including sildenafil and tadalafil, are estab- lished, effective, and well-tolerated therapy in patients with group ph, either alone or as combination thera- py with other vasodilators. – pde is are associated with improvements in pulmonary vascular remodeling, improvements in rv contractility, and antiproliferative effects. they are also associated with improvements in exercise capacity and reduced rates of clinical events in patients with group ph. – these trials did not specifically select patients with rhf. in smaller studies, pde i treatment has been dem- onstrated to improve exercise capacity, exercise hemo- dynamics, and hrqol in patients with hfref and sec- ondary ph (group ) – ; however, these results await validation in large-scale multicenter randomized clinical trials. a multicenter trial of pde i treatment in patients with hfpef failed to demonstrate improvement in exer- cise capacity or clinical status. this trial did not specif- ically select patients with evidence of ph or rhf. these agents are well tolerated and generally not associated with clinically significant reductions in bp, although decreases in bp may be accentuated by concomitant medications such as nitrates. this combination is specif- ically contraindicated. although treatment with pde is is beneficial in patients with group ph, their role in patients with isolated rhf or ph caused by lh disease remains uncertain. endothelin receptor antagonists endothelin- , a potent vasoconstrictor, is implicated in the pathogenesis of pah, and endothelin receptor an- tagonists targeting endothelin receptors type a and b have been extensively studied in group ph. whereas these studies did not explicitly select patients with ph with rhf, endothelin receptor antagonists are nonethe- less associated with improvements in hf symptoms, exercise capacity, hemodynamics, and time to clinical worsening in group patients. – phase ii studies of endothelin receptor antagonist administration in patients with hfref have documented short-term hemodynamic benefits. , unfortunately, phase iii hfref trials failed to demonstrate improve- ments in morbidity or mortality in this population, d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines and increases in hepatic aminotransferases were re- ported. , because of hepatic congestion commonly seen in rhf, monitoring of liver function should be per- formed routinely in patients receiving endothelin recep- tor antagonist therapy. management of crhf in chd despite the significant contribution of rhf to prema- ture morbidity and mortality in the acute chd popula- tion, no adequately powered clinical trials have been completed to elucidate the role of medical therapies in this population. furthermore, although many patients with acute chd have rhf, these patients have typically been excluded from adult hf clinical trials. thus, most standard hf therapies must be regarded as lv specific until proven otherwise. rhf resulting from pressure and volume loading may be seen after repair of tof, pul- monary atresia, ebstein anomaly, and pulmonary val- votomy for congenital ps. a thorough evaluation at a center specializing in hf of patients with chd is recom- mended to identify and correct any hemodynamically- significant lesions contributing to rhf. specific pop- ulations of patients with chd with rhf are reviewed briefly, including those with systemic rvd (in whom the rv is subaortic) in patients with a -ventricle circulation, those with a single-ventricle fontan palliation circula- tion in whom the single ventricle is of rv morphology, and patients with a subpulmonary rv who are at risk for rv failure resulting from chronic ps or insufficiency. comprehensive guidelines for the management of chd are available elsewhere. , patients with a systemic rv generally include those who have d-tga that has been corrected with an atrial switch (mustard or senning) procedure or those with congenitally corrected l-tga. a major determinant of survival in patients with a systemic rv is progressive systolic dysfunction of the systemic ventricle, which is often associated with progressive atrial-ventricular (tricuspid) valve regurgitation. worsening atrial- ventricular regurgitation can lead to deterioration in systemic ventricular function. , atrial-ventricular valve repair or replacement can improve the course of disease, particularly if performed before a decline in systemic rvef. , , cardiac resynchronization therapy has also been proposed in patients with chd and systemic rvd. – these preliminary studies have demonstrated markers of clinical improvement and improvement in rv function, although larger, longer-term studies are necessary. few studies have addressed the role of standard hf therapies in this population. given the physiological and anatomic dif- ferences in the rv, the response of the rv to standard hf therapies remains uncertain. a number of small single-center reports suggest a potential clinical benefit of β-blockade in patients with systemic rv, including improvement in symptoms and functional capacity, less systemic atrial-ventricular valve regurgitation, and potential beneficial effects of reverse rv remodeling. , , however, in the largest clinical trial to date, carvedilol did not improve hf outcomes or echocardiographic measures of ventricular function in a randomized, double-blind, placebo-controlled study of children with systolic hf, including those with a sys- temic rv. indeed, there was instead a nonsignificant trend toward worsening of ventricular function in those patients who had a systemic rv and were treated with the β-blocker. unfortunately, this study was not pow- ered to fully address this question. thus, routine use of β-blocker therapy cannot at this time be recommended in patients with systemic rvd and, if used, should be done so with caution. furthermore, given an increased risk of sinus node dysfunction after atrial switch proce- dures in patients with d-tga and a risk of heart block in those with l-tga, β-blocker use requires close obser- vation in these populations. there have been a number of small prospective ran- domized trials of angiotensin- receptor blocker , and angiotensin-converting enzyme inhibitor , , administration in patients a systemic rv secondary to d-tga or l-tga. these trials have demonstrated mixed results and generally have not demonstrated a clear benefit or renin-angiotensin-aldosterone system inhi- bition in this population. in a study of patients with atrial switch repair for d-tga, randomization to eplerenone was not associated with improvement in mri parameters or rv function, although there was a trend toward improvement in biomarkers of collagen turnover. patients with a single-ventricle circulation in whom the single ventricle is of rv morphology are at greater risk of hf than patients whose single ventricles are of lv morphology. manifestations of a failing fontan cir- cuit are variable and may include systemic venous con- gestion, protein-losing enteropathy, plastic bronchitis, and cirrhosis. it is important to evaluate for potentially reversible conditions of hf in patients with a fontan op- eration such as a correctable mechanical obstruction. even small pressure gradients in the low-pressure fon- tan circuit can be deleterious to the single-ventricle circulation. diuretics remain the mainstay therapy. in a multicenter randomized trial in infants with a single ventricle, enalapril did not improve ventricular func- tion, hf severity, or somatic growth, although the duration of follow-up was too short to assess longer- term benefits. similarly, a randomized, double-blind, placebo-controlled crossover trial of enalapril did not demonstrate improvement in functional capacity or he- modynamic measurements. a crossover trial of oral sildenafil in patients with a fontan circulation likewise did not lead to increased maximal oxygen consump- tion, although ventilatory efficiency was improved. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es small series of patients with protein-losing enteropathy report a benefit of mineralocorticoid receptor antago- nism with high-dose spironolactone. , in a separate report, short-term therapy with spironolactone led to evidence of improved endothelial function and im- provement in cytokine profiles in patients with a fontan operation. data, however, remain limited. finally, in patients with a pulmonary rv and chd, for example, those after repair of tof with a transan- nular patch or after repair of pulmonary atresia with an rv-pa conduit, the risk of rv dilation and rv failure persists. few studies have shown definitive efficacy of any of the standard heart rate therapies in this patient population. in a study of adults with repaired tof, the β-blocker bisoprolol did not improve echocardiographic or mri parameters of rv size and function, and in a randomized trial of the angiotensin-converting enzyme inhibitor ramipril in repaired tof and pulmonary regur- gitation, rvef was not increased, although in a sub- group of patients with restrictive rv physiology, ramipril decreased lv end-systolic volume index and increased lvef. in summary, current data do not support the rou- tine administration of standard hf drug therapies to patients with chd with either a single rv or systemic rv or in patients with a pulmonary rv at risk for rv failure. patients with chd and hf should be cared for by clinicians with specific expertise in hf in the set- ting of chd. select patients may be considered for a trial of these lv-specific hf therapies with close moni- toring in these settings. , , ultimately, many ap- propriately selected patients with refractory symptoms require consideration for heart or heart-lung trans- plantation. mcs and transplantation for rhf mcs is reserved for patients refractory to optimal medi- cal management of acute or chronic rhf. appropri- ate timing of intervention to prevent delays in institut- ing mcs therapy is critical to its success in optimizing patient outcomes and preventing unnecessary morbid- ity and mortality. mcs is used for bridge to recovery; bridge to heart, lung, or heart-lung transplantation; or, in unique cases, as destination therapy (ie, permanent use). recent literature suggests that % to % of patients with acute forms of rhf may recover sufficient function to allow mcs device explantation. – thus, appropriate application of mcs is essential to maximize the possibility of myocardial recovery. the appropri- ate type of mcs device used for rhf is determined by whether the pathogenesis is a primary rv insult or is the result of disease of the pulmonary vasculature or lv (figure  ). typically, primary pathogeneses of the rv lend themselves to application with temporary implant- able or percutaneous vads. arhf secondary to obstructive diseases of the pul- monary vasculature may be more appropriately treated with extracorporeal membrane oxygenation (ecmo) as opposed to an rvad because increased pulmonary blood flow from an rvad will further increase pap. there is a risk that when ph is present, excessive fur- ther increase in pap could precipitate pulmonary hem- orrhage. arhf caused by lhf is often more suitably treated with temporary or durable mcs support of the lv, alongside temporary or durable rv support if need- ed (biventricular assist device support). not all patients with biventricular dysfunction need biventricular sup- port. however, determining which patients can be ade- quately supported by isolated lv support is challenging figure . mechanical circulatory support options based on the pathogenesis of right ventricular (rv) failure. a challenge in the management of patients with biventricu- lar dysfunction is discriminating between patients who have primary rv involvement necessitating true biventricular sup- port and those with right-sided heart failure caused primar- ily by left-sided heart disease. among this latter group, left ventricular (lv) mechanical circulatory support alone may provide adequate cardiac support given secondary unloading of the rv after lv decompression. clinician experience and estimations of the probability of recovery guide decisions on the need for durable vs temporary support devices. the pres- ence of pulmonary disease also influences device selection. bivad indicates biventricular assist device; ecmo, extracor- poreal membrane support; lvad, left ventricular assist device and rvad, right ventricular assist device. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines and requires the expert review of a multidisciplinary hf team experienced with mcs. patients with biventricu- lar dysfunction who receive isolated lv support should be closely monitored for clinical deterioration. evidence of worsening rhf should lead to rapid deployment of rv mechanical support. common indications for right- sided mcs use in arhf include arhf after lvad, heart transplantation, rvmi, and myocarditis. temporary support temporary mcs device options include newer percuta- neous devices designed specifically for rv support and include the impella rp (abiomed inc, danvers, ma) and tandemheart pvad – with the protek duo cannula (tandemlife, inc, pittsburgh, pa) (figure  ). the impella rp is a microaxial temporary extracorporeal vad that is placed percutaneously through the femoral vein and positioned with the distal tip in the pa. the microaxial pump positioned within the catheter drains blood from the ra and pumps it into the pa. the ef- ficacy and safety of this device were investigated in recover right (the use of impella rp support system in patients with right heart failure: a clinical safety and probable benefit study), a prospective, multicenter, single-arm outcomes trial. two groups of patients with rhf were enrolled, the first after lvad placement and the second consisting of a mixture of patients af- ter cardiotomy, after transplantation, and with acute mi. the primary outcome was a combined end point of either survival at days or hospital discharge or survival to next therapy (ie, transplantation or surgical rvad). thirty patients were enrolled with % survival to discharge. the major complication was postopera- tive bleeding, which occurred in . % of patients. the protek duo cannula is a dual-lumen coaxial cannula positioned, via the internal jugular vein, with its distal tip in the pa and connected to an extracorpo- real centrifugal blood pump. because of its internal jug- ular cannulation site, this configuration allows ambula- tion during support. these devices provide an option for mcs because of the ease of device insertion and removal, and they obviate the need for surgical ster- notomy. these devices have corresponding variations designed specifically to support the lv so that biventric- ular assist device support can be achieved completely with a percutaneous option. , ecmo represents a viable alternative for acute mcs for arhf that is either caused by primary rvd or is a consequence of pa disease, including in the presence of systemic oxygen desaturation. there has been a sig- nificant adoption of ecmo in adults. the benefits of ecmo are that it can be applied percutaneously at the bedside for initiation of emergent support, it provides biventricular support, and it addresses pathogeneses of the pulmonary system that are not addressed with isolated rv support. the more typical support configu- ration includes femoral venous drainage and arterial outflow to the femoral artery. this configuration can be achieved either percutaneously or by surgical pro- cedure. other configurations, including internal jugular cannulation for venous drainage and arterial outflow to the axillary artery, are also feasible to facilitate am- bulation. an rvad-like configuration with ra inflow to pa outflow with an oxygenator has also been de- scribed. the complications of ecmo have been well chronicled in the literature. , intermediate support surgical options of intermediate-term rv support re- main important, particularly for patients experiencing postcardiotomy failure to wean from cardiopulmo- nary bypass after open heart surgery, including heart transplantation, or for arhf after implantation of a durable lvad. , , , , surgical implantation of an rvad involves cannulation of the ra or rv for venous drainage and pa for arterial outflow. the cannulas are figure . mechanical circulatory support options for acute right ventricular (rv) support. la indicates left atrium; lv, left ventricle; pa, pulmonary artery; ra, right atrium; rvad, right ventricular assist device; and va- ecmo, veno-arterial extracorporeal membrane oxygenation. adapted with permission from kapur et al. copyright © , american heart association. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es connected to an extracorporeal centrifugal flow pump such as the centrimag (abbott medical, abbott park, il). , numerous reports detailing the use of a surgi- cally implanted temporary rvad after lvad have dem- onstrated that this strategy is effective in supporting the patient through severe arhf during the postop- erative period. however, patients requiring rvad af- ter implantation of a durable lvad have consistently worse outcomes related to bleeding complications and hepatic and renal dysfunction compared with those who do not need an rvad. , , although effective, a surgical rvad typically requires a repeat sternotomy if not placed at the same time as the lvad, and a sec- ond surgery is usually required to remove the device at the time of rv recovery. long-term support long-term, durable mcs device options for irrecover- able forms of rhf are limited, and destination therapy for chronic advanced rhf is not well studied. wheth- er mcs with newer, durable, implantable continuous- flow vads could be of benefit in patients with refrac- tory crhf with a contraindication to transplantation is unknown. the off-label use of durable mcs devices in the setting of rhf caused by chronic ph is controver- sial, and rigorous data on its efficacy in this setting are lacking. typically, durable devices used for long-term or per- manent rv support have been designed for lv support, and their use for the rv represents an off-label or un- approved indication. the most frequently used durable vad for chronic rv support has been the heartware hvad (medtronic, inc, minneapolis, mn), which is a small, continuous-flow centrifugal device with mag- netic and hydrodynamic levitation of the internal im- pellor. small, single-center observational series have demonstrated successful application of this device for long-term rv support. typically, the durable rvad has been used in conjunction with a durable lvad. the in- flow cannula of the device has been positioned within the diaphragmatic surface of the rv, anterior surface of the rv, or ra, with outflow connected to the pa. however, the use of durable continuous-flow rotary pumps for rv support raises many issues of feasibility, including the increased risk of thrombus generated in the venous system and embolizing to the pump. the total artificial heart (tah) represents an alter- native therapy for biventricular support for the failing rv and lv. the most used tah is the syncardia tah- t (syncardia systems, llc, tucson, az). , the syn- cardia tah-t is a pneumatically-driven pulsatile device currently approved for bridge to transplantation in the united states. the device is available in - and -cm sizes and is being investigated for destination therapy indication. the use of the tah may be advantageous over biventricular assist device support options in clini- cal situations such as arvc, restrictive cardiomyopa- thies, biventricular failure with significant intraventricu- lar thrombus burden, very large body size, and failed transplantation. transplantation in patients with advanced refractory crhf, transplan- tation can be considered after the exclusion of all re- versible causes of crhf and careful assessment of comorbidities, including cachexia, cardiac cirrhosis, chronic kidney disease, protein malnutrition, and other potential contraindications to transplantation. in pa- tients with ph and crhf (rap > mm hg and a cardi- ac index < . l·min− ·m− ), prognosis is generally poor, and referral for transplantation should be considered. in carefully selected patients with crhf from severe pulmonary vascular disease, heart-lung or double-lung transplantation can be considered. , , , , outcomes for isolated heart transplantation are generally excellent, and -year survival is ≈ % in the most recent era for all patients according to registry data. , however, the presence of rvad support be- fore heart transplantation is associated with a relative mortality hazard of . after transplantation. simi- larly, outcomes have improved after lung transplanta- tion, with a -year survival of ≈ % for all patients re- ported in the most recent era. palliative interventions balloon atrial septostomy (bas) represents a percuta- neous option to treat rhf caused by severe ph, creat- ing a surgical right-to-left shunt to unload the rv. the associated decrease in systemic oxygenation must be outweighed by the increased oxygen delivery mediated by the increased co. bas is typically used as a bridge to lung transplantation or as a palliative measure in refrac- tory ph. preoperative optimization of filling pressures is crucial, and periprocedural inotropic support may be necessary. bas is contraindicated in severe rhf and should not be offered to patients with rap > mm hg, significant hypoxemia (< % on room air), or pvr in- dex > dynes·s·cm− /m . surgical shunt placement between the left pa and descending aorta (potts shunt) has also been described as a palliative intervention for refractory ph. surgical management of valvular lesions tv surgery tr is a common valve disorder; however, data clarify- ing indications for interventions or outcomes after tv d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines repair or replacement are less robust compared with disorders of the aortic or mitral valves. severe tr (ef- fective regurgitant orifice > . cm ) can result in sig- nificant symptoms and mortality but remains under- treated. , , despite the morbidity and mortality associated with significant tr, patients are rarely re- ferred for isolated surgical intervention, and most sur- geries of the tv are performed in the context of other planned cardiac procedures. patients with severe tr and signs or symptoms of crhf are classified as having stage d tv disease. recent data have demonstrated that isolated tv surgery can be performed with accept- able risk if patients undergo intervention before the onset of advanced hf or severe rvd. patients with rhf caused by severe tr from implantable cardiovert- er-defibrillator or pacemaker leads should also be con- sidered for surgical evaluation. in the setting of concomitant valve disease, severe tr of either a primary or functional nature may not improve after treatment of the left-sided valve lesions and reduction of rv afterload. the addition of tv re- pair during surgery for left-sided valve disease does not add appreciable risk, whereas reoperation for severe, isolated tr after left-sided valve surgery is associated with a perioperative mortality rate of up to %. these observations have prompted clinicians toward a higher rate of tv intervention during surgery for left- sided valve disease. moderate or even mild degrees of functional tr left uncorrected at the time of left-sided valve surgery may progress over time in approximately a quarter of patients and result in reduced long-term functional outcome and survival. risk factors for per- sistence or progression of tr include tv annular dila- tation > mm or mm/m on transthoracic echo- cardiogram, significant rvd or dilatation, significant tricuspid leaflet tethering, atrial fibrillation or ph at the time of left-sided valve surgery, rheumatic or functional origin of the mitral disease, or history of rhf. the overwhelming majority of cases requiring sur- gery for tr are amenable to repair. singh et al demon- strated that tv repair is associated with improved peri- operative, midterm, and event-free survival compared with tv replacement for patients with organic tricuspid disease. repair was associated with greater recurrence of tr, although reoperation rates and functional class were similar. thus, repair should be pursued whenever possible. repair should include an annuloplasty ring because it is associated with improved survival and event-free survival compared with other techniques not using an annuloplasty ring (eg, de vega technique). the durability of tv repair may be limited, even in cir- cumstances when an annuloplasty ring is used, because of increased preoperative tv leaflet tethering height and area, low lvef, and increased rv pressure. these factors are associated with a greater degree of tr dur- ing follow-up. patients with significant tethering, sig- nificant distortion of the tv, significant rvd, or severe ph may require tv replacement to avoid long-term fail- ure of repair and worse clinical outcome. percutaneous options for tv replacement may become available in the future. , although the impact of concomitant tv repair at the time of lvad implantation on long-term outcomes remains poorly defined, anecdotally, most operators err toward addressing moderate or worse tr at the time of lvad surgery. – recommendations from the aha/american college of cardiology guide- line for tv surgery are provided in figure  . tricuspid stenosis surgery for severe tricuspid stenosis (ts) is generally performed in conjunction with surgery for left-sided valve disease, most commonly mitral valve stenosis. ts is usually caused by rheumatic heart disease; car- cinoid disease is a less common cause. tv surgery for relief of symptomatic ts is preferred over percutane- ous balloon tricuspid commissurotomy because most cases of severe ts have important concomitant tr (rheumatic, carcinoid, or congenital). indications for surgery for ts include stage c or d ts characterized by t / ≥ milliseconds and valve area < . cm with or without the presence of symptoms. repair for primary ts is feasible but has a higher rate of need for reoperation. pulmonary insufficiency significant pv regurgitation (pr) is uncommon but is most typically observed after surgery for tof or other congenital lesions. residual pr after repair of tof is initially well tolerated but eventually contributes to rv enlargement, rvd, decreased exercise tolerance, in- creased incidence of arrhythmias, and increased risk of sudden death. , , pr is less commonly seen in association with infective endocarditis or carcinoid syndrome. secondary pr after long-standing ph and annular dilation is uncommon. primary treatment of pr in this setting should focus on the cause(s) of elevated pap. surgery for pr is considered when symptoms or signs of rvd have occurred and pr is severe. surgery is generally recommended for asymptomatic severe pr in the setting of severe rv dilation or dysfunction (car- diac mri–derived rv end-diastolic volume index > ml/m , rv end-systolic volume index > ml/m , rvef < %) or symptomatic atrial and ventricular arrhyth- mias. , , current guidelines support surgery for se- vere pr along with ( ) moderate to severe rvd (class iia; level of evidence b), ( ) moderate to severe rv enlargement (class iia; level of evidence b), ( ) symp- tomatic or sustained atrial and ventricular arrhythmias (class iia; level of evidence c), or ( ) moderate to se- d ow nloaded from http://ahajournals.org by on a pril , may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es konstam et al evaluation and management of right-sided heart failure vere tr (class iia; level of evidence b). transcatheter pv replacement is also now possible. in patients with concomitant severe tr undergoing transcatheter pv re- placement, transcatheter pv replacement resulted in a clinically relevant reduction in tr that persisted over years of follow-up. pulmonic stenosis ps is typically caused by chd. acquired types are less common and include carcinoid disease or obstructing vegetations from endocarditis or obstructing tumors. ps may be treated with either percutaneous balloon pv commissurotomy or valve replacement. surgical ther- apy, as opposed to percutaneous therapies, is recom- mended for patients with severe ps and associated hy- poplastic pulmonary annulus, severe pr, subvalvular ps, or supravalvular ps. surgical therapy is also generally preferred for a dysplastic pv or when there is associated severe tr or the need for a surgical maze procedure (class i; level of evidence c). conclusions and future directions we must continue to enhance our understanding of pathophysiology. it is remarkable how misunderstood are some basic concepts of right-sided heart dysfunc- tion among practicing clinicians and the impact that such misunderstanding can have on appropriate pa- tient management. acute right-sided heart syndromes such as acute rv infarction or acute pe must be rec- ognized principally as syndromes of impaired lh filling caused by direct ventricular interdependence to avoid excessive volume administration and to turn more rapidly to rv mechanical unloading and support. like- wise, central and renal venous engorgement must be recognized as at least as important as impaired for- ward output in the pathogenesis of the cardiorenal syndrome. our management of these conditions remains subop- timal, and new therapies, pharmacological and device based, should be sought. for example, as we continue to grow in our understanding of the interdependency of lh function, right-sided heart function, and renal he- modynamics, it may be possible to develop drugs and devices tailored to alleviation of diuretic resistance and the cardiorenal syndrome. ultimately, however, it may be time that matters most because unrecognized and undertreated rhf inevitably results in sequelae of end- organ damage caused by chronic congestion and acute malperfusion. early identification is critical to improve care targeting this complex syndrome, which remains frequently misjudged given the diverse pathways and pathological processes leading to its condition. figure . indications for surgery for tricuspid regurgitation (tr). tricuspid annular (ta) dilation is defined by > mm on transthoracic echocardiography (> mm/m ) or > mm on direct intraoperative measurement. lv indicates left ventricular; phtn, pulmonary hypertension; rv, right ventricular; tv, tricuspid valve; and tvr, tricuspid valve replacement. reproduced with permission from nishimura et al. copyright © , american heart association. d ow nloaded from http://ahajournals.org by on a pril , konstam et al evaluation and management of right-sided heart failure circulation. ; :e –e . doi: . /cir. may , e clinical statem ents and guidelines we should improve our ability to distinguish among rhf caused by lhf, pulmonary vascular obstructive dis- ease, and intrinsic rv pathology through novel hemody- namic indexes and biomarkers of load-dependent and load-independent forms of rhf. although we currently define pulmonary vascular obstruction as either fixed or reversible, perhaps the notion of fixed pathology mere- ly connotes our pathophysiological naiveté. improved understanding of the pathobiology of pulmonary mi- crovascular disease will lead us to novel approaches for reversing fixed pathology. although we have come a long way in rv imag- ing, more advances are warranted, particularly in re- lating measures of systolic and diastolic performance to indexes of load. although we have made enormous strides in advanced mcs, we continue to struggle with patients who have biventricular disease. there is room for considerable advance in biventricular support tech- nologies, particularly durable devices with applicability to broader patient populations. we have seen progressive advances in our under- standing and managing of rhf, but we have much more to learn. the future holds many diagnostic and therapeutic advances that will markedly expand our ability to tackle these many complex clinical challenges. article information the american heart association makes every effort to avoid any actual or po- tential conflicts of interest that may arise as a result of an outside relationship or a personal, professional, or business interest of a member of the writing panel. specifically, all members of the writing group are required to complete and submit a disclosure questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest. this statement was approved by the american heart association science advisory and coordinating committee on october , , and the american heart association executive committee on december , . a copy of the document is available at http://professional.heart.org/statements by using either “search for guidelines & statements” or the “browse by topic” area. to purchase additional reprints, call - - or e-mail kelle.ramsay@wolterskluwer.com. the american heart association requests that this document be cited as follows: konstam ma, kiernan ms, bernstein d, bozkurt b, jacob m, kapur nk, kociol rd, lewis ef, mehra mr, pagani fd, raval an, ward c; on behalf of the american heart association council on clinical cardiology; council on car- diovascular disease in the young; and council on cardiovascular surgery and anesthesia. evaluation and management of right-sided heart failure: a scientific statement from the american heart association. circulation. ; :e – e . doi: . /cir. . the expert peer review of aha-commissioned documents (eg, scientific statements, clinical practice guidelines, systematic reviews) is conducted by the aha office of science operations. for more on aha statements and guidelines development, visit http://professional.heart.org/statements. select the “guide- lines & statements” drop-down menu, then click “publication development.” permissions: multiple copies, modification, alteration, enhancement, and/ or distribution of this document are not permitted without the express permis- sion of the american heart association. instructions for obtaining permission are located at http://www.heart.org/heartorg/general/copyright-permission- guidelines_ucm_ _article.jsp. a link to the “copyright permissions request form” appears on the right side of the page. disclosures writing group disclosures writing group member employment research grant other research support speakers’ bureau/ honoraria expert witness ownership interest consultant/ advisory board other marvin a. konstam tufts medical center none none none none none none none michael s. kiernan tufts medical center none none none none none abbott*; medtronic* none daniel bernstein stanford university department of defense (grant to explore mechanisms of defective angiogenesis in right ventricular failure)† none none none regencor* none none biykem bozkurt baylor college of medicine and medvamc none none none none none none none miriam jacob cleveland clinic foundation heart and vascular institute none none none none none none none navin k. kapur tufts medical center none abiomed†; cardiac assist†; st. jude (preclinical research)†; maquet (translational research)† abiomed†; heartware*; maquet*; st. jude* none none abiomed*; cardiac assist*; maquet*; st. jude* none robb d. kociol beth israel deaconess medical center dcri (steering committee– connect–hf)* hfsa (executive board, speaker at meeting) * aha spotlight series–hf* none none none none eldrin f. lewis brigham & women’s hospital amgen†; novartis†; sanofi† none none none none novartis† none (continued ) d ow nloaded from http://ahajournals.org by on a pril , mailto:kelle.ramsay@wolterskluwer.com may , circulation. ; :e –e . doi: . /cir. e cl in ic al s ta te m en ts an d gu id el in es konstam et al evaluation and management of right-sided heart failure mandeep r. mehra brigham and women’s hospital and harvard medical school none heart failure society of america (president)*; international society for heart and lung transplantation (editor- in-chief of the journal of heart and lung transplantation)† none none nupulse, inc† abbott*; johnson and johnson (janssen)*; medtronic*; mesoblast*; portola* none francis d. pagani university of michigan health system cardiac surgery none none none none none none none amish n. raval university of wisconsin none none none none none none none carey ward duke university none none none none none none none this table represents the relationships of writing group members that may be perceived as actual or reasonably perceived conflicts of interest as reported on the disclosure questionnaire, which all members of the writing group are required to complete and submit. a relationship is considered to be “significant” if (a) the person receives $ or more during any -month period, or % or more of the person’s gross income; or (b) the person owns % or more of the voting stock or share of the entity, or owns $ or more of the fair market value of the entity. a relationship is considered to be “modest” if it is less than “significant” under the preceding definition. *modest. †significant. reviewer disclosures reviewer employment research grant other research support speakers’ bureau/ honoraria expert witness ownership interest consultant/advisory board other robert p. frantz mayo clinic none none none none none actelion (unpaid)*; arena pharmaceuticals*; bayer (unpaid)*; abbott*; novartis*; united therapeutics (unpaid)* none jason n. katz university of north carolina none none none none none none none robert l. kormos university of pittsburgh none none none none none none none gregory d. lewis massachusetts general hospital nhlbi (investigation of markers of right ventricular dysfunction)†; nhlbi (evaluation of metabolic disease in relation to pulmonary hypertension in heart failure)* bayer (clinical trial research)† none none none ironwood* none myung park houston methodist debakey heart & vascular center none none bayer* none none actelion*; bayer* none this table represents the relationships of reviewers that may be perceived as actual or reasonably perceived conflicts of interest as reported on the disclosure questionnaire, which all reviewers are required to complete and submit. a relationship is considered to be “significant” if (a) the person receives $ or more during any -month period, or % or more of the person’s gross income; or (b) the person owns % or more of the voting stock or share of the entity, or owns $ or more of the fair market value of the entity. a relationship is considered to be “modest” if it is less than “significant” under the preceding definition. *modest. †significant. writing group disclosures continued writing group member employment research grant other research support speakers’ bureau/ honoraria expert witness 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/j.ijcard. . . . . oosterhof t, van straten a, vliegen hw, meijboom fj, van dijk ap, spijkerboer am, bouma bj, zwinderman ah, hazekamp mg, de roos a, mulder bj. preoperative thresholds for pulmonary valve replacement in patients with cor- rected tetralogy of fallot using cardiovascular magnetic resonance. circulation. ; : – . doi: . /circulationaha. . . . jones tk, rome jj, armstrong ak, berger f, hellenbrand we, cabalka ak, benson ln, balzer dt, cheatham jp, eicken a, mcelhinney db. transcatheter pulmonary valve replacement reduces tricuspid regurgita- tion in patients with right ventricular volume/pressure overload. j am coll cardiol. ; : – . doi: . /j.jacc. . . . d ow nloaded from http://ahajournals.org by on a pril , doi: . /s - ( ) - promoter and intronic variants affect the transcriptional regulation of the human dopamine transporter gene tiffany a. greenwood and john r. kelsoe* department of psychiatry, university of california, san diego and san diego va health care system, san diego, ca , usa received march ; accepted april abstract we have attempted to identify regions involved in the transcriptional regulation of the dat (hugo approved symbol slc a ) gene that may harbor functional variants predisposing to several neuropsychiatric disorders by examining haplotypes of various � and intronic regions for their effect on expression in a dopaminergic cell line. a . -fold difference in regulatory activity was observed between haplotypes of the proximal promoter/intron region, representing the two previously identified � clades. although we found no effect on transcription with inclusion of the - and -repeat alleles of the � vntr, introns , , and appear to contain enhancer elements capable of increasing expression approximately -fold with respect to the promoter constructs. differences in expression were also observed between two alleles of intron . these results thus suggest that it may be the particular combination of polymorphisms in a haplotype across the gene that ultimately affects dat gene expression. © elsevier inc. all rights reserved. keywords: dopamine transporter; dopamine; bipolar disorder; genetic variation; single-nucleotide polymorphism; gene expression; transcriptional regulation the dopamine transporter (dat) mediates the active reuptake of dopamine from the synapse and thereby plays a key role in the regulation of dopaminergic neurotransmis- sion. dopamine is an important mediator of motor behav- iors through projections to the striatum and of emotional and attentional behaviors largely through projections to a variety of limbic targets. cocaine, amphetamine, and other stimulants block the action of dat, thus increasing synap- tic dopamine concentrations. the neuropsychiatric effects of these drugs, as well as a wide array of other data, argue for the importance of dopamine in many neuropsychiatric illnesses. a better understanding of the elements controlling the expression of this gene may provide insight into the pathophysiology of these disorders. the dat gene is expressed exclusively in the central nervous system, primarily in midbrain dopaminergic neu- rons of the substantia nigra and ventral tegmental regions [ ]. this highly restricted pattern of dat gene expression is presumably regulated by a unique combination of positive and negative regulatory factors, which have thus far re- mained elusive. characterization of the � flanking se- quence of the human dat gene (dat ; hugo approved symbol slc a ) has provided some insight as far as which sequences or regions may be involved in transcriptional regulation. in vitro experiments have demonstrated that proximal dat sequences constitute a strong core promoter capable of initiating expression without obvious neurospeci- ficity [ , ]. the presence of a strong nonspecific promoter suggests the existence of silencing elements that define the specific expression of dat , an idea supported by the ob- served silencing of basal proximal promoter activity in reporter gene constructs with the inclusion of � flanking sequences [ , ]. enhancer elements also appear to play a role in regulating the expression of the dat gene. the transcriptional activity of dat gene constructs has been shown to be enhanced in the presence of the nuclear recep- tor nurr , an interaction that appears to be mediated by an as yet unidentified nurr -responsive element located within kb upstream [ , ]. although regulatory elements have pre- * corresponding author. fax: � - - - . e-mail address: jkelsoe@ucsd.edu (j.r. kelsoe). r available online at www.sciencedirect.com genomics ( ) – www.elsevier.com/locate/ygeno - / /$ – see front matter © elsevier inc. all rights reserved. doi: . /s - ( ) - dominantly been localized in the � flanking regions of genes, there are a growing number of examples of intronic and � sequences that play a significant role in defining the tissue-specificity of gene expression [ – ]. in vitro analyses of intron of the dat gene have revealed a putative neural-specific silencer in this region [ ]. however, the existence of more � regulatory elements has not been investigated. the dat gene has been implicated in a variety of neuropsychiatric disorders. cook et al. [ ] first reported the association of a -bp repeat in the � untranslated region of the gene with attention deficit hyperactivity disorder (adhd). this association has since been supported by several independent studies [ – ]. although less ro- bustly, dat has also been implicated in cocaine-induced paranoia [ ], alcoholism [ ], the severity of alcohol with- drawal [ , ], schizophrenia [ ], and parkinson’s disease [ , ]. we have previously reported evidence for linkage of bipolar disorder to the dat locus [ ]. our subsequent attempt to identify possible functional variants and anony- mous markers for linkage disequilibrium (ld) analyses led to the initial identification of sequence variants across the gene in individuals from the two families with suggestive evidence of linkage to bipolar disorder [ ]. analysis of the ld structure across the gene revealed the presence of two haplotype blocks defining the � (promoter through intron ) and � (exon through exon ) regions of the gene [ ]. we used this segmental ld structure to construct a limited number of haplotypes for use in association studies of bi- polar disorder. transmission/disequilibrium test analysis of a sample of parent-proband triads revealed a strong association (empirical p � . ) for a particular set of haplotypes comprising five snps deriving from the � re- gion of dat , exon through exon [ ]. the absence of nonsynonymous coding sequence variants in any of the individuals examined, in combination with the ld data, suggested the presence of one or more regulatory variants. these data are consistent with other reports of comprehen- sive mutational analyses of the gene that suggest the pres- ence of regulatory variants [ ]. however, the segmental nature of the observed ld impeded our attempts to refine the region of interest further within the � block of the dat gene using genetic methods. for these reasons, we chose to examine the implicated regions of the gene for the presence of functionally active transcriptional regulatory elements that might be candidate regions for disease susceptibility variants. we have con- structed a series of reporter plasmids containing different intronic and upstream segments and examined their effect on transcription in a dopaminergic cell line. we now present functional data that support the role of multiple variants in the transcriptional regulation of the dat gene. we have analyzed two common haplotypes comprising promoter and intron snps, as well as alleles of several � intronic regions and the widely studied vntr in exon of dat . these results demonstrate the potential impact of haplo- types on dat gene transcription. results we have previously examined the haplotype structure of the gene and found a segmental distribution of linkage disequilibrium consistent with the presence of separate � and � haplotype blocks [ ]. fig. a illustrates the cladistic structure of the haplotypes identified in this previous report. the existence of two distinct � clades exhibiting very little recombination may be an indication that haplotypes of the � regulatory regions of dat have differential effects on gene expression, which has served to maintain them in the population. at least two, and possibly three, � clades also exist within the dat gene, again raising the possibility of a functional difference between haplotypes of these clades. we therefore chose to assay a representative haplotype from each � clade, as well as several polymorphic � intronic regions and the widely studied vntr in exon , to deter- mine whether functional differences exist between different haplotypes in these gene regions. in a study finding suggestive evidence for linkage to bipolar disorder, two extended pedigrees were identified as segregating a similar haplotype at the dat locus [ ]. these two families, old order amish pedigree and family of the ucsd bipolar collection, both derive from southeastern pennsylvania and share a common ancestry. subsequent genotyping of the previously reported snps and vntr revealed a single haplotype, gaagagc- atgggg t, to segregate exclusively with illness in fam- ily [ ]. a similar haplotype differing only at one snp in intron , gaagaacatgggg t, was found to seg- regate with bipolar disorder in at least a portion of the amish pedigree. as it is possible that these haplotypes share a functional variant that predisposes to bipolar disorder, we chose a representative from each family for a comparison of the “affected” versus “unaffected” haplotypes for these in- dividuals. partial haplotypes for these individuals are shown in fig. . to determine whether haplotypes of the two � clades differentially affect dat gene regulation, we cloned these regions from two subjects, each of whom had been previ- ously determined through extensive sequencing to be ho- mozygous for one of the � haplotypes. the gaaga hap- lotype was cloned from an affected subject, , representative of family , and the acgag haplotype was cloned from an unaffected control subject, . the puta- tive � regulatory regions for analysis were chosen based on the results of previous analyses of the dat promoter region and intron [ ] and inserted upstream of the lucif- erase reporter gene to generate three constructs for each haplotype. a detailed diagram of the dat genomic regions cloned into these promoter plasmids is shown in fig. a. previous analysis of a sample of parent-proband triads t.a. greenwood, j.r. kelsoe / genomics ( ) – fig. . (a) illustration of the haplotype structure of the � (promoter through intron ) and � (exon through exon ) regions of the dat gene, indicating the previously identified clades in each region [ ] and the � and � haplotypes that segregate with illness in the two families with linkage to dat , ucsd family and the old order amish. only haplotypes present in at least % of chromosomes are shown, and snp positions are as indicated in the lower portion of (b). (b) diagram of the dat gene showing the positions of all previously characterized snps and the three partial haplotypes present in the representative individuals of the two families with linkage to dat . only the alleles present in the promoter/intron , exon /intron , intron , and vntr constructs are indicated. t.a. greenwood, j.r. kelsoe / genomics ( ) – fig. . (a) details of the design of the three dat promoter constructs from each subject. p- designates promoter constructs extending from � to � , p- designates promoter constructs extending from � to � , and p- designates promoter constructs extending from � to � . for each type of promoter construct, the alleles of , representing the gaaga � clade, and , representing the acgag � clade, were cloned from genomic dna. (b) diagram of the design of the � and � dat gene constructs showing the sequential insertion of each genomic region. the three dat promoter regions shown in (a) were cloned into the multiple cloning region upstream of the luciferase reporter gene in the pgl basic vector using the kpni (k) and bglii (b) sites in pgl basic and an aatii (a) site in the core promoter. the four dat � regions were cloned into the sali (s) site downstream of the luciferase gene in the respective p- promoter constructs. t.a. greenwood, j.r. kelsoe / genomics ( ) – revealed a significant association with a haplotype compris- ing snps from the � region (exon through exon ) of the dat gene [ ]. subsequent analysis of an independent sample of parent-proband triads revealed more modest evidence for association with the region between introns and (data to be presented separately). as these regions exhibit an overlap defined by three snps deriving from exon and intron , it is conceivable that a functional variant conferring a susceptibility to bipolar disorder exists within this region of the dat gene. therefore, the two exon /intron alleles of subject , defined by the previously identified atg and agg haplotypes, were cloned into the p- plasmid to simulate the naturally occurring haplotype structure in (see fig. b). the atg-defined allele segregates with illness as part of the affected haplotype in both family and the old order amish and was shown to be preferentially transmitted with illness in our initial analyses of the triads. we also cloned the gga-defined allele from a representative amish sub- ject, , into the p- plasmid to simulate the alter- native haplotype in this individual and to provide for a comparison between haplotypes representing the two pri- mary � clades. the � vntr polymorphism in exon of dat has been widely employed in association studies of dat , and although there have been reports of functional activity as- sociated with this repeat, these data are inconsistent and have largely employed nonhomologous cell lines, as well as nonhomologous promoters [ – ]. to determine whether alleles of the vntr possess regulatory activity, the - and -repeat alleles were each cloned into the p- and p- promoter plasmids. the choice of additional � regions for analysis was based on the availability of a large degree of polymorphic variability in the region as determined by extensive se- quencing of these regions in . introns and were previously found to contain numerous polymorphisms and were thus chosen for assays of regulatory activity. the two alleles of intron were cloned into the p- plasmid to simulate the haplotypes in . as intron could not be cloned in its entirety due to its large size, the region sur- rounding a -bp insertion/deletion polymorphism was cloned into the p- plasmid. fig. b details the design of all � and � dat gene constructs, and table details all of the polymorphisms contained in each construct, in- cluding those snps that have been used to define the hap- lotypes. a comparison of reporter activity driven by each pro- moter segment in the sn cells is shown in fig. a. significant differences in expression were observed be- tween the three promoter plasmids derived from (f( , ) � . , p � . ), as well as between the three promoter plasmids derived from (f( , ) � . , p � . ). the p- plasmids containing the proximal bp of the dat promoter displayed high levels of activity com- pared to the sv promoter used as a control (pgl promoter), . -fold for p- and . -fold for p- . inclusion of an additional kb of flanking se- quence in the p- plasmids served to reduce expression in these constructs relative to the p- plasmids, . -fold (p � table summary of regions cloned and variants present in all dat reporter plasmidsa plasmid region cloned polymorphic variants present in plasmid p- core promoter p� t p- core promoter p� a p- � region through core promoter p� t, p� t p- � region through core promoter p� c, p� a p- � region through intron p� t, p� t, i � t, i � a, i � a, i � a, i � g, i � a, i � c p- � region through intron p� c, p� a, i � g, i � c, i � g, i � g, i � a, i � g, i � t vntr- exon -repeat vntr allele vntr- exon -repeat vntr allele vntr- exon -repeat vntr allele vntr- exon -repeat vntr allele i -atcg exon /intron e � a, i � t, i � c, i � g i -agcg exon /intron e � a, i � g, i � c, i � g i -ggaa exon /intron e � g, i � g, i � a, i � a i -ggaaa intron i � g, i � g, i � a, i � a, i � a i -taggg intron i � t, i � a, i � g, i � g, i � g i -del intron i � deletion i -ins intron i � cagggctggtgtaca insertion a the regions cloned into each plasmid are indicated, along with a complete list of the polymorphic variants represented by the different alleles at each locus in the relevant subjects employed in this study. for each � plasmid, the � variants are as indicated for the p- promoter plasmids of the corresponding individual id in the plasmid name. variants indicated in bold represent those that were previously reported for the association studies of bipolar disorder [ ] and the ld studies of dat [ ]. t.a. greenwood, j.r. kelsoe / genomics ( ) – fig. . (a) comparison of reporter gene activity for the dat gene promoter constructs. the luciferase gene was placed under the control of the indicated segments from the � region of the dat gene. data are expressed as the mean � standard deviation from three independent transfection experiments. analysis by anova and subsequent tukey post hoc tests revealed a significant difference (p � . ) between all promoter constructs from each subject, as well as between subjects for each promoter region (*). (b) effects of alleles of the � dat gene regions on reporter gene expression. the alleles of the vntr and introns , , and were subcloned downstream of the firefly luciferase gene driven by � sequence � to � from either or . the panels show expression, indicated as relative light units (rlu), of the � reporter plasmids compared to p- (left) or p- (right) activity. these data represent the mean � standard deviation of three to four independent transfection experiments. each intronic region was analyzed with the appro- priate p- plasmid by one-way anova and subsequent tukey post hoc tests. * indicates significance (p � . ) compared to either p- or p- . ** indicates significance compared to i -del (p � . ). . ) and . -fold (p � . ) for p- and p- , respectively. the inclusion of intron further decreased expression of the p- plasmids with a . -fold decrease seen between p- and p- (p � . ) and a . -fold decrease between p- and p- (p � . ). this represents an -fold reduction of activity between p- and p- (p � . ) and a . -fold reduction of activity between p- and p- (p � . ). significant differences in expression between alleles of each promoter region were also observed. a -fold difference in expression was observed between the two alleles of the core promoter, p- and p- (f( , ) � . , p � . ). this observed difference in regulatory activity was reduced to . -fold with the inclusion of the � flanking sequence in the p- constructs (f( , ) � . , p � . ) and . -fold with the further inclusion of intron in the p- constructs (f( , ) � . , p � . ). however, the trend remained between all constructs with the gaaga-representative al- leles of having higher expression than the acgag- representative alleles of . these results thus indicate the presence of negative regulatory elements both immedi- ately upstream of the dat gene and within intron , as well as apparent allelic differences in regulatory activity between the two � clades. fig. b shows the activity of reporter constructs contain- ing the indicated � regions and alleles compared to the respective p- promoter plasmids into which they were cloned. inclusion of the - and -repeat alleles of the vntr in exon did not result in a significant change in expression compared to the respective p- promoter plas- mids, nor were there differences in expression between the alleles of the vntr. by contrast, all alleles of the exon /intron region were found to increase expression greater than -fold with respect to the p- plasmids into which they were cloned (f( , ) � . , p � . for the atcg and agcg alleles; f( , ) � . , p � . for the ggaa allele). although significant differences in activity were observed between the atcg and the agcg alleles of exon /intron in combination with the � region from and the ggaa allele of exon /intron in combination with the � region of ( . -fold, p � . and . -fold, p � . , respectively), this most likely reflects the difference in activity of the two promoter alleles rather than a differ- ence between the alleles of intron . analysis of the alleles of intron also revealed significant differences in expres- sion compared to the p- promoter plasmid (f( , ) � . , p � . ); however, no significant difference in activity was observed between these two alleles. allelic differences in expression were, however, observed for in- tron . the deletion allele of intron , which segregates with the affected haplotype in family , was found to increase expression approximately -fold (p � . ) with respect to the p- promoter plasmid and displayed a . -fold (p � . ) higher activity than the allele contain- ing the -bp insertion. it thus appears as though a combi- nation of positive and negative regulatory elements in both the � and the � ends of the gene influence dat transcrip- tion and may also play a role in determining its exceptional tissue specificity, although the elements involved remain to be identified. discussion in summary, we have demonstrated the presence of tran- scriptional regulatory elements in both the � upstream and the � intronic regions of the dat gene. two of the regions, the � promoter region and two alleles of intron , also displayed differences in activity between different hap- lotypes present in the caucasian population. these suggest candidate genomic regions that may include sequence vari- ants that contribute to functional variation in gene expres- sion and possible susceptibility to human disease. however, more studies are needed to define further potential regula- tory elements within these regions, identify the transcription factors that bind to those elements, and determine whether differential binding of those factors impacts dat gene regulation. some attention in recent years has been focused on defining the � regulatory elements involved in controlling the expression of the dat gene [ , ]. however, these studies have employed nonhomologous cell types for their analyses of gene expression. therefore, further character- ization of the previously implicated � regulatory regions in a more homologous system was warranted. the analyses of the dat � region presented here indicate a strong core promoter and suggest the presence of repressor elements both in the � flanking sequence and within intron , some- what consistent with the results of others [ , ]. the exis- tence of two common � clades exhibiting very little recom- bination poses the question of whether differences in regulatory activity have served to maintain these clades in the population. these studies provide evidence to suggest that functional differences do exist between haplotypes of the two � clades, with haplotypes of the gaaga clade, which segregate in family and the amish, displaying a significant increase in expression over haplotypes of the acgag clade. one snp, p� , located in the core promoter bp � of exon has been recently identified and is the only polymorphism common to all � plasmids in this study (data to be presented separately). this snp is also present in the bp immediately � of the transcription start site, the only stretch of highly conserved regulatory sequence between the human and the murine dat genes [ ]. it is thus possible that alleles of this snp result in the differential binding of a transcription factor, thereby influ- encing dat gene expression, although this remains to be determined. the vntr in the � untranslated region of the dat gene has been the focus of much attention as a potential regulatory element. although neither allele of the vntr seemed to have an effect on dat gene expression in these t.a. greenwood, j.r. kelsoe / genomics ( ) – analyses, a recent study by miller and madras [ ] found an approximate . -fold greater activity for the -repeat allele of the vntr in comparison with the -repeat allele. this is in contrast to the findings of fuke and colleagues [ ], who reported the -repeat allele to have a greater degree of expression than the -repeat allele. however, these obser- vations were made using heterologous promoters and cell lines that lack endogenous dat expression. a study by michelhaugh and colleagues [ ] found the -repeat allele to be capable of enhancing expression in the same cell line employed in these studies, sn , yet a heterologous promoter was used and the -repeat allele was not tested. the situation is further complicated by the in vivo findings of heinz and colleagues [ ], who found an association between the -repeat allele of the vntr and reduced dat protein availability, and those of jacobsen and colleagues [ ], who reported the -repeat allele to be associated with a reduction in dat protein. additionally, a recent study by mill and colleagues [ ] observed an association between the number of -repeat alleles and increased dat expres- sion in vivo. taken together, these studies suggest that the vntr may play a role in mrna stability, rather than transcriptional regulation, a possibility not explored in these analyses. thus, although the data presented here represent the first in vitro functional analyses of the dat vntr using the homologous dat promoter in a cell line that expresses the dat gene, and are therefore likely to be more accurate than previous studies of this polymorphism, further investigation in both in vitro and in vivo systems will be required before any conclusions about the functionality of this polymorphism can be drawn. it is also possible that the � vntr is not functional itself, but is in linkage disequilibrium with a nearby func- tional polymorphism. our data suggest the intron inser- tion polymorphism, which is in complete linkage disequi- librium with the -repeat allele of the vntr, as a possibility. however, the insertion variant is relatively rare ( % allele frequency in this sample), suggesting that it does not likely explain the reported disease associations. there are several limitations of the methods employed in these studies. first of all, this is an in vitro model, which may not actually reflect transciptional regulation in vivo. therefore, the use of a transgenic mouse may be necessary to characterize fully the mechanisms of dat gene regula- tion. the cells used for these studies are also derived from mouse substantia nigra, and although there is high coding sequence conservation between the human and the mouse dat genes, there is very little conserved regulatory se- quence [ ]. therefore, a mouse cell line, even one that expresses dat, may not be ideal for studying the regulation of the human dat gene. another important issue is that these constructs are an incomplete assembly of dat gene regions. since most of the gene is absent, it is likely that many crucial cis-regulatory elements may not be present in the constructs and the observed expression may not reflect the actual regulation of the gene. this problem could be circumvented through the use of a bacterial artificial chro- mosome containing the entire dat gene fused to a reporter gene. finally, these studies analyzed dat gene regulation only in the basal state. it is thus possible that a cellular signal is required for induction of dat gene expression, and abnormalities in gene regulation may be evident only in the presence of such a signal. there may also be an issue with the potential introduc- tion of bias resulting from the initial selection of snps that could be unambiguously genotyped by as-pcr in our pre- vious studies [ , ]. such a selection bias may have af- fected our cladistic analyses, ultimately affecting our choice of haplotypes for functional analysis. however, since of the initially attempted snps were amplified successfully, this is not likely to be of great concern. although our work has focused on the role of dat in the susceptibility to bipolar disorder, as described above, dat has also been associated with numerous other disor- ders, suggesting that variation in this gene may have a general impact on a broad range of neuropsychiatric func- tion. as dat has been more consistently observed to be associated with adhd than with bipolar disorder, in which it may play only a minor role in susceptibility, it is possible that dat may be preferentially associated with a subset of bipolar patients who also have adhd or other attentional deficits. it is thus likely that the same genetic variability that we have observed in bipolar disorder and the functional differences we report here are also relevant to adhd and other neuropsychiatric disorders. few in vitro studies have assessed the relevance of com- binations of multiple snps or specific haplotypes in gene expression. however, pharmacogenetic analyses of haplo- types of the � -adrenergic receptor promoter and coding region provide precedence for differential effects conferred by different haplotypes [ ]. this study suggested that it may be the unique interactions of multiple snps within a haplotype that ultimately affect biological phenotype, whereas individual snps may have poor predictive power as pharmacogenetic loci. the studies presented here of hap- lotypes at the dat gene may also reflect the effect of the interaction of multiple snps in a haplotype on gene expres- sion. these results suggest the presence of allelic heteroge- neity at the dat locus and that many snps may each contribute a small effect on gene regulation. there may be several � regulatory elements that, in combination with � elements, differentially regulate dat gene expression. these results thus highlight the likely importance of non- coding sequence variants in complex traits. materials and methods plasmid construction. all plasmids were constructed based on the pgl basic luciferase reporter plasmid (promega, madison, wi, usa), which lacks eukaryotic promoter and enhancer sequences. genomic dna segments for the con- t.a. greenwood, j.r. kelsoe / genomics ( ) – structs were amplified from one of three subjects: , a subject with schizoaffective disorder, bipolar type from family of the ucsd family set; , the proband of old order amish family with a diagnosis of bipolar i disorder; and , a normal control. though has a diagnosis of schizoaffective disorder, bipolar type, this fam- ily is primarily segregating typical bipolar i disorder. this individual was chosen based on the large amount of rese- quencing data available. these subjects were diagnosed using structured interviews and consensus best estimate review following irb-approved informed consent proce- dures as described previously [ , ]. the designs of the � and � constructs are illustrated in fig. . the � regions for analysis were chosen based on the results of previous analyses of the dat promoter region and intron [ ]. these putative � regulatory regions were cloned from the genomic dnas of the two subjects, and , who had previously been determined to be ho- mozygous for one of the two identified � haplotype clades, gaaga and acgag, respectively [ ]. these regions were then inserted sequentially into the kpni and bglii sites in the multiple cloning region upstream of the luciferase reporter gene to generate three constructs for each subject: one containing the core promoter extending from � to � in the genomic sequence ( p- , p- ), another extending from � to � and encompassing the core promoter with approximately kb of � flanking sequence reported to contain both enhancer and repressor elements ( p- , p- ), and a final construct extending from � to � , representing the core promoter, � flank- ing region, and intron , which contains a putative neuron- specific silencer element ( p- , p- ). each of the four selected � regions were cloned into the sali site located downstream of the luciferase gene in the promoter/intron constructs. each intronic allele was cloned into the p- plasmid containing the appropriate � haplotype to simulate the haplotype structure observed in the individual from whom they were cloned. all variation was known as these introns had been previously sequenced in their entirety in the relevant individuals. alleles of intron and a portion of exon were amplified from the genomic dnas of subjects and and cloned into the p- and p- plasmids, respectively. the two iden- tified alleles of intron were amplified from genomic dna and cloned into the p- plasmid. a portion of intron containing a polymorphic -bp insertion was amplified from genomic dna to generate - and -bp fragments, representing the wild-type and inserted alleles, respectively, that were cloned into the p- plasmid. the vntr in exon ( – in dat cdna sequence nm_ ) [ ] was amplified from genomic dna to generate - and -bp fragments, representing the -repeat and -repeat alleles, respectively, each of which was cloned into the p- and p- plasmids. cell culture. transient transfection studies were performed using sn cells, an immortalized mouse embryonic substantia nigra-derived cell line that has been shown to endogenously express the mrnas for several dopaminergic neuron-specific markers, including dat [ , ]. the cells were grown at °c in a % co humidified atmosphere in dulbecco’s modified eagle’s medium, high glucose, con- taining % heat-inactivated fetal bovine serum and sup- plemented with . % d-glucose (sigma, st. louis, mo, usa), . mm l-glutamine, u/ml penicillin (invitrogen life technologies), and �g/ml streptomycin. all re- agents, with the exception of that indicated, were obtained from invitrogen life technologies (grand island, ny). transient transfections and luciferase assays. sn cells were plated on -well plates in . ml growth medium h prior to transfection and subsequently transfected at approximately – % confluence with equimolar con- centrations of all dat reporter plasmids. each well was transfected with . �g total plasmid dna complexed with �l of lipofectamine reagent (invitrogen life technologies) according to the manufacturer’s protocol. the transfection medium was replaced after h with growth medium. the plasmid prltk containing the re- nilla luciferase reporter gene driven by a herpes simplex virus thymidine kinase promoter was cotransfected ( ng) in all experiments as an internal control for normalization of transfection efficiency. the pgem plasmid was used as a carrier to keep the total dna constant between wells. in addition to dat constructs, the pgl promoter vector, a firefly luciferase construct driven by an sv promoter, and the pgl basic vector were employed as controls. all vectors were obtained from promega. every condition was performed in triplicate, and each experi- ment was repeated three to four times. cell extracts were assayed for firefly and renilla lucif- erase activity h following transfection using the dual- luciferase reporter assay system (promega) according to the manufacturer’s protocol. all relative light unit counts were in the linear range of � to � . all data are expressed as the means � standard deviation of three to four independent experiments. all comparisons between con- structs within a group were analyzed by one-way analysis of variance, unless otherwise indicated, followed by tukey post hoc analysis for pairwise comparisons between specific plasmids. acknowledgments this work was supported by grants to j.r.k. from the department of veterans affairs and the national institute of mental health (mh , mh ); the va mental ill- ness research, education and clinical center of visn ; the ucsd mental health clinical research center (mh ); and the ucsd general clinical research cen- t.a. greenwood, j.r. kelsoe / genomics ( ) – ter (m rr ). j.r.k. is a founder and holds equity interest in psynomics, inc. references [ ] m. bannon, j. granneman, g. kapatos, the dopamine transporter: potential involvement in neuropsychiatric disorders, in: f. bloom, d. kupfer (eds.), psychopharmacology: the fourth generation of progress, raven press, new york, , pp. – . 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( ) – . t.a. greenwood, j.r. kelsoe / genomics ( ) – 	promoter and intronic variants affect the transcriptional regulation of the human dopamine transporter gene 	results 	discussion 	materials and methods 	plasmid construction 	cell culture 	transienttransfectionsandluciferaseassays 	acknowledgments 	references send orders for reprints to reprints@benthamscience.ae clinical practice & epidemiology in mental health, , , - - / bentham open clinical practice & epidemiology in mental health content list available at: www.benthamopen.com/cpemh/ doi: . / research article the sardinian puzzle: concentration of major psychoses and suicide in the same sub-regions across one century alberto bocchetta* and francesco traccis department of biomedical sciences, section of neurosciences and clinical pharmacology, university of cagliari, cagliari, italy received: july , revised: november , accepted: november , abstract: background: sardinia, the second largest mediterranean island has long been considered a privileged observatory for the study of several medical conditions. the peculiar epidemiology of mood disorders and suicide across sardinian sub-regions has long intrigued clinicians and researchers. objective: the principal aim of the present study was to test whether the geographical distribution of suicides committed in sardinian over the last three decades are comparable with the geographical origin of patients hospitalized up to half a century ago. method: the distribution of the municipalities of origin of the patients hospitalized in sardinia between and for schizophrenia, bipolar disorder, and depression was reanalyzed and compared with the distribution of municipalities where suicides were committed between and . data were also analyzed by the altitude above the sea level and by the population size of the municipalities. results: there was a significant variation of hospitalization and suicide rates across sardinian sub-regions. the sub-regions of origin of the patients hospitalized for schizophrenia and bipolar disorder correlated with each other (p = . ). both hospitalizations and suicides were more incident in municipalities with a higher altitude and a smaller population size. the incidence of hospitalizations and suicides correlated significantly with each other both at the municipality (p = . x - ) and at the sub-region level (p = . x - ). conclusion: the present study confirms the peculiar geographical distribution of major psychoses and suicide in sardinia. the two phenomena appear to have been correlated for as long as one century. keywords: mood disorders, bipolar and related disorders, schizophrenia spectrum and other psychotic disorders, suicide, hospital, psychiatric, altitude, population size, sardinia. . introduction sardinia, the second largest mediterranean island with a current population of . million, has long been considered a privileged observatory for the study of medical conditions. islands are often intriguing for epidemiologists and geneticists because they may represent geographically isolated pockets with unique populations. just to give an * address correspondence to this author at the department of biomedical sciences, section of neurosciences and clinical pharmacology, university of cagliari, san giovanni di dio hospital , via ospedale , cagliari, italy; tel: + ; fax: + ; http://benthamopen.com http://crossmark.crossref.org/dialog/?doi= . / &domain=pdf http://www.benthamopen.com/cpemh/ http://dx.doi.org/ . / major psychoses and suicide in sardinia clinical practice & epidemiology in mental health, , volume e-mail: bocchett@unica.it example regarding the psychiatric field, the series of studies started in the s by erik strömgren in the island of bornholm, denmark are among the most frequently cited epidemiological genetic works in the history of psychiatry [ ]. in his doctoral thesis, strömgren provided a complete census of psychoses existing in bornholm in , and the census was repeated half a century later [ ]. the peculiar epidemiology of mood disorders and suicide across sardinian sub-regions has long intrigued clinicians and researchers. the first attempts to survey the incidence of mental disorders in sardinia municipalities were published in the s [ , ]. in the s, athanasios koukopoulos noted that a strikingly high number of his patients with bipolar disorder came from one small village in sardinia. after first consulting with strömgren, koukopoulos tried to undertake an epidemiological study there, but the progress in this unfunded community survey was limited [ ]. our group started to study bipolar and related disorders in sardinia in the s. the high prevalence of g pd deficiency in sardinia prompted us to survey g pd activity in our population of patients with the initial aim of identifying pedigrees for an x-linkage study [ ]. unexpectedly, we found an excess of g pd deficiency in the subgroups of patients with bipolar spectrum disorders [ - ]. we suggested that the uneven distribution of g pd deficiency throughout sardinia, depending on the once malaria endemicity, may explain in part why bipolar syndromes appear to be enriched in some areas, as already suggested by the two s surveys of psychiatric hospitalizations [ , ]. in the meantime, we surveyed also heterozygous β-thalassemia as a phenotypic marker to be used in a linkage study with chromosome p , which was popular in psychiatric genetics at that time. unexpectedly, similarly to g pd deficiency, we found excessive proportions of heterozygous β-thalassemia among subgroups of patients with bipolar spectrum disorders, especially those with psychotic symptoms [ , , ]. one possible explanation of such disproportions is that the two genetic conditions typical of mediterranean areas have pleiotropic effects and play a role in psychiatric syndromes, even though a bias due to geographical stratification cannot be ruled out. with the advent of dna molecular markers, our group undertook several genetic association studies of candidate loci for bipolar illness, but results were not substantial [ - ]. over the last decades, we have also investigated clinical characteristics of bipolar spectrum disorders and suicide behavior within families of sardinian patients [ , ]. more recently, we focused again on the geographical distribution of mental illness across sardinian sub-regions. for example, we found higher suicide mortality rates in municipalities whose lithium content in stream sediments is lower (submitted for publication). given the known relationship between suicide and major psychiatric illness, the aim of the present study was to test whether the geographical distribution of suicides committed in sardinia over the last three decades may be comparable with the geographical origin of patients hospitalized in the two sardinian mental hospitals up to half a century ago. . methods . . reappraisal of data on former psychiatric hospitalizations in sardinia ( - ) the aforementioned survey by camba and rudas [ ] regarded all clinical records of patients hospitalized between and in the two provincial mental hospital of cagliari and sassari, which were instituted in . patients were classified by birthplace. sardinian municipalities in amounted to . the paper provided geographical charts showing the incidence of hospitalizations for various disorders across the municipalities of origin. in addition, the cumulative incidence rates were provided regarding the so-called historical regions of sardinia. the latter subdivision is still in use for some purposes, even if not officially corresponding to the current administrative regions. it reflects the ancient subdivisions dating back to medieval times, mostly based on the absence of geographical barriers, the relative language homogeneity, and the tendency towards endogamy. the survey studied , hospital records, which can be considered a very representative sample of severe psychiatric disorders in sardinia in the first half of the xx century. in the original survey, the rates for single municipalities were calculated taking into account the average number of residents during the period under study. the overall sardinian population between and averaged , , . the diagnoses were based on classifications in force at that time in italy. the authors considered the following nine nosographic classes (corresponding current diagnoses are shown in parentheses): ) schizophrenic syndromes; ) paranoia and delusional syndromes; ) oligophrenic syndromes (intellectual disability); ) epilepsy; ) alcohol-related mailto:bocchett@unica.it clinical practice & epidemiology in mental health, , volume bocchetta and traccis disorders and toxic psychoses; ) dysthymias (bipolar disorder); ) psychoneurotic syndromes (major depression); ) progressive paralysis (general paresis or paralytic dementia due to neurosyphilis); ) dementia syndromes. it must be noted that infectious brain diseases and epilepsy might led to psychiatric hospitalizations because specific medications were not available yet. . . data on suicides ( - ) the italian national institute of statistic (istat) provides the database of all causes of death from medical certification sorted by codes according to the world health organization international classification of disease (icd- and icd- ). we extracted data on suicides occurred in sardinian municipalities during the periods - and - . data regarding single municipalities were not available for the years and . suicides corresponded to icd- categories e -e (in the - period), and to icd- categories x -x (in the - period). demographic data were extracted from the italian national institute of statistics—istat web site (http://demo.istat.it/index.html), in which population data were obtained from population register offices of each italian municipality. data available from the register are estimates in the intercensal period (ten years) and inferred by elaborating data concerning the last census and the demographic flows, such as births and deaths, in the decades considered. we calculated the cumulative incidence of suicide per residents for each municipality according to the average residential population over the period from to . sardinia currently consists of municipalities whose population ranges from one hundred to , inhabitants. many hamlets have become independent since the survey on psychiatric hospitalizations, when municipalities amounted to [ ]. the principal variation in population size over the last decades has regarded larger cities and coastal towns whose population has increased, whereas the majority of smaller villages has maintained a similar population size. the overall sardinian population size has varied from , , ( census) to , , ( census). to avoid bias potentially associated with outliers, we also calculated cumulative suicide incidence in sardinian sub- regions, similar to those used in the survey on psychiatric hospitalizations [ ]. we chose sub-regions based on a slight modification of the plan for the reassessment of the best administrative subdivisions published in in a deliberation by the sardinian government (regione autonoma della sardegna: deliberazione giunta regionale, dicembre , n. / ). . . population size of municipalities and altitude we calculated the cumulative incidence of hospitalizations for schizophrenia, bipolar disorder, and depression between and and the cumulative suicide incidence with a view to the population size of the municipalities and their altitude above the sea level. . . correlation between hospitalizations ( - ) and suicides ( - ) we compared the incidence of hospitalizations for the three principal psychiatric disorders (schizophrenia, bipolar disorder, depression) between and and the incidence of suicides recorded between and , using two levels of geographical subdivisions (municipalities and historical sub-regions). . results . . reappraisal of data on former psychiatric hospitalizations in sardinia ( - ) with new calculations according to the original survey [ ], the cumulative incidence of individuals hospitalized for any diagnosis in the two sardinian mental hospitals between and was . per residents. the incidence rates per residents for the single diagnostic subgroups were the following: schizophrenia = . ; delusional syndromes = . ; intellectual disability = . ; epilepsy = . ; alcohol-related disorders and toxic psychoses = . ; bipolar disorder = . ; major depression = . ; neurosyphilis = . ; dementia = . . the distribution by area of origin was similar between schizophrenia, bipolar disorder, and depression. there was an “epicenter” of municipalities with higher incidence for such disorders in the south east area of sardinia. the distribution http://demo.istat.it/index.html major psychoses and suicide in sardinia clinical practice & epidemiology in mental health, , volume regarding the remaining diagnostic subgroups did not reveal any particular pattern. the epicenter was graphically evident in the charts included in the original publication depicting the historical sub-regions. according to our new calculations, the overall cumulative incidence of hospitalizations for schizophrenia, bipolar disorder, and depression between and in sardinia was . per residents ( % ci = . - . ). these represented % of all the hospitalizations recorded. there was a threefold variation between the birthplace sub-region with the highest rate (barbagia di seulo = . per residents; % ci = . - . ) and the birthplace sub-region with the lowest rate (sulcis = . per residents; % ci = . - . ). the hospitalization rates for schizophrenia and bipolar disorder across the historical sub-regions correlated with each other (r = . ; p = . ). the rates of hospitalizations for schizophrenia, bipolar disorder, and depression were lower for the individuals born in towns with more than , residents (that represented % of all residents) ( . per residents; % ci = . - . ) compared to the individuals born in smaller municipalities ( . per residents; % ci = . - . ). there was a positive correlation between the hospitalization rates for schizophrenia, bipolar disorder, and depression and the altitude of the municipalities (multiple r = . ; p = . x - ). . . data on suicides recorded between and the overall number of suicides committed in the sardinian municipalities during the study period ( - ) amounted to , corresponding to a cumulative incidence of . per residents ( % ci = . - . ). there was a five-fold variation between the sub-region with the highest rate (barbagia di seulo = . per residents; % ci = . - . ) and the sub-region with the lowest rate (baronie = . per residents; % ci = . - . ). suicide rates were lower for the individuals born in towns with more than , residents (that represented % of all residents) ( . per residents; % ci = . - . ) compared with the individuals born in smaller municipalities ( . per residents; % ci = . - . ). there was a positive correlation between the suicide rates and the altitude of the municipalities (multiple r = . ; p = . x - ). . . correlation between hospitalizations ( - ) and suicides ( - ). when examining the municipalities existing in sardinia in , the incidence of hospitalizations recorded between and for the three principal psychiatric disorders (schizophrenia, bipolar disorder, and depression) and the incidence of suicides recorded between and correlated significantly (r = . ; p = . x - ). similar results were obtained by examining the sardinian historical sub-regions (r = . ; p = . x - ) (fig. ). r a te p e r r e si d e n ts the sardinian subregions (ordered by decreasing values of suicide rates) correlation between hospitalization and suicide rates across the sardinian sub-regions suicide rate hospitalization rate linear trendline fig. ( ). for graphical purposes, data were ordered by decreasing values of suicide rates. clinical practice & epidemiology in mental health, , volume bocchetta and traccis . discussion the availability of data on past hospitalizations and recent suicides divided by municipality has allowed the principal interesting observation from the present study that major psychoses and suicide have had similar distributions within sardinian sub-regions across one century. a secondary observation was that the correlation with altitude and the rural/urban ratio were similar between hospitalizations and suicides. we must underscore that both mental illness and suicide are complex phenomena with many underlying factors. their shared geographical distribution, as found in the present study, may depend on sociocultural, genetic and/or environmental aspects, but it can provide an initial step aiming at identifying new potential tiles in the sardinian puzzle. recent studies have confirmed that isolated populations can provide interesting hints in the psychiatric field, as was for example the case of the traditional amish community [ ]. the amish study of major affective disorder (asmad), initiated in the s has tracked several multi-generation pedigrees with high prevalence of bipolar spectrum disorders [ ]. all suicides for a -year period ( to ) were ascertained [ ]. the majority ( %) of suicide cases were diagnosed with a major affective disorder and were situated in multigenerational families with heavy loading for bipolar, unipolar, and other affective-spectrum illnesses. the population originating from some sardinian sub-regions can be considered as isolated as the amish community, and may also provide interesting data. interestingly, a recent study of the amish families combining microsatellite and high-density single nucleotide polymorphism (snp) genotypes with whole-genome sequence data implicated dozens of rare alleles that may interact to determine risk for bipolar disorder [ ]. other data from the amish study suggest potential roles of functional variation of neuronal potassium channels or copy number variants which may be contributing factors in the phenotypic presentation and heterogeneity of mental illness [ , ]. thus, it has become evident, as also suggested from other genetic studies that single genetic variants may result in a wide range of psychopathological presentations [ - ]. the latter observation challenges the validity of current nosographic classifications and may even be consistent with the results from the sardinian survey of hospitalizations showing similar distributions between schizophrenia and mood disorders. the incidence rates of hospitalizations for schizophrenia appear at least two times greater than those of bipolar disorders, that is at odds with current nosology, but it must be noted that the presence of psychotic symptoms in patients with affective episodes often resulted in a diagnosis of schizophrenia up to the advent of current criteria (at least up to , when dsm-iii began to be used also in italy) and to the introduction of lithium therapy in the s. the comparable geographical distribution between hospitalizations and suicide rates found in the present study is consistent with the evidence that mental illness is present among > % of suicides [ , ]. the lifetime risk of suicide is estimated to be % in patients with mood disorders [ ], % in people with bipolar disorder [ , ], and % in people with schizophrenia [ ]. to date, the interpretation of the reasons of the variable geographical distribution of mental illness and suicides have largely been influenced by the perspective of the xix century french pioneer sociologist Émile durkheim. for example, in his book “suicide” published in [ ], durkheim focused on family and social factors, including marital status, religion, employment, and social integration. one of the principal issues has been the rural/urban ratio in suicide rates. in the present study, we found that both hospitalizations and suicides were more incident in small villages. similar patterns are being observed in recent studies of other populations [ , ]. another interesting aspect shared with studies from other countries is the positive correlation between suicide and altitude above the sea level, that has been attributed to the effects of metabolic stress associated with mild hypoxia in individuals with mood disorders [ - ]. besides the risk factors shared with other populations, our hypothesis is that some genetic and/or environmental peculiarities may contribute to the geographical distribution of mental illness and suicide in sardinia. for example, our series of the aforementioned studies suggested that g pd deficiency and heterozygous β-thalassemia, two hematological conditions typical of the mediterranean area, can have pleiotropic effects, including a potential role in bipolar-spectrum disorders [ - ]. with regard to suicide, we also suggested a role of low cholesterol concentrations, major psychoses and suicide in sardinia clinical practice & epidemiology in mental health, , volume which may depend on either genetic factors and/or specifically mediterranean dietary habits [ ]. more recently, we suggested that comorbidity with thyroid autoimmunity, which is very prevalent in sardinia [ ] may be associated with psychiatric manifestations including suicidality [ , ]. with particular regard to the geographic distribution across sardinian sub-regions, we would like to mention our recent finding that the concentration of lithium in stream sediments correlates inversely with suicide mortality (manuscript in preparation). a protective effect of naturally occurring lithium against suicide has already been suggested by research groups from several countries studying lithium content in drinking water (for review see [ ]). the relationship between lithium content in drinking water and suicide may also have complex interactions with altitude [ ], which is was in turn associated with hospitalization and suicide rates in the present study. conclusion the present study confirms prior suggestions that there is a peculiar geographical distribution of major psychoses and suicide in sardinia. the two phenomena appear to have been correlated for as long as one century. these data can be the base for further studies aiming at unraveling the complex pathogenesis of major psychoses and suicide. one potential approach, similar to that already used in the amish study [ ], would be to search for candidate genetic variants carried by affected members of families originating from specific sardinian isolates. ethics approval and consent to participate not applicable. human and animal rights no animals/humans were used for studies that are base of this research. consent for publication not applicable. conflict of interest the authors declare no conflict of interest, financial or otherwise. acknowledgements the authors wish to thank dr marianna tosi (italian national institute of statistics) for access to data on suicide mortality in sardinian municipalities and ms enrica mosca for editing the manuscript. references [ ] schioldann j, strömgren ls. erik robert volter strömgren, november - 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[http://dx.doi.org/ . /yic. ] [pmid: ] [ ] helbich m, blüml v, leitner m, kapusta nd. does altitude moderate the impact of lithium on suicide? a spatial analysis of austria. geospat health ; ( ): - . [http://dx.doi.org/ . /gh. . ] [pmid: ] © bocchetta and traccis. this is an open access article distributed under the terms of the creative commons attribution . international public license (cc-by . ), a copy of which is available at: https://creativecommons.org/licenses/by/ . /legalcode. this license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://dx.doi.org/ . /yic. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /gh. . http://www.ncbi.nlm.nih.gov/pubmed/ https://creativecommons.org/licenses/by/ . /legalcode 	the sardinian puzzle: concentration of major psychoses and suicide in the same sub-regions across one century 	[background:] 	background: 	objective: 	method: 	results: 	conclusion: 	 . introduction 	 . methods 	 . . reappraisal of data on former psychiatric hospitalizations in sardinia ( - ) 	 . . data on suicides ( - ) 	 . . population size of municipalities and altitude 	 . . correlation between hospitalizations ( - ) and suicides ( - ) 	 . results 	 . . reappraisal of data on former psychiatric hospitalizations in sardinia ( - ) with new calculations 	 . . data on suicides recorded between and 	 . . correlation between hospitalizations ( - ) and suicides ( - ). 	 . discussion 	conclusion 	ethics approval and consent to participate 	human and animal rights 	consent for publication 	conflict of interest 	acknowledgements 	references brief report common variation in the lmna gene (encoding lamin a/c) and type diabetes association analyses in , subjects katharine r. owen, christopher j. groves, robert l. hanson, william c. knowler, alan r. shuldiner, steven c. elbein, , braxton d. mitchell, philippe froguel, , maggie c.y. ng, , juliana c. chan, weiping jia, panos deloukas, graham a. hitman, mark walker, timothy m. frayling, andrew t. hattersley, eleftheria zeggini, , and mark i. mccarthy , for the international type diabetes q consortium* mutations in the lmna gene (encoding lamin a/c) under- lie familial partial lipodystrophy, a syndrome of monogenic insulin resistance and diabetes. lmna maps to the well- replicated diabetes-linkage region on chromosome q, and there are reported associations between lmna single nucleotide polymorphisms (snps) (particularly rs ; h h) and metabolic syndrome components. we exam- ined the relationship between lmna variation and type diabetes (using six tag snps capturing > % of common variation) in several large datasets. analysis of , u.k. diabetic case and , control subjects revealed no signif- icant associations at either genotype or haplotype level: the minor allele at rs was no more frequent in case subjects (allelic odds ratio [or] . [ % ci . – . ], p � . ). in u.k. trios, family-based association analyses revealed nominally significant overtransmission of the major allele at rs (p � . ), which was not corroborated in other samples. finally, genotypes for , additional subjects from the international q consortium revealed no consistent case-control or family-based asso- ciations with lmna variants. across all our data, the or for the rs minor allele approached but did not attain significance ( . [ . – . ], p � . ). our data do not therefore support a major effect of lmna variation on diabetes risk. however, in a meta-analysis including other available data, there is evidence that rs has a modest effect on diabetes susceptibility ( . [ . – . ], p � . ). diabetes : – , o nly a limited number of genes with reproduc- ible evidence of association with type diabe- tes have been described. one emerging theme is the frequency with which rare mutations in these same genes display causal involvement in mono- genic forms of diabetes or insulin resistance ( ). conse- quently, there are good grounds for considering genes causing monogenic forms of disease as especially promis- ing candidates with regard to susceptibility to common forms of type diabetes. mutations in the lmna gene cause one form of familial partial lipodystrophy (fpld) ( ), a monogenic syndrome of extreme insulin resistance characterized by abnormal fat distribution, dyslipidemia, hypertension, hepatic ste- atosis, and diabetes. lmna codes (by alternate splicing) for two major protein products, lamin a and c. as constit- uents of the nuclear envelope, these have both structural and regulatory functions ( ). lmna mutations (at sites other than those underlying fpld) are responsible for a range of pathologies (the “laminopathies”) affecting mul- tiple cell types ( ). the structure-function relationships underlying these diverse phenotypes are unclear. equally, the mechanisms whereby lmna mutations lead to fpld are not understood, though loss of lmna binding to the sterol responsive element binding protein may explain the disturbed adipocyte differentiation and development ( ). consequent diversion of dietary-derived triglycerides into ectopic sites (liver and skeletal muscle) likely under- lies the profound insulin resistance. similar mechanisms from the oxford centre for diabetes, endocrinology and metabolism, university of oxford, oxford, u.k.; the phoenix epidemiology and clinical research section, national institute of diabetes and digestive and kidney diseases, phoenix, arizona; the division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, maryland; the endocrinology section, medical service, central arkansas veterans healthcare system, little rock, arkansas; the division of endocrinology and metabolism, department of internal medicine, college of medicine, university of arkansas for medical sciences, little rock, arkansas; the cnrs umr , institut de biologie de lille, lille, france; the faculty of life sciences, imperial college, london, u.k.; the department of medicine, university of chicago, chicago, illinois; the department of medicine and therapeutics, chinese university of hong kong, shatin, hong kong sar; the shanghai diabetes institute, department of endocrinology and metabolism, shanghai jiaotong university no. people’s hospital, shanghai, china; the wellcome trust sanger institute, hinxton, u.k.; the centre for diabetes and metabolic medicine, bart’s and the london queen mary’s school of medicine and dentistry, london, u.k.; the department of medicine, university of new- castle, newcastle, u.k.; the institute of clinical and biomedical science, peninsula medical school, exeter, u.k.; and the wellcome trust centre for human genetics, university of oxford, oxford, u.k. address correspondence and reprint requests to katharine owen, clinical lecturer, oxford centre for diabetes, endocrinology and metabolism, uni- versity of oxford, churchill hospital, old road, headington, oxford ox lj, u.k. e-mail: katharine.owen@drl.ox.ac.uk. received for publication july and accepted in revised form november . *a complete list of the international type diabetes q consortium is available in the online appendix. additional information for this article can be found in an online appendix at http://dx.doi.org/ . /db - . fpld, familial partial lipodystrophy; hrc, human random control; maf, minor allele frequency; snp, single nucleotide polymorphism. doi: . /db - © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. see accompanying brief reports, p. and . diabetes, vol. , march are increasingly implicated in the pathogenesis of insulin resistance, which characterizes type diabetes ( ). lmna’s credentials as a type diabetes candidate are enhanced by prior genetic data. lmna maps within the well-replicated area of type diabetes linkage on chromo- some q – , which has generated powerful signals in european, east-asian, and native-american pedigrees ( , ). additionally, there have been several recent associ- ation studies, most concentrating on a coding variant in exon (rs ; h h). as this codon is directly adjacent to the lamin a/c alternate splice site, even synonymous dna sequence variation has the potential to modulate relative expression of lmna products. initial reports in indigenous north american popula- tions ( , ) suggested the minor allele of rs was associated with increased bmi and central obesity. how- ever, the largest published study of this variant ( ) ( , pima indians, % with diabetes) detected no association with diabetes, bmi, lipid parameters, insulin sensitivity, or �-cell function. subsequent data from the same group indicated a possible association with abdominal adipocyte size ( ). likewise, a small japanese study found no association between rs and diabetes ( ). a more extensive survey of common variation within lmna (six tag single nucleotide polymorphisms [snps] including rs ) in the amish family study (n � , % with type diabetes) reported that rs was associated with metabolic syndrome and triglyceride levels but not diabe- tes ( ). most recently, analyses of appropriately large danish samples ( ) have provided the most convincing evidence yet that the minor allele at rs is associated with type diabetes and that other lmna variants show (at least nominally) significant associations with metabolic and anthropometric traits. the present study sought to exam- ine these interesting, but inconsistent, findings with re- spect to type diabetes susceptibility in analyses of , u.k. subjects and, through the international q consor- tium, a further , samples from populations with the strongest evidence of linkage to the lmna region. first, we performed a large-scale case-control analysis in , u.k. samples (table ). we included as case subjects probands, all ascertained for positive family history, from the diabetes u.k. warren sibpair collec- tion; , type diabetic subjects from the mrc/diabetes u.k. case resource, ascertained for type diabetes diag- nosed before age years; and exclusively british/ irish probands from the warren trios resource. as control subjects, we examined u.k. subjects (human random control [hrc]�), from the hrc resource plus non-hrc samples from the same source (ecacc, salisbury, u.k.), and , from the british birth cohort of . all cases were diagnosed with diabetes based on biochemical evidence of hyperglycemia and/or require- ment for oral agents or insulin. subtypes other than type diabetes were excluded using clinical, genetic, and immu- nological criteria (all are gad antibody negative). glucose tolerance status is not known for any of the control subjects. all subjects were unrelated and of british/irish- european origin. further details of ascertainment, subject characteristics, and validation of these samples are pro- vided in the online appendix (available at http://dx.doi.org/ . /db - ). using pairwise tag selection approaches ( ) applied to u.k. control genotype data for lmna-region snps (minor allele frequency [maf] � %) generated by the q consor- tium (see below), we prioritized six tag snps (threshold r � . ) for genotyping. three mapped upstream of the lmna coding region (rs [maf . ], rs [maf . ], and rs [maf . ]), one in the large first intron (rs [maf . ]), and two were synonymous snps (rs [d d] in exon [maf . ] and rs [h h] in exon [maf . ]). rs was included as a proxy for a q consortium snp (rs ), which failed assay redesign (mutual r of one in the ceu component of hapmap). snp positions and linkage dis- equilibrium relationships are summarized in online appen- dix figure a. using hapmap phase data where available (rs , rs , and rs ) plus hapmap prox- ies for rs and rs (identified using the q consortium genotypes), we estimate that these snps capture � % of common variation at an r � . across the -kb region (containing hapmap snps), which spans lmna and its putative regulatory regions. table characteristics of the u.k. subjects studied case samples control samples probands from sibpair families warren case subjects probands from parent-offspring trios* birth cohort hrc resource n , , male (%) . . . . . age at examination (years) . � . . � . . � . not available not known age at diagnosis (years) . � . . � . . � . not applicable not applicable bmi (kg/m ) . ( . – . ) . ( . – . ) . ( . – . ) not available not known waist-to-hip ratio (males) . ( . – . ) . ( . – . ) . ( . – . ) not available not known waist-to-hip ratio (females) . ( . – . ) . ( . – . ) . ( . – . ) not available not known treatment (ins/oha/diet)† (%) / / / / / / not applicable not applicable data are mean � sd or geometric mean (sd range). *results given for all trios probands (n � ). of these, were of british/irish origin ( % male; age at diagnosis . � . years; bmi . kg/m � . – . �). †treatment at the time of ascertainment. ins, insulin; oha, oral hypoglycemic agent. common variation in lmna and type diabetes diabetes, vol. , march genotyping was performed at kbiosciences (hoddes- don, u.k.) using a fluorescence-based competitive allele- specific (kaspar) assay (details available from the authors upon request). call rates for all snps exceeded % overall (with no snp in any sample � %). genotyping perfor- mance was evaluated against stringent quality control criteria, including a discrepancy rate on duplicate geno- typing � . %; there were no mendelian inconsistencies observed in families and no departure from hardy- weinberg equilibrium (all p � . ) in control subjects. genotype counts by subgroup are shown in online appendix table b. in the absence of heterogeneity be- tween case and control subgroups (p � . ), our primary analyses used pooled case and control data. analyses were conducted with both inclusion (to maximize power) and exclusion (to preserve the independence of the family- based analyses) of the british/irish warren trio probands. genotype frequency comparisons were imple- mented in statxact (cytel corporation, cambridge, ma) using the cochran-armitage trend test (additive model) supplemented by recessive analyses where the maf was � %. in the case-control study (table ), only a single snp, rs , displayed nominal evidence (uncorrected p � . ) of association with type diabetes (odds ratio [or] per additional copy of allele c: . [ % ci . – . ], cochran-armitage test, p � . ). however, inclusion of the warren trio probands rendered this association nonsignificant (p � . ). notably, the minor allele of rs showed no significant association with type diabetes (all case vs. all control subjects: . [ . – . ], p � . ). stratification by sex did not alter the findings for any snp. lmna haplotypes were inferred using the expectation- maximization algorithm implemented in helixtree (boze- man, mt) (online appendix table c). haplotype trend regression ( ) revealed no evidence for haplotypic asso- ciations (p � . ). family-based association tests (table ) were per- formed in all , members of the full set of parent- offspring trio pedigrees (see online appendix). the transmission disequilibrium test, implemented in un- phased ( ), indicated overtransmission of the common allele (t) at rs (p � . ) but no evidence of departure from expectation for any other allele or haplo- type. estimates of overall significance (i.e., global tests of whether any of the individual snps or haplotypes showed transmission disequilibrium, based on , permuta- tions) were not significant for either single-point (p � . ) or haplotypic ( . ) analyses. using lmna genotypes from all , members of the warren sibpair families, there was no indication that table case-control analysis of combined groups snp ncbi build position genotype combined case subjects w sp � w c (n � , ) w tp (n � ) combined control subjects (n � , ) case-control: w sp � w c vs. controls case-control: including w tp cochran armitage test recessive test* cochran armitage test recessive test* rs tt , ( . ) ( . ) , ( . ) . . . . tc ( . ) ( . ) ( . ) cc ( . ) ( . ) ( . ) rs tt ( . ) ( . ) ( . ) . — . — tc ( . ) ( . ) , ( . ) cc ( . ) ( . ) ( . ) rs aa , ( . ) ( . ) , ( . ) . — . — ag ( . ) ( . ) ( . ) gg ( . ) ( . ) ( . ) rs tt , ( . ) ( . ) , ( . ) . . . . tc ( . ) ( . ) ( . ) cc ( . ) ( . ) ( . ) rs tt , ( . ) ( . ) , ( . ) . . . . tc ( . ) ( . ) ( . ) cc ( . ) ( . ) ( . ) rs cc , ( . ) ( . ) , ( . ) . — . — ct ( . ) ( . ) ( . ) tt ( . ) ( . ) ( . ) data are n (%). *recessive test used where maf was � %; considers common allele as recessive. ncbi, national center for biotechnology information; w c, warren case subjects; w sp, warren sibpair probands; w tp, warren trio probands. table family-based association in u.k. trios rs rs rs rs rs rs minor allele c c g c c t t/nt* / / / / / / p . . . . . . analyses are by single-point transmission disequilibrium test. the global p value (p � . ) addresses the null hypothesis that there is no departure from expectation across the set of six single-point tests. *transmission/nontransmission (t/nt) of minor allele to offspring. permutation p � . . k.r. owen and associates diabetes, vol. , march common variants were contributing to the q linkage signal previously observed in these pedigrees (p � . , using the program lamp, which tests the extent to which associated snps can account for regional linkage [ ]). in addition, using anova approaches (spss version ) in the case samples, we found no evidence that lmna snps were associated with age of diagnosis of diabetes, bmi, or waist-to-hip ratio, after logarithmic transformation to nor- mality where appropriate (see online appendix table d for rs data; other data not shown). next, genotype data gathered by the international type diabetes q consortium (see online appendix), in the course of efforts to map susceptibility variants within the replicated linkage region on chromosome q, allowed us to extend our lmna analysis in , samples from the q case-control study ( , non-u.k.; u.k.) and pima family study (n � ). the q consortium has to date attempted genotyping of snps (online appendix figure a and table e) spanning the lmna region in these samples. the q case-control study includes some of the u.k. samples included in the analyses described above (warren sibpair probands and hrc�: these were the only u.k. samples typed for more than the six tag snps) and amish and pima samples included in previous publi- cations ( , ). genotypes were gathered as part of three -plex illumina golden gate bundles ( ). single-point (cochran-armitage test using stata version ) and haplo- type-based (haplotype trend regression using helixtree) analyses of these data revealed no consistent associations between lmna snps and type diabetes (data not shown). analyses of the tag snps typed in the q consor- tium samples (rs , rs , rs , rs , rs , and rs ) confirmed no association with type diabetes in the amish, pima, u.k., shanghai, or hong kong case-control datasets. nominal associations for rs in the utah sample (p � . ), and for rs (p � . ) and rs (p � . ) in the french (additive model), were not substantiated in other samples. combined analysis of information from all seven datasets (using the mantel-haenszel meta-analysis method under recessive, dominant, and additive models) showed no convincing association of lmna tag snps with type diabetes (online appendix table f). notably, rs was not associated with type diabetes in any of the samples (online appendix table g). finally, a further pima samples from the original linkage pedigrees ( and online appendix data) were, with individuals from the case-control sample, analyzed using family-based association methods. these pima samples included type diabetic subjects (diagnosed � years), nondiabetic siblings (aged � years), and parents (to reconstruct family relationships). family- based association analyses under the additive model were performed using binomial generalized estimating equa- tions to control for family membership ( ). again, no lmna snps were associated with type diabetes (all p � . ). for reasons stated earlier, lmna is a logical choice of candidate to investigate for association with multifactorial type diabetes. in this study, we have been unable to show any compelling evidence of association with any of the snps typed. it is noteworthy that the nominally significant results at rs in the case-control and family-based analyses lie in the opposite direction. the estimate of the combined or (including all the nonoverlapping data re- ported in the present study), was calculated using the inverse variance method ( ) to allow proper adjustment for nonindependence in some of the datasets (e.g., amish). in this meta-analysis, the effect of rs on diabetes risk approached but did not attain nominal significance: allelic or . ( % ci . – . ), p � . . the strongest evidence supporting an association be- tween the minor allele of rs and type diabetes risk comes from a large study of danish subjects ( ). in comparison of , case and , control subjects, the observed or was . ( % ci . – . ). while our study fails to replicate this association, the or estimates from the two studies show substantial overlap in their cis. ascertainment effects, as well as sampling error, may have contributed to modest differences in the effect size esti- mates. many of the u.k. case subjects were selected for positive family history and/or early disease onset, maneu- vers expected to boost effect size estimates compared with the less-selective danish case ascertainment. however, differences in control ascertainment may have had a small effect in the opposite direction. the danish control sub- jects are confirmed as normoglycemic, while glycemic status is unknown for the u.k. control subjects. however, given the relatively low prevalence of diabetes in middle- aged u.k. subjects ( ), the magnitude of the dilution of effect size engendered by such misclassification can be shown to be extremely modest ( ). meta-analysis provides one route to improved specifica- tion of true effect sizes. combining all the case-control data in the present study with the previous japanese report ( ) (using inverse variance method, not including the previous amish and pima data, given overlap with the current study), the per-allele or for the minor allele at rs reaches . ( % ci . – . ), p � . . further, if the danish case-control data ( ) are included (contrib- uting % of the total , genotypes), the evidence in favor of a type diabetes susceptibility effect at rs increases substantially ( . [ . – . ], p � . ). while our data cannot be considered to provide replication (p � . ) of the association reported by wegner et al. ( ), the fact that this combined analysis generates a more signifi- cant result than that seen in either study alone indicates that the u.k. data provides some support for the danish findings, particularly when one factors in the strong bio- logical candidacy of lmna. these data again illustrate the tremendous difficulties that exist in the detection, replication, and interpretation of association analyses for variants with modest suscepti- bility effects. if the true effect size of rs is an or of . , then even a study of , case-control pairs has only % power (given a liberal 	 � . ). indeed, reaching strin- gent genome-wide significance (p � � ) for such a variant would require analysis of � , case-control pairs. in addition, such modest effects need to be distin- guished from spurious association signals on a similar scale that may be generated as a result of artifact (e.g., informative missingness) or biological effects such as cryptic population stratification. acknowledgments this study was funded by diabetes u.k. (collection of u.k. samples and u.k. genotyping) and supported by grants u -dk , r -dk , r -dk , k -dk , and r -dk from the national institute of diabetes and digestive and kidney diseases (niddk); grant t - ag from the national institute of aging (nia); and common variation in lmna and type diabetes diabetes, vol. , march intramural funds. its contents are solely the responsibility of the authors and do not necessarily represent the official views of the niddk and nia. e.z. is a wellcome trust research development fellow (wellcome trust ). we thank oluf pedersen, torben hansen, and col- leagues for sharing data prepublication. a full list of funders and personal acknowledgments are provided in the online appendix. references . gloyn al, mccarthy mi: the genetics of type diabetes. in diabetes best practice and research compendium. barnett ah, ed. philadelphia, elsevier, , p. – . cao h, hegele ra: nuclear lamin a/c r q mutation in canadian kindreds with dunnigan-type familial partial lipodystrophy. hum mol genet : – , . gruenbaum y, margalit a, goldman rd, shumaker dk, wilson kl: the nuclear lamina comes of age. nat rev mol cell biol : – , . jacob kn, garg a: laminopathies: multisystem dystrophy syndromes. mol genet metab : – , . lloyd dj, trembath rc, shackleton s: a novel interaction between lamin a and srebp : implications for partial lipodystrophy and other laminopa- thies. hum mol genet : – , . petersen kf, shulman gi: etiology of insulin resistance. am j med :s – , . mccarthy mi: growing evidence for diabetes susceptibility genes from genome scan data. curr diab rep : – , . ng mc, so wy, cox nj, lam vk, cockram cs, critchley ja, bell gi, chan jc: genome-wide scan for type diabetes loci in hong kong chinese and confirmation of a susceptibility locus on chromosome q – q . diabetes : – , . hegele ra, cao h, harris sb, zinman b, hanley aj, anderson cm: genetic variation in lmna modulates plasma leptin and indices of obesity in aboriginal canadians. physiol genomics : – , . hegele ra, huff mw, young tk: common genomic variation in lmna modulates indexes of obesity in inuit. j clin endocrinol metab : – , . wolford jk, hanson rl, bogardus c, prochazka m: analysis of the lamin a/c gene as a candidate for type ii diabetes susceptibility in pima indians. diabetologia : – , . weyer c, wolford jk, hanson rl, foley je, tataranni pa, bogardus c, pratley re: subcutaneous abdominal adipocyte size, a predictor of type diabetes, is linked to chromosome q – q and is associated with a common polymorphism in lmna in pima indians. mol genet metab : – , . murase y, yagi k, katsuda y, asano a, koizumi j, mabuchi h: an lmna variant is associated with dyslipidemia and insulin resistance in the japanese. metabolism : – , . steinle ni, kazlauskaite r, imumorin ig, hsueh wc, pollin ti, o’connell jr, mitchell bd, shuldiner ar: variation in the lamin a/c gene: associa- tions with metabolic syndrome. arterioscler thromb vasc biol : – , . wegner l, andersen g, sparsø t, grarup n, glümer c, borch-johnsen k, jørgensen t, hansen t, pedersen o: common variation in lmna increases susceptibility to type diabetes and associates with elevated fasting glycemia and estimates of body fat and height in the general population: studies of , danish whites. diabetes : – , . carlson cs, eberle ma, rieder mj, yi q, kruglyak l, nickerson da: selecting a maximally informative set of single-nucleotide polymorphisms for association analyses using linkage disequilibrium. am j hum genet : – , . zaykin dv, westfall ph, young ss, karnoub ma, wagner mj, ehm mg: testing association of statistically inferred haplotypes with discrete and continuous traits in samples of unrelated individuals. hum hered : – , . dudbridge f: pedigree disequilibrium tests for multilocus haplotypes. genet epidemiol : – , . li m, boehnke m, abecasis gr: joint modeling of linkage and association: identifying snps responsible for a linkage signal. am j hum genet : – , . zeggini e, damcott cm, hanson rl, karim ma, rayner nw, groves cj, baier lj, hale tc, hattersley at, hitman ga, hunt se, knowler wc, mitchell bd, ng mc, o’connell jr, pollin ti, vaxillaire m, walker m, wang x, whittaker p, kunsun x, jia w, chan jc, froguel p, deloukas p, shuldiner ar, elbein sc, mccarthy mi: variation within the gene encoding the upstream stimulatory factor does not influence susceptibility to type diabetes in samples from populations with replicated evidence of linkage to chromosome q. diabetes : – , . hanson rl, ehm mg, pettitt dj, prochazka m, thompson db, timberlake d, foroud t, kobes s, baier l, burns dk, almasy l, blangero j, garvey wt, bennett ph, knowler wc: an autosomal genomic scan for loci linked to type ii diabetes mellitus and body-mass index in pima indians. am j hum genet : – , . farook vs, hanson rl, wolford jk, bogardus c, prochazka m: molecular analysis of kcnj on q as a candidate gene for type diabetes in pima indians. diabetes : – , . petitti d: statistical methods in meta-analysis. in meta-analysis, decision analysis and cost-effectiveness analysis: methods for quantitative synthesis in medicine. petitti d, ed. oxford, u.k., oxford university press, , p. – . national centre for social research: health survey for england . london, department of epidemiology and public health at the royal free and university college medical school, . colhoun hm, mckeigue pm, davey smith g: problems of reporting genetic associations with complex outcomes. lancet : – , k.r. owen and associates diabetes, vol. , march chemometrics: data analysis for the laboratory and chemical plant | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /tech. .s corpus id: chemometrics: data analysis for the laboratory and chemical plant @article{booth chemometricsda, title={chemometrics: data analysis for the laboratory and chemical plant}, 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(j nucl cardiol ; : – .) key words: myocardial perfusion imaging: spect Æ infarction Æ myocardial Æ computed tomography (ct) Æ coronary artery disease Æ diagnostic Æ prognostic application see related editorial, pp. – introduction the principal cause of morbidity and mortality in all industrialized nations is coronary artery disease (cad). in approximately half of these patients, the initial pre- sentation of cad is either myocardial infarction (mi) or death. unrecognized mi leads to less effective man- agement and increased major adverse cardiac events (mace). population-based studies have revealed that one-fourth of mis demonstrated by q waves on the ecg are clinically unrecognized and estimates of the fre- quency of unrecognized mi detected in the framingham study may well underestimate the true prevalence of the condition. the -year mortality from an unrecognized mi has been estimated to be % to % , comparable to or even higher than in patients with diagnosed mi. given the implications of unrecognized mi, studies are needed to address screening strategies, risk stratification, and the role of post-infarction therapies in these patients. nuclear myocardial perfusion imaging (mpi) has been widely used as a non-invasive method for the diagnosis of cad. in comparative studies with autopsy findings, thallium (tl)- myocardial single-photon emission computed tomography (spect) has been from the division of cardiology, a los angeles biomedical research institute at harbor ucla medical center, torrance, ca. division of cardiology, b stanford university school of medicine, stanford, ca. received for publication apr , ; final revision accepted oct , . reprint requests: mohit gupta, md, division of cardiology, los angeles biomedical research institute at harbor ucla medical center, w carson st., torrance, ca ; mohit_gupta @ yahoo.com. - /$ . copyright � the author(s). this article is published with open access at springerlink.com doi: . /s - - - shown to accurately assess the extent of mi and myo- cardial viability for the selection of therapy and for predicting prognosis. - although rest tc- m-meth- oxyisobutyl isonitrile (mibi) underestimates the extent of viable myocardium compared with tl- imaging, however several studies show that sublingual adminis- tration of nitroglycerin before a rest tc- m-mibi study improves the detection of defect reversibility in exer- cise-induced perfusion defects and thus improves the assessment of myocardial viability in comparison with the standard tc- m-mibi exercise–rest protocol. , coronary artery calcium (cac) score measured through non-contrast cardiac computed tomography (cct) scanning has been shown to predict future coronary events in both asymptomatic and symptomatic patients. non-contrast cct has also been used for assessing the pericardial fat, liver fat, and determining the field of view for cct angiography (ccta) to reduce radiation dose. the potential role of non-enhanced cct to detect chronic mi, however, has not been well studied. areas of chronic left ventricular (lv) infarction are readily apparent on non-contrast cct as regions of hypo-atten- uation either due to deposition of fibrous or adipose tissue , or probably reduced capillary density. we conducted this study to evaluate the accuracy and feasibility of using non-contrast cct to detect chronic mi in patients with irreversible perfusion defect on mpi. we also define the optimal cutoff value for lv myocardium hounsfield unit (hu) to detect chronic mi. methods study population our study included consecutive symptomatic patients (with exertional angina or dyspnea) who were referred for coronary cac scanning at our institution after undergoing an mpi study. patients with reversible perfusion defects on mpi were excluded. subjects with arrhythmia, renal failure, or hemodynamic instability were excluded. the characteristics of the study population are summarized in tables and . demographic data and clinical history were retrieved from the clinical records for all patients in the study group. information about any evidence of previous clinical mi was also collected. mi in these patients was diagnosed by elec- trocardiogram findings (st-segment elevation or depression, pathologic q waves, new onset of left bundle branch block) and abnormal levels of cardiac enzymes. non-contrast cct acquisition and analysis all ccts were performed using -multidetector ct scanner (ge healthcare). following a scout radiograph of the chest (antero-posterior and lateral), the coronary arteries were imaged with - contiguous and . -mm slices during mid- diastole using ecg-triggering during a -s breath hold. cac scoring acquisition was reconstructed at % of the r–r interval with the use of axial planes and multiplanar recon- struction. all the non-enhanced cct images were assessed by two experienced readers for presence and location of mi on advantage windows (aw) . workstation (general electric medical systems, milwaukee, wi, usa). longitudinal views of axial images were evaluated from base to apex to assess the septal, antero-apical, and lateral lv wall; coronal images were used to assess the inferior wall. mi on non-enhanced cct was visually detected based on the areas of hypo-attenuation (dark) in the myocardium. hypo-attenua- tion was assessed by the consensus of two experienced observers who blinded to the results of the mpi. the mean hu of normal and hypo-attenuated area were measured in the septal, antero-apical, lateral, and inferior lv walls. a standard region of interest (roi) of cm was used for the normal myocardium; and for areas with small hypo-attenuation, smaller roi was used (figure ). mpi acquisition and analysis all the mpi scans were performed by the referring phy- sicians at their respective offices. a standard imaging protocol as endorsed by asnc was used for all patients. all patients underwent rest and stress imaging with technetium- m-se- stamibi (mibi). patients who could exercise underwent treadmill stress using the bruce protocol to reach % of maximum predicted heart rate. patients who could not exercise underwent pharmacological stress using dipyridamole at mg/kg/min infused over min. patients were asked to terminate beta-blocker h prior to testing when desirable. for resting studies, patients were injected with - mci of technetium- m-sestamibi and spect images were acquired - min after resting injection. all spect studies were acquired on a commercially available camera and computer using a -degree arc, high-resolution parallel-hole collima- tor; and stops with s per stop for a total imaging time of min. all images were acquired using a matrix. table . demographics of the total study pop- ulation (n = ) age (years) . ± . sex (m/f) ( . %)/ ( . %) risk factors hypertension ( . %) cholesterol ( . %) diabetes ( . %) smoker ( . %) family history ( . %) race caucasian ( . %) hispanic ( . %) asian ( . %) african american ( . %) journal of nuclear cardiology gupta et al volume , number ; – non-contrast cct can accurately detect chronic mi patients underwent symptom-limited exercise bruce protocol. at peak heart rate, - mci of technetium- m-sestamibi (dose was weight based) was injected. patient then underwent post-stress spect image acquisition. the post-stress and rest mibi scans were interpreted using visual assessment of perfusion abnormalities by the referring physicians. all the readers were nuclear board cer- tified and blinded to the results of ct. the short axis data were displayed in polar map format, with the maps divided into segments. the segments were allocated to the territories of the different coronary arteries as previously described. for the sake of comparison with ct, the nuclear study results were then extrapolated into a -region model with the lv wall divided into septal, antero-apical, lateral, and inferior as done on cct. gating was not performed in all the mpi studies and thus regional wall motion was not assessed. statistical analysis categorical variables are presented as frequencies and percentages, and continuous variables as mean ± standard deviations (sd). mpi served as the reference standard for the diagnosis of regional mi. the area under the receiver operator characteristics (roc) curve was calculated for mi assessed by non-contrast cct to identify irreversible perfusion defect detected by mpi. diagnostic performance and predictive value of non-contrast cct for the diagnosis of regions showing mi table . characteristics of the groups with normal and irreversible perfusion defect on mpi variable normal myocardial perfusion (n ) irreversible perfusion defect (n ) p-value age (years) ± ± . gender (male) . hypertension % % . hypercholesterolemia % % . diabetes mellitus % % . family history of chd % % . smoking status % % . known cad % % . septal lv wall hu ± ± . antero-apical lv wall hu ± ± . lateral wall hu ± ± . inferior wall hu ± ± . hu, hounsfield unit. figure . (a) axial, non-contrast cct image demonstrating thin curvilinear hypo-attenuation (arrows) within distal septal, apical, and distal lateral lv myocardium in subendocardial distribution. (b) axial, contrast-enhanced cct image of the same patient shows thinning of associated portions (arrows) of the lv myocardium. (c) sample placement of roi within infarcted and normal myocardium on non-contrast cct image to assess mean hu. roi mean hu = - . (infarct), roi mean hu = (normal myocardium). gupta et al journal of nuclear cardiology non-contrast cct can accurately detect chronic mi january/february compared with the reference standard was evaluated on a per patient level and per region level and expressed as sensitivity, specificity, positive predictive value (ppv), and negative pre- dictive value (npv) and their corresponding % confidence intervals. the mean hu of normal and hypo-attenuated area in the septal, antero-apical, lateral, and inferior lv walls were compared to their corresponding areas in mpi and a second roc was used to determine the myocardial hu threshold to detect mi using non-enhanced cct. intra- and inter-observer variability was calculated using intra-class correlation coeffi- cient (icc) in a two-way mixed effects model, and bland & altman models. all statistical analyses were performed using pasw . (spss institute inc., chicago, il, usa). results a total of patients were studied, including patients ( %) with an irreversible perfusion defect on mpi and patients with a normal mpi. the mean age for this patient cohort was . ± years and ( %) were males. out of the patients with irreversible perfusion defect on mpi, non-contrast cct accurately detected infarct in subjects. thus, on a per patient level and using mpi as the reference standard, non-contrast cct showed a sensitivity of % ( % ci, . - . ), specificity of % ( % ci, . - . ), npv of % ( % ci, . - . ), and a ppv of % ( % ci, . - . ), to detect regions of mi. on a per region level, non-contrast cct showed a sensitivity of % ( % ci, . - . ), specificity of % ( % ci, . - . ), npv of % ( % ci, . - . ), and a ppv of % ( % ci, . - . ) to detect regions of mi on mpi (table ). there was good agreement in the identification of hypo- enhanced regions between mpi and non-contrast cct (figures and ). the mean hu of the regions correctly identified as infarct or normal was calculated to be . ± . and . ± . (p \ . ), respectively. based on a roc curve, a reference hu of . can be used to detect infarcts on non-contrast cct with a sensitivity of . % and specificity of . % (figure ). in a sub-study analysis, intra- and inter-observer variability of patients with and without irreversible perfusion defect detected by mpi was assessed using visual and corresponding hu criteria methods on cct. the intra- and inter-observer variability of cct mea- sured normal and hypo-attenuation segments were low (coefficient of variability of % for intra-observer and % for inter-observer variability). furthermore, there was good intra- and inter-observer agreement to assess normal vs hypo-attenuated segments in ct (icc: . and . , respectively, p = . ) (table ). in a subgroup of patients, with clinical evidence of prior mi diagnosed on the basis of electrocardiogram findings or abnormal levels of cardiac enzymes (as described in ‘‘methods’’ section), hypo-attenuation on cac scan was present in patients yielding a sensi- tivity of % to detect chronic mi. out of these patients with clinical mi, irreversible perfusion defects on mpi were present in patients. discussion contrast-enhanced ccta has been shown to be comparable to mpi and contrast-enhanced cardiac magnetic resonance (cmr) in detecting fixed perfusion defect or scar as areas of late contrast enhancement. - hypo-enhanced regions on contrast-enhanced ccta have been shown to closely relate to delayed enhance- ment of iodinated contrast. , the aim of this study was to evaluate the ability and feasibility of non-contrast cct scans to detect chronic mi in patients with irre- versible perfusion defect on mpi. chronic mi is characterized by a process of ven- tricular remodeling, fatty replacement (rarefaction), and in rare cases calcification. small studies have docu- mented that the presence of fat and/or calcification in the lv-free wall on non-contrast cct is consistent with mi. , , subsequently, areas of infarction are readily apparent on non-contrast cct as regions of hypo- attenuation either due to reduced capillary density or deposition of adipose tissue. , ct imaging features of mi include thin linear or curvilinear fat attenuation within lv myocardium, most commonly sub-endocar- dial, often associated with lv wall thinning, and/or calcification. furthermore, the presence of lv myo- cardial fat deposition in sites of myocardial scarring has table . accuracy of non-contrast cct to detect chronic mi as compared to irreversible perfusion defects on mpi as the reference sensitivity ( % ci) specificity ( % ci) ppv ( % ci) npv ( % ci) per patient analysis . % ( . – . ) . % ( . – . ) . % ( . – . ) . % ( . – . ) per region analysis . % ( . – . ) . % ( . – . ) . % ( . – . ) . % ( . – . ) journal of nuclear cardiology gupta et al volume , number ; – non-contrast cct can accurately detect chronic mi figure . (a) axial, non-contrast cct image demonstrating an infero-septal (arrowheads) and anterior (arrow) wall infarct. (b) coronal, non-contrast cct image confirming the infero-septal (arrowheads) infarct. lead artifact makes the assessment of infarct difficult in this view. (c) nuclear mpi of the same patient demonstrating the fixed perfusion defects. gupta et al journal of nuclear cardiology non-contrast cct can accurately detect chronic mi january/february been reported to be associated with severe heart failure. the preliminary results of our study demonstrate that the non-contrast cct performed for standard cac scoring has an excellent agreement with mpi in detecting irreversible perfusion defects. despite a moderate specificity of %, cac showed an excellent sensitivity of % to detect irreversible perfusion defects on. the attenuation levels in the infarcted myocardium were lower than the normal myocardium ( . ± . and . ± . , respectively) and the dif- ference was statistically significant (p \ . ). we also determined the optimal cutoff value for lv myo- cardium hu to detect chronic mi as . , yielding a sensitivity of . % and specificity of . % based on the roc curve. this cutoff can be used as an objective method of diagnosing mi on cct in daily clinical practice with high accuracy (auc— . , % ci, . - . ). cac score measured through non-contrast cct scanning has been well validated to predict future cor- onary events in both asymptomatic and symptomatic patients. this study highlights a novel clinical utility of non-contrast cct in addition to assessment of overall burden of atherosclerosis measured by cac, and pro- vides evidence that it can accurately detect chronic mi. we illustrate the importance of complete review of the left ventricle apart from calcium scoring as an addition to the role of non-contrast ct. this application can further be used to detect occult scars in asymptomatic population undergoing cac scan for risk stratification. silent or unrecognized mi, which was once thought to be an unusual presentation of cad, is now widely recognized as a prominent manifestation of the disease process. past cohort studies have demonstrated that %- % of mis are clinically unrecognized. these asymptomatic infarctions escape detection until ulti- mately an ecg is performed for screening or other clinical purposes. the true prevalence of unrecognized mi may be even higher, owing to the insensitivity of q waves. despite its benign clinical presentation the prognosis for unrecognized mi is about as serious as that for recovered symptomatic infarction for all cause and cardiovascular mortality. thus, there is a need for a cost effective screening mechanism for early diagnosis of prior mi to promote early management and poten- tially improved outcomes. although other imaging techniques like late gado- linium enhancement (lge) on contrast-enhanced cmr can detect and characterize mi that is missed by clinical history, ecg, conventional wall motion, , or nuclear figure . area under roc curve for accuracy of non-contrast cct to detect mi diagnosed by mpi. figure . roc curve for accuracy of hu criterion to detect mi on non-contrast cct. table . intra- and inter-observer variability to detect mi on non-contrast cct variable d % ci icc p-value intra-observer . - . to . . . inter-observer . - . to . . . d, mean ratio difference; icc, intra-class correlation coefficient. journal of nuclear cardiology gupta et al volume , number ; – non-contrast cct can accurately detect chronic mi scintigraphic techniques, non-contrast cct is a less expensive, non-invasive method for detecting unrecog- nized mi. there is no incremental radiation or scanning required beyond the cac scan, so one can simulta- neously evaluate for both prior mi and cac with one scan, potentially adding incremental value to the cac scan at no cost. the presence of lge on cmr has been shown to provide incremental prognostic value to mace and cardiac mortality beyond common clinical, angio- graphic, and functional predictors. , these occult myocardial scars have been associated with a high risk of future cardiac events, and therefore identify a sub- population of patients who may benefit from more intensive medical or revascularization treatment strate- gies. similarly, the presence and extent of mi as detected by a non-contrast cct could also be a predictor of mace and cardiac death. the presence of hypo- attenuation may provide incremental prognostic value beyond traditional risk factors and markers of subclini- cal atherosclerosis. non-contrast cct may also represent a better standard for unrecognized mi than ecg for future population-based studies. whether these findings can result in better patient outcomes by guiding management decisions, such as implantation of icds, coronary revascularization, or intensive medical treat- ment, requires additional study. limitation the primary limitation of this study is that we used irreversible perfusion defects on standard rest mibi as the reference for detecting chronic mi on cac scan. however, it should be noted that rest tc- m-mibi underestimates the extent of viable myocardium com- pared with tl- imaging, and thus some of the irreversible perfusion defects might represent hibernat- ing viable myocardium. thus, in this study some of the true positives on cac scan could actually have been false positives. sublingual administration of ntg before a rest tc- m-mibi study would be a better reference standard with improved assessment of myocardial viability. although mpi has been validated as an accurate tool to identify the presence of mi, it is known to give a large number of false-positive results, primarily because of attenuation artifacts. also, smaller infarcts might be underestimated or missed by mpi due to its limited resolution. thus, some non-contrast cct scans may have been classified as false negatives or false positives due to false nuclear results. cmr imaging has been proven to be more sensitive than mpi in the detection of small and occult mis. therefore, additional studies using cmr with delayed enhancement as the reference stan- dard are warranted. conclusion this study demonstrates that the non-contrast cct has an excellent agreement with mpi in detecting chronic mi. further prospective studies are warranted to evaluate the role of non-contrast cct in infarct assessment for the management of at-risk patients. acknowledgment none of the authors have received any funding for this study from any institution. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. references . kannel wb, schatzkin a. sudden death: lessons from subsets in population studies. j am coll cardiol ; : b- b. . kannel wb, cupples la, gagnon dr. incidence, precursors and prognosis of unrecognized myocardial infarction. adv cardiol ; : - . . yano k, maclean cj. the incidence and prognosis of unrecog- nized myocardial infarction in the honolulu, hawaii, heart program. arch intern med ; : - . . kannel wb, abbott rd. incidence and prognosis of 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	abstract 	background 	methods 	results 	conclusion 	introduction 	methods 	study population 	non-contrast cct acquisition and analysis 	mpi acquisition and analysis 	statistical analysis 	results 	discussion 	limitation 	conclusion 	acknowledgment 	references << /ascii encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma . ) /calrgbprofile (srgb iec - . ) /calcmykprofile (iso coated v % \ eci\ ) /srgbprofile (srgb iec - . ) /cannotembedfontpolicy /error /compatibilitylevel . /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /perceptual /detectblends true /detectcurves . /colorconversionstrategy /srgb /dothumbnails true /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel /emitdscwarnings false /endpage - /imagememory /lockdistillerparams true 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left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ management and conservation article argali abundance in the afghan pamir using capture–recapture modeling from fecal dna richard b. harris, department of ecosystem and conservation science, university of montana, missoula, mt , usa john winnie, jr., department of ecosystem and conservation science, university of montana, missoula, mt , usa stephen j. amish, department of biological sciences, university of montana, missoula, mt , usa albano beja-pereira, centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, - vairão, portugal raquel godinho, centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, - vairão, portugal vânia costa, centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, - vairão, portugal gordon luikart, department of biological sciences, university of montana, missoula, mt , usa, and centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, - vairão, portugal abstract estimating population size in a mark–recapture framework using dna obtained from remotely collected genetic samples (e.g., feces) has become common in recent years but rarely has been used for ungulates. using dna extracted from fecal pellets, we estimated the size of an argali (ovis ammon) population that was believed to be isolated from others within the big pamir mountains, afghanistan, an area where access was difficult and expensive. we used closed-capture models to estimate abundance, and pradel models to examine closure assumptions, both as implemented in program mark. we also made visual counts of argali in the big pamirs, allowing comparison of count indices of abundance with modeled estimates. our model-averaged estimate for female argali in the big pamir was ( % ci – ), which was about % higher than our best assessment using uncorrected visual counts. however, mark–recapture models suggested that males were not a closed population; thus, we were unable to provide a meaningful estimate of overall population size. males either suffered much higher mortality than females during the sampling period, or, more likely, males moved in and out of the big pamir area. although information from dna did not provide a clear overall population estimate, it suggested that the big pamir was not isolated from other argali populations, which could not have been confirmed with visual observations alone. estimating argali population size using mark–recapture models and fecal dna is feasible but may be too expensive for frequent monitoring of large and remote populations. our study demonstrates the importance of sex identification and separate abundance estimation for each sex, especially if movement ecology differs by sex. key words abundance estimate, afghanistan, argali, fecal samples, mark–recapture, noninvasive sampling, ovis ammon. estimating population size using well-established statistical techniques that deal with imperfect detectability is generally considered preferable to uncorrected counts (anderson , williams et al. , white ). yet for many wide-ranging ungulate species, particularly those inhabiting remote, mountainous habitats, uncorrected index counts remain the staple (e.g., magomedov et al. , harris and loggers , schaller and kang ). the most common classes of models to deal with imperfect detect- ability are distance sampling, and physically marking animals and later recapturing or resighting them (capture– mark–recapture [cmr]). for mountain ungulates, both distance sampling and cmr are logistically difficult to apply without gross violations of crucial assumptions, very small samples sizes, or both. although having an estimate of abundance is not necessarily as important as having a measure of population trend or an understanding of the important covariates of population vigor, there are circum- stances in which simply having a population estimate is important (sensu caughley ). conservation and management options for small or isolated populations often depend on knowing the approximate population size, particularly when these populations are of interest to legal or illegal hunters. the argali (ovis ammon) is the epitome of a species for which obtaining a population estimate, as differentiated from an abundance index, remains a largely unresolved challenge. counts of individual argali are more easily obtained than for many other species, so visual counts have usually been the basis of abundance assessments. however, argali are also capable of long-distance movements and usually move away from observers at distances far in excess of those that allow individual recognition. argali are group-living ungulates, but group size and composition are usually fluid, with individuals often leaving or joining groups daily (schaller , fedosenko and blank , harris ). with the exception of a few distinctive older rams or occasional animals with deformities or distinctive markings, argali within broad age and sex classes look alike. this, together with the difficulty of approaching argali, makes identification of individuals by direct observation subject to great uncertainty. thus, although argali are easy to count, interpreting these counts as population sizes is fraught with error: some animals go undetected in any survey (thus biasing e-mail: rharris@montana.com present address: ecology department, montana state university, bozeman, mt , usa present address: flathead lake biological station, university of montana, polson, mt , usa journal of wildlife management ( ): – ; ; doi: . / - the journal of wildlife management n ( ) counts low), whereas argali roaming behavior may cause duplicate counts of individuals (thus biasing counts high). estimating abundance with cmr models using dna microsatellites for individual identification has attracted interest in recent years (lukacs and burnham a). using dna is attractive because it can often be obtained without physically handling individual animals (taberlet et al. ). advances in field study design (boulanger et al. , ) and the ability of models to account for both field and laboratory limitations (lukacs and burnham b, petit and valiere , lukacs et al. , knapp et al. ) continue to be made. the use of cmr with remotely collected samples (sensu garshelis ) to estimate abundance has become common among some taxa but remains uncommon for others. bears (ursus spp.) pose particular problems to investigators interested in abundance, but bears can easily be induced to provide hair samples, so dna-based cmr has become a standard part of bear biologists’ tool kit (e.g., bellemain et al. , solberg et al. , kendall et al. ). most other uses of dna-based cmr have been for other carnivores, including canids (creel et al. , prugh et al. ), felids (perez et al. , ruell et al. ), and mustelids (wilson et al. , mulders et al. , williams et al. ). in theory, remotely based cmr estimation could be used for any species (e.g., raptors, primates; rudnick et al. , guschanski et al. ), but its use for free-ranging artiodactyls has thus far been uncommon (but see fickel and hohmann , valiere et al. ). as part of a broader study of the conservation status of argali in the islamic republic of afghanistan, our objectives were to estimate abundance of argali (o. ammon polii, ‘‘marco polo sheep’’) in a portion of the wakhan district within badakhshan province locally termed big pamir. the big pamir was historically reserved as a hunting area for king zahir of afghanistan and during the late s and s was named the pamir-i-buzurg (big pamir) wildlife reserve and operated by the afghan tourist organization (petocz , ). based on previous surveys and anecdotal information, we suspected that argali in the big pamir range had declined considerably from their abundance during the s and that argali were largely isolated (petocz , petocz et al. ; g. b. schaller, wildlife conservation society, unpub- lished report). there were no recent reports of argali inhabiting areas to the south of the big pamir (either within afghanistan’s wakhan district or in the hindu kush range forming the border with pakistan) or elsewhere to the west within badakhshan province (habibi , fitzherbert and mishra ). similarly, reports suggested only occasional individual sightings of argali to the east of the big pamir to approximately u n, u e, where a larger, seemingly more robust population was known to inhabit the little pamir mountains some km away (g. b. schaller, unpublished report; b. habib, wildlife conservation society, unpublished report). it was unknown whether argali in the big pamir were demographically or genetically linked with animals to the north of the panj (amu darya) river in tajikistan. study area the wakhan corridor was a – -km-wide section of afghanistan extending southwest to northeast in the far northeast of the country (fig. ). bordered on the north by tajikistan, china on the east, and pakistan to the south, the region geographically termed the pamir knot, formed by the confluence of the pamir, hindu kush, and karakoram mountain ranges, was characterized by high, broad valleys (pamirs) bordered by steep, rugged mountains. the approximately , -km big pamir study area (fig. ) was located roughly at the midpoint of the wakhan corridor and bordered tajikistan to the north. valleys bordered by high ridges ascend from the panj river (which downstream is called the amu darya) in the northwest (approx. , m) to the crest of the big pamir range that rises to . , m to the southeast. at their lowest, near the panj river, the mountains and ridges are generally rounded, punctuated by heavily eroded, steep gullies. to the south- east, at higher elevations, the terrain is rugged and steep, with occasional small lakes and ponds in the upper valleys and numerous glaciers and permanent snowfields on higher mountain slopes and peaks. south- and southwest-facing slopes were dominated by sparse grass (primarily agrostis, poa, festuca, and agropyron spp.), and sage (artemisia spp.) communities, with sedge (carex and kobresia spp.) meadows in wetter sites (d. bedunah, university of montana, unpublished data). north- and northeast-facing slopes were similar but tended to be wetter and contained more extensive areas of sedge meadows. except for occasional, small sedge meadows at wet sites, there was little vegetation above , m, and we did not see argali at elevations . , m. weather data for the study area are scarce, but the library of congress country studies reported that the wakhan corridor received , mm of rainfall annually and classified the area as arid to semi-arid. this assessment accords with our experiences in the field: rain was rare, brief, and light, from spring through fall, and snows were light, rarely accumulating to depths . cm in the winter. the big pamir was used extensively by wakhi livestock herders, primarily from late spring to early fall, although one herding operation remained year-round, high in the aba khan valley near the northeast boundary of the study area. domestic sheep and goats were typically grazed in the mid- to lower elevations ( , m to , m), whereas cattle and yaks were grazed near the heads of the valleys, often to elevations in excess of , m. methods field procedures we conducted field work (visual counts and fecal sampling) in discrete sessions: june– july (summer ), november– december (autumn ), january– february (winter ), and may– july (summer ). long intervals between sampling sessions were not ideal for cmr estimation; however, they were necessitated by logistical constraints and other research harris et al. n argali abundance in the afghan pamir objectives. we conducted field work on foot, horseback, or yak-back. because argali moved frequently through difficult terrain and our own movements were circumscribed by the valley systems separated by steep ridges, we made no attempt to impose a standardized geographic sampling regime. instead, we attempted to survey for argali within all major drainages of the big pamir during each field session, generally walking to high vantage points to search for animals during the early morning and late afternoon. we adopted a noninvasive approach to cmr estimation, using dna extracted from fecal samples as individual marks. we collected fecal pellets from each pellet group when we encountered fecal pellets we were reasonably certain were freshly deposited by argali. we only collected pellets adjacent to each other within the group, reducing to inconsequential the probability of a sample containing dna from . argali. we avoided collecting from pellet groups scattered over more than approximately . m or that appeared to have been deposited while the animal was moving. low quality samples with malformed or broken pellets were not extracted. we did not sample fecal pellets that appeared to have been produced by lambs of the year; thus, our abundance estimates from dna-based cmr modeling apply to animals l year of age. we took global positioning system (gps) locations for each sample, unless samples were within approximately m of an existing gps fix, in which case we recorded the same location, and noted the date, time, and name of the collector. we stored fecal pellets in sterile -cm centrifuge tubes with securely fitting screw-tops to which sporks (plastic, ovoid-shaped protuber- ances with fork-like tines attached to the inside of the cap) were attached, allowing individual handling of each sample without risk of contamination (evergreen scientific, los angeles, ca). we added approximately parts % ethanol for each part fecal material. we counted observed argali and attempted to assess whether animals were unique from others previously observed during that session. when possible, we classified animals as adult females (age l yr), lambs (either sex), yearlings (either sex), and adult males (age l yr). we made these counts while collecting fecal samples as well as while making observations related to assessing argali figure . afghanistan, showing the location of the wakhan district in northeast part of the country, separating tajikistan (to the north) from pakistan (to the south). inset: wakhan district, showing relative locations of the big pamir and little pamir ranges and waghjir valley. arrow identifies the big pamir study area, where we studied argali, summer to summer , roughly corresponding with the boundary (light outline) of the former pamir-i-buzurg wildlife reserve. the journal of wildlife management n ( ) productivity, habitat use, and responses to disturbance. because we had no way to quantify the uncertainty surrounding duplicate counts arising from movements and the formation of new and dissolution of existing groups, we developed upper and lower bounds to the number of argali we observed in each session. for the upper bound, we deleted only observations of animals that we observed temporally and geographically so closely to other observa- tions of the same sex and age class that we felt certain they represented identical individuals; we counted all other observations. for the lower bound, we deleted observations that could plausibly have resulted from movements of previously recorded animals. neither index accounted for animals we did not visually observe during the session. genotyping genetic work was conducted in laboratories. we conducted initial work at centro de testagem molecular (ctm/cibio), portugal, where we extracted dna from fecal samples using the dneasytm blood kit (qiagen, valencia, ca) modified to include an initial wash of one fecal pellet for minutes in ml of lysis buffer ( . m tris-hcl, . m edta, . m nacl, % n-lauroyl sarcosine, ph . ). we used approximately ml of the lysis buffer directly in the extraction protocol as if the sample were blood (as in maudet et al. ). at ctm, we co-amplified microsatellite dna markers in multiplex polymerase chain reaction (pcr) amplifica- tions (mp : maf , adc; mp : maf , fcb , and fcb ; mp : glycam, krt , and lif) using the qia multiplex mix (qiagen). we performed amplifications on a mjr dyad ptc dna thermal cycler following qia mix protocol for cycles at annealing temperatures ( u c, u c, and u c for mp , mp , and mp , respectively). total reaction volume was ml, including ml of the qiagen pcr master mix, ml of primer mix, and ml of dna. we accomplished reactions with primers for each locus, following the m -tailed primer method (oetting et al. ). fluorescently labeled dna fragments were visualized on an abi xl dna analyzer (applied biosystems, foster city, ca) and chromatograms were analyzed using genemapper . software (applied biosystems) by independent experts at scoring micro- satellite profiles. we initially screened all samples twice with mp to quantify the quality of nuclear dna. we selected samples that showed reliable amplifications and matching genotypes to continue the genotyping process. we inde- pendently regenotyped selected samples to times. we conducted the remaining lab work and genetic analysis at the university of montana conservation genetics laboratory, missoula, montana, usa. we repeated markers initially run in portugal as a data quality check, and we screened additional microsatellite loci, plus the amelogenin sex identification locus (pidancier et al. ). we optimized a multiplex and one pcr and performed - ml reactions on mjr ptc thermocyclers using touch- down profiles. each reaction contained . ml of template dna, . ml of qia multiplex mix (qiagen), and either ml of primer mix, or ml of pm forward and reverse primers. we used touch-down profiles with cycles, one with an initial annealing temperature of u c stepping down to u c and another starting at u c and stepping down to u c. we visualized fluorescently labeled dna fragments on an abi xl automated capillary sequencer (applied biosystems) in the murdock dna sequencing facility at the university of montana. we determined allele sizes using the abi gs liz ladder (applied biosystems). chromatograms were viewed and analyzed using genemapper software v . (applied bio- systems) by independent researchers. we determined sex by pcr amplification of the amelogenin gene as in pidancier et al. ( ). we obtained pcr products (approx. base pairs [bp] and approx. bp) for males but only the longer product for females. we based consensus genotypes on multiple sample runs and the following rules: ) for a sample to be heterozygous at a locus we had to observe both alleles twice and ) for a sample to be homozygous, we had to observe the allele times. we randomly chose % of samples for re-extraction and repeat genotyping to monitor for errors; we detected no genotype differences or errors. due to the large size of fragments at the amelogenin locus, we determined con- sensus genotypes as above with the following changes: we provisionally accepted heterozygotes (m) if we observed a male band only once; we classified homozygotes where we observed only the female band , times (e.g., of independent pcrs) as of unknown sex, and we accepted as males genotypes where we observed only the male band l times. we ran principal correspondent analysis (pcoa) and multilocus genotype matching in genalex (peakall and smouse ) to identify outliers due to potential tube mishandling (in the lab or field), genotyping errors, or non- argali samples and to identify recaptures. we computed amplification success rate, false allele rate, and allelic drop- out rate as in luikart et al. ( ). we found loci contributing significantly more unique individuals than expected with dropout (mckelvey and schwartz ). we estimated expected heterozygosity, tested for gametic (linkage) disequilibrium, and assessed departures from hardy–weinberg proportions (using exact tests and a markov chain) as implemented in genepop . (ray- mond and rousset ). we computed the probability that randomly drawn, unrelated individuals would have identical genotypes (probability of identity [pid]) with api-calc (ayres and overall ). estimation of abundance we used closed capture modeling in program mark to estimate population size, selecting among plausible models of capture variation. except for one reference model, we assumed throughout that recapture probability would not differ from probability of initial capture (i.e., c p) because we could envision no situation in which our sampling activity would affect probability of subsequent capture (i.e., we included no models that allowed for a behavioral harris et al. n argali abundance in the afghan pamir response to initial capture). we examined models in which capture probability varied by sex and by session. we also examined a reduced model in which we classified sessions into low and high capture effort (sessions and were low effort and sessions and were high effort according to total no. of samples collected in each session, see below). because we occasionally obtained recaptures within our defined capture sessions, we also estimated abundance in a session-free context using program capwire (miller et al. ). program capwire uses a continuous sampling approach and thus potentially can make use of recaptures occurring within a given capture session. to examine the assumptions of geographic and demo- graphic population closure (which, under our sampling design, were confounded and could not be distinguished), we examined the fit of a series of pradel models (pradel ) to the same data. in addition to unconstrained pradel models (for open populations), we systematically con- strained each model by fixing the apparent survival term (q) to one (thus closing the population to subtractions) and the recruitment term ( f ) to zero (thus closing the population to additions). our interest was not in either of these demographic terms (or in the derived parameter, the finite per capita rate of increase, l), but rather in the relative fit of models with differing closure assumptions to our data. we used both akaike’s information criterion corrected for small sample size (aicc) and likelihood ratio tests to examine the weight of evidence for closure (cooch and white ). results visual counts of argali varied, depending on the amount of time we spent in the field, habitats used by the animals, weather conditions, and our level of uncertainty in judging observations to be duplicates of animals previously tallied. our counts of yearlings were not always reliable, depending on the distance between the animals and observer. counts of adult females (table ) varied from as few as in summer (with all possible duplicates removed) to as many as in summer (allowing for some possibility of duplication). counts of adult males varied from as few as (in autumn ) to as many as (in summer ). we typically were unable to classify – % of animals we observed (table ). we knew of no way to objectively extract one best estimate from these data. however, assuming rough closure among females, taking the lower bounds of the autumn and summer counts as only slightly conservative, and adding half the number of yearlings (assuming a : sex ratio) from these time periods, our direct observations suggested about females aged l year present in the big pamir during – . fecal sampling, dna extraction, and genotyping we collected fecal samples ( in summer , in autumn , in winter , and in summer ) we judged to be freshly deposited by argali, of which samples yielded consensus genotypes at l of loci. amplification success was relatively low ( %), but false allele ( %) and allelic drop-out ( %) rates were also relatively low (luikart et al. ). to improve precision, we dropped problematic loci (adc, lif) and required remaining samples to share l of loci in common. we dropped lif because . % of consensus genotypes were missing data at this locus. we removed adc from the analysis because dropout found it identified significantly (p , . ) more unique individuals than expected. these measures to improve precision reduced our genetic sample size to consensus genotypes. genotyping success was higher in winter than other seasons (although handling and storage differences may also have influenced genotyping success). we identified multilocus genotypes from samples analyzed ( %), genotypes from samples ( %), genotypes from samples ( %), and genotypes from samples ( %) in summer , autumn , winter , and summer , respectively. we identified unique genotypes from these consensus genotypes. we removed outlier individuals identified by pcoa because they were closely related to samples collected in tajikistan from urials (ovis orientalis). of the remaining genotypes, we could not reliably classify to sex based on the amelogenin sex identification locus. for of these , however, our field knowledge of the fecal pellets in question was sufficient to assign a sex (i.e., only one sex known to be in the vicinity of the specific area within a few days of the collection), leaving for which we table . number of argali, classified by sex and age, that we visually observed during each session, big pamir mountain range, afghanistan, summer through summer . upper row in each case represents a conservative estimate of the number of unique animals seen, removing groups from the cumulative total when duplication was a possibility, and may be underestimates; lower row in each case represents the maximum number of unique animals we observed, removing only certain duplicate observations, and thus are most likely overestimates. counts during winter were conducted by a field assistant who did not provide sufficient details to derive lower and upper bounds and thus are not reported here. sampling period type of estimate argali classification ad f yearlings lambs ad m unclassified total summer lower bound upper bound autumn lower bound upper bound summer lower bound upper bound the journal of wildlife management n ( ) could not determine sex. our raw data for all subsequent cmr analysis (and min. population sizes) thus consisted of uniquely identified argali ( f, m). of the sex- identified consensus genotypes, none were mismatched by one allele, one pair was mismatched by one allele at loci, and one pair was mismatched by one allele at loci. heterozygosity was high (mean expected heterozygosity . ; range . – . among loci), as was the number of alleles per locus (x̄ . , range – ). probability that randomly selected, unrelated individuals would have iden- tical genotypes (pid) was low ( . for loci, . for loci), as was probability of randomly selected siblings having identical genotypes ( . , . for and loci, respectively). marker fcb deviated from hardy–weinberg proportions (fis . , p , . ), probably due to a null allele. one pair of loci revealed gametic disequilibrium (maf and fcb , p , . ). abundance estimation we tallied captures (including recaptures but excluding intrasession recaptures, see below) of the individually identified argali, which formed the basis of closed-capture estimation. recaptures among the sessions were uncom- mon. we captured of the identified females only once, we recaptured once (i.e., captured them twice), and recaptured one twice. of the identified males, we recaptured only animals after initial identification (we recaptured animal once and animals twice, table ). in addition, we tallied intrasession recaptures that did not contribute to estimates using program mark: in summer , in autumn , in winter , and in summer . the top ranked closed-capture model of population abundance allowed capture probability to vary by both sex and session (table ). the second ranked model was similar, with session categorized by effort. other models enjoyed less support. model averaging produced a point estimate of . female argali, with a % confidence interval of . – . (ci coverage/estimate . ). approximately % of total variance was attributed to model uncertainty. the same procedures produced a model- averaged point estimate of . male argali ( % ci . – . ). we suspected this estimate for males was positively biased because ) we never visually observed more males than females in the big pamir study area during any sampling session, ) our molecular analyses identified more females than males, and ) although as yet unstudied for argali, males of similar species generally have lower survival than females, suggesting that females should outnumber males, at least among adults (toı̈go and gaillard ). thus, we conducted post hoc pradel modeling to investigate closure. pradel modeling with various assumptions regarding closure did not result in an unambiguous signal but generally supported the hypothesis that females could be acceptably modeled in a closed-population framework but males could not. in most comparisons, models with q, f, or both constrained for males (i.e., no additions or subtractions for m) ranked lower than corresponding unconstrained models (table ; with models ranked by deviance rather than aicc, because constrained models will tend to have lower aicc due to having fewer parameters, independent of model fit). likelihood ratio tests comparing constrained with uncon- strained models for males were not significant, but these tests are known to lack power, particularly when the number of sessions is low (e.g., only in our case) and capture heterogeneity is present. model comparisons provided little evidence to reject the hypothesis that females were a closed population during the time period, although the weakness of our tests must be kept in mind. table . captures and recaptures of identified argali during the sessions, big pamir, afghanistan, – (excluding recaptures within sessions). sessions are : summer , : autumn , : winter , : summer . the last column also includes one male captured in sessions , , and ; and one male and one female captured during sessions , , and . session sex initial capture recaptured in session total captured during session f m f m f m f m table . top ranked candidate models of probability of capture (p) of female argali in the big pamir mountains, afghanistan, – , based on akaike’s information criterion (aicc), model weights (wi), number of parameters (k ), deviance, and their point estimates of the number of females and standard errors. estimates of male abundance are not included in this table. modela daicc wi k deviance point estimate f se p c + (g + s) . . . . . p c + (g + effort) . . . . . p c + (g) . . . . . p c + (s) . . . . . p c + (.) . . . . . p c (effort) . . . . . weighted average . % ci . – . a abbreviations: c probability of recapture, g sex, s session (capture occasion), effort occasions grouped by similar numbers of fecal pellets collected, (.) constant. harris et al. n argali abundance in the afghan pamir in capwire, equal catchability models were always more preferred (using likelihood ratio tests) over the innate rates (i.e., mixture) models. including all recaptures (regardless of session or distance from other captures, n captures) resulted in an estimate of female argali ( % ci – ; ci coverage/estimate . ), lower than the model averaged estimate using closed captures in program mark, but with a narrower confidence interval. however, we noted that the spatial distribution of within- session recaptures differed significantly (kolmogorov–smir- nov -sample test of equality, p , . ) from that of between-session recaptures (fig. ). in fact, of within-session recaptures occurred when sampling from groups of argali that had been bedded at one site (and thus assigned an identical location). we considered that recaptures obtained under such circumstances violated the independence assumption underlying all continuous time models that we know of, and we thus rejected this estimate as having false precision. removing within-session recap- tures that had identical locations (i.e., were not indepen- dent) and rerunning program capwire resulted in an estimate of female argali ( % ci – ; ci coverage/estimate . ), higher than suggested by our closed-capture models. because our exploration of closure using pradel models suggested that males were not a closed population, we made no attempt to estimate male abundance using capwire. discussion although argali are not difficult to count through direct ground-based observations, interpreting surveys based on count tallies is fraught with difficulty. beyond the well- explored issue that not all animals will be detected, argali move more quickly than ground-based observers, who must therefore deal with the possibility of tallying the same individuals multiple times. capture–mark–recapture estima- tion, using dna obtained from fecal samples, offers a potential solution to these problems. our closed-capture abundance point estimate for females (n̂ , % ci – ) was about % higher than our best interpolation of our series of visual counts and considerably higher than most counts obtained during individual field sessions. more importantly, our estimate was based on models we under- stood and assumptions we could articulate. equally importantly, our exploration of closure assump- tions provided evidence that male abundance varied among time periods. our closed capture models thus overestimated the number of males within the sample area, but we lacked information with which to identify a superpopulation to which the estimate might apply (kendall ). we hypothesize that most variation in male abundance was due to movements in and out of the big pamir area rather than mortality. undetected poaching and natural mortality no doubt occurred during the study, and one would expect mortality rates of males to exceed those of females. our data, however, gave us no indication that mortality was substantially greater among males than females. we observed directly (table ) and captured (table ) more males in summer than in summer , contrary to what we would expect if males were declining throughout the time period. shortly after our summer session (and prior to our autumn session in which we tallied few m), b. habib and z. moheb of our survey team observed – male argali (depending on assumptions about duplicate counts) but no females, during a week in the waghjir valley, about km southeast of the big pamir. we can reasonably assume that argali males, unencumbered table . support for pradel open population models of argali population abundance in the big pamir mountains, afghanistan, – , with selected parameters constrained to produce closed or partially closed models. fully closed models have apparent survival (q) constrained to . (no subtractions) and recruitment ( f ) constrained to . (no additions). models are shown in pairs, in ascending order of deviance. shown are akaike’s information criterion (aicc), number of parameters (k ), deviance, and results of likelihood ratio tests, x , df (difference between the pair of models), and the probability of obtaining a x value this large or larger if there is no difference in model fit. model aicc k deviance x df p m q, open, rest closed . . . . both sexes closed . . m open, f closed . . . . both sexes closed . . both sexes open . . . . both sexes closed . . f open, m closed . . . . both sexes closed . . figure . distances (binned to nearest kilometer) between remote recaptures of the same individual argali, big pamir study area, wakhan district, afghanistan, summer through summer (f: shaded bars; m: open bars). (a) recaptures within a given sampling session. (b) recaptures among sampling sessions. the journal of wildlife management n ( ) by lambs and interested in maximizing mating opportunities during rut, travel greater distances than do females, as males do in most polygynous artiodactyls. we cannot identify sources or destinations of itinerant males (nor exact time periods of movement) but find the overall data persuasive that males moved in and out of the big pamir freely. thus, even had we a field method that overcame the issues of imperfect detectability and possible duplicate counting, an estimate of male abundance based solely on direct observa- tions could easily have misled us into interpreting it as meaningful for the big pamir range in isolation. our study design was compromised by logistical con- straints (it being expensive and time-consuming to mount expeditions to the area), along with competing research objectives (e.g., argali habitat use during specific seasons). ideally in closed-capture cmr estimation, sampling sessions would be shorter, separated by briefer intervening time intervals, and more numerous than we report here. had we been able to conduct multiple capture sessions within a shorter time span during which males remained on the study area, we might have avoided the problem of population closure and thus succeeded in generating population estimates for both sexes. for males, such an estimate would have been meaningful only for that limited time duration, however. because we lacked information on when males moved into and out of the area, we had no way to optimize the timing of our cmr work, even had logistics not been limiting. the robust design (pollock , kendall and pollock ), employing secondary sampling sessions nested within primary sessions, would likely have solved these problems and yielded better information, but we lacked the resources to intensify our sampling sufficiently to implement it. remotely obtained genetic data do not necessarily con- form to traditional designs of mark–recapture studies, in which a capture may occur, at most, once during each session. continuous-session models that make use of within session recaptures such as capwire offer the potential for increased precision (miller et al. , lukacs et al. , puechmaille and petit , robinson et al. ). in our case, however, we found that most ( %) within-session recaptures actually contained no new information. because they originated from fecal samples deposited in the same location (and at a similar time) as other samples from that individual, they reflected our inability to distinguish . defecation of an individual from those of multiple individuals, rather than anything about the abundance of animals. had we ignored this spatio-temporal independence problem, our results would have contained falsely high precision. after removing within-session recaptures that were not independent, program capwire produced an estimate with slightly less precision than model averaged closed captures in mark. we urge investigators to consider whether samples collected closely in time and (or) space are truly biologically independent. if not, the ability of traditional closed-capture models (e.g., in mark) to consider heterogeneity in capture probability explicitly remains an advantage. although we were disappointed that we did not develop an abundance estimate for both sexes in the big pamir range, we were heartened to find evidence that our initial assumption of population isolation was evidently wrong. further genetic information from this population (g. luikart, university of montana, unpublished data) suggests considerable gene flow with argali in other areas, notwith- standing that these are apparently separated from the big pamir by argali-free regions. most analyses of mark–recapture data consider males and females separately because they are likely to differ in capture probability. however, genetic data, particularly from low- quality samples such as feces, sometimes allow discrimination among more individuals (based on microsatellites) than it can reliably identify to sex. there may thus be a temptation to increase overall sample size by ignoring sex, considering males and females together. our study reminds investigators that in addition to differing in capture probability (and thus adding undesirable variation to n̂), sexes may differ in their conformance with closure assumptions. abundance assess- ments of species that are sexually dimorphic behaviorally should consider them separately. these insights from molecular data came at some cost. because other aspects of our argali study (e.g., g. luikart, unpublished data; j. winnie, jr., university of montana, unpublished data) required considerable field work and employed many of the same personnel, estimating the added cost of our dna-based cmr estimate over unadjusted visual counts is not straightforward. including the costs of supplies, shipping, and personnel, a reasonable estimate is approximately us$ /sample. a stand-alone survey de- signed to obtain similar data would have to budget for field work and personnel over and above this. obtaining better precision than we did would require larger samples, more sampling sessions, or both. however, given the remote nature of the area and the added expenses associated with handling animals, the use of traditional mark–recapture or telemetry studies to answer the questions of population size or sex- biased movement patterns would be even more expensive. much of the laboratory costs involved development and optimization of genotyping assays (in each of independent laboratories). future analyses would probably cost less (e.g., us$ /sample). costs will likely continue to decline with technological advancements (beja-pereira et al. ). further, future argali analyses might be conducted with less replication if higher quality samples could be collected, for example by sampling only in the winter or when fecal pellets are better formed and contain better-quality dna (maudet et al. , luikart et al. ; for review see beja- pereira et al. ). although collecting high quality fecal samples for genetic analysis in remote areas can be a challenge, it is also easy to integrate into field surveys without many of the additional costs associated with traditional mark–recapture or telemetry studies. management implications both cmr estimates and our visual counts confirmed that the number of argali occupying the big pamir range is small harris et al. n argali abundance in the afghan pamir and that this portion of argali range is appropriately viewed as a serious conservation concern. further monitoring and careful management is needed. although petocz ( ) considered the big pamir to have contained . adult females in the early s, differences in estimation methods lead us to caution against interpreting our estimate as necessarily indicating a dramatic decline since then. capture–mark–recapture modeling, as well as asso- ciated genetic information (g. luikart, unpublished data) suggested that argali inhabiting the big pamir are not highly inbred or genetically isolated. with such low numbers, genetic (and possibly occasional demographic) linkage with other populations, including those in tajiki- stan or china, has probably functioned to help maintain this population. remote (noninvasive) estimation of abundance using cmr models and genetic data from fecal samples is increasingly feasible and is often the only reliable option for wide-ranging ungulates living in difficult habitats such as argali. such estimates overcome many of the problems inherent in uncorrected visual counts and in traditional cmr ap- proaches. however, although fecal pellets are easy to collect, rigorous laboratory analyses are costly (especially when initial optimization is required), and statistical analyses must be considered carefully. we suspect our approach will be useful primarily for populations of particular concern, which typically will be small and well defined. acknowledgments our study was part of the afghanistan biodiversity conservation program of the wildlife conservation society (wcs), supported by the united states agency for international development (usaid). g. luikart was partially supported by the portuguese american foundation for development and centro de investigação em biodiver- sidade e recursos genéticos-universidade do porto. r. godinho and a. beja-pereira were supported by post- doctoral grants (sfrh/bpd/ / and sfrh/ bpd/ / , respectively) from the portuguese science foundation. for field assistance we thank b. habib, z. moheb, sabir, and a. khairzad. we also thank s. ostrowski, d. bedunah, s. nikzad, z. ejlasi, i. farahmand, q. sahar, k. sediqi, k. sidiqi, g. sediq, a. ahamad, r. king, and l. yook for their able assistance. we thank a. dehgan, p. zahler, p. smallwood, p. bowles, and a. simms for advice and administrative assistance, and g. white and p. lukacs for analytical help. literature cited 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a. burke, c. l. cheesman, and r. j. delahay. . estimation of badger abundance using faecal dna typing. journal of applied ecology : – . associate editor: latch. harris et al. n argali abundance in the afghan pamir qtl influencing blood pressure maps to the region of pph on chromosome q - in old order amish qtl influencing blood pressure maps to the region of pph on chromosome q - in old order amish wen-chi hsueh, phd; braxton d. mitchell, phd; jennifer l. schneider, bs; michael j. wagner, phd; callum j. bell, phd; elizabeth nanthakumar, ms; alan r. shuldiner, md background—hypertension is a major risk factor for coronary heart disease, stroke, congestive heart failure, renal insufficiency, and peripheral vascular disease. although the genetic contribution to variation in blood pressure is well recognized, the specific genes involved are mostly unknown. we carried out a genome-wide scan to identify loci influencing blood pressure in the old order amish population of lancaster county, pennsylvania. methods and results—blood pressures were measured in adult participants from families recruited without regard to blood pressure. we performed a quantitative linkage analysis by using microsatellite markers. in multipoint analysis, strong evidence for linkage was observed with both diastolic (lod . ; p . ) and to a lesser extent systolic (lod . ; p . ) blood pressure in the region of chromosome q - . peak evidence for linkage occurred at map positions and cm from pter for diastolic and systolic blood pressure, respectively. conclusions—a gene linked to familial primary pulmonary hypertension has recently been mapped to this same region, suggesting the intriguing hypothesis that other (attenuated) mutations in this same gene may influence variation in systolic and diastolic blood pressure in this population. (circulation. ; : - .) key words: blood pressure n amish n genetics n hypertension, pulmonary hypertension is one of the most common chronic diseasesin the united states, affecting . % of adults to years of age and nearly three quarters of african americans and one half of whites to years of age. it is a major risk factor for coronary heart disease, stroke, congestive heart failure, end-stage kidney disease, and peripheral vascular disease and consequently results in tremendous disability and mortality rates. variation in blood pressure is influenced by both genetic and environmental factors. , although several rare simple mendelian forms of hypertension have been described, no underlying cause or transmission pattern can be readily discerned in the vast majority of patients with hypertension. most family studies indicate that genes account for % to % of the variation in blood pressure levels. – to date, however, there has been little progress in identifying the specific genetic defects responsible for the common forms of hypertension. to identify specific loci influencing variation in blood pressure, we conducted a genome-wide scan in the old order amish (ooa), a genetically isolated white population char- acterized by large family sizes. methods the ooa population originated in western europe (mainly swit- zerland), when followers of jacob ammann split from their parent anabaptist sect and emigrated to the united states to escape religious prosecution over a -year period beginning in . approximately pioneer couples settled in lancaster county, pennsylvania, and may be considered founders of the present group of the lancaster amish. the number of ooa in the area is . today, and nearly all surviving members can be linked to a single -generation pedigree. the ooa are a rural-living population and are characterized by their eschewal of technological innovation and strong interest in their ancestry and genealogical relationships. furthermore, there is considerable homogeneity in the amish life- style. the majority of men are farmers and the women are mostly homemakers. fewer than % of the ooa report that they currently smoke, and nearly % of our study participants reported not having any leisure physical activities. alcohol consumption is minimal. the ooa do not practice birth control, and family members usually eat their meals together. with the support of the amish community, recruitment for the amish family diabetes study began in early with the goal of identifying susceptibility genes for type diabetes and related traits. the study protocol was approved by the institutional review board at the university of maryland school of medicine, and informed consent was obtained from each study participant. with the help of liaisons from the ooa community, we identified individuals with type diabetes. these probands and their family members $ years of age were recruited into the study. between february and february , subjects received examinations at the amish diabetes research clinic in strasburg, pennsylvania. appointments were made in advance by home visit, since the amish do not use telephones or cars. at the clinic, study subjects received an extensive interview regarding their personal medical history and family history received september , ; revision received january , ; accepted january , . from southwest foundation for biomedical research, san antonio, tex (w.-c.h., b.d.m., j.l.s.); glaxowellcome, inc, research triangle park, nc (m.j.w.); axys pharmaceuticals, la jolla, calif (c.j.b., e.n.); and the university of maryland school of medicine, baltimore (a.r.s.). correspondence to braxton d. mitchell, department of genetics, southwest foundation for biomedical research, po box , san antonio, tx - . e-mail bmitchel@darwin.sfbr.org © american heart association, inc. circulation is available at http://www.circulationaha.org d ow nloaded from http://ahajournals.org by on a pril , of diabetes. anthropometric measurements and a -hour, -g oral glucose tolerance test were also performed. systolic blood pressure (sbp) ( st phase) and diastolic blood pressure (dbp) ( th phase) levels were obtained in duplicate with the use of a standard sphygmomanometer with the patient sitting for $ minutes and were recorded to the nearest mm hg. body mass index (bmi) was calculated as weight (kg) divided by height squared (m ). mean blood pressure and prevalence of hypertension (sbp $ mm hg or dbp $ mm hg or current use of antihyperten- sive medications) were compared between the amish population and a representative sample of the overall white population in the united states, as assessed by the national health and nutrition examination survey (nhanes) iii, conducted during to . dna was extracted from leukocytes, and a screening set of highly polymorphic microsatellite short tandem repeat markers was genotyped from the abi prism linkage mapping set (perkin- elmer). the mean marker heterozygosity was . , ranging from . to . . the average interval between markers was . cm, and the largest gap between markers was . cm, occurring on chromosome . quantitative trait linkage analysis was carried out with the use of a variance components methodology, in which we partitioned vari- ation in blood pressure into components attributable to environmen- tal covariates, the additive effects of genes (ie, residual heritability), and a specific quantitative trait locus, or qtl (ie, the linkage component). these analyses were conducted with the use of maxi- mum likelihood procedures as implemented in the solar software package. the additive genetic effect was modeled as a function of the expected genetic covariances between relatives, and the qtl effect was modeled as a function of the identity by descent relationships at the marker locus. the hypothesis of linkage is evaluated by the likelihood ratio test, in which one evaluates whether the locus-specific effect is significantly . (ie, ho: s qtl versus ha: s qtl. ). both multipoint and -point linkage analyses were carried out. sbp and dbp were analyzed separately, and in each analysis, we simultaneously adjusted for the effects of sex and sex-specific age and age . individuals currently taking antihyperten- sive medications (n ) were excluded from analysis. thus, the total number of individuals included for linkage analysis was . we derived the distribution of nominal lod scores under the null hypothesis of no linkage empirically by simulation. to generate this distribution, we simulated an unlinked marker locus with equifre- quent alleles, assigned genotypes to each founder, and then dropped genotypes down through the pedigree based on mendelian expecta- tions and the founder genotypes. the simulated unlinked marker had approximately the same information content (ie, heterozygosi- ty %) as the markers used in the genome scan. we then conducted linkage analysis of blood pressures with the simulated unlinked marker. the unlinked marker locus was simulated with the use of pap software, and the linkage analysis on each simulated data set was carried out with the use of the solar software program. we conducted replicates and defined the probability of obtaining a false-positive result as the proportion of replicates for which we obtained a specified lod score or higher. these probabil- ities were then converted into lod scores by first converting them into x values, and then dividing the x statistic by ( zloge ). all lod scores presented in this article were obtained from this simulation. although all subjects can be related by tracing their ancestors back multiple generations, to reduce computational difficulties, we di- vided the sample into discrete families, ranging in size from to individuals. the sample included a large number of relative pairs, including parent-offspring pairs, sib-pairs, avuncular (aunt/uncle-niece/nephew) pairs, and first-cousin pairs. results the mean age of the participating study subjects was . . years, and mean levels of sbp and dbp were . . mm hg and . . mm hg, respectively. mean bmi was higher in women than in men ( . . versus . . kg/m ). mean blood pressures for men and women are shown in figure for both the ooa and the overall us white population. among men, blood pressures were similar in the ooa compared with the overall us white male population, but ooa women tended to have slightly higher blood pressures than the overall us white female population. in general, the prevalence of hypertension in the ooa women ( . %) was comparable to that in the us white women, although the prevalence in ooa men ( . %) is slightly lower than in us white men. the age-specific prevalence rates of hypertension in the ooa are shown in figure . the level of hypertension control in the ooa was low, with only % of hypertensive subjects currently under treatment, compared with . % in the overall us white population. heritabilities of sbp and dbp were . . (p, . ) and . . (p, . ), respectively, indi- cating that a substantial portion of the variation in these traits is attributable to additive genetic factors. detailed results from our multipoint genome-wide scan are shown in figure . (these results may also be obtained from the sfbr web site at http://www.sfbr.org/sfbr/departments/genetics/genepid/). the maximum lod scores were . and . for dbp and figure . sex-specific mean blood pressure levels in ooa and national health and nutrition examination survey (nhanes) iii white population. figure . sex-specific prevalence of hypertension in ooa. hsueh et al qtl influencing blood pressure d ow nloaded from http://ahajournals.org by on a pril , sbp, respectively, both occurring in the same region on chromosome q, ' to cm from pter. on only other chromosome did we obtain a lod score as high as . (lod score . for dbp on the pter end of chromosome ). results from the multipoint linkage analysis for chromo- some are shown in figure . peak evidence for linkage for both sbp and dbp occurred in the region bounded by markers d s and d s . the -lod unit support interval (ie, the region corresponding to the peak lod score minus ) for the region encompassing the dbp qtl included a -cm interval flanked by markers d s and d s , and that for sbp included a -cm interval flanked by markers d s and d s . the addition of diabetes and bmi as covariates did not substantially alter these results (adjusted lod scores . and . for dbp and sbp, respectively, at these same marker positions). there was no evidence for linkage of the dichotomous trait, hypertension, to chromo- some q markers (data not shown), although the power to dtect linkage to the dichotomous trait was low in this sample. a second region of suggestive linkage to sbp (lod . ) was also observed on chromosome , ' cm from pter on the short arm. a conditional analysis was performed to determine whether the effect of allele-sharing at this second locus accounted for a significant portion of the trait variation, after accounting for allele-sharing at the locus on chromo- some q. this hypothesis was evaluated by the likelihood ratio test, in which we compared the likelihood of a -locus model (ie, qtl effects at chromosome p and q) with that of a -locus model (qtl effect at chromosome q only). the likelihood of the -locus (conditional) model was only marginally better than that of the likelihood of the single- locus model, with the marginal lod score of the p locus estimated to be only . . the -point linkage analyses provided substantial support- ing evidence for linkage of both sbp and dbp to the -cm region on chromosome q. three markers were typed within the region of linkage: d s (at . cm from pter), d s (at . cm from pter), and d s (at . cm from pter). the -point lod scores associated with these markers were (for dbp and sbp, respectively) . and . for d s ; . and . for d s ; and . and . for d s . discussion the results from our analyses provide strong evidence for the presence of a gene on the long arm of chromosome that influences variation in systemic blood pressure. this conclu- sion is bolstered by the fact that linkage was detected in this same region to both dbp and sbp and that consistent evidence for linkage across multiple markers in this region was obtained in the -point analyses. the evidence for linkage, as determined by simulation studies, was p . for dbp and p . for sbp (corresponding to lod scores of . and . for dbp and sbp, respectively). we detected virtually no other linkage signals for either trait anywhere else in the genome. considering that the correlation between sbp and dbp in this population is . , it is possible that a locus on chromosome q influences variation in both traits. there are, however, almost certainly additional loci elsewhere in the genome that contribute to variation in either sbp or dbp (or both). despite the unique background of the ooa, epidemiolog- ical aspects of blood pressure variation in this population appear very similar to the overall us white population. for example, the distribution of blood pressure levels and the prevalence of hypertension in the amish are comparable to those of the us white population. furthermore, as in other populations, blood pressure variation in the amish has a significant familial component, but there is no clear mode of inheritance. since the lancaster county ooa arose from ' founding couples who migrated to the united states from western europe in the early to mid s, and a subset of the overall us gene pool also originated from this region of europe, we hypothesize that common gene variants that contribute to blood pressure variation in the amish are likely to comprise a subset of those that are relevant in the overall us and european white populations. it is possible that the unique characteristics of the ooa population may favor detection of these blood pressure genes since there may be a smaller number of genetic variants segregating in this popu- lation, each contributing a greater proportion of variation in blood pressure. in addition, the relatively homogenous life- style of the amish may further make these gene variants easier to detect, since they will account for a larger compo- nent of the trait variation. proof of these hypotheses will require the identification of specific gene variants through positional cloning or positional candidate approaches. linkage has previously been reported between hyperten- sion (and/or blood pressure) and several functional candidate genes, including angiotensinogen on chromosome q - , , a b-adrenergic receptor and dopamine receptor type a on chromosome q . -qter, lipoprotein lipase on chromosome p , genes encoding the b and g subunits of epithelial sodium channel on chromosome p , and angiotensin-converting enzyme on chromosome q . , two recent genome-wide linkage studies using discordant sib-pairs found several linkage signals on p - . , q . - , q . - . , and q . - . and regions containing markers d s , d s , d s , d s , and d s . however, there was no evidence in our analysis for linkage of any of these regions to blood pressure variation. linkage to blood pressure variation has been reported in several studies with rodent models, – but none of these maps to human chromosome . the fact that we failed to detect linkage to any of these regions in the ooa can be attributed to any of a number of factors, including the possibility that the prior results were false-positives and the lack of power of our study to detect linkages observed in other populations. we estimated the power of our sample to detect qtl effects that accounted for % to % of the phenotypic variation in blood pressure in our population. these results, obtained by simulation, re- vealed that we would have % power (at lod$ ) to detect a qtl that accounted for % of the phenotypic variation, % power to detect a qtl accounting for % of the phenotypic variation, and % power to detect a qtl accounting for % of the phenotypic variation. thus, even if these other gene effects did exist, our power to detect them would be low. circulation june , d ow nloaded from http://ahajournals.org by on a pril , our power to detect linkage to the qualitative trait, hyperten- sion, was far lower, as the number of affected sib-pairs in our sample was , ( %) of whom come from only sibships, thus making the number of independent sibships substantially lower. there are, in addition, substantial differences in study design and analytic strategies between our studies and others that may also account for the failure of our study to detect linkages reported by others. for example, some of the published studies (except for recent discordant pair analy- ses , ) did not test for linkage genome-wide but rather evaluated evidence for linkage only to a set of hypertension candidate genes, none of which were on chromosome q. some studies have evaluated evidence for linkage to the dichotomous trait hypertension only, whereas others have reported results for sbp only. , although it is likely that there may be genes with pleiotropic effects on all traits, it is also possible that there are genes whose influence is primarily on of these traits. a further key difference among published studies is the age distribution of the population. different genes may express their effects at different ages, or their effects may be expressed only in the presence of other figure . hsueh et al qtl influencing blood pressure d ow nloaded from http://ahajournals.org by on a pril , age-related factors, as, for example, long-term smoking exposure and/or hormonal profile. in our study, the majority of subjects were $ years of age, whereas at least other genome scan studies have focused primarily on younger subjects. , the high proportion of postmenopausal women in our sample may have added an additional source of variability into our study, since different genes may influence blood pressure regulation in the premenopausal and post- menopausal states. recently, different groups have independently localized a gene for familial primary pulmonary hypertension (pph) to a -cm region on q - , an interval corresponding closely to the peak region of linkage in our analysis. , pph is a rare disease characterized by elevated pulmonary artery pressures in the absence of a secondary cause. the disorder leads to right ventricular failure and, in the absence of treatment, death. young women are at higher risk for the disorder, and an estimated % of pph cases are inherited in an autosomal dominant fashion with reduced penetrance. in , ni- chols et al identified families in which familial pph was segregating and reported a maximum multipoint lod score of . , occurring at the map position of marker d s . on our map, this marker falls within the interval flanked by markers d s and d s , the markers that flank the figure . lod scores for genome-wide scan of sbp and dbp levels. solid curve indicates sbp; dashed curve, dbp. tick marks indicate the marker locations. agt indicates angioten- sinogen; pph , primary pulmonary hypertension ; adra b, a b adrenergic receptor; lpl, lipoprotein lipase; scnn b and scnn g, epithelial sodium channel, b and g subunits; and dcp , dipeptidyl carboxypeptidase (angiotensin i– converting enzyme). circulation june , d ow nloaded from http://ahajournals.org by on a pril , peak region of linkage in our analysis. in fact, nichols et al place the location of marker d s at . cm telomeric to marker d s , a position corresponding to position . cm from pter on the amish map. thus, our peak signal for dbp in multipoint analysis occurred at a position , cm away from the point of peak linkage reported by nichols et al, and our peak signal for sbp occurred at a position ' cm away. at about the same time, morse et al observed significant evidence for linkage (lod . ) with markers in this same region in a single family with autosomal dominant pph. these investigators mapped the pph locus to a -cm region flanked by markers d s and d s , with peak evidence for linkage also occurring at marker d s , but with additional markers (including d s ) on the centro- meric side of d s also providing equally strong evidence for linkage. to date, pph has not been cloned, nor is its function known. an autoimmune component to the underlying cause of pph has been suggested on the basis of reported associations between pph and several autoimmune disorders , and with specific hla alleles. , the candidate region encompassing pph contains other known genes that could influence vascular wall function, such as parathyroid receptor and insulin growth factor binding proteins and . in addition, a cluster of immunoglobulin superfamily genes that encode integrin sub- units av, a , and b has been localized to this region. – to our knowledge, there have been no ooa families with pph. nevertheless, it is intriguing to speculate that the qtl identified in our analysis of blood pressure variation in the amish may, in fact, be pph , or a linked regulator of this gene. although pph is a rare disease (with only families identified in the united states since as having . affected family members), it is possible that other defects in this gene may produce a phenotype of systemic blood pressure elevation by affecting systemic endothelial vascula- ture and/or function. the results of this genome-wide scan to detect blood pressure genes have revealed the presence of a qtl on chromosome q in the ooa that influences dbp and perhaps also sbp. the point of peak linkage coincides closely with the location of pph . the identification of this gene, whether it turns out to be pph or a closely linked gene, should enhance our understanding of the cause of hypertension and perhaps lead to novel strategies for the prevention and 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from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ pone. .. genetic mapping and exome sequencing identify variants associated with five novel diseases erik g. puffenberger , *, robert n. jinks , carrie sougnez , kristian cibulskis , rebecca a. willert , nathan p. achilly , ryan p. cassidy , christopher j. fiorentini , kory f. heiken , johnny j. lawrence , molly h. mahoney , christopher j. miller , devika t. nair , kristin a. politi , kimberly n. worcester , roni a. setton , rosa dipiazza , eric a. sherman , james t. eastman , christopher francklyn , susan robey-bond , nicholas l. rider , , , stacey gabriel , d. holmes morton , , , kevin a. strauss , , clinic for special children, strasburg, pennsylvania, united states of america, department of biology and biological foundations of behavior program, franklin & marshall college, lancaster, pennsylvania, united states of america, the broad institute, boston, massachusetts, united states of america, department of biology, swarthmore college, swarthmore, pennsylvania, united states of america, department of pathology and laboratory medicine, school of medicine and public health, university of wisconsin, madison, wisconsin, united states of america, college of medicine, university of vermont, burlington, vermont, united states of america, lancaster general hospital, lancaster, pennsylvania, united states of america abstract the clinic for special children (csc) has integrated biochemical and molecular methods into a rural pediatric practice serving old order amish and mennonite (plain) children. among the plain people, we have used single nucleotide polymorphism (snp) microarrays to genetically map recessive disorders to large autozygous haplotype blocks (mean = . mb) that contain many genes (mean = ). for some, uninformative mapping or large gene lists preclude disease-gene identification by sanger sequencing. seven such conditions were selected for exome sequencing at the broad institute; all had been previously mapped at the csc using low density snp microarrays coupled with autozygosity and linkage analyses. using between and patient samples per disorder, we identified sequence variants in the known disease-causing genes slc a and flvcr , and present evidence to strongly support the pathogenicity of variants identified in tubgcp , brat , snip , cradd, and hars. our results reveal the power of coupling new genotyping technologies to population-specific genetic knowledge and robust clinical data. citation: puffenberger eg, jinks rn, sougnez c, cibulskis k, willert ra, et al. ( ) genetic mapping and exome sequencing identify variants associated with five novel diseases. plos one ( ): e . doi: . /journal.pone. editor: andreas r. janecke, innsbruck medical university, austria received september , ; accepted november , ; published january , copyright: � puffenberger et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. funding: this research was supported by the following: an award to franklin & marshall college from the howard hughes medical institute’s undergraduate science education program; the eyler endowment for biochemistry at franklin & marshall college; mr. sam lombardo; franklin & marshall college; grants from the national human genome research institute of the national institutes of health (nih), u hg , to the broad institute for the large scale sequencing program; and dhm through a gift from the john d. and catherine t. macarthur foundation. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. competing interests: the authors have declared that no competing interests exist. * e-mail: epuffenberger@clinicforspecialchildren.org introduction the plain populations of pennsylvania are descended from small groups of swiss immigrants who organized into multiple endogamous demes that have remained genetically isolated over the last – generations [ , ]. certain recessive disorders are highly concentrated in plain sects [ , ]. the overwhelming majority (. %) of affected individuals are homozygous for their respective pathogenic variant, which resides within a relatively large, homozygous haplotype block. we have exploited this knowledge to map dozens of recessive conditions using low- density (i.e. , and , marker) single nucleotide polymorphism (snp) microarrays with as few as two patients [ ]. this is an efficient, low-cost strategy [ ]. the ease of genetic mapping is counterbalanced by the difficulty of disease gene identification. shared homozygous blocks among affected individuals tend to be large (mean . mb) and contain dozens or hundreds of genes [ , , , , , , ]. large gene lists are significant obstacles, particularly if expression and functional data provide few clues to prioritize the list. since , we have mapped loci for genetic disorders within amish and mennonite demes. for ( %) of these, we could not identify the causative gene as no pathogenic variants were found after sequencing all high-priority candidate genes within the mapped interval. exome sequencing has recently been shown to expedite disease gene discovery [ , , , , , , , , , , , , , , , , ]. in a pilot study to investigate the utility of exome sequencing, the clinic for special children and the broad institute initiated a collaboration to combine thorough phenotyping, autozygosity mapping, and exome sequencing. within months, we identified pathogenic variants for seven disorders, six of them novel. to delineate the functional consequences of these variants, we designed and executed studies of mutant protein expression and function. this work highlights the extraordinary potential of next generation technologies for the investigation of monogenic disease among appropriately selected individuals, families, and communities. it provides a realistic model for using next generation sequencing strategies in everyday clinical practice. plos one | www.plosone.org january | volume | issue | e results phenotypes the phenotypes are summarized in table . detailed descriptions of each disorder are presented below. infantile parkinsonism-dystonia syndrome. the pheno- type and gene defect for infantile parkinsonism-dystonia have been described elsewhere [ ]. briefly, our patient developed irritability and feeding difficulties soon after birth. generalized rigidity and dystonia developed during early infancy, impeding motor development, and evolving into severe rigid parkinsonism by late childhood. the proband cannot speak or use her hands to communicate, and it has thus been difficult to assess cognitive function or thought content. brain structure is normal. in cerebrospinal fluid, homovanillic acid (hva) is elevated, - hydroxyindoleacetic acid ( hiaa) is normal, and the concentration ratio of hva to hiaa is . – . mol:mol (normal – . mol:mol). treatment with haloperidol, tetrabenazine, levodopa-carbidopa, trihexyphenidyl, and tyrosine restriction have been ineffective. lethal neonatal rigidity and multifocal seizure syndrome. episodic jerking begins in utero. newborns have small heads ( . to sd below normal for age), overlapping cranial sutures, small or absent fontanelles, and depressed frontal bones. hands are fisted and extreme axial and limb rigidity prohibit volitional movements and tendon reflexes. brief focal jerks of the tongue, face and arms are prominent soon after birth and occur in a nearly continuous sequence throughout each child’s short life. neuroimaging is normal or reveals mild hypoplasia of the frontal lobes. electroencephalograms show bilateral medium- high voltage spikes over temporal and central regions, frequent multifocal seizures, background slowing, and no posterior rhythm. seizures are only partially responsive to anticonvulsants and not affected by high-dose pyridoxine. affected children have stagnant head growth, remain visually inattentive, do not feed independently, and make no developmen- tal progress. they have frequent spontaneous apnea and bradycardia that uniformly culminates in cardiopulmonary arrest before age months. the brain of a child who died at weeks of age weighed grams (expected for age +/ g) but was otherwise normally developed. primary lesions were localized to most regions of the corpus striatum and cerebral cortex with relative sparing of the anterior caudates and parietal lobes. lesions consisted of neuronal loss associated with a striking microglial reaction and proliferation of alzheimer type astrocytes (figure , a–c). microcephaly with chorioretinopathy. the phenotype was originally described by victor mckusick [ ]. all patients are born with microcephaly, a sloping forehead, diminutive anterior fontanelle, and sutural ridging (figure , a). head circumference is more than standard deviations below normal at birth and remains so into adulthood (figure , b). affected neonates transition well and feed normally. children walk independently between and months of age and language emerges at an appropriate age but remains rudimentary. cognitive impairment ranges from moderate (adult mental age – years) to severe (adult mental age , months); intelligence quotients of two young patients were and (normal ). two of nine patients ( %) have epilepsy: one started having drop attacks in late childhood and another developed nocturnal epilepsy as an adult. magnetic resonance imaging shows diffuse pachygyria (figure , c). the cerebral hemispheres are small relative to the cerebellum, which has a hypoplastic vermis (figure , d). although myelin volume appears reduced, it has normal signal quality. the surface table . phenotype summary. disease group omim clinical synopsis infantile parkinsonism-dystonia m – infantile-onset rigidity, dystonia, or chorea dopamine non-responsive parkinsonism progressive frontal lobe degeneration lethal neonatal rigidity and seizure syndrome a – in utero myoclonic spasms neonatal-onset intractable focal seizures congenital rigidity dysautonomia (hypothermia, apnea, bradycardia, sids) mental retardation, non-syndromic m – delayed language development mild-moderate mental retardation microcephaly with chorioretinopathy m congenital pachygyric microcephaly global developmental delay chorioretinopathy and retinal detachment posterior column ataxia with retinitis pigmentosa m impaired propioception retinitis pigmentosa symptomatic epilepsy and skull dysplasia a – global developmental delay intractable epilepsy skull dysplasia usher syndrome a – retinitis pigmentosa progressive sensorineural hearing loss episodic psychosis doi: . /journal.pone. .t exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e area of the corpus callosum is approximately half that of an age- matched control child ( . cm versus . cm ). visual impairment becomes evident during the first year of life. the retina and choroid are underdeveloped and have focal defects that reveal bare sclera. just posterior to the equator of the eye, much of the retina has a scalloped appearance that suggests focal areas of arrested development. the more anterior parts of the retina, near the periphery and pars plana, have a grayish hue and figure . corticobasal degeneration in the brain of an infant who died from a homozygous brat mutation. (a) throughout frontal, occipital and temporal cortex, there is marked neuronal loss, gliosis with astrocytes (arrowheads) and swollen oligodendroglia. the arrow indicates a perivascular microcalcification (superior frontal gyrus, deep cortex, ). (b) the anterior hippocampus is smaller than expected and there is neuronal loss and gliosis in zone ca- (sommer’s sector), demarcated from the ca- sector by the dotted line ( ). (c) at magnification, the putamen shows a paucity of neurons, abundant alzheimer type astrocytes (arrowhead) and scattered microglial nodules (arrow). heterologous overexpression of n-terminal flag-tagged human brat (d) and hbrat c. _ insa (e) in mouse imcd cells. wild-type brat localizes to the nucleus and cytoplasm of mimcd cells. mutant brat (c. _ insa) does not localize to the nucleus and instead forms punctate aggregations in the cytoplasm. similar results were obtained in harpe- cells (data not shown). (f) rt-pcr demonstrating the stability of overexpressed human brat transcripts (, . kb) in harpe- cells. a b-actin amplicon (, bp) was used as a loading control on the same gel. (g) western blot of lysates from human arpe- cells transiently transfected with wt hbrat displaying flag-hbrat fusion protein at , kda or with hbrat c. _ insa displaying the truncated flag-hbrat mutant fusion protein at , . kda (flag-tag and linker = . kda). b-actin was labeled as a loading control. doi: . /journal.pone. .g exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e diminutive vasculature similar to retinopathy of prematurity. condensations of vitreous may attach to the retina in transition regions between scalloped and gray tissue, marking points of traction for retinal detachment. non-syndromic mental retardation. the phenotype is typical of that described for other forms of non-syndromic mental retardation. all milestones are mildly delayed and cognitive function remains significantly impaired, precluding independent living and self-care. speech is rudimentary but articulate. affected individuals are not autistic. posterior column ataxia and retinitis pigmentosa. the axpc phenotype has been described elsewhere [ ]. tunnel-like visual loss and photophobia begin early in childhood when fundoscopy reveals signs of non-spiculated retinitis pigmentosa and cellophane maculopathy. as vision deteriorates throughout adolescence, patients might develop posterior subcapsular cataracts. motor milestones are slightly delayed (independent ambulation by months). sensory ataxia, wide-based gait, and rombergism emerge by age . signs of sensory neuropathy include pan-areflexia, stocking- glove loss of vibration and position sense, astereognosia, agraphesthesia, and blunted sensation of applied force (e.g. accidentally crushing paper cups). muscle tone, power, and electromyography are normal. mri reveals t signal hyperinten- sity running the length of the dorsal spinal cord. sensory sural nerve action potentials and h waves are absent. some patients develop focal epilepsy marked by interictal focal spike-wave discharges. cognitive function is normal. symptomatic epilepsy and skull dysplasia. affected neonates are hypotonic and feed poorly. dysmorphic features that evolve over time include a bulbous nose, wide mouth and tongue, broad jaw with protuberant angles, short hands, short tapered fingers, and broad thumbs (figure , a). affected children have severe psychomotor delay and do not learn to walk or speak. many retain use of their hands to scoot, maneuver a wheelchair, or gesture. some children show behavioral responses to language, but they do not socially engage or follow verbal commands. examination typically reveals a subdued child with strabismus, slow horizontal nystagmus, hypotonia, and weak or absent tendon reflexes. magnetic resonance imaging reveals ventriculomegaly, thin corpus callosum, white matter abnormalities, and an undulating or ‘‘lumpy’’ skull surface (figure , e). the cortical ribbon follows the irregular skull contour (figure , e–f). multifocal spike-wave discharges from central, occipital, and figure . microcephaly and chorioretinopathy due to a homozygous tubgcp mutation. (a) an affected infant has marked microcephaly (. sd below normal), a receding forehead, diminutive anterior fontanelle, and sutural ridging. she has cognitive delay and visual impairment but is socially engaged. (b) head circumference and length plots for mennonite microcephaly patients. (c) brain magnetic resonance imaging (mri) shows diffuse pachygyria, normal myelination, and (d) a hypoplastic cerebellar vermis. doi: . /journal.pone. .g exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e temporal regions typically begin by months of age and are accompanied by focal or generalized seizures that can manifest as dystonic posturing, drop attacks, myoclonic jerks, or generalized tonic-clonic events. multiple intractable seizure types can afflict an individual patient. physical anomalies found in some patients include subglottic stenosis, aortic stenosis, bicuspid aortic valve, umbilical hernia, and hydrocele. usher syndrome. growth and development are normal during infancy. visual impairment becomes evident during early childhood with the emergence of fine horizontal nystagmus, light aversion, and optic pallor. as vision deteriorates, fundoscopic exam reveals marked attenuation of retinal arteries and veins, pigmentary changes and a cellophane-like reflex that produce ‘‘bull’s eye’’ maculae and diffuse pigmentary stippling of the peripheral retinae, consistent with retinitis pigmentosa. this constellation suggests a combination of optic nerve disease, retinal dystrophy, and cone dysfunction. patients are typically blind by the second or third decade of life but the pace of visual deterioration is highly variable. we do not have auditory data from affected newborns, but some auditory function is present during infancy and deteriorates during early childhood; all five evoked auditory waveforms are absent by age . amplifiers or cochlear implants can partially restore hearing. patients have delayed gross motor development, hyperactive patellar tendon reflexes, mild truncal ataxia, and a wide-based gait. in contrast, upper limb coordination (allowing for visual impairment) and reflexes, peripheral nerve function, strength, tone, and intelligence are normal. based on the current classification scheme, this condition is most consistent with the type iii variant of usher syndrome, which is characterized by progressive vision and hearing loss during early childhood years. infectious illnesses may provoke vivid visual hallucinations (the charles bonnet syndrome). these attacks begin during early childhood and may be accompanied by nonsensical speech, inappropriate laughter, repetitive eye blinking, or psychomotor agitation. in one case, acute psychosis merged into a deep catatonia that lasted several days. hallucinations typically respond to anti-psychotic medications (e.g. haloperidol, thorazine) and are sometimes associated with transient myopathy (elevated serum creatine kinase). rarely, children die suddenly and unexpectedly during an illness. these are presumably cardiac events, but routine figure . symptomatic epilepsy and skull dysplasia due to a homozygous snip mutation. (a) two affected brothers presented with severe psychomotor delay, intractable seizures, bulbous nose, wide mouth and tongue, broad jaw with protuberant angles, short hands, short tapered fingers, and broad thumbs. (b,c) brain mri (b, axial t ; c, coronal t ) mri showed enlarged ventricles, a thin corpus callosum, hypomyelination, and an irregular, undulating skull surface. (d) mouse flag-snip (wt) fusion protein, when transiently overexpressed in mimcd cells, localizes to the nucleus in a punctate pattern consistent with transcriptional complexes. (e) mouse flag-snip (p.glu gly) localizes to the nucleus, but with a more aggregated distribution. (f) top – reverse-transcriptase pcr from three wild-type msnip -transfected samples and four c. a.g msnip -transfected samples. msnip amplicon – bp. mgapdh (loading control) – bp amplicon. bottom – western blot of lysates from mimcd cells transiently transfected with wt (lanes wt & ) or c. a.g (lanes p.glu gly & ) msnip displaying the flag-msnip fusion protein at , kda. the -kda non-specific band was used as a loading control. data shown are two out of four replicate sets of transfections. doi: . /journal.pone. .g exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e electrocardiogram and -hour holter monitor results have been normal. genetic mapping we chose to study patients representing the seven discrete phenotypes described above (table ). all patients were originally genotyped using , ( k) and/or , ( k) single nucleotide polymorphism (snp) microarrays as previously de- scribed [ , , , , , , ]. using between and samples, we genetically mapped five disorders using k snp arrays; four of these were localized with ease using multiple affected children from separate sibships (figure , panels e–h). for two conditions, a shared homozygous block could only be identified at k snp resolution (see below). shared autozygous blocks were an average of . mb (range . – . mb) and contained a mean of (range – ) genes. the genetic mapping studies are summarized in table . for infantile parkinsonism-dystonia syndrome, the proband was one of the first patients to be examined at the csc nearly years ago. a similarly affected sister died, but a lymphoblastoid cell line was available for study. no other affected individuals were known in the mennonite population. dna samples were isolated from the proband, her parents, six unaffected siblings, and her deceased sister’s cell line. autozygosity mapping of the two affected individuals identified a single, large block of homozygosity on chromosome (figure , panel a, yellow peak). analysis of the unaffected siblings excluded this homozygous block, but identified genomic regions greater than mb in size consistent with linkage in the family (figure , panel a, red peaks, and panel b). none of these regions had large homozygous blocks at k snp resolution. three affected individuals from three separate sibships with lethal neonatal rigidity and multifocal seizure syndrome were genotyped figure . genetic mapping of seven plain disorders. the results of autozygosity mapping using affymetrix genechip k or k snp microarrays are plotted for each disorder. the x-axis depicts chromosomal location on autosomes. yellow peaks represent the number of contiguous homozygous snps shared by affected individuals and the purple peaks depict location scores. (a) autozygosity mapping of two affected individuals identified a single, large block of homozygosity on chromosome (yellow peak). genotyping of unaffected siblings excluded this homozygous block, but identified genomic regions greater than mb in size (red peaks) that were consistent with linkage in the family. (b) list of genomic regions consistent with linkage in the single nuclear family with infantile parkinsonism-dystonia syndrome. panels c–h provide mapping plots for the other disorders. for two disorders (c,d), k microarrays were used after k microarrays failed to unequivocally localize the disease gene. the other four disorders (e–h) were mapped with k microarrays. doi: . /journal.pone. .g table . genetic mapping summary. disease sample size chr snp start snp stop size (mb) genes infantile parkinsonism-dystonia many – – – – lethal neonatal rigidity and seizure syndrome rs rs . mental retardation, non-syndromic rs rs . microcephaly with chorioretinopathy rs rs . posterior column ataxia with retinitis pigmentosa rs rs . symptomatic epilepsy and skull dysplasia rs rs . usher syndrome rs rs . doi: . /journal.pone. .t exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e t a b le . e x o m e v a ri a n t s u m m a ry . a v e ra g e a u to s o m a l v a ri a n ts p e r s a m p le in fa n ti le p a rk in s o n is m - d y s to n ia s y n d ro m e (n = ) l e th a l n e o n a ta l ri g id it y a n d m u lt if o c a l e p il e p s y (n = ) m ic ro c e p h a ly w it h c h o ri o re ti n o p a th y (n = ) n o n -s y n d ro m ic m e n ta l re ta rd a ti o n (n = ) p o s te ri o r c o lu m n a ta x ia a n d re ti n it is p ig m e n to s a (n = ) s y m p to m a ti c e p il e p s y a n d s k u ll d y s p la s ia (n = ) u s h e r s y n d ro m e (n = ) n o v e l a n d h o m o z y g o u s s n p s s n p s in m a p p e d in te rv a l s n p s in m a p p e d in te rv a l s n p s in m a p p e d in te rv a l s n p s in m a p p e d in te rv a l s n p s in m a p p e d in te rv a l s n p s in m a p p e d in te rv a l t o ta l n o v e l n o v e l a n d h o m o z y g o u s t o ta l l in k e d in te rv a ls t o ta l n o v e l t o ta l n o v e l t o ta l n o v e l t o ta l n o v e l t o ta l n o v e l t o ta l n o v e l - f la n k in g - u t r - u t r in tr o n m is s e n s e , n o n s e n s e r e a d -t h ro u g h s p li c e s it e s y n o n y m o u s , m ir n a ig r o th e r in d e ls * * * * t o ta l , *n o v e lt y fo r in d e ls m e a su re d a g a in st d b s n p o n ly , a u to m a te d in d e l c a ll e r d o e s n o t d e te rm in e zy g o si ty . d o i: . /j o u rn a l.p o n e . .t exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e originally with k snp arrays. no significant shared blocks of homozygosity were identified. as the patients were from different yet related pennsylvania amish demes, we suspected that the shared homozygous block might be small and thus below the resolution of a k microarray. we subsequently genotyped these patients at k resolution and mapped the disease locus to chromosome p (figure , panel c). in , a microcephalic mennonite baby was evaluated at the csc and thought to have the same disorder first described by victor mckusick in (microcephaly with chorioretinopathy, omim ) [ ]. we were able to locate and genotype four affected individuals from mckusick’s original study as well as another patient related to our index case. genotyping at k snp resolution was required to identify a small . mb shared block of homozygosity in the subtelomeric region of chromosome q (figure , panel d). for both disorders that required higher density arrays, a dearth of snps on the k microarray and high recombination rates in these subtelomeric regions complicated mapping. exome sequencing prior to exome analysis, we sequenced between and candidate genes for each condition and found no pathogenic variants. as defined here and throughout the paper, novelty of dna sequence variants was determined by absence in dbsnp and the genomes project. all putative pathogenic exome variants described below were confirmed by sanger sequencing in the affected individuals used for genetic mapping. in addition, siblings and parents were also sequenced, when available, to confirm appropriate segregation of the allele within the family. we developed an unlabeled probe melting analysis for each putative pathogenic variant and genotyped population-specific controls for these variants. for each disorder, over population-specific chromosomes were screened, the allele frequencies ranged from – . %, and no homozygous controls were identified. the exome variant data are summarized in tables and . infantile parkinsonism-dystonia syndrome. exome sequencing was performed on the single living patient. the data were constrained by first tabulating all homozygous, novel variants. nineteen novel homozygous variants were identified: synonymous, missense, -utr, and splice site (table ). of the potentially pathogenic changes, only one localized to a region consistent with linkage in the family, slc a ivs + g.t. the slc a gene encodes the dopamine transporter, a known cause of infantile parkinsonism-dystonia syndrome (omim ). direct sequencing revealed both parents were heterozygous for this change, both affected individuals were homozygous, and the unaffected siblings were either heterozygous or homozygous wild-type. this confirmed the linkage block that was identified on chromosome by snp genotyping. genotyping of old order mennonite control samples identified no carriers (table ). lethal neonatal rigidity and multifocal seizure syndrome. exome data from two patients revealed nine variants in the mapped interval, but only one, brat c. _ insa, was novel (table ). genotyping by unlabeled probe melting analysis for old order amish control samples identified carriers ( . %)(table ). further confirmatory evidence for the pathogenicity of this variant was provided by two unrelated old order amish infants from different demes in wisconsin and kentucky who had an indistinguishable clinical phenotype and were homozygous for the brat c. _ insa variant. their samples were tested for the brat variant after local physicians contacted our clinic for clinical guidance. microcephaly and chorioretinopathy. exome data from a single affected patient identified sequence variants within the mapped interval. only one was novel, tubgcp c. t.g (table ). an unlabeled probe melting analysis in old order mennonite control samples identified carriers ( . %)(table ). this read-through variant is predicted to incorporate extra amino acids at the c-terminus of tubgcp and/or may accelerate mrna degradation via non-stop mediated decay. non-syndromic mental retardation. exome sequencing of affected individuals identified homozygous sequence variants within the mapped interval. only a missense variant in cradd (c. g.c; p.gly arg) was novel (table ). polyphen- predicted this change to be ‘‘probably damaging’’ (score = . ). an unlabeled probe melting analysis detected carriers among old order mennonite control individuals ( . %) (table ). posterior column ataxia and retinitis pigmentosa. exome data from a single axpc patient revealed coding variants in the mapped interval; were non-synonymous and only one missense variant was novel, flvcr c. a.g (p.asn asp) (table ). polyphen- predicts the p.asn asp change to be ‘‘probably damaging’’ (score = . ). an unlabeled probe melting analysis for this variant detected heterozygotes out of old order mennonite control chromosomes ( . %) (table ). the pathogenicity of this variant and several others in flvcr has recently been confirmed independently by others [ ]. symptomatic epilepsy and skull dysplasia. exome data on three affected individuals from two sibships revealed table . pathogenic variant summary. disease sample size gene gene variant protein variant population-specific allele frequency infantile parkinsonism-dystonia slc a ivs + g.t – . % ( / ) lethal neonatal rigidity and seizure syndrome brat c. _ insa – . % ( / ) mental retardation, non-syndromic cradd c. g.c p.gly arg . % ( / ) microcephaly with chorioretinopathy tubgcp c. t.g p.ter gly . % ( / ) posterior column ataxia with retinitis pigmentosa flvcr c. a.g p.asn asp . % ( / ) symptomatic epilepsy and skull dysplasia snip c. a.g p.glu gly . % ( / ) usher syndrome hars c. a.c p.tyr ser . % ( / ) doi: . /journal.pone. .t exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e figure . overexpression of mouse cradd in mimcd cells. (a) wild-type n-terminal flag-cradd localizes to the cytoplasm and nucleus. (b) mutant n-terminal flag-cradd (p.gly arg) localizes to the cytoplasm and nucleus in a manner that is indistinguishable from the wild-type localization. (c) western blot of lysates from mimcd cells transfected with wt flag-cradd and wt v -mpidd, wt v -mpidd, or flag-cradd (p.gly arg) and wt v -mpidd. blot was labeled with anti-v monoclonal antibody. note that full-length v -mpidd ( . kda) was cleaved into a . kda n-terminal fragment, suggesting normal autocatalytic activity. (d) co-transfection of flag-cradd (wt) and wt v -pidd death domain (dd) results in uniform colocalization of the fusion proteins throughout the cytoplasm and nucleus (yellow). (e) overexpressed v -mpidd dd (wt) localizes to the cytoplasm and the nucleus. (f) co-overexpression of flag-cradd (p.gly arg) with v -mpidd dd (wt) results in dense aggregations of flag- cradd in the cytoplasm. (g) co-transfection of flag-mcradd (wt) and v -mpidd (wt) results in relatively uniform colocalization of the fusion proteins throughout the cytoplasm (yellow); punctate flag-mcradd aggregation is also evident (green). flag-mcradd (wt) localizes to the nucleus (green), v -mpidd does not (red). (h) overexpressed v -mpidd (wt) localizes to the cytoplasm as previously reported (berube et al., ). (i) co- overexpression of flag-mcradd (p. gly arg) with v -mpidd (wt) results in dense aggregations of flag-mcradd in the cytoplasm, decreased mcradd:mpidd colocalization, and a relative loss of localization of mcradd to the nucleus. green fluorescence – anti-flag m antibody ( : ); red fluorescence – anti-v antibody ( : ); blue fluorescence – dapi-labeled nuclei ( . mg/ml); western blots – anti-flag m antibody ( : ). (j) co- immunoprecipitation (co-ip) of v -tagged mouse pidd death domain (dd) with flag-tagged mouse cradd dd in mimcd cells. wild-type flag- cradd dd co-immunoprecipitates wild-type pidd dd; the flag-cradd dd p.gly arg variant does not. the upper blots were labeled with the anti- flag m antibody ( : ). the lower blots of the same lysates/eluates were labeled with the anti-v antibody ( : ). doi: . /journal.pone. .g exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e homozygous variants in the mapped region. of these, only one was novel, snip c. a.g (p.glu gly) (table ). polyphen- predicts the p.glu gly substitution to be ‘‘probably damaging’’ (score = . ). an unlabeled probe melting analysis for this variant identified carriers among old order amish controls ( . %) (table ). usher syndrome. exome data from two patients revealed homozygous variants within the mapped interval. only one variant, hars c. a.c (p.tyr ser), was novel (table ). unlabeled probe melting analysis detected carriers among old order amish controls ( . %) (table ). further evidence for pathogenicity was provided by an old order amish patient who was from an unrelated deme in ontario, canada. this amish settlement arose from a separate migrational event from europe than the lancaster county settlement. the patient had an identical phenotype to our patients and was homozygous for the hars c. a.c variant. in vitro studies the brat gene encodes brca -associated protein required for atm activation- (brat ) [ ]. wild-type human brat displays prominent nuclear and diffuse cytosolic localization (figure , d) and its c-terminus (amino acids – ) interacts with brca [ ]. the c. _ insa change is a frameshift variant that is predicted to alter the amino acid sequence after lys by introducing a stop codon that prematurely truncates the protein at leu ( . kda vs. wild-type . kda) (figure , f), abolishes its nuclear localization, and renders the protein unstable in human arpe- cells (figure , e–g). similar results were obtained with mouse brat (data not shown). abundance of the c. _ insa variant was . . % (n = ) lower than that of wild-type human brat for flag-fusion proteins overexpressed in human arpe- cells (figure , g). these data suggest that the amish variant destabilizes the protein and may disrupt an interaction between brat and brca that is required for nuclear localization of brat . knockdown of brat results in p -induced apoptosis independent of dna damage [ ]. the destabilization of brat observed might underlie the catastroph- ic epilepsy and corticobasal neuronal degeneration observed in affected infants [ , , , , ]. cradd. cradd (aka raidd) is a caspase-recruitment- domain (card) and death domain containing adaptor protein. it links pidd (p -induced protein with death domain) and caspase- in the formation of the piddosome required for caspase- activation during apoptosis [ , , , ]. the cradd c. g.c mutation alters a highly conserved residue (p.gly arg) within the cradd death domain. we overexpressed wild-type and mutant (p.gly arg) murine cradd in mouse inner medullary collecting duct cells (mimcd ) and found no significant difference in protein localization (figure , a– b) [ ]. however, mutant p.gly arg cradd formed large aggregates when co-overexpressed with wild-type pidd (figure , i) or the pidd death domain (figure , f). we found that overexpressed pidd localized to cytosol (figure , h) as described previously [ ] and was autocatalytically cleaved as expected (figure , c) [ ]. these data suggest that the p.gly arg mutation alters one of the interaction surfaces of the cradd death domain to decrease affinity for the pidd death domain and increase homotypic binding to the cradd death domain [ ]. this is further supported by co-immunoprecipitation assays demonstrating that wild-type cradd dd co-immunoprecipitates pidd dd, but cradd dd p.gly arg does not (figure , j). in rat pc- cells and cultured sympathetic neurons, similar perinuclear cradd aggregations are associated with caspase- activation and initiation of apoptosis [ ]. in humans, alteration of caspase- initiated apoptosis (resulting from disruption of the piddosome) during nerve growth factor mediated proliferation of synaptic contacts may lead to inappropriate synaptic pruning that results in cognitive impairment. snip . wild-type snip (smad nuclear interacting protein ) contains an n-terminal nuclear localization signal [ ], bridges c- myc activity with cbp/p activity during development, and competitively inhibits tgf-b and nf-kb signaling [ , ]. snip glu is highly conserved and the p.glu gly mutation is in the c-terminus where snip interacts with c-myc [ ], smad and smad [ , ]. reverse transcriptase-pcr suggests that the snip c. a.g (corresponding to human snip c. a.g) transcript is expressed at levels comparable to wild-type (figure , f). we transiently overexpressed wild-type and mutant snip in mimcd cells. wild-type protein localized to the nucleus with a punctate appearance, consistent with its involvement in transcrip- tional complexes [ , ] (figure , d). however, snip p.glu gly (corresponding to human p.glu gly) had a more aggregated appearance (figure , e) and western blotting proved it unstable (figure , f); its band density was . . % sd lower than wild-type (n = independent transfections each). decreased abundance of snip likely results in decreased c- myc activity and increased tgf-b and nf-kb signaling. disruption of c-myc or cbp/p signaling in mice can result in abnormal development of the brain, skull, craniofacial bones, and distal limbs [ , , , , ]. inappropriate increases in tgf- b/smad signaling and in nf-kb signaling in the mouse brain have been shown to independently initiate epileptogenesis [ , ]. hars. histidyl-trna synthetase (hars/hisrs) is a homodimeric class iia aminoacyl trna synthetase that charges trna with the amino acid histidine [ ]. the p.tyr ser change rests in the interface between the catalytic domain and anticodon binding domain suggesting that it may alter anticodon recognition and/or catalytic activity. overexpression of wild-type and mutant (p.tyr ser) murine hars in mimcd cells did not reveal any significant differences in localization (figure , a– b). in chinese hamster ovary (cho) cell lysates, both wild-type and mutant murine protein dimerized properly with endogenous hars and were expressed at qualitatively similar levels (data not shown). our data suggest that the p.tyr ser change reduces the maximal forward reaction velocity (vmax) of the enzyme for aminoacylation of human trnahis with histidine nearly two-fold (figure , c). discussion next generation sequencing technologies promise to expedite disease gene discovery and allowed us to identify known and novel pathogenic variants in our patients. although costly, exome sequencing is practical as it interrogates the . % of the genome that contains approximately % of pathogenic variants [ , , , , , , , , , , , , , , , , ]. to assess the utility of exome sequencing in an active clinical setting, we selected patient samples representing different genetic conditions. each disorder had previously been mapped to a chromosomal locus and candidate gene sequencing failed to identify the pathogenic variant. for six disorders, an average autozygous block of . mb ( . % of the human genome) contained only one novel homozygous variant and rendered disease gene identification straightforward. even in the case of infantile parkinsonism-dystonia syndrome, for which autozygosity and linkage mapping were only partially informative, a manage- exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e able list of candidate variants was assembled simply by assuming mutation homogeneity. prior mapping data and thorough knowledge of the patient implicated a single variant from this list (slc a ivs + g.t). for four of the conditions, more than one affected individual was available for exome sequencing. even in the absence of mapping data, the identification of the putative pathogenic variant would still have been unambiguous. when we examined the shared, novel homozygous variants in affected individuals, we found one, and only one, that was not homozygous in any other (unaffected) individuals in the study. thus, the assumption of mutation homogeneity obviates the need for snp genotyping and mapping; we reach the same conclusion by exome sequencing of multiple affected individuals without the added time and expense of snp genotyping. the number of novel homozygous variants in each individual was surprisingly small. average inbreeding coefficients of % and . % in the lancaster amish and mennonite populations, respectively, suggested that a small but significant fraction of variation will be homozygous. on average, we found only novel homozygous variants per sample across the exome. of these variants, only were predicted to be potentially pathogenic (missense, nonsense, splice site). this represents . % of all novel variants per exome. for the two disorders where a singleton was sequenced, we identified only potentially pathogenic novel variants which were homozygous in the patient but in no other figure . overexpression of mouse hars in mimcd cells. (a) wild-type n-terminal flag-hars localizes to the cytoplasm. (b) mutant n- terminal flag-hars (p.tyr ser) localizes to the cytoplasm in a manner that is indistinguishable from the wild-type localization shown in a. transfected and non-transfected cells were labeled with anti-flag m monoclonal antibody and alexafluor -conjugated anti-mouse igg (green fluorescence). (c) reaction velocity vs. human trna his concentration for histidine aminoacylation of trna his by wild-type murine hars (hars) and p.tyr ser (y s) hars. scale bar = mm in a and b. doi: . /journal.pone. .g exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e samples. since our total sample size was small ( individuals), we expect that future studies which leverage accumulated exome data will allow us to sequence single individuals to identify rare, uniquely homozygous pathogenic variants. in the outbred population, a strategy that scans for homozygosity or compound heterozygosity for novel variants in the same gene should yield equally manageable candidate gene lists. among the fifteen individuals studied, we found different novel autosomal sequence variants, roughly % of which have pathogenic potential. we infer that . % of these variants were non-pathogenic changes as they were homozygous in one or more unaffected individuals. as more amish and mennonite exomes are analyzed within a clear clinical context, our ability to determine pathogenicity will improve. the exome data also provided a broader view of the genetic disease burden within these populations. we have catalogued known pathogenic sequence variants within the plain populations that should be detectable by exome sequencing. of these, were represented in at least one individual. on average, each individual harbored . known plain pathogenic alleles (range, – ). we also compared our exome results against the human gene mutation database (hgmd) [ ]. carrier status for hgmd mutations, that cause phenotypes not yet encountered in the plain populations, was detected in our patients. these data permit us to generate a more comprehensive molecular differential diagnosis list when faced with a new clinical phenotype. it is notable that several hgmd- dm mutations were homozygous in one or more patients, casting some doubt on the pathogenicity of these variants. similar results have been reported elsewhere and highlight the need for better curation of mutation databases [ ]. critics will argue that we have failed to exclude all possible variants due to incomplete coverage. our sequencing metrics show excellent, albeit incomplete, exome coverage. on average, . % and . % of the targeted exome was sequenced to a depth of and , respectively. while this is a potential hazard, our study design minimized this risk. prior mapping analyses narrowed the focus to a vanishingly small . % of the genome. within these mapped regions, we discovered only one novel homozygous variant. this is significantly better coverage and stronger evidence than we and others have demonstrated for disease gene identification prior to the advent of exome sequencing. addition- ally, our snp filtering strategies might be questioned; dbsnp is polluted with many pathogenic variants and their numbers continue to grow as data accrues. our current conservative strategy used dbsnp and the genomes project to filter exome variants. the risk to our analyses is relatively small since we study very rare and highly penetrant alleles. nonetheless, in our population and elsewhere, local population-specific variant databases will prove most useful for inferring pathogenicity. pathogenicity is difficult to prove, but for four conditions we provide ample functional data to demonstrate abrogation of protein function. in vitro studies of protein localization and function in mammalian cells provide further confirmation that the homozygous variants identified were indeed pathogenic. the predicted consequence of the brat c. _ insa frameshift variant is a truncated protein at amino acid position . overexpression of this truncated protein abolished nuclear localization and demonstrated protein instability ( . % decrease relative to wild-type). an alternative disease mechanism, nonsense- mediated mrna decay, has not been investigated. others have shown that knockdown of brat results in p -induced apoptosis [ ]. this is consistent with the neurodegeneration observed in patients with the brat variant. when transfected into mouse imcd cells, the mouse counterpart of the cradd c. g.c variant disrupts interaction with mouse pidd (it’s normal binding partner) and forms dense aggregates when co- expressed with wild-type mouse pidd. this is in contrast to a pattern of uniform colocalization of wild-type cradd and pidd throughout the cytoplasm and nucleus. mouse snip protein, when transiently overexpressed in imcd cells, localizes to the nucleus in a punctate pattern consistent with transcriptional complexes, while mutant mouse snip (p.glu gly, correspond- ing to human p.glu gly) localizes to the nucleus, but with a more aggregated distribution. western blotting proved this structure unstable. for the hars c. a.c variant, we demonstrate that reaction velocity (vmax) for aminoacylation of human trnahis with histidine is reduced nearly two-fold. while these studies cannot prove pathogenicity beyond a shadow of doubt, the totality of evidence is compelling and strongly suggests that we have identified the disease-causing alleles. the association between flvcr and slc a variants and disease has previously been established. we provide no further evidence for pathogenicity of the tubgcp c. t.g variant. however, the primary micro- cephalies result from disruption of the centrosomal complex during mitosis, reducing the neural progenitor pool during development ( – ). centrosomal proteins such as cdk rap and cenpj/cpap interact with the c-tubulin ring complex (c- turc) to regulate microtubule nucleation ( – ). tubgcp (gcp ) is a component of the human c-turc ( ), where it is required for cdk rap to activate microtubule nucleation ( ). we predict that defects in tubgcp , cdk rap and cenpj cause primary microcephaly by similar mechanisms. all seven variants described in this paper interfere with neurological development. functional studies of brat and snip will expand our understanding of epilepsy and also deepen our knowledge of dna damage repair and transcriptional regulation in cortical development and neuronal survival. selective degeneration of photoreceptors and dorsal column afferents caused by flvcr mutations suggest an unusual vulnerability of these cells to deranged heme transport, or may reveal an altogether different function of the flvcr protein. despite its ubiquitous expression, a defect in aminoacylation by hars selectively damages elements of afferent sensory systems and, by unknown mechanisms, predisposes to episodic psychosis and sudden death. abnormalities of neuroblast proliferation and migration caused by tubgcp mutations fit nicely with our existing knowledge about centrosomal complexes, microtubular arrays, and cortical growth, but also introduce new questions about the diverse brain morphologies linked to various specific tubulin-associated proteins and the role of these proteins in early eye development. finally, further studies on the connection between cradd and general intelligence will certainly change our understanding of the microanatomical and molecular bases of cognition. small focused studies as described herein will be a steady engine of progress for understanding the specific connec- tions between genes and the human brain. ultimately, genomics can only shape medical practice within the context of regional particulars and clinical facts. our local, patient- and family-based approach to gene discovery stands in stark contrast to the prevailing model of genomic research, where the people who produce genotype data are frequently separated from those who collect and analyze clinical facts, and both struggle to translate genetic knowledge into primary care. although we are focused on specific regional populations ( , ), these studies reveal concepts of broad biological and economic relevance ( , ). the discovery of rare, highly penetrant alleles among small social groups may prove more useful than large genome-wide association studies exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e for revealing the basic genetic foundations of complex disease, particularly when these alleles can be viewed against a background of population-specific genetic variation. even at current prices, microarray analyses to detect copy number abnormalities coupled with exome sequencing are an order of magnitude cheaper than the standard workup for a complex patient at a tertiary medical center. thoughtful and appropriately scaled application of these genetic technologies to other regional populations should yield similar economic and clinical benefits in the years ahead. materials and methods the study was approved by institutional review boards at lancaster general hospital and the broad institute. all parents consented to participation. patients were from old order amish or mennonite populations, received medical care at the clinic for special children (csc), and presented with a distinctive recessive clinical phenotype (table ). genetic mapping genetic mapping of the seven disorders presented in this study was performed as previously described [ , , , , , , ] with the exception that two disorders required higher density arrays (affymetrix k) to establish chromosomal location. single- nucleotide polymorphism (snp) genotyping was performed with the genechip mapping k and k assay kits (affymetrix, santa clara, ca, usa) as previously described. data were analyzed in microsoft excel spreadsheets (microsoft corporation, redmond, wa, usa) that were custom formatted at the clinic for special children. snp positions came from affymetrix genome annotation files and genotype data came from the affymetrix genechip human mapping k xba and k xba arrays. data analyses were designed for rapid identification of genomic regions that were identically homozygous between all affected individuals. these analyses assumed mutation and locus homogeneity. two- point lod scores were calculated for each genotyped snp using an approach similar to broman and weber [ ]. cumulative two-point lod scores for blocks of homozygous snps were considered the location score for that region, providing a relative measure that a specific homozygous block harbored the disease gene. genotype data from healthy population-specific (amish or mennonite) females were used for estimation of snp allele frequencies. exome sequencing exome sequencing was performed using the agilent sureselect all exon kit (v. , mb) as previously described [ ]. a solution hybrid selection methodology was used to isolate exomic dna, which was subjected to sequencing on the illumina ga-ii platform. briefly, dna oligonucleotides, corresponding to bp of target sequence flanked by bp of universal primer sequence, were synthesized in parallel on an agilent microarray, then cleaved from the array. the oligonucleotides were pcr amplified, then transcribed in vitro in the presence of biotinylated utp to generate single-stranded rna ‘‘bait.’’ genomic dna was sheared, ligated to illumina sequencing adapters, and selected for lengths between – bp. this ‘‘pond’’ of dna was hybridized with an excess of bait in solution. the ‘‘catch’’ was pulled down by magnetic beads coated with streptavidin, then eluted, and sequenced on the illumina ga-ii. massively parallel sequencing data were processed by the sequencing platform at the broad institute using two consecutive pipelines. the first pipeline, called ‘‘picard’’, utilized the reads and qualities produced by the illumina software for all lanes and libraries and produced a single bam file (http://samtools. sourceforge.net/sam .pdf) representing a sample. the final bam file stored all reads with well-calibrated qualities and alignments to the genome. the second pipeline, called genome sequencing analysis, then performed post-processing and analysis of the data including snp identification, small insertion and deletion identification, local realignment of insertion or deletion containing reads, gene annotation and filtering with common polymorphisms ( genomes, dbsnp build ). the details of our sequencing data processing have been described elsewhere [ ]. inter-exome analyses were performed by importing variant call data into a filemaker pro database (filemaker, inc., santa clara, ca) and querying mapped intervals for novel variants shared among affected individuals. candidate disease gene cloning total rna extracted with trizol (invitrogen, carlsbad, ca) from wild-type mouse tissues, cultured mouse imcd cells, or human arpe cells was reverse transcribed with superscript ii reverse transcriptase (invitrogen). gene specific primers were used to amplify full-length cdna (coding sequence, cds) for the candidate disease genes by the polymerase chain reaction (pcr). mouse pidd was amplified from pcdna . ( )/mpidd (a gift from s. benchimol, york university, toronto, ontario, canada). constructs were ligated into d-topo gateway entry vectors (invitrogen) for cloning, and site-directed mutagenesis (quik- change ii, agilent, santa clara, ca) was used to introduce the mutations discovered by exome sequencing. wild-type and mutant constructs (confirmed by sanger sequencing of the full-length inserts) were subcloned into pcag/flag/rfc/a and/or pcag/v /rfc/a (gift of q. zhang and e.a. pierce, university of pennsylvania, philadelphia, pa) for expression of n-terminal flag- and/or v -fusion proteins in mammalian cells. immunohistochemistry for protein localization studies, mimcd cells grown to – % confluence on glass coverslips in six-well plates were transfected with flag and/or v -fusion protein expression vector plasmid dna using lipofectamine (invitrogen). cells were grown for – h post-transfection and then fixed for min in % paraformaldehyde in phosphate buffered saline (pbs), permeabilized for min with . % triton x- in pbs, blocked for min with % bovine serum albumin in pbs with . % triton x- , and incubated with primary antibodies for h at room temperature (rt). following a wash in pbs, cells were incubated in secondary antibodies for h at rt. nuclei were counterstained with dapi ( , -diamidino- -phenylindole – . mg/ml; santa cruz biotechnology, santa cruz, ca) and coverslips were mounted with fluoromount g (electron micros- copy sciences, ft. washington, pa). fluorescent images were captured with a leica dm rb microscope. primary antibodies – monoclonal anti-flag m igg ( : ; sigma, st. louis, mo) and/or monoclonal anti-v igg a ( : ; invitrogen). secondary antibodies – dylight -conjugated goat anti-mouse igg ( : ; jackson immunoresearch, west grove, pa), alexafluor - conjugated goat anti-mouse igg ( : ), and/or alexafluor - conjugated goat anti-mouse igg a ( : ; invitrogen). western blotting, co-immunoprecipitation, and rt-pcr western blotting was used to assess relative protein overexpres- sion levels. briefly, % confluent mimcd cells in six-well or -cm plates transfected as described above were lysed in either lds sample buffer (invitrogen) or in lysis buffer (cellyticm, sigma) containing protease inhibitor and phosphatase inhibitor cocktails (roche applied science, indianapolis, in). lysates were exome sequencing and rare monogenic diseases plos one | www.plosone.org january | volume | issue | e sonicated briefly and cleared by centrifugation at rpm for – min. following sds-page, proteins were blotted to nitrocellulose, and the membranes were blocked for h at rt with % nonfat dry milk in tris buffered saline (tbs) with . % tween- . membranes were then incubated with primary antibody (anti-flag m – : or anti-v – : ) overnight at uc, washed in tbs with . % tween- , and then incubated with secondary antibodies for h at rt. secondary antibodies – horseradish peroxidase (hrp)- conjugated goat anti-mouse igg ( : ) and hrp-conjugated anti-biotin ( : ). proteins were detected with enhanced chemiluminescence using lumiglo (cell signaling technology, danvers, ma) and chemiluminescence signals were imaged on biomax light film (kodak, rochester, ny). band densities were measured with imagej (http://rsbweb. nih.gov/ij/) following calibration with a nist calibrated step tablet (kodak ek ). co-immunoprecipitation was carried out using the flag-m affinity resin according to the manufacturer’s protocols (sigma cellmm /f ) with the following exception: cleared lysates were incubated at uc for h prior to overnight incubation on the resin. reverse transcriptase-pcr was used to assess relative transcript levels for genes overexpressed in cultured mimcd cells. cells were transfected on -cm plates and cultured as described above. total rna was harvested, incubated with dnase ( u/mg rna) at uc for min, and reverse transcribed as described above. a flag-tag specific forward primer and gene specific reverse primers were used to determine the transcript levels for the overexpressed gene relative to endogenous gapdh or b-actin expression using – cycles of pcr. total transcript levels (endogenous+overexpressed) for the gene of interest were deter- mined with gene specific primer pairs for short ( – bp) amplicons relative to gapdh expression. aminoacylation assays murine hars and the variant p.tyr ser hars were expressed in chinese hamster ovary (cho) cells as flag-tagged enzymes and affinity purified. enzymes were stored at uc in mm tris hcl, ph . , mm nacl, % glycerol at a concentration of mm and diluted to mm in cold reaction buffer ( mm hepes ph . , mm kcl, mm mgcl and mm dtt) just before use. human trnahis, which has an identical sequence to murine trnahis, was prepared from human placenta as described, with minor modifications [ ]. isolated placental rna was stored at uc in mm na cacodylate (ph . ). this preparation yields approximately mg rna/g tissue, of which . % ( . pmol/a unit) can be aminoacy- lated with histidine by murine hars. kinetics assay enzyme assays were performed under steady state conditions for atp and histidine, with the concentration of trna varied from approximately . to km, as previously described [ , ]. briefly, atp ( . mm), [ h] histidine mixed with histidine ( mm, % radiolabeled), pyrophosphatase ( ng/ml) and buffer ( mm hepes ph . , mm kcl, mm mgcl and mm dtt) were mixed with placental trna ( . to mm, of which . % could be histidylated). the reaction was initiated by addition of enzyme ( nm), and quenched by spotting on filter paper pre-soaked with % trichloroacetic acid. after washing away unbound radiolabeled histidine, the histidy- lated trna was quantified by scintillation counting. the time course reactions were analyzed using kintek explorer software, yielding a velocity versus substrate curve from which kinetic parameters were determined. acknowledgments we acknowledge the broad biological samples, genotyping, and sequencing platform for excellent data 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) core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core articles the spirituality of reform in the late medieval church: the example of nicolas de clamanges christopher m. bellitto where was your church before luther? claims for the antiquity of protestantism examined s. j. barnett what children did not learn in school: the intellectual quickening of young americans in the nineteenth century r. laurence moore lending a hand to labor: james myers and the federal council of churches, - elizabeth fones-wolf and ken fones-wolf putting the amish to work: mennonites and the amish culture market, - david weaver-zercher books review essays africans' religions in british america, - jon butler religious goods elizabeth mckeown book reviews and notes brooten, bernadette j., love between women: early christian responses to female homoeroticism david g. hunter jefford, clayton n. with kenneth j. harder and louis d. amezaga jr., reading the apostolic fathers: an introduction kenneth d. snyder petersen, joan m., trans, and ed., handmaids of the lord: holy women in late antiquity and the early middle ages caroline t. schroeder amidon, philip r., s.j., trans., the church history ofrufinus of aquileia: books and william g. rusch stewart, columba, cassian the monk david brakke long, marceau and francois monnier, eds., la france, i'eglise quinze siecles deja thomas kselman somerville, robert and bruce c. brasington, comm. and trans., prefaces to canon law books in latin christianity: selected translations, — kathleen g. cushing markus, r. a., gregory the great and his world carole straw daley, brian e., s.j., trans, and intro., on the dormition of mary: early patristic homilies stephen j. shoemaker core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core backus, irena, ed., the reception of the church fathers in the west: from the carolingians to the maurists mickey l. mattox venarde, bruce l., women's monasticism and medieval society: nunneries in france and england, - catherine peyroux watt, d., ed., medieval women in their communities constance h. berman antonopoulou, theodora, the homilies of the emperor leo vi z. j. kosztolnyik phillips, jonathan, ed., the first crusade: origins and impact george beech coureas, nicholas, the latin church in cyprus, - charles a. frazee gilmour-bryston, anne, inrro. and trans., the trial of the templars in cyprus: a compete english edition james m. powell dale, thomas e. a., relics, prayer, and politics in medieval venetia: romanesque painting in the crypt of aquileia cathedral meredith j. gill tyerman, christopher, the invention of the crusades james a. brundage housley, norman, ed. and trans., documents on the later crusades, - priscilla baumann da milano, fra nicola, m. michele mulchahey, ed., collationes de beata virgine: a cycle of preaching in the dominican congregation of the blessed virgin mary at imola, - gavin ferriby lansing, carol, power and purity: cathar heresy in medieval italy maureen c. miller allmand, christopher, ed., the new cambridge medieval history. vol. : c. -c. marcia l. colish kieckhefer, richard, forbidden rites: a necromancer's manual of the fifteenth century jeffrey burton russell watt, diane, secretaries of god: women prophets in late medieval and early modern england nicholas watson oliva, marilyn, the convent and the community in late medieval england sharon k. elkins french, katherine l., gary g. gibbs, and beat a. kumin, eds., the parish in english life, - dan g. danner parker, douglas h., the praier and complaynte of the ploweman unto christe john r. griffin muir, edward, ritual in early modern europe todd e. johnson burns, j. h. and thomas m. izbicki, eds., conciliarism and papalism francis oakley wengert, timothy j., human freedom, christian righteousness: philip melanchthon's exegetical dispute with erasmus of rotterdam erikarummel den hollander, a. a., de nederlandse bijbelvertalinge, - irvinb. eorst marissen, michael, lutheranism, anti-judaism and bach's "st. john passion" kyle c. sessions de leon-jones, karen silvia, giordano bruno and the kabbalah: prophets, magicians, and rabbis rebecca moore whiting, robert, local responses to the english reformation stanford lehmberg ogier, d. m., reformation and society in guernsey matthew pinson mccullough, peter e., sermons at court: politics and religion in elizabethan and jacobean preaching susan wabuda doerksen, daniel w., conforming to the word: herbert, donne, and the english church before laud annabelles. wenzke maltby, judith, prayer book and people in elizabethan and early stuart england peter iver kaufman cope, esther s., ed., prophetic writings of lady eleanor davies teresa feroli worcester, thomas, seventeenth-century cultural discourse: france and the preaching of bishop camus james c. deming core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core greaves, richard l., dublin's merchant-quaker: anthony sharp and the community of friends, - marcella biro barton jago, judith, aspects of the georgian church: visitation studies of the diocese of york, - myron c. noonkester hindmarsh, d. bruce, john newton and the english evangelical tradition between the conversions of wesley and wilberforce w. r. ward mcleod, hugh, piety and poverty: working-class religion in berlin, london and new york, - david martin davie, martin, british quaker theology since thomas d. hamm swatos, william h., jr. and loftur reimar gissurarson, icelandic spiritualism: mediumship and modernity in iceland s. k. johannesen heschel, susannah, abraham geiger and the jewish jesus arnold eisen melloni, a., ed., vatican ii in moscow ( - ): acts of the colloquium on the history of vatican ii moscow, march -april , john d. basil reese, thomas ]., inside the vatican: the politics and organization of the catholic church sandra yocum mize fulop, timothy e., and albert j. raboteau, eds., african-american religion: interpretive essays in history and culture dennis c. dickerson kuklick, bruce and d. g. hart, eds., religious advocacy and american history chris r. armstrong shriver, george h. and bill j. leonard, eds., encyclopedia of religious controversies in the united states lisa j. pruitt claghorn, george s., ed., the works of jonathan edwards. vol. : letters and personal writings wayne s. hansen carroll, bret e., spiritualism in antebellum america stephen prothero startup, kenneth moore, the root of all evil: the protestant clergy and the economic mind of the old south kurt o. berends sarna, jonathan d., ed., minority faiths and the american protestant mainstream mary farrell bednarowski hamstra, sam, jr. and arie j. griffioen, eds., reformed confessionalism in nineteenth-century america: essays on the thought of john williamson nevin paul westermeyer bealle, john, public worship, private faith: sacred harp and american folksong daniel w. patterson flynt, wayne, alabama baptists: southern baptists in the heart of dixie george h. shriver launius, roger d. and linda thatcher, eds., differing visions: dissenters in mormon history douglas d. alder wider, sarah ann, anna tilden: unitarian culture and the problem of self-representation margaret bendroth albanese, catherine l., a cobbler's universe: religion, poetry and performance in the life of a south italian immigrant david l. salvaterra weisenfeld, judith, african american women and christian activism: new york's black ywca, - cecilia a. moore wilson, charles reagan, judgment and grace in dixie: southern faiths from faulkner to elvis beth barton schweiger siry, joseph m., unity temple: frank lloyd wright and architecture for liberal religion randall balmer hawkins, merrill m., jr., will campbell: radical prophet of the south peter a. huff harding, vincent, martin luther king: the inconvenient hero dwight n. hopkins friedland, michael b., lift up your voice like a trumpet: white clergy and the civil rights and antiwar movements, - james findlay core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core merton, thomas, and patrick hart, o.c.s.o., ed., the other side of the mountain: the end of the journey mark s. massa aldridge, marion d. and kevin lewis, eds., the changing shape of protestantism in the south michael e. williams sr. wright, stuart a., ed., armageddon in waco: critical perspectives on the branch davidian conflict richard kyle demerath, n. j., ill, peter dobkin hall, terry schmitt, and rhys h. williams, eds., sacred companies: organizational aspects of religion and religious aspects of organizations r. stephen warner janz, denis r., world christianity and marxism daniel m. bell jr. mckenna, joseph c , finding a social voice: the church and marxism in africa ogbu u. kalu megged, amos, exporting the catholic reformation: local religion in early-colonial mexico carlos m. n. eire drogus, carol ann, women, religion, and social change in brazil's popular church anna l. peterson fletcher, jesse c , bill wallace of china gary j. bekker gilhus, ingvild saelid, laughing gods, weeping virgins: laughter in the history of religion john corrigan coakley, sarah, ed., religion and the body amy hollywood kienzle, beverly mayne and pamela j. walker, eds., women preachers and prophets through two millennia of christianity catherine a. brekus johnson, elizabeth a., friends and prophets: a feminist theological reading of the communion of saints jill raitt kyle, richard, the last days are here again: a history of the end times stephen j. stein collinge, william j., historical dictionary of catholicism richard gribble, c.s.c. mcgonigle, thomas d. and quigley, james f., a history of the christian tradition: from the reformation to the present robert f. rea pellicia, guerrino and giancarlo rocca, eds., dizionario degli istituti di perfezione. vol. : saba-spiritualii pellicia, guerrino and giancarlo rocca, eds., dizionario degli istituti di perfezione. vol. : spiritualita-vezelay bernard mcginn bendix, regina, in search of authenticity: the formation of folklore studies seanmccloud young, lawrence a., ed., rational choice theory and religion: summary and assessment lynn robinson books received core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ assessment and treatment of post patent ductus arteriosus ligation syndrome assessment and treatment of post patent ductus arteriosus ligation syndrome afif f. el-khuffash, md, dce, frcpi , amish jain, md , , , dany weisz, md , luc mertens, md , and patrick j. mcnamara, md, mrcpch , , , objective to compare differences in tissue doppler imaging, global longitudinal strain (gls), and cardiac troponin t (ctnt) between infants with low (< ml/kg/min) and high (> ml/kg/min) left ventricular (lv) output hour after duct ligation and assess the impact of milrinone treatment on cardiac output and myocardial performance. study design lv function was assessed preoperatively and and hours postoperatively. infants were catego- rized into a low-output or a normal-output group based on the echocardiographic assessment of lv output at hour. results thirty infants with a mean gestation of . weeks were enrolled. lv basal lateral s , basal septal s , and basal right ventricular s were lower in the low-output group (n = ) at hour postoperatively, with no significant difference in gls (low-output � . % vs high-output � . %, p >. ) or ctnt between the groups. patients in the low-output group were treated with milrinone, and by hours lv performance recovered to levels comparable with the high output group. ctnt values increased at hours in the whole cohort with no significant difference between the groups. conclusion tissue doppler imaging and gls provide novel insights and further characterization of myocardial performance immediately after patent ductus arteriosus ligation. a reduction in tissue doppler-derived lv systolic velocity may further help in monitoring cardiac performance after patent ductus arteriosus ligation and for monitoring the effects of treatment. (j pediatr ;-:---). t he decision to undergo patent ductus arteriosus (pda) ligation is subject to much debate and controversy, in part related to the high morbidity in survivors less than g. postligation cardiac syndrome (plcs) complicates the postoperative course in %- % of preterm infants. plcs is a clinical entity characterized by hypotension requiring inotropic support, and/or ventilation/oxygenation failure, usually occurring - hours after ligation. , several studies have investigated the physiologic mechanisms contributing to cardiorespiratory instability after pda ligation and demonstrated a decrease in shortening fraction and ejection fraction associated with an increase in systemic vascular resistance (svr) and a sudden reduction in preload. , the sudden increase in svr is not well tolerated by the preterm myocardium. , our group recently demonstrated that a left ventricular output (lvo) < ml/kg/min on echocardiography evaluation hour after ligation is predictive of developing plcs over the subsequent few hours. early administration of milrinone to infants with low lvo reduces the incidence of plcs from % to %. conventional echocardiographic measures of left ventricular (lv) systolic performance, including fractional shortening and ejection fraction, do not predict plcs. previously, we demonstrated that pda ligation resulted in significant changes in myocardial tissue doppler measures and myocardial strain imaging. the aim of the present study was to characterize lv performance using tissue doppler imaging (tdi) and speckle tracking echocardiography (ste) in neonates with high (> ml/min/kg) vs low (< ml/min/kg) lvo hour after pda ligation. we hypothesized that the tissue doppler and strain measures at hour postoperatively would be lower in infants with low lvo compared with those with a greater lvo. we also performed a longitudinal evaluation of tdi- and ste-derived estimates of lv systolic performance in the group with low lvo treated with milrinone. bp ctnt fio gls lv lvedc lvo map methods from the department of pediatrics, the rotunda hospital, dublin, ireland; department of pediatrics, all preterm neonates with birth weights between and g and gestation between and weeks who were admitted to the hospital for sick children mount sinai hospital; department of physiology, university of toronto; physiology and experimental medicine, hospital for sick children; department of pediatrics, sunnybrook health sciences centre; the labatt family heart center, the hospital for children; and departments of neonatology and pediatrics, the hospital for sick children, toronto, canada funded by the physicians’ services incorporated foun- dation, ontario, canada. the authors declare no conflicts of interest. - /$ - see front matter. copyright ª elsevier inc. all rights reserved. http://dx.doi.org/ . /j.jpeds. . . blood pressure cardiac troponin t fraction of inspired oxygen global longitudinal strain left ventricular lv end-diastolic circumference left ventricular output mean airway pressure pda patent ductus arteriosus plcs postligation cardiac syndrome pwd pulsed-wave doppler rv right ventricular ste speckle tracking echocardiography svr systemic vascular resistance tdi tissue doppler imaging vti velocity time integral delta: _given name delta: _surname delta: _given name delta: _surname delta: _given name http://dx.doi.org/ . /j.jpeds. . . the journal of pediatrics � www.jpeds.com vol. -, no. - for pda ligation were considered eligible for inclusion. infants with major congenital abnormalities, cardiac lesions other than pda, or a patent foramen ovale were excluded from this study. referrals for pda ligation are triaged according to the clinical and echocardiography findings via use of the pda staging system. all infants are anesthetized with fentanyl before the procedure at a dose ranging between and mg. our institutional approach to the postoperative manage- ment of preterm infants after ligation is as follows. a routine echocardiography assessment is performed hour postoper- atively to assess lvo. infants with lvo less than ml/kg/ min are considered at high risk of developing plcs and are started on . mg/kg/min intravenous infusion of milrinone treatment. a bolus of ml/kg of . % saline is coadminis- tered for the first hour to prevent low diastolic blood pressure (bp). infants who develop systolic hypotension (defined as a value less than the third percentile for any given gestation) in the postoperative period were treated with an intravenous infusion of - mg/kg/min dobutamine. infants with diastolic hypotension (defined as a value less than the third percentile for any given gestation) were treated with boluses of . % saline and/or intravenous infusion of - mg/kg/ min dopamine. adrenocorticotropic hormone (cosyntropin) testing was performed on all patients before surgical intervention. intravenous hydrocortisone . mg/kg every hours was administered to all neonates with intractable hypotension and postadrenocorticotropic hormone cortisol < mmol/l despite cardiovascular treatment. the cohort was divided into low (lvo < ml/kg/min) and high (lvo > ml/kg/min) lvo groups based on the hour postoperative echocardiogram. the study was approved by the institutional research ethics board, and written parental consent was obtained. neonatal birth and postnatal demographics (antenatal steroid administration, apgar scores, gestational age and weighs at birth, respiratory distress syndrome, surfactant administration, and ventilation days) were recorded. details of medical and surgical treatment for the pda also were collected. serial evaluation of hemodynamic (eg, heart rate, bp, arterial ph, and base deficit) and respiratory variables (eg, fraction of inspired oxygen [fio ], mean airway pressure [map]) was performed. oxygenation index was calculated during the time points via the following formula: oxygenation index = (map � fio � ) pao . studies were performed with the use of a vivid or e ultrasound scanner (ge healthcare, milwaukee, wisconsin) with a - or -mhz neonatal probe, respectively. each infant underwent echocardiography assessments: - hours preoperatively (preoperatively), hour postoperatively before the administration of milrinone if needed ( hour postoperatively), and - hours postoperatively ( hours postoperatively). all studies were conducted in accordance with our standardized functional protocol, which follows recently published guidelines , and included tdi and ste imaging methods. all studies were stored as raw data for offline analysis (echopac; ge, milwaukee, wisconsin). offline measurements were conducted after the infants were discharged from the unit. all infants underwent a preoperative complete echocardiographic assessment by a pediatric cardiologist to exclude congenital heart disease. we obtained the following measurements by using conventional echocardiography methods that have previously been described , : assessment of pda: ( ) pda diameter measured at the pulmonary end ( d imaging); and ( ) the direction and peak velocity of flow across the shunt (pulsed-wave doppler [pwd]). markers of volume loading: ( ) left atrial to aortic root ratio (m-mode); ( ) mitral valve e and a velocities and the e wave to a wave ratio (pwd); and ( ) lv end-diastolic diameter (m-mode). measures of myocardial performance: ( ) lv and right ventricular (rv) outputs (pwd + d measurement); ( ) shortening fraction (m-mode); ( ) ejection fraction measured by simpson biplane method (ejection fraction) – ( d imaging); ( ) tissue doppler velocities (s , e , and a ) of the mitral, septal, and tricuspid annuli; ( ) isovolumic contraction and isovolumic relaxation times measured by tdi; and ( ) lv global longitudinal strain (gls). lvo was calculated by measuring the blood velocity at the aortic valve annulus from an apical -chamber view via a pwd and the diameter of the aortic annulus from a parasternal long-axis view by measuring the distance between the visible hinges of the aortic valve in early systole. from the pwd tracing, the velocity time integral (vti) was calculated. the aortic cross-sectional area was calculated using the formula: p � aortic diameter / . lvo was then indexed to weight as (ml/kg/min) = (aortic cross-sectional area � vti � heart rate)/weight. the heart rate was calculated from the r-r interval obtained from electrocardiogram tracing of the aortic dopplerflowimage.svrwascalculatedbyusingthefollowing formula: (mean systemic bp � mean tricuspid valve inflow pressure gradient)/lvo. we also measured lv mean velocity of circumferential fractional shortening. it is determined by the following method : velocity of circumfer- ential fractional shortening = (lvedc � lv end-systolic circumference)/(lvedc � ejection time corrected for heart rate [ejection time/orr interval]), where lvedc = lv end-diastolic circumference. tissue doppler velocities were obtained from the apical -chamber view. we used a pwd sample gate of mm at the level of the annuli while always maintaining an angle of < � between the pulsed-wave cursor and the longitudinal plane of motion. the sector width was minimized to improve the frame rate and temporal resolution. on the tissue doppler traces we measured peak systolic (s ), early diastolic (e ), and late diastolic (a ) velocities. the isovolumic contraction and relaxation times and lv systolic time were measured (figure ; available at www.jpeds.com). for strain analysis, d grayscale images were recorded from the apical -, -, and -chamber views. gls was calculated based on the segmental values as previously described by our group. image acquisition was carried out by of investigators (a.k., a.j., and d.w.). offline image analysis was carried el-khuffash et al http://www.jpeds.com table i. patient demographics and clinical and pda characteristics before ligation low lvo, n = high lvo, n = p value gestation, weeks . [ . - . ] . [ . - . ] . birth weight, g [ - ] [ - ] . gestation at ligation, weeks . [ . - . ] . [ . - . ] . weight at ligation, g [ - ] [ - ] . postdelivery age at ligation, days [ - ] [ - ] . male ( %) ( %) . cesarean delivery ( %) ( %) . -minute apgar score [ - ] [ - ] . antepartum hemorrhage ( %) ( %) . preeclampsia ( %) ( %) . chorioamnionitis ( %) ( %) . antenatal steroids . partial ( %) ( %) complete ( %) ( %) days on invasive ventilation [ - ] [ - ] . necrotizing enterocolitis ( %) ( %) . intraventricular hemorrhage (iii/iv) ( %) ( %) . nsaids treatment ( %) ( %) . hemoglobin, g/dl [ - ] [ - ] . ph . [ . - . ] . [ . - . ] . oxygen requirement, % [ - ] [ - ] . map, cm h o [ - ] [ - ] . pda diameter before ligation, mm . [ . - . ] . [ . - . ] . maximum pressure gradient, mmhg [ - ] [ - ] . nsaids, nonsteroidal anti-inflammatory drugs. values are expressed as median [iqr] or absolute value (%). - original articles out by investigators (a.k. and d.w.). the intra- and inter- observer reliability of tdi and ste in the preterm population undergoing pda ligation was previously reported by our group using the same observers. the components of the lvo calculation (heart rate, aortic vti, aortic diameter) were entered into an electronic database, and lvo was calculated post-hoc. as a result, the observers were unaware of the lvo grouping assignment at the time of the analysis. cardiac troponin t (ctnt) measurements were taken immediately after the preoperative echocardiogram, hour postoperatively, and after the -hour postoperative echocardiogram. ctnt increases in the presence of a pda in premature infants and is predictive of adverse neurodevelopmental outcomes associated with a pda. ctnt is a measurement of myocardial damage that also may be associated with the hemodynamic loading. the preoperative and postoperative blood samples were performed with routine blood work and an additional . ml of blood was required per sample. the samples were tested using the roche bedside cardiac reader (cobas h poc system; roche diagnostics limited, rotkreuz, switzerland). for low levels, the troponin reader provides a range in which the true value is: less than . mg/l or between . and . mg/l. a level greater than . mg/l is shown as an absolute value. we represented a level < . as “ ” and a level between . and . as “ . .” statistical analyses we presented data as means � sd for normally distributed variables and as medians and iqr for nonparametric data. continuous variables between the groups were compared with a student t test or a mann-whitney u test as appropriate. categorical data were compared using the c test or fisher exact test as appropriate. we compared clinical and echo data between the groups across the time points using -way anova with repeated measures. we assessed the correlation between various echocardiography measures using pearson correlation coefficient for normally distributed data and spearman correlation coefficient for skewed data. we accepted a p value of less than . as significant. the statistical analysis was performed using spss version (spss institute, chicago, illinois). results thirty infants were included in this study. nineteen infants had lvo less than ml/kg/min (low lvo group) and were started on an intravenous infusion of milrinone. eleven infants had a lvo greater than ml/kg/min (high lvo group) and did not require milrinone. with the exception of the -minute apgar score, which differed by a single point only between the groups, there were no differences in baseline neonatal demographics, clinical or pda characteristics between the groups (table i). none of the infants was on inotropic agents before ligation. in the low-lvo group, there was a greater proportion of infants requiring an escalation of fio > % ( [ %] vs [ %], p = . ), assessment and treatment of post patent ductus arteriosus lig in the postoperative period. similarly, there was a nonsignificant trend of infants in the low lvo group needing high-frequency oscillation ( [ %] vs [ %], p = . ) accompanied by an increase in map > % ( [ %] vs [ %], p = . ). only ( %) of those infants also required inotropic agents and therefore met the criteria for plcs. both developed a systolic bp less than the third percentile for gestation between and hours postoperatively and received intravenous dobutamine treatment. none of the high lvo group developed plcs. none of the infants in our cohort received hydrocortisone. we identified an increase in median (iqr) preoperative ctnt level from . mg/l ( . - . ) to . mg/l ( . - . ) at hours after surgical intervention (p < . , -way anova). there were no differences in ctnt between groups at any time point. a decrease in lvo (p < . , -way anova) was demonstrated in the entire cohort after pda ligation (table ii). the magnitude of the reduction from pre-operative values was greater in the low-lvo group (table iii; available at www.jpeds.com). there was, however, recovery in lvo by hours in milrinone-treated patients (low-lvo group) to a level comparable with infants in the high-lvo group. an increase in svr was seen in both groups hour after pda ligation (p < . , -way anova), although the magnitude of the increase was greater in the low-lvo group (table ii). by hours, svr decreased in milrinone-treated patients (low-lvo ation syndrome http://www.jpeds.com table ii. conventional echocardiography markers preoperative hour postoperatively hours postoperatively group anova time anovalow lvo high lvo low lvo high lvo low lvo high lvo heart rate ( ) ( ) ( ) ( ) ( ) ( ) . . mean bp ( ) ( ) ( ) ( ) ( ) ( . )* . . oxygenation index . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . . lvo, ml/kg/min ( ) ( ) ( )* ,† ( )* ( )* ( )* . <. rvo, ml/kg/min ( ) ( ) ( ) ( ) ( ) ( ) . . shortening fraction (%) ( ) ( ) ( )* ( )* ( )* ( )* . <. ejection fraction (%) ( ) ( ) ( )* ( )* ( )* ( )* . <. vcfs, circ/s . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . . lvedd, mm . ( . ) . ( . ) . ( . )* . ( . )* . ( . )* . ( . )* . <. la:ao . ( . ) . ( . ) . ( . )* . ( . )* . ( . )* . ( . )* . <. mitral valve e:a ratio . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . . mitral valve e wave, cm/s . ( . ) . ( . ) . ( . )* . ( . )* . ( . )* . ( . )* . <. mitral valve vti . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . ( . ) . <. svr, mmhg/kg/min ( ) ( ) ( )*,† ( )* ( )* ( )* . <. e:a, e wave to a wave; la:ao, left atrial to aortic root; lvedd, lv end-diastolic diameter; ns, not significant (p > . ); vcfs, lv mean velocity of circumferential fractional shortening (corrected for heart rate). values are presented as means (sd). two-way, repeated-measures anova was used to compare the difference in values across the time points (anova p). pairwise comparisons were only performed, and the p value displayed, if the -way, repeated-measures anova test yielded significant results. *p < . vs baseline. †p < . vs low-risk infants. the journal of pediatrics � www.jpeds.com vol. -, no. - group) to levels comparable with the high-lvo group (table ii). lv systolic function and ejection fraction decreased in the entire cohort after pda ligation, but there were no intergroup differences. tissue doppler velocities were measurable in all infants across the evaluation time points. isovolumic timing measurements were not possible in ( %) studies because of the difficulty in identifying the start and end of systolic and diastolic waves. gls measurements were not possible in ( %) scans because of poor image quality. there were early postoperative intergroup differences in tissue doppler-derived systolic velocities. at hour, lv s , septal s , and rv s were lower in low-lvo group compared with the high-lvo group (table iv and figure ). the table iv. gls and tdi estimates of myocardial performance preoperative hour post postopera low lvo high lvo low lvo hig lv lateral tdi, cm/s s . ( . ) . ( . ) . ( . )*,† e . ( . ) . ( . ) . ( . )† a . ( . ) . ( . ) . ( . )† septal tdi, cm/s s . ( . ) . ( . ) . ( . )*,† e . ( . ) . ( . ) . ( . )† a . ( . ) . ( . ) . ( . )† rv tdi, cm/s s . ( . ) . ( . ) . ( . )*,† e . ( . ) . ( . ) . ( . )† a . ( . ) . ( . ) . ( . ) lv gls, % � . ( . ) � . ( . ) � . ( . )† � lv event times lv systolic time, ms ( ) ( ) ( )† ivct, ms ( ) ( ) ( )† ivrt, ms ( ) ( ) ( )* ,† ivct, isovolumic contraction time; ivrt, isovolumic relaxation time; lvet, lv ejection time. values are presented as means (sd). repeated-measures anova was used to compare the differenc the p value displayed, if the -way, repeated-measures anova test yielded significant results. *p < . vs low-risk infants. †p < . vs baseline. magnitude of change in systolic velocity from preoperative to hour after intervention was only significant for lv lateral s and septal s (table iii). there was complete recovery by hours in lv systolic velocities in the low-lvo group who received milrinone to levels comparable with the high-lvo group. gls decreased in both groups after surgical intervention, with a nonsignificant trend towards lower values in the low-lvo group (� . � . % vs � . � . %, p > . ). by hours, gls in the low-lvo group who received milrinone returned to a level comparable with the high- lvo group (tables iii, iv, and figure ). a negative correlation between svr and tissue doppler systolic velocities of the lv and septal walls (r = � . , tivley hours postoperativley group anova time anovah lvo low lvo high lvo . ( . )† . ( . )† . ( . )† . <. . ( . )† . ( . )† . ( . )† . <. . ( . ) † . ( . ) † . ( . ) † . <. . ( . ) † . ( . ) † . ( . ) † . <. . ( . )† . ( . )† . ( . )† . <. . ( . )† . ( . )† . ( . )† . <. . ( . ) . ( . ) . ( . ) . . . ( . ) † . ( . ) † . ( . ) † . <. . ( . ) . ( . ) . ( . ) . . . ( . ) � . ( . )† � . ( . ) . <. ( )† ( )† ( )† . <. ( )† ( )† ( )† . <. ( ) † ( ) † ( ) † . <. e in values across the time points (anova p). pairwise comparisons were only performed, and el-khuffash et al figure . lv, septal, and rv s velocities and lv gls in the groups across the time points. strain values are represented as a positive value for the purposes of graphical representation. the solid line represents the low-lvo group, and the dotted line represents the high-lvo group. the error bars represent the se. *depicts a significant difference between the groups at that time point ( -way anova with repeated measures). - original articles p < . and r = � . , p < . , respectively) was present at the preoperative and -hour evaluation. there was a weaker negative correlation between svr and tissue doppler diastolic velocities of the lv and septum: lv e (r = � . , p = . ), lv a (r = � . , p = . ), and septal e (r = � . , p < . ). there was no correlation between svr and septal a (r = � . , p = . ). there was a negative correlation between svr and gls (r = � . , p < . ). there was a positive correlation between lvo and lv s (r = . , p < . ), septal s (r = . , p < . ), and gls (r = . , p < . ). there was a weak positive correlation between rv s and lvo (r = . , p = . ). similarly, there was a positive correlation between lvo and lv e (r = . , p < . ), lv a (r = . , p < . ), septal e (r = . , p < . ), and septal a (r = . , p = . ). discussion reference ranges for tissue doppler velocities in extremely low birth weight infants are emerging. - it is difficult to perform a direct comparison between preoperative tdi assessment and treatment of post patent ductus arteriosus lig velocities in this cohort and previously published data, because most neonatal studies of tdi were performed in the infant’s first week of life. nevertheless, the preoperative tdi systolic and diastolic velocities in this cohort were all generally greater than those presented in the literature for similar gestational-age infants. this finding may relate to the impact of a high-volume shunt present in our population increasing preload. it is also worth noting that after pda ligation, infants in the low-lvo group had tdi velocities that were lower than the previously published values. we also observed significant changes in lv gls in both groups. although the magnitude of the change was larger in the lower output group, it did not reach statistical significance. this may relate, at least in part, to the greater variability of the strain measurements compared with the tissue doppler measurements or to the small study sample size. the effect of the acute changes on tissue doppler velocities is likely attributable to the load-dependency of the measures rather than a true decrease in lv contractility (intrinsic myocardial function). evaluation of true myocardial contractility is not feasible with current echocardiography techniques, which ation syndrome the journal of pediatrics � www.jpeds.com vol. -, no. - provide information on myocardial systolic/diastolic performance alone. longitudinal changes in lv function measured by tissue doppler and strain measurements appear to be influenced by the acute changes in loading conditions. there was a significant negative linear relationship between peak systolic tissue doppler velocitiesand svrmeasurements. the same negative correlation was observed between the absolute value of gls and svr, suggesting that those markers of myocardial performance are strongly influenced by afterload changes. although we accepted the limitations of the svr calculation based on echocardiography, it was interestingto note that thelow-lvo group hadamuch greater svr than the high-lvo group. in a study in animals, researchers demonstrated that strain was sensitive to acute changes in afterload and that strain rate measurements seem to be less affected. strain rate measurements are, however, technically more difficult and are highly frame rate dependent. in preterm infants with greater heart rates, assessment of strain rate is technically challenging. lv diastolic velocities (e and a ) were lower after pda ligation, but an intergroup difference was not seen. early tissue doppler velocities are influenced by different factors, including early relaxation, restoring forces determined by the amount of shortening during systole, and lengthening load which represents preload. only lv isovolumic relaxation time at hour was different with longer interval duration in the low-lvo group, which may relate to the acute volume unloading causing early relaxation abnormalities or may be a consequence of increased afterload. diastolic function may contribute to the evolution of low lvo after ligation. the lack of intergroup differences in most measures of diastolic function suggests that the magnitude of the change in diastolic function (and preload) play a lesser role in the etiology of low lvo. the weak correlation between lvo, svr, and the diastolic tdi variables further supports this reasoning. rv diastolic velocities were less influenced by ligation. it has been suggested that the reduction in lv systolic function post duct closure may relate to myocardial injury related to ischemia. for this reason, we included the use of biomarkers in the current project. ctnt levels were unchanged hour after pda ligation but increased at hours. the levels were not different between the groups. the lack of intergroup differences at and hours suggests that myocardial ischemia is not a contributory factor in the etiology of low lvo. however, it is possible that the delayed increase in ctnt at hours is a consequence of its sudden exposure to increased svr, analogous to the increase in plasma troponin that occurs in the transitional period. , the nature of the elevation in ctnt may relate to the release of cytosolic ctnt not bound to the myofibril. administration of milrinone to patients with low lvo was associated with recovery in lvo, lv lateral, and septal s velocities to levels comparable with the high-lvo group. milrinone, a phosphodiesterase iii inhibitor, increases tissue bioavailability of cyclic adenosine monophosphate, resulting in vasodilation, positive inotropy, and afterload reduction. we previously demonstrated that the immediate postopera- tive administration of intravenous milrinone to infants with low lvo results in a reduction in the risk of development of plcs. the presumed benefits of milrinone treatment were thought to relate predominantly to its vasodilator and afterload-reducing effects. in this study, we found that intravenous milrinone was associated with longitudinal improvement in tdi- and ste-derived indicators of systolic function by hours. although milrinone is known to possess a lusitropic effect, with the exception of septal e , treatment was not associated with recovery in diastolic veloc- ities in the low lvo group by hours of life. this finding suggests that the predominant effect of milrinone is afterload reduction coupled with an improvement in systolic function rather than lusitropy. there were, however, infants in the low-output group who developed plcs despite treatment with milrinone. it was not possible to further investigate factors contributing to the development of plcs in these patients because of the low event rate. the physiologic nature of disease and causal factors in neonates with plcs, despite milrinone treatment, requires further evaluation in larger groups. there were no obvious differences in any echocardiography markers of systolic performance between these infants and the rest of treatment cohort. there are several limitations to the study design. first, because the study is not randomized and lacks a control group of high-risk infants that did not receive milrinone, it is not possible to be certain that the changes seen were related to treatment and not independent temporal changes. second, the sample size is small, which prohibits making any meaningful statements of treatment effect. third, as mentioned previously, it is not possible to accurately measure lv afterload in these patients. also, the calculation of svr is dependent on lvo, and thus may not represent true vascular resistance in this population. tdi and ste provide novel insights and further character- ization of lv systolic performance immediately after pda ligation. treatment with milrinone appears to have a positive influence on lv systolic function, although this needs confirmation in a prospective randomized controlled trial. n submitted for publication dec , ; last revision received feb , ; accepted mar , . reprint requests: dr patrick j. mcnamara, md, hospital for sick children, university of toronto, university avenue, toronto, ontario mg x . e-mail: patrick.mcnamara@sickkids.ca references . kabra ns, schmidt b, roberts rs, doyle lw, papile l, fanaroff a. neurosensory impairment after surgical closure of patent ductus arteriosus in extremely low birth weight infants: results from the trial of 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measured. table iii. comparison of the change in lvo, svr, and function parameters between the groups hour postoperatively � preoperatively p value hour postoperatively � hour postoperatively p valuelow lvo high lvo low lvo high lvo lvo, ml/kg/min � ( ) � ( ) . + ( ) + ( ) . svr, mmhg/kg/min + ( ) + ( ) . � ( ) ( ) . shortening fraction, % � ( ) � ( ) . + ( ) ( ) . ejection fraction, % � ( ) � ( ) . + ( ) � ( ) . vcfs, circ/s � . ( . ) � . ( . ) . + . ( . ) � . ( . ) . lv s , cm/s � . ( . ) � . ( . ) . + . ( . ) . ( . ) . lv e , cm/s � . ( . ) � . ( . ) . . ( . ) . ( . ) . lv a , cm/s � . ( . ) � . ( . ) . . ( . ) � . ( . ) . septal s , cm/s � . ( . ) � . ( . ) . + . ( . ) � . ( . ) <. septal e , cm/s � . ( . ) � . ( . ) . + . ( . ) ( . ) . septal a , cm/s � . ( . ) � . ( . ) . + . ( . ) ( . ) . gls, % � . ( . ) � . ( . ) . + . ( . ) . ( . ) . comparisons are made between preoperative to hour postoperative and between hour postoperative to hours postoperative. a positive sign (+) depicts an increase in the parameter between the compared time points. a negative sign (�) depicts a reduction in the value of the parameter between the compared time points. the journal of pediatrics � www.jpeds.com vol. -, no. - .e el-khuffash et al 	assessment and treatment of post patent ductus arteriosus ligation syndrome 	methods 	statistical analyses 	results 	discussion 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ microsoft word - tree-d- - _edited_v .docx genomics in conservation: case studies and bridging the gap between data and application brittany a. garner, , ,‡, brian k. hand, ,‡,* stephen j. amish, , louis bernatchez, jeffrey t. foster, kristina m. miller, phillip a. morin, shawn r. narum, stephen j. o’brien, gretchen roffler, william d. templin, paul sunnucks, jeffrey strait, , kenneth i. warheit, todd r. seamons, john wenburg, jeffrey olsen, and gordon luikart , flathead lake biological station, fish and wildlife genomic group, division of biological sciences, university of montana, polson, mt , usa wildlife program, fish and wildlife genomic group, college of forestry and conservation, university of montana, missoula, mt , usa institut de biologie intégrative et des systèmes (ibis), université laval, québec, qc g v a , canada department of molecular, cellular, and biomedical sciences, university of new hampshire, durham, nh , usa molecular genetics laboratory, pacific biological station, nanaimo, bc v t n , canada southwest fisheries science center, national marine fisheries service, national oceanic and atmospheric administration, la jolla shores drive, la jolla, ca , usa columbia river inter-tribal fish commission, hagerman fish culture experiment station, -f national fish hatchery road, hagerman, id , usa theodosius dobzhansky center for genome bioinformatics, st petersburg state university, sredniy prospect, st petersburg, russia alaska department of fish and game, division of wildlife conservation, rd street, douglas, ak , usa                                                              ‡these authors contributed equally. © . this manuscript version is made available under the elsevier user license http://www.elsevier.com/open-access/userlicense/ . / gene conservation laboratory, alaska department of fish and game, raspberry road, anchorage, ak , usa school of biological sciences, monash university, melbourne, vic australia washington department of fish and wildlife, molecular genetics laboratory, capitol way n., olympia, wa , usa conservation genetics laboratory, east tudor road, ms , anchorage, ak , usa *correspondence: brian.hand@umontana.edu (hand, b.k.). keywords: conservation genomics; population genomics; conservation biology; next-generation sequencing; conservation practice; genetic monitoring; natural resource management.         we agree with shafer et al. [ ] that there is a need for well-documented case studies of the application of genomics in conservation and management as well as increased communication between academics and natural resource managers. however, we challenge shafer et al.’s [ ] relatively pessimistic assertion that ‘conservation genomics is far from seeing regular application’. here we illustrate by examples that conservation practitioners utilize more genomic research than is often apparent. in addition, we highlight the work of nonacademic laboratories [government and nongovernmental organizations (ngos)], some of which are not always well represented in peer-reviewed literature. finally, we suggest that increased agency–academic collaboration would enhance the application of genomics to real-world conservation and help conserve biodiversity. there is substantial controversy and confusion surrounding the definition of ‘genomics’ versus traditional genetic approaches. here we address this by expanding shafer et al.’s [ ] definition to include a broad- and narrow-sense definition to better illuminate the different ways that genomics contributes to conservation practice. we define broad-sense conservation genomics as the use of new genomic techniques and genome-wide information to solve problems in conservation biology (as in shafer et al. [ ] and allendorf et al. [ ]). our narrow-sense definition also requires the use of approaches that are conceptually and quantitatively different from traditional genetics that would be impossible using genetic data alone (e.g., detecting genome- wide adaptation, use of transcriptomics, epigenetics, using annotated genomes). this narrow- sense definition includes using hundreds to thousands of mapped or gene-targeted marker loci in combination with recent computational and conceptual approaches such as mapping runs of homozygosity, comparing neutral versus adaptive patterns of population structure or gene flow,         testing for signals of selection to assess adaptation, and testing assumptions of neutrality (e.g., before estimating effective population size or gene flow patterns). narrow-sense genomic approaches have been used for diverse conservation applications including identifying conservation units, assessing gene flow, and detecting local adaptation (table s in the supplementary material online). we agree with shafer et al. [ ] and others [ ] about the general and serious concern of erroneous identification of adaptive loci and their subsequent use (or misuse) in conservation practice. however, we remain cautiously optimistic given the recent efforts to use putatively adaptive loci to inform management practices. for instance, genome-wide scans using diversity array technology (dartseq) in gimlet trees (eucalyptus salubris) generated neutral and putatively adaptive snp markers used to uncover distinctive molecular lineages signaling adaptation to different environments. these genome-wide scans offered enhanced precision otherwise unavailable with traditional genetics or phenotypic traits alone [ ] (table s ). such novel insights are important in seed choice for the ecological restoration of gimlet trees, a keystone species in the great western woodlands of australia, in the wake of wildfires [ ]. in many broad-sense studies, next-generation sequencing (ngs) has enabled the discovery of management-informative markers that are subsequently screened in populations of conservation concern. for example, state management agencies in washington and idaho, usa used ngs to discover markers of introgression from hatchery broodstock into wild populations of salmonid fishes [ , ]. other applications of broad-sense conservation genomics are evident (table s ) and have been enabled by recent ngs and snp genotyping technologies [ ] (http://biorxiv.org/content/early/ / / / ). these approaches allow genome-wide         discovery and genotyping of highly informative markers, making cost-effective monitoring feasible using relatively small marker sets (e.g., – markers) [ ]. decreases in costs (e.g., sequencing, library prep, bioinformatics) are sparking the application of ngs to a broader set of conservation questions and taxa where funding is relatively more limited. in addition to the examples above, genomic data are currently applied in conducting parentage analyses in pacific lampreys (lampetra tridentata) and monitoring for disease in tasmanian devils (sarcophilus harrisii) [ , ] and fish (table s ). power analyses and cost- savings comparisons of using snps versus microsatellite markers in conservation genomics would be of great benefit, but such analysis is beyond the scope of this letter. however, using genomic approaches has been shown to provide more statistical power than microsatellites and to cost as low as % of traditional sanger sequencing prices [ , , ] (table s ). we have included multiple case studies from salmonids because these species are of great conservation concern due to their ecological, commercial, and cultural importance in many northern pacific rim river systems. for example, ~ % or more of salmonid populations in the columbia river basin (usa–canada) have been extirpated and many remaining populations are listed as endangered or threatened under the endangered species act (esa) or the species at risk act in canada because of, for example, over-harvesting, habitat degradation, pollution, and hydrological dams [ ]. therefore, more money and time is being spent on these species than other taxa due to their multiple conservation concerns (e.g., climate change, hybridization, over- harvesting). there are ~ nonacademic laboratories (e.g., federal, tribal, ngo, state agencies) using genomic data to work mostly or exclusively on salmonids in the pacific northwest of north america. shafer et al. [ ] insufficiently acknowledged one of the most significant contributions of genomics to conservation by not fully highlighting the work of these         laboratories, particularly the alaska department of fish and game (adfg), a leader in snp and ngs tool development and application. adfg genotypes approximately fish annually for management using broad-sense conservation genomic approaches, showing that conservation genomics in salmonids is an example of what is possible when adequate resources are devoted to the issue [ ] (table s ). we highlight recent applications of genomics in real-world management where some are published, but many similar studies are not published or widely disseminated. some nonacademic laboratories have relatively limited incentive to publish or are delayed due to urgent deadlines reinforced by political, legislative, or legal constraints. for example, some agency laboratories produce reports or declarations used in litigation or the planning of harvest regulations or introductions (e.g., hatchery fish management plans), which can delay scientific publication. nonacademics could potentially publish more by collaborating with academic groups who have strong incentives to publish (e.g., to ‘publish or perish’). academics could in turn achieve greater conservation impact by working closely with practitioners who can provide benefits such as large sample and data collections, funding and field staff, collection permits, and high-throughput genomics platforms. while research and publications from some nonacademic laboratories are often underappreciated or delayed, they can help the conservation biology community to understand the extent and feasibility of applying genomics to conservation. we hope by highlighting case studies we will expand discussions and applications of genomic techniques in conservation and encourage the closing of gaps between nonacademic laboratories and academia. acknowledgments         the authors thank the following people for providing comments, feedback, and case studies: margaret byrne, todd cross, taylor wilcox, robb leary, sally aitken, jon ballou, bob lacy, eric peatman, luciano beheregaray, katherine ralls, brett addis, and kathy belov. they thank james and lisa seeb for extensive contributions to earlier versions of the manuscript. they also thank fred allendorf for help in developing the initial idea of a narrow-sense definition and subsequent advice on defining narrow sense (e.g., in table s ). they especially thank aaron shafer, michael bruford, and michael schwartz for encouraging publication and improving earlier versions the manuscript with a shared vision of enhancing the contribution of genomics in conservation. g.l. and b.k.h. were partially supported by grants from the nsf (deb ), nasa (nnx ab g), and montana fish wildlife and parks.         references shafer, a.b.a. et al. ( ) genomics and the challenging translation into conservation practice. trends ecol. evol. , – allendorf, f.w. et al. ( ) genomics and the future of conservation genetics. nat. rev. genet. , – steane, d.a. et al. ( ) genome-wide scans reveal cryptic population structure in a dry- adapted eucalypt. tree genet. genomes , warheit, k.i. ( ) measuring reproductive interaction between hatchery-origin and wild steelhead (oncorhynchus mykiss) from northern puget sound populations potentially affected by segregated hatchery programs, washington department of fish and wildlife steele, c.a. et al. ( ) a validation of parentage-based tagging using hatchery steelhead in the snake river basin. can. j. fish aquat. sci. , – narum, s.r. et al. ( ) genotyping-by-sequencing in ecological and conservation genomics. mol. ecol. , – campbell, n.r. et al. ( ) genotyping-in-thousands by sequencing (gt-seq): a cost effective snp genotyping method based on custom amplicon sequencing. mol. ecol. resour. , – hess, j.e. et al. ( ) use of genotyping by sequencing data to develop a high-throughput and multifunctional snp panel for conservation applications in pacific lamprey. mol. ecol. resour. , – miller, w. et al. ( ) genetic diversity and population structure of the endangered marsupial sarcophilus harrisii (tasmanian devil). proc. natl acad. sci. u. s. a. , –         lemmon, a.r. et al. ( ) anchored hybrid enrichment for massively high-throughput phylogenomics. syst. biol. , – gustafson, r. et al. ( ) pacific salmon extinctions: quantifying lost and remaining diversity. paper , us department of commerce habicht, c. et al. ( ) harvest and harvest rates of sockeye salmon stocks in fisheries of the western alaska salmon stock identification program (wassip), – . special publication no. - , alaska department of fish and game jc nainby-luxmoore. ellis - van creveld syndrome (chondroectodermal dysplasia syndrome) in a gurkha family jr army med corps ; l : - case report ellis - van creveld syndrome, (chondroectodermal dysplasia syndrome) in a gurkha family captain j c nainby-luxmoore ba, mb, bs, ramc se ni or hou se officer in paed iatri cs srilish miliwry hospital, hon g kong, sfpo • summary: the first reported case of ellis - van c reveld syndrome in a gurkha child is described , and the implications of th e syndrome in this ethnic group are briefly considered. lntroduction this autoso ma l recessive syndrome \vas first described by e jli s a nd va n crevcld in , t hey reported three children with the fo llowing co ngeni ta l ab normaliti es: ec todermal dysplasia (affectin g the hair , teeth and nails), polydact yly , cho ndrodyspla sia , and co nge nital '-'morbus cordis". the largest si ngle stud · of this syndrom e was presented by mckui sk in \v he n be reported cases in a highl y inbre d amish population in the usa. it has , however, never been previously documented in a nepalese fa mil y. case report a fe ma le chil d was delivered by a year old gurkha corporal 's wife , para i , gravida . e moth er's fir st child , a boy. had hee n born e ighteen mo nth s previo usly by for ceps de live ry in nepa l, but had di ed at mo nths of age due to me ningiti s. during thi s seco nd pregnanc y she was admitted to hospital twice, once a t weeks (by dates) because of po lyhydramnios, and then again at weeks becau se of abdominal pain , possihle ante par tum haemorrhage a nd premature labour. labo ur st arted spo ntaneously a t + weeks by dates ( weeks by sca n) . the firs t stage took hou rs s minutes and was latterly aug me nted by oxytoci n; th e second stage took minutes. th e baby's birth weight was . kg, and she was give n illu cus extrac tion a nd facial oxygen. apgar scores were at i minute, at minutes and at minutes. she was t ben tra nsferred to an in cubator. genera l clini ca l examination revea led multipl e t;!x terna l ab no rmalities. the limbs were short a nd the chest was small (fig. i). six fingers were present o n each hand , and short simian palmar creases with in cur vin g linle fi ngers were also present bilaterall y (fig . ) . clefts we rt;! noted bil atera ll y between the great and second toes, a nd a ll the nail s were sma ll and dysplast ic (fig. ). two inci sor teeth were clearly presen t in both uppe r and lowe r jaws (fig. ). there was a lso a n ejection s)'sto li c murmur which was loudes t at the left stern a l cdge , but the re were no sign s o f cardi ac failure. the passage of a nasogas tri e tube exclud ed oesophageal a tresia. she rece ived regu lar o ral feeds, but sixtee n ho urs after delivery she developed profound hypoglycae mia. thermoreg ul atory di sturban ce a nd cardia.e failure. d espite a ll res usei tati vt;!effort s, she died six hours la te r. ra dio logica l examinati on after death co nfirmed prematurit y, and revealed a normal vau lt and sp ine, a nd no rmal ribs . t he re was, howeve r, symmetrica l short e ning of both t he proxi mal a nd distal bones of the limbs, \vhich we re ot he rwise norma ll y mode lle d . u ln ar sided polydactyly was also co nfi rmed. i at postmortem exam in a tion the pericardiu m was . no rmal. a nd tbe heart was of norma l size. there was, however. a common atrioventricular orifice and a singl e o utl et from the hea rt. this truncus arteriosus supplied both the systemic and pulmonary circulation s. th e main sys te mi c veins and arte ries a ppeared to have a normal ~ confi guration . chest and short extremities. • g u e st. p ro te cte d b y co p yrig h t. o n a p ril , b y h ttp ://m ilita ryh e a lth .b m j.co m / j r a rm y m e d c o rp s: first p u b lish e d a s . /jra m c- - - o n o cto b e r . d o w n lo a d e d fro m http://militaryhealth.bmj.com/ • t • j c nainby-luxmoo re fig. . post mortem view of the right hand showing polydact)!ly . fig. . post mortem view of the dorsum or the right ha nd .. showing nail dysplasia and polydactyly. uiscussion ... e llis - van c reveld syndrom e carrie s a mortality of about fi fty per ce nt in ea rl y infancy because o f th e card iac ab no rma li ties. most of those \ .... ho s ur vive are o f normal int e lli gence a nd h,we an adu lt h eight of to inches. the re a re, however, freque nt d e nt al problems and a lso lim itatio ns in h a nd function . • a s the sy ndro me has no t prev io us ly bee n report ed in a ne palese family , it s impli ca tion s for these fannin g peo ple have neve r h ee n cons idere d. whil e the short stat ure wo uld be o f .litt le signi ficance in a populat io n of s ma ll peo ple, th e ah no rmaliti es of the h and s and the • possibilit y o f me ntal re tardation would not be socia ll y accep tab le. neve r thel ess, ne palese culture h o ld s the ability tu raise a la rge family in high es teem, despit e hig h infant mo rtali ty, and ma le childre n recei ve markedl y fig. . post mortem ,"'iew of the mouth showing the upper two teeth. pre fe re ntial care. t hi s famil y is, the re fo r e currentl y of low s tatus becau se bo th o f th e ir babi es so far ha ve died. thl: autusoma l recessive nature of th e co ndition mea ns that they a re a t hi g h ri s k of h aving ano ther affec kd child a nd wou ld obvio usly benefi t from antenata l diag n osis , as describe d in papers by hobb in s et al\ ma ho ney et al and sui et al , usin g tcchniqu t:s such a s rea l im e ultrasou nd .. hid fe toscopy. th ese are not , un for tuna te ly, ava il hhle in nepa l. the question therefore arose as to w·hethe r the fa mil y shou ld h e told of t he (x, chance of s u bsequent ba bies bein g affecled. in view of the social mores , it was fe lt that they wo 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. bu i t . el al. prena ta l di ag no sis of chondrocctodcrm at dysp lasia with fetoscopy. prclla/ diagll. ; ( ): - y. g u e st. p ro te cte d b y co p yrig h t. o n a p ril , b y h ttp ://m ilita ryh e a lth .b m j.co m / j r a rm y m e d c o rp s: first p u b lish e d a s . /jra m c- - - o n o cto b e r . d o w n lo a d e d fro m http://militaryhealth.bmj.com/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message 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left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ © r a d c l i f f e va s c u l a r access at: www.verjournal.com peripheral artery disease wearable technology refers to the broad category of compact electronic devices that can be incorporated into clothing or accessories. these devices are used in the clinical and non-clinical environment and they offer multiple applications that help maintain physiological and psychological wellbeing. wearable technologies not only facilitate self- tracking for the consumer but also allows for remote monitoring and analysis by a third party, such as a healthcare provider. the internet of things is a network of real-world objects that have the ability to communicate data and sense the status of each object and the surrounding environment. through the combined use of wearable devices and internet of things technology, patients and healthcare providers may be able to track, monitor and analyse various clinically relevant measurements. this could lead to real-time monitoring of disease progression as well as treatment adherence. this concept feeds into the premise of telemonitoring, which is defined as the use of telecommunication devices to remotely monitor patients at a distance. of note, telemonitoring is broadly synonymous with biotelemetry, which involves the transmission of health data from one location to another where the data can be interpreted and used to affect healthcare decision-making. , telemonitoring is useful in collecting biological, environmental or physiological data in a remote setting when direct observation is not possible. common clinically pertinent measurements such as blood pressure (bp), blood glucose and lung function have been successfully tracked using telemonitoring technologies. – the use of telemonitoring allows patients to be managed at home, thereby helping to overcome barriers concerning access to healthcare, such as travel, time and costs. these technological advancements may prove useful in the management of cardiovascular disease (cvd). cvd is a major global health burden and accounts for a significant proportion of hospital admissions, as well as up to one-third of all deaths globally. however, given the importance of conservative management and lifestyle modifications, wearable technology may be able to disrupt the way in which medical therapy and lifestyle modification advice is delivered to this patient cohort. therefore, in this review, we aim to summarise the current applications of wearable and telemonitoring technologies in facilitating the management of cvd outside the hospital setting. current wearable technologies the broad definition of wearable technologies encompasses a variety of devices, such as head-mounted displays, clothing with smart technology, fitness trackers, smart watches and biosensors. fitness trackers (activity monitors) allow the long-term daily monitoring of physical activity in a real-world setting and usually take the form of wrist bands, ankle bracelets or clip-ons. fitness trackers use sensors (pedometers or accelerometers) to detect movement and are therefore able to measure physical activity. electronic displays on devices allow quick and easy viewing of exercise progress and goals. many devices are able to transfer data to computer or smartphone apps, allowing remote access to data. abstract wearable devices and telemonitoring are becoming increasingly widespread in the clinical environment and have many applications in the tracking and maintenance of patient wellbeing. interventions incorporating these technologies have been used with some success in patients with vascular disorders. wearable fitness monitors and telemonitoring have been used in the community to mobilise patients with peripheral vascular disease with good results. additionally, wearable monitors and telemonitoring have been studied for blood pressure monitoring in patients with hypertension. telemonitoring interventions incorporating electronic medication trays and ingestible sensors have also been found to increase drug adherence in hypertensive patients and ultimately improve health outcomes. however, wearable and telemonitoring interventions often face problems with patient adherence, digital literacy and infrastructure. further work needs to address these challenges and validate the technology before widespread implementation can occur. keywords peripheral vascular disease, wearable technology, personalised medicine, telemedicine, digital health disclosure: the authors have no conflicts of interest to declare. received: november accepted: february citation: vascular & endovascular review ; :e . doi: https://doi.org/ . /ver. . correspondence: viknesh sounderajah, department of surgery and cancer, imperial college london, london w ny, uk. e: vs @imperial.ac.uk open access: this work is open access under the cc-by-nc . license which allows users to copy, redistribute and make derivative works for non- commercial purposes, provided the original work is cited correctly. the role of wearable technologies and telemonitoring in managing vascular disease calvin chan, viknesh sounderajah, amish acharya, pasha normahani, colin bicknell and celia riga department of surgery and cancer, imperial college london, london, uk mailto:vs @imperial.ac.uk https://creativecommons.org/licenses/by-nc/ . /legalcode va s c u l a r & e n d o va s c u l a r r e v i e w peripheral artery disease luley et al. used aipermotion (aipermon) fitness trackers as part of an intervention to increase physical activity in patients with metabolic syndrome and found significant improvements in weight loss and markers of the metabolic syndrome. in a randomised controlled trial, frederix et al. used triaxial accelerometers (yorbody) to monitor physical activity and set daily step-count prescriptions for patients with coronary artery disease enrolled in hospital-based cardiac rehabilitation. this study found a significant improvement in lung function assessments and a trend towards fewer rehospitalisations in the intervention group. kirk et al. conducted a meta-analysis examining the use of wearable devices to alter physical activity behaviour in adults with chronic cardiometabolic disease and found a positive impact on physical health. smart watches have many of the same functions as smartphones, such as mobile applications, internet connectivity and gps. similar to fitness trackers, smart watches can also incorporate movement and exercise sensing functions, but their functionality is expanding. for example the apple watch (apple) is currently being validated by clinical trials for detection of af and other abnormal heart rhythms. wearable biosensors are attached to the body for long continuous periods of time and have diagnostic and monitoring applications. the wearable biosensor (philips medical systems) is a lightweight, wireless self-adhesive biosensor that can detect vital signs – ecg, respiratory rate, skin temperature – and movement data – posture, fall detection, step count. a study conducted by braem et al. found that the use of this wearable device as a tool for screening activity levels in patients undergoing transcatheter aortic valve implantation was feasible, but noted some concerns regarding the reliability of the data collection. continuous glucose monitoring can provide real-time blood glucose data otherwise unobtainable by conventional intermittent sampling. glucowatch (cygnus) is a non-invasive wearable continuous glucose monitor that extracts and samples glucose through intact skin via reverse iontophoresis. a validation study by tierney et al. found blood glucose readings from the device were clinically acceptable. however, a randomised trial by chase et al. found no improvement in glycaemic control with the glucowatch, which was, in part, attributed to poor device adherence due to skin irritation. , peripheral artery disease management wearable devices with the ability to monitor movement may be particularly useful in the management of patients with peripheral artery disease (pad). pad is a debilitating condition that results in significant walking impairment and poor quality of life. current recommended first-line management for pad is supervised exercise therapy, with patient mobilisation crucial to prevent disease progression and reduce hospital admissions. , despite this, patients often face challenges in accessing therapy due to cost, travel and time constraints. this contributes to the low uptake and adherence with such programmes. , several studies have investigated the efficacy of home-based exercise programmes incorporating fitness trackers and telemonitoring as an alternative to supervised exercise (table ). – duscha et al. used wrist-worn commercially available fitness trackers (fitbit charge, fitbit) to monitor daily physical activity and set exercise prescriptions based increasing patients’ step count each day. patients wore fitness trackers continuously during waking hours and daily step count was synchronised with smartphones where it could be remotely viewed. this allowed investigators to not only monitor physical activity, but also offer personalised feedback via phone calls. patients using the fitness tracker intervention were found to have significant improvement in claudication distance, maximum walking distance and steps walked per day, compared with baseline. similarly, normahani et al. incorporated the nike+ fuelband (nike) fitness tracker as part of a home-based exercise intervention for patients with pad. instead of setting exercise prescriptions using step count, ‘fuel points’ were used, which measure overall activity and movement. online accounts on the nike+ website allowed activity data to be reviewed at follow-up visits and exercise prescriptions could be programmed into patient devices. the study found significant increases in walking ability and quality of life in patients using the device, compared with those attending a weekly -hour supervised exercise session over a -month period. in addition to using the ae xl pedometer (accusplit) to track daily physical activity and weekly phone consultations, mays et al. incorporated an element of coaching into their study. this allowed them to identify local barriers and encourage exercise adherence. this involved an assessment of a patient’s local area using google street view to identify walking routes and community resources, such as benches on which to rest. moreover, they identified potential barriers to walking, such as discontinuous pavements. , this resulted in significant improvements in walking ability for the intervention group compared with baseline and usual care. the use of smartphone applications may also be beneficial for the remote monitoring and management of pad. ata et al. developed vasctrac, an apple smartphone app that enabled remote collection of medical and physical activity data. vasctrac is able to record measures of daily physical activity (steps walked, distance walked, flights of stairs climbed and maximum continuous number of steps) using the built-in accelerometer in apple smartphones. patients were sent notifications through the app to perform twice-weekly -minute walk tests which allows tracking of changes in walking ability. study results are yet to be published. blood pressure monitoring hypertension is a well-known risk factor for cvd. environmental factors can often limit the usefulness of conventional bp measurements because of phenomena such as white-coat, masked and nocturnal hypertension. home measurement is a superior method of determining bp and wearable devices are well established for this purpose. apart from traditional oscillometry-based bp measurement (involving an inflatable cuff), wearable devices also use other non- invasive methods of eliciting measures of bp such as tonometry, volume clamp, pulse wave velocity and pulse transit time (table ). the bpro (healthstats) is a cuffless, wrist-bound bp measurement system that works via arterial applanation tonometry. the wrist monitor incorporates a small protruding force sensor that rests over the radial artery and captures the arterial pulse waveform. the device requires initial calibration to bronchial bp using a standard oscillometry-based bp monitor. in a validation study, the device was found to meet the accuracy criteria for systolic and diastolic bp in both european society of hypertension protocol and association for the advancement of medical instrumentation (aami) standards under stationary conditions. however, in a study comparing accuracy of the bpro against intra- arterial bp measurement in post-operative patients, there were wearable technologies and telemonitoring for vascular disease va s c u l a r & e n d o va s c u l a r r e v i e w table : characteristics and main findings of studies incorporating wearable devices for management of peripheral artery disease study study design study duration study size (n) wearable device patient population wearable intervention additional interventions control main findings dacha et al. rct weeks fitbit charge exercise limited by ic, abpi < . daily walking prescription, increasing every weeks electronic pad information, weekly tips, phone consultations physician advice, pad book significant improvement in walking ability and vo max, increase in steps per day and exercise intensity endicott et al. ps months fitbit one veterans, symptomatic ic daily walking prescription clinic consultations every weeks na significant increase in steps per day gardner et al. rct weeks stepwatch (modus) symptomatic ic, abpi < . walking three times per week, increasing duration biweekly min consultation biweekly -month set, physician advice significant improvement in walking ability, walking speed, qol and vo max gardner et al. rct weeks stepwatch (modus) symptomatic ic, abpi < . walking three times per week, increasing duration evert weeks min consultation every weeks -month set, light resistance training significant improvement in walking ability, walking speed, qol and vo max mays et al. , rct weeks accusplit ae xl symptomatic ic, previous endovascular therapy walking three times per week initial -week set ( × per week), weekly phone consultations physician advice significant improvement in walking ability. mcdermott et al. rct months fitbit zip abpi < . exercise five times per week, increasing duration and intensity four initial set sessions then weekly phone consultations no intervention no improvement in walking ability or qol. significant improvement in exercise frequency nicolaï et al. rct months pam personal activity monitor stage ii pad walking feedback and encouragement set ( – mins per week), wam scores used to give feedback on walking effort outside set set ( – × per week), physician advice significant improvement in walking ability and qol. no difference compared with set only normahani et al. rct months nike+ fuelband symptomatic ic, stenosis on ultrasound daily activity targets: ‘fuel points’ set ( hour/ week), clinic consultations at , and months -month set ( hour per week) significant improvement in walking ability and qol tew et al. rct weeks yamax sw- digiwalker clinically diagnosed ic walking self-regulation, goal to reach > , steps/day structured ic education (sedric), phone consultation at weeks pad information leaflet significant improvement in walking ability and qol. no change in steps/day abpi = ankle–brachial pressure index; ic = intermittent claudication; na = not available; pad = peripheral artery disease; ps = prospective study; qol = quality of life; rct = randomised controlled trial; sedric which is structured education for rehabilitation in intermittent claudication; set = supervised exercise therapy; wam = wearable activity monitor. table : characteristics and validation study findings for wearable blood pressure monitoring devices wearable device calibration measurements provided method of measurement level of validation bpro (medtach) required with validated oscillometry-based device sbp, dbp, raix applanation tonometry • meets aami and esh standards of accuracy compared with sphygmomanometer under stationary conditions. • inaccurate agreement in sbp against intra-arterial measurement in post-operative patients; device affected by slight movement. • fair agreement with a brachial cuff-based device under ambulatory conditions; volunteers instructed to hold still during measurements. • inaccuracies in estimating raix. seismo-watch required with validated oscillometry-based device sbp, dbp pulse transit time acceptable dbp measurements when volunteers were static. va s c u l a r & e n d o va s c u l a r r e v i e w peripheral artery disease significant inaccuracies according to aami standards in systolic and mean bp between the methods, which was attributed to patient movement. a -hour study comparing the bpro to conventional arm- bound oscillometry-based bp monitoring in normotensive and prehypertensive volunteers found fair agreement between the two devices. however, during bp measurements the volunteers were asked to keep still and the bpro was unable to obtain almost % of the scheduled measurements. in addition to bp measurement, radial augmentation index (raix) can be calculated from radial wave pulse measurements from the bpro. the raix has been proposed as an estimate of central bp and a useful parameter of vascular function and ageing. although there were discrepancies in raix measured by bpro versus a reference device, vardoulis et al. developed and validated a novel wrist-bound tonometer that produced accurate raix readings compared with a reference hand- held tonometer. , the seismowatch is an example of a wrist-worn bp monitor that functions via pulse transit time. to obtain bp measurements, the watch face contains an accelerometer which is pressed for a short duration on the sternum to obtain a seismocardiogram and determine timing of left ventricular ejection. this is compared with a distal reading from a wrist-obtained photoplethysmogram to obtain the pulse transit time and, therefore, an estimate of bp. in a small feasibility study, the device was found to obtain acceptable mean and diastolic bp, but required subjects to perform measurements while static. similarly, ogink et al. investigated the feasibility of a cuffless bp monitor (checkme pro, viatom) based on pulse transit time to measure systolic bp of hypertensive patients at home. the study found that the device produced reproducible bp readings and had good adherence due to its ease of use. however, the device was limited by its inability to measure diastolic bp and failure to measure bp % of the time. boubouchairopoulou et al. validated a cuffless bp monitor (freescan, maisense) that obtained readings via pulse transit time. in large successive feasibility and validation studies (a total of patients were involved in the four feasibility studies), the device was found capable of obtaining systolic and diastolic bp readings when compared with a standard mercury sphygmomanometer. however, accurate readings required a device sensor upgrade and careful initial device calibration in a research setting. further work is needed to investigate the validity of this prototype device in a clinical setting. the heartguide (omron corp) is a novel smartwatch with the ability to monitor bp. it uses the traditional oscillometry-based principle where the strap of the wrist-worn device acts as a bp cuff and allows bp measurement for up to days on a single charge. it has additional abilities such as data synchronisation to an online account as well as step count and heart rate monitoring. however, this device is yet to be validated. best medical therapy compliance monitoring non-adherence to medication in hypertensive patients is a contributing factor to poor bp control and is associated with higher risks of vascular events, hospitalisation and increased healthcare costs. davidson et al. developed and validated a telemonitoring intervention involving electronic medication trays, bluetooth-enabled bp monitors and sms messaging. the electronic medication tray (maya, medminder) reminded patients to take their dose at the prescribed time first with a blinking light, then a -minute chime and finally an automated phone call or sms. patients were reminded to use the bluetooth-enabled bp monitor (ua- plusbt, a&d engineering) via sms, and readings were sent from the device to a provided smartphone and a remote data repository via cellular network. if bp measurements were above a predefined threshold, patients were instructed via phone to obtain additional measurements and their physician was alerted. patients in the telemonitoring group had significant lower systolic and diastolic bp across the study’s duration and a significantly greater proportion achieved bp control. frias et al. validated a system involving ingestible sensors and wearable sensor patches (proteus discover, proteus digital health) for patients with uncontrolled hypertension and type diabetes. the medications of patients in the intervention group were co- encapsulated with ingestible sensors. once swallowed, the ingestible sensor would activate and send a signal to the adhesive sensor patch. data from the patch was then transmitted to an online data repository via a smartphone app. data could be viewed by patients on the mobile app and remotely by their physicians. the mobile app also prompted patients to take their medications at the prescribed times. there were significant improvements in systolic bp and a larger proportion of patients achieved their bp goal in the intervention group. in terms of diabetes, however, there was no significant difference in hba c or fasting plasma glucose between intervention and control groups. physicians with access to the online data made approximately three times more medical decisions per participant and patients with uncontrolled hypertension in the intervention group were more likely to be given a medication adjustment or adherence counselling. however, a comparison of adherence to medication could not be assessed due to the intrinsic design of the study. table : cont. wearable device calibration measurements provided method of measurement level of validation checkme (viatom) required with validated oscillometry-based device sbp pulse transit time good reproducibility of results but weak correlation with cuff-based bp monitors. device failed to produce measurements % of the time. freescan (maisense) required with validated oscillometry-based device sbp, dbp pulse transit time produced valid sbp and dbp measurements that met aami accuracy standards. required careful calibration. heartguide (omron) not required sbp, dbp, pulse rate, physical activity oscillometry none aami = association for the advancement of medical instrumentation; dbp = diastolic blood pressure; esh = european society of hypertension; raix = radial augmentation index; sbp = systolic blood pressure. wearable technologies and telemonitoring for vascular disease va s c u l a r & e n d o va s c u l a r r e v i e w discussion the nhs long term plan pushes for a digital transformation where digitally enabled care will become mainstream. as the nhs shifts towards a digital-first approach for patient consultations, there must be robust and validated methods for remote patient monitoring to aid analysis and decision-making by clinicians. concurrently, wearable technologies are increasingly ubiquitous, with an estimated million devices connected to the internet. , these trends are set to continue, with shipments of wearable devices expected to almost double from to , increasing from million to . million. as these technologies become more widespread, clinicians have the responsibility to be early adopters, harnessing the potential they possess especially regarding their telemonitoring properties. however, before these technologies can be implemented clinically, a number of issues must first be addressed. compliance and the digital divide the digital divide is the difference between those who possess technological skills and those who do not. cardiovascular diseases most commonly manifest in older patients, the cohort with the lowest rates of smartphone adoption and digital literacy. it has been suggested that older adults are reluctant to use mobile electronic devices due to factors such as lack of knowledge, disinterest or vision impairment. this potentially limits the use of wearable technology in this population. poor adherence has also been demonstrated in other groups. several studies included in this review mentioned limitations of technology due to know-how or non-use. ogink et al. found that only % of participants correctly performed bp measurements with their cuffless monitor after instruction. however, the authors suggested that education using a set procedure or an instructional video may increase correct usage. , , nicolaï et al. found that % of patients in the wearable intervention group did not use the prescribed fitness tracker for the duration of the study or at all. similarly, endicott et al. reported % non-use of fitness trackers by participants. although adherence to the use of fitness trackers was very good (> %) in a series of trials by gardner et al., it was noted that participants were volunteers. , this exposes the study to selection bias, where patients who were more comfortable with technology or were more motivated were more likely to participate. further work is needed to determine digital literacy in vascular patients. this could be achieved through validated literacy questionnaires such as eheals for ehealth literacy or mdpq- for mobile device proficiency. , thus, appropriate wearable and telemonitoring interventions may be used and adequate coaching given to participants. digital infrastructure the expansion of wearable and telemonitoring interventions is limited by infrastructure. hovey et al. investigated several practical aspects that negatively affected telemonitoring. suboptimal internet connectivity, especially in rural areas, affected data collection and patient adherence. there were hardware issues that needed to be diagnosed remotely and required replacement equipment, thereby reducing patient access and increasing costs. software issues were identified that could affect large-scale implementations of telemonitoring. for successful wearable and telemonitoring interventions, there is a need for robust and reliable infrastructure. larger-scale extended studies could be conducted to validate the large-scale extension of a wearable or telemonitoring intervention. cost the up-front cost of novel technology is often cited as prohibitive to widespread adoption. in fact, wearable and telemonitoring interventions may have cost benefits compared with conventional management. with regards to pad management, a standard -month supervised exercise programme was estimated to cost about £ –£ per patient in a study. in comparison, fitness trackers can cost as little as £ per unit, although this can be considerably higher with more advanced devices. furthermore, this cost can be offset by the reduction in travel expenses for the patient. these interventions additionally allow the individual tailoring of exercise requirements for each patient, which cannot be achieved with group supervised exercise. similarly, telemonitoring hypertension interventions may be cost- efficient. low adherence to antihypertensive therapy was estimated to cost us$ , per patient over a -year period. in a study, davidson et al. estimated the cost of their telemonitoring intervention (electronic medication tray, sms encouragement, bluetooth bp monitor and associated support staff) to be us$ per month for patients who owned a smartphone and us$ per month for those who did not. investigators noted that the average cost of an emergency department visit to be us$ , and the intervention group had a % reduction in visits compared with those receiving standard care, therefore giving a us$ , cost saving over a -month period. in addition, as telemonitoring can potentially identify patients with white-coat hypertension. it may avoid overtreatment and associated medication costs. there is a need, however, for further work to formally evaluate cost-effectiveness of wearable and telemonitoring interventions in cardiovascular patients. conclusion wearable technologies are becoming increasingly prevalent and there is some evidence that wearable and telemonitoring interventions may be beneficial for managing vascular patients and keeping them out of hospital. with healthcare moving towards a digital future, it is inevitable that wearable devices and telemonitoring will become increasingly widespread in the clinical environment. more work is needed to validate these technologies with regards to digital literacy of patients, cost- effectiveness and supporting digital infrastructure before widespread implementation. . tehrani k, michael a. wearable technology and wearable devices: everything you need to know. wearable devices magazine . http://www.wearabledevices.com/what-is-a- wearable-device (accessed march ). . iqbal mh, aydin a, brunckhorst o, et al. a 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https://www.longtermplan.nhs.uk/online-version/chapter- -digitally-enabled-care-will-go-mainstream-across-the-nhs https://www.longtermplan.nhs.uk/online-version/chapter- -digitally-enabled-care-will-go-mainstream-across-the-nhs https://www.statista.com/statistics/ /electronic-wearable-fitness-devices-worldwide-shipments https://www.statista.com/statistics/ /electronic-wearable-fitness-devices-worldwide-shipments https://doi.org/ . /mce. . https://doi.org/ . /iuser. . https://doi.org/ . /iuser. . http://www.ncbi.nlm.nih.gov/pubmed/ https://doi.org/ . / https://doi.org/ . / https://doi.org/ . /jmir. https://doi.org/ . /jmir. https://doi.org/ . /tmj. . https://doi.org/ . /j.ejvs. . . op-brai .. letter to the editor rsrc loss-of-function variants cause mild to moderate autosomal recessive intellectual disability marcello scala, , , majid mojarrad, , , saima riazuddin, karlla w. brigatti, zineb ammous, julie s. cohen, heba hosny, muhammad a. usmani, mohsin shahzad, sheikh riazuddin, , valentina stanley, atiye eslahi, , richard e. person, hasnaa m. elbendary, anne m. comi, laura poskitt, vincenzo salpietro, , , queen square genomics, jill a. rosenfeld, katie b. williams, dana marafi, fan xia, marta biderman waberski, maha s. zaki, joseph gleeson, erik puffenberger, henry houlden and reza maroofian ucl queen square institute of neurology, university college london, london, uk department of neurosciences, rehabilitation, ophthalmology, genetics, maternal and child health, university of genoa, genoa, italy pediatric neurology and muscular diseases unit, irccs istituto giannina gaslini, genoa, italy department of medical genetics, faculty of medicine, mashhad university of medical sciences, mashhad, iran medical genetics research center, mashhad university of medical sciences, mashhad, iran genetic center of khorasan razavi, mashhad, iran department of otorhinolaryngology head and neck surgery, school of medicine, university of maryland, baltimore, md , usa clinic for special children, strasburg, pa, usa community health clinic, topeka, in, usa departments of neurology and pediatrics, kennedy krieger institute, johns hopkins medical institutions, baltimore, md, usa national institute of neuromotor system, cairo, egypt center for genetic diseases, shaheed zulfiqar ali bhutto medical university, pakistan institute of medical sciences, islamabad, pakistan national centre of excellence in molecular biology, university of the punjab, lahore , pakistan department of neuroscience, rady children’s institute for genomic medicine, howard hughes medical institute, university of california, san diego, ca, usa medical genetics research center, faculty of medicine, mashhad university of medical sciences, mashhad, iran genedx, gaithersburg, maryland, usa clinical genetics department, human genetics and genome research division, national research centre, cairo , egypt department of molecular and human genetics, baylor college of medicine, houston, tx, usa department of pediatrics, university of wisconsin hospitals and clinics, madison, wi, usa genetics and molecular biology branch, national human genome research institute, national institutes of health, bethesda, maryland, usa correspondence to: reza maroofian queen square institute of neurology university college of london queen square, wc n bg, london, uk e-mail: r.maroofian@ucl.ac.uk sir, we read with great interest the article by perez et al. ( ) on the emerging condition of intellectual disability caused by biallelic pathogenic variants in rsrc (arginine and serine rich coiled-coil ). previous genome wide associ- ation studies (gwas) by potkin et al. ( , ) had vc the author(s) ( ). published by oxford university press on behalf of the guarantors of brain. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. doi: . /brain/awaa brain : page of | d ow nloaded from https://academ ic.oup.com /brain/advance-article-abstract/doi/ . /brain/aw aa / by u c l, london user on a pril http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - suggested a possible involvement of rsrc in non-syn- dromic intellectual disability and gene regulatory networks in schizophrenia. however, the results of these studies remained elusive and were not confirmed by a subsequent gwas study by schizophrenia working group of the psychiatric genomics consortium ( ). meanwhile, berndt et al. ( ) identified rsrc as a new locus influ- encing height through a genome-wide meta-analysis. the first family in which a homozygous rsrc variant clearly segregated with non-syndromic intellectual disability was only recently reported by maddirevula et al. ( ). a pos- sible role of rsrc in major depressive disorder has been further suggested by a meta-analysis on three large gwas performed by li et al. ( ). serine and arginine-rich (sr) proteins are evolutionary con- served co-regulators of constitutive and alternative pre- mrna splicing. the longest rsrc transcript (nm_ . ) includes exons (fig. a) and encodes a -amino-acid sr-related protein of kda (srrp ) (fig. b), localized to the nuclear speckled domain. interacting with other splicing regulators, rsrc plays a rele- vant role in the second step of pre-mrna splicing. in add- ition, it could be involved in post-splicing mrna processing, shuttling between the nucleus and cytoplasm (cazalla et al., ). rsrc further promotes pias -mediated sumoylation of the pleiotropic transcription factor oestrogen receptor b (erb), acting as transcriptional regulator (chen et al., ). the involvement of rsrc in cancer was first suggested by the identification of the recurrent ptplb-rsrc in- frame gene fusion in nasopharyngeal carcinoma by valouev et al. ( ). teplyuk et al. ( ) showed that rsrc is a target gene of the microrna- b (mir- b), an oncogenic microrna that is highly expressed in glioblastoma and rep- resents a candidate for the development of targeted thera- pies. more recently, several studies have implicated rsrc in cancer predisposition and progression. the rsrc in- tronic polymorphism rs g a has been associated with neuroblastoma susceptibility (mcdaniel et al., ; tang et al., ). a possible role of rsrc in non-small- cell lung cancer tumorigenesis and progression has been hypothesized based on the data from rna sequencing data of tumour-educated platelets (sheng et al., ). rsrc has been also shown to suppress gastric cancer cell prolifer- ation and migration through the regulation of pten expres- sion, acting as tumour suppressor (yu et al., ). the first rsrc pathogenic variant segregating with intel- lectual disability was reported by maddirevula et al. ( ) in three affected siblings from a consanguineous malaysian family (supplementary table ). the homozygous truncating variant c. c t (p.arg *) (nm_ . ) resulted in a full loss of function, causing the natural knockout of the gene. all the reported patients showed developmental delay and variable degree of intellectual disability. one sub- ject also suffered from febrile seizures. brain mri was nor- mal in the -year-old male (patient ), whereas temporal lobe atrophy was found in his -year-old brother (patient ). no distinctive neurological features or facial dysmorphism were observed. more recently, perez et al. ( ) reported five further individuals from consanguineous bedouin kin- dred with early developmental delay, intellectual disability, hypotonia, behavioural abnormalities, and mild facial dys- morphic features. brain mri was normal (supplementary table ). exome sequencing revealed the homozygous c. c t (p.arg *) in rsrc (nm_ . ) in all patients, a nonsense variant leading to nonsense-mediated mrna decay. rsrc knock-down sh-sy y cells showed impaired alternative splicing. specific differential expression of genes associated with intellectual disability, hypotonia, schizophrenia, and dementia was also observed, supporting the pivotal role of rsrc in transcriptional regulation (perez et al., ). we report additional subjects from seven consanguin- eous families with intellectual disability, behavioural abnor- malities, and facial dysmorphism, harbouring homozygous rsrc loss-of-function variants (table and supplementary table ). the families were of different ancestries (european/ middle eastern, saudi, egyptian, old order amish, pakistani, and persian) (supplementary fig. ). the collabor- ation among the involved study centres was managed through genematcher (sobreira et al., ). after informed consent was obtained from the parents, photographic mater- ial was collected and genetic testing through exome sequenc- ing was performed. genetic methods are provided in detail in the supplementary material. trio-exome sequencing was con- ducted in patient , the family quartet was sequenced for patients and , and proband-exome sequencing was per- formed in all the remaining individuals, followed by variants validation through sanger sequencing. no relevant single nu- cleotide variant was identified in the siblings of amish ances- try (patients – ), who were further studied through comparative genomic hybridization (cgh) array. five rsrc sequence variants (including an intragenic duplica- tion) and two partial deletions were identified. in all the enrolled subjects, global psychomotor develop- mental delay and mild-to-moderate intellectual disability were observed. twelve patients were diagnosed with vari- able behavioural disorders (table and supplementary table ). neurological examination revealed generalized hypotonia in all patients and five of them had a history of hypotonia at birth. decreased deep tendon reflexes were found in six patients ( %). five patients showed gait ataxia, which was associated with bradykinesia in patient . truncal ataxia was observed in patient . seizures occurred in six individuals ( %), including four cases of fe- brile seizures. afebrile generalized tonic-clonic seizures in addition to febrile seizures were observed in patients , , and . patient experienced three episodes of generalized tonic-clonic seizures before becoming seizure-free. his eeg showed occasional epileptiform discharges in the centrofron- tal and in the left parietocentral regions during sleep. no re- current epileptic phenotype or peculiar eeg features were recognizable in our cohort. psychomotor regression was not observed in any case. the most common dysmorphic | brain : page of letter to the editor d ow nloaded from https://academ ic.oup.com /brain/advance-article-abstract/doi/ . /brain/aw aa / by u c l, london user on a pril https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data figure genetic findings and clinical pictures of rsrc patients. (a) schematic diagram of the longer rsrc transcript (nm_ . ) consisting of nucleotides in exons. the deletions encompassing exons and – are represented by diagonal green lines. single nucleotide variants are shown in red (previously reported patients) or in green (this study). (b) the rsrc protein (np_ . ) consists of amino acids encompassing an rs domain rich in arginine (r) and serine (s) that mediates the interactions with letter to the editor brain : page of | (continued) d ow nloaded from https://academ ic.oup.com /brain/advance-article-abstract/doi/ . /brain/aw aa / by u c l, london user on a pril features were deep-set eyes, broad nasal base, and ogival palate (fig. c). associated congenital anomalies were ex- tremely variable, ranging from simple pes planus to redun- dant skin. other isolated clinical features included mitral valve prolapse, recurrent respiratory infections in the first year of life, and tracheomalacia. when available, brain mri did not reveal any distinctive finding. non-specific mild cere- bral atrophy was observed in patients and , whereas en- largement of subarachnoid spaces was found in patients and . delayed myelination, dysmorphic lateral ventricles, and unilateral focal polymicrogyria were observed in patient . in our cohort, we identified homozygous rsrc variants or deletions leading to loss of function (table and supplementary table ). variants’ nomenclatures are given with reference to the rsrc transcript nm_ . . the four amish siblings (patients – ) carried an -kb dele- tion encompassing exons – of rsrc and the entire mlf gene. although mlf is involved in haematopoiesis and leukemogenesis, a possible pathogenic role for this gene in some of the dysmorphic features and congenital anomalies in the amish patients cannot be excluded (yoneda-kato et al., ; nakamae et al., ). a second -bp dele- tion involving exon of rsrc was identified in the sib- lings from the pakistani family (patients and ). both these rearrangements are novel and are not reported in clinvar and decipher databases (supplementary table ). the three patients from the second pakistani family (patients – ) carried the splicing variant c. - g a. this amino acid change is predicted to cause aberrant splicing through the alteration of the splice acceptor site at exon of rsrc (supplementary table ). in the remaining fami- lies, homozygous stop-gain or start-loss variants were iden- tified. the -year-old female of european ancestry (patient ) harboured the intragenic duplication c. _ dupagaaaag (p.glu argfs* ). the stop- gain variants c. c t (p.gln *) and c. c t (p.arg *) were identified in the siblings from the persian (patients – ) and middle east (patient ) families, re- spectively. in the egyptian family (patients – ), the variant c. g t (p.met ?) causing a start loss and leading to full loss of function (null variant) was found. all the variants fully segregated with the phenotype and were not found in the most common genome databases, including the genome aggregation database (gnomad), iranome, greater middle east variome project (gme variome), and our database of in-house control exomes. the only exception was the splicing variant c. - g a, which was observed in heterozygous state in of in gnomad (allele frequency . ). furthermore, the start loss variant affecting the same nucleo- tide of the c. g t (p.met ?) variant observed in our egyptian family is reported in heterozygous state in gnomad (genomes allele frequency . ). however, neither variant has been reported in homozygous state in healthy individuals. the analysis of sequence conservation through genomic evolutionary rate profiling (gerp) score revealed good conservation of the affected residues and in silico prediction analysis through cadd score calculation revealed high-deleterious scores (supplementary table ). all variants were predicted to be damaging or likely damaging by several bioinformatic tools (e.g. sift, mutationtaster, and human splice finder) and were classi- fied as pathogenic (class ) or likely pathogenic (class ) according to the american college of medical genetics and genomics (acmg) guidelines (richards et al., ). this study supports the idea that rsrc pathogenic var- iants cause a non-syndromic disorder characterized by mild- to-moderate intellectual disability, generalized hypotonia, and variable neurological and behavioural features (fig. d). despite a few minor dysmorphic features being observed (es- pecially deep-set eyes and broad nasal base), a consistent re- current facial gestalt could not be recognized. furthermore, the extremely variable associated non-neurological features were not suggestive of a syndromic condition. even though seizures are common in rsrc patients, most of them only figure continued other rs-rich proteins involved in splicing regulation (sf /asf and u af ) and a coiled coil domain required for rsrc /erb interaction as well as the enhancement of erb sumoylation. the residues k and k are necessary for rsrc sumoylation by sumo and the e ligases pias and pias . pathogenic amino acid changes reported in previous papers and identified in this study are shown in red and green, respectively. (c) sequential pictures from selected patients. patients from the amish family (patients – ) show prominent forehead, deep set eyes, depressed nasal bridge, protruding ears, and overbite with drooling. redundant skin is evident in patient . the persian patient (patient ) shows straight eyebrows with mild synophrys, deep set eyes, and protruding ears. in the first pakistani family (patients and ) dysmorphic features include straight eyebrows with mild medial flaring, deep set eyes, wide nasal base, short philtrum, uplifted earlobes, and prominent chin. subjects from the second pakistani family (patients – ) also show straight eyebrows with mild synophrys and deep set eyes, in addition to protruding ears with uplifted lobes (patient ). patients from the egyptian family (patients – ) show thick eyebrows with medial sparing, deep set eyes, and prominent columella. (d) graphic illustrations of the most common clinical findings in rsrc patients in our cohort: the bar graph shows the percent distribution of the cardinal features of rsrc -related intellectual disability, with the grey lines representing not available data; the pie charts show the percentage distribution of developmental delay/intellectual disability and the different behavioural abnormalities observed in rsrc patients. gene transcript and protein details available at: https://www.ensembl.org (rsrc - , transcript id enst . ), https://www.nextprot.org (nx_q iz ), https://www.uniprot.org (q iz ), https://www.proteomicsdb.org (q iz ). adhd = attention def- icit hyperactivity disorder; asd = autism spectrum disorder; dd = developmental delay; id = intellectual disability; n/a = not available; pt = patient; sct = sluggish cognitive tempo; tt = temper tantrums. | brain : page of letter to the editor d ow nloaded from https://academ ic.oup.com /brain/advance-article-abstract/doi/ . /brain/aw aa / by u c l, london user on a pril https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data https://www.ensembl.org https://www.nextprot.org https://www.uniprot.org https://www.proteomicsdb.org t a b le i s u m m a r y o f g e n e ti c fi n d in g s a n d c li n ic a l fe a tu r e s o f r s r c p a ti e n ts f a m il y f a m il y i (a m is h ) f a m il y ii (p e r s ia n ) f a m il y ii i (p a k is ta n i) f a m il y iv (s a u d i) f a m il y v (e u r / m e ) f a m il y v i (p a k is ta n i) f a m il y v ii (e g y p ti a n ) m a d d ir e v u la e t a l, (m a la y s ia n ) p e r e z e t a l, (b e d o u in ) p a ti e n t (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) (i i- ) p a ti e n ts p a ti e n ts a g e /s e x y /f y /m y /m m o /m y /m y /f y /f y /m y /m y /m y /f y /f y /m y /f y /f y /f y /f – y / m , f . – y / f, m h o m o zy g o u s r s r c v a ri a n ts [n m _ . ] c . , , _ , , d e la c . c t (p .g ln *) c . , , _ , , d e lb c . c t (p .a rg *) c . _ d u p a g a a a a g (p .g lu a rg fs * ) c . - g a c . g t (p .m e t ?) c . c t (p .a rg *) c . c t (p .a rg *) c o n sa n g u in it y + + + + + + + + + + + + + + + + + + ( ) + ( ) d y sm o rp h ic fe a tu re s + + + + + + + – – + + + + + – – – – + ( ) g lo b a l d d /i d + + + + + + + + + + + + + + + + + + + + + + + + + + + + ( ) + / + + ( ) s p e e c h d e la y n /a + + + n /a n /a n /a n /a n /a + + + + – – + – + ( ) + ( ) b e h av io u ra l a b n o rm a li ti e s – a s d a d h d , a s d – a s d a s d a s d a d h d a d h d – s c t a g g a g g s c t a s d a g g a s d – t t ( ), a s d ( ), a d h d ( ) h y p o to n ia + + + + + + + + + + + + + + + + + + + + + + + + + – + ( ) m o v e m e n t d is o rd e rs – g a it a ta x ia g a it a ta x ia – g a it a ta x ia g a it a ta x ia – – – t ru n c a l a ta x ia b ra d y -k in e si a , a ta x ic g a it – – – – – – – f in e m o to r im p a ir m e n t ( ) s e iz u re s – – – – n /a – f s , g t c s f s – g t c s – f s , g t c s f s , g t c s n /a – – f s f s ( ) f s ( ), e p il e p sy ( ) m u sc u lo -s k e le ta l a b n o rm a li ti e s – p p, c v p p, c v – – – – p p p p – p p, c v , sh o rt to e s p p, sh o rt fi ft h to e s c v , g e n u v a lg u m p p p p p p – f o o t d e fo rm it ie s ( ) – b ra in m r i n p s s p s s n /a m c a n n n n l e ft te m p o ro - p a ri e ta l a tr o p h y n n /a n /a n /a n n n t e m p o ra l lo b e s a tr o p h y ( ) n ( ) a d h d = a tt e n ti o n d e fi c it h y p e ra c ti v it y d is o rd e r; a g g = a g g re ss iv e ; a s d = a u ti sm sp e c tr u m d is o rd e r; c v = c u b it u s v a lg u s; d d = d e ve lo p m e n ta l d e la y ; e u r = e u ro p e a n ; f s = fe b ri le se iz u re s; g t c s = g e n e ra li ze d to n ic -c lo n ic se iz u re s; id = in te ll e c tu a l d is a b il it y ( + , m il d ; + + , m o d e ra te ); m c a = m il d c e re b ra l a tr o p h y ; m e = m id d le e a st e rn ; m o = m o n th s; n = n o rm a l; n /a = n o t av a il a b le ; p p = p e s p la n u s; p s s = p ro m in e n t su b a ra c h n o id sp a c e s; s c t = sl u g g is h c o g n it iv e te m p o ; y = y e a rs . a h g ; -k b d e le ti o n e n c o m p a ss in g e x o n s – o f r s r c a n d w h o le m lf . b h g ; -b p d e le ti o n e n c o m p a ss in g e x o n o f r s r c . letter to the editor brain : page of | d ow nloaded from https://academ ic.oup.com /brain/advance-article-abstract/doi/ . /brain/aw aa / by u c l, london user on a pril suffer from febrile seizures, lacking a distinctive epileptic phenotype. definite epileptic seizures were only diagnosed in patient and in a case reported by perez et al. ( ). this patient suffered from focal seizures with impaired awareness occasionally progressing to tonic-clonic and was treated with valproic acid. behavioural abnormalities are frequent, although extremely variable and ranging from attention deficit hyperactivity disorder (adhd) to autism spectrum disorder (asd) (fig. d). besides the non-specific neuroradiological abnormalities observed in our patients, bilateral symmetric temporal lobe atrophy has been described in a single case by maddirevula et al. ( ). even though further neuroimaging studies will be necessary, the limited data available support the lack of a peculiar neuroradiological phenotype. according to these observations, biallelic rsrc variants should be consid- ered the cause of a non-syndromic intellectual disability mainly associated with generalized hypotonia and behav- ioural disturbances. facial dysmorphism and other minor clinical features have limited diagnostic relevance, likewise neuroimaging is of limited value. thanks to the remarkable advances in gene discovery achieved through exome sequencing, the large group of known genes causing non-syndromic intellectual disability is rapidly expanding with relevant impact on diagnosis and pa- tient management. our findings support the pathogenic role of biallelic loss-of-function rsrc variants in autosomal re- cessive intellectual disability, in addition to contributing to the phenotypic delineation of this emerging condition. global developmental delay, mild-to-moderate intellectual disability, behavioural abnormalities, and generalized hypotonia repre- sent the cardinal features of rsrc -related intellectual dis- ability. other neurological features may be less frequently observed, especially hyporeflexia and febrile seizures. further studies will help clarify the possible role of neuroimaging in the diagnostic process. in conclusion, we suggest that the in- volvement of rsrc should be considered in the differential diagnosis in intellectually disabled children with hypotonia and behavioural disturbances, and that rsrc should be included in next generation sequencing (ngs) panels for in- tellectual disability. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. web resources the following urls were used for data presented herein: clinvar; https://www.ncbi.nlm.nih.gov/clinvar combined annotation dependent depletion (cadd); http:// cadd.gs.washington.edu decipher; https://decipher.sanger.ac.uk ensembl; https://www.ensembl.org/index.html gene cards; http://www.genecards.org gene matcher; http://www.genematcher.org genome aggregation database (gnomad); http://gnomad. broadinstitute.org greater middle east (gme) variome project; http://igm. ucsd.edu/gme/ human splice finder; http://www.umd.be/hsf iranome; http://www.iranome.ir mutalyzer; https://mutalyzer.nl mutation taster; http://www.mutationtaster.org nextprot; https://www.nextprot.org online mendelian inheritance in man; http://www.ncbi.nlm. nih.gov/omim proteomics db; https://www.proteomicsdb.org pubmed; http://www.ncbi.nlm.nih.gov/pubmed refseq; https://www.ncbi.nlm.nih.gov/refseq sift; https://sift.bii.a-star.edu.sg the genomes browser; http://browser. genomes. org/index.html the greater middle east (gme) variome project; http://igm. ucsd.edu/gme/index.php uniprot; https://www.uniprot.org ucsc human genome database; http://www.genome.ucsc. edu varsome; https://varsome.com acknowledgements the authors would like to thank the patients’ families for their support and consent to the publication of this study. this research was conducted as part of the queen square genomics group at university college london, supported by the national institute for health research university college london hospitals biomedical research centre. funding the work at university of maryland, baltimore, usa was supported by national institute of neurological disorders and stroke (ninds) (r ns ) (to s.r.). this study was funded by the medical research council (mrc) (mr/ s x/ , mr/s / , g ), the national institute for health research university college london hospitals biomedical research centre, rosetree trust, ataxia uk, multiple system atrophy trust, brain research uk, sparks great ormond street hospital charity, muscular dystrophy uk (mduk), muscular dystrophy association (mda usa). competing interests j.c. is consultant to invitae. r.e.p. is an employee of genedx, inc. the department of molecular and human genetics at baylor college of medicine receives revenue from clinical genetic testing completed at baylor genetics. | brain : page of letter to the editor d ow nloaded from https://academ ic.oup.com /brain/advance-article-abstract/doi/ . /brain/aw aa / by u c l, london user on a pril https://www.ncbi.nlm.nih.gov/clinvar http://cadd.gs.washington.edu http://cadd.gs.washington.edu https://decipher.sanger.ac.uk https://www.ensembl.org/index.html http://www.genecards.org http://www.genematcher.org http://gnomad.broadinstitute.org http://gnomad.broadinstitute.org http://igm.ucsd.edu/gme/ http://igm.ucsd.edu/gme/ http://www.umd.be/hsf http://www.iranome.ir https://mutalyzer.nl http://www.mutationtaster.org https://www.nextprot.org http://www.ncbi.nlm.nih.gov/omim http://www.ncbi.nlm.nih.gov/omim https://www.proteomicsdb.org http://www.ncbi.nlm.nih.gov/pubmed https://www.ncbi.nlm.nih.gov/refseq https://sift.bii.a-star.edu.sg http://browser. genomes.org/index.html http://browser. genomes.org/index.html http://igm.ucsd.edu/gme/index.php http://igm.ucsd.edu/gme/index.php https://www.uniprot.org http://www.genome.ucsc.edu http://www.genome.ucsc.edu https://varsome.com supplementary material supplementary material is available at brain online. references berndt si, gustafsson s, mägi r, ganna a, wheeler e, feitosa mf, et al. genome-wide meta-analysis identifies new loci for an- thropometric traits and provides insights into genetic architecture. nat genet ; : – . cazalla d, newton k, cáceres jf. a novel sr-related protein is required for the second step of pre-mrna splicing. mol cell biol ; : – . chen l, li w, qiu w, ren w, li q, han b, et al. rsrc sumoylation enhances sumoylation and inhibits transcriptional activity of estrogen receptor b. febs lett ; : – . li x, luo z, gu c, hall ls, mcintosh am, zeng y, et al. common variants on q . , q . and p . are associated with major depressive disorder. neuropsychopharmacology ; : – . maddirevula s, al zahrani f, anazi s, almureikhi m, ben-omran t, abdel-salam gmh, et al. gwas signals revisited using human knockouts. genet med ; : – . mcdaniel ld, conkrite kl, chang x, capasso m, vaksman z, oldridge da, et al. common variants upstream of mlf at q and within cpz at p associated with neuroblastoma. plos genet ; : e . nakamae i, kato jy, yokoyama t, ito h, yoneda-kato n. myeloid leukemia factor stabilizes tumor suppressor c/ebpa to prevent trib -driven acute myeloid leukemia. blood adv ; : – . perez y, menascu s, cohen i, kadir r, basha o, shorer z, et al. rsrc mutation affects intellect and behaviour through aberrant splicing and transcription, downregulating igfbp . brain ; : – . potkin sg, macciardi f, guffanti g, fallon jh, wang q, turner ja, et al. identifying gene regulatory networks in schizophrenia. neuroimage ; : – . potkin sg, turner ja, fallon ja, lakatos a, keator db, guffanti g, et al. gene discovery through imaging genetics: identification of two novel genes associated with schizophrenia. mol psychiatry ; : – . richards s, aziz n, bale s, bick d, das s, gastier-foster j, et al. standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology. genet med ; : – . schizophrenia working group of the psychiatric genomics consortium. biological insights from schizophrenia-associated genetic loci. nature ; : – . sheng m, dong z, xie y. identification of tumor-educated platelet biomarkers of non-small-cell lung cancer. ono targets ther ; : – . sobreira n, schiettecatte f, valle d, hamosh a. genematcher: a matching tool for connecting investigators with an interest in the same gene. hum mutat ; : – . tang j, liu w, zhu j, zhang j, wang fh, liang jh, et al. rsrc and cpz gene polymorphisms with neuroblastoma susceptibility in chinese children. gene ; : – . teplyuk nm, uhlmann ej, gabriely g, volfovsky n, wang y, teng j, et al. therapeutic potential of targeting microrna- b in estab- lished intracranial glioblastoma: first steps toward the clinic. embo mol med ; : – . valouev a, weng z, sweeney rt, varma s, le qt, kong c, et al. discovery of recurrent structural variants in nasopharyngeal carcin- oma. genome res ; : – . yoneda-kato n, look at, kirstein mn, valentine mb, raimondi sc, cohen kj, et al. the t( ; )(q . ; q ) of myelodysplastic syn- drome and acute myeloid leukemia produces a novel fusion gene, npm-mlf . oncogene ; : – . yu s, gautam n, quan m, gao y. rsrc suppresses gastric cancer cell proliferation and migration by regulating pten expression. mol med rep ; : – . letter to the editor brain : page of | d ow nloaded from https://academ ic.oup.com /brain/advance-article-abstract/doi/ . /brain/aw aa / by u c l, london user on a pril https://academic.oup.com/brain/article-lookup/doi/ . /brain/awaa #supplementary-data 	awaa -tf 	awaa -tf 	awaa -tf testing the gene or testing a variant? the case of tcf l mark o. goodarzi , , and jerome i. rotter , , , g iven that susceptibility to type diabetes ap- pears in large measure due to genetic makeup, investigators have spent years of effort trying to identify genes that influence type diabetes risk. an important goal of gene identification is to improve our understanding of the pathophysiology of diabetes, leading to new measures of diagnosis, prevention, and treatment. a diabetes gene is considered identified when variants in that gene (more specifically, variation in dna sequence between individuals) are found to be associated with type diabetes and/or its pathophysiologic abnormal- ities such as insulin resistance or secretion. tcf l (transcription factor -like ) was identified as a gene for type diabetes in ( ). notably, its effect on diabetes (relative risk . – . ) is substantially larger than previously established diabetes genes (e.g., peroxisome proliferator–activated receptor � [pparg] and �-cell in- wardly rectifying k� channel kir . [kcnj ], relative risk � . each). most studies of tcf l have focused on the genetic variants that were implicated in the original report ( ), largely ignoring the remainder of the gene. in this issue of diabetes, investigators took the alternative approach of examining variants across the entire gene, which allowed them to discover a completely novel variant in tcf l that affects diabetes risk ( ). we believe this approach to genetic association studies is of great merit, as described below. in the report that first identified tcf l as a diabetes gene, a microsatellite (dg s ) was highly associated with type diabetes (in three caucasian cohorts), as were five single nucleotide polymorphisms (snps) in linkage disequilibrium (ld). of those five snps, two (rs and rs ) were identified as most strongly associated with type diabetes; subsequent reports determined snp rs had the greatest effect ( , ). deep resequencing of exons and the surrounding region has not identified any variants with stronger effect ( ). over articles subse- quently described the role of tcf l in multiple cohorts, firmly establishing the gene’s place among diabetes genes ( ). quantitative phenotypic associations ( , , ) and its role in proglucagon gene expression ( ) suggest that tcf l modulates diabetes risk via impaired insulin se- cretion. further support that tcf l influences insulin secretion comes from an article recently published in diabetes, wherein genetic variation in tcf l was shown to influence efficacy of sulfonylureas (agents that promote insulin secretion) but not efficacy of metformin (insulin sensitizer) ( ). the majority of the subsequent studies followed the lead of the original study, genotyping either the one or two most associated snps, or all five, with or without the microsatellite dg s . these variants are in a haplotype block that encompasses dg s and includes part of intron , all of exon , and part of intron ( ). in the few instances where more extensive snp genotyping was performed, it was usually centered around dg s ( , , ). as a result, snp rs (specifically the t-allele) has been firmly established as increasing risk of type diabetes in multiple european populations and in caucasians, in general, and including diverse groups such as mexicans, amish, indian asians, and moroccans ( ). some studies of individuals of african descent were not able to document association of tcf l with diabetes ( , ), while others, including a study in this issue of diabetes, were positive for rs ( , ). unlike in caucasians, the two most associated snps exhibit weak ld in africans, allowing determination of the greater role of rs than rs in diabetes susceptibility. the functional role that this intronic snp may play is still unknown. furthermore, it does not explain the linkage signal that originally led investigators to chromosome q ( ), raising the possibility that other variants in the region (in tcf l or other genes) may predispose to type diabetes. other plausible candidate genes do exist on chromosome q ( , ). the near-exclusive focus on rs and snps in ld with it has probably delayed the discovery of other vari- ants in tcf l that may affect risk of type diabetes. in asian populations, the frequencies of snps rs and rs are quite low. nevertheless, association with these snps was identified in two large japanese cohorts (� , cases each), where the minor allele frequency (maf) ranged from to % ( , ). the large size of these cohorts provided adequate power for discovery despite the rarity of the variants examined. this was fortuitous; had the allele frequencies or sample sizes been slightly lower, false-negative studies would have been the likely result, even though the gene and those variants are pro- ducing risk in these populations. from the division of endocrinology, diabetes and metabolism, cedars-sinai medical center, los angeles, california; the medical genetics institute, cedars-sinai medical center, los angeles, california; the department of medicine, university of california, los angeles, los angeles, california; the department of pediatrics, university of california, los angeles, los angeles, california; and the department of human genetics, university of california, los angeles, los angeles, california. address correspondence and reprint requests to mark o. goodarzi, md, phd, cedars-sinai medical center, division of endocrinology, diabetes and metabolism, beverly blvd., becker b- , los angeles, ca . e-mail: mark.goodarzi@cshs.org. received for publication july and accepted in revised form july . ld, linkage disequilibrium; maf, minor allele frequency; snp, single nucleotide polymorphism. doi: . /db - © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. commentary diabetes, vol. , october in the study of chinese han ( case and control subjects) reported in this issue ( ), if the investigators had followed the usual strategy of looking only at the original associated snps, they would have found no associations. in chinese, the rarity of rs (maf – . %) would require , cases to detect an association with an effect size similar to that in caucasians ( ). fortunately, these investigators tested the entire tcf l gene rather than only particular variants of prior interest. in addition to genotyping snps previously associated with type diabe- tes, they used information from hapmap ( ) to select a set of snps to capture the majority of snps in the gene with maf � %. the “classic” snps showed no associa- tion; however, snp rs at the � end of the gene, as well as haplotypes including this snp, were associated with type diabetes. the comprehensive approach of this study led to this identification of a tcf l snp not in ld with rs that may affect type diabetes risk. this demonstrates the utility of the global gene approach. given the narrow focus of prior studies of tcf l , no other study to date has examined this recently identified � variant. those groups with large diabetic cohorts should go back and examine this variant and its neighbors for a role in type diabetes, which is feasible given that rs occurs with appreciable frequency in all hapmap populations except yorubans. hapmap also reveals that this snp is not well captured by other snps (at r � . ), indicating that its effect is likely to be missed if the snp itself is not genotyped. the only other study to take a global approach to tcf l (challenging with this large [� kb] gene) was carried out in mexican americans ( ). this study did not genotype rs but did geno- type rs , also in the � end, with negative results. notably, one of the recent genome-wide association stud- ies for type diabetes found that rs was associated with type diabetes (a finding eclipsed by the stronger association of rs ld group snps) ( ). this latter observation and the data of chang et al. ( ) suggest the potential functional importance of variation in the � end of the gene, which may influence alternative splicing of tcf l , in which there exist many alternative splice sites ( ). clearly this end of the gene deserves the kind of attention that the upstream region of rs has received. when follow-up gene marker studies focus only on the original associated variants, they amount only to replica- tion studies that will not result in novel discoveries unless they examine a related but different phenotype, e.g., response to treatment ( ), or extend the result to addi- tional populations ( ). to test the entire gene, compre- hensive genotyping such as that carried out by chang et al. ( ) is necessary. this is particularly important if the replication cohort is not of the same ethnic group as the original cohort. comprehensive global genotyping leads to a complete understanding of the snp frequencies and haplotype structure, which may account for differences in association results. given the availability of the hapmap database, and the falling price of genotyping, investigators now more readily have the option to test the entire gene (or region) rather than only a particular variant. references . grant sf, thorleifsson g, reynisdottir i, benediktsson r, manolescu a, sainz j, helgason a, stefansson h, emilsson v, helgadottir a, styrkars- dottir u, magnusson kp, walters gb, palsdottir e, jonsdottir t, gud- mundsdottir t, gylfason a, saemundsdottir j, wilensky rl, reilly mp, rader dj, bagger y, christiansen c, gudnason v, sigurdsson g, thor- steinsdottir u, gulcher jr, kong a, stefansson k: variant of transcription factor -like (tcf l ) gene confers risk of type diabetes. nat genet : – , . chang yc, chang tj, jiang yd, kuo ss, lee kc, chiu kc, chuang lm: association study of the genetic polymorphisms of the transcription factor -like (tcf l ) gene and type diabetes in the chinese population. diabetes : – , . saxena r, gianniny l, burtt np, lyssenko v, giuducci c, sjogren m, florez jc, almgren p, isomaa b, orho-melander m, lindblad u, daly mj, tuomi t, hirschhorn jn, ardlie kg, groop lc, altshuler d: common single nucleotide polymorphisms in tcf l are reproducibly associated with type diabetes and reduce the insulin response to glucose in nondiabetic individuals. diabetes : – , . helgason a, palsson s, thorleifsson g, grant sf, emilsson v, gunnars- dottir s, adeyemo a, chen y, chen g, reynisdottir i, benediktsson r, hinney a, hansen t, andersen g, borch-johnsen k, jorgensen t, schafer h, faruque m, doumatey a, zhou j, wilensky rl, reilly mp, rader dj, bagger y, christiansen c, sigurdsson g, hebebrand j, pedersen o, thorsteinsdottir u, gulcher jr, kong a, rotimi c, stefansson k: refining the impact of tcf l gene variants on type diabetes and adaptive evolution. nat genet : – , . florez jc: the new type diabetes gene tcf l . curr opin clin nutr metab care : – , . damcott cm, pollin ti, reinhart lj, ott sh, shen h, silver kd, mitchell bd, shuldiner ar: polymorphisms in the transcription factor -like (tcf l ) gene are associated with type diabetes in the amish: replica- tion and evidence for a role in both insulin secretion and insulin resistance. diabetes : – , . florez jc, jablonski ka, bayley n, pollin ti, de bakker pi, shuldiner ar, knowler wc, nathan dm, altshuler d: tcf l polymorphisms and progression to diabetes in the diabetes prevention program. n engl j med : – , . yi f, brubaker pl, jin t: tcf- mediates cell type-specific regulation of proglucagon gene expression by beta-catenin and glycogen synthase kinase- beta. j biol chem : – , . pearson er, donnelly la, kimber c, whitley a, doney as, mccarthy mi, hattersley at, morris ad, palmer cn: variation in tcf l influences therapeutic response to sulfonylureas: a godarts study. diabetes : – , . watanabe rm, allayee h, xiang ah, trigo e, hartiala j, lawrence jm, buchanan ta: transcription factor -like (tcf l ) is associated with gestational diabetes mellitus and interacts with adiposity to alter insulin secretion in mexican americans. diabetes : – , . scott lj, bonnycastle ll, willer cj, sprau ag, jackson au, narisu n, duren wl, chines ps, stringham hm, erdos mr, valle tt, tuomilehto j, bergman rn, mohlke kl, collins fs, boehnke m: association of transcrip- tion factor -like (tcf l ) variants with type diabetes in a finnish sample. diabetes : – , . humphries se, gable d, cooper ja, ireland h, stephens jw, hurel sj, li kw, palmen j, miller ma, cappuccio fp, elkeles r, godsland i, miller gj, talmud pj: common variants in the tcf l gene and predisposition to type diabetes in uk european whites, indian asians and afro-caribbean men and women. j mol med : – , . elbein sc, chu ws, das sk, yao-borengasser a, hasstedt sj, wang h, rasouli n, kern pa: transcription factor -like polymorphisms and type diabetes, glucose homeostasis traits and gene expression in us partici- pants of european and african descent. diabetologia : – , . sale mm, smith sg, mychaleckyj jc, keene kl, langefeld cd, leak ts, hicks pj, bowden dw, rich ss, freedman bi: variants of the transcription factor -like (tcf l ) gene are associated with type diabetes in an african-american population enriched for nephropathy. diabetes : – , . goodarzi mo, lehman dm, taylor kd, guo x, cui j, quinones mj, clee sm, yandell bs, blangero j, hsueh wa, attie ad, stern mp, rotter ji: sorcs : a novel human type diabetes susceptibility gene suggested by the mouse. diabetes : – , . lehman dm, fu dj, freeman ab, hunt kj, leach rj, johnson-pais t, hamlington j, dyer td, arya r, abboud h, goring hh, duggirala r, blangero j, konrad rj, stern mp: a single nucleotide polymorphism in mgea encoding o-glcnac-selective n-acetyl-�-d glucosaminidase is as- sociated with type diabetes in mexican americans. diabetes : – , . hayashi t, iwamoto y, kaku k, hirose h, maeda s: replication study for the association of tcf l with susceptibility to type diabetes in a japanese population. diabetologia : – , . horikoshi m, hara k, ito c, nagai r, froguel p, kadowaki t: a genetic tcf l : gene or variant diabetes, vol. , october variation of the transcription factor -like gene is associated with risk of type diabetes in the japanese population. diabetologia : – , . the international hapmap consortium: the international hapmap project. nature : – , . lehman dm, hunt kj, leach rj, hamlington j, arya r, abboud he, duggirala r, blangero j, goring hh, stern mp: haplotypes of transcription factor -like (tcf l ) gene and its upstream region are associated with type diabetes and age of onset in mexican americans. diabetes : – , . sladek r, rocheleau g, rung j, dina c, shen l, serre d, boutin p, vincent d, belisle a, hadjadj s, balkau b, heude b, charpentier g, hudson tj, montpetit a, pshezhetsky av, prentki m, posner bi, balding dj, meyre d, polychronakos c, froguel p: a genome-wide association study identifies novel risk loci for type diabetes. nature : – , . duval a, rolland s, tubacher e, bui h, thomas g, hamelin r: the human t-cell transcription factor- gene: structure, extensive characterization of alternative splicings, and mutational analysis in colorectal cancer cell lines. cancer res : – , m.o. goodarzi and j.i. rotter diabetes, vol. , october wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top 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the posi- tion of the head in the anterior-posterior plane. in none of the others could we demonstrate a vestibular (otolith) mecha- nism. despite these negative observations for a clear otolith influence upon head tremor electrophysiological measurements have shown that otolith spinal reflexes may be modulated by both head and body position. thus there still remains the possibility that a change in head or body position may in itself modify vestibular influences upon body movement. s mossman l cleeves l findley mrc human movement and balance unit, section of neuro-otology, national hospital for neurology and neurosurgery, london, uk correspondence to: dr mossman, department of neurology, wellington hospital, private bag, wel- lington south, wellington, new zealand. denny-brown d. clinical symptomatology of diseases of the basal ganglia. in: vinken p, bruyn g, eds. handbook of clinical neurology, vol . amsterdam: north holland publishing co, : - . bronstein a, rudge p, beechey a. spasmodic torticollis following unilateral viii nerve le- sions: neck emg modulation in response to vestibular stimuli. j neurol neurosurg psy- chiatry ; : - . cleeves l, findley l, marsden d. odd tremors. in: marsden d, fahn s, eds. movement disorders . butterworths (in press). podivinsky f. torticollis. in: vinken p, bruyn g, eds. handbook of clinical neurology, vol . amsterdam: north holland. : - . gresty m, barratt h, page n, rudge p. analysis of downbeat nystagmus. otolith vs semi- circular canal influences. arch neurol ; : - . dieterich m, brandt t, fries w otolith func- tion in man. brain ; : - . probable cases of mast syndrome in a non-amish family complicated forms of hereditary spastic paraplegia are rare. a year old flemish woman was admitted with a clinical picture of slowly progressive spastic paraplegia, dys- arthria, presenile dementia and mild atheto- sis. at the age of her gait became shuffling. before the age of she used a walking stick, since the age of years she has needed a walking frame and from the age of she has become more wheelchair bound. during the third decade (maybe earlier) dysarthria, apa- thy and negativism appeared. towards her th year, urinary incontinence began. on admission, aged , significant bradyphrenia and comprehension difficulties were noted and during the following years she presented a further mental deterioration. she is now bedridden and her speech restricted to rare, usually inappropriate, single syllable answers, which are sometimes repeated. she has diffi- culty swallowing fluids. she often shows spontaneous repeated slow turning of the head to the right and left and mild tortuous movements of the shoulders. except for a divergent strabismus there are neither oculo- motor abnormalities, nor fundoscopic anomalies. there is a slight bilateral facial weakness. the fine motor hand skills are lost , = proband; * = birth order in this sibship is uncertain figure pedigree without clear paresis of the upper limbs. the strength of leg muscles measures about to / . there were neither sensory deficits nor cerebellar signs. deep tendon reflexes in the upper limbs were increased slightly. knee jerks were unusually brisk and without clo- nus; ankle jerks were decreased. plantar responses were extensor. snout reflex and bilateral palmomental reflexes were present whilst corneomandibular reflexes were absent. the following laboratory investigations showed no significant abnormalities: routine blood examination (except for intermittent elevation of glucose with normal haemoglo- bin a c ), creatine kinase, copper, lipids, very long chain fatty acids ratios c /c and c /c , vitamin e, vitamin b , folate, cortisol, adrenocorticotropic hor- mone, thyroid hormones, arylsulfatase a and hexosaminidase a + b; the csf protein was mg/l with normal electrophoretic pat- tern. the eeg showed mild general slowing. nerve conduction studies and needle electro- myography showed a mild axonal polyneur- opathy. the somato-sensory evoked potentials demonstrated a slightly prolonged central conduction time. an electrocardio- gram was normal. mri of the brain showed diffuse cortico-subcortical atrophy, periven- tricular hyperintensities, thin corpus callo- sum and less marked atrophy of the brainstem and cerebellum. light microscopic and electron microscopic examination of conjunctiva and skin showed some mem- branous cytoplasmic body-like inclusions (professor j j martin, dr c ceuterick-de groote, university hospital antwerp). the family history revealed two similar cases (figure). there was no known con- sanguinity. the monozygotic twin has an almost identical medical history and clinical picture. she is able to stammer a few simple words. her answers are sometimes slightly more appropriate, particularly for old memories. her knee jerks and ankle jerks are both unusually brisk, and the plantar responses are extensor. one brother died at the age of . the medical records and relatives des- cribed difficulties with walking from the age of (maybe earlier). during the following decades he presented a progressive spastic paraparesis, dysarthria, mental deterioration and urinary and faecal incontinence; no deficits of sensation or coordination were demonstrated. there was dysphagia in his last years. death was due to pneumonia. the pedigree suggests an autosomal reces- sive inheritance. the neurodegenerative syn- drome in this family seems fully comparable to the mast syndrome, described in in an ohio amish isolate by cross and mcku- sick.`'- there appears to be no similar cases that have been described outside the amish population. marc d'hooghe department of neurology, algemeen ziekenhuis st j an, ruddershove , brugge, belgium cross he, mckusickva. the mast syndrome: a recessively inherited form of presenile de- mentia with motor disturbances. arch neurol (chic) ; : - . harding ae. classification of hereditary ataxias and paraplegias. lancet ;i: - . mckusick va. mendelian inheritance in man. baltimore: the johns hopkins university press, : . ultrasensitive tsh assay and anti-par- kinsonian treatment with levodopa we have recently reported the association of parkinson's disease and hyperthyroidism in a group of patients.' in that report, symp- toms of parkinson's disease were always significantly exacerbated by the development of hyperthyroidism and improved by its successful treatment.' we proposed that hyperthyroidism should be suspected in all parkinsonian patients when their condition deteriorates.' since clinical diagnosis of thy- rotoxicosis is difficult in parkinsonian patients, they should have a comprehensive thyroid examination and, if there is the slightest suspicion of hyperthyroidism, a hor- monal evaluation of thyroid function (free t , ultrasensitive tsh). thyroid hormone levels (t and t ) have been found to be normal in parkinsonian patients untreated or treated with levodopa. however, a decreased response of thyro- tropin (tsh) after stimulation by trh (thyrotropin releasing hormone) has been reported in parkinsonian patients treated with levadopa. such a decreased tsh response after trh stimulation is observed during hyperthyroidism, and is sometimes the only hormonal abnormality, especially in elderly patients with autonomous thyroid nodules. thus the decreased tsh response after trh-stimulation in patients treated with levodopa could be responsible for a false diagnosis of hyperthyroidism. these results, however, were obtained before the ultra- sensitive tsh determination with a mono- clonal antibody assay was available. a low ultrasensitive tsh level has the same sig- nificance as a decreased tsh response to trh, indicating an increased negative feed- o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn n p .b m j.co m / j n e u ro l n e u ro su rg p sych ia try: first p u b lish e d a s . /jn n p . . . -a o n d e ce m b e r . d o w n lo a d e d fro m http://jnnp.bmj.com/ letters to the editor table ultrasensitive tsh assay and anti-parkinsonian treatment with levodopa. means (sd). levodopa treated normal patients controls ranges (n = ) (n = ) p age (years) - ( ) - ( ) - freet (pmol/l) - - - - ( - ) - ( - ) - freet (pmol/l) - - ( ) - ( ) ultrasensitivetsh (mu/i) - - - ( - ) - ( - ) - free t , free t and ultrasenstiivetsh levels in levodopa treated parkinsonian patients compared with age- matched controls, mean (sd). ns: not significant by student's t test. back of thyroid hormones on pituitary tsh secretion. thus basal ultrasensitivetsh level has become an important tool to detect hyperthyroidism, replacing the trh-stim- ulation test. the aim of our study was to determine whether chronic treatment with levodopa was able to lower the basal tsh levels, measured by an ultrasensitive assay, in parkinsonian patients and thus be responsible for a false diagnosis of hyperthyroidism in such patients. thirty two parkinsonian patients, mean (sd) age ( ) years; range: - treated with levodopa ( - mg/day to mg/day) for one to months were studied. these patients were clinically euthyroid, had a normal clinical thyroid examination and normal levels of free t . blood samples were drawn at : am in fasting patients, from to hours after the last levodopa dose, for determination of free t , free t and ultra- sensitive tsh. these patients were not receiving any other drugs known to modify thyroid function. thirty age matched con- trols were also studied, for comparison [mean (sd) - ( ) years; range: - ]. each patient and each control had titres of antithyroglobulin and antimicroso- mial autoantibodies less than / and / respectively (haemagglutination assay). free t and free t were measured using radioimmunoassay (ria amersham). ultrasensitive tsh was measured by radio- immunoassay, using a monoclonal antibody (ria behring). the sensitivity of this tsh assay is - mu/l, the intra-assay and inter- assay coefficient of variations are - % and % respectively. comparisons of means were made with student's unpaired t test after verification of a normal distribution for the data. free t [mean (sd) - ( ) pmol/l; range: - ] and free t [mean (sd) - ( - ) pmol/l; range: - - ] levels were not significantly different in parkinsonian patients treated with levodopa than controls (table). the ultrasensitive tsh level was not significantly different in levodopa treated parkinsonian patients [mean (sd) - ( ) mu/l; range: - ] from controls [mean (sd) - ( - ) mu/l; range: - ] (table). among the patients, none had an ultrasensitive tsh level below the normal range. ultrasensitive tsh level was not shown to be influenced by levodopa doses since no difference was noted between patients treated with less than mg/day (n = ) and those treated with more than mg/day (n = ) [ - ( ) vs - ( - ) mu/l; not significant]. furthermore, dura- tion of levodopa therapy did not appear to modify ultrasensitive tsh level since no difference was observed between patients treated for less than months (n = ) and those treated for more than months (n = ) [ - ( - ) vs - ( - ) mu/l; not significant]. clinical diagnosis of hyperthyroidism is often difficult in patients with parkinson's disease because symptoms such as tremor, weight loss and sweating are common to both diseases, because thyrotoxicosis only pro- duces few symptoms in elderly patients, and because the parkinson's syndrome predom- inates in most cases. ' thus hormonal evaluation of thyroid function appears to be very helpful, for diagnosis of hyperthyroid- ism, in parkinsonian patients. ultrasensitive tsh assay had been a great improvement in hormonal thyroid function evaluation, lead- ing to easier detection of hyperthyroidism. a low ultrasensitive tsh level, with normal thyroid hormones, is frequently seen in eld- erly patients with autonomous thyroid nodules. such patients require a thyroid scintigraphy to detect hyperfunctioning nodules, allowing the diagnosis of hyperthy- roidism.' before the ultrasensitive tsh assay was available, an absence of tsh response aftertrh stimulation was the only method of detecting such hyperthyroid patients. as a decreased response of tsh aftertrh had been reported in parkinsonian patients treated with levodopa, it remains to be determined whether basal tsh levels, measured by ultrasensitive assay were mod- ified by levodopa treatment. in our study, basal tsh levels, measured by an ultra- sensitive assay, in parkinsonian patients trea- ted with levodopa, were not found to be lower than in age matched controls. no patient treated with levodopa had an ultra- sensitive tsh level below the normal range. furthermore, in patients treated with levo- dopa, ultrasensitivetsh level was influenced neither by the levodopa dose nor by the duration of levodopa treatment. in conclusion, chronic anti-parkinsonian therapy with levodopa does not modify basal tsh levels measured by an ultrasensitive- assay. thus ultrasensitive tsh evaluation is as efficient a method to detect hyperthyroid- ism in patients treated with levodopa as in the general population. b verges, m giroud, g vaillant, b verges-patois, jm brun, r putelat university hospital (hopital du bocage) dijon, france correspondence: drverges, service de medecine , hopital du bocage, b.p. dijon cedex, france verges b, giroud m, richard a, giroud- baleydier f, vaillant g, lorcerie b, brun jm, putelat r. parkinson's disease after antithy- roid treatment. lancet ;ii; . johannessen ac, boye a, parkenberg h. thy- roid function in patients with parkinson's disease. acta neurol scand ; : - . spaulding sw, burrow gn, donabedian r,van woert m. l.dopa suppression of thyrotropin releasing hormone response in man. clin endocrinol metab ; : - . davis pj, davis fb. hyperthyroidism in patients over the age of years. medicine ; : . caradoc-davies th. resolution of dyskinesia and the "on-off' phenomenon in thyrotoxic patients with parkinson's disease after anti- thyroid treatment. bmj ; : - . lavy s, marks es, abramsky . parkinsonism and hyperthyroidism. europ neurol ; : - . cadwell g, gow sm, sweeting um, et al. a new strategy for thyroid function testing. lancet ;i:i - . bruce s, rangedara dc, lewis rr, cordless d. hyperthyroidism in elderly patients with atrial fibrillation and normal thyroid hormone measurements. j royal soc med ; : - . vestibular and ventilatory dysfunction in sensory and autonomic neuropathy associated with primary sjogren's syn- drome subacute sensory neuropathy was described by denny-brown in association with carci- noma' and has recently been described in association with primary sj gren's syndrome (ss). the primary pathology is in the dorsal root ganglia and consists of lymphocytic infiltration and neuronal cell destruction. we describe a patient with primary ss with sensory and autonomic neuropathy and the previously undescribed clinical features of vestibular and ventilatory dysfunction. a year old man presented with difficulty with walking for one month. he also com- plained of pain and tingling in the feet and hands, patchy areas of abnormal sensation on the trunk and perioral paraesthesiae. he had experienced grittiness and dryness of the eyes and a dry mouth for months and had keratoconjunctivitis sicca on ophthalmolog- ical examination about months before his neurological presentation. his pupils reacted sluggishly to light but the visual fields and optic fundi were normal. there was reduced sensation in all divisions of the fifth cranial nerve bilaterally, although there was some sparing of the perioral region, and the cor- neal reflexes and corneal sensation were reduced. in the limbs the power was normal. the deep tendon reflexes were absent. in the upper limbs there was reduction of pinprick and light touch sensation in a distal distribu- tion and loss of vibration sense and proprio- ception up to and including the wrists. in the lower limbs, pinprick sensation was reduced in the toes, light touch was absent up to the thighs and vibration sensation was absent below the iliac crests. there was severe impairment of proprioception up to and including the knees. romberg's sign was positive and the gait was severely ataxic. there was bilateral punctate keratitis in the interpalpebral distribution and the schir- mer's test was mm on the right and mm on the left (normal > ). on later examina- tion, the schirmer's test revealed no lacrima- tion from either eye. the significant results of investigations were that the ra latex test was negative, anticardiolipin antibody level was (normal < ) and smooth muscle antibody titre was : . screening for other autoantibodies, including extractable nuclear antigens, was negative. csf protein was initially mg/l (normal < ), csf igg was mg/l (normal - ) and csf igg/albumin was (normal < i ). on a later occasion the csf protein level was , mg/l. csf electrophoresis did not reveal oligoclonal bands. motor nerve conduction velocity was normal but sensory action potentials were absent in the right median, ulnar and sural o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn n p .b m j.co m / j n e u ro l n e u ro su rg p sych ia try: first p u b lish e d a s . /jn n p . . . -a o n d e ce m b e r . d o w n lo a d e d fro m http://jnnp.bmj.com/ improper adjustment for baseline in genetic association studies of change in phenotype | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . / corpus id: improper adjustment for baseline in genetic association studies of change in phenotype @article{mcardle improperaf, title={improper adjustment for baseline in genetic association studies of change in phenotype}, author={p. f. mcardle and b. w. whitcomb}, journal={human heredity}, year={ }, volume={ }, pages={ - } } 	p.f. mcardle, b.w. whitcomb 	published 	medicine 	human heredity objective: in studies of associations between genetic factors and outcomes where change in phenotype is of interest, proper modeling of the data, particularly the treatment of baseline trait values, is required to draw valid conclusions. methods: the authors compared models of blood pressure response to a cold pressor test with and without inclusion of baseline blood pressure as a regressor and evaluate the resultant biases. results: adjustment for baseline presents a potential source of bias… expand view on pubmed karger.com save to library create alert cite launch research feed share this paper citations methods citations view all figures, tables, and topics from this paper 	 figure 	 table 	 table 	 figure 	 table 	 figure 	 table view all figures & tables 	mental association 	estimated 	population parameter 	genetic association studies 	baseline dental cement citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency the impact of adjusting for baseline in pharmacogenomic genome-wide association studies of quantitative change 	a. oni-orisan, tanushree haldar, + authors thomas j. hoffmann 	medicine 	npj genomic medicine 	 	 	pdf 	view excerpts, cites methods save alert research feed should studies of diabetes treatment stratification correct for 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capacity on genetic association studies of late-onset alzheimer's disease 	d. fardo, l. gibbons, shubhabrata mukherjee, m. m. glymour, p. crane 	medicine 	alzheimer's & dementia 	 	 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency the effects of measurement error in response variables and tests of association of explanatory variables in change models. 	n. yanez, r. kronmal, l. shemanski 	mathematics, medicine 	statistics in medicine 	 	 	pdf save alert research feed when is baseline adjustment useful in analyses of change? an example with education and cognitive change. 	m. m. glymour, j. weuve, l. berkman, i. kawachi, j. robins 	psychology, medicine 	american journal of epidemiology 	 	 	pdf save alert research feed a structural approach to selection bias 	m. hernán, s. hernández-díaz, j. robins 	psychology, medicine 	epidemiology 	 	 , 	pdf save alert research feed the genetic response to short-term interventions affecting cardiovascular function: rationale and design of the heredity and phenotype intervention (hapi) heart study. 	b. mitchell, p. mcardle, + authors a. shuldiner 	medicine 	american heart journal 	 	 	 save alert research feed quantifying biases in causal models: classical confounding vs collider-stratification bias 	s. greenland 	psychology, medicine 	epidemiology 	 	 save alert research feed change from baseline and analysis of covariance revisited. 	s. senn 	psychology, medicine 	statistics in medicine 	 	 	pdf save alert research feed marginal structural models and causal inference in epidemiology 	j. robins, m. hernán, b. brumback 	mathematics, medicine 	epidemiology 	 	 , 	pdf save alert research feed confounding in health research. 	s. greenland, h. morgenstern 	psychology, medicine 	annual review of public health 	 	 	pdf save alert research feed basic methods for sensitivity analysis of biases. 	s. greenland 	medicine 	international journal of epidemiology 	 	 	pdf save alert research feed genetic influences on blood pressure with the cold-pressor test: a twin study 	a. busjahn, h. faulhaber, r. viken, r. rose, f. luft 	medicine 	journal of hypertension 	 	 save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception 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a monday, march , provide valuable insights into its mode of action. the molecular framework of dfbp resembles that of levosimendan, thus it was chosen to mimic levosimen- dan to establish how the ctnc-ctni binding equilibrium is modulated. we have utilized d { h, n} hsqc and d { h, c} hsqc nmr spectroscopy to examine the binding of dfbp to cntnc�ca þ in the absence and presence of ctni - and of ctni - to cntnc�ca þ in the absence and presence of dfbp. the results show that dfbp and ctni - bind cntnc�ca þ concurrently and the affinity of dfbp for cntnc�ca þ is increased ~ - - fold by ctni - . we are in the process of determining the nmr solution structure of cntnc�ca þ�ctni - �dfbp. this structure will contribute to the understanding of the mechanism of action of levosimendan in the therapy of heart disease. it will also provide a structural basis for the design of ca þ- sensitizing drugs in general. -pos board b decreased fatigue tolerance in diaphragm muscle of slow troponin t knockdown mice hanzhong feng, bin wei, j.-p. jin. northshore university healthsystem and northwestern university feinberg school of medicine, evanston, il, usa. the loss of slow skeletal muscle troponin t (tnt) results in a severe type of nemaline myopathy in the amish (anm). the genes encoding tnt and tropo- nin i (tni) are closely linked in pairs in which the -enhancer region of the slow tnt gene overlaps with the cardiac tni gene. in a mouse line with the en- tire cardiac tni gene deleted, a partial destruction of the slow tnt gene pro- moter produces a knockdown effect. by crossing with transgenic mouse lines that over-express a core structure of cardiac tni (ctni-nd) under the control of cloned alpha-mhc promoter, we rescued the postnatal lethality of the car- diac tni gene-deleted mice with no detrimental cardiac phenotypes or leaking expression in non-cardiac tissues. the double transgenic mice exhibited de- creased expression of slow tnt mrna and protein in adult diaphragm muscle. functional analysis of isolated muscle strips showed that the slow tnt deficient (stnt-kd) diaphragm had significantly decreased fatigue tolerance evident by the faster decrease in force and slower rate of recovery as compared with that in wild type controls. as a consequence of slow tnt deficiency, the stnt-kd di- aphragm muscle contained a higher proportion of fast tnt, decreased slow tni with increased fast tni, and decreased type i myosin with increased type ii my- osin. consistent with the switch toward fast myofilament contents, the stnt- kd diaphragm muscle produced higher specific tension in twitch and tetanic contractions as well as shorter time to develop peak tension in twitch contrac- tions. the decreased fatigue tolerance of stnt-kd diaphragm muscle explains the terminal respiratory failure seen in virtually all anm patients and this dou- ble transgenic mouse model provides a useful experimental system to study the pathogenesis and treatment of anm. -pos board b troponin isoforms and stretch-activation of insect flight muscle uros krzic , gian de nicola , vladimir rybin , annalisa pastore , kevin leonard , wolfgang linke , belinda bullard . embl, heidelberg, germany, nimr, london, united kingdom, ebi, cambridge, united kingdom, university of muenster, muenster, germany, department of biology, university of york, united kingdom. oscillatory contraction of insect indirect flight muscle (ifm) is activated by si- nusoidal length changes. work done by oscillating fibres is measured from the area of loops on a length-tension plot. at [ca þ] above mm, progressively less oscillatory work is produced because fibres contract isometrically and are unable to relax fully after each cycle of oscillation. periodic stretches during oscillations activate fibres through the action of tnc f , which binds one ca þ in the c-lobe. activation of isometric contraction by ca þ acts through f , which binds ca þ in both n- and c-lobes. lethocerus ifm fibres substituted with f gave oscillatory work, which did not decline at high [ca þ], while fibres substituted with f produced more isometric tension as [ca þ] was increased. varying proportions of f and f gave maximal work with an f :f ratio of : , which is higher than the in vivo ratio of : . the structure of f , and the interaction with tni, were determined by nmr. the n-lobe of f is in the closed conformation in apo and ca þ- bound forms and does not bind tni. unexpectedly, the c-lobe is open in both states, and binds the n-terminal domain of tni independently of ca þ. the affinity of f and f for a complex containing tropomyosin, tnt and tnh (lethocerus tni) were measured by isothermal calorimetry in the presence of ca þ. the affinities of f and f for the complex were . mm and nm respectively. this difference is likely to be due to a single tni binding site on f and two sites on f . stretch may be sensed by an extended c-terminal domain of tnh, and transmitted to the c-lobe of f , resulting in a change in the interac- tion of the tni inhibitory domain and actin. -pos board b tracking of qdot conjugated titin antibodies in single myofibril stretch experiments reveals ig-domain unfolding at physiological sarcomere lengths anika grützner , pallav kosuri , julio m. fernandez , wolfgang a. linke . university of münster, münster, germany, columbia university, new york, ny, usa. the mechanical characteristics of titin in muscle sarcomeres were previously studied by us in single myofibril stretch experiments, where the extensibility of i-band titin segments was usually measured under static conditions. here we investigated the behavior of i-band titin during and after stretch of single rabbit psoas myofibrils in real-time. the focus was on titin’s proximal ig-do- main region, whose stretch dynamics were analyzed by labeling the myofibrils specifically in the n a-titin domain using antibody-conjugated quantum dots, which stained the periphery of the myofibril but did not enter the myofilament lattice. qdot labels were tracked to obtain the stretch-dependent change in epi- tope distance (across z-disc) and sarcomere length (sl) over time. in contrast to what was expected from the current titin extensibility model, at sarcomere lengths of . and . mm, titin’s proximal ig-domain region elongated contin- uously, in proportion to the half i-band length. already at ~ . mm sl the prox- imal ig-segment length exceeded the value expected if all ig-domains remain folded. our results suggest that ig-domains unfold in parallel with pevk-titin extension at physiological sarcomere lengths and under relatively low forces. by reducing the antibody-qdot concentration, we succeeded in observing titin ig-domain dynamics in myofibrils at the single-molecule level. -pos board b constitutive phosphorylation of cardiac myosin binding protein-c increases the probability of myosin cross-bridge interaction with actin brett a. colson , tanya bekyarova , matthew r. locher , carl w. tong , daniel p. fitzsimons , patricia a. powers , thomas c. irving , richard l. moss . university of wisconsin medical school, madison, wi, usa, illinois institute of technology, chicago, il, usa. protein kinase a-mediated (pka) phosphorylation of cardiac myosin binding protein-c (cmybp-c) accelerates the kinetics of cross-bridge cycling and ap- pears to relieve the tether-like constraint of myosin heads imposed by cmybp-c (colson et al., , circ res., : - ). we favor a mechanism in which phosphorylation of the pka sites in cmybp-c modulates cross- bridge kinetics by regulating the proximity and interaction of myosin with actin. to test this idea, we used synchrotron low-angle x-ray diffraction and mechanical measurements in skinned myocardium isolated from a mouse model with phosphomimetic substitutions in cmybp-c, i.e., the ctsd mouse. the substitutions were introduced by transgenic expression of cmybp-c with ser-to-asp mutations on a cmybp-c null background. western blots showed that expression of ctsd cmybp-c was % of wild-type (wt), and the heart weight to body weight ratio was similar ( . . mg/g) in ctsd and wt mice. expression of wt cmybp-c on the knockout background served as con- trol (i.e., the ctwt mouse). skinned myocardium from ctsd and ctwt mice exhibited similar maximum active forces (mn/mm : . . vs . . ), ca þ-sensitivities of force (pca : . . vs . . ), and maximum rates of force development (ktr, sec - : . . vs . . ; kdf, sec - : . . vs . . ). i /i intensity ratios and d lattice spacings determined from equatorial reflections from ctsd and ctwt myocardium were used to determine the effect of constitutive cmybp-c phosphorylation on the distribution of cross-bridge mass between the thick and thin filaments and on interfilament lattice spacing. the results suggest that interactions between cmybp-c and the s domain of myosin heavy chain are dynamically regulated by phosphorylations in the cmybp-c motif. (aha-predoctoral fellowship (bac); nih-hl-r - ) -pos board b obscurin interacts with a novel isoform of myosin binding protein c-slow to regulate the assembly of thick filaments maegen a. borzok, rebecca hu, amber l. bowman, john strong, robert j. bloch, aikaterini kontrogianni-konstantopoulos. university of maryland, baltimore, md, usa. obscurin is a multidomain protein composed of adhesion and signaling do- mains that plays key roles in the organization of contractile and membrane structures in striated muscles. we used adenoviral-mediated gene transfer to overexpress its extreme nh -terminus in developing myofibers, followed by immunofluorescence and ultrastructural methods to study its effects in sarco- merogenesis. we found that overexpression of obscurin’s second immunoglob- ulin domain (ig ) inhibits the assembly of a- and m-bands, but not z-disks and 	decreased fatigue tolerance in diaphragm muscle of slow troponin t knockdown mice 	troponin isoforms and stretch-activation of insect flight muscle 	tracking of qdot conjugated titin antibodies in single myofibril stretch experiments reveals ig-domain unfolding at physiological sarcomere lengths 	constitutive phosphorylation of cardiac myosin binding protein-c increases the probability of myosin cross-bridge interaction with actin 	obscurin interacts with a novel isoform of myosin binding protein c-slow to regulate the assembly of thick filaments dresang.indd a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r low primary cesarean rate and high vbac rate with good outcomes in an amish birthing center abstract purpose recent national guidelines encourage a trial of labor after cesarean (tolac) as a means of increasing vaginal births after cesarean (vbacs) and decreasing the high us cesarean birth rate and its consequences ( national institute of health consensus statement and american college of obstetricians and gynecologists revised guideline). a birthing center serving amish women in southwestern wisconsin offered an opportunity to look at the effects of local culture and practices that support vaginal birth and tolac. this study describes childbirth and perinatal outcomes during a -year period in lafarge, wisconsin. methods we undertook a retrospective analysis of the records of all women admitted to the birth center in labor. main outcome measures include rates of cesarean deliveries, tolac and vbac deliveries, and perinatal outcomes for deliveries between and . results the cesarean rate was % ( of ), the tolac rate was %, and the vbac rate was % ( of ). there were no cases of uterine rupture and no maternal deaths. the neonatal death rate of . of , was comparable to that of wisconsin ( . of , ) and the united states ( . of , ). conclusions both the culture of the population served and a number of fac- tors relating to the management of labor at the birthing center have affected the rates of cesarean delivery and tolac. the results of the lafarge amish study support a low-technology approach to delivery where good outcomes are achieved with low cesarean and high vbac rates. ann fam med ; : - . doi: . /afm. . introduction t he cesarean rate in the united states has risen from . % in to . % in to % in to . % in . , a study calculates that if trends continue, the cesarean rate will be . %. the primary cesarean rate was . %. the vaginal birth after cesarean (vbac) rate—the percentage of pregnant women who give birth vaginally after a previous cesarean—has fallen from a high of . % in to . % in . the world health organization (who) and healthy people have suggested the ideal cesarean rate should be around %. , the national institutes of health consensus conference on vbac highlighted high-grade evidence that maternal mortality risk is decreased by vbac compared with a repeat cesarean ( . vs . of , ). data suggest decreasing the primary cesarean delivery rate and increasing the vbac rate as key strategies to decrease the us cesarean rate. amish communities ascribe religious and cultural value to child- bearing. , contraception, including sterilization, may be prohibited. advanced maternal age and grand multiparity are common. a study of james deline, md lisa varnes-epstein, mhs, pa-c, cpm lee t. dresang, md mark gideonsen, md laura lynch john j. frey iii, md amish birthing center, la farge, wisconsin university of wisconsin school of medicine and public health, madison, wisconsin confl icts of interest: the authors include the practi- tioners who founded and operate the birthing center. corresponding author jim deline, md la farge medical clinic-vmh w snow st lafarge, wi jdeline@vmh.org a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r l o w c e s a r e a n , h i g h v b a c r at e s f o r a m i s h , deliveries in an ohio amish community found that % of births were to women aged years or older, and the median number of children for women older than years was . . health insur- ance of any kind and participation in such programs as social security, medicare, and medicaid are rare. although generalizations can be made about amish communities, each community is different. this study focused on a southwest wisconsin amish community serviced by the lafarge birthing center. amish women of southwest wisconsin generally give birth at home attended by an unlicensed birth attendant, mother, mother-in-law, or neighbor. formal education for men and women is through th grade, and women do not work outside the home. amish culture prohibits electricity, telephone service, and car ownership, leading to delays in care for pregnancy complications. responding to a community request, the lafarge medical clinic developed a low-cost, com- munity-based birthing center in the offi ce staffed by a family physician and certifi ed professional midwife to provide a safer alternative for amish women. registered nurses, midwifery interns, or trained laypeople with clinical skills also attended the births. this study describes a -year experience with amish women giving birth in the lafarge birthing center (a photograph of which is shown in supplemental figure , available at http://annfammed.org/ content/ / / /suppl/dc ). methods the lafarge birthing center was developed as an alternative to home birth for amish women. originally, higher-risk patients were encouraged to give birth in the hospital. with time it was apparent that most higher- risk patients were having their babies at home rather than the hospital. after discussion of risks and benefi ts, these patients were increasingly allowed an attempted birthing center delivery. patients were asked to review and sign a consent form contrasting treat- ments available at the birth center vs hospital, by which they agreed to abide by birth attendants’ deci- sion for hospital transfer at any time. women wanting a tolac were advised of uterine rupture risk, and verbal consent was obtained. women not accepted for birthing center delivery included those with known placenta previa or brisk third-trimester bleeding, severe preeclampsia, nonfrank breech presentation not ame- nable to external version, and presentation of a fi rst twin in a nonvertex position. although birthing center prenatal care was encouraged, women who had planned home births could be considered for transfer to the birthing center before or during labor. women evalu- ated in the birthing center and then sent to the hospital for reasons including birth center contraindications described above were included in birthing center statis- tics. lafarge birthing center practices are outlined in table . an ambulance service near the birthing center allowed prompt transport. at vernon memorial hospi- tal (vmh), minutes from the birthing center, births were attended by family physicians and midwives; a general surgeon performed cesareans. the vmh cesar- ean rate from through was . %. table . lafarge birthing center practices condition management previous cesarean trial of labor expected by women and supported by clinician breech or trans- verse lie external cephalic version offered from to weeks’ gestation; if unsuccessful or declined, vaginal breech delivery offered if frank breech; if unstable lie, managed in labor preterm labor previable fetus delivered at birth center (included in mortality statistics); viability until weeks’ gestation transferred to tertiary care hospital; to weeks’ gestation transferred to local hospital postdates because of poor dating, no induction unless oligohydramnios (offi ce sonogram weekly starting weeks); amniotic fl uid documented as “inadequate, adequate, or excessive” with induction recommended for inadequate elective or social induction none twins vaginal delivery offered unless twin a was not in vertex position labor dystocia with normal maternal and fetal status no cesarean until at least hours of no cervical change in active labor and on oxytocin arrest of descent no cesarean unless no progress after pushing for more than hours in primipara and - hours in multipara mother; allowed to push even longer if signs of progression and normal maternal and fetal status electronic fetal monitoring none; instead, fetal status monitored using structured intermittent auscultation vacuum and forceps no forceps; vacuum assist when indicated according to also guidelines labor pain massage, hot packs, continuous clinician presence and encouragement; husband present in all cases; no intravenous narcotics, no epidurals postpartum hemorrhage prevented or treated early by active management of rd stage, one- on-one nursing care with uterine assessment every minutes in fi rst hour, aggressive intravenous fl uid therapy, oxytocin, misoprostol, and methylergonovine oxytocin used for labor induction, augmentation, active management of the rd stage of labor, and treatment of postpartum hemorrhage prenatal education none beyond that taught at prenatal visits discharge criteria maternal stable vital signs, bleeding controlled; intravascular volume replaced; infant stable. minimum stay hours; generally to hours also = advanced life support in obstetrics. a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r l o w c e s a r e a n , h i g h v b a c r at e s f o r a m i s h all fetal malpresentations were confi rmed by ultrasound examination. women wanting external cephalic version in the birthing center were advised that immediate surgical capabilities to address proce- dure complications were lacking, and verbal consent was obtained. women with frank breech babies were allowed attempted vaginal delivery after a discus- sion that included risks of cord prolapse and head entrapment. for women with twins, birthing center delivery criteria included fi rst twin presenting in a vertex posi- tion and more than weeks’ gestation. women were counseled on risks of a twin delivery without operative capabilities and offered hospital transfer. women in premature labor were appropriately counseled and offered transfer to a tertiary center if their pregnancy was less than weeks or to vmh if their pregnancy was between and weeks; some refused. when a fetus was not viable or extremely preterm, patients generally preferred birthing center delivery. these infants are included in the mortality statistics. no analgesics were administered during labor at the birthing center. not using analgesics was the cultural norm, as was continuous support throughout labor. postpartum hemorrhage was prevented or treated early by active management of third-stage, one-on-one nursing care with uterine assessment every minutes in the fi rst hour, aggressive intravenous fl uid therapy, oxytocin, misoprostol, and methylergonovine. all women attending the birthing center were uninsured. they were charged $ per delivery regardless of length of stay or supplies used (except for rh immune globulin, for which they were charged separately). the charge included a postpartum visit for mother and baby and the newborn screening tests. we entered data from to birthing center records for all women admitted for labor into an excel database for analysis. the birth records were written in a uniform format throughout the study period. the data were de-identifi ed, and we converted dates into time periods to preserve confi dentiality. this study was exempted by the university of wisconsin institu- tional review board. data entered included demographics, duration of stages of labor, transfer details, cesarean statistics, and perinatal outcomes. the denominator for rates (inclu- sion criteria) was all women admitted in labor to the birthing center, including those who planned to give birth there and those who were transferred in from home. for women who gave birth more than once at the birthing center, each pregnancy was included. women who were transferred to a hospital before labor for conditions including those listed above were not counted when determining rates (exclusion crite- ria). perinatal deaths were defi ned as the sum of fetal deaths plus neonatal deaths; fetal death was defi ned as any death beyond weeks’ gestation but before deliv- ery, and neonatal death as death after birth but before days of life. we investigated birthing practices for the entire service area. by reviewing birthing center records for all deliveries between and , we determined that % of women giving birth at the birthing center were from vernon and monroe counties. we then reviewed birth certifi cates for all amish vernon and monroe county births between and at the register of deeds offi ce, identifi ed the birth location, and calculated the percentage of infants born at home, at the birthing center, at small local hospitals, and at tertiary hospitals. we reviewed all vmh records of amish births between and to determine whether women were referred from the birthing center or home and whether the referral was before or during labor. results southwest wisconsin amish women give birth to most children at home. of , births in vernon and mon- roe counties from to , ( %) occurred in the home, ( %) occurred at the birthing center, ( %) took place in small community hos- pitals, mostly vmh, and ( %) occurred at tertiary hospitals with obstetricians on staff. of vmh amish births from through , % of mothers were transferred from the birthing center in labor, % were transferred from attempted home births, % were transferred from the birthing center antenatally, and % ( patients) were self-referred to vmh for prena- tal care and delivery. from until , women were attended for deliveries at the birthing center. figure shows the age and figure the parity of women in the study at the time of each delivery. ninety-six deliveries ( %) were of women com- ing to the birthing center after planning a home birth (table ). this group accounted for % ( of ) of cesarean deliveries and % ( of ) of perinatal deaths. table lists indications and frequency for transfers from the birthing center to the hospital. transfer from the birthing center to the hospital occurred for % ( of ) of women planning go have their babies at the birthing center and % ( of ) of women who transferred from home to the birthing center. the overall cesarean rate was % ( cesareans of deliveries). the cesarean rate was % ( of a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r l o w c e s a r e a n , h i g h v b a c r at e s f o r a m i s h deliveries) for women planning a clinic birth and % ( of deliveries) for women who transferred from home. details of other cesarean deliveries are docu- mented in table . the vbac rate was % ( of deliveries). of the tolacs % ( of deliveries) took place with women who had had previous cesarean, % ( of deliveries) were of women who had had previ- ous cesarean deliveries; no tolacs were attempted for women with or more prior cesarean deliveries. patients in labor with fetal malpresentation included those who failed or declined external version or who transferred to the birthing center without prenatal care. of these women, were primagravida and were multigravida with breech presentation. two patients with frank breech presentations chose cesarean; had nonfrank breech presentations, and cesarean deliv- ery was performed. three mul- tiparous women had successful intrapartum version and vaginal delivery. of the remaining patients, all with frank breech presentation desiring trial of labor, had a successful vaginal breech delivery, and required cesarean for lack of descent. seven of successful breech pre- sentations with vaginal delivery were in nulliparous patients. ten women had twins with the fi rst baby in the vertex posi- tion; mother was at weeks’ gestation and refused hospital transfer, chose tolac, and pregnancy was previable. of the sets of viable twins, were in figure . age (n = deliveries) of women at the time of each delivery. n o . o f m o th e rs age, years < years = . % - years = % ≥ years = . % < - - - - - > figure . parity (n = deliveries) of women at time of each delivery. n o . o f m o th e rs no. of births nulliparous = ( %) para = ( . %) para - = ( . %) para ≥ = ( . %) a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r l o w c e s a r e a n , h i g h v b a c r at e s f o r a m i s h vertex/vertex and were in vertex/breech positions. one woman gave birth to the fi rst twin vaginally and had the second twin by cesarean delivery because of an arm presentation. maternal complications included women with prolonged rupture of membranes, with cord prolapse, with preterm delivery, with gestational hyperten- sion, with severe preeclampsia, with shoulder dys- tocia, with postpartum hemorrhage, with placental abnormalities, and with complex lacerations. medical and surgical complications during the pregnancies are outlined in supplemental table , available at http://annfammed.org/ content/ / / /suppl/dc . there were fetal deaths ( . of , births) and neo- natal deaths ( . of , births) (see supplemental table , avail- able at http://annfammed. org/content/ / / /suppl/ dc ). of the fetal deaths, had multiple fetal anomalies, and were previabile. of the neonatal deaths, were extremely prema- ture, had a genetic syndrome, had multiple anomalies, and had macrosomia and encephalopathy (but no dystocia and no neonatal hypoglycemia). nine babies had a -minute apgar of less than ; had no long-term issues, has cerebral palsy, and died of encephalopathy and seizures. discussion our study shows that a low cesarean and high vbac rate with good maternal and neonatal outcomes can be obtained in a nonhospital setting with skilled birth clinicians and basic resources. the lafarge birthing center cesarean rate was % ( of ), the tolac rate was %, and the vbac rate was % ( of ). there were no cases of uterine rupture or mater- nal death. the risk of uterine rupture with tolac at table . reason for and number of transfers from home to birthing center or hospital reason transfer to and delivery at birthing center (n = ) transfer to and delivery at hospital (n = ) total transfers (n = ) failure to progress prolonged rupture of membranes postdates with inadequate labor midwife unavailable malpresentations absent or decreased fetal movement third trimester bleeding preterm labor othera a bleeding vulvar varix; undiagnosed twins at weeks’ gestation, refused hospital transfer, delivered, but infants needed transfer and did well; arrived without notice or prenatal care; severe preeclampsia, refused hos- pital transfer; home birth canceled after husband’s death. table . reason for transfers from birthing center to hospital reason no. intrapartum transfers (n = ) malpresentation arrest of descent or dilation prolonged induction preterm third-trimester bleeding preeclampsia other postpartum transfers of infant (n = ) anoxic encephalopathy (cord prolapse) jaundice (day admission) preterm twins ( wk), refused intrapartum transfer multiple anomalies encephalopathy with seizures (macrosomic infant) genetic syndrome with seizures and hypotonia ( siblings with same) postpartum transfers of mother (n = ) postpartum hemorrhage (bleeding controlled but requiring transfusion) extensive perineal lacerations retained placenta table . characteristics of cesarean deliveries for all lafarge birthing center births characteristic no. of births cesarean delivery no. (%) total births ( ) cesarean rate by planned delivery location planned home delivery with intrapar- tum transfer to birthing center ( ) planned birthing center delivery ( ) cesarean by gravidity primagravida ( ) multigravida ( ) indication for cesarean malpresentation ( ) labor dystocia ( ) cord prolapse ( ) failed induction with prolonged rupture of membranes ( ) failed induction for preeclampsia ( ) a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r l o w c e s a r e a n , h i g h v b a c r at e s f o r a m i s h term is per , , so it is not unexpected that there would be no uterine ruptures in tolacs. the neonatal mortality rate of . of , was com- parable to that of wisconsin ( . of , in ) and the united states ( . of , in ). cesarean rate multiple cultural and intrapartum factors contributed to the low rate of cesarean delivery among women using the birthing center in this amish community. these factors likely included infrequent inductions, active management of labor, external cephalic ver- sion, vaginal breech delivery, vaginal delivery of twins, encouragement of tolac, and continuous labor support without electronic fetal monitoring or epidural analgesia. the clinician mix and medicolegal climate were also quite different in the lafarge birth- ing center, with community-based physicians and staff who knew the community and culture well and were respected for issues other than childbirth. multiparity, a low induction rate, and use of evidence-based guidelines for diagnosing failure to progress and arrest of descent contributed to the low rate ( of ) of cesarean delivery for labor dystocia. elective induction was never offered, and postdates induction was recommended only if an ultrasound examination showed oligohydramnios. allowing at least (rather than ) hours of adequate contractions with no cervical change before diagnosing failure to progress results in a lower cesarean rate with no worsening of maternal or fetal outcomes. in addition, during the second stage of labor, arrest of descent was not diagnosed so long as progress was being made and maternal and fetal status was reassuring. this approach has been documented in multiple studies to lower cesarean rates without compromising outcomes. external cephalic version was offered by clinicians and accepted by women. the american congress of obstetricians and gynecologists (acog) recom- mends “all women near term with breech presentations should be offered a version attempt.” in non-amish settings, external cephalic version is an underused option in the management of malpresentation. vaginal breech delivery was offered for appropri- ate candidates. in the united states, vaginal breech delivery rates dropped precipitously after the term breech trial showed increased neonatal mortal- ity with vaginal breech delivery. subsequent analysis years later by the same authors found no neonatal or developmental risk with vaginal breech delivery, and acog changed its policy to again support vagi- nal breech delivery for appropriate candidates. the society of obstetricians and gynecologists of canada conclude that “careful case selection and labor manage- ment in a modern obstetrical setting may achieve a level of safety similar to elective caesarean section.” the premoda study of , cesarean and , planned vaginal deliveries for breech in belgium and france found that % of women planning vaginal breech delivery were successful with no worsening of neonatal outcomes. many clinicians, however, have abandoned the practice of vaginal breech delivery and now feel they do not have the experience to start offering this option again. vaginal delivery was offered for full-term twins when the fi rst twin was in a cephalic position. tolac for twin delivery is supported by acog. although acog supports vaginal delivery of twins when the fi rst twin is in the cephalic position, cesarean is fre- quent even for cephalic presentation of twins in many hospitals. clinician experience may be a factor. all deliveries were attended by a family physician or nurse midwife. clinician mix was identifi ed as a reason for lower cesar- ean rates in indian health service (ihs) studies. - in zuni-ramah hospital, more than % of births were attended by family physicians. at the santa fe indian hospital, % of births were attended by nurse midwives, and those attended by obstetricians had an increased risk of cesarean (odds ratio = . ; p = . ) after controlling for medical and obstetric factors. tuba city cited a high percentage of deliveries by midwives as a reason for its lower cesarean rate. financial incentives for cesarean delivery may affect cesarean rates in certain systems. at the lafarge birthing center, there was no fi nancial incen- tive for operative delivery. additionally, malpractice concerns may increase non-amish cesarean rates. in one survey, % of obstetricians reported that mal- practice concerns have caused them to increase the number of cesareans they perform. most amish do not believe in litigation and trust clinicians willing to work with their beliefs about childbirth and work in an uninsured, cash economy. finally, and importantly, cultural beliefs and his- tory are important factors in what women expect in the birthing process. amish often prefer out-of-hospital and low-technology births for reasons including “reduced cost, increased comfort and privacy and a chance for a ‘more natural birth.’” these beliefs make it easier for clinicians caring for amish women to follow evidence- based guidelines and avoid unnecessary surgery. higher vbac rate in our study, % of births after a previous cesarean were vaginal, in sharp contrast with the general us rate of % from . the birthing center’s high vbac rate contributes substantially to its low overall a n na l s o f fa m i ly m e d i c i n e ✦ w w w. a n n fa m m e d . o r g ✦ vo l . , n o. ✦ n ov e m b e r / d e c e m b e r l o w c e s a r e a n , h i g h v b a c r at e s f o r a m i s h cesarean rate. the higher vbac rates in ihs hospitals and the lafarge birthing center likely have to do with clinicians and patients. although the clinicians in the lafarge birthing center universally offer tolac to women who had a previous low-transverse cesarean, many clinicians elsewhere do not. even when tolac is offered, it may be presented in a way that guides women not to choose it; “several studies suggest that how risk is presented and communicated by providers may have a powerful effect.” the high vbac rate of success in this study was achieved not only by support of vbac by the attend- ing clinicians but also by a high acceptance of tolac by the women. although some women wanting an elective repeat cesarean delivery may have self-referred to a hospital, no woman who came to the birth center chose a repeat cesarean over a tolac. in other popu- lations, a much higher percentage of women who are offered a tolac choose a cesarean delivery. desire for a partner’s involvement, a sense that vaginal birth can be empowering, improved maternal-infant bond- ing, greater ease with breastfeeding, and expectation of an easier recovery have all been identifi ed as reasons that women choose a tolac. these reasons can be promoted in offi ce visits, public service announce- ments, and other venues in changing cultural attitudes toward greater support for vbac. labor support may also be a factor in the low cesar- ean rate at the lafarge birthing center. a cochrane review of trials involving , women found that women with continuous support in labor were less likely to have a cesarean delivery, instrumental vaginal birth, or a baby with a low -minute apgar score. at the birthing center, the physician, midwife, or nurse was at the mother’s bedside throughout each labor. maternal-fetal outcomes the positive outcomes cannot be explained by low-risk populations. women giving birth at the lafarge birth- ing center were at higher risk than the general us population in terms of higher rates of advanced mater- nal age and grand multiparity (figures and ), as is the case in other amish communities. as in ihs hospitals, deliveries in our study resulted in a low primary cesarean rate and a high vbac rate. at %, our cesarean rate is much lower than the national rate of % and closer to the rates of . %, . %, and . % from ihs hospitals. the vbac rate of % is much higher than the national rate of . %. national birth center standards subsequent to the national birth center study (nbcs), many birthing centers and organizations developed policies of not attempting vbacs. the nbcs of , women attempting vbac in birthing centers found uterine ruptures ( . %), hysterec- tomy ( . %), infants with a -minute apgar of less than ( . %), and fetal deaths ( . %). the study concluded that “birth centers should refer women who have undergone previous cesarean deliveries to hospitals for delivery.” in the lafarge community, most women with a previous cesarean refuse hospital delivery. tolac is offered at the birthing center as a safer alternative to tolac at home. it is uncertain whether any of the perinatal deaths or other bad out- comes could have been avoided if the babies had been delivered in a hospital rather than a birthing center. without the birthing center, mothers would probably would have given birth at home rather than the hospi- tal, without a physician or nurse in attendance. we do not argue that tolac in birthing centers should be adopted for the general us population. we do believe our experience offers important lessons that, in the right circumstances and practice environ- ment, cesarean rates can be decreased and vbac rates increased in a safe and evidence-based manner. the lafarge birthing center does offer a model of care sensitive to cultural norms that highly value the health of the childbearing woman. to read or post commentaries in response to this article, see it online at http://www.annfammed.org/content/ / / . key words: birthing centers; wisconsin; prenatal care; cesarean section; pregnancy; natural childbirth submitted june , ; submitted, revised, january , ; accepted february , previous presentations: findings were presented at a poster session at the annual spring conference of the society of teachers of family medicine, april -may , , new orleans, louisiana. references . wall e, roberts r, deutchman m, heston w, atwood l, ireland b. trial of labor after cesarean (tolac), formerly trial of labor versus elective repeat cesarean section for the woman with a previous cesar- ean section: american academy of family physicians; . http:// www.annfammed.org/content/suppl/ / / / . . .dc / tolac_ _guideline.pdf. . national institutes of health consensus development conference panel. national institutes of health consensus development confer- ence statement: vaginal birth after cesarean: new insights march - , . obstet gynecol. ; ( ): - . . hamilton b, martin j, ventura s. births: preliminary data for . national statistics reports. vol . hyattsville, md: national center for health statistics; 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( pt ): - . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ letters table . clinical characteristics according to alms genotype amino acid type dm p ngt p asp/asp asp/his his/his asp/asp asp/his his/his n . ( . )a bmi (kg/m ) . ± . . ± . . ± . . . ± . . ± . . ± . . fpg (mmol/l) . ± . . ± . . ± . . . ± . . ± . . ± . . h glucose (mmol/l) n.a. n.a. n.a. . ± . . ± . . ± . . fpi (pmol/l) . ± . . ± . . ± . . . ± . . ± . . ± . . homa ir . ± . . ± . . ± . . . ± . . ± . . ± . . arg/arg arg/ser ser/ser arg/arg arg/ser ser/ser n . ( . )a bmi (kg/m ) . ± . . ± . . ± . . . ± . . ± . . ± . . fpg (mmol/l) . ± . . ± . . ± . . . ± . . ± . . ± . . h glucose (mmol/l) n.a. n.a. n.a. . ± . . ± . . ± . . fpi (pmol/l) . ± . . ± . . ± . . . ± . . ± . . ± . . homa ir . ± . . ± . . ± . . . ± . . ± . . ± . . lys/lys lys/arg arg/arg lys/lys lys/arg arg/arg n . ( . ) a bmi (kg/m ) . ± . . ± . . ± . . . ± . . ± . . ± . . fpg (mmol/l) . ± . . ± . . ± . . . ± . . ± . . ± . . h glucose (mmol/l) n.a. n.a. n.a. . ± . . ± . . ± . . fpi (pmol/l) . ± . . ± . . ± . . . ± . . ± . . ± . . homa ir . ± . . ± . . ± . . . ± . . ± . . ± . . data are means±sem. a differences in genotype and allele (p value) distributions were tested by fisher’s exact tests. p values represent values obtained after linear regression analysis with correction for age, sex and bmi lack of association between gene variants in the alms gene and type diabetes mellitus association studies were carried out with type diabetic pa- tients (n= ) and age-matched normoglycaemic subjects (n= ) randomly chosen from a population-based study in the netherlands [ ]. glucose tolerance status was confirmed by ogtt in all subjects as described previously [ ]. all participants were between and years and of caucasian origin to avoid bias. allele frequencies of gene variants in the alms gene were compared between type diabetic subjects and matched controls using fischer’s exact tests. anova or linear regression analysis was carried out to test associations with other diabetes related pa- rameters like bmi, glucose and insulin concentrations. a p value of . or less was considered statistically significant. we carried out a database search to identify genetic variation in the coding region of the gene. recently it has been shown that disease-associated gene variants are found more frequently in the coding regions of the gene compared to the non-coding re- gions [ ]. therefore we have limited our study to these gene variants. a total of snps in the alms gene were described in the public snp database which is available via the internet (http://www.ncbi.nlm.nih.gov/snp/, jan ). seven of these were in the coding regions of the alms gene. two of these seven gene variants were silent, asn (aac-aat) and leu (ctg-ttg). the other variants were at positions gly (gly-val, gga-gta), asp (asp-his, gat-cat), asp (asp-ala, gac-gcc), arg (arg-ser, agg- doi . /s - - - received: february / revised: april published online: june © springer-verlag serum insulin response and increased risk of type diabe- tes. diabetes : – . frayling tm, hattersley at, mccarthy a et al. ( ) a putative functional polymorphism in the igf- gene. diabe- tes : – . winarto a, miki t, seino s, iwanaga t ( ) morphologi- cal changes in pancreatic islets of katp channel-deficient mice: the involvement of katp channels in the survival of insulin cells and the maintenance of islet architecture. arch histol cytol : – corresponding author: a. t. hattersley, centre for molecular genetics, peninsula medical school, barrack road, exeter, ex ax, uk to the editor: recently the gene responsible for the alström syndrome has been identified [ , ]. it was shown that muta- tions in this large gene, encoding for a protein of amino acids, associate with alström syndrome cases. alström syn- drome is an autosomal recessive disease; characterized by reti- nitis pigmentosa, type diabetes mellitus, obesity and sensori- neural deafness (omim ). furthermore patients fre- quently have cardiomyopathy, insulin resistance and dyslipida- emia. since many of these features are also seen in type dia- betes mellitus patients this gene is a potential candidate gene for type diabetic patients and associating co-morbidities. this led us to search for gene variants in the alms gene in type diabetes, obesity and insulin resistance. letters agt) and lys (lys-arg, aag-agg). the gene variants at gly and asp and at arg and leu were in complete linkage disequilibrium with each other (n= and n= respectively). gene variants at position asp and asn were not detected in our cohorts. therefore we limited our studies to the gene variants at positions asp , arg and lys , which were subsequently studied in detail in the association studies (table ). genotypes were determined by pcr-rflp based methods in each individual after validation of the detection method by direct sequencing. the observed frequencies were all in hardy-weinberg equi- librium (data not shown). genotype and allele frequencies were not significantly different between patients and control subjects for either of the gene variants (all p> . , table ). furthermore we did not observe significant associations with other parameters like bmi, glucose or insulin concentrations during ogtt (all p> . , table ). insulin resistance as cal- culated by the homa insulin resistance index (homa ir) was also not significantly different between the different geno- types (p > . , table ). trends observed in the initial cohort could not be replicated in an independent population-based sample from the rotterdam study (type dm, n= and ngt, n= , data not shown) [ ]. a priori power calculations showed that we had an percent power to detect differences in allele frequency around percent. we have also recon- structed haplotype combinations of the different gene variants using the phase v . program [ ]. we observed six different haplotype combinations, however; they were not significantly different between the cases and controls (p> . , data not shown). also the reconstructed haplotypes did not associate with diabetes related parameters (data not shown). this report describes association studies with gene variants in the coding region of the alms gene in type diabetes. the absence of significant associations suggest that the vari- ants observed in our studies are not major factors in the patho- genesis of type diabetes mellitus or obesity. furthermore we found no evidence for a disease-associated haplotype. we con- clude that known gene variants in the coding regions of the alms gene are not associated with type diabetes, bmi or other diabetes related parameters in a population based study in the netherlands. further studies in other (larger) cohorts and with gene variants in other parts of this large gene locus are necessary to fully investigate the role of this gene in the pathogenesis of type diabetes mellitus and/or obesity. acknowledgements. the authors would like to thank the par- ticipants for their cooperation. the work was in part supported by a grant from the dutch diabetes foundation (dfn). l. m. ‘t hart, j. a. maassen department of molecular cell biology, leiden university medical center, leiden, the netherlands j. m. dekker, r. j. heine, j. a. maassen on behalf of the hoorn study, vu university medical center, institute for research in extramural medicine (emgo), amsterdam, the netherlands references . collin gb, marshall jd, ikeda a et al. ( ) mutations in alms cause obesity, type diabetes and neurosensory de- generation in alstrom syndrome. nat genet : – . hearn t, renforth gl, spalluto c et al. ( ) mutation of alms , a large gene with a tandem repeat encoding amino acids, causes alstrom syndrome. nat genet : – . mooy jm, grootenhuis pa, vries h de et al. ( ) preva- lence and determinants of glucose intolerance in a dutch caucasian population. the hoorn study. diabetes care : – . botstein d, risch n ( ) discovering genotypes underly- ing human phenotypes: past successes for mendelian dis- ease, future approaches for complex disease. nat genet [suppl]: – . hofman a, grobbee de, jong ptvm de et al. ( ) deter- minants of disease and disability in the elderly: the rotter- dam elderly study. eur j epidemiol : – . stephens m, smith nj, donnelly p( ) a new statistical method for haplotype reconstruction from population data. am j hum genet : – corresponding author: dr. j. a. maassen, on behalf of the hoorn study, vu university medical center, institute for research in extramural medicine (emgo), amsterdam, the netherlands e-mail: j.a.maassen@lumc.nl autoantibodies in type and type diabetes in the old order amish of lancaster county, pennsylvania to the editor: autoantibodies to ia- (ia- a) and gad (gada) have been widely used to identify individuals with type diabetes [ ]. up to % of newly diagnosed type pa- tients have ia- a and/or gada. the number of type patients doi . /s - - -z received: february / revised: april published online: june © springer-verlag with these autoantibodies is considerably lower, usually between to % for ia- and to % for gad. of non-diabetic control populations % or less have these autoantibodies. autoantibodies are of particular interest in genetically ho- mogenous populations as in finland [ ] and sardinia [ ] since they could provide epidemiological and mechanistic insights in- to the role of these antibodies in the pathogenesis of diabetes. such a genetically unique population also exists in the united states. the old order amish emigrated from western europe to the united states at the beginning of the th century, ap- proximately families settled in lancaster county, pennsyl- vania [ ]. the old order amish are a genetically isolated and well-defined closed caucasian population, with a high degree of consanguinity, and virtually no outsiders marrying into the community. type diabetes has been studied in this population [ ], but autoimmune diabetes has not been examined. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") 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volume, first page, author or article title). http://europepmc.org/abstract/med/ this month in the journal editors’ corner this month in the journal kathryn d. bungartz and robin e. williamson mutant ick in a multisystem syndrome lahiry et al., pp. – intestinal cell kinase (ick) is a member of the cyclin- dependent kinase family of proteins. kinases are respon- sible for phosphorylating other proteins within the body and thereby modifying their activity. kinases play an important role in numerous cell processes, including proliferation, apoptosis, and cell cycling. despite its name, ick is expressed in all adult human tissues and is evolutionarily conserved. using homozygosity mapping and sequencing, lahiry et al. identify ick mutations in six members of a large old order amish family affected by a multisystem neonatal lethal disorder. lahiry and colleagues define this recessive disorder as endocrine- cerebro-osteodysplasia (eco). as the name implies, eco manifests within the endocrine system, the brain, and the skeletal system. although eco does share similarities with syndromes such as majewski and hydroethalus, it appears to be a distinct entity caused by ick mutations. crohn disease structure chapman et al., pp. – many complex traits and diseases are made up of a combi- nation of simpler phenotypes, or subphenotypes. in an effort to simplify complex phenotypes for genetic-associa- tion analyses and create more homogeneous case datasets, researchers often look at subphenotypes when studying a disease. although this allows for easier classification, results can be complicated by the fact that many subphe- notypes are correlated with one another. an association between a genetic variant and a subphenotype may indi- cate that the variant has a direct effect on that subpheno- type, or perhaps the variant affects a second subphenotype that, in turn, modifies the first. chapman et al. report the methods they develop to distinguish direct effects from indirect effects. the authors use simulations to demon- strate the advantages of their methods over using univar- iate analysis and then look for associations between crohn disease subphenotypes and gene variants. by evaluating the location of disease and behavior of disease along with the genotype of five variants that have previously been found to be associated with crohn disease, chapman et al. report which variants have direct effects on certain crohn subphenotypes. similarly, the authors identify the am which aspects of the disease are highly correlated with one another. survey of nonsense-snp variation yngvadottir et al., pp. – nonsense mutations are alterations that introduce a stop codon into a genetic transcript. this type of mutation often leads to a truncated and usually nonfunctional protein product. thus, nonsense mutations are widely viewed as disruptive, and many human diseases are the result of such alterations. however, the persistence of such mutations within the genome implicates certain nonsense mutations in improved fitness. yngvadottir et al. investigate the prevalence of nonsense mutations and thereby shed light on recent human evolution. their findings indicate that the average person differs from other individuals by about genes as a result of the nonsense snps analyzed. very few of these nonsense snps were shown to represent disease-causing alleles. resequencing of one nonsense-snp-containing gene, magee , revealed a slight favor for the truncated version of the correspond- ing chb protein, implying positive selection of the nonsense snp. taken together, these findings encourage an alternate view on the effects of nonsense mutations. genetic genealogy and the utah pedigrees gitschier, pp. – as an increasing amount of genetic data is deposited into repositories, there is a concern about how best to protect the identities of those participating in genetic studies. here, gitschier reports her efforts to determine how much work is involved in gaining family information about indi- viduals who contributed material for the ceu hapmap samples. because the y chromosome is inherited relatively intact through the male lineage, shared y-chromosome haplotypes can infer direct relationships. because the ceu samples were collected from utah and many of the partici- pants were part of the population of the latter-day saints (lds), the author starts by determining the y haplotoypes and assembling pedigrees for two leaders of the latter-day saints (lds) from publicly available genealogy informa- tion. she then compares these y haplotypes to the ceu hapmap samples. none of the samples is a descendent of either of the lds leaders, but when the ceu haplotypes science editor, ajhg; deputy editor, ajhg doi . /j.ajhg. . . . ª by the american society of human genetics. all rights reserved. erican journal of human genetics , – , february , are then compared to the haplotypes in the genealogy infor- mation, of the samples hit on perfect matches. from these matches, it is possible for gitschier to make predic- tions as to the surnames of those ceu hapmap samples. although the author does not pursue verifying the accuracy of these predictions, the findings support the need to develop careful ways to maintain anonymity of individuals contributing genetic samples. synaesthesia whole-genome scan asher et al., pp. – people who are affected with synaesthesia react in unex- pected ways to certain stimuli. for example, some patients perceive colors when they hear sound. the cognitive the american journal of human genetics , – , february effects of synaesthesia can vary, and the disorder can lead to dysfunction in language and numerical processing, but also to an improvement in memory and perception. brain imaging has revealed that unusual regions of the brain are activated in people with synaesthesia when they are exposed to specific stimuli. unraveling the mech- anisms behind synaesthesia will not only contribute to our understanding of the disorder but also add to our knowl- edge of normal brain function and perception. although the disorder has been recognized to have heritable quali- ties, precise knowledge of the pathways that are disrupted has been lacking. asher et al. report the results of their genome-wide linkage scan in families with auditory-visual synaesthesia. the loci identified might help to pinpoint genes involved in perception. , 	this month in the journal 	mutant ick in a multisystem syndrome 	lahiry etnbspal., pp. - 	crohn disease structure 	chapman etnbspal., pp. - 	survey of nonsense-snp variation 	yngvadottir etnbspal., pp. - 	genetic genealogy and the utah pedigrees 	gitschier, pp. - 	synaesthesia whole-genome scan 	asher etnbspal., pp. - wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ decoding susceptibility to respiratory viral infections and asthma inception in children international journal of molecular sciences review decoding susceptibility to respiratory viral infections and asthma inception in children james f. read , and anthony bosco ,* telethon kids institute, university of western australia, nedlands, wa , australia; james.read@telethonkids.org.au school of medicine, the university of western australia, perth, wa , australia * correspondence: anthony.bosco@telethonkids.org.au received: july ; accepted: september ; published: september ���������� ������� abstract: human respiratory syncytial virus and human rhinovirus are the most frequent cause of respiratory tract infections in infants and children and are major triggers of acute viral bronchiolitis, wheezing and asthma exacerbations. here, we will discuss the application of the powerful tools of systems biology to decode the molecular mechanisms that determine risk for infection and subsequent asthma. an important conceptual advance is the understanding that the innate immune system is governed by a bow-tie architecture, where diverse input signals converge onto a few core pathways (e.g., irf ), which in turn generate diverse outputs that orchestrate effector and regulatory functions. molecular profiling studies in children with severe exacerbations of asthma/wheeze have identified two major immunological phenotypes. the irf hi phenotype is characterised by robust upregulation of antiviral response networks, and the irf lo phenotype is characterised by upregulation of markers of tgfβ signalling and type inflammation. similar phenotypes have been identified in infants and children with severe viral bronchiolitis. notably, genome-wide association studies supported by experimental validation have identified key pathways that increase susceptibility to hrv infection (ormdl and chdr ) and modulate tgfβ signalling (gsdmb, tgfbr , and smad ). moreover, functional deficiencies in the activation of type i and iii interferon responses are already evident at birth in children at risk of developing febrile lower respiratory tract infections and persistent asthma/wheeze, suggesting that the trajectory to asthma begins at birth or in utero. finally, exposure to microbes and their products reprograms innate immunity and provides protection from the development of allergies and asthma in children, and therefore microbial products are logical candidates for the primary prevention of asthma. keywords: human rhinovirus; respiratory syncytial virus; systems biology; omics; multi-omics; asthma; wheeze; innate immunity; bow-tie architecture; early life . introduction human respiratory syncytial virus (hrsv) and human rhinovirus (hrv) are the leading cause of respiratory tract infections in infants and children [ , ]. most children experience mild or asymptomatic infection. however, in susceptible individuals, hrsv and hrv infections may result in severe disease [ ]. hrsv reaches almost universal infection by age and is the major causative agent of bronchiolitis in infants [ ]. hrv and hrv-c species, in particular, are major triggers for severe wheezing and asthma exacerbations in children, accounting for at least two-thirds of episodes [ – ]. bronchiolitis is the leading cause of hospitalisation for infants (< years) in the united states, contributing % of all infant hospitalisations [ , ]. moreover, it is estimated that hrsv is responsible for more than , emergency department (ed) visits and . million outpatient visits among young children (< years) annually in the us [ ]. there are more than . million asthmatic children in the us, int. j. mol. sci. , , ; doi: . /ijms www.mdpi.com/journal/ijms http://www.mdpi.com/journal/ijms http://www.mdpi.com http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /ijms http://www.mdpi.com/journal/ijms int. j. mol. sci. , , of and asthma exacerbations account for more than , ed visits, , hospitalisations and million gp visits [ ]. in addition to the burden of hrsv- and hrv-induced acute illness on health care resources, severe illnesses with either of these viruses are strongly linked to the development of persistent wheeze and asthma [ , , ]. what remains an open question is whether or not these viral agents drive the development of the various clinical manifestations of asthma and wheeze (e.g., atopic asthma, non-atopic asthma, and transient/persistent/recurrent wheezing) in susceptible individuals or alternatively unmask a pre-existing susceptibility in children who were already on a trajectory towards asthma. whilst both hrsv and hrv induce wheezing, hrv wheezing is a much stronger predictor of subsequent asthma than hrsv wheezing, especially in children with aeroallergen sensitisation [ ]. moreover, prevention of rsv infection in pre-term or high-risk infants with the monoclonal antibody palivizumab decreased recurrent wheezing but had no effect on atopic asthma or lung function [ – ]. oral prednisolone treatment of first-time wheezers with hrv decreased time to recurrence in the subgroup of children with high viral loads [ , ]. together, these data suggest that hrsv is a risk factor for non-atopic wheeze/asthma whereas hrv drives atopic asthma. given that asthma is a highly complex and heterogeneous disease, untangling the role of these viruses in the development of specific asthma phenotypes will require a detailed understanding of the underlying cellular and molecular mechanisms. omics enables the systematic investigation of the molecular states that underpin phenotypic states. when combined with the powerful tools of systems biology, which model genes as components of an interconnected system, it is possible to unveil the intervening biology that connects interactions between genes and environmental exposures with expression of disease for hypothesis generation, detection of biomarkers and endotypes, and prioritization of therapeutic targets [ – ]. as biological processes are under constant regulatory control [ ], a comprehensive understanding of health and disease will require an integrative perspective of how molecular features across multiple regulatory elements (dna sequence variation, open chromatin states, transcription factor binding, transcription of mrna, protein synthesis, and metabolites) work together to jointly contribute to emergent phenotypic states. . genetics the development of genotyping microarrays and next generation sequencing technologies has enabled the systematic investigation of associations between dna sequence variations and disease in human populations. the first genome-wide association study (gwas) for asthma identified the q - region, which is now the most significant and replicated susceptibility locus associated with asthma in the genome [ , ]. the q locus comprises asthma-associated snps, which span multiple candidate genes located in three regions divided into the core (ikzf , zpbp , gsdmb, and ormdl ), distal (gsdma) and proximal regions (erbb and pgap ). the core region is strongly associated with early onset asthma [ ], and the proximal region is associated with asthma in adults [ , ]. caliskan et al. [ ] investigated the relationship between q genotypes, virus-induced wheezing and asthma risk in early life. they found that the q genotypes were associated with hrv wheezing but not hrsv wheezing, and additionally that the association of these variants with asthma was only observed in those children who experienced hrv wheezing [ ]. these findings demonstrate that there is a specific interaction between q variants, hrv wheezing, and asthma risk in children. interestingly, studies from children raised in farming environments have demonstrated that the same q variants that are associated with risk for wheezing and asthma are also associated with protection from these conditions in children exposed to animal sheds [ , ]. this suggests the hypothesis that q variants increase susceptibility to the effects of hrv on asthma risk in the absence of protective microbial exposures [ ]. decoding the role of the q region in asthma pathogenesis is extremely challenging because the locus harbors multiple candidate genes and is characterised by strong patterns of linkage disequilibrium in european and asian populations [ ]. to address this issue, previous studies have focused on patterns of gene expression. ormdl and gsdmb are promising candidates in this regard because int. j. mol. sci. , , of expression levels of these genes are highly correlated with asthma risk genotypes at q in blood cells and are also induced by hrv infection [ ]. an alternative and elegant approach to pinpoint the putative causal genes is to study populations of african ancestry [ ], where patterns of linkage disequilibrium are less pronounced. employing this approach, ober and colleagues reported that only two snps in the q region were associated with asthma in african american children, and these snps were correlated with expression of gsdmb but not ormdl in airway epithelial cells [ ]. the q variants that correlated with ormdl and gsdmb in blood were not associated with asthma in african americans, suggesting that these snps were not central to asthma pathogenesis, and by inference, gsdmb is the leading candidate gene in the region. functional studies have also been employed to dissect the role of candidate genes on q in asthma. knockdown of ormdl in airway epithelial cells resulted in a marked reduction in expression of icam , which is the main receptor exploited by some hrv-a and hrv-b species for host cell entry, demonstrating a plausible functional link between ormdl and hrv wheezing [ ]. liu et al. [ ] demonstrated that silencing ormdl reduced replication of hrv- in airway epithelial and hela cells following in vitro infection. unlike the previous report, however, icam was not reduced following ormdl knockdown which may suggest the influence of ormdl occurs after viral binding [ , ]. ormdl is also important in the regulation of endoplasmic reticulum stress and the unfolded protein response, and the regulation of levels of ceramide and sphingosine -sulfate [ , ]. gsdmb is highly expressed in bronchial epithelial cells from subjects with asthma and expression is correlated with disease severity. overexpression of gsdmb in primary airway epithelial cells of human gsdmb transgenic mice induced -lipoxygenase and transforming growth factor-β (tgfβ) expression, and the mice developed increased airways hyperresponsiveness and airways remodeling [ ]. notably, tgfbr and smad , which mediate tgfβ signalling, are susceptibility loci identified in gwas for asthma [ ]. erbb mediates epithelial repair processes, and this function may be defective in subjects with asthma and co-opted by type inflammatory processes [ , ]. together, these data highlight the complexity of the q region, which likely contains multiple independent associations with asthma, variations across asthma endotypes and ethnic groups, and functional variations between blood and airway cells. bönnelykke et al. [ ] performed a gwas that focused on a severe asthmatic phenotype in children ( - yrs) characterised by recurrent severe exacerbations requiring hospitalisation, thereby avoiding the need for large sample sizes often required for more heterogeneous phenotypes [ ]. employing cases and controls conferred sufficient power to detect associations with the q locus, interleukin- (il- ), and a novel signal in the cadherin-related family member (cdhr ) gene [ ]. subsequently, bockhov et al. [ ] used microarray profiling to identify differentially expressed genes between epithelial cells which were susceptible versus resistant to hrv-c infection, identifying several membrane proteins (e.g., ccrl , il- rα, and cdhr ) that they hypothesized were potential candidates for hrv-c binding. ectopic expression of cdhr but not the other candidates in hela cells facilitated replication of an hrv-c-gfp reporter virus, and structural analysis identified putative binding sites for viral surface proteins which are conserved across all hrv-c subtypes [ ]. notably, the snp identified by bönnelykke and colleagues (cys →tyr, rs ) conferred approximately -fold greater hrv-c binding and a -fold increase in the number of infected cells [ , , ]. moreover, cdhr is highly expressed in differentiated ciliated epithelial cells, and cells that are homozygous for the asthma risk variant rs have -fold higher expression levels of the transcription factor foxj , a master regulator of ciliogenesis, which in turn accelerates the development of functional cilia [ , ]. this is an elegant example of how gwas combined with functional analyses can unlock the mechanisms that determine disease risk. exome sequencing is a promising approach to identify rare variants associated with disease, which would fall below the coverage offered by gwas. salas et al. [ ] sequenced the exome of paediatric cases (< years) with severe hrsv infection requiring hospitalisation. they found rare variants in olfactory receptor- and mucin-related genes (or u / , and muc , respectively) [ – ]. further, common disease-associated variants (maf > %) were detected in a similar complement int. j. mol. sci. , , of of genes (e.g., or c and muc ) as well as genes involved in antigen presentation (hla-dqa , hla-dpb ) [ ]. asgari et al. [ ] assessed infection susceptibility in infants (> year) hospitalised with severe viral respiratory infections, for which hrsv ( %) and hrv ( %) were the most common pathogens identified. exome sequencing revealed three rare loss-of-function variants in ifih (maf < . %), which encodes key viral rna-sensing receptor melanoma differentiation-associated gene (mda ) [ ]. mutant ifih isoforms were associated with reduced interferon-β (ifnβ) production and impaired atpase activity, and only proper function of ifih restricted hrv and hrsv replication [ ]. mda , a member of the rig-i-like receptor family, binds viral nucleic acid ultimately leading to the atpase-dependent transcription of type i interferon genes [ , ]. whilst gwas have furthered our understanding of the role of hrv in childhood asthma, there are limitations that are noteworthy. first, it is not possible to dissect complex disease mechanisms employing a single omics modality. second, susceptibility regions may contain multiple susceptibility genes with pleiotropic functions. third, gwas requires massive sample sizes to detect a large number of candidate genes, but the signals identified to date only account for a tiny fraction of the total predicted genetic variance. to address this issue, we would argue that novel perspectives and conceptual advances are required to advance the field. kitano and oda first argued that the innate immune systems is governed by a bow-tie architecture [ ]. as illustrated in figure , in a bow-tie structure, diverse input signals converge on a highly conserved core, which consists of a few non-redundant components, and this fans-out into diverse output signals. we previously proposed that genetic perturbation of the numerous input and output signals would create a lot of noise and complexity, but ultimately the whole system is controlled by a few core pathways [ ]. this concept has recently been extended to biological networks more generally into a new conceptual framework to understand the underlying genetic architecture of complex phenotypes. the “omnigenic” model proposes that a given complex phenotypes is dictated by; ( ) a minority contribution of variation by a small number of high-penetrance (“core”) genes, and ( ) a majority contribution of variation by a large number of low-penetrance (“peripheral”) genes [ ]. under this model, core genes have a direct effect on disease outcome. however, disease risk is driven by a large number of indirect effects from peripheral genes, acting within a highly interconnected regulatory network [ ]. we would also like to make the point that the core genes need not be polymorphic. adoption of the concept of the bow-tie architecture and the omnigenic inheritance model has potential to transform our understanding of the genetic architecture of complex inflammatory diseases. int. j. mol. sci. , , x for peer review of genes (e.g., or c and muc ) as well as genes involved in antigen presentation (hla-dqa , hla-dpb ) [ ]. asgari et al. [ ] assessed infection susceptibility in infants (> year) hospitalised with severe viral respiratory infections, for which hrsv ( %) and hrv ( %) were the most common pathogens identified. exome sequencing revealed three rare loss-of-function variants in ifih (maf < . %), which encodes key viral rna-sensing receptor melanoma differentiation- associated gene (mda ) [ ]. mutant ifih isoforms were associated with reduced interferon-β (ifnβ) production and impaired atpase activity, and only proper function of ifih restricted hrv and hrsv replication [ ]. mda , a member of the rig-i-like receptor family, binds viral nucleic acid ultimately leading to the atpase-dependent transcription of type i interferon genes [ , ]. whilst gwas have furthered our understanding of the role of hrv in childhood asthma, there are limitations that are noteworthy. first, it is not possible to dissect complex disease mechanisms employing a single omics modality. second, susceptibility regions may contain multiple susceptibility genes with pleiotropic functions. third, gwas requires massive sample sizes to detect a large number of candidate genes, but the signals identified to date only account for a tiny fraction of the total predicted genetic variance. to address this issue, we would argue that novel perspectives and conceptual advances are required to advance the field. kitano and oda first argued that the innate immune systems is governed by a bow-tie architecture [ ]. as illustrated in figure , in a bow-tie structure, diverse input signals converge on a highly conserved core, which consists of a few non-redundant components, and this fans-out into diverse output signals. we previously proposed that genetic perturbation of the numerous input and output signals would create a lot of noise and complexity, but ultimately the whole system is controlled by a few core pathways [ ]. this concept has recently been extended to biological networks more generally into a new conceptual framework to understand the underlying genetic architecture of complex phenotypes. the “omnigenic” model proposes that a given complex phenotypes is dictated by; ) a minority contribution of variation by a small number of high-penetrance (“core”) genes, and ) a majority contribution of variation by a large number of low-penetrance (“peripheral”) genes [ ]. under this model, core genes have a direct effect on disease outcome. however, disease risk is driven by a large number of indirect effects from peripheral genes, acting within a highly interconnected regulatory network [ ]. we would also like to make the point that the core genes need not be polymorphic. adoption of the concept of the bow- tie architecture and the omnigenic inheritance model has potential to transform our understanding of the genetic architecture of complex inflammatory diseases. figure . the innate immune system is governed by a bow-tie architecture. the bow-tie structure enables diverse input signals to converge on a few core pathways, which in turn drive functional responses through the actions of a large number of effector and regulatory molecules. we have figure . the innate immune system is governed by a bow-tie architecture. the bow-tie structure enables diverse input signals to converge on a few core pathways, which in turn drive functional int. j. mol. sci. , , of responses through the actions of a large number of effector and regulatory molecules. we have illustrated this concept by providing specific examples of key molecules associated with host responses to hrsv, hrv, and asthma risk. . epigenome the above studies highlight the utility of gwas to identify novel susceptibility loci. however, as genetic variation remains constant throughout life and does not vary between cell types, it can be difficult to pinpoint the timing and the cellular context of genetic effects. epigenetic mechanisms, on the other hand, are dynamic and programmed by environmental cues. epigenetic mechanisms are active prior to birth, enabling a unique opportunity to track the timing and trajectory of the early origins of disease [ , ]. an epigenome-wide study of dna methylation patterns in cord blood mononuclear cells (cbmcs) from subjects enrolled in a prospective birth cohort (n = ) found differentially methylated regions between children who did or did not develop asthma by age [ ]. construction of a molecular interaction network of the genes associated with the differentially methylated regions revealed clustering around regulatory (smad ) and pro-inflammatory (il- β) gene networks [ , ]. further, smad was hypermethylated in the cord blood of asthmatics compared to non-asthmatics, specifically among children born to asthmatic mothers [ ]. this finding was replicated in two independent cohorts and was not associated with cell type populations. devries et al. [ ] also demonstrated increased il- β following lps stimulation of cbmc of children born to asthmatic mother who will later develop asthma compared to non-asthmatics (independent of maternal asthma). as noted above, smad is a critical mediator of tgfβ signalling, which plays central roles in the regulation of immune responses and also in airways remodeling. importantly, the finding that smad methylation patterns are increased at birth in children who develop asthma at age suggests that progression towards asthma may begin in utero [ ]. the findings from large-scale gwas for asthma and related traits suggest that the genome comprises at least susceptibility loci, and that the bulk of these signals are located in non-coding regions of the genome [ , ]. by overlaying these gwas signals with epigenomic data derived from a broad range of cell types, it is possible to identify cell populations that have open chromatin states in these regions. employing this approach, multiple studies have demonstrated that candidate genes located near asthma risk loci are preferentially expressed in blood and lung tissue, and are strongly co-localised with tissue-specific regulatory regions, such as enhancers, in immune cells, especially cd t cells [ , , ]. however, it is noteworthy that the reference epigenomic data sets have limited coverage with respect to cell subpopulations (e.g., plasmacytoid dendritic cells) and molecular states (i.e., resting versus activated). epigenetic studies of nasal epithelial cells from asthmatic children have observed differential methylation in the hrv receptors cdhr and ldlr [ , ]. further, hrv-infected nasal epithelial cells from children ( – years) with asthma identified differential dna methylation in cpg, including in genes involved in the host immune response against viral infections, such as bat and neu [ ]. risk of severe lower respiratory tract infections, including hrsv, has been associated with increased dna methylation at birth in the enhancer region of prf , which encodes perforin- , an important mediator of cd + t cell and nk t cell-mediated cytotoxicity [ – ]. dna methylation of the enhancer region of prf was also observed in children ( – years) who have a history of severe hrsv infection compared to healthy controls [ ]. further, studies in experimental mouse models have demonstrated that epigenetic control of histones is important during hrsv infection. in hrsv-infected murine dendritic cells, kdm b demethylase alterations of h k methylation results in a dampened pro-inflammatory immune response, and an increased th response [ ]. in addition, methylation of h k by the histone methyltransferase smyd in regulatory t cells appears important for proper control of airway inflammation following hrsv infection [ ]. in human bronchial epithelial cells, ifnγ-dependent changes in histone methylation of h k of the promotor of rig-i, a pattern recognition receptor that detects viral double-stranded rna, results in upregulation of rig-i and int. j. mol. sci. , , of increased hrsv clearance [ ]. furthermore, inhibiting histone deacetylase in hrsv-infected airway epithelial cells suppresses hrsv infection and may alleviate virus-induced airway inflammation [ ]. together, these findings demonstrate that epigenetic regulation of key pathways involved in viral sensing (input signals, figure ) and immune effector function (output signals) influence risk for severe viral illness in children. . transcriptome type i and iii interferons induce a robust antiviral state in infected and surrounding cells and are essential for immunity to many viruses [ , ]. the dominant paradigm in the literature proposes that type i and iii interferons responses to hrv are deficient in subjects with asthma, providing a plausible explanation for the role of these viruses in exacerbations [ ]. however, this finding has not been consistently observed in every study, and most studies were performed using samples from adults [ ]. the childhood asthma study (cas) is a prospective birth cohort of children at high risk for asthma, which tracked all episodes of respiratory viral infection over the first years of life [ ]. production of interferon subtypes was assessed at the mrna level in cord blood mononuclear cells after stimulation with poly-ic. deficient production of type i and type iii but not type ii interferons (ifnγ) was observed in samples from % of the children, and this was associated with febrile lower respiratory tract infections in the first year of life and increased risk for development of persistent wheeze at age [ ]. notably, by four years of age, type i and type iii interferon responses were exaggerated in children who experienced febrile respiratory tract infections in the first year of life compared to children with wheezy infections, suggesting that deficient production of interferons in this subgroup was restricted to a specific temporal window in early infancy [ ]. a subset of infants are highly susceptible to acute viral bronchiolitis, and susceptibility to this illness is inversely related to post-natal age. we reasoned that the mechanisms that underpin the heightened susceptibility in infants could be elucidated by comparing immune response patterns during acute viral bronchiolitis in infants versus older children. we collected nasal scrapings and pbmc from children who were hospitalised with acute viral bronchiolitis and stratified the subjects into two age groups—infants (< months, n = ) and children ( months– years, n = ) [ ]. follow-up samples were collected at post-convalescence. hrsv was detected in % of the infants and % of the children, whereas detection rates for hrv were % and % in infants and children, respectively. in pbmc, response patterns in infants were characterised by upregulation of type i interferon-mediated antiviral responses. in contrast, response patterns in pbmc from older children were characterised by upregulation of immunoregulatory and growth factor signalling pathways (ptger , tgfβ, erbb , vegf, il- , areg, and hgf) and nk cell cytotoxicity. in the nasal mucosa, responses in both infants and children were dominated by type i and iii interferons, but the responses were much more intense in the infants. this intuitive picture of age-dependent differences in host immune responses can become somewhat distorted in the presence of high within-group variability. to address this issue, we employed a personalised analytical approach called n-of- pathways, which identifies dysregulated pathways within each individual subject, and can unmask covert immunophenotypes. two major phenotypes underlying acute viral bronchiolitis responses were discernible in pbmc—one phenotype demonstrated robust interferon responses, while the other displayed a general dampening of interferon and innate immunity and upregulation of type immunity and growth factor signalling [ ]. importantly, the emergent phenotypes were not restricted by age, although the hyperresponsive phenotype was enriched within infants. these data suggest for the first time that the pathogenesis of acute viral bronchiolitis may be driven by distinct immunophenotypes [ ]. weighted gene co-expression network analysis (wgcna) of gene expression profiles derived from nasal wash/swab samples is a powerful and unbiased technique to reconstruct the molecular networks that are mobilised during respiratory viral infections in children. employing this approach, we reported that irf , a master regulator of type i and iii interferon responses [ , ], was a major hub, linking interferon-mediated antiviral responses in asthmatic children with mild-moderate exacerbations [ ]. int. j. mol. sci. , , of to elucidate the role of irf in hrv responses, we employed sirna-mediated gene silencing to knockdown irf in airway epithelial cells. the data showed that knockdown of irf reduced the innate antiviral response to hrv infection and increased the expression of proinflammatory mediators (e.g., cxcl , il- , and il rl [ ]). the role of irf has also been investigated in an experimental mouse model of severe viral bronchiolitis. in this model, viral loads were markedly elevated in irf deficient mice, and this unleased the alarmins il- and hmbg , which induced type inflammation and airways remodeling [ ]. together, these data suggest that irf plays a dual role in respiratory viral infections, by promoting interferon-mediated antiviral responses and limiting the activation of alarmins and type inflammation. to explore the role of irf gene networks in children with more severe disease, we profiled gene expression in nasal swab samples from children (< years old) who presented to the emergency department with severe exacerbations of asthma or wheeze [ ]. hierarchical clustering delineated two major molecular phenotypes of hrv-induced wheeze, which were characterised by irf hi versus irf lo gene network patterns. children with the irf hi molecular phenotype were characterised by robust upregulation of type i interferon responses, whereas the irf lo phenotype lacked an irf signature and instead were characterised by upregulation of type inflammation (il- r, fcer g, arg , and serpinb ) and growth factor signalling pathways (tgfβ, csf , and egf) and downregulation of interferon-γ. importantly, the irf phenotypes were associated with distinct clinical features; irf hi children presented to emergency approximately two days after the onset of clinical symptoms, whereas irf lo children presented approximately days post-first symptoms (figure ). the irf lo phenotype was also associated with increased risk of admission to hospital and a shorter time to recurrence [ ]. int. j. mol. sci. , , x for peer review of investigated in an experimental mouse model of severe viral bronchiolitis. in this model, viral loads were markedly elevated in irf deficient mice, and this unleased the alarmins il- and hmbg , which induced type inflammation and airways remodeling [ ]. together, these data suggest that irf plays a dual role in respiratory viral infections, by promoting interferon-mediated antiviral responses and limiting the activation of alarmins and type inflammation. to explore the role of irf gene networks in children with more severe disease, we profiled gene expression in nasal swab samples from children (< years old) who presented to the emergency department with severe exacerbations of asthma or wheeze [ ]. hierarchical clustering delineated two major molecular phenotypes of hrv-induced wheeze, which were characterised by irf hi versus irf lo gene network patterns. children with the irf hi molecular phenotype were characterised by robust upregulation of type i interferon responses, whereas the irf lo phenotype lacked an irf signature and instead were characterised by upregulation of type inflammation (il- r, fcer g, arg , and serpinb ) and growth factor signalling pathways (tgfβ, csf , and egf) and downregulation of interferon-γ. importantly, the irf phenotypes were associated with distinct clinical features; irf hi children presented to emergency approximately two days after the onset of clinical symptoms, whereas irf lo children presented approximately days post-first symptoms (figure ). the irf lo phenotype was also associated with increased risk of admission to hospital and a shorter time to recurrence [ ]. figure . schematic representation of outcomes associated with irf hi and irf lo molecular phenotypes underlying severe exacerbations of asthma and wheeze. gene network diagrams reproduced with permission; originally published in the journal of immunology [ ], copyright © the american association of immunologists, inc. altman et al. [ ] collected nasal lavage samples from asthmatic children at baseline and at two timepoints ( – days, – days) after the onset of cold symptoms. wgcna was employed to elucidate gene network patterns and their dynamic states during viral and non-viral exacerbations. expression of a type i interferon response module (irf , stat , and stat ) was upregulated in virus positive subjects; expression of the module peaked at approximately days after cold onset and the expression intensity was increased in children with exacerbations compared to those without exacerbations. exacerbations responses were also associated with initial ( – days) enhanced epithelial-associated smad signalling and downregulation of lymphocyte-related pathways, followed by later upregulation of egfr signalling [ , ], mucus hypersecretion [ ], and eosinophil activation [ , ]. finally, the authors demonstrated that the ratio of expression between a type inflammatory module and the type i interferon response module at baseline could predict time to recurrence. figure . schematic representation of outcomes associated with irf hi and irf lo molecular phenotypes underlying severe exacerbations of asthma and wheeze. gene network diagrams reproduced with permission; originally published in the journal of immunology [ ], copyright © the american association of immunologists, inc. altman et al. [ ] collected nasal lavage samples from asthmatic children at baseline and at two timepoints ( – days, – days) after the onset of cold symptoms. wgcna was employed to elucidate gene network patterns and their dynamic states during viral and non-viral exacerbations. expression of a type i interferon response module (irf , stat , and stat ) was upregulated in virus positive subjects; expression of the module peaked at approximately days after cold onset and the expression intensity was increased in children with exacerbations compared to those without exacerbations. exacerbations responses were also associated with initial ( – days) enhanced epithelial-associated smad signalling and downregulation of lymphocyte-related pathways, followed by later upregulation of egfr signalling [ , ], mucus hypersecretion [ ], and eosinophil int. j. mol. sci. , , of activation [ , ]. finally, the authors demonstrated that the ratio of expression between a type inflammatory module and the type i interferon response module at baseline could predict time to recurrence. . microbiome it has been known for some time that children who are raised in traditional farming environments are protected from the development of allergies and asthma [ ]. to elucidate the underlying mechanisms, stein et al. [ ] conducted an elegant study to compare microbial exposure and innate immunity in amish and hutterite farm children. notably, the amish of indiana and the hutterites of south dakota have similar diets, lifestyles, and genetic backgrounds. however, the amish employ traditional farming methods and live on single-family farms in close proximity to their animals. in contrast, the hutterites live in large communal farms, and the animals are housed in large industrial complexes away from their homes. strikingly, the prevalence of asthma and allergies is approximately - -fold lower in amish children compared to hutterite children. house dust samples collected from amish homes differ from those of hutterite households, most notably with -fold higher levels of endotoxin (lipopolysaccharide (lps)) [ ]. in peripheral blood, proportions of neutrophils were increased, and proportions of eosinophils were decreased in amish compared to hutterite children. moreover, amish and hutterite children displayed distinct gene network patterns in peripheral blood leukocytes, which were characterised by upregulation of tnf- and irf -associated gene networks in the amish [ ]. finally, exposure of dust collected from amish but not hutterite households was sufficient to inhibit the development of asthma-related traits in an experimental mouse model, and this protection was dependent on the core innate immune signalling hubs myd and trif [ , ]. together, these data demonstrate that exposure to microbial products in early life can rewire innate immune programs and prevent the development of allergy and asthma. the protective “farm effect” is not restricted to farming practices per se, but rather is specifically determined by the microbial composition present. kirjavainen et al. [ ] reasoned that protective indoor microbiota from farm houses should confer similar protection in non-farm houses, irrespective of environmental and lifestyle factors. indeed, exposure to farm-like microbial relative abundance at age two months was associated with decreased risk of asthma development by six years of age in individuals who grew up in farm and non-farm households. thus, microbial compositional exposure in early life is a readily identifiable predictor of asthma development and therefore represents a modifiable intervention strategy [ ]. the host microbiome is a crucial determinant of innate immune maturation in early life. arrieta et al. [ ] found that reduced relative abundance of four bacterial genera in the gut microbiome in the first days of life marked infants at risk of asthma. decreased relative abundance of these bacteria was associated with reduced lps (endotoxin) biosynthesis within the microbiome. to further explore this finding, adult germ-free mice were inoculated with human faecal microbiota, with or without supplementation with the identified protective species. among the subsequent generation, mice born to supplemented mothers maintained the protective gut bacteria and exhibited lower airway inflammation following experimental challenge, characterised by reduced airway infiltration of immune cells and dampened pro-inflammatory cytokine production [ ]. the findings demonstrate that maternal exposure to specific microbes and their products can modulate risk of the development of asthma in the offspring. the airway microbiome is highly dynamic in early life, and matures to a stable, diverse profile later than microbial communities from other sites [ , ]. in the cas study, teo et al. [ ] examined the development of the infant (n = ) nasopharyngeal microbiome over the first year of life, capturing data during predefined asymptomatic periods and all symptomatic respiratory viral episodes. streptococcus-, moraxella-, and haemophilus-dominated profiles were all significantly more frequent during acute respiratory infections compared to asymptomatic periods and conferred a higher risk of more severe illness (presence of fever and/or infection spread to the lower airways) [ ]. in addition, high abundance int. j. mol. sci. , , of streptococcus colonisation was significantly more frequent in infants who developed wheeze at age . at years of age, these bacteria maintained their dominant colonisation and association with greater risk of respiratory viral infection and chronic wheeze, particularly alongside allergic sensitisation [ ]. importantly, the shift towards increased abundance of these bacteria frequently preceded viral detection and the onset of symptoms, suggesting that transient incursions of the airway microbiome with pathogenic microorganisms may destabilise homeostatic mechanisms and increase exacerbation risk [ , ]. the post-infection recovery of airway microbial populations appears important for future asthma risk. a multi-centre prospective cohort of infants (< year, n = ) hospitalised with bronchiolitis found that participants with increased airway colonisation with moraxella and streptococcus after viral clearance ( weeks post-hospitalisation) were at increased risk for recurrent wheeze at years of age [ ]. this was independent of the causative agent of viral bronchiolitis and no association was seen from index samples at the time of hospitalisation. moraxella and streptococcus genera were also prominent in the nasal microbiomes of older asthmatic children ( – years, n = ) followed over the fall season [ ]. moraxella-dominated microbiomes were prevalent among asthmatic children and were associated with increased risk of exacerbation and eosinophil activation, particularly in the younger participants. furthermore, in vitro inoculation of epithelial cells with m. catarrhalis provoked greater epithelial damage and inflammatory cytokine production [ ]. additionally, asthmatic airway microbiomes dominated by streptococcal species was associated with increased risk of hrv infection over the virus season. further studies investigating acute bronchiolitis have found an association of these bacterial species with the severity of hrsv infections and identified differences in airway microbial composition between hrsv and hrv infections [ – ]. taken together, exposure to microbes and their products can confer both risk or protection from the development of asthma-related traits and the triggering of exacerbations, depending on the timing, the location, and the specific microorganisms involved. accordingly, administration of specific microbes or their products in utero or in early infancy is a plausible strategy to reprogram innate immunity for the primary prevention of asthma and wheeze [ , – ]. . multi-omics multi-omic investigations provide unique insight into disease mechanics as they simultaneously assess the joint contribution of molecular features across multiple layers of biological regulation on a disease process or phenotype, and unveil emergent properties of biological systems. consequently, multi-omics studies have increased predictive power over single-omics analysis [ , ]. for example, zhou et al. [ ] demonstrated improved performance in classifying respiratory viral infection events from baseline when the classifier was built from the integration of multi-omic data. a key challenge of multi-omic research is the availability of computational tools to synthesise data from different omics platforms that are heterogeneous with respect to the scale, noise, and inherent bias of each platform. data integration methods are generally divided into two subgroups; integration of data derived from different cohorts of subjects using the same omics platform (horizontal), or alternatively integration of data derived from different omics modalities on the same subjects (vertical) [ – ]. vertical integration methods can be further divided into parallel or hierarchical methods; the former approach is agnostic to the data type, and the latter approach takes into account the sequence of information exchange in biological systems (e.g., →mrna→protein) [ ]. the application of multi-omic technologies to interrogate immune responses in early life is extremely challenging because there are limitations in the amount of sample that can be collected from infants. recently, novel sample processing protocols have been developed which have enabled the partition of very small blood volumes into separate aliquots that are sufficient for multi-omic studies. olin et al. [ ] applied a systems biology approach to investigate immune development over the first months of life in pre-term and term births, from as little as µl of blood. targeted serum proteomics and whole blood mass cytometry revealed striking differences in immune cell populations between cord blood and peripheral blood collected from newborns in the first week of life [ ]. int. j. mol. sci. , , of further, integrated topological analysis revealed distinct clustering according to term/pre-term birth status in newborns. importantly, immune cell and proteome profiles from week of life preterm and term participants converged to a shared trajectory within weeks, which was particularly evident in neutrophils and naïve cd + t cells [ ]. in contrast, transcriptomic analysis of a subset of individuals revealed gene transcription regulation was not equivalent at months between the individuals from term and preterm births, indicating that immune function may be altered even though cellular profiles have converged. additionally, faecal microbiome analysis generally displayed increasing diversity over the first months of life, although individuals who exhibited early dysbiosis were characterised by perturbed developmental immune trajectory. taken together, these data suggest that drastically different immune profiles at birth may converge to a stereotypic trajectory determined by a (sufficiently) large number of response-eliciting immune exposures [ ]. lee et al. [ ] investigated immune development over the first week of life with multi-omic data generated from > ml blood. transcriptomic, proteomic and metabolomic data, as well as cellular immunophenotyping and cytokine profiling, demonstrated dramatic biological change over the first week of life compared to baseline (day of life ). three separate integration strategies were employed; prior knowledge-based network construction (networkanalyst [ ]), multivariate biomarker detection (diablo [ ]), and multiscale, multifactorial response network (mmrn [ ]). the integration results did not overlap at the level of features/variables, although they did exhibit common biological themes from higher-level functional pathways, most notably type i interferon and neutrophil signalling and complement activation. furthermore, these findings were validated in an independent cohort, suggesting immune development over the first week of life is not random but follows a robust, shared trajectory, irrespective of interindividual variation [ ]. together, these studies have revealed that the neonatal immune system undergoes rapid developmental changes during the first few weeks of life. upregulation of key innate immune defense functions exemplified by the type i interferon pathway would presumably provide protection from respiratory viral infections during this crucial period of heightened susceptibility. . single-cell omics immune function relies on the coordinated activity of multiple populations of innate and adaptive immune cells, with surrounding structural cells in the local tissue microenvironment. the development of single-cell profiling enables the first dissection of complex biological processes at single-cell resolution. a promising application of scrnaseq is to establish cellular atlases which deeply profile blood/tissue in healthy versus disease states to provide a reference framework for future research [ – ]. for example, single-cell genomics led to the discovery of the pulmonary ionocyte and its role as the primary source of cftr gene activity in the airway epithelia, which is important for understanding the pathogenesis of cystic fibrosis [ , ]. villiani et al. [ ] employed scrnaseq to profile monocytes and dendritic cell (dcs) in healthy adult pbmc, defining a revised taxonomy of these critical cells in innate immunity. notably, identification of a novel dc subset, distinguished by axl and siglec expression, demonstrated homology to both conventional dcs and plasmacytoid dcs [ ]. in addition to the discovery of novel cell types and states, because cells are continuously undergoing changes in their functional state, and this process is not synchronised between cells, it is possible to extract dynamic information from single-cell profiles and project cells onto temporal (pseudotime) or developmental trajectories [ ]. during infections, dynamic interactions between infected cells, immune cells and other bystander cells are needed to clear the infection and restore normal homeostasis. ligands produced by “sender cells” are recognised by receptors on “receiver cells”, triggering downstream signalling events, which ultimately activates target genes. computational methods have been developed that leverage ligand–receptor expression patterns between cells to infer cell-to-cell communication networks that mediate immune activation [ ]. int. j. mol. sci. , , of krausgruber et al. [ ] employed rna-seq and atac-seq to elucidate the role of three structural cell populations (endothelial, epithelial, fibroblasts) across tissues in regulation of the immune response to viral infection. notably, by inferring cell-to-cell communication networks on the basis of ligand–receptor pairs, they identified substantial cross talk between structural and immune cells. moreover, there were substantial differences between immune gene profiles from the same structural cell types across different organs, indicating an underappreciated role of structural cells in the regulation organ-specific immune responses. importantly, integrative analysis of epigenetic and transcriptomic data revealed an epigenetic immune potential in structural cells, defined by gene expressed at low levels during homeostasis which were “epigenetically poised” for rapid upregulation [ ]. indeed, functional evaluation employing an in vivo viral challenge model demonstrated preferential activation of genes with unrealized potential, suggesting structural cells are epigenetically pre-programmed to rapidly respond to viral exposure. these findings highlight the potential for single-cell genomic approaches to generate expression atlases across asthma-relevant tissues and contexts, and leverage ligand–receptor network analyses to decipher immune/structural cross talk in an organ-specific and organism-wide manner. . conclusions hrsv and hrv are the most frequent agents of respiratory infection in infants and children and are major triggers of bronchiolitis and wheeze/asthma exacerbations, placing an enormous burden of global health care resources. the bow-tie architecture and omnigenic inheritance model represents an important conceptual advance that posits that a small number of core pathways drive disease processes through many input and output signals, which themselves contribute to disease risk through direct and indirect signalling paths [ , ]. omics investigation have begun to unveil the molecular details and intricate signalling pathways that govern the innate immune responses to respiratory viruses. assessment of gene expression profiles in children with severe viral-induced exacerbations of asthma/wheeze uncovered two distinct molecular phenotypes—one defined by high expression of irf and a robust interferon/antiviral response and the other characterised by low irf expression and upregulation of markers of tgfβ signalling and type inflammation [ ]. comparable phenotypes are also evident in infants and children who were hospitalised with acute viral bronchiolitis [ ]. the first asthma gwas identified the q locus as a major susceptibility locus for asthma, which is specifically associated with early-onset asthma in children who wheeze with hrv infection [ ]. subsequent gwas identified further candidates in diverse regions across the genome [ , , ]. importantly, functional validation of gwas candidate asthma risk genes have uncovered key roles in hrv infection susceptibility (ormdl and cdhr ) and tgfβ signalling (gsdmb, tgfbr , and smad ) [ , , , ]. furthermore, a subset of individuals display deficient type i and iii interferon responses at birth and this confers greater risk of developing febrile lower respiratory tract infections in the first year of life and persistent wheeze in early childhood [ ], indicating that the trajectory towards asthma begins at birth or in utero. in the first week of life, expression of the interferon system is rapidly upregulated and by inference the innate immune system is poised to respond to respiratory viral infections [ ], and in this context it is noteworthy that infants with severe viral bronchiolitis displayed exaggerated type i and iii interferon responses in the airways compared to older children [ ]. finally, early life exposures to microbes and their products are critical for training appropriate innate immune responses, and can confer protection from development of allergies and asthma in children [ ]. therefore, administration of microbial products in early life is a promising strategy for the primary prevention of asthma and wheeze. author contributions: writing, review and editing, j.f.r. and a.b.; 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[crossref] [pubmed] © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . /science.aah http://dx.doi.org/ . /nbt. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /s - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /s - - - http://www.ncbi.nlm.nih.gov/pubmed/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	genetics 	epigenome 	transcriptome 	microbiome 	multi-omics 	single-cell omics 	conclusions 	references varicella infection complicated by group a beta-hemolytic streptococcal retropharyngeal abscess case report varicella infection complicated by group a beta-hemolytic streptococcal retropharyngeal abscess christine m. clark, colin huntley, and michele m. carr college of medicine, the pennsylvania state university, university drive, hershey, pa , usa department of otolaryngology-head & neck surgery, thomas jefferson university, chestnut street, th floor, philadelphia, pa , usa division of otolaryngology-head and neck surgery, department of surgery and department of pediatrics, college of medicine, the pennsylvania state university, university drive, hershey, pa , usa correspondence should be addressed to michele m. carr; mcarr@hmc.psu.edu received may ; accepted august academic editor: nicolas perez-fernandez copyright © christine m. clark et al. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. an unimmunized -month-old child presented with a retropharyngeal abscess and coincident varicella infection. the abscess resolved with operative drainage. this is the first published report of this connection, although varicella is known to be associated with abscesses in general. practitioners should be aware that cervical abscesses may complicate varicella infections. . introduction cervical abscesses are frequently diagnosed and treated by otolaryngologists. as head and neck surgeons, we may be tempted to perform a directed physical exam targeted to the site of symptoms. however, this case illustrates the need for a complete assessment of the patient and the importance of keeping an open mind with regard to parsimonious explanations of a constellation of signs and symptoms. . case presentation a previously healthy -month-old amish female presented to her primary care physician with a -day history of right neck swelling, decreased oral intake, and decreased urine output. she developed a rash on the day of presentation, which prompted her parents to have her evaluated. there was no history of recent travel or sick contacts. she had no previous immunizations and was otherwise healthy. a ct of her neck, ordered by the primary care physician, suggested a retropharyngeal abscess, and she was subsequently trans- ferred to our tertiary care facility. on examination, she was afebrile and breathing effort- lessly. torticollis was noted. there was firm induration of the right neck, measuring approximately × cm in diameter. she was found to have edema of the right side of her soft palate and peritonsillar region, as well as an erythematous fullness on the posterior oropharyngeal wall. she had + tonsils, clear rhinorrhea with erythematous nasal mucosa, and normal tympanic membranes. a red macular rash was noted on her forehead, neck, abdomen, and back with accompanying vesicles on some of the lesions. her lungs were clear to auscultation bilaterally. she had a grade i of vi flow murmur that disappeared when supine. no abdominal masses were palpable, and her bowel sounds were normal. the ct scan revealed a hypodense multiloculated mass consistent with an abscess in the right retropharyngeal space, extending from the soft palate inferiorly to the angle of the mandible (figure ). she was admitted to the otolaryngology service and started on intravenous ampicillin-sulbactam. she was taken to the operating room the next morning for intraoral incision and drainage of the abscess. after induction of general anesthesia, a draining abscess was visualized on the posterior pharyngeal wall. an incision was made in the posterior pha- ryngeal wall under general anesthesia, but evacuation of pus was limited. an -gauge needle was used to aspirate several locations along the right side of her posterior pharyngeal wall from the nasopharynx to the hypopharynx. bacteriological hindawi publishing corporation case reports in otolaryngology volume , article id , pages http://dx.doi.org/ . / / http://dx.doi.org/ . / / case reports in otolaryngology figure : ct shows a hypodense area in the right parapharyngeal region, surrounded by an area of soft tissue thickening. there is minimal ring enhancement of the lesion. cultures from the abscess grew group a beta-hemolytic streptococcus. intraoperative scrapings were taken of her skin lesions and returned positive for varicella zoster antigen. she was continued on iv antibiotics until postoperative day two, at which time she was switched to oral amoxicillin- clavulanate for a total of days and discharged. two weeks later, the family reported by telephone that her rash had resolved and that she had resumed a normal diet and activities. . discussion complications of varicella infections differ greatly in their severity and in the tissues that they affect. abscess formation is a known but relatively rare sequela of acute varicella infection [ , ]. the location of abscesses in varicella is variable. a total of four patients with a history of varicella coincident with spinal epidural abscesses have been reported in the literature, with group a beta-hemolytic streptococcus and staphylococcus aureus as the underlying bacterial isolates from the abscesses in these cases [ – ]. other cases of varicella complicated by staphylococcal and streptococcal abscesses on the extremities and back as well as in the mediastinum have also been reported [ – ]. to the best of our knowledge, no cases of primary abscesses in the cervical region in association with varicella infection in pediatric patients have been previously identified. varicella (chickenpox) is a highly contagious, typically benign disease that generally afflicts children. it is caused by the varicella zoster virus, which is spread through direct contact with skin lesions or inhalation of viral particles. varicella infection usually presents with pustular lesions covering the trunk and head. more than % of people are exposed to this virus by age twenty, with most cases observed in children less than ten years of age [ , ]. since the implementation of the varicella vaccine in , there has been a significant reduction in the number of varicella cases and a % reduction in varicella associated hospitalizations [ ]. one in five of those vaccinated will become infected, but the symptoms in these patients are much less severe [ , ]. the patient described in this case had not received routine childhood immunizations, as this conflicted with the family’s amish belief system; however, this case underscores the importance of adhering to a correct vaccination schedule. as with most infections, varicella can be complicated by spread of infection to other tissues of the body. neonates, adults, and the immunocompromised are most vulnerable to complications of varicella [ ]. the primary infection with the varicella zoster virus creates a temporary depression of the immune system. this immune depression predisposes the individual to a secondary bacterial or viral infection [ , ]. some common complications of varicella include central nervous system dysfunction, pneumonia, and skin and soft tissue infections [ , , ]. along with these common complications of varicella, the immune depression caused by the primary varicella infection can also create a predisposition to tonsillitis [ ]. tonsillitis is an inflammation of the tonsils, which are lymphatic tissues located in the pharynx. tonsillitis can be either bacterial or viral in origin. group a beta-hemolytic streptococcus is the most common bacterial cause [ , ]. tonsillitis is also associated with various complications, such as dehydration, airway obstruction, and abscess for- mation in the potential spaces of the neck. these potential spaces, created by fascial layers, are the retropharyngeal, vascular, submandibular, submental, parapharyngeal, peri- tonsillar, and parotid spaces [ ]. abscess formation in these potential spaces is commonly caused by tonsillar infection [ , ]. the varicella infection seen in our patient may not have been the direct cause of her retropharyngeal abscess but still played a pivotal role in its formation. a combination of factors including the weakened immune system created by the primary varicella infection, the development of tonsillitis, and the proximity of the tonsils to the retropharyngeal space may have been vital in the development of the patient’s abscess. . conclusion this case is the first report of a retropharyngeal abscess occur- ring as a sequela of varicella infection in a pediatric patient. it underscores the need for a complete physical examination as well as the need to consider parsimonious explanations when disparate signs and symptoms occur together. competing interests the authors declare that there is no conflict of interests regarding the publication of this paper. references [ ] m. a. reynolds, b. m. watson, k. k. plott-adams et al., “epidemiology of varicella hospitalizations in the united states, – ,” the journal of infectious diseases, vol. , no. , pp. s –s , . 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[ ] s. b. millan and w. a. cumming, “supraglottic airway infec- tions,” primary care: clinics in office practice, vol. , no. , pp. – , . ped - .pmd volumen - número colestasias familiares: una causa común de hiperbilirrubinemia conjugada marcela godoy p. , humberto soriano b. . servicio de pediatría. unidad de gastroenterología pediátrica. complejo hospitalario san borja arriarán. . unidad de gastroenterología y nutrición pediátrica. departamento de pediatría. facultad de medicina. pontificia universidad católica de chile. abstract familial cholestasis: a comon cause of conjugated hyperbilurubinemia cholestasis is defined as a disorder affecting the production of bile resulting in the retention of its components in the liver and blood. in children, this disorder is almost always due to genetic alterations. functionally, cholestasis may be the result of hepatic failure to secrete bile due to decrease in transport, synthesis or biliary obstruction. extrahepatic cholestasis may be caused by biliary atresia and other obstructions of the bile ducts. intrahepatic cholestasis may be the result of several disorders including progressive familial intrahepatic cholestasis (pfic) types , and , an autosomal recessive disease due to mutations in the genes atp b , abcb and abcb respectively. pathophysiology and clinical presentation of this disease are now well understood. clinically, these patients may present with jaundice, itching, anorexia, and generally unwell. laboratory tests may disclose conjugated bilirubin over mg/dl or larger than % of total bilirubin. ursodeoxycholic acid, cholestiramine and biliary diversion may help in some of these conditions. ongoing research into the mechanisms of genetic cholestasis could be key to therapy. (key words: cholestasis, progressive familial intrahepatic cholestasis, genetic cholestasis syndromes, hepatobiliary transport). rev chil pediatr ; ( ): - resumen la colestasia corresponde a un trastorno en la formación y excreción de la bilis que provoca retención de sus componentes y daño en hígado y sangre. la colestasia en el niño casi siempre se debe a una alteración hepática secundaria a causas ahora mayormente conocidas a nivel molecular. desde el punto de vista funcional la colestasia resulta de una insuficiencia secretora del hígado debido a una disminución del flujo biliar por falla en los procesos de transporte o síntesis o a una obstrucción de la vía biliar. la colestasia extrahepática incluye la atresia de vías biliares y otras obstrucciones de la vía biliar. la colestasia intrahepática incluye las colestasias progresivas familiares pfic , y causadas por fallas en los genes atp b , abcb y abcb respectivamente. clínicamente pueden presentarse con ictericia, prurito, artÍculo de revisiÓn review article rev chil pediatr ; ( ): - trabajo recibido el de diciembre de , aceptado para publicación el de mayo de . correspondencia a: dr. humberto soriano b. e-mail: hsoriano@med.puc.cl revista chilena de pediatría - julio - agosto introducción la colestasia se clasifica en extrahepática causada por atresia de vías biliares, quiste de colédoco u otras obstrucciones e intrahepática (tabla ) , causada por colestasia intrahepática familiar progresiva (pfic) tipo , ó y coles- tasia intrahepática recurrente benigna (bric); y otras patologías que resultan en colestasia tales como fibrosis quística, déficit de alfa antitripsina, hemocromatosis neonatal, hipoti- roidismo y tirosinemia. las causas de colestasia en pediatría, sus síntomas clínicos, diagnóstico y tratamiento se resumen en la tabla - . las pfic es un grupo de colestasias cróni- cas de inicio precoz en niños que son la causa más frecuente de colestasia intrahepática y que en general progresan a cirrosis hepática. la primera descripción fue la enfermedad de byler en la comunidad amish en descendientes di- rectos de jacob byler y nancy kauffman , , en el condado de lancaster, cerca de filadelfia, ee.uu. posteriormente, se ve que esta enfer- medad puede presentarse en otro grupo étnico de niños que no pertenecían a la comunidad amish por lo que se denomina síndrome de byler . actualmente, se prefiere la nomencla- tura pfic , ó según el gen afectado - . clave para el diagnóstico de las colestasias por pfic y es que la hiperbilirrubinemia conju- gada se acompaña de ggt (gamma glutamil transpeptidasa) normal o baja porque no hay daño de conductos biliares. sin embargo, en la pfic , el mecanismo del daño es por falla en el trasporte biliar de los fosfolípidos protectores del daño por acción detergente de la bilis lo que resulta en daño de conductillos biliares y eleva- ción importante de la ggt. estas diferencias clínicas llevaron al estudio molecular de las causas de estas enfermedades y en los últimos años se han clonado y caracterizado los genes y transportadores res- ponsables de estos defectos que causan coles- tasia. específicamente los subgrupos se han nombrado como pfic /bric, pfic y pfic o por la consecuencias de la mutaciones descritas: fic (familial intrahepatic cholestasis ), bsep (bile salt export pump) y mdr (multi drug resistance regulator ) , , , - . a continuación se describen los diferentes tipos de colestasias intrahepáticas familiares (tabla ) , . colestasia intrahepática familiar progresiva tipo (pfic ) se caracteriza por presentarse con ictericia en neonatos de mes a , meses y con diarrea de inicio entre los a meses. la pfic tiene además manifestaciones extrahepáticas tales como síndrome de malabsorción, pancreatitis y nefrolitiasis. los exámenes de laboratorio se caracterizan por bilirrubinemia total de hasta mg/dl a mg/dl y de predominio conjugada, transaminasas veces el valor normal, ggt normal o disminuida, colesterol normal, eleva- ción de las sales biliares séricas promedio de umol (n < umol) y disminución de la concentración de sales biliares en bilis a . umol (n > . umol) - . en la pfic el gen afectado es atp / fic en el cromosoma q -q , , fic es una proteína integral de la membrana del canalículo biliar. fic si bien controla la secre- ción biliar no es un transportador de ácidos biliares. en pfic los mecanismos de inhibi- ción de la secreción biliar hepática todavía se están estudiando, pero serían por el efecto de fic en otros genes. las manifestaciones extrahepáticas podrían ser explicadas porque fic se expresa además del hígado en otros tejidos, como páncreas, estómago, intestino del- gado y riñón . se ha descrito que los pacientes soriano h. y col. anorexia y compromiso del estado general. desde el punto de vista del laboratorio las enfermedades colestásicas se caracterizan por hiperbilirrubinemia conjugada mayor a mg/dl o mayor a % de bilirrubina total. (palabras clave: colestasia, colestasia intrahepática familiar progresiva, síndromes colestásicos genéticos, transportador hepatobiliar). rev chil pediatr ; ( ): - volumen - número que han requerido trasplante hepático conti- núan con diarrea lo que sugiere un rol del transportador fic a nivel intestinal. una teo- ría para explicarlo sería que el defecto fic en intestino altera el ciclo enterohepático de sales biliares. en ratones homocigotos para la muta- ción g v en atp b , que es la misma que presentan los niños con pfic se evidenció desregulación del transportador ileal asbt (apical sodium bile salt transporter), originando una alta absorción intestinal de ácidos biliares con los que fueron alimentados , , . en la biop- sia hepática de pfic se observa colestasia intracanalicular y en la microscopia electrónica bilis granular típica de byler lo que es diagnósti- co . el tipo de mutación se correlacionan en general con la severidad de la presentación clínica, lo que podría explicar la presentación clínica como pfic o bric . es posible realizar diagnóstico prenatal de enfermedad colestásica hereditaria (pfic - y síndrome de alagille) mediante screening de mutaciones en dna obtenido de vellosidades coriónicas y muestras de amniocitos cultiva- dos . existe además la posibilidad de estudio me- diante gene chip para diagnóstico de mutacio- nes en pacientes con síndromes colestásicos intrahepáticos hereditarios además de pfic , y , síndrome de alagille y déficit de alfa antitripsina . colestasia intrahepática recurrente benigna (bric) fue descrita por primera vez por summerskill and walshe en , . se han publicado más de casos en el mundo . al igual que en la pfic , el gen involucrado en su patogenia es el fic , pero en estos individuos tiene penetrancia variable . además se ha descrito bric aso- ciado con mutaciones de bsep , . la modu- colestasias familiares tabla . causas de colestasia intrahepática a. desordenes de transportadores de membrana y secreción ) desordenes de secreción canalicular a) transportadores ácido biliar - deficiencia bsep - persistente progresiva (pfic ) - recurrente benigna (bric ) ( , ). b) tansporte fosfolípido-deficiencia mdr (pfic ) c) transporte ion cloro - fibrosis quística (cftr) ) desordenes complejos o multiorgánicos a) deficiencia del gen fic - persistente progresiva (pfic ) - recurrente, benigna (bric ) b) colangitis esclerosante neonatal c) artrogriposis - disfunción renal - síndrome colestásico b. desordenes de biosíntesis y conjugación de ácidos biliares ) deficiencia de -oxo- esteroide b reductasa ) deficiencia b-hidroxilasa – c - dehidrogenasa esferoidal isomerasa ) deficiencia oxysterol & hidroxilasa c. desordenes de embriogénesis y morfológicos ) síndrome alagille - ausencia de conductillos biliares ) arpkd enfermedad poliquística renal autosómica recesiva. (antiguamente síndrome de caroli) malformación de la placa ductal en límite de endoderma y mesoderma ) adpld enfermedad poliquística hepática autosómica dominante. similar a c , pero de presentación más tardía d. no clasificada-mecanismo desconocido abreviaturas: bsep (bile salt export pump); pfic (progressive familial intrahepatic cholestasis); bric (benign recurrent intrahepatic cholestasis); mdr (multi drug resistance regulator); cftr (cystic fibrosis transmembrane conductance regulator). modificada de balistreri-shneider , . revista chilena de pediatría - julio - agosto soriano h. y col. tabla . enfermedades colestásicas: clínica, diagnóstico y tratamiento enfermedad clínica prueba hepática examen diagnóstico tratamiento atresia de vías biliares ictericia bili t ± mg/dl ecografía abdominal kassai < - semanas acolia bili d ± mg/dl biopsia hepática tx hepático coluria ggt ± iu/i hida colangio intraoperatoria quiste de colédoco igual a igual a ecografía abdominal y de roux resección completa (carcinoma biliar) síndorme de alagille facies típica ggt elevada rx columna urso prurito Ácidos biliares elevados ecocardiograma colestiramina xantomas ex. oftalmológico tx hepático cardiopatía bx hepática (sin conductos biliares) fibrosis quística rn igual a electrolitos en sudor enzimas mayores cirrosis biliar esteatocrito urso focal gen cftr déficit alfa- -antitripsina ictericia ggt elevada alfa - - antitripsina tx hepático si es severo hepatoesplenomegalia ac. biliares elevados biopsia hepática síntomas pulmonares coagulopatía (acúmulo a at en tos, asma hepatocito) tirosinemia ictericia alfa fetoproteína succinilacetona en orina ntbc insuficiencia hepática > . ug/l hemocromatosis insuficiencia hepática ferritina > ng/ml biopsia glándula salival prevención en embarazo neonatal labio gamma ev cao déficit síntesis hiperbilirrubinemia Ác. biliares disminuidos espectrometría de masa existe tratamiento Ác. biliares conjugada en orina específico dependiendo del déficit hipotiroidismo hipotonía t libre levotiroxina retraso crecimiento y tsh desarrollo hernia umbilical galactosemia catarata galactosa total dieta sin galactosa hipoglicemia galactosa -p hepatoesplenomegalia uridiltransferasa hipotonía (screening neonatal ampliado) pfic , y se discute más adelante en tabla . ggt: gamma glutamil transferasa; hida: hepatic imido diacetic acid; ntbc ( -( -nitro- -fluoromethylbenzoyl)- , -cyclohexanedione); urso: Ácido ursodeoxicolico; cao: cocktail antioxidante. lación del fenotipo por otros genes asociados es materia de activa investigación. en bric la edad de presentación es variable de niño a adulto ( meses a años). esta enfermedad se caracteriza por episodios recurrentes de hiperbilirrubinemia conjugada con valores hasta veces el valor normal desencadenada por virosis en niños y adultos. la bric presenta recurrencia estacional en muchos pacientes (pri- mavera, verano) cada años. la duración del episodio puede ser de semanas a meses (promedio meses), pudiendo permanecer asintomático por un período variable ( mes a años). la mayoría de estos pacientes pre- senta prurito aunque el % puede no tenerlo. en la evaluación del primer episodio se debe incluir estudios serológicos para excluir hepati- tis viral aguda y crónica así como también otras causas de enfermedad hepática crónica. ade- más es necesario suspender todos los medica- mentos que pudieran causar colestasia como acido clavulánico, azatioprina, eritromicina y volumen - número clorpromazina - . es fundamental realizar ecografía abdominal para excluir patologías biliares obstructivas con dilatación de la vía biliar. en adolescentes o adultos la colangioreso- nancia o la colangiografía retrograda endos- cópica se pueden utilizar para excluir colangitis esclerosante u otras causas de alteraciones estructurales de árbol biliar. es importante rea- lizar una biopsia hepática para precisar el diag- nóstico y descartar otras patologías . la biop- sia hepática muestra una colestasia centrolo- bulillar sin lesión hepática o ductular a pesar de que puede observarse un leve infiltrado infla- matorio , , . la diferencia entre bric y las colestasias progresivas (pfic , y ) es que estas últimas progresan hasta llegar a cirrosis , . es muy raro que el bric progrese a fibrosis o requiera trasplante hepático . colestasia intrahepática familiar progresiva tipo (pfic ) en pfic la clínica es similar a la que presentan los pacientes con pfic . la edad de presentación es principalmente en neonatos (rango: mes a años) . presentan ictericia con hiperbilirrubinemia total en rango de a mg/dl, bilirrubinemia directa en rango de , a , mg/dl. alt en rango de a iu/l. al igual que en pfic la ggt y el colesterol son normales. en la ecografía abdominal pueden tener hepatoesplenomegalia leve y litiasis bi- liar , . la diferencia radica en que tienen una evolución más rápida a cirrosis e insuficiencia hepática. en pfic no se han encontrado otros órganos afectados. posterior al trasplante he- pático no desarrollan diarrea y recuperan la curva estatural . el gen afectado es abcb /bsep en cromosoma q . bsep participa en el trans- porte de ácidos biliares hacia la bilis a nivel de la membrana canalicular del hepatocito. la biop- sia hepática demuestra transformación gigante celular similar a la descrita en hepatitis neonatal . colestasia intrahepática familiar progresiva tipo (pfic ) la presentación clínica de pfic es dife- rente a pfic y , la edad de presentación es de mes a años , . sólo un tercio de los casos se diagnostican antes del año de vida y muy rara vez su debut es como colestasia neonatal en cuyo caso es parte del diagnóstico diferencial de la atresia de vías biliares, la cau- sa más frecuente de colestasia. la mayoría de los niños son diagnosticados por hepato- esplenomegalia o por complicaciones de su cirrosis hepática durante la infancia o adoles- colestasias familiares tabla . comparación de pfic , , y bric pfic pfic pfic bric transportador fic bsep mdr fic herencia autosómica autosómica autosómica autosómica recesiva recesiva recesiva recesiva gen atp b abcb abcb atp b cromosoma q -q q - q q - clínica sint. extrahepático si no no no prurito intenso intenso leve a moderado ausente % laboratorio ggt normal normal elevada normal colesterol normal normal normal o elevado normal Ác. biliares séricos elevado elevado elevado elevado (recaída) histología bilis de byler hepatitis células gigantes proliferación ductal colestasia centrolobulillar evolución cirrosis cirrosis cirrosis benigna revista chilena de pediatría - julio - agosto cencia . dentro de los exámenes de laborato- rio tienen hiperbilirrubinemia directa en rango de , mg/dl a , mg/dl, alt rango de iu/l a iu/l (n< iu/l) y ggt en rango de iu/l a iu/l (n < iu/l) . la ggt eleva- da en pfic sirve para diferenciarla de la pfic , pfic y bric en los que la ggt es normal o baja. la ggt es elevada en pfic pues es la única que causa daño endotelial de los conductos y conductillos biliares . en pfic el gen afectado es abcb / mdr en cromosoma q . el mdr es el transportador de fosfolípidos hacia la bilis. los fosfolípidos cumplen la función de solubilizar las sales biliares y proteger el epitelio ductal , . en la biopsia hepática se demuestra prolifera- ción ductal acompañada de fibrosis y cirrosis . en pacientes o familiares de pacientes con pfic se han reportado litiasis biliar, episodios de colestasia del embarazo y litiasis intrahe- pática , . fisiopatología de pfic , , y bric el componente principal de la bilis son los ácidos biliares que son sintetizados en el hígado a partir del colesterol; son tóxicos para las membranas por lo que deben ser transportados unidos a fosfolípidos y colesterol . los ácidos biliares a nivel de la membrana sinusoidal del hepatocito son transportados por ntcp (na+ taurocholate co-transporting polypeptide) que necesita bomba na/k atpasa para mantener el gradiente eléctrico intracelular adecuado y por oatps (organic anion transporting poly- peptides) independiente de la bomba na/k atpasa. al llegar a la membrana canalicular son transportados por bsep (bile salt export pump) a la bilis. el mdr (multi drug resistance regulator) es el transportador de fosfolípidos hacia la bilis. los fosfolípidos cumplen la fun- ción de solubilizar las sales biliares y proteger el epitelio ductal , , (figura ). la regulación de la actividad excretora de bilis se realiza por el sensor fisiológico de sales biliares denomina- do fxr (farnesoid x receptor) , , , , . los ácidos biliares (ácido cólico, ácido deoxicólico y ácido litocólico) activan a fxr y a otros recep- tores regulando el transporte y biosíntesis de los ácidos biliares y lípidos , . en el hepatocito fxr induce la expresión de bsep aumentando la excreción de sales biliares , e inhibe la expresión de ntcp con lo cual disminuye la entrada de sal biliar en el polo sinusoidal del hepatocito. además, fxr inhibe la expresión de enzimas involucradas en la síntesis de ácidos biliares a partir del colesterol, es decir, neosínte- sis . esta acción la realiza activando shp (short heterodimer protein o small heterodimer partner), una proteína que en el ileon inhibe la expresión de asbt (apical sodium bile salt transporter), transportador que se encarga de la reabsorción de sales biliares que posterior- mente llegan al hígado mediante sistema portal. el exacto rol de fic el gen de la pfic es desconocido, aunque estaría relacionado con la reducción en la expresión de fxr , lo que resulta en disminución del flujo biliar con acu- mulación de ácidos biliares. tratamiento de la colestasia el tratamiento de la colestasia va a depen- der de la causa. no todos los casos de colestasia tienen terapia específica. se debe suplementar con vitaminas liposolubles (a, d, e y k) y calcio en colestasia crónica puesto que la falta soriano h. y col. figura . transportadores de ácidos biliares. muestra el mecanismo fisiopatológico de los transportadores hepatobiliares involucrados en pfic - . en la membrana sinusoidal: ntcp (na+ taurocholate co- transporting polypeptide) y oatps (organic anion transporting poly- peptides) y en la membrana canalicular: bsep (bile salt export pump), mdr (multi drug resistance regulator) y fic (proteína que trasloca aminofosfolípidos). fe: fosfatidil etanolamina, fs: fosfatidil serina, sb: sales biliares, f: fosfolípidos. volumen - número de bilis resulta en malabsorción de grasa con saponificación que quela el calcio y reduce su absorción. en el tratamiento del prurito se ha utilizado colestiramina en dosis de a g/día que es una resina de intercambio aniónico que liga sales biliares en el intestino y aumenta su eliminación fecal y disminuye el prurito en pfic y . quizás por efecto de otros genes modu- ladores, la respuesta al ursodeoxicolico y a la colestiramina es a veces dramática y a veces su uso resulta en empeoramiento de los sínto- mas por lo que el tratamiento debe ser indivi- dual. colestasias familiares mecanismo de acción de Ácido ursodeoxicólico (udca) udca (bilis de oso usado en medicina china) es ampliamente usado para el tratamien- to de una variedad de enfermedades hepáticas colestásicas crónicas. en humanos alcanza el % del total de ácidos biliares. los primeros reportes de sus efectos beneficiosos fueron publicados en el año por leuschner et al . dentro de los mecanismos de acción de udca se describen la protección de los colangiocitos contra los efectos tóxicos de lo ácidos biliares , estimulación de la secreción figura . algoritmo diagnóstico de colestasia. revista chilena de pediatría - julio - agosto soriano h. y col. biliar y de los ácidos biliares hidrofóbicos e inhibición de la apoptosis de los hepatocitos , . a nivel de los transportadores hepatobiliares el udca estimula la expresión de bsep lo que facilita el transporte de sales biliares a nivel canalicular e inhibe asbt (apical na+ depen- dent bile salt transporter) a nivel intestinal lo que facilita la eliminación de sales biliares del organismo , . en resumen los efectos de udca son: colerético, inmunomodulador, cito- protector y antiapoptótico . la dosis de udca en niños con colestasia es mg/kg/día , . otros reportes sugieren a mg/kg/día , . udca en pfic sólo funcionaría en pa- cientes que tengan una mutación missense (ac- tividad residual de la proteína). los pacientes que no responden al tratamiento con udca deben ser considerados para el trasplante he- pático , o para la derivación biliar. la dosis adecuada de ursodeoxicólico en bric es desconocida , pero se ha utilizado dosis de mg/kg/día hasta la desaparición de la clínica y luego mg/kg/día hasta normaliza- ción de parámetros de laboratorio . su objetivo sería acortar el período de colestasia . el tudca (tauroursodeoxycholic acid) se ha uti- lizado en forma experimental. estimula la inser- ción de proteínas transportadoras en la mem- brana canalicular del hepatocito , . el trasplante hepático es el tratamiento más eficaz en pfic y para pacientes con enfer- medad progresiva o cirrosis establecida, en ge- neral en la primera década, con sobrevida so- bre el % a largo plazo. la derivación biliar externa parcial también existe como un trata- miento de respuesta variable para pacientes con pfic y . en aquellos pacientes con prurito intratable sin respuesta a tratamiento médico la derivación biliar externa parcial tiene efecto importante sobre la colestasia y el creci- miento de los niños, aunque no todos ellos se benefician de la cirugía. episodios de deshidra- tación después de efectuada la derivación de- ben ser considerados , . en resumen se han realizado grandes avan- ces en el diagnóstico y biología molecular de la colestasia intrahepática otrora hepatitis neonatal. estos avances auguran un futuro optimista para el manejo y tratamiento de estas enfermedades de alta morbimortalidad en niños. referencias .- balistreri w: intrahepatic cholestasis. j pediatr gastroenterol nutr ; : s -s . .- balistreri w, bezerra j, jansen p, karpen s, 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breaking new ground resource center purdue university west lafayette, in william e. field, ed.d. department of agricultural and biological engineering purdue university west lafayette, in introduction old order anabaptists, such as the amish and old order mennonites, hold a unique place in american agriculture. from the time they arrived in america during the late s until today, old order anabaptist communities have been inseparably tied to agriculture. their farming practices have also made a significant impression on the larger society, not only because of their unusual, often antiquated, agricultural technologies, but also because of their consistent agricultural successes. commentaries on amish farming practices during the s consistently praised them as being among the best farmers (cosgel, ), and earlier in this century kollmorgan stated that it is doubtful that any other socio-religious bodies have so consistently distinguished themselves in agricultural enterprise as the amish and mennonites ( ). another essential factor that distinguishes old order anabaptist agriculture from the remainder of the farming population is the anabaptist view that farming is not just a method of economic survival but also a primary means of preserving their entire culture. hostetler states that the charter of the amish requires that members make their living from farming, rural, or semi-rural occupations ( ). cosgel demonstrated that the amish were not always as productive as other farmers during the mid and late s, but he concluded that the reason for their lower productivity was their sacrifice of current income to increase their bequests to the next generation ( ). other researchers have shown that the more successful amish parents are at getting their children into farming, the more likely that the children will remain amish (ericksen, et.al., ). amish leaders decry dwindling farmland, and the corresponding difficulty with keeping children in farming, as one of their greatest frustrations (ericksen, et.al., ). although the old order anabaptists have such an inseparable link to agriculture, and even though by they had settlements in nearly half of the american states (friesen and friesen, ; redekop, ), centralized statistics regarding farm-related injuries and interventions among the old order anabaptists have been sparse. a review of over published farm accident or injury reports from more than states revealed none that specifically addressed work-related injuries within old order anabaptist communities or categorized data such that these cases could be identified. even in the three states with the largest amish and mennonite populations, ohio, pennsylvania, and indiana, this type of summary had not been done. to help bridge this information gap and to assist with developing an appropriate response, this paper focuses on the problem of farm-related injuries within old order anabaptist communities. basic cultural, historical, and religious beliefs are examined, farm-related fatality statistics are summarized, and best-practice intervention strategies are explored. the authors believe that current evidence suggests that certain old order anabaptist choices concerning farm safety issues may be directly related to their socio-religious beliefs. researchers who have explored the problem of farm-related injuries have largely ignored, or dismissed as irrelevant, the contributing roles played by personally held religious beliefs or religious worldviews held by certain groups of agricultural producers or the community in which they live. this omission is reflected in the near absence of published research on topics relating to agricultural safety and health in which even secondary attention is given to the religious beliefs of those impacted by farm injuries. even articles published by rural sociologists, economists, agricultural historians, geographers, and other social scientists have rarely addressed the relationship between religious beliefs and agricultural practices (swierenga, ). the few studies that have been done, however, appear to suggest that different religious views do, in fact, influence how farmers view agriculture, how they work and how they approach risk taking (winter, ; swierenga ; cosgel ; salamon, ; van den barr, ). as the topic of old order anabaptist farm-related injuries is examined, especially in relation to their religious beliefs, the authors also believe that important implications beyond old order communities will become apparent. for example, amish farmers not only use production methods similar to those used by the larger agricultural community years ago, but their overall worldview, based on a scriptural framework, is also similar to that of many american farmers of the nineteenth century and earlier. therefore, by taking a close-up view of old order anabaptists and the possible effects of their religious beliefs on farming practices, it may be possible to make inferences about some of the underlying beliefs and related behaviors of the larger agricultural population. such beliefs and behaviors may, in fact, have had their roots in religious principles but with time have become diluted as rural society has become more urbanized and the overall society influenced by secular humanism. in other words, old order communities may provide a "window to the past" not only in regard to the farming practices of earlier generations but also in reference to some of the underlying, shared beliefs of the larger agricultural community that affect aspects of farming such as risk taking. using such a lens may provide additional insight that would be helpful in designing more effective injury prevention strategies. though these issues cannot be fully addressed within this paper, they are worthy of further consideration. cultural background information as previously indicated, consolidated farm injury data concerning old order communities are rare. there are undoubtedly multiple reasons for the lack of information, and some probable contributors relate to cultural factors. within the rules of their society, the amish, especially, stress separateness from the larger culture (hostetler, ), and that results in little information about their activities being communicated to outsiders. in addition, this separation creates a tendency among the amish to shun assistance from those outside their communities (kollmorgan, ; kraybill, ). for example, they rarely participate in health or disability insurance programs, a common source of data on injury and disease. old order anabaptists are seldom a vocal political force, especially on the national level, and most amish do not pay social security taxes (kraybill, ) or participate in government farm programs (kollmorgan, ). consequently, there is little political pressure for federal or state governments to give special consideration to their situation, especially with respect to safety and health issues. finally, reporting agencies generally do not separate farm-related fatalities according to religious affiliation, even though old order communities are not solely religious groups: hostetler labels the amish with such terms as "little commonwealth," "sectarian society," and "folk society" ( ). kreps, et. al., classify the amish as a subculture rather than simply a religious group and identify four dimensions of their subcultural status apart from religious beliefs: historical, geographical, social, and economic dimensions( ). to better define the population being addressed and to further explore the effect of socio- religious beliefs on farm-related behavior, additional historical and cultural background information is provided below. today's old order anabaptist communities have common roots in the anabaptist movement that began in switzerland during the early th century. the first anabaptists separated from the roman catholic and reformed churches over issues such as the anabaptist refusals to participate in military activities or baptize infants. (the term "anabaptist," meaning "re- baptizer," was coined by opponents of the anabaptists because of their insistence on baptizing adults, even if they had been baptized as infants.) in , menno simmons broke from the state church in the netherlands for reasons similar to those of the swiss anabaptists, and his followers were known as mennonites. later, in , jacob amman began a separation from the swiss mennonites because of controversies concerning such issues as clothing and church discipline, and thus, the amish sect began (hostetler, ). the largest migrations to the united states by amish and mennonites occurred during the mid s, primarily to escape persecution in europe (hostetler, ). since that time, these groups have continued to differentiate themselves from each other in a series of debates on various doctrinal issues (umble, ). in general, the mennonites have taken a more moderate stance than the amish concerning contact with the world outside their communities, and many mennonite groups have moved toward mainstream american religious traditions (redekop, ). however, there remain groups of "old order mennonites" that resemble the amish in almost every respect (redekop, ), including strict group discipline, an emphasis on humility and nonresistance, the control of technological innovations (umble, ), and distinctive, plain clothing. the next step toward liberality on the mennonite belief continuum is a group that this paper identifies as conservative mennonites. members of this group may use agricultural technologies that are more modern than the amish, such as steel-wheeled tractors, and they may use automobiles, but they are, overall, less modern in their technologies than typical american farmers. because of the similarities between conservative mennonite, old order mennonite, and amish beliefs and practices, fatalities from all these groups are included in the statistical observations found in this paper. it should also be noted that there are other types of old order or conservative anabaptist communities in north america, such as the hutterites and the old order german baptists. some of these groups use traditional practices based on the use of horses, while others live simply but have adopted modern agricultural technologies and practices, and are know as innovative producers (hostetler, ). an ongoing review of farm-related fatalities within these communities has resulted in very few documented cases. several characteristics of old order anabaptist socio-religious beliefs are important factors when considering farm-related fatalities within their populations. these include selective use of technology, a higher than average birth rate and family size, attitudes toward child labor, and beliefs about death. amish and old order mennonite choices in regard to technology are some of the most conspicuous labels of their societies. in areas near amish settlements, horses pulling buggies or plows are familiar sights, and such common technologies as electricity and telephones are used with reticence. the amish contend that these technological choices help to keep their communities in tact (hostetler, ). because many of these methods also necessitate increased contact with animals, a higher than average number of farm injuries and fatalities involving animal behavior would be expected. and though some amish farm with steel-wheeled tractors, many use no tractors or only use them for stationary power; thus, amish fatalities caused by events such as tractor rollovers or pto entanglements should be sparse or nonexistent. the technological distinctions regarding most old order mennonites and conservative mennonites are less dramatic than with the amish since many of the former groups use steel-wheeled tractors for fieldwork (umble, ). however, horses and wagons are still used on some mennonite farms. the amish have large families and prohibitions against birth control (hostetler, ). the average number of live births per amish couple is seven, and the amish population doubles approximately every years (hostetler, ). in one survey of the amish in lancaster county, pennsylvania, % were under the age of eighteen (kraybill ). it is not uncommon to encounter amish families with or more offspring ranging from infants to married adults. in addition, teaching children the importance of work is a high priority for amish parents (hostetler, ). amish children are expected to begin helping with chores soon after they can walk, and children frequently begin driving teams of horses before the age of ten (kraybill, ). in addition, since work is seen as a calling from god and not just a job, and since it is a central fiber in the fabric of amish community life, the integration of children into the family workforce takes on a sacred dimension (kraybill, ). kraybill states: amish children assume daily chores by five or six years of age, and their responsibilities in the barn and house grow rapidly. they are not seen as economic burdens but welcomed as blessings from the lord and as members who will contribute their fair share to the family economy ( , p. ). these socio-religious beliefs and practices point toward the probability of higher than average child injury and fatality rates in regard to participation in work-related activities. however, old order beliefs and practices may, in fact, result in lower than average injury and fatality rates with respect to other hazardous activities in which amish children do not generally participate, such as riding in automobiles or operating atvs, motorcycles, or snowmobiles. the anabaptist view of death is also a significant factor in addressing farm fatalities among their populations. while death is not treated lightly among the amish, they accept the biblical view that life in this world is temporary and that eternity is infinitely more significant (hostetler, ). according to hostetler: for all people who cling to life and enjoy it to the full, death is the greatest menace. the amish, who profess not to have conformed to this world, turn to the promise of life beyond death. their belief in the divine order of all things, including immortality, is a source of comfort to the mourning family and community ( , p. ). the central anabaptist concept of "gelassenheit," or yielding to authority, involves submission to the will of a sovereign god (kraybill ), even in matters of death. this belief that "the best is yet to come" may make it difficult to motivate old order anabaptists into complying with safety efforts simply out of a fear of death or serious injury. anabaptist fatality statistics in order to begin constructing an empirically based picture of old order anabaptist farm injury patterns, and to see if their socio-religious beliefs may affect those patterns, ninety-five ( ) case reports of old order anabaptist farm fatalities were drawn from the records of purdue university, the ohio state university, pennsylvania state university, the commonwealth of kentucky cabinet for health services, the new york state department of health, finger lakes occupational health services (n.y.), the new york center for agricultural medicine and health, the university of wisconsin, and from newspaper articles and farm reports submitted by a variety of sources. the inquiry focused on identifying fatalities that occurred during the performance of, or exposure to, farm work or within the farm environment. in addition to farm- related fatalities, several deaths were documented which involved motor vehicle and buggy collisions, but these were not included in this analysis. for reporting purposes, children were considered individuals years of age and younger, a classification consistent with most agriculture-related injury statistics. part of the reason for this distinction was that persons under the age of years are not legally permitted to operate farm machinery unless they are formally trained or are working for their parents or guardians. furthermore, in the amish community, children, especially boys, do not generally attend school beyond the eighth grade or age , the minimum age for formal education mandated by most states. information regarding the cases was initially categorized according to the following factors: state of origin, i.e. ohio; year of fatality; age of victim; time of fatality; day of week of fatality; month of year of fatality; and finally, source of injury. in several instances, specific information, such as time of day or date of death, was not reported in the original case summaries. of the ninety-five ( ) cases identified as old order anabaptist farm-related fatalities, four cases ( %) occurred in the s, seventy-six cases ( %) occurred in the s, and fifteen ( %) occurred in the s. seventy-six ( ) of the fatality victims were identified in the reports as amish, were mennonite, and one victim's religious affiliation was undetermined. figure shows a breakdown of the cases in regard to the state from which the fatality report originated. the distribution of fatalities by state was generally reflective of the distribution of the old order population. the one noted exception was the lower number of cases reported from ohio, which has the second largest population of amish. gender as would be expected in a study of agricultural, work-related fatalities, the vast majority ( %) of the cases occurred among males. this figure, however, is a lower percentage than would be found in non-old order anabaptist farm communities, suggesting greater involvement of females in agricultural tasks. in two cases ( %), the sex of the victim was not reported. age of the cases in which the age of the victim was reported, % of the fatalities were to children fifteen years and under, and % occurred in children six years and under. in at least of the fatalities involving children fifteen years and under ( % of all fatalities where age was reported), the victim was directly engaging in work-related activities at the time of death ; the remaining occurred in a work-related environment. youth aged - comprised only nine ( %) of the fatalities. in % of the cases, the victims were between the ages of and . only five fatalities ( %) were documented that occurred in individuals over the age of years. the latter figure is dramatically different from the distribution in non-old order populations where those over the age of typically account for more than one third of reported fatalities (purschwitz, ; whitman, ). among females, all the victims were aged years and figure : fatality reports by state under, and fourteen of nineteen female victims ( %) were under the age of years. the average age of all fatality victims was . years; the median age was years. figure provides a representation of the overall age distribution of fatalities. source of injury there is little consensus among researchers on how to group agricultural injury data regarding the source of injury (purschwitz, ; murphy & yoder, ). of particular concern in this report was the role of secondary sources, especially animal behavior, as contributors to the fatalities. for example, in % of the cases the immediate source of injury was due to being run over by a wagon or other vehicle. such fatalities were sometimes attributable to human behavior such as a father backing over an infant or the victim losing his or her balance and falling off a wagon. however, runovers were often precipitated by a horse becoming unruly and throwing the vehicle out of control, thereby ejecting the victim into the path of the wagon wheels. consequently, it was important to distinguish between primary and secondary sources of injury, especially when animal behavior played a role. figure : fatalities by age the following categories were chosen in regard to the primary source of injury: being run over by a vehicle; direct animal behavior (i.e., being kicked or stepped on); falls; entanglement in farm machinery; suffocation; being crushed by or between heavy objects or machinery; drowning; being struck by an object; fire/explosion; heat stroke; tractor rollovers; unintentional firearms fatalities; and "other" fatalities. figure summarizes the fatality statistics for sources of injury. by far, the largest number of fatalities was attributed to being run over by vehicles. of all the fatalities, fell into this category ( %), and as would be expected in the old order anabaptist community, a large number of these ( ) involved horse drawn vehicles. unlike data reported on farm-related fatalities within the larger agricultural community in which well over % relate to agricultural tractors, only nine old order anabaptist cases, or %, involved tractors. in regard to secondary sources of injury, of the fatalities were attributable to human behavior and to indirect animal behavior . in one run-over case, the secondary sources were uncertain, and in another, a wagon tongue broke, causing the victim to be ejected from the vehicle and run over, thereby making machinery failure a secondary source of injury. in children years and under, being run over caused of fatalities ( %). only eight of the latter cases were attributable to indirect animal behavior, while were attributable to human behavior, either on the part of the child or an adult. in approximately eight cases, it appears that figure : fatalities by source of injury children were run over by adults or older siblings who were simply unaware of the child's location. in cases ( % of total fatalities), children were run over after falling from farm implements, wagons, or tractors they had been riding on or driving. the second most prevalent source of injury to both adults and children was direct animal behavior, such as being kicked, stepped on, or dragged by horses, mules, or cattle. sixteen cases ( % of total fatalities) were listed in this category. in one of these cases, horses dragged the victim when he became entangled in reins after a vehicle tongue broke, thereby making machinery failure a secondary source of injury. nine of the direct animal fatalities ( % of total fatalities) involved children years and under. in addition to the direct animal-related fatalities, sixteen cases ( % of total fatalities) were related indirectly to animal behavior such as bolting. therefore, % of all fatalities were directly or indirectly related to animal behavior. (see figure .) in nine cases ( %), the victims were crushed between or by heavy objects, and six of them were aged eight years and under. five of these children were crushed by heavy objects, and one was pinned between a tractor and a wagon. one of these fatalities was indirectly caused by animal behavior: horses bolted, thereby throwing a boy and two logs from a wagon, and the boy was crushed beneath the logs. the only adult victim was crushed between a skid steer loader bucket and frame when a hydraulic line broke. in regard to other sources of injury, entanglements and falls proved to be significant sources of injury, as they are in the general farm population (nsc, ). a total of five victims died by drowning; three drown in farm ponds, while the other two were small children who fell into horse tanks. all of the suffocation victims ( ) died in grain or feed bins. one fire victim was killed when a leaking propane heater exploded in a farm building; the other two were children who died in a barn fire after playing with matches in a hayloft. one old order mennonite youth was killed when a steel-wheeled tractor overturned. the lone firearms victim was an adolescent who was crawling through grass, was mistaken for a woodchuck, and was inadvertently shot by a figure : fatalities by animal behavior neighbor. in the "other" fatality category, one of the victims had been using a horse drawn implement; the horses were later found in the barn, while the victim was found in a field with head and chest injuries from which he later died. no specific source of injury was determined. in the remaining case classified as "other," the victim was reported to have experienced a seizure, came into contact with an electric fence, and suffered cardiac arrest. month recent statistics show that summer months and midwestern harvest times are peak periods for all agricultural fatalities (murphy & yoder, ). some reasons for this include increased exposure to work hazards during the growing season, longer workdays, and a larger number of workers, such as children out of school (murphy & yoder, ). fatalities documented in the old order anabaptist community followed a similar pattern, as shown in figure . june is usually a prime time for haymaking, corn planting, and crop cultivation among the amish. the pattern for fatalities according to month was similar between all cases and those involving children. in both groups, the number of fatalities for july appeared unexpectedly low. day of week sunday is normally the day with the lowest number of farm fatalities, (purschwitz, ; murphy & yoder, ). in addition, because of the anabaptist belief in a sabbath rest, it would be expected that the fewest farm fatalities would occur on sunday. that was indeed the case with the observed cases, though thursday was only slightly higher in fatalities. a possible reason for the sunday fatalities is the fact that livestock must still be cared for on sunday, and thus, some chores must be done. in the cases where the day of fatality was discernable, friday had the highest percentage of fatalities with %, followed by wednesday with %, saturday with figure : fatalities by month (children shown in white) %, and monday and tuesday with %. for children years and under, % were killed on saturday, % on wednesday, and % on friday and tuesday. in the childhood cases in which complete information was reported, approximately % of the fatalities occurred during the weekend period of friday evening through sunday. apart from the fact that children are out of school during this time period, another explanation for this finding is possibly the increased involvement of members of the old order anabaptist community in off-farm occupations, causing a greater intensity of farm work over the weekend period. figure shows fatalities by the day of week. fatality statistics observations though children are frequently the victims of fatal farm injuries in the overall population (field & tormoehlen, ; murphy, ; national committee for childhood agricultural injury prevention, ), the percentage of old order anabaptist childhood fatalities was unexpectedly high in the cases identified and summarized. the national safety council reports that % of agricultural work fatalities in were under years of age (nsc, ). (national safety council data is reported as " and under," whereas this report classified data as " and under".) in comparison, for the current survey, the percentage of fatalities involving children or adolescents under years of age was %. as previously indicated, the average age of victims in the current survey was . years. in contrast, the farm fatality summary for indiana, a state representing % of the cases currently being considered, revealed an average age of years (field, ). a study of farm-related fatalities in indiana revealed that % involved figure : fatalities by day of week (children shown in white) persons over years of age (purschwitz & field, ), while only one fatality victim identified in this summary was over years of age. it is possible that the high number of child fatalities may be related to the old order anabaptist beliefs concerning large families and the involvement of children in farming activities. however, several mitigating factors should also be considered in regard to the lower than average age of victims observed in this summary. first, the number of cases considered was too small to draw concrete comparisons between the amish and non-amish populations. in addition, national sources of injury data do not routinely collect data on workers under the age of (murphy & yoder, ); therefore, non-amish childhood farm-related fatalities might be understated. it is also possible that a younger than average retirement age among the old order anabaptist farmers further contributes to a lowering of the average fatality age. for example, hostetler reports that the amish may retire from active farming at age ( ). according to meyers, the tendency of amish farmers in northern indiana is to retire in their late s to allow other family members to get started in farming. however, they generally remain active on the farm or employed in other areas ( ). therefore, given these factors, it cannot be concluded that childhood farm-related fatality rates are higher among the amish than in the general population. it should also be noted that jones reported that overall mortality and morbidity appeared lower in the amish population than in the general population ( ). it is also inappropriate to compare child mortality rates between the old order anabaptist community and the overall farm population without taking into consideration the exposures non- amish children have to hazards typically not found in old order anabaptist communities. the incidence of fatalities associated with motor vehicles, motorcycles, atvs, snowmobiles, firearms, and other agents are much higher in the general population because such agents are rarely used by old order children. even activities such as football and other contact sports result in several deaths per year (nsc ). though amish children are required and encouraged to begin contributing to the family and community well-being at an early age, for religious and cultural reasons, they are prohibited or discouraged from pursuing most recreational activities that typically dominate the lives of many north american farm children. since a significant number of children were directly involved in work-related activities at the time of their deaths, it is evident that some amish children were attempting tasks too dangerous for their maturity levels or were being placed or allowed to be in hazardous situations (as is undoubtedly also the case with some non-amish children). however, it should be noted that hostetler perceives that in amish society parents attempt to create a safe environment for their children, protecting them from physical and moral danger ( ). hostetler and huntington's research also indicated that young children are encouraged to be useful but are not pushed to perform tasks beyond their abilities ( ). still, even if the typical amish farm family conforms to the standards of hostetler and huntington's comments, it is clear from the results of this summary that exceptions occur. it should also be stated that even though child labor is clearly beneficial to amish families and communities, it would be erroneous to conclude that the amish place low value on their children or simply use them for labor. hostetler states, "children are treasured. child care and nurture are the most important adult activities." ( ). erickson reiterates that the amish value children highly ( ). run-overs were a very high percentage of the overall fatalities. apart from small children being run over inadvertently, many of these fatalities occurred when a rider was ejected from a vehicle. it appears that old order farms could benefit from initiatives that have proven successful in the general farm population regarding tractor safety. these include warnings against extra riders and possibly the introduction of safety devices such as child passenger seats and safety belts in vehicles. animal behavior was a significant factor in many runovers in addition to the cases where animal behavior resulted directly in the death of the victim. approximately % of all fatalities were related directly or indirectly to animal behavior such as kicking or bolting. in contrast, according to the national safety council, only % of agricultural work fatalities in the overall farming community were attributable to animals in (nsc, ). this finding suggests the need for further consideration of possible injury prevention interventions, such as more intensive instruction about handling and training farm animals, more selective use of livestock, re- evaluation of certain work practices, and utilization of specialized livestock handling equipment. intervention strategies in order to effectively influence change among the amish, it is essential to understand their social structure and attitudes toward those outside their communities. drake and james point out that this is necessary for making education information compatible with doctrine and community, and applicable to the context of their lives ( ). in contrast to modern society, old order anabaptists view tradition as superior to change (kraybill, ), and as previously indicated, old order communities are generally resistant to assistance or interference from sources outside their church communities. usually, this simply means avoiding participation in activities and programs outside their groups. however, throughout their history in america, matters of conscience have periodically brought the amish into direct conflict with the rules of the larger society. they have frequently prevailed in their ability to practice their beliefs unhindered by the state or have negotiated acceptable compromises in such areas as military service, education, and taxation (kraybill ). some amish are currently seeking exemptions from child labor laws that restrict their youth from certain activities, such as working in sawmills. as kraybill states, "reluctant to demand rights and privileges, the amish will not, however, acquiesce on deeply held beliefs but will respectfully take a stand, even if it brings fines, imprisonment, prosecution, or migration…" ( ). it is also important to understand the old order anabaptist worldview that is rooted in the biblical doctrine of the two kingdoms: the kingdom of god, and the kingdom of the world (kraybill ). in general, those within the amish community (or similar groups) are viewed as belonging to the kingdom of god, while everyone outside their communities belongs to the kingdom of the world. the amish hold strongly to the belief that these two groups should remain as separate as possible so as not to contaminate the purity of their spiritual communities. these views create an inherent tension in attempts to establish cooperative relationships between the amish and outsiders, even on issues that might prove beneficial to both communities. this tension is especially evident in dealings with the government: old order anabaptists view it with suspicion because its use of force to gain compliance violates the anabaptist belief in non- violence (kraybill, ). however, exceptions to this strict separation of church and state, or church and other outside organizations, are possible. for example, in , lagrange county, indiana, the home of one of the nation's largest amish settlements, experienced a high number of amish farm-related fatalities, almost all children. in response, a series of meeting were held involving amish leadership, local county extension educators, concerned citizens, and purdue university's agricultural safety and health program staff. the result was the formation of the northern indiana family safety committee, a group of amish and non-amish leaders who address safety issues within the amish and the non-amish communities. in some of their first efforts, the committee sponsored a series of farm safety workshops specifically targeting the amish community. to date, approximately eleven of these events have been conducted, involving over members of the amish community. the committee also produced and distributed, both in indiana and other states, a culturally and spiritually sensitive farm safety coloring book called weed in our gardens for old order anabaptist children. approximately , copies of this book have been distributed to a wide variety of audiences, including nearly all second and third graders in the amish school districts in the lagrange county area. recently, the committee coordinated a program to install smv emblems and reflective tape on amish buggies throughout the community. they also facilitated the design and production of an amish buggy/auto safety brochure targeting tourists in highly amish areas. approximately , copies have been distributed in several states. in another effort to influence old order anabaptist children with farm safety information, penn state university's school of nursing developed an interactive board game called "amos and sadie's farm: a pathway to safety." the game quizzes players about safety issues, such as potentially dangerous aspects of haylofts, silos, and livestock. a variety of other cooperative intervention efforts were presented at the extension education in anabaptist communities conference in shipshewana, indiana, in may . among the recurring ideas presented was the concept that amish settlements have doctrinal similarities but also characteristics unique to the individual communities. thus, attitudes toward outsiders, and cooperative efforts with such parties, can vary significantly between settlements depending on the rules or "ordnung" of the individual communities. conference participants reiterated that the more professionals know about anabaptist doctrine and its impact on lifestyle, the more effectively they can interact with this population. also, according to extension educators at the conference, in dealing with the amish it is more effective to address them in informal settings instead of through structured programs or external services from state agencies. as kraybill states, "instead of secondary relationships, amish life revolves around primary, face to face social ties…" ( ). it is also important to avoid a patronizing attitude or the image of being an "outside expert," according to the extension educators at the conference. a second conference for professionals working with old order anabaptist communities is scheduled for march in holmes county, ohio, the location of the largest amish settlement in the country. the indiana state police have also initiated intervention strategies of conducting informational meetings with amish audiences and providing special training to law enforcement personnel in counties with amish populations. the focus of these meetings has been on highway safety and responding to emergency situations. recommendations . to better address the overarching issue of old order anabaptist farm-related fatalities, a centralized, ongoing database and a more uniform reporting procedure would be helpful for gathering and quantifying relevant data. this would enable the old order anabaptist community, and professionals working with them, to better understand the problem, draw more concrete conclusions, and develop more effective prevention strategies for reducing injuries and fatalities among their population. . efforts should be made to develop more farm safety materials that are culturally and religiously sensitive to the old order anabaptist community and that reflect their traditional agricultural practices. special attention should be given to material oriented toward children but also designed for use in a family setting to communicate safety messages. . appropriate farm safety information should be made available for dissemination through the channels most frequently used by the old order anabaptist community, such as their community schools, livestock auctions, and farm markets. . community-based farm and family safety committees should be encouraged in geographical regions with high concentrations of old order anabaptists. . in response to the large number of small children run over in circumstances where the driver was unaware of the child's location, the use of more clearly defined or fenced-in play areas should be considered. . the role of extra riders on horse drawn equipment needs further exploration to determine the actual risks involved and possible intervention strategies, such as passenger seats in buggies and wagons and improved seating on horse drawn implements. references cosgel, m.e. 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( ). assessing senior farmers' perceptions of tractor and machinery-related hazards. journal of agricultural safety and health, ( ): - . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ jnn .indd © s. karger ag, basel – / / – $ . / original paper j nutrigenet nutrigenomics ; : – the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations christopher r. d’adamo a valerie j. dawson b kathleen a. ryan c laura m. yerges-armstrong c richard d. semba d nanette i. steinle c, f braxton d. mitchell c, e alan r. shuldiner c patrick f. mcardle c a department of family & community medicine, university of maryland school of medicine, b university of maryland school of medicine, c department of medicine, university of maryland school of medicine, d wilmer eye institute, johns hopkins university school of medicine, e geriatrics research and education clinical center, baltimore veterans administration medical center, and f baltimore veterans affairs medical center, baltimore, md , usa keywords alpha-carotene · carotenoids · genome-wide association study · capn · capn · prkce · old order amish abstract background/aims: alpha-carotene is a provitamin a carotenoid present in fruits and vege- tables. higher serum concentrations of α-carotene have been associated with lower risk of cancer and all-cause mortality. previous studies have suggested that genetic variants influ- ence serum concentrations of provitamin a carotenoids, but to date no variants have been robustly associated with serum α-carotene concentrations. the aim of this study was to iden- tify genetic associations with serum α-carotene concentrations using the genome-wide as- sociation study (gwas) approach. methods: a gwas of serum α-carotene concentrations was conducted in old order amish adults who had consumed a -day controlled diet. linear regression models adjusting for age, gender, and family structure were utilized to evaluate associations between genetic variants and serum α-carotene concentrations. results: ge- nome-wide significant associations with α-carotene concentrations were observed for loci on chromosome q between the genes capn and capn (rs , p = . × – ), chro- mosome p in prkce (rs , p = . × – ), and chromosome q (rs , p = . × – ). conclusions: we identified novel loci associated with serum α-carotene concentrations among a population that consumed a controlled diet. while replication is nec- essary, the capn /capn locus provides compelling evidence for an association with serum α-carotene concentrations and may suggest a relationship with the development and pro- gression of cancers. © s. karger ag, basel received: october , accepted: october , published online: december , christopher r. d’adamo, phd university of maryland school of medicine west lombard st., east hall baltimore, md (usa) e-mail cdadamo @ som.umaryland.edu www.karger.com/jnn doi: . / j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel introduction the carotenoids are a class of pigments synthesized by plants and bacteria [ , ] . carot- enoids are not produced endogenously in humans and other animals and are obtained from the diet [ ] . only of the identified carotenoids are contained in foods, the most abundant carotenoid-containing foods being fruits and vegetables [ ] . approximately half of the dietary carotenoids can be found in human serum and tissues, with α-carotene, β-carotene, lycopene, lutein, zeaxanthin, and β-cryptoxanthin representing % of all the carotenoids in the human body [ ] . provitamin a carotenoids can be converted to retinol in the body and include β-carotene, α-carotene, and β-cryptoxanthin. Αlpha-carotene is obtained through the diet mainly from yellow-orange fruits and vegetables, especially carrots, pumpkin, and squash [ ] . although observational studies have consistently associated eating of carotenoid- containing fruits and vegetables with lower risk of a variety of chronic diseases, interven- tions with carotenoid-rich diets and supplementation have not shown consistent health benefits [ – ] . in fact, β-carotene supplementation has been associated with an increase in mortality among smokers, although it should be noted that a high daily dose of synthetic β-carotene was utilized in that study [ ] . the discrepancy between observational and clinical studies may be due in part to genetic differences in how circulating carotenoid concentrations and their physiological effects respond to carotenoid intake. alpha-carotene has not been studied as extensively as β-carotene, but it is believed to have numerous bene- ficial health effects. alpha-carotene is a potent antioxidant that prevents lipid oxidation, scavenges free radicals, and enhances gap junction communication [ ] . in vivo studies suggest that the ability of α-carotene to inhibit proliferation of cancer cells is approximately times more potent than that of β-carotene [ ] . in addition, α-carotene has been asso- ciated in observational and animal studies with a decreased risk for gastric cancers, liver and lung cancers, and other chronic diseases, including diabetes, cardiovascular disease, and chronic lower respiratory diseases [ , , ] . due in part to its antioxidant properties, low levels of α-carotene have also been associated with diseases that are worsened by excessive oxidative stress, such as glaucoma and atrophic gastritis [ , ] . furthermore, data from the national health and nutrition examination surveys revealed an inverse asso- ciation between serum α-carotene concentrations and the risk of all-cause mortality as well as mortality from cardiovascular disease, cancer, and all other causes not related to cardio- vascular disease or cancer [ ] . therefore, a greater understanding of genetic associations with circulating α-carotene concentrations in humans may have important public health implications. there is one previously reported genome-wide association study (gwas) of α-carotene concentrations. in a meta-analysis that combined results across diverse study populations ( n = , subjects total), a single locus was found to be associated with provitamin a carot- enoids at a genome-wide threshold for statistical significance. in this study, single nucleotide polymorphisms (snps) in the gene bco were associated with significantly higher circulating concentrations of β-carotene, and the same locus was associated to a lesser extent with α-carotene concentrations [ ] . bco catalyzes the first step of provitamin a carotenoids to vitamin a (retinol) in the small intestine. bco is a - ′ dioxygenase that performs a - ′ central cleavage that generates one molecule of retinol from each molecule of α-carotene. the association between snps in bco and α-carotene was weak ( p = . ), relative to its asso- ciation with β-carotene ( p = . × – ), and did not have convincing evidence of consistency in the original report ( p = . in the replication cohort). little else is known about genetic variants that may influence serum α-carotene concentrations, and no robustly associated genetic associations with this important micronutrient have been identified to date. j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel the goal of this study was to identify novel genetic associations with serum α-carotene concentrations. we studied a relatively genetically homogenous population of old order amish men and women living in lancaster county, pennsylvania. the study participants were administered a -day controlled diet, which helped reduce variability in serum α-carotene concentrations associated with varying intake of α-carotene and fat as well as other dietary factors that influence its absorption. we hypothesized that the controlled diet would enable us to more closely isolate the genetic influences of serum α-carotene concentrations. while relatively brief, the controlled diet helped reduce potential confounding of the relationship between genetic variants and circulating α-carotene concentrations introduced by varying dietary intake. methods study population the study sample was composed of the participants from the heredity and phenotype intervention (hapi) heart study who completed a controlled diet and had frozen blood samples obtained during a clinic visit on the final day of the controlled diet. the design of the hapi heart study has been described previously [ ] . briefly, the initial aim of the hapi heart study was to identify genetic and environmental determinants of the responses to intervention affecting cardiovascular risk factors. of the old order amish adults recruited into the hapi heart study, were administered the controlled dietary intervention and subse- quently provided a blood sample at the conclusion of the -day diet. there were samples of insufficient quality to measure serum α-carotene concentrations and participants with incomplete genotype data who were excluded from analysis. the study was approved by the institutional review board of the university of maryland school of medicine, and all participants provided written informed consent. controlled diet research staff prepared the controlled diet for study participants. a registered dietitian visited several old order amish households to obtain diet histories and observe meals and foods that were in their homes. all meals in the controlled diet were designed to be representative of the typical diets of old order amish adults and were delivered to the homes of study participants during the -day controlled diet period. study participants did not consume any prescribed or over-the-counter medications or dietary supplements during this -day period. the complete menus for the -day controlled diet that the study participants consumed are provided in the online supplementary material (see www.karger.com/doi/ . / ). the controlled diet contained an average of , kilocalories per day, with % from carbohydrate, % from fat, and % from protein. there was an average of mg of cholesterol per day in the diet. the diet contained approximately , μg of α-carotene per day, coming primarily from carrots, green beans, and lettuce. compliance with the controlled diet was assessed by comparing sodium, potassium, and creatinine levels from first morning urine samples obtained ( ) prior to consuming the -day controlled diet, ( ) on the final day of the -day controlled diet, and ( ) on the final day of a second -day controlled diet that was low in salt and consumed after the blood draw that was used to conduct the gwas. the compliance data have been reported in detail previously [ ] . in brief, while not a direct measure of carotenoid intake, the excreted sodium, potassium, and creatinine levels reflecting the varying salt content in the different diets suggest that compliance with the controlled diet in this study was excellent. serum micronutrient measurement frozen blood samples taken on the final day of the -day controlled diet were assayed for serum concen- trations of α-carotene, other key carotenoids (β-carotene, lycopene, lutein, zeaxanthin, cryptoxanthin), vitamin e (γ-tocopherol, α-tocopherol), and retinol (preformed vitamin a) at the johns hopkins university nutritional biochemistry laboratory. reverse-phase high-pressure liquid chromatography was utilized to assess serum α-carotene concentrations from each μl frozen blood sample [ ] . the intra- and inter- assay coefficients of variability for α-carotene were . and . %, respectively. j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel genotyping genotypes were obtained using either the affymetrix k or the affymetrix m snp chip v . by the genomics core laboratory at the university of maryland. genotyping calls were made separately for the chips using brlmm ( k array) and birdseed version ( m array), which is part of the birdsuite tools [ , ] . called genotypes were then synthesized and filtered by excluding markers with > % missing and extreme hardy-weinberg equilibrium ( p < × – ). a total of , snps passed quality control stan- dards, were called per each calling algorithm, were in common on both arrays, and had a minor allele frequency (maf) ≥ %. these snps were then passed to the imputation phase. imputation was conducted with mach using the hapmap ceu reference sample [ ] . an imputation quality score of ≥ % was used as a final quality control filter. variants with an imputed maf ≥ % were used in association analyses for a final analyzed snp count of , , . haploreg version . [ ] was utilized to pull data from encode and the roadmap epigenomic projects to annotate the functions of any variants associated with α-carotene concentrations in our analyses. the encode and the roadmap epigenomic projects provide data across a variety of tissue and cell types. for each associated variant, we looked for its predicted chromatin state across multiple cell types, its effect on regulatory motifs, and potential enrichment of cell type-specific enhancers. statistical methods descriptive statistics were computed to characterize the study sample at baseline and determine the mean serum α-carotene concentrations. we estimated the effect of genotype on α-carotene levels at each snp, adjusting for the effects of age and sex by utilizing a general linear model. genotype was coded as the number of copies of the reference allele ( , , or ), thereby corresponding to an additive genetic model. gwas analyses were performed using the mmap software, which accounts for family structure [ ] . statis- tical analysis was performed using a variance component approach to account for relatedness among study participants. this approach has been shown to provide valid estimates of regression parameters [ ] . we estimated that our sample provided % power to detect snps accounting for – % of trait variation at genome-wide thresholds for statistical significance ( p < × – ). our secondary aim of the statistical analysis was to perform replicative association testing of previously reported associations. ferrucci et al. [ ] previously reported associations with serum α-carotene, with one locus achieving genome-wide significance. we here report replicative association tests for rs at the bco locus. table . characteristics of our old order amish study population from lancaster county, pa after consuming a -day controlled diet characteristic all (n = ) female (n = ) male (n = ) p value age, years . ± . . ± . . ± . < . bmi . ± . . ± . . ± . < . lycopene, μmol/l . ± . . ± . . ± . . lutein, μmol/l . ± . . ± . . ± . . zeaxanthin, μmol/l . ± . . ± . . ± . . cryptoxanthin, μmol/l . ± . . ± . . ± . . Αlpha-carotene, μmol/l . ± . . ± . . ± . . Βeta-carotene, μmol/l . ± . . ± . . ± . . retinol, μmol/l . ± . . ± . . ± . . gamma-tocopherol, μmol/l . ± . . ± . . ± . . alpha-tocopherol, μmol/l . ± . . ± . . ± . . the student t test was utilized to calculate p values for differences in all variables between females and males. j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel results the baseline characteristics of the study sample are provided in table . there were men and women in the study. the participants had a mean age of . years and a mean bmi of . ; mean α-carotene concentrations were . μmol/l. the residual heritability of α-carotene concentrations after accounting for age and sex was estimated to be . ± . . a manhattan plot summarizing the results of the gwas is provided in figure . there was little evidence for genomic inflation (lambda = . ), as illustrated in figure . details of the genome-wide significant ( p < × – ) associations are given in table . chromosome –l o g (p ) gc lambda = theoretical p values (–log ) o b se rv ed p v al ue s (– lo g ) fig. . manhattan plot for the genome-wide association study of serum α-carotene concentrations in the study population following a -day controlled diet. the x axis represents the chromosomal position along the genome. the y axis shows the p value for the association test at each locus on the log scale. fig. . quantile-quantile plot of the genome-wide association study of serum α-carotene con- centrations. the axes plot the ob- served ( y axis) versus theoretical ( x axis) association p values on the log scale for all single nucleo- tide polymorphisms with a minor allele frequency > %. the old or- der amish are a closed founder population with little admixture expected. the genomic control (gc) lambda is estimated to be . , indicating little bias due to population stratification. c o lo r ve rs io n av ai la b le o nl in e c o lo r ve rs io n av ai la b le o nl in e j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel three novel loci with genome-wide significant associations with α-carotene concentra- tions were detected on chromosomes , , and . the strongest genetic evidence of an associ- ation exists on chromosome q . figure provides a regional association plot of the genome- wide significant association on chromosome . several snps in linkage disequilibrium tag this association. the minor allele at the lead snp (rs , p = . × – ) was associated with a . μmol/l increase in serum α-carotene, and this locus accounted for . % of the variation in α-carotene levels. the minor allele at the locus c is present in the old order amish (maf = . ) at frequencies similar to those of other populations (hapmap ceu = . ). two other genome-wide significant associations were identified, though there was little corroborating evidence from nearby snps at those loci. the strongest of the associations with α-carotene concentrations was a locus on chromosome p (snp = rs ; p = . × – ). each copy of the a allele was associated with a . μmol/l increase in serum α-carotene, and this locus accounted for . % of the variation in α-carotene levels. figure provides a regional association plot of the genome-wide significant association on chromosome . the maf at the locus a is present in the old order amish (maf = . ), though it is more frequent in other populations (hapmap ceu = . ). the rs variant is situated in an intronic region of the prkce gene. finally, we identified a genome-wide significant association with a locus on chromosome q (lead snp = rs ; p = . × – ). each copy of the g allele was associated with a . μmol/l increase in serum α-carotene, and this locus accounted for . % of the vari- ation in α-carotene levels. the minor allele at the locus g is present in the old order amish (maf = . ) as well as other populations (hapmap ceu = . ). figure provides a regional association plot of the genome-wide significant association on chromosome ; rs is in an intergenic region near the non-protein-coding rna linc . there are no obvious candidate genes in the region. we also performed look-ups for snps at the bco locus previously reported to be asso- ciated with serum β-carotene levels in the gwas meta-analysis reported by ferrucci et al. [ ] . there was no evidence of an association between this locus and α-carotene concentra- tions in our study population (lead snp: rs , p = . ). gwas was also performed for the other carotenoids (β-carotene, lycopene, lutein, zeaxanthin, cryptoxanthin), vitamin e (α-tocopherol, γ-tocopherol), and retinol that were assayed. in brief, there were no meaningful associations noted between retinol, vitamin e, or any of the other carotenoids and any other genetic loci ( p > × – , data not shown), with the exception of lycopene. serum lycopene concentrations among our study population were associated ( p = . × – ) with the variant rs on chromosome , located in the intron region of the setd gene [ ] . in addition, our results offered nominal support ( p = . × – ) for the association previously noted between scarb and serum lycopene levels, albeit with a different variant (rs ) in the region. table . genome-wide (p < × – ) associations with serum α-carotene concentrations snp chromo- some position gene maf coded allele beta (se) p value rs prkce . a . ( . ) . × – rs . a – . ( . ) . × – rs capn , capn . c . ( . ) . × – maf, minor allele frequency; se, standard error. j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel discussion we report the results of a gwas of serum α-carotene concentrations among a study sample that consumed a controlled diet. three genome-wide significant associations were identified, though all three require further replication from independent studies and fine mapping to help determine the potential functional effects of these associations. the capn / capn locus on chromosome has the strongest evidence for association due to the associ- ation being identified with relatively common genetic markers (maf ∼ %) and multiple snps. the other two loci, the prkce locus on chromosome and the chromosome q locus, lack strong collaborating evidence from nearby snps. while having associated snps in strong linkage disequilibrium is not a requirement of a causal genetic determinant, it lends supporting statistical evidence for a meaningful association and lowers the probability of an association being identified due to genotype error. we were unable to replicate the association previ- ously noted between a variant on bco and α-carotene concentrations. however, this is not surprising because this snp was more strongly associated with β-carotene than α-carotene concentrations and did not have strong replicative evidence in the initial publication [ ] . gwas performed on retinol, vitamin e (α-tocopherol, γ-tocopherol), and the other carot- plotted snps . r . . . rs susd capn capn fbxo degs c orf tp bp –l o g (p ) re co m b in at io n ra te (c m /m b ) . . . . . position on chromosome (mb) fig. . regional association plot of chromosome q . the x axis represents the chromosomal position with the location of genes at the locus annotated. the left y axis shows the p value for association tests at each lo- cus (dot) on the log scale. the right y axis provides recombination rates in centimorgans per megabase (cm/ mb) in the chromosomal region identifying recombination hotspots in the region (blue line). the most sig- nificantly associated variant in the region is indicated by a purple diamond. all other variants in the region are indicated with colored solid dots. the color reflects the linkage disequilibrium (measured in r ) between the variant and the top hit. variants in high linkage disequilibrium ( r > . ) are in red, variants in low link- age disequilibrium are in dark blue ( r < . ), with all other colors representing linkage disequilibrium in between ( . ≤ r ≤ . ), as described in the color legend in the upper left hand corner of the figure. c o lo r ve rs io n av ai la b le o nl in e j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel plotted snps . r . . . rs pigfepas loc rhoq cript prkce atp v e –l o g (p ) re co m b in at io n ra te (c m /m b ) . . . . . position on chromosome (mb) plotted snps . r . . . rs linc –l o g (p ) re co m b in at io n ra te (c m /m b ) . . . . position on chromosome (mb) fig. . regional association plot of chromosome q . see figure for details. fig. . regional association plot of chromosome p . see figure for details. c o lo r ve rs io n av ai la b le o nl in e c o lo r ve rs io n av ai la b le o nl in e j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel enoids (β-carotene, lycopene, lutein, zeaxanthin, cryptoxanthin) assayed in this study revealed no other meaningful genetic associations, with the exception of lycopene [ ] . our results introduce a potentially interesting relationship between capn /capn , α-carotene, and gastric cancer. calpains are a group of calcium-sensitive cysteine proteases that are ubiquitously expressed in mammals. the calpain system has been shown to be dysregulated in cancer [ ] . capn is a stomach-specific calpain whose expression is localized to gastric pit cells. functioning capn genes produce ncl- , a cysteine protease essential for mucosal defense [ ] . dysfunction of this protease and the pit cells in which they are expressed can lead to atrophic gastritis and metastatic gastric cancers [ ] . we determined that the genetic variant rs , found near capn , was associated with higher serum α-carotene concentrations. higher levels of α-carotene have been shown to reduce the risk of atrophic gastritis, a precancerous condition associated with gastric cancer [ ] . alpha-carotene protects the gastric mucosa by scavenging free radicals and preventing the initiation or prop- agation of lipid peroxidation reactions [ ] . while our findings suggest that there could potentially be a relationship between dysfunction of capn , lower circulating α-carotene concentrations, and gastric cancer mediated by reduced protection of the gastric mucosa from lipid peroxidation, this potential relationship will need to be evaluated in future studies. interestingly, though the relationship noted between the prkce locus and α-carotene concentrations relies on a single snp, prkce may also have implications in cancer as uncon- trolled chronic activation of prkce has been shown to lead to malignant tumor development [ ] . additional studies are clearly needed to confirm whether α-carotene concentrations play a role in mediating the oncogenic activity of these genes. the significant association on chromosome q is in a region with no obvious candidate genes. more work is required, and replication evidence needed, to verify this and all other associations reported here. there are several notable strengths of the study which may have resulted in successful identification of associated loci. this was the first gwas aimed at identifying genetic associa- tions with serum α-carotene concentrations that was conducted among participants who had consumed a controlled diet. while we did not assess serum micronutrient concentrations prior to the controlled diet, as determining the specific effects of this diet on α-carotene concentrations was beyond the scope of the current study, the controlled diet minimized the potential for confounding of the relationship between serum α-carotene and genetic variants due to differences in dietary intake among people consuming variable diets. the controlled diet enabled us to more closely isolate the genetic contributions to the variance in serum α-carotene. the diet was designed to be culturally appropriate, representative of the typical diet of our study population, and was delivered to study participants at their homes to support compliance. it should be noted that the controlled diet contained more α-carotene ( , μg), as is typical of the diet of our old order amish study population, than that of the average adult population of the united states ( μg), as expressed in the most recent publicly available national health and nutrition examination surveys data [ ] . urinary excretion tests suggested that adherence to the controlled diet was excellent. the old order amish study population also provided unique advantages in a study of this nature. the relationship structure of the old order amish enabled us to perform the first estimate of the heritability of serum α-carotene concentrations in humans. the old order amish are also a relatively homogenous population with respect to both genetics and lifestyle. the genetic homogeneity provided increased power to detect genetic variants associated with α-carotene, and the similar lifestyles further minimized potential sources of confounding of the study findings. there were also a number of key limitations to this study. the relatively small sample size ( n = ) provided power to detect associations of genetic variants with relatively large effect sizes only. despite our relatively small sample size, our study was able to identify novel loci associated with serum α-carotene concentrations. we believe that these successes j nutrigenet nutrigenomics ; : – doi: . / d’adamo et al.: the capn /capn locus on chromosome q is associated with variation in serum alpha-carotene concentrations www.karger.com/jnn © s. karger ag, basel may be attributable in part to the controlled diet administered to study participants prior to the blood draw and the relatively similar lifestyle habits of the old order amish study popu- lation which enabled us to more closely isolate the genetic contributions to serum α-carotene concentrations. while the novel association between a variant near capn and serum α-carotene concentrations, both of which have been associated with gastritis and gastric cancer, may provide the rationale for further study into the specific mechanisms of this rela- tionship, this study did not collect data on family history of gastric cancer or other markers of the disease, and thus no direct inference can be made. bioinformatics analysis utilizing haploreg demonstrated that some of the variants associated with α-carotene concentrations, or other variants in high linkage disequilibrium with them, have potential regulatory function by virtue of being associated with enhancer or promoter activity across multiple tissues. however, without more direct experimental evidence, it is not possible to ascribe a regulatory function of these snps to nearby genes (e.g., capn or capn ), nor the nearby genes to regu- lation of α-carotene concentrations. in summary, this was the first study to identify genome-wide significant associations with serum α-carotene concentrations. three novel genome-wide associations were noted with serum α-carotene levels: the capn /capn locus, prkce , and the chromosome q locus. these findings suggest that genetics may influence serum concentrations of α-carotene. further studies are needed to replicate these findings and to identify potential mechanisms underlying the relationships between the identified genetic loci, α-carotene concentrations, and clinical endpoints such as gastric cancer. acknowledgments this work was supported by the national institutes of health (u hl , p dk , r ag , and t dk ). the authors would also like to acknowledge the study participants for their partnership in research, the staff at the amish research clinic for their collaboration, and nga hong brereton, ms, rd, at johns hopkins university school of medicine for designing the controlled diet in this study and evaluating its nutrient content. disclosure statement the authors do not have any conflicts of interest to disclose. references   Á lvarez r, vaz b, gronemeyer h, de lera ar: functions, therapeutic applications, and synthesis of retinoids and carotenoids. chem rev ; : – .   li c, ford es, zhao g, balluz ls, giles wh, liu s: 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micronutrient antioxidants in gastric mucosa and serum in patients with gastritis and gastric ulcer: does helicobacter pylori infection affect the mucosal levels? j clin gastroen- terol ; : – . akita y: protein kinase c-epsilon (pkc-epsilon): its unique structure and function. j biochem ; : – . u.s. department of agriculture, agricultural research service: what we eat in america, nhanes – . nutrient intakes from food and beverages: mean amounts consumed per individual, by gender and age, in the united states, – . available from http://www.ars.usda.gov/sp userfiles/place/ /pdf/ /table_ _nin_gen_ .pdf (accessed may , ). maternal-pup interaction disturbances induce long-lasting changes in the newborn rat pulmonary vasculature maternal-pup interaction disturbances induce long-lasting changes in the newborn rat pulmonary vasculature yulia shifrin, sina sadeghi, jingyi pan, amish jain, andres f. fajardo, patrick j. mcnamara, , and jaques belik , physiology and experimental medicine program, the hospital for sick children research institute, toronto, ontario, canada; and department of paediatrics and physiology, university of toronto, toronto, ontario, canada submitted february ; accepted in final form september shifrin y, sadeghi s, pan j, jain a, fajardo af, mcnamara pj, belik j. maternal-pup interaction disturbances induce long- lasting changes in the newborn rat pulmonary vasculature. am j physiol lung cell mol physiol : l –l , . first published september , ; doi: . /ajplung. . .—the factors accounting for the pathological maintenance of a high pulmonary vascular (pv) resistance postnatally remain elusive, but neonatal stressors may play a role in this process. cross-fostering in the immediate neonatal period is associated with adult-onset vascular and behavioral changes, likely triggered by early-in-life stressors. in hypothesizing that fostering newborn rats induces long-lasting pv changes, we evaluated them at days of age during adulthood and compared the findings with animals raised by their biological mothers. fostering resulted in reduced maternal- pup contact time when compared with control newborns. at wk of age, fostered rats exhibited reduced pulmonary arterial endothelium-depen- dent relaxation secondary to downregulation of tissue endothelial nitric oxide synthase expression and tetrahydrobiopterin deficiency-induced uncoupling. these changes were associated with neonatal onset-in- creased ang ii receptor type expression, pv remodeling, and right ventricular hypertrophy that persisted into adulthood. the pulmonary arteries of adult-fostered rats exhibited a higher contraction dose response to ang ii and thromboxane a , the latter of which was abrogated by the oxidant scavenger tempol. in conclusion, fostering-induced neonatal stress induces long-standing pv changes modulated via the renin-angio- tensin system. neonatal stressors; pulmonary vasomotor tone; renin angiotensin sys- tem the transition from fetal to postnatal life is characterized by a rapid decrease in pulmonary vascular resistance (pvr), as a result of the birth-associated lung expansion, alveolar oxygenation, and co removal ( ). infants with the so-called persistent pulmonary hypertension syndrome of the newborn (pphn) either fail to show the physiological decline in pvr at birth or exhibit a postnatal increase in resistance ( ). neonatal stressors as one of the causative factors of this syndrome have been suggested ( ), but their pathogenesis is poorly understood. the renin-angiotensin (ang)-aldosterone system (raas) plays an essential role in systemic blood pressure regulation and is a likely modulator of the stress-induced vasomotor tone. raas primarily depends on the renal cell generation of renin, which in turn, promotes the liver and fat-derived angiotensinogen conver- sion to ang i. ang-converting enzyme (ace), mostly present in the lungs, converts ang i into the vasoactive form ang ii that signals through two main receptors: ang ii receptor types and (at and at , respectively) ( ). in the regulation of vascular resistance, at receptors are responsible for vasoconstriction and vessel-wall compliance, whereas at receptors modulate vasodi- lation ( , , , ). adding to the renin-angiotensin system complexity, ace promotes the conversion of ang ii into ang-( – ), which induces vasodilation via the mas receptor ( ). downregulation of ang-( – ) and/or its mas receptor is associ- ated with systemic hypertension ( ). a prenatal, maternal low-protein diet results in late-onset systemic hypertension in rats via a mechanism involving raas ( , ). this fetal programming is characterized by an enhanced vasomotor response to ang ii later in life, second- ary to the increased renal and vascular tissue at receptor expression ( ). such fetal programming is not limited to the systemic circulation, since a restrictive protein diet during gestation also induces an increase in lung ace activity in adult mice ( ). fetal sheep delivered by cesarean section have lower plasma ang ii levels when compared with animals born vaginally ( ), suggesting that birth-related stress is associated with higher concentration of this metabolite in the immediate neo- natal period. limited studies, however, previously addressed the impact of postnatal stressors on the regulation of vascular tone. maternal cross-fostering in the early postpartum period in rodents is associated with adult-onset systemic hypertension and metabolic dysfunction ( ), as well as behavioral abnor- malities ( ). changes in maternal pup-rearing behavior in rodents are known to promote neonatal humoral changes that result in altered stress response later in life ( ). thus there is reason to suspect that maternal cross-fostering via maternal- pup interaction disturbances results in neonatal stress. yet, the mechanism accounting for these changes and whether they are limited to the systemic circulation are presently unknown and the main focus of the present study. in hypothesizing that maternal cross-fostering promotes neo- natal pulmonary vasomotor changes via raas, we compara- tively evaluated control and fostered rat pups in the immediate postnatal period and later in life. the maternal behavior fol- lowing cross-fostering was assessed continuously during the st wk postpartum, and the factors potentially affecting the pulmonary vasomotor regulation were evaluated. cross-foster- ing promotes raas-dependent endothelial dysfunction, which results in short- and long-term pv changes. materials and methods animals time-bred sprague-dawley rats were studied from birth until adulthood. all procedures were conducted according to criteria estab- lished by the canadian council on animal care and were approved by address for reprint requests and other correspondence: j. belik, the hos- pital for sick children research institute, university ave., toronto, ontario, m g x , canada (e-mail: jaques.belik@sickkids.ca). am j physiol lung cell mol physiol : l –l , . first published september , ; doi: . /ajplung. . . - / copyright © the american physiological society http://www.ajplung.orgl downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . mailto:jaques.belik@sickkids.ca the animal care committee of the hospital for sick children research institute. cross-fostering and maternal behavior monitoring cross-fostering. female rats were bred and their litters culled to pups, randomly assigned in the immediate postpartum period to be either raised by their biological mothers (control group) or by a foster doe. cross-fostering was conducted as follows: two female rats were time bred such that their litters were born within � h of each other. on the morning of the second postpartum day, the mothers were swapped, such that they were no longer raising their biological pups. daily pup body weight was obtained during the first days of life by placing each pup on a laboratory scale with a . -g precision accu- racy. the mother and litter were maintained in a -h timed light-dark cycle quiet room, and the cages were cleaned twice/week. the mothers were fed a regular pellet diet, and water was provided ad libitum. maternal behavior. in a subset of animals, maternal behavior was videotaped continuously for the first postpartum days. footage was recorded continuously using two cameras positioned at different angles to ensure constant maternal-behavior tracking. the recorded videos were later scored by one of the investigators (s. sadeghi) without knowledge of the group assignment. maternal behavior was scored, as proposed by others ( ), in one of five categories and the precise duration of each category recorded. the distinct maternal behavior categories used in this study were as follows: ) arched-back nursing; ) blanket nursing (laying over the pups in a blanket position; ) passive nursing (passive nursing while in a supine or side position); ) grooming the pups; ) no pup contact. the distinct maternal behaviors are reported as a percentage of the total observation time over a -h period. organ bath studies and fulton index determination the rats were studied at , , and – (adult) days of age. the animals were euthanized with an overdose of pentobarbital sodium ( mg/kg ip). the lungs were quickly removed and maintained on an ice bed; the heart was excised and maintained in % buffered paraformaldehyde for future determination of the fulton index. the near-resistance (third to fourth generations) intrapulmonary arteries were dissected free and mounted on a wire myograph (danish myo technology a/s, aarhus, denmark). the muscle bath was filled with krebs-henseleit buffer solution (nacl, mm; nahco , mm; nahpo , . mm; kcl, . mm; mgso - h o, . mm; cacl , . mm; and dextrose, . mm), bubbled with air/ % co , and maintained at °c. after h of equilibration, the optimal vessel resting tension was determined by repeated stimulation with mm kcl until maximum active tension was reached. all subsequent force measurements were obtained at optimal resting tension. pv muscle force generation was evaluated by stimulating with the thromboxane a mimetic u or ang ii. contractile responses were normalized to the tissue cross-sectional area as follows: (width � diameter) � , expressed as milli-newtons/ square millimeter. the relaxant response to endothelium-depen- dent acetylcholine and the endothelium-independent nitric oxide (no) donor s-nitroso-n-acetyl-penicilamine were determined after vascular muscle precontraction with u ( � m). for the fulton index determination, the hearts were dissected to obtain the right-ventricular (rv) wall and left-ventricle plus sep- tum weights ( ). the data are expressed as the rv/left-ventricular plus septum-weight ratio and used as a measure of rv hypertro- phy. western blot analysis vascular endothelial cells were isolated from freshly dissected peripheral lung tissue by digesting with mg/ml collagenase type ii (sigma-aldrich, oakville, ontario, canada) for h at °c. the digest was then passed through a -�m cell strainer to remove tissue fragments, pelleted by centrifugation at g for min, and resuspended with % fbs (gibco, ontario, canada) in pbs contain- ing �l biotinylated rat anti-mouse cd antibody (bd pharmingen, san diego, ca). after incubation on ice for h, the endothelial cells were immobilized with streptavidin magnetic beads (new england biolabs, ipswich, ma). the endothelial cells were then placed on the easysep magnet (stemcell technologies, vancouver, bc, canada) for min and the unbound cells removed. bead-bound endothelial cells were lysed in mmol/l tris-hcl, ph . , lysis buffer contain- ing % triton x- and protease/phosphatase inhibitors (roche diagnostics canada, laval, quebec, canada) and centrifuged at , g for min. protein content was determined by the bradford method using the bio-rad protein assay (bio-rad laboratories, her- cules, ca). equal amounts of lysate proteins in laemmli buffer were separated by sds-page, transferred onto polyvinylidene fluoride membrane, and immunoblotted using the following antibodies: mouse endothelial no synthase (enos; : , ; bd biosciences, franklin lakes, nj), mouse dihydrofolate reductase (dhfr; : , ; cell signaling tech- nology, danvers, ma), mouse tubulin ( : , ; santa cruz biotech- nologies, santa cruz, ca), anti-mouse igg horseradish peroxidase (hrp) conjugated ( : , ; sigma-aldrich), and anti-rabbit igg hrp conjugated ( : , ; cell signaling technology). detection was performed with the enhanced chemiluminescence reagent (perkinel- mer, waltham, ma). band intensities were quantified using imagej software (national institutes of health, bethesda, md) and expressed relative to tubulin. cell lysates from control and cross-fostered ani- mals were run on the same gel for comparison, and nonessential lanes were removed during image processing. enos dimer/monomer ratio to ascertain for the enos coupling state, lung tissue was dissected and homogenized immediately in cold lysis buffer containing mmol/l tris-hcl, ph . , % triton x- , and protease/phosphatase inhibitors without boiling and separated on % lithium dodecyl sulfate-page maintained at °c. proteins were immunoblotted using enos antibody, as above described. the ratio of dimer over monomer expression was determined by measuring the western blot-respective band densities using imagej software. lung h o content the lung tissue h o content was measured as a surrogate marker of superoxide generation. for this, lung tissue was homog- enized in cold lysis buffer, followed by centrifugation at , g for min, and divided into two groups. for the first group, the lysate h o content was determined by the amplex red hydrogen peroxide/peroxidase assay kit (life technologies, carlsbad, ca), according to the manufacturer’s protocol. the second group of tissue homogenates was first preincubated with polyethylene gly- col-superoxide dismutase (peg-sod; u/ml) for min. absorbance of all samples was measured at � nm using polarstar omega microplate reader (bmg labtech, orten- berg, germany). the difference between the absorbance of peg- sod-treated and untreated samples represents sod-inhibitable h o tissue content. tissue total protein was determined by the bradford method using the bio-rad protein assay (bio-rad lab- oratories) and used for data normalization. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . lung ros generation lung tissue homogenates were divided into two groups. lucige- nin was added to the first group of samples to a final concentration of mm, and ml of the homogenate-lucigenin mixture was transferred to individual wells of an opaque, white -well plate. to confirm further the specificity of the measurement for super- oxide determination, the second group of tissue homogenates was preincubated with . mm ng-nitro-l-arginine methyl ester (l- name) for min at °c before adding lucigenin. the reaction was started by the addition of mm nadph. chemilumines- cence readings were recorded every min over a period of min in a polarstar omega microplate reader (bmg labtech). background signals from buffer were subtracted from homogenate signals, and the resulting value was further corrected by determin- ing the l-name-inhibitable values and normalized for protein concentration. real-time qpcr total rna was isolated from lung tissue using the purelink rna mini kit (life technologies), according to the manufacturer’s instruc- tions. first-strand cdna was synthesized from �g rna by super- script ii rt (life technologies) and amplified by real-time quantita- tive (q)pcr using sybr select master mix (life technologies). primers used are listed in table . for quantification, mrna expres- sion of the target gene was normalized to the expressed housekeeping gene gapdh. biopterin measurements tissue biopterin concentrations were determined by liquid chroma- tography electrospray tandem mass spectrometry. a triple quadrupole mass spectrometer api (applied biosystems/mds sciex, foster city, ca), operated in negative ionization mode with the turboionspray ionization probe source and coupled to an agilent hplc system (agilent technologies, palo alto, ca), was used. the method used a c column ( � mm id, �m particle size, Å pore size) with an isocratic solvent system of water with . % formic acid at . ml/min and a run time of min. tissue tetrahydrobiopterin (bh ) and dihydrobiopterin (bh ) concentration were measured after differential iodine oxidation, as described previ- ously ( ). echocardiographic assessment of pvr changes the inverse ratio of pulmonary arterial acceleration time (paat) to rv ejection time (rvet), as a surrogate of pvr, was measured by two-dimensional echocardiography/doppler ultrasound, as described previously in detail ( , , ). briefly, a short axis view at the level of the aortic valve was obtained, and the pulmonary artery was identified using color-flow doppler. the paat was measured as the time from the onset of systolic flow to peak pulmonary outflow velocity and the rvet as the time from onset to completion of systolic pulmonary flow. tricuspid annular plane systolic excursion (tapse) was measured by m-mode. an m-mode cursor was oriented to the junction of the tricuspid valve annulus and the rv free wall, using the apical four-chamber view. tapse was measured as the perpendicular dis- tance covered by the tricuspid annulus in systole in adult rats. for technical reasons, tapse measurement cannot be obtained in the newborn rat. measurements were obtained and averaged for three consecutive cardiac cycles. this parameter has been shown by others to be a very robust measurement of rv dysfunction in the rat model of pulmonary hypertension and is used routinely in clinical practice ( , , ). lung histology. a subset of -day-old and adult animals from both groups was prepared for lung histology, as we have described previ- ously ( ). briefly, the lungs were inflated with % paraformaldehyde at a constant pressure ( cm h o) and the pv perfused with heparinized physiologic buffer saline at an average pressure of mmhg and paraffin embedded. a -�m section was cut and stained with masson’s trichrome to allow for clear demarcation of the internal and external elastic lamina, as well as the muscle layer. with the aid of a computerized image analyzer system (openlab/improvision, perkinelmer), coupled with a fine-resolution microscope, the external and internal perimeters of each identifiable pulmonary artery were measured. the arterial lumen diameter was calculated as internal perimeter/�. the arterial muscle layer was quantified by measuring the medial area (external-internal arterial area in square millimeters). statistical methods data were first evaluated to determine gaussian distribution by skewness, kurtosis, and omnibus testing. normally, distributed data were analyzed by repeated-measures, two-way anova with multiple comparisons obtained by the tukey-kramer test or unpaired student’s t-test when appropriate. the mann-whitney u-test was used for nonparametric data. statistical significance was determined at p � . . all statistical analyses were performed with the number cruncher statistical system software (ncss, kaysville, ut). data are presented as means � se. results cross-fostering affects maternal-newborn interaction cross-fostering had no impact on the experimental pups’ breast-milk intake, as judged by the nonsignificant differences table . primers used for the rt-pcr assays target oligonucleotide sequence at f: ctcaagcctgtctacgaaaat gag r: tagatcctgaggcag ggtgaat at f: accttttgaacatggtgctttg r: gtttctctgggtctgtttgctc mas f: tgtgggtggctttcgattt r: attagacccccatgcatgtagaa ace f: cagcttcatcatccagttcc r: ctaggaagagcagcacccac nox f: tgaacaacagcactcaccaatgcc r: agttgttgaaccaggcaaaggcac nox f: ccagtgaagatgtgttcagct r: gcacagccagtagaagtagat nox f: accagatgttgggcctaggattgt r: agttcactgagaagttcagggcgt gapdh f: cccttcattgacctcaactacatg r: cttctccatggtggtgaagac at / , ang ii receptor types / ; ace, angiotensin-converting enzyme; nox / / , nadph oxidase / / ; f, forward; r, reverse. table . pup body weight at and days of age for control and experimental groups control fostered days body weight, g . � . ( ) . � . ( ) rv/body weight, � � . � . ( ) . � . ( )* lv 	 sept/body weight, � � . � . ( ) . � . ( )† adult body weight, g � ( ) � ( )† rv/body weight, � � . � . ( ) . � . ( )* lv 	 sept/body weight, � � . � . ( ) . � . ( )* values are means � se (number of pups). rv, right ventricular; lv, left ventricular; sept, septum. *p � . and †p � . compared with control animal values. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . in pups’ body weight at days of life (table ). yet, significant changes in maternal-pup interaction were docu- mented in fostered litters. as shown in fig. , during the first -h postcross-fostering initiation, the experimental pups ex- perienced significantly less maternal contact and grooming when compared with animals raised by their biological moth- ers. cross-fostering induces early-onset pv endothelium- dependent changes we next evaluated the cross-fostering effect on the rv wall mass by comparatively measuring the fulton index at days of age (fig. a) and later in adulthood (fig. b). at both ages, fostered pups exhibited higher index values when compared with same-age control animals. the rv/body weight ratio was also increased significantly in fostered when compared with control animals of both ages (table ). no sex-dependent group differences in either the fulton index or rv/body weight ratio were observed. lastly, the left ventricular 	 septum/body weight ratio was increased in the experimental animals when compared with control rats of both ages (table ). echocardiographic pvr assessment showed no group dif- ferences for -day-old and adult animals (fig. , a and b, respectively). adult-fostered rats, however, exhibited a signif- icant reduction in tapse, indicative of rv dysfunction, when compared with same-age control animals (fig. c), possibly accounting for the lack of group difference in pvr index later in life. the tapse measurement is not technically feasible at wk of age. the pulmonary arterial medial smooth muscle mass was evaluated in control and fostered animals at days of age and during adulthood (fig. ). at both ages, the fostered group’s mid-size and large diameter pulmonary arterial muscle mass was increased significantly when compared with control ani- mals. to evaluate the factors accounting for the cross-fostering effect on the pulmonary vasculature, we proceeded to study the animals’ pulmonary arterial smooth muscle contraction and relaxation potentials. following agonist-induced con- traction, a significant age-dependent group difference in dose response was observed (fig. , a and b). whereas no group differences were observed at days of age (fig. , a and c), adult pulmonary arteries exhibited a significantly increased (p � . ) dose response to ang ii and throm- boxane a analog u when compared with control animals (fig. , b and d). tempol, a reactive oxygen species (ros) scavenger, reduced the fostered adult ani- mals’ u -induced dose response to a level comparable with control rat values (fig. e). a distinct age-dependent difference in pulmonary arterial relaxation potential was also observed between groups (fig. ). compared with vessels from control-group animals, endothelium-dependent pulmonary arterial relaxation dose response was reduced significantly in -day-old fostered pups (fig. a) but not adult animals (fig. c). there was no significant group differences in endothelium-independent pulmonary arterial relaxation response at either age (fig. , b and d). r v / lv + s ep t r v / lv + s ep t control fostered . . . . ** control fostered . . . * days adult a b fig. . fulton index for control and fostered animals. the right ventricular/left ventricular 	 septum ratio (rv/lv 	 sept) was measured in control and fostered animals at days of life (a; n and , respectively) and during adulthood (b; n and , respectively). means � se. **p � . and *p � . compared with age-matched control animals by unpaired student’s t-test. ti m e (% of to ta l) no co nt ac t gr oo m in g ar ch ed -b ac k bl an ke t n ur sin g pa ss ive nu rs ing control fostered ** ** ** fig. . cross-fostering effect on the maternal-pup interaction. maternal behav- ior during the -h postcross-fostering (n ) or equivalent period for controls (n ) was determined by continuous video monitoring and offline scoring. methodological details are provided in the text. means � se. **p � . compared with control group by unpaired student’s t-test. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . fostering is associated with lung enos expression downregulation and uncoupling, as well as increased ros generation given the abnormal endothelium-dependent pulmonary ar- terial relaxation response of -day-old fostered pups, we proceeded to investigate the mechanism responsible for this change. we first hypothesized that cross-fostering may induce age-dependent changes in lung vascular endothelial cell enos expression and/or uncoupling. indeed, a marked decrease in lung endothelial cells’ enos expression was documented in fostered animals at wk of age but not later in adulthood when compared with control group animals (fig. ). we further evaluated the lung tissue ros generation for control and fostered animals at both ages. when compared with control animals, a -fold increase in lung h o content was documented in the -wk fostered pups, whereas no group difference was observed in adult animals (fig. a). to confirm further that cross-fostering enhanced enos- dependent lung ros generation, we measured tissue total and l-name-inhibitable superoxide levels at both ages using the lucigenin chemiluminescence assay (fig. b). compared with control animals, total chemiluminescence values were increased significantly in fostered animals at both ages. yet, an l-name-inhibitable, lucigenin-derived signal was significantly higher in fostered pups at days of age but not during adulthood. these data led us to interrogate whether enos uncoupling was responsible for the increased ros generation in fostered pups. compared with control pups, the enos dimer/monomer - - - > vessel diameter (µm) m us cl e la ye r (µ m ) vessel diameter (µm) m us cl e la ye r (µ m ) ** ** - - - > control fostered * * * newborn adult a b fig. . pv remodeling. pulmonary arterial muscle layer area from days of age and adult control (n and , respectively) and fostered (n for both ages) animals for vessels of different diameters (n � for each diameter/ group). *p � . and **p � . compared with control values. insets: typical mid-size diameter pulmonary arteries of control and fostered animals of both ages. p v r in de x p v r in de x . . . . ta p s e (c m ) control fostered days control fostered adult control fostered ** adult a b c fig. . echocardiographic measurements. the pulmonary vascular resistance (pvr) index for control and fostered animals at days of age (a; n for both groups) and adults (b; n for both groups) is shown, together with the tricuspid annular plane systolic excursion (tapse; c; control, n ; fostered, n ) in adult rats. **p � . compared with control values. see method- ology for detailed description of echocardiographic measurements. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . ratio, a marker of enos uncoupling, was reduced significantly at days of age in fostered animal (fig. a). fostering-induced enos uncoupling is related to dhfr- dependent bh deficiency amongst other factors, reduced bh availability pro- motes enos uncoupling ( ). to test whether cross-foster- ing-induced enos uncoupling was related to bh defi- ciency, we determined the lung tissue bh /bh content ratio at days of age in both groups. the lung bh /bh ratio was significantly lower (p � . ) in fostered pups when compared with control animals at wk of age (fig. b), indicating tissue bh deficiency. since dhfr expression regulates tissue bh /bh ratio by recycling bh into bh ( ), we proceeded to interrogate whether the fostering-induced lung bh /bh ratio changes were the result of a reduction in the pv endothelium dhfr content. two-week-old fostered pups had significantly (p � . ) lower dhfr levels in lung vascular endothelial cells when compared with same-age control animals (fig. a), whereas no significant group differences were found in adult animals (fig. b). cross-fostering is associated with changes in raas components expression based on the previously reported raas involvement in the programming of systemic hypertension in rodents ( ), we investigated whether a similar mechanism was operative in fostering-induced pv changes. firstly, we evaluated mrna expression of the ang ii receptors at and at and mas. when compared with control animals, at mrna expression was increased significantly in fostered pups at days of age and during adulthood (fig. , a and b, respectively), whereas no group differences in at (fig. log [u ] (m) fo rc e (u / k c l) - - - - . . . . fostered control log [ang ii] (m) fo rc e (a n g ii / k c l) fo rc e (u / k c l) fo rc e (u / k c l) fo rc e (a n g ii / k c l) - - - . . . . . control fostered log [u ] (m) - - - - . . . . fostered control ** ** ** ** ** ** ** log [ang ii] (m) - - - . . . . . fostered control ** ** ** ** ** days adulta b c d log [u ] (m) - - - - . . . . fostered - tempol control - tempol e fig. . pulmonary vasomotor contraction. va- socontraction dose response in near-resistance pulmonary arteries from control and fostered -day-old (n and , respectively) and adult (n and ) induced by ang ii (a and b) and thromboxane a analog (u ; c and d). tempol ( � m) preincubation normalized the u -induced dose re- sponse of the adult-fostered animals (e; n for both groups). data expressed as means � se and normalized to the response to mm kcl. **p � . compared with control animals by -way anova and tukey- kramer multiple comparison testing. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . , c and d) or mas (fig. , e and f) mrna expression were documented. possibly responsible for the fostering-induced lung at receptor upregulation was a decrease in its tissue ang ii content. since the lung ang ii levels are dependent on the expression/activity of ace, we proceeded to measure the enzyme expression. the lung ace mrna expression was reduced significantly in fostered animals at but not days of age when compared with control pups (fig. ). given the ros scavenger (tempol) effect at reducing the u -induced, dose-response magnitude in fostered rats to a level comparable with control animals, we speculated whether the nadph oxidase (nox) pathway was involved in the increased ros generation in the experimental group. we documented that nox , - , and - isoforms of mrna expres- sion were increased significantly in adult-fostered animals’ lungs when compared with control rat values (fig. ), sug- gesting that this pathway plays a role in fostering-induced ros generation. discussion in the present study, we showed that cross-fostering results in a significant reduction in maternal-pup contact without affecting the animals’ breast-milk intake and weight gain during the first wk of life. fostered animals exhibited pv changes and rv hypertrophy at wk of age that persisted into adulthood. the factors responsible for the fostering-induced pv changes involve raas and are age dependent. fourteen- day-old fostered pups showed increased ros generation and impaired enos-dependent pulmonary vasodilation secondary to reduced enos expression and bh deficiency-induced en- zyme uncoupling. these changes were associated with a tran- sient decrease in lung ace expression at wk of age and at receptor expression upregulation from days until adulthood. functionally, an increased pulmonary vasomotor response to u and ang ii was documented later in life in fostered animals, and ros scavenging normalized the u -induced enhanced vasocontraction. lastly, pv remodeling changes were present in fostered animals at days of age and during adulthood. there is evidence that adverse intrauterine conditions increase the risk of cardiovascular disease in humans ( , ). although the mechanism responsible for these changes is poorly understood, raas has been implicated in the fetal programming of late-onset systemic hypertension ( , ). in newborn rodents, maternal separation-induced stress results in attenuation of the hypercapnia response ( ), alterations in the hypothalamic-pituitary-adrenocortical axis ( ), be- havioral changes ( ), and ang ii-mediated, adult-onset systemic hypertension ( ). limited studies, however, ad- dressed the effect of neonatal stressors on the pulmonary vasculature. maternal pup-rearing behavior is known to modulate behavioral and hemodynamic regulation later in life in rodents ( , ). in the present study, we showed that cross-fostering in the immediate postpartum period resulted in reduced maternal-pup interaction when compared with the animals reared by their biological mothers. we here propose that the neglect-like behavior exhibited by fostered mothers induced a significant neonatal stress that resulted in pv changes within the first days of life that persisted until adulthood. the raas pathway is developmentally regulated and thus susceptible to early-life stressors. the newborn ang ii plasma levels are highest immediately after birth and gradually de- crease afterwards in humans ( ) and other mammals ( , , log [snap] (m) - - - - - - control fostered log [ach] (m) - - - - - - control fostered log [ach] (m) r el ax at io n (% o f m ax c on tr ac tio n) r el ax at io n (% o f m ax c on tr ac tio n) r el ax at io n (% o f m ax c on tr ac tio n) r el ax at io n (% o f m ax c on tr ac tio n) - - - - - - control fostered ** log [snap] (m) - - - - - - control fostered days adulta b c d fig. . pulmonary vasomotor relaxation. en- dothelium-dependent [acetylcholine (ach); a and c] and -independent [s-nitroso-n- acetyl-penicilamine (snap); b and d] vas- orelaxation dose response in u precon- tracted near-resistance pulmonary arteries from control and fostered -day-old (n and , respectively) and adult (n for both groups) animals. data expressed as means � se. **p � . compared with control animals by -way anova and tukey-kramer multiple comparison testing. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . , ). at receptors modulate the ang ii-dependent pul- monary vasomotor tone. inhibition of the at receptor in newborn pigs blunts the hypoxic pulmonary vasoconstriction response ( ). in rats, the lung tissue at mrna expression was reported by morrell et al. ( ) to be low early in life, whereas gao et al. ( ) showed that at receptor protein expression in the same tissue is highest during the fetal and neonatal period. this apparent discrepancy likely reflects the well-known lack of specificity of commercially available at , at , and mas receptor antibodies ( ). as such, in the present study, we chose instead to use rt-pcr to evaluate mrna expression changes of the distinct raas pathway components. ace expression in the rat lung is developmentally regu- lated and lower in the newborn when compared with adult animals ( ). lung ace activity has been developmentally studied in rodents and found to be low at birth and progres- sively increased with maturation ( ). raas has been widely recognized as an important player in the pathobiol- ogy of adult-onset pulmonary arterial hypertension ( , , ). chronic hypoxia ( )- and monocrotaline ( )-induced pulmonary hypertension in adult rats is associated with a reduction in ace activity. in the fetal period, the ace activity changes associated with pulmonary hypertension have been studied. in the nitrofen-induced prenatal rat model of diaphragmatic hernia and pulmonary hypertension, the lung ace activity increases when compared with con- trol animals ( ). a reduction in lung ace protein expres- sion, however, was documented in fetal mice subjected to hypoxemia ( ), a noxious stimulus capable of inducing pulmonary hypertension ( ). together, these studies sug- gest that ace expression and/or activity are susceptible to change during the fetal-neonatal period and may play a role in the pathogenesis of pulmonary hypertension. in the pres- ent study, the fostered pups’ total lung tissue ace mrna expression was reduced at wk but not days of life. this finding suggests that the cross-fostering-induced decrease in ace mrna expression led to the upregulation of at h o c on te nt (µ m ol /µ g pr ot ei n) control fostered control fostered ** days adult lu ce gi ni n (c he m ilu m in es ce nc e/ µg p ro te in ) * control fostered control fostered days adult total l-name inhibitable * * a b fig. . lung reactive oxygen species (ros) generation. control and fostered -wk-old and adult lung tissue ros measured as polyethylene glycol-super- oxide dismutase-inhibitable h o content (a) and total and ng-nitro-l-arginine methyl ester (l-name)-inhibitable lucigenin-dependent chemiluminescence (b). both assays’ data were normalized to tissue protein content. means � se. a: -wk-old (control, n ; fostered, n ), adult (control, n ; fostered, n ). **p � . , compared with samples of age-matched control animals by unpaired student’s t-test. b: n for both ages and groups. *p � . by -way anova and multiple comparison testing. control fostered fostered tubulin enos control en o s / tu bu lin en o s / tu bu lin ** control fostered control fostered enos tubulin days adult a b fig. . lung endothelial nitric oxide synthase (enos) expression. enos protein in lung endothelial cells of control and fostered -wk-old (a; n and , respectively) and adult (b; n for both groups) animals, normalized to tubulin expression. representative western blots are shown. all samples from control and cross-fostered animals were run on the same gel, and nonessential lanes were removed during image processing. **p � . compared with samples of age-matched control animals by unpaired student’s t-test. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . receptor expression at days of age via reduction in lung ang ii content during the st wk of life. it is interesting to note that although at receptor mrna expression is increased in fostered animals at both days of age and during adulthood when compared with controls, the pulmonary arteries’ ang ii-induced force response is only enhanced later in life. this likely reflects the reduced lung at receptor expression documented by others during the neonatal period ( ). similarly, when compared with same-age control rats, the adult-fostered animals’ enhanced pulmonary vasocontraction to u is likely mediated via nox-derived ros generation. this is so, since adult- fostered animals exhibited upregulated expression of nox isoforms, and incubation with the ros scavenger tempol abolished the enhanced, u -induced vasocontraction in fostered animals in a similar manner as reported by others ( ). it is known that ang ii induces endothelial dysfunction via a reduction in endothelial cell bh content, thus facilitating enos uncoupling and ros generation ( , , , ). bh is an essential cofactor for enos activity that is crucial for enos stabilization in its coupled form. bh is formed de novo via a gtp cyclohydrolase i pathway or via a dhfr enzyme-depen- dent alternative salvage pathway. previous studies in rodents showed that in the presence of dhfr dysregulation, the resulting bh deficiency and bh accumulation lead to enos uncoupling and are associated with systemic hypertension ( , , ). thus the present-study data suggest that the pulmonary arterial enos uncoupling and ros production in -day-old fostered animals were caused by a transient dhfr dysregula- tion. in the present study, we documented that fostering-induced pv remodeling was present at days of age and continued into adulthood (fig. ). such changes have been reported in association with raas upregulation in adult animal models of pulmonary hypertension and ameliorated by at blockade ( ). following monocrotaline-induced pulmonary hyperten- sion in adult rats, for instance, overexpression of ang-( – ) or d h fr / tu bu lin d h fr / tu bu lin control fostered ** control fostered dhfr tubulin control fostered fostered tubulin dhfr control days adult a b fig. . lung dihydrofolate reductase (dhfr) expression. control and fos- tered -wk-old (a; n for both groups) and adult (b; n for both groups) lung endothelial cell dhfr protein normalized to tubulin expression. repre- sentative western blots are shown. all samples were run on the same gel and nonessential lanes removed during image processing. **p � . compared with samples of control animals by unpaired student’s t-test. . . . en o s d im er / m on om er ** control fostered dimer monomer control fostered a b b h / b h * control fostered fig. . lung enos uncoupling and biopterin content. control and fostered -wk old rat lung tissue enos dimer/monomer ratio (a; n for both groups) and tetrahydrobiopterin (bh ) and dihydrobiopterin (bh ) ratio (b; n for both groups). a, inset: western blot is shown. all samples were run on the same gel, and nonessential lanes were removed during image processing. *p � . and **p � . compared with control samples by unpaired student’s t-test. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . ace attenuates the pv remodeling process and reduces rv hypertrophy ( ). the extent to which the pv endothelial, mechanical, and histological changes documented in fostered pups are indica- tive of the presence of pulmonary hypertension in these ani- mals merits further discussion. on one hand, the higher fulton index values in experimental animals, when compared with control rats, suggest a fostering-induced pvr increase. yet, we failed to confirm this finding by echocardiography, suggesting that if a group difference in pvr at either age is present, then the magnitude of pvr increase is small. nevertheless, the echocardiographic assessment of pvr relies on the paat and rvet measurements that are depen- dent on myocardial systolic performance. in fostered adult rats, tapse, an echocardiographic parameter of rv function ( ), was abnormally decreased when compared with control ani- mals (fig. ). reduced tapse heralds the presence of rv dysfunction in fostered adult rats. others have shown that rv remodeling, associated with increased afterload, occurs not only in response to pvr rise but also following enhanced pulmonary arterial stiffness ( ). thus the echocardiographic evidence of reduced tapse in fostered adult animals, in the absence of a group difference in pvr index, can be explained as follows. firstly, fostered animals exhibited pv remodeling that was likely associated with increased pulmonary arterial stiffness, thus possibly accounting for the reduced tapse values. secondly, in the swine pulmonary hypertension model, a decrease in tapse values was shown to be present in the early-adaptive rv-remodeling phase before the onset of heart failure ( ). a t (m r n a n or m al iz ed to g ap dh ) a t (m r n a n or m al iz ed to g ap dh ) m a s (m r n a n or m al iz ed to g ap dh ) a t (m r n a n or m al iz ed to g ap dh ) a t (m r n a n or m al iz ed to g ap dh ) m a s (m r n a n or m al iz ed to g ap dh ) ** ** control fostered control fostered days adulta f d b c e fig. . ang ii receptor expression in the lung. two-week-old (n /group) and adult (n /group) control and fostered lung tissue ang ii receptor types (at ; a and b) and (at ; c and d), and mas (e and f) receptor mrna, normalized to their respective mrna gapdh expression. means � se. **p � . compared with samples of age-matched con- trol animals by unpaired student’s t-test. . . . . age (days) control fostered **ac e m r n a n or m al iz ed to g ap dh fig. . lung angiotensin-converting enzyme (ace) expression. control and fostered lung ace mrna normalized to their respective mrna gapdh expression at and days of life. **p � . compared with samples of age-matched control animals by unpaired student’s t-test. l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . lastly, in adult subjects with progressive pulmonary hyperten- sion undergoing echocardiographic assessment, minimal in- creases in pulmonary arterial systolic pressure result in a significant decrease in tapse measurements ( ). taken to- gether, the reduced tapse values documented in fostered adult rats, when compared with the control animals, are sug- gestive of a progressive increase in rv afterload without a significant rise in pulmonary arterial pressure at the time the measurements were obtained. in summary, we demonstrated that cross-fostering interferes with the maternal-pup interaction and results in neonatal pro- gramming of pv changes. the mechanism involved in this process includes raas-mediated changes in the pv at receptor that promote endothelium dysfunction, ros genera- tion, and pv remodeling. figure outlines the pathways involved in the cross-fostering-induced pv changes. the present findings have experimental and translational significance. cross-fostering is commonly used to ensure ade- quate maternal milk supply when chronically subjecting new- born rodents to hypoxia ( ) or hyperoxia ( ) to induce pulmonary hypertension experimentally. the cross-fostering effects on the pulmonary vasculature ought to be distinguished from the chronic hypoxia/hyperoxia-induced pulmonary hy- pertension. translationally, disturbance of the parental-new- born interaction is inevitable in infants requiring intensive care. the impact of parental-newborn disruption on the transition from fetal to neonatal life and the pathogenesis of pphn merit further investigation. grants support for this work was funded by a grant from the canadian institutes of health research (mop ; to j. belik). disclosures no conflicts of interest, financial or otherwise, are declared by the authors. author contributions y.s. and j.b. conception and design of research; y.s., s.s., j.p., and a.f.f. performed experiments; y.s., s.s., j.p., a.j., a.f.f., p.j.m., and j.b. analyzed data; y.s., j.p., a.j., p.j.m., and j.b. interpreted results of experiments; y.s., s.s., and j.b. prepared figures; y.s. and j.b. drafted manuscript; y.s., a.j., p.j.m., and j.b. edited and revised manuscript; y.s., s.s., j.p., a.j., a.f.f., p.j.m., and j.b. approved final version of manuscript. ang ii and txa induced force maternal – pup interaction cross-fostering bh dhfrenos ang ii at r neonatal stress enos uncoupling pulmonary vascular changes txa (tp) r nox , , ros generation fig. . pathogenesis of fostering-induced pv changes. the diagram outlines the mechanism by which cross-fostering induces an increase in pvr during the neonatal period and adulthood. txa (tp) r, thromboxane a receptor. n o x (m r n a n or m al iz ed to g ap dh ) n o x (m r n a n or m al iz ed to g ap dh ) n o x (m r n a n or m al iz ed to g ap dh ) ** ** . . . . . . . . . . . . . ** control fostered a b c fig. . lung nadph oxidase (nox) expression. adult control animals (n ) and fostered rats (n ) nox isoforms (a), (b), and (c) mrna expression, normalized to their 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l neonatal stress and pulmonary vascular changes ajp-lung cell mol physiol • doi: . /ajplung. . • www.ajplung.org downloaded from journals.physiology.org/journal/ajplung at carnegie mellon univ ( . . . ) on april , . doi: . /j.cradex. . . case report epstein-barr virus-associated smooth muscle tumours in a patient with an immuno-osseous dysplasia p. humphriesa,*, g. daviesb, r. de bruyna departments of aradiology and bimmunology, great ormond street hospital for children, london, uk introduction epstein-barr virus (ebv)-related soft-tissue tumours are a rare occurrence in patients with a primary immunodeficiency. here we present a case of multiple synchronous ebv-driven leiomyomata in a child with a t-cell immunodeficiency associated with short-limbed dwarfism, and review the literature. case report a term female baby born via a normal delivery to consanguinous parents, presented with a lower respiratory tract infection in the neonatal period. at this time the patient was noted to have a low white cell count associated with short limbs. immunological investigation revealed lymphopenia and markedly reduced lymphocyte proliferation in keeping with a t-cell immunodefi- ciency. this episode of lower respiratory tract infection responded well to antibiotic therapy and the patient remained well until months of age. at months of age the patient developed jaundice and was noted to have haemoglobin of . g/dl, which was treated with transfusion. laboratory indices revealed an autoimmune haemolytic anaemia. this caused repeated anaemic episodes, requiring blood transfusions on multiple occasions. in an attempt to control the autoimmune haemolytic anaemia, therapy in the form of cyclosporin a and steroids were commenced. the autoimmune haemolytic anae- mia was controlled on this regimen until the age of years when it was noted that the patient was developing increasing abdominal distension and breathlessness. plain film radiography of the limbs was performed to investigate the nature of her limb shortening, which demon- strated generalized osteopenia, delayed bone age and symmetri- cal limb shortening. the metaphyses were noted to be flared, with under modelled, wide diaphyses and wide ribs. in the hands ivory and cone-shaped epiphyses were seen. these features were consistent with an immuno-osseous dysplasia, most likely to be metaphyseal chondrodysplasia mckusick type (figs. and ). an abdominal ultrasound was performed which showed hepatomegaly, with multiple well-defined reduced reflectivity focal lesions throughout both liver lobes, which were hypovas- cular on colour doppler. no biliary dilatation was seen and the remaining abdominal viscera had a normal ultrasound appear- ance. owing to her breathlessness a chest radiograph was performed which revealed a right lower zone soft-tissue density. computed tomography (ct) of the thorax was then performed, demonstrating multiple soft-tissue density masses within the lungs. percutaneous biopsy of both a chest lesion and a liver lesion was performed, revealing the lesions to be leiomyomata. the tumours were demonstrated to be positive for ebv using in- situ hybridization (figs. – ). this case presents a grave dilemma for future management. the optimal treatment for the congenital immunodeficiency and auto-immune haemolytic anaemia is ablation of the native bone marrow, followed by bone marrow transplant. the danger of this treatment strategy in this patient is the risk of unchecked proliferation of further smooth muscle tumours, which could become overwhelming. in patients with solitary ebv-associated leiomyomata, surgical excision is a viable option. in this patient with mutiple ebv-driven leiomyomata this is not a treatment option. currently the patient is not receiving treatment for the tumours, and they are not rapidly progressing. discussion this patient with an immuno-osseous dysplasia was found to have multiple ebv-associated smooth muscle tumours in both the liver and the lungs. the main differential diagnoses to be considered in terms of multiple hepatic lesions in an immuno- compromised child are infection, such as candida species and, in the setting of a post-transplant patient, post-transplant lymphoproliferative dis- ease (ptld). multiple haemangiomas in children have a wide range of imaging features and may demonstrate increased or decreased reflectivity in comparison with the remaining hepatic tissue, - /$ - see front matter q the royal college of radiologists. published by elsevier ltd. all rights reserved. doi: . /j.cradex. . . clinical radiology extra ( ) , – *guarantor and correspondent: p. humphries, department of radiology, great ormond street hospital for children, london wc n jh, uk. tel.: þ - - - ; fax: þ - - - . e-mail address: humphriespaul@hotmail.com making haemangiomas a further differential diagnosis. hepatic candidiasis is almost exclusively seen in immunocompromised patients and may present as a fever unresponsive to anti-microbial therapy, right upper quadrant pain and a raised alkaline phospha- tase. on imaging studies hepatic candidiasis is seen as multiple, small reduced reflectivity, or reduced attenuation, masses with a central area of lower echogenicity or attenuation. ptld is seen after both solid organ and bone marrow transplantation. pathologically this is manifest as a spectrum from mild lymphoid hyperplasia to malignant lymphoma, with a predilection for extra-nodal sites. on imaging studies ptld may be seen as a well-defined figure anteroposterior radiograph of the right leg demonstrating flared metaphyses, associated with wide diaphyses. figure anteroposterior radiograph of the left hand demonstrating ivory and cone epiphyses. figure ultrasound longitudinal section through the right lobe of the liver demonstrating a low reflectivity lesion between callipers and two further lesions indicated by asterisks. p. humphries et al. hepatic mass or as well-circumscribed chest nodules. ptld may be multifocal. ebv is thought to infect approximately % of individuals by early adulthood. the majority of infections are sub-clinical, others producing infec- tious mononucleosis clinically. after primary infec- tion the virus enters a latent stage and can reactivate at any time. a wide range of clinical entities is associated with ebv infection, classically including burkitts lymphoma and nasopharyngeal carcinoma. the association of ebv with smooth muscle tumours has been well recognized since the s, and smooth muscle tumours are the second most prevalent solid neoplasm in the paediatric acquired immunodeficiency syndrome (aids) popu- lation, the most prevalent being non-hodgkin’s lymphoma. ebv-associated smooth muscle tumours can be either benign (leiomyoma) or malignant (leiomyosarcoma) depending on the degree of mitotic activity and other factors including cellular atypia and necrosis. in addition to patients with aids, ebv-associated smooth muscle tumours have been well documen- ted after organ transplantation. – in one series of paediatric post-transplant malignancies, lymphoma was found to account for % of the total, followed by skin malignancy ( %). this series found non- kaposi’s sarcoma (including leiomyosarcomata) to account for % of the total. ebv-associated smooth muscle tumours are seen in patients with a wide variety of organ transplants including liver, heart, lung and renal. the most commonly associated organ transplant is liver, with the liver also being the most commonly affected organ (both native and transplant). in the paedia- tric post-transplant population smooth muscle tumours are multifocal, more frequent, occur earlier in the disease, and are more likely to involve the abdominal viscera than those occurring in adult patients after transplant. it is well recognized that there is an increased risk of malignant tumours in patients with con- genital immunodeficiency; however, the number of cases of ebv-associated smooth muscle tumours in patients with congenital immunodeficiency is small. cases have been reported in the literature of ebv-associated smooth muscle tumours in ataxia telangiectasia; a chromosomal breakage disorder with impaired dna repair mechanisms and a degree of immunodeficiency, and an ebv-associated leio- myosarcoma of the thyroid in a child with a congenital t-cell immunodeficiency. this patient had features of metaphyseal chon- drodysplasia mckusick type, one of a spectrum of immuno-osseous dysplasias. mckusick-type meta- physeal chondrodysplasia is inherited in an auto- somal recessive fashion, with incomplete penetrance. incidence is highest amongst the amish population of north america ( . : live births) and in finland ( : , live births). clinically mckusick presents as short-limbed dwarf- ism associated with immunological defects. the most common immunological defect is a general- ized immunodeficiency, seen in %; however b-cell and t-cell deficiencies have also been described. to our knowledge there has been no previous reports of ebv-associated smooth muscle tumours in the setting of an immuno-osseous dysplasia, and although uncommon, ebv-associated smooth muscle tumours should be considered in the figure axial ct section through the thorax (lung window settings) demonstrating multiple soft-tissue nodules throughout the right lung.figure axial ct section through the upper abdomen after intravenous contrast medium administration. mul- tiple low attenuation lesions are seen throughout the liver. epstein-barr virus-associated smooth muscle tumours in a patient with an immuno-osseous dysplasia differential diagnosis of multifocal hepatic lesions in immunocompromised patients. references . reyes c, abuzaitoun o, de jong a, et al. epstein-barr virus associated smooth muscle tumours in ataxia telangiectasia: a case report and review. hum pathol ; : — . . mcclain kl, leach ct, jenson hb, et al. association of epstein-barr virus with leiomyosarcomas in children with aids. n engl j med ; : — . . jenson hb, leach ct, mcclain kl, et al. benign and malignant smooth muscle tumours containing epstein-barr virus in children with aids. leuk lymphoma ; : — . . collins mh, montone kt, leahey am, et al. metachronous epstein-barr virus-related smooth muscle tumours in a child after heart transplantation: a case report and review of the literature. j pediatr surg ; : — . . lee es, locker j, nalesnik m, et al. the association of epstein- barr virus with smooth muscle tumors occurring after organ transplantation. n engl j med ; : — . . timmons cf, dawson db, richards cs, et al. epstein-barr virus-associated leiomyosarcomas in liver transplantation recipients. cancer ; : — . . penn i. de novo malignancy in paediatric organ transplant recipients. j pediatr surg ; : — . . purtilo dt, strobach rs, okano m, davis jr. epstein-barr virus associated lymphoproliferative disorders. lab invest ; : — . . tulbah a, al-dayel f, fawaz i, rosai j. epstein-barr virus- associated leiomyosarcoma of the thyroid in a child with congenital immunodeficiency: a case report. am j surg pathol ; : — . p. humphries et al. 	epstein-barr virus-associated smooth muscle tumours in a patient with an immuno-osseous dysplasia 	introduction 	case report 	discussion 	references the effect of truncated troponin components on activation of lethocerus flight muscle a wednesday, february , of cardiac troponin c’s defunct ca þ -binding site (site ). in this study, we combined in vitro and in situ structural and functional techniques to elucidate the role this mutation may play in the modulation of troponin’s function. we used nuclear magnetic resonance to solve the structure and characterize the backbone dynamics of the regulatory lobe of troponin c with this mutation. the overall structure and dynamics of troponin c was not significantly altered by l q; however there was a slight rearrangement of site making it more similar to trout cardiac troponin c, which also has a glutamine at position at residue and displays increased ca þ sensitivity. backbone dynamics mea- surements indicated that q was more flexible than l . the structure and function of l q was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. the structure and/or orientation of the regulatory lobe of troponin c was slightly perturbed by l q in relaxing conditions and was unaffected at activating ca þ concentrations. the ca þ sensitivity of the structural change and contractility were both unaltered by the l q mutation, suggesting that while this may cause a small change in the structure of troponin c, this does not translate to a large functional effect in cardiac muscle. -pos board b troponin i ser- phosphorylation sustains troponin ca d sensitivity in an acidic environment benjamin r. nixon, shane d. walton, jonathan p. davis, brandon j. biesiadecki. physiology and cell biology and davis heart and lung research institute, the ohio state university, columbus, oh, usa. a hallmark of cardiac ischemia is decreased intracellular ph which can affect a number of cellular processes. such an acidic environment alters cardiac troponin (tn) myofilament regulation to decrease ca þ sensitive force produc- tion. tn also undergoes cardiac ischemia-induced ampk troponin i (tni) ser- phosphorylation. we recently characterized the effects of tni ser- phos- phorylation demonstrating that it blunted the functional effects of canonical tni ser- / phosphorylation; however, the role of ser- phosphorylation in ischemia remains unknown. as an initial step, we sought to investigate the effect of acidic ph on myofilament regulation in the presence of tni ser- phos- phorylation alone and in combination with ser- / phosphorylation. we first investigated the effect of in vivo cardiac ischemia on levels of tni ser- and ser- / phosphorylation. exposure to minutes of regional ischemia re- sulted in elevation of both tni ser- and ser- / phosphorylation. next we determined the effects of tni ser- pseudo-phosphorylation (s d) on the myofilament by measuring troponin c (tnc) ca þ binding properties at normal and acidic ph. results demonstrate acidic ph decreases steady- state ca þ binding to tnc in reconstituted thin filaments across all tn (wt, s d, s / d, and s / / d) such that tni s d ca þ sensitivity at ph . is similar to wt at ph . decreasing the ph had no effect on ca þ disso- ciation such that compared to wt, s / / d remained fast while s d was slowed. we conclude that tni ser- phosphorylation imparts resistance to acidic ph-induced myofilament ca þ desensitization while retaining increased tn ca þ dissociation when in combination with ser- / phosphorylation suggesting the potential for an increase in force while maintaining accelerated ca þ dissociation. future investigations are aimed at examining the effect of tni ser- and ser- / phosphorylation on protease cleavage of tni. -pos board b deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue bin wei, yingru lu, j.-p. jin. physiology, wayne state university, detroit, mi, usa. loss of slow skeletal muscle troponin t (sstnt) due to a nonsense mutation at codon glu in exon of the tnnt gene causes a severe form of recessive nemaline myopathy (amish nemaline myopathy, anm). to investigate the pathogenesis and muscle pathophysiology of anm, we studied the phenotypes of partial and total loss of sstnt in tnnt gene targeted mice. an insertion of neomycinr cassette in intron of tnnt caused approximately % decrease in sstnt protein expression whereas deletion of exons - using cre-loxp approach resulted in total loss of sstnt as that seen in the muscle of anm pa- tients. in diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that sstnt deficiency resulted in significantly decreased levels of other slow fiber-specific myofilament proteins while fast fiber-specific myofilament proteins were increased. histology studies revealed that sstnt deficiency caused significant atrophy of type i slow fibers and a hypertrophic growth of type ii fast fibers. along with the slow fiber atrophy and the changes in myofilament protein isoform contents, sstnt deficiency in soleus muscle shifted the force-frequency relationship toward the fast muscle type and significantly reduced the tolerance to fatigue. sstnt deficient soleus muscle also exhibited a significant number of smaller size central nuclei type i fibers, indicating an adaptive regeneration. sstnt deficient mouse soleus muscle contained apparently normal number of spindles, in which intrafusal fibers were positive for type i myosin with a trend of atrophic morphology. the results demonstrate the essential function of sstnt in skeletal muscle and the causal effect of its loss on the pathology of anm. -pos board b attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp thomas j. longyear , matthew a. turner , brandon j. biesiadecki , joseph lopez , jonathan p. davis , edward p. debold . university of massachusetts, amherst, ma, usa, the ohio state university, columbus, oh, usa. repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, which defines fatigue. the decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains unclear. we sought to gain novel insight into the decrease in velocity by determining if the depressive effect of acidosis could be altered by ) introducing ca þþ -sensitizing mutations into troponin (tn) or ) by agents that directly affect myosin function, including inorganic phosphate (pi) and -deoxy-atp (datp) in an in vitro motility assay. acidosis reduced regulated thin filament velocity (vrtf) at both maximal and sub- maximal caþþ levels in a ph-dependent manner. a truncated construct of the inhibitory subunit of tn, r , and a caþþ-sensitizing mutation in the caþþ- binding subunit of tn, v q, increased vrtf at sub-maximal ca þþ under acidic conditions, but had no effect on vrtf at maximal ca þþ levels. in contrast, both mm pi and replacement of atp with datp reversed much of the acidosis- induced depression of vrtf at saturating ca þþ ( . . control, . . withpi, . . withdatp, . . withbothpi anddatp),withthecombined effectfully restoringthevrtf to thevalue under control conditions.interestingly, despite producing similar magnitude increases in vrtf, the combined effects of pi and datp were additive, suggesting different underlying mechanisms of action. these results suggest that the major mechanism by which acidosis slows vrtf is through directly slowing myosin’s rate of detachment from actin. -pos board b the effect of truncated troponin components on activation of lethocerus flight muscle belinda bullard , bogos agianian , gian-felice de nicola , annalisa pastore , kevin leonard . department of biology, university of york, york, united kingdom, molecular biology and genetics, democritus university of thrace, alexandroupolis, greece, national institute of medical research, london, united kingdom, european bioinformatics institute, cambridge, united kingdom. indirect flight muscle (ifm) of lethocerus is activated by periodic stretches at a constant priming concentration of calcium. the muscle is unusually stiff and stress is transmitted to the thick and thin filaments by kettin, which reinforces links between both filaments and the z-disc. the activating effect of stress on thin filaments is likely to affect troponin. the isoforms of troponin in ifm differ from those in other muscles. tnt has a c-terminal extension not present in vertebrate tnt; tnh is an isoform of tni with a c-terminal extension rich in pro and ala; tnc is present in two isoforms: f binds a single calcium in the c-lobe and is needed for stretch-activation; f binds one calcium in both n- and c-lobes and is needed for isometric force. under conditions of low ionic strength, native fibres have a force-pca curve that shows high calcium- sensitivity and low cooperativity (pca = . , nh = . ). fibres with f alone have a pca curve similar to that of cardiac muscle, (pca = . , nh = . ). a fragment of f without the n-lobe (f -ct) inhibits stretch-activation; therefore the n-lobe of f is necessary, although it does not bind calcium or tnh. f -ct is displaced by f and isometric force is restored, but not stretch-activation. we hope to show the effect of replacing endogenous troponin in fibres with a com- plex containing tnt truncated at the c-terminus, tnh with tni sequence but without the pro-ala extension, and either f or f . this will show how impor- tant the ifm isoforms of troponin are to the stretch-activation response. -pos board b changes in the orientation of the myosin light chain domain (lcd) associated with thick filament-based regulation of skeletal muscle luca fusi, zhe huang, malcolm irving. randall division of cell and molecular biophysics, king’s college london, london, united kingdom. the dependence of myosin lcd orientation on temperature, myofilament lattice spacing and sarcomere length was determined using fluorescence 	troponin i ser- phosphorylation sustains troponin ca + sensitivity in an acidic environment 	deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue 	attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp 	the effect of truncated troponin components on activation of lethocerus flight muscle 	changes in the orientation of the myosin light chain domain (lcd) associated with thick filament-based regulation of skelet ... wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ shepherdstown : race and faith shepherdstown : race and faith jack l. b. gohn the hopkins review, volume , number , winter , pp. - (review) published by johns hopkins university press doi: for additional information about this article [ access provided at apr : gmt from carnegie mellon university ] https://doi.org/ . /thr. . https://muse.jhu.edu/article/ https://doi.org/ . /thr. . https://muse.jhu.edu/article/ reviews solos, whipping off fouettés, unfolding her leg in a gorgeous supported développé, kicking out left and right, and whirling in a series of supported turns. diamonds was the last of balanchine’s “white ballets,” dance’s classical apparitions of heavenly perfection. as the finale of jewels, it transforms emeralds’ unfulfilled yearning and rubies’ symbiotic partnership into a grand vision of human potential, like that of william blake: what is it men in women do require? the lineaments of gratified desire. what is it women do in men require? the lineaments of gratified desire. the ideal and the actual fuse in jewels’ final moment, when the cavalier kneels to the ballerina one last time, and they divulge their dazzling secrets to a gratified world. —jay rogoff shepherdstown : race and faith i’ve been following the contemporary american theater festival for six seasons (often in these pages). produced each july in shepherdstown, wv, it focuses on new or nearly new full-length conventional plays (as distinct from musicals or from the “theater pieces” more associated with fringe theater) by contemporary american playwrights, mostly of the emerging variety (though there were productions of newer plays by the well-estab- lished sam sheppard and neil labute in recent seasons). in other words, this is a proving ground for fare that may be headed for off-broadway and then regional theater, not for either broadway or fringe festivals. catf provides thoroughly professional conditions: equity casts, top-notch sets, excellent acoustics, lavishness of costumes where it’s called for––in short (to borrow a phrase from the vocabulary of self-help books) it dresses the shows for the new york or regional theater job they want. there is a seriousness about the enterprise that might seem a bit conser- vative to some: no messing around with nontraditional casting, nothing fringe-y, limited fourth-wall violations. those things might appear to some to be major components of the future of the american theater, but catf exemplifies the present, a present which is not at all ready to con- sider itself vieux jeux. and in many ways, especially in the area of race, the festival aggressively pushes the envelope. about half the shows in the last two seasons, for instance, had major and often challenging racial themes, and many of the playwrights were nonwhite. this season, without a doubt, the most racially charged entry was the niceties, by eleanor burgess. the setup was simple: at a northeast- ern university that strongly resembles yale, janine, a middle-aged history the hopkins review professor, goes over a paper with zoe, an african american student. they differ, politely at first. zoe’s thesis is that the american revolution was a moderate one not because of the statesmanship of the founding fathers but because those who waged it had no desire to right the wrong of slav- ery and fix the fundamental problems of american society. janine demurs. she argues that historians must work with the pri- mary data available, and that everything not found in such data must be ignored. because nothing that might support zoe’s conclusions stands out in the primary data, she reasons, there is no good reason to subscribe to zoe’s conclusions. janine’s initial lines come across as measured, rational, and supremely composed. zoe’s initial riposte, almost as measured, is that this neat construct consigns us to relying entirely on the voices of white men, history’s winners, who had a nearly exclusive ability to create the record and were unreliable narrators. to zoe, janine’s utter dismissal of her theory ignores self-evident truths of human nature, which should be evidence enough. as the discussion grows more heated, leading to a crisis that leaks out of the professor’s office, it becomes both a proxy for and a microcosm of the larger disputes around race in our country. in the second act (i almost wrote “the second round”) zoe accuses janine of not being a suit- able teacher. when janine responds she earned her position, zoe reminds janine of all the reasons certain potential competitors may have fallen by the wayside on the way to earning that position: “[f]irst came years of slavery, and then came a hundred years of segregation, and then came a deliberate and systematic attempt to exclude black people from good school districts and good jobs and to lock them up or hunt them down for doing things white people do every day. i need you to say that whatever else it stands for, america has systematically persecuted one part of its population, in a way that benefits the other part. in a way that has benefit- ted you. . . . you won fair and square cuz everyone else had lead boots on.” the fight culminates with zoe demanding that janine make personal repa- rations for the illegitimate benefit she has received. with the positions of the parties so lucidly laid out, this rather shocking demand seems––less so. there is little doubt who burgess thinks the winner is. and that is a mistake. the unwritten rule for shows that are truly duels of ideas gener- ally provide that each side will get enough good lines so that the spectator can reasonably come out agreeing with either. the dispute in freud’s last session, for instance, could be called for either sigmund freud or c. s. lewis. burgess opts for the path less traveled and shows one of the women as the clear winner. the imperfection is more than that, however. the polemic victory for one of the characters means there should be no need to tilt the balance by any other means. nonetheless, burgess puts her thumb on the scale, and has the losing party also act corruptly at two or three points. it seems inconsistent with this party’s character everywhere else in the play. it would be better, i believe, if the winner had emerged on her own terms reviews from the clash of views and identities, making this a contest of admirable people fated by skin color and history alone to be adversaries. this is a minor flaw in a show that will send you out with your mind abuzz: it’s a clash of nearly perfectly opposed titans. and there are lots of different ways to deliver the lines. i look forward to seeing other produc- tions with other actresses; the parts are that juicy. (that said, robin walsh as janine and margaret ivey as zoe gave these roles a great sendoff.) the other two shows with race at or near their center lacked the nice- ties’ crystalline clarity. welcome to fear city, by kara lee corthron, was more ambitious, a look at the bronx years ago, a time and place where a lot of things happened, and one vitally important thing, hip-hop, came into being. other occurrences included the tightening of the financial screws on lower-middle-class black families in the bronx, and the shut- ting down of economic opportunity, with attendant impacts upon living arrangements, health, and emotional well-being. they included urban decay and what was euphemistically called urban renewal, which focused largely on the destruction of buildings, i.e. the cityscape within which black families were still trying to live their lives. also the rise of aggressive policing of minority young men via endless stops and searches. this mate- rial is all presented in the midst of a loose, black-family dramedy. hence, we see: e (dyllon burnside), a young man with underemployed mechanical aptitude, afflicted by gay impulses he does not want to deal with and the urge to make some kind of mark in a worse-than-indifferent world; e’s mother wanda (cherene snow), who can’t safely take in her family because of section housing rules but does it anyway, and whose respiratory problems mandate a visit to the er that her finances will not permit; e’s sister neesy (adrian kiser), academically gifted but not smart in love, who had followed a man to california only to be ditched, and has now stumbled home to support herself with topless waitressing; and e’s friend cheky (vincent ramirez), whose distinction is that he has a “j-o-b” as a ups deliveryman, but lives only for the block parties where he serves as a dj. their joint frustrations wind them all up tighter and tighter until they must find release. we see e slipping into nefarious activities connected with “urban renewal,” as he is observed sardonically by a rat (yaegel t. welch), and fighting to have his rap poetry attended to (his delivery is not very good). we see neesy flirting with another potential mr. wrong. we see wanda’s health declining. and we see cheky scrappily going on assembling his career, sparking dance parties with stolen electronic gear. meanwhile, fire is literally consuming the neighborhood. and in the midst of all this, we witness performances of this new rhyme chanted over rhythm tracks as the ensemble dances. we can feel how this artistic form responds to the pressure inside each of them. the end of the first act communicates the power of this aborning musical style in a performance that involves the audience especially well in the confined space of a small theater-in-the-round. this was a play, not a musical, but the proto-hip-hop performance was recognizably a first-half closer. the hopkins review the play would have worked fine if it had stopped there. the second act is not as strong, and, comparing what was on stage with what was in the script, it became apparent that that act was more of a work in progress than a finished product. among the defects was a lengthy transfiguration sequence, where the ensemble devolved first into a sort of enactment of white racist tropes, a minstrel show version of themselves, and then (if i’m understanding correctly) a sort of surreal essential version of them- selves, confused by gibberish talk. then there was a bring-to-date on the characters. finally, there was a kind of flash forward in which subjects like ferguson and black lives matter were conjured up, leading to a moment where one character exhorted the audience to declare its solidarity with raised fists––we did, and we walked out happy because we did. two observations about that raised-fist moment. first, as already described, it was the culmination of some sloppy playwriting. second, it still worked. the crowd with whom i saw the play, mostly senior and white like me, would not seem like an obvious target to have been solicited for the gesture, nor an obvious demographic for cooperating and joining in, especially when (to convey the request) the fourth wall was broken (which in itself always produces awkwardness). but even through the chaos, the show had built up a momentum and an appeal, especially through late iterations of song and dance, that transcended everyone’s identities. at that moment we all came from the bronx. also, we were crazy about those characters, and wanted to say a rousing goodbye to them. i hope the sloppiness gets fixed, though; corthron should lose the transfiguration and the deliberate gibberish talk near the end. i would also lose the ferguson and the black lives matter material, which is worth- while but badly anachronistic in a play intended to capture a moment years back. instead, i’d urge corthron to focus on her own title, or perhaps better on what lies behind it. there are two different kinds of fear referenced in the play. one is the fear that informed a real-life pamphlet further described by corthron in the program notes entitled welcome to fear city: a survivor’s guide for visitors. it was handed out to new york airport visitors. as the rat summarizes: “some corn-fed meatball from iowa is in fear city limits just by goin’ to broadway to see fuckin’ annie.” call it white fear for short. it is overblown and foolish. then there’s the black variety: e’s fear of asking a boss for a raise, and his fear of doing too much in his questionable cooperation with urban renewal, and wanda’s fear of going to the er. where exactly corthron is going with this theme, how- ever, is not clear, because black fear is not always unreasonable, and often responds reasonably to the objective situation. the four central characters all end up transcending something by the end. maybe fear is the wrong word for it. their transcendence is what mat- ters and what we admired. i am certain that the characters’ refusals to give up on themselves or on the bronx, expressed in, but not only in, the music, is what the audience was identifying with when it raised its collective fist. reviews in his recent memoir dispatches from pluto, british writer richard grant, trying to suss out race relations in the mississippi delta region, proposes this formula: “in the south whites didn’t mind how close blacks got, so long as they didn’t get too high socially and economically, and . . . in the north, it was the other way around.” the town of byhalia, a poor exurb of memphis, lies one county over from the delta, and the play to which evan linder has given the town’s name seems to reflect those same delta racial dynamics. this might be surprising, because in the annals of civil rights struggles, byhalia is mainly known for a traumatic moment in when a police killing of a young black man there ignited lengthy boycotts and protests, referenced in the play. but, at least by , the time of the play, things are much more nuanced, and enough water has flowed under the bridge so that a white character does not even recognize the name of the young black man who was shot. byhalia, mississippi depicts instead a place where blacks and whites can be close friends or lovers without anyone commenting on it much except when things go really wrong. it’s not giving away a great deal to say what goes wrong here, since that cat escapes from the bag in the second scene: interracial adultery leading to an unexpected biracial child. and a good deal of the play is given over to what one might call the geographic ques- tion: whether the white mother should even attempt to raise such a child in byhalia. but the bigger question is marital: can the white mother who cheated and her estranged husband (who cheated first) reunite despite all the hurt––and can that husband accept fatherhood under these circum- stances? the comic tone throughout suggests how these questions will be resolved, but, as in most romantic stories, getting there is the main fun. these are not generic romantic characters. the wife, laurel (jessica savage), describes herself as a “redneck momma,” and the pejorative label certainly fits her husband jim (jason babinsky) as well. their story is race- and class-specific. jim is a weed-smoking, not-really-employed guy who does not look like much of a catch, certainly not what his sardonic jesus- loving mother-in-law celeste (hollis mccarthy) was hoping for for her daughter. even with laurel’s job as a schoolteacher, she relies on celeste to pay the power bill. laurel summarizes the situation just before the baby is born: “things are not good, jim! . . . things are never going to be good. and you know what? . . . i’m good with things never being good. i’m fine with it.” but of course the revelation of the baby’s race and history is bound to destabilize even this already unstable structure of a marriage. if laurel is going to rescue it from complete collapse, she is going to require a great deal of centeredness and luck––and jim. the path back for this couple will bring jim into uneasy reliance upon his black best friend karl (yaegel t. welch), and laurel into confrontation with her old black frenemy ayesha, laurel’s boss’s wife (adrian kiser). in these encounters, playwright linder seems to be confirming but also refin- ing richard grant’s aperçu. face-to-face, the racial differences hardly need to be mentioned and play only a small role in how these characters deal the hopkins review with each other. but the social environment in which these pairs find each other matters a lot. there may not be room enough for someone like karl to stay friends with someone like jim. and ayesha can neither understand nor tolerate the prospect of laurel raising her half-black baby in byhalia. somehow the challenges posed by karl and by ayesha must be met. it emerges that the strongest card laurel has to play, with both ayesha and jim (and also with her mother) is simply her unflinching determina- tion to stay put in byhalia. there is no suggestion that there is any magic in byhalia itself; there may be magic, though, in just staying put and going on with one’s life plan, not deviating because of changed circumstances. the virtue of byhalia, mississippi lies precisely in its modesty. it pre- scribes no rules, apart from loving one another and telling the truth, for getting through a marital and race-inflected social crisis in a small town; it simply shows how one not-overwhelmingly admirable couple does it. and at that, the true secret here may just be the sitcom-like jokes. those, and the blackout line at the very end of the play, which may bring a lump to the throat. of course the modesty of the play’s ambitions preclude it from making the hard observations about race of the two previously mentioned entries, but it does enable some presentation of the way things may actually work out on the ground a lot of the time. there are worse aspirations. the most ambitious play in this year’s festival may have been the smallest, a one-woman show called wild horses, by allison gregory. this comically indulgent reminiscence of youth, à la ah, wilderness! or a christmas story, during its roughly hour-and-a-half running time, covers much ground. the narrator recalls two -hour stretches of her life as a -year-old, but in them we find ourselves encountering very distinct characters, and dealing with themes as diverse as first encounters with alcohol and sex, strains in a parental marriage, animal welfare, sibling rivalry, teenage friendships and what the passage of the years can do to them, and, most of all, the simultaneous wonder and danger of encounter- ing, as gregory summarizes in the program notes, a teenager’s dilemma of having “so many needs” and “so little power.” this heady mix presents its own combination of wonder and danger, a novel’s worth of content shrunk to the size of a play, and presented through a single performer. that performer, kate udall, did a jaw-drop- ping job keeping all the characterizations separate and making us fall in love with her characters. i have mentioned the festival’s sometimes elaborate sets: sometimes wonder can be evoked by the relatively modest. here, designers jesse dreikosen and sam transleau did wonders by parking a camper van at one end of a theater in the (three-quarters) round. as the audience entered, it encountered not only normal raked seating on three sides, but also a few tables and stools in the middle where some of the spectators sat, and, at the far end, the camper fitted out as a working refreshment stand serving audience members until the action began. after udall’s character, identi- reviews fied only as the woman, entered, the camper became a swiss army knife of adaptability, serving in turn as basement bar, the side of a house, stash for props, and situs of a wild experiment in driving by the narrator’s ear- lier, completely untrained self. meanwhile, the space between the specta- tors at the tables became a range the woman could freely roam, rapidly changing orientation so that the great annoyance of theater in the round, speakers facing away from spectators, was minimized. and because the play was presented as an act of raconteur-dom anyway, which presup- poses an audience, there was no fourth-wall problem when the woman interacted with audience members (asking them, for instance, to hold her purse or turning one of them into a quondam steering wheel). have any teenager’s real-life few hours really been so full of incident? probably not, and the compression does take a toll on dramatic verisi- militude. one audience member i spoke with on the way out was clearly troubled by this. it did not bother me because “turning the accomplish- ment of many years into an hourglass” is what theater does, and shoving some of those accomplishments closer together in the time represented is a traditional move. what matters here is not the strictness of the account (in real life a raconteur putting a satisfying tale together is often apt to take just such liberties with the timeframe). the point is the group portrait of the youngsters (the woman’s younger self, her partners in crime zabby and skinny lynny, the callow young men who pursue them or whom they pursue, and the woman’s big sister, aka the favorite) in all their confusion, pain, and, most important, their exuberance and their desire to meet life head-on, even if they do not really know what that meeting will demand or entail. if the compression did not bother me as a dramatic strategy, it did trouble me a bit as dilution of message––as with fear city. with so many themes wandering around in a single play, there are likely to be some underdeveloped issues and some tonal dissonances; the drama in the par- ents’ lives, for instance, seemed a bit too sketchy, lacking explanation or depth. and because of the dominant ruefully comical tone set by the narra- tor’s own adolescent experiences, it was not really possible to assess how we were supposed to respond to the parents’ separate trials, which could have been either tragic or not, based on the limited evidence presented. (we get it and can forgive, of course, that a teen’s self-preoccupied mind may tune out the pain among adults in close proximity, but a storyteller does not enjoy the same privilege; the audience’s curiosity about all the major characters should ordinarily be satisfied.) likewise, the animal wel- fare piece came with too few explanations. it looked as if the protagonist and her friends had stumbled on a major piece of villainy, but maybe not, and in any event we did not learn much about the putative perpetrators. still, these are minor carps. this was my favorite of a very strong field. and speaking of overstuffed plays, that charge can certainly be laid at the feet of everything is wonderful, by chelsea marcantel, although the stuffing here was more philosophical and closer to tragedy. at the heart of the hopkins review the issues was the tension between the benevolence that religions preach and their response to apostates, a tension particularly vexing with largely closed, self-involved faiths like the amish. the more closed a community is, the harder it becomes for that community to live up to the benevolence it preaches, particularly towards those who have challenged the integrity of the community by leaving it. protection of the community boundar- ies tends to trump benevolence. and there is a problem at the opposite extreme as well: solicitude for a community’s boundaries may lead to too-ready reconciliation with those who have violated its standards but remained in communion. marcantel has dramatized these paradoxes in the context of an amish community. on the evidence of the play, the amish are generally peaceful and forgiving; indeed, we are introduced at the outset to eric (jason babin- sky), a young outsider who through negligence has done a terrible wrong to the community, and comes seeking forgiveness and healing, receiving plenty of both. the treatment we witness eric receiving provides a striking counterpart or counterpoint, as the case may be, to the community’s treatment of two other young violators of its standards, miri (jessica savage) and abram (lucky gretzinger). the difference in the treatment they receive is partly owing to the nature of miri’s and abram’s respective transgressions, but also depends upon how they relate to the community’s boundaries. miri, having left the community, has been excommunicated, so that she cannot sit at her family’s dinner table or sleep under its roof or touch them, a penalty which is as keenly felt by her family as by her. yet, as we eventually learn, she left only when those community boundaries failed to provide her meaningful protection or support in the light of a wrong done to her. abram’s failing is not revealed at once, but he has always stayed within the group, and has in consequence received the community’s abso- lution in a way that seems far too easy. in short, we are witnessing a situation where community sanctions, perhaps rational in the abstract, lead to irrationally unequal consequences. dramatically, this conflict cries out for someone to defend the commu- nity, to justify its ways. yet for better or for worse, the community, the antagonist which has created this ethical mess, is not directly represented. instead, there are only the three members of miri’s estranged family: sister ruth (lexi lapp), mother esther (hollis mccarthy), and father jacob (paul deboy). when we see the community meting out its inequitable justice, we see it happening only through them, and they too are victimized by it. they do what they do simply because it is what is laid down in the ordnung, the group’s unwritten rules. it is a code as unequal to the tests presented to it as is the code of military justice which forces captain vere to hang a virtuous young man in billy budd. and the ordnung is just as unapproachable and unchangeable in its abstractness as that code. the story here is, thank goodness, not billy budd; the conclusion will not prove quite so bleak. however, that statement must be followed imme- reviews diately by the acknowledgment that it is not easy to figure out what hap- pens in the conclusion. i spoke with a number of members of the audience about it, and none of us could work it out. the script makes the obscurity a little clearer, but there seem to be limits even on the page. throughout the play, there are shifts back and forth between the present and the past, and part of the key to the end is that present and past occupy the stage together. things happen in that space that probably could not literally happen anywhere in the “real” fictional timeline. those things create the feeling of resolution, but perhaps without the play having fully earned it. clearly, what the characters need is an overthrowing of the ordnung, or at least the insertion of some exceptions to it, so that they can effectively forgive each other. nothing short of that will earn the feeling that the end- ing strives for. and it does not seem as if that has actually occurred in the world of the play, notwithstanding a sort of transfiguration of the entire ensemble in the show’s final moments. in reacting to the play as a whole, therefore, we need to take a step back from the conclusion. and fortunately we can. we do not need the last few minutes, or at least not this version of them. if the resolution enacted before us is wanting, the sketching out of the problem is beautifully done. we have been brought to the point where we can see clearly how religion has let its adherents down, and how the way past that disappointment lies in human connection, moral accountability, and forgiveness. whether these particular characters achieve it is not that important. the play may be set in an amish world, but dramas with many simi- larities could be set in catholic or jewish or muslim worlds, and probably among most other faiths. i do not read marcantel as indicting religion as such; she shows us how much groundedness and understanding faith gives, and not just what faith frequently takes away. every faith needs, and has, its own ordnung, but in order to live fully and well, marcantel seems to be saying, believers will always need to transcend it. and then, as the play hints, believers will also need to return to it. every faith journey will thus be a work in progress, forever. a faith journey taken to a different kind of extreme is the subject of david meyers’s we will not be silent, which imaginatively recreates the gestapo interrogation of sophie scholl, a young woman whose religiously inspired resistance to the third reich led to her execution by guillotine in february . this kind of confrontation is almost a genre. we have wit- nessed the scene in various ways at various times, but the essentials do not differ. there is always a table. there is always uncomfortable lighting. the inquisitor always has the full powers of the state at his back. the prisoner answering the questions is often restrained, sometimes under torture, usu- ally in fear for his or her life. and, given the situation and the nature of the prisoner, the outcome is usually a foregone conclusion. the state will win the legal contest, and the prisoner will pay with life or freedom. but on the stage in front of us, the prisoner and the interrogator are pri- marily fighting over something other than the prisoner’s survival, and for the hopkins review that reason the odds in the contest are not as lopsided as they may seem. the fight is over souls: not only the prisoner’s but the interrogator’s. and from a dramatic standpoint, this is the real struggle. in the play the interrogator is named kurt grunwald (paul deboy), although it would appear that he is based on a real-life gestapo investiga- tor named robert mohr. like the historical mohr, grunwald apparently tries to save scholl by having her inform on her brother. perhaps unlike mohr, grunwald also tries to give sophie (lexi lapp) a chance to go free by letting others take all the responsibility, though grunwald fully and correctly anticipates that she is unlikely to agree to saving her skin in that way. and this is the interesting twist: we do not know what kind of game grunwald is really playing. on the evidence presented to this point in the play, when he offers these outs to sophie, he might be serious or he might just be trying to provoke acts of self-sacrifice which will have the not-so- incidental effect of more firmly incriminating her. and that ambiguity as to grunwald’s strategy betokens an ambiguity about his motives, indeed about what he is going through. is grunwald actually a secret admirer of sophie’s heroism, unwilling to emulate her simply because he lacks her courage, or are his professions of empathy with her situation just a secret policeman’s trick? does he know the answer himself? the author does not tip his hand on this dilemma until the last three pages of the script. the genius of the play is how this ambiguity is handled up until those last three pages. there is a certain progression in such dramatic interroga- tions. we know it from examples like the interrogations of thomas more in a man for all seasons, and cromwell’s examinations of anne boleyn’s doomed associates in mike poulton’s dramatization of hilary mantel’s wolf hall, and the mossad dialogues with adolf eichman in evan weiner’s captors, danforth’s interrogation of john proctor in the crucible, and a thousand movies. it typically, if not invariably, includes stages such as denial by the accused, apparent exoneration, partial confession, attempts to win over the interrogator, self-doubt of the interrogator, promises of leniency attached to unacceptable conditions, existential crises on the part of the prisoner, and finally a reckoning, in which we learn which of the two has prevailed. the listed stages all occur here. and in every one but the last, the ambiguity is preserved and grows richer, because grunwald’s pressing of scholl for either a confession or a conviction could plausibly stem from a desire to make an example of her for the third reich, or a martyr of her for those who find the third reich horrifying. ultimately, just as the play establishes, scholl was executed a day after a brief trial. but her memory has been kept very much alive in today’s germany. so in real life she fulfills the exemplary function of martyrdom; in the world of the play, however, it seems most likely that her example will be forgotten. that risk of oblivion heightens the existential question confronting her: if by betraying her principles she could prolong her life, as opposed to adhering to her principles, dying, and having no impact reviews at all, which choice should she make? and this is not just her existential question: it is his as well. it would appear that grunwald has made the opposite choice. but has he? the very end of the play reopens that ques- tion. the theatergoer will not resolve these moral and logical dilemmas entirely, but will leave the theater breathless from identifying and working through them as far as he or she can. as these summaries hopefully establish, this was a challenging, thought-provoking, and frequently rousing selection of plays––i believe the festival’s strongest season on my watch, further confirmation, if any were needed, that today’s american playwrights are a versatile and pow- erful breed. ––jack l. b. gohn these comments appeared in substantially different form on the baltimore page of broadwayworld.com in july . megan marshall, elizabeth bishop: a miracle for breakfast (houghton mifflin harcourt, ), pp. in the nearly forty years since elizabeth bishop’s death in , her reputa- tion has grown to exceed that of any of her contemporaries or successors. her friend robert lowell stands nowhere close to the pinnacle he stood on at the time of his own death in , while john berryman, randall jarrell, anne sexton, or theodore roethke scarcely challenge bishop’s supremacy. the next generation of poets, born a decade or more later, for all their acknowledged technical and human accomplishments haven’t and prob- ably won’t win the special place accorded bishop. to put it flatly, she is the poet no one is permitted to condescend to, surely not to dislike. brett mil- lier’s sturdy -page biography of the poet appeared in ; the library of america has published in one volume everything of bishop’s except her incomplete, unpublished work, which has been collected in edgar allan poe and the juke-box. now megan marshall, who won a pulitzer some years back for her biography of margaret fuller, has ambitiously attempted a very personal account of bishop’s life, while singling out for brief com- mentary a score or more of, in marshall’s opinion, her best poems. what makes this book something other than a straight biography is the active presence in it of the biographer. marshall has chosen the somewhat risky procedure of alternating her account of bishop’s life with a parallel (much shorter) one of her own. a student at radcliffe in the s, she took writing courses from both lowell and bishop, and the six parts into which her book is divided are each prefaced by an account of her own relation to the scene in question. for example, the book begins not with bishop’s childhood, but with an account of a memorial service held for her at radcliffe shortly after her death. marshall gives us the picture of an audience waiting for the poet john ashbery, who is scheduled to kick off josp_ .qxd political soul-making and the imminent demise of liberal education martha nussbaum we may become powerful by knowledge, but we attain fullness by sympathy . . . but we find that this education of sympathy is not only systematically ignored in schools, but it is severely repressed. rabindranath tagore, “my school” achievement comes to denote the sort of thing that a well-planned machine can do better than a human being can, and the main effect of education, the achieving of a life of rich significance, drops by the wayside. meantime mind-wandering and wayward fancy are nothing but the unsup- pressible imagination cut loose from concern with what is done. john dewey, “democracy and education” i begin with three examples, which illustrate, in different ways, a profound crisis in education that faces us today, although we have not yet faced it. all illus- trate the loss of insights contained in the statements by tagore and dewey, two of our greatest educational reformers and thinkers about the role of education in a pluralistic democracy. . it is a hot march in new delhi. i am attending a conference on pluralism and the indian democracy at jawaharlal nehru university, most prestigious of indian universities. much of the conference is concerned with issues of educa- tion. all the papers on education focus on the content of required national text- books, which are to be memorized and regurgitated on examinations. the hindu right wants a content that supports their view of india’s history; the conference presenters, all opponents of the government then in power, inveigh against this aim and propose textbooks with a different, more nehruvian content. but nobody mentions the children: the stultifying atmosphere of rote learning in classrooms, the absence of critical thinking and all cultivation of imagination. tagore once wrote a fable called “the parrot’s training,” in which lots of smart people talk about how to educate a parrot, preparing a fine gilded cage and lots of fancy text- books. nobody notices that along the way the bird itself has died. the education debate at jnu, typical of the larger debate in india, reminds me of that story. . it is a surprisingly warm november in chicago, and i go across the midway to the lab school, the school where john dewey conducted his path- breaking experiments in democratic soul-making. the teachers are having a retreat, and i have been asked to address them on the topic of education for demo- cratic citizenship, something that i undertake with some trepidation because i am journal of social philosophy, vol. no. , summer , – . © blackwell publishing, inc. sure they all know so much more about this topic than i do. as i defend the legacy of dewey and introduce them to the very similar writings of tagore, i discover that i am not where i thought i was, the safe home of dewey’s ideas. i am on a battleground, where teachers who still take pride in stimulating children to ques- tion, criticize, and imagine are an embattled minority, increasingly suppressed by other teachers, and especially by wealthy parents, intent on testable results of a technical nature that will help produce financial success. when i present what i thought of as a very banal version of dewey’s vision, there is deep emotion, as if i have mentioned something precious that is being snatched away. . and finally: one week later i keep a phone appointment to talk with the head of the committee that is searching for a new dean for the school of edu- cation in one of our nation’s, and the world’s, most prestigious universities. here- after i will just refer to the university as x, and its president as y. z. they want my advice, or think that they want it. since, as a result of the first two incidents and many others of a similar nature, i am already alarmed about the future of the humanities and the arts in primary and secondary education, i lay out for this woman my views about education for democratic citizenship, stressing the crucial importance of critical thinking, knowledge about the many cultures and groups that make up one’s nation and one’s world, and the ability to imagine the situa- tion of another person, abilities that i see as crucial for the very survival of demo- cratic self-government in the modern world. to me, it seemed that i was saying the same thing i talk about all the time, pretty familiar stuff. but to this woman, it was utterly new. how surprising, says she, no one else i have talked to has mentioned any of these things at all. we have been talking only about how x university can contribute to scientific and technical progress around the world, and that is the thing that y. z. is really interested in. but what you say is very interesting, and i really want to think about it. she really seems to mean it. well, say i, i will be very happy to tell y. z. exactly what i think, and you can tell him that i’d welcome a call on this topic—although, i add, i am not sure that he would really understand what i am talking about in this case. she agrees that the talk would nonetheless be a good idea. i know y. z., and have a pretty cordial rela- tionship with him, so i actually think this conversation might happen. but i am not going to sit by the phone waiting. and i can just see it now: all over the world, initiatives sponsored by x and the huge money of x, focusing narrowly on sci- entific and technical training, producing many generations of useful engineers who have not a clue about how to criticize the propaganda of their politicians, and who have even less of a clue about how to imagine the pain that a person feels who has been excluded and subordinated. appiah’s book is splendidly argued and also splendidly written. i have little to disagree with concerning its proposal for a “rooted cosmopolitanism.” indeed, although he seems to think that we disagree, it is my belief that we really have very similar views. but i do think that there is a good deal more to be said on the topic of education, which he rightly makes central. i shall begin by setting out the one serious normative disagreement i have with appiah, insisting on the martha nussbaum importance of a distinction between “political liberalism” and “comprehensive liberalism” that does not figure in his argument—although he seems to lean, along with mill, toward the more comprehensive form. i shall then locate the issue of education within a political-liberal conception of political principles, where its role will look subtly different from its role in appiah’s autonomy-focused con- ception. i shall then make some claims about what is truly essential to public edu- cation for democratic citizenship, so construed. finally, i shall make some observations about the crisis that faces us. i. political and comprehensive appiah neglects a distinction that i believe central to good thought about political principles in a pluralistic society: namely, john rawls’s and charles larmore’s distinction between a liberalism that makes comprehensive recom- mendations for how lives should go, using comprehensive epistemological, ethical, and/or metaphysical doctrines, and a liberalism that carefully prescinds from reliance on any such comprehensive doctrines, out of respect for the plu- rality of different reasonable ways citizens may choose to live their lives. (that ethical reason of respect, not skepticism or neutralism for its own sake, is its reason for so prescinding.) appiah does refer to the relevant works of rawls and larmore, but he mischaracterizes them, i believe. he imputes to rawls the view that “governments should be neutral among different reasonable conceptions of the good life” ( ). so far so good, though he does not tell us why this degree of neutrality is recommended. but he then infers from this that “government should not interfere in the ethical dimensions of our lives” ( ). later, similarly, he states that “when antiperfectionists inculcate civic virtues . . . they do so for . . . consequential reasons. the state’s principal concern isn’t with the ethical success of our lives; it’s with the stability and survival of the political order” ( ). since rawls is the only antiperfectionist named in the preceding discus- sion, i take this as a characterization of rawls’s position. (later, in p. , he imputes a similar position to larmore.) the rawlsian state, however, is not ethically neutral. the political sphere is what rawls repeatedly calls a “partial moral conception;” he also characterizes it as a “module” that can be attached to the rest of one’s comprehensive doctrine by people who otherwise have very different views of the meaning and purpose of life. larmore makes similar claims, emphasizing very strongly that the polit- ical conception is not morally neutral. so rawls’s (and larmore’s) “method of avoidance” means only that the political ought to avoid grounding itself in any comprehensive doctrine outside itself: it ought to be both partial in scope and “freestanding,” not invoking comprehensive doctrines in its own justification. so it will not use notions such as those of the soul, or self-evident truth, that are controversial among the major comprehensive doctrines. it will, however, use (partial) ethical notions: a partial ethical conception, indeed, is exactly what it is. the imminent demise of liberal for rawls, as for larmore (and larmore’s own writing about rawls empha- sizes this point) the key ethical notion is that of equal respect, which grounds the political principles (the priority of liberty and the difference principle). it is for an ethical reason, the reason of equal respect, that the rawlsian state will pre- scind from employing comprehensive theological, metaphysical, and epistemo- logical doctrines, and also ethical doctrines about how lives go well in general. rawls adds, however, that the state will have to have some way of talking about issues of objectivity (avoidance of bias and so on), and some way of dealing with issues of motivation to engender support for itself over time; it will therefore use a political notion of objectivity and a “reasonable political psychology,” which try to avoid the most contentious issues that divide the major comprehensive doc- trines. whether he has succeeded in this task the reader must judge; i believe that he does succeed, and i try to develop the psychological doctrine further myself, writing about the political role of compassion, and of the political critique of disgust and stigmatization. all of this can, rawls believes, be endorsed as good, and not just for con- sequential reasons of stability, by people who hold many different comprehen- sive doctrines. that is what the idea of “overlapping consensus” is: the idea that people really affirm the political principles as an ethically rich part, or module, of their own comprehensive doctrine. the idea of an overlapping consensus is carefully distinguished from the idea of a mere modus vivendi focused on stabil- ity. whether such a consensus is possible is a question i will not discuss here. but it is a picture of political pluralism very different from the one that appiah paints. and it is the one i accept, though my specific account of the content of the political is slightly different from rawls’s. what this picture means is that the political is both thicker and narrower than it is in appiah’s sort of pluralism, which seems to me to be a thin sort of com- prehensive liberalism. on appiah’s view, the state can legitimately intervene to promote autonomous lives, so long as it does so with considerable restraint, focus- ing primarily on preventing and correcting obvious obstacles to such lives, such as misinformation, lack of self-control, and ignorance. thus, his view is similar to the views of j. s. mill and joseph raz, both comprehensive liberals, although appiah has a lot more sensitivity to issues of pluralism than mill and probably than raz. on the rawlsian view, by contrast, the state has a sphere that is nar- rower: that of ensuring continued support for the values that form part of the polit- ical conception itself, and of distributing to all citizens some important prerequisites of well-being, but stopping well short of any comprehensive doc- trine of well-being, however thin. it is important to see that the rawlsian state does not limit itself to cultivat- ing sentiments and attitudes required by the political conception and its replica- tion over time, although this will be one of its most important educational tasks. the state also, in rawls’s view and mine, has a further job, namely, giving all citizens some very important prerequisites of general well-being. politics is not martha nussbaum just about politics, it is about and for life. (here lies one of the major disagree- ments between rawls and habermas.) that is why the redistribution of wealth and income is so important: because these are prerequisites for people’s lives in general, not just for the sustenance of the political conception. rawls’s view, then, may overlap considerably with appiah’s in its actual recommendations, but its account of itself will be subtly different. describing the general prerequisites of well-being in a way that does not involve any comprehensive ethical doctrine is a difficult matter, concerning which rawls kept changing his mind until the end of his life. (he began by saying that primary goods such as income and wealth are all-purpose means to the good in all comprehensive doctrines. later, persuaded by thomas nagel and others that it simply was not true that income and wealth play a similar role in all the com- prehensive doctrines, he altered his view of primary goods, saying, instead, that they should be understood in connection with the kantian political conception of the person, which citizens accept for political reasons. he continued to insist, however, that the political must provide for more than itself. and so it remained somewhat unclear why primary goods should be understood primarily in con- nection with the political conception of the person. later still, in making revi- sions for a new edition of political liberalism, which he did not complete due to illness and death, he emphasized that the kantian flavor of the conception of the person troubled him, and he would prefer to recast the whole account in terms of a less divisive conception of reason. all this means that rawls had not fully solved the problem to which i refer, that of getting a conception of the prerequi- sites of well-being that could form part of the overlapping consensus.) my own preferred way of solving the problem is to state the general prerequisites in the form of a list of capabilities, not actual functionings, and to prescind very care- fully from any recommendations as to functioning. thus, the state will make it possible for all citizens to have decent health care, but it will not penalize unhealthy choices. it will give citizens the right to the free exercise of religion, but it will not recommend religious functioning. it will give political rights and opportunities to all, but avoid compulsory voting (which offends some religious doctrines). and so forth. i make exceptions for children in cases where the adult capability requires actual functioning as a child; in this way, once again, there may be considerable convergence between my political liberalism and appiah’s comprehensive liberalism in practice. the theoretical account, however, remains significantly different. the state that i favor should probably not use the notion of autonomy, a notion that has (and had in mill) strong historical links to the rejection of theis- tic sources of meaning. it should not announce, with mill, that non-autonomous lives (such as the lives mill thought calvinism urged one to cultivate, or lives in the military, or lives in a traditional community-based religion like that of the old order amish) are less worthwhile than lives that have thrown off the shackles of authority. but it may reasonably support a general capability for practical the imminent demise of liberal reasoning, understood in a more everyday way, both in order to support the polit- ical conception and in order to give citizens opportunities to think critically about their own way of life, at least enough so that exit options are available to them. supporting human capabilities without showing disrespect for comprehen- sive doctrines that urge people not to use some of these capabilities is a difficult balancing act, as wisconsin v. yoder shows us all. and yet it is plausible to think that the case was rightly decided, supporting sufficient public education to give amish children exit options and the critical thinking relevant to citizenship, without undermining utterly the comprehensive doctrine of the amish. mill would not like the decision, and i am not sure whether appiah would: much depends on how he defines the idea of autonomy, and how comprehensive he makes it. clearly, as rawls says, the state does not need to make it equally easy for all conceptions to accept the overlapping consensus; some will do so only with strain, and some may fail to gain adherents over time. but it will never say to the amish adult, or the soldier, or the roman catholic, “autonomy is a general good in human lives, and your life is therefore ‘pinched’ and ‘hidebound’ [i quote from mill’s discussion of calvinism in on liberty], less rich and satisfactory than the lives of many of your fellow citizens who have dared to throw off the shack- les of authority.” does appiah say this to the amish, or to the career soldier? i am not sure, but i suspect that the answer is yes. so: the political-liberal state narrows its claims. in another way, however, its claims may be considerably richer and deeper than appiah’s thin comprehensive liberalism suggests. anything that lies inside the political module is, ex hypoth- esi, affirmed by all citizens, at least all whose comprehensive doctrines affirm the key idea of equal respect that underlies the constitution, and the principles embed- ded in the constitution itself. so, the state does not have to pussyfoot around with these things: it can teach them flat out, as the best ideas to live with together, using whatever devices of imagery, rhetoric, and emotional suasion seem best suited to the task of imparting them to the young. it has no obligation to be fair to the opposing position. take race, for example. the constitution forbids dis- crimination on the basis of race. so, our schools should not simply teach that we have antidiscrimination laws; it should actively bring up small children as non- racist citizens, in the variety of ways that is familiar to us in daily life, since this is one thing our schools have learned to do pretty well: by not racializing the classroom; by strong opprobrium directed at racist behavior and speech in the classroom; by good teaching about the history of race, teaching that is in no way neutral, but which inculcates anger at racial injustice and hope for a world of racial harmony. small children will, as they so often do, put on plays in which some of them pretend to be people forced to sit in the back of the bus. they will see how they feel when they sit back there, and those emotions will be a topic of classroom discussion—not at all neutrally, since the ethical principle of non- racism is part of the political module that we share. the school will celebrate martin luther king jr.’s birthday, and it will not celebrate strom thurmond’s birthday. it will commemorate the life of rosa parks, and it will not commemo- martha nussbaum rate the life of george wallace—except, perhaps, as an example of regeneration and apology. this non-neutrality is no problem for political liberalism, since it is a partial ethical conception, and neutrality is not what it is about inside the polit- ical module. (about related things that lie outside the boundaries of the politi- cal/ethical conception, however, such as the existence or non-existence of god or gods, and whether god disfavors the racist, the classroom will be normatively silent, although it certainly may and should tell people about the variety of beliefs that is held in their society.) in rawls’s view, and my own, anyone who is willing to accept the basic polit- ical principles can accept all of this. there will be people in any society who do not accept its constitutional principles—who want to reintroduce slavery, for example, or to do away with our constitutional commitment to non-discrimina- tion. those people will not be suppressed, unless they violate the rights of others. they may speak freely, unless their speech poses an imminent threat of public disorder. but they are not going to be treated neutrally. the classroom will not give their views equal weight with the views that support the constitution. it is under no obligation to do so, since, once again, the political conception has a def- inite ethical content, and is not a form of neutralism. even when we are dealing with adults, these racists will be treated asymmetrically with those who do support the constitution: they will not be able to propose legislation that goes in their direction, since the relevant contradictory values are entrenched in the constitu- tion. they will have to amend the constitution itself—or even, in the case of india, call for an entirely new constitutional convention—if they want to change one of their society’s “basic features.” ii. educating citizens in a pluralistic society one thing that a society based upon equal respect needs to do most urgently is to teach young citizens that their society contains many religions and many ethnicities, and that we are all committed to the fair treatment of all of them. indeed, for both rawls and me any such society should teach that this commit- ment to equal respect is the source of our most basic political principles. under- standing this commitment is an essential prerequisite for citizenship, so it is part of the political “module” itself. we do not even need to reach the question of whether it is a consensus-worthy prerequisite of good life in general. this brings me to appiah’s treatment of mozert v. hawkins, the case in which a baptist mother requested an exemption for her children from a series of primary school readers that presented children with pictures of different american ways of life, appealing to them to imagine these different lives. at the outset of his dis- cussion of education, appiah states, and apparently endorses, mill’s view that the liberal state has two tasks, one related to individual autonomous lives, and one related to the good of society (p. ). in the analysis that follows, however, he focuses entirely on the first job, more or less dropping the second. (see “would severely diminish a child’s well-being,” p. ; “to protect the child’s growth to the imminent demise of liberal autonomy,” p. ; “to compromise the possibility of an autonomous adulthood,” p. ; “a child’s prospects for autonomy,” p. . in none of these contexts is the question of society’s good mentioned.) i believe that this neglect of the polit- ical part of the task skews his treatment of the issues involved in mozert. thus, appiah says that the case could “readily” have been resolved by allowing the children to opt out of the reading series: after all, they could have been tested on reading in some other way (p. ). this may be so, if the only issue were personal autonomy. but this is far from being the only issue. these students are going to become citizens in a pluralistic society, so they had better learn about the existence of other ways of life, and they had better be encouraged to imagine those other ways. the mother’s claim that the truths contained in the bible were all her children needed to know is just not reasonable from the point of view of citizenship and the political module. if she does not accept the constitutional principles, then we do not have to give her equal treatment in framing the curriculum, any more than we need give the racist equal treatment. (would appiah say that a racist parent should have an exemption from a reading curriculum that teaches non-racism?) and i think she really cannot be accepting our constitutional principles, when she says that the bible contains everything her children need to know. she is just wrong, from the point of view of the political conception: they need to know the history of race, and religious persecution, and many other facts about america not contained in the bible, if they are to participate in and sustain a political conception based on equal respect and the political principles to which that value gives rise. the school wisely decided that a democracy needs to teach children to think (respectfully and imaginatively) about other ways of life. that is a primary task of the public education system, namely to devise ways of inculcating in the young the values that sustain the political conception. the most significant opinion in the case said exactly this. citing another case in which the court had affirmed that the public schools “serve the purpose of teaching fundamental values ‘essential to a democratic society,’ including ‘tolerance of divergent political and religious views’,” the court then makes a rawlsian distinction between “civil tolerance” and “theological tolerance,” saying that the reading texts do not ask students to accept any particular theo- logical belief (including beliefs about the salvation of others) as true, but they do teach respect and understanding, a legitimate and indeed crucial civil (political) value. correctly, in my view, they hold that such a teaching cannot possibly be an unconstitutional burden on vicky frost’s free exercise of religion. for after all, it is required for the sustenance of the whole set of rights and liberties of which the first amendment is one part. in my view, we can say quite a lot about what education for citizenship in a pluralistic democratic society that is part of an interlocking world should look like. because i have written a book on this topic, i shall simply summarize, in order to turn to the current crisis. i focus, as before, on the humanities and social sciences, leaving the science curriculum for another time. in my book i focused martha nussbaum on liberal education in colleges and universities, but it was always my view that these values need to be cultivated appropriately by primary and secondary edu- cation. colleges will not get very far, unless students have begun much earlier. three values, i argue, are particularly crucial to citizenship in such a nation and for such a world. all, i now add, can be handily accommodated within the political module, as essential underpinnings for its most basic values—although all also have contributions to make to life more generally. the first is the capac- ity for socratic self-criticism and critical thought about one’s own traditions. although some parents may object to this sort of teaching, as they always have since the time of socrates, who lost his life on the charge of “corrupting the young,” we may give them socrates’s own answer: democracy needs citizens who can think for themselves, rather than deferring to authority, who can reason together about their choices rather than simply trading claims and counter-claims. socrates, appropriately, compared himself to a gadfly on the back of a noble but sluggish horse: he was waking democracy up, so it would conduct its business more responsibly. the second ability is the one that was involved in mozert, namely the ability to see oneself as a member of a heterogeneous nation, and world, understanding something of the history and character of the diverse groups that inhabit it. most of my book is taken up with spelling out the curricular dimensions of this one and the pedagogical pitfalls involved, so i shall say no more about it at present. the third ability seems to me possibly the most crucial: it is what i call “the narrative imagination,” the ability to think what it might be like to be in the shoes of a person very different from oneself, indeed a whole range of such persons. for this ability, we need literature and the arts. the great progressive educators of the twentieth century, dewey and tagore, understood this extremely clearly, building the arts into the very core of their curricula for democratic citizenship. (tagore did this through elaborate dance-dramas and other student productions, dewey more abstractly, leaving a lot to the teacher’s own ingenuity, though at the lab school he favored curricular requirements in the arts that are still, though barely, in place. ) both felt that the cultivation of imaginative sympathy was a key prop to good citizenship; that children had this ability to be tapped, if it was not killed off; but that it had to be made more sophisticated and precise through education. iii. democratic education on the ropes education of the type i recommend is doing splendidly in the place where i first studied it, namely the liberal arts portion of college and university curricula. indeed, it is this part of the curriculum, in institutions such as my own, that par- ticularly attracts philanthropic support, as rich people remember with pleasure the time when they read books that they loved, and pursued issues open-endedly. (some of their enthusiasm is related to the personal-life contribution of liberal education, some to its contribution to citizenship.) many nations whose univer- the imminent demise of liberal sity curricula do not include a liberal arts component are now striving to build one, since they acknowledge its importance in crafting a public response to the problems of pluralism their societies face. i have been involved in such discus- sions in the netherlands, in sweden, in india, in germany, in italy, in india and bangladesh. (whether reform in this direction will occur is hard to say: for liberal education has high financial and pedagogical costs. teaching of the sort i rec- ommend needs small classes, or at least sections, where students get copious feed- back on their writing assignments. european professors are not used to this idea, and would at present be horrible at it if they did try to do it; and bureaucrats are unwilling to believe that it is necessary to support the requisite number of faculty positions.) the abilities of citizenship are doing very poorly, however, in the most crucial years of children’s lives, the years known as k through . here the demands of the global market have made everyone focus on scientific and tech- nical proficiency as the key abilities, and the humanities and the arts are increas- ingly perceived as useless frills, which we can prune away to make sure our nation (whether it be india or the united states) remains competitive. to the extent that they are the focus of national discussion, they are recast as technical abilities themselves, to be tested by quantitative multiple-choice examinations, and the imaginative and critical abilities that lie at their core are typically left aside. at one time, dewey’s emphasis on learning by doing and on the arts would have been second nature in any american elementary school. now it is under threat even at the dewey laboratory school. national testing has already made things worse, as national testing usually does: for at least my first and third ability are not testable by quantitative multiple-choice exams, and the second is very poorly tested in such ways. (moreover, nobody bothers to try to test it even in that way.) whether a nation is aspiring to a greater share of the market, like india, or strug- gling to protect jobs, like the united states, the imagination and the critical fac- ulties look like useless paraphernalia, and people even have increasing contempt for them. thus in west bengal, tagore’s pathbreaking santiniketan school, which pro- duced amita sen, amartya sen, satyajit ray, and many more gloriously inde- pendent and imaginative world citizens, is now viewed with disdain as a school for problem children. long ago, jawaharlal nehru sent his daughter indira there, though she spoke little bengali. (and it was the only happy time at school she had, though she attended many famous schools.) today nobody from outside west bengal wants to go there. a parent’s glory is the admission of a child to the indian institute of technology. meanwhile, at the dewey lab school, the arts require- ment is being watered down under pressure of the drive for success on the part of parents and administrators. worse yet, the sheer burden of homework in all disciplines makes it impossible for children to enjoy the use of their critical and imaginative faculties. the united states has some resilience still, thanks to its traditions of local autonomy. thus, indian-american friends of mine wistfully compare the educa- martha nussbaum tion their children receive here (my example of the play about rosa parks comes from one such discussion) with the education they themselves never had in india, where rote learning rules the roost. but the united states is moving toward india, not vice versa. indeed, most outrageously and thoughtlessly, the united states is currently egging on other nations to emulate our worst, not best, traits. the phone call from x university was more upsetting to me than any other confrontation with the recent decline in democratic education: for x is currently investing a lot of money to set up educational outposts all over the world. these narrow ways of thinking about what is worth cultivating will, then, be exported widely, with great money attached. imagine the spectacle of y. z. telling the people of kolkata, who have their own rich and glorious traditions of democratic education, that x can provide wonderful new assistance toward the further destruction of precisely that tradi- tion. and yet exactly this is about to happen. imagine, too, the elites of kolkata lapping all this up, since the prestige of x is superior even to the prestige of the indian institute of technology, so much so that every controversial word of y. z. (and there are so many) makes the front pages of every national indian newspa- per. will y. z. even urge people to think about what makes democracy strong, and to cultivate abilities that strengthen it? not very likely, since the same y. z. has proposed eviscerating the core curriculum at x itself, in such a way that instead of one course in moral reasoning one could take two technical econom- ics courses, and in such a way that the humanities make up a much smaller frac- tion of the entire curriculum than previously. (he may not get his way, but that will not mean that he has broadened his conception of education.) what will we have, if these trends continue? nations of technically trained people who do not know how to criticize authority, useful profit-makers with obtuse imaginations. what could be more frightening than that? indeed, if you look to the indian state of gujarat, which has for a particularly long time gone down this road, with no critical thinking in the public schools and a concerted focus on technical ability, one can see clearly how a band of docile engineers can be welded into a murderous force to enact the most horrendously racist and antidemocratic policies. and yet, how can we possibly avoid going down this road? i believe that outrage is called for, on the part of every person who cares about the future of democracy in the world, and i think philosophers should be leading the expression of outrage. and yet, sad to say, virtually no prominent philosopher writes much about education (one of the great subjects of the pro- fession from plato to locke, rousseau, kant, mill, and sidgwick), and virtually no current philosopher writes on k through in a way that makes a public impact. (here i very much include myself.) appiah’s book is a welcome step back to a philosophical focus on public education. its brilliance and its literary quality suggest that it may reopen a wider debate in the united states, and possibly in other nations as well. the book does not go far enough, however, because it focuses on the personal rather than the the imminent demise of liberal social task, on autonomous lives rather than the prerequisites of citizenship. i call for a second volume, and for all of us to write on the topic in our own ways, with the sense of urgency that our situation demands. notes see my discussion of the indian situation in “education and democratic citizenship: beyond the textbook controversy”, in islam and the modern age ( ), – ; and a slightly different version, “freedom from dead habit,” the little magazine (new delhi) ( ), – . see my reply to his and other essays in for love of country, and the preface to the new edition of that volume. a fuller version of that preface, and the fullest statement of my view on the topic, is in “compassion and terror,” daedalus (winter ): – . a slightly different version, same title, in terrorism and international justice, ed. james sterba (new york: oxford university press, ), – . see my “political objectivity,” new literary history ( ), – . see the discussion of political liberalism in upheavals of thought, chap. and hiding from human- ity, chap. . i give a detailed account of the conception in rawls, justice, and tolerati: an intro- duction to political liberalism, forthcoming from columbia university press. thus, when appiah writes that the doctrine of perfectionism is “controversially” imputed to me and to sen ( n. ), i would rather write—of myself, since sen has not taken a position on this controversy—that it is “falsely” imputed. i have endorsed political liberalism very clearly in arti- cles dating back to and in two books, women and human development ( ) and frontiers of justice ( ). i am not aware of anyone who imputes perfectionism to me, but it may be so. here i am referring to correspondence between rawls and his columbia editor a copy of which is in my possession, and which i have mardy rawls’s permission to cite. see jerome schneewind, the invention of autonomy cambridge: cambridge university press, . putting it this way obscures a problem in rawls’s text that is actually very difficult, since the concept of “reasonable comprehensive doctrines” is actually given more than one definition. in one view, which i take to be the one rawls ought to affirm, a reasonable doctrine is one that accepts and affirms the political “module.” rawls also, however, has an independent characterization of the “reasonable” that involves other ideas such as comprehensiveness, consistency, and ordering of values. i believe that it was mistaken of him to offer this characterization, deep though the reasons for it are, and that it actually sweeps far further than he believes. it is not just new age religion that will be ruled out by these criteria, it is, arguably, christianity itself, since that religion is deliberately grounded on belief in a contradiction: the trinity is standardly understood to be a doctrine belief which humbles reason, because it cannot be given a logically coherent form. i discuss all this in my forthcoming book on pl, columbia university press forthcoming. rawls does not think the brandenburg test speech-protective enough: he recommends protecting all speech unless there is a constitutional crisis and the speech risks upsetting the constitution itself. madison had a similar view, in his debate with george mason. term taken from keshevananda bharati, the indian supreme court case that introduced this doctrine—after indira gandhi had gotten parliament to repeal the whole list of fundamental rights, and voters, thinking this a bad thing, turned her out of office. see mozert v. hawkins county board of education, f. d ( th cir. ): frost, the mother who brought the case, said that the bible “is the totality of my beliefs.” the case was heard by a three-judge panel; there was no majority opinion, though all three reached the same conclusion, because all three wrote separate opinions. i cite from judge lively’s, which was (since he was chief judge) the opinion of the court. the other case, bethel school district no. v. fraser, u.s. ( ), is long on theory but peculiar in content. it concerns a high school student who gave a speech on behalf of a friend who was running for student president; the speech had a mild sexual content that deeply shocked martha nussbaum the justices leading them to wax eloquent about matters of civil toleration—before they proceed to take away the student’s right to be valedictorian of his class! (the majority opinion, written by chief justice burger, refuses even to quote from the speech, saying it is offensive, but justice brennan, in dissent, quotes the entirety of it, and we see just how innocuous and stupid it really is.) this distinction goes back at least to rousseau’s on the social contract, where rousseau argues that societies are entitled to enforce both sorts of toleration, since “it is impossible to live at peace with those whom one believes to be damned.” experience would appear to have proven him wrong. cultivating humanity: a classical defense of reform in liberal education (cambridge, ma: harvard university press, ). we might add leonard elmhirst in england, whose dartington hall school was closely modeled on tagore’s santiniketan, where elmhirst had spent a good deal of time. at the cambridge school, in weston, massachusetts, where my daughter went to high school and where i am a trustee, the dewey values continue in their full force, in part because the boston area contains so many private schools that a dewey-style school can carve out a market niche and avoid the pressure toward conformity that lab, the leading private school in chicago, con- tinually faces. for details, see my the clash within: violence, hope, and india’s future (cambridge, ma: harvard university press, forthcoming ), chap. . one excellent book, philosophical in orientation though not by a professional philosopher, that should have received more attention is leon botstein’s jefferson’s children; but botstein, pres- ident of bard college, has recently been focusing all his attention on post-secondary education (and on his musical career); the book’s recommendations for the public schools have been little discussed. the imminent demise of liberal molecular basis for cytokine biomarkers of complex d microtissue physiology in vitro amish asthana and william s. kisaalita* cellular bioengineering laboratory, college of engineering, driftmier engineering center, university of georgia, athens ga , usa *corresponding author: kisaalita, w.s. (williamk@engr.uga.edu). keywords: biomarkers; d; three-dimensionality; cytokines; tissue engineering; drug discovery; material discovery; cell-based biosensors. teaser: cytokines make excellent early biomarkers for complex d microtissue physiology in vitro. they should be a powerful tool in establishing cell–cell and cell–material interactions in microenvironments that support physiologically more-relevant microtissue formation when validated. © . this manuscript version is made available under the elsevier user license http://www.elsevier.com/open-access/userlicense/ . / mailto:williamk@engr.uga.edu ‘physiologically more-relevant’ claims are readily made for cells cultured on any surface or in a scaffold that provides loosely defined d geometry. a set of tools to measure culture ‘ d-ness’ more accurately are needed. such tools should find applications in fields ranging from high- throughput identification of substrates for tissue engineering and regenerative medicine to cell- based screening of drug candidates. until now, these fields have not provided a consensus for the most promising place to initiate the search. here, we review recent advances in transcriptomic, proteomic, inflammation and oncology-related pathways, as well as functional studies that strongly point to cytokines as the most likely compounds to form the missing consensus. introduction the definition of d cell culture has recently been extended from simple spatial organization to providing a complete microenvironment that leads to the formation of a complex physiologically relevant microtissue. this new definition can be translated to mean better emulation of in-vivo- like functional competence in a way not achievable in monolayer cultures [ ]. interestingly, the crucial components of this microenvironment can be expressed in a d cartesian coordinate system (figure ), with the following microenvironmental factors (mefs) or three-dimensions of: (i) biochemical or chemical configuration; (ii) temporal dimensions and spatial (geometric d) architecture; and (iii) force and substrate physical properties. the three mefs are well supported by recent literature [ – ]. in the following four paragraphs, before proceeding, we provide four compelling reasons why searching for three-dimensionality biomarkers in required. first, if the resultant phenotypic characteristics are different between cells growing in d and those grown on any platform that provides a loosely defined d architecture, either at the nano- or micro-scale or their combinations, they are deemed to be physiologically more relevant. apart from the d matrix adhesion, initially proposed by cukierman et al. [ ] as a possible ‘diagnosis’ or indication for the three-dimensionality of a culture, the area of tissue engineering has not provided the knowhow basis on which these claims could be validated. as such, a consensus for the d state of a culture and the complex physiological relevance associated with it should be established. second, hit materials can be rapidly screened for future development via high-throughput combinatorial approaches. a library could be generated with information on polymers or other scaffolding substrates [ , ] useful in tissue engineering or cell-based drug discovery applications, providing d assays are available. the advancement of these assays or biosensors can potentially be governed by a cell–substrate interaction outcome [ ]. as previously pointed out, three-dimensionality biomarkers would provide the intellectual basis for material discovery platform development, where interactions with a substrate that produces cells that mimic in-vivo- like competence are desired [ ]. figure shows an illustration of a theoretical framework in which scaffold substrate discovery can be carried out in a hts [ ] format. as shown in figure a,b, poly(desamino tyrosyl-tyrosine ethyl ester carbonate) (pdtec) and poly (desamino tyrosyl- tryrosine octyl ester carbonate) (pdtoc) have a structurally similar backbone, but different side- chains (i.e. ethyl on pdtec and octyl of pdtoc). because of this, they show different properties, which lead to variations in cells grown on films ( d) of the polymers and their composites. when cultured on pdtec, cells displayed enhanced adhesion, spreading and proliferation in comparison with pdtoc [ , ]. if a balance between differentiation and proliferation is needed, then an optimum polymeric composition that satisfies the requirement might exist and readily be found using hts with a polymer blend combinatorial scaffold library. the library can be constructed using a fluid handling instrument represented by a dual syringe pump system (figure c) that would generate arrays of porogen-leached scaffolds of varying polymeric blends (figure d). in the proof-of-concept study of the system shown in figure , yang et al. [ , ] used fourier transform infrared spectroscopy (ftir) to verify scaffold polymer mixtures. extending such a study to the question of how well the polymer blends support the three-dimensionality of cultures within requires biomarkers measurable in hts readouts. third, it is essential to reduce the costs of d platforms to increase their accessibility for hts applications; simplification of the platform without affecting the physiologically relevant behavior of the cells can only be achieved with validated biomarkers. in recent work, the definition of three-dimensionality in cell culture has been extended to provide a complete microenvironment that leads to the formation of a complex physiologically relevant microtissue or better emulation of in-vivo-like functional competence in a way not achievable in monolayer cultures [ ] (see figure for more detailed illustration). the platform simplification can be achieved easily if the physiologically relevant outcome can be measured in terms of three- dimensionality biomarkers, as elaborated by lai et al. [ ]. as the architecture of the platform is simplified, it will be feasible, using validated biomarkers, to know when the trajectory toward complex physiological relevance (cpr) outcomes is being affected. fourth, cpr is a phenomenon that is generally expressed late in culture and is often associated with a combination of structural and functional attributes that are not quantitative. a biomarker that might be quantitative and expressed early in culture can act as an early indicator of the trajectory toward cpr outcomes. additionally, different techniques are typically employed to detect cpr in different tissue types and they are often incompatible with hts. therefore, a ubiquitous biomarker expressed early in culture would provide a single assay that can predict cpr outcomes in cells derived from many tissue types. taken together, the field of d culture requires validated biomarkers. the elephant in the room issue is whether such a thing exists. in this review, we bring together evidence from transcriptomic, proteomic, inflammation and oncology-related pathways, as well as cellular functional studies that strongly suggest that cytokines are the most likely entity to provide the badly needed biomarkers. timing of cpr and screening studies differences in resulting cell phenotypes between d and d platforms are necessary but not sufficient for suggesting cpr. it is therefore necessary to show conclusively that cells cultured on d platforms are emulating the functional and/or structural outcomes observed in vivo. the well-established or provisional cpr outcomes for cells belonging to the three types of tissues of major interest in preclinical drug discovery (i.e. epithelial, cardiac and neuronal) have been discussed in detail by asthana and kisaalita [ ]. although structural and/or functional cpr outcomes can be used to establish and validate three-dimensionality, there are additional questions or drawbacks beyond the late-in-culture expression that has been mentioned already. for example, a cpr outcome can be considered an endpoint measurement, suggesting that the culture has been on a trajectory, prior to this point, toward this desired in vivo emulation state. a natural question to ask is: when is the most appropriate time to use the microtissue in screening studies – before, after or at this point? it is well known that the viability of microtissues in vitro has a limited time. this raises the question of how long after observing cpr outcomes is the microtissue suitable for screening studies? assuming a cytokine expression profile early in culture accurately predicts cpr outcomes expressed later in culture, are the resulting microtissues suitable for screening studies before cpr outcome expression? answers to the above questions are crucial to the advancement and utilization of complex physiologically relevant d cell-based assays. to answer these questions or to establish the optimal performance time for a microtissue, with respect to meaningful assay results, biomarkers of three- dimensionality that reliably predict cpr outcomes and are expressed early in culture are a must- have. cohesivity: microtissue formation and cytokine production living organisms have the ability to perceive their environment and respond to changes in it. cells, being the building blocks of an organism, are also endowed with this power. their surrounding, being comparable to their own size, is termed the microenvironment and cells are affected by changes in it. if the cells are transitioned from a monolayer culture to a d environment how do they perceive this change? cells in d find themselves in the vicinity of homotypic neighbors leading to the formation of a loosely bound aggregate. a similar scenario is encountered in vivo during avascular tumor progression, early stages of inflammatory wound healing and development. these phenomena are similar in nature and are controlled by the same molecules: cytokines [ ]. in vitro, depending upon their nature (malignant, primary or stem), the cells cultured in d relate to any of these phenomena and therefore an increase in their cytokine expression is physiologically justified. cytokines are soluble low molecular weight extracellular protein mediators that typically act at short range between neighboring cells. they have important roles in intercellular regulation and mobilization of cells engaged in innate and adaptive inflammatory host defenses, cell growth and death, differentiation, angiogenesis, development and repair processes [ ]. they have been studied extensively for their role in inflammation, tumor progression and normal development [ – ]. cytokines and their associated receptors provide major signals for essential processes. abnormalities associated with them, their receptors or the signaling pathways they affect are involved in a wide array of diseases, particularly by promoting and perpetuating inflammation. based on the structural homologies of their receptors, cytokines have been assigned to various family groups and can be broadly classified into: colony stimulating factors, interleukins (il), interferons, transforming growth factors (tgfs), tumor necrosis factors (tnfs), platelet-derived growth factors (pdgfs) and chemokines. they act on cells expressing complementary receptors in autocrine and paracrine manners [ ]. they can control their own production as well as that of the others by initiating a feedback loop [ – ]. cytokine production and function is generally governed by the transcription factors nuclear factor (nf)-κb [ ] and activator protein (ap)- [ ] because they have binding sites in the promoter region of most of the cytokine genes. the extracellular- signal-regulated kinase (erk) [ ] and the janus kinase/signal transducers and activators of transcription (jak/stat) [ , ] pathways have also been widely implicated in their regulation. along with the downregulation of cytokines, discussed in a later section, another intriguing absence in the case of d monolayers is that of cell adhesion molecules (cams). this is substantiated by the microarray analysis done on many established cell lines and tissue-derived cells in which cams were found to be downregulated in conventional cultures [ ]. cams are transmembrane receptors found on the surface of the cell and are composed of three domains: (i) an extracellular domain that enables interactions either with similar cams (homophilic binding) or with different cams or the extracellular matrix (heterophilic binding); (ii) an intracellular domain that facilitates interactions with the cytoskeleton; and (iii) a transmembrane domain. cams can be classified into five protein families: immunoglobulin (ig) superfamily (igsf cams), integrins, cadherins, selectins and the lymphocyte homing receptor. their structure and function have been reviewed in detail elsewhere [ ]. in d cultures, formation of aggregates or microtissues is brought about by cell–cell and cell–ecm (extracellular matrix) interaction through cams [ ]. traditionally, integrins have been associated with cell–substrate adhesion whereas cadherins form cell–cell adherens junctions [ ]. however, recent studies carried out on cho cells [ ] and fibroblasts [ ] have shown that integrins play a part in forming cohesive cell–cell bonds in a d microenvironment leading to the formation of a microtissue. cohesivity seems to be brought about by integrin binding to the fibronectin (fn) matrix assembly [ ] present in the ecm of d cultures, but absent in monolayers. cellular crosstalk mediated by integrins and the subsequent intracellular pathways that they invoke might be related to the upregulation of cytokines in d. this is the main premise behind cytokines as the most likely family of compounds to provide the missing three-dimensionality biomarker consensus. in the remainder of this review, we provide more-detailed experimental evidence to support this proposition. we have organized our presentation thus: (i) d– d comparative transcriptomic and proteomic studies; (ii) integrin–fn studies: and (iii) signaling pathways involved as an overarching view that integrates the other experimental evidence. d– d culture comparative transcriptomic–proteomic upregulation of cytokines recently, many studies have shown that when cells are grown in a d culture their cytokine levels are elevated as compared with the traditional monolayer cultures (table ). we compared neural progenitor (np) cells grown on d substrates, d porous polystyrene scaffolds and as d neurospheres (in vivo surrogate) with respect to transcriptomic expression using the human whole genome u plus . genechip expression analysis (affymetrix, santa clara, ca) [ ]. the expression data are available on the geo site as series gse . an upregulation in the expression of cytokines as a group in d and neurospheres was observed. the numbers of probe-sets that were elevated in d and neurospheroid culture conditions were and , respectively. the difference in the number of upregulated probe-sets might be the result of an inability to regulate the diameter of neurospheres. many neurospheres were observed to be bigger than the pore size of the d scaffolds (e.g. three-times the maximum pore diameter of µm) [ ]. the core of the microtissue can experience hypoxia in large neurospheres, to the extent that genes not observed in d are upregulated. for example, macrophage inflammatory protein (mip)- gene, induced by hypoxia [ ], was found to be elevated in neurospheroids but not in d conditions in our study. a group of cytokines including angiopoietin-like (angtl )/cdt , armet/mesencephalic astrocyte-derived neurotrophic factor (manf), bone morphogenetic protein b/osteogenic protein (bmp b/op ), ccl /monocyte chemotactic protein- (mcp- ), fibroblast growth factor (fgf ), ghrelin (ghrl)/obestatin, il- , il- b/il- f , nephroblastoma overexpressed (nov)/ibp- , platelet-derived growth factor subunit b (pdgfb), stanniocalcin- (stc ), transforming growth factor alpha (tgfa) and vascular endothelial growth factor a (vegf-a) were commonly elevated in cells grown in polystyrene scaffolds and neurospheres. as such, any or a combination from this list has the potential to serve as biomarkers of three-dimensionality. we particularly focused on the above genes because there was less likelihood of them being upregulated as a result of hypoxic conditions that could be present in neurospheroids but not in d conditions. the functional classification of these cytokines suggested an underlying theme of development, which is physiologically relevant because progenitor cells in a d culture in vitro try to emulate embryonic development. the result of this study is in agreement with many other transcriptomic and proteomic studies where cytokines have been shown to be elevated in d cultures. these studies have been conducted on many different cell lines spanning various cell types including primary cells, fibroblasts, multi and pluripotent stem cells and cancer cells grown on different d platforms. the results are summarized in table . as shown, despite the fact that there are no neural cells in the studies cited in table , identical (il- ) or related (angtl , il- b and vegf-a) cytokine upregulation from our study has been reported by others from different cell types. in a recent review discussing how cells know where they are, lander [ ] has emphasized the importance of morphogen diffusion gradients. an upregulation of cytokines would result in a change in the diffusion gradient. because many cytokines (e.g. bmp b, pdgfb) are well known morphogens, this change in the diffusion gradient results in different cellular activity in d compared with d cultures. taken together, this shows that cytokines have the potential to validate the d platforms with respect to emulating the in vivo microenvironments. cytokines are suitable as biomarkers because they are secreted in the culture media and can be quantified without lysing by elisa [ ]. this will not be the first instance for the proposed usage of cytokines as biomarkers. owing to their robustness and versatility, cytokines have been proposed extensively to be used as markers for early detection of head, neck [ ] and ovarian cancers [ ], alzheimer’s disease [ ], heart diseases [ ] and prostatitis [ ], determining the potential activity of drugs early in clinical development [ ] and many more scenarios. hypoxia is another physiologically relevant characteristic of d microenvironments in vivo and in vitro [ ]. in tissues, the concentration of oxygen is dependent on the balance between oxygen supplied and consumed. this balance is well controlled in vivo by evenly distributed capillary networks, which are lacking in vitro. therefore the core of a homotypic d microtissue might become hypoxic as the size of the tissue increases. this event can cause induction of chemical signals (cytokines) from the cells for angiogenesis, which is similar to the way normal tissues respond to hypoxia where balanced signaling mechanisms lead to angioadaptation and vascular remodeling until the concentration of oxygen in tissue is back within its normal range [ ]. hypoxia-inducible transcription factors (hifs) are the molecular mediators of physiological hypoxia and the cellular response. under hypoxic conditions, hif- α and hif- β translocate to the nucleus [ ], where their dimerization takes place and they subsequently bind to target gene motifs called hypoxia-responsive elements (hres) resulting in altered gene expression [ ]. hif- α seems to regulate the production of vegf because its suppression has been shown to cause transcriptional inhibition of vegf [ ] and reduced vascular density [ ]. hypoxic conditions have also been shown to trigger ap- [ ] and nf-κb [ ], which affects the production of a myriad of cytokines (see table s in supplementary material online). the effect of hypoxia on cytokine production was further established by a study that showed higher levels of cytokines produced by hypoxic d co-cultures of cd and human umbilical vein endothelial cells (huvec) than normal cultures [ ]. in another study, by fischbach et al. [ ], hypoxia was found to be associated with upregulation in cytokine secretion in d tumors. however, comparing monolayer cultures under reduced oxygen concentration with d cultures indicated that the sole contribution of hypoxia is comparably small and that the joint effect of hypoxia and the d microenvironment is necessary to elicit increased expression of cytokines, particularly il- [ ]. intercellular crosstalk in d culture: integrin–fn interaction integrin α β is the only integrin that naturally assembles fn into a matrix and this can lead to the formation of an endogenous matrix. fn is a multifunctional component of the extracellular matrix, which exists in a dimeric state, with the two chains attached through disulfide bonds at the c terminus [ ]. every fn chain has a single cell-binding domain having an arginine- glycine-aspartic acid (rgd) sequence to which α β integrin specifically binds [ ]. fn matrix assembly structural make-up includes the dimeric structure of fn, the n-terminal assembly domain and fn-binding sites in the first two type iii repeats [ , ] and integrin binding to the rgd sequence in the cell-binding domain [ ]. interaction of integrin with fn promotes intermolecular association between the fn dimers, leading to the formation of fibrils, which further increases cell adhesion and cohesivity. this is consistent with the fact that monomers of fn could not lead to aggregate formation [ ] and this substantiates the notion that fn matrix assembly can support aggregate cohesivity by forming a scaffold or an organized d matrix, which leads to a functional linkage between cells. using α -, αv- and β -integrin-blocking antibodies [ ] caused a reduction in the amount of fn produced in fibroblast spheroids. this suggests that these integrins are involved in the expression of fn in spheroids but that the formation of tight compact spheroids is mediated through the α β integrin only. integrin α , α and β subunits are important for embryonic development in vivo, because their absence has been shown to be lethal to mouse embryos [ ]. it has also been observed that a perfect correlation exists between the cohesion of the germ layers in amphibian gastrulae and the spatial position of these integrins [ ]. moreover, injecting rgd peptide into amphibian blastulae has been shown to disrupt cell interactions with fn causing blockage of gastrulation and preventing formation of fn fibril meshwork involved in migration [ ]. if the interaction between integrin α β and fn is indeed responsible for the cohesivity of tissues, particularly very early in development when embryos are essentially microtissue aggregate-like, then it is possible that this particular interaction also has the capacity to provide cohesive forces to cells within a microtissue in vitro. compaction mediated by fn has also been shown to be of importance in the later stages of development. a correlation has been found between fn production and pre-cartilage mesenchymal condensation during the development of wings and leg buds in chick embryos [ ], with higher fn expression correlating with compaction of tissues. the interaction between integrin α β and fn has also been shown to generate the tractional force that is essential for retraction of d fn–fibrin clot matrices [ ]. this retraction process is crucial for early wound healing and tissue remodeling. it has been proposed that this intercellular cohesivity also contributes to clot retraction, akin to the apparent retraction or compaction observed for cho aggregates in response to increased concentrations of fn. ergo, this cell–cell cohesivity produced by integrin engagement with fn is probably responsible for triggering the intracellular signaling pathways that lead to the difference in cytokine production seen in d monolayers and d microtissues. these pathways and their key molecular regulators have been elaborated in the following section. evidence in support of involvement of integrin-mediated cell adhesion to fn in the production of cytokines has been provided by a study where microarray analysis done on myeloma cells growing on fn showed upregulation of many nf-κb-regulated genes [ ]. nf- κb controls the expression of many growth factors and cytokines as discussed below. some of the cytokines that were found to be upregulated, compared with control suspended cells, included tnfaip , ccl , il- and il- . another study by fischbach et al. [ ] showed how transition from a monolayer to a d environment led to an increase in the production of il- and vegf by breast cancer, glioblastoma and oral carcinoma cells in vitro and in vivo in a scid mouse model. the results from this study [ ] suggest that the d microenvironment plays a vital part in regulating the secretion of il- but that the combined effects of the d microenvironment, d cell morphology and d integrin (particularly α β ) engagement are required to fully regulate the production of il- by tumor cells. interestingly, engagement of integrins in d and spread morphology of cells in a d space led to a higher il- production than a conventional monolayer but less than d cultures, further emphasizing that spatial cues along with cell–ecm interactions and morphology of the cell regulate its cytokine secretion. also, cell morphology is an often neglected but an important parameter because alterations in morphology cause actin cytoskeleton remodeling that can lead to differential nf-κb signaling [ ] which controls cytokine production. integrin-mediated adhesion regulates many important intracellular signaling cascades. these pathways are most likely aberrantly regulated in d cultures because of the absence of an endogenous extracellular matrix assembly or cell–cell interactions. however, in a more physiologically relevant microenvironment, they can relay signals through a variety of adapter proteins that are localized at their cytoplasmic tails forming the focal adhesion complexes. integrin signaling is mediated through focal adhesion kinase (fak), rous sarcoma oncogene cellular homolog (src), adapter protein involved in oncogenesis (shc) and growth factor receptor-bound protein (grb ) to downstream kinases. the ras/raf/erk pathway is one of the most important pathways regulated by integrin-mediated adhesion. erk activates several transcription factors in the nucleus such as nf-κb, ap- , camp response element-binding protein (creb) and e amv virus oncogene cellular homolog (ets- ) that control the production of cytokines and survival and growth factors important for the cell. therefore, through proper activation of the erk pathway, integrins can indirectly regulate the production of cytokines, provided they interact with a tissue-mimicking environment having optimum spatial, biophysical and biochemical cues. the link between the microenvironment and gene expression focal adhesion kinase fak is a type of non-receptor tyrosine kinase that affects the dynamics of integrin-associated adhesion and the actin cytoskeleton that is coupled to it, through various molecular interactions. being a component of focal adhesion scaffolding, it performs protein–protein interaction adaptor functions at the loci of cell adhesion to the ecm and also relays adhesion and growth-factor- dependent cues into the cell body. many reports have linked upregulated fak expression with tumor growth [ , ]. the fak gene locus at q -q has also been shown to be a target of gene amplification during tumor progression [ ]. however, as far as cytokine production is concerned, the gene is controlled by fak through the ras/raf/erk signaling pathway and activation of its downstream transcription factors nf-κb and ap- . fak signaling has been directly implicated in vegf production by avascular tumors in vivo [ ]. fak expression and its catalytic activity as a result of y phosphorylation promotes the erk pathway, leading to an upregulation in vegf expression resulting in tumor neovascularization without any significant alteration in cell proliferation or anchorage-independent survival. inhibition of fak activity by stable fak c-terminal domain (frnk) expression led to reduced secretion of vegf compared with control in t breast carcinoma cells [ ]. cells expressing frnk showed formation of small tumors that were avascular, without exhibiting differences in tumor- associated apoptosis. it was observed that frnk led to the inhibition of a fak/grb /mapk signaling linkage controlling vegf production. this finding was further supported by point- mutations that affected fak catalytic activity or y phosphorylation, disrupting the ability of fak to promote erk- and vegf-associated tumor growth. reduction in vegf expression was also observed when fak expression was inhibited in prostate, breast and neuroblastoma cells. furthermore, a point mutation (frnk s- ) that inactivates frnk by disrupting its co- localization with integrins leads to the restoration of fak activity and vegf production [ ]. another major factor that activates vegf expression is the development of hypoxia within the core of the proliferating tumor [ ]. interestingly, hypoxia has also been found to increase fak tyrosine phosphorylation and bolster the linkage between fak and grb in cardiac myocytes in vitro [ ]. fak overexpression has also been associated with neovascularization of the retina in a mouse model of hypoxia-induced retinal angiogenesis [ ]. further evidence of fak involvement in vegf production is presented in the studies where overexpression of fak in vascular endothelial cells led to angiogenesis in transgenic mice [ ]. additionally, fak expression was found to be upregulated in angiogenic blood vessels within astrocytic-associated tumor stroma in comparison with normal brain endothelial cells [ ]. also, expression of fak within endothelial cells is essential for vasculogenesis during development [ ]. the pathway to cytokine upregulation: ras/raf/erk signaling and transcription factors the extracellular signals are relayed from the integrin via fak and src, leading to the activation of several intracellular signaling pathways, mainly the ras/raf/erk pathway (figure ). in the context of cytokine production, raf activity has been implicated in many studies. transformation of hematopoietic cells by activated raf genes has often been shown to result in the expression of granulocyte macrophage-colony stimulating factor (gm-csf), which acts as an autocrine growth factor [ , ]. nih- t cells expressing activated raf have shown increased secretion of heparin-binding epidermal growth factor (hbegf) [ ]. kaposi’s sarcoma transformed b cells that exhibit elevated expression of b-raf also show an increased production of vegf [ ]. it has been shown recently that the infectivity of kaposi’s sarcoma virus is increased by b-raf expression [ ] and induction of vegf production by b-raf might be a mechanism for this elevation in viral infection [ ]. moreover, raf activity leads to the subsequent activation of mitogen-activated protein kinase (mek) /erk which activates downstream erk which regulates the activation of transcription factors like nf-κb and ap- (figure ) that have been directly implicated in the expression of cytokines, mitogens and cell survival factors (see table s in supplementary material online). the involvement of these transcription factors is discussed in detail in the following subsection. the activity of raf is regulated positively through phosphorylation on s residues in its catalytic domain. all three members of the raf family (b- raf, raf- and a-raf) have the capability to phosphorylate and activate mek with different biochemical efficiencies [ ]. this step is also adhesion dependent and is brought about by activation of endogenous p -activated kinase (pak) by small gtpase rac induced by integrin- mediated adhesion to fn. b-raf, which also directly activates mek independently of raf- , is activated by integrin-bound focal adhesion complexes. however, raf- can also activate nf-κb through a mechanism independent of mek /erk by degradation of its inhibitor iκb via mek kinase (mekk ) [ ] (figure ). erk and erk activities are regulated positively through phosphorylation mediated by mek and mek and can directly activate many transcription factors like ets- , ap- and c- myc. erks can also regulate the phosphorylation and activation of the kda ribosomal s kinase (p rsk or rsk ), which in turn controls the activation of the transcription factor creb [ ]. moreover, it has also been observed that p rsk can lead to the activation of nf-κb by phosphorylating its inhibitor iκbα at ser causing its ubiquitination [ ] (figure ). the localization of activated erk is important in determining the cellular fate. to carry out the phosphorylation of its downstream transcription factors, erk needs to be translocated from the cytoplasm to the nucleus. this is an adhesion-dependent step and is brought about by pak activation by small gtpase rac, induced by integrin-mediated adhesion to fn. in cells lacking adhesion cues (suspended cells), erk activation can be achieved by overexpressing active raf ( w raf) or mek (mek -Δed) mutants. however, the signal is transmitted poorly to the nucleus because activated erk cannot translocate to the nucleus and fully activate its downstream transcription factors [ ]. most of the cytokines and growth factors are the target genes of nf-κb, ap- and ets (see table s in supplementary material online) which are in turn regulated by erk or other components of the mapk pathway (raf and p rsk) and so proper control and activation of the pathway is essential for production and regulation of the cytokines [ ]. nf-κb. nf-κb activation can be brought about by a variety of stimuli and, thus far, two nf-κb signaling mechanisms – classical and alternate – have been described [ ]. the main nf-κb dimer that is activated through the classical pathway is p :p . translocation of p :p to the nucleus leads to the transcription of many proinflammatory targets, such as cytokines, chemokines, proangiogenic factors, adhesion molecules, antiapoptotic proteins and inducible enzymes. nf-κb activity can be regulated either in an erk-independent fashion by raf- via membrane shuttle kinase mekk or in an erk-dependent manner by rsk (as discussed in the previous section). the activation of both these pathways (raf- /mekk or erk/rsk) is dependent on integrin-mediated adhesion to the ecm and is regulated by the microenvironment and therefore requires physiologically relevant microenvironmental cues to be properly activated. nf-κb was found to be upregulated in hepatic stellate cells (hscs) in d collagen i gel cultures [ ] and in mammary tumor cell spheroids, where its activation was also responsible for resistance to apoptosis [ ]. many studies have also correlated nf-κb activation with cytokine production in different d cultures. in most of these studies, cytokine production in control d monolayer cultures was either lacking or at basal levels. fibroblasts, when grown in spheroids, have shown an inverse correlation between nf-κb and iκb at a time point just before cytokine secretion [ ]. the degradation of iκb leads to the activity of nf-κb in spheroids, whereas monolayer levels remained unchanged and the correlation was further substantiated when the activity of nf-κb was found to be higher in d than monolayers at the same time points [ ]. also, changes in cell morphology can lead to production of cytokines because changes in the actin cytoskeleton lead to differential activation of nf-κb [ ]. such cytoskeletal changes are evident in transition from monolayer to a d culture platform [ ]. furthermore, it has been shown that fn is a potent activator of the nf-κb signaling pathway [ ] and α β - integrin-mediated formation of fn endogenous matrix assembly leading to cellular cohesivity in d cultures as described earlier. taken together, this information suggests the basis for upregulation of cytokine production in d cultures in comparison to d or monolayer cultures. ap- . the ap- transcription factor is not a solitary protein; it is instead made of various dimeric basic-region leucine zipper (bzip) proteins that belong to the jun, fos, maf and atf [ ] families. the activity of ap- is induced by various stimuli such as cytokines, growth factors, polypeptide hormones, neurotransmitters, bacterial and viral infections, cell–matrix interactions and many physical and chemical stresses. these stimuli lead to the activation of mitogen- activated protein kinase (mapk) signaling cascades [ ] that upregulate ap- activity through phosphorylation of distinct moieties. ap- activation controls the expression of several cytokines and cams. in monolayers, ap- activity might be impaired because the erk pathway is differentially regulated owing to the lack of optimum microenvironmental factors. however, in d cultures, in the presence of a physiologically relevant tissue mimicking microenvironment, ap- activity leads to cytokine production, depending on the cell type (malignant versus primary) and/or scenario (tumor progression or wound healing). this hypothesis is substantiated by the fact that recently ap- was found to be activated in spheroids of different cell types culminating in cytokine production. all three cell types tested – transformed human embryonic kidney (hek ) [ ], primary human foreskin fibroblasts (hff- ) and carcinogenic glioblastoma (t g) [ ] – showed activation of different pathways leading to spheroid formation, quiescence and survival but the signals converged to the activation of ap- resulting in higher cytokine production than control monolayer cultures. this shows that response to microenvironmental cues can be cell-line-specific in terms of pathways but the final outcome (autocrine cytokine signaling and survival in this case) is dependent on the same transcription factor, further highlighting its importance. furthermore, such autocrine and paracrine signaling is lacking in monolayer cultures owing to lack of proximity between cells and absence of cell–cell interactions and crosstalk. ets. ets represents a family of transcription factors, including ets- , ets- , elk- , sap , sap , e af, pea , pu , among others [ ]. the ets transcription factors regulate the expression of various other transcription factors like p [ ], c-fos [ ] and nf-κb [ ]. ets proteins usually activate their target genes through association with other transcription factors. for instance, ets- regulates the expression of gm-csf in jurkat t cells [ ] and mast cells by coordinately regulating the gm-csf promoter in partnership with nf-kb and/or ap- factors [ ]. it has been shown that ets- and ets- induce the expression of the il- promoter reporter gene construct in jurkat t cells on stimulation with pma (phorbol ester) and ionomycin [ ]. ets- also bound to an il- promoter reporter gene when transfected into the mouse d .g . th t cell clone, where it strongly cooperated with an adjacent ap- binding motif [ ]. also, co- transfection of ets- and ap- (fos/jun) proteins into d .g . cells was able to induce expression of the endogenous il- gene in the absence of exogenous activator molecules like pma or camp that normally induce il- expression in these cells [ ]. this cooperative activity is consistent with the production of cytokine in d cultures, where erk cascade- controlled ap- and ets activation is induced as a result of the presence of a physiologically relevant milieu and cellular crosstalk, synergistically driving cytokine production without the presence of any external induction molecule. furthermore, ets- binding sites have also been found in the promoters of many chemokines like cxcl (pf ) [ , ], ccl (mcp- ) [ ] and cxcl (il- ) [ ], and ets- knockout (ets- -/-) or silencing (sirna) has been shown to block the induction of these chemokines. concluding remarks we have methodically built a case for the potential of cytokines as biomarkers for three- dimensionality or d cultures. the fact that cytokines are secreted in media makes their detection easier, especially in hts readouts. additionally, cytokines seem to be expressed in a wide range of cells from the four tissue types (muscle, connective, epithelial and nerve), which suggests their good potential for ubiquity as opposed to being cell- or tissue-specific. also, their temporal expression suggests use of profiles as opposed to single-time-point measurements, increasing their robustness as biomarkers. for these reasons, cytokines are particularly attractive as biomarkers. as pointed out above, cytokines are broadly classified into seven subfamilies. in ongoing investigations in our lab (and possibly other laboratories), the question of which subfamily is most suitable to target for follow-up validation studies is being addressed. acknowledgments the authors acknowledge funding from the national science foundation grant ecs- . references . kisaalita, w.s. 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( ) is time an extra dimension in d cell culture? drug discov. today doi: . /j.drudis. . . http://dx.doi.org.proxy-remote.galib.uga.edu/ . /j.drudis. . . figure legends figure . schematic depiction of a total microenvironment ( d culture) supporting the formation of microtissue that exhibits ‘complex’ physiological relevance (cpr) or better emulation of the in vivo tissue functionality in a manner not possible in d cultures. the four factors shaping this microenvironment are: (i) chemical or biochemical composition; (ii) spatial (geometric d and topography); (iii) substrate biophysical properties; and (iv) temporal. chemical cues can be subdivided into short-range (e.g. cell–cell) and long-range (e.g. diffusible species like nutrients and growth factors). physical cues consist of shear as a result of fluid flow and/or physical properties of the material or scaffolding [ ]. spatial (a d space) and biochemical (protein coating) is of initial (t ) importance, whereas there seems to be a lesser degree of dependence on biophysical cues [ ]. as the time in culture increases (t ), the cells produce an endogenous extracellular matrix (ecm), which has a physiologically relevant fibrillar conformation as opposed to exogenously supplied protein coating [ ]. the cells send signals to neighboring cells for chemotaxis (e.g. cytokines), which is proposed herein for early biomarking of three-dimensionality as depicted in the intermediate circle. once on the trajectory, the d microtissues should yield endpoint physiologically relevant outcomes (structural and/or functional) known in vivo but absent in d formats (e.g. microvilli-lined bile canaliculi and secretion of albumin for hepatic platforms) [ ]. these outcomes can serve as the gold standard for formed microtissue emulation of its native counterpart. the microtissue can continue in a quasi-steady state until the size reaches a critical limit and oxygen and nutrient (biochemical factors) diffusion limitations lead to the initiation of a hypoxic phase, especially in constructs that cannot impose a physical constraint on the size of the microtissue [ ]. figure . hts scaffold material discovery conceptual framework. (a) chemical structure of pdtec. (b) chemical structure of and pdtoc. (c) combinatorial fabrication of ppdtec-pdtoc polymer blend scaffold library schematic. (d) porogen-leached and freeze-dried sample of scaffold library in a -well plate. adapted, with permission, from [ ]. figure . extracellular signal-regulated kinase (erk) and subsequent transcription factor activation via extracellular matrix (ecm)–integrin binding. upstream events in the signaling cascade like integrin-mediated adhesion to ecm lead to gtp loading of ras – in many ways like activation of src and phosphorylation of shc and grb . transmission of the signal to raf and subsequently to erk kinase (mek) and erk is mitigated in suspended cells as there exists an evident anchorage-dependent step between ras and raf. a similar anchorage-dependent signal relay seems to be present between raf and mek. these two activation steps are regulated by integrin-dependent p -activated kinases (paks). the activation of erk can be achieved through heterologous expression of active upstream components like raf ( w raf) or mek (mek -Δed) in suspended cells but still the signal is transmitted poorly to the nucleus [ ] because integrin activated rac regulates erk translocation to the nucleus along with controlling erk activation through pak. mek activation is also controlled by b-raf, which in turn is activated through engagement of focal adhesion kinase (fak) with the actin cytoskeleton and subsequent formation of focal complexes. there is also a close interplay between the erk pathway and nuclear factor (nf)-κb, with rsk [ ] and raf [ ] regulating its activation. in the nucleus, erk is involved in the activation of all the three subclasses of the ets family of transcription factors and formation of the ternary nucleoprotein complex between tcfs (elk- , net and sap- ) and serum response factor (srf) over the serum response element (sre) of the promoter. activation of rsk in the cytoplasm and translocation to the nucleus further activates camp response element-binding protein (creb) [ ]. abbreviations: egfr, epidermal growth factor receptor; src, rous sarcoma oncogene cellular homolog; shc, adapter protein involved in oncogenesis; grb , growth factor receptor-bound protein ; crk/c g, ct sarcoma oncogene cellular homolog/guanine nucleotide-releasing factor ; rap , repressor activator protein ; mek, mapk/erk kinase; rsk, ribosomal protein s kinase; tnfr , tumor necrosis factor receptor ; tim, t cell immunoglobulin- and mucin-domain-containing molecule; rip, receptor- interacting protein; traf , tnf receptor-associated factor ; traf , tnf receptor-associated factor ; ciap, cellular inhibitor of apoptosis protein; ikk, iκb kinase; ub, ubiquitin, iκb, inhibitor of nf-κb; mekk , mapk/erk kinase kinase ; nik, nf-κb-inducing kinase; msk , mitogen and stress activated kinase ; atf, activating transcription factor; cbp, creb binding protein; srf, serum response factor; erf, ets repressor factor; ap- , activator protein ; ets, e amv virus oncogene cellular homolog; elk, ets-like transcription factor. figure http://ees.elsevier.com/drudis/download.aspx?id= &guid=e - cb - - c b- f a &scheme= figure http://ees.elsevier.com/drudis/download.aspx?id= &guid=fc ee- e - -b aa-f f a c eff&scheme= figure http://ees.elsevier.com/drudis/download.aspx?id= &guid= aca -e - - - c dcb&scheme= table . transcriptomic–proteomic upregulation of cytokines in d cultures cell line cell type d scaffold cytokinesa refs fibroblasts primary (biopsy) collagen matrix il- [ ] c rat glial tumor cell line spheroids vegf [ ] mcas, skov/mg ovarian cancer cell lines spheroids by liquid overlay vegf [ ] imr- human fetal lung fibroblasts collagen – gag matrix il- , cxcl , cxcl , cxcl , cxcl , vegf, lif [ ] na melanoma spheroids on phema plates cxcl , il- , mip- a, angiopoetin like , cxcl , [ ] mg- , saos- human osteosarcoma si-hpmc polymer hydrogel il- , gm-csf [ ] r murine es cells cytomatrix rw-spinner culture bmp- , igf [ ] l hodgkin-lymphoma-derived cell line rada- oligopeptide matrix ccl , ccl , ccl , ccl , il- , tnf, tnfs , tnfs , inhba [ ] na melanoma spheroids on phema plates il- , vegf, angptl [ ] pdac pluripotent progenitor cell pecm il- , il- , mcp- [ ] hfsf- human foreskin fibroblasts spheroids il- β, il- , il- , il- , gm-csf, lif [ ] hfsf, crl- hes hal mrc- human foreskin fibroblasts embryonic skin fibroblasts adult lung fibroblasts embryonic lung fibroblasts spheroids on agarose plates ccl - , cxcl - , cxcl cxcl cxcl [ ] oscc u mda-mb oral squamous cell carcinoma glioblastoma breast cancer plg, rgd alginate, matrigel ® il- , vegf [ ] bmsc bone-marrow-derived mesenchymal stroma cells spheroids on agarose plates cxcl , wnt a, kitlg [ ] hmsc mesenchymal stem cells spheroids il- , lif, stanniocalcin- [ ] u glioma cell line collagen- coated plga d scaffolds vegf, bfgf [ ] plc, hepg hepatocellular carcinoma chitosan- alginate vegf, bfgf, il- [ ] oscc oral squamous cell carcinoma alginate vegf, il- [ ] u glioblastoma . . osteosarcoma silk fibroin scaffold vegf, il- [ ] mda-mb- breast cancer d fibroin scaffold vegf, il- [ ] abold indicates protein results, other results are only transcriptomic. a schematic depiction of a total microenvironment ( d culture) supporting the formation of microtissue that exhibits ‘complex’ physiological relevance (cpr) or better emulation of the in vivo tissue functionality in a manner not possible in d cultures. graphical abstract (for review) wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ thomas l, et al. bmj open ; :e . doi: . /bmjopen- - open access multicentre prospective observational study exploring the predictive value of functional echocardiographic indices for early identification of preterm neonates at risk of developing chronic pulmonary hypertension secondary to chronic neonatal lung disease laura thomas, , michelle baczynski, poorva deshpande, , ashraf kharrat, , sébastien joye, faith zhu, daniel ibarra- rios, prakesh s shah, , luc mertens, robert p jankov, xiang y ye, elaine neary, joseph ting, michael castaldo, philip levy, , aisling smith, afif f el- khuffash, regan e giesinger, patrick j mcnamara, dany e weisz, , amish jain , to cite: thomas l, baczynski m, deshpande p, et al. multicentre prospective observational study exploring the predictive value of functional echocardiographic indices for early identification of preterm neonates at risk of developing chronic pulmonary hypertension secondary to chronic neonatal lung disease. bmj open ; :e . doi: . / bmjopen- - ► prepublication history for this paper is available online. to view these files, please visit the journal online (http:// dx. doi. org/ . / bmjopen- - ). received september revised march accepted march for numbered affiliations see end of article. correspondence to amish jain; amish. jain@ sinaihealth. ca protocol © author(s) (or their employer(s)) . re- use permitted under cc by- nc. no commercial re- use. see rights and permissions. published by bmj. abstract introduction although chronic pulmonary hypertension (cph) secondary to chronic neonatal lung disease is associated with increased mortality and respiratory and neurodevelopmental morbidities, late diagnosis (typically ≥ weeks postmenstrual age, pma) and the use of qualitative echocardiographic diagnostic criterion (flat interventricular septum in systole) remain significant limitations in clinical care. our objective in this study is to evaluate the utility of relevant quantitative echocardiographic indices to identify cph in preterm neonates, early in postnatal course and to develop a diagnostic test based on the best combination of markers. methods and analysis in this ongoing international prospective multicentre observational diagnostic accuracy study, we aim to recruit neonates born < weeks pma and/or birth weight < g and perform echocardiograms in the third week of age and at weeks pma (early diagnostic assessments, eda) in addition to the standard diagnostic assessment (sda) for cph at weeks pma. predefined echocardiographic markers under investigation will be measured at each eda and examined to create a scoring system to identify neonates who subsequently meet the primary outcome of cph/death at sda. diagnostic test characteristics will be defined for each eda. pulmonary artery acceleration time and tricuspid annular plane systolic excursion are the primary markers of interest. ethics and dissemination ethics approval has been received by the mount sinai hospital research ethics board (reb) (# - - e), sunnybrook health sciences centre reb (# - ), nhs health research authority (iras ), university of iowa human subjects office/ institutional review board ( ), rotunda hospital research and ethics committee (rec- - ), and ubc children’s and women’s reb (h - ), and is under review at boston children’s hospital institutional review board. study results will be disseminated to participating families in lay format, presented to the scientific community at paediatric and critical care conferences and published in relevant peer- reviewed journals. trail registration number nct . introduction background chronic pulmonary hypertension (cph) is a debilitating and often life limiting condition strengths and limitations of this study ► in a large prospective cohort of extremely premature neonates, quantitative echocardiographic mark- ers are being acquired longitudinally using a stan- dardised methodology. ► simple and reliable point- of- care tests to charac- terise pulmonary vascular resistance (pulmonary artery acceleration time) and right ventricular func- tion (tricuspid annular plane systolic excursion) are being systematically evaluated and shared with the clinical and scientific community to facilitate early diagnosis of chronic pulmonary hypertension. ► a robust sample size calculation to guide the recruit- ment strategy and ‘blinded’ analysis of echocardio- grams is an integral part of the study design. ► the final diagnosis of chronic pulmonary hyperten- sion, against which new early diagnostic tests are being evaluated, is based on qualitative diagnostic criteria, as per current standard of care. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n m a rch . d o w n lo a d e d fro m http://bmjopen.bmj.com/ http://orcid.org/ - - - http://crossmark.crossref.org/dialog/?doi= . /bmjopen- - &domain=pdf&date_stamp= - - nct http://bmjopen.bmj.com/ thomas l, et al. bmj open ; :e . doi: . /bmjopen- - open access in which sustained vasoconstriction, vascular hypoplasia and arterial wall remodelling are associated with high and often progressive increases in pulmonary vascular resis- tance (pvr) and pulmonary arterial pressure, resulting in chronic exposure of the right ventricle to high afterload and ultimately leading to right ventricular (rv) failure. – the data from studies in adults and children indicate that early diagnosis and management focused on preservation of rv performance is a major determinant of treatment success and longevity. – recognising its importance, the us national heart, lung and blood institute has recently identified ‘determinants of rv function and failure’ as a key priority for research. in neonates, cph is inex- tricably linked with developmental disorders of the lung, most commonly chronic neonatal lung disease (cnld), a frequent complication following extreme preterm birth. it is characterised by inflammation and impaired alveolar development and pulmonary angiogenesis. – cnld accounts for ~ % of all cases of cph diagnosed during childhood and for the vast majority during infancy. in canada, cnld affects %– % of all extremely low birth weight neonates (elbw, birth weight < g) resulting in more than new cases each year, of which %– % will be diagnosed with cph during their stay in the neonatal intensive care unit (nicu). over the last decade, several retrospective and a few prospective studies consistently demonstrated an asso- ciation of cnld/cph with higher predischarge and postdischarge mortality, longer duration of mechanical ventilation oxygen therapy, hospitalisation and higher rates of adverse neurodevelopmental outcomes compared with elbw survivors with cnld alone. – the inci- dence, severity and resultant mortality of cph in cnld is generally proportional to the severity of parenchymal lung disease. a recent meta- analysis reported the pooled incidence of cph to range from % ( % ci: % to %) in mild cnld (ie, oxygen dependency ≥ days but not at weeks postmenstrual age, pma), to % ( % ci: % to %) in severe cnld (ie, oxygen dependency ≥ days and need for ≥ % oxygen or positive pressure ventilation at weeks pma). patients with cnld/ cph had higher odds of mortality versus only cnld (or: . ( % ci: . to . )). in stark contrast to the expanding literature on clinical outcomes associated with cph, there is little data to guide day- to- day manage- ment, including best practices regarding diagnostic and therapeutic strategies. to date, there is no treatment trial targeting cph in preterm neonates. while expert bodies have published consensus- based guidelines to provide some direction to clinicians, it remains unknown whether a particular approach can ameliorate the extra burden of disease imposed by cph on cnld. the optimal approach to the surveillance of cph remains unclear and is a major barrier to timely and effective diagnosis and treatment in this population. with this background, this project was envisioned to fill some of the key knowledge gaps in the diagnostic practices relating to cph in this population. importance of early identification of cases although cnld and its severity correlate with the risk of cph, the diagnosis of cnld is typically not made until weeks pma; currently, this is also the characteristic age when cph evaluation is undertaken clinically using echocardiography. late diagnosis, however, is a signifi- cant barrier for therapeutic trials to demonstrate clinical benefits. similarly, early prophylactic treatment of all extreme premature infants is an undesirable approach, as it exposes patients without disease to pharmacotherapies. this will adversely affect the risk- benefit profile for ther- apies, increase treatment costs and necessitate a larger sample size for clinical trials. several potential therapies are available, and, despite lack of trial data, are already being used in clinical practice, although often as rescue therapy for severe disease. day- to- day clinical care is significantly hampered by the absence of a diagnostic test that allows case identification early in disease course. in early stages, the disease may be more functional, deter- mined by sustained pulmonary vasoconstriction and rela- tively less by anatomical remodelling, and may be more amenable to treatments. thus, the overall aim of this study is to systematically develop quantitative echocardio- graphic diagnostic criteria which will allow for the iden- tification of neonates with significant pulmonary vascular disease early in postnatal life (figure ). a significant reduction in lag between disease onset and diagnosis will offer new avenues for testing therapeutic interventions; a necessary prerequisite for trials to have the maximum chance of success. need for quantitative echocardiographic diagnostic test for early cph diagnosis in premature neonates challenges in the clinical evaluation of at- risk premature neonates include associated parenchymal lung disease, which often masks the symptoms of evolving cph and rv dysfunction, non- applicability or delayed appearance of specific clinical signs (eg, raised jugular venous pres- sure and systemic venous congestion) and non- feasibility of a gold standard test (right heart catheterisation) due to small patient size and high risk for complications. – cardiac mri, a clinical reference investigation for the assessment of rv function in older patients, is cumber- some and not feasible during early disease in the majority of patients due to clinical instability and need for out- of- facility transport. when feasible, these investigations can only be performed sporadically, late in the disease course and are not suitable for longitudinal assessments. two- dimensional echocardiography is a safe, quick, well- tolerated, non- invasive bedside investigation which is ideally suited for longitudinal assessments. although it is considered the clinical investigation of choice for the assessment of heart function and pulmonary haemo- dynamics in neonates, it has not been utilised to its full potential in neonatal cph. previous studies have advanced our understanding of cph in neonates; however, their key limitation is the use of qualitative subjective diagnostic criteria—‘flat o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n m a rch . d o w n lo a d e d fro m http://bmjopen.bmj.com/ thomas l, et al. bmj open ; :e . doi: . /bmjopen- - open access interventricular septum’ in the majority of cases. this index represents a qualitative assessment for the loss of normal concave curvature of the septal wall between the right and the left ventricle; this is thought to reflect rv systolic pressure > / systemic systolic pressure. pres- ently, based on above studies, this criterion is used as the standard method to diagnose cph in nicus. though easily assessable in the majority of patients and specific for severe disease, it is unsuitable for early diagnosis or close monitoring of disease progression. for these consider- ations, these criteria are not usually relied on by clinicians in older patients. only one- third of neonates eventu- ally diagnosed with cph could be identified using these criteria in the fourth week of life. many quantitative echocardiographic markers relevant to the study of cph are now widely available for use in neonates, in partic- ular those representing rv function and pvr. several of these have been validated and shown to be of prognostic significance with clearly identified cut- off values for clin- ical use in adults with cph. this protocol seeks to address at least three of the domains to improve diagnostic preci- sion and clinical monitoring of cph in preterm infants recently identified by the paediatric pulmonary hyper- tension network. these areas are: ( ) the standardisa- tion of echocardiographic characterisation of cph, ( ) the use of echocardiography to identify a risk profile for early prediction of risk, diagnosis and follow- up and ( ) enhanced assessment of rv performance and afterload using novel quantitative echocardiographic techniques. this will be the first study systematically examining the predictive value of novel echocardiographic markers to develop a diagnostic test for early diagnosis of cph in the infants with gestational age (ga) < weeks or bw < g. research hypothesis and study objectives we hypothesise that quantitative echocardiographic indices of rv function and pvr will allow early diagnosis of cph prior to weeks pma in extreme premature infants; thus, reducing the time to diagnosis compared with contemporary clinical practice. more specifically, we hypothesise that pulmonary artery acceleration time (paat, a simple marker of pvr validated in adults and children against invasive measurements) and tricuspid annular plane systolic excursion (tapse, a routinely used marker of rv function) either alone or in combi- nation will be the most useful parameters for early case identification. the primary objective of this study is to evaluate the ability of relevant quantitative functional echocardio- graphic indices, in particular paat and tapse, to iden- tify preterm neonates with cph early in postnatal life, develop a diagnostic test based on the best combination of parameters and define its diagnostic characteristics (sensitivity, specificity, positive and negative likelihood ratios). our secondary objectives are ( ) to test the rela- tionship between the developed diagnostic test and clin- ical outcomes known to be affected by cph (mortality and respiratory morbidities). this is important for clin- ical corroboration of our new criteria and will provide the necessary preliminary data to enable sample size calculations for subsequent therapeutic trials, targeting improvement in clinical outcomes. ( ) to longitudinally figure in early stage of chronic pulmonary hypertension (cph), the disease is expected to be more functional, determined by sustained pulmonary vasoconstriction and relatively less by ‘fixed’ anatomical remodelling, hence, may be more amenable to treatments. the overall aim of this study is to develop new diagnostic criteria sensitive enough to identify extreme premature neonates with significant pulmonary vascular disease, who subsequently will be diagnosed with cph secondary to chronic neonatal lung disease. cnld, chronic neonatal lung disease; pvr, pulmonary vascular resistance o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n m a rch . d o w n lo a d e d fro m http://bmjopen.bmj.com/ thomas l, et al. bmj open ; :e . doi: . /bmjopen- - open access study the myocardial maturation pattern in extreme premature neonates and evaluate how it may be affected by occurrence of cph. this is important as preliminary work indicates that occurrence of cnld/cph may nega- tively impact cardiac maturational patterns in preterm infants. however, this observation needs further vali- dation and the relative impact of cnld versus cph on cardiac development is not known. ( ) to identify clin- ical risk factors associated with occurrence of cph in this cohort. ( ) to establish a large representative cohort of high- risk preterm neonates to pursue future long- term follow- up studies to understand their development and respiratory morbidities during childhood. methods and analysis design, setting and study population this study is a prospective, multisite, observational cohort study being conducted in five tertiary nicus across canada, the usa, uk and ireland. the study teams from the six participating sites have been trained in the stan- dardised procedure of enrollment, echocardiography and data collection. at its inception, the study was being conducted at two tertiary nicus in toronto, mount sinai hospital (start date november ) and sunnybrook health sciences centre (start date january ). subsequently, to help accelerate study completion, the following additional sites were recruited: liverpool women’s nhs foundation trust (start date november ), the rotunda hospital (start date november ), the university of iowa stead family children’s hospital (start date july ) and bc women’s hospital and health centre (start date september ). the study protocol is also currently under review at boston children’s hospital institutional review board. these additional sites were selected based on their knowledge in similar patient populations and study settings as the primary study sites and their similar echocardiography equipment, analysis software and presence of a formal in- house neonatal echocardiography programme led by an experienced neonatologist with formal training in neonatal echocardiography, all of whom are site collabo- rators in this study. infants are considered eligible for enrollment if they are born at ga < weeks and/or with a birth weight (bw) < g, and are cleared by the attending clinical team to approach for informed consent between and days of age. neonates with major congenital and/or genetic anomalies and congenital heart defects (with the excep- tion of patent ductus arteriosus (pda), patent foramen ovale (pfo), peripheral pulmonary artery stenosis or small (< mm diameter) ventricular septal defects) are not considered eligible for recruitment. recruitment and study procedures families of eligible neonates are approached for consent between and days postnatal age. all recruited neonates undergo three serial cardiopulmonary eval- uations: early diagnostic assessments (edas) at two predefined time points (study interventions, between and days of age (inclusive) and again between nd and rd weeks pma) and a standard diagnostic assess- ment (sda) at ≥ ± weeks pma (standard clinical care). each eda consists of collecting relevant clinical data and a focused echocardiogram to collect indices representa- tive of pvr and rv functions, while sda consists of an echocardiogram to allow assigning the final diagnosis of cph using the current clinical definition (figure ). each assessment is carried out by a cardiac sonographer or fellow trained in neonatal haemodynamics, with infants in a non- agitated state and is coordinated with any one of the routine handling times on that day. in the event of an intercurrent illness or procedure, such as sepsis, necro- tising enterocolitis or treatment for retinopathy, assess- ments are deferred until the neonate is deemed to have recovered by the attending neonatologist. blinded anal- ysis of all eda echocardiograms to measure parameters- under investigation (table ) is completed by a centralised trained senior research sonographer, at arm’s length from the study, who remains blinded to all clinical data. the echocardiogram done for sda will be analysed by sepa- rate personnel to categorise infant as cph or not based figure schematic representation of planned study interventions. each infant, after obtaining informed parental consent, will undergo two sequential early diagnostic assessments (edas) at predefined time points, followed by a standard diagnostic assessment (sda) to categorise study cohort as chronic pulmonary hypertension (cph) or no cph, as per the standard currently used clinical definition. blinded measurements will be performed for tricuspid annular plane systolic excursion (tapse, a marker of right ventricular function) and pulmonary artery acceleration time (paat, a marker of pulmonary vascular resistance) at both edas to calculate their early diagnostic characteristics (sensitivity, specificity and positive and negative likelihood ratios) to diagnose cph by comparing to eventual diagnosis made at sda. ga, gestational age; nicu, neonatal intensive care unit; pma, postmenstrual age. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n m a rch . d o w n lo a d e d fro m http://bmjopen.bmj.com/ thomas l, et al. bmj open ; :e . doi: . /bmjopen- - open access on the currently used definition, as noted above. if study participants have clinically indicated scans after weeks pma, the data regarding outcome of cph are collected. clinical data, including baseline demographics, respira- tory illness severity during first weeks of age and expo- sure to relevant but unrelated pre- existing morbidities and treatments are also collected in addition to data regarding common neonatal outcomes at discharge and from the infants’ - month follow- up neurodevelop- mental clinical assessment. at the end of the recruitment, all deidentified data will be sent for analysis to a statisti- cian blinded to all other aspects of this study. rationale for primary parameter selection paat and tapse are simple, reliable, bedside tests, which, in older patients, have been validated – and correlate with prognosis in cph. – the normal data for neonates are well established and these markers are commonly used in nicus, including study nicus, for longitudinal monitoring; however, no specific diagnostic cut- offs currently exist. in previous work, we have estab- lished the reproducibility and normal values for various markers of ventricular function and pvr in neonates; intraclass correlation coefficient for paat and tapse was . and . , respectively. we then confirmed the ability table echocardiographic variables measured on study echocardiograms index name description measurement study specific pulmonary artery acceleration time (paat) (primary index) a marker of pulmonary vascular resistance (inversely related). interval between the onset of ejection and peak flow velocity measured from a pulse wave doppler tracing obtained by placing a mm sample volume in the middle of the main pulmonary artery, at the tip of pulmonary valve leaflets. in addition, the ratio of total right ventricular (rv) ejection time to paat is also measured as a heart rate independent measure of pvr (directly related). tricuspid annular plane systolic excursion (tapse) (primary index) a marker of rv longitudinal systolic function. measure of the downward displacement of tricuspid annulus during contraction. from an apical four- chamber view by placing m- mode cursor through the tricuspid valve annulus. two- dimensional peak global end- systolic longitudinal strain (pls)—right ventricle (secondary index) a measure of myocardial deformation expressed as percentage change in length in systole from the baseline at end diastole. performed offline using software, which uses frame to frame tracking of the unique ultrasound speckles within the myocardial wall. longitudinal strain will be calculated for rv lateral and inferior wall from rv apical four chamber and three chamber views, respectively. fractional area change (fac- c) % (secondary index) a surrogate to ejection fraction and indicative of overall ‘pump function’. manual tracing of the rv endocardial borders at respective phases of the cardiac cycle. from an apical four chamber view the end systolic area (esa) and end diastolic area (eda) are measured. fac- c (%) =((eda- esa)/ eda)x tissue doppler imaging (tdi) (secondary index) allows measurement of velocities directly from the myocardium to assess longitudinal systolic and diastolic rv function. measure the peak systolic and diastolic velocities, and duration of various phases of a cardiac cycle. measurements obtained at the rv basal segment. rv peak systolic pressure (rvsp) (secondary index) a direct measure of pulmonary artery systolic pressure. calculated by measuring peak velocity (v) of tricuspid regurgitation using the modified bernoulli equation. rvsp= v +right atrial pressure. calculated whenever feasible. right atrial pressure is predefined as mm hg in all cases. assessment of shunts (secondary index) patent ductus arteriosus and patent foramen ovale/ atrial septal defect. visualised on colour doppler assessment. shunt diameter in millimetre and flow direction documented. flow patterns in main and branch pulmonary arteries (secondary index) presence of notching is considered a marker of high downstream resistance visualised on pulse wave doppler presence or absence of midsystolic notching in the doppler profile. pvr, pulmonary vascular resistance. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n m a rch . d o w n lo a d e d fro m http://bmjopen.bmj.com/ thomas l, et al. bmj open ; :e . doi: . /bmjopen- - open access of paat to detect reduction in pvr in healthy neonates during first day after birth. among rv markers, tapse was found sensitive to these changes, while no change was seen on septal flattening. study definitions cph: infants will be classified as having significant cph if they meet any of the following features at sda: . right ventricular systolic pressure (rvsp) ≥ . systolic blood pressure. . bidirectional (right- left in systole) or a right to left shunt across a pda. . a flat interventricular septal motion in end- systole. infants who meet the above criteria on edas will only be classified as cph if the findings persist until sda or if severity increases enough for clinicians to initiate cph treatment. cnld: infants are assessed at weeks pma to clas- sify the severity of cnld based on standard well- defined objective clinical criteria: . mild cnld: supplemental oxygen requirement for ≥ days but off all respiratory support at weeks pma or discharge, whichever is earlier. . moderate cnld: supplemental oxygen requirement for ≥ days and at weeks pma but fractional in- spired oxygen concentration (fio ) < . . . severe cnld: supplemental oxygen requirement for ≥ days and at weeks pma with fractional inspired oxygen concentration (fio ) ≥ . or dependent on any positive pressure ventilatory support. infants who are discharged from study nicu before weeks pma will be classified based on respiratory status at discharge. outcome measures primary clinical outcome the primary clinical outcome is final diagnosis of cph, made based on above definition at sda. our intention is to develop a risk score using a combination of echocar- diographic markers, at each eda, predicting the primary clinical outcome. a cut- off point of the risk score will then be determined using youden’s method to define a new diagnostic test at each eda. the sensitivity and specificity of the new diagnostic test for early prediction of cph will be estimated. secondary clinical outcomes key prematurity- related clinical outcomes including composite of predischarge mortality or cnld, cnld, total duration of need for respiratory support, length of hospital stay, discharge on home oxygen and neurodevel- opmental status at – months corrected age. these secondary clinical outcomes are those which are well known to be worsen in relation to the diagnosis of cph in preterm neonates. these will be used to corroborate and test the clinical validity of our newly identified early cph diagnostic test, as we anticipate that the infants identified as cph with the new diagnostic test developed by this study would demonstrate more adverse clinical outcomes than those without cph. statistical methods sample size calculation: the incidence of cph in the study population is esti- mated to at least %. the mean and sd or sensitivity and specificity of echocardiographic markers during the eda time points are not known, making specific sample size calculations non- feasible. presuming a disease prevalence of %, to estimate new diagnostic test with a sensitivity of % and an absolute precision of . , a sample size of approximate babies will be required. the actual incidence of cph at sda will be evaluated at the halfway stage in the recruited cohort, to confirm representative- ness of the estimated incidence. the sample size calcu- lation may be revised if the incidence in the recruited cohort is found to be significantly higher or lower than the estimate. analysis ► the study population will be summarised descrip- tively. the baseline infant characteristics (including bw z- score), clinical measurements and echocardio- graphic indices (ie, paat and tapse), as well as the presence of shunts such as atrial septal defect, at each eda will be compared between two groups: infants with/without cph using the χ test for categorical variables and the student t- test or wilcoxon rank- sum test for continuous variables, as appropriate. changes over time in echocardiographic indices will also be compared using two- way repeat measures analysis of variance. ► we will conduct a multiple logistic regression at each eda using backward variable selection procedure with variable stay criterion of p< . . all echocardio- graphic indices associated with cph identified in the bivariate analysis (p< . ) or based on clinical exper- tise, including the presence of shunts if relevant, will be included in the full model. denote the final model as logitpr ( cph = yes ) = a + a x + a x + . . . + akxk … ( ) where x , …, xk are the echocardiographic indexes that remain. a risk score (rscore) will then be defined as rscore= a + a x + a x + . . . + akxk . a new diagnostic test can be defined as dtest=(rscore <u ), where dtest= predicts cph and the cut- off u is determined based on the youdon method such that the sum of the sensitivity and specificity of dtest is maximised. the diagnostic characteristics such as sensitivity, the specificity, positive and negative predic- tive values of the new diagnostic test will be reported for each eda. we will also analyse each individual echocardiographic index under investigation (ie, paat and tapse) and report their diagnostic charac- teristics using similar methods. ► in addition, the association between the neonatal outcomes and the newly developed diagnostic test o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n m a rch . d o w n lo a d e d fro m http://bmjopen.bmj.com/ thomas l, et al. bmj open ; :e . doi: . /bmjopen- - open access will be assessed, by comparing the outcomes between infants with/without cph defined based on the new diagnostic test using χ test. multivariable regression analysis will also be performed to investigate the asso- ciation between the new diagnostic test and subse- quent occurrence of composite outcome of cnld or death, adjusted for potential confounders identified in the univariate analysis. similarly, the association between the outcomes and the key early- diagnostic echocardiographic parameter(s) identified in the analyses mentioned above will be examined. ► further, since male sex has consistently been shown to be associated with worse neonatal outcomes, we will also include an interaction term between sex and the combination of echocardiographic indices in the model ( ). a significant in the interaction indi- cates that the performance of the diagnostic test may vary between males and females. if this is the case, subgroup analysis stratified by sex will be conducted to develop a sex- specific diagnostic test for cph. ► to identify risk factors for diagnosis of early cph, we will also compare demographics and baseline vari- ables, including pre- existing morbidities, between patients who are diagnosed with cph using the newly developed diagnostic test and those who do not meet the criteria of early cph diagnosis. multivariable regression analysis will also be performed to investi- gate the association between cph diagnosis and base- line variables. ► intervariability for the diagnosis of cph as per currently practiced qualitative criteria will be reported using cohen’s kappa statistic in random deidenti- fied scans ( each with and without cph), while reli- ability for tapse and paat will reported as intraclass correlation coefficient, bland altman analysis and coefficient of variability. data management maternal and neonatal data are collected in a standardised format (data collection forms approved by the research ethics boards at all participating sites) and stored in a password protected electronic file on a secure server. for the purpose of confidentiality, the data are entered using the participant’s study number and all identifying infor- mation will be removed prior to data analysis. a separate log is maintained linking the study number to the infants’ identifying information. only the study coordinator at each site has access to this file. study echocardiograms are stored on a secure hospital network and measure- ments are performed offline using ge echopac soft- ware version (ge vingmed ultrasound as, horten, norway). some recruited infants may not have an sda due to discharge from the study nicus prior to reaching eligibility age. infants discharged in room air to commu- nity hospitals with no sda results will be presumed to have no cph, as cph is known to be rare in infants off respiratory support < weeks pma. however, infants discharged on respiratory support without sda will be excluded and additional participants will be recruited to ensure that all included infants have at least the first eda (third week of life) and outcome of death or cph. patient and public involvement a range of stakeholders and partners have been actively involved in the design and conduct of this observational study including healthcare providers in participating nicus, paediatric cardiologists, members of the cana- dian neonatal network (cnn), respiratory therapists, neonatal nurses and parent groups. their input was sought during protocol design regarding the selection of echocardiographic markers most suitable for clinical translation and their potential for early detection of cph. the burden of serial echocardiographic assessments on preterm neonates was also carefully assessed in consulta- tion with these groups. all stakeholders and partners are updated at regular intervals about the study’s progress in order to identify facilitators and barriers to study conduct and knowledge translation. following study completion, we plan to discuss future directions, including a multi- centre clinical trial based on study findings, in consul- tation with neonatologists, paediatricians, healthcare practitioners and parent- led community groups. ethics and dissemination this study was approved for the coordinating site (mount sinai hospital) by the research ethics board at mount sinai hospital on june (reb id# - - e). documented approval was obtained from the research ethics board/institutional review board for all partic- ipating centres prior to participant enrollment at that centre. parents, as the legal substitute decision- maker, have the opportunity to read the approved consent form and ask questions as needed. once all questions are answered and parents confirm their understanding of study procedures, parents are invited to provide voluntary written informed consent on behalf of their child. all coauthors will facilitate knowledge dissemina- tion among key stakeholders, as listed above, providing the network necessary to support future multicentre randomised clinical trials. as done previously, we will work with national (cnn, canadian paediatric society, children’s healthcare canada) and provincial (the provincial council of maternal and child health) child health advocacy organisations, using our research find- ings to drive evidence- informed changes in clinical prac- tice. the results of this study will also be disseminated to participating families in a lay format and communicated to a wider scientific and healthcare community through end of study meetings, presentations at national and international paediatric and critical care conferences and high- impact peer- reviewed publications. informa- tion regarding the use of specific echocardiographic diagnostic criteria to identify extreme preterm neonates with early significant pulmonary vascular disease will be o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / b m j o p e n : first p u b lish e d a s . /b m jo p e n - - o n m a rch . d o w n lo a d e d fro m http://bmjopen.bmj.com/ thomas l, et al. bmj open ; :e . doi: . /bmjopen- - open access shared with clinicians across various disciplines including neonatologists, cardiologists, trainees, developmental paediatricians and other community- based healthcare practitioners. study results are also intended to be shared with software companies of echocardiography technology in order to inform the development of equipment specific to the needs of preterm infants. finally, these findings will be communicated to policy- makers to identify priority areas of care, to reduce burden on families and to reduce costs to the healthcare system by organising tailored follow- up services for these neonates. author affiliations paediatrics, sinai health system, toronto, ontario, canada newborn and developmental paediatrics, sunnybrook health sciences centre, toronto, ontario, canada respiratory therapy, sinai health system, toronto, ontario, canada paediatrics, university of toronto, toronto, ontario, canada clinic of neonatology, lausanne university hospital, lausanne, switzerland neonatology, hospital infantil de mexico federico gomez, mexico city, mexico division of cardiology, department of paediatrics, the hospital for sick children, toronto, ontario, canada division of neonatology, department of paediatrics, university of ottawa, ottawa, ontario, canada micare research centre, sinai health system, toronto, ontario, canada neonatology, liverpool women's hospital nhs foundation trust, liverpool, uk neonatology, the university of british columbia, vancouver, ontario, canada boston children's hospital department of pediatrics, boston, massachusetts, usa pediatrics, harvard medical school, boston, massachusetts, usa neonatology, rotunda hospital, dublin, ireland pediatrics, the university of iowa stead family children's hospital, iowa city, iowa, usa acknowledgements we would like to thank all parents of infants who participated in this study and the department of paediatrics at mount sinai hospital, toronto for their support. contributors lt is the lead coordinator overseeing all study sites and is responsible for all patient recruitment and acquisition of data in toronto along with mb. pd, ak, sj, fz and di- r are involved in the completion of all study echocardiographic assessments in toronto. as is responsible for patient recruitment and the completion of study echocardiographic assessments in dublin. aj, dew, en, jt, mc, pl, afe- k, reg and pjm are the principal investigators at their respective sites and were all extensively involved in the study concept, design and completion of echocardiographic assessments. pss, xyy, lm and rpj are involved in the development of the analysis plan. all the authors have extensively reviewed the protocol/final manuscript. all authors read and approved the final manuscript. funding this work is supported by a four- year sickkids foundation- canadian institute of child health (skf- cihr) new investigator research grant (funding reference number: ) and conducted as a part of aj’s 'improving management of pulmonary hypertension and right heart function in neonates (imprint)' research programme, supported by funding from the heart & stroke foundation of canada (hsfc).the granting agencies had no role in the writing of the manuscript or in the decision to submit for publication. competing interests none declared. patient and public involvement patients and/or the public were involved in the design, or conduct, or reporting or dissemination plans of this research. refer to the methods section for further details. patient consent for publication not required. provenance and peer review not commissioned; externally peer reviewed. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by- nc . ) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non- commercial. see: http:// creativecommons. org/ licenses/ by- nc/ . /. orcid id amish jain http:// orcid. org/ - - - references ambalavanan n, mourani p. pulmonary hypertension in bronchopulmonary dysplasia. birth defects res a clin mol teratol ; : – . berger rmf, beghetti m, humpl t, et al. clinical features of paediatric pulmonary hypertension: a registry study. lancet ; : – . khemani e, mcelhinney db, rhein l, et al. pulmonary artery hypertension in formerly premature infants with bronchopulmonary dysplasia: clinical features and outcomes in the surfactant era. pediatrics ; : – . mourani pm, abman sh. pulmonary vascular disease in bronchopulmonary dysplasia: pulmonary hypertension and beyond. curr opin pediatr ; : – . abman sh, hansmann g, archer sl, et al. pediatric pulmonary 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http://bmjopen.bmj.com/ 	multicentre prospective observational study exploring the predictive value of functional echocardiographic indices for early identification of preterm neonates at risk of developing chronic pulmonary hypertension secondary to chronic neonatal lung disease 	abstract 	introduction 	background 	importance of early identification of cases 	need for quantitative echocardiographic diagnostic test for early cph diagnosis in premature neonates 	research hypothesis and study objectives 	methods and analysis 	design, setting and study population 	recruitment and study procedures 	rationale for primary parameter selection 	study definitions 	outcome measures 	primary clinical outcome 	secondary clinical outcomes 	statistical methods 	sample size calculation: 	analysis 	data management 	patient and public involvement 	ethics and dissemination 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - 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title). http://europepmc.org/abstract/med/ original article polymorphisms in the glucokinase-associated, dual-specificity phosphatase (dusp ) gene under chromosome q linkage peak are associated with type diabetes swapan kumar das, , winston s. chu, , terri c. hale, , xiaoqin wang, , rebekah l. craig, , hua wang, , alan r. shuldiner, philippe froguel, panos deloukas, mark i. mccarthy, eleftheria zeggini, sandra j. hasstedt, and steven c. elbein , linkage of type diabetes to chromosome q -q is well replicated across populations. in an initial -kb marker map ( markers) across the linked region, one of the two strongest associations observed in utah caucasians was at marker rs (p < . in pools, p < . in individuals). based on this association, we typed additional markers and screened for sequence variation in the nearby dusp gene. the strongest associations mapped to a highly conserved nongenic sequence just telomeric to rs and extended kb telomeric through the dusp gene and into the � end of the adjacent atf gene. no coding variant could explain the association in the dusp gene. an extended haplotype encompassing mark- ers from � , to � , bp relative to the atg start was more common in caucasian case ( . ) than control subjects ( . , p � . ) and was uniquely tagged by a -bp allele at either of two simple tandem repeat vari- ants or by the t allele at marker � , . markers � , and � , were nominally associated with type diabetes in african-american subjects (p < . ), but with different alleles. marker rs was strongly associated with postchallenge insulin levels among family members (p � . ), but sequence variation in this region was not associated with type diabetes in three other populations of european ancestry. our data suggest that sequences in or upstream of dusp may contribute to type diabetes susceptibility, but the lack of replication suggests a small effect size. diabetes : – , a genetic etiology underlying the high preva- lence of type diabetes is widely accepted. nearly genome scans and countless associ- ation studies have been conducted in multiple populations ( ), which in aggregate and in recent meta- analyses ( ) suggest that the genetic susceptibility to type diabetes will involve many susceptibility loci. genes identified to date have had small to moderate effects and have been difficult to replicate, and risk alleles have generally been marked by noncoding variants ( – ). re- ported associations likely account for only a small portion of the total type diabetes genetic susceptibility. linkage of type diabetes to chromosome q -q was described initially by our laboratory ( ) in northern euro- pean caucasians and in pima indians ( ), and it was subsequently replicated in british ( ), french ( ), and amish caucasians ( , ); in chinese han ( , ); and in supportive data in african americans from arkansas (s.c.e., unpublished observations). this well-replicated region of type diabetes linkage is characterized by an extraordinary gene density, including a plethora of strong candidate genes. we recently reported that the region encompasses at least two and possibly three linkage peaks ( ), and we reported previously that two other genes in this region were associated with type diabetes ( , ). both associations were observed in other populations ( – ), and they support a model of multiple susceptibil- ity loci accounting for the chromosome linkage signal. in an initial collaborative effort to fine-map type diabetes in caucasians, we typed single nucleotide polymorphisms (snps) over a -mb region (mean snp/ kb) that encompassed both major linkage peaks, using massarray maldi-tof mass spectrometry (seque- nom, san diego, ca) ( , , ) in pooled dna samples of case and control subjects of northern european ancestry ( ). we identified two prominent associations, one in intron of the calsequestrin gene ( ) and a second at marker rs (p � . ), which was verified by individual typing of the case and control subjects (p � from the division of endocrinology and metabolism, department of medi- cine, university of arkansas for medical sciences college of medicine, little rock, arkansas; the endocrine division, medicine and research services, central arkansas veterans healthcare system, little rock, arkansas; the division of endocrinology, diabetes and nutrition, department of medicine, university of maryland school of medicine, baltimore, maryland; the institut de biologie, institut pasteur de lille, lille, france; the wellcome trust sanger institute, oxford, u.k.; the oxford centre for diabetes, endocrinology and metabolism, university of oxford, oxford, u.k.; and the department of human genetics, university of utah health sciences center school of medicine, salt lake city, utah. address correspondence and reprint requests to steven c. elbein, md, professor of medicine, university of arkansas for medical sciences, endocri- nology j- /lr, john l. mcclellan memorial veterans hospital, w. th st., little rock, ar . e-mail: elbeinstevenc@uams.edu. received for publication october and accepted in revised form may . s.k.d. and w.s.c. contributed equally to the work reported in the manu- script. additional information can be found in an online appendix at http:// diabetes.diabetesjournals.org. airg, acute insulin response to glucose; cng, conserved nongenic region; fsigt, frequently sampled intravenous glucose tolerance test; snp, single nucleotide polymorphism; str, simple tandem repeat. doi: . /db - © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , september . ). based on these initial data, we evaluated additional snps from the public database across an -kb region surrounding the initial observation, and we narrowed the association to a -kb region that included dual-specificity phosphatase gene (dusp ) and the � end of the endoplasmic reticulum stress factor atf . we report studies to identify the associated variants in this region in caucasians and extend the analysis to african-american case and control subjects. we report that noncoding variants in and near the dusp gene, which has been shown to associate with and activate glucokinase in the liver ( ), are associated with type diabetes in both utah caucasian and arkansas african-american populations, although a subset of the variants failed to replicate in other european populations. research design and methods dusp gene variation was detected in unrelated caucasian individuals with type diabetes from utah families linked to the q region and nondiabetic (control) family members, and it was detected in diabetic members of african-american families used in the chromosome linkage studies and african-american control subjects. association studies were conducted in unrelated caucasian case subjects and unrelated caucasian control subjects, as described previously ( , ). to reduce geno- typing costs, some markers for initial genotyping were typed in pooled dna samples, as described in detail previously ( , ). african-american samples were ascertained in arkansas and included unrelated nondiabetic individ- uals who had no family history of type diabetes and type diabetic subjects who had at least diabetic first-degree relative. pooled dna was constructed from control individuals, case subjects with type diabetes and no nephropathy, and individuals with type diabetes and diabetic nephropathy ( ). for markers more recently tested, we first typed unrelated nondiabetic caucasian and nondiabetic african-american sub- jects for linkage disequilibrium before selecting markers for typing in the full population. family-based association studies were conducted on mem- bers of utah families used to define the original linkage, of which individuals were considered affected ( , ). we tested the physiological impact of associated markers rs and simple tandem repeat (str) marker � in individuals from the utah families who had undergone tolbutamide-modified frequently sampled intravenous glucose tolerance tests (fsigts) ( ) and an additional caucasian individuals from arkansas who had undergone insulin-modified (n � ) or tolbutamide-modified (n � ) fsigts. subjects ascertained in utah provided written informed consent under a protocol approved by the university of utah institutional review board. subjects studied in arkansas provided written informed consent under protocols approved by the university of arkansas for medical sciences human research advisory committee. dusp screening. we screened a total of , bp from each of the six exons, each intron, kb of the � flanking region, bp of the � flanking region, and the , -bp conserved nongenic region (cng) (fig. ) in caucasian and african-american individuals, using denaturing high-perfor- mance liquid chromatography (transgenomic, omaha, ne) with bidirectional sequence analysis of fragments showing altered chromatographic patterns. genotyping of sequence variants. snps were typed, using pyrosequencing on a psq- (biotage, uppsala, sweden) ( ). snps at a distance from the initial observation were typed first in pooled caucasian samples, and differ- ences between case and control frequencies � % were confirmed in individual samples. three snps not amenable to typing by pyrosequencing were typed in individuals by oligonucleotide ligation ( ). str and insertion deletion markers were detected on li-cor gr sequencers and scored, using gene imager version . software (scanalytic, rockville, md). for the noncoding regions, particularly in the dusp � flanking region, which was not highly conserved, we first determined linkage disequilibrium and allele frequencies in control samples. snps were chosen for typing in the full caucasian and african-american populations based on a minor allele frequency over % and selection of tagged snps, using the parameter r � . and the programs ldselect ( ) and tagger ( ) (supplemental table s, which is detailed in the online appendix [available at http://diabetes.diabetesjournals.org]). all snps included in this study were in hardy-weinberg equilibrium. quality control steps included reliable automated calls by pyrosequencing software and inclusion of at least duplicate samples for the full set. no assays failed the duplicate checks, and all variants typed in families showed mendelian inheritance. all members of families ascertained in utah were typed for five dusp variants: rs (� bp), the � bp str (rs ), rs (� bp), rs (� bp), and rs (� bp). african- american subjects were typed primarily for snps shown to be associated in caucasian individuals, based on linkage disequilibrium determined in unrelated african-american control samples. additionally, we have typed � ancestrally informed snps as well as � total snps in candidate genes in the african-american population to detect admixture ( ). these results did not support evidence for spurious association based on admixture among african- american subjects. cng. we searched the -kb region � to the dusp atg start for potential cngs that might represent regulatory sequences ( ), using the may build (build ) and the university of california santa cruz conservation tracks (genome.ucsc.org) for human (hg ), chimp (pantro ), mouse (mm ), rat (rn ), and dog (canfam ). no conservation was observed in nonmamma- lian species (supplemental fig. s). multiple alignments were assigned a conservation score using phastcons ( ), which is based on hidden markov models, and multipipmaker ( ). analysis of mrna expression in transformed lymphocytes. total rna was isolated from epstein-barr virus–transformed lymphocytes, and allele- specific expression of snp � was quantified from heterozygous individuals, as described previously ( , ). dusp mrna levels were measured from transformed lymphocyte rna by quantitative real-time pcr (rt-pcr), using sybr green, and normalized to s rna. typing of international type diabetes chromosome q consortium samples. we tested for replication of snps in the dusp region in the caucasian subset of the international type diabetes chromosome q consortium samples, including of the samples included in the utah studies reported here ( case and control subjects), samples from the amish family diabetes study ( case and control subjects) ( ), samples from a french diabetes study ( case and control subjects) ( ), and samples from the u.k. ( case and control subjects) ( ). genotyping was performed as part of a high-throughput, large-scale dense mapping effort on chromosome q of three bundles of , -plex assays, using the illumina golden gate assay ( ) performed at the wellcome trust sanger institute, that included snps noted to be associated in individual populations. the international type diabetes chromosome q consortium map included snps covering the region from , , to , , bp on chromo- some q (the region encompassing dusp ). snps rs (� in the utah study) and rs (not typed in the utah study) failed quality control steps in this multiplex assay and were excluded. quality control steps included blinded duplicates, analysis of plate-to-plate variation, and adher- ence to hardy-weinberg equilibrium. a subset of arkansas african-american samples was represented in the international type diabetes chromosome q consortium typing, but we report only the non-consortium typing of the full african-american case-control set. statistical methods. allele frequencies were compared, using the � or fisher’s exact tests or, in the case of the multiallelic str polymorphisms, the clump program ( ). we considered p � . to be significant, and we report uncorrected p values. secondary analyses of genotypic association were computed, using pearson’s � test and logit regression models under additive, dominant, and recessive models. pairwise linkage disequilibrium was esti- mated from the combined case and control population data, using the expectation maximization algorithm, from which d� and r were calculated, using haploview . ( ). extended haplotypes were estimated, using phase . . or haploview . programs ( , ). insulin sensitivity index (si) and acute insulin response to glucose (airg) were calculated from fsigt data, using the minmod program ( ) as described previously ( ). genotype effects on insulin secretion and insulin sensitivity were tested using general linear models with age, sex, bmi, glucose tolerance status, and family membership for utah families as covariates and factors ( ). for unrelated individuals from arkansas, we included the protocol (tolbutamide or insulin) as a covariate ( ). family-based association was calculated by a maximum likelihood implementation of the transmission disequilibrium test computed in the pedigree analysis package ( , ) and conducted on the full set of linkage families, including families not showing linkage to chromosome . family- based quantitative trait associations were tested, using the pedigree analysis package ( ), by comparing the maximized likelihood estimation of the three genotypic means to that if the three means were equal. a pearson’s � test statistic with degrees of freedom (df) was computed as twice the natural logarithm of the ratio of the two likelihoods. familiality was accounted for through a polygenic component, and sex, age, and bmi effects were estimated. for both insulin and glucose measures, only nondiabetic individuals were examined. insulin and glucose values were also examined as multivariate values, using all five oral glucose tolerance test time points simultaneously ( dusp is associated with type diabetes diabetes, vol. , september df) while allowing for correlations between them. this method included individuals for whom some time points were missing. transcription factor binding sites were predicted using the tfsearch version . program, which is based on the transfac database ( ). for analysis of international type diabetes chromosome q consortium data, between-group differences in allele frequency were evaluated on a population-specific basis, using standard contingency table methods, and exact p values were calculated using stata se version (stata, college station, tx). single-point data from the case-control samples were combined, using the mantel-haenszel fixed-effects method (stata se version ), and com- bined odds ratios were generated under dominant and recessive models. analyses in the amish took into account correlations among related individuals. results analysis of dups region in caucasians. based on the initial observation of an association at snp rs (location , , bp), we selected snps for -kb spacing extending from , , to , , bp and identified snps that could be typed successfully (mean interval . kb), including snps extending . kb centromeric and snps telomeric to snp rs (supplemental table s). the associated snps clustered primarily in the -kb region telomeric to rs ( , , to , , bp; may build ) and encompassing the dusp gene, a widely expressed dual- specificity phosphatase with homology to the yeast gene yvh ( ) that has been shown to regulate glucokinase ( ). the associated snps spanned a region of high sequence conservation among all mammalian species (cng) between , , and , , bp (supple- mental fig. s), the � boundary of which was , bp upstream from the dusp atg start (fig. ). we screened the full region from � kb upstream (a highly conserved, nongenic region) to kb downstream of the dusp atg start site (fig. ). we identified snps, including snps previously typed (supplemental table s), and, based on the observed associations in cauca- sians (table and fig. ), extended the screening to fig. . dusp region map in caucasians. the cng is shown as a shaded box, exons – as darkened blocks, and untranslated regions as lighter shaded boxes. the block structure based on cis of d� is outlined as triangles, and the shaded blocks represent linkage disequilibrium measured as r between pairs of variants. for purposes of this figure, both str polymorphisms (� and � ) were dichotomized to versus not alleles, as described in the text. bars above the figure are based on negative log of p values for tests of allelic association. s.k. das and associates diabetes, vol. , september african-american individuals. among caucasians, vari- ants were associated with type diabetes (p � . ), clustered primarily between � and � bp relative to the dusp atg start site ( / snps) and encom- passing the dusp gene and � flanking region (fig. ). an additional six variants showed a trend to an association with type diabetes (p � . ), of which three again fell in this interval. based on these results, we focused the study on the region from � to � kb. the data are summa- rized in table and graphically in fig. , and raw genotype counts are shown in supplemental table s. based on the confidence interval (ci) block definition ( ), the entire dusp gene, the � promoter, part of the cng, the � flanking region to � bp, and most associated variants were encompassed in one block (block ) (fig. and supplemental fig. s). most associated snps that fell outside of this interval were nonetheless in linkage dis- equilibrium with block snps by r (fig. ). however, rs (� ), which was the original observation, fell outside of the associated block and was not in strong linkage disequilibrium with the most significantly associ- ated variants. the associated region included two str variants at � and � . we tested the association in caucasians using the clump program ( ) (supplemental tables s and s); for both strs, the allele was significantly overrepresented in case subjects relative to control sub- jects. using the t test, uncorrected p values were . and . for strs � and � , respectively, with p values based on monte-carlo simulation of . and . , respectively. to facilitate further analyses, we di- chotomized both strs to versus x, where x was any other size. in post hoc analyses, we tested dominant and recessive models for all snps and for dichotomized strs. table summary of snps typed in caucasian individual samples for dusp region location name alleles case frequency control frequency p allelic p additive p recessive p dominant � rs g/c . . . . . . � rs a/g . . . . . . � rs c/g . . . . . . � rs g/a . . . . . . � rs c/g . . . . . . � rs c/t . . . . . . � rs t/c . . . . . . � rs a/g . . . . . . � rs t/c . . . . . . � rs g/a . . . . . . � novel bp del/ins . . . . . . � rs g/a . . . nd nd . � rs c/t . . . nd . . � rs str (x/ ) . . . . . . � rs t/c . . . . . . � rs c/t . . . . . . � rs c/g . . . . . . � rs g/a . . . . . . � rs c/t . . . . . . � rs c/t . . . . . . � rs t/a . . . nd nd . � novel ct ins/del . . . . . . � rs c/g . . . . . . � rs g/t . . . . . . � rs a/c . . . . . . � rs g/a . . . nd nd . novel c/g . . . . . . rs a/g . . . . . . rs g/a . . . . . . rs g/a . . . nd nd . rs g/t . . . . . . rs t/c . . . . . . rs c/g . . . nd nd . rs g/a . . . . . . rs str (x/ ) . . . . . . rs c/g . . . . nd . rs c/t . . . . . . rs g/t . . . . . . rs g/c . . . . . . rs g/a . . . . . . snps that were in complete linkage disequilibrium (r � ) were typed only in control samples and are not included. likewise, snps with low minor allele frequencies (� %) were generally not tested. raw counts are available in supplemental table , which is detailed in the online appendix. p values are shown for allelic tests, additive genotypic model (p additive), rare allele recessive model (p recessive), and rare allele dominant model (p dominant). nd, not determined where rare allele was uncommon and hence homozygotes were not observed. dusp is associated with type diabetes diabetes, vol. , september the associated sequence variants were most consistent with a dominant or additive model for the minor allele (table and supplemental table s). haplotype analysis in caucasians. we first analyzed the haplotypes for each of the four blocks defined by d� cis ( ) (fig. and supplemental fig. s), using the phase . program to permit analysis without recoding of the str alleles ( ). in block , the three-marker haplotype � , � , and � was associated with type diabetes (p � . for global; p � . for combination atg present at . in case subjects and . in control subjects). block (supplemental fig. s) extended from � to � and encompassed variants, of which showed nominal association (p � . ). however, visual inspection suggested an area of linkage disequilibrium extending from snp � to snp � (supplemental fig. s) and encompassing variants. hence, we re- peated the haplotype analysis with all variants with minor allele frequency � . . a single haplotype was overrepre- sented in case subjects ( . ) versus control subjects ( . ), was the most common single haplotype ( vs. % for the next most common), and was uniquely tagged by the -bp allele at either the � or � str or by the t allele at snp , bp (rs ) (supplemental table s). when we again dichotomized both strs ( vs. x, as noted above), only eight haplotypes were ob- served. the single-risk haplotype differed from the other two common (frequency over %) haplotypes at every position (table ). similar conclusions were reached when haplotypes were constructed from a five-variant moving window (supplemental table s). the most significant ad hoc snp combination, which included snps from three blocks but excluded the strs, was � , � , � , and � (global p � . ). association and haplotype analysis in african amer- icans. to test for replication and to narrow the associated region, we examined snps (� to � ) in african-american individuals (supplemental figs. s and s). we selected snps and the � str for minor allele frequencies � % and based on linkage disequilib- rium (r � . ) to test in the full sample (table ). variants � and � were nominally associated with type diabetes, but in both case subjects, the allele frequencies and associated alleles differed from cauca- sians (fig. and supplemental table s). we observed alleles at � (from to bp), which showed nominal association with type diabetes: clump analysis ( ); t and t tests p � . and p � . , respectively; empirical p values of . and . , respectively, with , replicates. at snp � (table ), the minor t allele was -fold less common among african americans ( . in type diabetic case subjects) than among cauca- sian case subjects; the major c allele was overrepresented in type diabetes. only of haplotypes for the block including dusp showed a nominal association (p � . ) with type diabetes (haplotypes and ) (supple- mental table ). metabolic and family analyses of dusp variants in caucasians. we tested the � str and snp � in members of utah families ( ). as observed in the larger population, the � -bp allele was found only in individuals with the � t allele; thus, we classified the sample as -t homozygotes; -t/x-c heterozy- gotes, where x was any other str allele; and x/c homozy- gotes. marginal means did not differ by genotype for insulin sensitivity (si), airg, or disposition index (si � airg). in a second population of unrelated glucose- tolerant caucasian individuals from arkansas ( homozy- gous for the low-risk haplotype, heterozygous, and homozygous for the high-risk haplotype), we likewise found no evidence for an effect of dusp haplotype on si, airg, or disposition index. we used simwalk ( ) to estimate haplotypes for markers � , � , � , � , and � in members of the previously described utah families ( , ). no variant or haplotype showed excess transmission from parents to affected offspring ( , ), even when restricted to the specific families that generated the linkage signal in this region ( , ). furthermore, the � -bp allele did not explain the linkage signal, using the gist (geno- type-ibd sharing test) ( ). in contrast, the � snp was significantly associated with fasting (p � . ), -min (p � . ), -min (p � . ), and -min (p � . ) insulin, and particularly the multivariate measure combining all five postglucose challenge insulin levels (p � . ). snps � and � showed a similar albeit lesser association with the multivariate insulin mea- sure (p � . ). no snp was associated with fasting or postchallenge glucose levels, however. transformed lymphocytes were available from most family members. among individuals heterozygous for transcribed snp � , equal amounts of cdna were observed from both alleles, and total dusp message levels did not differ between t/t homozygotes and c/c homozygotes (dusp –to– s ratios . 	 . t/t table dusp region haplotypes in caucasians haplotype case frequency control frequency � p tccgcc cgagagttg cgg . . . . ctgatt gtccgagca ttc . . . . tccgct cgacaggtg cgg . . . . ctgatt gtccgagca ctc . . . . tccgcc cgacagttg cgg . . . . tccgct ctacgggca ctc . . . . ctcatt gtacgggcg ctc . . . . tccgcc cgacagttg cgg . . . . table shows the haplotypes from the str at � through snp � , with only the common (minor allele frequency � . ) variants included. the two str variants (in bold) are encoded as bp � , and all other alleles � (� and � ). for the indel variant at � , deletion is encoded as and insertion as . the table includes variants: � , � , � , � , � , � , � , � , � , � , � , � , � , � , � , � , � , � , � , � , � , and � . s.k. das and associates diabetes, vol. , september vs. . 	 . c/c). replication in other chromosome q–linked popula- tions. to determine whether dusp snps were associ- ated with type diabetes in other populations, we examined three additional populations of european ances- try with linkage to the q -q region: amish ( case and control subjects), french ( case and control subjects), and british ( case and control subjects) caucasians. although nominal associations were noted at snps � and � (p � . ) on meta- analysis of all populations of european ancestry, no asso- ciation was found when utah samples were excluded (p � . ). genotype data are shown in supplemental table s. discussion chromosome q -q is among the best-replicated re- gions of linkage to type diabetes, with evidence for linkage in multiple caucasian populations, pima indians, chinese, and, in unpublished studies from our laboratory, african americans. previous studies from our laboratory strongly support the existence of multiple susceptibility genes contributing to the overall linkage signal and possi- bly to the replication of linkage on q -q . our current fig. . allele distribution in caucasian and african-american case and control subjects at str � . dusp � str shows the large differences in allele frequencies by ethnicity. numbers are shown as the frequency of each allele. total number of chromosomes were for african-american diabetic subjects, for african-american control subjects, for caucasian diabetic subjects, and for caucasian control subjects. z, african-american case subjects; `, african-american control subjects; o, caucasian case subjects; p, caucasian control subjects. table summary of variants fully typed in african-american case and control subjects location major/minor allele case minor allele frequency control minor allele frequency � p � a/g . . . . � � /x . . . . � c/t . . . . � t/c . . . . � a/g . . . . � t/g . . . . c/g . . . . c/t . . . . t/a . . . . c/g . . . . c/t . . . . c/g . . . . a/g . . . . a/g . . . . c/g . . . . c/t . . . . c/t . . . . variants typed in african-american families are shown by location, as in table and fig. . major and minor alleles are switched in african americans from caucasian frequencies for some snps. note that � has been dichotomized for this table into - and -bp alleles versus all others. the actual distribution is shown in fig. . � , the � value for allelic association ( df); p value, the p value from � test. dusp is associated with type diabetes diabetes, vol. , september studies have focused on the largest linkage peak, centered at mb, encompassing markers crp and apoa , and including the calsequestrin gene (casq ; mb), for which we ( ) and fu et al. ( ) recently reported an association. that association seemed unlikely to account for the entire linkage signal, and in the current study we followed up on an association at snp rs , . mb away from the casq gene. this region included only three genes. fcrlm , which is � to rs and in a region without associated snps, is a member of the fc receptor family involved in antibody-dependent cell cytotoxicity. the dual-specificity phosphatase (dusp ) gene is widely expressed and shows broad conservation even in yeast. indeed, the human dusp gene can complement a yeast mutant ( ). the entire gene and most of a � cng were included in a single haplotype block (fig. ), and within this block most of the common variants were associated with type diabetes in utah caucasians (fig. and table ). although we found some evidence among caucasians for associated snps in the � end of the atf gene, these were clearly outside the dusp haplotype block and not in linkage disequilibrium with the dusp snps (data not shown). atf is an essential activator of endoplasmic reticulum stress and the unfolded protein response ( ) and, based on recent data suggesting a role of endoplasmic reticulum stress in type diabetes, insulin resistance, and impaired insulin secretion ( ), a strong functional candidate. work is in progress to fully evaluate the large atf gene, but we focused the current study on dusp as the more proba- ble candidate based on the clustering of associated snps and strs, the cng, and evidence for association (albeit with different alleles) in african-american subjects. dusp was pulled from a rat hepatic cdna library with a yeast two-hybrid system, using glucokinase as bait, and identified as the glucokinase-associated protein ( ). dusp (glucokinase-associated protein) accelerated glu- cokinase activity in a manner suggesting functional signif- icance. hence, dusp may modulate glycolysis in the liver and pancreatic -cell through dephosphorylation of glucokinase in the cytoplasm. the wide distribution and relatively high levels of expression suggest that dusp may have other, as yet unidentified, roles. because we identified no coding variants that could explain the asso- ciation in dusp , we propose that the associated variant or variants, mainly in highly conserved regions, may be regulatory. the � str, which showed strong associ- ations in utah caucasians and more modest associations in african americans, is within a very highly conserved region of � kb, kb upstream of dusp (supplemental fig. s). the allele, which was associated with type diabetes in caucasians, eliminates the binding site for the transcription factor sry (sex-determining region y), but this factor appears unlikely to have a role in type diabetes. based on the strong conservation across mam- malian species, this region may act as an enhancer for dusp , atf , or a chromosome gene beyond this region of association. given the size of the cng, we considered that the region might be transcribed, but we were unable to detect a transcript in hepg cells or transformed lymphocytes (unpublished data). the � microsatellite is not in a conserved region, but it contains multiple copies of the predicted binding site for the homeobox transcription factor cdxa (cdx ; caudal type homeobox transcription factor ), which is widely ex- pressed. the multiple cdxa binding sites and lack of conservation suggest that the � variant may not itself be the causative variant. in contrast, snp � , at the � end of dusp , is at the downstream end of a region of conservation among mammals. the common c (g) allele is conserved in dogs, chimpanzees, and humans, and it binds the basic helix-loop-helix transcription factor th / e . that factor in turn interacts with key pancreatic -cell genes to regulate insulin gene transcription. altered binding might impact transcription of either dusp upstream or atf downstream in a tissue-specific man- ner. however, opposite alleles are associated in cauca- sians and african americans, which appears inconsistent with a causative variant. finally, although str � and snp � show the strongest associations with type diabetes and are in the most conserved regions among mammals, the strongest quantitative trait association was with snp � farther upstream and post– glucose load insulin levels. this snp was the location of the original observation, but it was no longer the strongest association with type diabetes once additional case and control subjects were typed. replication of association with complex disease genes has been difficult, and, consequently, concerns have been raised about the power to detect and replicate complex disease associations ( ). multiple apparently convincing associations in smaller samples have failed to replicate in very large populations ( – ). both of our study popula- tions are relatively small, although our previously de- scribed association at the pklr locus has held up in the larger international type diabetes chromosome q consortium data set ( ). thus, the associations found here may well be spurious. facts favoring a role for dusp in type diabetes include the strong candidacy based on a previous study ( ); the finding of associated dusp region markers in a second population, albeit with less significance and with different alleles; and the association of snps in this region with postchallenge insulin levels in unaffected family members. nonetheless, several aspects of our extensive analyses did not support a direct role of dusp . first, we could not find a difference in allelic expression in transformed lymphocytes for a marker (snp � ) that is in strong linkage disequilibrium with the risk haplotype, nor did dusp expression differ among homozygotes at this snp. this finding is inconsistent with a cis-acting regula- tory variant, and may suggest involvement of a gene other than dusp . alternatively, altered dusp regulation may be tissue specific. second, the only physiologic con- sequence of dusp region variants was on the insulin response to an oral glucose load, but detailed measures of insulin sensitivity and insulin response to intravenous glucose were not altered. other challenges, such as a graded glucose infusion or measurement of hepatic glu- cose function, may be required to understand the physio- logic importance of dusp variants. third, the associated variants were not transmitted in excess from parents to affected offspring, nor could we explain the linkage signal using the gist program. such tests have also failed for pklr alleles and casq variants ( , ). for variants with a minor allele frequency of . , using all available utah families, we have % power to detect a transmission rate of . from a heterozygous parent to affected offspring, which translates to a relative risk of . under a dominant and . under a recessive model. finally, and perhaps most importantly, analysis of snps in this region, including the associated snp � , showed s.k. das and associates diabetes, vol. , september no association with type diabetes in the european-based populations from the international type diabetes chro- mosome q consortium when utah samples were not included. notably, these studies did not include the two str variants that show the strongest associations in the utah population. although typed snps, particularly � (rs ), should have served as a proxy for the -bp allele, the possibility remains that these studies did not type the functional variants. in summary, we have performed a detailed analysis of markers in a � -kb region surrounding the dusp and atf genes. we report evidence for an association with type diabetes in utah caucasians, with some evidence for association in african americans recruited in arkan- sas, albeit with different alleles. the most strongly associ- ated snps are in highly conserved regions, thus suggesting a possible regulatory role. snps in this region are also associated with insulin levels after an oral glucose chal- lenge. however, attempts to replicate the snp associa- tions in a meta-analysis of data from the three other european populations with linkage of type diabetes to chromosome q -q showed no significant association. interactions between dusp region snps and other chromosome variants or variants elsewhere in the ge- nome might reconcile these differences, as might the typing of the str variants in other populations. additional studies will be needed to determine whether the associa- tions observed in utah caucasians are spurious or play a role in insulin response to glucose and susceptibility to type diabetes. acknowledgments this work was supported by grants from the national institutes of health/national institute of diabetes and digestive and kidney diseases (niddk; no. dk ) and by the research service of the department of veterans affairs. subject ascertainment was supported in part by grants from the american diabetes association, and sub- ject ascertainment and metabolic studies 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n, orho-melander m, schayer s, sun m, almgren p, lindblad u, tuomi t, gaudet d, hudson tj, daly mj, ardlie kg, hirschhorn jn, altshuler d, groop l: association testing in , people fails to confirm the association of the insulin receptor substrate- g r polymorphism with type diabetes. diabetes : – , . florez jc, agapakis cm, burtt np, sun m, almgren p, rastam l, tuomi t, gaudet d, hudson tj, daly mj, ardlie kg, hirschhorn jn, groop l, altshuler d: association testing of the protein tyrosine phosphatase b gene (ptpn ) with type diabetes in , people. diabetes : – , . winckler w, graham rr, de bakker pi, sun m, almgren p, tuomi t, gaudet d, hudson tj, ardlie kg, daly mj, hirschhorn jn, groop l, altshuler d: association testing of variants in the hepatocyte nuclear factor � gene with risk of type diabetes in , people. diabetes : – , s.k. das and associates diabetes, vol. , september untitled common variants in the slco b locus are associated with bilirubin levels and unconjugated hyperbilirubinemia serena sanna ,{, fabio busonero ,{, andrea maschio , patrick f. mcardle , gianluca usala , mariano dei , sandra lai , antonella mulas , maria grazia piras , lucia perseu , marco masala , mara marongiu , laura crisponi , silvia naitza , renzo galanello , gonçalo r. abecasis , alan r. shuldiner , , david schlessinger , antonio cao and manuela uda ,� istituto di neurogenetica e neurofarmacologia del consiglio nazionale delle ricerche, monserrato, cagliari, italy, division of endocrinology, diabetes and nutrition, university of maryland school of medicine, baltimore, md, usa, clinica pediatrica, ospedale regionale delle microcitemie, dipartimento di scienze biomediche e biotecnologie, università degli studi di cagliari, cagliari, italy, center for statistical genetics, department of biostatistics, university of michigan, ann arbor, mi , usa, veterans administration medical center, geriatric research and education clinical center, baltimore, md, usa and laboratory of genetics, national institute on aging, baltimore, md, usa received february , ; revised march , ; accepted april , bilirubin, resulting largely from the turnover of hemoglobin, is found in the plasma in two main forms: uncon- jugated or conjugated with glucuronic acid. unconjugated bilirubin is transported into hepatocytes. there, it is glucuronidated by ugt a and secreted into the bile canaliculi. we report a genome wide association scan in sardinian individuals for total serum bilirubin levels. in addition to the two known loci previously involved in the regulation of bilirubin levels, ugt a (p . ) and g pd (p . ), we observed a strong association on chromosome within the slco b gene (p . ). our findings were replicated in an independent sample of sardinians and in subjects from the old order amish (combined p < ). we also show that slc b variants contribute to idiopathic mild unconju- gated hyperbilirubinemia. thus, slc b appears to be involved in the regulation of serum bilirubin levels in healthy individuals and in some bilirubin-related disorders that are only partially explained by other known gene variants. introduction bilirubin results from hemoglobin catabolism (and to a lesser extent from other heme-containing proteins) within the reticulo-endothelial system. bilirubin is transported into the liver, where specific carrier proteins belonging to the organic anion protein transporter family ( ) are thought to mediate its uptake. in hepatocytes, bilirubin is glucuronidated by (udp)-glucuronosyltransferase and thereby transformed into a water soluble conjugated molecule ( ). conjugated bilirubin is actively secreted into the bile canaliculi by a membrane atp-dependent transporter, designated multidrug resistance-associated protein (mrp ) ( ). in plasma, biliru- bin occurs primarily unconjugated tightly bound to albumin, or conjugated as a water-soluble glucuronide. high levels of unconjugated bilirubin in plasma may indicate increased pro- duction by hemolysis, ineffective erythropoiesis or decreased conjugation, whereas conjugated hyperbilirubinemia reflects decreased excretion by damaged biliary duct or hepatic parenchymal cells. † s.s. and f.b. equally contributed to this work. � to whom correspondence should be address. tel: þ ; fax: þ ; email: manuela.uda@inn.cnr.it # the author . published by oxford university press. all rights reserved. for permissions, please email: journals.permissions@oxfordjournals.org human molecular genetics, , vol. , no. – doi: . /hmg/ddp advance access published on may , results within the sardinia study we conducted a genome-wide association (gwa) analysis ( , ) in sardinians to ident- ify genetic factors affecting a series of quantitative traits, including serum bilirubin levels (see material and methods). the strongest associations were observed on chromosome , in the promoter of ugt a (uridine diphosphoglucuronyl- transferase) gene (rs , p ¼ . � , fig. and supplementary material, table s ), a major and well-known modifier of bilirubin levels ( ), and on the x chromosome near the g pd (glucose- -phosphatedehydrogenase) gene (rs , p ¼ . � , fig. and supplementary material, table s ). in addition, a third locus on chromosome p . reached genome-wide significance (fig. ). the strongest p-value was observed at snp rs (p ¼ . � , supplementary material, table s ) downstream of the -utr of the slco b (solute carrier organic anion transporter family, member b ) gene ( ) (fig. ); each copy of the minor allele c was respon- sible, on average, for an increase of . mg/dl in bilirubin levels. the second ranked marker in this region was rs (p ¼ . � ), located in intron of slco b , in modest ld with rs (r ¼ . ), and responsible for a slightly larger effect ( . mg/dl per copy of the minor allele g). our genotype strategy was designed to take advantage of the relatedness of the sample. in fact we gen- otyped first a small number of markers ( k genechip) in largest families to identify shared stretches of chromosome between individuals, and then genotyped with a denser map (the k chip) a subset of individuals, selected to be dispersed along the known pedigrees with an overlap of individuals genotyped with both platforms. next a within-family imputa- tion method was used to predict missing genotypes (see material and methods). the two markers, rs and rs , belong to the k snp array and k snp array, respect- ively, and thus were imputed the first in the majority of the samples, the second in only one-fourth. to evaluate the effect of the different number of genotyped and imputed samples, we directly genotyped both markers in individuals for whom only imputed genotypes were available. using all available gen- otypes, the association test showed snp rs as more representative of total bilirubin variation (p ¼ . � , see table and supplementary material, table s ). interestingly, when including this marker in the model, snp rs still showed some evidence for association (p ¼ . ). further- more, we observed a significant interaction between the two markers (p ¼ . � ). using all available genotypes, we were able to infer the complete haplotypes of individuals for which the wet-genotype for both markers were available and we observed that only haplotypes carrying both positive or both negative alleles were significant (hgc p ¼ . � and hct p ¼ . � , see table ). notably, we observed that the haplotype frequencies were similar to the minor allele frequency of the two variants (hgc freq ¼ . and hct freq ¼ . ), and that each haplotype was highly correlated with one variant (hgc and rs cor ¼ . , hct and rs cor ¼ . ) (table ). those results were also replicated in the amish popu- lation, where the only two haplotypes associated with bilirubin levels were those carrying both positives or both negatives alleles. each haplotype was highly correlated with one of the two markers (table ). thus the two snps, rather than having independent effects, are likely imperfectly tagging a third common variant of which the allele responsible for an increase in bilirubin levels would be probably associated with the hgc haplotype, and the other allele with the hct haplotype. figure . genome-wide scan of serum total bilirubin. the figure summarizes association p-values (additive test) for all snps that passed quality control tests (n ¼ , ). position of ugt a, slco b and g pd genes are annotated. red dotted line marks bonferroni threshold ( . � ). human molecular genetics, , vol. , no. we also evaluated the effect of possible confounding factors related to the particular hematological features of the sardi- nian population, and repeated the association tests including as covariates mean corpuscular hemoglobin, mean corpus- cular volume, total hemoglobin, b-thalassemia carrier status and g pd levels ( , ). none of those factors affected the association at the three loci. as expected, adjustment for g pd levels reduced the association at the g pd locus, some- what strengthening the ugt a signal (supplementary material, table s ). in addition, we observed that the associ- ation of ugt a and slco b appeared stronger in women than in men, although we found no significant difference in the magnitude of the effect between sexes. in contrast, the associ- ation with the x-linked g pd locus was stronger in men, most likely because of the major impact of g pd deficiency in males. in fact, there was no difference in association between g pd genotype and bilirubin when g pd level was taken into account (supplementary material, table s ). finally, none of the tests for epistasis between the top snps at the three loci was significant after correction for multiple testing (supplementary material, table s ). additional studies are required to confirm this observation. our findings at the slco b locus were successfully repli- cated in an independent sample of sardinians, previously described and reported as sardinia stage ( ), with an effect in the same direction as that observed in the gwa sample (p , � , table ). moreover, we also repli- cated the association in individuals enrolled in the amish heredity and phenotype intervention (hapi) heart study ( ) (p , � , table ), suggesting that variants in the slco b influence bilirubin levels not only in sardi- nians but also in other populations of caucasian origin. com- bined, the evidence for association at this locus was p , � (table ). as shown in fig. , although our results point to the slco b gene, weaker association encompasses a genomic region of kb harboring, in addition to slco b , four more genes in the same family of membrane transport pro- teins. among them, slco b ( ) has been previously impli- cated in bilirubin liver uptake ( ) and associated with bilirubin levels in asian populations. in this gene two coding variants, namely val ala/rs and asp asn/rs , have been described as major determi- nants of bilirubin variation ( , ), and they weakly correlated with rs (r ¼ . and r ¼ . , respectively) and with rs (r ¼ . and r ¼ . , respectively). to compare their effects with polymorphisms at slco b we used inferred genotypes based on hapmap ( ) ceu (utah residents with ancestry from northern and western europe) haplotypes available from previous studies ( ), and tested for association with total, conjugated and unconjugated bilirubin. only nominal association with total bilirubin was detected at these coding variants (p ¼ . and p ¼ . � , table ). in addition, when including rs and figure . evidence of (a) association with serum total bilirubin and (b) linkage disequilibrium around the slco b locus. (a) all snps in the slco b region ( kb) are plotted with association p-values (additive test) compared with physical position. genomic position and gene annotations refer ncbi build map. snps are colored according to their linkage disequilibrium with the top variant (rs ). blue line represents the recombination rate (in cm/mb). black diamonds represent combined p-values from sardinia and sardinia stage cohorts. (b) patterns of linkage disequilibrium (r ) for the ceu hapmap popu- lation (utah residents with european ancestry ( )) are plotted and colored as indicated in the palette. human molecular genetics, , vol. , no. rs as covariates in the model, snps at the slco b locus still remain highly significant (supplementary material, table s ), supporting the hypothesis that the association signal observed in the slco b is not modulated by an indirect effect of these coding variants. furthermore, since the snps tested in the gwas covered % of the common variants in the slco b at r . . , it is unlikely that other snps in this gene may have a stronger effect than those in the slco b locus. the differential contribution of the two transporters may be owing to population-specific genetic features. in asians, for example, snp rs is monomorphic. interestingly, we observed that snps in slco b were mostly associated with conjugated bilirubin (p ¼ . � for rs ), whereas no association was observed with unconjugated bilirubin (p ¼ . , table ). in contrast, snp rs in slco b showed a stronger association with unconjugated bilirubin (p ¼ . � ) compared with conjugated bilirubin (p ¼ . ). a similar pattern of association was observed in the sardinia stage sample (table ), where snp rs was directly genotyped. again, the association with unconjugated bilirubin at snps rs and rs remained significant after account- ing for the two snps in the slco b gene (supplementary material, table s ). conjugated bilirubin was not available in the amish cohort, and thus a parallel analysis could not be performed. we next tested for any effect of the ugt a gene on the association of bilirubin levels with the slco b gene. includ- ing snp rs , the top marker on the ugt a locus in the model, the association at rs and rs did not disappear but rather increased (data not shown), suggesting that slco b explains part of the variability not accounted for by ugt a polymorphisms. to explore this possibility further, we evaluated whether this locus may be responsible for idiopathic mild hyperbilirubinemia in patients lacking mutations in the ugt a gene. hence, we genotyped table . summary of association results for snps in theslco b locus in all cohorts marker study n allele high/low freq effect (stderr) effect (stderr) mg/dl p-value rs stage sardinia g/c . . ( . ) . ( . ) . � stage sardinia stage g/c . . ( . ) . ( . ) . � old older amish g/c . . ( . ) . ( . ) . � overall . � rs stage sardinia c/t . . ( . ) . ( . ) . � stage sardinia stage c/t . . ( . ) . ( . ) . � old older amish a c/t . . ( . ) . ( . ) . � overall . � the tables summarize the association results in all cohorts. for each marker, frequency and effect estimates are given with respect to the ‘high frequency’ allele. association parameters (effect, stderr, p-value) are relative to the model where the trait is transformed, thus the effect estimated in this model represents the change in standard deviation units. effect in mg/dl is also given for each marker. sardinia results are relative to the imputed genotypes inferred from affymetrix and taqman genotypes. a old older amish sample refers to snp rs , r ¼ . with rs in ceu hapmap population, for which additional genotyped individuals were available. table . association analysis of haplotypes resulting from all possible combinations of alleles at markers rs and rs haplotype rs rs a freq effect b stderr p-value correlation c with rs rs a sardinia hcc c c . . . . . . hct c t . . . . � . . hgc g c . . . . � . . hgt g t . . . . . . old older amish hcc c c . . . . . . hct c t . . . . . . hgc g c . . . . . . hgt g t . – – – – – a for the amish sample, rs (t/c alleles) was genotyped instead of rs (c/t alleles). b effect size and standard error are given in standard deviation units. c correlation is given in absolute value. human molecular genetics, , vol. , no. healthy individuals and unrelated patients with hyperbilir- ubinemia (defined as serum total bilirubin . . mg/dl) ( ) from the sardinia sample for the mutation in the tataa element of the promoter region of the ugt a gene ( ). we observed a % enrichment in the frequency of the g allele of snp rs in the hyperbilirubinemic patients compared with the healthy subgroup (p ¼ . ; table ). notably, most of the difference was attributable to patients homozygous for the wild-type allele, a(ta) taa at the ugt a locus (n ¼ , p ¼ . ), rather than to patients car- rying at least one copy of the mutated allele a(ta) taa (n ¼ , p ¼ . ) ( ). thus one can infer that slco b not only acts in the general population, but also affects mild hyperbilirubinemias. discussion gwa analysis for serum bilirubin levels on sardinian revealed two striking signals in the ugt a and the g pd genes. the association with ugt a is well known. in fact, mutations in this gene are responsible for the crigler – najjar syndrome as well as the more common mild unconjugated hyperbilirubinemia of gilbert syndrome ( ). the association of g pd with bilirubin levels was also expected, given the high frequency of deficiency in g pd in the sardinian popu- lation; this condition is indeed associated with a shorter life span of red blood cells, resulting in a modest increase in bilir- ubin levels ( ). furthermore, the results revealed an additional locus on chromosome p . . the strongest associ- ated marker in this region, an intronic snp of the slco b gene, was successfully replicated in an independent sardinian sample and in a cohort of amish individuals. notably, this gene is highly expressed in the basolateral membrane of hep- atocytes and is a member of the oatp family encoding the organic anion transporter polypeptide na þ -independent, oatp b ( ). the oatp proteins are expressed in a variety of tissues, including gut, brain, kidney and liver where they play important roles in drug absorption and distri- bution and excretion, with a wide spectrum of substrates that include both endogenous and xenobiotic compounds ( ). thus slco b is a plausible candidate gene responsible for table . association of slco b with hyperbilirubinemia n c/c c/g g/g c g genotypic test p-value allelic test p-value healthy . . . . . hyper-bilirubinemia . . . . . . . hyper (ta /ta ) . . . . . . . hyper (ta /ta , ta /ta ) . . . . . . . the table describes the allele and genotype frequencies in a data set of unrelated healthy (total bilirubin , mg/dl) and hyperbilirubinemia patients, screened for the ugt a a(ta) taa mutation. hyperbilirubinemia was defined as total bilirubin . . mg/dl. p-value , . are highlighted in bold. table . association with total, conjugated and unconjugated bilirubin of snps in the slco b and slco b genes locus marker study freq total bilirubin conjugated bilirubin unconjugated bilirubin effect (stderr) p-value effect (stderr) p-value effect (stderr) p-value slco b rs (c) sardinia a . . ( . ) . . ( . ) . � . ( . ) . sardinia stage . . ( . ) . . ( . ) . . ( . ) . old order amish b . . ( . ) . � – na – na pcombined . . � . slco b rs (g) sardinia a . . ( . ) . � . ( . ) . � . ( . ) . sardinia stage – – na – na – na old order amish c . . ( . ) . – na – na pcombined . . � . slco b rs (g) sardinia . . ( . ) . � . ( . ) . . ( . ) . � sardinia stage . . ( . ) . � . ( . ) . . ( . ) . � old order amish . . ( . ) . � – na – na pcombined . � . � . � the table summarizes association results with total, conjugated and unconjugated bilirubin for snp rs and snp rs , polymorphisms in the slco b gene previously known to be associated with neonatal hyperbilirubinemia. they are coding mutations, with the following aminoacid changes: val ala and asp asn, respectively. effect size and standard error are given in standard deviation units. conjugated and unconjugated bilirubin were not available in the amish cohort. a snps rs and rs were imputed in all sardinia samples based on hapmap ceu haplotypes (see material and methods). quality of imputation was r ¼ . and r ¼ . , respectively. b snp rs was not genotyped in the amish cohort, and rs (r ¼ . ) was analyzed as best proxy among genotyped snps. c snp rs was not genotyped in the amish cohort, and rs (r ¼ . ) was analyzed as best proxy among genotyped snps. human molecular genetics, , vol. , no. changes in bilirubin levels. interestingly, another solute carrier organic anion transporter, slco b , previously implicated in liver bilirubin uptake ( ), maps in the same region, but only weaker association was observed at this locus. in particular, we found that snps at slco b were mostly associated with conjugated bilirubin, although no association was detected with unconjugated bilirubin. although we cannot exclude the possibility of a differential contribution of the two transporters in a population-specific fashion, the results produced in this study indicate that although the slco b gene regulates unconjugated serum bilirubin, slco b appears to act on conjugated bilirubin. slco b , with an high affinity for glucuronated bilirubin ( ), may contribute (together with mpr ) to reverse transport from hepatocytes to plasma in normal conditions, and very likely also in cholestasis when the major export pump mpr is down- regulated ( ). further studies are necessary to identify the slco b - specific causative variants and then to clarify the functional role of both oatp genes on conjugated and unconjugated bilirubin. finally, the slco b snps identified here were also found to be associated with unconjugated hyperbilirubine- mia of unknown origin. speculatively, they may also act as modifiers of the clinical course of some phenotypes, as already shown for ugt a polymorphisms in hemolytic anemia, and anemia resulting from ineffective erythropoiesis, and neonatal jaundice ( , , ). thus, our findings not only reveal an additional regulator of bilirubin levels in the general population but may also contribute to the identification of genetic variants necessary to predict and prevent severe clinical manifestations in hematological and hepatic disorders linked to hyperbilirubinemia. material and methods sample description we recruited and phenotyped individuals, males and females, ages – years, from a cluster of four towns in the ogliastra province of sardinia ( ). during physical exam- ination, a blood sample was collected from each individual, and divided into two aliquots. one aliquot was used for genomic dna extraction and the second ( ml peripheral blood without anticoagulant) to characterize several blood phenotypes, including evaluation of serum total and conju- gated bilirubin levels, measured with express plus (bayer) equipment, according to manufacturer’s instruction. the normality range was . – . and – . mg/dl for total and conjugated bilirubin, respectively. hyperbilirubinemia was defined when total bilirubin serum concentration exceeded . mg/dl ( ). gwas genotyping we genotyped from the whole sample individuals selected to represent the largest available families, regardless of their phenotypic values ( ). specifically, were geno- typed with the k affymetrix mapping array set and , with the k mapping array set, with individuals genotyped with both arrays. this genotyping strategy allowed us to examine the majority of our cohort in a cost-effective manner because genotypes for the snps that passed quality- control checks could be propagated through the pedigree via imputation ( , ). bilirubin measurements were available for individuals among the genotyped. a total of snps passed initial quality-control checks ( ) and were tested for association. although the statistical analysis was ongoing, an additional million autosomal markers from hapmap imputed data became available. repeating the gwa analysis using this larger data set of markers detected no additional loci, and the top markers at chromosomes and loci were confirmed. further details of the strategy for imputation and data analysis are given in the statistical analysis section below. replication we designed a parallele custom chip (from affymetrix) to replicate the regions associated with bilirubin levels as well as other traits studied in the sardinia project. genotyping was performed in sardinian, selected within the sardi- nia sample and independent from the individuals included in the gwa scan (kinship coefficient ¼ with those directly typed with the k chip), and indicated here as sardinia stage data set ( ). snps rs and rs , were included in the parallele custom chip, and also genotyped in additional individuals using taqman snp genotyping assay (applied biosystems) according to the protocol. the concordance rate between inferred and wet genotypes was . and . % for rs and rs (over n ¼ and n ¼ samples), respectively. snp rs , not included in the custom chip was genotyped in the sardinia stage data set using taqman technology. mutations in the tataa element of the promoter region of the ugt a gene were analyzed by direct sequen- cing (abi-prism ; applied biosystems, usa) of genomic dna. statistical analysis for individuals genotyped with a sparse map, we used a modi- fied version of the lander – green algorithm ( ) to estimate ibd sharing at the location of the snps being tested and ident- ify stretches of haplotype shared with close relatives who were genotyped at higher density and probabilistically infer missing genotypes ( ). our inference approach allowed us to account for uncertainty in genotype assignment, estimating, instead of the most likely genotype, an expected genotype score, repre- senting the expected number of copies of a reference allele (a fractional number between and ). the genotype scores were then used in the family-based association test for evalu- ating the additive effect of each marker, as described else- where ( , ). owing to computational constraints, we divided large pedigrees into sub-units with ‘bit-complexity’ of or less (typically, – individuals) before all the ana- lyses. the association test fits a simple regression model and uses a variance component approach to account for correlation between different observed phenotypes within each family. to evaluate association on the x chromosome, we modeled a polygenic variance component shared according to an human molecular genetics, , vol. , no. x-linked kinship coefficient in addition to the usual autosomal polygenic variance component ( , ). prior to all analyses, traits were normalized using quantile normalization to avoid inflation of type i error rates; gender, age and age squared were included as covariates. in the sardinia stage the association with bilirubin levels was tested as in the sardinia gwa sample. to infer haplo- types we used all available genotypes and merlin software. we created a variable for each haplotype coded as , or , representing the number of copies carried by an individual. a linear regression model was fitted for each haplotype. epis- tasis and haplotype-specific tests were performed in the gwa sample using the r kinship package. old order amish the hapi heart study was initiated in . participants of the hapi heart study comprised adults from the old order amish community of lancaster county, pa, who were recruited over a -year period. the study aims and recruitment details, including ascertainment criteria, have been described previously ( ). physical examinations were conducted at the amish research clinic in strasburg, pa. a blood sample was obtained for measurement of bilirubin levels. bio- chemical assays were performed by quest diagnostics (horsham, pa, usa). genotypes for snps rs , rs (proxy of rs ; r ¼ . ), rs (proxy of rs ; r ¼ . ) and rs (proxy of rs ; r ¼ . ), were obtained from a previous gwas performed using the k affymetrix mapping array set. statistical analysis was performed using in house developed software. in brief, we performed a measured genotype approach utilizing a t-test of the beta coefficient for the snp variable. we included sex, age and age squared as fixed cov- ariates in the model and a polygenic component modeled as a random effect to account for the full generation pedigree of the amish. the bilirubin trait was normalized using quantile normalization to avoid inflation of type i error rates; gender, age and age squared were included as covariates, as in the sar- dinia gwa sample. supplementary material supplementary material is available at hmg online. acknowledgements we thank the many individuals who generously participated in this study, monsignore piseddu, bishop of ogliastra, the mayors and citizens of the sardinian towns (lanusei, ilbono, arzana and elini), and the head of the public health unit asl , the province of ogliastra for their volunteerism and cooperation. in addition, we are grateful to the mayor and the administration in lanusei for providing and furnishing the clinic site. we thank the team of physicians angelo scuteri, marco orrù, maria grazia pilia, maria cristina spada, danilo fois, liana ferreli, francesco loi, and nurses paola loi, monica lai and anna cau who carried out participant physical exams, and monica balloi who recruited the volunteers. conflict of interest statement. none declared. funding the work was supported by the following national institutes of health grants: hl to g.r.a., hg to g.r.a., national institute of aging contracts no -ag- - to the sardinia (‘progenia’) team and -ma- to the university of michigan (g.r.a).the old order amish study was supported by national institutes of health research grant u hl , the university of maryland general clinical research center, grant m rr , the clinical nutrition research unit of maryland p dk , and the baltimore veterans administration medical center geriatrics research. author contributions g.r.a, d.s., a.c., a.r.s. and m.u. designed the study; r.g., a.s., d.s. and m.u. provided material and reagents; f.b., a.m., g.u., m.d., s.l. and a.m. performed the genotyping; m.g.p., l.p., m.m., m.m., a.r s. performed characterization of phenotype and data collection; s.s., p.f.m., analyzed the data; s.n., l.c., f.b., a.r.s., p.f.m. contributed to draft the manuscript; s.s., d.s., a.c. and m.u. wrote the paper. references . hagenbuch, b. and gui, c. 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( ) the genetic response to short-term interventions affecting cardiovascular function: rationale and design of the heredity and phenotype intervention (hapi) heart study. am. heart j., , – . . abe, t., kakyo, m., tokui, t., nakagomi, r., nishio, t., nakai, d., nomura, h., unno, m., suzuki, m., naitoh, t. et al. ( ) identification of a novel gene family encoding human liver-specific organic anion transporter lst- . j. biol. chem., , – . . konug, j., cui, y., nies, a.t. and kepplera, d. ( ) novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. am. j. physiol. gastrointest. liver physiol., , – . . zhang, w., he, y.j., gan, z., fan, l., li, q., wang, a., liu, z.q., deng, s., huang, y.f., xu, l.y. et al. ( ) oatp b polymorphism is a major determinant of serum bilirubin level but not associated with rifampicin-mediated bilirubin elevation. clin. exp. pharmacol. physiol., , – . . nozawa, t., nakajima, m., tamai, i., noda, k., nezu, j., sai, y., tsuji, a. and yokoi, t. ( ) genetic polymorphisms of human organic anion transporters oatp-c (slc a ) and oatp-b (slc a ): allele frequencies in the japanese population and functional analysis. j. pharmacol. exp. ther., , – . . the international hapmap consortium ( ) a second generation human haplotype map of over . million snps. nature, , – . . sanna, s., jackson, a.u., nagaraja, r., willer, c.j., chen, w.m., bonnycastle, l.l., shen, h., timpson, n., lettre, g., usala, g. et al. ( ) association of human height with genetic variants in the gdf - bfzb locus: genome-wide scans and replication. nat. genet., , – . . cappellini, m.d., martinez di montemuros, f., sampietro, m., tavazzi, d. and fiorelli, g. ( ) the interaction between gilbert’s syndrome and g pd deficiency influences bilirubin levels. br. j. haematol., , – . . cui, y., könig, j., leier, i., buchholz, u. and keppler, d. ( ) hepatic uptake of bilirubin and its conjugates by the human organic anion transporter slc a . j. biol. chem., , – . . trauner, m., arrese, m., soroka, c.j., ananthanarayanan, m., koeppel, t.a., schlosser, s.f., suchy, f.j., keppler, d. and boyer, j.l. ( ) the rat canalicular conjugate export pump (mrp ) is down-regulated in intrahepatic and obstructive cholestasis. gastroenterology, , – . . origa, r., galanello, r., perseu, l., tavazzi, d., domenica cappellini, m., terenzani, l., forni, g.l., quarta, g., boetti, t. and piga, a. ( ) cholelithiasis in thalassemia major. eur. j. haematol., , – . . abecasis, g.r., cherny, s.s., cookson, w.o. and cardon, l.r. ( ) merlin-rapid analysis of dense genetic maps using sparse gene flow trees. nat. genet., , – . . lander, e.s. and green, p. 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( ) family based association tests for genome wide association scans. am. j. hum. genet., , – . human molecular genetics, , vol. , no. thomson ucla ucla previously published works title developmental disorders. permalink https://escholarship.org/uc/item/ g pg journal current opinion in neurology, ( ) issn - authors jeste, shafali s geschwind, daniel h publication date - - doi . /wco. peer reviewed escholarship.org powered by the california digital library university of california https://escholarship.org/uc/item/ g pg https://escholarship.org http://www.cdlib.org/ editorial currentopinion developmental disorders copyrig - copyright � wolte shafali s. jeste and daniel h. geschwind university of california, los angeles (ucla) semel institute of neuro- science and human behavior, david geffen school of medicine, los angeles, california, usa correspondence to shafali s. jeste, ucla center for autism research and treatment, semel institute, room - b, westwood plaza, los angeles, ca , usa. tel: + ; e-mail: sjeste @mednet.ucla.edu curr opin neurol , : – doi: . /wco. when a child receives a diagnosis of a neurodeve- lopmental disorder, the first questions posed by the caregivers reflect the tremendous uncertainty surrounding treatment and outcome: ‘what is my child’s prognosis? which types of treatments will work most effectively for my child? how can we maximize his/her potential?’ historically, the pauc- ity of evidence regarding mechanisms of disease, diagnostic stratification, and potential treatment targets in these heterogeneous conditions has necessitated answers that are nonspecific and clini- cally unsatisfying. decisions regarding treatment targets and intervention strategies rely not on the specific cause or clinical presentation of a child’s neurodevelopmental disorder, rather on diagnostic categorization, standardized test scores, and access to services. over the past several years, however, advances in genetics, animal models, mechanism- driven biomarker development, and targeted inter- vention design have illuminated the path toward the realization of personalized medicine in neuro- developmental disorders. in this issue, these invited articles review the state of the field in neurodevelopmental disorders by focusing not on diagnostic classifications, but on themes that are common across this group of dis- orders that can provide insight into disease mech- anisms and targeted treatments. ebrahimi-fakhari and sahin (pp. – ) consider several genetic syndromes that cause autism spectrum disorder (asd) and intellectual disability, namely, fragile x syndrome, tuberous sclerosis complex, angelman syndrome, and phelan-mcdermid syndrome. the authors highlight the remarkable fact that these genetically and phenotypically distinct disorders share common pathways to abnormal brain devel- opment, namely, in the dysregulation of synapse formation and structure. this work highlights the theme that is emerging from the genetics of asd, that there is convergence on molecular and neuro- developmental pathways, despite extreme genetic heterogeneity in the condition. lázaro and golshani (pp. – ) emphasize the high value of mouse models of these high- risk genetic syndromes. despite being separated by million years of evolution from humans, mouse models have taught us much about the ht © wolters kluwe rs kluwer health, inc. all rights rese specific molecular, cellular, and circuit-level impair- ments that lead to the core deficits of asd, namely, repetitive behaviors and social communication impairments. although it was not known a priori how conserved the brain systems serving social cognition and repetitive behavior were in mice, mice harboring human mutations show remarkable parallels with the human conditions in many cases, including social deficits and repetitive behaviors. after careful characterization of the mouse pheno- type, rationally designed treatments for asd can be tested, with the goal of establishing efficacy at the brain and behavioral level, and understanding mechanisms, both of which would then translate to a targeted treatment for the patient. the following three clinical articles bridge mol- ecular mechanisms to patients. jeste, frohlich and loo (pp. – ) discuss the critical insights that can be gained from electrophysiological biomarkers in asd and attention deficit hyperactivity disorder (adhd), not only for diagnosis but also for risk prediction and treatment monitoring. they argue that electrophysiological measures of connectivity and signal complexity may serve as more direct measurements of the underlying mechanism of dis- ease in subpopulations of children within diagnostic categories and may inform risk status before a clinical diagnosis can be made. as we gain a greater understanding of the sensitivity of these biomarkers to both typical and atypical development, outcome measures and treatment response monitoring will be redefined from being purely behavioral to those that combine behavior with measures of brain function. prediction of atypical development and, more specifically, asd has become an area of great interest, particularly as behavioral and biological measures of risk can now be quantified in the first r health, inc. all rights reserved. rved. www.co-neurology.com mailto:sjeste@mednet.ucla.edu mailto:sjeste@mednet.ucla.edu developmental disorders year of life. brian, bryson and zwaigenbaum (pp. – ) provide a timely discussion about early markers of asd, emphasizing the challenges surrounding optimal timing and strategies in inter- ventions for infants at risk. greater precision in the prediction of atypical development necessitates more studies in early intervention, both in specific targets and in appropriate outcome measures. finally, kasari (pp. – ) provides a thought- provoking analysis of the current state of behavioral intervention research in asd and related develop- mental disorders. embracing the theme of individ- ualized treatment, kasari asserts that behavioral interventions in asd must evolve from the wide- spread ‘one package’ approach (in which children with a range of phenotypes receive the same menu of services) into a modular approach (in which a series of flexible, evidence-based strategies are tail- ored to the specific needs of a child). she emphasizes the need for rigorous and innovative methodology in clinical trials to test the efficacy of these tailored interventions. for personalized, or precision, medicine to suc- ceed, patients must be treated based on their specific biological and clinical profile, often informed by the presence of specific causal mutations or complex genetic risk factors. information from biomarkers and behavioral data can be combined with genetic data to identify the ‘subtype’ of the disorder, its trajectory, and treatment. the road from gene to mechanism to behavior is being traveled now by many investigators, including studies in rare dis- orders such as timothy syndrome [ ], tuberous sclerosis complex, and cortical dysplasia-focal epi- lepsy [ ]. a recent example of this is the identifi- cation of the contactin-associated protein-like (cntnap ) mutation in the amish, recapitulation of the major phenotypes in a mouse model, and recent drug screening that identifies oxytocin path- ways as a potential therapeutic avenue in this dis- order, with oxytocin treatment reversing the social impairments found in the mouse model of this copyright © wolters kluwer www.co-neurology.com disorder. one lesson from these studies is that one size does not fit all; treatments may need to be tailored to the specific conditions in which they will be most effective based on model systems data. two terms recur throughout these reviews: ‘heterogeneity’ and ‘convergence’. although seem- ingly contradictory, they are, in fact, complemen- tary, and they capture the neurobiological essence of neurodevelopmental disorders. although the causes of these disorders are extremely hetero- geneous, a multiplicity of causes seems to lead to convergent disruptions in brain development. a wide range of genetic and environmental factors, and their complex interactions, dysregulate com- mon pathways in asd and adhd, pathways that then serve as promising targets for treatment and prevention. however, given the dynamic nature of development and the constant interplay of biology with environment, these common pathways can still result in a wide range of symptoms and pheno- typic expression, thereby challenging the imple- mentation of effective treatment strategies. acknowledgements none. financial support and sponsorship this work is funded by national institute of mental health grant k mh (jeste) and national insti- tute of child health and human development grant p hd - (geschwind and jeste). conflicts of interest there are no conflicts of interest. references . paşca sp, portmann t, voineagu i, et al. using ipsc-derived neurons to uncover cellular phenotypes associated with timothy syndrome. nat med ; : – . . strauss ka, puffenberger eg, huentelman mj, et al. recessive symptomatic focal epilepsy and mutant contactin-associated protein-like . n engl j med ; : – . health, inc. all rights reserved. volume � number � april human itch e ubiquitin ligase deficiency causes syndromic multisystem autoimmune disease core metadata, citation and similar papers at core.ac.uk provided by elsevier - publisher connector report human itch e ubiquitin ligase deficiency causes syndromic multisystem autoimmune disease naomi j. lohr, jean p. molleston, ,* kevin a. strauss, , , wilfredo torres-martinez, eric a. sherman, robert h. squires, nicholas l. rider, , kudakwashe r. chikwava, oscar w. cummings, d. holmes morton, , , and erik g. puffenberger , , ubiquitin ligases play an important role in the regulation of the immune system. absence of itch e ubiquitin ligase in mice has been shown to cause severe autoimmune disease. using autozygosity mapping in a large amish kindred, we identified a linkage region on chromosome and selected candidate genes for screening. we describe, in ten patients, identification of a mutation resulting in trun- cation of itch. these patients represent the first reported human phenotype associated with itch deficiency. these patients not only have multisystem autoimmune disease but also display morphologic and developmental abnormalities. this disorder underscores the importance of itch ubiquitin ligase in many cellular processes. we describe ten old order amish children with organome- galy, failure to thrive, developmental delay, dysmorphic features, and autoimmune inflammatory cell infiltration of the lungs, liver, and gut. extensive testing failed to reveal a diagnosis. we used single-nucleotide polymor- phism (snp) autozygosity mapping to localize the disease gene to chromosome q and subsequently found that all affected patients were homozygous for a truncating mutation in itch (mim ), which codes for an e ubiquitin ligase. to our knowledge, we provide the first direct evidence connecting itch deficiency to human disease. the primary function of ubiquitin is to target proteins for degradation in the protesome. ubiquitination of target proteins is important for immune regulation. – ubiquitin tagging affects antigen processing by antigen-presenting cells and promotes immunological tolerance by changing signaling components to shift the balance away from acti- vation and toward anergy. – in mice, mutations of the e ligase itch cause fatal autoimmune disease characterized by histiocyte and lymphocyte infiltration of lungs, liver, kidneys, and heart. , our findings have broad implica- tions for the study of autoimmunity in humans and under- score the important role of ubiquitination in the develop- ment of other organ systems. three related amish children were evaluated for poor growth, developmental delay, hepatosplenomegaly, diar- rhea, and chronic lung disease. seven additional relatives (indiana¼ , pennsylvania¼ , new york¼ ) with a similar phenotype were identified during a field study and by personal correspondence. institutional review boards at indiana university/clarian hospitals and at lancaster general hospital approved the collection of clinical infor- clinical genetics and metabolism, department of pediatrics, university of c pediatric gastroenterology, hepatology, and nutrition, indiana university sch burg, pa, usa; department of medical genetics, indiana university school gastroenterology, hepatology, and nutrition, children’s hospital of pittsburgh of medicine, indianapolis, in, usa; franklin & marshall college, lancaster, pathology, children’s hospital of philadelphia, philadelphia, pa, usa *correspondence: jpmolles@iupui.edu doi . /j.ajhg. . . . ª by the american society of human the ameri mation and dna for genetic mapping. all patients demon- strated multiple lines of common ancestry, and each was the product of a consanguineous marriage (figure ). characteristic clinical features were dysmorphic facies, failure to thrive, hepatomegaly, splenomegaly, multi- system autoimmune disease, and delayed motor develop- ment (table ). the patients ranged from months to years of age; the mean age was . years, and the median age was years. growth was stunted, and patients dis- played relative macrocephaly (typically at the th percen- tile or above), weight and height below the rd percentile (z-score less than � ), and body mass index below normal (z-score less than � ). six of ten patients required gastro- stomy tube placement within the first year of life after having failed to gain weight with oral feedings, although only two had overt symptoms of malabsorption. distinc- tive craniofacial features included frontal bossing, dolicho- cephaly, orbital proptosis, flattened mid-face with a promi- nent occiput, small chin, and low, posteriorly rotated ears (figure ). the liver was typically – cm below the costal margin, and the spleen was – cm below the costal margin. the remainder of the physical examination was significant for global hypotonia and campto- or clinodac- tyly. all children were delayed in gross motor skills (they often did not walk until – years of age) and cognitive skills (they usually required special help in school). several of the children were treated for recurrent infections. complete blood counts, when available, did not demon- strate abnormal white blood cell counts, hematocrits, platelet counts, absolute neutrophil counts, or absolute lymphocyte counts except during acute infections. three patients had autoimmune hepatitis resulting in ala- nine aminotransferase (alt) and aspartate aminotransferase olorado at denver, aurora, co, usa; department of pediatrics, section of ool of medicine, indianapolis, in, usa; clinic for special children, stras- of medicine, indianapolis, in, usa; department of pediatrics, division of , pittsburgh, pa, usa; department of pathology, indiana university school pa, usa; lancaster general hospital, lancaster, pa, usa; department of genetics. all rights reserved. can journal of human genetics , – , march , https://core.ac.uk/display/ ?utm_source=pdf&utm_medium=banner&utm_campaign=pdf-decoration-v mailto:jpmolles@iupui.edu figure . pedigree demonstrating autosomal-recessive inheritance of traits from several common ancestors information obtained from a community genealogy book. unaffected siblings (both carriers and noncarriers) are not displayed because of space constraints. of the individuals tested, carriers include the parents of vii- , the father of vii- (no record of testing the mother), the mother and three siblings of vii- and vii- (no record of testing the father), two brothers of vii- and vii- , and one sister of vi- . (ast) levels that were – times the upper limit of normal. one had isolated anti-liver/kidney microsomal (lkm) anti- bodies, one had both lkm and antinuclear antibodies (ana), and a third had anti-neutrophil cytoplasmic anti- bodies (panca). liver biopsies on two children showed table . clinical and autoimmune features seen in itch-deficient pa clinical features patient number vii- vii- vi- vii- hepatomegaly and/or splenomegaly þ þ þ þ failure to thrive þ þ þ þ developmental delay þ þ þ þ dysmorphic features þ þ þ þ relative macrocephaly þ þ þ þ chronic lung disease þ þ - þ hypotonia þ þ þ þ autoimmune disease þ - þ - –hypothyroidism þ - þ - –hepatitis - - - - –enteropathy þ - - - –diabetes mellitus - - - - current age or age at death (in years) currently living? (yes or no) y y y n the american journal of human genetics , – , march dense periportal mixed inflammatory infiltrate with inter- face activity consistent with autoimmune hepatitis (figures a and b). in one of these children, there was substantial bridging fibrosis associated with profound loss of hepato- cytes. one child had improvement in transaminases in tients percent affectedvii- vii- vii- vi- vii- vii- þ - þ þ þ þ % þ þ þ þ þ þ % þ þ þ þ þ þ % þ þ þ þ þ þ % þ þ þ þ - þ % þ þ þ þ þ þ % - - þ ? þ ? % þ - þ þ þ - % - - þ - þ - % þ - þ - þ - % - - þ - - - % - - - þ - - % . . y y y y n n , figure . the two index patients photos were used with the family’s consent. figure . histology showing the effects of itch deficiency in multiple tissues (a) a mid-power view of the liver in a subject with autoimmune hepatitis shows a dense mixed inflammatory infiltrate throughout the periportal region and an accompanying cholangitis and reac- tive proliferation of bile ducts. (b) mid-power view of the liver in another subject with autoim- mune hepatitis shows a mixed inflammatory cell infiltrate in the periportal region. (c) lung showing a patchy expansion of the interstitium by lymphocytes and clusters of macrophages in the alveolar airspaces. there is no fibrosis. (d) higher magnification of the lung exhibiting a mild interstitial lymphocytic infiltrate. the central alveolar airspace contains a collection of macrophages (arrow). (e) although liver architecture is intact in a subject with normal liver enzymes but hepatomegaly, some multinucleated hepato- cytes are present, and periportal hepatocytes contain large lafora- body-like inclusions (arrow); there is no inflammation. (f) an electron micrograph of the same specimen as in e reveals heterogeneous cytoplasmic material (bounded by a dotted line): small droplets of fat and endoplasmic reticulum displace mito- chondria eccentrically (inset shows a normal hepatocyte for comparison). response to corticosteroids, one died of lung disease before autoimmune hepatitis was treated, and a third is currently under evaluation. two children had autoimmune enteropathy that re- sulted in chronic diarrhea. one of these children had anti-enterocyte antibodies, panca, and anti-smooth- muscle antibodies (sma), whereas another had anti-enter- ocyte antibodies only. both children had intestinal biop- sies that showed lymphocytic inflammation of the lamina propria; one had severe villous blunting (not shown). the child with mild inflammation improved on low-dose steroids, whereas the second improved after being treated with an array of immunosuppressives, including cortico- steroids, rapamycin, azathioprine, and tacrolimus. four of ten children developed hypothyroidism. auto- antibodies were found in the three children who were tested: two tested positive for anti-thyroid peroxidase (tpo), and one tested positive for anti-thyroglobulin. biochemical abnormalities resolved with appropriate thyroid hormone replacement. one subject was diagnosed with type diabetes mellitus; autoantibodies were not measured. nine of ten children had chronic lung disease, often characterized as asthma. five children had chronic lung opacities seen on chest radiography (n ¼ ) or chest computed tomography (n ¼ ). three had a chronic oxygen requirement, and one child required tracheostomy placement and home ventilator support. lung disease was treated with bronchodilators, antibiotics, and occasionally, the ameri corticosteroids with apparent response. a wedge lung biopsy of one severely affected child revealed mononuclear infiltrates expanding the interstitium and clusters of macrophages filling the alveolar airspaces (figures c and d). these findings are somewhat difficult to classify but seem to fit best as a cellular, nonspecific interstitial pneumonitis. three children died of respiratory failure. intermittent hepatomegaly was seen in several children in the absence of elevated serum transaminases. a liver biopsy in one such child was read as normal, but another demonstrated large pink inclusions in the cytoplasm of periportal hepatocytes after hematoxylin and eosin stain- ing; electron microscopy of these cells, but not other hepatocytes, revealed cytoplamic accumulations of large amounts of smooth endoplasmic reticulum (figures e can journal of human genetics , – , march , and f). a third child with intermittent marked elevations in transaminases had steatohepatitis on one biopsy and giant cell hepatitis without typical autoimmune features on another. total genomic dna from whole blood was isolated with the puregene dna isolation kit (qiagen, valencia, ca, usa) according to the manufacturer’s protocol. snp geno- typing was performed with the genechip mapping k assay kit (affymetrix, santa clara, ca, usa) as previously described. in brief, ng of double-stranded genomic dna was digested with xbai, the restriction fragments were ligated to adapters, and the ligated products were amplified in quadruplicate with a generic primer. pcr products were purified, fragmented with dnase, labeled with terminal deoxynucleotidyl transferase, and finally hybridized to a genechip human mapping k array (xbai , . ). microarrays were then washed with a fluidics station, incubated, and scanned with the gene- chip scanner enabled by genechip gcos software (affymetrix). data analyses ascertained genomic regions identically homozygous among affected individuals and assumed mutation and locus homogeneity. we calculated cumula- tive two-point lod scores (location scores) for each shared block of homozygous snps to determine the relative likeli- hood that the shared homozygous region harbored the disease gene. we used snps bounding a shared block to construct a candidate gene list by using the ncbi genome browser. for each gene, we assessed function and expres- sion to generate a priority list for sequencing. suitable candidates were subjected to polymerase chain reaction (pcr) amplification and sequencing of the coding regions and adjacent intron-exon boundaries. pcr primers were designed with primer . a ml reaction was prepared with u of taq polymerase (qiagen, valencia, ca), mm each of datp, dctp, dgtp, and dttp, . ml of pcr buffer (qiagen), and ng of genomic dna. we used a perkin-elmer or applied biosystems geneamp thermocycler to cycle the reaction for cycles ( s at �c, s at �c, s at �c), and this was fol- lowed by an min incubation at �c. pcr products were sequenced with the bigdye terminator cycle sequencing protocol (applied biosystems). extension products were then size-fractionated on an abi genetic analyzer. coding exons and adjacent intronic regions from candi- date genes from patient sequences were compared to the human reference sequence and dbsnp (ncbi) so that path- ogenic variants could be detected. genome-wide autozygosity mapping for five patients identified a large homozygous block in the pericentro- meric region of chromosome (figure a). the mb region was bounded by snps rs and rs and contained known or hypothetical genes. five of these candidate genes were sequenced on the basis of their predicted function. prominent autoimmune disease in one patient directed candidate gene sequencing to itch because of similar autoimmune findings in itch�/� the american journal of human genetics , – , march mice. sequence analysis of itch revealed a homozygous single basepair insertion in exon (c. _ insa) (figure b). this frameshift mutation was predicted to truncate itch at amino acid position (figures c and d). all affected individuals were homozygous for this variant, and among those tested, their parents were heterozygous. none of the nine heterozygous siblings or parents were symptomatic or dysmorphic. among unaffected siblings tested from five families, none were homozygous for the mutation. we developed a simplep- robe pcr assay for itch c. _ insa to facilitate rapid carrier and diagnostic testing. genotyping of random lancaster county old order amish identified no carriers. this is not unexpected because all affected individuals descend from the indiana amish, a group genetically distinct from the lancaster county amish. we did not have banked dna we could analyze from the indiana amish. human itch deficiency results in a complex phenotype that affects physical growth, craniofacial morphology, muscle development, and immune function. the conse- quences of itch deficiency in humans appear to be similar to those in itch�/� mice. nine of the ten affected children had chronic lung disease, and respiratory failure caused death in three. this lung disease, similar to that seen in mice, was probably a result of immune dysregulation. pathology results revealed a nonspecific interstitial pneu- monitis that has been linked previously to autoimmune disease. , five other patients had autoimmune disease in other organ systems. itch attaches ubiquitin to substrate proteins. ubiqui- tination of the t cell receptor mediates its downregula- tion, and downstream signaling events are altered by ubiquitination of proteins such as junb, which may inhibit il- production and thus t cell proliferation. dysfunction of e ligases, which catalyze the final step of ubiquitin attachment, can lead to indiscriminate t cell activation and loss of tolerance to self-antigens. , in mice, mutations of the e ligase itch cause fatal autoim- mune disease characterized by histiocyte and lymphocyte infiltration of the lungs, liver, kidneys, and heart. , ‘‘itchy’’ mice have a skewed th phenotype and dysfunc- tional b cell responses. the mice are small, dysmorphic, and pruritic and have elevated levels of circulating lymphocytes, immunoglobulins, and anti-nuclear anti- bodies. both antigen processing and t cell anergy are abnormal in affected animals, , and multiple organs are infiltrated with lymphocytes, particularly autoreactive b cells, that lead to fatal lung disease early in life. , , human itch deficiency causes disease beyond the immune system, consistent with the role of protein ubiquitination in cell trafficking, functional activation, proteosomal degradation, and programmed cell death. in humans, itch is strongly expressed in the gi tract, pancreas, neuronal cells, and lymphoid tissue but is present in many other tissues (protein atlas; see web resources). the craniofacial abnormalities that lead to , figure . genetic mapping and gene mutation in itch-deficient patients (a) genetic mapping to chromosome p . -q . a block of contiguous snps on chromosome provided overwhelming evidence for identity-by-descent in five affected old order amish patients. (b) the itch sequencing trace; sequencing of the coding exons of itch identified a single base-pair insertion in exon . panel shows sequence from a control sample demonstrating normal exon sequence. panel depicts sequence from an obligate heterozygote showing the single base-pair insertion. panel illustrates homozygosity for the c. _ insa mutation (the red a denotes the mutation). (c and d) the imputed effect of the insertion mutation on itch translation. the mutation causes a frameshift predicted to misincor- porate eight amino acids in the peptide chain and cause a premature termination at codon . the truncated itch protein lacks the three ww domains as well as the hect domain that contains the e interaction (active) site. the distinctive facies in all of these children suggest a devel- opmental role for itch in nonhematopoietic cells. the organomegaly and occasional cytoplasmic inclusions suggest possible storage of nonubiquitinated protein in the cells. ubiquitin-related protein accumulation in the brain has been associated with some cns diseases and may provide an explanation for psychomotor delays seen in affected children. a recent report suggests that itch might regulate nod signaling, which could explain the bowel inflammation in two of these patients. the ameri the link between itch deficiency and immune dysregu- lation has implications for treatment. screening for auto- immune disease is important in these children, and auto- immune manifestations in liver, intestine, and lung sometimes respond to immunosuppressive treatment. in patients who can be diagnosed early in life, such therapy could halt or slow progression of the disease. moreover, exciting oncology research in recent years has focused on drugs that might affect ubiquitin pathways and thus allow more specific molecular therapies for the disorder. the can journal of human genetics , – , march , severe phenotype in a few of the patients even raises consideration of bone marrow transplantation as a poten- tial therapy. the discovery of involvement of a ubiquitin ligase in autoimmune disease has significance beyond this isolated amish community. the disease demonstrates the vital importance of ubiquitin pathways in the cell signaling that controls t cell responsiveness in humans. the fact that only some of the itch-deficient patients had severe autoimmune disease suggests that, not surprisingly, other genetic modifiers or environmental factors contribute to t cell anergy. on the other hand, it is notable that other ubiquitin ligases in the cell fail to complement the defect enough to restore a normal phenotype. further studies will be needed to clarify the pathophysi- ology of itch deficiency. morphologic examination of the affected tissues might further clarify the site of substrate accumulation or the specific subcellular site of toxicity. functional assays will be necessary for determining the fate of ubiquitinated substrates such as jun in the cell and for studying downstream signaling pathways such as those involving notch. detailed studies of immunolog- ical function, t cell regulation, and autoimmunity are underway. our report describes an approach to the elucidation of a novel genetic disorder within an isolated population. prior to our investigations, several of these children had undergone extensive medical evaluations. the cause of their illness had not been found. the molecular diagnosis that relates the disease process to a mutation in the immune regulatory gene itch provides a new biological framework within which clinical observations and selected laboratory data can be used for studying the natural history of this disorder and evaluating therapies. as in our previous reports about genetic mapping in the old order amish and mennonite populations, this approach repre- sents a cost-efficient way to discover the genetic basis of disease. supplemental data supplemental data include one table and can be found with this article online at http://www.cell.com/ajhg. acknowledgments naomi lohr was funded by an american academy of pediatrics resident research grant. the authors thank vicki haviland-wil- hite and sharon mcpheeters for expert secretarial assistance. they also thank the amish families who shared their medical histories and allowed physical examinations and blood draws. jean molleston receives research funding support from hoffman laroche. received: november , revised: january , accepted: january , published online: february , the american journal of human genetics , – , march web resources the urls for data presented herein are as follows: online mendelian inheritance of man (omim), http://www.ncbi. nlm.nih.gov/omim/ genome browser, http://www.ncbi.nlm.nih.gov/ single nucleotide polymorphism database, http://www.ncbi.nlm. nih.gov/snp/ protein atlas, http://www.proteinatlas.org/ references . anandasabapathy, n., ford, g.s., bloom, d., holness, c., para- gas, v., seroogy, c., skrenta, h., hollenhorst, m., fathman, c.g., and soares, l. ( ). grail: an e ubiquitin ligase that inhibits cytokine gene transcription is expressed in aner- gic cd þ t cells. immunity , – . . mueller, d.l. ( ). e ubiquitin ligases as t cell anergy factors. nat. immunol. , – . . safford, m., collins, s., lutz, m., allen, a., huang, c.t., kowal- ski, j., blackford, a., horton, m.r., drake, c., schwartz, r.h., and powell, j.d. ( ). egr- and egr- are negative regulators of t cell activation. nat. immunol. , – . . bilodeau, p.s., urbanowski, j., winistorfer, s., and piper, r. ( ). the vps p-hse p complex binds ubiquitin and mediates endosomal sorting. nat. cell biol. , – . . lu, q., hope, w., brasch, m., reinhard, c., and cohen, s. ( ). tsg interaction with hrs mediates endosomal trafficking and receptor down-regulation. proc. natl. acad. sci. usa , – . . jeon, m.-s., atfield, a., venuprasad, k., krawczyk, c., sarao, r., elly, c., yang, c., arya, s., bachmaier, k., su, l., et al. ( ). essential role of the e ubiquitin ligase cbl-b in t cell anergy induction. immunity , – . . matesic, l., copeland, n., and jenkins, n. ( ). itchy mice: the identification of a new pathway for the development of autoimmunity. in immunology, phenotype first: how muta- tions have established new principles and pathways in immu- nology. current topics in microbiology and immunology, b. buetler, ed. (heidelberg: springer-verlag), pp. – . . matesic, l.e., haines, d., copeland, n., and jenkins, n. ( ). itch genetically interacts with notch in a mouse autoim- mune disease model. hum. mol. genet. , – . . puffenberger, e.g., hu-lince, d., parod, j.m., craig, d.w., dobrin, s.e., conway, a.r., donarum, e.a., strauss, k.a., dunckley, t., cardenas, j.f., et al. ( ). mapping of sudden infant death with dysgenesis of the testes syndrome (siddt) by a snp genome scan and identification of tspyl loss of function. proc. natl. acad. sci. usa , – . . katzenstein, a.l.a., and myers, j.l. ( ). nonspecific inter- stitial pneumonia and the other idiopathic interstitial pneu- monias: classification and diagnostic criteria. am. j. surg. pathol. , – . . kinder, b.w., collard, h.r., koth, l., daikh, d.i., wolters, p.j., elicker, b., jones, k.d., and king, t.e. jr. ( ). idiopathic nonspecific interstitial pneumonia lung manifestation of undifferentiated connective tissue disease? am. j. respir. crit. care med. , – . . lorick, k.l., yang, y., jensen, j.p., iwai, k., and weissman, a.m. ( ). studies of the ubiquitin proteasome system. in , http://www.cell.com/ajhg http://www.ncbi.nlm.nih.gov/omim/ http://www.ncbi.nlm.nih.gov/omim/ http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/snp/ http://www.ncbi.nlm.nih.gov/snp/ http://www.proteinatlas.org/ current protocols in cell biology, chapter , unit . (hoboken, nj:: john wiley and sons). . bachmaier, k., krawczyk, c., kozieradzki, i., kong, y.y., sasaki, t., oliveira-dos-santos, a., mariathasan, s., bouchard, d., wakeham, a., itie, a., et al. ( ). negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor cbl-b. nature , – . . parravicini, v., field, a.-c., tomlinson, p.d., basson, m.a., and zamoyska, r. ( ). itch �/� ab and gd t cells independently contribute to autoimmunity in itchy mice. blood , – . . zhang, m., veselits, m., o’neill, s., hou, p., reddi, a.l., berlin, i., ikeda, m., nash, p.d., longnecker, r., band, h., and clark, m.r. ( ). ubiquitinylation of igb dictates the endocytic fate of the b cell antigen receptor. j. immunol. , – . . hershko, a., ciechanover, a., and varshavsky, a. ( ). the ubiquitin system. nat. med. , – . . martinez, a., potero-otin, m., pamplona, r., and ferrer, i. ( ). protein targets of oxidative damage in human neuro- the ameri degenerative disease with abnormal protein aggregates. brain pathol. published online august , . . /j. - . . .x. . tao, m., scacheri, p.c., marinis, j.m., harhaj, e.w., matesic, l.e., and abbott, d.w. ( ). itch k -ubiquitinates the nod binding protein, rip , to influence inflammatory signaling pathways. curr. biol. , – . . nalepa, g., rolfe, m., and harper, j.w. ( ). drug discovery in the ubiquitin-proteasome system. nat. rev. drug discov. , – . . burt, r.k., slavin, s., burns, w.h., and marmont, a.m. ( ). induction of tolerance in autoimmune disease by hematopoi- etic stem cell transplantation: getting closer to a cure? blood , – . . strauss, k.a., and puffenberger, e.g. ( ). genetics, medi- cine, and the plain people. annu. rev. genomics hum. genet. , – . . eicher, b.j., and schartz, m.n. ( ). das schwatz buch descendants of john j. schwartz – (gordonville, pa: print shop), pp. . can journal of human genetics , – , march , 	human itch e ubiquitin ligase deficiency causes syndromic multisystem autoimmune disease 	acknowledgments 	web resources 	references deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue a wednesday, february , of cardiac troponin c’s defunct ca þ -binding site (site ). in this study, we combined in vitro and in situ structural and functional techniques to elucidate the role this mutation may play in the modulation of troponin’s function. we used nuclear magnetic resonance to solve the structure and characterize the backbone dynamics of the regulatory lobe of troponin c with this mutation. the overall structure and dynamics of troponin c was not significantly altered by l q; however there was a slight rearrangement of site making it more similar to trout cardiac troponin c, which also has a glutamine at position at residue and displays increased ca þ sensitivity. backbone dynamics mea- surements indicated that q was more flexible than l . the structure and function of l q was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. the structure and/or orientation of the regulatory lobe of troponin c was slightly perturbed by l q in relaxing conditions and was unaffected at activating ca þ concentrations. the ca þ sensitivity of the structural change and contractility were both unaltered by the l q mutation, suggesting that while this may cause a small change in the structure of troponin c, this does not translate to a large functional effect in cardiac muscle. -pos board b troponin i ser- phosphorylation sustains troponin ca d sensitivity in an acidic environment benjamin r. nixon, shane d. walton, jonathan p. davis, brandon j. biesiadecki. physiology and cell biology and davis heart and lung research institute, the ohio state university, columbus, oh, usa. a hallmark of cardiac ischemia is decreased intracellular ph which can affect a number of cellular processes. such an acidic environment alters cardiac troponin (tn) myofilament regulation to decrease ca þ sensitive force produc- tion. tn also undergoes cardiac ischemia-induced ampk troponin i (tni) ser- phosphorylation. we recently characterized the effects of tni ser- phos- phorylation demonstrating that it blunted the functional effects of canonical tni ser- / phosphorylation; however, the role of ser- phosphorylation in ischemia remains unknown. as an initial step, we sought to investigate the effect of acidic ph on myofilament regulation in the presence of tni ser- phos- phorylation alone and in combination with ser- / phosphorylation. we first investigated the effect of in vivo cardiac ischemia on levels of tni ser- and ser- / phosphorylation. exposure to minutes of regional ischemia re- sulted in elevation of both tni ser- and ser- / phosphorylation. next we determined the effects of tni ser- pseudo-phosphorylation (s d) on the myofilament by measuring troponin c (tnc) ca þ binding properties at normal and acidic ph. results demonstrate acidic ph decreases steady- state ca þ binding to tnc in reconstituted thin filaments across all tn (wt, s d, s / d, and s / / d) such that tni s d ca þ sensitivity at ph . is similar to wt at ph . decreasing the ph had no effect on ca þ disso- ciation such that compared to wt, s / / d remained fast while s d was slowed. we conclude that tni ser- phosphorylation imparts resistance to acidic ph-induced myofilament ca þ desensitization while retaining increased tn ca þ dissociation when in combination with ser- / phosphorylation suggesting the potential for an increase in force while maintaining accelerated ca þ dissociation. future investigations are aimed at examining the effect of tni ser- and ser- / phosphorylation on protease cleavage of tni. -pos board b deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue bin wei, yingru lu, j.-p. jin. physiology, wayne state university, detroit, mi, usa. loss of slow skeletal muscle troponin t (sstnt) due to a nonsense mutation at codon glu in exon of the tnnt gene causes a severe form of recessive nemaline myopathy (amish nemaline myopathy, anm). to investigate the pathogenesis and muscle pathophysiology of anm, we studied the phenotypes of partial and total loss of sstnt in tnnt gene targeted mice. an insertion of neomycinr cassette in intron of tnnt caused approximately % decrease in sstnt protein expression whereas deletion of exons - using cre-loxp approach resulted in total loss of sstnt as that seen in the muscle of anm pa- tients. in diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that sstnt deficiency resulted in significantly decreased levels of other slow fiber-specific myofilament proteins while fast fiber-specific myofilament proteins were increased. histology studies revealed that sstnt deficiency caused significant atrophy of type i slow fibers and a hypertrophic growth of type ii fast fibers. along with the slow fiber atrophy and the changes in myofilament protein isoform contents, sstnt deficiency in soleus muscle shifted the force-frequency relationship toward the fast muscle type and significantly reduced the tolerance to fatigue. sstnt deficient soleus muscle also exhibited a significant number of smaller size central nuclei type i fibers, indicating an adaptive regeneration. sstnt deficient mouse soleus muscle contained apparently normal number of spindles, in which intrafusal fibers were positive for type i myosin with a trend of atrophic morphology. the results demonstrate the essential function of sstnt in skeletal muscle and the causal effect of its loss on the pathology of anm. -pos board b attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp thomas j. longyear , matthew a. turner , brandon j. biesiadecki , joseph lopez , jonathan p. davis , edward p. debold . university of massachusetts, amherst, ma, usa, the ohio state university, columbus, oh, usa. repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, which defines fatigue. the decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains unclear. we sought to gain novel insight into the decrease in velocity by determining if the depressive effect of acidosis could be altered by ) introducing ca þþ -sensitizing mutations into troponin (tn) or ) by agents that directly affect myosin function, including inorganic phosphate (pi) and -deoxy-atp (datp) in an in vitro motility assay. acidosis reduced regulated thin filament velocity (vrtf) at both maximal and sub- maximal caþþ levels in a ph-dependent manner. a truncated construct of the inhibitory subunit of tn, r , and a caþþ-sensitizing mutation in the caþþ- binding subunit of tn, v q, increased vrtf at sub-maximal ca þþ under acidic conditions, but had no effect on vrtf at maximal ca þþ levels. in contrast, both mm pi and replacement of atp with datp reversed much of the acidosis- induced depression of vrtf at saturating ca þþ ( . . control, . . withpi, . . withdatp, . . withbothpi anddatp),withthecombined effectfully restoringthevrtf to thevalue under control conditions.interestingly, despite producing similar magnitude increases in vrtf, the combined effects of pi and datp were additive, suggesting different underlying mechanisms of action. these results suggest that the major mechanism by which acidosis slows vrtf is through directly slowing myosin’s rate of detachment from actin. -pos board b the effect of truncated troponin components on activation of lethocerus flight muscle belinda bullard , bogos agianian , gian-felice de nicola , annalisa pastore , kevin leonard . department of biology, university of york, york, united kingdom, molecular biology and genetics, democritus university of thrace, alexandroupolis, greece, national institute of medical research, london, united kingdom, european bioinformatics institute, cambridge, united kingdom. indirect flight muscle (ifm) of lethocerus is activated by periodic stretches at a constant priming concentration of calcium. the muscle is unusually stiff and stress is transmitted to the thick and thin filaments by kettin, which reinforces links between both filaments and the z-disc. the activating effect of stress on thin filaments is likely to affect troponin. the isoforms of troponin in ifm differ from those in other muscles. tnt has a c-terminal extension not present in vertebrate tnt; tnh is an isoform of tni with a c-terminal extension rich in pro and ala; tnc is present in two isoforms: f binds a single calcium in the c-lobe and is needed for stretch-activation; f binds one calcium in both n- and c-lobes and is needed for isometric force. under conditions of low ionic strength, native fibres have a force-pca curve that shows high calcium- sensitivity and low cooperativity (pca = . , nh = . ). fibres with f alone have a pca curve similar to that of cardiac muscle, (pca = . , nh = . ). a fragment of f without the n-lobe (f -ct) inhibits stretch-activation; therefore the n-lobe of f is necessary, although it does not bind calcium or tnh. f -ct is displaced by f and isometric force is restored, but not stretch-activation. we hope to show the effect of replacing endogenous troponin in fibres with a com- plex containing tnt truncated at the c-terminus, tnh with tni sequence but without the pro-ala extension, and either f or f . this will show how impor- tant the ifm isoforms of troponin are to the stretch-activation response. -pos board b changes in the orientation of the myosin light chain domain (lcd) associated with thick filament-based regulation of skeletal muscle luca fusi, zhe huang, malcolm irving. randall division of cell and molecular biophysics, king’s college london, london, united kingdom. the dependence of myosin lcd orientation on temperature, myofilament lattice spacing and sarcomere length was determined using fluorescence 	troponin i ser- phosphorylation sustains troponin ca + sensitivity in an acidic environment 	deficiency of slow skeletal muscle troponin t causes atrophy of type i slow fibers and decreases tolerance to fatigue 	attentuating the depressive effect of acidosis with mutations in troponin and with -deoxy-atp 	the effect of truncated troponin components on activation of lethocerus flight muscle 	changes in the orientation of the myosin light chain domain (lcd) associated with thick filament-based regulation of skelet ... structural, transport and optical properties of (la . pr . ) . ca . mno nanocrystals: a wide band-gap magnetic semiconductor dalton transactions paper cite this: dalton trans., , , received th november , accepted th december doi: . /c dt j www.rsc.org/dalton structural, transport and optical properties of (la . pr . ) . ca . mno nanocrystals: a wide band-gap magnetic semiconductor satyam kumar,a g. d. dwivedi,b shiv kumar,a r. b. mathur,c u. saxena,d a. k. ghosh,a amish g. joshi,c h. d. yangb and sandip chatterjee*e (la . pr . ) . ca . mno system has been synthesized via a sol–gel route at different sintering tempera- tures. structural, transport and optical measurements have been carried out to investigate (la . pr . ) . - ca . mno nanoparticles. raman spectra show that jahn–teller distortion has been decreased due to the presence of ca and pr in a-site. magnetic measurements provide a curie temperature around k and saturation magnetization (ms) of about . µb/mn at k. x-ray photoemission spectroscopy study suggests that mn exists in a dual oxidation state (mn + and mn +). resistivity measurements suggest that charge-ordered states of mn + and mn +, which might be influenced by the presence of pr, have enhanced insulating behavior in (la . pr . ) . ca . mno . band gap estimated from uv-vis spectroscopy measurements comes in the range of wide band gap semiconductors (∼ . ev); this makes (la . pr . ) . ca . mno a potential candidate for device application. introduction hole doped manganite systems have been generating curiosity among scientists and engineers for more than years because of their unprecedented and wide range of properties, e.g., ferromagnetism, insulator-metal transition and colossal magnetoresistive properties. the physics behind all these pro- perties is even more challenging and has attracted attentions of theoreticians and experimentalists with almost equal authority. – manganite materials have been used in different electronic devices such as magneto-tunable photocurrent devices, resistive switching devices, and spin hall magneto- resistive devices. – the parent compound lamno is known to be an antiferromagnetic insulator, while divalent cation (ca +, sr + and ba +) doping induces paramagnetism with polaron type conductivity at high temperatures, and metallic ferromagnetism below the curie temperature tc. the origin of ferromagnetism has been attributed to the double-exchange interaction between the valence electronic states of mn +–o− – mn +. – the double-exchange mechanism along with the effect of lattice distortion is believed to be responsible for the occurrence of colossal magnetoresistance (cmr). , as an outcome of the double-exchange interaction and the lattice distortion, a large spin splitting of the conduction band (majority and minority sub-bands) takes place in the ferro- magnetic state. – governed by the hund’s rule, these sub- bands are separated by few electron-volts energy, which depends on a-site doping. the large spin splitting produces the half-metallic properties of the material because at fermi level, charge carriers with only one spin direction (up/down) are present, whereas there is a gap in the density of states for the carriers with the other spin direction (down/up). hole doped manganite systems in the nano-range act in a different way, i.e., ferromagnetic ordering and metal-insulator transition are well separated from each other. transport pro- perties basically depend on boundary conditions. in nano- range, surface to volume ratio increases; thus, the boundary effect becomes more pronounced in transport properties. due to more boundaries, charge carriers face much more scatter- ing, and as a result the resistivity of the system increases. however, the double exchange interaction between mn + and mn + is not affected by the boundary conditions, and therefore it is not affected much in the nano-phase. magnetic properties of ca doped prmno (pr −xcaxmno ) materials at low temperatures are governed by mn +/mn + ordering, intra- and inter-mn exchange interactions and struc- tural distortions produced by the ca + cations. prmno has an antiferromagnetic ground state. with ca doping, a ferromagnetic ground state develops for x = . . the charge-ordering of the mn-ions occurs over a wide range from . < x < . with an adepartment of physics, banaras hindu university, varanasi- , india bdepartment of physics, national sun yat-sen university, kaohsiung- , taiwan ccsir-national physical laboratory, dr. k.s. krishnan road, new delhi- , india ddepartment of physics, mahila mahavidyalaya, banaras hindu university, varanasi- , india edepartment of physics, indian institute of technology (banaras hindu university), varanasi- , india. e-mail: schatterji.app@iitbhu.ac.in this journal is © the royal society of chemistry dalton trans., , , – | p ub li sh ed o n j an ua ry . d ow nl oa de d by n at io na l p hy si ca l l ab or at or y (n p l ) on / / : : . view article online view journal | view issue www.rsc.org/dalton http://crossmark.crossref.org/dialog/?doi= . /c dt j&domain=pdf&date_stamp= - - http://dx.doi.org/ . /c dt j http://pubs.rsc.org/en/journals/journal/dt http://pubs.rsc.org/en/journals/journal/dt?issueid=dt ordering temperature near k. , pr . ca . mno shows insulating behavior in both the paramagnetic and ferromagnetic states because of the charge-ordered state of mn + and mn +. magnetic and transport properties of manganites have been studied with great interest and enthusiasm by scientists and will be continued to be studied in the future because of the vast range of properties these systems possess which make them desirable from an application point of view. however, studies have not been done concerning their optical properties because these systems show either insulator (large band gap; typically > ev) behavior or metallic (no band gap) behavior, which makes them less interesting for optical studies. in this manuscript, we have doped % pr in the la site of la . ca . mno system, which shows metallic ferromagnetic behavior below k. the idea behind the doping of pr is that the pr . ca . mno system shows ferromagnetic charge- ordered insulator behavior. we are expecting that with the doping of pr, the semiconducting behavior will increase because of the stabilization of charge-ordering, along with the sustaining magnetic order of the system. along with a strong magnetic moment, if we can induce a semiconducting nature, these systems can be used as a better candidate to improve magnetic semiconducting devices. to realize this pheno- menon, we have used la . ca . mno nanoparticles, which show better semiconducting nature along with ferromagnetic ordering as compared to its bulk counterpart. in addition, the doping of pr may also increase its semiconducting behavior without disturbing the magnetic ordering. we are using par- ticularly % of pr doping in the la site because excess doping results in a completely insulating phase, while doping less than % decreases the band gap significantly, and both the cases are not favorable for application purpose. experimental section (la . pr . ) . ca . mno samples were synthesized using the sol–gel method. analytical grade metal oxides pr o , la o , and caco powders were taken in stoichiometric proportions ( . mol: . mol: . mol) and dissolved in nitric acid (hno ) and thoroughly mixed for minutes. the prepared solution of metal nitrates along with mn(no ) · h o ( mol) were mixed in aqueous solution of citric acid [c h o ] ( . % purity) with stirring, to obtain a homogeneous precursor solu- tion. citric acid serves as the fuel for the reaction. in this solu- tion, ethylene glycol was added drop wise with stirring. citric acid, ethylene glycol and metal nitrates were precisely taken in a : : molar ratio. the precursor solution was dried at °c for h in an oven to obtain xerogel and the swelled xerogel was kept at °c for h to dry. after grinding the xerogel, powders were sintered at , , °c for h under air to obtain (la . pr . ) . ca . mno nanoparticles. phase purity of the as-prepared (la . pr . ) . ca . mno samples were checked with a model: philips x’pert prox-ray diffractometer using cukα radiation (λ = . Å). transmission electron microscopy measurements were done with a jeol- (japan). raman spectra were obtained with a renishaw micro- raman spectroscope in the range of cm− – cm− using . nm ar+ laser as the excitation source. fourier transform infrared (ftir) spectroscopy measurements were done with an ftir spectrometer (spectrum one, perkin elmer instrument, usa) in the range of – cm− with a resolu- tion of cm− . magnetic measurements were done using a superconducting quantum interference device [magnetic prop- erty measurement system (mpms) xl- , quantum design inc.]. room temperature x-ray photoemission spectroscopy measure- ments were done using a omicron multiprobe surface analysis system operating at an average base pressure of ∼ × − torr with a monochromatic alkα line at . ev. the total energy resolution, estimated from the width of the fermi edge, was about . ev for monochromatic alkα line with photon energy . ev. resistivity measurements were done via -probe method using a keithley source meter. the optical absorp- tion spectra were measured in the range of – nm using a uv-vis spectrometer (shimadzu). results and discussion structural analysis x-ray diffraction patterns of (la . pr . ) . ca . mno sintered at °c, °c and °c have been shown in fig. . all the three samples are in single phase (without any secondary phase) and crystallize in orthorhombic pbnm space group like their extreme parents la . ca . mno and pr . ca . mno . we have determined average crystallite size (d) of all the three samples sintered at °c, °c and °c from the scherrer’s formula: d ¼ : λ β cos θ where λ = . Å, wavelength of x-ray used, β is full width at half maximum (fwhm) of bragg’s reflection plane and θ is fig. x-ray diffraction pattern of (la . pr . ) . ca . mno nanoparti- cles sintered at °c, °c and °c. paper dalton transactions | dalton trans., , , – this journal is © the royal society of chemistry p ub li sh ed o n j an ua ry . d ow nl oa de d by n at io na l p hy si ca l l ab or at or y (n p l ) on / / : : . view article online http://dx.doi.org/ . /c dt j half of the angle of the corresponding reflection plane. we have used the most intense peak ( ) as a reference plane to calcu- late the crystallite size, which is observed to be about nm, nm and nm for the samples sintered at °c, °c and °c, respectively. we have also estimated the strain using the williamson–hall method. the average strain values for different sizes of (la . pr . ) . ca . mno are given in table . to study the surface morphology and microstructure of (la . pr . ) . ca . mno nanocrystals, low-resolution trans- mission electron microscopy (tem) measurements have been performed. due to the strong magnetic moment, particles are strongly agglomerated with each other to make irregular shapes and sizes, which makes it rather difficult to distinguish one from the other. estimated average grain sizes of the three samples sintered at °c, °c and °c is observed to be about nm, nm and nm respectively, which is con- sistent with the x-ray diffraction results, which suggests increase in crystallite sizes with increasing sintering tempera- ture (fig. (a, c and e)). selected area electron diffraction (saed) images show increase in crystalline nature of (la . pr . ) . ca . mno system with increase in sintering temperature (fig. (b, d and f)). (la . pr . ) . ca . mno sin- tered at °c shows much clearer and brighter bragg’s spots than the rest of the two systems sintered at °c and °c, which indicates the better crystalline nature of this system. x-ray diffraction, low-resolution tem and saed measurements have confirmed the single crystalline nature of our samples with almost homogeneous distribution. raman spectroscopy to investigate crystal structure, lattice distortions and defects, raman spectroscopy is considered to be the most powerful non-destructive technique. we doped % pr in the la-site of la . ca . mno to obtain magnetic semiconductor materials. to study the effect of doping on the crystal struc- ture and lattice distortions, we obtained raman spectra of (la . pr . ) . ca . mno nanoparticles sintered at °c, °c and °c, which have been shown in fig. . in the orthorhombic rare-earth manganites, raman modes become active due to deviations from the ideal cubic perovskite struc- ture. group theory analysis of the lamno structure suggests normal modes. among all these modes, only modes are raman active and the remaining modes are infrared (ir) active. Γoptical ¼ ð ag þ b g þ b g þ b gÞraman þ ð au þ b u þ b u þ b uÞir table particle sizes estimated from x-ray diffraction and tem, and average strain estimated using williamson–hall method samples average strain (estimated using williamson–hall method) particle size (nm) (estimated from xrd data) particle size (nm) (estimated from tem) (la . pr . ) . ca . mno sintered at °c . × − (la . pr . ) . ca . mno sintered at °c . × − (la . pr . ) . ca . mno sintered at °c . × − fig. low-resolution transmission electron micrographs and their corresponding selected area electron diffraction images of (la . pr . ) . ca . mno nanoparticles sintered at °c (a, b), °c (c, d) and °c (e, f). fig. raman spectra of (la . pr . ) . ca . mno nanoparticles sin- tered at °c, °c and °c. dalton transactions paper this journal is © the royal society of chemistry dalton trans., , , – | p ub li sh ed o n j an ua ry . d ow nl oa de d by n at io na l p hy si ca l l ab or at or y (n p l ) on / / : : . view article online http://dx.doi.org/ . /c dt j these raman active modes can be divided into sym- metric modes, asymmetric stretching modes, bending modes, rotation/tilt modes of the octahedral, and the remaining modes are associated with the motion of a-site (la/pr/ca) cations. all these raman modes become active because of fundamental distortions from the ideal perovskite structure, namely, rotations of mno octahedra around the cubic [ ]c and [ ]c axes, jahn–teller distortion, and a-site (la/pr/ca) shift from its position in the ideal perovskite lattice. the raman spectra of (la . pr . ) . ca . mno sintered at °c, °c and °c do not show any phase changes due to change in sintering temperature (fig. ). we observed distinct raman modes in – cm− range; first, around cm− , second around cm− and third around cm− . the raman mode around cm− assigned as b g( ) is related to the symmetric stretching vibration of oxygen in mno octahedra. however, a very broad raman mode around cm− can be divided into two distinct modes, namely, cm− (ag( )) and cm − (b g( )), related to the jahn–teller type asymmetric stretching mode and sym- metric bending modes of mno octahedra, respectively. dediu et al. reported that pr . ca . mno shows no variation in raman spectra above the charge-ordered transition tempera- ture (tco), while below tco, raman mode near cm − has been divided into two modes: ag( ) and b g( ). in our case, due to the presence of ca and pr in a-site, the jahn–teller dis- tortion at room temperature further suppressed, and the raman mode related to this becomes relatively less promi- nent. consequently, the modes around cm− are not con- siderably distinguishable. the raman mode around cm− denoted as ag( ) is related to the tilting of mno octahedra. raman modes observed in the range of – cm− are pre- dominantly because of the vibrations of a-site cations. fourier transform infrared spectroscopy to get information about the molecular and functional species present on the surface and to further investigate lattice vibration present in our system, we used fourier transform infrared (ftir) spectroscopy. ftir measurements of (la . pr . ) . ca . mno sintered at °c, °c and °c have been shown in fig. . in the finger-print region, the band around cm− corresponds to the characteristic mn–o bond. this confirms that each sample strongly contains mn–o bond and a change in the bond length of mn–o–mn, because of the internal motion, is responsible for the band for- mation. stretching vibration is responsible for the change in mn–o–mn length, while bending vibration involves the change of mn–o–mn bond angle. the peaks at and cm− corresponds to the asymmetric stretching vibrations of cvc bond and symmetric stretching of cvo bond in citrate (which formed in the solution and may be present on the surface of nano-crystals). magnetic properties to investigate the magnetic properties of (la . pr . ) . - ca . mno nanoparticles sintered at °c, °c and °c, we performed temperature dependent field cooled magnetization measurements (m–t) from k to k at oe magnetic field (fig. ). m–t measurement of all the samples show that ferromagnetic transition occurs around k and particle size is not showing any significant effect on curie temperature, but it is considerably affecting the mag- netic moment. (la . pr . ) . ca . mno sintered at °c, °c and °c show magnetic moments of . emu gm− , . emu gm− and . emu gm− , respectively, at k. at a low magnetic field, the (la . pr . ) . ca . mno sintered at °c shows a higher magnetization value than the (la . pr . ) . ca . mno sintered at °c; however, at a fig. fourier transform infrared spectroscopy of (la . pr . ) . - ca . mno nanoparticles sintered at °c, °c and °c. fig. field cooled temperature dependent magnetization measure- ments of (la . pr . ) . ca . mno nanoparticles sintered at °c, °c and °c at oe magnetic field down to k. inset figure shows the m–h hysteresis loop up to t magnetic field at k. paper dalton transactions | dalton trans., , , – this journal is © the royal society of chemistry p ub li sh ed o n j an ua ry . d ow nl oa de d by n at io na l p hy si ca l l ab or at or y (n p l ) on / / : : . view article online http://dx.doi.org/ . /c dt j higher field (la . pr . ) . ca . mno sintered at °c shows a higher saturated magnetic moment. calculated satur- ation magnetization (ms) of (la . pr . ) . ca . mno is observed to be around . µb/mn (with % of mn + and % of mn +). m–h measurements at k show that t magnetic field gives saturation magnetization of . µb/mn, . µb/mn and . µb/mn for (la . pr . ) . ca . mno sintered at °c, °c and °c, respectively (inset fig. ). mag- netic moment observed for (la . pr . ) . ca . mno sintered at °c is very close to the calculated magnetic moment . µb/mn. this shows that with increasing particle size, mag- netic response increases consistently and approaches the mag- netic moment of bulk (la . pr . ) . ca . mno . this confirms the strong ferromagnetic character of our samples below k. the ferromagnetic behavior of a similar (la . pr . ) . - ca . mno has been reported earlier, which is consistent with our result. x-ray photoemission spectroscopy x-ray photoemission spectroscopy (xps) study has been done to investigate the chemical state of (la . pr . ) . ca . mno nanoparticles sintered at °c, °c and °c (fig. ). a survey scan of (la . pr . ) . ca . mno sintered at °c, °c and °c confirms the presence of la, pr, ca, mn and o on the surface (fig. (a)). high resolution xps core level spectra of ca p, o s, and mn p regions have been shown in fig. (b), (c) and (d) respectively. in the ca p region, peaks observed at . ev and ev have been denoted as ca p / and ca p / . the spin–orbit splitting energy is . ev, which indicates that the ca exists in + oxidation state. in o s region, two peaks at . ev and . ev are attributed to the contribution of the crystal lattice oxygen and adsorbed oxygen, respectively. the adsorbed oxygen has a tendency to bind with oxygen vacancies (vo). in the mn p region, the two fig. (a) survey scan x-ray photoemission spectroscopy (xps) for (la . pr . ) . ca . mno nanoparticles sintered at °c, °c and °c. high resolution core level xps of (b) ca p region, (c) o s region, (d) mn p region for (la . pr . ) . ca . mno nanoparticles sintered at °c, °c and °c. (e) deconvoluted peaks of mn p region for (la . pr . ) . ca . mno nanoparticles sintered at °c as a representative. (f) valence band spectra of (la . pr . ) . ca . mno nanoparticles sintered at °c, °c and °c. dalton transactions paper this journal is © the royal society of chemistry dalton trans., , , – | p ub li sh ed o n j an ua ry . d ow nl oa de d by n at io na l p hy si ca l l ab or at or y (n p l ) on / / : : . view article online http://dx.doi.org/ . /c dt j peaks are located at . and ev, which belong to mn p / and mn p / , respectively. the peaks of mn p / and mn p / can be deconvoluted into two peaks each. the deconvoluted peaks of mn p region for (la . pr . ) . ca . mno sintered at °c have been shown as a representative in fig. (e). the deconvoluted peaks of mn p / at . ev and . ev (and mn p / at . ev and . ev) represent mn + and mn +, respectively. this confirms that mn exists in two oxi- dation states (+ and + ), which participate in the double exchange interaction to give ferromagnetic ordering in the system. valence band spectra have been shown in fig. (f) for the discussed nanoparticles. the two most intense peaks around . ev and . ev are due to the strong hybridization of mn d(t g) and o p states. a weak emission near fermi level have been detected, which belongs to mn d(eg) states. similar type of features had been reported earlier in manganite materials, , which supports our result. resistivity measurement la . ca . mno shows insulator-metal transition around k but pr . ca . mno shows charge ordered type insu- lating behavior at low temperatures. the primary reason behind the formation of charge-ordered state in pr . ca . mno is competition between double exchange and super exchange among the core spins of mn and the coulom- bic interaction between the electrons of different orbitals of the same mn-site. , fig. shows the variation of resistivity (ρ) with respect to temperature for (la . pr . ) . ca . mno sintered at °c, °c and °c. the (la . pr . ) . - ca . mno system shows insulator type behavior at higher temperatures because of the development of charge-ordered states in the nano-crystalline system due to the presence of ample amount of pr in a-sites and it is playing a dominant role in the transport behavior of the system at higher tempera- tures; however, at low temperatures, double exchange inter- action becomes more dominant and the system starts to behave as a metal. the insulator-metal transition temperature (tim) and resistivity (ρ) of nanoparticles depend on the sinter- ing temperature of the system and decrease with increase of sintering temperature. in other words, insulator-metal tran- sition temperature depends on the particle size of the nano- crystals. as we have seen in x-ray diffraction analysis and low- resolution transmission electron microscopy analysis, particle size increases with increase in sintering temperature. due to the increase in particle (grain) size, the effect of grain bound- ary reduces and consequently the charge carrier faces less scat- tering from grain boundaries. this factor also improves the double exchange interaction mechanism and the system starts to show metal-insulator transition at higher temperatures, and the resistivity of the system also decreases significantly. the resistivity of (la . pr . ) . ca . mno sintered at °c, °c and °c can be well fitted by ρ = ρ exp(ea/ kbt) for the nearest-neighbour hopping of small polarons (fig. ), where kb is the boltzmann’s constant and ea is the activation energy. the activation energy (ea) for the samples was calculated using the small polaron theory, and values are given in table . with increase in sintering temperature, the resistivity decreases and ea increases. the linear fit shows that thermally activated band conduction is the dominant mechan- ism in the high-temperature region. the deviation from the linear fit indicates that the thermal activation mechanism is not valid in the low-temperature region. the variable-range- hopping (vrh) conduction of polarons has been found to dominate in this temperature region. the conduction mechan- ism due to the variable range hopping of polaron at low temp- erature can be described by the mott’s equation – ρ(t) = ρ exp[t /t] / , where ρ and t are constants and are given by ρ = {[ παkbt/n(ef)] / }/( e νph) and t = α /[kbn(ef)], where νph (∼ s− ) is the phonon frequency at debye temperature, n(ef) is the density of localized electron states at the fermi level, and α is the inverse localization length. using the above equations, a linear plot is expected from ln(ρt− / ) versus ( /t) / for vrh conduction. the linear fit of ln(ρt− / ) versus ( /t) / plot (see inset fig. ) indicates that vrh is the dominant mechanism of conduction below a certain temperature th. the th values are k, k and k for the samples sintered at °c, °c and °c. the average hopping distance (r) and average hopping energy (eh) at k have been calcu- lated from r = [ / παkbtn(ef)] / and eh = /[ πr n(ef)]. other conditions for vrh conduction are that the value of αr > and that eh > kbt. both of these conditions are satisfied in all the studied samples (see table ). it is not possible that the variation of particle sizes can change the n(ef), which in effect can account for the large decrease of t . however, the decrease of t may indicate the increase of localization length α − which leads to electronic delocalization. ultraviolet-visible spectroscopy to investigate the optical absorbance and evaluate the optical band gap of (la . pr . ) . ca . mno , we obtained ultraviolet- visible (uv-vis) spectra. uv-vis spectroscopy measurements have been done for (la . pr . ) . ca . mno sintered at °c, °c and °c (fig. ). uv-vis spectra of all the fig. variation of resistivity with temperature of (la . pr . ) . - ca . mno nanoparticles sintered at °c, °c and °c. paper dalton transactions | dalton trans., , , – this journal is © the royal society of chemistry p ub li sh ed o n j an ua ry . d ow nl oa de d by n at io na l p hy si ca l l ab or at or y (n p l ) on / / : : . view article online http://dx.doi.org/ . /c dt j three samples can be divided into three parts: (i) a sharp absorption edge around nm (ultraviolet region), (ii) an exponential decay region near the absorption edge (ultraviolet to visible region) and (iii) a long smooth extended region (visible to infra-red region). the optical absorption edge has been analyzed as follows: αhν / αðhν � egÞn where n is equal to and for direct and indirect transitions, respectively, while absorption coefficient ‘α’ can be calculated from the following equation: αðνÞ ¼ : � ða=dÞ where ‘a’ is the optical absorbance, and ‘d’ is the thickness of the sample. the variations of (αhν) with photon energy hν for (la . pr . ) . ca . mno sintered at °c, °c and °c have been plotted in fig. . (αhν) varies linearly for a very wide range of photon energy (hν), which suggests direct type of transitions in these systems. the intercepts of these plots on the energy axis give the energy band gaps of the systems. direct band gaps of (la . pr . ) . ca . mno sin- tered at °c, °c and °c determined from these plots are . ev, . ev and . ev, respectively. the decrease in band gap (red-shift) with increasing sintering temperature can be attributed to increased particle sizes, which have been related to the increased metallic behavior in fig. variation of ln ρ as function of /t for (la . pr . ) . - ca . mno nanoparticles sintered at °c, °c and °c in low temperature region (for t > k, k and k, respectively). inset: ln(ρt− / ) vs. t− / in lower temperature region (for t < k, k and k, respectively, and above the metal-semiconductor tran- sition temperature). the linear fit indicates variable range hopping con- duction is active in this temperature range. table values of various parameters of variable-range-hopping mechanism of (la . pr . ) . ca . mno and activation energy from small polaron theory samples t (k) α (cm− ) (inverse localization length) r (cm) (average hopping distance) activation energy (ev) (from spt) hopping energy eh (ev) (from vrh) (la . pr . ) . ca . mno sintered at °c . × . × . × − . . × − (la . pr . ) . ca . mno sintered at °c . × . × . × − . . × − (la . pr . ) . ca . mno sintered at °c . × . × . × − . . × − fig. ultraviolet-visible spectra of (la . pr . ) . ca . mno nano- particles sintered at °c, °c and °c. dalton transactions paper this journal is © the royal society of chemistry dalton trans., , , – | p ub li sh ed o n j an ua ry . d ow nl oa de d by n at io na l p hy si ca l l ab or at or y (n p l ) on / / : : . view article online http://dx.doi.org/ . /c dt j resistivity analysis. the observed band gaps of these systems appear in the range of wide band gap semiconductors and these values are even more than the band gap of zno ( . ev) and gan ( . ev). – in addition, these systems show much better magnetic ordering and magnetic moment than any known diluted magnetic semiconductors, which might prove significant in applications as magnetic semiconductors. the absorption coefficient near the band edge decays expo- nentially with photon energy (fig. ) and this dependence can be written as follows: α ¼ α exp hν eu � � where ‘α ’ is a constant and eu is urbach energy, defined as the width of the localized states (related to the amorphous state) present in the forbidden gap. this exponential depen- dence on photon energy may arise due to the random fluctu- ations associated with the small structural disorder present within the system. conclusion x-ray diffraction, low-resolution tem, saed and x-ray photoe- mission spectroscopy (xps) measurements have been done to confirm the phase formation, crystal quality and chemical con- stituents of our (la . pr . ) . ca . mno systems. crystallite size and grain size calculated from xrd and tem, respectively, confirm the nano-crystalline structure of our systems. saed images show that the system becomes more crystalline with increasing sintering temperature. the raman spectra of (la . pr . ) . ca . mno show typical vibration modes of perovskite structures. raman study confirms the stretching (b g( )), bending (b g( )) and tilting (ag( )) modes of oxygen in mno octahedra, which play a significant role in structural dis- tortion along with jahn–teller distortion (ag( )) mode, but due to the presence of pr and ca in a-site, jahn–teller distortion decreases, which is evident from the reduction of ag( ) mode. ftir measurements further confirm the presence of character- istic mn–o stretching vibration mode near cm− , which is responsible for the structural distortion, magnetic and trans- port properties of this system. magnetization measurement shows that the ferromagnetic ordering occurs around k and the system shows a saturation magnetization of . µb/ mn, which is very close to the calculated value of the bulk sample. xps measurement confirms that mn exists in dual oxi- dation state (mn + and mn +), which contributes to double exchange interaction and ferromagnetic ordering. valence band spectra show two intense peaks around . ev and . ev, which is due to strong mn d(t g)–o p hybridization. resistivity measurement shows that due to the doping of pr in a-sites, the system started to behave like a charge ordered insu- lator and insulator-metal transition decreased up to k. with the increase of particle size (by increasing sintering tempera- ture), this insulator-metal transition temperature improves to the higher temperature side. band gap estimated from uv-vis measurement appears in the wide band gap semiconductor range (∼ . ev), which is higher than zno ( . ev) and gan ( . ev) systems. we believe that with wide band gap and strong magnetic properties, the (la . pr . ) . ca . mno system will certainly prove to be a potential candidate for mag- netic semiconductor device application. acknowledgements sc is grateful to the funding agencies dst (grant no.: sr/s / cmp- / ), csir (grant no.: ( )/ /emr-ii) and brns, dae ((grant no.: / p/ /brns) for financial support. satyam kumar is grateful to ugc for financial support. authors gratefully acknowledge prof. o. n. srivastava, prof. r. s. tiwari, prof. p. c. srivastava and biophysics lab for their help in providing experimental facilities for tem, xrd, uv-vis and ftir, respectively. references g. jonker and j. van santen, physica, , , . e. o. wollan and w. c. koehier, phys. rev., , , . r. von helmolt, j. weeker, b. hopzapfel, l. schulz and k. samwer, phys. rev. lett., , , . s. jin, t. h. teifel, m. mccormack, r. a. fastnacht, r. ramesh and l. h. chen, science, , , . j. vogier, physica, , , . p. schiffer, a. p. raniirez, w. bao and s. w. cheong, phys. rev. lett., , , . a. p. ramirez, j. phys.: condens. matter, , , . j. m. coey, m. viret and s. von molnar, adv. phys., , , . fig. variation 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the restriction fragment length polymorphism (rflp), which is defined by an identifiable portion of dna, a probe, which can be labelled and made to combine with the same sequence of the dna in the genome after this has been cut into fragments by restriction enzymes. probes are selected so that they attach to one site only. if linkage can be established by a consistent tendency to cosegregation between a marker gene and a disease gene it permits certain conclusions. it confirms the mendelian nature of the disease and identifies the chromosome on which a major predisposing locus lies. in principle it provides the basis for locating a short segment of chromosome, thereby reducing the potential genes to a mere hundred or so, one of which must be the offender. it also provides, in principle, the means of detecting unaffected gene carriers. linkage investigation in amish families the amish are the descendants of early eighteenth-century immigrants from the swiss-german border area to the united states who have preserved their identity to such a degree that they constitute a genetic isolate. extensive pedigrees of this group have been documented and one, which showed transmission of affective disorder, was the subject of the linkage studies carried out by egeland and her colleagues ( ). these began with markers (kidd et al. ), as a result of which some hopeful candidate markers were defined. to confirm this the appropriate procedure would be to use the candidate markers, or markers closely linked to them, on other families. one substantial attempt failed (kidd et al. ). this is not surprising, as such highly informative ' address for correspondence: dr d. c. watl, churchway, stone, aylesbury hpi rg. ii psm at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core editorial: locating the gene(s) for affective disorder families showing segregation consistent with dominant inheritance are rare; so rare, in fact, that these families with apparent mendelian inheritance in manic-depressive illness could reasonably be explained by the joint effects of chance and a generalized predisposition based on allelic variation at many loci. in a further attempt at confirmation of the suggested linkage they had observed (egeland et al. ) the authors used the same families with some additional family members and employed additional markers known to be closely linked to their candidate locus. the results of this procedure hardly support the authors' conclusion that i t ' provided convincing evidence for the localisation of a gene that is implicated in the aetiology of a common clinical disorder' for two reasons. the first reason for caution is that no allowance was made in the calculation in the first study for the use of a large number of markers ( ), as opposed to a single marker, which increases the possibility that cosegregation of a particular marker and the gene in question may occur by chance. the second is that the first round of linkage tests defined a set of cosegregating segments of dna which may or may not include the putative dominant locus. consequently, further tests using markers known to be on these segments can give only limited information on whether or not these segments contain this putative locus. as there were some additional family members in this second study (egeland et al. ) and their candidate locus was uninformative in some individuals, some supporting evidence was provided but it is not clear if it was sufficient to justify the strong claims of linkage which were made. the effect of multiple markers if we had two dice, one normal and the other bearing sixes on all faces, which we will term the ' linked' die, and one die had been selected at random and tossed, revealing a six on its upper face, which alone was visible, then t h e ' odds ratio' favouring the linked die would be : \ or : . if a single die were tossed twice to yield two sixes it would be : and if four times : . these are odds ratios, i.e. the odds that the die being tossed was the 'linked' die compared with the odds that it was the normal die. since addition is easier than multiplication it is usual to take logarithms, and to term the logarithm of the odds ratio a ' l o d ' , an acronym for log-odds. as log ( ) is - these odds ratios give lods of - , - and . in human linkage we are testing for identity over chromosomes, excluding the sex chromosomes, and these not only differ in size but usually undergo a crossing-over during the meiotic processes which produce gametes. if we ignore these problems initially, and consider chromosomes of similar size within which recombination is rare, then we can model this by a set of dice, one all sixes; defining a linkage can then be compared to detecting the ' linked' die by tossing one of the dice a number of times. the odds against will be : before we have made a throw. four throws of sixes will favour a 'linkage' with odds of : . but the initial odds against are : giving total odds of / : or about : favouring the 'linked' die. in human linkage it is usually assumed reasonable to infer linkage if the odds ratio on a test is over : (i.e. the lod is over - ). these are not probabilities, in the usual statistical sense, which relate to a range of possibilities, but likelihoods, and merely refer to the relative likelihood of two possibilities, in this case tight linkage and non-linkage. the : convention stems from morton ( ), who advanced it in relation to tests of a single marker; it has since become a conventional guideline, well tested in practice and found a useful compromise between error and indecision. where more than one marker is used, several opportunities for the marker to show in an affected individual are provided and the likelihood of this happening by chance is therefore increased. if a number of marksmen, instead of a single one, fire at the same target the chance of a hit is increased irrespective of whether any additional skill is provided by the increase in participants. a bet spread over a number of runners will attract lower odds than one backing only a winner. in the case of linkage similar allowance must be made for multiple test. with testing of many markers the criterion of credibility based on the number of opportunities provided for a marker to show in an affected at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core editorial: locating the gene(s) for affective disorder individual must be increased if the chance of inferring a false linkage is to be kept within acceptable limits. a simple solution often adopted is to divide the odds ratio by the number of markers, which is satisfactory for small numbers of markers, but it over-compensates or, in statistical terms, is 'conservative', so that at least it errs on the side of caution. this procedure is equivalent to adding the logarithm of the number of tests to the acceptance criterion. for example with tests, since log ( ) is , we would need a criterion of a lod of , rather than . this conclusion can also be derived by a more complicated argument (kidd & ott, ; ott, ). an exact analysis gives similar results (thompson, ). multiple markers in the amish studies in neither of the amish studies was allowance made for the effect of multiple markers. the genuine strength of evidence is impossible to infer from the published data, due in part to the publication of a pedigree falsified to protect potential depressives from identifying themselves and, by concluding that they carry a pathogenic gene, compounding their innate tendency to depression. if the key information, the phenotypes of all affected members, had been published then an independent assessment of the evidence might have been possible by the reader using more robust even though less efficient methods. these include direct counts of cosegregating pairs of alleles and, since this was one large family, of consistent allelic association. as it is, the joint problems of whether a form of depression consequent upon a single gene exists among the amish and, if so, whether the position of the gene on the short arm of chromosome has been inferred with reasonable precision, remain unclear. in view of the limited number of amish clarification may not be possible until a further generation has had time to develop psychiatric symptoms. multiple markers in hla association studies failure to appreciate the consequences of using multiple markers in one family is common. it is, for example, one of the reasons for the falsely optimistic predictions of the cost of linkage studies in botstein et al. ( ). a similar situation arose in studies of the association of disease with alleles at the hla locus. whereas in linkage studies the problem is the number of loci at which markers are available or informative, in hla association it arises from the large number of alleles. in one study of association of hla with affective disorder a positive association was reported (govaerts et al. ). no correction for the number of alleles had been made; when this was done the apparent association declined to well within the likely vagaries of chance (mendelwicz et al. ). it is clear, therefore, from both theory and practical demonstration that linkage will be mistakenly inferred from random assortment when no allowance has been made for multiple tests. j. h. edwards and david c. watt r e f e r e n c e s kidd, k. k. & ott, j. ( ). power and sample size in linkage studies. human gene mapping , . botstein, d., white, r. l., skolnick, m. & davis, r. w. ( ). mendelwicz, j., verbanck, p , linkowski, p. & govaerts, a. ( ). construction of a genetic linkage map in man using restriction hla antigens in affective disorders and schizophrenia. journal of fragment length polymorphisms. american journal of human affective disorders , - . genetics , . morton, n. e. ( ). sequential tests for the detection of linkage. egeland, j. a., gerhard, d. s., pauls, d. l., sussex, j. n., kidd, american journal of human genetics , - . k. k., allen, c. r., hostetter, a. m. & housman, d. e. ( ) ott, j. ( ). linkage probability and its approximate confidence bipolar affective disorders linked to dna markers on chromosome interval under possible heterogeneity. genetic epidemiology supp. . nature , . , . govaerts, a., mendelwic/, j. & verbanck, p. ( ). manic- thompson, e. a. ( ). interpretation of lod scores with a set of depressive illness and hla. tissue antigens , . marker loci. genetic epidemiology , . kidd, k. k., gerhard, d. s., kidd, j r , housman, d. & egeland, j. a. ( ). recombinant dna methods in genetic studies of affective disorders. clinical neuropharmacology , supp. i, . at https://www.cambridge.org/core/terms. https://doi.org/ . /s downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available https://www.cambridge.org/core/terms https://doi.org/ . /s https://www.cambridge.org/core the trumpeter issn - volume , no. ( ) loren wilkinson critical review chad wriglesworth, ed. distant neighbors: the selected letters of wendell berry and gary snyder. counterpoint, . distant neighbors is an important book for many reasons, not least because it is of that increasingly rare genre, the epistolary portrait of a long, deep friendship. (perhaps, in the dubious future of books, there will be volumes titled the selected emails of . . . but they are not likely to breathe the warmth, leisure, and rootedness of this forty-year correspondence!) the greater importance of this record of a friendship is that wendell berry and gary snyder are both leaders in one of the most important counter-revolutions of our time. that is the growing recognition that, despite the placelessness of urbanization, globalization, commodification, and the internet, we are most fully human when we are rooted in a place. through their many books, articles, stories, and poems, berry and snyder have helped many people to see that we are each members of communities that include not only people but plants, animals, soil, and weather. we have rather lamely labeled this complex set of relationships “the environment”—hence the popular name for this diverse revolution, “the environmental movement.” the first letters in this correspondence are dated , at about the time that my wife, mary ruth, and i began reading berry and snyder seriously, as guides to our own thinking about how to live wisely on the earth. “the environment” back then was a word that had only begun to be used in its current (and highly inadequate) sense, as a synonym for the great gift we call “nature” or “creation.” we had first encountered wendell berry in an essay reprinted in in that venerable, counter-culture bible the whole earth catalog. the piece was called “think little,” and it expressed with great clarity what we had begun vaguely to feel and try to live out. after pointing out the tendency of the recent civil rights and peace movements to degenerate into faddish posturing (mainly because they were about bad things done by other people), berry wrote: the environmental movement rises close to home. every time we draw a breath, every time we drink a glass of water, every time we eat a bite of food, we are suffering from it. nearly every one of us, nearly every day, is contributing to the ruin of this planet. a protest meeting on the issue of environmental abuse is not a convocation of accusers, it is a convocation of the guilty. this realization ought the trumpeter issn - volume , no. ( ) loren wilkinson to clear the smog of self-righteousness that has almost conventionally hovered over these occasions and let us see the work that is to be done. the astringent words about guilt and righteousness resonated deeply with our own christian understanding (though we have gradually come to understand, partly through berry’s writings, how much more the christian way is about joy and gratitude than it is about guilt!). mary ruth and i encountered “think little” again in a book called a continuous harmony, which we used as a text in an environmental studies program we started in at seattle pacific college. in my teaching in that program i was also helped and troubled by a book of poetry and essays by gary snyder, turtle island, published in (it won the pulitzer prize for poetry). in its preface he explains the title: turtle island—the old/new name for the continent, based on many creation myths of the people who have lived here for millennia, and reapplied by some of them to north america in recent years. also, an idea found world-wide, of the earth, or cosmos even, sustained by a great turtle or serpent-of-eternity. at the end of the collection in an essay titled “the wilderness,” which draws widely on north american, hindu, and buddhist mythology and practice, snyder acknowledges, “the voice that speaks to me as a poet, what westerners have called the muse, is the voice of nature herself, whom the ancient poets called the great goddess, the magna mater.” thus there is no “god” other than nature: nature is god. a native oregonian, i found snyder helpful first because, as one who had grown up in the northwest, he wrote vividly, and at first-hand, of the damage done—mainly through logging—to my own place. (i was and am less familiar with berry’s strip-mined and eroded kentucky.) but also, he began to connect me to the ancient, almost-obscured practices, traditions, and myths of “the people who have lived here for millenia.” he began to make me more aware of the importance of those “old ways” (the title of a later book by snyder). as one who was trying to work out an ethic of care for the earth as part of the gospel (the “good news”) of the christian story—i found snyder troubling because he clearly (along with most of the then-emerging “environmentalist” community) understood the christian story to be part of the problem. in his view, it encouraged an ethic of human wendell berry, “think little,” the berry center, http://www.berrycenter.org/think-little-wendell-berry/. gary snyder, turtle island (new york: new directions, ), introductory note. ibid., . the trumpeter issn - volume , no. ( ) loren wilkinson dominion and exploitation, stressing a transcendent god and a (correspondingly) transcendent “man” made in his image. if there was to be a way forward, snyder argued, it lay in recovering “the old ways” of those native people, to whom the divine was resident in all things and people. a serious buddhist, he found equivalents of native american mythology more fully spelled out in the animism underlying japanese and hindu myths. in the years that followed i have read most of the work of both writers with great interest and profit. in , when i joined a group of scholars at calvin college in a project to articulate a christian theology of environmental ethic (later published as the book earthkeeping: stewardship of creation), snyder wrote a note of encouragement, saying he had been trying without much success for years to get americans to care for the earth from a native and buddhist starting point; maybe we would have more luck from a christian foundation. we appreciated the encouragement. snyder, in important works like the practice of the wild, continues to draw on what he considers to be remnants of neolithic wisdom, captured in native american mythology, and in the animism he detects beneath zen buddhism. and berry, in his poetry and fiction—and especially in essays like “the gift of good land” and “christianity and the survival of creation”—has made clear the biblical roots of his thought. these biblical dimensions have been made even clearer through more recent interpreters like ellen davis, in scripture, culture and agriculture: an agrarian reading of the bible, and norman wirzba, in from nature to creation. i begin this review with these personal reflections not simply because they explain why this book is so important to me, but because hopefully they give some indication of why the long, friendly dialogue between these two thinkers is so important today for all who are concerned with (as pope francis recently put it in his great encyclical) “care for our common home.” berry and snyder continue to be among the most important voices influencing that movement, which is attempting to reshape and restore our relationship with the earth. both write passionately about their commitment to the local: the need to know and be at home in our own communities. the letters often reflect their delight in and gratitude for this deep agreement. in , after reading snyder’s newly published turtle island, berry wrote, “your poems offer a sort of companionship that is of the greatest importance to me. i feel the differences of pope francis, laudato si’: on care for our common home, may , http://w .vatican.va/content/ francesco/en/encyclicals/documents/papa-francesco_ _enciclica-laudato-si.html. the trumpeter issn - volume , no. ( ) loren wilkinson our origins and educations—and the similarity of our hopes and aims. that is good” ( ). a little later ( ) berry writes, “your words, wherever i find them, give me the sense of being spoken not just to but for. what a relief!” ( ). those words appear in a letter in which berry thanks snyder for what he wrote when praising three memorial poems, which berry had just published in honour of a recently departed neighbour and mentor. snyder’s words were —poems that go off the edge of the world to visit the dead: our teachers still. brave poems—they send me back to my own life. refreshed; work joyfully in impermanence! the harmony between the two men is re-enforced by their common vocation: to be both poets and farmers. yet the two bodies of poetry that they have produced are very different from each other. their farming is different as well. berry is working to recover and sustain traditional agrarian life in the fertile hills of kentucky; snyder is trying something rather different, to homestead and “re-inhabit” the dry foothills of the sierra nevada, in a way consistent with the hunter/gatherer culture of the nearly vanished native people. yet as berry writes, after his first visit to kitkitdizze, snyder’s homestead in california (referring to his description of the visit in an article for organic gardening): it’s clear from what i wrote, i hope, how much i was pleased and moved by what i saw there. i’m only sorry we couldn’t stay longer, and will hope to come back again before too long. we are neighbors—distant neighbors. ( ) berry’s fine phrase “distant neighbours” gives the title to this collection, and both words are important. the “neighbourliness” is the most obvious characteristic. the letters reflect on every page the deep similarities in the way both men see the world: from their delight in the different weathers and creatures of their respective places, to their pleasure in learning to farm those places, to distress at the forces in the culture that ignore, threaten, or trivialize every attachment to place. as snyder wrote to berry in a letter (complaining about the claims made by a doctoral student who, writing a thesis comparing the work of the two men, argued that there were irreconcilable differences between them): “i’m not sure if anything is [‘between you and me’] except wendell berry, three memorial poems (berkeley: sand dollar books, ), back cover. the trumpeter issn - volume , no. ( ) loren wilkinson distance and differing plant communities and climates. that’s how it feels to me anyway” ( ). yet the distance is real, and both men recognize it. appreciating that distance is one of the challenges—and pleasures—of reading this collection. in his introduction, chad wriglesworth, whose thorough, scholarly, but unobtrusive editing wonderfully honours the letters, matches snyder’s words about their closeness with another quote from a letter: dear gary, i think it would be both surprising and disappointing if we agreed more than we do. if we agreed about everything, what would we have to say to each other? i’m for conversation. ( ) in a footnote expanding that quote wriglesworth cites berry’s words from an interview: “gary snyder and i agree on a lot of things, but his point of view is different from mine and it has been immensely useful to me. some differences make for binocular vision” ( n). some of the distance (like the spatial distance between kentucky and california) is ultimately superficial. snyder is the more obviously “counter-cultural” figure. slightly older than berry, he is often associated with the “beat generation” and the “san francisco renaissance.” along with allen ginsberg and others he participated in the “six gallery” reading that heralded a new style of counter-cultural poetry; he is the figure on whom “japhy ryder,” the hero of jack kerouac’s dharma bums, is modeled. his experience in a japanese zen monastery, in india, and in various communal living arrangements mark him as an early hippy. these early years are in distinct contrast to berry’s more conventional path, from his many-generation-rooted kentucky childhood, through college, graduate school (he studied with wallace stegner at stanford, in a program that included ken kesey), his life-long marriage to tanya, his university teaching, and his return to the farming community where he grew up. in his very helpful introduction to the letters, wriglesworth includes two photos from a poster advertising the event in san francisco where they first read their poetry together. his words on the two photos are revealing: berry is shown smiling in the afternoon sun of frankfort, kentucky, where local citizens have rallied to oppose the construction of a dam on the red river. he is a clean-cut protestor. he wears a sweater and has a blade of kentucky blue-grass leisurely resting on his lower lip. the other the trumpeter issn - volume , no. ( ) loren wilkinson photograph, taken at roughly the same time, shows snyder working at kitkitdizze. he is a long-haired voice of the counterculture. he wears a denim workshirt, an earring, and a goatee. he looks directly into the camera and—like berry—is connected to place by a blade of grass held between his lips. the two men speak from different regions and backgrounds, yet the placement and content of the two photos illustrate their common bond. (xix) one of the differences between snyder and berry is more significant—but still not fundamental. berry is champion of what could be called a new agrarianism: a way of life which recognizes that for all people “eating is an agricultural act,” whether they recognize it or not. thus he is deeply critical of the “industrialization” of our food production, a trend that results in the double tragedy of, in the city, both alienating most urban dwellers from connection with their food and condemning them to inferior food and, in the country, destroying, through capital-intensive, technologized agriculture the knowledge of sustainable mixed farming and the communities able to support it. this defense of an older style of farming is central to all his work, from the extended argument of the unsettling of america to the laments of his novels, which tell the story of the declining vigour of the fictionalized “port william” of his home region in kentucky. snyder, on the other hand, while agreeing with berry’s critique of modernity, is convinced that it does not go deep enough, or back far enough in time. if the ideal relation to the land in berry’s work is represented in small agricultural communities, in snyder it is in the hunting/gathering communities that preceded agriculture and from which it emerged. the primary value for snyder is not well-tended agricultural land but wilderness: the infinitely rich complex of naturally occurring systems that sustain soil and forests in their fertility. thus, if berry has become the spokesman for agrarianism, snyder is the spokesman instead for bioregionalism: the health of wild ecosystems before they were “tamed” by agriculture. his thought is clearly set out in an article titled “good, wild, sacred,” published in practice of the wild: our idea of good land comes from agriculture. here “good” (as in good soil) is narrowed to mean land productive of a small range of favoured cultivars, and thus it favours the opposite of “wild”: the cultivated. to raise a crop you fight the bugs, shoo the birds, and pull the weeds. the wild that keeps flying, creeping, burrowing in—is sheer frustration. yet wild nature cannot be called unproductive, and no plant in the almost endless mosaics of micro and macro communities is ever out of place. the trumpeter issn - volume , no. ( ) loren wilkinson for hunting and gathering peoples for whom the whole spread of richness, the wild natural system is also their economy, a cultivated patch of land might seem bizarre, and definitely not good, at least at first. gathering people draw on the whole field, ranging widely daily. agricultural people live by a map constructed of highly productive nodes (cleared fields) connected by lines (trails through the scary forest)—a beginning of linear. both men are aware of this substantial difference. thus, when berry writes to snyder in , asking for his permission to dedicate to snyder his own book on poets and poetry, standing by words, he does so apologetically: my worry about the dedication is that it might make the book an embarrassment to you. . . . because we have a good deal in common, our differences are instructive and clarifying and useful to me. sometimes my awareness of where you are standing gives me a sort of binocular vision. because, for instance, my point of view is pretty much that of a christian—a forest christian—it seems likely that you will want or need to disagree to some extent with my book. if by dedicating the book to you, i made you feel that you should not disagree, i would look on that as a deprivation. ( – ) in his reply snyder passes over berry’s reference to his christian perspective (which constitutes the most substantial difference, which we will shortly consider). he focuses instead on their differing understandings of their preferred relationship of people and land: good grief, i’d be delighted and honoured to have such a book dedicated to me. i never thought we would have to agree about everything anyway—clearly you are pro-agrarian, and i am pro-hunting and gathering, which only shows i am even less credible, in the twentieth century, than you are. ( ) this difference between agrarian and hunter/gatherer occurs early in the correspondence and touches on the deeper philosophical and theological differences. thus in , after having finished reading what remains berry’s most in-depth defense of the agrarian, the unsettling of america, snyder writes: i think your points are clear and well-taken. the question i ask myself is: what next? . . . the best intentions in the world will not stop the inertia of a heavy gary snyder, “good, wild, sacred,” in the practice of the sacred (new york: north point, ), . the trumpeter issn - volume , no. ( ) loren wilkinson civilization that is rolling on its way. as poets, our politics mostly stand back from that flow of topical events; and the place where we do our best work is in the unconscious, or myth-consciousness of the culture. . . . what you and i are really talking about, is reviving the value system and integrity and authenticity that belongs to the neolithic. the neolithic mind-set has been struggling to retain itself—in terms of what has been called “folk-culture”—against the taxing powers of governments ever since. ( – ) in a return letter berry cautiously agrees with snyder’s argument that for the best models of right relationship to place, we must go back to pre-civilizational times. but in so doing, he introduces the more fundamental difference between them, one which they never really resolve: the difference between hebrew/christian and animist or neolithic sources for their thought. i think you’re right about the neolithic. we may have had a few chances since then to recover that integrity and authenticity on somewhat different terms, but each time we blundered—maybe, each time, by failing to see which way technical innovation would take us, hence never limiting that. and so we must go to the neolithic for examples. of course, innovation was slow then and they didn’t have to deal with it. for dealing with it we have to consider the amish, not so much for anything they have achieved but for their uncanny instinct about limits, about the connection between spirit and tools. where did they get it? i wish i understood. i’m sure the bible can get you to where the amish are, but why didn’t it get the rest of us there? ( ) berry’s interest in the amish continues. a few months later (september ) he writes to snyder, after spending a day with an amish convert, “i now have a list of amish social principles which i want to give you” ( ). berry sums up the most important of these several years later in an essay subtitled “twenty-seven propositions about global thinking and the sustainability of cities”: if we could think locally, we would take far better care of things than we do now. the right local questions and answers will be the right global ones. the amish question “what will this do to our community” tends toward the right answer for the world.” wendell berry, “out of your car, off your horse: twenty-seven propositions about global thinking and the sustainability of cities”, in sex, economy, freedom & community (new york: pantheon books, ), . the trumpeter issn - volume , no. ( ) loren wilkinson berry’s words, “i’m sure the bible can get you to where the amish are, but why didn’t it get the rest of us there?” reflect an emerging theme in berry’s work: the roots of his agrarian philosophy in biblical principles. and discussion of those roots—in comparison with snyder’s neolithic roots—evoke the longest and most substantial letters of the whole collection. the theme appears first as berry thanks snyder for his explication of “right occupation,” a part of the buddhist eightfold path, early in : thanks for your note on right occupation. did i tell you what i wanted it for? i’m to speak in april at southern baptist theological seminary in louisville, and i’m going to argue that a christian doctrine of right occupation is implicit in the bible—that in fact it is almost explicit there, in passages that i have been quite a while locating and connecting. my argument will begin with the conditions surrounding the gift of the promised land in the pentateuch and wind up with a passage in romans where paul says that not just mankind but all creation awaits a resurrection by which it will be delivered from corruption and glorified. if i can make it all stick together, it will be an argument against the otherworldly values that have caused or allowed so much christian abuse of “nature.” ( ) berry’s address to the baptists is later published as “the gift of good land” in the sierra club journal. when snyder read it, he wrote: i found the article very useful, re-opened my thinking—jumping over and beyond genesis—(too bad jehovah seemed to overlook the fact that people were already there living in the promised land, though—). ( ) the criticism implicit in snyder’s sarcastic comment on “the promised land” is passed over for the time being. but snyder’s discomfort with christianity emerges directly the following spring (april , ) in his response to a draft of the first of berry’s “sabbath” poems. (berry has elsewhere said that he wrote these from the discipline of walking in the woods on sundays instead of going to church, where he was never comfortable. in a later letter berry admits, “i still see no church i could be at home in. i am a solitary christian—a most paradoxical creature” ( ). the “sabbath” poems often draw on the language of christian worship; some are almost prayers, and address “the lord.” snyder wrote in response: reading sabbaths. i can enjoy the poems, but not the theology—i am truly convinced both from study and personal practice that there is no one central informing and organizing “lord” to invoke—and that the center is everywhere, the trumpeter issn - volume , no. ( ) loren wilkinson simultaneously, and, also, that human and non-human are not qualitatively removed. ( – ) after some positive and negative comments on individual poems, and a warning to avoid “hymnal language-tone,” snyder closes: yr [sic] old buddhist buddy gary snyder thus lays out the three parts of his “a-theology” succinctly: ) atheism: there is nothing that can meaningfully be called “god”; ) monism: there is no difference between centre and circumference: “the center is everywhere”; and naturalism: we are simply part of nature: there is no special place for the human. berry’s response sets up a pattern that he follows in his other letters on this crucial subject. first, he tries to define his own position in a way to include and welcome snyder’s buddhism—indeed he signs his letter your friend, wendell (buddhist) [sic] ( ) more directly he writes: i wonder if it might not be possible to define a western theology sufficiently responsive to biblical tradition that would not alienate a buddhist such as yourself. the question may be merely naïve, yet i think thomas merton had it much in mind. ( ) after this suggestion that it might be possible to get beyond their differences, berry continues, as he does in other letters, with the more pointed part of his response. he points out that the christianity that snyder is rejecting is a caricature: it overlooks the richness of the christian view: “i too believe that ‘the center is everywhere simultaneously.’ . . . you can find good biblical support for an argument—for instance— that god is at once transcendent and immanent” ( ). after a six-week delay, snyder writes an extended response, in which he makes his criticism of christianity more explicit: my problems with christianity are two: one is basically theological, the place in the bible where the lord says the living creatures of the earth are “meat” for our use and the distinction from the beginning which is hardened into dogma by the church between creator and creation. it is heretical for a christian to aspire to be completely one with the maker. (june , , pp. – ) the trumpeter issn - volume , no. ( ) loren wilkinson he suggests too that he could “get along with christianity”—but that “my mother, being raised in a strict southern town, gave me more than my share of atheist hostility, i know, and my marxist leanings of my twenties reinforced it.” he continues by referring to some of his own experience of rapprochement between zen and christianity, especially in his own zen teacher, robert aitken. (part of the charm of this whole collection is illustrated in the fact that snyder can move easily from this deep discussion of zen and christianity to asking berry’s advice about tractors versus horses, “wondering if what we can do with a good heavy duty rototiller is hold the line between horses and a tractor for a while and make it do until we can enlarge our grass-growing capacity without a tractor. . . . can a somewhat larger, sturdier rototiller do useful work around the place beyond just small gardens [june , , p. ]?”) berry’s response, three weeks later, is extensive—the second longest letter of the whole correspondence (june , ; pp. – ). beginning by answering snyder’s question about rototillers, he moves into the argument. as in the earlier exchange, he goes far in agreeing with snyder—saying for example that like himself snyder reads the bible “as the outcropping of something timeless, unhuman and true—all encrusted with shards, artifacts and fossils of what you call ‘civilization.’” but he adds that he does not seek explanations for wonders. “i believe wonders have happened because i see that they happen.” but he is quite critical over what he considers to be snyder’s shallow reading of both the bible and the whole christian tradition. he points out that the genesis giving of meat for food is in the context of a covenant god makes with all creatures; that “the book of job is a ferocious indictment of the assumption that the world is man’s meat,” and argues that the idea of “atonement” suggests that it is not heretical to imply a unity between the christian and the maker. the discussion continues, interspersed with many other topics, over several letters and years. ultimately, the two men seem to agree to disagree, and don’t dwell any more on the differences, which is probably a good thing. the last significant appearance of the animism/theism argument is in berry’s response (august , , pp. – ) to snyder’s book, the practice of the wild. berry praises the book, but has two substantial criticisms, which highlight the abiding difference in their approach. the first is to the essay “good, wild, sacred,” in which snyder says that agriculture is the beginning of a “linear” kind of thinking which distances us from nature. to this berry the trumpeter issn - volume , no. ( ) loren wilkinson complains, “i think you see agriculture too exclusive of wildness” and proceeds to give some examples of the harmonious relationship of the two. berry’s second criticism is more substantial, and echoes his earlier complaint that the christianity that snyder dismisses is a simplistic caricature: your definition of “the ideology of monotheism” strikes me as not only far too general and simple, but improbable too. the allegation of uniformity becomes uncomfortable, it seems to me, in the presence of almost any pair of examples you can name. he proceeds to name several such pairs of “monotheist” thinkers: dante and chaucer, billy graham and e. f. schumacher, dorothy day and jerry falwell, thomas merton and the pope. “also,” he continues, “it is impossible to reconcile the idea of centralization with the anti-state principles of the anabaptists and other sects, or with the baptist doctrines of “the priesthood of all believers.” berry concludes his response by saying, the theological parts of your book brought me to a question that i would like to know your answer to. doesn’t polytheism (which you seem to allow) finally imply monotheism? or, to put it a different way, could a uni-verse (a coherent or integrated nature) have been created by a committee of deities of limited and contending powers? he then refers to a section of spencer’s faerie queen in which spencer speaks of the christian creator in terms of “the greatest goddesse,” which accomplishes, he says, “both a profound criticism of classical polytheism and a very significant elaboration of christian monotheism.” the points berry raises here are substantial. if snyder responds to them, this collection does not reflect it. when one looks back over the long exchange, it is hard to avoid the conclusion that snyder cannot easily answer berry’s criticisms. so to preserve the friendship they abandon the argument. nevertheless—it is sad that despite his long conversation with berry on this crucial topic, snyder (as at least one recent poem, “stories in the night” makes clear) has not moved from his earlier simplistic understanding of the biblical picture of creation and our place in it. i will not be the only reader who hopes this conversation between them continues. in any case—it is misleading to write an article on this fine book that dwells on ideas and disagreements. what comes through most clearly—despite their disagreements, and their pain of living in an unwise time and culture—is delight in the sheer gift of the the trumpeter issn - volume , no. ( ) loren wilkinson world, however they understand the mystery of what it is. the delight comes first. two excerpts from near the end of the correspondence make the point. in september of snyder tells berry this story: how do you like this: a grey squirrel which has now for ten days been regularly climbing into our apple trees, first the gravenstein, then the jonathan, now the pippin so that they fall on the ground and then immediately a four-point buck comes along and eats some of the apples. and the rest of the time he lounges in the shade of the trees. what a deal! ( ) to which berry replies a few days later, “i love your story of the squirrel and the deer. long live interracial cooperation!” ( ) and in the final exchange of the correspondence, responding to a picture on the cover of farming magazine with berry, a horse, and a mule, snyder responds with the single question: “but why is it horses have small ears and big feet & mules have big ears & small feet—?” ( ). berry dutifully answers with a brief lesson in mule genetics. then he concludes with his own squirrel story, about a birdhouse taken over by flying squirrels. “sometimes one of the squirrels comes to the opening and regards me, and i regard him or her back. i am unsure how either of us is affected by this” ( ). those are the last words in the collection. presumably these two friends are still writing such things to each other. the correspondence is packed not only with deep thinking, but also with keen seeing, good humour—and genuine friendship. long may it continue! i return in conclusion to reflect on the larger significance of this collection of letters. speaking as one who has been reading and appreciating both writers since before the correspondence began, i can attest to the way in which it provides a kind of overview of the history of two wings of the environmental movement, agrarianism and bioregionalism, from two of its most important voices. to illustrate the deeper importance of the disagreements at the centre of the correspondence, i conclude with two brief examples. when, in a reflective retreat the leaders of a local environmental organization were asked to identify what story or stories they understood their work to be a part of, most found the question difficult to answer. “that we are a part of nature” was the clearest answer. but, unable to define nature as anything more than “what is happening in the universe” they found it difficult, when challenged, to find in this a basis for action or education. for if humanity—whether destructive or conserving—is simply the latest thing the random action of nature has produced, then there is no reason to be concerned about the trumpeter issn - volume , no. ( ) loren wilkinson the consequences of any human action such as global warming, habitat destruction, and species extinction. these are just what nature is doing. yet that view of nature—as a purposeless, impersonal process that has by chance produced intelligence, consciousness, and conscience—seems to be the one within which they are operating. there is little room for hope in such a vision. despair is a common environmentalist problem. but the idea that nature could be better understood as creation, in which human creatures have a special role of relationship and responsibility to both the earth and its creator—was unpalatable to this group of environmentalist leaders, as it is (as these letters show) to snyder. a second instance of the relevance of these letters comes from conversation with a university professor of environmental studies who is an expert in ecological restoration. but in conversation and in his published work he acknowledges a deep problem. we have a good handle on the science of climate change and ecological degradation. but no science can tell us what “restoration” means, especially when it is now impossible to restore what has been destroyed. thus there is a kind of philosophical vacuum at the centre of the environmental movement: the situation seems to require a larger narrative. “environmentalism” is an ethic in search of a religion, a larger story in which our actions are coherent, meaningful, and right. there really are only two larger narratives available. one is the monist one, which snyder finds in neolithic hunter-gatherer cultures: in this view nature is nothing more than what has been purposelessly happening. the other is the christian one which underlies berry’s work: the premise that we live in a universe in which personality and responsibility are real, rooted in a creator who is both other than the universe and intimately present to it, and whose character underlies, justifies, and invites human love, care, and stewardship. the gift of these letters is that both berry and snyder, beginning where all humans begin, with the miraculous given of the world—whether we call it “nature” or “creation”— have lived such thoughtful, articulate lives, though motivated by different narratives. thus they are indeed “distant neighbors.” their rich body of writing, from within these different narratives, invites us into that neighbourliness, and these letters invite us into their work. loren wilkinson biomed centralbmc oral health bmc oral health , research article elevated antibody to d-alanyl lipoteichoic acid indicates caries experience associated with fluoride and gingival health martin levine* , robert l brumley , , kevin t avery , willis l owen and donald e parker address: department of biochemistry and molecular biology, university of oklahoma health sciences center, p.o. box , oklahoma city ok usa, department of community dentistry, university of oklahoma health sciences center, p.o. box , oklahoma city ok usa, department of biostatistics and epidemiology, university of oklahoma health sciences center, p.o. box , oklahoma city ok usa and gensys technologies inc., little farm dr white lake, mi e-mail: martin levine* - martin-levine@ouhsc.edu; robert l brumley - jaluckey@aol.com; kevin t avery - kevin-avery@ouhsc.edu; willis l owen - willis-owen@ouhsc.edu; donald e parker - don-parker@ouhsc.edu *corresponding author background: acidogenic, acid-tolerant bacteria induce dental caries and require d-alanyl glycerol lipoteichoic acid (d-alanyl lta) on their cell surface. because fluoride inhibits acid-mediated enamel demineralization, an elevated antibody response to d-alanyl lta may indicate subjects with more acidogenic bacteria and, therefore, an association of dmft with fluoride exposure and gingival health not apparent in low responders. methods: cluster analysis was used to identify low antibody content. within low and high responders (control and test subjects), the number of teeth that were decayed missing and filled (dmft), or decayed only (dt) were regressed against fluoride exposure in the water supply and from dentrifice use. the latter was determined from gingival health: prevalences of plaque (pl) and bleeding on probing (bop), and mean pocket depth (pd). age was measured as a possible confounding cofactor. results: in high responders, dmft associated with length of exposure to fluoridated water (f score), pl and bop (r = . , p < . ), whereas in low d-ala-igg responders, dmft associated with pl, age, and pd (r = . , p < . ). bop correlated strongly with number of decayed teeth (dt) in high responders (r = . , p < . ), but poorly in low responders (r = . , p < . ). the strength of the pd association with dmft, or of bop with dt, in high responders significantly differed from that in low responders (p < . ). conclusion: caries associates with gingival health and fluoridated water exposure in high d-alanyl lta antibody responders. background over the last years, the widespread usage of fluoridated water and fluoridated dentrifices have been cited as major reasons for a decline in caries since the early s [ ], and for the appearance of a significant association be- tween oral hygiene and caries experience [ – ]. an inverse published: february bmc oral health , : received: october accepted: february this article is available from: http://www.biomedcentral.com/ - / / © levine et al; licensee biomed central ltd. verbatim copying and redistribution of this article are permitted in any medium for any purpose, provided this notice is preserved along with the article's original url. page of (page number not for citation purposes) http://www.biomedcentral.com/ - / / http://www.biomedcentral.com/ bmc oral health , http://www.biomedcentral.com/ - / / relationship exists between salivary fluoride concentra- tion and caries experience in the deciduous and perma- nent dentition [ ], but fluoride concentration is excluded from most caries prediction models [ , ]. acids in bacte- rial plaques cause caries in pits, fissures and interdental re- gions of teeth, but they also enhance the inhibitory effect of fluoride on demineralization, confounding the ability to predict caries from the salivary fluoride concentration [ , ]. the greater the consumption of dietary sucrose, the great- er the fall in ph and fraction of acidogenic, acid tolerant bacteria in tooth adherent plaques [ , ]. the number of these bacteria (mostly mutans streptococci and lactoba- cilli), and the fluoride content, discriminate between se- vere and mild caries in – year-old children [ , ]. acid-tolerant bacteria require d-alanyl glycerol lipotei- choic acid (d-alanyl lta) in their membranes and cell surfaces [ ]. d-alanyl lta is made by esterifying car- boxyl-activated d-alanine to glycerol in membrane lta by means of a d-alanyl-carrier enzyme, dcp [ ]. strains of streptococcus mutans in which dcp is inactive do not in- itiate growth at below ph . and make glycerol lta with- out d-alanine [ ]. in the dcp active strains, soluble d- alanyl lta is extruded into culture fluid in vitro[ , ] or plaque in vivo[ ]. the d-alanyl esters are stable at ph . at °c, but hydrolyze to free d-alanine and lta with a half-life of . h at ph . [ ]. healthy gingival sulci have a ph of . – . and inflamed sulci a ph of . – . [ ]. about % of young adults have serum igg antibodies that precipitate with d-alanyl lta, but not with d- alanine-free lta [ , ]. it is likely that plaques induce these igg antibodies from gingival sulci that contain more acid-tolerant bacteria. an elevated igg antibody response to d-alanyl lta may therefore indicate the subjects in whom an inhibitory effect of fluoride on caries is en- hanced. the fluoride concentrations of plaque and saliva are related to whether the drinking water is fluoridated [ ] and to oral hygiene, which nearly always involves us- ing a fluoridated dentrifice. the aim of this study was therefore to determine whether elevated antibody re- sponders to d-alanyl lta show a association of dmft with fluoride exposure and gingival health not apparent in low responders. methods subjects antibody was obtained from blood from four sources: ) dental students, ) patients seeking dental treat- ment, ) volunteer blood donors (volunteers), and ) siblings aged through from six amish families. the dental students and patients were attending the uni- versity of oklahoma health sciences center between and . the volunteers and amish family mem- bers were attending centers elsewhere in the us at the same time. all subjects consented to provide blood for an- tibody analysis according to local institutional review board procedures (see acknowledgements). the student, patient and volunteer populations ( subjects) were used to determine what igg concentration constituted an elevated antibody response to d-alanyl lta, to ensure that these antibodies were not unique to dental or okla- homa populations and to examine whether the antibody concentration was sex or age-associated. the amish family siblings were selected to determine the frequency of high antibody concentration in children and young adults. each sibling had at least one parent high responder to in- crease the likelihood of exposure to an antibody-associat- ed oral microbiota from birth. the clinical study participants consisted of dental stu- dents ( . % male) and patients ( . % male) who were medically healthy. all had or more natural teeth and were aged > and < years. of these participants, dental students and patients provided information that permitted an estimate of exposure to fluoridated wa- ter: residence(s) from birth through age in the fluoridation census. subjects not using the public water supply, or resident outside of the us for more than months, were excluded. exposure to water fluoridation scored for each of five -year age cohorts: – , – , – , – and – to give a fluoride exposure score (f score) of (no exposure) to (complete exposure) de- scribed previously [ ]. most dental students had mild caries and gingivitis and most patients had moderate to severe caries and gingivitis. exceptionally healthy or exceptionally diseased subjects were therefore increased compared to a similar number of subjects obtained as a random sample. this wide distribu- tion of clinical measurements provided more stable esti- mates (narrower confidence intervals) of regression coefficients (β) than would be obtained from a similar number from a random survey of the general population. regression lines are more robust when a greater range of measurements is used [ ]. clinical measurements dental caries experience was the number of decayed, missing and filled teeth (dmft), excluding third molars and teeth reported missing for other reasons. decayed teeth (dt) were also enumerated separately from missing and filled teeth (mft). dt indicates a combination of de- lay in seeking therapy and faster development of new cav- ities. fluoridated dentrifice use is related to oral hygiene but not toothbrushing frequency in adults aged as in the present clinical study [ ] and young enough to have likely used fluoridated dentrifices from early childhood. sensi- page of (page number not for citation purposes) bmc oral health , http://www.biomedcentral.com/ - / / tive staining for plaque accumulation [ ] was therefore used with measures of gingivitis and pocket depth at the mesio-buccal, buccal, disto-lingual and lingual surfaces of the six teeth employed for the simplified oral hygiene in- dex [ ], substituting adjacent teeth as necessary ( sites sampled). gingivitis was determined by whether a site bled within sec of gentle probing, bop [ , ] and pocket depth by measuring the distance (mm) from the free gingival margin to the base of the sulcus or pocket. finally, each subject was asked to suck an erythrosin tablet for sec and the sites examined for stained plaque [ ]. for each subject, the mean prevalences of plaque (pl) and bleed- ing on probing (bop), and the mean pocket depth (pd), were calculated across all sampled sites. the clinical meas- urements were made by two experienced clinicians who were calibrated for this study. the clinicians agreed strongly with respect to all measurements (correlation co- efficients, r > . ; p < . ) except gingival bleeding in- dex, for which a weaker correlation was noted (r = . , p < . ). the data reported are the mean measurements from the clinical examiners. antigen purification d-alanyl lta, but not d-alanine-free lta, is present in culture filtrates of streptococcus mutans gs [ ]. bacteria were grown at °c in trypticase soy broth to late station- ary phase ( h), when the maximal amount of antigen is extruded into the culture fluid [ ]. after centrifugation to remove the bacteria, culture fluid ( ) was concentrated -fold over a ym diaflo membrane filter (amicon corp., beverley, ma). d-alanyl lta in the concentrate was detected by immunoelectrophoresis, using a standard hu- man serum identified previously [ ]. d-alanine-free lta does not react with this serum igg [ , , ]. the d-ala- nyl lta was purified by passing the concentrated culture fluid over a × . cm sephacryl column in . m nacl buffered with . m sodium acetate ph . antigen in the fractions was collected. after equilibrating with mm so- dium acetate buffer ph . , it bound to a short sephacryl s- and eluted by adding mm nacl as described pre- viously [ ]. measuring antibody content and determining high and low responders igg antibody content was measured by enzyme-immu- noassay employing a fast assay screening test system at room temperature [ ]. pegs protruding from a lid were placed over a -well plate or trough containing ml of µg/ml d-alanyl lta in acetate buffer ph for h (bec- ton dickinson, lincoln park, nj). the pegs were blocked with ml of phosphate buffered saline (pbs) ph . in . % tween- and immersed in wells containing . ml serum. after overnight incubation, excess igg antibody from the serum was washed away by thrice transferring the pegs to troughs containing ml of pbs containing . % tween (pbs-tween) for min each time. the pegs were then immersed for h in a trough containing ml of anti-human igg f(ab' ) fragment conjugated to alkaline phosphatase in pbs-tween and developed with nitrophenyl phosphate (sigma chemical co. st louis, mo). the concentration of antigen-specific igg in standard se- rum was obtained by measuring the optimal amount of protein immunoprecipitated [ ], and a standard curve of absorbance against concentration was obtained (fig. ). the greatest range of absorbance occurred when sera were measured at a dilution of : [ ]. replicate antibody assays were performed on each serum and the concentra- tions read off the standard curve. the antibody concentra- tions are shown ranked in fig. . data analyses the amount of igg antibody to d-alanyl lta varies with no obvious cutoff (fig. ). however, the sera containing precipitating antibody should tend to have high igg anti- body contents. the igg antibody measurements were al- ternatively divided into clusters, using the unweighted pair group method with arithmetic averages [ ] and nt- sys, a package of multivariate statistical computer pro- grams [ ]. a low response supremum was obtained by taking the antilog of the mean igg content of the non-pre- cipitating sera plus one standard deviation, or the antilog of the highest igg content of the cluster grouping contain- ing the least antibody. figure graph of absorbance at nm against log ng/ml of antibody to d-alanyl lta. vertical lines indicate the standard deviation of the measurements. page of (page number not for citation purposes) bmc oral health , http://www.biomedcentral.com/ - / / the effect of age was determined after splitting the sub- jects into decile cohorts (table ) and comparing the frac- tion of high antibody responders in each cohort. the youngest cohort was composed of amish family siblings who were younger than any dental students, patients or volunteers. a multiple linear regression procedure was utilized to ex- amine the relationship of caries (dmft) with age, f score, and measures of gingival health obtained in this study: pl, bop, and pd. the regression on dmft was used: ) to es- timate the partial regression coefficients (β coefficients) within the high and low antibody response group; ) to test each β coefficient for significance after accounting for the effects of the other four variables; and ) to examine for significant differences in β coefficients between the an- tibody response groups. a β coefficient is interpreted as the change in disease response (dmft) per unit change in one of the independent variables after adjusting for all the other independent variables in the model. within each antibody response group, the multiple regression coeffi- cient (r ) provided an estimate of the proportion of vari- ance in dmft explained by the combination of variables tested. stepwise regression then identified the best esti- mate of the variance in dmft that was explained by mul- tiple variables in the separate and combined high and low responder groups. these multiple regression analyses were repeated using gingivitis (bop) as the dependent variable and ohpi, pd, dt and mft and age as independ- ent variables. all of the clinically examined subjects were included because f score was not an independent variable for bop. to ensure that obtained relationships were robust, influ- ential points (outliers) were identified using a statistic (dffits) which measured the change in coefficients caused by removing the data for each subject. if this change exceeded √(p/n), where p was the number of in- dependent variables and n the number of samples [ ], the point was influential. repeating the regressions with all the influential points removed determined the degree to which these points had affected the results. results definition of high antibody response igg antibodies were initially detected in sera irrespective of whether d-alanyl lta was immunoprecipitated. ex- cluding the amish family group, there were subjects whose sera failed to immunoprecipitate antigen (detected by immunoelectrophoresis). the mean igg antibody con- centration (log ng/ml) was . ( . standard deviation, s.d.) compared with . ( . s.d.) for subjects whose sera did precipitate antigen ('t' test p < - ). high responders therefore had a log antibody content (ng/ml) that exceeded the mean plus standard deviation of non- precipitating serum (log ng igg /ml > . ). however, low igg concentrations formed a cluster whose supremum (log ng igg antibody/ml) was . , which corresponds to . µg/ml (left side of fig. ). subjects were therefore classified as low responders if their log igg antibody con- tent exceeded . rather than . . the odds ratio for a serum from a high antibody responder immunoprecipi- tating d-alanyl lta was times greater than for a low re- sponder. figure graph of ranked antibody contents. the cut-off points sepa- rates high from low responders (see methods). table : age decile cohorts for determining changes in fraction of high antibody responders cohort no. decile anumber in cohort high responders – b c . % – b . % – . % – . % – . % – c . % – c . % a subjects after excluding all amish family members and of the dental student, patient and volunteer subjects whose age was not recorded. bthe amish family siblings < years comprised cohort and those > years were included within cohort . ccomparison of cohorts , and with the remainder: x = . , p = . (not sig- nificant). page of (page number not for citation purposes) bmc oral health , http://www.biomedcentral.com/ - / / high antibody response, age and gender of the students, patients and volunteers, did not have their age recorded. high responders had a mean age of . years ± . s.d. ( subjects) and low responders a mean age of . years ± . s.d. ( subjects). table shows the fraction of high responders in different age cohorts. there was a high responder frequency of – % from age through . the reduced frequencies of high response in childhood and old age were not significant. however, within the amish family offspring high igg re- sponders were significantly older. table shows that the high responder offspring had a mean age of . years compared with . years for low responders. this signif- icant difference ('t' statistic = . , degrees of freedom, d.f. = ; p < . ) was due to few high responder children and young teenagers and a slightly greater fraction of sib- lings aged – years who were high responders ( %) compared with the general population. high antibody responders accounted for a similar fraction of subjects irrespective of whether they were in the clinical study, or dental students, or patients (table ). men were . % of the students patients and volunteers whose sex was recorded. men had also a greater frequency of high response . % vs . % and a greater mean log ng/ml igg antibody content, . ± . standard deviation (s.d.) vs . ± . s.d. neither of these differences were significant (x = . , p = . ; 't' statistic = . , p = . ). in serum samples from subjects aged between and , the igg antibody concentrations were essen- tially the same after months as originally estimated (squared correlation coefficient, r > . , p < . ). the results indicate that, for subjects aged – , age and sex had little effect on the frequency of high d-alanyl lta an- tibody response. association of dmft with gingival health and fluoride in high and low responders table lists the variables tested for association with dmft and the observed β coefficients in high and low antibody responders. it was immediately apparent that the β coeffi- cients from pl were similar in both groups, whereas those from bop and f score were only significant in high re- sponders and those from pd were only significant in low responders. comparison of the differences in β coeffi- cients between high and low responders, column (col- umn – column ), indicated relationships of dmft to pocket depth that were significantly different and relation- ships of dmft to f score that were almost significantly different, p = . (table , column ). stepwise regression confirmed the similar associations of dmft with plaque prevalence in both high and low igg antibody responders, but significant associations with only f score and bop prevalence in high responders and table : age of siblings from families with at least one high re- sponder parent high responders low responders familya ages ages f+m- , . , , , f+m+ , , , , , f+m+ , , , , f?m+ none , , , , f+m- , , , f+m' , , , , , bmean age (s.d)c . ( . ) . ( . ) af, father; m, mother; +, high responder; -, low responder, ? not known. bmean age of the high and low responders cs.d., standard devi- ation. table : fraction of high antibody responders in or not in the clin- ical study. subject group number % high responders dental students in clinical studya . patients in clinical studya . same-age subjects not in clinical study . other subjects not in clinical studyc . all subjects . asee first section of materials & methods. bsame-age subjects not in the clinical study were . % dental students, . % patients and . % volunteers. cother subjects were a mixture of patients and vol- unteers: . % older (ages > and < years), . % younger (ages > and < years), and the remainder age unknown. table : changes in the equality of the partial β coefficients for as- sociation of tested variables withcaries severity in high and/or low respondersa. variablea high responders bn = low responders n = difference (hi – low) f score c- . . d- . pl d . d . . bop c . . . pd - . c . c- . age . d . - . avariable names are defined under "clinical measurements" in the methods bn = number of subjects cp < . for value of constant or variable (β) or for all values in indicated model. dp > . & < . . if no subscript, p > . ehls: high response = , low response = . page of (page number not for citation purposes) bmc oral health , http://www.biomedcentral.com/ - / / with only pd and age in low responders. in high respond- ers, dmft increased as plaque and bop prevalences in- creased and fell as fluoride exposure increased. the equation obtained was: dmft = . + . pl + . bop - . fscore (r = . , f statistic = . , p < . ). by contrast, in low responders, dmft increased with plaque prevalence, pocket depth and age. the equa- tion was: dmft = - . + . pd + . age + . pl (r = . , f statistic = . , p < . ). within each equation, the constant and the respective β coefficients were significant (p < . ), except for the β coefficient of age in low responders (p = . ). high responders re- ceiving fluoridated drinking water for all years of child- hood (f score = ), had a significantly lower dmft than those never receiving fluoridated water (f score = o): dmft = . ± . (s.d.) vs . ± . (s.d.); 't' test p < . . this was not true of low responders in whom the difference between f score and f score was not signif- icant (dmft = . ± . vs . ± . ; 't' test p = . ). when antibody was ignored in stepwise regression (con- trol), dmft increased with age, pl and bop, and de- creased with f score: dmft = - . + . age + . pl + . bop - . fscore (r = . , f statistic = . , p < . ). pl and bop were individually significant. f score and age were borderline, p = . and . respec- tively, and pd was not significant, being entirely replaced with age. despite the subjects increasing to (from or ) the strength of association was similar to that of low responders only. association of dt with gingivitis in high and low responders because caries experience associated with fluoride and gingival health in high responders, poor gingival health should increase the number of decayed teeth (dt) more than in low responders. when bop was regressed against the variables in table , only dt (column ) differed sig- nificantly between the high and low responders. although dt alone significantly correlated with bop in both re- sponse groups (p < . ), it associated with bop much more strongly in high responders (r = . ) than in low responders (r = . ). fig. shows the respective corre- lations, and also the patient data (filled circles) skewed by few healthy subjects and the student data (unfilled circles) skewed by few moderate and no severely diseased sub- jects. clearly, combining students and patients strength- ened the respective associations (β coefficients). stepwise regression indicated that, excluding dt, bop associated with pl and pd similarly (bop = - . + . pl + . pd in high responders and bop = - . + . pl + . pd in low responders; r = . and . respectively; p < . ). however, dt explained more variance (bop = - . + . pl + . dt, r = . , p < . ) in high responders, and less variance (bop = - . + . pl + . dt, r = . , p < . ) in low responders. influential points (outliers), whose presence might have affected the strength and significance of these complex re- gression analyses, were identified in five low responders and two high responders. when these subjects were omit- ted, the respective regression coefficients or their signifi- cance were little changed, indicating that the different, partial, linear regression coefficients in high or low re- sponders were not artifacts of influential or outlying points. figure graph of number of decayed teeth against gingival bleeding index. results are provided separately for the dental students (o) and patients (•). data from more than one subject are superimposed. regression line equation, high responders (upper graph): dt = . bop - . regression line equa- tion, low responders (lower graph): dt = . bop - . page of (page number not for citation purposes) bmc oral health , http://www.biomedcentral.com/ - / / discussion this study has demonstrated that igg antibodies to d-ala- nyl lta are widespread in us adults. the fraction of high responders was essentially constant from early adulthood through middle age, but reduced in children (< years) and old age (> years). within the adults (ages – years) a change from low to high response or vice versa was found unlikely from repeated measurements over – months. a similar lack of change in this igg antibody con- centration was reported – months after an additional similarly aged patients had received oral hygiene ther- apy in another study [ ]. finally, the family studies es- tablished that a high antibody response was probably induced during the mid-teenage years. in order to apply the results of this study to children and young teenagers, longitudinal studies of the antibody response in relation to age and the clinical measurements in this study may need to be undertaken. despite few investigations of caries risk in – year old subjects compared with a younger or older group [ ], the association of dmft with pl in this study agrees with that obtained from year old norwegians [ ]. plaque (sim- plified oral hygiene index measurement) accounted for % of the variance in number of decayed/filled teeth sur- faces in that study, and for % of the dmft variance within all clinically examined subjects in this study (ignoring age, antibody and all other variables). other studies have shown that the amount of fluoride applied from dentrifices is measured better from oral hygiene or plaque accumulation, as in this study, and not from the reported frequency of dentrifice use [ , ]. finally, because subjects aged more than are unlikely to have used fluoridated toothpastes until later in life, they were omit- ted to avoid confounding the results. the rationale behind this study was that acidic plaque en- vironments increase the amount of d-alanyl lta and pro- mote its immunogenicity. accordingly, caries-protection by fluoride in the water supply and dentrifices was strong in high igg antibody responders, accounting for just over % of the variance in dmft. in addition, gingivitis (bop prevalence) associated strongly and significantly with the number of decayed (untreated) teeth, suggesting that pre- venting gingivitis increased fluoride exposure from dentri- fices and reduced the number of decayed teeth. increased exposure to fluoridated water, and dentrifices associated with good gingival health, may result in fluoride inhibit- ing enamel remineralization at the acidic plaque ph likely present in high responders. in low responders, the fewer antibodies to d-alanyl lta suggest less colonization by acid-tolerant bacteria and a weaker cariogenic attack. dmft associated with age, as re- ported for other subjects whose sera did not precipitate d- alanyl lta [ ], and also with pocket depth. an increase in pocket depth is caused by periodontopathic bacteria that associate with an alkaline environment in the sulci over many years [ ] and a microbiota that is neither acid- ic nor acid-tolerant [ ]. the coefficient of dmft associa- tion with pd in low responders therefore differed significantly from high responders within whom f score and gingival health were stronger covariates. conclusions an increased mutans streptococcal challenge accompany- ing low plaque ph (high antibody response to d-alanyl lta) allows much of the variation in caries experience to be controlled by water fluoridation and by the use of fluoridated dentrifices associated with maintaining oral health. high igg antibody responders are therefore better protected from caries in an optimally fluoridated environ- ment. the concept that fluoride protects better from caries in a low ph environment [ ] was recently used to ex- plain why there is a poor association between caries expe- rience and ph fall after a % sucrose rinse [ ]. in low responders, increased fluoride exposure from dentriflce use to maintain oral health, or from water fluoridation, associate relatively poorly with caries experience. al- though this study has indicated that the igg antibodies to d-alanyl lta do not become elevated until after age , when much caries may have already developed, they may be elevated to a lower level in children who eventually be- come high responders. the d-alanyl lta antibody re- sponse is not detectable in saliva (unpublished studies), but it can be measured from only a thumb-prick of blood. longitudinal studies of the d-alanyl lta response in chil- dren could improve current efforts to predict caries sus- ceptibility by relating it to fluoride or the fluoride ion product for fluoroapatite in saliva and the ph change after a sucrose rinse [ , , , ]. table : changes in the equality of the partial β coefficients for as- sociation of tested variables withgingivitis (bop) in high and/or low responders. variablea high responders n = low responders n = difference (hi – low) pl d . c . - . pd c . c . - . dt c . . c . mft . . . age . - . . avariable names are defined under "clinical measurements" in the methods bn = number of subjects. cp < . for value of constant or variable (β). dp > . & < . . if no subscript, p > . . page of (page number not for citation purposes) bmc oral health , http://www.biomedcentral.com/ - / / competing interests none declared acknowledgements this investigation was largely supported by usphs research grant r de- from the national institute of dental research, national insti- tutes of health, bethesda. we sincerely thank dr. w. bias, immunogenetics laboratory, johns hopkins university, baltimore and dr. f. bach, immuno- biology research center, university of minnesota medical school, minne- apolis for donations of human serum from their patients; e. carter and d. leflore for technical assistance; s. pitts, j. chowning and dr. a. cucchiara, computing center, university of oklahoma health sciences center, for computing assistance; drs. r. reynolds, m. martin and l. coggins dept. of oral diagnosis for clinical assistance. references . marthaler tm, o.mullane dm, vibric c, moller dm: symposium re- 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kishpaugh j, kirk d: numerical taxonomy systems of multivariate statistical programs. stonybrook, ny, suny press . belsley da, kuh e, welsch re: regression diagnostics: identify- ing influential data and sources of collinearity. new york, j. wiley and sons . levine m, ronda-lapolla s, owen wl, socransky ss: antibody- based diagnostic for 'refractory' periodontitis. j clin periodontol , : . dong ym, pearce ei, yoe l, larsen mj, gao xj, wang jd: plaque ph and associated parameters in relation to caries. caries res , : - publish with biomed central and every scientist can read your work free of charge "biomedcentral will be the most significant development for disseminating the results of biomedical research in our lifetime." paul nurse, director-general, imperial cancer research fund publish with bmc and your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in pubmed and archived on pubmed central yours - you keep the copyright editorial@biomedcentral.com submit your manuscript here: http://www.biomedcentral.com/manuscript/ biomedcentral.com page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ http://www.biomedcentral.com/manuscript/ http://www.biomedcentral.com/manuscript/ http://www.ncbi.nlm.nih.gov/pubmed/ http://www.pubmedcentral.nih.gov/ 	elevated antibody to d-alanyl lipoteichoic acid indicates caries experience associated with fluor... 	background 	methods 	results 	discussion 	conclusions 	competing interests 	acknowledgements 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ linear regression model for predicting patient- specific total skeletal spongiosa volume for use in molecular radiotherapy dosimetry james m. brindle , a. alexandre trindade , amish p. shah , derek w. jokisch , phillip w. patton , j. carlos pichardo , and wesley e. bolch , department of nuclear and radiological engineering, university of florida, gainesville, florida; department of statistics, university of florida, gainesville, florida; m.d. anderson cancer center orlando, orlando, florida; department of physics and astronomy, francis marion university, florence, south carolina; department of health physics, university of nevada–las vegas, las vegas, nevada; and department of biomedical engineering, university of florida, gainesville, florida the toxicity of red bone marrow is widely considered to be a key factor in restricting the activity administered in molecular radio- therapy to suboptimal levels. the assessment of marrow toxicity requires an assessment of the dose absorbed by red bone mar- row which, in many cases, requires knowledge of the total red bone marrow mass in a given patient. previous studies demon- strated, however, that a close surrogate—spongiosa volume (combined tissues of trabecular bone and marrow)—can be used to accurately scale reference patient red marrow dose es- timates and that these dose estimates are predictive of marrow toxicity. consequently, a predictive model of the total skeletal spongiosa volume (tssv) would be a clinically useful tool for im- proving patient specificity in skeletal dosimetry. methods: in this study, male and female cadavers were subjected to whole- body ct scans. manual image segmentation was used to es- timate the tssv in all active marrow–containing skeletal sites within the adult skeleton. the age, total body height, and ct-based skeletal measurements were obtained for each ca- daver. multiple regression was used with the dependent vari- ables to develop a model to predict the tssv. results: os coxae height and width were the skeletal measurements that proved to be the most important parameters for prediction of the tssv. the multiple r value for the statistical model with these parameters was . . the analysis revealed that these parameters predicted the estimated the tssv to within approx- imately % for of the cadavers and to within approxi- mately % for all cadavers in this study. conclusion: although the utility of spongiosa volume in estimating patient- specific active marrow mass has been shown, estimation of the tssv in active marrow–containing skeletal sites via patient- specific image segmentation is not a simple endeavor. however, the alternate approach demonstrated in this study is fairly simple to implement in a clinical setting, as the input measurements (os coxae height and width) can be made with either pelvic ct scanning or skeletal radiography. key words: bone marrow; radionuclide therapy; spongiosa vol- ume; active bone marrow; radiation dosimetry j nucl med ; : – the objective of molecular radiotherapy treatment plan- ning is to determine the appropriate activity level of the therapy agent to be administered to an individual patient so as to ensure an effective response in the targeted diseased tissue while minimizing toxicities to nontargeted normal or- gans and tissues. the dose-limiting toxicity most frequently encountered in molecular radiotherapy, in particular, radio- immunotherapy, is myelosuppression for protocols that do not provide a priori for hematopoietic stem cell support ( ). dose–response relationships determined from data in human clinical trials therefore are sought for use in antici- pating marrow toxicity in future patients, with the assump- tions that consistent dosimetric analyses are conducted ( , ) and that other biologic modifying factors are consid- ered ( ). recent and comprehensive reviews of the marrow dosimetry techniques currently used in clinical medicine may be found in articles by stabin et al. ( ), sgouros ( ), and siegel ( ). once marrow activity levels are assessed in a patient, through either blood-based ( ) or image-based ( ) methods, estimates of doses absorbed by red (hematopoietically active) bone marrow may be made by use of radionuclide s values for skeletal tissues assigned to a reference patient or ana- tomic phantom ( – ). when the radiolabeled agent does not bind to marrow cellular components, reference phantom s values may be used for skeletal dosimetry without an ex- plicit need to assign or estimate the total red bone marrow (rbm) mass in the individual patient, as shown by shen et al. ( ). as discussed by siegel ( ), however, a total rbm mass estimate is required when marrow or bone tissue is targeted by the therapy agent. the conventional approach used in nuclear medicine is to make this estimate under the received jul. , ; revision accepted aug. , . for correspondence or reprints contact: wesley e. bolch, phd, advanced laboratory for radiation dosimetry studies (alrads), department of nuclear and radiological engineering, university of florida, gainesville, fl - . e-mail: wbolch@ufl.edu total skeletal spongiosa volume model • brindle et al. presumption that the total rbm mass within the skeleton is scaled linearly with the total body mass, as follows: ðmrbmÞpatient � ðmrbmÞphantom tbmpatient tbmphantom � � : eq. in equation , mrbm is the mass of the total rbm in either the patient or the reference phantom and tbm is the total body mass of the same individual or phantom. stabin et al. ( ) noted, however, that lean body mass might be a more appropriate scale for rbm mass estimates because lean body mass excludes the mass associated with storage fat, as follows: ðmrbmÞpatient � ðmrbmÞphantom lbmpatient lbmphantom � � : eq. in , studies by bolch et al. ( ) and by shen et al. ( ) independently suggested the use of trabecular spongiosa volumes (combined tissues of bone marrow and bone trabec- ulae in cancellous bone) as clinically useful scales for ref- erence s values in skeletal dosimetry. using -dimensional ( d) image-based radiation transport in the femoral and humeral heads, bolch et al. ( ) demonstrated close agree- ment between patient-specific and spongiosa volume–scaled energy-dependent radionuclide s values for electron energies above ; kev. shen et al. ( ) further showed signifi- cantly improved correlations between rbm absorbed dose and observed myelotoxicity when the dose estimates were adjusted by patient-specific measurements of spongiosa volumes within the lumbar vertebrae. both of these studies made the implicit assumption that ratios of rbm masses in skeletal region x are closely approximated by corresponding ratios of their trabecular spongiosa volumes, as follows: ðmrbmÞxpatient ðmrbmÞxphantom ðsvÞxpatient ðmvfÞ x patient ðcfÞ x patient ðrtamÞ ðsvÞxphantom ðmvfÞ x phantom ðcfÞ x phantomðrtamÞ � ðsvÞxpatient ðsvÞxphantom : eq. in equation , sv is the spongiosa volume, mvf is the marrow volume fraction of the spongiosa, cf is the marrow cellularity factor (fraction of marrow volume that is hema- topoietically active), and r is the tissue density. equation demonstrates that the ratio of spongiosa volumes is equiv- alent to the ratio of red marrow masses under the condition that the marrow volume fraction and the marrow cellularity of the patient are faithfully represented in the reference patient. the studies of bolch et al. ( ) and watchman et al. ( ) demonstrated that reference skeletal models can now be made to explicitly account for marrow cellularity in reference patient radionuclide s values, although this is not yet current clinical practice. furthermore, mri techniques are available to noninvasively assess marrow cellularity in individual patients ( – ). consequently, cancellation of the cellularity factor terms in equation can be justified by proper selection of the reference s values matched to the patient’s marrow cellularity assessed throughout the skel- eton. cancellation of the marrow volume fraction terms must still be justified, but the marrow volume fraction term has been shown to be important only for very low-energy– emitting radionuclides (mean b-energies of , kev) ( ). the purpose of the present study was to develop a clini- cally feasible regression model for predicting in a given pa- tient the cumulative volume of trabecular spongiosa found across all major skeletal sites known to contain rbm in an adult ( ). accordingly, we defined the total skeletal spongiosa volume (tssv) in the present study as encom- passing the axial skeleton (trunk and head) and the prox- imal femora and humeri of the appendicular skeleton (as the distal extremities contain primarily inactive, or yellow, bone marrow in the adult). tssv data for the regression analysis were taken from detailed image segmentation of whole-body ct scans of cadavers ( males and females). dependent fitting parameters included the total body height and skeletal size measurements, with particular attention to sites used clinically for direct imag- ing of marrow activity uptake in molecular radiotherapy ( ). materials and methods cadaver and image acquisition with the cooperation of the state of florida anatomic board at the university of florida, cadavers were selected for whole- body ct. selection criteria included a trauma-related cause of death or a death that did not result in prolonged or progressive disease. in both cases, the skeletal structure of the individual was believed not to be altered from its normal age-dependent state. the body donation records used in cadaver selection do not con- tain the total body masses either before or after death. addition- ally, the embalming process often prevents an accurate total body mass estimate after receipt of an individual by the anatomic board. accordingly, premortem total body masses were estimated through visual inspection by the senior laboratory technician. these data, however, were for internal reference only and were not used in the regression analysis. with the cooperation of the department of radiology at the university of florida, each cadaver was subjected to a series of whole-body ct scans acquired with a siemens sensation ct scanner. the slice thickness for the first cadavers was mm, and the in-plane pixel resolution was mm. the number of slices to be analyzed in the -mm datasets was on the order of , . in an effort to reduce analysis time to a more manageable magnitude, the slice thickness was increased to mm for the remaining cadavers in the study. the in-plane pixel resolution for the -mm datasets ranged from to mm. spongiosa volume estimation image segmentation of the ct datasets was manually performed with a user-written interactive data language (idl version . ; itt visual information solutions, inc.) software program ( ). this program allows the user to create a computer file consisting of the journal of nuclear medicine • vol. • no. • november contours of each slice superimposed on ct image slices. within each slice of the contour file, an interactive pen display was used to outline selected skeletal regions for each ct slice. the interior of the outlined skeletal region was assigned a segmentation or tag value within the contour file; each tag value corresponded to a unique color and skeletal site (for example, cranium, ribs, and cervical vertebrae), as shown in figure . the numbers of voxels with the same tag value in the contour file were then summed and multiplied by the single voxel dimensions to produce a regional tissue volume. details of this program are given in greater detail in articles by nipper et al. ( ) and shah et al. ( ). figure displays sets of transverse slices through of the cadavers in the study. figures a and b show slices in the upper section of the skull, where spongiosa is segmented within the frontal and left/right parietal bones. figures c and d display an inferior view of the skull (just below the nose) showing sections of the mandible, the occipital and temporal bones, and the sphe- noid and nasal sinuses. the bones of the upper facial region (sphe- noid, ethmoid, lacrimal, nasal, zygomatic, and maxillary) were not segmented in the present study because these regions present very thin sections of spongiosa that were in many cases difficult to discern from the sinus cavities at the ct image resolution used for the head and because their combined spongiosa volumes account for less than % of the total cranial spongiosa volume and conse- quently less than % of the tssv ( ). figures e and f corre- spond to the upper torso near the transition between the cervical and the thoracic vertebrae. figures g and h display a view of the central region of the torso, where the rib head is joined to the thoracic vertebral body. note that at this level, the humeri are not segmented, as they contain only yellow marrow in the adult. finally, figures i and j provide a view of the pelvic regions of the body corresponding to the second sacral vertebrae. figure j demonstrates that neither the sacral foramina nor regions of sacral vertebral fusion (upper white region) are included in the assess- ment of the sacral spongiosa. spongiosa volumes were obtained for ct datasets containing different skeletal sites to determine the impact that various slice thicknesses have on spongiosa volume determinations. the slice thicknesses of the first and second ct datasets were and mm, respectively. the mean percentage difference between the volume estimates was . %, with an associated % confidence limit of . %. spongiosa volumes were manually segmented and estimated for the active marrow–containing skeletal sites, as defined by the international commission on radiological protection (icrp) ( ) and listed in table . the tssv, determined by summing the individual skeletal site spongiosa volumes, was used in the regression analysis. the accuracy of the manual segmentation process was previously investigated by brindle et al. ( ) using figure . comparison of original ct images and correspond- ing segmented images of skeletal spongiosa within transverse views through study cadaver. (a and b) superior region of skull (pink: cranium). (c and d) inferior regions of skull (pink: cranium; blue: mandible). (e and f) upper torso region (orange: humeral heads; navy blue: clavicles; cyan: scapulae; teal: cervical vertebrae; magenta: ribs). (g and h) midtorso region (green: sternum; yellow: thoracic vertebrae; cyan: scapulae; magenta: ribs). (i and j) pelvic region (red: ossa coxae; light blue: sacrum). table skeletal sites (and quantities) used for spongiosa volume estimation* skeletal site (no. of bones) % of total body active marrow cranium ( ) . mandible ( ) . humeral heads ( ) . clavicles ( ) . scapulae ( ) . sternum ( ) . ribs ( ) . cervical vertebrae ( ) . thoracic vertebrae ( ) . lumbar vertebrae ( ) . sacrum ( ) . proximal femora ( ) . ossa coxae ( ) . *table is adapted from table - in international commission on radiological protection. basic anatomical and physiological data for use in radiological protection: reference values. new york, ny: international commission on radiological protection; . icrp publication . copyright , international commis- sion on radiological protection. total skeletal spongiosa volume model • brindle et al. polyvinyl chloride pipe phantoms in which the pipe wall repre- sented regions of cortical bone and the pipe lumen represented spongiosa. for the region considered to be a surrogate for trabec- ular spongiosa, the mean percentage error at a resolution equiv- alent to that in vivo was . %. the time required to segment the skeletal sites in cadaver ranged from to h. shen et al. ( ) similarly commented that determining the volumes of spongiosa within skeletal sites containing active marrow would be a labor-intensive process. consequently, the manual segmentation effort was divided among several individuals in our research study team. brindle et al. ( ) reported a study in which individuals segmented a variety of skeletal sites, and the variations among their volumetric data were shown to be not statistically significant (p . . ). before being assigned cadavers, all individuals received an orientation review and were asked to complete the segmentation of a single skeletal site (for example, excised femoral head scanned ex vivo). the segmentation work by all individuals was reviewed by the first author to identify any anatomic discrepancies. an instructional packet provided to the segmenters outlined potentially difficult anatomic regions, such as the interface between the base of the skull and the first cervical vertebra, and guidance was provided when an anatomic question arose. anatomic measurements one anthropometric measurement and numerous image-based skeletal measurements were obtained for each of the cadavers. because of uncertainties in total body mass estimates, the sub- ject’s height was the only anthropometric measurement used in this study, as it could be easily measured on the cadaver either directly or through viewing of the ct image set. a report by the forensic anthropology center at the university of tennessee was used as a guide for an initial set of skeletal measurements ( ). the intent was to find skeletal measurements within anatomic regions of the ct scans that could be used to quantify the cumulated activity in a patient undergoing molecular radiotherapy by spect/ct, pet/ct, or planar imaging. a review of the lit- erature indicated that biokinetics measurements of a particular radiolabeled agent are often quantified in skeletal sites such as the sacrum, lumbar vertebrae, femoral heads, and the iliac crest ( , ). table summarizes the parameters measured in each cadaver and provides a brief description of how each measurement was obtained. each measurement was obtained twice, and the average was used in the regression analysis. several of the image-based skeletal measurements were obtained from the anterior–posterior scout image from each cadaver’s ct scan. consequently, these measurements were more properly projected skeletal dimensions. table anthropometric measurements obtained for use in multiple regression analysis parameter abbreviation measurement (cm) height ht total body height os coxae width oc.w maximum width of os coxae viewed in ct scout image (projection) os coxae height oc.h average of maximum heights of left and right sides of os coxae viewed in ct scout image (projection) os coxae length oc.l average of maximum lengths of left and right sides of os coxae viewed in transverse plane of d ct dataset bitrochanteric breadth bi.b distance between exterior portions of greater trochanters viewed in ct scout image (projection) anterior sacral height ash distance from anterior sacral promontory to apex of sacrum viewed in sagittal plane of d ct dataset sacral width s.w maximum width of sacrum viewed in transverse plane of d ct dataset l thickness l .t thickness of l vertebra viewed in sagittal plane of d ct dataset; measurements were made parallel to anterior surface of vertebral body and approximately . cm into vertebral body s breadth s .b distance between most lateral points on superior surface of s viewed in transverse plane of d ct dataset femoral head perimeter p average of maximum perimeters of left and right femoral heads viewed in sagittal plane of d ct dataset feret’s diameter fd measurement is based on femoral head perimeter; this measurement is referred to as caliper length, as it represents longest distance between any points along selected boundary; in this study, perimeter measurement served as this boundary maximum height of femoral head max.h maximum height of femoral head viewed in sagittal plane of d ct dataset; each measurement represented average for left and right femoral heads maximum width of femoral head max.w maximum width of femoral head viewed in sagittal plane of d ct dataset; each measurement represented average for left and right femoral heads humeral head breadth hh distance between exterior portions of right and left proximal humeral heads viewed in ct scout image (projection) femoral height fh maximum height of femoral bones viewed in ct scout image (projection); each measurement represented average for left and right femoral bones the journal of nuclear medicine • vol. • no. • november nevertheless, clinical implementation of the final regression model is greatly facilitated by use of these projected dimensions without the need for d reconstruction or multiple -dimensional imaging of the patient’s skeletal anatomy. statistical analysis all statistical analyses were performed with microsoft excel and the r statistical software package ( ). multiple regression was used to develop a statistical model for the prediction of the tssv. initially, the intent was to develop sex-dependent models, but a decision was made to pool the sexes because of the sample size constraints. a matrix of scatter plots was used to qualitatively assess the colinearity among the predictor variables. the linear relationships between each predictor variable and the response variable, tssv, were also assessed with the scatter plot matrix. the linear model to be fit was the following: tssv b b x b x bi xi . . . bn xn e: eq. in equation , b represents the intercept term, bi represents the parameter estimates for the predictor variables xi included in the model, and e is the error associated with the model. a number of statistical methods exist to aid in the selection of a parsimonious subset of predictor variables for inclusion in the final regression model. the stepwise method sequentially adds and removes predictor variables to the model on the basis of their contributions to the overall fit ( ). a second approach examined the use of the adjusted r criteria. the multiple r value is a measure of the success of the regression equation at explaining the relationship between the tssv and the included predictor vari- ables, on a scale of – ( , ). however, selection of the model with the highest multiple r value is not advantageous because that model will always contain all of the possible predictor vari- ables. the adjusted r value is a version of the multiple r value adjusted for the number of predictor variables in the model ( ). the adjusted r value will increase or decrease according to the importance of a predictor variable added to the model and there- fore lends itself for use in the variable selection process. two other predictor variable selection methods considered were the corrected akaike information criterion (aicc) and the bayes information criterion (bic). these methods have their genesis in sophisticated information–theoretic principles and are gaining popularity as model selection tools in the biological sciences ( ). their information–theoretic basis makes them well suited to situations in which the predictive capabilities of the final model are of paramount importance, as is the case in this study. further- more, they are consistent criteria in the sense that if the pool of candidate models were to indeed contain the true model, then aicc and bic would correctly identify it in the limit of increasing sample size. the approach involves considering all possible subset models that can be constructed from the pool of available pre- dictor variables and calculating the aicc and bic statistics for each model. models with the smallest values for these statistics are considered optimal according to each respective criterion. (the aicc adjusts and corrects the original aic for small sample sizes.) once a final regression model was selected, a type of cross- validation that we refer to as a ‘‘leave-one-out’’ analysis was con- ducted. this analysis entails splitting the data into sets. in general, the first (usually larger) set is used for fitting the regres- sion equation, and the second set is used to assess how well the regression equation can predict future values ( ). in the present study, cadaver was removed from the original set of . the predictors selected for the final model and the data from the re- maining cadavers were used to fit a regression equation. the resulting equation was then used to predict the total spongiosa volume of the cadaver removed from the original set of . this process was repeated for every cadaver so that the final model was validated times. a % prediction interval for the total spongiosa volume of each cadaver omitted in the leave-one-out analysis was also computed. the true value of a specific individ- ual’s tssv is expected to be contained in this prediction interval % of the time. results the anatomic measurements as well as the ages of the cadavers are shown in table . table shows each of the coefficient estimates and associated p valuesfor the predictor variable chosen by the model selection method. the stepwise regression approach produced the following model: tssv b b ðoc:wÞ b ðoc:hÞ b ðmax:wÞ e: eq. in equation , the os coxae width is represented by oc.w, oc.h represents the os coxae height, max.w represents the maximum width of the femoral head (viewed in the sagittal plane), and e represents the error associated with the model. the multiple r value for this model was . . the adjusted r value approach resulted in a model with of the same predictor variables (oc.w and oc.h) as those found in the model produced by the stepwise regres- sion approach. the adjusted r value approach produced the following model: tssv b b ðoc:wÞ b ðoc:hÞ b ðmax:wÞ b ðmax:hÞ e: eq. the multiple r for the model obtained with the adjusted r value approach was . . with the aicc method, the following model was pro- duced: tssv b b ðoc:wÞ b ðoc:hÞ e: eq. the multiple r value for the model obtained with the aicc approach was . . the bic approach selected the same model parameters. after the findings were reviewed, a final recommended model consisting of the os coxae width (oc.w) and the os coxae height (oc.h) was selected, as given in equation . with the final model selected, the leave-one-out analysis was performed. using a model with only the os coxae width and the os coxae height, we calculated the predicted tssv for each cadaver on the basis of the data from the other cadavers. table shows the ct-measured tssv for each total skeletal spongiosa volume model • brindle et al. cadaver along with the respective predicted tssv and the percentage differences between the values. the percent- age differences ranged from . % to . %. the mean percentage difference was . %, with an associated % confidence limit of . %, and the median was . %. the prediction intervals associated with the predicted tssv for each cadaver are also shown. the percentage distribution of the tssv by skeletal site is shown in table for the cadavers studied. these values were fairly consistent with the percentage distribu- tions of total bone marrow in icrp reference male and female adults; the latter values are given as the normalized ratios of the percentage distributions of active bone marrow cellularity and reference marrow cellularity ( ). exact comparisons of the percentage distribution of tssv cannot be made, as regional values for the marrow volume fraction remain undefined in icrp reference adults. discussion from a statistical viewpoint, the final regression model containing the os coxae height and width was chosen for several reasons. with a sample size of , the number of predictor variables included in the final model should not be more than about . pedruzzi et al. ( ) indicated that the sample size or the number of events per variable included in the model should be at least to preserve the model’s validity. models with a number of events per variable of less than have been shown to introduce bias into the regression coefficients ( ). table results of various model selection methods selection method multiple r variable coefficient coefficient estimate coefficient p stepwise . intercept b , . , . oc.w b . . oc.h b . . max.w b . . adjusted r . intercept b , . , . oc.w b . . oc.h b . . max.w b . . max.h b . . aicc and bic . intercept b , . , . oc.w b . . oc.h b . , . table values for predictor variables obtained from cadavers for use in multiple regression analysis* cadaver age at death (y) ht oc.w oc.h oc.l bi.b ash s.w l .t s .b p fd max.h max.w hh fh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . *abbreviations are explained in table . all measurements are given in centimeters. the journal of nuclear medicine • vol. • no. • november both the aicc and the bic model selection approaches identified only the os coxae height and the os coxae width as the optimal set of predictors. the stepwise selection ap- proach chose additional variable, the maximum width of the femoral head, and the adjusted r approach selected the maximum height of the femoral head in addition to these . the multiple r values for these models varied from . with the adjusted r approach to . with the aicc approach. the use of only the os coxae height and the os coxae width did not reduce to a substantial degree the pre- dictive capability of the model relative to those of models with more parameters. however, it is possible that subse- quent studies with larger numbers of cadavers will find or both of the maximum femoral head measurements to be important predictors as well. the leave-one out analysis suggested that the use of the os coxae width and the os coxae height was adequate in predicting the tssv. the mean percentage difference be- tween the measured tssv and the predicted tssv was found to be . %. for of the cadavers, the percent- age difference was found to be less than %. a paired t test revealed that the mean percentage difference was not table regional percentage distribution of trabecular spongiosa by skeletal site* skeletal site % active bone marrowy % marrow cellularityz % total bone marrow (icrp adult) mean sd % tssv (present study) cranium . . . . mandible . . . . scapulae . . . . clavicles . . . . sternum . . . . ribs . . . . cervical vertebrae . . . . thoracic vertebrae . . . . lumbar vertebrae . . . . sacrum . . . . ossa coxae . . . . femora (proximal) . . . . humeri (proximal) . . . . *for comparison, data on percentage distribution of total bone marrow for icrp reference adult (male or female; age, y) are shown. ydata are from table in international commission on radiological protection ( ). zdata are from table in international commission on radiological protection ( ). table segmented and predicted tssvs, percentage errors, and prediction intervals tssv (cm ) prediction interval (cm ) cadaver segmented predicted % difference lower upper , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . , . , . . , . , . total skeletal spongiosa volume model • brindle et al. significantly different from (p . ). the % predic- tion interval for the predicted total spongiosa volume cap- tured the total spongiosa volume in of the cadavers. the data for cadaver did not fall inside the prediction interval. upon closer inspection of the data, we found that this cadaver had a somewhat larger tssv than would be expected on the basis of the os coxae width and the os coxae height. in any case, a prediction success rate of of ( %) means that the regression model was performing as expected. it should be noted that the utility of the final model, which contains the os coxae height and the os coxae width parameters, was assessed only with a certain sample pop- ulation. the sample population had a mean os coxae width of . cm and a mean os coxae height of . cm, with % confidence limits of . and . , respectively. the os coxae width measurements ranged from . to . cm, and the os coxae height measurements ranged from . to . cm. less reliable predictions of the tssv would therefore be expected for individual patients with os coxae dimensions outside these ranges. not surprisingly, the parameters associated with the os coxae were central to predicting the tssv. for all ca- davers in the present study, the os coxae was the skeletal site with the largest spongiosa volume, comprising an aver- age of % of the tssv in the adult for skeletal sites known to house hematopoietic bone marrow in the adult. in the study of brindle et al. ( ), the os coxae height had an r value of . and correlated fairly well with the total pelvic spongiosa volume; the os coxae width had an r value of . and thus did not correlate very well with the total pelvic spongiosa volume. in the present study, the observations were similar, as the os coxae width had an r value of . and the r value for the os coxae height was . . however, when the parameters were considered together in a regression model, they predicted the tssv fairly well. one potential explanation is that the os coxae is a relatively thin bone and that its height and width are thus characteristic of its overall size. another factor that was considered in the selection of this model was the ease of obtaining necessary measure- ments. implementation of any technique, model, or process into a clinical environment should be practical, and a model based on a complex measurement scheme would likely be time-consuming for both the patient and the clinician. mea- surements of the os coxae width and height can be made quickly and easily with a pelvic ct scan, ct scout image, or even a skeletal radiograph of the pelvis. imaging with either pet or spect is performed for several radionuclide therapy schemes to quantify a patient’s biokinetics ( – ). with the development of pet/ct and spect/ct, not only would biokinetics information be available but also the ct scan would provide a means to obtain the necessary ana- tomic measurements of the os coxae height and width and thus provide a reasonable estimate of the tssv for scaling reference marrow masses for dosimetric evaluation for that patient. if planar imaging were used, then a simple pelvic radiographic could easily suffice to obtain these skeletal size measurements. the os coxae width and os coxae height measurements are truly projection measurements. in order to better characterize the pelvis, the length of the os coxae was also measured with volumetric ct data (not the scout image); pelvic tilt was explicitly considered in the supine positioning of the ca- davers during ct. not surprisingly, the os coxae length was highly correlated with the projected os coxae height (corre- lation coefficient r . ). because the os coxae height was highly correlated with the os coxae length and the difficulty associated with obtaining the os coxae height measurement is minimal (compared with the os coxae length), the os coxae height was the parameter selected for statistical modeling. given a patient-specific estimate of the tssv with equa- tion , one may derive a corresponding estimate of the patient-specific rbm mass with equation by using the tssv for the reference phantom. although the icrp does provide total and regional values for rbm mass and mar- row cellularity for its reference adult male and female ( ), corresponding values for marrow volume fraction and spongiosa volume are not defined in icrp publication ( ). shen et al. ( ) encountered the same issue in assigning reference patient values for spongiosa volume for the l –l vertebrae, and these authors instead resorted to the use of average patient data. in a similar manner, we present in table approximate reference values for tssv based on averages of the cadaver total body height ( cm of the icrp reference height). the height of the icrp reference male is cm, and of the male cadavers in the present study had heights within cm of this value. their average height was . cm, and their average tssv was , . cm . the height of the icrp reference female is cm, and of the female cadavers in the present study had heights within cm of this value. their average height was . cm, and their average tssv was , . cm . conclusion the objective of the present study was to develop a clin- ically viable means for estimating the tssv within a given patient undergoing molecular radiotherapy. currently, the mass of a patient’s active marrow is estimated from that of the icrp reference adult scaled by the ratio of phantom anthro- pometric parameters to patient anthropometric parameters, such as total body mass or lean body mass. other studies have suggested the use of more anatomically based scaling param- eters such as the volume of trabecular spongiosa in a given skeletal region or throughout the entire body. conceptually, the spongiosa volume in active marrow–containing skeletal sites would be a better parameter to use for scaling reference active marrow masses. in the present study, multiple linear regression with the measured tssv and ct-based skeletal size measurements from cadavers resulted in the development the journal of nuclear medicine • vol. • no. • november of a predictive model of the tssv in individual patients. although a leave-one-out analysis illustrated the ability to use simple os coxae measurements (height and width) to predict the tssv to within approximately % accuracy, the use of these estimates to improve correlations of dosim- etry with observed marrow toxicity remains to be evaluated. expanding the cadaver database of the present study will potentially increase the predictive nature of the model and may offer the ability to predict spongiosa volumes separately in male and female patients. acknowledgments the authors thank the following individuals for their assistance in performing the manual image segmentation: stephanie adams, daren cato, steve frederick, daniel harrington, deanna hasenauer, matt hough, kayla kielar, wendy kresge, andrea knowlton, jared lyons, paul moore, and scott wyler. this work was supported by grants ro ca and f ca to the university of florida from the national cancer institute. references . macey d, williams l, brietz h, lui a, johnson t, zanzonico p. a primer for radioimmunotherapy and radionuclide therapy. madison, wi: medical physics publishing; 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: – . . roeske jc, lujan a, reba rc, penney bc, diane yamada s, mundt aj. incorporation of spect bone marrow imaging into intensity modulated whole- pelvic radiation therapy treatment planning for gynecologic malignancies. radiother oncol. ; : – . . desai ag, thakur ml. radiopharmaceuticals for spleen and bone marrow studies. semin nucl med. ; : – . . datz fl, taylor a jr. the clinical use of radionuclide bone marrow imaging. semin nucl med. ; : – . . international commission on radiological protection. basic anatomical and physiological data for use in radiological protection: reference values. new york, ny: international commission on radiological protection; . icrp publication . total skeletal spongiosa volume model • brindle et al. a paired-image radiation transport model for skeletal dosimetry amish p. shah, phd ; wesley e. bolch, phd , ; didier a. rajon, phd ; phillip w. patton, phd ; and derek w. jokisch, phd department of biomedical engineering, university of florida, gainesville, florida; department of nuclear and radiological engineering, university of florida, gainesville, florida; deparment of neurosurgery, university of florida, gainesville, florida; department of health physics, university of nevada–las vegas, las vegas, nevada; and department of physics and astronomy, francis marion university, florence, south carolina toxicity of the hematopoietically active bone marrow continues to be a primary limitation in radionuclide therapies of cancer. improved techniques for patient-specific skeletal dosimetry are thus crucial to the development of dose–response relationships needed to optimize these therapies (i.e., avoid both marrow toxicity and tumor underdosing). current clinical methods of skeletal dose assessment rely heavily on a single set of bone and marrow cavity chord-length distributions in which particle energy deposition is tracked within an infinite extent of trabec- ular spongiosa, with no allowance for particle escape to cortical bone. in the present study, we introduce a paired-image radia- tion transport (pirt) model that can provide a more realistic -dimensional geometry for particle transport of the skeletal site at both microscopic and macroscopic levels of its histology. methods: ex vivo ct scans were acquired of the lumbar ver- tebra and right proximal femur excised from a -y male ca- daver (body mass index, . kg m� ). for both skeletal sites, regions of trabecular spongiosa and cortical bone were identi- fied and segmented. physical sections of interior spongiosa were then taken and subjected to nuclear magnetic resonance (nmr) microscopy. voxels within the resulting nmr microim- ages were segmented and labeled into regions of bone trabec- ulae, endosteum, active marrow, and inactive marrow. the pirt methodology was then implemented within the egsnrc radia- tion transport code, whereby electrons of various initial energies are simultaneously tracked within both the ex vivo ct macro- image and the nmr microimage of the skeletal site. results: at electron initial energies greater than – kev, a divergence in absorbed fractions to active marrow is noted between pirt model simulations and those estimated under infinite spongiosa transport techniques. calculations of radionuclide s values un- der both methodologies imply that current chord-based models used in clinical skeletal dosimetry can overestimate dose to active bone marrow in these skeletal sites by � %– % for low-energy �-emitters ( p, er, and lu), by � %– % for intermediate-energy �-emitters ( sm, re, and sr), and by � %– % for high-energy �-emitters ( p, re, and y). conclusion: the pirt methodology allows for detailed model- ing of the d macrostructure of individual marrow-containing bones within the skeleton, thus permitting improved estimates of absorbed fractions and radionuclide s values for intermedi- ate-to-high �-emitters. key words: skeletal dosimetry; marrow dose; nuclear magnetic resonance microscopy; radionuclide s value; absorbed fraction j nucl med ; : – the skeletal system represents one of the more complex challenges in internal dosimetry. this distributed organ, with its wide variety of bone sizes and configurations, encompasses the hematopoietic tissues of the active (red) bone marrow as well as the osteogenic tissues of the en- dosteum, both of which are relevant targets for short-term deterministic and long-term probabilistic radiation effects. of primary importance is the -dimensional ( d) micro- scopic architecture of the bone trabeculae, which separate and define the marrow cavities. for short-ranged radiations (�-particles and lower-energy �-particles), knowledge of this d microstructure is necessary and sufficient for accu- rate computation of particle transport through these skeletal tissues. for longer-ranged radiations (such as intermediate- to high-energy �-particles), further consideration should be given to the d macrostructure of the skeletal site, including the location and extent of cortical bone into which escaping particles may penetrate. the vast majority of initial studies in skeletal dosimetry were conducted at the university of leeds by spiers and his students ( – ). spiers was the first to recognize that the anisotropic structure of trabecular bone required a unique method for characterizing the trabecular geometry as needed for accurate skeletal dosimetry of �-emitters ( , ). consequently, he and his students constructed an optical bone-scanning system that measured linear chord-length distributions across -dimensional radiographs of excised bone tissue slices. using these frequency distributions of linear chord lengths through both bone trabeculae and mar- row cavities, the fraction of a particle’s kinetic energy deposited in each tissue type was estimated. spiers and his students obtained chord-length distributions in the lumbar received jul. , ; revision accepted sep. , . for correspondence or reprints contact: wesley e. bolch, phd, advanced laboratory for radiation dosimetry studies, department of nuclear and ra- diological engineering, university of florida, gainesville, fl - . e-mail: wbolch@ufl.edu the journal of nuclear medicine • vol. • no. • february vertebrae for several subjects, as well as at several skeletal sites of a . -y child ( sites), a -y child ( sites), and a -y man ( sites) ( – ). in many ways, the chord-length distribution data measured for the -y man has served to define many of the skeletal attributes of reference man as defined by the international commission on radiological protection (icrp) ( , ). furthermore, all skeletal dosim- etry models published and presently used in clinical dose assessment are fundamentally reliant on this single set of adult chord-length distributions ( – ). in this technique, radiation particles are effectively trans- ported within an infinite region of trabecular spongiosa (defined as the combined tissues of the bone trabeculae, endosteum, and marrow cavities). models of skeletal do- simetry used in current clinical practice, such as the ecker- man and stabin model ( ) of mirdose and its successor codes ( ), belong to a class of models called cbist (chord-based infinite spongiosa transport) and do not account for particle escape to cortical bone. consequently, absorbed fractions to skeletal tissues are potentially overes- timated in cbist models for higher-energy �-emitters. one of the first attempts to account for energy loss to cortical bone was made by spiers’ doctoral student j.r. whitwell ( , ). she introduced a trabecular equilibrium factor, qtrab, to account for the finite extent of the spongiosa. this correction factor was determined for several radionu- clides of interest in radiation protection and for each of the skeletal sites for which chord-length distributions were obtained in the -y male subject. for y, the highest correction noted by whitwell was for the parietal bone (qtrab � . ), whereas the lowest was for the head of the femur (qtrab � . ). nevertheless, these values of qtrab were determined using simplified geometries for both spon- giosa and cortical bone (e.g., planes and spheres). in a more recent study by patton et al. ( ), nuclear magnetic resonance (nmr) microscopy was applied to the study of the d microstructure of bone trabeculae within the femoral and humeral heads of subjects: a -y man, an -y woman, and a -y woman. to account for energy lost to cortical bone, an ex vivo ct scan of the excised femoral or humeral head was obtained before spongiosa sectioning. from spatial measurements on the ct images, a spheric region of spongiosa was constructed surrounded by a spheric shell of cortical bone. electrons of various initial energies were thus transported (via the esg radiation transport code) simultaneously within the nmr microimage (constructed of voxels of bone and marrow) and within a stylized model of the femoral or humeral head. compari- sons were subsequently made between energy-weighted ab- sorbed fractions to active marrow under particle transport in either (a) an infinite extent of spongiosa or (b) the stylized model of the bone site. patton et al. demonstrated that, without explicit consideration of energy loss to cortical bone, radionuclide s values for p and y could potentially overestimate active marrow dose by % and %, respec- tively, in the femoral head—values that exceeded the % corrections predicted by whitwell. this tendency to over- estimate dose to active marrow under infinite spongiosa transport had also been demonstrated by jokisch et al. ( ) for the thoracic vertebra, in which the physical extent of the vertebral spongiosa was delineated in a stylized model of the vertebral body (e.g., truncated circular cylinder). due to their geometric complexity, however, no attempt was made to include the vertebral processes in the stylized vertebral model (which account for up to � % of vertebral spon- giosa). in the present study, we significantly extend the skeletal modeling approach originally explored by jokisch et al. ( ) and patton et al. ( ) to fully account for the d macro- structural dimensions of skeletal sites within which dose estimates are desired. a paired-image radiation transport or pirt model for skeletal dosimetry is introduced, in which radiation particles are tracked simultaneously within different segmented digital images: (a) an ex vivo ct image of the entire skeletal site outlining regions of trabec- ular spongiosa, cortical bone, and surrounding tissues and (b) an ex vivo nmr microscopy image of the interior bone trabeculae and marrow cavity microstructure representative of that found in spongiosa volumes of the larger ct image. the pirt model is demonstrated within skeletal sites obtained from a single male cadaver: the l vertebra and the right proximal femur. in addition, representative site- specific s values are calculated and compared with those obtained under particle transport within infinite regions of spongiosa for a variety of radionuclides of interest in skel- etal imaging and therapy. materials and methods cadaver selection candidate subjects for study were obtained through the state of florida anatomic board located on the university of florida (uf) campus. cadaver selection criteria included (a) an age between and y (representative of typical radionuclide therapy patients), (b) a body mass index (bmi) of . – kg m� (centers for disease control and prevention–recommended healthy range), and (c) a cause of death that would preclude significant skeletal dete- rioration. the subject identified was a -y male, approximately kg in total mass and cm in total height at the time of death (bmi, . kg m� ). the subject died suddenly of complications associated with cardiomyopathy. in vivo ct before bone harvesting, the male cadaver was subjected to whole-body imaging via multislice helical ct at a pitch necessary to reconstruct contiguous -mm axial slices. the images were acquired on a siemens sensation unit within the department of radiology at uf shands hospital. image reconstruction was per- formed with a bone filter at an in-plane pixel resolution of � �m. the ct image sets were then transferred to workstations within the advanced laboratory for radiation dosimetry studies (alrads) in the uf department of nuclear and radiological engineering for image processing and data storage. the in vivo ct scans provided image data for (a) selecting the anatomic region from which the bone site would be harvested and (b) constructing paired-image model in skeletal dosimetry • shah et al. d anatomic models of skeletal sites where bone harvesting (and thus ex vivo ct) might be incomplete (e.g., facial bones of the skull). bone harvesting and ex vivo ct after detailed review of the whole-body in vivo ct images, bone harvesting was conducted. thirteen major skeletal sites were taken from the male cadaver, including the entire vertebral column and both proximal femora. once each skeletal site was excised, it was cleaned of excess tissue, bagged, labeled, and stored frozen until ex vivo ct could be scheduled. after harvest, ex vivo ct was conducted at higher resolution ( . -mm slice thickness with an in-plane resolution of . � . mm) than permitted for in vivo scans. the ex vivo ct scans provided image data for (a) identi- fying the location and extent of trabecular spongiosa to be sec- tioned for nmr microscopy, (b) quantifying volumes of trabecular spongiosa and cortical bone within the bone site, and (c) construct- ing d anatomic models of the bone site for subsequent pirt simulations. after detailed review of the ex vivo ct scans, physical sections of trabecular spongiosa were taken from each bone site. sections represented as large a region of spongiosa as possible, given the constraints of the bone shape and the nmr imaging system (e.g., cuboidal samples taken from a spherically shaped femoral head). marrow-intact sections of spongiosa were bagged, labeled, and kept frozen until nmr microimaging sessions could be arranged. for the lumbar vertebra, cuboidal sections (roughly, . � . � . cm on edge) were cut from the vertebral body repre- senting � % of the total vertebral body spongiosa. for the right proximal femur, cuboidal sections were cut from the femoral head (� % of total spongiosa within the head) and sections were cut from the femoral neck (� % of the total spongiosa within the neck). image segmentation of spongiosa and cortical bone regions to create tomographic anatomic models for use in internal dosimetry, radiation transport codes must be able to decipher the boundaries of each tissue region for which an independent dose assessment is to be made. limitations of ct image acquisition can result in an overlap of gray-scale values for tissues of interest, thus precluding the use of simple automated methods of boundary definition. in the present study, the program ct_contours was adopted for use in segmenting spongiosa and cortical bone within each ex vivo ct image set ( ). this program is based on inter- active data language version . and can output labeled contour files in a variety of formats, including binary files for egsnrc ( ) and ascii text for mcnp ( ). ct_contours displays the current ct information as well as a color overlay of the contours being edited. the contours can be created using a variety of tools, including basic thresholding, pixel growing, voxel growing, region growing, and manual segmentation. the voxels contained in the individual contours are filled with the desired segmentation value, generating volumes of voxels with identical tag values. in the present study, these volumes represent individual regions of either trabecular spongiosa or cortical bone within the skeletal site. ct_contours was written to have the option of displaying the images using different filters, including gaussian smoothing ( � , � , or � ), median ( � , � , or � ), roberts edge detection, sobel edge detection, prewitt edge detection, iso- tropic edge detection, histogram equalization, adaptive histogram equalization, sharpening, and kuwahara ( � or � ) filtering. by altering regions of a separate contour dataset, the desired segmentation can be performed. ct_contours was designed so that roi creation or modification can be performed in the trans- verse, sagittal, or coronal plane. nmr microscopy of trabecular spongiosa nmr microscopy of trabecular bone for the purposes of skeletal dosimetry has been discussed previously ( , – ). nmr imag- ing requires physical sectioning of the excised sample and diges- tion of the marrow tissues. samples of trabecular bone sections are first immersed and suspended within a circulating solution of sodium hypochlorite for � h. the samples are then rinsed in water and reimmersed in a new solution. this process is repeated up to times depending on the size of the sample. visual inspec- tion is used to determine the number of repetitions needed. to ensure that water completely fills all marrow cavities, each sample is placed in a container filled with gd-doped water under vacuum. while immersed, the sample is placed in a smaller container needed for insertion into the magnet. this imaging container is then sealed and taken to the advanced magnetic resonance im- aging and spectroscopy facility at the uf mcknight brain insti- tute for nmr microscopy. nmr microscopy images in the present study were acquired on a bruker -cm wide-bore imaging spectrometer, operated at a -mhz proton resonance frequency ( -t magnetic field strength). the system is fitted with a small gradient set (for microimaging), consisting of -axes magnetic-field gradients, with a maximum gradient amplitude of g/cm in all orthogonal directions. a -mm-diameter quadrature birdcage coil of -mm length is used to obtain the best signal-to-noise ratio (snr). for all imaging sessions, a d rare (rapid acquisition with relaxation enhancement) spin-echo pulse sequence is used to obtain fully d images of the samples. fields of view are typically . � . � . cm with matrix dimensions of � � . the resulting spatial resolution of the d images is thus � � �m. smaller voxel dimensions can be achieved at uf (� �m), but at the cost of smaller sample sizes and increased imaging time (to preserve snr). postacquisition image processing, including gray- level thresholding, voxel segmentation, and d median filtering, have been reported previously ( , ). for use in radiation trans- port simulations, interior rois are taken to avoid physical distor- tions (bone saw tearing) or imaging distortions (nmr aliasing) at the edges of the sectioned specimen. voxel-based infinite spongiosa transport (vbist) model after nmr microscopy of our skeletal samples, a series of vbist models were created to approximate (via d transport) the results of current cbist models. first, marrow voxels within the binary nmr microscopy image are further labeled into voxels of active (red) marrow and inactive (yellow) marrow at a predeter- mined value of marrow cellularity. this process has been outlined previously by shah et al. and is based on microscopy measure- ments of the spatial distribution of adipocytes within normal bone marrow biopsies covering a broad range of marrow cellularities ( ). skeletal endosteum is further defined as a -�m tissue layer at the bone-marrow interface as previously described by jokisch et al. ( ). the resulting -tissue d model of trabecular spongiosa is coupled to the egsnrc radiation transport code ( ) for electron and �-particle transport simulations. source tissues include the trabecular active marrow (tam), trabecular bone surfaces (tbs), and trabecular bone volume (tbv). bone surface sources are the journal of nuclear medicine • vol. • no. • february approximated as a . -�m layer on the marrow side of the bone- marrow voxel interface. target tissues include the tam and the trabecular bone endosteum (tbe). once a given electron reaches the physical edge of the d nmr microscopy image, that particle is reintroduced to the image at a corresponding location at its opposing edge. the processes of particle transport within the image of spongiosa and its reintroduction are continued until all initial kinetic energy is expended. particle histories are continued ( , – , , ) until coefficients of variation on the absorbed fraction are � %. it is noted, however, that results given here for our voxel-based infinite spongiosa transport (ist) model are only approximate to those given from chord-based ist models. in previous studies by jokisch et al. ( , ), the authors question the sampling independence of the marrow and bone chord-length distributions within existing cbist models and suggest a d joint distribution might be more appropriate to describing the full d microarchitecture of particle transport within the spongiosa re- gions of trabecular bone. pirt model: l vertebra in contrast to the vbist model, the pirt model supplements the d microscopic histology provided by the nmr microscopy image with the d macroscopic histology given in the correspond- ing ex vivo ct image. the latter provides additional data for particle transport, including (a) the spatial extent of the trabecular spongiosa (e.g., vertebral processes and body) and (b) the spatial extent of the surrounding cortical bone (which laterally encom- passes the vertebral body, forms the lamina separating the verte- bral processes, and is absent at the superior and inferior body-disk interfaces). a schematic of the pirt model for the l vertebra of the -y man is given in figure . the ex vivo ct image is shown in the top left, in which segmented regions of spongiosa and cortical bone surfaces are highlighted in orange and white, respectively. two representative transverse slices are shown (top middle and top right), where regions of spongiosa (orange) and cortical bone (light blue) are again differentiated. superimposed over the entire ex vivo ct image is a d array of the replicate cuboidal nmr microscopy images, each representing the d microstructure of the individual bone trabeculae and marrow cavities. a d rendering of the nmr microimage is thus shown in the bottom left of figure . finally, a single transverse slice through the nmr microimage is shown in the bottom right, displaying individual voxels of bone (black) and total marrow (white). in the egsnrc implementation of the vertebral pirt model, individual electrons are tracked simultaneously within the coordi- nates of the nmr microimage (indicating locations in tbv, tbe, tam, or trabecular inactive marrow [tim]) and the coordinates of the ct macroimage (indicating locations in spongiosa, cortical bone volume [cbv], or surrounding tissues—muscle, soft tissue, or vertebral disks). elemental compositions and mass densities assumed within the pirt model were taken from report of the international commission on radiation units and measurements ( ) (see table ). when the particle is shown to leave the spongiosa of the ct macroimage, tracking within the nmr mi- croimage is halted and the particle is transported within a homo- geneous region of cortical bone defined only by the larger voxels of the ex vivo ct macroimage. upon particle escape from outer surface of the bone site, particle tracking is terminated. in cases in which the particle leaves cortical bone and reenters the interior spongiosa, particle tracking in the nmr microimage is resumed. the pirt model is thus far more anatomically realistic than is the geometry provided by either the cbist or vbist model, espe- cially for higher-energy, longer-ranged electrons and �-particles. the principal approximation inherent within the pirt model is that the trabecular microstructure given by the physical section of spongiosa (as imaged via nmr) is uniform across all ct-seg- figure . schematic of pirt model constructed for l vertebra. table elemental tissue compositions (% by mass) and mass densities used in either vbist or pirt model of skeletal dosimetry tissue or region h c n o trace mass density (g cm� ) tam . . . . . p, . s, . c, . k, . fe . tim . . . . . na, . s, . c . tbe . . . . . na, . p, . s, . c, . k . tbv . . . . . na, . mg, . p, . s, . ca . cbv . . . . . na, . mg, . p, . s, . ca . surrounding tissues . . . . . na, . p, . s, . c, . k . tam � “adult red marrow”; tim � “adult yellow marrow”; tbe � “adult icru- soft tissue (male)”; tbv � “adult cortical bone”; cbv � “adult cortical bone” (appendix a of icru report ) ( ). paired-image model in skeletal dosimetry • shah et al. mented regions of spongiosa within the skeletal site. as a result, the trabecular microstructures of the various vertebral processes (spinal, superior articular, and transverse) are implicitly assumed to be approximated by that imaged within the vertebral body. in cases in which more than one physical section of spongiosa has been imaged by nmr, the pirt model can be rerun using differ- ent nmr microimages. the resulting microimage-specific ab- sorbed fraction profiles can thus be averaged either uniformly or weighted by the volume of spongiosa sectioned. finally, it is noted that the pirt model permits explicit consideration of a cortical bone volume (cbv) as a potential radioactivity source—a feature not permitted within the cbist or vbist model of skeletal dose. pirt model: proximal femur a corresponding schematic of the pirt model for the right proximal femur of the -y male subject is shown in figure . in adults, hematopoiesis occurs primarily within the proximal epiph- ysis of the femur and, thus, the macrostructural model (shown in the top right of fig. and given by the ex vivo ct) is terminated inferiorly at the point where the lesser trochanter merges anatom- ically with the femoral diaphysis. as with the university of leeds chord-length measurements for their -y man, the biomechanics and, thus, the trabecular microstructure are notably different within the femoral head and femoral neck; consequently, d nmr mi- croscopy images were taken separately from the head and neck regions of the proximal femur. representative transverse nmr image slices are shown in the bottom middle and bottom right of figure . for each tissue source region in the model (tam, tbv, or tbs), different transport simulations are performed— one in which electrons are started within the spongiosa of the femoral head (orange voxels of the ex vivo ct transverse slice) and one in which electrons are started within the spongiosa of the femoral neck (red voxels of the ex vivo ct transverse slice). in each case, only the corresponding nmr microscopy image is used within the pirt model (head or neck microimage). final absorbed fraction results for the entire proximal femur are taken as mass weighted averages of results from the head-only and neck-only spongiosa source transport calculations. table displays the various source and target tissue masses for both the proximal femur and lumbar vertebra pirt dosimetry models (given as the product of their segmented volume and the reference densities of table ). the bottom row of table gives values of marrow volume fraction (mvf) defined as the fraction of all voxels within the nmr microimage that are assigned to marrow tissues after image thresh- olding. here it is noted that the mvf of the femoral head is . %, whereas it is . % within the femoral neck. the mvf within the l vertebral body, however, was measured at %. results absorbed fractions to active marrow within l vertebra figure displays values of electron-absorbed fraction to active (red) bone marrow within the l vertebra of the -y male subject. figure a corresponds to an assumption of % marrow cellularity (no voxels of adipose tissue are labeled within the nmr microimage), whereas figure b corresponds to an assumed marrow cellularity of % (ref- erence value in both icrp publications and ( , )). in each graph, solid lines indicate energy-dependent ab- sorbed fractions obtained from pirt model simulations, whereas dashed lines indicate those derived from vbist model simulations. for either model and at both cellulari- ties, source tissues are considered: tam (diamonds), tbs (triangles), and tbv (circles). at source energies below � kev, the model types yield essentially equivalent results, as boundary effects at the spongiosa– cortical bone interface (within the pirt model) play a negligible role in modifying the pattern of energy deposition to active marrow voxels (as seen within the vbist model). model equivalency is noted to extend to figure . schematic of pirt model constructed for right proximal femur. table tissues masses used in pirt model ( % marrow cellularity) tissue region or quantity l vertebra femoral head femoral neck proximal femur tam (g) . . . . tbe (g) . . . . tbv (g) . . . . cbv (g) . . mvf* (%) . . *mvf � marrow volume fraction: ratio of total marrow voxels to total voxels in binary d nmr microscopy images of excised cube of trabecular spongiosa. the journal of nuclear medicine • vol. • no. • february electrons of � -kev initial energy when emitted within the volume of the bone trabeculae (tbv sources). as the electron initial energy increases above – kev, energy deposition to active marrow as predicted under vbist model simulations increasingly overpredicts that given by the more anatomically realistic pirt model. as previously noted for chord-based skeletal models under either cbist or vbist simulations, absorbed fractions asymptotically approach a limited value independent of the source tissue ( , , ). at % cellularity, the vbist model absorbed fraction to active marrow approaches a value of . at high electron energies, whereas it ap- proaches a limiting value of . at % cellularity ( % of . ). similarly, absorbed fractions to active marrow pre- dicted under pirt model simulations also converge in a source-independent manner, but this convergence value is energy dependent as more and more electron energy is lost to the surrounding cortical bone (and potentially surround- ing tissues). with the pirt model results serving as the local standard, percentage errors in the self-absorbed frac- tion to active marrow given by the vbist model are % at kev, % at mev, and % at mev. corresponding percentage errors are %, %, and % for tbs sources and %, %, and % for tbv sources. these percentage errors are roughly equivalent at both marrow cellularities. absorbed fractions to active marrow within proximal femur figure displays values of electron-absorbed fraction to active marrow for tam, tbs, and tbv sources located within the spongiosa of the right proximal femur of the -y male subject. figures a and b correspond to marrow figure . electron-absorbed fractions to active bone marrow within l vertebrae for source tissues: tam, tbv, and tbs. data shown by solid lines are from pirt model, whereas those given by dashed lines are from vbist model. data for a correspond to % marrow cellularity, whereas those for b correspond to icrp reference cellularity of % ( ). figure . electron-absorbed fractions to active bone marrow within proximal femur for source tissues: tam, tbv, and tbs. data shown by solid lines are from pirt model, whereas those given by dashed lines are from vbist model. data for a correspond to % marrow cellularity, whereas those for b correspond to icrp reference cellularity of % ( ). paired-image model in skeletal dosimetry • shah et al. cellularities of % and %, respectively, where the latter is the default cellularity for the upper femur given in icrp publications and ( , ). in each graph, the individual absorbed fraction profiles for electron sources in the femoral head and in the femoral neck have been averaged according to the total mass of source tissue in the head and neck regions of the proximal femur, respectively. in figure b, the ordinate has been expanded to better view differences in modeling results at high electron energies. at the lowest energy considered ( kev), a value of 	(tam tam) � . is seen under both vbist and pirt simulations. patterns of divergence between the modeling ap- proaches (vbist vs. pirt) in the proximal femur are seen to occur at lower energies compared with those found within the l vertebra (� kev for tam sources, � kev for tbs sources, and � kev for tbv sources). furthermore, it is seen that at mev (the highest energy considered), full convergence of the absorbed fraction to active marrow under both vbist and pirt model simula- tions has not yet been reached for the source regions. nevertheless, the energy-independent (vbist) and energy- dependent (pirt) patterns of convergence are still evident at electron initial energies of mev. with the pirt model results serving as the local standard, percentage er- rors in self-absorbed fraction to active marrow ( % cel- lularity) given by the vbist model are % at kev, % at mev, and % at mev. corresponding percentage errors are %, %, and % for tbs sources and %, %, and % for tbv sources. these percentage errors are � %– % higher when the marrow cellularity of the proximal femur is reduced to % (fat fraction of � %). absorbed fractions to endosteal tissues figure displays values of absorbed fraction to the trabecular endosteal tissues defined as a -�m layer of soft tissue on the marrow side of the bone–marrow interface within the nmr microimages. figure a gives results for tbs, tbv, and tam electron sources emitted within the l vertebra containing bone marrow at % cellularity. figure b shows data for these same source tissues within the right proximal femur at % marrow cellularity. in both graphs, the ordinate scale is expanded to a maximum value of . to facilitate viewing model differences at higher energies. at the lowest energy considered ( kev), a value of 	(tbe tbs) � . is seen under both vbist and pirt simulations. at each energy for each model, higher absorbed fractions are noted for electron sources on the trabecular surfaces, whereas lower absorbed fractions are seen for electron sources emitted within the active bone marrow. intermedi- ate absorbed fractions are shown for bone volume sources that peak in value at a source energy of � kev in both skeletal sites. as expected, vbist model simulations ap- proach energy- and source-independent convergence values at high electron initial energies ( . in the l vertebra, and . in the proximal femur), whereas source-indepen- dent convergence values for the pirt model are shown to continually decline with increasing source energy above mev. this decline is slightly more prominent in the l vertebra than seen in the proximal femur and is accountable in part by cortical bone losses within the vertebral pro- cesses. in these anatomic regions of the vertebra (which encompass � % of total vertebral spongiosa), the surface- to-volume ratio of trabecular spongiosa is higher than that found in the vertebral body and, thus, electron escape to cortical bone is greater for individual electron emissions. discussion as a further means of comparing the vbist and pirt model results, radionuclide s values were calculated for a wide range of �-particle emitters of interest in skeletal figure . electron-absorbed fractions to trabecular bone endosteum within l vertebra (a) and proximal femur (b) for source tissues: tam, tbv, and tbs. data shown by solid lines are from pirt model, whereas those given by dashed lines are from vbist model. the journal of nuclear medicine • vol. • no. • february tissue imaging and radionuclide therapy. absorbed fractions to active bone marrow given in figures a and b and figures a and b, along with tissue mass data of table and �-particle energy spectra from eckerman et al. ( ), were used to calculate s values under the mird schema for different radionuclides. ratios of the s value based on vbist-model absorbed fractions to that using pirt-model absorbed fractions are displayed in table for both skeletal sites and at both % and icrp reference marrow cellu- larities. for low-energy �-emitters such as p, er, and lu, absorbed fractions given by the vbist model simu- lations overestimate radionuclide s values for tam, tbs, and tbv sources by only %– % in the l vertebra. higher errors are noted in the proximal femur, particularly for bone trabeculae volume sources (ratios of . – . ). for radio- nuclides at intermediate �-energies (eave of – kev), s value ratios range from . to . in the l vertebra and from . to . in the proximal femur. for radionuclides in the highest �-energy range (eave of – kev), s value ratios range from . to . in the l vertebra and from . to . in the proximal femur. it is reasonable to assume that similar errors are also present in radionuclide s values derived from chord-based models ( , ), which, as in the vbist simulations of the present study, assume an infinite region of spongiosa during particle transport. before the full development of the pirt methodology given here, the uf alrads research group had attempted to correct for energy loss to cortical bone by applying a stylized model of the skeletal site macrostructure. for ex- ample, in the study by patton et al. ( ), a spheric region of spongiosa surrounded by a spheric shell of cortical bone was applied to the femoral heads of different individuals based on ct image analysis. in that study, it was demon- strated that infinite spongiosa transport yielded radionuclide s values for p that were � %– % higher than those in which cortical bone energy loss was accounted for via stylistic modeling of the femoral head. for the higher- energy y, the infinite spongiosa transport results gave s values %– % higher. in the present study, however, the full d histologic macrostructure of the proximal femur (head as well as neck and trochanter regions) is treated within the pirt model simulations. corresponding correc- tions to infinite spongiosa transport are shown in the present study (by the pirt model) to be significantly higher (up to . for p and up to . for y) than indicated previously by patton et al. ( ) for the femoral head. these larger corrections are attributed to enhanced particle energy loss at spongiosa regions of the pirt femur model: the femoral neck, the trochanters, and the bottom interface of the model (where particles are lost to inactive marrow of the femoral diaphysis; fig. ). these regions of enhanced electron es- cape were not present within the spheric femoral head model of the patton et al. study. improved macrostructural modeling of the skeleton via the pirt model methodology will potentially lead to im- provements in correlations between marrow dose estimates and observed patient myelotoxicity. for example, clinical studies of the bone pain palliation agents sm-ethylenedi- table ratio of radionuclide s value for active marrow (tam) target as given by voxel-based vbist model to that given by pirt model radionuclide eave (kev) emax (kev) l vertebra: % cellularity proximal femur: % cellularity tam source tbs source tbv source tam source tbs source tbv source p . . . . . . er . . . . . . lu . . . . . . sm . . . . . . re , . . . . . . sr , . . . . . . p , . . . . . . re , . . . . . . y , . . . . . . l vertebra: % cellularity proximal femur: % cellularity radionuclide eave (kev) emax (kev) tam source tbs source tbv source tam source tbs source tbv source p . . . . . . er . . . . . . lu . . . . . . sm . . . . . . re , . . . . . . sr , . . . . . . p , . . . . . . re , . . . . . . y , . . . . . . paired-image model in skeletal dosimetry • shah et al. aminetetramethylene phosphonate ( sm-edtmp) ( – ) and re-hydroxyethylidene diphosphonate ( re-hedp) ( – ) have shown patient marrow toxicities that were lower than expected based on marrow dose estimates from standard cbist skeletal dose models (e.g., mirdose and mirdose ). although various studies have been ini- tiated to explain these discrepancies, including improve- ments in activity uptake quantification ( , ), the data of table indicate that perhaps values of marrow dose were simply overestimated in these studies, as the standard clin- ical models do not properly account for particle escape from marrow-filled regions of spongiosa. for both bone surface and volume sources, infinite spongiosa transport is shown in table to overestimate the femoral marrow self-dose by %– % for sm and %– % for re, whereas the vertebral marrow self-dose is overestimated by % for sm and % for re. conclusion a pirt model for skeletal dosimetry is presented in which electrons and �-particles are tracked simultaneously within different segmented digital images: (a) an ex vivo ct image of the skeletal site with segmented regions of trabecular spongiosa, cortical bone, and surrounding tissues and (b) an ex vivo nmr microscopy image of the interior bone trabeculae and marrow cavity microstructure represen- tative of that found within spongiosa regions of the ex vivo ct image. example dose calculations under the pirt meth- odology within the l vertebra and right proximal femur of an adult -y male subject demonstrate a divergence from standard infinite spongiosa transport (vbist) methods at energies as low as – kev, depending on the source tissue and skeletal site. calculations of radionuclide s val- ues under both methodologies imply that current chord- based models used in clinical skeletal dosimetry may over- estimate dose to active bone marrow in these skeletal sites by � %– % for low-energy �-emitters ( p, er, and lu), by � %– % for intermediate-energy �-emitters ( sm, re, and sr), and by � %– % for high-energy �-emitters ( p, re, and y). higher errors are noted for bone-volume seekers, whereas lower errors are seen for source emissions within the active bone marrow. though the proximal femur and lumbar vertebra are investigated in the present study, potentially larger errors in skeletal do- simetry are presumed to exist in skeletal sites with dispro- portionately smaller volumes of spongiosa (e.g., ribs, cra- nium, and sternum). the pirt methodology supersedes previous stylized modeling attempts by the uf alrads research group to account for the finite spatial extent of trabecular spongiosa and the presence of cortical bone. this approach thus ren- ders obsolete any need for mathematic modeling of the either simple or complex bone site geometries. furthermore, the technique increases the prospects for expanded avail- ability of reference skeletal dosimetry models for both gen- ders and of individuals of varying stature and skeletal size for use in radionuclide therapy treatment planning of cancer in which marrow toxicity is of concern. acknowledgments this work was supported in part by grant ca from the national cancer institute and grant de-fg - id from the u.s. department of energy with the university of florida. references . spiers fw. radiotherapeutic physics. ii. dosage in irradiated soft tissue and bone. br j radiol. ; : – . . spiers fw. dose to trabecular bone from internal beta-emitters. in: snyder ws, ed. first international congress of radiation protection. vol. . rome, italy: pergamon press; : – . . spiers fw. a review of the theoretical and experimental methods of determining radiation dose in bone. br j radiol. ; : – . . spiers fw. beta particle dosimetry in trabecular bone. in: mays cw, jee wss, lloyd rd, stover bj, dougherty jh, taylor gn, eds. delayed effects of bone-seeking radionuclides. salt lake city, ut: university of utah press; : – . . spiers fw. dosimetry at interfaces with special reference to bone. in: booz j, ebert hg, eds. symposium on microdosimetry. report eur . ispra, italy: european atomic energy commission; : – . . spiers fw. determination of absorbed dose to bone and red bone marrow. in: cloutier r, edwards c, snyder w, eds. medical radionuclides: radiation dose and effects. aec symposium series . oak ridge, tn: u.s. atomic energy commission; : – . . beddoe ah. the microstructure of mammalian bone in relation to the dosim- etry of bone-seeking radionuclides [thesis]. leeds, u.k.: department of medical physics, university of leeds; . . beddoe ah, darley pj, spiers fw. measurements of trabecular bone structure in man. phys med biol. ; : – . . whitwell jr. theoretical investigations of energy loss by ionizing particles in bone [thesis]. leeds, u.k.: department of medical physics, university of leeds; . . whitwell jr, spiers fw. calculated beta-ray dose factors for trabecular bone. phys med biol. ; : – . . icrp. report on the task group on reference man. icrp publication . oxford, uk: international commission on radiological protection; : – . . icrp. basic anatomical and physiological data for use in radiological pro- tection: reference values. publication . new york, ny: international com- mission on radiological protection; : – . . eckerman kf. aspects of the dosimetry of radionuclides within the skeleton with particular emphasis on the active marrow. in: schlafke-stelson at, watson ee, eds. proceedings of the fourth international radiopharmaceutical dosimetry symposium. conf- . oak ridge, tn: oak ridge associated universities; : – . . bouchet lg, bolch we, howell rw, rao dv. s values for radionuclides localized within the skeleton. j nucl med. ; : – . . bouchet lg, jokisch dw, bolch we. a three-dimensional transport model for determining absorbed fractions of energy for electrons in trabecular bone. j nucl med. ; : – . . eckerman kf, stabin mg. electron absorbed fractions and dose conversion factors for marrow and bone by skeletal regions. health phys. ; : – . . stabin mg, eckerman kf, bolch we, bouchet lg, patton pw. evolution and status of bone and marrow dose models. cancer biother radiopharm. ; : – . . stabin mg. mirdose: personal computer software for internal dose assessment in nuclear medicine. j nucl med. ; : – . . patton pw, rajon da, shah ap, jokisch dw, inglis b, bolch we. site-specific variability in trabecular bone dosimetry: considerations of energy loss to cortical bone. med phys. ; : – . . jokisch dw, bouchet lg, patton pw, rajon da, bolch we. beta-particle dosimetry of the trabecular skeleton using monte carlo transport within d digital images. med phys. ; : – . . nipper j, williams j, bolch w. creation of two tomographic voxel models of pediatric patients in the first year of life. phys med biol. ; : – . the journal of nuclear medicine • vol. • no. • february . kawrakow i. accurate condensed history monte carlo simulation of electron transport. i. egsnrc, the new egs version. med phys. ; : – . . briesmeister jf. mcnp: a general monte carlo n-particle transport code. la- -m. los alamos, nm: los alamos national laboratory; . . patton pw, jokisch dw, rajon da, shah ap, myers sl, bolch we. skeletal dosimetry via nmr microscopy: investigations of sample reproducibility and signal source. health phys. ; : – . . shah ap, patton pw, rajon da, bolch we. adipocyte spatial distributions in bone marrow: implications for skeletal dosimetry models. j nucl med. ; : – . . bolch we, patton pw, shah ap, rajon da. considerations of anthropomorphic, tissue volume, and tissue mass scaling for improved patient specificity of skeletal s values. med phys. ; : – . . bolch we, patton pw, rajon da, shah ap, jokisch dw, inglis b. consider- ations of marrow cellularity in d dosimetric models of the trabecular skeleton. j nucl med. ; : – . . jokisch dw, patton pw, inglis ba, et al. nmr microscopy of trabecular bone and its role in skeletal dosimetry. health phys. ; : – . . jokisch dw, patton pw, rajon da, inglis ba, bolch we. chord distributions across d digital images of a human thoracic vertebra. med phys. ; : – . . icru. photon, electron, proton and neutron interaction data for body tissues. report . bethesda, md: international commission on radiation units and measurements; . . icrp. basic anatomical and physiological data for use in radiological pro- tection: the skeleton. icrp publication . oxford, u.k.: international com- mission on radiological protection; : – . . eckerman kf, westfall rj, ryman jc, cristy m. availability of nuclear decay data in electronic form, including beta spectra not previously published. health phys. ; : – . . turner h, claringbold p. a phase ii study of treatment of painful multifocal skeletal metastases with single and repeated dose samarium- -edtmp. eur j cancer. ; : – . . collins c, eary jf, donaldson g, et al. samarium- -edtmp in bone metas- tases of hormone refractory prostate carcinoma: a phase i/ii trial. j nucl med. ; : – . . farhanghi m, holmes ra, volkert wa, logan kw, singh a. samarium- - edtmp: pharmacokinetic, toxicity and pain response using an escalating dose schedule in treatment of metastatic bone cancer. j nucl med. ; : – . . kucuk no, ibis e, aras g, et al. palliative analgesic effect of re- hedp in various cancer patients with bone metastases. ann nucl med. ; : – . . giannakenas c, kalofonos hp, apostolopoulos dj, zarakovitis j, kosmas c, vassilakos pj. preliminary results of the use of re- -hedp for palliation of pain in patients with metastatic bone disease. am j clin oncol. ; : – . . breitz hb, fisher dr, wessels bw. marrow toxicity and radiation absorbed dose estimates from rhenium- -labeled monoclonal antibody. j nucl med. ; : – . . van rensburg aj, alberts as, louw wk. quantifying the radiation dosage to individual skeletal lesions treated with samarium- -edtmp. j nucl med. ; : – . . brenner w, kampen wu, kampen am, henze e. skeletal uptake and soft-tissue retention of re-hedp and sm-edtmp in patients with metastatic bone disease. j nucl med. ; : – . paired-image model in skeletal dosimetry • shah et al. wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ book reviews tion to technological detail. t good reference material but fox ing. the index is appalling and; to list dyskinesia, dystonia, on- receptors. some can be found ings if you search for them. it such blemishes in an otherwist tion, but i think uk readers prefer gerald stem's recent though not confined to therapy its practical issues, and is a mo: work of reference. his makes for clarity-that is all except th r boring read- author himself points out, th inter alia, fails may be destroyed while th -off effects, or remains intact and the term "t as sub-head- "brain stem death" seems moi is sad to find the best way to practise e useful colla- anticipate problems before the at least, will way, complications are preve text, which effects if they occur are mi embraces all book approaches the subject re satisfactory manner. i am sure neurosurgei and in particular trainees, jms pearce valuable. it should sell well d relatively expensive for its another edition along the sam vascular topics should be cor future. some pitfalls and problems in neurosurgery. series: progress in neurological surgery, vol. . editor: j c de villiers. (pp ; price: £ . .) basel, karger, . this book is the thirteenth in the series "progress in neurological surgery." unlucky for some, perhaps, but the thir- teenth edition which covers transphenoidal operations, frontal and temporal basal tumours, csf shunts, spinal intramedullary lesions, lumbar discs, and brain death, should not prove a disappointment to the editor, dr de villiers. "pitfalls" are interpreted as a complication which may result from incorrect interpretation of clinical signs and investiga- tions, or occur as a result of the operative procedure. the first chapter is an excellent review of the problems encountered with the radiology of pituitary tumours and transphenoidal sur- gery. the text accurately describes the nasal complications following operation (usually omitted from other descriptions) and advises on the ways of minimising these. surpris- ingly, the author fails to mention the need for steroid cover during an anaesthetic or operative procedure in these patients with potential pituitary insufficiency. there follows a rather disappointing chapter on frontal and basal tumours. the radiological examples are all based on angiography, air encephalography and early generation ct scanning. much information with useful advice is buried within a very detailed text which makes heavy reading. again there is no emphasis on the need for steroid cover during operation on suprasellar tumours. shunt complications bedevil us all. a chapter on the ways of minimising these provides practical advice. perhaps the text should have included some mention of the advantages and dis- advantages of the newer variable pressure valves. the next chapter provides helpful infor- mation on spinal intramedullary tumours, incorporating a detailed description of sur- gical technique. a subsequent chapter on lumbar disc prolapse is full of gems often omitted from more standard texts. interest- ing text, however, inevitably contains con- troversy. i was surprised by an introductory sentence stating that "confirmatory tests" (i.e. confirming the clinical findings) "will be required by the majority". surely in this day and age, all patients should undergo at least a ct scan if not myelography? classic root signs do not necessarily result from disc protrusion at the expected level. the "out- patient" operative procedure for disc removal will also raise some eyebrows. the final chapter on the diagnosis of cerebral death by the editor is a model of e title. as the ie "cerebrum" ie brain stem brain death" or re appropriate. surgery is to ty arise. in this nted and their inimised. this in a pragmatic ons at all levels, , will find it lespite seeming size. perhaps e lines covering nsidered in the kw lindsay pain syndromes in neurology. edited by howard l fields. (pp ; price: £ . .) guildford, butterworth scientific ltd. isbn - - - . . much of the most severe chronic intractable pain seen in pain clinics is neuropathic rather than nociceptive pain and whereas mechan- isms of pain with an intact nervous system have been extensively investigated, mechan- isms of neuropathic pain are poorly under- stood, and have received much less attention. this book is particularly concerned with these mechanisms of neuropathic pain and with treatment, but also considers peripheral nociceptive mechanisms and cancer pain, much of which has a nociceptive basis. the editor, fields, opens with a succinct over- view of pain transmission in the normal and damaged nervous system. this is an excellent introduction for the newcomer to this subject. raja and colleagues next consider hyper- algesia and receptor sensitisation, covering both neurophysiological and chemical aspects. devor and rappaport review experimental peripheral nerve injury. perhaps the single most important fact to emerge from all the work on peripheral nerve injury is that damage to sensory axons frequently leads to ectopic impulse generation, and this is a likely basis for at least some of the pain experienced with such lesions. burchiel considers the effects of deafferentation and this is linked with a discussion of the place of the dorsal root entry zone lesion operation described by nasholt. a prominent effect of deafferenta- tion is disinhibition of central neurones with the development of chronic abnormal repetitive neuronal discharges. this explains the common failure ofablative surgery. more damage may lead to more pain. a separate chapter by watson is devoted to post herpetic neuralgia, dealing with clinical features and treatment rather than possible underlying mechanisms of pain. the involvement of the sympathetic nervous system in peripheral nerve injury and in the obscure reflex sympathetic dystrophy syndromes is discussed in two chapters. roberts and kramis consider mechanisms and offer several interesting ideas but we are far from understanding these abnormal states. payne provides a clinical description of reflex sympathetic dystrophy and considers treatment options. the frequent total failure of treatment is under-emphasized here. asbury surveys pain in peripheral neuropathies, drawing attention to the different mechanisms. in a masterly review, tasker considers the place of surgery for pain, both of nociceptive and neuropathic type. stimulation procedures in spinal cord and brain are also discussed and there is brief mention of spinal and intraventricular opiate installation. the literature is comprehensively reviewed and the author admits, with honesty, the influence of personal bias. the unpredictability of results and variability of success of the same procedure in different surgical hands are the two main messages which emerge here. por- tenoy considers cancer pain, in which the multiplicity of possible causes and mechan- isms is stressed and thus the need for careful clinical assessment. the final chapter, again by portenoy deals with the drug treatment of chronic pain. neurologists and neurosurgeons will find much of interest and relevance to their clin- ical practice here. the authors have been well chosen and present difficult subjects clearly, and the book has the advantage ofbeing fairly short. it should find a place in all departmen- tal libraries. the book is not complete in its coverage; those wanting to find full accounts of pain of myelopathic, brainstem, or thalamic origin will be disappointed. these topics are only briefly considered in the chapter by tasker. perhaps correctly, in a book of this length, the emphasis is on the more common painful neurological condi- tions and this is a small criticism. overall, the book can be highly recommended. jw scadding plasticity and morphology of the cen- tral nervous system. a challenge for psychiatry of the nineties. edited by c l cazzullo, e sacchetti, g conte, g inver- nizzi and a vita. (pp ; price: dfi . ; us$ . ; uk£ . ). dordrecht: kluwer academic publishers group, . isbn - - - the title of this book is misleading. it is not about neuroanatomy at all, which is what one might expect. it is a heterogeneous collection ofpapers presented at a conference on schizo- phrenia in milan in . the majority ofthe papers are not even about schizophrenia. there is one on phenylketonuria, one on eating disorders and nine (out of ) on affective disorders. the topics covered include the genetics of depression among the amish population in pennsylvania, ct scan findings in schizophrenics, the description of a new battery of psychometric tests and glucose- -phosphate dehydrogenase deficiency in psychosis. altogether it is a curious mixture. equally puzzling is why it has taken three years for the book to be published, because it is not printed but an amalgam of different type-written manuscripts, a process which i had always assumed speeded up publication. more serious, though, is the fact that some of these contributions would not have passed the refereeing process of even a moderately pres- tigious journal. with five editors available one might have expected a higher standard. in fact, bypassing the refereeing process is not always a bad thing. i have long believed that the reason why we rarely seen papers of the quality of those written by german, french and british neurologists in the first o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jn n p .b m j.co m / j n e u ro l n e u ro su rg p sych ia try: first p u b lish e d a s . /jn n p . . . o n d e ce m b e r . d o w n lo a d e d fro m http://jnnp.bmj.com/ apoc deficiency: from mice to man news and commentary apoc null mutation affects lipoprotein profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . apoc deficiency: from mice to man marten h hofker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . european journal of human genetics ( ) , – ; doi: . /ejhg. . ; published online july during the s and early s, thegreat majority of genes involved in lipoprotein metabolism were cloned, includ- ing the genes encoding the apolipoproteins. these proteins form the protein moiety of the lipoproteins and are essential for lipid meta- bolism. subsequently, genetic variants that were associated with human dyslipidemias were identified. in parallel, the function of these genes has been thoroughly studied in knockout and transgenic mouse models. generally, these rodent models recapitulated the human findings, and proved to be highly useful in studying the etiology of cardio- vascular disease. in particular, mice with deficiencies of low-density lipoprotein recep- tor (ldlr) and apolipoprotein e replicating familial hypercholesterolemia and type iii hyperlipidemias are commonly used as the background for studying the pathogenesis of atherosclerosis. in several instances, mouse models were generated to understand gene function in the absence of the human-equivalent mutation. this is the case for apolipoprotein c (apoc ). apoc has been established as an inhibitor for lipoprotein lipase, a gene that hydrolyzes triglycerides to generate free fatty acids before their uptake by muscle and adipose tissue (reviewed in jong et al ). mice with a high-level expression of human apoc on a background of ldlr deficiency proved to be an excellent model for familial combined hyperlipidemia, because they are disturbed in the breakdown of triglycerides. in contrast, mice lacking apoc show increased activity of lpl, which causes hypo- triglyceridemia and protection from post- prandial hypertriglyceridemia. from these mice studies, it became clear that a deficiency of apoc could cause a healthier lipoprotein profile, which is associated with protection from cardiovascular diseases. however, in the absence of apoc -deficient subjects, this hypothesis was difficult to test directly. to make the situation more complex, apoc is located in a gene cluster – together with genes encoding the apolipoproteins a , a and a . genetic evidence showed that sequence variation in the promoters and regu- latory elements of apoc and apoa inde- pendently affects triglyceride levels. however, there is strong functional interaction between the genes in this cluster. in the absence of structural mutations in single genes, it has proven to be complicated to genetically dissect the independent functions of these genes as has been done in rodent models. a recent paper in science has now identi- fied a null allele of apoc and thereby substantially strengthened the evidence that heterozygous deficiency of the apoc gene in humans leads to reduced levels of tg and is associated with reduced arterial plaque formation. one of the reasons why the detection of apoc deficiency in man took so long is that it is associated with a healthier lipoprotein profile, and such individuals are usually not screened for mutations in lipoprotein genes. the study by pollin et al ( ) included healthy participants who were studied for their postprandial response. this response was tested in old order amish indivi- duals by giving the volunteers a high-fat drink and measuring the subsequent trigly- ceride levels after – h. subsequently, all these individuals were genetically investigated using a genome-wide association screen with the k array set of affymetrix (santa clara, ca, usa). one snp with a relatively low allele frequency ( . ) was strongly asso- ciated with low fasting triglyceride levels and low postprandial triglyceride levels. this snp (rs ) was located at a distance of kb from the apoc gene cluster. a second snp (rs ), located at a distance of kb from the cluster, with an allele frequency of . , showed a much weaker association and was moderately correlated with rs . on the basis of the relative proximity of the snps and the apoc gene cluster, and given the phenotype that pre- dicted a function for apoc , this gene was sequenced, and a c - t substitution was found in exon , which predicted a premature stop codon at position of the amino-acid sequence. it is remarkable that a marker lying kb away from the apoc gene cluster was identified as the most strongly linked snp and that a marker at a closer location was less strongly associated. it is likely that this fact can be explained because the study was carried out in an isolated population. the genetic structure of this population has been extremely well documented, and permitted the conclusion that the gene mutation was introduced in the early s. hence, the gwas study should be regarded as an ‘iden- tity by decent (ibd)’ approach, rendering a very large region around the markers as the candidate region for the mutation screen. so what is the evidence that the apoc gene mutation is the genuine defect in this study and not any other gene mediating the postprandial response in this subject? the method used implies that genes in a large region around the s marker are candidate genes; in addition, it cannot exclude mutations in non-coding sequences. in this case, all the evidence is strongly in favor of a function for apoc to harbor the culprit mutation. other genes with an estab- lished function in lipoprotein metabolism include the other genes in the apoc gene cluster, and abcg . furthermore, other genes of interest include pcks and inter- leukin receptor a, but these genes currently have no documented function in postpran- dial lipoprotein metabolism. therefore, the apoc null mutation found in the old order amish population, residing close to a strongly associated snp, in combination with a wealth of information from rodent studies and also human genetic studies provides a strong basis for the main conclusion of this paper that the apoc gene is the causal gene. nevertheless, to formally prove this finding, it will be necessary to professor dr mh hofker is at the department of pathology and medical biology, medical biology section, molecular genetics, university medical center groningen, antonius deusinglaan , av groningen, the netherlands. tel: + - - / ; fax: + - - ; e-mail: m.h.hofker@med.umcg.nl european journal of human genetics ( ) , – & macmillan publishers limited all rights reserved - / $ . www.nature.com/ejhg http://dx.doi.org/ . /ejhg. . http://www.nature.com/ejhg obtain sequence information for the entire genetic interval. in the absence of such an excellent functional candidate gene, the study presented here would have required a more than substantial resequencing effort, as the genetic intervals to be sequenced in iso- lated populations tend to be large. nonethe- less, isolated populations are frequently enriched for interesting mutations, and could also facilitate follow-up studies to further understand the genotype–phenotype relationship’ jong mc, hofker mh, havekes lm: role of apocs in lipoprotein metabolism: functional differences between apoc , apoc and apoc . arterioscler thromb vasc biol ; : – . masucci-magoulas l, goldberg ij, bisgaier cl et al: a mouse model with features of familial combined hyper- lipidemia. science ; : – . maeda n, li h, lee d, oliver p, quarfordt sh, osada j: targeted disruption of the apolipoprotein c-iii gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia. j biol chem ; : – . talmud pj, hawe e, martin s et al: relative contribution of variation within the apoc /a /a gene cluster in determining plasma triglycerides. hum mol genet ; : – . pollin ti, damcott cm, shen h et al: a null mutation in human apoc confers a favorable plasma lipid profile and apparent cardio protection. science ; : – . news and commentary european journal of human genetics 	apoc deficiency: from mice to man 	references case reports journal of medical genetics, , , - a new alopecia/mental retardation syndrome summary three cousins are reported in an inbred family with a mental retardation/ alopecia syndrome. the occurrence of mental retardation with other easily recognisable features is of importance for the identification of rare recessive syndromes. total alopecia is a useful marker as there have been only a few conditions in which its association with mental retardation has been noted. moynahan' reported a family in which members had epilepsy, oligophrenia, and scalp alopecia, and shokeir reported a separate condition with universal alo- pecia, mental retardation, and pyorrhoea. the present family differs from those previously described and the purpose of this paper is to draw to the attention of geneticists a hitherto undescribed recessive syndrome. case report the patient is a -year-old male from the middle east. he was delivered after a normal birth at weeks and weighed g. at birth a mild talipes of the left foot was noted. he was given oxygen but intubation was not necessary and he was home within a week. no seizures have occurred at any stage, but developmental milestones have all been markedly delayed. he is now walking but has very little speech. head circumference and weight are below the nd centile. no dysmorphic facial features were noted (fig ) but there was a striking absence of hair on the scalp and neither eyebrows nor eye- lashes were present. some hair was noted at birth but after the ritual shaving of the head at days the hair did not regrow. occasionally a single hair appeared on the scalp but soon fell out. the only hair that could be sampled (a single strand) was submitted for histological examination and found to be normal. the rest of the examination was negative. it can be seen (fig ) that the patient himself has one normal male sib but his mother's sister's child ( - ) is similarly affected (bald from birth and received for publication january . fig the proband. ( )l fig pedigree offamily. mentally retarded) as is his father's aunt's daughter (ii- ). iii , although not seen personally, is said to wear a wig to cover her complete baldness. discussion shokeir reported a family with dominant inherit- ance of mental retardation and alopecia. twelve persons were affected over four generations but not all were retarded (eight out of the ). seven of the had epilepsy but the onset was late ( years in the proband). the alopecia was universal, involving' the eyelashes and pubic hair, but was not total. the hair was thin and brittle and histologically showed thinning, uneven growth, and occasional beading. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n f e b ru a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ case reports inheritance was dominant and therefore unlike the present family. moynahan' reported male sibs with mental subnormality, epilepsy (onset at to years), and alopecia which involved the scalp only. the hair later regrew. the mode of inheritance in this family is difficult to ascertain as the boy's father only grew hair at the age of years and the boy's mother's sister was bald until the age of years. the regrowth of the hair, the restriction of the baldness to the scalp, and the epilepsy differentiate moynahan syndrome from the one described here. a similar condition in two sibs whose parents were con- sanguineous was reported by perniola et al. deafness occurred in both. there are at least three other syndromes in which hair loss is associated with mental retardation. menkes disease can be excluded as the hair is kinky and not totally absent. prognosis for life is poor and seizures are frequent. inheritance is x linked recessive. in monilethrix the hair is normal at birth but is then lost within the first few months. red pustular lesions on the scalp may be present. alopecia is seldom total and through the pustules brittle hairs which break easily emerge. the nails and teeth are abnormal and mental retardation is only occasionally a feature. in the hair-brain syndrome (amish brittle hair syndrome) those affected had short stature and relatively mild mental retardation. all the affected had hair but it was brittle and broke easily. total alopecia was not a feature and inheritance was recessive. in the condition described in this paper, the alopecia was total and involved all areas of normal hair growth. it probably represents a distinct entity. m baraitser, c carter, and e m brett* clinical genetics unit and *department of neurology, the hospital for sick children, great ormond street, london wcin jh. references moynahan ej. familial congenital alopecia, mental retardation with unusual electroencephalograms. proc r soc med ; : - . shokeir mhk. universal permanent alopecia, psycho- motor epilepsy, pyorrhoea and mental subnormality. clin genet ; : - . perniola t, krajewska g, carnevale f, lospalluti m. congenital alopecia, psychomotor retardation, convul- sions in two sibs of a consanguineous marriage. j inher met dis ; : - . requests for reprints to dr m baraitser, clinical genetics unit, the hospital for sick children, great ormond street, london wcin jh. a female infant with features of mohr and majewski syndromes: variable expression, a genetic compound, or a distinct entity? summary a female child, the offspring of a consanguineous mating, had a cleft palate, tongue tumours, hypoplastic tibiae, and poly- syndactyly. the relationship to the mohr and majewski syndromes is discussed. variable expression is a common problem en- countered by those who seek to define syndromes. the best method to determine the extent of the phenotypic expression of a gene defect is to study the variability within sibships, but in rare disorders this is not always possible. it is then necessary to depend on somewhat arbitrary definitions. the mohr and majewski syndromes each behave as a probable autosomal recessive disorder with a relatively distinct phenotype. temtamy and mc- kusick reported two subjects whose clinical features fulfilled the definition of both syndromes. we report a female infant, the second child of consanguineous parents, in whom combined features of both syndromes were seen. case report the patient was born on . . at weeks' gestation weighing g. she was the second child of first cousin pakistani moslem parents. the mother and father were and years old, respectively. a sib was normal and there was no relevant family history. the child (fig ) was noted to have low set ears, micrognathia, and shallow orbits causing mild proptosis. there was a high arched palate with a posterior cleft and fleshy tumours were present on the underside of the tongue. bilateral postaxial polysyndactyly was noted in the hands with poly- syndactyly, severe talipes equinovarus, and distal shortening of both lower limbs. at months her weight of - kg was well below the rd centile. she fed poorly and had frequent chest infections. her development was slightly delayed. radiological examination revealed severe bilateral tibial dysplasia (fig ), whereas the chest x-ray did not show significant rib shortening. there was a normal ,xx karyotype. received for publication july . o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n f e b ru a ry . d o w n lo a d e d fro m http://jmg.bmj.com/ functional cardiac fibroblasts derived from human pluripotent stem cells via second heart field progenitors article functional cardiac fibroblasts derived from human pluripotent stem cells via second heart field progenitors jianhua zhang , , ran tao , katherine f. campbell , , juliana l. carvalho , , edward c. ruiz , gina c. kim , eric g. schmuck , amish n. raval , andré monteiro da rocha , , todd j. herron , , josé jalife , , james a. thomson , & timothy j. kamp , , cardiac fibroblasts (cfs) play critical roles in heart development, homeostasis, and disease. the limited availability of human cfs from native heart impedes investigations of cf biology and their role in disease. human pluripotent stem cells (hpscs) provide a highly renewable and genetically defined cell source, but efficient methods to generate cfs from hpscs have not been described. here, we show differentiation of hpscs using sequential modulation of wnt and fgf signaling to generate second heart field progenitors that efficiently give rise to hpsc-cfs. the hpsc-cfs resemble native heart cfs in cell morphology, proliferation, gene expression, fibroblast marker expression, production of extracellular matrix and myofibroblast transformation induced by tgfβ and angiotensin ii. furthermore, hpsc-cfs exhibit a more embryonic phenotype when compared to fetal and adult primary human cfs. co-culture of hpsc-cfs with hpsc-derived cardiomyocytes distinctly alters the electrophysiological properties of the cardiomyocytes compared to co-culture with dermal fibroblasts. the hpsc- cfs provide a powerful cell source for research, drug discovery, precision medicine, and therapeutic applications in cardiac regeneration. https://doi.org/ . /s - - - open department of medicine, school of medicine and public health, university of wisconsin-madison, madison, wi , usa. stem cell and regenerative medicine center, university of wisconsin-madison, madison, wi , usa. center for arrhythmia research, department of internal medicine, cardiovascular medicine, university of michigan, ann arbor, mi , usa. frankel cardiovascular regeneration core laboratory, ann arbor, mi , usa. department of genomic sciences and biotechnology, catholic university of brasilia, brasilia distrito federal, brazil. fundación nacional centro nacional de investigaciones cardiovasculares carlos iii (cnic), melchor fernández almagro, madrid, spain. regenerative biology division, morgridge institute for research, madison, wi , usa. department of cell and regenerative biology, school of medicine and public health, university of wisconsin-madison, madison, wi , usa. correspondence and requests for materials should be addressed to j.z. (email: jz @medicine.wisc.edu) or to t.j.k. (email: tjk@medicine.wisc.edu) nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications () :,; http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - http://orcid.org/ - - - mailto:jz @medicine.wisc.edu mailto:tjk@medicine.wisc.edu www.nature.com/naturecommunications www.nature.com/naturecommunications c ardiac fibroblasts (cfs) compromise a significant fraction of cells in the heart estimated using lineage tracing to be about % of the nonmyocyte cells in mouse heart , and cfs contribute to heart function, homeostasis, and structure in multiple ways. the production and remodeling of extracellular matrix (ecm) are the best known functions of cfs. secretion of growth factors and complex mixtures of regulatory molecules in extracellular vesicles provide another important way that cfs regulate cardiac function. during development, signaling from cfs is necessary to stimulate cardiomyocyte proliferation . cfs are intimately involved in many of the dysregulated signaling pathways linked to heart disease such as β-adrenergic signaling in heart failure . activated cfs and resulting pathological fibrosis can lead to impaired mechanical function associated with heart failure as well as abnormal electrical activity linked to arrhyth- mias and sudden cardiac death . not surprisingly, with this range of cardiac-specific effects, evidence has emerged that cfs are tissue-specific cells distinct from other fibroblasts in the body . given the role of cfs in cardiac biology and disease, investi- gators have isolated and studied primary cfs from surplus patient material obtained at the time of cardiac surgery, cardiac biopsy, or autopsy. however, disease- and patient-specific cardiac tissue samples are not always available. for example, many patients with inherited heart disease typically do not undergo cardiac surgical interventions. thus, a robust, reproducible, and patient-specific source of cfs is desirable. because human plur- ipotent stem cells (hpscs) can renew indefinitely in culture, hpscs provide an unlimited source of genetically identical hpsc- cfs in contrast to primary tissue sources of cfs. prior studies have identified fibroblasts as a low-abundance cell type present following protocols optimized to differentiate hpscs to cardio- myocytes (hpsc-cms) , . in addition, recent investigations have defined protocols to generate epicardial cells from hpscs that subsequently can be differentiated into cfs – . however, effi- cient differentiation of hpscs to well characterized cfs has not been described. during embryonic development, cfs arise from multiple sources. the epicardium is considered the major source of cfs found in the ventricular myocardium – with epicardial cells undergoing an epithelial-to-mesenchymal transition requiring expression of tcf to generate cfs , , , . lineage tracing with the epicardial marker, tbx , demonstrated tbx -expres- sing fibroblasts compromised only one-third of the cfs in embryonic and adult heart ; however, a more recent investiga- tion with a wt -cre mouse showed up to % of cfs in the adult heart were derived from the epicardium . another significant population of cfs is derived from the endocardium at the time of endocardial cushion formation by an endothelial-to- mesenchymal transition , , and endocardial cells are gener- ated in part from the second heart field progenitors (shfps) . lastly, a small fraction of fibroblasts are derived from neural crest lineages . in the present study, we develop a protocol to generate hpsc- cfs via shfps using sequential inhibition of gsk β to activate canonical wnt signaling followed by stimulation with bfgf. the differentiated hpsc-cfs exhibit cell morphology, growth, gene expression, fibroblast markers, ecm production, and myofibro- blast transformation similar to native human cfs. results progenitors arise during cardiac differentiation of hpscs. biphasic modulation of the wnt signaling pathway with small molecules is sufficient to direct the differentiation of monolayer cultured hpscs to cms , . activation of canonical wnt sig- naling by gsk β inhibition stimulates hpscs to sequentially form mesoderm and cardiac progenitors that are subsequently induced to differentiate to cms by inhibition of wnt signaling. we first determined the stage-specific mesodermal and cardiac progenitor populations generated by this small molecule (giwi) protocol (fig. a). flow cytometry demonstrated that the early meso- dermal marker brachyury (bry) was expressed in almost all cells after h of treatment with chir. expression of bry was present for another h before it was downregulated after day (fig. b, supplementary fig. a). cd was robustly expressed in hpscs, but its expression gradually declined during the differentiation. most of the cells expressed cd at days – (> %), but the expression level of cd was lower than in hpscs (fig. b, supplementary fig. b). this brydown/cd low progenitor stage at day – first showed upregulation of mesp and gata mrna expression which are expressed in cardiac mesodermal progenitors (fig. c), followed by the upregulation of cardiac transcription factors isl , nkx - , and tbx indicating com- mitment of cardiac progenitors in the giwi protocol (fig. c). the apelin receptor (aplnr) is expressed in mesodermal pro- genitors including lateral plate mesodermal cells specified to be cardiac progenitors as well as aplnr+ cells that have the potential to give rise to mesenchymal stem cells (mscs) and endothelial cells – . aplnr expression was first seen at day , and aplnr+ cells peaked at % of the cells on day and then rapidly declined (fig. b, supplementary fig. c). kdr+/pdgfrα+ cells have been identified as cardiac progenitor cells (cpcs) that can be differentiated mainly to cms in the cardiac differentiation of hpscs . we found the kdr+/pdgfrα+ cpcs were mainly generated on day – (fig. b, supplementary fig. d). these stage-specific progenitors were reproducibly gen- erated from other hpsc lines using the giwi protocol (supple- mentary fig. ). fgf promotes differentiation of shfps and cfs. because prior studies have demonstrated that fibroblast growth factor (fgf) signaling contributes to the generation of cardiac mesodermal progenitors and exerts a dominant effect over bmp signaling directing cardiac mesodermal progenitors to nonmyocyte fates , , we tested the impact of fgf signaling on the stage- specific progenitors present in the giwi protocol to promote differentiation of cfs rather than cms (fig. a). we examined a range of concentrations of bfgf ( – ng/ml) added at different cardiac progenitor stages in a defined medium (cfbm, supple- mentary table ) for fibroblast differentiation. following days of differentiation, cells were analyzed by flow cytometry using a specific fibroblast antibody (mouse anti-human fibroblast, clone te- ) and an antibody to sarcomeric myosin (mf ) expressed in cms (fig. b, supplementary fig. ). changing the medium and adding bfgf on day or day effectively prevented the generation of mf + cms, and there was a concentration- dependent increase in fibroblast marker positive cells, which peaked at ng/ml bfgf added on day of differentiation (fig. b). higher concentrations of bfgf, when added on day or day , resulted in significant cell death after day of differ- entiation and no cells survived to days (fig. b). applying a fixed concentration of ng/ml of bfgf to cardiac mesodermal progenitors in a context-dependent fashion generated large population of fibroblasts from multiple hpsc lines which we refer to as hpsc-cfs (fig. c). we focused our effort on the protocol adding ng/ml of bfgf in cfbm medium starting on day following gsk β inhibition (gi) which we describe as the gifgf protocol (protocol i in fig. a). to understand the impact of bfgf on the differentiation of day cardiac mesodermal progenitors in the gifgf protocol, we examined the temporal gene-expression article nature communications | https://doi.org/ . /s - - - nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications pattern for developmentally regulated transcription factors and associated genes using quantitative real time polymerase chain reaction (qrt-pcr) (fig. a, supplementary fig. ). the early mesodermal gene t was transiently upregulated after the gsk β inhibitor (chir) treatment peaking at day . mesp and gata started to express on day of differentiation following the expression of t, indicating the formation of cardiac mesodermal progenitors (fig. a). next, the cardiac-related transcription factors were upregulated following bfgf treatment including isl , tbx , gata , nkx - , hand (fig. a, supplementary fig. ). the expression of isl , gata , and hand remained high through days of differentiation. interestingly, the pattern of expression is consistent with the formation of shfps given the prominent expression of isl , tbx , gata , and hand (fig. b) – . the results are also consistent with the demon- strated role of fgf signaling driving differentiation of pharyngeal mesoderm to shfps . these results contrast the cardiomyocyte- optimized giwi protocol in which transcription factors b a iwpchir day confluent hpscs cmsbry+ brydown cd low rpmi+b no insulin aplnr+ kdr+ pdgfra+ rpmi+b (complete) c neg ctrl b ry fsc fsc fsc kdr c d a p l n r p d g f r α day day % % % % % % % % % day day day day . t mesp gata isl nkx – tbx . r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n . . . . . . . . . . . . . . . . . . . d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d fig. identification of progenitors in cardiac differentiation of hpscs. a schematic method for the small molecule protocol using gsk β inhibition with chir followed by wnt inhibition with iwp (giwi protocol) to efficiently differentiate hpscs to cardiomyocytes (cms) and the associated markers for stage- specific progenitors. b flow cytometry of stage-specific progenitors labeled by brachyury (bry), cd , apelin receptor (aplnr), kdr, and pdgfrα in early differentiation (day – ) of the giwi protocol. no primary antibody controls and isotype controls were performed for each time point, and the day , no primary antibody control (neg ctrl) is shown as an example. c qrt-pcr showing the expression of relevant mesodermal and cardiac-related transcription factors in the progenitor stages of the giwi protocol (day – , n = technical replicates). tbx , d expression was not detectable. data are mean ± sem. the data are from df - - t hipsc line nature communications | https://doi.org/ . /s - - - article nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications www.nature.com/naturecommunications associated with first heart field (fhf) progenitors including tbx , hand , and tbx are more prominently expressed (fig. b). in addition, the ion channel gene, hcn , which is recognized as the fhf progenitor marker , was minimally expressed in the gifgf protocol compared to the giwi protocol (fig. b). furthermore, the cardiac transcription factors isl , nkx - , and gata , which are expressed in both fhf and shf, were differentially expressed in the gifgf and giwi protocols (fig. b). particularly, isl expression persisted longer in gifgf protocol compared to an early peak in expression in the giwi protocol on day then declining. in contrast, nkx – expression peaked early in the gifgf protocol at day and rapidly downregulated after day compared to the giwi protocol where nkx - expression increased after day and is greatest at day . tcf , a bhlh transcription factor required for the formation of cf in mouse heart , was progressively upregulated from day – in the gifgf protocol (fig. a, supplementary fig. ). however, we did not observe a significant upregulation of (pro)epicardial genes such as tbx and wt with the fgf directed cf differentiation (fig. a, supplementary fig. ), consistent with the findings that fgf signaling inhibits (pro) epicardium differentiation from the cardiac mesoderm . we also examined the emt markers of snai and snai in the gifgf protocol and found an early upregulation of snai (day – ) and a late upregulation of snai (day – ) (fig. a). thy (cd ) expression was high in the undifferentiated hpscs but down- regulated during cf differentiation (fig. c). given that high bfgf concentrations can be supportive of maintenance of pluripotency of hpscs, we examined the expression of the pluripotency gene oct and showed it to be completely downregulated during the cf differentiation (fig. c), similar to the downregulation observed in cardiomyocyte differentiation protocols , , , . finally, we examined the cell lineage-specific gene-expression pattern. vim, commonly expressed in fibro- blasts, was greatly upregulated during cf differentiation in contrast to tnnt , a cardiomyocyte-specific gene, which is minimally expressed in the gifgf protocol (fig. d). further- more, postn, a gene expressed in cfs , was also greatly upregulated by days differentiation in the gifgf protocol, whereas the general fibroblast-associated ddr gene showed a smaller fold change compared to postn (fig. d). to screen for gene expression typical of endothelial or smooth muscle cells, we examined pecam (cd ) and myh (smooth muscle myosin heavy chain) expression and found there was very low expression of pecam and essentially no expression of myh in the gifgf protocol (fig. d). based on the gene-expression data showing shf genes were significantly upregulated upon bfgf addition in the gifgf protocol, we used flow cytometry to examine the temporal expression of islet and the recently identified shf marker cxcr (cd ) in the gifgf protocol compared to the giwi protocol (fig. a, supplementary fig. ). islet expressing cells first were detected on day of both protocols, but bfgf signaling resulted in the majority of cells being islet positive in the gifgf protocol relative to a smaller fraction in the giwi protocol during the progenitors stages (day – ). furthermore, islet /cxcr double positive cells were the majority of cells ( %) on day in the gifgf protocol and were only scarcely detected in the giwi protocol. after days of differentiation, we evaluated for the different cell lineages present and confirmed the giwi protocol gives rise to predominantly hpsc-cms (mf +) and the gifgf protocol gives rise to predominantly hpsc-cfs (fibroblast te- +) with a small population of smooth muscle cells (sm-mhc+) in the giwi protocol only (fig. b, supplementary fig. ). no daya b c i ii iii i m f f ib ro b la st ( % ) fibroblast - - h ii iii chir rpmi+b no insulin iwp cfbm bfgf bfgf (ng/ml) % % % % % % % % % % % % . % . % . % . % % % . % % % % . % . % no cells no cellsno cells fig. effect of bfgf on hpsc-derived mesodermal and cardiac progenitors. a schematic for testing the concentration-dependent effect of bfgf addition to stage-specific progenitors generated by the giwi protocol beginning bfgf on day , , or , corresponding to labeled protocols i, ii, and iii. gray lines indicate rpmi medium+b without insulin supplement; blue lines indicate cardiac fibroblast basal medium (cfbm); green lines indicate chir treatment; red lines indicate bfgf treatment; orange line indicates iwp treatment. b flow cytometry analysis of day differentiated cells from protocols i, ii, iii labeled by human fibroblast marker (clone te- antibody) and cardiomyocyte marker (mf antibody for sarcomeric myosin). c average percentage of fibroblast population differentiated from hesc line h (n = , biological replicates) and hipsc line df - - t (n = , biological replicates) at days. data are mean ± sem article nature communications | https://doi.org/ . /s - - - nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications cd + endothelial cells were detected under these conditions (fig. b, supplementary fig. ). we sought to further define the lineage hierarchy of the hpsc- cfs by evaluating for expression of a marker associated with vascular endothelial progenitors, tie , and the endocardial- specific transcription factor during early cardiac development, nfatc . we also examined the time course of expression of islet as endocardial populations have been described from islet - positive and -negative populations in the developing mouse heart . despite the persistent expression of islet detected from day to day in the gifgf protocol, no significant tie - positive cells were observed (fig. c, supplementary fig. ). evaluation for nfatc expression from day to day also showed no significant population of nfatc -positive cells (supplementary fig. ). a time course of the generation of hpsc-cfs during the gifgf protocol shows the appearance of te- + cells starting on day and increasing in abundance to day consistent with the generation of hpsc-cfs from islet / cxcr double positive shfps (fig. d). in summary, the gifgf protocol generates an enriched pool of shfps that give rise to hpsc-cfs without going through an evident endocardial progenitor cell stage (fig. e). comparison of hpsc-cfs, primary cfs, and dermal fibro- blasts. we next compared the properties of hpsc-cfs with commercially available primary human fetal ventricular cfs (hfv-cfs) and adult ventricular cfs (hav-cfs), and adult dermal fibroblasts (hdfs). the morphology of hpsc-cfs, hfv-cfs, and hav-cfs maintained in similar media was comparable with spindle-shaped and polygonal cells present, but distinct from hdfs which were larger, flatter and more irregular in shape (fig. a). the hpsc-cfs and hfv-cfs were more proliferative than hav-cfs and can propagate for more than passages, while hav-cfs could only grow ~ passages before undergoing senescence (fig. b). the expression of cd (thy ), which has been used as a cell surface marker for some fibroblast popula- tions, was uniformly expressed on hdfs and maintained over multiple passages. in contrast, expression of cd was present on only a fraction of hpsc-cfs, hfv-cfs and hav-cfs (~ %) and was present on a progressively smaller percentage of cells with passaging. notably, the hpsc-cfs, hfv-cfs, and hav-cfs showed a similar change in expression of cd over passages (fig. c, supplementary fig. ). the hdfs were uniformly positive (~ %) for the intracellular fibroblast marker (clone te- ) during passaging, and the hpsc-cfs, hfv-cfs, and hav-cfs showed that – % of the cells were positive at early passage. the te- -positive cells increased to % with subsequent pas- saging and then declined as the cfs approached senescence (fig. c supplementary fig. ). we tested labeling vimentin, an intermediate filament protein expressed in fibroblasts, and show that all of the clone te- fibroblast antibody labeled cells from hpsc-cfs, hfv-cfs, hav- cfs, and hdfs populations also uniformly expressed vimentin, whereas hpsc-cms did not express vimentin (fig. a, supple- mentary figs. and ). the surface markers of pdgfrα (cd a) and pdgfrβ (cd b) have been reported to label a portion of primary mouse and human cfs , and so we also examined their expression in hpsc-cfs, hfv-cfs, hav-cfs, hdfs, and hpsc-cms by flow cytometry. pdgfrα was expressed in more than % of the cells in all the fibroblast populations, but not in hpsc-cms (fig. b, supplementary fig. ). pdgfrβ was . snai a snai tcf wt tbx hey hand tbx isl tbx tbx hcn nkx - gata mesp t . r e la tiv e e xp re ss io n . . . . . . d d d d d d d d fhf shf . b . . . . gifgf giwi gifgf giwi gifgf giwi gifgf giwi gifgf giwi gifgf giwi gifgf giwi gifgf giwi gifgf giwi . . . . . d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d . . . . . . . . . . . . . . . . . . . . . hcn hand isl nkx - gata hand tbx dual tbx tbx r e la tiv e e xp re ss io n . . . . . . . . . . r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n . . . . . r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n c oct thy . . . . . . . r e la tiv e e xp re ss io n . . . . r e la tiv e e xp re ss io n d d d d d d d d d d d d d d d d vim tnnt postn ddr pecam myh . . . . . . cardiomyocyte/fibroblast markers nonmyocytes lineage markers r e la tiv e e xp re ss io n r e la tiv e e xp re ss io n d d d d d d d d d d d d d d d d d fig. analysis of cardiogenic gene expression during gifgf protocol. a qrt-pcr showing the expression and hierarchy of transcription factors in the time course of the gifgf protocol (day – ). b qrt-pcr showing the expression patterns of the transcription factors in fhf and shf in the gifgf protocol in comparison with the giwi protocol. c qrt-pcr showing the expression time course of cd and oct in gifgf protocol (day – ). d qrt-pcr showing the expression of markers for fibroblasts (vim, postn, and ddr ), cardiomyocytes (tnnt ), and nonmyocyte lineages (pecam and myh ) in the time course of the gifgf protocol (day – ). the qrt-pcr time course data are from consecutive samples from the same differentiation round with n = technical replicates for each point. data are mean ± sem. the data are from df - - t hipsc line nature communications | https://doi.org/ . /s - - - article nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications www.nature.com/naturecommunications also expressed in the majority of the hpsc-cfs and hfv-cfs, as wells as hdfs ( %), but with a relatively lower percentage ( %) in hav-cfs. a very small population of pdgfrβ+ cells which may represent nonmyocytes in the hpsc-cms preparation (fig. b). immunolabeling of the cell preparations was performed to evaluate for other proteins associated with fibroblasts. the transcription factor gata has previously been reported to be expressed in cfs but not in dfs , and we found the te- -labeled fibroblasts from hpsc-cfs, hfv-cfs, and hav-cfs were all gata positive in contrast to hdfs which showed no immunolabeling for gata . hpsc-cms were negative for the fibroblast marker te- , but positive for gata (fig. c). there were no ctnt+ cells detected in any of the fibroblast cell lines, but hpsc-cms preparation showed abundant ctnt+ cells (fig. c). periostin is a matricellular protein associated with fibroblasts that is expressed in mouse cfs during embryonic development and downregulated in adult mouse cfs except following injury , . furthermore, a recent study demonstrated that periostin is expressed in cfs differentiated from hpsc- derived epicardial cells . because periostin is a secreted protein, flow cytometry is problematic for detection of expressing cells, so we performed q-pcr to examine postn expression. all of the fibroblast populations examined expressed postn, but hpsc- cm did not show expression (fig. d). immunolabeling for fsp , another protein associated with fibroblasts, was detected in the majority of cells from all fibroblast cell lines and likewise, the ecm proteins collagen i and fibronectin were expressed across all the fibroblast preparations (supplementary fig. ). thus, while hpsc-cfs share some properties with hdfs, they are most similar to hfv-cfs and hav-cfs in their morphology and marker expression, particularly in the pattern of expression of cd and gata . gene expression of hpsc-cfs is most similar to primary cfs. to characterize the gene-expression pattern in hpsc-cfs, we performed rna-seq on hpsc-cfs and compared the total mrna transcripts with those of hav-cfs, hdfs, hpsc-cms, and hpscs. analysis of gene expression across all the samples showed the greatest similarity in the abundance of overall transcripts ( , ) between hpsc-cfs and primary hav-cfs calculated by euclidean distance (fig. a). bioinfomatics analysis using the online tool amigo for cardiac-related genes ( ) demonstrated cardiac factors were enriched in hpsc-cfs, hav-cfs, and hpsc- cms with hpsc-cfs and hav-cfs showing the greatest similarity (fig. b). for example, bmp , gata , and sox were similarly expressed in hpsc-cfs and hav-cfs, but not expressed in hdfs. however, other key cardiac genes showed greater expression in hpsc-cfs compared to hav-cfs, including hand , hey , isl , and nkx - (fig. b). we also analyzed the ecm-related gene expression in all samples given the role of ecm production in fibroblast biology. high expression of collagens (col a , col a , col a , col a , col a , col a , col a , col a , col a , and col a ), fibronectin (fn ), the matrix metalloproteinases , , and (mmp , mmp , and mmp ), b c d chir cfbmrpmi+b no insulin bfgf mp cmp shfp cfhpsc e day . % % % % % % % % % % % % % % % . % . % . % . % . % . % . % . % % . % % . % % . % % . % % . % % . % % . % % . % % . % % . % is le t is le t m f tie fibroblast cxcr giwi giwi day gifgf day gifgf . % % . % . % . % % % . % % . % % . % . % . % % % . % % % % % . % . % . % . % % % % . % % . % m f s m -m h c c d fibroblast fibroblast fsc day day day day day day day day day day day day day day day day day day day day day day day day day day day a day fig. evaluation of progenitors and cardiovascular lineages in cf and cm differentiation. a flow cytometry analysis of cells on day – in the gifgf and giwi protocols for fhf/shf marker islet and shf marker cxcr . for day – , the protocols are identical, diverging on day . b flow cytometry analysis of the day differentiated cells from the gifgf and giwi protocols for fibroblasts (anti-human fibroblasts, clone te- ), cms (mf ), smooth muscle cells (sm-mhc), and endothelial cells (cd ). c flow cytometry analysis of cells throughout the gifgf protocol for expression of islet and the endothelial progenitor marker, tie . d flow cytometry analysis of cells throughout the gifgf protocol for expression of the fibroblast marker (anti-human fibroblasts, clone te- ). e schematic method of the gifgf protocol and stage-specific progenitors in differentiation of hpscs to cfs. gray lines indicate rpmi medium +b without insulin supplement; blue lines indicate cardiac fibroblast basal medium (cfbm); green lines indicate chir treatment; red lines indicate bfgf treatment. mp mesodermal progenitor, cmp cardiac mesodermal progenitor, shfp second heart field progenitor, cf cardiac fibroblast. the data are from df - - t hipsc line article nature communications | https://doi.org/ . /s - - - nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications and tissue inhibitor of metalloproteinases , , and (timp , timp , and timp ) were seen in all fibroblasts (fig. c). how- ever, some of the genes, including col a , col a , col a , and col a were more highly expressed in hpsc-cfs and hav-cfs than hdfs, and mmp was expressed only in hdfs. cluster analysis for the overall ecm-related gene expression again demonstrated hpsc-cfs and hav-cfs were the most clo- sely related cell populations studied (fig. c). to compare the hpsc-cfs and hav-cfs in more detail, we performed gene ontology (go) analysis using the online functional annotation tool (david bioinformatics resources . , niaid/ nih) for the differentially expressed genes (fig. d, supplementary data and ). genes classified as extracellular region or extracellular region part are highly enriched in both hpsc-cfs and hav-cfs. genes involved in membrane proteins are enriched in hpsc-cfs, whereas genes involved in cell–cell and cell–ecm attachment are enriched in hav-cfs. interestingly, genes involved in pattern specification process, anterior/posterior pattern formation, and embryonic morphogenesis are enriched in hpsc-cfs consistent with the hpsc-cfs being more embryonic in phenotype (fig. d). to further examine the identity of hpsc-cfs, we compared our rna-seq data with a recent publication which performed rna-seq for gene-expression analysis of primary human fetal and adult cfs . unsupervised cluster analysis of ten repre- sentative genes differentially expressed in human fetal and adult cfs identified by jonsson et al. , demonstrated that hpsc-cf cluster most closely with hfv-cfs relative to the hav-cfs (supplementary fig. a). furthermore, unsupervised cluster analysis of the cardiac factors ( genes) showed the most similarity between the two hav-cfs, then grouped with the hfv-cfs and hpsc-cfs subsequently. the key cardiac factors expressed in hpsc-cfs that we identified above including bmp , gata , sox , hand , hey , isl , and nkx - (fig. b) are all expressed in hfv-cfs. furthermore, hey , isl , and nkx - showed higher expression in hfv-cfs than in hav-cfs in the jonsson et al. study , similar to the expression pattern in hpsc-cfs relative to hav-cfs in our data, consistent with a more embryonic phenotype of the hpsc- cfs (supplementary fig. b, data ). interestingly, the transcription factor tbx has been identified in adult mouse and human cfs as a key marker , but tbx is expressed to significantly lower level in fetal human cfs . our data also showed tbx expression is significantly lower in hpsc-cfs (supplementary data ), consistent with a more embryonic phenotype of the hpsc-cfs. ecm gene expression was similar across all the samples (supplementary fig. c). in summary, these gene-expression data demonstrate hpsc-cfs are most closely related to human native cfs relative to the other cell types studied, and the gene expression pattern is consistent with the hpsc-cfs having a more embryonic cf phenotype. . e + hpsc-cfa b c hpsc-cf hpsc-cfhfv-cf hfv-cfhav-cf hdfhav-cf hfv-cf hav-cf p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p hdf . e + . e + . e + . e + . e + . e + . e + . e + to ta l c e lls ( × ) . e + . e + . e + . e + . e + . e + . e + . e + . e + . e + . e + . e – t h a w p p p p p p p passage # in subculture p p p p p p p p p p % c d + c e lls % f ib ro b la st + c e lls passage # in subculture passage # in subculture fig. comparison of cf and df in morphology, proliferation and marker expression. a phase contrast images of hpsc-cfs, hfv-cfs, hav-cfs, and hdfs. b growth capacity of hpsc-cf, hfv-cf, and hav-cf during sequential passaging. c cd and the human fibroblast maker (clone te- ) expression examined by flow cytometry in hpsc-cfs, hfv-cf, hav-cfs, and hdfs during passaging. scale bars are μm. the hpsc-cfs are from hipsc line df- - - t. the primary hfv-cfs are from cell applications, inc. the hav-cfs are from lonza, nhcf-v. the hdf line is from skin biopsies from healthy donor nature communications | https://doi.org/ . /s - - - article nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications www.nature.com/naturecommunications hpsc-cfs produce d ecm scaffold. generation of ecm is a major function of cfs. therefore, we examined the ecm generated by the hpsc-cfs in comparison with the hfv-cfs, hav-cf, and hdfs following days of high-density cul- ture . to examine the ecm production and assembly, the fibroblast cultures were fixed, but not permeabilized, and immunolabeled with the antibodies to collagen type i and fibronectin. the nuclei were stained with hoechst. confocal imaging demonstrated that all of the fibroblast preparations robustly produced collagen i and fibronectin (fig. a). how- ever, d reconstruction of the confocal images revealed that the hpsc-cfs, hfv-cfs, and hav-cfs produced multicell layer cultures while the hdfs produced only monolayer cul- tures (fig. a, supplementary movies – ). the average depths of the multilayer hpsc-cf (~ µm) and the hfv-cf (~ µm) cultures were similar and greater than the hav-cf cultures (~ µm) and the monolayer hdf cultures (~ . µm) (fig. b). although extracellular fibronectin and collagen i were evenly distributed throughout the monolayer hdf cultures, fibronectin was concentrated in the top layers of all of the cf cultures in contrast to the distribution of collagen i throughout the depth of the multilayer cf cultures (fig. a). to confirm adequate antibody penetration and ecm protein labeling, multilayer hpsc-cf cultures were permeabilized before immunolabeling. the permeabilized hpsc-cf culture shows a distinct pattern of fibronectin immunolabeling with clear intracellular fibronectin detected as well as more fibrillar extracellular fibronectin concentrated in the upper layers (supplementary fig. , movie ). polarization of extracellular fibronectin has been observed in other in vitro cultured cell systems and attributed to the binding of cell produced fibro- nectin with soluble fibronectin in the serum containing medium to generate insoluble fibronectin concentrated at the apical surface of the cultures – . overall, these results demonstrate that high-density culture of the fibroblast pre- parations results in robust production of ecm proteins, but hdfs exhibit contact inhibition and limited proliferation forming a monolayer relative to hcfs that generate multilayer b pdgfrβ p d g f r α n e g ct rl . % % . % . % % % . % . % . % % . % . % . % . % % . % . % % . % . % d c a hpsc-cf hfv-cf hav-cf hdf hpsc-cm hpsc-cf hfv-cf hav-cf hdf hpsc-cm secondary only . . r e la tiv e e xp re ss io n . . hpsc-cf hfv-cf hav-cf hpsc-cmhdf fibroblast gata ctnt gata hpsc-cf postn hfv-cf hav-cf hdf hpsc-cm vim+ . % vim– . % vim+ . % vim– . % vim+ . % vim+ . % vim+ . % vim– . % vim– . % vim– . % fig. representative markers expression in cf, df and hpsc-cm. a histogram plot of flow cytometry analysis for vimentin. b flow cytometry analysis of co-labeling for pdgfrα and pdgfrβ. c immunolabeling by antibodies for fibroblast (clone te- ), ctnt and gata . d postn expression examined by qrt- pcr (n = technical replicates). data are mean ± sem. scale bars are μm. all fibroblasts samples used in the measurement are from passages to . the hpscs are df - - t hipscs. the primary hfv-cfs are from cell applications, inc. the hav-cfs are from lonza, nhcf-v. the hdf line is from skin biopsies from healthy donor article nature communications | https://doi.org/ . /s - - - nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications hpsc-cm , , , , hpsc hpsc-cf hdf hav-cf – – – euclidean distance cardiac factors extracellular matrix log log – – – a c b hpsc-cf hav-cf go term (category) count p-value go term (category) count p-value extracellular region part (cc) . e- extracellular region (cc) . e- extracellular region (cc) . e- extracellular region part (cc) . e- intrinsic to membrane (cc) . e- extracellular matrix (cc) . e- pattern specification process (bp) . e- cell adhesion (bp) . e- anterior/posterior pattern formation (bp) . e- biological adhesion (bp) . e- sequence-specific dna binding (mf) . e- pattern binding (mf) . e- transcription factor activity (mf) . e- polysaccharide binding (mf) . e- cell adhesion (bp) . e- vasculature development (bp) . e- embryonic morphogenesis (bp) . e- blood vessel development (bp) . e- d hp sc -c m hp sc hp sc -c f hd f ha v- cf hp sc -c m hp schd f ha v- cf hp sc -c f hp sc -c m hp sc hd f hp sc -c f ha v- cf mmp eln col a col a lamb lamc matn mmp col a mmp col a mmp col a col a col a mmp col a fbln col a mmp col a col a fbn col a col a fn col a col a col a col a col a timp col a col a fbln mmp mmp cthrc timp col a tnc fbln fbln lama lama col a col a col a bp matn col a col a lamb lamb lamc fbn col a timp col a col a col a lama col a col a mmp col a col a lama col a col a mmp b mmp col a mmp mmp vtn col a lama col a col a col a mmp o timp col a fbn mmp matn lamc mmp mmp mmp col a mmp col a mmp mmp mmp lamb mmp akap ankrd bmp camk camk d cav cited ctnnb erbb fgf fgfr gata gata gata hand hand hdac hes hey hey heyl igf isl jup lmcd maml mef a mef c nedd l nkx - nos notch notch nr a parp pou f prox rbpj rest sall slc a smad snai sox sox sox srf t tbx tbx tgfb tgfbr tp wnt a wt zc h a zfpm zfpm fig. gene expression by rna-seq in cf compared to df, hpsc-cm, and hpsc. a dendrogram showing the similarity in the abundance of overall transcripts ( , genes) across samples of hpsc-cf, hav-cf, hdf, hpsc-cm, and hpsc that was calculated by euclidean distance. b heatmap of cardiac factors ( genes) expression across samples of hpsc-cf, hav-cf, hdf, hpsc-cm, and hpsc organized by unsupervised cluster analysis. c heatmap of extracellular matrix related gene expression ( genes) across samples of hpsc-cf, hav-cf, hdf, hpsc-cm, and hpsc presented by unsupervised cluster analysis. d gene ontology (go) analysis using the functional annotation tool (david bioinformatics resources . , niaid/nih) for the differentially expressed genes in hpsc-cf and hav-cf (supplementary data and ) with calculated p values shown. the hpsc line used for rna-seq are df - - t hipscs. the hav-cfs are from lonza, nhcf-v, and hdfs are from a line. the tpm values from the rna-seq were used for the analysis nature communications | https://doi.org/ . /s - - - article nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications www.nature.com/naturecommunications cultures. furthermore, the thickness of the hpsc-cf cultures is most similar to hfv-cf cultures and significantly greater than the thickness of hav-cfs, which is consistent with the hpsc-cfs exhibiting a more embryonic cf phenotype. hpsc-cfs can transform to myofibroblasts. in the setting of injury or disease, cfs can convert to myofibroblasts and con- tribute to remodeling of the heart. tgfβ signaling is a major stimulus for the transformation of cfs to myofibroblasts – . angiotensin ii (ang ii) is another agonist that stimulates myo- fibroblast conversion and fibrotic effects in cfs but typically not in dfs , . to evaluate for myofibroblast formation induced by both tgfβ and ang ii, we cultured the cfs in dmem+ % fbs for h in the presence or absence of tgfβ or ang ii. the hpsc-cfs, hfv-cfs, and hav-cfs grown with optimized fibro- blast media exhibited low expression of α-smooth muscle actin (smalow) measured by both flow cytometry and immunocy- tochemistry. in contrast, hdfs in the confluent culture exhibited a smahigh population by flow cytometry (fig. , supplementary fig. ). totally, h of culture in dmem+ % fbs led to the emergence of a smahigh population in all of the cf cultures. furthermore, application of tgfβ increased the smahigh population for the hpsc-cfs, hfv-cfs, and hav-cfs, and the sma immunolabeling showed the strong sma expressing stress fibers (fig. ). in contrast, the tgfβ -treated hdfs showed the overall increase of sma expression and a larger smalow popu- lation measured by flow cytometry, and the sma immunolabel- ing also showed the increased sma expression in the stress fibers (fig. ). the cell morphology of all the cfs from baseline con- ditions changed significantly from spindle-shaped, small cells to larger, more elongated cells (fig. ). thus, all of the cf cultures exhibited comparable patterns of transformation to myofibro- blasts based on the formation of a smahigh population with prominent stress fibers in response to tgfβ . the expression pattern of sma also changed in hdfs in response to tgfβ , but the pattern of response was distinct from cfs as measured by flow cytometry (fig. ). ang ii treatment induced the smahigh population as observed with tgfβ treatment in both hpsc-cfs and hfv-cfs, but in hdfs ang ii resulted in a decrease in the smahigh population. comparable results were observed in response to and nm of ang ii (supplementary fig. ). co-culture of hpsc-cfs effects hpsc-cm electrophysiology. given that cfs can influence the electrophysiological properties of the native heart, we examined the functional impact of the co- culture of hpsc-cfs, hav-cfs, or hdfs with hpsc-cms monolayers. various ratios of fibroblasts and hpsc-cms were co-cultured, and representative images confirm different co- culture proportions using immunofluorescent labeling for ctnt (green) to identify hpsc-cms and dapi to label the nuclei of all cells (fig. a). in functional experiments, co-cultured mono- layers were labeled with a membrane potential sensitive dye (fluovolt) to quantify the electrophysiological effects of co- cultured fibroblasts. time–space plots of action potential propa- gation showed the differential impact of each fibroblast subtype on hpsc-cm monolayer function (fig. b). cultures of % hpsc-cms showed regular spontaneous automaticity with uni- form conduction across the cultures (supplementary movie ). comparison of and % fibroblast co-culture demonstrated that the spontaneous rate was significantly faster for hpsc-cf co- culture compared to progressively slower rates for hav-cf and hdf co-cultures (fig. b, c, supplementary movie a–c). impulse conduction velocity was slowed by co-cultures with % of any of the fibroblast populations, although to a greater extent in the hdf co-cultures (fig. b, c, supplementary movie a–c). interestingly, at a ratio of % hpsc-cms: % fibroblasts, we observed fibrillatory conduction patterns in all of the monolayer co-cultures with hpsc-cfs ( / ) and in half of the monolayers with hav-cfs ( / ), while the same ratio of hdfs did not induce this arrhythmic pattern in any hpsc-cm monolayer co-cultures tested ( / ) (fig. b, c, supplementary movie a–c). given the different spontaneous rates observed for the various conditions, we used field stimulation to control rate and constructed action potential duration (apd) restitution curves for the monolayer co- cultures. the % hpsc-cm and % hpsc-cf co-cultures showed the shortest apd s over the range of paced rates (fig. d). these data demonstrated that the electrophysiological hpsc- cf hfv- cf hav- cf hdf collagen i fibronectin nuclei t h ic kn e ss ( μm ) * a p ro je ct io n s id e v ie w b * * * ns hpsc-cf hfv-cf hav-cf hdf fig. ecm production in cf and df cultures. a confocal imaging of high density cultures of hpsc-cf, hfv-cf, hav-cf, and hdf immunolabeling for extracellular collagen i and fibronectin. z-scans are presented as projection images in the top panels and d reconstructions showing side views in lower panels. scale bars in projection are μm. scale bars in side view of hpsc-cf and hfv-cf are μm, hav-cf are μm, and hdf are μm. b thickness of the d ecm scaffolds measured from d reconstructions of hpsc-cf (n = ), hfv-cf (n = ), hav-cf (n = ), and hdf (n = ) biological replicates. the box plots summarize the biological replicates with the box enclosing from first to third quartile, middle square indicating mean, line in box indicating median, and whiskers indicating outliers. *p < . , one-way anova with post hoc bonferroni test article nature communications | https://doi.org/ . /s - - - nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications properties of the hpsc-cm monolayers are distinctly modulated by co-cultures with the different populations of fibroblasts. discussion in the present study, we demonstrate that stage-specific activation of wnt and fgf signaling promotes the efficient differentiation of hpscs via shfps to cfs. the hpsc-cfs share characteristics with native human cfs based on gene expression, cell morphology, proliferation, ecm production, myofibroblast transformation, and the distinct modulation of hpsc-cm elec- trophysiology. although cell type-specific markers for cfs are not known, the combination of different markers present in the hpsc-cfs including expression of pdgfrα, vimentin, fsp , and labeling with human fibroblast antibody along with gata expression are consistent with a cf phenotype. overall, we conclude that this monolayer-based protocol using defined con- ditions allows the scalable generation of shf-derived hpsc-cfs. in mammalian cardiac development, cfs arise from epicardium, endocardium, and neural crest progenitor populations . these different fibroblast populations are concentrated in different regions of the heart and exhibit distinct contributions to cardiac develop- ment such as the role of endothelium-derived cfs in formation of the cardiac cushion and ultimately cardiac valves. the three embryological origins of cfs suggest that distinct fibroblast popu- lations can be differentiated from hpscs. the mesoderm-directed differentiation protocol argues against neural crest ectoderm derived cfs. recent studies have described the differentiation of hpscs to epicardial cells that can give rise to cfs – . these studies provided evidence for generation of epicardial cells based on the expression of characteristic markers including wt , tbx , and aldh . in the present study, we found a different pattern of gene expression induced by fgf signaling in the developing progenitor populations with strong evidence for a shfp population marked by persistent isl expression, high hand , and transient expression of cxcr . the shfps in development can give rise to endo- cardium via a tie expressing angiogenic progenitor or a tie − but isl + population. the endocardium is a specialized endothelial population commonly distinguished by the expression of nfatc . our studies also did not reveal clear expression of tie or nfatc during intermediate stages, implying the differentiation of endocardial cells may require different signaling pathways than the fgf signaling in the gifgf protocol. however, given the strong signal for shf genes there is evidence for shfps and the expression of an endothelial-to-mesenchymal transition related gene in the gifgf protocol, snai , suggests the hpsc-cfs may have passed through a transient endocardial intermediate. alternatively, the shfps may directly give rise to cfs as has been suggested for shf- derived endocardial cells in which some endocardial cells arise via an endothelial/endocardial progenitor population and others arise directly from shfps . consistent with our findings of the shf- derived hpsc-cfs, a recent study using genetic lineage tracing in mouse models demonstrated that shfps in vivo can give rise to cfs as can esc-derived shfps in vitro . the maturity of lineages derived from hpscs remains a critical question in applications. for example, hpsc-cms generated from existing protocols are relatively immature exhibiting an embryonic/ fetal cm phenotype based on a number of key properties including tgfβ , h s m a n u cl e i p h a se s m a h is to g ra m tgfβ , h confluent ctr ng/ml ng/ml confluent ctr ng/ml ng/ml s m a n u cl e i p h a se s m a h is to g ra m neg . low high . neg . low high neg . low high neg . low high . neg . low high neg . low high low high . neg . low high neg . low high neg . neg . low high neg . low high neg . low high h p s c -c f h fv -c f h a v -c f h d f fig. myofibroblast transformation of cfs and dfs. analysis of α-smooth muscle actin (sma) expressing cells to assay for myofibroblast transformation from hpsc-cf, hfv-cf, hav-cf, and hdf in culture. all fibroblast cultures in passages – were treated with ng/ml tgfβ for h in dmem+ % fbs medium. expression of sma was measured by both flow cytometry (upper panel) and immunolabeling (lower panel). phase contrast images of representative cultures are shown in the middle panel. phase contrast images and sma immunofluorescence images are from the same biological samples, but not the same field of view. scale bars are μm. the hpsc-cfs are derived from df - - t hipscs. the hfv-cfs are from cell applications, inc. the hav-cfs are from lonza, nhcf-v, and hdfs are from a line nature communications | https://doi.org/ . /s - - - article nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications www.nature.com/naturecommunications cell size and structure, myofilament composition, electrophysiology and metabolism – . in the case of hpsc-cfs, the issue of maturity is equally relevant. however, the cf-specific developmental mile- stones are not well defined. we observed the hfv-cfs and hpsc- cfs have a greater proliferative capacity than hav-cfs, a more uniform expression of pdgfrβ, and the ability to generate thicker d high density cultures. furthermore, the gene expression pattern in the hpsc-cfs from our data is more similar to human fetal cfs relative to adult cfs from a recently published study . although these functional assays and gene expression studies are consistent with hpsc-cfs having a more embryonic phenotype, there are other possible explanations given the multiple sources of cfs that may exhibit differences in their relative abundance in fetal and adult human heart. the ability to differentiate hpscs to hpsc-cfs provides an unlimited supply of cfs for a range of research in cardiac biology and disease as well as potential clinical applications in tissue engineering and cardiac regeneration. nevertheless, future studies will be needed to fully appreciate the properties of hpsc-cfs and how they relate to the heterogeneous and incompletely defined cfs present in the native heart. methods human psc culture. human ipsc line df - - t and esc lines h and h - tnnt -gfp were used in this study (wicell research institute, cell line name: ips df - - t; wa ; h -htnnt -pgz-d , respectively). hpscs were cultured on matrigel (gfr, bd biosciences) coated -well plates in either mtesr (df - - t, h ) medium (stem cell technologies) or e (h - tnnt -gfp) medium (thermofisher). cells were passaged using versene solution (invitrogen) every – days. cells were washed with dpbs without ca ++/mg++ (gibco) and incubated with ml/well versene solution at °c for mins. aspirating versene solution and adding ml/well mtesr or e medium to gently pipette up and down and scrape to cell clusters. cell clusters a b c % hpsc-cf % hdf % cm cm cm cm cm cm s s s d % hav-cf cm cm % hpsc-cf % hdf % hav-cf apd restitution curves cycle length (ms) a p d ( m s) % hpsc-cms % hpsc-cms/ %hpsc-cfs % hpsc-cms/ %hdfs f ib ri lla to ry co n d u ct io n ( / ) f ib ri lla to ry co n d u ct io n ( / ) *** *** *** *** ** ** * ctnt dapi / / % h df % h av -c f % h ps c- cf % h df % h av -c f % h ps c- cf % c m / / % h df % h av -c f % h ps c- cf % h df % h av -c f % h ps c- cf % c m s p o n ta n e o u s ra te ( h z) c o n d u ct io n ve lo ci ty ( cm /s ) % hpsc-cm / % hpsc-cm/hpsc-cf / % hpsc-cm/hpsc-cf / % hpsc-cms/hpsc-cf % hpsc-cms/ %hav-cfs fig. effect of cfs or dfs on hpsc-cm electrophysiology in co-culture. a confocal images of different ratios hpsc-cms and hpsc-cfs in culture together with immunolabeling for ctnt to identify hpsc-cms and dapi labeling for all cells present. scale bars are µm. b time–space plots of optical mapping experiments. postacquisition analysis of optical recordings was done by plotting the changes of fluorescence over time along a single line of each monolayer, similar to a confocal line scan. differences in the spontaneous rates of activation are evident as are altered patterns of conduction with fibrillatory conduction observed with % hpsc-cf and hav-cf co-cultures with hpsc-cm. c average spontaneous beating rate and action potential propagation velocity for % hpsc-cm monolayers (n = ) compared to co-cultures of percentages and cell types indicated (red, hpsc-cf % (n = ) and % (n = ); green, hav-cf % (n = ) and % (n = ); blue, hdf % (n = ) and % (n = )). statistical comparisons were made between different fibroblast populations at each fixed mixture with anova and tukey’s post hoc testing for significant differences as shown, ***p < . ; **p < . ; and *p < . . d average action potential duration at % of repolarization (apd ) restitution curves generated by pacing indicated preparations over a range of different cycle lengths and measuring optical apd . the hpsc-cf and hpsc-cms are derived from h -ctnt-gfp hescs. the hav-cfs are from lonza, nhcf-v, and hdfs are from skin biopsy from a healthy adult donor. data are mean ± sem with n representing number of separate monolayers (biological replicates) tested article nature communications | https://doi.org/ . /s - - - nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications www.nature.com/naturecommunications were pellet down by centrifugation at rpm for mins, and resuspended in mtesr or e medium, and plated in -well plate at the split ratio of : to : . mycoplasma tests were routinely performed (monthly) for all the cell culture in this study. differentiation of hpscs to cms using giwi protocol. human pscs were dissociated with ml/well versene solution at °c for min, and seeded on matrigel coated -well plates at the density of . – . × cells/well in mtesr medium supplemented with μm rock inhibitor (y- ) (tocris). cells were cultured for – days in mtesr medium until reaching % confluence when differentiation started (day ). at day , the medium was changed to . ml rpmi+b without insulin (gibco) and supplemented with µm chir (tocris), and cells were treated in this medium for h (day ). after day , medium was changed to ml rpmi+b without insulin and cells were cultured in this medium for h (day - ). seventy-two hours (day ) after addition of chir , a combined medium was prepared by collecting . ml of medium from the wells and mixing with same volume of fresh rpmi+b without insulin medium and supplemented with µm iwp (tocris), and cells were treated with iwp in the medium for h (day - ). the medium was changed to ml rpmi+b without insulin on day . on day the medium was changed to rpmi+b with insulin (gibco), and the cells were fed every other day and cultured until day for flow cytometry analysis. differentiation of hpscs to cfs. human pscs were dissociated with ml/well versene solution (gibco) at °c for min, and seeded on matrigel (gfr, bd biosciences) coated -well plates at the density of . – . × cells/well in mtesr medium supplemented with μm rock inhibitor (y- ) (tocris). cells were cultured for – days in mtesr medium with medium changes daily until they reached % confluence when differentiation started (day ). at day , the medium was changed to . ml rpmi+b without insulin and supplemented with µm chir (tocris) and cells were treated in this medium for h (day ). after day , the medium was changed to . ml rpmi+b without insulin (gibco) and cells were cultured in this medium for h (day ). after day but within h (before day ), the medium was changed to . ml of the cfbm medium (supplementary table ) supplemented with ng/ml bfgf (wicell research institute). cells were fed with cfbm+ ng/ml bfgf every other day until day when they were used for flow cytometry analysis and passaged. culture of human cfs and dfs. the hpsc-differentiated cfs (hpsc-cfs), pri- mary human fetal ventricular cardiac fibroblasts (hfv-cfs, cell applications, inc., cryopreserved hcf, fetal: - f, lot ) and adult ventricular cardiac fibro- blasts (hav-cfs, lonza, nhcf-v, cc- , lot ) and primary dermal fibroblasts (hdfs, healthy donors, a and a lines, generated with informed consent as approved by uw-madison health science irb, protocol h- - ) were used in this study. hpsc-cfs at days of differentiation were dissociated with . % trypsin-edta (gibco) and plated in -well noncoding plastic plates at the density of , cells/well in ml of fibrogro medium (millipore emd, scmf ) plus % fbs (gibco). the hpsc-cfs were fed every other day with ml fibrogro+ % fbs medium and passaged every – days using . % trypsin- edta. the plating density for passaging hpsc-cfs was cells/cm . the hfv- cfs were thawed at the plating density of cells/cm in the human cf growth medium (cfgm, cell applications, inc., – ) and the hav-cfs were thawed at the plating density of cells/cm in fgm- medium (lonza, cc- ) in noncoding plastic t flasks. the media were changed the next day after thaw and cells were fed every other day afterward. the hfv-cfs were cultured in both the cfgm and fibrogro+ % fbs media and were passaged every – days using . % trypsin-edta. the hav-cfs were cultured with the fgm- medium and were passaged every – days using . % trypsin-edta. the subsequent plating density for passaging after thaw was and cells/cm for hfv-cfs and hav- cfs, respectively. the primary hdfs were thawed in dmem high glucose with glutamax supplement (gibco - ) plus % fbs (gibco ) in non- coding plastic t flasks at the density of cells/cm . the hdfs were cultured in the dmem high glucose+ % fbs with medium change every other day and were passaged every – days using . % trypsin-edta. the subsequent plating density for passaging hdfs was cells/cm . all fibroblasts were cryopreserved in % dmem high glucose with glutamax supplement (gibco - ), % fbs and % dmso. flow cytometry. cells were dissociated by incubation with . % trypsin-edta (gibco) plus % chick serum (sigma) for min at °c. cells were vortexed to disrupt the aggregates followed by neutralization by adding equal volume of eb medium . cells were counted and . – million of cells were used for labeling. for labeling surface markers, cells were labeled either before, or after fixed in % paraformaldehyde at °c water bath for min in the dark. for labeling intra- cellular markers, cells were fixed in % paraformaldehyde and permeabilized in ice- cold % methanol for min on ice. cells were washed once in facs buffer (dpbs without ca++/mg++, . % bovine serum albumin (bsa), . % nan ), centrifuged and resuspended in about μl facs buffer with (for intracellular markers) or without . % triton x- (sigma). for labeling surface markers, appropriate amount of the conjugated antibodies according to the manufacturer’s instruction were added to the cells in facs buffer to make the final volume of μl with facs buffer, and incubated at room temperature in dark for min. cells were washed once with ml facs buffer and resuspended in – μl facs buffer for analysis. for labeling intracellular markers, the primary antibody was diluted in μl /sample facs buffer plus . % triton x- and aliquoted to each sample for a total sample volume of μl. samples were incubated with the primary antibodies overnight at °c. please refer to supplementary table for the primary antibodies used in this study. for the secondary antibody labeling, cells were washed once with ml facs buffer plus . % triton x- after the primary antibody labeling, centrifuged, and supernatant discarded leaving ~ μl. second- ary antibody specific to the primary igg isotype was diluted in facs buffer plus . % triton x- in a final sample volume of μl at : dilution. samples were incubated at room temperature in dark for min, washed in facs buffer and resuspended in – μl facs buffer for analysis. data were collected on a facscalibur (beckton dickinson) and attune nxt (thermofisher) flow cyt- ometers and analyzed using flowjo. immunocytochemistry. for imaging with evos microscope (life technologies), cells were cultured in either -well or -well plates. cells were fixed with % paraformaldehyde for min at room temperature, and permeabilized in . % triton x- (sigma) for . – h at room temperature. after fix and permeabili- zation, cells were blocked with % nonfat dry milk (bio-rad) in . % triton x- solution and incubated for . – h at room temperature on a rotator followed by two washes with dpbs. primary antibodies (supplementary table ) were added in % bsa in dpbs solution with . % triton x- , and incubated overnight at °c. cells were washed with . % tween in dpbs twice and dpbs twice. secondary antibody specific to the primary igg isotype were diluted ( : ) in the same solution as the primary antibodies and incubated at room temperature for h in dark on a rotator. cells were washed with . % tween in dpbs twice and dpbs twice. nuclei were labeled with hoechst or dapi. quantitative rt-pcr. cell samples were collected using . % trypsin–edta (invitrogen) to remove the cells from cell culture plates. total rna was purified using qiagen rneasy® mini kit. any genomic dna contamination was removed by rnase-free dnase set (qiagen) with the rneasy columns or by dnase i (invitrogen) treatment for min at room temperature. ng of total rna was used for oligo (dt) —primed reverse transcription using superscript™ iii first- strand synthesis system (invitrogen). quantitative rt-pcr was performed using taqman pcr master mix and gene expression assays (applied biosystems, supplementary table ) in triplicate for each sample and each gene. . μl of cdna from rt reaction was added as template for each q-pcr reaction. the expression of genes of interest was normalized to gapdh. rna-seq and data analysis. cell samples were collected using . % trypsin–edta (invitrogen) to remove the cells from cell culture plates. total rna was purified using qiagen rneasy® mini kit. possible genomic dna con- tamination was removed by rnase-free dnase set (qiagen) with the rneasy columns. duplicates for each sample were submitted for rna-seq. total rna was qualified with the life technologies qubit fluourometer (q ) and agilent bioanalyzer (g ca). one-hundred nanograms of total rna was used to prepare indexed cdna libraries with the ligation mediated sequencing (lm-seq) protocol . final indexed cdna libraries were pooled with thirty uniquely indexed lm-seq cdna libraries per lane and sequenced on an illumina hiseq with a single bp read and a bp index read. base-calling and demultiplexing were performed using casava (v . . ). sequences were filtered and trimmed to remove low quality reads, adapters, and other sequencing artifacts. the remaining reads were aligned to refseq genes extracted from the illumina igenomes refer- ence, selecting only those with “nm_” annotations. bowtie (v . . ) was used for alignment, allowing two mismatches in a bp seed . reads with more than alignments were excluded from further analysis. rsem (v . . ) was used to esti- mate isoform and relative gene expression levels (transcripts per million or “tpm”) . genes for specific functional groups were selected by searching online tool amigo (http://amigo.geneontology.org) using key words and manually con- firmed. heatmaps were generated using pheatmap_ . . in r version . . . the similarity heatmap showed the euclidean distances between samples which were calculated using r dist function on normalized read counts. ecm production and confocal imaging. confluent hpsc-cfs, primary hfv-cfs and hav-cfs, and hdfs were dissociated with tryple express (gibco) and seeded in μ-dish mm ibidi dish (cat# ) at the density of . × cells/dish. cells were grown for days with medium change every other day. hpsc-cfs and primary hfv-cfs were grown with fibrogro medium (millipore emd, scmf ) plus % fbs gibco, primary hav-cfs were grown in fgm- media (lonza), and hdfs were grown in dmem high glucose with glutamax supple- ment (gibco) plus % fbs (gibco) medium. on day , cells were fixed with % paraformaldehyde for min at room temperature, and stored in dpbs at °c. the ecm proteins including collagen and fibronectin were co-labeled with anti- col a (col- ) (santa crutz biotechnology, sc- ) and anti-fibronectin nature communications | https://doi.org/ . /s - - - article nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications http://amigo.geneontology.org www.nature.com/naturecommunications www.nature.com/naturecommunications (a- ) (santa crutz biotechnology, sc- ) antibodies without permeabiliza- tion of the cells to examine the ecm using the immunolabeling protocol in the ‘immunocytochemistry’ section. the cellular fibronectin and sma expression were examined in the permeabilized cells and the antibodies were co-labeled using the same immunolabeling protocol in the “immunocytochemistry” section. cells were imaged using the confocal microscope (leica tcs sp system). co-cultures of hpsc-cms and hpsc-cfs, hav-cfs or hdfs. hpsc-cms were cultured with hpsc-cfs, hav-cfs, or hdfs at varying percentages: , , , , or %. these co-cultures were plated at a density of , cells per µl drop of eb medium ( % dmem/f , . mmol/l nonessential amino acids, mmol/l l-glutamine, . mmol/l β-mercaptoethanol, % fbs and μmol/l blebbistatin) on × mm pieces of polydimethylsiloxane silicone sheeting (smi; saginaw, mi) coated with matrigel ( μg/ml, corning) and grown for days kept at °c in % co . at this point the media was changed to rpmi (gibco) supplemented with b (gibco) and replaced every days for days prior to experimentation. optical mapping. optical action potentials and time–space plots were recorded using fluovolt™ membrane potential probe (f , life technologies) – . after a minute incubation period, monolayers were transferred to hank’s balanced salt solution (hbss with calcium and magnesium, gibco) for optical mapping recording and kept at °c for the duration of the experiment. all monolayers containing hpsc-cms displayed spontaneous pacemaker activity which was recorded using a ccd camera (red-shirt little joe, scimeasure, decatur, ga, × pixels) with the appropriate emission filters and led illumination . a total of s movies were taken at fps (labwindows acquisition, national instruments, austin, tx, usa) which were amplified, filtered and digitized for offline analysis. briefly, time sequence plot graphs of fluorescence intensity over time were generated from average action potentials and used for calculation of action potential duration after signal filtering. in addition, optical mapping movies were processed with scroll to generate acti- vation maps and calculation of conduction velocity after providing spatial calibration to the software. repetitive stimuli (duration, ms; strength, twice diastolic threshold) was applied by field stimulation using silver electrodes. monolayers were paced faster than the spontaneous beating rate, which was different depending on the co-culture with hpsc-cfs, hav-cfs, or hdfs (fig. c, d). stimulation frequency ranged from to . ms cycle length. statistics. sample size varies and was determined based on anticipated scatter of the data, but formal power calculations were not done given the lack of previous comparable datasets from which to estimate variance. data are presented as mean ± standard error of the mean. technical and biological replicates are as indicated for each dataset. for datasets with normal distributions, statistical significance was determined by student’s t test for two groups or one-way anova for multiple groups with post hoc test using bonferroni and tukey methods. statistical analysis was performed using origin, v , p < . was considered statistically significant. reporting summary. further information on research design is available in the nature research reporting summary linked to this article. data availability the rna-seq data for this study are deposited in geo with the accession code gse . all other relevant data generated in this manuscript that support the findings of this study are available upon request from the authors. received: july accepted: april references . pinto, a. r. et al. revisiting cardiac cellular composition. circ. res. , – ( ). . ieda, m. et al. cardiac fibroblasts regulate myocardial proliferation through beta integrin signaling. dev. cell , – ( ). . d’souza, k. m. et al. g protein-coupled receptor kinase- is a novel regulator of collagen synthesis in adult human cardiac fibroblasts. j. biol. chem. , – ( ). . kohl, p. & gourdie, r. g. fibroblast-myocyte electrotonic coupling: does it occur in native cardiac tissue? j. mol. cell cardiol. , – ( ). . furtado, m. b. et al. cardiogenic genes expressed in cardiac fibroblasts contribute to 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from coordenação de aperfeiçoamento de pessoal de nível superior and fundação de amparo à pesquisa do distrito federal. the work was funded by nih r hl (t.j.k.); nih u hl (t.j.k.); s rr (t.j.k.); and the uw institute for clinical and translational research, grant ul tr , from the clinical and translational science award of the ncats/nih. author contributions t.j.k. and j.z. conceived the study and wrote the paper. j.z. designed and carried out the experiments, and analyzed the data. r.t., g.c.k. and j.l.c. did most of the cell culture and differentiation and the pcr. k.f.c., a.m.d., t.j.h. and j.j. designed, performed the co-culture work and analyzed the data. e.c.r., e.g.s. and a.n.r. performed the ecm production. j.a.t. contributed to conception, writing, and funding of the project. additional information supplementary information accompanies this paper at https://doi.org/ . /s - - - . competing interests: t.j.k. is a consultant for cellular dynamics international. e.g.s. and a.n.r. are co-founders and have a financial interest in cellular logistics inc. the remaining authors declare no competing interests. reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ journal peer review information: nature communications thanks nadia rosenthal and other anonymous reviewer(s) for their contribution to the peer review of this work. peer reviewer reports are available publisher’s note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article’s creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article’s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/ . /. © the author(s) nature communications | https://doi.org/ . /s - - - article nature communications | ( ) : | https://doi.org/ . /s - - - | www.nature.com/naturecommunications https://doi.org/ . /s - - - https://doi.org/ . /s - - - http://npg.nature.com/reprintsandpermissions/ http://npg.nature.com/reprintsandpermissions/ http://creativecommons.org/licenses/by/ . / http://creativecommons.org/licenses/by/ . / www.nature.com/naturecommunications www.nature.com/naturecommunications 	functional cardiac fibroblasts derived from human pluripotent stem cells via second heart field progenitors 	results 	progenitors arise during cardiac differentiation of hpscs 	fgf promotes differentiation of shfps and cfs 	comparison of hpsc-cfs, primary cfs, and dermal fibroblasts 	gene expression of hpsc-cfs is most similar to primary cfs 	hpsc-cfs produce d ecm scaffold 	hpsc-cfs can transform to myofibroblasts 	co-culture of hpsc-cfs effects hpsc-cm electrophysiology 	discussion 	methods 	human psc culture 	differentiation of hpscs to cms using giwi protocol 	differentiation of hpscs to cfs 	culture of human cfs and dfs 	flow cytometry 	immunocytochemistry 	quantitative rt-pcr 	rna-seq and data analysis 	ecm production and confocal imaging 	co-cultures of hpsc-cms and hpsc-cfs, hav-cfs or hdfs 	optical mapping 	statistics 	reporting summary 	references 	references 	acknowledgements 	author contributions 	competing interests 	acknowledgements [pdf] the association of coronary artery calcification and carotid artery intima-media thickness with distinct, traditional coronary artery disease risk factors in asymptomatic adults. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /aje/kwn corpus id: the association of coronary artery calcification and carotid artery intima-media thickness with distinct, traditional coronary artery disease risk factors in asymptomatic adults. @article{rampersaud theao, title={the association of coronary artery calcification and carotid artery intima-media thickness with distinct, traditional coronary artery disease risk factors in asymptomatic adults.}, author={e. rampersaud and l. bielak and afshin parsa and haiqing shen and w. post and k. ryan and p. donnelly and j. rumberger and p. sheedy and p. peyser and a. shuldiner and b. mitchell}, journal={american journal of epidemiology}, year={ }, volume={ }, pages={ - } } 	e. rampersaud, l. bielak, + authors b. mitchell 	published 	medicine 	american journal of epidemiology coronary artery calcification (cac) and common carotid artery intima-media thickness (cimt) are measures of subclinical vascular disease. this - study aimed to characterize the associations among coronary artery disease risk factors, cac quantity, and cimt and to estimate shared genetic and environmental contributions to both cac and cimt among asymptomatic amish adults in lancaster county, pennsylvania. heritability for cac quantity and cimt, adjusted for age and sex, was . (p… expand view on pubmed academic.oup.com save to library create alert cite launch research feed share this paper citationshighly influential citations background citations methods citations results citations view all figures, tables, and topics from this paper 	 table 	 figure 	 table 	 figure 	 table view all figures & tables 	cdisc sdtm coronary artery disease risk terminology 	arteriopathic disease 	coronary artery calcification 	vascular calcification 	vascular diseases 	carotid arteries 	ldl cholesterol lipoproteins 	waist circumference 	mental association 	common carotid artery 	paget's disease, mammary citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency the effect of age and risk factors on coronary and carotid artery atherosclerotic burden in males-results of the heinz nixdorf recall study. 	m. bauer, s. moehlenkamp, + authors r. erbel 	medicine 	atherosclerosis 	 	 save alert research feed association between coronary artery calcium score and carotid atherosclerotic disease. 	q. zhao, xinhuai wu, + authors z. cai 	medicine 	molecular medicine reports 	 	 	 save alert research feed segment-specific carotid intima-media thickness and cardiovascular risk factors in koreans: the healthy twin study 	k. lee, j. sung, + authors y. song 	medicine 	european journal of preventive cardiology 	 	 save alert research feed coronary calcium score and thickness of carotid middle intima in patients with suspicion of ischemic heart disease 	yudeisy rodríguez hernández, vladimir mendoza-rodríguez, + authors aníbal gonzález trujillo 	medicine 	 	pdf 	view excerpt, cites background save alert research feed causes of changes in carotid intima-media thickness: a literature review 	b. qu, tao qu 	medicine 	cardiovascular ultrasound 	 	 	 	view excerpt, cites background save alert research feed association of systemic inflammatory activity with coronary and carotid atherosclerosis in the very elderly. 	w. freitas, l. a. quaglia, + authors a. sposito 	medicine 	atherosclerosis 	 	 	highly influenced 	view excerpts, cites results and background save alert research feed correlation of intima media thickness of carotid arteries with coronary artery disease 	dr. a. khare 	 	pdf save alert research feed meta-analysis of genome-wide association studies from the charge consortium identifies common variants associated with carotid intima media thickness and plaque 	j. bis, m. kavousi, + authors c. o’donnell 	medicine, biology 	nature genetics 	 	 	pdf save alert research feed genetics of coronary artery calcification among 	m. wojczynski, m. li, + authors m. reilly 	geography 	 save alert research feed genetic ancestry is associated with measures of subclinical atherosclerosis in african americans: the jackson heart study 	samson y. gebreab, p. riestra, + authors s. davis 	medicine 	arteriosclerosis, thrombosis, and vascular biology 	 	 	 save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency determinants of coronary artery and aortic calcification in the old order amish 	w. post, l. bielak, + authors b. mitchell 	medicine 	circulation 	 	 	 	view excerpts, references background save alert research feed potential of quantity of coronary artery calcification to identify new risk factors for asymptomatic atherosclerosis. 	j. e. maher, j. raz, l. bielak, p. sheedy, r. schwartz, p. peyser 	medicine 	american journal of epidemiology 	 	 	pdf save alert research feed association of coronary heart disease incidence with carotid arterial wall thickness and major risk factors: the atherosclerosis risk in communities (aric) study, - . 	l. chambless, g. heiss, + authors l. clegg 	medicine 	american journal of epidemiology 	 	 , 	pdf save alert research feed genetic and environmental contributions to atherosclerosis phenotypes in men and women: heritability of carotid intima-media thickness in the framingham heart study 	c. fox, j. polak, + authors c. o’donnell 	medicine 	stroke 	 	 	 save alert research feed the determination of carotid intima medial thickness in adults--a population-based twin study. 	l. swan, d. birnie, g. inglis, j. connell, w. hillis 	medicine 	atherosclerosis 	 	 save alert research feed association of traditional risk factors with coronary calcification in persons with a family history of premature coronary heart disease: the study of the inherited risk of coronary atherosclerosis. 	a. m. valdés, m. wolfe, h. tate, w. gefter, a. rut, d. rader 	medicine 	journal of investigative medicine : the official publication of the american federation for clinical research 	 	 save alert research feed differential associations of traditional and non-traditional risk factors with carotid intima-media thickening and plaque in peritoneal dialysis patients 	angela yee-moon wang, s. s. ho, + authors c. lam 	medicine 	american journal of nephrology 	 	 save alert research feed heritability of carotid intima-media thickness: a twin study. 	j. zhao, f. cheema, + authors v. vaccarino 	medicine 	atherosclerosis 	 	 	 save alert research feed heritability of coronary artery calcium quantity measured by electron beam computed tomography in asymptomatic adults 	p. peyser, l. bielak, + authors p. sheedy 	medicine 	circulation 	 	 	pdf save alert research feed relation of thoracic aortic and aortic valve calcium to coronary artery calcium and risk assessment. 	n. wong, m. sciammarella, + authors d. berman 	medicine 	the american journal of cardiology 	 	 	view excerpt save alert research feed ... ... related papers 	abstract 	figures, tables, and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue oral health status, knowledge, and practices in an amish population vol. , n o . . summer i oral health status, knowledge, and practices in an amish population robert a. bagramian, dds, drph chairman sena narendran, bds, ddph, msc a. mahyar khavari department of prevention and health care school of dentistry university of michigan a n n arbor, mi - abstract this study was conducted in the summer of to assess the oral health status, knowledge, and practices of an amish population in southwest michigan. dental caries experience, periodontal health, and oral hygiene status were recorded using decayed, missing, and filled surfaces (dmfs), periodontal index (pi), and sim- plified oral hygiene index (ohi-s). data on oral health knowledge and practices were collected by interviews using a structured questionnaire. results showed sig- nificantly lower levels of disease among amish. dmfs scores for - -year-old amish children were almost half that of the us general population (nidr - ). pi score of all ages combined was . , which was . times lower than a national sample ( - ). lower levels of disease in amish could be related to their way of life and dietary patterns. a relatively higher level of unmet need for prosthodontic care, inadequate oral health knowledge, and barriers to dental care in the study population emphasize the need for dental public health and health education programs. key words: amish, caries, periodontal disease, oral hygiene, oral health knowledge, oral health practices the oral health of numerous contemporary a n d in- digenous populations has been studied in different na- tions ( - ). these studies were both anthropological ( - ) a n d epidemiologic ( - ) in nature. in the united states, national surveys d o n e periodically have docu- mented the oral health o f american citizens ( - ). further, in the us the oral health of a group of native americans, the pima indians, has been investigated recently ( - ). there is a lack of documentation of oral health status of the amish population, which differs from the general population primarily in its life-style and dietary habits. amish are a g r o u p of settlers from send correspondence and reprint requests to dr. bagramian. this paper was presented a t the american association for dental re- search annual meeting, march - , , washington, dc. manu- script received: / / ; returned to authors for revision: / ; ac- cepted for publication: / / . northern europe w h o first arrived in the united states during the latter half of the th century ( ). in the us, the old order amish number over , persons. over percent of the amish population live in pennsylvania, ohio, a n d indiana; more than per- cent of these live in the counties of lancaster (pennsyl- vania), holmes (ohio), a n d lagrange-elkhart (indiana) ( ). the amish settlement in southwestern michigan constitutes approximately , people originally from northern indiana. the amish life-style is dependent o n farmland; settlers moved from lndiana to michigan in the late s in search of more available land at cheap- e r cost. the unit of a n amish society is the church district, which is determined by the number o f people who can be accommodated in a farmhouse for religious services a n d h o w far o n e can travel conveniently by horse and buggy to attend. the districts are ruled over by lay clergy called bishops w h o are chosen by l o t ( ). the present investigation appears to be the first oral health study of this g r o u p ( - ). the association between diet a n d dental caries has been well documented ( - ). the amish have a unique life-style, generally abstaining from processed food items, a n d relying o n homegrown foods. since a s much a s percent or more o f the sugar consumed in developed countries is in hidden form ( ), i t could be suggested that dietary habits of amish might have a beneficial effect o n their dental health. the purpose of this study was to assess ( ) the oral health status of a selected amish population, ( ) their knowledge a n d attitude to oral health, a n d ( ) their dental health behavior. methods the study population was composed o f subjects from amish families living in southwest michigan. the local bishop was contacted concerning participa- tion in the study. initially families were contacted, of whom ( %) agreed to be included in the study. the families contacted for the study were those within one church district in southwestern michigan. of the families that consented, all of the members present at the time of examination agreed to participate in the i journal of public health dentistry study. this sample may not be representative of the amish population due to the small numbers of partici- pants in the study and potential selection bias. there were subjects younger than years of age with males and females, and older than years of age including males and females. dental examinations included caries experience, oral hygiene status, and periodontal health and were carried out in the summer of by an oral epidemiologist with the help of an experienced recorder. dental examinations were conducted in natural light with subjects seated on a straight-back chair using a mouth mirror and no. explorer. diagnostic criteria for dental caries were the same as those used by the national institute of dental research, nidr ( ). softness or definite break in continuity of enamel was a prerequisite for a tooth to be considered carious. each tooth space was recorded only once. the reason for a missing tooth, either because of caries or for any other reason, was assigned by the examiner based on the caries experience of the respective subject. wherever necessary, teeth were probed to confirm the presence or absence of dental caries. radiographs were not used. caries experience of each subject was record- ed using decayed, missing, and filled surfaces (dmfs). periodontal disease status was assessed by applying the periodontal index (pi) of russell ( ), with criteria based on signs of periodontal inflammation, pocket formation, and loss of function. each tooth in the mouth was scored; scores were totaled and then divid- ed by number of teeth present. this mean score repre- sents the periodontal condition of the teeth in the mouth. the pi was used in this study to allow compari- sons with existing national and international data. the mesiobuccal area of every permanent tooth was exam- ined for periodontal pockets using a modified probe, which has a mm black band around the shank starting at mm from the tip and ending at mm from the tip. a score of four was given if the metal tip was no longer visible when the probe was inserted to the depth of the pocket and the black band remained visible. a score of six was given if both metal tip and the black band was submerged in the pocket. the simplified oral hygiene index (ohi-s) was ap- plied to measure oral hygiene status ( ). ohi-s has two component parts, debris and calculus, each having a range of scores from zero to three. six representative surfaces were scored in each mouth and a mean was derived for each person by dividing the total scores by six. data on oral health knowledge and practices were collected by face-to-face interviews. a survey instru- ment was developed and pretested among family members of the amish bishop ( n = ). the question- naire included questions, four open-ended and precoded. the responses to open-ended questions were coded before the statistical analyses. the number of choices to precoded questions ranged from two to eight. forty-eight percent of the questionnaires were related to oral health knowledge, percent to dental health practices, and percent to general items of inter- est such a s sources of drinking water. the average time for the interviews was minutes, with a range of to minutes. results dental caries. caries experience ranged from . dmfs for children under years to . for adults over years (table ). approximately percent of the population under years was caries-free. distribution of dmfs scores was not normal as indicated by relative- ly higher standard deviations. the finding of a higher standard deviation (compared to the mean) is often encountered in many epidemiologic studies following the recent decline in dental caries in children. the dis- tribution of dmfs scores by age and sex showed fe- males with higher scores than males in all groups ex- cept - years. analysis by age group indicated that, with increasing age, contribution from decayed sur- faces (d) to dmfs scores remained constant, while those from missing (m) and filled surfaces (f) in- creased. relative contributions of decayed (d), missing (m), and filled (f) to dmfs scores are shown in table . the ratio of decayed d to dmf surfaces was . percent, indicating very little unmet needs of dental caries in this population. the ms/dmfs ratio of . percent reflects a high missing component, and also a high level of unmet need for prosthodontic care. the fs/dmfs ratio for the - -year-old age group in the study popu- lation was . percent. oral hygiene status. oral hygiene scores ranged from . in children under to . in adults over years. the ohi-s scores shown in table indicate a higher score for males compared to the females. there appears to be a typical decline in ohi-s scores around the time of puberty for both sexes followed by an in- crease in scores after age . table caries experience (dmfs) by age < - - - + . ? . . ? . . ? . . ? . . ? . table ratios of decayed, missing, and filled surfaces to dmfs status of surface mean?sd ratio d . ? . ds/dmfs (x ) = . % f . . fsldmfs (x )= . % m . ' . ms/dmfs (x ) = . % dmf . ? . - vol. , n o . , summer i table mean ohi-s scores by age and sex males females age (yrs) n ohi-sksd n ohi-s sd ~~ ~ ~~~ < . . . . - . . . . - . k . . -c . - . k . . . + . . . . table mean pi scores by age and sex males females age (yrs) n pi'sd n pi k sd <i . * . . k . - . + . . * . - . k . . f . - . k . . f . + . k . . e . periodontal health. periodontal index scores for this population were consistently low, a s shown in table . periodontal index scores increased with age from a low of . for children under years of age to . for adults over . as with ohi-s scores, males generally had a higher pi than females. of the adults examined for periodontal pockets using a specially marked probe, only two subjects had periodontal pockets. one subject had two mm pockets and the other had two pockets that were more than mm deep. both subjects were over years of age. dental health behavior. almost two-thirds of this amish population reported brushing their teeth less than once a day, while only . percent brushed twice or more a day; . percent reported never having brushed their teeth. knowledge. in general, respondents exhibited inad- equate knowledge of the available preventive methods for dental diseases. an open-ended question on pre- vention of periodontal diseases showed that percent of respondents were unaware of any preventive prac- tices for controlling periodontal disease. to a precoded question on prevention of dental caries, percent agreed that fluoride is helpful in preventing tooth de- cay, while percent did not know the beneficial role of fluoride. almost all who agreed on the cariostatic role of fluoride, however, obtained this information from their dentists. treatment needs. the majority ( %) of the amish in this study perceived themselves as possessing good dental health. only percent believed their teeth were in poor condition. about one-third of the population did not perceive themselves as having good dental health status and felt the need for dental treatment. dental visits. the study population appeared to consist mostly of irregular dental attenders. among the respondents, more than one-third ( %) reported visit- ing a dentist every six months to one year, percent visited every to months, and percent visited less frequently than every two years. in other words, nearly half of the study population was made up of irregular attenders of dental care. financial barriers seemed to be the most important factor for amish not seeking dental care regularly. about percent of the respondents felt dental services were expensive, percent cited pain as a barrier to their being regular attenders, and availability of dental serv- ices was a problem for percent. about one-third ( percent) did not perceive themselves as needing dental care. discussion given the poor oral hygiene status and lack of ade- quate knowledge concerning dental health and prac- tices, a s well a s existing barriers to seeking dental care, it was surprising that levels of dental diseases were so low in the amish population. such low levels of dental diseases could be explained by their dietary habits and possibly herd immunity (collective resistance to the dis- eases displayed by the community). a comparison of dental caries experience between the amish and us children aged ( , ) indicated dmfs scores of amish children to be approximately one-half to one-third of non-amish. amish children in the present study had a dmfs score of . , while non- amish children of similar age groups had dmfs values of . in - and . in - ( ,lz). some of the differences between the amish and non-amish may be explained by the secular decline in dental caries first evident in - and probably continuing during the s. low levels of dental caries found in amish children could be attributed to infrequent snacking habits and consumption of sugar only at meal times. the latter concept is in agreement with the landmark vipeholm study ( ) on the association of sugar and dental caries. amish homes d o not have the usual food products such as prepared, precooked, frozen, and baked products that can be purchased in supermarkets. although these families reported eating three large meals per day in- cluding dessert containing a large amount of sugar, they are not in the habit of constantly snacking between meals, like the general public. analysis of well water revealed a low fluoride level, thus ruling out fluoride as a factor in low levels of dental caries seen in the population. the amish d o not purchase any type of health insur- ance plan, including dental insurance ( ). since their population consists primarily of irregular dental at- tenders, a high d/dmfs ratio, indicating unmet restor- ative needs, was expected in amish children. o n the contrary, the d/dmfs ratio was only percent and the dmfs scores were mainly made u p of missing (m) and filled (f) components. the f/dmfs ratio for - -year- olds in this study was . percent, compared to . i journal of public health dentistry percent in non-amish children from nidr region ( ). factors such as financial barriers, lack of dental insurance, and availability of dental services might have contributed to the difference in the filled surface (fs) component of dmfs between these two groups. the low level of treatment needs for dental caries in the study population should be interpreted cautiously. this low d/dmfs ratio may be d u e to a selection bias in the sample, with the dentally conscious amish more likely to have consented to participate in the study. the high m component could also have contributed to the low d/dmfs ratio. a study on a larger scale is needed before this finding of low d/dmfs ratio can be general- ized to the entire amish population. the relatively high m/dmfs ratio is similar to that of the new zealand population that participated in the international collaborative study ( ). the amish, like new zealanders, are predominantly farmers and face certain barriers to seeking dental care. they may be reluctant to invest their time in traveling, waiting, and receiving treatment. also restorative care would in- volve follow-up visits for completion and replacement of restorations. along with the time factor, financial barriers and a lack of availability of dental services might have contributed to the relatively high m/dmfs ratio. a high m component and edentulousness appear to be the social norm among new zealanders, as seen in the international collaborative study. we are not certain whether the amish possess similar social and cultural values; thus, there is a need for further re- search on sociological and cultural aspects of the amish population. %although these families reported eating three large meals per day, including dessert containing a large amount of sugar, they are not in the habit of constantly snacking between meals, like the general public." ohi-s scores of the amish were higher than those of the non-amish who participated in the nchs survey of - . in addition, caucasians aged ( - ) had . times ( . vs . ) higher mean pi scores than this study population ( ). the high ohi-s and low pi scores suggest that certain features in the oral flora as well as the immune system of the amish may be re- sponsible for their being less susceptible to periodontal diseases. the oral flora of the amish should be a subject of further research. the finding that females in general have better oral hygiene and periodontal health status than males is not unique to this study. the small sam- ple size in the present study warrants caution in gener- alizing to the rest of the amish population the finding of better oral hygiene and periodontal health in the amish females than males. the lack of knowledge and attitudes concerning den- tal health can be attributed to the low level of literacy of the amish. the amish d o not encourage education of their children, who only attend their own schools to the eighth grade. in addition, without electricity and tele- phones, there is relatively little communication with the outside world. the amish have limited contact with the media since they d o not possess radios o r televi- sions. this study showed a high level of unmet need for prosthodontic care in the amish population. nearly one-half of those who participated in the study w e r e reported to be irregular attenders of dental care. all these factors indicate that a n urgent need for dental public health programs-preventive, curative, and ed- ucational-for this population. this study is unique in that there has apparently been no documentation of dental health status in the amish previously. this is d u e primarily to the life-style of the amish, who are reluctant to have contact with groups other than themselves and d o not easily consent to participate in any study. members of the amish community d o not wish to have their life-styles threat- ened by contact with the outside world. this is part of the basis for not having modern conveniences such a s electricity, radio, and tv. access to the population was very difficult and required contact and discussions with the designated bishop of the community. limitations of this study include small numbers of subjects and selection bias, which indicate the sample may not be representative. the conditions under which dental examinations were conducted limited the use of certain dental indices. biases might also have been in- troduced by refusals and "non-contact" prior t o the study as well as during the interviews. the lack of representativeness of the study population is often a limitation in such studies. a representative sample would entail not only a large investment of resources, but also cooperation of subjects, which may not be possible in a detached community such a s the amish. references i. leigh rw. dental pathology of indian tribes o f varied environ- mental and food conditions. am j phys anthropol ;x: l -y . . stewart td. dental caries in peruvian skulls. am j i'hys anthro- . hardwick jl. the incidence and distribution of caries throughout the ages in relation to an englishman's diet. u r dent j ; : - . . mehta fs, shroff bc. aspects of dental diseases in thc indian aborigines. int dent j ; : - . . russell al. world epidemiology and oral health. in: krcshover sj, mcclure j f (eds). environmental variables in oral discase. p u b no . washington, dc: american association for the advance- ment of science, . . schamschula rg, barmes de, keyes ph, gulbinat w. i'revalencc and interrelationship of root caries in lufa, i'apua n e w guinca. community dent oral epidemiol ; : - . . donnelly cj, thomson la, stiles hm, brewer c, n c ~ l j v , brun- elle ja. plaque, caries, periodontal diseases, and acculturation among yanomamo indians, venezuela. community dent oral epidemiol ; : - . . cohen l k . dental care delivery in seven nations. international collaborative study and dental manpower systems in rchtion t o oral health status. in: ingle jj, blair p (eds). international dental care delivery systems. battle creek, mi: wk kellogg foundation, . pol ; : - . vol. , n o . , summer i . todd je, walker am, dodd p. adult dental health. vol . united kingdom . london: hmso . . nordling h, tala h. oral health in finland. helsinki: national board o f health, . . harvey cr, kelley j e (eds), u s public health service, national center for health statistics. statistics: decayed, missing and filled teeth among persons - years, united states - . dhhs pub no (phs) - . washington, dc: government printing office, . . u s department o f health and human services. the prevalence of dental caries in united states children. national dental caries prevalence survey. nih pub no (nih) - . washington, dc: government printing office, . . l(ie h, carlos jp. survey results previewed. j am dent assoc . shlossman m, knowler d, pettitt a, arevalo, genco rj. type i diabetes and periodontal disease [abstract]. j dent res ; (spec iss): . . genco rj, shlossman m, zambon jj. immunologic studies of periodontitis patients with type diabetes mellitus [abstract]. j dent res ; (spec lss): . . hostetler ja. amish society. rd ed. baltimore, md: j o h n s hop- kins university press, . . mckusick va. the amish. endeavor ; : - . ; - . . bagramian ra, khavari am, narcndran s. oral health assess- ment in a selected amish population [abstractl. j dent res ; (spec iss): . . bagramian ra, khavari am, ward m. correlation of dental caries and s mutans in a rural amish population [abstract]. j dent res ; (spec iss): . . gustafsson be, quensel ce, lanke ls, et al. the vipeholm den- tal caries study. the effects o f different levels of carbohydrate intake o n caries activity in individuals observed for five years. acta o d o n t scand ; : - . . takeuchi m. epidemiological study on dental caries in japanese children before, during, and after world war . int dent j . harris r. biology of the children of hopewood house, bowral, australia, . observations on dental caries experience extending over five years ( - ). j dent res ; : x - . . viton a. the outlook for sugar and sugar-containing foods. fo licht's int sugar rep ; : - , . russell al. a system of classification and scoring for prevalence surveys of periodontal disease. j dent res ; : - . . greene jc, vermillion j r . the simplified oral hygiene index. j am dent assoc ; : - . . kelly je, harvey cr. basic data on dental examination findings of persons - years, united states, - . dhew pub no (phs) - , series , no . ~ - . linkage studies suggest a possible locus for bipolar disorder near the velo-cardio-facial syndrome region on chromosome linkage studies suggest a possible locus for bipolar disorder near the velo-cardio-facial syndrome region on chromosome herbert m. lachman, john r. kelsoe, * ronald a. remick, a. dessa sadovnick, mark h. rapaport, margaret lin, beverly a. pazur, anne marie a. roe, takuya saito, and demitri f. papolos department of psychiatry, program of behavioral genetics, albert einstein college of medicine, bronx, new york department of psychiatry, university of california, san diego and department of veterans affairs medical center, san diego, california department of psychiatry, st. paul’s hospital, vancouver, canada department of genetics, university of british columbia, vancouver, canada velo-cardio-facial syndrome (vcfs) is a congenital anomaly characterized by mul- tiple dysmorphisms, cleft palate, cardiac anomalies, and learning disabilities, that re- sults from a microdeletion of chromosome q . an increased prevalence of psychiat- ric illness has been observed, with both schizophrenia and bipolar disorder com- monly being diagnosed. for these reasons, the vcfs region is an interesting candidate region for bipolar disorder. we examined this region in bipolar families from three populations: families from the general north american population (university of california, san diego/university of british columbia, ucsd/ubc), three larger families from new york, and a portion of old order amish pedigree . three microsatellite markers spanning cm around the vcfs region were genotyped in all the families. a maximum lod score of . was obtained in the ucsd/ubc families under a dominant model at d s . in the combined family set, maximum lod scores of . and . were obtained at this marker under domi- nant and recessive models, respectively. four additional markers were subsequently typed in selected positive families, and yielded positive lods at of markers span- ning cm in this region. nonparametric, multipoint analyses using the affected pedi- gree member (apm) method also yielded suggestive evidence for linkage in both the ucsd/ubc family set (p = . ) and in the combined families (p = . ). affected sib- pair analyses were similarly positive in the ucsd/ubc families (p = . ), and in the combined families (p = . ). these results are suggestive of a possible locus for bipolar disorder near the vcfs region on chro- mosome . am. j. med. genet. : – , . © wiley-liss, inc. key words: bipolar disorder; genetic linkage; velo-cardio-facial syndrome; chromosome ; vcfs; manic depressive ill- ness introduction bipolar disorder (manic depressive illness) is a com- mon psychiatric illness characterized by recurrent epi- sodes of mania and depression. twin, family, and adop- tion studies show that the vulnerability to develop bi- polar disorder is inherited [nurnberger et al., ; bertelsen et al., ; nurnberger and gershon, ]. however, despite years of investigation, the exact mode of inheritance is not well-understood. although some family studies have supported the existence of autoso- mal-dominant major loci, a simple mendelian pattern of inheritance is usually not observed [spence et al., ; rice et al., ; sham et al., ; faraone et al., ]. the mode of inheritance in bipolar disorder is an important concern since linkage analysis, which is used to identify the chromosomal position of disease- causing alleles as a prelude to positional cloning, is most successfully exploited for single-gene defects. evi- contract grant sponsor: nimh; contract grant number mh ; contract grant sponsor: ucsd mental health clinical research center; contract grant number mh ; contract grant sponsor: ucsd general clinical research center; contract grant number mo rr . *correspondence to: john r. kelsoe, department of psychiatry , university of california, san diego, la jolla, ca - . received june ; revised september american journal of medical genetics (neuropsychiatric genetics) : – ( ) © wiley-liss, inc. dence for linkage to the short arm of chromosome in an old order amish bipolar pedigree and the x- chromosome in several israeli families was provided in some early linkage studies [egeland et al., ; baron et al., ]. however, these findings have not been replicated, and the strong support for chromosome linkage was substantially diminished following further analysis of the original pedigrees [kelsoe et al., ; berrettini et al., ; gershon, ; baron et al., ]. a recent genome-wide search also failed to pro- vide convincing evidence for linkage to any chromosom- al locus [coon et al., ]. recently, using nonpara- metric methods of statistical analysis [weeks and lange, ; blackwelder and elston, ; risch, ] in conjunction with conventional lod score analy- sis, evidence was obtained for linkage to chromosomes and in subsets of bipolar families [berrettini et al., ; straub et al., ; gurling et al., ; stine et al., ; freimer et al., ]. more recently, evi- dence for linkage has been reported on p , q , q , and p [ginns et al., ; blackwood et al., ]. however, these findings await replication. in the search for genes for complex illnesses such as bipolar disorder, an alternative to a labor-intensive whole-genome survey is to analyze specific candidate genes or loci. although it is difficult to target appropri- ate candidate genes in bipolar disorder, a number of dopaminergic and adrenergic receptor gene loci have been investigated. so far, no evidence for linkage has been reported for these candidate genes [holmes et al., ; mitchell et al., ; de bruyn et al., ]. can- didate loci in bipolar disorder could also be targeted if one could identify clinical entities that are caused by well-characterized chromosomal defects that are also associated with psychiatric illness. for this reason, we investigated chromosome q , which is involved in velo-cardio-facial syndrome (vcfs). vcfs is a phenotypically heterogeneous congenital anomaly, that is usually characterized by cleft palate, typical facial appearance, cardiac abnormalities, and learning disability [shprintzen et al., ]. in addi- tion, approximately two dozen minor clinical features have been described including scoliosis, renal aplasia, t-cell deficiency, and hypocalcemia. fluorescent in situ hybridization (fish) and molecular analysis indicate that vcfs is caused by microdeletions on chromosome q , with a defined minimally deleted region of ap- proximately – megabases [scambler et al., ; lindsay et al., ; morrow et al., ]. a number of behavioral disorders have been identi- fied in patients with vcfs during early childhood, in- cluding separation anxiety disorder, attention-deficit disorder, school phobia, obsessive-compulsive disorder, mood disorders, and paranoid disorder [shprintzen et al., ; chow et al., ; goldberg et al., ]. as these patients reached adolescence, approximately % of them developed psychotic symptoms [shprint- zen et al., ]. in a recent study of patients with vcfs, were given a diagnosis of either schizophrenia or schizoaffective disorder [pulver et al., c]. an additional patients were found to have alcohol abuse, obsessive-compulsive disorder, social phobia, or adjust- ment disorder with depressed mood. in addition, in a study of patients with schizophrenia, were found to have q microdeletions consistent with vcfs [lindsay et al., ]. however, an ongoing evaluation of adolescents with vcfs suggests a pattern of symp- toms that falls more within the bipolar disorder spec- trum [papolos et al., ]. since deletion of a gene on chromosome q ap- pears to be a factor in the development of severe psy- chiatric illness in vcfs patients, some of whom appear to have bipolar disorder, we wanted to determine whether this region could also be responsible for bipo- lar vulnerability in the general population. we there- fore used highly polymorphic dinucleotide repeat markers that flank the vcfs region on chromosome q to determine whether linkage could be estab- lished in multiplex families with bipolar disorder. we here report linkage evidence suggestive of a locus for bipolar disorder in this region. materials and methods family ascertainment and diagnosis families for this study were ascertained from four sites: san diego, vancouver, new york, and pennsyl- vania. the size and clinical characteristics of these families are shown in table i. thirteen of these fami- lies derive from an ongoing collaboration between the university of california, san diego (ucsd) and the university of british columbia (ubc). these families represent a collection of small-to-medium size families from the general population selected for a bipolar pro- band, and at least two other members with bipolar or recurrent unipolar affective disorder. families in san diego were selected from ucsd hospitals and clinics, and from the ucsd mental health clinical research center outpatient clinic. families in vancouver were ascertained from the ubc mood disorders clinic [sa- dovnick et al., ]. family members at these two sites were interviewed using the structured clinical interview for dsm-iii-r (scid) [spitzer et al., ] by clinician interviewers who had undergone the same table i. characteristics of bipolar families ucsd/ubc new york amish number of families total subjects average members per family . . bipolar i/schizoaffective bipolar ii recurrent major depression single-episode major depression other psychiatric diagnosis average affecteds per familya . . unilineal familiesb parent of origin (paternal/maternal/other)c / / / / / / aaffecteds per family is based on a definition of affected which includes bipolar i, bipolar ii, schizoaffective and recurrent major depression. bfamilies are broadly defined as unilineal if at no mating do both parents have diagnoses of bipolar, schizoaffective or recurrent depression disor- ders. cfamilies in which both maternal and paternal transmission is present or in which the parent of origin cannot be established are classified as other. lachman et al. extensive training in the use of the instrument. diag- noses were determined by review of the scid inter- view, information from at least one family informant, and medical records, by a committee of psychiatric cli- nicians who were blind to both genotypes and family membership. diagnostic reliability was monitored both within-site and between sites by annual blind review of videotaped scid interviews, and was consistently high. an exception is family , which was ascertained from the nimh intramural program in bethesda. in this case, diagnoses were made in similar fashion, but interviews were conducted by two of us (j.r.k. and m.h.r.) using the sads-l [endicott and spitzer, ] modified for dsm-iii-r diagnoses. a portion of old order amish pedigree was also included in this study. the advantages of the old or- der amish for study, and the methods used in ascer- tainment and diagnosis, have been reviewed elsewhere [hostetter et al., ; egeland et al., ]. briefly, subjects were interviewed using the sads-l, and re- search diagnostic criteria (rdc) [spitzer et al., ] diagnoses were made by a panel of psychiatric clini- cians blind to genotype or family membership. infor- mation from the interview, from at least one family informant, and from medical records was incorporated in this process. a portion of pedigree was selected as more likely to be unilineal in transmission of affec- tive disorder and was included in this study. three families from new york were selected for high density of affective illness from albert einstein college of medicine hospitals and clinics. all family members were interviewed using a version of the sads-l modi- fied in order to obtain additional information regarding bipolar disorders (sads-lb). the interview data and medical records were reviewed by a panel of two clini- cal research psychiatrists and a senior research social worker who adhered to rdc criteria for developing consensus diagnoses. this diagnostic panel was blind to genotypic information. the ucsd and ubc families derive from similar general north american populations, and were ob- tained through carefully coordinated ascertainment and diagnostic procedures. for these reasons, these families have been treated as one population in our analyses. the three resulting populations (ucsd/ ubc, amish, and new york) are compared in terms of a variety of clinical characteristics in table i. they differ primarily in that the amish family is larger, and that the amish and new york families have more af- fecteds per family. the ucsd/ubc families have more subjects with bipolar ii disorder. the ucsd/ubc and amish families also have a greater ratio of bipolar to unipolar diagnoses than do the new york families. genotyping the region of chromosome surrounding the vcfs deletion site was examined using seven ca repeat mi- crosatellite markers, which have been described in sev- eral recent maps of chromosome [gyapay et al., ; murray et al., ; morrow et al., ]. their order, approximate map distances, and relationship to the vcfs region are illustrated in figure . two of these markers, d s and d s (heterozygosi- ties . and . ), flank an approximately -cm region of chromosome q that contains the vcfs deleted region [morrow et al., ]. the other five markers, d s , d s , d s , d s , and d s (heterozygosities . , . , . , . , and . , re- spectively), span an approximately -cm region flank- ing these two markers. in an initial screen, three of these markers (d s , d s , and d s ) were examined in all families. subsequently, the other four markers were genotyped in selected families in order to confirm positive results from the first screen, or to obtain more linkage information from poorly informative families. high molecular weight dna was extracted from pe- ripheral blood leukocytes using standard techniques. primer pairs were obtained from research genetics (huntsville, al). the forward primers were end- labeled using polynucleotide kinase (boehringer- mannheim, indianapolis, in) and p-atp. a polymer- ase chain reaction (pcr) was carried out in ml using – ng of genomic dna, pmoles of labeled forward primer, pmoles of unlabelled reverse primer, unit of taq polymerase (boehringer-mannheim), mm of each nucleotide, and buffer supplied by manufacturer. cycling conditions included an initial -min denatur- ation step at °c followed by cycles of °c for min, °c for min, and °c for sec, with a final fig. . map of vcfs region on chromosome . marker order and po- sition of vcfs deletion region are derived from physical mapping data from overlapping yeast artificial chromosomes (yacs) [morrow et al., ]. recombination fractions, indicated at left, derive from an integra- tion of several linkage maps [gyapay et al., ; murray et al., ], and must be considered an approximation. chromosome locus for bipolar disorder -min extension. denaturing buffer ( . ml) contain- ing % formamide, mm edta, ph . , and . % each of bromophenol blue and xylene cyanol was added to the reaction and, following heat denaturation, a – - ml aliquot was loaded onto either a % or % acryl- amide gel containing m urea. to ensure quality con- trol, a set of common dna samples was used for each gel. in addition, several samples were included from gels that had been previously analyzed. the samples were electrophoresed at watts for – . hr. follow- ing electrophoresis, the gels were dried and exposed to x-ray film for a period of – hr. autoradiograms were analyzed blind to diagnosis. linkage analyses the power of the pedigree collection was estimated using the program simlink [ploughman and boehnke, ]. pedigree data were simulated under an assump- tion of linkage between the disease and a biallelic marker whose true recombination fraction was either . or . . fifty percent heterogeneity was also as- sumed in the simulation. the simulated data were then analyzed under an assumption of heterogeneity at a variety of recombination fractions in order to obtain the estimated mean maximum lod score. parametric linkage analyses were conducted using the mlink program from the linkage . program package [lathrop et al., ]. analyses employed a definition of ‘‘affected’’ that included bipolar i, bipolar ii, schizoaffective, and recurrent major depression di- agnoses. one subject had a diagnosis of schizophrenia and was considered to have an unknown affection sta- tus. subjects with other psychiatric diagnoses were also considered unknown. three models of genetic transmission were tested as shown in table ii. in the table, model is a high-penetrance dominant model derived from a segregation analysis in the old order amish, and was used in previous studies of this popu- lation [egeland et al., ; kelsoe et al., ]. models and are moderate-penetrance dominant and reces- sive models, respectively. age-dependent penetrance was modeled using five liability classes which in- creased linearly from age to a maximum at age . allele frequencies were obtained from the genome da- tabase (johns hopkins university) or from published reports of these markers [gyapay et al., ; buetow et al., ], except for d s , where they were es- timated from control chromosomes, and d s , where allele frequencies were assumed equal. the homog program was used for analyses under an as- sumption of heterogeneity [ott, ]. nonparametric analyses were performed using an identity-by-state approach, the affected pedigree mem- ber method (apm) [weeks and lange, ], and an identity-by-descent approach, i.e., the affected sib-pair method. both analyses employed the same diagnostic model as the lod score analyses. for the apm analyses, the f(p) /sqr weighting function for allele fre- quency was used because it has been shown to have the greatest statistical power, and it is recommended by the designers of the test. empirical p values were es- timated by simulation using the sim, simmult, and hist programs from the apm package. a multipoint apm analysis incorporated the three markers d s , d s , and d s , using map dis- tances as indicated in figure . affected sib-pair analy- ses were conducted using the sibpal program [s.a.g.e., ]. in these analyses, only results from affected sib pairs were considered, and sample size was corrected for nonindependence of multiple pairs de- rived from the same sibship. pedigrees and genotypic data will be provided to in- terested investigators upon request. results power analyses of the family collections were first performed by simulation in order to determine the ap- proximate lod scores likely to be obtained if a marker was linked to bipolar disorder. the results of these simulation studies are summarized in table iii for the combined families, and for the ucsd/ubc families where the highest actual lod scores were obtained. only under the recessive model at u . was the estimated maximum lod > . for the combined fami- lies. under the dominant models, estimated mean maximum lods under linkage and % heterogeneity ranged from . – . for the combined families, and from . – . for the ucsd/ubc families. table iv summarizes the lod scores for each popula- tion and for the combined family set for the three mark- ers used in the initial screen. a maximum lod score of . was obtained at d s in the ucsd/ubc fami- lies under model . lod scores of . and . were also obtained at this marker in this population under models and , respectively. in the amish family, a maximum lod of . was obtained at d s under model . at d s in the combined families, lod scores of . and . were obtained under models and , respectively. for both the ucsd/ubc families and the combined families, these lod scores are compa- rable to or exceed those expected from the simulation studies under model . the maximum lod score for d s was . in the ucsd/ubc families under table ii. models for linkage analysis* model q mode f f f . dominant . . . . dominant . . . . recessive . . . *q frequency of disease gene; f penetrance of aa, where a is the disease allel; f penetrance of aa; f penetrance of aa. table iii. statistical power of the family collection population model estimated mean maximum lod score under linkage u . u . ucsd/ubc . . . . . . combined . . . . . . lachman et al. model . no notable positive lods were obtained at d s in any of the populations, nor in the new york families at any of the three markers. analyses under heterogeneity using homog yielded results similar to those under homogeneity. under model at d s , maximum lod scores under heterogeneity were . in the ucsd/ubc families, and . in the combined families. no significant evidence of heteroge- neity was detected. examination of lods in individual families indicates that positive lods are found in multiple families and at multiple markers. this argues against the results be- ing an isolated chance positive, and lends further sup- port to the possibility of real linkage. as seen in table v, positive lods at d s are found primarily in the ucsd/ubc families , and , and in amish pedigree (family ). the other two markers included in the primary screen, d s and d s , also yielded slightly positive lods ranging from . – . in the three ucsd/ubc families , , and . families with positive lods were examined in more detail using the four markers in the secondary screen. positive lods were detected with three of these markers in the three ucsd/ubc families , and . the highest indi- vidual lods obtained were . at d s in family , and . at d s in family . the expected lod scores for each of the family-model combinations as de- termined in the simulation studies are also presented in table v. in each case, except for the amish family, these positive lod scores exceed those expected under linkage. it is of interest that positive lods were ob- table iv. lod scores for chromosome markers* locus population model theta . . . . . . . d s ucsd/ubc − . − . − . . . . . . . . . . . . − . − . − . − . − . − . . amish − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . . . . . new york − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . total − . − . − . − . − . − . − . − . − . − . − . − . . . − . − . − . − . − . − . − . d s ucsd/ubc − . − . − . − . − . . . − . − . − . − . . . . − . − . − . − . − . − . − . amish − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . . . . . new york − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . total − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . − . d s ucsd/ubc . . . . . . . . . . . . . . . . . . . . . amish − . − . − . − . − . − . − . − . − . − . − . − . − . − . . . . . . . . new york − . − . − . − . − . − . − . − . − . − . − . − . − . − . . . . . . . . total . . . . . . . . . . . . . . . . . . . . . *maximum lod scores > . are in boldface. table v. positive lod scores in individual families locus familya model theta lod expected lod (simulated)b d s . . d s . . d s . . d s . . d s . . d s . . d s . . d s . . d s . . d s . . d s . . d s . . afamilies , , and are from the ucsd/ubc set; family is from the new york set; and family is old order amish pedigree . bestimated mean maximum lod score for the given model and recombina- tion distance, as determined by simulation using simlink. chromosome locus for bipolar disorder tained under both dominant and recessive models for different family-marker combinations. this would be consistent with allelic heterogeneity at a disease locus. overall, positive lods were obtained with markers spanning the entire -cm interval examined, and the two lods over . came from markers separated by cm and flanking the vcfs region. these results were further explored using the non- parametric affected pedigree member (apm) method, as shown in table vi. the results were consistent with those obtained in the lod score analyses. in the com- bined families, only d s yielded suggestive evi- dence of linkage in single-locus analyses (p . ). a multipoint apm analysis incorporating all three mark- ers in the primary screen yielded results similarly sug- gestive of linkage (p . ). stronger evidence was seen in the ucsd/ubc families alone, with suggestive evidence of linkage obtained at both d s (p . ) and d s (p . ). the multipoint analy- sis in this population yielded the strongest results for this test (p . ). similar results were obtained using the affected sib- pair method as summarized in table vii. results sug- gestive of linkage were obtained at d s in the combined family set (p . ), as well as in the ucsd/ubc families (p . ) and the amish family (p . ) examined separately. no positive evidence was obtained at the other two markers from the initial screen. since this region of chromosome has also been implicated in linkage studies of schizophrenia, we con- sidered the hypothesis that the disease susceptibility locus might predispose more generally to psychosis, rather than specifically to bipolar disorder. to test this hypothesis, the lod score analyses were repeated, con- sidering as affected only those subjects who manifested marked psychotic symptoms. only three families were informative for linkage under this definition of affec- tion status (families , , and ). the maximum lod score obtained was . for family , at d s under model . therefore, only very limited support for this hypothesis could be obtained. rather, the sugges- tive evidence for linkage to bipolar disorder comes al- most exclusively from nonpsychotic subjects. discussion these data are consistent with the hypothesis that a bipolar susceptibility gene may exist on chromosome q in a subset of affected families. however, none of these results reach the conventional level for statistical significance of a lod score of . or , : odds in favor of linkage. for this reason, conclusions regarding these data must be qualified as only suggestive of linkage. the finding of positive lods in multiple families at mul- tiple markers in this region does, to some extent, argue against this being an isolated false-positive. further- more, some have argued that in examining a candidate gene with a specific a priori hypothesis, a less stringent lod score criterion is indicated. ott, [ ] however, argues that given the history of candidate gene studies, and the fact that they are usually conducted as part of a genome survey, the conventional level of . should be retained. in either case, given the complexities and uncertainties of the mode of transmission of bipolar disorder, the meaning of linkage results of this magni- tude is unclear. nevertheless, these results are interesting and war- rant further investigation in other collections of bipolar families. published reports to date in which chromo- some q markers have been examined in bipolar fami- lies indicate no evidence for linkage [coon et al., ; detera-wadleigh et al., ]. however, the markers used in these studies did not adequately evaluate the vcfs deleted region. further studies of bipolar fami- lies using microsatellite markers in this region are re- quired. these results are also intriguing in the light of recent reports of possible linkage of schizophrenia to markers at q . – q [pulver et al., b; las- seter et al., ]. though these results have not been uniformly replicated, the possibility of a common sus- ceptibility locus for psychosis should not be completely excluded [pulver et al., a; polymeropoulos et al., ; coon et al., ]. although this region of chromosome was analyzed in the context of vcfs, the marker that consistently yielded the maximal lod scores was d s , which is approximately cm telomeric to the vcfs deleted re- gion. in general, negative lod scores were obtained for markers d s and d s , which flank the com- monly deleted region. this could suggest that chromo- some q has two bipolar susceptibility loci, one in the vcfs deleted region, which is involved in the psy- chiatric manifestations of vcfs, and another near d s . consistent with this view are recent findings regarding one candidate gene for the psychiatric mani- festations of vcfs, catechol-o-methyltransferase (comt). comt is one of the major enzymes respon- sible for catecholamine inactivation [axelrod and tom- table vi. affected pedigree member analyses population locus statistica empirical p valueb combined d s . . d s . . d s . . multipointc . . ucsd/ubc d s . . d s . . d s . . multipointc . . amish not significant new york not significant athese apm statistics were calculated using the /sqr function for weighting of allele frequency, which yields an intermediate weighting fac- tor and is recommended by the authors of the analysis program. bempirical p values were calculated by simulation using , replicates and the programs sim, simmult, and hist. cmultipoint apm incorporates the above three markers and map distances, as indicated in figure . table vii. affected sib-pair analyses at d s population no. pairs proportion of alleles i.b.d. p ucsd/ubc . . amish . . new york not significant combined . . lachman et al. chick, ], and is deleted in most, if not all, patients with vcfs [winqvist et al., ; grossman et al., ; scambler et al., ]. we recently characterized a low-activity variant of comt that is associated with bipolar spectrum disorders occurring in vcfs, espe- cially rapid-cycling bipolar disorder [lachman et al., a,b]. however, recent findings from our laboratory suggest that the low-activity variant does not exert a major gene effect in bipolar disorder occurring in the general population [lachman et al., ]. one inter- esting candidate gene that maps close to d s is the g-protein alpha subunit, gz, which is a member of a gene family considered to be a feasible candidate for bipolar disorder [lachman and papolos, ; schreiber and avissar, ; blatt et al., ]. in summary, we examined the vcfs region in bipolar families using seven microsatellite markers, and found results suggestive, but not conclusive, of linkage. in a genetically heterogeneous condition, con- firmation of linkage and the extraction of informative mapping data may require an analysis of hundreds of families. acknowledgments h.m.l. is supported by the carmel hill fund and is a recipient of the stanley foundation research award from the national alliance for the mentally ill. d.f.p. is a recipient of an nimh physician scientist career development award. this work has also been sup- ported in part by the department of veterans affairs, the nimh (grant mh ), the ucsd mental health clinical research center (grant mh ), and the macarthur foundation (ucsd and ubc). portions of this work were also supported by the ucsd general clinical research center (grant m rr ). some of the analyses were performed using the program package s.a.g.e. (grant p rr ). the authors thank drs. raju kucherlapati and bernice morrow for mapping data from the vcfs region and for access to unpublished markers in this region. we also acknowl- edge the generous assistance of dr. janice egeland in providing clinical information regarding the amish sample, and assistance in the diagnostic assessment of one of the new york families. jean endicott and joh- nine cummings also provided assistance in assessing one of the new york families. we thank dr. j. chris- tian gillin for the support of the ucsd mental health clinical research center, and drs. maureen morison and arvin mirow for assistance with ascertainment and diagnosis of the ucsd families. references axelrod j, tomchick r ( ): enzymatic o-methylation of epinephrine and other catechols. j biol chem : – . baron m, risch n, hamburger r, mandel b, kushner s, newman m, drumer d, belmaker rh ( ): genetic linkage between x- chromosome markers and bipolar affective illness. nature : – . baron m, freimer nb, risch n, lerer b, alexander jr, straub re, asokan s, das k, peterson a, amos j, endicott j, ott j, gilliam tc ( ): diminished support for linkage between manic depressive illness and x-chromosome markers in three israeli pedigrees. nat genet : – . berrettini wh, goldin lr, gelernter j, gejman pv, gershon es, detera- wadleigh s ( ): x-chromosome markers and manic-depressive ill- ness. rejection of linkage to xq in nine bipolar pedigrees. arch gen psychiatry : – . berrettini wh, ferraro tn, goldin lr, weeks de, detera-wadleigh s, nurnberger ji, gershon es ( ): chromosome dna markers and manic-depressive illness: evidence for a susceptibility gene. proc natl acad sci usa : – . bertelsen a, harvald b, hauge m ( ): a danish twin study of manic- depressive disorders. br j psychiatry : – . blackwelder wc, elston rc ( ): a comparison of sib-pair linkage tests for disease susceptibility loci. genet epidemiol : – . blackwood dhr, he l, morris sw, mclean a, whitton c, thomson m, walker mt, woodburn k, sharp cm, wright af, shibasaki y, st. clair dm, porteous dj, muir wj ( ): a locus for bipolar affective disorder on chromosome p. nat genet : – . blatt c, eversole-cire p, cohn vh, et al. 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// // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ j clin exp dent. ; ( ):e - . amish children’s oral health e journal section: community and preventive dentistry publication types: research oral health and medical conditions among amish children masahiro heima , marc-allen harrison , peter milgrom dds, phd, assistant professor, department of pediatric dentistry, school of dental medicine, case western reserve university bs, dmd, department of pediatric dentistry, school of dental medicine, case western reserve university dds, professor, northwest center to reduce oral health disparities, university of washington correspondence: department of pediatric dentistry school of dental medicine case western reserve university euclid ave. cleveland, oh - usa masahiro.heima@case.edu received: / / accepted: / / abstract background: the amish are a growing population who live a traditional, rural way of life, which makes them less accepting of modernism. most amish live in poverty and are detached from modern health care. in addition, the recent change of their lifestyle has been reported, such as consuming a nontraditional diet and the usage of electronic devices. as a result, their lifestyle change may have impacted their oral health. however, since only a single report about oral health among amish children has been published approximately three decades ago it has not yet been updated. this study describes oral health among amish children and their medical conditions during visits to a mobile dental unit (mdu). material and methods: the dental records of all patients (n= ) who visited a mobile dental unit were reviewed, which covers year from may , , the first date of service. the following factors were taken into conside- ration during the review process: parental perceptions of their children’s oral health care, dental care experiences, and general health information. results: fifty-four ( . %) children, ages to , have never received dental treatment before visiting the mdu; the average number of untreated decayed teeth was . . in spite of this, most parents rated their children’s oral health as good or very good ( . %). the high cost and long distance travel associated with routine, professio- nal dental care makes it difficult for children to maintain good oral hygiene. our analysis revealed that bleeding disorders were more prevalent among this gene pool compared to the nation at large; however, asthma was less common. conclusions: there are oral and general health disparities among amish children. there is a lack of awareness among amish parents with regard to their children’s oral health. key words: amish, child, dental caries, mobile health units. doi: . /jced. http://dx.doi.org/ . /jced. article number: http://www.medicinaoral.com/odo/indice.htm © medicina oral s. l. c.i.f. b - eissn: - email: jced@jced.es indexed in: pubmed pubmed central® (pmc) scopus doi® system heima m, harrison ma, milgrom p. oral health and medical conditions among amish children. j clin exp dent. ; ( ):e - . http://www.medicinaoral.com/odo/volumenes/v i /jcedv i p .pdf j clin exp dent. ; ( ):e - . amish children’s oral health e introduction the amish are a growing group of protestant christians, and they are distinct from non-amish by the simplicity of their lifestyle and detachment from the conveniences of modern technology, such as electricity and motori- zed machines ( ). although some amish have begun to use modern medical therapy, others still strongly prefer alternative medicine, traditional remedies, homeopathic and natural treatments due to their religious practices and beliefs ( - ). for this reason, immunization rates among amish children were low; % of parents indicated that their children have received at least one immunization; % of parents completed the vaccination exemption form for their children. a contributing factor to the low immunization rate among children is the fear that parents have with regard to the adverse effects of the vaccines, re- gardless of health care accessibility ( ). another essential factor affecting amish health is marriage, which traditio- nally occurs only between members within their commu- nity. consequently, many cases of rare genetic disorders, such as cohen syndrome, ellis-van creveld syndrome, have also been reported in the amish communities ( ). today, the amish have adopted many aspects of modern society: adults often work outside of their communities, and shop at stores near their place of work. as a result, the amish currently consume a more non-traditional diet ( - ). although, societal and parental influences directly impact children’s oral health ( ), current oral health of amish children is poorly understood. a single study in clinically examined children under years of age residing in one amish community in michigan ( ). although no radiographs were taken and convenience sampling was employed, the author con- cluded that amish children had a surprisingly low dental caries prevalence in spite of inadequate oral health care knowledge. the report attributed the low rate of sugar diet. however, this circumstance might have changed and there is no updated report based on our knowledge . despite a growing number of amish families using modern health care today, health disparities still exist, mainly due to their religious customs and beliefs. at the request of this amish community, our pediatric dentistry team operated a dental mobile unit (dmu) for year starting in may . four days per month the dmu served the amish community of geauga coun- ty in ohio. our patient sample was primarily attained through local advertisements, newspapers and referrals from a medical clinic. this report explains the oral and general health concerns among the amish children who participated in the mo- bile dental unit. material and methods -study design: retrospective chart review study. all charts from the first date of service, may , , through may , , from the geauga county site were reviewed. the state of ohio is the most populated amish location in the united of states and geauga county is the second most amish populated county in ohio ( ). in geauga county, in the year , the ratio of dentist to popula- tion was : ; it was lower than the national weighted average which has a ratio of : ( ). according to the united states census bureau, there were , people, age to and births in geauga county. the per capita income of geauga coun- ty in was $ , , and . % of children under age was living in poverty ( ). -data elements: the oral and general health record infor- mation for each child was reviewed, which included infor- mation on each child’s parental education level, insurance, and home address. parents completed a -item question- naire addressing the following: “how long has it been sin- ce your child last visited the dentist? (within the past year, - years ago, more than years ago, never have been, i don’t know)”; “during the past months, was there a time when your child needed dental care, but could not get it at that time?” ; “what was the main reason the child couldn’t get care? (could not afford, dentist did not accept medicaid/ insurance, difficulty in getting the appointment, no way to get there, other reason, i don’t know)”; “please describe the condition of your child’s teeth (very good, good, poor)”; “during the past months did your child have a toothache? (yes, no, don’t know)? ” the flesch-kincaid grade rea- ding level of the questions was . . standardized dental caries assessments, including tac- tile-visual examination and bite-wing radiographs were completed by pediatric dentists or residents, and recor- ded at the surface level on a tooth chart. outcomes were abstracted from the tooth chart: deca- yed primary teeth (dt), primary teeth with either decay or filling (dft), decayed permanent teeth (dt), and deca- yed, missing, or filled (dmft) permanent teeth. we also abstracted general health information, which was above category of the american society of anesthesiologists physical status classification system, which include “pa- tients with mild systemic disease”, like asthma ( ). statistical analysis: descriptive analysis was performed for amish children and their parents in the areas of ge- neral health, dental accessibility and dental care. the amish distance to travel from home to dental treatment was tracked using google maps (google inc., califor- nia, usa.) outcomes were grouped by children’s ages: - , - , - , and ≥ years old. tests regarding the differences of parents’ perception and the actual den- tal caries care index (dt + dt) were analyzed by using the analysis of variance (anova) tests. all statistical analyses were conducted by ibm spss statistics . the institutional review board of our institute appro- ved the research protocol. j clin exp dent. ; ( ):e - . amish children’s oral health e results -characteristics of patients and parents: we reviewed charts; six children’s charts were excluded from analyses because they were incomplete. the questionnai- res were mainly completed by amish mothers ( . %). most parents ( / ) did not complete high school; parents did not answer this question. the mean (sd) age of the children was . ( . ) years with a range from to ; . % were male. . % ( ) were abo- ve the category physical status (medically compromi- sed at least “mild systemic disease”) ( ), including ( . %) with bleeding problems, and ( . %) had asthma. table shows the distribution of general health, which the dentists encountered. n= frequency percent healthy . general health problems * . asthma . hemophilia or bleeding problems . allergy to medications . heart problems . gastroesophageal reflux disease . other health problems . epilepsy . mental illness (including: adhd, add) . diabetes . table . general health problems reported by the parents of amish children. *: children have single problem and children had multiple problems. -oral health care service accessibility: only . % ( ) children visited a dentist in the past year. about one- quarter ( / , . %) had never seen a dentist (mean (sd) age = . ( . ) years). the average (sd) travel dis- tance was . ( . ) km; % traveled more than . km. most were uninsured ( . %) and were not enrolled in medicaid, which is a governmental social health care program for families and individuals with limited re- sources, even though it was available to them. almost a third ( / , . %) reported they could not get dental care for their children when they needed it in the past months; among those, the most frequent reason was “i could not afford the dental visit” ( / , . %). -oral health: table shows the dental health of amish children. only children were caries free. the typical amish child had . untreated decayed primary or per- manent teeth and . % of the parents ( / ) reported the child had a toothache in the last months. across age categories more than % of children have untrea- ted decayed teeth. nevertheless, most parents rated their children’s health as good or very good ( / , . %). our clinical observation is that parents were not very concerned about their child’s teeth. we have provided many permanent tooth extractions and dentures for teen patients. an amish girl told us “my mom and my sisters had a denture when they were teenagers. many amish lost their teeth when they were young.” -parental perception of their children’s oral health and the number of untreated dental caries: the analysis of variance (anova) was used to test the differences bet- ween children’s number of untreated decayed teeth and parental perception of their children’s oral health: very good (n= )”, “good (n= )”, and “poor (n= )”. the mean (sd) of children’s number of untreated deca- yed teeth were . ( . ), . ( . ) and . ( . ) respectively. it indicated a significantly different number of decayed teeth among the three parental perceptions (f= . , p= . ) and tukey hsd post-hoc multiple comparisons indicated “very good” group has signifi- cantly less caries than others (vs. “good” p= . , vs. “poor” p= . ). however, the number of decayed teeth between “good” and “poor” groups were very similar (p= . ). discussion although we gathered our data from patient charts, we found amish children had high levels of untreated tooth decay. it was consistent with the disparities of children’s oral health seen in other rural communities in the u.s. these families must travel long distances by non-auto- mobile transportation to seek care, and care is often not readily available because of their financial difficulties. families use well water to drink and do not access fluo- ridated water. although the recruitment of participants is different between convenient population and patients of the mobile dental unit, these clinical findings seemed j clin exp dent. ; ( ):e - . amish children’s oral health e age dt dft dt dmft - years old total n= valid n mean . . sd . . minimum maximum % children caries free: . % ( ) % children with untreated decay in primary teeth: . % ( ) - years old total n= valid n mean . . . . sd . . . . minimum maximum % children caries free: . % ( ) % children with untreated decay in primary teeth: . % ( ) % child with untreated decay in permanent teeth: . % ( ) - years old total n= valid n mean . . . . sd . . . . minimum maximum % children caries free: % ( ) % children with untreated decay in primary teeth: . % ( ) % child with untreated decay in permanent teeth: . % ( ) ≥ years old total n= valid n mean . . sd . . minimum maximum % children caries free: % ( ) % child with untreated decay in permanent teeth: % ( ) table . oral health of amish children in geauga county, ohio seen by the mobile clinic (n= ). in stark contrast to the report from a similar amish community ( ). there are some possible reasons poor oral health exists among amish patients: amish are fatalistic ( ) and do not view preventive care as a priority ( ), which is con- sistent with reports that many amish children are not vaccinated ( ). further, amish are willing to suffer from their diseases and lean on the providence of god, and they believe god is the ultimate healer. amish cultural norms eschew accepting governmental health supports ( ), and as a result they are left with the option to pay expensive professional dental care fees out-of-pocket. furthermore, we could not see any association between dental accessibility (travel distance) and the prevalence of amish children’s dental caries in preliminary analy- ses. however, it is the fact that the families have to travel long distances for care. at last, parental education level and awareness levels of their child’s oral condition are important factors in children’s dental caries prevalen- ce ( ), however, we found many amish parents were unaware of the actual state of their teeth. we are not able to conclude that the high prevalence of dental caries in amish children was caused by the life changes, poverty, failure to seek preventive care, chan- ging diet, or selection bias. however, compared with the u.s. population as a whole, the caries (dft) prevalence j clin exp dent. ; ( ):e - . amish children’s oral health e for to year olds was fold higher ( . % in amish vs. . % in national data ( )) and similar to rates pu- blished for alaska native or american indian children who live in non-fluoridated isolated villages, which are similar to the amish community; the reported untreated dental caries prevalence among alaska native children with decayed primary teeth (dt) is % among to year olds. the rate of untreated teeth among older children was % among - year olds, % of children among - year olds (dt), % among - year olds (dt), and % among - year olds (dt) ( ). another re- port also showed . - . % of - year old alaska na- tive or american indian children had tooth decay ( ). thus, the general magnitude of the rates is plausible. we encountered more medically compromised amish children at the mobile dental unit site for the amish than usual. many amish children suffer from conditions from gene abnormalities ( ). the rate of “bleeding problems,” such a von willebrand disease and hemophilia b, were fold higher ( . % in amish vs. % in national data) ( , ). dentists who care for amish children may be the first health care provider to recognize a bleeding di- sorder. on the other hand, physicians who provide care of bleeding disorder might recognize poor oral health in their amish patients. in contrast, the prevalence of asth- ma ( %) was lower than the national rate ( national health interview survey data, . %), perhaps reflec- ting fewer environmental challenges ( ). this report provides a rare snapshot of concerns regarding oral health in a single amish community: ) there are nota- ble disparities in dental access and oral health, and ) high rates of major health problems among amish children who visited the mobile dental unit. the next step in this study is to conduct a population based research in of amish oral health problems to eliminate selection bias so that we can understand the dynamics of their oral health care. references . young center for anabaptist and pietist studies ec. amish po- pulation trends - , -year highlights elizabethtown [available from: http://www .etown.edu/amishstudies/population_ trends_ _ .asp. accessed: - - . (archived by webci- te® at http://www.webcitation.org/ ycgokaqi) . kolacz nm, jaroch mt, bear ml, hess rf. the effect of burns & wounds (b&w)/burdock leaf therapy on burn-injured amish patients: a pilot study measuring pain levels, infection rates, and healing times. journal of holistic nursing: official journal of the american holistic nurses’ association. ; : - . . medina-marino a, reynolds d, finley c, hays s, jones j, soyemi k. communication and mass vaccination strategies after pertussis out- break in rural amish communities-illinois, - . j rural health. ; : - . . gillum dr, staffileno ba, schwartz ks, coke l, fogg l, rei- ling d. cardiovascular disease in the amish: an exploratory study of knowledge, beliefs, and health care practices. holistic nursing practi- ce. ; : - . . von gruenigen ve, showalter al, gil km, frasure he, hopkins mp, jenison el. complementary and alternative medicine use in the amish. complementary therapies in medicine. ; : - . . wenger ok, mcmanus md, bower jr, langkamp dl. underim- munization in ohio’s amish: parental fears are a greater obstacle than access to care. pediatrics. ; : - . . strauss ka, puffenberger eg. genetics, medicine, and the plain people. annu rev genomics hum genet. ; : - . . stoltzfus v. amish agriculture: adaptive strategies for economic survival of community life. rural sociology. ; : . . hill jo, wyatt hr, peters jc. energy balance and obesity. circula- tion. ; : - . . hostetler j. folk and scientific medicine in amish society. human organization. ; : - . . fisher-owens sa, gansky sa, platt lj, weintraub ja, soobader mj, bramlett md, et al. influences on children’s oral health: a concep- tual model. pediatrics. ; :e - . . bagramian ra, narendran s, khavari am. oral health status, knowledge, and practices in an amish population. j public health dent. ; : - . . ohio department of health. geauga county [available from: http://publicapps.odh.ohio.gov/oralhealth/reportsdisplay.aspx? report=bohsreport&format=pdf&countyname=geauga&repor tversion= . (archived by webcite® at http://www.webcitation. org/ z rm ni ). . geauga county. geauga county profile statistical and de- mographic data. in: services dojaf, editor. geauga county: geauga county; . p. . . american society of anesthesiologists. american society of anaesthesiologists physical status classification system. . [avai- lable from https://www.asahq.org/resources/clinical-information/asa- physical-status-classification-system. accessed: - - . (archi- ved by webcite® at http://www.webcitation.org/ klz bbeo)]. . adams z. health survey of the lancaster county plain commu- nities and propensity score matching for asthma in amish women . . clay r. culture clash hemaware: national hemophilia founda- tion; [available from: http://www.hemaware.org/story/culture- clash . accessed: - - . (archived by webcite® at http://www. webcitation.org/ ycisc yp). . feldens ca, rosing ck, dos santos bz, cordeiro mm. pattern of fluoride-containing dentifrice use and associated factors in preschool children from ijui, south brazil. oral health prev dent. ; : - . . national institute of dental and craniofacial research, national institutes of health. dental caries (tooth decay) in children (age to ): national institute of dental and craniofacial research; [available from: http://www.nidcr.nih.gov/datastatistics/finddatab- ytopic/dentalcaries/dentalcarieschildren to .htm#table . (archi- ved by webcite® at http://www.webcitation.org/ yg ycla ). . klejka j, swanzy m, whistler b, jones c, bruce mg, hennessy tw, et al. dental caries in rural alaska native children--alaska, [ ]. available from: url:http://www.cdc.gov/mmwr/pre- view/mmwrhtml/mm a .htm. accessed: - - . (archived by webcite® at http://www.webcitation.org/ yeayta q). . phipps kr, ricks tl, manz mc, blahut p. prevalence and severity of dental caries among american indian and alaska native preschool children. j public health dent. ; : - . . sharathkumar a, hardesty b, greist a, salter j, kerlin b, heiman m, et al. variability in bleeding phenotype in amish carriers of hae- mophilia b with the c-->t mutation. haemophilia. ; : - . . crookston k, rosenbaum l, gober-wilcox j. coagulation, he- reditary bleeding disorders, hereditary bleeding disorders - general. bingham farms, mi: pathologyoutlines.com, inc.; [available from: url:http://www.pathologyoutlines.com/topic/coagulationhe- reditarybleedinggeneral.html. accessed: - - . (archived by webcite® at http://www.webcitation.org/ z ela ya). . centers for disease control and prevention. lifetime asthma pre- valence percents by age, united states: national health interview survey, [cited march , ]. available from: http:// www.cdc.gov/asthma/nhis/ /table - .htm. (archived by webci- te® at http://www.webcitation.org/ z mpd b). j clin exp dent. ; ( ):e - . amish children’s oral health e acknowledgments this mobile dental unit was supported by ronald mcdonald house charities of northeastern ohio and university hospitals rainbow ba- bies & children’s hospital.this study was supported by the summer research program of the school of dental medicine at case western reserve university and clinical and translational science award (ctsc) - ul tr . the authors thank dr. lawrence p. greksa, a human population biologist expert in the field of old order amish, for his valuable input in this study. conflict of interest all authors declare no conflicts of interest with respect to the authors- hip and publication of this article. short report disruption of the cntnap gene in a t( ; ) translocation family without symptoms of gilles de la tourette syndrome jose m belloso , , , iben bache , miriam guitart , maria rosa caballin , christina halgren , maria kirchhoff , hans-hilger ropers , niels tommerup and zeynep tümer*, department of cellular and molecular medicine, wilhelm johannsen centre for functional genome research, the panum institute, university of copenhagen, copenhagen, denmark; laboratori de genètica, udiat-centre diagnòstic, hospital de sabadell, corporació sanitària parc taulı́, fundació parc taulı́ institut universitari uab, sabadell, spain; unitat d’antropologia, universitat autònoma de barcelona, cerdanyola, spain; department of clinical genetics, rigshospitalet, copenhagen, denmark; max-planck institute for molecular genetics, berlin, germany caspr is a member of neurexin superfamily, members of which are transmembrane proteins that mediate cellular interactions in the nervous system. recently, truncation of the cntnap gene coding for the caspr protein has been suggested to be associated with the gilles de la tourette syndrome, a neurological disorder characterized by motor and vocal tics, and behavioral anomalies. in this study, we describe a familial balanced reciprocal translocation t( ; )(q ;q . ) in phenotypically normal individuals. the q breakpoint disrupts the cntnap gene, indicating that truncation of this gene does not necessarily lead to the symptoms of the complex gilles de la tourette syndrome. european journal of human genetics ( ) , – . doi: . /sj.ejhg. ; published online march keywords: caspr ; chromosome ; cntnap ; cortical dysplasia-focal epilepsy syndrome; gilles de la tourette syndrome; translocation introduction cntnap encodes contactin-associated protein-like (caspr ), which is localized to the juxtaparanodal regions at the node of ranvier of myelinated axons. caspr is colocalized and physically associated with shaker-type voltage-activated kþ channels (kv ). studies with caspr -null mice suggest that caspr is necessary for localization and/or maintenance of kv channels. the cntnap gene resides at q -q . and consists of exons, covering an approximately . -mb genomic region (figure a). representing . % of chromosome , it is one of the largest genes in the human genome. mutations in cntnap have been associated with two distinct neurological disorders: cortical dysplasia-focal epilepsy syndrome (cdfe, omim ) and gilles de la tourette syndrome (gts, omim ). cdfe is characterized by cortical dysplasia, focal epilepsy, relative macrocephaly, and diminished deep-tendon reflexes. homozygous frame-shift mutations of cntnap have been detected in cdfe patients in the old order amish community. heterozygotes did not present symptoms, suggesting a recessive condition. gts is a chronic received november ; revised february ; accepted february ; published online march *correspondence: dr z tümer, department of cellular and molecular medicine, faculty of health sciences, wilhelm johannsen center for functional genome research, university of copenhagen, blegdamsvej , kbh n, denmark. tel.: þ ; fax: þ ; e-mail: zeynep@imbg.ku.dk european journal of human genetics ( ) , – & nature publishing group all rights reserved - / $ . www.nature.com/ejhg neuropsychiatric disorder characterized by intermittent and involuntary motor and vocal tics beginning in child- hood. this phenotype is associated frequently with behavioral, emotional and cognitive problems that are often more incapacitating than the tics. gts is a complex disorder with a strong heritable component. sequence variations of slitrk have been detected in a few gts patients, but the genetic component in most gts cases is yet unknown. in a family with a complex chromosome rearrangement, the cntnap gene was truncated by one of the breakpoints in family members with gts and cntnap was therefore suggested to be a candidate gene for this disorder (figure a). in this study, we describe a three-generation family with a balanced t( ; ) translocation. the chromosome q breakpoint disrupts the cntnap gene, but the transloca- tion carriers do not present clinical features of gts. materials and methods cytogenetic analyses, fish and cgh metaphase chromosomes were obtained from peripheral blood lymphocytes and the karyotype of the patient was determined by g-banding. fluorescence in situ hybridiza- tion (fish) was performed using bacterial artificial chro- mosome (bac) clones from the rpci- library (human genome browser) according to standard procedures. high- resolution comparative genome-hybridization (cgh) was carried out as described previously. results and discussion the family presented in this study (index case iii; , figure b) was detected through a systematic re-examina- tion of balanced reciprocal translocation carriers in den- mark. the family history revealed that the translocation in the family was first recognized after the birth of a boy (v; ) with multiple congenital malformations and an unbalanced karyotype, ,xy,der( )t( ; )(q ;q . ) (figure b). cytogenetic analyses showed that his father (iv; ), grandmother (iii; ) and great grandfather (ii; ) were all carriers of an apparently balanced translocation with the karyotype ,xx/xy, t( ; )(q ;q . ). subsequently iv; was investigated and she was shown to be carrier of the same unbalanced translocation as found in v; (figure b). iv; had multiple malformations, severe mental retardation and did not have any language development. she had scoliosis and could take only a few steps with support, but otherwise could not walk. she had a single kidney, hearing loss, ptosis and vision loss because of cataract and affected optic nerve. she did not have any sense of hunger and therefore she was fed through a tube. she had a cheerful and extrovert personality. she died at the age of years. the other patient (v; ) with the same unbalanced translocation is presently years old; he also has multiple congenital malformations, severe mental retardation, no language development and scoliosis. how- ever, he cannot walk even with support. he has myopia, otherwise his vision is normal. he has an introverted personality. the index case (iii; ) had hypermetropia and she developed pre-eclampsia during her second gestation. all the balanced translocation carriers were otherwise phenotypically normal. none of the balanced transloca- tion carriers showed any signs of gts, assessed according to the dsm-iv diagnostic criteria. fish analyses of the balanced translocation carrier iii; using bac clones from the rpci- library (human genome browser) revealed that the chromosome q breakpoint was within the bac clone rp - a , which is located entirely within the genomic region covered by the cntnap gene. the closest non-spanning proximal bac clones were rp - o and rp - n , and the closest non-spanning distal bac clones were rp - o and rp - c . the breakpoint was thus localized to a region of approximately -kb (chromosome position, . . – . . . human genome browser, march assembly) within intron of the cntnap gene (figure a). the q . breakpoint was within the overlapping region of three bac clones rp - p , rp - b , and rp - i . this breakpoint was localized to an figure (a) schematic representation of the two chromosome breakpoints truncating the cntnap gene. the white bar indicates the breakpoint region described by verkerk and colleagues in a gts patient with inv( )(p q ),ins( ; )(q -q ;p p ) and the black bar indicates the breakpoint region of the present case with t( ; ). the horizontal line represents the genomic region covered by the gene and the vertical lines represent the exons; (b) the pedigree of the t( ; )(q ;q . ) translocation family. n, normal karyotype; filled symbol, unbalanced translocation ,xy/xx, der( )t( ; )(q ;q . ); symbol with dot, translocation carrier. arrow indicates the index case. truncation of cntnap without tourette syndrome jm belloso et al european journal of human genetics approximately kb region (chromosome position, , , – , , , human genome browser) within a hypothetical gene (ak ). cgh analysis did not reveal loss or gain of any chromosome region (data not shown). a homozygous mutation of cntnap has been asso- ciated with cdfe in the old order amish community, but it is unknown whether cntnap mutations result in cdfe in other populations. however, the present case supports the finding that disruption of a single allele of cntnap does not lead to cdfe, in line with the suggested recessive- inheritance. verkerk and colleagues suggested cntnap as a candidate gene for gts based on the finding of a truncating chromosome breakpoint (figure a). the authors hypothesized that disruption of cntnap would lead to decreased gene expression, which in turn would affect localization and/or maintenance of kv channels. this would influence conduction and/or re-polarization of action potentials in certain brain regions sensitive to these changes, resulting in a diminished inhibition of unwanted tics as observed in gts. the absence of gts symptoms in the present translocation family indicates that disruption of cntnap does not necessarily lead to gts. however, taking into consideration the complex etiology of gts with a probable polygenic character, this finding does not strictly exclude the involvement of caspr in gts pathogenesis. acknowledgements we are grateful to the family for their cooperation. we thank minna becher, karen friis henriksen, anita niebuhr, kirsten winther for their skillful technical assistance. wilhelm johannsen centre for functional genome research is established by the danish national research foundation. online information online mendelian inherance in man (omim) (http:// www.ncbi.nlm.nih.gov/entrez/query.fcgi?db ¼ omim). human genome browser, ucsc genome bioinformatics (http:// genome.ucsc.edu/cgi-bin/hggateway). references rasband mn: it’s ‘juxta’ potassium channel!. j neurosci res ; : – . poliak s, gollan l, martinez r et al: caspr , a new member of the neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with k+ channels. neuron ; : – . poliak s, salomon d, elhanany h et al: juxtaparanodal clustering of shaker-like k� channels in myelinated axons depends on caspr and tag- . j cell biol ; : – . nakabayashi k, scherer sw: the human contactin-associated protein-like gene (cntnap ) spans over mb of dna at chromosome q . genomics ; : – . strauss ka, puffenberger eg, huentelman mj et al: recessive symptomatic focal epilepsy and mutant contactin-associated protein-like . new eng j med ; : – . verkerk ajmh, mathews ca, joosse m et al: cntnap is disrupted in a family with gilles de la tourette syndrome and obsessive compulsive disorder. genomics ; : – . american psychiatric association: diagnostic and statistical manual of mental disorders, fourth edition (dsm-iv), washington dc, . pauls dl: update on the genetics of tourette syndrome. adv neurol ; : – . abelson jf, kwan ky, o’roak bj et al: sequence variants in slitrk are associated with tourette’s syndrome. science ; : – . kirchhoff m, gerdes t, rose h, maahr j, ottesen am, lundsteen c: detection of chromosomal gains and losses in comparative genomic hybridization analysis based on standard reference intervals. cytometry ; : – . bache i, hjorth m, bugge m et al: systematic re-examination of carriers of balanced reciprocal translocations: a strategy to search for candidate regions for common and complex diseases. eur j hum genet ; : – . truncation of cntnap without tourette syndrome jm belloso et al european journal of human genetics 	disruption of the cntnap gene in a t( ; ) translocation family without symptoms of gilles de la tourette syndrome 	introduction 	materials and methods 	cytogenetic analyses, fish and cgh 	results and discussion 	acknowledgements 	note 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ _c .indd www.pharmacist.com december • pharmacy today review go to www.pharmacist.com and take your test online for instant credit. objectives: to introduce historical and sociocultural influences on health and health care decisions that should be considered by pharmacists and other health professionals when serving amish patients and to describe the roles of pharmacists in working with amish populations, as an example of culturally and linguistically appropriate care. setting: community independent pharmacy in arthur, il, from to . practice description: reflections of a pharmacist–owner whose community prac- tice serves a sizeable amish population. case summary: the old order amish are a religious group that values health and actively participates in its health care decisions. the amish possess a strong sense of community responsibility and often seek advice of friends, family, and community in health care decisions. their explanatory models of health and illness differ, in some respects, from the larger american society. the amish are open to the use of folk medi- cine, complementary and alternative medicine, and conventional care when deemed nec- essary. they are receptive to health care information and explanations of options from trusted sources and use increased self-care modalities, including herbal remedies. results: knowledge of salient cultural differences is important, but care should be given to avoid stereotyping patients because amish rules and customs differ across dis- tricts. culturally competent pharmacist care should be individualized based on patient needs and in consideration of aspects of differences in amish cultures and districts. when serving amish patients, special consideration should be given to addressing poten- tial barriers to health care use, such as unique dialects, affordability issues for largely cash-paying customers, lower prenatal care use, and lower vaccination rates. conclusion: enhanced awareness and sensitivity to amish lifestyles and beliefs can lessen misconceptions and minimize barriers that interfere with optimal provision of patient-centered pharmacy care and services. by working through established com- munity norms, building trust, and effectively applying cultural competency techniques, pharmacists can best serve the amish communities. keywords: amish, community pharmacy, cultural competence, barriers, alternative medicine. pharmacy today. (dec); ( ): – . pharmacists’ considerations when serving amish patients stephanie y. crawford, aimée m. manuel, and bruce d. wood review © by the american pharmacists association • all rights reserved. • printed in u.s.a. stephanie y. crawford, phd, is associate professor and associate head, depart- ment of pharmacy administration, and aimée m. manuel is student pharmacist, college of pharmacy, university of illinois at chicago. bruce d. wood, bs, is pharma- cist–owner, dicks pharmacy, arthur, il. correspondence: stephanie y. crawford, phd, department of pharmacy adminis- tration, college of pharmacy, university of illinois at chicago, s. wood st. (mc ), chicago, il . fax: - - . e-mail: crawford@uic.edu continuing education credits: see learning objectives below and assess- ment questions at the end of this article, which is acpe universal program number - - - -h -p in apha’s edu- cational programs. the ce examination form is located at the end of this article. to take the ce test for this article online, go to www.pharmacist.com/educa- tion and follow the links to the apha ce center. disclosure: the authors declare no con- flicts of interest or financial interests in any product or service mentioned in this article, including grants, employment, gifts, stock holdings, or honoraria. acknowledgments: to theresa binion, executive director for the arthur-amish country visitor center, for reviewing background information pertaining to the amish residing in the arthur, il, area. funding: article development sup- ported in part by a grant from the project export center for excellence in rural health, the national center for rural health professions, the university of illi- nois college of medicine at rockford, and the national institutes of health national center on minority health and health disparities. the contents of this article are solely the responsibility of the authors and do not necessarily represent the of- ficial views of the sponsors. published concurrently in pharmacy today and the journal of the american pharmacists association (available online at www.japha.org). abstract pharmacy today • december www.pharmacytoday.org review go to www.pharmacist.com and take your test online for instant credit. review a ccording to the american pharmacists association code of ethics for pharmacists, the primary obligation of a pharmacist is individual patient care, and service obli- gations sometimes extend beyond the individual to the com- munity and society. ethical guidelines also note the need for pharmacists to respect individual and cultural differences. cultural competence involves the ability of individuals and systems to deliver culturally and linguistically appropri- ate services, interventions, and care. , aspects of culture include race and ethnicity, gender, age, religion, socioeco- nomic status, geographic region, disability, sexual orienta- tion, and health beliefs, among others. cultural competency techniques (at the systems level), designed to reduce health disparities, have been described elsewhere – and are sum- marized in table . table provides brief descriptions of the applicability of the techniques to amish populations, which will be discussed in this article. the effective use of cultural competency techniques should result in ( ) greater knowl- edge of illness, disease, and treatments; ( ) improved com- munications; ( ) increased trust; and ( ) improved under- standing of patient beliefs and expectations. the amish require special considerations from health pro- fessionals because of unique aspects of their culture, which varies in different districts across the country. currently, a limited body of published information is available for health professionals treating the amish. a better understanding of amish perspectives on health and health care practices can help provide patient-centered care to this population. because of shortages of health professionals in many rural locations (including communities near amish settle- ments), necessary health care services may be unavailable or limited. dickinson et al. reported that % of amish respondents in a small wisconsin survey stated that they would use the services of a pharmacist, if one were avail- able. using reports in the literature and the experience of a pharmacist practitioner, the current article introduces his- torical and sociocultural influences on health care decisions that should be considered by pharmacists and other health care practitioners when serving the amish community and describes the role of the pharmacist in working with amish patients. although relatively few pharmacists will interact with amish patients on a routine basis outside of localized regions with heavy populations, this report aims to increase understanding of the community and to illustrate the appli- cability of general cultural competency recommendations to a specific subpopulation. at a glance synopsis: enhanced understanding of amish beliefs and culture can help pharmacists serving amish com- munities to minimize barriers that may interfere with optimal care. the reflections of a pharmacist–owner whose community practice serves a sizeable amish pop- ulation and observations from the literature are provided to inform pharmacists of special considerations when serving the amish. unique dialects, affordability issues for mainly cash-paying customers, lower prenatal care use, and lower vaccination rates may be potential barri- ers to health care use in this population. the amish use folk medicine, complementary and alternative medicine, and, when deemed necessary, conventional care. they are receptive to health care information and explana- tions of options from trusted sources and use increased self-care modalities, including herbal remedies. analysis: culturally competent pharmacy care should be individualized in consideration of aspects of differences in cultures and rules among amish dis- tricts. to effectively counsel patients on therapeutic use and safety and to check for potential drug interactions, pharmacists serving amish communities should strive to be nonjudgmental about folk care and knowledge- able of herbal remedies and vitamins. the amish may be receptive to vaccination outreach programs if the topic is openly discussed and individual concerns are addressed. outsiders to amish communities need to establish rela- tionships in order to be accepted, usually by first consult- ing and working with community church leaders through established local community structures. collaborative efforts then can be established with other health profes- sionals serving the amish community. learning objectives n list four potential outcomes of effective cultural competency techniques. n provide six examples of cultural competency techniques that are applicable to amish cultures. n state health beliefs and explanatory models of health and illness accepted in amish cultures. n differentiate the three sectors of the health care system that may be used by amish people. n name four reasons for lower vaccination rates among the amish. n identify examples of how pharmacists can help alleviate logistical, communication, and cost barriers to effective health care provision for amish patients. www.pharmacist.com december • pharmacy today review go to www.pharmacist.com and take your test online for instant credit. review table . summary of cultural competency techniques and applicability in serving amish patients cultural competency techniquesa applicability to amish populations language assistance services (interpreter services or bilingual providers) individuals may speak english and/or a blended english–german dialect (e.g., penn- sylvania dutch). language assistance services may be needed if patients have lim- ited english proficiency, although many amish community members are bilingual. recruitment, retention, and training of staff who reflect community diversity hire and train staff who are respectful of, sensitive to, and knowledgeable of patient health beliefs, practices, and illnesses. coordination of efforts with traditional healers consider possible patient use of alternative care and folk care providers (e.g., brauchers) and remedies. collaboration with community health workers attempt to work with district bishops and other church leaders through established local structures to disseminate information in the amish community; collaborate with other health professionals, as appropriate, in offering preventive and educa- tional services locally. use of appropriate health interventions develop and/or disseminate culturally and linguistically appropriate health promotion and education materials. inclusion of family and community members decisions about illness and health behaviors often emanate from discussions with family and community members; work with the patient and his or her support group (as appropriate and allowable). immersion into culture make staff available to patients in one-on-one interactive counseling sessions, invite amish community members to service functions, and learn about the culture and community. administrative and organizational accommodations establish accommodating service hours and on-call policies; consider pharmacy delivery service to amish community; create organizational climate of respect and cooperation. asources of competency techniques: references – . setting located in east-central illinois, the town of arthur has a popula- tion of approximately , , and its surrounding farmland area is settled by the largest concentration of amish people in the state. the community was founded by a small number of families in and is currently home to more than , individuals. the local amish have one church, but different amish church districts are located around arthur (personal communications, theresa binion, november ), spanning miles east and west and miles north and south of town. each district has its own set of rules (ordnung), which govern customs and practices. dicks pharmacy, a community independent pharmacy, is located in the town of arthur. two full-time equivalent (fte) pharma- cists (including the owner), one fte pharmacy technician, two fte clerks, and six part-time clerks are employed at the phar- macy. dicks pharmacy dispenses an average of prescriptions per day and is open from : am to : pm monday through saturday (closing : pm on saturdays). the original pharmacy was established in the s or s. bruce wood began working at the pharmacy in and became the pharmacist proprietor years later. wood has a notable his- tory of community and professional service and has engaged in civic activities for the greater arthur community, including serving as current president of the local chamber of commerce. approximately % to % of the patients and customers at his pharmacy are amish. based on years’ experience, his per- spectives and advice in serving the amish community are sum- marized and presented as a first-person account in the following section and, where possible, throughout the article as an adjunct to the literature. although one individual’s observations cannot be considered generalizable to the larger population, relevance and practical applicability are augmented by including a pharmacist practitioner’s reflections. wood’s introductory reflections on serving the amish “we have a typical, old-fashioned community pharmacy. cus- tomer service is what we stress the most. at dicks pharmacy, our experience with serving the amish has been pretty similar as with serving our other ‘english’ (as the amish refer to us) customers. the amish that live in our area are very concerned with their health and that of their children as well. one might note pharmacy today • december www.pharmacytoday.org review go to www.pharmacist.com and take your test online for instant credit. review that there seems to be a higher incidence of certain disease states in this group over others (for example, whooping cough). aside from vaccinations, we do see that the amish are quite active in receiving medical care. in arthur, many amish receive traditional (conventional) care; however, some do use the other methods of alternative care. “when seeking care, the amish do not necessarily need to go to the doctor’s office and often practice self-care. they call me a lot (sometimes early in the morning before the store opens), often asking, ‘do you have [medication or other treatment].’ they call to find out my recommendations for themselves or their families. they also call seeking advice for veterinary medications. on the farms, they have more use for veterinary preparations. “in the pharmacy, we counsel the amish the same way as the english. for all of our patients, we interact on a one-on-one basis regarding how medications should be taken and how patients can take care of themselves. “the amish customers that we serve are primarily cash- paying customers. a few of our customers require the assistance of the church to pay some more outstanding bills. we also have some amish customers that do have a form of insurance. i have really enjoyed serving the local amish community in the arthur area.” when asked about the training, if any, that his staff (pharma- cists, pharmacy technicians, and clerks) receive before interact- ing with amish patients, wood added the following: “i tell staff that they (the amish) are very honest. they are willing to learn. they are interested in taking their meds. most of the people hired have lived in the community a long time and respect the amish traditions.” brief history and background of the amish the amish originated from the anabaptist movement of the swiss brethren. a number of people fled switzerland in to escape religious persecution by protestants and catholics. , these people were called anabaptist christians because they rebaptized their adult followers. from switzerland, the movement spread to germany, france, and the netherlands. the anabap- tists believed in the separation of church and state and a commit- ment to pacifism and nonviolence and were considered radical. a key leader in the anabaptist religion was menno simons, whose followers were called mennonites. members of the mennonite religious group became less strict in their ways of life over time and began to adopt more modern practices. some followers, however, had different biblical interpretations and did not believe in the laxity of practices, which resulted in embracing too many worldly conveniences. among these was a swiss–german men- nonite named jacob ammann (sometimes spelled as amman). in , a conservative group of mennonites broke off from the religion to follow ammann—they became known as amish. , the amish migrated to america, first settling in a pennsylvania colony in the early th century. , three major amish groups exist: the old order amish and the more progressive amish mennonites, which include the beachy amish (e.g., car ownership and use of electricity from public utili- ties allowed) and new order amish (e.g., use of electricity and telephones in homes and use of modern farm equipment may be permitted). the old order amish is the largest and strictest (most conservative) group in terms of traditional beliefs and practices. as the most populous group and the most iconic with societal perceptions of conduct, any reference to the amish from this point forward in the article refers to the old order amish. the populations of different amish communities can total hundreds or thousands of individual members. amish community structures consist of settlements, church districts, and affilia- tions. settlements are composed of proximate households. the size of settlements may range from small, including only a few households and their religious leader, to large settlements that might encompass several counties. self-governing church dis- tricts (i.e., congregations) are established within geographic areas of each settlement. the size of congregations depends on how many families can gather at a farm dwelling or home. each autonomous congregation typically consists of about to families, and every church district is led by a bishop—the chief authority who decides how conservative the congregation will be by clarifying the code of conduct, religious practices, and rules (ordnung) of social behavior. , , , approximately , amish congregations exist. groups of church districts with similar dis- cipline structure can commune together, and these congregations represent an affiliation. amish behaviors and practices should not be generalized or stereotyped in a singular image, as diversity exists among groups within amish communities. amish culture varies by location; different bishops may approve different prac- tices across respective church districts. each district may differ across the united states in terms of religious practices, customs, conduct, dress, buggy styles, and other rules. noted concentrations of amish communities in north amer- ica are located in states and ontario, canada, with the great- est numbers of amish people and districts in ohio, pennsylvania, and indiana. in , professor donald kraybill (elizabethtown college) estimated that the amish american population totaled , old order and new order amish, including children; adult baptized members were estimated to total , . the amish are considered to be conservative christians who live a simple life in geographic and social separation (though not seclu- sion) from the predominant american culture. the basic amish tenets include integrity, order, responsibility, obedience (to par- ents, church, and god), nonresistance, and consideration of the human body as god’s temple. the amish lifestyle the amish believe in conserving their traditions and practices, and they reject or avoid unnecessary material possessions. they are well known for living a simple life with minimal technology www.pharmacist.com december • pharmacy today review go to www.pharmacist.com and take your test online for instant credit. review in the fast-changing world around them. the amish dress plainly and have a limited use of modern conveniences for fear of becom- ing too worldly. technology can be adopted if approved by the district bishop, based on group discussions with congregation elders about its effect on the community way of life. electricity is rarely, if ever, used in amish homes. electric generators are used in some amish communities for work reasons, such as welding, milk production, and battery recharging. the amish generally travel by walking and riding a bicycle or horse-drawn buggy. they may hire a driver or take a bus or taxi for distance travel. in an emergency, they may have a non-amish person drive them, for example, to the nearest hospital. the amish neither watch television nor have telephones inside their homes because those are means of bringing the outside world into the home, which is verboten. use of business, public, or communal phones may be permitted by a district bishop, espe- cially for outgoing calls. attitudes toward phone ownership are changing, with reluctance, in some communities as more amish have access to cell phones and their multiple capabilities. the amish settle in rural communities, and farming is the historical way of life for amish households. as farmland has grown more scarce, other sources of income and livelihood have grown. more amish men have found jobs in bookstores and voca- tional crafts, such as woodworking/carpentry, canning, carriage making, performing repair work, and working in farm equipment stores and print shops. , amish women work around the house (e.g., tending the garden, cooking natural foods from the garden and farm, making clothes). the amish also sell homemade fur- niture, quilts, produce, and prepared foods and other goods near their homes or in nearby markets; this trade is fueled by curiosity and demand from tourist shoppers and others. , most amish children are formally educated only through the eighth grade, which is deemed a sufficient level to live the amish lifestyle. , the amish resist sending their older children to high school because of concerns that outsiders (i.e., non-amish peo- ple) and higher education will negatively alter their thinking and behaviors. the lifestyle and culture of the amish directly affect their views on sickness and health care. wood’s reflections. “they (the amish) leave school after a limited number of years of formal education, but they continue to read voraciously. amish families read a great deal in the evenings. reading is a normal activity in the home; they do not watch tele- vision or play video games. they also read a lot on health care. they listen attentively to advice and are avid readers most of the time. they will do research on all kinds of things, including their medications, and they definitely ask questions.” amish health beliefs and health care practices the amish value health, are health conscious, and generally rec- ognize when someone is sick. their explanatory models of illness and treatment approaches, however, differ in some respects from those that are traditionally used by people in the larger american society. the amish do not perceive illness as a frailty but rather as part of life. they believe that good health, both mental and physical, is a gift from god that they can help achieve by working hard, living simply and cleanly, and eating a well-balanced diet. in amish terms, a healthy person is one who “gets up early, gets enough rest, needs fresh air, eats as naturally as possible, wants to work, wants to be healthy, helps to create a happy atmosphere, accepts what one has and goes on, does not worry; at least not too much, has faith, and has not too much stress.” compared with the traditional biomedical perspective, the amish possess a holis- tic outlook on health and healing. the amish are actively involved on a daily basis in decision making and actions about health and treatment of illness. many amish may take food supplements, vitamins, and eat natural or organic food. a strong sense of soli- darity, responsibility, and caring for their own exists within amish communities. they promote individual, family, and community well-being. three sectors of the health care system may be used by the amish. in the terminology used by the national center for complementary and alternative medicine, these sectors include conventional care and complementary and alternative medicine (including biologically based therapies, energy/biofield therapies, and manipulative and body-based therapies). these practices are commonly categorized as folk care, alternative care, and professional care in literature on the amish. , , no aspect of professional medical or other health care is forbidden among the amish; however, individuals may show more reluctance or hesi- tancy toward using certain services, and health care practices may vary among districts. the amish generally embrace use of conventional medicine in treating incapacitating and traumatic diseases and infections. with nondebilitating chronic illnesses, treatment modalities include conventional care, folk care, and complementary and alternative medicine. folk care and complementary and alternative care folk care includes the influences of the popular sector (i.e., seek- ing advice on illness from family, friends, and community mem- bers). when a member of the amish community becomes ill, the family members generally weigh their options, including folk care and alternative medicine, before seeking professional care. this pattern has led to misconceptions that the amish shun pro- fessional care. folk care is indigenous in amish communities, and treatments are passed down through generations. examples of folk care may include heavy use of teas, chelation therapies, onions, mustard plasters, camphor applications, herbs, and, increasingly, commercial herbal remedies. , (depending on the source, herbal remedies are included under both folk care and alternative care.) the amish often grow gardens of herbs and teas to use as curative medicine. if approved in the district, folk care is generally provided in the home or amish community, using home remedies or the services of brauchers, who are amish pharmacy today • december www.pharmacytoday.org review go to www.pharmacist.com and take your test online for instant credit. review healers. practitioners serving amish communities should strive to be nonjudgmental about folk remedies and knowledgeable on herbal remedies and vitamins so that they will be able to counsel patients on therapeutic use and safety and check for potential interactions. the amish may be hesitant to discuss their use of herbal remedies, home remedies, and alternative care. wood’s reflections. “we realize that the amish today do take herbal remedies, but getting them to tell us that they are using something other than the prescribed medications is often difficult (as with the general population). most of the time, we just have to ask what else they are using for this or that condition, and then they will spill the beans about what else they are taking. unfortu- nately, we do not always ask if they are taking something else. as with any of our patients, all we can do is counsel them on what we know about their conditions, medications, and lifestyles.” the term braucher comes from the word brauche, which means folk-healing art. some amish believe that individuals inherit the power of healing through touch. brauchers are amish practitioners who generally use their warm hands to touch (or place near the person’s body) and physically manipulate the prob- lem area in an attempt to draw out the illness. brauchers do not have prescriptive authority, although they can recommend herbs and many folk remedies. brauchers sometimes refer to them- selves as braucher–chiropractors; however, they are not licensed practitioners (and are not recognized to practice chiropractic medicine by the state) but may have observed aspects of chiro- practic treatments from others. in an article from , emanuel stoltzfus, a full-time self-described braucher–chiropractor, said that he discovered one day that he had “electric” in his hands and could feel where a person was sick and would treat accord- ingly. brauchers learn the techniques of the art of healing from a member of the opposite sex upon promising to keep the art a secret. brauchers are generally seen when amish patients do not know what is wrong or want to avoid drug therapies. , amish parents often take their ailing babies and small children to visit the braucher because the children are too young to readily describe their pain or other symptoms. the braucher is said to be able to touch the child and pull out the pain. brauchers prac- tice their folk art using physical manipulations, charms, incanta- tions, and sacred rituals. in past years, aspects of traditional european occult practices (e.g., sorcery and “black magic”) may have routinely been included in the practices of amish brauchers and other community healers; such practices have largely been abandoned. , mixed feelings exist within the community about the prac- tice of brauche, with some amish questioning the practice and/ or expressing skepticism and embarassment ; however, many amish believe that brauchers can help some people. , in certain districts, brauchers may be the preferred care provider because they are easily accessible and less expensive and are trusted as members of the community. brauchers and other folk care pro- viders do not charge a fee (for historical reasons and to avoid the appearance of practicing medical therapies without a license), but donations are accepted and sometimes expected. in addition to money, donations may include brokered goods or services. , , wood’s reflections. “personally, i have only once been approached by the amish about what a braucher had told them, and that was a number of years ago, so i do not remember the exact details at all. if i remember correctly, the information that they had been given was fairly accurate, and we did not have a problem assisting the family member in what they were look- ing for. the use of brauchers in this area does not appear to be very high.” the executive director for the arthur-amish country visitor center provided further insight, stating that the use of a braucher does not exist in the arthur, il, area (personal com- munications, theresa binion, november ). some authors include powwow healers (including brauchers and informal family/friends who use sympathy curing or faith healing) under the category of alternative healers, but most categorize as folk care providers. examples of alternative care services that may be used by the amish include traditional chiro- practic care, lay midwifery, reflexology, massage, foot treatments, homeopathy, iridology, and herbal therapists. , alternative care may be sought regularly by the amish for body adjustments, treat- ment of back pain, and other maladies for which patients believe it may be effective. alternative care providers charge a fee for ser- vices, which is perceived to be less expensive than physicians. conventional care professional or conventional care is offered by health care practi- tioners who have been formally educated and trained in the domi- nant medical and health system. most amish members pay their bills in cash for services and care rendered at hospitals and by physicians, nurses, dentists, pharmacists, and other providers in the traditional professional sector. most amish families have a physician whom they visit when they recognize the need for scientific medical practice. reasons for choosing a family physician extend beyond medical knowl- edge and include family tradition, proximity, recommendation by others, affordability, trust, sympathy, and integrity. , con- ventional care is generally sought by the amish in cases of high fever and when surgery or stitches, hospital care, or prescription medication are needed. compared with non-amish, the amish are much less likely to seek professional care for preventive medicine because of lack of convenience, high costs, and a perception that it is not necessary. conventional care is most often sought when an emergency occurs. the amish will visit hospital emergency departments in cases of severe abdominal pain, chest pains, lacerations, frac- tures, and traumatic injuries, among other urgent conditions. , farm accidents resulting in severe injuries are commonly seen on an emergency basis. children are often the victims of these trau- matic injuries (e.g., falls, machinery accidents, animal injuries) www.pharmacist.com december • pharmacy today review go to www.pharmacist.com and take your test online for instant credit. review in amish communities because they begin working the farm at a young age. a number of emergency visits and deaths also result from lack of protections with respect to buggy and automobile accidents. , prenatal care medical means of birth control reportedly are not practiced in amish communities, and average households include six or seven children. , , most amish women visit a physician for initial confirmation of a pregnancy diagnosis. however, most amish women do not seek professional prenatal care throughout the pregnancy. if prenatal care is initiated, it is often late in the preg- nancy, such as the third trimester. reasons for minimal prenatal care use include ( ) amish views of uncomplicated pregnancies as valued normal states that do not require medical interventions and ( ) logistical barriers pertaining to transportation difficul- ties, child care arrangements, and costs. many amish women prefer to deliver their babies at home using house-call physicians, nurse midwives, or lay midwives and other alternative care pro- viders. if the expectant mother shows serious symptoms that could result in a high risk of complications, the delivery will be scheduled to take place in a hospital. , wood’s reflections. “all of them (expectant amish mothers) seem to get prenatal vitamins. they are more aware of the situ- ation than they were in years past. they know the healthier their bodies, the healthier their babies. the amish buy more breast shield, breast pumps, and nursing pads than others, which may be indicative that they breast-feed more often.” hereditary disorders in addition to an increased incidence of twinning, some dis- tinct amish communities in different regions show a higher prevalence of certain chromosomal and genetic abnormalities (e.g., ellis-van creveld syndrome or dwarfism, cartilage–hair hypoplasia, pyruvate kinase deficiency anemia, hemophilia). the hereditary disorders result in part from common blood- lines of offspring from endogamous unions among members of a relatively closed society. , genetic screening and counsel- ing have been advocated for high-risk populations when early diagnosis may decrease morbidity and mortality. pharmacists and other health professionals could serve as sources of refer- ral to medical and public health specialists in genetics, when deemed appropriate. such initiatives, however, are controver- sial and may have psychological and ethical implications. most amish would reject prenatal screening. efforts are undertaken within amish communities to prevent genetic diseases and to understand their causes and outcomes. professional care is not always sought for such genetic conditions because they are viewed as god’s will by the amish, although some communities are accepting of promising gene therapy. many amish may have treatable autoimmune thyroid diseases. wood’s reflections. “many amish (we serve) have hereditary thyroid diseases and need thyroid replacement medicines. they may start see the appearance of thyroid problems in their late teens or early s, and often the amount of replacement therapy can be very extensive.” vaccination rates while not prohibited by their religious doctrine, many amish people do not get vaccinated, which places them at increased risk for acquiring vaccine-preventable diseases. outbreaks of rubella, childhood tetanus, pertussis, measles, poliovirus infec- tions, and haemophilus influenzae type b (hib) have occurred in amish communities at disproportionately high rates. , , , for example, fry et al. found full childhood hib vaccination coverage in two pennsylvania amish communities to be only % and %, whereas % coverage occurred in the non-amish comparison group. on the other hand, yoder and dworkin found that % of respondents from households in an illinois amish settlement self-reported that all of their children had been vac- cinated (though these reports could not be validated with health records), which was attributed in part to learning about the low vaccination rates and outbreaks experienced in other amish communities. despite isolated success stories, vaccination cov- erage levels among the amish tend to be very low. wood’s reflections. “we have seen a few outbreaks of whoop- ing cough in the past years. the amish do not get vaccinated for this disease, nor do they get boosters as adults. i have not noticed any differences in vaccination rates in the arthur com- munity in recent years.” the reasons most amish do not get their children vac- cinated include lack of recognized need for vaccines, fears of adverse effects/vaccine safety for their children, religious or philosophical objections, and lack of priority in life. , , , another reason pertains to logistical barriers (e.g., need to travel long distances to get vaccinated, limited transportation in general, inconvenient clinic hours). , efforts can be made by pharmacists and other health professionals to help promote vaccination. the amish may be receptive to vaccination outreach pro- grams if the topic is openly discussed and efforts are put forth to address individual concerns. developing culturally and lin- guistically appropriate educational materials would be useful, and information approved by the district bishops and other church leaders can appear in local community newsletters and other readily utilized publications. clinical and public health professionals in amish communities could also try to set up immunization drives within amish communities so that travel to the clinic or physician’s office would not be as burdensome, which would eliminate one of the logistical barriers. local pharmacists can partner with local physicians, nurses, and/ or public health departments in attempts to contact church leaders to educate them on the greater benefits (and potential adverse effects) of vaccination. pharmacy today • december www.pharmacytoday.org review go to www.pharmacist.com and take your test online for instant credit. barriers and opportunities to pharmacist care and services the lack of available transportation is a logistical barrier that may limit access to medical and pharmacy care for amish patients. to help alleviate that problem, wood said, “we added delivery ser- vice at the pharmacy when i took over in the s. the service is available for all patients, but mainly exists for the amish and elderly patients, who use the service extensively. the delivery service was established to make life easier and to ensure that patients obtain their medications (new and refill) in a timely man- ner. there is no charge for prescription delivery. we deliver to five different counties over about a -mile radius (from arthur, il) on a weekday basis, starting each day at : pm.” language language barriers could be a challenge for health professionals serving an amish community. most often, pennsylvania dutch (also known as pennsylvania german or deitsch)—a blended dialect of german and english—is spoken in amish communi- ties, at home, and at church. , children will learn english at school, but often preschool-aged children only speak pennsyl- vania dutch, if that is the language spoken in their home. the amish refer to the non-amish in their surrounding area as “the english” because that is the language they typically use. , , in rural areas, finding translators is difficult; therefore, health professionals must be as descriptive and complete as possible when counseling patients with limited english proficiency. dem- onstrations (of correct medication use, for instance) often help. if language is a barrier, having patients repeat to the health care provider the counseling information that he or she heard, in order to help ascertain whether the communicated message was under- stood in the manner intended, may be helpful. wood’s reflections. “the previous pharmacy owner spoke some german, and the original owner in the s spoke fluent ger- man. the amish patients learned to watch what they said in the store in the presence of the former proprietors since they knew that the staff understood their personal conversations. i know virtually no german, but their [the amish] english is usually very good; they learn both languages.” costs of conventional care high costs can present another barrier to health care among the amish. the amish strive to live plain, uncomplicated lives, including the means of paying for health care. many amish self- pay for health care services and products. they are reluctant to accept any government insurance programs because of lack of trust in the knowledge sources and lack of community consensus and clear approval by the district bishops. amish may perceive the buying of health insurance as a lack of faith. some who work for non-amish employers may accept health insurance coverage, but most amish tend to eschew social security benefits or other financial assistance from the government, and most frown on commercial health insurance. this pattern is slowly changing. wood’s reflections. “the local members of the amish com- munity are some of the most trusted customers. they are more honest than the ‘english’ and more likely to pay their bills in full. today, with the increase in members of the amish community getting jobs outside of the family farm due to the scarcity of the farmland, insurance is more common. “i find it interesting that the percentage of amish with medi- care is certainly less than those in the general population, but higher than i had suspected as well. in this area, many of the amish who are ineligible for social security benefits do qualify as beneficiaries for illinois cares rx (an income-based state pre- scription drug assistance program for qualified individuals age years or older, as well as other eligible beneficiaries, including some disabled). they are taking advantage of that plan, much like the rest of the population, but they do not get assigned to a medicare part d company because they (currently) do not qualify for medicare.” delaying or refusing a necessary visit to a physician or other health professional because of cost concerns or failing to obtain needed medications can cause patients to become sicker, which could result in even more costly and more complicated medical therapies. in line with reliance on support of the church and community, some districts form their own insurance fund, known as church aid or amish aid, to which each family contributes an initial amount and contributes again when someone is in need. , amish members accept financial assistance from these commu- nity funds, if they exist, only when they are personally unable to pay medical expenses. even then, some individuals in the amish communities will go without health care services if they believe the burden for the community would be too much. to help mini- mize costs, amish people may venture outside of the united states (especially to mexico) to obtain more affordable health care ser- vices. the amish honor their financial obligations and pay their debts in a timely manner. providers (e.g., hospitals, physicians) serving amish populations may make special arrangements for these cash-paying customers, including discounted rates for ser- vices. if cost is a barrier, pharmacists may have an opportunity to recommend a less-expensive generic substitute or therapeuti- cally similar agent. in some communities, pharmacy owners allow members of the amish community to charge prescriptions to the family for later (e.g., monthly) cash payment. building trust health care practitioners have an important opportunity to build trusting relationships with their amish patients so that the patients feel at ease when asking questions and seeking help. amish members welcome provision of health information from trusted sources. , this desire for information facilitates oppor- tunities for pharmacists in amish communities to counsel and educate patients, including welcomed explanations of options that can be considered. outsiders to amish communities need www.pharmacist.com december • pharmacy today review go to www.pharmacist.com and take your test online for instant credit. to establish relationships in order to be accepted, usually by first consulting and working with community leaders through estab- lished local community structures. collaborative efforts then can be established with other health professionals serving the amish community. wood’s reflections. “each month on the third thursday, we host the lincolnland visiting nurses blood screenings. each month, there are usually or so people wanting one of the various screenings they offer. there is a charge for the cholesterol screen- ings, but blood pressure and blood sugar tests are administered free of charge. over half of the people seen in this -hour time slot are normally of the amish faith.” culturally competent care as aspect of patient-centered care the knowledge of salient cultural differences when serving cross- cultural patient populations is needed to provide competent phar- macy practice. however, all individuals within amish cultures do not think or act the same, and care must be given to ensure that these patients are not stereotyped based on intrinsic and extrin- sic cultural aspects. within the setting described in this article, each district in the arthur, il, area adheres to its own rules, including use of home remedies and conventional health care services (personal communications, theresa binion, november ). universal application of the described general amish char- acteristics to all patients in the subpopulation may result in sub- optimal health care decisions and actions. individualized patient care by pharmacists is needed to help patients make the best use of their medications, promote safety, and avoid unintended and harmful effects. conclusion to our knowledge, this is the first article to address pharma- cists’ considerations when serving amish patients. bruce wood’s reflections supported much of the information culled from the literature review. pharmacists in all settings play an important role in ensuring that patients get good health care. special con- siderations may need to be made by health care practitioners when serving the amish because of their culture, health beliefs, and lifestyles. culturally competent pharmacist care should be individualized based on patient needs and in consideration of aspects of differences in amish cultures and districts. increased understanding and accommodation of amish beliefs and culture can help pharmacists and other health care practitioners better serve the community and minimize barriers that may interfere with optimal care provision. references . american pharmacists association. code of ethics for pharmacists. accessed at www.pharmacist.com/am/tem- plate.cfm?section=search &template=/cm/htmldisplay. cfm&contentid= , november , . . american college of clinical pharmacy, o’connell m, korner e, et al. cultural competence in health care and its implications for pharmacy. part . overview of key concepts in multicultural health care. pharmacotherapy. ; : – . . shaw-taylor y. culturally and linguistically appropriate health care for racial or ethnic minorities: analysis of the u.s. office of minority health’s recommended standards. health policy. ; : – . . brach c, fraser i. can cultural competency reduce racial and ethnic health disparities: a review and conceptual model. med care res rev. ; (suppl ): – . . dickinson n, slesinger d, raftery p. a comparison of the perceived health needs of amish and non-amish families in cashton, wisc. wis med j. ; : – . . yoder j, dworkin m. vaccination usage among an old-order amish community in illinois. pediatr infect dis j. ; : – . . amish country marketing. arthur … “the heart of the illinois amish country.” accessed at www.illinoisamishcountry.com, october , . . amish country marketing. the amish. accessed at www. illinoisamish.net/amish/amishmain.htm, december , . . wood b. dicks pharmacy. accessed at www.dicks-pharmacy. com, may , . . brewer j, bonalumi n. health care beliefs and practices among the pennsylvania amish. j emerg nurs. ; : – . . hostetler j. amish society. th ed. baltimore, md: johns hop- kins university press; . . wenger a. cultural context, health and health care decision making, . j transcult nurs. ; : – . . amish country marketing. amish religion. accessed at www. amishillinois.com/religion/amish_religion.htm, november , . . weyer s, hustey v, rathbun l, et al. a look into the amish cul- ture: what should we learn? j transcult nurs. ; : – . . campanella k, korbin j, acheson l. pregnancy and childbirth among the amish. soc sci med. ; : – . . young center for anabaptist and pietist studies, elizabeth- town college. amish population growth - highlights. accessed at www .etown.edu/amishstudies/population_ trends_ _ .asp, november , . . nolt s, meyers t. plain diversity: amish cultures and identities. baltimore, md: johns hopkins university press; . . von gruenigen v, showalter a, gil k, et al. complementary and alternative medicine use in the amish. complement ther med. ; : – . . graham l, cates j. health care and sequestered cultures: a perspective from the old order amish. j multicult nurs health. ; : – . . miller k, yost b, flaherty s, et al. health status, health condi- tions, and health behaviors among amish women: results from the central pennsylvania women’s health study (cepawhs). women’s health issues. ; : – . . hostetler j. amish life. scottdale, pa: herald; . . patton m. genetic studies in the amish community. ann hum biol. ; : – . . centers for disease control and prevention. pertussis outbreak in an amish community: kent county, delaware, septem- ber -february . mmwr morb mortal wkly rep. ; : – . . national center for complementary and alternative medi- cine. what is cam? accessed at http://nccam.nih.gov/health/ whatiscam, may , . pharmacy today • december www.pharmacytoday.org review go to www.pharmacist.com and take your test online for instant credit. review ce credit: to obtain . contact hours of continuing education credit ( . ceus) for “pharmacists’ considerations when serving amish patients,” complete the assessment exercise, fill out the ce examination form at the end of this article, and return to apha. you can also go to www.pharmacist.com and take your test online for instant credit. ce processing is free for apha mem- bers and $ for nonmembers. a statement of credit will be awarded for a passing grade of % or better. pharmacists who complete this exercise successfully before december , , can receive credit. the american pharmacists association is accredited by the accreditation council for pharmacy education as a provider of continuing pharmacy education. the acpe universal program number assigned to the program by the accredited provider is - - - -h -p. “pharmacists’ considerations when serving amish patients” is a home-study continuing education program for pharmacists developed by the american pharmacists association. . weaver-zercher dl. writing the amish: the worlds of john a. hostetler. university park, pa: penn state press; . . miller l. the role of a braucher-chiropractor in an amish com- munity. mennon q rev. ; : – . . vitale m, rzucidlo s, shaffer m, et al. the impact of pediatric trauma in the amish community. j pediatr. ; : – . . rhodes d, hupcey j. farm injuries among old order amish children. clin excell nurse pract. ; : – . . aaland m, hlaing t. amish buggy injuries in the st century: a retrospective review from a rural level ii trauma center. am surg. ; : – . . mcardle p, pollin t, o’connell j, et al. does having children extend life span? a genealogical study of parity and longevity in the amish. j gerontol a biol sci med sci. ; : – . . ross l, moon m. ethical issues in genetic testing of children. arch pediatr adolesc med. ; : – . . grady d. at gene therapy’s frontier, the amish build a clinic. the new york times. june , . . allen e, hsueh w, sabra m, et al. a genome-wide scan for autoimmune thyroiditis in the old order amish: replication of genetic linkage on chromosome q . -q . . j clin endocrinol metab. ; : – . . centers for disease control and prevention. poliovirus infec- tions in four unvaccinated children: minnesota, august-octo- ber . mmwr morb mortal wkly rep. ; : – . . fry a, lurie p, gidley m, et al. haemophilus influenzae type b disease among amish children in pennsylvania: reasons for persistent disease. pediatrics. ; :e . . salmon d, haber m, gangarusa e, et al. health consequences of religious and philosophical exemptions from immuniza- tion laws: individual and societal risks of measles. jama. ; : – . . mccollum m. the uninsured: uncovered–and unfazed. hosp health netw. ; ( ): . www.pharmacist.com december • pharmacy today review go to www.pharmacist.com and take your test online for instant credit. review assessment questions instructions: you may take the assessment test for this program on paper or online. for each question, circle the letter on the answer sheet corresponding to the answer you select as being the correct one. there is only one correct answer to each ques- tion. please review all your answers to be sure that you have circled the proper letters. to take the ce test for this article online, go to www.pharmacist.com and click education. once you are on the education welcome page, search for this article with the search function, using “ce” and a keyword. follow the online instructions to take and submit the assessment test. this ce will be available online at www.pharmacist.com after december , . you can also find it on www.pharmacytoday.org. . which of the following is not an example of a cultur- ally competent technique that could be used in serv- ing amish patients? a. language assistance if patients speak deitsch and demonstrate limited english proficiency b. universal application of knowledge about amish health beliefs to all amish patients c. outreach and working with church leaders through established local structures to disseminate information d. consideration of possible use of folk care and alterna- tive care by amish patients . what term is often used by the amish to describe people who are not amish? a. the outsiders b. the americans c. the english d. the seculars . what amish group is the most conservative regarding traditional beliefs and practices? a. amish mennonites b. old order amish c. new order amish d. beachy amish . what amish group is the most populous in the united states? a. amish mennonites b. old order amish c. new order amish d. beachy amish . which of the following is correct? a. all old order amish adhere to the same social behav- iors and practices. b. the majority of pharmacists interact with at least one amish patient routinely. c. the largest amish population is located in the state of new york. d. local bishops interpret the rules of conduct within each amish church district. . what is the highest level of formal education for most amish? a. sixth grade b. eighth grade c. ninth grade d. twelfth grade . which of the following is not correct regarding amish health beliefs and practices? a. the amish view illness as an individual frailty. b. the amish read a great deal about health care and ask questions about their medications. c. amish people are health conscious and actively involved in health care decisions. d. the amish possess a holistic perspective on health and illness. . which of the following aspects of well-being is/are promoted in the amish culture? a. community well-being b. family well-being c. individual well-being d. all of the above alternatives are correct. . which of the following is not an example of folk care in the amish culture? a. health advice from family and community members b. teas used for health purposes c. chelation therapies d. traditional chiropractic care . which of the following is generally forbidden in old order amish culture? a. use of telephones in homes b. use of vitamins and herbal remedies c. professional medical care d. vaccinations pharmacy today • december www.pharmacytoday.org review go to www.pharmacist.com and take your test online for instant credit. review . which of the following is not correct regarding brauchers? a. brauchers are also known as braucher–chiropractors. b. brauchers are licensed by the state and granted pre- scriptive authority. c. brauchers may recommend herbal remedies. d. brauchers are preferred healers in some amish com- munities. . which of the following does not typically charge a fee for services? a. brauchers b. homeopaths c. lay midwives d. herbal therapists . the amish generally do not seek conventional care for which of the following? a. traumatic injuries b. infections c. prenatal physician care beyond confirmation of preg- nancy d. prescription medications . which of the following is correct? a. amish women routinely practice medical means of birth control. b. most amish are amenable to prenatal screening to detect hereditary disorders. c. amish women tend to shun use of prenatal vitamins. d. many amish need thyroid hormone replacement thera- pies because of increased prevalence of some thyroid disorders. . which of the following is not a reason for low vaccina- tion rates among amish people? a. lack of recognized need for vaccines b. fear of adverse effects c. religious doctrine prohibiting vaccines d. transportation difficulties . which of the following would not be an appropriate method for promoting vaccination use among amish people? a. developing linguistically appropriate educational mate- rials on vaccines for inclusion in community newslet- ters b. establishing immunization drives within amish com- munities to minimize travel burdens c. partnering with local health departments and/or health providers to educate local church leaders about vac- cines d. distributing comprehensive, technical product infor- mation on vaccines from product manufacturers . which of the following languages is not typically spo- ken or learned by school-aged amish children? a. english b. french c. pennsylvania dutch d. pennsylvania german . which of the following is not correct regarding pay- ment for health care services among the amish? a. many amish pay for health care out of pocket. b. many amish are reluctant to use government insurance programs; however, these attitudes are slowly chang- ing. c. the amish refuse all types of commercial health insur- ance. d. some amish church districts form their own insurance programs. . which of the following is not correct? a. the amish insist on paying for services at the time of service provision and would not agree to paying over time. b. the amish honor their financial obligations and pay their debts. c. even if their church is willing to pay, amish people may go without needed health care services if they believe the community burden would be too great. d. amish individuals may seek health care options outside the united states to minimize costs. . which of the following is not correct? a. pharmacists should build trusting relationships with members of the local amish community in order to be accepted as health care practitioners. b. knowledge of relevant cultural and district differences is an important aspect of providing competent pharma- cist care. c. all old order amish adhere to the same philosophies, and pharmacists should treat all members the same. d. individualized patient-centered pharmacist care is needed by amish patients. www.pharmacist.com december • pharmacy today review go to www.pharmacist.com and take your test online for instant credit. . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d . a b c d ce examination form ce assessment questions—answers please circle your answers (one answer per question). participant information name address cit y state zip e-mail work phone home phone how long did it take you to read the program and complete this test? _____ hours ____ minutes my signature certifies that i have independently taken this ce examination: excellent poor please rate the following items. . overall quality of the program . relevance to pharmacy practice . value of the content please answer each question, marking whether you agree or disagree. . the program met the stated learning objectives: agree disagree after reading this ce article, the pharmacist should be able to: 	 •	list four potential outcomes of effective cultural competency techniques. ❏ ❏ 	 •	provide	six	examples	of	cultural	competency	techniques	that	are	applicable	to	amish	cultures. ❏ ❏ 	 •	state health beliefs and explanatory models of health and illness accepted in amish cultures. ❏ ❏ 	 •	differentiate the three sectors of the health care system that may be used by amish people. ❏ ❏ 	 •	name four reasons for lower vaccination rates among the amish. ❏ ❏ 	 •	identify examples of how pharmacists can help alleviate logistical, communication, and cost barriers to effective health care provision for amish patients. ❏ ❏ . the program increased my knowledge in the subject area. ❏ ❏ . the program did not promote a particular product or company. ❏ ❏ impact of the activity the information presented (check all that apply) : . ❏ reinforced my current practice /treatment habits ❏ will improve my practice /patient outcomes ❏ provided new ideas or information i expect to use ❏ adds to my knowledge . will the information presented cause you to make any changes in your practice? ❏ yes ❏ no . how commit ted are you to making these changes? (very commit ted) (not at all commit ted) . do you feel future activities on this subject mat ter are necessary and /or important to your practice? ❏ yes ❏ no program evaluation pharmacists’ considerations when serving amish patients follow-up as part of our ongoing quality-improvement effort, we would like to be able to contact you in the event we conduct a follow-up survey to assess the impact of our educational interventions on professional practice. are you willing to participate in such a survey? ❏ yes ❏ no this ce will be available online at www.pharmacist.com af ter december , . to receive . contact hours of continuing education credit ( . ceu), please pro- vide the following information: . type or print your name and address in the spaces provided. . mail this completed form for scoring to: american pharmacists association—ce exam p.o. box baltimore, md - . ce processing is free for apha members. if you are not an apha member, please enclose a $ handling fee for grading the assessment instrument and issuing the statement of credit. a statement of credit will be awarded for a passing grade of % or bet ter. if you fail the exam, you may retake it once. if you do not pass the second time, you may no longer participate in this continuing pharmacy education program. please allow weeks for processing. pharmacists who complete this exercise success- fully before december , , may receive credit. the american pharmacists association is accredited by the accredi- tation council for pharmacy education as a provider of continuing pharmacy education. the acpe universal program number assignedto the program by the accredited provider is - - - -h -p. pharmacy today • december www.pharmacytoday.org classifiedadvertising classifi edadvertising for more great jobs—go to www.pharmacist.com may • pharmacy today cvs caremark redefi nes pharmacy by integrating the personalized reach of the nation’s largest retail pharmacy with the innovative delivery technology of the nation’s premier pharmacy benefi ts management provider. the result is health care that has the ability to enhance the lives of millions of patients in the united states. we leverage the tremendous resources of cvs caremark to provide our pharmacists limitless possibilities to grow personally and professionally. we seek only the best pharmacists to join our team and advance the quest to deliver outstanding health care every day. everyday redefi ning health care... cvs caremark is an equal opportunity employer supporting a drug-free work environment. of�$ �%sjwf�t� ppotpdlfu
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? j a n u a r y n v o l u m e , n u m b e r p h a r m a c y t o d a y n w w w . p h a r m a c i s t . c o m nilotinib for cml heart health update rxwiki: pharmacy facts by the keystroke supervalu pharmacist sherry butler brings small-town service to patients pqa paves the way for high-quality health care disciplinary action for tpn error icaac : infectious disease update for pharmacists p visit www.pharmacytoday.org ° j a n u a r y n v o l u m e , n u m b e r p h a r m a c y t o d a y n w w w . p h a r m a c i s t . c o m nilotinib for cml heart health update rxwiki: pharmacy facts by the keystroke supervalu pharmacist sherry butler brings small-town service to patients pqa paves the way for high-quality health care disciplinary action for tpn error icaac : infectious disease update for pharmacists visit www.pharmrmacytoday.org v p y y g ° j a n u a r y n v o l u m e , n u m b e r p h a r m a c y t o d a y n w w w . p h a r m a c i s t . c o m nilotinib f or cml heart hea lth update rxwiki: p harmacy f acts by the key stroke super valu pha rmacist sherry bu tler brings small- town serv ice to pati ents pqa pave s the way for high-quali ty health c are disciplina ry action f or tpn error icaac : inf ectious diseas e updat e for ph armaci sts p visit www.p harmacytod ay.org ° and did you know, as a practicing pharmacist, you are eligible to receive a full-year subscription to pharmacy today, absolutely free? be in the know by joining thousands of informed pharmacists who subscribe to pharmacy today and receive the regularly featured mtm tip, continuing education, product showcase, ismp error alert, otcs today, counseling corner tip, and much more! did you know… some things can’t be measured by degrees as the profession of pharmacy becomes more specialized and more demanding, the board of pharmaceutical specialties offers a unique opportunity to demonstrate advanced clinical profi ciency in fi ve major practie areas: nuclear pharmacy, nutrition support pharmacy, pharmacotherapy, oncology pharmacy, and psychiatric pharmacy. visit the bps web site for details. board of pharmaceutical specialties www.bpsweb.org th street, nw, suite | washington, dc - pharmacy today • december www.pharmacytoday.org c olor-coded schemes for ophthalmic products have produced look-alike problems for nurses and pharmacists for many years. at the request of ophthalmol- ogists seeking a method to help differen- tiate eye products on their office trays, the american academy of ophthalmology (aao) endorsed a color-coding scheme in that was later approved by fda for use by manufacturers. this voluntary scheme is based on therapeutic class; for example, anti-infective ophthalmic containers and carton labels use a tan background, mydriatics and cycloplegics use red, miotics use green, beta-blockers use yellow or blue, and so on. for details, refer to www.aao.org /about /policy/ upload/color_codes_for_topical_ocu- lar_medications.pdf. although aao intended to reduce errors with this policy, color coding instead often makes items in the same therapeutic class much more difficult to differentiate. because of similar and highly stylized corporate logos, fonts, package sizes, and color combinations, designs that seem to work well in an office setting or patient’s home do not necessarily translate to pharmacies or other clinical locations. as a result, the usp–ismp medication error reporting program has long received reports of mix-ups among items within each class. no to grab and go errors with ophthalmic prod- ucts can occur with dispensa- tion or administration of these products on nursing units, in ophthalmology clinics, and in hospital and ambulatory care pharmacies; however, ophthalmologists continue to endorse the system. “the sys- tem results in a time saver for the physi- cian who can read the label on the drugs once a day—at the beginning of the day,” said an aao spokesperson at a fda center for drug evaluation and research hearing on the issue. this “grab and go” approach to selecting medications with- out fully reading the label can lead to medication errors. despite ismp’s efforts over the years to convince aao, fda, and drug manu- facturers that color coding can lead to errors, no changes have been made to improve safety. confusion among these products continues, as evidenced by a recently reported mix-up between vials of bausch & lomb cyclopentolate % and tropicamide % (figure ). bausch & lomb is not alone in its adherence to the color-coding scheme. for example, for many years, merck’s timoptic (timolol) ophthalmic contain- ers had different colored caps, depend- ing on the drug’s concentration; light blue represented . %, while yellow was . %. however, merck recently started using yellow caps for both con- centrations based on an aao-endorsed update to the color-coding system. the old system recommended either blue or yellow caps for beta-blockers; the new scheme assigns yellow caps to beta- blockers and dark blue caps to beta- ismperroralert caution regarding color-coded eye meds blocker combinations. mix-ups have occurred with simi- lar packaging between ophthalmic and otic drops as well, where ear drops are instilled into the eyes. a community pharmacy reported that a prescription was written for ocuflox (ofloxacin) . % ophthalmic drops ml. the prescription was entered into the pharmacy order entry system cor- rectly and days after the eye drop was dispensed, the child’s mother called and said she had noticed the phrase “for use in the ears only” on her child’s eye drops. she was advised to discontinue the otic drops that had been given to her in error, and the ophthalmic preparation was dispensed. carton coding not enough cartons of alphagan p (brimonidine tar- trate—allergan) ophthalmic solution use color effectively to differentiate between the two available strengths of . % and . % (figure ). however, the enclosed bottles are not color coded and appear virtually identical. as a result, they could easily be replaced in the incorrect carton, increasing the risk of error. alphagan p is an alpha- adrenergic agonist indi- cated for lowering intraocular pressure in patients with open-angle glaucoma or ocular hypertension. ismp has contacted allergan to ask that the actual bottles be color coded to match the cartons. ismp has also requested that the size of the text denot- ing the strength be increased. in the meantime, outpatient areas, patient care facilities, and patients should avoid stor- ing alphagan p bottles outside of their cartons. —institute for safe medication practices the reports described in this column were received through the usp–ismp medication errors reporting program (merp). errors, close calls, or hazardous conditions may be reported on the institute for safe medication practices (www.ismp.org) or u.s. pharmacopeia (www.usp.org) web sites or communicated directly to ismp by calling -fail-saf ( - - ) or e-mailing ismpinfo@ismp.org. the topics in this column are covered in greater detail in medication errors, nd edition, written by ismp president michael r. cohen, bpharm, ms, scd. the book may be purchased from apha at www.pharmacist.com or by calling – – . figure . a patient recently received atropine instead of cyclopentolate. figure . color helps differentiate strength on cartons but is missing from bot tles. the american pharmacists association (apha) and the national association of chain drug stores (nacds) are pleased to continue to offer a four-part webinar series presenting cutting-edge topics related to pharmacy-based immunization programs. the webinar series will highlight innovations in pharmacy- based immunization related to practice, advocacy, and science. pharmacy-based immunization webinar series: innovations in practice, advocacy, & science wednesday, january , , : p.m. est federal and state regulatory update: increasing opportunities for pharmacist-provided immunizations thursday, february , , : p.m. est challenges of pharmacy-based immunization services: key operational issues tuesday, march , , : p.m. est emergency preparedness and the role of immunizing pharmacists wednesday, april , , : p.m. est innovations in vaccine science more details and registration coming soon… visit www.nacds.org/immunization or www.pharmacist.com for more detailed program information in the near future! the national association of chain drug stores (nacds) foundation and the american pharmacists association (apha) are accredited by the accreditation council for pharmacy education (acpe) as providers of continuing pharmacy education. each webinar offers participants the opportunity to earn . contact hours ( . ceu) of continuing pharmacy education credit. statements of continuing pharmacy education credit may be printed online by visiting the nacds foundation's ce center and completing an evaluation for each program. then save the date now for these free live ce webinars! attention pharmacists do you immunize? educate? advocate? supported, in part, by independent educational grants from: new so kids can feel better sooner. new kids-eeze ® chest relief hones in and relieves chest congestion without overmedicating. unlike multi-symptom remedies, kids-eeze chest relief with guaifenesin targets chest congestion without overmedicating. and only kids-eeze chest relief comes in a convenient, great-tasting chew with patented taste-masking technology. which means no more messy spoons or yucky medicine aftertaste, so it’s easy on parents and kids. recommend it today, and you’ll be helping kids and moms both breathe easy. one symptom. one remedy. equals a ton of happy moms. one symptom. one remedy. equals a ton of happy moms. kids-eeze.com wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ journal club kidney international ( ) j o u r n a l c l u b http://www.kidney-international.org © international society of nephrology kidney international ( ) , – . doi: . /ki. . shorter sleep duration associated with coronary artery calcification the design of phase iv clinical trials in nephrology has made a decided move toward the use of intermediate outcomes. while this is an advantage in that generally fewer subjects need to be enrolled and followed for shorter periods of time, the inherent caveat lies in the validation of the intermediate outcome as a true surrogate of the hard clinical outcomes such as mortality or cardiovascular events. many argue that the validation of coronary artery calcification (cac) among persons with abnormal kidney function is not complete. a new article by king et al. provides insight into the investigation of the potential mechanisms of its genesis. in this observational cohort study, healthy participants from a sin- gle site in the coronary artery risk development in young adults (cardia) study were included. cac was measured by computed tomography in – and – . participants had no detect- able calcification at baseline, and their incidence of new calcification was the primary outcome. sleep metrics (wrist actigraphy-measured duration and fragmentation, daytime sleepiness, overall quality, self- reported duration) along with potential confounders (age, sex, race, education, apnea risk, smoking status) and mediators (lipids, blood pressure, body mass index, diabetes, inflammatory markers, alcohol consumption, depression, hostility, self-reported medical conditions) were measured and examined for association with incident calcifi- cation. approximately % developed calcification over the -year observation period. longer sleep duration was significantly associated with a decreased risk of new calcification (p = . ). this association persisted in models adjusted for demographics and for medical history including apnea risk, as well as known mediators of calcification such as lipid and blood pressure parameters. the authors suggest inflam- matory and hormonal mediators as causes of both cac and decreased sleep duration, and decreased sleep duration resulting in transient decreases in glucose tolerance as a cause of cac. these findings sug- gest that a renewed interest in sleep may benefit our patients’ health in addition to their quality of life. additionally, we must continue to be objective about our understanding of the pathophysiology of arterial calcification as we scrutinize metrics such as the cac score as sur- rogate markers. (jama ; : – ) lynda szczech jama ; : – . met and the epidermal growth factor receptor cooperatively regulate final nephron number kidney development occurs by the interactions between the ureteric bud and the adjacent metanephric mesenchyme. while the ureteric bud branches to form the collecting ducts, the more proximal parts of the nephron develop via mesenchymal–epithelial transforma- tion of the metanephric mesenchyme cells in contact with the tips of ureteric bud branches. the result of this interplay is that the final nephron number is determined by the signals that regulate ureteric bud branching. a number of these signals have been identified, but there is little doubt that more remain to be discovered. during kid- ney development, hgf is expressed by the metanephric mesenchyme, whereas its receptor (met) is present on both ureteric bud and mes- enchymal cells. in vitro work with neutralizing antibodies against hgf showed inhibition of ureteric bud branching, suggesting an impor- tant role for hgf. however, loss of either hgf or met expression in the mouse led to death by approximately embryonic day , and early kidney development appeared normal. because ureteric bud branch- ing continues through embryonic day , the exact role of hgf–met signaling in the development of the collecting system and nephron number remained unclear. ishibe et al. now use cre-loxp technology to address this question. they found that deletion of the met recep- tor selectively in the collecting system (met fl/fl;hoxb -cre) did not result in abnormal morphology, but the kidneys of these mice had a % reduction in nephron number at weeks of age and glomerular hypertrophy by year of age (figure). examination of these collecting ducts revealed sustained upregulation and activation of the egf recep- tor. moreover, addition of egf to embryonic kidneys (with deleted met in the ureteric bud) grown in vitro rescued the decrease in ureteric bud branching observed in vivo. finally, mice lacking both met and egfr signaling in the collecting duct had a marked decrease in ureteric bud branching, small kidneys, renal failure, and early death. thus, met and the egf receptor act cooperatively to regulate branching of the ureteric bud and nephron number. (development ; : – ; doi: . / . /dev. ) juan oliver vasopressin signaling pathway in primary cilia of renal epithelia the primary cilium is a solitary, sensory organelle projecting from the apical surface of many cells. most renal tubular epithelial cells of deletion of met in collecting duct (metfl/fl;hoxb -cre) reduces nephron number. (a) glomeruli at and weeks from metfl/fl;hoxb -cre and met+/+;hoxb -cre (control) mice. (b) quantification of glomerular surface area from kidney sections. (c) representative image of isolated glomeruli from metfl/fl;hoxb -cre and control mice. (d) quantification of total glomeruli per mouse in metfl/fl;hoxb -cre and control. pu b lis he d b y th e co m pa ny o f b io lo gi st s © http://www.kidney-international.org http://www.biologists.com/web/cob_copyright.html kidney international ( ) j o u r n a l c l u b the mammalian nephron, with the exception of intercalated cells in the collecting duct, contain a single primary cilium, which responds to fluid flow or mechanical bending by eliciting cytosolic calcium + signals. the signaling pathways for the regulation of ciliary-located channel function are unknown. in a recent publication, raychowd- hury et al. report the presence of type vasopressin receptor (v r) in primary cilia of llc-pk cells (renal epithelial cells derived from pig kidneys). while hormone receptors in renal tubular epithelial cells have been mostly located to the basolateral membrane of the cells, several receptors, such as v r and parathyroid hormone receptor, as well as the vasopressin type receptor, have been found in the luminal aspect of tubular epithelia. the function of these luminal receptors remains unclear. the authors demonstrated the presence of v r in the cilia by the spontaneous fluorescence of a v r-gfp chimera (figure) and confirmed it by immunocytochemi- cal analysis in llc-pk cells stained with anti-v r antibodies and in llc-pk cells overexpressing a v r-flag chimera, with an anti- flag antibody. they also demonstrated that ciliary v r colocalized with adenylyl cyclase in all cell types tested. finally, they demon- strated functional coupling of the ciliary receptors with adenylyl cyclase by measuring cyclic adenosine monophosphate (camp) production in isolated cilia and by testing vasopressin-induced cation-selective channel activity either in reconstituted lipid bilay- ers or by membrane-attached patch clamping: the addition of either vasopressin (trans) or forskolin (cis) stimulated cation-selective channel activity in ciliary membranes. these results provide the first demonstration of v r in primary cilia of renal epithelial cells, and a functional camp-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium. (am j physiol renal physiol ; : f –f ; doi: . /ajprenal. . ) juan oliver stk is a hypertension susceptibility gene essential hypertension (eh) is widely believed to involve multi- ple genes with variant alleles. as with other complex disorders, manifestation of eh in any single individual is likely dependent on a variety of genetic and environment factors, making identifi- cation of eh susceptibility genes in the general population a major challenge. in contrast, studies on the genetic basis of rare blood pressure monogenetic disorders have identified mutations in genes affecting a single physiological pathway: renal salt transport. although such studies highlight the importance of extracellular fluid volume to blood pressure regulation, the underlying genetic basis for eh remains poorly understood. to identify common eh susceptibility genes, wang et al. conducted a genome-wide asso- ciation analysis in the old order amish, a closed population with descendents from a small number of founders that has a relatively homogeneous lifestyle, making it ideal for identifying genes that underlie complex diseases. they analyzed genotype data for systo- lic blood pressure and diastolic blood pressure in subjects. this approach identified a novel hypertension susceptibility gene, stk (a serine/threonine kinase gene), in which common vari- ants were associated with blood pressure levels. they confirmed this association in an independent amish and four non-amish caucasian samples, including the diabetes genetics initiative, framingham heart study, gennet, and hutterites. functional studies in cells showed that stk interacted with wnk kinases and cation-chloride cotransporters, mutations that are known to cause monogenic forms of blood pressure disorders. the authors also found that stk was expressed in the distal nephron (fig- ure), where it may interact with these proteins. although none of the associated single-nucleotide polymorphisms altered pro- tein structure, they identified and experimentally confirmed a highly conserved element with in vitro transcription activity as a functional candidate for the association. thus, variants in stk may influence blood pressure by increasing stk expression and consequently altering renal na+ excretion, thus unifying rare and common blood pressure-regulating alleles in the same physi- ological pathway. (proc natl acad sci usa ; : – ; doi: . /pnas. ) juan oliver localization of v r in primary cilia of renal epithelial cells. v r was localized to the primary cilia of confluent wild-type llc-pk renal epithelial cells (wt) and further confirmed in cells expressing either the v r-flag or the v r-gfp chimera. cells were also stained with mouse anti-acetylated tubulin antibody (ac-tubulin; red). dapi fluorescence (blue) is shown for contrast. u se d w it h p er m is si o n fr o m a m j ph ys io l r en al p hy si ol immunolocalization of spak in kidney. (a) spak strongly localizes to the thick ascending limb (tal) of the loop of henle and the cortical collecting duct (ccd). (b) immunolocalization of aquaporin in the same section as in a as a segment-specific marker for ccds. (c) immunolocalization of spak in the distal convoluted tubules (dct). (d) immunolocalization of sodium chloride cotransporter as a segment-specific marker for dcts. r ep ri n te d w it h p er m is si o n fr o m p ro c n at l a ca d sc i u sa http://cancerweb.ncl.ac.uk/cgi-bin/omd?adenosine+monophosphate 	shorter sleep duration associated with coronary artery calcification 	met and the epidermal growth factor receptor cooperatively regulate final nephron number 	vasopressin signaling pathway in primary cilia of renal epithelia 	stk is a hypertension susceptibility gene mennonites, amish, and the american civil war (review) mennonites, amish, and the american civil war (review) sean a. scott ohio history, volume , , pp. - (review) published by the kent state university press doi: for additional information about this article [ this content has been declared free to read by the pubisher during the covid- pandemic. ] https://doi.org/ . /ohh. . https://muse.jhu.edu/article/ https://doi.org/ . /ohh. . https://muse.jhu.edu/article/ bo ok reviews of these men joined harrod and bowman on the unsuccessful ohio foray, and subsequently “capt. wm. harrod, with others, went up the ohio to red stone in the two keel boats &c. and took along several bones & tusks got at the big bone lick.” what became of these specimens remains unknown, but this was an important early effort at “mining” the deposits at big bone. similarly, neither jillson nor hedeen mentions thomas rodney’s well-doc- umented stop there on october , , less than a week after meriwether lewis’s collecting visit for thomas jefferson. rodney not only took measure- ments of several of the remaining fossil bones but speculated (incorrectly) that no animals larger than bison ever existed there, larger bones being “only fossil concretions.” rodney himself collected a mastodon tooth and a tusk fragment, but these were lost when his boat sank at natchez. curiously, hedeen merely reproduces without explanation a photograph of three paleo-indian points “collected” at big bone lick. elsewhere, he has written that these were discovered in the lowest level of the big bone de- posits during william clark’s excavations for thomas jefferson and are “now likely” housed at the cincinnati museum center. the significance of associated paleo-indian artifacts and megafauna at big bone lick should certainly be acknowledged but so too should the considerable debate exist- ing about the precise provenance and pedigree of these artifacts. such strictures aside, this book provides ample context for the present big bone lick state park museum and should remain a standard resource for many years. ja m e s l . m u r p h y the ohio state university mennonites, amish, and the american civil war. by james o. lehman and steven m. nolt. (baltimore: johns hopkins university press, . xiv, pp. cloth $ . , isbn - - - .) when war engulfed virginia in the summer of , mennonite christian good was conscripted into the state militia. after good experienced battle and failed to discharge his rifle, his perturbed captain asked him why he . henry hill, “bowman’s campaign— ,” ohio archaeological and historical publica- tions ( ): . . simon gratz, “thomas rodney,” pennsylvania magazine of history and biography . ( ): ‒ . . stanley hedeen, natural history of the cincinnati region. cincinnati museum center scientific contributions no. (cincinnati: the center, ), . in the same publication, these artifacts are captioned only as “most likely [discovered] during william clark’s expedition” (plate ). ohio history had refused to shoot at union soldiers in clear view. the committed pacifist reportedly responded, “they’re people; we don’t shoot people” ( ). while most protestants north and south wholeheartedly supported their governments and sent scores of soldiers to fight in the civil war, members of anabaptist sects struggled to balance the conflicting duties of citizen- ship in both an earthly state and a heavenly kingdom. as religious outsiders who traced their origins to the radical fringe of the protestant reformation, many mennonites and amish had settled in america to avoid compulsory military service in europe. more significant than challenging their civic loyalty and threatening their commitment to the principles of pacifism, the civil war forced them to engage the political and cultural mainstream and adapt to the contingencies of war in order to protect their distinctive and countercultural religious beliefs. in a compelling narrative, lehman and nolt effectively recount the unique circumstances of american anabaptists in three geographic regions. in vir- ginia’s shenandoah valley, conscripted pacifists could secure an exemption from military service by paying a $ commutation fee or furnishing a substitute. although a few mennonites fought for the confederacy, many more attempted to flee north and avoid supporting the rebellion. during the autumn of , the union’s hard war policy resulted in the destruction of several mennonite farms, and these southern conscientious objectors suf- fered at the hands of rebels who confiscated their crops and livestock and federals who destroyed what remained. pennsylvania mennonites, in contrast, benefited from political ties to radical republican senator thaddeus stevens, who helped his constituents secure military exemptions from the state militia without paying a commu- tation fee. most pennsylvania pacifists realized that political participation was essential to the preservation of their religious privileges. for several reasons, midwestern mennonites and amish lacked the po- litical connections of their pennsylvania brethren. many pacifists drafted in ohio and indiana paid a $ commutation fee or hired substitutes to avoid service. several ohio anabaptists supported the democratic party, and townships with large pacifist populations in holmes and wayne counties produced large majorities for clement vallandigham in the gubernato- rial contest. however, by the campaigns of , many midwestern menno- nites had adopted the apolitical stance espoused by john funk, who began publishing the herald of truth in elkhart, indiana, and asserted in its pages that a consistent application of pacifist principles prevented a conscientious objector from voting, seeking public office, or purchasing a substitute. most mennonites and amish, the authors conclude, remained true to their spiritual convictions despite the turbulent conditions of civil war. by highlighting the struggles of these religious outsiders who strived to keep bo ok reviews the church distinct from the world, lehman and nolt have produced an insightful study that further elucidates the centrality of religion for a proper understanding of the civil war. s e a n a . s c o t t ouachita baptist university politician extraordinaire: the tempestuous life and times of martin l. davey. by frank p. vazzano. (kent, ohio: kent state university press, . xiv, pp. cloth $ . , isbn - - - - .) with his biography of martin davey, frank vazzano sets out to tell the “good story” of a “very, very interesting man” (x). he accomplishes this goal admi- rably as an “unabashed story teller” and as an historian. certainly, no one knows davey better than the author who has written three articles about davey’s terms as governor, all of which appeared in ohio history. an effective biography not only examines the subject’s life but also the nature of place and time and the meaning of change. as the title indicates, vazzano understands this well. vazzano is especially successful in describing davey’s life and times during his formative years and early career during the late nineteenth and the early twentieth centuries. born of an immigrant father obsessed with the apparently quixotic quest for scientific tree care and raised in poverty in small town kent, ohio, davey grew to manhood as a gifted salesman and businessman, community booster, and brilliant politician. vaz- zano captures the transformation of davey during changing times as he turns his father’s vision into a successful business with customers across much of the nation and helps “boost” kent from a nineteenth-century village with dirt streets to a modern community with all the amenities of public services. vazzano successfully captures the emergence of modern america and the nature of local politics. barely out of his teens, davey is traveling to cleveland to sell his father’s book, the tree doctor, and then new york to promote the davey tree expert company. he boldly cultivates captains of industry, wins the tree care of the capitol grounds, and uses advertising to creatively expand the reach of the business. at the same time, vazzano aptly shows davey mastering local politics: the network of friendships and personal alliances, the use of patronage to reward friends and define politi- cal power, the necessity of strong organization, and the efficacy of public speaking. he was elected mayor of kent, served well, and, as countless poli- ticians before and after him, used that base to win higher office. although one of ohio’s most successful politicians in the twentieth cen- tury who served multiple terms as mayor, in congress, and as governor of ohio, davey remains elusive as a political leader. vazzano documents davey’s brilliance and limitations as a politician; his organizational skills, his tireless wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ pteridines € eur - euro £ gbp - pound $ usd - dollar en english deutsch × your purchase has been completed. your documents are now available to view. × changing the currency will empty your shopping cart. confirm cancel pteridines official journal of the international society of pteridinology issn: - first published: april , editor-in-chief: dietmar fuchs impact factor: . 	overview 	latest issue 	issues 	ranking 	submit 	editorial about this journal objective pteridines is an open acess international quarterly journal dealing with all aspects of pteridine research. pteridines are heterocyclic fused ring compounds involved in a wide range of biological functions from the color on butterfly wings to cofactors in enzyme catalysis to essential vitamins. of the pteridines, , , , -tetrahydrobiopterin is the necessary cofactor of several aromatic amino acid monoxygenases, the nitric oxide synthases and glyceryl ether monoxygenase (gemo). neopterin plays an essential role in the immune system and is an important biomarker in laboratory medicine for diseases such as hiv, cardiovascular disease, malignant tumors, among others. topics 	neopterin, dihydroneopterin, monapterin 	biopterin, tetrahydrobiopterin 	folates, antifolates, riboflavin 	phenylalanine, tyrosine, phenylketonuria, serotonin, adrenalin, noradrenalin, l-dopa, dopamine, related biogenic amines 	phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, nitric oxide synthases (inos), alkylglycerol monooxygenase (agmo), dihydropterin reductase, sepiapterin reductase 	homocysteine, mediators of inflammation, redox systems, iron article formats original articles, reviews, mini reviews, commentaries, opinions on controversial subjects > information on submission process your benefits your benefits: 	unique interdisciplinary forum devoted to conjugated and unconjugated pteridines 	basic and clinical approach to the topic combining the fields of chemistry, biochemistry, clinical chemistry, and laboratory medicine 	international, authoritative editors and reviewers boosting the prestige of published papers 	free language-correction services for authors from non-english speaking regions 	worldwide dissemination and unrestricted access to all published articles 	promotion of each published article 	secure archiving by de gruyter and the independent archiving service portico 	no submission charges pteridines is the official journal of the international society of pteridinology (isp). history pteridines was founded in and has been continued at de gruyter since . abstracting and indexing pteridines is covered by the following services: 	baidu scholar 	biobase 	cabells journalytics 	case 	chemical abstracts service (cas) - caplus 	chemical abstracts service (cas) - scifinder 	chronos hub 	cnki scholar (china national knowledge infrastructure) 	cnpiec - cnplinker 	dimensions 	doaj (directory of open access journals) 	ebsco (relevant databases) 	ebsco discovery service 	embase 	genamics journalseek 	gooa 	google scholar 	index copernicus 	j-gate 	journal citation reports/science edition 	journalguide 	journaltocs 	kesli-ndsl (korean national discovery for science leaders) 	meta 	microsoft academic 	mysciencework 	naver academic 	naviga (softweco) 	primo central (exlibris) 	proquest (relevant databases) 	publons 	qoam (quality open access market) 	readcube 	reaxys 	scimago (sjr) 	scopus 	semantic scholar 	sherpa/romeo 	summon (proquest) 	tdnet 	text mining 	ulrich's periodicals directory/ulrichsweb 	wanfang data 	web of science - science citation index expanded 	worldcat (oclc) 	yewno discover supplementary materials 	 author''s ethical statements 	 editorial policy 	 data sharing policy 	 article processing charges 	 instruction for authors 	 privacy statement 	 guidelines for reviewers 	 open access license 	 publication ethics statement topics 	biochemistry 	human biology 	life sciences 	molecular biology external links 	journal archive 	open access statement 	crosscheck plagiarism screening 	online submission of manuscripts 	open access statement 	crosscheck plagiarism screening 	privacy statement volume issue january unable to retrieve citations for this document × retrieving citations for document... laboratory diagnostic value of neopterin measurements in patients with covid- infection dietmar fuchs, magnus gisslen january , page range: - more cite open access pdf pdf abstract the new coronavirus sars-cov- was identified to be responsible for the covid- pandemic. there are striking differences in the response to infection, some people develop no or mild symptoms, while other outcomes are severe of even fatal. for those covid- patients who require hospitalization, prognostic markers could help clinicians to identify patients with a poor outcome early. the serum levels of the immune activation marker neopterin have already been shown to be of prognostic value in patients with sars-cov- and a similar pattern can be observed for sars-cov- . this comment discusses the biochemical circuits that support the clinical value of neopterin measurements in covid- patients. unable to retrieve citations for this document × retrieving citations for document... norepinephrine was superior in death risk reducing and hemodynamics compared to dopamine in treatment of patients with septic shock xudong lu, xianghua xu, yueying wu february , page range: - more cite open access pdf pdf abstract background to investigate the clinical effects of norepinephrine versus dopamine in treatment of septic shock by pooling the data form open published clinical trials. material and methods the clinical trials relevant to norepinephrine versus dopamine in treatment of septic shock were electronically searched in the databases of pubmed, embase, the cochrane library, web of science, google scholar and cnki. the original data related to the treatment effects such as death risk, oxygen metabolism and hemodynamics index were extracted from the included original studies. the death risk was pooled by the effect size of relative risk (rr), the oxygen metabolism and hemodynamics index were pooled by standard mean difference (smd) and the corresponding % confidence interval ( %ci). the publication bias was evaluated by begg's funnel plot and egger's line regression test. results thirteen clinical trials were included in the meta-analysis. the pooled results demonstrated the death risk was significantly decreased (rr= . , %ci: . to . , p= . ) in septic shock patients who received norepinephrine compared to those receiving dopamine. the hr (smd=− . , %ci: − . to − . , p< . ) and cardiac index (smd=− . , %ci: − . to − . , p< . ) were lower in norepinephrine group compared to dopamine group. the systemic vascular resistance index (smd= . , %ci: . to . , p< . ) in norepinephrine group was higher than those of dopamine group with statistical difference. the begg's funnel plot and egger's line regression test (t=− . , p= . ) showed no publication bias. conclusions based on the present evidence, norepinephrine was superior to dopamine in the aspects of death risk reducing and hemodynamics. unable to retrieve citations for this document × retrieving citations for document... clinical significance of serum homocysteine as a biomarker for early diagnosis of diabetic nephropathy in type diabetes mellitus patients bin ye, xiangying zhu, zhifu zeng, xiaozhen ji, meixia ji march , page range: - more cite open access pdf pdf abstract objective the aim of this study was to investigate the clinical significance of serum homocysteine (hcy) as a biomarker for early diagnosis of diabetic nephropathy (dn) in type diabetes mellitus (t dm) patients. methods fifty-five t dm patients with dn and t dm patients without dn were prospectively recruited from january to may in our hospital. the serum hcy was tested by electrochemiluminescence assay in dn and t dm groups and compared. the diagnostic efficacy of serum hcy as a biomarker for early diagnosis of dn was evaluated by calculating the diagnostic sensitivity, specificity and area under the roc curve (auc). results the serum levels of hcy were . ± . and . ± .  μmol/l for dn and t dm patients, respectively, with statistical difference ( t = . , p < . ). in the dn group, the serum hcy levels for patients with hyperfiltration, intermittent proteinuria, microalbuminuria, macroalbuminuria and uremic were . ± . , . ± . , . ± . , . ± . and . ± .  μmol/l, respectively, which indicated that serum hcy levels in dn were higher than those of t dm patients and correlated with patient’s renal damage. using the serum hcy level as the reference, the diagnostic sensitivity, specificity and auc were . ( . – . )%, . ( . – . )% and . ( . – . )%, respectively, with the cutoff value of . between dn and t dm. the serum hcy also had relatively good differential diagnostic efficacy between different dn stages with high sensitivity, specificity and auc. conclusion serum hcy was obviously elevated in dn compared to t md and correlated with the renal damage severity, which can be applied as a potential serological marker for early diagnosis of dn. unable to retrieve citations for this document × retrieving citations for document... the urinary biopterin in autism spectrum disorder aleksandra waligóra, aleksandra damasiewicz-bodzek, piotr gorczyca, sławomir waligóra, krystyna tyrpień-golder march , page range: - more cite open access pdf pdf abstract objective the aim of the study was to determine whether biopterin is present in significantly lower quantities in urine samples of patients with autism spectrum disorder (asd) compared to healthy individuals. methods the concentration of biopterin in urine samples was measured by elisa using commercially available kit. the study involved children aged – years with asd and healthy children aged – years. results significantly lower biopterin concentration was observed in autistic patients compared to the control group. however, no significant difference was observed between mild, moderate, and severe asd. conclusion one of the potential causes of decrease in urinary biopterin levels may be tetrahydrobiopterin (bh ) deficiency, which has extensive and serious health consequences for the nervous system. the results of measuring biopterin as a fully oxidized form of bh may suggest that biosynthesis or regeneration of bh may be decreased in children with asd. on the other hand, decreased urinary biopterin levels in children with asd may be due to bh overuse, a good regeneration process, and decreased urinary excretion; and abnormalities in bh metabolism appear to be related to the aetiology of asd or may be due to asd. volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue - 	 issue 	 issue volume ( ) 	 issue - 	 issue - volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue - 	 issue 	 issue volume ( ) 	 issue - 	 issue 	 issue special issue: th international symposium on pteridines and folates, antalya, turkey, may – , volume ( ) 	 issue - 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue volume ( ) 	 issue volume ( ) 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue - volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 	 issue 	 issue 	 issue 	 issue volume ( ) 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(tianjin lung cancer institute, tianjin medical university general hospital, tianjin, china) editorial office publisher de gruyter poland bogumiła zuga a str. - warsaw, poland t: + editorial contact jędrzej daszkiewicz, managing editor jedrzej.daszkiewicz@degruyter.com​ open access details 	online issn: - 	 type: journal 	 language: english 	 publisher: de gruyter 	 first published: april , 	 publication frequency: issue per year 	 audience: researchers in the fields of chemistry, biochemistry, clinical biochemistry, metabolic disorders, clinical chemistry, clinical immunology, immunology, laboratory medicine, biology, molecular biology, molecular medicine, nutrition, phytomedicine 	contact us 	customer service 	human resources 	press 	contacts for authors 	career 	how to join us 	current vacancies 	working at de gruyter 	open access 	articles 	books 	funding & support 	for authors 	publish your book 	publish your journal article 	abstracting & indexing 	for libraries & trade partners 	electronic journals 	ebooks 	databases & online reference 	metadata 	our partner publishers 	rights & permissons 	repository policy 	free access policy 	about de gruyter 	de gruyter foundation 	our locations 	help/faq 	privacy policy 	terms & conditions 	legal notice © walter de gruyter gmbh wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ proc. nad. acad. sci. usa vol. , pp. - , december genetics identification of a recent recombination event within the human ( -globin gene cluster (meiosis/genetic linkage/chromosome /haplotypes) daniela s. gerhard*, kenneth k. kiddt, judith r. kiddt, janice a. egelandt, and david e. housman* *center for cancer research, massachusetts institute of technology, cambridge, ma ; tdepartment of human genetics, yale university school of medicine, new haven, ct ; and tdepartment of psychiatry, university of miami, miami, fl communicated by edward a. adelberg, august , abstract in a detailed study of inheritance of dna se- quence polymorphism in a large reference pedigree, an indi- vidual was identified with an apparent genetic recombination event within the human j globin gene duster. analysis of the haplotypes of relevant individuals within this pedigree suggest- ed that the meiotic crossing-over event is likely to have oc- curred within a . -kilobase-pair region of the fglobin gene cluster. analysis of other dna markers closely linked to the ( - globin gene cluster-segment of chromosome (d s ) and loci for insulin, the cellular oncogene c-ha-ras, and pre- proparathyroid hormone-confirmed that a crossover event must have occurred within the region of chromosome be- tween d s and the (-globin gene cluster. it is suggested that the event observed has occurred within a dna region compatible with recombinational "hot spots" suggested by population studies. genetic recombination in mammals is a process that has been under study for many decades. attempts to identify the molecular basis of this process have been hindered by the high complexity of mammalian genomes. to precisely ana- lyze meiotic recombination mechanisms, it would be ex- tremely desirable to identify the dna segments that recently have undergone crossing-over at meiosis. to achieve this goal, it is necessary to have available a significant number of genetic markers distributed over a chromosomal dna seg- ment of kilobase pairs (kbp) or less. at present, howev- er, most genetic markers are distributed at such great dis- tances along a mammalian chromosome that precise delinea- tion of the site of a crossover is quite difficult. one region of the human genome that is relatively favorable for a detailed analysis is the , globin gene cluster (hbbc) of human chro- mosome . within a -kbp region starting . kbp ' of the e-globin gene and extending kbp beyond the ' end of the ,b-globin gene, there are at least polymorphic restriction enzyme recognition sites ( ). the question of whether recombination occurs with equal frequency at all sites along the chromosome is one of signifi- cance in understanding the molecular basis of meiotic re- combination. evidence suggesting that frequency of recom- bination is not evenly distributed within this region has been obtained through population studies of these polymorphic loci. antonarakis, orkin, and their collaborators ( , ) have analyzed hbbc haplotypes in various human populations and demonstrated significant linkage disequilibrium among some alleles within the cluster. their data suggest that re- combination events are not evenly distributed within the re- gion. these authors propose that crossovers occur with a relatively high frequency within a localized region of kbp ' of the f globin gene. in the course of linkage studies of a large multigenera- tional pedigree, we analyzed the segregation of several re- striction fragment length polymorphisms (rflps) on the short arm of chromosome , including several in the -glo- bin gene region. this analysis led to the identification of an individual with one chromosome that appears to be a prod- uct of a recombination within the l -globin gene region itself. since detailed analysis of the structure of such recombinant chromosomes should give insight into the distribution of and mechanisms underlying meiotic crossing-over, we report here our initial analyses of this event. materials and methods pedigree studied. the pedigree reported on here is part of the old order amish pedigree no. previously studied by egeland and collaborators ( , ). lymphoblast and fibro- blast cell lines have been established on individuals in this kindred by the institute for medical research (camden, nj). preparations of dna. dna was extracted from cultured cells by standard procedures ( ). analysis of dna polymorphisms. dnas were digested with different restriction enzymes that have been shown to identify polymorphic sites when hybridized with a given probe on a southern blot. the digests were performed with - units/pg of dna overnight under conditions specified by the manufacturer. dnas were run on appropriate-per- centage gel, transferred as described by southern ( ) to ny- lon-based filter, and probed with nick-translated probes ( ). definitions of loci studied. globin haplotypes for each in- dividual were characterized by the use of the following re- striction enzymes in the southern hybridization procedure. the hbbc sites were: (i) the hincii site ' to the e-globin gene ( ); (ii) the hindiii sites present in the intervening se- quence (ivs ) of both g% and ayglobin genes ( , ); (iii) the hincii sites present in the q,p -globin gene and ' to the & -globin gene ( ); (iv) the taq i site ' to the -globin gene called "e" ( ); (v) the hinfl site ' to the f globin gene ( ); (vi) the rsa i site ' to the ,b-globin gene ( ); (vii) the hgiai site in the first exon of the fglobin gene ( ); (viii) the ava ii site in the ivs of the fbglobin gene ( ); (ix) the hindiii site ' to the j globin gene homologous to probe prk- isolated by r. kaufman as described in ref. ; and (x) the bamhi site ' to the fbglobin gene ( ). the four other marker loci used in the study are hrasi (human oncogene c-ha-ras-j), d s (segment of chro- mosome ), ins (insulin gene), and pth [gene for prepro- parathyroid hormone, which is enzymatically cleaved to parathyroid hormone (pth)]. the hrasj locus was typed on dnas digested with bamhi ( , ) and is characterized abbreviations: ins, insulin locus; hrasi, designation for human locus of oncogene c-ha-ras-j; pth, designation for locus of prepro- parathyroid hormone; ivs, intervening sequence; cm, centimor- gans; kbp, kilobase pairs; hbbc, f globin gene cluster. the publication costs of this article were defrayed in part by page charge payment. this article must therefore be hereby marked "advertisement" in accordance with u.s.c. § solely to indicate this fact. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. natl. acad sci usa ( ) y c ar e i o e lt +---- + -a - +-++ ++q -+-+++ +- f +_+ - a +---- + + b -++-+ +-d -++-++ - + e - ++ -+ + - + e i~ ~ .i i i . i i i a a c c / a a c d +___-- + - a -+- ++ + + + c efbbti eb e e f e f e e' a'ae a' e' a' +---- + - e -++-+ + - + c a c c c a a- e e e c a' a a t ir e ~'r + _ __+ + + .___+ - fig. . globin haplotypes of key members of the sibship. partial globin haplotypes are shown for each relevant member. +, presence of a restriction enzyme recognition site; -, absence of the restriction enzyme recognition site. individual x exhibits a unique fglobin haplotype not exhibited by her parents, individuals v and w. the f -globin haplotypes of v and w can be inferred by the / -globin haplotypes of their parents, individuals r and s and t and w, respectively. intact transmission of the f globin gene region of the parents of x to the remaining siblings was observed and indicated by letter designation in table . the designation refers to the bamhi polymorphism ' to the j-globin structural gene; all other sites near the f-globin gene were not segregating in the relevant individual. by variously sized insertion sequences. three alleles were distinguishable. the d s locus was typed on taq i-di- gested dna; the two alleles were defined by presence or absence of the polymorphic taq i site ( ). pth was typed on pst i-digested dna; two alleles were defined by presence or absence of the polymorphic pst i site in the third exon ( ). ins was studied with both sac i and pvu ii. the allelic definitions for this restriction fragment length polymorphism are discussed below with the results and discussion. the es- timated map distances of these markers from the globin clus- ter are centimorgans (cm) to ins, cm to hrasi, cm to d s , and - cm to pth ( - ) (see fig. ). results fig. illustrates the family structure for the sibship in which the -globin haplotype of interest was observed. a possible explanation for an inconsistent globin haplotype would be nonpaternity for individual x; however, previously typed polymorphic marker systems, including hla, (refs. and ; unpublished results) are in complete agreement with the biological relationships shown in fig. . based on the mark- ers alone, the odds of the identified parentage to a random paternal gamete for individual x exceed . x . all other dna polymorphisms studied were consistent with parental genotypes, further increasing the odds that the attributed pa- ternity is correct. these results, which led us to reject non- g ayr hloc a kb paternity as an explanation for the unexpected hbbc haplo- type in individual x, are consistent with previous studies of the general nature of this community ( , ) and personal knowledge (j.a.e.) of this cohesive family unit. the availability of all four grandparents of the core sibship has made the establishment of linkage phase in the two par- ents (v and w in fig. ) unequivocal for the markers in the hbbc as well as for the other loci studied. six different hbbc haplotypes are observed segregating in this pedigree. polymorphic loci within the hbbc are depicted in fig. . eight of the polymorphic sites shown in fig. were used to establish the globin haplotype shown in fig. . haplo- types a and e are each represented twice in the grandpar- ents. the two parents (v and w in fig. ) have the genotypes a/c and a'/e, respectively. these genotypes predict four possible types of offspring: namely, aa', a'c, ce, and a'e. all four combinations are represented; two of the offspring were a'c, four were aa', one was ae, and four were ce. individual x has a genotype that could not have arisen by transmission of the intact / globin gene region carried by the parental chromosomes from her father or her mother. several explanations appeared initially compatible with the data: (i) a recombination within the p -globin gene region, (ii) a gene conversion event within the , globin gene region, (iii) a new mutation at the bamhi site in the germ cell of either individual x's father or mother, and (iv) an artifact due *a hind i |ini hinft l it borm hi toqi hgiai hindm zen fig. . map of the hbbc. arrows indicate the polymorphic restriction enzyme recognition sites._, coding regions; r, -globin pseudo- gene. genetics: gerhard et al d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. natl. acad sci usa ( ) a pth *- f a i - cm d nowm ins hrasi - cm b fig. . (a) order of the five loci studied as determined by linkage analysis. recombination distance between markers on the short arm of chromosome was estimated by two-point analysis (refs. and ; unpublished results). the heavy dot represents the centromere. (b) the predicted orientation of the genes within the hbbc with respect to the centromere. to a mutation at the bamhi site in the cultured lymphoblast cell line from which the dna of individual x was obtained. to address the fourth possibility, the typing of individual x was repeated on dna derived from an independent source. a skin biopsy obtained from individual x was used to derive dna from cultured fibroblasts. the results of this determination were identical to results with dna derived from cultured lymphoblast cells of individual x; the bamhi typing was heterozygous. during the course of these studies, determination of the key -globin polymorphisms were per- formed two to four times to eliminate any possible typing error in the / globin haplotypes. to distinguish among the remaining three possibilities, we attempted to establish whether the chromosome carrying the + - - - - - - - f-globin haplotype had undergone an ob- ligate crossover within the region of lip carrying the hbbc. data were obtained for four markers known to be closely linked to the hbbc ( , , ). these markers and their estimated map distance from the hbbc are shown in fig. a. table gives the genotypes of the relevant individuals in the sibship for these markers. it is possible to determine link- age phase for all of the markers for the two parents in fig. (v and w, fig. ). these assignments are compatible with the genotypes of the other children of this couple (table ). both parents of individual x are heterozygous (+/-) for the dls dna segment. the genotypes of the grandpar- table . genotypes of five loci of family members analyzed genotypes individual pth hbbc d s hrasi ins r +/+ a/b +/+ / / s +/+ cid nd / / t -/- fie +/- / / u +/- a'/e' +/- / / v +/+ a/c +/- / / w -/- a'/e +/- / / x +/- +/+ / / +/- c/a' +/+ / / +/- a/a' +/- / / +/- c/e +/- / / +/- c/e -/- / / nd c/e +/- / / +/- c/e +/- / / +/- a/a' +/- / nd nd a/e -/- / / +/- a/a' +/- / / +/- a/a' +/- / / +/- c/a' +/+ / / nd, not determined. ents of individual x establish the phase of the d s alleles in the mother of individual x. individual x's maternal grand- mother (individual u) transmitted hbbc haplotype a' to her daughter. since u was homozygous for the presence of the taq i site in d s , we must conclude that w carried the + allele for d s and hbbc a' on one chromosome and the - allele for d s and hbbc e on the other chromosome. this assignment is consistent with all typing of the sib- lings of individual x. individual v, the father of individual x, was also heterozygous at d s . typing of his other offspring establishes that in v the hbbc haplotype a is as- sociated with the absence of the taq i site while haplotype c is associated with the presence of this taq i site. individual x is homozygous for the presence of the taq i site. the ge- notype of individual x at each of the polymorphic sites of the f -globin gene region except the ' bamhi site and at d s requires an obligate paternal crossover between the hbbc and d s . in contrast to the other sites in the hbbc, no crossover is required between the ' bamhi site and d s . even if the bamhi site had been converted from - to + by a gene conversion event, a crossover between d s and the hbbc would still have been required by the data. further evidence supporting the occurrence of a pater- nal crossover within this region is provided by analysis of genotypes at the hrasj locus. at the hrasi locus, the father was homozygous for the smallest of the three bamhi fragments- , -while the mother was heterozygous, hav- ing one copy of the smallest bamhi fragment and one copy of the largest bamhi fragment- , . the haplotypes shown in fig. support the view that the maternal chromosome lip was transmitted intact without crossover in the region be- tween the hbbc and hrasj. the most parsimonious ex- pth c gr a k e diis hrasi ins i , //+ + i+ -++ -+ + + x - cd -if + - ......................? + - -::::: ::: ::::,: .....:, .. '. -:- ,-, '.'..:.. ft fig. . haplotypes of the parents w and v and individual x determined from typings of all kindreds in this sibship. +, restric- tion enzyme site is present; -, restriction enzyme site is absent. the typings were done on dna digested with enzymes indicated in ma- terials and methods. hras allele i is a . -kbp bamhi fragment, while allele is . kbp. the three alleles of ins are: , bp; , bp; and , bp. genetics: gerhard et al ...... d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. natl acad scd usa ( ) planation of the genotype of individual x is thus a single pa- ternal crossover event occurring between the taq i site at the ' side of the -globin gene ("e") and the bamhi site at the ' side of the -globin gene. this interpretation of the data specifies the orientation of the genes within the hbbc with respect to the other markers on lip. if a crossover has occurred within the -globin gene region, then the d s dna sequence must be closest to the ' end of the hbbc. the data are thus most consistent with the gene order shown in fig. b. if, however, a gene conversion event was responsible for the bamnhi genotype of individual x, then no inference can be made regarding the gene order given in fig. b. the polymorphic site ' to the ins gene is due to insertion of sequences that are made up of -bp repeats ( ). in the general population, a wide range of size variation is observed in this region of the dna. this variation is exhibited by di- gestion of the dna with sac i and hybridization to a probe that contains a segment of the ' region of ins. within the segment of the old order amish population that we have studied, considerably less variation in length of this dna segment is observed than in the general population. to clear- ly identify sequence length variation in ins, we used the probe phins , which includes the region directly adjacent to the variable dna segment ( ). this identifies dna frag- ments from this region, which vary in length between = bp and = bp. allele i is " bp, allele is - bp, and allele is - bp. the father and the mother of individual x are heterozygous for this locus. these alleles are assigned to the paternal and maternal chromosomes as shown in fig. . the fact that individual x is homozygous for allele of ins also supports the conclusion that a paternal crossover oc- curred between ins and the ' cluster of sites in the hbbc but that no crossovers occurred between ins and d s or between ins and the bamhi site ' of the hbbc. typing of the pth locus did not give additional informa- tion since the father was homozygous for the presence of the polymorphic pst i site, while the mother was homozygous for the absence of this site. individual x is, as expected, het- erozygous for this site. in an attempt to further delineate the site of the crossover, we typed relevant individuals in the sibship for a number of reported polymorphisms around and within the hbbc. the hgiai, hinfl, rsa i, and ava ii sites (fig. and materials and methods) proved to be uninformative because both the father and mother of individual x were homozygous for the same allele in each case. discussion the data presented here demonstrate either a crossover event within the fglobin gene cluster or a gene conversion event encompassing a segment of the cluster. previous stud- ies based on population genetic methods have suggested that recombination within the cluster may be relatively much more frequent between the qif - and f-globin genes than in the - kbp on either side ( , ). this conclusion is based upon the observation of nonrandom associations of alleles at the various polymorphic sites. alleles within the cluster of polymorphic sites stretching from ' of the e-globin gene to ' of q pl-globin pseudogene show strong linkage disequilib- rium; similar linkage disequilibrium exists for the alleles in the cluster of sites from ' of the f-globin gene to kbp downstream. no linkage disequilibrium exists between these two subclusters. since recombination is the primary evolu- tionary mechanism for reducing linkage disequilibrium, the implication of the population data is that recombination rate is relatively much higher between the two subclusters than within either subcluster. thus, on an evolutionary time scale, this region has been implicated as a "hot spot" for recombination (see ref. for review). chakravarti et al. ( ) have used population genetic theory and the observed link- age disequilibripm in the hbbc to estimate the rate of re- combination in the ' subcluster, in the ' subcluster, and in the small "hot spot" between them. their calculations, based upon two-locus linkage disequilibrium theory for sites on opposite sides of the "hot spot," is . . in a previous study, stamatoyannopoulos et al. ( ) estimated the recom- bination between the s-globin structural gene and -thalasse- mia to be . . assuming that our observations represent a single crossover event, we can estimate the recombination rate between the qip- and ,-globin loci. we have typed other branches of this old order amish pedigree and a large vene- zuelan pedigree ( ) for these same loci ( , ). a modified version of liped ( , ) gave a maximum lod score (loga- rithm of odds of linkage) of . at = . + . ; the two-support-unit confidence interval ( ) extends from = . to = . . thus, our estimate of the recombination frequency in this interval is not significantly greater than chakravarti's. all three of these estimates are significantly higher than that predicted on distance in nucleotides alone. we do not have as clear an expectation for the rate of gene conversion. in no other case in our study have we observed an anomalous transmission of a multisite haplotype, but it is difficult to calculate the exact number of nonconversions definitely observed. we can say that, if the ' bamhi site was converted, we also observed an independent crossover event between the hbbc and d s . this second event has a prior probability of only % since the map distance involved is cm ( , ). although this probability is low, it is not sufficiently low to reject the hypothesis. if we accept the hypothesis that a crossover within the hbbc has been observed, it is of interest to note that the crossover falls within the region suggested to be a hot spot for recombination. the identification of additional polymor- phic sites in the region of interest would clearly be of utility in delineating the site of the crossover. the most direct ap- proach to this problem would be to isolate dna from the crossover region from the relevant individuals by recombi- nant dna techniques and to identify dna sequence poly- morphism directly by dna sequencing methods. it would be of interest to compare the data obtained here with results in other well-mapped regions of the human genome. the most obvious candidate for such an analysis would be the human major histocompatibility complex (mhc). crossover individuals have already been identified in this region by immunological methods. sites of recombi- nation also have been studied in the murine mhc. data from this region of the mouse genotne indicate that specific hot spots for recombination exist within this region ( ). com- parison of lna sequences at sites of recombination in the two species would make it possible to determine whether crossover sites within the human and mouse mhc regions exhibit sequence' homology to sites of crossing-over in the hbbc. the data presented here allow a prediction to be made on the orientation of the hbbc with respect to other markers in the adjacent region of chromosome . the data presented here are most consistent with the organization of this region of chromosome as shown in figs. b and . in this inter- pretation, the e globin gene region of the hbbc is closest to pth, and the globin gene is closest to ins, hrasj, and d s . note added in proof. we have completed the genotype analysis of the restriction fragment length polymorphism associated with the rsa i site . kbp ' of the bamhi site ( ). the genotype of individ- ual x is +/- at this rsa i site individual v is +/- at this site, while individual w is +/+. typing of remaining members of the sibship assigns the - allele for individual v to the c haplotype for hbbc. this result is consistent with the occurrence of a crossover genetics: gerhard et al d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , proc. natl acad sci usa ( ) within the region indicated in this paper. if a gene conversion has occurred, it must have extended over a region of at least . kbp. we wish to thank the following people: h. kronenberg for ppthm , g. bell for phins , c. tabin for pej . , r. white for padj- , and s. antdnarakis, h. kazazian, r. kaufman, and b. forget for the hbbc probes. this work was supported in part by grants gm and ca to d.e.h.; ns , mh , and mh to k.k.k.; mh to j.a.e.; and a damon run- yon-walter winchell cancer fund fellowship drg- to d.s.g. . collins, f. s. & weissman, s. m. 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( ) nature (london) , - . genetics: gerhard et al d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , journal of medical genetics ( ). , . familial hypertrichosis cubiti: hairy elbows syndrome peter beighton from division of medical genetics, johns hopkins hospital, baltimore, u.s.a. genetically determined hypertrichosis is uncom- mon, but several forms of familial hairiness have been described. in some kindreds the unusual hairiness has been generalized, while in others, the hypertrichosis has been confined to a specific site on the body. the purpose of this paper is to report the occur- rence of undue hairiness of the elbow regions in members of an amish family, and to discuss the genetic significance of the condition. no previous descriptions of hypertrichosis of this distribution could be found in the literature, and it is therefore suggested that the condition be termed familial hypertrichosis cubiti or the hairy elbows syndrome. case report a white girl was born in after an uneventful pregnancy and delivery. unusual hairiness of the elbow regions was observed soon after her birth. this hair increased in length and thickened in texture during infancy, reaching a maximum at the age of , before be- ginning to regress. apart from her elbow regions, she was not unduly hairy in early life, though a good head of hair was apparent in a photograph which had been taken at the age of weeks. like many members of her family, the patient was of small stature, but her general health was otherwise normal. examination in revealed a pleasant intelligent girl with hypertrichosis involving the lower third of the upper arm and the upper third of the forearm. this hair was long, dark, and coarse, but the underlying skin was normal (fig. ). the hair of her head and eye- brows and her pubic and axillary hair were normal for her age, and no other localized hypertrichosis was pre- sent. her finger-nails were short, but not dysplastic, and her teeth were unremarkable (fig. ). she was only cm. tall, but her bodily proportions were nor- mal. no other abnormality was detected on physical examination. received november . present address: st. thomas' hospital, london s.e. . her younger brother, born in , was also noted to have hairy elbows soon after birth. he was also small in stature, and, like his sister, his hypertrichosis became worse in infancy and then regressed in later childhood. hair was present over the spine during his early years, but it had disappeared by the time that he was examined in . two younger brothers had phenylketonuria, and a younger sister was apparently normal. none of these sibs had any hypertrichosis (fig. ). her father was also hairy, with thick eyebrows, a force- ful beard, and hairy arms. however, these findings could not be considered to be outside the accepted range of normality. he stated that he had been more hairy during childhood, and that his hypertrichosis usually increased during the winter. his finger-nails and teeth were normal, and he was otherwise well. the paternal grandfather, and several of her father's sibs, were also said to be somewhat hairy, but precise details were not available. her kindred were members of the pennsylvania com- munity of old order amish, a highly inbred religious isolate. both of her parents were of small stature, pro- bably because they were heterozygotes for the weill- marchesani syndrome, and the patient's own dwarfism could possibly be attributed to the same abnormal gene. discussion excessive generalized hairiness may be a mani- festation of hyperactivity of the adrenal cortex, or of an underlying metabolic disorder, such as porphyria or a mucopolysaccharidosis. widespread hairiness is also the major feature of hypertrichosis universalis congenita, which is inherited as an autosomal domi- nant trait (gates, ). localized hypertrichosis may occur in association with a naevus, or over a spina bifida. for instance, miss belle carter, the 'lady with a mane' had an area of hypertrichosis associated with a naevus pilosus over the upper dorsal spine (danforth, ). circumscribed hypertrichosis may be spora- dic, and rare familial varieties have also been o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ familial hypertrichosis cubiti: hairy elbows syndrome fig. . the patient and her brother at the respective ages of and years. their elbow regions are remarkably hairy. described (butterworth and strean, ). the burgomaster of graunau, who was killed in when his luxuriant beard caught in his horse's stirrup (beigel, ), probably had a sporadic form of hypertrichosis, while one variety of familial localized hypertrichosis was exemplified by the seven sutherland sisters, who were depicted by durant and durant ( ). these long-haired ladies, in addition to performing as vaudeville artistes, made a fortune from advertising hair tonic! it is of interest that the hypertrichosis of the affected sibs increased during infancy, and then re- solved in later childhood, as this sequence of events has previously been noted in hypertrichosis uni- versalis congenita (cockayne, ). it is also noteworthy that their nails were short, but not fig. . the finger-nails of the affected sibs are short, but not dysplastic. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ peter beighton - -,---it- -i t e fig. . the affected brother and sister in , with two of their unaffected younger sibs. dysplastic, as abnormalities of the nails sometimes occur in association with this type of hypertrichosis (pillsbury, shelley, and kligman, ). abnor- malities of the teeth and gums have also been encountered in affected subjects, but these changes are not invariable concomitants. the patients' short stature was probably not related in any way to their hypertrichosis, as various forms of familial dwarfism are not uncommon in the amish community. there was no evidence of systemic ramifications, and the patient's localized hypertrichosis seems to be nothing more than an interesting curiosity. however, the occurrence of the condition in out of sibs suggests a genetic aetiology. as the kin- dred was highly inbred, it is tempting to postulate an autosomal recessive mode of inheritance. on the other hand, the presence of hairiness in the father and several of his relations might indicate that the disorder is due to a dominant gene, which is very variable in its clinical expression. this dilemma may be resolved when the affected children raise their own families. summary two amish sibs were born with unusual hairi- ness of their elbow regions. this form of localized hypertrichosis, familial hypertrichosis cubiti, has not been previously described. their finger-nails were short, but not dysplastic, and no other systemic ramifications were present. i am grateful to professor v. a. mckusick for his per- mission to examine the patients, and for his guidance during the preparation of the case report. the author was supported by a grant from the national foundation for birth defects (crcs- ). reerences beigel, h. ( ). ueber abnorme haarentwickelung beim menschen. virchows archiv fur pathologische anatomie und physiologie und fur klinische medizin, , - . (cited by gates, .) butterworth, t., and strean, l. p. ( ). clinical genodermato- logy, p. . williams and wilkins, baltimore. cockayne, e. a. ( ). inherited abnormalities of the skin and its appendages, pp. - . oxford university press, london. danforth, c. h. ( ). studies on hair with special reference to hypertrichosis. archives of dermatology and syphilogy, , - . durant, j., and durant, a. ( ). pictorial history of the american circus, pp. - . a. s. barnes, new york. gates, r. r. ( ). human genetics, vol. , pp. - . mac- millan, new york. pillsbury, d. m., shelley, w. b., and kligman, a. m. ( ). dermatology, p. . w. b. saunders, philadelphia. o n a p ril , b y g u e st. p ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / j m e d g e n e t: first p u b lish e d a s . /jm g . . . o n ju n e . d o w n lo a d e d fro m http://jmg.bmj.com/ compound heterozygous or homozygous truncating mybpc mutations cause lethal cardiomyopathy with features of noncompaction and septal defects article compound heterozygous or homozygous truncating mybpc mutations cause lethal cardiomyopathy with features of noncompaction and septal defects marja w wessels , , johanna c herkert*, , , ingrid m frohn-mulder , michiel dalinghaus , arthur van den wijngaard , ronald r de krijger , michelle michels , irenaeus fm de coo , yvonne m hoedemaekers and dennis dooijes familial hypertrophic cardiomyopathy (hcm) is usually caused by autosomal dominant pathogenic mutations in genes encoding sarcomeric or sarcomere-associated cardiac muscle proteins. the disease mainly affects adults, although young children with severe hcm have also been reported. we describe four unrelated neonates with lethal cardiomyopathy, and performed molecular studies to identify the genetic defect. we also present a literature overview of reported patients with compound heterozygous or homozygous pathogenic mybpc mutations and describe their clinical characteristics. all four children presented with feeding difficulties, failure to thrive, and dyspnea. they died from cardiac failure before age weeks. features of left ventricular noncompaction were diagnosed in three patients. in the fourth, hypertrabeculation was not a clear feature, but could not be excluded. all of them had septal defects. two patients were compound heterozygotes for the pathogenic c. dup p.(trp fs) and c. c t p.(arg *) mutations, and two were homozygous for the c. dup and c. c t mutations. all patients with biallelic truncating pathogenic mutations in mybpc reported so far (n = ) were diagnosed with severe cardiomyopathy and/or died within the first few months of life. in % ( / ), septal defects or a patent ductus arteriosus accompanied cardiomyopathy. in contrast to heterozygous pathogenic mutations, homozygous or compound heterozygous truncating pathogenic mybpc mutations cause severe neonatal cardiomyopathy with features of left ventricular noncompaction and septal defects in approximately % of patients. european journal of human genetics ( ) , – ; doi: . /ejhg. . ; published online october introduction hypertrophic cardiomyopathy (hcm) is a major cause of sudden cardiac death in people younger than age years under physical stress, and a major cause of mortality and morbidity in the elderly. it has an estimated prevalence of in individuals. in ~ % of cases, a pathogenic variant in one of the sarcomeric contractile protein genes is found. , familial hcm is usually an autosomal dominant disorder that is transmitted due to heterozygous pathogenic gene mutations with incomplete penetrance. variants in genes encoding proteins involved in the sarcomere, cytoskeleton and z-disk, in calcium handling, and in mitochondrial and lysosomal functions have been associated with hcm. to date, different pathogenic mutations have been found in genes that encode sarcomeric proteins, such as β-cardiac myosin heavy chain (myh ) and cardiac myosin-binding protein-c (mybpc ). interfamilial and intrafamilial clinical variability in hcm is high, and it is difficult to establish genotype–phenotype correlations. in addition to the primary genetic defect, the effects of modifier genes or additional sarcomeric gene variants may contribute to the phenotypic expression of hcm. childhood-onset cardiac hypertrophy is also genetically determined in the majority of cases, and two-thirds of familial cases of childhood-onset cardiac hypertrophy are caused by a pathogenic mutation in one of the sarcomeric protein genes. we describe four unrelated children with severe cardiomyopathy, features of ventricular noncompaction, and septal defects due to compound heterozygosity or homozygosity for truncating pathogenic mutations in mybpc , resulting in early death. materials and methods clinical diagnosis four unrelated families with an index patient with severe neonatal cardiomyo- pathy were studied, after obtaining informed consent. clinical evaluation included clinical history and physical examination, electrocardiography (ecg) and d and m-mode echocardiography. the clinical diagnosis of hcm is made when there is a hypertrophied (often asymmetric), non-dilated left ventricle on echocardiography (lvw ≥ mm) in the absence of other cardiac or systemic diseases. in children, diagnosis is made on the basis of left ventricular wall thickness two or more standard deviations above the normal population mean department of clinical genetics, erasmus medical center, rotterdam, the netherlands; department of genetics, university of groningen, university medical center groningen, groningen, the netherlands; department of pediatric cardiology, erasmus medical center, rotterdam, the netherlands; department of clinical genetics, maastricht university medical center, maastricht, the netherlands; department of pathology, erasmus medical center, rotterdam, the netherlands; department of cardiology, erasmus medical center, rotterdam, the netherlands; department of neurology, erasmus medical center, rotterdam, the netherlands; department of medical genetics, university medical center utrecht, utrecht, the netherlands *correspondence: jc herkert, department of genetics, university of groningen, university medical center groningen, hanzeplein , rb groningen, the netherlands. tel: + ; fax: + ; e mail: j.c.herkert@umcg.nl these authors contributed equally to this work. received february ; revised august ; accepted september ; published online october european journal of human genetics ( ) , – & macmillan publishers limited all rights reserved - / www.nature.com/ejhg http://dx.doi.org/ . /ejhg. . mailto:j.c.herkert@umcg.nl http://www.nature.com/ejhg for body surface area. noncompaction of the left ventricle was diagnosed based on three echocardiographic criteria defined by jenni et al, including ( ) a thick noncompacted (nc) endocardial layer in the end systole on parasternal short- axis views (ratio nc/c ) with numerous, excessively prominent trabecula- tions and deep intertrabecular recesses, ( ) these recesses were perfused on color doppler studies, and ( ) a predominantly apical localization. pathologic studies microscopic examination and electron microscopy of cardiac septal autopsy material of patient were performed with standard techniques (hematoxylin and eosin staining, magnification × ). molecular analysis genomic dna of the patients was isolated from blood samples or fibroblasts. all coding regions and intron–exon boundaries of the mybpc gene were analyzed by direct sequencing analysis as described. in addition, mlpa analysis of the mybpc gene was performed (salsa mlpa kit p , mrc holland, amsterdam, the netherlands) to detect possible genomic rearrangements. (mybpc exon numbering according to ng_ . , dna variants were numbered according to reference sequence lrg_ (identical to reference sequence nm_ . ) using hgvs nomenclature (www.hgvs.org)). variants have been submitted to the leiden open variation database (http://databases. lovd.nl/shared/genes/mybpc (patient ids: – ). literature review we searched the literature for clinical and molecular data of patients with neonatal cardiomyopathy and biallelic mybpc variants, focusing on english- language articles published in pubmed and embase between and january . we identified articles in which the title/abstract included one of the following terms: double mybpc mutations, biallelic mybpc mutations, homozygous or compound heterozygous mybpc mutations or neonatal hypertrophic cardiomyopathy. we then assessed all the references cited by these articles. in our analysis, we only used reports if pathogenic homozygous or compound heterozygous mybpc variants were identified. results table summarizes the characteristics of the patients and their parents. patient patient was hospitalized at age weeks because of feeding problems and cyanosis. x-thorax showed a grossly enlarged heart. echocardio- graphy revealed a moderately dilated left ventricle with severe systolic dysfunction. the apical wall of the left ventricle was excessively thickened with prominent hypertrabeculation. the left and right atria showed mild dilatation. a small secundum atrial septal defect (asd) was also present. within weeks the x-thorax and echocardiography revealed further enlargement of the heart (heart–thorax ratio ± . ) with severe left ventricular hypertrophy and deep trabeculations in the apex (interventricular septum in diastole (ivsd) mm, z+ ; left ventricular posterior wall in diastole (lvpwd) mm, z+ ) and a fractional shortening (fs) of the left ventricle of % (left ventricular internal dimension (lvid) / ). the child died from cardiac failure at age weeks. macroscopic examination of the heart revealed severe cardiomegaly and dilatation with a total weight of g (normal table clinical and molecular characteristics of the four patients and their parents patient gender male male female female gestational age (weeks) birth weight (g) age of diagnosis (weeks) noncompaction lv lv not evident lv structural defect asdii vsd ofo asdii pathology myofibrillar disarray lvnc not performed not performed not performed viral serology normal normal normal normal metabolic screening normal normal normal normal age of death (weeks) mybpc genotype c.[ dup];[ c t] p.[(trp fs)];[(arg *)] c.[ dup];[ c t] p.[(trp fs)];[(arg *)] c.[ dup];[ dup] p.[(trp fs)];[(trp fs)] c.[ c t];[ c t] p.[(arg *)];[(arg *)] family history (scd, hcm) positive unknown positive positive parental consanguinity no no yes yes father genotype c.[ c t];[= ] p.[(arg *)];[(= )] not performed c.[ dup];[= ] p.[(trp fs)];[(= )] c.[ c t];[= ] p.[(arg *)];[( = )] phenotype echocardiography normal (age years) not performed normal normal ecg repolarization abnormal not performed normal incomplete rbbb follow-up ivs mm (age years) not performed low ecg voltages (age years) unknown mother genotype c.[ dup];[= ] p.[(trp fs)];[( = )] not performed c.[ dup];[= ] p.[(trp fs)];[(= )] c.[ c t];[= ] p.[(arg *)];[( = )] phenotype echocardiography normal (age years) not performed normal hcm (age years) ecg normal (age years) not performed normal lvh, abnormal repolarization follow-up ivs mm (age years) not performed ivs mm (age years) unknown abbreviations: asd, atrial septal defect; ecg, electrocardiography; hcm, hypertrophic cardiomyopathy; ivs, intraventricular septum; lv, left ventricle; lvnc, left ventricular noncompaction; ofo, open foramen ovale; scd, sudden cardiac death; vsd, ventricular septal defect. biallelic pathogenic mybpc mutations in neonates mw wessels et al european journal of human genetics www.hgvs.org (http://databases.lovd.nl/shared/genes/mybpc (http://databases.lovd.nl/shared/genes/mybpc weight at this age, g). right ventricular thickening was noted, especially of the posterior wall. the anterior wall of the left ventricle was severely thickened and showed abnormal trabeculation and multiple intertrabecular recesses as seen in noncompaction cardio- myopathy. secundum asd was confirmed. no other congenital malformations were found. microscopic examination of cardiac tissue (figure a) showed myofibrillar disarray in both the ventricular septum and the left ventricular wall. hypertrophic myocytes with a diameter of – μm (normal μm) and multiple vacuoles on electron microscopy suggested glycogenosis (figure b). as the echocardiographic images and ecg did not suggest pompe disease and urine oligosaccharide analysis was normal, no α-glucosidase enzyme or molecular assay was performed. mybpc mutation analysis revealed compound heterozygosity for the pathogenic mutations c. dup p.(trp fs) and c. c t p.(arg *). both pathogenic mutations are known founder muta- tions within the dutch hcm population. , neither parent had cardiac symptoms. initial echocardiography revealed no abnormalities in the -year-old mother, who was heterozygous for c. dup, nor in the -year-old father, heterozygous for c. c t. ecg in the father showed mild repolarization abnormalities (st elevation of . mm in v , followed by a negative t in v –v ). re-evaluation after years revealed moderate septal hypertrophy (hcm) with an interventricular septum of mm in the mother, and interventricular septal measurements at the upper limit of the normal range ( mm) in the father. the family history of both parents was positive for hcm and sudden death (figure a). patient patient had feeding problems in the first weeks of life and dyspnea and mild hypotonia were noticed at age weeks. a grossly enlarged heart was observed on the x-thorax (heart–thorax ratio . %) and low oxygen saturation blood levels were found. echocardiography revealed moderately dilated ventricles with severe diastolic and systolic dysfunction. hypertrabeculation with flow perfused intertrabecular recesses was present in both ventricles, including the apical walls (figure ). an apical muscular ventricular septal defect (vsd) was visualized, although deep recessal flow complicated interpretation of the images. unfortunately, owing to the critically ill status of the patient, there are no reliable m-mode measurements of the left ventricle. a muscle computed tomography scan showed no signs of atrophy, and a muscular biopsy revealed no congenital myopathy. his condition deteriorated at age weeks and he died. in this case too, we identified compound heterozygosity for the pathogenic c. dup p.(trp fs) and c. c t p.(arg *) mutations in the mybpc gene. the parents agreed neither to an autopsy on the child nor to cardiologic evaluation for themselves or pedigree analysis. patient the third proband presented with severe feeding problems and signs of cardiac decompensation at age weeks. cardiomegaly was seen on chest x-ray, and echocardiography showed hcm with dilated ventricles. m-mode measurements were unavailable. noncompaction was not a clear feature, but could not be excluded. there was a small foramen ovale and mitral valve insufficiency. the condition of the patient deteriorated owing to a viral infection and she died at age weeks. the parents did not give permission for autopsy and, at that time, molecular analysis of genes involved in cardiomyopathy was not available. cardiac analyses of first-degree relatives were normal. eleven years later, a cousin of the father presented with cardiac arrest due to hcm. he had a heterozygous pathogenic c. dup p.(trp fs) mutation in mybpc . we subsequently confirmed homozygosity for the c. dup variant in dna isolated from stored fibroblasts from the deceased proband. both parents of the proband were heterozygous for this truncating variant (figure b). cardiac re-evaluation showed only slightly reduced ecg voltages in the father (age ) and borderline hypertrophy (septal thickness mm) in the mother (age ). patient patient presented with dyspnea, hypotension, feeding difficulties, and failure to thrive at age weeks. echocardiography showed severe hcm with features of noncompaction, and severe diastolic and systolic left ventricular dysfunction (ivsd mm, z+ ; lvpwd mm, z+ , fs % (lvid / ) and left ventricular mass g/m ). a secundum asd with a left–right shunt was also observed. one week later she died after an unsuccessful resuscitation. homo- zygosity for the c. c t p.(arg *) nonsense mutation in mybpc was found. both parents were heterozygous for this pathogenic mutation. her mother was diagnosed with postpartum hcm with diastolic dysfunction, confirmed by cardiac mri, at age . echocardiography of the father was normal; ecg showed a right bundle branch block. a paternal uncle of patient had died aged months of unknown cause (figure c). figure (a) microscopic postmortem examination of heart muscle from patient . hypertrophic myocytes with myofibrillar disarray typical of hcm due to sarcomeric protein variations were present, albeit without a significant amount of interstitial fibrosis (hematoxylin and eosin staining, magnification × ). (b) electron micrograph showing a large, irregular vacuole. biallelic pathogenic mybpc mutations in neonates mw wessels et al european journal of human genetics literature review the results of our literature review are summarized in table , showing cases (including our four cases) of double-truncating pathogenic mutations, all presented neonatally or in early childhood. fifteen died before the age of year; the others had either undergone transplantations or were severely affected. structural defects were detected in cases, but neither hypertrabecularization nor deep intertrabecular recesses were described in any of the previously reported cases. discussion we describe four unrelated newborns with severe cardiomyopathy due to compound heterozygosity or homozygosity for pathogenic truncat- ing mutations in the mybpc gene. all of them died soon after birth. although initially their cardiomyopathies were described as severe atypical hcm, hcm with features of left ventricular noncompaction (lvnc) was considered after re-evaluation of serial ultrasounds and pathologic examination in three of them. like hcm, lvnc is genetically heterogeneous. , recently, lvnc was shown to be mainly caused by heterozygous variants in genes encoding sarcomeric c.[ dup];[=] † m c.[ dup];[ c>t] c.[ c>t];[=] † y † y† y † y † y † y † y † y † y† y c.[ c>t];[=] c.[ c>t];[=] patient † w c.[ dup];[ dup] c.[ dup];[=]c.[ dup];[=] † w c.[+];[=] c.[+];[=] c.[+];[=] c.[+];[=] c.[+];[=] c.[+];[=] c.[+];[=]c.[+];[=] † y patient † † w c.[ c>t];[ c>t] c.[ c>t];[=] c.[ c>t];[=] c.[ c>t];[=]c.[ c>t];[=] † m † y† y patient symbol definitions clear symbol carrier sudden death cardiomyopathy cardiac symptoms hcm hcm with features of lvnc figure pedigrees of patients , , and . figure echocardiographic studies in patient showing cardiomyopathy with lvnc. the four-chamber view shows excessive trabeculation (arrows) at the apical right ventricular wall, whereas the left ventricular wall also showed numerous deep trabeculae and recesses. biallelic pathogenic mybpc mutations in neonates mw wessels et al european journal of human genetics table patients with cardiomyopathy due to variants in the mybpc genea mybpc /variant mybpc /variant phenotype structural severity age at first study reference compound heterozygous or homozygous variants in mybpc c. c a p.(pro his) c. c a p.(pro his) hcm moderate/severe years nanni et al c. g a p.(arg his) c. g a p.(arg his) hcm severe years nanni et al c. c t p.(arg trp) c. g c p.(gly arg)b hcm c d van driest et al c. _ del p.(pro fs) c. g a p.(glu lys) hcm c d van driest et al c. c t p.(ala val) c. c t p.(ala val) hcm severe years garcia-castro et al c. c a p.(pro his) c. a g p.(asp gly) hcm severe years ingles et al c. g c p.(glu gln) c. c t p.(ala val) hcm severe years ingles et al c. c t p.(gln *) c. a c p.(his pro) hcm mild symptoms years richard et al c. c t p.(gln *) c. a c p.(his pro) childhood hcm mild symptoms years richard et al c. c g p.(thr ser) c. + t g childhood hcm before years morita et al c. c t p.(arg trp) c. g a p.(ser asn) childhood hcm before years morita et al c. t c p.(ile thr) c. _ del p.(asp del) childhood hcm before years morita et al c. + g t c. g t p.(arg leu)e hcm moderate years otsuka et al c. del p.(ser fs) c. t c p.(cys arg)e childhood hcm severe years otsuka et al c. del p.(gln fs) c. t c p.(leu pro) neonatal hcm/lvnc weeksw days dellefave et al c. a g p.(asp gly) c. c a p.(pro his)e hcm severe years maron et al c. g c p.(glu gln) c. c t p.(ala val)e hcm severe years maron et al c. a g p.(asp gly) c. c a p.(pro his)e none years maron et al c. a t p.(glu val)d c. t c p.(cys arg)e hcm severe years maron et al c. c t p.(gln *) c. g a p.(arg his)f hcm moderate years girolami et al c. g a p.(glu lys) c. - g a neonatal hcm monthw days marziliano et al c. g a p.(glu lys) c. - g a neonatal hcm daysw days marziliano et al c. c t p.(ala val) c. c t p.(ala val) childhood hcm severe years garcia-castro et al c. c t p.(arg trp) c. g a p.(ser asn) childhood hcm moderate years saltzman et al c. c t p.(arg trp) c. g c p.(glu gln) childhood hcm severe, scd years saltzman et al c. c t p.(arg trp) c. g c p.(glu gln) childhood hcm severe years saltzman et al c. c t p.(arg trp) c. g a p.(gly arg) childhood hcm severe years saltzman et al c. c t p.(arg trp) c. g a p.(gly arg) childhood hcm yearsw years saltzman et al c. c t p.(arg trp) c. g a p.(gly arg) childhood hcm moderate years saltzman et al c. - _ - del c. t c p.(leu pro) hcm moderate years bashyam et al c. del p.(ile fs) c. g a p.(glu lys) hcm moderate years bashyam et al c. + g ag c. + g ag hcm severe years ortiz et al c. + g ag c. + g ag hcm severe years ortiz et al double-truncating mutations in mybpc c. c t p.(gln *) c. c t p.(gln *) neonatal hcm monthsw ? richard et al c. + g a c. dup p.(trp fs) neonatal hcm weeksw days lekanne deprez et al c. c t p.(arg *) c. del p.(glu fs) neonatal hcm vsd weeksw weeks lekanne deprez et al c. + t g c. + t g neonatal hcm vsd weeksw o weeks zahka et al c. + t g c. + t g neonatal hcm monthsw ? zahka et al c. + t g c. + t g neonatal hcm vsd severe newborn zahka et al c. + t g c. + t g hcm severe months zahka et al c. + t g c. + t g neonatal hcm vsd weeksw newborn zahka et al c. + t g c. + t g hcm severe months zahka et al c. + t g c. + t g hcm severe months zahka et al c. + t g c. + t g neonatal hcm monthsw weeks zahka et al c. + t g c. + t g neonatal hcm pda, asd monthsw weeks zahka et al c. + t g c. + t g neonatal hcm vsd severe newborn zahka et al c. c t p.(arg *) c. c t p.(arg *) hcm/skeletal myopathy severe weeks tajsharghi et al c. + t g c. + t g neonatal hcm vsd weeks- w months newborn- weeks xin et al ( patients) c. c t p.(arg *) c. dup p.(trp fs) hcm asd weeksw weeks this study c. c t p.(arg *) c. dup p.(trp fs) neonatal hcm vsd weeksw weeks this study c. dup p.(trp fs) c. dup p.(trp fs) hcm ofo weeksw weeks this study c. c t p.(arg *) c. c t p.(arg *) hcm asd weeksw weeks this study abbreviations: asd, atrial septal defect; ofo, open foramen ovale; pda, patent ductus arteriosus; scd, sudden cardiac death; vsd, ventricular septal defect. mutations in italic are functional -alleles. athe pathogenicity of some of the missense variants listed here is uncertain. bthe mean allele frequency of this variant is . %; therefore, this is probably a polymorphism. cmore severe hcm and a higher incidence of myectomy compared with patients with single pathogenic mybpc mutations. ddiagnosis at a younger age (between . and . yrs) compared with patients with single pathogenic mybpc mutations. enot clear whether these variants are in cis or in trans. fa third variant in myh was found. gc. + g a (ivs + g a) is an intronic variant not affecting the canonical consensus splice site. given the later age of onset, this is probably not a truncating mutation, but a hypomorphic allele leading to less efficient normal splicing. consistent with this notion both patients had no septal defect. wdeceased. biallelic pathogenic mybpc mutations in neonates mw wessels et al european journal of human genetics proteins, including myh , actc , and tnnt . , this study confirms our previous results demonstrating that pathogenic variants in another sarcomeric protein gene, mybpc , can also lead to lvnc (omim number # , lvnc ). , mybpc mutations in hcm lead to an altered primary contractile function. whether contractile dysfunction is the mechanism that links mutant sarcomere protein to the morphologic features of lvnc is still uncertain. in the majority of patients with familial cardiomyopathy owing to a variant in one of the genes encoding sarcomeric proteins, a single autosomal dominant pathogenic mutation is found. in contrast, our four patients were compound heterozygotes (two patients) or homo- zygous (two patients) for two truncating mybpc variants, suggesting a cumulative effect. pathogenic mutations in the mybpc gene are one of the most common genetic causes of hcm in many popula- tions, found in – % of individuals with hcm. , autosomal dominant variants in the mybpc gene, which are mostly truncating pathogenic mutations and sometimes missense variants, give rise to hcm with an age of onset after the third decade, moderate left ventricular hypertrophy, and a favorable prognosis. as variants in mybpc (and myh ) are the most common genetic cause of familial hcm, compound heterozygous or homozygous variants should be considered in a neonate who presents with severe hcm or lvnc, even in the absence of symptoms in family members. this is illustrated by patients and who were compound heterozygotes for the two most common dutch pathogenic founder mutations for hcm: c. dup p.(trp fs) and c. c t p.(arg *). , patients and were homozygous for the c. dup p.(trp fs) and c. c t p.(arg *) mutations, respectively. the pathogenic c. dup p.(trp fs) mutation accounts for approximately one-fifth of all hcm cases in the netherlands. it is an important founder mutation in the dutch hcm population and is also present in other populations. , , it creates a new aberrant splice donor site leading to skipping of exon , resulting in a frameshift after p.gln and a premature stop codon. no truncated protein product from the c. dup p.(trp fs) allele could be detected in the sarcomere-using antibodies, suggesting that the truncated protein was unstable, or the aberrant transcript was degraded by cell surveillance mechanisms such as nonsense-mediated decay. the c. c t p.(arg *) nonsense mutation was found in three of our patients and is located in exon , leading to a premature stop codon and protein truncation beyond domain c of mybpc . it is thought to lead to reduction in mybpc owing to protein instability and/or loss of the c-terminus of mybpc that binds myosin thick filaments and titin, which is required to incorporate mybpc normally into the a-band of the sarcomere. consequently, the mybpc protein was not expected to be incorporated into the sarcomere in any of these four patients. this may explain the early and severe presentation of their cardiomyopathy. in larger series, ~ – % of adult hcm patients prove to be compound or double heterozygotes for two disease-causing variants in the same or different sarcomeric protein genes. , however, most of these are missense variants and their pathogenic nature is not always easy to establish. in these adult cases, a more severe hcm phenotype is generally seen, characterized by an earlier age of onset around the second decade or during childhood (table ). , , , – in a recent study on sarcomeric protein gene variants in childhood-onset hcm, out of children ( %) had compound variants. this suggests that a gene–dosage effect might be responsible for manifesta- tions at a younger age. as table shows, all patients with two functional null alleles died within the first year of life. patients with a missense variant and a truncating pathogenic mutation, or patients with two missense variants in mybpc , had a milder phenotype and an onset of cardiomyopathy at a more advanced age. this might be explained by some residual activity of the mybpc protein. all cases with biallelic truncating mybpc mutations present as neonates. nevertheless, we do expect prenatal onset of hcm in these patients. one of the reasons that prenatal presentation has not yet been reported, could be that prenatal ultrasounds in the netherlands, as well as in most other european countries, are not performed at a regular basis after weeks of gestation. also, mutation analysis of sarcomeric genes is not always performed in pregnancies of fetuses with cardiomyopathy, and some of these fetuses might die before a molecular diagnosis can be made. in addition, prenatal circulation is different from postnatal circulation, after the fetus is disconnected from the umbilical vessels. owing to the loss of tremendous blood flow through the placenta, the systemic vascular resistance at birth doubles. as resistance rises, aortic and left ventricular pressure increase. the neonatal heart adapts (remodels) to sudden increased systolic pressures following birth, by increasing ventricular wall thickness and stiffness (ie tensile strength). this is a result of a twofold increase in the number of fibroblasts and the formation, compaction, and alignment of collagen fibrils that envelop myocytes. we hypothesize that absence of mybpc protein would impair this process, leading to hcm with features of noncompaction in neonates. few neonatal cases with severe cardiomyopathy owing to homo- zygous or compound heterozygous truncating pathogenic mutations in mybpc have been described. , – a homozygous truncating pathogenic splice site variant c. + t g in mybpc was reported in three neonates with severe hcm who all died at an average age of – months in a consanguineous old order amish pedigree with severe hcm. this variant was also reported in another cohort of neonates with severe infantile hcm of old order amish descent, suggesting a founder effect. of note, several of the affected amish neonates with homozygous mybpc truncating variants also pre- sented with septal defects including apical muscular vsd, asd, and patent ductus arteriosus. septal defects were also present in the neonates with severe hcm owing to compound heterozygous truncating pathogenic mutations described by lekanne et al and in three of our cases. different congenital heart malformations (septal defects, patent ductus arteriosus, aortic aneurysm, and ebstein anomaly) have been reported in families with pathogenic mutations in sarcomeric protein genes, including mybpc , myh , myh , myh , and actc . , – in children with noncompaction cardi- omyopathy, tsai et al showed that % had a congenital heart defect. these data suggest that sarcomeric cardiac muscle proteins are not only involved in cardiomyopathies but also in congenital heart malformations. table shows that only patients with double- truncating pathogenic mutations have a septal defect, suggesting that functional mybpc protein has a crucial role in septal development. septal defects have not been reported in patients with missense variants, or with a missense variant in combination with a truncating variant. in conclusion, the absence of functional mybpc from the sarcomere can lead to a phenotype of severe hcm with features of ventricular noncompaction and septal defects, which appears to be lethal in the postnatal period. conflict of interest the authors declare no conflict of interest. biallelic pathogenic mybpc mutations in neonates mw wessels et al european journal of human genetics acknowledgements we thank the families and the referring physicians. we thank dr kp dingemans for providing an electronic micrograph, t de vries lentsch for graphical support, dr f van den heuvel for re-evaluating an echocardiography, and dr jp van tintelen and j senior for editing the text. maron bj, gardin jm, flack jm, gidding ss, kurosaki tt, bild de: prevalence of hypertrophic cardiomyopathy in a general population of young adults. echocardio- graphic analysis of subjects in the cardia study. coronary artery risk development in (young) adults. circulation ; : – . alcalai r, seidman jg, seidman ce: genetic basis of hypertrophic cardiomyopathy: from bench to the clinics. j cardiovasc electrophys ; : – . bos jm, ommen sr, ackerman mj: genetics of hypertrophic cardiomyopathy: one, two, or more diseases?. curr opin cardiol ; : – . lopes lr, rahman ms, elliott pm: a systematic review and meta-analysis of genotype- phenotype associations in patients with hypertrophic cardiomyopathy caused by sarcomeric protein mutations. heart ; : – . daw ew, chen sn, czernuszewicz g et al: genome-wide mapping of modifier chromosomal loci for human hypertrophic cardiomyopathy. hum molec genet ; : – . morita h, rehm hl, menesses a et al: shared genetic causes of cardiac hypertrophy in children and adults. n engl j med ; : – . grenier ma, osganian sk, cox gf et al: 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wessels mw, willems pj: mutations in sarcomeric protein genes not only lead to cardiomyopathy but also to congenital cardiovascular malformations. clin genet ; : – . biallelic pathogenic mybpc mutations in neonates mw wessels et al european journal of human genetics 	compound heterozygous or homozygous truncating mybpc mutations cause lethal cardiomyopathy with features of noncompaction and septal defects 	introduction 	materials and methods 	clinical diagnosis 	pathologic studies 	molecular analysis 	literature review 	results 	patient 	patient 	patient 	patient 	literature review 	discussion 	acknowledgements 	references untitled le journal canadien des sciences neurologiques letter to the editor to the editor samhd mutations are also responsible for aicardi– goutiÈres in the cree population keywords: encephalopathy, aicardi–goutières syndrome, cree encephalitis aicardi–goutières syndrome (ags) is a progressive inflammatory encephalopathy with multi-organ involvement. there are seven genes known to be responsible for ags (trex , rnaseh a, rnaseh b, rnaseh c, samhd , adar, and ifih ). the most common is trex , typically presenting as neonatal encephalopathy with cystic leukoencephalopathy and calcifications on brain imaging. affected infants may present with hepatosplenomegaly, transaminitis, and thrombocytopenia and are often presumed to have a congenital viral infection. however, many children with ags present within the first weeks of life following a normal neonatal period. clinical manifestations include developmental regression, difficulty feeding, micro- cephaly, spasticity, seizures, and sterile pyrexias. first described in cree children from a northern quebec village, cree encephalitis is thought to be an immune condition possibly triggered by a prenatal viral or retroviral infection in a genetically predisposed population. currently, only trex mutations have been implicated in cree encephalitis. a pathogenic variant affecting samhd has not been described in cree children. we report a case of a cree boy with phenotypic severe ags and a homozygous samhd mutation. consent was obtained from his biological parents. the ags phenotype due to the common muta- tion in the cree population has traditionally been referred to as cree encephalitis but is allelic to ags in some patients. therefore, cree encephalitis is likely part of the ags spectrum of disorders. our patient is a -year-old boy born at term by caesarean section to a healthy gravida- /para- mother after an uncompli- cated pregnancy and delivery. prenatal ultrasounds were normal. he required no resuscitation, and there were no postnatal complications. to the parents, he appeared developmentally nor- mal for the first months of life with normal head control and feeding. however, he never learned to smile, coo, or reach for objects. between the age of and months, he had an arrest of gross motor development and lost head control. he developed spastic quadriplegia with opisthotonus. at months, he required a gastrostomy tube for feeding, through which he is now exclusively fed. at months, he developed myoclonic seizures. he was diagnosed with glaucoma at years and is cortically blind. he has several hospital admissions for aspiration pneumonia and status epilepticus. other issues included hypertension, secundum atrial septal defect, and recurrent fevers of unknown origin. physical examination at . years revealed severe micro- cephaly (< %), spastic quadriplegia with severe contractures of all limbs, spontaneous clonus, dystonic posturing, upgoing plantar responses bilaterally, and hepatosplenomegaly. he demonstrated small violaceous palpable lesions on his nose and right great toe. currently, at age , he smiles in response to his parents’ voice and occasionally coos, but he has no spontaneous movements. he continues to have intermittent fevers without infections. his seizures are under control. both parents are of cree origin and reside in northeastern ontario along james bay, and are from different communities. the mother had two male cousins via her maternal aunt with developmental delay and seizures, both of whom died at age months. no other family member has had neurological issues. head mri at age year showed tiny periventricular white matter and basal ganglia t hyperintensities (figure ). findings on t -weighted imaging included frontal predominant periven- tricular hyperintensities extending into the subcortical u-fibers, anterior temporal lobe and bifrontal subcortical cysts, frontal encephalocele, cavum septum pellucidum, abnormal myelination of external capsules and the internal capsule, and a thin corpus callosum, as well as brainstem and cerebellar atrophy. these findings persisted at months with further cortical and brainstem atrophy. there was no enhancement on gadolinium. mr spec- troscopy demonstrated lactate peak and an increased choline- to-naa ratio. no vessel imaging was acquired. chromosomal microarray done at age year was normal. initial testing for cree encephalitis (trex ) followed by testing for rnaseh a, cree leukoencephalopathy (eif b ), leukodystro- phies, and fibroblasts for respiratory chain analysis were negative. he was then tested for an ags panel including trex , rnaseh a/b/c, and samhd , and was found to have a homozygous sequence variant at c t > a; p.leu gln in samhd , which has not been previously reported in the literature. both parents are carriers of this variant. in-silico analysis was done using four different programs (align gvgd, sift, mutationta- ster, and polyphen- ). based on the current available information, samhd :c. t> a is classified as a “variant of uncertain significance” (vus, acmg ). evidence supporting pathogenicity (pm , pp , pp ), falls short of being sufficient to classify the variant as “likely pathogenic” (acmg ). a different amino acid substitution has been reported at the same position (c. t> c, p.leu pro) in a case of gastric adenocarcinoma (cosmic id: cosm ). based on the in-silico fathmm algorithm, this variant is classified as “pathogenic.” this evidence (pm ) was not included in the final acmg classification because the pathogeni- city of the c. t > c (p.leu pro) variant was based entirely on in-silico predictions. if this additional information is considered (pp ), the samhd :c. t> a variant would be classified as “likely pathogenic” (acmg ). homozygous and compound heterozygous mutations in samhd were first implicated in ags patients in . approximately % of ags is caused by mutations in samhd (including missense and null mutations as well as large deletions). . the majority of these patients have a normal neonatal course with neurological symptoms presenting in the first year of life. samhd mutations have been found in amish populations, maltese patients, ashkenazi jews, and patients of northern european, north american, asian, and australian descent. the phenotypic similarities between ags and cree encepha- litis are well-described. currently, only trex mutations have been identified in cree encephalitis. to our knowledge, there are no reports of samhd mutations in cree children. the canadian journal of neurological sciences https://www.cambridge.org/core/terms. https://doi.org/ . /cjn. . downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /cjn. . https://www.cambridge.org/core several conditions are associated with ags, including glau- coma, autoimmune diseases, familial chilblain cardiomyopathy, intracerebral vasculitis, and arthropathy. among ags patients, those with samhd mutations have a higher likelihood of devel- oping glaucoma and chilblains. they are also the only group to develop intracerbral vasculopathy and arthropathy. in a series of five patients with samhd mutations, all had both a cerebral arteriopathy and peripheral vasculopathy with chilblains, and two had leukocytoclastic vasculitis on skin biopsy. these findings were convincing for a role for samhd in systemic inflammation. therefore, for this subgroup of ags, anti-inflammatory therapies and immunosuppression may help prevent disease progression. patients with ags display increased interferon activity in cerebrospinal fluid and serum. this was not investigated in our patient due to his deteriorating condition. currently, in canada, trex testing for the common mutation is typically done first-line in patients suspected of having cree encephalitis. if negative, an ags panel (including samhd ) or individual samhd testing should be performed. a wider genetic screen of the cree population may help elucidate the carrier rate of samhd in this population. if additional cases are identified, it would assist in defining the pathogenicity of the variant. this testing would be particularly important, as ags with samhd mutations can have potential complications, including glaucoma and arteriopathy, requiring anticipatory screening and treatment. fur- thermore, immunosuppressive therapies may play a role in the management of this subgroup of ags patients. acknowledgments we thank dr. john waye for his support in writing this manuscript and the patient’s family for their permission to report his case. disclosures ashraf kharrat, jennifer mackenzie, and sunita venkateswaran do not have anything to disclose. ashraf kharrat department of paediatrics, university of toronto toronto, ontario, canada figure : a through c, age months, t w and t flair axial images demonstrating temporal and frontal cysts; d through f, age months, t w and t flair axial images demonstrating temporal and frontal cysts and abnormal myelination status; g, age months, t w axial image demonstrating left glaucoma; h, age months, t w axial image demonstrating ongoing cerebral atrophy; i, age months, t w axial image demonstrating basal ganglia calcifications. the canadian journal of neurological sciences https://www.cambridge.org/core/terms. https://doi.org/ . /cjn. . downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /cjn. . https://www.cambridge.org/core jennifer mackenzie department of paediatrics, mcmaster university hamilton, ontario, canada sunita venkateswaran division of paediatric neurology, department of paediatrics university of ottawa, ottawa, ontario, canada references . black dn, watters gv, andermann e, et al. encephalitis among cree children in northern quebec. ann neurol. ; ( ): - . . crow yj, black dn, ali m, et al. cree encephalitis is allelic with aicardi–goutières syndrome: implications for the pathogenesis of disorders of interferon alpha metabolism. j med genet. ; ( ): - . . rice gi, bond j, asipu a, et al. mutations involved in aicardi– goutières syndrome implicate samhd as regulator of the innate immune response. nat genet. ; ( ): - . . crow yj, chase ds, lowenstein schmidt j, et al. characterization of human disease phenotypes associated with mutations in trex , rnaseh a, rnaseh b, rnaseh c, samhd , adar, and ifih . am j med genet a. ; a( ): - . . xin b, jones s, puffenberger eg, et al. homozygous mutation in samhd gene causes cerebral vasculopathy and early onset stroke. proc natl acad sci u s a. ; ( ): - . . straussberg r, marom d, sanado-inbar e, et al. a possible geno- type–phenotype correlation in ashkenazi jewish individuals with aicardi–goutières syndrome associated with samhd mutation. j child neurol. ; ( ): - . . ramesh v, bernardi b, stafa a, et al. intracerebral large artery disease in aicardi–goutières syndrome implicates samhd in vascular homeostasis. dev med child neurol. ; ( ): - . . lebon p, badoual j, ponsot g, goutières f, hémeury-cukier f, aicardi j. intrathecal synthesis of interferon-alpha in infants with progressive familial encephalopathy. j neurol sci. ; ( – ): - . le journal canadien des sciences neurologiques volume , no. – november https://www.cambridge.org/core/terms. https://doi.org/ . /cjn. . downloaded from https://www.cambridge.org/core. carnegie mellon university, on apr at : : , subject to the cambridge core terms of use, available at https://www.cambridge.org/core/terms https://doi.org/ . /cjn. . https://www.cambridge.org/core 	outline placeholder 	figure a through c, age months, t w and t flair axial images demonstrating temporal and frontal cysts; d through f, age months, t w and t flair axial images demonstrating temporal and frontal cysts and abnormal myelination status; g, age months [pdf] from genotype to phenotype: nonsense variants in slc a are associated with decreased serum sulfate and increased serum aminotransferases | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /g . . corpus id: from genotype to phenotype: nonsense variants in slc a are associated with decreased serum sulfate and increased serum aminotransferases @article{tise fromgt, title={from genotype to phenotype: nonsense variants in slc a are associated with decreased serum sulfate and increased serum aminotransferases}, author={christina g tise and j. perry and leslie e. anforth and mary a pavlovich and j. backman and k. ryan and j. lewis and j. o'connell and l. yerges-armstrong and a. shuldiner}, journal={g : genes|genomes|genetics}, year={ }, volume={ }, pages={ - } } 	christina g tise, j. perry, + authors a. shuldiner 	published 	medicine, biology 	g : genes|genomes|genetics using genomic applications to glean insights into human biology, we systematically searched for nonsense single nucleotide variants (snvs) that are rare in the general population but enriched in the old order amish (amish) due to founder effect. we identified two nonlinked, nonsense snvs (r x and w x) in slc a (allele frequencies . % and . % in the amish; enriched . -fold and . -fold, compared to the outbred caucasian population, respectively). slc a encodes the apical sodium… expand view on genetics g journal.org save to library create alert cite launch research feed share this paper citationsbackground citations view all figures and topics from this paper 	 figure 	 figure 	 figure 	 figure 	 figure view all figures & tables 	transaminases 	nonsense mutation 	slc a gene 	acetaminophen 	calcium 	sulfates, inorganic 	adverse reaction to drug 	chemical and drug induced liver injury 	bone mineral density test 	nucleotides 	genome-wide association study 	intestinal wall tissue 	nonsense mediated mrna decay 	anions 	physiological aspects 	gene frequency citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count 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beck, + authors a. ruhe 	biology, medicine 	plos one 	 	 	highly influential 	pdf 	view excerpts, references background save alert research feed pathogenetics of the human slc transporters. 	p. dawson, d. markovich 	biology, medicine 	current medicinal chemistry 	 	 	view excerpts, references background save alert research feed plasma and urinary sulfate determination in a cohort with autism 	f. bowling, h. heussler, a. mcwhinney, p. dawson 	biology, medicine 	biochemical genetics 	 	 save alert research feed atelosteogenesis type ii is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (dtdst): evidence for a phenotypic series involving three chondrodysplasias. 	j. hästbacka, a. superti-furga, w. wilcox, d. rimoin, d. cohn, e. lander 	biology, medicine 	american journal of human genetics 	 	 	view excerpts, references background save alert research feed in a shake of a lamb's tail: using genomics to unravel a cause of chondrodysplasia in texel sheep. 	x. zhao, s. onteru, + authors m. rothschild 	biology, medicine 	animal genetics 	 	 	view excerpt, references background save alert research feed disruption of nas sulfate transport function in mice leads to enhanced acetaminophen‐induced hepatotoxicity 	s. lee, p. dawson, a. hewavitharana, p. n. shaw, d. markovich 	chemistry, medicine 	hepatology 	 	 	highly influential 	 	view excerpts, references background save alert research feed identification of the finnish founder mutation for diastrophic dysplasia (dtd) 	j. hästbacka, a. kerrebrock, + authors e. lander 	biology, medicine 	european journal of human genetics 	 	 	pdf 	view excerpt, references background save alert research feed slc a and slc a ko models reveal physiological roles of anion transporters 	d. markovich 	 	 	pdf 	view excerpt, references background save alert research feed inactivating papss mutations in a patient with premature pubarche. 	c. noordam, v. dhir, + authors w. arlt 	medicine 	the new england journal of medicine 	 	 	view excerpts, references background save alert research feed ... ... related papers 	abstract 	figures and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue book reviews j. howard kauffman a n d l e o driedger, tlze merzlzorzite mosaic: identity nlzd model-rzization (scottdale: herald press, ). paperback, pages. in j. howard kauffman and leland harder published the results of the first major survey of north american mennonite and brethren in christ church members. melzrzolzite mosaic reports and interprets the data gathered in a second parallel survey in , seventeen years after the first study. with about two-thirds of the questions identical to those asked in , valid comparisons can be made and trends discerned. the five denominations studied in both surveys (mennonite church, general conference mennonite, mennonite brethren, brethren in christ, and evangelical mennonite church) include some , of the , baptized mennonites in north america. data were gathered from over members by a -page question- naire administered in group settings and analyzed with the aid of current computer technology. the survey developed a profile of religious beliefs, attitudes and practices of members of the participating denominations. in addition to validating and up-dating that profile, the study sought to identify trends "that would provide clues regarding the impact of modernization" ( , ). it also added questions to explore in-group identity, male-female roles, and other aspects not included in . most of the trends revealed by the study were not unexpected. mennonite brethren have changed most and brethren in christ least in the intervening years ( ). but "similarities between these five bodies [remain] more significant than their differences" ( ). and, of course, on most items the whole range of responses was represented in each denomination. the five groups are very close to each other when compared with the largerecumenical scene in north america. from time to time the authors make helpful comparisons of their findings with similar data from surveys of other religious bodies (e.g. reginald bibby in canada) or national samples (e.g. gallup poll). the tables provide a wealth of summarized data. the authors have presented this information in very accessible form and added helpful interpretation in each case. especially valuable are the tables of correlations made possible by computer analysis. conference and congregational leaders as well as those involved in mennonite educational and community organizations will benefit greatly from a study of the presented data. if the authors have not identified as many practical implications as some readers would like, the information is there to draw one's own conclusions. the study gives major attention to the impact of modernization. in an occasional lapse from social scientific detachment it even becomes the "onslaught" of modernization ( , ). relating it to technological society, the authors use five b o o k re iew.s dimensions or indicators of modernization: education, urbanization, mobility, occupational rank, and income. all five denominations have become more urban and increased in educational attainment since (mbs most; bics least); professional, business and clerical ranks have grown, while farm and labour segments are down: urbanization has increased mobility. that "objective" sense, then, mennonites have clearly modernized during the past years. an important question is how this nlodernization will have affectedmennon- ite identity: "beliefs, values, attitudes, and behaviour" ( ). how far will it have led in the direction of greater secularization, individualism, and materialism ("con- comitants of modernization") as social theory predicts it should? the answer is unclear. in fact, contrary to expectation, higher education andsocio-economic status correlate negatively with secularism and individualism ( , ). sinularly surprising is the finding that the age distribution of those scoring high on all three concomitants of modernization is not lineas, but concave, with teenagers and the over group higher than the three intermediate groups ( ). this raises important questions for the authors. is the assumption that secularism, individualism, and materialism are concomitants of modernization wrong? do mennonites simply not fit the expected pattern? ( ) readers may further wonder: did the questionnaire gather adequate data, and were the scales of "secularism" etc., appropriately defined? to that the authors might well respond in the words of kauffman and harder in their report of the survey: of course, the validity of such findings depends much on the way the questions were asked and the scales defined. there is a sense, however, in which their meaning transcends the researchers' ability to control the instrument. church members responded to over three hundred questions and gave a wide range of answers on many subjects. when a conglomerate of information is fed into the computer and i t reports plus and minus correlations of various magnitudes, we are pushed to make sense of the reports whether or not we have defined the factors in advance (arlnbnptists fo~rr- cerlt~rr-ies later-, ). some interesting findings: scores on "devotionalism" were unchanged from ; ( ) opposition to abortion has significantly increased for all of the options offered, from pregnancy resulting from rape to "does not want baby;" ( ) farmers are the category in which the highest percentage has a household income below $ , ; ( ) males inore strongly support women in leadership roles than do females in of categories, from "worship leader" ( - %) to "conduct ordinations" ( - %)( ). chapter , comparing the five denominations, shows mcs and gcs to be the most compatible with each other, although fewer than one fourth of either group favours "uniting with another mennonite or bic group" ( ). in a number of areas there appears to be a considerable gap between attitude (what people believe) and behaviour (what they do). thus, % agreed that "members of our denomination should vote in public" elections ( % in ) but only % did so in recent years ( % in ) ( ). fifteen percent believed that youth should refuse to register with the draft, but only % would take that position if faced with the draft ( ). tables to in chapter show a consistent spread between "moral attitudes" and "moral behaviour," with attitudes generally but not always rating higher. the authors might have given more attention to this than their brief comments ( f). my few criticisms are not meant to detract from the value of this study. the historical contexts plrovided in the introduction of a number of the chapters are usually carefully stated. elsewhere imprecise remarksjar unnecessarily: e.g.that the evangelical mennonites split away from the "old" mennonites ( ) when the latter had been equated with the mcs on the previous page; or the curious juxtaposing of menno simons and thomas muentzer ( ) when menno and jan van leyden or even hubmaier would have been much more appropriate. similarly, survey results are usually interpreted carefully and precisely, avoiding unwarranted cause-effect language. elsewhere less care is evident: e.g. "with urbanization came greater interest in education" ( ) [vice versa?] or "agrarian lifestyle brought with it large extended families, strongly patriarchal." when the categories "liberal" and "conservative" are used in comparing mennonite groups or positions, the results are often unenlightening. kauffman and driedger ( of) introduce the terms carefully and give them specific content from the survey questions. even so it becomes problematic to identify those who hold strongly to pacifism as "liberal" ( ) when that belief is identified as a central element of anabaptist orthodoxy. identity and modernization may not have been the most central questions to address in this survey, as some of the ambiguous results suggest. nevertheless, the book contains an enormous wealth of information to stimulate further study, interpretation and extrapolation. it will undoubtedly become a basis for many important decisions within the five conference bodies in the decade ahead. adolf ens canadian mennonite bible college c.j.dyck, william e. keeney, and a.j.beachey, eds., tlze writings of dirk plzilips (classics of tlze radical refor~~zatio~z, volume ) ( scottdale and waterloo: herald press, ). pages. the writings of dirkphilips are generally little known, even though his name is familiar to students of mennonite history as an important co-worker of menno simons, and, as translators and editors have insisted, his writings are more accessible than those of menno in the sense of being more clearly written. in fact, dirkphilips (perhaps more correctly dirk philipsz) writings have been available in a useful translation since , that by a. kolb and published by j.f.funk (reprinted in bonk reliebc~s ). a german translati of the eizcl~iridioi~ oclei- hrrizrlduclzleii~ was published in the usa in i . it is chiefly this book that was made available in english by kolb. the editors of the classics series decided on anew translation of this work and others by dirk philips for several reasons, perhaps most importantly because of their desire to "annotate or contextualize the documents within the framework of dirks' life and thought." there is no doubt that this volume adds valuable information to the texts thenlselves, thus making these works available to a new, more general readership. from alimited sampling withinits more than pages it can beestimatedthat the translations, which came through several hands and benefitted from that editorial process, are very good. their improvement on the earlier one is chiefly in the revision and reformulation of the very numerous biblical quotations. the text itself is not as problematic as that of menno and the editors have wisely included some original t e r n s in parentheses (wederdooper, p. ), since the reader is entirely dependent on the translators in the absence of the original. it is not so clear which bible translation has been used, surely something which should be clarified in the introduction. and so the reader will find the quotation on page : "what did our arrogance profit us, ourrichness, oui-ostentatiousness"(wisdom of solomon : ), when a good, contem- porary translation, the neb, renders: "what can we show for all our wealth and arrogance?" other writings beside the eizclziridion are included in this generous volume, several individual tracts, letters and even two songs. these hymns understandably do not scan well and it is a shame that these brief texts could not be reproduced in the original as well, perhaps in photocopy. technically, the volume is well put together, with an introductory biography by william keeney and excellent anno- tation and footnoting. the review copy had some unclear printing in the early pages and the single portrait is not even of xerox quality. but apart from such minor criticisms there is much reason to be grateful for a readable and useful presentation of the works of one of the outstanding leaders of the anabaptist movement. victor g. doerksen university of manitoba clarence b a u m a n , tlze spiritual legacy of harzs derzck. irzterpretcrtiorz arzd trarzslation of key texts (leiden: e.j.bril , ). pages. to those who have read and heard clarence bauman, either in his doctoral opus gewrrltlosigkeit, his volume on the sermon on the mount or on other occasions, it will not come as a surprise to see this translation of, and commentary on, the writings of hans denck, writings which have always been key texts forbauman. after speaking with him as a young graduate student in germany it wasnatural for me to acquire denck's writings in the fellmann edition and to read vorz delel- vvc~rw~ lieb for myself. many students of professor bauman will have been similarly affected. clarence bauman's teaching and writing bespeak a world of spiritual experience which we refer to as mystical. his voice encourages us to be still and to listen, to contemplate, to explore spiritual resources within as did the mystics of earlier times. of hans denck he says: hans denck represents the contemplative genius of the anabaptist movement at its highest and best. no understanding of the anabaptist vision is complete without coming to terms with the uniqueness of denck's intellectual spirituality: its inner dynamic, its medieval context, its mystic content, and its jewish roots ( i ) . this edition begins with a forty-page essay on denck's life and thought, a clear, well-documentedrecordof this brief ( years) life and adiscussion ofdenck's spirituality, emphasizing and explaining his "jewish biblical mysticism" ( ). bauman concludes: denck's spiritual consciousness appears to transcend all the fixed categories with which he has hitherto been identified. he was too undogmatic to beanevangelical reformer and too quietistic to be an anabaptist radical. he was too biblical to be a rationalist and too theological to be a humanist: tooecumenical to be asectarian and too christological to be a unitarian. his faith was too articulate for pietism, his hope too evangelicalfor liberalism, and hislove too universal forconservatism. his heart was tooethically- mindedforan individualisticspiritualist,andhis spirit was toomystically orientedfor asocial activist. his mentality was toodynamic to be doctrinally fixed, and his spirituality was too radical to be existentially safe. his jewishness was too prophetic to be tolerated, and his christianity was too authentic to be endured-almost as though his faith were too real to be true and his life too true to be real ( ). part i contains the chosen texts, with an introduction and the german original facing its translation. the originals are reprinted with their annotation from the fellmann edition (quellen zirzd forsclzzirzgerz z ~ t r refor zntiorzsgescizicizte x x i v ) . they include the major texts with the exception of the micah commentary, which cannot be certainly ascribed to denck. a part i deals with some latin poems, polemics and the micah commentary. the beautifully made volume is completed by a bibliography, glossary and indices. a translation of denck's major texts by edward furcha has been available in two editions, a rather basic one from and a revised edition in . furcha's translations are, as he says, "aimed at transmitting the sense of a given passage in good english without unduly violating the original" ( , vi). a coinparison of selected passages indicates that both furcha and bauman have done well with a difficult text, but that there are significant differences. where furcha aims at "good english,"bauman clearly opts for faithfulness to thegerman original. forexa~nple, denck's reference to himself, nirz au~a~tseligernlerzsclz, is rendered by furcha as "a pitiable man." baulnan says: "a poor in spirit person." one might have preferred "a person poor in spirit," but bauman's faithfulness is almost that of the medieval interlinear version. a footnote in bauman's edition then expands on this interesting word, pointing out that this paradoxical formulation ("blessed-poor") derives from book rnliervs the beatitudes of jesus. furcha's text is not always successful in arriving at "good english;" for example, he uses the pronoun "she" in speaking about love (von dei- mi-en lieb), but naturally cannot be consistent in that usage ( ). in contrast with furcha, bauman's work gives the reader the wherewithall to approach and to understand the german original, for which the original, the annotation and the translation are all necessary. this has resulted in a splendid, if expensive book. clarence bauman himselfwouldlikely call the price of the volume "siindhaft teuer" and one would be inclined to agree, although one should also bear in mind that the word teuei- means valuable as well as expensive. victor g. doerksen university of manitoba ursula lieseberg, studieu rltrn m l i l - ~ r e r l i e d der tiiufei- inz . jnlzrlzundel? (european university studies )(frankfurt/main: p e t e r l a n g , ). pages. this recent study is "a first attempt to define the still relatively unfamiliar genre of the german mastyr songs of the anabaptists and to introduce these songs with regard to content, structural characteristics and function" (back cover). although lieseberg has drawn on all the significant existing sources of anabaptist studies (e.g. wolkan, liliencron, h.s.bender, rosella duerksen and others) she makes two rather remarkable observations: .thereis adearth ofresearch on the poetry of th century anabaptist songs, in comparision with the research on prose texts, and . the martyr song is to a great extent still a little understood species of sacred song. basic to this study is lieseberg's definition of the miii-tyr-er-lied: "...a song in strophic narration about a martyr event, written in the th or th century by lutheran or anabaptist authors." this definition rules out the inclusion of other kinds of hymns as martyr songs which were written by persons who later met a martyr's death. with this definition as a basis, and with an impressive fund of fresh documentary material at her disposal (see liter-crtun~e~eichr~is, pp. - ), the author, using selected lnartyr hymns (see ver,eichliis der- lieder, pp. - ), developed the outline, structure and content of this thesis. lieseberg presents a tri-partite classification of lnartyr songs: . by sources: lutheran, anabaptist, hutterite; . by period: early ( - ) , middle ( - ). late ( - ), beginning with the first lnartyr song by mastinluther andending with the last known song in this genre in ; . according to priinary and secondary song texts. primary songs dealt with the death of conte~nporary martyrs, usually well known to the author. secondary songs concerned biblical and early church martyrs, as well as a few "special" martyr songs of a general nature. in chapter six lieseberg uses pages to present a clear and comprehensive study of the structural features, narrative and stylistic elements of typical martyr songs. a comparison with other studies, for example yoder's " years with the ausbu~zcf" and rosella duerksen's dissertation on anabaptist hymnody ( ), as well as various articles would help us to recognize the additional new information and the clear manner in which this fascinating new material has been presented here. in chapter nine the author makes a little excursion into the music (melodies and singing styles) of the martyr songs. only five pages aredevoted to this rich field. the information is largely a summary of some of the information given in much greater detail by wolkan and rosella duerksen. in the section dealing with present singing practices among north american hutterites the reviewer is left to wonder why such a significant resource as helen martens' "die lieder der hutterer" was not consulted. the extensive commentaries which lieseberg provides on individual songs is a rich source of information for hyrnnologists and authors of handbooks to hymnals. a case in point is the commentary on wer clz~isto jetzt v~~ill~folge~l rznclz (who now would follow christ in life, mh , the first martyr song of the anabaptists. this was a popular hymn sung by swiss anabaptists and hutterites alike, the latter having adopted it in thirteen hymn books with frequent textual variants. the thesis includes five very valuable indices: ) an alphabetical list of the songs used in the research. for each song lieseberg lists hymnals, codices and other source material in which the song appears. ) index of first lines, providing page references to hymns dealt with throughout the study. ) index to the martyrs referred to in the study. ) index to melodies. ) an index of the nineteen different rhyming schemes used in martyr songs. the final five pages, a facsimile reproduction of hutterite songs written in fraktur, add a delightful artistic touch to the paperback volunle. in this study ursula lieseberg has provided sources and opened doors for further meaningful research in sixteenth and seventeenth century anabaptist hymnody in general and martyr songs in particular. for this we are gratefully indebted. the author was born in loetzen, east psussia, in . she studied germanistik, anglistilc and general linguistics at kiel university. this thesis was written in fulfillment of her master's degree in . george wiebe canadian mennonite bible college winnipeg book r e ~ d e l ~ ~ s g e r a l d r. brunk, ed., meizno siinons. a renppr-nisnl. essays in h o n o r of irvin b. h o r s t o n t h e th anniversary of t h e f~~rzdnnzeiztboek (harrisonburg, virginia: eastern m e n n o n i t e college, ). hardcover, pp. this collection of essays includes the papers presented at a conference on menno simons held on march - , , at eastern mennonite college and seminary. the book honours the well-known anabaptist and menno silnons scholar irvin b. horst who for many years taught at eastern and at the university of amsterdam. included in the collection are the following scholars and their essays: myron augsburger (on horst as a scholar and churchman), walter klaassen (on the relevance of menno simons today and menno research), sjouke voolstra (on themes in menno's early theology), helmut isaak (on menno's vision of god's kingdom), pieter visser (onmenno simons printed, readanddebated), ma-jan blok (on discipleship in menno), abraham friesen (on menno and miinster), and irvin horst (on the meaning of menno today). augsburger's essay on horst and iuaassen's biographical essay were not presented at the conference but were added for the book. the collection concludes with a bibliography of horst's writings. while most of the essays are generally of a scholarly nature, the tone of some is personal, even humorous (augsburger, friesen), and some are celebratory and didactic in intent and purpose (horst, klaassen). like horst's life-long work, the essays reflect scholarship in the service of the church and faith. and this is certainly in the spirit of menno simons, who was always wary of the "learned ones" and scholarship detached from faith. time and again the authors refer to the ground-laying work of harold s. bender, who with his "anabaptist vision" inspired horst and a whole generation of anabaptist scholars and churchmen. horst expresses his appreciation for bender's vision when he writes: "...as amiddle class group ofchristians living in aprosperous society, we tend to regard discipleship as a contingent issue rather than a central one ... as christians we avoid confrontation by favouring religious expressions that stress piety rather than ethical demands' '(p. ). all writel-s, while acknowledging menno's evangelical theology and practice, at least imply that the dutch anabaptist leader would be averse to what is known as fundamentalist evangelicalism today. does the book advance our knowledge of menno simon's life and work? the purpose of the essays is to restate, reappraise and emphasize some of the themes and issues in menno's early writings, especially in his f~oirhn~entboek. we still do not know ~lluch about menno's early life, including whether he received his training in a monastery or in a latin school. with regard to the authorship and date of tize blrrsl~he~~zy ofjokrz ofle)lde~l, friesen argues persuasively that menno wrote this work in , before he officially joined anabaptism, but he does not explain why menno does not mention polygamy among the sins of john of leyden. it is unfortunate that there is not a single woman among the contributors of essays, although some writers, not all, use inclusive language (klaassen, for example, refers to congregational ministers alternately as "he" and "she"). this absence of female scholars in the book (surely there must have been female students whom horst taught) is all the more unfortunate when it is considered that menno simons addressed in his writings both "brothers" and "sisters," took the side of a woman in her struggle against the harsh rules of the ban and shunning, and, as augsburger surmises, even commissioned "elizabeth dirks as the first woman to serve in a deaconess role in the church" (p. ). mennonite study groups and pastors will find this bookhelpful. it isrelatively free of misprints. page xii, line , should read "as" not "we," and page , line , should read "pragmatism." the following sentence on page is obscure: "in it he offers pre-eminently an evangelical basis for the preaching of the people." the author here refers to the fzazdanzeiztboek of menno simons. harry loewen the university of winnipeg james r. coggins, jolztz srnytlz's congregation. englislz sepnmtisnz, mennonite bzjluetzce, and tlze elect nntioa. no. in the studies in anabaptist and mennonite history series (waterloo, ontario/scottdale, pa: herald press, ). hardcover, pages, $ . (cdn.). in the first decade of the seventeenth century, during the reign of king james i of england, some puritan separatists together with their spiritual leader john smyth (c. - ) left for amsterdam in search of religious freedom. soon after their arrival on dutch soil the group split into three factions. the largest faction, led by john robinson, moved to leiden and later as pilgrim fathers to new england, where they influenced america through congregationalism. the group which remained true to john smyth in joined the waterlander mennonites in amsterdam. the smallest group, a mere ten members, adhered to thomas helwys and returned to england, establishing there the first baptist congregation. coggins' book, based on his ph.d. dissertation at the university of waterloo, deals with the john smyth congregation as a group, describing and interpreting its leaders, their theology, and the congregation's struggles, divisions, and search for divine truth. while the book recounts the stories of all three factions, its focus is on john smyth and his group's relationship to the dutch mennonites. in six chapters the author deals with issues that were important to both the smyth group and the mennonites, including the relationship between church and state, religious freedom and tolerance, authoritarianism in church leadership, nationalism and patriotism, predestination and election, and believer's baptism. this fascinat- ing story is not only well told but it also contributes to a better understanding of the early mennonite-baptist connection. book r e ~ ~ i e w s drawing upon primary andsecondary sources, coggins corrects some earlier held views and misconceptions with regard to the relationship between the smyth group and the dutch mennonites. for example, by means of a careful analysis of the purpose, date and content of hans de ries's "short confession" and john smyth's "defense" of the "short confession" against a certain critic's attack, coggins is able to establish that the alliance between the smyth group and the waterlander mennonites was concluded in , two years before smyth died, and not in as some had formerly believed. what happened after smyth's death was a merger or complete union between the two bodies (pp. - ). of significance is also the effect that this alliance had upon the dutch mennonites. since the mennonites like the smyth group had a congregational form of church government and as autonomous congregations were loosely allied with all other dutch congregations, it was difficult to come to a decision and consensus with regard to accepting the english congregation. when the waterlander and high german mennonite congregations favored an alliance with the smyth group but the frisian and flemish mennonites opposed it, the mennonite alliance known as the "pacified brotherhood came to an end. the clash between the more tolerant and liberal waterlanders and the more conservative and exclusive mennonites resulted in the breakup. the alliance between the english and the mennonites also had a decided effect upon members in smyth's congregation. led by thomas helwys, a small group was not willing to join the mennonites. english nationalism and culture, an inability to speak dutch, and some aspects of mennonite theology, notably christology, separation of church and state and nonresistance, made it difficult for the helwys group to go along with smyth's decision to join the mennonites. while the historical narrative is well researched and carefully interpreted, coggins' treatment of some theological issues is less satisfactory. for example, in his discussion of "mennonite christology" coggins leaves the impression that there was a particular and normative mennonite christology. while it is true that melchior hoffman, menno simons and some of their followers adhered to a "celestial f l e s h view of the incarnation of christ, there were many other anabaptists andmennonite% particularly in southern europe and among the waterlanders, who rejected this view. moreover, menno's christology certainly grew out of his concept of al'pure church," but that does not necessarily mean that there is a connection between menno's "celestial flesh" theology and mennonite "perfectionism" and "spiritual arrogance" (p. ). coggins' detailed analysis of hans de ries's "short confession" and smyth's "defense" makes it quite clear that the mennonite concern for holiness and righteous living derivedfro~n many specific new testament passages on the subject and not necessarily from a narrow theology of incarnation. similarly, coggins speaks of tl e anabaptist and tlze mennonite "view of the state" (p. ), implying that all anabaptists and mennonites separated church and state, refused to serve in government, and upheld the principle of nonresistance. a study of sixteenth century anabaptism indicates that there were at least two major positions with regard to church-state relations: the schleitheim separatist position and the balthasar hubmaier position, which accepted christians' involvement in government and their serving with the sword. it is thus incorrect, or toogeneral at best, to state that "from the beginning, anabaptist attitudes toward the state were based on a concept of separation" (p. ). incidentally, hubmaier whom baptists today consider their anabaptist hero, is not mentioned at all in this study. the book includes six appendices, including "members of the john smyth congregation," "others associated with the smyth congregation," "the robinson congregation," "the helwys congregation," "letter of thomas helwys to the mennonites," and an excellent english translation of the latin "defense of the ries's confession." copious notes for each chapter, an extensive list of primary and secondary sources, and a useful general index complete this study. the text is relatively free of typographical errors and misprints. in the sebastian franck quotation on page the word should be "sects" not "sentences." harry loewen the university of winnipeg heinrich sawatsky, merzrzonite tenzplers, translated by victor g. doerksen (winnipeg: cmbc publications and the manitoba mennonite historical society, ). paperback, xvi, pp. heinrich goerz, metzizotzite settlenzerzts irz crirnen, translated by john b. toews (winnipeg: cmbc publications and the manitoba mennonite historical society, ). paperback, xiii, pp. these two books bring to three the total number of books in the echo verlag series of volumes to be translated andpublished. the first volume was tlze k~ldarz settlernerzt ( -see review in jms, vol. , ). the very dissimilar nature of the two books makes it difficult to do justice to both in the same review. memzorzite settlerne~zts irz crirnea was written by someone who hadnoteven livedin the crimea and thereforerelied on the reflections of others. although, as the translator remarks, there is "a sense of the authentic and the graphic," there is nevertheless also a sense of distance. considerable detail concerning the settlements, their geography, agriculture, industry, institutions and religious leaders is given. some of the information can be found in much more detail in other accounts (e.g., the stories of some of the prominent leaders such as the unruhs and the kroekers). there is also very little drama in the accounts. book rei~iews sawatsky's book on the templer movement is of a very different nature, although it too lacks drama. first, it is told by someone who was only two years old when the temple church (friends of jerusalem in russia) was organized in in gnadenfeld, molotschna. sawatsky therefore was connected with the movement virtually from its beginnings until the book was published in (sawatsky died in ). no one could have understood the inside story better. sawatsky was a student of p. m. friesen in halbstadt, spent the years ( ?) to in palestine where he met his future wife, went back to the caucasus for fifteen years, then moved to palestine where he helped establish a new settlement in , was banished to germany for a short period after world war i, then came back to palestine and eventually was forced to spend his final years (after the establishment of the state of israel) in australia! it is impossible to imagine the drama and hardship of such an experience. it would be most interesting to compare this story with the story of franz bartsch, a participant in the exodus to asia (urlser auszug rzaclz mittelasierz, echo verlag no. ). in fact, sawatsky's bookreveals much less of the drama, passion and hardship than bartsch's, although it must certainly have been there. sawatsky always remains quite distant and the events scarcely betray any transcendent dimensions. does this in itself say something about the nature of the templer leaders, many of whom were among the "best and the brightest" of the mennonite colonists in russia? on the whole, the religious and theological dimensions of the templer movement are scarcely touched on. the concluding essay (which was not in the original german edition), purports to address the issue under the title "the battle for existence and the pursuit of the kingdom of god." this is basically an autobiographical account with a wealth of interesting detail, but there is little that helps us to understand the religious pulse of the movement. helpful additions to the original books include maps of the regions in russia which relate to the content of the books. some pictures have also been added to the templer volume. the quality of the reproductions of the photographs from the original volumes is not always up to the standards possible. a few typographical errors can be discovered (e.g., "kirshcenhardhof," p. , merzrzorlite terizplers; "selbstschutsz," pp. , , merzrzorzite s e t t b n e n t s ) . it is also unfortunate that a consistent spelling of "templer" has not yet been arrived at (s.v., "templar movement," by v. g . doerksen in me v). the binding of the templer volume was also poor. the quality of the translations of both books is excellent and should make it possible for many english-only readers to become more familiar with important dimensions of the mennonite experience. abe dueck centre for mennonite brethren studies r o y d e n l o e w e n , blu??ze?zort. a me~z~zo?zite conznzuizi~ in trmuitior? - (steinbach, manitoba: t h e b l u m e n o r t m e n n o n i t e historical society, . s e c o n d printing ). hardcover, pp. in an attempt to return to the early anabaptist mennonite faith and practice, the kleine gemeinde (today evangelical mennonite church), led by klaas reimer in , was the first group among the russian mennonites to break away from the old congregations. this reforming group objected to the increasing materialism, secularism and worldliness among their co-religionists and insisted that simplicity, humility, non-violence and christian love in all areas of life were the characteristics of true christianity. opposed anddespised by the larger mennonite body, the kleine gemeinde, together with other conservative mennonites, left russiain the s for canada, hoping to establish their ideal community there. blumenort nearsteinbach, manitoba, became the early kleine gemeinde centre in canada. loewen's massive book on the village of blumenort is a kaleidoscopic historical portrait of a typical mennonite community in southern manitoba. by means of community and church records, letters and interviews, the author, himself a farmer and historian living in the blumenort area, has reconstructed a community history which will be of interest to the people of the area and to the historian and sociologist of mennonite and ethnic studies. to read the story of blumenort is to read the history of the mennonites who came to manitoba between and . loewen deals in great detail with the religious, social, cultural and economic factors which shaped andeventually changed the blumenortcommunity. beginning with the religious conservatismof the community in which even the reading of more liberal mennonite newspapers was proscribed and the first automobiles were rejected, the author traces the blumenorters' gradual acceptance of new methods of fanning, more up-to-date education, and eventually modem conveniences that the late twentieth century provided. especially after the second world war the community registered a higher standard of living, more openness to "worldly" influences, and an interaction with non-mennonites. while loewen does refer to external events and factors which influenced the faith and life of blumenort, he is most sketchy with regard to the larger canadian and world context in which his story unfolds. fewer details about blumenort and its people and greatercontextualizing would not haveenlarged the volume but would haveenhanced this valuable history. also in an attempt to be as objective as possible, loewen seems to shy away from an analysis and evaluation of the conservative views and values of the blumenort community. in an epilogue written for the reprinting of the book, loewen states that while the community has changed (first seminary-educated pastor, greater orienta- tion toward the city of winnipeg and steinbach, % of the households bearing non-mennonite names), conservative life values are still dominant. there is an antipathy toward such issues as divorce, alcohol consumption, dancing, and abortion. in the federal election % of the areas people voted in favour of the conservative candidate jake epp. loewen concludes: " ... blumenort's people were book revie s still organizing their lives with reference to family lines, ethnic identity and rural values" (p. ). this book, together with esther epp-tiessen's altonn and peter zacharias' reitzlnnd, helps us to understand and appreciate more fully the "kanadier" mennonite contribution to the multicultural mosaic of canada. harry loewen the university of winnipeg peter a n d elfrieda d y c k , up.fi-onz tlze rubble (scottdale, pa/ waterloo, ont.: herald press, ). paperback, pages. up fronz tlze r~lbble tells the story of the work of peter and elfrieda (klassen) dyck in europe during and immediately after the second world war. as representa- tives of the mennonite central committee, they devoted themselves to the task of aiding victims of the war. the book is unique in that it is written jointly, each one serving as narrator at different points in the story. the style is conversational, the authors claiming that they are not writing history nor an autobiography, but telling a story - that of their involvement in the lives of thousands of refugees. since peter and elfrieda have reported extensively on their experiences in the congregations of north america, the story is well known to an older generation of mennonites, but not to the youngergeneration born since those turbulent times some years ago. the informal narrative style make the book accessible to a wide reading audience, and it is indeed a dramatic and compelling tale which the authors relate. peter was sent to england on behalf of the mennonite central committee in on what was originally tobe aone-year assignment. elfrieda hadalso been sent to england to work as a nurse. both were assigned to work in a convalescent boys' home, and they married in . in they were transferred to the netherlands to begin the mcc relief programme there. refugees of mennonite background from the soviet union began to find their way to the netherlands, seeking asylum on the basis of the fact that their forebears had left this country in the sixteenth century. as more refugees tried to enter the country, officials began to resist, and dyck tells of promising authorities on behalf of mcc that the refugees would be looked after and moved at the earliest opportunity to another country. to allow this temporary asylum he had to produce adocument for these people withoutpapers; the menno pass, a document in three languages, was devised, and this allowed russian mennonites of dutch descent to enter the netherlands. as a sizeable number of mennonite refugees from the soviet union began to find their way to berlin, the dycks were asked to go there to direct refugee operations. a camp was established, but plans had to be made to move the group out of berlin, since they had no wish to be re-patriated to the soviet union. a provision of the yalta agreement was that refugees be re-patriated following the war, and the russians were insisting that soviet citizens be returned. dyck was promised by the u.s. military that they would transport the refugees by train from berlin through the soviet zone, provided permission was granted by the soviets. a ship was waiting in bremerhaven to transport the refugees to paraguay - one country willing to open its doors to the homeless group. after several delays permission was eventually granted and the group travelled without major incident through the soviet zone. there has been much discussion around this "miracle," the fact that the soviet authorities actually signed the papers. one rumour, dismissed as "sheer nonsense" by dycli, was that the russian official, sokolovsky, was drunk and didn't know what he was doing when he signed the papers. dyck does not give an explanation for the decision to sign the permission papers. a thoroughly researched account by t.d. regehr ("anatomy of a miracle," jms, vol. , ) concludes that it was the "fundamentally decent and humanitarian refugee policy officially enunciated slightly more than a year before the rescue of the mennonite refugees in berlin that made possible the rescue." this policy stipulated that soviet refugees would only be turned over to the authorities if they had voluntarily become involved with the enemy forces. regehr also attributes the fact that the train was allowd to go through the soviet zone to the amicable and practical way in whichlieutenant general clay, the american military official, and marshall sokolovsky dealt with many problems that arose between the two occupying powers. peter and elfrieda, sometimes elfrieda alone, accompanied several shiploads of refugees to new homes in south america. the latter part of the book is an account of the challenge of helping people on difficult voyages and in settling in the challenging conditions of uruguay and the paraguayan chaco. peter and elfrieda bring to life the horror and suffering of countless people uprooted by a terrible war and its dreadful aftermath. one is impressed by the dedication, resourcefulness and courage of these two people. also impressive is the account of the supporting role played by the mennonite central committee. we should read this book, lest we forget. ruth vogt winnipeg, manitoba harry huebner, ed., the clzul-clz as theological conznzu~zity: essays ilz hoizo~~l- of david schl-oedel- (winnipeg: c m b c publications, ). xviit- pp. this is an outstanding festsclzl-ift - scholarly, interesting and well-edited. rodney sawatsky, president ofconradgrebelcollege, waterloo, ontario, opens the volume with a speech which was prepared for the occasion of honouring dr. book rel~ietvs schroeder. at times light-hearted and personal but always insightful, it radiates friendship and respect. it also contains some weighty themes-the meaning of suffering, the significance of scholarship, and the concern for peace. particular attention is directed to the cornmunal centre of christian existence, the church, understood as the "hermeneutical community." schroeder's scholarly contribution is evaluated as follows: "what makes his theology so important to us in themennonite church is that his is a theology ofthe church forthe church" (p. ) in which message and lifestyle are mutually supportive. then, as a quiet but visible proof of the significance of schroeder, there follows "a selection from david schroeder's presentations and writings," pp. - . it is an impressive list. the first of the subsequent essays is schroeder's own, entitled "once you were no people " - a carefully analyzed interpretation of the centre of i peter, with particular attention to holiness and service in obedience. the editor is to be congratulated for including this luminous text. the second part of the festschrifi, devoted to historical theology, opens with "theology of the hermeneutical community in anabaptist-mennonite thought" by adolf ens. he follows the lead of george h. williams: "there is one principle or practice-group study and reverent d i s p u t a t i o n o m m o n to the entire radical reformation which goes far to explain the spirit of the movement as a whole" (pp. - ). contrasting such an approach with the practice of magisterial reformers and the spiritualists, ens goes a long way to establish the uniqueness of the anabaptist "hermeneutical community." this study should stimulate further research, which will need to look at the sources in their original languages as well as to pay closer attention to other perspectival issues, notably church discipline and martyrdom. waldemar janzen's "a canonical rethinking of the anabaptist-mennonite new testament orientation" offers a refreshing evaluation of the traditional mennonite view of the old testament, subordinated to the new testament. this approach, thinks janzen, has resulted in a significant neglect, notably of the theological understanding of family and land. in the first instance the stress of the individual faith decisions has hindered more explicit covenantal concerns, specifi- cally in reference to the children of believers who have remained outside thechurch. in the second instance, "the rich land theology of the old testament has scarcely been addressed by our ancestors other than in a spiritualized form" (p. ). the third part of the festsclzr-(fi, identified as "biblical theology," begins with a brilliant statement by mary h. schertz: "interpretation as discipleship: luke as model." to those of us who have read and celebrated-and sometimes only celebrated-dietrich bonhoeffer's tlze cost of discipleslzip, "discipleship" may have drifted into a superlative category. professor schertz makes it very clear that "the synoptic disciples at times appear to be something less than saints" (p. ). she also makes clear that there are times when the gospel is proclaimed "through the words and the actions of the poor, the outcast, the outsider, the women" (p. ). in this way the basis for true discipleship is seen in grace rather than in accomplished perfection. william klassen, "the voice of the people in the biblical story of faithfulness," as always profound and charming, underscores the christian indebt- edness to the bible for the "commitment to basic human rights, justice and peace." here klassen observes that the bible has always acknowledged the reality of human "unfaithfulness" (p. ). hence the meaning of grace is that god's love is not earned by human faithfulness. from within such a context klassen deals with the role of the community in the formulation and the interpretation of the bible. he concludes with the challenge that the local congregation needs to be a dialogical participant in the formulation of theology. in the fourth part, "contemporary theology ," the first offering is by a. james reimer, "how modem should theology be? the nature and agenda of contem- porary theology." here we encounter an overview of what the author regards as significant contemporary developments. he discusses at some length hans jonas and modem technology, gregory baum and social justice, andseveral other thinkers on pluralism. perhaps the reviewer failed to grasp the full depth of this chapter- assuming that it had something to say in the first place. lydia harder, "discipleship reexamined: women in the hermeneutical community," argues that there should be "no privilege of the powerful," but instead "mutual dialogue and counsel" (p. ). while i appreciate her rejection of the traditional misrepresentation of the discipleship of women as mere submission, i think that it might have been helpful to reflect on the administrative structure of the hermeneutical community. the fifth part, "theological ethics," begins with "a critical analysis of narrative ethics" by duane k. friesen, in which critical attention is devoted to stanley hauerwas. the conclusion is forceful: "the fact is that the categories of narrative ethics do not give very precise guidance for our decisions" (p. ). "christian pacifism and the character of god" by harry huebner views pacifism as the only authentic perspective for acceptable theological thinking. having with some care and respect reviewed a variety of alternatives, huebner quickly offers his central insight: "in christ jesus we come to know not merely another side of god, but we come to know the very character of god" (p. ). a variety of texts which have led other interpreters to other conclusions, however, have remained unexamined. moreover, to state that dietrich bonhoeffer was "in prison for his political peace activities" (p. ) is to offer a half-truth. bonhoeffer was executed for participation in an attempt on the life of adolf hitler. indeed, bonhoeffer soughtpeace, but was charged with doing it through other than peaceful means. the concluding sixth part of the festsclzl-ift is somewhat oddly entitled "practical theology." indeed, practical concerns are present, but such was the case in the preceding chapters as well! peter c. erb in "traditional spirituality and mennonite life" offers a truly vintage contribution-wise, insightful, stimulating. it reflects in depth on several weighty religious issues: "the problem of return" to one's religious roots found in an age which widely differs from the present: "defining spirituality" as a concern with the interplay of "spiritual life, love and the book revie\~j.s trinity;" the problem of "spirituality and the baptism of adultsm-probably the most searching and potentially the most controversial statement; concerns with action and contemplation; and "the spirituality of the gospel of peace." finally, james n. pankratz reflects on "mennonite identity and religious pluralism" in a mennonite perspective. having known dr. schroeder personally as a highly respected, valued and loved colleague during the occasions of his teaching directly at the university of manitoba, i can attest that agreat friend and genuine scholar has been well celebrated by this festsclzrif. it should have a wide circulation and an appreciative readership. egil grislis, university of manitoba p e t e r brock, freedom fro violence, sectarian norzr-esistarzce fi- tlze middle ages to tlze great war- (toronto/buffalo/london: university of t o r o n t o press, ). hardcover, pp, $ . . peter brock's book freedom fronz v i o l e ~ ~ c e is part of a trilogy. the other two studies are the qllakerpeace testirnorzy ( ) and freedonz fronz w a r ( ). other books by brock on the subject of pacifism are: pncifisilz in tlze uilited states: from tlze colorzinl era to tlze first world war ( ), t~i)entietlz-cer~t~lv pacifisnz ( ), and pacifisriz irz europe ( ). brock's writing combines skilful summarizing of historical events and probing analysis of issues and their development. on the subject of pacifism brock has gained an enviable reputation surpassed by none. with so many books on pacifism some repetition can hardly be avoided, but generally brock's studies are amazingly fresh, interesting, and informative. this holds true for freedom frori~ violer~ce as well. the pacifist groups included in this book are the following. the medieval waldenses and the followers of petr chelcicky in czechoslovakia; the early swiss and german anabaptists, including the hutterites; the polish anabaptists and the socinians; the dutch, german, french, russian and american mennonites; and the american seventh-day adventists and plymouth brethren. brock ends his study with the beginning of the first world war. about two-thirds of the book, fifteen chapters out of twenty-three, deals with anabaptist-mennonite pacifism. the author shows correctly that anabaptist pacifism, initiated by the swiss brethren and codified in the schleitheim confession of , formed not only the basis but also the norm for later mennonite faith and practice. intending to restore the faith of the early christian church, the anabaptist- mennonites made rejection of sword power, separation of church and state, and the ethic of love in all human relationships the centre of their confessional life. this, according to brock, differentiated the pacifism of the mennonites from that of the medieval groups, including the so-called "vocational pacifism" of monks and some heretical individuals and communities. while the "vocational pacifists" renounced war because of their order or status in society, the mennonites as followers of christ demanded nonresistance of all their members. mennonite pacifism, however, was not consistently sustained throughout the centuries, nor in all the countries in which mennonites came to dwell. social and political pressures and assimilation often weakened, diluted and sometimes even destroyed the earlier pacifism of many mennonites. this happened first in the netherlands, then in prussia and russia, and more recently in modern germany. those mennonites who in spite of governmental pressures sought to uphold their traditional nonresistance emigrated to the americas, particularly the united states, canada and some latin american countries where they were exempt from serving in the army. it thus becomes clear from brock's study that mennonites were able to preserve their pacifism primarily in countries where because of their thrift, skills and economic contribution to society, they were tolerated and allowed to practice their traditional faith. the chapter on canadian-mennonite nonresistance is the shortest chapter in the book, a mere six pages in length. for a fuller treatment of pacifism in canada the reader might turn to the excellent study by thomas socknat, wit~zess against war: pacifisr z itz carzada - (univ. of toronto press, ). this book argues convincingly that it was the so-called "historic peace churches," including the mennonites and hutterites, that established the pacifist tradition in this country. the more modem non-mennonite peace groups treated in brock's study are the seventh-day adventists and the plymouth brethren. while their contribution to the pacifist tradition is significant and similar to that of the mennonites, it is, according to brock, of a different kind. together with the jehovah's witnesses and the christadelphians, two groups not treated in this book, these "eschatological pacifists," as brock has called them, profess a kind of interim ethic. as brock points out, correctly i think, these groups "required their members to become conscientious objectors while at the same time supporting participation in fighting at some final day of reckoning when christ would descend from heaven to overthrow the wicked ....[ tlhese types are all peripheral to the main stream of pacifism" (p. ). nevertheless, i believe that especially the pacifism of the jehovah's wit- nesses requires to be investigated and analyzed by historians of brock's stature. it was the jehovah's witnesses who in hitler's germany suffered more on account of their pacifism than any other group, including the mennonites. in fact, among the mennonites in nazi germany there was not a single recorded mennonite who because of his pacifism went to jail or was executed by the government, whereas there were between , and , jehovah's witnesses who refused to serve in hitler's army, , of whom were sent to concentration camps and more than either died in prison or were executed (see eberhard rohm, sterde~zfiirclerz friecler~, , p. ). the book includes an appendix on "christian pacifism in denmark and sweden to ," an annotated "bibliographical postscript," citing the literature on pacifism which has appeared since the book had gone to press, copious notes, twenty-four book titles for further reading, and a useful index. the book's selling price may be a bit steep for the average student, but for the interested reader and student of the subject brock's book is definitely a must. harry loewen the university of winnipeg r o s s t. b e n d e r a n d alan p.f. sell, eds., baptisni, peace and tl?e state in tlze reformed and merzrzorlite traditiorzs (waterloo, ont.: wilfrid laurier university press, ). hardcover, xi + pages. $ . the context of ecumenical dialogue between the streams of the christian tradition has shifted notably in the last generation. in the s and s, some of us, under the spell of both rationalistic self-confidence and pietistic enthusiasm, and encouraged by the remarkable changes in the roman catholic church, anticipated that the divisions which had proved such a handicap to the proclamation of the gospel would perhaps be overcome in our lifetime. with the shift of the christian centre of gravity to what in the time of superpower confrontation we called the "third world," the resolution of disputes which arose among western europeans four centuries priviously has moved to the background, while issues which most of us have regarded as more global and urgent have takencentre stage in the ecumenical drama. fortunately, the dialogue on unresolved issues from the reformation of the sixteenth century has not been forgotten. indeed, the conduct, if not the conclusion, of this dialogue may be crucial to the wholesome participation of those of us with european roots in the tasks of the global christian community. both the acrimony of the reformation disputes and the persecution and bloodshed with which they were too often linked represent demons which will trouble us until they are exorcised. this exorcism will no doubt require of us at least as much intellect and energy as the original debates received from those who conducted them. in this generation the dialogue has been unhitched from discussio~ls of institutional mergers, and from an immediate concern for the total ecumenical vision. rather, issues dividing one particular stream from another have been taken up in an atmosphere of patient civility and modest expectation. the results have had a low public profile but have been admirable and encouraging. we may celebrate, for example, the achievements of the lutheran-roman catholic dialogue as reflected in the three volumes of lutl~er- s arzcl crrtlzolics in dicrloglre , edited b y paul c. empie and others, and published by augsburg/fortress press. the volume being reviewed here is another example of this worthwhile kind of dialogue between two streams of the christian tradition. the occasion behind this book was a consultation held in october at the university of calgary under the sponsorship of that university's institute for humanities. it was initiated by alan p.f. sell, incumbent of the chair of christian thought in that institution andformerly theological secretary of the world alliance of reformed churches. inspired by the reformed-mennonite consultation held in strasbourg in , dr. sell sought and received support from both the warc and the mennonite world conference for a north american sequel. this co-operation of the churches and the academy brought together an exceptionally fine (although unfortunately all male) collection of scholars from canada and the united states. as the title suggests, the dialogue focused on three topics which have typically been at issue between mennonites and the reformed churches: baptism, peace, and the proper relationship between church and state. six papers - two on each topic - form the first and largest part of the book. although reformed and mennonite views are thus presented alternately, almost as if to structure the contrast between them, the tone of the presentations is irenic without being unfaithful to the presenters' roots. charles west records his own ambivalence about pedobaptism, and reminds us of reformed theologian karl barth's verdict against it. marlin miller, while making a strong case for what he understands to be the "best" mennonite view of baptism, recogilized the wide diversity of mennonite baptismal practice. harry loewen lifts up somewhat neglected dimensions of the anabaptist heritage, such as the thought of balthasar hiibinaier and pilgram marpeck, to show that there are points of contact with the reformed concern for responsibility for civil justice. iain nicol presents a clear summary of calvin's critique of the anabaptists on this topic, but concludes with questions about the contemporary adequacy of calvin's views. to this reviewer, the middle two papers were the most complex and interesting. max stackhouse links the topic of peace to persistent questions about church, family and state, breaking through stereotypes of opposition between the two traditions to a recognition of unresolved issues which will require us to draw upon the "plurality of gifts" given to our churches. howard john loewen points to the need for continuing re-interpretation of the mennonite tradition of social ethics, attempting to show that contemporary mennonites are articulating an approach to peace which is, within the terms of h.r. niebuhr's well-lcnown typology, transfor- mational, a typification which niebuhr himself believed was best expressed by the reformed churches. dialogue on these topics, of course, involves more than reformed and mennonite perspectives, and it raises interpretational questions of concern to the whole academy. both the conference and the booli recognized this wisely. part two of the booli gives us the responses of catholic philospher hugo meynell, united church historian torn sinclair-faulkner, and two baptist scholars: sociologist har-ry h. hillar and theologian andrew d. macrae. the first two, in particular, add book rel~irt~'.r the lustre of imaginative writing to an aready well-crafted set of presentations. in fact, the whole bookis amazinglyreadable for being the proceedings of aconference. further, the editing and printing are both of exceptional quality. this reader found only ony typographical ersor (p. ). even the five-page conclusion which, with a list of nine recommendlt' ions to the sponsoring denominations, makes up the brief third part of the book is unusually well-written. it goes beyond mere summary of what was said, charting a course for the future of the dialogue in compelling fashion.we are not told who wrote the conclusion. the two editors and the calgary institute for the humanities are to be commended for organizing this fruitful dialogue, and for presenting it to us in this well-crafted book. we may hope that this achievement will encourage further, and broader, dialogue. john badertscher the university of winnipeg titus p e a c h e y and l i n d a g e h m a n peachey, seelcitzg peace (intercourse, pa: g o o d b o o k s , ). there are some things that story does better than other kinds of discourse. it can, for instance, cany truth into the heart more directly than either essay or sermon. seeking peace is, for thisreason, abookfor the heart. it is an omnibus of stories (more than ) with a common theme: how do mennonites around the world close the gap between what they profess to believe about non-violence and how they live out that profession? the focus is pretty well narrowed to the mennonite response to war- related destruction and violence. such response, in these stories, most frequently is the refusal of one of three activities: taking up arms, employment which aids the war industry, or paying war taxes. (incidentally, some readers may be surprised, as i was, to learn that war tax refusal is not just a recent stance, but one taken by anabaptists in the th century.) individual stories vary in intensity and impact on the reader. some are merely sketches, less than a page, while others are more fully developed, allowing the characters to take on life and the described events to exert a degree of dramatic force on the reader. it's the cumulative effect of story piled on story that eventually compels the reader, creating for her the sensation of being sussounded by a veritable cloud of witnesses. these witnesses, like those of hebrews, are not restricted to one historical era, and, although most livedllive in america, collectively they represent the world. they speak with a rich diversity of voices, reaffirming, for the most part, what the lancaster, pennsylvania, mennonites said in november, : "we find no free- dom in giving, or doing, or assisting in any thing by which men's lives are destroyed or hurt" (p. ). individually, the stories represent varying degsees of success in working out the mennonite, amish and hutterian teaching on non-violence. for instance, the controversial experiment in self-defence undertaken by russian mennonites after the revolution stands in contrast to zairian pastor kobangu thomas's refusal to take a gun during the volatile events in zaire during the early s. pastor thomas could say, "i icnow my hands are clean of human blood." siegfried bartel, on the other hand, came to his peace position nfrer- serving as captain in the german army in world war . conviction came when he heard the russian soldiers singing the same carols his soldiers were singing in german; and when, in the polish corridor, he realized that, "icould have killed my cousin!" during the same war, marian claassen franz, growing up in rural kansas, observed how her mother invited gesman prisoners, who helped on the family farm, into their home for lunch. for mary, the american guards accompanying these prisoners were more frightening than the "enemy." the irony is intensified when a guard falls asleep while supervising the pows and a jeep full of military superiors arrives. the pows walce the guard in time to save him from the wrath of his superiors. at firstreading, i was puzzled by the organizationof the book: the stories jump from continent to continent, from century to century. why aren't they grouped according to time and place, or around specific wars? i came to the conclusion that the collage effect deliberately chosen by the authors was appropriate. by placing in sequence elena munoz, a latin american mennonite struggling to live out peace principles in a compromising work situation; paul and loretta leatherman, modem americans deliberately withholding war taxes; and jan smits, a sixteenth centuryanabaptist martyr, the authors once more make their point: the way of peace demands sacrifice from followers of christ, regardless of time and place. at the end of the book, there are indices that organize the stories according to general issue, geography, and era. and the bibliography will be useful for readers whose appetite has been whetted for more information on the anabaptist experience and peace theology. the authors, titus peachey and linda gehman peachey, have long been acquainted with peace issues. from - they served as directors of mennon- ite central committee relief and development programmes in laos. following their return to the u.s. they completed a study of military-related industries in lancaster county, pennsylvania, titled wor-ldpeace begirls irz lnncnster: at present they serve as co-executive secretaries for mennonite central committee. u.s. peace section. sarah klassen winnipeg, manitoba book re ~irn .s you doll't get to be a saint. p o e m s by patrick friesen (winnipeg: t u r n s t o n e press, ). patrick friesen has established himself as a poet with a distinctive voice in canadian letters. his explorations of experience which is of necessity mennonite but not necessarily mennonite has been sensitive and profound. in the past several years he has also extended his range by recasting his powerful poem the sh~rr lillg as a stage production, and this season his play tlze raji had a very successful run at the prairie theatre exchange in winnipeg, demonstrating that friesen's compelling lines have dramatic as well as lyrical intensity. his new book of poems is named after its last poem, a conversation of sorts at the margin of death, but this title characterizes rather well what is a collection of diverse pieces, including texts for a multi-media performance (a hcl rlfill ofrnir~), a dance collaboration (nmzn), a radio reading (sillgel-), as well as several groups of new poems. apart from his ventures into new avenues of expression, the poems of patrick friesen remain keen and taut because of his unending pursuits of the lifelines of his experience. dennis lee refers to his "furious equipoise" on the cover, which is a fittingly dramatic collage by the artist esther warkov. my own favorites among the many splendid poems are those evoking those earlier memories which have become his frequent preoccupations, like: rowing home sundays after a week of the hammer after ladders and beams and skyline father collapsed in a sprawl like an idling motor about to stall like a tamarack leaning into a fall like a bundle of clothes or a doll it frightened me as if he was dying as if the strength of his arms was flowing through his fingers into the air as if the continent of his body was sinking away as if his breath was a sigh from the end of the world i watched the hesitant rise and fall of his chest i imagined his red heart slow-pulsing the blanket slid from his shoulders and he turned against the cold reaching blindly in his sleep for comfort he seemed a wounded lord i wanted to bury his name take him into my arms and carry him to the river to find the boy he was i wanted to row him home in poems such as thesepatrickfriesen not only shows adeep love of language but his language betrays layers of his own love, which he is exploring. as he says in his "biography": i'm a child in a northern tree still climbing at the sky in a brief review it is impossible to represent the varieties of friesen's style. readers will find an intriguing variation on t.s. eliot in the poem "godly w o r l d , a text which would have appeared here, were it not for fear of the dreaded f-word in this journal. another great gift of this poet is his recognition of the meaning of silence, the silence which so essentially surrounds and accompanies the poet's word. he brings his own tribute here to silence if it's chosen silence is not an enemy it is where words begill and end silence is the heart's chamber between beats we have reason to be grateful to the turnstone press for producing this and the other poetry collections which have allowed a gifted mennonite generation to find a free voice and an understanding audience which is by no means limited to the mennonite world. victor g. doerksen university of manitoba s a r a h klassen, violeizce and mercy. p o e m s (windsor: netherlandic press, ). paper, pages. $ . where violence is dismemberment, mercy consist of making whole again. while many poets mirror the fragmentation of violence, sarah klassen ap- proaches it from a desire to heal. violerzce nr d mercy, her second collection of poetry, speaks of the desire to soar above this broken earth and see it whole. i came away from this book saddened by her evocations of murder and v i c t i m - zation, yet grateful for the courage and faith with which she shares her vision, and grateful, as well, for the dedication with which she attends her work. these poems are carefully and lovingly crafted. klassen handles free verse with admirable discipline. the rhythm of her lines is simultaneously natural and dramatic; the imagery is vivid. violer~ce arzd mercy opens with poems about the complex relationship between a teacher of literature and her students. klassen is very aware of the failure of language to bridge the gaps between us. the teacher can only "dispense connectives" ("language arts"). even the confessional journals of the students, filled with their private terroranddesperation, bring theirteacher only slightly closer to them: ~ o o k r e i i e \ ~ ~ s ... there is nothing i can do. nothing except point out perhaps the lack of punctuation, circle the misspelled words with a soft pencil, join all disconnected fragments ... ("evidence") it is a brilliant touch, that "soft" pencil -as though tenderness could only be bestowed through marks on the page. language and human mercy, it seems, are often inadequate for the violence of the world. but the poet refuses to despair. this theme carries through into many of the poems of domestic and military violence which follow. "while waitingfor war" captures the "long white silence" and "terrible emptiness" of january , as george bush's deadline approached and the world prepared for war. the stanzas are numbered one to ten, recalling the grim countdown of those days, the inevitability, the willfulness, the premeditation. but there is more faith than anger here. the poem speaks of prayer and of petition. "eachcedar tree holds snow in its upliftedarms," as if in supplication. klassen treats this difficult theme with humility and with a sense of wonder. there is no righteousness here, no pronouncements, only an invitation to question: "what will you beg for?" viole~lce and mercy closes with a series of poems about leonardo da vinci, his "obsession with motion" and his "desire/ to master each tiny nerve/ and artery, each bone,/ the stubborn direction of blood" ("anatomy"). the artist's desire to masterthe impossible resonates sweetly and sorrowfully with the natureof i<lassen's own work. for da vinci longed to devise a way to fly and a way to paint "the face ofchrist" ("you, leonardo"). klassen's lastpoem, "venice, from space," speaks to da vinci from the future: "you should hear us now, leonardo, laughing as the earth spins away from our feet." listen. you can hear us singing like the meadowlark children wait for in spring on the wet stubble. in the diamond air its gold throat swells. every desperate feather trembling. art, perhaps, will have some small triumph, will lift us up above the earth for a moment, will lend us vision. that's all we can ask of a poet - and sarah klassen gives it to us with sure skill and considerable grace. catherine hunter the university of winnipeg a r m i n w i e b e , mlli-der irz gz~terztlznl. a sclzrzeppa kjrznls mystery (winni- peg: turnstone press, ). the wide success of armin wiebe's sah~ation of yclsclz siellzelzs was asurprise and something of a shock in the mennonite constituency. what many could not understand was that its apparently convolutedflat german syntax could be read and enjoyed by ordinary welt~~zensclze~z, even across canadaon the cbc! and then there was its "questionable" narrative, claiming to deal with salvation, no less. it was the realization by many readers that this claim was justified-in its own way-that created a kind of aura of profundity around this work. no one was sure how serious, how important, how good this book really was. already critics are suggesting that yasch sienzelzs will occupy a pivotal place in the transitional history of canadian mennonite writing because of its successful merging of low german and low canadian. all of this makes things very difficult as armin wiebe presents a new novel, and it is well that mur-del-irz gurentlzal is clearly identified as abookof another genre. whether we call themdetective stories, who-done-its orpot boilers, it should beclear that we have no business looking for metaphysics (or even metalinguistics) in what proposes to be the first in a series of schneppa kjnals mysteries, in spite of the fact that a bible is mentioned in the first sentence. wiebe, having found a locale and an idiom, turns them loose and leads the reader on a more or less merry chase through a series of complications which lead neither to salvation nor damnation (unless that be confusion). this will leave mennonite readers very unfulfilled. of course, a private eye or snooper is not supposed to bring about salvation, but a solution of some kind is calledfor. and in thatregard perhaps wiebe has scatteredabout too many false leads and clues and has really not succeeded in tying up the right ones. for some readers like me the asides and reflections of armin wiebe are among the best parts. the schneppa becomes something of a seer (or mystic?) when he ruminates about the windows of the soul: don't ask me how i know this, or how i know it's true, or if i know it's true. of course, you know what comes in through the windows of the soul, but the guy who said theeyes are the window of the soul left a lot of windows boarded up, i think, at least when it comes to knowing what is true. what is true is what you make of it. i f enough people make the same thing of it as you do, then it's true. if you make something different of it than everybody else, then you're crazy. truth is no use without believers. of course, it helps if you take the boards off all the windows so as much light as possible comes in ( ). for mennonites bibles, zippered or unzipped, are serious subject matter, as are other items such as women and cars, and many readers will not share schneppas view of them. maurice mierau in a pmirie fire review quite arbitrarily attributes this view to wiebe himself. finally, it is aquestion of whether the mennonite reader (and others too) will suspend herlhis belief long enough to enjoy what is nothing book re jie .s more or less than (dare i say it?j aromp through the southern manitobacountryside. victor g. doerksen university of manitoba sara stainbaugh, tlze sign of tlze fox (intercourse pa: good boolts, ). the author of the fine first novel, i hear tlze r e q e r ' s song, here presents another pennsylvania story, but one which is quite different from her lyrical earlier work. the sign o f the fox is a much busier novel, very readable but scarcely as memorable; this does seem to be a problem with second novels, as stambaugh's mentor, rudy wiebe, may recall (what was the title of his second novel?). sara stambaugh writes about a locale which she obviously "remembers" well. the fox is an inn in lancaster county run by a german immigrant family (carpenters, who were zimmermann), whose heavy-handed head, squire will carpenter, confronts the surrounding mennonite folk with tough, entrepreneurial realism (the "world") and places the neighboring family of the heroine in a suitable posture of suffering for their quiescent faith. together with his slick lawyer son, carpenter outmaneuvers gideonlandis in an attempt to obtain the rights to land and a quarry which will become valuable as the railway is built through the area. the carpenter family and its aspirations form an important part of the novel, much more than a backdrop, so much so in fact that the mennonite "angle" is almost lost from view at times. this certainly gives the novel a more substantial social framework than is often the case in ethnic literature, but the complexity of plot and character is too muchforthe short novel and various individuals who might well have grown on the reader remain confined to epithets and even clichcs. this is evident not only in the stock characters who spit and drink, or the complaining cook, but much more disturbingly when the author includes episodes like that of the escaping slave, making the fox an involuntary stop on the underground freedom railroad. there are simply too many such disparate elements, from american national politics and corruption to divorce, suicide and single parenthood. such freight is too much even for jake, the jovial waggoner, and certainly for a novel of pages. the heroine, gideon's attractive and dutisul daughter, catherine, is syrnpa- thetically drawn in spite of being attractive and dutiful. she suffers the fate of many heroines who are long-suffering, and is saved for a twentieth-century reader only by the author's careful understatement. even so, the "happy ending" strongly supports the contention that there is more soap here than that manufactured by the hardworking heroine. knowing the kind of strong, lyrical writing of which this author is capable, one must express the hope that this too-busy tale will be a brief waystation enroute to more substantial depictions of the human condition. meanwhile, this story may serve a younger readership very well. victor g. doerksen university of manitoba victor peters a n d j a c k thiessen, pla~~tdietsche jesclziclzten, gesl~rflcke-i~~ter-vievvs- elziihlzl~zgerz ( marburgilahn: n.g. elwert verlag, ). paperback with dustjacket, pp., c a n . $ . . this book is acollection of interviews and stories written in the low german dialect of the netherlandic mennonites who emigrated to america via prussia and russia since . the interviews have been conducted mostly with mennonite seniors in southern manitoba, but several took place in mexico and elsewhere. they provide interesting side-lights on events of history involving mennonites since world war i, describing personal views and reactions in the words of those who experienced and survived those events. one interview is with a canadian doctor of scottish descent who lived and practised among mennonites and learned to speak their language better than many. among the more than forty short stories by the authors, most tend toward the humorous, many with thought-provoking undercurrents. one story about "sotknacka" derksen and his misadventure among the beams of the neighbour's hay-loft first appears in peters' interview with nikolaus neufeld (p. ) and then later in changedforin inthiessen'sstory "adam, wobist du?" (p. ). it is stroi~gly reminiscent of the stories entitled "icasper-ohm un ick" written in the low german of mecklenburg by john brinckman in the s and since republished. readers who stem from the first migration of mennonites from the molotschna colony of the ukraine to american in the s, as does this writer, will notice that the low german of victor peters differs from that of jack thiessen, and that the language of both differs from ours. victor was born in the old colony (chortitza) and speaks in the old colony manner of that period. thiessen's father is from the old colony but jack was born in canada. his speech resembles more that of the molotschna colonists. a minor example of the difference is found in the index where peters writes "jeschichten" whereas thiessen writes "jeschichte." the plauidietsch of both authors reflects those changes that infiltrated the dialect in russia under the influence of intensified use of high german and russian among mennonites there in the period after . a problem for writers of plautdietsch is that it has no generally-accepted written form. consequently, the authors have made choices in spelling with which some may not agree, but which one now cannot fairly criticize. however, readers will wonder why inconsistencies in spelling which detract from easy reading were not eliminated at the proofreading stage. the index to short stories lists a number of titles consisting of a name followed by "en aundre menschen." it is quite understandable that a character named in atitle interacts with other peoplein the story, but tolist asstory titles aseries of names, each followed by "and other people" eight times in a row seems overly repetitious. this book is the culmination of years of work by two capable authors. their interviews and stories cover a broad spectrum of subject matter and points of view. a glossary of terms with translations into english and german, together with over fifty pictures of people and places round out those dimensions. since it is written in mennonite low german, it will be of particular interest to those who speak or study the language. reuben epp, kelowna, b.c. paton yoder, tradition atzd traizsitiorz: ai zislz mennoi~its ciiid old orcier- aiuish - (scottdale, pennsylvania: herald press, ). pp. hardcover. at a gathering of amish ministers in the spring of in wayne county, ohio, a turning point was reached for the amish church which would come to be known as the great schism. this date maslced the beginning of the distinction between the old order amish on the one hand, and other amish congregations, who would eventually join with the mennonite church. this historic gathering was not, in fact. an isolatedevent. it was one of aseriesof similarmeetings, beginning in , which were a response to conflicts that had been emerging for some time between conservatives and change-minded factions among the amish. these gathelings had, however. themselves seemed to have theeffect of polarizingexpectations. they thus led to the presentation, by i ministers representing conservative congregations, of a position paper at the gathering which they deemed to be right and biblical, and therefore non-negotiable. the events leading up to this historic series of meetings, as well as the subsequent progress of the two emerging factions, are described in considerable detail by paton yodcr. yoderhirnselfhas amish mennoniteroots. he has also taught american history for the past fol-ty years. yoder treats the great schism as a focal point. several introductory chapters set the stage by providing an overview of early amish history in north america; an era which has been neglected by other amish scholars. some valuable glimpses into the amish way of life in the early nineteenth century, their faith, and their traditions is also provided. the account of the early struggles of the amish in north america may be of interest, as well as the descriptions of the structure of the amish ministry. for example, a detailed account is provided of the origin and use of the practice of choosing ministers by lot. yoder does not simply present an idealized image of amish life during this period. his descriptions also convey some of the stresses and conflicts that were experienced. for instance in describing the practice of ordination by lot, he includes the story of a john stolzfus of the buffalo valley congregation, who upon being informed that the lot had fallen on him, fainted, frothed at the mouth and could not be revived for half a day. one limitation of this work lies in its focus on the political activities of a relatively small group of church leaders. this is particularly true with reference to the great schism and its consequences. while references are made to broader social forces, they seem to become secondary to the personalities and activities of a relatively small group of men who were organizers and participants in the various meetings, and whose correspondence forms the basis for yoder's analysis. within this context, ambiguities also arise regarding the actual issues which divided the amish during this period. for example, frequent references ase made in the preceeding discussion to the practice of stream baptism as a point of contention. however, we note that it is not even mentioned in the position paper of . yoder states that this omission was deliberate, but then does not tell us why it was not included. yoder does nevertheless contribute to our understanding of the amish way of life through his elaboration of the historical precedents of the amish as they exist today. this historical background provides us with some important insights into the emergence and development of this unique and distinctive culture. paul redekop the unversity of winnipeg book re~zie~c,s hical notes donovan e. smucker, tlze sociology of mero~onites, h~rtterites ar~cl ai~zish. a bibliograplzj~ cvitlz anr~otations, vol~rrize ii, - . (waterloo: wilfrid laurier university press, ). this bibliography provides an annotatedsurvey andanalysis of the "sociological literature" concerning "the highly va-ied mennonites, the com~nunal hutterites and the semi-communal anti-industrial amish." (p. ix) while not complete and attimes not clear as to whether an entry is of a sociological, literary or historical nature, this sequel to smucker's earlier volume ( ) is a welcome addition to mennonite bibliography. the volume includes titles of published books, pamphlets, graduate theses and articles, as well as unpitblished sources. the special issue of tlze g e n i z ~ ~ ~ - c a ~ ~ a d i a ~ ~ yearbook, vol. i, edited by hartmut froeschle and lothar zimmesmann (toronto: historical society of mecklenburg upper canada, ), includes , entries of titles of histol-ical, cultural and literary studies by german-canadian and mennonite writers. this is an invaluable bibliography for anyone working in german-canadian andcanadian- mennonite research. tlze radical refolnzatiorz, edited and translated by michael g. baylor (cambridge university press, j, includes writings by the most important radical reformers including: thomas miintzer, andreas karlstadt, conrad grebel, felix mantz, hans denck, hans hut, michael sattler, balthasal-hubmaier, hans hergot, and an anonymous writer. the appendices include the following documents: "the eleven miihlhausen articles," "the twelve articles," "the memlningen federal constitution," "the document of articles of the black forest peasants," "the forty-six frankfurt articles," and michael gaismair's "te~ritorial constitution for tyrol." this collection, which includes a valuable introduction and helpful biographical notes, is a welcome text for radical reformation studies. john s. oyer androbert s. kseider, mirrorofthe m a r ~ r s . storiesofco~rrage, irzspirirlgly retold o f / century arzabaptists wlzo gave their lives,for tlzeir.faitlz (intercourse, pa: good books, ). this is an attractive paperback introducing the reader to the massive mar-tyrs mirror of thieleman j . van braght. the illustrations, reproduced from the original work, and the text in modern english will appeal to both young and older readers. thomas p. socknat, witrzess agairzst war. pac(fisriz in ccirlackr - (toronto: university oftoronto press, j, argues convincingly that peace groups such as the mennonites, hutterites and jehovah's witnesses laid the foundation for the pacifist tradition in canada. the author shows that members of the united church of canada and various women's groups were most active during the two world wars and how they suffered for their pacifist stance. donald wiebe, tlze imny of tlzeolog~~ arzd the nature of religio~ls tl~ouglzt (mcgill-queen's university press, ), argues that theology as a rational, academic discipline, rather than being supportive of religion, actually undermines and even destroys religious faith. this study draws upon sources in philosophy, history, anthropology and sociology, thus appealing to scholars involved in these disciplines. wiebe seems to confirm what anabaptist-mennonites have felt all along: that the "learned ones" are dangerous to christian faith. merzrlorziie wtdtirzg in cnrzcrdcr, a special issue of tlze new, qll~i-ter-ly: nelv dii.ections in cnrzadinrz writing, vol. x , numbers and (springisummer ), guest-edited by hildi froese tiessen, includes the following: articles by e.f. dyck, peter erb, jeff gundy, lorraine janzen kooistra, and henry wiebe; stories by h o l d dyck, sarah klassen, anne konrad, andreas schroeder, sara stambaugli, jack thiessen, armin wiebe, rudy wiebe, and others; poems by di brandt, patrick friesen, jeff gundy, sarah klassen, douglas schulz, david waltner-toews and others; and translations by a reimer and andreas schroeder. included also is the editor's interview with the poet patrick friesen. tlze balarlcirlg of the cloucls, pnirltirlgs of mary klassen, by john unrau (winnipeg: windflower communications, ). beautifully combines picture and text, theillustratedstory, by herson, ofa mennoniteartist who followed her vocation with courage. a painter "bowled over" by the work of lawren harris, who developed her own powerful and varied landscape style - while being a wife, mother and all the things expectedin the generation before the current one. excellent reproductions and an eloquent text make this a fine and unique book. one cannot do justice in a brief bibliographical note to merzrlor~ite f ~ ~ r . r ~ i t ~ ~ r e . a migrarlt trnditioil ( i - ) (intercourse, pa: good books, ), hardcover, pp., $ . u.s., written and compiled by reinhild kauenhoven janzen and john m. janzen. this book, which includes figures of full-colour and black and white photographs of mennonite furniture and other household items, is a delight for the eyes and mind. art lovers, historians, and other persons interested in things mennonite will find this artistic and scholarly book a pure pleasure. the pictures, the text, the endnotes and the binding are of the highest quality. the editors wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ ellis van creveld is required for postnatal craniofacial bone development a cc ep te d a rt ic le ellis van creveld is required for postnatal craniofacial bone development mohammed k. badri , , honghao zhang , yoshio ohyama , sundharamani venkitapathi , nobuhiro kamiya , , haruko takeda , manas ray , greg scott , takehito tsuji , tetsuo kunieda , yuji mishina , and yoshiyuki mochida ,* department of molecular and cell biology, henry m. goldman school of dental medicine, boston university, albany street, boston, ma , usa department of pediatric dentistry and orthodontics, college of dentistry, taibah university, al-madinah al-munawarah, saudi arabia department of biologic and materials sciences, school of dentistry, university of michigan, n. university ave., ann arbor, mi - , usa national institute of environmental health sciences, tw alexander drive, research triangle park, nc , usa unit of animal genomics, giga-r & faculty of veterinary medicine, university of liège, avenue de l’hôpital, -liège, belgium graduate school of environmental and life science, okayama university, okayama city, japan correspondence to: yoshiyuki mochida, d.d.s., ph.d. albany street, w d, boston, ma , usa. tel: + - - - , fax: + - - - , email: mochida@bu.edu running title: role of evc in craniofacial bone development grant sponsors: nih/nidcr; de (y.mo.), nih/nidcr; de (y.mi.). page of the anatomical record this is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the version record. please cite this article as doi: . /ar. . this article is protected by copyright. all rights reserved. http://dx.doi.org/ . /ar. http://dx.doi.org/ . /ar. a cc ep te d a rt ic le abstract ellis-van creveld (evc) syndrome is a genetic disorder with mutations in either evc or evc gene. previous case studies reported that evc patients underwent orthodontic treatment, suggesting the presence of craniofacial bone phenotypes. to investigate whether a mutation in evc gene causes a craniofacial bone phenotype, evc knockout (ko) mice were generated and cephalometric analysis was performed. the heads of wild type (wt), heterozygous (het) and homozygous evc ko mice ( -, - and -week-old) were prepared and cephalometric analysis based on the selected reference points on lateral x-ray radiographs was performed. the linear and angular bone measurements were then calculated, compared between wt, het and ko and statistically analyzed at each time point. our data showed that length of craniofacial bones in ko was significantly lowered by ~ % to that of wt and het, the growth of certain bones, including nasal bone, palatal length and premaxilla was more affected in ko, and the reduction in these bone length was more significantly enhanced at later postnatal time points ( and weeks) than early time point ( week). furthermore, bone-to-bone relationship to cranial base and cranial vault in ko was remarkably changed, i.e. cranial vault and nasal bone were depressed and premaxilla and mandible were developed in a more ventral direction. our study was the first to show the cause-effect relationship between evc deficiency and craniofacial defects in evc syndrome, demonstrating that evc is required for craniofacial bone development and its deficiency leads to specific facial bone growth defect. keywords cephalometric analysis, craniofacial bone, ellis-van creveld syndrome, evc , knockout (ko) mouse page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le introduction ellis-van creveld (evc) syndrome (omim # ) is an autosomal recessive genetic disorder showing chondro-ectodermal dysplastic dwarfism characterized by short limbs, short ribs and postaxial polydactyly (kurian et al., ). this rare medical condition has been first reported in by drs. richard ellis and simon van creveld (ellis and van creveld, ). the unique intra-oral clinical manifestations in evc patients include; a shallow sulcus, the presence of broad/multiple labial frena, missing and dysmorphic teeth, taurodontism and incomplete root formation and malocclusion (ghosh et al., ; kurian et al., ). this syndrome is most prevalent in the amish population of lancaster county, pennsylvania, occurring in / , live births, whereas the birth prevalence in non-amish population is estimated to be / , , (kurian et al., ). weyers acrofacial dysostosis (wad; also known as curry-hall syndrome, omim # ) is described as an allelic variant of the evc syndrome with an autosomal dominant inheritance, but shows a milder phenotype (weyers, ; curry and hall, ). in partnership with the amish, mutations associated with evc syndrome were first identified in evc gene, however, these mutations did not account for all the evc cases tested (ruiz-perez et al., ), suggesting the presence of genetic heterogeneity in this syndrome. bovine chondrodysplastic dwarfism (bcd) has been reported in many cattle breeds (jones et al., ; jayo et al., ; agerholm et al., ). affected calves show an autosomal recessive trait and characteristic changes similar to those of chondrodysplasia in human (julian et al., ). the bcd locus in japanese brown cattle was mapped to the distal end of bovine chromosome by linkage analysis (yoneda et al., ), and disease-specific mutations were identified (takeda et al., ). we previously identified the causative gene for bcd and page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le designated as limbin (lbn) gene (takeda et al., ). later, a similar mutation has been identified in evc patients (galdzicka et al., ) where it is designated as evc gene, identified as the human ortholog of lbn, indicating the biological importance of evc /lbn protein function in skeletal tissue development. it is now known that evc and wad patients have mutations in either evc or evc gene, both of which are located on human chromosome p in a head-to-head configuration (ruiz-perez et al., ; galdzicka et al., ; ruiz-perez et al., ; ye et al., ). evc patients exhibit craniofacial bone growth and developmental phenotypes, i.e. defective skull growth pattern such as enlarged skull, depressed nasal bridge, mandibular prognathism, skeletal class iii (maxillary deficiency and mandibular prognathism), and skeletal open bite (ellis and van creveld, ; goor et al., ; susami et al., ), while some studies described that the face appeared to be normal (varela and ramos, ; hanemann et al., ). such inconsistent reports may be in part due to the following reasons; ) no gene mutation(s) were identified in these earlier case reports and ) there is little information regarding cephalometric measurements and analysis in these patients. this raises a question whether evc or evc mutation affects the craniofacial bone development. it has been previously reported that evc knockout (ko) mice exhibited chondrodysplasia affecting chondrocyte differentiation. although the report showed the presence of evc expression in the lateral nasal, maxillary and mandibular processes during prenatal craniofacial development, craniofacial bone phenotype has not been described (ruiz- perez et al., ). as we previously reported that evc is expressed in cranial bone and facial primordia during mouse development (takeda et al., ), evc likely plays an important role in craniofacial development. to support this notion, more recently, evc ko mice were generated by replacing the first exon with an egfp cassette and showed cranial base phenotype page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le at embryonic day (e) . (caparros-martin et al., ). however, the study was limited to show only the presence of impaired development of the intrasphenoidal synchondrosis and any other craniofacial defects have not been described in detail, partly because these mice do not survive to an appropriate post-natal age (caparros-martin et al., ). this remains the cause- effect relationship between evc deficiency and craniofacial phenotype(s) unclear and thus, a new animal model by which post-natal craniofacial bone phenotype(s) can be analyzed is warranted. very recently, we reported that a new mouse model of evc /lbn ko mouse by introducing a premature stop codon in exon mimicking the mutation found in bcd cattle in exon (takeda et al., ). the objective of this study was thus to characterize craniofacial skeletal morphology of the evc ko mouse. materials and methods ethical approval all experimental animals used in this study had access to food and water ad libitum and housed under regular light cycles. at the end point of the experiment, animals were euthanized by carbon dioxide gas in an uncrowded chamber followed by a physical method to ensure death. all animal procedures were approved by the iacuc at boston university medical campus (boston, ma, usa) and university of michigan (ann arbor, mi, usa) and performed in accordance with the nih guide for the care and use of laboratory animals. evc knockout (ko) mouse generation and genotyping of evc ko mice ( svev c bl/ j mixed strain) were previously reported (zhang et al., ). in brief, a targeting vector was designed for evc ko page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le mice by introducing a premature stop codon in exon followed by internal ribosomal entry site (ires) fused to β-galactosidase (lacz). the targeting cassette was introduced to ab . embryonic stem (es) cells and the correctly targeted es cell clones were identified by southern blot and genomic pcr strategies. after the germline transmission, resulted heterozygous (het) evc mice were intercrossed to generate homozygous evc ko mice as well as the control wild- type (wt) and het mice. the deficiency of evc protein in evc ko mouse-derived primary chondrocytes was verified by western blot analysis (data not shown). sample preparation the heads of evc wt, het and ko mice were collected, dissected, fixed with % formaldehyde and stored in pbs at ºc until use. the samples were divided into three groups based on the genotypes and age of animals. the -week group included wt (n= ), het (n= ) and ko (n= ) mice at postnatal day from to (p -p ), the -weeks group wt (n= ), het (n= ) and ko (n= ) at p -p , and the -weeks group wt (n= ), het (n= ) and ko (n= ) at p -p . radiographic imaging each head of mice was oriented in a reproducible head position to ensure a true sagittal and horizontal view image by using a specific mold and incorporating a grid background to orient the skulls on a horizontal line that passes through the tip of the nasal bone anteriorly and the most posterior point on the cranial vault (po) posteriorly. x-ray images of the heads were taken using a standard imaging machine (prostyle-intra; planmeca, il, usa) with a digital x-ray sensor (schick cdr with size sensor; sirona, ny, usa) at the source to image distance page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le of inches, kv of energy, ma of intensity and . seconds of exposure time. object magnification was minimized through direct positioning of the sample onto the film. the radiographic images ( x dpi digital resolutions) were scanned and used for cephalometric analysis. cephalometric analysis linear and angular cephalometric parameters were used based on the previous report (engstrom et al., ) with some modifications, i.e. additional linear and angular parameters including nasal bone height and angulation in relation to premaxilla. based on fourteen reference points identified (table ), thirteen linear and twenty-two angular bone measurements (table ) were calculated by the cdr dicom software (sirona, ny, usa). all data in the graphs were presented as the mean values ± sd. the average of all the linear bone measurements in each genotype was calculated at each time point by the total of linear measurements of all the craniofacial bones divided by the total number of bones and displayed as (average skull linear measurements). values of linear measurements of each individual bone in ko at each time point were normalized to those in wt and are shown as (ko/wt ratios; %). statistical analysis the mann-whitney u test was used to identify the significant difference with the p-value of less than . marking the significance. results page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le phenotype of the homozygous evc ko mice generated evc ko mice had phenotypic changes similar to those seen in evc patients as well as the evc knockout mouse model (ruiz-perez et al., ). while het evc mice showed no discernible phenotype similar to wt, ko mice demonstrated short body length showing dwarfism (fig. a, left) and signs of ectodermal dysplasia of hair, teeth and nails, some of these phenotypes were briefly described previously (zhang et al., ). impairment of bone growth in evc ko mice during postnatal craniofacial bone development as evc ko mice appeared to exhibit smaller skull size comparing to wt (fig. a, right boxed panels), the head samples were subjected to lateral cephalometric radiography to measure the craniofacial bone structures. specific reference points were defined according to the anatomical locations of specific landmarks (engstrom et al., ) (fig. b) and used to analyze the linear bone measurements and the angular bone relations (fig. c) in order to investigate which bones are affected due to evc deficiency during postnatal development. at week, the values of nasal bone length (n-a), total skull length (po-a), cranial base length (so-e), neurocranial length (po-e), viscerocranial height (e-mu), mandibular corpus length (mn-id), and mandibular lingual alveolar bone length (ml-bl) in ko were statistically smaller than those in wt and/or het (fig. a). at weeks, in addition to the reduced bone length found at week, almost all of other linear bone measurements in ko were also significantly smaller than those in wt and het (fig. b). in -week-old ko mice, the values of the viscerocranial length (premaxilla); both anterior and posterior to the upper incisors (e-pr and e-bu), and palatal length (mu-bu) became significantly smaller than those in wt and/or het page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le (fig. b). however, the value of nasal bone height (a-pr) was higher in ko than wt and het, and ml-bl showed no difference among the three genotypes (fig. b). at weeks, ko mice had statistically smaller linear bone measurements than wt and het for n-a, e-pr, e-bu, po-a, mu- bu, so-e, and po-e (fig. c). on the other hand, no significant differences were seen for a-pr, e-mu, mn-id, and ml-bl lengths between ko and both wt and het (fig. c), although the mandibular length (mn-id, ml-bl) in ko was shorter than wt and het. linear measurements for the incisors showed that the erupted upper incisors length (pr-iu) was significantly shorter in ko than wt and/or het at any time points tested (fig. ). the erupted lower incisors length (ii-id) was significantly shorter in ko than wt and het at and weeks (figs. a&b), while there was no significant difference at weeks (fig. c). there were no statistical differences in linear bone measurements between wt and het at any time points tested. abnormal bone-to-bone relation in evc ko mice angular bone relations demonstrate bone-to-bone relationships and establish the spatial relations and positions of the facial bones to those of the cranial base and cranial vault. we next compared angular bone relations between evc wt, het and ko mice at three different ages. at week, the values of angular bone relations of nasal bone to cranial base (anl/soel), premaxilla to cranial base (prel/soel, buel/soel), and premaxilla to cranial vault (prel/poel) in ko were significantly smaller than those in wt and/or het, and those of maxilla-premaxilla to cranial base (muprl/soel), maxilla-premaxilla to cranial vault (muprl/poel), mandibular corpus to cranial vault (mnidl/poel), and mandibular corpus to cranial base (mnidl/soel) in ko were significantly higher than those in wt (fig. a). the differences became significantly obvious at and weeks (figs. b & c). page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le at weeks (fig. b), the values of angular measurement in nasal bone to cranial base and to cranial vault (anl/soel and anl/poel) in ko were significantly decreased when compared to those in wt and het. moreover, the value of nasal bone to premaxilla (anl/prnl) was increased in ko comparing to wt and het. these two altered relations demonstrated that the nasal bone was depressed. the values of premaxilla to cranial base and to cranial vault (prel/soel, buel/soel, prel/poel, and buel/poel) in ko were more significantly decreased than those in wt and het, demonstrating a rotation of the premaxilla in a ventral direction. moreover, the values of palatal plane to cranial base and to cranial vault (muprl/soel, mubul/poel and muprl/poel) in ko were significantly increased compared to wt and/or het, indicating the ventral rotation of this plane consistent with the rotation of the premaxilla. the values of mandibular corpus to cranial vault and to cranial base (mnidl/poel, mlbll/poel, mnidl/soel, and mlbll/soel) were markedly increased in ko comparing to wt and/or het, indicating rotation in a clockwise/ventral direction. dental relations showed that the upper incisor to cranial vault relationship (iuel/poel) in ko was decreased compared to wt (fig. b) showing a rotation in a clockwise/ventral direction. in addition, the upper incisors were more anteriorly inclined (muprl/priul) (less curved), while the lower incisors were more posteriorly inclined (mliil/idlil and mibil/idlil) in ko than wt and/or het. at weeks, the results of angular bone relations were mostly consistent with those found at weeks, i.e. the values of cranial vault to cranial base (poel/soel), nasal bone to cranial bone and to cranial vault (anl/soel, anl/poel), and maxilla-premaxilla to cranial vault (mubul/poel) in ko was significantly decreased as compared to wt and het. similar to the data at weeks, the values of premaxilla to cranial base and to cranial vault (prel/soel, buel/soel, prel/poel, and buel/poel) in ko were significantly decreased as compared to page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le those in wt and het, indicating ventral (downward and backward) rotation of the premaxilla. the value of maxilla-premaxilla to cranial base (mubul/soel) became significantly smaller at weeks. in addition, the value of nasal bone to premaxilla (anl/prnl) was significantly increased in ko comparing to het. on the other hand, there was no statistical difference of maxilla-premaxilla to cranial base and to cranial vault, upper incisors to cranial base, and upper and lower incisor inclination (muprl/soel and muprl/poel, iuel/soel, muprl/priul and mliil/idiil) between ko, wt and het. the values of mandible to cranial vault (mnidl/poel and mlbll/poel) were markedly increased in ko as compared to wt and het, indicating rotation in a ventral direction/diverge relation. the values of mandibular corpus to cranial vault and to cranial base (mnidl/poel, mlbll/poel, mnidl/soel, and mlbll/soel) were markedly increased in ko comparing to wt and het, indicating rotation in a ventral direction. abnormal bone-to-bone relations are not due to decreased skull size in evc ko mice the evc ko mice were smaller (fig. ), thus one possible argument is that the differences in angular bone relations described in fig. are simply due to the reduction in bone length as observed by linear measurement (fig. ). thus, we normalized the linear measurements as follows. we compared the values of linear measurement between -week-old-ko and - week-old-wt only in bone length, but not incisor length (thus, pr-iu and ii-id were omitted in fig. a and angle measurements including these two length were also eliminated in fig. b) as incisor length is likely affected by both bone and tooth developmental processes. most of their linear measurements such as the length of nasal bone (n-a), viscerocraial bone (e-pr, e-bu), total skull (po-a), cranial base (so-e), neurocranial bone (po-e) and viscerocranial height (e- mu) were not statistically different between -week-old-ko and -week-old-wt (fig. a). the page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le length of palatal length (mu-bu), nasal bone height (a-pr), and mandibular lingual alveolar bone (ml-bl) in -week-old-ko was greater than that of -week-old-wt, and the length of mandibular corpus length in -week-old-ko was smaller than that of -week-old-wt (mn-id) (fig. c, indicated by red lines). angular bone relations were then compared between these mice. as shown in fig. b, although -week-old-ko had the same size of the nasal bone (n-a) and premaxilla (e-pr and e-bu) as -week-old-wt, the angles of nasal bone to premaxilla (anl/prnl), and premaxilla-maxilla to cranial base and to cranial vault (muprl/soel, muprl/poel) were significantly larger in -week-old-ko, and those of premaxilla to the cranial base and to cranial vault (prel/soel, prel/poel) were significantly smaller in -week-old-ko comparing to -week-old-wt (smaller/larger angle measurements were indicated by blue lines in fig. c), indicating the ventral rotation or clockwise depression of the premaxilla and palatal plane. these data clearly demonstrate that evc deficiency lead to defective bone development not only in size but also spatial bone-to-bone relations particularly affecting premaxilla and maxilla. impairment of nasal and palatal length growth in evc ko mice to further identify postnatal bone growth phenotype in evc ko mice, we first analyzed the average skull linear measurements (fig. a). the data demonstrated the high resemblance of profile of values between wt, het and ko with a constant decrease of ~ % in ko during the postnatal development (fig. a). secondly, ko/wt ratios in each bone were calculated at each time point tested. the results demonstrated that the ko/wt ratios of nasal bone and palatal length were significantly decreased (~ % decrease at week, ~ % at weeks, and ~ - % at weeks for both bones) over the postnatal development period, indicating that the growth in page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le these bones was severely affected (fig. b). on the other hand, the ko/wt ratios of mandibular bone were markedly increased during the development, indicating there may be a compensatory mechanism in this bone development (fig. c). there were no significant changes of ko/wt ratios in other bones (fig. d). discussion in this study, we have characterized and analyzed the craniofacial morphology in a mouse model of evc syndrome, evc /lbn ko mice. our data indicated that specific craniofacial bone structures in evc ko mice were affected in the areas of nasal bone, total skull, cranial base, cranial vault, premaxilla, maxilla and palate showing the significant reduction in their size. on the other hand, mandible exhibited a different growth pattern; i.e. significantly smaller at and weeks, however, there was no statistical difference at weeks (figs. and ). expectedly the expression pattern of evc is strongly associated with these affected craniofacial bone areas including meckel’s cartilage (mkb, ymo; unpublished data). however, it is not fully clear at this point as to why there was an increase of mandible length postnatally in ko. unlike other bones in the craniofacial area, the body of mandible is initially formed from condensing mesenchyme through intramembranous ossification where meckel’s cartilage serves as a template (ramaesh and bard, ). subsequently mandible increases its size through endochondral ossification of condyle (shen and darendeliler, ). based on the high levels of evc expression in the meckel’s cartilage, it is possible to speculate that impact of loss of evc is more prominent during early stage of mandibular growth than postnatal growth when the mandible condylar growth mainly takes place (shen and darendeliler, ). it has been previously reported in some evc cases that the zone of proliferative cartilage was affected page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le (smith and hand, ) and chondrosternal epiphyseal plates showed delayed maturation (peraita-ezcurra et al., ). this observation was further supported by the characterization of bcd cattle (with evc /lbn mutation), i.e. the long bones of the affected animals have insufficient endochondral ossification with irregularly arranged chondrocytes and partial disappearance of the epiphyseal growth plates (takeda et al., ). these findings suggest that the craniofacial abnormalities by evc deficiency may be caused by abnormal chondrocyte function including meckel’s cartilage. further studies are clearly warranted to investigate the chondrogenesis in craniofacial bone structures such as skull base. in support with our findings, the previous cephalometric analysis reported that an evc patient had the downward growth of mandible and open bite (waldrigues et al., ). another evc case showed that the patient had a small size of maxilla and cranial base and that the analysis indicated retruded maxilla and steep and diverge mandible with the ramus rotated counter-clockwise. although the angular relation of nasal bone was not described in the evc patient, it was mentioned that the patient had depressed base of the nose (susami et al., ). consistently, angular measurements in ko where nasal bone, premaxilla and mandible were involved showed similar bone relations to these clinical cases. to the best of our knowledge the current study was the first to demonstrate that evc deficiency causes the craniofacial defects likely seen in evc patients. our results demonstrated that anterior cross bite may seem to be consistent phenotype in evc ko mice at postnatal stages (figs. b and c). at this point, it is not fully clear whether evc patients have this particular occlusion phenotype, while many of previous literatures described that they have malocclusion (varela and ramos, ) or susami et al. reported a female case of evc showing an open bite and an angle class iii molar relation (susami et al., page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le ). it is noteworthy to address that the anterior cross bite phenotype in evc ko mice evidently affects the feeding behavior, which potentially contributes to the bone size difference as a secondary effect. in conclusion, our data indicate that evc is indispensable for preserving the normal craniofacial bone size as well as bone-to-bone relations during murine postnatal craniofacial bone development and lack of evc affects these relations leading to specific bone growth impairment. conflict of interest all authors declare no conflicts of interest in this paper. literature cited agerholm js, arnbjerg j, andersen o. . familial chondrodysplasia in holstein calves. j vet diagn invest : - . caparros-martin ja, valencia m, reytor e, pacheco m, fernandez m, perez-aytes a, gean e, lapunzina p, peters h, goodship ja, ruiz-perez vl. . the ciliary evc/evc complex interacts with smo and controls hedgehog pathway activity in chondrocytes by regulating sufu/gli dissociation and gli trafficking in primary cilia. hum mol genet : - . curry cj, hall bd. . polydactyly, conical teeth, nail dysplasia, and short limbs: a new autosomal dominant malformation syndrome. birth defects orig artic ser : - . ellis rw, van creveld s. . a syndrome characterized by ectodermal dysplasia, polydactyly, chondro-dysplasia and congenital morbus cordis: report of three cases. arch dis child : - . page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le engstrom c, linde a, thilander b. . craniofacial morphology and growth in the rat. cephalometric analysis of the effects of a low calcium and vitamin d-deficient diet. j anat : - . galdzicka m, patnala s, hirshman mg, cai jf, nitowsky h, egeland ja, ginns ei. . a new gene, evc , is mutated in ellis-van creveld syndrome. mol genet metab : - . ghosh s, setty s, sivakumar a, pai km. . report of a new syndrome: focus on differential diagnosis and review of ellis-van creveld, curry-hall, acrofacial dysostosis, and orofacial digital syndromes. oral surg oral med oral pathol oral radiol endod : - . goor d, rotem y, friedman a, neufeld hn. . ellis-van creveld syndrome in identical twins. br heart j : - . hanemann ja, de carvalho bc, franco ec. . oral manifestations in ellis-van creveld syndrome: report of a case and review of the literature. j oral maxillofac surg : - . jayo mj, leipold hw, dennis sm, horton wh. . bovine dwarfism: clinical, biochemical, radiological and pathological aspects. zentralbl veterinarmed a : - . jones th, mcclintock ae, smith gf, williams g. . achondroplasia in british friesians. vet rec : . julian lm, tyler ws, gregory pw. . the current status of bovine dwarfism. j am vet med assoc : - . kurian k, shanmugam s, harsh vardah t, gupta s. . chondroectodermal dysplasia (ellis van creveld syndrome): a report of three cases with review of literature. indian j dent res : - . peraita-ezcurra m, martinez-garcia m, ruiz-perez vl, sanchez-gutierrez me, fenollar-cortes m, velez-monsalve c, ramos-corrales c, pastor i, santonja c, trujillo-tiebas mj. . ellis-van creveld syndrome in a fetus with rhizomelia and polydactyly. report of a case diagnosed by genetic analysis, and correlation with pathological andradiologic findings. gene : - . ramaesh t, bard jb. . the growth and morphogenesis of the early mouse mandible: a quantitative analysis. j anat : - . ruiz-perez vl, blair hj, rodriguez-andres me, blanco mj, wilson a, liu yn, miles c, peters h, goodship ja. . evc is a positive mediator of ihh-regulated bone growth that localises at the base of chondrocyte cilia. development : - . ruiz-perez vl, ide se, strom tm, lorenz b, wilson d, woods k, king l, francomano c, freisinger p, spranger s, marino b, dallapiccola b, wright m, meitinger t, page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le polymeropoulos mh, goodship j. . mutations in a new gene in ellis-van creveld syndrome and weyers acrodental dysostosis. nat genet : - . ruiz-perez vl, tompson sw, blair hj, espinoza-valdez c, lapunzina p, silva eo, hamel b, gibbs jl, young id, wright mj, goodship ja. . mutations in two nonhomologous genes in a head-to-head configuration cause ellis-van creveld syndrome. am j hum genet : - . shen g, darendeliler ma. . the adaptive remodeling of condylar cartilage---a transition from chondrogenesis to osteogenesis. j dent res : - . smith hl, hand am. . chondroectodermal dysplasia (ellis-van creveld syndrome); report of two cases. pediatrics : - . susami t, kuroda t, yoshimasu h, suzuki r. . ellis-van creveld syndrome: craniofacial morphology and multidisciplinary treatment. cleft palate craniofac j : - . takeda h, takami m, oguni t, tsuji t, yoneda k, sato h, ihara n, itoh t, kata sr, mishina y, womack je, moritomo y, sugimoto y, kunieda t. . positional cloning of the gene limbin responsible for bovine chondrodysplastic dwarfism. proc natl acad sci u s a : - . varela m, ramos c. . chondroectodermal dysplasia (ellis-van creveld syndrome): a case report. eur j orthod : - . waldrigues a, grohmann lc, takahashi t, reis hm. . ellis-van creveld syndrome. an inbred kindred with five cases. rev bras pesqui med biol : - . weyers h. . [a correlated abnormality of the mandible and extremities (dysostosis acrofacialis).]. fortschr geb rontgenstr : - . ye x, song g, fan m, shi l, jabs ew, huang s, guo r, bian z. . a novel heterozygous deletion in the evc gene causes weyers acrofacial dysostosis. hum genet : - . yoneda k, moritomo y, takami m, hirata s, kikukawa y, kunieda t. . localization of a locus responsible for the bovine chondrodysplastic dwarfism (bcd) on chromosome . mamm genome : - . zhang h, takeda h, tsuji t, kamiya n, rajderkar s, louie k, collier c, scott g, ray m, mochida y, kaartinen v, kunieda t, mishina y. . generation of evc /limbin global and conditional ko mice and its roles during mineralized tissue formation. genesis. : - . page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le figure & table legends figure . gross morphology of evc ko mice and cephalometric analysis. (a) gross morphology of evc wild type (wt) and knockout (ko) mice at weeks. note that the body length and skull size in ko are smaller than those in wt mouse. the craniofacial images of these mice were magnified and shown on the right panels (wt; dotted open box, ko; indicated by a black open box). (b) lateral radiographic images of -week-old-wt (left panel) and -ko (right panel) with the selected reference points (see table for abbreviation). (c) cephalometric analysis was performed by measuring the linear distances and bone angles (table for abbreviation) based on the selected reference points. figure . linear bone measurements of evc mice. comparison of linear bone measurements between wt, het and ko mice at , and weeks. (a) at week, most bone measurements were significantly smaller in ko, except for the premaxilla (e-pr, e-bu), palate (mu-bu) and nasal bone height (a-pr) comparing to wt and/or het. *p < . . (b) at weeks, all the linear bone measurements in ko were significantly smaller than wt and/or het, except for nasal bone height (a-pr) and mandibular lingual measurement (ml-bl). *p < . . (c) at weeks, ko mice showed significant decrease in most linear bone measurements comparing to wt and het, except for nasal bone height (a-pr) and mandibular bone measurements (mn-id and ml-bl). *p < . . figure . angular bone measurements of evc mice. comparison of angular bone measurements between wt, het and ko mice at , and weeks. (a) at week, the nasal page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le bone (anl/soel), premaxilla (prel/soel, buel/soel, and prel/poel), palatal plane (muprl/poel), and mandible relations in ko were different from controls. *p < . . (b,c) at and weeks, differences in bone relations between ko and wt and/or het were enhanced. major changes in the bone relations included nasal bone (anl/soel, anl/poel), premaxilla (prel/soel, buel/soel, prel/poel, buel/poel), palatal plane (muprl/soel, muprl/poel), and mandible (mnidl/poel, mlbll/poel, mnidl/soel, mlbll/soel) to the cranial base and/or vault in ko as compared to wt and het. in addition, at weeks there was a decrease in the relation of the cranial vault to cranial base (poel/soel) in ko. no differences in morphometric bone relations between wt and het. *p < . . figure . abnormal bone-to-bone relations are not due to decreased skull size in evc ko mice. comparison of linear bone measurements between -week-old-wt and -week-old- ko mice. (a) no statistical difference was found in the linear bone measurements of n-a, e-pr, e-bu, po-a, so-e, po-e and e-mu between wt and ko, while the length of mu-bu, a-pr, and ml-bl was significantly higher in ko. * p< . . (b) the value of premaxilla relation (prel/poel) in ko was significantly decreased from that in wt, although the bone measurements in (e-pr) showed no differences, indicating that the changes in angular bone relation was not entirely due to the bone size. (c) illustration of the evc ko mouse with reference points and lines in the cephalometric analysis. red solid lines indicate the linear measurements which were significantly different between -week-old-ko and -week-old-wt identified in fig. a. angles in blue lines indicate the angular measurements which were significantly different between -week-old-ko and -week-old-wt identified in fig. b. page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le figure . growth impairment of specific craniofacial bones in evc ko mice. (a) the profile of averaged linear bone measurements. the average of all the linear bone measurements in each genotype was calculated at each time point by the total of linear measurements of all the craniofacial bones divided by the total number of bones and displayed as (average skull linear measurements). the profile of averaged measurement values during postnatal development in ko was similar to that in wt and het, while the value in ko was lowered by . % at week, . % at weeks, and . % at weeks compared to wt, and lowered by . % at week, . % at weeks and by . % at weeks comparing to het. the values in wt and het were essentially same. (b, c, d) values of linear measurements of each individual bone in ko at each time point were normalized to those in wt and are shown as (ko/wt ratio, %). (b) the ko/wt ratios of nasal (n-a) and palatal (mu-bu) bones were markedly decreased at and weeks. (c) although mandibular bone growth was lowered at week, the ko/wt ratio of this bone was significantly improved at weeks and further increased at weeks. (d) the change of ko/wt ratios of other craniofacial bones (e-pr, e-bu, po-a, so-e, po-e, e-mu, ml-bl) was within ~ % of the range. n-a; nasal bone length, mu-bu; palatal length, mn-id; mandibular corpus length, ml-bl; mandibular lingual alveolar bone. table . list of reference points based on the anatomical landmarks for morphometric measurements. table . list of abbreviations for linear distances and angles used in this study. an asterisk (*) indicates a new linear or angular measurement added to the current study. all other measurements were adapted from engstrom et al (engstrom et al., ). l; line. page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le figure . gross morphology of evc ko mice and cephalometric analysis. (a) gross morphology of evc wild type (wt) and knockout (ko) mice at weeks. note that the body length and skull size in ko are smaller than those in wt mouse. the craniofacial images of these mice were magnified and shown on the right panels (wt; dotted open box, ko; indicated by a black open box). (b) lateral radiographic images of - week-old-wt (left panel) and -ko (right panel) with the selected reference points (see table for abbreviation). (c) cephalometric analysis was performed by measuring the linear distances and bone angles (table for abbreviation) based on the selected reference points. x mm ( x dpi) page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le figure . linear bone measurements of evc mice. comparison of linear bone measurements between wt, het and ko mice at , and weeks. (a) at week, most bone measurements were significantly smaller in ko, except for the premaxilla (e-pr, e-bu), palate (mu-bu) and nasal bone height (a-pr) comparing to wt and/or het. *p < . . (b) at weeks, all the linear bone measurements in ko were significantly smaller than wt and/or het, except for nasal bone height (a-pr) and mandibular lingual measurement (ml-bl). *p < . . (c) at weeks, ko mice showed significant decrease in most linear bone measurements comparing to wt and het, except for nasal bone height (a-pr) and mandibular bone measurements (mn-id and ml-bl). *p < . . x mm ( x dpi) page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le figure . angular bone measurements of evc mice. comparison of angular bone measurements between wt, het and ko mice at , and weeks. (a) at week, the nasal bone (anl/soel), premaxilla (prel/soel, buel/soel, and prel/poel), palatal plane (muprl/poel), and mandible relations in ko were different from controls. *p < . . (b,c) at and weeks, differences in bone relations between ko and wt and/or het were enhanced. major changes in the bone relations included nasal bone (anl/soel, anl/poel), premaxilla (prel/soel, buel/soel, prel/poel, buel/poel), palatal plane (muprl/soel, muprl/poel), and mandible (mnidl/poel, mlbll/poel, mnidl/soel, mlbll/soel) to the cranial base and/or vault in ko as compared to wt and het. in addition, at weeks there was a decrease in the relation of the cranial vault to cranial base (poel/soel) in ko. no differences in morphometric bone relations between wt and het. *p < . . x mm ( x dpi) page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le figure . abnormal bone-to-bone relations are not due to decreased skull size in evc ko mice. comparison of linear bone measurements between -week-old-wt and -week-old-ko mice. (a) no statistical difference was found in the linear bone measurements of n-a, e-pr, e-bu, po-a, so-e, po-e and e- mu between wt and ko, while the length of mu-bu, a-pr, and ml-bl was significantly higher in ko. * p< . . (b) the value of premaxilla relation (prel/poel) in ko was significantly decreased from that in wt, although the bone measurements in (e-pr) showed no differences, indicating that the changes in angular bone relation was not entirely due to the bone size. (c) illustration of the evc ko mouse with reference points and lines in the cephalometric analysis. red solid lines indicate the linear measurements which were significantly different between -week-old-ko and -week-old-wt identified in fig. a. angles in blue lines indicate the angular measurements which were significantly different between -week-old-ko and -week- old-wt identified in fig. b. x mm ( x dpi) page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le figure . growth impairment of specific craniofacial bones in evc ko mice. (a) the profile of averaged linear bone measurements. the average of all the linear bone measurements in each genotype was calculated at each time point by the total of linear measurements of all the craniofacial bones divided by the total number of bones and displayed as (average skull linear measurements). the profile of averaged measurement values during postnatal development in ko was similar to that in wt and het, while the value in ko was lowered by . % at week, . % at weeks, and . % at weeks compared to wt, and lowered by . % at week, . % at weeks and by . % at weeks comparing to het. the values in wt and het were essentially same. (b, c, d) values of linear measurements of each individual bone in ko at each time point were normalized to those in wt and are shown as (ko/wt ratio, %). (b) the ko/wt ratios of nasal (n-a) and palatal (mu-bu) bones were markedly decreased at and weeks. (c) although mandibular bone growth was lowered at week, the ko/wt ratio of this bone was significantly improved at weeks and further increased at weeks. (d) the change of ko/wt ratios of other craniofacial bones (e-pr, e-bu, po-a, so-e, po-e, e-mu, ml-bl) was within ~ % of the range. n-a; nasal bone length, mu-bu; palatal length, mn-id; mandibular corpus length, ml-bl; mandibular lingual alveolar bone. x mm ( x dpi) page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le table list of reference points based on the anatomical landmarks for morphometric measurements. x mm ( x dpi) page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. a cc ep te d a rt ic le table list of abbreviations for linear distances and angles used in this study. an asterisk (*) indicates a new linear or angular measurement added to the current study. all other measurements were adapted from engstrom et al (engstrom et al., ). l; line. x mm ( x dpi) page of john wiley & sons, inc. the anatomical record this article is protected by copyright. all rights reserved. untitled s n p d i s c o v e r y : n e x t g e n e r a t i o n s e q u e n c i n g next-generation rad sequencing identifies thousands of snps for assessing hybridization between rainbow and westslope cutthroat trout p a u l a . h o h e n l o h e , * s t e p h e n j . a m i s h , † j u l i a n m . c a t c h e n , * f r e d w . a l l e n d o r f † a n d g o r d o n l u i k a r t †‡§ *center for ecology and evolutionary biology, university of oregon, eugene, or - , usa, †fish and wildlife genomics group, division of biological sciences, university of montana, missoula, mt , usa, ‡flathead lake biological station, university of montana, polson, mt , usa, §cibio, centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, - vairão, portugal abstract the increased numbers of genetic markers produced by genomic techniques have the potential to both identify hybrid individuals and localize chromosomal regions responding to selection and contributing to introgression. we used restric- tion-site-associated dna sequencing to identify a dense set of candidate snp loci with fixed allelic differences between introduced rainbow trout (oncorhynchus mykiss) and native westslope cutthroat trout (oncorhynchus clarkii lewisi). we distinguished candidate snps from homeologs (paralogs resulting from whole-genome duplication) by detecting exces- sively high observed heterozygosity and deviations from hardy–weinberg proportions. we identified candidate spe- cies-specific snps from a single illumina sequencing lane containing barcode-labelled individuals. published sequence data and ongoing genome sequencing of rainbow trout will allow physical mapping of snp loci for genome-wide scans and will also provide flanking sequence for design of qpcr-based taqman� assays for high-throughput, low-cost hybrid identification using a subset of – loci. this study demonstrates that it is now feasible to identify thousands of infor- mative snps in nonmodel species quickly and at reasonable cost, even if no prior genomic information is available. keywords: conservation biology, genome duplication, homeolog, invasive species, population genomics, threatened salmonids received october ; revision received november ; accepted november introduction hybrid zones and invasive species provide some of the richest natural experiments in evolutionary biology. hybridization and invasion are also important in conser- vation biology and are among the most serious threats to the persistence of many native species (allendorf & luik- art ; sax et al. ). interspecific hybridization can break up co-adapted gene complexes, disrupt local adap- tation and lead to genomic extinction (rhymer & sim- berloff ; allendorf et al. ). rainbow trout (rbt, oncorhynchus mykiss), the most widely introduced salmonid fish in the world (halverson ), produce fertile offspring when crossed with cut- throat trout (oncorhynchus clarkii). introgression often continues until a hybrid swarm is formed and all native cutthroat genomes are lost (allendorf & leary ). such introgression poses a serious threat to all remaining subspecies of cutthroat trout in western north america (trotter ). westslope cutthroat trout (wct, o. c. lew- isi) are native to the interior of north-western north america (fig. a). hybridization with non-native rbt is the leading factor contributing to the decline of geneti- cally pure wct populations throughout their range, and nonhybridized populations now persist in < % of their historic range (shepard et al. ). currently, only – diagnostic loci are commonly used to study introgression in these taxa, which is not sufficient for individual-level testing for admixture (allendorf et al. ). the development of a dense gen- ome-wide set of single-nucleotide polymorphism (snp) markers in rbt and wct would allow localization of chromosomal regions under selection and detection of rbt alleles rapidly spreading across wct populations (e.g. fitzpatrick et al. ). a smaller set of – loci to correspondence: paul hohenlohe, fax: ; e-mail: hohenlo@uoregon.edu molecular ecology resources ( ) (suppl. ), – � blackwell publishing ltd molecular ecology resources ( ) (suppl. ), – doi: . /j. - . . .x be used in high-throughput qpcr assays would allow identification of individuals with no introgression, at high statistical power and low cost, for use in brood stock development and translocations with only nonhybrid- ized (‘pure’) individuals. a particular challenge in developing snp markers in salmonids is a recent (� – mya) genome duplica- tion, ancestral to all salmonids, including both wct and rbt (allendorf & danzmann ; koop et al. ). it can be difficult to distinguish true snps from duplicate regions, or homeologs, that differ by only one or a few nucleotide positions. here, we use restriction-site-associ- ated dna (rad) sequencing (baird et al. ), a next- generation sequencing technique that generates short sequence reads at thousands of regions adjacent to restric- tion endonuclease recognition sites across the genomes of multiple individuals. we currently lack a reference gen- ome sequence against which to align the sequence reads to identify homeologs. however, the number of nucleo- tide positions genotyped across multiple individuals, combined with barcoding of individual samples, allows us to screen through thousands of candidate loci and to distinguish candidate snps from homeologs on the basis of observed patterns of heterozygosity, prior to any fur- ther snp validation steps. for example, homeologous regions that are fixed for one or more nucleotide differ- ences should appear as heterozygotes in most individuals genotyped. putative loci showing an excess of heterozy- gosity (e.g. proportion of heterozygotes > . for a bi-alle- lic locus, or substantial deviation from hardy–weinberg proportions) can be excluded from further analysis. we have two goals in this study: (i) to identify a set of (> ) snp markers that are fixed between rbt and wct to use in further rad sequencing studies to exam- ine the genomic patterns of selection and introgression between rbt and wct and (ii) to use a subset ( – ) of these markers to design qpcr-based assays (e.g. snp chips) that can be applied at relatively low cost to a large number of samples, including degraded and historic dna, to distinguish hybrids and quantify genome-wide average levels of introgression in multiple populations. methods we collected genomic dna from a total of individuals from populations in interior north-west north amer- ica (fig. b): wct, coastal rbt (including from a hatchery population and from introduced populations) and inland rbt (redband trout, oncorhynchus mykiss gairdnerii). we prepared rad libraries according to etter et al. (in press). each individual sample was barcoded with a unique six-nucleotide sequence, and all samples were run in a single lane on an illumina genome ana- lyzer ii. we filtered out low-quality reads and those that lacked a correct barcode (see emerson et al. for fur- ther details). we pooled the reads from all individuals and aligned them against each other, allowing a maxi- mum of two nucleotide mismatches among reads at a putative rad tag locus. we then applied a maximum- likelihood algorithm (hohenlohe et al. ) to estimate the diploid genotype for each individual at each nucleo- tide position. this genotyping method is designed to account for the sampling and sequencing error inherent in rad sequencing, assigning a genotype only if a likelihood pacific ocean rbt (a) (b) wct yct n fig. range and sampling locations of rainbow and cutthroat trout. (a) historic range of rainbow (rbt), westslope cut- throat (wct) and yellowstone cutthroat (yct) trout in western north america. (b) enlargement of box in (a) to show sampling locations of origin for wct (dots) and rbt (stars; grey for redband, white for introduced coastal rbt and black for hatchery rbt). three of the locations for coastal rbt are in close proximity and appear as a single star. molecular ecology resources ( ) (suppl. ), – � blackwell publishing ltd p . a . h o h e n l o h e e t a l . ratio test is significant at a level of a = . . this implicitly results in a threshold for depth of sequencing coverage, so that some loci in some individuals will not be assigned a genotype simply because of the sampling variance among alleles and loci in a sequenced rad library. for example, if all reads are identical at a nucleo- tide site (i.e. no apparent sequencing errors), a minimum of three reads is required to call an individual a homozy- gote; if the reads are evenly split between two alternative nucleotides, a minimum of two high-quality reads of each allele is required to call an individual a heterozygote (hohenlohe et al. ). sequencing error and sampling variance across alternative alleles increase these implicit thresholds, so that in practice read depth needs to be substantially higher to call most genotypes (see below). software to conduct these steps of the analysis on high- throughput, short-read sequence data is available online at http://creskolab.uoregon.edu/stacks. we then applied a number of filters to identify candi- date diagnostic snps for detecting hybridization and other population-level studies. we focused on loci geno- typed for at least of the individuals assayed (i.e. missing up to four individuals, thus a minimum of seven wct or nine rbt) and on rad tags containing only one bi-allelic, putative snp within the bp of potentially variable sequence. the presence of no more than one bi-allelic snp ensures pcr primer and probe fidelity for qpcr assay design. it also helps screen out homeologs, as many duplicate regions are likely to have diverged at more than one nucleotide position within the -bp rad tag sequence (seeb et al. ). at this point, we excluded additional putative snp loci as homeologs by examining the pattern of observed heterozygosity within each species. at each putative locus, we calculated expected heterozygosity as hexp = ) p ni(ni ) ) ⁄ n(n ) ), where ni is the count of allele i in the sample and n = p ni, and observed heterozygosity hobs as the proportion of individuals that appear heterozygous at the locus. we also calculated fis = ) (hobs ⁄ hexp), which provides a measure of the deviation of genotype frequencies (i.e. observed heterozygosity) from hardy–weinberg proportions. we applied two stringent filters based on these measures: putative loci with hobs > . within either species and those with fis < . within either species were eliminated. results rad sequencing generated a total of just over million single-end, -bp reads, prior to any quality filtering. sequence reads have been deposited in the ncbi sequence read archive (accession no. sra . ). each read provided bp of genomic sequence after trimming the barcode, of which bp may contain snps (the remaining bp compose the partial restriction site, which is constant across all reads). reads were filtered for overall quality and presence of a barcode and then aligned with each other to create a catalogue of putative loci, or rad tags. a total of . million reads contributed to this alignment, for a mean depth of . · per individual per tag (one wct individual had a mean coverage of . · because of low dna concentration, while the other individuals ranged from . · to . ·; variation among individuals is often observed in a pooled rad sequencing library). further filtering identified candidate snps (table ). a total of putative rad tags had exactly one bi-alle- lic putative snp and were genotyped in at least of the individuals. for these putative tags, the mean depth of coverage per individual was . ·. this list included putative snps that were likely homeologs, based on excess observed heterozygosity (fig. ). we removed tags because hobs > . in both species ( ) or in one of the two species ( ), plus an additional tags at which fis < . in both species ( ) or in one of the two species ( ). these screens produced a total of candidate snp markers (table ). of these, were fixed within each species, providing candidate species-specific snps to distinguish hybrids and estimate levels of introgres- sion (table ). design of qpcr taqman� assays for a subset of these loci requires additional flanking sequence of approxi- mately bp on each side of the snp. we searched for the species-specific rad tag loci (using both alleles of each locus) in a database of longer sequences produced by sequencing of a reduced representation genomic library in rbt (sánchez et al. ), using the alignment software bowtie (langmead et al. ). the rbt allele for candidate diagnostic rad tag loci aligned to one table counts of putative loci after each step of filtering (each row incorporates all filters above), and final counts of candidate snps after filtering category count % of total filtering steps ( ) total putative rad tag loci . ( ) ‡ snp w ⁄ in rad tag . ( ) genotyped in ‡ samples . ( ) exactly snp in rad tag . ( ) bi-allelic snp . ( ) observed heterozygosity £ . in each species . ( ) fis ‡ . in each species . candidate snps total . fixed between species . polymorphic within rbt . polymorphic within wct . molecular ecology resources ( ) (suppl. ), – � blackwell publishing ltd s n p d i s c o v e r y : n e x t g e n e r a t i o n s e q u e n c i n g or more sequences from sánchez et al. ( ) with no nucleotide mismatches in the -bp rad tag sequence. in addition, four rbt alleles and one wct allele aligned to these sequences with just one mismatch, and nine rbt alleles and one wct allele aligned with two mismatches. in total, this provides flanking sequence to design prim- ers and validate qpcr assays for of our candidate diagnostic snps. genomic sequence contigs from a gen- ome sequencing project in rbt using a bac library will also be available in (m. miller, u. oregon, unpub- lished data), providing flanking sequence for many more of our diagnostic snps. we also found a large number of candidate snps within each species useful for future studies in population genetics and landscape genomics (schwartz et al. ) (table ). discussion many populations and species of salmonids are of con- servation and management concern (waples & hendry ). development of genetic tools in these fish has been hampered in part by their relatively large genome con- taining substantial duplicate regions and by the lack of a reference genome sequence. our application of rad sequencing here alleviates some of these problems and facilitates the development of snp markers. first, rad sequencing identifies such a large number of putative loci that stringent screens can be applied to narrow the list of candidate snps. second, we were able to barcode indi- vidual samples, run all samples together in a single high- throughput illumina lane and obtain sufficient depth of sequencing coverage to identify individual-level diploid genotypes (as in hohenlohe et al. ). this allowed us to screen out homeologs on the basis of observed hetero- zygosity among individuals. such an approach is not possible in a pooled population sample, which can be used to estimate population-level allele frequency but not observed heterozygosity (sánchez et al. ). the presence of homeologs is apparent in our data (fig. ). in both species, a significant number of putative fig. frequency histograms of population genetic statistics for putative snp loci. counts include only those loci that passed filtering step in table and that are polymorphic within each species ( in rbt and in wct). (a,b) rbt. (c,d) wct. (a,c) expected (grey) and observed (black) heterozygosity at each putative snp. (b,d) fis at each putative snp. dashed boxes enclose likely homeologs that were excluded in steps and of filtering (table ). numbers along the x-axis indicate lower and upper boundaries of each bin, but note that loci with either hobs or fis exactly equal to . are grouped into a separate bin in each plot. molecular ecology resources ( ) (suppl. ), – � blackwell publishing ltd p . a . h o h e n l o h e e t a l . loci have hobs = . , while the maximum hexp is . for bi-allelic loci in a large sample of individuals. in wct, the distribution of hobs is bimodal, with a second peak at hobs =� . (fig. c). we do not expect loci to be in hardy–weinberg proportions even within either of the two species, because individuals were sampled from multiple populations. however, this should produce a deficit of heterozygotes (fis > ), rather than an excess, because of the wahlund effect. consistent with this expectation, the majority of loci in both species show fis > and low values of hobs, including many loci at which no individuals were heterozygous (hobs = ) despite the detection of two alleles within the species (fig. a,c). in contrast, the observed excess of heterozygotes (hobs > . or fis < ) likely indicates homeologous pairs of loci that were incorrectly combined into a single puta- tive locus during initial construction of the rad tag cata- logue. other processes could also produce an excess of heterozygotes, such as selection for heterozygotes. how- ever, this selection would have to be quite strong to pro- duce hobs values approaching . , and loci under such strong selection would make poor candidates for gen- eral-purpose population genetic markers. in addition, of the putative snps that we removed because of high observed heterozygosity, % had hobs > . in both spe- cies. this is consistent with homeologous regions that diverged prior to the split between rbt and wct, approximately ma (allendorf & leary ). finally, our primary goal in this study was to identify snp mark- ers fixed between the species. this provided a further conservative screen for homeologs by eliminating candi- date snps with any observed heterozygosity within either species. several factors can increase confidence in the discrimi- nation of true single-locus snps from homeologs using observed heterozygosity and deviation from hardy– weinberg expectations. for example, increasing the number of individuals sampled would provide a better estimate of genotype and allele frequencies. increasing the depth of coverage, and reducing variance in coverage across individuals, would improve statistical confidence in assigning diploid genotypes to each individual (ho- henlohe et al. ). increased coverage could also allow one to use the expected double number of sequence reads from homeologs compared to true snps to distinguish the two. however, there is substantial variance in num- ber of reads across loci and across individuals, and we did not attempt to use this approach here. variance in read depth among individuals can be minimized, but not eliminated, by making every effort to use equal dna quality and quantity among samples in a pooled sequencing run (etter et al. in press). in a nonmodel spe- cies, a rough expectation of depth of coverage can be cal- culated using an estimate of genome size and the expected frequency of restriction cut sites given a partic- ular restriction enzyme. for the maximum-likelihood genotyping method used here in samples from an out- bred population, a mean coverage depth of – · should allow genotyping at most sites across most individuals. applying next-generation sequencing techniques as an initial screen to identify snps, followed by more tradi- tional genotyping techniques across larger samples of individuals, is likely to become commonplace in conser- vation genetics (allendorf et al. ). in this study, we used single-end illumina sequencing and have taken advantage of existing published data as well as ongoing work to gather flanking sequence for qpcr assay design. one alternative is to conduct paired-end rad sequenc- ing for marker development, in which overlapping paired-end sequences (e.g. bp per read) at sufficient depth of coverage can provide much longer contigs (e.g. – bp) containing candidate snps and sufficient flanking sequence for direct pcr primer development (j. davey, u. edinburgh, unpublished data). other tech- niques, such as sequencing of rad tags, could also be used to generate longer flanking sequence around putative diagnostic snps. from this point, further research is needed to validate candidate snps through re-sequencing or direct snp genotyping. for example, expected patterns of mendelian inheritance can be tested from pedigree samples. rad sequencing can be limiting in some respects: it requires at least lg of high-quality genomic dna per sample (etter et al. in press), and like any genomic approach, bioinformatic analysis comprises a substantial portion of the work. nonetheless, if sufficient dna sam- ples can be gathered for a relatively small number of individuals and used in the initial marker-development stage in as little as a single illumina sequencing lane, as we have performed here, other techniques that require smaller amounts of low-quality dna can be applied to a larger sample of individuals. bioinformatic tools con- tinue to be developed for rad and similar genomic data types and are available online (e.g., http://creskolab.uor- egon.edu/stacks). our results suggest that it is now feasi- ble to identify thousands of informative snps in nonmodel species for a reasonable price even if no prior genomic information is available. acknowledgements we thank r. leary (montana fish, wildlife and parks), c. muhl- feld (us geological survey) for samples and advice, and w. cre- sko and m. miller (both u. oregon) for helpful comments on this research. pah received support from national science founda- tion grants ios- and deb- and national insti- tutes of health grant r gm - a to w.a. cresko. jmc molecular ecology resources ( ) (suppl. ), – � blackwell publishing ltd s n p d i s c o v e r y : n e x t g e n e r a t i o n s e q u e n c i n g received support from nih nrsa ruth l. kirschstein post- doctoral fellowship f gm - and from nih grant r rr to j. postlethwait. gl and fwa were partially supported by the nsf grant deb- . gl also was funded by the walton family foundation and portuguese science foundation grants (ptdc ⁄ bia-bde ⁄ ⁄ ; and ptdc ⁄ cvt ⁄ ⁄ ), and nsf grant (deb deb- ). conflict of interest the authors have no conflict of interest to declare and note that the sponsors of the issue had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. references allendorf fw, danzmann rg ( ) secondary tetrasomic segregation of mdh-b and preferential pairing of homeologues in rainbow trout. genetics, , – . allendorf rf, leary rf ( ) conservation and distribution of genetic variation in a polytypic species: the cutthroat trout. conservation biol- ogy, , – . allendorf fw, luikart g ( ) conservation and the genetics of populations. blackwell publishers, oxford, uk. allendorf fw, leary rf, spruell p, wenburg jk ( ) the problems with hybrids: setting conservation guidelines. trends in ecology and evolu- tion, , – . allendorf fw, hohenlohe pa, luikart g ( ) genomics and the future of conservation genetics. nature reviews genetics, , – . baird na, etter pd, atwood ts et al. ( ) rapid snp discovery and genetic mapping using sequenced rad markers. plos one, , e . emerson kj, merz cr, catchen jm et al. 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( ) a salmonid est geno- mic study: genes, duplications, phylogeny and microarrays. bmc genomics, , . langmead b, trapnell c, pop m, salzberg sl ( ) ultrafast and mem- ory-efficient alignment of short dna sequences to the human genome. genome biology, , r . rhymer jm, simberloff d ( ) extinction by hybridization and intro- gression. annual review of ecology and systematics, , – . sánchez cc, smith tpl, wiedmann rt et al. ( ) single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library. bmc genomics, , . sax df, stachowicz jj, brown jh et al. ( ) ecological and evolutionary insights from species invasions. trends in ecology and evolution, , – . schwartz mk, luikart g, mckelvey ks, cushman s ( ) landscape ge- nomics: a brief perspective. chapter in: spatial complexity, informat- ics and animal conservation (eds cushman sa, huettman f), pp. – . springer, tokyo. seeb je, pascal ce, grau ed et al. ( ) transcriptome sequencing and high-resolution melt analysis advance snp discovery in duplicated sal- monids. molecular ecology resources, , this issue. shepard bb, may be, urie w ( ) status and conservation of westslope cutthroat within the western united states. north american journal of fisheries management, , – . trotter p ( ) cutthroat: native trout of the west, nd edn. university of california press, berkeley, ca. waples rs, hendry ap ( ) special issue: evolutionary perspectives on salmonid conservation and management. evolutionary applications, , – . molecular ecology resources ( ) (suppl. ), – � blackwell publishing ltd p . a . h o h e n l o h e e t a l . david c. lindberg and ronald l. numbers (eds.), when science and christianity meet. chicago and london: university of chicago press, . pp. xii+ . isbn - - - . &pound; . , $ . (hardback). alan h. goodman, deborah heath and m. susan lindee (eds.), genetic nature/culture: anthropology and science beyond the two-culture divide. berkeley, los angeles and london: university of california press, . pp. xvii+ . isbn - - - . £ . , $ . (paperback). doi: . /s genetic nature/culture explores the relationship between anthropology and genetics in the cur- rent age of geneticization. the book is divided into four parts. the first considers the formation of knowledge about human genetics and the ways in which people are recruited (or forced) into networks of knowledge creation. the second part focuses on animals and their role in making knowledge. the third part looks at how groups of people redefine knowledge to fit their culture. the fourth part examines the genetics of race. a major theme running throughout is ethics: in genetics, in anthropology, and more generally in the power relationships surrounding knowledge. as such, the book speaks more directly to science studies professionals than to historians of science. however, some of the issues surrounding the genetics of today also apply to the genetics – and more widely to the science – of the past. the book therefore provides ideas for writing non-standard histories of science that address present concerns. the opening essays use historical cases to investigate the ethics of using people to create knowledge. the first tells the story of the major human population-genetics project that james neel conducted during the s and s. in the second, susan lindee discusses how victor mckusick recruited amish people into a network of informants about the health of community members. her essay can be profitably compared with that of donna haraway, in the second part, on dogs. haraway shows that animal fanciers have collected information about inheritance which geneticists have neither the inclination nor the means to collect. at the same time, as she reveals, fanciers have had to enrol scientists to turn their information into knowledge. when haraway’s analysis is considered alongside lindee’s story of a geneticist recruiting people to gain information, it appears that only geneticists can create genetic knowledge, but that they often use information gained from others to do so. another interesting comparison between these two essays concerns the question of audience, of whom the knowledge was created for – a second theme that runs throughout the book. lindee points out that while mckusick took advantage of the amish’s interest in health issues, the results of his research were not intended for amish eyes; similarly, haraway shows that many fanciers did not want to recognize their stock as diseased. in the book’s animal-oriented second part, haraway’s essay is placed alongside those of sarah franklin and jonathan marks. while har- away considers animal genetics in connection with fancying, franklin looks at that science in the context of agriculture, and marks in the context of comparative anthropology. none of these contexts have received much consideration by historians of genetics. the general lesson i drew from these essays was that historians need to pay more attention to the roles that non-geneticists have played in creating genetic knowledge. we have begun to investigate the people and animals book reviews uczcido textbox marie, j; ( ) alan h. goodman, deborah heath and m. susan lindee (eds.). genetic nature / culture: anthropology and science beyond the two-culture divide, berkeley, los angeles and london: university of california press, . pp xvii+ . isbn - - - . £ . , $ , (paperback). british journal for the history of science , - . 
 that geneticists have studied, but have much to learn about what fanciers, agriculturalists and anthropologists have contributed. where the first half of the book deals with knowledge creation, the second half turns to the meanings given to knowledge and the uses to which it is put. in the third part, the focus is on cultural meanings among traditionally opressed groups. for example, chaia heller and arturo escobar look at how the people of the pacific rainforest have reinterpreted biodiversity to include different ways of living, in order to preserve their land (and other) rights. joan fujimura and himla soodyall’s essays go further, arguing that cultural meanings can be the reason for knowl- edge formation. in fujimura’s example, japanese scientists believe that the results of genetics are more compatible with eastern culture than western. in soodyall’s case, the very act of doing anthropological genetics in south africa is seen to restore a sense of pride to the south african people. the final essays, on race, looks at the question of whom knowledge serves and the surrounding social, ethical and legal issues. we learn, for example, that though studies of disease in african americans might be supposed to benefit the health care of that group, its social situation means that its members tend to lack access to health care, so that being defined as high risk for certain diseases in fact threatens access to health insurance and employment. in troy duster’s contribution, he argues that, while we can disprove the existence of biological races, denying their social existence amounts to denying racial discrimination. despite historians’ protestations that science must be studied because it affects many aspects of everyday life, the use and meanings surrounding genetic knowledge have so far been little examined. this book is not a history of genetics, anthropology or other sciences. however, in discussing genetics from a wide range of perspectives (anthropological, methodological, ethical), it suggests that perhaps historians should broaden their view of genetics, from one concentrating on a single scientific discipline, to one which encompasses the multiple and diverse influences of genetics on individuals’ lives. jenny marie max planck institute for the history of science book reviews identification of type diabetes genes in mexican americans through genome-wide association studies m. geoffrey hayes, anna pluzhnikov, kazuaki miyake, ying sun, maggie c.y. ng, cheryl a. roe, jennifer e. below, raluca i. nicolae, anuar konkashbaev, graeme i. bell, , nancy j. cox, , and craig l. hanis objective—the objective of this study was to identify dna polymorphisms associated with type diabetes in a mexican- american population. research design and methods—we genotyped , single nucleotide polymorphisms (snps) in mexican americans with type diabetes and random mexican amer- icans from starr county, texas, using the affymetrix genechip human mapping k set. allelic association exact tests were calculated. our most significant snps were compared with results from other type diabetes genome-wide association studies (gwass). proportions of african, european, and asian ancestry were estimated from the hapmap samples using struc- ture for each individual to rule out spurious association due to population substructure. results—we observed more significant allelic associations than expected genome wide, as empirically assessed by permu- tation ( below a p of � � [ . expected]). no significant differences were observed between the proportion of ancestry estimates in the case and random control sets, suggesting that the association results were not likely confounded by substruc- ture. a query of our top � % of snps (p � . ) revealed snps in or near four genes that showed evidence for association (p � . ) in multiple other gwas interrogated: rs and rs near ubqlnl and or h on chromosome , rs and rs in or near ralgps on chromosome , and rs near egr on chromosome . conclusions—we identified several snps with suggestive evidence for replicated association with type diabetes that merit further investigation. diabetes : – , d iabetes continues to pose a substantial and increasing burden of morbidity and mortality on society, especially among minority popula- tions. in the u.s., � million people have diabetes, of which one-third remain undiagnosed and most ( – %) have type diabetes ( ). by , rates of diagnosed diabetes are projected to more than double to million, with fully one-third of children born in the year expected to develop diabetes over their lifetime ( ). minority populations, such as mexican americans, have a disproportionate incidence of diabetes ( – ). for example, the mexican-american population from starr county, texas, has the highest diabetes-specific morbidity and mortality of any county in texas, yet it is only the rd largest of texas’ counties. age-specific prevalences are three- to fivefold higher than the general u.s. popula- tion ( , ), and in the last two decades alone there has been a % increase in type diabetes prevalence in those aged � years in this population. population studies, pedigree investigations, molecular studies, and animal models consistently implicate a sub- stantial role for genes in determining risk for type diabetes (see , ). these studies also establish that no simple genetic model adequately explains risk for diabe- tes. rather, there are likely to be multiple genes with small to modest effects that interact with each other and with environmental factors to affect susceptibility ( – ). this view of the genetics of diabetes is able to explain both its population and familial aggregation and implies that we are looking for genes whose effects are neither necessary nor sufficient to cause disease. a great deal of effort has been expended in identifying genes underlying the risk for type diabetes, including genome linkage scans (see , ), candidate gene studies (e.g., ), and, more recently, genome-wide association studies (gwass) ( – ). to date, such studies have yielded several replicated type diabetes–associated risk genes including capn , cdkal , cdkn a, hhex, hnf a, igf bp , kcnj , pparg, slc a , and tcf l ( – ), but none account for a large proportion of the risk of developing type diabetes in the particular population under study nor are any seen universally across all populations. again, this suggests that many more type diabetes susceptibility genes remain undiscovered. over the past decade, we have conducted genome-wide linkage scans on mexican-american families from starr county, texas, to localize genes conferring risk to type diabetes and were successful in positionally cloning the capn gene as a type diabetes susceptibility locus ( , ). given the increased power in association studies over linkage studies ( ) for complex genetic diseases from the department of medicine, university of chicago, chicago, illinois; the department of human genetics, university of chicago, chicago, illinois; and the human genetics center, university of texas health sciences center, houston, texas. address correspondence and reprint requests to nancy j. cox, phd, department of medicine, university of chicago, s. maryland ave., mc , chicago, il . e-mail: ncox@bsd.uchicago.edu; or craig l. hanis, phd, human genetics center, university of texas health science center at houston, p.o. box , houston, tx . e-mail: craig.l. hanis@uth.tmc.edu. received for publication april and accepted in revised form september . published ahead of print at http://diabetes.diabetesjournals.org on sep- tember . doi: . /db - . additional information for this article can be found in an online appendix at http://dx.doi.org/ . /db - . brlmm, bayesian robust-fitting linear model with mahalanobis distance classifier; dgi, diabetes genetics initiative; dm, dynamic modeling; fdr, false discovery rate; fhs, framingham heart study; gel, genotype calling algorithm using empirical likelihood; gwas, genome-wide association study; hwe, hardy-weinberg equilibrium; ld, linkage disequilibrium; maf, minor allele frequency; poa, proportion of ancestry; snp, single nucleotide poly- morphism. © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. original article diabetes, vol. , december such as type diabetes, we conducted a gwas of a � -member case-control set to identify additional genomic regions harboring type diabetes susceptibility loci in the starr county population. we present the results of this type diabetes gwas, the first in a non-caucasian population, along with supporting evidence for replication from available gwass, primarily the three accompanying this one ( , , ). research design and methods this study was completed in a mexican-american population from starr county, texas. we selected as unrelated cases individuals who represent the youngest age-at-onset individuals from the multiplex families in our previous linkage studies and for whom we have the richest phenotypic data. the comparison individuals are not true control subjects in that their diabetes status is unknown. rather, they are a representative sample of unrelated individuals drawn from a random survey of starr county. of this case and random control set, and individuals were analyzed (see “quality control” below) and are described in table . an overlapping cohort (online appendix table [available at http://dx.doi.org/ . /db - ]) of individuals (including of the individuals analyzed) was used to verify genotypes before single nucleotide polymorphism (snp) selection for fol- low-up replication. diabetes was classified based on earlier national diabetes data group recommendations ( ), namely, previously diagnosed diabetes and current or sustained use of glucose-lowering medications, fasting glucose � mg/dl on more than one occasion, or a -h postload glucose of � mg/dl. individuals were considered to have type diabetes unless they were diagnosed before age years, had a bmi � kg/m , and had used insulin continuously since diagnosis. genotypying. genomic dna was isolated from lymphocytes and quantified by picogreen. the genotyping assay was performed according to the manu- facturer protocols (affymetrix, santa clara, ca) by the functional genomics core facility at the university of chicago. in brief, ng dna was digested with the restriction enzymes xbai and hindiii, followed by adaptor ligation. the dna fragments were then amplified, fragmented, labeled, and hybridized overnight to the affymetrix genechip human mapping k xbai and hindiii arrays. the arrays were scanned with the affymetrix g scanner and analyzed with affymetrix genechip dna analysis software to generate hybridization intensity files and subsequent dynamic modeling (dm) algorithm– derived genotypes. case and random samples were dispersed randomly throughout the plates to eliminate the possibility of spurious associations due to systematic differ- ences in genotyping conditions between experiments. genotypes were called using the default affymetrix dm algorithm and two improved algorithms, (gel) ( ) and bayesian rlmm (brlmm) ( , ). after removal of mono- morphic markers, we analyzed genotypes for , autosomal snps of the possible , snps interrogated on the array. we anticipate analyzing x chromosome polymorphisms at a later date. genotyping in verification sets was performed using taqman assays on the abi prism ht sequence detection system. statistical methods. we examined the case-random control cohort for evidence of related individuals that went undetected during sample collection using plink ( ). pairs with identity-by-descent estimates � . were trimmed, preferentially keeping case rather than control subjects and individ- uals with higher genotype call rates if the pair was a case-case or control- control. fisher’s exact tests for allelic associations and departures from hardy- weinberg equilibrium (hwe) were calculated for all polymorphic snps. we did not remove any of the snps for strict quality-control reasons but, rather, cataloged quality-control indicators for each snp and considered them during the interpretation of the data. we observed that our most significant snps, those with p values between . � � and . � � , had highly significant departures from hwe (p � . ) in random control subjects or call rates � . , so we subsequently focused our attention on those that surpassed these thresholds. we also set a minor allele frequency (maf) � . criterion, as the allelic associations at snps below this threshold are largely driven by differences in a small number of individuals. we anticipate following-up rare polymorphisms with significant evidence for association separately at a later date. a total of , snps passed these criteria (fig. ). false discovery rates (fdrs) were estimated by conducting the allelic association test in , permutations (permuting the case and random labels) and tabulating the p values at given thresholds. we also conducted logistic regressions between type diabetes status and genotypes under an additive model, with and without a proportion of european ancestry covariate. this was not meant as a substitute for the allelic associations but simply to provide a reasonable approach to investigate how the estimated proportions of ancestry might affect the results when included as a covariate. all statistical analyses were performed using r (available at http://www.rproject.org). measures of linkage disequilibrium (ld) were calculated using gold ( ). using a population prevalence of %, we estimated that a case-random study was sufficiently powered ( %) to detect a genotype relative risk of � . under dominant, recessive, and additive models in the mid-range of allele frequencies ( ). assessing admixture proportions. we compared the full set of genotypes for the , snps in the mexican-american subjects (ma group) and in the unrelated hapmap samples ( europeans from utah from the centre d’etude du polymorphisme humain [ceu group]; yoruba from ibadan, nigeria [yri group]; and asians [asn group] including japanese subjects from tokyo [jpt group] and han chinese from beijing [chb group]) as proxies for native americans (see online appendix). the asian hapmap samples were chosen as proxies because no k data exist for an appropriate native american population once thought to be ancestral to the mexican americans under investigation here. this leaves the asian samples as the most appropriate proxy. after removing snps either not typed or monomorphic in all four populations (ceu, yri, asn, and ma groups), we divided the remaining , snps into equal subsets (by taking every th snp) to reduce the degree of ld between snps (median intermarker distance � kb in the subsets). to estimate genome-wide proportions of ancestry (poas) for each individual, we ran structure ( ) for each of the subsets using the hapmap populations as learning samples (fixed population identity) and subsequently averaged the estimated poas across the subsets. the structure runs were conducted under an admixture model with default parameter settings of , burn-in replications and , estimating replications after burn in. altering the prior migration probability from . to . had little effect on the results, and we present the poa estimates for the . runs herein. in silico replication. we entered into a consortium to share results with three other groups analyzing type diabetes gwas data in three distinct populations (amish, pima indians, and framingham heart study [fhs]) (tables and ), each with different study designs but using the same genotyping platform ( , , ). each group requested summary data for their top � , snps following criteria specific to each group in the type diabetes k gwas consortium and shared the same for the other groups’ best signals. we requested summary data for our top , most significant high-quality snps (those with p � . and passing the quality-control thresholds described above). we directly compared our fisher’s exact tests for allelic associations to type diabetes in the mexican americans with the type diabetes association tests under an additive model in the amish, the type diabetes association tests by generalized estimating equations and family-based association tests in the fhs, and the case-control and within- family association tests in the pima indians. we considered a mexican- american type diabetes–associated snp to be in silico replicated if it was associated at p � . in the same direction (i.e., the same allele was associated with type diabetes) in at least one other k gwas (fig. ). we also queried our data against the march prereleased data from a similar study in a scandinavian cohort (diabetes genetics inititative [dgi]) (available at http://www.broad.mit.edu/diabetes/) but conducted with a denser genotyping platform. we compared our top , association signals with any snp reaching nominal significance (p � . ) in the other gwass that were within kb and had r � . in either the hapmap europeans (ceu group) or asians (asn group). again, we considered a mexican-american type diabetes–associated snp to be in silico replicated if another snp with r � . in the ceu or asn groups to the mexican-american type diabetes– associated snp was associated at p � . in the same direction (i.e., the same allele was associated with type diabetes) in the dgi gwas (fig. ). table descriptive statistics for the individuals with type diabetes in the primary gwas set from starr county, texas n sex (n female) age (years) . � . age at diagnosis (years) . � . fasting glucose (mg/dl) . � . a c (%) . � . bmi (kg/m ) . � . data are means � sd, unless otherwise indicated. type diabetes gwas in mexican americans diabetes, vol. , december results quality control. we selected for subsequent analysis the xbai and hindiii chip experiment with the highest call rate for each individual and two or less discordant geno- type calls for the snps duplicated on the two chips. of random control and case subjects for which genotyping was attempted, and , respectively, met these criteria. the mean per-chip call rate using the dm algorithm was � %, although the xbai chip performed slightly better than the hindiii chip ( . and . %, respectively). for both chips, � % of experiments had call rates � % ( . % for xbai and . % for hindiii). using the dm algorithm calls, we observed significant (p � . ) departures from hwe in a substantial number of snps ( . % all samples, . % random subjects only, and . % case subjects only). this is largely attributable to snps with excess homozygosity, consistent with nonran- dom missing data (heterozygotes have more “no-calls” since their intermediacy between the two homozygote classes renders them more difficult to call than the two homozygote classes). using gel, both increased the call rate ( . % mean xbai call rate with . % of experiments having � % call rates and . % mean hindiii call rate with . % of experiments having � % call rates) and reduced nonran- dom missing data by increasing the proportion of hetero- zygote genotype calls (online appendix table ), which subsequently reduced the number of snps showing signif- icant (p � . ) departures from hwe ( . % all samples, . % random subjects only, and . % case subjects only). with either genotype calling algorithm (gel or dm), there was no substantial case-random control difference throughout the majority of the distribution of per-chip genotype call rates, although there were some outliers in the tails of the distribution (online appendix fig. ). for comparative purposes, we also called the genotypes with the brlmm algorithm, which again yielded an in- creased proportion of heterozygotes (online appendix table ). in contrast to gel and dm, which call the genotypes for each chip experiment individually, brlmm normalizes the intensity patterns across all chip experi- ments, and therefore it is recommended that only chips with dm call rates � % be used. to do this would require chip-genotyping experiments ( . %) to be removed from consideration, substantially reducing our power. we experimented with lowering the dm algorithm call rate threshold and found that brlmm overcompensates for missing genotypes in the heterozygote class and increases the proportion of heterozygotes to unrealistic levels in chips with dm call rates � % (online appendix fig. ). given the limited number of samples under investigation, the marginal increase in genotype calls using brlmm over gel and the high concordance rates between gel and brlmm (online appendix tables and ), we decided to report results using the gel algorithm to retain maximal power. allelic associations. the chromosomal distribution of fisher’s exact test p values for the , snps passing our quality-control thresholds are presented in fig. . a total of , had allelic association p � . and are presented in online appendix table . the best (p � � ) snps (table ) survey different regions of the genome and are in or near ankrd , dyrk , epb l , grik , hpse , ica , ifng, nxph , or d , sdf l , sorbs , spry , slc a , and tmeff . two adjacent snps on the affymetrix genechip human mapping k set (rs and rs ) on chromosome , in and near ankrd , respectively, are in perfect (r � ) ld with each other and are associated at p � � . our most significantly associated snp, rs , has a p value of fig. . schematic of analysis and in silico replication plan. m.g. hayes and associates diabetes, vol. , december . � � , approximately one order of magnitude below a conservative bonferroni correction for multiple tests ( . / , � . � � ). we note that none of the most significant signals are snps with low mafs ( . – . ; we excluded snps with mafs � . ). while this observation is not unexpected given the reduced power for detecting susceptibility loci with allele frequencies at the tail of the maf distribution, it remains noteworthy since nonrandom patterns of missing data and other genotyping errors not detected in quality-control analysis often lead to snps with low mafs being disproportionately found among those with the most significant p values, which are subse- quently poorly replicated. using permutations, we empirically estimated the fdr at various thresholds (online appendix table ) and found that we observe many more significant allelic associations than expected genome wide. for our best signals, those meeting a p � � significance threshold, the fdr is estimated to be %. this suggests that – of the snps will likely turn out to be false-positives. we also compared the distribution of allelic association p values against a uniform distribution (online appendix fig. ). our ob- served distribution begins to depart from the expected uniform one at approximately p � � , suggesting an appropriate threshold for investigating in silico replication in order to prioritize snps for follow-up. ancestry estimates in case and random control sam- ples. the starr county mexican-american population is a relatively homogeneous ( . % hispanic by self-report [available at factfinder.census.gov]) yet highly admixed population with contributions to the contemporary gene pool from individuals of spanish, native american, and african ancestry. previous estimates using classical mark- ers suggest ancestry proportions of , , and %, respec- tively ( ). since population substructure can yield spurious case-control associations, we investigated the patterns of ancestry in the case and random control subjects used in the gwas. we observed no significant difference between the subsets (online appendix fig. ), which permitted us to average the admixture proportions over them. the ancestry estimates observed using the k snp sets ( % european, % asian, and % african) fig. . fisher’s exact test �log (p values) for tests of association between the high-quality autosomal snps (snps with hwe departure p > . in random subjects, call rates > . , and maf > . ) and type diabetes affection status. table snps most significantly associated with type diabetes dbsnp rs chr. position* gene* allele / control frequency (a ) case frequency (a ) p† or† rs c/g . . . � � . rs tmeff a/g . . . � � . rs spry /ankrd c/t . . . � � . rs slc a a/g . . . � � . rs ica /nxph a/g . . . � � . rs sdf l g/t . . . � � . rs ankrd a/c . . . � � . rs dyrk /ifng c/t . . . � � . rs hpse c/t . . . � � . rs or d c/t . . . � � . rs sorbs g/t . . . � � . rs epb l a/t . . . � � . rs grik a/g . . . � � . rs a/g . . . � � . *affymetrix netaffx annotation; †allele vs. . chr., chomosome. type diabetes gwas in mexican americans diabetes, vol. , december were consistent with the previous estimates from classical markers. more importantly, for the purposes here, esti- mates of the proportion of african, asian, and european ancestry for the case and random control subjects were indistinguishable from each other (fig. ). formal compar- isons by q-q plots show no significant differences in the case and random control distributions of ancestry propor- tions (online appendix fig. ), suggesting that spurious associations due to different ancestries of the case and random control subjects are unlikely. we used these poa estimates as covariates in logistic regressions between type diabetes status and genotype. the poa estimates indicate that ) there is very little difference from one individual to the next in the african poa and ) the difference in poa estimates per individual lie along an asian versus european axis of variation. this suggests that the poa variation could be efficiently cap- tured by using the european or asian poa as a covariate, and we chose to use the former. including the ceu group covariate had little impact on the association results. the p value for nearly all snp � genotype regressions in- creased or decreased by less than one-half an order of magnitude (online appendix fig. ). we did not observe any highly significant regressions disappearing after in- cluding the ceu group poa covariate, again suggesting that spurious associations due to different ancestries of the case and random samples are highly unlikely. instead, the difference in the regression p values distributions was skewed toward increased significance when using the european poa as a covariate. verification. before genotyping any snp in a larger collection of individuals for replication, we wanted to first verify the association in the same set of individuals using a different genotyping platform (taqman). to identify the most robust snps for verification genotyping, we selected a subset of snps from the highly associated snps (table ) that met our quality-control criteria (hwe de- parture p � . in random subjects, call rates � . , and maf � . ) using both the dm and gel algorithms. all snps remained significant at a p � . ( / p � � , / p � � , and / p � � ), with the exception of rs (near sdf l), which dropped to p � . (online appendix table ). since the overall genotyping concor- dance between the genotyping platforms was . % (per- marker range . – . %), the decrease in allelic association significance is not a function of differential genotyping but rather the increase in sample size. in silico replications in other k type diabetes gwass. a total of snps (online appendix table ) associated in the mexican-american subjects (p � . ) had the same allele associated (p � . ) in one of the other k gwass ( , , ). at the more stringent p � . level (table ), six were replicated in the amish, three were replicated in the pima indians (all by case- control tests), and four were replicated in the fhs (one by generalized estimating equations alone and three by fam- ily-based association test alone). these included snps in or near the following genes: ralgps and angptl (chromosome ); lcorl, ncapg, and csn (chromo- some ); htr and adrb (chromosome ); utrn (chromosome ); lingo (chromosome ); egr (chro- mosome ); ubqlnl and or h (chromosome ); and rora (chromosome ). of these, one was replicated in multiple studies: rs *t (p � . ; odds ratio [or] . ) near ubqlnl and or h in the amish (p � . ; . ) and fhs (p � . ; hazard rate ratio . ). additionally, two nonredundant snps (r � . ) in or near ralgps were independently replicated in the amish (rs *g; p � . and or . in mexican americans; p � . and or . in amish) and pima indians (rs *g; p � . and or . in mexi- can americans; p � . and or . in pima indian case-control subjects). replication in non- k type diabetes gwass. we also observed snps associated (p � . ) with type diabetes in the mexican americans in high ld in either the hapmap europeans or asians, also showing evidence for association with type diabetes in a gwas (p � . ) in a scandinavian cohort (dgi; online appendix table ). four of these are significant in the mexican americans at a more stringent p � . level and are located in or near actn on chromosome , gdnf and egflam on chro- mosome , egr on chromosome , and a nongenic region on chromosome (table ). replication in more than one other gwas. we inves- tigated the intersection of the in silico replications in the other gwas examined and found that six snps associated in mexican americans (p � . ) replicated in multiple studies (p � . ). snps in or near gypc (chromosome ), egr (chromosome ), and a nongenic region (chromo- some ) replicated in the pima indians and dgi, dbc (chromosome ) in the pima indians and fhs, and phldb (chromosome ) in the amish and pima indians (table ). rs *t in or near mgc and chd was found to decrease risk in the three (amish, pima indians, and dgi) of four comparative cohorts as well as the mexican americans. an additional region on chromo- some contains two redundant snps (r � . ) that show evidence for replication: rs in or near ubqlnl and or h is replicated in the amish and fhs and nearby rs is replicated in the dgi study. this is in addition to the multiple ralgps replications discussed above. fig. . estimates of ancestry proportions in case and random control subjects. the genome-wide average ancestral proportions of ancestry for each individual is plotted in a triangular matrix in which each point of the triangle represents % ancestry for the indicated ancestral population. the average proportion of each ancestral population in the starr county mexican americans is listed after each ancestral popula- tion. red � case subjects; blue � random control subjects. m.g. hayes and associates diabetes, vol. , december t a b l e s n p s as so ci at ed w it h ty p e d ia b et es in m ex ic an a m er ic an s (p � . ) w it h re p li ca ti o n in at le as t o n e o th er k g w a s d b s n p rs c h r. p o si ti o n * g en e* a ll el e / m ex ic an a m er ic an s (p va lu e) † m ex ic an a m er ic an s (o r )† a m is h (p va lu e) ‡ a m is h (o r )‡ p im a in d ia n s (c c p )§ p im a in d ia n s (c c o r )§ p im a in d ia n si b s (p )� p im a in d ia n si b s (o r )� f h s g e e (p )¶ f h s f b a t (p )# f h s h r r ** rs u b q l n l /o r h c /t . . . . . . . rs r o r a c /t . . . . rs l i n g o g /t . . . . rs a /c . . . . rs c /g . . . . rs c s n a /c . . . . rs h t r /a d r b c /t . . . . rs r a l g p s a /g . . . . rs e g r a /g . . . . rs r a l g p s /a n g p t l a /g . . . . rs l c o r l /n c a p g c /g . . . . rs u t r n c /t . . . . *a ff ym et ri x n et a ff x an n o ta ti o n ; †a ll el e vs . ; ‡g en o ty p e vs . ; §c as e- co n tr o l te st s, al le le vs . ; �w it h in -f am il y te st s, al le le vs . ; ¶ ge n er al iz ed es ti m at in g eq u at io n s (g e e s) , al le le vs . ; #f am il y- b as ed as so ci at io n te st (f b a t ), al le le vs . ; ** h az ar d ra te ra ti o (h r r ), al le le vs . . c h r. , ch ro m o so m e. t a b l e r ep li ca ti o n o f m ex ic an -a m er ic an s n p s (p � . ) w it h n o n - k t yp e d ia b et es g w a s re su lt s m ex ic an a m er ic an (d b s n p rs ) m ex ic an a m er ic an (c h ro m o - so m es ) m ex ic an a m er ic an (p o si ti o n )* m ex ic an a m er ic an (g en e) † m ex ic an a m er ic an ( / ) m ex ic an a m er ic an (p )‡ m ex ic an a m er ic an (o r )‡ d g i (d b s n p rs ) d g i (a ll el e) d g i (c h ro m o - so m es ) d g i (p o si ti o n )* d g i (p ) d g i (o r ) h ap m ap (r ) c e u a s n rs a c t n c /t . . rs t . . n /a n /a rs e g r a /g . . rs g . . n /a n /a rs g d n f /e g f l a m c /t . . rs c . . . . rs c /t . . rs t . . . . *p o si ti o n in m ar ch u n iv er si ty o f c al if o rn ia s an ta c ru z b u il d ; †a ff ym et ri x an n o ta ti o n s; ‡a ll el e vs . . n /a , n o t ap p li ca b le (i .e ., id en ti ca l s n p ). type diabetes gwas in mexican americans diabetes, vol. , december discussion we have carried out a gwas of type diabetes in mexican americans from starr county, texas. we observed a number of allelic associations showing replication in one of the other gwas, and a limited number of which show multiple lines of evidence for replication. the association signals that appear to be the most robust would be the three that are significant at p � � in the mexican- american subjects and are replicated (p � . ) in at least two of four other gwass interrogated (rs and rs near ubqlnl and or h on chromosome , rs and rs in or near ralgps on chromosome , and rs near egr on chromosome ). these snps and many other significantly associated snps will be prioritized for further follow-up genotyping in a larger mexican-american case-random control cohort. our fdr estimate suggests that if we followed up the associations significant at the p � � threshold, a little less than half would not be false-positives. the broad replication of these three signals meeting this significance threshold suggests that they may be true rather than false-positive associations, but confirmation of such will await the results of the follow-up genotyping in the more numerous mexican-american case-random sample cohort. even though our most promising snps may turn out to be false-positives, it is tempting nonetheless to query whether any of these putative type diabetes susceptibility genes identified in this gwas have supporting biological evi- dence for their candidacy as type diabetes genes. of the genes implicated and discussed above, no direct links to a diabetes-related phenotype were found. given the large amount of data generated in a gwas, one might naively think that this study represents a comprehensive interrogation of the human genome for type diabetes susceptibility genes, but this is simply not true ( ). although the mean intermarker distance for the , snps genotyped on the affymetrix genechip human mapping k set is only . kb, the k platform does not completely cover the genome given the patterns of ld and uneven snp density ( ). nowhere is this more evident than searching for associations at previously iden- tified and replicated type diabetes genes. for example, the mexican-american population under investigation here is the same in which capn was identified through positional cloning studies subsequent to genome linkage scans. however, the nearest snps to capn on the k platform are and kb in either direction, well beyond the ld block in which capn resides. the results for other “known” type diabetes genes in our study are presented in online appendix table . like capn , there are no snps on the affymetrix genechip human mapping k set near hnf a or kcnj and hhex. the previously identified type diabetes–associ- ated variant (rs ) in pparg is included on the k set but is not associated with type diabetes in mexican americans (p � . ). for tcf l , the snp (rs ) in highest ld (r � . ) with the previously identified type diabetes–associated variant (rs ) also shows no significant associations to type diabetes in the mexican- american subjects (p � . ). the snps in or near two genes (igf bp and slc a ), previously identified in other gwass as containing type diabetes risk alleles, show no evidence of association and have modest ld between the previously associated variant and the snps on the k platform. however, we did observe significantt a b l e s n p s as so ci at ed w it h ty p e d ia b et es in m ex ic an a m er ic an s (p � . ) w it h re p li ca ti o n in m o re th an o n e o th er g w a s d b s n p rs c h r. p o si ti o n * g en e* a ll el e ( / ) m ex ic an a m er ic an (p )† m ex ic an a m er ic an (o r )† a m is h (p )‡ a m is h (o r )‡ p im a in d ia n ca se - co n tr o l (p )§ p im a in d ia n ca se - co n tr o l (o r )§ p im a in d ia n si b s (p )� p im a in d ia n si b s (o r )� f h s g e e (p )¶ f h s f b a t (p )# f h s h r r ** d g i (r s) d g i (p )† d g i (o r )† rs u b q l n l /o r h c /t . . . . . . . rs r a l g p s a /g . . . . rs e g r a /g . . . . . . rs r a l g p s /a n g p t l a /g . . . . rs d b c a /g . . . . . . rs p h l d b a /g . . . . . . rs g y p c c /g . . . . rs . . rs m g c /c h d c /t . . . . . . rs . . rs c /t . . rs . . rs c /g . . . . rs . . *a ff ym et ri x n et a ff x an n o ta ti o n ; †a ll el e vs . ; ‡g en o ty p e vs . ; §c as e- co n tr o l te st s, al le le vs . ; �w it h in -f am il y te st s, al le le vs . ; ¶ ge n er al iz ed es ti m at in g eq u at io n s (g e e s) , al le le vs . ; #f am il y- b as ed as so ci at io n te st (f b a t ), al le le vs . ; ** h az ar d ra te ra ti o (h r r ), al le le vs . . c h r. , ch ro m o so m e. m.g. hayes and associates diabetes, vol. , december t a b l e c o m p ar is o n o f fo u r g w a st u d ie s f h s m ex ic an a m er ic an p im a c /c p im a fa m il y a m is h n , ca se / co n tr o l ca se / co n tr o l si b sh ip s ca se / co n tr o l t yp e p o p u la ti o n , fa m il y- b as ed c as e/ co n tr o l c as e/ co n tr o l f am il y c as e/ co n tr o l e th n ic it y n o n -h is p an ic w h it e m ex ic an a m er ic an a m er ic an in d ia n a m er ic an in d ia n n o n -h is p an ic w h it e m al e/ fe m al e / c as e / / / / c o n tr o l / / / / m ea n ag e (y ea rs ) . � . c as e . � . . � . . � . . � . c o n tr o l — . � . . � . . � . m ea n b m i (k g/ m ) . � . c as e . � . . � . . � . . � . c o n tr o l — . � . . � . . � . q u an ti ta ti ve gl yc em ic tr ai ts an al yz ed f p g , tf p g , a c , f i, h o m a -i r , g u tt is i _ f a s t g , gl u co se a u c , in su li n a u c , h o m a -i r , in su li n se cr et io n in d ex n o te s ca se s o f in ci d en t d ia b et es c o n tr o ll ed fo r ad m ix tu re a ge o f o n se t � ye ar s s ib sh ip s o ve rl ap w it h c /c c as es h av e (� ) f h ; ac ti ve li fe st yl e s n p s an al yz ed , , , , , s n p s fa il ed , m a f � % ; , h w e p � . ; , ca ll ra te � % , m a f � % ; , h w e p � . ; , ca ll ra te � % , m a f � % ; , h w e p � . ; , ca ll ra te � % an d /o r er ro r ra te � % in d u p li ca te sa m p le s , m a f � % ; , h w e p � . ; , ca ll ra te � % an d /o r er ro r ra te � % in d u p li ca te sa m p le s , m a f � % ; , h w e p � . ; , ca ll ra te � % p va lu e th re sh o ld s s tr at eg y : p � . in al l th re e gl u co se tr ai ts (f p g , tf p g , an d a c ) o r in al l in su li n tr ai ts (f i, h o m a -i r , an d g u tt ) o r tw o gl u co se an d tw o in su li n tr ai ts o r in ci d en t ty p e d ia b et es ; s tr at eg y : p � . p ri m ar y an al ys is : p � . fo r ty p e d ia b et es ; in si li co re p li ca ti o n : p � . in si li co re p li ca ti o n : p � . in co m b in ed w it h in -f am il y an d ca se -c o n tr o l an al ys is (w ei gh te d to gi ve p ri o ri ty to w it h in -f am il y te st ); a d d it io n al ge n o ty p in g: p � . in co m b in ed w it h in -f am il y an d ca se -c o n tr o l an al ys is as ab o ve in si li co re p li ca ti o n : p � . in co m b in ed w it h in -f am il y an d ca se -c o n tr o l an al ys is (w ei gh te d to gi ve p ri o ri ty to w it h in -f am il y te st ); a d d it io n al ge n o ty p in g: p � . in co m b in ed w it h in -f am il y an d ca se - co n tr o l an al ys is as ab o ve p ri m ar y an al ys is : p � . fo r ty p e d ia b et es ; in te rn al re p li ca ti o n : ty p e d ia b et es p � . an d o n e gl u co se tr ai t (f a s t g o r g a u c ) o r o n e in su li n tr ai t (i s i, ia u c , o r h o m a -i r ) p � . r ep li ca ti o n co h o rt , u n re la te d f h s p ar ti ci p an ts (n o n -o ve rl ap p in g) o ve rl ap p in g in d iv id u al s fo r s n p ve ri fi ca ti o n n o n -o ve rl ap p in g p im a in d ia n s: , ca se / , co n tr o l n o n -o ve rl ap p in g p im a in d ia n s: , ca se / , co n tr o l n o n d ia b et ic a m is h p ar ti ci p an ts ( co n tr o l fr o m p ri m ar y ty p e an al ys is ) in si li co re p li ca ti o n t d k c o n so rt iu m � d g i k t d k c o n so rt iu m � d g i k t d k c o n so rt iu m � d g i k t d k c o n so rt iu m � d g i k t d k c o n so rt iu m � d g i k t d , ty p e d ia b et es . type diabetes gwas in mexican americans diabetes, vol. , december associations with snps in cdkal and cdkn a (p � . ) but only at snps not in ld with the originally associated snp, so these could not be considered direct replication of the original signal but may point to other variation contributing to risk of type diabetes in mexican americans. the lack of difference in the poa estimates between the case and random samples speaks not just to a reduced likelihood of spurious associations due to substructure but also to a larger issue. given the high prevalence of type diabetes among native americans, it has been previously hypothesized that the high prevalence of type diabetes in mexican americans may be due to their native american ancestry ( , ). in support of this hypothesis is our estimate that � % of the contemporary mexican-ameri- can gene pool is native american derived. given the prevalence of diabetes among native americans, the pre- dicted prevalence in mexican americans parallels that expected based on this degree of admixture ( ). how- ever, if type diabetes in mexican americans was largely native american derived, a higher proportion of asian (proxy for native american) ancestry would have been observed in the case subjects than in the random control subjects; we did not observe this. the poas are genome-wide estimates. we assume these may be highly variable from one genomic region to the next, so it remains possible that for any given gene associated with type diabetes in mexican americans, it is the native american– derived variant that is the risk allele. we also noted that the difference in the distribu- tions of the regression p values was skewed toward increased significance when using the european poa as a covariate. this suggests that we may be able to exploit this when admixture mapping methods are used in the future. in conclusion, we observed many snps associated with type diabetes, some of which were replicated in at least one of four other gwass we queried. this study repre- sents our initial examination of the mexican-american k gwas data; more sophisticated approaches will follow, including a meta-analysis of four k gwass. it may also be that subsequent investigations of this gwas table replication evidence across four gwa studies study snp chr. position (hg ) gene gene name omim initial finding fhs rs p . pde b camp-specific phos- phodiesterase g-allele protective for type diabetes (hr . 	 . – . , cox p � . ) fhs rs q . kb from nearest gene n/a minor t-allele at snp rs protective against diabetes (hr . 	 . – . , cox p � . ) fhs rs q . ms a membrane-spanning -domains subfamily a member t-allele associated with lower fpg in fhs (fbat p � . ) amish rs p . grb growth factor receptor bound protein g-allele protective for type diabetes (or . , p � . ) amish rs p . fhit fragile histidine triad gene t-allele increased type diabetes risk (or . , p � . ) pima rs p . fancf fanconia anemia, complementation group f a-allele increased type diabetes risk (or . , p � . ) pima rs q . ztbt zinc finger and btb domain containing- a-allele increased type diabetes risk (or . , p � . ) ma rs p ubqlnl ubiquilin t-allele protective for type diabetes (or . , p � . ) ma rs q ralgps ral gef with ph domain and sh binding motif g-allele protective for type diabetes (or . , p � . ) ma rs q . egr early growth response g-allele protective for type diabetes (or . , p � . ) chr., chromosome. continued on following page m.g. hayes and associates diabetes, vol. , december with haplotypes or genes as the unit of investigation, rather than snps, will prove to be more informative. nonetheless, we have highlighted several interesting puta- tive type diabetes genes for follow-up in the hopes that it may further elucidate the etiology of type diabetes and identify new avenues for both the treatment and preven- tion of this complex disease. acknowledgments this study was supported in part by u.s. public health service grants dk- , dk- , dk- , dk- , and hl- and a gift from the kovler family founda- tion. m.g.h. was supported by a mentor-based fellowship from the american diabetes association. we thank laura martinolich, xinmin li, edwin cook, and carole ober for providing technical assistance with the affymetrix genotyping assays. we also thank the k type diabetes consortium members and authors of the three other k gwas studies ( , , ) for valuable discussion and comments regarding the data, analysis, and manuscript preparation. references . centers for disease control and prevention: chronic disease prevention: preventing diabetes and its complications. atlanta, ga, u.s. department of health and human services, centers for disease control and preven- tion, . flegal km, ezzati tm, harris mi, haynes sg, juarez rz, knowler wc, perez-stable ej, stern mp: prevalence of diabetes in mexican americans, cubans, and puerto ricans from the hispanic health and nutrition examination survey, – . diabetes care : – , . hamman rf, marshall ja, baxter j, kahn lb, mayer ej, orleans m, murphy jr, lezotte dc: methods and prevalence of non-insulin-dependent diabetes mellitus in a biethnic colorado population: the san luis valley diabetes study. am j epidemiol : – , . hanis cl, ferrell re, barton sa, aguilar l, garza-ibarra a, tulloch br, garcia ca, schull wj: diabetes among mexican americans in starr county, texas. am j epidemiol : – , . samet jm, coultas db, howard ca, skipper bj, hanis cl: diabetes, gallbladder disease, obesity, and hypertension among hispanics in new mexico. am j epidemiol : – , . hanis cl, boerwinkle e, chakraborty r, ellsworth dl, concannon p, stirling b, morrison va, wapelhorst b, spielman rs, gogolin-ewens kj, shepard jm, williams sr, risch n, hinds d, iwasaki n, ogata m, omori y, petzold c, rietzch h, schroder he, schulze j, cox nj, menzel s, boriraj vv, chen x, lim lr, lindner t, mereu le, wang yq, xiang k, yamagata k, table continued qt internal replication external replication k t d consortium external replication k dgi same protective allele associated with higher gutt isi and lower fpg, mfpg, a c, and homa-ir (gee p � . ) fpg and homa-ir (p � . ) no rs in moderate ld (r � . ); consistent trends in fpg (p � . ) and homa-ir (p � . ) same protective allele associated with lower fpg and mfpg in the fhs sample (gee p � . and . , respectively) no t-allele protects from type diabetes in mexican americans (or . , nominal p � . ) and in the amish (or . , nominal p � . ) with similar trends in the pimas rs in perfect ld (r � . ); minor t-allele shows opposite nominal trend in increasing risk of type diabetes (or . , p � . ) same no t-allele was nominally protective from diabetes in the mexican americans (or . , p � . ) and in the pimas (or . , p � . ) rs in perfect ld (r � . ); minor t-allele shows consistent nominal trend in protection from type diabetes (or . , p � . ) same protective allele associated with lower gauc (p � . ) no no not with rs ; six additional grb snps in ld (r . – . ) are associated with type diabetes (p � . ) same risk allele associated with increased gauc (p � . ) no mexican-american type diabetes or . 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} } 	m. enomoto, a. jain, + authors j. belik 	published 	medicine 	american journal of physiology. lung cellular and molecular physiology inhaled nitric oxide (no) and other cgmp- or camp-dependent pulmonary vasodilators are often used in combination for the treatment of the persistent pulmonary hypertension of the newborn syndrome. there is in vitro evidence to indicate that no downregulate the pulmonary vascular response to cgmp-dependent agonists raising concern as to whether a synergistic effect is observed when employing a combined strategy in newborns. hypothesizing that a synergistic effect is absent, we evaluated newborn… expand view on pubmed ajplung.physiology.org save to library create alert cite launch research feed share this paper citationsbackground citations methods citations results citations view all figures and topics from this paper 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure 	 figure view all figures & tables 	cyclic gmp 	sildenafil 	pulmonary hypertension 	vasodilator agents 	nitric oxide 	acetylcysteine 	colforsin 	infant, newborn 	hypoxia 	hypertensive disease 	persistent fetal circulation syndrome 	heat shock proteins 	vasodilation disorder 	heat-shock response 	pulmonary artery structure 	 -chloro-cyclic adenosine monophosphate 	myocytes, smooth muscle 	guanylate cyclase 	down-regulation 	hypersensitivity desensitization 	penicillamine 	muscle, smooth, vascular 	smooth muscle (tissue) citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency enhanced no-dependent pulmonary vasodilation limits increased vasoconstrictor sensitivity in neonatal chronic hypoxia. 	joshua r sheak, laura weise-cross, ray j dekay, b. walker, n. l. jernigan, t. resta 	medicine 	american journal of physiology. heart and circulatory physiology 	 	 	 save alert research feed soluble guanylate cyclase modulators blunt hyperoxia effects on calcium responses of developing human airway smooth muscle. 	r. britt, m. thompson, + authors y. prakash 	biology, medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	 save alert research feed therapeutic potential of soluble guanylate cyclase modulators in neonatal chronic lung disease. 	g. t. wagenaar, p. hiemstra, r. gosens 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf 	view excerpt, cites methods save alert research feed maternal-pup interaction disturbances induce long-lasting changes in the newborn rat pulmonary vasculature. 	y. shifrin, sina sadeghi, + authors j. belik 	biology, medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf save alert research feed update on novel targets and potential treatment avenues in pulmonary hypertension. 	john c huetsch, k. suresh, m. bernier, l. shimoda 	biology, medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	 	view excerpt, cites background save alert research feed roles of heat shock protein toward hypoxia-inducible factor α ( hif- α ) blockade in newborn rats with hypoxia-induced pulmonary hypertension 	l. wang, mingxia li 	 	pdf 	view excerpt, cites background save alert research feed hydrogen peroxide promotes gastric motility in the newborn rat 	a. f. fajardo, curtis sobchak, y. shifrin, j. pan, t. gonska, j. belik 	medicine 	pediatric research 	 	 	pdf 	view excerpt, cites results save alert research feed gastric and pyloric sphincter muscle function and the developmental-dependent regulation of gastric content emptying in the rat. 	curtis sobchak, a. f. fajardo, y. shifrin, j. pan, j. belik 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	pdf save alert research feed recent advances in the mechanisms of lung alveolarization and the pathogenesis of bronchopulmonary dysplasia. 	diogo m silva, c. nardiello, agnieszka pozarska, r. morty 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf 	view excerpt, cites background save alert research feed antenatal sildenafil administration to prevent pulmonary hypertension in congenital diaphragmatic hernia (stop-ph): study protocol for a phase i/iib placenta transfer and safety study 	f. russo, a. benachi, + authors j. deprest 	medicine 	trials 	 	 	 	view excerpt, cites background save alert research feed ... ... references showing - of references sort byrelevance most influenced papers recency prolonged treatment of porcine pulmonary artery with nitric oxide decreases cgmp sensitivity and cgmp-dependent protein kinase specific activity. 	w. perkins, d. o. warner, k. a. jones 	biology, medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf save alert research feed acute vasodilator effects of rho-kinase inhibitors in neonatal rats with pulmonary hypertension unresponsive to nitric oxide. 	p. mcnamara, p. murthy, + authors r. jankov 	medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf 	view excerpts, references methods and background save alert research feed desensitization of no/cgmp signaling in smooth muscle: blood vessels versus airways 	f. mullershausen, a. lange, e. mergia, a. friebe, d. koesling 	biology, medicine 	molecular pharmacology 	 	 	pdf 	view excerpt, references background save alert research feed postnatal maturation of phosphodiesterase (pde ) in piglet pulmonary arteries: activity, expression, effects of pde inhibitors, and role of the nitric oxide/cyclic gmp pathway 	l. moreno, b. losada, + authors f. perez-vizcaino 	medicine 	pediatric research 	 	 	pdf 	view excerpts, references background save alert research feed development and mechanism of a specific supersensitivity to nitrovasodilators after inhibition of vascular nitric oxide synthesis in vivo. 	s. moncada, d. rees, r. schulz, r. palmer 	chemistry, medicine 	proceedings of the national academy of sciences of the united states of america 	 	 	pdf 	view excerpt, references results save alert research feed reduction in soluble guanylyl cyclase-specific activity following prolonged treatment of porcine pulmonary artery with nitric oxide. 	w. perkins, miwa taniguchi, d. o. warner, e. chini, k. a. jones 	chemistry, medicine 	american journal of physiology. lung cellular and molecular physiology 	 	 	pdf 	view excerpt, references background save alert research feed mechanisms of relaxant activity of the nitric oxide-independent soluble guanylyl cyclase stimulator bay - in rat tracheal smooth muscle. 	haroldo a. toque, fabíola z.t. mónica, rafael p. morganti, g. de nucci, e. antunes 	chemistry, medicine 	european journal of pharmacology 	 	 	view excerpt, references results save alert research feed no-independent regulatory site on soluble guanylate cyclase 	johannes-peter stasch, e. becker, + authors m. schramm 	biology, medicine 	nature 	 	 	view excerpt, references results save alert research feed ng‐nitro‐l‐arginine methyl ester attenuates vasodilator responses to acetylcholine but enhances those to sodium nitroprusside 	v. ralevic, r. mathie, b. alexander, g. burnstock 	chemistry, medicine 	the journal of pharmacy and pharmacology 	 	 save alert research feed sildenafil improves alveolar growth and pulmonary hypertension in hyperoxia-induced lung injury. 	f. ladha, s. bonnet, f. eaton, k. hashimoto, g. korbutt, b. thébaud 	medicine 	american journal of respiratory and critical care medicine 	 	 	view excerpt, references background save alert research feed ... ... related papers 	abstract 	figures and topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ original article activating transcription factor (atf ) sequence polymorphisms in type diabetes and pre-diabetic traits winston s. chu, swapan kumar das, hua wang, juliana c. chan, panos deloukas, philippe froguel, , leslie j. baier, weiping jia, mark i. mccarthy, maggie c.y. ng, , coleen damcott, alan r. shuldiner, eleftheria zeggini, and steven c. elbein activating transcription factor (atf ) is located within the region of linkage to type diabetes on chromosome q - q and is a key activator of the endoplasmic reticulum stress response. we evaluated single nucleotide poly- morphisms (snps) spanning > kb in people, from which we selected snps for evaluation in caucasian case subjects from utah and between and control subjects. six snps showed nominal associations with type diabetes (p � . – . ), including the nonsynonymous snp rs (m v) in exon and rs in the � flanking region. only rs remained significant on permutation testing. the associations were not replicated in african-american case subjects and control subjects, nor were atf snps associated with altered insulin secretion or insulin sensitivity in nondiabetic cau- casian individuals. no association with type diabetes was found in a subset of snps in caucasian (n � , ), pima indian (n � ), and chinese (n � ) samples. allelic expression imbalance was found in transformed lympho- cyte cdna for � untranslated region variants, thus sug- gesting cis-acting regulatory variants. atf does not appear to play a major role in type diabetes, but further work is required to identify the cause of the allelic expres- sion imbalance. diabetes : – , t he endoplasmic reticulum is a membranous lab- yrinthine network in eukaryotic cells that is the site for the synthesis, folding, assembly, and posttranslational modification of proteins. the endoplasmic reticulum includes a highly conserved system of proteins that facilitates protein folding and processing, protects cells from the toxic effects of accumulating unfolded proteins (endoplasmic reticulum stress), and, when these measures fail, initiates apoptosis. this system, known as the unfolded protein response, is activated by accumulat- ing unfolded proteins ( – ). the endoplasmic reticulum stress and unfolded protein response pathways are partic- ularly important in secretory cells, including pancreatic �-cells, hepatocytes, and adipocytes ( ). in the presence of endoplasmic reticulum stress, the transcription factor activating factor (atf ) serves as a key proximal sensor and is transported to the golgi complex, where it is cleaved by proteases to yield an active cytoplasmic do- main that functions to upregulate chaperone proteins. considerable data have implicated endoplasmic reticu- lum stress in diabetes pathogenesis. in akita mice, aber- rant proinsulin processing induces endoplasmic reticulum stress and results in �-cell apoptosis ( ), and mutations in other proximal sensors impair the endoplasmic reticulum stress response and lead to �-cell apoptosis and diabetes in experimental models and humans. endoplasmic reticu- lum stress involvement in liver and adipose are activated in obese mice ( ), and improved protein folding alleviated insulin resistance and improved glucose tolerance ( ). atf includes exons and spans kb on chromo- some q - , a region of well-replicated linkage to type diabetes in eight different populations ( , ). thus, atf is a strong positional and functional candidate gene for type diabetes. furthermore, thameem et al. ( ) recently reported that the coding variant pro ala (p a) was nominally significantly associated with type diabetes (p � . ). additionally, we recently described an associ- ation of variants in the gene dusp , just upstream of atf , with type diabetes in caucasian and african- american subjects ( ). to test the hypothesis that se- quence variants in or near atf contribute to type diabetes susceptibility and to defects in insulin secretion and insulin resistance, we typed single nucleotide polymorphisms (snps) spanning the atf gene in a northern european caucasian population. we expanded the study by examining a subset of snps in seven from the division of endocrinology, department of medicine, university of arkansas for medical sciences and central arkansas veterans healthcare system, little rock, arkansas; the department of medicine and therapeutics, chinese university of hong kong, shatin, hong kong, special administrative region (sar), china; the wellcome trust sanger institute, hinxton, u.k.; the institut de biologie de lille, lille, france; the faculty of life sciences, imperial college, london, u.k.; the phoenix epidemiology and clinical research section, national institute of diabetes and digestive and kidney diseases, phoenix, arizona; the shanghai diabetes institute, department of endocrinology and metabolism, shanghai jiaotong university no. people’s hospital, shanghai, china; the oxford centre for diabetes, endocrinology and metabolism and wellcome trust centre for human genetics, university of oxford, u.k.; the department of medicine, university of chicago, chicago, illinois; and the division of endocrinology, diabetes and nutrition, univer- sity of maryland school of medicine, baltimore, maryland. address correspondence and reprint requests to steven c. elbein, md, professor of medicine, university of arkansas for medical sciences, endocri- nology j- /lr, john l. mcclellan memorial veterans hospital, w. th st., little rock, ar . e-mail: elbeinstevenc@uams.edu. received for publication september and accepted in revised form december . w.s.c. and s.k.d. contributed equally to this work. additional information for this article can be found in an online appendix at http://dx.doi.org/ . /db - . airg, acute insulin response to glucose; fsigt, frequently sampled intra- venous glucose tolerance test; snp, single nucleotide polymorphism. doi: . /db - © by the american diabetes association. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked “advertisement” in accordance with u.s.c. section solely to indicate this fact. diabetes, vol. , march additional populations (u.k., french, and amish cauca- sians; hong kong and shanghai chinese; pima indians; and arkansas african americans) included in the interna- tional type diabetes q consortium. nonsynonymous snps and snps showing the best evidence for an associ- ation with type diabetes were tested for an association with insulin sensitivity (si) and insulin secretion and for an association with type diabetes in an arkansas african- american population. finally, we sought evidence that variation in or near atf acting in a cis fashion might alter the expression ratio between alleles. research design and methods the primary study populations are summarized in table . the primary study population comprised unrelated caucasian case subjects and unre- lated caucasian control subjects ascertained from utah and arkansas for northern european ancestry, as described previously ( , ). case subjects were diabetic on treatment for type diabetes and had a diabetic first-degree relative; control subjects had normal glucose tolerance tests and no first- degree relative with type diabetes. a subset of members of the control population was used to determine linkage disequilibrium and to select tag snps (see below). the same case subjects and a subset of the control subjects ( control individuals) were genotyped as part of the international type diabetes q consortium ( ). confirmatory association studies were conducted in seven additional populations representing four ethnic groups included in the international type diabetes q consortium ( ): african-american individuals from arkansas ( case subjects and control subjects), samples from the amish family diabetes study ( case subjects with diabetes or impaired glucose tolerance and control subjects), samples from the french diabetes study ( case subjects and control subjects), samples from the u.k. ( and , respectively), samples from the pima indian study ( and ), samples from the hong kong chinese study ( and ), and samples from the shanghai chinese study ( and ). this resource is described in detail elsewhere ( ). additional african-american samples were tested in confirmatory genotyping studies conducted in arkansas for a total population of control subjects with normal fasting and/or postchal- lenge glucose levels and no family history of type diabetes and type diabetic subjects who also had a diabetic first-degree relative and diabetes diagnosis before age years. the consortium pima indian samples overlap with those published previously for analysis of atf ( ). physiological studies were conducted in members of utah families ascertained for two diabetic siblings; each individual had undergone a tolbutamide-modified frequently sampled intravenous glucose tolerance test (fsigt) ( ). a second population of unrelated caucasian individuals from arkansas underwent either a tolbutamide-modified (n � ) or an insulin-modified ( . units/kg, n � ) fsigt ( ). subjects ascertained in utah provided written informed consent under a protocol approved by the university of utah institutional review board. subjects studied in arkansas provided written informed consent under protocols approved by the univer- sity of arkansas for medical sciences institutional review board. other consortium samples were collected under approved procedures for local institutional review boards. snp genotyping. we evaluated snps selected from public ncbi (national center for biotechnology information) databases ( ) and spanning from kb upstream to kb downstream of the atf atg start site ( , , – , , bp; ncbi build ) (supplemental table s, which can be found in an online appendix [available at http://dx.doi.org/ . /db - ]). snps were selected from three sources: ) snps typed as part of an initial -kb map across the region in the utah sample; ) known exonic variants (l l, m v, p p, s s, p a, and s p) in the atf gene; and ) snps that fell within this region that were typed as part of a dense linkage disequilibrium map by the international type diabetes q consortium ( ). we selected additional snps from hapmap ( ) to fill in gaps identified based on initial hapmap linkage disequilibrium measures. these snps were then typed in at least utah caucasians to select a subset of snps with a minor allele frequency � % and that fully captured the hapmap phase snps (r � . ). coding snps and snps already typed in the full utah case-control set were forced in. additional snps from the consortium snps that were required to capture the common variants were typed in the remaining control samples. the consortium snps were typed using -plex illumina golden gate assays (illumina, san diego, ca) ( ). all other snps in the utah and arkansas samples were typed using either pyrosequencing on a psq- (biotage, uppsala, sweden) or oligonucleotide ligation assays. for snps initially typed by the international type diabetes q consortium but expanded to addi- tional control subjects, typing was accomplished by pyrosequencing but with at least samples overlapping with the illumina golden gate assay. additionally, full typing of seven snps and partial typing of two additional snps showed excellent correspondence between pyrosequencing and illu- mina golden gate assays. snps were selected for full typing using the tagger program in haploview version ( ). we typed snps in case and control subjects; an additional snps typed by the international type diabetes q consortium but not required to capture the common caucasian variants were typed in case and control subjects and included in all analyses. nondiabetic caucasian subjects who had undergone detailed metabolic studies were typed for nonsynonymous exonic snps or snps shown to be strongly associated in the caucasian case-control cohort. african americans were typed for the consortium snps, as well as exonic snps and snps showing an association in caucasians, but no attempt was made to fully capture the common african-american variation based on hapmap yoruban samples. allelic expression imbalance. total rna was isolated from epstein-barr virus–transformed lymphocytes, and allele-specific expression of two � untranslated region snps was quantified from heterozygous individuals as described elsewhere ( ). briefly, total rna was converted to cdna using random primers. both cdna and genomic dna were typed using the same assay by pyrosequencing, using a psq- machine (biotage) with a universal biotinylated primer. peak heights for the two alleles were quantified in both cdna and genomic dna using allele quantification software (biotage), which was designed for quantification of pooled genotypes and which provides a ratio (percentage of total peak summing to %) for each allele. allelic expression imbalance was considered to be present if the cdna ratio fell outside the % ci determined from the dna ratio in heterozygous individu- als, in which the expected ratio is ( % for each allele). statistical methods. the primary analyses compared genotypes using the armitage trend test, which tests the null hypothesis: p � / (p ) � q � / (q ), where p and q are heterozygous case and control subjects, respec- tively, and p and q are rare homozygous case and control subjects, respectively. the test, which is distributed as � with degree of freedom, retains the power of an allelic association but remains valid in the presence of deviations from hardy-weinberg equilibrium. we considered an uncorrected two-sided p � . to be nominally significant. secondary, exploratory analyses of genotypic association were conducted under additive, dominant, and recessive models using � or fisher’s exact tests. tests of association and hardy-weinberg equilibrium were conducted using the definetti program ( ). pairwise linkage disequilibrium measures were estimated from the genotype data using the em (expectation maximization) algorithm imple- mented in haploview version . or . ( ). haplotype case-control associ- ation was tested within each haplotype block using haploview version . , which tests the frequency of each haplotype in case versus control subjects; corrected p values are subsequently estimated by permutation tests ( , replicates). haplotype blocks were established using haploview version . with the solid spline of linkage disequilibrium definition. si and acute insulin response to glucose (airg) were calculated from table summary of primary study populations population study description sex (m/f) bmi (kg/m ) age (years) caucasian case/control case/control control / , case / . ( . – . ) . � . african-american case/control case/control control / , case / . ( . – . ) . � . utah caucasian metabolic family-based metabolic study / . ( . – . ) . � . arkansas caucasian metabolic population-based metabolic study / . ( . – . ) . � . data are arithmetic means for normal variables and geometric means for skewed variables (bmi), means ( % ci) transferred to the linear scale from the ln-transformation, or means � sd. w.s. chu and associates diabetes, vol. , march fsigt data using the minmod ( ) or minmod millennium programs ( ). genotype effects on insulin secretion and si were tested using general linear models with sex and genotype as factors and bmi and age as covariates. for the utah study, glucose tolerance status and family membership were included as additional factors, whereas for unrelated individuals from arkan- sas, protocol type (tolbutamide or insulin) was included as an additional factor. for analysis of consortium data, between-group differences in allele frequency were evaluated on a population-specific basis using standard contingency table methods and exact p values calculated using stata se version (stata, college station, tx). single-point data from the case-control samples were combined using the mantel-haenszel fixed-effects method (stata se version ), and combined odds ratios (ors) were generated under dominant models for each allele. studies in the amish population accounted for family structure. results from snps spanning . kb (	 , to , bp relative to the atg start) (fig. ), we selected snps for full evaluation in the utah northern european caucasian case-control sample (denoted utah caucasian in the ta- bles); nonsynonymous variants were forced into inclusion. the typed snps fell into five linkage disequilibrium fig. . atf region map in caucasians. a total of exons are shown as darkened blocks. bars above the figure denote �log of p values for tests of allelic association in the utah caucasian sample without correction for multiple hypothesis testing. arrows denote snps typed in the full consortium sample. fig. . linkage disequilibrium structure of typed markers in the utah caucasian sample. image is taken from haploview . for the snps typed by the international type diabetes q consortium and/or in house. blocks are defined using the solid spline of linkage disequilibrium. shades of gray refer to the strength of pairwise linkage disequilibrium based on r , and numbers refer to pairwise d� values. black squares indicate r , white squares are very low values of r . squares without numbers indicate d� values of . , and all lower values are indicated. atf polymorphisms in type diabetes diabetes, vol. , march blocks of sizes , , , , and kb (fig. and supplemental figs. s and s). we found a nominal association with of snps (uncorrected p values from allelic association of . – . ) (table and fig. ), including a nonsynony- mous snp in exon (rs , m v, p � . ) and a noncoding snp in the � flanking region (rs , p � . ). two other nonsynonymous variants in exon , rs (p a) and rs (s p), were more common in control subjects, but differences were not significant (p � . ). eight snps were modestly out of hardy-weinberg equilibrium, even after assay redesign, in either case or control subjects (p � . for rs to p � . in rs ) (supplemental table s), including several snps with a nominal association with diabetes (rs , p � . in case subjects for deviation from expected hardy-weinberg proportions; rs , p � . for deviation from expected proportions in control subjects). for both rs and rs , the minor allele was overrepresented in case compared with control subjects (table ). other nominally associated variants included intronic snps rs (p � . ) and rs (p � . ) and � flanking snps rs (p � . ) and rs (p � . ) (table ). linkage disequilibrium was similar in case and control subjects, and no haplotype was more strongly associated than individual snps (supplemental table s). we sought to replicate the modest associations in seven additional samples, including caucasian ( , case and , control subjects, excluding the utah sample), chi- nese ( case and control subjects), african-ameri- can ( and , respectively), and pima indian ( and ) populations using consortium snps. no snp was associated in individual samples (data not shown), by meta-analysis of all samples or meta-analysis of caucasian samples without the utah contribution (supplemental ta- ble s), nor did we find a trend to an association for the three nonsynonymous snps, rs , or other snps associated in utah caucasians in african-american diabetic and control subjects (supplemental table s). similarly, the three nonsynonymous snps showed no trend to association with type diabetes in the amish family diabetes study (data not shown). defects in endoplasmic reticulum stress response could contribute to impaired insulin secretion and/or insulin action. we tested the nonsynonymous variants m v (rs , exon ), p a (rs , exon ), and s p (rs , exon ) and the most strongly associated � flanking snp (rs ) in two caucasian populations: a family-based study of subjects from utah and a population-based study of individuals from arkansas. no snp altered si or insulin secretion, measured as either airg, the ability of the �-cell to compensate for insulin resistance (disposition index: si � airg), or airmax (the maximum insulin secretory capacity) (supplemental ta- bles s and s). we tested for cis-acting variants by testing the allelic expression balance in cdna prepared from epstein-barr virus–transformed lymphocytes from caucasian individu- als who were heterozygous for the � untranslated snps rs and rs . the allelic ratio in cdna for snp rs fell outside the % cis established from dna from heterozygotes (ratio . , % ci . – . ) in of heterozygous individuals (� expected), with a range of . – . (p � 	 ) (fig. ), and in of heterozygotes for rs ( % ci in genomic dna from individuals of . – . , p � . ) (fig. ). discussion as a key transcriptional activator at the head of the unfolded protein response, atf is a strong positional and functional candidate for type diabetes, particularly given recent animal data supporting a role for endoplasmic reticulum stress response in peripheral and hepatic insulin resistance ( ) and insulin secretion ( ). the three com- mon nonsynonymous snps m v (rs ), p a (rs ), and s p (rs ) are potentially func- tional. both exon variants are nonconservative substitu- tions, and the three nonsynonymous snps are located in the nh -terminal cytoplasmic-localized transcription acti- vation domain of atf , which is essential for the activa- tion of unfolded protein response. a recent study in pima indians reported borderline associations of p a (rs ) and s p (rs ) with type diabetes (p � . ) and reduced insulin response to oral glucose ( ). we did not find an association with type diabetes for snps rs (p a) and rs (s p), which are much less common in caucasians, but we did find a nominal association with type diabetes for m v (rs ). however, no coding variant remained signif- icant when p values were estimated by permutation test- ing. furthermore, no snp altered glucose homeostasis traits in our population or was associated with type diabetes in our african-american population. snp rs (p a) was directly typed by the international type diabetes q consortium, and other consortium snps were excellent proxies for m v (rs ) and s p ( ); hence, an association with type diabe- tes was excluded in � , caucasian case and , caucasian control subjects. if atf coding variants have an effect on diabetes susceptibility, the effect size is likely small. the power of this study is limited for markers of effect comparable to the potassium channel kcnj e k ( ) or ppar� p a variants ( ). in the utah sample, over the range of minor allele frequencies observed in this study, we have � % power at p � . to detect a difference in allele frequency of – % between caucasian case and control subjects, corresponding to an or of . – . , or approximately the range observed for the well-replicated tcf l diabetes gene ( ). considering the consortium caucasian sample of , case and , control subjects, we had nearly % power to replicate the utah associa- tions at snps rs , rs , rs , and rs . power to detect changes in si ranged from % to detect a % drop in log-transformed si for common alleles (rs ) to % to detect a % fall in si for an uncommon snp, such as rs . hence, small changes would not have been detected without a considerably larger sample. clearly, our power to detect ethnic-specific variants in chinese or african-american populations is low, in the former case because of the small sample, and in the latter because of the low likelihood that we captured all common variants with the selected snps. based on hapmap phase snps, we selected our markers for the detailed utah caucasian study to capture % of the variation; hence, we are unlikely to have missed common variants. based on the markers typed in the utah caucasian set, the markers typed by the interna- tional type diabetes q consortium captured all but five snps (rs , rs , rs , rs , and rs ) at r � . . snps rs and rs showed nominal associations in the utah population and w.s. chu and associates diabetes, vol. , march table summary of snps typed in caucasian individual samples for atf region location name gene location allele (� strand) maf or ( % ci) pcase control 	 rs �flanking g/a . . . ( . – . ) . 	 rs �flanking c/a . . . ( . – . ) . 	 rs �flanking g/c . . . ( . – . ) . 	 rs �flanking g/a . . . ( . – . ) . rs * intron t/c . . . ( . – . ) . rs intron a/c . . . ( . – . ) . rs * intron c/t . . . ( . – . ) . rs intron t/c . . . ( . – . ) . rs * intron t/c . . . ( . – . ) . rs * exon (l l) c/t . . . ( . – . ) . rs exon (m v) a/g . . . ( . – . ) . rs * exon (p p) t/c . . . ( . – . ) . rs * intron a/t . . . ( . – . ) . rs * intron a/g . . . ( . – . ) . rs * exon (p a) c/g . . . ( . – . ) . rs exon (s p) c/t . . . ( . – . ) . rs intron g/a . . . ( . – . ) . rs * intron t/c . . . ( . – . ) . rs * intron g/a . . . ( . – . ) . rs * intron a/g . . . ( . – . ) . rs * intron g/a . . . ( . – . ) . rs intron a/g . . . ( . – . ) . rs intron t/a . . . ( . – . ) . rs * intron g/a . . . ( . – . ) . rs * intron g/c . . . ( . – . ) . rs * intron a/g . . . ( . – ) . rs * intron c/t . . . ( . – . ) . rs * intron g/t . . . ( . – . ) . rs * intron c/t . . . ( . – . ) . rs * intron t/c . . . ( . – . ) . rs * intron a/g . . . ( . – . ) . rs * intron a/t . . . ( . – . ) . rs * intron a/g . . . ( . – . ) . rs intron g/a . . . ( . – . ) . rs * intron c/t . . . ( . – . ) . rs * intron c/t . . . ( . – . ) . rs * intron t/a . . . ( . – . ) . rs * intron c/a . . . ( . – . ) . rs * intron a/g . . . ( . – . ) . rs intron c/t . . . ( . – . ) . rs * intron c/t . . . ( . – . ) . rs * intron t/c . . . ( . – . ) . rs * intron g/a . . . ( . – . ) . rs * intron g/a . . . ( . – . ) . rs * intron g/a . . . ( . – . ) . rs * intron a/c . . . ( . – . ) . rs * intron c/t . . . ( . – . ) . rs * intron t/g . . . ( . – . ) . rs * intron a/g . . . ( . – . ) . rs * intron g/a . . . ( . – . ) . rs intron a/g . . . ( . – . ) . rs * intron a/g . . . ( . – . ) . rs * intron a/g . . . ( . – . ) . rs * intron c/a . . . ( . – . ) . rs * intron g/a . . . ( . – . ) . rs �utr a/g . . . ( . – . ) . rs �utr g/a . . . ( . – . ) . rs �flanking t/c . . . ( . – . ) . rs �flanking g/a . . . ( . – . ) . rs * �flanking c/g . . . ( . – . ) . rs �flanking a/g . . . ( . – . ) . rs * �flanking g/a . . . ( . – . ) . rs �flanking c/a . . . ( . – . ) . rs * �flanking c/t . . . ( . – . ) . this table summarizes the association data in utah and arkansas caucasians. snps that were in complete ld (r � ) were typed only in control samples and are not included in this table (see supplemental table s). likewise, snps with low minor allele frequencies (mafs) (� %) were generally not tested unless nonsynonymous. raw counts are available in supplemental table s. alleles are listed as the major/minor allele. ors were taken from the allelic association test considering the minor allele as the risk allele. p values are shown for genotypic association by armitage’s trend test. *snps also genotyped by the international type diabetes q consortium in eight populations but shown here only for the utah sample. locations are given with respect to the a of atg start as and first base upstream as 	 , based on ncbi build (university of california santa cruz, may ). atf polymorphisms in type diabetes diabetes, vol. , march thus might become significant in a larger population. we have not resequenced the atf gene, given the low likelihood that additional common variants exist that were not captured by the snps that we chose to type. however, we have likely not captured rare variants in the atf gene, if these exist. theoretically, rare exon variants may be present and may contribute to type diabetes. several facts support a role for atf variation in disease. several snps, particularly those in strong linkage disequilibrium with m v (rs ), had genotypes that departed from hardy-weinberg equilibrium expectations, despite assay redesign, and may suggest genetic selection. snp rs , which showed the strongest association, is predicted to alter the binding of transcription factor cdxa, and the minor allele of snp rs in the � untranslated region, which showed allelic imbalance, is predicted to create binding sites for transcription factors pbx- and c/ebp
. most significantly, we found evidence for cis-acting sequence variants in the gene based on the highly significant allelic expression imbalance. because the expression ratios were both above and below the % cis in different individuals, the causative cis-acting variant is likely in incomplete linkage disequilibrium with rs and rs . additional studies, including allelic imbalance in other tissues, are needed to identify the likely causative variant and to determine whether the expression levels alter diabetes risk. the true functional variant, which may be considerably distant, may not have been tested for association with type diabetes in this study. in conclusion, we have thoroughly evaluated a strong chromosome candidate gene. the strongest association in utah caucasians is in the � flanking region (p � . based on , permutations), but it could not be confirmed in other populations. both the � untranslated snp and the most strongly associated � flanking region snp alter transcription factor binding, and thus may be functional, but an undiscovered cis-acting regulatory vari- ant appears more likely to explain the allelic expression imbalance. hence, the possibility remains that a regulatory variant that alters atf message levels also contributes to defective insulin action or insulin secretion. acknowledgments this work was supported by grants from national insti- tutes of health (nih)/national institutes of diabetes and digestive and kidney diseases (niddk) (dk ) and by the research service of the u.s. department of veterans affairs. subject ascertainment was supported in part by grants from the american diabetes association and nih/ niddk (dk ). subject ascertainment and metabolic studies were supported by general clinical research center grant m rr from the national center for research resources (nih) to the university of arkansas for medical sciences. studies of the international type diabetes q consortium were supported primarily as a supplement to niddk award u -dk and by nih/ niddk grant dk (to m.i.m.). e.z. is a wellcome trust career development fellow. amish studies were supported by grant r dk from the nih/niddk (to a.r.s.). fig. . allelic expression imbalance in atf gene. scatter plot showing allele-specific expression in dna (�) and cdna (‚) for cdna samples at rs and samples for rs . both cdna ratios are significantly different from the genomic dna ratios using the same assay. w.s. chu and associates diabetes, vol. , march references . kaufman rj: orchestrating the unfolded protein response in health and disease. j clin invest : – , . rutkowski dt, kaufman rj: a trip to the er: coping with stress. trends cell biol : – , . schroder m, kaufman rj: the mammalian unfolded protein response. annu rev biochem : – , . ron d: proteotoxicity in the endoplasmic reticulum: lessons from the akita diabetic mouse. j clin invest : – , . ozcan u, cao q, yilmaz e, lee ah, iwakoshi nn, ozdelen e, tuncman g, gorgun c, glimcher lh, 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reynisdottir i, benediktsson r, manolescu a, sainz j, helgason a, stefansson h, emilsson v, helgadottir a, styrkars- dottir u, magnusson kp, walters gb, palsdottir e, jonsdottir t, gud- mundsdottir t, gylfason a, saemundsdottir j, wilensky rl, reilly mp, rader dj, bagger y, christiansen c, gudnason v, sigurdsson g, thor- steinsdottir u, gulcher jr, kong a, stefansson k: variant of transcription factor -like (tcf l ) gene confers risk of type diabetes. nat genet : – , atf polymorphisms in type diabetes diabetes, vol. , march mapping a gene involved in regulating cholesterol absorption the journal of clinical investigation volume , number , september , – http://www.jci.org mapping a gene involved in regulating dietary cholesterol absorption the sitosterolemia locus is found at chromosome p shailendra b. patel,* gerald salen, ‡ hideki hidaka, § peter o. kwiterovich, jr., i anton f.h. stalenhoef, ¶ tatu a. miettinen,** scott m. grundy,* mi-hye lee,* jeffrey s. rubenstein, ‡‡ mihael h. polymeropoulos, ‡‡ and michael j. brownstein §§ *center for human nutrition, university of texas southwestern medical center, dallas, texas - ; ‡ department of medicine, umd-new jersey medical school, newark, new jersey , and gasteroenterology research laboratory, new jersey veterans health care system, east orange, new jersey ; § third department of medicine, shiga university of medical science, seta-tsukinowa-cho, otsu, shiga - , japan; i department of pediatrics, the johns hopkins hospital, the children’s medical and surgical center, baltimore, maryland - ; ¶ department of medicine, division of general internal medicine, university hospital nijmegen, hb nijmegen, nijmegen, the netherlands; **department of internal medicine, helsinki university central hospital, helsinki, finland; ‡‡ gene mapping unit, laboratory of genetic disease research, national human genome research institute, and §§ section on genetics: people investigating genes, national institute of mental health/national human genome research institute, bethesda, maryland abstract the molecular mechanisms regulating the amount of di- etary cholesterol retained in the body as well as the body’s ability to selectively exclude other dietary sterols are poorly understood. studies of the rare autosomal recessively inher- ited disease sitosterolemia (omim ) may shed some light on these processes. patients suffering from this disease appear to hyperabsorb both cholesterol and plant sterols from the intestine. additionally, there is failure of the liver’s ability to preferentially and rapidly excrete these non-cho- lesterol sterols into bile. consequently, people who suffer from this disease have very elevated plasma plant sterol lev- els and develop tendon and tuberous xanthomas, acceler- ated atherosclerosis, and premature coronary artery dis- ease. identification of this gene defect may therefore throw light on regulation of net dietary cholesterol absorption and lead to an advancement in the management of this impor- tant cardiovascular risk factor. by studying well-charac- terized families with this disorder, we have localized the ge- netic defect to chromosome p , between microsatellite markers d s and d s (maximum lodscore . , u . ). ( j. clin. invest. . : – .) key words: genetics • sitosterolemia • linkage analyses • chromosomal localization introduction sitosterolemia is a lipid disorder first described by bhatta- charyya and connor in ( ). the disease is characterized by the presence of tendon and tuberous xanthomas, premature atherosclerotic disease, absence of a family history of prema- ture coronary artery disease, and normal to occasionally ele- vated plasma cholesterol levels ( ). premature fatal myocar- dial infarction was the presenting feature in the next three families reported with this condition ( ). sitosterolemia shares several clinical characteristics with the well-characterized ho- mozygous familial hypercholesterolemia (fh), including ex- pression of tendon xanthomas in the first ten years of life, and development of aortic stenosis and premature atherosclerosis in the first several decades. however, homozygotes for sito- sterolemia usually have normal to moderately elevated total sterol levels, in contrast to the profound hypercholesterolemia in fh homozygotes. sitosterolemia is further distinguished from fh by its autosomal recessive mode of inheritance and the diagnostic elevation of plasma phytosterols. this condition may be underreported, as the detection of plasma phytosterols requires the use of hplc or capillary glc ( ). sitosterol is clearly the major plant sterol species, hence the name sitoster- olemia, although many other plant sterols are also significantly elevated in the plasma. the term phytosterolemia may there- fore be preferable. segregation analyses of a large amish ped- igree showed that the trait showed an autosomal recessive pat- tern of inheritance ( ). the true prevalence of this disorder is not known, but . individuals with this condition have been reported worldwide ( ). under normal circumstances, our diets contain almost equal amounts of cholesterol and plant sterols. however, only – % of total dietary cholesterol and , % of total plant sterols are retained by the body ( ). the liver excretes most of the absorbed noncholesterol sterols rapidly into bile, almost unchanged, such that the net absorption of these sterols is al- most negligible ( ). in sitosterolemia, affected individuals show an increase in the absorption of total dietary sterols, with failure to discriminate between different sterol species and a failure to excrete absorbed noncholesterol species rapidly into bile ( – ). this leads to greatly expanded body pool size of both cholesterol and sitosterol. clinical studies of affected in- dividuals show that, in addition to the defects of absorption and excretion of sterols, whole body cholesterol biosynthesis is depressed ( , , ). treatments that normally result in an in- crease in cholesterol biosynthesis in normal individuals fail to do so in affected individuals ( ). additionally, analyses of liver and intestinal biopsies from affected individuals show that activities of many of the enzymes involved in cholesterol biosynthesis pathway are depressed ( , – ), suggesting that intracellular sterol pools are being sensed as “replete.” how- ever, the expression of the ldl receptor in liver and intestinal biopsies was found to be elevated, suggesting discordant regu- address correspondence to shailendra b. patel, y . , center for human nutrition, ut southwestern medical center, harry hines blvd., dallas, tx - . phone: - - ; fax: - - ; e-mail: spatel@crcdec.swmed.edu received for publication may and accepted in revised form july . . abbreviations used in this paper: fh, familial hypercholester- olemia; srebp, sterol regulatory element binding protein. patel et al. lation ( ). the structural genes for ldl receptor and some of the enzymes along the cholesterol biosynthetic pathway have been excluded as sites of the defect ( ). similarly, transcrip- tional factors that are thought to play a major role in regulat- ing many of these genes, sterol regulatory element binding proteins (srebps), have also been excluded ( ). to identify the genetic defect in sitosterolemia, a genome-wide scan and linkage analyses of ten well-characterized pedigrees was un- dertaken. methods pedigrees. all probands were identified on the basis of clinical find- ings of tendon and tuberous xanthomas, the suspicion of sitoster- olemia as a diagnosis, confirmed by the diagnostic elevation of the plasma sitosterol levels determined by either hplc or capillary gas- liquid chromatography as previously described. pedigrees , , , , , , and have been previously described ( , , – ). informed consent was obtained from all participants. al- though individuals . and . are dead, their dna was avail- able from fibroblast cultures established before death. individuals . , . , . , . , . , . , . , . , and . were not available for genotyping. all of these, except . , are unaffected siblings, as determined by plasma sitosterol levels. genotyping. genomic dna was extracted from whole-blood fi- broblast cultures or transformed lymphoblastoid cell lines, as previ- ously described ( ). genome-wide linkage analyses were performed using primer sets from the weber version screening set (research genetics, birmingham, al). reverse primers from each set were end-labeled with g - p-atp, using standard techniques. pcr amplifi- cation of the microsatellite repeats was performed as previously de- scribed ( ), and the products separated by denaturing urea-acryl- amide gels, dried and analyzed by phosphorimager. additional markers were analyzed using fam-labeled primers (research ge- netics) and the pcr products analyzed by gel separation and fluori- metric detection using a fluorimager (molecular dynamics, sunny- vale ca). allele sizing was determined using ceph family dna, - and - . linkage analyses. linkage analyses were performed using the computer package linkage ( , ). the preplink function was recompiled as a macro to run on a macintosh using a microsoft excel worksheet. this checks the pedigrees for misinheritances of the alleles and generates an appropriately formatted output file. two- point linkage was performed using mlink, running on a dec sta- tion / , ultrix v . operating system. autosomal recessive in- heritance and a disease prevalence of : were specified. allele frequencies were obtained from the ceph family database. multi- point analysis was performed using genehunter ( ). results we have assembled families with the diagnosis of sitoster- olemia, some of which have been reported previously (fig. ). there are two japanese families (pedigrees and ), one amish family (pedigree ), one asian indian family (pedi- gree ), one finnish family (pedigree ), and one dutch family (pedigree ); the remaining families are from the united states, and are of caucasian origins (pedigrees , , and ). dna from affected individuals, unaf- fected siblings, and parents was available for genotyping. the plasma sitosterol levels in affected individuals, their obli- gate heterozygous parents, unaffected siblings (from all except pedigree ), and unrelated normal controls are shown in fig. . sitosterol levels in pedigree have been reported previ- ously ( ). we have also included plasma sitosterol levels from other known sitosterolemia individuals on file in our data- bases. as can be seen, the diagnosis of affected status can be made definitively. based solely on the plasma sitosterol levels, the heterozygous and normal control states are indistinguish- able. total plasma sterol levels in affected individuals may only be marginally elevated relative to control populations and do not correlate with the presence of accelerated atherosclero- sis, suggesting that elevated cholesterol per se is not responsi- ble for the increased cardiovascular risk. a genome-wide scan was performed using a microsatellite screening set at an average interval of cm, using families , , , , , and . all of the autosomal chromo- somes were examined. a significant two-point lodscore ( . , u . ) was obtained with marker d s . for the remain- ing autosomes, the highest maximum lodscore obtained was . , u . , for one telomeric marker on chromosome (data not shown). mapping markers at an average density of -cm intervals, using all of the families, gave a maximum lod- score of . , u . , with the marker d s (table i). a multipoint analysis is shown in fig. . by comparing the shared haplotypes within sibling pairs and examining for informative recombination events, we located the disease locus between markers d s and d s (fig. ). this region spans al- most cm. family showed no informative recombination events within the area of interest. family had one affected child, and his other two siblings did not share any haplotypes with the affected individual. areas of haplotype sharing were made within pedigrees only and are shown by the closed areas. the unshared regions, by virtue of a recombination event on the maternal or the paternal chromosome, are shown in open figure . sitosterolemia pedigree trees. in the pedigrees assembled for this study, affected individuals are shown by filled symbols. only those individuals for whom both se- rum sitosterol values and dna were available were included in the analyses. a total of parents, affected individuals, and unaf- fected siblings were genotyped. mapping a gene involved in regulating cholesterol absorption areas. in families and , two recombination events were detected. these were inferred for family because the mother was not available for genotyping. for family , dis- tinct haplotypes could be constructed for both parents. for all of the pedigrees analyzed, haplotype analyses showed that all of the affected individuals share the region between d s and d s . markers d s and d s are very close to each other ( , . cm), and their order has not been deter- mined accurately. no evidence of genetic heterogeneity has been detected in our pedigrees. as sitosterolemia appears to be very rare and the disease locus in pedigrees drawn from dif- ferent racial backgrounds maps to the same region, the likeli- hood that mutations in different genes along a common meta- bolic pathway are responsible for causing the same or similar disease is reduced. discussion the disease locus for sitosterolemia maps to p , to an inter- val no larger than cm, spanned by the microsatellite mark- ers d s and d s . because the families used for these analyses are of different racial origins and no evidence of ge- netic heterogeneity was found, these results would suggest that there is only one locus that is responsible for causing this rare disease. although this region is too large for application of po- sitional cloning, a number of cdnas have been mapped to this region and could be considered as candidate genes. for exam- ple, the genes for -hydroxyanthranilate , -dioxygenase, di- oxin-inducible cytochrome p ( cyp b ), sodium/calcium exchanger precursor, lutropin-choriogonadotropic hormone receptor and fsh receptor, amino acid transporter protein, phosphatidylinositol-glycan biosynthesis class f protein, a - kd leucine-rich protein, genes homologous to the drosophila figure . plasma sitosterol levels in affected individuals, their obli- gate heterozygous parents, unaffected siblings, and normal controls. plasma sitosterol levels were determined by capillary glc or hplc for all of our pedigree volunteers shown above as well as for nor- mal individuals chosen at random. additionally, data on file on other families not included in this study are also shown. note the break in the y-axis. in general, most unaffected individuals had plasma sito- sterol levels , mg/dl. of the three parents and three siblings who had values higher than mg/dl, all of these were measured by hplc; these values may reflect the different technique used. none was higher than mg/dl. all of the affected individuals had plasma sitosterol values . mg/dl, and many of these values were obtained while the patients were in treatment. table i. two-point lodscores between microsatellite markers and sitosterolemia recombination fraction, u locus . . . . . z max u max d s - ∞ . . . . . . d s - ∞ . . . . . . d s . . . . . . . d s . . . . . . . d s - ∞ . . . . . . d s - ∞ . . . . . . d s - ∞ . . . . . . d s - ∞ . . . . . . two-point lodscores were calculated with an autosomal recessive pat- tern of inheritance, a phenocopy rate of , and a disease prevalence of : , . significant lodscores ( . . ) are in boldface. figure . multipoint and haplotype analyses of infor- mative markers to localize the sitosterolemia gene. mul- tipoint analysis between markers d s and d s was performed as described in methods. the multipoint analysis is shown graphically on the right and the haplotype analysis on the left. only those haplotypes that had an informative re- combination are shown. the allele assignments are as indi- cated. the maximal area of localization is indicated by the shaded rectangle. the upper border is marked by the marker d s , which is recombinant in pedigrees and . the lower border is defined by recombination events in pedigrees and for marker d s . for family , the mother was not available for genotyping and her haplotypes were therefore inferred. patel et al. melanogaster dosage compensation regulator, serum protein mse , tis b protein, and calmodulin all map to this re- gion. more than unidentified transcripts also have mapped to this region. of the known genes that map to this region, none seem likely candidates genes based on their known and/ or proposed functions, which are based on homology. normal individuals show almost no net dietary absorption of non-cholesterol sterols. this may result from a highly selec- tive intestinal absorptive process, allowing cholesterol, but not other sterols, to enter the body, or by a very rapid and prefer- ential clearance of these noncholesterol sterols by the liver into bile, giving the appearance of selective sterol absorption. equally feasible is that both of these mechanisms may be oper- ative. the identification of the gene in sitosterolemia is there- fore likely to be very informative of these processes. the other pathological features of sitosterolemia are in- creased foam cell formation, leading to widespread tendon and tuberous xanthoma formation with accelerated atherosclero- sis. although the mechanisms by which atherosclerosis is ac- celerated are not fully worked out, sitosterol and cholesterol per se are unlikely to be the key sterols that lead to the cascade of differential gene expression changes and foam cell forma- tion. sitosterol infusions in rats or man do not recapitulate the discordant gene regulation ( ), and the plasma levels of cho- lesterol in many affected individuals with accelerated athero- sclerosis are frequently not elevated ( , ). however, as many other sterol species such as shell-fish sterols ( ), are also ab- sorbed, and many of these also are partially metabolized by the body to oxy-sterols ( , ), any one of these compounds could be responsible for these changes ( ). it is known that cholesterol needs to be metabolized before it can be “sensed” by the cell. considerable advances have been made in identify- ing transcriptional factors that regulate intracellular choles- terol homeostasis, namely srebps, as well as the novel mechanisms by which srebps are activated. however, the in- tracellular sterol “sensor” has not been definitively identified. although a number of sterol species have been shown to act as potent repressors of this pathway, both the exact sterol species that is sensed and the intracellular compartment that this oc- curs in have not been elucidated. while we believe that the gene product defective in sitosterolemia is primarily involved in selective transport of cholesterol across the plasma mem- brane, the pattern of discordant gene regulation observed sug- gests that these two pathways are linked. the elucidation of the gene defective in sitosterolemia may therefore shed some light on these processes. acknowledgments we thank john poindexter for help with linkage analyses. we also thank all of our pedigree members for their continued cooperation. this work was supported by the medical research service, de- partment of veterans affairs, washington, dc, us public health service grant hl- (g. salen); by a grant-in-aid ( ) from the ministry of education, science, and culture, japan (h. hidaka); by an american heart association scientist development award n (s.b. patel); by the southwestern medical foundation, dallas, tx, and the moss heart foundation, dallas, tx. references . bhattacharyya, a.k., and w.e. connor. . beta-sitosterolemia and xanthomatosis. a newly described lipid storage disease in two sisters. j. clin. invest. : – . . salen, g., s. shefer, l. nguyen, g.c. ness, g.s. tint, and v. shore. . sitosterolemia. [review]. j. lipid res. : – . . bjorkhem, i., and k.m. boberg. . inborn errors in bile acid biosyn- thesis and storage of sterols other than cholesterol. in the metabolic basis of inherited disease. vol. . c.r. scriver, a.l. beaudet, w.s. sly, and d. valle, editors. mcgraw-hill, inc., new york. – . . beaty, t.h., p. kwiterovich, jr., m.j. khoury, s. white, p.s. bachorik, h.h. smith, b. teng, and a. sniderman. . genetic analysis of plasma sito- sterol, apoprotein b, and lipoproteins in a large amish pedigree with sitoster- olemia. am. j. hum. genet. : – . . salen, g., e. ahrens, jr., and s.m. grundy. . metabolism of beta- sitosterol in man. j. clin. invest. : – . . miettinen, t.a. . phytosterolaemia, xanthomatosis and premature atherosclerotic arterial disease: a case with high plant sterol absorption, im- paired sterol elimination and low cholesterol synthesis. eur. j. clin. invest. : – . . bhattacharyya, a.k., w.e. connor, d.s. lin, m.m. mcmurry, and r.s. shulman. . sluggish sitosterol turnover and hepatic failure to excrete sito- sterol into bile cause expansion of body pool of sitosterol in patients with sito- sterolemia and xanthomatosis. arterioscler. thromb. : – . . gregg, r.e., w.e. connor, d.s. lin, and h. brewer, jr. . abnormal metabolism of shellfish sterols in a patient with sitosterolemia and xanthomato- sis. j. clin. invest. : – . . salen, g., v. shore, g.s. tint, t. forte, s. shefer, i. horak, e. horak, b. dayal, l. nguyen, a.k. batta, et al. . increased sitosterol absorption, de- creased removal, and expanded body pools compensate for reduced cholesterol synthesis in sitosterolemia with xanthomatosis. j. lipid res. : – . . salen, g., g.s. tint, s. shefer, v. shore, and l. nguyen. . increased sitosterol absorption is offset by rapid elimination to prevent accumulation in heterozygotes with sitosterolemia. arterioscler. thromb. : – . . nguyen, l.b., s. shefer, g. salen, g.c. ness, g.s. tint, f.g. zaki, and i. rani. . a molecular defect in hepatic cholesterol biosynthesis in sitoster- olemia with xanthomatosis. j. clin. invest. : – . . nguyen, l.b., g. salen, s. shefer, g.s. tint, v. shore, and g.c. ness. . decreased cholesterol biosynthesis in sitosterolemia with xanthomatosis: diminished mononuclear leukocyte -hydroxy- -methylglutaryl coenzyme a reductase activity and enzyme protein associated with increased low-density li- poprotein receptor function. metab. clin. exp. : – . . nguyen, l.b., g. salen, s. shefer, j. bullock, t. chen, g.s. tint, i.r. chowdhary, and s. lerner. . deficient ileal -hydroxy- -methylglutaryl co- enzyme a reductase activity in sitosterolemia: sitosterol is not a feedback inhib- itor of intestinal cholesterol biosynthesis. metab. clin. exp. : – . . patel, s.b., a. honda, and g. salen. . sitosterolemia: exclusion of genes involved in reduced cholesterol biosynthesis. j. lipid res. : – . . salen, g., p. kwiterovich, jr., s. shefer, g.s. tint, i. horak, v. shore, b. dayal, and e. horak. . increased plasma cholestanol and alpha-saturated plant sterol derivatives in subjects with sitosterolemia and xanthomatosis. j. lipid res. : – . . berger, g.m., w.m. deppe, a.d. marais, and m. biggs. . phy- tosterolaemia in three unrelated south african families. postgrad. med. j. : – . . berger, g.m., r.j. pegoraro, s.b. patel, p. naidu, l. rom, h. hidaka, a.d. marais, a. jadhav, r.p. naoumova, and g.r. thompson. . -hydroxy -methylglutaryl coenzyme a reductase is not the site of the primary defect in phytosterolemia. j. lipid res. : – . . hidaka, h., t. nakamura, t. aoki, h. kojima, y. nakajima, k. kosugi, i. hatanaka, m. harada, m. kobayashi, a. tamura, et al. . increased plasma plant sterol levels in heterozygotes with sitosterolemia and xanthomato- sis. j. lipid res. : – . . hidaka, h., h. sugiura, t. nakamura, h. kojima, m. fujita, n. sugie, h. okabe, y. nishio, h. maegawa, a. kashiwagi, et al. . b -sitosterolemia with generalized eruptive xanthomatosis. endocr. j. : – . . lathrop, g.m., j.m. lalouel, c. julier, and j. ott. . strategies for multilocus linkage analysis in humans. proc. natl. acad. sci. usa. : – . . terwillinger, j.d., and j. ott. . handbook of human genetic link- age. johns hopkins university press, baltimore. . kruglyak, l., m.j. daly, m.p. reeve-daly, and e.s. lander. . para- metric and nonparametric linkage analysis: a unified multipoint approach. am. j. hum. genet. : – . . kwiterovich, p., jr., p.s. bachorik, h.h. smith, v.a. mckusick, w.e. connor, b. teng, and a.d. sniderman. . hyperapobetalipoproteinaemia in two families with xanthomas and phytosterolaemia. lancet. : – . . shefer, s., g. salen, j. bullock, l.b. nguyen, g.c. ness, z. vhao, p.f. belamarich, i. chowdhary, s. lerner, a.k. batta, et al. . the effect of in- creased hepatic sitosterol on the regulation of -hydroxy- -methylglutaryl- coenzyme a reductase and cholesterol alpha-hydroxylase in the rat and sito- sterolemic homozygotes. hepatology. : – . . smith, l.l., and b.h. johnson. . biological activities of oxysterols. free radical biol. med. : – . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ pq p proc. natl. acad. sci. usa vol. , pp. – , december genetics a genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the old order amish edward i. ginns*†, pamela st. jean‡§, robert a. philibert*§, marzena galdzicka*, patricia damschroder-williams*, bonnie thiel‡, robert t. long*, loring j. ingraham*, harnisha dalwaldi*, melissa a. murray*, melissa ehlert*, sharon paul*, brian g. remortel*, ashima p. patel*, maria c. h. anderson*, car y shaio*, elaine lau*, inna dymarskaia*, brian m. martin*, barbara stubblefield*, kathleen m. falls¶, john p. carulli¶, tim p. keith¶, cathy s. j. fanni, lucy g. lacy**, cleona r. allen**, abram m. hostetter**, robert c. elston‡, nicholas j. schork‡, janice a. egeland**, and steven m. paul*†† *clinical neuroscience branch, intramural research program, national institute of mental health, bethesda, md ; ‡department of epidemiology and biostatistics, case western reserve university, cleveland, oh ; ¶genome therapeutics corporation, waltham, ma ; idepartment of epidemiology and social medicine, albert einstein college of medicine, new york, ny ; **department of psychiatry, university of miami school of medicine, miami, fl ; and ††departments of psychiatry, pharmacology, and toxicology, indiana university school of medicine and lilly research laboratories, eli lilly and co., indianapolis, in communicated by seymour s. kety, national institute of mental health, bethesda, md, october , (received for review may , ) a bstr act bipolar affective disorder (bpad; manic- depressive illness) is characterized by episodes of mania andyor hypomania interspersed with periods of depression. compelling evidence supports a significant genetic component in the sus- ceptibility to develop bpad. to date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing bpad. to determine whether there could be protective alleles that prevent or reduce the risk of developing bpad, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration old order amish pedigrees exhibiting an extremely high incidence of bpad. we have found strong evidence for a locus on chromosome p at d s (maximum genehunter-plus nonparametric linkage score . , p . ; sibpal pempirical value < ) and suggestive evidence for a locus on chromosome q at d s (maximum genehunter-plus nonparametric linkage score . , p . ; sibpal pempirical value < ) that are linked to mental health wellness. these findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of bpad and perhaps other related affective disorders. bipolar affective disorder (bpad) afflicts approximately % of the population and is associated with a high risk of suicide ( ). twin, family, and adoption studies have provided strong evidence for an important genetic component in the susceptibility to develop bpad ( ), but unlike for other common medical ill- nesses, robust biological markers have not been identified for bpad, and genetic linkage studies have had to rely on categorical diagnoses. genetic heterogeneity, phenocopies, genotyping er- rors, and the complexities of performing and interpreting statis- tical analyses may contribute to some of the inconsistences observed in these linkage studies ( – ). however, because the inheritance of bpad is probably multifactorial, the possible involvement of multiple genetic components of small effect andyor the occurrence of major allelic effects only in epistasis must be considered. we previously have suggested ( ), that in addition to susceptibility alleles, there could be alleles that reduce the risk of developing bpad in a manner similar to that reported for other complex genetic disorders. if model-based linkage analyses are used, a false-negative linkage finding could result when individuals inherit disease susceptibility alleles but do not manifest the phenotype because of the presence of protective alleles. the inclusion of individuals who inherit susceptibility alleles but do not manifest disease because of protective alleles, or of individuals who inherit protective alleles but nevertheless manifest the disease, also will reduce the power of model-free (allele-sharing) analyses. thus, regardless of whether model- based or model-free analyses are used, wellness or protective alleles could have a significant impact on linkage analyses. ascertainment of psychiatric disorders and health among several large multigenerational old order amish pedigrees cov- ers a period of more than years. throughout this longitudinal study, procedures for assessing and diagnosing subjects have remained constant and have included a thorough evaluation of all bipolar i (bpi) probands and their relatives ( – ). morbid risk analyses have demonstrated a high prevalence of affective disor- der among first-degree relatives of bipolar probands in these families ( ). because of the long-term, longitudinal nature of the study, the unaffected, mentally healthy individuals in these fam- ilies also were followed, many for a period of years past the age of risk for bpad. consequently, rather than limit our genome- wide search to identifying susceptibility loci for the disease phenotype (bpad), we tested the hypothesis that protective alleles may contribute to the absence of psychiatric illness (i.e., mental health wellness) in unaffected family members in these high-risk pedigrees. because the mode of inheritance of any gene(s) modifying the relative risk for affective disorder is un- known ( ) we relied exclusively on model-free linkage analyses. we now report strong evidence for linkage of dna markers on chromosome p to mental health wellness in relatives at high risk for, but who did not develop, major affective disorder in several large multigenerational old order amish pedigrees with an extremely high incidence of bpad. the publication costs of this article were defrayed in part by page charge payment. this article must therefore be hereby marked ‘‘advertisement’’ in accordance with u.s.c. § solely to indicate this fact. © by the national academy of sciences - y y - $ . y pnas is available online at www.pnas.org. abbreviations: bpad, bipolar affective disorder; npl, nonparametric linkage; bpi, bipolar i; gh-plus, genehunter-plus; ibd, identical by descent. †to whom reprint requests should be addressed at: clinical neuro- science branch, national institute of mental health, building , room b ee , convent drive, msc , bethesda, md - . e-mail: ginnse@irp.nimh.nih.gov. §p.st.j. and r.a.p. contributed equally to this work. d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , materials and methods diagnostic assessment. our genetic-epidemiologic study of bpad among the old order amish in southeastern pennsylvania has been described in detail ( ), including the methods for ascertainment and diagnostic documentation with informed con- sent ( , ). diagnoses were made, by using strict research diagnostic criteria ( ), by a five-member psychiatric review board whose members were blind to pedigree membership, diagnostic opinions, treatment data from abstracted medical records, and genetic marker status. twenty-five nuclear families were devel- oped based on one or more confirmed cases of bpi, which formed the structure of pedigrees , , and . our present report uses all of these earlier subjects plus additional expansions, especially in pedigree , so that our overall study now contains samples, including those from bpi individuals. the unaffected individuals (mentally well or healthy) are those for whom all sads-l (schedule for affective disorders and schizo- phrenia-lifetime version) interview responses were negative, and no contradictory reports were given by family informants. any individuals for whom some symptomatology was identified, even if it did not meet criteria for a formal diagnosis, were labeled as unknowns in our linkage analyses. the method used for this longitudinal study is ethnographic and culturally appropriate to the field setting. several members of each nuclear family with a bpi proband (bpi nuclear family) are seen annually. those diagnosed with bpi or other major affective disorder undergo a yearly course-of-illness update. parents of each bpi patient are visited regularly and have proven to be accurate informants. at least one unaffected sibling (control sample) of the married bpi patients has been interviewed yearly since in connection with a prospective study of children at risk for bpad. hence, at least three members and occasionally all members of each bpi nuclear family have been evaluated yearly. individuals are interviewed anew with the complete sads-l schedule whenever any abnormal mental or emotional symptoms are identified by the follow-up mechanisms. the long-term, systematic follow-up of the families in our study has demonstrated that onset of illness in the old order amish usually is reported by multiple informants. patient samples. blood samples were generally collected after each first-degree relative (including parents, siblings, and children older than age ) of the bpi probands had been interviewed with the complete sads-l schedule. samples were obtained with written informed consent and coded to maintain confidentiality. the phlebotomist was kept blind to pedigree relationships and diagnostic status. lymphoblastoid cell lines, on an average of eight members of each nuclear family, were established at the coriell institute for medical research, camden, n.j. andyor the clinical neuroscience branch, intramural research program, national institute of mental health, bethesda, md. the nigms human genetic mutant cell repository catalogue ( ) contains updated pedigree and diagnostic information for several of the amish pedigrees used in our study. genotyping. genomic dna was obtained from peripheral blood samples andyor immortalized lymphoblastoid cell lines as described ( ). the order of typed markers on our mapping panels was obtained from the genetic location database ( ). dna panels for pcr were in a -microtiter plate format, and aliquoted by using a biomek robot (beckman instruments). the dna samples were pcr-amplified separately with each of the dna markers and then the pcr products were multiplexed, six markers per lane, for electrophoresis using genescan software on abi instruments (applied biosystems division, perkin– elmer). the genotypes subsequently were analyzed with geno- typer. dna from several individuals was represented multiple times in the genotyping panels as controls to evaluate the consistency of genotypes across different gels. genetic analysis software ( ) was used to identify problem- atic marker data, and a utility written in spss (spss, chicago) generated a list of samples that needed to be rerun because of inheritance discrepancies or unreadable signals. we maximize the useful information by repeating the genotypingyanalysis cycles until all possible dna marker genotypes are obtained. once genotyping for a marker was finished, the data were reanalyzed with g.a.s., and observed allelic mutations and other noninher- itances were zeroed out in the data file and noted. histograms indicating the marker allele size bins were generated. fastlink ( ) was used to reanalyze the data before further statistical analyses. linkage analyses. model-free linkage analyses were con- ducted by using the two-point affected sib pair analysis program s.a.g.e. sibpal ( ) and the multipoint analysis program gene- hunter-plus (gh-plus) ( ). because there were a few sib- ships with incomplete marker information, marker allele fre- quencies were estimated from the entire old order amish family data set by using a maximum likelihood method implemented in the program mendelyuserm ( , ). sibpal was used to identify markers showing an excess of alleles shared identical by descent (ibd) among unaffected, mentally healthy sib pairs. under the null hypothesis of no linkage between a trait and marker, sib pairs would be expected to share on the average % of alleles ibd, but when a trait and marker are linked, ibd sharing will be increased in both affected and unaffected sibpairs. because sibpal assumes marker allele frequencies appropriate for random samples, it underestimates the proportion of alleles shared ibd by concordant sib pairs when there is linkage. multipoint analyses by using the model-free linkage program gh-plus produced nonparametric linkage (npl) scores along points at the chromosomal region of interest. two scoring func- tions are available in gh-plus: ibd sharing can be assessed among concordant relative pairs (nplpairs) or it may be assessed among larger groups of concordant relatives (nplall). our anal- yses were conducted by using the nplall statistics as kruglyak and colleagues ( ) have demonstrated that the nplall statistic results in a more powerful test than the nplpairs statistic. results first, we analyzed our genome-wide scan dataset looking for evidence of chromosomal loci linked to mental health wellness (the absence of any psychiatric illness). in these analyses, only mental health wellness, in individuals who were over years of age and had a first-degree bpi sibling in their family (pedigrees , , , and ), was the linkage phenotype of interest (concordantly unaffected pairs) by using sibpal. of more than dna markers, only five markers representing three chro- mosome regions had test statistics that were sufficiently outlying and that were likely to represent significant linkage results. of the markers on chromosome p, d s , which is located in the vicinity of the bpad susceptibility locus reported by blackwood et al. ( ), had an empirical sibpal p value , (nominal p value , ). the marker d s on chromosome q had an empirical sibpal p value (nominal p value ) on chromosome q, two dna markers (d s and d s ), located over an approximately -cm region, each had a nominal p value , (sibpal; simulations were not performed). to supplement standard criteria for assessing the significance of our linkage analysis results, we used graphical techniques (fig. ) and the empirical assessment of p values ( – ). if each marker assessed in a pairwise linkage analysis is unlinked to the trait, then the p values associated with those markers should be uniformly distributed. in addition, the test statistics used to generate these p values (for instance, t tests in the case of sibpal) should follow an appropriate distribution. a plot (generated by using proc chart, sas) of the test statistics obtained from each pairwise linkage analysis is shown in fig. . the plot in the inset depicts a line that should be linear if all markers are unlinked. as seen in fig. , there are outlying test statistic values that likely represent false null hypotheses, that is, evidence for significant linkage results. in addition, in the inset to genetics: ginns et al. proc. natl. acad. sci. usa ( ) d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , fig. , the small upturned portion of the p value plot near values of p represents departures from uniformity and hence most likely reflects false null hypotheses. because of the effort required to investigate the significance of these findings and the previous evidence for a bpad-related locus on chromosome ( ), we chose to examine dna markers on chromosome first for linkage to mental health wellness. to evaluate the findings on chromosomes p and q in more detail, we genotyped the subpedigrees and nuclear families containing at least one sibling with bpi (table ) by using additional dna markers in these interesting regions. compared with our previous report ( ) a larger number of individuals was included in these analyses (table ). in this report, model-free linkage analyses using sibpal and gh-plus ( ) were per- formed by using mental health wellness as the linkage phenotype (tables and ). in our analyses, individuals having a psychiatric diagnosis other than bpi, as well as those having psychiatric symptoms but no diagnosis, were classified in the unknown category for affected status. in the amish study sample of bpi patients (n ) the mean and median ages of onset (research diagnostic criteria) are and years, respectively. hence, in all analyses we used a conservative age cutoff of years to define family members with the unaffected wellness phenotype. we also examined the influence of different age cutoffs for defining well individuals and the contribution of different subpedigrees (fam- ilies from pedigrees , , , and versus only families from pedigree ) on the test statistics for linkage. well indi- viduals younger than the specified age cutoff were considered to have an unknown affected status in the analyses. on chromosome p, the maximum multipoint npl values (gh-plus; including only individuals . age years) were . (p . ) for pedigree and . (p . ) for all pedigrees, respectively. the maximum multipoint npl values (gh-plus; including individuals . age years) for markers on chromosome q were . (p . ) for pedigree and . (p . ) for all pedigrees, respectively. the gh-plus log p value as a function of the map position at these locations on chromosome is shown in fig. . sibpal test statistics for markers on chromosomes p and q are shown in tables and . on chromosome the lowest (nominal) p values obtained from the sibpal test statistics were for markers d s ( p; p , ) and d s ( q; p ). the maximum multipoint npl values (gh-plus; including individ- uals . age years) for markers on chromosome q were . for pedigree and . for all pedigrees, respectively. to obtain empirical p-values, we simulated genotype data by randomly assigning marker alleles to the founders and then assigning alleles to their descendants following mendelian inher- itance. allowing for consanguineous matings, the entire family structure was used in marker assignment, thus taking into account all relationships between individuals in the dataset. for each simulation, after marker assignment, the pedigrees were trimmed down to that of the nuclear families used in the linkage analysis. sibpal then was run on the trimmed dataset, and test statistics for concordant and discordant sib pairs were obtained. the true p value is simply estimated as the proportion of replicates in which the simulated statistic is greater than or equal to the observed statistic, i.e., the probability that the observed result or something more extreme would be obtained by chance alone. simulations were conducted for markers on chromosomes p and q. for each marker, , replicates were obtained. the empirical p value on chromosome p clearly meets the proposed criteria of signif- icance for linkage ( ). discussion if alleles exist that are associated with mental health wellness, then the identification of chromosome regions containing these alleles would be enhanced by studying the genetically at risk, mentally healthy members of large, multigenerational pedigrees like our old order amish families. however, in trying to identify protective or wellness alleles, one must recognize that there are phenocopies that need to be considered in our analyses. despite the extremely high risk for developing disease, some individuals are undoubtedly well because they do not inherit any (or all) of the requisite susceptibility alleles for bpad. because the age of greatest liability for onset of bpad in the old order amish is from early teens through years of age, the misspecification of the well phenotype for individuals who eventually will develop bpad would be greatest through this age period. in these old order amish families susceptibility alleles for bpad probably occur in very high frequency. an important step in demonstrating that there are protective alleles will be to show that there are mentally healthy individuals who share marker alleles that should increase the risk of developing bpad, and yet, in the presence of protective alleles, these individuals do not manifest bpad. for many of the markers, )̂, an underestimate of the proportion of alleles shared ibd in well sibpairs, increases with increasing age, i.e., a more stringent definition of the well phenotype. for example, with respect to marker d s on p, )̂ is . , . and . for age cutoff points of , , and years, respectively. this finding suggests that increasing the age for inclusion elimi- nates some age-related well phenocopies. fig. . plot of test statistics obtained from the pair-wise linkage results. (inset) a cumulative plot of p-values whose linearity would reflect uniformity in p-values associated with multiple linkage results whose null hypotheses were all true (see text). the outlying test statistics and p-values (denoted by arrows) were associated with markers, d s (t . ), d s (t . ), d s (t . ), d s (t . ), and d s (t . ). table . old order amish subjects included in linkage analysis subjects analysis categories mentally healthy unknowns pedigrees , , , $ years old $ years old $ years old $ years old pedigree only $ years old $ years old $ years old $ years old the category of unknowns includes individuals of unknown phenotype, individuals with psychiatric diagnoses other than bpi, and individuals who are mentally healthy but are younger than the particular age cut-off used in analyses. bpi individuals are not included in the unknown phenotype category. in pedigrees , , , and , people were diagnosed with bpi, eight with bpii, with recurrent depressive disorder, two with unipolar depressive disorder, and with other psychiatric illness. in pedigree alone, people were diagnosed with bpi, two with bpii, with major depressive disorder, and five with other psychiaric illness. note: the individuals used in these linkage analyses represent only a subset of the entire amish bipolar pedigrees because only nuclear families and subpedigrees containing a sibling with bpi were included. genetics: ginns et al. proc. natl. acad. sci. usa ( ) d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , it is conceivable that virtually all cases of affective disorder in these families are caused by a common set of susceptibility alleles. the wellness or protective loci that we tentatively have identified could harbor alleles that prevent the manifestation of a bpad spectrum phenotype, which also could include major depressive disorder. in our analyses the strongest evidence for protective alleles comes from pedigree , suggesting that such alleles may be more likely in this branch of the family. however, highly significant test statistics and multipoint logarithm of odds scores (using gh-plus) also are observed when pedigrees , , , and are used for analyses (fig. a and b). the decreased sharing in proportion of alleles ibd for discordant pairs provides further support for the existence of alleles associated with the absence of affective disorder (mental health wellness) in these families (data not shown). in addition, epistatic interactions between alleles also could prevent or delay onset of an illness such as major depressive disorder from developing into bpad. in- deed, as we increase the age-of-risk cutoff for defining the well phenotype from to years in our linkage analyses, the number of mentally healthy members decreases as expected, yet the evidence for linkage increases (data not shown). there is some debate on the analysis of sibling pairs as to whether the use of inbred sibling pairs results in an increased number of false-positives if allele-sharing-based statistical meth- ods are used ( ). however, the arguments that (i) inbred sibling pairs are likely to share more genes than noninbred sibling pairs (i.e., have a kinship factor greater than . ) and (ii) that greater regions of the genome would show significant deviations from the expected noninbred sibling sharing value of . are incorrect when one is merely considering an analysis of sibling pairs involving only the transmission of alleles from parents to off- spring. the transmission of alleles from parents to offspring will follow mendelian ratios, and thus the null values for , , or ibd sibling allele sharing in any population will be . , . , and . , whenever only parental and sibling genotype information is used. however, if the origin of the parental alleles is taken into consideration, then there will be greater information about alleles shared by sibling pairs from inbred populations. for example, this increased information has the potential to resolve ambiguities in the sharing of alleles transmitted from homozygous parents, because the two copies of the allele in an inbred homozygous parent could be ibd. this information also could help resolve alleles shared by siblings identical in state into alleles shared ibd, showing that alleles transmitted to two offspring from different parents may be copies of the same allele because of the related- ness of the parents. if genealogy is taken into account, then the increased ability to resolve ambiguities in allele sharing would result in greater power in the analysis of inbred sibling pairs ( ). ultimately, if inbreeding exists in a population from which sibling pairs have been gathered, but one ignores genealogical information by merely studying the transmission of alleles from parents to offspring, then no increase in false-positive linkage results will occur, because mendel’s law applies to inbred as well as outbred parent-offspring allele transmission studies. on the contrary, a decrease in power may result from inbred sibling pair analyses because spouses may manifest greater homozygosity and therefore provide less informative genotypes for parent- offspring-based linkage studies. genetic mapping of complex disorders with multifactorial inheritance could be especially difficult if, in addition to suscep- tibility alleles, individuals inherit protective alleles that prevent or reduce the risk of manifesting the disease phenotype. even though model-based linkage analyses that do not allow for a multifactorial component are of only limited usefulness in these circumstances, they are still used frequently. in these instances, a false-negative linkage finding (type error) could result when table . results of sibpal analysis of p markers marker pedigree pedigrees , , , p̂ (s.e.) p-value p̂ (s.e.) p-value nominal simulated nominal simulated d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . . . (. ) . . d s . (. ) . np . (. ) . np d s . (. ) . . . (. ) . . d s . (. ) . . . (. ) . . d s . (. ) , np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) , np . (. ) . np afma za . (. ) , np . (. ) . np d s . (. ) , np . (. ) , np d s . (. ) . . . (. ) . . d s . (. ) . . . (. ) . . d s . (. ) , . . (. ) , . d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) , , . (. ) , , afm zg . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . . . (. ) . . d s . (. ) , np . (. ) , np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) , np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . . . (. ) . . d s . (. ) . . . (. ) . . d s . (. ) . . . (. ) . . p̂ is the estimated proportion of alleles shared identical by descent. np, simulations not performed. genetics: ginns et al. proc. natl. acad. sci. usa ( ) d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , individuals inherit disease susceptibility alleles but do not man- ifest the phenotype because of the simultaneous presence of protective alleles. if model-based methods are used, it is impor- tant to provide a reasonably low estimate of penetrance and include a multifactorial component in the model. in the initial stages of analyzing a disorder like bpad, which most likely displays multifactorial inheritance, robust model-free (allele sharing) methods are usually more useful than model- based linkage analysis ( ). concordant individuals should dem- onstrate excess allele sharing, even with the occurrence of phe- nocopies, genetic heterogeneity, high frequency of susceptibility alleles, and incomplete penetrance. individuals who inherit sus- ceptibility alleles but do not manifest disease because of protec- tive alleles and individuals who inherit protective alleles but nevertheless manifest the disease will reduce the power of these analyses. thus, regardless of the type of linkage analysis per- formed, the presence of protective alleles could have a major impact on identifying susceptibility loci. although the idea that protective alleles could modify (or even prevent) a behavioral phenotype like bpad is relatively novel, there are examples where such protective alleles can affect the expression or inheritance of other mendelian and multifactorial disorders. the severity of sickle cell anemia is influenced by genes that increase the amount of circulating fetal hemoglobin ( ). similarly, the genotype of the chemokine receptor ccr dra- matically influences the kinetics of hiv- infection, where most individuals who are homozygous for a -bp deletion in the ccr gene encoding the coreceptor for macrophage-tropic hiv- are protected from virus infection ( ). in alzheimer’s disease, apolipoprotein (apo) e , in contrast to apoe , appears to reduce the risk of developing the disease and may protect individuals who inherit a disease-associated apoe allele ( ). in an extended italian family, apo a-imilano protects against the development of both clinical and pathologic signs of atheroscle- rosis, despite significantly elevated plasma triglycerides and a markedly decreased level of high density lipoprotein-cholesterol ( ). in the nonobese diabetic mouse model of human autoim- mune insulin-dependent diabetes mellitus, partial protection from disease is provided by resistance alleles occurring singly at either the idd or idd non-major histocompatibility complex loci, whereas epistatic interaction between resistance alleles at these two loci produces nearly complete protection from diabetes ( ). there are several mechanisms by which wellness or protective alleles could affect the clinical manifestations of bpad in the old order amish. one possibility is that dominant acting protective alleles, either singly or acting together in epistasis, could prevent or modify the bpad phenotype. the variable penetrance of illness or its heterogeneous clinical manifestations could result from resistance or protective alleles that alone provide only partial protection, while together with other genes produce epistatic interactions, resulting in a greater degree of modification of the phenotype. alternatively, there also could be cellular target molecules, e.g., mood effectors, having forms that are either resistant or susceptible to the genetic andyor environmental susceptibility factors for bpad. individuals having resistant mood effectors would be protected from the effects of suscepti- bility alleles andyor environmental factors that result in the bpad phenotype. in contrast, individuals with sensitive forms of these mood effectors would be vulnerable to developing the bpi phenotype when requisite bpad susceptibility alleles andyor environmental factors are present. if epistatic interactions are required for manifestation of the effects of either susceptibility or protective alleles, the existence of resistant and sensitive forms of cellular effectors or protective alleles would be most apparent in families (or populations) where there is a high density of affected individuals such as the old order amish in the present study. regardless of the mechanism, the presence of wellness or pro- tective alleles would have a significant impact on linkage analyses as evidenced by preventing the appearance of the bpad phe- notype (or its presentation as a forme fruste) in individuals who are otherwise genetically predisposed to developing illness. thus, we suggest that the identification of chromosomal loci harboring genes that contribute to the clinical manifestations of bpad will likely require a multilocus approach that considers both additive and subtractive influences of alleles on the bpad phenotype. the involvement of protective or wellness alleles in determining the manifestation of the bpad phenotype would provide an attractive, testable explanation for at least some of the difficulty encountered in searches for bpad susceptibility alleles. table . results of sibpal analysis of q markers marker pedigree pedigrees , , , p̂ (s.e.) p-value p̂ (s.e.) p-value nominal simulated nominal simulated d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . . . (. ) . . d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . . . (. ) . . d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . . (. ) . d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . . . (. ) . . d s . (. ) . . . (. ) . . d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np d s . (. ) . np . (. ) . np p̂ is the estimated proportion of alleles shared identical by descent. np, simulations not performed. genetics: ginns et al. proc. natl. acad. sci. usa ( ) d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , the test statistics from our analyses for alleles linked to the absence of psychiatric illness in the old order amish are at least as significant as those reported for any susceptibility locus, and further investigations into the significance of these findings in the inheritance of bpad are warranted. the identification and characterization of protective alleles and their gene products could lead to the development of a more rational and direct approach to effective therapy for affective disorders. we thank the amish study psychiatric board (drs. j.n. sussex, j.j. schwab, j. endicott, d.r. offord, and a.m. hostetter) and the amish families for more than years of participation; j.r. engle for adminis- trative help; alma becker for phlebotomy; the coriell institute for medical research for establishing and maintaining amish study collec- tion of cell lines; azita kashani, misha schmilovish, and yu-hsien lee for technical assistance; b. kuhns for help with manuscript preparation; and dr. seymour kety and many other colleagues for critical and helpful discussions. the amish study ( – ) was supported by national institute of mental health grant mh to j.a.e., with additional support from the stanley foundation, national alliance for the mentally ill ( – ). support to r.c.e., n.j.s., and p.st.j. was, in part, through national center for research resources grant rr , and to r.c.e. through national institute of general medical sciences grant . a portion of this work was supported by a cooperative research and development agreement (crada) with eli lilly and company. . goodwin, f. k. & jamison, k. r. 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( ) nat. genet. , – . . franceschini, g., sirtori, c. r., capurso, a. nd, weisgraber, k. h. & mahley, r. w. ( ) j. clin. invest. , – . . wicker, l. s., todd, j. a., prins, j. b., podolin, p. l., renjilian, r. j. & peterson, l. b. ( ) j. exp. med. , – . . young, a. ( – ) genetic analysis software (g.a.s.), ver- sion . (oxford university, oxford). fig. . model-free linkage analysis of wellness by using gh-plus. map position is in kosambi centimorgans. the log p was calculated by using p values generated by gh-plus on the assumption that the npl score is standard normally distributed. a log p of . corresponds asymptotically to a logarithm of odds score of . . only mentally healthy individuals years of age or older were classified as being well (see text). (a) log p for markers on chromosome p: dotted line, pedigree only; and solid line, pedigrees , , , and . (b) log p for markers on chromosome q: dotted line, pedigree only, and solid line, pedigrees , , , and . genetics: ginns et al. proc. natl. acad. sci. usa ( ) d o w n lo a d e d a t c a rn e g ie m e llo n u n iv e rs ity o n a p ri l , serveur académique lausannois serval serval.unil.ch author manuscript faculty of biology and medicine publication this paper has been peer-reviewed but dos not include the final publisher proof-corrections or journal pagination. published in final edited form as: in the absence of a copyright statement, users should assume that standard copyright protection applies, unless the article c ontains an explicit statement to the contrary. in case of doubt, contact the journal publisher to verify the copyright status of an article. title: triglyceride and hdl: the entangled pair. authors: amati f, widmann c journal: current opinion in lipidology year: oct volume: issue: pages: - doi: . /mol. http://dx.doi.org/ . /mol. tg and hdl: the entangled pair francesca amati, christian widmann department of physiology, faculty of biology and medicine, university of lausanne corresponding author: professor christian widmann rue du bugnon, lausanne, switzerland phone: + fax: + conflict of interest statement: the authors report no conflict of interest acknowledgement: f.a. is a recipient of a swiss national science foundation ambizione grant (pz p - ) and is supported by the leenaards foundation and baspo switzerland. cw is a member of the european cooperation in science and technology (cost) action “hdlnet” (bm ). cw is supported by the swiss national science foundation ( a_ and izlsz _ ). two recent publications from the new england journal of medicine (nejm) [ ; ] echoed in international and national news agencies (agence france presse [afp]; dallas news). the afp journalist highlighted the importance of high triglycerides (tgs), but not low levels of high-density lipoprotein (hdl) cholesterol, as a risk factor for cardiovascular diseases (cvds). are hdl levels actually not relevant for cvds in contrast to what the current guidelines are advocating [ ]? let us take a look at the data provided by the nejm studies. in the first study [ ], members of the triglycerides and high-density lipoprotein (tg and hdl) working group sequenced the exome of persons and then tested the association of plasma tg with rare sequence variants. the gene most strongly associated with plasma tg encoded apolipoprotein c (apoc ). carriers of apoc variants with lower tg levels displayed lower apoc plasma levels and reduced risk of cvds. functionally, it makes sense that low apoc levels favor increased tg levels because apoc is an inhibitor of the lipoprotein lipase that participates in the catabolism of tgs [ ]. as transfer of tg to hdls accelerates their turn-over [ ] reduced tg levels are also expected to positively affect hdl levels. additionally, hydrolysis of tg-rich lipoproteins provides material for hdl synthesis. via this effect, lower apoc can also contribute to elevation in hdl levels [ ]. it came therefore as no surprise that the nejm study reported increased hdl levels in loss-of-function apoc carriers. such an association between loss-of-function of apoc with reduced tg levels and increased hdl levels had in fact been described earlier in the amish population [ ] but the global impact of this mutation on cvd was not determined. the second nejm study [ ] applied a reverse approach. the authors initially demonstrated an association of tg levels and cvd risk and then determined that loss-of-function mutations in apoc , which they found associated with low tg levels, were also predictive for lower risks of cvd. in this case again, an inverse relationship was observed between tg and hdl levels. in conclusion, the two nejm studies nicely demonstrated the impact of a tg- modulating protein (apoc ) on cvd risks but, as in most observational studies, cannot isolate the individual contributions of entangled biological parameters, such as tgs and hdls, in the development of cvds. in particular, the data reported in these studies cannot be used as evidence that tg levels causally determine cvd risks, as the authors in fact carefully pointed out (but not the journalist). finally, in the context of hdls and diabetes, recently reviewed in depth [ ; ], a new observational study reports that low hdl levels predict earlier and greater intensity treatment with oral hypoglycemic agents and insulin in type diabetic individuals [ ]. this work is in line with a large number of studies reporting that hdl levels predict incidence of type diabetes development. its novelty stems from the finding that in individuals with established diabetes, low hdl levels predict hastened deterioration of glucose control. here again, causality cannot be inferred. as hdl and tg levels are so closely entangled, tgs may as well contribute to beta cell dysfunction in diabetic patients as reported in a recent retrospective study [ ]. reference list · . loss-of-function mutations in apoc , triglycerides, and coronary disease. n.engl.j.med. , in press. this study reports the association of mutations in the apoc gene that are associated with decreased tg levels and reduced risk of coronary heart disease. this paper show the power of exon sequencing in identifying rare variants/mutations that has the potential to be causal for the phenotypic traits of a disease. compared to genome wide association studies, such approach, by identifying mutations in candidate proteins, provide researchers molecular and structural information to generate hypothesis- driven functional experiments to investigate the causal mechanisms of the studied disease. · . jorgensen ab, frikke-schmidt r, nordestgaard bg, tybjaerg-hansen a: loss-of-function mutations in apoc and risk of ischemic vascular disease. n.engl.j.med. , in press. in contrast to the previous study, this work did not start with exon sequencing of a cohort of patients, but rather with the establishement of a link between high tg levels and cvd risks. then a candidate gene approach was undertaken that showed that mutations in the apoc gene, associated with reduced tg levels, were also associated with reduced ischemic vascular disease risks. this and the previous study are fine examples of genetic studies that can pinpoint the importance of genes in the aetiology of a diseas. . vollenweider p, von eckardstein a, widmann c: hdls, diabetes, and metabolic syndrome. handb.exp.pharmacol. , in press. . von eckardstein a, widmann c: hdl, beta cells and diabetes. cardiovasc.res. , in press. . pollin ti, damcott cm, shen h, ott sh, shelton j, horenstein rb, post w, mclenithan jc, bielak lf, peyser pa, mitchell bd, miller m, o'connell jr, shuldiner ar: a null mutation in human apoc confers a favorable plasma lipid profile and apparent cardioprotection. science. , : - . . waldman b, jenkins aj, davis tm, taskinen mr, scott r, o'connell rl, gebski vj, ng mk, keech ac: hdl-c and hdl-c/apoa-i predict long-term progression of glycemia in established type diabetes. diabetes care . . shimodaira m, niwa t, nakajima k, kobayashi m, hanyu n, nakayama t: serum triglyceride levels correlated with the rate of change in insulin secretion over two years in prediabetic subjects. ann.nutr.metab. , : - . further recommended reading churilla jr, johnson tm, curls r, richardson mr, boyer wr, devore sr, alnojeidi ah: association between alcohol consumption patterns and metabolic syndrome. diabetes metab.syndr. , : - . raising hdl levels is proposed to be a therapeutical approach in the context of cvds and diabetes. this notion is based on the inverse relationship observed between hdl levels and risks of developing such diseases. the present study confirms the association between alcohol consumption and hdl levels. it also indicates that individual consuming alcohols are less likely to develop a metabolic syndrome. considering alcohol, when moderately consumed, as therapeutic beneficial raises ethical and medical issues however. nevertheless, it is certainly worthwhile to experimentally assess, at the molecular level, how alcohol exerts its beneficial effect as this may lead to the identification of molecules that could be targeted for the development of anti-atherogenic and anti-diabetogenic drugs. kurano m, hara m, tsuneyama k, sakoda h, shimizu t, tsukamoto k, ikeda h, yatomi y: induction of insulin secretion by apolipoprotein m, a carrier for sphingosine -phosphate. biochim.biophys.acta . the hdl components mediating their beneficial effects in pancreatic beta cells are not definitely characterized yet. apom, an apolipoprotein found in about % of hdls, has the capacity to bind sphingosine- -phosphate (s p). this latter molecule is able to activate various signals in cells, including anti- apoptotic pathways. the present study indicates that apom favors insulin secretion in a s p receptor-dependent manner. it will be interesting in future studies to determine if apom-containing hdl particles are the only hdl particles that mediate beneficial effects in beta cells. 	serveur académique lausannois serval serval.unil.ch 	author manuscript 	faculty of biology and medicine publication 	published in final edited form as: title relationship between structural and stress relaxation in ablock-copolymer melt author(s) patel, aj; narayanan, s; sandy, a; mochrie, sgj; garetz, ba;watanabe, h; balsara, np citation physical review letters ( ), ( ) issue date - - url http://hdl.handle.net/ / right copyright american physical society type journal article textversion publisher kyoto university relationship between structural and stress relaxation in a block-copolymer melt amish j. patel, suresh narayanan, alec sandy, simon g. j. mochrie, bruce a. garetz, hiroshi watanabe, and nitash p. balsara , department of chemical engineering, university of california, berkeley, california , usa argonne national laboratory, argonne, illinois , usa department of physics, yale university, new haven, connecticut , usa othmer department of chemical & biological sciences & engineering, polytechnic university, brooklyn, new york , usa institute for chemical research, kyoto university, uji, kyoto - , japan materials sciences division and environmental energy and technologies division, lawrence berkeley national laboratory, university of california, berkeley, california , usa (received february ; published june ) the relationship between structural relaxation on molecular length scales and macroscopic stress relaxation was explored in a disordered block-copolymer melt. experiments show that the structural relaxation time, measured by x-ray photon correlation spectroscopy is larger than the terminal stress relaxation time, measured by rheology, by factors as large as . we demonstrate that the structural relaxation data are dominated by the diffusion of intact micelles while the stress relaxation data are dominated by contributions due to disordered concentration fluctuations. doi: . /physrevlett. . pacs numbers: . .+e, . .eq, . .lg polymer materials offer unique insight into the molecu- lar underpinnings of macroscopic mechanical properties because the consequences of molecular motion, which are generally difficult to measure directly, can be readily ob- served in relatively simple stress relaxation experiments [ ]. a quantity of central importance is the so-called longest relaxation time, which characterizes the terminus of the stress relaxation function. in simple systems such as linear homopolymer melts, this relaxation time reflects the time scale for the diffusion of entire polymer chains across length scales comparable to molecular dimensions. in the case of heterogeneous single-phase systems such as disor- dered block-copolymer melts, the relationship between molecular motion and stress relaxation is complicated due to the spontaneous formation and disappearance of transient structures such as concentration fluctuations, mi- celles, and concomitant interfaces of varying widths [ ]. stress relaxation in such systems is coupled to both mo- lecular motion and the relaxation of these transient struc- tures. an interesting consequence of this coupling is that structural relaxation can, in principle, be much slower than the terminal stress relaxation. in this letter we present the first quantitative measurements of both stress and structural relaxation in a block-copolymer melt. the experimentally determined characteristic times for structural relaxation are – orders of magnitude larger than those for stress relax- ation. thus, the use of the term, ‘‘longest relaxation time’’, that is often used to describe terminal relaxation in disor- dered block copolymers [ , ] is, perhaps, not appropriate. relaxation processes are quantified by the wave-vector (q) dependent dynamic structure factor, s�q;t�. s�q;t� is the spatial fourier transform of the density-density auto- correlation function h�a�r ; t��a�r; �i, where �a�r; t� is the concentration of chain segments of type a at position r and time t. while techniques such as dielectric relaxation [ , ] give insight into relaxation processes, s�q;t� can be mea- sured directly by techniques such as light or x-ray photon correlation spectroscopy (lpcs or xpcs) [ – ], or quasi- elastic neutron scattering [ ]. a large majority of mea- surements of s�q;t� have been made using lpcs wherein the length scales ( �=q) that are accessed are in the range of nm [ ]. on this length scale, structural relaxation is almost always dominated by diffusion. in contrast, our measurements using xpcs enable the quantification of structural relaxation on molecular length scales (� nm). the goal of this letter is to identify the struc- tural relaxation process that dominates the xpcs signal from our diblock-copolymer melt, and to explore the rela- tionship between structural and stress relaxation. a polystyrene-block-polyisoprene copolymer, si( – ), was synthesized and characterized by methods described in ref. [ ]. the weight-averaged molecular weights of the polystyrene and polyisoprene blocks were : kg=mol and : kg=mol, respectively. the copolymer melt exhibits an order-disorder transition from hexagonally packed cylin- ders to disordered micelles at todt � � �c [ ]. the static and dynamic structure factors were measured by small-angle x-ray scattering (saxs) and xpcs on the - id-i beam line at the advanced photon source (aps), argonne national laboratory. figure shows the static saxs intensity profiles, i�q�, [where q �� �=��sin��= �, � � : �a is the wave- length of the x rays and � is the scattering angle] obtained at temperatures between �c and �c. in ref. [ ], we presented a thorough analysis of this data set, proving the presence of disordered micelles in this temperature range. recent theoretical computations by wang et al. [ ] enable estimation of the volume fraction of micelles in these systems. using self-consistent field theory to compute the structure factor of block-copolymer melts with disordered prl , ( ) p h y s i c a l r e v i e w l e t t e r s week ending june - = = ( )= ( ) - © the american physical society http://dx.doi.org/ . /physrevlett. . micelles, they showed that the peak in the structure factor, q�, moves to lower q values as the micelle volume fraction increases. if we assume that our melt has no micelles at �c (the upper limit of our temperature window where we do not observe any signature of micelles) and attribute all of the changes in q� with decreasing temperature (inset of fig. ) to the presence of micelles, we conclude that the upper bound for the micelle volume fraction �m is . [ ]. even though the micelle concentration is small, the saxs signal at q� is strongly affected by the presence of micelles due to their large scattering power [ ]. xpcs was used to measure s�q;t� in the vicinity of q�. if a single process dominates the relaxation of concentration fluctuations, i.e. s�q;t�� exp��t=�struc�, the x-ray scatter- ing intensity time correlation function, g �q;t�, is given by g �q;t�	 hi�q; �i�q;t�i hi�q;t�i � ke� t=�struc�q�; ( ) where �struc�q� is the characteristic time for structural relaxation. in this letter, we focus on �struc�q � q �� (changes in �struc with q were within experimental error in the narrow range of q values where the xpcs signal could be measured) which we shall refer to as �struc from now on. typical measurements of g �q �; t� for si( – ), shown in fig. , are in agreement with eq. ( ). the curves in fig. are least squares fits of eq. ( ) from which we estimate �struc. our measurements do not rule out the presence of faster relaxation processes that are below the lower limit of our time window. rheological measurements on si( – ) were conducted using an ares rheometer (rheometrics inc.). our mea- surements covered the frequency (!) range from . to rad s� . figure shows the results of the frequency (!) dependence of the storage and loss shear moduli (g and g ) of our sample at selected temperatures after time- temperature-superposition (tt) [ , ] of the high-! data. the high-! moduli reflect single chain dynamics (as in- ferred from similarity with homopolymer data [ ]). the low-! moduli are similar to those for the relaxation of concentration fluctuations giving branched master curves of g and g after the tt superposition [ ]. the rheological terminal relaxation time �tr ��g =!g �!! is attributed to this relaxation process. in fig. , the values of �tr obtained by rheology are compared to �struc. it is evident that �struc is – orders of magnitude larger than �tr. the temperature dependences of �struc and �tr are similar but not identical. the decrease in �struc with temperature is less pronounced than that of �tr. stress relaxation in polymers is due to a multitude of relaxation processes, occurring at various time and length scales. xpcs measurements provide a direct measure of the time scale for one of these processes. it has been argued that for systems with peaks in s�q�, relaxation times near the critical point would be longest at q � q� [ ]. pioneering experiments by anastasiadis and co-workers have shown that this is true for disordered diblock copoly- mers [ , ]. this provides the qualitative reason for our observation regarding the relative magnitudes of �tr and �struc. quantitative relationships between �tr and �struc are contained in the theoretical work of fredrickson and larson [ ]. in this theory, a fokker-planck equation is used to describe the response of a system to simple shear flow, within a gaussian hamiltonian approximation. the complex modulus g� � g ig (i � ������� � p ) is given by . . . . . . g °c t (seconds) . . . . . . g °c . . . . . . g °c fig. . typical intensity-intensity time autocorrelation func- tions, g �q �; t� at �c, �c, and �c. . . . . . °c °c °c °c °c i (c m - ) q (Å- ) . . . . . q * (Å - ) temperature (°c) fig. . saxs intensity, i�q�. the peak in the saxs intensity (q�), indicated by the vertical bars, shifts to lower q values as micelle volume fraction increases. the inset shows q� as a function of temperature. prl , ( ) p h y s i c a l r e v i e w l e t t e r s week ending june - g��!�� kbt! � r g z xc x = s �x� !� i �!�x� � @s� �x� @x � dx; ( ) where rg is the chain radius of gyration, x 	�qrg� , xc 	 �qcrg� , qc 	 �=l, l is the statistical segment length, s�x� is the static structure factor, and �!�x�, the rate at which order parameter fluctuations of wave vector q relax at thermal equilibrium, is given by �!�x�� xg�x�ns� �x�= �, where � is the molecular relaxation time, n is the number of monomers per chain, and g�x� is the debye function: g�x�	 x �x e�x � �. the con- nection between xpcs and rheological measurements is provided by the fact that if fluctuations dominated the xpcs relaxation �struc � = �!�x�. we use the term �fl to refer to the structural relaxation time based on the fredrickson-larson theory, which is given by �fl � �tr x�g�x��ns� �x�� �rxc x = ns� �x� g�x� � @s�x� @x � dxrxc x = g �x� � @s�x� @x � dx � : ( ) the application of eq. ( ) to the disordered micelle phase of block copolymers is not possible because the hamiltonian and the structure factor for this system [ ] are much more complicated than those used in ref. [ ]. however, the effect of simple (nonmicellar) concentration fluctuations on �fl can readily be computed using well- established expressions for s�q� [ ], and the measured value of �tr. we take the measured odt temperature of our system as the spinodal temperature and compute s�q� using the leibler theory [ , ]. this enables determination of all of the parameters on the right hand side of eq. ( ). the temperature dependence of �fl is shown by the dashed curve in fig. . the temperature dependencies of �fl and �struc are similar and �fl is greater than �tr at all tempera- tures. the fredrickson-larson theory thus provides a qualitative explanation of our observation that �struc is greater than �tr. the magnitude �struc at a given temperature is, however, a factor of larger than that of �fl. this indicates the need for further analysis. our analysis of the xpcs data thus far ignores the presence of micelles. the decay of g �q �; t� of a micellar phase can, in principle, reflect either the dissolution of micelles or the diffusion of intact micelles. the time scale �se for the diffusion of an intact micelle over a distance equal to its radius rh (
 �=q �) can be readily calculated on the basis of the stokes-einstein relationship, �se � �r h�eff�t� kbt ; ( ) where �eff is the effective viscosity of the medium, kb is the boltzmann constant, and t is the temperature [ ]. in general, �eff differs from the rheologically measured ter- minal viscosity, � , because the micelle diffusion itself contributes to � . however, since our micelles are dilute (�m < : ), we may safely assume that �eff 
 � in the evaluation of �se using eq. ( ). in fig. , we compare the �se thus calculated with the experimentally determined �struc. the agreement between �struc and �se at all tempera- tures is remarkable, considering the uncertainty in rh and . . structural relaxation terminal relaxation fredrickson-larson fluctuation relaxation re la xa tio n t im e ( se co n d s) temperature (°c) fig. . comparing the structural relaxation time, �struc with the rheological terminal relaxation time, �tr. note that �struc is to orders of magnitude larger than �tr. the dashed curve is the structural relaxation time as predicted by the fredrickson-larson theory, �fl. - °c °c °c lo g (g '/p a ) log(ωa t ) - . . . . . . lo g ( a t /r a d - s ) temperature (°c) . . . . - °c °c °c lo g (g "/ p a ) log(ωa t ) (a) (b) fig. . time-temperature superposed rheological data on si( – ) from �c to �c. (a) storage modulus g �!�. (b) loss modulus g �!�. the inset in (a) shows the shift factor, at as a function of temperature. prl , ( ) p h y s i c a l r e v i e w l e t t e r s week ending june - the fact that the stokes-einstein relationship applies to rigid spheres diffusing in a homogeneous continuum. we can thus assert that the decay of g �q �; t� is dominated by micelle diffusion, and that the micelle lifetime in our temperature window must be greater than the �struc values given in fig. . the frequency at which we expect to observe the rheological consequences of micelle diffusion, ! � �=�struc, was within our experimental frequency window at all temperatures. however, the micelles are dilute (�m < : ) and thus their diffusion does not con- tribute significantly to the measured relaxation. understanding equilibrium dynamics on molecular length scales affects our understanding of many other phenomena in block copolymers. an example of such a phenomenon is the kinetics of the disorder-to-order tran- sition. in some cases [ , ], large discrepancies have been reported between measured and estimated grain growth rates, if the rheological relaxation times are used in the estimation. our results indicate that local dynamics on molecular length scales can be significantly slower than those estimated from rheological measurements. in conclusion, we have used xpcs and rheology to find the relationship between structural and stress relaxation in a block-copolymer melt that contains disordered micelles. we have found that the structural relaxation time can be larger than the longest stress relaxation time by factors as large as . a comparison of the stokes-einstein micelle diffusion time with �struc reveals that the structural relaxa- tion mode is dominated by the diffusion of intact micelles. we gratefully acknowledge hyeok hahn and hany eitouni for their help with the experiments, and glenn fredrickson for educational discussions. financial support was provided by the national science foundation (decs and dmr ). the aps is supported by the u.s. doe under contract no. w- - -eng- . a. j. p. gratefully acknowledges additional support from tyco electronics. s. g. j. m. was supported by nsf dmr . [ ] j. d. ferry, viscoelastic properties of polymers (wiley, new york, ). [ ] f. s. bates, j. h. rosedale, and g. h. fredrickson, j. chem. phys. , ( ). [ ] w. g. kim, b. a. garetz, m. c. newstein, and n. p. bal- sara, j. polym. sci., part b: polym. phys. , ( ). [ ] b. r. chapman, m. w. hamersky, j. m. milhaupt, c. kostelecky, t. p. lodge, e. d. von meerwall, and s. d. smith, macromolecules , ( ). [ ] m. e. baur and w. h. stockmayer, j. chem. phys. , ( ). [ ] h. watanabe, o. urakawa, and t. kotaka, macro- molecules , ( ). [ ] s. h. anastasiadis, curr. opin. colloid interface sci. , ( ). [ ] s. g. j. mochrie, a. m. mayes, a. r. sandy, m. sutton, s. brauer, g. b. stephenson, d. l. abernathy, and g. grubel, phys. rev. lett. , ( ). [ ] p. falus, m. a. borthwick, and s. g. j. mochrie, phys. rev. lett. , ( ). [ ] a. arbe, u. buchenau, l. willner, d. richter, b. farago, and j. colmenero, phys. rev. lett. , ( ). 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[ ] at a given temperature t the quench depth, " ���s � ��=�s, is calculated as " �� : =�s�� =todt � =t� using � �� : : =t [ ] and �s � ��todt�. s�q� is then calculated for si( – ) using � � �s� �"�, polystyrene volume fraction f � : , chain length n � , and t � �c. [ ] e. e. dormidontova and t. p. lodge, macromolecules , ( ). [ ] y. matsumiya and h. watanabe, macromolecules , ( ). [ ] t. q. chastek and t. p. lodge, macromolecules , ( ). structural relaxation micelle diffusion re la xa tio n t im e ( se co n d s) temperature (°c) fig. . comparing �struc to the stokes-einstein diffusion re- laxation time, �se. prl , ( ) p h y s i c a l r e v i e w l e t t e r s week ending june - aggressive cd + epidermotropic cutaneous t-cell lymphoma associated with homozygous mutation in samhd case series aggressive cd epidermotropic cutaneous t-cell lymphoma associated with homozygous mutation in samhd miesha merati, do, a douglas j. buethe, md, b kevin d. cooper, md, b kord s. honda, md, b heng wang, md, c,d and meg r. gerstenblith, md b richmond heights, cleveland, and middlefield, ohio from of me me ne ra fund confl corre de me key words: cutaneous t-cell lymphoma; sam domain and hd domainecontaining protein . abbreviations used: ags: aicardi-goutieres syndrome ctcl: cutaneous t-cell lymphoma samhd : sam domain and hd domain- containing protein sams: stroke, aneurysm, moyamoya, and stenosis sle: systemic lupus erythematosus introduction mutations in the samhd (sam domain and hd domainecontaining protein ) gene are impli- cated in sams (stroke, aneurysm, moyamoya, and stenosis) association, , also described as aicardi- goutieres syndrome type (ags- ). we present a patient with homozygous germline mutations in samhd who subsequently had a cd aggressive epidermotropic cutaneous t-cell lymphoma (ctcl). case report a -year-old amish woman with intellectual disability and arthritis caused by the homozygous mutation c. - a [ g in the samhd gene (identified by xin et al ), presented with fever, cervical lymphadenopathy, and a diffuse rash. the rash began weeks before presentation and consisted of reddish-brown scaly plaques on the chest, abdomen, and back, which were treated with topical steroids by her primary physician. she then became acutely ill and was admitted to the medical intensive care unit of our institution for circulatory shock and hypoxemic respiratory failure. on examination by the dermatology service, the patient had diffuse, red-brown, circinate, coalescing, scaly plaques involving the trunk, proximal extrem- ities, and face (fig , a). a -cm -cm red-purple, exophytic tumor was noted on the right posterior thigh (fig , b). joint examination found deformities of the bilateral hands without active synovitis. laboratory evaluation was notable for normocytic university hospitals richmond medical center a ; department dermatology, case western reserve university school of dicine, uh seidman cancer center, university hospitals case dical center, clevelandb; ddc clinic, center for special eds children, middlefield c ; and department of pediatrics, inbow babies and children’s hospital, cleveland. d ing sources: none. icts of interest: none declared. spondence to: meg r. gerstenblith, md, department of rmatology, case western reserve university school of dicine, uh seidman cancer center, university hospitals anemia, thrombocytopenia, elevated erythrocyte sedimentation rate, elevated c-reactive protein, positive antinuclear antibodies with : , titer, positive antiedouble-stranded dna antibodies, and positive antichromatin antibodies. blood, lower respiratory, and urine cultures were negative. computed tomography of the neck, chest, abdomen, and pelvis showed bilateral pulmonary infiltrates and cervical, axillary, retroperitoneal, and gastro- hepatic lymphadenopathy. skin biopsy from the abdomen showed significant exocytosis of lymphocytes with mildly enlarged hyperchromatic nuclei without significant cyto- plasm. some lymphocytes were surrounded by clear halos. there was also a moderate superficial and mid-dermal interstitial and periadnexal lymphocytic infiltrate (fig ). flow cytometry from affected skin found a population of atypical t cells ( % of all events) that were cd , cd , cd �, cd , cd , cd dim, cd dim, cd dim, cd �, hla-dr�, cd ro�, and cd ra�. the cells were t-cell case medical center, cleveland, oh . e-mail: meg.gersten blith@uhhospitals.org. jaad case reports ; : - . - � by the american academy of dermatology, inc. published by elsevier, inc. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc- nd/ . /). http://dx.doi.org/ . /j.jdcr. . . delta: _given name delta: _surname delta: _given name delta: _surname delta: _given name delta: _surname mailto:meg.gerstenblith@uhhospitals.org mailto:meg.gerstenblith@uhhospitals.org http://creativecommons.org/licenses/by-nc-nd/ .� / http://creativecommons.org/licenses/by-nc-nd/ .� / http://dx.doi.org/ . /j.jdcr. . . fig . aggressive cd epidermotropic ctcl. cutaneous findings on initial presentation from the abdomen (a) and right posterior thigh (b). fig . aggressive cd epidermotropic ctcl. histopa- thology from lesional skin shows significant exocytosis of normal-size atypical lymphocytes. the lymphocytes stain with antibodies against cd and cd rb, without significant cd or cd staining. (hematoxylin-eosin stain; original magnification: .) jaad case reports july merati et al receptor (tcr)-a/b positive and did not react with any antibodies used in the tcr-vb analysis, consis- tent with an abnormal clonal t-cell population. peripheral blood flow cytometry found no evidence of a peripheral lymphoproliferative disorder. tcr-g chain gene rearrangement from affected skin was negative. based on these results, an aggressive cd epidermotropic ctcl was diagnosed. lymph node biopsy was declined, as it would not change management, and because of severe thrombocyto- penia, it represented an unnecessary risk to the patient. the patient’s family declined treatment with systemic chemotherapy, and she subsequently died under hospice care. discussion sams association was first described in the old order amish in . the clinical phenotype is highly heterogeneous, ranging from normal cogni- tive development to severe intellectual impairment and early death from recurrent strokes. it is likely that ags- and sams association might be the same entity, although most patients from the amish cohort, particularly the mildly affected patients, do not fit the clinical spectrum of ags- . the clinical features of ags- include progressive encephalopa- thy, basal ganglia calcification, white matter changes, and cerebrospinal fluid abnormalities of lymphocy- tosis and elevated interferon alfa levels. - the samhd protein degrades the intracellular pool of deoxynucleoside triphosphates, maintaining genomic stability and the innate immune system. samhd is upregulated in response to viral infections and is a restriction factor for hiv- infection. absence of samhd causes an imbalance in dna precursors, which acts as a danger signal to the innate immune system, triggering a type i interferon immune response. activation of the innate immune system plays a central role in sams association/ags- and may lead to autoimmunity. many of the symptoms of sams association overlap with autoimmune conditions such as vasculitis, systemic lupus erythematosus (sle), rheumatoid arthritis, and mixed connective tissue disease, and ags is associated with systemic autoimmunity and sle. , our patient met of american college of rheumatology classification criteria required for the diagnosis of sle (arthritis, thrombocytopenia, positive antinuclear antibodies and antiedouble-stranded dna antibodies). genomic instability caused by samhd muta- tions may lead to increased mutagenesis and cancer development. samhd somatic mutations have been identified in % of refractory cases of chronic lymphocytic leukemia and in lung adenocarcinoma; jaad case reports volume , number merati et al glioblastoma; and breast, pancreatic, and colorectal cancers. recently, clifford et al and de silva et al found decreased samhd expression in patients with sezary syndrome and with advanced-stage mycosis fungoides. aggressive cd epidermotropic ctcl is a rare subtype associated with a poor prognosis. median expected lifespan from diagnosis is . to months, with an % chance of -year survival. tumor markers cd � and cd appear to predict a more aggressive clinical course. our patient’s ctcl was cd and cd . although cd ctcl is often clonal, our patient did not have tcr-g gene rearrangement. potential explanations include insufficient representation of the clone, genetic instability leading to deletion of portions of the tcr-g gene, or other unknown factors. the absence of prior reports of ctcl in patients with homozygous mutations of samhd could relate to the rarity of ags- or sams association and the reduced lifespan caused by recurrent cerebrovascular accidents seen in these patients. along with de silva et al, our case provides further evidence for a possible link between samhd deficiency and ctcl. additional studies to further characterize the role of samhd in the pathogenesis of ctcl are indicated. author contributions: drs gerstenblith and merati, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. study concept and design: drs merati, buethe, cooper, honda, wang, gerstenblith acquisition, analysis, and interpretation of data: drs merati, buethe, cooper, honda, wang, gerstenblith drafting of the manuscript: drs merati, buethe, cooper, honda, wang, gerstenblith critical revision of the manuscript for important intellectual content: drs merati, buethe, cooper, honda, wang, gerstenblith references . xin b, jones s, puffenberger eg, et al. homozygous mutation in samhd gene causes cerebral vasculopathy and early onset stroke. proc natl acad sci. ; ( ): - . . xin b, li w, bright a, hinze c, wang h. reply to du moulin, et al. cerebral vasculopathy is a common hallmark in individuals with samhd mutations. proc natl acad sci. ; ( ): e . . rice gi, bond j, asipu a, et al. mutations involved in aicardi-goutieres syndrome implicate samhd as regulator of the innate immune response. nat genet. ; ( ): - . . thiele h, du moulin m, barczyk k, et al. cerebral arterial stenoses and stroke: novel features of aicardi-gouti�eres syndrome caused by the arg x mutation in samhd are associated with altered cytokine expression. hum mutat. ; ( ):e -e . . ramesh v, bernardi b, garone c, et al. intracerebral large artery disease in aicardi-gouti�eres syndrome implicates samhd in vascular homeostasis. dev med child neurol. ; ( ): - . . ramantani g, kohlhase j, hertzberg c, et al. expanding the phenotypic spectrum of lupus erythematosus in aicardi-gouti�eres syndrome. arthritis rheum. ; : - . . kretschmer s, wolf c, k€onig n, et al. samhd prevents autoimmunity by maintaining genome stability. ann rheum dis. ; ( ):e . . clifford r, louis t, robbe p, et al. samhd is mutated recurrently in chronic lymphocytic leukemia and is involved in response to dna damage. blood. ; ( ): - . . de silva s, wang f, hake ts, porcu p, wong hk, wu l. downregulation of samhd expression correlates with promoter dna methylation in s�ezary syndrome patients. j invest dermatol. ; ( ): - . . berti e, tomasini d, vermeer mh, meijer cj, alessi e, willemze r. primary cutaneous cd -positive epidermotropic cytotoxic t cell lymphomas: a distinct clinicopathological entity with an aggressive clinical behavior. am j pathol. ; ( ): - . http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref http://refhub.elsevier.com/s - ( ) - /sref 	aggressive cd + epidermotropic cutaneous t-cell lymphoma associated with homozygous mutation in samhd 	introduction 	case report 	discussion 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ genome-wide association study identifies multiple loci associated with both mammographic density and breast cancer risk the harvard community has made this article openly available. please share how this access benefits you. your story matters citation lindström, s., d. j. thompson, a. d. paterson, j. li, g. l. gierach, c. scott, j. stone, et al. . “genome-wide association study identifies multiple loci associated with both mammographic density and breast cancer risk.” nature communications ( ): . doi: . /ncomms . http://dx.doi.org/ . /ncomms . published version doi: . /ncomms citable link http://nrs.harvard.edu/urn- :hul.instrepos: terms of use this article was downloaded from harvard university’s dash repository, and is made available under the terms and conditions applicable to other posted material, as set forth at http:// nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of- use#laa http://osc.hul.harvard.edu/dash/open-access-feedback?handle=&title=genome-wide% association% study% identifies% multiple% loci% associated% with% both% mammographic% density% and% breast% cancer% risk&community= / &collection= / &owningcollection / &harvardauthors= b f a b de cc ab&department http://nrs.harvard.edu/urn- :hul.instrepos: http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa http://nrs.harvard.edu/urn- :hul.instrepos:dash.current.terms-of-use#laa genome-wide association study identifies multiple loci associated with both mammographic density and breast cancer risk sara lindström , , deborah j. thompson , , andrew d. paterson , jingmei li , gretchen l. gierach , christopher scott , jennifer stone , julie a. douglas , isabel dos-santos- silva , pablo fernandez-navarro , , jajini verghase , , , paula smith , , judith brown , , robert luben , nicholas j. wareham , ruth j.f. loos , , john a. heit , v. shane pankratz , aaron norman , ellen l. goode , julie m. cunningham , mariza deandrade , robert a. vierkant , kamila czene , peter a. fasching , , laura baglietto , , melissa c. southey , graham g. giles , , kaanan p. shah , heang-ping chan , mark a. helvie , andrew h. beck , nicholas w. knoblauch , aditi hazra , , , david j. hunter , , , peter kraft , , , marina pollan , , jonine d. figueroa , fergus j. couch , , john l. hopper , per hall , douglas f. easton , , , norman f. boyd , celine m. vachon ,†, and rulla m. tamimi , , ,† program in genetic epidemiology and statistical genetics, harvard school of public health, boston, ma, , usa department of epidemiology, harvard school of public health, boston, ma, , usa centre for genetic epidemiology, university of cambridge, cambridge cb rn, uk department of public health and primary care, university of cambridge, cambridge cb rn, uk program in genetics and genome biology, the hospital for sick children, toronto, ontario, m g x , canada human genetics, genome institute of singapore, singapore, , singapore hormonal and reproductive epidemiology branch, national cancer institute, bethesda, md , usa department of health sciences research, mayo clinic, rochester, mn , usa correspondence to: rulla tamimi, channing division of network medicine, department of medicine, brigham and women’s hospital and harvard medical school, longwood avenue, boston, ma , usa, rulla.tamimi@channing.harvard.edu. †these authors jointly supervised this work author contributions s.l., d.j.t., a.d.p., j.l, g.l.g, j.s., j.l.h., p.h., d.f.e., n.f.b., c.m.v., and r.m.t., designed the study. s.l., d.j.t., a.d.p., j.l, j.s., c.s. j.a.d, p.f.n, j.v, p.s., a.h.b., and n.w.k. performed the statistical analysis. j.a.d., i.d.s.s., j.b., r.l., n.j.w., r.j.f.l., j.a.h., v.s.p., a.n., e.l.g., j.m.c., m.d., r.a.v., k.c., p.a.f., l.b., m.c.s., g.g.g., k.p.s., h.p.c., m.a.h., a.h., d.j.h., p.k., m.p., j.d.f., f.j.c., j.l.h., p.h., d.f.e., n.f.b., c.m.v., and r.m.t provided samples and data. s.l., d.j.t., a.d.p., j.a.d., j.l.h., n.f.b., c.m.v and r.m.t drafted the manuscript. all authors contributed to the final paper. competing financial interests the authors declare no competing financial interests. nih public access author manuscript nat commun. author manuscript; available in pmc april . published in final edited form as: nat commun. ; : . doi: . /ncomms . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t centre for genetic origins of health and disease, university of western australia, perth, wa , australia department of human genetics, university of michigan medical school, ann arbor, mi , usa faculty of epidemiology and population health, london school of hygiene and tropical medicine, london, wc e ht, uk cancer and environmental epidemiology unit, national center for epidemiology, carlos iii institute of health, madrid , spain consortium for biomedical research in epidemiology & public health (ciber en epidemiología y salud pública – ciberesp) , spain plastic surgery unit, royal free hospital, london, uk medical research council (mrc) epidemiology unit, institute of metabolic science, university of cambridge, cambridge cb rn, uk the icahn school of medicine at mount sinai, the charles bronfman institute for personalized medicine, the mindich child health and development institute, new york, ny , usa division of cardiovascular disease, department of medicine, mayo clinic, rochester, mn , usa department of laboratory medicine and pathology, mayo clinic, rochester, mn , usa department of medical epidemiology and biostatistics, karolinska institutet, stockholm , sweden department of gynecology and obstetrics, erlangen university hospital, friedrich alexander university of erlangen-nuremberg, comprehensive cancer center erlangen-emn, erlangen, germany university of california at los angeles, department of medicine, division hematology/ oncology, david geffen school of medicine, los angeles, ca , usa cancer epidemiology centre, cancer council victoria, melbourne , australia centre for epidemiology and biostatistics, melbourne school of population and global health, the university of melbourne, , australia department of pathology, university of melbourne, melbourne , australia department of radiology, university of michigan medical school, ann arbor, mi , usa department of pathology, beth israel deaconess medical center and harvard medical school, boston, ma , usa channing division of network medicine, brigham and women’s hospital, boston, ma , usa department of biostatistics, harvard school of public health, boston, ma , usa department of oncology, university of cambridge, cambridge, cb rn, uk lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t campbell family institute for breast cancer research, ontario cancer institute, toronto, ontario m g m , canada abstract mammographic density reflects the amount of stromal and epithelial tissues in relation to adipose tissue in the breast and is a strong risk factor for breast cancer. here we report the results from meta-analysis of genome-wide association studies (gwas) of three mammographic density phenotypes: dense area, non-dense area and percent density in up to , women in stage and an additional , women in stage . we identify genome-wide significant (p< × − ) loci for dense area (areg, esr , znf , lsp /tnnt , igf , tmem b, sgsm /mkl ), non- dense area ( p . ) and percent density (prdm , p . , tmem b). four of these regions are known breast cancer susceptibility loci, and four additional regions were found to be associated with breast cancer (p< . ) in a large meta-analysis. these results provide further evidence of a shared genetic basis between mammographic density and breast cancer and illustrate the power of studying intermediate quantitative phenotypes to identify putative disease susceptibility loci. introduction variations in the appearance of the mammogram reflect differences in breast fibroglandular tissue that appears white or radio-dense, and fat that appears black or non-dense. after adjustment for age and body mass index (bmi), the proportion of the total breast area that is dense (percent density (pd)) is a strong risk factor for breast cancer , and both dense (da) and non-dense areas (nda), are also independently associated with breast cancer risk , . pd, da and nda are all highly heritable ( . – . ) , , but to date few genetic loci associated with mammographic density have been identified – . here we report results from a two-stage (discovery and replication stages) gwas of da, nda and pd, respectively. we identify genome-wide significant (p< × − ) loci for dense area (areg, esr , znf , lsp /tnnt , igf , tmem b and sgsm /mkl ), non- dense area ( p . ) and percent density (prdm , p . , tmem b). our results add to the growing body of evidence that mammographic density and breast cancer risk share a genetic component. results our discovery phase included eleven studies with gwas data (methods, supplementary note ) comprising a total of , women. study subjects were predominantly post- menopausal women of european ancestry participating in the markers of density (mode) consortium. mammographic density was measured using cumulus (supplementary table ) and , ( %) of the subjects were breast cancer cases. all studies were imputed to hapmap phase ii before meta-analysis (supplementary table ). for each snp, we combined study-specific p-values and direction of association using the metal software . we assessed promising snps for replication in up to , women from eleven different studies (supplementary table , supplementary note ). lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t for da (n= , ), no snp reached genome-wide significance in the discovery phase (supplementary figs. and ). however, through replication analysis (supplementary table ), we identified seven independent loci significantly associated (p< × − ) with da (table , supplementary figs. and ) including areg, esr , znf , lsp /tnnt , igf , tmem b and sgsm /mkl . the areg gene is a member of the epidermal growth factor family that promotes growth of normal epithelial cells and variants strongly correlated with our top snp rs in this region have previously been associated with breast size . although we observed the strongest association for rs , another snp (rs ) located kb away and in weak linkage disequilibrium (ld) with rs (r-sq= . , d’= . ) also reached genome-wide significance (supplementary table ). we investigated these two snps further in , women from the nhs, bbcc, mcbcs and mmhs studies for whom we had individual-level genotype data. both snps were associated with da in this dataset when analyzed separately (β=− . , p= . for rs and β= . , p= × − for rs ). including both snps in the same model attenuated the signal for both snps (β=− . , p= . for rs and β= . , p= . for rs ). thus, it is possible that these two snps are either a proxy for another yet unidentified causal snp or that they represent two independent causal snps. interestingly, rs is located in a weak enhancer region in human mammary epithelial cells (hmec). snps in esr have earlier been associated with breast cancer risk − and rs identified here is in strong ld with the breast cancer snp rs (r-sq= . , d’= . ) and in moderate ld with snps previously associated with breast size . the rs snp in the znf region has been associated with both pd and breast cancer risk but this is the first time it has been found to be associated with da specifically. we observed multiple snps in the znf gene associated with da and since multiple independent snps in znf are associated with breast cancer , , we conducted conditional analyses to identify potential independent signals. in particular, snps rs (r-sq= . , d’= . with rs ) and rs (r-sq= . , d’= . with rs ) showed genome-wide significant associations with da. after adjusting for rs , the associations for both rs (p= . × − before and p= . after adjustment) and rs (p= . × − before and p= . after adjustment) were attenuated. additional analyses in larger datasets will be necessary to determine if there are multiple independent snps in this region. we identified a rare (minor allele frequency (maf)= . ) snp kb upstream of igf that was associated with da. igf is a candidate gene for breast cancer risk and is hypothesized to be involved in breast development. indeed, circulating levels of igf- are associated with breast cancer risk . we also confirmed previous findings that rs in the known breast cancer gene lsp is associated with da and also observed a genome-wide significant association for a weakly correlated snp rs (r-sq= . , d’= . ). both these snps have been associated with breast cancer risk and the recently published icogs analysis of breast cancer found that lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t rs is the snp most strongly associated with breast cancer at the lsp locus. large- scale fine-mapping efforts are needed to pinpoint the causal variant(s). snp rs (tmem b) was associated with both da and pd. a correlated snp rs (r-sq= . , d’= . ) located in the pla g gene has previously been associated with cutaneous nevi . interestingly, two recent independent studies recently reported a link between cutaneous nevi and breast cancer , and it is possible that this link can be partly explained through a shared genetic origin between cutaneous nevi and mammographic density. the snp rs (sgsm /mkl region) is in moderate ld (r-sq= . , d’= . ) with rs that has been previously associated with breast cancer . we also observed several nearby snps located in the tnrc b and mkl genes that were associated with da. however, these snps did not remain significant after adjusting for rs . for nda (n= , ), multiple snps at p . reached genome-wide significance in the discovery phase (supplementary figs. and ); this region has previously been associated with breast size , (table , supplementary figs. and ). replication analysis (supplementary table ) confirmed this region (top snp rs , combined p= . × − ), and this snp was also associated with pd on a genome-wide significant level. for pd (n= , ), the only two regions that reached genome-wide significance in the discovery stage were the previously identified znf and q loci (supplementary figs. and ). through replication analysis (supplementary table ), we identified three new loci (p< × − ) that mapped to prdm , p . and tmem b (table , supplementary figs and ). rs ( p . ) was also significantly associated with nda and rs (tmem b) with da on a genome-wide significance level. snp rs is located in prdm , a gene involved in regulation of endothelial cell proliferation, survival and differentiation. interestingly, we observed a borderline association (p= . × − ) between rs and da (supplementary table ). we also observed two snps in znf , rs and rs , that reached genome-wide significance but their associations were attenuated when adjusting for the known pd snp rs . as with da, analysis in larger datasets will be needed to assess the possibility of multiple independent snps in this region. we used data from the encode project to identify potential overlap between snps in regions associated with mammographic density phenotypes and regulatory elements in mammary tissue (supplementary table ). we identified multiple snps in these regions that were in strong ld (r-sq≥ . ) with the lead snps and mapped to regulatory regions as defined by dnase i hypersensitive site (dhs) or enhancer histone marks in mammary tissue for the esr , igf , tmem b, sgsm /mkl and p . regions. in particular, several snps including rs (proxy for rs ) in the esr region map to a dhs in the breast mcf- and hmec cell lines. snp rs has previously been shown to disrupt a partially methylated cpg sequence within a known ctcf binding site . interestingly both rs and rs (proxy for rs ) in the tmem b lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t region are in regions that bind ctcf. ctcf is believed to play genome-wide role in transcriptional regulation and chromatic structure. in addition, rs also mapped to enhancer histone marks and dhs in hmec cell lines. based on these data, rs and rs are intriguing candidates for further follow up. we also identified snps in these regions that bind several proteins implicated in breast cancer including gata , esr , foxa , yy , rad , smc , gr and egr . to explore potential function of identified snps further, we assessed their association with gene expression levels in adipose tissue and lymphoblastoid cell lines (lcl) . the da and pd snp rs (tmem b) was associated with expression of maff and ankrd in lcl (p< . ) and baiap l in adipose tissue (p< . ). rs (sgsm /mkl ) was associated with sgsm expression in both adipose tissue and lcl (p< . ). we also examined if any of these snps (or proxies) were associated with transcript levels in breast cancer tumors using data from the cancer genome atlas (tcga). we conducted both cis (within mb of the transcription start or end site) and trans (genome-wide) eqtl analysis. although we did not identify any significant pathways in gene-set enrichment analysis, we identified some significant eqtls with a raw p< . (supplementary table ). interestingly, rs in the tmem b region that showed up in the encode analysis was also associated with multiple transcript levels in tcga. to investigate if snps associated with mammographic density phenotypes are also associated with breast cancer, we accessed data from the game-on (http:// gameon.dfci.harvard.edu) and icogs breast cancer meta-analysis based on , cases and , controls (table ). eight out of nine snps were associated with breast cancer risk (p< . ), four of which have already been reported to be associated with breast cancer on a genome-wide significance level (esr , znf , lsp and sgsm /mkl ) – , , . four additional snps (prdm , p . , igf and tmem b) were nominally associated with breast cancer (p< . , table ) and indicate potential new breast cancer susceptibility loci. among the eight snps associated with both mammographic density phenotypes and breast cancer, six snps showed consistent direction between the mammographic density and breast cancer association, whereas sgsm /mkl and p . showed conflicting direction of associations with breast cancer in relation to the mammographic density association. we conducted snp-breast cancer association analyses with and without adjusting for mammographic density (supplementary table ) in up to , breast cancer cases and , controls for whom we had mammographic density data on. we did not observe strong evidence that mammographic density mediates the snp-breast cancer association, but we note that our low sample size limits our ability to draw conclusions from these analyses. the snps identified here explain only a small fraction of the variance of da ( . %), nda ( . %) and pd ( . %). we generated phenotype-specific genotype scores and estimated the difference in density associated with each density-increasing allele carried. the score- specific differences per allele were . cm for da, . cm for nda and . % for pd, respectively. it is noteworthy that two out of three snps associated with pd were associated with either da or nda and that there is overlap between our findings here and two recent gwas of a correlated but distinct phenotype, breast size , . this was also partly reflected in our gwas analyses (supplementary table , supplementary figs. – ). lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t http://gameon.dfci.harvard.edu http://gameon.dfci.harvard.edu discussion in this two-stage gwas of mammographic density phenotypes we identified genome-wide significant loci for all three phenotypes investigated: dense area, non-dense area and percent density. four of the identified regions are known breast cancer susceptibility loci, and four additional regions were found to be associated with breast cancer (p< . ) in a large meta- analysis. these four mammographic density snps represent putative novel breast cancer loci. while the majority of density related snps we identified showed associations that were consistent in direction with breast cancer risk, there were two snps that were inconsistent. rs (sgsm /mkl ) was strongly associated with both dense area and breast cancer risk but in opposite directions. interestingly, there is accumulating data that mkl may have both tumor inhibiting and tumor promoting roles depending on the cellular context. recently, it was shown that the mkl signaling pathway was activated in er-cell lines and silenced in er+ cell lines . additionally, the mkl breast cancer snp has been shown to be associated with triple negative breast cancer . rs has also been associated with sgsm (involved in signal transduction pathway) expression in both lcl and adipose tissue. while these data suggest that this snp influences expression levels that may affect breast cancer risk, it is unclear how well these tissues represent expression in normal breast tissue. the differing effects of this pathway dependent on the er status of the tissue suggest that understanding the cellular environment is important. although the underlying biology is still not well understood it suggests that it is possible that rs affects mammographic dense area and breast cancer risk through different mechanisms associated with different target genes. the majority of women included in our study were postmenopausal at the time of the mammogram. this single assessment of breast density will reflect both the formation of dense tissue early in life, as well as, influences such as age- related and lactation related involution. the apparent opposing directions of this locus on dense area and breast cancer risk may suggest important biologic differences of the effect of this snp on breast tissue and breast cancer risk by factors we are unable to assess in the current study (e.g, age, menopausal status). similarly, rs was also associated with apparent opposing directions on non-dense area and breast cancer risk. again, this may reflect true biologic differences over the life course. for example, it has been demonstrated that adiposity during early life is inversely associated with breast cancer , while postmenopausal bmi is positively associated with breast cancer . there are some weaknesses with our study that should be mentioned. first, we used the hapmap project as imputation panel which prohibited us from assessing the contribution of rare variants. future genetic studies of mammographic density phenotypes should use more dense imputation panels such as the genomes that will provide a more complete coverage of the genome. moreover, it is possible that the causal variant(s) within each mammographic density gwas region was not captured here. pin-pointing the causal variants will require not only denser genotyping and/or sequencing of these regions but also larger sample sizes. another weakness with our study is that it was not designed or adequately powered to test if mammographic density mediates snp effects on breast cancer. lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t future large studies with both mammographic density and breast cancer data should assess such mediation effects. in summary, we report multiple loci associated with mammographic density phenotypes. we identified six da-specific loci of which five showed an association with breast cancer and one pd-specific locus also associated with breast cancer. we also report an additional locus associated with da, pd and breast cancer risk as well as a locus associated with nda, pd and breast cancer risk. these results confirm previous observations that mammographic density phenotypes and breast cancer risk share genetic origin and biological pathways . despite the smaller sample size in this mammographic density gwas (n= , in the discovery and n= , in the replication phase) compared with recent large-scale breast cancer studies (n= , in the discovery and n= , in the replication phase) , our ability to identify known as well as putative novel breast cancer loci by studying mammographic density phenotypes demonstrates the power of using quantitative intermediate phenotypes to discover new disease loci. methods ethics statement each study obtained informed consent from patients and had relevant ethics and institutional approvals from the following institutions. brigham and women’s hospital (nhs), harvard school of public health (nhsii), norwich district ethics committee (epic-norfolk), karolinska institutet (sasbac), mayo clinic (mbcfs, mayo vte, mcocs, mmhs, mcbcs), university health network, toronto, canada (tor), eastern multicentre research ethics committee (sibs), instituto de salud carlos iii (ddm-spain), university of melbourne (amdtdss), university of michigan and university of maryland (ooa), the cancer council victoria ethic commitee (mccs), friedrich-alexander university erlangen-nuremberg (bbcc), nci special studies institutional review board (pbcs) and national research ethics committee (nrec) east of england – cambridge south (search). study design we conducted a meta-analysis of twelve gwas of mammographic density (supplementary note ). for da and nda, we had gwas data from eleven studies and for pd we had gwas data from twelve studies. to follow up promising snps (p< . ) (supplementary tables – ), we conducted replication efforts using data from three different sources: icogs, iselect and in silico look-ups in gwas data. we assessed a total of snps that showed suggestive associations with da, nda or pd for replication. we pursued replication of snps that were included on the icogs array and genotyped additional snps in , women using a customized iselect array. for the replication analysis, we also included data from the old order amish (ooa, n= , ) gwas and for the da analysis, the australian md twins and sisters study (amdtss) gwas (n= ). lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t genotyping, quality control and imputation study participants were genotyped on various genotyping platforms, and standard quality control filters for call rate, hardy-weinberg equilibrium p-value, and other measures were applied to exclude individuals and genotyped snps. to generate a common set of snps for meta-analysis, all studies were imputed to hapmap phase ii (supplementary table ). imputed genotype dosage values (estimated reference allele count with a fractional value ranging from to . ) were generated for approximately . million snps. snps with an imputation quality score < . (as defined by the rsqr_hat value in mach, the proper_info in impute and the information content (info) measure in plink)) or a minor allele frequency < . were excluded. gwas analysis primary association analysis was performed separately within each study. all studies except the toronto/melbourne (tor) and amdtss used linear regression assuming an additive inheritance model. for imputed snps, the estimated number of effect alleles (ranging from to ) was used as a covariate. to account for the family structure in minnesota breast cancer family study (mbcfs) and sisters in breast screening (sibs), we used the “multic” package as implemented in r. multic uses a linear mixed effects model, whereby the genetic relatedness among individuals is incorporated into the covariance structure of the random effects , . the relationships between subjects within the sibs study were adjusted for using the mmscore option within probabel, based on the estimated genomic kinship matrix . the fixed effect is used for the tests of association and covariate adjustment. the tor and amdtss used logistic regression where women in the % top percentile of percent mammographic density (tor) or dense area (amdtss) were defined as “cases” and women in the bottom % percentile were defined as “controls”. as the included data from the nurses’ health study (nhs) were generated using two different genotyping platforms, they were analyzed as two separate studies. similarly, data from the singapore and sweden breast cancer study (sasbac) were obtained through two separate genotyping efforts and therefore analyzed separately. all studies adjusted their analysis for age and bmi. additional study-specific adjustment factors are described in supplementary table . study-specific genomic inflation factors ranged between . and . . meta-analysis meta-analysis was based on summary statistics from the participating studies. for each snp, we combined study-specific p-values and direction of association using the metal software . weights were proportional to study-specific genomic inflation factors and sample size. to account for the extreme sampling scheme in the tor study, we up- weighted the study with a scale factor of . . for a snp to be considered in the meta- analysis, we required genotyping data from at least , women. we used cochran’s q statistic to test for heterogeneity across studies. replication analysis candidate snps were followed up through replication genotyping and in silico look-ups. we obtained replication data from three separate sources: through the icogs genotyping lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t effort, through a customized iselect array and through look-ups in gwas of mammographic density in the ooa and amdtss studies (supplementary note ). in total, we successfully pursued and obtained replication data for snps (supplementary tables – ) selected based on their suggestive association (p< . ) with at least one of the density phenotypes. we also included the breast cancer snps rs , rs , rs and rs since they were associated with at least one mammographic density phenotype at p< . . we extracted genotype data on snps for , women for whom we had both icogs and mammographic density data. for snps that were not included on the icogs array but had a proxy (r-sq≥ . ) on the icogs array, we included the proxy instead. we also genotyped additional snps in , women from the nurses’ health study ii (nhsii), the mayo mammography health study (mmhs), the mayo clinic breast cancer study (mcbcs) and the melbourne collaborative cohort study (mccs) using a customized iselect array. we excluded subjects with call rates < % (n= ) and two subjects (out of included duplicates) that showed multiple discordances leaving , subjects for analysis. remaining duplicates had concordance > %. in addition, we also included association results from the ooa (n= , ) and amdtss (n= for the da analysis) gwas where available. to account for the extreme sampling scheme in amdtss, we up-weighted this study with a scale factor of . . in total, our replication sample size for snps included on the icogs array was , women and the sample size for snps included on the iselect , women. assessment of regulatory functions for identified snps we used the encode data to assess if any of the identified mammographic density snps or their proxies (r-sq≥ . in genomes ceu population) are located in regulatory regions. look-ups were made using the haploreg and regulomedb software. we also investigated if identified mammographic density snps or their proxies were associated with gene expression in cis in adipose tissue and lymphoblastoid cell lines (lcl) in the muther data by accessing the genevar database. to further explore the regulatory properties of the mammographic density snps, we conducted eqtl analyses on mammographic density snps and their proxies (r-sq≥ . ) using data from the cancer genome atlas (tcga). we identified eqtls using beqtl (manuscript under review, http://beqtl.org) that robustly assesses the association between snp genotypes and mrna transcript levels using linear regression with bootstrap. we assessed a total of snps and a total of , transcripts among estrogen receptor positive cases and , transcripts among estrogen receptor negative cases. to robustly define the correlation between snp genotype and gene expression level, the % confidence interval and median of the t- statistic for the correlation coefficient were estimated via statistical bootstrap. for the bootstrap procedure, case resampling was performed n*log(n) times where n is the total number of cases. we computed p-values from the median t-statistic obtained in linear regression. functional gene set analysis was performed using david , (http:// david.abcc.ncifcrf.gov/) for the set of transcripts achieving a raw p-value less than . in the eqtl analysis. lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t http://beqtl.org http://david.abcc.ncifcrf.gov/ http://david.abcc.ncifcrf.gov/ breast cancer association analysis we looked up the association between mammographic density snps and breast cancer in the icogs + game-on breast cancer gwas meta-analysis. the game-on meta- analysis , , , can be found at (http://gameon.dfci.harvard.edu) and is based on eleven breast cancer gwas. in total, the reported breast cancer associations for the replicated mammographic density snps were based on , breast cancer cases and , controls. we conducted logistic regression analysis with and without adjustment for mammographic density including up to , breast cancer cases and , controls from the nhs, nhsii, mcbcs, mmhs, bbcc, sasbac and mccs studies. supplementary material refer to web version on pubmed central for supplementary material. acknowledgments this study was supported by ca , ca , ca , ca , ca , ca , ca , ca , ca , ca , k lm and x hg from nih; genotyping services for the ooa study were provided by the center for inherited disease research (cidr), which is fully funded through a federal contract from the national institutes of health to the johns hopkins university, contract number hhsn ; the breast cancer research foundation, breast cancer research fund; cancer research uk; märit & hans rausing’s initiative against breast cancer; susan komen foundation; agency for science, technology and research of singapore (a*star); david f. and margaret t. grohne family foundation; campbell family institute for breast cancer research; david f. and margaret t. grohne family foundation; ontario ministry of health and long term care; fashion footwear charitable foundation of new york/qvc presents shoes on sale; fis pi from the spain’s health research fund and epy / collaboration agreement between astra-zeneca and the carlos iii institute of health; elizabeth c. crosby research award, gladys e. davis endowed fund, and the office for vice president of research at the university of michigan. epic-norfolk was funded by research programme grant funding from cancer research uk and the medical research council with additional support from the stroke association, british heart foundation, department of health, research into ageing and academy of medical sciences. the sibs study was supported by programme grant c /a and project grants from cancer research uk [grant numbers c / ]. search is funded by a programme grant from cancer research uk [c /a ]. the polish breast cancer study was supported (in part) by the intramural research program of the national institutes of health, national cancer institute. the breast cancer meta-analysis is supported by the game-on drive (ca ) and bcac initiatives. the authors thank the bcac, game-on and drive initiatives for generously sharing breast cancer association results for selected snps. the authors also thank the investigators in bcac, practical, cimba and ocac for access to the icogs data for the replication analysis. bbcc was funded in part by the elan program of the medical faculty, friedrich-alexander university erlangen- nuremberg. we thank all the individuals who took part in these studies and all the researchers, clinicians, technicians and administrative staff who have enabled this work to be carried out. the ooa study investigators thank the members of the amish community for their generous support and participation, the staff at the amish research clinic for their dedicated recruitment and fieldwork efforts, the members of dr. margarita shultz’s radiology clinic for their expert mammography services, the staff at the center for inherited disease research for their exceptional genotyping services, drs. alan shuldiner and braxton mitchell at the university of maryland for their guidance and help with our fieldwork, and terry gliedt, jennifer greene nidetz, kristen maas, cris van hout, james macdonald, chris plotts, lubomir hadjiiski, and chuan zhou at the university of michigan for their technical assistance with data management, entry, and analysis and film digitization and scoring. the pbcs would like to thank pei chao and michael stagner from information management services (silver spring, md) for data management support; laurie burdette, amy hutchinson, and jeff yuenger from the nci core genotyping facility for genotyping support; the participants, physicians, pathologists, nurses, and interviewers from participating centers in poland for their efforts during field-work; drs. jola lissowska and ewa wesolowska from the maria sklodowska-curie memorial cancer center and institute of oncology, warsaw, poland for their lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t http://gameon.dfci.harvard.edu assistance with mammogram retrieval; dr. norman boyd from the university of toronto for providing the mammographic density assessments; and drs. louise brinton, montserrat garcia-closas, beata peplonska, and mark sherman for their contributions to the study design. the authors thank paul pharoah and the search and epic teams. the icogs project would not have been possible without the contributions of the following: paul pharoah, kyriaki michailidou, manjeet k. bolla, qin wang (bcac), andrew berchuck (ocac), rosalind a. eeles, ali amin al olama, zsofia kote-jarai, sara benlloch (practical), georgia chenevix-trench, antonis antoniou, lesley mcguffog and ken offit (cimba), joe dennis, alison m. dunning, andrew lee, and ed dicks, craig luccarini and the staff of the centre for genetic epidemiology laboratory, javier benitez, anna gonzalez-neira and the staff of the cnio genotyping unit, jacques simard and daniel c. tessier, francois bacot, daniel vincent, sylvie laboissière and frederic robidoux and the staff of the mcgill university and génome québec innovation centre, stig e. bojesen, sune f. nielsen, borge g. nordestgaard, and the staff of the copenhagen dna laboratory, and julie m. cunningham, sharon a. windebank, christopher a. hilker, jeffrey meyer and the staff of mayo clinic genotyping core facility. funding for the icogs infrastructure came from: the european community’s seventh framework programme under grant agreement n° (health-f - - ) (cogs), cancer research uk (c /a , c /a , c /a , c /a , c /a , c /a , c / a ), the national institutes of health (ca ) and post-cancer gwas initiative ( u ca , u ca and u ca – the game-on initiative), the department of defence (w xwh- - - ), the canadian institutes of health research (cihr) for the cihr team in familial risks of breast cancer, komen foundation for the cure, the breast cancer research foundation, and the ovarian cancer research fund. the results published here are in part based upon data generated by the cancer genome atlas project established by the nci and nhgri (dbgap study accession: phs .v .p ). information about tcga and the investigators and institutions who constitute the tcga research network can be found at http:// cancergenome.nih.gov/. references . vachon cm, et al. mammographic density, breast cancer risk and risk prediction. breast cancer research: bcr. ; 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[pubmed: ] lindström et al. page nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t lindström et al. page t a b le s n p s as so ci at ed w it h m am m og ra ph ic d en se a re a (d a ), n on -d en se a re a (n d a ) an d pe rc en t de ns it y (p d ). l ea d s n p c h r: p os it io n g en e a ll el es m a f s ta ge z -s co re p s ta ge + β (s e) z -s co re p p ( h et ) s n p s as so ci at ed w it h m am m og ra p h ic d en se a re a rs : a r e g a /g . − . . × − − . ( . ) − . . × − . − . . × − rs : e s r t /a . . . × − . ( . ) . . × − . . . × − rs : z n f g /a . − . . × − − . ( . ) − . . × − . − . . × − rs : l s p t /c . . . × − . ( . ) . . × − . . . × − rs : ig f a /g . − . . × − − . ( . ) − . . × − . − . . rs : t m e m b c /a . − . . × − − . ( . ) − . . × − . − . . rs : s g s m , m k l a /c . − . . × − − . ( . ) − . . × − . − . . × − s n p s as so ci at ed w it h m am m og ra p h ic n on -d en se a re a rs : n /a c /t . − . . × − − . ( . ) − . . × − . − . . × − s n p s as so ci at ed w it h m am m og ra p h ic p er ce n t d en si ty rs : p r d m g /a . . . × − . ( . ) . . × − . . . rs : n /a c /t . . . . ( . ) . . × − . . . × − rs : t m e m b c /a . − . . × − − . ( . ) − . . × − . nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t lindström et al. page l ea d s n p c h r: p os it io n g en e a ll el es m a f s ta ge z -s co re p s ta ge + β (s e) z -s co re p p ( h et ) − . . h g v er si on , in cl ud es n ea rb y ge ne s, m aj or a ll el e (r ef er en ce a ll el e) /m in or a ll el e (e ff ec t al le le ), m in or a ll el e f re qu en ci es ( m a f ) as i n th e g en om es p ro je ct . β a nd s ta nd ar d er ro r es ti m at es w er e ob ta in ed u si ng f ix ed -e ff ec ts m et a- an al ys is o f cr os s- se ct io na l st ud ie s (i .e . s tu di es t ha t an al yz ed d en si ty p he no ty pe s as a q ua nt it at iv e tr ai t) i n un re la te d in di vi du al s. m am m og ra ph ic d en si ty p he no ty pe s ar e sq ua re -r oo t tr an sf or m ed . p -v al ue f or h et er og en ei ty b et w ee n st ud ie s. nat commun. author manuscript; available in pmc april . n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t n ih -p a a u th o r m a n u scrip t lindström et al. page t a b le b re as t ca nc er a ss oc ia ti on s fo r m am m og ra ph ic d en si ty s n p s ba se d on a m et a- an al ys is o f , b re as t ca nc er c as es a nd , co nt ro ls . m am m og ra ph ic d en si ty p h en ot yp e s n p c h r g en e a ll el es z -s co re , m am m og ra ph ic d en si ty as so ci at io n ( de n si ty p h en ot yp e ) b re as t c an ce r a ss oc ia ti on o r ( % c i) p b re as t c an ce r a ss oc ia ti on d en se a re a rs a r e g a /g − . ( d a ) . ( . – . ) . rs e s r t /a . ( d a ) . ( . – . ) . × − rs l s p t /c . ( d a ) . ( . – . ) . × − rs ig f a /g − . ( d a ) . ( . – . ) . rs s g s m /m k l a /c − . ( d a ) . ( . – . ) . × − p er ce nt d en si ty rs p r d m g /a . ( p d ) . ( . – . ) . d en se a re a an d p er ce nt d en si ty rs z n f g /a − . ( d a ) . ( . – . ) . × − rs t m e m b c /a − . ( d a ), − . ( p d ) . ( . – . ) . n on -d en se a re a an d p er ce nt d en si ty rs n /a c /t − . ( n d a ), . (p d ) . ( . – . ) . × − r ef er en ce a ll el e/ e ff ec t al le le d a = de ns e ar ea ; p d = pe rc en t de ns it y; n d a = n on -d en se a re a p -v al ue b as ed o n a fi xe d ef fe ct s m et a- an al ys is . e st im at es a nd s ta nd ar d er ro rs f or e ac h br ea st c an ce r st ud y w er e ca lc ul at ed u si ng l og is ti c re gr es si on . nat commun. author manuscript; available in pmc april . wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ diplomarbeit / diploma thesis titel der diplomarbeit / title of the diploma thesis „gender roles in amish literature: an analysis of how gender roles are portrayed by non-amish and amish writers in amish romance novels “ verfasst von / submitted by mag.rer.soc.oec. christine pünner angestrebter akademischer grad / in partial fulfilment of the requirements for the degree of magistra der philosophie (mag.phil.) wien, / vienna, studienkennzahl lt. studienblatt / degree programme code as it appears on the student record sheet: ua studienrichtung lt. studienblatt / degree programme as it appears on the student record sheet: lehramtsstudium uf mathematik uf englisch betreut von / supervisor: univ.-prof. mag. dr. alexandra ganser-blumenau acknowledgments: i would like to thank my family, especially my husband and my son, for bearing with me while writing and finishing this thesis in these last months. i am sorry for not taking as much time for you as i should have, and i promise that this will change in the days ahead. thank for your patience and support. i would also like to thank my supervisor, univ.-prof. mag. dr. alexandra ganser- blumenau for her help with literature suggestions, helpful hints in structuring this thesis and most of all, for her patience and understanding, as the completion took longer than expected. table of contents . introduction .......................................................................................................... . the amish ............................................................................................................ . . a brief history of the amish ............................................................................ . . a short overview of amish concepts and customs......................................... . . the history of amish literature ..................................................................... . theoretical background ..................................................................................... . . the literary genre of amish romance novels ............................................... . . gender ........................................................................................................ . . . the concept of gender .......................................................................... . . . gender stereotypes .............................................................................. . . . gender roles among the amish ............................................................ . gender roles in amish serial novels ................................................................... . . a lancaster county saga by wanda brunstetter ( ) ............................. . . . main female and male characters and their gender roles ..................... . . . other female/male gender roles ............................................................ . . healing grace series by adina senft ( ) ............................................... . . . main female and male characters and their gender roles ..................... . . . other female/male gender roles ............................................................ . . lancaster burning series by linda byler ( ) ......................................... . . . main female and male characters and their gender roles ..................... . . . other female/male gender roles ............................................................ . conclusion ......................................................................................................... bibliography .............................................................................................................. appendix ................................................................................................................... english abstract .................................................................................................... deutsche zusammenfassung ................................................................................ . introduction within the last decades the amish people in the united states have been moved into the focus of american media. they continue to be the topic of hollywood movies such as witness ( ), for richer or poorer ( ), plain truth ( ), saving sarah cain ( ), amish grace ( ), and pee-wee’s big holiday ( ). furthermore, they are repeatedly the subject of tv shows like for weddings ( ), breaking amish ( - ), and return to amish ( ) and – especially since the late s – romantic fictional novels (igou ). the typical elements that can be found in contemporary amish romances are the concepts of rumspringa , shunning , and evangelical piety (weaver-zercher ). the internal struggle of the amish heroine – or sometimes hero – between following her or his tradition and leaving the amish church seems to be a never-ending storyline that continues to attract readers throughout the united states, especially those of christian faith. a further element of amish fictional novels is the withdrawal from the world that is offered to the reader, especially because the stories take place in a contemporary and real setting (shelley ). no matter how fascinating these novels might seem to their readers, the question arises whether these representations of amish men and women, if portrayed by non- amish authors, are similar to those described by amish writers or simply correspond to wishful thinking. due to the fact that the amish do not hold publishing in high esteem, most of the fictional literature of the last decades stems from non-amish writers. fortunately, this situation has changed within the last ten years, when amish writers such as linda byler and lena yoder started writing amish biographies or novels. apart from these amish writers, there are also those authors who have grown up within the amish church but left their community in later years. their inside experience offers a less stereotypical view of amish men and women. before insight is given into the way amish gender roles are represented by non- amish as well as amish writers, a short account of the amish people’s history will be provided in order to understand their tradition and way of life more clearly. see chapter . . the amish . . a brief history of the amish the origin of the amish people, also called plain people, lies in the christian reformation movement that took place in europe in the fifteenth and sixteenth centuries. huldrych zwingli, one of the two famous swiss reformers, was joined by a man named felix manz. because of the fact that zwingli continued to baptize babies, “felix and some others broke away and founded a church in which only adults were baptized” (igou ). similar movements took place in the netherlands and in german-speaking central europe. they argued that baptism “should be a mark of voluntary commitment and therefore fitting only for those who understood the implications of a disciplined life” (nolt - ). as these believers, who had already been baptized as babies before the reformation movement started, now supported the baptism of adults, they were called anabaptists, meaning re-baptizers (nolt ). the amish consider these anabaptists as their ancestors and a collection of stories of these martyrs can be found in the ausbund, a hymnal that was first printed in and is still in use in amish worship services (igou ). the reformation did not only affect germany and switzerland, but also spread throughout other parts of europe. menno simmons, who converted to anabaptism in , became another well-known leader and writer (cooper ). the believers following his teachings were called the mennonites and during the s, they mainly lived in switzerland, alsace, northern germany, southern germany, and holland (igou ). the name amish stems from jacob ammann, who was one of the elders of the mennonites in alsace at the end of the th century. at that time, a conflict arose between him and hans reist, the elder of the swiss mennonites, concerning certain issues regarding the church. among those was the frequency of communion in church services, the question of shunning and banning, as well as the issue of clothing. the people in alsace were strict in this respect, promoting plain and uniform dress, whereas the swiss mennonites were more liberal. finally, in the fall of , this conflict resulted in a separation of the swiss and the alsace believers, and in the course of time, jacob ammann’s followers became known as the amish mennonites, shortened to “amish” (igou - ). in essence, there were nine points in which the amish differed from the other mennonites at that time. to be able to understand their lifestyle and culture as it is described in the literature that will be discussed in this thesis, some of these nine points are quoted here as they are listed in igou’s book (igou ). . ammann believed in the literal shunning of those who are excommunicated. . ammann did not believe the true-hearted (those who gave food and shelter to the anabaptists) should be considered as christians. . ammann felt the need for stronger discipline in dress and everyday living. . he felt that men should cut their hair fairly short and let their beards grow; the others followed the prevailing fashions of letting their hair grow very long and shaving their beard. several attempts were made to unite these groups again, but none of these succeeded at that time. in the eighteenth and nineteenth centuries, two waves of amish immigration to the united states took place, with most of them settling in pennsylvania at first. now they live in more than settlements in thirty states, as well as in one canadian province (weaver-zercher ). for more than a century, the amish in america lived in unity. but around , various tensions arose as new inventions such as photography and new styles in clothing developed. some amish settlements were more progressive and in order to keep the unity among the various groups, a ministers’ meeting was held in june of and another one in . however, these meetings only resulted in unity among the progressive bishops, who consequently formed a new group, the “amish- mennonites” (igou - ). those groups who decided to keep to the old rules were henceforth called the “old order amish”. in the first half of the twentieth century, another separation occurred, initiated by moses m. beachy. he allowed modernity to enter his community, favored a less strict discipline, and separated from the old order amish. these groups were later called the “beachy amish” (johnson-weiner ). in the following illustration, an overview on amish roots is given: fig. . anabaptist-amish timeline (nolt ) it is the old order amish who are at the center of the growing number of amish novels with their horse-and-buggies, their plain and sober clothing, and long beards. according to the amish, each tradition, restriction, or rule is based on the bible and often the result of some controversial issue the church faced in the past. as one amishman once explained, “if we see or experience something that is not good for us spiritually, we will discipline ourselves to do without” (igou ). to hold up their standard of nonconformity, the amish put a lot of emphasis on discipline (igou ). concerning the media, only print media such as books, magazines and newspapers are accepted among the old order amish. they have their own publications about community news such as the budget and the message, which differ from outside publications insofar as they put a focus on reporting news that convey the normal rather than writing about events which disrupt the normal (cooper ). other well- known publications that are popular among the amish are family life, young companion, and blackboard bulletin. these magazines focus on literature concerning their own values and are plain, black and white, without photos or fancy graphics, simply containing text and line drawings (igou ). . . a short overview of amish concepts and customs rumspringa rumspringa is the pennsylvania dutch word for “running around” and it is used to describe the time when amish teenagers are sixteen or seventeen and are sent out into the world. this time is a sort of ritual, where parents expect their children to test their own faith before they come back to the community and get baptized or married. in contrast to popular belief, the teenagers are not sent out by their parents to try out all kind of deviant behavior. also the way the parents handle their teenage offspring during that time varies from family to family. furthermore, the youngsters still continue to live with their families, but are simply allowed more independence than before. nevertheless, they are expected to help full time on the family farm or in the household – either in their own family or at a relative’s home. they often spend their weekends not at home but meet with amish friends of their own age (nolt - ). these cliques are also known as die youngie (young folks). it should be mentioned that during this period the amish teenagers are not at risk of being shunned, because they have not been baptized yet and are therefore not members of the amish church (nolt ). shunning the practice of shunning is another part of the amish church that is a common theme in amish fiction. however interesting it might be to readers, it does not happen very often within an amish community. now what does shunning actually mean? the amish, as believers in the bible, have certain rules and customs that members – those who have been baptized – have to obey and follow. if someone falls short and disobeys, the opportunity for confession is given. only if individuals do not show any remorse, do not want to confess, or show through their actions that they want to leave the church, they are excommunicated. the reason why excommunication or shunning, as it is called, does not happen that often, is that those people who really want to leave the amish usually do not get baptized in the first place (nolt ). when shunning happens, “members will avoid the person in certain symbolic ways, such as not sharing a meal together and not entering into business contracts with them” (nolt ). depending on the situation that leads to shunning, shunned people might feel betrayed and get bitter. therefore, the consequences are of a more severe nature than in those communities where a positive relationship with other amish people is maintained. from an amish viewpoint, shunning “punishes the evildoer not in the spirit of revenge, but to bring him to repentance” (igou ), meaning that it is possible that the so-called bann (shunning) can be reversed. ordnung the term ordnung describes a moral road map of an amish community and offers a guideline as to how the community members should lead their lives. these guidelines are meant to be a helpful resource in areas of contemporary life where the bible, that the amish see as their highest authority, offers no clear instructions. ordnung directs as well as limits what a person should do, including for example the means of transportation, the style of clothing, or types of technology used at home (nolt ). in contrast to other religious regulations, such as the jewish mishnah or the islamic hadith, ordnung is not written down but passed on orally to the next generation ( ). furthermore, it is not a fixed set of rules but responds both to developments in the real world and to the particular circumstances a person might find her- or himself in. for example, lay people enjoy a bit more flexibility that a bishop of an amish community, who has more responsibility and should serve as a role model to other members ( ). one or more of these three concepts – rumspringa, shunning, and ordnung – are usually used as tensions in amish literature, as they are often a source of conflicts and developments among the protagonists of a story. for example, there might be the amish teenager questioning the ways of his upbringing, or a shunned family member struggling with the distance to his family and friends, or a young girl feeling limited in her personal development due to community rules. therefore, these three customs also create tension in one way or another in the three serial novels discussed in this paper. . . the history of amish literature as already mentioned, print media is the only form of media that is accepted among old order communities. besides the bible, they have their own magazines such as die botschaft and family life (cooper ), publications that inform their readers about local news, every-day issues that concern their daily lives and problems, and sometimes short fictional stories that try to represent their way of life. die botschaft, for example, “employs dialect and description of outsiders as ‘strangers’ and ‘english’, which reinforces the solidarity of readers” (cooper ). a non-amish reader would probably describe its articles as boring. apart from these internal newspapers that are well-known and respected among the amish communities, not much other literary effort has been made throughout the th century. however, the first time the amish received a greater audience in print media was when helen reimensnyder martin wrote the novel sabina: a story of the amish in . her book includes the typical elements of a rural surrounding, rumspringa and romance for the first time (weaver-zercher ). this combination of themes proved to be quite successful in amish literature decades later. the story revolves around the amish protagonist sabina, who is supposed to marry a dull amish farmer, because this is what her despotic father has planned. as time goes by, sabina meets augustus, who is not amish, and they fall in love with each other. however, the heroine and the hero do not end up together, but sabina marries the amish farmer. although the lives, customs and the appearance of the plain people are vividly described in the book, the amish are not put on a pedestal; rather, they are portrayed as people of bad manners, primitive, and lacking sophistication (stekovic ). three years later, cora gottschalk welty took up the amish topic again with her novel the masquerading of margaret. this time the female and male protagonists are both outsiders, but they meet while staying in an amish community. margaret habecker, a wealthy new yorker, visits her amish relatives and decides to dress like them during her stay. at the same time, robert jackson, also english, resides at margaret’s relatives’ home. both are positively impressed by the simple lifestyle and experience a transformation in their own lives. finally, they become a couple, marry, and although they return to the city, their encounter with the old order community has changed their attitudes forever (weaver-zercher ). in contrast to sabina, welty’s novel portrays the amish in a positive way, and it again includes the elements of rurality and romance. just like martin’s book, it offers the readers an inside glimpse into a different life, but at the end, the protagonists can return to their normal surroundings and do not really have to interfere with the unknown (w eaver-zercher ). furthermore, these two novels are a good example of the dialectic manner how the amish are represented: they are either portrayed as “american saints” or “denigrated as a fallen people” (weaver-zercher ). this pattern can repeatedly be observed in works of fictional literature that have been written about the plain people until the present day. in , the third notable amish novel, ruth lininger dobson’s straw in the wind, was published. the story is set in a northern indiana amish community and portrays an egoistic and authoritarian amish bishop called moses bontrager, who forces his will on his wife and daughters. in the end, his wife dies from overwork, one daughter is forced to marry someone she does not love, and the other one is prohibited from marrying the man she loves. the book was a success and was even awarded the hopwood prize from the university of michigan (weaver-zercher ). the mennonite and amish communities, however, were shocked about this negative description of amish leaders and one of them decided to take action and protest against it in a literary way. this man was joseph w. yoder. in , he wrote: “being amish born, raised amish and still belong [sic] to the amish church, i wish to protest against the many misrepresentations of [sic] that novel” (qtd. in weaver-zercher ). consequently, he started writing the story of his mother, rosanna of the amish. it is about the life of his mother rosanna, who was born into an irish family living in america but was adopted by an amish woman after her mother died in childbirth. in his book, yoder includes lengthy illustrations of amish culture and family life to positively portray his community and, unlike other writings, covers the whole span of his mother’s life. in the midst of the twentieth century, another element was added to amish novels by clara bernice miller. she was the first author to include evangelicalism into her stories. from now on, most novels do not only describe rural life and the protagonists’ struggle with amish tradition, but also focus on a personal experience with christ, including born-again salvation as well as evangelism (weaver-zercher ). for example, in katie, which was published in , the heroine starts questioning her image of god. by reading christian romance novels, something her family frowns upon, “katie learns the evangelical message of personal salvation” ( ). miller, who was raised amish and later joined a more liberal mennonite church, hoped that her books would be read by the old order people. her idea was that her books would motivate the amish to base their christianity not on their traditions alone but to search for a more personal experience of god. this amish “mission movement” started in the s and continues until today. at the end of the twentieth century, beverly lewis followed in miller’s footsteps when writing amish romance novels, the most popular among them being the shunning ( ). about three years later, wanda brunstetter followed suit and used amish fictional novels to portray an amish protagonist so that he or she would set an example on how to become a better christian (stekovic ). both lewis and brunstetter are not amish themselves, but by referencing research about the amish, they “position themselves as credible insiders, thus seeking to mitigate the charge of factual errors and to gain authenticity – and hence popularity and sales” (graybill, chasing ). nevertheless, stekovic ( ) claims that striking differences can be noticed between novels depending on the author’s knowledge of amish culture and traditions. apart from these authors who try to add authenticity to their work by either doing research on the amish themselves or by referring to existing research, there is a small group of amish writers who have published their own books. among these is joseph w. yoder, who has been mentioned before, as well as emma king, who, in , wrote about a tragic event that unsettled her family. in , brad igou, despite being non-amish, undertook the task of collecting several years’ worth of texts by amish that had been published in their magazine family life. he sorted the various contributions by topic and gave the book the appropriate title the amish in their own words. in the course of the last decade, however, two remarkable new and truly amish releases have conquered amish literature. the first one was written by linda byler, who started writing amish novels in and has published more than twenty of them until today. as graybill (chasing ) reports, “byler still composes by hand, writing thoughts by pencil in composition books. her writing is candid, humorous and true to her life as an old order amish woman in more isolated pennsylvania communities.” the second notable author is lena yoder, whose first book was a diary called my life as an amish wife ( ). apart from these two distinct groups of writers – those who are either truly amish or total outsiders – another branch of authors has joined the market in recent years. they are in a certain way a subgroup of amish writers, because they were either raised in amish families but later in life left the community such as jerry eicher ( ), ira wagner ( ), emma gingerich ( ) and misty griffin ( ) or joined the amish faith like marlene miller ( ). these books are all autobiographies and allow the reader an inside glimpse into amish life. as different as the background and motives of all these authors might be, they have one thing in common: in one way or another, they write about a community where gender difference is at least obvious in the members’ outward appearance. consequently, the question arises whether these outward differences are also reflected in the character and behavior of males and females in amish literature. therefore, the focus of this paper is on the following research question: how are gender roles described by various novelists writing about the amish and does the writer’s background influence these descriptions? in order to answer these questions, three recent serial novels have been chosen. two of them were written by non-amish authors: a lancaster county saga ( ) by wanda brunstetter and healing grace ( ) by adina senft. the third one, lancaster burning ( ), was written by the amish writer linda byler. . theoretical background . . the literary genre of amish romance novels the three works discussed in this paper all belong to the subgenre of amish romance novels. as the term romance novel suggests, this genre comprises elements of a novel and a romance. but what exactly is the difference between these two genres, or, on the other hand, what do these two have in common? although it is not easy to define these two terms clearly, one attempt has been made by richard chase, who asserts that “[t]he main difference between the novel and the romance is the way in which they view reality” (qtd. in post ). according to him, the events in a novel will usually be more plausible and closer to reality than in a romance, which often describes astonishing events that serve a more symbolic cause. furthermore, the characters in a romance tend to be ideal and somehow abstract, whereas in novels they are more important and more closely observed and depicted ( ). a similar explanation has been given by clara reeve in , when “[s]he defined the novel as ‘a picture of real life and manners, and of the times in which it was written,’ whereas the romance ‘in lofty and elevated language, describes what has never happened nor is likely to’” (qtd. in regis ). when romance and novel are now combined, one could basically define this genre as a “prose fiction love story”, which includes the themes “love”, “a happy ending”, and the importance of the heroine (regis ). these themes are then translated into narrative events, which are “courtship” and “betrothal”, which regis explained like this: heroines and heroes in love conduct a courtship—that is the action in the novel that expresses the love noted by all of the critics. courting couples become betrothed—that is the action that leads to the universally endorsed happy ending. the wedding itself is often omitted, but it is always promised in a betrothal. ( ) regis continues to expand this basic definition of a romance novel by stating eight essential narrative elements that are usually included in this genre: in one or more scenes, romance novels always depict the following: the initial state of society in which heroine and hero must court, the meeting between heroine and hero, the barrier to the union of heroine and hero, the attraction between the heroine and hero, the declaration of love between heroine and hero, the point of ritual death, the recognition by heroine and hero of the means to overcome the barrier, and the betrothal. these elements are essential. ( ) sometimes, additional elements such as a bad character, who is converted to goodness, a scapegoat, who does not really appear in the actual events, or a wedding might occur in the romance novel, but they remain optional ( ). turning now to the subgenre of amish romance novel, further elements can be identified. one predominant feature is religion, thereby putting the amish romance novel in the category of inspirational fiction. faith and culture always play an important part in the protagonists’ lives and influence their thoughts, feelings and actions. the recurring literary element of a rural setting for the storyline shows traces of regional fiction, too (weaver-zercher ). furthermore, the heroine usually struggles in one way or another for self-fulfillment and finding herself ( ). overall, the amish romance novel is “the province of purity culture” ( ), not containing sexual elements, refraining from modernity on various levels and thereby, it “becomes for its readers a tripartite literature of chastity: chaste texts about chaste protagonists living within a chaste subculture” ( ). in her book “the thrill of the chaste”, diane weaver-zercher quotes from an article about amish fiction in publishers weekly the following “recipe for an amish novel”: take one young woman (sarah, katie, or rebecca), one young man (jacob, daniel, samuel). add one, or more, problems: someone is and unmarried. someone has a family secret. someone is tempted by life outside the amish community. someone’s heart has been broken. mix together with one daed, one mamm, assorted siblings. (optional: add grossdawdi and/or grossmammi). bake together for pages till resolved. garnish with pennsylvania dutch glossary or recipes or quilt pattern. (qtd. in weaver-zercher - ) although written in a rather cynical way, this recipe nevertheless includes core elements that can be found in most amish romance novels: the occurrence of pennsylvania dutch dialect in the dialogues, a love story, a character questioning and finally finding faith in god, and the importance of relationships and family. in addition to these themes that usually appreciate amish community values and tradition, sigrid cordell also observes that “gothic tropes of confinement and escape” (cordell ) are often part of the storyline. as the amish reject modern technology, these novels are somehow set in an earlier time period, which is a common element in gothic romance ( ), and as the ordnung emphasizes rules and regulations, an atmosphere of confinement is created ( ). . . gender . . . the concept of gender the term gender originates from social studies and can be generally defined as describing female or male characteristics of a person within a society or culture. it has to be distinguished from the term sex, which mainly defines humans by their biological features, namely their reproductive organs and structures. within the study of gender, however, there have been various viewpoints on this issue ever since this subject area has been researched. at the beginning of the twentieth century, the underlying idea was “that male-female psychological differences were natural, deep-seated, and of profound personal and social consequence” (shields and dicicco ). in the s, researchers tried to establish a measurement scale for masculine and feminine characteristics to reveal differences between males and females, which did not prove very reliable ( ). in fact, studies conducted at the end of the th century “revealed the multidimensionality of gender – that is, ‘gender’ encompasses distinct factors that cannot be used to predict or make generalizations about gender-related attitudes or behaviors” (shields and dicicco ). . . . gender stereotypes the encyclopedia of women and gender ( ) defines gender stereotypes as “organized, consensual beliefs and opinions about the characteristics of women and men and about the purported qualities of masculinity and femininity” ( ). these beliefs do not only describe the traits of women and men, but also prescribe certain ways of behavior, telling them who they are expected to be ( ). the traditional gender roles that most of us are aware of, follow a patriarchal model, where men are the privileged gender. they are considered as “rational, strong, protective, and decisive”, whereas women are seen as “emotional, weak, nurturing, and submissive” (tyson ). even though these traditional views are constantly being challenged in our western culture, they are still in effect today, excluding women from leadership positions and careers in engineering, and often paying them lower wages for doing the same job ( ). the concept of patriarchy is based upon the belief that the biological difference between the sexes implies gender difference, stating that “women are innately inferior to men” and therefore not capable of the same tasks as men or lack certain abilities such as logical thinking ( ). feminists do not negate the biological differences, but object the view that women thus have an inferior status. they draw a clear line between the terms sex and gender ( ). apart from the fact that stereotypical gender roles discriminate women, also men are negatively influenced by this patriarchal ideal. for example, they are expected to be strong and emotionally stable, and are not supposed to show any weaknesses such as crying or being afraid. furthermore, the failure to economically support his family can have a humiliating effect on a man, because it implies that he cannot fulfill his role as provider ( ). even though the patriarchal model has been challenged in our world today by women acquiring leadership positions and pursuing careers outside their homes, the underlying patriarchal gender roles are still in effect, as tyson explains with this example: in upwardly mobile, middle-class american culture today, the woman on the pedestal is the woman who successfully juggles a career and a family, which means she looks great at the office and over the breakfast table, and she’s never too tired after work to fix dinner, clean house, attend to all her children’s needs, and please her husband in bed. ( ) as it can be seen, the stereotypical gender roles are not a concept of the past, but still persist in our modern society. . . . gender roles among the amish as the amish reject technology and modernity, their social life and culture remain traditional, too. at first sight, they follow a patriarchal form of society, and gender roles throughout the various communities are generally clearly defined. broadly speaking, amish men are the ones who carry the main responsibility for the financial support of the family. their role is to be the head of the family and its spiritual leader. women are expected to support their husbands and to take care of the household and the children. the idea behind these gender roles is rooted in the bible, which talks about a divine hierarchy (johnson-weiner ). this hierarchy “places the man at the head of the household just as christ is the head of the church” ( ). however, the bible also states that the church is seen as the body of christ and “that in christ there is no male or female” ( ), thereby giving men and women equal importance. the equality women share with men as christians has even a higher status than her subordination to men in the communal hierarchy ( ). as a result, men and women in old order amish communities are connected to and influence each other by sharing responsibilities in daily activities. in fact, there are “patterns of social interaction that give women power and responsibility equal to that of men in ensuring the survival of the church community” (johnson-weiner ). both, men and women take part in informal discussions concerning the church community. within a family, men and women have different tasks – the amish wife takes care of the children and the household while the husband is in charge of farming and other economic dealings. in reality, however, they often share their tasks, helping each other with their responsibilities ( ). as igou ( ) quotes from a family life (december ), the amish have defined the roles like this: woman was not taken from man’s head for him to lord over her. she was not taken from his feet so that he could trample on her or kick her around. but she was taken from his side, close to his heart, thus being a loving partner to help him with life’s responsibilities [gen. : - ]. among [her duties] are allowing the husband to be the head of the house, converting a house into a home, and assuming the responsibilities of motherhood. certainly the submission of the wife does not mean slavish subjection, but a joyful working together for a common cause. ( ) it can be seen, that amish women do not consider their role as housekeeper as inferior, but rather as complementary to their husband’s role as bread-winner. even though husband and wife seem to follow traditional gender roles concerning their tasks, the underlying assumption that the social status of man and woman is not equal does thus not stand up to scrutiny. another amish woman describes her marriage in family life (february ) like this: “[…] i began to realize we can complement each other. in many areas we compromise. it is essential to give in to each other, but it is not necessary to lose one’s individual identity.” (igou ) as it can be noted, women in amish society are not subject to an inferior status but see men and women’s gender roles as complementary to each other. this division of roles while working together and sharing equal importance is an inherent characteristic of the old order amish communities., however, as some communities adapted to modernity over time and also some old order men started getting engaged in working off the farm, the relationship between men and women changed. on the one hand, women’s work stayed relatively constant, but the husbands could not help as much at home anymore. this resulted in a higher formal hierarchy with males becoming more dominant (johnson-weiner ). to deal with this new situation accordingly, a minister writing in family life admonishes community members to bear in mind what the scriptures say: whereas wives are counseled to ‘submit yourselves unto your own husbands,’ husbands are reminded to be ‘patient, forgiving, tender, appreciative, and sympathetic … reasonable, considerate, compassionate, courteous, and gentlemanly.’ (johnson-weiner ) through allowing modernization to enter some amish communities, these groups separated from the old order amish, forming the beachy amish and fellowship churches. johnson-weiner observes that within these new-found churches the belief that man should dominate woman has grown stronger. consequently, women do no longer have as much influence on church decisions as before: they are only allowed to discuss issues with their husbands at home, but in church, the husband speaks for his wife and additionally, as a couple they only have one vote together – women do not vote at all ( - ). furthermore, the beachy and fellowship doctrine holds that god created men and women “as different types of beings, with specific and distinctive roles” ( ). therefore, the gender roles in these communities are far more fixed and more clearly defined than in old order communities. observing this development, it is interesting to note that women of more traditional amish communities have a higher social status and more rights than those in more progressive ones. it seems as if the more intimate interaction with modern american society has challenged amish men to emphasize their role as head of the household. dress codes one important feature that reflects particular gender roles is the strict dress code in amish communities. through their clothing the members distinguish themselves from the surrounding culture (graybill, meanings ). the regulation concerning their outfit are far more restrictive for women than for men, “allowing men to ‘pass’ more easily in the outside world” ( ). women have to wear plain dresses as well as a head covering. those long-sleeve dresses come in ordinary colors, include full skirts and are covered by an apron. the dress should reflect womanly virtues such as “submission, gentleness, motherliness, patience, and meekness” ( ). as amish themselves describe, wearing this outfit makes them feel “less susceptible to harm” ( ) and it also “provides [them with] internal motivation for upright, virtuous living” ( ). their hair is never cut, but they wear it in a bun under their head covering. men, on the other hand, wear dark-colored suits, straight-cut coats, broad trousers with suspenders and solid-colored shirts. another part of their attire are black or straw broad-brimmed hats and after marriage, they grow beards. despite their clothing, women engage in activities such as playing softball or working in the garden. compared to men’s clothing, which emphasizes agility and physical effectiveness, women are obviously restricted in their body movements by their dresses, which according to kaiser should emphasize stability and, to a certain extent, attractiveness (graybill, meanings ). summarizing these amish traditions and old order customs, the following gender roles and attributes can be observed: women are expected to be family-oriented, patient, modest, upright, traditional, and hard-working. if married, they should support their husband and take care of the children. even though they should have a submissive attitude, they share an equal status with their husband, when decisions need to be made. amish men, on the other hand, are supposed to be the financial providers of the family. therefore, they should be hard-working, strong, responsible, and traditional. nevertheless, they are also described as family-oriented, respectful and gentlemanly, taking care of their wives’ and children’s needs as well. in the following literature analysis, the gender roles in three amish serial novels will be closely studied in order to answer the following questions: how are female and male roles defined in the novels? do the characters follow a patriarchal gender role model or do they rather support the complementary model of amish communities? do characters reflect traits from opposite genders and how do others react to them? how is the relationship between women and men portrayed? and finally, is there a difference in gender role representation between the two non-amish authors, wanda brunstetter and adina senft, and the amish writer linda byler? . gender roles in amish serial novels . . a lancaster county saga by wanda brunstetter ( ) wanda brunstetter started writing amish fiction a few years after beverly lewis. her husband, richard, grew up in a mennonite church and has amish ancestors. they spent a lot of time among amish communities and befriended members of the old order people. to gain credibility in her writing, brunstetter took time to research the amish communities and claims that “many of her books are well-read and trusted by the amish” (wanda e. brunstetter). the six-part series a lancaster county saga was released in and revolves around a young amish couple in lancaster county, meredith and luke stoltzfus. struggling financially, luke agrees to learn a new trade at a relative’s business in indiana in order to take it over in the future. on his way to his relative, he has to change bus in philadelphia. at a diner close to the bus stop, a homeless man watches luke as he takes out his wallet to pay for his meal. the vagabond notices that luke has many bills in his wallet and plans to rob him. back at the bus stop he takes his chance when luke enters the restroom and beats him senseless. as he also discovers luke’s bus ticket, he exchanges his clothes with luke’s and continues the bus travel with luke’s documents. the bus has a deadly accident and burns down completely. while luke’s family believes that luke is dead, luke himself ends up in hospital with no memories of his name and his past. after his physical recovery, an older christian couple with two young granddaughters offers him a new home until he can remember his past life. meanwhile, meredith, who believes that luke has died, gives birth to their first child and is courted by another young amish man. at the same time, luke is about to fall in love with one of his host’s granddaughters. finally, in the sixth part of the series, luke’s memory returns and he travels back home – just in time before meredith marries jonah miller, her new suitor. . . . main female and male characters and their gender roles luke stoltzfus right at the beginning of the story, the reader is confronted with the couple’s financial struggle that obviously has a direct effect on their marriage. luke does not seem as attentive towards meredith as he had been before, and he tries to keep to himself (brunstetter, goodbye ). luke feels it is his duty to provide for his young family as is described in the first chapter: “on more than one occasion, meredith had suggested that she look for a job, but luke wouldn’t hear of that. he insisted that it was his job to provide for them.” ( ). by using the phrases “on more than one occasion” and “he insisted”, the tension between the couple is highlighted, and a vivid picture of a tight financial situation is created. furthermore, the image of luke as a patriarch, who is not willing to admit his failure in his role as breadwinner, is reinforced: “luke’s folks had offered to help out financially, but luke had turned them down” ( ). his responsibility weighs heavily on him and he seems unwilling to thoroughly discuss the situation with his wife, but insists on being the head of the household: “[…] until we get on our feet again, we should leave well enough alone.” “but luke, if you knew —” “mir sin immer am disch bediere iwwer eppes.” he frowned. „and i’m gettin‘ tired of it.” “it does seem like we’re always arguing about something,” she agreed, “and i don’t like it, either.” “then let’s stop arguing and talk about something else.” irritation edged luke’s voice. “you can be so eegesinnich sometimes,” she muttered, looking away. “i’m not being stubborn; i’m being practical. and as far as i’m concerned, this discussion is over!” ( ) this lively description of their dialogue shows luke’s dominance: he frowns, speaks with irritation in his voice and finally ends the discussion, ignoring his wife’s criticism. meredith, however, does not speak clearly, but is “muttering” and “looking away”. with these phrases, brunstetter paints a picture of an inferior woman, who simply gives up on stating her opinion equal. although the male protagonist is described as dominant, in a short flashback the reader understands that the situation was different before: “[…] until recently they’d always discussed things and made important decisions together” ( ). this indicates that as long as everyday life is without any challenges, luke treats his wife as an equal counterpart. but as soon as his male role as provider is attacked, equality does not count any more. however, as the financial situation grows worse and luke realizes that something needs to be done, he remembers that such an important decision as investing all their finances into a new trade cannot be made by him alone – it has to be in agreement with his wife: how would she take the news that he wanted to withdraw money from their dwindling bank account to learn a new trade he wasn’t even sure he’d be any good at? not only that, but would meredith be okay with him being gone for a few weeks until he learned this new skill? ( - ) [luke:] “i shouldn’t be gone more than a few weeks, and i’d really like your blessing on this new venture.” ( ) as brunstetter writes the story from a third-person omniscient perspective, the reader cannot only observe luke’s actions and conversation, but also his thoughts. these convey that he really cares about his wife’s opinion on his enterprise. he does not only want to reach an agreement with his wife, but is also not ashamed of admitting a wrong and apologizes to his wife when he says: “’i know i’ve been kinda hard to live with lately, and …’ he paused and reached for her hand. ‘es dutt mir leed.’” ( ). through using pennsylvania dutch dialect, a feeling of intimacy is created. moreover, the reader can also observe that luke really wants to please his wife and fulfill her wishes as well. although at first he denies her wishes for a new paint inside the house because of financial restrictions, he plans to reconsider this decision on his way to his uncle: “i’m gonna tell her” and “i’m sure she’ll be pleased” ( ). this shows an inner struggle between feeling responsible as a bread-winner and his desire to be a considerate and respectful husband, who wants to show respect towards his wife, accepting her as a partner in their marriage. one – for amish male characters – rather unusual characteristic, at least as their culture prescribes, is luke’s desire to know about the world: the bus he’d be transferring to in philadelphia wouldn’t be leaving the station until : a.m., which meant he had plenty of time to get a bite to eat and buy a newspaper, so he’d have something to read. luke was always interested to see what was going on in the rest of the world and wanted to check for any articles on the weather to see if the storm had reached the areas where he’d be traveling. ( ) but he does not only want to know about the news in the world, but is also interested in modern inventions, showing that he has an innate desire to explore new things: luke was also captivated seeing airplanes whiz across the sky. if he was looking in the right spot, occasionally he’d see a satellite move silently across the night sky. i can’t even imagine what it’s like to be up in the sky like that, looking down at the earth, he thought. ( ) during the period of his amnesia, he even goes as far as riding in a hot-air balloon, suggesting that the amish culture and their traditional beliefs suppress the explorative nature of men – which the male protagonist easily gives up when he finally returns to his amish family and community. apart from this unusual attribute, brunstetter describes her male protagonist as the paramount example of a young male amish. luke is hard-working, considerate, always friendly, helpful and respectful: luke had become a friend to many in the community. he’d exuded confidence, and even to strangers, he had seemed comfortable talking about most any subject. he’d been open minded and straightforward, and it was those qualities that people had liked about him. he’d had an infectious personality and had made many friends over the years because of it. folks just gravitated toward him, […] (silence ) he had respected his parents and been a good son to them. meredith had never heard him say an unkind word about either one of his folks. ( ) even during the period of his amnesia, his character remains that of a responsible and sensible young man who treats his surroundings carefully and respectfully, not wanting to owe anything to anybody (“it didn’t seem right for him not to pay,” brunstetter, pieces ). he is still described as someone caring for the world around him, even for the little things: susan smiled. it was refreshing to meet a man with such a tender spirit. she wondered if he’d ever had any pets of his own. if he had, he’d probably treated them with the same gentleness he was using on this kitten right now, as it purred and rubbed its small head against eddie’s [i.e. luke’s] hand. the kitten was definitely content lying there in his lap. (pieces ) luke’s attending to the kitten could be regarded as a metaphor to his “tender spirit”: his gentleness is reflected in the kitten – a small fragile being – purring and rubbing its head against his hand, reinforcing the picture of luke as a sensible, considerate man. “he and your grandpa are outside, shoveling snow off the driveway so you and anne can get your cars out of the garage. it’s a good thing you both have afternoon shifts, because the roads should be cleared by then.” susan smiled. “luke’s thoughtful, isn’t he, grandma?” (vows ) again, luke’s helpfulness and gentlemanly behavior is shown – he does not only aid the old man but offers his help to make life easier for the young women. to sum it up, luke stoltzfus is portrayed in a very positive way. only at the beginning he shows signs of dominance and patriarchy, but as the story unfolds, only his good traits are focused on – as if he had learned a lesson after the tragic accident that happened to him. meredith stoltzfus turning to the female protagonist of the novel series, the reader can detect the expected amish female attributes right from the beginning. meredith is portrayed as being modest, submissive and responsible, not given to emotional outbursts. […] luke felled blessed. she wasn’t the type to ask for much, and buying some paint was really no big expense – that is, until now. (goodbye ) he also knew she kept herself extra busy around the house so she wouldn’t fret so much about him being out of a job. ( ) meredith hesitated. then she gave a slow nod. “all right, luke, if that’s what you think is best.” although i’m really not sure it’s the right decision for us, she silently added. ( ) “i’d like to [travel with you, luke], but i don’t think we should spend the money for an extra bus ticket. besides, luke will be busy learning his new trade, so i think it’ll be better for both of us if i stay home.” ( ) these four quotations illustrate the hierarchical nature of the relationship between meredith and luke: luke feels blessed, because meredith is modest, stays busy in order to not think about his unemployment, and supports his decision to take their money and learn a new trade at his uncle’s place, although she is skeptical about his move. apart from these expected feminine attributes, there is one feature in meredith that stands out – her stubbornness and willingness to do and manage things on her own. after luke has left to learn a new trade at his uncle’s place the female protagonist is determined to manage life on her own, even though it is a typical amish feature that family members take care of and depend on each other: just then, dad entered the room. “are you comin’ home with us?” he asked, looking at meredith. she shook her head. “i appreciate the offer, but i’d prefer to stay here.” dad looked over at mom and said, “this daughter of ours is an eegesinnisch one, jah?” (goodbye ) [meredith’s father:] “[…] my daughter can be stubborn sometimes, and i know she wants to make it on her own.” (hope ) “you don’t have to worry about any of those things, because i’m ready to go home,” meredith blurted out. “it’s quieter there, and since i’m feeling better now, i’m sure i can manage fine on my own.” ( ) “i’m glad he [jonah] was there, but if i had been alone when the pains got bad, i would have somehow made my way out to the phone shack and called for help,” meredith said in her own defense. ( ) meredith jaw clenched. she didn’t want anyone babysitting her, but if she didn’t agree, mom would insist that she stay with the family. ( ) brunstetter paints a vivid picture of how the rather timid and submissive female from the beginning of the story suddenly rises up and stands her ground: she “blurts out”, her “jaw clenches” as she feels being compared to a little girl, who needs a babysitter. her eagerness to manage life on her own brings out her hard-working attitude. as johnson-weiner described the role of women in amish communities, the female protagonist is an example for the women’s tendency to not be idle but hard-working to provide for the family. for meredith, work does not only support her financially but also psychologically after she receives the news of her husband’s presumed death. she tries to be strong by not giving in to grief and depression. every night meredith made a mental note of what she wanted to accomplish the next day. as long as she did that, she felt like she had a reason to get out of bed each morning. (silence ) forcing herself to think about something else, meredith began to fret because she couldn’t be at home doing the things she’d planned in preparation for the baby. […] she’d always been the type to keep her hands busy, and doing nothing was so frustrating. (hope ) her culture and religious background, however, put her in an emotional conflict. on the one hand, she wants to be independent, supporting herself as a widow; on the other hand, she is afraid that this self-reliance might be interpreted as pride and feels sorry for it. in this way, she is experiencing an inner struggle between the desire to be independent as a woman and to conform to traditions and fulfilling her expected female role – one that is dependent on other people. and it is one of the older women – her grandmother – who reminds her of the community’s values: “i guess i’m full of hochmut, or i would have asked for help painting the baby’s room.” “you should never be too proud to ask. that’s what families are for, you know.” grandma took a sip of tea and winked at meredith. (hope ) on the outward, she often complies with the expectations that are imposed on her, but when the author writes about meredith’s thoughts, the reader becomes aware that inwardly, she is not the submissive and obedient woman she seemed at first sight. she has a deep-seated desire to make decisions on her own: “that’s right, and i’m happy to say that i’m doing much better now and was more than ready to come home.” “i’m glad to hear that, but you need to take it easy and not try to do too much,” he [jonah] warned. meredith stiffened. first mom, then laurie, and now jonah? why did everyone think they needed to tell her what to do? ( ) through the enumeration “first mom, then laurie, and now jonah” followed by “everyone”, the reader can take a hold of meredith’s anger at being told what to do. by using this narrative device of a climactic enumeration, the author succeeds in portraying, the character’s aggravation can easily be felt. this kind of feeling and behavior is rather characteristic of a modern woman than an amish traditional one. as the story evolves, meredith starts thinking it might be good to start doing new things and not remain where she has always been. one reason is her sister laurie, who is about to marry a mennonite man and leave the amish community with him. the other reason is an excursion to a county fair where people can take a short ride in a hot air balloon. meredith does not actually venture to take a ride in it, a fact which she regrets it later on. meredith wished now that she’d taken a ride in the balloon, just to see what it was like way up there. she’d never been an adventurous person, but maybe it was time to step out of her comfort zone and start taking some chances. (revelation, ch. ) these desires are reflected in neriya-ben shahar’s study ( ) that describes amish women as “gatekeepers”, people who protect traditions and values by taking care what influences are allowed to enter the community, as well as “agents-of- change”, people who carefully explore new things and practice self-control in the use of it. on the one hand, meredith is fascinated by the thought of flying, but at the same time, she knows she should not do it and keep to her traditions instead. she continues to prove her responsible and considerate character when she and jonah decide to start courting. assuming that her parents-in-law might not accept this easily, she wants to inform them first. [meredith:] “no, not yet. it’ll be hard telling luke’s folks that jonah and i will be courting, but i know i have to. i wouldn’t want them to hear about it from someone else. it would hurt them deeply.” (revelation, ch. ) meredith loved sadie and elam and wanted their approval. (vows ) as the story unfolds, one can observe how the female protagonist changes her behavior. while she is described as modest, gentle and submissive as long as her husband is at home, her attitudes change as she believes herself to be a widow and therefore responsible for her own and her child’s life. this moment in the romance, which regis calls “point of ritual death” ( ), offers meredith the chance to break out of her tradition and aim at self-fulfillment. however, this phase of her life only lasts until a new suitor wins her heart and she turns back to her traditional roots. due to the omniscient narrative perspective, the reader, however, knows that her first husband is still alive and feels the need to detain the female from marrying another, supporting her desire for independence. but in the end, the tension is released by luke himself, who appears on meredith’s doorstep in the very last moment. with his return, meredith also returns to her appropriate amish gender role. jonah miller jonah miller is the third main amish character in the novel and makes his debut in the second volume of the series. he is about the same age as meredith and luke and still single. for some years, he has been living in another state and now comes back to his hometown to work in his father’s business. it becomes evident that he shares a close relationship with his parents, nevertheless he desires a family of his own – as is expected of him as a young amish male. it would be great working alongside dad again – just like he’d done since he was a teenager, when dad first taught him how to make and repair amish and other types of buggies. jonah and his dad not only had a close father-son relationship, but they were linked in a working relationship, too, and both took their work seriously. […] he sure couldn’t stay living with mom and dad forever, and he didn’t wish to remain single indefinitely. (silence ) another typical attribute can be seen in his expectations concerning his future wife. jonah imagines her as being the one taking care of the home to turn it into a comfortable place, just as he saw his mother doing it during his childhood. he obviously appreciates the traditional amish gender roles, where the women are responsible for the household and the children: he dreamed of a spouse who would fill their home with love – a place where jonah knew he belonged as soon as he walked through the door. his mom had a way of putting her heart into every room in her house, and anyone entering could actually feel the welcome. (silence ) in this paragraph, brunstetter describes jonah’s attitudes toward his future wife: she should be the one making the house a home; none of his thoughts touch the idea that he might have a part in this as well: in his imagination, it’s solely the woman who is responsible for the emotional well-being of family members, just as his mother did when he grew up. to emphasize this image, the author uses phrases such as “fill with love”, and “put one’s heart into it” to talk about the woman’s tasks, in order that the man can “belong” and “feel welcome”. however, jonah does not appear to be a dominant male character. he is rather caring and helpful and wants the best for the people close to him. even though he was interested in meredith years before, he accepted the fact that she married someone else because he cared more for her happiness than his own. being jealous, angry or depressed was no option for him, because his christian belief taught him to accept this situation as god’s will. ever since jonah was a boy, he’d been sensitive to others, especially when they needed help in any way. (silence ) “[…] jonah seems like a caring young may. i’m sure he’ll fit in well with our community.” (hope ) jonah had been disappointed at first but consoled himself with the thought that meredith deserved to be happy. he’d been hoping she might be the girl for him but had learned to accept it as god’s will when she’d fallen in love with someone else. (silence ) as the story unfolds and jonah hears about luke’s death, his sacrifice of giving up meredith seems to be quickly forgotten. knowing that he cannot rush things because she is still mourning, he still uses every opportunity of being of help to her. once again, his caring and helpful attitude can be observed, at the same time, however, he does not really worry about what others in the community might think. even when his parents remind him of it, he chooses to follow his own desires instead of submitting to his parents’ advice. this kind of stubbornness and individualistic behavior can be observed in the following conversations: jonah tapped his foot impatiently. this inquisition was getting worse. “if you must know, i’m planning to stop by the kings’ place to see how meredith is doing.” deep wrinkles formed across mom’s forehead. “do you think that’s a good idea?” “why wouldn’t it be?” jonah asked. he had an inkling of what she was going to say next but hoped she wouldn’t. “well, some folks might get the wrong idea.” (hope ) “i know what the bible says,” mom flapped her hand. “but meredith is the friend you had an interest in after you came back from sarasota with stars in your eyes.” “well, you two enjoy your day,” jonah said, giving no reply to mom’s last comment. (hope ) dad tapped his foot as he stared at jonah. “you’ve been goin’ over there a lot lately. aren’t ya worried about what others will say?” jonah tipped his head. “what is it you think they might say?” dad cleared his throat real loud. “do i need to remind you that meredith’s a young widow, and she’s expecting a boppli besides?” a rush of heat spread across jonah’s face. “exactly what are you saying, dad?” he asked. “i just feel you oughta be concerned about what others may think. some folks could get the idea that you have it in mind to make meredith your wife.” oh, great, jonah thought. now i’m in for one of dad’s long lectures. i’d better put an end to this before it gets started. (hope ) the way brunstetter describes jonah’s conversations with his parents leaves the impression of a teenager who is reprimanded by his parents. the use of the hyperbole – “inquisition” – creates tension between jonah and his parents, as do his seemingly naïve answers: “why wouldn’t it be?” and “exactly what are you saying?”, which are not really answers but counter questions that indicate his unwillingness to agree to his parents’ point of view. interestingly, jonah does not admit his feelings for meredith to his parents – he behaves is if their concerns about his motives were completely unfounded. amish communities and especially families are usually described to be very close, and important matters such as courting and marriage are commonly discussed within the family. in jonah’s case, he keeps his feelings to himself and is not ready to discuss them with his parents or even to seek their counsel. as can be noted, he follows a rather individualistic attitude instead of the traditional amish one. finally, when meredith’s appropriate time of mourning has passed, he openly states his intentions – first to meredith and then to others. “what do you think, meredith?” jonah questioned. “are you comfortable with me courting you right now, or would you rather wait a few more months?” meredith shook her head. “i don’t want to wait. i think once sadie sees how good you are with levi and realizes you’re not trying to take luke’s place she’ll accept the idea.” jonah’s eyebrows pulled together. “maybe i should have a talk with her—try to make her see how much i care about you and levi and that i only want what’s best for you. i’d like to assure her that even though we’ll be courting, i have no intention of changing how often they can see their grandson. i would never come between them and levi.” (vows ) being accepted by meredith, jonah’s sensitive character comes into play again. he does not want luke’s parents to object to him, thereforehe strives for their approval. his good intentions are revealed in more than one expression. by a certain climax in his speech he shows himself as a considerate young man: he wants to “try to make her see”, he would “like to assure her”, has “no intention of changing” and “would never come between them and levi”. again, the close ties within the amish community have an influence on this male character, his individualistic way of thinking and behavior are set aside as all circumstances seem to have turned out to his favor. although the reader is aware that meredith’s husband is not dead, the character of jonah is described in such a way that – just like meredith – one starts to sympathize with him. due to his constant gentlemanly and helpful attitude, one cannot but like this young amish man. when, however, luke suddenly returns, the reader, on the one hand, is relieved and glad about the happy ending of meredith and luke, but, on the other hand, feels sorry for jonah. the author, however, takes care of this situation as well and mentions in the epilogue that jonah, who moves to illinois, finds a girlfriend as well (vows ). . . . other female/male gender roles in addition to the three main characters in brunstetter’s lancaster county saga, there are three older amish couples and their relationships that will be analyzed. these are the parents of luke, meredith, and jonah, and they all try to influence the decisions of the main protagonists on different levels. they all live in the same amish community and are about the same age; their characters, their interaction as husband and wife, and their involvement in meredith’s and jonah’s lives, however, are rather diverse. sadie and elam stoltzfus (luke’s parents) at the beginning of the novel series, sadie stoltzfus is described as a happy amish mother, who loves her nurturing role and only has the best interest of her children in mind. she is welcoming, everyone loves being around her, thus she represents the perfect amish female model. luke’s mother was always so cheerful. in fact, sadie’s radiant smile was contagious, and at the age of sixty-seven, she still had the cutest little dimples. just being around her made meredith feel at ease. (goodbye ) brunstetter paints a harmonious picture of sadie by consistently using positive adjectives such as “cheerful”, “radiant” and “cute”. and when she states that despite her age, “she still had the cutest little dimples” the impression is given that she has always been a very happy woman. furthermore, sadie is portrayed as very creative, because she writes little poems about her feelings, her faith, and her role as a mother. obviously being a mother is very important to her and she is aware of the responsibility that comes with it. she considers it her duty to be a role model to others. sadie had written a poem about being a mother, which she’d shared with meredith this morning before the service. it had almost been meredith’s undoing as she’d listened to sadie read the poem in a quavering voice: “a mother wants her faith to give hope to her child; stability and trust in a world gone wild. a mother’s faith should be handed down; in the next generation it will be found. a mother’s faith must be steadfast and sure; so her children will desire to be like her.” (silence ) the repetition of “a mother” in sadie’s poem underlines the significance this role has for her. reciting these lines, she feels emotion, probably because the weight of a mother’s responsibility dawns on her. when tragedy strikes and her son luke is pronounced dead, her character surprisingly changes. as long as sadie thinks that meredith will stay single after the delivery of her and luke’s baby, she can cope with the death of her son, comforting herself in the knowledge that she will play an important part in her grandchild’s future. however, when she realizes that meredith has a new suitor, she becomes jealous, thereby losing all the good character traits such as friendliness, responsibility and a welcoming attribute as described before: a few seconds later, luke’s mother, sadie, stepped down from the buggy and secured her horse at the hitching rail. a frown creased her brow as she approached the garden. was she upset about something? had she come with bad news? “wie geht’s?” meredith asked. “i’m fine,” sadie said curtly. “i just came by to see how you’re doing.” she glanced at jonah, frowned slightly, and then quickly looked away. meredith thought sadie’s behavior was a bit strange; she was usually quite warm and friendly. (pieces - ) her whole countenance changes, instead of cute dimples and a smile, she “frowns” and talks “curtly” – a complete contrast to the description at the beginning of the story. thereby a change in her character can be noted. sadie discusses her feeling with her husband elam, hoping that he will agree with her point of view. however, she does not really seek his advice as would be expected in an amish couple’s discussion, but she rather tries to force her point of view on him. elam, however, does not give in, but clearly states his point of view. outwardly, she reminds him of his role as the head of the household by laying a hand on his shoulder, encouraging him to do something, but when he is not willing to follow her advice, she draws back to herself and decides to do what she wants without his consent. “jah, and i don’t think it’s right that he’s been hanging around there so much. i’m afraid he’s trying to take luke’s place, and it’s way too soon for meredith to be seeing another man.” she placed her hand on elam’s shoulder. “i think you should do something about it, and the sooner the better.” “what do you want me to do, sadie?” elam asked, shrugging his shoulders. “am i supposed to barge into the buggy shop and demand that jonah stay away from meredith?” “that might not be a bad idea,” she said with a nod. elam looked at her and frowned as he slowly shook his head. “if jonah and meredith are meant to be together, there’s nothing either one of us can do about it.” sadie’s lips compressed while she tapped her foot. “we’ll just have to see about that.” (pieces ) elam realizes sadie’s feelings of jealousy and is aware that he has to interfere. he tries to stop her plans with ironic statements. in this way he is portrayed as a good- natured and humorous man. but nevertheless, he openly states his opinion when he realizes that his wife’s attitude will probably cause problems and hurt not only meredith but also their future relationship with their grandchild. elam grunted warily. „you [sadie] need to mind your own business. we’ve been over this before, and it’s time you realized that meredith has her own life to live. if her future includes jonah someday, then you’ll just have to accept it.” (revelation ch. ) by observing this couple throughout the novel, it is interesting to note that elam fits well into the typical amish male role – head of the household, responsible and family- oriented -, whereas sadie follows the amish feminine attributes of being gentle, family-oriented, motherly and submissive only as long as there are no troubles. as soon as she experiences the fear of losing influence on her family, her submission to her husband becomes less important than her own will and her feelings. honesty and uprightness are of no significance anymore and she acts against her husband’s advice. thereby, she does not follow the ideal of an amish couple that complements each other and discusses important issues together. i’m glad elam’s visiting his friend joe today, sadie thought as she hitched her horse, daisy, to the buggy. if he knew i was going over to confront jonah about seeing meredith, he’d probably tell me i shouldn’t go and that it’s none of my business what jonah does. (pieces ) through the narrator’s omniscient perspective, the reader becomes aware of sadie’s emotions and plans. she knows her actions are not approved of by her husband, but her feelings of anger and jealousy seem to overcome her. although in the end, she finally admits her wrong behavior, it is interesting to notice that in an emotionally strenuous situation, sadie does not confide in her husband in order to seek advice; rather, she only seeks approval of her point of view. elam, however, does not really know how to cope with the situation and attempts to get to the root of the problem, leaving the impression that this couple is not used to talking things over thoroughly. luann and philip king (meredith’s parents) in his physical appearance, philip king represents the typical hard-working amish male: “[…] her dad was in tip-top health. his arms were still muscular from all the farmwork he’d done over the years, and meredith found comfort in his warm embrace.” (goodbye ). just as his physical stature offers comfort, he is also described as a loving father who has an open ear for his children’s problems (silence ). spending time with his family plays an important role in his life and although he knows that he has to take care of the financial situation of the family, he would never neglect his family. she was glad dad hadn’t taken on another stand at the crossroads market, like he’d talked about doing a few months ago. he worked hard enough as it was, and it was difficult for the family to have him gone so much. it was important for a father to spend time with his wife and children, not only for the family’s sake but for his own, as well. (pieces ) the burden of financial responsibility weighs hard on him, and his friendly nature only then turns into a more dominant one when his wife luann mentions their tight budget. he does not start an argument, but nevertheless clearly states that this criticism hurts his feelings and shows doubt in his abilities as the breadwinner of the family. this behavior is different than that of sadie and elam. philip king openly talks about his feeling, whereas sadie stoltzfus only tells her husband what she wants to be done and not how she feels about a situation: “first off, we don’t need another horse, and second, i don’t need the reminder of how bad off we are financially.” dad rubbed the side of his slightly crooked nose and frowned. “when you say things like that, luann, it makes me feel like a failure – like i can’t provide well enough for our family.” (hope ) luann’ and philip’s relationship is obviously a very affectionate one. after more than three decades of marriage, they are still fond of each other and enjoy having fun and joking around when the family gets together. this short dialogue creates the mood of a caring and affectionate home: luann’s sixteen-year-old daughter, kendra, wrinkled her nose and glared at arlene. “it’s not polite to lick your fingers, sister.” “maybe not,” luann’s husband, philip, put in, “but this chicken is finger lickin’ good.” he swiped his tongue over his fingers and grinned at luann’s mother, who gave him a wide smile in return. (hope - ) meredith’s mother luann is described as the ideal amish wife and mother. she is friendly, has a welcoming spirit (revelation ch. ), is motherly and taking care of her children well (silence ). in contrast to luke’s mother sadie, she keeps her opinion about meredith decision to marry jonah to herself, even though she does not feel comfortable with the idea. in this respect, she shows self-control and does not allow her feelings to control her actions. sarah and raymond miller (jonah’s parents) jonah’s parents sarah and raymond are also described as a happy older couple. raymond is a hard worker and has taught his son jonah his trade of making and repairing buggies (silence ). while jonah lives with his parents, father and son work together, helping each other. sarah shares a close relationship with her son, too, and is eager to see him getting married. if necessary, she is ready to help him achieve this goal, as can be read when she meets luann: “luann didn’t voice her opinion, but it sounded to her like sarah might be trying to plan her son’s future, which she didn’t think was a good thing at all” (silence ). in sarah’s mind, women are better off if they are married, especially when they have children as it is in meredith’s case. a woman raising her child alone does not seem to be right to her – love between the husband and wife is not as important as the mere fact that a man is there for the child as well. “that’s wunderbar. maybe after a suitable time, she’ll get married again, like our daughter did,” sarah’s emerald-green eyes shimmered as she smiled. luann slowly shook her head. “meredith loved her husband very much, and she’s taken his death quite hard, so i doubt she would ever marry again.” “but don’t you suppose after some time has passed, if the right man came along, she might get married – for the sake of the boppli, if for no other reason?” sarah asked. (silence - ) in this short dialogue she proposes a new marriage for meredith twice, as if this were the only achievable goal for meredith. in this conversation, sarah is portrayed as an excited matchmaker, her excitement made visible when her “emerald-green eyes shimmered as she smiled.” however, sarah as well as raymond place value on what others in the community think about them and their son. therefore, they are both sceptical when jonah starts spending quite a lot of time helping meredith. sarah does not directly address this topic, but desires to find out what is going on. similar to sadie, she beats around the bush instead of directly discussing the issue at hand. “ja, and we’re glad he’s here with us. oh, by the way, jonah, where all are you going today?” mom asked as jonah’s hand touched the doorknob. “for one thing, i’ll be stopping by the shoe and boot store,” jonah replied. he wished he didn’t have to answer to mom, it made him feel like a schoolboy. “that shouldn’t take all day,” she said. “are there some other places you’ll be stopping, as well?” “stop badgering the boy, sarah,” dad said, flapping the end of his newspaper at mom. “it shouldn’t matter where he’s going.” (hope ) she appears rather annoyed und curious as she keeps asking her son what he plans to do. however, her eager curiosity is directly addressed and stopped by her husband raymond. thus, he fulfills his role as the head of the household and puts his wife in her place. furthermore, he directly addresses his son to remind him what kind of behavior is expected of him concerning meredith’s circumstances and his family’s reputation in the amish community. similar to luann and philip, this couple talks openly with each other. however, just as with sadie and elam, the wife is the more active partner, wishing to influence her son’s future, just as sadie wants to intervene in meredith’s life. both husbands, elam as well as raymond, are rather annoyed be their wives’ behavior. . . healing grace series by adina senft ( ) adina shelley writes amish women’s fiction under the name of adina senft. she grew up in a plain house church – but not an amish one – in canada and moved to california in her twenties. (stories with spirit) even though she did not grow up amish, she has a lot of friends among the amish who read and help her with writing her novels. furthermore, she spends one week in lancaster county each year to talk with amish women and to listen to their conversations in order to do research for her amish novels. (destination amish) the healing grace serial novel contains three volumes, herb of grace, keys of heaven and balm of gilead. the story revolves around amish widow sarah yoder who raises her two teenage sons – stepson simon and son caleb – alone and tries to provide a home for them in the village of willow creek. recently, henry byler, an ex-amish who grew up in the amish community of willow creek but never joined the church, decides to move there again after inheriting his aunt’s farm. he starts his pottery business on the old farm and soon becomes friends with sarah’s younger son caleb. simon, together with his friend joe byler, decides to move out west to look for work and to get to know life from a different perspective. in the meantime, sarah is encouraged by ruth lehman, an amish dokterfraa, to learn from her to become a herbal healer, because she has a gift for growing plants. as time goes by, sarah’s relatives try to encourage a relationship between her and silas, a visiting amish man. their attempts are not successful, because sarah becomes emotionally attached to henry although she does not want to admit it, knowing that he left the amish church. henry’s pottery business turns out successful and he gets to know ginny, an english inn-owner in willow creek, and falls in love with her. they plan to get married, but that changes when the company who commissions his work wants to market him as amish. henry struggles with his amish roots, his respect for the amish community, and the chance to leave his past behind forever by binding himself to an english woman. in the end, he realizes his strong ties to his background and his love for sarah and returns to his community again. simon and joe also return from their working journey to colorado. joe already courted priscilla, a young amish girl, before they left – although priscilla was interested in simon at first. when they return, these two young men start competing for priscilla, but she holds fast to her decision to be courted by joe. . . . main female and male characters and their gender roles sarah yoder the main protagonist of the novel trilogy is sarah yoder, an amish widow in her mid- thirties, who raises her seventeen-year-old stepson simon and her fourteen-year-old son caleb alone. although she is supported by her and her husband’s family, she is determined to provide for her small family on her own. as she is quite successful in planting and cultivating herbs, ruth, an older amish woman who has great knowledge in herbs, teas and salves, offers to pass on this knowledge to her, thus enabling her to make a living and be a help to the community. her outward appearance is described as modest and as being a woman who follows the amish dress code. she carefully observes it, especially when meeting outsiders like henry. she leaped to her feet and smoothed down her dress with one hand while she pulled her duchly up with the other. she tried to tie the knot more tightly, because she certainly couldn’t talk to an englisch man bareheaded. (herb ) from the beginning, it is obvious that sarah wants to be a respectful woman and often restrains herself from speaking in an unruly matter. in her conversation with ruth, for example, senft writes: “ruth was older, and enjoyed a position of respect in several communities – both very good reasons for sarah to school her tongue to a soft answer.” (herb ) even when she feels attacked, she is aware of her surroundings and the respect that is due to others: it was all sarah could do not to dash in there and give oran yost a piece of her mind for talking to her son like that. but she could not. it was not her place to upbraid a man in his own shop, in front of the other men – especially not a man in a position of authority over simon. it would shame simon and show her to be an interfering busybody who didn’t know how to submit – which was not the case. (herb ) as one can see, she is well aware of her position as a woman in a patriarchal society and of the fact that a woman should be ready to yield and to be submissive in order to follow the beliefs of the amish community (herb ). nevertheless, sarah continues to struggle with this attribute of restraint as can be seen from the narrator’s omniscient perspective: inwardly, sarah wants to “dash in”, “give a piece of her mind” and “upbraid a man”, creating the image of a very emotional female who does not want to accept a wrong. in more than one situation, sarah is quick to speak her mind, only to regret it moments later, knowing that this is not the becoming behavior of an amish woman. the reason for her frank speaking is often care for other people and not to appear as a know-it-all. “shouldn’t his boys be helping, too?” the question shot out of sarah’s mouth before she could stop it. “i’m sorry. it’s none of my business how he and ella bring up their family.” (keys ) “you’re not having dinner with ginny?” came out of her mouth before she even realized the thought was lurking in her mind, ready to spring like a kitten upon any wisp of passing common sense. (keys ) here, senft makes use of a metaphor to describe sarah’s forward attitude. in her vivid description, sarah is portrayed as someone outspoken, who has a lot of thoughts going through her mind. “you were thinking out loud one day?” corinne’s forehead was furrowed with concern. “you were talking with linda and advising her to leave her home with ella and arlon? oh, sarah.” sarah wilted under the pain in her mother-in-law’s face. “she couldn’t conceive. i thought it would be for the best – that if she had calm and quiet and her own home, she might be able to.” “who says we ain’t calm and quiet?” benny burst out. “we have prayer time same as anybody else.” “i’m sorry, benny. i judged your family,” sarah whispered. “it was prideful of me and presumptuous and i beg you to forgive me.” (keys - ) feeling compelled to help others by speaking out causes sarah to struggle against pride, an attitude that is undesirable among the amish. therefore, she often finds herself in the conflict between acting on what seems to be right in her eyes and showing humility despite her feelings. consequently, she struggles with her bad conscience and the expected traditional behavior when she says: “i did not show oran yost the humility i should have, and because of it, you’ve lost your apprenticeship.” (herb ). she herself judges her outspoken attribute as stubbornness and only sees humility as a way out of the conflict (herb ). in sarah’s eyes, the others can hardly ever be blamed for troubles in her life, and she is quick to put the blame and guilt on herself (herb ). her role as a mother is one that she does not take lightly, and she is eager to teach her sons the obedience that is expected. obviously, her sons are well trained by her, because they readily obey knowing that there will be consequences if they refuse to. “caleb yoder, it is only one thirty in the afternoon. you disobeyed me earlier, and this is the price you must pay. […]” he knew better than to argue any more, because she could think of an endless series of jobs that would keep him working late into the evening, […] (herb ) this hyperbole of “an endless series of jobs” highlights sarah’s strictness as a mother. the task of disciplining simon and caleb is not always easy for her, but she knows and exercises this responsibility, having the result in mind and not the situation at hand. however, she does not always correct and control them, being fully aware that teenagers sometimes need to have the chance to experience things on their own. sarah grasped at her common sense, which was being flooded by the maternal urge to hold him close. “you’re seventeen – i suppose it’s time for you to get out and see a little of the world.” (herb ) again, senft uses the literary device of hyperbole “flooded by the maternal urge” to stress sarah’s motherly nature. sarah enjoys spending time with her boys and loves to see them happy. she is aware that a mother’s task includes both – love and discipline. sarah herself says, “we mothers don’t stop loving our sons just because they’re disobedient.” (balm ). her sons are also used to help at home, although both also have jobs somewhere else. her boys were busy every day and some evenings, too, so sarah hoarded saturday nights like a miser, hugging them close to her heart and savoring them before she let the minutes fall through her fingers. (balm ) spending family time with her sons is precious to her, as is exemplified by the comparison of “hoarding saturday nights like a miser”. in figurative language, senft describes the intimate and affectionate relationship between sarah and her sons. her sons’ willingness to work is not of their own making but can clearly be traced back to the way sarah and her husband educated them. when sarah realizes that simon becomes lazy after returning from his journey to colorado, she quickly makes up her mind to intervene and talk with him about it: even if all he did was walk over to milk his grandfather’s cows morning and evening, she’d be satisfied, but he had to keep his hands busy somehow, or the devil would. (balm ) again, her determined stance to see her son working is expressed by the metaphor “or the devil would”, showing that she is aware how important work is for a young man. even though simon is only sarah’s stepson she takes her responsibility in educating him seriously. on the one hand, she values him as a man in her household, even as the man of the house, now that he is of age and her husband dead for five years. but on the other hand, she is still aware of her role as mother to him. therefore, when simon openly criticizes her friendship with henry, she puts him in his proper place. she knew very well what simon was thinking. but it wasn’t his place to bring it up – he might be the man of the house, but she was still his mother, and there were certain things that respect taught a young man not to say. but she had a thing or two to say to him, and this seemed like a god given opportunity. (balm ). although sarah sometimes regrets her tendency to be outspoken towards other people and is determined to be humbler and more restrained, she is nevertheless an independent and self-reliant woman. being a single mother is not always easy for her, but she is determined to manage her family and household on her own. therefore, when her relatives come up with the idea to set her up with silas lapp in order to provide her with a husband again, she does not welcome the idea at all. when sarah glanced at her mother-in-law to see if she wanted to begin clearing, there was a look on corinne’s face that she had never seen before. she was gazing at silas lapp as though that new idea had occurred to her in the last minute or so. sarah did not want to know anything about it. she stood and began to clear the dishes herself. (keys ) the narrative style of these short last two sentences indicates sarah’s anger about her mother-in-law’s matchmaking ideas. she wants to distance herself from being driven into a relationship she is not in favor of. even when other relatives try to steer her in the direction of courting silas using religious argumentation, sarah remains determined at not allowing herself to be set up but to decide on her own about her future. this attitude contradicts the ideal of an amish woman who strives to get married and have a husband by her side instead of staying single and independent. “but these things aren’t up to us, are they?” fannie said. “if god has revealed to us his choice of mate, it’s up to us to be willing, isn’t that so?” “yes, but we must have a conviction, too,” sarah countered. “the only conviction i’ve had is that i need to learn more about the healing path he has set me on, and that is taking all my time and thought. there is none to spare for – for men.” zeke chuckled. “i never met a woman who couldn’t make a little time for a man. or a lot. it’s funny how many things can be put aside when there’s courting to be done.” (keys ) it is interesting to note that the female protagonist is not easily convinced to give up her single status merely because there is a possible marriage candidate. the simple fact that her sons need a father is not sufficient to think about a new husband. since sarah’s first marriage was based on a relationship of love, she wants nothing less for a new one. and as her feelings for silas are not more than amicable, she is determined to keep their relationship on that level. therefore, she tries her best to convince him about that in a respectful, polite, but distinct manner. [silas:] “what i see more than that is a heart with a care for the people of god, and that’s more difficult to teach. i like that about you, sarah. it tells me that you have a big capacity to love.” oh, no. that word could not come into the conversation under any circumstances. “i love my family, certainly. my sons. and my husband, still.” “your husband?” he said gently. “corinne tells me it has been five years.” “ja. six, in the fall.” “he was a good man.” “the best.” “but your youngest boy, i would think he needs a father.” oh, now, this really was forward. “he is close to his daed, and he has several uncles close by to stand in that place for him.” (keys ) the author uses the omniscient perspective to allow a glimpse into sarah’s thoughts. she obviously is not interested in silas, when her thoughts are “oh, no”, and she tries to put him off with short, simple, but clear answers. as the amish are also called the “plain people”, it is customary for them to keep things simple and plain. their dresses are of a plain color and also within their home things are kept simple and practical (graybill, meanings ). concerning her dress, sarah complies with these regulations. however, as she starts her herb planting, a sudden urge of creativity takes hold of her and she plans her garden in an untypical manner. “she would probably regret not being able to hoe a straight line, but my goodness, she’d been hoeing straight lines all her life. maybe it was time for a little creativity – a little fun.” (herb ) by doing so, she is fully aware that her amish community might not understand her touch of creativity, but “that henry might be the only person in the settlement who would appreciate her craziness” (herb ). the relationship between sarah and henry throughout the three volumes of the serial novel proves to be a constant struggle for the female character between her amish beliefs and traditions and her feelings as a woman being interested in a man. at the beginning, the situation seems to be unambiguous for sarah: “henry byler was not amish, and therefore a single woman couldn’t cast her eyes in his direction.” (herb ). by foreshadowing, “a single woman couldn’t cast her eyes in his direction”, senft already provides a hint into the direction the storyline is going. however, as her son caleb quickly befriends henry, sarah is forced to act against her beliefs and finds herself more than once involved with him: “and somehow, despite all her good intentions, she found herself sitting in an englisch restaurant opposite an englisch man with her disobedient son beside her, feeling verhuddelt and … and hungry” (herb ). following her amish belief, she continually reminds herself that he is a “worldly man, a man who had turned his back on god” (herb ), being well aware that others in the community will talk negatively about her if she enters any kind of relationship – be it professional or personal – with him. nevertheless, being a woman who cares – especially about her patients – she casts her anxieties and social norms away and approaches him. “[…] i hope that you might do something. for me.” oh goodness, she sounded like a fifteen-year-old talking to a boy for the first time. worse, a blush was rising in her face and she could do absolutely nothing about it. (herb ) half of her wanted to run away down the path and take refuge in her own house. but the other half – the half that concerned itself for her patients – made her follow him out of the barn. (herb ) the way her thoughts are described lets sarah appear like a young girl who has fallen in love. the colloquialism of her thoughts – “oh goodness” – allows the reader to anticipate that sarah’s feelings towards henry are more than general friendliness. sooner or later, sarah realizes that she is becoming emotionally attached to henry – a feeling that probably seems decent and comprehensible to any other woman. but for her, it is not that uncomplicated because the social construct of amish religion and regulations breathes down her neck. the mere fact that henry left the amish before being baptized has formed a “wide gulf” between them: an amish woman could be neighborly with an englisch man. the amish were friendly to everyone. but henry was more than englisch. […] he and sarah might share a fence line, but between them there was a great gulf fixed … and any feelings that might have gone beyond friendship could never cross that gulf. (keys ) the author uses genette’s zero focalization to create tension between the two main characters. in sarah’s mind, a love relationship with henry is absolutely impossible due to her cultural confinement. these restrictions lead to an emotional struggle in her life, and she is forced to make a decision between her feelings and her traditional surroundings. therefore, true to her role as an amish widow, she turns to her god to receive help overcoming her feelings. instead of enjoying a new relationship, her traditions force her to overcome any emotional attachment: “ach, what have i done? i can’t afford these feelings, lord. i can’t have any feelings at all for a man who does not choose you. help me. please help me to overcome this craziness and set my heart on you, where it should be.” (herb ) the repetition of her pleading reinforces the struggle that she is in. her resolution not to see him or think about him anymore does not work out because henry visits her again, needing help. in her mindset, henry turns into “her greatest temptation” (balm ) and a test from god to prove her strength ( ). the idea to leave her community and lifestyle behind to follow the feelings of her heart are completely unimaginable for her. she tried to imagine living right here in this house, so close to her family, and not being amish. not going to church every other sunday, not having the deep fellowship, love, and acceptance that came with each one submitting him- or herself to god’s will. her imagination shuddered away from the picture. (balm ) her thoughts gain momentum by the anaphora “not going” and “not having” and show how deeply sarah is involved in her faith and her tradition. she maintains her position until the end of the novel and tells henry directly that their relationship can only be successful if he turns back to god and to the amish community: “but henry we can’t. it’s wrong. you must give your heart to god before you share it with me – you know it and i know it . . . and god knows it.” (balm ). again, senft uses an anaphora – “know it” – to add intensity to sarah’s determination to stay amish. again, her faith plays a key role in their situation and finally prevails, allowing sarah to remain a faithful plain woman and being rewarded with henry as her husband who is won over by her determined mindset. to sum it up, this female character is portrayed as a responsible and traditional amish woman, who nevertheless has a strong will and does not allow others – neither family or a man – to dictate the decisions she has to make. she is also the one who sets the conditions on which she is ready to enter a new relationship, which might be rather unusual for an amish woman. henry byler the male protagonist of the story, henry byler, is a character that enters the novel as an english man with amish roots. the reason why he left the amish faith behind was the overwhelming amount of rules and regulations his community taught him and the accompanying image of a god who was a “watchful, frightening being who cared so deeply about the widths of hat brims and the shapes of buggies and the truth of every thought that flitted through a boy’s mind” (keys - ). senft uses these exaggerations of dress codes and thought control to enhance henry’s distaste of amish rules. nevertheless, he does not only carry bad memories within himself, but also positive ones, like the one of his aunt, who appointed him as an heir of her farm: he didn’t think he could handle going through an old lady’s things. not because of male diffidence, but because outside of mamm, she [aunt sadie] was the only one of his relatives he had loved – and been loved by in return. (herb ) when henry arrives at the farm he has not yet decided about his next steps, and by this metaphor of “a man caught between two worlds, like a guy with a foot in two canoes, both moving apart and a huge splash inevitable” (herb ) one can sense the character’s inner turmoil. in contrast to the typical amish male who is hard- working, strong, and responsible, he appears at first insecure, indecisive and led by feelings he cannot classify himself. nor would it [change] until he made up his mind as to what he was going to do. had he really given up his denver apartment, loaded up his things, and come blasting across the country for … what? nostalgia? the prospect of a home that had been welcoming and warm in memory, but in cold reality was … amish? (herb ) rhetorical questions show his insecurity about his decision to move into an amish neighborhood, and the juxtaposition of warm memory and cold reality emphasizes his mixed emotions. on the other hand, he shows a lot of other characteristics that are expected of an amish man, such as helpfulness, politeness and gentleness. he is quick to help sarah carrying heavy things (herb ), willing to give a ride, when the need is there (herb ), opening the car door for a woman (herb ) and is quick to offer comfort when he realizes that sarah is in distress: “’sarah,’ he breathed, and she realized that he had reached across the table to take both her hands in his big, warm ones. ‘if you need to cry, don’t be brave. just let it go.’” (herb ). and even though henry does not know at the beginning what his next steps should be, he knows that he alone is responsible for making a living and that hard work is the way to achieve this goal: that was his goal, wasn’t it? to sell his mugs, his plates, his bowls, into the homes and kitchens of folks in this township. that was it. ubiquity had a bad rap. from where he stood, it looked pretty good. it looked like a living. (herb ) before his move to the amish community, his dream was to become an artist. but circumstances require plain, unexciting work to earn money, made clear by the enumeration of “his mugs, his plates, his bowl”. as he starts being successful in his pottery business, he faces the challenge to reach a higher level of publicity for his work. at first, henry embraces the opportunity, but when it is revealed that this success can only be achieved by bringing discredit on the amish community, his upright character prevails: “’i told you how i want the story slanted. i want it to be about art – with the amish focus on community over individuals being the reason i left the church. about how my art has its roots in a focus on the land.’” (balm ). although he deliberately left the amish faith and traditions behind, he still shows character and a clear value system. he is not willing to be successful and earn his living at any price, even though he knows that this decision will have a negative effect on his fiancée ginny, who could increase her income at her bed and breakfast through the media coverage about henry. again, he is faced with a decision between two worlds: either following the upright attitude he still believes in or neglecting this value system in order to gain a higher profit. but his past attitudes prevail – no matter the costs. his family and neighbors are more important to him than a boost of his career (balm ). henry glared at him in a way that made the smile falter. “either you change the focus of this episode or you find yourselves another subject,” he said with deliberate emphasis. “i’m not a man who issues ultimatums, but i’m at my limit here. this goes against everything i believe in.” (balm ) despite the fact that henry byler cares about his amish family and neighbors, he continues being a maverick as far as his personal life is concerned. he is afraid to let others know what is going on inside of him, though he still seems to be touched by the compassionate approach of the plain people around him. however, he is afraid of their asking him questions and judging the decision he made as a youth, when he decided to leave the amish traditions. “i was engaged some years ago, but it didn’t work out.” he clamped his lips shut on a subject that was so private that the very own words hurt in his mouth. (herb ) but her [barbara byler’s] voice was gentle, the voice of a deeply compassionate woman, and henry found his throat closing up. (herb ) he had no intention of joining them [at the amish sunday service], blast it. but his relatives and neighbors would probably rejoice if they knew he felt the pull, like the one the moon exerted on the tides – the combination of guilt and memory and social expectation that demanded he knuckle under and submit to god’s will. (herb ) by way of the metaphor of the moon and tides, the author describes how strong the old ties are. just as the influence of the moon cannot be separated from the tides, so henry’s past as an amish cannot be separated from his life. nevertheless, he continues to fight against the past without showing to anyone. even towards sarah, with whom he shares an understanding and a common tendency to creativity, he conducts himself in a distant way as soon as his private and personal affairs are concerned. he enjoys friendship with her as long as the amish beliefs and culture do not come into the picture. “sarah, i like and respect you. but i have to lay down some boundaries here. my personal life is off-limits, if all you’re going to do is lecture me about an ordnung that means nothing to me anymore. it will only cause offense between us, and i don’t want that. we’re neighbors and friends, and i’d like to keep it that way.” (herb ) in this speech, henry uses i-messages to openly state his feelings about his relation to the amish faith. it is the first time he states his position clearly. but as soon as he speaks his mind bluntly, he regrets it. “henry had never silenced a woman before. he found he didn’t like it much.” (herb ). in this instance, henry’s respect towards women can be noticed. it is not in his nature to treat women as inferior beings or to feel dominant towards them. on the contrary, he desires to receive advice from sarah, more than from his fiancée ginny. that indicates that he esteems the close companionship and exchange of opinions between amish couples. “you don’t need my opinion, henry,” she said softly, […] “i wouldn’t have asked for it if i didn’t.” his eyes were haunted, his face drawn, as though he hadn’t slept the night before – or the one before that. “you always tell me the truth, even when it might not be strictly in accordance with the ordnung.” her lips tipped up in a smile of acknowledgment. “i need to hear what you think.” (balm ) [henry talking with ginny] “are you saying i’m not qualified to talk this over with?” well, maybe he wasn’t – not on that level. but he did his taxes every year, just like everyone else. “the only qualification i really have is that i’m going to be your husband, and wives and husbands talk over big decisions.” she sighed. “right. like you talked over the show with me before you told them once and for all you wouldn’t do it.” she had him there. and who had he talked it over with? really talked? sarah, that was who. the one name he couldn’t bring into this conversation at any cost. (balm ) another role that henry takes on is to be a defender of the youth. he is quick to understand and to find an excuse for their behavior, remembering how he struggled with keeping all the rules when he was their age. however, he does not thoughtlessly defend their actions when an accident happens, because he is aware that they had no bad intentions and even assumed responsibility for their wrongdoing. in this way he wants to demonstrate that sometimes amish parents’ punishment is exaggerated. for example, when sarah starts to get angry at caleb, he “did his best to head it off at the pass. after all, he had been complicit in the plan. it wasn’t fair that poor caleb should take all the heat.” (herb ). and some time later, when he takes caleb out for lunch – without caleb informing his mother – and sarah starts scolding her son, “henry put out a placating hand. ‘don’t be too hard on him, sarah. i told him i’d take him to lunch. it only seemed fair, after he gave up his morning for me.’” (herb ). nevertheless, he encourages young people to be honest with their parents, thereby revealing a fatherly disciplining role without being harsh. just like a parent, he suddenly shows a strong character by admonishing young people to be truthful and willing to apologize when they had done something wrong. “i’ll remember that. but what i’d really like to know is how you’re going to explain this to your mother, simon.” he hunched over his plate defensively. “i’ll think of something.” “like maybe the truth?” he suggested. (herb ) when a group of teenagers is involved in a secret hoedown on his fields, they accidentally start a fire. some of the young amish people are quick enough to quench it. nevertheless, the police arrive together with henry as the owner of the field. since no harm is done, henry does not want to press charges against them (herb ), and later on he goes as far as to take a stand for them in front of their families. finally, henry spoke up. “it seems there was a hoedown, with the band playing up on a flatbed trailer. from what the firemen tell me, the generator’s exhaust manifold failed and a spark caught the grass. these kids here put it out with ice and water from the drink coolers.” “is that true?” paul asked his son. “ja,” joe said. “me and simon were trying to beat it out with our coats but it wasn’t working. it was pris, who thought of the ice.” […] “but since no real damage was done, and they came and got their tractor just before i left to come here, i let the matter rest with an apology.” (herb - ) as insecure as he might be concerning his own life and future, he feels a certain need to interfere when injustice might be done. according to him, the amish young people that were caught in the field put the fire out and were not the ones causing it, therefore they should not be punished (herb ). another interesting situation arises when a family from new york spends their holidays in lancaster county. the two boys of the family are obviously not very well- educated, but over time eric, the younger one, shows a real interest in henry’s pottery business. without his parents’ knowledge, he asks henry to teach him pottery and as henry discovers eric’s talent, he helps him to finish a project to be admitted to an art school. eric even goes as far as putting the blame for some stolen item on someone else, simply so that his family will stay in lancaster county for some more days. when henry finds out about this, he openly confronts eric, just like any responsible father would do with his child. rather than answer, henry cocked an eyebrow at eric. “want to let your dad in on what’s been going on?” eric shook his head. “i think you’re going to have to, if this is how you planned to stall them long enough for the clay to dry. bad plan, son. you owe priscilla here an apology.” (keys ) the decision that eric can stay at sarah’s place to finish his project for art school, forces henry and sarah to work together. through this cooperation, henry’s fatherly role is awakened to a greater extent. now, his disciplinary actions are not his solitary decision any longer, but they demand to be based on their common agreement. sarah follows the pattern of an amish wife who allows the man to deal with the child if the situation asks for it. and henry readily takes this place, just like an amish father would. sarah yoder was nothing more than a friend – despite that odd moment the other night when she had done a very amish thing and yielded the man’s place to him in dealing with eric. it had been a very long time since he’d experienced that very feminine submission – and since he’d left church at nineteen, he hadn’t had a chance to experience it much to begin with. (keys ). as the author phrases it, a woman’s submission to him is odd for henry, and he can’t remember experiencing it in the non-amish world. what surprises him even more is his instinctive reaction to it by “taking the lead in teaching a boy” (keys ). this behavior follows the typical male role of an amish husband and father. as the story unfolds, this is not an isolated incident, but henry’s parental instinct is aroused again, when amish run-away jesse gets stranded at his place. as jesse is sick, sarah comes to nurse him back to health. again, henry steps in and assumes responsibility. she could have handled jesse, even if it had meant wedging his jaws open with the spoon and pouring the tea down his throat. she was not the mother of two teenage boys for nothing. but not for the first time, she was grateful that henry’s first instinct was to back her up – to demand obedience on her behalf when hers was the place of authority. (balm ) in summary, it can be stated that the character of henry byler follows the example of an amish man in more than one respect. he is gentlemanly, respectful, hard-working, and family-oriented when handling young people. arriving in lancaster county, he appears to be career-oriented, a characteristic that dissolves in the course of the story, retransforming him into a traditional member of the amish community. only his insecurity at the beginning and his creativity do not fit into the stereotypical male gender role of the plain people. priscilla mast priscilla mast is a sixteen-year-old amish girl in the middle of exploring the courting and dating business. at first, she is interested in sarah’s stepson simon, but as he does not reciprocate her feelings, her affection is won by joe byler. at the beginning of the novel, she behaves like probably a lot of teenage girls, whether amish or not. she likes to withdraw from her bustling family – “[her room] contained the things she valued most – privacy and a place to dream.” (herb ) –, tends to be driven by her emotions, and dreams about entering a relationship with simon who has not shown any interest in her yet. but she keeps on daydreaming and uses situations to her advantage to get closer to him, even though this behavior is not becoming for an amish girl. too late, she remembered that she should be trying to look as if she were waiting for someone else, or pulling up her socks in her gumboots, or anything but standing there like a grinning fool, waiting for him. mamm said that when it came to boys, only a forward girl walked up to them and started a conversation, […] (herb ) using the omniscient narrator’s perspective, the author portrays priscilla as a love- struck teenage girl who is aware of her foolish actions. in her own thoughts she compares herself to a “grinning fool”, but simply cannot help herself behaving like she does. furthermore, she even shows some male characteristics by desiring to be adventurous – “the fact that interesting and exciting were not words you’d use to describe the lives of the youngie in willow creek didn’t mean she wouldn’t be ready for them.” (herb ), and by her stubbornness to prove her parents that she is not a little girl anymore: “[…] she could still go to a band hop. if she did, they’d have to see that she wasn’t twelve anymore, that she could make her own decisions.” (herb ). on the surface, priscilla follows the plain amish dress code, inwardly however, she is rather concerned about her outward appearance. the feminine amish attribute of modesty does not truly apply to her, although her influence on her looks is restricted to the cultural regulations. a couple of hours later, the dresses were finished. priscilla put hers on and turned this way and that in front of the glass doors of der echschank, the corner cabinet, where mamm kept her wedding china. it was the only surface in the house large enough that you could see most of your reflection at once, since the bathroom mirror was only big enough to see your face and hair. the dress fit nicely and she liked the smooth touch of the fabric on her shins. (keys ) with this detailed description of the place where priscilla can admire her outfit, the reader is able to feel the limitations this teenager faces as well as the simple joy about her clothing. as far as her handling of young men is concerned, she is neither patient nor upright at the beginning. she makes use of tricks and even of another young man to arouse jealousy in simon. her conscience, however, convicts her of her wrongdoing, indicating that priscilla is still in a state of developing her character – a character that will eventually be more conform with the amish expectation. she’d been a complete idiot to agree to let joe byler take her home, when all she wanted to do was be with simon. had he even seen her leave with joe? and had she really sunk so low that she wanted that – wanted to make him jealous? (herb ) turning to priscilla’s traits that are more in line with typical amish females, there is her appreciation of friendships and community with other girls or women. just like her mother, she enjoys the exchange with her female friends and does not plan to neglect these relationships even when she is courting or when she will be married some time later (balm - ). additionally, she is very family-oriented, and it is relevant to her to let her family take part in her life. the thought of not staying in touch with them over a longer period of time is unthinkable for her. for instance, if she had been planning to travel a long distance to return to people who cared about her, she would have found out train schedules and bus schedules, decided on them, bought a ticket, and been able to write and tell her family that she would be arriving on this day at this time in this place. […] but boys? could they really be as oblivious as this to the simple pleasure of looking forward to something? (balm ) as far as a serious relationship to a young man is concerned, she finally outgrows her teenage attitudes, and blames them on pride and greed – “she had walked into a trap she had set for herself out of pride and greed – why be satisfied with one boy courting you when you could have two with a wave of your hand?” (balm ). priscilla then assumes responsibility and confesses to joe that she allowed simon to kiss her. when joe wants to take revenge on simon for it, priscilla stops him and says: ““i’m the one who’s in the wrong, so i’m the one to talk to him.” (balm ) – again showing that she has outgrown her childish ways and become a responsible young woman. furthermore, her attitudes towards a prospective husband have changed: this whole subject of courtship was a puzzle, for certain. at least she was feeling fairly settled about joe. […] joe didn’t make her heart leap – or only a little anyway. it was his steadiness she appreciated. […] it was nice to be liked just for being yourself. (keys ) the way her feelings toward joe are expressed here, is in stark contrast to her earlier attitudes. while she was looking for excitement and adventure before, she now feels “fairly settled about joe” and he “makes her heart leap only a little”. nevertheless, the author creates the image that her stance is now more desirable, because she enjoys being “liked just for being herself” as if she has now reached a safe haven. as it is customary among amish that couples discuss things and make decisions together, priscilla starts to desire and enjoy that intimacy with joe. it provides her with a sense of importance – as expressed in the comparison “second only to his father” – and appreciation: pris had never felt this sense of humility and gladness before – being a young man’s confidante (second only to his father) on a matter as important as how he would make his living was a completely new experience. it made her feel like a grown-up. (balm ) the character of priscilla mast is a vivid description of an amish girl who develops from an emotional, adventurous girl into a responsible, traditional, but still amiable young woman, who appreciates her female role in her community. . . . other female/male gender roles silas lapp silas lapp, a male amish character that appears for the first time in the second part of the series, comes from a different amish community and is about the same age as sarah. he is described as the embodiment of a typical amish man, as one character in the book says, “i’m glad to see it, too. he’s not a man to put himself forward, is silas. he’s modest, good to his parents, and works hard, just as a man should.” (keys ). as sarah’s relatives plan to set her up with silas, they continue to praise his virtues. “that’s the kind of man he is,” fannie said with satisfaction. “as i said – considerate, thoughtful, and on the lookout for chances to help, especially with a young widow who is also thoughtful and looking for ways to help.” (keys ) “apparently. he has a tender, faithful heart, and it has taken him a long time to recover.” (keys ) with these rather exaggerated statements about silas – “especially with a young widow” and “a long time to recover” – the author emphasizes the intentions of sarah’s relatives to set her up with this amish man. by looking at silas’s actions, the reader notices that he is helpful, indeed, even ready to choose women’s work over men’s – “to her surprise, instead of heading out to the barn as well, silas took a dish towel out of the drawer and leaned on the counter next to the drain rack.” (keys ). but as the story unfolds, it becomes obvious that his motive behind his actions is a desire to win sarah and to use every possible chance to spend time with her. and although his relatives claim that “he’s not a man to put himself forward” (keys ), this does not hold true when he approaches sarah very directly: “but your youngest boy, i would think he needs a father.” oh, now, this really was forward. “he is close to his daed, and he has several uncles close by to stand in that place for him.” (keys ) silas is not only very direct, but he also turns out to be the kind of man who thinks that women must have a husband to be able to raise their children. his words might be polite – “i would think” –, but their content is very direct. in sarah’s eyes, this is a quite provoking attitude and she quickly counters his argument. his forwardness can also be seen when he asks herself out just a short time after they have met, a characteristic that does not go in line with amish men’s humbleness and modesty. “i’ll be here for a few days,” he said. “perhaps we might go for a drive and i can tell you more.” sarah swallowed her surprise at his forwardness; he hadn’t seemed like that kind of man. (keys ) his forwardness is depicted in his speech: rather than asking her, he suggests what they could do and what he wants to do. he only speaks from his perspective and does not ask for her opinion at all. within the plot of the romance novel, the character of silas lapp serves the purpose of being the rival in love. however, the danger he presents is not substantial as the author describes him in a rather unsympathetic and dominant way. simon yoder simon is the son of sarah’s deceased husband whom she raised as if he was her own son. he is seventeen at the beginning of the story and in the middle of his rumspringa years. nevertheless, he is thoughtful and hardworking (balm ), and “a passionate young man who thought and felt much more than he ever said” (balm ). one amish male attitude he has a problem with is humbleness. his stepmother sarah observes more than once that simon has a problem with pride. “the trouble is, much as i love my boy, he has his faults. and one of them is pride. i’m not talking behind his back – he knows it as well as i do. he thinks that all he has to do to win priscilla’s heart is crook his finger and smile at her.” (balm ) [sarah:] “[…] it’s not the size of the sin that matters, it’s the attitude and unwillingness behind it. and simon, you and i both know that you have a little struggle with pride. […]” (balm ) his weakness of being proud goes hand in hand with his dominant attitude. this trait is partly due to the mere fact that his father died five years before and he feels responsible for the family now. the older he gets the more does he adopt the role of man of the house, even though he is still insecure about his own future. furthermore, he cannot discern when he should take responsibility and when to obey his mother. her boy straightened, his face flushing even as his hands came out of his pockets. “someone has to stand in dat’s place, and you’ve told me often enough that it’s me.” “that is true for many things, but not this one.” (herb ) she knew very well what simon was thinking. but it wasn’t his place to bring it up – he might be the man of the house, but she was still his mother, and there were certain things that respect taught a young man not to say. (balm ) in spite of his juvenile restlessness and some rebellious moments, simon is a family- oriented young man. he does not only respect his stepmother but cherishes her. he does not want to leave for his working journey to colorado without her blessing (herb ), and he shows his affection to her openly – “and to her surprise, he wrapped his strong young arms around her and gave her a bear hug – something he hadn’t done since he was caleb’s age.” (herb ). however, he only does so privately and not in public since such a behavior would not be appropriate for a young man (herb ). furthermore, he has a strong sense of belonging to his family and his community. when he writes a letter from colorado, he mentions his desire to be at home and be part of the amish community more than once: and while they’re real nice here and treating me like i’m actually doing the job they hired me to do, it’s not home. i don’t know what joe’s got in his mind, but i’m thinking that when the snow flies in october, i’ll get back on that train and come home. i miss you and caleb and grandma and grandpa and the family. […] it feels funny not going to church. guess i’m not cut out for rumspringing and the english life the way some people are. (keys ) in summary, it can be stated that the author portrays simon as an amish young man who still needs to find his purpose in life, and therefore mainly serves as an example for an amish teenager in his phase of rumspringa. he is strong willed and adventurous, explores the world outside his home town but realizes in the end that he belongs to his community. jacob yoder the last notable character to be discussed is jacob yoder, sarah’s father-in-law. although he does not appear in the novel frequently, he nevertheless serves as a stereotypical example for an older amish man and thereby supports the amish setting of the story. by introducing this character, the author creates some comic relief in the story, especially when she describes his outward appearance as seen through the eyes of a young english boy: from what caleb said, eric thought that jacob yoder was some human incarnation of an old testament prophet, and hardly had the courage to speak at all in the same room in case he got zapped by lightning. (keys ) by this rather humorous hyperbole she portrays jacob yoder as an example of an amish patriarch whose sheer presence seems to be awe-inspiring. this is partly due to his size and the typical long beard: yesterday morning, her father-in-law had set the englisch boy to simple tasks in the milking parlor, and sheer awe of his size and his beard and the way his kindly gaze still managed to pin you down had apparently made eric decide that obedience was the only way he’d survive the experience. (keys ) the amusing juxtaposition of jacob’s kindly gaze that still demands total obedience already indicates that jacob yoder is not a dominant and overly austere person. he definitely expects respect, but he has a kind heart and is ready to help when help is needed: “she had been lucky that michael was such a good father – and that his father had stepped in to fill that place when michael had been taken from her” (keys ). this character fulfills the role as a representative of the amish community, and while other characters have their weaknesses and flaws, he seems to stand out as a person of respect and credibility: jacob was here to preside at the table in michael’s place. his humility was his greatest strength. no one could be offended – not even trent parker – when jacob explained their customs in such a gentle but firm way. (keys ). by focusing on his humility and his gentle but firm manner, no place is left for criticism. even though he is hardly involved in the plot, this figure serves as an anchor for amish faith, values, and traditions. . . lancaster burning series by linda byler ( ) linda byler grew up in an amish community and still is an active member of the amish church in her hometown in central pennsylvania. she started writing amish novels in and continues publishing books. she lives with her husband, children, and grandchildren and besides writing novels, she is also a columnist for a weekly newspaper in the amish community (book series in order). in a short introductory video about herself, she states that she gets the ideas for her characters from people she has met throughout her life, and that it is her goal to depict amish life “as it really is” in contrast to the ideal that people have about the amish (meet linda byler). the lancaster burning series consists of three books: fire in the night ( ), davey’s daughter ( ) and the witness ( ), and is set in lancaster county, pennsylvania. the heroine of the story is sarah byler, a young amish woman and the daughter of david and malinda byler. since her childhood, sarah has been drawn to matthew stoltzfus and now, as a young woman, she is desperately in love with him. matthew, however, has started dating rose, sarah’s friend. the framework plot of the novel is barn fires that break out on various properties at an interval of several months, beginning at the byler’s home. right from the beginning, it is clear that these fires must have been caused by an arsonist and did not happen by accident. while the community discusses if and what should be done to stop the arsonist, sarah struggles with her love life. when rose breaks up with matthew, she believes her dreams to come true, as matthew focuses his attention on her. however, as he himself is unsatisfied with amish life, he wants to leave the community and his family’s faith. at the same time, another young amish man, lee glick, is drawn to sarah, but knows about her attachment to matthew. matthew finally decides to leave and to join a missionary group that works in haiti. he asks sarah to come with him, and while she ponders the possibility of leaving everything behind, she is suddenly informed that matthew has married one young woman of the missionary group and gone to haiti with her. in the midst of her emotional struggles, sarah is asked to take over the local amish school as a teacher. she accepts this job, and there she also meets lee again, because his young nephews attend the school. https://www.bookseriesinorder.com/linda-byler/ https://www.youtube.com/watch?v=wjmnrijlzeu as they grow closer, lee and sarah finally decide to start dating, but right at that time, news spread about matthew that his wife has died and that he is probably coming back home. again, sarah hopes for matthew’s attention. then, another barn fire breaks out and sarah, who is close by, tries to rescue the horses from the barn. while doing so, a beam explodes over her and knocks her to the ground. she is rescued but suffers severe burns in her face from the flames, and it seems that her career as a teacher as well as a prospective wife and mother is over. however, it turns out, that lee, in contrast to matthew, does not care as much about her looks as about her as a person, and she finally realizes that he is the love of her life. in the end, the arsonist is caught, and peace is restored in the community. . . . main female and male characters and their gender roles sarah byler sarah, the nineteen-year-old daughter of david and malinda byler is the heroine of the novel. she is portrayed as strong, hard-working young woman as can be seen in this passage: she was a farm girl, her arms rounded, strong, and muscular. so the fifty- pound bags of flour and sugar were no problem, the endless rolling of pie crusts no big deal. she smiled easily and was always friendly and helpful to the other workers. (lancaster burning ) whatever she does, she does with all of her heart, and by using words like “truly” and “devotion” the writer reinforces this trait: “sarah truly loved her job now, and her devotion showed in her willingness to take on any task” ( ). concerning her outward appearance, she seems to be rather pretty, especially seen through the eyes of lee, one of her suitors: the grace with which she moved! not very many girls could carry off this unconsciousness of themselves, an innocence born of a good upbringing. she was like a calm pool of water in a hidden corner of a forest – ethereal, transparent, cool, and untouched. ( ) in this passage, linda byler makes use of metaphors and hyperboles to describe the good traits of sarah. lee perceives her as calm, cool, and innocent, and in his eyes, she is rather a supernatural being, indicating that she is unreachable for him. this exaggerated image creates some humor for the reader, because the inner emotional turmoil of sarah concerning her love life has been vividly portrayed before: they [rose and matthew] were the perfect match, and sarah was happy for matthew. so happy, in fact, that she cried great tears of happiness that puddled into a rushing river of misery. ( ) the juxtaposition of “tears of happiness” and “river of misery” reflects sarah’s emotions. on the one hand, she desires to be a loyal friend to rose and matthew, on the other hand, her own feelings are hurt. because of her amish belief she tries to fight jealousy but is not very successful. she earnestly desires to find the love of her life, the narrator creating a very intense image by depicting her thoughts like this: “so she yearned, said nothing, spoke of a love only to herself, and hoped someday, somewhere, god would have mercy and fulfill the want she harbored” ( ). in this respect, sarah is portrayed in the way of a stereotypical woman whose main goal in life is to be married and have a family: motherhood was an eagerly awaited event for sarah. she looked forward to cozily sitting together with other young mothers, comparing experiences about births and babies, raising children, the various ways of canning and freezing, cooking and baking. it was an objective for every young girl, and sarah was no different. ( ) all the verbs used in this passage deal with the tasks of an amish woman and generate the impression of a welcoming, comfortable home. to emphasize the importance of this woman’s goal, the author makes use of the following alliteration: “marriage, managing a home, and motherhood was the only route, chosen for her by her parents’ wishes” ( ). sarah, however, keeps on struggling with her unrequited love for matthew. she has a very close relationship to her mother and they repeatedly talk about this issue, but to no avail: “mam had stood by her side. they’d talked, reasoned, shared their feelings, and grew close. but at the end of the day, she still had to sit on the slippery banks of the muddy river called misery – and simply deal with it” ( ). again, the metaphor of the “river called misery” creates the impression of hopelessness. however, the heroine is not willing to let outsiders know about her misery, or to allow them to pity her for being still single; rather, she shows a certain sense of humor by making ironic remarks as can be seen in this encounter with an older amish woman: she decided to stand her ground [when asked why she doesn’t date yet]. “i have dreams of becoming an old maid and having my own dry goods store.” her words carried well, reaching mam’s ears as she ducked behind a washer to conceal her wide grin and jiggling shoulders. ( ) when she finally has private moment with matthew, where he behaves as if he were interested in her – although he is still courting rose – she cannot really enjoy it, because she is quickly overcome by guilt: sarah looked up, the sting clouding her vision, as he [matthew] looked down into her pain-filled eyes. it wasn’t her fault that he didn’t look away, she chided herself later when she felt so guilty and brash and so bold and so . . . well, stupid. it wasn’t her fault that as he had laid the burdock leaf across her hand, his hands were shaking more than a little. […] when she stood up, he was much too close, and she wished he’d move away. ( ) by this anaphora “it wasn’t her fault” her inner struggle is intensified, she does not want to appear as a brash young woman. it is obvious that she still knows what the appropriate behavior of an amish female should be, and even though her feelings for matthew are very intense, she is not willing to give up her attitudes completely. that proves her to be a strong-willed young woman. when she finally comes to her senses, – after she heard about matthew marrying someone else although shortly before he had asked her to start dating – she is outspoken and direct as can be seen in this dialogue with matthew’s mother: clearly, sarah spoke, her words precise, well placed, ringing. “hannah, in your opinion, nothing has ever been matthew’s fault. his whole life has been spent atop the pedestal you provided for him.” “sarah! don’t be so . . . why, sarah, i hardly know you like this!” “well, you can get to know me if you want. if not, that’s fine with me. i have been dragged through the muck by matthew for the last time, hannah. and you, too.” ( ) in figurative language the author describes the heroine’s feelings about matthew’s behavior towards her. nevertheless, that period in her life has led her to question her background and her culture. for a short time, she thinks about leaving her family and community and she ponders the advantages and disadvantages of doing so: “the inner conflict was an unbearable thing. she could never leave dat” ( ). obviously, family relations are more important to her, especially the one with her parents: “she couldn’t make a choice of such magnitude in a few hours. it would break her parents’ heart. the church would eventually cast her away, shun her.” ( ) furthermore, the thought of being shunned seems unimaginable to her. but it is not fear that makes her decide to stay, but rather the realization that she simply feels at home where she is right now: well, i’m just too amish. home canned pumpkin, bean soup, white cape and apron pinned to her dress, the uniform of the unmarried woman. she had still not fully recovered from the wonder of the possibility of leaving, the excitement of actually being able [to] go and do something out of the ordinary. […] no, she would not want to leave, truly. ( ) nevertheless, this incident shows her that she is free to choose, that she is an independent woman, able to make her own choices. sarah chooses the traditional, esteeming its values and security: it was an appreciation of heritage, a rich experience of lives lived before theirs – the stories, the respect for birth, life, and death, for marriage and raising children. it was continuing to live upright humble lives and existing in harmony amid a world filling with more and more confusing and unwanted technology. ( ) the author chooses the omniscient narrator’s perspective to stress the positive traits of amish living. in this juxtaposition she compares amish life to appreciation, respect, uprightness and harmony, whereas the world outside is described as confusing and unwanted. thus, sarah decides to remain within her tradition, as it offers her stability and harmony. due to the fact that sarah rejects lee twice because of matthew, she has to take the initiative in order to experience a happy ending: “if god gave her one last chance before tomorrow, she’d take it” ( ). as she does take initiative, she does not care what others might think of her, being very spontaneous, expressed in short rhetorical questions: “then, a wild thought. what did it matter? who would care? they were two mature adults, no longer silly teenagers shamelessly flirting” ( ). because of all that occurred within the last year, sarah sees herself as matured, as a woman who knows what she wants. her relationship to lee perfectly fits sarah’s choice: “[…] this was slow, easy, secure. it was bliss borne of knowledge, knowing the wait was all a part of god’s plan” ( ). nevertheless, there are strong feelings between them, indicating that sarah’s choice of lee is one of love and not one of reason: “sarah watched lee emerge from the door of the diesel shed and experienced a strange thrill, an intuition, or was it only her imagination? would he feel it, seeing her there […]” ( ). linda byler portrays her heroine as an amish young woman, who develops her character in the course of events. from being a teenager being hopelessly in love with the wrong man, she develops into a strong, young woman who embraces her role as an amish female on purpose. she does not feel inferior to men but enjoys what she is doing and finds stability and security in the amish tradition and culture. matthew stoltzfus mathew stoltzfus is the villain in this romance novel. he is a few years older than sarah and grew up in the same amish community. the author describes him as “tall, dark, and built like a wrestler” ( ), this simile already indicating that he does not really fit into the amish culture. he is well aware of his good looks and knows that a lot of girls admire him. sarah’s sister priscilla sees him as “arrogant, a flirt, and not even worth her time” ( ) and sarah’s mother remarks, “matthew is less than ambitious. he flirts with any girl who will look at him, and there are plenty of them. i’m not convinced he loves sarah at all” ( ). more than once, someone states that matthew is a flirt, who just loves to be loved by the girls. not only that, he does not follow any rules of propriety, as can be seen in this scene: matthew’s arm went out, for only a second, a half circle about her waist. blushing furiously, priscilla yanked at the cake, grasped it, and pivoted out of his way. matthew laughed and looked down at sarah. “boy, your little sister is growing up!” he whistled, watching her retreating form. ( ) this behavior does not fit into the stereotypical role of an amish man, so the reader can easily imagine that he will probably leave the community. to him, women are inferior and if he could, he would exclude them wherever they seem to be a danger for him. for example, when sarah defeats him in a game of ping-pong, he reacts like this: “matthew’s voice was low and harsh. ‘yeah, well. it might be a good idea to eliminate the girls playing against the guys, and you know it.’” ( ). by depicting his voice as brutal and mean, the image of matthew as a bad guy with no good intentions is reinforced. it seems as if the only thing that could keep him among the amish community is his date rose. so when she breaks up with him, there is nothing to hold him back: “[…] i told her [rose] if she broke up with me i was going to go english. i mean it. if i can’t have rose, i’m not going to stay amish. there’s no point in it” ( ). the way he talks, he reminds one of a stubborn little child, posing threats on grown-up people: he always got his own way with everything, and when he couldn’t have his gorgeous rose – the one time he had ever hit a brick wall – he acted like the spoiled brat he’d always been and just left, taking it out on everyone. it was his battered ego, his pride that was driving him. ( ) towards sarah, he explains his desire to leave in a more sophisticated way: “he wanted to go away. he wanted to travel, see the world. he needed to get away on a spiritual quest” ( ). he claims to be on a path of self-finding rather than being wounded in his vanity. sarah realizes, that deep in his heart, he “did not want to be amish. he didn’t appreciate the heritage of his family, the old linage of conservatism, the traditions, the way of life” ( ). the simple enumeration of amish values emphasizes the missing emotional connection of matthew with the amish culture. whenever the whole community gets together after another barn fire to help the affected family, matthew is not part of it and even mocks those who are there to help: matthew listened and then responded kindly but with a sort of petulance, inquiring about her [sarah] having to be there. […] “every time there’s a scene, and that seems to be happening with a certain regularity, there you are in the middle of it. why?” his tone was high, anxious, mocking. […] “it’s my duty. my parents always stress that.” “well, mine don’t.” there was nothing to say in response. ( ) the attribute of petulance, and his high, mocking tone paint a picture of ruthlessness, and by asking why sarah is always there, his complete ignorance of community values such as helpfulness or care becomes obvious. this is in stark contrast to his claim of desiring to go on a spiritual quest. his thoughtlessness and vanity are further exposed in the way he talks about people with special needs: “yeah, well. one of these days the old boy [levi] will kick the bucket. mongoloids don’t often live to be forty, do they?” sarah opened her mouth in reply but was stopped short by priscilla’s clipped tones. “he’s not a mongoloid, matthew. that word is outdated, taboo. he has down syndrome. we hope he’ll live to be a hundred. you have no idea how much our family enjoys him. he’s the star of the household. but you wouldn’t know, because he doesn’t look nice to you.” ( ) as the author makes use of colloquialism when matthew is talking, this character appears even less sympathetic. besides being rude, he is furthermore depicted as lazy, remaining “maddeningly absent” ( ) when everyone else in the community is helping together. his mother hanna is the only one who still excuses his behavior: hannah got up, saying she’d go get coffee and wake matthew. it was embarrassing, the way he slept. sarah had no idea matthew was still in bed. my, he was quite a sound sleeper. ( ) to sum up, the character of matthew is everything an amish man should not be. he is portrayed as dominant, flirtatious, insensitive, lazy and proud and, nevertheless, amish. he serves the purpose of the rascal in the novel and thereby emphasizes the importance of the good traits of amish men. lee glick lee glick is another young amish man and the hero of the romance novel. he encompasses all the good traits of a young amish. the narrator brings him into play without mentioning his name at first, thereby creating tension in the story: “this exchange [between sarah and her brother levi] was not lost on the blond youth, who watched sarah and forgot to eat his cake. he’d never seen eyes that color” ( ). he is fascinated by sarah right from their first meeting, which is conveyed by his action. besides being blond, his features are described through sarah’s eyes, when they meet for the first time: “she looked at him fully for the first time ever, the blue of his eyes taking her completely off guard. his eyebrows were perfect, like wings. his nose was stubby and wide but somehow also just right” ( ). the adjectives “perfect” and “just right” indicate a deeper meaning than the mere description of his facial features. it also serves the purpose of foreshadowing, that this young man might be “just right” for sarah. furthermore, he is “also very good-looking, in a blond, non-matthew way” ( ). the author begins already at this point to compare lee and matthew. concerning his character, lee is portrayed as humorous and friendly: “he turned and smiled easily, unselfconsciously. the humor on his open face was genuine, a magnet that drew her eyes to his” ( ). the adjectives “open” and “genuine” paint the image of an upright character, someone who has no bad intentions. and his impression is compared with the simile of “a magnet”, hinting at the attraction that begins between sarah and lee already at this point of the novel. furthermore, he is thoughtful – “the blond guy had jumped up and offered his hand to help her up. not very many boys were so thoughtful” ( ) –, as well as kind and helpful: “[h]e was always one of the first ones on the scene at barn raisings, one of the last to leave. it seemed as if that was all he did – work for other people and help them out” ( ). the image of lee as a thoroughly good man is reinforced by this statement of him being always among the first who come to help and staying until the end. this constancy and patience is also in effect concerning his love for sarah. because he knows that her feelings are captivated by matthew, he decides to stay in the background and not to rush things: when a guy named matthew ducked under the net and teased her [sarah], the look of raw adoration she gave him cemented his resolve to stay in the background. ( ) “i’ll be honest, sarah, okay? if you say it makes it easier, do you mean i may have a chance someday?” she was going to say, “don’t wait for me, lee.” she really was. what she said was, “your eyes are so blue. they remind me of a . . . a . . . this is dumb, lee.” she gave a low laugh. “your eyes make everything easy. they’re calm.” “thank you, sarah.” ( ) he proves strong in his patience, and he does not force his feelings on her. nevertheless, he is honest and clearly speaks his mind. he wants her to make a decision on her own, respecting her free will as a woman and is willing to wait until she chooses, and he outrightly tells her so: “when matthew comes back, you’ll be completely unfettered. you alone must choose” ( ). he goes even further and says, “but i think i honestly love you so much that i want only your happiness. if i have to give you up for you to find true happiness, i will. it won’t be easy, but i’ll do it. for you” ( ). this kind of unselfishness is mentioned in the novel more than once, it seems to be part of his lifestyle, “he lived a life of unselfishness, always giving his time and energy to others” ( ). the more sarah gets to know lee, the more he reminds her of her own father, who is paramount example for an amish male (see chapter . . ): she realized that lee spoke, and thought, along the same lines as her own revered father. ( ) “lee, pulling off to the side of the road and allowing cars to pass – that puts you in the same highly-respected category as my father. thank you. not all men will do that.” ( ) this similarity excites respect in sarah, just as she reveres her own father. this comparison puts lee in the category for a perfectly fitting husband for her, one who will keep the old tradition and the lifestyle she cherishes. after sarah’s accident in the fire, lee is also the one who does not care about her scars. even though she struggles with her scarred face, he does not: “lee had told her she was beautiful, which she obviously was not. and he had told her she was an amazing person, which she obviously wasn’t either” ( ). this repetition shows his ability to look beyond outward appearance, and that he really cares about her inner values, when he explains her: “(…) you can’t know how bad it really is. it will disgust you.” “but sarah. that has nothing to do with love. if a man truly loves a woman, he accepts her just the way she is. her size, her looks, what she wears, scars, whatever – it’s all insignificant.” ( ) the perfection and the goodness of lee, however, culminate at the end of the novel, when sarah talks with her sisters about him: “you mean, he never says no?” ruthie shrieked. “i wouldn’t know when.” “he never gets angry? not even when he’s stressed? snappy?” anna mae asked in disbelief. “i have never seen him that way.” ( ) with the character of lee glick, linda byler has created a real knight in shining amour. he is the epitome of the ideal amish man. he is helpful, thoughtful, kind and good-hearted. he respects women and their wishes, is hard-working and hardly ever stressed or angry. all in all, the complete opposite of the villain matthew. . . . other female/male gender roles david byler david byler is sarah’ father and has had his share of difficulties in his life. his firstborn son levi was born with down-syndrome, which caused david to examine his heart. at that time, he believed that he “must have done something wrong for god to send them a ‘retarded’ son” ( ). but in the course of his life, levi becomes a center of joy for the whole family. as the byler’s barn is the first one to burn down, david’s faith is challenged again, as can be seen in this dramatic description: “dat was a dark tragic figure now, so human and pitiful, somehow so unable” ( ). he seems to be far away from a strong amish believer. yet, besides being a farmer, he is an ordained minister of the community, “carrying the burden of being a servant of the lord, striving to keep peace and unity among his people – the small district of twenty-some families – protecting his flock from “the wolf”, the ways of the world” ( ). the way his task is depicted – “carrying the burden and striving to keep peace” – makes it obvious that it is not always easy. although he is a person in leadership, his character resembles the one of a sensitive man: “dat was the kindhearted one, the dreamer who colored your days with different shades of jokes, laughter, smiles, little sayings, or poems” ( ). the phrase “colored your day with different shades” indicates that he is someone who positively influences the lives of the people around him. furthermore, he is portrayed as a thoughtful and affectionate father, who goes to great lengths to make his children happy. for example, when priscilla loses her horse in the barn fire, he shows deep compassion and is willing to pay a high price for a new horse: dat saw priscilla’s fear, slowly laid his german bible aside, and went to her. gripping her arms, he looked into her terror-stricken eyes and gave her a small shake. “priscilla!” his voice was kind but firm. as if roused from a faint, she blinked, looked at dat, then fell against him. as she sobbed out her pain and anguish, his arms came around her, his head laid on her hair. ( ) what a precious father! she knew that he wouldn’t stop bidding until he had procured the one object that would successfully erase the hurt and the pain the arsonist had inflicted with a small rasp of his lighter. ( ) the determination of david at the auction expressed by “he wouldn’t stop bidding” proves that he is strong-willed, whenever a goal needs to be achieved. although sensitive, he still has his beliefs and does not waver in them. concerning the arsonist, he holds fast to the amish belief of not taking revenge but to forgive. even when a greater part of the community prefers to stand up and take action to find out who started the barn fires, he still holds fast to forgiveness: “that’s just it, malinda. it’s not our way. it’s not. we are a nonresistant people. or used to be. to my way of thinking, we do not fight back. god allowed the arsonist to accomplish this […]” ( ). by the anaphora of “it’s not”, his decision is confirmed. however, that does not mean, that it is easy for him, as this metaphor describes: “but in the still of the night, he’d wrestled with his own personal angel” ( ). especially, when sarah is severely injured in the last fire, his steadfast stand is challenged: […] the only emotion he felt, after a long while, was anger. unresolved, boiling anger, as hot as the falling beam that had hit sarah. it had scraped and burned her shoulders and back raw, like ground beef. […] it was his fault. he’d clung to the old ways. how much of it was tradition, how much was pride, and how small was the slice of heartfelt conviction? […] how could he forgive an arsonist who had burned his daughter at the stake? ( ) in this passage, the author makes use of an anaphora again: “how much, how much, how small”, intensifying the image of david struggling with his faith and his tradition. during these challenging times, his wife malinda is the support that he needs, and they are obviously on equal terms, complementing and helping each other in any situation: “when dat reached across the tabletop and grasped mam’s hand, he told her she was all the support he needed and thanked her for being strong” ( ). at the end of his emotional fight, he finally returns to his steadfast belief in forgiveness. he is upright and open with his community and tells them about his feelings: “’i wanted to literally beat up the person who did this. i am ashamed to tell you what i wanted to do. but there is only one way through this, and it’s forgiveness.’” ( ). furthermore, when the arsonist is caught and brought to justice, he lets actions follow his belief, indicating that he does not only talk about forgiveness, but is actually acting on it: when he rose to go, dat did not shake hands, he simply pulled the young man into an embrace and released him. keeping a hand on his shoulder, he said firmly. “we forgive you. be a man now, and change your ways. you’ll come out of this a better person.” ( ) david byler is not only a traditional amish man, hard-working, god-fearing, respectful, thoughtful and working together with his wife on an equal level, he also serves as a symbol for amish tradition and belief in the novel. although shaken himself, he is still an anchor for his community throughout the story. malinda byler malinda byler is the wife of david byler and sarah’s mother. she is described as a “comely older woman with years of compassion and hard work molding her features” (lancaster burning ), being a “hard-core realist” ( ), and no matter what happens in her life or in the lives of her family, “[n]o one like mam to bring you straight back to reality, plunk you down in the middle of it, and put you to work” ( ). even though she experiences more than one tragic event in the course of the story, she nevertheless tries to remain steadfast, not allowing hurt and grief to overtake her: that was mam. she was the best manager in lancaster county, hannah said. sarah watched, amazed at her mother’s ability to deal with the responsibility of the day’s demands after only a few hours of sleep. ( ) when sarah is severely injured, malinda stays at her bedside to do what needs to be done. this behavior is portrayed with the short, clear sentences in this passage: “mam never wavered. there were no tears. her daughter was awake, she was calling for her, and she had a duty to perform” ( ). duty seems to be more important to her than giving in to emotions, and the other’s well-being is more important than her own as she says, “those times she didn’t think of herself, her weariness. she just went ahead and did her duty, the same as she would do now” ( ). the author calls malinda a “great traditionalist” ( ) and a woman who knows her duty and her position: what set a minister’s wife apart? nothing, and yet a great deal depended on her support. she needed to supply quantities of it, always striving to build her husband’s confidence and his service by guta-rote (sound advice) as the lesser vessel. at the same time she was a powerful ally, and sarah knew the power of her mother’s prayers better than anyone. ( ) in this passage the juxtaposition of “lesser vessel” and “powerful ally” indicates that malinda is not simply a submissive wife. on the contrary, as she is a powerful ally to her husband, she obviously complements him, and both are working together to have a positive impact on the community. their roles might be different, but it is their relationship and companionship that makes them powerful and influential. concerning sarah’s attraction to matthew, malinda acts as a wise mother, who knows when to speak and when to be quiet. but she always lets her daughter know that she loves her and is available to her: “mam saw the strained smile and the increasing despair, but she decided to let sarah busily weave her web of unhappiness until she was ready to talk” ( ). however, when she feels that the right moment has arrived, she speaks openly and directly: “so what should i do to change things?” sarah asked. “stop thinking he wants you. he doesn’t. he’s dating rose. he’s a flirt.” like rocks thrown at her, mam’s words hurt, and sarah winced, now painfully aware of her mother’s honesty. this was so unlike her soft-spoken mother. ( ) the simile of “like rocks thrown at her” show how clear malinda’s words are, and portray her as an upright woman, who knows when the truth needs to be told. and especially because she is usually “soft-spoken”, her words of truth have an even stronger impact. besides, her realistic attitude and her experience in handling her children, malinda is still a woman who enjoys beauty and a nice home: though she beamed and smiled and her eyes twinkled as she secretly anticipated her wonderful “new” house, she always kept her head bowed and tried to be humble – but she really wasn’t. ( ) she was, after all, a minister’s wife, and her house needed to be in the ordnung (within the rules) as befitted the wife of a leader in the church. but, oh, how she adored it! she scattered hand-woven rugs made from cast-off clothing, enjoying the charm the vibrant colors added, and went about her days with a song in her heart, surrounded by the things she loved. ( ) as the reader can notice here, malinda thoroughly enjoys her new house, although at the same time she is aware that this might be interpreted as pride. she only “tried to be humble”, while she actually was totally excited about the home they had renovated. thus, she can also be seen as a woman who enjoys pleasures in her life. not only that, she certainly has a sense of humor and enjoys a good laugh as well: “mam lamented her total lack of restraint but finally conceded to it, sitting down and laughing till she had to remove her glasses, wipe her eyes, and take a deep breath” ( ). the detailed description of how she laughs enhances the sympathy she already gained with the reader and makes her even more loveable. as one can see, malinda byler is a strong, traditional and realistic woman, who remains steadfast through the troubled times of her life. she exudes wisdom and kindness, but also great love for the people around her. towards her husband, she is a submissive wife but also a strong companion by his side. within the confines of her family, however, she behaves very emotional, too, and enjoys the simple pleasures of life. hanna stoltzfus the character of hanna stoltzfus is an unusual portray of an amish woman. she is the mother of matthew, and while she is described as “kind” and “understanding” at first ( ), and outwardly follows the stereotypical image of an amish mother who loves cooking and pampering family and friends with her meals, a closer look shows a different picture. for example, she arrives with lots of home-cooked food at a barn raising, but obviously does it mainly to be observed and admired: “always running the show, being the boss, and then the minute the real chores started, she sat there in all her glory. it just irked mamie” ( ). she is a woman who does want others to see her only in a positive way. for any fault or failure she easily finds an excuse. when her son matthew leaves the amish and marries an english woman, although he has just started courting sarah, hanna reacts like this: “’hannah said he’s so soft-hearted, he just couldn’t tell you. he knew you would take it hard. he’s just so kind, hannah said.’” ( ). with this anaphora, the excuses she makes for her son, appear even more ridiculous and show how much she ignores the wrongs of her son. compared to her husband, who feels ashamed of his son’s decisions, she praises her son even more: he [elam] carried the true humility of having a son gone astray, but hannah bore her pride for her son like a misplaced banner, speaking loudly about his missionary work in haiti without an ounce of modesty in her bearing. ( ) with the simile “like a misplaced banner”, the author highlights the fact that this behavior, which might be totally understandable in the modern world, does not fit into the amish setting. instead of being a mother who tries to raise her child, sometimes encouraging and disciplining it, “matthew was firmly glued to the pedestal of his mother’s pride, slowly turning from flesh and blood into various metals, hardening into an idol she would worship her whole life long” ( ). this image that hannah has of her son leads so far that she “was always hopeful that matthew was pleased and not too put out by her poor management” ( ), revealing that in her eyes her son somehow has taken the position a husband might have. throughout the novel, her view of her son does not change. and she does not recognize her mistakes, but in the end blames her husband for the wrong choices her son has made: hannah berated her husband for just sitting behind his paper. she thought he should be admonishing matthew, the way other fathers did. it was no wonder he wasn’t amish, and now it was hard to tell who he would marry. ( ) the interesting feature about the character of hanna is that she does not change her behavior at all as the story unfolds. she shows only a few typical amish female traits – being helpful, kind and an expert in the kitchen –, but there is hardly any humbleness or modesty; but she rather craves for validation and likes to put the blame on others. . conclusion in this concluding chapter of the thesis, the findings of the three serial novels concerning the gender roles of the characters portrayed in the books will be summarized. these findings will be compared to each other, especially in regard to the question, how these gender representations differ between the novels written by non-amish authors compared to the ones of the amish author linda byler. furthermore, the relationship between female and male protagonists in the selected romance novels will be compared. to begin with, the focus will be on the heroines. meredith in a lancaster county saga ( ) fits into the scheme of a typical young amish woman. in contrast to the other two heroines, she is about twenty years old, has recently married luke and is not looking for a prospective partner – at least not on the outset. the situation changes a short time later, when she believes her husband luke has died. she is portrayed as kind, friendly and modest, having a close relationship with her husband, but also as submissive, when realizing that luke is stressed. her behavior takes a turn, when she believes herself to be a widow, and she suddenly stands her ground, wanting to be self-sufficient. in this respect, she is similar to sarah yoder in the healing series ( ). sarah yoder is already a widow at the beginning of the romance, however, she is in her mid-thirties, raising two teenage sons. her husband died five years before, so she has learned already to take care of her family, although she receives help from her relatives. sarah has no intentions of finding another husband, she rather focuses on making a living as a herbal healer. sarah yoder is portrayed as a kind and helpful woman, too, but from the beginning on she wants to be self-sufficient. she is also rather outspoken and driven by the desire to help other people. meredith, on the other hand, appears more like a woman who needs to be helped, although she does not want to admit it. the third heroine sarah byler (lancaster burning ) differs from the two other female protagonists inasmuch as she is a nineteen-year-old girl hoping to find the right husband. she is also described as friendly, kind and helpful and cherishes amish values and traditions. similar to sarah yoder, she is someone ready to hel p and support other people, therefore also focused a lot on others, even though she is in a deep emotional conflict concerning the love of her life, matthew. concerning him, one could say, she is submissive, but not because she sees it as her amish trait as woman but rather because she is ready to do everything necessary to win his attention. although she does not allow other people to treat her unkindly, and she knows how to hold her ground, this attitude vanishes when matthew appears on the scene. all three women show a certain stubbornness: meredith, when she wants to take care of her herself, sarah yoder in raising her two sons on her own, and sarah byler when she is not listening to any warnings about matthew. to sum up, all three female main characters show the stereotypical feminine features of being kind, friendly and helpful. in addition, the kind of work they do stays within the confines of typical female jobs: sewing, planting, baking and working as a teacher. at the same time, they all use their circumstances to prove that they can stand their ground, nevertheless, they happily enter a partnership, as soon as the right husband appears on the scene. although meredith and sarah yoder question their background a little bit and quickly realize that they are happy within the amish confines, sarah byler compares the amish and the english world on a deeper level and even for a short time thinks about leaving together with the man she believes to be in love with. sarah yoder, on the other hand, knows that she will only enter a relationship with henry, if he comes back to the amish faith. this indicates that she is deeper rooted in her tradition than sarah byler, who is much younger and still has to find herself. turning to the male protagonists of the story, they differ more widely in the three novels. in a lancaster county saga ( ), meredith’s husband luke, as well as her suitor jonah are both hard-working, traditional, friendly, and helpful young men. both of them desire to have a family and be the breadwinners. luke shows some dominant attitudes when he realizes that he cannot provide for his family and keeps on denying the right to start looking for a job as well to meredith. in the healing series ( ) the two opponents are henry and silas. these characters are more diverse than the men in a lancaster county saga ( ). henry is a former amish and is rather insecure, constantly drawn between his former life and the outside world, nevertheless he shows typical amish features such as taking over the role of a responsible father, who disciplines young people if needed. at the same time, he shows a lot of sympathy for the amish teenagers who overstep traditional limits, excusing their behavior. furthermore, he is polite, gentlemanly and thoughtful. in contrast to henry, silas always was and still is amish, and is determined in finding a suitable wife. although he behaves rather politely, he nevertheless shows traces of feeling superior to women, firmly believing that a woman cannot raise children without a husband. he is quite forward with sarah, not readily accepting that his feelings are not reciprocated. the highest contrast in male gender roles, however, can be found in lancaster burning ( ). here, the author describes the young amish man named matthew, who is flirtatious, selfish and lazy. he does not consider women to be on equal levels with men but uses them for his own pleasure. in stark contrast to him is lee, who is portrayed as thoughtful, kind and patient, always ready to be of help, where help is needed. he respects women and does not see them as inferior. furthermore, outward appearance is not as important to him as the character of a person. in the portrayal of these two young amish men, linda byler as the amish author, differs greatly from the other two authors, whose male protagonists fit into a rather stereotypical amish gender role. when we look at the relationships between men and women in the stories, the heroine and the hero are almost always on equal level. hardly any trace of dominance of the men over the women can be found, except in a lancaster county saga ( ), where at the beginning, luke rejects the idea of his wife looking for a job, because he thinks it is his duty alone to provide for them. and he even quenches any further discussion about this issue. however, he apologizes to her later, and discusses his new plans with her. still, the impression remains that meredith does not really have a say in this, as he is determined to pursue his plan. in healing grace ( ) it is rather the woman, sarah yoder, who appears to be the stronger. her steadfastness causes henry to return to the amish faith, if he wants to enter a relationship with her. during some incidents, however, where they are both involved in educating and training a teenager, sarah allows and expects henry to take the place of leadership. when looking at lancaster burning ( ), sarah and lee are a couple that really complements each other, not one of them being superior to the other. the villain matthew, however, obviously feels superior to women and does not consider them having equal rights. apart from the lovers in the novels, there are some older couples portrayed, whose relationship is either very harmonic or rather unbalanced. the parents of sarah yoder and the parents-in-law of her deceased husband michael (healing grace ) are both portrayed as harmonic couples, there is no unusual interaction to be noted among them. they love each other, respect each other and discuss important issues together. furthermore, the fathers as head of the household do not show any superior behavior. in wanda brunstetter’s serial novel (a lancaster county saga ), there is one couple that stands out: sadie and elam stoltzfus. at first sight they appear as a happy and harmonic couple, but soon it turns out that sadie, knowing that her husband would not approve of her actions, secretly follows her own plans. there might be outward submission, but her actions tell another story. apart from sadie and elam in brunstetter’s romance novel, there is also one rather untypical couple in linda byler’s book: the parents of matthew, hannah and elam stoltzfus. hannah obviously spoiled her son matthew during his childhood and teenage years and excuses every misbehavior on his part. but rather than disciplining him and being ashamed of their son’s actions, she continues to take a stand for him. apart from her bevahior, her husband elam does not intervene at all and shows no traces of being the head of the household. he simply lets his wife do whatever she wishes. thus, there is neither any sign of leadership and submission in their marriage, nor any evidence that they discuss things at home and complement each other. one figure that appears in senft’s as well as in byler’s novel, is the amish patriarch. in healing series ( ), sarah’s father-in-law jacob yoder fulfills this role not only in his outward appearance (tall and with a long beard) but also in his character, who radiates strength and wisdom, and disciplines others in kindness. he is the epitome and guiding figure for the amish tradition and faith. a similar character is found in lancaster burning ( ) in the person of david byler, the father of sarah. throughout the story, he is described as the kind, friendly and wise bishop of the community, who speaks openly to the members of the congregation, reminding them of the amish values of forgiveness and non-resistance. furthermore, he not only preaches about these values, but follows them in his own life, no matter how hard it seems to be. to sum up, the female main characters in all three romance novels – no matter if written by a non-amish or amish author – portray rather stereotypical amish gender roles except for their desire to be self-sufficient at least until the right husband appears. the thoughts and emotional struggles of the heroine are, however, most intensely described by non-amish writer linda byler. furthermore, she is the author who describes the greatest variety of characters, among them such, who do not fit into a stereotypical amish gender role like matthew or hanna stoltzfus. as far as this can be said about a fictional romance novel, linda byler portrays her characters in the most realistic way, including a wide range of different personalities. in contrast to the amish writer, wanda brunstetter and adina senft stay mainly within the stereotypical gender roles in the depiction of their characters. bibliography primary literature brunstetter, wanda e. goodbye to yesterday: part . uhrichsville, oh: barbour publishing, . ebook file. brunstetter, wanda e. the silence of winter: part . uhrichsville, oh: barbour publishing, . ebook file. brunstetter, wanda e. the hope of spring: part . uhrichsville, oh: barbour publishing, . ebook file. brunstetter, wanda e. the pieces of summer: part . uhrichsville, oh: barbour publishing, . ebook file. brunstetter, wanda e. a revelation in autumn: part . uhrichsville, oh: barbour publishing, . kindle file. brunstetter, wanda e. a vow for always: part . uhrichsville, oh: barbour publishing, . ebook file. byler, linda. lancaster burning: trilogy. new york, ny: good books, . senft, adina. balm of gilead. new york, ny: faithwords, . senft, adina. herb of grace. new york, ny: faithwords, . senft, adina. keys of heaven. new york, ny: faithwords, . secondary literature book series in order. n.d. aug. <https://www.bookseriesinorder.com/linda- byler/>. cordell, sigrid. “loving in plain sight: amish romance novels as evangelical gothic”. journal of amish and plain anabaptist studies . 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( ): - . nolt, stephen m. the amish: a concise introduction. baltimore, md.: john hopkins university press, . post, robert c. “a theory of genre: romance, realism, and moral reality”. american quarterly (fall ): - . regis, pamela. a natural history of the romance novel. philadelphia, pa.: university of pennsylvania press, . shelley, adina. “it’s not about the bonnet: the appeal of amish fiction”. romance writers’ report (oct. ). (http://shelleyadina.com/extras/) shields, stephanie a., and elaine c. dicicco. “the social psychology of sex and gender: from gender differences to doing gender.” psychology of women quarterly (fall ): - . Štekovič, maja. ”crossing cultural frontiers: representations of the amish in american culture”. acta neophilologica . ( ): - . stories with spirit. mckay, memphis, . aug. . <https://shelleyadina.com/about>. visser, irene. “prototypes of gender.” women’s studies international forum . ( ): - . wanda e. brunstetter. dylnicki, ron, . aug. . <https://wandabrunstetter.com/about/>. weaver-zercher, valerie. thrill of the chaste: the allure of amish romance novels. baltimore, md.: john hopkins university press, . worell, judith. encyclopedia of women and gender. san diego, ca.: academic press . http://shelleyadina.com/extras/ appendix english abstract the amish people have been moved into the focus of the american media in the last decades, not only through movies or television shows but also through books of various genres. especially among readers of christian faith, novels about the old order people have gained high popularity, the amish’s simple lifestyle providing a welcome alternative to the fast-paced lifestyle of the modern world. apart from their outward appearance – women wearing long-sleeve dresses and head coverings, men with long beards and broad trousers with suspenders – they seem to still follow a patriarchal society model with the women staying at home, taking care of the children, whereas the men carry the responsibility of the breadwinner. within the last decades, novels about the amish were mainly written by non-amish authors. only about ten years ago, this situation changed, when linda byler, an amish mother and grandmother, started writing fictional novels, too. the focus of this thesis is to examine how gender roles are portrayed in amish romance novels by non-amish writers compared to amish writers. in order to analyze this question three serial novels have been chosen: wanda brunstetter’s a lancaster county saga ( ), adina senft’s healing grace ( ) and linda byler’s lancaster burning trilogy ( ). although there are a lot of similarities, the novels show that the non-amish authors mostly follow the stereotypical amish gender roles in the portrayal of their characters with only a few small exceptions; whereas linda byler portrays a far wider range of female and male characters that do not always fit into a typical feminine or masculine amish role. deutsche zusammenfassung in den letzten jahrzehnten wurden die amischen in den amerikanischen medien immer populärer, sei es durch filme oder fernsehserien oder durch bücher unterschiedlicher gattungen. vor allem bei der christlichen leserschaft werden romane über die amischen immer beliebter, da der einfache ländliche lebensstil eine willkommene abwechslung zur schnelllebigen, modernen welt bietet. die amischen fallen nicht nur durch ihr äußeres erscheinungsbild auf – die frauen mit langärmeligen kleidern und kopfbedeckung, und die männer mit bärten, breiten hosen und hosenträgern –, sondern auch durch ihr patriarchalisches gesellschaftsmodell. die frauen kümmern sich um haushalt und kinder, während die männer die hauptversorger der familie sind. die meisten romane über die amischen, die in den letzten jahrzehnten geschrieben wurden, wurde von autoren verfasst, die nicht den amischen angehörten. diese situation änderte sich vor etwa zehn jahren, als unter anderem linda byler, eine amische mutter und großmutter, begann, ebenfalls romane zu verfassen. der schwerpunkt dieser diplomarbeit liegt nun darauf, zu analysieren, wie die geschlechterrollen in amischen liebesromanen dargestellt werden, insbesondere darauf, ob in dieser darstellung ein unterschied zwischen autorinnen, die nicht den amischen angehören, und jenen, die tatsächlich amische sind, besteht. zur durchführung dieser analyse wurden die folgenden drei romanserien ausgewählt: wanda brunstetters a lancaster county saga ( ), adina senfts healing grace ( ) and linda bylers lancaster burning trilogy ( ). obwohl es in den büchern viele Ähnlichkeiten gibt, so zeigen die romane dennoch, dass die nicht amischen autorinnen größtenteils den typischen amischen geschlechterrollen in der darstellung ihrer charaktere folgen – mit einigen wenigen ausnahmen –, wohingegen linda byler ein weitaus größeres spektrum an personen beschreibt, die nicht immer in das bild einer typischen amischen frau oder eines typischen amischen mannes passen. op-brai .. brain a journal of neurology mutations in b galnt (gm synthase) underlie a new disorder of ganglioside biosynthesis gaurav v. harlalka, ,* anna lehman, ,* barry chioza, ,* emma l. baple, ,* reza maroofian, harold cross, ajith sreekantan-nair, david a. priestman, saeed al-turki, meriel e. mcentagart, christos proukakis, louise royle, radoslaw p. kozak, laila bastaki, michael patton, karin wagner, roselyn coblentz, joy price, michelle mezei, kamilla schlade-bartusiak, frances m. platt, matthew e. hurles and andrew h. crosby institute of biomedical and clinical science, university of exeter medical school, st. luke’s campus, heavitree road, ex lu, exeter, devon, uk child and family research institute, the university of british columbia, west th ave, vancouver, bc, v z h , canada department of ophthalmology and vision science, university of arizona school of medicine, tucson, az , arizona, usa department of pharmacology, university of oxford, oxford, ox qt, uk wellcome trust sanger institute, wellcome trust genome campus, hinxton, cambridge, cb sa, uk medical genetics unit, floor , jenner wing, st. george’s university of london, cranmer terrace, london sw re, uk department of clinical neuroscience, institute of neurology, university college london, queen square, london wc n bg, uk ludger ltd., culham science centre, oxfordshire, ox eb, uk kuwait medical genetics centre, ghanima alghanim building, maternity hospital, sulaibikhat, postal code: , kuwait windows of hope genetic information centre, holmes county, walnut creek, oh ohio, usa division of neurology, the university of british columbia, ubc hospital s – wesbrook mall, v t b , canada *these authors contributed equally to this work. correspondence to: prof. andrew h. crosby, institute of biomedical and clinical science, university of exeter medical school, st. luke’s campus, heavitree road, ex lu, exeter, devon, uk e-mail: a.h.crosby@exeter.ac.uk correspondence may also be addressed to: prof. frances platt, department of pharmacology, university of oxford, oxford, ox qt, uk, e-mail: frances.platt@pharm.ox.ac.uk glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. this disease, because of defects in the first step of ganglioside biosynthesis (gm synthase), results in a severe epileptic disorder found at high frequency amongst the old order amish. here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from kuwait, italy and the old order amish. our genetic studies identified mutations in b galnt (gm synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of gm in affected subjects in association with a predictable increase in levels of its precursor, gm , a finding that will greatly facilitate diagnosis of this condition. with the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously doi: . /brain/awt brain : ; – | received june , . revised july , . accepted july , . advance access publication october , � the author ( ). published by oxford university press on behalf of the guarantors of brain. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. at s aid b usiness s chool on january , http://brain.oxfordjournals.org/ d ow nloaded from http://brain.oxfordjournals.org/ http://brain.oxfordjournals.org/ unidentified family of ganglioside deficiency diseases exist. the study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies. keywords: ganglioside biosynthesis; b galnt ; amish; spg ; hereditary spastic paraplegia abbreviation: gsl = glycosphingolipid introduction glycosphingolipids (gsls) are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons (schnar, ). deficiencies in their catabolic pathways give rise to excessive intralysosomal accumulation of these lipids associated with a large and well-studied group of inherited disorders, the lysosomal storage diseases (platt and walkley, ; ballabio and gieselmann, ). these disorders typically involve progres- sive neurodegeneration and, for the majority of these diseases, no effective treatment is available (platt and lachmann, ). although many gsl catabolic defects have been defined to date (ginzburg et al., ), very few are known to result from a defect in gsl biosynthesis and previous reports of potential gan- glioside biosynthesis disorders based on biochemical evidence have not been proven genetically (fishman et al., ). proven dis- orders include a defect in serine palmitoyltransferase long chain subunit (sptlc ), which catalyzes the first step in sphingolipid biosynthesis leading to downstream ceramide and sphingolipids biosynthesis, which is associated with hereditary sensory and auto- nomic neuropathy (hsan ) (bejaoui et al., ). however, these patients retain the ability to synthesize gsls by using l-alanine as the precursor instead of serine. this results in the production of an atypical class of deoxy-sphingolipids. a more selective defect has also been described, termed ‘gm synthase deficiency’, which was previously the only proven defect in ganglioside biosynthesis. this novel human disease is a severe epileptic disorder identified amongst families of the old order amish community (simpson et al., ). its existence led us to consider that other human diseases, not yet identified, may exist that result from defects in enzymes that function at different steps in the ganglioside biosyn- thetic pathway (lannert et al., ; daniotti and iglesias- bartolome, ). as gangliosides are conserved lipids, expressed at high levels in the nervous system of mammals, we hypothesized that any such conditions would likely display neurological (particu- larly neurodegenerative) phenotypes. in the current study, we investigated the genetic and molecular basis of an unusual neurodegenerative phenotype, best described as a complex form of hereditary spastic paraplegia. the hereditary spastic paraplegias are among the most genetically diverse of all inherited mendelian disorders, with distinct loci and genes identified to date. they comprise a large group of neuro- degenerative diseases characterized clinically by the presence of lower limb spasticity, and pathologically by the retrograde degen- eration of the longest nerve fibres in the corticospinal tracts and posterior columns. the genes known to underlie hereditary spastic paraplegia seem to encode molecules with a diverse range of cel- lular functions, implicating numerous defective biological processes in the pathogenesis of this disease (blackstone et al., ). we previously mapped a gene for a complex autosomal recessive form of hereditary spastic paraplegia (spg ) in a large kuwaiti family (family , fig. a) to a � mb region of chromosome q (wilkinson et al., ). in the current study, we determined that mutations in b galnt , encoding gm synthase, are re- sponsible for this condition. we also identified mutations in this gene in an italian-canadian family as well as families from the old order amish community, in which the only other known disorder relating to defective ganglioside biosynthesis has also been described. materials and methods subjects blood or buccal samples were obtained from affected children, parents and unaffected family members with informed consent. dna and rna were extracted by standard procedures. a single . -mm diameter skin biopsy was taken from individuals from family iv: , iv: (affected) and iv: (unaffected), and family vii: and vii: (affected). all samples were obtained with approved informed consent. all affected individuals underwent a detailed physical examination. exome and targeted regional sequencing exome sequencing was undertaken in a single affected individual from each family, using the sureselect all exons mb target enrichment system (agilent) and sequencing on a hiseq (illumina) with bp paired end reads. single nucleotide variants were called using two programs (gatk, samtools) while insertion-deletions (indels) were called using (gatk and dindel). all variants were annotated using dbsnp ( ) and the genomes pilot study. single nucleotide polymorphism effect predictor (vep) was used for predicting the vari- ant consequences on the protein structure based on ensembl (version ). both single nucleotide variants and indels called by the three programs were then merged into a single file for downstream analysis. variants were filtered out if the read depth was � or �, if the consensus quality was or if the single nucleotide polymorph- ism quality was . genotyping and linkage analysis single nucleotide polymorphism microarray genotyping was performed using illumina human cytosnp- v . (family ) and affymetrix . new disorder of ganglioside biosynthesis brain : ; – | at s aid b usiness s chool on january , http://brain.oxfordjournals.org/ d ow nloaded from http://brain.oxfordjournals.org/ http://brain.oxfordjournals.org/ single nucleotide polymorphism (family ) arrays. marker saturation was carried out using existing and newly generated microsatellite markers (primer sequences available on request). alleles were size- fractionated using % polyacrylamide gels and dna was visualized by silver staining. mutation analysis unique sequence intronic primers were designed to amplify all poten- tially pathogenic variants identified within the critical region. purified pcr amplification products were sequenced using dye-terminator chemistry and electrophoresed on an abi xla capillary sequencer (applied biosystems). mutation analysis was carried out using mutation surveyor software v . (softgenetics). both strands of each product were sequenced. fibroblast culture human forearm fibroblasts were cultured in dulbecco’s modified eagle medium with % foetal bovine serum and antibiotics, after standard protocols in cm flasks. cells were dissociated from flasks for analysis by means of min incubation with . % trypsin with . % edta at �c. fibroblast pellets were taken up in . ml milli-q water and freeze-thawed three times. gsl extraction, isolation, aa-labelling and hplc were performed as previously described (neville et al., ). exoglycosidase digests labelled glycans released from fibroblast gsls were digested for h at �c in a total volume of ml with sialidase a, �-galactosidase, b-galactosidase and b-hexosaminidase (prozyme/glyko) according to manufacturer’s instructions. following digestion, labelled glycans were separated from enzyme protein using corning� costar� spin-x� centrifuge tube filters before hplc, as above. maldi mass spectrometry released glycans were permethylated (ciucanu and costello, ) then analysed on a shimatzu resonance maldi instrument using , -dihydroxybenzoic acid (dhb) matrix per-o-methylation of neutral carbohydrates directly from aqueous samples for gas chromatography and mass spectrometry analysis (ciucanu and caprita, ). samples were analysed with two different mass range settings: – and – da. results genetic studies we previously detailed the clinical features of a large kuwaiti family with a complex neurodegenerative phenotype (wilkinson figure (a) pedigrees of families (kuwaiti), (italian) and (old order amish). segregation of c. dup (exon ; p.leu thrfs* ), c. g c (exon ; p.lys asn) and c. g a (exon ; p.arg his) variants throughout each family with mutant allele indicated by ‘ + ’ and wild-type allele indicated by ‘�’, as well as the sequence chromatogram for each variant. (b) the position of each sequence variant with regard to domain architecture of the protein where ‘a’ refers to ‘glycosyl transferase, family domain’, and ‘b’ indicates ‘nucleotide-diphospho-sugar transferases superfamily domain’. (c) species conservation alignments for the regions affected by each variant. | brain : ; – g. v. harlalka et al. at s aid b usiness s chool on january , http://brain.oxfordjournals.org/ d ow nloaded from http://brain.oxfordjournals.org/ http://brain.oxfordjournals.org/ et al., ) and a clinical description of the additional italian-canadian and old order amish families is supplied in the supplementary material. the most prominent clinical phenotype common to these families is progressive weakness and spasticity, affecting the lower extremities, plus or minus the upper extremities. all affected individuals also manifest apparently non- progressive cognitive impairment. additional features included cerebellar signs, sensory polyneuropathy, pes cavus, stereotypies, emotional lability and psychiatric illness. seizure activity was reported in two of four amish individuals. where neuroimaging was undertaken no abnormalities were reported. therefore, a clin- ical diagnosis of complicated hereditary spastic paraplegia had been assigned to these families, although there are additional fea- tures present in some affected individuals that provide evidence of a broader phenotype associated with gm synthase deficiency. to robustly position the gene responsible for this condition, we first refined the extent of the disease locus previously defined in the kuwaiti family using microsatellite markers to chr : – , a � . mb region containing � genes (data not shown). in order to identify the causative mutation we designed a custom � tiling bait array to capture a targeted region encompass- ing this region. the target size was � . mb after repeat masking, generating . gb of data with a mean depth of . after filtering and exclusion of novel sequence variants found to be present in homozygous state in kuwaiti controls, only a single likely deleterious variant remained as potentially causative. this was a frameshift in- sertion in the last exon of the beta- , -n-acetyl-galactosaminyl transferase (b galnt ) gene (fig. ; chr :g. dup; nm_ . : c. dup, p.leu thrfs* ), which may there- fore be likely to encode a transcript that escapes nonsense-mediated messenger rna decay surveillance mechanisms and produces a polypeptide with an altered and elongated c-terminus (fig. ). dideoxy sequence analysis revealed that the variant cosegregated with the disease phenotype, with affected individuals being homo- zygous for the variant, and parents and all unaffected offspring being heterozygous carriers (fig. ). in parallel with this targeted regional sequencing approach, whole exome sequencing was undertaken using dna from a single affected individual from two additional families with a clinically similar presentation to the kuwaiti family. the first of these families, who originate from a small village in the frosinone region of central italy, have two affected offspring from a consanguineous union (family , fig. ). after filtering of variants only two novel likely deleterious sequence variants were found to be present in genes located in shared homozygous regions between the two affected cases detected by a complementary affymetrix . single nucleotide polymorphism microarray scan (data not shown), both of which cosegregated with the disease phenotype as deter- mined by dideoxy sequence analysis. one was located in or s (nm_ . : c. c t; p.pro leu), an olfactory recep- tor gene considered an unlikely candidate to cause this condition. the second variant affects a stringently conserved residue in b galnt (fig. ; chr :g. g c; nm_ . :c. g c; p.lys asn) and is predicted to be damaging by polyphen- , sift and provean. the second dna sample subject to exome sequencing was from a single affected individual from an extended amish pedigree (family , fig. ). this identified another homozygous likely deleterious sequence alteration in b galnt (fig. ; chr :g. c t; nm_ . : c. g a; p.arg his), located in � mb region of homozygosity delimited by markers rs and rs (data not shown) shared by four affected individuals in three amish nuclear families. this variant cosegregates with the disease phenotype as determined by dideoxy sequence analysis and is predicted deleterious by polyphen- , sift and provean. it was found to be present in a heterozygous state in two out of amish control subjects, which is not unexpected of a founder mutation present in the community. the amish variant is not listed in the genomes genomic database (comprising chromosomes) and only a single heterozygous carrier of european origin was identified out of chromosomes analysed in the national heart, lung and blood institute exome sequencing project database (esp si release). neither the b galnt c. dup frameshift nor c. g c variants are present in publicly accessible genomic databases, or in ethnically matched control chromosomes. biochemical studies as the b galnt variants identified were predicted to abolish gm synthase activity of the encoded polypeptide, we investi- gated the biochemical effect of the mutation in cultured skin fibro- blasts from an affected brother and sister from the italian family, as these were available for investigation. gm ganglioside is a sialylated gsl that is synthesized in the golgi apparatus as part of a complex biosynthetic pathway (supplementary fig. ). gm synthase is a galnac transferase responsible for synthesizing gm , ga and gd (pohlentz et al., ). if gm synthase were deficient in the affected individuals, no gm , ga , gd or down- stream derivatives would be synthesized (blue box, supplementary fig. ). to test this hypothesis, fibroblasts from both affected in- dividuals were analysed by generating fluorescently-labelled gsl- derived oligosaccharides and performing hplc (fig. ) (neville et al., ). we confirmed the glycan structures of the gsl- derived oligosaccharides in healthy individuals by comparing them with commercially available authentic standards, by perform- ing exoglycosidase digestion and by confirming structures by mass spectrometry (supplementary figs and and supplementary table ). a representative profile of fibroblast gsls from an un- affected individual (fig. a) showed that the major gsl species detectable were laccer, gb , gm , gm and gb . hplc profiles from the two affected patients’ fibroblasts (family individuals iv: and iv: ) were identical to each other, but differed in several ways from unaffected fibroblasts. firstly, there was an increase in the level of gm relative to gb (fig. c). secondly, there was a large reduction in the gm peak (authenticated by mass spec- trometry, supplementary fig. , in the unaffected patient fibro- blasts), leaving only a minor peak ‘x’, in affected patient cells. to determine the structure of peak ‘x’, we performed sialidase-a di- gests on both affected and unaffected fibroblast samples. as ex- pected, the gm glycan was digested to lac, confirming this structure in both samples. in the unaffected fibroblasts the gm peak was converted to asialo gm (ga ) confirming it as gm (fig. b). peak ‘x’ in the affected patients (fig. c) was also sensitive to sialidase digestion (fig. d). however, in contrast with the unaffected sample (fig. b), it did not lead to the new disorder of ganglioside biosynthesis brain : ; – | at s aid b usiness s chool on january , http://brain.oxfordjournals.org/ d ow nloaded from http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/ http://brain.oxfordjournals.org/ production of ga and instead, was converted into either gb or laccer, ruling out the presence of gm in the affected patient fibroblasts (fig. d). to establish more specific structural informa- tion, preparative hplc was performed collecting individual peaks (fig. c). when peak ‘x’ was digested with sialidase-a, the peak shifted to a retention time consistent with gb , revealing the structure to be a previously unreported novel gsl namely sialy- lated gb . to explain this, we hypothesize that in the absence of functional gm synthase in the patient cells there will be a lack of downstream gsls as substrates for sialyltransferase, st galii, and this may be the cause of the ectopic sialylation of gb . absolute quantities of each individual gsl peak were determined and are summarized (supplementary table ). the two affected siblings showed remarkably similar absolute amounts of gsls whereas the control sample had � % less, in total, suggesting that the patients may have not only altered composition but also increased gsl levels. analysis of more samples will be required to verify this observation. in addition to the sialidase digests, further digests with �-galactosidase, b-galactosidase and b-hexosamini- dase confirmed the presence of gb , laccer and gb glycan structures in both samples (data not shown). similar biochemical findings were seen when analysing fibroblasts from the amish pedigree (individual ix: ; supplementary fig. ). discussion in the cns of mammals, ganglioside structures and their expres- sion levels are highly conserved. four gangliosides predominate, namely gm , gd a, gd b and gt b (schnaar, ). insights into the functions of gangliosides have come in part from studying engineered mice deficient in key glycosyltransferases required for ganglioside biosynthesis (schnaar, ). for example, when b galnt (gm synthase, the enzyme deficient in the patients in the current study) is deficient in the mouse, none of the major brain gangliosides are synthesized. instead, the simple gan- gliosides gm and gd are present at higher than normal levels, as they are synthesized before the biosynthetic block (takamiya et al., ; kawai et al., ). indeed, total gsl levels are comparable in both wild-type and engineered mice, with increased levels of ‘pre-block’ simple gsls compensating for the lack of complex ganglioside biosynthesis (sun et al., ) illustrating tight control of total gsl levels. as gd does not bind to myelin-associated glycoprotein (mag), a key mediator of myelin stability, and gm binds poorly to myelin-associated glycoprotein (collins et al., ), these mice have defective axon:myelin sta- bility leading to progressive neuropathy, similar to that seen in mag-deficient mice (sun et al., ). wallerian axonal figure representative hplc profiles for -aa labelled glycans released from gsls isolated from cultured human skin fibroblasts. unaffected patient (control; a and b). affected sister/sibling (c and d) in the italian family. b and d show the peak shifts, indicated with dashed arrows, following digestion of the labelled glycans with sialidase a. peak ‘x’ (in c) is a previously unreported gsl, sialylated gb . | brain : ; – g. v. harlalka et al. at s aid b usiness s chool on january , http://brain.oxfordjournals.org/ d ow nloaded from http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/ http://brain.oxfordjournals.org/ degeneration occurs in b galnt mice leading to impaired motor coordination and diminished hind-limb reflexes (sheikh et al., ). in addition, the mice displayed abnormal structures at the nodes of ranvier, leading to electrophysiological defects charac- terized by reduced conduction velocities in motor nerves (susuki et al., ). it remains to be determined the extent to which the clinical features of the patients in this study and the engineered gm synthase mouse converge, but loss of complex gangliosides could potentially disrupt axon:myelin stability in both mouse and man. to date, only one other human inherited disease resulting from a proven ganglioside biosynthetic defect has been described, namely gm synthase deficiency present amongst the old order amish (simpson et al., ). this presents clinically as a severe neurological syndrome, which in most patients involves intractable seizures, but not in the mouse model (yamashita et al., ), likely because of compensation through o-series gsl biosynthesis (supplementary fig. ). here we describe a second human neurological disease involving a defect in the adja- cent enzyme acting in the ganglioside biosynthetic pathway in families of different ethnic backgrounds, including the amish in which both known ganglioside disorders are present. all families show a phenotype consisting mainly of progressive spastic para- paresis, together with variable learning difficulties, although this doesn’t appear to be progressive. clinical examination and nerve conduction suggest a mild peripheral neuropathy in some of the cases. the siblings in family share several unusual traits in add- ition to paraplegia and intellectual disability: congenital cervical spinal narrowing, dysmorphological features (prognathism, wide mouth, deep-set eyes), and tumours (ovarian teratoma and colonic adenocarcinoma). these features may be merely coinci- dental, may relate to another genetic disorder, or more likely be connected to this disorder of ganglioside biosynthesis. the earlier report of a patient with the biochemical hallmarks of gm syn- thase deficiency at post-mortem (fishman et al., ) remains genetically unproven. whether that report describes a more severe phenotype arising from gm synthase deficiency remains unclear, although this may be unlikely as the mutations described in the current study and by boukhris et al. ( ) are all predicted to result in complete loss of enzyme activity, which has been con- firmed biochemically in families and in the current study. consequently it therefore remains impossible to determine the relationship between the cases of proven gm synthase deficiency in the current study, and the historic case report. however, the biochemical assay we employed may be of use diagnostically in the future in individuals with clinical features reminiscent of the complex form of hereditary spastic paraplegia described here. with the description now of two neurological human diseases involving defects in two sequentially acting enzymes involved in the ganglioside biosynthetic pathway, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exists. the study of patients and animal models of these disorders will pave the way for greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the future development of effective therapies to treat patients afflicted with these devastating disorders. web resources the urls for data presented herein are as follows: gatk; http://www.broadinstitute.org/gatk/about/citing-gatk vep; http://www.ensembl.org/info/docs/variation/vep/index. html samtools; http://samtools.sourceforge.net nhlbi exome sequencing project (esp); http://evs.gs.washing- ton.edu/evs/ online mendelian inheritance in man; http://www.ncbi.nlm. nih.gov/omim pubmed; http://www.ncbi.nlm.nih.gov/pubmed/ gene; http://www.ncbi.nlm.nih.gov/gene clustalw ; http://www.ebi.ac.uk/tools/msa/clustalw / polyphen- ; http://genetics.bwh.harvard.edu/pph / sift; http://sift.jcvi.org/ provean; http://provean.jcvi.org/seq_submit.php genecards; http://www.genecards.org/ the genomes browser; http://browser. genomes.org/ index.html the ensembl project; http://www.ensembl.org/index.html the national center for biotechnology; http://www.ncbi.nlm. nih.gov/ ucsc human genome database; http://www.genome. ucsc.edu accession numbers the databank accession number for b galnt reported in this paper is nm_ . . acknowledgements the authors are grateful to the families described herein for participating in this study, in particular the amish families for supporting the windows of hope genetic project (consent obtained from all participants or their parents or guardians with ethical approval from the institutional review board, st. george’s, university of london, uk; wandsworth research ethics committee, london, uk; the clinical research ethics boards of university of arizona, usa and the university of british columbia, canada). funding the study was supported by the medical research council grants (g ; g ), wellcome trust (wt ), newlife foundation, research to prevent blindness, mizutani foundation for glycoscience and the rare disease foundation (krz ). supplementary material supplementary material is available at brain online. new disorder of ganglioside biosynthesis brain : ; – | at s aid b usiness s chool on january , http://brain.oxfordjournals.org/ d ow nloaded from http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://www.broadinstitute.org/gatk/about/citing-gatk http://www.ensembl.org/info/docs/variation/vep/index.html http://www.ensembl.org/info/docs/variation/vep/index.html http://samtools.sourceforge.net http://evs.gs.washington.edu/evs/ http://evs.gs.washington.edu/evs/ http://www.ncbi.nlm.nih.gov/omim http://www.ncbi.nlm.nih.gov/omim http://www.ncbi.nlm.nih.gov/pubmed/ http://www.ncbi.nlm.nih.gov/gene http://www.ebi.ac.uk/tools/msa/clustalw / http://genetics.bwh.harvard.edu/pph / http://sift.jcvi.org/ http://provean.jcvi.org/seq_submit.php http://www.genecards.org/ http://browser. genomes.org/index.html http://browser. genomes.org/index.html http://www.ensembl.org/index.html http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/ http://www.genome.ucsc.edu http://www.genome.ucsc.edu http://brain.oxfordjournals.org/lookup/suppl/doi: . /brain/awt /-/dc http://brain.oxfordjournals.org/ http://brain.oxfordjournals.org/ references ballabio a, gieselmann v. lysosomal disorders: from storage to cellular damage. biochim biophys acta ; : – . bejaoui k, wu c, scheffler md, haan g, ashby p, wu l, et al. sptlc is mutated in hereditary sensory neuropathy, type . nat genet ; : – . blackstone c, o’kane cj, reid e. hereditary spastic paraplegias: membrane traffic and the motor pathway. nat rev neurosci ; : – . boukhris a, schule r, loureiro jl, lourenço cm, mundwiller e, gonzalez ma, et al. alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia. am j hum genet ; : – . ciucanu i, caprita r. per-o-methylation of neutral carbohydrates directly from aqueous samples for gas chromatography and mass spectrometry analysis. anal chim acta ; : – . ciucanu i, costello ce. elimination of oxidative degradation during the per- o-methylation of carbohydrates. j am chem soc ; : – . collins be, kiso m, hasegawa a, tropak mb, roder jc, crocker pr, et al. binding specificities of the sialoadhesin family of i-type lectins. sialic acid linkage and substructure requirements for binding of myelin- associated glycoprotein, schwann cell myelin protein, and sialoadhesin. j biol chem ; : – . daniotti jl, iglesias-bartolome r. metabolic pathways and intracellular trafficking of gangliosides. iubmb life ; : – . fishman ph, max sr, tallman jf, brady ro, maclaren nk, cornblath m. deficient ganglioside biosynthesis: a novel human sphingolipidosis. science ; : – . ginzburg l, kacher y, futerman ah. the pathogenesis of glycosphingo- lipid storage disorders. semin cell dev biol ; : – . kawai h, allende ml, wada r, kono m, sango k, deng c, et al. mice expressing only monosialoganglioside gm exhibit lethal audiogenic seizures. j biol chem ; : . lannert h, gorgas k, meissner i, wieland ft, jeckel d. functional or- ganization of the golgi apparatus in glycosphingolipid biosynthesis— lactosylceramide and subsequent glycosphingolipids are formed in the lumen of the late golgi. j biol chem ; : – . neville dc, coquard v, priestman da, te vruchte dj, sillence dj, dwek ra, et al. analysis of fluorescently labeled glycosphingolipid- derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling. anal biochem ; : – . platt fm, lachmann rh. treating lysosomal storage disorders: current practice and future prospects. biochim biophys acta ; : – . platt fm, walkley su. lysosomal defects and storage. in: platt fm, walkley su, editors. lysosomal disorders of the brain. oxford: oxford university press; . p. – . pohlentz g, klein d, schwarzmann g, schmitz d, sandhoff k. both ga , gm , and gd synthases and gm b, gd a, and gt b synthases are single enzymes in golgi vesicles from rat liver. proc natl acad sci usa ; : – . schnar rl. neuroglycobiology. oxford, uk: oxford university press; . schnaar rl. brain gangliosides in axon-myelin stability and axon regen- eration. febs lett ; : – . sheikh ka, sun j, liu y, kawai h, crawford to, proia rl, et al. mice lacking complex gangliosides develop wallerian degener- ation and myelination defects. proc natl acad sci usa ; : – . simpson ma, cross h, proukakis c, priestman da, neville dc, reinkensmeier g, et al. infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of gm synthase. nat genet ; : – . sun j, shaper nl, itonori s, heffer-lauc m, sheikh ka, schnaar rl. myelin-associated glycoprotein (siglec- ) expression is progressively and selectively decreased in the brains of mice lacking complex gangliosides. glycobiology ; : – . susuki k, baba h, tohyama k, kanai k, kuwabara s, hirata k, et al. gangliosides contribute to stability of paranodal junctions and ion channel clusters in myelinated nerve fibers. glia ; : – . takamiya k, yamamoto a, furukawa k, yamashiro s, shin m, okada m, et al. mice with disrupted gm /gd synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system. proc natl acad sci usa ; : – . wilkinson pa, simpson ma, bastaki l, patel h, reed ja, kalidas k, et al. a new locus for autosomal recessive complicated hereditary spastic paraplegia (spg ) maps to chromosome p . - q . j med genet ; : – . yamashita t, hashiramoto a, haluzik m, mizukami h, beck s, norton a, et al. enhanced insulin sensitivity in mice lacking ganglioside gm . proc natl acad sci usa ; : – . | brain : ; – g. v. harlalka et al. at s aid b usiness s chool on january , http://brain.oxfordjournals.org/ d ow nloaded from http://brain.oxfordjournals.org/ http://brain.oxfordjournals.org/ pii: - ( ) - cell, vol. , - , april , , copyright © by cell press mutations in the proteolytic enzyme calpain cause limb-girdle muscular dystrophy type a isabelle richard,* odile broux,* val rie allamand,* frangoise fougerousse,* nuchanard chiannilkulchai,* nathalie bourg,* lydie brenguier, * catherine devaud,* patricia pasturaud,* carinne roudaut,* dominique hillaire,* maria-rita passos-bueno,t mayana zatz,t jay a. tischfield,~ michel fardeau,§ charles e. jackson,ii daniel cohen,# and jacques s. beckmann*# *g n~thon , rue de i'lnternationale evry france tdepartment de biologia instutio de bioci~ncias universidad de s~.o paulo s~o paulo - brazil tdepartment of medical and molecular genetics indiana university school of medicine indianapolis, indiana - §lnstitut national de la sant~ et de la recherche medicale unit~ centre national de la recherche scientifique unite a , rue du fer & moulin paris france iihenry ford hospital detroit, michigan #fondation jean dausset centre d'etudes du polymorphisme humain , rue juliette dodu paris france summary limb-girdle muscular dystrophies (lgmds) are a group of inherited diseases whose genetic etiology has yet to be elucidated. the autosomal recessive forms (lgmd ) constitute a genetically heterogeneous group with lgmd a mapping to chromosome q . -q . . the gene encoding the muscle-specific calcium-activated neutral protease (canp ) large subunit is located in this region. this cysteine protease belongs to the family of intracellular calpains. fifteen nonsense, splice site, frameshift, or missense calpain mutations cosegregate with the disease in lgmd a families, six of which were found within la rdunion island patients. a digenic inheritance model is proposed to account for the unexpected presence of multiple independent mutations in this small inbred population. finally, these results demonstrate an enzymatic rather than a structural protein defect causing a muscular dystro- phy, a defect that may have regulatory consequences, perhaps in signal transduction. introduction the term limb-girdle muscular dystrophy (lgmd) was first proposed by walton and nattrass ( ) as part of a classi- fication of muscular dystrophies. lgmd is characterized by progressive symmetrical atrophy and weakness of the proximal limb muscles and elevated serum creatine ki- nase. the symptoms usually begin during the first two decades of life, and the disease gradually worsens, often resulting in loss of walking ability or years after onset (bushby, ). yet the precise nosological definition of lgmd still remains unclear. consequently, various neuro- muscular diseases such as facioscapulohumeral, becker muscular dystrophies, and especially spinal muscular at- rophies have occasionally been classified under this diag- nosis. these issues highlight the difficulty in undertaking an analysis of the molecular and genetic defect(s) involved in this pathology. both autosomal dominant and recessive transmission have been reported, the latter being more common with an estimated prevalence of - (emery, ). the local- ization of a gene on chromosome (lgmd a, mim ; beckmann et al., ) has provided proof for the genetic basis of one form of recessive lgmds. subse- quent genetic analyses confirmed this chromosome localization (young et al., ; passos-bueno et al., ). the latter group also demonstrated genetic hetero- geneity of this disease, while a recent study localized the lgmd b gene to chromosome (bashir et al., ). yet there is evidence that at least one other locus is involved, since genetic heterogeneity was demonstrated in the in- bred indiana amish lgmd kindreds (allamand et al., a). the nonspecific nosological definition, the relatively low prevalence, and genetic heterogeneity of this disorder limit the number of families that can be used to restrict the genetic boundaries of the lgmd a interval. no cytoge- netic abnormalities have been reported. immunogenetic studies of the dystrophin-associated protein complex (mat- sumara et al., ) and cytoskeletal or extracellular ma- trix proteins (e.g., tom~ et al., ) failed to demonstrate any deficiency, in addition, there is no known specific physiological feature or animal model to suggest a candi- date gene. thus, there was no alternative to a positional cloning strategy. detailed genetic and physical maps of the q . - q . lgmd a region were established. construction and analysis of a - mb yeast artificial chromosome (yac) contig (fougerousse et al., ) permitted the mapping of polymorphic markers within this interval and to nar- row the lgmd a region to between d s and d s . furthermore, extensive analysis of linkage disequilibrium suggested a likely position for the gene in the proximal part of the contig (allamand et al., b). cdna selection (tagie et al., ) for muscle-expressed sequences encoded by this interval led to the identification of five known genes and newly expressed sequences, core metadata, citation and similar papers at core.ac.uk provided by elsevier - publisher connector https://core.ac.uk/display/ ?utm_source=pdf&utm_medium=banner&utm_campaign=pdf-decoration-v cell table . pcr primers for the localization of the canp gone pcr product size (base pairs) position within primer name primer sequence ( '- ') the cdna cdna genomic dna annealing temperature (in celsius) c a n p - i n . a atggagccaacagaactgac - canp -in .m gtatgactcggaaaagaaggt - canp -inl .a taagcaaaagcagtccccac - can p qnl .m ttgctgttcctcactttcctg - can p - a . a gtttcatctgctgcttcgtt - canp - a .m ctggttcaggcatacatggt - canp -exlter.a ttctttatgtggaccctgagtt - canp -exlter.m acgaactggatggggaact - all potential c a n d i d a t e g e n e s (chiannilkulchai et al., ). o n e of these, p r e v i o u s l y c l o n e d by s o r i m a c h i et al. ( ), a p p e a r e d also to b e a functional c a n d i d a t e gone, as it e n c o d e s a m u s c l e - s p e c i f i c protein, c a n p ( n a m e d for cal- pain large p o l y p e p t i d e l ), w h i c h b e l o n g s to the c a l p a i n f a m i l y (or c a l c i u m - a c t i v a t e d neutral p r o t e a s e [canp]; ec . . . ). c a l p a i n s a r e n o n l y s o s o m a l intracellular cysteine prote- ases (murachi, ; s u z u k i and ohno, ; croall a n d d e m a r t i n o , ). t h e m a m m a l i a n c a l p a i n s include t w o u b i q u i t o u s proteins, canp and c a n p , as well as t w o s t o m a c h - s p e c i f i c proteins ( s o r i m a c h i et al., a) a n d canp . t h e u b i q u i t o u s e n z y m e s consist of h e t e r o d i m e r s with distinct large s u b u n i t s a s s o c i a t e d with a c o m m o n small s u b u n i t (murachi, ), all of w h i c h are e n c o d e d by different g e n e s (ohno et al., ). the association of tissue-specific l a r g e s u b u n i t s with a small s u b u n i t has not yet b e e n d e m o n s t r a t e d . t h e large s u b u n i t s of c a l p a i n s can be s u b d i v i d e d into four d o m a i n s (ohno et al., ). d o m a i n s i and iii, w h o s e functions r e m a i n u n k n o w n , s h o w no h o m o l o g y with known proteins. t h e f o r m e r , h o w e v e r , m i g h t be i m p o r t a n t for the regulation o f the proteolytic activity (imajoh et al., ). d o m a i n ii s h o w s similarity with o t h e r cysteine proteases, w h i c h s h a r e histidine, cys- teine, and a s p a r a g i n e r e s i d u e s at their active sites (sori- m a c h i et al., ). d o m a i n iv c o m p r i s e s four ef hand structures t h a t are potential c a l c i u m - b i n d i n g sites. in addi- tion, three u n i q u e regions with no k n o w n h o m o l o g y a r e p r e s e n t in the muscle-specific canp protein, n a m e l y ns, is , and is , the latter c o n t a i n i n g a n u c l e a r t r a n s l o c a t i o n signal (sorimachi et al., ). t h e s e r e g i o n s m a y be im- p o r t a n t for the m u s c l e - s p e c i f i c function of canp . w e have d e t e r m i n e d the g e n o m i c o r g a n i z a t i o n of the h u m a n canp g e n e , w h i c h consists of e x o n s a n d ex- t e n d s o v e r kb, kb of w h i c h have b e e n s e q u e n c e d . a s y s t e m a t i c s c r e e n i n g of this g o n e in l g m d f a m i l i e s led to the identification of different mutations, establishing t h a t m u t a t i o n a l events in canp are r e s p o n s i b l e for a g e n o m i c s t r u c t u r e of t h e c a n p gene ~ ~/i~f i ~ ..~i .i ill i ~l.t~_l li~,li~ji[i i li ii • x / b ecor, restrlction _ kb map ~ "~t~ ~[ ~ ~ ~,, ~ ~,~ e e ee e ee e e e i ,;~ i i i [ ii i l i c c o s m i d m a p , g • b l l • t b • f l l • • • a • g • • • g a • • = al ~. figure . genomicorganizationofthecanp gone (a) the canp gone covers a kb region of which kb were sequenced. introns and exons are drawn to scale, exons being indi- cated by numbered vertical bars. intron is the largest one and remains to be fully sequenced as well as intron . positions of intragenic mi- crosatellites are indicated by asterisks. closed and cross-hatched arrows indicate the orienta- tion of alu and mer repeat sequences, re- spectively. (b) ecori restriction map. an ecori (e) restric- tion map was established with cosmids from this region. the location of the canp gone is indicated by a closed bar. the size of the corresponding fragments are indicated and underlined when determined by sequence analysis. (c) cosmid map of the canp gone region. cosmids, from a library constructed by sub- cloning yac g , are presented as lines. overlaps are based on sequence-tagged site information from a cosmid contig established by i. r. et al. (unpublished data). dots on lines indicate positive sequence-tagged sites, which are boxed in rectangles. a minimum of three cosmids covers the entire gone. mutations in canp cause lgmd a a ~ c ~ g t t ~ = ~ c q q c c t ~ c c a c o ~ = ~ t ~ c c t t q ~ ~ ~ c t = = = ~ c = = ¢ t c . = q = q a = c ¢ t = c ¢ ~ = t q = t g ~ = q t ~ a ~ = t t ¢ c c t = t e c t = ~ t q = ¢ t t t t t ~ t l t t t t t t . . . . . . . ~ = c - . g . . t t ~ g a c c t c c ~ . t ~ c c c t ~ c ~ l g c g t t i t ~ i ~ t ~ t ~ t t ~ t t t ~ / a t ~ / g ~ g t ~ t t g ~ l g t c t g r i t a - $ j . . c t ~ c c t a c l c ~ t a ~ c g t c l g ~ t o c t ~ c t t c t t t c ~ g t g t c ~ c t t ~ ¢ t ~ g . t a ~ t t t ~ t t t t c t g ~ s ~ c c - • g ~ c t t ~ t ~ . a a t g t c c c ~ ¢ ¢ ~ ~ t c t c t & © ~ ~ g t c t ~ c t t ~ c t c t ~ t . c t ~ l r ~ t ~ t t c t t ~ ~ e o ' ~ o ~ ~ q ~ v w k ~ p ~ z c e n ~ r f ~ z d g a n r c d ~ o s o ~zo ~ o s s o s ~ o ~ o e~.~ ~ ) o s ~ o eso s ~ o ~ o v z z ~ o • z z ] o z ~ s o z z v o o z z ~ o • z ~ z o ~ z z o o ~ o s d x ~ o ~ w ~ v s v n ~ ¢ ~ w v e ~ c s . ' . ~ ~ c ~ n r z ~ s ~s~o z s ~ o * ~ o x :e ~ a ¢ s ~ o a l ~j k o r ~" ~ y r .~. s e ^ ~ s x *r • z s ~ c ~ r ~ ' r c c c ~ . c ~ , r r c c ~ c , r c . c c , r c c c ~ c ~ . , . c ~ , r c ^ ¢ c ¢ ~ c c ¢ c c x c ~ * c ¢ ~ u ; c c c c ~ c ~ c x ~ c ~ e ~ o ~ s ~ o z s s o zs~o ~ o o p k o • ( ; s r a p q p = s s o o b s ~ ~ o ~ s f ~ " z ~ k o o z l ( i v t ~ " ~ ~ c i ~ ~ c ~ c c ' ~ c c t ~ t c ~ " ~ / c ~ t g ~ / ~ l t c l c t ~ / g " t t t ~ g t t t ~ c c c t = t a t t t ¢ ~ a l g c c ~ c t ~ a c c t ~ a l ~ g l c c ~ g ~ a ~ c c c c t l ~ g g c t t c c . ~ c c t ~ t ~ l t ~ t g t t o c t c o t c ~ t c t t l ¢ ¢ c c c ~ c c ~ c c t t g l t ~ / ~ / t c " t g c c t a l ~ z g c e t ~ . c c c t t t l g " ~ . t g ~ / t ~ g g ~ l ~ c c c t t ~ l t c c ¢ ~ t t g c ~ t t t ~ a ~ g l ~ g t ' : / c " t g e c t c c ~ l g t e e ~ " ~ c c ~ q ~ c t n ~ t t c t g ~ ' e . ~ l ~ g ~ . t c ~ q c t t . ¢ ¢ t ~ g c ~ c t a q ~ c ~ t ¢ ~ ¢ ¢ ~ c c g g t g ~ c t ~ g ~ c c t c © t t ~ t ~ c t l ; a ~ a q ~ c t c ~ t ~ t t ~ c q c t g c c ~ c ~ t g g g c ~ l l ~ l ¢ ~ g c a c t g g g t t c t l c t c t t q ~ g t a a a c t e . ~ g t ~ g t c c c c t t l / t g t t t t ~ t t ~ t c t ~ t t t a g a t a t c a g c ~ t g ~ t g a c c g a l t g ~ ¢ t t c ~ t ~ © c t a t ~ ¢ c ~ l g ~ ~ g e l ~ t g ~ a " a ~ l ~ t c t t ~ / " a t t t t t t ~ t ~ t g c c t a ~ / c t a t t t c t g ~ l ~ t a a l / a a t g ~ / c t c ~ / a t l c ~ i a ~ / c t ~ / t t t ~ t t t g c ~ ¢ ~ c t c t g ~ / a c ~ t g g ~ t ~ t a t c t ~ t " ~ / g ~ t c c t ~ ' t g t c t t c ~ = c ~ t t t c c t t c t t ~ t ~ / c ~ c " ~ g c ~ g ~ g g ~ t ~ c ~ t ~ " e t ~ t g ~ a ~ g t m ~ g " ~ t ~ t ~ " t t ~ t ~ g t ~ c t t ~ t t t t c ~ t ~ t t a t a t t c ¢ t ~ t ~ a t ~ a ~ a t t t t ~ t t t t ~ ` c c ~ t ~ t t t ~ c ~ a t t ~ a t t ~ g g ~ c ~ t ~ t t g a a t ~ figure . sequence of the human canp cdna and flanking ' and ' genomic regions (a) and (c) show the polyadenylation signal and putative caat and tataa sites, which are boxed. the putative spl (position - to - ), maf -binding sites (position - to - ), and carg box (position - to - ) are in bold. the alu sequence present in the ' region is underlined. (b) shows the corresponding amino acids, indicated below the sequence. the coding sequence between the atg initiation codon and the tga stop codon is bp, encoding for an amino acid protein. the adenine in the first methionine codon has been assigned position . locations of introns within the canp gene are indicated by arrowheads. nucleotides that differ from the pre- viously published ones are indicated by asterisks. l g m d a . finally, this demonstrates a case of muscular dystrophy resulting from an enzymatic rather than a struc- tural defect. r e s u l t s l o c a l i z a t i o n of c a n p w i t h i n t h e l g m d a interval c d n a capture using y a c s from the lgmd a interval al- lowed the identification of positional candidate genes. canp was one of two transcripts that showed muscle- specific expression as evidenced by northern blot analysis (chiannilkulchai et al., ). t h e canp g e n e had pre- viously been localized to chromosome (ohno et al., ). primers (table ; figure ) designed from different parts of the published h u m a n c d n a s e q u e n c e (sorimachi et al., ) were used to position the g e n e in a region previously defined as q . -q . (richard et al., ) and, more precisely, on three y a c s ( g , g , and g ) localized in this region, between d s and d s , in a candidate region suggested by linkage dis- equilibrium studies (allamand et al., b). t h e s e primers were also used to screen a cosmid library derived from y a c g (i, r. et al., unpublished data). five cosmids were identified. experiments with different primer pairs (table ) established that these cosmids cover all canp exons except exon and that a second group of four cosmids contain this exon (figure c). a minimal set of three overlapping cosmids ( g - b - f l l ) covers the entire gene. d n a from these cosmids was used to construct an ecori restriction map of this region (figure b). t h e c a n p g e n e s e q u e n c e most of the sequences were obtained through shotgun sequencing of partial digests of cosmid f l l subcloned in m and bluescript vectors and by walking with internal primers. the s e q u e n c e assembly was in agreement with the restriction map of the cosmids. s e q u e n c e s of exon and adjacent regions were obtained by sequencing cos- mid d n a or polymerase chain reaction (pcr) products from human genomic dna. t h e first and eighth introns are still not fully sequenced, but there is evidence that they may be between and kb and about kb in length, respectively (based on hybridization of restriction fragments; data not shown). t h e entire g e n e spans more than kb. t h e determined canp s e q u e n c e completes the pub- lished human c d n a s e q u e n c e (sorimachi et al., ). it contains the missing bases corresponding to the n-terminal amino acids (figure b). it also differs from the published sequence at positions, three of which occur at third base positions of codons and preserve the encoded amino acid sequence. t h e other eight differ- ences lead to c h a n g e s in amino acid composition (figure ). a s these different exons were sequenced repeatedly on at least distinct genomes, we are confident that the sequence shown in figure represents an authentic cell o h%iman imp tvi sasvap rtaaeprspgpvp hpao skateagggnp s g i y saii srnfp i i gvkektf~q~hkkclekk~ zat ...... pt ..... g .............. g.t ...... h.g ................................... pig cow ioo ¢ o l ..................................... g .......... d ..... l .......... er ....... . . . . . t . . . . . . . . . . . . . . . d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... t ..... k .......... r .............................................. r ....... d .......... e.r.r .......... e ......... r.r , o ~ e | d s d ldprgs d e rp trt i i pvoy~ rmac~vrg ~v~gldevp f ~ e k v ~ l " ~ q ~ w k d ....... a..d..s...v ....................... e.al .... . ...................... g... • . . . .ev. .d ...... v. . .f ................... e.al .......................... s. . , • .i. . .ev. .d .... m.v. . .f ................... e.aly ......................... s. . . o | ~,isfvdkdekarlqhqvte ,lz~, j~ye~ y hf tkli~tada~qs d klqtwtvsvn~gc s~gcr~ .......................... d..v ................ e ............................ .......................... d ................... e ....................... tg... ..y ............... ~) o ............................................... n ........................... .................................... r .......... n ........................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . . . . . . . . . . . . . . n . ......... r ......... e ........................ m ........ k ............... r ..... | p i i fvs d ran snkelgvdoe see gkgkt spdkokos popop gs s doe s e eooofrni f kqiagddme~cade~kk .................... a .... d..g .... ge .... r..ht .............. r ............... n ........................ qd ....... ek..k.e. snt ....... @ o o ] v~ntvvnkhkd l kth~ tle s crsmi alm~d gs gklnle~h h~nki kawqk~fkhy~td q s~t i nsy~m~h~ .............. q ........ . ........... r .......... k ............... h ........... ~) aoo vnd~hlnnqlydi i t~adkhnnni d~d s~i c~v~g~raf haf d kd gd n iklnvle~ltmya ......... s ............................................................. figure . alignmentsofaminoacid sequences of the muscle-specific calpains the human canp protein is shown on the first line. the three muscles-specific se- quences (ns, is , and is ) are underlined, and the three active site residues are indicated by asterisks. the second line corresponds to the rat sequence (swissprot accession number p ). the third and fourth lines show the deduced amino acid sequences encoded by pig and cow expressed sequence tags (gen- bank/embl accession numbers u and u , respectively). amino acids that are conserved among all known calpains are boxed in black. a period indicates that the same amino acid is present in the homologous sequence, and letters refer to the variant amino acid. positions of missense mutations are given as encircled numbers above the corre- sponding amino acid. sequence rather than minor polymorphic variants. further- more, these modifications increase the local similarity with the rat canp amino acid sequence (sorimachi et al., ), although the overall similarity is still %. the atg numbered in figure b is presumed to be the translation initiation site based on homology with the rat canp and is within a sequence with five nucleotides out of eight in common with the kosak consensus se- quence (kosak, ). putative caat and tata boxes were observed or (caat) and or bp (tata) upstream of the initiating atg codon (bucher, ). pcr amplification on muscle cdna libraries excluded the downstream tata site since it was contained within the amplified products (data not shown). a gc-box binding the spl protein (dynan and tjian, ) was identified at position - . consensus sequences corresponding to potential muscle-specific regulatory elements were identi- fied (figure a). these include a myocyte-specific en- hancer-binding factor (mef )-binding site (gosset et al. ), a carg box (serum response elements observed in muscle-specific genes and in genes inducible by growth hormones; minty and kedes, ), and four e boxes (con- tact sites for basic-helix-loop-helix proteins found in mem- bers of the myod family; blackwell and weintraub, ). the functional significance of these putative transcription factor-binding sites in the regulation of canp gene ex- pression remains to be established. two aauaaa were identified, and bp down- stream of the tga stop codon. the sequencing of a partial canp cdna containing a poly(a) tail demonstrated that the first aauaaa is the polyadenylation signal. the latter is embedded in a region well conserved with the rat canp sequence and is followed after bp by a gt cluster, pres- ent in most genes ' of the polyadenylation site (birnstiel et al., ). the ' untranslated region of the canp mrna is bp long. considering the most likely pro- moter location, the predicted length of the cdna should therefore be approximately bp. the dna and protein sequences of the human canp gene show high degree of conservation with other calpains (figure ), in particular rat (swissprot accession numbers j and p ), bovine (genbank accession num- ber u ), and porcine (genbank accession number u ) homologous sequences. high local similarities between the human and rat dna sequences are even observed at the ' ( %) or in different parts of the ' un- translated regions (over %) (data not shown), sugges- tive of evolutionary pressures in their untranslated regions. genomic organization of the canp gene a comparison of the published canp human cdna (sod- machi et al., ) with the corresponding genomic se- quence led to the identification of exons ranging in length from bp (exon ) to bp (exon ), with a mean size of bp (see figure a). the size of introns ranges from bp to about - kb for intron . the intron-exon boundaries (table ) exhibit close adherence mutations in canp cause lgmd a table . sequences at the intron-exon junctions score score splice donor site (%) intron (%) splice acceptor site exon , c t c c g g t g a g t . , . , g c t a g g t a g g a . . , , t c c a g g t g a g g , . , , g c t a a g t a a g c . . , , t t g a t g t a a g t . . . . c c c g g g t g t g t , . . . a t g a g g t a a g c . . . . g a t a g g t a g g t . . . . t t c t g g t g a g t . . . . c c c a g g t g g g a . . . . a c g a g g t g t g t , . . . a a g a g g t a t a g . . . . t c t g a g t g a g t . . . . c a g t g g t g a g t . . . . c c a a g g t a g g t . . . . c a c a g g t g t c t . . . . g a g a t g t g a g t . . . .caaacgtgagt . . . t g g a t g t a t c c .. .ggcaggtggga . . .cgcaggtgctg . . . g t t c a g t a a g t . . .tggaggtaaag .. . ~lntron ~ . . ~lntron ~ . ~lntron -,- . *-intron ~ . ~lntron ~ . . ~lntron ~ ~lntron -* . *-intron ~ . ~lntron ~ *-intron ~ . . *-intron ~ ~lntron ~ . ~lntron ~ -,-intron ~ , ~lntron ~ ~lntron ~ ~lntron -* , *-intron ~ *-intron ~ ,--intron -* ~lntron ~ "~-intron ~ . ~lntron -* ., t t t t t g t t t c a c a g g a a a t . . . g t g t c t g c c t g c a g g g g a c . . . a c g c t t c t g t g c a g t t c t g . . . a t c c t c t c t c t a a g g c t c c . .. c c a t c g g g c c t c a g g a t g g . . . t t a c t g c t c t a c a g a c a a t . . . . t c t g t g t g c t t a a g g t c c c . . . . c a t t t t c c c a c c a g a t g g a . . t t c c a a c c t c t c a g g a t g t . . , t t c t g g g g g t g c a g a t a c t . . . t g t t t c t t c t c a a g g t t c c . . . t c c c c a t c t c t c a g a t g c a . . . t g t a t t c c t c a c a g g g a a g . . . c t t t t c t t a t g c a g a a a a a . . . c c t c c t c t c t c c a g c c c a t . . . t t g t g c c t c c a c a g c c a c a . . . c c c t t c c t c c t c a g g a c a t . . . c t c c a t c c c c c c a g a c a a g . . . c c t c c c t c c t c c a g a c a g a . . . t t t t c t a t t g c c a g a a a t a . . . g g t c c c c t c c a c a g g a t t c . . . g c a t t c t t t c a c a g g a g c t . . , g g g a c t t c t t t c a g t g g c t . exon ( bp) ~ exon ( bp) ~ exon ( bp)-* exon ( bp) ~ exon ( bp) ~ exon ( bp) ~ exon ( bp) -~ exon ( bp) ~ exon ( bp) ~ exon ( bp) ~ exon ( bp) ~ exon ( bp) ~ exon ( bp)-* exon ( bp)--* exon ( bp)--* exon ( bp)-* exon ( bp)-* exon ( bp)~ exon ( bp) ~ exon ( bp)~ exon ( bp) ~ exon ( bp)~ exon ( bp)~ exon ( bp)~ a score expressing adherence to the consensus was calculated for each size according to shapiro and senapathy ( ). sequences of exons and introns are in uppercase and lowercase, respectively. sizes of exons are given in parenthesis. to ' a n d ' s p l i c e site c o n s e n s u s s e q u e n c e s (shapiro a n d s e n a p a t h y , ). w h e n t h e g e n o m i c s e q u e n c e w a s sub- mitted to g r a i l analysis ( u b e r b a c h e r a n d mural, ), e x o n s w e r e c o r r e c t l y r e c o g n i z e d , four were not identi- fied, six were i n a d e q u a t e l y defined, and t w o w e r e too small to be r e c o g n i z e d (data not shown). as a l r e a d y noted, the canp g e n e has three u n i q u e s e q u e n c e blocks, ns (amino acid residues - ), is (res- idues - ) , a n d is (residues - ) . it is interest- ing to note that each is s e q u e n c e , as well as the n u c l e a r translocation signal (residues - ) inside is , is es- sentially f l a n k e d b y introns (figure ). t h e e x o n - i n t r o n o r g a n i z a t i o n of the h u m a n canp is similar to that re- p o r t e d for the c h i c k e n canp (the only o t h e r large subunit calpain g e n e w h o s e g e n o m i c structure is k n o w n ; emori et al., ). four microsatellite s e q u e n c e s w e r e identified (see fig- ure a). t w o of t h e m are in the distat part of t h e first intron: an (at)~ a n d a p r e v i o u s l y identified n o n p o l y m o r p h i c m i x e d - p a t t e r n microsatellite, d s ( f o u g e r o u s s e et al., ). a (ta) (ca) (ga)~ w a s identified in the s e c o n d intron, and g e n o t y p i n g of unrelated c e n t r e d'etudes du p o l y m o r p h i s m e h u m a i n (ceph) individuals revealed t w o alleles (with f r e q u e n c i e s of . and . ). t h e fourth microsatellite is a m i x e d (ca),(ta)m r e p e a t present in the ninth intron. t h e latter and t h e (at), r e p e a t h a v e not been i n v e s t i g a t e d for p o l y m o r p h i s m s . o n e mer r e p e a t a n d m e m b e r s of the alu f a m i l y were identified in the canp g e n e (see f i g u r e a), which has, thus, on a v e r a g e o n e alu e l e m e n t p e r . kb. expression of the canp gene t h e pattern of tissue specificity was i n v e s t i g a t e d b y north- ern blot hybridization, t h e r e is no e v i d e n c e for the exis- t e n c e of an a l t e r n a t i v e l y s p l i c e d form of canp , although a e x o n s met inn'on p o s i t i o n s b canp protein ~ protein domaln_s i nonsense [b mutations m [m m p. frameshlft b mutations b r m _ _ splice site ir , ,~ . t i mutauon [ " " ' ' j ~ i j ' ] j i l t ~ t~ ~ f f i [ r t o n ii ]ii i v cysteine pro~ase domain ca + binding dommn ( ef-hands) r l l x tt x ddg delca delagac ....... dela • , - ag-->aa ag-->tcatct figure . distribution of the mutations along canp protein (a) positions of the introns are indicated by vertical bars. numbers written below refer to the corresponding amino acids. ( ) schematic representation of the canp protein, its four domains (i, ii, ill, and iv), and muscle-specific sequences (ns, is , and is ). the three active site residues are underlined. the positions of missense mutations are indicated by dots. the effects of nonsense and frameshift mutations are illustrated as truncated lines, representing the extent of protein synthesized. the out-of-frame sequence is shown by hatched lines. names of the families carrying these mutations are indicated to the left of the lines. cell table . pcr primers for the analysis of the canp gene pcr product size annealing (in base p a i r s ) temperature primer name primer sequences ( '- ') amplified region (genomic dna) (in celsius) canp -pro.a ttcagtacctcccgttcacc promoter canp -pro.m gatgcttgagccaggaaaac canp -exl.a ctttccttgaaggtagctgtat exon canp -exl .m gaggtgctgagtgagaggac canp -ex .a actccgtctcaaaaaaatacct exon canp -ex .m attgtccctttacctcctgg canp -ex .a tggaagtaggagagtgggca exon canp -ex .m gggtagatgggtgggaagtt canp .ex .a gaggaatgtggaggaaggac exon canp -ex .m ttcctgtgagtgaggtctcg canp -ex .a ggaactctgtgaccccaaat exon canp -ex .m tcctcaaacaaaacattcgc canp -ex .a gttccctacattctccatcg exon canp -ex .m gttatttcaacccagaccctt canp -ex .a aatgggttctctggttactgc exon canp -ex .m agcacgaaaagcaaagataaa canp -ex .a gtaagagatttgccccccag exon canp -ex .m tctgcggatcattggttttg canp -exg.a ccttcccttcttcctgcttc exon canp -ex .m ctctcttccccacccttacc canp -exl .a cctcctcacctgctcccata exon canp -exl .m tttttcggcttagaccctcc canp -exl .a tgtggggaatagaaataaatgg exon canp -exl .m ccaggagctctgtgggtca canp -ex .a ggctcctcatcctcattcaca exon canp -exl .m gtggaggagggtgagtgtgc canp -ex .a tgtggcaggacaggacgttc exon canp -ex .m ttcaacctctggagtgggcc canp -ex .a caccagagcaaaccgtccac exon canp -ex .m acagcccagactcccattcc canp -ex .a ttctcttctcccttcaccct exon canp -exl .m acacacttcatgctctctaccc canp -ex .a ccgcctattcctttcctctt exon canp -exl .m gacaaactcctgggaagcct canp -ex .a acctctgacccctgtgaacc exon canp -exl .m tgtggatttgtgtgctacgc canp -ex .a cataaatagcaccgacaggga exon canp -exl .m gggatggagaagagtgagga canp -ex .a tcctcactcttctccatccc exon canp -ex .m accctgtatgttgccttgg canp -ex .a ggggattttgctgtgtgctg exon - canp -ex .m attcctgctcccaccgtctc canp -ex .a cacagagtgtccgagaggca exon canp -ex .m ggagattatcaggtgagatgcc canp -ex .a cagagtgtccgagaggcaggg exon - canp -ex .m cgttgacccctccaccttga canp -ex .a gggaaaacatgcaccttctt exon canp -ex .m tagggggtaaaatggaggag c a n p - p a . a actaactcagtggaataggg polyadenylation signal c a n p - p a . m ggagctaggatagctcaat this c a n n o t be e x c l u d e d . a transcript of a b o u t . - . kb w a s d e t e c t e d in skeletal m u s c l e m r n a (chiannilkulchai et al., ). this size therefore further s u p p o r t s the position - as t h e functional t a t a box. mutation screening canp fulfils both positional a n d functional criteria to be a c a n d i d a t e g e n e for lgmd . we therefore s y s t e m a t i c a l l y s c r e e n e d l g m d families for the p r e s e n c e of n u c l e o t i d e c h a n g e s in canp using a c o m b i n a t i o n of h e t e r o d u p l e x (keen et al., ) and direct s e q u e n c e analyses. pcr p r i m e r s were d e s i g n e d to specifically amplify t h e e x o n s and splice junctions as well as t h e regions con- taining putative c a a t and t a t a b o x e s a n d t h e p o l y a d e - nylation signal (table ). p c r p r o d u c t s m a d e on d n a of lg md patients w e r e then s u b j e c t e d either to h e t e r o d u p l e x analysis or direct s e q u e n c i n g , d e p e n d i n g on w h e t h e r t h e mutation, b a s e d on h a p l o t y p e analysis, w a s e x p e c t e d to be h e t e r o z y g o u s or h o m o z y g o u s , respectively. it w a s oc- c a s i o n a l l y n e c e s s a r y to clone the pcr p r o d u c t s to identify the mutations precisely (i.e., for m i c r o d e l e t i o n s or inser- tions and for s o m e heterozygotes). d i s e a s e - a s s o c i a t e d m u t a t i o n s are s u m m a r i z e d in t a b l e , a n d their position mutations in canp cause lgmd a table . canp mutations in lgmd a families families amino (r(~union nucleotide nucleotide acid effect of restriction site protein exon haplotype) position change position mutation change domain mutation b " cga~tga m ctg~cag m , m c~tu~-*ca m , m ggg~gag m cgg--*cg - rl ", r , r ", r , r ", - g~a r ", r ", r (i, ii) m " gtg-*ggg m tgg-*tag m cgg--*tgg r " (vii) cgg~ccag r (viii) - deletion ac r , r ( ) agc--*ggc a*, b ", m cgg--*cag b - deletion agac - b , r (iv) - ag~tcatct arg~stop -- ii r x leu~gin - ii l q frameshift -- ii aa gly-*glu - ii g e frameshift without smal ii ag abberant -- ii - g--*a splicing val~gly - ii v g trp-*stop without bstnl, ii t x without ecori, without scrfl arg--*trp iii r t arg-*gin without mspl ill r q frameshift iv aca ser-*gly without alul iv $ g arg-*gin -- iv r q frameshift -- iv aagac frameshift -- iv ag~tcatct the first letter of the family code refers to the origin of the population (b, brazil; m, metropolitan france; r, isle of la r union; a, amish; the corresponding haplotypes for la r union island families are numbered in parentheses). families that are homozygous for mutation are indicated by asterisks. positions are numbered on the basis of the cdna and protein sequences starting from atg and the first methionine residue, respectively. the mutated nucleotides are underlined. a l o n g t h e p r o t e i n is s h o w n in f i g u r e . e a c h m u t a t i o n w a s c o n f i r m e d b y h e t e r o d u p l e x a n a l y s i s , s e q u e n c i n g of b o t h s t r a n d s in s e v e r a l m e m b e r s of t h e f a m i l y o r e n z y m a t i c d i g e s t i o n w h e n t h e m u t a t i o n m o d i f i e d a restriction site. s e g r e g a t i o n a n a l y s e s of t h e m u t a t i o n s , p e r f o r m e d o n d n a s f r o m all a v a i l a b l e m e m b e r s of t h e f a m i l i e s , c o n - f i r m e d t h a t t h e s e s e q u e n c e v a r i a t i o n s a r e on p a r e n t a l c h r o m o s o m e s c a r r y i n g a lgmd a m u t a t i o n . t o a s s e s s t h e p o s s i b i l i t y t h a t m i s s e n s e s u b s t i t u t i o n s m i g h t b e poly- m o r p h i s m s , t h e i r p r e s e n c e w a s s y s t e m a t i c a l l y t e s t e d in a c o n t r o l p o p u l a t i o n : n o n e of t h e m u t a t i o n s w a s s e e n a m o n g c o n t r o l c h r o m o s o m e s f r o m t h e c e p h r e f e r e n c e f a m - ilies. chromosome ascertained families t h e initial s c r e e n i n g for c a u s a t i v e m u t a t i o n s w a s per- f o r m e d on f a m i l i e s f r o m t h e l a r u n i o n i s l a n d ( b e c k m a n n et al., ), f r o m t h e old o r d e r a m i s h of n o r t h e r n i n d i a n a ( y o u n g et al., ), a n d f r o m brazil ( p a s s o s - b u e n o et al., ). la r~union island families g e n e a l o g i c a l s t u d i e s a n d t h e g e o g r a p h i c o r i g i n of t h e f a m - ilies f r o m la r u n i o n w e r e s u g g e s t i v e of a s i n g l e f o u n d e r effect. g e n e t i c a n a l y s e s a r e , h o w e v e r , i n c o n s i s t e n t w i t h this h y p o t h e s i s a s t h e f a m i l i e s p r e s e n t h a p l o t y p e h e t e r o - g e n e i t y , w i t h at l e a s t six d i f f e r e n t c a r r i e r c h r o m o s o m e s ( a l l a m a n d e t al., b). in t h e c o u r s e o f this w o r k , d i s t i n c t m u t a t i o n s c o r r e s p o n d i n g to s i x h a p l o t y p e s h a v e b e e n i d e n t i f i e d ( t a b l e ). y e t t h e s e m u t a t i o n s a r e a b s e n t in s o m e of t h e m i n o r h a p l o t y p e s . thus, s o m e of t h e m m u s t still c o n t a i n at l e a s t o n e o t h e r u n i d e n t i f i e d m u t a t i o n . in f a m i l y r , e x o n s , , a n d s h o w e d e v i d e n c e for s e q u e n c e v a r i a t i o n u p o n h e t e r o d u p l e x a n a l y s i s (figu re ). s e q u e n c i n g of t h e a s s o c i a t e d p c r p r o d u c t s r e v e a l e d a p o l y m o r p h i s m in e x o n , a a--*g m u t a t i o n in e x o n t r a n s f o r m i n g s e r - to g l y c i n e in t h e s e c o n d ef h a n d ( s e e f i g u r e ), a n d a a g - * t c a t c t f r a m e s h i f t m u t a t i o n in e x o n c a u s i n g p r e m a t u r e t e r m i n a t i o n a t n u c l e o t i d e w h e r e an i n - f r a m e s t o p c o d o n o c c u r s (see f i g u r e ). t h e e x o n m u t a t i o n a n d t h e p o l y m o r p h i s m in e x o n f o r m an h a p l o t y p e t h a t is a l s o e n c o u n t e r e d in f a m i l y r . a f f e c t e d i n d i v i d u a l s in f a m i l y r a r e h o m o z y g o u s o v e r t h e e n t i r e lgmd a i n t e r v a l ( a l l a m a n d et al., b). se- q u e n c i n g of t h e p c r p r o d u c t s of e x o n r e v e a l e d a g---a t r a n s i t i o n at b a s e of t h e c d n a r e s u l t i n g in a s u b s t i t u - t i o n o f g l u t a m i n e for a r g - ( f i g u r e ) i n s i d e d o m a i n ill, a r e s i d u e t h a t is h i g h l y c o n s e r v e d t h r o u g h o u t all k n o w n c a l p a i n s . this m u t a t i o n , d e t e c t a b l e b y loss of an m s p l r e s t r i c t i o n site, is p r e s e n t o n l y in this f a m i l y a n d in no o t h e r e x a m i n e d l g m d a f a m i l i e s o r u n r e l a t e d c o n t r o l s ( d a t a not s h o w n ) . in f a m i l y r , h e t e r o d u p l e x a n a l y s i s f o l l o w e d b y se- i ~ t i l l e e e e e l i e l e i e figure . representative mutations identified by heteroduplex analysis examples of mutations detected by heteroduplex analysis. lanes c represent control samples. exon numbers (e) are indicated below the gels. pedigree b displays the segregation of two different muta- tions in exon . cell a e x o n a a t c c c c g a t t t a a iilvvvl ,,', n o r m a l [,i .î l li~ lli~,"~ m sequence/~y,.,i~_ .."~ ~.~"i~"'_ i~ a a t c c c t g a t t t a c g a - > t g a a r g s t o p b e x o n s a g c t g g t g c g g c t i i ~ j, , i i i i normal a g c t g g g g c g g c t g t g - > g g g va g l y c e x o n d e x o n c c a t g c g g t a c g c s e q u e n c e :'~ ,. r. / i t:~av i; . . . . ~,.., c c a t g c a g t a c g c ,jtv]',:i c g g - > c a g a r g g i n c c a t g c a g t a c g c c g g -> c a g a r g g i n figure . sequence of homozygous mutations sequences from a healthy control are shown above the mutant se- quences present in axons (a), (b), (c), and (d). asterisks indicate the position of mutated nucleotides. the consequences on codon and amino acid residues are indicated at the left of each panel together with the name of the family. t c c t c c g g g t c t t ~ i i , ?. . n o r m a l ,.,, a i! :: ~: ! r s e q u e n c e i ~ i~ ~ i~-'~ i~,'~ t c c t c c a g g t c t t c g g - > c a g a r g g l n quencing of the pcr products of an affected child revealed a bp deletion in exon (see figure ; table ). one ac out of three is missing at this position of the sequence, producing a stop codon at position of the cdna se- quence (see figure ). since both mutation detection enhancer heteroduplex analysis and direct sequencing failed to reveal the muta- tions in the two major haplotypes (i and ii), an attempt was made to uncover aberrant splicing of the muscle-specifi c calpain mrna from these patients by taking advantage of the illegitimate transcription in lymphocytes (chelly et al., ). the pcr products corresponding to exons - showed the presence of a band about bases longer than expected. fine pcr mapping localized the mutation at the junction of exons and , while sequencing of the corresponding products (figure ) revealed an acceptor splice site mutation (ag~aa), resulting in the utilization of an alternative site within intron , bp upstream of exon . the same mutation was seen in haplotypes i and ii (table ), even though they differ in out of markers. it is still unclear whether these are related to one another or a coincidence of two independent recurrent mutational n o r m a l c d n a s e q u e n c e exon / exon / p r o t h r a r g [fhr ile lie c o / ~ c c g o a c a a t c a t t s p l i c e site m u t a t i o n exon < bo -~ exon p r o t h r a r g iglu stop *l c c g a c c c g o g a a t a g c t a c a a t a c a a t c a t t . :~: i / figure . sequence analysis of the junction between exons and of canp of illegitimately transcribed mrnas the corresponding sequence from a normal muscular canp cdna (above) or from a lg md a patient (below) are shown, together with the corresponding amino acids. the asterisk indicates the g ~ a transition responsible for the loss of the normal acceptor site leading to the use of a cryptic splice site within intron . this aberrant splicing results in the presence in the mrna of an additional bp intronic sequence, represented by an arrow, and of a stop codon as indicated above the sequence. events. the presence of these mutations was confirmed on genomic dna. the net result is presumably the loss of canp activity since they lead to premature termination by a stop codon that is adjacent to the aberrant splice site (figure ). amish families as expected, owing to multiple consanguineous links, the examined northern indiana amish lgmd a patients were homozygous for the carrier hapiotype (allamand et al., b). a g ~ a missense mutation was identified at nucle- otide within exon (see figure ), transforming arg- to glutamine. this residue, which is conserved throughout all members of the calpain family in all species, is located in domain iv of the protein within the third ef hand at the helix-loop junction (see figure ). this muta- tion was encountered in a homozygous state in all patients from chromosome - inked amish families. we also verified that this nucleotide change was not present in patients from the six southern indiana amish lgmd fami- lies for which the chromosome locus was excluded by linkage analyses (allamand et ai., a), thus confirming the genetic heterogeneity of this disease in this genetically related isolate. brazilian families as a result of consanguineous marriages, two brazilian families (b and b ) are homozygous for extended lgmd a carrier haplotypes (allamand et al., b). af- fected individuals from family b were shown to have the same exon mutation as the amish lgmd a pa- tients (see figure ), but embedded in a completely differ- ent haplotype (allamand et al., b). in family b , the patients carry a c ~ t transition in exon in a homozygous mutations in canp cause lgmd a table . canp polymorphisms amino acid restriction site location families nucleotide position nucleotide change position change promoter m - t~c -- -- m act~acc ddel~secl m , m o ttc~ttt without mboll r , r atc~att -- intron rll, r , r , m , m + deletion a -- -- see table legend for details. state, replacing arg- with a tga stop codon (see fig- ure ), thus presumably leading to a very truncated and inactive protein (see figure ). analysis of other lgmd families having validated the role of canp in the chromosome ascertained families, we next examined by heteroduplex analysis lgmd families for which linkage data were not informative. these included one brazilian (b ) and metropolitan french pedigrees. additional mutations were uncovered enabling the ascertainment of eight more lgmd a families. heteroduplex bands were revealed for exons , , , , , , , , and the promoter region of one or more pa- tients (see figure ). of all sequence variants, were identified as pathogenic mutations (five missense, one nonsense, and four frameshift mutations) and four as poly- morphisms. altogether, two morbid alleles were identified in five families, one in three, and none in three others (table ). identical mutations were uncovered in appar- ently unrelated families. the mutations shared by families m and m or by m and m , respectively, are likely to be the consequence of independent events since they are embedded in different marker haplotypes. in con- trast, it is likely that the point mutation present in exon of the amish and in the m kindreds corresponds to the same mutational event, as both chromosomes share a common four-marker haplotype (d s -d s - d s -d s ) around canp (unpublished data), possibly reflecting a common ancestor. the same holds true for the exon substitution shared by families b and r . in addition to the polymorphisms present in exon in families r and r (position ) and in the intragenic microsatellites, four additional neutral variations were de- tected (table ). discussion several lines of evidence implicate the canp gene in the etiology of lgmd a. this gene is localized inside the mb lgmd a interval, in the region suggested by linkage disequilibrium studies (allamand et al., b). southern blot experiments (ohno et al., ) and sequence-tagged site screening (data not shown) suggest that there is but one copy per genome of this member of the calpain family. transcription studies suggested that it is an active gene rather than a pseudogene, and its muscle-specific pattern of expression is consistent with the phenotype of this disor- der (sorimachi et al., ; chiannilkulchai et al., ). a minimum of independent mutational events were identified in families from different ethnic and geographic groups. these represent different mutations, distrib- uted throughout the gene (figure ), that cosegregate with the disease in lgmd a families. the discovery of two nonsense, one splice site, and five frameshift mutations in canp supports the hypothesis that a deficiency of this gene product causes lgmd a. all eight mutations result in a premature in-frame stop codon, leading to the produc- tion of truncated and presumably inactive proteins (figure ). evidence for the morbidity of the missense mutations come from several sources: their relative high incidence among lgmd a patients, the failure to observe these mu- tations in control chromosomes, and the occurrence of mutations at evolutionarily conserved residues, in regions of documented functional importance, or both (sorimachi et al., ). of seven mutations, four change an amino acid that is conserved in all known members of the calpain family in all species (figure ). two of the remaining muta- tions affect less conserved amino acid residues, but are located in important functional domains: v g is four residues before the asparagine at the active site; $ g within the second ef hand may impair the calcium- dependent regulation of calpain activity or the interaction with a small subunit (figure ). several missense muta- tions change a hydrophobic residue to a polar one or vice versa (table ), possibly disrupting higher order struc- tures. the r union paradox only four different mutations were identified by hetero- duplex and sequence analyses in the families of la r~- union, despite the fact that all exons were scanned. even- tually, the use of illegitimate transcription allowed us to uncover aberrant splicing, leading to the recognition of the same splice site mutation in haplotypes i and ii (table ). thus, five different mutations have been identified so far in this population. but given that none of them was found on the remaining carrier haplotypes, there should be at least one other (as yet unidentified) mutation. the presence of at least six different mutations among patients from the la r~union island demonstrates the va- lidity of the suggestion based on haplotype analysis of a multifounder effect (ailamand et al., b). this is, how- cell ever, an unexpected result given the multiple consanguin- eous links in these families. indeed, the affected patients of la r~union all belong to a small genetic isolate, pre- sumed to derive from a single ancestor who immigrated to this island in the s. these patients were all thus expected to carry the same lgmd a mutation. the occur- rence of multiple independent events in other small popu- lations is not unprecedented (e.g., bach et al., ; rod- ius et al., ; heinisch et al., ), although no satisfactory explanation has been forwarded so far. thus, the presence of multiple mutations in the la r~- union population needs to be reconciled with the reported low prevalence of this disease, a problem we refer to as the r~union paradox. to begin, the global prevalence of lgmd a could just be much more common than initially presumed, but this seems highly unlikely. another hypothesis assumes that heterozygous healthy individuals could benefit from a se- lective advantage (as in -thalassemia), resulting in a high local prevalence. this hypothesis, however, seems also unlikely since one would not expect such selective pres- sures to lead to these results in such a limited timespan (i.e., years at most). we therefore speculate that this condition, which has this far been considered as a monogenic disorder, may reflect a more complex inheritance pattern, in which ex- pression of the calpain mutations would be dependent on genetic background (nuclear or mitochondrial). consider, for instance, a digenic model: only in the presence of spe- cific alleles at a permissive second unlinked locus (e.g., a compensatory, partially redundant, regulatory, or modifier gene) will there be expression of calpain mutations. since one would need mutations at both loci to be affected, the disease prevalence would remain low. under this model, members of the la r~union island community would, as a result of genetic drift, have a disease-associated allele at the hypothesized second locus at high frequency (or even fixed in this small population), conditions that would explain the apparent complete penetrance of the calpain mutations. the complete penetrance of this disease in the amish and in the other described lgmd a pedigrees would also be under control of the second locus. if this model is true, there may be fewer selective pres- sures against the appearance of canp mutations, as a result of the conditional penetrance. in other words, the frequency of the calpain variants in the overall population can be much higher than initially deduced, based on the estimates of the prevalence of the disease under a simple monogenic model. assume, for instance, recessive, inde- pendent, and fully penetrant expression at two autosomal loci, with similar frequencies for both deleterious alleles. considering the reported prevalence ( - ; emery, ) and the estimated genetic heterogeneity, in persons would be a carrier for a deleterious calpain allele (and in would carry deleterious alleles at both loci). the frequency of calpain variants could thus approach that of the cystic fibrosis gene. if we assume the allele frequency at the second locus to be, respectively, . or . , this would give us one lgmd a carrier per or individu- als. but, since expression of the trait would require the simultaneous presence of both sets of deleterious alleles, the overall prevalence remains, as expected, on the order of -~. under this model, some of the families for which the role of the lgmd a locus was previously excluded, based on linkage analyses assuming simple monogenic inheri- tance, might be authentic lgmd a families, reflecting dif- ferential segregation of these two unlinked genes. the digenic inheritance model th us predicts that in a number of kindreds, there will be healthy individuals with two mutant calpain genes. to test the validity of this model, we are currently performing a systematic screening for the pres- ence of calpain mutations in dna from affected probands from all small nuclear families that cannot be identified as lgmd a on the basis of linkage analyses. we will then assess the cosegregation of the mutations in these pedi- grees, searching for asymptomatic carriers of two mutant calpain genes. an alternative possibility assumes that the calpain muta- tions are pathogenic only in a specific mitochondrial con- text. it is worthwhile remembering that the mitochondria are central suppliers of energy and could therefore influ- ence the fate of muscle cells. the mitochondrial model predicts a monogenic inheritance pattern within nuclear families (all sibs share the same mitochondrial genome). in other pedigrees, however, we should fail to get expres- sion of the disease in individuals carrying calpain muta- tions in a nonpermissive mitochondrial genetic back- ground. it is important to stress that the observations in favor of the proposed departure from simple monogenic inheri- tance are not unprecedented. there has been the report of digenic inheritance of retinitis pigmentosa (kajiwara et al., ). and there are a number of additional descrip- tions of traits for which expression is clearly influenced by the presence of unlinked mutations (e.g., oppenheim et al., ). the concomitant involvement of two or more loci in the lgmd a phenotype may have some immediate practical consequences. since the current estimates for genetic heterogeneity are based on a simple monogenic model, these would be truly inadequate under digenic or more complex inheritance models. in addition, if one of the latter turns out to be true, its impact on genetic counseling will also need to be carefully evaluated. clearly, the mechanism underlying the results reported here may have major bearing on our comprehension of simple genetic traits. they may imply that one may occa- sionally face similar situations in other inbred human popu- lations, such as finland. the proposed di- or oligogenic model could also explain some of the failures to reproduce known or expected human phenotypes in mouse gene knockout experiments, since the genetic backgrounds allowing for expression of the corresponding traits may not have been present. one particularly convincing example is the work of rudnicki et al. ( ) on myod and myf- transgenic mice, where only double knockout animals ex- press a pathological phenotype. another example are the i g f l knockouts, which show variable survival rates de- pending on the genetic background (liu et al., ). one mutations in canp cause lgmd a prediction of this model is that the mouse canp knock- outs may also fail to have a pathological phenotype. c a n p a n d l g m d a identification of canp as a defective gene in lgmd a suggests a novel pathological mechanism leading to a muscular dystrophy in which this condition is caused by mutations affecting an enzyme and not a structural com- ponent of muscle tissue. this result is to be contrasted with all known other muscular dystrophies, such as duchenne and becker (bonilla et al., ), severe childhood autoso- real recessive (m atsumara et al., ), fu kuyama (matsu- mara et al., ) , merosin-deficient congenital muscular dystrophies (tom~ et al., ), and primary adhalin defi- ciencies (roberds et al., ). the understanding of the lgmd a phenotype needs to take into account the fact that there is likely to be no active canp protein in several patients, a loss compatible with the recessive manifestation of this disease. simple models in which this protease would be involved in the degradation or destabilization of structural components of the cytoskel- eton, extracellular matrix, or dystrophin complex must therefore be ruled out. furthermore, there are no signs of such alterations by immunohistochemical studies on lgmd muscle biopsies (matsumara et al., ; tome et al., ). likewise, since lgmd a myofibers are ap- parently not different from others that are dystrophic, it seems unlikely that this calpain plays a role in myoblast fusion, as proposed for ubiquitous calpains (wang et al., ). alternative hypotheses must therefore be forwarded. one could imagine that the canp participates in the acti- vation of an enzyme or other protein involved in muscle metabolism, or it could be responsible for the catabolism of protein compounds in the muscle; the absence of such activity would result in a toxic accumulation of these com- pounds and eventually in the degeneration of muscle fi- bers. we favor another hypothesis wherein the canp protein plays an active role in transduction of a signal, a hypothesis that takes the previously described properties of the ubiquitous calpain and of canp into account. indeed, studies of canp expression (sorimachi et al., b) reported that its mrna is abundant (its expression being -fold higher than that of the ubiquitous calpains) and specific to fully differentiated myotubes and myofi- bers. yet attempts to detect this protein failed (sorimachi et al., b). this is interpreted as a consequence of its extremely rapid turnover mediated by autocatalysis, possibly reflecting the need for precise regulation of its activity. when the canp protein was expressed and measured in transfected cos and l myoblast cells, it was shown to have a nuclear localization (sorimachi et al., b), possibly mediated by the nuclear translocation signal in the is region. this result suggests that this cellu- lar compartment could be its natural site of action. the calpains have been proposed as having regulatory rather than degradative role, mediated by restricted proteolysis of specific proteins (wang et al., ; suzuki and ohno, ; croall and demartino, ). in fact, ubiquitous cal- pains have been reported to regulate transcription by spe- cific cleavage of c-jun and c-fos (hirai et al., ) and by controlling nf-~<b activities through proteolysis of its ikb-~ inhibitor (miyamoto et al., ). these reports sug- gest that canp protein could be also involved in the control of gene expression by regulating the turnover or activity of transcription factors or of their inhibitors (e.g., members of the id family; benezra et al., ). the finding that a defective calpain underlies the patho- genesis of lgmd a may prove useful for the identification of other loci involved in neuromuscular diseases. other forms of lgmd may indeed be caused by mutations in genes whose products are calpain substrates or in genes involved in the regulation of canp expression. tech- niques such as the two-hybrid selection system (fields and song, ) could lend themselves to the isolation of the natural protein substrate(s) of this calpain and thus p o t e n t i a l l y help to identify candidate loci. these ap- proaches, inasmuch as they will lead to the identification of the other partners responsible for the lg md phenotype, could eventually also clarify the molecular genetic basis of the inheritance pattern of lgmd a. with the caveat of the as-yet-unclarified inheritance mode, identification of mutations in the canp gene could provide means for direct prenatal or presymptomatic diag- nosis and carrier detection in families in which both muta- tions have been identified. gene-based classification of lgmd a families should prove useful for the differential diagnosis of this disorder and for the establishment of phe- notype-genotype relationships. the fact that morbidity re- sults from the loss of an enzymatic activity raises hopes for novel pharmacotherapeutic prospects for this neuro- muscular disorder or other myopathies. finally, the evi- dence of pathological consequences of loss of a tissue- specific proteolytic activity, having a possible regulatory role, suggests that this may be a process of general signifi- cance. experimental procedures description of the patients the lgmd a families analyzed included three brazilian families (passos-bueno et al., ), interrelated nuclear families from la reunion (beckmann et al., ), french metropolitan families, and amish families (jackson and carey, ). the majority of these families were previously ascertained by linkage analysis to be- long to the chromosome group (beckmann et al., ; young et al., ; passos-bueno et al., ). however, some families from metropolitan france, as well as one brazilian family (b ), had insig- nificant iod scores (less than . ) for chromosome . sequencing of cosmid c g - f and ecori restriction map of cosmids cosmid fll (figure c) was subcloned and sequenced following conventional procedures. the sequences were analyzed and align- ments performed using the xbap software of the staden package, version . (staden, ). gaps between sequence contigs were filled by walking with internal primers. ecori restriction map of cosm ids was performed essentially as described in sambrook et al. ( ). analysis of canp in lgmd a patients human dna ( ng), obtained from peripheral blood lymphocytes, was used per pcr (fougerousse et al., ; table ). heteroduplex analyses were performed on p.i of pcr products essentially as in keen et al. ( ). for sequence analysis, the pcr products were subjected to dye-dideoxy sequencing, after purification through micro- cell table . pcr primers for the illegitimate transcription of the canp gene pcr product size annealing position within on cdna temperature primer name primer sequence ( '-- ') the cdna (in base pairs) (in celsius) canp -ala cagctcggtttttaagatgg - to - canp -a m cgtccacccactctccatag - canp -bla aagttccccatccagttcgt - canp -b m gaagcttgtcagactgcaga - ,, canp -c a gtcacgcctactctgtcacg - canp -c m gcttttgcttatcagggctt - can p -d a ttgaaaataccatctccgtg - canp -d m ggggtaaaatggaggaggaa - canp -a a ctttccttgaaggtagctgtat - to - canp -a m aagatccctgcgtagttttcg - canp -b a atggagccaacagaactgac - canp -b m ggccgtgaggttgcagatct - canp -c a aggtggagtggaacggttct - canp -c m gctccttgttgctgtttgct - canp -d a ccatcatcttcgtttcggac - can p -d m gggtgaaactgaaatcctga - con devices (amicon). when necessary, depending on the nature of the mutations (e.g., frameshift mutation or for some heterozygotes), the pcr products were cloned using the ta cloning kit from invitrogen. total cellular rna was extracted from lymphoblastoid cell lines by the rnazol method (bioprobe systems). reverse transcription was performed under standard conditions using p_g of rna in a total volume of p~l containing u of rnasin (promega) and u of superscript reverse transcriptase (bethesda research laboratories), we performed cycles of pcr ( s at °c, s at °c, and min at °c) on p.i of the cdna sample using one of four sets of primers (a , b , c , and d ; table ) to obtain four overlapping frag- ments spanning the whole canp coding sequence. the pcr product ( pl) was then subjected to a second round of pcr amplification using nested primers (a , b , c , and d ; table ). the pcr amplification products were analyzed by agarose gel electrophoresis and by direct double-stranded dna sequencing. acknowledgments correspondence should be addressed to j. s. b. we gratefully ac- knowledge the help of drs c. auffray, p. m. conneally, d. fugman, d. caterina, i. cadjee, n. fonknechten, j. weissenbach, and p. bucher. we thank dr. h. cann for critical reading of the manuscript and dr. e. bakker for suggesting the involvement of the mitochondrial genome. above all, we are particularly indebted to all patients, to their families, to their clinicians, and to b. barataud without whose participa- tion and encouragement this work would never have seen the light. this research was supported by grants from the association fran~:aise contre les myopathies. we also acknowledge the muscular dystrophy association, fundacao de amparo a pesquisa do estado de s~o paulo (fapesp), and programs de apoio de desenvolvimento cientifico e tecnologico (padct) for their support for the collection of the ameri- can and brazilian families. received november , ; revised february , . references allamand, v., broux, o., bourg, n., richard, i., tischfield, j., hodes, m. e., conneally, p. m., fardeau, m., jackson, c. e., and beckmann, j. s. 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( ). locating protein-coding regions in human dna sequences by a multiple sensor-neural network approach. proc. natl. acad. sci. usa , - . cell walton, j. n., and nattrass, f. j. ( ). on the classification, natural history and treatment of the myopathies. brain , - . wang, k. w., villalobo, a., and roufogalis, b. d. ( ). calmodulin- binding proteins as calpain substrates. biochem. j. , - . young, k., foroud, t., williams, p., jackson, c. e., beckmann, j. s., cohen, d., conneally, p. m., tischfield, j., and hodes, m. e. ( ). confirmation of linkage of limb-girdle muscular dystrophy, type- , to chromosome . genomics , - . genbank accession numbers the accession numbers for the sequences reported in this paper are x ( ' sequence), x ( ' sequence), and x (cdna se- quence). j contemp med sci | vol. , no. , autumn : – research aim this study aimed to evaluate the association between stk snp rs and some cardiovascular risk factors in hypertension patients in holy kerbala city, iraq. materials and methods we included hypertensive patients with no signs and symptoms of renal impairment and another control subjects. the links between genotype and hypertension were examined. then, the snp related variances in the blood pressure and remaining cardiovascular risk factors were studied. results there is no significant association between stk rs and hypertension in current study. however allele a showed a significant association in hypertensive patient compared with control group particularly in female gender. the hypertensive patients showed a significant higher result in age, bmi, fbs, total cholesterol, and stg, ldl-c and lower level in hdl-c. conclusion the association between the snp rs ofstk and hypertension was not significant in current study in kerbala population of iraq. furthermore, only allele a showed a significant association with hypertension in females group. further studies needed to clarify the effect of other stk variants on these cardiovascular risk factors. keywords hypertension, bmi, stk , ldl-c, polymorphism relationship between serine/threonine kinase gene polymorphisms with some cardiac biomarkers in hypertensive patients fadhil jawad al-tu’ma,a zena abdul-ameer mahdia & hassan mahmood abo almaalib introduction the pathogenesis of essential hypertension is believed to be multifactorial and under the influence of multiple genetic and environmental factors. genes associated with blood pressure (bp) are not sufficiently known as yet. a recent genome wide association study examined single nucleotide polymorphisms (snps) in american old order amish and identified snps in the serine/threonine kinase (stk ) that were associated with hypertension. the stk encodes a serine/threonine kinase known as a ste -related proline rich kinase (spak), one of ste family. these kinases were shown to interact with a new discovered serine/threonine kinases (wnk kinases) and cation-chloride co-transporters, and it was suggested that their functional change may cause bp dysregulation. furthermore the spak is also related to cytoskeletal rearrangement, mitogen activated protein (map) kinases, and inflammation. the associations between the above-mentioned snps and dif- ferent stk snps and bp were confirmed in other cauca- sian and chinese cohorts. , however, the influence of stk polymorphism on bp has been inconsistent, and the effects of the variants have not been found in other large studies. , although a few studies have been conducted to replicate the results in the asian population, the association between stk variants and hypertension is still unclear among the asians. here we aimed to examine the effect of rs of stk on hypertension in a population of karbala prov- ince, iraq, using hypertensive and control subjects. the primary purpose was to evaluate the association between the snps and the risk of hypertension in females and males. in addition, we investigated the effects of the snps on other cardiovascular risk factors. materials and methods subjects in current study, hypertensive patients were studied who were all diagnosed previously and some were taking treatment along with the healthy controls. and the control subjects had to have systolic bp < mmhg and diastolic bp < mmhg, without using any blood pressure lowering agents. patients were selected from the out-patient department of al-hussein teaching hospital, al-hussein medical city, holy karbala, iraq. the history, blood pressure, cardiac biomarkers and body mass index of all the subjects were investigated and measured. on the day of enrolment, clinical data including demo- graphic variables, medical history, and antihypertensive med- ications were recorded for all study samples. bp was measured by trained nurses at the right brachial artery using a mercury sphygmomanometer. subjects had minutes of rest in the supine position before the measurements, and the average of at least three measurements were used in the study. venous blood samples were collected after an overnight fast, and sam- ples were analyzed within hours of collection. all analyses were conducted by the hospital laboratory. genotyping genomic deoxyribonucleic acid (dna) was extracted from ml of peripheral venous blood by genomic dna extrac- tion kit, from bioneer, south korea. in order to detect allele a, tetra primer amplification refractory mutation system- polymerase chain reaction (arms-pcr) has been used. the detection of the allele g was carried out by using arms pcr reaction for stk rs (figs. , ). the pcr was carried out using an eppendorf gradient issn - adeparment of biochemistry, college of medicine, university of karbala, holy karbala city, iraq. bbranch of clinical laboratory science, college of pharmacy, university of karbala, holy kerbala city, iraq. correspondence to zena abdul-ameer mahdi (email: z.al-hadedy@outlook.com). (submitted: october – revised version received: november – accepted: november – published online: autumn ) j contemp med sci | vol. , no. , autumn : – relationship between stk and hypertension research fadhil jawad al-tu’ma et al. thermocycler. the reaction was carried out according to the following program: • °c for minutes (initial denaturation), cycles of the following: • °c for seconds (denaturation) • °c for seconds (annealing) • °c for second (extension) • °c for minutes (final extension) and °c (hold phase) the pcr products were separated by . % agarose gel electrophoresis and the run at v for – minutes and the gel was transferred to uv trans-illuminator for visualiza- tion under ultraviolet light. bands for the required product sizes were obtained. bands for the required product sizes of stk gene were obtained as outer primers bp, allele-a bp and allele-g bp (table ). biochemical parameters fasting blood sample of ( ml) collected by venipuncture placed into plain tubes and allowed to clot for – minutes, table . four primers for stk rs stk- ctcatggaatta aaggattattaggataacg inner forward stk- aacactctcacaagaagagatcccagtg outer forward stk- cacattttggcagtgtttggacagct inner reverse stk- ctcccaggtcgttttcaaacaaaaataa outer reverse table . characteristics of the study subjects parameters hypertension mean ± sd control mean ± sd p value age (years) . ± . . ± . . bmi (kg/m ) . ± . . ± . . sbp (mmhg) . ± . . ± . . dbp (mmhg) . ± . . ± . . fbs (mg/dl) . ± . . ± . . serum urea (mg/dl) . ± . . ± . . s. creatinine (mg/dl) . ± . . ± . . s. albumin (g/l) . ± . . ± . . s. cholesterol (mg/dl) . ± . . ± . . s. tg (mg/dl) . ± . . ± . . s. hdl-c (mg/dl) . ± . . ± . . s. ldl-c (mg/dl) . ± . . ± . . s. vldl-c (mg/dl) . ± . . ± . . fig. electrophoresis band for allele g of stk gene. fig. electrophoresis band for allele a of stk gene. centrifuged, and the separated serum was used for further measurement of fasting blood sugar, urea, creatinine, albumin, and lipid profile. statistical analysis group differences in the categorical variables, genotypes, were assessed by chi-square test, and continuous variables were examined by student’s t-test. results were expressed as mean ± sd and -tailed value of p < . was considered statistically significant. all data were analyzed using statistical package for the social sciences (spss) . version. results the characteristics of population of this study are presented in table . the average age was . ± . years for the patients and . ± . years for the controls. the hypertensive patients have higher bmi, fbs, with significant p = . and high total cholesterol, triglyceride, ldl-c, and low high den- sity lipoprotein-cholesterol (hdl-c) levels. relationship between genotype of stk and hypertension (fig. ) genotype distributions of stk polymorphisms were almost the same between normotensive controls and hypertensive patients (table ). fig. the stk rs genotype in patients and control groups. j contemp med sci | vol. , no. , autumn : – research relationship between stk and hypertensionfadhil jawad al-tu’ma et al. table . the stk alleles in hypertension and control groups sex alleles result hypertension n (%) control n (%) p value female allele a negative ( . ) ( . ) . positive ( . ) ( . ) male allele a negative ( . ) ( . ) . positive ( . ) ( . ) female allele g negative ( . ) ( . ) . positive ( . ) ( . ) male allele g negative ( . ) ( . ) . positive ( . ) ( . ) table . the stk rs genotypes distribution in hypertension aa n (%) ag n (%) gg n (%) p value sex female ( . ) ( . ) ( . ) . male ( . ) ( . ) ( . ) chronic diseases hypertension ( . ) ( . ) ( . ) . control ( . ) ( . ) ( . ) systolic sbp > mmhg ( . ) ( . ) ( . ) . sbp < mmhg ( . ) ( . ) ( . ) diastolic dbp > mmhg ( . ) ( . ) ( . ) . dbp < mmhg ( . ) ( . ) ( . ) relationship between studied alleles and hypertension (fig. ) the relationship between hypertension risk and alleles of stk in males is presented in table . indicated no associa- tion between genotypes and hypertension in any group which reached statistical significance. the only significant associa- tion was in females with allele a. relation among studied variants, blood pressure, and cardiovascular risk factors to start with, we have to clarify that variants gg has been detected in one sample hence all this variants statistics depended on one sample which would have been much better having more than one case for accurate outcome. although it has not been statistically proven, all ag vari- ants in total sample and hypertensive patients have higher sys- tolic bp than other variants. also, an obvious relationship has been clarified for the ag variants with elevated s. urea, s. creatinine, s. total cholesterol, s. tg and s. ldl-c compared to other variants in total, hyper- tensive, and control groups with significant p value in the con- trol group of serum urea. moreover, ag variant patients have lower s. hdl-c in comparison to the other variants in all groups (table ). discussion in the current study, we found no significant association between snp stk rs and hypertension in the case control study of iraqi populations. although there was no association of this snp on adjusted bps, there was a significant association between allele a of stk rs and hyper- tension group. also, there was a significant association between stk genotype and age, systolic bp and albumin this relationship was more obvious in females. on the other hand, the polymorphisms of stk were reported to be associated with bp first in caucasian samples. in a meta-analysis combining four studies, wang et al., , showed the effect of snps of rs and rs on bp. another snp of stk , rs , was also related to the hypertension prevalence in women of two cohorts in swedes. in the chinese population, two snps of stk (rs and ) were found to be associated with hypertension. in the results of current study, the associations between stk snps and hypertension or bp were not significant. although the reason for the difference is not clear, several pos- sibilities may be suggested. first, all the samples were of iraqis resident in karbala holy city, and the stk -bp relationship may be weak in this population. the influence of stk polymorphism on bp has not been consistently confirmed. in two large gwas, using more than , people, the effects of stk snps on bp were not found. , in addition, in one british and one chinese study, var- iants of stk were not associated with bp. recently, rhee et al. reported four previously reported snps associated with salt-sensitivity in koreans. however, in that study, no pol- ymorphism of stk showed significant effects after adjusting of confounding factors. second, although stk gene encodes for a protein that plays a role in bp regulation, its final effects may be too small to be detected consistently in every cohort study. then, as the population size of this study is not big enough, it cannot be completely ruled out that some variants with impacts may not have obtained statistical significance. in this study, the influence of stk variants on the risk factors was clearer in women. this finding is in accordance with prior studies that have shown gender dependency of stk effect. in a study by fava et al. the association of stk rs variant and hypertension was evident only in women. sex differences in hypertension may be attributed to multiple factors such as lifestyle, diet, and genetic polymor- phism. interestingly, it has been documented that spak expression is affected by both androgen and oestrogens in fig. the stk alleles in hypertension and control groups. j contemp med sci | vol. , no. , autumn : – relationship between stk and hypertension research fadhil jawad al-tu’ma et al. table . genotype of the studied variants and cardiovascular risk factors risk factors alleles total hypertension control mean ± s.d. mean ± s.d. mean ± s.d. age (years) . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± bmi . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± systolic bp . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± diastolic bp . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± fbs . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± s. urea . . . aa . ± . . ± . . ± . ag . ± . . ± . . ±. gg . ± . . . ± s. creatinine . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± s. albumin . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± s. cholesterol . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± s. tg . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± s. hdl-c . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± continued j contemp med sci | vol. , no. , autumn : – research relationship between stk and hypertensionfadhil jawad al-tu’ma et al. s. ldl-c . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± s. vldl-c . . . aa . ± . . ± . . ± . ag . ± . . ± . . ± . gg . ± . . . ± table . continued risk factors alleles total hypertension control mean ± s.d. mean ± s.d. mean ± s.d. human prostate cancer cells. nevertheless, further studies are needed to clarify the underlying mechanism of gender depend- ency in stk effect on cardiovascular risk factors. the population studied in the initial gwas from wang et al. is american old order amish, which is a closed founder population emigrated from switzerland in the early s. the study populations in articles of fava et al. and donner et al. are from sweden and finland. those populations are all cau- casians and close in geographical location. when we checked the details of the positive snps (rs , rs , and rs ) in wang’s gwas, we found that the minor allele frequency differs greatly between europeans and asians. to determine whether stk is a common candidate gene for hypertension, we set to find out whether it is associated with hypertension in iraqi population or not. there were studies for two snps (rs and rs ) within stk in chinese population, which were assumed functional snps based on a multispecies sequence arrangement. however, neither snp was significantly associated with hypertension in the chinese population. as the result was quite different from fava’s replication study and these result con- firmed our result. five genome-wide significant snps genotyped in this study did not show a positive association with hyperten- sion in the chinese population. similarly, cunnington et al. also did not find any association between the three snps (rs , rs , and rs ) within stk and systolic bp or diastolic bp from a cohort of british caucasians. furthermore, in fava’s study, and after exclusion of individuals from malmo preventive project cohort, who also par- ticipated in the malmo diet and cancer study, g allele of rs was no longer associated with hypertension. thus, it is still questionable whether stk can be accepted as a common susceptibility gene for hypertension. in a european study carried out in , the stk gene snp rs were coincidently associated significantly with systolic and diastolic blood pressure, (p < . ). in another study in tharparkar, pakistan population, they found an insignificant association of this polymorphism with ehtn (p = . ). they looked at the prevalence of stk snp and observed the prevalence of frequencies of reference and rare alleles and also observed the concordance with hwe. the reference allele a showed higher frequency than the rare allele g (p = . ). these findings highlight the weaker association of stk gene with ehtn in the study population. conclusion it was found that a presence of significant association between the studied snp and hypertension, particularly in females, through allele a. however, the snp showed genotype-related differences in blood urea, creatinine, albumin and lipid profile levels. the current study is the first to systemically analyse the association between stk variants and multiple cardiovas- cular risk factors.  references . j. kuneš, j. zicha. the interaction of genetic and environmental factors in the etiology of hypertension. center for cardiovascular research and institute of physiology, academy of sciences of the czech republic, prague, czech republic. physiol res. ; (suppl. ):s –s . . huan t, esko t, peters mj, pilling lc, schramm k. schurmann c, et al. a meta- analysis of gene expression signatures of blood pressure and hypertension. plos genet. mar; ( ):e . doi: . /journal.pgen. . pmid: . wang y, o’connell jr, mcardle pf, wade jb, dorff se, shah sj, et al. from the cover: whole-genome association study identifies stk as a hypertension susceptibility gene. proc natl acad sci u s a. jan; ( ): – . doi: . /pnas. pmid: . delpire e, gagnon kb. spak and osr : ste kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells. biochem j. jan; ( ): – . doi: http://dx.doi.org/ . / bj pmid: . fava c, danese e, montagnana m, sjögren m, almgren p, engström g, et al. serine/threonine kinase is a candidate gene for primary hypertension especially in women: results from two cohort studies in swedes. j hypertens. mar; ( ): – . doi: . /hjh. b e - b c pmid: . chen ly, zhao wh, tian w, guo j, jiang f, jin lj, et al. stk is an independent risk factor for male hypertension in han chinese. int j cardiol. jan; ( ): – . doi: . /j.ijcard. . . pmid: . newton-cheh c, johnson t, gateva v, tobin md, bochud m, coin l, et al. genome-wide association study identifies eight loci associated with blood pressure. nat genet. jun; ( ): – . doi: . /ng. pmid: . levy d, ehret gb, rice k, verwoert gc, launer lj, dehghan a, et al. genome-wide association study of blood pressure and hypertension. nat genet. jun; ( ): – . doi: . /ng. pmid: . sayers ew, barrett t, benson da, boltan e, bryant sh, canese k, et al. database resources of national center for biotechnology information. nucleic acids res. jan; :d –d . doi: . /nar/gkq pmid: . cunnington ms, kay c, avery pj, mayosi bm, koref ms, keavney b. stk polymorphisms and blood pressure: an association study in british caucasians and assessment of cis-acting influences on gene expression. bmc med genet. dec; : . doi: . / - - - pmid: j contemp med sci | vol. , no. , autumn : – relationship between stk and hypertension research fadhil jawad al-tu’ma et al. . niu wq, zhang y, ji kd, gao pj, zhu dl. contribution of five top whole-genome association signals to hypertension in han chinese. j hum hypertens. apr; ( ): – . doi: . /jhh. . pmid: . rhee my, yang sj, oh sw, park y, kim ci, park hk, et al. novel genetic variations associated with salt sensitivity in the korean population. hypertens res. may; ( ): – . doi: . /hr. . pmid: . ruixing y, jinzhen w, shangling p, weixiong l, dezhai y, yuming c. sex differences in environmental and genetic factors for hypertension. am j med. sep; ( ): – . doi: . /j.amjmed. . . pmid: . qi h, labrie y, grenier j, fournier a, fillion c, labrie c. androgens induce expression of spak, a ste /sps -related kinase, in lncap human prostate cancer cells. mol cell endocrinol. sep; ( ): – . doi: http:// dx.doi.org/ . /s - ( ) - pmid: . loung vasandas umedani, bushra chaudhry (department of biological and biomedical sciences, aga khan university, karachi, pakistan),vikram mehraj (unite mixte de recherche , centre national de larecherchescientifique, france), muhammad ishaq (department of biochemistry, united medical and dental college, ibrahim hydri, korangi creek, karachi, pakistan) . the role of the microbiome in food allergy: a review children review the role of the microbiome in food allergy: a review christina l. nance , , roman deniskin , , veronica c. diaz , , misu paul , , sara anvari , and aikaterini anagnostou , ,* baylor college of medicine, section of pediatric immunology, allergy and retrovirology, houston, tx usa; clnance@texaschildrens.org (c.l.n.); rxdenisk@texaschildrens.org (r.d.); veronica.diaz@bcm.edu (v.c.d.); misu.paul@bcm.edu (m.p.); sara.anvari@bcm.edu (s.a.) texas children’s hospital, department of pediatrics, section of immunology, allergy and retrovirology, houston, tx , usa * correspondence: aikaterini.anagnostou@bcm.edu received: april ; accepted: may ; published: may ���������� ������� abstract: food allergies are common and estimated to affect % of children and % of adults in the united states. they pose a significant burden—physical, economic and social—to those affected. there is currently no available cure for food allergies. emerging evidence suggests that the microbiome contributes to the development and manifestations of atopic disease. according to the hygiene hypothesis, children growing up with older siblings have a lower incidence of allergic disease compared with children from smaller families, due to their early exposure to microbes in the home. research has also demonstrated that certain environmental exposures, such as a farming environment, during early life are associated with a diverse bacterial experience and reduced risk of allergic sensitization. dysregulation in the homeostatic interaction between the host and the microbiome or gut dysbiosis appears to precede the development of food allergy, and the timing of such dysbiosis is critical. the microbiome affects food tolerance via the secretion of microbial metabolites (e.g., short chain fatty acids) and the expression of microbial cellular components. understanding the biology of the microbiome and how it interacts with the host to maintain gut homeostasis is helpful in developing smarter therapeutic approaches. there are ongoing trials evaluating the benefits of probiotics and prebiotics, for the prevention and treatment of atopic diseases to correct the dysbiosis. however, for routine use of probiotics as an intervention for preventing allergic disease is not currently recommended. a new approach in microbial intervention is to attempt a more general modification of the gut microbiome, such as with fecal microbiota transplantation. developing targeted bacterial therapies for food allergy may be promising for both the treatment and prevention of food allergy. similarly, fecal microbiota transplantation is being explored as a potentially beneficial interventional approach. overall, targeted bacterial therapies for food allergy may be promising for both the treatment and prevention of food allergy. keywords: allergic disease; microbiome; hygiene hypothesis; food allergy; asthma; atopy; atopic dermatitis; short chain fatty acids; t regulatory cells; dysbiosis; immune tolerance; g-protein coupled receptors; prebiotics; probiotics; fecal microbiota transplantation . introduction food allergies are estimated to affect approximately % of us children [ ] and % of us adults [ ]. food allergy is defined as an adverse immune response to a food and can be either ige-mediated (e.g., urticaria and anaphylaxis) or non-ige mediated (e.g., food protein-induced enterocolitis syndrome and eosinophilic esophagitis) [ ]. food allergies pose a significant disease burden, which includes children , , ; doi: . /children www.mdpi.com/journal/children http://www.mdpi.com/journal/children http://www.mdpi.com https://orcid.org/ - - - https://orcid.org/ - - - https://orcid.org/ - - - http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /children http://www.mdpi.com/journal/children children , , of physical, dietary, social and psychological effects [ , ]. currently, there is no cure for food allergies and the underlying cause has yet to be elucidated. ige-mediated food allergy has been associated with both immune dysregulation and impaired gut epithelial integrity. in addition, there is increasing interest in a potential link between food allergy and the gut microbiome [ ]. the microbiome consists of many microorganisms, including bacteria, fungi and viruses, as well as their genomic elements. there is increasing evidence that the human microbes residing in the airways, gastrointestinal tract and skin play an important role in normal health and disease states [ ]. there are three different microbe–host relationships: (a) pathogenic, when the microbes harm the host; (b) commensal, when microbes coexist with the host without providing benefit or causing harm; and (c) symbiotic, when microbes and the host are mutually beneficial to each other [ ]. mucosal surfaces that line our gastrointestinal tract are continuously exposed to trillions of bacteria that form a symbiotic relationship and impact host health and disease. there are numerous studies evaluating the interactions between the host and microbiome including the dynamic changes in the commensal bacterial population, secretion, and absorption of metabolites. imbalances in this relationship, or dysbiosis, contribute to the development of diseases such as inflammatory bowel disease, asthma, type diabetes, cardiovascular disease, metabolic syndrome, obesity and allergic diseases such as asthma and food allergy [ , ]. in , david strachan coined the term “hygiene hypothesis” to explain the rise in atopic disorders. the hygiene hypothesis states that early childhood exposure to microorganisms protect against the development of allergic disease. a lack of exposure to these microbes due to reduced household sizes, improvements in household amenities, and higher standards of personal hygiene may have resulted in the impairment of immune tolerance and thus the rise in allergic disorders [ ]. the hygiene hypothesis later evolved into the “old friends” hypothesis [ ]. the “old friends” hypothesis provided a more detailed explanation between microbes and the development of inflammatory disorders. the “old friends” hypothesis proposed that microorganisms and their host have co-evolved symbiotically over thousands of years thereby allowing the organisms to play a key role in shaping the host immune response. a disruption in the symbiotic relationship could lead to immune dysregulation and promote allergic disease [ ]. . methods we performed a non-systematic review of published literature on “food allergy” and the “microbiome” from – using the pubmed database. we included clinical trials, meta-analyses, randomized controlled trials, reviews and systematic reviews in the english language. we identified key studies from the last years, with special emphasis on the last decade to ensure we included recent research. . the gut microbiome the gastrointestinal (gi) microbiome is a diverse community of bacteria, archaea, fungi, protozoa and viruses that colonize the mammalian gi tracts. the national institute of health (nih) human microbiome project identified more than microorganisms in the intestine. these microorganisms encompass > species with essential physiological roles. the composition and concentration of these microbes differs along the tract and is affected by the host diet [ , ]. these bacteria have coevolved with humans over millennia and have a symbiotic relationship—humans consume prebiotic fiber, which is metabolized by resident microbes in the gut to create short chain fatty acids (scfa). scfas, in turn, regulate immune responses [ ]. the modern use of antimicrobial medications and the consumption of high-sugar, low-fiber diets have adversely affected the host–microbe interaction. there is emerging scientific evidence that the imbalance in the microbial flora (dysbiosis) contributes to altered host metabolism and predisposes humans to developing atopic diseases and an increased susceptibility to infections [ ]. children , , of . . host–microbiome interactions advances in technology have enhanced our understanding of the interplay between the gut microbiome and health and human disease. these include high-sensitivity means to study microbial communities in any type of ecosystem. although the terms microbiota and microbiome are used interchangeably, microbiota are defined as the microbial taxa associated with complex organisms such as humans, whereas the microbiome is the catalog of these microbes and their genes [ ]. furthermore, for gut microbiota and their metabolic byproducts impact the host and regulate homeostasis by providing essential nutrients, affecting the health of the intestinal epithelial barrier, and regulating host innate and adaptive immune processes [ ]. the disruption of a balanced microbiome can lead to immunological dysregulation with diseases such as inflammatory bowel disease (ibd), obesity and allergic diseases (including asthma and food allergy). an altered gut microbiome affects microbiota-derived products and metabolites, including pro- and anti-inflammatory materials [ , ]. protection against microbial invasion is due to the intestinal barrier. this barrier has multiple lines of defense including commensal bacteria, which competitively inhibit the colonization of pathogenic bacteria and the production of metabolically protective compounds [ ]. . . early life microbiome the timing of host–microbe interactions in early life appears to be important. the gut microbiome changes during the course of life, with the most rapid changes occurring in early life [ , ]. it is known that the overall diversity of the human gut microbiota increases steadily from birth until around years of age, remains relatively stable throughout adulthood, and then declines in later years [ ]. in adults, – % of the gut microbiome is stable, with the degree of stability varying between phyla [ ]. additionally, for gut microbiome changes dramatically during pregnancy; intrinsic factors (e.g., stress) and extrinsic factors (e.g., diet, and drugs) influence the composition and activity of the gut microbiome throughout life [ , ]. . the microbiome and atopic diseases there is growing evidence that the microbiome contributes to the development and manifestations of allergic disease. according to the hygiene hypothesis, mentioned previously, children growing up with older siblings have a lower incidence of allergic disease, compared with children from smaller families, likely due to their early exposure to environmental microbes in the home [ ]. additionally, factors associated with a decreased risk of developing allergic disease later in life include being born via vaginal delivery, being breastfed during the first months of life, pet ownership, and the absence of early antibiotic exposure. by contrast, growing up in an urban, westernized environment is associated with increased rates of asthma, atopic dermatitis and food allergy [ ]. . . effect of environmental exposures on the composition of the gut microbiome and atopic diseases certain environmental exposures prenatally and during early life are associated with a diverse bacterial experience and reduced risk of allergic sensitization. the pasture study, a birth cohort study, showed an association between living in a farming environment with an increased environmental endotoxin exposure and a reduced incidence of asthma. the parsifal and gabriela studies also demonstrated that there was a lower prevalence of asthma and other atopic diseases in children living on farms compared to suburban environments [ , ]. a diversity of microbial exposure was noted to be inversely associated with the risk of asthma in children growing up on farms in central europe and being exposed to a greater diversity of environmental fungi and bacteria [ ]. similarly, wlasiuck et al. reported that the effect on asthma was largely due to exposure to cows, livestock, and straw, and the consumption of raw cow’s milk [ ]. children , , of . . infant microbiome the infant microbiome is shaped by race, diet, delivery method, and even the maternal microbiota of the gut and reproductive system during pregnancy [ , ]. the infant microbiome begins during fetal development with exposure to the uterine microbiome and meconium [ , , ]. during fetal life, for fetal gut microbiota resembles the microbiota of amniotic fluid, which is colonized by bacteria. this includes organisms belonging to the enterobacteriaceae family and bacteria from the firmicutes phylum (such as lactobacillus species, clostridium species and bacillus species) [ , ]. the method of delivery influences the infant’s microbiome as infants born by cesarean section (c-section) had microbiotas similar to the skin microbiota with staphylococcus, corynebacterium and propionibacterium as the dominant species [ , ]. bacteroides colonization is also delayed in infants born by c-section. cesarean section has been associated with a higher risk of allergic rhinitis, asthma and autoimmune disorders such as celiac disease [ ]. after birth, for microbiome is transiently dominated by enterobacteriaceae and staphylococcus. [ ]. savage et al. demonstrated that breastfeeding, and not formula feeding, was the dietary factor most consistently associated with the infant’s intestinal microbiome. breastfed infants have higher numbers of bifidobacterium species compared to formula fed infants. the latter group have higher clostridium species and bacterioides species [ ]. as solid foods are introduced, bacteria from the firmicutes phylum colonize the gut, and by about three years of age firmicutes and bacteroides are the main colonizers of the child gut microbiota. neonates have a limited capacity to initiate a th response, and the fetus is generally very th -directed immunologically. close immunologic interaction between the mother and fetus might lead to a th -skewed state noted in infants who develop allergic disease based on the evidence that non-allergic mothers have a lower th response from mid to late gestation compared to mothers with atopy [ , ]. the bacterial colonization of the gut is also important in the differentiation of t helper (th) cells into th , th , t regulatory cells (tregs) and th cells. furthermore, intestinal bacteria such as lactobacillus, bifidobacterium, bacteroides and clostridium, as well as their metabolites, induce peripheral treg cells that control inflammation in the gut and lungs. th cells produce anti-inflammatory cytokines that improve barrier function and confer protection against pathogens. . . atopic dermatitis and the microbiome multiple studies have linked the microbiota of the respiratory tract, gastrointestinal tract and skin to allergic disease. the composition and diversity of the microbiome is different across body areas, with the gi tract showing the largest number and diversity of microbes [ ]. interestingly, for effects of microbial dysbiosis are not restricted to the local tissue environment. for example, for skin microbiota may affect the gut and gastrointestinal system [ ]. atopic dermatitis (ad) is characterized by an abnormal skin barrier, a result of multiple factors (e.g., genetic, environmental and immunologic). ad may predispose to the development of other atopic conditions, such as food allergy, allergic rhinitis and asthma (this process is known as the “atopic march”). epithelial dysfunction linking the microbiome and immune dysregulation can lead to the atopic march. for example, disruption in the skin barrier allows increased allergen permeability and sensitization in addition to colonization by pathogenic organisms [ ]. as a result of this, a th -type response is induced, causing further breakdown of the epithelial barrier. the th response is generalized and affects distant sites such as the intestinal and respiratory tracts [ ]. in ad, dysbiosis is characterized by reduced skin bacterial diversity, with increased s. aureus and decreased s. epidermis. colonization and infection with s. aureus has been linked to increased ige responses and severity of ad disease [ ]. commensal microbes, on the other hand, such as s. epidermidis, have the potential to inhibit s. aureus growth and improve the skin barrier by improving tight junctions and producing antimicrobial peptides [ ]. exposure to food allergens via a disrupted skin barrier has been shown to be a risk factor for food allergy [ ]. additionally, ad patients show increased intestinal permeability and a defective (“leaky”) gut barrier, enabling food allergen penetration and sensitization via the gut. a large-scale birth cohort study showed e. coli and children , , of c. difficile overgrowth in infants with ad; this was associated with a decreased number of beneficial bacteria, abnormal gut barrier function and loss of immune tolerance [ ]. . . asthma and the microbiome in asthma, living in an environment with diverse microbial flora has been shown to be protective against allergic inflammation and disease. stein et al. examined two separate agricultural populations in the us, for amish and the hutterites. both groups have similar lifestyles, but different farming practices—the amish follow traditional practices, and the hutterites use industrialized practices [ ]. the investigators showed that the prevalence of asthma and allergic sensitization was four and six times lower in the amish, respectively. additionally, median endotoxin levels in amish house dust were . times higher compared to those in the hutterite houses. differences in the immune response were also observed between the two populations—the genes most expressed in the amish children were related to anti-inflammatory effects (limited activity of pathways depending on nf-kβ), whereas those expressed in the hutterites were related to the activation of nf-kβ [ ]. the authors concluded that differences in the amish farming environment, with increased microbiome exposure, appeared to be protective against asthma through the modulation of the innate immune response [ ]. . the gut microbiome and food allergy the gut microbiome has gained significant interest in recent years due to its many immuno-modulatory properties and role in mucosal tolerance. gut dysbiosis likely precedes the development of food allergy, and the timing of such dysbiosis appears to be critical [ ]. multiple microbial orders have been implicated in food allergy, most commonly clostridiales and lactobacillales (beneficial effects), as well as bacteroidales and enterobacteriales (beneficial and detrimental effects have been described) [ ]. studies evaluating the profile of the gut microbiome have revealed unique microbial differences in patients with food allergies compared to healthy patients, and these studies have provided evidence that the dysbiosis of the microbiome precedes the development of food allergy [ ]. since food allergy is a complex and heterogenous disease and studies often involve very different patient populations and enrolment criteria, for results have not been consistent across various research projects. there has been, however, an increasing interest in investigating microbiome signatures related to food allergy. . . microbiome signatures in food sensitization and food allergy a study of children with ad examined fecal microbiome signatures for food allergy. sixty-two children presented with ad and food allergy, whereas children had ad only (no food allergy). there were no differences in microbial diversity between the two groups. however, there were differences in microbial species; children with ad and food allergy had relatively more e. coli and b. pseudocatenulatum and less b. breve, b. adolescentis, f. prausnitzii and a. muciniphila than children with ad and no food allergy [ ]. in infants, microbial colonization is complete within the first month of life; significant changes occur subsequently during weaning to solid foods. the first two years of life are crucial for the further development of the intestinal microbiota, with the first months of life considered to represent a critical window of interaction between the infant gut microbiota and the immune system [ ]. delayed colonization with commensal bacteria or alterations in the microbiota profile during infancy may predispose to the development of immune-mediated disorders such as allergic diseases [ ]. early colonization with potentially more pathogenic bacteria (e.g., c. difficile or s. aureus) has been linked to food allergy development, whereas colonization with more beneficial bacteria (e.g., bifidobacteria) is seen more often in non-allergic children [ ]. a us study collected and analyzed intestinal microbiome samples from infants at – months of age—the study examined infants without food sensitization, with food sensitization, without food allergy and infants with food allergy in this cohort [ ] the investigators showed that the genera children , , of haemophilus, dialister, dorea and clostridium were underrepresented in infants who were sensitized to food, whereas the genera citrobacter, oscillospira, lactococcus and dorea were underrepresented in infants with food allergy. no differences were found in microbial diversity between the groups of sensitized and food-allergic versus the infant controls (non-sensitized and non-food allergic) [ ]. a smaller study from taiwan evaluated differences in the gut microbiota between children with food sensitization and healthy controls. the mean age of subjects was months (range: – months). the investigators reported lower diversity of the total microbiota and the bacterial phylum bacteroidetes in children with food sensitization. additionally, for number of bacteroidetes bacteria was significantly decreased and that of firmicutes was significantly increased compared with those in the healthy children. at the genus level, there were significant increases in the numbers of sphingomonas, sutterella, bifidobacterium, collinsella, clostridium sensu stricto, clostridium iv, enterococcus, lactobacillus, roseburia, faecalibacterium, ruminococcus, subdoligranulum and akkermansia in the food-sensitized children. the above group showed significant decreases in the numbers of bacteroides, parabacteroides, prevotella, alistipes, streptococcus and veillonella [ ]. . . microbiome signatures in egg and cow’s milk allergies in terms of investigating specific food allergies, a total of children aged – months (median age: . months) were enrolled from five different centers in the united states with the aim of characterizing the gut microbiomes of egg-allergic subjects and controls [ ]. in this study, children had egg allergies and displayed enrichment of the lachnospiraceae and streptococcaceae families, while leuconostocaceae were enriched in the healthy control group (non-food allergic children). additionally, egg sensitization was associated with greater gut microbiome diversity and the lachnospiraceae and ruminococcaceae genera. interestingly, neither compositional differences in the gut microbiome nor differences in gut bacterial diversity could predict the resolution or persistence of egg allergy at age years. [ ]. by contrast, a separate study investigating the association between early life gut microbiota composition and the resolution of cow’s milk allergy reported that enrichment of clostridia and firmicutes was noted in the infant gut microbiome (at – months of life) of subjects whose milk allergy resolved later in childhood [ ]. feehly et al. took this work a step further by colonizing germ-free mice with feces from either healthy infants or infants with cow’s milk allergy (cma) [ ]. the mice were subsequently sensitized with b-lactoglobulin (cow’s milk allergen) and the mucosal adjuvant cholera toxin. the investigators reported that mice colonized with feces from cow’s milk allergic infants were highly susceptible to anaphylaxis in response to a beta-lactoglobulin allergen challenge; a drop in core body temperature was noted as an indicator for anaphylaxis in the mice. all of the mice that received microbiota from the healthy infant donors were protected from an anaphylactic response to a beta-lactoglobulin challenge. microbial analysis showed no difference in community diversity and evenness between the healthy and cma-colonized mouse groups, but a clostridial species, anaerostipes caccae, was identified as protective against an allergic response to food. these findings underscore the importance of commensal bacteria in regulating responses to dietary antigens and potentially preventing allergic responses to food [ ]. interestingly, increased gut microbial diversity is suggested to be protective of allergic disease by some investigators, but not by others. this discrepancy may suggest that there is not always a beneficial association between increased microbiome diversity and atopic disease. additionally, for role of the microbiome cannot be captured by a single dimension (e.g., alpha diversity), but involves more complex interactions between different taxa and their metabolic effects [ ]. . nutrition and the microbiome in food allergy nutrition is an important environmental factor in early life and may influence the immune system’s maturation and development in multiple ways. the first oral exposure to food allergens has the potential to mediate unresponsiveness and lead to oral tolerance, as seen in recent research studies on peanut allergy [ ]. this observation has resulted in a significant change in infant feeding children , , of practices in the us and worldwide, with most westernized countries now recommending allergenic food introduction at age months and certainly within the first year of life [ ]. . . dietary factors and the microbiome a variety of dietary factors appear to play a key role in the development of food allergy. roduit et al. investigated the feeding practices during the first year of life of children in a birth cohort study, while also prospectively collecting data on environmental factors and allergic disease development. they reported that an increased diversity of complementary food introduced in the first year of life was inversely associated with food allergy and food sensitization development up to years of age. a dose–response effect was also noted with each additional food item introduced. additionally, for investigators showed that increased food diversity was associated with an increased expression of forkhead box protein (foxp ), a marker for regulatory t cells (tregs), suggesting a protective effect of a diverse diet against food allergy development. by contrast, children with a low food diversity score showed a reduced expression of foxp [ ]. . . the “nutrition–gut microbiome–physiology axis” host diet and nutrition are key in maintaining a healthy microbiome. mckenzie et al. describe the “nutrition–gut microbiome–physiology axis” as an essential link between diet, gut microbiota and allergic disease [ ]. short chain fatty acids (scfas) are metabolites produced by intestinal bacteria through the fermentation of non-digestible fibers. there is long-standing evidence that links high-fiber diets to better health outcomes [ ]. by contrast, for western diet is “pro-inflammatory”, due to its high fat, low fiber and highly processed nature, which appear to contribute to gut dysbiosis. scfas have been highlighted as key signaling molecules enabling “cross-talk” between the gut microbiome and the host, with emerging evidence of their importance in food allergy [ ]. roduit et al. analyzed scfa levels in fecal samples from children at the age of year from a birth cohort. they reported that children with the highest levels of butyrate and propionate at the age of one year were less likely to suffer from asthma at the ages of and years. these children also showed significantly less allergic sensitization, and decreased risks of a food allergy and allergic rhinitis diagnosis. the authors suggest that increasing scfa levels may be an option for preventing allergic disease in children [ ]. more recently, cait et al. examined whether bacterial butyrate production in the gut during early infancy is protective against the development of atopic disease in children [ ]. the investigators found that the microbiome of infants who went on to develop allergic sensitization later in childhood lacked genes encoding key enzymes for both carbohydrate breakdown and butyrate production [ ]. . the microbiome and underlying mechanisms in food allergy the crosstalk between gut microbiota, host epithelial cells, and immune cells making up the gut-associated lymphoid tissue (galt) is complex. hence, having a tolerogenic phenotype depends on maintaining a balanced repertoire of intestinal microorganisms, a heathy gut epithelial barrier, and appropriate immune cell recruitment/activation. imbalances in this host–microbe axis cause the breakdown of immune tolerance to food antigens that leads to allergic disease. in general, for microbiome affects food tolerance via the secretion of microbial metabolites and the expression of microbial cellular components (pathogen-associated molecular patterns, pamp) [ ]. . . scfa interaction with host epithelial and immune cells intestinal microorganisms such as faecalibacterium prausnitzii, clostridium leptum and eubacterium rectal produce a large repertoire of metabolites including short-chain fatty acids (scfa), aryl hydrocarbon receptor (ahr) ligands and polyamines [ ]. the importance of scfas in immune tolerance has been extensively characterized at the genetic, biochemical and clinical levels. the major scfas are butyrate, propionate, acetate and valerate, which are metabolic byproducts of the microbial children , , of fermentation of scfas, present in varying concentrations throughout the gut but most abundant in the colon [ ]. total scfa levels are also affected by the host genotype/ethnicity [ ]. scfas directly engage g-protein coupled receptors (gpcr) on intestinal epithelial cells (e.g., gpr , gpr , gpr a and olfr ) or can passively diffuse through the cell membrane to inhibit histone deacetylases (hdac) in epithelial and intestinal immune cells [ , ]. the downstream effect on enterocytes is regulating the expression of genes involved in energy metabolism, cell proliferation and differentiation, and fortifying the epithelial barrier (tight junctions and mucus production) [ ]. scfas also effect gut inflammatory and tissue repair processes by altering nlrp inflammasome and autophagy activity [ , ]. scfas have direct immune-modulatory effects in the intestinal milieu and peripherally [ , ]. scfas induce gut dendritic cells (dc) to express retinal aldehyde dehydrogenase (raldh), which produces retinoic acid from vitamin a [ ]. secreted retinoic acid upregulates expression of the gut-homing integrins α β on peripheral regulatory t cells (treg). treg cells constitute %– % of the cd t cell population in intestinal lamina propria, and they promote immune tolerance by suppressing bystander t cells [ ]. separately, scfas regulate host antibody responses by altering metabolic pathways in b cells. scfas increase intracellular concentrations of acetyl-coa and enhance oxidative phosphorylation, glycolysis and fatty acid synthesis [ ]. the net effect is an increased production of iga and igm. additionally, butyrate stimulates the production of protective cytokines (ifnγ and il- ) in peripheral blood mononuclear cells (pbmcs) [ ]. il- induces the expansion of treg cells and also suppresses t helper (th ) and th cells, which are anti-pathogenic but pro-inflammatory cell types. finally, in vitro studies show that scfas inhibit hdac expression in human macrophages and pbmcs and downregulate the expression of inflammatory cytokines (il- , il- and tnfα) [ , ]. the butyrate inhibition of hdac promotes the differentiation of monocytes into macrophages and induces antimicrobial activity in vivo [ ]. overall, scfas are essential for maintaining immune homeostasis in the gut by regulating protective and inflammatory responses. in vivo and clinical studies have validated the importance of scfas in food allergy. for example, vonk et al. demonstrated that butyrate supplementation enhances oral immunotherapy (oit) desensitization in a murine model of cow’s milk allergy (cma) [ ]. in humans, in studies with pediatric patients with cma, for ingestion of hydrolyzed formula containing a butyrate-producing probiotic l. rhamnosus gg (lgg) increased fecal butyrate levels and was associated with the acquisition of immune tolerance [ ]. furthermore, compared to healthy children, children with atopic disease have lower levels of fecal butyrate and valeric acid [ ]. children who developed tolerance to peanut and egg had an increased frequency of il- -expressing tregs with concomitant higher blood levels of il- [ ]. consistent with the in vitro findings is the observation that butyrate supplementation increases the number of activated (foxp +)treg cells. in children, developing tolerance to cma correlates with a higher frequency of treg cells and a reduced proliferation of milk-specific t cells [ ]. by contrast, germ-free mice and antibiotic-treated mice who develop allergic disease have lower numbers of colonic treg cells [ ]. similarly, patients with dysregulation in foxp + treg (immune dysregulation, polyendocrinopathy, x-linked; ipex) develop food allergies among other gi and immune pathologies [ ]. . . polyamines, ahr ligands and gut homeostasis polyamines (pa) are very reactive polycationic molecules mostly derived from the diet (e.g., soybeans, mushrooms, beef and pork). pas are also produced de novo by commensal bacteria (bacteroides species) and host cells [ ]. spermidine, spermine, putrescine and cadaverine are the predominant pas produced by gut bacteria. polyamines are essential for maintaining the gut epithelial barrier function (upregulating junctional proteins, e.g., occludins and e-cadherins) in in vitro studies [ ]. the role of polyamines in preventing food allergies is limited but an active topic of investigation [ – ]. aryl hydrocarbon receptors (ahr) are ubiquitously expressed transcription factors that sense xenobiotic ligands. ahr ligands include tryptophan metabolites derived from microbiota and dietary children , , of macronutrients. the activation of the ahr signaling pathway is important for maintaining gut health including a direct effect on mucosal immunity [ ]. for example, intestinal ahr signaling is important for intestinal crypt stem cell differentiation [ ]. additionally, exogenous ahr ligands activate intestinal intraepithelial lymphocytes and affect epithelial barrier function [ ]. furthermore, in animal studies, mice fed a diet deficient in ahr ligands had lower fecal iga compared to mice consuming a regular chow diet [ ]. in murine studies on peanut allergy, activating ahr pathways decreases peanut sensitization by shifting the naïve t cell population to a treg phenotype [ , ]. to our knowledge, there are currently no clinical studies evaluating the functional effect of ahr ligands on food tolerance. . microbiome and food allergy treatments in the horizon . . prebiotics and probiotics in food allergy with the evidence to support that the gut microbiome plays a role in the development of food allergies, for investigation and understanding of microbiome therapy has been of interest in providing a potential avenue for food allergy prevention and treatment. microbiome therapy entails the use of both prebiotics and probiotics. the term prebiotics was first defined in , and since that time, for definition of prebiotics has evolved. currently, prebiotics are described as a “substrate” that is selectively utilized by host microorganisms, leading to specific changes in the composition and/or activity of the gastrointestinal microbiota, thereby providing beneficial health effects [ ]. additionally, prebiotics include non-digestible compounds, such as oligosaccharides or soluble fermentable fibers that are selectively utilized and promote the growth of beneficial microorganisms and improve health. prebiotics pass through the upper gastrointestinal tract undigested and serve to stimulate the activity and/or growth of microorganisms colonized in the large intestines [ ]. by contrast, probiotics are live microorganisms that provide beneficial health benefits to the host. when given in adequate doses, probiotics may provide health benefits through the modulation of immune responses. when paired together, products containing both prebiotics and probiotics are known as symbiotics. a few studies have provided evidence that the composition of the gut microbiota can influence the development and course of food allergy. therefore, for use of prebiotics, probiotics or synbiotics has been investigated for their potential benefits on the host, in order to prevent and/or treat food allergies. . . . prebiotics the consumption of prebiotics has been suggested to provide more favorable microbial colonization patterns and potentially allow for the development of tolerance and the prevention of allergy. there are limited studies investigating the role of prebiotic supplementation on food allergy prevention. a systematic review in examined whether prebiotics given to infants could prevent infant sensitization to dietary allergens [ ]. the authors of the systematic review concluded that prebiotic supplementation could potentially prevent eczema in infants up to two years of age; however, neonatal prebiotic supplementation did not prevent the development of food allergies [ ]. overall, more research is needed to evaluate the role of prebiotics in the development of allergic disease, including food allergies. . . . role of probiotics in preventing food sensitization with regard to probiotics, few clinical trials have investigated whether the use of probiotics has a beneficial effect on preventing the development of atopy and food sensitization. in , a systematic review by cuello-garcia et al. evaluated randomized trials, with trials (n = ) evaluating probiotic use during pregnancy, trials (n = ) evaluating the use of probiotics in breastfeeding mothers and trials (n = ) evaluating the use of probiotics in infants. the authors concluded that probiotics reduced the risk of eczema when taken during the last trimester of pregnancy, during children , , of breastfeeding or during infancy; however, for evidence was not strongly supported. additionally, for prevention of food allergies and other allergic conditions was not observed with probiotic use during pregnancy, breastfeeding and/or when given to infants [ ]. the world allergy organization (wao) also conducted a systematic review of randomized controlled trials of probiotics for allergy prevention. the wao concluded that there was weak evidence to support that probiotic supplementation could reduce the risk of developing allergic disorders in pediatric patients; however, a small risk reduction for eczema prevention could be observed with probiotic use [ ]. a meta-analysis examined trials in order to determine whether prenatal and postnatal probiotic administration could reduce the risk of atopy and food sensitization. the authors of the meta-analysis examined trials and concluded that with a combined approach of maternal supplementation during pregnancy and infant supplementation of probiotics, a beneficial effect was observed in preventing the development of atopy and reducing the risk of food sensitization (rr . , % ci: . – . ); however, for protective effect was not observed when probiotics were not given during both periods [ ]. . . . role of probiotics in accelerating tolerance to foods there have also been studies investigating the use of probiotics in patients with a confirmed food allergy diagnosis. in , a double-blind randomized, placebo-controlled trial evaluating infants with food challenge-confirmed cow’s milk allergies examined whether extensively a hydrolyzed formula supplemented with both probiotics lactobacillus casei and bifidobacterium lactis for months could accelerate tolerance to cow’s milk in infants with cow’s milk allergy. no difference was observed in the development of cow’s milk tolerance between the treatment ( %) and placebo groups ( %) [ ]. by contrast, a subsequent study examined infants with food challenge-confirmed cow’s milk allergies who received either extensively hydrolyzed casein formula (ehcf) or ehcf supplemented with lactobacillus gg (lgg). ehcf supplemented with lgg augmented the development of tolerance to cow’s milk protein after months and months of treatment compared to that in the control group [ ]. additionally, for same investigators examined the effects of the supplementation of ehcf with/without lgg for years in children with ige-mediated cow’s milk allergy. the authors demonstrated that the infants with ige-mediated cow’s milk allergy receiving the ehcf supplemented with lgg had developed cow’s milk tolerance over the course of the -year treatment period. the incidence of developing other atopic conditions was reduced in patients receiving ehcf with lgg [ ]. . . . probiotics in peanut allergy probiotics have also been examined in patients with peanut allergy undergoing peanut oral immunotherapy. in , tang et al. reported the first results of the effects of probiotics in patients receiving peanut oral immunotherapy (poit). the study compared peanut allergic children receiving daily poit with the probiotic lactobacillus rhamnosus cgmcc . and peanut allergic children receiving daily poit with placebo (maltodextrin) for a total of months. the study’s primary outcome was to evaluate sustained unresponsiveness to weeks following the discontinuation of poit treatment. the authors reported that . % of the probiotic and poit group achieved sustained unresponsiveness compared to . % of the placebo and poit group (p < . ). additionally, . % of those receiving the probiotic and poit were desensitized compared to . % of those receiving the placebo (p < . ) [ ]. the follow-up study, years following treatment cessation, reported that % of the subjects who received the probiotic and poit treatment were consuming peanuts compared to % of the patients who received the placebo and poit. additionally, for subjects who received the probiotic and poit had smaller peanut skin prick wheal sizes and significantly higher peanut sigg /sige ratios compared to subjects who received the placebo and poit [ ]. although both studies suggested that probiotics could help patients achieve peanut tolerance, for research was limited by the absence of a probiotic-only or poit-only control group. the evidence at this time for probiotics, as either a preventative or therapeutic agent for food allergies, remains low, and probiotics are currently not recommended for routine use in food allergy prevention or treatment. children , , of . . . ongoing studies on synbiotics currently, an ongoing clinical trial (tempo) investigating partially hydrolyzed infant formula with and without the added synbiotics bifidobacterium breve m- v plus the prebiotics oligofructose, long-chain inulin and acidic oligosaccharides is being examined in infants at risk of developing allergy. the primary outcome of the study will be to measure the levels of bifidobacterium breve in stools at weeks of age, and the secondary outcome will be to assess ige-mediated allergic manifestations in the blood up to weeks of age (clinicaltrials.gov/ ct / show/ nct ). in addition to the tempo study, a similarly designed study, known as the maestro study, is investigating the effect of a partially hydrolyzed infant formula with added synbiotics on the development of allergic manifestations in infants at high risk of developing allergy up to the age of months (clinicaltrials.gov/ ct / show/ nct ). finally, for ongoing double-blind prospective trial (presto) has been evaluating the development of tolerance to cow’s milk at , and months, in infants with ige-mediated cow’s milk allergy, following months of dietary management with an amino acid formula (aaf) supplemented with and without synbiotics (short and long chain fructo-oligosaccharides and bifidobacterium breve m- v). (trialregister.nl/ trial/ ). . . metagenomics and metabolomics novel methodologies and approaches are now being implemented in the field of the microbiome as it relates to food allergy. these involve the analysis of diseased versus healthy microbial populations by metagenomics, metabolomics or other techniques to uncover key commensal microbial factors that influence disease pathology, gut homeostasis and the immune response [ ]. limited information was garnered in initial studies in this field. this includes harvesting and expanding microbiotia using culture-based approaches. these experiments provided limited information as the vast majority of microbiota cannot be grown in culture media. more recent studies have used next-generation sequencing, including s rrna sequencing and metagenomic sequencing. the metagenome is the collective assembly of genomes from an environment (e.g., for gut) [ ]. this enables a more comprehensive and culture-free profiling of taxa from collected specimens [ , ]. shotgun metagenomic sequencing, in which the total dna of an ecosystem is sequenced, provides a broad and deep characterization of all types of microbiota. it provides coverage and resolution of the lowest taxonomic levels (e.g., species) and the potential for the functional annotation of genes and proteins [ ]. metabolomics represents one of the meta-omic approaches to study gut microbiota functions. metabolomics is the collective array of metabolites present in a biological sample [ ]. untargeted metabolomic profiling has emerged as a powerful technique to dissect altered pathways contributing to complex diseases. metabolomics uses high throughput techniques to characterize and quantify small molecules in several biofluids, such as feces, urine, plasma, serum and saliva. the use of metabolomics is considered a powerful top-down systems biology approach, and it is essential to reveal the genetic–environment–health relationship, as well as the clinical biomarkers of diseases such as atopic disorders [ ]. ultimately, these types of studies can identify beneficial bacteria related to a targeted disease based on their metabolomic “fingerprint”. these specific bacteria can be cultured and then introduced into the patient to treat disease. . . fecal microbiota transplantation (fmt) a new approach in microbial intervention is to attempt a more general modification of the gut microbiome using fecal microbiota transplantation (fmt). fmt is a type of bacteriotherapy (the purposeful use of bacteria or their products to treat illness), where stool (consisting of diverse microbe populations) from a healthy donor is transferred into the gi tract of a diseased individual with the goal of treating a particular disease. fmt may be performed by different routes, including colonoscopy, a nasogastric tube, or oral capsules [ , ]. pathogenic or opportunistic microbes are out-competed by the newly introduced bacteria to re-shape the gut microbiome to benefit the host [ ]. clinicaltrials.gov/ct /show/nct clinicaltrials.gov/ct /show/nct trialregister.nl/trial/ children , , of compared to probiotic treatments that contain a few bacterial species, fmt involves thousands of bacterial species native to the gi. fmt increases microbial diversity, restoring the gut microbiota community structure and diversity to the level of a healthy person [ ]. in humans, fmt for gut diseases has been well-studied for clostridium difficile infection (cdi) [ , ] and has also been applied to antibiotic-resistant gastrointestinal infections [ ]. fmt has been successfully used to treat patients with clostridium difficile colitis with communities from healthy donors transferred to those with the condition [ , ]. in ground-breaking studies in murine models, for identification of specific microbiota was elucidated to launch fmt clinical trials in humans with food allergies. studies showed that fmt using stools taken from healthy human infants and transplanted into a food-allergic mouse model protected them from anaphylaxis after allergen exposure, whereas anaphylaxis was not abrogated when fmt was performed with stools taken from food-allergic infants [ ]. other mouse studies, investigating clusters impacted by dysbiosis in infants, utilized a type of mouse, il raf , that is genetically prone to food allergy [ , , ]. using fmt bacteriotherapy containing commensal clostridiales strains with or without five bacteroidales strains both prevented and treated food allergy. in addition, bacteriotherapy led to the upregulation of ror gamma treg cells. there was also a decrease in total and ova-specific serum ige, il- production and gata expression by treg cells. protection from anaphylaxis was also noted when treating these mice with a single clostridial species, subdoligranulum variabilis, which was also impacted by the dysbiosis in food-allergic infants [ , ]. in a different food allergy mouse model, another species of clostridia known as anaerostipes caccae was protective against allergic reaction [ ]. the research advances in the microbiome field have moved food allergy therapy into the arena of microbiome-related human clinical trials. fmt is an intriguing gut microbiome manipulation strategy to consider given that findings from murine models support that fecal transfer is an effective mode of altering allergic outcomes [ ]. a small phase i open-label trial to evaluate the safety and tolerability of oral encapsulated fmt administered in an open-label manner over days for the treatment of peanut allergy in adult subjects ( – years) is underway (nih clinicaltrials.gov #nct ). the agent being tested is a screened-donor inoculum of frozen fecal material [ , ]. a study of the next-generation microbial transfer of rationally selected strains based on preclinical findings by atarashi et al. is in a phase i clinical trial for the treatment of peanut allergy (nih clinicaltrials.gov #nct ). ve is a combination of dormant bacteria given as a capsule to subjects pretreated with vancomycin to facilitate the colonization of the transferred bacteria. in this ongoing study, ve or placebo will be given with peanut oral immunotherapy [ ]. . conclusions in conclusion, for microbiome plays an important role in early immunological development, and it is stipulated that early infancy may represent a key window for interventions that aim to manipulate the microbiome for the benefit of the human host. microbial colonization begins shortly after birth, and multiple factors contribute to the development of normal flora. these factors include genetics, maternal–fetal interactions, for place and mode of delivery, infant feeding methods and the use of antibiotics. a negative family history of atopy, vaginal delivery, home birth, colostrum, breast milk (as a source of commensal bacteria) and the avoidance of antibiotic use in early life are all considered protective against food sensitization and allergy. by contrast, a positive family atopic history, caesarean delivery, hospital delivery, bottle-feeding and the use of antibiotics early in life are considered risk factors for atopic disease and food allergy development. certain environmental exposures, such as growing up in a farming environment, have also been shown to protect from the development of atopic disease. additionally, there is both direct and indirect scientific evidence for the role of the microbiome and their metabolic byproducts such as short chain fatty acids in food allergy development. this underscores the important role of the microbiota in shaping the intestinal immune phenotype and provides invaluable insight into developing novel treatment strategies to restore symbiosis. studies that have children , , of directly measured microbial diversity and composition in populations with and without food allergy have provided direct evidence that gut microbiota differ in individuals with food allergy. advances in immunotherapy have highlighted the immunologic influence of commensal microbiota on oral tolerance in recent years. as a result, there is potential for the application of microbiota and/or microbial-derived products in food allergy treatment. the routine use of probiotics as an intervention for preventing allergic disease, with the exception of eczema in high-risk infants, is not currently recommended. there are ongoing trials evaluating the benefits of probiotics and prebiotics, in addition to the optimal strains and dosages needed, as well as the duration of probiotic administration and how they may contribute to the prevention or treatment of atopic diseases. developing targeted bacterial therapies for food allergy may be promising for the treatment and prevention of food allergy. understanding the biology of the microbiome and how it interacts with the host to maintain gut homeostasis will be key to developing smarter therapeutic approaches. author contributions: a.a. conceived the manuscript theme and created the initial draft structure of the manuscript. all authors contributed to the writing of the manuscript. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. conflicts of interest: a. anagnostou has institutional research contract funding from aimmune therapeutics. s. anvari has institutional research contract funding from dbv technologies and aimmune therapeutics. the other authors declare no conflict of interest in relation to this work. abbreviations ahr aryl hydrocarbon receptor dbpc double-blind placebo controlled peanut challenge dc dendritic cells raldh retinal aldehyde dehydrogenase ehcf extensively hydrolyzed casein formula fmt fecal microbiota transplantation gpcr g-protein coupled receptors hdac histone deacetylases lgg lactobacillus gg pbmcs peripheral blood mononuclear cells pa polyamines poit peanut oral immunotherapy scfas short chain fatty acids t regs t regulatory cells th t helper th t helper th 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[crossref] [pubmed] © by the authors. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). http://dx.doi.org/ . /j.jaci. . . http://dx.doi.org/ . /md. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.jaci. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.jaci. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /j.jaci. . . http://www.ncbi.nlm.nih.gov/pubmed/ http://www.sciencedirect.com/science/article/pii/s http://www.sciencedirect.com/science/article/pii/s http://dx.doi.org/ . /s - ( ) -x http://dx.doi.org/ . /fimmu. . http://dx.doi.org/ . /j.iac. . . http://dx.doi.org/ . /j.gtc. . . http://dx.doi.org/ . /cpt. http://www.ncbi.nlm.nih.gov/pubmed/ http://dx.doi.org/ . /s - - -z http://www.ncbi.nlm.nih.gov/pubmed/ http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	methods 	the gut microbiome 	host–microbiome interactions 	early life microbiome 	the microbiome and atopic diseases 	effect of environmental exposures on the composition of the gut microbiome and atopic diseases 	infant microbiome 	atopic dermatitis and the microbiome 	asthma and the microbiome 	the gut microbiome and food allergy 	microbiome signatures in food sensitization and food allergy 	microbiome signatures in egg and cow’s milk allergies 	nutrition and the microbiome in food allergy 	dietary factors and the microbiome 	the “nutrition–gut microbiome–physiology axis” 	the microbiome and underlying mechanisms in food allergy 	scfa interaction with host epithelial and immune cells 	polyamines, ahr ligands and gut homeostasis 	microbiome and food allergy treatments in the horizon 	prebiotics and probiotics in food allergy 	prebiotics 	role of probiotics in preventing food sensitization 	role of probiotics in accelerating tolerance to foods 	probiotics in peanut allergy 	ongoing studies on synbiotics 	metagenomics and metabolomics 	fecal microbiota transplantation (fmt) 	conclusions 	references research article changes in renal function in elderly patients following intravenous iodinated contrast administration: a retrospective study ali alsafi, zaid alsafi, amish lakhani, and nicola h. strickland radiology department, imperial college healthcare nhs, hammersmith hospital, du cane road, london w hs, uk correspondence should be addressed to ali alsafi; ali.alsafi @alumni.imperial.ac.uk received january ; revised february ; accepted february ; published march academic editor: keiji tanimoto copyright © ali alsafi et al. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. background. contrast-induced nephropathy (cin) is a recognised complication of intravascular administration of iodinated contrast media (icm). previous studies suggest a higher incidence in the elderly, but no large study has assessed this to date. we set out to assess changes in creatinine in elderly inpatients following computed tomography (ct) examination and compare those who received intravenous contrast to those who did not. methods. using the radiology information system in two teaching hospitals, inpatients over the age of seventy who had a ct examination and a baseline creatinine were identified and their follow-up creatinine levels were analysed. elderly inpatients who underwent a noncontrast ct over the same period were used as controls. results. elderly inpatients who received icm were compared with controls. . % of patients who received icm developed acute kidney injury (aki) compared to . % of inpatient controls (𝑃 < . ). patients with higher baseline egfr had a higher incidence of post-ct aki. conclusions. the incidence of post-ct aki is higher in patients who received iv icm compared to those who did not; the difference may be partly attributable to contrast-induced nephropathy. this suggests that the incidence of cin in the elderly may not be as high as previously thought. . background intravascular administration of iodinated contrast media (icm) is used in a variety of diagnostic and therapeutic pro- cedures. while icm are generally safe, only resulting in minor side effects, they can result acute renal impairment known as contrast induced nephropathy (cin) [ ]. the majority of cases of cin are self-limiting, although occasion- ally chronic renal failure ensues necessitating long-term renal replacement. even when transient, cin is associated with increased length of hospital stay and significant morbidity [ , ]. diabetes mellitus, preexisting renal failure, congestive heart failure, and hypotension, dehydration as well as the use of ace inhibitors, diuretics, and nonsteroidal anti- inflammatory drugs have all been identified as important risk factors for developing cin [ , ]. hydration in high-risk patients remains the most important preventative measure [ , ]. many attempts at defining cin exist, but the most widely accepted definition is a % rise in serum creatinine concen- tration from baseline or an absolute increase by 𝜇mol/l over the to hours following icm administration, once other causes of renal impairment have been excluded [ , ]. the use of ct in clinical medicine has exponentially increased over the past two decades, including managing elderly patients, who are thought to be particularly suscep- tible to developing cin [ – ]. the true incidence of cin in the elderly is not known, as most studies addressing the issue assumed that acute kidney injury (aki) following icm administration is in fact cin [ – ]. the results of these studies are therefore likely to be an overestimate, as they failed to compare the incidence of aki in their patient population to that of a control group. aki itself is difficult to define, hindawi publishing corporation radiology research and practice volume , article id , pages http://dx.doi.org/ . / / radiology research and practice but a practical definition is that of a rapid deterioration in renal function resulting in the inability to maintain fluid, electrolyte, or acid-base homeostasis [ ]. cin has come recently under question as a clinical entity, with suggestions that its incidence and significance have been grossly overestimated. one study even questions the existence of cin, particularly in patients with preexisting normal renal function [ – ]. in this study, we sought to retrospectively evaluate changes in renal function in elderly patients who underwent a contrast enhanced ct examination and compare to controls who had a noncontrast ct. the aim was to estimate the incidence of post-ct aki in the elderly. . methods using the radiology information system (ris) in two teach- ing hospitals, inpatients over the age of seventy who had a contrast enhanced computed tomography (ct) examina- tion between august and august were identified. demographic and examination data were recorded. using the hospitals’ pathology results systems, the most recent creatinine (cr) values prior to the contrast examination were recorded. follow-up cr values – hours following the examination were also noted. the most recent cr prior to the ct examination (up to six weeks) was taken as the baseline against which changes in cr were compared. egfr was calculated using the modified diet in renal disease (mdrd) formula [ ]. information relating to prehydration, posthydration, and administration of nac was not readily available and not recorded, although our standard in-hospital practice is to pre- and posthydrate high-risk patients. we excluded patients who had no baseline creatinine, those who had no blood tests between and hours following the ct examination, and those on haemodialysis, peritoneal dialysis, or hemofiltration. patients who had a nephrostomy or renal stent placement within days of the study were also excluded. patients who had a contrast head ct receiving ml of omnipaque were excluded. this group constitutes a small number of patients with a signif- icantly lower contrast dose to the remainder of the study population. all elderly inpatients who underwent a noncontrast examination over the same period were used as controls with the same exclusion criteria. these patients were also identified using the ris system. those who developed aki of an order comparable to that seen in cin, ( % rise in serum crea- tinine concentration from baseline or an absolute increase by 𝜇mol/l) were identified. the incidence of aki was calculated for the icm and control group. institutional review board approval was obtained and informed consent was waived prior to commencing this retrospective study. . . statistical analysis. two-tailed fisher’s exact test was used to compare categorical data, while two-tailed student’s t-test was used to compare numerical data. 𝑃 values < . were considered significant. odds ratio was calculated using table : demographic data and baseline egfr (ml/min/ . m ). body ct (𝑛 = ) controls (𝑛 = ) 𝑃value average age (range) . ( – ) ( – ) < . ∗ males (%) ( . %) ( . %) . # females (%) ( . %) ( . %) baseline egfr> ( %) ( . %) . # ∗unpaired student’s 𝑡-test; #fisher’s exact test. table : number of patients developing post-ct aki in the icm and control groups with varying baseline egfr. (egfr in ml/min/ . m ). baseline egfr icm group (%) controls (%) > ( . %) (𝑛 = ) ( . %) (𝑛 = ) – ( . %) (𝑛 = ) ( . %) (𝑛 = ) – ( . %) (𝑛 = ) ( . %) (𝑛 = ) < ( . %) (𝑛 = ) ( . %) (𝑛 = ) total ( . %) (𝑛 = ) ( . %) (𝑛 = ) graphpad prism . for mac os x (graphpad software inc., san diego, ca, usa). . results and discussion inpatients over the age of had a contrast enhanced ct examination between august and , including body ct examinations and head ct examinations; the latter were excluded. a further patients were excluded because they were receiving renal replacement therapy and one patient was excluded because of a recent nephrostomy. patients had a baseline creatinine and at least one further creatinine measurement – hours following their ct. none of the patients had more than one contrast enhanced examination during the study period. over the same period, inpatients underwent a ct examination without contrast. of these patients were excluded: because they were receiving renal replacement, who had no baseline creatinine, one who had acute kidney injury predating the ct examination, and who did not have a creatinine level measured – hours following their ct examination. the remaining cases were used as controls. there was no significant difference in sex or baseline egfr between the two groups, although the control group was slightly older than the icm group. (demographics and baseline egfr are shown in tables and resp.) in the to hours following intravenous adminis- tration of – ml of omnipaque , . % ( / ) of patients with a baseline egfr > ml/min/ . m devel- oped aki compared with . % ( / ), . % ( / ), and . % ( / ) of those with egfr – ml/min/ . m , – ml/min/ . m , and < ml/min/ . m , respec- tively (figure ). the relative risk for developing post-ct aki in patients who had – ml of icm with a baseline egfr < ml/min/ . m compared to those with an radiology research and practice < – – > ## baseline egfr ml/min/ . m icm group po st -c t a k i ( % ) controls ∗ ∗∗ figure : percentage of patients developing post-ct aki in the icm group compared to controls. using fisher’s exact test: ∗𝑃 < . and ∗∗𝑃 < . , comparing the icm group versus controls and ##𝑃 < . comparing to patients with a baseline gfr > ml/min/ . m . egfr > ml/min/ . m was . ( % ci . to . , 𝑃= . ). the relative risk for developing post-ct aki in inpatients who had – ml of icm compared with controls is . ( % ci . to . ,𝑃< . ). intravenous contrast media (icm) are widely used in diagnostic and interventional radiology; their use, however, is associated with up to % of hospital-acquired aki [ ]. cin is a cause of significant morbidity and mortality in hospital patients and is thought to be an important cause of aki in this population alongside sepsis, hypoperfusion, major surgery, and nephrotoxic medication [ , – ]. increasing age is an independent predictor for developing cin with a fivefold increase in risk in patients over the age of seventy [ ]. previous studies, including macdonald et al’s recent large meta-analysis, showed no difference in incidence of post- ct aki, between contrast and control groups. these studies assessed significantly younger patient populations than ours (median patient age of versus in our study) and their results may not be generalizable to an older age group [ , , ]. davenport et al. on the other hand demonstrated that iv icm is an important risk factor, albeit not the only one, in developing post-ct aki in an adult patient population [ ]. our results extend these conclusions to a significantly older population, which may be even more susceptible to cin. two studies estimated the incidence of cin in the elderly at % and %, respectively. neither study accounted for other causes of aki nor did they compare with controls [ , ]. to our knowledge, this is the first study to compare the incidence of post-ct aki in elderly patients who received iv icm with controls who had nonenhanced examinations. while the excess aki in the icm group is, at least partly, likely attributable to contrast administration, there may be inherent differences between the two groups, with patients not receiving icm being perceived as high-risk and therefore undergoing noncontrast examinations. the fact that the average baseline egfr, age, and gender distribution are similar in the two groups does not support this. furthermore, a significant number of the noncontrast studies were head ct examinations ( %), suggesting that the disease processes for which the two groups presented to hospital may be different, with the icm group being more likely to have systemic, rather than neurological, symptoms and therefore more likely to develop aki. this is merely speculative but may partly account for the differences seen in conjunction with true cin. if this is the case, the incidence of cin may even be lower. . limitations although we did not assess for risk factors other than preexisting renal impairment, by using a control population with similar demographics who were inpatients during the same study period, the difference in incidence of aki may be partly attributable to the administration of iv icm. our results apply to an elderly hospital inpatient popu- lation and may not be generalizable to a healthier outpatient population, whose risk of cin is perhaps even lower. . conclusions the incidence of aki in our elderly inpatient population was . % after administration of – ml of intravenous icm compared with . % in inpatient controls. the difference between the two groups may be, at least partly, attributable to icm. caution, however, needs to be exercised in elderly patients with low baseline egfr (< ml/min/ . m ) as post-ct aki approaches % in this group. abbreviations cin: contrast induced nephropathy icm: iodinated contrast media ct: computed tomography aki: acute kidney injury egfr: estimated glomerular filtration rate cr: creatinine nac: n-acetylcysteine mdrd: modified diet in renal disease ris: the radiology information system. conflict of interests the authors declare that there is no conflict of interests regarding the publication of this paper. radiology research and practice references [ ] p. parfrey, “the clinical epidemiology of contrast-induced nephropathy,” cardiovascular and interventional radiology, vol. , supplement , pp. s –s , . 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[ ] r. j. bruce, a. djamali, k. shinki, s. j. michel, j. p. fine, and m. a. pozniak, “background fluctuation of kidney function versus contrast-induced nephrotoxicity,” the american journal of roentgenology, vol. , no. , pp. – , . submit your manuscripts at http://www.hindawi.com stem cells international hindawi publishing corporation http://www.hindawi.com volume hindawi publishing corporation http://www.hindawi.com volume mediators inflammation of hindawi publishing corporation http://www.hindawi.com volume behavioural neurology endocrinology international journal of hindawi publishing corporation http://www.hindawi.com volume hindawi publishing corporation http://www.hindawi.com volume disease markers hindawi publishing corporation http://www.hindawi.com volume biomed research international oncology journal of hindawi publishing corporation http://www.hindawi.com volume hindawi publishing corporation http://www.hindawi.com volume oxidative medicine and cellular longevity hindawi publishing corporation http://www.hindawi.com volume ppar research the scientific world journal hindawi publishing corporation http://www.hindawi.com volume immunology research hindawi publishing corporation http://www.hindawi.com volume journal of obesity journal of hindawi publishing corporation http://www.hindawi.com volume hindawi publishing corporation http://www.hindawi.com volume computational and mathematical methods in medicine ophthalmology journal of hindawi publishing corporation http://www.hindawi.com volume diabetes research journal of hindawi publishing corporation http://www.hindawi.com volume hindawi publishing corporation http://www.hindawi.com volume research and treatment aids hindawi publishing corporation http://www.hindawi.com volume gastroenterology research and practice hindawi publishing corporation http://www.hindawi.com volume parkinson’s disease evidence-based complementary and alternative medicine volume hindawi publishing corporation http://www.hindawi.com wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ - - - .fm boston university openbu http://open.bu.edu department of medicine med: medicine papers - - nicotinic acetylcholine receptor subunit variants are associated with blood pressure; findings in the old order amish and replication in the framingham heart study mcardle, patrick f, sue rutherford, braxton d mitchell, coleen m damcott, ying wang, vasan ramachandran, sandy ott, yen-pei c chang, daniel levy, nanette steinle. "nicotinic acetylcholine receptor subunit variants are associated with blood pressure; findings in the old order amish and replication in the framingham heart study" bmc medical genetics : . ( ) https://hdl.handle.net/ / boston university biomed centralbmc medical genetics ss open acceresearch article nicotinic acetylcholine receptor subunit variants are associated with blood pressure; findings in the old order amish and replication in the framingham heart study patrick f mcardle , sue rutherford , braxton d mitchell , coleen m damcott , ying wang , vasan ramachandran , sandy ott , yen- pei c chang , daniel levy and nanette steinle* address: department of medicine, university of maryland school of medicine, baltimore, md, usa, department of genetics, southwest foundation for biomedical research, san antonio tx, usa, division of cardiology, boston university school of medicine, boston ma, usa and national heart, lung, and blood institute, bethesda, md, usa email: patrick f mcardle - pmcardle@medicine.umaryland.edu; sue rutherford - srutherf@sfbrgenetics.org; braxton d mitchell - bmitchel@medicine.umaryland.edu; coleen m damcott - cdamcott@medicine.umaryland.edu; ying wang - ywang@medicine.umaryland.edu; vasan ramachandran - vasan@bu.edu; sandy ott - sott@som.umaryland.edu; yen- pei c chang - cchang@medicine.umaryland.edu; daniel levy - levyd@nhlbi.nih.gov; nanette steinle* - nsteinle@medicine.umaryland.edu * corresponding author abstract background: systemic blood pressure, influenced by both genetic and environmental factors, is regulated via sympathetic nerve activity. we assessed the role of genetic variation in three subunits of the neuromuscular nicotinic acetylcholine receptor positioned on chromosome q, a region showing replicated evidence of linkage to blood pressure. methods: we sequenced chrna , chrnd and chrng in amish subjects from the amish family diabetes study (afds) and identified variants. we then performed association analysis of non-redundant variants (n = ) in the complete afds cohort of , individuals, and followed by genotyping blood pressure-associated variants (n = ) in a replication sample of , individuals from the framingham heart study (fhs). results: the minor allele of a synonymous coding snp, rs in chrng, was associated with higher systolic blood pressure in both the amish (p = . ) and fhs populations (p = . ) (minor allele frequency = . in both populations). conclusion: chrng is currently thought to be expressed only during fetal development. these findings support the barker hypothesis, that fetal genotype and intra-uterine environment influence susceptibility to chronic diseases later in life. additional studies of this variant in other populations, as well as the effect of this variant on acetylcholine receptor expression and function, are needed to further elucidate its potential role in the regulation of blood pressure. this study suggests for the first time in humans, a possible role for genetic variation in the neuromuscular nicotinic acetylcholine receptor, particularly the gamma subunit, in systolic blood pressure regulation. published: july bmc medical genetics , : doi: . / - - - received: february accepted: july this article is available from: http://www.biomedcentral.com/ - / / © mcardle et al; licensee biomed central ltd. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ . ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. page of (page number not for citation purposes) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids= http://www.biomedcentral.com/ - / / http://creativecommons.org/licenses/by/ . http://www.biomedcentral.com/ http://www.biomedcentral.com/info/about/charter/ bmc medical genetics , : http://www.biomedcentral.com/ - / / background hypertension is one of the most important risk factors for cardiovascular disease, and end-stage renal disease. glo- bally hypertension is the leading risk factor for morbidity and mortality [ ], having multifactorial causal agents, including genetic and environmental components. we previously reported a genome wide scan of subjects from the amish family diabetes study that provided strong evi- dence for linkage to diastolic (lod = . ; p = . ) and systolic (lod = . ; p = . ) blood pressure on chromosome q -q [ ]. follow-up analyses with additional markers resulted in an increase in the lod score to . (p = . ) for diastolic blood pressure (figure ). initial fine mapping of the region with a densely spaced set of single nucleotide polymorphisms (snps) showed association between several snps in chrnd and systolic blood pressure (sbp). this region also contains chrng and chrna , which encode subu- nits of the same nicotinic acetylcholine receptor that form the neuromuscular nicotinic acetylcholine receptor. systemic blood pressure is tightly regulated through sym- pathetic nerve activity. feedback information about sys- temic blood pressure is conveyed to the brainstem by baroreceptor afferents. accumulating evidence suggests that perturbations in these neuronal mechanisms may play a role in hypertension [ ]. indeed, hypertensive indi- viduals tend to have increased sympathetic nerve activity [ , ] and a functional change in the sensitivity of the baroreceptor reflex [ ]. in the spontaneously hypertensive rat, abnormalities in cholinergic activity were shown to be involved in the development of hypertension [ ]. nico- tinic acetylcholine receptors play an important role in processes involving regulation of systemic blood pressure [ ], and may also play an important role in neural devel- opmental processes including cell differentiation, out- growth and survival [ ]. the nicotinic acetylcholine receptor has two main recep- tor subtypes, the neuronal (ganglionic) receptor found in the central nervous system and the neuromuscular recep- tor found in the neuromuscular junction of somatic mus- cle. the neuromuscular nicotinic acetylcholine receptor variance components linkage analysis of str markers spaced approximately every – cm across the blood pressure linkage interval on chromosome qfigure variance components linkage analysis of str markers spaced approximately every – cm across the blood pressure linkage interval on chromosome q. lod = . (p = . ) for dbp; lod = . (p = . ) for sbp. page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / consists of two alpha subunits, a beta, a delta and either a gamma subunit in fetal muscle or an epsilon subunit in adults [ - ] polymorphisms in the alpha and delta sub- units have been shown to confer increased risk of myasthenia gravis [ - ]. the gamma subunit of the nicotinic acetylcholine recep- tor is reported to be important for neuromuscular devel- opment [ ] and ligand binding [ ]. disruption of the gamma subunit, in vitro, prevents the correct localization of the receptor in cell membranes [ ]. the gamma subu- nit contributes to neuromuscular signal transduction and is also important for neuromuscular organogenesis [ , ]. rare mutations in chrng have been shown to cause escobar syndrome, a condition manifested by orthopedic and cranial anomalies [ , ]. to identify common genetic variants in the nicotinic acetylcholine receptor genes on chromosome q, each gene was sequenced in subjects enrolled in the amish family diabetes study (afds). variants were then geno- typed in the entire afds sample and tested for association with blood pressure levels. associated variants were geno- typed in an outbred caucasian population from the fram- ingham heart study as a replication sample. to assess whether associations relate to regulation by the postgan- glionic nicotinic acetylcholine receptor, we genotyped replicated variants in a second sample of amish and tested for association with response to a cold pressor stress test. we provide the first evidence that a common genetic var- iant in chrng is associated with systolic blood pressure. methods samples primary amish sample our principle sample consisted of old order amish (ooa) individuals from lancaster county, pa. the ooa are a genetically closed founder population originating from western europe and who immigrated to central pennsylvania in the early s [ ]. they are particularly well suited for genetic studies of complex diseases and traits because they maintain a relatively uniform lifestyle and tend not to use prescription medications. family members tend to eat meals together and the amish diet is largely similar across families. in addition, farming is still the primary occupation and most ooa maintain a life- style of high physical activity levels. the homogenous life- style of the amish reduces the variability of many environmental influences and thereby enhances the power to detect the genetic susceptibility to complex dis- eases. our primary amish sample consisted of , partici- pants of the afds. the afds began in with the goal of identifying susceptibility genes for type diabetes and related traits [ ]. probands were individuals identified with type diabetes and family members years of age and over were recruited into the study. the , individ- uals recruited into the study were members of pedi- grees. the study protocol was performed either in the individual's home or in the amish research clinic in strasburg, pennsylvania. multiple phenotypes, including blood pressure measurements, were obtained using a standardized protocol. after the subject rested for at least minutes, a single observer recorded systolic (first phase) and diastolic (fifth phase) blood pressure (sbp and dbp respectively) to the nearest mmhg in duplicate using a standard sphygmomanometer. the use of anti-hypertensive medication was < % among the afds subjects. we accounted for hypertension medication use in two ways. first we performed the anal- ysis using only those subjects who were not prescribed anti-hypertension medication. this approach has the pos- sibility of biasing the results because we are removing those with the highest (untreated) blood pressures from the analysis. since the use of anti-hypertension medica- tion is not as common in the amish as the general popu- lation, this bias is reduced. second, we adjusted for medication use by adding a constant to the observed blood pressure measurements, mmhg to sbp and mmhg to dbp. this approach was suggested by cui et. al. [ ] and later validated by tobin et. al. [ ]. framingham heart study sample we genotyped bp-associated variants in a replication sam- ple from an independent caucasian population from framingham, ma. the framingham heart study began in to investigate the causes of heart disease [ ]. men and women (n = , ) between the ages of and years were recruited and followed prospectively over time. beginning in , offspring of the original cohort were recruited as part of the framingham offspring study [ ]. an additional , individuals were recruited for this study, consisting of offspring and spouses of offspring of the original cohort. our sample consisted of , unre- lated individuals who were randomly selected from the framingham offspring study as per the protocol estab- lished by the collaboration between the national heart, lung, and blood institute and boston university. each participant underwent a physician administered exam where blood pressure measurements were taken and a dna sample was collected. data on blood pressure meas- urements were collected every four years for members of the offspring cohort. for the analysis in this report, we used data from visits which took place from february to september . the participants ranged in age from to years at the time of this visit. of the , randomly selected individuals, , have both a sbp and dbp measurement recorded. anti-hypertension medica- page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / tion use in the framingham cohort was more common ( . % in our sample) and was accounted for by the methods described above. amish sample – cold pressor test variants associated with blood pressure in both the initial amish sample and the framingham sample were geno- typed in a second sample of amish who were participants of the heredity and phenotype intervention (hapi) heart study. the hapi heart study was initiated to identify genes that interact with specific environmental exposures to influence cardiovascular risk factors [ ]. as part of this study, we administered a . -minute cold pressor test (cpt) to participants from extended, old order amish families from lancaster county, pennsylvania. of the participants of the cpt in the hapi heart study, were also participants of the afds. in the hapi heart study, we utilized repeated blood pressure measurements taken before, during, and after the cpt to derive physio- logically relevant response traits such as reactivity to and recovery from the cold pressor stimulus. individuals remained semi-reclined until their blood pressure was sta- ble. the right hand and wrist to the ulnar styloid was sub- merged in ice water for . minutes. blood pressure and heart rate were measured at , , , , , , . , , , and minutes by use of an appropriate fitting automated blood pressure cuff. cpt reactivity was defined as the change in blood pressure levels from baseline to min- utes after immersion. cpt recovery was defined as the change in blood pressure from minutes to minutes after immersion. the change in blood pressure (slope) was calculated as sum((bp-mean(bp))(t-mean(t))/ sum((bp-mean(bp)) ) for all non-missing blood pressure measurements. all subjects were withdrawn from their medications (only subjects were on anti-hypertensive medications) for days prior to testing. the amish studies and fhs were approved by respective institutional review boards, and conform to the declara- tion of helsinki. all subjects provided informed consent. dna sequencing and genotyping genetic variants in chrna , chrnd and chrng were identified through direct sequencing of non-first degree relatives from the afds. this sequencing set pro- vides % power to detect at least one copy of the minor allele for single nucleotide polymorphisms (snps) with allele frequencies of greater than or equal to %. we sequenced all exons, splice junctions (~ bp of the intronic flanking sequence), and untranslated regions. both strands were sequenced on an abi dna sequencer and analyzed using sequencher v . (gene codes corporation, ann arbor, mi). initial sequencing identified variants, of which were not annotated with rs numbers in dbsnp and were subsequently not con- firmed. (sequence and primer information is available at http://medschool.umaryland.edu/endocrinology/afd spublic.asp. follow the links "publication list" and "sup- plemental data.") three snps (rs , rs , rs ) were in perfect linkage disequilibrium in the sequencing set of individuals with three other snps identified (rs , rs , rs respec- tively) and therefore represented redundant information. only the later set of snps were genotyped in the complete sample. one snp (rs ) failed our quality control procedures. thus genotypes for snps were available for association testing in the complete afds sample. geno- typing was carried out using the manufacturer's protocol on the snpstream ultra high throughput platform (beck- man coulter, fullerton, ca) and the pyrosequencing psq hs a system (pyrosquencing, uppsala, sweden). the genotype error rates based on blind replicates were – %. all genotype frequencies conformed to hardy-wein- berg expectations with the exception of rs (p = . ). statistical methods analysis performed on the genotypes for the afds and hapi heart study utilized a variance component frame- work, which modeled the genotype as a fixed effect and controlled for sex, age and age squared. each variant's additive, dominant and recessive effects were estimated using separate regression models. the variance compo- nent approach models the correlations between the trait of interest (the dependent variable) and dummy variables coding the genotype effect (independent variable) condi- tional on fixed covariates and the residual correlations among individuals implied by the pedigree structure. spe- cifically, the covariance between each pair of individuals within the pedigree was estimated as a function of their degree of relationship, the trait heritability, and the phe- notypic variance of the trait. the analysis was performed using the solar software package [ ]. simple linear regression was used to test for the associa- tion between genotype and blood pressure in the fram- ingham sample. three genetic models (additive, dominant and recessive) were tested regardless of which model was identified in the ooa. an alpha level of < . was considered as strong evidence of association. results sample characteristics characteristics of the three samples are shown in table . the average age of the primary sample, the afds, was . years, while the mean age of the framingham partic- ipants was . years. the use of anti-hypertensive medi- cation in the ooa sample was rare ( . %) as compared to page of (page number not for citation purposes) http://medschool.umaryland.edu/endocrinology/afdspublic.asp http://medschool.umaryland.edu/endocrinology/afdspublic.asp bmc medical genetics , : http://www.biomedcentral.com/ - / / the population based sample from fhs ( . %). the afds sample contained ( . %) individuals with diabetes. the participants in the hapi heart study tended to be younger (mean age = . years) and were relatively more healthy (lower bmi and resting blood pressure) than either the afds or fhs. linkage disequilibrium block structure figure shows the linkage disequilibrium (ld) plots for variants genotyped in the complete afds sample. there is little ld between the four snps in chrna . chrng snp rs is correlated with rs (r = . ) and in strong ld with chrnd snp rs (r = . ). snps rs and rs span the inter-genetic region between chrnd and chrng. there is also significant long range correlation between chrnd snp rs and chrng snp rs (r = . ), which are sepa- rated by . kb. association with blood pressure table shows the results from the association analysis to blood pressure adjusted for age, sex and familial related- ness in the afds sample. the values given are p-values for the association analysis after removing those on anti- hypertensive medication from the analysis. very similar results were found when accounting for medication in the analysis as described in the methods (data not shown). evidence of association with blood pressure was present for several variants including variants whose most sig- nificant model had strong evidence for association (p ≤ . ) with either sbp or dbp (table ). as shown in fig- ure , these associations may not be independent signals as some of the associated snps are in linkage disequilib- rium. five bp-associated variants (rs , rs , rs , rs , and rs ) were genotyped in the framingham sample and association results are shown in table . the association with rs was the only snp showing significant association with blood pres- sure in the framingham sample. this snp is a c -> t (arg -> arg ) synonymous coding polymorphism in the last exon of chrng. although not statistically significant, the rs snp was modestly associated with baseline sbp in the hapi heart study (p = . ). however, the allele associated with higher mean sbp levels in afds and fhs was also associated with higher mean sbp levels in hapi. after tak- ing into account individuals that were in both the afds and hapi cohorts, we combined the amish samples and found rs remained strongly associated with sbp (p = . ). there was no association between rs and any of the response traits (change in blood pressure during the reactivity time period, change in blood pres- sure during recovery time period) to the cold pressor test. figure shows the distribution of blood pressure by rs genotypes in the afds, fhs and the hapi heart study samples. in both the ooa and fhs popula- tions, the individuals with the tt genotype at snp rs have the highest age and sex adjusted residual sbp. the tt genotype is associated with a – mmhg increase in blood pressure over the age and sex predicted values. this effect of – mmhg remained in each sam- ple after adjusting for bmi which provides evidence that rs is acting in pathways not mediated by body mass. discussion evidence from animal models supports the role of the nic- otinic acetylcholine receptor in blood pressure regulation in the central nervous system. the potential role in blood pressure regulation of acetylcholine receptors at the neu- ral muscular junction remains to be elucidated. it has been shown that spontaneously hypertensive rats, have a lower number of nicotinic acetylcholine receptors in spi- nal cord membranes than age-matched normotensive rats [ ] furthermore, studies have shown rats with varying blood pressure levels maintain relatively constant num- table : sample characteristics for the afds, fhs, and the hapi heart study. afds fhs hapi n sex (% male) . % . % . % mean (sd) age (yrs) . ± . . ± . . ± . mean (sd) bmi (kg/m ) . ± . . ± . . ± . hypertension medication use (%) . % . % * mean (sd) sbp/dbp (mmhg) . ± . . ± . . ± . those on medication removed . ± . . ± . . ± . mean (sd) sbp/dbp (mmhg) . ± . . ± . * constant added to those on medication . ± . . ± . * anti-hypertension medications were withdrawn days prior to testing and therefore no hapi subjects were on medications at the time of study. page of (page number not for citation purposes) bmc medical genetics , : http://www.biomedcentral.com/ - / / page of (page number not for citation purposes) linkage disequilibrium block structure for chrna , chrnd and chrng in the old order amishfigure linkage disequilibrium block structure for chrna , chrnd and chrng in the old order amish. values given are r-squared times . table : association analysis of polymorphisms in chrna , chrnd and chrng with blood pressure in the afds. marker polymorphism minor allele frequency mean sbp/dbp by genotype (mmhg) p-value homozygous for major allele heterozygous homozygous for minor allele sbp dbp chrna rs intron . / / / . . rs exon : his his . / / / . . rs intron . / / / . . rs intron . / / / . . chrnd rs intron . / / / . . rs intron . / / / . . rs exon utr . / / / . . chrng rs intron . / / / . . rs intron . / / / . . rs intron . / / / . . rs intron . / / / . . rs exon : arg arg . / / / . . values given are p-values from the most significant genetic model (additive, dominant, recessive), controlling for age and sex. results are given after removing those on antihypertensive medication from the analysis. bmc medical genetics , : http://www.biomedcentral.com/ - / / bers of nicotinic receptors. this indicates that blood pres- sure does not drive receptor number but rather that the lower receptor number might be a primary event in hyper- tension [ ]. we investigated the association between blood pressure regulation and genetic polymorphisms in genes encoding three subunits of the neuromuscular nicotinic acetylcho- line receptor located on chromosome q. we demon- strated significant association with dbp and genetic variation in chrna and between sbp and several poly- morphisms in chrnd and crhng in ooa subjects from the afds. however, blood pressure-associated vari- ants in chrna and chrnd were not replicated in a sample from the framingham heart study, perhaps indi- cating that these associations were false positive signals or, less likely, that these variants are only relevant to blood pressure determinants in the amish. by contrast, we did demonstrate replication of the association of the chrng snp rs with sbp. the replicated rs is a c- > t synonymous coding snp in the last exon of the gamma subunit of the nicotinic acetylcholine receptor. in our caucasian populations, the tt genotype at this locus was associated with – mmhg higher sbp than age and sex predicted values. a trend with a similar effect size for association between sbp and rs was detected in an independent ooa cohort from the hapi heart study. although not of statistical significance in the hapi cohort, the fact that the subjects are on average younger and healthier than subjects in the afds may influence the association result for rs in this cohort. to place our findings in the context of potential impact on general health, we estimated the change in year risk of myocar- dial infarction or death using the framingham risk score calculator available online through the national heart, lung, and blood institute http://hp .nhlbihin.net/ atpiii/calculator.asp. for a man age years or older, or a woman age or older who has normal cholesterol and does not use tobacco and is not currently taking medica- tion to lower blood pressure, each incremental rise in sbp of mm hg would increase the year framingham risk of myocardial infarction or death by approximately %. although the polymorphism we identified is synony- mous, it may influence expression by altering pre mrna splicing, mrna stability, or efficiency of translation [ ]. alternatively, rs may not be causative but rather may be in linkage disequilibrium with a causative, as yet, ungenotyped polymorphism. while this is possible, it is unlikely that such a polymorphism exists in a coding region since all exons were sequenced. further, it is possi- ble that the association identified is a false positive. one reason for false positives is population stratification and is unlikely to be a significant concern in this study because we are using relatively homogenous caucasian popula- tions, particularly the amish, whose ancestry since emi- gration to the new world is well characterized. the replication of our findings for rs in two distinct populations is further evidence against a false positive finding. hales and barker posed the notion that fetal genotype and intra-uterine environment influence susceptibility to chronic diseases later in life [ ]. our findings are consist- ent with this hypothesis, demonstrating association between genetic variation in a gene expressed during embryogenesis and adult hypertension. additional work will be necessary to discern how this receptor subunit, cur- rently understood to be expressed during fetal develop- ment, impacts blood pressure in adulthood. it is also possible that this receptor may be expressed during adult- hood at levels that are below current detection thresholds, or expressed transiently during periods of metabolic stress. neoteric models to test these hypotheses will need to be developed to further our understanding of the role of chrng in blood pressure regulation. conclusion our study suggests for the first time in humans, a possible role for genetic variation in the neuromuscular nicotinic acetylcholine receptor, particularly the gamma subunit, in table : replication of association in a sample of caucasians enrolled in the fhs. marker minor allele frequency mean sbp/dbp by genotype (mmhg) p-value homozygous for major allele heterozygous homozygous for minor allele sbp dbp rs . / / / . . rs . / / / . . rs . / / / . . rs . / / / . . rs . / / / . . * values given are p-values from the most significant genetic model (additive, dominant, recessive), controlling for age and sex. results are given after removing those on antihypertensive medication from the analysis. page of (page number not for citation purposes) http://hp .nhlbihin.net/atpiii/calculator.asp http://hp .nhlbihin.net/atpiii/calculator.asp bmc medical genetics , : http://www.biomedcentral.com/ - / / page of (page number not for citation purposes) mean of age and sex adjusted residual blood pressure by rs genotype for the afigure mean of age and sex adjusted residual blood pressure by rs genotype for the a. amish family diabetes study; b. the framingham heart study; and c. the heredity and phenotype intervention (hapi) heart study. sbp residual in solid bars, dbp residual in hatched bars. standard error is represented by vertical lines. a. amish family diabetes study - - tt tc cc r e s id u a l b lo o d p re s s u re n= n= n= sbp p = . dbp p = . b. framingham heart study - - tt tc cc r e s id u a l b lo o d p re s s u re n= n= n= sbp p = . dbp p = . c. hapi heart study - - tt tc cc r e s id u a l b lo o d p re s s u re n= n= n= sbp p = . dbp p = . bmc medical genetics , : http://www.biomedcentral.com/ - / / sbp regulation. additional analyses of these polymor- phisms in other ethnic populations, as well as studies of the effect of genetic polymorphisms on expression and function of this nicotinic acetylcholine receptor are needed to further elucidate its potential role in the regula- tion of blood pressure. competing interests the authors declare that they have no competing interests. authors' contributions pfm, sr, yw, cmd and so carried out the molecular genetic studies, and participated in the sequencing. pfm drafted the manuscript. y–pcc and bdm participated in the design of the study. pfm and bdm performed the sta- tistical analysis. ns conceived of the study, and partici- pated in its design and coordination and provided significant contributions toward writing the manuscript. dl and vr led the studies in the framingham cohort and provided guidance in the approach to the analysis in the framingham cohort. all authors read and approved the final manuscript. acknowledgements this work was supported by research grants r hl , r dk , u hl , the university of maryland general clinical research center, grant m rr , the johns hopkins university general clin- ical research center, grant m rr , general clinical research centers program, national center for research resources (ncrr), and the maryland clinical nutrition research unit (p dk ), nih; the american heart association; and the baltimore veterans administration geriatric research and education clinical center (grecc). the framing- ham heart study is supported by contract nih n -hc- . we gratefully acknowledge alan shuldiner who initiated and maintains the amish family diabetes study and the hapi studies. we are also grateful to our amish liaisons and field workers and the extraordinary cooperation and support of the amish community without which these studies would not have been possible. we acknowledge and are grateful for the contribu- tions of the framingham heart study participants and investigators as well. references . kearney pm, whelton m, reynolds k, muntner p, whelton pk, he j: global burden of hypertension: analysis of worldwide data. 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available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in pubmed and archived on pubmed central yours — you keep the copyright submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp biomedcentral r, herzog w, weir mr, peyser pa, shuldiner ar: the genetic response to short-term interventions affecting cardiovascu- lar function: rationale and design of the heredity and pheno- type intervention (hapi) heart study. am heart j , : - . . almasy l, blangero j: multipoint quantitative-trait linkage anal- ysis in general pedigrees. am j hum genet , : - . . khan im, printz mp, yaksh tl, taylor p: augmented responses to intrathecal nicotinic agonists in spontaneous hypertension. hypertension , : - . . khan im, youngblood kl, printz mp, yaksh tl, taylor p: spinal nic- otinic receptor expression in spontaneously hypertensive rats. hypertension , : - . . parmley jl, chamary jv, hurst ld: evidence for 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http://www.biomedcentral.com/ 	abstract 	background 	methods 	results 	conclusion 	background 	methods 	samples 	primary amish sample 	framingham heart study sample 	amish sample - cold pressor test 	dna sequencing and genotyping 	statistical methods 	results 	sample characteristics 	linkage disequilibrium block structure 	association with blood pressure 	discussion 	conclusion 	competing interests 	authors' contributions 	acknowledgements 	references 	pre-publication history gender differences in first and secondhand smoke exposure, spirometric lung function and cardiometabolic health in the old order amish: a novel population without female smoking research article gender differences in first and secondhand smoke exposure, spirometric lung function and cardiometabolic health in the old order amish: a novel population without female smoking robert m. reed *, mark t. dransfield , michael eberlein , michael miller , , , giora netzer , , mary pavlovich , , toni i. pollin , , steven m. scharf , alan r. shuldiner , , , don sin , braxton d. mitchell , , university of maryland school of medicine, division of pulmonary and critical care medicine, baltimore, maryland, united states of america, university of alabama school of medicine, division of pulmonary and critical care medicine, birmingham, alabama, united states of america, university of iowa school of medicine, division of pulmonary and critical care medicine, iowa city, iowa, united states of america, university of maryland school of medicine, division of endocrinology, diabetes, and nutrition, baltimore, maryland, united states of america, department of veterans affairs and veterans affairs medical center baltimore geriatric research education and clinical center (grecc), baltimore, maryland, united states of america, university of maryland school of medicine, department of epidemiology and public health, baltimore, maryland, united states of america, university of british columbia respiratory medicine; vancouver, british columbia, canada * rreed@medicine.umaryland.edu abstract due to their relatively homogeneous lifestyle and living environment, the amish offer a novel opportunity to study the health associations of tobacco smoke exposure, particularly sec- ondhand smoke. we hypothesized that secondhand smoke exposure is associated with worse pulmonary and cardiometabolic health. we examined cross-sectional data on amish study participants, including tobacco use and secondhand smoke exposure from family members included in the study. thirty-four percent of amish men reported ever smok- ing. of this proportion, % used cigars, % cigarettes, and % pipes. less than % of women reported ever smoking. smoking was associated with lower spirometric lung func- tion, higher body mass index, lower hdl cholesterol, higher heart rate, lower ankle-brachial index, and larger aortic diameter in men. a greater number of sources of secondhand smoke exposure (defined from the total of spouses, parents, and siblings who smoke) was associated with higher body mass index (p = . ) and with higher fasting glucose in men (p = . ), but not in women (p = . for sex*secondhand smoke interaction). second- hand smoke exposure was also associated with reduced hdl cholesterol only in women (p = . ) and a lower heart rate only in men (p = . ). smoking habits among the old order amish are notable for the absence of female participation and a high proportion of cigar and pipe use. smoking is associated with decreased spirometric indices of lung func- tion and increased cardiovascular risk in this population and secondhand smoke exposure is associated with a greater burden of risk factors for cardiovascular disease. sex plos one | https://doi.org/ . /journal.pone. march , / a a a a a open access citation: reed rm, dransfield mt, eberlein m, miller m, netzer g, pavlovich m, et al. ( ) gender differences in first and secondhand smoke exposure, spirometric lung function and cardiometabolic health in the old order amish: a novel population without female smoking. plos one ( ): e . https://doi.org/ . / journal.pone. editor: olga y. gorlova, dartmouth college geisel school of medicine, united states received: august , accepted: march , published: march , copyright: this is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. the work is made available under the creative commons cc public domain dedication. data availability statement: phenotypic and genotypic data on the study participants are to be shared through dbgap in accordance with nih data sharing policies, but the specific database used in this study include direct integration with the amish pedigree. as such, individuals in the study could be directly identified within the dataset, and the consent forms signed do not permit such sharing. given the unique ancestral relationships among amish participants and publicly available https://doi.org/ . /journal.pone. http://crossmark.crossref.org/dialog/?doi= . /journal.pone. &domain=pdf&date_stamp= - - http://crossmark.crossref.org/dialog/?doi= . /journal.pone. &domain=pdf&date_stamp= - - http://crossmark.crossref.org/dialog/?doi= . /journal.pone. &domain=pdf&date_stamp= - - http://crossmark.crossref.org/dialog/?doi= . /journal.pone. &domain=pdf&date_stamp= - - http://crossmark.crossref.org/dialog/?doi= . /journal.pone. &domain=pdf&date_stamp= - - http://crossmark.crossref.org/dialog/?doi= . /journal.pone. &domain=pdf&date_stamp= - - https://doi.org/ . /journal.pone. https://doi.org/ . /journal.pone. https://creativecommons.org/publicdomain/zero/ . / https://creativecommons.org/publicdomain/zero/ . / differences in correlations could reflect differences in exposure patterns, mechanisms, or susceptibilities. introduction tobacco smoking is the leading preventable cause of death in the united states, responsible for an estimated , deaths annually, including , deaths attributable to lung cancer and coronary heart disease related to secondhand smoke exposure[ ]. secondhand smoke consists of main-stream smoke (the smoke that is inhaled and then exhaled by the smoker) and side-stream smoke (smoke that wafts directly from the burning tobacco). secondhand smoke exposure has been associated with increased risks of copd[ ], lung cancer[ ], stroke [ ], as well as various fetal and pediatric health issues including low birth weight and infant death[ , ]. while uncertainty remains, emerging data suggest secondhand smoke may also contribute to diabetes[ ], obesity[ ], and hypertension[ , ]. epidemiologic issues complicate correlative studies of the effects of tobacco smoke expo- sure and make the amish a population well-suited for such investigation. socioeconomic sta- tus and educational levels are associated with both smoking behavior[ ] and cardiovascular risk[ ], but within the amish these and other lifestyle factors are relatively homogeneous[ ]. another key benefit to studying secondhand smoke in the amish is that smoking is virtually nonexistent among amish women[ , ]. in populations including female smokers, it is diffi- cult to disentangle the effects of secondhand smoke from the exposures uniquely associated with maternal smoking such as intrauterine fetal exposure from a mother who smokes, high- intensity side-stream smoke exposure during infancy, and exposure through breastmilk[ ], which are exposures absent in the amish. cigars and pipes represent the predominant forms of tobacco smoked by the amish[ ] and these create a greater degree of side-stream smoke than do cigarettes[ ], thus presenting potential for a significant burden of secondhand tobacco exposure on the family members of those who smoke. as such, the amish represent a useful population in which to examine the associations of secondhand smoke exposure. for these reasons, we examined the health characteristics associated with smoking patterns in a cross sectional sample of the old order amish of lancaster county to consider the hypoth- esis that smoke exposure in this novel population would be associated with pulmonary, cardiac, and metabolic derangements. materials and methods data source this report is based on data obtained in three community surveys of cardiovascular health in the amish between and [ , ]. all studies were conducted with participant informed consent and with approval from the institutional review board of the university of maryland. inclusion criteria and definitions enrollment in each survey was limited to adults (age � years). the majority (n = ) par- ticipated in a wellness screening program offered to the community beginning in and open to all amish adults. an additional were recruited into a study that limited enrollment tobacco smoke in the amish plos one | https://doi.org/ . /journal.pone. march , / genealogical records (i.e. anabaptist genealogical data base), limited data may be provided in order to protect participant confidentiality. de-identified data is available upon request to melanie daue (mdaue@som.umaryland.edu). funding: robert m. reed is supported in part by the flight attendants medical research institute. michael eberlein is supported by a pilot grant from the institute for clinical and translational science (icts) at the university of iowa via the national institutes of health (nih) clinical and translational science award (ctsa) program, grant ul tr - . dr. mitchell is funded in part by the department of veterans affairs and veterans affairs medical center baltimore geriatric research, education and clinical center. this work was made possible by funding from: r hl - , u gm - , nih k hd - , r hl , u hl and the university of maryland general clinical research center (grant m rr ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. competing interests: the authors have declared that no competing interests exist. https://doi.org/ . /journal.pone. mailto:mdaue@som.umaryland.edu to age � years and excluded pregnant women[ ]. the remaining participated in a study of longevity conducted between – that enrolled participants � years of age, as well as their offspring and the spouses of their offspring[ ]. amish who participated in multiple studies were included only once in these analyses, preferentially using data from the wellness screening program. diabetes was defined by self-report, use of a diabetic medication, or fasting glucose � . mmol/, and obesity was defined by a body mass index � . smoking history was ascertained through self-report using questionnaires administered by research coordinators. “ever smoking” was defined by either reporting any cigarette, pipe, or cigar use, or by answering affirmatively to having ever smoked regularly. all secondhand smoking analyses were limited to participants who had never smoked. family and spousal smoking was characterized based on the self-reported smoking status of enrolled family mem- bers. we examined secondhand smoke exposure in both dichotomous (exposed/not exposed) terms as well as in ordinal terms according to a score relating to the number of family mem- bers reporting a history of smoking. for dichotomous analyses a study participant was consid- ered exposed to secondhand smoke if either the participant’s father, husband, or brother reported himself to be a current or an ex-smoker. to quantify the number of sources of sec- ondhand smoke, we counted the number of smokers in the nuclear family, assigning point if the participant’s father smoked, point if the participant’s husband smoked, and point if one or more of the participant’s brothers smoked. we then generated a secondhand smoking expo- sure category score ranging from to , based on the sum of these points. for this analysis, the secondhand smoke exposure category was set to missing if the smoking status of either the father or the husband of the participant was unknown.the number of parental sources of sec- ondhand smoke exposure has previously been shown to correlate with cardiovascular mea- sures[ ]. assessments blood samples were taken while fasting and processed in clia (clinical laboratory improve- ment amendments) certified centers. spirometry was performed in accordance with ats stan- dards[ ] without bronchodilator, and interpreted in terms of reference values for a caucasian cohort [ ]. vascular health was assessed using ankle brachial index (abi) and aortic ultra- sound measures on fasting individuals over age . mean abi was obtained by averaging mea- sures from the left and right side. aortic ultrasound measures were performed using a c - probe with a vivid e, ge laptop ultrasound device. longitudinal and transverse images were recorded at a minimum of locations along the descending aorta, and the maximum outer-to- outer measure was identified. statistical analysis data are presented as counts and percentages or as medians with interquartile range (iqr). in order to control for non-independence of observations due to family clustering effects, we per- formed regression analyses using the linear mixed model software as implemented in mmap (mixed models analysis for pedigrees), which accounts for relatedness of individuals (family structure) through inclusion of relationship matrix as a random effect[ ]. we also used mmap to examine interaction factors for sex in linear models to complement sex-stratified analyses. all other statistical analyses were performed using stata sc software (stata- corp-lp, college station, tx). despite examination of multiple outcome variables a bonfer- roni correction was considered unnecessary based on suggested criteria for use [ ]. for all analyses, two-tailed p< . was considered significant. tobacco smoke in the amish plos one | https://doi.org/ . /journal.pone. march , / https://doi.org/ . /journal.pone. results the study group included adult amish individuals, of whom % were men. four per- cent of the group were diabetic and % were obese. a history of having ever smoked was reported by ( %) men, and (< %) women. of men who reported having ever smoked, % also reported current smoking. among smokers, cigar smoking was most common ( %), followed by cigarette ( %), and then pipe ( %). median cigarette pack-years smoked (one pack a day for a year) was . (iqr . to . ), cigar-years ( cigar a day for a year) . (iqr . to ), and pipe-years (one pipe bowl a day for a year) . (iqr . to . ). heavy cigarette smoking was uncommon, with % of smokers reporting > pack-years of lifetime cigarette smoking. using previously proposed cigarette-per day equivalency estimates based on nicotine content for pipes and the small cigars most commonly smoked among the lancaster amish[ ], a > pack-year “equivalent” was reported by % of cigar smokers and % of pipe smokers. firsthand tobacco exposure in men a history of having ever smoked was associated with older age (median vs. years, p< . ), and higher bmi ( vs. , p< . ) (table ). smoking was associated with lower fev /fvc%predicted ( vs. %, p< . ), and this difference was independent of bmi. the association between higher bmi and smoking persisted after controlling for age differences, and appeared to be a result of both higher weights (p< . ) as well as shorter table . health related correlates of smoking in old order amish from lancaster county never smoking men (n = ) ever smoking men (n = ) p never smoking women (n = ) age at evaluation (yrs) ( to ) ( to ) < . ( to ) bmi (kg/m ) ( to ) ( to ) < . * ( to ) height (cm) ( to ) ( to ) . * ( to ) weight (kg) . ( . to . ) . ( . to . ) < . * . ( . to . ) waist to hip ratio . ( . to . ) . ( . to . ) . * . ( . to . ) fev (% predicted) ( to ) ( to ) . † ( to ) fvc (% predicted) ( to ) ( to ) . † ( to ) fev /fvc (%predicted) ( to ) ( to ) . † ( to ) ldl-c (mmol/l) . ( . to . ) . ( . to . ) . ‡ . ( . to . ) hdl-c (mmol/l) . ( . to . ) . ( . to . ) . ‡ . ( . to . ) tg (mmol/l) . ( . to . ) . ( . to . ) . ‡ . ( . to . ) heart rate by ekg ( to ) ( to ) < . ‡ ( to ) fasting glucose (mmol/l) . ( . to . ) . ( . to . ) . ‡ . ( . to . ) sbp (mmhg) ( to ) ( to ) . ‡ ( to ) mean abi (n = ) . ( . to . ) . ( . to . ) . ‡ . ( . to . ) aorta (cm) (n = ) . ( . to . ) . ( . to . ) . ‡ . ( . to . ) data are expressed as medians (iqr) or as percentages. unadjusted p values were obtained using wilcoxon rank sum. adjusted p values were obtained using linear regression and were limited to men. *adjusted for family structure and age. †adjusted for family structure. ‡adjusted for family structure, age, and bmi. abi: ankle-brachial index; bmi: body mass index; dbp: diastolic blood pressure; ekg: electrocardiogram; fev : forced expiratory volume in second; fvc: forced vital capacity; ldl-c: low density lipoprotein cholesterol; hdl-c: high density lipoprotein cholesterol; sbp, systolic blood pressure; tg; triglycerides. https://doi.org/ . /journal.pone. .t tobacco smoke in the amish plos one | https://doi.org/ . /journal.pone. march , / https://doi.org/ . /journal.pone. .t https://doi.org/ . /journal.pone. statures (p = . ). after controlling for age and bmi, smoking was associated with lower high density lipoprotein cholesterol (hdl-c) levels (p< . ), higher triglyceride levels, higher resting heart, lower mean ankle-brachial index, and higher aortic size. secondhand tobacco exposure: dichotomous approach evaluation of secondhand smoke exposure was evaluated in dichotomous terms separately in never-smoking men and women (data not shown). differences in spirometric lung function associated with secondhand smoke were not appreciable in men, but were detected in women. the magnitude of these differences was small, with a mean difference (assessed by student’s t- test) in fev % of . % (p = . ) and fvc% of . % (p = . ). these differences persisted after adjustment for bmi. analyses adjusted for age and bmi also showed secondhand smoke to be associated with significantly lower hdl-c in women (p = . ), but not men (p = . ). secondhand tobacco exposure: ordinal approach characteristics of nonsmokers according to number of secondhand smoking sources are pre- sented in table . forty percent of women and % of never-smoking men had no family member identified as a source of smoke exposure. potential family sources of exposure in women was primarily from fathers ( %), followed by siblings ( %) and spouses ( %). of never-smoking men, % had fathers with a smoking history and % had siblings with a smoking history. no men had more than two sources because none of their spouses smoked. the patterns of exposure in this subgroup were representative of the exposures in the entire group. in women, secondhand smoke exposure was associated with older age (p < . ) and lower hdl cholesterol (age-adjusted p = . ). in men, secondhand smoke exposure was associated with older age (p < . ), higher heart rate (age-adjusted p = . ), and higher fasting glucose (age-adjusted p = . ). table shows the association between secondhand smoking score and cardiovascular risk factors in the combined set of men and women. with adjustment for age and sex, secondhand smoking score was significantly associated with higher bmi (p = . ). after adjusting for age, sex, and bmi, secondhand smoking score remained significantly associated with lower hdl-c (p = . ). secondhand smoking score was also significantly associated with higher fasting glucose levels in men (age and bmi-adjusted p = . ), but not women (sex�second- hand smoking score interaction p = . ). no association was detected between secondhand smoke exposure and abi or aortic size in either sex-stratified (data not presented) or com- bined set. these vascular measures were made only in participants � years of age, and analy- ses were limited by the relatively small sample sizes. discussion we found that both smoking and secondhand tobacco exposures in a novel amish population were associated with cardiovascular risk factors. our data provide evidence supporting an association between secondhand smoke exposure and higher glucose levels, higher bmi, lower hdl-c, lower heart rates, and lower spirometric measures of lung function. these associa- tions demonstrated notable gender differences. first hand smoke exposure the amish study group presented here included only male smokers with a relatively high fre- quency of cigar ( %) and pipe ( %) use. for context, smoking preferences in the general us population favor cigarettes ( % of smokers) with far fewer smokers using cigars ( % of tobacco smoke in the amish plos one | https://doi.org/ . /journal.pone. march , / https://doi.org/ . /journal.pone. table . health related correlates to categories of passive smoking in old order amish study participants. no smoke exposure source of secondhand smoke exposure sources of secondhand smoke exposure sources of secondhand smoke exposure p* women n = n = n = n = age at evaluation (yrs) ( to ) ( to ) ( to ) ( to ) < . bmi (kg/m ) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . height (cm) ( to ) ( to ) ( to ) ( to ) . weight (kg) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . waist to hip ratio . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . fev (% predicted) ( to ) ( to ) ( to ) ( to ) . fvc (% predicted) ( to ) ( to ) ( to ) ( to ) . fev /fvc (% predicted) ( to ) ( to ) ( to ) ( to ) . ldl-c (mmol/l) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . hdl-c (mmol/l) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . tg (mmol/l) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . heart rate by ekg ( to ) ( to ) ( to ) ( to ) . fasting glucose (mmol/l) . ( . to . ) . ( . to . ) . ( . to . ) . ( . to . ) . sbp (mmhg) ( to ) ( to ) ( to ) ( to ) . dbp (mmhg) ( to ) ( to ) ( to ) ( to ) . men n = n = n = n = age at evaluation (yrs) ( to ) ( to ) ( to ) < . bmi (kg/m ) ( to ) ( to ) ( to ) . height (cm) ( to ) ( to ) ( to ) . weight (kg) . ( . to . ) . ( . to . ) . ( . to . ) . waist to hip ratio . ( . to . ) . ( . to . ) . ( . to . ) . fev (% predicted) ( to ) ( to ) ( to ) . fvc (% predicted) ( to ) ( to ) ( to ) . fev /fvc (% predicted) ( to ) ( to ) ( to ) . ldl-c (mmol/l) . ( . to . ) . ( . to . ) . ( . to . ) . hdl-c (mmol/l) . ( . to . ) . ( . to . ) . ( . to . ) . tg (mmol/l) . ( . to . ) . ( . to . ) . ( . to . ) . heart rate by ekg ( to ) ( to ) ( to ) . fasting glucose (mmol/l) . ( . to . ) . ( . to . ) . ( . to . ) . sbp (mmhg) ( to ) ( to ) ( to ) . (continued ) tobacco smoke in the amish plos one | https://doi.org/ . /journal.pone. march , / https://doi.org/ . /journal.pone. smokers) or pipes ( % of smokers)[ ]. as cigar and pipe users typically inhale less deeply, such smoking involves more side-stream rather than main-stream smoke inhalation, and thus it more closely resembles secondhand smoke than the first-hand smoking of cigarettes[ , ]. despite this and modest smoking habits overall, adjusted analyses demonstrated associations between smoking and detectable differences in spirometric lung function, bmi, hdl-c, and heart rate. furthermore, smoking was associated with subclinical cardiovascular disease as evi- denced by lower ankle-brachial index and larger aortic size. this supports prior assertions that a large proportion of cardiovascular risk attributable to tobacco exposure occurs at low levels of exposure[ , ]. prior observations have suggested that this low threshold for effect may be attributable primarily to prothrombotic effect[ , ]. our data would suggest a low threshold for other mechanisms as well. table . (continued ) no smoke exposure source of secondhand smoke exposure sources of secondhand smoke exposure sources of secondhand smoke exposure p* dbp (mmhg) ( to ) ( to ) ( to ) . data are expressed as medians (iqr) or as percentages. *p values were obtained using mmap software controlling for pedigree and age (with the exception of age and spirometric measures, which were controlled for pedigree alone). abi: ankle-brachial index; bmi: body mass index; dbp: diastolic blood pressure; ekg: electrocardiogram; fev : forced expiratory volume in second; fvc: forced vital capacity; ldl-c: low density lipoprotein cholesterol; hdl-c: high density lipoprotein cholesterol; sbp: systolic blood pressure; tg: triglycerides. https://doi.org/ . /journal.pone. .t table . correlation between health measures and ordinal categories of passive smoking in old order amish study participants from lancaster county. adjusted for β (se) p interaction p (sex*smoke exposure) fev (liters) age, height, sex, bmi - . ( . ) . . fvc (liters) age, height, sex, bmi - . ( . ) . . fev /fvc age, height, sex, bmi . ( . ) . . bmi (kg/m ) sex, age . ( . ) . . waist to hip ratio sex, age . ( . ) . . height (cm) sex, age - . ( . ) . . weight (kg) sex, age . ( . ) . . hdl-c (mmol/l) sex, age, bmi - . ( . ) . . hdl-c (mmol/l) sex, age, bmi, tg - . ( . ) . . ldl-c (mmol/l) sex, age, bmi - . ( . ) . . tg (mmol/l) sex, age, bmi - . ( . ) . . glucose (mmol/l) sex, age, bmi . ( . ) . . sbp (mmhg) sex, age, bmi - . ( . ) . . dbp (mmhg) sex, age, bmi - . ( . ) . . heart rate sex, age, bmi - . ( . ) . . mean abi (n = ) sex, age, bmi . ( . ) . . aorta (n = ) sex, age, bmi . ( . ) . . n = . p values were obtained using regression methods including controls for family structure. abi: ankle-brachial index; bmi: body mass index; dbp: diastolic blood pressure; fev : forced expiratory volume in second; fvc: forced vital capacity; ldl-c: low density lipoprotein cholesterol; hdl-c: high density lipoprotein cholesterol; sbp: systolic blood pressure; tg: triglycerides. https://doi.org/ . /journal.pone. .t tobacco smoke in the amish plos one | https://doi.org/ . /journal.pone. march , / https://doi.org/ . /journal.pone. .t https://doi.org/ . /journal.pone. .t https://doi.org/ . /journal.pone. pulmonary associations of secondhand smoke our study found a . % lower spirometric lung function (fev %) associated with secondhand smoke in women. the power to detect this difference in the female group was %, whereas power was only % to detect this difference in the never-smoking men. as such, the lack of signal in men may represent a limitation of power rather than a true gender difference. power in our study as well as in prior studies could be impaired due to issues of nondifferential mis- classification bias related to ascertainment of smoke exposure, potentially resulting in both diffi- culty detecting effect as well as underestimation of effect size. this might particularly affect studies assessing only one source of tobacco exposure, such as spousal smoking[ – ]. the surgeon general’s review states, “the evidence is suggestive but not sufficient to infer a causal relationship between chronic secondhand smoke exposure and a small decrement in spiromet- ric lung function in the general population[ ]” notably, a meta-analysis involving cross sec- tional studies reported an estimate similar to ours (- . %, %ci - . to - . %)[ ]. our results bolster that observation and should reduce uncertainty around the association between second- hand smoke exposure and small decrements in spirometric lung function. cardiometabolic associations of secondhand smoke the cardiovascular risk associated with secondhand smoke exposure is striking and approaches that of smoking[ , ]. studies demonstrate a consistent – % increased risk for coronary disease associated with secondhand smoke[ ]. the best established mechanisms underlying this effect include endothelial dysfunction and a prothrombotic effect[ ]. our findings suggest the possibility of a significant role for mechanisms less clearly established in secondhand smoking, but recognized in association with active smoking. such effect media- tors include hdl-c and bmi. we found lower hdl-c levels associated with secondhand smoke exposure in women only. svendsen described a cohort of never-smoking men in whom hdl-c levels were not asso- ciated with spousal smoking status[ ]. another study described lower hdl-c in female ado- lescents exposed to secondhand smoke from their parents, but no such association existed in males[ ]. notably, smoking is clearly associated with reduced hdl-c levels in men[ ], including in our cohort. as such, it would seem that both men and women manifest reduction in hdl-c levels in response to smoke exposure, but women may have a greater susceptibility for this particular effect. while our study design does not facilitate determination of mecha- nism, it is likely that the differences observed between men and women result from both differ- ences in exposure as well as susceptibility. differences in susceptibility are supported by biologic plausibility. smoke exposure has been shown to influence sex hormone profiles in women[ ], and sex hormone profiles in turn correlate with levels of hdl-cholesterol, independently of smoking[ ]. we found more familial sources of secondhand smoke to correlate with higher bmi in both men and women without apparent differential effect according to gender. a higher bmi has been associated with secondhand smoke in a number of large studies[ , , – ]. it has been suggested that tobacco smoke is a source of a variety of “obesogens”, including polycyclic aromatic hydrocarbons, which are produced by incomplete combustion of organic materials [ ]. these polycyclic aromatic hydrocarbons are produced from other sources as well, and may function synergistically with secondhand smoke to promote obesity[ ]. heart rate in our cohort was higher in smoking men, lower in men exposed to secondhand smoke, and unassociated with secondhand smoke exposure in women. our findings in smokers reflect prior observations consistent with increased adrenergic tone associated with smoking [ ]. the observation of paradoxically lower heart rates in men in and absence of association tobacco smoke in the amish plos one | https://doi.org/ . /journal.pone. march , / https://doi.org/ . /journal.pone. women exposed to secondhand smoke is not easily explained, although a murine model has shown estrogen to modulate chronotropic effects of nicotine[ ]. notably, the possibility of confounding attributable to use of medications that would affect the heart rate is possible but unlikely in our cohort as medication use is not common in the amish, with rates of beta blocker use under %[ ]. smoking has been associated with insulin resistance and increased risk for the development of type diabetes in both men and women[ , , , ]with an apparent dose-reponse relationship. secondhand smoke has also been associated with increased risk for type diabetes[ , ]. a large meta-analysis showed a stronger correlation between smoke exposure and elevated glucose levels in men[ ], possibly suggestive of a differential susceptibil- ity according to sex. supporting this possibility, we found more family sources of secondhand smoke exposure to be associated with higher glucose levels in men, but not women. strengths and limitations our methods for assessment of secondhand smoke exposure involved questioning family members about their smoking history and then extrapolating exposure through pedigree rather than questioning study participants about secondhand smoke exposure directly. while this approach mitigates recall bias, it led to some data points missing due to nonrandom inabil- ity to capture all family members (older participants were less likely to have parents enrolled) that was not possible to meaningfully address through imputation. the methods also did not permit differentiation between acute exposure versus chronic or past exposure. it is likely that the different categories of potential sources comprising the exposure score reflect exposures occuring primarily at different periods of life such that exposure from a father would be more likely to occur during childhood whereas exposure from a spouse would more likely represent a more recent or current source. power was insufficient to meaningfully distinguish differ- ences between these potential sources of exposure. workplace exposure was also not captured in our data and represents a possible source of misclassification bias that would likely be non- differential and thus bias towards the null in analyses stratified by sex, but could represent a source of differential exposure between men and women.finally, the cross-sectional nature of our study is suceptible to survivorship bias. we would, however, expect mortality related to smoke exposure to preferentially affect those with high exposure or susceptibility and as such would result in underestimation of the true magnitude of effect. conclusions in a unique population controlling for maternal tobacco use, smoking is associated with sub- clinical cardiovascular disease and secondhand smoke exposure primarily from cigar and pipe use is associated with a greater burden of risk factors for cardiovascular disease. sex differences are suggested in the correlation between smoke exposure and hdl-c levels, and glucose levels. acknowledgments the authors thank the amish research coordinators and study participants for making this work possible. author contributions conceptualization: rmr mtd me mm gn sms ds bdm. data curation: rmr mp tip ars bdm. tobacco smoke in the amish plos one | https://doi.org/ . /journal.pone. march , / https://doi.org/ . /journal.pone. formal analysis: rmr mp bdm. funding acquisition: rmr tip ars bdm. investigation: rmr mp tip ars bdm. methodology: rmr gn bdm. project administration: rmr mp tip ars bdm. resources: rmr tip ars bdm. supervision: rmr tip ars bdm. writing – original draft: rmr. writing – review & editing: rmr mtd me mm gn mp tip sms ars ds bdm. references . services usdohah, prevention cfdca. the health consequences of smoking— years of progress: a report of the surgeon general. office of the surgeon general. . . hagstad s, bjerg a, ekerljung l, backman h, lindberg a, ronmark e, et al. passive smoking exposure is associated with increased risk of copd in never smokers. chest. ; ( ): – . https://doi. org/ . /chest. - pmid: . dockery dw, trichopoulos d. risk of lung cancer from environmental exposures to tobacco smoke. cancer causes 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[pdf] local symmetry breaking in sno nanocrystals with cobalt doping and its effect on optical properties. | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /c nr a corpus id: local symmetry breaking in sno nanocrystals with cobalt doping and its effect on optical properties. @article{roy localsb, title={local symmetry breaking in sno nanocrystals with cobalt doping and its effect on optical properties.}, author={s. roy and amish g joshi and s. chatterjee and a. ghosh}, journal={nanoscale}, year={ }, volume={ }, pages={ - } } 	s. roy, amish g joshi, + author a. ghosh 	published 	materials science, medicine 	nanoscale x-ray photoemission spectroscopy (xps), x-ray diffraction (xrd) and transmission electron microscopy (tem) have been used to study the structural and morphological characteristics of cobalt doped tin(iv) oxide (sn -xcoxo ; ≤ x ≤ . ) nanocrystals synthesized by a chemical co-precipitation technique. electronic structure analysis using x-ray photoemission spectroscopy (xps) shows the formation of tin interstitials (sni) and reduction of oxygen vacancies (vo) in the host lattice on co doping… expand view on pubmed rsc.org save to library create alert cite launch research feed share this paper citationsbackground citations view all topics from this paper 	nanocrystalline materials 	cobalt 	oxygen 	transmission electron microscopy 	doping in sports 	luminescence 	x-ray photoelectron spectroscopy 	spectroscopy, fourier transform infrared 	viola <angiosperm> 	computed tomography scanning systems 	tin citations citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency influence of oxygen vacancy defects and cobalt doping on optical, electronic and photocatalytic properties of ultrafine sno -δ nanocrystals 	z. dohčević-mitrović, v. araújo, + authors m. nikolić 	materials science 	 	pdf 	view excerpts, cites background save alert research feed structural, optical, electrical and room temperature gas sensing characterizations of spin coated multilayer cobalt-doped tin oxide thin films 	mohamed a. basyooni, y. eker, m. yılmaz 	materials science 	 	 save alert research feed role of size, alio-/multi-valency and non-stoichiometry in the synthesis of phase-pure high entropy oxide (co,cu,mg,na,ni,zn)o. 	nandhini j. usharani, rajat shringi, harshil sanghavi, s. subramanian, s. s. bhattacharya 	materials science, medicine 	dalton transactions 	 save alert research feed large-area spray deposited ta-doped sno thin film electrode for dssc application 	r. ramarajan, nandarapu purushothamreddy, + authors d. joseph 	materials science 	 save alert research feed tungsten doped stannic oxide transparent conductive thin film using 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and magnetic properties of co-doped sno powders synthesized by the coprecipitation technique 	a. bouaine, n. brihi, g. schmerber, c. ulhaq-bouillet, s. colis, a. dinia 	materials science 	 	 save alert research feed optical and structural properties of nd doped sno powder fabricated by the sol–gel method 	karima bouras, j. rehspringer, + authors a. slaoui 	materials science 	 	 	 save alert research feed realizing ferromagnetic ordering in sno and zno nanostructures with fe, co, ce ions. 	k. verma, r. k. kotnala 	materials science, medicine 	physical chemistry chemical physics : pccp 	 	 save alert research feed structural and optical properties of cu doped sno nanoparticles: an experimental and density functional study 	p. chetri, b. saikia, a. choudhury 	materials science 	 	 save alert research feed structural characterization of nanocrystalline sno by x-ray and raman spectroscopy 	l. abello, b. bochu, a. gaskov, s. koudryavtseva, g. lucazeau, m. roumyantseva 	chemistry 	 	 save alert research feed strong room-temperature ferromagnetism in co +-doped tio made from colloidal nanocrystals. 	j. d. bryan, s. heald, s. chambers, d. r. gamelin 	chemistry, materials science 	journal of the american chemical society 	 	 	pdf save alert research feed ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- 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http://europepmc.org/abstract/med/ ellis-van creveld syndrome and weyers acrodental dysostosis are caused by cilia-mediated diminished response to hedgehog ligands american journal of medical genetics part c (seminars in medical genetics) c: – ( ) a r t i c l e ellis–van creveld syndrome and weyers acrodental dysostosis are caused by cilia-mediated diminished response to hedgehog ligands victor l. ruiz-perez* and judith a. goodship** ellis –van creveld syndrome (evc; omim ) is a recessive disorder comprising chondrodysplasia, polydactyly, nail dysplasia, orofacial abnormalities and, in a proportion of patients, cardiovascular malformations. weyers acrodental dysostosis (weyers; omim ) is an allelic dominant disorder comprising polydactyly, nail dysplasia, and orofacial abnormalities. evc results from loss-of-function mutations in evc or evc , the phenotype associated with the mutations in these two genes being indistinguishable. three convincing causative mutations have been identified in patients with weyers acrodental dysostosis, which are clustered in the last coding exon of evc and lead to production of a truncated protein lacking the final amino acids. localization and function of evc and evc are inferred from studying the murine orthologs. both evc and evc proteins localize to the basal bodies of primary cilia and analysis of an ellis –van creveld mouse model, which includes the limb shortening and tooth abnormalities of evc patients, has demonstrated hedgehog signaling defects in the absence of evc. the loss of evc has not been studied directly, but hedgehog signaling is impaired when a mutant murine evc weyer variant is expressed in vitro. we conclude that the phenotypic abnormalities in evc and weyers syndrome result from tissue specific disruption of the response to hh ligands. � wiley-liss, inc. key words: ellis –van creveld syndrome; weyers acrodental dysostosis; evc; evc ; hedgehog how to cite this article: ruiz-perez vl, goodship ja. . ellis–van creveld syndrome and weyers acrodental dysostosis are caused by cilia-mediated diminished response to hedgehog ligands. am j med genet part c semin med genet c: – . introduction ellis–van creveld syndrome ellis – van creveld syndrome (chondro- ectodermal dysplasia) was recognized as a distinct syndrome more than years ago when ellis and van creveld reported three children with chondrodysplasia, nail dysplasia, oral abnormalities, poly- dactyly and, in two of the three, a heart murmur [ellis and van creveld, ]. these authors recorded that intelligence in these three children was normal and noted that the parents of two of the three cases were consanguineous indicating recessive inheritance. the bone abnormalities in evc consist of disproportionate short stature with shortening of the limbs and narrow chest. generally birth length is below the third centile and height continues to be below the third centile to adulthood [mckusick et al., ]. however, this is not always the case, for example, out of affected individuals in one of the large pedigrees used to map the disorder were said to be of normal height in their population [da silva et al., ]. the limb shortening is acromesomelic. affected individuals are unable to make a tight fist because shortening of the phalanges is also disproportionate with the distal phalanges being more affected than the proximal phalanges. genu valgum is a frequent though not constant finding. in addition to the chondrodys- plasia there are skeletal patterning abnormalities; bilateral type a postaxial polydactyly of the hands is present in almost all affected individuals, and poly- dactyly of the feet is present in approxi- mately % of cases. polydactyly is typically postaxial and it is unusual to dr. victor l. ruiz-perez is a tenure scientist at the instituto de investigaciones biomedicas, consejo superior de investigaciones cientı́ficas-universidad autónoma de madrid, madrid and his research group is also a member of the networking center of biomedical research in rare diseases (ciberer). his research interests are in rare diseases and the etiology of growth plate malformations. professor judith goodship is a clinical geneticist at the northern genetic service, newcastle upon tyne hospitals nhs foundation trust, uk. her research group is in the institute of human genetics, newcastle university and her research interests are developmental disorders and the etiology of cardiovascular malformations. grant sponsor: spanish ministry of science and innovation; grant number: saf- ; grant sponsor: ramon areces foundation; grant sponsor: european union; grant number: lshm-ct- - . *correspondence to: victor l. ruiz-perez, instituto de investigaciones biomédicas, csic-uam, arturo duperier, , madrid, spain. e-mail: vlruiz@iib.uam.es **correspondence to: judith a. goodship, institute of human genetics, newcastle university, international centre for life, central parkway, newcastle upon tyne ne bz, uk. e-mail: j.a.goodship@ncl.ac.uk doi . /ajmg.c. published online october in wiley interscience(www.interscience.wiley.com) � wiley-liss, inc. have more than six digits to a limb. there are occasional reports of inter- digital polydactyly of hands or feet and syndactyly between fingers or toes can occur. nail dysplasia is a distinctive, almost invariant finding, though again it was not reported in the brazilian pedigree that contributed to mapping the disorder [da silva et al., ]. the radiographic features of evc include postaxial hexadactyly, cone- shaped epiphyses of the phalanges and fusion of the carpals, especially the capitate and hamate, in later childhood and adolescence (fig. ). in the majority of cases there is shortening of the ribs and progressive distal shortening of the limbs (fig. ). in the pelvis there are hook-like protrusions or spikes from the medial and lateral borders of the acetabulum giving rise to the terms ‘‘trident aceta- bulum’’ (fig. ). the appearances of the ribs and the pelvis become more normal in later childhood and adolescence. the proximal tibial metaphyses is slanted with hypoplasia of the lateral part of the proximal tibial epiphysis (fig. ) giving rise to a genu valgum deformity which becomes more obvious as the child grows. there is little information on the radiographic appearance in the fetus. all individuals with evc have oral features (fig. ) [hattab et al., ; cahuana et al., ; mostafa et al., ]. there is often a notch in the center of the upper lip due to tethering of the upper lip to the gingival margin. there are multiple labial-gingival fre- nulae and in some cases partial ankylo- glossia can lead to a bifid appearance when the tongue is protruded. natal teeth are not uncommon. teeth are small and abnormal in shape, conical or with abnormal cusp pattern, and position. there are abnormalities in eruption of primary and permanent teeth, usually delayed though premature eruption of permanent teeth has also been reported. hypodontia is quite frequently observed, typically absence of primary and/or permanent maxillary and mandibular incisors, though super- numary teeth and fused teeth also occur. clefts of the mandibular and/or maxil- lary alveolar ridge may be seen at the lateral incisor region. tooth enamel may be hypoplastic. although teeth and nails are abnormal, evc is not a generalized ectodermal dysplasia as skin and hair are normal. congenital heart defects were recognized as being a feature of the condition from the earliest reports and occur in approximately % of evc patients [giknis, ; digilio et al., ]. the commonest cardiac mal- formations are atrioventricular canal defects and common atrium, which each account for approximately a third of the cardiac defects. additional congenital heart defects were recognized as being a feature of the condition from the earliest reports and occur in approximately % of evc patients. the commonest cardiac malformations are atrioventricular canal defects and common atrium, which each account for approximately a third of the cardiac defects. malformations reported in combination with atrioventricular canal defects are hypoplastic left ventricle and left supe- rior vena cava. additional malforma- tions reported in combination with common atrium are hypoplastic left ventricle, left superior vena cava, vsd, transposition of the great arteries and tricuspid atresia. the abnormalities in the remaining third of patients with a cardiac malformation are atrial or ven- tricular septal defects either as isolated anomalies or in combination with additional malformations including pul- monary stenosis, aortic coarctation, figure . radiological abnormalities in ellis –van creveld syndrome. a: short ribs, short long bones, and acetabular spikes with narrow sacrosciatic notches. b: postaxial polydactyly, short middle, and distal phalanges with cone shaped epiphyses, and accessory carpals with carpal fusion. c: postaxial polydactyly, short distal phalanges, and broad hamate. d: absence of lateral component of proximal tibial epiphyses and sloping of lateral tibial metaphyses giving rise to genu valgum deformity. american journal of medical genetics part c (seminars in medical genetics) article left superior vena cava, and anomalous pulmonary return. there has been a case report of single ventricle with asd. right isomerism sequence and situs inversus have also been reported. this incidence and spectrum of malfor- mations in the literature is very similar to that in a group of patients in whom we identified evc or evc mutations [tompson et al., ]. cardiovascular malformations (cvm) were present in / patients with evc mutations, comprising avsd, partial avsd, common atrium, primum asds, and complex cardiac defect. cardiovascular malfor- mations were present in / patients with evc mutations, comprising avsds, partial avsds, common atrium, primum asd, secundum, unspecified asd, complex cardiac malformation and coarctations of the aorta. thus there is no apparent differ- ence in the pattern or incidence of cvm between patients with evc and evc mutations. prognosis in evc is related to the severity of cardiovascular malforma- tions and in the absence of cardiovascular malformations lifespan is normal with reports of families including affected individuals in their seventh and eighth decade. there have been a number of reports of affected males having children but no reports of affected females having children [mckusick et al., ; da silva et al., ; mostafa et al., ]. parents of evc-affected individuals do not have features of the condition. weyers acrodental dysostosis weyers [ ] described the radiological findings in three infants with postaxial polydactyly and oral abnormalities similar to, but milder than, those reported in evc. in contrast to evc in which a large number of case reports followed the first description, relatively few weyers families have been described weyers described the radiological findings in three infants with postaxial polydactyly and oral abnormalities similar to, but milder than, those reported in evc. in contrast to evc in which a large number of case reports followed the first description, relatively few weyers families have been described. [curry and hall, ; roubicek and spranger, ; ye et al., ; zannolli et al., ]. weyers is transmitted as a dominant disorder, and it was mapped to the same chromosomal region as evc in a family with eight affected individuals from four generations who had postaxial polydactyly, nail dystrophy, mild short stature, and tooth abnormalities. the proband of this family, in addition to polydactyly, nail dystrophy, and tooth abnormalities, had disproportionate short stature and a cardiovascular mal- formation [howard et al., ]. this proband is the only case described in a dominant pedigree with a cardiovascular malformation. it is not clear whether the proband in this family extends the phenotypic spectrum of weyers syn- drome or whether he has evc. evc and evc mutation analysis in the proband and mildly affected individuals in this family would clarify this. in the families reported, height has generally been in the lower half of the normal range and there have been no reports of genu valgum. whilst postaxial polydactyly type a has been reported in hands and feet, it has not been always been present in the hands and seems more common in the feet, which is the converse of the situation in evc. polydactyly type b and – toe syndactyly have also been reported [ye et al., ; zannolli et al., ; zannolli et al., ; valencia et al., in press]. the onychodystrophy and oral manifestations are very similar to those reported in evc. identification of the evc genes the gene for evc was mapped to chromosome p using nine amish figure . oral abnormalities in a patient with ellis– van creveld syndrome. congenital absence of the incisors, conical shaped teeth and multiple frenulae. article american journal of medical genetics part c (seminars in medical genetics) families along with single families from ecuador, mexico, and brazil the gene for evc was mapped to chromosome p using nine amish families along with single families from ecuador, mexico, and brazil. [polymeropoulos et al., ]. the positional cloning exercise that followed led to identification of mutations in a novel gene (evc) in seven evc families including an intronic change (ivs þ g>t) in the amish pedigree that was subsequently demonstrated to affect splicing [ruiz-perez et al., ; the positional cloning exercise that followed led to identification of mutations in a novel gene (evc) in seven evc families including an intronic change (ivs þ g>t) in the amish pedigree that was subsequently demonstrated to affect splicing. tompson et al., ]. however, no mutations were identified in this gene in the other families used to map the disorder or in two additional consangui- neous families that were homozygous across the region. furthermore, on systematic screening of evc in a panel of samples we identified causative mutations in only a third of affected individuals. thus we continued to clone and investigate additional genes in the region. meanwhile takeda and coworkers mapped autosomal recessive bovine chondrodysplastic dwarfism in japanese brown cattle to the region orthologous to human chromosome p [yoneda et al., ] and after excluding bovine evc went on to identify two mutations in a contiguous novel gene that they named limbin, lbn, which accounted for the bovine pheno- type [takeda et al., ]. mutations in the human ortholog, evc , were takeda and coworkers mapped autosomal recessive bovine chondrodysplastic dwarfism in japanese brown cattle to the region orthologous to human chromosome p and after excluding bovine evc went on to identify two mutations in a contiguous novel gene that they named limbin, lbn, which accounted for the bovine phenotype. mutations in the human ortholog, evc , were identified in evc patients shortly thereafter. identified in evc patients shortly there- after [ruiz-perez et al., ; galdzicka et al., ]. evc and evc are in divergent orientation in close proximity to each other; for example, in humans the transcription start sites are separated by bp, and this genomic organiza- tion has been conserved through evolu- tion, being present not only in vertebrates but also in amphioxus, sea urchin and snail (chris ponting, personal communication). initially we thought that the two genes were not homologous as no resemblance was detected in classical blast searches but further psi-blast analysis has revealed homology indicating that they arose from an ancient duplication event (chris ponting, personal communication). evc encodes a novel amino acid protein and evc encodes a novel amino acid protein. analysis of the predicted peptide sequence shows that both proteins have multiple coiled-coil regions, that evc has a predicted trans- membrane domain at its n-terminus and that evc has a predicted trans- membrane domain at the n-terminus with a second transmembrane domain (amino acids – ) but no other recognized motifs. function of evc and evc an evc mouse model was generated by replacing the first exon of evc with a lacz cassette fused to the first atg of the gene [ruiz-perez et al., ]. this strategy created a null allele that mimics the effect of the mutations seen in evc patients, most of which introduce nonsense codons with the transcripts predicted to undergo nonsense-medi- ated decay and hence absence of protein. as the lacz reporter was driven by the evc promoter the evc targeted allele was also used to obtain information about the evc expression pattern. x-gal staining of evc þ/� and evc �/� mouse embryos showed lacz expression in the orofacial region at e . and in the orofacial mesenchyme and all the form- ing cartilage elements from e . . lacz expression is also present in developing nails. thus x-gal staining correlated with the phenoyptic abnormalities of evc. in addition to the tissues stained by x-gal, which should correspond with those having the highest levels of evc transcription, rt-pcr analysis and immunostaining have demonstrated that evc was expressed at low levels in most cells. while the animals heterozygous for the targeted allele were normal, mice lacking evc phenocopied the human condition. like evc patients, the mice had short limbs, short ribs, and dental abnormalities but did not have polydactyly or obvious cardiovascular malformations. radiographic measure- ments clearly demonstrated that the skeletal shortening was more pro- nounced in the distal part of the limbs. on histological analysis of the long bones, there was epiphyseal shortening and delayed periosteal induction with the reduction in length of the growth plate being mainly due to a shortening of the columns of proliferative chondro- cyte though the size of the hypertrophic american journal of medical genetics part c (seminars in medical genetics) article region is also reduced. this appearance was consistent with defective ihh signaling and analysis of the expression of indian hedgehog (ihh) and its down- stream targets by in situ hybridization demonstrated normal ihh expression but diminished mrna levels of the ihh downstream targets, ptch , gli , and pthrp. in vitro studies treating murine embryonic fibroblasts (mefs) and chondrocytes with the hh agonist purmophamine confirmed that hedge- hog signal transduction was defective in cells lacking evc [ruiz-perez et al., ]. however not all aspects regulated by ihh in the growth plate are equally affected. chondrocyte proliferation which is reduced to approximately % in ihh mutants was shown by brdu labeling to be normal in the evc knock- out mice. the reduced proliferation rate in ihh mutants is due to increased levels of gli r as proliferation is restored in the ihh; gli double knockouts [hilton et al., ; koziel et al., ]. gli r was shown not to be increased in evc knockout mice explaining why chondrocyte prolifera- tion was unaffected in these mice. comparable studies have not been reported for mice lacking evc but we deduce that evc also plays a role in hedgehog signaling as the phenotype in patients lacking evc is indistin- guishable from the phenotype of patients lacking evc. furthermore, hedgehog signaling was disrupted when a weyers mutant version of evc was expressed in vitro providing evidence for its involvement in hedgehog signaling [valencia et al., in press]. consistent with their role in hh signaling, on immunofluorescence microscopy both proteins localized to the base of primary cilia [ruiz-perez et al., ; sund et al., ]. this is of particular interest as hedgehog signaling is mediated through primary cilia [huangfu et al., ; huangfu and anderson, ; haycraft et al., ]. evc is not required for ciliogenesis as cilia appear normal in the mouse mutant [ruiz-perez et al., ]. however, the precise role of these two proteins in hedgehog signal transduction remains to be elucidated. genotype–phenotype: evc evc and evc mutations each account for approximately half of patients with evc, the phenotype associated with mutations in each gene being indistin- guishable. initially it had been thought that there may be further locus hetero- geneity, but mutations were identified in all cases in a panel of evc patients containing a high proportion of con- sanguineous cases screened recently evc and evc mutations each account for approximately half of patients with evc, the phenotype associated with mutations in each gene being indistinguishable. initially it had been thought that there may be further locus heterogeneity, but mutations were identified in all cases in a panel of evc patients containing a high proportion of consanguineous cases screened recently. [tompson et al., ; valencia et al., in press]. thus any additional genes account for only a small proportion of cases. in both genes the majority of mutations have been nonsense muta- tions or frameshift mutations that intro- duce a nonsense codon, and for these it is likely that transcripts undergo non- sense-mediated decay (fig. a). in total independent evc mutations have been reported, eight of which have been seen in more than one family (table i). whilst the majority have introduced directly or indirectly non- sense codons, approximately a quarter have occurred within consensus splice sites. investigation of a homozygous mutation present in only one parent revealed segmental uniparental disomy of a region on the short arm of chromosome in one patient [tompson et al., ]. there has been one deletion removing the last exons and another one removing the last . six missense changes or in-frame deletions have been reported in evc: p.k del, p.s p, p.t _g del, p.l _l del, p.l p, and p.q h. the protein effect of the c. t>a mutation which changes the first atg to aag is unknown. in total independent evc mutations have been reported associated with the evc phenotype, of which have been seen in more than one family (table ii). three changes have been at consensus splice sites and four have been deletions of more than one exon. cdna analysis of the del- e -c. showed that it led to skipping of exons and resulting in an in frame deletion p.a _m del that removes amino acids [tompson et al., ]. cdna was not available to study the effects of the other three deletions. three missense changes have been reported in evc : p.d y, p.i r, and p.a v. two mutations, p.s rfsx and p.s vfsx , introduce nonsense codons in the final coding exon of evc and are thus predicted to produce truncated proteins. the mechanism by which the missense and in-frame deletions in the proteins lead to the phenotype has not been studied. an evc family with a homozygous contiguous gene deletion removing both evc and evc , c orf and stk b has recently been reported [temtamy et al., ]. all three affected individuals in the family had postaxial polydactyly of hands and feet, nail dysplasia, the characteristic pattern of bone shortening, genu valgum, and dental anomalies. none of the three affected individuals had a cardiovascular malformation. all three had borderline intelligence, which could be due to deletion of both evc and evc or due to loss of one of the other two genes removed by the deletion or possibly, though less likely, due to an unrelated recessive disorder in this consanguine- article american journal of medical genetics part c (seminars in medical genetics) ous family. the fact that the skeletal, orofacial, and cardiac phenotypes were typical in this family suggests that neither gene compensates for the other, that is, there is no functional redundancy. the same chromosomal deletion was found in conjunction with a splice site muta- tion in a patient of different ethnic origin who had classical evc and normal intelligence [temtamy et al., ]. the observation that none of the carriers of this deletion had phenotypic features of evc, formally excludes evc/evc digenic inheritance as a mechanism for evc. finally, interpretation of a previ- ously reported evc mutation warrants comment. a father and daughter, not with classical evc but with postaxial polydactyly of the hands, a partial atrioventricular canal with common atrium and agenesis of the upper lateral incisors with enamel hypoplasia, were reported as having a heterozygous mis- sense change, p.r q [ruiz- perez et al., ]. this change has subse- quently been reported as a rare poly- morphism, rs , that is more common in african populations and thus does not account for their phenotype. genotype–phenotype: weyers acrodental dysostosis weyers commented on the similarity between the oral and hand and feet abnormalities in the patients that he described and those observed in evc [weyers, ]. a heterozygous frame- shift c. delc in the last coding exon of evc reported in a chinese pedigree with six living affected individuals in different generations with weyers acro- dental dysostosis provided definitive evidence that the conditions are allelic [ye et al., ]. this mutation changes leucine to tyrosine and introduces a stop in the next codon. mutations have recently been identified in three further weyers families (table iii) [valencia et al., in press]. in one caucasian family, the mutation segregating in the family is c. delc, identical to that reported in the chinese family. in the remaining two families the changes were found at nucleotide , c. t>a and c. t>a, which also truncate the protein at l , a striking clustering of mutations. of these two mutations, one segregated in the family and the other occurred as a de novo event providing further evidence for its path- ogenicity. the initial description of the evc gene included investigation of a child who had classical evc whose father’s height was on the second centile and who had dysplastic nails and widely spaced conical-shaped teeth but did not have polydactyly [spranger and tariver- dian, ]. this father was found to carry evc p.s p, and it was suggested that this accounted for his clinical features which were consistent with weyers syndrome [ruiz-perez et al., ]. however, this is one of the few recurrent evc mutations and no other figure . distribution of the mutations described in evc and evc . panel a illustrates the exon composition of evc and evc with the mutations found in each exon in ellis– van creveld patients shown above. code: nonsense mutations are represented by red circles, microinsertions by blue triangles, microdeletions by green inverted triangles, missense mutations by light blue squares and splice site by violet rhombi. large deletions spanning more than one exon are represented by arrows which cover the deleted exons. the stk b-evc deletion spanning both genes is also represented. the picture is drawn so that p telomere is on the left. panel b indicates the position of the evc recessive mutations (above) and weyers dominant mutations (below) exon of evc . american journal of medical genetics part c (seminars in medical genetics) article table i. mutations identified in evc exon/intron nucleotide change protein effect number of cases refs. exon c. t>a p.m ? intron c. þ g>a intron c. � a>g exon c. dela p.n ifsx exon c. c>a p.y x intron c. þ _ ga>ac intron c. � g>c exon c. dupt p.i yfsx exon c. delt p.d tfsx exon a c. t>a p.l x exon c. dupt p.e x , exon c. _ del p.k del , exon c. dupa p.r kfsx exon c. t>c p.s p , intron c. � t>g exon c. c>t p.r x , , exon c. g>t p.e x intron c. þ g>a , exon c. _ del p.t _g del exon c. delt p.l rfsx exon c. g>t p.e x exon c. _ del p.q rfsx exons – ex _ del intron c. þ g>c exons – ex _ del exon c. g>t p.e x exon c. delc p.a vfsx intron c. � a>g exon c. c>t p.q x exon c. _ del p.l _l del exon c. t>c p.l p , intron c. þ g>t intron – exon del_ivs (� to þ ) exon c. _ dupca p.r tfsx exon c. c>t p.q x exon c. _ dupccgg p.a pfsx intron c. þ t>g exon [c. _ del; c. _ del] p.t qfsx exon c. delg p.m wfsx exon c. c>t p.q x exon c. g>c p.q h some changes have been adapted to the current human genome variation society specifications for describing sequence changes (http://www.hgvs.org/mutnomen/). mutations are named using nm_ . as the reference sequence taking the a of the atg translation initiation codon as nucleotide . references code: , ruiz-perez et al. [ ]; , ruiz-perez et al. [ ]; , galdzicka et al. [ ]; , ye et al. [ ]; , tompson et al. [ ]; , temtamy et al. [ ]; , ulucan et al. [ ]; , sund et al. [ ]; , valencia et al. [in press]. a this mutation introduces a stop codon when the rs c>t polymorphism at c. is a t. this is the case for the reference sequence used in the original report (af ) of this mutation. article american journal of medical genetics part c (seminars in medical genetics) table ii. mutations identified in evc in evc patients exon/intron nucleotide change protein effect number of cases refs. exon c. _ dupggcgg p.s gfsx exon c. dupt p.k x exons – ex _ del introns – del_ivs þ _ivs - intron c. þ t>c exon c. g>t p.d y exon c. c>t p.q x , exon c. t>g p.i r , exon c. dela p.h pfsx exon c. delg p.g efsx exon c. a>t p.k x exon c. _ del p.l pfsx exon c. c>t p.r x exon c. _ del p.r kfsx exon c. _ dupga p.s gfsx exon c. _ del p.l rfsx exon c. _ del p.g dfsx exon c. c>t p.q x exon c. c>t p.q x exon c. c>t p.q x intron –exon del- ex _c. p.a _m del exons – ex _ del exon c. dela p.m cfsx exon c. dela p.k nfsx exon c. dupt p.k x exon c. c>t p.r x exon c. dupc p.q pfsx exon c. c>t p.q x exon c. g>t p.e x exon c. _ dupaggcc p.v rfsx exon c. c>t p.r x exon c. c>t p.r x exon c. g>a p.w x exon a c. g>t p.e x exon c. c>t p.q x exon c. dela p.s afsx exon c. dupa p.r kfsx exon c. delg p.g afsx exon c. c>t p.a v exon c. c>t p.q x exon c. g>t p.e x intron c. þ g>a exon c. _ del p.g rfsx intron c. þ t>c , exon c. delc p.s rfsx , , exon c. dupt p.s vfsx some changes have been adapted to the current specifications of the human genome variation society for describing sequence changes (http://www.hgvs.org/mutnomen/). the mutations are named using nm_ . as the reference sequence taking the a of the atg translation initiation codon as nucleotide . reference code: , ruiz-perez et al. [ ]; , ruiz-perez et al. [ ]; , galdzicka et al. [ ]; , ye et al. [ ]; , tompson et al. [ ]; , temtamy et al. [ ]; , ulucan et al. [ ]; , sund et al. [ ]; , valencia et al. [in press]. a this mutation differs form the original report as the amino acid at position is e instead of q. american journal of medical genetics part c (seminars in medical genetics) article carriers of this mutation have been reported as having a phenotype. in view of the findings in subsequent weyers families, it would be interesting to study evc in this man. functional studies of evc exon mutations it is intriguing that three of the five mutations that have been identified in the last exon of evc have been associated with the weyers phenotype and are dominant mutations whilst the other two mutations have been identified in evc pedigrees with no phenotypic manifestations in the heter- ozygous parents or siblings (fig. b). it is intriguing that three of the five mutations that have been identified in the last exon of evc have been associated with the weyers phenotype and are dominant mutations whilst the other two mutations have been identified in evc pedigrees with no phenotypic manifestations in the heterozygous parents or siblings. the two evc-associated mutations are of the weyers mutations. given that these five mutations occur in the final exon, the corresponding transcripts are predicted to escape nonsense-mediated decay. this has been confirmed by cdna analysis for the c. delc (p.s rfsx ) evc-associated muta- tion and for the c. delc weyers- associated mutation for which primary fibroblasts were available. the effect on hh signaling of these two changes have been investigated in vitro [valencia et al., in press]. the equivalent murine ver- sions of these two truncated proteins and wild-type evc were expressed in nih t mouse fibroblasts along with a luciferase reporter of hedgehog signal- ing, and the response to the hedgehog agonist sag assayed. the hedgehog pathway was fully active in cells express- ing wild-type evc and cells expressing the protein mimicking the evc muta- tion, however, there was almost no activation of the hh responsive luci- ferase reporter in the cells expressing the protein mimicking the weyers mutation indicating that expression of this protein disrupts hedgehog signaling. this is in keeping with manifestation of a pheno- type in individuals heterozygous for the weyers mutation but not in individuals heterozygous for the evc mutation. the mechanism by which removal of the last amino acids disturbs the function of the normal protein produced from the other chromosome, whilst deletion of the last amino acids does not is not yet known. in the absence of antibodies that detect evc on western blot analysis of proteins from human fibroblasts, pro- duction of protein corresponding to the transcripts containing the c. delc and c. dupt recessive changes has not been demonstrated and it remains possible that the exon evc mutations lead to less stable truncated proteins than the weyers changes and thus to a reduction in evc protein as for the majority of evc associated mutations. nevertheless the cluster of mutations observed in association with the dominant weyers phenotype indicates that the final amino acids of evc are necessary for normal protein function. conclusion the data from the analysis of the evc knockout and the in vitro studies using evc dominant mutations indicate that evc and evc are required together for a normal level of response to hedge- hog ligands in certain tissues. the two proteins do not have redundant func- tions as the phenotypes resulting from the loss of evc, the loss evc or the loss of evc and evc are equivalent. it has been demonstrated that the bone abnormalities in evc result from diminished response to indian hedge- hog. the two phenotypes that have been shown to result from mutations within indian hedgehog itself differ from the evc skeletal phenotype. brachydactyly a results from heterozygous mutations restricted to a specific region of the n- terminal active fragment of indian hedgehog that has been shown to be a calcium-binding site essential for inter- action with its receptor and cell-surface partners [mclellan et al., ; byrnes et al., ]. modeling a brachydactyly a mutation in the mouse has con- firmed that impaired interaction with the receptor decreases signaling effi- ciency and also increases the range of ihh signaling in the developing digit [gao et al., ]. in brachydactyly a the middle phalanges are most affected in contrast to the progressively distally shortening that occurs in evc. the absence of long bone and rib abnormal- ities in patients with brachydactyly a implies that the evc phenotype cannot be completely explained by diminished signaling efficiency and increased range of signaling. acrocapitofemoral dy- splasia is a recessive disorder featuring short stature, brachydactyly and narrow thorax but in which the most striking table iii. mutations identified in evc in weyers patients exon nucleotide change protein effect number of cases refs. exon c. delc p.l yfsx , exon c. t>a p.l x exon c. t>g p.l x the mutations are named using nm_ . as the reference sequence taking the a of the atg translation initiation codon as nucleotide . reference code: , ruiz-perez et al. [ ]; , ruiz-perez et al. [ ]; , galdzicka et al. [ ]; , ye et al. [ ]; , tompson et al. [ ]; , temtamy et al. [ ]; , ulucan et al. [ ]; , sund et al. [ ]; , valencia et al. [in press]. article american journal of medical genetics part c (seminars in medical genetics) radiological abnormalities are in the head of the femur [hellemans et al., ; mortier et al., ]. in contrast, the head of the femur is relatively normal in evc patients. furthermore whilst the typical knee deformity in evc is genu valgum, in acrocapitofemoral dysplasia the knee deformity is genu vara. eluci- dating the precise role of evc and evc in modulating response to hedge- hog ligands remains a challenge. the orofacial and nail abnormalities observed in evc are expected to be the consequence of diminished response to sonic hedgehog signal (shh) in tissues in which evc expression is high. the oral frenulae seen in evc patients are very similar to those seen in oro-facio-digital syndrome type (ofd ). as hedgehog signaling is mediated through primary cilia and as the protein mutated in ofd plays a role in ciliogenesis [ferrante et al., ] it is likely that some aspects of the ofd oral phenotype are due to aberrant response to sonic hedgehog. the cardiovascular defects found in evc patients are also likely to arise from disruption of shh signaling as atrioven- tricular septal defects are seen following conditional abrogation of shh in the mouse [goddeeris et al., ]. evc is grouped together with jeune syndrome and the short rib-polydactyly syndromes (srp) i– iv on the basis of their overlapping radiological pheno- types. mutations in ift , which encodes an intraflagellar transport pro- tein, account for a small proportion of patients with jeune syndrome [beales et al., ] and mutations of the gene encoding a second cilia-related protein, dync h , which is a component of the cytoplasmic dynein complex, have recently been identified in some patients with the jeune phenotype and patients with short rib-polydactyly type [dagoneau et al., ]. the mechanism by which mutations in these cilia-related proteins lead to chondro- dysplasia is likely to be aberrant response to hedgehog signaling. putting together these findings and considering the central role of ihh signaling in the growth plate, the chondrocyte primary cilia is a crucial organelle for skeletal development. acknowledgments this work was funded by the spanish ministry of science and innovation (saf- ), ramon areces founda- tion and the european union (lshm-ct- - ). we thank dr. michael wright and dr. mostafa for providing us with the photograph shown in figures and , respectively. references beales pl, bland e, tobin jl, bacchelli c, tuysuz b, hill j, rix s, pearson cg, kai m, hartley j, johnson c, irving m, elcioglu n, winey m, tada m, scambler pj. . ift , which encodes a conserved intraflagellar transport protein, is mutated in jeune asphyxiating thoracic dystrophy. nat genet : – . byrnes am, racacho l, grimsey a, hudgins l, kwan ac, sangalli m, kidd a, yaron y, lau yl, nikkel sm, bulman de. . brachydactyly a- mutations restricted to the central region of the n-terminal active fragment of indian hedgehog. eur j hum genet : – . cahuana a, palma c, gonzales w, gean e. . oral manifestations in ellis-van creveld syndrome: report of five cases. pediatr dent : – . curry cj, hall bd. . polydactyly, conical teeth, nail dysplasia, and short limbs: a new autosomal dominant malformation syn- drome. birth defects orig artic ser : – . da silva eo, janovitz d, de albuquerque sc. . ellis-van creveld syndrome: report of cases in an inbred kindred. j med genet : – . dagoneau n, goulet m, genevieve d, sznajer y, martinovic j, smithson s, huber c, baujat g, flori e, tecco l, cavalcanti d, dele- zoide al, serre v, le merrer m, munnich a, cormier-daire v. . dync h mutations cause asphyxiating thoracic dys- trophy and short rib-polydactyly syndrome, type iii. am j hum genet : – . digilio mc, marino b, ammirati a, borzaga u, giannotti a, dallapiccola b. . cardiac malformations in patients with oral-facial- skeletal syndromes: clinical similarities with heterotaxia. am j med genet : – . ellis rwb, van creveld s. . a syndrome characterized by ectodermal dysplasia, poly- dactyly, chondro-dysplasia and congenital morbus cordis. arch dis chil : – . ferrante mi, zullo a, barra a, bimonte s, messaddeq n, studer m, dolle p, franco b. . oral-facial-digital type i protein is required for primary cilia formation and left-right axis specification. nat genet : – . galdzicka m, patnala s, hirshman mg, cai jf, nitowsky h, egeland ja, ginns ei. . a new gene, evc , is mutated in ellis-van creveld syndrome. mol genet metab : – . gao b, hu j, stricker s, cheung m, ma g, law kf, witte f, briscoe j, mundlos s, he l, cheah ks, chan d. . a mutation in ihh that causes digit abnormalities alters its signalling capacity and range. nature : – . giknis fl. . single atrium and the ellis-van creveld syndrome. j pediatr : – . goddeeris mm, rho s, petiet a, davenport cl, johnson ga, meyers en, klingensmith j. . intracardiac septation requires hedge- hog-dependent cellular contributions from outside the heart. development : – . hattab fn, yassin om, sasa is. . oral manifestations of ellis-van creveld syn- drome: report of two siblings with unusual dental anomalies. j clin pediatr dent : – . haycraft c, zhang q, song b, jackson w, detloff p, serra r, yoder b. . intraflagellar transport is essential for endochondral bone formation. development : – . hellemans j, coucke pj, giedion a, de paepe a, kramer p, beemer f, mortier gr. . homozygous mutations in ihh cause acrocapitofemoral dysplasia, an autosomal recessive disorder with cone-shaped epiphyses in hands and hips. am j hum genet : – . hilton m, tu x, cook j, hu h, long f. . ihh controls cartilage development by antago- nizing gli , but requires additional effectors to regulate osteoblast and vascular develop- ment. development : – . howard td, guttmacher ae, mckinnon w, sharma m, mckusick va, jabs ew. . autosomal dominant postaxial polydactyly, nail dystrophy, and dental abnormalities map to chromosome p , in the region con- taining the ellis-van creveld syndrome locus. am j hum genet : – . huangfu d, anderson k. . cilia and hedge- hog responsiveness in the mouse. proc natl acad sci usa : – . huangfu d, liu a, rakeman a, murcia n, niswander l, anderson k. . hedgehog signalling in the mouse requires intraflagellar transport proteins. nature : – . the mechanism by which mutations in these cilia-related proteins lead to chondrodyspla- sia is likely to be aberrant response to hedgehog signal- ing. putting together these findings and considering the central role of ihh signaling in the growth plate, the chondrocyte primary cilia is a crucial organelle for skeletal development. american journal of medical genetics part c (seminars in medical genetics) article koziel l, wuelling m, schneider s, vortkamp a. . gli acts as a repressor downstream of ihh in regulating two distinct steps of chondrocyte differentiation. development : – . mckusick v, egeland j, eldridge r, krusen d. . dwarfism in the amish i. the ellis- van creveld syndrome. bull johns hopkins hosp : – . mclellan js, zheng x, hauk g, ghirlando r, beachy pa, leahy dj. . the mode of hedgehog binding to ihog homologues is not conserved across different phyla. nature : – . mortier gr, kramer pp, giedion a, beemer fa. . acrocapitofemoral dysplasia: an auto- somal recessive skeletal dysplasia with cone shaped epiphyses in the hands and hips. j med genet : – . mostafa mi, temtamy sa, el-gammal ma, mazen im. . unusual pattern of inheritance and orodental changes in the ellis-van creveld syndrome. genet couns : – . polymeropoulos mh, ide se, wright m, good- ship j, weissenbach j, pyeritz re, da silva eo, ortiz de luna ri, francomano ca. . the gene for the ellis-van creveld syndrome is located on chromosome p . genomics : – . roubicek m, spranger j. . weyers acrodental dysostosis in a family. clin genet : – . ruiz-perez vl, ide se, strom tm, lorenz b, wilson d, woods k, king l, francomano c, freisinger p, spranger s, marino b, dallapiccola b, wright m, meitinger t, polymeropoulos mh, goodship j. . mutations in a new gene in ellis-van creveld syndrome and weyers acrodental dysostosis. nat genet : – . ruiz-perez v, tompson s, blair h, espinoza- valdez c, lapunzina p, silva e, hamel b, gibbs j, young i, wright m., et al. . mutations in two nonhomologous genes in a head-to-head configuration cause ellis- van creveld syndrome. am j hum genet : – . ruiz-perez vl, blair hj, rodriguez-andres me, blanco mj, wilson a, liu yn, miles c, peters h, goodship ja. . evc is a positive mediator of ihh-regulated bone growth that localises at the base of chon- drocyte cilia. development : – . spranger s, tariverdian g. . symptomatic heterozygosity in the ellis-van creveld syndrome? clin genet : – . sund kl, roelker s, ramachandran v, durbin l, benson dw. . analysis of ellis van creveld syndrome gene products: implica- tions for cardiovascular development and disease. hum mol genet : – . takeda h, takami m, oguni t, tsuji t, yoneda k, sato h, ihara n, itoh t, kata sr, mishina y, womack je, moritomo y, sugimoto y, kunieda t. . positional cloning of the gene limbin responsible for bovine chondrodysplastic dwarfism. proc natl acad sci usa : – . temtamy sa, aglan ms, valencia m, cocchi g, pacheco m, ashour am, amr ks, helmy sm, el-gammal ma, wright m, lapunzina p, goodship ja, ruiz-perez vl. . long interspersed nuclear element- (line )- mediated deletion of evc, evc , c orf , and stk b in ellis-van creveld syndrome with borderline intelligence. hum mutat : – . tompson sw, ruiz-perez vl, wright mj, goodship ja. . ellis-van creveld syndrome resulting from segmental unipar- ental disomy of chromosome . j med genet :e . tompson s, ruiz-perez v, blair h, barton s, navarro v, robson j, wright m, goodship j. . sequencing evc and evc identifies mutations in two-thirds of ellis- van creveld syndrome patients. hum genet : – . ulucan h, gül d, sapp jc, cockerham j, johnston jj, biesecker lg. . extending the spectrum of ellis van creveld syndrome: a large family with a mild mutation in the evc gene. bmc med genet : . valencia m, lapunzina p, lim d, zannolli r, bartholdi d, wollnik b, al-ajlouni o, eid ss, cox h, biuoni s, et al. (in press). widening the mutation spectrum of evc and evc : ectopic expression of weyers variants in nih t fibroblasts disrupts hedgehog signalling. hum mutat. weyers h. . a correlated abnormality of the mandible and extremities (dysostosis acrofacialis). fortschr geb rontgenstr : – . ye x, song g, fan m, shi l, jabs ew, huang s, guo r, bian z. . a novel heterozygous deletion in the evc gene causes weyers acrofacial dysostosis. hum genet : – . yoneda k, moritomo y, takami m, hirata s, kikukawa y, kunieda t. . localization of a locus responsible for the bovine chondrodysplastic dwarfism (bcd) on chro- mosome . mamm genome : – . zannolli r, buoni s, viviano m, macucci f, d’ambrosio a, livi w, mazzei ma, mazzei f, sacco p, volterrani l, vonella g, orsi a, zappella m, hayek j. . polydactyly with ectodermal defect, osteo- penia, and mental delay. j child neurol : – . article american journal of medical genetics part c (seminars in medical genetics) the rural midwest since world war ii ed. by j. l. anderson (review) the rural midwest since world war ii ed. by j. l. anderson (review) jeff bremer middle west review, volume , number , fall , pp. - (review) published by university of nebraska press doi: for additional information about this article [ this content has been declared free to read by the pubisher during the covid- pandemic. ] https://doi.org/ . /mwr. . https://muse.jhu.edu/article/ https://doi.org/ . /mwr. . https://muse.jhu.edu/article/ book reviews s and s midwest. while his enthusiasm for and commitment to midwestern literature should be commended, his study generalizes too much and fails to truly articulate valuable differences and nuance that can illustrate the complexity of emotions and ideas that these writers bring to the region. sara kosiba troy university montgomery, alabama j. l. anderson, ed., the rural midwest since world war ii. dekalb: northern illinois university press, . pp. $ . , paper. this outstanding collection of essays provides a comprehensive survey of an area that is often overlooked by scholars. the rural midwest is a region of idealized innocence, where the sentimental notions of american culture have created a society that produces superman and garrison keillor’s lake wobegon. the mythology of the modern midwest— that of a peaceful ru- ral place where community values remain in the face of a materialistic and selfi sh world— stands in stark contrast to the images of excess that often defi ne places such as southern california and new york city. this bucolic world is mostly gone, if it ever existed at all, and the forces of globaliza- tion, deindustrialization, agricultural consolidation, and demographic change have transformed the most american of all regions. this book, edited by j. l. anderson, provides a much needed historical correction to the stereotypes of an agricultural utopia populated by white americans driving pickup trucks. today’s rural midwest is a highly diverse place, rapidly evolving due to immigration and economic modernization and increasingly dependent on the federal government. most of the mid- west’s population is urban, and less than ten percent of rural people live on farms. most farm income does not even come from agriculture, but from off- farm employment. the rural midwest since world war ii contains a rich variety of essays explor- ing how this region has changed in the past seven decades. useful chapters on ecology and environmental history open the book, followed by essays on manufacturing, government policy, women, children and ethnic and reli- gious minorities. a brief introduction and a concluding overview put the re- gion in its historical and sociological context. there is not a weak chapter in middle west review • vol. no. this book, and its authors— pamela riney- kehrberg, debra reid, and david danbom, among others— are all noted scholars in their fi elds. j. l. anderson’s introduction makes clear that the recent history of the rural midwest contradicts the supposedly static world of small towns and farms. serious challenges, such as depopulation, have joined with social and economic divisions to endanger the survival of key aspects of midwest- ern life. the wrenching farm crisis of the s demonstrated the dangers of farming. shuttered small town main streets provided stark reminders of the negative consequences of capitalism’s creative destruction. as the rural world of the mid- twentieth century faded, the federal government became increasingly important. rural life also became more like that of the nation overall, thanks to improved transportation and communication. suburbs and exurbs have transformed the countryside, as urban workers chose to live away from cities and commute to work. the distinctiveness of rural life has faded since the s. chapters clearly summarize recent scholarship, while keeping historical debates limited. each chapter is about twenty- fi ve pages and would supply plenty of material for undergraduate discussion. the book is also a great place for historians to mine information for lecture material; detailed foot- notes offer fruitful ideas for further research. chapters on the environment lay an excellent foundation for later essays. while midwestern society has become less homogenous since world war ii, the land has become more so. the countryside became more simplifi ed, james pritchard argues, and widespread agriculture has modifi ed the geography. river improvements and habitat destruction have transformed the region. other changes have infl uenced the ecology, including crop monoculture, regular antibiotic use in animals, and a pervasive reliance on fertilizers. even as many farmers turned to corn or soybeans, others grew new crops, from cherries to vege- tables to sugar beets. the demand for workers helped pull nonwhite labor- ers into the midwest and changed the demography of rural areas. the midwest experienced great economic change after world war ii. while the phrase “rust belt” suggests the departure of industry from ur- ban areas, some industry moved into rural spaces. meatpacking and etha- nol production partially replaced traditional agricultural- related industry. improved highways and the growth of trucking helped shift manufacturing away from cities. wilson warren’s essay “beyond the rust belt” sprawls across several centuries and is less focused on the post- period, but it places industrialization in its long- term context. cornelia and jan flora book reviews deftly survey the impact of social and economic change on rural communi- ties, tracing the effects of a shrinking and aging population. cultural ho- mogeneity declined along with many main streets, and the acceptance of ethnic diversity increased. anderson explains the important role of the government in rural areas in his essay on the subject. the new deal and the second world war ex- panded its reach and power. federal farm policy has had a major role in ag- riculture and farmers have debated the merits of subsidies for decades, ac- cepting them in hard times and discussing their demise in boom times. in addition to the farm bill, electrifi cation, road construction, educational as- sistance, research and other programs have helped sustain communities. essays by jenny barker devine on farmwomen and pamela riney- kehrberg on children explain how life for these groups changed after world war ii. women’s work, on and off the farm, helped support fami- lies. they took on roles as farm operators and managers, just as the lives of their children changed as well. rural childhood increasingly came to mir- ror that of their suburban counterparts. farm kids worked for their par- ents, but they gained access to a national youth culture of sports, televi- sion, fi lm, and scouting. the fi nal chapters trace the postwar history of various ethnic and re- ligious groups— african americans, latinos, and the amish. the black population of the midwest was limited, at only about one percent of to- tal population. debra reid explains how rural black midwesterners faced the same challenges that african americans faced nationwide. black mi- grants usually bypassed the rural midwest and headed to northern cities. white landowners were usually not interested in selling or leasing land to african americans. white supremacy also left blacks facing economic dis- advantage and cultural isolation. in , for example, there were forty- seven black farmers in iowa. farming was the “whitest of occupations,” reid contends. the latino population of the midwest has boomed in the past several decades, pushed away from border states by poor education systems, dangerous barrios, and racial intolerance. in some midwestern states latino numbers doubled in the s. meatpacking and farm labor brought thousands to the region. widespread “white fl ight” from areas of latino settlement proves that this group was not easily folded into the re- gion, historian jim norris insists. a fi nal chapter on the amish ably docu- ments the historical experience of this oft- romanticized group. the rural midwest since world war ii is a superb collection of essays that middle west review • vol. no. smartly reviews the history and culture of this neglected region. it helps to fi ll a historiographical gap and gives scholars a starting point for future research. it is also one of the best written and most comprehensive edited works that this reviewer has read. anyone who studies, or cares about, this region will want to have a copy on his or her bookshelf. jeff bremer iowa state university ames, iowa wilfred m. mcclay and ted v. mcallister, eds., why place matters: geography, identity, and civic life in modern america. new york: encounter books, . pp. $ . . the essays in this volume edited by wilfred m. mcclay, a historian at the university of oklahoma, and ted v. mcallister, a professor of public poli- cy at pepperdine university, emerged out of conferences on the subject of place in march and march at pepperdine’s school of public poli- cy in malibu, california. a book focusing upon the importance of place in people’s lives will be of special interest to readers of a journal devoted to middle western history and culture. although the only reference to the midwest in the index is to andrew r. l. cayton and peter s. onuf ’s the midwest and the nation ( ), several of the sixteen authors whose essays appear in the volume hail from the re- gion: joseph a. amato, whose essay focuses on local history; philip bess, who writes on urban design and the built environment; and yi- fu tuan, who focuses on place, space, identity, and the home. amato is the only one to directly address the theme of regional history, which he fi xes fi rm- ly within a spatial matrix starting with family and local history and pro- ceeding to regional, national, and even global levels of analysis. drawing upon his extensive research and previous books on local and regional his- tory and concentrating especially upon southwestern minnesota and the area around the town of marshall, his essay crackles and sparks with fertile ideas that help to illuminate matters of place, environment, region, and as- sociated matters. students of midwestern history and culture will gain much, too, from the other essays included in this wide- ranging collection. former chair- man of the national endowment for the arts dana gioia’s exuberant de- untitled impaired culture generated cytotoxicity with preservation of spontaneous natural killer-cell activity in cartilage-hair hypoplasia glenn f. pierce, charlotte brovall, bernice z. schacter, and stephen h. polmar, departments of pathology and pediatrics, case western reserve university school of medicine, cleveland, ohio a b s t r a c t recent studies of cartilage-hair hypo- plasia (chh), a form of short-limbed dwarfism, have shown that all affected individuals have a cellular pro- liferation defect that results in a cellular immunode- ficiency. however, only a minority of chh individuals suffer from severe, life-threatening infections. for this reason, relevant immune defense mechanisms that may be responsible for maintaining intact host defen- ses in the majority of chh individuals were studied. spontaneous and allogeneic culture-induced (mixed lymphocyte response-mlr) specific and nonspecific (nk-like) cytotoxic mechanisms were analyzed and correlated with lymphocyte subpopulations present in chh and normal individuals. spontaneous natural- killer (nk) activity was present at or above normal levels, but culture-induced specific cytotoxicity and nk-like cytotoxicity as well as nk-like activity by t cell lines were significantly reduced in chh individ- uals. the generation of radiation-resistant cytotoxicity, which normally occurs during allogeneic mlr, was markedly diminished in chh, and was correlated with the decreased proliferation observed in chh cultures. preservation of spontaneous nk activity and loss of all forms of culture-induced cytotoxicity was associated with an increase in the proportion of lymphocytes bearing a thymic independent nk phenotype (okm + okt - fcy+ low-affinity e+), and a significant de- crease in thymic derived okt + cytolytic t cell sub- populations in chh individuals. therefore, an intact cellular cytotoxic effector mechanism has been iden- tified in chh (i.e., nk activity). natural cytotoxicity may be of importance in maintaining host resistance to viral infections despite diminished thymic-derived effector mechanisms in cartilage-hair hypoplasia. address reprint requests to dr. s. h. polmar, department of pediatrics, washington university school of medicine, p.o. box , st. louis, mo . received for publication july and in revised form december . introduction cartilage-hair hypoplasia (chh)' is an autosomal re- cessive form of short-limbed dwarfism, which is found in increased frequency in the old order amish of the united states, as well as in the finnish population ( , ). it is characterized by short stature, sparse unpig- mented hair, and cellular immunodeficiency ( - ). chh individuals have decreased numbers of circu- lating b and t lymphocytes, negative delayed-type hypersensitivity reactions, and severely depressed pro- liferative responses to b and t cell specific antigens and mitogens. impaired interleukin (il ) production and utilization have been found in studies on contin- uous t cell lines (ctcl), suggesting that the presence of a gc phase defect in the activation of chh t cells ( ). the amish cartilage-hair hypoplasia subjects inves- tigated in our previous studies were healthy and in- fection free, in spite of their significant in vivo and in vitro immunologic abnormalities. only a minority of chh individuals suffer severe, sometimes fatal, viral infections ( - ). thus, we sought to identify im- munological mechanisms present in chh that might account for this apparent paradox. cytotoxicity against virally infected altered-self has been demonstrated by okt + okt + hla-restricted t cytotoxic cells as well as by fc'yy natural killer (nk) cells ( - ). our previous studies noted decreased numbers of okt + (pan-t), okt + (helper/inducer), and okt + (sup- pressor/cytotoxic) t lymphocytes, and a large increase in the proportion of okm + lymphocytes in chh in- ' abbreviations used in this paper: chh, cartilage-hair hypoplasia; cml, cell-mediated lympholysis; ctcl, con- tinuous t cell lines; eae , sheep erythrocytes pretreated with -amino-ethylisothiouronium; fcy', cells bearing receptors for the fc portion of igg; il , interleukin ; mlr, allo- geneic mixed lymphocyte reaction; nk, natural killer; pbmc, peripheral blood mononuclear cells. j. clin. invest. © the american society for clinical investigation, inc. * - / / / / $ . volume june - dividuals ( ). since many okm + lymphocytes me- diate spontaneous nk activity ( - ), and okt + t cells are responsible for proliferation and specific al- logeneic cell-mediated lympholysis (cml) in the mlr ( - ), we have evaluated and compared spontaneous nk activity with allogeneic culture-induced specific cml and nonspecific (nk-like) cytotoxicity in chh and normal individuals. we found that spontaneous nk activity was preserved in chh. however, allo- geneic culture induced specific and nk-like cytotox- icity were markedly diminished in chh. in addition, the induction of radiation-resistant cytotoxicity in the mlr did not occur in lymphocyte cultures from chh patients. since most chh individuals are healthy and survive well into adulthood, these findings suggest a clinically significant role for spontaneous nk cytotox- icity in host defense. methods patients. the nine amish chh individuals (mean age . yr, range - ) who participated in these studies were originally diagnosed by field teams from the johns hopkins hospital, under the direction of dr. v. a. mckusick, using physical examinations, family histories, pedigree analyses, and, in some cases, radiographic and hair sample analyses ( , ). all individuals were clinically healthy at the time of study. six affected subjects had a history of severe, prolonged varicella infection, and four of these individuals also had histories of severe repeated chronic respiratory tract infec- tions. none of the patients studied received vaccinia or polio immunizations in childhood. eight patients had blond body hair, and in three it was exceptionally sparse (lanugo). age- and sex-matched unrelated normal individuals, as well as four unaffected siblings, served as controls. blood samples were drawn in accordance with the guidelines established by the institutional review board of university hospitals of cleveland. cell separation. peripheral blood mononuclear cells (pbmc) were obtained from heparinized whole blood by ficoll-hypaque density gradient centrifugation. pbmc were washed three times, counted, and used for the nk assay and the mixed lymphocyte culture induced cytotoxicity studies. for surface marker analysis, macrophages were removed from pbmc by adherence to plastic in % fetal calf serum (fcs) in rpmi and % co for min. nonadherent cells, designated as peripheral blood lymphocytes (pbl), were decanted and used immediately for cell surface antigen studies. for surface marker analysis of purified t cells, pbmc were rosetted with -amino-ethylisothiouronium- treated sheep erythrocytes (eaet), and eaet+ cells were iso- lated on ficoll-hypaque gradients, as previously described ( ). cell recovery after separation procedures was similar for both chh and normal individuals. fluorescence staining. subpopulation analysis was per- formed using monoclonal antibodies against cell surface de- terminants, as previously described ( ). briefly, okt (pan- t), okt (helper/inducer), and okt (suppressor/cyto- toxic) were purchased from ortho pharmaceutical, raritan, nj. okt (suppressor/cytotoxic), okial (anti-ia), okm , and directly fluorescein-labeled okt , okt , okt , okm were generously provided by dr. patrick kung (ortho phar- maceutical). incubation of pbl and eaet+ cells in % fcs with latex particles ( . gm, difco laboratories, detroit, mi) for min at °c was performed before staining to identify re- sidual macrophages. for staining, - x pbl or eaet+ cells were incubated in duplicate with unlabeled monoclonal antibody for min at °c, washed three times in % bovine serum albumin, then incubated with rhodamine-conjugated goat anti-mouse igg (cappel laboratories, inc., cochran- ville, pa) for min at °c. nonspecific fluorescence was assessed by use of a mouse monoclonal igg of irrelevant specificity, and was usually < %. sheep erythrocyte (e), eaet, and fcy rosettes were de- termined on duplicate samples. for e and eaet rosettes, ul of % (vol/vol) untreated or aet-treated sheep eryth- rocytes, respectively, were added to x lymphocytes in % fcs. cells were centrifuged at g, incubated at °c for min, and viable cells (> %) were read under epi- fluorescence after the addition of acridine orange-ethidium bromide ( ). fc-y rosettes were determined after overnight incubation of x lymphocytes in serum-free media with gl of % igg-coated ox erythrocytes, as previously de- scribed ( ). culture of cml effectors. one-way mlr were initiated by culturing pbmc in round-bottomed tubes in -ml vol at a cell concentration of x /ml with either an equal number of irradiated ( , rad) autologous or allogeneic pbmc in rpmi containing % pooled ab serum. after d, proliferation was assessed in well microcultures by tritiated thymidine incorporation ([ hjtdr; . mci/well, ci/mmol, new england nuclear, boston, ma), as described previously ( ). after d, effector cells were washed, counted, and diluted for the cytotoxic assays. cytotoxicity targets. autologous and allogeneic pbmc targets were maintained in culture for a total of d in % ab serum in the absence of mitogens until they were re- quired for the cytotoxic assays. the erythroleukemia cell line k was maintained in exponential growth culture. cytotoxic assays. fresh or cultured effector pbmc were placed in round-bottomed wells of a microtiter tray with x chromium-labeled (> mci/mg, mci/ml, new england nuclear) target cells at effector to target cell (e/t) ratios of : , : , and . : in ml rpmi and % fcs. the targets used were autologous or allogeneic pbmc, or k . the tray was spun at g for min, incubated for h at °c in % co in air, and then centrifuged at g for min. aliquots ( ul) of the supernatant were then removed and placed in glass tubes for counting in a gamma counter. cytotoxic activity was quantitated as percentage of lysis: % lysis cpm (experimental release) -cpm (spontaneous release) x . cpm (total release) - cpm (spontaneous release) spontaneous release of "chromium from target cells was determined in the presence of medium alone, whereas total release was determined by lysis in % sodium dodecyl sul- fate. spontaneous release from targets was always < %. activity was linear over the range of effector-to-target ratios used. cytotoxicity for autologous or allogeneic targets in autologous mlr-cml cultures was generally less than % of the cytotoxicity found in primed allogeneic mlr cultures directed against allogeneic targets. autologous cytotoxicity in primed allogeneic cultures was less than % of the al- logeneic cytotoxicity. radiation-resistant killing. aliquots of fresh or cultured pbmc were irradiated ( , rad with a cobalt source) to g. f. pierce, c. brovall, b. z. schacter, and s. h. polmar assess radiation-resistant killing, as previously described ( ). irradiated cells were treated identically to nonirradiated cells, except that radiation resistance experiments were per- formed using e/t ratios of : only. intraassay coefficients of variation among triplicate samples were < % for unir- radiated and irradiated samples. ctcl. cell lines were initiated as previously described ( ). briefly, t cell blasts derived from phytohemaglutinin stimulated pbmc were maintained in continuous prolifer- ation by feeding at -d intervals with % (vol/vol) inter- leukin- -containing medium. il was prepared from an ir- radiated, pooled mlr from allogeneic individuals cul- tured in the presence of . ug/ml pha-m (difco labo- ratories). after h, the supernatant was harvested, filtered through a . -am filter, and stored at - °c until used. supernatants were tested for il potency in a ctcl mi- croassay, as previously described ( ). after d of culture, ctcl from two chh and two normal individuals were tested for cytotoxicity against k targets as described above. statistical analysis. a two-tailed unpaired student's t test was used to evaluate significance levels. critical values of the sample correlation coefficient, r, were determined in a two-tailed test. results cytotoxic mechanisms in chh and normal indi- viduals. spontaneous nk activity was at or above normal levels in all chh individuals studied (fig. a). fresh pbmc from eight chh individuals lysed . ± . % (mean±se) of k target cells in the -h nk assay at a : e/t ratio, compared with . ± . % lysis by fresh pbmc from eight normal individuals (fig. ia). the killing was linear for all e/t ratios tested, and no significant differences were found be- tween the two groups. in contrast, minimal mlr-induced specific cell me- diated lympholysis could be generated in chh indi- viduals ( . ± . % at e/t : ), whereas pbmc from normal subjects sensitized in an mlr lysed . ± . % of the targets at the same e/t ratio (p < . , fig. la). three chh individuals had no detectable cml. decreased mlr-induced proliferation was found against each of the allogeneic individuals used as spe- cific targets in the cml assay (chh = , ± cpm, normal controls = , ± , cpm; p < . ; fig. b). this was not due to a lack of hla-d locus differences, since in this series, as in previous studies ( , ), the proliferative response to a pool of allo- geneic individuals was significantly diminished (chh = , ± cpm; normal = , ± cpm; p < . ). nk-like cytotoxicity against k was also assayed in the -d allogeneic mlr cultures. nk-like activity against k was significantly decreased in chh in- dividuals, compared with normals (p < . , fig. a). thus, chh individuals showed a markedly de- creased specific cml, as well as nk-like cytotoxicity l pnms\wllll' _llm~__s ank nkc- i-.. lll omeeemmm ike mlr -cml % cytotoxicity (e/t : )) b mlr cpm "h-tdr (x -) figure cytotoxic mechanisms present in eight chh (solid bars) and eight normal (striped bars) individuals. (a) spontaneous nk cytotoxic activity was measured on day against k . culture-induced nonspecific (nk-like, p < . ) and specific (mlr-cml, p < . ) cytotoxicity was measured after d using k or mlr stimulator cells, respectively. (b) allogeneic mlr-induced proliferation was assessed in microcultures after d (p < . ). after d of allogeneic culture, but showed normal spontaneous activity in the nk assay. radiation sensitivity and the generation of radia- tion-resistant killing. previous studies revealed that radiation sensitivity of spontaneous nk activity from healthy control subjects was under the control of an x-linked gene, with radiation resistance found in % of caucasian subjects ( ). nk-like and specific killing by mlr-generated effector cells prepared from ra- diation-sensitive subjects was found to be radiation resistant. therefore, it was of interest to determine the relative ability of chh effectors to develop radiore- sistance in allogeneic mlr cultures. spontaneous nk activity was sensitive to irradiation in all chh indi- viduals tested, as well as in radiosensitive control sub- jects (data not shown). culture-induced cml effectors from chh individuals retained minimal cytotoxic ac- tivity after irradiation, compared with those from nor- mal individuals, p < . (table i). in contrast, the amounts of radiosensitive cytotoxicity of chh and normal cml effectors were not significantly different. similarly decreased radiation resistance was also found for nk-like activity against k induced in allogeneic cultures in chh cells, compared with normal cells p < . , (table ii). radiation-sensitive nk-like activity was comparable in chh and normal cells. the gen- eration of radioresistant nk-like and specific cytotox- icity was correlated with allogeneic mlr-induced pro- liferation in seven chh and normal individuals (pro- liferation vs. specific radioresistant cytotoxicity, chh: r = . , p < . ; normal: r = . ; p < . ; pro- cytotoxic activity in cartilage-hair hypoplasia table i radiation resistance of cml activity in chh and nornml individuals cytotoxic activity' cytotoxic activity' radiation radiation normal radiation radiation chit subject total resistanti sensitive§ subject total resistant sensitive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mean±sem . ± . . ± . . ± . . ± . . ± . . ± . pl" < . < . ns - % 'cr release. radiation-resistant cytotoxic activity is the cytotoxic activity remaining after effector cells have been irradiated with , rad. § radiation-sensitive cytotoxic activity is the difference between the total cytotoxic activity minus the radiation-resistant activity. comparison of chh vs. normal by two-tailed unpaired student's t tests. liferation vs. radioresistant nk-like activity; chh: r = . , p < . ; normal: r = . , p < . ). lymphocyte subpopulation analysis. it was of in- terest to relate the cytotoxic activities present in chh pbmc to the lymphocyte subpopulations known to mediate cytotoxicity that are present in these individ- uals. in earlier work ( ), we found a decrease in okt + (pan-t), okt + (helper/inducer), and okt + (sup- pressor/cytotoxic) t lymphocytes, normal proportions of eaet+ cells, and a marked increase in percentages of okm + lymphocytes in chh. the monoclonal an- tibody analyses of pbl of the chh and normal in- dividuals in the present study is shown in table iii and are similar to our previous findings ( ). okt + cells, reactive with a monoclonal antibody that iden- tifies a subset of suppressor/cytotoxic t cells similar but not identical to the okt + subpopulation, were also decreased in chh individuals (p < . ). e+ cells were significantly lower, but eaet+ cells, which con- tain more low-affinity rosette-forming cells, were in normal range, in spite of a marked decrease in okt + cells in chh patients, compared with controls. in purified eaet+ cell fractions made from chh pbmc, okt + cells were decreased (p < . ), and table ii radiation resistance of nk-like activity in chh and normal individuals cytotoxic activity' cytotoxic activity' radiation radiation normal radiation radiation chh subject total resistanti sensitive§ subject total resistant sensitive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mean±sem . ± . . ± . . ± . . ± . . ± . . ± . pl" < . < . ns % 'cr release. radiation-resistant cytotoxic activity is the cytotoxic activity remaining after effector cells have been irradiated with , rad. § radiation-sensitive cytotoxic activity is the difference between the total cytotoxic activity minus the radiation resistant activity. comparison of chh vs. normal by two-tailed unpaired student's t tests. g. f. pierce, c. brovall, b. z. schacter, and s. h. polmar table iii surface marker analysis of subpopulations in chh and normal individuals % positive peripheral blood lymphocytes' subjects okt okt okt okt okm eleaett chh (n = ) ± ± ± ± ± ± ± normal (n = ) ± ± ± ± ± ± ± p§ < . < . < . < . < . < . ns ° the absolute lymphocyte count, calculated from the wbc and differential, for chh individuals was + /mm ; normals ± ; p < . . t e rosettes were determined using untreated sheep erythrocytes; eaet rosettes were determined using sheep erythrocytes pretreated with aet. both were read following a -min incubation at °c. § significance levels were determined by two-tailed unpaired student's t tests. both fc-y' and okm + lymphocytes were significantly increased (p < . , table iv). in chh individuals, there was a strong positive correlation between the increased percentages of fc"y cells and okm + cells (r = . , p < . ). in normal subjects, fcy'y and okm + cells represented only a small proportion of the total lymphocytes. in chh, nk activity was sig- nificantly correlated with the number of okm + cells (r = . , p < . ), as well as with the percentage of fc-y' cells (r = . , p < . ). in normal subjects, all nk activity does not appear to be mediated by okm + cells ( , ). thus, no significant correlations would be expected and none were detected. therefore, it appears that the subpopulation bearing an nk cell phenotype (fcy' okm + okt - okt - low affinity e+) is proportionally increased in chh individuals, whereas thymus derived okm - okt + okt + eaet+ cells, which mediate cytotoxic t-cell activity, are selectively depleted. these findings are consistent with the cytotoxicity studies in chh in which nk activity is preserved while cml is markedly reduced. table iv subpopulation analysis of eaet + cells in chh and normal individuals % positive eaet + cells' subjects fc t okml okt chh (n = ) ± § ± ± normal (n = ) ± ± ± p§ < . < . < . * eaft cells were obtained after rosetting pbmc with aet-treated sheep erythrocytes. t fcy rosettes were determined after overnight incubation with igg-coated ox erythrocytes in serum-free media. § significance levels were determined by unpaired two-tailed t tests. anti-k activity generated in ctcl. analysis of ctcl confirmed the absence of culture-generated nk-like activity in chh individuals. ctcl depen- dent on il were established simultaneously from two normal and two chh subjects. both chh ctcl showed diminished cytotoxicity against k targets when tested after d of culture (table v). no okm + or fc"y cells were detected in either chh or normal ctcl cultures. cells from both groups were greater than % eaet+ and okt +. both chh and normal ctcl were - % okt + and % okt + and mor- phologically resembled -d phytohemaglutinin blasts. okial detected - % of the cells from both groups of individuals. as in allogeneic mlr-cml cultures, radioresistant cytotoxicity could not be generated in chh ctcl (table v). discussion marked impairment of lymphocyte proliferation has been described in cartilage-hair hypoplasia ( - ). in table v radiation resistance of nk-like cytotoxic activity of ctcl from chh and normal individuals cytotoxic activity' radiation radiation ctcl total resistant i sensitivel chh . ± . . ± . . ± . chh . ± . . + . . ± . normal . ± . . ± . . ± . normal . ± . . ± . . ± . % "cr release. radiation-resistant cytotoxic activity is the cytotoxic activity re- maining after effector cells have been irradiated with , rad. § radiation-sensitive cytotoxic activity is the difference between the total cytotoxic activity minus the radiation-resistant activity. cytotoxic activity in cartilage-hair hypoplasia this paper, we report that cellular cytotoxicity effector mechanisms are impaired but not constitutively ab- sent. culture-induced specific and nk-like cytotox- icity mediated by okt + t-cells is proliferation dependent, and was found to be absent or markedly diminished in chh cultures. however, proliferation- independent spontaneous natural cytotoxicity is intact in chh. individuals with cartilage-hair hypoplasia appear to have a selective loss of high-affinity e-ro- setting cells which are okt +, whereas low-affinity e rosette-positive cells bearing the okm and fc-y receptor markers are preserved. spontaneous nk activity against k has been shown to be mediated by fcy' okm + okt - e+ and e- (null) large granular lymphocytes (lgl) ( - , - ). in contrast, allogeneic mlr-induced specific cytotoxicity is mediated by the okt + t /t + t lym- phocyte subpopulation ( - ), whereas the pheno- type of the cells mediating nk-like culture-induced cytotoxicity has not been established. however, recent studies by lopez-botet et al. ( ) suggest that nk-like activity is mediated by an il -responsive okt + t- cell blast. seeley et al. ( ) found that mlr-cml and nk-like against k were mediated by different cell types. they, and others, also reported that the anti- k culture-induced killing was not mediated by fc-y' cells, in contrast to spontaneous nk activity ( - ). our results support the concept that the cells mediating spontaneous nk activity are distinct from nk-like and mlr-cml, since (a) nk-like and specific cytolysis could not be generated in significant amounts in chh individuals with impaired t cell function after culture, but nk activity was found in freshly isolated pbmc, and (b) fcy' and okm + cells decrease rap- idly in mlr cultures, as culture-induced cytotoxicity increases, and are not present on ctcl, which bear strictly t-cell phenotypes (okt +t + or okt +t +). using clonally selected specific cytotoxic ctcl, others have also demonstrated the t cell nature of nonspecific anti-k cytotoxicity ( , ). moretta et al. ( ) re- ported three out of four anti-k clones were okt + and all were e+ and fcy-. the inability of chh individuals to recruit okt +/ t + cells to function in both hla-restricted (mlr- cml) and nonrestricted killing (nk-like) is due, at least in part, to decreased production and utilization of interleukin by chh t cells ( ). the clonal ex- pansion of mlr-generated specific cml is dependent upon helper factors (il ) synthesized by some okt + cells during the mlr sensitization ( , , ). radio- resistant nonspecific cytotoxicity can be recruited by il -containing media ( ). however, decreased il production by chh okt + cells cannot entirely ex- plain the persistent radiosensitivity and severely de- pressed levels of anti-k activity by chh ctcl, since excess exogenously supplied il permits the re- cruitment of radioresistant effectors in normal lines, which themselves do not produce il . the failure of chh pbmc and ctcl to develop culture-induced radioresistance supports the concept of defective re- cruitment due to an intrinsic defect in chh t cells, which is also associated with decreased responsiveness to il ( ). in contrast, thymic-independent sponta- neous nk activity does not require cell proliferation or soluble recruitment factors. interestingly, the athymic nude mouse also has normal spontaneous nk activity and diminished culture generated cytolysis. however, nu/nu mice appear to have an isolated il production deficit, and can generate cytolytic t cell effectors when exogenous il is provided ( ). the in vivo role of spontaneous nk activity, as well as antibody-dependent cellular cytotoxicity, which is mediated by the same cell population, remains to be elucidated. deficient spontaneous nk activity has been found in x-linked lymphoproliferative syndrome ( ) and chediak-higashi syndrome (c-hs) ( ). in both diseases, as well as in the beige mouse model of c-hs, the selective loss of nk effectors is associated with an increased susceptibility to infections and lym- phoproliferative disorders ( ). only a minority of in- dividuals with cartilage-hair hypoplasia have a history of severe viral infections (usually varicella, vaccinia, or polio) as well as recurrent, persistent respiratory infections, and in only a minority of these individuals is the outcome lethal (unpublished observations). most chh individuals are clinically healthy and survive well into adulthood despite profound in vitro cellular immune dysfunction ( , ). moreover, rates of lym- phoproliferative and autoimmune diseases do not ap- pear to be increased in chh individuals (unpublished observations). spontaneous natural cytotoxicity has been proposed to mediate host-defense against certain viral infections (i.e., vaccinia, cytomegalovirus, mea- sles) ( - ), as well as immune surveillance against malignancies ( ), and our studies of chh support this theory. our observations in chh lend support to the importance of nk activity in human host defenses and raise questions regarding the relative importance of proliferation-dependent t cell mediated cytotoxic mechanisms. thus, cartilage-hair hypoplasia provides a unique human model of lifelong partial cellular im- munodeficiency for the analysis of the in vivo require- ments for immunocompetence. acknowledgments we thank dr. patrick kung and dr. gideon goldstein for their advice relating to some of the immunofluorescence studies. this study was supported in part by national institutes of health grants al , ai , and rr . glenn f. pierce was supported by medical scientist training pro- gram grant gm . g. f. pierce, c. brovall, b. z. schacter, and s. h. polmar references . mckusick, v. a., r. eldridge, j. a. hostetler, u. ruang- wit, and j. a. egeland. . dwarfism in the amish. ii. cartilage-hair hypoplasia. bull. johns hopkins hosp. : - 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. . roder, j. c., t. haliotis, m. klein, s. korec, j. r. jett, j. ortaldo, r. b. herberman, p. katz, and a. s. fauci. . a new immunodeficiency disorder in humans in- volving nk cells. nature (lond.). : - . . herberman, r. b., and j. r. ortaldo. . natural killer cells: their role in defenses against disease. science (wash. dc). : - . cytotoxic activity in cartilage-hair hypoplasia codas syndrome is associated with mutations of lonp , encoding mitochondrial aaa+ lon protease article codas syndrome is associated with mutations of lonp , encoding mitochondrial aaaþ lon protease kevin a. strauss, , , , ,* robert n. jinks, , erik g. puffenberger, , , sundararajan venkatesh, kamalendra singh, , iteen cheng, natalie mikita, jayapalraja thilagavathi, jae lee, stefan sarafianos, abigail benkert, , alanna koehler, anni zhu, victoria trovillion, madeleine mcglincy, thierry morlet, matthew deardorff, , a. micheil innes, chitra prasad, albert e. chudley, , irene nga wing lee, and carolyn k. suzuki , codas syndrome is a multi-system developmental disorder characterized by cerebral, ocular, dental, auricular, and skeletal anomalies. using whole-exome and sanger sequencing, we identified four lonp mutations inherited as homozygous or compound-heterozygous combinations among ten individuals with codas syndrome. the individuals come from three different ancestral backgrounds (amish- swiss from united states, n ¼ ; mennonite-german from canada, n ¼ ; mixed european from canada, n ¼ ). lonp encodes lon protease, a homohexameric enzyme that mediates protein quality control, respiratory-complex assembly, gene expression, and stress responses in mitochondria. all four pathogenic amino acid substitutions cluster within the aaaþ domain at residues near the atp-bind- ing pocket. in biochemical assays, pathogenic lon proteins show substrate-specific defects in atp-dependent proteolysis. when ex- pressed recombinantly in cells, all altered lon proteins localize to mitochondria. the old order amish lon variant (lonp c. c>g[p.arg gly]) homo-oligomerizes poorly in vitro. lymphoblastoid cell lines generated from affected children have ( ) swollen mitochondria with electron-dense inclusions and abnormal inner-membrane morphology; ( ) aggregated mt-co , the mtdna-encoded subunit ii of cytochrome c oxidase; and ( ) reduced spare respiratory capacity, leading to impaired mitochondrial pro- teostasis and function. codas syndrome is a distinct, autosomal-recessive, developmental disorder associated with dysfunction of the mitochondrial lon protease. introduction cerebral, ocular, dental, auricular, skeletal (codas) syn- drome (mim ) was first described in as a distinctive constellation of developmental delay, craniofa- cial anomalies, cataracts, ptosis, median nasal groove, delayed tooth eruption, anomalous cusp morphology, malformed helices, hearing loss, short stature, delayed epiphyseal ossification, metaphyseal hip dyplasia, and vertebral coronal clefts. codas is a rare disease; only three additional cases (from brazil, canada, and france) were reported between and . – autosomal- recessive inheritance of codas syndrome was first sug- gested by shebib et al., who identified the phenotype within an endogamous mennonite community, and it was corroborated by the observations of innes et al. we used whole-exome and sanger sequencing to study ten individuals with codas syndrome from three distinct ancestral backgrounds: amish-swiss from the united states (n ¼ ), mennonite-german from canada (n ¼ ), and clinic for special children, strasburg, pa , usa; lancaster general hosp dations of behavior program, franklin and marshall college, lancaster, pa genetics, new jersey medical school, rutgers, the state university of new jerse university, cleveland, oh , usa; department of molecular microbiolo missouri, columbia, columbia, mo , usa; auditory physiology and psy ton, de , usa; division of human genetics, the children’s hospital of p man school of medicine, university of pennsylvania, philadelphia, pa research institute, cumming school of medicine, university of calgary, ca pediatrics, children’s health research institute and western university, lond university of manitoba, winnipeg, mb r a s , canada; department of mb r a s , canada these authors contributed equally to this work *correspondence: kstrauss@clinicforspecialchildren.org http://dx.doi.org/ . /j.ajhg. . . . � by the american societ the amer mixed european (german, scottish, irish, english) from canada (n ¼ ). our analysis revealed four different mutations within lonp (refseq accession number nm_ . [isoform ]; [mim ]). these muta- tions were either homozygous (lonp c. c>g [p.arg gly] and lonp c. c>t [p.pro ser]) or compound heterozygous (lonp c. c>a/c. c>t [p.ser tyr/p.ala val]) (table ). in humans, lonp encodes the atp-dependent mitochondrial lon protease, which belongs to the aaaþ superfamily of atpases associ- ated with various cellular activities. aaaþ proteins share a conserved atpase module and mediate diverse cellular pro- cesses such as dna replication, membrane fusion, signal transduction, and transcriptional regulation. – lon is an atp-driven proteolytic machine and is highly conserved from archaea to mammals. in human mito- chondria, lon is the simplest of four energy-dependent proteolytic systems that include clpxp, m-aaa, and i-aaa. mutations of clpp (mim ), which encodes the proteolytic component of clpxp, are associated with ital, lancaster, pa , usa; department of biology and biological foun- , usa; department of microbiology, biochemistry, and molecular y, newark, nj , usa; department of chemistry, case western reserve gy and immunology, christopher bond life sciences center, university of choacoustics research laboratory, dupont hospital for children, wilming- hiladelphia, philadelphia, pa , usa; department of pediatrics, perel- , usa; department of medical genetics and alberta children’s hospital lgary, ab t n n , canada; medical genetics program, department of on, on n c v , canada; department of pediatrics and child health, biochemistry and medical genetics, university of manitoba, winnipeg, y of human genetics. all rights reserved. ican journal of human genetics , – , january , mailto:kstrauss@clinicforspecialchildren.org http://dx.doi.org/ . /j.ajhg. . . http://crossmark.crossref.org/dialog/?doi= . /j.ajhg. . . &domain=pdf table . four pathogenic lonp alleles among ten individuals with codas syndrome origin of sample n ancestry lonp variants previously reportedallele a allele a pennsylvania amish-swiss arg gly arg gly canada mennonite-german pro ser pro ser shebib et al., canada mixed european ser tyr ala val ain every case, genotyping showed allele segregation consistent with recessive inheritance. perrault syndrome type (mim ), and mutations of spg , which encodes a subunit of m-aaa, are linked to hereditary spastic paraplegia (mim ). in humans, lon functions as a protease, chaperone, and dna-binding protein. it contributes to mitochondrial proteostasis by eliminating misfolded and oxidatively damaged pro- teins – and supports cell viability during proteotoxic, hypoxic, and endoplasmic-reticulum stress. – lon also degrades key regulatory proteins of mitochondrial metabolism, for example alas- and star, rate-limiting enzymes of heme and steroid hormone biosynthesis, respectively. , independent of its proteolytic activity, lon influences function of the mitochondrial genome and respiratory chain. its chaperone-like function promotes assembly of respiratory-chain complexes within the mitochondrial inner membrane, , , and knockout of lon-encoding genes in yeast and mammals leads to mtdna deletions and depletion, respectively. , , lon binds single- stranded mtdna in a sequence- and strand-specific manner – and associates preferentially with the mtdna control region where replication and transcription are initiated. here, we establish a link between lonp and codas syndrome in humans and discuss potential mechanisms linking the dysfunction of mitochondrial lon to diverse clinical manifestations. subjects and methods subjects we studied a total of ten individuals from two countries (united states, n ¼ and canada, n ¼ ). all had the core develop- mental, ocular, dental, auricular, and skeletal features of codas syndrome. – detailed phenotype studies were conducted on four children homozygous for lonp c. c>g, a founder mutation among the pennsylvania amish (figures and ). the study was approved by the institutional review board of lancaster general hospital. parents consented to research on their affected children on their behalf and signed a separate con- sent to reproduce photographs. affected children were evaluated by routine clinical methods, including bone radiographs, . t magnetic resonance imaging, slit-lamp and dilated indirect ophthalmoscopy, middle-ear tympanometry, distortion-product otoacoustic emissions, evoked auditory brainstem responses, and direct laryngoscopy. the american journal of human genetics , – , january molecular genetics genetic mapping of codas syndrome was performed as previ- ously described. – in brief, snp genotyping was performed with the genechip mapping k assay kits (affymetrix). data were analyzed in microsoft excel spreadsheets that were custom formatted at the clinic for special children. snp positions came from affymetrix genome-annotation files, and genotype data came from the affymetrix genechip human mapping k xba arrays. data analyses were designed for rapid identification of genomic regions that were identically homozygous between affected individuals. exome sequencing was performed as previously described. – a solution-hybrid-selection methodology was used for isolating exomic dna, which was subjected to sequencing on the illumina ga-ii platform. in brief, dna oligonucleotides, corresponding to bp of target sequence flanked by bp of universal primer sequence, were synthesized in parallel on an agilent microarray, then were cleaved from the array. the oligonucleotides were amplified by pcr, then transcribed in vitro in the presence of bio- tinylated uridine- -triphosphate so that single-stranded rna ‘‘bait’’ would be generated. genomic dna was sheared, ligated to illumina sequencing adapters, and selected for lengths between and bp. this ‘‘pond’’ of dna was hybridized with an excess of bait in the solution. the ‘‘catch’’ was pulled down by magnetic beads coated with streptavidin, eluted, and sequenced on the illumina ga-ii. massively parallel sequencing data were processed by the sequencing platform at the broad institute via two pipelines. the first, called ‘‘picard,’’ utilized the reads and qualities produced by the illumina software and produced a single bam file represent- ing each sample. the second pipeline, called genome sequencing analysis, then performed post-processing and analysis of the data, including snp identification, small-insertion and -deletion identi- fication, local realignment of insertion- or deletion-containing reads, gene annotation, and filtering with common polymor- phisms ( genomes, dbsnp build ). the details of the sequencing-data processing have been described elsewhere. for inter-exome analyses, variant call data were imported into a file- maker pro database, and rare and novel variants shared among affected individuals were queried. molecular modeling a homology model of the human mitochondrial lon hexamer was generated from the crystal structure of b. subtilis lon (pdb m a). , in brief, we generated a homology model of a single lon subunit that lacks its amino-terminal mitochondrial-targeting sequence (mts) (see figures b– d) by using three independent molecular modeling software tools: prime (schrodinger), modeller (see web resources), and the i-tasser online server at the , figure . codas phenotype (a and b) a year-old child who is homozygous for lonp c. c>g (p.arg gly) and who has characteristic physical features of codas syndrome. features include (a) broad skull and flattened midface; ptosis; grooved nasal tip; anteverted nares; and (b) helix hypoplasia (‘‘crumpled’’ ears). (c) laryngoscopy of an affected child revealed airway narrowing secondary to paretic, atrophic vocal cords (inset: normal vocal cords for comparison). (d) dense bilateral nuclear cataracts develop rapidly between and months of age in all codas-syndrome-affected amish individuals. (e and f) scoliosis and genu valgum progress with advancing age. (g) despite previous reports of developmental delay in children with codas syndrome, timely ophthalmologic and audiologic inter- vention appear to be critical developmental determinants; at age years our oldest subject has a vocabulary of more than words, reads at the first grade level, communicates effectively by signing, and has advanced drawing and writing skills. university of michigan. we obtained the final model of the lon subunit by averaging three models. we obtained the lon hexamer by superimposing the structure of one subunit onto the crystal structure of b. subtilis lon (pdb m a). cdna constructs human lonp cdna (refseq nm_ . ) that was amplified from arpe- (human adherent retinal pigment epithelium- ) total rna by rt-pcr (superscript ii reverse transcriptase, life tech- nologies; phusion dna polymerase, new england biolabs) was cloned into pentr/d-topo (life technologies), and codas mutations were introduced by site-directed mutagenesis (quik- change ii, agilent) (table s ). for mammalian expression of lon proteins with c-terminal v or flag tags, the lonp cdna was cloned in frame into pcdna . /v -dest or pcdna . /flag- dest by recombination from pentr/d-topo/lonp constructs with lr clonase ii (life technologies). for bacterial expression, cdnas encoding lon proteins that lacked the first amino acids (containing the predicted mts) were sub-cloned into pet c(þ) as well as into pproex- ; the latter constructs encoded a his tag fused to the n terminus. all amino acid numberings were assigned on the basis of the full-length lon precursor. purification of recombinant lon wild-type and pathogenic lon variants were overexpressed in ro- setta (de ) (novagen) and purified as previously described. – the amer lon constructs were cloned into either pet c(þ) (figure ) or proex (hexa-histidine tag ) (figure s ). for figure , lon-positive fractions were pooled and concentrated, and protein concentra- tions were determined via the bradford assay, for which bsa was used as a standard and uv absorbance was set at nm, and us- ing the equation ε ¼ w( ) þ y( ) þ c( ), where ε is the molar absorptivity at nm and w, y, and c are the numbers of tryptophan, tyrosine, and cysteine residues, respectively. , protein concentrations were further corrected on the basis of pro- tein-band optical density normalized to lonwt in coomassie- stained sds-page gels (image j). recombinant steroidogenic acute regulatory protein (star) and mitochondrial transcription factor a (tfam) were purified as described previously. , enzymatic assays peptidase and protease assays fmoc-protected amino acids, boc-abz, fmoc-protected lys-wang resin, and hbtu (n,n,n ,n -tetramethyl-o-( h-benzotriazol- -yl) uronium hexafluorophosphate, o-(benzotriazol- -yl)-n,n,n ,n - tetramethyluronium hexafluorophosphate) were purchased from advanced chemtech and novabiochem. peptide substrate s was purchased from genscript. peptide synthesis of s was per- formed with fmoc solid-phase synthesis methodologies as described previously. peptidase activity was measured on a fluo- romax- spectrofluorimeter (horiba group). all reactions mixtures contained mm hepes (ph . ), mm mg(oac) , mm dtt, ican journal of human genetics , – , january , figure . radiographic features of codas syndrome (a) sagittal t (upper) and axial t (lower) images of an affected -year-old amish child show mild diffuse cortical atrophy, an immature pattern of myelination, and hypoplasia of the corpus callosum. (b) skeletal radiographs show metaphyseal dysplasia most evident at hip joints (upper) and valgus knees with both hypoplasia and delayed ossification of epiphyses (lower). (c) severe scoliosis is evident early in childhood, and coronal clefts are observed at various levels of the vertebral column. (d–g) prenatal imaging of a -week-old fetus with codas syndrome revealed polyhydramnios, a two-vessel umbilical cord, midfacial hypoplasia, (d) crumpled helices, (e) a balanced atrioventricular canal, (f) common atrium, omphalocele, (g, arrow) and absence of left lower long bones, which were replaced by a relatively well-formed foot attached directly to the hip. (g, arrowhead) the asterisk in (g) marks the left palpebral fissure for orientation. nm wild-type and mutant lon proteins, and . mm s . to avoid problems from the inner filter effect, we used s , a mixed substrate consisting of % fluorescently labeled peptide s [y(no )rgitcsgrqk(abz)] and % non-fluorescent analog of the peptide s (yrgitcsgrqk(ac)). after the mixture equili- brated at �c for min, the reaction was initiated with mm atp. we measured the amount of hydrolyzed peptide by deter- mining the maximum fluorescence generated per micromole of peptide after complete digestion by trypsin. the steady-state rate of the reaction was determined from the tangent of the linear portion of the time course. this rate was converted to an observed rate constant (kobs) by division of the rate by the enzyme concen- tration. at least three identical experiments were performed. peptidase assays using the fluorescent dipeptide substrate rhodamine , bis-(cbz-l-alanyl-l-alanine amide) (aa -rh , anaspec), were performed in quadruplicate ( ml) in -well plates as previously described. lon ( nm monomer) or no- enzyme controls were incubated in a reaction buffer ( mm nacl, mm hepes [ph . ], and mm mgcl ) containing aa -rh ( mm) and atp ( mm) for hr at �c, after which fluorescence was measured at an excitation and emission of and nm, respectively, with a perkin elmer victor v. the american journal of human genetics , – , january the relative fluorescence units (rfu) of the background (no- enzyme control) measurements were subtracted, and the resultant values were normalized to percent activity of the no-drug reac- tions. data were fit to -parameter dose-response curves using graphpad prism , and the error bars represent the sd of four repli- cate reactions. at least three identical experiments were per- formed. star and tfam purification and degradation assays were performed as previously described. , , , atpase assay the atpase activity of wild-type and mutant lon variants was measured in quadruplicate reactions ( ml) in a -well plate via the adp-glo assay (promega) according to the manufacturer’s recommendations. all steps were performed at room temperature. purified human lon ( nm monomer) was incubated in a reaction buffer ( mm tris-hcl [ph . ], mm mgcl , and . mg/ml bsa) with ultra pure atp ( mm; promega) for min. adp-glo� reagent ( ml) was added for min followed by kinase detection substrate ( ml) for min, which was linear with [adp]. relative luminescence units (rlu) were measured on a perkin elmer victor v, and background (no-enzyme control) rlu were subtracted. at least three identical experiments were performed. , figure . location of amino acids that are mutated within mitochondrial lon in codas syndrome (a) domain structure of the human lon subunit. mitochondrial targeting sequence (mts), substrate recognition/binding (n) domain, atpase domain (aaaþ), and a pro- tease domain (p). red arrows indicate the position of pathogenic codas mutations. (b) homology model of human mitochon- drial lon. the model shows the position, within a single lon subunit (shown in amber), of amino acids that are altered in codas syndrome. (c and d) the positions of amino acids pro and arg (yellow), which are altered in proteins encoded by codas homozygous alleles. adp is shown occu- pying the atp- and adp-binding pocket (a green dashed line represents a proline-induced kink in the helix; a yellow dotted line represents a salt bridge; starbursts represent hydrophobic interac- tions). (c) the pro -induced kink pro- motes hydrophobic interactions between ala , leu , leu , and val . (d) the positions of arg as well as pro , glu , and the atp- and adp-binding site are located on the same lon subunit (amber). arg and glu form salt bridges with glu and lys , respectively, which are located on the adjacent lon subunit (green). cell culture arpe- cells, human embryonic kidney (hek t) cells, hela cells (atcc, manassas, va), and mouse auditory sensory epithe- lium (ub/oc- ) cells (gift from matthew holley, university of sheffield, uk) were cultured in dmem:f , dmem, eagle’s mem, or mem with glutamax and u/ml interferon-gamma (life technologies), respectively, each with % fetal bovine serum (fbs; sigma or life technologies) at �c/ % co . ep- stein-barr virus (ebv)-transformed b-lymphoblastoid cell lines (lcls) were generated from two amish codas-syndrome- affected probands homozygous for lonp c. c>g (p.arg - gly) as well as from their respective heterozygous parents (lineberger comprehensive cancer center, university of north carolina). lcls were cultured in rpmi (atcc formulation) with % fbs at �c and % co . genotyping of lcls was performed by sanger sequencing (the pennsylvania state university nucleic acid facility). in brief, superscript iii reverse transcriptase (life technologies) and oligo dt primers were used for reverse transcrip- tion of cdna from lcl total rna (trizol, life technologies). cdna was then amplified by the polymerase chain reaction (pcr) with lonp -specific primers (table s ) and purified for dna sequencing with qiagen pcr purification spin columns. lon knockdown in hela, pc , and hek t cells was performed as previously described (and as also described in supplemental data). mitochondrial localization wild-type and mutant lon variants were overexpressed by tran- sient transfection (fugene , promega) in arpe- , hela, and ub/oc- cells cultured on glass coverslips. after – hr, cells were incubated with mitotracker red cmxros ( nm) at �c/ % co for min, washed with pbs (ph . ), and fixed in % paraformaldehyde in pbs for min at �c. cells were per- meabilized in acetone at � �c for min, washed with pbs, and the amer blocked in % bovine serum albumin (bsa) with . % triton x- in pbs for min. immunofluorescence detection of lon- v fusion proteins was conducted as described elsewhere. immunoblotting and immunoprecipitation cells were lysed in the following buffers as indicated: in ripa buffer ( mm tris-hcl [ph . ], mm nacl, . % octylphe- noxypolyethoxyethanol ca- , . % sodium deoxycholate, and . % sodium dodecyl sulfate) supplemented with mm edta, mm naf, mm na vo , and protease-inhibitor cocktail (roche); triton x- buffer ( mm tris, [ph . ], mm nacl, and . % triton x- ) supplemented with halt protease- and phosphatase-inhibitor cocktail (thermoscientific); or urea buffer ( mm triethylammonium bicarbonate buffer [teab, sigma] [ph . ], m urea) supplemented with protease-inhibitor cocktail and phosphatase-inhibitor cocktail (sigma). when the ripa or triton x- buffer was used, cells were incubated for min on ice, then centrifuged at , rpm for min at �c so that lysates would be cleared. for the urea buffer, cells were son- icated with microprobe for s followed by a s pulse, and this was repeated – times before centrifugation at �c cleared lysates. the protein concentration of cell extracts was measured with the bradford assay and then normalized. immunoblotting and co-immunoprecipitation were performed as described else- where; washes following immunoprecipitation contained % tween- . the following antibodies were employed: rabbit anti- lon ( : , custom made and affinity purified as previously described) or rabbit anti-lon ( : , novus, h - d p); rabbit anti-clpx ( : , , custom made by dr. irene lee); rabbit anti-aconitase ( : , provided by dr. luke szweda, okla- homa medical research foundation); mouse anti-clpp ( : , , abcam ); rabbit anti-pink ( : , abcam ); rabbit anti-mt-cytochrome oxidase subunit ii ( : , , abcam ); ican journal of human genetics , – , january , figure . enzymology, cellular expression, and homo-oligomerization of pathogenic lon proteins (a) atp-dependent degradation of the fluorogenic peptide substrate s . the rates of s cleavage were determined with recombinant lon proteins ( nm monomer) and . mm of s ( . -fold km) in buffer. the degradation reactions were initiated by the addition of mg-atp at �c for – s. s cleavage was monitored as an increase in fluorescence at an excitation and emission of nm and nm, respectively (three or more replicates). error bars represent sem. (b) star ( . mm) or tfam ( mm) were combined in buffer with mg-atp, and reactions were initiated by the addition of lon ( nm) at �c. aliquots were removed at time points, reactions were terminated with reducing sample buffer, and aliquots were analyzed by sds-page and coomassie brilliant blue staining. imagej was used for determining the percent star and tfam degraded after a min incubation period with the same lon variant (no replicates). (c and d) overexpression of (c) wild-type lon-v and (d) p.arg gly-v (green ¼ v immunofluorescence) in arpe- cells counter- stained with mitotracker red (red) and dapi (blue). nontransfected cells display only red mitotracker fluorescence. (e) coimmunoprecipitation (ip) of lon-v by lon-flag (co-overexpressed in hek t cell lysates) with anti-flag m antibody. immunoblotting (ib) with anti-flag and v antibodies followed. oxphos antibody cocktail ( : , mitoscience ); goat anti- actin ( : , , ; santa cruz); mouse anti-b-actin (sigma, : , , ); rabbit anti-mt-cytochrome b ( : , , aviva systems biology arp _p ); mouse monoclonal anti-v ( : , , life technologies) or mouse anti-flag m ( : , sigma); and hrp-conjugated secondary antibodies (cell signaling technologies or santa cruz biotechnology). immunoblots were developed by chemiluminescence. transmission electron microscopy lcls were prepared for transmission electron microscopy (tem) as described previously. , in brief, cells were pelleted by centrifuga- tion at , rpm for min, washed with pbs, re-pelleted, and re- suspended in . % glutaraldehyde and % paraformaldehyde in . m pbs overnight at �c. lcls were then post-fixed in % osmium tetroxide for hr at �c and dehydrated with ethanol; infiltration with spurr’s resin (electron microscopy sciences) fol- lowed. infiltrated lcls were collected by centrifugation and embedded directly into beem capsules, which were gently centri- fuged so that the cells would be driven to the bottom of the capsule. ultrathin sections ( - nm) were cut with glass knives, the american journal of human genetics , – , january mounted on naked copper grids ( mesh), and stained with a saturated solution of uranyl acetate in % ethanol for min, fol- lowed by reynold’s lead citrate for min. differences in the fre- quency of abnormal mitochondrial morphology between proband and parent cells were analyzed by two-way anova with bonfer- roni post-hoc correction (spss, ibm). quantitative pcr (qpcr) determination of mtdna copy number genomic dna ( ng) from peripheral blood lymphocytes and lcl cells was used for amplification of both the mtdna- encoded mt-cyb gene and the nuclear-dna-encoded app gene as a reference control. genomic dna was amplified in reactions ( ml; applied biosystems; universal pcr master mix) with the following taqman primers. forward: app, -tttttgtgtgct ctcccaggtct- ; and mt-cyb, -gcctgcctgatcctccaa at- ). reverse: app -tggtcactggttggttggc- ; and mt cyb, -aaggtagcggatgattcagcc- . taqman probes: app, -[ fam]ccctgaactgcagatcaccaatgtggtag[tam]- ; and mt-cyb, -[ fam]caccagacgcctcaaccgcctt [tam]- an ab rt-pcr system (applied biosystems) and , standard qpcr conditions were employed: �c for min, cycles at �c for s, and �c for min. all reactions were performed in triplicate. relative quantitation of the mtdna copy number was calculated by the ddct method. mitochondrial oxygen-consumption measurements proband and parent cells ( /well) in a ml unbuffered assay medium (dmem with mm glucose, mm pyruvate, and mm glutamine) were plated in poly-d-lysine-coated seahorse xf -well plates ( mg/ml) and centrifuged so that cells were adhered to the plates. after the uniformity of cells plated in each well was ensured, plates were transferred to a �c incubator without co for min, ml warm assay medium was added, and then plates were left in the incubator for another min. each plate was transferred to the xf analyzer, and the oxygen consumption rate (ocr) was measured at baseline. sequential injections were then performed with the following: oligomycin ( mm), an inhibitor of complex v (the atp synthase); fccp ( nm), an uncoupler that dissipates the membrane potential across the mitochondrial inner membrane; and rotenone ( mm), an inhibitor of complex i (nadh dehydrogenase), which blocks the transfer of electrons from complex i to ubiquinone. the spare respiratory capacity (src), which is the maximum ocr after fccp injection, was calculated as a percentage of baseline ocr. the mean, standard deviation, and statistical significant difference between proband and mother cells were calculated by student’s t test in microsoft excel. results molecular genetic studies we initiated a genetic mapping study involving two amish children who were affected with codas syndrome. affy- metrix genechip mapping k dna microarrays were initially used for genotyping, but no significant regions of homozygosity were detected. exome sequencing was performed, and variants were filtered so that those with minor allele frequencies (mafs) > % in either genomes or the exome variant server or with maf > % in our own internal amish variant database were excluded. analysis of variants detected in codas-affected individuals revealed a single shared homozygous allele, lonp c. c>g (p.arg gly), located at physical position chr : , , (ncbi grch , hg ) the gene mapped to a region of chromosome with poor coverage on the affymetrix k array, hence our inability to conclusively map the phenotype. the presence of lonp c. c>g was confirmed by sanger sequencing, and we verified proper segregation by genotyping parents and siblings. using an unlabeled probe to genotype amish controls by high-resolution melt analysis, we found a surprisingly high population allele fre- quency of . %. we next acquired additional codas-syn- drome dna samples accompanied by clinical data from collaborators in canada and designed primers to amplify and sequence all exons of lonp . the first sample was from a mennonite child from manitoba, canada, who was the subject of a previous publication. she was the amer found to be homozygous for lonp c. c>t (p.pro ser). the second sample was from a child of mixed european ancestry (german, scottish, irish, and en- glish) from ontario, and she was found to be compound heterozygous for lonp c. c>a (p.ser tyr) and c. c>t (p.ala val)(table ). phenotype associated with codas syndrome eight children from the old order amish community of pennsylvania were homozygous for lonp c. c>g (table ). three died of laryngeal obstruction in the first days of life, and one with a tracheostomy died from pneu- monia during early infancy. four remaining children (ages . – . years, two female) required tracheostomies to sur- vive infancy; only one child was subsequently decannu- lated. all three surviving children who underwent sedated laryngoscopies had paretic, atrophic vocal cords, glottic narrowing, chronic sialorrhea, and swallowing dysfunc- tion. three of four surviving affected children were nour- ished exclusively by gastrostomy tube. lonp c. c>g homozygotes had physical features characteristic of codas syndrome. – these included broad skull and flattened midface, helix hypoplasia (‘‘crumpled’’ ears), ptosis, grooved nasal tip, anteverted nares, and with advancing age, short stature, scoliosis, genu valgus, and pes valgus (figures a– f). teeth erupted late with cusp-tip extensions. dense bilateral nuclear cata- racts developed rapidly between and months of age. audiological testing showed impaired tympanic mem- brane mobility (type b pattern), normal otoacoustic emissions and neural synchrony, and low-frequency conductive hearing loss. the two oldest individuals had a mixed pattern involving mild-to-moderate sensory hear- ing loss in the – khz range. all affected children had hypotonia, developmental delay, and variable intellectual disability, some of which was remediable. one child with delayed ambulation was able to walk independently when knee-ankle-foot orthoses were applied at age years. this same child had early audi- ological intervention (myringotomy tubes and amplifica- tion). by age , he had a vocabulary of more than words, could read at the first grade level, could communi- cate effectively by signing, and had advanced drawing and writing skills (figure g). neuroimaging of one affected child (age years) was notable for prominent cortical sulci, symmetric ventriculo- megaly, subcortical hypomyelination, and a thin corpus callosum (figure a). skeletal radiographs showed meta- physeal dysplasia (most evident at hip joints) and both hy- poplasia and delayed ossification of epiphyses (figure b). two children had cervical radiographs that showed dens hypoplasia and, in one case, synostosis between the odon- toid and c . scoliosis was evident within the first few years of life, and coronal clefts could be observed at various levels of the vertebral column (figure c). two surviving lonp c. c>g homozygotes had an imperforate anus, in one case associated with omphalocele ican journal of human genetics , – , january , table . phenotype of codas-syndrome-affected children homozygous for lonp c. c>g (p.arg gly) agea . b . . . gender f m f m cerebral hypotonia and motor delay þ þ þ þ intellectual disability þ þ n/a n/a epilepsy þ � � � ocular ptosis þ þ þ þ cataractsc þ þ þ þ dental delayed tooth eruption þ þ n/a n/a enamel dysplasia þ þ n/a n/a auricular scapha and helix dysplasia (‘‘crumpled’’ ears) þ þ þ þ conductive hearing loss n/a þ þ þ sensorineural hearing loss þ þ � � skeletal short stature þ þ þ þ metaphyseal dysplasia þ þ þ þ epiphyseal hypoplasia and delayed ossification þ þ þ þ scoliosis þ þ þ þ genu valgus þ þ þ þ pes valgus þ þ n/a n/a vertebral coronal clefts þ þ n/a n/a dens hypoplasia þ þ n/a n/a other grooved nasal tip þ þ þ þ anteverted nares þ þ þ þ tracheostomy þ þ þ þ vocal-cord paresis � þ þ þ endocardial cushion (atrial septum defect)d � þ þ � gastresophageal reflux � þ � þ gastrostomy tube feeding � þ þ þ omphalocele � � � þ imperforate anus þ � � þ rectovaginal fistula þ � � � cryptorchidism n/a þ n/a � tongue hemiatrophy � þ � þ abbreviations are as follows: f, female; m, male; n/a, not applicable because age or clinical data were not available. alimited clinical data are available for four of eight children who had codas syndrome and died in the perinatal period as a result of respiratory the american journal of human genetics , – , january and in the other with a rectovaginal fistula. omphalocele was also observed in two codas-affected siblings who died during infancy. one of two affected males had unilat- eral cryptorchidism. two affected children had striking hemiatrophy of the tongue, presumably because of hypo- plasia or aplasia of the ipsilateral hypoglossal nerve; both also had vocal cord paresis and chronic sialorrhea. given the diverse roles of lon in mitochondrial homeo- stasis, we screened affected individuals for biochemical indices of mitochondrial dysfunction. plasma lactate ( . . mm; reference range . . mm) and alanine ( mm; reference range mm) were normal in children with codas syndrome, as were all urine-tricarboxylic-acid-cycle intermediates, including citrate, alpha-ketoglutarate, succinate, fumarate, and ma- late (data not shown). prenatal presentation of codas syndrome one unborn female amish infant with codas syndrome had a prenatal ultrasound at weeks’ gestational age. esti- mated weight ( , g; st centile) and femur length ( . cm, th centile) were low; estimated head circumfer- ence was normal ( . cm; rd centile). the study was notable for the presence of polyhydramnios, a two-vessel umbilical cord, midfacial hypoplasia, crumpled helices, retrognathia, a balanced atrioventricular canal, omphalo- cele (containing intestine, stomach, and part of the liver), and mild thoracic scoliosis and the absence of left lower long bones, which were replaced by a relatively well- formed foot attached directly to the hip (figures d– g). she was stillborn at weeks’ gestation. location of amino acids altered in codas syndrome within the homology model of lon the lon holoenzyme is composed of six identical subunits that each consist of four domains: an mts, a substrate recognition and binding (n) domain, an atpase (aaaþ) domain, and a proteolytic (p) domain (figure a). the amino terminal mts targets the newly synthesized cytosolic polypeptide to the mitochondrial matrix, where removal of the mts produces mature lon. the aaaþ domain contains walker boxes a and b that mediate atp binding and hydrolysis, and the p domain active site contains a ser -lys dyad required for peptide-bond hydrolysis. using the crystal structure of b. subtilis lon as a tem- plate, we established a homology model of human mito- chondrial lon, which has a homohexameric ring-shaped arrangement. all four lon variants identified in codas complications (these children are not represented in table ). two had ompha- locele. bdied at . years of age as a result of accidental trauma. cdense nuclear cataracts develop rapidly between and months of age. dtwo surviving children with codas syndrome have atrial septal defects; two who died perinatally had atrioventricular canal defects. the total incidence of congenital heart disease was %. , syndrome resulted in amino acid substitutions within the aaaþ domain. ser , arg , and ala are located at the interface between lon subunits and mediate subunit interactions. pro is at the atp-binding site (figures a and b). replacements of pro with serine (p.pro ser) and arg with glycine (p.arg gly) are pathogenic in the homozygous state (table ), allowing homo-oligomeric composition of the holoenzyme to be inferred. pro is on an alpha-helix near the atp-binding pocket and is involved in subdomain interactions (figure c); it gener- ates an alpha-helix kink that facilitates hydrophobic inter- actions between leu , ala , leu , and val . these interactions most likely maintain the geometry of the atp-binding pocket. arg contributes to intersubu- nit interactions by forming a salt bridge with glu on the neighboring subunit (figure d). arg is near glu , which also forms a salt bridge with lys in the aaaþ domain of the neighboring subunit (figure d). the p.arg gly substitution most likely weakens intersu- bunit interactions and reduces stability of the holoenzyme. recombinant codas proteins show substrate- specific defects in enzymatic activity individual codas variants were purified as recombinant proteins from bacteria, and their respective peptidase, pro- tease, and atpase activities were compared with those of wild-type lon. we measured atp-dependent peptidase activity by using the fluorogenic decapeptide reporter substrate s . relative to wild-type protein, all four codas variants (p.ser tyr, p.pro ser, p.arg gly, and p.ala val) showed decreased energy-dependent peptidase activity; cleavage of s ranged from %– % of wild-type activity (figure a). for the amish p.arg gly variant, we also determined atp-independent peptidase activity by using the fluores- cent dipeptide reporter aa -rh , which is cleaved by wild-type lon. in the absence of atp, p.arg gly and wild-type lon showed significantly increased cleavage of aa -rh . however, in the presence of atp, peptidase ac- tivity of wild-type lon was augmented -fold in compari- son to minimal augmentation of p.arg gly, and direct determination of atpase function showed that p.arg - gly was % less active than wild-type lon (data not shown). finally, we examined atp-dependent proteolysis of p.pro ser and p.arg gly by using two purified recom- binant natural lon substrates: star , , and tfam , (figures b and s ). compared to wild-type lon, which degraded %– % star, p.pro ser and p.arg gly degraded only %– % star during a min incubation. in contrast, tfam degradation by both p.pro ser and p.arg gly was comparable to that of the wild-type ( %– % of tfam was degraded during min incubation)(figure b). control experiments confirmed that tfam degradation was dependent on both atp and lon (figures s c and s d). the efficiency with which the amer p.pro ser and p.arg gly degrade different proteins is probably influenced by the substrate’s secondary or ter- tiary structure (i.e., whether the substrate is tightly or loosely folded). for example, folded and unfolded tfam have been shown to be in equilibrium with one another. these results suggest that in codas syndrome, lon-mediated degradation of some but not all protein sub- strates is impaired. impaired homo-oligomerization of transiently expressed r g to characterize the behavior of codas variants in cultured cells, we engineered fusion proteins with c-terminal epitope tags to distinguish them from endogenous lon. when transiently expressed in arpe- , ub/oc- , or hela cells, codas and wild-type lon were expressed at similar levels (figures , s , and s ). all four codas vari- ants localized to mitochondria, as determined by double labeling with immunofluorescence and mitotracker red (figures , s , and s ). to determine whether p.arg gly was able to oligo- merize with wild-type or other p.arg gly lon, we per- formed co-immunoprecipitation experiments by using recombinant proteins with different epitope tags (e.g., flag and v epitopes) (figure e), and we transiently co-expressed p.arg glyflag or wild-typeflag with either p.arg glyv or wild-typev in hek t cells. both wild- typeflag and p.arg glyflag co-precipitated wild-typev ; p.arg glyflag co-precipitated significantly less wild- typev (figure e). co-immunoprecipitation of p.arg - glyv by p.arg glyflag was barely detected, suggesting impaired homo-oligomeric assembly of p.arg gly or disassembly of an unstable complex during the experi- mental procedure. normal mtdna copy number and lon substrate levels in immortalized codas lymphoblast cell lines we generated ebv-immortalized b- lcls from the periph- eral blood of two amish family trios, each consisting of a homozygous p.arg gly/p.arg gly proband and his or her unaffected heterozygous p.arg gly/wt parents. no difference in lonp transcript abundance was observed by rt-pcr in proband versus parent cells (figure s ). immunoblot analysis of whole-cell extracts demonstrated that lon protein abundance was virtually identical in cell lines from the proband, mother, and father (figure a). there was no compensatory upregula- tion of clpx or clpp, subunits of another mitochondrial aaaþ protease, and we found no change in the abundance of mitochondrial aconitase or tfam (figure a), which are natural lon substrates under conditions of oxidative stress or mtdna depletion, respectively. , recent work sug- gests that lon is involved in degrading the pten-induced putative kinase (pink ). , however, an increase in pink abundance was not detected in proband or parent cells, not even in the presence of proteasome inhibitors ican journal of human genetics , – , january , figure . lon protein levels and mtdna copy numbers in co- das-syndrome-affected probands are similar to those of their unaffected heterozygous parents (a) proteins were extracted from lcls generated from codas- syndrome-affected probands and their parents. triton x- lysis buffer was used, and mg of the extract was immunoblotted for the proteins as shown. (b) quantitative pcr (qpcr) was performed so that the relative mtdna copy number in codas-syndrome-affected probands and their respective mothers and fathers could be determined with genomic dna isolated from the peripheral blood lympho- cytes from the family members as shown. qpcr was performed with taqman probes and primers for the mtdna-encoded mt- cyb gene and the nuclear dna-encoded app gene as a reference. the relative quantity of mtdna copy number was calculated by the ddct method. error bars represent the maximum and mini- mum relative quantitation. mg or epoxomicin (data not shown), which stabilize pink in vitro. , in lower and higher eukaryotes, knocking out lon leads to reduced mtdna copy-number or mtdna dele- tions. , , , we performed mtdna copy-number anal- ysis and mtdna sequencing by using total genomic dna isolated from peripheral blood lymphocytes obtained from three homozygous p.arg gly probands and their unaffected heterozygous parents. quantitative pcr showed no consistent difference of mtdna copy number between probands and parents (figure b). similar results were obtained with genomic dna isolated from proband and parental lcls (figure s ). using genomic dna iso- lated from peripheral blood lymphocytes for deep sequencing of mtdna exposed no deletions or significant differences between mtdna sequences of each proband and that individual’s mother (table s ). abnormal mitochondrial morphology in codas lymphoblast cell lines transmission electron microscopy of proband lcls re- vealed enlarged mitochondria with swollen intra- or inter- cristal compartments, uniform vesicular structures, and electron-dense intramitochondrial inclusions (figures a and c), suggestive of abnormal inner-membrane topol- ogy. , by contrast, parental lcls showed typical mito- chondrial morphology with well-organized cristae and only occasional swollen cristal spaces (figures b and d, and s ). approximately . % (sem) of mitochondria (n ¼ ) examined from two codas probands displayed the american journal of human genetics , – , january abnormal mitochondrial morphology (p < . ), whereas only . . % of paternal (n ¼ ), . . % of maternal (n ¼ ), and . . % (n ¼ ) of homozy- gous-normal lcl mitochondria were abnormal. there was no significant difference between maternal and paternal mitochondria (p ¼ . ). reduced mt-co and src in codas mitochondria lon has been shown to mediate protein quality control and remodeling of cytochrome c oxidase, which is com- plex iv of the mitochondrial respiratory chain. , , we examined the abundance of various protein subunits belonging to respiratory chain complexes i-v and detected a specific and substantial reduction of mtdna-encoded subunit ii of cytochrome c oxidase (mt-co ) in proband cells (figure a). in contrast, proband and parental lcls had similar abundances of other mtdna-encoded (mt- cyb, mt-co ) and nuclear-encoded (ndufb , sdhb, uqcrc , atp a ) respiratory chain subunits. one possible explanation for reduced mt-co abun- dance in codas cell lines was its poor solubility in the protein extraction buffer employed, which contained . % triton x- . to address this, we used cell-lysis buffer with m urea, which effectively solubilizes most aggre- gated proteins and inclusion bodies. when cells were ex- tracted with this strong denaturant, similar abundances of mt-co were observed in proband and parental lcls. urea did not alter solubility of other mtdna- or nuclear- encoded respiratory-chain subunits (figure a). insolubility of mt-co might be caused by the failure of p.arg gly to properly degrade and/or assemble this sub- unit into complex iv. to determine whether mt-co is a natural substrate of lon, we knocked down the protease in different cell lines. mt-co is highly hydrophobic and difficult to purify; we therefore used a cellular system rather than directly testing purified lon activity against that of mt-co . inducible or transient knockdown of lon was associated with increased mt-co abundance in hela, hek t, and pc cells (figure b). taken together, these results suggest that mt-co is a natural lon sub- strate, and that p.arg gly’s failure either to degrade or to properly assemble this protein in codas cells results in its aggregation within mitochondria. after observing the reduced solubility of mt-co , we investigated the efficiency of mitochondrial respiration. normalized basal mitochondrial oxygen consumption and atp-linked respiration were similar in proband and parental cells, but codas cells had significantly lower src when mitochondrial membrane potential was dissi- pated by the uncoupler fccp (figure c). discussion pathophysiology of codas syndrome mitochondrial lon is a multi-functional enzyme with diverse roles that include ( ) elimination of misfolded and oxidatively damaged proteins, – , , ( ) selective , figure . abnormal mitochondrial ultra- structure in codas cells (a–d) transmission electron micrographs were generated from cell lines of (a and c) two codas-syndrome-affected chil- dren homozygous for p.arg gly (gly /gly ) and (b and d, respectively) their heterozygous unaffected mothers (arg /gly ). (a and c) in codas cells, % of mitochondria (n ¼ cells, p < . ) display abnormal morphology, including swollen intracristal space and/or vesiculated cristae and electron-dense ag- gregations (white arrows). the mitochon- drion below the asterisk in (a ) appears to be in transition toward the vesiculated state when it is compared to the normal mitochondrion to the left of the asterisk. (b and d) the mothers’ cells display normal mitochondrial morphology (e.g., arrowheads); % show swelling of the intracristal space (n ¼ cells, p < . ) (n ¼ nucleus). scale bars represent mm in (a–d) and . mm in magnified regions (a –d ). the fathers’ cells show normal mitochondrial morphology, and only . % show cristal-space swelling (n ¼ ) (figure s ). degradation of key rate-determining mitochondrial pro- teins in response to metabolic flux and cell stress, – , , ( ) chaperone-like assembly of protein complexes within the respiratory chain, , and ( ) regulation of mitochondrial gene expression. homozygous loss of lon in mice results in embryonic death associated with poor growth and reduced mtdna copy number, indicating the vital importance of this protein in mammals. in contrast, heterozygous mice mature similarly to wild- type. these findings, combined with our enzymatic studies, support the idea that allele combinations giving rise to codas syndrome must result in alteration and not complete abrogation of lon function. we speculate that the complex pathophysiology of codas syndrome most likely reflects dysregulation of multiple lon-dependent proteins and pathways. there are clinical similarities between codas syndrome and other mitochondrial disorders, including hypotonia, motor delay, ptosis, and sensorineural hearing loss. , – however, many classic clinical and biochemical hall- marks , , of mitochondrial dysfunction were not observed in codas children, and several codas manifes- tations, including postnatal cataracts, skeletal and dental abnormalities, and conductive hearing loss, are atypical of mitochondrial disease. such complications might reflect lon-dependent processes that are currently unknown. moreover, tissue-specific defects could arise because of dif- ferential histological dependence on lon as a protease, a the american journal of human g chaperone, and/or a dna-binding protein. for example, some tissues might be more dependent on lon to degrade oxidatively damaged pro- teins, whereas others might depend critically on as-yet- unknown lon-mediated pathways. the reduced src of codas lcls suggests limited capacity to boost atp pro- duction in response to high energy demands, such as oc- curs during increased cellular stress and work load. further investigations using tissues with metabolic demands higher than those of lymphoblastoid cells are needed if we are to delineate the physiological relevance of mecha- nistic defects in mitochondrial function and morphology associated with codas syndrome. pathogenic amino acid changes in codas syndrome cluster in the aaaþ domain our structural analysis of homozygous codas variants is consistent with observed alterations of their enzyme activ- ity in vitro. proline is located within an a helix near the atp-binding pocket and generates a kink in this a helix (figure c). removal of the kink at pro might alter key hydrophobic interactions with four other residues— ala (on the same helix as pro ), leu , leu , and val —that likely help to shape the active site (figure c). the p.pro ser substitution could disrupt this kink, changing active site geometry or flexibility of the aaaþ module (figure c) and decreasing the protein’s capacity for atp binding and hydrolysis. accordingly, we found reduced atp-dependent peptidase (figure a) and substrate-specific protease (figure b) activities of the p.pro ser variant. enetics , – , january , figure . alterations in the structure and function of the electron transport chain in proband lcls (a) for extraction of cellular proteins, lysis buffer containing . % triton x- (tx ) or m urea (urea) was used. immunoblotting was performed for proteins as shown. mt-co abundances were selectively reduced in codas cells but were recovered in the presence of m urea. (b) mt-co is a protein substrate of mitochondrial lon. a hela cell line with a stably integrated anhydrotetracycline (atc)-inducible shrna targeting lon was grown in the presence or absence of atc ( ng/ml) for days. hek t cells and pc cells were transiently transfected with a sirna targeting the untranslated regionof lon or a control sirna and cultured for days. cell extracts ( mg) were immunoblotted for lon, mt-co , or actin as the loading control. (c) basal normalized oxygen-consumption rate was established at min (representing %), followed by addition of oligomycin (oligo, mm); fccp ( nm); and finally rotenone ( mm). a student’s t test showed that cells from codas-syndrome-affected probands had significantly (p < . ) lower spare mitochondrial respiratory capacity in the presence of the uncoupler fccp. error bars represent sd. arg also makes an important intersubunit contact by forming a salt bridge with glu from the aaaþ module of the neighboring subunit (figure d). this salt bridge, together with one formed between glu and lys , appears to be critical for stabilizing the lon complex. p.arg gly might thus destabilize the lon complex and explain reduced homo-oligomerization, atpase activity, and peptidase activity of this variant (figure ). functional consequences of homozygous lon p.arg gly in human cells our studies using recombinant protein and codas lcls indicate that the p.arg gly pathogenic variant has altered activity. purified p.arg gly has reduced atpase and peptidase activities and degrades some natural sub- strates (e.g., tfam) but not others (e.g., star) (figure b). substrate-restricted protease activity might reflect poor conformational resilience or stability of the p.arg gly holoenzyme, as suggested by co-immunoprecipitation experiments (figure e). in this study, we show that mt-co is upregulated when lon is knocked down in three different cell types, suggest- ing that mt-co is a lon substrate. in codas cells, we observed insolubility and aggregation of mt-co (figure a) but not mt-co , which is similarly hydropho- bic. previous work shows that wild-type lon plays a role in the assembly of the cytochrome c oxidase complex and in its remodeling the during adaptive switching between normoxic and hypoxic conditions. , , work in yeast and mammalian cells suggests that lon promotes cyto- chrome c oxidase assembly through a chaperone function independent of its protease activity. , the american journal of human genetics , – , january the failure of p.arg gly to degrade or chaperone mt-co (and possibly other inner-membrane proteins yet to be identified) might promote protein aggregation and explain the morphological and bioenergetic changes observed in codas cell mitochondria (figures and ). in this study, we examined immortalized b-lcls generated from codas-syndrome-affected probands and their par- ents. because this cell type is not principally affected in codas syndrome, future studies are aimed at employing primary cells and tissue samples as well as cell lines engi- neered with specific codas lonp mutations. natural history and treatment of codas syndrome the high allele frequency (maf ¼ . %, carrier fre- quency ¼ . %) of lonp c. c>g among the amish was unexpected. of all the known pathogenic alleles in the lancaster county amish population, the lonp variant has the second-highest carrier frequency, eclipsed only by that of a founder mutation for ellis-van creveld syndrome (evc; mim ; refseq nm_ . ); c. þ g>t; carrier frequency ¼ . %). the eight known amish codas-syndrome-affected individuals comprise six sibships. in two sibships, three affected children died shortly after birth. one affected fetus was stillborn. given the high allele frequency and perinatal mortality, the amish codas variant might result in a high rate of fetal demise. this could explain an apparent discrepancy between the relatively low birth incidence of codas syndrome and the high allele frequency of lonp c. c>g in the amish population. accurate prenatal diagnosis of codas syndrome has important clinical implications (figure ). newborns , with codas syndrome are likely to struggle through the neonatal transition. for syndrome variants (e.g., p.arg - gly) associated with neonatal vocal-cord paresis, the ability to anticipate and prevent perinatal asphyxiation can be life saving. three of eight ( %) amish codas- syndrome-affected individuals died of airway complica- tions shortly after birth, and one was stillborn. surviving children required intubation, mechanical respiratory support, and tracheostomy. incomplete cardiac septation occurred in % of p.arg gly homozygotes and mani- fested in two cases ( %) as a complete atrioventricular defect. thus, supportive cardiopulmonary care for babies with a suspected or confirmed diagnosis of codas syndrome requires tertiary-level subspecialty services and must begin immediately after birth. a careful, staged approach to subsequent management can allow for rela- tively good cognitive outcome and for independence in affected individuals. supplemental data supplemental data include six figures and two tables and can be found with this article online at http://dx.doi.org/ . / j.ajhg. . . . acknowledgments we thank kimberly jacob, prashant patel, ana quiroz, and jennie silver for technical assistance and d. streck from the molecular genetics laboratory at the rutgers new jersey medical school for next-generation mtdna sequencing. r.n.j., e.g.p., and k.a.s. were supported by howard hughes medical institute undergradu- ate science education awards and . r.n.j. was also supported by the center for research on women and newborn health and by connectcare . i.l. was supported by national science foundation grant che- . c.k.s. was supported in part by national institutes of health grants r gm and r ns and also by the nj health science foundation. the clinic for special children is supported by charitable donations from the communities it serves. received: november , accepted: december , published: january , web resources the urls for data presented herein are as follows: i-tasser online protein structure and function predictions, zhang laboratory, university of michigan, http://zhanglab.ccmb.med. umich.edu/i-tasser/ modeller, https://salilab.org/modeller/ online mendelian inheritance in man (omim), http://www. omim.org refseq: ncbi reference sequence database, http://www.ncbi.nlm. nih.gov/refseq/ sali laboratory, university of california san francisco, http:// salilab.org/index.html samtools, http://samtools.sourceforge.net/ the amer references . shebib, s.m., reed, m.h., shuckett, e.p., cross, h.g., perry, j.b., and chudley, a.e. ( ). newly recognized syndrome of cerebral, ocular, dental, auricular, skeletal anomalies: codas syndrome—a case report. am. j. med. genet. , – . . de almeida, j.c., vargas, f.r., barbosa-neto, j.g., and llerena, j.c., jr. ( ). codas syndrome: a new distinct mca/mr syndrome with radiological changes of spondyloe- piphyseal dysplasia. another case report. am. j. med. genet. , – . . innes, a.m., chudley, a.e., reed, m.h., shuckett, e.p., hildes- ripstein, g.e., and greenberg, c.r. ( ). third case of cerebral, ocular, dental, auricular, skeletal anomalies (codas) syndrome, further delineating a new malformation syndrome: first report of an affected male and review of literature. am. j. med. genet. , – . . marlin, s., ducou le pointe, h., le merrer, m., portnoi, m.f., chantot, s., jonard, l., mantel-guiochon, a., siffroi, j.p., garabedian, e.n., and denoyelle, f. 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( ). helix kinks are equally prevalent in soluble and membrane proteins. proteins , – . ican journal of human genetics , – , january , 	codas syndrome is associated with mutations of lonp , encoding mitochondrial aaa+ lon protease 	introduction 	subjects and methods 	subjects 	molecular genetics 	molecular modeling 	cdna constructs 	purification of recombinant lon 	enzymatic assays 	peptidase and protease assays 	atpase assay 	cell culture 	mitochondrial localization 	immunoblotting and immunoprecipitation 	transmission electron microscopy 	quantitative pcr (qpcr) determination of mtdna copy number 	mitochondrial oxygen-consumption measurements 	results 	molecular genetic studies 	phenotype associated with codas syndrome 	prenatal presentation of codas syndrome 	location of amino acids altered in codas syndrome within the homology model of lon 	recombinant codas proteins show substrate-specific defects in enzymatic activity 	impaired homo-oligomerization of transiently expressed r g 	normal mtdna copy number and lon substrate levels in immortalized codas lymphoblast cell lines 	abnormal mitochondrial morphology in codas lymphoblast cell lines 	reduced mt-co and src in codas mitochondria 	discussion 	pathophysiology of codas syndrome 	pathogenic amino acid changes in codas syndrome cluster in the aaa+ domain 	functional consequences of homozygous lon p.arg gly in human cells 	natural history and treatment of codas syndrome 	supplemental data 	acknowledgments 	web resources 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ efficacy of paracetamol on patent ductus arteriosus closure may be dose dependent: evidence from human and murine studies copyright © international pediatric research foundation, inc. articles basic science investigation nature publishing group background: we evaluated the clinical effectiveness of variable courses of paracetamol on patent ductus arteriosus (pda) closure and examined its effect on the in vitro term and preterm murine ductus arteriosus (da). methods: neonates received one of the following three paracetamol regimens: short course of oral paracetamol (scop), long course of oral paracetamol (lcop), and intrave- nous paracetamol (ivp) for – d. pressure myography was used to examine changes in vasomotor tone of the preterm and term mouse da in response to paracetamol or indomethacin. their effect on prostaglandin synthesis by da explants was measured by mass spectroscopy. results: twenty-one preterm infants were included. no changes in pda hemodynamics were seen in scop infants (n = ). the pda became less significant and eventually closed in six lcop infants (n = ). pda closure was achieved in eight ivp infants (n = ). on pressure myograph, paracetamol induced a concentration-dependent constriction of the term mouse da, up to % of baseline (p < . ), but required > µmol/l. indomethacin induced greater da constriction and suppres- sion of prostaglandin synthesis (p < . ). conclusion: the clinical efficacy of paracetamol on pda closure may depend on the duration of treatment and the mode of administration. paracetamol is less potent than indo- methacin for constriction of the mouse da in vitro. there is a need to explore alternatives to current therapeutic approaches of patent ductus arteriosus (pda) in preterm infants for several reasons. first, medical closure of the pda is limited to nonselective nonsteroidal anti-inflammatory drugs (nsaids), namely indomethacin and ibuprofen, but these only achieve a closure rate of – %. second, there remain concerns regarding the safety of nsaids. third, nsaid drugs are contraindicated in infants with feeding intolerance, nec- rotizing enterocolitis, intestinal perforation, intraventricular hemorrhage, concomitant use of corticosteroids, and severe hyperbilirubinemia ( ). there is an increasing interest in the use of paracetamol for pda closure in preterm infants with the recent publication of several case reports ( – ). however, the enthusiasm to proceed with interventional trials needs to be tempered as there remains a paucity of pharmacodynamic data on this treatment in preterm infants. the dose–response relationship between paracetamol and pda closure needs pro- spective evaluation. case reports suggest that paracetamol may be an effective agent in inducing ductal closure and is probably not associ- ated with serious side effects. the vasoconstrictive effect of paracetamol on ductal tissue is achieved through inhibition of the peroxidase moiety, thereby inhibiting prostaglandin production ( ). oral and intravenous (i.v.) paracetamol have demonstrated efficacy in pda closure in a variety of situations, particularly when conventional nsaids are either contrain- dicated, or have failed to achieve pda closure ( , , , ). those studies however, used paracetamol in a heterogeneous group of infants with limited echocardiography data on the characteris- tics of the pda. in this study, we retrospectively examined the effectiveness of three distinct clinical regimes of paracetamol administration in preterm infants, which provide escalating drug levels (phase i). in addition, we performed an in vitro evaluation of the dose-responsiveness of paracetamol in the term and preterm murine ductus arteriosus (da) (phase ii). results human study a total of infants were included in the study; specifically, infants received a short course of oral paracetamol (scop), received a long course of oral paracetamol (lcop), and   infants received a course of intravenous paracetamol (ivp) (table ). the pda remained open in all neonates who received scop. there was neither clinical improvement nor change in the echocardiography markers of hemodynamic significance following treatment (table ). all infants eventually required pda ligation. seven infants received a lcop (lcop group, received october ; accepted february ; advance online publication july . doi: . /pr. . department of pediatrics, the rotunda hospital, dublin, ireland; department of pediatrics, mount sinai hospital, toronto, ontario, canada; division of neonatology, department of pediatrics, vanderbilt university medical center, nashville, tennessee; division of clinical pharmacology, department of medicine, vanderbilt university school of medicine, nashville, tennessee; department of neonatology, the hospital for sick children, toronto, ontario, canada; department of physiology and experimental medicine, hospital for sick children, toronto, ontario, canada. correspondence: patrick j. mcnamara (patrick.mcnamara@sickkids.ca) efficacy of paracetamol on patent ductus arteriosus closure may be dose dependent: evidence from human and murine studies afif el-khuffash , amish jain , david corcoran , prakesh s. shah , christopher w. hooper , naoko brown , stanley d. poole , elaine l. shelton , ginger l. milne , jeff reese and patrick j. mcnamara , pediatric research volume | number | september http://www.nature.com/doifinder/ . /pr. . mailto:patrick.mcnamara@sickkids.ca) copyright © international pediatric research foundation, inc. paracetamol and pda closure articles ta b le . in d iv id u al p at ie n t c h ar ac te ri st ic s an d re sp o n se to p ar ac et am o l t re at m en t c as e g es ta ti o n (w ee ks ) b ir th w ei g h t ( g ) d ay o f tr ea tm en t c o rr ec te d g es ta ti o n (w ee ks ) r es p ir at o ry su p p o rt a t s ta rt o f t re at m en t pd a d ia m et er (m m ) en te ra l fe ed s d ay s o f tr ea tm en t pr ev io u s n sa id u se c o n tr ai n d ic at io n to n sa id s pd a re sp o n se pd a li g at io n sh o rt c o u rs e o f o ra l p ar ac et am o l / / ve n ti la te d . fu ll n o n e r en al fa ilu re n o c h an g e ye s / / ve n ti la te d . fu ll in d o m et h ac in n o n e n o c h an g e ye s / / ve n ti la te d . fu ll in d o m et h ac in n o n e n o c h an g e ye s / / ve n ti la te d . fu ll in d o m et h ac in n o n e n o c h an g e ye s / / c pa p . fu ll in d o m et h ac in n o n e n o c h an g e ye s lo n g c o u rs e o f o ra l p ar ac et am o l / , / c pa p . fu ll in d o m et h ac in n o n e r es tr ic ti o n n o / / ve n ti la te d . fu ll in d o m et h ac in n o n e r es tr ic ti o n n o / / ve n ti la te d . fu ll n o n e r en al f ai lu re c lo su re n o / / c pa p . fu ll in d o m et h ac in n o n e r es tr ic ti o n n o / / c pa p . fu ll in d o m et h ac in n o n e r es tr ic ti o n n o / , / ve n ti la ti o n . fu ll in d o m et h ac in n o n e n o c h an g e ye s / , / c pa p . fu ll in d o m et h ac in n o n e r es tr ic ti o n n o in tr av en o u s p ar ac et am o l / , / ve n ti la te d . m in im al n o n e se ve re iv h c lo su re n o / / ve n ti la te d . fu ll ib u p ro fe n n o n e c lo su re n o / / ve n ti la te d . ib u p ro fe n n ec /p er fo ra ti o n c lo su re n o / / ve n ti la te d . n o n e iu g r c lo su re n o / / ve n ti la te d . fu ll ib u p ro fe n n o n e r es tr ic ti o n n o / / ve n ti la te d . fu ll ib u p ro fe n n o n e r es tr ic ti o n n o / / ve n ti la te d . fu ll ib u p ro fe n n o n e r es tr ic ti o n n o / , / ve n ti la te d . ib u p ro fe n n ec c lo su re n o / / ve n ti la te d . n o n e pe rf o ra ti o n r es tr ic ti o n n o fu ll fe ed s ar e d ef in ed a s an e n te ra l i n ta ke >   m l/ kg /d . m in im al fe ed s ar e d ef in ed a s <  m l/ kg /d . e ac h d ay o f p ar ac et am o l t re at m en t c o m p ri se d   m g /k g /d in fo u r d iv id ed d o se s. c pa p, c o n ti n u o u s p o si ti ve a ir w ay p re ss u re ; i u g r, in tr au te ri n e g ro w th re st ri ct io n ; i vh , i n tr av en tr ic u la r h em o rr h ag e; n ec , n ec ro ti zi n g e n te ro co lit is ; n sa id , n o n st er o id al a n ti -in fla m m at o ry d ru g s; p d a , p at en t d u ct u s ar te ri o su s. volume | number | september pediatric research copyright © international pediatric research foundation, inc. articles el-khuffash et al. table ). following a -d course, pda closure was achieved in one patient and there was a reduction of ductal diameter and an improvement of the echocardiography markers of pda significance in five infants (table ). all six infants demon- strated clinical improvement and were successfully weaned from respiratory support. there was no response to treatment in one infant who required pda ligation. nine infants received ivp treatment (ivp group). of these, five achieved immediate pda closure and three infants dem- onstrated a significant reduction of pda diameter (table ). subsequent pda closure was achieved in the three infants prior to discharge without any need for further intervention. the remaining infant had a nonsignificant pda on discharge. two infants died during their hospital stay due to unrelated causes (pulmonary hypoplasia and cystic periventricular leu- komalacia). none of the deaths occurred during paracetamol therapy. none of the infants in the cohort had elevated liver enzyme or developed liver toxicity. none of the pdas success- fully closed following lcop or ivp reopened after treatment. in vitro study the isolated mouse da is more sensitive to indomethacin than paracetamol. exposure to paracetamol did not produce a significant change in the diameter of the ex vivo preterm table . echocardiography characteristics before and after treatment of the groups short course of oral paracetamol long course of oral paracetamol intravenous paracetamol pre post p pre post p pre post p pda diameter (mm) . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . . ( . – . ) ( – . ) < . left atrium:aortic ratio . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . . ( . – . ) . ( . – . ) . ivrt (msec) ( – ) ( – ) . ( – ) ( – ) . ( – ) ( – ) . lvo (ml/min/kg) ( – ) ( – ) . ( – ) ( – ) . ( – ) ( – ) . lvedd (mm) ( – ) ( – ) . ( – ) ( – ) . ( – ) ( – ) . data are presented as medians (interquartile range). ivrt, isovolumic relaxation time; lvo, left ventricular output; lvedd, left ventricular end diastolic velocity; pda, patent ductus arteriosus. figure . paracetamol-induced constriction of the ex vivo ductus arteriosus. the ductus arteriosus (arrow) of fetal mice (a) was sharply excised from sur- rounding tissues. the ductus of term (day ) and preterm (day ) fetuses were removed along with a vascular segment extending from the base of the pulmonary artery to the transverse aortic arch (b) (shown with μm dissecting pins). this vascular bloc was mounted on glass pipette tips and secured by tying back the branch pulmonary arteries and the aortic arch, in order to isolate the entire length of the ductus for myography studies (c). pressure myography was performed to study ductus tone and examine drug-induced changes in lumen diameter (d). after pressurization and equilibration, mounted vessels were briefly exposed to mmol/l kcl to verify contractile potential, then allowed to return to baseline tone. cumulative exposure to increasing paracetamol demonstrated concentration-dependent constriction of the day ductus (d). constricted vessels were then exposed to sodium nitroprusside, a potent nitric oxide donor, to document vasodilatory capacity. terminal exposure to kcl ensured viability of the preparation at the end of each experiment. time ( min intervals) paracetamol wash wash wash − − − − − − − snp kci kci l u m e n d ia m e te r (µ m o l/l ) a d b c pediatric research volume | number | september copyright © international pediatric research foundation, inc. paracetamol and pda closure articles da. indomethacin caused a small but significant constriction of the ductus with increasing concentration (figure a). in contrast, indomethacin produced marked constriction of the isolated mouse ductus at term gestation, with complete clo- sure of the vessel lumen at the highest concentrations studied (figure b). paracetamol also caused significant concentra- tion-dependent constriction of the term ductus. the magni- tude of paracetamol-induced constriction was less than half of indomethacin-induced constriction at each concentration. ex  vivo lumen closure was not observed in paracetamol treated vessels. indomethacin inhibits ductus-specific prostaglandin produc- tion. due to the limited quantity of preterm ductus tissue and because isolated preterm vessels had only modest response to inhibitors (figure a), only the excised ductus of term gesta- tion mice was assayed for prostaglandin synthesis. we observed significant reduction in -keto prostaglandin f α (pgf α), the stable metabolite of prostacyclin (pgi ), and prostaglandin e (pge ) in response to indomethacin treatment (figure ). a reduced trend in prostaglandin (pg) synthesis was noted in paracetamol-treated explants, but did not reach significance in this particular assay. thromboxane b (txb ) and prosta- glandin d (pgd ) levels were below the detection limits of the instrument; interfering substances with similar m/z character- istics prevented accurate interpretation of pgf α results. discussion in this study, we demonstrated that the responsiveness of the da to paracetamol may depend on the method of adminis- tration, duration of treatment, and the dose of the drug. the response to paracetamol treatment was highly variable, rang- ing from no response in infants receiving scop, to near-com- plete closure in the majority of patients who received ivp, which suggests that a critical paracetamol level is required to achieve maximal therapeutic effect. the complete ineffective- ness of scop is in contrast to other recent reports assessing the efficacy of oral paracetamol on ductal closure ( , , , ). there were, however, differences in the treatment cohort that may explain the discordance. unlike our cohort, neonates had a higher gestational age at birth ( up to vs.  wk), pda diameter was smaller ( . – . vs. .  mm and greater), and treatment was initiated at an earlier postnatal age ( – vs. d). in addition, no echocardiography information was available to appraise the hemodynamic impact of the pda. it is possible that the pda was of borderline hemodynamic significance and may have closed spontaneously. our experience with a longer course of oral paracetamol was more positive with avoidance of the need for pda ligation in all but one patient. this may relate to achieving a higher serum drug level or may be a func- tion of more prolonged exposure to the biological effects of figure . response of the ex vivo ductus arteriosus to paracetamol and indomethacin. the isolated ductus of preterm mice (a) displayed limited response to increasing concentrations of paracetamol (black squares, n = ) whereas indomethacin (white circles, n = ) induced a modest, sig- nificant constriction (*p < . ). the isolated ductus of term gestation mice (b) showed significant concentration-dependent constriction in response to either drug (**p < . ). indomethacin (white circles, n = ) was more effective than paracetamol (black squares, n = ) at term gestation (†p < . ). each drug was more potent at term than preterm gestation (p < . ). mean ± sem. ** ** † % c h a n g e f ro m b a se lin e − − − − − − − − − − − log (m) * % c h a n g e f ro m b a se lin e − − − − − − − − − − − a b figure . inhibition of ductus arteriosus prostaglandin synthesis. freshly isolated term gestation ductus explants were incubated in the presence of drug or the appropriate vehicle. prostaglandin synthetic activity was measured by the formation of -keto pgf α, the stable metabolite of prostacyclin (pgi ) (a), or pge (b) after exposure to exogenous µmol/l arachidonic acid for  min. vessels treated with paracetamol (para) had insignificant reduction in prostaglandin synthesis compared to vehicle (water). indomethacin-treated vessels (indo) produced significantly less pgi and pge compared to controls (ethanol). *p < . . water etoh para indo . . f o ld c h a n g e . water etoh para * * indo . . f o ld c h a n g e . a b volume | number | september pediatric research copyright © international pediatric research foundation, inc. articles el-khuffash et al. paracetamol. oncel et al. ( ) achieved % closure rate in infants who received ivp. although gestational age and weight at birth were comparable, advanced age at treatment (median of vs. d) and larger pda diameter ( . ( . – . ) vs. . ( . – . )) in our cohort may account for the discrepancy in treatment success rate between the two studies. paracetamol serum levels after  h of an i.v. course are higher than those achieved after  h of an oral course of  mg/kg/d as reported by yurttutan et al. ( ) ( . ( – ) vs. µg/ml ( – )). the benefits of ivp have not been uniform. roofthooft et al. ( ) reported giving paracetamol at a dose of  mg/kg/d to infants ( with i.v. and with oral paracetamol) with a median (range) gestation of / ( / – / ) at a median age of d. pda failed to close in seven of those infants with all needing ligation. pda closure was achieved in only one infant, with one infant exhibiting pda restriction and another infant died from gastrointestinal problems. none of the infants developed liver toxicity. similarly, another case series of three infants reported failure of pda closure in all patients following iv paracetamol (  mg/kg every  h of unspecified duration), necessitating surgical ligation. two infants developed transient raised liver enzymes ( ). more recently in a randomized controlled trial, oncel et al. compared the efficacy of oral paracetamol (at a dose of  mg/kg every  h for d) with that of oral ibuprofen (at a dose of  mg/kg followed by two doses of  mg/kg  h apart) in a group of preterm infants less than ,  g. the treatment was instituted between and  h of life. the group found that paracetamol achieved similar pda closure rates to ibuprofen ( . vs. . %) with no significant differences in reopening rates ( ). this study suggests that the timing of treatment is also an important consideration as early pdas may be more responsive to paracetamol than the ones treated beyond the second week of life. in the animal experiment, unlike indomethacin, paracetamol did not have an ex vivo constricting effect on the preterm duc- tus, and vessels treated with paracetamol had insignificant reduction in prostaglandin synthesis compared to vehicle (water). indomethacin-treated vessels produced significantly less pgi and pge compared to controls (ethanol). we previ- ously showed that the preterm mouse da was less affected by – mol/l indomethacin than the term gestation da ( ). the current study examined the ductus response to an extended range of indomethacin concentrations and demonstrates a marked upregulation in sensitivity of the ductus to prosta- glandin inhibition with advancing gestation. this finding is in agreement with previous clinical reports ( , ) and obser- vations in other animal species ( ). momma et al. ( ) also demonstrated that indomethacin (when administered to the maternal rat) possesses a more potent da-constricting effect in fetal rats when compared to paracetamol. paracetamol, however, also possessed a significant but milder constrictive effect. the results of the clinical observation by roofthooft ( ) highlighted above support the finding of our animal study. the difference in response between the human and mouse preterm ductus may represent different expression of the peroxidase moiety of prostaglandin h synthetase enzyme responsible for metabolizing arachidonic acid ( ). the differences may also relate to the fact that the majority of human neonates who received paracetamol were previously treated with nsaids, which may modulate the responsiveness of ductal tissue through mechanisms that have not been delineated. it was not possible to mimic the human situation using the in vitro experimental design. pge is the most important factor in the regulation of da tone during fetal da development. it is generated through the combined actions of prostaglandin h synthase- /- (pghs- /- ) and microsomal pge synthase enzymes on arachidonic acid. pge acts on g protein-coupled e-prostanoid receptors causing vasodilation of ductal tissue ( ). the inhibition of pghs- leads to reduced pge levels and ductal constriction ( ). current practice exploits this mechanism by the use of nonselective pghs inhibitors such as indomethacin and ibu- profen to close ducts postnatally ( , ). paracetamol is also thought to be a potent pghs inhibitor and reduces the levels of pge . the paracetamol trend of lowering pge levels in our study further supports this mechanism. however, there may be a graded inhibition effect, which is related to gestation. both clinical and animal evidence suggest that the inhibitory effect of paracetamol on pge is not present at lower gestations. our study was limited by the small number of infants and the lack of paracetamol drug levels in the clinical study. as a result, we could not demonstrate that the escalating dosing regimen described in our infant cohort translated into an esca- lating drug level. we based this assumption on the graded clin- ical response to the varying regimens. in addition, the ex vivo effect of paracetamol on preterm and term mice das may not mirror the in vivo effect in humans, and the result of this needs to interpret with caution. in conclusion, the routine use of paracetamol for pda clo- sure in preterm infants cannot be recommended until ran- domized controlled trials of its efficacy are completed. those rcts should be supported by robust pharmacokinetic data designed to determine the optimal dosing regimen, duration of treatment, and mode of administration. the safety of the use of paracetamol to close pdas in preterm infants also warrants further investigation. methods human study study setting. this was a retrospective review of all infants who received paracetamol for pda closure in two tertiary neonatal inten- sive-care units between january and june : mount sinai hospital, and the rotunda maternity hospital, dublin, ireland. one center (mount sinai hospital) administered either scop of  mg/ kg every  h for  h or lcop of  mg/kg every  h for d (tyle- nol oral suspension liquid  mg/  ml, johnson & johnson, toronto, ontario, canada). this was done on an ad hoc basis due to the various reports of the differing regimens at the time. neonates at the second center (rotunda) received ivp of  mg/kg every  h for a minimum of  h until pda closure was confirmed on echocardiography or up to a maximum of d (paracetamol  mg/ml solution for infu- sion, fresenius kabi, cheshire, uk). in both centers, the decision to administer paracetamol treatment to neonates with a hemody- namically significant pda was either after failure of two courses of either ibuprofen or indomethacin or if there were contraindications to medical treatment. liver enzymes were measured before and after pediatric research volume | number | september copyright © international pediatric research foundation, inc. paracetamol and pda closure articles treatment. paracetamol was only commenced if liver function tests were normal. clinical and echocardiography data. the following clinical infor- mation was collected for every infant enrolled: gestation and birth weight; previous use of nsaid; contraindications to nsaid use; age and weight at paracetamol treatment; duration of paracetamol treat- ment; and reason for pda treatment. treatment of the pda was at the discretion of the attending neonatologist. clinical outcomes within d of treatment were also collected and included pda ligation, wean- ing of ventilator or continuous positive airway pressure support, and death. in both centers, echocardiography assessment of pda significance was performed before and after paracetamol treatment using ge vivid i (ge medical, milwaukee, wi), or phillips hd ultrasound system (andover, ma). pda treatment with paracetamol was com- menced if there was clinical concern and echocardiography evidence of a large pda (> .  mm) accompanied by echocardiography evi- dence of pulmonary over circulation or systemic hypoperfusion ( ). the following echocardiography parameters were collected before and after treatment: pda diameter (measured in d at the pulmo- nary end) and shunt direction, mean pressure gradient across the pda, and markers of pulmonary over-circulation (left atrium: aortic (la:ao) ratio; isovolumic relaxation time; left ventricular output and left ventricular end diastolic velocity). the institutional ethics committees of mount sinai hospital and the rotunda hospital approved the retrospective clinical review. in vitro study animals and tissues. due to the limited availability and viability of human ductus tissues, the response of the ex vivo term and pre- term mouse da to indomethacin and paracetamol was examined. experiments were conducted in accordance with national institutes of health animal care standards and were approved by the institutional animal care and use committee at vanderbilt university medical centre. adult female cd mice (charles river, raleigh, nc) were bred with fertile males of the same strain to produce timed pregnan- cies. the morning of finding a vaginal plug was considered day of pregnancy. preterm (day ) and term (day ) gestation da were surgically isolated and either mounted in microvessel perfusion chambers as previously described ( ), or plated for drug inhibition studies. pressure myography. pressure myography was used to directly study vasomotor tone of the isolated da. myography chambers were placed on inverted microscopes equipped with a digital image capture sys- tem (ionoptix, burlington, vt) to record intraluminal diameter. vessels were studied in deoxygenated ( % n , %co ) krebs buffer that was modified (in mmol/l: nacl, nahco , . kcl, . mgso , . kh po , . dextrose, and . cacl ) to maintain a sta- ble ph ( . – . ) and relative hypoxia in the vessel bath (dissolved oxygen content = . – . %; measured pao = – torr). mounted vessels were pressurized using a column of krebs buffer and allowed to equilibrate, after which the pressure was increased in a stepwise manner to approximate mean arterial pressure ( mmhg, preterm; mmhg, term). vessels were then challenged with two doses of mmol/l kcl in krebs buffer to test vessel reactivity and to determine maximum constriction values. vessels that failed to constrict were excluded from further study. after wash out with krebs buffer, ves- sels were exposed to increasing concentrations ( – to – mol/l) of paracetamol or indomethacin in the perfusion bath. changes in vessel diameter were continuously monitored by video recording; lumen diameter was plotted in real-time to determine the concen- tration-specific effects of each drug. following each drug dose, ves- sel diameters were allowed to plateau ( –  min) before addition of the next dose. at the end of each drug study, the nitric oxide donor, sodium nitroprusside (snp) was added to demonstrate the vasodila- tory capacity of the ductus after drug-induced vasoconstriction; then compounds were washed off and vessels returned to resting baseline tone (figure ). after the final wash out, vessels were challenged with mmol/l kcl at the completion of each study to demonstrate via- bility of the vessel preparation. paracetamol was prepared in water; indomethacin was dissolved in ethanol but did not exceed . % in the final bath concentration. measurement of prostaglandins. the ductus of term fetal mice (day , n = litters) were sharply excised from surrounding tissues, split open along the longitudinal axis, and placed in a -well plate con- taining serum-free dulbecco's modified eagle's medium media + % penicillin–streptomycin (three vessels per well). vessels were then exposed to paracetamol ( − mol/l), indomethacin ( .  ×  − mol/l), or vehicle controls (water or ethanol, respectively) for  min at °c, after which the conditioned media was collected for analysis. pg, including pge , pgd , pgf α, txb and the prostacylin metabolite -keto-pgf α, were measured in the conditioned media by a stable isotope dilution assay utilizing gas chromatography/negative ion chemical ionization-mass spectrometry as previously described ( – ). statistical analysis. continuous data were presented as medians and interquartile ranges, and categorical data as absolute values and per- centages. independent data were compared using mann–whitney u-test, and paired data were compared using wilcoxon signed-rank test. multiple groups were compared using the kruskal–wallis test. clinical variables were not compared statistically. for the animal data, individual drug responses were analyzed by repeated mea- sures anova; interdrug comparison was analyzed using two-way anova. post hoc analysis (tukey) was performed for studies that were significant. we considered a p value of < . as significant. we used spss (version . ) to perform 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be dose dependent: evidence from human and murine studies 	main 	results 	human study 	in vitro study 	discussion 	methods 	human study 	in vitro study 	references [pdf] impact of prevailing thiamin levels on thiamin pyrophosphate uptake in pancreatic acinar cells: do the shuttle! | semantic scholar skip to search formskip to main content> semantic scholar's logo search sign increate free account you are currently offline. some features of the site may not work correctly. 	doi: . /ajpgi. . corpus id: impact of prevailing thiamin levels on thiamin pyrophosphate uptake in pancreatic acinar cells: do the shuttle! @article{kennedy impactop, title={impact of prevailing thiamin levels on thiamin pyrophosphate uptake in pancreatic acinar cells: do the shuttle!}, author={lindsey kennedy and h. francis and g. alpini}, journal={american journal of physiology. gastrointestinal and liver physiology}, year={ }, volume={ }, pages={ g -g } } 	lindsey kennedy, h. francis, g. alpini 	published 	chemistry, medicine 	american journal of physiology. gastrointestinal and liver physiology thiamin is a water-soluble b vitamin that regulates critical cellular processes, such as oxidative energy metabolism, adenosine triphosphate (atp) production, and mitochondrial function; thus, it is referred to as the energy vitamin ([ ][ ], [ ][ ]). based on these known roles of thiamin,  view on pubmed physiology.org save to library create alert cite launch research feed share this paper citations view all topics from this paper 	thiamine pyrophosphate 	paclitaxel 	p bacteriophage artificial chromosomes 	adenosine triphosphate 	acinar cell of pancreas 	ethanol 	nervous system disorder 	mitochondrial diseases 	necrosis 	butyl phosphorotrithioate 	pyruvate dehydrogenase 	microcephaly 	oxidative stress 	thiamine deficiency 	email 	thiamine diphosphate catabolic process 	in vitro [publication type] 	large 	scientific publication 	structure of parenchyma of lung 	import into cell 	thiamine-phosphate kinase activity one citation citation type citation type 	all types 	cites results 	cites methods 	cites background has pdf publication type author more filters more filters filters sort by relevance sort by most influenced papers sort by citation count sort by recency adaptive regulation of pancreatic acinar mitochondrial thiamin pyrophosphate uptake process: possible involvement of epigenetic mechanism(s). 	subrata sabui, v. subramanian, rubina kapadia, h. said 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	 save alert research feed references showing - of references sort byrelevance most influenced papers recency adaptive regulation of pancreatic acinar mitochondrial thiamin pyrophosphate uptake process: possible involvement of epigenetic mechanism(s). 	subrata sabui, v. subramanian, rubina kapadia, h. said 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	 	view excerpts, references background save alert research feed inhibition of pancreatic acinar mitochondrial thiamin pyrophosphate uptake by the cigarette smoke component -(methylnitrosamino)- -( -pyridyl)- -butanone. 	p. srinivasan, e. thrower, f. gorelick, h. said 	chemistry, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	highly influential 	 	view excerpts, references background save alert research feed thiamine deficiency in cultured neuroblastoma cells: effect on mitochondrial function and peripheral benzodiazepine receptors 	l. bettendorff, g. goessens, + authors t. grisar 	biology, medicine 	journal of neurochemistry 	 	 	pdf 	view excerpt, references background save alert research feed thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells 	marjorie gangolf, j. czerniecki, + authors l. bettendorff 	biology, medicine 	plos one 	 	 	pdf 	view excerpt, references background save alert research feed the compartmentation of phosphorylated thiamine derivatives in cultured neuroblastoma cells. 	l. bettendorff 	chemistry, medicine 	biochimica et biophysica acta 	 	 	view excerpt, references background save alert research feed adaptive regulation of intestinal thiamin uptake: molecular mechanism using wild-type and transgenic mice carrying hthtr- and - promoters. 	j. reidling, h. said 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	pdf 	view excerpt, references background save alert research feed chronic alcohol exposure affects pancreatic acinar mitochondrial thiamin pyrophosphate uptake: studies with mouse - cell line and primary cells. 	p. srinivasan, s. nabokina, h. said 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	highly influential 	pdf 	view excerpts, references background save alert research feed relative contribution of thtr- and thtr- in thiamin uptake by pancreatic acinar cells: studies utilizing slc a and slc a knockout mouse models. 	v. subramanian, s. subramanya, h. said 	biology, medicine 	american journal of physiology. gastrointestinal and liver physiology 	 	 	pdf 	view excerpt, references background save alert research feed the evidence that the dnc (slc a ) is not the mitochondrial deoxyribonucleotide carrier. 	jonghoon kang, d. samuels 	biology, medicine 	mitochondrion 	 	 	view excerpt, references background save alert research feed oxidative stress is associated with region-specific neuronal death during thiamine deficiency. 	n. calingasan, w. j. chun, l. park, k. uchida, g. gibson 	biology, medicine 	journal of neuropathology and experimental neurology 	 	 	pdf 	view excerpt, references background save alert research feed ... ... related papers 	abstract 	topics 	 citations 	 references 	related papers stay connected with semantic scholar sign up about semantic scholar semantic scholar is a free, ai-powered research tool for scientific literature, based at the allen institute for ai. learn more → resources datasetssupp.aiapiopen corpus organization about usresearchpublishing partnersdata partners   faqcontact proudly built by ai with the help of our collaborators terms of service•privacy policy the allen institute for ai by clicking accept or continuing to use the site, you agree to the terms outlined in our privacy policy, terms of service, and dataset license accept & continue received: . . accepted: . . published: . . phenotypic variation in patients with homozygous c. g>t mutation in evc gene: report of two mexican families with ellis-van creveld syndrome abcdef marisol ibarra-ramirez abc luis daniel campos-acevedo be jose lugo-trampe aef laura e. martínez-garza cd , víctor martinez-glez cd , maría valencia-benitez cd , pablo lapunzina cdef , víctor ruiz-peréz corresponding author: marisol ibarra-ramírez, e-mail: m.ibarrar @gmail.com conflict of interest: none declared case series patient: — final diagnosis: ellis van creveld syndrome symptoms: conical teeth • polydactyly • short stature medication: — clinical procedure: — specialty: pediatrics and neonatology objective: rare disease background: ellis-van creveld syndrome is an autosomal recessive chondro-ectodermal dysplasia characterized by dispro- portionate short stature, limb shortening, narrow chest, postaxial polydactyly and dysplastic nails and teeth. in addition, % of cases present congenital heart defects. ellis-van creveld syndrome is predominantly caused by mutations in the evc or evc ( p ) genes, with only a few cases caused by mutations in wdr . case report: here, we report on two mexican families with patients diagnosed with ellis-van creveld syndrome. family includes four patients: three females of , , and years of age and a -year old male. family has only one affected newborn male. all patients exhibited multiple features including hypodontia, dysplastic teeth, ex- tra frenula, mild short stature, distal limb shortening, postaxial polydactyly of hands and feet, nail dystrophy, and knee joint abnormalities. only two patients had an atrial septal defect. in all cases, molecular analysis by sanger sequencing identified the same homozygous mutation in exon of evc, c. g>t, which leads to a premature stop codon. conclusions: the mutation c. g>t has been previously reported in another mexican patient and it appears to be a recur- rent mutation in mexico which could represent a founder mutation. the large number of patients in this case allows the clinical variability and spectrum of manifestations present in individuals with ellis-van creveld syn- drome even if they carry the same homozygous mutation in a same family. mesh keywords: ellis-van creveld syndrome • genes, recessive • rare diseases full-text pdf: https://www.amjcaserep.com/abstract/index/idart/ authors’ contribution: study design a data collection b statistical analysis c data interpretation d manuscript preparation e literature search f funds collection g department of genetics, faculty of medicine, autonomous university of nuevo león, monterrey, nuevo león, mexico institute of medical and molecular genetics (ingemm), hospital la paz institute for health research (idipaz), madrid, spain ciber rare diseases, carlos iii health institute, madrid, spain institute of biomedical research “alberto sols” (iibm), spanish national research council (csic), autonomous university of madrid (uam), madrid, spain e-issn - © am j case rep, ; : - doi: . /ajcr. this work is licensed under creative common attribution-noncommercial-noderivatives . international (cc by-nc-nd . ) background ellis-van creveld syndrome (evc; omim: ) is an auto- somal recessive chondro-ectodermal dysplasia, first described by ellis and van creveld in [ ]. this syndrome is charac- terized by disproportionate short stature, with shortening of limbs, narrow chest, post-axial polydactyly, dysplastic nails, conical shaped teeth, and multiple frenula. in addition, congen- ital heart defects occur in % of cases [ – ]. the incidence of this condition is about . cases per , live births [ ], although in some populations, like the amish community of pennsylvania, it is as high as / , [ ]. the large majority of ellis-van creveld syndrome cases are as- sociated with mutations in either evc or evc which are adja- cent genes located on chromosome p [ – ]. the evc and evc proteins form a protein complex that localizes at the base of primary cilia and which acts as a positive regulator of hedgehog (hh) signaling, especially in chondrocytes [ – ]. using model organisms, it has been shown that evc and evc are essential for perichondrium formation and for the prolifer- ation and differentiation of chondrocytes [ , ]. the clinical pre- sentation of patients with mutations in either evc or evc is indistinguishable, and is characterized by variable expressivi- ty even in individuals of the same family. about % of ellis- van creveld syndrome patients have been reported with no mutations in evc or evc [ ]. recently, three of these cas- es were found to have mutations in wdr gene, which en- codes a protein that is part of the ift complex a, which is in- volved in the formation and maintenance of cilia and which also regulates hh signaling [ ]. here, we report the clinical characterization of ellis-van creveld syndrome patients from two mexican families and the molecular identification of the causing mutation. case report in the case of family , the clinical history includes affected in- dividuals of three generations (figure ). the parents of these patients (iii- and iii- ) were non-consanguineous and healthy, although two members from the paternal lineage had ellis- van creveld syndrome (ii- and iii- ). the paternal grandpar- ents (ii- and ii- ) were consanguineous. patients iv- , iv- and iv- were females of , , and years of age, respec- tively and patient iv- was a seven-year-old male. iv- was a miscarriage. iv- male and iv- female both died at one-year- old from unknown causes. it is important to mention that this family belonged to a very low income population from rural ar- eas, where there was not access to hospitals or medical spe- cialist, thus making it difficult to acquire medical records and which also explains the delay in diagnosis. all affected individuals showed multiple clinical features (table , figure ) including hypodontia, dysplastic teeth, multi- ple frenula, mild short stature, distal limb shortening, post-axial polydactyly of hands, and nail dysplasia. chest x-rays showed abnormal morphology of the clavicle without narrow chest or short ribs. patient iv- had an atrial septal defect (asd) shown by echocardiogram, while the other patients had a structur- ally normal heart. abdominal ultrasound was performed and reported normal in all patients. in the case of family , the patient was a newborn male, born at weeks of gestation by cesarean section delivery with a birth weight of , g (fifth percentile) and a length of cm (– sd who growth charts). the mother and father were and years old respectively, were both healthy and un- related, with no relevant family history. the patient had short stature, distal limb shortening, multiple frenula, post-axial polydactyly of hands and feet, nail dysplasia, and micropenis. figure . pedigree of family . the pedigree shows presence of ellis-van creveld syndrome patients in three generations. i evc synrrome patients heterozygous ii iii iv ibarra-ramirez m. et al.: phenotypic variation in patients with homozygous c. g>t mutation… © am j case rep, ; : - this work is licensed under creative common attribution-noncommercial-noderivatives . international (cc by-nc-nd . ) iv- iv- iv- iv- newborn (family ) age years old years old years old years old newborn sex female female female male male weight . kg (– . ds) . kg (– . ds) . kg (– . ds) . kg (– ds) . kg (p ) length . cm (– . ds) . cm (– . ds) . cm (– . ds) . cm (– . ds) cm (– sd) ofc . cm (p ) . cm (p ) . cm (– . ds) . cm (p – ) . cm (p ) spam cm cm cm cm . cm congenital heart defects asd – – – asd/ductus arteriosus postaxial polydactyly in hands + +s + + + postaxial polydactyly in feet – – +s – +s fusion of capitate and hamate + + + + – genu valgum + – + + – nail dystrophy + + + + + conical shaped teeth + + + + – multiple frenulae + + + + + long narrow chest + + + + + table . clinical features of ellis-van creveld syndrome patients. asd – atrial septum defect; ofc – occipital-frontal circumference; s – polysyndactyly. echocardiogram showed asd of mm and ductus arteriosus. abdominal and pelvic ultrasound were both normal (table ). the patient died at five months of age of unknown causes. the molecular analysis using sanger sequencing of evc and evc in the proband of family (iv- ) revealed a homozygous mutation in exon of evc, nm_ . : c. g>t, which causes a premature stop codon p.(glu ter). the same mutation was confirmed in homozygosis in the oth- er affected siblings (iv- , iv- , iv- ) and was found in the het- erozygous state in the parents (iii- and iii- ). as no dna of the newborn patient of family was available, mutation anal- ysis was performed for his parents. both parents were found to carry the same nm_ . : c. g>t mutation in the heterozygous state. thus, we inferred that this mutation pres- ent as homozygosis in their affected son. discussion evc and evc are two ciliary proteins that are not required for ciliogenesis, but are essential mediators of hh signaling. accordingly, ellis-van creveld syndrome is included in the path- ological group of ciliopathies [ ]. so far, more than mu- tations have been reported in evc and evc , most of which introduce premature stop codons and therefore are predicted to lead to non-functional truncated proteins [ , ]. ellis-van creveld syndrome is a rare condition with only around cases reported [ ]. however, there are very few mexican cas- es reported. we have performed clinical and molecular analysis of a group of patients from two independent mexican families and have found that all the patients have a homozygous muta- tion in exon of evc, nm_ . : c. g>t, which intro- duces a premature stop codon p.(glu ter). interestingly, the same mutation was previously reported by valencia et al. [ ] in another mexican patient who was described as a compound heterozygote having in addition to the c. g>t change an- other nonsense mutation in evc exon , also in the exac brows- er the only carrier of this variant is a latino [ ]. family showed a non-classic pedigree of autosomal recessive inheritance. however, after the molecular analysis, this pattern of inheritance could be explained by ethnic inbreeding and the consanguinity of the grandparents. a high frequency of ellis-van ibarra-ramirez m. et al.: phenotypic variation in patients with homozygous c. g>t mutation… © am j case rep, ; : - this work is licensed under creative common attribution-noncommercial-noderivatives . international (cc by-nc-nd . ) figure . clinical features of patients with ellis-van creveld syndrome. (a, b) patient iv- and iv- showing conical shaped teeth and multiple frenulae. (e–g) showed postaxial polydactyly of hands or feet, syndactyly and nail dysplasia. (h, i). x-rays of the hands showed postaxial polydactyly and syndactyly, short middle, and distal phalanges with cone shaped epiphyses and carpal fusion. (j) radiological abnormalities of the patient iv- : short ribs and cardiomegaly, due to asd. (c, k) the x-rays of the patient iv- showed lateral tibial metaphysis slanted giving rise to genu valgum deformity. (d, l) male newborn from family present shortening of limbs, narrow chest, and the radiological abnormalities of the short ribs and short longs bones. a e h i j k l f g b c d creveld syndrome has been reported in inbreeding communi- ties like the amish population [ ]. our patients from family belonged to a very small rural population of less than , people, and it is likely that inbreeding and founder mutation may account for the increased frequency of cases in this family. the analysis of carrier frequency of the c. g>t mutation in this population would provide valuable information about the prevalence of this recurrent mutation and would facilitate offering appropriate genetic counseling. parents of family were not consanguineous or related to family . they live in a metropolitan area and were, never- theless, carriers of the same mutation. the geographic region of origin of the family reported by valencia et al. [ ] is un- known; therefore, we cannot determine whether that patient was from a community near to the place where family lived. from these data, we can conclude that c. g>t is a recur- rent mutation in mexico. the clinical features of the patients from our report were con- sistent with the previous literature. the affected individuals from family all had disproportionate short stature, which ranged from cm to cm (patients iv- , iv- , and iv- ) and similarly the newborn male of family was below normal length at birth ( cm, – sd) [ , ]. the phalanges of these pa- tients also showed disproportionate shortening, with the dis- tal phalanges more affected than the proximal phalanges [ ]. nail dysplasia, which is one of the cardinal features of ellis-van creveld syndrome, was detected in all the patients. patients iv- , iv- , and iv- had severe genu valgum, which is also fre- quent in patients with ellis-van creveld syndrome. bilateral postaxial polydactyly type a in hands (figure ), conical teeth, and multiple frenula were observed in all patients, however, only patient iv- and the child of family had feet polysyndactyly. previous studies have reported polydactyly of the feet in only % of the cases [ ]. patient iv- (family ) and the patient of family had an asd, which is the most common heart defect in ellis-van creveld syndrome. heart abnormalities are observed in approximately % of ellis-van creveld syndrome patients and are a major cause of morbidity in this syndrome [ ]. hh signaling, that is affected when evc and evc are mutated, has been shown to play an important role in intracardiac sep- tation [ ]. thus, it is very likely that altered hh signaling un- derlies the cardiac malformations reported in ellis-van creveld syndrome patients [ ]. ibarra-ramirez m. et al.: phenotypic variation in patients with homozygous c. g>t mutation… © am j case rep, ; : - this work is licensed under creative common attribution-noncommercial-noderivatives . international (cc by-nc-nd . ) conclusions the large number of cases in family illustrated the clinical variability and spectrum of manifestations present in individuals with ellis-van creveld syndrome even if they carry the same homozygous mutation. to our knowledge, this is the first re- port of two ellis-van creveld syndrome mexican families de- scribed with both clinical and molecular findings. references: . ellis rw, van creveld s: a syndrome characterized by ectodermal dyspla- sia, polydactyly, chondro-dysplasia and congenital morbus cordis: report of three cases. arch dis child, ; : e . ruiz-perez vl, goodship ja: ellis-van creveld syndrome and weyers ac- rodentaldysostosis are caused by cilia-mediated diminished response to hedgehog ligands. am j med genet c semin med genet, ; c( ): – . muenster oj, berdon w, mcmanus c et al: ellis-van creveld syndrome: its history. peadiatric radiol, ; : – . valencia m, tabet l, yazbek n et al: ellis-van creveld syndrome: mutations uncovered in lebanese families. case rep genet, ; : . mckusick va, egeland ja, eldridge r, krusen de: dwarfism in the amish i. the ellis-van creveld syndrome. bull johns hopkins hosp, ; : e– . polymeropoulos mh, ide se, wright m et al: the gene for the ellis-van creveld syndrome is located on chromosome p . genomics, ; : – . ruiz-pérez, vl, ide se, strom tm et al: mutations in a new gene in ellis-van creveld syndrome and weyers acrodentaldysostosis. nature genet, ; : – . ruiz-perez vl, tompson sw, blair hj et al: mutations in two nonhomolo- gous genes in a head-to-head configuration cause ellis-van creveld syn- drome. am j hum genet, ; ( ): – . ruiz-perez vl, blair hj, rodriguez-andres me et al: evc is a positive medi- ator of ihh-regulated bone growth that localizes at the base of chondro- cyte cilia. development, ; : – . caparrós-martin ja, de lucca a, cartault f et al: specific variants in wdr cause a distinctive form of ellis-van creveld syndrome by disrupting the recruitment of the evc complex and smo into the cilium. hum mol genet, ; : – . dorn kv, hughes ce, rohatgi r: a smoothened-evc complex trans- duces the hedgehog signal at primary cilia. dev cell, ; ( ): – . yang c, chen w, chen y, jiang j: smoothened transduces hedgehog signal by forming a complex with evc/evc . cell res, ; : – . zhang h, takeda h, tsuji t et al: generation of evc /limbin global and con- ditional ko mice and its roles during mineralized tissue formation. genesis, ; : – . d’asdia mc, torrente i, consoli f et al: novel and recurrent evc and evc mutations in ellis-van creveld syndrome and weyers acrofacial dyostosis. eur j med genet, ; ( ): – . huber c, cormier-daire v: ciliary disorder of the skeleton. am j med genet c semin med genet, ; c: – . valencia m, lapunzina p, lim d et al: widening the mutation spectrum of evc and evc : ectopic expression of weyer variants in nih t fibroblasts disrupts hedgehog signaling. hum mutat, ; ( ): – . lek m, karczewski kj, minikiel ev et al., exome aggregation consortium: analysis of protein-coding genetic variation in , humans. nature, ; ( ): – . baujat g, le merrer m: ellis-van creveld syndrome. orphanet j rare dis, ; : . goddeeris mm, rho s, petiet a et al: intracardiac septation requires hedge- hog dependent cellular contributions from outside the heart. development, ; : – . hills cb, kochilas l, schimmenti la, moller jh: ellis-van creveld syndrome and congenital heart defects: presentation of an additional cases. pediatr cardiol, ; : – ibarra-ramirez m. et al.: phenotypic variation in patients with homozygous c. g>t mutation… © am j case rep, ; : - this work is licensed under creative common attribution-noncommercial-noderivatives . international (cc by-nc-nd . ) cir .. m a j o r a r t i c l e unrecognized ingestion of toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in north america kenneth boyer, , dolores hill, ernest mui, kristen wroblewski, theodore karrison, j. p. dubey, mari sautter, a. gwendolyn noble, , shawn withers, charles swisher, peter heydemann, , tiffany hosten, jane babiarz, daniel lee, paul meier, , ,* rima mcleod, , , , and other members of the toxoplasmosis study groupa department of pediatrics, and division of pediatric infectious diseases, rush university medical center, chicago, illinois, and united states department of agriculture, agricultural research services, animal and natural resources institute, animal parasitic diseases laboratory, beltsville, maryland; department of ophthalmology and visual sciences, and department of health studies, university of chicago, illinois; department of ophthalmology, and division of pediatric neurology, children's memorial hospital, westchester, illinois; department of neurology, rush university medical center, chicago, illinois; department of biostatistics, columbia university, new york, new york, deceased; department of pediatrics, division of infectious diseases, university of chicago, illinois; division of infectious diseases, university of chicago, illinois; and department of medicine, division of infectious diseases, michael reese hospital and medical center, chicago, illinois (see the editorial commentary by linn, on pages – .) background. congenital toxoplasmosis presents as severe, life-altering disease in north america. if mothers of infants with congenital toxoplasmosis could be identified by risks, it would provide strong support for educating pregnant women about risks, to eliminate this disease. conversely, if not all risks are identifiable, undetectable risks are suggested. a new test detecting antibodies to sporozoites demonstrated that oocysts were the predominant source of toxoplasma gondii infection in north american epidemics and in mothers of children in the national collaborative chicago-based congenital toxoplasmosis study (ncccts). this novel test offered the opportunity to determine whether risk factors or demographic characteristics could identify mothers infected with oocysts. methods. acutely infected mothers and their congenitally infected infants were evaluated, including in-person interviews concerning risks and evaluation of perinatal maternal serum samples. results. fifty-nine ( %) of mothers of congenitally infected infants in ncccts had primary infection with oocysts. only % of these mothers identified significant risk factors for sporozoite acquisition. socioeconomic status, hometown size, maternal clinical presentations, and ethnicity were not reliable predictors. conclusions. undetected contamination of food and water by oocysts frequently causes human infections in north america. risks are often unrecognized by those infected. demographic characteristics did not identify oocyst infections. thus, although education programs describing hygienic measures may be beneficial, they will not suffice to prevent the suffering and economic consequences associated with congenital toxoplasmosis. only a vaccine or implementation of systematic serologic testing of pregnant women and newborns, followed by treatment, will prevent most congenital toxoplasmosis in north america. if mothers of infants with congenital toxoplasmosis could reliably identify exposure to toxoplasma gondii, it would provide strong support for eliminating this dis- ease by educating pregnant women about risk factors. conversely, if not all risk factors are recognized, an un- detectable environmental risk for pregnant women that cannot be eliminated by education alone may be present. one author (k. b.) conducted in-person interviews of a cohort of north american mothers of infants with congenital toxoplasmosis regarding recognized risk factors [ ]. serum samples from of these mothers were obtained in the perinatal period when their con- genitally infected infants were born and have been received april ; accepted july ; electronically published october . *dr. meier died during the review process. aother members of the toxoplasmosis study group are listed at the end of the text. correspondence: rima mcleod, md, toxoplasmosis center, university of chicago, s maryland ave, chicago, il (rmcleod@midway.uchicago.edu). clinical infectious diseases ; ( ): – � the author . published by oxford university press on behalf of the infectious diseases society of america. all rights reserved. for permissions, please e-mail: journals.permissions@oup.com. - / / - $ . doi: . /cid/cir unrecognized ingestion toxoplasma gondii oocysts d cid : ( december) d at u . of f lorida h ealth s cience c enter l ibrary on d ecem ber , http://cid.oxfordjournals.org/ d ow nloaded from http://cid.oxfordjournals.org/ http://cid.oxfordjournals.org/ stored by the national collaborative chicago-based congenital toxoplasmosis study (ncccts). recently, these serum sam- ples were tested with a new assay to determine whether anti- bodies to the sporozoite form of t. gondii were present [ ]. antibodies to sporozoites are present only when humans or other animals have been infected with t. gondii oocysts, formed in cats. antibodies to sporozoites are not present when the host is infected with bradyzoites in tissue cysts [ ]. tissues are in- fectious when consumed in undercooked meat [ ]. we recently found, by testing serum samples for this antibody to a -kda sporozoite protein, that oocyst infection also occurred in per- sons in north american epidemics (ie, at a riding stable in atlanta [ ], at a research laboratory, in an amish family [ ] in the united states, and in victoria, canada [ ]) and in mothers of infants in the ncccts [ ]. here, we correlate presence of this serum antibody to sporozoites with a mother’s recognition of risk factors for acquisition of t. gondii, either from materials contaminated with oocysts or bradyzoites during the months before delivery, demographic features, or presence and mani- festations of clinical illness in mother and child after delivery. methods national collaborative chicago-based congenital toxoplasmosis study mothers seventy-six acutely infected mothers in the ncccts who transmitted t. gondii to their fetuses in utero provided serum samples for testing near the time when their infected child was born between and . these mothers have been de- scribed in other publications of the ncccts, including the amish and the victoria, canada, mothers [ , , , – ]. our study was conducted according to the ethical standards for hu- man experimentation established in the declaration of helsinki, with the prior approval of the institutional review board of the university of chicago and in accordance with health insurance portability and accountability act regulations. informed con- sent was obtained from all adult participants and from parents or legal guardians of minors. questions asked concerning risk factors in addition to providing serum samples for serologic testing, the mothers were questioned about their possible exposure to common vehicles of t. gondii transmission, specifically cats, oocyst-contaminated soil, and meat not cooked to well-done. each mother was asked whether she owned a cat, had cleaned a cat’s litter box, had gardened, had come into contact with a sandbox, or had very close, sustained, hours-long contact with a new kitten. for example, mother kept her new kitten in bed with her at night and was scratched by this kitten while playing with it. the interviewer classified the type of exposure to cats for each mother. cleaning a litter box, gardening, coming into contact with a sandbox, or very close, sustained contact with a kitten were defined as significant risk factors. owning an in- door cat fed dry or canned food is considered a cat-associated risk factor, but not necessarily one that has substantial risk for exposure to oocysts for the cat owner. the mothers were also asked whether they had prepared or consumed raw or under- cooked meat or other foods that may have harbored the parasite, such as raw eggs or unpasteurized dairy products. details about the type and frequency of the exposure and when it occurred during the pregnancy were noted. any symptoms of illness during pregnancy that could indicate infection, such as influenza-like symptoms, fever, night sweats, headache, or lymphadenopathy, were also recorded. demographic information including ma- ternal age, place of residence, race/ethnicity, method of pay- ment for care, and the variables needed to calculate the family’s hollingshead index (a measure of socioeconomic status) were obtained. evaluation of mother and child socioeconomic limitations were circumvented by providing complimentary travel and accommodations for all patients participating in the study. each patient’s medical history and neuroradiological scans were reviewed. for the infants, eval- uations were conducted by adult and pediatric specialists in infectious diseases, neurology, developmental psychology, developmental pediatrics, pediatric ophthalmology, audiol- ogy, and neuroradiology. hematologic laboratory tests, in- cluding complete blood cell count, differential white blood cell count, and platelet counts were also performed. the evalua- tions were conducted at predetermined ages: birth and , . , , . , , , and years [ – ]. toxoplasma gondii serologic testing of mother and child toxoplasma gondii immunoglobulin g assays the biomérieux direct agglutination assay [ ] was used to measure immunoglobulin g (igg) in serum samples from the pregnant amish women. the sabin-feldman dye test was used to measure igg in all other samples tested [ ]. the dye test is based on the observation that living parasites incubated with normal serum become swollen and stain deeply blue when methylene blue is added. exposure to antibody-containing se- rum results in thin, distorted unstained parasites when the dye is added because of lysis of the organisms secondary to activation of complement. toxoplasma gondii–specific immunoglobulin m assays the immunoglobulin m (igm) enzyme-linked immunosorbent assay (elisa) [ ] was used to test serum samples from the mothers in the ncccts. the igm elisa [ ] was used and, beginning on april , igm immunosorbent agglutination assay (isaga) [ ] was used to test newborns’ serum samples for igm-specific antibodies to t. gondii in the infants in our d cid : ( december) d boyer et al at u . of f lorida h ealth s cience c enter l ibrary on d ecem ber , http://cid.oxfordjournals.org/ d ow nloaded from http://cid.oxfordjournals.org/ http://cid.oxfordjournals.org/ patient cohort [ ]. the igm isaga is more sensitive than the igm elisa for detection of igm antibody specific to t. gondii in the infant [ ]. serologic testing for the persons in the atlanta epidemic was performed as described elsewhere [ ]. other t. gondii–specific serologic testing during the later years of the study, immunoglobulin a (iga) elisas were performed for ncccts mothers and their chil- dren [ ]. in addition, differential agglutination tests [ ] were performed for pregnant women in the ncccts, and avidity assays [ ] were also performed. determination of presence of antibody to sporozoites this assay was performed as described elsewhere [ ]. statistical methods correlations between the presence of serum antibody to the -kda sporozoite protein and demographic characteristics, perceived risk factors, and clinical findings were examined using fisher exact test for categorical variables and the wilcoxon rank-sum test [ ] for continuous or ordinal variables. the sensitivity, specificity, and positive and negative predictive val- ues of the perceived risk factors were calculated by treating the presence of serum antibody as the gold standard, or true indicator of exposure to oocysts. exact confidence intervals for proportions were based on the binomial distribution. all statistical analyses were performed using stata software, version (statacorp) [ ]. results high rate of exposure to oocysts in national collaborative chicago-based congenital toxoplasmosis study mothers seventy-eight percent of ncccts mothers tested, including mothers who were part of an epidemic in victoria, canada, and the mother in an amish family, were infected by spor- ozoites. lack of correlation of birth year or time of year with oocyst exposure neither year (figure ) nor month of the year (figure ) when the infant was born was clearly associated with exposure to oocysts. table . correlation of antibody to sporozoites in maternal serum samples and maternal demographic characteristics demographic factor overall (n ) positive (n ) negative (n ) p value ses scorea . ( . ); (n ) . ( . ); (n ) . ( . ); . agea . ( . ); . ( . ); . ( . ); . hometown size b . rural suburban urban caucasianb . abbreviation: ses, socioeconomic status. a numbers are mean (standard deviation); median. b numbers are percentages. figure . distribution of mothers with and without antibody to sporozoites in each year, – . unrecognized ingestion toxoplasma gondii oocysts d cid : ( december) d at u . of f lorida h ealth s cience c enter l ibrary on d ecem ber , http://cid.oxfordjournals.org/ d ow nloaded from http://cid.oxfordjournals.org/ http://cid.oxfordjournals.org/ demographic characteristics and exposure to oocysts socioeconomic status, age, race (white vs other), and hometown size classified as rural, urban, or suburban did not differentiate those who did or did not have antibodies to sporozoites (table ). women exposed to sporozoites were from throughout the united states (figure ). sensitivity and specificity of risk factors in identifying mothers infected by oocysts although some persons could be identified who had substantial exposure to a cat and likely oocyst exposure (eg, a mother who had moved into a house where dozens of cats had used the basement to defecate, and the mother had cleaned this basement while pregnant), the correlation between cat exposure and having antibody to t. gondii sporozoites was not robust (table , table ). in addition, none of the mothers who were exposed during the water-borne victoria epidemic [ ] recognized cat exposure. two of these mothers reported exposure to meat not cooked to well-done. it is of interest that the serum samples from mothers of ncccts children who were in this epi- demic had antibody to sporozoites, although several could only identify a rare meat exposure. there are no published data documenting oocyst exposure for other persons in the epidemic. at present, serum samples from other persons in this epidemic have not been tested using our sporozoite assay. maternal illness and oocyst exposure presence of specific symptoms or total number of symptoms did not correlate significantly with acquisition of oocysts by mothers (figure ). severity of illness in the infant and oocyst exposure neither the severity of illness in the infant nor the presence of hydrocephalus correlated significantly with presence of maternal serum antibody to sporozoites (figure ). maternal and infant serologic tests the magnitude of serologic tests for t. gondii–specific igg, igm, and iga in mothers and infants did not correlate significantly with oocyst exposure. the acetone-fixed parasite titer in the differential agglutination test was higher in the serum samples from mothers with antibody to the -kda sporozoite protein (p . ; table ). discussion a new method to determine whether infection with t. gondii was acquired from oocysts in the preceding months was figure . lack of correlation between presence of antibody to sporozoites in maternal serum samples and month of birth of child. d cid : ( december) d boyer et al at u . of f lorida h ealth s cience c enter l ibrary on d ecem ber , http://cid.oxfordjournals.org/ d ow nloaded from http://cid.oxfordjournals.org/ http://cid.oxfordjournals.org/ reported recently [ ]. this assay determined that oocyst in- fection induces an antibody specific to an -kda sporozoite protein [ ]. this antibody response differentiates between in- fections acquired by ingesting oocysts and infection caused by tissue cysts in pigs, mice, and humans [ ]. analysis of serum samples for presence of antibody to an -kda sporozoite protein can determine whether t. gondii infection was caused by the acquisition of oocysts for – months after acquisition of the infection [ ]. all persons received a diagnosis on the basis of serologic testing from the us reference laboratory. for adults (ie, for acutely infected mothers), these tests are highly reliable, es- sentially % sensitive, and specific in this reference laboratory. in an analysis of serum samples obtained in the united states and canada, oocysts were found to be the predominant source of t. gondii infection in % of the mothers in our ncccts cohort and in north american epidemics, including an out- break in victoria ( of mothers), of persons in an amish community, in all persons in a laboratory accident, and in a stable in atlanta ( [ %] of persons) [ ]. these findings indicate that oocysts are a major and often unrecognized source of infection in mothers of children with congenital toxoplas- mosis and in epidemics in the united states. it is important to both inform susceptible individuals and the general populations that meat should be cooked thoroughly, fruits and vegetables should be washed before consumption, and contact with oocysts should be avoided, particularly when changing cat litter. nonetheless, the data reveal that ownership of a cat is not necessary to acquire t. gondii, because it has been shown that oocyst exposure is not always associated with cat ownership or with recognition of risk factors. in the united states, there are an estimated million feral cats and million domestic cats [ ], which allows for environmental contami- nation by oocysts to be the source of infection even for those who do not own cats. hill et al recently determined that there was a low prevalence of tissue cysts currently in retail meats in the united states, including beef, chicken, and pork [ ]. the low frequency of infected meat suggests that cats potentially were a cause for much t. gondii infection in the united states. analysis of the correlation between the antibody specific to the sporozoite protein in serum samples from infected mothers and recognized risk factors indicates that exposure to oocysts was not identified in % of those who actually acquired the infection by this route. although it is beneficial for physicians to figure . lack of correlation of antibody to sporozoites in maternal serum samples with hometown location and hometown size. mothers with serum antibody to sporozoites and cysts are distributed across rural, urban, and suburban regions in the united states. table . correlation of antibody to sporozoites in maternal serum samples and recognition of exposure to uncooked meat versus material contaminated by oocysts from cats overall (n ) positive (n ) negative (n ) p value at least significant cat risk factor a,b . any risk factora . a numbers are percentages. b significant cat exposure is defined in methods as cleaning a cat’s litter box, gardening, coming into contact with a sandbox, or very close, sustained contact with a kitten. owning an indoor cat fed dry or canned food is considered a risk factor associated with cats, but not necessarily one that has substantial risk for exposure to oocysts for the cat owner. unrecognized ingestion toxoplasma gondii oocysts d cid : ( december) d at u . of f lorida h ealth s cience c enter l ibrary on d ecem ber , http://cid.oxfordjournals.org/ d ow nloaded from http://cid.oxfordjournals.org/ http://cid.oxfordjournals.org/ advise pregnant women to avoid exposure to cat feces, not all exposure is from recognized sources; most appeared not to be. thus, although education may help to prevent some cases of toxoplasmosis, it will not be sufficient to prevent a substantial proportion of these infections, because acquisition can be un- detected and recognized risk factors are frequently absent. because t. gondii infection in the united states is diagnosed without a systematic, gestational serologic screening program, it largely presents as severe disease in the infant. there are sub- stantial clinical manifestations detected in the newborn period [ – ] that lead to diagnosis. the route of acquisition in the mother did not correlate with severity of symptoms in the in- fant. this has varied only slightly during the past decades (r. mcleod, k. boyer, e. mui, k. wroblewski, t. karrison, m. sautter, g. noble, s. withers, c. swisher, p. heydemann, d. lee, d. burrowes, e. holfels, m. mets, p. latkany, w. mieler, table . specificities, sensitivities, and negative and positive predictive values of attributing risk of exposure based on acutely infected mothers of children enrolled in national collaborative chicago-based congenital toxoplasmosis study specificity a % ci sensitivity b % ci npv c % ci ppv d % ci at least cat risk factor – – – – ( / ) ( / ) ( / ) ( / ) at least significant cat risk factore – – – – ( / ) ( / ) ( / ) ( / ) abbreviations: ci, confidence interval; npv, negative predictive value; ppv, positive predictive value. a specificity indicates the percentage of individuals negative for sporozoite antibody who did not identify any cat risk factors. b sensitivity is the percentage of persons positive for antibody who identified at least cat risk factor. c npv is the percentage of persons not identifying any risk factors who were negative for antibody to the -kda sporozoite protein. d ppv is the percentage of persons identifying a risk factor who were positive for antibody to the sporozoite protein. e significant cat exposure is defined in methods as cleaning a cat’s litter box, gardening, coming into contact with a sandbox, or very close, sustained contact with a kitten. owning an indoor cat fed dry or canned food is considered a risk factor associated with cats, but not necessarily one that has substantial risk for exposure to oocysts for the cat owner. figure . lack of correlation of antibody to sporozoites in maternal serum samples with presence of maternal illness. mothers' symptoms include lymphadenopathy, fever, night sweats, headache, and influenza-like illness. d cid : ( december) d boyer et al at u . of f lorida h ealth s cience c enter l ibrary on d ecem ber , http://cid.oxfordjournals.org/ d ow nloaded from http://cid.oxfordjournals.org/ http://cid.oxfordjournals.org/ d. patel, p. meier, unpublished data). acquisition from oocysts, as shown by presence of antibodies to sporozoites, also has not changed over time. there may be differences in other countries or regional dif- ferences in the united states that are not reflected in our cohort. incidence in different populations may vary significantly, as shown by the national health and nutrition examination survey data showing %– % chronic infection rates among persons in various regions of the united states, with more in- fection in the southern states and an association with poverty. another example is that this assay determined that approxi- mately % of pregnant women with acute acquired toxoplas- mosis during gestation in chile had antibody to sporozoites [ ], in contrast to % of mothers in the present series. another example of differing incidence in different geographic regions or populations in the united states is evident in an amish community in lancaster, pennsylvania. serum sample from an amish child from the ncccts whose mother had antibody to oocysts was tested. in addition, serum samples from the child’s mother and siblings were tested. all but the congenitally infected infant and her -year-old sibling were acutely infected and had antibody to oocysts. because of concern for other pregnant women and their children in the lancaster amish community, pregnant women from this community provided serum samples for t. gondii igg testing. fifty-nine of these women were seropositive ( %), and women ( %) were t. gondii igg figure . lack of correlation of antibody to sporozoites in maternal serum samples and manifestations of infection in the newborn infant. asevere disease is defined in references [ ]. table . correlation of presence of antibody to sporozoites in maternal serum samples and pattern of antibody response to toxoplasma gondii in mother and/or child at birth of child antibody assay overall (n ) positive (n ) negative (n ) p value iga elisa a mother (n ) . ( . ); . . ( . ); . . ( . ); . . child (n ) . ( . ); . . ( . ); . . ( . ); . . igm elisaa mother (n ) . ( . ); . . ( . ); . . ( . ); . . child (n ) . ( . ); . . ( . ); . . ( . ); . . igg dta mother (n ) ( ); ( ); ( ); . child (n ) ( ); ( ); ( ); . igm isaga a child (n ) . ( . ); . ( . ); . ( . ); . aca (n ) ( ); ( ); ( ); . hsa (n ) ( ); ( ); ( ); . abbreviations: ac, results with acetone-fixed antigen; dt, sabin-feldman dye test; elisa, enzyme-linked immnosorbent assay; hs, results with formalin-fixed antigen; iga, immunoglobulin a; igg, immunoglobulin g; igm, immunoglobulin m; isaga, immunoglobulin-m immunosorbent agglutination assay. a numbers are mean (sd); median. unrecognized ingestion toxoplasma gondii oocysts d cid : ( december) d at u . of f lorida h ealth s cience c enter l ibrary on d ecem ber , http://cid.oxfordjournals.org/ d ow nloaded from http://cid.oxfordjournals.org/ http://cid.oxfordjournals.org/ seronegative. one of the igg-seropositive women had igm antibody at low titer. the serum samples from women in the amish community were tested later for antibody to spor- ozoites. the serum samples from those who had no anti- bodies to t. gondii were seronegative controls. all women who were t. gondii igg seropositive and igm seronegative were considered chronically infected persons for development of the assay [ ]. none of these women had antibody to the sporozoite protein, and only had t. gondii–specific igm antibodies, in- dicating that their infection was remote in time [ ]. other seronegative and chronically infected seropositive controls also were described in hill et al [ ]. severe damage may occur in immunocompromised patients or in fetuses infected during early pregnancy, underscoring the key role of host factors in the pathogenicity of t. gondii. how- ever, data suggest that other factors also play a role in the disease. for example, the higher incidence and severity of ocular tox- oplasomsis in tropical areas (south america and africa), com- pared with nontropical areas (europe and north america), may be associated with acquisition of infection from oocysts rather than tissue cysts, exposure to a higher parasite load because of- diet or living conditions, and genetic differences in strain viru- lence or host susceptibility to toxoplasmosis. the new test detecting antibodies to sporozoites contained in oocysts de- scribed here will be important in the field of toxoplasmosis be- cause it offers the opportunity to better know the epidemiology of toxoplasmosis by identifying the source of infection (cysts vs oocysts). it can also pave the way for a better understanding of the role of the life-cycle stage in the pathogenicity of t. gondii. the predominance of maternal acquisition of oocysts and the lack of recognition of oocyst exposure indicates that environ- mental contamination by oocysts contributes substantially to the acquisition of t. gondii and consequent disease in north america. not only are risk factors commonly associated with acquisition of t. gondii often unrecognized by those infected, but our analysis indicates that socioeconomic status, maternal clinical presentations, and ethnicity were not reliable predictors of acquisition of t. gondii oocysts. thus, only systematic screening of pregnant women and/or a vaccine have the po- tential to prevent the fetal disease caused by acquisition of t. gondii during gestation by women in north america. notes acknowledgments. we thank the families who generously worked with us in this study; joseph mccammon for his assistance with this manuscript; j. s. remington and the palo alto research institute and stanford uni- versity in palo alto, california, for performing the serologic testing used to diagnose ncccts patients and amish mothers; michael kirisits and douglas mack for processing samples; frank jagdis, andrew burnett, patrick mcleod, marianna wilson, holmes morton, and donna robinson for patient referral, identification, and evaluation; and c. loss and c. munoz-zanzi for development of the sporozoite assay. financial support. this work was supported by the national institute of allergy and infectious diseases (r ai to r. m.); the research to prevent blindness foundation, the stanley foundation and medical re- search institute ( r- to r. m.), the united states department of agriculture, ars (to d. h.); and gifts from the blackmon, brennan, cornwell, cussen, dougiello, jackson, kapnick, kiewiet, koshland, langel, lipskar, mann, morel, rooney-alden, rosenstein, samuel, and taub families. the funding source and study sponsors had no involvement in the study design; the collection, analysis, and interpretation of data; the writing of the report; or the decision to submit the paper for publication. potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. other members of the toxoplasmosis study group include dianna bardo, delilah burrowes, audrey cameron, ellen holfels, paul latkany, douglas mack, john marcinak, james mcauley, marilyn mets, sanford meyers, william mieler, dushyant patel, jeanne perkins, peter 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pathogenesis, diagnosis and treatment. ama arch ophthalmol ; : – . . wilcoxon f. individual comparisons by ranking methods. biometrics ; : – . . statacorp. stata statistical software: release . college station, tx: statacorp lp, . . levy jk, crawford pc. humane strategies for controlling feral cat populations. j am vet med assoc ; : – . . hill de, chirukandoth s, dubey jp. biology and epidemiology of toxoplasma gondii in man and animals. anim health res rev ; : – . unrecognized ingestion toxoplasma gondii oocysts d cid : ( december) d at u . of f lorida h ealth s cience c enter l ibrary on d ecem ber , http://cid.oxfordjournals.org/ d ow nloaded from http://cid.oxfordjournals.org/ http://cid.oxfordjournals.org/ wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . 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murders and the cultural fetishization of “amish forgiveness” darcy metcalfe department of religious studies, university of iowa, iowa city, la , usa; darcy-metcalfe@uiowa.edu received: july ; accepted: september ; published: september ���������� ������� abstract: in the days and weeks following the west nickel mines amish school murders, hegemonic u.s. cultural discourse largely fetishized the amish response of forgiveness in revealing ways. within this discourse, the terrorist attacks of september were referenced in articles and commentaries which sought to weigh the moral value of forgiveness in response to extreme violence. in this way, understandings of amish forgiveness were largely “strip-mined” from the nickel mines community and “transported wholesale” to other counter-cultural settings. in dominant u.s. capitalistic and consumeristic culture, amish forgiveness quickly became a fluctuating material commodity that was fetishized in ways which revealed the destabilized moral consciousness of a nation. dominant cultural discourse exposed this destabilization while it also worked to interrogate it. i conclude that the fetishization of forgiveness following the amish school murders reflected collective concerns that reached far beyond the immediate context of the nickel mines amish community. the u.s. cultural fetishization of forgiveness revealed, instead, a cultural consciousness that desperately sought relief from the chaos and confusion of what it means to be a citizen of nation that exists in and by the normativity of extreme violence. keywords: amish; forgiveness; revenge; fetishism; violence; september ; religious ethics . introduction my primary argument is that hegemonic u.s. cultural discourse fetishized the amish response of forgiveness in the days and weeks following the west nickel mines amish school murders. the fetishization of forgiveness was a phenomenon that was produced, in part, from vital ethical indeterminacies of the word “forgiveness” as it moved across cultural boundaries of contrary determinations: amish culture and dominant u.s. culture. this fetishization revealed a cultural consciousness that desperately sought relief from the chaos and confusion of what it means to be a citizen of nation that exists in and by the normativity of extreme violence. what u.s. cultural discourse underestimated or ignored in the weeks following the amish school murders also reveals the potency of the normativity of violence within dominant culture. in order to clearly articulate these contentions, it is helpful first to begin with a brief review of what happened on the day of the amish school murders, followed by notable responses in the days immediately following. . the west nickel mines amish school murders on october , charles carl roberts iv executed a calculated attack on young girls in a rural amish community. he strategically overtook a one-room schoolhouse near nickel mines, pennsylvania. prior to the attacks that morning, he walked his children to the school bus stop, left suicide notes for each family member at his home, and then filled a borrowed truck with supplies he had stored for the murders. religions , , ; doi: . /rel www.mdpi.com/journal/religions http://www.mdpi.com/journal/religions http://www.mdpi.com http://www.mdpi.com/ - / / / ?type=check_update&version= http://dx.doi.org/ . /rel http://www.mdpi.com/journal/religions religions , , of roberts bought supplies throughout the previous week and kept them in a shed near his home. that morning he stopped at an amish-owned valley hardware store to purchase plastic zip-ties to add to his supply stash. in the borrowed truck, he packed a -mm handgun, a -gauge shotgun, a - rifle, a stun gun, six-hundred rounds of ammunition, a tube of lubricating jelly, a hammer, nails, wrenches, binoculars, earplugs, batteries, a flashlight, a candle, tape, two-by-four and two-by six wood planks, and an extra set of clothing. around : , roberts backed his truck up to the front entrance of the schoolhouse, entered with a semi-automatic handgun, and ordered everyone to the floor. the teacher managed to escape with her mother, who was visiting for the day, and ran to a nearby farm for help. roberts then bound ten girls with zip ties, ages six to thirteen, and bound them to one another. he ordered every boy and every adult to leave. he pulled the blinds and nailed the doors shut. roberts then called his wife. he confessed to her that when he was twelve-years-old he had molested two female relatives between the ages of three and five. he told his wife that he had recently began fantasizing about molesting young girls again (spicher kasdorf , p. ). however, police arrived at the school before roberts had opportunity to carry out his plans of sexual assault. at : , gunfire rang out from inside the schoolhouse and police officers rushed the building. before roberts turned his handgun on himself, he shot all the bound girls at close range. five of the girls died. the other five girls, although critically injured, survived. the immediate response of this amish community to these murderous acts was forgiveness as it is commonly practiced in old order amish faith and history (kueny and cardenas ). in this article, i will explore the u.s. cultural fetishization of forgiveness in the days and weeks following the amish school murders. i explore why and how “forgiveness” became a fetishized cultural commodity of this event and how the destabilized moral consciousness of a nation uniquely shaped the cultural discourse surrounding these murders. . “amish forgiveness” forgiveness is a virtue that is defined differently by various scholars and distinct fields of study. aristotle, in his discussion of virtues and vices relative to anger, asserts that a virtuous person “is not revengeful, but rather tends to forgive” (nicomachean ethics, aristotle and brown , iv, a ). an aristotelian ideal perceives forgiveness to be a virtue as it is a mean of anger when anger is governed by reason. this idea of forgiveness as virtuous persists in many contemporary understandings of forgiveness. psychologist everett l. worthington, jr. studies forgiveness and notes that forgoing revenge is often a primary component of forgiveness. worthington gives specific attention to practices of forgiveness within religious traditions. he suggests that a person who values religion tends to perceive the world through religious values (worthington ). for the amish, forgiveness is a highly valued and foundational religious virtue that is vital to how the amish perceive the world. worthington uses the terms “decisional” and “emotional” forgiveness to describe the different ways forgiveness is often extended. he writes, “decisional forgiveness promises not to act in revenge or avoidance, but it doesn’t necessarily make a person feel less forgiving” (worthington , p. ). working in tandem, “emotional forgiveness” involves a long-term process that includes replacing harmful and detrimental emotions connected to the offender with positive emotions. for the nickel mines amish, one may contend that decisional forgiveness included the religious community’s initial commitment to the virtue of forgiveness, while emotional forgiveness includes an ongoing work of forgiveness which addresses the emotions that remain. worthington maintains that with “decisional forgiveness” details of these events are primarily taken from amish grace: how forgiveness transcended tragedy by donald b. kraybill, steven m. nolt, and david l. weaver-zercher, and from “‘amish forgiveness,’ silence, and the west nickel mines school shooting,” by julia spicher kasdorf. religions , , of a person initially decides to forgo any claim to revenge and chooses, instead, to treat the offender in a way that values that person’s humanity (worthington , p. ). forgiveness is commonly defined as what takes place when a person who has been wronged or injured chooses to forgo payback or any claim to revenge upon the offender (kraybill donald b. and weaver-zercher , p. ). in the case of the west nickel mines amish school murders, the amish community immediately chose to forgo any form of payback. instead, “gracious words came first, quickly followed by gracious acts–words and acts offered in good faith that kind feelings would eventually replace bitter ones” (kraybill donald b. and weaver-zercher , p. ). within hours of the attack, members of the amish community visited members of roberts’ family, verbally stating their forgiveness of roberts with an assurance of the amish community’s commitment to accompany the family in this time of immense grief. when roberts’ family gathered that saturday for his burial at the cemetery of georgetown united methodist church, more than half of the seventy-five mourners were amish (kraybill donald b. and weaver-zercher , p. ). a relationship between the roberts family and this amish community existed long before these horrific events, and this relationship continued to grow and extend far beyond the events of october. . a nation’s response to “amish forgiveness” within the first week of the west nickel mines amish school attack, over two-thousand global news stories appeared, many which focused upon the forgiveness extended by this amish community (doblmeier martin ). religious leaders and writers in the united states quickly offered commentaries that both scrutinized and praised the amish community’s response. the overall spirit of the commentaries was one of praise, but there were also many strong critiques of the moral nature of such a seemingly instantaneous practice of forgiveness. many of the articles that appeared in the weeks that followed the murders grappled with various religious and moral concerns and questions. does the amish christian demand to forgive conflict with moral imperatives that secure a thriving and safe society? is forgoing the right to revenge such a violent and egregious offense morally suspect? can such practices of forgiveness be genuine in nature? within these articles, diverse perspectives on forgiveness were offered based on differing ethical persuasions. religion was often evoked in support of various ethical stances. diana butler bass wrote a brief article ten days after the tragedy entitled, “what if the amish were in charge of the war on terror?” in which she critiques the morality of the u.s. war response to the september attacks. she writes: what if the amish were in charge of the war on terror? what if, on the evening of sept. , , we had gone to osama bin laden’s house (metaphorically, of course, since we didn’t know where he lived!) and offered him forgiveness? what if we had invited the families of the hijackers to the funerals of the victims of / ? what if a portion of the september th fund had been dedicated to relieving poverty in a muslim country? what if we dignified the burial of their dead by our respectful grief? she continues: so, here’s my modest proposal. we’re five years too late for an amish response to / . but maybe we should ask them to take over the department of homeland security. after all, actively practicing forgiveness and making peace are the only real alternatives to perpetual fear and a multi-generational global religious war. (butler bass ) butler bass goes on to praise the amish community’s response of forgiveness as an example of the “christian practice of forgiveness,” as she encourages the u.s. government to follow these christian practices with direct implementation in foreign policy. seven days after the murders, catholic writer and nun joan chittister wrote “what kind of people are these?” praising the amish response as “the christianity we all profess” but the amish religions , , of practice (chittister ). like butler bass, chittister compares the amish response to the amish school murders to ways the united states chose to respond after / , although she stops short of imploring the u.s. government to follow suite. she writes: the real problem with the whole situation is that down deep we know that we had the chance to do the same. after the fall of the twin towers we had the sympathy, the concern, the support of the entire world. you can’t help but wonder, when you see something like this, what the world would be like today if, instead of using the fall of the twin towers as an excuse to invade a nation, we had simply gone to every muslim country on earth and said, “don’t be afraid. we won’t hurt you. we know that this is coming from only a fringe of society, and we ask your help in saving others from this same kind of violence.” “too idealistic,” you say. maybe. but since we didn’t try, we’ll never know, will we? instead, we have sparked fear of violence in the rest of the world ourselves. so much so, that they are now making nuclear bombs to save themselves. from whom? from us, of course. (chittister ) the common reference to / during the aftermath of the west nickel mines amish school murders is vital to note. jonathan kooker details how september was frequently mentioned in news stories and publications that focused specifically on understandings of “amish forgiveness” (kooker ). the / reference is especially intriguing when considering the prevalence of school shootings within u.s. culture. in the decade before the amish school murders, there were many school shootings reported in mainstream media, such as the columbine high school massacre ( ), the thurston high school shooting ( ), the westside middle school shooting ( ), the frontier middle school shooting ( ), the sand diego state university shooting ( ), and the santana high school shooting ( ). twenty days before the amish school shooting, there was a school shooting at duquesne university that injured five people. ten days before there was a school shooting at platte canyon high school in bailey, colorado in which the perpetrator took six girls hostage and sexually assaulted them before killing one of the teenage girls and himself. six days before, there was another school shooting at weston high school in cazenovia, wisconsin in which the principal was killed. however, these events were not often mentioned in the immediate cultural commentary that sought to connect to the tragedy of the amish school murders. what was mentioned in cultural discourse more often was / . why? given the context of the amish school murders, at the surface it would seem more plausible for cultural discourse to evoke other similar events of extreme violence in u.s. schools, but this was not the case. instead, this reference to / by dominant cultural discourse was consistently evoked primarily to analyze or question the moral merit of forgiveness in extreme circumstances. the amish community also referred to / in the aftermath of the shootings, although the usage was distinct and unlike usage in u.s. cultural discourse. “amish searching for healing, forgiveness after ‘the amish / ′” is an article that was published three days after the school murders (burke ). this article highlights the specific reference of the nickel mines amish community to the school murders as the “amish / .” the amish community did not reference this event as “the amish columbine” or “the amish platte canyon.” instead, the connection was made to / . this distinct reference within the amish community underscores the magnitude of how the community viewed its own violent violation and devastation. it highlights the cataclysmic nature of how these events were interpreted by the amish community. although u.s. cultural discourse and the amish community referenced / in different ways, i contend this shard reference is important to note. the shared reference to / between two vastly different cultures served as a vital point of connection at which human pain seems to have been translated and its meaning shaped and communicated between amish and u.s. cultures. the amish / reference also reveals the purposely segregated nature of this community within u.s. borders. religions , , of while the amish are u.s. citizens, there is also a degree of “set-apartness” that is important to the community’s way of life. this “set-apartness” creates the space for the community to abide by moral and religious ideals that predominately counter dominant u.s. cultural ideals. in u.s. cultural discourse, “ / ” was used in the days following the amish school murders to convey a sense of devastation and loss that seemed inarticulable in any other way. i contend that in this manner, “ / ” was shaped into a unique cultural point of connection across amish and u.s. cultures. its usage reflected a shared and palpable sense of violent disorientation and panicked chaos that reached across the boundaries of communities that were largely countercultural. what “ / ” conveyed in this usage was, in part, a shared (albeit very different) experience of seeking meaning and stability after life-altering acts of senseless and extreme violence. the shared experience of disorientation and despair reconfigured the expansive boundaries of these countercultural worlds in a way that viscerally connected people and the materialities of human pain. there was also a shared realization of the need for a change in foundational understandings of what it means “to be” in the world. it was impossible to return to what once was. one amish fire official who responded to the emergency call on the day of the school murders acknowledged that life could never resume as it once had been. the community had to find “a new normal” in order to continue to live in the world in a way that had meaning (kraybill donald b. and weaver-zercher , p. ). in multiple articles and commentaries in days immediately following the amish school shootings, “ / ” was configured into a primary point of connection and communication between drastically different cultures. however, the reference that, by far, garnered the most attention in the articles and commentary that followed the murders was “forgiveness.” when used by dominant us. cultural discourse, the reference to / was primarily used to question the morality of forgiveness in the most violent and horrific circumstances. the word “forgiveness” became the primary material marker of the west nickel mines amish school murders. one of my primary questions in this article is, “why?” after all, this is not an unfamiliar response from the amish community. amish history is replete with stories of the extreme practice of forgiveness compared to commonly held social standards of practice. in this case, what caused the collective consciousness of a nation to be so captivated, obsessed even, with the practice of forgiveness? what dominant cultural values and moral norms were destabilized and interrogated by the amish response of forgiveness? there were so many pivotal points of fact that were largely overlooked or underemphasized by media and social commentary in the days and weeks that immediately followed the amish school murders. there were numerous significant factors that convened in that tragic day’s events which expose primary contributing components that can converge to create a culture in which such horrors occur. as julia spicher kasdorf observed, this event was a violent crime “against girls who were separated from adults and boys for sexual assault and death” (spicher kasdorf , p. ). however, the prevalence of violence against girls and women, particularly at the hands of white men, was not a topic that garnered much attention. the ease with which roberts acquired firearms and ammunition was not a topic of concern. the normativity of sexual/physical violence and the exploitation of young girls was mostly a moot point. rather, the concern of a nation became the practice of “amish forgiveness.” why? . anabaptist history and practices: what was underestimated in national discourse although the nickel mines amish community’s response of forgiveness is a practice drastically removed from dominant u.s. socio-cultural practices, forgiveness is foundational to what it means to be amish. the amish tradition was birthed as part of the anabaptist tradition during the reformation in europe. the reformation was a time of religious-socio-political-cultural upheaval in which figures such as martin luther and john calvin sought to drastically change what they perceived as the powerful and corrupt catholic church—the church from which they would eventually separate. from religions , , of these reformers arose another group which sought to move the work of reformation beyond the church institution and more concretely into daily life and community. this new group believed that only baptism by consent was valid. child and infant baptism, as practiced in the catholic and reformed church, was declared invalid due to the absence of the individual’s capability to consent. these new reformists received the disparaging moniker anabaptists, which means “rebaptizers,” and they quickly found themselves condemned as heretics by both protestant and catholic church leaders. although the anabaptist movement was never large, it accounted for forty to fifty percent of all western european christians who were martyred for religious practice during the sixteenth century (kraybill donald b. and weaver-zercher , p. ). the anabaptists eventually sought refuge from persecution in the colonies of the new world. the ethos of amish life is captured well in the german word gelassenheit. roughly translated, gelassenheit means yielding oneself to a higher authority (amish studies: the young center n.d.). habits and practices of this religious life and culture include commitment to community rather than individualism; submission and obedience to christ’s commands and church leaders; humility; separation rather than integration into modern society; and non-resistance and peaceful resolution of conflict, which includes the practice of forgiveness. forgiveness is much more than a decision, emotion, or process in amish life and history. forgiveness is a way of being in the world, which, from the amish view, is demanded by the christian faith. this practice of forgiveness is rooted in the belief that in order to be forgiven by god, one must forgive others, no matter how egregious the offense. old order amish communities, such as the nickel mines amish, intentionally instill forgiveness in their children through “multiple networks and strategies” (kueny and cardenas , p. ). forgiveness is part and parcel of the spirit of peace and love in which the amish choose to exist and operate in the world. this history and these specific cultural/moral/religious habits and practices are paramount to understanding the nickel mines amish community’s response to the attack and murder of their children. i contend that, in most cases, in the weeks following the murders, the national discourse largely underestimated the countercultural value system from which forgiveness had emerged. the amish response at nickel mines was “not so much the ‘best of america’ as it was an expression of love by a people who every day challenge many of the values the rest of us hold dear” (kraybill donald b. and weaver-zercher , p. ). as kasdorf so aptly explains, the responses that come freely and immediately from this amish community are entirely consistent with deeply ingrained cultural patterns and theological convictions (spicher kasdorf , p. ). . conclusions: the cultural fetishization of amish forgiveness hegemonic u.s. cultural discourse fetishized the amish response of forgiveness in the days and weeks following the west nickel mines amish school murders. the fetishization of forgiveness was a phenomenon that was produced, in part, from ethical indeterminacies of the word “forgiveness” as it moved across cultural boundaries of contrary determinations. in order to develop this argument more fully, it is helpful to give a working definition of “fetish” in this context. although lengthy, the following definition from william pietz is particularly beneficial. he writes: the fetish is always a meaningful fixation on a singular event; it is above all a “historical” object, the enduring material form and force of an unrepeatable event. this object is “territorialized” in material space (an earthly matrix), whether in the form of a geographical locality, a marked site on the surface of the human body, or a medium of inscription or configuration defined by some portable or wearable thing . . . this reified, territorialized historical object is also “personalized” in the sense that beyond its status as a collective social object it evokes an intensely personal response from individuals. this intense relation to the individual’s experience of his or her own living self through an impassioned response to the fetish object is always incommensurable with (whether in a way that reinforces or undercuts) religions , , of the social value codes within which the fetish holds the status of material signifier. it is in these “disavowals” and “perspectives of flight” whose possibility is opened in the clash of this incommensurable difference that the fetish might be identified as the site of both the formation and the revelation of ideology and value-consciousness. (pietz , pp. – ) in the days and weeks following the amish school murders, “forgiveness” became the fixated point of u.s. cultural discourse and the medium of inscription that materially marked this horrific event. in u.s. cultural discourse, “forgiveness” also evoked an intensely “personal” response which highlighted a critical incommensurability of foundational moral codes as they drastically differed between cultures. “forgiveness” was ultimately a material signifier bound to contraries, and this signifier became the discursive point at which a destabilized nation began to question and interrogate its own values, alongside its own violent nature and aggression in the world. “forgiveness” in the written word—in the thousands of news articles focused on amish forgiveness—became a term of derridean différance that revealed and interrogated a disrupted cultural consciousness. the fetishization of forgiveness marked the space where codes and their logics broke down across cultural boundaries. in the aftermath of / , the u.s. war machine promptly pursued a new war on terror in the name of the nation’s historically claimed values of “freedom,” “liberty,” and “justice.” the exposed fragility of these values awakened a collective cultural consciousness to the reality that even those who wield the greatest degree of militaristic and economic power globally are not immune to the most extreme and devasting acts of violence. juxtaposed to this u.s. response, the nickel mines amish were a peaceful, and mostly segregated people who responded to extreme violence and cruelty in a manner that was completely counter-cultural to the dominant social values and cultural norms of the u.s. in the aftermath of the amish school murders, the nation witnessed that even the most peaceful people are not immune to the most extreme and devasting acts of senseless violence. both the peaceful and the powerful are not immune. within this space, the u.s. cultural fetishization of forgiveness worked to bring order to the emotional/spiritual/psychological upheaval of what it means “to exist” and live in a world in which extreme and senseless violence is entirely normative. what u.s. cultural discourse underestimated or ignored in the weeks following the amish school murders reveals the potency of the normativity of violence within dominant culture. as mentioned in the first section of this article, kasdorf reminds us that this event was a violent crime “against girls who were separated from adults and boys for sexual assault and death” (spicher kasdorf , p. ). however, what was not widely present in u.s. cultural discourse in the days and weeks following the murders were other concerns, such as the prevalence of white male perpetrators in the u.s.; the deficiencies of federal hate crime legislation to protect women and girls; the normativity of violence and sexual exploitation of girls and women; or the ease with which citizens can purchase and stockpile weapons and ammunition. none of these horrific realities became a primary point of concern in national discourse. ultimately, it was not the violence itself that was most noted. what was most noted in dominant u.s. cultural discourse, what was most foreign to a nation’s way of life and moral values, was “forgiveness.” in this way, it seems that the fetishization of forgiveness in cultural discourse was not about the amish at all. the fetishization revealed and interrogated a national consciousness that was violently awakened to its own mortality on the morning of september . funding: this research received no external funding. conflicts of interest: the author declares no conflict of interest. references amish studies: the young center. n.d. communal values. available online: https://groups.etown.edu/ amishstudies/social-organization/communal-values/ (accessed on july ). aristotle, w. d. ross, and lesley brown. . the nicomachean ethics. oxford: oxford university press. https://groups.etown.edu/amishstudies/social-organization/communal-values/ https://groups.etown.edu/amishstudies/social-organization/communal-values/ religions , , of burke, daniel. . amish search for healing, forgiveness after ‘the amish / ’. religious news service. available online: https://web.archive.org/web/ /http://www.religionnews.com/articleofweek . html (accessed on july ). butler bass, diana. . what if the amish were in charge of the war on terror? sojourners. available online: https://sojo.net/articles/what-if-amish-were-charge-war-terror (accessed on july ). chittister, joan. . what kind of people are these? the national catholic reporter . available online: http://www.nationalcatholicreporter.org/fwis/pc .htm (accessed on july ). kooker, jonathan. . amish forgiveness: an amish lesson for the rest of us? the friends journal : – . kraybill donald b., steven m. nolt, and david l. weaver-zercher. . amish grace: how forgiveness transcended tragedy. san francisco: john wiley & sons. kueny, angela, and sandra cardenas. . old order amish teaching children forgiveness. in perspectives on forgiveness: contrasting approaches to concepts of forgiveness and revenge. edited by susan divietro and jordan kiper. leiden: koninklijke brill, pp. – . doblmeier martin. . the power of forgiveness. alexandria: journey films, inc. pietz, william. . the problem of the fetish, i. res: anthropology and aesthetics : – . [crossref] spicher kasdorf, julia. . ‘amish forgiveness,’ silence, and the west nickel mines school shooting. crosscurrents : – . worthington, everett l., jr. . understanding the values of religious clients: a model and its application to counseling. journal of counseling psychology : – . [crossref] worthington, everett l., jr. . forgiving and reconciling: bridges to wholeness and hope, rev. ed. downers grove: intervarsity press. © by the author. licensee mdpi, basel, switzerland. this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license (http://creativecommons.org/licenses/by/ . /). https://web.archive.org/web/ /http://www.religionnews.com/articleofweek .html https://web.archive.org/web/ /http://www.religionnews.com/articleofweek .html https://sojo.net/articles/what-if-amish-were-charge-war-terror http://www.nationalcatholicreporter.org/fwis/pc .htm http://dx.doi.org/ . /resv n ms http://dx.doi.org/ . / - . . . http://creativecommons.org/ http://creativecommons.org/licenses/by/ . /. 	introduction 	the west nickel mines amish school murders 	“amish forgiveness” 	a nation’s response to “amish forgiveness” 	anabaptist history and practices: what was underestimated in national discourse 	conclusions: the cultural fetishization of amish forgiveness 	references wp-p m- .ebi.ac.uk params is empty sys_ exception wp-p m- .ebi.ac.uk no params is empty exception params is empty / / - : : if (typeof jquery === "undefined") document.write('[script type="text/javascript" src="/corehtml/pmc/jig/ . . /js/jig.min.js"][/script]'.replace(/\[/g,string.fromcharcode( )).replace(/\]/g,string.fromcharcode( ))); // // // window.name="mainwindow"; .pmc-wm {background:transparent repeat-y top left;background-image:url(/corehtml/pmc/pmcgifs/wm-nobrand.png);background-size: auto, contain} .print-view{display:block} page not available reason: the web page address (url) that you used may be incorrect. message id: (wp-p m- .ebi.ac.uk) time: / / : : if you need further help, please send an email to pmc. include the information from the box above in your message. otherwise, click on one of the following links to continue using pmc: search the complete pmc archive. browse the contents of a specific journal in pmc. find a specific article by its citation (journal, date, volume, first page, author or article title). http://europepmc.org/abstract/med/ doi: . / am. j. hum. genet. : – , autosomal dominant postaxial polydactyly, nail dystrophy, and dental abnormalities map to chromosome p , in the region containing the ellis–van creveld syndrome locus timothy d. howard, alan e. guttmacher, , wendy mckinnon, mridula sharma, victor a. mckusick, and ethylin wang jabs , departments of pediatrics and surgery and department of medicine, center for medical genetics, johns hopkins school of medicine, baltimore; and departments of pediatrics and medicine, vermont regional genetics center, university of vermont college of medicine, burlington summary we have studied a four-generation family with features of weyers acrofacial dysostosis, in which the proband has a more severe phenotype, resembling ellis–van crev- eld syndrome. weyers acrofacial dysostosis is an auto- somal dominant condition with dental anomalies, nail dystrophy, postaxial polydactyly, and mild short stature. ellis–van creveld syndrome is a similar condition, with autosomal recessive inheritance and the additional fea- tures of disproportionate dwarfism, thoracic dysplasia, and congenital heart disease. linkage and haplotype analysis determined that the disease locus in this pedigree resides on chromosome p , distal to the genetic marker d s and within a -cm region flanking the genetic locus d s . this region includes the el- lis–van creveld syndrome locus, which previously was reported to map within a -cm region between genetic markers d s and d s . either the genes for the condition in our family and for ellis–van creveld syn- drome are near one another or these two conditions are allelic with mutations in the same gene. these data also raise the possibility that weyers acrofacial dysostosis is the heterozygous expression of a mutation that, in ho- mozygous form, causes the autosomal recessive disorder ellis–van creveld syndrome. received june , ; accepted for publication october , ; electronically published november , . address for correspondence and reprints: dr. ethylin wang jabs, center for medical genetics, johns hopkins school of medicine, north wolfe street, baltimore, md - . e-mail: ewjabs@welchlink.welch.jhu.edu � by the american society of human genetics. all rights reserved. - / / - $ . introduction weyers acrofacial dysostosis (curry-hall syndrome; omim [http://www.ncbi.nlm.nih.gov/omim/]) is an autosomal dominant condition characterized by hypotelorism, an abnormal mandible, incisors abnormal in shape and number, a single central incisor, conical teeth, postaxial polydactyly type a or type b, hypo- plastic or dysplastic nails, and short stature. it was first reported in three unrelated cases (weyers ), and three additional families have since been described (curry and hall ; roubicek and spranger ; shapiro et al. ). ellis–van creveld syndrome (omim [http:// www.ncbi.nlm.nih.gov/omim/]) (ellis and van creveld ) is a rare autosomal recessive disproportionate dwarfism that is most prevalent in the amish population (mckusick et al. ). it is characterized by lip defects (oral frenula), dental abnormalities (neonatal teeth, hy- podontia, and premature tooth loss), cardiac malfor- mations (atrial septal defect and single atrium), geni- tourinary anomalies (epispadias and hypospadias), skeletal abnormalities (postaxial polydactyly type a, brachydactyly, fusion of the capitate and hamate, genu valgum, and distal limb shortening), and nail dysplasia (mckusick et al. ; biggerstaff and mazaheri ; taylor et al. ). weyers acrofacial dysostosis has some features in common with, but is distinct from, ellis–van creveld syndrome (table ) (gorlin et al. , pp. – ). the two syndromes are dissimilar in mode of inheritance and phenotypic severity. linkage analysis of families with ellis–van creveld syndrome previously mapped the locus to a -cm region between the genetic loci d s and d s on chro- mosome p . (polymeropoulos et al. ). the max- imum two-point lod score (zmax) observed was . (recombination fraction [v] of . ) with a genetic marker for the msx homeobox gene. subsequent sequencing of both msx exons in two patients and one obligate carrier of ellis–van creveld syndrome ruled out the pos- am. j. hum. genet. : – , table phenotype of the family and related syndromes proband (iv: ) other affected family members ellis–van creveld syndrome jeune asphyxiating thoracic dysplasia weyers acrofa- cial dysostosis short stature � mild � mild mild abnormal frenula � � � � � natal teeth � � � � � hypodontia � � � � � conical teeth � � � ? � mandibular anomalies � � � � �/� retinal degeneration � � � � � postaxial polydactyly hands and feet hands and feet hands; sometimes feet �/�; if present, usu- ally hands and feet hands and/or feet onychodystrophy � � � � � thoracic dysplasia � � � � � cardiac findings patent ductus arter- iosus, ventricular septal defect � atrial septal defect, atrioventricular sep- tal anomaly � � hepatic/pancreatic abnormalities � � � � � renal abnormality � � � � � radiological findings flat acetabular roof with pointed pel- vic prominence � fifth carpal in distal row of wrist, multi- ple ossification cen- ters in hamate, flat acetabular roof with pointed pelvic prominence flat acetabular roof with pointed pelvic prominence � lethal in newborns � � �/� often � inheritance ? autosomal dominant autosomal recessive autosomal recessive autosomal dominant chromosomal linkage p p p ? p ? note.—a plus sign (�) indicates present; a minus sign (�) indicates absent; a plus/minus sign (�/�) indicates sometimes present; and a question mark (?) indicates unknown. sibility that mutations in the coding region are causative of this condition (ide et al. ). it has been suggested that isolated postaxial polydac- tyly, which occurs in these two conditions, may be a heterozygous manifestation of ellis–van creveld syn- drome (fryns ; goldblatt et al. ). another sim- ilar condition with postaxial polydactyly as a clinical feature is jeune syndrome (omim [http:// www.ncbi.nlm.nih.gov/omim/]) (jeune et al. ; pir- nar and neuhauser ; langer ). the pelvis and limbs are similar in ellis–van creveld and jeune syn- dromes, but nail dystrophy, abnormal frenula, and car- diac abnormalities are not found in the latter condition. in addition, renal involvement is a frequent feature of jeune syndrome and is not found in ellis–van creveld syndrome. postaxial polydactyly also has been observed to segregate as a single trait. linkage of an autosomal dominant form of postaxial polydactyly type a to chro- mosome p -q . recently was reported in a five- generation indian family with no other clinical findings (radhakrishna et al. ). we have studied a family in which one individual (the proband) had findings that are most typical of ellis–van creveld syndrome. the paternal relatives, however, pre- sented with postaxial polydactyly, short stature relative to unaffected sibs, and tooth abnormalities segregating in an autosomal dominant manner. these findings are similar to those reported for weyers acrofacial dysostosis (curry and hall ; roubicek and spranger ; shapiro et al. ). we performed linkage analysis of this condition and mapped it to chromosome p in a region that encompasses the locus for ellis–van creveld syndrome. patients and methods patient population genomic dna was isolated from blood samples or lymphoblast cultures from available family members, by use of the blood and cell culture dna kit (qiagen). all these family members were examined clinically by one of the authors (a.e.g.). howard et al.: polydactyly maps to chromosome p case presentation the proband (fig. ; individual iv: in fig. ) was the -lb -oz male product of an uncomplicated term preg- nancy of a -year-old mother and her nonconsanguin- eous -year-old husband. the initial physical exami- nation of the newborn was significant for a narrow chest, four-extremity postaxial polydactyly type a, short limbs, and swelling secondary to forceps delivery because of face presentation. as this swelling receded, examination of the upper lip revealed multiple frenula with shallow sulcus. neonatal cardiology evaluation demonstrated a large patent ductus arteriosus that closed spontaneously at age d and a small muscular ventricular septal defect. radiographs were remarkable for short ribs and a flat acetabular roof with pointed pelvic prominence. the proband was ventilator dependent for the first month of life but otherwise had an uncomplicated new- born course. he remained on oxygen supplementation by nasal cannula until mo of age. at ∼ mo of age, the proband developed a hemangioma over the left chest that began to involute after his first birthday. by ∼ years of age, his fingernails and toenails had become dys- trophic. he has decreased range of motion of the distal interphalangeal joints of his hands, and his thumbs ap- pear to be mildly digitalized. dental evaluation has re- vealed a number of symmetrically absent primary teeth and dental pitting. renal evaluation demonstrated no structural or functional abnormalities. he generally has enjoyed excellent health and normal development. height has been at slightly below but paralleling the th percentile, weight has been at approximately the th percentile, and head circumference has been at the th– th percentile. the proband’s father has a history of postaxial poly- dactyly type a, radial fifth-digit clinodactyly, and ony- chodystrophy, as do at least seven other of the father’s relatives (see fig. ). chromosome analysis was per- formed on the affected father (iii: in fig. ) and revealed a normal ,xy karyotype. in the affected individuals, the polydactyly involves all four extremities, with the extra digits on the feet usually more fully formed than those on the hands. the individuals also may be slightly shorter in stature than their sibs. for instance, the pro- band’s father (iii: ) is feet inches tall, and his affected brothers are feet inches (iii: ) and feet inches (iii: ), whereas their unaffected brothers are feet inches (iii: ) and feet inches (iii: ) and their unaf- fected sister (iii: ) is feet inches. some of these affected individuals have unusual labial frenula or shal- low labial sulci. several, but not all, of the affected family members have had dental abnormalities: the paternal great-aunt (ii: ) is reported to have conical-shaped teeth; the proband’s father (iii: ) has tooth agenesis; a paternal uncle (iii: ) had a pear-shaped tooth that was removed; and a first cousin (iv: ) has bilateral fusion of primary mandibular canine and lateral incisors and several con- ical-shaped primary teeth. the same cousin also has a prominent raphe extending over the middle of her phil- trum, from vermilion border to nasal root. the family history includes the proband’s mother (iii: ) being feet inches tall and having a history of urinary reflux. her family’s history is negative for short stature, polydactyly, onychodystrophy, congenital heart disease, or dental abnormalities. pcr amplification and sequence analysis for the nine genetic markers listed in table , pcr was performed with dna from each family member, in -ml reactions consisting of ng genomic dna, mm kcl, mm tris-hcl, mm each dntp, . mm mgcl , . mg bsa/ml, . mmol each primer, – ng one g[ p]-datp end-labeled primer, and . u taq dna polymerase (boehringer mannheim). amplifica- tion parameters were as follows: min at �c; cycles of s at �c, s at the annealing temperature, and s at �c; followed by cycles of s at �c, s at the annealing temperature, and s at �c; and a final extension of min at �c. the annealing tem- perature for each genetic marker was as described in the genome database (http://www.gdb.org/), with the ex- ceptions that the temperature for d s was increased from �c to �c and that for d s was decreased from �c to �c. the pcr products were separated on a . % polyacrylamide sequencing gel and were de- tected by autoradiography. the msx gene was sequenced by, first, amplification of both exons with two sets of pcr primers derived from the published sequence (hewitt et al. ). to amplify exon , the forward primer ′-ctgctgaca- tgacttctttgc- ′ and the reverse primer ′-tgg- gttctggctactacctg- ′, which amplify a -bp fragment, were used. pcr reactions for exon were performed in -ml volumes consisting of – ng genomic dna, mm tris-hcl (ph . ), mm kcl, . mm mgcl , mm each dntp, . mm each pri- mer, % dimethyl sulfoxide, and . u taq dna poly- merase (boehringer mannheim). pcr parameters were cycles of min at �c, min at �c, and min at �c. exon was amplified with the forward primer ′-ggctgatcatgctccaatgctt- ′ and the re- verse primer ′-tacagcaccagggctggagg- ′, yielding a -bp fragment. pcr reactions were per- formed as described for exon , with cycling parameters of min at �c, min at �c, and min at �c. pcr products were run on % nusieve gels (fmc bioproducts) and were extracted. the dna, isolated with the gel extraction kit (qiagen), was directly se- figure clinical photographs of affected family members. a, proband (iv: in fig. ), front and lateral view. note the short stature, abnormal configuration of the chest, hands with nail dysplasia of the thumbs (bottom right), and postaxial polydactyly in infancy (bottom left). b, father of the proband (iii: in fig. ), front and lateral view. note the proportional stature, normal chest, and onychodystrophy. his postaxial polydactyly had been surgically removed. howard et al.: polydactyly maps to chromosome p figure linkage- and haplotype-analysis data for the studied family with features of weyers acrofacial dysostosis and ellis–van creveld syndrome. for each individual evaluated, the alleles for the genetic markers are given below the symbol; individuals without as- signed alleles were not available for analysis. the proband is indicated with an arrow. the boxed areas include the alleles that are either certainly or possibly inherited from an affected parent and show the largest possible region in which the disease locus may lie. table linkage analysis of postaxial polydactyly, nail dystrophy, and dental abnormalities to chromosome p marker lod score at v � zmax vmax. . . . . . . d s . . . . . . . . . d s �. �. �. �. �. �. . . . d s . . . . . . . . . msx . . . . . . . . . d s . . . . . . . . . d s . . . . . . . . . d s . . . . . . . . . d s �� . . . . . . . . d s �� . . . . . . . . quenced by the johns hopkins genetic resources core facility, by use of the specific pcr primers. linkage and haplotype analysis nine genetic markers, spanning cm (polymero- poulos et al. ), were used for linkage analysis (table ). the ilink and mlink options of the linkage software package (version . ) (ott ) were used to calculate lod scores by use of an ibm personal com- puter. the disease was modeled as an autosomal dom- inant, fully penetrant disorder. the allele frequencies for each marker were set as equal. for the haplotype anal- ysis, the order of the genetic markers was determined from the publicly available yac physical map from the stanford university genome center (ftp:// shgc.stanford.edu/pub/hgmc/yac�data) and from the order described elsewhere (polymeropoulos et al. ). results phenotype of family members as table indicates, the proband and a number of his paternal relatives have features similar to those observed in weyers acrofacial dysostosis, ellis–van creveld syn- drome, and jeune syndrome. of these conditions, the proband most closely fits ellis–van creveld syndrome, on the basis of his short stature, four-extremity postaxial polydactyly type a, dystrophic nails, abnormal labial frenula and sulcus, hypodontia with poor enamel, tho- racic dysplasia, congenital heart disease, neonatal pelvic x-ray significant for flat acetabular roof with pointed pelvic prominence, and survival postinfancy. if one as- sesses the family when the proband is excluded, however, the autosomal dominant mild short stature, four-extrem- ity postaxial polydactyly type a, onychodystrophy, hy- podontia, and abnormal frenula, with which the affected members present, closely resemble weyers acrofacial dysostosis. linkage to chromosome p and haplotype analysis the proband in this family has features most consis- tent with ellis–van creveld syndrome. therefore, we evaluated this family by using nine genetic markers from near the ellis–van creveld locus on chromosome p . (table ) (polymeropoulos et al. ). evidence for link- age to this region was observed, with lod scores . and no recombination for two of the markers, d s and d s . the highest two-point lod score was . with the genetic marker d s . one recombi- nation event, between the genetic markers d s and d s , was detected in individual iii: (fig. ), de- fining the most proximal boundary for the region con- taining the disease locus. recombinants that would have indicated the most distal boundary for the location of the gene were not detected within this -cm region. using the lod score . , we calculated the % con- fidence interval for the disease locus to be cm prox- imal and distal of d s . however, the genetic dis- tance between d s and d s , which flank d s , is only cm (polymeropoulos et al. ); thus, the recombinant (detected in individual iii: ) lim- its the proximal boundary to a maximum of cm from am. j. hum. genet. : – , d s . therefore, the region most likely to contain the gene is a -cm interval. this region encompasses the -cm critical region, between d s and d s , for the ellis–van creveld syndrome locus, re- ported elsewhere (polymeropoulos et al. ). hap- lotype analysis provided similar results, with the critical region found to reside distal to d s on the basis of recombination events detected in individual iii: (fig. ). sequencing of the msx candidate gene a candidate gene that maps within the critical region is the homeobox gene msx . the mouse homologue, hox- , is expressed in the neural crest, the developing mandible and teeth, the embryonic heart, and the limb buds (robert et al. ), all of which are developmental regions affected in ellis–van creveld syndrome. in ad- dition, a mutation in msx was recently reported in a family with selective tooth agenesis (vastardis et al. ), which may be relevant to the dental abnormalities observed in both ellis–van creveld syndrome and wey- ers acrofacial dysostosis. both exons of msx from the proband (iv: ) and his affected paternal uncle (iii: ) were sequenced. no mutations were detected, suggesting that changes in the msx coding region are not re- sponsible for the clinical phenotype of this family. this result is consistent with the recent report that no mu- tations were detected in either of the two msx exons in patients with ellis–van creveld syndrome (ide et al. ). discussion we have studied a four-generation family segregating a condition with features of both weyers acrofacial dy- sostosis and ellis–van creveld syndrome. the disorder in affected members of this family, including in the pro- band, demonstrated linkage to a chromosome p re- gion estimated to be cm and defined by recombi- nation only proximally between loci d s and d s . the linkage-analysis data clearly show that, in this family, the candidate region for the condition encompasses the ellis–van creveld gene. the mapping of these two diseases to the same chromosomal region might represent any one of several phenomena. one pos- sibility is that all affected members of this family have either an unreported autosomal dominant condition or weyers acrofacial dysostosis with variable expression, with the proband being the most severely affected. thus, the proband’s condition would be a genocopy for el- lis–van creveld syndrome. a second possibility is that the proband is a double heterozygote, with a mutation for the condition inherited from his father and a mu- tation for weyers acrofacial dysostosis, ellis–van cre- veld syndrome, or jeune syndrome inherited from his mother. a final possibility is that the proband has ellis–van creveld syndrome and that his “affected” paternal rel- atives are symptomatic heterozygotes. although some have questioned this interpretation (see omim [http://www.ncbi.nlm.nih.gov/omim/]) because no het- erozygous effects of ellis–van creveld syndrome were observed in the amish population (mckusick et al. ), reports exist of several families in which heter- ozygotes exhibit findings similar to those in the family reported here (fryns ; goldblatt et al. ; spran- ger and tariverdian ). in fact, a similar family, con- sisting of a proband with ellis–van creveld syndrome and her father, who had features of weyers acrofacial dysostosis (mild short stature, nail dysplasia, and teeth abnormalities but no polydactyly), has been reported (spranger and tariverdian ). the features observed in our proband’s paternal relatives could be the phe- notype resulting from one specific allele of the ellis–van creveld syndrome gene, an allele that differs from the allele affected in the amish population. the proband may have inherited this allele from his father and a dif- ferent mutant allele from his mother, leading to the full ellis–van creveld phenotype. although no mutations were detected in the coding region of msx in the two members of this family who were tested, it is conceivable that mutations in the msx regulatory elements may be responsible for the condition in this family and, perhaps, for ellis–van creveld syndrome. previous studies, using deletion analysis of the mouse msx promoter, identified regions that were important for distinct spatial and tem- poral expression patterns (mackenzie et al. ). another candidate gene in the critical region for the condition in our family is that for fibroblast growth- factor receptor (fgfr ). mutations in fgfr have been reported in patients with achondroplasia (rousseau et al. ; shiang et al. ), hypochondroplasia (bel- lus et al. ), thanatophoric dysplasia (tavormina et al. ; rousseau et al. ), and craniofacial and limb abnormalities resembling crouzon, pfeiffer, or sae- thre-chotzen syndromes (meyers et al. ; bellus et al. ). fgfr maps to chromosome p . (thompson et al. ) and is a potential candidate gene for the condition in this family or for weyers acrofacial dysostosis, but it is distal to the critical region for the ellis–van creveld locus (polymeropoulos et al. ). other genes located on human chromosome p (those for dopamine receptor d b [drd b], zinc finger pro- tein– [znf- ], dopamine receptor d [drd ], and protein s- p [s p]; the myosin light-chain reg- ulatory gene [myl ]; and homeobox gene h [hmx ]), identified in the syntenic region of mouse chromosome (those for phosphodiesterase b, cgmp- specific, rod, beta [pdeb]; diacylglycerol kinase, delta howard et al.: polydactyly maps to chromosome p [dagk ]; iduronidase, alpha-l [idua]; ldl-related protein-associated protein [lrpap ; alpha- -macro- globulin receptor-associated protein]; g-protein-coupled receptor kinase- [drosophila]–like [gprk l]; alpha- c-adrenergic receptor [adra c]; adducin, alpha subunit [add ]; huntingtin [hd]; and casein, beta [csn ]) (debry and seldin ; http:// www.ncbi.nlm.nih.gov/omim/homology/), or associ- ated with relevant mouse models are not obvious can- didates, because their developmental expression pat- terns, function, or phenotypic effects differ significantly from the features present in the affected members of the family in this study. the concept of a single gene causing both dominant and recessive conditions, as is possibly the case reported here, is not uncommon. mutations in the genes for rho- dopsin (rosenfeld et al. ), von willebrand factor (zimmerman and ruggeri ), thyroid hormone re- ceptor–beta (takeda et al. ), globin-beta (thein et al. ), and collagen types a (pruchno et al. ), a (de paepe et al. ), and a (christiano et al. , ) have been found in both autosomal dom- inant and recessive diseases. for example, a much more severe osteogenesis imperfecta phenotype was found in two sibs homozygous for mutations in the collagen type a gene than was found in their heterozygous parents or sibs (de paepe et al. ). this finding is similar to those for the family studied here, in which the hetero- zygous carriers are affected more mildly than the pre- sumed homozygote (the proband). a potential mecha- nism for different mutations in the same gene, causing autosomal dominant and recessive inheritance patterns, is that certain mutations in the dominant form lead to dominant-negative effects, as is the case in generalized resistance to thyroid hormone (takeda et al. ; hay- ashi et al. ). in our family, a mutation in one gene may have a dominant-negative effect in the heterozy- gotes, leading to weyers-acrofacial-dysostosis–like fea- tures, but this mutation in combination with a second mutation would lead to loss of function in the homo- zygote, resulting in the ellis–van creveld phenotype. ex- pression analysis and examination of the regulatory re- gion of msx and of other candidate genes that later may be mapped to chromosome p in affected mem- bers of this family will help to determine the etiology of this condition. acknowledgments the work was supported by nih grants de and de , outpatient general clinical research center grant rr , and shriners hospital for crippled children re- search grant project . references bellus ga, gaudenz k, zackai eh, clarke la, szabo j, fran- comano ca, muenke m ( ) identical mutations in three different fibroblast growth factor receptor genes in auto- somal dominant craniosynostosis syndromes. nat genet : – bellus ga, mcintosh i, smith ea, aylsworth as, kaitila i, horton wa, greenhaw ga, et al ( ) a recurrent mu- tation in the tyrosine kinase domain of fibroblast growth factor receptor causes hypochondroplasia. nat genet : – biggerstaff rh, mazaheri m ( ) oral manifestations of the ellis–van creveld syndrome. j am dent assoc : – christiano am, greenspan ds, hoffman gg, zhang x, tamai y, lin an, dietz hc, et al ( ) a missense mutation in type vii collagen in two affected siblings with recessive dys- trophic epidermolysis bullosa. nat genet : – christiano am, ryynanen m, uitto j ( ) dominant dys- trophic epidermolysis bullosa: identification of a gly-to-ser substitution in the triple-helical domain of type vii collagen. proc natl acad sci usa : – curry cj, hall bd ( ) polydactyly, conical teeth, nail dys- plasia, and short limbs: a new autosomal dominant mal- formation syndrome. birth defects : – de paepe a, nuytinck l, raes m, fryns j-p ( ) homo- zygosity by descent for a col a mutation in two sibs with severe osteogenesis imperfecta and mild clinical ex- pression in the heterozygotes. hum genet : – debry rw, seldin mf ( ) human/mouse homology rela- tionships. genomics : – ellis rwb, van creveld s ( ) a syndrome characterized by ectodermal dysplasia, polydactyly, chondrodysplasia and congenital morbus cordis: report of three cases. arch dis child : – fryns jp ( ) postaxial polydactyly as heterozygote mani- festation in ellis–van creveld syndrome? 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